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P 90-180 d
Buchin et al. (1998) Gaya et al. (1990) Ordonez et al. (1999)
Mendia et al. (2000)
240 d: 240 days of ripening. Water: water-soluble nitrogen. pH 4.6, pH 4.6 soluble nitrogen. 12% TCA: 12% trichloroacetic acid-soluble nitrogen. PTA: nitrogen soluble in 5% phosphotungstic acid. FAAs: free amino acids.
Lipolysis
The level of lipolysis and its impact on cheese quality depend on the cheese variety. It is limited in Swiss-type and semi-hard cheeses, where a level higher than 0.25-1.5% (Cheddar, depending on the commercial quality), 1.5% (Gouda), 1% (Emmental) or 2% (Comte), is considered to induce flavour defects such as rancid (Choisy etal., 1997). In contrast, lipolysis is much more extensive in some hard Italian, mould-ripened (5-20%), blue-veined (18-25%) and goat-milk cheeses, where lipolysis is essential for typical flavour formation. Lipolysis in cheese is due to the activity of indigenous milk lipoprotein lipase (LPL), to lipases or esterases of starter bacteria or the native microflora, or to added pregastric esterases (Deeth and Fitz-Gerald, 1983; Gripon, 1993). Table 4 summarises the differences between FFAs obtained in R, P or MF cheeses.
In semi-hard and hard, uncooked cheeses, the total amount of FFAs appears to be lower in P than in R cheeses (Table 4); more than 50% lower in Cheddar (McSweeney etal., 1993; Shakeel-Ur-Rehman etal., 2000b,c) and 38% lower in Manchego (Gaya etal., 1990). This was attributed to the inactivation of LPL by pasteurisation. This inactivation varies according to the severity of the heat treatment since heating for 15 s at 70 or 75 ~ results in a residual activity of 2% and 0%, respectively (Andrews etal., 1987). In the study of McSweeney etal. (1993), lipolysis in MF Cheddar cheeses was comparable to that in P cheeses; so it is likely that lipases of the native microflora (NSLAB in this case) could also play a role. According to Choisy et al. (1997), LPL would have a lesser impact on the lipolysis of R cheeses than microbial or technological enzymes. Some fatty acids do not originate from lipolysis, e.g., only fatty acids with six or more carbon atoms
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Raw Milk Cheeses
come exclusively from lipolysis. The proportion of short-chain fatty acids in total FFAs varies according to the extent of lipolysis of the cheeses; it can reach 50-80% (mol/mol) in hard-cooked cheeses. Thus, the validity of global measurement to estimate lipolysis in such cheeses is questionable, and it can be concluded that the examination of individual FFAs is more informative. The higher level of lipolysis in R compared with P cheeses affects different fatty acids: all FFAs from C6 to C16 in Cheddar cheeses (Shakeel-Ur-Rehman et al., 2000a), all the major fatty acids in a goats' milk hard cheese (Buffa et al., 2001a), only short-chain (C4-C8) fatty acids in Cheddar (Shakeel-Ur-Rehman etal., 2000c) and only C6, C8 and C18:1 in Canestrato Pugliese cheese (Albenzio et al., 2001). All the major fatty acids in Cheddar were affected to the same extent by pasteurisation or MF (McSweeney et al., 1993). In the study by Chavarri et al. (2000), the amounts of short-chain FFAs (C4 to C10) and of C16:1, C18:0, C18:1 in Idiazabal were affected by pasteurisation. But, the effect of pasteurisation varied according to the season; amounts of FFA were higher in R cheeses made in winter and spring, but lower in those made in summer. In the study by Buchin et al. (1998) on R and P Morbier-type, semi-hard cheeses, no differences were observed in the levels of C6 to C9 fatty acids. The attribution of the observed differences in lipolysis, either to LPL or to microbial lipases, is difficult. Inactivation of LPL was suggested by Gaya etal. (1990). Chavarri et al. (1998) found that depending on the period of the trial, LPL was inactivated by 73-95%, by pasteurisation of milk for the manufacture of Idiazabal. Short-chain fatty acids were released preferentially during ripening, showing microbial participation in lipolysis, but the balance between the different FFAs was not affected by pasteurisation, indicating that microbial lipolysis was not significantly affected by pasteurisation (Chavarri et al., 2000). Albenzio et al. (2001) attributed the slight differences in lipolysis between R and P cheeses to differences in lipase and esterase activities of the milk microflora (NSLAB). Shakeel-Ur-Rehman et al. (2000a) also attributed the differences observed in Cheddar to the activity of NSLAB, because NSLAB were highest in the most highly lipolysed cheeses. However, agreement between NSLAB counts and FFA levels did not occur in all cheeses, so the authors supposed that not only number but also species or strains play a role. In hard cheeses, such as Cheddar, where moulds are not present, LAB could be the main cause of lipolysis (Choisy et al., 1997). In particular, starter LAB may contribute to the release of fatty acids, because they have lipase and esterase activities (Deeth and Fitz-Gerald, 1983).
327
The role of starter LAB in the observed differences is also questionable; modification of their activity due to interaction with the natural microflora cannot be excluded. Such differences could be expected in soft cheeses, but the studies on this type of cheese show contradictory results. In goats' milk mould-ripened cheeses, Morgan et al. (2001) found higher lipolysis, determined by a global method, in R cheeses than in P cheeses. The extent of lipolysis in mature cheeses was related to that of the milk. Pasteurisation was supposed to inactivate LPL. But, in this study, pasteurisation also induced lower moisture cheeses, which could reduce the activity of the lipolytic surface microflora. In contrast, Sousa and Malcata (1997) found higher lipolysis in P cheeses made from ovine milk (Serra) than in R cheeses, despite higher levels in the fresh cheese. However comparison between the two types of cheese was made difficult by a higher fat retention in P than in R cheeses, and the amounts of FFAs were expressed per weight of cheese. Thus, in both studies, interpretation of the results is difficult because of the difficulties in controlling the cheese composition. In Swiss-type cheeses, the level of C6 could be a good indicator of the extent of lipolysis, as suggested by Kuszdal-Savoie (Choisy et al., 1997). In the studies of Bouton and Grappin (1995), Beuvier et al. (1997) and Demarigny et al. (1997), the variations in C4 followed those of C6, which indicates that, in these cases, C4 was mainly from lipolysis, and not from the growth of clostridia on lactate. Bouton and Grappin (1995) and Beuvier et al. (1997) found no differences in these two acids in cheeses (R, P, MF or P with the addition of the natural microflora), whereas Demarigny et al. (1997) found that R cheeses tended to be more lipolysed than MF cheeses, depending on the season of the year and the age of the cheese. In the latter cheeses, lipolysis may have been of microbial origin. On the one hand, the heating of the milk in the vat, performed in this type of technology, would inactivate the LPL (Chamba and Perreard, 2002), which would explain why no differences were observed between P and MF cheeses (Beuvier et al., 1997). On the other hand, thermophilic starter LAB and propionibacteria have lipolytic activity in Swisstype cheeses (Deeth and Fitz-Gerald, 1983). According to Chamba and Perreard (2002), the lipolytic activity of propionibacteria is far greater than that of starter bacteria. Moreover, it could be strain-dependant, which could also explain the variability in the above results. Volatile compounds
Comparisons of the volatile compounds in R and P or MF cheeses, have been undertaken only recently and are not very numerous. The same volatile compounds
328
Raw Milk Cheeses
were present in each type of cheese within the same study, whether the microflora was present or removed from the milk. Differences were observed in the levels of many volatile compounds, R cheeses generally having higher levels than P or MF cheeses, with exceptions in certain chemical families.
Volatile fatty acids (VFAs) Volatile fatty acids in cheese are the products of various metabolic pathways, mostly microbial. Acetic acid may have different origins, e.g., from the catabolism of lactose, citrate, amino acids, or the propionic fermentation (PF). Propionic acid is one of the end products of lactose or lactate fermentation by propionibacteria. Butyric acid can originate from the catabolism of triglycerides, but also from lactate fermentation by clostridia. Hexanoic acid (C6) is found only in triglycerides and is liberated by lipolysis (its behaviour is described under Lipolysis and catabolism of fatty acids in cheese. Branched-chain VFAs result from catabolism of the amino acids, e.g., 2-methyl propanoic (isobutyric), 2-methyl butanoic, 3-methyl butanoic (isovaleric) acids from valine, isoleucine and leucine, respectively. These compounds are major volatile compounds in cheese, and participate significantly in the flavour of many cheese varieties, as suggested by results from olfactometry: acetic acid has a characteristic vinegar flavour, propionic acid a pungent, fruity one, butyric, isobutyric and isovaleric acids have similar flavours, associated with cheesy, sweaty, rancid and sour notes (Yvon and Rijnen, 2001; Curioni and Bosset, 2002). Table 5 summarises the differences between VFAs obtained in R, P or MF cheeses. The PF is one of the major fermentations that occur in Swiss-type cheeses (Curioni and Bosset, 2002). In the studies of Bouton and Grappin (1995), Beuvier et al. (1997), Demarigny et al. (1997) and Eliskases-Lechner et al. (1999), the presence of the raw milk microflora was associated with higher levels of acetic and propionic acids and lower levels of lactic acid in the ripened cheeses. These results showed a greater PF in R cheeses, due to the presence of the native propionibacteria, which are eliminated by pasteurisation or microfiltration. It should be noted that no propionibacteria were added to the cheeses studied, as required in the manufacture of Bergk~se or Comte. In contrast, in a hard cheese such as Emmental, in which propionibacteria are used as starters to ensure a high level of PE the effect of native populations of propionibacteria seems to disappear. Buchin et al. (2002) compared R and MF Emmental-type cheeses: the total population of propionibacteria tended to be lower in R cheeses from midripening, with a subsequent lower level of propionic acid at the end of ripening.
Generally, acetic acid is more important in Swisstype cheeses made from R milk than in those from P or MF milk (Bouton and Grappin, 1995; Demarigny et al., 1997; Eliskases-Lechner et al., 1999). This was related to the presence of propionibacteria and facultatively heterofermentative lactobacilli (FHL) in the R milk. In Swiss-type cheeses, the level of butyric acid tended to be higher in R than in MF cheeses (Demarigny et al., 1997). Because no butyric acid bacteria were found in these cheeses, the origin of this compound could not be butyric fermentation. It is likely that it was liberated from triglycerides by the lipolytic activity of the microorganisms present in the cheese, particularly propionibacteria (Chamba and Perreard, 2002). Caproic acid (C6) followed the same trend as butyric acid, but the differences between R and MF cheeses were less marked (see 'Lipolysis'). In contrast, Bouton and Grappin (1995) and Beuvier et al. (1997) observed no differences between R, P, MF or P cheeses with added native microflora. The level of isovaleric acid was higher in R cheeses than in P or MF cheeses (Beuvier et al., 1997; Demarigny et al., 1997). The occurrence of this compound was correlated with FHL, propionibacteria or enterococci populations, and at least the former two populations may be involved (Langsrud and Reinhold, 1973; Paulsen et al., 1980; Thierry and Maillard, 2002). However, Buchin et al. (2002) found that the production of isovaleric acid was positively correlated with the level of propionibacteria, but negatively with the levels of starter LAB, native mesophilic lactobacilli or enterococci. The influence of the native microflora on VFA production was also observed in other cheese varieties, e.g., Cheddar (Shakeel-Ur-Rehman et al., 2000a,c), Raclette (Klantschitsch et al., 2000) and Morbier-type cheese (Buchin et al., 1998). In Cheddar, several VFAs, e.g., acetic, propionic, butyric, valeric and caproic acids, were found at higher levels in R than in P cheeses. Moreover, the levels of all these compounds increased at a ripening temperature of 8 ~ compared with 1 ~ and the levels in cheeses of the different acids, except caproic, increased with the proportion of raw milk in several blends of R and P milk. The authors attributed all these differences to the presence of NSLAB in the raw milk, because their presence and growth in cheese were closely related to the production of fatty acids. NSLAB were mostly eliminated by pasteurisation, and the higher the R:P milk ratio or the higher the ripening temperature, the higher was the number of these bacteria in cheese, at all times during ripening. The influence of the presence of the native microflora on the production of acetic and propionic acids in a semi-hard, Morbier-type cheese was confirmed by Buchin et al. (1998) (comparison of R and P cheeses), by Klantschitsch et al. (2000) in Raclette (comparison of R, P and MF cheeses) and by
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Raw Milk Cheeses
Ortigosa et al. (2001) in ovine Roncal cheese (comparison of R, P and P + Lb. casei). In Cheddar cheese, the variations in VFAs originating from amino acid catabolism were not the same as those of other VFAs. In a study by Shakeel-Ur-Rehman et al. (2000c), 2- and 3-methyl butanoic acids were found at higher levels in P cheeses than in R cheeses, and in another of their studies (Shakeel-Ur-Rehman et al., 2000a), 2-methyl propanoic and 3-methyl butanoic acids were found at higher levels in cheeses ripened at low temperature (1 ~ compared to 8 ~ These compounds correlated negatively with the number of NSLAB in the cheeses, so the authors hypothesised that NSLAB have a minor role in the formation of these compounds or that NSLAB broke them down further.
Carbonyl compounds Tables 6 and 7 summarise the differences between ketones and aldehydes found in R, P or MF cheeses. Diacetyl (2,3-butanedione) and acetoin (3-hydroxy2-butanone) are products of the metabolism of citrate by Lc. lactis spp. lactis bv. diacetylactis, Leuconostoc spp. or some Lactobacillus species (McSweeney and Sousa, 2000). They are important aroma compounds in numerous varieties of cheese (Curioni and Bosset, 2002). Nevertheless, they tend to decrease during ripening. In 6-month-old Cheddar, diacetyl is present in quantities too low to participate in flavour (Urbach, 1997). In cheese, diacetyl can be converted into acetoin, then 2,3butanediol and 2-butanone and finally 2-butanol (Urbach, 1993). It is notable that 2-butanone and 2-butanol, in contrast to diacetyl and acetoin, generally increase during ripening, like other methyl ketones and secondary alcohols (Bosset and Liardon, 1985; Barlow etal., 1989; Urbach, 1993; Carbone|l etal., 2002). Therefore, it is likely that the transformation of diacetyl into the more reduced compounds progresses throughout ripening due to the enzymatic activities of microorganisms, so the raw milk microflora can be expected to influence these compounds. Indeed, diacetyl and acetoin were found in lower quantities in R cheeses than in P or MF cheeses in semi-hard (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000a,c; Ortigosa et al., 2001) or Swiss-type cheeses (Buchin et al., unpublished). The contrasting result was observed in only one study on Morbier-type cheese (Buchin et al., unpublished). The quantities of 2-butanone in ripened cheese were affected differently by the presence of the raw milk microflora. Ortigosa et al. (2001) found less 2-butanone in Roncal cheese made from raw ewes' milk than in cheese made from P milk, but the opposite was true for the Morbiertype cheeses studied by Buchin etal. (1998). These contradictory results could be expected because the amount of 2-butanone in a ripened cheese arises from
the balance between its formation and its reduction to 2-butanol. It is difficult to establish a correlation between the presence of microbial populations and the level of 2-butanone at a given time of ripening, because of the continuous production and degradation of this compound. To follow the evolution of microbial growth and the level of 2-butanone throughout the ripening period may provide valuable information. In contrast, as expected, 2-butanol was more abundant in R cheeses than in P cheeses in all studies (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000c; Ortigosa et al., 2001). The other carbonyl compounds found in cheeses are methyl ketones and aldehydes. Their characteristics have been summarised in a recent review by Curioni and Bosset (2002). Methyl ketones originate from the B-oxidation of fatty acids by microorganisms. Their aromatic impact is of primary importance in blue and surface mould-ripened cheeses, but they are also likely to have an influence on the flavour of other varieties, e.g., 2-heptanone in Emmental, Gruyere and Grana Padano. This ketone is typical of blue cheese flavour, whereas the others have fruity, floral or musty notes. Straight-chain aldehydes originate from the oxidation of unsaturated fatty acids. They are characterised by green, fatty odours. The branched-chain aldehydes with four or five carbon atoms originate from the catabolism of the amino acids, valine (2-methyl propanal), leucine (2-methyl butanal) and isoleucine (3-methyl butanal). In cheese, they have green or malty notes. As previously noted, the variations of these compounds in the presence of the raw milk microflora differed according to the study. In the studies of ShakeelUr-Rehman etal. (2000c) and Buchin etal. (1998) on Cheddar and Morbier-type cheese, respectively, R cheeses contained less of the different methyl ketones and aldehydes than P cheeses. This is in contrast with the results of the studies of Shakeel-Ur-Rehman et al. (2000a) comparing R and P Cheddar, and Buchin et al. (2002) comparing R and MF Morbier-type cheeses. The results of Buchin et al. (2002) showed less ketones but more aldehydes in R Swiss-type cheeses compared to MF ones. The explanation for these contrasting results may be the same as that for the products of pyruvate metabolism. Methyl ketones and aldehydes are intermediate products in the degradation of fatty acids or amino acids. Due to the enzymatic activities of microorganisms in cheese, methyl ketones are progressively reduced to 2-alkanols, aldehydes are oxidised to acids or reduced to n-alkanols. Therefore, their levels depend on the balance between production and degradation, which is linked to the degree of maturity of the cheese. The maturity of the cheeses for each of the studies may be
Raw Milk Cheeses
Table 6
Ketones in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses
Cheese
Emmental
Cheddar
Cheddar
Propanone 3-Hydroxy-2butanone (acetoin) 2,3-Butanedione (diacetyl) 2-Butanone
R < MF R < MF
R
R
3-Methyl 2butanone 2-Pentanone 2,3-Pentanedione 4-Methyl 2pentanone 3-Methyl 2pentanone Cyclopentanone 2-Hexanone 4-Methyl cyclohexanone Cyclohexanone 2-Heptanone 3-Methyl 2heptanone 5-Methyl 2heptanone 6-Methyl 2heptanone 6-Methyl-5-hepten2-one 2-Octanone 2-Nonanone 8-Nonen-2-one 2-Decanone 2-Undecanone 2-Dodecanone 2-Tridecanone 2-Pentadecanone Acetophenone Reference
a b c d
331
Semi-hard
Semi-hard
Roncal
n.d. a
R
n.d. R > MF
n.d. n.d.
R < MF
R
n.d.
R < MF
R>P
R>MF
R
P R
P
Semi-hard
R> P R>P
Semi-hard
Roncal
n.d. R > P (240 d) b n.d.
n.d.
ShakeeI-UrRehman et al. (2000c)
ShakeeI-UrRehman et al. (2000a)
Buchin et aL (1998)
Buchin et al. (unpublished)
Ortigosa et al. (2001)
a n.d no difference. b 240 d: 240 days of ripening.
compounds, or indirect, by modifying the composition of the cheese, with the production of precursors of volatile compounds or of molecules that influence chemical reactions or the activity of other microorganisms. In particular, the activities of the indigenous populations can interfere with those of the starter bacteria. Within the complexity of the native milk microflora, it is difficult presently to establish the role of each population, at the species and at the strain level. It is likely that many of the metabolic pathways producing volatile compounds are strain-dependant (Yvon and Rijnen, 2001), which would make their elucidation all the more difficult. The development of molecular techniques for the discrimination of microbial populations at the strain level could be very beneficial to such studies. This situation underlines the importance of maintaining a high diversity of strains in the milk, to retain the diversity of the molecules produced.
Table 11
Lactones in raw (R) and pasteurised (P) milk cheeses
Cheese
Cheddar
Cheddar
-,/-Octanolacton e y-Decanolactone -,/-Dodecanolactone y-Hexadecanolactone y- Decan olacton e y-Dodecanolactone y-Dodecenolactone
PR 10b< P> R n.d. n.d. n.d. R< P R< P
n.d. a R> P R> P n.d. n.d. R> P R> P
Reference
ShakeeI-UrRehman et aL (2000c)
ShakeeI-UrRehman et aL (2000a)
a n.d.: no difference. b PR10: mix of 90% pasteurised milk with 10% of raw milk.
Sensory Aspects In order to avoid any ambiguity, due to the different use of the same terms by different authors, we have chosen to define sensory perceptions as follows: odour is perceived by the nose, with no introduction of the food into the mouth, while flavour is the perception of the food during mastication, either retronasaly or by the tongue (five basic tastes: sweet, acid, bitter, salty, umami). Flavour/odour
Table 12 summarises the differences between the flavour and odour attributes reported for R, P or MF cheeses. Raw milk cheeses ripen faster than cheeses made from milk, the microflora of which has been removed. As a consequence, R cheeses tend to develop a stronger odour/flavour at the same age than those made from P or MF milk (Johnson et al., 1990b; Lau et al., 1991). This has been observed in all types of cheese studied: Cheddar (McSweeney et al., 1993; Shakeel-Ur-Rehman et al., 2000a,b), Manchego (Gaya et al., 1990; FernandezGarcia et al., 2002; Gomez-Ruiz et al., 2002), Raclette (Gallmann and Puhan, 1982), other hard and semi-hard cheeses (Lau et al., 1991; Van den Berg and Exterkate, 1993; Buchin etal., 1998; Skie and ArdO, 2000), Bergk~se (Ginzinger et al., 1999a), Swiss-type cheeses (Bouton and Grappin, 1995; Beuvier etal., 1997; Demarigny et al., 1997) and soft goats' milk cheese (Morgan et al., 2001). In all cases, this phenomenon seems to be directly linked to the activity of the indigenous microflora of the milk. In Cheddar, it has been attributed, in part, to the presence of NSLAB (composed mainly of lactobacilli, but also of pediococci and micrococci) in the raw milk, which are the major part of the natural microflora of this variety of cheese
336
Raw Milk Cheeses
Table 12
Characteristic flavour and odour attributes of raw (R), pasteurised (P) and microfiltered (MF) milk cheeses
Cheese variety
Raw milk
Bergk&se
Odour: intense Flavour: intense Flavour: intense, typical, acid, pungent Flavour: intense, pungent, salty Odour: intense, creamy/milky, fruity/sweet, acid/sharp, pungent Flavour: intense, sour/ acid, sulphur/eggy, bitter, rancid, unclean Only cheeses ripened at 8 ~ (vs 1 ~ Odour: intense, acid Flavour: intense, sour Odour: intense, fruity/ sweet, pungent Flavour: sour/acid Flavour: intense, of acid milk, of rind, Flavour: of fresh milk, fruity, of garlic, spicy, animal, chemical, rancid, bitter, pungent Flavour: intense, animal, spicy, sour
Swiss-type Swiss-type Cheddar
Cheddar
Cheddar
Semi-hard cheese, Morbier-type
Semi-hard round-eyed cheese Roncal
Idiazabal
Idiazabal
Odour: intense (120 d), animal (240 d) Flavour: characteristic, pungent (240 d), animal Aftertaste :intense Odour: characteristic, pungent, sour Flavour: characteristic, pungent, salty Aftertaste: characteristic, pungent Flavour: characteristic, creamy, pungent, acid
Pasteurised milk
Microfiltered milk
Flavour: bitter Flavour: bitter Flavour: acid, bitter, salty Odour: musty
Reference
Ginzinger et al. (1999a) Bouton and Grappin (1995) Beuvier et aL (1997) ShakeeI-UrRehman et aL (2000a)
ShakeeI-UrRehman et aL (2000b) ShakeeI-UrRehman et aL (2000c) Buchin et aL (1998)
Skie and Ard5 (2000)
Odour: animal (120 d)
Ortigosa et aL (2001)
Flavour: torrefied (240 d) Odour: sweet
Mendia et aL (1999)
Flavour: sweet, bitter, sour Aftertaste: bitter, sour Flavour: sweet
Ordonez et aL (1999)
Odour: sweet
(McSweeney et al., 1993). In Swiss-type cheeses (Beuvier et al., 1997), flavour intensity was correlated with counts of FHL, propionibacteria and enterococci, which occur naturally in the raw milk. In pasteurised milk cheeses, denaturation of enzymes and whey proteins by the heat treatment may also be involved; the aggregation of whey proteins on the surface of the caseins micelles also prevents proteolysis of the caseins. This difference in maturity is enhanced by the temperature of ripening and depends on the age of the cheese (Klantschitsch et al., 2000; Shakeel-Ur-Rehman et al., 2000b).
Besides the intensity of flavour, differences in the flavour profile of cheese can be observed. The flavour of the ripened cheese is richer and more complex when the indigenous microflora is present in the milk to be processed. Some observations are constant from one study to another, whereas others vary. In almost all studies comparing cheeses made from raw milk and raw milk after elimination of the microflora, the R cheeses received a higher score for the pungent attribute. Similarly, acid, sour or rancid characteristics were also generally higher in these cheeses. It is likely that these sensory attributes are related to the presence of volatile and FFAs (Curioni and Bosset, 2002;
Raw Milk Cheeses
Gomez-Ruiz et al., 2002). In general, R cheeses are characterised by more 'strong' attributes, such as animal, garlic, spicy, sulphur and unclean. All these characteristics of R cheeses are linked to the notion of higher maturity, expressed from a sensory point of view, but also revealed by physico-chemical patterns, i.e., a greater degree of proteolysis, a higher content of most volatile compounds, and sometimes greater lipolysis (Fig. 2). The distribution of milder attributes, such as fruit, milk or sweet, differs with the study; they can be characteristic of cheeses made either from R or P milk. In Idiazabal cheese, Ordonez et al. (1999) found a relationship between the sweet taste and the amounts of free proline and asparagine, which were higher in P cheeses. Fruity notes may be linked to some methyl ketones such as 2-nonanone (Gomez-Ruiz et al., 2002) or esters (Ortigosa et al., 2001; Gomez-Ruiz et al., 2002). Milky notes are characteristic of diacetyl and acetoin (GomezRuiz et al., 2002). The relationship between bitterness and the presence of the microflora depends on the variety of cheese. When differences were observed in relation to the milk treatment, R semi-hard cheeses were more bitter than P (Buchin etal., 1998; Shakeel-Ur-Rehman etal., 2000a) or MF cheeses (Buchin et al., 2002). In contrast, hard cheeses made from R milk were less bitter than those made from MF or P milk (Bouton and Grappin, 1995; Beuvier et al., 1997; Mendia etal., 1999; Ginzinger et al., 1999a). On the one hand, it seems that the presence of the indigenous microflora is involved,
Axis 3 14 %
2heptanone 2,3peb.tar ~edione i I Ax~s 1 9 dia~tyl ~ 43% 9 aceto.~ ! '..... 2pentan#ne 3m/~ butana ...... 3me 2penta.aOne// ..............................9........heptan/e "Hk
Figure 2 Distribution of volatile compounds and flavour attributes (additional variables, italicized and boldfaced) within R ( . ) and P (O) semi-hard Morbier-type cheese using principal component analysis C2, C3, C5: acetic, propionic, valeric acids (from Buchin et al., 1998).
337
because P and MF cheeses were similar and differed from R cheeses. On the other hand, the heat treatment is likely to play a role. In the study by Beuvier et al. (1997), where R, R MF and P + indigenous microflora (PR) milks were processed, the most bitter cheeses were P and PR. Bitterness is attributed mainly to the presence of hydrophobic peptides, resulting from the hydrolysis of caseins, mostly ORS1- and 6-. Bitterness in cheese results from the balance between the production of bitter peptides by the action of rennet (preferentially in semi-hard cheeses), plasmin (preferentially in hardcooked cheeses), bacterial proteinases and peptidases, and their further degradation by bacterial peptidases. The role of the respective proteolytic systems and their interactions are known to differ according to the cheese variety (Bergere and Lenoir, 1997). In Bergk/ise, a Swisstype cheese, Ginzinger etal. (1999a) found more hydrophobic peptides in P cheeses, which were also more bitter than R cheeses. This distribution of peptides was confirmed in Cheddar by Lau et al. (1991). According to Gomez et al. (1997), bitterness of peptide origin is more likely to be masked by other flavour components in R than in P cheeses. Bergere and Lenoir (1997) pointed out that other components such as indole, amino acids, amines, amides, long-chain ketones or monoglycerides could contribute to the bitter taste of cheese. Thus, Ordonez et al. (1999) found a relationship between bitterness and the amounts of arginine and aromatic amino acids in Idiazabal, though no differences in bitterness were found between R and P cheeses. It is likely that the presence of the R milk microflora affects the flavour characteristics in two ways: on the one hand, acceleration of ripening by faster metabolic pathways, and, on the other hand, the occurrence of a greater variety of metabolic pathways, specific to particular strains of bacteria, which is also influenced by the microbial diversity. The acceptability of R cheeses is also dependent on the cheese variety. Cheddar cheese made from raw milk is, in general, of lower quality than that made from pasteurised milk (Johnson et al., 1990b; McSweeney et al., 1993). In the study of Shakeel-Ur-Rehman et al. (2000b), R Cheddar cheese received higher flavour scores than P cheeses, but this was dependent on the ripening temperature, as higher temperatures (8 ~ instead of 1 ~ led to defects in R cheeses after 6 months. In the study by Morgan et al. (2001), soft goat's milk cheeses had more flavour defects when made from raw than from pasteurised milk, in relationship to microflora and lipolysis levels. Moreover, although the 'goat' flavour of these cheeses is linked to the liberation of particular fatty acids (Le Quere et al., 1996), no differences were found in this attribute.
338
Raw Milk Cheeses
According to Klantschitsch et al. (2000), the quality of Raclette cheese in relation to raw milk is related to ripening temperature and time; to avoid flavour and openness defects, R cheeses should be ripened for less than 90 days at 11 ~ or 60 days at 14 ~ whereas P or MF milk cheeses can be ripened at 17 ~ for 90 days. These differences in acceptability are of course related to the speed of ripening, since the presence of the native microflora accelerates biochemical transformations in the cheese. The difference in maturity, and hence in the occurrence of defects, is more perceptible in soft or semi-hard cheeses, because of their high moisture content; biochemical activities are favoured by the presence of water. Thus, besides the elimination of pathogens, pasteurisation is useful in this type of cheese to obtain a longer shelf-life by slowing the ripening and delaying the occurrence of flavour defects. The consumer of these cheeses is used to the milder flavour provided by pasteurised milk, and may regard the stronger flavour of R cheeses as a defect. It is likely that these varieties of cheese made from raw milk would be appreciated mostly by 'connoisseurs'. Conversely, Swiss-type cheeses, hard Italian cheeses (Johnson et al., 1990b; Bouton and Grappin, 1995), or hard Spanish ovine cheeses, like Idiazabal (Ordonez et al., 1999; Chavarri et al., 2000), are preferred when made from raw milk. Because of their low moisture content, hard cheeses ripen more slowly than soft or semi-hard ones. The presence of the natural microflora in the raw milk may have a lesser influence on the speed of ripening and on the shelf-life of these cheeses. The use of raw milk does not induce defects, and may even reduce some, e.g., bitterness. Moreover, the more complex flavour provided by raw milk may be appreciated by the consumer of hard cheeses. The loss of microflora and, to a lesser extent, of native milk enzyme activities, in pasteurised milk, affects the typical flavour of these cheeses. In Idiazabal cheese, the level of the sensory scores was related to the level of lipolysis, the less lipolysed cheeses being rated 'rather mild', suggesting that this cheese requires a minimum level of lipolysis to develop its characteristic flavour (Chavarri et al., 2000). In hard Italian cheeses such as Romano, Parmesan or Asiago, the inhibition of milk lipase (LPL) in pasteurised milk may be detrimental to the development of typical flavour (Johnson et al., 1990b). In goats' milk cheeses, the preservation of LPL activity can be important for the development of the 'goat' flavour, linked to the liberation of typical goat-flavoured fatty acids from glycerides (Le Qu~r~ et al., 1996). According to Ordonez et al. (1999) and Chavarri et al. (2000), the characteristic Idiazabal flavour is related to the extent of proteolysis. In Swiss-type cheeses, Bouton and Grappin (1995) found a relationship between the extent of primary proteolysis
and the flavour intensity, whereas the typical flavour was related to the concentration of propionic acid. The presence of the raw milk microflora contributes to the sensory diversity of raw milk cheeses. This has been supposed by Shakeel-Ur-Rehman et al. (200Oh) for Cheddar cheese, and shown in Swiss-type cheese models by Beuvier et al. (1997) and Demarigny et al. (1997). There is a higher heterogeneity in the sensory characteristics of cheeses when the native microflora was retained than when it was removed from milk (Fig. 3). The diversity of R cheeses is likely to depend on the level but also on the nature of the strains present in the microflora. Whether the strains in themselves have different metabolic potentialities or interfere by affecting the activity of starter bacteria has not yet been elucidated. Nevertheless, Bouton and Grappin (1995) have shown an interaction between the composition of starter mixtures and the raw milk microflora in the biochemical transformations and sensory characteristics of Swiss-type cheeses. Thus, whatever the mechanisms involved, the preservation of the microbial diversity in raw milk seems to contribute to the diversity of cheeses such as Swiss-type cheeses, particularly Comte. This diversity in the sensory characteristics is a point of major interest in the production of PDO cheeses. Texture
The texture of cheeses is the macroscopic expression of the structure of the cheese matrix, i.e., its composition and organisation. The texture is formed during two
Axis 2 17%
ABxis 1
{
.~_
~ I _ C N
Acid
Pungent) C3 Salted Aroma 9 1 6 2
P+bact
y-CN
Figure 3 Distribution of physico-chemical, microbiological and flavour criteria (additional variables, italicized and boldfaced) within raw (R), pasteurised (P), microfiltered (MF) and pasteurised + microorganisms contained in retentate (P + Bact) milk using principal component analysis. MesoLb: mesophilic lactobacilli; Entero:enterococci; PAB: propionibacteria; C2: acetic acid; C3: propionic acid; iC5: isoavaleric acid; PTA: PTA-soluble N (from Beuvier et aL, 1997).
Raw Milk Cheeses
stages of cheese processing: manufacturing and ripening. The events that occur during these two steps are different in nature. During manufacture, the cheese matrix is formed. It begins with coagulation of the milk, where the caseins organise themselves into a network, entrapping fat globules, water pockets and gas bubbles. The initial structure of the network is thus determined by the composition of the milk, and also by the technological conditions of coagulation: renneting parameters and work in the vat which influence the moisture content of the curd. The network is then modified by acidification due to the fermentation of lactose, that begins in the vat and continues in the mould. Acidification influences the extent of mineralisation of the caseins, and thus their hydration as well as their interactions. During ripening, changes occur in the matrix through the influence of the loss of water and proteolysis. Proteolysis begins with coagulation in the vat; this is essentially primary proteolysis, i.e., internal hydrolysis of casein molecules, by the coagulant or indigenous enzymes of milk, such as plasmin. Secondary proteolysis occurs essentially during ripening, by the action of peptidases of microorganisms. Proteolysis weakens the structure of the casein matrix. It can be easily supposed that removal of the native microflora from raw milk may alter the texture of subsequent cheeses by two major mechanisms. On the one hand, the heat treatment of the milk used to destroy the microflora may alter the structure of the casein matrix by denaturation of whey proteins or the loss of water, or modify the proteolysis patterns by denaturation, activation or modified retention of enzymes. On the other hand, the elimination of most of the indigenous microflora, either by heating or microfiltration, may modify the biochemical changes in cheeses, in particular proteolysis (Grappin and Beuvier, 1997). Among all the articles in which R, P or MF cheeses were compared, few deal with cheese texture. Some work resulted in no differences related to the treatment of milk: no clear differences between R/P/MF milks (McSweeney et al., 1993) and R/P milks (Shakeel-UrRehman et al., 1999) in Cheddar cheese, no sensory textural differences between R/MF milk Swiss-type cheeses (Bouton and Grappin, 1995) or rheological differences between R/P Bergk~se cheese (Ginzinger et al., 1999a). The comparison of R and P cheeses from a texture point of view is difficult, because of the differences in the behaviour of milk during the coagulation step, due to the heat treatment. Depending on the cheesemaking procedures, contradictory findings have been reported, in terms of moisture, on the compositional differences of
339
cheeses (Lau etal., 1990; Buffa etal., 2001b), or pH (Buffa et al., 2001b). Texture differences are thus difficult to interpret. The results of Beuvier et al. (1997) seem to indicate that in Swiss-type cheeses, sensory texture characteristics of R cheeses are influenced by both the heat treatment of milk and the activity of the indigenous microflora. They showed in a comparison of R, P, MF and P cheeses to which the indigenous microflora contained in microfiltration retentate had been added, that R cheeses had a firmer and more granular texture. Proteolysis appears to be the main factor responsible for differences in texture between R and P cheeses. According to Shakeel-Ur-Rehman et al. (2000a), the increase in chymosin retention and in plasmin activity by heat-treatment of milk is the major cause of texture differences between R and P Cheddar cheeses ripened at 1 or 8 ~ While the temperature influenced all texture descriptors, milk treatment influenced only rubberiness (P > R) and graininess ( R > P). They attributed the texture characteristics mostly to differences in water-soluble N (WSN) (Shakeel-Ur-Rehman et al., 2000b), in which enzymes such as chymosin or plasmin have more influence than the activity of the indigenous microflora. Gaya et al. (1990) found a lower fracturability, elasticity and hardness in Manchego cheese made from R ewes' milk than in P cheeses, whatever the ripening time (2 or 4 months) and the ripening temperature (between 8 and 16 ~ They attributed these differences to higher secondary proteolysis in R cheeses, measured by pH 4.6-, TCA- and PTA- soluble N. Buffa et al. (2001b) studied the rheological characteristics of goats' milk semi-hard cheeses made from R or P milk. R cheeses were firmer, less fracturable, and more cohesive than P ones. These characteristics were attributed to the levels of moisture and WSN: the lower the moisture and more intact the caseins, the less the fracturability and deformability. Fracture stress was higher for R cheeses, i.e., a lower fracturability than the P cheeses. This parameter was correlated with the levels of moisture and WSN: the less the moisture and more intact the caseins, the less the fracturability. Fracture strain, which describes the deformability of cheese, was higher for R cheeses, but only at one day. It could be due to the higher pH of these cheeses at this stage, water being partly absorbed to hydrate the negative charges formed in caseins with high pH values. This parameter has the same correlation with moisture and WSN as previously- deformability decreases when the hydration of proteins decreases and when elastic structural elements disappear. The microstructure of R cheeses was more regular, with a closed protein matrix, and smaller and more uniform fat globules, whereas
340
Raw Milk Cheeses
P cheeses had an open structure with irregular cavities. As a consequence, differences in colour were observed. Rosenberg etal. (1995) measured the viscoelastic characteristics, G' (storage modulus) and G" (loss modulus), of Cheddar cheeses. These parameters were higher in R cheeses than in P cheeses ripened for 8 months. In P cheeses, they were found to be related to the extent of proteolysis; a higher G' signified a higher elastic behaviour of the matrix with the accumulation of proteolysis products. The authors explained this observation by the binding of water by the ionic groups liberated by the cleavage of peptide bounds. This relation with the extent of proteolysis was not observed in R cheeses, maybe because of different proteolytic activities during the ripening of these cheeses, as revealed by differences in peptide composition. Mendia et al. (1999) found more graininess and firmness and less creaminess and elasticity in R ewes' milk Idiazabal cheeses than in P cheeses. These differences were attributed to the slower maturation of P cheeses. This was confirmed by the fact that the differences diminished with increases in ripening time, and were thought to be linked to the moisture content. For certain types of cheese consumed mainly in a melted form, such as Raclette, it is more interesting to evaluate the texture characteristics of the cheese after melting. Melting properties were evaluated in Raclette cheeses made from R, P or MF milk and mixtures of the three types of milk in different proportions (Klantschitsch et al., 2000). R cheeses had a longer consistency than P/MF cheeses after 90 days ripening. According to the authors, this is related to the proteolysis patterns, proteolysis 'in width', pH 4.6 N/TN (lower in MF) leading to longer consistency and higher viscosity, proteolysis 'in depth' (NPN/TN) leading to shorter consistency. The viscosity did not differ between the cheeses. The firmness of melted cheese was also higher in R than in P/MF cheeses after 90 days ripening, with a score indicating insufficient melting quality. Fat separation increased more rapidly with ripening time in R than in P cheeses. Softening and dropping points were in the range for good melting quality in all cheeses ripened at 11 or 14 ~ but only in the MF cheeses ripened at 17 ~ The effect of the microflora on the melting quality of Raclette is dependant on the ripening temperature and time; a high temperature (17 ~ is detrimental when using raw milk, whereas, in the case of microfiltered milk, it is useful to accelerate ripening. In all these studies, the lack of microbial investigations made it difficult to establish a relationship between the microbial populations, whether of indigenous or starter origin, and the characteristics of texture. Nevertheless, when the observed differences were
attributed to the secondary proteolysis, microbial activity was involved.
Conclusion
Microbial communities play an essential role in the control of sensory qualities of cheese. They are more diverse and complex in R cheeses for which milk undergoes no treatment to reduce the microflora. They contribute to the development of a typical cheese taste and flavour. Diversity of the sensory qualities is a specific feature of R cheese. Elimination of the raw milk microflora by pasteurisation or microfiltration definitively leads to different cheeses from a sensorial point of view. Still, is it necessary to have raw milk that is sufficiently rich in terms of quantity and diversity of microorganisms? As outlined at the beginning, the improvement in hygienic practices on farms has led to a 'clean' raw milk, with low microbial counts (Odet, 1999). Raw milk with a low level of microbes could induce a reduction in sensorial diversity of cheese due to a reduction of microbial diversity. Indeed, Dasen et al. (2003) have observed that the strain diversity of mesophilic lactobacilli in raw milk experimental Cheddar cheese was close to that observed in industrial Cheddar cheese manufactured with pasteurised milk. The former was made from raw milk with a total of around 10 000 cfu m1-1. The fact that raw milk tends to be more and more microbiologically 'clean' implies that there is a risk that the sensorial differences between R and P cheeses will be erased. Some experiments in progress, particularly in France, aim to evaluate dairy farming practices, including milking practices, on the raw milk microflora in terms of quantity and diversity. Recently, Michel et al. (2001) observed links between milking practices and the bacteriological quality of milk, showing that it is possible to manage the microbial quality of milk on the farm to promote the technologically 'useful' microflora, while maintaining pathogens at a low level. This is a good way to keep the natural microflora in R cheese production, in terms of quantity and diversity, in order to preserve their sensorial diversity. To add selected microorganisms could enhance the aroma of cheese, but the cheese would have a more uniform flavour, a characteristic which is not sought by both the producers and the consumers, because diversity of flavour is considered a special feature of traditional R cheeses (Grappin and Beuvier, 1997). Otherwise, according to Montel (2002), microbial communities may play a key role in the microbiological safety of R cheese. This potential role is supported by several studies in which cheeses or milk, with a more complex microflora, were less contaminated by
Raw Milk Cheeses
L. monocytogenes than those with a less diversified flora (Brouillaud-Delattre et al., 1997; Eppert et al., 1997). Thus, well-monitored microbial diversity, from farm to cheese, by acting as a barrier against pathogens, may be a trump card for cheese safety (Montel, 2002). According to Stanton et al. (1998), cheeses, because of their high fat content and their texture, could offer protection to the living microorganisms contained within them, especially at the m o m e n t of their passage into the gastrointestinal tract of the consumer. More and more studies demonstrate the beneficial effects on health of strains of microorganisms and give hope for other discoveries in R cheeses, which are rich in microorganisms (Bouton, 2001; Moreau and Vuitton, 2002). The preservation of the microbial diversity in raw milk, essential to obtain cheeses with greater sensorial diversity, more and more appreciated by (European) consumers, potentially useful to fight against pathogens and potentially useful for health, is a challenge for milk and cheese producers, and researchers, to take up over the next years.
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Bertozzi, L. and Panari, G. (1994). Cheeses with Appellation d'Origine Contn31ee (AOC): factors that affect quality. Int. Dairy J. 3,297-312. Beuvier, E. (1990). Influence du traitement thermique du lait en fonction des conditions de stockage et de maturation sur la flaveur d' un fromage a pate pressee cuite. These de 1' Universite de Franche-Comte, no. 188. Beuvier, E., Berthaud, K., Cegarra, S., Dasen, A., Pochet, S., Buchin, S. and Duboz, G. (1997). Ripening and quality of Swiss-type cheese made from raw, pasteurized or microfiltered milk. Int. Dairy J. 7, 311-323. Bosset, J.O. and Liardon, R. (1985). The aroma composition of Swiss Gruyere cheese. III. Relative changes in the content of alkaline and neutral volatile components during ripening. Lebensm. Wiss. Technol. 18, 178-185. Bouton, Y. (2001). Raw milk cheese characteristics and their positive effects on health. Caseus Int. 1, 54-62. Bouton, Y. and Grappin, R. (1995). Comparaison de la qualite de fromages/t pate pressee cuite fabriques/~ partir de lait cru ou microfiltre. Lait 75, 31-44. Bouton, Y., Guyot, P., Beuvier, E., Tailliez, P. and Grappin, R. (2002). Use of PCR-based methods and PFGE for typing and monitoring homofermentative lactobacilli during Comte cheese ripening. Int. J. Food Microbiol. 76, 27-38. Brouillaud-Delattre, A., Maire, M., Collette, C., Mattei, C. and Lahellec, C. (1997). Predictive microbiology of dairy products: influence of biological factors affecting growth of Listeria monocytogenes. JAOAC Int. 80, 913-919. Buchin, S., Delague, V., Duboz, G., Berdague, J.L., Beuvier, E., Pochet, S. and Grappin, R. (1998). Influence of pasteurization and fat composition of milk on the volatile compounds and flavor characteristics of a semi-hard cheese. J. Dairy Sci. 81, 3097-3108. Buchin, S., Beuvier, E., Duboz, G. and Tessier, L. (2002). Raw milk microflora and flavour characterictics of ripened cheeses: variations caused by technology. Poster in Congrilait 26th IDF World Dairy Congress, Paris, 24-28 September. Buffa, M., Guamis, B., Pavia, M. and Trujillo, A.J. (2001a). Lipolysis in cheese made from raw, pasteurized or highpressure-treated goats' milk. Int. Dairy J. 11, 175-179. Buffa, M.N., Trujillo, A.J., Pavia, M. and Guamis, B. (2001b). Changes in textural, microstructural, and colour characteristics during ripening of cheeses made from raw, pasteurized, or high-pressure-treated goats' milk. Int. Dairy J. 11,927-934. Callon, C., Duthoit, E, Millet, L. and Montel, M.-C. (2001). Les leuconostocs: flore lactique caracteristique du fromage d'AOC Salers au lait cru. Affiche ECO10. 11 ~ Reunion du Club des bacteries lactiques, 14-16 Novembre, Bordeaux. Canillac, N. and Mourey, A. (1993). Sources of contamination by Listeria during the making of semi-soft surfaceripened cheese. Sci. Aliment. 13,533-534. Carbonell, M., Nunez, M. and Fernandez-Garcia, E. (2002). Evolution of the volatile components of ewe raw milk La Serena cheese during ripening. Correlation with flavour characteristics. Lait 82,683-698.
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Raw Milk Cheeses
Centeno, J.A., Rodrfguez-Otero, J.L. and Cepeda, A. (1994a). Microbiological studies of Arzua cheese (NW Spain) throughout cheesemaking and ripening. J. Food Safety 14, 229-241. Centeno, J.A., Rodrfguez-Otero, J.L. and Cepeda, A. (1994b). Changes in the protein profile of Arzua cheese (NW Spain) during ripening. Milchwissenchaft 49, 319-322. Centeno, J.A., Varela, J.A., Almena, M. and Rodriguez-Otero, J.L. (1996). Effects of various Micrococcus strains on the ripening and organoleptic characteristics of Arzua cow's milk cheese. Z. Lebens. Unters. Forsch. 203,546-552. Centeno, J.A., Menendez, S., Hermida, M. and RodrfguezOtero, J.L. (1999). Effects of the addition of Enterococcus faecalis in Cebreiro cheese manufacture. Int. J. Food Microbiol. 48, 97-111. Chamba, J.E and Perreard, E. (2002). Contribution of propionic acid bacteria to lipolysis of Emmental cheese. Lait 82, 33-42. Chavarri, E, Santisteban, A., Virto, M. and de Renobales, M. (1998). Alkaline phosphatase, acid phosphatase, lactoperoxidase, and lipoprotein lipase activities in industrial ewe's milk and cheese. J. Agric. Food Chem. 46, 2926-2932. C.havarri, E, Bustamante, M.A., Santisteban, A., Virto, M., Albisu, M., Barron, L.J.R. and de Renobales, M. (2000). Effect of milk pasteurization on lipolysis during ripening of ovine cheese manufactured at different times of the year. Lait 80,433-444. Choisy, C., Desmazeaud, M., Gripon, J.C., Lamberet, G. and Lenoir, J. (1997). La biochimie de l'affinage, in, le Fromage, A. Eck and J.C. Gillis, eds, Tec et Doc Lavoisier, Paris. pp. 86-161. CNIEL (2002). Eeconomie laitiere en chiffres, Paris. p. 69. Cogan, T.M. and Rea, M.C. (1996). Artisanal European cheeses. Ed. European Commission- Directorate-General XII, Brussels. EUR16788. Coppola, S., Blaiotta, G., Ercolini, D. and Moschetti, G. (2001). Molecular evaluation of microbial diversity occurring in different types of Mozarella cheese. J. Appl. Microbiol. 90, 414-420. Curioni, P.M.G. and Bosset, J.O. (2002). Key odorants in various cheese types as determined by gas chromatographyolfactometry. Int. Dairy J. 12,959-984. Dasen, A., Berthier, F., Grappin, R., Williams, A.G. and Banks, J. (2003). Genotypic and phenotypic characterization of the dynamics of the lactic acid bacterial population of adjunct-containing Cheddar cheese manufactured from raw and microfiltered pasteurised milk. J. Appl. Microbiol. 94(4), 595-607. De Angelis, M., Corsetti, A., Tosti, N., Rossi, J., Corbo, M.R. and Gobbetti, M. (2001). Characterization of non-starter lactic acid bacteria from Italian ewe cheeses based on phenotypic, genotypic and cell wall protein analyses. Appl. Environ. Microbiol. 67, 2011-2020. De Buyser, M.-L., Dufour, B., Maire, M. and Lafarge, V. (2001). Implication of milk and milk products in food-borne diseases in France and in different industrialised countries. Int. J. Food Microbiol. 67, 1-17. Deeth, H.C. and Fitz-Gerald, C.H. (1983). Lipolytic enzymes and hydrolytic rancidity in milk and milk products, in,
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Biochemistry of Cheese Ripening: Introduction and Overview P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland
Introduction As discussed in 'Cheese: An Overview', Volume 1, rennetcoagulated cheeses are ripened (matured) for a period ranging from 2 weeks (e.g., Mozzarella) to 2 or more years (e.g., Parmigiano Reggiano or extra-mature Cheddar) during which the flavour and texture characteristic of the variety develop. Ripening usually involves changes to the microflora of the cheese, including death and lysis of the starter cells, development of an adventitious non-starter microflora and, in many cheeses, growth of a secondary microflora (e.g., Propionibacterium freudenreichii subsp, shermanii in Swiss cheese, moulds in mould-ripened varieties and a complex Gram-positive bacterial microflora on the surface of smear-ripened cheeses). The metabolic activity of the secondary microflora often dominates flavour development, and in some cases, e.g., whitemould cheeses, the texture, of varieties in which they grow. The microbiology of cheese during ripening is discussed in 'The Microbiology of Cheese Ripening', Volume 1. As discussed in 'Rheology and Texture of Cheese', Volume 1, ripening usually involves the softening of cheese texture, as a consequence of the hydrolysis of the casein matrix, changes in the waterbinding ability of the curd and changes in pH (which may cause other changes such as the migration and precipitation of calcium phosphate). The flavour of cheese curd immediately after manufacture is rather bland and indeed it can be difficult to differentiate the flavours of certain varieties at this stage. During ripening, cheese flavour develops due to the production of a wide range of sapid compounds by the biochemical pathways described below. Volatile flavour compounds are of particular importance to cheese flavour and are discussed in 'Sensory Character of Cheese and its Evaluation', Volume 1. Quantification of the volatile flavour compounds of cheese are described in 'Instrumental Techniques', Volume 1. Biochemical reactions which occur in cheese during ripening are usually grouped into four major categories: (1) glycolysis of residual lactose and catabolism of lactate, (2) catabolism of citrate, which is very important in certain varieties, (3) lipolysis and the
catabolism of free fatty acids and (4) proteolysis and the catabolism of amino acids (Fig. 1). These reactions are discussed in 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1. Since the biochemistry of cheese ripening is complex, the purpose of this chapter is to present an overview of the principal biochemical pathways which contribute to cheese ripening and to discuss the role of the principal ripening agents in cheese and the acceleration of cheese ripening. Aspects of cheese ripening common to many varieties are discussed in 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1; ripening of specific varieties is discussed in the relevant chapters in Volume 2.
Glycolysis of Residual Lactose and Catabolism of Lactate Since cheeses are fermented dairy products, the metabolism of lactose to lactate is essential in the manufacture of all varieties. Cheese curd contains a low level of residual lactose which is metabolised rapidly early in ripening to lactate which may be catabolised subsequently via a range of pathways. Catabolism of lactate probably occurs in all cheeses and is particularly important in surface mould-ripened varieties (e.g., Camembert) and in Swiss cheese. These reactions were reviewed by Fox et al. (1990, 1993) and McSweeney and Sousa (2000) and are discussed in detail in 'Metabolism of Residual Lactose and of Lactate and Citrate', Volume 1. The pathway through which lactose is metabolised depends on the starter type (see 'Starter Cultures: General Aspects', Volume 1; Cogan and Hill, 1993; Fox et al., 2000; McSweeney and Sousa, 2000; Broome et al., 2003). The final step in the glycolysis of lactose is the conversion of pyruvate to lactate which is catalysed by lactate dehydrogenase (LDH). Depending on
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
348
Biochemistry of Cheese Ripening: Introduction and Overview
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Figure 1 General overview of the biochemical pathways which operate in cheese during ripening.
the type of LDH (D- or L-LDH) in the cell, D- (e.g.,
Lb. delbrueckii subsp, bulgaricus), L- (e.g., Lactococcus, Sc. thermophilus) or D/L- (e.g., Lb. helveticus) lactate is the end product of glycolysis which converts 1 mol of lactose to 4 mol of lactate with the production of 4 mol of ATR Unlike most lactic acid bacteria (LAB), Leuconostoc spp. use the phosphoketolase pathway to metabolise lactose; the end products of this pathway are lactate, ethanol and CO2 and thus differ from that of the glycolytic pathway9 Although essential for cheese manufacture, the metabolism of lactose to lactate is essentially complete at the end of manufacture or during the early stages of ripening. Most lactose in milk is lost in the whey and that which is retained in the curd is metabolised rapidly after drainage. However, the activity of the starter is greatly reduced at the end of manufacture or soon thereafter due to the combination of low pH, high NaC1 and lack of a fermentable carbohydrate. The inhibition of acid production is particularly abrupt in dry-salted varieties (e.g., Cheddar) where NaC1 concentration reaches equilibrium much faster than in brine-salted cheeses. Fresh cheese curd contains a low level of lactose which, in the case of Cheddar cheese, is reduced to trace levels within one
month of ripening by the (albeit reduced) activity of the starter or by the action of the non-starter lactic acid bacteria (NSLAB). Lactate contributes to the flavour of cheese, particularly early during maturation, but the major effect of acidification on flavour development is indirect since, together with the buffering capacity of the curd, it influences pH and thus the growth of the secondary flora and the activity of ripening enzymes. Lactate is an important substrate for a range of reactions which contribute positively or negatively to cheese ripening. L-Lactate, produced by Lactococcus, can be racemised to DL-lactate by the NSLAB flora in Cheddar and Dutch-type cheeses. DL-Lactate is less soluble than k-lactate, resulting in the formation of Ca-D-lactate crystals which appear as white specks on the surface of the mature cheese. Lactate can also be metabolised to acetate and CO2 by some members of the NSLAB flora, although this oxidative pathway is relatively minor in cheese due to its low oxidationreduction (redox) potential (c. - 2 5 0 mV) and is limited by the availability of 02. Late gas blowing is a defect in certain hard and semi-hard varieties caused by the anaerobic catabolism of lactate to butyrate and H2
Biochemistry of Cheese Ripening: Introduction and Overview
by Clostridium tyrobutyricum. This problem can be overcome by good hygiene, addition of NaNO3 or lysozyme or by the physical removal of the spores by bactofugation or microfiltration. However, catabolism of lactate is particularly important in Swiss and surface mould-ripened cheeses. In the former, lactate is catabolised by Propionibacterium freudenreichii subsp, shermanii to propionate, acetate, H20 and CO2. Propionate and acetate contribute to the flavour of Swiss cheese; CO2 migrates through the curd to points of weakness where it collects to form the large eyes characteristic of Swiss-type cheese. The oxidative catabolism of lactate to H20 and CO2 by Penicilliurn camemberti at the surface of Camembert and Brie-type cheeses is of great indirect importance to their ripening. The catabolism of lactic acid causes a large increase in the pH of the surface of these cheeses which leads to a pH gradient from the surface to the core and to the migration of lactate towards the surface. The high pH at the surface causes precipitation of calcium phosphate, which, in turn, causes the migration of calcium and phosphate to the cheese surface. These changes lead to the characteristic softening of surface mould-ripened cheese which, when mature, have an almost liquid-like consistency. Oxidative metabolism of lactate is also of significance at the surface of smear-ripened cheeses (e.g., Tilst or Limburger) where, early in ripening, yeasts deacidify the surface which encourages the growth of the Gram-positive bacteria characteristic of the surface. Oxidative metabolism of lactate probably also occurs in Blue cheese but its effect is less important than in surface mouldripened cheese since P. roqueforti is distributed throughout the cheese and thus gradients do not develop across the cheese mass.
Lipolysis and Metabolism of Fatty Acids Studies in which milk fat was replaced with other lipids have demonstrated that milk fat is essential for the development of the flavour of Cheddar and probably all other ripened cheeses. As in all high-fat foods, lipids present in cheese can undergo hydrolytic or oxidative degradation; the latter is generally considered not to be important in cheese, primarily due to its low redox potential. Lipolysis in cheese during ripening is discussed in detail in 'Lipolysis and Catabolism of Fatty Acids in Cheese', Volume 1. As discussed by McSweeney and Sousa (2000) and Collins et al. (2003a), lipases in cheese originate from a number of sources. Milk contains an indigenous lipoprotein lipase (LPL), which contributes to lipolysis in cheese during ripening. Lipoprotein lipase activity is more important in cheese made from raw milk than
349
in that made from pasteurised milk since the enzyme is extensively inactivated by pasteurisation. Rennet paste, used as coagulant in certain Italian cheese varieties, contains a potent lipase, pregastric esterase, which is responsible for lipolysis in cheeses such as Provolone and the Pecorino varieties. Lactic acid bacteria are weakly lipolytic, but their enzymes have been shown to contribute to the low level of lipolysis characteristic of Cheddar cheese (Collins etal., 2003b). Likewise, Pr. Jreudenreichii subsp, shermanii possesses a lipase which, together with enzymes from the thermophilic starter organisms, contributes to the low level of lipolysis in Swiss cheese. Penicillium roqueforti produces potent extracellular lipases which are responsible for the extensive lipolysis characteristic of Blue cheese. P. camemberti and the complex Gram-positive surface microflora of smear cheeses also produce extracellular lipases which contribute to lipolysis in surface-bacterial or white mould-ripened varieties. The level of lipolysis in cheese is determined using various non-specific techniques (e.g., solvent extraction and titration of the fatty acids with alcoholic KOH or by the formation of coloured Cu soaps) or by quantitation of individual fatty acids, usually by gas chromatography (see Collins et al., 2003a). Fatty acids have a direct impact on the flavour of many cheese varieties. In particular, C4-C10 acids are strongly flavoured. Levels of fatty acids vary considerably between varieties. Many internal bacterially ripened varieties (e.g., Edam, Swiss and Cheddar) contain low levels of fatty acids (c. 200-1000 mg kg-1). Very high levels of fatty acids are found in Blue cheese (c. 30 000 mg kg-1). In addition to their direct role in cheese flavour, fatty acids are important precursors for the production of other volatile flavour compounds during ripening (Fig. 2). Fatty acid esters are produced by reaction of fatty acids with an alcohol; ethyl esters are most common in cheese. Thioesters are formed by reaction of a fatty acid with a thiol compound formed via the catabolism of sulphur-containing amino acids. Fatty acid lactones are cyclic compounds formed by the intramolecular esterification of hydroxyacids; 7- and 8-1actones contribute to the flavour of a number of cheese varieties. The principal class of volatile flavour compounds in Blue cheese is n-methyl ketones (alkan2-ones) which are produced from fatty acids by partial ]3-oxidation. n-Methyl ketones may be reduced to the corresponding secondary alcohols. Fatty acid catabolism is summarised in Fig. 2 and is discussed in detail in 'Lipolysis and Catabolism of Fatty Acids in Cheese', Volume 1. Volatile fiavour compounds in cheese, including those derived from fatty acids, are usually quantified using gas chromatography-mass spectrometry (GC-MS; see 'Instrumental Techniques', Volume 1).
350
Biochemistry of Cheese Ripening: Introduction and Overview
Triglycer~
O,cf o/
%f
>/ J
OH
I
O
II u~zv'C--OH
y- or ~-hydroxy fatty acids Partial glycerides ~ H20
Fo-1 ~ II ~
C
-
-
O
~ , , w , c,o
7- or ~-Iactones
O
H
Fatty acids R-SH
Thiols V
CH3CH2OH
Ethanol
/ ~
C
-
-
S
-
II
O
R
Thioesters
co•Padla113-oxidation O
0
~,~J~',r~,r~,r~,r~
II
c --OCH2CH 3
~-J~,~,~r,,~-,~
II c --CH 3
Alkan-2-ones
Ethyl esters
1 ~,*~-j~-,~~
OH
I
C H _ C H3
Alkan-2-ols Figure 2
Pathways for the production of flavour compounds from fatty acids during cheese ripening.
Proteolysis and Catabolism of Amino Acids Proteolysis is the most complex, and in most varieties, the most important biochemical event which occurs during cheese ripening. Proteolysis has been discussed in reviews by Grappin et al. (1985), Rank et al. (1985), Fox (1989), Fox et al. (1993, 1994, 1995), Fox and McSweeney (1996), McSweeney and Sousa (2000) and Sousa et al. (2001) and is covered in detail in 'Proteolysis in Cheese during Ripening', Volume 1. Proteolysis is very important for cheese texture by hydrolysing the para-casein matrix which gives cheese its structure and by increasing the water-binding capacity of the curd (i.e., to the new ot-carboxylic and or-amino groups produced on cleavage of peptide bonds). Proteolysis may indirectly affect texture by increasing pH through the production of NH3 following amino acid catabolism.
Peptides may have a direct impact on cheese flavour (some are bitter) or they may provide a brothy background flavour to cheese. However, recent research has indicated that the major role of proteolysis in cheese flavour is in the production of amino acids which act as precursors for a range of catabolic reactions which produce many important volatile flavour compounds (see McSweeney and Sousa, 2000; Yvon and Rijnen, 2001). In most cheese varieties, the initial hydrolysis of caseins is caused by the coagulant and to a lesser extent by plasmin and perhaps somatic cell proteinases (e.g., cathepsin D) which result in the formation of large (water-insoluble) and intermediate-sized (watersoluble) peptides which are subsequently hydrolysed by the coagulant and enzymes from the starter and non-starter flora of the cheese. The production of
Biochemistry of Cheese Ripening: Introduction and Overview
small peptides and amino acids is caused by the action of microbial proteinases and peptidases, respectively. Preparations of selected aspartyl proteinases are used to coagulate milk. Chymosin (EC 3.4.23.4) is the principal proteinase (88-94%) in traditional calf rennets, the remainder being pepsin (EC 3.4.23.1) (Rothe et al., 1977). Although, the principal role of the coagulant in cheesemaking is to coagulate milk, some activity is retained in the curd, depending on factors such as coagulant type, cooking temperature and pH at drainage, and contributes to proteolysis in many varieties (Creamer et al., 1985). Plasmin (fibrinolysin; EC 3.4.21.7) is the dominant indigenous proteinase in milk and is produced from its inactive precursor, plasminogen, by a system of plasminogen activators (PA). Inhibitors of plasmin and of PA are also present in milk. Plasmin, which is optimally active at pH 7.5 and 37 ~ is most active in high-cook cheeses due to denaturation of inhibitors and increased activation of plasmin and in cheeses in which the pH increases during ripening (e.g., Blue cheese or the surfaces of white-mould and smearripened varieties). Plasmin is most active on [3-casein, hydrolysing it at three sites to produce the y-caseins and some proteose peptones. Milk contains somatic (white blood) cells, which contain lysosomes, which in turn, contain many proteolytic enzymes. To date, cathepsin D (see review by Hurley et al., 2000) and cathepsin B (Magboul et al., 2001) have been confirmed in milk. Lactic acid bacteria (Lactococcus, Lactobacillus, Streptococcus) possess very comprehensive proteolytic systems that have been studied extensively and reviewed (e.g., Fox and McSweeney, 1996; Kunji et al., 1996; Law and Haandrikman, 1997; Christensen etal., 1999). Lactic acid bacteria possess a cell envelope-associated proteinase (PrtP or lactocepin), 3-4 intracellular proteinases, intracellular oligoendopeptidases (PepO, PepF), a number of aminopeptidases (PepN, PepC, PepG, PepA, PepL), a pyrolidone carboxyl peptidase (PCP), a dipeptidylaminopeptidase (PepX), a proline iminopeptidase (PepI), an aminopeptidase P (PepP), a prolinase (PepR), a prolidase (PepQ), general dipeptidases (PepV, PepD, PepDA) and a general tripeptidase (PepT). They also possess oligopeptide, di/tripeptide and amino acid transport systems (Fig. 3). This proteolytic system is necessary to enable the LAB to grow to high numbers in milk (109-1010 cfuml-1), which contains only low levels of small peptides and amino acids. PrtP contributes to the formation of small peptides in cheese, probably by hydrolysing larger peptides produced from Otsl-casein by chymosin or from [3-casein by plasmin, whereas the aminopeptidases, dipeptidases and tripeptidases (which are intra-
Cellenvelopeassociated / proteinase /' (CEP,PrtP, / L
Endopeptidases
(PepO,PepF)
Aminope~idases
\
\
351
\
CaseinlmCinoaci H dsE ~ i ~E.( ~Se ~Ii .~.~i.D. IA ~~E I. / TransportDi,tripeptides systems Oligopeptides Figure 3
Summary of the proteolytic system of Lactococcus. The proteolytic systems of other lactic acid bacteria are generally similar.
cellular) are responsible for the release of free amino acids after the cells have lysed. Non-starter lactic acid bacteria, although present initially at low numbers (<50 cfu g-1 in Cheddar made from pasteurised milk, and probably in other cheeses), grow at a rate largely governed by ripening temperature to reach - 1 0 r cfu g-1 within 4 weeks and remain relatively constant thereafter. The activity of the NSLAB appears to supplement the proteolytic action of the starter. Non-starter lactic acid bacteria in cheese are discussed in 'The Microbiology of Cheese Ripening', Volume 1. In many cheese varieties, a secondary microflora (secondary starter) is added intentionally and/or encouraged to grow by environmental conditions and has a diverse range of functions, depending on the organisms used. A number of different LAB, e.g., strains of Lactobacillus, have been added to Cheddar cheese with the objective of improving flavour or accelerating ripening. These have proteolytic systems similar to those of other species of LAB. Brevibacteriurn linens is the best-studied smear microorganism; it secretes an extracellular proteinase and aminopeptidase, and possesses a number of intracellular peptidases, which contribute to proteolysis at the surface of smear-ripened cheeses (see Rattray and Fox, 1999). Penicillium roqueforti produces potent extracellular aspartyl and metalloproteinases and various peptidases which are major contributors to the extensive proteolysis found in Blue cheese. P camernberti secretes active metallo and aspartyl proteinases which contribute to proteolysis in Camembert and Brie-type cheeses. The final products of proteolysis are amino acids, the concentration of which depends on the cheese variety. The concentration of amino acids in cheese at a given stage of ripening is the net result of the liberation of amino acids from the caseins by proteolysis and
352
Biochemistry of Cheese Ripening: Introduction and Overview
their catabolism or transformation into other amino acids by the cheese microflora. The principal amino acids in Cheddar cheese are Glu, Leu, Arg, Lys, Phe and Ser. Medium and small peptides contribute to a brothy background flavour in many cheese varieties; short, hydrophobic peptides are bitter. Amino acids contribute directly to cheese flavour as some amino acids taste sweet (e.g., Gly, Ser, Thr, Ala, Pro), sour (e.g., His, Glu, Asp) or bitter (e.g., Arg, Met, Val, Leu, Phe, Tyr, lie, Trp). However, research in the last decade has shown that accelerating proteolysis does not necessarily accelerate flavour development, suggesting that the production of amino acids is not the rate-limiting step in the development of cheese flavour. It is now generally believed that the principal role of proteolysis in the production of flavour compounds is the liberation of amino acids as precursors for a complex series of catabolic reactions that produce many important volatile flavour compounds. Amino acid catabolism was reviewed by Yvon and Rijnen (2001), and is discussed in detail in 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1 and summarised in Fig. 4. Amino acid catabolism appears to proceed via two major p a t h w a y s - transaminase action and elimination reactions. Transaminases catalyse the transfer of the or-amino group from an amino acid to
Casein
Coagulant Plasmin
an (x-keto acid (usually ot-ketoglutarate) with the production of the corresponding amino acid and an o~-keto acid corresponding to the amino acid substrate (cf. Fig. 5 for leucine). The second pathway, which is initiated by elimination reactions, is particularly important in the production of volatile sulphur compounds from the side chain of methionine. In addition, decarboxylases remove the carboxylic acid group of amino acids to produce amines, some of which have physiological effects (see 'Toxins in Cheese', Volume 1). Decarboxylases may also act on ot-keto acids to produce aldehydes, which in turn may be oxidised to carboxylic acids or reduced to primary alcohols. The or-amino group of amino acids may be removed by the action of deaminases, with the formation of a carboxylic acid and ammonia. In addition, the side chains of amino acids may be degraded by the action of various lysases (see 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1).
Ripening A g e n t s in C h e e s e Agents from five, and possibly six, sources are involved in the ripening of cheese: 9 enzymes from the coagulant; 9 indigenous milk enzymes;
Large and small polypeptides
I Starterproteinases Small peptides
I Starterandnon-starterpeptidases Small peptides 4- iAmino acids"
/
~
Deamina~es NH3 ~ / Decarboxylasse ~>~o~-Ketoacidl/Lyases ~.~ C02 Acids KTransaminases
Carbonyls ~
r
acid
o~-Ketoacid 2 Various compounds (e.g., sulphur compounds) Figure 4 Summary of proteolysis and amino acid catabolism in cheese during ripening.
Amines
Biochemistry of Cheese Ripening: Introduction and Overview
O'---C--C--OH
I iH2
r
H2N--CH--C--OH
I Glutamic acid I CH2 I C----O I OH CH~
C----O
I OH
I
OH2
I I CH--CH 3 OH2
y
o...c...
I
TRANSAMINASE
OH
CO2
o II O---C--C--OH
H2N--CH--C--OH
I
353
o~c,.~ox~,s~
OH2
I
CH--CH 3
I CHa Leucine
-Methylbutanol
I OH2
I
CH--CH31
i H-CH3
cm
~y
2-Keto-4-methylpentanoic acid
3-Met
O~cIOH
a,
Ibutan
I IOH--OH 3 I GIla CHe
3-Methylbutanoic acid
0o2~/ C--OH
NH3
I
CHe
NH2
I
erie
I ICH--CH3 I CH3
I CHe I CH--CH 3 I GH3
CHe
4-Methylpentanoic acid
3-Methylbutylamine Figure 5 Catabolism of leucine initiated by transaminase, deaminase or decarboxylase action and volatile flavour compounds which may be formed from this amino acid. Similar catabolic pathways operate for the other branched-chain aliphatic amino acids (isoleucine and valine).
9 starter bacteria and their enzymes, which are released after the cells have died and lysed; 9 enzymes from secondary starters (e.g., Propionibacterium freudenreichii subsp, shermanii, Gram-positive bacteria on the surface of smear-ripened cheese, yeasts and moulds, such as Penicillium roqueforti and P. camemberti), which are of major importance in some varieties; 9 non-starter bacteria, i.e., organisms that either survive pasteurisation of the cheese milk or gain access to the pasteurised milk or curd during manufacture; and, in certain circumstances, 9 exogenous enzymes added to accelerate cheese ripening. There has been interest for about 40 years in developing model systems in which to quantify the contribution of each of these agents to cheese ripening. The techniques developed eliminate one or more of the above agents, thereby enabling its role to be assessed, directly or indirectly.
Non-starter bacteria may be eliminated by using an aseptic bucket milking technique, developed by Perry and McGillivray (1964); the teat cups and clusters were chemically sterilised and the bucket steam-sterilised. Cows were screened for the bacteriological quality of their milk and animals with counts <100 cfum1-1 selected; prior to milking, their udders were cleaned with a quaternary ammonium solution. An essentially similar approach was used by O'Keeffe etal. (1976a), who obtained milk with a total bacterial count <500 cfu m1-1. Kleter and de Vries (1974) included a cooling coil between the cluster and the bucket and succeeded in achieving counts averaging 46 cfu m1-1. This approach was also used by Visser (1976). Reiter et al. (1969) withdrew milk aseptically by means of a teat cannula, but the quantities obtained (11) were sufficient to produce cheeses of only about 100 g. More recently, it has been our experience (McSweeney et al., 1994; Lynch et al., 1996, 1997) that special precautions for milking are unnecessary; good quality raw milk pasteurised at 78 ~ for 15 s is suitable for aseptic cheesemaking.
354
Biochemistry of Cheese Ripening: Introduction and Overview
Having collected low-count milk, a heating step is usually used to reduce bacterial counts further. Perry and McGillivray (1964) used batch pasteurisation (68 ~ • 5 min) in a steam-jacketed cheese vat. Chapman et al. (1966), who did not use an aseptic milking technique, used HTST pasteurisation (71.6 ~ • 17 s) to produce low-count milk. Reiter et al. (1967), Kleter and de Vries (1974) and Visser (1976, 1977a) also used HTST pasteurisation. An LTLT regime (63 ~ • 30 min) was used by Reiter etal. (1969) and O'Keeffe etal. (1976a). Le Bars et al. (1975) used a UHT treatment and offset the ill-effects of the high heat treatment on the rennetability of milk by using a higher rennet concentration, a higher setting temperature and adding CaC12. Tyndallisation (three successive cycles of heating at 75 ~ x 5 min) or treatment with H 2 0 2 followed by catalase was used by Roberts et al. (1995) to treat milk for the production of aseptic cheese curd. Both treatments were successful, although H 2 0 2 caused the development of oxidised off-flavours. Cheese with a controlled microflora must be manufactured under aseptic conditions. Enclosed vats equipped with integral rubber gauntlets were used by Mabbitt etal. (1959) and modified by Perry and McGillivray (1964) to include pressurised or sterile air. Chapman etal. (1966) and Reiter etal. (1967, 1969) used a similar technique. Le Bars et al. (1975) made cheese in an aseptic room (5 • 3 m) with a filtered air supply and the cheesemakers were clothed in sterile garments. O'Keeffe etal. (1975, 1976a,b), McSweeney et al. (1994) and Lynch et al. (1996, 1997) made cheese in 20-1 vats set in thermostatically controlled water baths in a laminar air-flow unit. If the cheese curd is to be acidified chemically, antibiotics should be added to the cheesemilk to inhibit the growth of any surviving (or contaminating) bacteria. Nisin, penicillin and streptomycin were used by Le Bars et al. (1975) and O'Keeffe et al. (1976a). Addition of antibiotics is probably necessary to achieve aseptic starter-free cheese. Acidification of cheese curd to --pH 5, which is an essential element of cheese manufacture, is normally achieved by in situ production of lactic acid by a culture of LAB (starter). If the contribution of starter to cheese ripening is to be assessed, the use of starter must be avoided and acidification is then accomplished by pre-formed acid or acidogen. Early workers used dilute acid for direct acidification but encountered difficulties in controlling the pH. Mabbitt et al. (1955) largely overcame this problem by using an acidogen, gluconic acid-8-1actone (GDL), which hydrolyses to gluconic acid at a predictable rate in aqueous solutions. O'Keeffe et al. (1975) found that GDL, used as recommended by Mabbitt etal. (1955), caused
excessively rapid acidification, leading to extensive demineralisation of the casein micelles. Demineralisation was considered to be responsible for the excessively rapid rate of proteolysis observed in chemically acidified cheese but this may have been due to increased retention or activity of rennet in over-acid curd (Creamer et al., 1985). O'Keeffe et al. (1975) overcame excessively rapid acidification by using incremental addition of lactic acid to mimic the pH drop during cooking, followed by the addition of GDL to the curd after whey drainage. Roberts et al. (1995) developed an aseptic system for making cheese curd which was then used to produce slurries. The system consisted of a 1L cylindrical polypropylene 'vat' fitted with a lid and a stainless steel cutter/stirrer. The milk was coagulated, cut, cooked and stirred in the sealed 'vat'. At the end of cooking, the lid was replaced by a steel mesh screen through which the whey was drained off. Role of rennet in cheese ripening
The manufacture of rennet-free cheese is necessary if the contribution of rennet to ripening is to be assessed. Since rennet must be used to form a para-casein curd, the approach usually adopted is to inactivate the rennet after it has completed the first stage of rennetinduced coagulation. Four techniques have been developed to achieve this objective. Visser (1976) used cheesemilk which had been depleted of Ca and Mg by treatment with an ion-exchange resin; at the reduced Ca concentration, the enzymatic phase of renneting could be completed without coagulation. The rennet was subsequently inactivated by heat treatment (72 ~ • 15s), the milk cooled to 5 ~ and CaC12 added. To induce coagulation, the renneted milk was heated dielectrically to avoid agitation. Cheesemaking was then completed in aseptic vats. This technique was used by Visser (1977a,b,c) and Visser and de Groot-Mostert (1977). Porcine pepsin is very unstable at pH values near or above neutrality. O'Keeffe etal. (1977) used porcine pepsin as coagulant; after the gel had formed, it was cut and the pH of the curds-whey mixture raised to "-7 using NaOH; this technique has been used subsequently by Lane et al. (1997) with satisfactory results. Mulvihill et al. (1979) demonstrated the potential of piglet chymosin for the manufacture of rennet-flee cheese; this enzyme hydrolyses bovine K-casein but appears to be inactive on o%1- or [3-caseins or to be inactivated rapidly during the early stages of cheesemaking. Its use in cheesemaking was demonstrated in small-scale experiments. Meinardi et al. (1998) developed methodology and equipment for the production of rennet-flee cheese
Biochemistry of Cheese Ripening: Introduction and Overview
using pH-inactivated pepsin. Pasteurised milk supplemented with CaC12 was cooled to 6 ~ in a cylindrical glass vessel (15 1) equipped with a series of glass tubes through which water could be circulated to control the temperature and to heat the contents of the vessel without stirring. Porcine pepsin was added and the first stage of rennet action allowed to progress. The coagulant was inactivated by titrating the milk to pH 7.8 using NaOH and holding at this pH for 45 min. The milk was adjusted to pH 7.0 using HC1; starter was then added and addition of HC1 continued until the milk reached pH 6.5. Shakeel-Ur-Rehman et al. (1999) added pepstatin A (isovaleryl-Val-Val-statine-Ala-statine), a potent inhibitor of aspartyl proteinases, to the curds-whey mixture during cooking; results indicated that this compound very effectively inhibited chymosin action in cheese during ripening. Immobilised rennets have been suggested as another approach to making rennet-free curd (e.g., Fox et al., 1993) but leaching of the enzyme from the solid support makes this technique unsuitable. Furthermore, properly immobilised rennets are unable to coagulate milk as the PhemMet bond of K-casein may not be able to reach the active site of immobilised chymosin and the rate of diffusion of large casein micelles to immobilised rennet is very slow compared to the rate of diffusion of the small chymosin molecule towards the casein micelle (Beeby, 1979). Plasmin
Eliminating the proteolytic activity of plasmin presents more difficulties than eliminating the action of the coagulant. The contribution of plasmin to proteolysis in cheese has been assessed indirectly, i.e., in cheese from which all other agents have been eliminated (e.g., Visser and de Groot-Mostert, 1977). Plasmin is inhibited by soybean trypsin inhibitor which should be suitable for the inhibition of plasmin activity in cheese but no studies to evaluate this approach have been reported. The high heat stability of plasmin and the finding that its activity is increased by high cooking temperatures (Farkye and Fox, 1990) suggest that a model system could be developed in which aseptic curd is produced, the rennet denatured by a suitable cooking temperature and the curd acidified by GDL; such a system would allow plasmin to act in isolation. 6-Aminohexanoic acid (AHA) is an inhibitor of plasmin and/or plasminogen activators but does not inhibit chymosin or bacterial peptidases. It was used by Farkye and Fox (1991) to assess the role of plasmin in Cheddar cheese made without aseptic precautions and with a normal lactic acid starter, y-Casein bands on electrophoretograms were less intense in cheeses c o n -
355
taining AHA than in the control, suggesting that plasmin plays a role in Cheddar cheese ripening. It was necessary to use a high concentration of AHA to inhibit the plasmin in cheese curd and this appeared to cause increased syneresis and consequently reduced the moisture content of the cheese. Further, since AHA contains N, the background level of soluble N was increased greatly. Several specific irreversible inhibitors of serine proteinases were described by Harper et al. (1985) who recommended dichloroisocoumarin; as far as we are aware, none of these inhibitors have been used in studies on cheese. Since most of the potential plasmin activity in cheese is in the form of its inactive precursor, plasminogen, it is possible to increase plasmin activity in cheese by activation of plasminogen to plasmin using exogenous plasmin inhibitors. Barrett et al. (1999) used urokinase to activate plasminogen to plasmin, while Upadhyay et al. (unpublished) used streptokinase, a plasminogen activator produced by the mastitis pathogen, Streptococcus uberis. In both studies, the rate of proteolysis was accelerated on activation of plasminogen to plasmin. Since plasmin associates with the casein micelles, most exogenous plasmin added to milk is retained in the curd, unlike many exogenous enzymes added to cheese milk, which are lost in the whey. Studies in which exogenous plasmin was added to milk include Farkye and Fox (1992), Farkye and Landkammer (1992) and O'Farrell et al. (2002). Increasing the level of plasmin in milk increased the rate of primary proteolysis, but did not greatly increase the production of secondary proteolysis products. Cathepsin D
Cathepsin D is a lysosomal proteinase found at low levels in milk and which has a very similar specificity on the caseins to chymosin (McSweeney et al., 1995). Thus, it is difficult to assess the role of cathepsin D in proteolysis in rennet-coagulated cheeses due to the presence of the much greater amount of chymosin (or other coagulant). The hydrolysis of Otsl-casein to Otsl-CN (f24-199) in 'rennet-free' cheeses (e.g., Lane et al., 1997) and in high-cook varieties such as Swiss (in which much of the rennet is inactivated; see for example Cooney et al., 2000) has been attributed to the action of cathepsin D. However, it is also possible that the production of Otsl-CN (f24-199) in these cheeses was due to a low level of residual chymosin activity rather than cathepsin D which is largely inactivated on pasteurisation (Hayes et al., 2000). However, clear evidence for a minor role for cathepsin D in proteolysis in cheese during ripening has emerged from the study of Quarg, an acid-curd cheese (Hurley et al., 2000) and
356
Biochemistry of Cheese Ripening: Introduction and Overview
a pickled Feta-type cheese made from ultrafiltered milk in which no rennet was used in manufacture (Wium et al., 1998). Other indigenous enzymes
As discussed by Fox (2003), milk contains about 60 indigenous enzymes, of which about 20 have been isolated and characterised in detail. However, the contributions of only plasmin, cathepsin D and lipoprotein lipase (see Proteolysis in Cheese during Ripening and 'Lipolysis and Catabolism of Fatty Acids in Cheese', Volume 1) to cheese ripening have been investigated. It is possible that other indigenous enzymes, e.g., xanthine oxidase, sulphydryl oxidase or acid phosphatase may contribute to cheese ripening, although these enzymes have not been studied in this context. Starter enzymes
Advances in the genetics of LAB have permitted study of the roles of specific bacterial enzymes in cheese during ripening. The first enzyme to be studied in this way was lactocepin (cell envelope-associated proteinase, PrtP) of Lactococcus. The gene for this enzyme is plasmid-encoded, and therefore it is easy to produce PrtP- mutants. The role of this enzyme has been studied by comparison of cheese made with PrtP + or PrtP- strains (e.g., Farkye etal., 1990; Law et al., 1993; Lane and Fox, 1997; Broadbent et al., 2002) or cheeses made with control strains and starters with enhanced PrtP activities (e.g., Law et al., 1993). The principal role of PrtP during cheese ripening appears to be the degradation of intermediate-sized peptides produced from the caseins by the action of chymosin or plasmin. The genes for peptidases are chromosomally encoded and therefore more sophisticated techniques are required to prepare mutants with different peptidase genes than those that were used to produce PrtP- strains. Strains deficient in specific peptidases (e.g., Christensen et al., 1995; Meyer and Spahni, 1998), strains which overproduce specific peptidases (e.g., McGarry et al., 1994; Christensen et al., 1995) or strains which express peptidase genes from other organisms (e.g., Wegmann et al., 1999; Luoma et al., 2001; Courtin et al., 2002; Joutsjoki et al., 2002) have been developed. Although the objective of many of these studies has been to study the role of peptidases in nitrogen metabolism in LAB in milk, some (e.g., McGarry etal., 1994; Christensen etal., 1995; Courtin et al., 2002) have made cheeses using these mutant strains and thus information is available on the roles of peptidases in cheese during ripening which appears to degrade polypeptides to shorter peptides, usually with the release of amino acids.
Recent research has suggested that the principal role of proteolytic enzymes is the production of amino acids as precursors for a range of catabolic reactions which produce volatile flavour compounds. The genetics of enzymes involved in amino acid catabolism is now an active area of research and it is expected that mutant strains of LAB which are deficient in, or overproduce, specific amino acid catabolic enzymes will be produced in the near future. Cheese made using such strains or their wild types as starters would give very clear insights into the role of specific amino acid catabolic enzymes in the development of cheese flavour. NSLAB and their enzymes
Non-starter lactic acid bacteria affect cheese quality and almost certainly contribute to the intensity of flavour, although sometimes they may cause offflavours in cheese. The role of NSLAB in cheese ripening has been studied actively, principally with a view to explaining the differences observed between cheese made from raw or pasteurised milk. Comparison of raw and pasteurised milk cheese (e.g., Lau et al., 1990, 1991; McSweeney et al., 1993; Shakeel-Ur-Rehman et al., 1999) generally showed that raw milk cheese ripens more quickly and develops a stronger flavour than pasteurised milk cheese. Descriptive sensory analysis of the flavour profiles of raw and pasteurised milk Cheddar cheese (Muir et al., 1997) showed that raw milk cheese is more intensely flavoured than pasteurised milk cheese but has higher ratings for certain atypical flavours and is more variable. The role of NSLAB in cheese has been studied by physically removing them by microfiltration (e.g., McSweeney et al., 1993; Bouton and Grappin, 1995; Beuvier et al., 1997; Roy et al., 1997) or by inhibiting their growth by the addition of antibiotics at salting (e.g., Walsh etal., 1996; Shakeel-Ur-Rehman etal., 1999) or by the use of bacteriocin-producing starters (Fenelon etal., 1999). Several investigators have added selected strains of NSLAB to pasteurised milk as adjuncts (e.g., Broome et al., 1990; Muir et al., 1996). Cheese has been made under controlled bacteriological conditions in an attempt to prevent NSLAB gaining access to the cheese from the environment (e.g., McSweeney etal., 1994; Lynch etal., 1996, 1997). Raw milk (Shakeel-Ur-Rehman et al., 2000c) or MF retentate from raw skim milk (Beauvier et al., 1997) has been added to pasteurised cheesemilk as an inoculum of NSLAB. Blends of as little as 1% raw milk with 99% pasteurised milk influence the quality of cheese (Shakeel-Ur-Rehman et al., 2000c). Since the rate of growth of NSLAB is strongly affected by temperature (see Folkertsma et al., 1996), Shakeel-Ur-Rehman
Biochemistry of Cheese Ripening: Introduction and Overview 357 et al. (2000a,b) successfully prevented the growth of NSLAB in raw milk Cheddar by ripening at 1 ~ The results of these studies suggest that the differences observed between cheese made from raw and pasteurised milk are due principally to heat-induced changes to the NSLAB microflora, although pasteurisation largely inactivates the indigenous lipoprotein lipase, which results in a reduced level of lipolysis in pasteurised milk cheese. Since the proteolytic systems of NSLAB are generally similar to those of other LAB, they appear to contribute to proteolysis in a similar way to the starter, but to a lesser extent since maximum NSLAB numbers in cheese (often c. 107-108 cfu g - l ) are lower than maximum numbers of starter (c. 109-1010 cfu g-l).
Acceleration of Cheese Ripening Cheese ripening is a slow, and consequently an expensive, process. The expense of cheese ripening arises principally from the inventory cost associated with holding a large amount of cheese in storage and the capital cost of providing a ripening facility adequate to hold sufficient cheese during ripening. The temperature and, in certain cases, the relative humidity of ripening rooms must be controlled, adding to the cost of cheese ripening. Thus, acceleration of cheese ripening has received considerable attention in the scientific literature. This topic has been reviewed by Fox (1988/89), E1 Soda and Pandian (1991), Wilkinson (1993), Fox et al. (1996) and Upadhyay and McSweeney
(2003). Various approaches have been used to accelerate the ripening of cheese, including the use of an elevated ripening temperature, addition of exogenous enzymes or attenuated starters, use of adjunct cultures, use of genetically modified starter bacteria and high-pressure treatments. As novel processing technologies become available, it is likely that they will find an application to accelerate cheese ripening. Certain approaches (in particular the use of attenuated starters and adjunct cultures) are also used commercially to intensify the flavour of hard cheese and low-fat variants thereof, without necessarily reducing ripening time. Enzyme-modified cheeses are products in which a cheese-like flavour develops rapidly in a base material of young cheese (or sometimes caseinates) using cocktails of exogenous enzymes (and sometimes LAB) (see Kilcawley et al., 1998; 'Cheese as an Ingredient', Volume 2). The simplest and the most successful approach to accelerate ripening studied to date is an elevated ripening temperature (e.g., Folkertsma et al., 1996). Modification of the ripening temperature is used to control the rate of flavour development in hard cheese,
and ripening at an elevated temperature (e.g., c. 16 ~ results in the rapid development of flavour, although problems can occur with texture but this is not a serious drawback if the cheese is to be used in certain ingredient applications. Recent advances in the genetics of LAB and a greater understanding of the role of specific enzymes in the generation of volatile flavour compounds in cheese during ripening will facilitate the development of genetically modified starter strains to enhance flavour development.
Acknowledgements The author wishes to express his sincere thanks to Ms Niamh O'Sullivan, Ms. Patricia O'Connell and Ms Anne Cahalane for their assistance in preparing the typescripts of this and the other sub-chapters on cheese ripening.
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opment in Gouda cheese. 1. Description of cheese and aseptic cheesemaking techniques. Neth. Milk Dairy J. 31, 120-133. Visser, EM.W. (1977b). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 2. Development of bitterness and cheese flavour. Neth. Milk Dairy J. 31,188-209. Visser, EM.W. (1977c). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 3. Protein breakdown: analysis of the soluble nitrogen and amino nitrogen fractions. Neth. Milk Dairy J. 31,210-239. Visser, EM.W. and de Groot-Mostert, A.E.A. (1977). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 4. Protein breakdown: a gel electrophoretical study. Neth. Milk Dairy J. 31,247-264. Walsh, E.M., McSweeney, P.L.H. and Fox, P.E (1996). Inhibition of the growth of non-starter lactic acid bacteria in Cheddar cheese using antibiotics. Int. Dairy J. 6, 425--431. Wegmann, U., Klein, J.R., Drumm, I., Kuipers, O.P. and Henrich, B. (1999). Introduction of peptidase genes from Lactobacillus delbrueckii subsp, lactis into Lactococcus lactis and controlled expression. Appl. Environ. Microbiol. 65, 4729-4733. Wilkinson, M.G. (1993). Acceleration of cheese ripening, in, Cheese: Chemistry, Physics and Microbiology, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 523-556. Wium, H., Kristiansen, K.R. and Qvist, K.B. (1998). Proteolysis and its role in relation to texture of Feta cheese made from ultrafihered milk with different amounts of rennet. J. Dairy Res. 65,665-674. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11,185-201.
Metabolism of Residual Lactose and of Lactate and Citrate P.L.H. McSweeney and P.R Fox, Department of Food and Nutritional Sciences, University College, Cork, Ireland.
M e t a b o l i s m of L a c t o s e in C h e e s e During the manufacture of cheese curd, lactose is converted to lactic acid (mainly the L-isomer) by the starter bacteria (see 'Starter Cultures: General Aspects', Volume 1). In the case of Cheddar-type cheeses, most of the lactic acid is produced in the vat before salting and moulding whereas for most other varieties, acidification occurs mainly after the curds have been placed in moulds (see Fox et al., 2000). For many common varieties, the pH of the curd reaches - 5 . 0 - 5 . 3 within - 1 2 h from the start of cheesemaking. The rate and extent of acidification has a major impact on cheese texture via &mineralization of the casein micelles (see Creamer etal., 1985, 1988; Lawrence et al., 1987; Fox et al., 1990) and on cheese proteolysis owing to the increased susceptibility of &mineralized casein micelles to proteolysis (O'Keeffe et al., 1975) and/or greater retention of chymosin at low pH (Holmes et al., 1977; Stadhouders et al., 1977; Visser, 1977; Creamer et al., 1985; Garnot et al., 1987). However, such aspects will not be considered here. Although - 9 8 % of the lactose is removed in the whey as lactose or lactate during the manufacture of Cheddar (Huffman and Kristoffersen, 1984), Cheddar cheese curd contains 0.8-1.0% lactose at milling. Under normal circumstances, this residual lactose is metabolized quickly, predominantly to L-lactate, mainly through the activity of the starter bacteria (see 'Starter Cultures: General Aspects', Volume 1). The complete and rapid metabolism of the lactose and its constituent monosaccharides in cheese curd is essential for the production of good quality cheese since the presence of a fermentable carbohydrate may lead to the development of an undesirable secondary flora (see Fox et al., 1990, 2000). In Cheddar cheese, the residual lactose is fermented at a rate and to an extent dependent on the salt-in-moisture (S/M) content of the curd (Turner and Thomas, 1980). Lactococcus lactis subsp, cremoris is more salt-sensitive than Lc. lactis subsp, lactis, which in turn is more sensitive than non-starter lactic acid bacteria (NSLAB; Turner and Thomas, 1980). Since some NSLAB are facuhatively heterofermentative, the
% S/M may determine the products of lactose fermentation post-manufacture. At low S/M concentrations and low populations of NSLAB, residual lactose is converted mainly to Dlactate by the starter. At high populations of NSLAB, e.g., at a high storage temperature, considerable amounts of D-lactate are formed, partly by fermentation of residual lactose and partly by isomerization of Dlactate (Turner and Thomas, 1980). At high S/M levels (e.g., 6%) or at low NSLAB populations the concentration of lactose falls very slowly and changes in levels of lactate are slight. The quality of cheese is strongly influenced by the fermentation of residual lactose, as is evident from the data of O'Connor (1974). The pH decreases after salting, presumably due to the continued action of the starter at S/M levels <5%, but at higher level of S/M, starter activity decreases abruptly, as indicated by the high level of residual lactose and high pH. The quality of the cheeses also decreases sharply at > 5 % S/M (O'Connor, 1974). In Cheddar cheese made from milk supplemented with lactose to a concentration of 8% (-2.5% lactose in the cheese curd), lactose persisted and the pH continued to fall throughout a 9-month ripening period; the cheese had a harsh, strong over-acid flavour (Waldron, 1997; Shakeel-ur-Rehman and Fox, unpublished). In certain varieties (e.g., Edam, Gouda, Samsoe, Havarti), some whey is removed during curd manufacture and replaced with hot water. Probably, the initial function of this step was to cook the curds (on farms lacking steam-generating facilities and jacketed cheese vats) but it removes some lactose and reduces the ratio of lactose to buffering substances and hence acts to control pH (Walstra et al., 1993). Dutch-type cheese curd contains up to - 1 . 4 % lactose at pressing but this decreases to <0.1% after - 1 2 h and to undetectable levels after brining. The lactose is fermented by the starter to Dlactate. The curd for some Cheddar-type cheeses (washedcurd Cheddar and Monterey Jack) is washed to reduce its lactose content. The lactose in washed-curd Cheddar is exhausted rapidly, the pH, which remains higher
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
362
Metabolism of Residual Lactose and of Lactate and Citrate
than normal for Cheddar, rises during ripening (in common with most varieties) and the cheese has a mild clean flavour (Waldron, 1997; Shakeel-ur-Rehman and Fox, unpublished). The fermentation of lactose in Swiss-type cheeses is quite complicated (see Mocquot, 1979; Turner etal., 1983; Fox et al., 1990). Typically, 30 min after moulding, Emmental contains --1.7% lactose which is metabolized rapidly by Streptococcus thermophilus to a low level within 12 h, with the production of up to 0.8% L-lactate (Turner et al., 1983). Only the glucose moiety of lactose is metabolized by Sc. thermophilus and, consequently, galactose accumulates to a maximum of---0.7% at ---10 h. The lactobacilli metabolize lactose and galactose to a mixture of D- and L-lactate, reaching ---0.35 and 1.2%, respectively, at day 14, by which time all sugars are normally completely metabolized. Thereafter, the concentrations of L- and D-lactate change little until the cheese is transferred to the hot room, when the propionic acid bacteria begin to grow. Changes in the concentrations of lactose, galactose, glucose, D- and L-lactate and their degradation products in Swiss-type cheese were studied by Turner et al. (1983) and are shown in Fig. 1. Changes
to Lactate During Ripening
Lactate is an important substrate for a series of reactions in cheese during ripening (Fig. 2):
9 in most cheeses, L-lactate is racemized to D-lactate by the NSLAB flora; 9 lactate is catabolized in Swiss-type cheese by Propionibacteriurn freudenreichii subsp, shermanii which is important for the development of characteristic eyes and flavour; 9 lactate is catabolized to CO2 and H20 by Penicillium camemberti in surface mould-ripened cheeses, such as Camembert and Brie, which is important for texture development; 9 in the presence of 02, some members of the NSLAB flora, particularly pediococci, can oxidize lactate to formate and acetate; 9 lactate can be metabolized anaerobically by Clostridium tyrobutyricum leading to defects known as 'late gas blowing'. Racemization of lactate
The concentrations of lactate in Camembert, Swiss, Cheddar and Dutch-type cheeses are 1.0, 1.4, 1.5 and 1.2%, respectively (Raadsveld, 1957; O'Connor, 1974; Turner and Thomas, 1980; Thomas and Pearce, 1981; Turner et al., 1983; Karahadian and Lindsay, 1987; Walstra et al., 1993). Turner and Thomas (1980), Thomas and Pearce (1981) and Tinson et al. (1982) showed that experimental and commercial Cheddar cheese contains a considerable concentration of D-lactate, which could 10
P+l/2h
P+4h
P+10h
1 day
1.8[ ~-
et}
-9 .~_~~_ O
1.5
_
m
~ ~
9
actose
--
c"
O
~12
Q-
9
O ~-
O
~
O
~ 0.9
J
c .s
~- 0.60 cO
0 0.3-
P r o p i o n a ~
-
6-
12
18
24
'
hours
days Time from start of manufacture
Figure 1 Relationship between lactose and lactate metabolism, growth of propionic acid bacteria and production of propionate and acetate in Swiss cheese (Turner et aL, 1983).
Metabolism of Residual Lactose and of Lactate and Citrate
363
DL-Lactate
=~ ~..0 d- "~ |
Butyrate, H2 .._~ ~ '_,.,, o;.
O ~
~, ~..._
..-,, ~9"
fiU'" . _~,it~aO~ Propmnate, acetat
.,..
tie
§ "~elp~/| Formate, acetate, C O 2
(5) \"%%. 002, H20
Figure 2 Pathways by which lactate is metabolized in cheese during ripening. (1) racemization, (2) metabolism by Propionibacterium freudenreichfi subsp, shermanfi in Swiss cheese, (3) oxidative metabolism of lactate, (4) conversion to formate, ethanol and acetate and (5) anaerobic metabolism of lactate to butyrate and H2 which leads to late gas blowing.
be formed from residual lactose by lactobacilli or by racemization of L-lactate. Racemization of L-lactate is likely to occur more rapidly in cheese made from raw milk than in pasteurized milk cheese due to higher numbers of NSLAB and a more diverse nonstarter microflora in the former. Commercial Gouda contains a relatively low proportion of D-lactate, probably due to the short ripening time. The level of L- or D-lactate in Camembert is very low (Gripon, 1993) due to the catabolism of lactate by the mould, as discussed below. Racemization presumably involves oxidation of I_-lactate by L-lactate dehydrogenase (LDH) to pyruvate which is then reduced to D-lactate by D-LDH. Except in cases where the post-milling activity of the starter is suppressed, racemization is likely to be the principal mechanism (Thomas and Crow, 1983). Racemization of lactate is a major change in cheese during ripening, transforming up to approximately half the lactate or ---0.7% of the cheese mass (Thomas and Crow, 1983).
H
H
I
I /C~'tllOH HOOC
L(+)-Lactic acid
~PCH3
D(-)-Lactic acid
Thomas and Crow (1983) showed that pediococci isolated from cheese racemize L-lactate more actively than lactobacilli; all 27 pediococci isolated from Ched-
dar cheese and P. pentosaceus N CDO 1220 were capable of converting L-lactate to D-lactate, eventually producing a racemic mixture, while only 5 of 16 Lactobacillus isolates were capable of racemizing L-lactate, at much slower rates and to a lesser extent than the pediococci. However, pediococci constitute only a small proportion of the microflora of Cheddar cheese (Jordan and Cogan, 1993; Crow et al., 2001) and thus racemization of lactate in Cheddar and similar cheeses is presumably mainly a consequence of the growth of non-starter lactobacilli. Both lactobacilli and pediococci possess L(+)-LDH and D(-)-LDH, both of which are NAD + dependent. Racemization of L-lactate by cell suspensions of both pediococci and lactobacilli is pH dependent (optima: 4.0-5.2 and 4.5-6.0, respectively) and is retarded by an NaC1 concentration >5% or >2% for pediococci and lactobacilli, respectively (Thomas and Crow, 1983). Racemization of lactate in a Cheddar cheese inoculated with pediococci was complete after ---19 days, while it required ---3 months in a control cheese with a much lower number of NSLAB, especially pediococci (Thomas and Crow, 1983). The racemization of L-lactate is probably not significant from the favour viewpoint. However, Ca-lactate may crystallize in cheese, causing undesirable white specks, especially on cut surfaces (Pearce et al., 1973; Severn et al., 1986; Dybing et al., 1988). Such crystals are harmless, but they may cause consumers to reject cheese as being mouldy or containing foreign bodies (Dybing et al., 1988). The solubility of Ca-DL-lactate is lower than that of pure Ca-L-lactate (Thomas and Crow, 1983; Dybing et al., 1988) and hence racemization of lactate favours the development of crystals in cheese. Dybing etal. (1988) calculated that the amount of available lactate in cheese can potentially create enough Ca-lactate pentahydrate to exceed its
364
Metabolism of Residual Lactose and of Lactate and Citrate
solubility only slightly at 0 ~ Thus, crystal formation is favoured if microbial metabolism increases the concentration of D- relative to L-lactate, due to the lower solubility of Ca-DL-lactate. Crystal growth requires nucleation centres which may be bacterial cells, microcrystals of calcium phosphate or undissolved CaCO3. Increased levels of residual lactose, which favour the growth of NSLAB, can facilitate production of Ca-lactate crystals (Pearce et al., 1973; Sutherland and Jameson, 1981). Likewise, factors which increase the release of casein-bound Ca (e.g., low pH or high salt which causes the ion-exchange of Na + for Ca2+; Dybing et al., 1988) or reduce the solubility of Ca-lactate (e.g., a lower ripening temperature) favour crystal formation.
Oxidation of lactate
Lactate can be metabolized by LAB, depending on strain, to acetate, ethanol, formate and CO2 (see Fox et al., 2000). Pediococci, if present in cheese together with high concentrations of 02, produce 1 mol of acetate and 1 mol of CO2 and consume 1 mol of 02 per mol of lactate utilized (Thomas et al., 1985). The pH optimum for oxidation is 5-6 and depends on the lactate concentration. The concentration of lactate in cheese exceeds that required for optimal oxidation, and lactate is not oxidized until all sugars have been exhausted. However, the oxidation of L-lactate to acetate occurs to a very limited extent in cheese wrapped in film due to the low level of 02 available. The oxidative activity of suspensions of starter and NSLAB isolated from cheese on lactose, lactate, citrate, amino acids and peptides was studied by Thomas (1986). Starter bacteria were active mainly on lactose, with low activity on enzyme-hydrolysed casein; Lb. casei oxidized citrate, while Lb. plantarum, Lb. brevis and P. pentosaceus oxidized lactose, peptides, L- and D-lactate, but not citrate. These results suggest that the oxidation of lactate to acetate in cheese depends on the NSLAB population and on the availability of 02, which is determined by the size of the block and the oxygen permeability of the packaging material (Thomas, 1987). Acetate, which may also be produced by starter bacteria from lactose (Thomas et al., 1979), citrate or from amino acids by starter bacteria and lactobacilli (Nakae and Elliott, 1965), is usually present at high concentrations in most, or all, cheeses and is considered to contribute to cheese flavour, although a high concentration may cause off-flavours (Aston and Dulley, 1982).
Oxidative metabolism of lactate in surface mould-ripened varieties
The catabolism of lactate is very extensive in surface mould-ripened varieties, e.g., Camembert and Brie. The concentration of lactate in these cheeses at day 1 is ---1.0%, produced mainly or exclusively by the mesophilic starter, and hence, presumably, is L-lactate. Secondary organisms quickly colonize and dominate the surface of these cheeses (Addis et al., 2001), initially Geotrichum candiclum and yeasts (e.g., Kluyveromyces lactis, Debaryomyces hansenii and Saccharomyces cerevisiae; Gripon, 1993), followed by a dense growth of Penicillium carnemberti (Mollimard et al., 1995) and, particularly in traditional manufacture, by low numbers of Gram-positive organisms similar to those found on the surface of smear-ripened cheeses, which do not colonize the cheese surface until the pH has increased to >5.8 (see 'Surface Mould-ripened Cheeses', Volume 2). G. candidurn and P. camemberti rapidly metabolize lactate to CO2 and H20, causing an increase in pH. Deacidification occurs initially at the surface, resulting in a pH gradient from the surface to the centre and causing lactate to diffuse outwards. When the lactate has been exhausted, P. carnemberti metabolizes proteins, producing NH3 which diffuses inwards, further increasing the pH. The concentration of calcium phosphate at the surface exceeds its solubility at the high pH and precipitates as a layer of Ca3(PO4)2 on the surface, thereby causing a calcium phosphate gradient within the cheese, resulting in its outwards diffusion; reduction of the concentration of calcium phosphate in the interior helps to soften the body of the cheese (Fig. 3). In addition to softening the texture, changes to the cheese matrix may influence cheese flavour by changing the rates of migration or release of flavour compounds (Engel et al., 2001). The elevated pH stimulates the action of plasmin, which, together with residual coagulant, is responsible for proteolysis in this cheese rather than proteinases secreted by the surface microorganisms, which, although very potent, diffuse into the cheese to only a very limited extent, although peptides or other low molecular weight compounds produced by them at the surface may diffuse into the body of the cheese (Sousa and McSweeney, 2001; Churchill et al., 2003). The combined action of increased pH, loss of calcium (which affects to the integrity of the protein network) and proteolysis are necessary for the very considerable softening of the body of Brie and Camembert (see Noomen, 1983; Lenoir, 1984; Karahadian and Lindsay, 1987; Sousa and McSweeney, 2001). Changes which occur in Camembert-type cheese during ripening are indicated in Fig. 3.
M e t a b o l i s m of Residual Lactose and of Lactate and Citrate
365
Growth of Penicillium camemberti at surface
:'
Ammonia produced at surface by proteolysis diffuses into cheese
~ ; " ~ [ , u II , ..~ ,. , ~igh pH
Lactate metabolized at surface Ca3(PO4) 2precipitates
.~ .....
;~ "' ,~,~
.
Cheese softens from surface towards
Low pH
, . ..
: '~ ~
"'
............. Migration of soluble Ca, P043-and lactate towards surface
.O
score
,,
...~.~~
' ,
........
~
,.,,!,~; ' ~
.D E
.)
~.
d
~r
"~
'2 ., ....
~" <"
.
'
Figure 3 Schematic representation of the changes which occur in Camembert-type cheese during ripening as a consequence of the growth of Penicillium camemberti at the surface.
A n a e r o b i c m e t a b o l i s m of lactate by Clostridium tyrobutyricum
Gas (CO2 or H2) production by microorganisms may occur in cheese during ripening and may be desirable (e.g., eye production in Swiss and Dutch-type cheeses) or a defect. Organisms responsible for gas production in cheese are summarized in Table 1.
Table 1 Major microbial groups responsible for producing gas in cheese (modified from Mullan, 2000)
Gaseous Microorganism
Substrate
product(s)
Clostridium tyrobutyricum Lactobacillus casei Lactobacillus brevis
Lactate
OO2, H2
Citrate Lactose Urea
002
Lactose Lactose Citrate
002, H2
Lactose/citrate
CO2
Lactose/citrate
CO2
Lactate
CO2
Streptococcus thermophilus Coliforms Yeasts Citrate-positive lactococci Leuconostoc mesenteroides Leuconostoc dextranicum Propionibacterium freudenreichii subsp, shermanfi
CO2 CO2
CO2 CO2
Late gas blowing and accompanying off-flavours are defects associated with certain hard cheeses resulting from the anaerobic metabolism of lactate (or glucose) by Clostridium tyrobutyricum (and perhaps other clostridia; see Ingham et al., 1998) to butyrate and H2 (Fox et al., 1995; Klijn et al., 1995; Fig. 4); Cl. tyrobutyricum preferentially utilizes D-lactate in a mixture of both isomers (Huchet et al., 1997). Late gas blowing is a problem only in certain varieties, principally those that are brinesalted, owing to the time lag for NaC1 to reach an inhibitory level throughout cheese (Kleter et al., 1984). Cheddar cheese is not susceptible to late gas blowing mainly because it is dry-salted. The combined effects of pH, lactate, glycerol and NaC1 on the growth of vegetative cells of Cl. tyrobutyricum were studied by Huchet et al. (1995) who found that the growth of this organism is very sensitive to changes in these parameters within the ranges found during the manufacture of Emmental cheese (pH, 5.3-5.9; NaC1 level, 0-0.6%; lactic acid concentrations, 0-1.6%; aw, 0.965-0.99). Late gas blowing may be avoided by minimizing spore numbers in milk by good hygiene and avoiding the feeding of silage (Driehuis and Elferink, 2000). Germination of spores and the growth of the vegetative cells may be inhibited by the use of lysozyme or NO3- or perhaps by biological control (e.g., Carminati et al., 2001, who used co-inocula of Sc. thermophilus), or in the case of processed cheese, by long-chain polyphosphates (Loessner et al., 1997). Spores may be removed from milk by bactofugation or microfihration (see McSweeney and Sousa, 2000).
366
Metabolism of Residual Lactose and of Lactate and Citrate
H HOOCH OH
O
H
OH
Glucose Pi
NAD
Butyrate
CoA~[,,~ Acetyl-CoA
~
NADH2~
iY
2Pi
~ ~ 2ADP
AcetyI-P
ADP~
2ATP
Acetate
ATP
NADH2
O ~,,,,,~
O - ~ ' ~ ~176- ' ,
S.CoA
2
Butyryl-CoA
O
Pyruvate
\J
NAD OH
2 " " ~ ~O1 7 6 Lactate
NAD 2H2 NADH2 1~ 0
2 CoA
Fd+H2
~ s . C O A
H2o.
O
Crotonyl-CoA
OH
,~",v~
2 " ' ' " s"c~ Acetyl-CoA
0~0
O
CoA
s.CoA
L-I~-Hydroxybutyryl-CoA
NAD
NADH2
Acetoacetyl-CoA
Figure 4 Pathwayfor the formation of butyrate and H2 from glucose, lactose or lactate by Clostridium tyrobutyricum(Fd: ferredoxin) (McSweeney and Sousa, 2000).
Bactofugation of milk, an increased level of NaC1 in cheese and a reduced ripening temperature are effective measures for preventing or reducing gas production by Clostridium spp. (Su and Ingham, 2000). Lactate metabolism by Propionibacterium
In Swiss-type cheese, Propionibacterium freudenreichii subsp, shermanii metabolizes lactate to propionate, acetate, CO2 and H20 (Piveteau, 1999).
3 CH3CH(OH)COOH---*2 CH3CH2COOH+ CH3COOH+ CO2+ H20 Lactic acid propionic acid aceticacid The CO2 generated migrates through the cheese mass to points of weakness where it accumulates as eyes, a characteristic feature of Swiss cheese. Carbohydrate metabolism in Swiss cheese is summarized in
Fig. 1. Since propionibacteria are very sensitive to NaC1 (Richoux et al., 1998), Swiss-type cheeses contain a low level of salt. Eye development in Swiss cheese, which is discussed in 'Cheese with Propionic acid Fermentation', Volume 2 and by Steffen et al. (1993) and Polychroniadou (2001), depends mainly
on: 9 Rate and quantity of CO2 production. 9 Number and size of loci suitable for future eye development. 9 CO2 pressure and diffusion rate. 9 Cheese texture and temperature (Steffen etal., 1993). Relatively little of the total amount of CO2 produced by the propionic acid fermentation in Swiss cheese remains trapped in the eyes; in a cheese of c. 80 kg,
Metabolism of Residual Lactose and of Lactate and Citrate
120 1 CO2 are produced during ripening. Approximately 60 1 remain dissolved in the cheese mass, - 4 0 1 are lost from the cheese and only c. 20 1 remain in the eyes (Steffen et al., 1993). L-Lactate is metabolized preferentially to D-lactate by propionic acid bacteria (Crow, 1986) to reach 0.2% after ---20 days in the hot room (Turner et al., 1983). In fact, the concentration of D-lactate continues to increase to --0.4% during the early days in the warm room, before being metabolized by propionic acid bacteria. Increasing the number of starter lactobacilli accelerates sugar metabolism and causes higher concentrations of both D- and L-lactate but suppresses the growth of propionibacteria (due to a lower pH in the cheese) and thus delays the production of propionate and acetate. In the absence of lactobacilli or with Gal- lactobacilli, galactose accumulates and no D-lactate is formed. Therefore, the proportion of lactobacilli in the starter probably influences the production of CO2 and volatile acids.
Citrate Metabolism
The relatively low concentration of citrate in milk ( - 8 mmol 1-1) belies the importance of its metabolism in many cheeses made using a mesophilic culture (for reviews see Cogan, 1985; Cogan and Daly, 1987; Fox et al., 1990; Cogan and Hill, 1993; 'Starter Cultures: General Aspects', Volume 1). Approximately 94% of the citrate in milk is soluble and most of it is lost in the whey; however, the concentration of citrate in the aqueous phase of cheese is - 3 times that in whey (Fryer et al., 1970), presumably reflecting the concentration of colloidal citrate; Cheddar cheese contains 0.2-0.5% citrate. Citrate is not metabolized by most strains of Lc. lactis subsp, lactis or Lc. lactis subsp, cremoris, but is metabolized, with the production of diacetyl, acetate, acetoin and CO2, by citrate-positive (Cit +) strains of lactococci (formerly referred to as Lc. lactis subsp, lactis biovar diacetylactis or Streptococcus diacetilactis) in which the trait is plasmid-encoded, and Leuconostoc mesenteriodes subsp, cremoris and Ln. lactis. It is not metabolized by Sc. thermophilus or by thermophilic lactobacilli (see Fox et al., 2000). Citrate is not used as an energy source by Cit + lactococci or Leuconostoc spp., but it is metabolized very rapidly in the presence of a fermentable carbohydrate by the pathway outlined in Fig. 5. Enzymes in this pathway have been characterized (see Cogan and Hill, 1993; O'Sullivan et al., 2001). The CO2 produced on citrate metabolism is responsible for the characteristic eyes of Dutch-type cheese and for the undesirable openness and
367
floating curd defects in Cheddar and Cottage cheese, respectively. Due mainly to the formation of diacetyl, citrate metabolism is very significant in aroma/flavour development in Cottage cheese (e.g., Antinone et al., 1994), Quarg (Mohr et al., 1997), and many fermented milks, particularly cultured 'buttermilk' (Ulberth, 1991; Gaafar, 1992; Laye et al., 1993; Hernandez et al., 1995; Rankin and Bodyfeh, 1995). Diacetyl also contributes to the flavour of Dutch-type and Cheddar cheeses (McGugan, 1975; Manning, 1979a,b; Dacremont and Vickers, 1994; Christensen and Reineccius, 1995; Milo and Reineccius, 1997). However, diacetyl is produced in small amounts (<0.11 mmol 1-1 in milk); acetoin production is usually 10-50 times greater. According to the pathway shown in Fig. 5, 1 mol acetate should be produced from 1 mol citrate. However, studies suggest that c. 1.2 mol acetate are actually produced per mol citrate used; the excess probably results from small amounts of acetate produced on the metabolism of sugars. Acetate produced from citrate may also contribute to cheese flavour. There are few data on the production of 2,3-butanediol by starters (see Fox et al., 2000). In cheese with a controlled microflora, Fryer et al. (1970) showed that in cheese made using Lc. lactis subsp, crernoris, citrate remained constant at 0.2% up to 3 months, but decreased to 0.1% at 6 months. Cheese made using Lc. lactis subsp, cremoris plus a Cit + strain of Lactococcus contained no citrate at 3 months. Although Lb. casei could metabolize citrate in milk, the concentration of citrate in cheese made using Lc. lactis subsp, cremoris and Lb. casei decreased at about the same rate as in cheese made with Lc. lactis subsp, cremoris alone. Although the pathway shown in Fig. 5 is probably the major route for the metabolism of citrate by LAB, the possibility that lactate may be formed from the pyruvate produced from citrate cannot be overlooked. Three out of four strains of Cit + Leuconostoc growing on glucose plus citrate produced no diacetyl or acetoin and more lactate than could be accounted for in terms of the amount of glucose used, suggesting that pyruvate derived from citrate was being reduced to lactate (Cogan, 1987). Citrate may be metabolized by some strains of facuhatively heterofermentative lactobacilli, which are components of the NSI~B flora, to acetoin, acetate and probably diacetyl (Palles et al., 1998) by the same pathway as Cit + lactococci and Leuconostoc spp. (Fig. 5). Thomas (1987) showed that citrate in Cheddar cheese decreased slowly to almost zero at 6 months, presumably as a result of metabolism by lactobacilli which became the major component of the NSLAB flora. Inoculation of cheese milk with Lb. plantarum accelerated the depletion of citrate. Pediococci did not appear to utilize citrate.
368
Metabolism of Residual Lactose and of Lactate and Citrate
OH
|
|
o o
o
| OH OHi
J~k
Citrate
i;-I OH
_..o
.LL
_..o
Citrate ~ . ~ H
H
H
o
J~
OH
lyaseI
Lactose
Acetate
o
o
| o~~/~'-,.~ o| o
Oxalacetate
Acetolactate decarboxylase OH
NAD +
0
Lactate
NADH
CO2
O
TPP
-"
0
Lactatedehydrogenase
Other metabolic pathways
C02
o
"-
Pyruvate
~
Acetaldehyde TPP
OH
o-
~ e A~
Acetyl-CoA ~ , "
\
Diacetyl synthase
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~
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~
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2,3-Butanediol Figure 5 Pathways for citrate metabolism in citrate-positive strains of and Hill, 1993).
Lactococcusand Leuconostocspecies (modified from Cogan
Metabolism of Residual Lactose and of Lactate and Citrate
References Addis, E., Fleet, G.H., Cox, J.M., Kolax, D. and Leung, T. (2001). The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses. Int. J. Food Microbiol. 69, 25-36. Antinone, M.J., Lawless, H.T., Ledford, R.A. and Johnston, M. (1994). Diacetyl as a flavor component in full-fat Cottage cheese. J. Food Sci. 59, 38-42. Aston, J.W. and Dulley, J.R. (1982). Cheddar cheese flavour. Aust. J. Dairy Technol. 37, 59-64. Carminati, D., Perrone, A. and Neviani, E. (2001). Inhibition of Clostridium sporogenes growth in mascarpone cheese by co-inoculation with Streptococcus therrnophilus under conditions of temperature abuse. Food Microbiol. 18, 571-579. Christensen, K.R. and Reineccius, G.A. (i995). Aroma extract dilution analysis of aged Cheddar cheese. J. Food. Sci. 60, 218-220. Churchill, M.M., Hannon, J.A. and McSweeney, P.L.H. (2003). Proteolysis at the surface of Tilsit cheese. Milchwissenschaft 58,293-296. Cogan, T.M. (1985). The Leuconostocs: milk products, in, Bacterial Starter Cultures ,for Foods, S.E. Gilliland, ed., CRC Press, Boca Raton, FL. pp. 25-40. Cogan, T.M. (1987). Co-metabolism of citrate and glucose by Leuconostoc spp.: effects on growth, substrates and products. J. Appl. Bacteriol. 63,551-558. Cogan, T.M. and Daly, C. (1987). Cheese starter cultures, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, P.E Fox, ed., Elsevier Applied Science, London. pp. 179-250. Cogan, T.M. and Hill, C. (1993). Cheese starter cultures, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, General Aspects, 2nd edn, RE Fox, ed., Chapman & Hall, London. pp. 193-256. Creamer, L.K., Lawrence, R.C. and Gilles, J. (1985). Effect of acidification of cheese milk on the resultant Cheddar cheese. NZJ. Dairy Sci. Technol. 20, 185-203. Creamer, L.K., Gilles, J. and Lawrence, R.C. (1988). Effect of pH on the texture of Cheddar and Colby cheese. NZ J. Dairy Sci. Technol. 23, 23-35. Crow, V.L. (1986). Utilization of lactate isomers by Propionibacterium freudenreichii subsp, shermanii: regulatory role for intracellular pyruvate. Appl. Environ. Microbiol. 52, 352-358. Crow, V., Curry, B. and Hayes, M. (2001). The ecology of non-starter lactic acid bacteria (NSLAB) and their use as adjuncts in New Zealand Cheddar. Int. Dairy J. 11, 275-283. Dacremont, C. and Vickers, Z. (1994). Concept matching technique for assessing importance of volatile compounds for Cheddar cheese aroma. J. Food Sci. 59,981-985. Driehuis, E and Elferink, SJ.W.H.O. (2000). The impact of the quality of silage on animal health and food safety: a review. Vet. Q. 22,212-216. Dybing, S.T., Wiegand, J.A., Brudvig, S.A., Huang, E.A. and Chandan, R.C. (1988). Effect of processing variables on the formation of calcium lactate crystals on Cheddar cheese.J. Dairy Sci. 71, 1701-1710. Engel, E., Tournier, C., Salles, C. and Le Quere, J.L. (2001). Evolution of the composition of a selected bitter Camem-
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bert cheese during ripening: Release and migration of tasteactive compounds. J. Agric. Food Chem. 49, 2940-2947. Fox, RE, Lucey, J.A. and Cogan, T.M. (1990). Glycolysis and related reactions during cheese manufacture and ripening. CRC Crit. Rev. Food Sci. Nutr. 29,237-253. Fox, RE, Singh, T.K. and McSweeney, P.L.H. (1995). Biogenesis of flavour compounds in cheese, in, Chemistry of Structure~Function Relationships in Cheese, E.L. Malin and M.H. Tunick, eds., Plenum Publishing Corp., New York. pp. 59-98. Fox, RE, Guinee, T.P., Cogan, T.M. and McSweeney, P.L.H. (2000). Fundamentals of Cheese Science, Aspen Publishers, Gaithersburg, MD. Fryer, T.E, Sharpe, M.E. and Reiter, B. (1970). Utilization of milk citrate by lactic acid bacteria and 'blowing' of filmwrapped cheese. J. Dairy Res. 37, 17-28. Gaafar, A.M. (1992). Volatile flavour compounds of yoghurt. Int. J. Food Sci. Technol. 2 7, 87-91. Garnot, P., Molle, D. and Piot, M. (1987). Influence of pH, type of enzyme and ultrafiltration on the retention of milk clotting enzymes in Camembert cheese. J. Dairy Res. 54, 315-320. Gripon, J.C. (1993). Mould-ripened cheeses, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, Major Cheese Groups, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 111-136. Hernandez, E.J.G., Estepa, R.G. and Rivas, I.R. (1995). Analysis of diacetyl in yoghurt by two new spectrophotometric and fluorometric methods. Food Chem. 53, 315-319. Holmes, D.G., Duersch, J.W. and Ernstrom, C.A. (1977). Distribution of milk clotting enzymes between curd and whey and their survival during Cheddar cheesemaking. J. Dairy Sci. 60,862-869. Huchet, V., Thuault, D. and Bourgeois, C.M. (1995). Development of a model predicting the effects of pH, lactic acid, glycerol and sodium chloride content on the growth of vegetative cells of Clostridium tyrobutyricum in a culture medium. Lait 75,585-593. Huchet, V., Thuault, D. and Bourgeois, C.M. (1997). The stereoselectivity of the use of lactic acid by Clostridium tyrobutyricum. Food Microbiol. 14, 227-230. Huffman, L.M. and Kristoffersen, T. (1984). Role of lactose in Cheddar cheese manufacture and ripening. NZ J. Dairy Sci. Technol. 19,151-162. Ingham, S.C., Hassler, J.R., Tsai, Y.W. and Ingham, B.H. (1998). Differentiation of lactate-fermenting, gas-producing Clostridium spp. isolated from milk. Int. J. Food Mircobiol. 43, 173-183. Jordan, K.N. and Cogan, T.M. (1993). Identification and growth of non-starter lactic acid bacteria in Irish Cheddar cheese. It. J. Agric. Food Res. 32, 47-55. Karahadian, G. and Lindsay, R.C. (1987). Integrated roles of lactate, ammonia and calcium in texture development of mold surface-ripened cheeses. J. Dairy Sci. 70, 909-918. Kleter, G., Lammers, W.L. and Vos, E.A. (1984). The influence of pH and concentration of lactic acid and NaC1 on the growth of Clostridium tryobutyricum in whey and cheese. 2. Experiments in cheese. Neth. Milk Dairy J. 38, 31-41. Klijn, N., Nieuwenhof, EEJ., Hollwerf, J.D., Vanderwaals, C.B. and Weerkamp, A.H. (1995). Identification of Clostridium
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tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification. Appl. Environ. Microbiol. 61, 2919-2924. Lawrence, R.C., Creamer, L.K. and Gilles, J. (1987). Texture development during cheese ripening. J. Dairy Sci. 70, 1748-1760. Laye, I., Karleskind, D. and Morr, C.V. (1993). Chemical, microbiological and sensory properties of plain nonfat yoghurt. J. Food Sci. 58, 991-995. Lenoir, J. (1984). The surface flora and its role in the ripening of cheese. Bulletin 171, International Dairy Federation, Brussels. pp. 3-20. Loessner, M.J., Maier, S.K., Schiwek, P. and Scherer, S. (1997). Long-chain polyphosphates inhibit growth of Clostridium tyrobutyricum in processed cheese spreads. J. Food Prot. 60, 493-498. Manning, D.J. (1979a). Cheddar cheese flavour studies. II. Relative flavour contributions of individual volatile components. J. Dairy Res. 46, 523-529. Manning, D.J. (1979b). Chemical production of essential flavour compounds. J. Dairy Res. 46, 531-537. McGugan, W.A. (1975). Cheddar cheese flavour. A review of current progress. J. Agric. Food Chem. 23, 1047-1050. McSweeney, P.L.H. and Sousa, M.J. (2000). Biochemical pathways for the production of flavour compounds in cheese during ripening: a review. Lait 80, 293-324. Milo, C. and Reineccius, G.A. (1997). Identification and quantification of potent odorants in regular fat and low-fat mild Cheddar cheese. J. Agric. Food Chem. 45, 3590-3594. Mocquot, G. (1979). Reviews of the Progress of Dairy Science: Swiss-type cheese. J. Dairy Res. 46, 133-160. Mohr, B., Aymes, E, Rea, M.C., Monnet, C. and Cogan, T.M. (1997). A new method for the determination of 2-acetolactate in dairy products. Int. DairyJ. 7, 701-706. Mullah, W.M.A. (2000). Causes and control of early gas production in Cheddar cheese. Int. J. Dairy Technol. 53, 63-68. Nakae, T. and Elliott, J.A. (1965). Volatile fatty acids produced by some lactic acid bacteria. I. Factors influencing production of volatile fatty acids from casein hydrolysate. J. Dairy Sci. 48, 287-292. Noomen, A. (1983). The role of surface flora in the softening of cheeses with a low initial pH. Neth. Milk Dairy J. 37, 229-232. O'Connor, C.B. (1974). The quality and composition of Cheddar cheese: effect of various rates of salt addition. III. Ir. Agric. Creamery Rev. 27(1), 11-13. O'Keeffe, R.B., Fox, P.E and Daly, C. (1975). Proteolysis in Cheddar cheese: influence of the rate of acid production during manufacture. J. Dairy Res. 42, 111-122. O'Sullivan, S.M., Condon, S., Cogan, T.M. and Sheehan, D. (2001). Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1. FEMS Microbiol. Lett. 194, 245-249. Palles, T., Beresford, T., Condon, S. and Cogan, T.M. (1998). Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum. J. Appl. Microbiol. 85, 147-154. Pearce, K.N., Creamer, L.K. and Gilles, J. (1973). Calcium lactate deposits on rindless Cheddar cheese. NZ J. Dairy Sci. Technol. 8, 3-7.
Piveteau, P. (1999). Metabolism of lactate and sugars by dairy propionibacteria: a review. Lait 79, 23-41. Polychroniadou, A. (2001). Eyes in cheese: a concise review. Milchwissenschaft 56, 74-77. Raadsveld, C.W. (1957). The course of lactose breakdown in Dutch cheese. Neth. Milk DairyJ. 11,313-328. Rankin, S.A. and Bodyfelt, EW. (1995). Solvent desorption dynamic headspace method for diacetyl and acetoin in buttermilk. J. Food Sci. 60, 1205-1207. Richoux, R., Faivre, E. and Kerjean, J.R. (1998). Effect of salt content on lactate fermentation by Propionibacterium freundenreichii in small scale Swiss-type cheeses. Lait 78, 319-331. Severn, D.J., Johnson, M.E. and Olson, N.E (1986). Determination of lactic acid in Cheddar cheese and calcium lactate crystals. J. Dairy Sci. 69, 2027-2030. Sousa, M.J. and McSweeney, P.L.H. (2001). Studies on the ripening of Cooleeney, an Irish farmhouse Camemberttype cheese. Ir. J. Agric. Food Res. 40, 83-95. Stadhouders, J., Hup, G. and van der Waals, C.B. (1977). Determination of calf rennet in cheese. Neth. Milk Dairy J. 31,3-15. Steffen, C., Eberhard, E., Bosset, J.O. and Ruegg, M. (1993). Swiss-type varieties, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, Major Cheese Groups, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 83-110. Su, Y.C. and Ingham, S.C. (2000). Influence of milk centrifugation, brining and ripening conditions in preventing gas formation by Clostridium spp. in Gouda cheese. Int. J. Food Microbiol. 54, 147-154. Sutherland, B.J. and Jameson, G.W. (1981). Composition of hard cheese manufactured by uhrafihration. Aust. J. Dairy Technol. 36, 136-143. Thomas, T.D. (1986). Oxidative activity of bacteria from Cheddar cheese. NZJ. Dairy Sci. Technol. 21, 37-47. Thomas, T.D. (1987). Acetate production from lactate and citrate by non-starter bacteria in Cheddar cheese. NZ J. Dairy Sci. Technol. 22, 25-38. Thomas, T.D. and Crow, V.L. (1983). Mechanism of o(-)lactic acid formation in Cheddar cheese. NZ J. Dairy Sci. Technol. 18, 131-141. Thomas, T.D. and Pearce, K.N. (1981). Influence of salt on lactose fermentation and proteolysis in Cheddar cheese. NZ J. Dairy Sci. Technol. 16, 253-259. Thomas, T.D., Ellwood, D.C. and Longyear, M.C. (1979). Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures. J. Bacteriol. 138, 109-117. Thomas, T.D., McKay, L.L. and Morris, H.A. (1985). Lactate metabolism by Pediococci isolated from cheese. Appl. Environ. Microbiol. 49,908-913. Tinson, W., Radcliff, M.E, Hillier, A.J. and Jago, G.R. (1982). Metabolism of Streptococcus thermophilus. 3. Influence on the level of bacterial metabolites in Cheddar cheese. Aust. J. Dairy Technol. 37, 17-21. Turner, K.W. and Thomas, T.D. (1980). Lactose fermentation in Cheddar cheese and the effect of salt. NZ J. Dairy Sci. Technol. 15,265-276.
Metabolism of Residual Lactose and of Lactate and Citrate
Turner, K.W., Morris, H.A. and Martley, EG. (1983). Swisstype cheese. II. The role of thermophilic lactobacilli in sugar fermentation. NZJ. Dairy Sci. Technol. 18, 117-124. Ulberth, E (1991). Headspace gas-chromatographic estimation of some yoghurt volatiles. J. Ass. Off. Anal. Chem. 74, 630-634. Visser, EM.W. (1977). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 1. Description of cheese and
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aseptic cheesemaking technique. Neth. Milk Dairy J. 31, 120-133. Waldron, D.S. (1997). Effect of Lactose Concentration on the Quality of Cheddar Cheese. MSc Thesis, National University of Ireland, Cork. Walstra, R, Noomen, A. and Geurts, TJ. (1993). Dutch-type varieties, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, Major Cheese Groups, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 39-82.
Lipolysis and Catabolism of Fatty Acids in Cheese Y.F. Collins, Teagasc, Dairy Products Research Centre, Ireland P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland M.G. Wilkinson, Department of Life Sciences, University of Limerick, Ireland
Introduction Lipids present in foods may undergo oxidative or hydrolytic degradation (McSweeney and Sousa, 2000). However, lipid oxidation does not occur to a significant extent in cheese, probably due to its low redox potential ( - 2 5 0 m V ) (Fox and Wallace, 1997; Fox et al., 2000; McSweeney and Sousa, 2000). Enzymatic hydrolysis (lipolysis) of triglycerides to fatty acids and glycerol, mono- or di-glycerides is considered to be essential for flavour development in cheese (McSweeney and Sousa, 2000). Lipolysis is an important biochemical event during cheese ripening and has been studied extensively in varieties such as Blue and hard Italian cheeses where extensive lipolysis occurs and is a major pathway for flavour generation. However, in the case of cheeses such as Cheddar and Gouda, in which the level of lipolysis during ripening is low, the contribution of lipolysis to cheese quality and flavour has received relatively little attention. Free fatty acids (FFAs) are important precursors of several volatile compounds which contribute to flavour (McSweeney and Sousa, 2000; Collins et al., 2003a,b).
Lipolytic Agents in Cheese Lipolytic enzymes may be classified as esterases or lipases, which are distinguished according to three main characteristics: length of the hydrolysed acyl ester chain, physico-chemical nature of the substrate (whether emulsified or not) and enzymatic kinetics (esterases have classical Michaelis Menten-type kinetics while lipases, since they are active only at an interface, obey interracial Michaelis Menten-type kinetics) (Chich etal., 1997; Villeneuve and Foglia, 1997; Deeth and Touch, 2000). Unfortunately, the terms 'esterases' and 'lipases' are often used interchangeably in the scientific literature. In general, lipolytic enzymes are specific for fatty acids esterified at the sn-1 or sn-3 positions of triglycerides (Deeth and Touch, 2000). Initially, triglycerides are hydrolysed to 1,2- and 2,3-diglycerides and later to
2-monoglycerides; butyrate, as well as the other shortand medium-chain acids, are located mainly at the sn-1 and sn-3 positions in milk lipids and thus are preferentially released by lipolytic enzymes (Parodi, 1971; Christie, 1995; Deeth and Touch, 2000). Lipases in cheese originate from six possible sources: 9 9 9 9 9 9
milk rennet paste starter bacteria secondary starter microorganisms non-starter lactic acid bacteria (NSLAB) exogenous lipase preparations
(Deeth and Fitz-Gerald, 1995; Fox and Wallace, 1997; McSweeney and Sousa, 2000). Milk contains a very potent indigenous lipoprotein lipase (LPL) (Olivecrona and Bengtsson-Olivecrona, 1991; Fox and Stepaniak, 1993; Fox etal., 1993; Olivecrona et al., 2003). The enzyme consists of 450 amino acid residues, with an overall molecular mass of 55 kDa and in milk exists as a dimer of identical subunits. The enzyme has an isoelectric point above pH 9, with a positive charge at physiological pH (Olivecrona and Bengtsson-Olivecrona, 1991). The lipase is of blood origin and is involved in the metabolism of plasma triglycerides; its presence in milk is due to leakage through the mammary cell membrane. Bovine milk contains 10-20 nmol lipase/l which, under optimum conditions (37 ~ pH 7, in the presence of an apolipoprotein activator, apo-CII), could theoretically release sufficient FFAs within 10 s to cause perceptible hydrolytic rancidity in milk (Walstra and Jenness, 1984). This does not occur under normal circumstances as LPL and fat are compartmentalized, c. 90% of the LPL in milk is associated with the casein micelles while the triglycerides in milk occur as globules surrounded by a lipoprotein membrane, the milk fat globule membrane (MFGM). If the MFGM is damaged, e.g., due to agitation, foaming, homogenization, inappropriate milking or milk-handling techniques, significant lipolysis may occur, resulting in off-flavours
Cheese: Chemistry, Physics and Microbiology, Third edition- Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
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Lipolysis and Catabolism of Fatty Acids in Cheese
in cheese and other dairy products (Fox et al., 2000). Lipoprotein lipase has a preference for medium-chain triglycerides (MCT) with a 2-fold faster rate of hydrolysis of emulsions containing triC6:0, triC8:0, triC10:0 or triC12:0 compared to long-chain triglyceride (LCT) emulsions containing triC16:0, triC18:0, triC18:l, triC18:2, triC18:3 or triC20:0 (Deckelbaum et al., 1990). The different hydrolysis rates were attributed to higher concentrations of MCT at the emulsion surface due to higher mobility of such triglycerides compared with LCT emulsions (Deckelbaum et al., 1990). Lipoprotein lipase is relatively non-specific for fatty acid type but is specific for the acids at the sn-1 and sn-3 positions of mono-, di- and tri-glycerides (Olivecrona et al., 1992). Therefore, short- and medium-chain fatty acids are released preferentially by LPL. In raw milk cheeses, LPL activity is significant. According to Deeth and Fitz-Gerald (1983), it is generally accepted that high-temperature short-time (HTST) pasteurization (72~ for 15s) very extensively inactivates the enzyme. However, it may contribute to lipolysis in pasteurized milk cheese, as heating at 78 ~ • 10 s is required for its complete inactivation (Driessen, 1989). Commercial rennet extracts are free from lipolytic activity, but rennet paste, used in the manufacture of some hard Italian varieties (e.g., Provolone and Pecorino cheeses), contains a lipase, pregastric esterase (PGE) (Nelson etal., 1977; Hamosh, 1990; Fox, 2003). Although the term 'esterase' suggests that this enzyme acts only on water-soluble esters, it is in fact able to hydrolyse water-insoluble triglycerides containing long-chain fatty acids and hence 'esterase' is a misnomer as this enzyme is a lipase (Hamosh, 1990). Pregastric esterase is highly specific for short chain acids esterified at the sn-3 position (Nelson et al., 1977; Fox and Stepaniak, 1993; Fox, 2003). Pregastric esterases from different species are optimally active at 32-42 ~ pH 4.8-5.5 in presence of 0.5 M NaC1. The enzyme has an isoelectric point above pH 9 with a positive charge at physiological pH (see Fox, 2003). Suckling stimulates the secretion of PGE by glands at the base of the tongue, and it is washed into the abomasum by the milk. Rennet paste is prepared from the abomasa of calves, kids or lambs slaughtered after suckling. The abomasum is partially dried and ground into a paste, which is slurried in milk before being added to cheesemilk (Fox and Stepaniak, 1993). Some interspecies differences in specificity have been reported for calf, kid and lamb PGEs which result in slight differences in the flavour characteristics of cheese, depending on the source of PGE (Nelson et al., 1977; Fox and Stepaniak, 1993). Due to concerns regarding the hygienic quality of rennet pastes, research has been focused on identifiying exogenous
lipases which could be blended with rennet extracts to produce substitutes for rennet pastes. Most lipases investigated are unsuitable due to incorrect specificity; certain fungal lipases (e.g., a lipase secreted by Rhizomucor miehei) may be acceptable alternatives to rennet paste (Fox, 1988). Lipases and esterases of LAB appear to be the principal lipolytic agents in Cheddar and Dutch-type cheeses made from pasteurized milk (Fox et al., 2000). Evidence for this comes from studies on aseptic starterfree cheeses acidified with gluconic acid-8-1actone, where very low levels of FFAs are released during ripening (Reiter et al., 1967) and from the relationship between autolysis of starter cells and FFA levels during ripening (Collins et al., 2003a). Lactic acid bacteria possess esterolytic/lipolytic enzymes capable of hydrolysing a range of derivatives of FFAs, tri-, diand mono-glyceride substrates (Holland and Coolbear, 1996; Chich et al., 1997; Fox and Wallace, 1997; Liu et al., 2001). Despite the presence of these enzymes, LAB, especially Lactococcus and Lactobacillus spp. are weakly lipolytic in comparison to species such as Pseudomonas, Acinetobacter and Flavobacterium (Stadhouders and Veringa, 1973; Fox etal., 1993; Chich et al., 1997). However, because they are present at high numbers over an extended ripening period, LAB are responsible for the liberation of significant levels of FFAs in many cheese varieties which do not have a strongly lipolytic secondary flora. Lipases and esterases of LAB appear to be intracellular and a number have been isolated and characterized (Chich et al., 1997; Castillo et al., 1999; Liu et al., 2001). The presence of lipases and esterases has been demonstrated (Piatkiewicz, 1987) in nine strains of lactococci, including Lc. lactis subsp, lactis, citratepositive lactococci and Lc. lactis subsp, cremoris. [3-Naphthyl laurate (C12:0) and [3-naphthyl acetate (C2:0) were the substrates used to determine lipase and esterase activities, respectively; esterase activity was higher than lipase activity in all strains. Kamaly et al. (1990) reported the presence of lipases in the cell-free extract of a number of strains of Lc. lactis subsp, lactis and Lc. lactis subsp, cremoris; these lipases were, in general, optimally active at 37 ~ and at pH 7-8.5. Lc. lactis subsp, cremoris showed higher lipolytic activity on tributyrin and milk fat emulsions than Lc. lactis subsp, lactis. The activity of all lipases was stimulated by reduced glutathione and low (c. 2%) concentrations of NaC1 but inhibited by high NaC1 concentrations (c. 20%). Chich et al. (1997) reported esterolytic activity on [3-naphthyl butyrate by an intracellular extract of Lc. lactis subsp, lactis N CDO 763; the enzyme was active on p-NP esters from C2 to C12, with pH and temperature optima 7.0-8.0 and 55 ~
Lipolysis and Catabolism of Fatty Acids in Cheese
El-Soda et al. (1986) found intracellular esterolytic activities in four species of lactobacilli: Lb. helveticus, Lb. delbrueckii subsp, bulgaricus, Lb. delbrueckii subsp. lactis and Lb. acidophilus. All lactobacilli showed activity on p-nitrophenyl (p-NP) derivatives of fatty acids up to C5:0; Lb. delbrueckii subsp, lactis and Lb. acidophilus showed the highest esterolytic activity. None of the microorganisms tested hydrolysed o- and p-NP derivatives of C6:0 to C14:0. Khalid and Marth (1990) quantified the lipolytic activity of Lb. casei L-7, Lb. casei L-14, Lb. plantarum L-34 and Lb. helveticus L-53 on milk fat, olive oil and tributyrin emulsions; the three substrates were hydrolysed by all lactobacilli with the exception of Lb. casei L-7, which did not hydrolyse olive oil. According to Lee and Lee (1990), esterolytic and lipolytic enzymes are released by lysis of Lb. casei subsp, casei LLG cells. Maximum lipolytic activity was observed at pH 7.2 and 37 ~ and activity was inhibited by Ag + and Hg 2+ and stimulated by Mg 2+ and Ca 2+ (Lee and Lee, 1990). Lb. fermentum contains a cell surface-associated esterase which is optimally active on C4:0 but which can hydrolyse 6-naphthyl esters of fatty acids from C2:0 to C10:0 (Gobbetti et al., 1997). Gobbetti et al. (1996) reported the purification of an intracellular lipase from a strain of Lb. plantarum isolated from Cheddar cheese. This enzyme had a molecular mass of 65 kDa and was optimally active at pH 7.5 and 35 ~ it was relatively heat stable at 65 ~ but was irreversibly inactivated on heating at 75 ~ for 2 min. The enzyme was most active on tributyrin, with less activity on trilaurin and tripalmitin and no activity on triolein. When activity was determined on [3-naphthyl esters from C2 to C18:1, the enzyme was most active on [3-naphthyl butyrate, with significant activity on [3-naphthyl esters of acetate, caproate, caprylate and laurate, and low activity on [3-naphthyl caprate, myristate, palmitate, stearate and oleate. Liu et al. (2001) identified three intracellular esterases in Streptococcus thermophilus, two of which were purified to homogeneity and designated esterase I and II, with a molecular mass of--~34 and 60 kDa, respectively Differences in substrate specificity between esterases I and II were noted; esterase I hydrolysed p-NP esters of short chain acids from C2 to C8 while esterase II hydrolysed C2-C6 p-NP esters only. Both enzymes had maximum activity on p-NP butyrate. Only esterase I was tested on a range of glyceride substrates; it hydrolysed di- and mono-glycerides containing fatty acids up to C14:0 but tributyrin was the only triglyceride it could hydrolyse. The impact of cheese composition on esterase I activity on p-NP butyrate substrate indicated that activity was reduced by decreasing pH in the range 5.5-8.0, and decreasing water activity in
375
the range aw 0.99-0.80. Interestingly, esterase activity increased on increasing the NaC1 concentration from 3.7 to 7.5%, w/v; this stimulatory effect of NaC1 on esterase activity had not been reported previously. The genetic characterization of lipolytic enzymes of LAB by Fernandez et al. (2000) confirmed the intracellular nature of a tributyrin esterase in Lc. lactis subsp, cremoris B1014. These workers showed that the 744 base pair estA gene encoded for a 258 amino acid protein of molecular mass --~29 kDa; however, this gene did not encode for a signal sequence required for extracellular secretion. Cloning of the gene and up to 170-fold overproduction of this enzyme was possible using a nisincontrolled expression system which allowed detailed characterization of the specificity and kinetics of the enzyme. The esterase showed highest activity on short-chain p-NP-esters of fatty acids, with highest activity on p-NP hexanoate (C6:0). This enzyme was not active on p-NP esters of fatty acids of chain length longer than C12:0. Tributyrin was readily hydrolysed while activity was also detected on phospholipids. However, increasing the concentration of phospholipids to levels favouring micelle formation did not lead to an increase in enzyme activity through interfacial activation, confirming the esterolytic nature of this enzyme. Since the esterases/lipases of LAB appear to be intracellular, they must be released into the cheese matrix through cell autolysis for maximum efficiency. However to date, few studies have been undertaken to establish whether a relationship exists between the extent of autolysis of LAB and lipolysis in various cheese varieties. Early work by Walker and Keen (1974) showed that Cheddar cheese made with Lc. lactis subsp, cremoris AM2 developed higher levels of oddnumbered C3--C15 methyl ketones than cheese made with Lc. lactis subsp, cremoris HP which indicated that properties of the starter strain may influence the concentrations of these compounds in cheese. However, these workers did not monitor cell viability or autolysis in cheese during ripening. Wilkinson et al. (1994) demonstrated that Lc. lactis subsp, cremoris AM2 is more autolytic than Lc. lactis subsp, cremoris HP and that secondary proteolysis is higher in Cheddar cheese made using the former strain. Collins et al. (2003a) studied the influence of starter autolysis on lipolysis during a 238-day ripening period, as measured by the release of FFAs (C4:0-C18:3) in Cheddar cheese made using Lc. lactis subsp, cremoris AM2 or Lc. lactis subsp. cremoris HP as starter. These workers found that cheese made using the highly autolytic starter, Lc. lactis subsp, cremoris AM2, developed significantly higher levels of caprylate (C8:0), myristate (C14:0), palmitate (C16:0) and stearate (C18:0) during ripening than
376
Lipolysis and Catabolism of Fatty Acids in Cheese
cheese made with the less autolytic strain, Lc. lactis subsp, cremoris HE Cell-free extracts prepared from both strains had generally similar levels of activity on a triolein emulsion (lipase) or a p-NP butyrate (esterase) and these workers suggested that there is a relationship between early autolysis of these starter bacteria and lipolysis, possibly as a result of elevated release of intracellular lipolytic activity in cheese. Freitas et al. (1999) assayed proteolytic and lipolytic activities of Enterococcus faecium, Ec. faecalis, Lb. plantarum and Lb. paracasei and three species of yeasts (Debaryomyces hansenii, Yarrowia lipolytica and Cryptococcus laurentii) isolated from Picante cheese. High lipolytic activity was reported for Y lipolytica, using tributyrin as substrate; the other species studied were less lipolytic. Brevibacterium linens is a constituent of the microflora of surface smear-ripened cheeses (e.g., Limburger) which undergo a significant level of lipolysis during ripening. Lipolytic activity has been demonstrated in B. linens using emulsified olive oil as substrate (San Clemente and Vadehra, 1967). Sorhaug and Ordal (1974) reported esterolytic and lipolytic activities in five strains of B. linens using tributyrin and olive oil as substrates. Welch Baillargeon et al. (1989) reported lipase activity in three strains of Geotrichum candidum. Emulsified esters of oleic or palmitic acid were used as substrates; optimum pH and temperature for lipolytic activity were pH 7 and 37 ~ respectively. Lipases of different strains of G. candidum may be classified either as type A strains, which are not highly specific for unsaturated substrates with cis-9 double bonds, or type B strains, which are specific for cis-9 18:1 (Jacobsen and Poulsen, 1995). In mould-ripened cheeses, such as Brie, Camembert and Roquefort, Penicillium spp. are significant agents of lipolysis (Gripon, 1993; McSweeney and Sousa, 2000). P. roqueforti possesses two lipases, one with a pH optimum of 7.5-8, the other with a more alkaline pH optimum (9-9.5) (Morris and Jezeski, 1953; Kman etal., 1966; Niki et al., 1966). The lipase with the lower pH optimum is more active on tricaproin while that with the higher pH optimum is more active on tributyrin (Menassa and Lamberet, 1982). P. camemberti produces an extracellular lipase, which is optimally active on tributyrin at pH 9 and 35~ (Lamberet and Lenoir, 1976). Propionic acid bacteria are between 10 and 100 times more lipolytic than LAB (Knaut and Mazurek, 1974; Dupuis, 1994). Using emulsified tributyrin as substrate, Oterholm et al. (1970) showed that P. freudenreichii subsp, shermanii possesses an intracellular lipase with pH and temperature optima of 7.2 and 47 ~ The maximum rate of hydrolysis of triglycer-
ides was observed on tripropionin (C3:0), followed in order by tributyrin (C4:0), tricaproin (C6:0) and tricaprylin (C8:0). Hydrolysis of substrates in solution was low in comparison to hydrolysis of emulsified substrates, suggesting that the enzyme is a lipase. Dupuis et al. (1993) screened a number of strains of propionic acid bacteria for both esterolytic and lipolytic activities on actetate, propionate and butyrate esters and tributyrin. Intracellular fractions prepared from strains grown in liquid media showed both esterase and lipase activities. In the study of Dupuis et al. (1993) evidence was provided for the presence of an extracellular esterase; however, the extent to which this activity may have been due to cell lysis was not determined. Kakariari et al. (2000) purified an intracellular esterase from P. freudenreichii subsp, freudenreichii. Cell-free supernatant, cell wall fractions and a sonicated intracellular extract were assayed for esterase activity. However, in contrast to Dupuis et al. (1993), esterase activity was not found in the cell-free medium, or in the cell wall fractions. The enzyme purified by Kakariari et al. (2000) was therefore either cytoplasmic or cell membrane-associated.
Catabolism of Fatty Acids and Other Reactions In cheese, FFAs are precursors of many important flavour and aroma compounds, such as methyl ketones, lactones, esters, alkanes and secondary alcohols. Pathways for fatty acid catabolism are summarized in Fig. 1. Methyl ketones (alkan-2-ones) are the most important flavour components in Blue cheese and are present at very high concentrations. Heptan-2-one and nonan2-one are the predominant methyl ketones in Blue cheese; their concentrations in Gamonedo increased during the early part of ripening to a maximum at 60 days, after which levels began to decrease (Gonzalez de Llano et al., 1990). Methyl ketones are formed in Blue cheese by the action of Penicillium roqueforti (Urbach, 1997). P. camemberti and G. candidum may also produce methyl ketones (Lamberet et al., 1982; Cerning et al., 1987; Molimard and Spinnler, 1996). Penicillium spores, as well as vegetative mycelia, can produce methyl ketones (Chalier and Crouzet, 1998). Metabolism of fatty acids by Penicillium spp. involves four main steps corresponding to the early stages of [B-oxidation (Fig. 2). Initially, fatty acids are released by lipases, followed by the oxidation of FFA to [3-ketoacids and decarboxylation to alkan-2-ones, of one less carbon atom than the parent FFA; alkan-2-ones may be reduced to the corresponding secondary alcohol (alkan-2-ol). It has been suggested (Dartey and Kinsella, 1973; Kinsella and Hwang, 1976a) that FFAs are not the
Lipolysis and Catabolism of Fatty Acids in Cheese
377
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only precursors of methyl ketones; they can also be formed from ketoacids naturally present at low concentrations in milk fat or by oxidation of monounsaturated fatty acids (Kinsella and Hwang, 1976b). The rate of production of methyl ketones in cheese is affected by temperature, pH, physiological state of the mould and the concentration of FFAs. Spores of E roqueforti can oxidize fatty acids containing 4-12 carbon atoms to methyl ketones, with octanoic acid being most rapidly oxidized. Oxidation of longer chain FFAs containing 16 or 18 carbon atoms has also been reported (Chalier and Crouzet, 1993). Mycelia oxidize FFAs over a wide pH range, with an optimum between pH 5 and 7, which is similar to the pH of mature Blue cheese (Dwivedi and Kinsella, 1974; Gripon, 1993).
Dumont etal. (1974a,b,c) identified 11 methyl ketones in Camembert cheese. Methyl ketones with even-numbered carbon chains appeared late in ripening and were present at low levels, except in very mature cheese. Heptan-2-one is present at significant concentrations in Parmigiano-Reggiano (Meinhart and Schreier, 1986). In full-fat Cheddar cheese, levels of heptan-2-one, nonan-2-one and undecan-2-one increased for approximately 14 weeks, after which levels decreased; concentrations of methyl ketones in low-fat Cheddar cheeses were lower than the levels in full-fat cheese (Dimos, 1992), perhaps due to lower levels of FFAs in the former (Dimos et al., 1996). Engels et al. (1997) compared the volatile compounds in the watersoluble fraction of seven cheese varieties (Gouda,
378
Lipolysis and Catabolism of Fatty Acids in Cheese
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002
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Figure 2 Pathway for the catabolism of fatty acids by Penicillium spp.
Proosdij, Gruyere, Maasdam, Edam, Parmesan and Cheddar). Nine ketones, mostly methyl ketones, were identified. Dirinck and De Winne (1999) reported levels of heptan-2-one and nonan-2-one ranging from 2903 to 3210 ng g- ] and from 1841 to 1960 ng g- 1, respectively, in Gouda cheese; levels varied between 3332 and 3598 ng g-1 and 1768 and 1986 ng g-l, respectively, in Emmental. In another study, pentan-2-one and heptan2-one were the most abundant methyl ketones in aged ewes' milk cheese (14 samples were analysed, nine of which were Manchego); mean levels were 737 and 368 ixg kg-1, respectively (Villasefior et al., 2000). Secondary alcohols can be formed in cheeses by enzymatic reduction of methyl ketones (Engels et al., 1997). P. roqueforti is responsible for the reduction of methyl ketones to secondary alcohols (e.g., 2-pentanol, 2-heptanol and 2-nonanol) in Blue cheese (Martelli, 1989). Gonzalez de Llano et al. (1990) reported that 2-heptanol and 2-nonanol are the main secondary alcohols in artisanal Gamonedo Blue cheese. Production of 2-propanol from acetone and 2-butanol from butanone has been reported in Cheddar cheese (Urbach, 1993), while Thierry et al. (1999) reported an increase in the concentrations of secondary alcohols in the aqueous phase of Emmental during ripening. A great diversity of esters, formed by the reaction of a FFA with an alcohol, is present in cheese (Molimard and Spinnler, 1996). While methyl, ethyl, propyl and butyl esters of FFAs have been reported in various cheese varieties (Meinhart and Schreier, 1986), ethyl
esters predominate (Arora et al., 1995). Esterification reactions resulting in the production of esters occur between short- to medium-chain fatty acids and ethanol, derived from lactose fermentation or from amino acid catabolism. Ethyl acetate may also arise from esterification of ethanol with acetyl-coenzyme A (Yoshioka and Hashimoto, 1983). Holland etal. (2002) suggested that esters are formed in cheese during ripening by the transesterification of a FFA from partial glycerides to ethanol. Thirty eight esters were identified by Meinhart and Schreier (1986) in Parmigiano-Reggiano cheese; ethyl acetate, ethyl octanoate, ethyl decanoate and methyl hexanoate were the most abundant. Methyl and ethyl esters have been found at high levels in artisanal Blue cheese (Gonzalez de Llano et al., 1990). Fourteen esters were found in Emmental cheese by Imhof and Bosset (1994) and Rychlik et al. (1997). Ethyl esters of fatty acids and smaller quantities of methyl esters have also been identified in Manchego cheese (Villasefior etal., 2000). Thioesters are formed when FFAs react with sulphydryl compounds (Molimard and Spinnler, 1996) and may be formed by the action of a wide range of microorganisms associated with cheese (Lamberet et al., 1997). Lactones are cyclic compounds formed by the intramolecular esterification of hydroxy fatty acids through the loss of water to form a ring structure (Christie, 1983; Molimard and Spinnler, 1996) and may be produced by heating their precursor hydroxyacids (Eriksen, 1976). or- and [3-1actones are highly reactive and unstable (Fox and Wallace, 1997) but ~/and 8-1actones (5- and 6-membered rings, respectively) are stable and have been identified in cheese (Eriksen, 1976). It has been reported that the mammary gland of ruminants has a 8-oxidation system for fatty acid catabolism (see Fox et al., 2000) which may produce the precursor hydroxy acids for the production of lactones, which may also be formed from keto acids after their reduction to hydroxyacids (Wong et al., 1975). The presence of large amounts of high molecular weight lactones has been reported in rancid Cheddar cheese, which has led to the suggestion that pathways other than the release of hydroxy acids from triglycerides may contribute to the formation of lactones (Wong et al., 1975). Dodecalactone may be formed by P. roqueforti spores and vegetative mycelia from long-chain unsaturated fatty acids (C18:1 and C18:2) (Chalier and Crouzet, 1992). Lactone precursors (hydroxy FA) may also be generated by the action of lipoxygenases and other enzymes present in members of the rumen microflora (Dufoss~ et al., 1994).
Lipolysis and Catabolism of Fatty Acids in Cheese 379 Production of lactone precursors in milk is influenced by several factors, including feed, season, stage of lactation and breed (see Fox et al., 2000). The sweet-flavoured y-dodecanolactone and y-dodec-Z-6-enolactone occur at much higher levels in milk from grain-fed than from pasture-fed cows (Urbach, 1997). 8-Dodecalactone and 8-tetradecalactone are the principal lactones in 75 day-old Blue cheese (Jolly and Kosikowski, 1975). In Cheddar cheese, lactone levels increased most rapidly early in the ripening period and were present at concentrations well above their flavour thresholds (Jolly and Kosikowski, 1975). Wong et al. (1975) reported levels of 1.5, 0.8, 4.9 and 8.9 I~g g-1 of 8C10, yC12, 8C12 and 8C14, respectively, in Cheddar cheese at 14 months. Several lactones have been identified in Parmigiano-Reggiano cheese; quantitatively, the most important lactone is 8-octalactone (Meinhart and Schreier, 1986). y-Decalactone, 8decalactone, y-dodecalactone and 8-dodecalactone have been found in Camembert cheese (Gallois and Langlois, 1990). Aldehydes may be formed via the catabolism of amino acids (see 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1). However, some straightchain aldehydes, e.g., butanal, heptanal and nonanal, may be formed by the [3-oxidation of unsaturated fatty acids. Gruyere and Parmesan have high levels of FFAs and high concentrations of straight chain aldehydes which have 'green grass-like' aromas (Moio et al., 1993).
Contribution of Lipolysis and Catabolism of FFA to Cheese Flavour Lipids play a major role in the quality of cheese: 9 They affect cheese rheology and texture (see 'Rheology and Texture of Cheese', Volume 1). 9 They influence flavour by: - Acting as a source of fatty acids which in turn may be catabolized to other flavour compounds, e.g., methyl ketones, esters, thioesters and lactones. - A c t i n g as a solvent for sapid compounds produced from lipids or other precursors. 9 Many reactions occur at the fat-water interface. Reduced-fat cheese lacks typical flavour and contains lower concentrations of FFAs than full-fat cheese, supporting the theory that FFAs are important to cheese flavour (Foda et al., 1974; Olson and Johnson, 1990; Dimos et al., 1996; Wijesundera et al., 1998). Cheddar cheese manufactured from milk in which the fat was replaced by vegetable or mineral oils has also been reported to develop atypical flavours (Foda et al., 1974; Wijesundera and Watkins, 2000). Foda et al.
(1974) found that cheese containing vegetable fats had very little Cheddar flavour, and cheese containing mineral oil had only a slight Cheddar flavour. Cheese containing milk fat gave the best results but its flavour was still inferior to that of cheese made from whole milk. These results suggest that the MFGM, which is replaced on homogenization of the milk fat, may have important enzymes or other factors which play a role in the development of Cheddar flavour. It is also plausible that the interface between the lipid and aqueous phases in the cheese is important for flavour development. However, Wijesundera and Drury (1999) reported no significant difference in Cheddar cheese flavour intensity between cheeses made from whole milk or from milk reconstituted from skim milk and cream or anhydrous milk fat. Long-chain FFAs ( > 1 2 carbon atoms) are considered to play a minor role in cheese flavour due to their high perception thresholds (Molimard and Spinnler, 1996). Short- and intermediate-chain FFAs (C4:0-C12:0) have considerably lower perception thresholds and each gives a characteristic flavour note. Butanoic acid contributes 'rancid' and 'cheesy' flavours; hexanoic acid has a 'pungent', 'Blue cheese' flavour note, while octanoic acid has a purported 'wax', 'soap', 'goat', 'musty', 'rancid' and 'fruity' note. Depending on their concentration and perception thresholds, volatile fatty acids can either contribute positively to the aroma of the cheese or to a rancidity defect. The flavour effect of FFAs in cheese is affected by pH. In cheeses with a high pH, e.g., smear-ripened and Blue cheeses, the flavour impact of fatty acids may be affected due to neutralization of the cheese (Molimard and Spinnler, 1996). In general, the flavour threshold of methyl ketones is quite low. Flavour threshold values determined in water vary widely; for heptan-2-one, ranging from 0.0009 to 3 mg kg -1 and for propan-2-one ranging from 40.9 to 500mg kg -1 (Molimard and Spinnler, 1996). Octan2-one, nonan-2-one, decan-2-one, undecan-2-one and tridecan-2-one are described as having 'fruity', 'floral' and 'musty' notes, while heptan-2-one has a Blue cheese note (Rothe et al., 1982). The mushroom and musty notes of methyl ketones are important contributors to the flavour of Camembert cheese (Molimard and Spinnler, 1996). According to Eriksen (1976), lactones have a strong flavour; although the aromas of lactones are not cheese-like, they may contribute to overall cheese flavour (see Fox et al., 1993, 2000; Fox and Wallace, 1997) and have been reported to contribute to a buttery character in cheese (Dirinck and De Winne, 1999). 8-Lactones have low flavour thresholds compared to other volatile flavour compounds (O'Keefe et al., 1969)
380 Lipolysis and Catabolism of Fatty Acids in Cheese and are generally characterized by very pronounced, fruity notes ('peach', 'apricot' and 'coconut') (Dufoss~ et al., 1994). 8-Lactones have generally higher detection thresholds than y-lactones; thresholds for y-octalactone, y-decalactone and ~/-dodecalactone are 7-11 txg kg -1 in water and are even lower for shorter chain lactones (Dufoss~ et al., 1994). In a survey of various cheese varieties, Engels et al. (1997) found high concentrations of ethyl butanoate in cheeses with a 'fruity' note, e.g., Gruyere, Parmesan and Proosdij. This fruity flavour is considered undesirable in Cheddar cheese (Urbach, 1997; McSweeney and Sousa, 2000). Arora et al. (1995), who analysed the odour-active volatiles in the headspace of Cheddar cheese, found that most of the esters separated had a 'buttery'/'fruity' aroma. However, thioesters formed by the reaction of short-chain fatty acids with methional imparted a characteristic 'cheesy' aroma to Cheddar cheese. According to Lamberet et al. (1997), S-methyl thioesters contribute a characteristic flavour to various smear-ripened soft cheeses (e.g., Tilsit, Limburger and Havarti). Secondary alcohols may contribute to cheese flavour (Arora et al., 1995). Propan-2-ol, butan-2-ol, octan-2-ol and nonan-2-ol are present in most soft cheeses and are typical components of the flavour of Blue cheeses (Engels et al., 1997). Moinas et al. (1975) found that heptan-2-ol and nonan-2-ol represented 10-20% and 5-10%, respectively, of all aromatic compounds in Camembert cheese. Oct-l-en-3-ol has the odour of raw mushroom with a perception threshold of 10 Ixg kg -1, and has been proposed as one of the key compounds in the aromatic note of Camembert cheese (Molimard and Spinnler, 1996).
Patterns of Lipolysis in Various Cheese Varieties The level of lipolysis in cheese varies considerably from moderate (e.g., Cheddar, Cheshire, Caerphilly) to extensive (e.g., mould-ripened, hard Italian and surface bacterially-ripened (smear) varieties) (de Llano et al., 1992; McSweeney and Fox, 1993; Fernandez-Garcia et al., 1994; Fox and Wallace, 1997; Fox et al., 2000; McSweeney and Sousa, 2000). Concentrations of FFAs reported for a number of cheese varieties are shown in Table 1. Excessive lipolysis is considered undesirable in many varieties (e.g., Dutch-type cheeses, Cheddar, Emmental) and cheeses containing even a moderate concentration of FFAs may be considered as rancid by some consumers (Fox et al., 1993). However, limited lipolysis is thought to be desirable in these varieties. In Emmental cheese, moderate levels of FFAs, in the range 2-7 g kg -1, are liberated during ripening and
make an important contribution to the characteristic flavour and aroma of both raw and pasteurized milk Emmental cheese (Zerfiridis et al., 1984; Steffen et al., 1993; Chamba and Perreard, 2002). Recent data for lipolysis in Emmental cheese manufactured from raw or microfihered milk, with or without various strains of propionic acid bacteria, indicate that FFAs are released during ripening by the lipolytic activity of propionic acid bacteria and starter and that the concentration of FFAs produced in the cheese is specific to the strain of Propionibacterium used for cheesemaking (Chamba and Perreard, 2002). The highest levels of lipolysis have been observed in mould-ripened cheeses; 5-10% of total triglycerides are hydrolysed in Camembert and up to 25% in Blue cheeses (Anderson and Day, 1966; Gripon et al., 1991; Gripon, 1993). Extensive lipolysis occurs in mouldripened cheeses without rancidity due to neutralization of fatty acids as the pH increases during ripening (Gripon, 1993). The most important lipolytic agents in mould-ripened cheeses are the enzymes of Penicilliurn spp., although a high proportion of free oleic acid in Camembert has been attributed to the action of the lipase of Geotrichum candidum (Gripon, 1993). In the manufacture of Danablu cheese, raw milk is separated and the resulting cream is homogenized and held before pasteurization. This process damages its MFGM and activates the LPL; additionally, homogenization reduces fat globule size and increases total fat globule surface area, providing a larger lipid-serum interface for LPL activity (Nielsen, 1993). Extensive lipolysis is also characteristic of many Italian varieties (e.g., Grana Padano and Parmigiano-Reggiano) (Woo and Lindsay, 1984; Bosset and Gauch, 1993). Arnold et al. (1975) found a direct relationship between the flavour intensity of ripened Romano-type cheese and its butyric acid content. Many Italian varieties are manufactured from raw milk (e.g., Parmigiano-Reggiano, Grana Padano, Provolone) which leads to higher levels of lipolysis in the ripened cheese due to the action of LPL. Pregastric esterase is responsible for extensive lipolysis in cheeses made using rennet paste, resulting in the characteristic 'piccante' flavour of these varieties (Fox et al., 2000; McSweeney and Sousa, 2000). High levels of lipolysis occur in smear cheeses (e.g., Limburger) (Woo et al., 1984). Brevibacterium linens is a major constituent of the surface microflora of bacterial smear-ripened cheeses and produces active lipolytic enzymes (Reps, 1993).
Measurement of Lipolysis Various quantitative techniques have been developed to monitor release of FFAs in cheese. The 'copper soaps' method is a colorimetric method enabling the
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Lipolysis and Catabolism of Fatty Acids in Cheese
determination of the total level of FFAs (IDE 1991). Copper soaps of fatty acids are selectively transferred to an organic phase; a coloured complex is then formed between the copper soaps and sodium diethyl dithiocarbamate, the intensity of which is related to the concentration of FFAs. While the method is sensitive and rapid (IDE 1991), the total level of FFAs is only estimated. Quantification of the total level of FFAs by extraction and titration of FFAs with alcoholic KOH gives an acid degree value (ADV), defined as the number of milliequivalents of alkali required to neutralize the FFAs in 100 g of fat. Gas chromatography (GC) has been the method most commonly used to quantify levels of individual FFAs in cheese and is the dominant technique for the routine analysis of FFAs; the flame ionization detector is robust with a wide and dynamic range, enabling accurate FFA quantification. In a review of the determination of FFAs in milk and milk products ODE 1991) methods for analysis of FFAs by GC were grouped under three headings. In the first group, FFAs are analysed directly and this group includes the method of Nieuwenhof and Hup (1971). Free fatty acids are isolated on an alkaline silica gel column, the eluate is concentrated and FFAs are quantified directly by GC. However, the silicic acid column used by Nieuwenhof and Hup (1971) was later shown to induce hydrolysis of the fat. The method of Woo and Lindsay (1982) is also included in this group; this method involves removal of lactic acid by a partition pre-column, followed by isolation of FFAs on a modified silicic acid-potassium hydroxide arrestant column. Free fatty acids are then separated in formic acidmobilized elutes on a glass column packed with diethylene glycol succinate (DEGS-PS) by GC. This GC procedure enables rapid separation and quantification of FFAs and has been used by others (Woo and Lindsay, 1984; Woo et al., 1984) to quantify individual FFAs in various cheeses. The second group of GC methods includes those of Iyer et al. (1967), McNeill and Connolly (1989) and Fontecha et al. (1990). Solvent is initially used to extract the lipid fraction from the cheese, followed by the preparation of methyl esters of FFAs and the separation and quantification of methyl esters of FFAs by GC. In general, the third group of GC methods quoted in Table 1 involves solvent extraction of the lipid fraction from the cheese, followed by separation of FFAs by, e.g., saponification (Martinez-Castro et al., 1986; Martin-Hernandez et al., 1990; MartinHern~ndez and Juarez, 1992; de la Feunte et al., 1993; Poveda et al., 1999), separation using chromatographic columns (Deeth et al., 1983; Lesage et al., 1993), or methylation to fatty acid methyl esters (FAME) (Ha and Lindsay, 1990; Kinderlerer et al., 1996; Partidario
et al., 1998; Partidario, 1999). The method of Dulley and Grieve (1974) uses steam-distillation followed by GC to quantify volatile fatty acids. The method described by de Jong and Badings (1990) is now a commonly used procedure for the quantification of FFA in cheese. Accurate and rapid determination of FFAs from C2:0 to C20:0 is achieved by direct separation of underivatized FFAs using capillary GC. In brief, the method involves extraction of FFA using ether/heptane from a cheese paste containing anhydrous sodium sulphate and H2SO 4. Extracted FFAs are then isolated using alumina or an anionexchange method (aminopropyl columns) and separation of FFAs (C2:0-C20:0) is achieved by capillary GC. Few studies have compared the different methods of FFA quantification from the same sample. According to IDF (1991), isolation of FFAs from the sample material is a vital step in any method. FFAs must be isolated from both the aqueous phase and the fat phase. Organic solvents are used in the extraction procedures of the majority of the methods referenced. According to IDF (1991), it is difficult to combine good extraction of short chain FFAs from the aqueous phase together with a good extraction from the fat phase. While these extraction methods commonly involve the use of internal standards of fatty acids dissolved in an organic solvent, this does not guarantee the completeness of the extraction procedure. Chavarri et al. (1997) compared two methods for sample preparation for the determination of FFAs in ewes' milk cheese by GC. In method 1, after fat extraction, FFAs were separated from triacylglycerides by aminopropyl bonded-phase chromatography. The fraction containing FFAs was then injected directly onto the gas chromatograph. In method 2, extracted fat was treated with tetramethylammonium hydroxide and the methyl ester derivatives were then formed in the injector. It was concluded that FFAs should be separated from the triacylglycerides before derivatization and chromatographic analysis, particularly for samples in which a minor fraction of the triacylglycerides has been hydrolysed to FFAs. Three gas chromatographic methods for the analysis of FFAs in cheese were compared by Ard/5 and Polychroniadou (1999). Method 1 included the preparation and methylation procedures described by Martinez-Castro et al. (1986) and Martin-Hern~indez etal. (1988). This involves diethyl ether extraction of fat followed by methylation with tetramethylammonium hydroxide, which results in an upper layer containing methyl esters from glycerides and a lower layer which holds the tetramethylammonium soaps of FFAs. The upper layer may be
Lipolysis and Catabolism of Fatty Acids in Cheese
used to analyse the fatty acid composition of glycerides. For FFA analysis, the bottom layer is separated, washed with ethyl ether and adjusted to pH 9. The methyl esters are then analysed by programmed GC, as described by Juarez et al. (1992). N2 or He was used as carrier gas; the column was a silica capillary column with silicone, containing 50% phenyl and 50% cyanopropyl groups as the stationary phase. Injecting samples at 300 ~ gave the best quantitative results. The derivitisation technique tested resulted in no loss of the volatile components, because the FFAs were injected in the form of soaps. Method 2 involved the extraction of fats with diethyl ether, fixation of FFAs onto an Amberlyst resin, methylation of FFAs followed by quantification of methyl esters of FFAs by GC using an FID detector. Method 3 involves extraction of fat using heptane with the addition of sodium sulphate and sulphuric acid, followed by overnight storage of samples. On the day of analysis, the sample is extracted using a mixture of diethyl ether and heptane, the mixture is then added to a NH2 Sep-Pak Vac cartridge. The cartridge is rinsed using a chloroform/isopropanol mixture, FFAs are eluted using a diethyl ether/formic acid mixture and subsequently analysed by GC. ArdO and Polychroniadou (1999) reported on the accuracy and recovery for method 1 only. The accuracy of method 1 was checked using two types of cheese; one was a fresh cheese with a low FFA content and the other a cheese that had undergone moderate lipolysis. Reproducibility values for the fresh cheese were comparable to those reported by Woo and Lindsay (1982) for Cheddar cheese; however, they were lower than those reported by McNeill and Connolly (1989) and Deeth et al. (1983) also for Cheddar cheese. Accuracy was substantially higher for cheese with a high FFA content and there was an improvement in the overall coefficient of variation. Recovery was examined by adding standard mixtures of FFAs to a sample from a cheese of known FFA content. Recoveries ranged from 91% for butanoic acid (C4:0) to 103% for octadecanoic acid (C18:0). It was concluded that this technique provides a fast and reliable method of FFA analysis in cheeses with a low FFA level and particularly for those with a high FFA level. High performance liquid chromatography (HPLC) may also be used as a method for FFA analysis (Marsili, 1985). High performance liquid chromatography can operate at ambient temperature, ensuring relatively little risk to sensitive functional groups (Christie, 1997). In the method of Kilcawley et al. (2001), C2:0, C3:0 and C4:0 were recovered by steam-distillation and quantified by ligand-exchange, ion-exclusion HPLC. C6:0 to C18:3 are derivatized to bromophenacyl esters follow-
385
ing solvent extraction and separation is achieved by reversed-phase HPLC. Bills and Day (1964) used a silicic acid column to separate FFAs, which were then quantified by titration with potassium hydroxide, using phenolphthalein as an indicator. Removal of CO2 from the air stream when titrating is vital to maintain the stability of the end point. In this method, the air stream was freed from CO2 by bubbling through 20% KOH, and IDF (1991) recommend titration under nitrogen. It was later shown that the resin used induced fat hydrolysis (McNeill and Connolly, 1989). When using a titration method to quantify FFAs, the eluate of each FFA must be titrated separately and the method is not as accurate as more sophisticated GC or HPLC methods. At present, the most suitable methods for the determination of lipolysis in cheese use GC and HPLC; levels of FFAs and FFA profiles may be determined. There is potential for increased use of established technologies, such as gas chromotographylmass spectroscopy (GC/MS) and emerging technologies, e.g., liquid chromotography/mass spectroscopy, for the measurement of FFAs. GC/MS provides very accurate qualitative and quantitative measurement of FFAs as well as the other volatile components of dairy products (see 'Instrumental Techniques', Volume 1).
References Anderson, D.E and Day, E.A. (1966). Quantitation, evaluation and effect of certain microorganisms on flavor compenents of Blue cheese..]. Agric. Food Chem. 14, 241-245. Ard0, Y. and Polychroniadou, A. (1999). Analysis of free fatty acids, in, Laboratory Manual for Chemical Analysis of Cheese, Publication Office of the European Communities, Luxembourg. Arnold, R.G., Shahani, K.M. and Dwivedi, B.K. (1975). Application of lipolytic enzymes to flavor development in dairy products.J. Dairy Sci. 58, 1127-1143. Arora, G., Cormier, E and Lee, B. (1995). Analysis of odoractive volatiles in Cheddar cheese headspace by multidimensional GC/MS/sniffing. J. Agric. Food Chem. 43, 748-752. Bills, D.D. and Day, E.A. (1964). Determination of the major free fatty acids of Cheddar cheese. J. Dairy Sci. 47, 733-738. Bosset, J.O. and Gauch, R. (1993). Comparison of the volatile flavour compounds of six European 'AOC' cheeses by using a new dynamic headspace GC-MS method. Int. Dairy J. 3,359-377. Castillo, I., Requena, T., Fernandez de Palencia, P., Fontecha, J. and Gobbetti, M. (1999). Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731. J. Appl. Microbiol. 86, 653-659.
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Lipolysis and Catabolism of Fatty Acids in Cheese
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Proteolysis in Cheese during Ripening V.K. Upadhyay, P.L.H. McSweeney, A.A.A. Magboul and P.F. Fox, Department of Food and Nutritional Sciences, University College, Cork, Ireland
Introduction As discussed in 'Biochemistry of Cheese Ripening: Introduction and Overview', Volume 1, proteolysis is the most complex and, in most varieties, the most important of the three primary biochemical events which occur in cheese during ripening. Because of its importance, the subject has been reviewed extensively (Grappin et al., 1985; Rank et al., 1985; Fox, 1989; Fox and Law, 1991; Fox etal., 1993, 1994, 1995b, 1996; Fox and McSweeney, 1996, 1997; Sousa et al., 2001). Proteolysis contributes to: 9 The development of cheese texture: - via hydrolysis of the protein matrix of cheese; - via a decrease in aw through changes to water binding by the new carboxylic acid and amino groups liberated on hydrolysis of peptide bonds. These groups are ionized at the pH of cheese and thus bind water; - indirectly via an increase in pH caused by the liberation of ammonia from amino acids produced by proteolysis. 9 Flavour and perhaps the off-flavour of cheese, - directly by the production of short peptides and amino acids, some of which have flavours; - indirectly by the liberation of amino acids which act as substrates for a range of catabolic reactions which generate important volatile flavour compounds (see 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1); - by facilitating the release of sapid compounds from the cheese matrix during mastication. Proteolysis in cheese during ripening is catalysed by proteinases and peptidases from six sources: 9 The coagulant: The enzymes involved depend on the type of coagulant used (chymosin, pepsin, fungal acid proteinases, plant acid proteinases). Residual coagulant activity retained in the curd is the major source of proteolytic activity in most cheeses except pasta-filata varieties and those with a high cook temperature in which enzymes from this source are denatured extensively.
9 The milk: A number of indigenous proteinases are present in milk, the most important of which is plasmin, which is produced from an inactive precursor, plasminogen. The action of plasmin is of particular importance in pasta-filata and high-cook cheeses (since it is a heat-stable enzyme) and in cheeses the pH of which increases during ripening (since the pH optimum of plasmin is c. 7.5). Somatic cells, recruited into milk to flight mastitic infection, contain lysosomes which contain a number of proteinases, including cathepsins D and B. 9 Starter lactic acid bacteria (LAB): The starter LAB contain a cell envelope-associated proteinase (CEE lactocepin, PrtP) which contributes to ripening principally by hydrolysing intermediate-sized and short peptides produced from the caseins by the action of chymosin or plasmin. The starter is the principal source of peptidases in cheese, which are responsible for the hydrolysis of short peptides and the liberation of amino acids. 9 Non-starter lactic acid bacteria (NSEAB): All ripened cheeses contain an adventitious secondary microflora which grows during ripening (see 'The Microbiology of Cheese Ripening', Volume 1). The proteinases and peptidases of NSLAB are generally similar to those of starter LAB and contribute to ripening in a similar fashion. 9 Secondary starter: Many cheese varieties are characterized by the development of a secondary microflora which is added to or deliberately encouraged to grow (e.g., Propionibacterium freudenreichii subsp, shermanii in Swiss-type cheese, Penicillium roqueforti in Blue cheese, P. camemberti in Camembert and Brie-type cheeses and a complex Grampositive bacterial microflora on the surface of smear cheeses). Most of these microorganisms possess potent enzyme activities, including proteinases and peptidases, which contribute greatly to proteolysis in these varieties. 9 Exogenous proteinases and peptidases: Exogenous proteolytic enzymes have been studied as a means of accelerating ripening, accentuating flavour
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-i
Copyright 9 2004 Elsevier Ltd All rights reserved
392 Proteolysisin Cheese during Ripening or debittering cheese. With the exception of enzymemodified cheese (see 'Cheese as an Ingredient', Volume 2), exogenous proteinases and peptidases are not used widely in cheese manufacture, although the action of these enzymes can dominate patterns of proteolysis in cheeses in which they are used. The use of exogenous proteinases to accelerate cheese ripening was discussed by Upadhyay and McSweeney (2003). The relative importance of enzymes from each of these sources depends on the cheese variety. Techniques used to assess the relative importance of proteolytic enzymes from different sources were discussed by Fox et al. (1993) and are outlined in 'Biochemistry of Cheese Ripening: Introduction and Overview', Volume 1. This chapter will focus on the principal proteolytic enzymes found in cheese and their role in cheese during ripening, with particular emphasis on their specificity on the caseins. Most attention will be paid to enzyme systems common to many cheese varieties (i.e., enzymes from the coagulant, milk and starter). Patterns of proteolysis in individual cheese varieties and the enzymes of specific secondary microorganisms are also discussed in some chapters of Volume 2.
Enzymes from the Coagulant As discussed in 'Rennets: General and Molecular Aspects', Volume 1, chymosin (EC. 3.4.23.4), the principal proteinase in traditional rennets used for cheesemaking, is an aspartyl proteinase of gastric origin, secreted by young mammals. The principal role of chymosin in cheesemaking is to hydrolyse the Phel05~Metl06 bond of the micelle-stabilizing protein, K-casein, as a result of which the colloidal stability of the micelles is destroyed, leading to gelation at temperatures >'--20 ~ (see 'Rennet-induced Coagulation of Milk', Volume 1). Most of the rennet added to cheesemilk is removed in the whey but some is retained in the curd and plays a major role in the initial proteolysis of caseins in many cheese varieties. From --0 to 30% of the coagulant added to the cheesemilk is retained in the curd, depending on enzyme type, cooking temperature, pH at draining and the moisture content of curd. In Cheddar cheese, - 6 % of the chymosin added to the milk is retained in the curd, but the amount increases with decreasing pH at whey draining (Holmes et al., 1977; Creamer etal., 1985). Pepsins, especially porcine pepsin, are more pH-sensitive than chymosin and hence the amount of these coagulants retained in cheese curd is very strongly dependent on the pH of the milk at setting and shortly thereafter; in fact, increasing the pH of the curds-whey mixture to - 7
after coagulation of milk by porcine pepsin is one of the methods used to produce rennet-free cheese curd (see 'Biochemistry of Cheese Ripening: Introduction and Overview', Volume 1). Only 2-3% of Rhizomucor rennets is retained in Cheddar cheese curd and appears to be independent of pH (Creamer et al., 1985). In high-cooked cheeses, e.g., Emmental, chymosin is extensively denatured (Matheson, 1981; Singh and Creamer, 1990; Boudjellab et al., 1994), and makes relatively little contribution to ripening. The action of chymosin on the B-chain of insulin indicates that it is specific for hydrophobic and aromatic amino acid residues (Fish, 1957). Relative to many other proteinases, chymosin is weakly proteolytic; indeed, limited proteolysis is one of the characteristics to be considered when selecting proteinases for use as rennet substitutes (Fox, 1989; Dalgleish, 1993). The primary chymosin cleavage site in the bovine milk protein system is the Phel05mMetl06 bond in K-casein which is many times more susceptible to chymosin than any other bond in milk proteins. Ors1-, CXs2- and [3-caseins are not hydrolysed during milk coagulation but may be hydrolysed in cheese during ripening. A number of authors (Pelissier et al., 1974; Creamer, 1976; Visser and Slangen, 1977; Carles and Ribadeau-Dumas, 1984) have investigated the hydrolysis of [3-casein by chymosin. In solution in 0.05 M Na acetate buffer, pH 5.4, chymosin cleaves [3-casein at seven sites: Leu192--Tyr193 > Ala189--Phe190 > Leu165~Ser106 = Gln107~Ser168 = Leu163--Ser164 > Leu139--Leu150 = Leu127~Thr128 (Visser and Slangen, 1977). The parameters, KM and kcat, for the action of chymosin on the bond Leut92~Tyr193 are 0.075 mM and 1.54 s -1, respectively, for micellar ]B-casein and 0.007 mM and 0.56 s -1 for the monomeric protein (Caries and Ribadeau-Dumas, 1984). NaC1 inhibits the hydrolysis of [3-casein by chymosin to an extent dependent on pH; hydrolysis is strongly inhibited by 5% and completely by 10% NaC1 (Mulvihill and Fox, 1978). The primary site of chymosin action on oLsl-casein is Phe23~Phe24 (Hill et al., 1974; Caries and RibadeauDumas, 1985; McSweeney et al., 1993b). Cleavage of this bond is believed to be responsible for the initial softening of cheese texture (de Jong, 1976; Creamer and Olson, 1982) and the small peptide (%1-CN f l - - 2 3 ) is hydrolysed rapidly by starter proteinases. The specificity of chymosin on Otsl-casein in solution was studied by Pelissier et al. (1974), Mulvihill and Fox (1979a), Pahkala et al. (1989) and McSweeney etal. (1993b). In 0.1 M phosphate buffer, pH 6.5, chymosin cleaves Otsl-casein at Phe23mPhe24, Phe28 Pro29, Leu40~Ser41, Leu149~Phe150, Phe153~ Tyr154,
Proteolysis in Cheese during Ripening 393 Leu156--Asp157, Tyr159--Pro160 and Trp164--Tyr165 (McSweeney et al., 1993b). These bonds are also hydrolysed at pH 5.2 in the presence of 5% NaC1 (i.e., similar to the conditions in many cheese varieties), and, in addition, LeUll--PrOl2, Phe32--Gly35, LeUl01-LySl02, Leu142--Ala144 and Phe179--Ser180. The rate at which many of these bonds are hydrolysed depends on the ionic strength and pH, particularly LeUl01--LySl02 which is cleaved far faster at the lower pH. The kcat and KM for the hydrolysis of Phe23--Phe24 bond of OLsl-casein by chymosin is 0.7 s -~ and 0.37 mM, respectively (Caries and Ribadeau-Dumas, 1985). The hydrolysis of OLsl-casein by chymosin is influenced by pH and ionic strength (Mulvihill and Fox, 1977, 1980). OLs2-Casein appears to be relatively resistant to proteolysis by chymosin; cleavage sites are restricted to the hydrophobic regions of the molecule (sequences 90-120 and 160-207), i.e., Phe88--Tyr89, Tyr95-Leu96, Gln97--Tyr98, Tyr98--Leu99, Phe163--Leu164, Phe174--Ala175 and Tyr179--Leu180 (McSweeney et al., 1994b). Although para-n-casein has several potential chymosin cleavage sites, it does not appear to be hydrolysed either in solution or in cheese (Green and Foster, 1974). Presumably, this reflects the relatively high level of secondary structure in K-casein compared to the other caseins (see Swaisgood, 1992); it would be interesting to investigate the hydrolysis of para-n-casein by chymosin or pepsin in the presence of a high concentration of urea (pepsin is active in 8 M urea). Good quality veal rennet contains about 10% bovine pepsin (EC 3.4.23.1; Rothe et al., 1977) but many 'calf rennets' contain up to 50% bovine pepsin. The proteolytic products produced from Na-caseinate by bovine pepsin are similar to those produced by chymosin (Fox, 1969), although, as far as we are aware, the specificity of bovine or porcine pepsins on bovine caseins has not been determined rigorously. However, Mulvihill and Fox (1979b) compared the hydrolysis of bovine, ovine, caprine and porcine [3-caseins by chymosins and pepsins from these species. The large peptides produced (detectable by urea-polyacrylamide gel electrophoresis) suggested generally similar specificities for chymosins and pepsins although differences were apparent in the short (pH 4.6-soluble) peptides. Pepsins were more proteolytic than the corresponding chymosins. For many years, the supply of calf rennet has been insufficient to meet demand, and much effort has been expended on searching for suitable rennet substitutes for cheesemaking (see Green, 1977; Phelan, 1985). Several proteinases have been assessed but only bovine pepsin and proteinases from Rhizomucor pusillus,
R. miehei and Cryphonectria parasitica have been used extensively in commercial practice (Phelan, 1985; van den Berg, 1992); blends of calf rennet and porcine pepsin (50:50) have been used in the past with generally satisfactory results. Of these rennets, that from R. miehei is the most widely used (under various trade names) and gives generally satisfactory results. The gene for R. miehei proteinase has been cloned in Aspergillus oryzae, resulting in a rennet containing less contaminating proteolytic enzymes. This coagulant was found to be acceptable for the manufacture of Cheddar cheese (Chen et al., 1994). C. parasitica proteinase is considerably more proteolytic than chymosin (Tam and Whitaker, 1972), especially on [3-casein, and is rarely used for cheesemaking except for high-cooked varieties (e.g., Swiss), in which the proteinase is extensively denatured by the high cook temperature. The specificity of many of these enzymes on the oxidized B-chain of insulin was summarized by Green (1977). Preliminary studies on the hydrolysis of sodium caseinate by fungal rennets were reported by Tam and Whitaker (1972) and later by Phelan (1985). Rea (1997) compared hydrolysates of sodium caseinate by chymosin, R. miehei proteinase and C. parasitica proteinase; the specificities of the three enzymes on the caseins were clearly very different (C. parasitica was particularly active on [3-casein). However, the bonds cleaved by the fungal proteinases have not been reported (except that the primary cleavage of K-casein by C. parasitica proteinase is at Serl04--Phel05, rather than Phel05--Metl06 which is cleaved by chymosin and R. miehei proteinase (Drohse and Foltmann (1989)).
Indigenous Proteinases in Milk Plasmin Milk contains a number of indigenous proteinases, of which plasmin (EC 3.4.21.7) is the most important with respect to cheese ripening. Plasmin is a trypsinlike serine proteinase with pH and temperature optima of "-7.5 and 37 ~ respectively. Plasmin is secreted into blood as its inactive zymogen, plasminogen, which is then activated to plasmin, the principal function of which in blood is to degrade fibrin clots. A complex plasminogen/plasmin system exists in blood, comprised of plasmin, plasminogen, plasminogen activators (PA), plasmin inhibitors, and inhibitors of PA, all of which are also present in milk. A diagrammatic representation of the plasmin/plasminogen system in milk is shown in Fig. 1. In milk, plasminogen, plasmin and PA are associated with the casein micelles while plasmin inhibitors and inhibitors of PA are in
394
Proteolysis in Cheese during Ripening
P l a s m i n o g e n activator inhibitors
P l a s m i n o g e n activators 4- . . . . . . . . .
Plasminogen
v
-- Plasmin*- . . . . . . . . . .
Casein
"
- Plasmin inhibitors
,, Polypeptides
Figure 1 Plasmin/plasminogen system in milk (adapted from Bastian and Brown, 1996).
the serum phase. Plasmin has trypsin-like specificity, showing preference for bonds of the type Lys--X and, to a lesser extent Arg~X, and acts on caseins in the order [3 ~ OLs2>> e~sl, while K-casein appears to be resistant to hydrolysis by plasmin (Bastian and Brown, 1996). [3Casein has 15-17 (depending on the genetic variant) potential plasmin-susceptible bonds, but only three are hydrolysed at significant rates in milk, Lys28mLys29, LySl05--Hisl06 and LySl07---Glul08, hydrolysis of which results in the release of yl-CN (~-CN f29-209), y2-CN ([3-CN f106---209), y3-CN (~-CN f108-209), proteose peptone (PP)8 fast ([3-CN fl-28), PP8 slow [3-CN (f29-105) and (f29-107) and PP5 (~-CN fl-105 and 1-107) (Fig. 2). In solution, plasmin hydrolyses [3-casein at LysgF----Va198,LySll3mTyrll4, LySlogmVal170, Lys176~Ala177, Arg183mAsp184, Arg202~Gly203 as well as at Lys28~Lys20, Lyslos~HiSlo6 and LySlO7---Glul08 (Fox et al., 1994). O~s2-Casein is also a good substrate for plasmin. In buffered systems, plasmin hydrolyses Ots2-casein at Lys21~Gln22, Lys24~Asn25, Lys149~Lys150, LySlso~Thrls1, Lys181mThr182, Lys188~Ala189,
Lys197mThr198 and Argll4mASnll5 (Le Bars and Gripon, 1989; Visser et al., 1989), but it has not yet been determined whether peptides resulting from cleavage at these sites are produced in milk or dairy products, although this is likely since the concentration of e~s2-casein, which is a poor substrate for chymosin, decreases in cheese during ripening (Fox and McSweeney, 1997). Otsl-Casein is less susceptible to hydrolysis by plasmin than [3-casein (Andrews and Alichanidis, 1983). However, Le Bars and Gripon (1993) identified seven Lys~X and four Arg~X plasminsusceptible bonds in CZsl-casein while McSweeney et al. (1993c) identified 12 Lys~X and 5 Arg~X plasminsusceptible bonds in e~sl-casein (Lys3mHis4, Lys7--Hiss, Arg22~Phe23, Lys34--Glu35, Lys36--Lys37, Lys58~ Gln59, Lys79mHisso, Arg90mTyrgl, Arg]oo~LeUl01, LySlo2--LySl03, LySlo3~Tyrl04, Lyslos~Vall06, arg119~ LeUl20, Lys124~Glu125, Gln131mLys132, ArglslmGln152, Lys193~Thr194), including all those reported by Le Bars and Gripon (1993). It is likely that ~,-caseins originate as a result of hydrolysis of Ots]-casein by plasmin (Aimutis
L,s,o,TiSlLs,1,TTr,,,
1 Lys2o-~--Lys
Arg183--~-AsP184 209
YSl07~GUlo8 Products Proteose peptones Known
PP8f PP8s PP8s PP-T PP5 PP5
"l-Caseins
Probable
13-CNfl-28 13-CNf29-105 13-CNf29-107 13-CNf29-113 13-CN f1-105 13-CNf1-107
13-CN f106-113 13-CN f108-113 13-CN f114-183 13-CN f106-183 13-CN f108-183 13-CN f1-113 (unlikely)
Figure 2 Hydrolysis of 18-casein by plasmin (adapted from Fox et
aL,
1994).
~,I-CN (13-CNf29-209) ~,2-CN (13-CNf106-209) -/3-CN (13-CNf108-209) ~,-CN (13-CNf114-209) ~,-CN (13-CNf184-209)
Proteolysis in Cheese during Ripening and Eigel, 1982; O'Flaherty, 1997). The resistance of K-casein to hydrolysis by plasmin is probably due to the carbohydrate moieties attached to its C-terminal region (Doi et al., 1979). Since they are associated with the casein micelles, plasmin, plasminogen and PA are incorporated into the cheese curd but plasmin inhibitors and inhibitors of PA are present in the cheese whey (Bastian and Brown, 1996). The contribution of plasmin to proteolysis varies depending on cheese type. Farkye and Fox (1990) found that plasmin activity in cheese was in the order Emmental>Blamey= Romano-type> Gouda> Cheddar> Cheshire cheeses. Many factors (e.g., cooking temperature, pH during ripening) contribute to this difference in plasmin activity in cheese. Plasmin is the primary proteolytic agent in Swiss-type cheese due to the fact that these cheeses are cooked at a high temperature (-55 ~ at which most of the coagulant, chymosin, is inactivated, while plasmin, which is a heat-stable enzyme, survives cooking. At high cooking temperatures, activation of plasminogen occurs, probably due to heat-induced inactivation of inhibitors of PA and of plasmin (Farkye and Fox, 1990). Hence, differences in plasmin activity between Swiss and Cheddar cheeses are due to differences in the cooking temperature used in the manufacturing protocols. Plasmin also plays a major role in ripening of mould-ripened (e.g., Camembert) and smear-ripened (e.g., Tilsit) cheese varieties. During ripening of Camembert-type cheese, catabolism of lactic acid and deamination of amino acids with the production of NH3 by the mould on the surface of the cheese result in an increase in the pH of the surface layer to --~7.0. Gradually, the pH of the interior of the cheese also increases due to the outward migration of lactic acid and the inward migration of NH3 (see 'Metabolism of Residual Lactose and of Lactate and Citrate', Volume 1). The increased pH facilitates the action of plasmin, which contributes significantly to proteolysis in these cheeses (Gripon, 1993). The pH of the surface layer of smearripened cheeses also increases during ripening, which facilitates plasmin action on the caseins and influences the quality of these cheese varieties (O'Farrell et al., 2002). Due to the significance of plasmin to proteolysis during the ripening of many cheese varieties, a number of attempts have been made to increase plasmin activity in cheese using different approaches. Farkye and Fox (1992) added exogenous plasmin to cheesemilk for Cheddar cheese. The level of water-soluble N in cheese enriched with plasmin was - 2 0 % higher than in the control cheeses. As expected, increased plasmin activity did not increase the level of phosphotungstic acid-soluble N, which is mainly due to free amino acids. The authors claimed superior organolep-
395
tic quality for the plasmin-enriched cheese. Somers et al. (2002) enriched milk for the manufacture of Mozzarella-type cheese with plasmin; greater hydrolysis of [3-casein by plasmin was observed in the experimental cheeses compared to the control, but enzyme treatment did not affect the composition or functionality of the cheese. Primary proteolysis (as measured by levels of pH 4.6-soluble N and urea-polyacrylamide gel electrophoresis, PAGE), was accelerated in smearripened cheese made from plasmin-enriched milk (O'Farrell et al., 2002). Farkye and Fox (1991), who added 6-aminohexanoic acid, a plasmin inhibitor, to stirred-curd Cheddar cheese, found slower degradation of [3-casein compared to control cheese. Successful attempts have been made to accelerate proteolysis in cheese by increasing plasmin activity by the use of exogenous PA (e.g., urokinase) in the manufacture of Cheddar (Barrett et al., 1999), Swiss (Bastian et al., 1997), ultrafiltered Havarti and Saint Paulin (Bastian et al., 1991) cheeses. Bastian et al. (1991) manufactured Havarti and Saint-Paulin cheese by traditional or ultrafiltration (UF) technology and added urokinase and KIO3, individually and in combination, to UF retentate before cheesemaking. Addition of urokinase increased plasmin activity in UF Havarti and Saint-Paulin cheese and increased proteolysis. Swiss cheese manufactured from milk containing 0.05 UL-1 or 0.5 UL -1 urokinase showed increased plasmin activity and faster degradation of [3-casein than control cheese (Bastian et al., 1997). The use of exogenous urokinase in the manufacture of Cheddar cheese resulted in activation of plasminogen and acceleration of proteolysis compared to control cheese (Barrett et al., 1999). Streptokinase, an extracellular protein secreted by Streptococcus uberis, forms a 1:1 complex with plasminogen, inducing a conformational change in plasminogen, which renders the proteinase active without prior proteolytic cleavage. The active streptokinase-plasminogen complex is then able to convert other plasminogen molecules to plasmin by proteolysis (Johnsen et al., 2000). Streptokinase activated most of the plasminogen in milk and increased plasmin activity in experimental Cheddar cheese compared to controls (V.K. Upadhyay, unpublished). Proteolysis in the cheese was accelerated as indicated by an increased level of pH 4.6-soluble nitrogen, breakdown of [3-casein and the production of hydrophobic peptides. In an alternative approach, V.K. Upadhyay (unpublished) used a starter strain, which had been genetically modified to produce streptokinase, for the manufacture of Cheddar cheese; plasminogen was activated to plasmin, and proteolysis in the cheese during ripening was accelerated.
396
Proteolysis in Cheese during Ripening
All the approaches for increasing plasmin activity in different cheese varieties have accelerated proteolysis. However, plasmin activity is not rate-limiting for flavour development in cheese during ripening as plasmin is responsible for the production of large to intermediate-sized peptides (Farkye and Fox, 1992), which do not contribute directly to flavour. However, plasmin may have an indirect role in cheese flavour by producing intermediate-sized peptides which are hydrolysed further by lactocepin and starter peptidases, ultimately to free amino acids, which are important precursors of volatile flavour compounds (see 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1). Other indigenous proteinases
During mastitis, there is an increase in the somatic cell count of milk as a result of increased transfer of leukocytes from blood to milk. Somatic cells contain many active proteinases, including cathepsins B, D, G, H, L and elastase (Kelly and McSweeney, 2003). The presence of cathepsins D and B in milk is confirmed. However, it is likely that other lysosomal proteinases are also present in milk. Cathepsin D is an aspartic proteinase located in the lysosomes of mammalian cells and has pH and temperature optima of 4.0 and 37 ~ respectively. Cathepsin D is synthesized on the rough endoplasmic reticulum as its inactive form, procathepsin D. Procathepsin D is converted autocatalytically to pseudocathepsin D which is then converted to mature active cathepsin D by further proteolysis, mediated by cysteine proteinases. Larsen and Petersen (1995) purified five molecular forms of cathepsin D, having apparent molecular masses of 46, 45, 43, 39 and 31 kDa, from bovine milk; the 46 and 45 kDa forms corresponded to procathepsin D (the reason for this variability in molecular mass is unknown; Hurley et al., 2000a), 43 kDa to pseudocathepsin D and 39 and 31 kDa to mature single-chain and heavychain cathepsin D, respectively (Fig. 3). However, Larsen and Petersen (1995) did not detect a band that corresponding to light chain cathepsin D (14 kDa), probably due to lack of sensitivity of their experimental methods. The concentration of cathepsin D in skimmed milk and whey is estimated to be 0.4 and 0.3 p~g m1-1, respectively, indicating that it is a serum protein (Larsen et al., 1996). Therefore, little cathepsin D would be expected in cheese. Furthermore, cathepsin D only partially survives heat treatment at 55 ~ for 30 min (45% survival) or HTST pasteurization (72 ~ • 15 s) (8% survival) (Larsen et al., 2000" Hayes et al., 2001). Hydrolysis of the caseins by cathepsin D has been studied in model systems. Kaminogawa et al. (1980) reported that cathepsin D partially purified from milk
ProcathepsinD
l
1q
1 I
Pseudocathepsin D
46 kDa
I
I
346 I
I
P27~
43kDa
3461
1
39kDa
346
31 kDa
346
I
i
I
Cathepsin D (Single chain) 102 Cathepsin D (Heavy chain)
1 14kDa 99
Cathepsin D (Light chain)
Figure 3 Molecular forms of cathepsin D present in bovine milk (adapted from Larsen and Petersen, 1995).
hydrolyses Otsl-casein with the production of a peptide with a molecular mass similar to Otsl-CN (f24-199), a peptide that is produced from %l-casein by chymosin (Fig. 4a) (McSweeney et al., 1993b). Bonds in %lcasein susceptible to the action of cathepsin D include Phex3~Phe24, Phe24~Va125, Leu98~Leu99 and Leu149~ Phe150 (Larsen et al., 1996). Cathepsin D hydrolyses [~-casein to peptides similar to those produced by chymosin (Fig. 4b); susceptible bonds include Phe52~ Alas3, Leu58~Va159, Pro81~Va182, Ser96~Lys97, Leu125~Thr]26, Leu]27~Thr128, Trp]43~Met144, Phe157~Pro158, Ser161~Val162, Leu165~Ser166, LeUlg]~Leu192, Leu192~Tyr]93 and Phe205~Pro206. However, hydrolysates of %2-casein produced by cathepsin D differ markedly from those produced by chymosin and have very few peptides in common when analysed by urea-PAGE and reversed phase-high performance liquid chromatography (RPHPLC) (McSweeney et al., 1995). Cathepsin D-sensitive bonds in Ots2-casein include Leu99~Tyrloo, Leu123~ Asn124, Leu180~Lys181 and Thr182~Val]83 (Larsen etal., 1996). Cathepsin D hydrolyses K-casein at Leu32~Ser33, Leu79~Serso and Phelos~Metl06. Two cleavage sites for cathepsin D on ot-lactalbumin have been identified (Leu52~Phe53 and Trplo4~LeUl05), while [3-1actoglobulin appears to be resistant to this enzyme (Larsen et al., 1996). Although the specificity of cathepsin D is similar to that of chymosin, it coagulates milk poorly (McSweeney et al., 1995). It is very difficult to assess the contribution of cathepsin D to proteolysis in cheese made with rennet due to the low level of cathepsin D and the masking effect by the much larger level of chymosin in cheese. Wium et al. (1998) reported that Otsl-CN (f24-199) was produced in a Feta-type cheese made without the addition of rennet from pasteurized, homogenized, ultrafiltered milk, acidified using gluconic acid-g-lactone (GDL),
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Proteolysis in Cheese during Ripening 399 which was attributed to the action of cathepsin D. Hurley et al. (2000b) made Quarg, from raw skim milk, pasteurized skim milk or raw skim milk with added pepstatin (a potent inhibitor of aspartyl proteinases) by acidification using GDL. Reversed phase-high performance liquid chromatography of the water-soluble fraction of the cheeses made from raw or pasteurized skim milk showed peptides eluting at 46-48 min, but these peptides were absent from the profiles of pepstatin-treated cheese, suggesting indigenous aspartyl proteinase activity in Quarg, presumably cathepsin D. Cathepsin D activity may contribute to a low extent to the degradation of Otsl-casein in high-cooked cheese varieties (e.g., Swiss-type, Parmigiano-Reggiano) in which most of the chymosin is inactivated. The presence of cysteine proteinase activity in bovine milk has been reported (Suzuki and Katoh, 1990; O'Driscoll etal., 1999). Mammalian cysteine proteinases (cathepsins B, H, L and I) are lysosomal enzymes that are synthesized as proenzymes (MW 37-55 kDa), which are later converted to catalytically active forms (MW 23-30 kDa) (Barrett and Kirschke, 1981; Zeece et al., 1992; Kirschke etal., 1998). Magboul et al. (2001) resolved five fractions (fI-fV) with cysteine proteinase activity by ion-exchange chromatography of acid whey on Q-Sepharose. Fractions fiII and fV were the most active and were capable of hydrolysing Otsz- and [3-caseins. Immunoblotting of fllI with antibodies to the bovine lysosomal cysteine proteinase, cathepsin B, indicated the presence of cathepsin B in fill. Partially purified fill retained 20% of its original activity when heated at 72 ~ • 30 s. It is possible that cathepsin B may contribute to cheese ripening but further research is required to determine its role, if any, and the distribution of this enzyme in milk. Cathepsin G is a serine proteinase with a molecular mass of 24-26 kDa. In solution, cathepsin G readily hydrolyses o%1-and [3-casein extensively, including some sites that are very close, or identical, to chymosin cleavage sites; it produces Otsl-CN (fl-23) by cleavage of the Phe23mPhe24 (Fig. 4a) (Considine et al., 2002). Many cathepsin G cleavage sites in Otsl- and [3-caseins are also close to those of plasmin. Cathepsin G has broad specificity on o%1-and ]3-caseins and hence it is possible that it may contribute to proteolysis in cheese made from high SCC milk; however the presence of this enzyme in milk has not been demonstrated. Elastase, a serine proteinase with a molecular weight of 29.5 kDa, is an important lysosomal enzyme in somatic cells, although its presence in milk has not been demonstrated. Elastase can hydrolyse a wide variety of proteins, including Otsl- and [3-caseins and has many cleavage sites in common with chymosin,
plasmin and the lactocepin of Lactococcus (Considine et al., 1999, 2000). Hence, if elastase is present in milk, it may contribute to proteolysis in cheese during ripening. Of all the indigenous enzymes discussed above, only plasmin and, to a lesser extent, cathepsin D, have been studied in detail. Research to establish the presence of the other enzymes in milk, and to elucidate their role in proteolysis in cheese during ripening is needed.
Proteolytic Enzymes of LAB Lactic acid bacteria are fastidious organisms that have complex amino acid requirements (Law et al., 1976; Morishita et al., 1981; Chopin, 1993). The concentrations of amino acids in milk are below the nutritional requirements for the growth of the auxotrophic LAB to high cell populations. When growing in milk, their complex proteolytic system degrades mainly caseins into small peptides and amino acids which fulfil their nutritional requirements and inadvertently contribute to the flavour of fermented dairy products (Law and Mulholland, 1995; Steele, 1995). Many components of the proteolytic system of LAB have been purified and characterized and most of the corresponding genes have been cloned and sequenced (for reviews of the extensive literature on this subject, see Pritchard and Coolbear, 1993; Bockelmann, 1995; Exterkate, 1995; Poolman et al., 1995; ]uillard et al., 1996; Kunji et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999). The best-studied proteolytic system among the LAB is that of Lactococcus followed by themophilic Lactobacillus spp. because of their economic importance as starter cultures in dairy fermentations. The proteolytic systems of mesophilic lactobacilli, which dominate the non-starter microflora of Cheddar, Dutch-type and probably most cheeses during ripening 0ordan and Cogan, 1993; Williams and Banks, 1997; 'The Microbiology of Cheese Ripening', Volume 1), have received less attention. The main components of the proteolytic system of LAB are proteinases (mainly the cell envelope-associated proteinase, CEP, or lactocepin; EC 3.4.21.96), although intracellular proteinases have been reported; Muset et al., 1989; Akuzawa et al., 1990), amino acid and peptide transport systems, and a range of intracellular peptidases. During the growth of LAB in milk, the initial step in casein degradation is performed by lactocepin and the short peptides produced are taken up by the cell via peptide transport systems (Juillard et al., 1995). Further degradation to amino acids is catalysed by a number of intracellular peptidases (Kunji etal., 1996; Law and Haandrikman, 1997; Christensen etal., 1999). The starter stops growing in cheese curd soon after the end of manufacture due to the low pH, increasing NaC1
400
Proteolysis in Cheese during Ripening
concentration, low temperature and lack of a fermentable carbohydrate substrate (see 'The Microbiology of Cheese Ripening', Volume 1). However, its enzymes play a very important role in proteolysis during ripening, particularly when intracellular enzymes are released from the cell following lysis. The rate of secondary proteolysis is higher in cheese made with fast-lysing than that with slow-lysing starter strains (Wilkinson et al., 1994; O'Donovan etal., 1996; Morgan etal., 1997; Martfnez-Cuesta et al., 2001; Hannon et al., 2003). Proteinases from LAB
Immunogold-labelling and genetic studies have shown that lactocepin is located outside the lactococcal cell (Hugenholtz et al., 1987; de Vos and Siezen, 1994). Calcium is necessary for stable attachment of lactocepin to the cell envelope; the proteinase is released by incubation of the cells in a calcium-free buffer, a characteristic which is usually exploited as the first step in isolation procedures (Mills and Thomas, 1978; McSweeney et al., 1993a). Lactocepins have a molecular mass of c. 140 kDa and a pH optimum of 5.5-6.5 (Law and Haandrikman, 1997). The lactocepins from a number of Lactococcus strains have been characterized biochemically and genetically; they are homologous with the subtilisin family of serine proteinases, with similar catalytic domains (Pritchard and Coolbear, 1993; Kok and de Vos, 1994; Kunji et al., 1996; Law and Haandrikman, 1997; Siezen, 1999; 'Starter Cultures: General Aspects', Volume 1). The lactocepins and subtilisins have a conserved active site triad, consisting of aspartic acid, histidine and serine. A region of 107 residues which includes the active site and the substrate-binding region is highly conserved (Law and Haandrikman, 1997). Lactocepins of Lactococcus were initially classified into two broad groups, Pl- and Piii-type proteinases (Tan etal., 1993). Pi-Type enzymes (e.g., produced by Lc. lactis subsp, cremoris HP and Wg2) degrade [3-casein rapidly but act only slowly on Otsl-casein whereas Pill-type proteinases (e.g., AM1 and SK11) hydrolyse [3-casein differently to Pi-type strains and rapidly hydrolyse C~sl- and K-caseins (Visser et al., 1986; Fox and McSweeney, 1996; Law and Haandrikman, 1997). Although this broad classification scheme remains useful, it soon became apparent that the lactocepin of certain strains of Lactococcus had a specificity intermediate between PI- and Phi-type enzymes. The nucleotide sequence of the gene encoding all lactocepins is very similar (>98% homology; Kok et al., 1988; Vos etal., 1989a,b) and alteration of a few amino acid residues in the enzyme can alter its specificity. Exterkate et al. (1993) proposed a classification
scheme for lactocepins based on their specificity on the peptide, Otsl-CN (fl-23), which is released early in cheese ripening by the action of chymosin (Fig. 5). The specificity of the lactocepins from a number of strains of Lactococcus on Ors1-, OLs2-, 13- and K-caseins is summarized in Figs 6-9. The primary role of lactocepin is to degrade the caseins to provide short peptides to support the growth of the lactococcal cells in milk. However, its role in cheese ripening is different. Peptides isolated from Cheddar cheese, the N- or C-terminus of which corresponds to the specificity of lactocepin, do not contain a major chymosin or plasmin cleavage site (Fox and McSweeney, 1996), suggesting that chymosin or plasmin act first and that lactocepin then hydrolyses the resulting intermediate-sized peptides. A number of authors have investigated the action of lactocepins on peptides produced from the caseins by the action of chymosin (Fig. 10). Cell envelope-associated proteinases with properties similar to the lactococcal lactocepins have also been isolated from a number of strains of Lactobacillus (see Kunji et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999). Although much less well studied than the lactocepins, Lactococcus spp. also possess intracellular proteinases. Muset et al. (1989) isolated an intracellular metalloproteinase from Lc. lactis subsp, lactis NCDO763 which was optimally active at pH 7.5 and 45~ and exhibited thermolysin-like specificity. Akuzawa et al. (1990) identified four intracellular proteinases with caseinolytic activity. The enzymes with activity on casein ranged in molecular mass from 12 to 160 kDa and were optimally active at pH 5.5-7.0. Two enzymes were metalloproteinases, one had a serine catalytic mechanism and one was a thiol proteinase. The authors also obtained eight fractions with activity on benzgloxycarbonyl-L-Phe-L-Arg-7(4-methyl) coumarylamide but none of the eight fractions was able to hydrolyse casein. Three intracellular proteinases (P1, dimeric, M r = 1 2 4 k D a ; P2, monomeric, Mr = 64 kDa and P3, monomeric, Mr = 47 kDa) were demonstrated in the cytoplasmic fraction of the lactocepin-negative strain, Lc. lactis subsp, cremoris MG1363, by Stepaniak et al. (1996). P1 was a metalloproteinase while P2 and P3 were serine proteinases. The enzymes were optimally active at pH 7.0 and 35 ~ (P1) or pH 7.5 and 45 ~ (P2, P3). Peptidases
While the role of lactocepin when the cell is growing in milk is the degradation of caseins to oligopeptides,
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the hydrolysis of these peptides (after internalization into the cell) to amino acids is catalysed by peptidases. Many different peptidases from LAB have been characterized biochemically and genetically (see Kun]i et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999; Siezen et al., 2002). The biochemical properties of the peptidases from cheese-related bacteria characterized to date are shown in Table 1. While the role of some of these peptidases (e.g., endopeptidases) is the degradation of oligopeptides to shorter peptides, exopeptidases function to release one or two amino acids at a time from short peptides. Based on their substrate specificity, peptidases are classified into different groups, as shown in Fig. 11.
Endopeptidases Several endopeptidases have been reported in lactococci and lactobacilli (Table 1), most of which are monomeric metallopeptidases. On the basis of substrate specificity, LAB appear to possess three types of endopeptidases (Monnet etal., 1994). PepO is a monomeric metallopeptidase with a molecular mass of ---70 kDa. It is capable of efficiently hydrolysing Metenkephalin, bradykinin, substance P, glucagon, oxidized B-chain of insulin and several casein fragments but not di-, tri- or tetra-peptides. PepO was the first endopeptidase for which the gene was sequenced (Mierau et al., 1993). The pepO gene is located immediately downstream of the genes for the oligopeptide transport system, indicating that the two systems are physiologically linked (Tynkkynen et al., 1993). Another oligopeptidase, designated PepE specifically cleaves the Phe--Ser bond in bradykinin and was purified from Lc. lactis subsp, lactis NDCO 763; its gene (pepF) was cloned and sequenced (Monnet et al., 1994). This enzyme is a monomeric metallopeptidase of "~70 kDa and is capable of hydrolysing peptides containing 7-17 amino acids with broad specificity but not smaller or larger peptides. PepF is unable to hydrolyse Metenkephalin, which is a good substrate for PepO.
A gene (pepE) encoding a thiol-dependent endopeptidase has been isolated from Lb. helveticus CNRZ32 (Fenster etal., 1997). The deduced amino acid sequence of PepE showed high homology with PepC from Lb. delbrueckii subsp, lactis DSM7290 (Klein et al., 1994a), Lb. helveticus CNRZ32 (Fernandez et al., 1994; Vesanto et al., 1994), Sc. thermophilus CNRZ302 (Chapot-Chartier et al., 1994) and Lc. lactis subsp, cremoris AM2 (Chapot-Chartier et al., 1993). Fenster et al. (1997) isolated and characterized recombinant PepE; the general properties of this enzyme indicated that it was different from the other metallo-endopeptidases characterized from LAB.
Di- and tripeptidases Tripeptidases (PepT) purified from LAB are generally di- or tri-meric metallopeptidases (Table 1) with broad specificity, capable of hydrolysing tripeptides with acidic, basic or neutral N-terminal amino acid residues. A broad-specificity general dipeptidase, PepV, which hydrolyses only dipeptides, is found in LAB (Kunji et al., 1996; Law and Haandrikman, 1997). A number of dipeptidases with similar properties have been purified and characterized from strains of Lactococcus and Lactobacillus (see Table 1). Most of the dipeptidases isolated from LAB are monomers with a molecular mass in the range 40-55 kDa (Table 1). With the exception of a dipeptidase from Lb. helveticus 53/7, which was reported to have a thiol catalytic mechanism (Vesanto et al., 1996), all the dipeptidases characterized to date are metallopeptidases (Table 1). All dipeptidases of LAB show broad specificity and are capable of hydrolysing all dipeptides except those containing a proline residue. Carboxypeptidases Carboxypeptidases are exopeptidases which catalyse the hydrolysis of peptides from the C-terminal. No carboxypeptidase activity has been detected in lactococci but some activity towards N-terminal-blocked
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Proteolysis in Cheese during Ripening
411
Endopeptidases (PepO, PepF)
Exopeptidases Aminopeptidases (PepN, PepA, PepC, PepL)
Iminopeptidase (Pepl)
1.0,..0..Q_ Pyrolidonyl carboxylylpeptidase (PCP)
X-Prolyldipeptidylaminopeptidase(PepX)
Carboxypeptidase
-
Dipeptidases (PepV, P e p D )
Prolidase(PepQ)
Prolinase(PepR)
|
Tripeptidase (PepT)
Figure 11 Schematic representation of the action of peptidases found in lactic acid bacteria.
peptides has been reported in strains of lactobacilli (Abo-Elnaga and Plapp, 1987; E1 Soda et al., 1987a,b). There are no reports on the purification and characterization of a carboxypeptidase from Lactobacillus or other LAB.
Aminopeptidases The most thoroughly studied exopeptidase from LAB is the general aminopetidase, PepN. In most strains studied, this enzyme is a monomeric metallopeptidase of 85-98 kDa. PepN is a broad specificity aminopeptidase; in addition to p-nitroanilide (pNA) derivatives of amino acids, the enzyme is capable of hydrolysing a wide range of peptides differing in both size and
amino acid composition (Arora and Lee, 1992; Miyakawa et al., 1992; Tan et al., 1992a,b; Niven et al., 1995; Sasaki et al., 1996). Substrates with a hydrophobic or basic amino acid residue at the N-terminal are hydrolysed preferentially. The ability to hydrolyse peptides containing hydrophobic amino acids suggests its potential as a debittering enzyme. The addition of PepN from Lc. lactis subsp, cremoris Wg2 was found to be effective in reducing the bitterness of tryptic digests of [3-casein (Tan et al., 1993). The manufacture of cheese using PepN-negative mutants resulted in increased bitterness (Baankreis, 1992). Generally, PepN does not hydrolyse substrates with Glu, Asp or Pro at the N-terminal or dipeptides containing Pro
412
Proteolysis in Cheese during Ripening
(Tan etal., 1991; Arora and Lee, 1992; Miayakawa et al., 1992; Tan et al., 1993). However, PepNs from Lb. delbrueckii subsp, bulgaricus B14 (Wohlrab and Bockelmann, 1993) and Lb. helveticus SBT 2171 (Sasaki et al., 1996) hydrolysed Pro-containing substrates. Specificity studies indicated that the PepN from Lc. lactis subsp, cremoris Wg2 was active on oligopeptides with a preference for peptides with six amino acid residues (Niven et al., 1995). PepC in LAB is a metal-independent general aminopeptidase (Kunji et al., 1996; Table 1). PepCs from Lactococcus and Lactobacillus strains characterized so far are muhimeric thiol aminopeptidases which are inhibited by p-chloromercuribenzoate and iodoacetamide (Neviani et al., 1989; Wohlrab and Bockelmann, 1993; Fernandez de Palencia et al., 1997). In both cases, the subunit molecular mass of the enzyme is ---40-50 kDa. PepC shows broad specificity, with particularly high activity on synthetic substrates containing a hydrophobic amino acid but exhibits little activity on peptides with positively charged amino acid residues (Neviani et al., 1989; Wohlrab and Bockelmann, 1993; Fernandez de Palencia et al., 1997; Mistou and Gripon, 1998). A gene (pepG) encoding a novel cysteine aminopeptidase and with a high degree of similarity to PepC has been identified in Lb. delbrueckii subsp, lactis DSM7290 by Klein et al. (1997). These authors over-expressed the pepG gene in E. coli and compared the enzyme to PepC; although both enzymes were structurally related, they had different substrate specificities. Lactococcal glutamyl/aspartyl aminopeptidase (PepA) is a muhimeric metallopeptidase with a subunit molecular mass of 38-43 kDa (Table 1). PepA is a narrow-specificity peptidase which releases only an Nterminal Glu or Asp from di-, tri- and oligo-peptides with up to ten amino acid residues (Exterkate and de Veer, 1987; Niven, 1991; Bacon et al., 1994). Glutamate is a well-recognized flavour enhancer and therefore the role of PepA in the development of flavour in cheese may be of great importance. Studies on mature Cheddar cheese have shown that glutamate is important for Cheddar cheese flavour (McGugan et al., 1979; Aston and Creamer, 1986; Engels and Visser, 1994; Fox et al., 1994). However, the precise role of PepA in the development of cheese flavour is unclear. Under certain conditions, the N-terminal glutamyl residue of a peptide can undergo spontaneous intramolecular cyclization, forming an N-terminal 2-pyrrolidone5-carboxylic acid (PCA; pyroglutamate residue) (Law and Haandrikman, 1997). An N-terminal PCA residue has been found in bitter peptides produced from [3casein by the lactocepin of Lc. lactis subsp, cremoris HP (Visser etal., 1983). Pyrrolidone carboxylyl peptidase
(PCP) is an aminopeptidase capable of releasing a PCA residue from peptides and proteins (Kunji et al., 1996). This enzyme is present in lactococcal strains and has been partially characterized from Lc. lactis subsp, cremoris HP (Baankreis, 1992). Two serine peptidases with a molecular mass of 25 and 80kDa and PCAp-nitroanilide hydrolase activity were identified in Lc. lactis subsp, cremoris HP using non-denaturing gel electrophoresis (Baankreis, 1992). The presence of more than one leucyl aminopeptidase in LAB has been reported (Atlan et al., 1989; Blanc et al., 1993; Banks et al., 1998). A gene encoding a specific leucyl aminopeptidase (pepL) in Lb. delbrueckii subsp, lactis DSM 7290 has been cloned and sequenced (Klein et al., 1995). PepL has a molecular mass of 35 kDa (Table 1) and it preferentially hydrolyses dipeptides (and some tripeptides) with an N-terminal leucyl residue. Sequence alignments of PepL with prolinases from Lb. helveticus and B. coagulans and an iminopeptidase from Lb. delbrueckii subsp, lactis and Lb. delbrueckii subsp, bulgaricus showed 46, 21.5, 25.5 and 25.5% homology, respectively. Two aminopeptidases, with characteristics similar to PepL, were purified from Lb. sake IATA115 and Lb. curvatus DPC2024 by Sanz and Toldra (1997) and Magboul and McSweeney (1999b), respectively. The former was a monomer with a molecular mass of 35-36 kDa and maximum activity at pH 7.5 and 37 ~ while the latter was a dimer with a subunit molecular mass of---32 kDa and optimum activity at pH 7.0 and 40 ~ The 20 N-terminal amino acid residues of the PepL from Lb. curvatus DPC2024 showed 50, 80 and 95% homology with PepL from Lb. delbrueckii subsp. lactis DSM 7290 (Klein et al., 1995), the prolinase from Lb. helveticus CNRZ32 (Dudley and Steele, 1994) and the prolinase from Lb. rhamnosus 1/6 (Varmanen et al., 1998), respectively.
Proline-specific peptidases Caseins, the major proteins in bovine milk, are rich in the imino acid, proline. Because of its unique structure, specialized peptidases are required to hydrolyse peptide bonds involving proline, thus making peptides accessible to the action of other peptidases (see review by Cunningham and O'Connor, 1997). Several proline-specific peptidases with distinct substrate specificities have been found in LAB. X-Prolyl dipeptidyl aminopeptidase (PepX) is a peptide hydrolase capable of releasing X-Pro and sometimes X-Ala dipeptides from the N-terminal of oligopeptides. Due to its unique specificity, PepX is the best characterized of the proline-specific peptidases. The enzyme has been demonstrated in several genera of LAB and isolated from a number of strains and
Proteolysis in Cheese during Ripening characterized (Table 1). All PepXs purified from LAB have a serine catalytic mechanism and most are dimeric proteins with a native molecular mass of 117-200 kDa (Table 1); however, a high molecular mass endopeptidase (---350 kDa) with PepX activity and able to hydrolyse Otsl-casein was isolated and characterized by Stepaniak et al. (1998a). Increasing the proportion of pepX-negative mutants in a starter culture reduced the organoleptic quality of the resultant cheese but did not increase bitterness (Baankreis, 1992). Meyer and Spahni (1998) studied the role of PepX from Lb. delbrueckii subsp, lactis by using PepXnegative mutants. This enzyme influenced proteolysis and the sensorial characteristics of Gruyere cheese but it was not essential for the growth of the microorganism in milk (Meyer and Spahni, 1998). Proline iminopeptidase (PepI) catalyses the release of an N-terminal proline residue from di-, tri- and oligo-peptides. PepI from Lc. lactis subsp, cremoris HP (Baankreis and Exterkate, 1991) is the only iminopeptidase that has been purified from Lactococcus. This enzyme is a dimeric metallopeptidase with a native molecular mass of 110 kDa (Table 1). In contrast, the iminopeptidases purified from Lb. helveticus LHE-511 (Miyakawa et al., 1994b) and Lb. casei subsp, casei LLG (Habibi-Najafi and Lee, 1995) were monomeric thiol peptidases which were slightly inhibited by the serine protease inhibitor phenylmethyl sulphonyl fluoride. The molecular mass of the enzymes from Lb. helveticus and Lb. casei was estimated as 70 and 46 kDa, respectively. In addition to these two iminopeptidases, a PepI was purified from Lb. delbrueckii subsp, bulgaricus CNRZ 397 by amplification and expression of the gene in E. coli (Gilbert et al., 1994). The purified enzyme was characterized as a trimeric serine peptidase with a subunit molecular mass of 33 kDa (Table 1). Prolinase (PepR) is a specific dipeptidase which hydrolyses dipeptides with the sequence Pro-X. PepR from Lb. helveticus CNRZ32 was purified and biochemically characterized by Shao et al. (1997) and found to have a relatively broad specificity. The PepR from Lb. rhamnosus 1/6 (Varmanen et al., 1998), in addition to its prolinase activity, hydrolysed the aminopeptidase substrates, Pro-[3NA, Leu-[3NA and Phe-[3NA. Prolidase (PepQ) is an X-Pro-specific dipeptidase. With the exception of PepQ from Lb. helveticus CNRZ32, which is a homodimer with a subunit molecular mass of 45 kDa, most PepQs characterized to date are monomeric metallopeptidases with a native molecular mass of ---42 kDa. These enzymes hydrolysed most X-Pro dipeptides with the exception of Gly-Pro and Pro-Pro (Kaminogawa et al., 1984; Femandez-Espki et al., 1997b; Morel et al., 1999). However, PepQs isolated from Lc. lactis subsp. cremoris AM2 (Booth et al., 1990a) and Lb. delbrueckii
413
subsp, lactis DSM7290 (Stuckey et al., 1995), hydrolysed di- and tripeptides that did not contain Pro, in addition to Pro-X dipeptides. Aminopeptidase P (PepP) is a specific aminopeptidase that catalyses the removal of the N-terminal amino acid from oligopeptides having the sequence X-Pro-Pro-(X)n or X-Pro-(X)n (Kunji et al., 1996). The enzyme has been purified from strains of Lactococcus and is a monomeric metallopeptidase with a molecular mass of 41-43 kDa (Table 1). Provided that the peptide contains the above sequences, PepP is capable of releasing the N-terminal amino acid from oligopeptides up to 11 residues long. This enzyme also hydrolyses peptides with Ala in the penultimate position but at a slower rate (McDonnell et al., 1997).
Enzymes from Secondary Starter Microorganisms Enzymes of LAB play an important role in the secondalT proteolysis in internal-ripened cheese varieties, and hence contribute significantly to the development of flavour and aroma. In mould-ripened, smear-ripened and Swiss-type cheeses, microorganisms other than LAB play a pivotal role in the development of characteristic flavour and texture. The ripening of these cheese varieties involves complex biochemical reactions, which are discussed in detail in Volume 2. While the enzymes of LAB have been well studied and characterized, there have been fewer studies on organisms associated with mould-ripened or smear-ripened cheese varieties or on enzymes from Propionibacterium freudenreichii subsp. shermanii. The microbial flora of surface mould-ripened and blue-veined cheese, such as Camembert and Roquefort, includes yeasts (e.g., Kluyveromyces lactis, Saccharomyces spp. and Debaryomyces hansenii), moulds (Geotrichum candidum, Penicillium spp.), lactococci, lactobacilli, micrococci, staphylococci, coryneform bacteria and coliforms. Penicillium spp. are major components of the microflora and their enzymes play an important role in cheese ripening. Proteolytic systems of P camemberti and P roqueforti are somewhat similar; both synthesize an aspartyl proteinase, a metalloproteinase, an acid carboxypeptidase and an alkaline aminopeptidase ('Surface Mould-ripened Cheeses' and 'Blue Cheese', Volume 2). The aspartyl proteinase from P camemberti hydrolyses Otsl-casein faster than [3-casein or K-casein (Gripon, 1993). Acid proteinases of P. camemberti and P roqueforti have similar action on [3-casein and hydrolyse Lys97mVa198, Lys99mGlu100 and Lys29~Ile30 bonds at a faster rate than other bonds in [3-casein (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982). Metalloproteinases of
414
Proteolysis in Cheese during Ripening
both species have similar properties and have a pH optimum in the range 5.5-6.0. Chrzanowska et al. (1995) purified an aspartic proteinase from the culture filtrate of P. camemberti by a two-step purification procedure. The proteinase had a molecular mass of 33.5 kDa and an optimum pH of 3.4 on haemoglobin. The enzyme showed specificity towards peptide bonds containing an aromatic or hydrophobic amino acid residue in the B-chain of insulin. Besides these proteinases, P. roqueforti has a carboxypeptidase, which has an optimum pH of 3.5 and releases acidic, basic or hydrophobic amino acids (Gripon, 1993). Geotrichurn candidum also synthesizes extracellular and intracellular proteinases, but the contribution of these enzymes to cheese ripening is less than that of enzymes from Penicillium spp. (Gripon, 1993). The bacterial microflora of surface cheeses, such as Tilsit, Limburger, MOnster or Taleggio at the beginning of ripening is dominated by yeasts and moulds, which are acid and salt tolerant, but at the end of ripening, bacteria of the genera Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus and Corynebacterium dominate (Eliskases-Lechner and Ginzinger, 1995; Valdes-Stauber et al., 1997; 'Bacterial Surface-ripened Cheeses', Volume 2). Growth of B. linens on the cheese surface is thought to play an important role in the development of the characteristic colour, flavour and aroma of smear surface-ripened cheese varieties (Rattray and Fox, 1999) and hence, its enzymes have been characterized. Extracellular enzymes of B. linens include proteinases, aminopeptidases and esterases, the biochemical properties of which vary because of wide inter-strain differences within the species. Brezina et al. (1987) partially purified four extracellular proteinases from B. linens, with pH and temperature optima of 5.0-8.0 and 50 ~ respectively. Hayashi et al. (1990) purified five extracellular proteinases from B. linens F (designated A, B, C, D and E), having a molecular mass of 37, 37, 44, 127 and 325 kDa, respectively, as determined by size exclusion chromatography (SEC). Proteinases A and B were stable at 35 ~ for 1 h and had a temperature optimum of 40 ~ while proteinases C, D and E were stable at 45 ~ for 1 h and had a temperature optimum of 50 ~ All five proteinases were optimally active at pH 11.0 and were serine proteinases. The production of multiple forms of the extracellular proteinases by B. linens ATCC 9172 is a result of aggregation of subunits and autocatalytic degradation (Buchinger et al., 2001). An extracellular serine proteinase partially purified from a strain of B. linens (Laktoflora 200), had a molecular mass of 52-55 kDa, as determined by SDSPAGE, and pH and temperature optima of 7.0-8.5 and
45 ~ respectively (Juh~isz and Sk~irka, 1990). A thermostable proteinase was partially purified from B. linens IDM 376; it had molecular mass of 18.5 kDa and pH and temperature optima of 7.5 and 67.5 ~ respectively, on azocasein (Clancy and O'Sullivan, 1993). An extracellular serine proteinase purified from B. linens ATCC 9174 had a molecular mass of 126 kDa, as determined by SEC and was optimally active at pH 8.5 and 50 ~ (Rattray et al., 1995). It hydrolysed Otsl-casein at His8mGln9, Ser161mGly162 and either Glnlr2mTyr173 or Phe23m Phe24 (Rattray et al., 1996) and [3-casein at Serls~Ser19, Glu20~Glu21, Gln56~Sers7, Gln72~Asn73, Leu77~Thr78, Alal01~ Met102, Phe119~Thr120, Leu139mLeu140, Ser142~Trp143, His145~Gln146, Gln167~Ser168, Gln175~Lys176, Tyr180~Pro181 and Phe190~Leu191 (Rattray et al., 1997). One of the five extracellular enzymes of B. linens ATCC 9172 was purified to homogeneity by Tomaschov~i et al. (1998) using ion-exchange chromatography and native preparative PAGE. The enzyme had nearly identical properties to the serine proteinase of /3. linens ATCC 9174 purified by Rattray etal. (1995). Its molecular mass was estimated to be 56 kDa by SDSPAGE and pH and temperature optima were 8.0 and 50 ~ respectively. B. linens also produces extracellular aminopeptidases, intracellular peptidases and proteinases. Sorhaug (1981) reported the presence of intracellular dipeptidase activity in six strains of B. linens. The presence of three extracellular aminopeptidases in B. linens (Laktoflora 200), having pH and temperature optima of 7.0-9.0 and 30 ~ respectively, was reported by Brezina et al. (1987). Two extracellular aminopeptidases, designated A and B, with a molecular mass of estimated to be 150 and 110 kDa, respectively, and pH and temperature optima of 9.3 and 40 ~ respectively, were purified from B. linens F by Hayashi and Law (1989). Ezzat et al. (1993) reported the presence of cell wall proteinases and dipeptidase activities in B. linens CNRZ 944. The authors partially purified the cell wall proteinase, which had maximum activity at pH 6.5 and 40 ~ An intracellular aminopeptidase from B. linens ATCC 9174, with a molecular mass of 59 kDa, as determined by SDSPAGE, and 69 kDa by SEC, was reported by Rattray and Fox (1997). The enzyme was optimally active at pH 8.5 and 35 ~ Curtin et al. (2002) showed aminopeptidase, dipeptidase and tripeptidase activities in brevibacteria, corynebacteria, staphylococci and brachybacteria, isolated from smear surface-ripened cheeses, Tilsit and Gubeen. Species of the genus Arthrobacter are major components of the microflora of surface mould-ripened cheeses, such as Brie and Camembert and red-smear
Proteolysis in Cheese during Ripening 415 cheeses. However, the enzymes of Arthrobacter have not been well studied. Smacchi et al. (1999a) purified two extracellular serine proteinases from A. nicotianae 9458, with molecular masses of about 53-55 and 70-72 kDa, as determined by SDS-PAGE. The enzymes were optimally active at 55-60 and 37 ~ respectively. Both enzymes were optimally active in the pH range of 9.0-9.5 and preferentially hydrolysed [3-casein over Otsl-casein. An extracellular PepI from A. nicotianae 9458 with a molecular mass of about 53 kDa, was purified and characterized by Smacchi et al. (1999b). The enzyme was optimally active at 37 ~ and 8.0. Some Micrococcus spp. are very proteolytic and produce extracellular proteinases and intracellular proteinases and peptidases (Fox et al., 1993). Nath and Ledford (1972) reported that extracellular proteinases from certain micrococci preferentially hydrolysed Otslcasein; production of extracellular proteinase was also reported by Garcia de Fernando and Fox (1991). Bhowmik and Marth (1989) purified and characterized an aminopeptidase, with broad substrate specificity, from M. freudenreichii ATCC 407. Propionibacterium spp. are weakly proteolytic, but they are highly peptidolytic, especially on proline-containing peptide bonds, thus contributing to the characteristic flavour of Swiss-type cheeses (see 'Cheese with Propionic Acid Fermentation', Volume 2). Biochemical characteristics of peptidases from propionic acid bacteria have been reviewed by Gagnaire et al. (1999). A PepX with a molecular mass of 84 kDa and pH and temperature optima of 7.0 and 40 ~ respectively, was purified and characterized from P. freudenreichii subsp, shermanii NCDO 853 by Fernandez-Espla and Fox (1997). Endopeptidases have been isolated from P. freudenreichii subsp, shermanii and characterized (Table 1) (Tobiassen et al., 1996; Stepaniak et al., 1998b).
Patterns of Proteolysis in Cheese The pattern of proteolysis in many varieties may be summarized as follows: the caseins are hydrolysed initially by residual coagulant activity retained in the curd and by plasmin (and perhaps other indigenous proteolytic enzymes) to a range of large and intermediate-sized peptides which are hydrolysed by proteinases and peptidases from the starter LAB, NSLAB and perhaps secondary microflora to shorter peptides and amino acids. However, the pattern and extent of proteolysis varies considerably between varieties due to differences in manufacturing practices (particularly cooking temperature), which cause differences in moisture content, residual coagulant activity, activation of plasminogen to
plasmin, and possibly the development of a highly proteolytic secondary microflora and ripening time. The extent of proteolysis (i.e., the degree to which the caseins and peptides therefrom are hydrolysed and measured by the development of water- or pH 4.6soluble N) in cheese varies from very limited (e.g., Mozzarella) to very extensive (e.g., Blue) and is summarized for many varieties in Table 2. The pattern of proteolysis (i.e., the relative concentrations of different peptides and amino acids) is very variable and is essentially unique to a particular variety. The differences in soluble N content are due to differences in moisture content, temperature and pH, length of ripening, cooking temperature and pH at draining (Fox and McSweeney, 1996) and is mainly due to the action of chymosin and to a lesser extent of plasmin (Fox and McSweeney, 1997). A short ripening period ( - 3 weeks) and extensive denaturation of chymosin during the high temperature ( - 7 0 ~ stretching step during the manufacture of Mozzarella cheese explain the low level of soluble N, whereas extensive proteolysis is characteristic of Blue cheese and some smear-ripened varieties, caused by the action of chymosin, plasmin and proteinases from their characteristic secondary microflora. In addition, differences in the action of these proteolytic agents cause differences in peptide profiles. Primary proteolysis is similar during the ripening of most cheeses; chymosin hydrolyses the Phe23--Phe24 bond of Otsl-casein (Hill et al., 1974; Caries and Ribadeau-Dumas, 1985) except in cheeses that are cooked at a high temperature ( - 5 5 ~ e.g., Swiss cheese), in which plasmin is the principal proteolytic agent. In blue-veined cheeses, after sporulation, enzymes from P. roqueforti hydrolyse Otsl-CN (f24-199) and other peptides, changing the peptide profile (Gripon, 1993). Analysis of the water-insoluble fraction of various cheeses by urea-PAGE gives insight into the differences in peptide profile between cheeses (Fig. 12). In many cheeses, Otsl-casein is hydrolysed faster than [3-casein (Sousa et al., 2001). In Blue-veined cheeses, both Ors1- and [3-caseins are completely hydrolysed at the end of ripening. In Swiss-type cheeses, [3-casein is hydrolysed faster than Otsl-casein, with concomitant increases in y-caseins, indicating a role of plasmin and denaturation of chymosin during cooking. However, Ot~l-CN (f24-199) is produced slowly in Swiss cheese, indicating either the survival of some chymosin during cooking or the activity of indigenous milk acid proteinase, cathepsin D (Gagnaire et al., 2001), which has specificity similar to chymosin (Hurley et al., 2000a). In the case of Camembert-type cheese, about - 2 0 % of total N is soluble at pH 4.6 (Khidr, 1995) (Table 2) and the pattern of proteolysis is similar to Cheddar cheese (Fig. 12). During the ripening of Mozzarella
416
Proteolysis in Cheese during Ripening Table 2 Soluble N as % of total nitrogen in different cheese varieties Cheese
Age
SN/'IN %
References
Mozzarella
25 days
4-5
Quarg Gouda
4 weeks 6 weeks 24 weeks 1 month
Somerset al. (2002) O'Reilly et al. (2002) Guinee et al. (1998) Mara and Kelly (1998) Messens et al. (1999) Exterkate and Alting (1995) Michalski et aL (2003) Sousa and McSweeney (2001) Khidr (1995)
Swiss Feta
16 weeks 2-6 months
--~12 12-13 23-25 Surface 15-17 Core 9-12 Surface---20 Core ---12 16-17 17-20
Mahon Cheddar
<2 months 4-6 months
19-20 20-25
Tilsit
28 weeks
Parmesan Gorgonzola Danablu
24 months
Surface 24-25 Core 22-23 31-35 43-46 50-53
Camembert
1 month
cheese, Otsl-CN (f24-199) is produced slowly and y-caseins more rapidly, indicating weak chymosin activity and fairly high plasmin activity (Kindstedt, 1993). Plasmin and Lactobacillus proteinases are responsible for extensive proteolysis in Parmigiano-Reggiano cheese that is ripened for a long period (---24 months) at an elevated temperature (---18-20 ~ (Battistotti and Corradini, 1993). The high cooking temperature used during the manufacture of Parmigiano-Reggiano cheese denatures most of the chymosin.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
~~~WW
U m ~Wlmem ~'~
Figure 12 Urea-polyacrylamide gel electrophoretograms waterinsoluble fraction of a selection of cheese varieties. Lane 1 Na caseinate, lane 2 Cheddar, lane 3 extra-mature Cheddar, lane 4 Cheshire, lane 5 Red Leicester, lane 6 Double Gloucester, lane 7 Emmental, lane 8 Leerdammer, lane 9 Jarlsberg, lane 10 Vorarlberger Bergkase, lane 11 Edam, lane 12 Gouda, lane 13 Norvegia, lane 14 Parmesan, lane 15 Parmesan (from McGoldrick, 1996).
Cooney et al. (2000) Michaelidou et aL (2003) Sarantinopoulos et aL (2002) Katsiari et aL (2000) Moatsou et al. (2002) Taborda et aL (2003) ShakeeI-Ur-Rehman and Fox (2002) Barrett et aL (1999) ShakeeI-Ur-Rehman et aL (1998) Lane et al. (1997) Churchill et al. (2003) Careri et al. (1996) Zarmpoutis et aL (1997)
Several peptides from Cheddar, Parmigiano-Reggiano, Blue, Swiss and Feta cheeses have been isolated and characterized. Of these varieties, the peptide profile of Cheddar cheese is the best characterized and is summarized in Figs 13 and 14. All the principal water-insoluble peptides in Cheddar cheese are produced either from e~sl-casein by chymosin or from [3-casein by plasmin (McSweeney et al., 1994a). After cheese manufacture, residual chymosin acts on Phe23--Phe24 of e~sl-casein to produce the large C-terminal peptide Otsl-CN (f24-199) and a small peptide C~sl-CN (fl-23) (Fox and McSweeney, 1997). Otsl-CN (f24-199) is hydrolysed by chymosin at LeUl01mLySl02 (Fig. 15) and more slowly at Phe32mGly33, Leu109~Glu110, Phe28~Pro29 and Leu40~Ser41; Otsl-CN (f24-199) is also hydrolysed slowly by plasmin at LySl03~Tyrl04 and LySl05mVall06 of Otsl-CN (f24-199). The large C-terminal peptides OtslCN (f24-199), Otsl-CN (f33-199), Otsl-CN (f102-199), Otsl-CN (f110-199), OLsl-CN (f99-199), Otsl-CN (f104-199) and O~sl-CN (f106-199) have been identified in the water-insoluble fraction of Cheddar cheese (McSweeney et al., 1994a; Mooney et al., 1998). The bond Trp164~Tyr165, which is hydrolysed rapidly in solution by chymosin (McSweeney et al., 1993b), does not appear to be hydrolysed in cheese, perhaps due to intermolecular interactions. Peptide Otsl-CN (fl-23) is hydrolysed at the bonds Gln9--Glylo, Gln13~Glu14, Glu14--Val15 and Leu16--Asn17 by lactocepin (Fox and
Proteolysis in Cheese during Ripening
417
A 199
1
199
24
24
186 199
99
24
102
24
102 33
~*
199 191
104 60
199
106
199 110
199
70
156 121 80
199
*
129
1
209
1 1
189/192 * 29 29 ~
209 *
30 ~ *
106 108
209 209
Figure 13 Principal water-insoluble peptides derived from OLsl-casein (A) and 13-casein (B) isolated from Cheddar cheese by McSweeney et al. (1994a) and Mooney et al. (1998) (from Sousa et al., 2001).
McSweeney, 1996). The peptides Otsl-CN (fl-9), Otsl-CN (f1-13) and Otsl-CN (fl-14) accumulate and dominate the RP-HPLC chromatogram of water-soluble fraction of Cheddar cheese (Fig. 15). Although the bond Leu192mTyr193 of [B-casein in solution is very susceptible to chymosin, it is hydrolysed very slowly in cheese, probably due to the effect of ionic strength which promotes hydrophobic interactions between susceptible regions of [3-casein molecules (Fox and McSweeney, 1997). The cleavage of Leu192mTyr193 in cheese is undesirable, as ~3-CN (f193-209) is very hydrophobic and bitter (Visser et al., 1983). Plasmin preferentially hydrolyses [B-casein at Lys28--Lys29, Lysl05mHiSl06 and Lysl0T--Glul08, producing yl-, Y2-, y3-caseins and PPs (5, 8 fast and 8 slow); "y-caseins are present in the water-insoluble fraction of Cheddar (McSweeney et al., 1994a; Lane and Fox, 1999; McGoldrick and Fox, 1999) and many other cheeses. No large peptides originating from Ots2-casein have been identified in Cheddar cheese (Mooney et al., 1998) and only four small peptides have been identified in the water-soluble fractions (Singh et al., 1995, 1997). Water-soluble peptides are characteristic of particular cheese varieties and are related to the specificity of the starter and non-starter proteinases and peptidases (Fox
and McSweeney, 1996). In terms of number, most of the peptides in the water-soluble fraction of Cheddar cheeses originate from N-terminal half of 6-casein (particularly from residues 53 to 91) and a small number from the N-terminal half of Otsl-casein. However, many of these peptides are present at low levels in cheese, and the water-soluble fraction of Cheddar cheese is dominated by a relatively small number of peptides, originating from Otsl-casein. Peptides in the water-soluble fraction of Cheddar cheese do not contain intact plasmin or chymosin cleavage sites and hence probably arise, not directly from the caseins, but rather from larger precursor peptides (produced by chymosin or plasmin) by the action of lactocepin or other microbial enzymes. Little work has been done on pH 4.6-soluble peptides in blue-mould cheese, in which extensive proteolysis occurs. Gonzalez de Llano et al. (199 I) studied the production and identification of phosphotungstic acid (PTA)-soluble peptides in Gamonedo blue cheese. Low molecular mass peptides from the PTA-soluble fraction were isolated and their amino acid composition was determined. The isolated peptides contained 7 - i 0 amino acids and the major amino acids were Ser, Glu, Gly/Thr, Ile and Leu (Gonzalez de Llano et al. 1991).
418
Proteolysis in Cheese during Ripening
24--
91__~ 931i06 85--92 93--? 85~95 85--91
?
26/35 25-34 25 35
751? 75~? 75~? 75~?
25 39 25--30 24-34
115 - - 1 2 4 115-121 110~
180--188
?
70--76 70~?
Cleavage sites of cr
24 - - 2 9 13/14 23/24 37/38 8/9/10 16/17118 33/34
74/75
84/85 88189 98/99
LL J, !lJ~, J,J, TTTTT 1 1
? II--i I0~
?
1
13
1
- 14
56~?
25B31 25~ ? 26--32
?
I --9
26 - -
34
8 5 - - 91 92~ 93~
44--?
18~?
~
~L
~L~
TT TTT
~
191/92 ~
T
199
? ?
102--
40~ ? 41--? 41~?
157/58 156/57 142/43 149/50 169/70 139140 148/49 161/62
149150 153/54 164/65 179/80 ! 56/57 158159 Cleavage sites of chymosin
105~
24--30
17--?
~
TT 98/99 101/02
23124125 40/41 28/29 32/33
1--?
130/31 121/22
envelope proteinasr of Lactococcus spp
?
?
175--182 176--?
191--197 204--207
Cleavage sites of cell envelope proteinasr of Lactococcus spp
115/16
79180 88/89
TT 21/22
61 61--
24/25
137138
T 114/15
71 70
149/50/51
36 2 5 - 2 7 ~ 41
53 57 57~68 57 57 57 5 7 ~ 58--? 59--? 9 54 ~
1~7 I--6
43~? 30--36 30~? 7 19 2 8 - - ? 29~39 7 18 29 37 8 14 29--33 8 ~ 2 3 29~? 53 10 - - 1 5 29 7 ~ 1 5 29 7 ,, 14 28/29
I ~ ? I I 7--?
7--? 10--? DF permeate
207
T 197/98 T 181/82 188/89
92 93 .95 % 60 60 60 60168
TT T
182183 178/79 174/75 197/98 166/67 186/87188 203/04
Cleavage sites of plasmin
57 57 57 57
6/7/8 16/17
150/51
57
57
96 97 93 91 72 66 73 73 73 69~'92 69
91 94
91
70
107/08 105/06 113114
,,69
TTT TTTTT TT
177--191 171--?
105
Cleavage sites of plasmin
183/84
~
T
TT
43/44 52/53 56157 68/6974/75 93194 101102 45/46/47 54/55 58/59 105/06 57 67 8 2 ~ ? 105 107 69 B 7 7 59 ,,,? 78-88 102--? 59 68 78--93 ~ / ? 69 7"~----88 69 89 53--? 69 ,91 43--? 57--? 69 ,, 92 53~? 69 83 69 85 45--52 6 9 ~ 9 3 69--84 97--108 69--82 58~?69~80 6 9 ~ 1 0 4 57 93 57 73 91 98 73--78 73--89 59 92 59 76 59 95 59 94
T TNiTTT TTTT
T z~
151152 160/61 175/76 207/08 165/66/67/68/69 190/91/92/93/94 182/83 Cleavage sites of cell envelope proteinase of Lactococcus spp
42-----52 29........_.9
193--206
158--? 1771?
Figure 14 Water-soluble peptides derived from O~sl-casein (A), OLs2-casein (B) and 13-casein (C) isolated from Cheddar cheese by Singh et aL (1994, 1995, 1997), Breen et al. (1995) and Fernandez et aL (1998). The principal chymosin, plasmin and lactocepin cleavage sites are indicated (from Sousa et aL, 2001).
Proteolysis in Cheese during Ripening
419
Chymosin 199
1
23
Chymosin 24
gL
, ~ kc-CEP
i ..........................................................................................
199
102
J~
199
Further hydrolysis products
Figure 15 Schematic representation of the early proteolysis of OLsl-casein during the ripening of many cheeses and the location of peptides produced on a urea-polyacrylamide gel electrophoretogram and a reverse-phase HPLC elution profile.
Low molecular weight peptides formed in Parmigiano-Reggiano cheese during ripening were isolated and identified by Addeo etal. (1992, 1994, 1995) using fast atom bombardment-mass spectrometry. Oligopeptides originating from regions 1-20 and 6-28 of [3-casein, five phosphopeptides originating from the region 64-84 of Otsl-casein, three phosphopeptides from the region 1-21/24 of Ots2-casein and one peptide from C-terminal part of Ots2-casein were identified. Several water-soluble peptides in Feta were identified by Michaelidou et al. (1998), including Otsl-CN (fl-14), (f4-14), (f24-30), (f24-32), (f40-49), (f91-98), (f102-109), [3-CN (f164-180), (f191-205) and K-CN (f96-105). Gagnaire et al. (2001) identified 91 peptides in aqueous phase of Emmental cheese, 52 of which originated from Otsl-casein, 29 from [3-casein, 9 from Ots2-casein and 1 from K-casein. Significant concentrations of amino acids, the final products of proteolysis, occur in all cheeses that have been investigated. Levels of free amino acids in a number of cheese varieties are given in Table 3. Relative to the level of water-soluble N, Cheddar contains low concentrations of amino acids; the principal amino acids are Glu, Leu, Arg, Lys, Phe and Ser. ParmigianoReggiano contains a very high concentration of amino acids which contribute to the characteristic flavour of this cheese (Resmini et al., 1988). Many amino acids have characteristic flavours (see McSweeney et al., 1997); although none has a cheese-like flavour, it is believed that they contribute to the savory taste of mature cheese. However, the principal role of amino acids in flavour development is as precursors of volatile flavour compounds produced by the range of catabolic reactions
as discussed in 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1.
Methods for Monitoring Proteolysis in Cheese A range of analytical techniques have been developed to study proteolysis in cheese and have been reviewed (IDE 1991, 1999; McSweeney and Fox, 1993, 1997; Fox et al., 1995a; Wallace and Fox, 1998). These methods for assessment of proteolysis in cheese can be classified in two categories, namely non-specific and specific methods. Non-specific methods, which give information about the extent of proteolysis and the activity of proteolytic agents, include determination of the nitrogen soluble in, or extractable by, various solvents or buffers (see Christensen etal., 1991; McSweeney and Fox, 1997; Ard0, 1999) or the measurement of reactive groups (e.g., NH2-groups) (McSweeney and Fox, 1997; Wallace and Fox, 1998). Soluble nitrogen is usually determined by the macro-Kjeldahl method, which is a highly repeatable, but time-consuming and potentially dangerous technique (Wallace and Fox, 1998). A number of techniques for quantifying proteolysis are based on cleavage of the peptide bond, which results in the formation of a new amino group which can react with several chromogenic (e.g., 2,4,6-trinitrobenzenesulphonic acid or ninhydrin) or fluorogenic (e.g., o-phthadialdehyde or fluorescamine) reagents (McSweeney and Fox, 1997; Wallace and Fox, 1998). Non-specific methods give a general idea of proteolysis, but give no information about the specific peptides produced or degraded during ripening. Specific
420
Proteolysis in Cheese during Ripening
Table 3
Concentration of amino acids (mg kg-1 cheese) in different cheese varieties (based on Fox and Wallace, 1997)
Amino acid
Cheddar
Edam
Emmental
Cys Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg
44.7 1532.8 416.3 3144.1 336.2 306.86 356.2 1096.4 434.8 2774.1 464.1 1472.6 1127.2 1096.4
20.5 144.5 71.1 351.7 153.8 34.6 68.8 167.4 60.3 48.1 426.6 89.2 291.9 49.7 245.5 130.5
166.5 688.5 548.9 2680.5 2535.2 430.8 568.2 1561.5 502.7 1051.1 1794.9 285.9 1279.1 868.2 2219.8 19.2
techniques (i.e., electrophoresis and chromatography) have been used extensively to resolve, isolate and identify the peptides that are produced during cheese ripening (Fox et al., 1995a; McSweeney and Fox, 1997; Otte et al., 1999; Singh et al., 1999). In addition, these techniques are used to determine peptide profiles of cheese extracts; data obtained using these techniques are often analysed using multivariate statistical techniques (e.g., Pripp etal., 1998, 1999, 2000a,b; Molina et al., 1999; Shakeel-Ur-Rehman et al., 1999). Urea-PAGE is a powerful tool for monitoring proteolysis during the early stages of cheese maturation and for comparing casein hydrolysis patterns in cheeses manufactured from the milk of different species (Marcos et al., 1979; Sousa and Malcata, 1997). Urea-PAGE is widely used to monitor proteolysis as it resolves proteins based on a combination of charge and mass while sodium dodecylsulphate (SDS)-PAGE, which is used more widely in biochemistry, is less suitable for studying proteolysis in cheese because this technique resolves proteins based on size and the caseins have similar molecular masses. Peptides separated by SDSor urea-PAGE can be isolated by excision of the bands or by electroblotting (McSweeney et al., 1994a; Singh etal., 1995; Sousa and Malcata, 1998a,b) and the N-terminal sequence of isolated peptides determined. Isolation of peptides by electroblotting has been used widely to study proteolysis in cheese (e.g., Singh et al., 1995, 1997; Gouldsworthy et al., 1996; Ferranti et al., 1997; Broadbent et al., 1998). Capillary electrophoresis (CE) is reported to be an excellent technique for resolving the caseins (including different genetic variants), peptides derived therfrom
ParmigianoReggiano 3241.9 4033.3 4459.5 14489.0 2115.6 2260.2 6011.9 2351.5 5205.2 7290.4 2054.7 4314.9 10091.0 791.4
Gorgonzola
Danablu
1380 1020 530 1570 3940 2320 390 1140 2220 780 1300 2910 850 1590 800 3050 280
1160 300 190 1020 1730 530 160 340 610 500 300 1530 520 680 610 1540 510
and whey proteins (Otte et al., 1997). Peptide profiles obtained by CE supplement the information obtained by reversed-phase high performance liquid chromatography (RP-HPLC) (Otte etal., 1997; Molina etal., 1998). Capillary electrophoresis has been used to study: 9 proteolysis in Cheddar (Strickland etal., 1996), Mozzarella, Feta and Danbo (Otte etal., 1997), Tilsit (Bockelmann et al., 1998), Roncal (Irigoyen et al., 2000), Danbo cheeses (Sorensen and Benfeldt, 2001) and Serpa, a raw ewes' milk cheese (Roseiro et al., 2003); 9 the effect of the amount of rennet on proteolysis and texture in Feta cheese made from ultrafiltered milk (Wium et al., 1998); 9 the effect of different strains of Penicillium roqueforti on the ripening of blue-veined cheese (Larsen et al., 1998); 9 the effect of added proteinases and level of starter cultures on the formation of biogenic amines in Manchego cheese made from raw milk (Fern~indezGarc~a et al., 1999). A number of chromatographic techniques, such as ion-exchange chromatography, SEC and RP-HPLC have been used to fractionate milk proteins or to fractionate cheese extracts for the purification of peptides, or less commonly, as analytical techniques to generate peptide profiles (see McSweeney and Fox, 1997). Ion-exchange and SEC are suitable for the fractionation of large caseinderived peptides. High performance ion exchange chromatography and HP-SEC have the advantage of speed and reproducibility. RP-HPLC is a very good method for resolving water-soluble peptides and has been used to characterize and compare the degree of proteolysis in
Proteolysis in Cheese during Ripening cheeses of various ages and quality, and to study the effect of various cheesemaking parameters on proteolysis (Singh et al., 1999). Reversed phase-high performance liquid chromatography has been used extensively to characterize peptides in casein hydrolysates (e.g., Le Bars and Gripon, 1989, 1993; McSweeney and Fox, 1993) as well as in studies on proteolysis of the caseins in cheese during ripening (e.g., Gonzalez de Llano et al., 1991; Addeo et al., 1992, 1994; McSweeney et al., 1994a; Lynch et al., 1997; Sousa and Malcata, 1998a; McGoldrick and Fox, 1999; Shakeel-Ur-Rehman et al., 2000; Katsiari et al., 2001; Trujillo et al., 2002; Poveda et al., 2003). Numerous peptides from cheese have been purified by a combination of chromatographic procedures and subsequently identified, usually by Edman degradation and mass spectrometry, leading to a better knowledge of the proteolytic pathways in cheese during ripening (Singh et al., 1999; Gagnaire et al., 2001). Free amino acids in cheese have been analysed using amino acid analysers based on ion-exchange chromatography, with post-column ninhydrin derivatization and photometric detection at 5 7 0 n m and 440 nm for primary and secondary amino acids, respectively. This method is relatively simple, accurate and quantitative and requires little sample preparation (BCltikofer and ArdO, 1999). Alternatively, fluorescent amino acid derivatives (e.g., dansyl, OPA or N(9-fluorenylmethoxycarbonyl)) can be prepared and separated and quantified by RP-HPLC; amino acids can also be quantified by gas chromatography but this method is rarely used (McSweeney and Fox, 1997). In addition to the above methods, new techniques have been evaluated for indirect measurement of proteolysis. During ripening, the texture of cheese undergoes major changes due to proteolysis, and in recent years, the use of new techniques such as fluorescence spectroscopy and ultrasound have been investigated to monitor changes in protein structure in different types of cheese during maturation. Fluorescence spectroscopy has the advantages of high sensitivity and rapidity for the characterization of molecular interactions and reactions. Tryptophan can be used as an intrinsic probe for monitoring changes in protein structure during cheese ripening as all the major proteins of bovine milk contain at least one tryptophan residue, which has a characteristic excitation in the region 280-295 nm and broad emission spectra (Hebert et al., 2000). Fourier transform infrared spectroscopy (FTIR) has been used to measure: 9 the levels of protein, fat and moisture in different cheeses (McQueen et al., 1995); 9 differences between soft cheese varieties (Herbert et al., 2000) and different Emmental-type cheeses (Picque et al., 2002);
421
9 changes in protein structure during cheese ripening (Mazerolles et al., 2001), protein/protein and protein/fat interactions and their relation to the texture of soft cheeses (Dufour et al., 2001); 9 cheese melting and its correlation with rheological properties (Karoui et al., 2003); 9 the stability of processed cheese (Christensen et al., 2003b). Mazerolles et al. (2001) investigated changes in the amide I and amide II regions of the FTIR spectra and in the tryptophan fluorescence spectra of 16 experimental semi-hard cheeses, varying in moisture, protein, fat and degree of maturity. The data obtained from mid-infrared and fluorescence spectral data were analysed by PCA, and correlations between spectral data and chemical composition as well as correlations between mid-infrared and fluorescence spectral data were found by canonical correlation analysis (CCA) methods. PCA and CCA of data helped to discriminate between samples at different stages of ripening. Application of low intensity ultrasonics in the food industry has increased during the last decade because it is non-destructive, rapid and cost-effective (McClements, 1997). Ultrasonics have been used in the dairy industry to monitor milk coagulation during cheesemaking (Gunasekaran and Ay, 1996; O'Donnell et al., 1996), to determine the maturity of Mahon, a Spanish semi-soft cheese variety (Benedito et al., 2000a), to determine structural defects in Parmesan cheese (Orlandini and Annibaldi, 1983) and to determine the physical properties of Cheddar cheese (Cho et al., 2001). The use of ultrasonic devices to monitor the maturity of Cheddar cheese non-destructively has been reported. Benedito et al. (2000b) ripened blocks of Cheddar cheeses at 5 or 12 ~ the ultrasonic velocity increased during maturation and decreased with increasing ripening temperature. Cho etal. (2001) measured ultrasound velocity and relative attenuation in Cheddar cheese using a noncontact piezoelectric ultrasound system and correlated results with the physical properties of Cheddar cheese (such as failure strain, failure stress, Young's modulus and toughness) using multi-linear neutral network analysis. Besides the above applications, ultrasonics can also be used to detect cracks in cheese and to assess eye distribution and size in Emmental cheese (Benedito et al., 2002). Research done so far has been limited to a few varieties of cheeses; research is required for other cheese varieties with different physical properties and ripening behaviour (e.g., mould-ripened cheeses, smearripened cheeses or cheese with eyes) for a clearer understanding of the interaction of ultrasound and cheese and subsequent application of ultrasound as a tool for monitoring ripening.
422
Proteolysis in Cheese during Ripening
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F u r t h e r hydrolysis p r o d u c t s
Plate 10 Schematic representation of the early proteolysis of asl-casein during the ripening of many cheeses and the location of peptides produced on a urea-polyacrylamide gel electrophoretogram and a reverse-phase HPLC elution profile. (See page 419.)
(a)
(b)
:i ~'+:::~~':; '~
,,j ', ,, ....,
-
"~""~_~11 ~l'/m
,,,
Plate 11 Vane rheometer probe before (a) and during (b) shear test on process cheese. Photos courtesy of Truong and Daubert (2000) Gel Consultants Inc. (See page 531 .)
Catabolism of Amino Acids in Cheese during Ripening A.C. Curtin and P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland
Introduction Catabolism of amino acids plays a major role in flavour development in cheese during ripening (McSweeney and Sousa, 2000; Yvon and Rijnen, 2001; Smit et al., 2002; 'Sensory Character of Cheese and its Evaluation' and 'Cheese Flavour: Instrumental Techniques', Volume 1). In particular, the catabolism of sulphur-containing amino acids (principally methionine), aromatic amino acids and branched-chain amino acids to flavour, or perhaps off-flavour, compounds has received considerable attention. The major aroma compounds produced from these amino acids are listed in Table 1. Recently, many authors have studied amino acid catabolism by cheese-related bacteria, including lactococci, non-starter lactic acid bacteria (NSLAB) and smear bacteria. However, it is important to remember that results obtained under laboratory conditions may not be representative of actual activity under cheeseripening conditions, as cheese constantly undergoes changes which may not be replicated in laboratory experiments. The pathways for amino acid catabolism remain to be characterised fully although much work has been done recently on LAB and Brevibacterium linens (see Yvon and Rijnen, 2001). There appear to be two major pathways by which amino acids are catabolised (Yvon and Rijnen, 2001; Fig. 1). The first series of reactions is initiated by the action of an aminotransferase which transfers the amino group from amino acid A to an ot-keto acid B (usually ot-ketoglutaric acid) and results in the production of an ot-keto acid corresponding to amino acid A and a new amino acid corresponding to ot-keto acid B (usually glutamic acid), ot-Keto acids produced by the transamination of aromatic amino acids, branched-chain amino acids and methionine may be degraded further to other compounds by enzyme-catalysed reactions or by chemical reactions. The second major series of reactions by which amino acids are catabolised is initiated by the action of amino acid lyases which cleave the side chains of amino acids. These pathways are particularly important for the catabolism of aromatic amino acids and methionine. Other pathways by which amino acids may
be catabolised include the production of amines by decarboxylases and the production of NH3 by deaminases. There are also specific pathways for the metabolism of threonine, aspartic acid, glutamic acid and arginine. Because of their importance to cheese flavour, the catabolism of methionine, branched-chain and aromatic amino acids will be discussed in separate sections below.
Transamination of Amino Acids Aminotransferases (EC 2.6.1.x) catalyse the transfer of the amino group from an or-amino acid to an ot-keto acid (Hemme et al., 1982). The enzymes have broad substrate specificity and can catalyse reversible transamination reactions (Weimer et al., 1999). Transamination is the first step in the degradation of amino acids by lactococci. In LAB, catabolism of aromatic amino acids, branched-chain amino acids and methionine is initiated by transamination since degradation occurs only in the presence of an ot-keto acid which acts as an amino group acceptor (see Yvon and Rijnen, 2001). In the context of cheese-related microorganisms, aminotransferases of LAB and B. linens have been researched most intensively; however, enzymes of Propionibacteriurn freudenreichii have also been studied (Thierry et al., 2002). In a study on the enzyme activities of a range of bacteria present on the surface of smear cheese (brevibacteria, corynebacteria, staphylococci and brachybacteria), Curtin etal. (2002) found methionine aminotransferase activity in only a strain of Staphylococcus equorum. Aminotransferases are pyridoxal-5'-phosphate (PLP)dependent enzymes (Hemme et al., 1982; Weimer et al., 1999; McSweeney and Sousa, 2000). The transamination reaction occurs in two steps. The first step involves transfer of the amino group of the amino acid to PLP to yield an ot-keto acid and an enzyme-bound pyridoxamine-5'-phosphate. In the second step, the amino group is transferred from pyridoxamine5'-phosphate to an ot-keto acid to produce an amino acid and to regenerate PLP. The amine acceptor is
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
436
Catabolism of Amino Acids in Cheese during Ripening
Table 1 Name and chemical nature of the major aroma compounds derived from methionine, branched-chain amino acids and aromatic amino acids (adapted from Yvon and Rijnen, 2001) Amino acid
Aldehydes
Alcohols
Carboxylic acids
Leucine
3-Methylbutanal/ isovaleraldehyde 2-Methylbutanal 2-Methylpropanal/ isobutyraldehyde Phenylacetaldehyde, benzaldehyde OH-phenylacetaldehyde, OH-benzaldehyde Indole-3-acetaldehyde, 3-Methylthiopropanal/methional
3-Methylbutanol
Phenylethanol
3-Methylbutanoic acid/ isovaleric acid 2-Methylbutanoic acid 2-Methylpropanoic acid/ isobutyric acid Phenylacetic acid
OH-phenylethanol
OH-phenylacetic acid
p-Cresol, phenol
Tryptophol 3-Methylthiopropanol
Indole-3-acetic acid 3-Methylthiopropionic acid
Skatole, indole Methanethiol
Isoleucine Valine Phenylalanine Tyrosine Tryptophan Methionine
2-Methylbutanol 2-Methylpropanol
usually ot-ketoglutaric acid. Tamman e t a l . (2000) observed that nine lactobacilli isolated from a 3-yearold Cheddar were able to transaminate amino acids only if exogenous ot-ketoglutarate was supplied. It
Thiols/Misc.
appears that the activity of transaminases may also rely on the presence of other compounds. For example, Amarita e t a l . (2001) found that the methionine aminotransferase activity of 29 L a c t o b a c i l l u s strains
Aromatic amino acids Branched-chain amino acids Methionine
Aminotransferase
ELIMINATION
Met
Hydroxyacid
0~"
Benzaldehyde (Phe) Hydroxybenzaldehyde (Tyr) Indole-3-acetate (Trp)
Alcohol
MGL CBL CGL
/ u~-,,,%~.~..~- . . . . "-'.,~.....
Aldehyde =
Tyr
TPL
Tr )
I
TIL
AcyI-CoA
DH
" . , .
Alcohol
,
Methanethiol
Carboxylic acid "
Indole Phenol
Methyl thioester
Ester
Dimethyldisulphide Dimethyltrisulphide Cresol, skatole (Tyr, Trp) Figure 1 Schematic diagram of pathways for amino acid catabolism found in different microorganisms and some chemical reactions (dotted lines) occurring in cheese during ripening (modified from Yvon and Rijen, 2001). AT: aminotransferase, HA-DH: hydroxyacid dehydrogenase, oL-KADH: oL-keto acid dehydrogenase, oL-KADC: oL-keto acid decarboxylase, aldDH: aldehyde dehydrogenase, alcohol DH: alcohol dehydrogenase, MGL: methionine-.y-lyase, CGL: cystathionine--,/-lyse, CBL: cystationine-13-1yase, TPL: tyrosinephenol lyase, TIL: tryptophan-indole lyase.
Catabolism of Amino Acids in Cheese during Ripening
increased in the presence of glucose in the reaction mixture. Williams et al. (2002) studied the effects of rate and stage of growth, amino acid type and the presence of glucose on aminotransf[erase activity in strains of NSLAB from Cheddar cheese. Aminotransf[erase activity off the cell-free extract of two strains of Lb. paracasei was maximal at, or close to, pH 6.0 and 30 ~ but activity was detected under conditions similar to those in cheese during ripening. The effect off NaC1 on activity at pH 8 and 30 ~ differed with leucine or phenylalanine as substrate. Glutamic acid was formed most efficiently from ot-ketoglutarate using aromatic, branched-chain or sulphur-containing amino acids as substrate. Martinez-Cuesta et al. (2002) found that aminotransf[erase activity was increased in cells treated with a bacteriocin, lacticin 3147, to increase the permeability of the cell wall to amino acids. Aminotransf[erases have been studied in several cheese-related bacteria. An aminotransf[erase from Lc. lactis subsp, cremoris NCDO763, with a pH optimum of 6.5-8, was found to be PLP-dependent but metal ionindependent (Yvon et al., 1997). The enzyme acted on leucine, methionine and aromatic amino acids. One off the two substrates off the reaction it catalyses (amino acid or keto acid) must have a hydrophobic group attached to the [3-carbon off the compound. This enzyme was responsible for the biosynthesis of phenylalanine and tyrosine, but it also had a catabolic role when high concentrations off aromatic amino acids were present. Yvon et al. (1997) suggested that the enzyme is involved in the catabolism off leucine, methionine, phenylalanine, tyrosine and tryptophan, and also in the synthesis off the aromatic amino acids. Engels (1997) purified two aminotransf[erases from Lc. lactis subsp, cremoris B78. The enzymes were active on methionine, leucine, isoleucine, valine and phenylalanine. Both transaminases were dimeric proteins, had a high temperature optimum and an alkaline pH optimum. The enzymes were found to catalyse the conversion of methionine to L-methyhhio-2-ketobutyric acid. Two aminotransf[erases were also isolated from Lc. lactis subsp, lactis $3 by Gao and Steele (1998). The enzymes were PLP-dependent methionine aminotransf[erases. Both also had activity on aromatic amino acids and leucine. The aminotransf[erases were active under cheese-ripening conditions and produced compounds that may be precursors of off-flavour compounds in cheese, e.g., p-hydroxyphenylpyruvic acid produced from tyrosine can breakdown chemically to p-cresol. Dias and Weimer (1998a) detected high levels of methionine aminotransf[erase activity in several strains off lactococci but found only slight activity in two strains of B. linens. This is in contrast to earlier findings of Lee et al. (1985) who observed aromatic aminotransf[erases
437
in 23 coryneforms isolated from cheese. These authors reported that the aminotransferase(s) of B. linens were inducible while the enzymes of other coryneform strains tested were constitutive. The aromatic aminotransferase of B. linens 47 was studied by Lee and Desmazeaud (1985). The inducible enzyme was responsible for removal of the amino group of amino acids [or their use as sole nitrogen sources by the bacterium; o~-ketoglutarate was preferred as the amino group acceptot over pyruvate. An aminotransferase from Lc. lactis LM0230 which acts on branched-chain amino acids was cloned and sequenced by Atiles et al. (2000). The enzyme was active on methionine and phenylalanine in addition to the three branched-chain amino acids. Sequence analysis showed high homology with other branched-chain aminotransferases. Transamination by Lb. helveticus was studied by Klein et al. (2001). The bacterium was incubated with a mixture of amino acids (phenylalanine, tyrosine, methionine, leucine, valine, isoleucine) in ratios similar to those in Emmental cheese. The authors concluded that transamination was the first and main step in the conversion of amino acids by Lb. helveticus, with phenylalanine and tyrosine being converted most efficiently. The transamination of branchedchain amino acids by Lb. paracasei has also been studied (Hansen et al., 2001). It is clear that many bacteria found in cheese are capable of amino acid transamination but what role do these reactions play in cheese flavour development? It has been suggested that the rate-limiting step in flavour development is the conversion of free amino acids to aroma compounds. Yvon et al. (1998) added ot-ketoglutarate to St Paulin-type cheese in an effort to accelerate flavour development. The level off glutamate in cheese increased when ot-ketoglutarate was added but the level off some amino acids, including leucine, phenylalanine, tyrosine and valine, decreased. The addition of ot-ketoglutarate increased the degradation of methionine. However, after six weeks of ripening, large amounts of the resulting ot-keto acids, produced by transamination, remained in the cheese. Thus, these authors suggested that aminotransf[erase activity was not limiting, but that subsequent steps in the f[ormation of aroma compounds may have been limiting. Banks et al. (2001) supplemented Cheddar cheese with ot-ketoglutarate to enhance amino acid catabolism during ripening, oL-Ketoglutarate (20 g kg -1 milled curd) was added as a mixture with the salt. It was observed that the levels off leucine, phenylalanine, valine, threonine, methionine, alanine and isoleucine decreased on addition of ot-ketoglutarate. Sensory analysis showed that the addition of ot-ketoglutarate caused statistically significant changes in aroma intensity, creamy character and fruity notes. In fact, the
438
Catabolism of Amino Acids in Cheese during Ripening
aroma intensity of the 12-week-old cheese supplemented with ot-ketoglutarate was equal to that of a 24-week-old control Cheddar. Supplementation of Cheddar cheese with ot-ketoglutarate caused statistically significant effects on the production of certain volatile flavour compounds. Shakeel-Ur-Rehman and Fox (2002) supplemented Cheddar cheese with ot-ketoglutarate, pyruvate or PLP and reported a beneficial effect of added ot-ketoglutarate and pyruvate on flavour. Cheese supplemented with 1 g ot-ketoglutaric acid per kg of curd was considered to be as mature at 60 days as 90-day-old commercial Cheddar cheese. Addition of ot-ketoglutaric acid to cheese also promoted syneresis during pressing of the curd but did not affect plasmin or chymosin activity. In an effort to overcome limitations in the concentration of ot-ketoglutarate, Rijnen et al. (2000) investigated the possibility of using lactic acid bacteria capable of producing ot-ketoglutarate from glutamate in cheese, which can be converted to ot-ketoglutarate by glutamate dehydrogenase. The authors cloned the glutamate dehydrogenase gene (gdh) from Peptostreptococcus asaccharolyticus into Lc. lactis, and followed the conversion of amino acids to aroma compounds in the Ch-Easy model. It was observed that the gdh + strain produced ot-ketoglutarate from glutamate under cheese-ripening conditions and this allowed transamination of aromatic amino acids and branched-chain amino acids. The gdh + strain could be used as an alternative to the addition of exogenous ot-ketoglutarate to cheese to increase amino acid catabolism. A number of groups are now working on the genetics of amino acid-catabolising enzymes with a view to developing strategies for controlling the development of cheese flavour. Rijnen et al. (1999a) characterised the gene (araT) encoding the lactococcal aromatic aminotransferase. It was shown that araT is transcribed as a single gene. This enzyme is essential for the catabolism of aromatic amino acids and is involved in the conversion of leucine and methionine. It also plays a role in the biosynthesis of phenylalanine and tyrosine. Subsequently, Rijnen et al. (1999b) inactivated the lactococcal araT gene and studied the possibility of controlling flavour development by directing the degradation of amino acids by starter bacteria in cheese. These authors followed the production of aroma compounds and the degradation of amino acids in St Paulin-type cheese using radiolabelled amino acids and quantified volatile products by GC-MS. c~-Ketoglutarate was added to half the cheeses to enhance transamination. It was observed that inactivation of the lactococcal araT gene did not significantly affect the levels of volatile compounds produced from branched-chain amino acids or methionine
during ripening, perhaps because lactococci produce more than one aminotransferase with overlapping specificities (Engels, 1997; Gao and Steele, 1998). Cheeses containing added ot-ketoglutarate were described as 'more odorous' than the control. In cheeses supplemented with ot-ketoglutarate and made with an araTnegative starter, the formation of aroma compounds was lower than in control cheeses. Since many of the aroma compounds produced from aromatic amino acids, such as phenethanol, phenol and indole, contribute to offflavours in cheese, the use of araT-negative starter strains may prevent the development of certain offflavours in cheese. Amino acids react chemically with carbonyl compounds to form azomethines; if the carbonyl compound has an electron-withdrawing group adjacent to the carbonyl group (e.g., a dicarbonyl), then transamination and decarboxylation occur. This process is known as the Strecker degradation, through which aldehydes are formed (Belitz and Grosch, 1987).
--C--O I --C----O
4-' 9 --C--N--CHR--U.
+ H2N--CHR--COOH
Dicarbonyl
I~J --CU
Amino acid
~NO__H
002 H20 H mC m NH2
I
--C=O
~
+
O'--CHa
~
- - C - - N "-- CHR
II
--C--OH
Strecker aldehyde
Transamination via the Strecker degradation forms the same volatile aldehydes as formed by enzymecatalysed transamination, although by a different reaction mechanism. Strecker degradation plays an important role in flavour development of many foods since dicarbonyls are produced by the Maillard reaction. There have been reports of Strecker aldehydes in cheese (e.g., Dunn and Lindsay, 1985) but the extent to which this chemical reaction occurs in cheese is unclear since the same aldehydes are produced from a pathway initiated by the action of aminotransferases. The keto acids produced as a result of transamination of methionine, branched-chain amino acids or aromatic amino acids serve as precursors of aroma compounds (Yvon and Rijnen, 2001), which can be formed by enzymatic or chemical reactions. Four main
Catabolism of Amino Acids in Cheese during Ripening 439 degradation pathways for ot-keto acids are used by cheese-related microorganisms (Fig. 1): 9 ot-Keto acids may be reduced to the corresponding hydroxyacid by the action of 2-hydroxyacid dehydrogenases, which have been found in LAB (see Yvon and Rijnen, 2001). Although hydroxyacids are not important flavour compounds, their production reduces the levels of ot-keto acids available for other reactions. ot-Keto acids derived from branched-chain amino acids, aromatic amino acids and methionine may also be decarboxylated to the corresponding aldehydes, although this pathway is not important in most LAB. Aldehydes produced by this pathway may be oxidised to the corresponding carboxylic acid by aldehyde dehydrogenases or reduced to alcohols by alcohol dehydrogenases. ot-Keto acids produced by transamination may also be oxidatively decarboxylated to carboxylic acids by the action of ot-keto acid dehydrogenases, generating acyl-CoAs which are hydrolysed, releasing carboxylic acids (Yvon and Rijnen, 2001). However, this pathway does not appear to be common in microorganisms found in cheese. 9 ec-Keto acids may degrade chemically. Phenyl pyruvate and hydroxyphenyl pyruvate (produced from Phe and Tyr, respectively) may be converted to benzaldehyde and hydroxybenzaldehyde, respectively. Likewise, indole-3-pyruvate, which is produced from Trp, is quite unstable and degrades to indole acetic acid, indole-3-aldehyde and skatole. Non-enzymatic degradation of ot-heto-y-methylthio butyrate, produced from methionine, to methanethiol has also been reported (Gao et al., 1998). 9
9
Production of Volatile Sulphur Compounds by Amino Acid Catabolism Volatile sulphur compounds are found in most cheeses and are important components of flavour (Fox and McSweeney, 1996). Since methionine is present in the caseins at a higher concentration than cysteine, sulphur compounds in cheese presumably originate principally from methionine. The pathways for the production of various flavour compounds from methionine are shown in Fig. 2. Weimer et al. (1999) discussed some of the sulphur compounds in cheese and their importance to flavour. They reported that dimethyldisulphide does not contribute to flavour, while dimethyltrisulphide is a flavour compound. The occurrence of these compounds is related to methanethiol content and the low redox potential of the cheese. Dimethylsulphide and dimethyldisul-
phide can be produced from methanethiol but it is unclear how dimethyltrisulphide is produced in cheese (McSweeney and Sousa, 2000). Methional ([3-methyl mercaptopropionaldehyde), which is considered to be part of Cheddar cheese aroma (Weimer et al., 1999), can be degraded to methanethiol either spontaneously or by decarboxylation. Methanethiol is a volatile compound with a 'putrid faecal-like' aroma at high concentrations. However, at low concentrations, it contributes to the characteristic aroma of cheese (Weimer et al., 1999). It is also a precursor of other volatile sulphur compounds which contribute to the garlic aroma of smear-ripened cheese (Hemme et al., 1982). Starter cultures, flavour adjunct bacteria and non-starter bacteria may form methanethiol in cheese from methionine (Fig. 2). Researchers continue to study the production of methanethiol and its role in cheese flavour. Dias and Weimer (1999) investigated the production of volatile sulphur compounds in Cheddar cheese slurries. It was observed that the production of total volatile sulphur compounds was four times higher in slurries acidified by Lc. lactis subsp, cremoris $3 than in those chemically acidified with gluconic acid-g-lactone. Addition of brevibacteria and methionine-y-lyase (MyL) to the slurries increased the production of volatile sulphur compounds. They concluded that adjunct cultures and enzymes are required to produce volatile sulphur compounds above their flavour threshold. Molimard and Spinnler (1996) also believed that coryneform bacteria, especially B. linens, are key agents in the production of sulphur compounds in surface mould-ripened cheese. The production of sulphur compounds by LAB was investigated by Seefeldt and Weimer (2000). Lactococci are auxotrophic for methionine while lactobacilli are auxotrophic for both cysteine and methionine. In addition, it was observed that lactococci possess greater cystathionine lyase activity than lactobacilli. The cell-free extract of both lactococci and lactobacilli was able to produce volatile sulphur compounds, including methanethiol, dimethyldisulphide and dimethyltrisulphide. Five bacteria from cheese were analysed by Bonnarme et al. (2001a) for enzymes involved in the production of methanethiol. B. linens had the highest demethiolating activity. S. equorum and M. luteus showed demethiolating activity but they produced only trace amounts of volatile sulphur compounds; all bacteria examined formed methanethiol. The ability of 7 Lb. casei and 22 Lb. plantarum strains to produce flavour compounds from methionine was investigated by Amarita et al. (2001). Several enzyme activities were studied: methionine aminotransferase, hydroxyacid dehydrogenase, methionine
440
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Catabolism of Amino Acids in Cheese during Ripening
lyase and amino acid decarboxylase. About 25% of the strains were capable of transaminating methionine to ot-keto-y-methylthiobutyrate. Dehydrogenase activity was observed to increase in the presence of glucose, the influence of which may have been due to the supply of energy by the proton motive force generated by lactate efflux. No methionine lyase or amino acid decarboxylase activities were detected. Amarita etal. (2002) studied the production of methional in a cheese slurry system by Lc. lactis IFPL730. The authors reported a decrease in the concentration of methional, and a corresponding increase in 3-methylthiopropanol. It was concluded that the conversion of methional to other volatile compounds, including 3-methylthiopropanol, contributes to cheese flavour. Catabolism of methionine by five cheese-ripening bacteria and five yeasts was investigated by Bonnarme et al. (2001b). For both yeasts and bacteria, dimethyldisulphide was the major sulphur compound produced. In addition, the microorganisms produced different amounts of methanethiol. G. candidum produced a low concentration of S-methyl thioacetate, and was the only yeast with the ability to produce S-methyl thioesters. Berger etal. (1999) observed that G. candidum, a fungus found commonly on the surface of smearripened cheese, can produce sulphur-containing flavour compounds; dimethyldisulphide was the main sulphur compound produced by the ten strains studied. The majority of the strains also produced methanethiol and dimethyltrisulphide. Four strains which produced sulphides could generate S-methyl thioesters. The authors concluded that G. candidum may play an important role in development of flavour in smear-ripened cheese. The pathways for the production of flavourful sulphides by G. candidum were subsequently studied by Demarigny et al. (2000). Two pathways were used to produce sulphides from methionine. The first pathway, which operated at high methionine concentrations, was initiated by the action of y-demethiolase and resulted in the production of methanethiol, dimethyldisulphide and dimethyltrisulphide. The other pathway operated at low methionine concentrations and resulted in the production of dimethylsulphide. Bonnarme et al. (2001a) found that G. candidum cultures produced three S-methylthioesthers (S-methylthioacetate, S-methylthiopropionate and S-methylthiobutyrate) from methionine. It was also found that G. candidum metabolised k-methionine using different pathways; L-methionine demethiolating activity was constitutive while L-methionine aminotransferase and 4-methylthio-2-ketobutyrate demethiolating activities were not.
441
Methanethiol can be formed from methionine by a number of enzyme-catalysed reactions. Methanethiolproducing capacity (MTPC) was found to varying degrees in LAB and brevibacteria (Weimer et al., 1997). B. linens BL2 had the highest MTPC. Cell-free extracts had no measurable MTPC under cheeselike conditions. Dias and Weimer (1998a) examined the conversion of methionine to thiols by lactococci, lactobacilli and brevibacteria. Again, MTPC was not detected under cheese-ripening conditions (pH 5.2, 5% NaC1), using methionine as a substrate, but had activity on cystathionine was found under such conditions. The authors found that lactococci and lactobacilli catabolise methionine using cystathionine [3- and y-lyases while Brevibacterium spp. use an MyL. According to the authors, the cystathionine lyases from lysed lactococci make an insignificant contribution to the production of volatile sulphur compounds from methionine in cheese during ripening. Amarita et al. (2001) did not detect methionine lyase activity in 29 strains of Lactobacillus. No cystathionine lyase or methionine lyase activity was detected in the strains of Lactococcus investigated by Gao et al. (1998). Methionine aminotransferase activity was found to be responsible for initiating methionine catabolism. The production of methanethiol from methionine by B. linens CNRZ918 was studied by Ferchichi etal. (1985), who found greatest methanethiol production at pH 8 in the case of both the rod and coccus forms of this microorganism. MTPC decreased as the proportion of coccoid forms increased. Amarita et al. (2002) used a model system to study the ability of Lc. lactis to produce methional and other compounds from methionine. Slurries containing resting cells and the intracellular fraction from Lc. lactis IFPL730 showed the highest production of methional at the outset of incubation, with a concomitant increase in the production of 3-methylthiopropanol. Sensory analysis of slurries indicated the characteristic aroma of methional (cooked potato-like) in samples containing ot-keto-y-methylthiobutyrate and the intracellular fraction from Lc. lactis IFPL730. On extended incubation, the intensity of methional aroma decreased but samples developed a cheese-like aroma. S-methylthioesters are important flavour compounds in surface-ripened cheese. Their specific flavour depends on their chain length and configuration (Weimer et al., 1999). B. linens GC71 is capable of esterifying acetic, propionic and methyl branched-chain acids with methanethiol to produce thioesters (Lamberet etal., 1997a). Lamberet et al. (1997b) compared the S-methyl thioester-synthesising ability of some cheese-related
442
Catabolism of Amino Acids in Cheese during Ripening
bacteria, including coryneforms, Micrococcaceae, Lactococcus and Leuconostoc. The strains of B. linens tested had a tendency to produce branched-chain thioesters, which was not observed in other COlTneforms tested. Lactococci were shown to have poor ability to synthesise these compounds. S-methyl thioacetate was produced by G. candidum, Saccharomyces cerevisiae, Debaryornyces hansenii, Kluyverornyces lactis and Yarrowia lipolytica (Arfi et al., 2002). These authors also reported that L-methionine aminotransferase, L-methionine and 4-methyhhio2-oxobutyrate decarboxylase activities were present in all the yeasts studied. Lyases involved in the catabolism of methionine
Methionine-y-lyase or methioninase (EC 4.4.1.11) is a PLP-dependent enzyme which catalyses the conversion of methionine to ot-ketobutyrate, methanethiol and ammonia (Soda et al., 1983; Tanaka et al., 1985). The enzyme plays an important role in the bacterial metabolism of methionine.
O
II
H2N--CH--C--OH
O
II
I OH 2
I OH2
I S
I
O--C--C--OH
=
I
+ H3C - - SH + NH 3
OH 2
I OH 3
OH 3 Methionine
ot-Ketobutyrate
Methanethiol Ammonia
Methionine-y-lyase have been isolated from several bacteria, including Pseudornonas ovalis (Tanaka et al., 1976), Aeromonas sp., Ps. putida (Nakayama etal., 1984), B. linens CNRZgl8 (Ferchichi etal., 1985), B. linens NCDO739 (Collin and Law, 1989), Trichornonas vaginalis (Lockwood and Coombs, 1991) and B. linens BL2 (Dias and Weimer, 1998b). The optimum pH for activity is generally 7.5-8. The enzyme is PLP-dependent and usually composed of four identical subunits, apart from the enzyme of Ps. ovalis which is composed of two non-identical subunits (Tanaka et al., 1976). The MyL of Ps. ovalis can also degrade various 13- and y-substituted amino acids in addition to L-methionine, and can perform both c~,y and ot,[3 elimination reactions. The enzymes from Ps. ovalis and Aeromonas spp. can act on derivatives of L-methionine and L-cysteine in addition to L-methionine (Tanaka et al., 1976; Nakayama et al., 1984). The MyL of Ps. putida ICR3460 performed or,yand y-replacement reactions of L-homocysteine and its
S-substituted derivatives (Nakayama et al., 1984). The enzyme from Trichomonas vaginalis catabolised homocysteine, ethionine and methionine by or,y-elimination (Lockwood and Coombs, 1991). The MyL of B. linens BL2 is active under cheeseripening conditions (Dias and Weimer, 1998b). Unlike the release of intracellular proteolytic and lipolytic enzymes, the release of which has been shown to be necessary for their activity in cheese, the influence of amino acid catabolic enzymes on ripening may be linked to the ability of the cells to resist lysis and remain metabolically active during ripening. However, the role of enzyme release by cell lysis in the production of flavour compounds by amino acid catabolism is unclear and requires more study. The reactions catalysed by cystathionine-~-lyase (C[3L) and cystathionine-y-lyase (CyL) are shown in Fig. 3. C[3L (EC 4.4.1.8) catalyses the conversion of cystathionine to homocysteine, pyruvate and ammonia while the products of CyL are cysteine, ammonia and e~-ketobutyrate (Weimer et al., 1999). Aubel et al. (2002) studied the genetics of the C[3L of Lb. delbrueckii subsp. bulgaricus N CDO 1489 and concluded that its physiological role is probably in the biosynthesis of methionine. Dwivedi et al. (1982) isolated a C[3L from E. coli which had a pH optimum of 9-10 and contained 1 mol of PLP per enzyme subunit. The gene coding for cystathionine lyase (mete) of E. coli was cloned by Laber et al. (1996) who also constructed a strain of E. coli which over-produced this enzyme. The C[3L of Lc. lactis subsp, cremoris B78 is reported to be a tetramer of identical ---40 kDa subunits (Alting et al., 1995). The enzyme catalyses the ot,[3-elimination reaction but is able to catalyse the or,y-reaction also. It was active at the pH and salt concentration of normal Gouda cheese. Unlike the MyL of B. linens BL2, lysis of cells was required for full activity. Fernandez et al. (2000) cloned and characterised the rnetC gene encoding C[3L from Lc. lactis strains B78 and MG1363. The proteins encoded by the genes were similar and had high homology to other PLP-dependent enzymes. Enzyme activities were determined in strains which overproduced C[3L activity or in which this activity was deleted. Results showed that the product of the gene rnetC is essential for the degradation of cystathionine but that at least one other lyase contributes to methionine degradation by an ot,y elimination. Dobric et al. (2000) reported the nucleotide sequence of the gene for the C[3/yL of Lc. lactis subsp, cremoris MG1363. The enzyme was unique as it could perform either ot,[3- and or,yelimination reactions on the same substrate. Yamagata et al. (1993) studied the CYS3 gene encoding the CyL of Saccharomyces cerevisae. No detectable
Catabolism of Amino Acids in Cheese during Ripening
O H2N
Cystathionine-~-Iyase O
II
H2N ~ C H
--C ~
>
OH
I
H2N
iCH m C ~ O H II 0
O
II CH m C ~ O H
"3L"
O--C ~C
I
I
i H2
i H2
SH
OH3
Cysteine
S
443
II ~ O H
+
a-Keto butryate
NH3
Ammonia
O H2N
Cystathionine-/3-1yase
,
>
Cystathionine
II
OH - - C ~
OH
-i-
C~OH
H3C
+
NH3
I 0
I CH2 I CH2 I
SH
Homocysteine
Pyruvate
Ammonia
Figure 3 The reactions catalysed by cystathionine 13-1yaseand cystathionine ,y-lyase.
homology was found between the CYS3 of S. cerevisae and the cysE gene of E. coli. HzS and methanethiol were the only volatile sulphur compounds found during the degradation of L-cysteine and L-methionine by C~/L of Lc. lactis subsp, cremoris SK11 (Bruinenberg et al., 1997). Lb. fermentum DT41 was isolated from the starter for traditional Parmesan cheese. A PLP-dependent C~/L was isolated from this strain by Smacchi and Gobbetti (1998). It was composed of four identical --~35 kDa subunits and was optimally active at 37 ~ and pH 8. The enzyme was reported to retain activity under cheese-ripening conditions. A 160 kDa homotetrameric C~/L was purified from Lb. reuteri DSM20016 by de Angelis et al. (2002). The enzyme was optimally active at pH 8 and 35 ~ and catalysed the conversion of a range of amino acids, including methionine. This organism, together with other lactobacilli, was used as an adjunct in the manufacture of Canestrato Pugilesetype cheese and cheeses containing an adjunct composed of Lb. fermentum DT41, and Lb. reuteri DSM 20016 had the highest levels of methanethiol, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide.
Catabolism of Aromatic Amino Acids Tryptophan
Pathways for the catabolism of tryptophan are shown in Fig. 4. Gummalla and Broadbent (1999) studied tryptophan catabolism by lactobacilli used as adjunct
cheese cultures to investigate the contribution of lactobacilli to off-flavour development. The main mechanism for tryptophan catabolism by Lb. casei involved its conversion to indole lactic acid (ILA) by a series of transamination and dehydrogenation reactions with indole-3-pyruvic acid as the sole intermediate. The results suggest that non-starter and adjunct lactobacilli may have important roles in secondary reactions involving indole-3-pyruvic acid and other aromatic metabolites. Tryptophan catabolism by B. linens BL2, under both optimum (pH 6.5, 25 ~ with agitation) and cheeselike (pH 5.2, 15 ~ 4% NaC1) conditions, was studied by Ummadi and Weimer (2001). At optimum temperature and pH, B. linens BL2 could degrade tryptophan by various routes simultaneously but under cheeselike conditions, the bacterium did not catabolise it. Cells grown under either conditions did not show tryptophanase, tryptophan decarboxylase or tryptophan 2-monooxygenase activities. Gao et al. (1997) investigated the catabolism of aromatic amino acids in lactococci. They reported that the first step in the catabolism of tryptophan by eight lactococcal strains is due to aminotransferase activity. Aminotransferase activity on L-tyrosine and L-phenylalanine was also found. Tyrosine
Tyrosine is a precursor of several compounds in cheese - tyramine formed by decarboxylation, p-cresol and phenol formed by an atypical Strecker degradation and p-hydroxyphenyl pyruvate formed by aminotransferase
444
Catabolism of Amino Acids in Cheese during Ripening
NH3
H
Deaminase
CH2 / CH2
/
002
Docarboxylaso CH2 \ ~NH,,_~ ~ C ~CH HO \\ 0
Indole-3-propionate
HO~(~\
o
H
CH2 \ Tryptamine OH2 \NH2
Tryptophan o~-Keto acid Aminotransferase
Amino acid H
CHe \ HOt ~"~'C~o O
H
OH2 \ _~CH HOIL<\ \OH 0
Indole-3-pyruvate | !
t
H
Indole-3-1actate
OH2
\
HoIC~o
Indole-3-acetate ! i
t
H
OH3
Skatole (3- met hyl- 1H-i ndole)
Figure 4 Pathways for the catabolism of tryptophan.
activity (McSweeney and Sousa, 2000), as shown in Fig. 5. Tyramine, a biogenic amine, can cause monamine intoxication (see 'Toxins in Cheese', Volume 1). Phenylalanine
Phenylmethanol, phenylethanol, phenylpropane, methylphenyl hydroxyacetate, phenylacetaldehyde,
phenylpyruvate and phenylethyl acetate are flavour compounds derived from phenylalanine and have been found in cheese or model systems (Adda et al., 1982; Dunn and Lindsay, 1985; Jollivet et al., 1992). Enzymes involved in the breakdown of pheylalanine are aminotransferases, I_-amino acid oxidases, L-pheylalanine ammonia lyases, L-aromatic amino acid decarboxylases and phenylalanine dehydrogenases. Phenylpyruvate is
Catabolism of Amino Acids in Cheese during Ripening
OH
-~
NH3
445
CO 2
OH
OH
Decarboxylase
Deaminase OH2 I H2N ~ C H - - C - - O H
CHe
I
OH2 I CH2
ii
C--OH II O
I
O
NH2
Tyrosine
Tyramine
p-Hydroxy phenylpropionate ot-Keto acid
Aminotransferase Amino acid OH
I
CHe I
O----C--C--OH II 0
p-Nydroxy phenylpyruvate OH A
t
f
OH2 I HO--CH-C--OH II O
t
~ OH
<>
OH
I
p-Hydroxy phenyl lactate
II
CH2
I C--OH
O
o.
<)
p-Hydroxy benzaldehyde H3C
II O
p-Hydroxy phenyl acetate
p-Cresol
Figure 5 Pathways for the catabolism of tyrosine.
formed by the action of an aminotransferase and it can be degraded to phenethanol and benzaldehyde (Fig. 6). During the first week of Camembert ripening, actively growing yeast cells produced phenethyl alcohol from L-phenylalanine (Lee and Richard, 1984). Several compounds with a phenyl group were also identified when the ability of B. linens to produce volatile compounds in liquid cultures was examined (Jollivet etal., 1992). The compounds included phenylmethanol, phenylethanol, phenylpropane and methylphenylhydroxyacetate. The formation of benzaldehyde from phenylalanine by Lb. plantarum is initiated by an aminotransferase
(Nierop Groot and de Bont, 1998). Phenylpyruvic acid was an intermediate in the reaction and its conversion to benzaldehyde was catalysed by Mn (II) which was present at high levels in these cells (Nierop Groot and de Bont, 1999) (Fig. 7). The low pH, low oxygen concentration and low temperature of cheese during ripening would not favour this chemical reaction. However, the authors pointed out that the reaction may still make a significant contribution because of the long ripening time of certain cheeses. Klein etal. (2001) also attributed the production of benzaldehyde from indole pyruvate, which was formed by transamination of phenylalanine by Lb. helveticus, to a chemical reaction.
446
Catabolism of Amino Acids in Cheese during Ripening
002
NH3
Docarboxylaso
Deaminase CH2 I OH2
CH2 I H2N - - CH - - C - - OH II O
I
C--OH II 0
OH2 I CH2
I NH2
Phenylalanine
Phenylethylamine
Phenylpropionate
(~
oeKetoacid
Aminotransferase
Amino acid
CH2 I
O----C--C--OH II
j/
O Phenylpyruvate
t
t s
t s
t
A
s
CH2 I HO--CH--C--OH II O Phenyl lactate OH2
C--H II 0
I OH2
Benzaldehyde
I
C--OH II O
OH2
I
OH
Phenylethanol Phenylacetate Figure 6 Pathwaysfor the catabolism of phenylalanine.
An inducible aromatic amino acid aminotransferase was isolated from B. linens 47 by Lee and Desmazeaud (1985), who detected several metabolites of aromatic amino acid catabolism, including phenylpyruvate, phenylacetate, 4-hydroxyphenyl acetate, indolepyruvate and indoleacetate, in the growth medium using HPLC. Hayashi et al. (1993) characterised an aromatic amino acid aminotransferase from E. coli. The haloenzyme contained 1 mol PLP per mol and could catalyse the transamination of a variety of neutral amino acids, in addition to aromatic amino acids. Two aromatic amino acid aminotransferases have also been purified from Lc. lactis subsp, lactis $3 and characterised (Gao and Steele, 1998). Both were active
under cheese-ripening conditions. The authors observed that p-hydroxyphenylacetic acid (pHPA) could be produced from tyrosine; pHPA can degrade chemically to p-cresol. The enzymes could also produce indole-3-acetic acid from tryptophan which could be converted to skatole. It was concluded that further research is required to confirm the role of aromatic amino acids in the development of off-flavours in cheese. An aminotransferase from Lc. lactis NCDO763 catalysed the transamination of L-amino acids only (Yvon et al., 1997). Using ot-ketoglutarate as an amino group acceptor, the resting cells of this organism produced phenylpyruvate, phenylacetate and phenylactate from phenylalanine. The authors suggested
Catabolism of Amino Acids in Cheese during Ripening
0
II
H2N~CHmC ~
I
CH2
o
0 OH
o
II
c
HO
OH
c
CH2 Metal ion/alkaline pH
Phenylalanine
C
II
C
OH
II CH
I
Aminotransferase
447
I
Enol tautomer
Phenylpyruvate
Chemically 02
COOH
I
CHO
OH2
Benzaldehyde
COOH
I
HC
OH
Phenylacetic acid Mandelic acid
COOH
I
C~-----O
Phenylglyoxylic acid
Figure 7 Proposed mechanism for benzaldehyde production by both enzymatic and chemical steps (modified from Nierop Groot and De Bont, 1998).
that the aminotransferase could have an important role in flavour development during ripening as it transaminates amino acids (leucine, phenylalanine, tyrosine, tryptophan, methionine) which are precursors of aroma compounds. Gummalla and Broadbent (2001) studied the catabolism of tyrosine and phenylalanine by Lactobacillus adjuncts. It was observed that in the two Lb. casei and two Lb. helveticus strains examined, the specific activity of phenylalanine aminotransferase was higher than that of tyrosine aminotransferase. The strains were incubated under conditions similar to those in cheese during ripening to assess catabolism. No decarboxylase activity was detected during 20 days of incubation but aminotransferase and dehydro-
genase activities were observed. Micellar electokinetic capillary chromatography was used to show that the four lactobacilli catabolised tyrosine to produce p-hydroxyphenyl lactic acid and p-hydroxyphenyl pyruvic acid under conditions similar to those in ripening cheese. Phenyl lactic acid, phenyl acetic acid and benzoic acid were produced from phenylalanine under conditions similar to those in cheese.
C a t a b o l i s m of B r a n c h e d - C h a i n A m i n o Acids Branched-chain amino acids are degraded by aminotransferases to ot-keto acids (Fig. 8). The aminotransferase of Lc. lactis subsp, cremoris N CDO 763 was most
448
Catabolism of Amino Acids in Cheese during Ripening
O
O
II
-CH--C--
H2N
I I CH--CH3 I CH3
CHe
Leucine
O H2N - -
CH--C
II
OH
I I CHe I CH3
CH--CH3
Isoleucine
O H2N
O---C
OH
II
CH--C--OH
I I CH3
CH--CH3
Valine
A M I N O T R A N S F E R A S E
>
II
C
OH
I I CH--CH 3 I CH3 CHe
o~-Ketoisocaproate
O
II
O--C--C--OH
>
I I CHe I CH3
CH--CH 3
o~-Keto-13-methylvalerate
O O-~-C
>
II
C
OH
I CH--CH 3 I CH3 o~-Keto-isovalerate
Figure 8 Transamination of the branched-chain amino acids to their corresponding c~-keto acids.
active on leucine, although it was also, but less, active on the aromatic amino acids (Yvon et al., 1997), while that of Lc. lactis subsp, cremoris B78 catalysed the transamination of valine, isoleucine and leucine (Engels, 1997). A branched-chain aminotransferase from Lc. lactis subsp, cremoris NCDO 763 was characterised by Yvon et al. (2000). The enzyme catalysed the transamination of the three branched-chain amino acids and was active under cheese-ripening conditions, although it had pH and temperature optima of 7.5 and 35-40 ~ respectively. Since the enzyme has a role in the degradation of isoleucine and valine, as well as in the transamination of leucine and methionine and was active under conditions similar to those found in cheese during ripening, the authors concluded that the enzyme was involved in flavour development. The branched-chain aminotransferase of Lc. lactis LM0230 has been cloned and sequenced (Atiles et al., 2000). The enzyme has broad specificity, being active on isoleucine, leucine, valine, methionine and phenylalanine.
Lb. paracasei subsp, paracasei LCO 1 produces at least one aminotransferase, capable of transaminating branched-chain amino acids, which was most active on isoleucine and leucine (Hansen et al., 2001). Responsesurface methodology showed that leucine concentration had a negligible effect on aminotransferase activity, while too high a concentration of ot-ketoglutarate could inhibit the enzyme. Ayad et al. (2001a) studied the effects of combining selected lactococci on flavour formation in milk. A chocolate-like flavour was produced by a combination of Lc. lactis subsp, cremoris NIZO131157 and Lc. lactis subsp, cremoris SKl l0. The authors speculated that this flavour was due to branched-chain aldehydes produced from branched-chain amino acids. Subsequently, Ayad etal. (2001b) studied the flavour-generating ability of wild lactococci isolated from artinsanal and non-dairy sources (fermented raw goats', sheep's and cows' milk, as well as from soil, grass, silage and the udder) in milk and in a cheese model. The authors believed that these wild
Catabolism of Amino Acids in Cheese during Ripening
strains may be able to produce more flavour compounds in cheese than the industrial strains currently used in cheesemaking. The majority of wild strains produced different flavours from industrial strains. Methylated alcohols and methylated aldehydes, probably produced from branched-chain amino acids, were the main volatile compounds formed. It was concluded that wild strains could be used for the development of new cheeses or to alter the flavour of existing types of cheese. However, since the non-dairy wild strains had no proteolytic activity, they would be unable to grow in and acidify cheese milk and would have to be combined with industrial starters. The catabolism of leucine by propionic acid bacteria was investigated by Thierry et al. (2002). P.freudenreichii catabolised leucine to ot-ketoisocaproic acid, but only if ot-ketoglutarate was present. The bacterium also converted ot-ketoisocaproic acid to isovaleric acid via oxidative decarboxylation by ot-ketoacid dehydrogenase activity yielding an acyl-CoA derivative which was then converted to the acid. The authors noted that the catabolism of branched-chain amino acids by P. freudenreichii was different to the catabolism of branched-chain amino acids by lactococci.
Dearninases There are two types of deamination involving redox reactions (Hemme et al., 1982), differing according to the nature of hydrogen acceptor: 9 Dehydrogenases (EC 1.4.1) which utilise NAD + as the co-enzyme. The general reaction catalysed by these enzymes is: L-amino acid + H20 + NAD + ---* ot-keto acid + NH4 + + NADH These reactions can produce compounds such as pyruvic acid and ot-ketoglutaric acid from alanine and glutamic acid, respectively. 9 Oxidases which use oxygen as hydrogen acceptor. L-amino acid oxidases (EC 1.4.3.2) produce ot-keto acids according to the following reaction: L-amino acid + 02--+ ot-keto acid + NH3 + H202 L-amine oxidases (EC 1.4.3.6) according to the reaction:
form aldehydes
Amine + O2--+ aldehyde + NH3 + H202 Ammonia, a product of these deamination reactions, is an important constituent of the flavour of cheeses such as Camembert, Gruyere and Comte and
449
contributes to an increase in pH during ripening (McSweeney and Sousa, 2000). Microorganisms from the smear surface have deaminating ability, e.g., G. candidum (see Fox and Wallace, 1997), while B. linens produces large quantities of ammonia from serine, glutamine, asparagine and threonine. However, most strains of coryneform bacteria from smear cheese were found to have low deaminating activity except on serine, glutamine and asparagine (Hemme et al., 1982). Williams et al. (2001) studied the deaminating ability of LAB isolated from mature Cheddar. Deaminase activity was not widespread in the isolates but this may have been due to the insensitivity or lack of specificity of the assay method used.
Decarboxylases Decarboxylation is the conversion of an amino acid to the corresponding amine with the removal of CO2. Decarboxylases generally have an acid pH optimum (---pH 5.5) and usually require PLP as a coenzyme (Hemme et al., 1982). Amines generally have strong and often unpleasant aromas, as evident in certain smearripened cheese types (Fox and McSweeney, 1996). In addition, many amines (e.g., tyramine, histamine, tryptamine, putrescine, cadaverine and phenylethylamine) cause adverse physiological effects ('biogenic amines'; see 'Toxins in Cheese', Volume 1). The relative concentration of amines in cheese depends on the type of cheese and its microflora (McSweeney and Sousa, 2000). The relative concentration of some amines does not compare with that of the parent amino acid, which may be due to differences in the rates of conversion of amino acids (Adda et al., 1982). Most amines in cheese can be formed by decarboxylation, as is the case with the production of tyramine from tyrosine and histamine from histidine. However, the formation of secondary and tertiary amines cannot be explained readily (Fox and McSweeney, 1996). Joosten (1988) studied factors that affect the concentrations of biogenic amines formed in cheese. It was observed that in Gouda cheese, a higher pH, combined with a storage temperature of 21 ~ caused an increase in concentration of histamine, as did low saltin-moisture. Starter type and pasteurisation of milk did not appear to affect the formation of histamine. The role of non-starter bacteria in the formation of biogenic amines in cheese was examined by Joosten and Northolt (1987) who investigated the decarboxylase activity of bacteria including lactobacilli, enterococci, enterobacteriaccae and pediococci. Some strains of lactobacilli could form biogenic amines in cheese. Since the number of enterococcal cells required to produce significant amounts of tyramine is rarely reached
450
Catabolism of Amino Acids in Cheese during Ripening
in cheese, these bacteria are not important for amine formation in Dutch cheese, although this may not be true for certain artisanal cheeses for which enterococci are a major part of the starter. The authors concluded that non-starter lactobacilli were the most important agents in Dutch cheese for the formation of biogenic amines. This is in agreement with the findings of Broome et al. (1990) who reported that the concentrations of tyramine and histamine in cheeses inoculated with lactobacilli were twice as high as in control cheeses, indicating that decarboxylases of lactobacilli have a role in their production. Novella-Rodriguez et al. (2002a) studied the effect of defined-strain starters on the production of amines in goats' milk cheese during ripening. The main amines found were tyramine (94.59 mg kg- 1), putrescine and tryptamine. The effect of high hydrostatic pressure on the production of amines in goats' milk cheese was studied by Novella-Rodriguez et al. (2002b) who found maximum production of amines when the cheeses were treated at 50 MPa for 72 h; rates of production were lower when cheeses received higher pressure treatments (400 MPa for 5 min or 400 MPa for 5 min followed by 50 MPa for 72 h) and in the untreated control cheeses. In addition to being involved in the production of amines, B. linens is able to reduce the amounts of histamine and tyramine in cheese during ripening (Leuschner and Hammes, 1998). During the four weeks of ripening of Munster cheese, B. linens reduced the histamine and tyramine content by 55-70%. Degradation of amines occurs at the surface of the cheese but the concentration of amines on the surface and interior differed only slightly after inoculation with B. linens LTH456. It was suggested that the concentration gradient was removed by diffusion of amines, leading to a decrease in the concentration of biogenic amines in the interior of the cheese. Lactobacilli used as cheese starter adjuncts were incubated by Gummalla and Broadbent (1999) in a defined medium containing L-tryptophan under carbohydrate starvation (CS), or under near-cheese ripening conditions (a chemically defined medium containing 4% salt, at pH 5.2). The specific activity of the tryptophan decarboxylases from Lb. casei strains was lower than those of the corresponding enzymes from Lb. helveticus strains. Generally, activity in either strain did not vary significantly with time or incubation conditions. Twenty-two Lb. plantarurn strains and seven strains of Lb. casei had no decarboxylase activity on methionine (Amarita et al., 2001). The combined effects of temperature, pH and salt on the growth of E. faecalis EF37, its proteolytic activity and its ability to produce biogenic amines were studied by
Gardini et al. (2001) who observed that 2-phenylethylamine accounted for more than half of the total content of biogenic amines. The production of biogenic amines was found to be independent of the incubation temperature and in general, was very low at the higher NaC1 concentration and was increased by lower pH. Roig-Sagues et al. (2002) studied the ability of 694 strains of bacteria isolated from Spanish artisanal cheeses to produce histamine and tyramine. Tyramineforming activity (mainly by enterococci and some other LAB) was found more frequently than histamine-forming activity, which was formed mainly by enterobacteria, but also by small numbers of other LAB. Most of the tyramine-forming strains of LAB were isolated from cheeses containing the highest levels of tyramine. However, histamine-forming LAB were generally isolated from samples with a low level of histamine. The amount of tyramine found in the samples was significantly higher than that of histamine. The distribution of aromatic L-amino acid decarboxylases in 326 bacteria (four species of E. coli, Erwinia herbicola, Serratia plymuthicum, two species of Proteus, Alcaligenes faecalis, Bacillus natto, Achrombacter hartlebii, 11 species of Micrococcus, one Staphylococcus, three Sarcina spp., Brevibacterium ammoniagenes, Bacterium cadaveris and three Pseudomonas spp.) was studied by Nakazawa etal. (1977). Micrococcaceae were observed to have the highest decarboxylase activity on L-tryptophan, S-hydroxy-L-tryptophan and L-phenylalanine. The amino acid decarboxylase of M. percitreus was reported by this author to be involved in synthesis of aromatic amines such as dopamine and tyramine. A histidine decarboxylase, which did not require PLP as a coenzyme, has been purified from Lactobacillus 30a (Chang and Snell, 1968). The substrates of the enzyme were found to have a heterocyclic nitrogen atom at the same position relative to its alanyl side chain which may be important in the formation of the enzyme-substrate complex. Jetten and Sinskey (1995) studied a decarboxylase isolated from a strain of Corynebacterium glutamicum with activity on oxaloacetate. The enzyme, which catalysed the decarboxylation of oxaloacetate only, a key intermediate in carbon metabolism, had optimum activity between pH 7.0 and 7.5. A glutamate decarboxylase was isolated from Lb. brevis IFO 12005 by Ueno et al. (1997) and was found to be a dimer. Temperature and pH optima were 30 ~ and 4.2, respectively. The enzyme could not decarboxylate any other amino acid assayed. Lucas and Lonvaud-Funel (2002) purified the tyrosine decarboxylase of Lb. brevis lOEB 9809. The enzyme had features typical of pyridoxal phosphate-dependent amino acid decarboxylases although this enzyme was
Catabolism of Amino Acids in Cheese during Ripening
not related by sequence homology to any known tyrosine decarboxylase.
Catabolism of Other Amino Acids Goux et al. (1995) investigated aspartate catabolism in an effort to understand ammonia generation by E. coli. It was reported that arginine may be an intermediate in aspartate catabolism, and may also be an intermediate for ammonia production from aspartate during nitrogen-limited growth. Hayashi et al. (1993) compared an aspartate aminotransferase with the aromatic amino acid aminotransferase of E. coli. Both enzymes were composed of two identical "--43.5 kDa subunits, and contained one molecule of PLP per subunit. An aspartate aminotransferase isolated from Lc. lactis LM0230 was cloned and characterised by Dudley and Steele (2001). It was determined using homologous recombination that a mutation in the Asp biosynthetic pathway prevented this strain from growing in milk. According to Kaneoke et al. (1993), at least seven I_-arginine degradation pathways are known, and in some species, more than one of these pathways can be operational. These authors studied the arginine oxygenase pathway in two coryneforms, Arthrobacter globiformis IFO 12137 (ATCC 8010) and B. helvolum IFO 12073. This pathway involves four enzymes and produces succinate from L-arginine (Fig. 9). This pathway in the coryneforms studied is not identical to other pathways reported, e.g., the pathways of Pseudomonas aeruginosa and Streptococcus faecalis. E. coli utilises the ammonia-producing succinyl transferase pathway for arginine catabolism, and to a lesser degree, the arginine decarboxylase pathway (Schneider et al., 1998).
Arginine
Agmatine
Citrulline
N-carbamoylputrescine
.12 Ornithine
~
Putrescine
Figure 9 Pathways of arginine metabolism in bacteria (Arena and Manca de Nadra, 2001). (1) Arginine deiminase, (2) Catabolic ornithine transcarbamylase, (3) Arginine decarboxylase, (4) Agmatine deiminase, (5) Agmatinase, (6) N-carbamoylputrescine hydrolase, (7) Ornithine decarboxylase, (8) Anabolic system.
451
Lactic acid bacteria isolated from wine can catabolise arginine by at least two pathways (Arena et al., 1999). The arginine deiminase pathway produces orthinine, CO2 and NH3 via three enzymatic reactions. The enzymes involved in this pathway are arginine deiminase (EC 3.5.3.6), catabolic ornithine transcarbamoylase (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2) (Champomier Verges et al., 1999). Alternatively, the arginase-urease pathway leads to the production of urea. Arginine deiminase, ornithine transcarbamoylase and carbamate kinase from the sourdough microorganism, Lb. sanfranciscencis CB1, were isolated by de Angelis et al. (2003). The enzymes had acidic pH optima and were optimally active at 30-37 ~ Interestingly, arginine has been proposed as a possible growth substrate for the secondary microflora of Swiss cheese (Laht et al., 2002); calculations showed that ATP available from the metabolism of arginine to ornithine was theoretically sufficient to support the growth of non-starter bacteria to populations of 108 cfu g-1 The catabolism of threonine, asparagine, arginine and glutamate in cheese has attracted some study. Aminotransferase or dehydrogenase activities catabolise glutamate to produce ot-ketoglutarate, while y-aminobutyrate is formed from glutamate by the action of a decarboxylase. Threonine is converted to acetaldehyde and glycine (McSweeney and Sousa, 2000). The specific pathways for the catabolism of other amino acids (e.g., glycine, alanine and serine) by cheese-related microorganisms have attracted little attention.
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Kaneoke, M., Shiota, K., Kusunose, M., Shimizu, E. and Yorifuji, T. (1993). Function of the arginine oxygenase pathway in utilization of L-arginine-related compounds in Arthrobacter globiformis and Brevibacterium helvolum. Biosci. Biotechnol. Biochem. 57, 814-820. Klein, N., Maillard, M.-B., Thierry, A. and Lortal, S. (2001). Conversion of amino acids into aroma compounds by cell-free extracts of Lactobacillus helveticus. J. Appl. Microbiol. 91,404-411. Laber, B., Clausen, T., Huber, R., Messerschmidt, A., Egner, U., Muller-Fahrnow, A. and Pohlenz, H.D. (1996). Cloning, purification and crystallisation of Escherichia coli cystathionine [3-1yase. FEBS Lett. 379, 94-96. Laht, T.M., Kask, S., Elias, P., Adamberg, K., Paalme, T. (2002). Role of arginine in the development of secondary microflora in Swiss-type cheese. Int. Dairy J. 12, 831-840. Lamberet, G., Auberger, B. and Bergere, J.L. (1997a). Aptitude of cheese bacteria for volatile S-methyl thioester synthesis. I. Effect of substrate and pH on their formation by Brevibacterium linens GC171. Appl. Microbiol. Biotechnol. 47, 279-283. Lamberet, G., Auberger, B. and Bergere, J.L. (1997b). Aptitude of cheese bacteria for volatile S-methyl thioester synthesis. II. Comparison of coryneform bacteria, Micrococcaceae and some lactic acid bacteria starters. Appl. Microbiol. Biotechnol. 48,393-397. Lee, C.W. and Desmazeaud, M.J. (1985). Utilisation of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase. Arch. Microbiol. 140,331-337. Lee, C.W. and Richard, J. (1984). Catabolism of L-phenylalanine by some microorganisms of cheese origin. J. Dairy Res. 51, 461-469. Lee, C.W., Lucas, S. and Desmeaud, M.J. (1985). Phenylalanine and tyrosine catabolism in some cheese coryneform bacteria. FEMS Microbiol. Lett. 26, 201-205. Leuschner, R.G.K. and Hammes, W.P. (1998). Degradation of histamine and tyramine by Brevibacterium linens during surface ripening of Munster cheese. J. Food Prot. 61, 874-878. Lockwood, B.C. and Coombs, G.H. (1991). Purification and characterisation of methionine -y-lyase from Trichomonas vaginalis. Biochem. J. 279,675-682. Lucas, P. and Lonvaud-Funel, A. (2002). Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis lOEB 9809. FEMS Microbiol. Lett. 211, 85-89. Martinez-Cuesta, C., Ruquena, T. and Pelaez, C. (2002). Effect of bacteriocin-induced cell damage on the branched-chain amino acid transamination by Lactococcus lactis. FEMS Microbiol. Lett. 217, 109-113. McSweeney, P.L.H. and Sousa, M.J. (2000). Biochemical pathways for the production of flavour compounds in cheese during ripening: a review. Lait 80,293-324. Molimard, P. and Spinnler, H.E. (1996). Compounds involved in the flavor of surface mould-ripened cheeses: origins and properties. J. Dairy Sci. 79, 169-184. Nakazawa, H., Sano, K., Kumagai, H. and Yamada, H. (1977). Distribution and formation of aromatic L-amino
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Sensory Character of Cheese and its Evaluation C.M. Delahunty, Department of Food and Nutritional Sciences, UniversityCollege Cork, Ireland M.A. Drake, Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina, USA
Introduction A remarkable variety of cheeses are made in all parts of the world where milk is produced. Cheeses are consumed for their highly regarded nutritional value, and enjoyed for their complex and varied eating quality. The sensory characteristics of cheeses, which determine their eating quality, are properties that are perceived by the human senses, predominantly during consumption. These properties can be described as appearance characteristics, flavour characteristics and texture characteristics. However, cheeses are complex foods, produced using milk from different animals, by many different techniques, and are presented in a variety of sizes, shapes, packages or coatings. Some cheeses are produced in small quantities, such as farmhouse types, sold in local markets and consumed by a relatively small number of people. Others are produced in large quantities in very large automated facilities, may find their way to markets in many different countries and are consumed by very many people. Some cheeses are ripened or matured for years before they are consumed; others are consumed young or unripened. Cheeses may have moulds of different types growing on their surface, may be pierced to allow blue moulds grow within the cheese, or include ingredients such as herbs and/or spices. This considerable diversity in cheesemaking practice, and the number of stages that any single cheese undergoes during its production, results in a wide variety of cheeses each of which has complex sensory characteristics. Sensory evaluation of cheese is absolutely necessary to determine the relative merits of cheesemaking procedures and the influence of measured composition on specific sensory characteristics of cheese. Sensory evaluation is also needed to determine the influence of sensory characteristics on the eating quality of cheese and its consumer acceptability. However, the complexity of cheese presents a considerable challenge for its sensory evaluation. This chapter will focus on human perception of sensory characteristics, on the advantages and disadvantages of sensory evaluation methods, on the intensity
and quality of the sensory characteristics of cheeses, and on the relationships between cheesemaking, cheese composition, cheese sensory characteristics and consumer acceptability of cheese.
A Definition of Sensory Character Sensory characteristics of cheeses are human responses to perceptions of stimuli that are experienced with the cheeses, and can generally be described using terms defined within the categories of appearance, flavour and texture. Sensory characteristics result from interactions of the human sensory modalities of vision, touch, olfaction, gustation and mouthfeel with stimuli induced by rheological, structural and chemical components of the cheese. Sensory characteristics are perceived by consumers when they observe, manipulate, smell and take cheese into the mouth for consumption, and are subsequently expressed as a behavioural response using actions or descriptive terms. A majority of sensory characteristics are complex and are stimulated by the association of many different properties of the cheese, with different sensory modalities acting together. It is this complexity, or component balance, that hinders attempts to adequately represent cheese sensory character using instrumental or chemical analyses. In addition, and unfortunately from the sensory scientists' point of view, consumers differ from one another. Sensory perception, and particularly its communication, differs between individuals as a result of physiological, psychological, social and cultural differences.
Sensory Characteristics and Cheese Preferences Cheese quality has been defined for many years by manufacturers as cheese produced reliably and economically (Muir et al., 1995a). In the past, limited choices were available to consumers and as a result of this
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Sensory Character of Cheese and its Evaluation
limited experience, the consumer's palate was less discerning. Today, cheese markets are international, and cheesemakers compete openly for consumers, offering them an eve>widening choice. Cheese consumers are more affluent and many have tasted or regularly consume a diversity of cheese types, leading them to become increasingly discerning. These consumers now define the quality standard for cheeses, which is ultimately determined by eating quality. The eating quality of cheese, or a consumer's liking for cheese, is an integrated response. The stimuli are the sensory characteristics, perceived before and during consumption. However, the response is influenced by other individual consumer-related factors that include sensory abilities, past experiences with cheese, what is expected from a cheese and when and where it will be consumed. Expectations are based on experience, but are created for a specific product by marketing, packaging and familiarity. Finally, liking for, and satisfaction with, a cheese is determined by the context in which it is consumed, and its appropriateness for that context (e.g., would one wish to consume Epoisses for breakfast?). Eating quality determines consumer acceptability and willingness to repeat purchase. Highly regarded eating quality is not that found in a cheese with no defects, but that which offers unique and appealing characteristics consistently. The producer of the cheese with the most acceptable sensory characteristics, if he is aware of this and can ensure that the market presentation of his cheese matches its sensory character, will have an advantage in the market. The concepts of 'healthy eating' and 'conscientious eating' (e.g., vegetarian and vegan diets) and environmentally friendly eating are now increasingly important to consumers. To meet these consumers' expectations, cheese producers are challenged to produce new, wholesome products that taste as good as traditional alternatives. This task is proving difficult as dietary guidelines for healthy eating may recommend reducing the intake of ingredients that provide desirable sensory character, such as fat or salt. The production of reduced- and low-fat cheeses to replace traditional types is an example of such consumer-driven product development. However, the majority of new low-fat cheeses do not meet the sensory quality requirements of discerning consumers (Mistry, 2001). This is because fat is not just a provider of desirable sensory character, but it is also important for cheese texture and body, for the development of compounds responsible for flavour and for the release of flavour compounds during consumption. It will be difficult to improve the eating quality of these cheeses unless eating quality is understood better.
Cheesemaking and the Variety of Sensory Character The sensory characteristics of a cheese at the time of its consumption reflect the milk from which it was produced (e.g., a goats' milk cheese is distinct from a cows' milk cheese), the processes used in its production and the physical and the chemical changes that occurred during maturation (e.g., proteolysis breaks down proteins to amino acids during cheese maturation, which may subsequently act as substrates for the formation of volatile compounds (see 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1)). Milk from the cow, sheep, goat, buffalo or other animals can be used as raw material, and its qualities are determined by breed, diet and stage of lactation. Treatment of milk before cheese production, particularly pasteurisation, can kill micro-organisms and reduce enzyme activity that could otherwise contribute to the development of sensory character during maturation. During cheese production, the coagulant used to form curds, the amount of salt added, the type of starter culture and the use of adjunct cultures will determine sensory characteristics. Finally, the maturation time and the temperature of maturation may be varied. The sensory characteristics of different types of cheese, and the potential variety that may be achieved, are determined by the choices the producer makes at each of the stages in production. Sensory characteristics of many different cheeses are described in the literature and in specialist cheese books. However, the sensory characteristics of relatively few types have been defined, standardised and measured objectively using sensory science methods. Lack of objective knowledge makes it difficult to compare accurately the sensory characteristics of different cheese types, but more importantly, as the cause of sensory characteristics is only partially known, it is difficult to compare accurately cheese appearance, texture and flavour research carried out in different laboratories. Tables 1 and 2 present terms used to describe the appearance, texture and flavour characteristics of cheeses that have been defined and standardised in an objective way. Table 3 presents terms used for descriptive sensory analysis by other researchers, but that have not been defined and standardised adequately. Similar terms are used in many cases even though each descriptive language referenced was developed independently by different research groups. In addition, in many cases, similar terms have been used to describe dominant characteristics of different cheese types. This comparison suggests that even though a remarkable variety of cheese types are produced, that potentially exhibit a wide variety of sensory characteristics, it should
Sensory Character of Cheese and its Evaluation
457
Table 1 Terms used to describe the appearance and texture of cheese using descriptive analysis methods. Terms in this list were developed and defined by trained panels, and in many cases standard materials that help to illustrate the term are provided. Cheeses studied were low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari etaL, 2003), natural and processed cheeses (Drake etaL, 1999a; Gwartney etaL, 2002), ten different types of cheese (Lawlor and Delahunty, 2000), Cheddar and Camembert (Cooper, 1987) and Mozzarella cheeses (Brown et aL, 2003)
Term Appearance Chalky Colour/colour intensity
Mottling Mouldy Open/openness Shiny
Texture Adhesiveness Chewy Cohesiveness Creamy/creaminess
Crumbly/crumbliness
Crustiness Curdiness Degree of breakdown Dry Firm/firmness
First-bite sticky Fracturability at first bite Grainy
Hardness Mealy Moist Mouth-coating Oily Rate of recovery Residual mouthfeel Residual smoothness of mouth coating
Definitiona
Resembling chalk in appearance The colour of Cheddar ranging from pale yellow to orange, the palest yellow representing the start of the scale The colour of cheese ranging from white to orange The evenness of colour shading within the cheese sample, with the most uniform coloured cheese being free from mottling, marbling or any other deficiencies in colour The degree of mouldiness/visible mould growth in the cheese structure The extent to which the interior of the cheese (that is the cut surface) is open, this encompasses cracks, pinholes, irregular-shaped holes and any other openings The extent to which the surface of the cheese is shiny, glossy, moist or sweaty-looking, as opposed to looking matt or dull The degree to which the chewed mass sticks to mouth surfaces, evaluated after five chews Requiring a good deal of mastication, toffee-like texture. Degree of chewing needed to break up the cheese The degree to which the chewed mass holds together, evaluated after five chews The extent to which the texture has broken down to a creamy semi-liquid texture, assessed between tongue and palate during mastication The feeling associated with heavy whipping cream (e.g., >30% fat content) The extent to which the cheese structure breaks up in the mouth, assessed during the first 2-3 chews The feeling in the mouth when the sample falls apart quickly in mouth during mastication The force required to break through the crust of the cheese when taking the first bite, assessed using the front teeth The extent to which a curdy or mealy texture is perceived in the mouth during mastication The amount of breakdown that occurs in the sample as a result of mastication, evaluated after five chews The degree of dryness or moistness sensed in the mouth during mastication Ranging from soft to firm. The extent of resistance offered by the cheese, assessed during the first five chews using the front teeth The force required to squeeze a cube (1.5 • 1.5 • 1.5 cm) of cheese flat between the first finger and thumb The amount of force required to take the first bite of cheese, assessed using the front teeth The amount of force required to completely bite through the cheese, assessed using the molars Sticky sensation experienced during the first bite Completely bite through the sample with the molars and evaluate the degree to which the sample fractures The extent to which granular structures are formed as the sample breaks down, perceived in the second half of chewing The feeling of coarse particles in the mouth during mastication The force required to bite the sample (first bite) The feeling in mouth when the sample breaks down in small pieces and it is difficult to gather them for swallowing The perceived moisture content of the cheese. Ranging from dry to moist The extent to which the cheese has a moist or wet texture around the palate during mastication The extent to which the cheese coats the palate and teeth during mastication The degree of coating on the tongue and the palate during mastication Oily, fatty, greasy mouth-feel of any kind Depress sample between thumb and first finger 30%, evaluate the speed or rate at which the sample returns to its original shape The degree of 'bittiness' or graininess in the mouth just before swallowing The degree of smoothness felt in the mouth after expectorating the sample
continued
458
Sensory Character of Cheese and its Evaluation
Table 1 continued Term
Definition a
Rubbery/rubberiness
The extent to which the cheese returns to its initial from after biting, assessed during the first 2-3 chews The degree of springiness experienced while biting the sample Of the nature of slime, soft, glutinous or viscous substance, soft, moist and sticky The smoothness of the cheese against the palate as it breaks up during mastication The degree to which the chewed mass surface is smooth, evaluated after five chews Yielding easily to pressure, easily moulded, pliable, easily spreadable Depress sample between thumb and first finger 30%, evaluate the total amount of recovery of the sample The stickiness of the cheese against the palate and around the teeth during mastication Overall sensation of stickiness during mastication The mouthfeel associated with consuming very viscous fluids like heavy whipping cream or honey
Slimy Smooth/smoothness Softness Springiness Sticky/stickiness Viscous
a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the meaning of each definition is unchanged.
Table 2 Terms used to describe the flavour of cheese using descriptive analysis methods. Terms in this list were developed, defined and referenced using standard materials by trained panels. Cheeses studied were: Cheddar (Murray and Delahunty, 2000b; Drake et aL, 2001), low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari et al., 2003), aged natural cheese of many types (Heisserer and Chambers, 1993), ewes' milk cheese (B~.rcenas et al., 1999) and cheese flavours (Stampanoni, 1994) Term
Definition a
S t a n d a r d b, c
Acid/yoghurt, acidic
The taste on the tongue associated with acids (citric, lactic... ) A sour, tangy, sharp, citrus-like taste. The fundamental taste sensations of which lactic and citric acids are typical Flavours indicating age in Cheddar cheese
0.35-0.86 g lactic acid/100 g Ricotta Fermented milk Natural yoghurt Citric acid (0.2% in water)
Age Ammonia Animal, animalic Astringent
Balanced
Bell pepper Biting
Bitter
Blue Brine
Brothy
The combination of aromatics reminiscent of farm animals and barnyards The complex of drying, puckering, shrinking sensations in the oral cavity causing contraction of the body tissues A mouth-drying and harsh sensation Mellow, smooth, clean. In equilibrium, wellarranged or disposed, with no constituent lacking or in excess Aroma associated with freshly cut green peppers The slightly burning, prickling and/or numbness of the tongue and/or mouth surface Fundamental taste sensation of which caffeine or quinine are typical A chemical-like taste The combination of aromatics associated with the saturated brine used during traditional ewes' milk cheesemaking Aromatics associated with boiled meat or vegetable stock soup
Aged Cheddar cheese (1 yr or older) Ammonia solution (0.25% in water) 4-Methyl-octanoic acid (2% in PG d) 1-Phenyl-2-thiourea (5000 mg/kg in PG) Alum (0.1% in water) Tea, six bags soaked in watere for 3 h Tannic acid (0.05% in water) Mild Cheddar
Methoxy pyrazines (5 #g/kg) Freshly cut bell pepper Horseradish sauce
Caffeine (0.02, 0.06 or 0.08% in water) Tonic water, quinine (0.01% in water) Octan-2-one (1% in PG) Ewes' milk cheese brine at room temperature
Canned potatoes Low-sodium beef broth cubes Methional (20 mg/kg)
Sensory Character of Cheese and its Evaluation
459
Table 2 continued Term
Definition a
Butter milk Buttery
B
Fatty, buttery tasting, of the nature of, or containing butter The aromatics commonly associated with natural, fresh, slightly salted butter Aroma rising from butter at room temperature
Butyric, butyric acid
Capric acid Caramel
Caseinate Catty Cheddary
Cheese rind Cooked, cooked milk
Cottage cheese Cowy/phenolic
Creamy
Dairy fat Dairy sour Dairy sweet Decaying animal Diacetyl Earthy Fatty Faecal Fermented Fermented fruity / winey
Flavour intensity Free-fatty acid Fresh fish
Sour flavour, similar to baby vomit The aromatics reminiscent of baby vomit; is sour and cheesy m
The taste and aromatics associated with burnt sugar or syrup; toffee made from sugar that has been melted further Aroma associated with tom-cat urine The taste and aromatics associated with typical Cheddar Typical aroma and taste of sharp/mature Cheddar cheese m
Aromatics associated with cooked milk The combination of sweet, brown flavour notes and aromatics associated with heated milk m
Aromas associated with barns and stock trailers, indicative of animal sweat and waste Fatty, creamy tasting, of the nature of, or containing cream The oily aromatics reminiscent of milk or dairy fat The sour aromatics associated with dairysoured products The sweet aromatics associated with fresh dairy products The aromatics reminiscent of decaying animal material Aromatics associated with diacetyl m
Aroma associated with complex protein decomposition The combination of aromatics reminiscent of red wine in general; it is sweet, slightly brown, overripe and somewhat sour The overall intensity of flavour in the sample, from mild to strong Aromatics associated with short chain fatty acids The aromatics associated with fresh fish
S t a n d a r d b, c
Pasteurised butter milk Unsalted butter Lightly salted butter Pasteurised cooking butter Diacetyl (1% in PG) Diacetyl in vaseline oil (several concentrations) Butyric acid, 2500 mg/kg in vaseline oil =SSf. 2 ml SS + cotton in 60-ml flask Butyric acid (10 000 mg/kg in PG) Butyric acid (1% in PG) Capric acid (pure) Condensed milk 3-Hydroxy-2-methyl-4-pyrone (2% in PG)
Sodium caseinate powder 2-Mercapto-2-methyl-pentan-4-one (20 mg/kg) Processed cheese Mature Cheddar cheese
Cheese rind (Tilsit mild, pasteurised full fat) Skim milk heated to 85 ~ for 30 min Evaporated milk UHT milk 3.6% fat, cooked for 10 min Cottage cheese 25% fat p-Cresol (160 mg/kg), bandaids
Mascarpone cheese ,y-Decanolactone (0.1% in PG) UHT Cream 35% fat Whipping cream Unsalted butter Sour cream Vitamin D milk Dimethyl disulfide (bottom notes only; 10 000 mg/kg in PG) Diacetyl (20 mg/kg) Geosmin (0.001% in PG) Palm kernal fat Indole, skatole (20 mg/kg) Fermented milk, 12% fat Burgundy cooking wine
Butyric acid (20 mg/kg) Elodea
(an aquatic plant) growing in water continued
460
Sensory Character of Cheese and its Evaluation
Table 2
continued
Term
Definitiona
Standard b, c
Fruity
The taste and aromatic blend of different fruity identities The aromatics associated with different fruits
Goaty
The aromatics reminiscent of wet animal hair; it tends to be pungent, musty and somewhat sour
Canned fruit salad (in syrup) trans-2-Hexenal (10 000 mg/kg in PG) Canned fruit cocktail juice Fruit of the forest yoghurt Ethyl butyrate (0.1% in PG) trans-2-Hexenal. 300 mg/kg in vaseline oil = SS. 3 ml SS + cotton in 60-ml flask Fresh pineapple Ethyl hexanoate (20 mg/kg) Hexanoic acid (5000 mg/kg in PG)
Green-grass Methyl ketone / blue Milkfat /lactone
Milky Mouldy, mouldy/musty
Mushroom
u
Aroma associated with blue-vein cheeses Aromatics associated with milkfat
The aromatics commonly associated with ewes' raw milk The combination of tastes and aromatics generally associated with moulds; they usually are earthy, dirty, stale, musty and slightly sour Aromas associated with moulds and/or freshly turned soil The taste and aromatics associated with raw mushrooms
Musty
Aroma of a damp room or very old book
Nutty
The aromatics reminiscent of several dry fruits such as pecans, walnuts and hazelnuts The non-specific nut-like taste and aromatics characteristic of several different nuts, e.g., peanuts, hazelnuts and pecans The nut-like aromatic associated with different nuts
Overall intensity
Strength of the stimuli perceived by the nose Strength of global stimuli originated by the volatiles released during mastication and perceived on the olfactory receptors via the retronasal route
Oxidised
Aroma associated with oxidised fat The fruity aromatic associated with pineapple
Pineapple
cis-3-Hexenol(1% in PG) 2-Octanone (40 mg/kg) Fresh coconut meat Heavy cream 5-Dodecalactone (40 mg/kg) Ewes' milk raw Pasteurised milk, 3.6% fat 2-Ethyl-l-hexanol (10 000 mg/kg in PG) 2-Ethyl-l-hexanol (40 mg/kg) Stilton cheese 2,4,6 Trichloroanisole (1% in PG)
Button mushroom (raw) Brown mushrooms (chopped, raw) 1-Octen-3-ol (0.5% or 1% in PG) 3-Octanol (10 000 mg/kg in PG) 3-Octanol. 5-10 mg/kg in vaseline oil = SS. 3 ml SS + cotton in 60 ml flask Cola infusion in ethanol (pure) Damp room Very old book Wheat germ 2 g Walnuts + 2 g hazelnuts, minced in 60-ml flask (mixed particulates to be sampled) Mixed crushed nuts 2-Acetyl-pyridine (0.01% in PG) Lightly toasted unsalted nuts Unsalted wheat thins Roasted peanut oil extract Roasted peanuts, ground hazelnuts, ground almonds, 1:1:1 1000-73 nut base by Givaudan-Roureg (10% in PG) 4 g cheese aroma/100 ml of pasteurised ewes' milk 0.5-3.5 g cheese aroma/100 g Quark 91549-24 by Givaudan Roureg 91483-24 by Givaudan Roure 91428-24 by Givaudan Roure 91125-73 by Givaudan Roure 10418-71 by Givaudan Roure 2,4 Decadienal, 20 mg/kg 4-Pentenoic acid (10 000 mg/kg in PG) Canned pineapple chunks
Sensory Character of Cheese and its Evaluation
461
Table 2 continued Term
Definition a
Standardb, c
Prickle/bite
Chemical feeling factor of which the sensation of carbonation on the tongue is typical A bland, shallow and artificial taste. Made by melting, blending and frequently emulsifying other cheeses
Soda water
Processed
Propionic acid Pungent
Rancid
Rennet Rosy/floral Salty
Sauerkraut Scorched Sharp
Smokey, smoky
Soapy
Sour
Soya sauce
m
A physically penetrating sensation in the nasal cavity. Sharp smelling or tasting, irritating Irritative, burnt and/or penetrating sensation in the interior of the mouth
The taste and aroma associated with sour milk and oxidised fats. Having the rank unpleasant aroma or taste characteristic of oils and fats when no longer fresh The aromatics associated with natural lamb rennet Aroma associated with flowers Fundamental taste sensation of which sodium chloride is typical Fundamental taste sensation elicited by salts Fundamental taste sensation produced by aqueous solutions of several products such as sodium chloride The aromatics associated with fermented cabbage Aroma associated with extreme heat treatment of milk proteins The total impression associated with the combination of aromatics that are sour, astringent and pungent Total impression of penetration into the nasal cavity The perception associated with aged and ripened cheeses, from flat to sharp The penetrating, dark brown, acrid aromatic of charred wood Aroma and taste of hickory-smoked ham The penetrating smoky taste and aromatics, similar to charred wood Tainted by exposure to smoke Perception of any kind of smoke odour (hickory, apple, cherry, mesquite or artificial flavouring) A detergent-like taste and smell. Similar to when a food is tainted with a cleansing agent Fundamental taste sensation elicited by acids Fundamental taste sensation of which lactic and citric acids are typical The aromatics that are reminiscent of soy sauce; they are sour, slightly brown and pungent
Cheese strings (a processed cheese snack) Propionic acid (1% in PG) A ratio of 1 part sour cream to 0.68 parts horseradish sauce Danish blue cheese Ammonia (1% in PG) 0.5 g cayenne/100 ml water, boiled in water for 5 min, 1.5 ml of filtration/10 g Quark Cheese stored at 21 ~ for 4 days Butyric acid (0.1% in PG)
Natural lamb rennet (33% NaCI) 2-Phenethylamine, 20 mg/kg Sodium chloride (0.25, 0.5, 0.75 or 1% in water) Pecorino Romano sheep cheese, 1200 mg NaCI/100 g Quark
Dimethyl disulfide (top notes only; 10 000 mg/kg in PG) Milk heated to 121 ~ for 25 min Propionic acid (100 000 mg/kg in PG) 5000 mg/kg of propionic acid in Vaseline oil = SS. 2 ml SS + cotton in 60 ml flask
Oil of cade Hickory smoked ham Applewood cheese Guaiacol (0.5% in PG) Guaiacol in vaseline oil (several concentrations) Liquid smoke flavouring. 40 #1 + cotton in 60-ml flask Lauric acid (pure) Mellow processed Cheddar Citric acid (0.08% in water) Lactic acid (0.05 and 0.085% in water) Soya sauce
continued
462
Sensory Character of Cheese and its Evaluation
Table 2 continued Term
Spicy/pungent Strength Sulfur Sweaty
Sweet
Toasty Umami Vinegary Waxy, waxy/crayon
Whey Yeasty
S t a n d a r d b, c
Definition a m
The overall intensity of aroma and flavour, the degree of mildness and maturity Aromatics associated with sulphurous compounds The aromatics-associated reminiscent of perspiration-generated foot odour; sour, stale, slightly cheesy and is found in unwashed gym socks and shoes Fundamental taste sensation of which sucrose is typical Fundamental taste sensation elicited by sugars Fundamental taste sensation produced by aqueous solutions of several products such as sucrose or fructose The combination of sweet aromatics produced after food toasting or cooking Chemical feeling factor elicited by certain peptides and nucleotides Aroma described as acidic, fermented and sweaty by the panelists The sweet aromatic that is associated with waxed paper or wax candles Aromatics associated with medium chain fatty acids Aromatics associated with Cheddar cheese whey Aromatics associated with fermenting yeast
Yoghurt
Valeric acid (1% in PG) English blue Stilton cheese Boiled mashed egg. H2S bubbled through water; struck match Isovaleric acid (10 000 mg/kg in PG) Isovaleric acid (0.1% in PG) Isobutyric acid (5% in PG) Cheese stored at 30 ~ for 3 h Sucrose (1,3, 4 or 5% in water) Condensed milk 1.2 g sucrose/100 g Quark
Cooked condensed milk Ciclotene (several concentrations in water) Monosodium glutamate (1% in water) Combination of acetic, butyric and propionic acids Decanoic acid (pure) Capric acid, lauric acid or decanoic acid (100 mg/ml) Fresh Cheddar whey Whey powder Raw yeast dough Yeast in 3% warm sucrose water Yoghurt, 3.2% fat
a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the meaning of each definition is unchanged. b Units of measurement are changed to a standard format where possible. c Publications referenced often provided brand names of food standards used. Brand names are not provided in this table as it is recognised that many of these will only be of interest to readers in their country of origin. In addition, as some publications referenced are now more than 10 years old, products may have changed. d Propylene glycol. e Volume of water not given in publication referenced. f Stock solution. g Codes refer to commercially available flavour mixtures that can be provided by Givaudan Roure, Switzerland.
be possible to develop and standardise a terminology that can be used universally, and for all cheese types, eventually leading to a much-improved understanding about the eating quality of cheese.
The Human Senses and the Sensory Properties of Cheese Cheese appearance
Humans are highly visual creatures and allow vision to dominate other sensory modalities. Vision is the perception of shape and texture, size and distance, brightness,
colour and movement. Appearance characteristics of cheese are assessed visually, usually prior to consuming the cheese, or when preparing the cheese for consumption by cutting or spreading. Appearance characteristics include colour, presence of eyes or holes (or openness), mould, rind, and visual texture (Tables 1 and 3). In addition, appearance includes a cheese's market image (e.g., size, shape, packaging), as most cheese is purchased in this form (Murray and Delahunty, 2000a). Appearance characteristics create sensory expectations, or expectations of how the cheese will 'taste', and as vision can dominate other sensory modalities, visual aspects of cheese can often have a strong influence on
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Sensory Character of Cheese and its Evaluation
the perception of other characteristics that, general experience has taught us, are related (even if they may not be physically related). For example, many consumers believe that a coloured cheese is more intensely flavoured than its uncoloured equivalent (Bogue et al., 1999). Cheese texture
Texture can be defined as the attribute of a cheese resulting from a combination of physical properties, including size, shape, number, nature and conformation of the constituent structural elements, that are perceived by a combination of the senses of touch (tactile texture), vision (visual texture) and hearing (auditory texture). For example, the 'softness' of a cream cheese can be assessed visually upon cutting the cheese, by proprioceptive sensations when spreading the cheese, and finally by tactile sensations in the mouth during consumption. During mastication and consumption, texture perception occurs in the superficial structures of the mouth, around the roots of the teeth and in the muscles and tendons. Cheese texture characteristics frequently described include firmness, rubberiness, crumbliness, graininess, cohesiveness and adhesiveness (Tables 1 and 3). Cheese flavour
Flavour is most often defined as the integrated perception of olfactory, taste and chemesthesis (or trigeminal) stimuli. Flavour perception begins prior to consumption when a consumer can smell a cheese, but is finally perceived during consumption when compounds that stimulate the olfactory system in the nose, the taste system in the mouth and the trigeminal system in the mouth and nose are released from the cheese and become available to receptors. A large number of flavour characteristics have been described in cheese. Some that have been defined and standardised for application in descriptive sensory evaluations are listed in Table 2. Smell or aroma is usually the first aspect of flavour encountered by a consumer. The stimuli for smell are air-borne compounds of volatile substances that allow them to travel from their source to the olfactory receptors, where perceptions are created that are endowed with distinctive smells. Volatile stimuli are released from cheese into the air, and may be delivered to the nose orthonasally, often consciously, by sniffing (e.g., when one opens a cheese package or removes a trier from the cheese for evaluation). Volatile compounds may also be released into the buccal cavity air during consumption, where they are delivered to the nose retronasally without any conscious effort. Many hundreds of different volatile
compounds, each with a distinctive aroma character, have been identified in cheese, and these provide the largest contribution to the diversity of cheese flavours. Compounds identified in cheeses include fatty acids, methyl, ethyl and higher esters, methyl ketones, aliphatic and aromatic hydrocarbons, short- and longchain alcohols, aromatic alcohols, aldehydes, amines, amides, phenols and sulphur compounds (Maarse and Visscher, 1996). Much of what we commonly refer to as 'taste' is incorrectly localised smell detection. The significant contribution of aroma to flavour can be easily demonstrated if one pinches the nose shut whilst eating, effectively blocking air circulation through the nasal passages. Then, a familiar cheese, e.g., Cheddar, will not be recognised, and can easily be confused with one that would otherwise be easily distinguished, e.g., Gruyere. Taste is another aspect of flavour. Tasting occurs in the oral cavity, primarily on the tongue, but also on the soft palate. The primary stimuli for taste are nonvolatile compounds, and these must make contact with the taste receptors. This contact creates perceptions that endow four distinctive taste qualities, referred to as sweet, salty, sour and bitter. A fifth taste, 'umami', has been accepted more recently, particularly in Japan and other cultures where it is the most familiar and the most easily perceived. Compounds that contribute directly to cheese taste include lactic acid (sour), sodium chloride (salty), mineral salts of potassium, calcium and magnesium (salty) and free amino acids and peptides of varying types (sweet, bitter, umami) (Warmke et al., 1996; Engel et al., 2000). The last aspect of flavour is chemesthesis. This term is used to describe the sensory system responsible for detecting chemical irritants. Detection is more general than that of taste and smell and occurs primarily in the eyes, nose and mouth. The perception is closely related to the somato-sensory characteristics of pain and temperature change, and provokes a strong behavioural response. The fizz of carbon dioxide (CO2), the cooling sensation of menthol and the burning sensation of chilli are perhaps the best examples of how chemical irritation can provide additional character that is very much desired in a wide range of food products. With regard to cheese, the pungency, the prickle/bite and the sharpness of mature Cheddar are examples of perceived chemical irritation (Table 2). Sensory interactions
Cross-modal sensory interactions also occur, adding complexity to the perception and description of sensory character. Consumers rarely distinguish between stimuli of different sensory modalities (unless trained to do so), and generally describe the integrated sensation as 'taste'.
Sensory Character of Cheese and its Evaluation
However, the factors that cause apparent cross-modal sensory interactions are not always the same and can be difficult to comprehend. A first cause of apparent sensory interactions when perceptible components of a cheese are studied together can be interactions between the components of the cheese prior to introduction to the senses. For example, changing the fat content or salt content of a cheese can influence the physical chemistry of the cheese matrix dramatically, changing the partition coefficients of volatile compounds, and therefore releasing volatiles from the cheese matrix (Delahunty and Piggott, 1995). As a cheese matures, the protein composition changes significantly due to proteolysis, and this may change the binding ability of the cheese for specific volatile compounds (Delahunty and Piggott, 1995). A second cause of sensory interaction is termed a halo effect, and is caused by learning to place greater reliance on one sensory modality over another to make behavioural decisions. This effect was referred to in the context of appearance, as it is most obvious by the dominance, or bias, of the visual sense over the taste or olfactory sense. It can be demonstrated by confusing familiar colour and flavour combinations, or by varying colour intensity beyond expectation (Clydesdale, 1993). With regard to cheese, an influence of added colour on consumers' perception of flavour has been reported (Bogue et al., 1999). A true cross-modal sensory interaction is one where the function of one sense (e.g., threshold measures, concentration-response functions) is changed by stimulation of another sense. This type of interaction can occur at receptor level, where one component blocks access to the receptor by another (e.g., increasing viscosity may coat the tongue and reduce access of tastants to taste receptors (Lynch et al., 1993)), or where stimulation by both components results in neural convergence as receptor sites are in close proximity and are served by the same nerve (e.g., capsaicin desensitisation reduces perceived taste intensity (Karrer and Bartoshuk, 1995)). The extent of these types of interactions in cheese, and their effect, is not known. Taste-aroma interactions are also observed and appear to be true interactions even though the physiology of the senses of olfaction and taste is independent. In this case, interaction is believed to occur centrally at a cognitive level where stimulus integration takes place (Stevenson et al., 1999). Taste-odour interactions have been observed in many different types of food and are easily demonstrated in model food studies (Noble, 1996). When volatile compounds are introduced to the oral cavity in the absence of taste-active compounds, they are generally perceived to be of low intensity and are described as bland in character. In cheese, it is most likely that the flavour impact of specific volatile compounds will be pronounced (and become familiar) only
467
when perceived in combination with appropriate tasteactive compounds, such as lactic acid, mineral salts, free amino acids and bitter peptides typically present in cheese (Frank and Byram, 1988). In addition, variations in taste quality and intensity, for example an increase in sourness (i.e., acidity), or an increase in bitterness, will affect how aroma is perceived (although volatile composition may be unchanged) and give the impression that overall flavour has changed considerably. Flavour-texture interactions are also observed widely. The precise nature of many of these interactions is not known, although structural components, such as proteins, can bind volatile compounds; rheology and structure can also influence mass transfer of non-volatile and volatile compounds to the surface of a cheese bolus where they will be released and become available for perception; fat can coat the receptor surface of the tongue, effectively blocking taste transduction (Lynch et al., 1993) and finally, interactions may occur at a cognitive level during perception integration in a way similar to taste-odour interactions (Weel etal., 2002). Texture-flavour interactions can also be influenced by individual consumer physiology, such as mastication behaviour and saliva flow rate and volume.
Sensory Methods Used to Evaluate Cheese Many reported studies on cheesemaking, cheese composition and cheese microbiology had the objective of controlling or improving sensory characteristics such as appearance, flavour and texture. However, it is difficult to compare the success of these studies as the final sensory character was often measured inappropriately. In many studies, judgements of overall sensory quality (i.e., a grade of 'good' or 'bad'), rather than objective measurements of the perceived intensity of specific sensory characteristics, were carried out to determine the influence of cheese composition, counts of micro-organisms, or control of a cheesemaking variable on flavour or texture quality. Although standard procedures may be followed, e.g., International Dairy Federation standards (IDE 1997), quality judgements are biased by the individual(s) who makes them. In addition, and of greater importance, traditional quality judgements do not allow the application of statistical analyses that would enable relationships between cheese study variables and specific sensory characteristics to be determined. The unaware reader of the literature can very easily confuse measurements of overall sensory quality with descriptions of sensory difference, as it is often reported, for example, that a specific cheesemaking procedure produced cheeses that 'tasted' similar, when in fact they were judged to be of similar quality (i.e., had no defects). Cheeses judged to be of similar quality by the same judge may differ
468
Sensory Character of Cheese and its Evaluation
significantly in sensory characteristics (Delahunty and Murray, 1997). The American Society for Testing and Materials (ASTM) Committee E-18 on Sensory Evaluation of Materials and Products has defined sensory evaluation as 'a scientific discipline used to evoke, measure, analyse and interpret reactions to the characteristics of foods and materials as they are perceived by the senses of sight, taste, touch and hearing'. A key distinction between sensory evaluation and other chemical and instrumental analytical techniques, is that different techniques can be used to evoke, measure and interpret sensory characteristics that have very different objectives and outcomes. Sensory evaluation can be carried out to determine whether cheeses exhibit defects or other undesirable characteristics, whether a difference in overall sensory character can be detected between two or more cheeses, whether specific differences in sensory characteristics can be perceived, to quantify the intensity of one or more sensory characteristics, to quantify the onset, maximum intensity and decline of a sensory characteristic, and to determine whether consumers find the cheeses to be acceptable or not, based on their sensory characteristics. The distinctions in sensory evaluation methodology can be broadly classified as quality scoring, discrimination testing, descriptive testing, time-intensity testing and consumer acceptability testing, respectively. There are some excellent texts that outline sensory tests in detail (Piggott, 1988; Stone and Sidel, 1993; Lawless and Heymann, 1998; Meilgaard et al., 1999).
Grading and quality scoring The manufacture of cheese of consistent quality is extremely difficult due to the number of production factors that ultimately contribute to eating quality (see 'Factors that Affect the Quality of Cheese', Volume 1). In addition, cheeses are susceptible to a large number of defects that can originate in milk, transfer to the cheese curd during making and storage, result from microbial contamination or develop during maturation if the composition at manufacture is not controlled. However, to maintain consumer confidence and loyalty towards a cheese, it is very important to control its quality. In addition, as consumers are becoming more brand-conscious, they become less-accepting of variations in sensory characteristics that traditionally would not be considered defects, and expect to find a cheese with near-identical appearance, flavour and texture in the package each time. To test instrumentally for all possible flavours and structural properties that contribute to eating quality would be an extremely laborious task, and may not achieve success. For example, many compounds that contribute to flavour are present
in concentrations below the detection limit of even the most sophisticated instruments. Quality scoring, grading or judging against specified defects on standardised scorecards (Bodyfelt et al., 1988) is the traditional and still most widely used type of formal sensory evaluation in the cheese industry. Cheese grading is carried out to classify the potential of a cheese to develop a satisfactory character during maturation, and to maintain quality at the point of sale. Grading standards generally specify a scoring system, where top grade is awarded a maximum score, and points are deducted when defects are found. For example, the IDF provides standard scorecards for cheese, and specifies a scale that ranges from 5, representing the highest possible quality, to 0, representing the lowest possible quality (IDE 1997). Each point deducted from the scale is supported by a list of defects that merit the deduction. The defect list that accompanies each score on the scale aims to provide objectivity to the evaluation. The US cheese grading system and the American Dairy Science Association (ADSA) cheese-judging ballot operate in a similar manner (Bodyfelt et al., 1988). Tables 4 and 5 show the United States Department of Agriculture (USDA) standards for grades of Cheddar cheese, effective since 1956, which provide guidelines for the award of four g r a d e s - AA, A, B or C. Table 6 shows the ballot used by the ADSA to judge Cheddar cheese quality. McBride and Muir (1999) recently reviewed grading systems used for Cheddar cheese in Australia, United Kingdom, United States, Canada, the IDF and New Zealand. In addition, chapters in recent textbooks by Kosikowski and Mistry (1997) and Robinson and Wilbey (1998) review in detail methods of cheese grading and defects found in cheese. Kosikowski and Mistry (1997) described the sequence of cheese quality judgement. One or more expert evaluators, who have detailed product knowledge built up over many years and maintain a standard in memory of what the ideal product is in terms of sensory characteristics, carry out this evaluation. These experts have the ability to relate their recognition of specific defects to the cause of that defect and to weight the influence of each defect at different levels of severity and how they detract from overall product quality. The overall exterior of a cheese is first judged to determine if it appears deformed or soiled in any way. The rind or surface is judged next as it may be discoloured, cracked or irregular. Internal appearance is judged following cutting, or directly from a cheese trier, as it may have holes, cracks, spots or other opening defects, and colour may be uneven, mottled or dull. Odour, which may be uncharacteristic in many ways, is judged prior to placing a cheese in the mouth,
Sensory Character of Cheese and its Evaluation
469
Table 4 Specifications for Grade AA and Grade A Cheddar cheese (United States Department of Agriculture, Agricultural Marketing Service, Dairy Division)
Detailed specifications for US Grade AA Fresh or current
Medium cured
Cured or aged
(a) Flavour : Fine and highly pleasing. May be lacking in flavour development or may possess slight characteristic Cheddar cheese flavour. May possess a very slight feed flavour, but shall be free from any undesirable flavours and odours.
Fine and highly pleasing. Possesses a moderate degree of characteristic Cheddar cheese flavour. May possess a very slight feed flavour but shall be free from any undesirable flavours and odours.
Fine and highly pleasing characteristic Cheddar cheese flavour showing moderate to well-developed degrees of flavour or sharpness. May possess a very slight feed flavour but shall be free from any undesirable flavours and odours.
A plug drawn from the cheese shall be firm, appear smooth, waxy, compact, close, flexible and translucent, but may have a few mechanical openings if not large and connecting. May be slightly or not entirely broken down. May possess not more than one sweet hole per plug but shall be free from other gas holes.
A plug drawn from the cheese shall be firm, appear smooth, waxy, compact, close, and translucent but may have a few mechanical openings if not large and connecting. Should be free from curdiness and possess a cohesive velvet-like texture. May possess not more than one sweet hole per plug but shall be free from other gas holes.
Shall have a uniform, bright attractive appearence; practically free from white lines or seams. May be coloured or uncoloured, but if coloured it should be medium yellow-orange.
Shall have a uniform, bright attractive appearance; practically free from white lines or seams. May show numerous tiny white specks. May be coloured or uncoloured, but if coloured it should be medium yellow-orange.
Bandaged and paraffin dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess very slight mould under bandage and paraffin, and the following other characteristics to a slight degree: Soiled surface and surface mould. The cheese shall be even and uniform in shape. Rindless. Same as for current, except very slight mould under wrapper or covering permitted.
Bandaged and paraffin dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess the following characteristics to a slight degree: Soiled surface and mould under bandage and paraffin; and surface mould to a definite degree. The cheese shall be even and uniform in shape. Rindless. Same as for medium.
(b) Body and texture: A plug drawn from the cheese shall be firm, appear smooth, compact, close and should be slightly translucent, but may have a few small mechanical openings. The texture may be definitely curdy or may be partially broken down if more than 3 weeks old. Shall be free from sweet holes, yeast holes and gas holes of any kind. (c) Colour : Shall have a uniform, bright attractive appearance; practically free from white lines or seams. May be coloured or uncoloured but if coloured it should be a medium yellow-orange.
(d) Finish and appearance: Bandaged and paraffin-dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess soiled surface to a very slight degree. The cheese shall be even and uniform in shape. Rindless. The wrapper or covering shall be practically smooth, properly sealed with adequate overlapping at the seams or by any other satisfactory type of closure. The wrapper or covering shall be neat and adequately and securely envelop the cheese. May be slightly wrinkled but shall be of such character as to protect fully the surface of the cheese and not detract from its initial quality. Shall be free from mould under wrapper or covering and shall not be huffed or lopsided.
continued
470
Sensory Character of Cheese and its Evaluation
Table 4
continued
Detailed specifications for US Grade A Fresh or current
Medium cured
Cured or aged
(a) Flavour: Shall possess a pleasing flavour. May be lacking in flavour development or may possess slight characteristic Cheddar cheese flavour. May possess very slight acid, slight feed but shall not possess any undesirable flavours and odours.
Shall possess a pleasing characteristic Cheddar cheese flavour and aroma. May possess a very slight bitter flavour and the following flavours to a slight degree: Feed and acid.
Shall possess a pleasing characteristic Cheddar cheese flavour and aroma with moderate to well-developed degrees of flavour or sharpness. May possess the following flavours to a slight degree: Bitter, feed and acid.
A plug drawn from the cheese shall be reasonably firm, appear reasonably smooth, waxy, fairly close and translucent but may have a few mechanical openings if not large and connecting. May be slightly curdy or not entirely broken down. May possess not more than two sweet holes per plug but shall be free from other gas holes. May possess the following other characteristics to a slight degree: Mealy, short and weak.
A plug drawn from the cheese should be fairly firm, appear smooth, waxy, fairly close and translucent but may have a few mechanical openings. Should be free from curdiness. May possess not more than two sweet holes per plug but shall be free from other gas holes. May possess the following other characteristics to a slight degree: Crumbly, mealy, short, weak and pasty.
Shall have a uniform, bright attractive appearance. May have slight white lines or seams. May be coloured or uncoloured but if coloured, it should be a medium yellow-orange.
Shall have a uniform, bright attractive appearance. May have slight white lines or seams and numerous tiny white it should be a medium specks. May be coloured or uncoloured, but if coloured, it should be a medium yellow-orange.
Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly but may possess very slight mould under bandage and paraffin and the following other characteristics to a slight degree: Soiled surface, surface mould, rough surface, irregular bandaging, lopsided and high edges. Rindless. Same as for current, except very slight mould under wrapper or covering permitted.
Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly but may possess the following characteristics to a slight degree: Soiled surface, rough surface, mould under bandage and paraffin, irregular bandaging, lopsided and high edges; and surface mould to a definite degree. Rindless. Same as for medium.
(b) Body and texture: A plug drawn from the cheese shall be firm, appear smooth, compact, close and should be slightly translucent but may have a few mechanical openings if not large and connecting. May possess not more than two sweet holes per plug but shall be free from other gas holes. May be definitely curdy or partially broken down if more than 3 weeks old. (c) Colour: Shall have a fairly uniform, bright attractive appearance. May have slight white lines or seams or be very slightly wavy. May be coloured or uncoloured but if coloured, it should be a medium yellow-orange. (d) Finish and appearance: Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly, but may possess the following characteristics to a very slight degree: Soiled surface and surface mould; and to a slight degree: Rough surface, irregular bandaging, lopsided and high edges. Rindless. The wrapper or covering shall be practically smooth, properly sealed with adequate overlapping at the seams or by any other satisfactory type of closure. The wrapper or covering shall be neat and adequately and securely envelop the cheese. May be slightly wrinkled but shall be of such character as to fully protect the surface of the cheese and not detract from its initial quality. Shall be free from mould under the wrapper or covering and shall not be huffed but may be slightly lopsided.
Sensory Character of Cheese and its Evaluation
471
Table 5 Specifications for Grade B and Grade C Cheddar cheese (United States Department of Agriculture, Agricultural Marketing Service, Dairy Division)
Detailed specifications for US Grade B Fresh or current
Medium cured
(a) Flavour: Should possess a fairly pleasing Should possess a fairly pleasing charactercharacteristic Cheddar cheese flavour, but istic Cheddar cheese flavour and aroma. may possess very slight onion and the May possess very slight onion and the following flavours to a slight degree: Acid, following flavours to a slight degree: Flat, flat, bitter, fruity, utensil, whey-taint, yeasty, yeasty, malty, old milk, weedy, barny and malty, old milk, weedy, barny and lipase; lipase; the following to a definite degree: feed flavour to a definite degree. Feed, acid, bitter, fruity, utensil, and whey-taint.
(b) Body and texture: A plug drawn from the cheese may possess the following characteristics to a slight degree: Coarse, short, mealy, weak, pasty, crumbly, gassy, slitty and corky; the following to a definite degree: Curdy open, and sweet holes. (c) Colour: May possess the following characteristics to a slight degree:Wavy, acid-cut, mottled, salt spots, dull or faded; and definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural
(d) Finish and appearance: Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess very slight mould under bandage and paraffin. The following characteristics to a slight degree: Soiled surface, surface mould, defective coating, checked rind, huffed, weak rind, and sour rind; and to a definite degree: Rough surface, irregular bandaging, lopsided and high edges.
Rindless. The wrapper or covering shall be fairly smooth and properly sealed with adequate overlapping at the seams or by other satisfactory type of closure. The wrapper or covering shall be fairly neat and adequately and securely envelop the cheese. May be definitely wrinkled but shall be of such character as to protect the surface of the cheese and not detract from its initial quality. Shall be free from mould under wrapper or covering but may be slightly huffed and slightly lopsided.
Cured or aged
Should possess a fairly pleasing characteristic Cheddar cheese flavour and aroma, with moderate to well-developed degrees of flavour or sharpness. May possess very slight onion and the following flavours to a slight degree: Flat, yeasty, malty, old milk, weedy, barny, lipase and sulfide; the following to a definite degree: Feed, acid, bitter, fruity, utensil, and whey-taint.
A plug drawn from the cheese may possess the following characteristics to a slight degree: Curdy, coarse, gassy, slitty, and corky; the following to a definite degree: Open, short, mealy, weak, pasty, crumbly, and sweet holes.
A plug drawn from the cheese may possess the following characteristics to a slight degree: Gassy, slitty, the following to a definite degree: Open, sweet holes, short, mealy, weak, pasty and crumbly.
May possess a very slight bleached surface; and the following characteristics to a slight degree: Wavy, acid-cut, mottled, salt spots, dull or faded and definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural.
May possess the following characteristics to a slight degree: Wavy, acid-cut, mottled, salt spots, dull or faded; and definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural.
Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess the following characteristics to a slight degree: Surface mould, mould under bandage and paraffin, checked rind, huffed, weak rind, and sour rind; the following to a definite degree: Soiled surface, rough surface, irregular bandaging, lopsided, high edges and defective coating.
Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess the following characteristics to a slight degree: Checked rind, huffed, weak rind, and sour rind; the following to a definite degree: Soiled surface, surface mould, mould under bandage and paraffin, rough surface, irregular bandaging, lopsided, high edges and defective coating. Rindless. Same as for medium.
Rindless. Same as for current, except slight mould underwrapper or covering permitted.
continued
472
Sensory Character of Cheese and its Evaluation
Table 5 continued Detailed specifications for US Grade C Fresh or current
Medium cured
Cured or aged
(a) Flavour: May possess the following flavours to a slight degree: Sour, metallic, onion; and to a definite degree: Acid, flat, bitter, fruity, utensil, whey-taint, yeasty, malty, old milk, weedy, barny, and lipase; feed flavour to a pronounced degree.
May possess the following flavours to a slight degree: Onion and sulfide; and to a definite degree: Flat, sour, metallic, sour, metallic, yeasty, malty, old milk, weedy, barny and lipase; and to a pronounced degree: Feed, acid, bitter, fruity, utensil, and whey-taint.
May possess slight onion and the following flavours to a definite degree: Flat, yeasty, malty, old milk, weedy, barny, lipase and sulfide; and to a pronounced degree: Feed, acid, bitter, fruity, utensil and whey-taint.
(b) Body and texture: A plug drawn from the cheese may possess the following characteristics to a definite degree: Curdy, coarse, corky, crumbly, mealy, short, weak, pasty, gassy, slitty, pinny; and to a pronounced degree: Open and sweet holes. The cheese shall be sufficiently compact to permit the drawing of a plug.
A plug drawn from the cheese may be slightly curdy and may possess the following other characteristics to a definite degree: Coarse, corky, gassy, slitty and pinny; and to a pronounced to a pronounced degree: Open, sweet holes, short, weak, pasty, crumbly and mealy. The cheese shall be sufficiently compact to permit the drawing of a plug.
A plug drawn from the cheese may possess the following characteristics to a definite degree: Gassy, slitty, pinny; and to a pronounced degree: Open, sweet holes, short, weak, pasty, crumbly and mealy. The cheese shall be sufficiently compact to permit the drawing of a plug.
(c) Colour: May have a slight bleached surface and possess the following other characteristics to a definite degree: Wavy, acid-cut, mottled, salt spots, dull or faded; and seamy to a pronounced degree. May be coloured or uncoloured but if coloured, may be definitely unnatural. The colour shall not be particularly unattractive.
May possess the following characteristics to a definite degree: Wavy, acid-cut, mottled, salt spots, bleached surface, dull or faded; and seamy to a pronounced degree. May be coloured or uncoloured but if coloured may be definitely unnatural. The colour shall not be particularly unattractive.
Same as for medium.
Bandaged and paraffin dipped. May possess very slight rind rot and the following other characteristics to a slight degree: Cracks in rind; soft spots and wet rind; and to a definite degree: Surface mould, mould under bandage and paraffin, huffed; and to a pronounced degree: Checked rind, weak rind, sour rind and huffed; and to a pronounced degree: Soiled surface, rough surface, defective coating, irregular bandaging, lopsided and high edges. Rindless. Same as for current, except definite mould under the wrapper or covering permitted.
Bandaged and paraffin dipped. May possess the following characteristics to a slight degree: Rind rot, cracks in rind; and to a definite degree: Checked rind, weak rind, sour rind, wet rind, soft spots and huffed; and to a pronounced degree: Rough surface, soiled surface, surface mould, mould under bandage and paraffin, defective coating, irregular bandaging, lopsided and high edges. Rindless. Same as for medium.
(d) Finish and appearance: Bandaged and paraffin dipped. May possess the following characteristics to a slight degree: Cracks in rind, soft spots and wet rind; and mould under bandage and paraffin; and to a definite degree: Soiled surface, surface mould, defective coating, checked rind, weak rind, sour rind, and huffed; and to a pronounced degree: Rough surface, irregular bandaging, lopsided and high edges. Rindless. The wrapper or covering shall be fairly smooth and properly scaled with adequate overlapping at the seams or by other satisfactory type of closure. The wrapper or covering shall adequately and securely envelop the cheese. May be definitely soiled and wrinkled but shall be of such character as to protect the surface of the cheese and not detract from its initial quality. May have slight mould under the wrapper or covering and may be definitely huffed and lopsided.
Sensory Character of Cheese and its Evaluation
473
Table 6 American Dairy Science Association ballot for judging the quality of Cheddar cheese. A score of 10 is awarded if the judge cannot find fault with the flavour of the cheese. A score of 5 is awarded if a judge cannot find fault with the body and texture of the cheese. When scores of 9 or less, or 5 or less, for flavour or body and texture, respectively, are awarded, the cause for deduction of marks should be indicated
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Sensory Character of Cheese and its Evaluation
and usually immediately upon opening a packed cheese, cutting a coated cheese or removing a trier plug from a cheese. Flavour judgement is made next, when a sample of cheese is placed in the mouth, chewed and moved around and then expectorated. As for odour, numerous uncharacteristic flavours may be detected in defective cheese, and in addition a cheese that is over-salty or very bitter may be considered defective. Finally, but sometimes simultaneously, body and texture are judged. Defects such as over-hardness, crumbliness, mealy and sticky are judged, most often by working a cheese between the thumb and the fingers. Table 7 presents a list of cheese sensory quality characteristics, which are mostly defects recognised internationally and are described in the IDF standard (IDE 1997). It should be noted that a characteristic considered to be a defect in one cheese type may be very much desired in another (e.g., the acceptable hardness of Parmigiano-Reggiano would be considered a defect in Cheddar), and therefore judges must take this into account, and evaluate based on their experience of each cheese type individually. In addition, it may be that a characteristic found in the same cheese type produced in two different countries may be considered defective in one country, but acceptable in another. This will be related to the experience of the cheese consumer in each country, which can be very different. However, cheeses produced in automated facilities today are much less likely to suffer from significant defects due to improved hygiene practices at
all stages of milk handling and cheesemaking, beginning on the farm. In addition, control over cheesemaking has improved significantly in recent years. Cheese grading or quality scoring provides a rapid and simple way quickly to assess overall sensory quality, but does not adequately take into account so-called 'non-quality' related differences in sensory characteristics that give the cheese of individual producers, or regions of production, a distinctive taste. Traditional 'quality criteria' are changing as product ranges expand (e.g., to include low-fat cheeses); variety of cheeses is much greater, and differentiation is increasingly made by purposely developing distinctive sensory characteristics, such as those now given to cheeses by the use of adjunct cultures. Sensory characteristics that are not traditionally considered defects, but which can also differ from one cheese to another, are now also important in determining eating quality for the discerning consumer. What is a negative attribute to one consumer may be a desirable attribute to another consumer. Also, although the characteristics that expert judges seek are those that their market demands, their assessments do not always coincide with those of consumers. It is now well documented that the consumer and the expert opinions of quality often differ. For example, McBride and Hall (1979) found that consumers' preferences among twelve cheeses, ranging from poor to good quality, were not correlated with their official grade scores. Finally, the current cheese-grading practice does not measure accurately the intensity of a given defect, and
Table 7 Terms used by cheese graders to describe sensory characteristics of cheeses that determine quality with particular emphasis on defects (IDF, 1997; Robinson and Wilbey, 1998) Exterior appearance Rind/surface
Appearance interior: Openings
Appearance interior: Colour
Consistency, body and texture
Flavour, odour and taste
Concave, convex, deformed (bulged), dirty, oblique, soiled, too flat, too high, vaulted (blown) Corroded, cracked, discoloured, dry, fatty, holes, incorrect mould, irregular mould, mould under covering, rotten, rough, smear under covering, smeary, speckled, spots of mould, thick, thin, too little mould, too little smear, too much smear, wet, wrinkled Blown, close, collapsed, cracks, distorted, foreign material, foreign mould, glossy openings, hoop side mould, many holes near the surface, nesty openings, no holes, not typical, pin-holed, spots of putrification, too few, too large, too many, too small, uneven, unevenly mouldy Bleached near the surface, bright, brownish, dirty, discoloured, grey, marbled, mottled, natural, pale/dull, red colour near the surface, speckled, streaky, strong, two-coloured, unevenly coloured, weak, yellow Brittle, chalky, close, coarse, crumbly, curdy, dripping, dry, elastic, firm, flaky, friable, gassy, granular (grainy), greasy, gritty, gummy, hard, harsh, hoop side sift, layered, leathery, long, lumpy, mealy, pasty, runny, rough, short, smeary, smooth, soft, soggy, spongy, springy, squeaky, sticky, stringy, thin (watery), tight, tough, uneven, wet Acid, alcoholic, ammoniacal, aromatic, bitter, bland, burnt, buttery, butyric acid, chemical, clove, cooked, cowy, creamy, ethereal, feedy, fermented, fishy, flat, flowery, foreign flavour, foul, foetid, fruity, garlic, goaty, harsh, malty, metallic, mild, mouldy, musty, musty-flat, nutty, off, oily, oniony, over-ripe, pale, peardrop, putrid, rancid, resinous, rich, ripe, sandy, salty, sharp, soapy, sour, spicy, stale, strong, superfine, sulphide, sweaty, sweet, tangy, tallowy, uncharacteristic, unclean, weedy, yeasty
Sensory Character of Cheese and its Evaluation
therefore further statistical analyses that determine the extent to which cheeses differ, and that mathematically relate composition to defect intensity, are not appropriate. It is important to note that there are still industry situations where grading or quality scoring may be appropriate due to a large number of products that must be assessed in a short period of time. However, these sensory tools were not designed to be quantitative or representative of the entire cheese sensory profile and are not ideal tools for research or marketing. Discrimination tests
Sensory discrimination tests differ from quality scoring tests in that they involve direct comparisons of cheeses to determine whether there is either an overall difference between them or whether they differ for a specific and designated characteristic. The most commonly used discrimination tests include the Paired Comparison (ISO, 1983a), Duo-Trio (ISO, 1991), Triangle (ISO, 1983b) and Ranking tests (I50, 1988). In the Paired Comparison test, two cheeses are presented for comparison with one another and assessors are asked whether they differ; generally, a difference for one specific sensory characteristic is tested. In the Duo-Trio test, assessors are asked which of the two products is the most similar to a third reference product, allowing a common reference to be used again and again. This test has obvious advantages for quality control, although it is not possible to maintain a consistent cheese reference over time. In the Triangle test, assessors are presented with three cheeses and asked to choose which is the most different from the other two. In the Ranking test, four to six cheeses are generally presented for comparison of a single-designated attribute, and the assessor is asked to rank them in order of increasing intensity of that attribute. In best practice, the assessors are forced to make a choice each time for all discrimination tests, thus eliminating response bias. Whether a difference exists or not is determined statistically, based on the number of choices a panel of assessors makes for each cheese in the test, using binomial tables or Chi-squared tests. Therefore, discrimination tests are the most objective and the most sensitive of sensory tests. An additional advantage of these tests is that they do not require well-trained assessors. The only requirement is that all assessors are reasonably sensitive and recognise and understand the designated attribute in a common way. W h e n compared with the traditional quality scoring methods, these discrimination procedures are by far better suited to application to research problems, they follow good sensory evaluation principles and do not encounter problems in scaling and statistical analyses.
475
For this reason, their principles should now be added to quality scoring methods in an attempt to introduce comparability between the scores of one judge and another. It is also common practice to carry out discrimination tests on cheeses to determine whether a difference exists prior to further testing by more costly methods that aim to describe and quantify differences. Descriptive analyses
A majority of scientists who study cheese are interested in understanding the fundamental reasons why a cheese 'tastes' as it does, not just whether the cheese is acceptable, and for this purpose quality control sensory methods are of little value. Descriptive sensory analysis refers to a collection of techniques that seek not only to discriminate between the sensory characteristics of a range of cheeses, but also to determine a quantitative description of all the sensory differences that can be identified. For example, Figs la and lb illustrate quantitative differences in perceived flavour, measured using descriptive analysis, between two hard Swiss cheeses and two Blue cheeses, respectively. All cheeses may be profiled in this way, providing objective and reproducible sensory descriptions of cheeses and providing a basis for determining what characteristics are influenced by changes in cheesemaking practice or composition, and also what characteristics are important for consumer acceptance. The most commonly used descriptive analysis methods for all food types include the Flavour Profile Method (Cairncross and Sj6strom, 1950), Texture Profile Method (Brandt et al., 1963), Quantitative Descriptive Analysis (QDA) T M (Stone et al., 1974), the Spectrum T M method (Meilgaard et al., 1999), Quantitative Flavour Profiling (Stampanoni, 1993a,b), and Free-Choice Profiling (Langron, 1983; Thompson and MacFie, 1983). A review of descriptive sensory analysis, which details advantages, disadvantages and applications of each of the methods referred to above was published recently by Murray et al. (2001). Each descriptive method has three stages to its implementation. The first involves selecting a panel to conduct the sensory evaluations, the second, establishing terminology or a vocabulary, by which to describe a products' sensory characteristics and the third, quantifying these sensory aspects. However, for each method, the process is somewhat different. In the cheese industry, as there is a strong tradition of judging that is linked to extensive knowledge of cheese, then it is a wise approach that seeks to build on this knowledge rather than to reinvent the wheel. If the investment in descriptive sensory testing is for the long term, then the Spectrum T M method, or a similar one, is preferable.
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Using this method, a group of cheese experts develop and define a descriptive language using a series of universal intensity scales upon which assessors score their perceptions (Drake et al., 2001; Drake and Civille, 2003). The sensory panels that will use the method, often at more than one research site, are then extensively trained. When trained, individual assessors must be able to discriminate between cheeses using each attribute in the descriptive language, repeat their assessments and agree with other panel members on the size and the direction of differences in cheese attributes. The advantages of this descriptive analysis technique are that one panel can be trained readily on several cheese types since one intensity scale is used, different types of cheese can be compared directly and panel scaling is less prone to drift with time (Drake and Civille, 2003). In addition, this approach is objective and allows comparison of results between panels, between laboratories, and from one time to another. For example, if one wishes to study the maturation of a cheese over time, then one must ensure that the differences observed in the results between 3, 6 and 9 months are related specifically to changes that occur in the cheese and not to changes in the performance of the sensory panel. If a cheese type is to be evaluated not very often, or the sensory panel available will not specialise in cheese only, or resources are limited, then the QDA T M approach may be preferable. Using this method, the panel of assessors develop and define the language themselves whilst tasting a wide range of the test cheeses (Murray and Delahunty, 2000b). Assessors must agree with other panel members on the meaning of terms in the descriptive vocabulary and repeat their assessments, but are not required to agree on how to use the attribute scales to rate intensity. When this method is used instead of the Spectrum T M method, it is more difficult to compare the results from one study with those from another in absolute terms. Free-Choice Profiling (FCP) is another useful descriptive analysis method (Williams and Langron, 1984). This method allows the use of untrained assessors, or consumers, to profile the sensory characteristics of cheese. Each assessor may use an individual descriptive vocabulary that they have developed themselves, and which they then readily understand, and data are analysed using Generalised Procrustes Analysis (GPA; Arnold and Williams, 1986). Free Choice Profiling has been used to describe Cheddar cheese (Jack et al., 1993; O'Riordan et al., 1998), Parmigiano-Reggiano (Parolari et al., 1994) and ewes' milk cheeses (Barcenas et al., 2003). The advantages of FCP are that accurate discrimination between cheeses in terms of perceived sensory characteristics can be achieved in a very short
477
time and at a relatively little cost, and that discrimination is based on a large selection of informative words that consumers use and with which they are familiar with. The main disadvantage is that it is difficult to correlate perceived intensity of sensory characteristics obtained in this way, as they are too numerous and imprecise, and there is no consensus vocabulary. To obtain improved accuracy, sensory panels used for descriptive analyses generally comprise of 10-12 assessors instead of a smaller number of experts (with the exception of FCP where 15-20 assessors are needed). These assessors are screened for sensory acuity and relative interest (Stone and Sidel, 1993). A panel or group of individuals is used as factors such as age, saliva flow and onset of fatigue vary between assessors. Assessors also vary in sensitivity to particular stimuli, and it is highly probable that they also vary in their concentration-response functions (Lawless et al., 1994; Williams, 1994). In addition, temporary illness or psychological bias can cause day-to-day changes in sensory ratings. The key point of objective descriptive analysis is that it should be reproducible and independent of consumer preferences. Unlike traditional quality methods that use scorecards, there is no judgment of 'good' or 'bad' as this is not the purpose of the evaluation. The trained sensory panel operates as an instrument and generates quantitative data analogous to instrumental data. As with any instrument, replication is required. Two guidelines have been published dealing with cheese texture (Lavanchy et al., 1994) and the aroma and flavour of cheese (B~rodier et al., 1997a). These guidelines are very valuable as they define descriptive vocabularies, and then detail a procedure for evaluation of each characteristic, including the use of universal scales that are standardised at a number of points with common food references. In addition, they provide translations of many descriptive characteristics of cheese in Spanish, French, Italian, English and German. However, it is important to note that sensory lexicons or languages are not finite and will continue to evolve with time, usage and application. Time-intensity sensory analyses The sensory methods discussed above do not account for the dynamics of flavour release from cheeses that occurs during their consumption. Nor do they account adequately for changes to cheese texture, which occur progressively during mastication and breakdown of a cheese in the mouth. When using conventional sensory procedures, particularly descriptive analyses, assessors 'time-average' their responses to arrive at a single intensity value. This looses much useful information such as
478
Sensory Character of Cheese and its Evaluation
rate of onset of stimulation, time and duration of maximum intensity, rate of decay of perceived intensity, time of extinction and total duration of the entire process (Lee and Pangborn, 1986). To determine most details about sensory characteristics, changes in sensory character that occur during cheese consumption (which can take up to 30 s for a 'bite-sized' piece) can be measured using time-intensity methodology (Lee and Pangborn, 1986), or in the case of texture, using progressive profiling (Jack et al., 1994). Time-intensity methods are useful for the study of new cheese types, such as low-fat cheeses, as the reduction in fat content not only influences sensory character development, but also the breakdown of the cheese in the mouth during consumption and the rate of release of compounds that contribute to flavour. For example, in a study of Cheddar cheese flavour, the time taken to reach maximum intensity for 'sharpness', 'bitterness' and 'astringency' was consistently longer in reduced-fat than in full-fat Cheddar and, more importantly, the rate of flavour release was greater (Shamil et al., 1991/92). Temporal differences in perception indicate an altered flavour balance, caused by reducing the fat content of the cheese, which may be important in consumer acceptability. Delahunty et al. (1996a) showed that a 'fruity' note, which might be considered an off-flavour (Aston et al., 1985; Urbach, 1993), became a dominant flavour characteristic sooner during consumption and at a much greater intensity in a low-fat Cheddar-type cheese than in the full-fat equivalent. Delahunty et al. (1996b) also demonstrated that improved relationships between volatile composition and perceived sensory characteristics could be achieved by relating time-intensity sensory data with dynamic volatile compound release data. Jack et al. (1994) found that the texture of Cheddar cheese was perceived to be relatively coarse and crumbly earlier in the chewing sequence, but became increasingly smooth and creamy as chewing progressed. In addition, other more subtle or specific cheese-dependent changes occurred as breakdown in the mouth progressed. It was hypothesised that knowledge of these dynamic changes in texture character is important for understanding consumer acceptability. Consumer acceptability testing
Trained sensory panels should not be asked to express a preference as their expert knowledge will introduce bias. To determine the eating quality of cheese, a naive consumer panel or subjective assessors are used. Ideally, these assessors will be regular consumers of the product type under test or represent the target market for the product. Such consumers bring their subjective experience to this test, for although their preferences
will be based on the sensory characteristics tested, they will be referring to past eating experience. In addition, when one considers that the target market may be children, elderly consumers, consumers in another country or consumers from a culture virtually unknown to the producer, then it becomes clear that the internal expertise in a company or organisation cannot hope to predict acceptability adequately. Consumer acceptability testing makes use of rating scales that measure relative dislikes and likes (e.g., the ninepoint hedonic scale (Peryam and Girardot, 1952)), discrimination tests based on preference (e.g., paired preference, ranked preference) or just right scales that ask a consumer how they feel about the designated sensory characteristic. It is recommended that a minimum of 50-60 targeted consumers be used for consume> sensory testing, and a greater number than this if one expects segmentation of preferences (MacFie and Hedderly, 1993). One of the biggest challenges in consumer research is the clarification of consumer language. Consumers may use terms that are ambiguous, have multiple meanings, are associated with 'good' or 'bad' or are combinations of several terms. Integrated terms, such as 'creamy', are often used by consumers to represent a combination of positive attributes. Determining exactly what attribute or attributes 'creamy' refers to (flavour or texture or mouthfeel) have been the subject of many studies relating consumer and trained sensory panels (Mela, 1988; Elmore et al., 1999; Bom Frost et al., 2001). Dacremont and Vickers (1994a,b), who used concept matching to clarify consumer perception of Cheddar cheese flavour, found that the concept of Cheddar cheese flavour is a consumer concept and probably varies widely among consumers, as does Cheddar cheese flavour itself. However, the number of consumers questioned was small and further studies with larger consumer groups, and with demographic information, including types (brand, age) of Cheddar cheese normally consumed, would provide additional clarification.
Influence of Cheesemaking Variables on Sensory Character During the past ten years or so, there have been numerous reports of the application of descriptive sensory analysis to determine accurately the influence of cheesemaking variables, e.g., maturation time and temperature, starter culture or use of adjunct cultures, on the sensory characteristics of cheese (Table 3). Studies of Cheddar cheese maturation have found that, overall, the intensity of odour, flavour and aftertaste is determined by the length (Piggott and Mowat,
Sensory Character of Cheese and its Evaluation
1991; Muir and Hunter, 1992a) and the temperature of maturation (Hannon et al., 2003). However, flavours such as milky/buttery and creamy decrease in intensity, while flavours such as sour, bitter, rancid and pungent increase in intensity (Piggott and Mowat, 1991; Muir and Hunter, 1992a; Hannon et al., 2003). Some textural changes, e.g., firmness, are controlled by the cheesemaking procedure and cheese composition, whereas mouth-coating character is related to maturation time (Piggott and Mowat, 1991; Muir and Hunter, 1992a). Hort and Le Grys (2001), who also studied Cheddar, found that springiness decreased, and crumbliness and creaminess increased as maturation progressed. Banks et al. (1993) and Fenelon et al. (2000) used descriptive analysis to determine the sensory properties of low-fat Cheddar cheese, and to compare these with the sensory properties of full-fat Cheddars. Fenelon et al. (2000) found that there were some differences in flavour characteristics related to fat content that were present regardless of the age of cheese. Fullfat cheeses were consistently more buttery, creamy and caramel-like. Adhikari et al. (2003) found that low-fat and full-fat Swiss cheeses, and low-fat Cheddar cheeses were dry and crumbly. Factory and farmhouse Cheddars have also been compared using descriptive sensory analysis (Muir etal., 1997a; Murray and Delahunty, 2000c); farmhouse cheeses were found to have a greater diversity in sensory characteristics. In addition, cheeses produced from pasteurised milk were found to be clearly different from those produced from unpasteurised milk, with the unpasteurised milk cheeses being more diverse in sensory character and more intensely flavoured (Grappin and Beuvier, 1997; Muir et al., 1997a; Murray and Delahunty, 2000c). Numerous studies have used descriptive sensory analysis to address the role of specific adjunct cultures or starter culture enzyme systems in Cheddar cheese flavour (Drake et al., 1996, 1997; Muir et al., 1996; Delahunty and Murray, 1997; Lynch et al., 1999; Banks et al., 2001; Broadbent et al., 2002). Muir et al. ( 1 9 9 6 ) demonstrated that starter culture type and adjunct determined the sensory character of cheese. However, they also found direct and interactive effects of composition. More recently, O'Riordan and Delahunty (2003a,b) found that starter culture type led to consistent differences in sensory characteristics between Cheddar cheeses, but that composition led to significant variation within batches of cheese made using the same starter culture. Delahunty and Murray (1997) also demonstrated differences between Cheddar cheeses based on starter culture type, although these cheeses were awarded the same grade score (Fig. 2). Descriptive sensory analysis has been used to determine the impact of yeast extract and milk standardisation
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with milk protein concentrate on reduced-fat Cheddar cheese flavour (Shakeel-ur-Rehman et al., 2003a,b,c) and of smoking parameters on cheese flavour (Shakeelur-Rehman et al., 2003d). There have been many studies of cheese types other than Cheddar, and to discuss them all would be impossible within the scope of this chapter. Of most interest are studies of Comte cheese using a flavour-descriptive vocabulary developed by B~rodier et al. (1997b; published in French). This lexicon has been used to identify naturally existing cheese geo-regions within France (Monnet et al., 2000). In addition, Virgili et al. (1994) used descriptive analysis to study the sensory-chemical relationships in Parmigiano-Reggiano cheese. Descriptive analysis of cheese texture has been conducted recently on a variety of cheeses, on cheeses of different fat contents and on fat replacers (Drake and Swanson, 1996; Drake et al., 1999a; Lobato-Calleros et al., 2001; Madsen and Ardo, 2001; Gwartney et al., 2002). In these studies, descriptive sensory analysis was used to differentiate cheeses and/or the impact of various treatments. A sensory texture language, like a cheese flavour language, is also not necessarily finite. The language will continue to be refined, particularly as additional cheeses are studied or as additional instrumental studies are conducted. The texture languages used by Drake et al.
480 Sensory Character of Cheese and its Evaluation (1999a) and Gwartney et al. (2002) were merged into one complete language by Brown et al. (2003).
Towards a Universal Cheese-Sensory Language As mentioned previously, some of the key advantages of using descriptive sensory vocabularies with definitions and references are the ability to communicate accurately results between multiple research groups or to reproduce research results at different sites. Hirst et al. (1994) compared the evaluation of cheese between trained British and Norwegian panels using independently developed sensory languages. Cross-cultural differences were attributed to the observed discrepancies in term usage and sample differentiation. More recently, ring trials at seven sites across the European Union were conducted and a core sensory language for evaluation was developed (Hunter and McEwan, 1998; Nielsen and Zannoni, 1998). While similar patterns of differentiation among samples by panels that use different languages are expected (particularly if the vocabularies are comprehensive and the panellists highly trained), standardised language with definitions and references improves communication, cross panel validation and subsequent application of descriptive analysis results to instrumental or consumer data. Further, other sources of variation potentially exist in comparing panel results at different sites within the same country using the same language. Drake et al. (2002) reported on the performance of three descriptive panels trained at different sites by different panel leaders on a previously developed and standardised cheese descriptive language (Drake et al., 2001). Panels were able to communicate accurately attribute differences between cheeses. However, differences were observed between these panels in scale usage and attribute recognition. These differences were attributed to the differences in panel leadership and the duration of panellist training. In a similar study, Martin et al. (2000) compared odour profile results of two panels. Language, scale and method of presentation were standardised. Results obtained from the two panels were similar. However, differences between attribute intensities were reported and were attributed to differences in the experience and/or perception of individual panellists. As with the conclusions of Drake etal. (2002), strong panel-leader interaction was recommended as a means of rectifying these differences, along with regular feedbacks between the two panels. As referred to previously, Tables 1, 2 and 3 present terms used for descriptive sensory analysis by different research groups for a wide variety of cheeses. In many cases similar terms have been used to describe dominant
characteristics of different cheese types, suggesting that it could be possible to develop and standardise a terminology that can be used universally and for all cheese types. The will to achieve this objective is much needed.
Relating S e n s o r y Characteristics to C o n s u m e r Preferences Preference mapping is a generic term given to a collection of techniques, which have emerged in recent years to quantify, analyse and interpret consumer preferences for products. A premise can be made that the preferences of a group of consumers of sufficient size (60 or more) will discriminate between comparable products based on intrinsic sensory differences, and that the degree and direction of discrimination will reflect the number and the intensity of sensory differences that can be perceived. Therefore, by simply quantifying and analysing preference, or acceptance for the range, a preference map reflecting sensory differences can be drawn. The preferences of individual consumers can be represented as a map loading, and areas of minimum and maximum preference can be identified. In addition, segmentation techniques, when used in tandem, can illustrate opportunities for a selection of optimised products within the same range (or sensory space). Analysis of consumer preference data in this way is referred to as internal preference mapping (McEwan, 1995; Schlich, 1995). When consumer preference evaluation of a set of cheeses is followed by the application of descriptive analysis to the same set of cheeses, this allows multivariate statistical analysis, e.g., using Partial Least Squares Regression (PLSR; Martens and Martens, 1986), and relation of descriptive properties that describe exactly what attributes are perceived and at what levels with the extent and direction of consumer preferences. This additional analysis facilitates interpretation of the internal preference map, and is referred to as external preference mapping (McEwan, 1995; Schlich, 1995). These techniques provide a powerful research tool for market analysis and new product development. One can extend the preference map by seeking technical extensions, or relationships between preferences, sensory characteristics and physical and chemical properties of products. One can also extend the preference map by seeking behavioural extensions, or by determining characteristics of the consumers and how they have developed their preferences and make their choice decisions. Preference mapping has been conducted with many products, including cheese (McEwan etal., 1989; Lawlor and Delahunty, 2000; Murray and Delahunty, 2000a,c; B~ircenas et al., 2001). Recently, Young et al.
Sensory Character of Cheese and its Evaluation 481 (2003) conducted preference mapping of Cheddar cheeses using consumers at two different locations (Oregon and North Carolina, USA). Seven Cheddar cheeses with distinct descriptive sensory properties were selected. Six distinct consumer clusters were identified, indicating a wide variability in consumer preferences even among one cheese type. Analysis of the consumer concept of 'aged cheese flavour' and 'young cheese flavour' indicated that consumers could differentiate between young and aged Cheddar cheeses and that these concepts were consistent with descriptive panel language. However, the consumer concept of 'Cheddar flavour' varied widely and was not pinpointed to specific descriptive cheese flavour terms. Lawlor and Delahunty (2000) conducted preference mapping with a diverse range of cheese types, and also found wide variability in consumer preferences. Although a Blue Shropshire cheese, described as coloured, mouldy and crumbly, was the least liked overall (162 consumers), it was preferred by two of seven segments of the consumer sample, representing 50% of the total questioned. On the other hand, a Gruyere cheese, described as fruity, sweet and firm, was preferred overall, but was the first choice of only one segment with 10 consumers.
Relating Sensory Perception to Chemical Components and Instrumental Measurements Relating defined sensory flavour and/or texture to specific instrumental tests or chemical compounds is an important and expanding area of research. Cheese flavour chemistry and texture analyses are addressed in detail in 'Cheese Flavour: Instrumental Techniques' and 'Rheology and Texture of Cheese' of Volume 1, but sensory characteristics of cheeses cannot be addressed without brief attention to this subject. Relating sensory perception to instrumental measurements is important because in certain cases an instrumental test would be more cost-effective and/or convenient than sensory testing. However, more importantly, establishment of key relationships between sensory perception and flavour chemistry or rheology provides the potential to link cheese flavour or texture to the technology of cheese production; this is a key issue in providing a consistent and high-quality product to the discerning consumer. Relating sensory language and chemical volatile compounds represents a challenge for several reasons. The relative concentration of a compound in a cheese is not necessarily a measure of its sensory impact due to different sensory thresholds and the effects of the food matrix on retention and release. The sensitivity and selectivity of the extraction technique must also be taken into account (Delahunty and Piggott, 1995).
Finally, only a small percentage of the volatile components in a food are odour-active (Friedrich and Acree, 1998; see also 'Cheese Flavour: Instrumental Techniques', Volume 1). Establishing these relationships is time-consuming and tedious. To use flavour as an example, extensive and relevant instrumental volatile analysis must be conducted, followed by gas chromatography-olfactometry (GC-O) and quantitative analysis to pinpoint volatiles of interest. On the sensory side, descriptive analysis with a defined and anchored language is required. Sensory threshold testing to confirm that key volatile compounds are above detection thresholds must be conducted, followed by descriptive sensory analysis of compounds in model systems across the concentration range found in the cheese to confirm the sensory response (Drake and Civille, 2003). It should also be noted that the perception of the cheese flavour is an integrated response to numerous mixed stimuli, including volatile compounds, nonvolatile compounds and structural properties. The perception of this stimulation is multi-modal, but simultaneous, and therefore very complex. Panelists tasted water-soluble extracts of Comte cheese to identify fractions, which had particular tastes, in an attempt to clarify the effect of peptides and amino acids on flavour (Salles etal., 1995). Preininger et al. (1996) used an unripened cheese matrix to evaluate both volatile and non-volatile flavour components of two Swiss cheese samples. A similar study was conducted on Emmental cheese and reduced-fat Cheddar cheeses (Rychlik etal., 1997; Suriyaphan etal., 1999). Suriyaphan etal. (2001) identified key chemical volatile components of cowy/barny and earth/bell pepper sensory perceived flavours in selected aged British Farmhouse cheeses. In this study, sensory properties were identified by descriptive sensory analysis, aroma volatiles were quantified by gas chromatography-mass spectrometry (GC-MS) and aroma properties described by GC-O. Suspected key volatiles were selected from GC-O data based on aroma properties and flavour dilution values. The selected aroma components were subsequently incorporated into mild (bland) cheese across the concentration range encountered in the Farmhouse cheeses and evaluated by descriptive analysis. Studies such as these provide convincing evidence of the contribution of particular compounds to flavour. Model systems have not as yet provided insights into the role of compound mixtures and the role of compounds at sub-threshold levels. These are complex issues and will require extensive future research. An alternative approach to determining the influence of composition on sensory character is to use multivariate statistical techniques, such as PLS, to determine
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relationships between compositional data and quantitative descriptive sensory data. This technique has the advantage of enabling comparison of all mathematically possible combinations of compositional variables with perceived intensity of one or more sensory characteristics, following theoretically the principle of the component balance theory (Mulder, 1952). The validity and value of relationships determined in this way will depend on the amount and type of compositional data collected, and the accuracy of both the compositional and the sensory data. Lawlor et al. (2001, 2002, 2003) determined predictive models using this technique for numerous flavour and texture attributes described in a wide variety of cheese types. Many studies have also been conducted to explore the relationships between sensory properties, compositional measurements and instrumental measurements of cheese texture (Wium et al., 1997; Bachmann et al., 1999; Drake etal., 1999b; Antoniou etal., 2000; Benedito etal., 2000; Truong etal., 2002) and to devise instrumental methods to assess more accurately or predict sensory properties of cheese (Sorensen and Jepsen, 1998; Breuil and Meullenet, 2001; Meullenet and Finney, 2002). Lawlor et al. (2001, 2002, 2003), using PLS, determined relationships between gross composition and perceived texture for a wide variety of cheeses, and found a number of consistent relationships. In particular, it was found that firmness was positively correlated with protein and mineral salt content, and negatively correlated with moisture and pH. Both hand and mouth terms can be used for sensory analysis of cheese texture (Drake et al., 1999c). In general, empirical texture tests and large-strain tests (compression) have been shown to correlate well with sensory bite terms (firmness, elasticity) although the correlation varies with cheese type, instrumental test and specific sensory term and definition. More recently, Brown et al. (2003) demonstrated specific knowledge gaps in relating sensory chewdown terms to rheological tests. Sensory rigidity and resiliency terms were correlated with rheological tests. However, chewdown terms such as 'degree of breakdown', 'cohesiveness', 'adhesiveness', 'smoothness of mass' and 'smoothness of mouth coating' were not related to instrumental tests. Additional work is needed to investigate the role that fundamental rheological tests can play in differentiating and relating to these important sensory texture parameters in cheese.
Conclusions The sensory characteristics of cheese determine the eating quality of cheese and consumer acceptability. The appearance, flavour and texture of cheese are extremely complex, not simply due to the very wide
diversity of cheese types that are produced, but also the many stages that any cheese goes through during its production and ripening. The complex composition and structure of cheese stimulate each of the human sensory modalities at approximately the same time, resulting in an integrated perception that a consumer responds to during and after cheese consumption. The dairy industry, including cheese production and marketing, has relied on outdated grading and judging methods for quality control and product development for many years. While these methods still have use, objective descriptive analysis techniques are increasingly being applied in cheese quality research in parallel with innovative studies of cheesemaking, cheese composition and consumer acceptability of cheese. Advances in the application of objective sensory science techniques have improved understanding of the relationships between these factors and the sensory attributes of cheese. However, direct comparison of research findings between different laboratories working with the same cheese type, and between studies on different types of cheese, will not be possible until such time as a universal language to describe cheese sensory character is defined and standardised.
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Papademas, P. and Robinson, R.K. (2001). The sensory characteristics of different types of halloumi cheese as perceived by tasters of different ages. Int. J. Dairy Technol. 54, 94-99. Parolari, G., Virgili, R., Panari, G. and Zannoni, M. (1994). Development of a vocabulary of terms for sensory evaluation of Parmigiano-Reggiano cheese by flee-choice profiling. Ital. J. Food Sci. 3, 317-324. Peryam, D.R. and Girardot, N.E (1952). Advanced taste test method. Food Eng. 24, 58-61. Piggott, J.R. (1988). Sensory Analysis of Foods, 2nd edn, Elsevier Applied Science, Barking, Essex. Piggott, J.R. and Mowat, R.G. (1991). Sensory aspects of maturation of Cheddar cheese by descriptive analysis. J. Sens. Stud. 6, 49-62. Preininger, M., Warmke, R. and Grosch, W. (1996). Identification of the character impact flavour compounds of Swiss cheese by sensory studies of models. Z. Lebensm. Unters. Forsch. 202, 30-34. Roberts, A.K. and u Z.M. (1994). A comparison of trained and untrained judges evaluation of sensory attribute intensities and liking of Cheddar cheeses. J. Sens. Stud. 9, 1-20. Robinson, R.K. and Wilbey, R.A. (1998). Cheesemaking Practice, R Scott 3rd edn., Aspen Publications, Inc., Gaithesburg, MD. Rychlik, M., Warmke, R. and Grosch, W. (1997). Ripening of Emmental cheese wrapped in foil with and without addition of Lactobacillus casei ssp. casei. III. Analysis of character impact flavour compounds. Lebensm. Wiss. Technol. 30,471-478. Salles, C., Septier, C., Roudot-Algaron, E, Guillot, A. and t~tievant, P.X. (1995). Sensory and chemical analysis of fractions obtained by gel permeation of water soluble Comte cheese extracts. J. Agric. Food Chem. 43, 1659-1668. Schlich, P. (1995). Preference mapping: relating consumer preferences to sensory or instrumental measurements, in, Bioflavour 95, P. Etievant and P. Schreier, eds, INRA, Paris. pp. 135-150. Shakeel-ur-Rehman, Farkye, N. and Drake, M.A. (2003a). Effects of standardization of whole milk with dry milk protein concentrate on the yield and ripening of reduced-fat Cheddar cheese. J. Dairy Sci. 86, 1608-1615. Shakeel-ur-Rehman, Farkye, N. and Drake, M.A. (2003b). Reduced-fat Cheddar cheese from a mixture of cream and liquid milk protein concentrate. Int. J. Dairy Technol. 56, 94-98. Shakeel-ur-Rehman, Farkye, N., Vedamuthu, E. and Drake, M.A. (2003c). A preliminary study on the effect of adding yeast extract to cheese curd on proteolysis and flavor development of reduced fat Cheddar. J. Dairy Res. 70, 99-103. Shakeel-ur-Rehman, Farkye, N. and Drake, M.A. (2003d). The ripening of smoked Cheddar cheese. J. Dairy Sci. 86, 1910-1917. Shamil, S., Wyeth, L.J. and Kilcast, D. (1991/92). Flavour release and perception in reduced-fat foods. Food Qual. Pref. 3, 51-60.
Sorensen, L.K. and Jepsen, R. (1998). Assessment of sensory properties of cheese by near-infrared spectroscopy. Int. DairyJ. 8,863-871. Stampanoni, C.R. (1993a). Quantitative flavour profiling: an effective tool in flavour perception. Food Marketing Technol. February, 4-8. Stampanoni, C.R. (1993b). The quantitative profiling technique. Perfum. Flavour. 18, 19-24. Stampanoni, C.R. (1994). The use of standardized flavor languages and quantitative flavor profiling technique for flavoured dairy products, J. Sens. Stud. 9, 383-400. Stevenson, R.J., Prescott, J. and Boakes, R. (1999). Confusing tastes and smells: how odours can influence the perception of sweet and sour tastes. Chem. Senses 24, 627-635. Stone, S. and Sidel, J.L. (1993). Sensory Evaluation Practices, 2nd edn, Academic Press, Inc., London. Stone, H., Sidel, J., Oliver, S., Woolsey, A. and Singleton, R.C. (1974). Sensory evaluation by quantitative descriptive analysis. Food Technol. 28, 24-34. Suriyaphan, O., Drake, M.A. and Cadwallader, K.R. (1999). Identification of volatile off-flavors in reduced-fat Cheddar cheeses containing lecithin. Lebensm. Wiss. Technol. 32,250-254. Suriyaphan, O., Drake, M.A., Chen, X.Q. and Cadwallader, K.R. (2001). Characteristic aroma components of British Farmhouse Cheddar cheese. J. Agric. Food Chem. 49, 1382-1387. Thompson, D.M.H. and MacFie, H.J.H. (1983). Is there an alternative to descriptive sensory assessment?, in, Sensory Quality in Food and Beverages: Definition, Measurement and Control, A.A. Williams and R.K. Atkin, eds., Ellis Horwood Ltd, Chichester. pp. 96-107. Truong, V.D., Daubert, C.R., Drake, M.A. and Baxter, S.R. (2002). Vane rheometry for textural characterization of Cheddar cheese: correlation with other instrumental and sensory measurements. Lebensm. Wiss. Technol. 35, 305-314. Urbach, G. (1993). Relations between cheese flavour and chemical composition. Int. Dairy J. 3,389-422. Virgili, R., Parolari, G., Bolzoni, L., Barbieri, G., Mangia, A., Careri, M., Spagnoli, S., Panari, G. and Zannoni, M. (1994). Sensory chemical relationships in ParmigianoReggiano cheese. Lebensm. Wiss. Technol. 27,491-495. Warmke, R., Belitz, H.D. and Grosch, W. (1996). Evaluation of taste compounds of Swiss cheese (Emmentaler). Z. Lebensm. Unters. Forsch. 203,230-235. Weel, K.G.C., Boelrijk, A.E.M., Alting, A.C., van Mil, P.J.J.M., Burger, J.J., Gruppen, H., Voragen, A.G.J. and Smit, G. (2002). Flavor release and perception of flavored whey protein gels: perception is determined by texture rather than by release. J. Agric. Food Chem. 50, 5149-5155. Wendin, K., Langton, M., Caous, L. and Hall, G. (2000). Dynamic analyses of sensory and microstructural properties of cream cheese, Food Chem. 71,363-378. Williams, A.A. (1994). Flavour q u a l i t y - understanding the relationship between sensory responses and
Sensory Character of Cheese and its Evaluation
chemical stimuli. What are we trying to do? The data, approaches and problems. Food Qual. Pref. 5, 3-16. Williams, A.A. and Langron, S.P. (1984). Use of free-choice profiling for evaluation of commercial ports. J. Sci. Food Agric. 35,558-568.
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Wium, H., Gross, M. and Qvist, K.B. (1997). Uniaxial compression of UF-Feta cheese related to sensory texture analysis. J. Text. Stud. 28,455-476. Young, N., Drake, M.A., Lopetcharat, K. and McDaniel, M. (2004). Preference mapping of Cheddar cheeses. J. Dairy Sci. 87, 11-i9.
Cheese Flavour: Instrumental Techniques J.-L. Le Qu~r~, Institut National de la Recherche Agronomique (INRA), Unite Mixte de Recherche sur les Ar6mes (UMRA), Dijon, France
Introduction The sensory properties of food are important determinants in the choice of foodstuffs by the consumer, and flavour plays a prominent role in this context. Flavour may be defined as the combination of taste and odour, sensations of pain, heat and cold (chemesthesis or trigeminal sensitivity), and tactile sensation. Sensory analysis is clearly the most valid means of measuring flavour characteristics. Applied to cheese flavour, sensory evaluation is a prominent descriptive tool which is used widely in dairy science and industry (Issanchou et al., 1997; see also 'Sensory Character of Cheese and its Evaluation', Volume 1). However, determining flavour also means analysing volatile compounds that are sensed in the nose at the olfactory receptors either via the orthonasal (odour) or retronasal (aroma) routes when foods are eaten, non-volatile compounds sensed on the tongue (taste), and compounds perceived as mouthfeel and texture. Instrumental analyses of flavour have been used primarily to analyse volatile components. The main reason for this is the major importance of aroma in the overall flavour of a food, as is easily demonstrated by the difficulties encountered by subjects attempting to identify a particular flavour if the air flow through the nose is prevented, and the fact that volatile components are more amenable to conventional instrumental analysis than non-volatile compounds. Therefore, since the early studies published in the 1960s and the 1970s (Dumont and Adda, 1972, and references cited therein), instrumental methods have concentrated on identification of aroma compounds (Mariaca and Bosset, 1997). Only recently, some significant efforts have been made to develop instrumental procedures to characterise non-volatile components in cheese which are responsible for cheese taste (Salles et al., 1995a; Salles and Le Qu~r~, 1998; Engel et al., 2000a,b; Le Quere and Salles, 2001). Instrumental analysis of aroma volatiles has been the subject of important specialised treatises (for the most recent literature on the subject, see Ho and Manley, 1993; Marsili, 1997; Mussinan and Morello, 1998; Stephan etal., 2000; van Ruth, 2001a; Reineccius, 2002, and specifically for instrumental analysis of
volatiles in milk and dairy products see Delahunty and Piggott, 1995; Mariaca and Bosset, 1997). Therefore, the part of this chapter that will be devoted to the analysis of cheese volatiles will focus on particular techniques adapted to the particular characteristics of cheese. Cheese flavour components result from the principal biochemical degradation pathways: glycolysis, lipolysis and proteolysis (see 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening' and 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1). The aroma compounds produced are mainly hydrophobic, or lipophilic, and consequently they tend to concentrate in the cheese fat according to their water/fat partition coefficient. Instrumental analysis of cheese volatiles must, therefore, consider, as a first step, an extraction method suitable for separating these volatiles from the cheese fat matrix. However, no single method yields a 'true' picture of a food aroma (Reineccius, 2002), and isolation and analysis of aroma remain challenging (Teranishi, 1998). Moreover, not only may the extraction step lead to artefacts, but the total volatile content in most cases is very difficult to relate to the flavour profile determined by a panel in sensory evaluation. Therefore, it appears much more efficient to concentrate efforts on the identification of those compounds that are really relevant to flavour. As no universally suitable extraction method exists, it appears essential to choose a method that yields an extract representative of the sensory properties of the food (Abbott et al., 1993; Eti~vant et al., 1994; Eti~vant and Langlois, 1998). Once this extraction method has been chosen, the next steps involve various forms of gas chromatography among which gas chromatography-olfactometry (GC-O) plays a prominent role in determining the key volatile compounds that contribute significantly to the flavour of the food (Leland et al., 2001), and gas chromatography-mass spectrometry (GC-MS), which is essential for the identification of those key odorants. Water-soluble extracts (WSE) from cheese have strong flavours (Biede and Hammond, 1979; McGugan
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et al., 1979; Aston and Creamer, 1986). Such extracts contain some volatile compounds (Le Quere et al., 1996; Engels et al., 1997; Le Quere and Salles, 2001), partly extracted by water according to their water/fat partition coefficient, although flavour compounds are generally more lipophilic than hydrophilic. However, the water extract mainly contains non-volatile compounds. This non-volatile, water-soluble fraction is composed of mineral salts, lactic acid, lactose, amino acids and peptides and has characteristic taste properties (Salles et al., 1995a). Amino acids and small peptides are considered to be mainly responsible for the taste characteristics of water-soluble extracts (McGugan et al., 1979; Aston and Creamer, 1986), their flavour impact being modulated by interaction with calcium and magnesium ions (Biede and Hammond, 1979). Moreover, it has been recognised for a long time that water-soluble, low molecular weight and mainly hydrophobic peptides, which accumulate during ripening as a result of proteolysis, are responsible for bitterness in cheese (Lowrie and Lawrence, 1972; Schalinatus and Behnke, 1975; Furtado, 1984; Lemieux and Simard, 1992). Some fundamental studies on model compounds have characterised the tastes of amino acids and low molecular weight peptides (Salles et al., 1995a and references cited therein); studies conducted on tastes in casein hydrolysates were reviewed by RoudotAlgaron (1996). However, until recently and apart from bitterness, no clear sensory data were obtained on water-soluble extracts from cheese. Although several hundred peptides have been isolated and identified from various types of cheese, only a few small peptides, that are suspected to be responsible for particular tastes, were isolated from the water-soluble fractions of various cheeses and identified (Salles et al., 1995a and references cited therein). However, no direct correlations between these peptides and the organoleptic properties of the fractions have been demonstrated, apart from bitterness. In fact, the watersoluble fraction of cheese generally has a very complex composition, and separation and identification of individual compounds are difficult. Moreover, most analytical techniques require the use of non-food-grade solvents or buffers that make sensory evaluation of sub-fractions difficult or impossible. Part of this chapter will focus on recent advances made to study and identify the taste-active components present in the water-soluble fraction of cheese. A general procedure for the preparation of fractions involves an extraction of grated cheese by water followed by a fractionation scheme, generally adapted from the fractionation protocol used to isolate cheese nitrogen fractions in the study of proteolysis in cheese during ripening (Fox et al., 1994; McSweeney and
Fox, 1997). However, as sub-fractions have to be evaluated sensorially to assess their relative sensory impact and try to link it to their chemical composition, a suitable eluent has to be used in the chromatographic steps. Water (Roudot-Algaron et al., 1993; Salles et al., 1995a; Molina et al., 1999) or water-food-grade ethanol mixtures (Lee and Warthesen, 1996a,b) have been used for this purpose in combination with gel permeation chromatography (GPC) or high-performance liquid chromatography (HPLC). The final identification step generally involves mass spectrometry (MS) and tandem mass spectrometry (MS/MS) of nitrogenous compounds isolated using HPLC, either in a standalone mode or coupled with a mass spectrometer (HPLC-MS) (Roudot-Algaron etal., 1993, 1994b; Sommerer et al., 2001). A specific method for the isolation of small peptides from cheese has been described (Sommerer et al., 1998a). As already outlined for cheese aroma, the relationships between all flavour compounds identified in a food and sensory perception experienced by consumers when eating this food are still not entirely clear. In fact, it is particularly difficult to predict a flavour perception as it is still not known how the various components combine to produce an overall sensory impression. Moreover, interactions between taste and aroma (Noble, 1996) and interactions of trigeminal sensations with taste and aroma (Green, 1996) occur and play an important role in overall flavour perception. However, methods that allow direct analysis of flavour molecules released in the mouth during consumption have been developed in recent years (Taylor and Linforth, 1996; Roberts and Taylor, 2000). Development of instrumental techniques and data obtained recently for volatile and non-volatile flavour compounds in cheese will be presented which may explain the link between flavour perception and cheese composition. Finally, specific instrumental techniques have been developed for the analysis of the complete flavour of cheese. The methods currently used in the quality control of food flavour are still usually based on sensory evaluation by a panel of experts. These panels are able to monitor the quality of a particular food, to detect defects and to compare samples for classification purposes. Nevertheless, obtaining results rapidly at low cost using instruments could be desirable. The so-called 'electronic noses' based on gas sensor technology, despite some important drawbacks for some of them (Schaller et al., 2000a), are theoretically able to perform some classification tasks (Schaller etal., 1998), and some applications for the analysis of cheese have been developed (Mariaca and Bosset, 1997; Schaller et al., 1999). However, two other global
Cheese Flavour: InstrumentaITechniques
analysis methods based on mass spectrometry seem more powerful and reliable for purposes of classification. One of these methods analyses total headspace using a mass spectrometer, without any prior GC separation (Vernat and Berdague, 1995). This method is often referred to as a mass-based electronic nose. Alternatively, headspace sampling may be replaced by solidphase microextraction (SPME) of food volatiles (Marsili, 1999). Both sampling methods, followed directly by mass spectrometry, have found applications for the rapid characterisation of cheese (Schaller et al., 2000b; Peres et al., 2001, 2002a). The second method is pyrolysis }nass spectrometry (Aries and Gutteridge, 1987), where a small food sample is pyrolysed at up to 500 ~ The resulting volatile fraction, characteristic of the flavour but also of the matrix composition, is analysed by a mass spectrometer. As with the other rapid instrumental methods for classification, a pattern or fingerprint is obtained for each sample, and extensive data treatment, either by conventional multivariate statistics or artificial neural networks, allows the construction of maps useful for classification and quality control purposes (Peres et al., 2002b).
Characterisation of Aroma (Volatiles) Sample treatment
Volatile aroma compounds in cheese, like in other foodstuffs, are hydrophobic, generally distributed in a heterogeneous manner throughout the matrix and present at low or even traces (<10 txg/kg) concentrations. Their analysis in cheese requires homogenisation of the sample prior to extraction, where isolation procedures adapted to lipophilic material dispersed in trace amounts in a high-fat food are required. A practice commonly used for cheese is freezing the sample in liquid nitrogen, followed by grating to a fine powder with a blender at low temperature. The rind is generally removed before sample homogenisation. The powder is then used for subsequent steps as such, or after dispersion and homogenisation in water, with possible pH adjustment, if necessary. Extraction methods
As already outlined, all the extraction procedures used to isolate the aroma fraction from the cheese matrix should be adapted to the analysis of trace levels of lipophilic material dissolved in a fatty phase, while minimising losses of highly volatile molecules and preventing modification of compounds or the formation of artefacts. Many techniques have been proposed for the extraction of volatile compounds from cheese, amongst
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which, the most traditional and popular methods, based on the volatility of aroma compounds, involve distillation. Although steam distillation methods, and particularly the simultaneous steam distillation/solvent extraction (SDE) technique (Chaintreau, 2001) are still used for dairy products, they have several drawbacks. Highly volatile compounds are recovered poorly, thermally sensitive compounds may disappear and artefacts may appear unless distillation is performed under a reduced pressure, with tight control of temperature. In either approach, at atmospheric pressure or under vacuum, the quality of the aroma extract finally obtained is dependent on the volatility of the aroma compounds and on their solubility in the solvent used. If steam distillation (also called hydrodistillation) is used without simultaneous solvent extraction, it is necessary to add a large quantity of water to the grated cheese to obtain a homogeneous slurry (c. 1 1 for 100 g of cheese). The distillate obtained is in fact a dilute aqueous solution of volatile compounds (Dumont and Adda, 1972). A subsequent extraction with large amounts of a suitable solvent, followed by a concentration step, is required. High-vacuum distillation techniques, on the contrary, produce small volumes of concentrated aqueous distillates (cheese moisture content only) that can be extracted with tiny volumes of an organic solvent. A typical experiment involves two steps. In the first step, the frozen grated cheese is transferred to a cone-shaped flask that is connected to a static vacuum (c. 10 Pa) renewed from time to time. Using sub-ambient temperature and rotation of the flask in order to break the continuously dehydrating surface of the sample, the volatiles are condensed with most of the cheese water in traps maintained at the temperature of liquid nitrogen (Dumont and Adda, 1972; Le Quere and Molimard, 2002). In the second step, the flask containing the dehydrated cheese powder is connected to a molecular distillation apparatus operating under a high vacuum (c. 10-2pa). In this step, also called 'cold-finger molecular distillation', the remaining water and volatiles are transferred directly to the surface of a cold condenser maintained at the temperature of liquid nitrogen and situated at a very short distance from the surface of the sample (Fig. 1). The condensed ice layer on the surface of the condenser contains less volatile and more lipophilic compounds. This fraction is combined with the aqueous distillate obtained in the first step. Since early workers (see Dumont and Adda, 1972 and references cited therein), high-vacuum distillation techniques have been applied to the extraction of aroma compounds from a large variety of cheeses (see Mariaca and Bosset, 1997 and references cited therein; Moio and Addeo, 1998; Moio et al., 2000). Working at
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C h e e s e Flavour: I n s t r u m e n t a I T e c h n i q u e s
K
L
J
E N
I
Figure 1 Apparatus used for cold-finger molecular distillation. A, I, traps for volatiles cooled with liquid nitrogen; E, round-bottom flask containing dehydrated cheese powder, equipped with a 'cold-finger' cooled with liquid nitrogen; F, H, J, K, L, high-vacuum stopcocks; M, connection to the high vacuum pumping system; N, guard trap cooled with liquid nitrogen.
ambient or even at sub-ambient temperature, the techniques prevent thermal degradation, but need a substantial amount of sample (c. 50-250 g) and are very time-consuming (a long distillation period up to a few hours). Moreover, the aqueous distillates have to be extracted with a suitable solvent (e.g., dichloromethane or diethyl ether) before performing further analysis. A chemical fractionation by controlling the pH of the aqueous distillate results in separation of the organic extracts into acid, neutral and basic fractions which may be analysed separately (Mariaca and Bosset, 1997; Reineccius, 2002). A nice example on Swiss Gruyere cheese was published 20 years ago (Liardon et al., 1982; Bosset and Liardon, 1984, 1985; Bosset et al., 1993). Specific methods for the analysis of volatile free fatty acids may be found in the literature (see for example Ha and Lindsay, 1990). Cheese volatiles may be extracted directly from samples by a solvent (e.g., diethyl ether). However, further steps are required to separate the aroma from the lipids that are also extracted very efficiently by the solvent. While a simple solvent extraction introduces some bias into an aroma profile, the following steps, which are necessary, may add more bias (Reineccius, 2002). For instance, separation of volatile and nonvolatile compounds that have dissolved in the solvent by distillation under high vacuum has been used by Grosch and co-workers in the study of Swiss (Preininger and Grosch, 1994; Preininger et al., 1994; Rychlik et al., 1997) and Camembert (Kubickova and Grosch, 1997) cheese and more recently by Qian and Reineccius (2002a) in a study on Parmigiano-Reggiano cheese. The risk in this case is that only the most volatile components are selected from an oil-rich phase (Reineccius,
2002). Therefore, direct solvent extraction methods that necessitate a subsequent distillation step under vacuum do not offer a significant advantage compared to other vacuum distillation methods. Dialysis techniques, that are based on molecular size differences and which separate molecules according to their ability to diffuse through a specific membrane at room temperature, may seem a good alternative. Reineccius and co-workers have used this technique (Benkler and Reineccius, 1979, 1980) and compared it to other methods for the isolation of volatiles from Cheddar cheese (Vandeweghe and Reineccius, 1990). More recently, Spinnler and co-workers reinvestigated the technique (Molimard and Spinnler, 1993) and applied it to the extraction of aroma volatiles in the study of the impact of the microflora on the aroma of Camembert-type cheese (Molimard, 1994; Spinnler et al., 1995). In order to eliminate adsorption and artefact formation, these authors used 1% water in the solvent, diethyl ether, to inactivate acidic sites on the perfluorosulphonic acid membrane. They also improved the dialysis yield by recycling the solvent using a distillation device to recycle the solvent attached to the dialysis cell (Fig. 2) in order to maintain a maximum concentration gradient between the two compartments of the cell (Molimard, 1994). Nevertheless, the method is time-consuming (72 h dialysis time), and its efficiency decreases dramatically as the number of carbon atoms of the aroma molecule increases (n > 10), modulated by their hydrophobicity (Molimard and Spinnler, 1993). A high-performance size-exclusion chromatographic method has also been described for the purification of aroma compounds from organic extracts of fat-containing food (Lubke et al., 1996). The method was applied
A
I IF i I
E m
E
m
Figure 2 Dialysis cell with solvent recycling device. A, B, cell compartments; C, round-bottom flask containing solvent to distil; D, condenser; E, magnetic stirrers; F, dialysis membrane.
Cheese Flavour: InstrumentaITechniques
successfully to the clean-up of a dichloromethane extract from goat cheese (L(ibke et al., 1996). The main interest in this size-exclusion chromatographic method is the limited number of injections necessary and the reduced final volume of the fractions, which in terms of final useful concentration, appeared significantly quicker and gave rise to less thermally induced artefacts and to reduced losses of the most volatile components than any other distillation method (Lobke et al., 1996). Headspace methods, either static or more often dynamic, also called 'purge-and-trap' methods, are popular techniques used to isolate volatiles from cheese. Although direct analysis of the equilibrium headspace would appear to be an ideal method to study aroma compounds, in terms of sensory representativeness and ease of use, static headspace techniques have severe limitations in terms of sensitivity, being restricted to the most volatile and abundant components (Mariaca and Bosset, 1997; Reineccius, 2002). Dynamic headspace, or 'purge-and-trap', methods are basically pre-concentration and enrichment techniques. They use stripping of the volatiles from the cheese samples, sometimes dispersed in water, with an inert gas. The volatiles are concentrated in a cold trap or adsorbed onto an inert support (adsorbing polymer, generally of the Tenax | type) and analysed by subsequent thermal desorption or elution by a suitable solvent (Mariaca and Bosset, 1997; van Ruth, 2001a; Reineccius, 2002). Although dynamic headspace methods minimise artefacts developed or introduced during sampling (van Ruth, 2001a), distortion of the aroma profile may result from the trapping of aromas (Reineccius, 2002), especially when polymeric adsorbents are used. However, despite the drawback of relatively poor sensitivity compared to other extraction methods, the main advantages of dynamic headspace techniques are the small amount of sample needed to perform the analysis (c. 20 g) and its speed (Le Quere and Molimard, 2002). The technique, even though it favours the isolation of the most volatile flavour compounds (Reineccius, 2002), has been applied widely to the analysis of cheese volatiles (see for example Arora et al., 1995; Canac-Arteaga et al., 1999a,b, 2000; Larrayoz et al., 2001; Rychlik and Bosset, 2001a,b). Recent comprehensive reviews on the technique include Wampler (1997) and Pillonel et al. (2002). A comparative study on the advantages of the use of dynamic headspace with cheese samples in the 'dry' form or in 'dispersed suspension' in water has been published recently (Larrayoz et al., 2001). The 'dry' method allowed the extraction of a greater number of compounds and in larger quantities, but a few compounds were extracted better using the 'suspension' technique (Larrayoz et al., 2001). Simultaneous distil-
493
lation extraction (SDE) was also used in this study and compared to dynamic headspace analysis. As expected, the authors concluded that the techniques were complementary; dynamic headspace extracted more highly volatile compounds and SDE was more efficient for phenols, free fatty acids, lactones and heavier aldehydes, ketones, alcohols and esters (Larrayoz et al., 2001). Interference from water in dynamic headspace that could be detrimental to the efficiency of the technique has been discussed in detail by Canac-Arteaga et al. (1999a,b, 2000) and Pillonel et al. (2002). Solid-phase microextraction, first developed for the extraction of volatile organic compounds in water, has been applied recently to the isolation of aroma compounds from food (Harmon, 1997; Pillonel et al., 2002; Reineccius, 2002). Solid-phase microextraction partitions analytes between a liquid or a vapour phase and a thin solid-phase adsorbent, of which there are several choices in terms of polarity and film thickness, coated on inert fibres, generally associated with a syringe which serves as a direct injection device (Harmon, 1997). The method, which is an equilibrium one, can be performed either in the direct extraction mode (immersion of the fibre in the sample matrix, generally in an aqueous solution or suspension) or in a headspace configuration. It can be automated very easily, but the extraction of the solutes depends on polarity, volatility, partition coefficients, sample volume, temperature and the nature of the adsorbentcoating material. Therefore, the technique exhibits a certain degree of selectivity, but with the advantages of sensitivity, ease of use, no solvent and small sample volume (Harmon, 1997; Pillonel et al., 2002; Reineccius, 2002). Solid-phase microextraction, used for the first time for the analyses of cheese volatiles by Chin et al. (1996), has since been used in some significant applications on cheese aroma (Dufour et al., 2001; Pillonel et al., 2002 and references cited therein). Analysing volatiles directly by immersing the fibre in highly complex matrices (as cheese) could damage the fibre, and SPME is, therefore, used almost always in the headspace mode. Comparison of direct SPME and headspace SPME of Camembert volatiles obtained after cryo-trapping of the aqueous phase under vacuum showed only a slight reduction in sensitivity using headspace SPME compared to direct SPME (Jaillais et al., 1999). The water-soluble extract (WSE) of cheese has been described for a long time as possessing a strong flavour (Biede and Hammond, 1979; Aston and Creamer, 1986; Engels and Visser, 1994; Salles et al., 1995a). Besides non-volatile materials responsible for taste, WSE also contains volatile compounds responsible for its intense aroma. Thus, water-soluble extracts of various
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Cheese Flavour: InstrumentaITechniques
cheeses, obtained by direct extraction with water followed by various centrifugation steps (Le Quere et al., 1996; Engels et al., 1997; Engel et al., 2002c) or by pressing to obtain an aqueous phase called 'cheese juice' (Salvat-Brunaud etal., 1995; Thierry etal., 1999), have been investigated for their volatile components. To be analysed using gas chromatography, WSEs were either extracted with a suitable solvent (Le Quere et al., 1996), submitted to dynamic headspace analysis (Engels et al., 1997; Thierry et al., 1999) or fractionated using nanofihration as the final membrane-filtration step (Engel et al., 2002c).
Representativeness As already outlined, because there is no universally applicable method, none of the extraction techniques described above yields an aroma isolate that truly represents either qualitatively or quantitatively the aroma profile of a food (Reineccius, 2002). This fact explains the frequently observed discrepancies between aroma analysis of a food extract and sensory analysis of the food itself. Therefore, the flavour analyst must choose the isolation procedure best suited to address the problem faced: determination of the complete aroma profile, identification of key odorants or off-flavours, monitoring aroma changes with time in foods or prediction of sensory properties (Reineccius, 2002). When the ultimate aim of a particular study is the identification of the compounds that are important for flavour (the key odorants), the most reliable results will be obtained if the odour of the extract resembles closely that of the food itself (Etievant et al., 1994; Etievant and Langlois, 1998). Different sensory methods, which necessitate a trained sensory panel, can be used to check the sensory representativeness of the food extract odours (Etievant et al., 1994). When an estimation of the relative importance of key constituents in a single sample is required, a similarity test is preferred. The panellists are asked to score the similarity of the odour of the extracts obtained by different methods to the odour of the food itself used as reference on an unstructured 10 cm scale. This approach was applied to three French and Swiss hardtype cheeses by Etievant et al. (1994) and Guichard (1995). It was shown that the distillates obtained at a pressure in the range 10-100 Pa had odours more similar to those of the cheeses than the distillates obtained at a lower pressure (10 mPa). This result means that strongly absorbed and less volatile flavour compounds, extracted only at lower pressure, may not be important for the odour of these cheeses. Similar results were obtained for extracts of Camembert cheese, showing clearly that the second step (molecular distillation operated under a high vacuum) is not necessary to obtain a representative distillate of the cheese odour.
When applied to goat milk cheese, this approach indicated that the best extract was obtained by a direct water extraction of the cheese volatiles (Le Quere et al., 1996). This result could perhaps be explained by the chemical and hydrophilic nature of the free fatty acids identified as key odorants of goat milk cheese (Le Quere et al., 1996; Salles and Le Qu~r~, 1998; Le Quer~ and Salles, 2001). A key point in these evaluations of representativeness is the choice of a suitable matrix for testing the olfactory character of the extracts. For cheese, the best results have been obtained when the extracts are added to an emulsion, i.e., a matrix similar to cheese in terms of fat composition (Etievant et al., 1994). Since, generally, a combination of techniques should be used to obtain a reasonably complete view of an aroma profile (Reineccius, 2002), it is noteworthy that sensory evaluation of headspace or SPME extracts by 'direct GC-olfactometry' (i.e., without a chromatographic column) has been demonstrated recently (Lecanu et al., 2002; Rega et al., 2003).
Identification of volatile aroma compounds using hyphenated GC techniques As aroma molecules are essentially volatile, the techniques used to analyse them are usually based on separation using high resolution gas chromatography (HRGC). Substantial progress has been made in this field during the last 20 years and several stationary phases are available which allow almost all separation problems to be overcome. Combined with universal or selective detectors, HRGC is clearly a fundamental technique, essential for all aroma identification studies. A comprehensive review on the use of HRGC for the analysis of milk and dairy products is available (Mariaca and Bosset, 1997). Other interesting comments on qualitative, including multidimensional GC (Wright, 1997), and quantitative aspects may be found in Marsili (1997), van Ruth (2001b) and Reineccius (2002). Among the hyphenated techniques that are coupled to HRGC, the one that uses the human nose as a detector and known as gas chromatography-olfactometry (GC-O, sometimes referred to as 'GC-sniffing'), has received considerable attention during the past 20 years in aroma research (see for example Blank, 1997; Leland et al., 2001; Reineccius, 2002). The selectivity of this specific detector is based only on the odorous properties of the individual compounds separated by HRGC. As the most abundant volatiles may have little, if any, odour of significance in a food (Mistry et al., 1997), GC-sniffing has been an invaluable tool for identifying target compounds in aroma extracts that are always very complex. The primary aim of this technique is to discriminate the odorous compounds from the many background volatile components. The so-called 'aromagram'
Cheese Flavour: InstrumentaITechniques
constructed from the chromatogram obtained by simply smelling a GC effluent (Blank, 1997; Reineccius, 2002) constitutes an interesting interface with sensory analysis, as odour descriptors sensed at the GC sniffing port can be compared to the descriptors generated by a sensory panel. This method is particularly efficient for identifying off-flavours. Selection of key odorants or character-impact compounds in a food is another objective of GC-sniffing. Quantitative approaches (the true GC-olfactometry) based on odour detection thresholds or on odour intensity have been developed and are the subject of specialised treatises (Mistry et al., 1997; Leland et al., 2001; van Ruth, 2001b; Reineccius, 2002). Three different methods have been developed for GC-O: dilution analyses based on determination of detection thresholds, detection frequency methods and intensity measurement methods. Original dilution methods, CHARM (for Combined Hedonic Aroma Measurement) analysis developed by Acree and co-workers (Acree et al., 1984) and Aroma Extract Dilution Analysis (AEDA) developed by Grosch and co-workers (Ullrich and Grosch, 1987) are essentially screening methodologies since the methods, based only on detection threshold determinations, violate certain sensory rules and psychophysical laws (Reineccius, 2002 and references cited therein). They can be used to determine those odorous compounds that are most likely to contribute to the odour of a food. Originally developed by McDaniel et al. (1990), the odour-specific magnitude estimation (OSME) method is basically a crossmodal technique aimed at measuring the perceived odour intensity of eluting volatiles. In OSME and other cross-modality matching methods (Guichard etal., 1995; Eti~vant et al., 1999), results are not based on odour detection thresholds, and only one concentration of the extract is evaluated by a panel, unlike dilution methods where several dilutions of the extract are evaluated. Results can be subjected to statistical analysis and more consistent results are obtained when panellists are trained (Callement et al., 2001). The detection frequency methods, originally developed by Roozen and co-workers (Linssen et al., 1993), and referred to as nasal impact frequency (NIF) or surface nasal impact frequency (SNIF) since the work of Chaintreau and co-workers (Pollien et al., 1997), also use a group of assessors who simply have to note when they detect an odour in a single GC run (i.e., also at only one concentration). Those GC peaks being detected as odorous by the greatest number of assessors are considered to be the most important. Not being based on real odour intensities, the method has important drawbacks, especially when all the odorous compounds are present above their sensory threshold for all the assessors (Reineccius, 2002).
495
There is no perfect GC-sniffing method for finding key odorants in foods. Each of the methods described above has its advantages and weaknesses. Only two studies have compared the methods in terms of performance (Le Guen et al., 2000; van Ruth and O'Connor, 2001). In both cases, the results obtained with the different techniques were found to be very similar and well correlated. Finally, the choice of a GC-O method depends on the objective of the study, on the quality of the panel and on the time scheduled for the analyses (Le Guen et al., 2000). Dilution techniques are clearly time-consuming, intensity methods require a trained panel (Le Guen et al., 2000; Callement et al., 2001) while detection frequency methods are the least demanding but also the least precise (Le Guen et al., 2000). The aim of any GC-O experiment is to determine the relative odour potency of volatiles present in an aroma extract or fraction and to prioritise compounds for identification. This identification step is done mainly through the use of another hyphenated technique that couples HRGC to mass spectrometry (GC-MS). For difficult identifications, GC coupled with Fourier transform infrared spectroscopy (GC/FTIR) provides an interesting complement to GC-MS (Le Quire, 2000). Mass spectrometry is also used for quantification purposes through the use of a stable isotope dilution assay (Milo and Blank, 1998; Blank et al., 1999 and references cited therein). Such a precise quantitation is required for the determination of odour activity values (OAVs) generally calculated when using AEDA (Grosch, 1994). Odour activity values, calculated as the ratio of concentrations to odour thresholds, despite their limitations in terms of psychophysical validity (Mistry et al., 1997), give a good indication of the respective contributions of key odorants to the aroma of foods. They are the basis of the first attempts at using recombination studies to validate impact odorants sensorially in model cheeses (Grosch, 1994). Aroma-recombination studies are the important last step in sensorially verifying the analytical data obtained by GC-O and for quantification of key odorants of food (Mistry etal., 1997). Either bland unripened cheese (Grosch, 1994; Preininger etal., 1996; Kubickova and Grosch, 1998a) or specially designed odourless model cheese systems (Smitet al., 1995; Salles et al., 1995b) have been used to incorporate potential key odorants. Thus, the importance of methional, 4-hydroxy-2,5-dimethyl-3(2H)-furanone and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone, acetic acid and propionic acid was confirmed as key compounds for the aroma of Emmental-type cheese (Preininger et al., 1996). The branched-chain volatile fatty acids, 4-methyloctanoic and 4-ethyloctanoic acids, were confirmed to be essential for the typical goaty note of goat cheese (Le Quere et al., 1996) and their
496 Cheese Flavour: InstrumentaITechniques retronasal aroma thresholds were determined in a cheese model (Salles and Le Quere, 1998; Le Quere and Salles, 2001; Salles et al., 2002). Finally, the odour profile of the aroma model built with a set of 11 potent odorants identified in a GC-O study of an extract from Camembert cheese (Kubickova and Grosch, 1997, 1998b), with four additional volatile compounds identified by headspace-GC-O, has been found to resemble closely the aroma of genuine French Camembert cheese (Kubickova and Grosch, 1998a; Grosch et al., 2001). The GC-O methods that have been developed during the past 20 years, combined with either aroma extracts, headspace or even SPME (Dufour et al., 2001), have facilitated the identification of potent odorants in various cheeses, including Swiss (Preininger and Grosch, 1994; Rychlik et al., 1997; Rychlik and Bosset, 2001a,b), Cheddar (Arora etal., 1995; Christensen and Reineccius, 1995; Dufour et al., 2001), ParmigianoReggiano (Qian and Reineccius, 2002a,b), Blue (Le Quere et al., 2002; Qian et al., 2002), Mozzarella (Moio et al., 1993), Grana Padano (Moio and Addeo, 1998) and Gorgonzola (Moio et al., 2000) cheeses.
Characterisation of Sapid (Non-Volatile) Flavour C o m p o u n d s Water-soluble extracts (WSE) of cheese The water-soluble extract (WSE) of cheese has been reported to possess a strong flavour (Biede and Hammond, 1979; McGugan etal., 1979; Aston and Creamer, 1986). Apart from some water-soluble volatile components responsible for aroma, a WSE of cheese contains mainly non-volatile components that have been considered to be responsible for the taste of cheese (McSweeney, 1997). It has been recognised for a long time that bitterness, which can limit cheese acceptability if too intense, is due to an excessive concentration of low molecular weight and mainly hydrophobic peptides, which accumulate during ripening as a result of proteolysis (Lemieux and Simard, 1992; McSweeney, 1997). Amino acids and small peptides were hypothesised to be mainly responsible for the basic taste of cheese (McGugan etal., 1979; Aston and Creamer, 1986; Engels and Visser, 1994), their flavour impact being supposedly influenced by their interaction with calcium and magnesium ions (Biede and Hammond, 1979). However, the exact role of medium- and smallsize peptides and free amino acids in cheese flavour has not been clearly demonstrated, although it is likely that they contribute to the background flavour of cheese (McSweeney, 1997). In fact, until recently and apart from bitterness, no clear sensory data have been available for
WSEs of cheese and no direct correlations between specific nitrogen-containing compounds and organoleptic properties of fractions have been demonstrated. Among the mineral salts present in the WSE of cheese, the compound responsible for the salty taste is almost always supposed to be NaC1 (McSweeney, 1997). The taste of most high molecular weight salts is known to be bitter rather than salty (McSweeney, 1997 and references cited therein). Acid taste is caused by H30 + and the principal acid in cheese is lactic acid. However, total lactate concentration does not seem to be a good index of cheese acidity as the pH may increase during ripening caused by the production of ammonia (McSweeney, 1997). Moreover, the perception of acidity in cheese was hypothesised to be influenced by the concentration of NaC1 (Stampanoni and Noble, 1991), and no correlation between acid taste and either cheese pH or the amount of lactic acid was found for the flavour of Swiss cheese (Biede and Hammond, 1979), while the acid flavour correlated positively with the levels of triand tetra-peptides and with amino acids (Biede and Hammond, 1979). It has also been hypothesised that short- and medium-chain fatty acids might contribute to the acid taste of cheese (McSweeney, 1997). Although this assumption seems reasonable for short chain acids (e.g., formic, acetic or propionic), their principal contribution to cheese flavour is to its aroma in the unionised form (RCOOH) (Le Qu~r~ et al., 1996; Salles and Le Quere, 1998; Qian and Reineccius, 2002b).
Extraction, separation, identification of sapid compounds in relation to their sensory properties The study of taste-impact compounds in cheese, or more precisely in its water-soluble fraction, involves the study of soluble low molecular weight material (i.e., small peptides, amino acids, organic acids, minerals, etc.) dispersed in a very complex mixture. As it is necessary to assess the relative sensory impact of potential taste-active compounds, a fractionation scheme suitable for subsequent sensory evaluation is needed, and non-food-grade solvents or buffers must be rejected. Commonly used procedures involve extraction of grated cheese with water, possibly completed by precipitation of caseins and large peptides at pH 4.6, leading to edible fractions with good recovery of nitrogenous compounds (Kuchroo and Fox, 1982). The fractionation scheme that follows is generally adapted from the fractionation protocol used for isolating cheese nitrogen fractions for the study of proteolysis (Fox et al., 1994; McSweeney and Fox, 1997). The following steps (Fig. 3) involve ultrafihration using membranes with 1, 3 or 10 kDa molecular weight cutoff or precipitation with 70% ethanol (Cliffe et al.,
Cheese Flavour: InstrumentaITechniques
497
Grated cheese Water extraction Homogenisation Centrifugation
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S e n s o r y evaluation Figure 3 Possible fractionation schemes used to isolate and evaluate non-volatile compounds from cheese.
1993). The ultrafiltered water-soluble or 70% ethanolsoluble extracts are then subjected to gel filtration chromatography (Fig. 3). Sephadex G10 (Engels and Visser, 1994; Roudot-Algaron et al., 1994a; Engels et al., 1995; Molina et al., 1999), G15 (Roudot-Algaron et al., 1993; Warmke etal., 1996; Kubickova and Grosch, 1998a), G25 (Cliffe et al., 1993; Salles etal., 1995a), or Toyopearl HW-40S (Salles et al., 1995a, 2000; Sommerer et al., 1998a, 2001) media have been used for this purpose, using pure water (generally), 0.01 M NaC1 (Engels and Visser, 1994), or aqueous 0.5 M acetic acid (Warmke etal., 1996; Kubickova and Grosch, 1998a) as eluent. The fractions obtained by gel permeation chromatography may be evaluated sensorially (Fig. 3) after freeze-drying and re-dissolution in water, possibly with pH adjustment. Alternatively, liquid chromatographic methods involving Sep Pak C18 cartridges eluted with a stepwise water-ethanol gradient (Engels and Visser, 1994; Engels et al., 1995) or HPLC
using a water/food-grade ethanol gradient (Lee and Warthesen, 1996a,b) have been used instead of gel filtration. This fractionation scheme was developed originally in order to identify small hydrophobic peptides supposedly responsible for taste characteristics such as bitter or umami (Mojarro-Guerra et al., 1991; Cliffe et al., 1993; Roudot-Algaron et al., 1993, 1994a). A dedicated liquid chromatographic purification method has been developed to isolate and identify oligopeptides from the WSE of goat milk cheese (Sommerer et al., 1998a, 2001). Systematic sensory evaluation of the final fractions allows target fractions to be determined that possess interesting tastes, and physicochemical assessment of these key fractions should permit the identification of those compounds that are really relevant to the flavour of cheese (Engels and Visser, 1994; Salles etal., 1995a). Using this approach, some recent studies have been dedicated to the taste of the WSE of various cheeses.
498
Cheese Flavour: InstrumentaITechniques
Low molecular weight peptides, with two to four amino residues, were identified in Vacherin Mont d'Or (Mojarro-Guerra et al., 1991). As there was not enough natural material available for sensory evaluation, commercially available analogous synthetic peptides were used in sensory experiments. The dipeptides tested were dissolved in tap water at a rather high concentration (50 mg/100 mL) and were found to be essentially bitter. However, neither quantitative nor threshold data were estimated and the importance of these peptides for the overall taste of the cheese was only an hypothesis (Mojarro-Guerra et al., 1991). In a study on Cheddar cheese, Cliffe et al. (1993) found bitter fractions in material thought to be large hydrophobic peptides while lower molecular weight fractions with savoury notes were thought to be small, more hydrophilic peptides and amino acids. The flavour of the WSE of Comt~ cheese was the subject of substantial efforts in the early 1990s. A great variety of small peptides was identified in these extracts (Roudot-Algaron et al., 1993, 1994a,b). Some of them were found to be essentially bitter (Roudot-Algaron et al., 1993), y-glutamyl dipeptides were found to be sour (Roudot-Algaron etal., 1994a), but all the identified compounds, including non-peptide material (Roudot-Algaron et al., 1993; Salles et al., 1995a), were found at a concentration much lower than their threshold values. Although possible synergistic effects between several molecules found at concentrations below individual threshold values cannot be a priori eliminated, these observations suggest that these components alone could not affect cheese flavour (Salles et al., 1995a). Umami taste was clearly identified in a fraction and easily explained by a substantial amount of monosodium glutamate which was found at a concentration ten times above its threshold value, while the concentrations of the other amino acids were all well below their thresholds (Sales et al., 1995a). Following the same methodology, Grosch and co-workers evaluated the taste compounds of Emmental cheese (Warmke et al., 1996). The contribution of individual free fatty acids, free amino acids, minerals, biogenic amines, lactic and succinic acids, and ammonia was estimated on the basis of taste activity values (TAVs), a concept analogous to the odour activity values (OAVs), and defined as the ratio of concentration to taste threshold. From these results, acetic and propionic acids were confirmed to be important contributors to the taste of Emmental cheese. Glutamic acid was the major taste compound in the fraction containing free amino acids while all the ions investigated might be involved in the taste of Emmental, as were also biogenic amines (tyramine and histamine), ammonia, lactic and succinic acids (Warmke et al., 1996).
However, taste evaluation of mixtures of compounds conducted in tap water suggested that the characteristic taste compounds of Emmental are acetic, propionic, lactic, succinic and glutamic acids, each in the undissociated form and/or as ammonium, sodium, potassium, magnesium and calcium salts, as well as chlorides and phosphates analogues (Warmke et al., 1996). A study conducted on a model based on unripened Mozzarella-type cheese confirmed the importance of acetic, propionic, lactic, succinic and glutamic acids, and sodium, potassium, calcium, magnesium, ammonium, phosphate and chloride ions to the taste of Emmental cheese (Preininger et al., 1996). The same approach applied to Camembert led to the conclusion that the important taste contributors for Camembert are acetic, butyric, 3-methylbutyric, caprylic and succinic acids, monosodium glutamate, ammonia and NaC1 (Kubickova and Grosch, 1998a). It was also found that the biogenic amine, cadaverine, and the rare amino acids, ornithine and citrulline, when present, are likely to contribute to the bitter taste of Camembert (Kubickova and Grosch, 1998a). The above results clearly indicated that only low molecular weight compounds found in the WSE contribute significantly to the taste of cheese, while small peptides do not seem to be key flavour compounds, as was previously hypothesised. A study on goat milk cheese led to the same conclusions (Sales and Le Quere, 1998; Salles et al., 2000; Le Quere and Sales, 2001). The taste of the various goat milk cheeses investigated was essentially due to mineral salts and lactic acid. Fractions rich in small peptides and free amino acids were found to be essentially tasteless when evaluated either in water (Sales et al., 2000) or in a model cheese (Sales and Le Quere, 1998; Le Quere and Sales, 2001). In a comparative study on cheeses made from cows', ewes' or goats' milk, Molina et al. (1999) concluded that, even though differences were found in the intensity and predominance of individual tastes in the fractions of the cheeses made from the milk of the three species, it was difficult to correlate the peptide pattern and the free amino acid content of cheese with the sensory evaluation of the fractions. However, synergistic effects on taste have been demonstrated between peptides, amino acids and mineral salts (Wang et al., 1996) and interactions between tastes in mixtures may exist (Breslin, 1996). Therefore, it appeared interesting to generalise the evaluation of model mixtures of compounds that have been identified and quantified in the WSE of cheese (Warmke et al., 1996; Kubickova and Grosch, 1998a). Moreover, fractionation of the WSE by gel filtration has two main limitations: poor resolution and the necessity of
Cheese Flavour: InstrumentaITechniques
tedious repetitive steps in order to obtain sufficient peptide material for sensory evaluation. To clarify the putative effect of the small water-soluble peptides on the taste of cheese, it was therefore necessary to develop a new isolation procedure. Nanofiltration using ionisable membranes with a molecular weight cut-off of 500 Da was used by Sommerer et al. (1998b). A nanofiltrate was prepared from the 1-kDa permeate obtained by ultrafiltration of the WSE (Fig. 3). A large proportion of mineral salts and a substantial proportion of amino acids were thus eliminated from the nanofiltration retentate in which the majority of small peptides were concentrated (Sommerer et al., 1998b). This relatively pure and edible peptide-containing fraction could be used in sensory analysis, after incorporation into a bland model cheese system (Salles etal., 1995b), on its own or with the addition of putative synergistic effectors such as mineral salts or amino acids (Sommerer et al., 1998b). Using omission tests (see Engel et al., 2002a,b, and references cited therein for a comprehensive review), it was shown that small peptides have no effect on the taste of goat milk cheese, and no additive or synergistic effects were found between those peptides and salts or amino acids (Sommerer et al., 1998b). This unexpected result has been confirmed after complete physicochemical assessment of the WSE from goats' milk cheese has allowed the development of a model mixture that was validated sensorially (Engel et al., 2000a). Using omission tests, the relative impact of WSE components on goat cheese taste has been determined (Engel et al., 2000b). Among the main taste characteristics of the WSE from goats' milk cheese (salty, sour and bitter), saltiness was explained by additive effects of Na +, K +, Ca 2+ and Mg 2+, sourness was due to synergism between NaC1, phosphates and lactic acid, and bitterness resulted entirely from CaCI2 and MgCI2. Amino acids, lactose and peptides had no significant impact on the taste properties of the WSE of goats' milk cheese (Engel et al., 2000b). The same procedure was applied recently to a specially selected bitter Camembert cheese (Engel et al., 2001a,b,c) and confirmed that the WSE from cheese contained taste-active compounds, the impact of which could be modulated by an effect of the cheese matrix (Engel et al., 2001a). Sourness of Camembert WSE was explained by an enhancing effect of NaC1 on the acid taste due to the concentration of H30 +, saltiness was due to NaC1 whereas bitterness was mainly due to the bitter peptides found in the fraction with a molecular weight in the range 500-1000 Da (Engel et al., 2001b). The intense proteolytic activity of the strain of Penicillium camemberti, specially selected to develop bitterness in this case, has been demonstrated to be responsible for the accumula-
499
tion of small (MW < 1000 Da) bitter peptides during ripening (Engel et al., 2001c).
Dynamic Methods for Flavour Characterisation Even if the 'best' extraction and identification methods are used, poor correlations are often found between the overall levels of flavour components (volatile and non-volatile) and sensory perception experienced by a consumer. In other words, it is not enough to know the exact composition of food in terms of flavour compounds to understand perfectly the perception of its flavour. In fact, the perception of flavour is a dynamic process (Piggott, 2000). During the consumption of food, the concentration of aroma compounds at the olfactory epithelium and of sapid compounds at the taste buds varies with time. Flavour components are released progressively from the food matrix during chewing. Kinetics of the release of flavour depends on the nature of the food matrix composition and of individual mastication pattern. Sensory methods, such as time-intensity, have been used to study the dynamicand time-related aspects of flavour perception (Piggott,
2000). Release of volatiles in vivo
Techniques which measure volatiles directly in the mouth or in the nose have been developed to obtain physico-chemical data that reflect the pattern of aroma molecules released from food and that are effectively present at the olfactory receptors during consumption (Linforth and Taylor, 1993; Taylor and Linforth, 1994). Among the various approaches aimed at sampiing aroma from the nose (nose-space), the collection of expired air samples on Tenax | traps (Fig. 4) provided the first robust results (Linforth and Taylor, 1993; Taylor and Linforth, 1994). When applied to
enaxtrap OC ' urnp, ,Ana,ys,s, Ill1, I
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Figure 4 Collection and analysis of expired air by Tenax trapping and GC-MS (reproduced from Roberts and Taylor (2000), with permission from the American Chemical Society).
500
Cheese Flavour: InstrumentaITechniques
Cheddar cheese (Delahunty et al., 1994), the 'buccal headspace' method demonstrated that, despite a similar composition of volatiles found with conventional headspace analysis, some cheeses, depending on their fat content, released a different balance of volatiles during consumption (Delahunty et al., 1996a). Gas chromatography-olfactometry of buccal headspace showed a number of volatile compounds which have been suspected to contribute primarily and most likely to Cheddar cheese flavour (Delahunty et al., 1996b). It was presumed that the buccal headspace extract was representative of the aroma compounds that a consumer perceives during consumption (O'Riordan and Delahunty, 2001). By overlapping the sampling time periods, release curves can be constructed and temporal changes reflecting relative concentrations of volatiles at a particular moment during consumption can be determined (Linforth et al., 1996). When applied to Cheddar cheese, 'temporal buccal headspace' results, obtained on an accumulated 'time-concentration' basis (four time periods: 15, 30, 45 and 60 s of cheese consumption), were correlated with sensory time-intensity data (Delahunty et al., 1996c). Time-course data confirmed the results of conventional analysis while providing improved sensory predictions from the instrumental results (Delahunty et al., 1996c). Mastication behaviour using electromyography and saliva production rates of individuals have also been measured during consumption of Cheddar cheese (Delahunty et al., 1998; O'Riordan et al., 1998). Combined to nose-space analysis and sensory evaluation using free choice profiling, these authors demonstrated that although there were differences in chewing styles and saliva production rates, the assessors' individual nose-space profiles were very similar for all Cheddar cheeses examined (Delahunty et al., 1998). Partial least-squares regression analysis allowed the most important flavour differences between cheeses to be predicted from the volatiles released during consumption (O'Riordan et al., 1998). Recently, atmospheric pressure ionisation-mass spectrometry (API-MS) has been developed to monitor aroma release during chewing (Taylor et al., 2000). Air from the nose (nose-space) is sampled directly into the API-MS source through an interface (Fig. 5), making real time breath-by-breath analysis possible (Linforth etal., 1996; Taylor and Linforth, 1996). Therefore, by combining time-intensity sensory studies with nose-space analysis, it is now possible to relate temporal parameters of aroma release to perception (Linforth etal., 2000). The method, reviewed in detail in specialised treatises (Roberts and Taylor, 2000; Taylor, 2002), has been applied recently to soft French
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cheeses (Salles etal., 2003). Three French mouldripened soft cheeses (Brie made from pasteurised milk, Camembert made from pasteurised milk and from raw milk) were evaluated by a panel of 15 assessors (Salles et al., 2003). Retronasal aroma profiles made by citation frequency of attributes revealed four main descriptors for the three cheeses. The sulphury note (cabbage/cauliflower/vegetable) was particularly intense for the Camembert cheeses, while the buttery/creamy note was important for the three cheeses studied; the mushroom attribute was less intense in the Camembert cheeses, and ammonia was perceived in all cheeses but was found particularly difficult to score by the panellists (Salles et al., 2003). Therefore, the three main aroma notes (sulphury, buttery and mushroom) were selected for subsequent time-intensity (TI) scoring (15 assessors evaluated each attribute, with three replicates of each cheese). Gas chromatography-olfactometry of the dynamic headspace sampling of the three cheeses allowed odour-active compounds to be identified, amongst which sulphur compounds (methanethiol, dimethylsulphide (DMS), S-methylthioacetate, dimethyldisulphide (DMDS), 2,4-dithiapentane, dimethyltrisulphide, 2,3,5-trithiahexane and dimethyltetrasulphide) could be related to the sulphury attribute scored by the panellists. However, API-MS nose-space experiments allowed the detection of only six compounds of which three contained sulphur ones (DMS, 5-methylthioacetate and DMDS). Simultaneous TI scoring of the sulphury note allowed a perfect superposition of the time-intensity curve with the release of the sulphur compounds (Fig. 6). The most significant perception and flavour release parameters allowed the three cheeses to be well discriminated by principal component analysis (PCA), showing a good agreement between perception scored by assessors and consistency in their release of aroma compounds while eating cheeses (Salles et al., 2003). Another PCA analysis showed a positive correlation for the sulphury note between the perception parameters derived from the TI curves and parameters derived from the aroma
Cheese Flavour: InstrumentaITechniques
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Figure 6 Flavour release from Camembert cheese for one assessor. Simultaneous time-intensity scoring of the sulphury/ cabbage attribute and API-MS analysis of S-methyl thioacetate, dimethyldisulphide (DMDS) and dimethylsulphide (DMS) in the nose-space.
release curves (Salles et al., 2003), as suggested by the characteristic curves presented in Fig. 6 for one panellist within one session. Non-volatiles in vivo
Development of methods to study flavour release has concentrated mainly on the volatile fraction, while only a limited number of studies have been devoted to the release of non-volatile compounds in the mouth. Conductivity measurements have been used to relate the release of salt during chewing to Cheddar cheese texture (Jack et al., 1995), and a similar approach with additional in-mouth measurement of pH has been used with a variety of foodstuffs, including Cheddar cheese (Davidson etal., 1998). However, in these approaches, the sensors available for in vivo measurements only give the best estimate for salt (non-specific to sodium) and acid release. Saliva sampling using cotton buds coupled to a direct liquid mass spectrometry procedure has been described to study the rate of release of sucrose (Davidson et al., 1999). Panellists were instructed to take a swab from a specific location on the tongue at different times during mastication using a cotton bud. The weight of saliva swabbed was measured and sucrose concentration was monitored using liquid-API-MS after extraction by a methanolwater solution (Davidson et al., 1999). A continuous
501
sampling technique using a motor-driven ribbon placed across the tongue while a panellist chews a food sample has also been described (Davidson et al., 2000). At the end of the eating process, the ribbon was cut into 5 cm lengths after estimation of the saliva weight adsorbed on the ribbon, each piece representing a certain time. Non-volatile components were extracted from the pieces of ribbon with a solvent and their concentration determined by direct liquid phase API- or electrospray (ES)-MS (Davidson et al., 2000). Temporal release of sucrose and glucose from biscuits, of sodium from potato crisps, of sucrose, glucose and fructose, citric and malic acids from fresh orange and finally minerals (sodium, calcium and potassium) from Cheddar cheese was monitored successfully (Davidson et al., 2000). The cotton bud technique has been applied recently to a model processed cheese in which aroma and non-volatiles compounds consistent with literature data had been incorporated (Pionnier et al., 2003). As it was demonstrated that with certain foodstuffs the increased frequency of sampling affected the chewing pattern (Davidson et al., 2000), each panellist produced only one saliva sample per mastication, at a time-consuming cost, however. Using ES-MS in negative ionisation mode, time-course release curves for minerals (sodium, calcium, magnesium and potassium), amino acids (leucine, phenylalanine, glutamic acid), organic acids (citric, lactic, propanoic and butyric) and phosphoric acid have been obtained (Pionnier et al., 2003). As a typical example, Fig. 7, shows release curves from cheese for phenylalanine, glutamic acid, leucine, phosphoric and lactic acids obtained for one assessor. The first conclusion that could be stressed from the analyses of the release curves is that individual physiological parameters (mainly mastication behaviour and salivation rate) are related more closely to the temporal release of taste compounds than to their physico-chemical properties (Pionnier et al., 2003). Model mouth systems
A number of mechanical devices which mimic in more or less detail the processes that occur in the mouth during eating 'model mouths' have been developed (Piggott, 2000 and references therein). These are often variants of dynamic headspace analysis, but their aim is to obtain time-resolved samples containing volatiles as similar as possible to those present during actual eating. The various parameters like temperature, air flow, mastication rate and addition of artificial saliva can be varied to study their effects on volatile flavour release. The main advantages of model mouths are the large quantities of food samples that can be handled, overcoming some sensitivity problems encountered
502 Cheese Flavour: InstrumentaITechniques
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Figure 7 Flavour release from a flavoured model cheese for one assessor. In mouth, time-course release of non-volatile compounds (concentration in g/100 g saliva) using the cotton bud technique. Data points measured in electrospray-MS in negative ionisation mode are the mean of three replicates. when monitoring volatiles at low concentrations (Taylor, 2002), and the suppression of inter-individual variations, always encountered when working with a panel, that can be detrimental to a robust interpretation of the data. Recently, using an imitation cheese preparation, the release of volatile flavour compounds from the Retronasal Aroma Simulator (RAS), originally developed by Roberts and Acree (1995), has been compared with flavour release in vivo using API-MS detection in both cases (Deibler et al., 2001). While delivering higher concentrations of volatiles than from human breath, the RAS gave a good approximation of time-averaged flavour release in the mouth, with volatile compounds present at similar ratios (Deibler et al., 2001). Volatiles in the RAS effluent from Cheddar, Brie and vanilla ice cream were measurable (Deibler et al., 2001). The model-mouth device originally developed by Roo7en and co-workers (van Ruth et al., 1994) has been used to investigate the relationships between the gross, non-volatile and volatile compositions and the sensory attributes of eight Swiss-type cheeses (Lawlor et al., 2002). Eight flavour attributes were found to be correlated with subsets of volatiles, amino acids, free fatty acids and gross compositional constituents with, for instance, the nutty flavour of Emmental that was positively correlated with the concentrations of propionic acid, ethyl acetate and 2-pentanone (Lawlor et al., 2002). Flavour release and flavour perception are dynamic processes and must be studied using dynamic methods (Piggott, 2000). Dynamic physico-chemical methods have been developed to study the parameters of flavour release from foods. Parallel increased applications of dynamic sensory methods provide a better understanding of food flavour, with important results obtained for
cheese flavour. However, further work is needed to improve our knowledge of various interactions arising at different levels in the process of food consumption: e.g., interactions between food ingredients (Delahunty and Piggott, 1995; Pionnier et al., 2002; Taylor, 2002), and interactions at the perceptual levels such as tastearoma interactions (Noble, 1996; Given and Paredes, 2002; Hollowood et al., 2002; Taylor, 2002), or trigeminal interferences (Green, 1996; Given and Paredes, 2002), as these play a fundamental role in overall flavour perception.
Global and Fast Assessment of Cheese Flavour The methods currently used to evaluate and control the quality of cheese flavour are still essentially based on sensory evaluation by a panel of experts. These trained panels are able to handle such difficult tasks like quality monitoring through descriptive analysis, off-flavour detection and comparison of samples for classification purposes. It could be interesting for such tasks to substitute humans by instruments that could give quicker answers at a reduced cost.
Electronic nose Evaluation of the complete aroma emitted from food using gas sensors, the so-called 'electronic noses', is now theoretically feasible (Hodgins, 1997; Schaller et al., ]998). Electronic noses are composed of arrays of non-specific gas sensors which are based on different physical principles (Hodgins, 1997; Schaller etal., 1998). The most common sensors are semiconducting metal oxides and conducting organic polymers, but
Cheese Flavour: InstrumentaITechniques
they all give rise to a response with a typical pattern. Therefore, pattern recognition software, using either standard statistics or artificial neural network technology, must be used for data treatment and final presentation of the results (Hodgins, 1997; Schaller et al., 1998). The electronic nose is particularly attractive for quality control applications where conformity/nonconformity answers are expected. Some discriminative studies have been conducted on cheese samples (Schaller et al., 1998 and references cited therein). Using metal oxide semiconductors, it was possible to distinguish between five Swiss cheese varieties (Mariaca and Bosset, 1997). However, some problems occurred with the repeatability of the system that could be possibly related to the product itself, the sampiing technique or the moisture content of the air used for sampling, precluding its use in routine tests (Schaller et al., 1998). Samples of Swiss Emmental cheese at different stages of ripening have been evaluated using different technologies over a period of one year (Schaller et al., 1999). The metal oxide semiconductors technology has given the best discriminative results. However, the sensors seemed to be damaged by short-chain fatty acids released from cheese. Conducting organic polymer sensors showed poor sensitivity to volatile components of cheese, the main problem being that these sensors are unstable (Schaller et al., 2000a). The other technologies tested were not sensitive enough to cheese volatile compounds and electronic noses containing these sensors showed poor discriminative power (Schaller et al., 1999). However, recently, the ripening of Danish Blue cheese was monitored by means of an electronic nose which contained 14 conducting polymer (polyaniline) sensors; results were found to be highly correlated to those of sensory analysis and GC-MS analysis of volatile compounds during a 5-12-week ripening period (Trihaas et al., 2003). The close control of the experimental sampling conditions (quality of dry air with a humidity <0.5% and equilibration time at controlled temperature) might explain this success (Schaller et al., 1998). Nevertheless, despite some success in some classification tasks when using perfectly controlled sampling conditions, electronic noses hardly meet the requirements of the food industry in terms of precision, reproducibility, sensitivity and stability. Of particular importance, the sensors are known to deteriorate over time or can be poisoned, therefore changing their response. Even with frequent calibration, the inherent weaknesses of the technique make the general applicability of the databases problematic. Moreover, these instruments cannot be used to identify single odorants or to differentiate samples with subtle differences in distinctive sensory attributes. Therefore, in off-flavour studies,
503
where identification of the off-flavour compound is a pre-requisite and in quality control assessment, they may be used successfully only after recognising their inherent weaknesses (Reineccius, 2002). Mass spectrometry-based systems
For classification purposes, two other global and fast analytical methods, based on mass spectrometry, have been used for dairy products and seem more powerful and reliable than electronic noses. The first consists of a global analysis of a headspace sample by a mass spectrometer operated in electron ionisation mode, without GC separation (Vernat and Berdague, 1995). The feasibility of the method was originally demonstrated for rapid classification of four rather different French cheeses (Vernat and Berdague, 1995). This method is often described as a 'MS-based electronic nose' (Schaller etal., 2000b). The mass patterns obtained, considered as fingerprints of the food products analysed, also need data treatment, either by conventional statistics or artificial neural networks. The technique has been used successfully to discriminate four Swiss Emmental cheeses differing in age (Schaller et al., 2000b), and Camembert-type cheeses according to their origin, manufacturing process or ripening stage (P~res et al., 2002a). Solid-phase microextraction may be used as a preconcentration technique instead of dynamic headspace analysis (Marsili, 1999). Applied to rapid characterisation of cheeses, SPME has been demonstrated to be a very efficient pre-concentration technique (Schaller et al., 2000b). In the task of discriminating Swiss Emmental cheeses ripened for different times, SPME has been found to be superior to dynamic headspace analysis in terms of repeatability, simplicity and compatibility with an autosampler (Schaller et al., 2000b). However, when applied to the characterisation of Camembert-type cheese (P~r~s et al., 2001), SPME yielded less satisfactory results than those obtained by dynamic headspace analysis (Peres et al., 2002a). The better performance of the dynamic headspace method in that case was attributed to the absence of signal drift (ageing of the SPME fibres causes drift, as demonstrated by Peres et al., 2001) and to automation of the injection of sample into the mass spectrometer. According to the authors, the protocol chosen for the analysis by dynamic headspace-MS was more efficient than SPME in terms of extraction yield, and reduced thermal, mechanical and chemical modification of the samples (P~r~s et al., 2002a). Developed in the 1980s for food applications, direct pyrolysis-MS is another method that delivers 'fingerprints' which can be used for classification/authentication
504
Cheese Flavour: InstrumentaITechniques
purposes (Aries and Gutteridge, 1987). With this method, a tiny sample is pyrolysed rapidly at up to 530 ~ and the resulting volatile fraction, characteristic of the flavour but also of the matrix breakdown, is analysed immediately by a mass spectrometer operated in low energy electron ionisation mode. Here again a mass pattern, this time rather complex, is obtained for each sample and several data pre-processing steps are often necessary to select a reduced number of mass fragments that allow satisfactory classification. Curiepoint pyrolysis-mass spectrometry with associated multivariate data analysis techniques is considered as a powerful classification tool in microbiology for the recognition of micro-organisms (Talon et al., 2002 and references cited therein) and food science (Aries and Gutteridge, 1987; Peres et al., 2002b and references cited therein). However, when applied to the discrimination of five Camembert-type cheeses, it appeared less competitive than SPME-MS or dynamic headspace-MS in terms of sample preparation and analysis time (P~res et al., 2002b). The main advantage of the method is that it provides a specific fingerprint of the cheese matrix which could be potentially related to textural parameters (Peres et al., 2002b). Recently, in a similar approach, the proton transfer reaction mass spectra (PTR-MS, another atmospheric pressure ionisation mode MS source) of the static headspace of Mozzarella cheese have been found to display comparable discrimination power to sensory descriptive analysis (Gasperi et al., 2001).
Concluding Remarks Cheese is a biochemically active product that undergoes many changes during ripening. The development of flavour is one of the consequences of these biochemical changes that occur over the entire ripening period. Modern instrumental methods allow for detailed analyses of volatile compounds, and some pertinent complementary sensory information can be obtained by combining gas chromatography with olfactometry. Recent developments have allowed the identification of the role of non-volatile components in the overall flavour of cheese. Nevertheless, the relationship between flavour-aroma and sapid compounds present in a foodstuff and sensory perception of that food by a consumer is not so easy to establish. It is still not well understood how the various flavour-active components combine to produce a particular sensory perception. Recent developments in dynamic instrumental methods that can follow the in vivo sequential release of the flavour molecules are valuable tools that can account for the balance of flavour compounds released, a balance that changes with time. With more complete and
accurate information, combined flavour chemistry and sensory evaluation should help understand the relationship between flavour stimuli and perceived flavour and explain the mechanisms of flavour perception. Authentication of cheese (for instance, varieties with protected designations of origins) is another challenge. Tools developed recently that combine analytical instrumentation for global assessment of flavour with multivariate data analyses have demonstrated their usefulness for classification purposes.
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Vernat, G. and Berdague, J.L. (1995). Dynamic headspacemass spectrometry (DHS-MS): a new approach to realtime characterization of food products. Proc. Bioflavour 95, Dijon, France. pp. 59-62. Wampler, T.P. (1997). Analysis of food volatiles using headspacegas chromatographic techniques, in, Techniques for Analyzing Food Aroma, R. Marsili, ed., Marcel Dekker, Inc., New York. pp. 27-58. Wang, K., Maga, J. and Bechtel, P. (1996). Taste properties and synergisms of beefy meaty peptide. J. Food 5ci. 61, 837-839. Warmke, R., Belitz, H.D. and Grosch, W. (1996). Evaluation of taste compounds of Swiss cheese (Emmentaler). Z. Lebensm. Unters. Forsch. 203,230-235. Wright, D.W. (1997). Application of multidimensional gas chromatography techniques to aroma analysis, in, Techniques for Analyzing Food Aroma, R. Marsili, ed., Marcel Dekker, Inc., New York. pp. 113-141.
Rheology and Texture of Cheese D.J. O'Callaghan and T.P. Guinee, Dairy Products Research Centre, Teagasc, Ireland
I n t r o d u c t i o n - Overview of Cheese Rheology and Texture Rheology of materials, e.g., cheese, may be defined simply as the study of their deformation and flow when subjected to a stress or strain. The rheological properties of cheese are those that determine its response to stress or strain, as applied, for example, during compression, shearing or cutting. In practice, such stresses and strains are applied to cheese during processing (e.g., portioning, slicing, shredding and grating) and consumption (slicing, spreading, masticating and chewing). The rheological properties include intrinsic characteristics such as elasticity, viscosity and viscoelasticity that are related primarily to the composition, structure and the strength of attractions between the structural elements of the cheese. The theological characteristics of cheese are quantified by rheological quantities that are measured in tests involving the application of stress or strain under defined experimental conditions. The output variables from these tests (e.g., creep, stress relaxation, compression tests), which may include change in dimensions over time, the ratio of stress-to-strain for certain strain levels, stress or strain required to induce fracture, enable the determination of quantities such as shear modulus, fracture stress and firmness. In lay terms, the behaviour of the cheese when subjected to these stresses and strains is referred to by descriptive terms such as hardness, firmness, springiness, crumbliness or adhesiveness. Owing to the variations in manufacturing conditions and composition, different cheese varieties exhibit a wide range of rheological behaviour, ranging from the viscous behaviour of soft cheese to the elastic behaviour of hard cheeses at low strain. The rheological properties of cheese are of considerable importance as they affect: its handling, portioning and packing characteristics; 2. its texture and eating quality, as they determine the effort required to masticate the cheese or alternatively the level of mastication achieved for a given level of chewing. The degree of chewing required may, in turn, influence the flavour/aroma properties and the suitability of the cheese for different consumer groups (e.g., children, aged); o
3. the use of cheese as an ingredient, as they influence its behaviour when subjected to different size reduction methods (such as shredding, grating or shearing) and how it interacts and blends with other ingredients in foods in which cheese is an ingredient. 4. its ability to retain a given shape at a given temperature or when stacked; 5. its ability to retain gas and hence to form eyes or cracks or to swell. Hence, the rheological properties of cheese are significant quality attributes of importance to the manufacturer, pre-packer, distributor, retailer, industrial user and consumer. The rheology of cheese is a function of its composition, microstructure (i.e., the structural arrangement of its components), the physico-chemical state of its components, and its macrostructure, which reflects the presence of heterogenities such as curd granule junctions, cracks and fissures. The physicochemical properties include parameters such as the level of fat coalescence, ratio of solid-to-liquid fat, degree of hydrolysis and hydration of the paracasein matrix, and the level of inter-molecular attractions between para-casein molecules. Hence, the rheological characteristics differ markedly with the cheese variety and age. The effect of variety on the rheological properties is readily apparent on comparison of an almost-flowable mature Camembert with a firm, brittle Parmesan or of a crumbly Cheshire cheese or with an elastic springy Swiss-type cheese or String cheese (Table 1). Similarly, the influence of age is clear on comparison of a young (e.g., < 1 - 2 months) rubbery Cheddar with a fully mature pliable Cheddar (Table 1). Cheese rheology and factors that affect it have been reviewed extensively (Sherman, 1969; Eberhard, 1985; Visser, 1991; van Vliet, 1991a; Rao, 1992; Prentice et al., 1993; Ustunol et al., 1995; Beal and Mittal, 2000; Fox et al., 2000; Madsen and ArdO, 2001; Guinee, 2003). In this chapter, the basic rheological characteristics of cheeses in general and the methods for their quantification will be examined. The effects of compositional and biochemical factors on the rheological properties of cheese are discussed in 'Cheese as an Ingredient', Volume 2. For detailed information on cheese texture,
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
512
Rheology and Texture of Cheese
Table 1
Rheological properties of raw cheese and their definitionsa
Cheese type displaying property
Rheological property
Definition
Elasticity (rubberiness)
Tendency of cheese to recover its original shape and dimensions upon removal of an applied stress Tendency to recover from large deformation (strain) after removal of deforming stress Tendency of hard cheese to crack, with very limited flow (confined to vicinity of crack); after fracture, the broken surfaces can be fitted to each other Tendency of hard cheese to fracture at a relatively low permanent deformation High resistance to deformation by applied stress
Springiness Elastic fracturability
Brittleness Firmness (hardness)
Toughness (chewiness)
The resistance of cheese to fracture until a relatively large deformation is attained A high resistance to breakdown upon mastication
Softness Plastic fracturability
Low resistance to deformation by applied force The tendency of cheese to flow on fracture
Shortness
The tendency to plastic fracture at a small deformation; low resistance to breakdown upon mastication The tendency to resist separation from another material with which it makes contact (e.g., another ingredient or a surface such as a knife blade or palate) The tendency to break down easily into small, irregularly shaped particles (e.g., by rubbing) The tendency to increase in apparent viscosity when subjected to an increasing shear rate (especially upon heating) The tendency to exhibit a decrease in apparent viscosity when subjected to an increasing shear rate
Longness
Adhesiveness (stickiness)
Crumbliness Shear thickening
Shear thinning
Swiss-type cheese, low-moisture Mozzarella Swiss-type cheese, low-moisture Mozzarella Parmesan, Romano, Gruyere
Romano, Parmesan Cheddar, Swiss-type cheese, Romano, Parmesan, Gouda Mozzarella, Swiss Mozzarella, String cheese, Halloumi Blue cheese, Brie, Cream cheese Mature Cheddar, Blue cheese, Chaumes, Raclette Camembert, Brie Mature Camembert
Cheshire, Wensyledale, Blue cheese, Stilton, Feta Cream cheese (when heated), 'creaming' of processed cheese products Quarg (especially at low temperatures, i.e., <4 ~
a Definitions modified from Szczesniak (1963a), van Vliet (1991a) and Fox et al. (2000).
the reader is referred to 'Sensory Character of Cheese and its Evaluation', Volume 1 and the following reviews: Szczesniak (1963a,b, 1998), Brennan (1988) and Rosenthal (1999).
Terminology of Rheology and Texture General rheological terminology The general terminology used to describe the rheology of materials has been discussed extensively (Sherman, 1983; Rao and Steffe, 1992; Whorlow, 1992; Collyer and Clegg, 1998; Sharma et al., 1999). The terms most commonly applied to the rheology of cheese are described in Table 1. Deformation and strain
Any rheological measurement involves deforming a sample of material by applying a force, e.g., by compression or by shear (Fig. 1). The displacement in response to the force at the point of application is known as deformation. The term 'deformation' used in this sense
does not imply permanent deformation but rather a change in shape (i.e., form) which may be temporary, permanent or partly recoverable. A series of instantaneous measurements of force and associated displacement describe the rheological characteristics of a material under the measurement conditions. The conditions which affect the force-displacement response include temperature, type of deformation (compression, extension, shear or pressure), level of deformation in relation to the elastic limit and fracture point of the material, rate of deformation, previous history of deformation. Strain may be defined as the fractional displacement that occurs under an applied stress. Stress
Stress is defined as the distribution of force over an area of a material. The 'area' over which a force is distributed may be a surface (e.g., the surface of a cylindrical sample exposed to a compression plate) or an imaginary section within a material (e.g., an internal fracture plane). The force applied at a surface is distributed throughout the material and is borne by the structural elements, e.g., in
Rheology and Texture of C h e e s e
Table 2
Rheological properties derived from stress/strain curves obtained from large strain deformation of cheese
Textural characteristic to which parameter is related
Rheological Property
Abbreviation
Interpretation
Elastic, or compression, modulus Apparent elastic modulus, or Deformability Modulus
E o-/e
Fracture stress
o-f
Fracture strain
ef
Measure of elasticity at low strain Ratio of stress to strain in a viscoelastic region below the fracture point Stress required for fracture and collapse of cheese mass beyond point of recovery. Deformation required to induce fracture
Firmness (maximum stress)
O'max
Fracture work
Wf
Stress to required to compress cheese sample to a given deformation The energy required to fracture the cheese
the case of cheese, the casein strands of the matrix and the occluded fat globules. The theological behaviour of the material is effected by the response of the structural elements to the applied stress. The initial response of a cheese sample to an applied stress is determined mainly by the para-casein matrix. At larger deformations, the moisture and fat phases, which are occluded in the matrix, contribute to the rheological response. Shear and normal modes of stress and strain
Two modes of stress can be applied on a surface, namely shear or normal. Shear stress is created when a force is applied parallel to the plane of a surface element, whereas normal stress is created by a force applied perpendicularly to a surface element. Normal stress, or, is defined as:
Elasticity Elasticity
Strength of cheese matrix
brittleness and "shortness" or "longness" of cheese firmness or hardness toughness
where A is the cross-sectional area over which the force (F) is applied (Fig. 1). Normal stress can occur in tension or compression. In a large strain compression situation, two expressions are used for normal stress, differing in respect of the calculation of area. Apparent stress is the applied force divided by the original crosssectional area of the sample, while true (or, more strictly, corrected) stress is the applied force divided by the instantaneous area of the sample, allowing for the fact that the sample spreads as it is compressed. When true stress is plotted against strain, a more distinct peak is observed around the fracture point (Fig. 2). However, the instantaneous area is not easily measured and is often approximated by a calculation based on constant volume (Ak and Gunasekaran, 1992). Shear stress, 1", is defined as:
F o'--
513
F I'--
A
A kPa 450<
>
7/
i F
4OO350300250-
!o (a)
200150-
o-t
100-
(b)
Figure 1 Deformation of a solid material by the application of a force, F, (a) in a direction normal to the surface (area, A), resulting in a compression deformation, or (b) tangential to the surface resulting in shear deformation. The stress is calculated as F/A; strain is calculated as zXL/Lo, where Lo is the original length of the sample of material.
50- / 0 r 0
i,
0'.2
0'.4
0'.6
0.8
Figure 2 Apparent stress, o-, and true stress, o-t, plotted against strain, ~, from force-displacement data obtained in the compression of Cheddar cheese on a texture analyser, Stable Micro Systems, model TA.HDi, showing a more distinct peak near the fracture point in the o-t plot.
514
Rheology and Texture of Cheese
where A is the cross-sectional area over which the shear force (F) is distributed (Fig. 1). Two alternative expressions for normal strain have been used in tensile or compression situations, namely Cauchy strain and Hencky strain. Cauchy strain (e), also referred to as strain, apparent strain or engineering strain, is defined as the deformation relative to the original sample dimension, i.e.,
impractical, as the transition from elastic to viscous behaviour is gradual. Throughout this text, strain is used in the engineering or Cauchy sense, unless otherwise stated. Shear strain, 3' is defined as: AL 3,=
Lo
AL Lo where Lo is the original height of the sample and AL is the displacement under applied stress, o" (Fig. 1). Hencky strain, sometimes referred to as natural strain or true strain, is defined as the natural logarithm of the ratio between the sample length upon application of force and the original length, i.e.,
8t
where Lo is the original sample length and L is the length under load. Hencky strain is thought to be more relevant than engineering strain in describing fluid (or non-recoverable) behaviour, e.g., squeeze flow patterns of deformation, as occur in large strain compression and in spreading cheese on a cracker. However, for small strains, the Hencky strain approaches the Cauchy strain (Fig. 3). The relationship between Cauchy and Hencky strain can be derived a s : ~?t =
- l n ( 1 - ~).
Ideally, engineering strain should be used for recoverable (elastic) deformations and true strain for nonrecoverable (viscous) deformations. Obviously, this is
Initial height, Lo: ,~,- _ _
Figure 3
Displacement, AL (mm)
Cauchy strain
Hencky strain
0 5 10 15 20 25
0 0.20 O.40 0.60 0.80 1.00
0.00 0.22 0.51 0.92 1.61 oo
Deformation of a sample of cheese, originally 25 mm high (Lo), under axial compression, showing equivalence between displacement (Z~L), Cauchy strain (e) and Hencky strain (st).
where AL is the shear (tangential) displacement on the application of shear stress, r. Compression testing is generally used for the rheological evaluation of cheese because of the relatively low tensile strength of most cheeses, e.g., compared to its compression strength. Exceptions include members of the pasta-filata family of cheeses, such as Mozzarella and Haloumi (see 'Pasta-Filata Cheeses', Volume 2), which when heated are able to undergo a high degree of stretching when pulled. As discussed in 'Pasta-Filata Cheeses' and 'Cheese as an Ingredient', Volume 2, this characteristic is associated with the presence of para-casein fibres which are formed during the exposure of the curds to high temperatures (e.g., 58-60 ~ at a low pH (e.g., 5.1-5.4) during manufacture and stretching. In practice, normal and shear stresses occur simultaneously during testing, size reduction at industrial level (e.g., comminution, shredding, grating) and consumption (mastication). In a compression test, a normal force is applied but fracture generally occurs as a consequence of shear stresses built up in the sample. Likewise, in a torsion test, a normal force must be applied to maintain sufficient contact between the sample and the plate delivering the shear stress. In general, the simplest fundamental rheological properties (e.g., u modulus, shear modulus) are defined for one mode of stress in one dimension, and for this reason, rheological measurements often attempt to confine stresses to one mode and one dimension. However, this is possible only in some low deformation situations, since stresses in one dimension tend to produce structural displacements, and hence stresses, in other dimensions and modes. Thus, it is not possible to create large deformations in one dimension in isolation, as for example during compression testing when deformations exceed the linear viscoelastic limit (see 'Fundamental Measurements: Oscillatory Rheometry for Linear Viscoelastic Measurements in Cheese'). However, cheese generally undergoes relatively large deformation during handling and consumption, and hence it is necessary to describe its rheological characteristics under large deformation conditions, e.g., during compression by the molar teeth ("-70%). It is difficult to measure shear and compression stresses simultaneously
Rheology and Texture of Cheese
in all dimensions under large strain deformation, and, consequently, much use is made of empirical or semiempirical methods to describe the rheology of cheese or other foods under large strain deformation conditions to which cheese is subjected in practice. In contrast, low strain linear viscoelastic tests, while giving precise rheological quantities (i.e., storage and loss modulo and indirect information on structure, tell little about the expected rheological behaviour of the cheese during processing and eating. Bagley and Christianson (1987) suggested a generalised approach to the measurement and interpretation of the rheological properties of foods aimed at dealing with the difficulties in describing behaviour that is highly viscous and highly elastic at the same time. With this approach, constants can be derived which enable the rheological property being measured in a given test, e.g., compression modulus at low strain during compression testing, to be related to a rheological property measured in another test, e.g., shear modulus in a torsion test or shear test. In practice, inhomogeneities and graininess in cheese can confound such comparisons and it is difficult to interpret the significance of such results (Bagley and Christianson, 1987). The relationship between stress and strata Stress and strain at a micro level result from an externally applied force at a macro level, the displacement at the point of application being the cumulative effect of a strain at every point along the length of the sample (Fig. 4). The relationship between stress and strain is characteristic of the material but depends on temperature, and for viscoelastic materials, on other factors including the time over which the stress is applied and the pre-test stress-strain history and the rate of strain (see 'Uniaxial Compression').
515
Under low e, solids, including some cheeses (e.g., a y o u n g - medium-aged, low-moisture, part-skim Mozzarella cheese), exhibit a simple linear relationship between o-and e which can be expressed in terms of various moduli. In compression or tensile testing, Young's Modulus (E) may be defined as: E=
O"
where o is the normal stress and e is the strain on the material. In shear tests, the shear modulus (G) is given by: G=
7"
3' where r is the shear stress and 3' is the shear strain. The above elastic moduli are intrinsic rheological characteristics of the material, that are independent of sample dimensions, time and strain rate. However, for most cheeses, the elastic region is small (e.g., 0.006; Guinee etal., 2000a,b) and of little consequence because most strains applied in practice are >>0.05. Bulk modulus and compressibility of cheese Isotropic stress, or pressure, is sometimes described as a third mode of stress. This usually occurs in fluid materials and is really a normal force applied equally in each of three dimensions. In general, the application of isotropic stress to a material results in a slight reduction in volume, or shrinkage. From the volume reduction, a bulk modulus (K) may be determined and is defined as: K=
PV ZXV
Sample surface
~176
oriht,l[ i,
~ -
roe
.... Sample interior .....
......iiiiiiiii'_;~St ress ) .............
......Stress.......Strain I I ........ii iiiiiiiii. Q
Strain l ..........i i @
ib:......................
(a)
(b)
4 The application of an external force over a surface area results in stress and strain throughout the sample, as illustrated in (a).The displacement at the surface of application is the cumulative effect of a strain at every point, e.g., X, along the length of the sample. For low strain deformations there is a linear relationship between stress and strain, and a modulus, equal to the ratio between them, may be determined (b). Figure
516
Rheology and Texture of Cheese
where P is the applied pressure, V is the initial volume of material and AV is the change in volume. There are simple relationships between compressibility, usually expressed in terms of bulk modulus (K), Young's modulus (E) and Poisson's ratio (/x) (Whorlow, 1992; Rosenthal, 1999): e = 3K(1
-
2~,)
and E=2G(1
+/,)
Since /x---0.5 for most cheeses (see Poisson effect in Glossary), these relationships simplify further to:
Rheological concepts applied to cheese Cheese structure
Cheese is essentially a concentrated protein gel, which occludes fat and moisture. Gelation is brought about by either of the following mechanisms (see 'Rennetinduced Coagulation of Milk' and 'Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels', Volume 1): 1. slow quiescent acidification (e.g., using a starter culture or food-grade acid and/or acidogen), at a temperature of 20-40 ~ to the isoelectric pH of casein, i.e., ---4.6; 2. sensitisation of the casein to calcium via the hydrolysis of the principal micelle-stabilising casein, K-casein, by added acid proteinases (i.e., rennets); or
E = 3G However, all of the above relationships apply only in the linear (i.e., elastic) region. The volume reduction up to the point of fracture is about 9% for Cheddar cheese (Calzada and Peleg, 1978). Cheese with a large vacuole volume (e.g., where eyes occupy a significant proportion of the volume, as in Swiss cheese), are more compressible and therefore have lower K values than cheeses without eyes. However, unlike their behaviour under normal or shear stresses, most cheeses are relatively non-compressible under isotropic stress. Consequently, from a practical point of view, their bulk modulus is of little interest, but may be of interest in the calculation of true stress in uniaxial compression (see 'Uniaxial Compression').
(a)
(b) , ,,
L
Force, F ( ~
at depth, y
Figure 5 Two situations where viscous forces are at work: (a) flow between parallel plates which move relative to each other; (b) flow in a pipe.
C-
.g O0
J Time
Viscous deformation
Flow is normally the result of shear displacement. Shear forces occur when a liquid flows inside a pipe or when a molten mass (e.g., melted cheese) flows along a surface (Fig. 5). In a fluid, strain is not recoverable and applied stress results in a continuously changing strain. A viscous material behaves as a fluid and responds to shear (stress), in terms of strain, in a time-dependent manner (Fig. 6). For an ideal viscous material, i.e., a Newtonian fluid, the rate of strain is proportional to applied stress (Fig. 7). The relationship between stress (r) and rate of strain (5'), or shear rate, is described by the coefficient of viscosity (r/): I"
Figure 6
Ideal viscous (Newtonian) response to constant applied stress, i.e., strain (3') increases at a constant rate (T).
10 r"-
~
6
~
4
9r - -
2
0 0
Figure 7
2
4 6 8 Stress, o-, arbitrary units
10
Relationship between shear stress (s) and shear rate (T) for an ideal (Newtonian)liquid.
Rheology and Texture of Cheese
3. a combination of acid and heat, e.g., heating milk to - 9 0 ~ at --pH 5.6. The micro-structure of milk gels and cheeses has been studied extensively (Hall and Creamer, 1972; Kalab and Harwalkar, 1974; Kimber et al., 1974; Kalab, 1977, 1979; de Jong, 1978; Green et al., 1981a,b, 1983; Green, 1990a,b; Kiely et al., 1992, 1993; Mistry and Anderson, 1993; Bryant et al., 1995; Desai and Nohing, 1995; Everett et al., 1995; Guinee et al., 2000a). The physico-chemical properties of the para-casein matrix and occluded components may be deduced from micro-structural observations, compositional analyses and theoretical considerations of the chemistry of the conversion of milk to cheese and partition of components (e.g., milk salts) between the whey and the cheese curd (Walstra and van Vliet, 1986). Natural rennet-curd cheese is essentially a particulate calcium phosphate-para-casein matrix, composed of interconnected and overlapping strands of partially fused para-casein aggregates (in turn formed from fused para-casein micelles). The integrity of the matrix is maintained by various intra- and inter-aggregate hydrophobic and electrostatic attractions. In young cheese, the matrix has an 'internal' structure consisting of a relatively loose network of clearly recognisable particles (para-casein micelles and aggregates of paracasein micelles) which are in contact with neighbouring particles over part of their surfaces. Ongoing fusion of para-casein particles during maturation leads to a gradual reduction in the extent of internal matrix structure, as reflected by the disappearance of interparticle boundaries and the formation of a more homogeneous mass (Kimber et al., 1974; de Jong, 1978). The para-casein network is essentially continuous, extending in all directions, although some discontinuities exist in the matrix at the micro- and macro-structural levels. Micro-structural observations made using transmission electron microscopy (TEM) suggest that hydrolysis of para-casein (e.g., by rennet) to watersoluble peptides results in parts of the matrix losing contact with the main para-casein network, an occurrence that leads to discontinuities or 'breaks' in the para-casein matrix at the micro-structural level (de Jong, 1978). Hence, it is noteworthy that ageing of Mozzarella for 50 days results in the degradation o f - 5 0 % Otsl-casein to Otsl-CN f 24-199 and an increase in the porosity of the defatted para-casein matrix, as observed using scanning electron microscopy (SEM) (Kiely et al., 1993). Discontinuities at the macro-structural level exist in the form of curd granule junctions or curd chip junctions (in Cheddar and related dry-salted varieties) (Kalab and Harwalkar, 1974; Kalab, 1979; Lowrie et al., 1982; Paquet and Kalab, 1988). Curd
517
granule junctions in low-moisture Mozzarella are well defined, - 3 - 5 p~m wide and appear as veins running along the perimeters of neighbouring curd particles (Kalab, 1977). Unlike the interior of the curd particles, the junctions are comprised mainly of casein, being almost devoid of fat. Factors that contribute to the formation of these junctions include leaching of the fat from the surface of the curd particles and dehydration of surface protein, during the cutting, acidification, cooking and pressing stages of cheese manufacture. Chip junctions in Cheddar and related dry-salted varieties are clearly discernible on examination of the cheese by light microscopy and, like curd granule junctions, have a higher casein-to-fat ratio than the interior. The difference in cheese composition at junctions, compared to the interior of the curd particles, probably leads to differences in the molecular attractions between contiguous para-casein layers in the interior and exterior of curd particles, and thus to differences in structure-function relationships. From a rheological viewpoint, the occurrence of structural discontinuities may result in the lack of tensile strength in many cheeses which in practical terms may be reflected as crumbliness, shortness, fracturability, e.g., Feta, Stilton and Cheshire. Discontinuities probably also contribute to poor replication of rheological measurements. The matrix encases fat globules (in varying degrees of coalescence), moisture, dissolved solutes and enzymes within its pores (Kimber etal., 1974; Laloy etal., 1996; Guinee etal., 2000a). Clumping and coalescence of fat globules occur during manufacture due to the combined effects of shear stress on the fat globule membrane and shrinkage of the surrounding paracasein matrix which forces the occluded globules into close contact. Evidence for fat clumping is provided by scanning electron micrographs which show fissures, or irregular-shaped openings, in the para-casein matrix, which remain after removal of fat during sample preparation (Mistry and Anderson, 1993; Bryant et al., 1995; Fig. 8). The frequency of these fissures decreases as the fat content is reduced, e.g., from 33.2 to 8.2%, w/w, fat (Mistry and Anderson, 1993; Guinee et al., 2000b). Major physico-chemical changes occur in the protein and fat phases of cheese during maturation. These include partial hydrolysis of the matrix comprising para-casein, increase in hydration of the para-casein, and coalescence of fat globules, resulting in the formation of fat pools (Fox et al., 1996, 2000; Guinee and Law, 2001; Guinee, 2002). These changes are mediated by the residual rennet, micro-organisms and their enzymes, and changes in mineral equilibrium between the serum and para-casein matrix. The type and level
518
Rheology and Texture of Cheese
(a)
(b)
. . . . . .
Figure 8 Scanning electron micrographs of Cheddar cheese, showing the continuous para-casein matrix (arrow heads) permeated by holes and fissures, corresponding to discrete, clumped or coalesced fat globules (solid arrows). Bar, i.e., 5 #m in (a) and 1 #m in (b) (from Guinee et al. (1998), reproduced with permission from the society of Dairy Technology).
of physico-chemical changes depend on the variety and composition of cheese and ripening conditions. These changes assist in the conversion of fresh 'green' curd to a mature cheese and markedly influence its rheological, textural, functional and flavour characteristics (see 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese During Ripening' and 'Sensory Character of Cheese and its Evaluation', Volume 1). Thus, a storage period is generally required before rennet-coagulated cheeses attain the desired rheological and textural attributes (e.g., fracturability, firmness, spreadability, brittleness) associated with the particular variety. Creep and stress relaxation in cheese
The time-dependent rheological behaviour of cheese has been studied (Visser, 1991; Ma et al., 1996; Pereira et al., 2001; Venugopal and Muthukumarappan, 2001). Creep is the time-related change in strain on application of a constant stress to a material such as cheese. Practical examples of creep occur when curd or cheese is compressed gradually under its own weight (e.g., Camembert), is pressed or stacked, e.g., during retailing. Creep (]) may be expressed in terms of strain or compliance, which is the ratio of strain to applied stress. When a constant stress, r, applied for a time, t, results in a strain, 3'(0, then the creep compliance is: J(t) =
3'(0 T
A creep curve for Cheddar cheese is shown in Fig. 9. Three characteristic regions can be identified. In the elastic region (A-B), 3" is instantaneous and fully reversible; in this region, the creep compliance is elastic (,]0). Viscoelastic deformation occurs in region B-C, where the material is partly elastic and partly viscous;
the creep compliance is retarded elastic (JR) and the recovery of the elastic component of 7 on the removal of r is delayed. In the viscous region (C-D), 3' increases linearly with time and permanent deformation occurs; the creep compliance is referred to as being Newtonian (JN). On removal of the stress at point D, the strain recovery curve shows three identifiable regions: an instantaneous elastic recovery (D-E), a delayed recovery (E-F), and an eventual flattening. The vertical distance from the fiat portion of the recovery curve to the time axis is the non-recoverable 3' per unit r, which is related to the amount of structural damage to the sample during the test. In the elastic region of the creep curve, the strands of the cheese matrix absorb and store the stress energy, which is instantly released on removal of r, enabling the cheese to regain its original dimensions. The extent and duration of the elastic region depends on the magnitude of r and the structural and compositional characteristics of the cheese. At 3' > critical strain, the structure of the cheese is altered via the breaking of bonds between structural elements, which are stressed beyond their elastic limit. Eventually, when the stress-bearing structural casein matrix has fractured, the cheese is said to flow. At short time scales and low r, most hard cheese varieties are essentially elastic, whereas after a long time, they flow, albeit very slowly, and do not recover to their original shape on removal of the stress. Failure to appreciate this characteristic can often lead to loss of shape (e.g., manifested by bulging, inclined surfaces) during storage, distribution and retailing, especially if cheeses of different consistencies are laid haphazardly upon each other. A stress relaxation test generally entails the instantaneous application of a constant deformation or strain, e (typically 0.10-0.20), by compression of the cheese sample between two parallel plates of a texture analyser (e.g., TA HDi Texture Analyser, Stable Micro Systems, Godalming, England; Instron Universal Testing Instrument (UTM); Instron Corporation, Massachusetts, USA.). On the application of e, or increases instantaneously to oro (i.e., zero-time value) but decays exponentially with time (t) (Shama and Sherman, 1973). The resultant or-time curve is used to determine the stress relaxation time, t, which may be defined as the time required for or to decrease to a fraction of Oro, e.g., t at which or = O-o/e, where e is the base of the natural logarithm. In a variation on such a test, Emmons et al. (1980) compressed full-fat (35%) and reduced-fat (17%) Cheddar cheeses, having a common level of moisture-in-non-fat-substance, at a constant speed to a strain of 0.2 and held the strain for 1 min. They
Rheology and Texture of Cheese
0.3
-
519
Recovery after removal of stress
Sustained constant stress
0.2 s (U .,,-, 00
-----------__Z
0.1
Non-recoverable strain J I, ,/ 0
50
100
150
200
250
300
Time, s
Figure 9 Creep-relaxation curve for mature Maasdammer cheese (fat, 29%, w/w, protein, 28%, w/w). A stress of 3700 Pa was applied to a cheese disc (diameter, 40 mm; height, 2.27 mm), placed between the parallel plates of a controlled strain rheometer (TA Carrri-Med csl2500) at 20 ~ and removed after 180 s. The curve is divided into regions indicating elastic, viscoelastic and viscous behaviour.
showed that the initial compression slope (or modulus of deformability), the relaxation slope and the residual force (after 1 min) were much higher for reduced-fat cheese, made from milk with or without homogenisation, than for full-fat cheese. Mechanical models of c h e e s e theology From its creep and stress-relaxation behaviour (Fig. 9), it can be inferred that cheese is a viscoelastic material. It exhibits elastic and viscous characteristics, but unlike true elastic or viscous materials, the relationship between stress and strain depends on the magnitude and the duration of the applied stress or strain. On the application of a low stress, that is sufficiently small so as not to induce permanent damage or fracturing (breaking of bonds between the structural elements) of the microstructure, for short times, cheese behaves as an elastic solid. However, a low stress applied over a relatively long time scale results in an increasing strain, a gradual failure of the structure and an eventual flow. Hence, the relationship between r (or o9 and T (or ~) is linear only at very low r and short time scales. The T at which linearity between r and 3' is lost is referred to as the critical strain (i.e., at the end of the linear viscoelastic range), which for most solidlike foods, including cheese, is relatively small, e.g., 0.02-0.05 (Walstra and van Vliet, 1982). The modelling of cheese rheology begins with simple relationships such as Hooke's Law for small displacements in the elastic region. In the region beyond the elastic limit, sometimes referred to as the elastoplastic region (i.e., where recovery following deformation is partial on removal of stress), modelling the rheology of cheese requires more complex models.
Mechanical models have been used to simulate creep and relaxation effects in materials (Rao, 1992; Tanner, 2000). The viscoelastic behaviour of cheese may be simulated by various mechanical models that contain different arrangements of dashpots (representing the fluid element) and springs (representing the elastic element) in series and/or in parallel. A simple model consisting of a spring in parallel with a dashpot is referred to variously as a Kelvin or Voigt element (Whorlow, 1992) or Kelvin-Meyer solid (Tanner, 2000) (Fig. 10). In contrast, a Maxwell element consists of a spring in series with a dashpot, which gives an exponentially decaying response to a suddenly applied constant strain (Fig. 11). Several models have been based on multiple Kelvin bodies in series, or Maxwell bodies in parallel, to simulate creep and stress relaxation, respectively, in viscoelastic solids (Whorlow, 1992); elements with a spectrum of time constants are employed in these models to approximate viscoelastic
(7
.L
Time
Figure 10 Kelvin model and its response to constant applied stress.
520
Rheology andTexture of Cheese
the force-displacement equations (Whorlow, 1992; Steffe, 1996).
Applied strain
of these models
Large strain deformation
Time Figure 11 A Maxwell model and its stress relaxation response to a constant applied strain.
behaviour (Fig. 12). Subramanian and Gunasekaran (1997b) showed that a model consisting of eight Maxwell elements could simulate the shear modulus over a wide dynamic range in low amplitude oscillation (0.1-20 Hz). Ma et al. (1996) showed that a six-element Kelvin model could simulate creep compliance in full-fat and reduced-fat Cheddar cheese. The Burgers body, which consists of a combination of Maxwell and Kelvin elements in series (Fig. 13), affords a close approximation to both the creep and stress relaxation behaviour of cheese. The mechanical representation of these models provides an intuitive guide to the nature of viscoelasticity and a simulation of rheological behaviour based on
(a)
(b)
rl Applied strain
9
89
"/-3
Figure 12 (a) Series of three Kelvin elements with a spectrum of time constants, which may be used to simulate creep and (b) A combination of Maxwell elements with a spectrum of time constants, which may be used to simulate relaxation behaviour in a viscoelastic solid.
Definitions and terminology Large strain measurement implies permanent deformation and measurement of non-linear rheological characteristics which are related to deformation of the microstructure. In contrast to linear viscocelastic deformation where applied strains are generally <0.05, large strain deformation may be defined as that which occurs at strains in the range of---0.1-0.9 during compression, and even at higher strains in the case of shear deformation (e.g., > > 1). Consideration of the forces that are applied to cheese from manufacture to consumption, indicates a very broad range of deformation. In some situations, the strains are of a relatively low magnitude and do not result in visible damage (e.g., during ripening, transport, retailing), while in others the strain results in fracture (e.g., during portioning) or complete disintegration of the cheese mass (e.g., comminution, as in shredding, grating, grinding, as for example in the preparation of cheese ingredients and in the manufacture of processed cheese products and cheese powders). Hence, in the current context, large strain deformation is arbitrarily subdivided into two regions, i.e., large strain deformation-elastoplastic (LSD-E; e.g., strains ---0.1-0.5; Fig. 14), where deformation does not result in fracture and the structure can partially recover, and large strain deformation-fracture (LSD-F; 0.3-0.9), where the cheese mass undergoes fracture or disintegrates and cannot recover. In the following discussion, the LSD-E and LSD-F regions will be treated jointly (Fig. 14). Measurement using texture analyser Large strain deformation testing of cheese usually involves the application of strains (e.g., ~ "-- 0.8) that result in fracture, by compression of the cheese sample
I Typical linear ] visco-elastic limit j I I
j_ Applied strain
I
( Typical extentof "~ |compression to which | I cheese is subjected in| | chewing and in | (poiitTypical fracture I L compression testing ) '
Time
0
0.40
0.80 Strain, A U L
Figure 13 Burgers four element model, which simulates creep and relaxation behaviour of cheese.
o
Figure 14 Range of strain in compression tests on cheese.
1.0
Rheology andTexture of Cheese
between two parallel plates of a texture analyser (Culioli and Sherman, 1976; Dickinson and Goulding, 1980; Creamer and Olson, 1982; Tunick et al., 1991; Guinee et al., 1996; Fenelon and Guinee, 2000; Truong et al., 2002). The cheese sample is placed on a base plate and is compressed at a fixed rate (typically 20 mm/min -1) to a pre-determined level (e.g., 75% of its original height) by the mobile plate (cross-head). However, the rate of compression used in various studies has differed widely, e.g., 5-500 mm/min -1 (Table 3). The force (F) developed during compression is recorded as a function of distance (or displacement); alternatively, the force may be converted to o-and the displacement to g. The resultant o-versus g curves for a range of hard rennet-curd cheeses (Fig. 15) typically show a number of distinct regions and enable the determination of a number of rheological parameters: 9 A-B; or increases proportionally with ~. The slope of this linear region defines the compression modulus, E (i.e., E = o-/~), which is of little practical significance in relation to cheese behaviour during processing or consumption, where strains are > >0.05. However, in the commercial grading of cheese, E may be an indication of springiness (e.g., where a grader sensorically monitors the resistance to small deformation, as in pressing the thumb into the outside of the cheese block; the force applied during this hand deformation is typically 18 N or o---- 40 kPa). 9 B-C, o-increases less than proportionally with ~. The slightly lower slope of the curve in this region compared to that in A-B is probably due to the formation of microcracks that do not spread throughout the sample but which allow some stress to be dissipated; 9 C-D, the slope of the o-/~ curve decreases markedly. The cheese begins to fracture at C, as cracks grow and spread throughout the entire sample at an increasing rate. Eventually, at D the rate of collapse of the stress-bearing para-casein matrix overtakes the build-up of o-within the matrix through further compression and a peak or, denoted as the fracture stress, is reached. The fracture stress, o-f, and strain, ~f, are measures of the stress and strain, respectively, required to cause complete fracture of the sample. Strength, or fracturability, is defined as the stress required to fracture the sample (at D), while toughness, or fracture work, is defined as the area under the curve up to the point of fracture. 9 D-E, o- decreases with further compression due to the collapse of the stress-bearing structure. The decrease in o-may be attributable to: (i) shattering of the samples into pieces that spread over the base plate, resulting in an increased surface area and (ii) the probable loss of contact between some
521
of the pieces of cheese and the base plate which results in dissipation of stress energy stored within the individual pieces. E-E cr increases as the cross-head begins to compress the fragmented pieces of cheese. The o- at the end of the compression (point F) is a measure of firmness, as judged in the first bite of mastication (Sherman, 1969; van Vliet, 1991a). The various quantities obtained from the o - ~ curve and their interpretation are given in Table 2. The application of a strain to a segment of cheese (e.g., cube or cylinder) and monitoring the resultant cr by a texture analyser, as above, is a typical method for measuring the large strain deformation behaviour of cheese. However, many variations of both the procedure of stress or strain application, and the levels, are possible. A so-called apparent elastic modulus can be calculated at a strain well below the fracture point, e.g., ~ --- 0.1, as the ratio between or and ~. A preferred term for this parameter is modulus of deformability, as the deformation in question may include some plastic flow (Ak and Gunasekaran, 1995; Johnston, 2000). However, such a parameter needs to be interpreted with caution as some apparent initial deformation may occur before complete contact is made between the compression plate and the sample surface, an occurrence that could lead to erroneous values. Fracture and work of fracture. Rheological behaviour over such a range of ~ in the form of shear or compression, can be explored in several ways, such as applying a gradually increasing ~, a fixed ~, a defined o followed by its removal, a gradually increasing ~ up to a point followed by its reversal. Stress-strain cycles, often referred to as bites (analogous to compression between the molar teeth during mastication), may be repeated at interval(s) or applied in a given sequence (e.g., pre-test compression). Depending on the level of applied strain, cheese exhibits a combination of rheological behaviours, such as non-linear elastic (e.g., region B-C, Fig. 15), sometimes referred to as viscoelastic, or inelastic (e.g., region D-E, Fig. 15), sometimes referred to as plastic behaviour.
Rheological Measurements in Cheese: Sensoric Methods The methods used to assess the rheological characteristics of cheese may be broadly classified as sensoric or instrumental, where instrumental methods can be categorised further as empirical or fundamental. The aim of sensoric methods, which are performed routinely by cheese graders, is to acquire an impression
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Rheology a n d T e x t u r e of Cheese
523
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m 200 150 C
100 50 0
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Figure 15 Large strain deformation test: typical stress-strain curve of a 6-month-old mature Cheddar cheese sample compressed to 25% of its original height; several regions of the curve are identifiable, based on slope variation (see text for details).
of how the texture of the cheese is perceived during consumption. Cheese texture may be defined as a composite sensory attribute resulting from a combination of physical properties that are perceived by the senses of touch (including kinaesthesis and mouthfeel), sight and hearing. The test conditions are arbitrary, frequently involving deformation which results in visual fracture, e.g., as when rubbing cheese between the fingers until it becomes pliable, three (finger) point bending of a cylindrical cheese plug or slice, and mentally gauging the force required to bend or break it. Alternatively, the cheese may be assessed by the application of forces or deformations which cause no visible fracture, e.g., pressing the ball of the thumb into the surface of a whole cheese and mentally assessing the degree of indentation or the force exerted on the fingers. In all cases, a sensory impression is formed and the grader assigns a score, based on one or more criteria, such as test conditions and response. The sensory properties of cheese, including texture, are discussed comprehensively in 'Sensory Character of Cheese and its Evaluation', Volume 1.
Table 4
Rheological Measurements in Cheese: Empirical Instrumental Methods A wide range of instrumental techniques is used for characterising the rheology of cheese (Table 4). Instrumental methods may be arbitrarily classified as empirical or fundamental. In general, the nature of the stresses and strains in empirical methods is less well defined than in fundamental methods. Moreover, unlike fundamental methods, the measurements obtained with some empirical methods are on an arbitrary scale (e.g., the ball compressor). Empirical instrumental m e a s u r e m e n t s
Many textural studies have involved rheological measurements to imitate the sensory evaluation of cheese texture. The aim of empirical tests is to measure a parameter, which experience indicates, or suggests, is related to the textural characteristics of the cheese. Hence, while the test conditions are arbitrary and the stresses and strains involved may not be well defined,
Typical rheological testing techniques applied to cheese
Test Oscillatory shear (parallel plate) Uniaxial compression Cone penetration Puncture Bending test Wire cutting test Torsion test a 4), diameter of cylinder.
Type of instrument used
Typical sample shape and dimensions a (mm)
Reference
Rheometer
Cylinder: 30 4) • 3
Ma et al. (1996)
Texture analyser or UTM Texture analyser or Instron, 30 ~ cone Texture analyser or Instron, 2-5 mm diameter needle Texture analyser Texture analyser Torsion gelometer
Cube: 25 x 25 • 25 Cube: 12.7 • 12.7 • 12.7
(see Table 3) Breuil and Meullenet (2001)
Cube: 12.7 • 12.7 • 12.7
Hennequin and Hardy (1993); Breuil and Meullenet (2001) Rosenthal (1999) Green et al. (1986) Truong and Daubert (2000)
Finger: 25 • 25 • 50 Finger: 25 • 25 • 50 Capstan: 19 ~max X 27.8
524
Rheology andTexture of Cheese
a value is obtained which gives some indication of the textural characteristics of the cheese and differentiates one sample from another. However, they provide only single datum values that are an overall measure of the many different facets of rheological behaviour. In these tests, a sample is compressed or penetrated in one or more bites, thereby simulating the compressive and penetrative actions of the teeth on cheese during mastication. Likewise, the action of a cheese grader who presses the ball of the thumb into the cheese is imitated by the ball-compressor test. Some empirical instrumental tests are discussed briefly below. Imitative t e s t s
Imitative instruments include the bite tenderometer and the denture tenderometer which measure the forces involved in chewing using strain gauges, and typically involve compression to 60% of the original height. In the Volodkevich bite tenderometer, which was designed to simulate the motions of mastication, a pair of tooth-like jaws, or wedges, compress a sample of about 6 mm thickness, imitating the squeezing and biting action of teeth (Szczesniak, 1963b). Later instruments used plungers to penetrate a sample, or parallel plates to compress a sample, e.g., to ---20-30% of its original height (Szczesniak, 1963b). Early devices for evaluating the hardness of cheese involved compression by a ball, in an instrument known as the ball-compressor, where deformation resulting from applying a fixed force for a specified time was measured (Szczesniak, 1963b). The action simulated that of a thumb pressing against cheese when making a sensory evaluation of the product. The General Foods Texturometer was designed to simulate the biting of food by the jaws and teeth (Friedman et al., 1963; Bourne, 1978). A food sample (---12.6 mm high) was loaded onto a fixed plate and then subjected to a deforming force by a tooth-shaped plunger, which was mounted on a hinge and actuated to simulate the vertical action of a human jaw. The area of the samples is at least that of the plunger base, which is available in sizes from 16 to 50 mm ~b. The instrument compresses samples to a height of 3.2 mm, i.e., 75% compression. When the plunger deforms the sample, strain gauges detect the movement of the plunger and a force-time trace is recorded and is known as a texture profile. The sample is subjected to two successive deformations (referred to as bites). The Texturometer has been superceded by uniaxial compression instruments, such as the Instron UTM, for the purpose of texture profile analysis. One distinction between the Texturometer and other instruments is that the Texturometer simulates the action of the human jaw, whereby the plunger decelerates as it
reaches the end of the compression stroke, and then accelerates upward as it withdraws. The usual practice with other instruments is compression at constant speed. Cutting tests Cutting tests measure the resistance to the passage of a knife or a wire through a cheese (e.g., Cherry-Burell Curd tension meter). As wire-cutting tests tend to be more fundamental, they are discussed in more detail in 'Fundamental Measurements: Large Strain Deformation'. Penetration tests Penetration tests involve measurement of the force required to insert a probe (cone or cylinder) a given distance into cheese, or alternatively the depth of penetration of a probe under a constant load for a given time. As the probe penetrates the sample, the cheese in its path is fractured and forced apart. The progress of the probe is retarded to an extent depending on the hardness of the cheese in its path, the adhesion of the cheese to its surface (which depends on the depth of penetration into the cheese and the thickness of the needle, or angle of the cone, used). Hennequin and Hardy (1993) used a cylindrical probe (5 mm diameter at a speed of 10 mm/min to a depth of 10 mm) to penetrate soft cheeses (e.g., Camembert, Coulommier, Munster) and found that the force at 10 mm penetration gave a high correlation with sensory firmness (r = 0.94, n = 19). They concluded that the technique is suitable as a rapid method for texture measurement in soft cheese. Breuil and Meullenet (2001) found a significant correlation between measurements obtained using a cone penetrometer (30~ or a 2-mm needle, and textural characteristics of a wide range of commercial cheeses (e.g., Colby, Edam, Cheddar, Mozzarella and Cream cheese) as measured by a sensory panel.
Fundamental Measurements: Oscillatory Rheometry for Linear Viscoelastic Measurements in Cheese Elastic shear (G') and loss modulus (G")
As discussed in 'General rheological terminology', there is a range of strain, typically <0.05, over which cheese recovers fully, although not instantaneously, when stress is removed. This behaviour is referred to as viscoelastic. Even at these low strains, ideal elastic (Hookean) or viscous (Newtonian) behaviour, represents two extremes, and cheeses, like most organic materials, show some characteristics of both. Hence, the accurate characterisation of cheese rheology requires the measurement of both elastic and viscous responses. In the elastic region, where there is a linear
Rheology
relationship between dynamic stress and strain, the behaviour is described as linear viscoelastic. Linear viscoelastic measurements are typically made by applying torsion, using oscillatory rheometry (van Vliet, 1991b). This involves using a precision actuator to apply a low oscillating strain to a sample (which could be a liquid, a 'soft' solid, or a rigid solid), and the measurement of the resultant stresses within the sample, using a sensitive transducer. Alternatively, a small stress is applied to the sample and the resultant strain is measured. The former is referred to as controlled strain rheometry, whereas the latter is known as controlled stress rheometry. Several geometries are possible for applying torsion, but the most convenient for a solid material, like cheese, are parallel circular plates. Parallel plates have the advantage that the sample can be easily prepared to fit the plates; moreover, plates with serrated surfaces minimise the risk of slippage, associated with fat liquefaction (Subramanian and Gunasekaran, 1997a; Guinee et al., 1999). With parallel plate geometry, the cheese sample, which is disc-shaped, is gripped between the plates, one of which is fixed, while the other applies a low-amplitude torsional harmonic motion (Fig. 16). At any time t, the angle of rotation, 0, of the oscillating plate is defined by:
andTexture
of Cheese
525
where w is the thickness of the sample. This displacement results in a strain, 3/(0, at any radius r:
3/-
sin 02t - 3/0 sin 02t
where 3/0 is the amplitude of 3/(0. (In this notation, 3/(0 is used interchangeably with 3/.) In general, the resultant oscillating stress is out of phase with the applied shear by a phase angle 6. The stress wave can be reconstructed as the sum of two sine waves, one in-phase with, and the other out-of-phase (by 90 ~) with, the strain wave (Fig. 17). Thus, r = "r' + r ' ' = %' sin 02t + to" cos 02t where to' and to" indicate the stress components which are in-phase and out-of-phase with the strain % and are related by the phase angle 6 (Fig. 18): !!
tan 6 =
1
The stress wave has an amplitude to, defined by:
0 = a sin rot ro -
where a is the maximum angle of rotation and o2 is the angular velocity. The shear applied by the plate varies throughout the sample, from zero at the central axis to a maximum at the edge. At a point on the oscillating plate, at a radial distance, r, from the axis, the displacement due to rotation by an angle 0 is r0, and the shear strain 3/is:
3/=
Two dynamic moduli, elastic shear modulus (or storage modulus), G', and viscous modulus (or loss modulus), G", may be defined from the relationship between r and % where,
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.__
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W
(a)
(c) u~t
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(b)
Radial distance from centre of sample
Figure 16 Schematic showing viscoelastic deformation of cheese during low-amplitude oscillation rheometry: (a) sample before test; (b) sample being subjected to a torsional shear displacement; (c) shear displacement as a function of radial distance from the central axis.
/ ~
~/' -
/z'~
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~
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Elastic, or in-phase, component, r o' sin ~t
-/
Loss, orout-ofphase, component, -
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Figure 17 Stress and strain traces in dynamic oscillation, showing phase delay, 6, between stress and strain (a), and resolution of in-phase and out-of-phase components of stress (b).
526
Rheology andTexture of Cheese (a)
,
,
~
Complex viscosity
(b) Loss component,
~
pp To
Elastic component, r o' Strain, %
Oscillatory shear also implies an oscillatory shear rate @), since
cos cot
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Figure 18 Trigonometrical representation of phase relationship between dynamic strain and dynamic stress in oscillatory measurement in viscoelastic region, (a), with vector direction indicating phase, as in (b).
ll
G
r/'= z
-
To w cos o)t - % cos wt
where 5'0 is, by definition, the amplitude of 5'. This enables a complex viscosity to be calculated, with viscous and elastic components, by dividing the appropriate component of shear stress by the shear rate. The 'viscous' component of viscosity (in-phase with shear rate) is: , T o II
and G
d j' = --~ (To sin wt)
II
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% = 3'0 o)
'Toll
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To
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G ll
To
~o
G'
Consideration of a spring-dashpot mechanical model, e.g., a Kelvin element, indicates that the same relationship between stress and strain applies (Fig. 19). However, it is noteworthy that the magnitudes of G' and G" for the spring and dashpot components of the mechanical model in the viscoelastic region differ markedly from those which characterise large strain deformation. Ma et al. (1996) reported decreases (of---50%) in G' and G" for full-fat and reduced-fat cheeses on increasing stress by a factor of 10. The tendency of 6 to approach a constant value as the strain was reduced towards zero shows that cheese behaves in a viscoelastic manner rather than an elastic manner at very low strains.
/ _ _ ~.. _
Thus, complex viscosity equals the complex (shear) modulus divided by the angular frequency, w, with the in-phase and out-of-phase components interchanged (Fig. 20). According to the Cox-Merz rule, the complex viscosity determined by dynamic rheometry is virtually identical to the (steady) viscosity when compared at numerically equal values of shear rate and w. Dynamic rheometry is often used for this purpose since it is easier to perform than viscometry on viscoelastic materials. Cylindrical geometry may be used in the case of liquids or gels, or cone-and-plate or parallel plate geometry in the case of more solid samples. It is preferable to operate in a shear-rate control mode to avoid the occurrence of
Stress r :~ Oscillatory strain % sincct
iOashot,, •
Shear rate,
(b)
(a)
"~o ii !
r/-
Loss component,
To
%
.,,
i
Elastic component, r o'
,, /-/ =
TO .
'TO
Figure 19 Kelvin element and stress response (r) to oscillatory
Figure 20 Trigonometrical representation of phase relationship
strain (e). The spring represents the elastic, or in-phase, component, G ' . The dashpot represents the loss, or out-of-phase component, G ' .
between shear rate and dynamic stress in oscillatory measurement in viscoelastic region (a), with corresponding components of complex viscosity illustrated in (b).
Rheology andTexture of Cheese
unpredictably large shear. The strain is kept low if one wishes to measure without structural damage to the sample but may be increased if one wishes to determine a fracture stress or to simulate a practical situation. Ma et al. (1996) found that elastic and loss moduli, G' and G", respectively, increase only to a second order with increasing frequency, i.e., by a factor of about 3 for a 1000-fold increase in frequency. This implies a 300-fold decrease in the viscosity components, r/' and r/', respectively (i.e., multiplying by 3/1000 --- dividing by 300), showing that the complex modulus is much more stable with respect to frequency for cheese than is complex viscosity. Various studies on different cheese varieties have indicated that G' and G" decrease, while # increases, with increasing temperature in the range 4-40 ~ the range normally encountered during consumption and mastication (Taneya et al., 1979; Home et al., 1994; Guinee et al., 2000b; Guinee, 2002). These changes, mark a transition from a cheese which is largely elastic in character at low temperature (#--- 12-16~ to one which is more viscous in character at the higher temperature (8--- 40 ~ and may be attributed to fat liquefaction. Tunick et al. (1990) made shear measurements on Cheddar and Cheshire cheeses in the range 20-40 ~ and fitted the Arrhenius equation to the complex viscosity in the form:
Aviscexp (Ev sc) where Avisc is a pre-exponential factor, Evisc is the activation energy, R is the gas constant and T is absolute temperature. A logarithmic form of this equation gives a linear plot:
6.5
527
+ Cheshire at 20 weeks
I
6.0
o Cheshire at 60 weeks
-x< 5.5 /k Cheddar at 60 weeks
5.0
4.5 4.0 20
30
40
Temperature, ~
Figure 21 Variation of complex viscosity of Cheddar and Cheshire cheese with temperature in the range 20-40 ~ Cheddar at 60 weeks, A; Cheshire at 20 and 60 weeks, O, +, respectively. Data taken from Tunick et aL (1990).
Fundamental Deformation
Measurements:
Large Strain
Large strain deformation measurements on cheese are usually undertaken using uniaxial compression, shear (or torsion), wire-cutting or bending. The methodology and instrumentation used for these measurements, and factors affecting the measurements, are discussed below. Uniaxial compression
The most common types of rheological measurement in cheese involve linear (uniaxial) displacement, e.g., using Instron UTM (Lee et al., 1978; Weaver et al., 1978; Imoto et al., 1979; Creamer and Olson, 1982; Visser, 1991; van Vliet, 1991b; Guinee etal., 1996; Pons and Fiszman, 1996; Fenelon and Guinee, 2000), the Stevens Response Compression Analyser (Stevens Group Ltd, Blackburn, UK) (Brennan, 1984), the TA.XT2 texture analyser or its derivatives from Stable Micro Systems (Truong et al., 2002; Xiong et al., 2002) (Table 3; Fig. 22).
(a) ~,~"
~
(b) ........................
where A = Evisc/(2.3 R) and B = log10 Avisc. Cheddar cheese had a higher activation energy (137 000 J/mol) than Cheshire (84 800J/mol) at 60 weeks of age, implying a greater sensitivity to temperature for the rheological properties of Cheddar cheese. However, the viscosity-temperature curves for the two cheeses crossed around 25 ~ i.e., Cheddar had a higher viscosity than Cheshire below 25 ~ and vice versa above 25 ~ (Fig. 21). The sensitivity of viscosity to temperature was found to decrease with age for Cheshire cheese (Fig. 21).
, ,
(c)
Figure 22 TA. HDI texture analyser (Stable Micro Systems) (a), and sample of Cheddar cheese before (b) and after (c) uniaxial compression to 20% of original height.
528
Rheology andTexture of Cheese
Measurements are generally made in compression mode rather than in tensile mode because (a) compression behaviour is relevant to sensory perception arising from chewing and mastication, (b) it is difficult to grip cheese samples to carry out a tensile test and (c) hard cheese has inhomogeneities which occur in random positions and directions with respect to sample dimensions, making it difficult to obtain reproducible results for tensile fracture. A compression measurement involves compression of a rectangular or cylindrical sample between parallel plates. Compression testing is more suited to large strain deformation than to linear viscoelastic deformation (low strain). This is because initial contact between the parallel plates and the sample usually involves some realignment of the sample surface due to imperfections in the surface smoothness, as a result of intrinsic macrostructural characteristics of the cheese (e.g., veins, cracks, openness). Moreover, the difficulty in fine precision cutting can give rise to lack of accuracy in low strain measurements (up to 0.5 mm of deformation). Ideally, the cheese sample may be conditioned by applying one or more low compression deformations (e.g., ~--- 0.05) prior to testing. A typical force-displacement curve, obtained by compression of a sample of Cheddar cheese at constant velocity to a strain of 0.8 (i.e., final height of the sample is 20% of original height), is shown in Fig. 23. Compared to other rheological methods for evaluating cheese, large-strain deformation compression offers several advantages: the strains applied are in the range of those applied to cheese during size-reduction operations as applied commercially; it is a dynamic method for which the calculated parameters (e.g., of, ef) depend on a range of stress-strain data accumu-
lated during the test; sample preparation does not require specialised cutting equipment; all cheeses, apart from soft cheeses with a very high sf, e.g., mature Camembert with an almost-runny consistency, can be prepared easily and tested; and the test method is simple and rapid. However, for reproducible results, sample shape and dimensions need to be precise, which can be difficult where cylindrical samples are acquired by pushing a cylindrical borer into a portion. As is common to all large strain deformation methods, a serious limitation is the difficulty in obtaining results for cheeses with eyes. Relationship between shear and normal stresses in uniaxial compression
A proper interpretation of uniaxial compression tests requires that the complexity of internal forces in a sample be understood. A sample undergoing uniaxial compression is distorted in various directions at any one time, e.g., vertically, horizontally and diagonally (Fig. 24). Even though the applied force is uniaxial, a combination of compressive and shear forces is created in different planes within the sample (Fig. 25). A fracture is more likely to be caused by shear rather than compression forces, since cheese is relatively incompressible. Thus, large strain uniaxial compression indirectly measures shear behaviour. Mohr's circle, a construction widely used in the analysis of the strength of materials, enables the shear and compression stresses (up to the fracture point) on an inclined plane, at any angle, O, to the horizontal to be calculated (Fig. 26). Analysis shows that the maximum normal s t r e s s (O'max = FIA) is twice the maximum shear stress (i.e., rmax = FI(2A)).
250
(a)
(b)
200 s Z
150
s SSS
I
ll C)
c) LL
$"t t
100 ii S 0
50 ('~
,,
0
Figure 23
,,
9
J
5
,
,
I
I
10 15 Displacement, mm
I
20
Force-displacement curve obtained from uniaxial compression of 6-month-old Cheddar cheese at 4 ~ on a TA.HDi texture analyser, showing the fracture point. The shaded area represents the fracture work or toughness.
Figure 24
Illustration of strains in relation to the principal axes of strain (vertical, y and horizontal, x) in a sample undergoing uniaxial compression. Compression along the y-axis results in a simultaneous extension along the x-axis. This coincides with a reduction in the angle of inclination, from ~1 to 82 of a diagonal line implying shearing along an inclined plane.
Rheology andTexture of Cheese
(a) Compression forces
(b) Shear forces
Frictional fo~
Figure 25 Compression (a) and shear forces (b) within a sample during a uniaxial compression test.
Effect of pre-test strain history Fracture stress decreases significantly if the sample has been cycled through successive compressions. This was shown for Cheddar, Cheshire and Leicester cheeses at 20 ~ by Dickinson and Goulding (1980). The effect was noticeable even when the previous strains were relatively low, e.g., o'f in Cheddar and Cheshire cheeses fell by --- 30% after 50 cycles of e - 0.1; the reduction in o-f in Leicester cheese was about 12% under the same conditions. However, pre-test strain history (50 cycles at - 0.2) produced no significant effect on ~f. This indicates that a history of recoverable deformation causes some internal structural weaknesses, which reduce the subsequent strength of the cheese, but the flow conditions under which fracture occurs are not affected. Effect of sample-machine interface conditions and sample dimensions The expression of compression characteristics in the form of stress-strain curves rather than force-displacement
Shear stress
.4
~~ - F/A
1~ Figure 26 Mohr's circle, for computing normal and shear stresses on any plane in a sample under uniaxial compression. Each point on the circle represents the normal (o-, x-axis) and shear (~-, y-axis) stresses which occur on a plane inclined at an angle 0. It can be inferred from the diagram that maximum .r occurs at 0 = 45 ~ and equals half of the uniaxially applied o-, i.e., at 0 = 0 ~ where F is the applied force and A is the cross-sectional area of the sample.
F/A,
529
curves is meant to remove the effect of sample dimensions. At low ~ values, the effect of sample dimensions may be eliminated in this way. However, for large e (>0.1), especially >el, the distribution of stress and strain within the sample depends on sample dimensions, as the sample may be deformed into an irregular shape, due to fracturing, barreling and squeezing. Squeezing flow is an intrinsic aspect of large strain uniaxial compression of cheese, i.e., as sample height is reduced, the cheese spreads in a lateral direction (Fig. 24). This implies that shearing takes place within the sample and that frictional shear forces occur at the points of contact between the sample and the compression plates. Friction can be reduced by lubrication of the contact surfaces with mineral oil or grease; in contrast, surface friction can be increased by the use of emery paper, or the surfaces can be bonded using glue, both of which eliminate slippage as a result of cheese 'sweating'. Because lubrication allows lateral movement at the contact surfaces during compression, the sample shows a slight tendency towards an hour-glass shape, as opposed to the relatively large barreling effect. Lubrication can reduce the stresses in squeezing flow by as much as 50% and increase the observed ef from - 0 . 4 5 to 0.55, in the case of Gouda cheese at 20 ~ with an aspect ratio (i.e., height/width) of unity and a cross-head speed of 500 mm/min (Culioli and Sherman, 1976). However, the frictional effect increases with cross-head speed. At a low cross-head speed (5 mm/min), lubrication decreased of b y - 2 0 % in Cheddar cheese where the aspect ratio was 0.35, with the effect becoming more pronounced (of the order of 20-30%) at e > ef (Casiraghi et al., 1985). In contrast, the bonding of the cheese surfaces to the compression plates (e.g., using cyanoacrylate ester adhesive) had relatively little effect on of, ef and O'max. A similar trend was found for Mozzarella and processed cheese spread (Casiraghi et al., 1985). At low compression plate speeds ( < 2 0 mm/min), friction had only a negligible effect in Gruyere and processed Mozzarella cheese with aspect ratios near unity (Charalambides et al., 2001). However, at aspect ratios <0.5, o- at a given e was slightly higher when the samples were not lubricated. Similar findings were reported by Ak and Gunasekaran (1992), using mineral oil as a lubricant on Cheddar cheese with aspect ratios of 0.65 and 1.0. Hence, accurate o'-e curves can be obtained from unlubricated testing provided the aspect ratio is sufficiently high, e.g., -> 1. Effect of deformation rate Due to viscous effects, o- at a given e (e.g., 0.1) and o-f depend on rate of deformation and increase by - 4 0 - 5 0 % per 10-fold increase in compression plate speed in cheeses at 4-20 ~ (Luyten et al., 1991a; Ak
530
Rheology andTexture of Cheese
and Gunasekaran, 1992; Pons and Fiszman, 1996; Xiong et al., 2002). ef is not significantly affected by deformation rate. Influence of shape Cylindrical and rectangular-shaped samples have been used in uniaxial compression (Table 3). For both shapes, only slight differences have been reported in the cr-e characteristics up to the fracture point (---40% compression). However, at e > el, compression of cubic samples resulted in significantly greater forces (and or) than cylindrical samples (Culioli and Sherman, 1976). For a given shape (e.g., cylindrical) and aspect ratio (e.g., unity), doubling the absolute dimensions had little effect on o-at a given deformation (Culioli and Sherman, 1976). Large strain shear measurements
A rheometer or a viscometer may be used also, in addition to a texture analyser, to apply large strain shear to cheese. In the rheometer, the parallel plate geometry already described (see 'Fundamental Measurements: Oscillatory Rheometry for Linear Viscoelastic Measurements in Cheese') for linear viscoelastic deformation, becomes applicable for large shear strain (i.e., torsion testing, where a shear is applied in rotational mode). A large shear strain may be applied also with a rheometer using samples of cheese with capstan geometry (Truong and Daubert, 2000, 2001; Truong et al., 2002). The effect of cutting the cheese into a capstan shape is that the greatest shear stress occurs at the cross-section of minimum radius (Fig. 27). Bowland and Foegeding (1999) used this technique to determine the fracture properties of model processed cheese. Torsion shear tests, as above, have been applied recently to cheese and offer few if any distinct advantages above large strain deformation in
(a)
~--..3
(b)
compression/extension mode. However, for highly deformable cheeses, e.g., flesh low-moisture part-skim Mozzarella and young reduced-fat cheeses, e.g., Cheddar, which generally do not undergo elastic fracture (i.e., where the sample breaks into distinct pieces), but rather plastic fracture, on compressing to strains of 0.7-0.8 (Fenelon and Guinee, 2000; Guinee et al., 2002), torsion shear may be useful in determining fracture stress and fracture strain. The latter parameters may be important in some cheeses, e.g., in the formation of cheese strings containing two different-coloured cheeses in a twisted helical (rope-like) configuration. However, preparation of capstan-shaped samples requires specialised milling equipment and is time-consuming. Vane rheometry has been used for large strain shear deformation tests in processed and natural cheeses, including Cheddar and Mozzarella. In this method a probe, typically with four blades or vanes, is inserted into a sample and rotated slowly at a constant rate (e.g., 10 cycles per minute), while the torque is measured (Fig. 28). The technique produces a shear stress versus strain characteristic with a distinct peak at the point of failure, i.e., fracture. Fracture stress is shear rate-dependent in both cases, being smaller by a factor of 2-3 with the vane technique than with the torsion technique. Using vane rheometry and torsion, it was possible to separate different cheese types on fracture stress/fracture strain 'texture maps', which were similar in both cases (Truong and Daubert, 2001). The vane method, which is rapid and does not require sample preparation, as in cutting, has been found to give trends comparable to those obtained using capstan geometry for processed cheese, Cheddar and Mozzarella (Truong and Daubert, 2001). However, disadvantages of vane rheometry may include the difficulty of inserting the vane without cracking the cheese mass (e.g., hard cheeses such as Romano or Parmesan, with a low fracture strain and soft cheeses with a low fracture strain, such as Feta), and variability of results for cheeses, such as Blue or Gouda, with macrostructural heterogeneities, such as cracks, veins, small openings and openness. Bending tests
Ic
2R
4
~ L 2r 4 2R
J q
Illustration of sample shapes used in torsion tests, using parallel plate geometry (a), and capstan geometry (b). The capstan shape is obtained by milling a cylindrical sample using
Figure 27
a purpose-built milling machine.
Hard cheeses can be subjected to bending tests which may involve three- or four-point loading (Whorlow, 1992; Rosenthal, 1999); a schematic for three-point bending of a 'finger-shaped' cheese sample is shown in Fig. 29. Such tests produce compression and tension on alternate sides of a neutral axis, with the maximum tensile strain occurring on the bottom surface of the sample and the maximum compressive strain on the top surface (Fig. 29). A force-displacement curve obtained from three-point bending of a cheese sample, e.g., Cheddar cheese (Fig. 30), enables the
Rheology andTexture of Cheese
(a)
531
(b)
~
,
,,,~.
,
. ..J":!
Figure 28 Vane rheometer probe before (a) and during (b) shear test on process cheese. Photos courtesy of Truong and Daubert (2000) Gel Consultants Inc. (See Colour plate 11 .)
estimation of fracture stress (o-f). With the assumption that the sample deforms into an arc shape on bending, the tensile strain (~) at any point on the bottom surface may be estimated for any displacement (y) as:
4~
E z
L 2 + 4312
(b)
~
~
.............Ei .......~ ~ '
\tension
Figure 29
Schematic of a bending test with three-point loading: (a) Geometry with cheese sample in place prior to testing, resting on the two support beams, C and D; (b) cheese sample being deformed by mobile beam E during testing.
where y is the displacement at any point of contact along the axis where the sample makes contact with the moving beam, H is the sample height and L is the span between the supporting beams (Fig. 29). Assuming that o-is proportional to e, the tensile or compressive stress (OM) at any point on either surface of the sample can be approximated, subject to assumptions about linearity, as:
O-M.d.
3FL 2WH 2
50
i 40 z d 30 o 0 LL 20
X\
10 0
i
0
Figure 30
1
5 10 Displacement, y, mm
15
Force-displacement curve for three-point bending of a 180-day-old Cheddar cheese sample (25 • 25 • 50 mm), ripened at 4 ~ The sample was deformed at a rate of 20 mm/min (by the mobile beam) on a TA.HDi texture analyser, using a three-point bend rig (model HDP/3PB) with 40 mm span between the supporting beams. A distinct fracture point (f) at point E1 (Fig. 29) on the bottom surface of the sample coincides with the maximum extension.
where F is the force (obtained from the force-displacement curve) and W is the width of the sample (Fig. 29). Even though the o--e behaviour departs from linearity before the point of fracture, the formula can be used to approximate the fracture stress (or). Since fracture is more likely to occur in tension than in compression, fracture behaviour in tensile mode can be compared more easily using bending tests than by using tensile tests, which are difficult to carry out because of the difficulty of grabbing a sample without deforming it. The fracture strain (el) obtained during threepoint bending may give a better estimation of the cutting behaviour of cheese than ef obtained from compression testing (see 'Uniaxial Compression'), as cutting involves a combination of tensile and shear strains. In compression testing, fracture is due to shear displacement.
532 RheologyandTexture of Cheese Wire-cutting Fracture energy during cutting is quantified by measuring the force required to push wires of different diameters at constant velocity through a cheese mass (Green etal., 1986; Marshall, 1990). Luyten etal. (1991b) investigated the fracture properties of Gouda cheese using wire-cutting. A typical force-time curve shows an initial rise in force, which reaches a maximum as the wire penetrates the sample surface. Once the surface has been broken, the force rapidly drops to a constant level, Fc, as the wire 'ploughs' through the sample. Fc increases somewhat with cutting speed (--- doubling for a 20-fold increase in speed) and with wire diameter (by 3-4 fold for 300 Ixm compared with 25 txm diameter). Since fracture develops around a crack, a specific fracture energy (Jim2), Rf, can be defined as the energy needed per unit area (of crack) to cause a fracture to spread. While it is not possible to determine specific fracture energy precisely, because of the inherent heterogeneity in cheese structure (see 'Cheese structure'), its order of magnitude can be determined by measuring cutting force using wires with a series of diameters and extrapolating to a diameter of zero. The specific fracture energy is calculated as:
Rf =
Fc0
d
where Fc0 is the cutting force, extrapolated to cutting with a wire of zero diameter, and d is the sample width, i.e., the length of wire in contact with the cheese (Luyten et al., 1991b). The fracture energy obtained with the wire-cutting method may give a more accurate prediction of the behaviour of cheese during cutting (e.g., portioning, slicing) than that obtained using large-strain shear or compression deformation tests.
Measurement of Time-Dependent Rheological Characteristics As has been stated (see 'Terminology of Rheology and Texture' and 'Fundamental Measurements: Large Strain Deformation'), the rheological behaviour of viscoelastic materials, like cheese, is generally influenced not only by instantaneous stress or strain, as in the case of 'ideal' materials, but also by rheological history of the material, i.e., the stresses and strains which have already been experienced. This is verified by natural occurrences, such as a gradual deformation of a pane of glass under its own weight in a window of an old building. Indeed the compression of rock or ice under gravitational force results in flow, albeit very slow, analogous to a creep experi-
ment. Similarly, hard cheeses can eventually bulge, especially if stacked. Such time-dependent behaviour may be measured in creep and stress-relaxation tests, and may be carried out by means of compression, tension or torsion in the viscoelastic region (e.g., e >0.1; cf. Fig. 9).
Effect of Sample Temperature on Large Strain Deformation Characteristics in Cheese Early research showed that increasing the temperature of Gouda cheese in the range 10-20 ~ reduced the value of el, o-f and O'max, as measured by compression to 80% using the Instron UTM (Culioli and Sherman, 1976). While o'f in Cheshire and Leicester cheeses decreased exponentially as the temperature was increased from 0 to 40 ~ the effect on fracture strain depended on the type of cheese; fracture strain for Cheshire cheese increased by ---2 over the range of temperature, while fracture strain for Leicester cheese was not affected by temperature (Dickinson and Goulding, 1980). Molander et al. (1990) reported a similar trend for o-f and O'max in 4-week-old Brie between 5 and 20 ~ however, in contrast to the results of Culioli and Sherman (1976), ef increased slightly on raising the temperature. The discrepancy between the latter studies in relation to strain may be attributable to differences in the degree of fat coalescence, proteolysis and therefore fat separation and slippage. On heating cheese to a temperature (30-60 ~ greater than those (e.g., 4-25 ~ normally associated with retailing, domestic refrigeration and consumption, compression results in squeezing flow behaviour (Ak and Gunasekaran, 1995), i.e., stress increases with strain as the cheese is squeezed between the plates and no fracture point is observed. The deformability modulus (initial slope of the stress-strain curve) showed an Arrhenius type of characteristic, decreasing exponentially with temperature from 18 kPa at 30 ~ to 3 kPa at 60 ~ Such a trend is expected, as milkfat is essentially fully liquid at 30 ~ (Norris et al., 1973). Indeed, heating cheese to 60 ~ in the absence of an applied stress generally results in flow of the part-molten cheese mass to an extent dependent on cheese type and heating time.
Techniques for Measurement of Viscosity In some situations, cheese products may occur in 'liquid' form, either in the course of processing or in their usage. Typical examples are processed cheese, cheese dips and cheese sauces. The viscosity of these products may be measured by a number of instruments, e.g., the
Rheology andTexture of Cheese
Bostwick consistometer, which has been used to give an empirical measurement of viscosity of a soft processed cheese spread (Rosenthal, 1999). In the latter instrument, a sample of the material being tested is placed in a cell and released by opening a simple guillotine slide gate, allowing the product to flow horizontally across a scale marked in centimeters. The length of flow in a given time period (usually 30 s), known as the Bostwick number, is taken as a measure of viscosity. Alternatively, viscosity can be measured under defined shear or low amplitude stress or strain in a rheometer, using different geometries such as concentric cylinders, a cone and plate, or parallel plates. Online measurements of viscosity of cheese products may be important, e.g., as an early measure of indicating the susceptibility of a formulation to 'creaming' (see 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). A range of commercial on-line viscometers are available for measuring viscosity in a continuous flow situation.
Terminology Applied to Cheese Texture Cheese texture may be defined as a composite sensory attribute resulting from a combination of physical properties that are perceived by the senses of touch (including kinaesthesis and mouth-feel), sight and hearing (Brennan, 1988). Thus, cheese texture is directly measurable only by sensory analysis. Sensory analysis requires definition and classification of textural attributes or descriptors. Descriptors applied to cheese texture have been grouped into mechanical, geometrical and other characteristics (Fig. 31). The mechanical characteristics are sensed as forces on the teeth, tongue and the mouth
generally during eating, and by hearing in the case of fracture, whereas geometrical characteristics are mostly sensed visually but may also be partly sensed by touch. The other characteristics are 'mouth-feel' qualities, described subjectively by terms such as hard, soft, firm, springy, crumbly, adhesive, moist or dry. These terms are thought to have significance in relation to consumer appeal and satisfaction (Szczesniak, 1963a). The mechanical characteristics, in turn, have been divided into five primary parameters and three secondary parameters (Table 5, Fig. 31). The secondary parameters are considered to be composed of various intensities of hardness and cohesiveness. The geometrical parameters are divided into two classes, i.e., those related to particle size and hardness, and those related to particle shape and orientation. Experience shows that panelists found hardness relatively easy to sense but that adhesiveness was much more difficult to judge (Halmos, 2000). Sensory texture terms, as distinct from rheological terms, have linguistic boundaries, i.e., they are susceptible to different interpretation in different languages (Lawless et al., 1997; Bourne, 2002). Some texturerelated characteristics can be measured by machines and these are not bound by language. These characteristics include hardness, cohesiveness, adhesiveness, elasticity, viscosity, brittleness, chewiness and gumminess, definitions for which are given in Table 5. The measurements give objective quantifiable data, provided the measurement conditions are well defined. Relationships between cheese texture and rheology
The Texture Profile Analysis (TPA) method, involving instrumental measurement using double bite compression, was developed to imitate the compressing action 9 visual appearance 9 sampling and slicingcharacteristics 9 spreading, creaming characteristics, pourability
Initial perception { (before placing in mouth)
9 analyticalcharacteristics
9 particle size, shape and size distribution 9 oil content; size, shape and size distribution of oil
Primary characteristics Initial perception on palate
particles 9 elasticity, cohesion 9 viscosity 9 adhesion (to palate)
l Secondary characteristics I
Mastication (high shearing
9 9 9 9 9
I Ze.iar characteristics
stress) Residual
masticatory impression
Figure 31
f
533
9
hard, soft brittle, plastic, crisp, rubbery, spongy smooth, coarse, powdery, lumpy, pasty creamy, watery, soggy sticky, tacky
9 greasy, gummy, stringy 9 melt down properties on palate
Classificationof food texture into primary, secondary or tertiary characteristics, based on Sherman (1969).
534
Rheology andTexture of Cheese
Table 5
Classification of the mechanical characteristics of cheese into primary and secondary parameters a
Primary parameters
Secondary parameters
Hardness - the force necessary to attain a given deformation Cohesiveness - strength of internal bonds making up the body of the product Elasticity- the rate at which a deformed material returns to its original form after the deforming force is removed Viscosity- rate of flow per unit force Adhesiveness - the work necessary to overcome the attractive forces between the surface of a food and surface of other materials with which it comes in contact, e.g., the teeth, palate and tongue
Brittleness - the force at which the material fractures Chewiness - the energy required to masticate a solid food, e.g., some cheeses such as Mozzarella, to a state ready for swallowing Guminess - the energy required to disintegrate a semi-solid food, e.g., some cheeses such as ripe Camembert, to a state ready for swallowing
a Modified from Szczesniak (1963a), Bourne (1978).
of molar teeth while masticating food in the mouth (Szczesniak, 1963a; Peleg, 1976; Bourne, 1978). Classification of the mechanical attributes of cheese texture, as described above, was designed with the aim of integrating sensory data for foods evaluated by trained panels, with texture-profile data obtained on the same foods using compression testing. For this purpose, objective rheological parameters, some of which correspond in name to the sensory-determined parameters, were defined (Table 5) and are known as TPA parameters (see 'Texture profile analysis'). While this classification system has been modified, the textural descriptors and their interpretation as devised by this classification scheme (Table 5) are still widely used in textural evaluation of food (Brennan, 1988; Drake et aI., 1999). Sherman (1969) proposed an alternative classification of food texture (Fig. 31). The characteristics contributing to the texture of cheese, and other foods, during eating have been classified as primary, secondary (e.g., adhesiveness) or tertiary (e.g., firmness) (Fig. 31). The primary characteristics, from which all others are derived, include the food's composition, its micro- and macro-structure, and its molecular properties. The secondary and tertiary categories of textural properties include many characteristics which are directly related to the rheological properties as it is subjected to various stresses and strains during eating, e.g., hardness, brittleness and adhesiveness (Sherman, 1969). According to this classification, the secondary characteristics are associated with initial perception in the mouth, i.e., upon contact with tongue, palate and teeth prior to mastication. Sherman (1969) claimed that the main characteristics sensed at this stage are elasticity (E), viscosity (r/) and adhesion to the palate, where elasticity is understood as the tendency to recover its shape after removal of the stress. Two of those characteristics, namely elasticity and viscosity, can together be represented by the Burgers mechanical model (see 'Cheese texture').
Texture profile analysis (TPA)
A system of rheological parameters (e.g., firmness, elasticity) related to texture and known as TPA was developed (Fig. 32; Table 6; Friedman et al., 1963). The rheological measurements were originally carried out using the General Foods Texturometer (see 'Imitative tests'), using double-bite compression (Bourne, 1978). Texture profile analysis parameters were later calculated from measurements using uniaxial doublebite compression at constant speed, using texture analysers including the Instron UTM (Breene, 1975; Bourne, 1978; Lee etal., 1978) and the texture analyser (TA series from Stable Micro Systems) (Halmos, 1997; Meullenet and Gross, 1999). Use of TPA to evaluate cheese texture
Szczesniak (1963b) found a curvilinear relationship between TPA hardness and an organoleptic rating of hardness. Casiraghi et al. (1989), working with five different Italian cheese varieties, including Grana Padano and Italico, showed that sensory hardness was highly correlated with instrumental hardness.
1st compression stroke
1
2nd compression stroke
b
0
Time
Figure 32
Typical stress trend during a double-bite compression test, from which TPA parameters are calculated (see Table 5).
Rheology andTexture of Cheese
Table 6
535
Texture profile analysis (TPA) parameters and physical definitions a
Terminologyb
Physical definition (TPA term)
Units
Fracturability Firmness Springiness (or elasticity) Cohesiveness Gumminess Chewiness Adhesiveness
Stress (or sometimes, force) to fracture point, H1 (Fig. 32) Stress (or sometimes, force) at a given deformation Percentage of deformation which is recovered between the first and second bites
Pa, kPa Pa or kPa
Area of second bite over area of the first bite (A2/A1) in Fig. 32 Hardness • Cohesiveness Hardness • Cohesiveness x Springiness Work necessary to pull the plunger (or compression plate) away from the sample (Area 3 in Fig. 32)
m
Pa, kPa Pa, kPa J/m3
a Sources: Bourne (1978), van Vliet (1991a), Szczesniak (1963a), Yang and Taranto (1982). b Fracturability was originally known as brittleness (Bourne, 1978), and firmness as hardness (Szczesniak, 1963a).
Green et al. (1985) found significant correlations between five sensory attributes (firmness, springiness, crumbliness, graininess and stickiness) and instrumental parameters (of and ef ). Hennequin and Hardy (1993) reported that TPA-hardness, i.e., force at 70% compression, also had a high correlation with sensory hardness (r = 0.78, n = 19, P < 0.001) for four soft cheeses. Halmos (2000) compared sensory and instrumental measurements of hardness, cohesiveness and adhesiveness of six cheeses with a wide range of texture (including Havarti, Swiss and Romano). The sensory measurements increased with the corresponding instrumental readings, apart from one parameter for Romano cheese, for which the cohesiveness as measured instrumentally was ranked higher than the corresponding sensory measurement. The significant correlations, which were characteristic of the overall study, confirm the value of objective measurements in support of sensory measurements. However, the deviation in the trend for the Romano cheese highlights the complexity of textural (i.e., tactile sensory) characteristics as compared with instrumental measurements. Antoniou et al. (2000) performed sensory and TPA analyses on 15 French cheeses (Munster, Emmental, Roquefort, Beaufort, Camembert, Reblochon, Pont l'Eveque, Brie de Meaus, Tomme de Savoie, Valencay, St Nectaire, Pyrenees Brebis, Blue d'Auvergne, Comte Vieux and Fourme de Salers). The cheeses fell into three compositional groups on the basis of moisture (means 34, 45 and 51%, w/w). This grouping carried through to the results of mechanical and sensory analysis. The mechanical (TPA) terms which were most significant in differentiating the groups were: force at 10% deformation, relaxation force (after holding sample for - 1 2 s at 10% compression), force at 80% deformation (hardness), fracture force and adhesiveness. The most significant sensory terms were: hardness, fracturability and chewiness. Some of the mechanical parameters were highly correlated with
each other (e.g., force at 10% deformation, fracture force and hardness). Likewise, some of the sensory parameters were inter-correlated, e.g., hardness with adhesiveness. In agreement with previous studies (Green et al., 1985; Casiraghi et al., 1989; Hennequin and Hardy, 1993; Halmos, 2000), sensory parameters were highly correlated with mechanical parameters, e.g., mechanical hardness with sensory hardness. It is noteworthy that the 10% compression measurements (a level of deformation that is mostly recoverable) predicted cheese texture (i.e., as judged in sensory terms) better than the 80% compression tests. Despite the significant correlations between some sensory textural parameters and rheological measurements, instrumental analysis of texture, e.g., using texture analysers, is not considered a complete substitute for sensory evaluation (see Halmos, 2000), because of several factors: complexity of mastication, differences between individuals in the perception of texture, effect of time of day upon perception of texture, and others. While instrumental methods alone cannot be relied upon to determine consumer acceptance, their value resides in their ability consistently to enable small changes in physical characteristics, which contribute to texture, to be quantified. Use of instrumental shear deformation to evaluate cheese texture
Three techniques for large strain shear deformation testing have been described (see 'Large strain shear measurements'). Truong et al. (2002) compared instrumental textural measurements on Cheddar cheese, as obtained using vane rheometry (shear), uniaxial compression (single bite) or TPA (double bite), with the corresponding sensory texture measurements. Instrumental texture maps of ten commercial Cheddar cheeses, generated by the vane method and by compression testing, clearly separated the cheeses and showed similar distribution patterns. Highly significant
536
Rheology andTexture of Cheese
correlations were found between vane parameters and TPA parameters (i.e., by uniaxial compression), and between TPA parameters and sensory texture parameters (by mouth). Correlations between vane parameters and sensory parameters were significant, but not as highly significant as between sensory and TPA parameters. The higher correlation between sensory texture and TPA texture could be due to the fact that TPA parameters were developed in conjunction with compression (i.e., General Foods Texturometer), while no corresponding texture-related parameters have been developed for torsional techniques, such as the vane method.
Conclusions The rheological properties of cheese have a large influence on its texture and behaviour during size reduction, and hence, its suitability as an ingredient (see 'Cheese as an Ingredient', Volume 2). Many factors influence the rheological properties, including m a n u facturing procedure, variety, composition and biochemical changes during ripening. The latter parameters have a major influence on the degree of hydration, or aggregation, of the para-casein matrix, which is the major structural element controlling deformation on the application of a stress. Many m e t h o d s are available for measuring the theological properties of cheese; some measure within the linear viscoelastic range to yield precise rheological quantities. In contrast, rheological m e a s u r e m e n t s made u n d e r large strain or stress yield quantities which are more empirical in nature, but which are typically related to the stresses and strains experienced during c o n s u m p t i o n and size reduction.
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Guinee, T.P., O'Callaghan, DJ., Mulholland E.O. and Harrington, D. (1996). Milk protein standardization by uhrafiltration for Cheddar cheese manufacture. J. Dairy Res. 63,281-293. Guinee, T.P., Fenelon, M.A., Mulholland, E.O., Kennedy, B.T., O'Brien, N. and Reville, W.J. (1998). The influence of milk pasteurization temperature and pH at curd milling on the composition, texture and maturation of reduced fat Cheddar cheese. Int. J. Dairy Technol. 51, 1-90. Guinee, T.P., Auty, M.A.E. and Mullins, C. (1999). Observations on the microstructure and heat-induced changes in the viscoelasticity of commercial cheeses. Aust. J. Dairy Technol. 54, 84-89. Guinee, T.P., Auty, M.A.E., Mullins, C., Corcoran, M.O. and Mulholland, E.O. (2000a). Preliminary observations on effects of fat content and degree of fat emulsification on the structure-functional relationship of Cheddar-type cheese. J. Text. Stud. 31,645-663. Guinee, T.P., Auty, M.A.E. and Fenelon, M.A. (2000b). The effect of fat content on the rheology, microstructure and heat-induced functional characteristics of Cheddar cheese. Int. Dairy J. 10, 277-288. Guinee, T.P., Feeney, E.P., Auty, M.A.A. and Fox, RE (2002). Effect of pH on calcium concentration on some textural and functional properties of Mozzarella cheese. J. Dairy Sci. 85, 1655-1669. Hall, D.M. and Creamer, L.K. (1972). A study of the submicroscopic structure of Cheddar, Cheshire and Gouda cheese by electron microscopy. NZ J. Dairy Sci. Technol. 7, 95-102. Halmos, A.L. (1997). Food texture and sensory properties of dairy ingredients. Food Aust. 49, 169-173. Halmos, A.L. (2000). Relationships between instrumental texture measurements and sensory attributes, in, Hydrocolloids - Part 2, Nishinari, K., ed., Elsevier, Amsterdam. pp. 431-444. Hennequin, D. and Hardy, J. (1993). Evaluation instrumentale et sensorielle de certaines proprietes texturales de fromage/t pate molle. Int. Dairy J. 3,635-647. Home, D.S., Banks, J.M., Leaver, J. and Law, A.J.R. (1994). Dynamic mechanical spectroscopy of Cheddar cheese, in, Cheese Yield and Factors Affecting its Control. Special Issue No. 9402. International Dairy Federation, Brussels. pp. 507-512. Hort, J. and LeGrys, G. (2000). Rheological models of Cheddar cheese texture and their application to maturation. J. Text. Stud. 31, 1-24. Hwang, C.H. and Gunasekaran, S. (2001). Measuring crumbliness of some commercial Queso Fresco-type Latin American cheeses. Milchwissenschaft 56,446-450. Imoto, E.M., Lee, C.-H. and Rha, C. (1979). Effect of compression ratio on the mechanical properties of cheese. J. Food Sci. 44,343-345. Innocente, N., Pittia, R, Stefanuto, O. and Corradini, C. (2002). Correlation among instrumental texture, chemical composition and presence of characteristic holes in a semi-hard Italian cheese. Milchwissenschaft 57, 204-208.
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Glossary Cauchy strain. See Engineering strain. Compliance. Symbol J, is the ratio of strain to stress. In the elastic region, J = 1/G', where G' is shear (or storage) modulus. Cox-Merz rule. This rule states that the (steady) viscosity versus shear rate curve is virtually identical to the viscosity versus frequency curve, determined by dynamic oscillation. Creep. The response to a constant applied (normal or shear) stress. Creep can be expressed in terms of strain or compliance. Creep compliance. The ratio of strain, y(t), resulting from an applied constant stress, Zc, to the stress, i.e., 7(t)/~'c. Creep modulus. The inverse of creep compliance, i.e.,
~-d~,(t). Deformability modulus. Slope of the stress-strain curve in an approximately linear region, typically up to a strain of ~0.10. Elastic material behaviour. An elastic deformation is one where the material recovers fully u p o n removal of applied stress without time dependency, i.e., recovery is instantaneous and complete u p o n removal of stress. Elastoplastic material behaviour. W h e n the stress in the material exceeds a certain limit, irreversible deformation results with negligible time dependency, i.e., partial recovery is instantaneous upon removal of stress; also k n o w n as elastoplastic deformation. Engineering strain. Deformation relative to original sample dimension, i.e., AL/Lo, is called engineering strain, or Cauchy strain, or strain. Engineering stress. The ratio between applied force, F, and original sample area, Ao, is k n o w n as engineering stress or stress. Fracture work. See Toughness. Kelvin element. Also k n o w n as a Voigt element or a Kelvin-Meyer solid. This is a mechanical model consisting of a spring in parallel with a dashpot. A number of such elements in series, with a spectrum of time constants, can be used to simulate creep compliance. Kinematic viscosity. This is the ratio between dynamic viscosity and density. Units: m2/s = 10 4 stokes. Linear behaviour. If one measured parameter varies in proportion to another, e.g., stress in proportion to a range of applied strain, their behaviour is described as linear and a modulus may be defined as the ratio between the parameters, e.g., Young's modulus. Linear viscoelastic deformation. Cheese and other organic materials exhibit a combination of elastic and viscous behaviour at low strains, i.e., they recover their shape upon removal of applied stress, but not instantly. The elastic and viscous effects can be determined using
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Rheology andTexture of Cheese
low-amplitude oscillatory rheometry. At low amplitudes of oscillation there is a constant relationship between the elastic and viscous components of complex modulus. Consequently, displacements of this type are referred to as linear viscoelastic deformation. Loss modulus. The ratio between the out-of-phase component of shear stress and shear strain (G" = r"lT) in a dynamic oscillatory measurement; also referred to as viscous modulus. Maxwell element. This is a mechanical model consisting of a spring in series with a dashpot. A combination of such elements in parallel, with a spectrum of time constants, may be used to simulate relaxation behaviour in a viscoelastic material. Modulus of deformability. See Deformability modulus. Poisson effect. W h e n a sample is compressed it bulges in the lateral direction, i.e., the cross-section increases with compression; this is the Poisson effect. The ratio between lateral strain and longitudinal strain is k n o w n as Poisson's ratio. Poisson's ratio equals 0.5 in the absence of a volume change, and is less than 0.5 for a compressible material. Shear modulus. The ratio between the in-phase components of shear stress and shear strain (G' = r'/T) in a dynamic oscillatory measurement; also referred to as storage, elastic, or in-phase, modulus. Storage modulus. See Shear modulus. Strength. The m a x i m u m stress a material withstands before it breaks (i.e., fractures) or flows (i.e., becomes plastic). Stress. See Engineering stress. Stress relaxation m o d u l u s . The stress that is required to maintain a constant deformation is observed, as a function of time (i.e., in a stress relaxation test). The ratio of shear stress to strain is k n o w n as stress relaxation modulus, or relaxation modulus. The relaxation modulus depends on the applied strain if the strain exceeds the limit of linear viscoelasticity. Thus, G(t, T) = r(t)/% Stress relaxation test. This test involves an initial application of (a normal or shear) strain at a constant rate up to a pre-determined level of strain and then measuring
the decay of stress as a function of time while holding the sample at constant strain; also k n o w n as a step strain transient test. Toughness. The work required to fracture; this is measured as the area under a force-deformation curve up to the point of fracture (Fig. 23). True strain. The accumulated strain during the applied loading, e' = In (ULo), where In is the natural logarithm, L is the sample length under load, and Lo is the original sample length, is k n o w n as the true strain, Hencky strain or natural strain. This is applicable where the strain is large and sample cross-section changes appreciably under the load. True strain is not used very much in cheese rheology. True strain can be related to engineering strain, e, using, e' = In (1 + e). True stress. The ratio between applied force, F, and actual area of cross-section, A', is termed true stress. Thus, O'true FIA', where A' is the actual area, taking the Poisson effect into account. Uniaxial compressive strength. The apparent stress at fracture, i.e., Fo/Ao, where Fo is the compression force at fracture and Ao is the initial cross-sectional area of the sample. Viscoelastic material behaviour. Where rheological behaviour can be resolved into elastic and viscous components, e.g., as represented by a Maxwell model. Viscoplastic material behaviour. In contrast to elastic behaviour, this is a time-dependent and irreversible deformation that occurs when a certain stress level has been exceeded, i.e., strain does not respond instantaneously to applied stress, but instead strain keeps on growing while the stress is applied and does not return to zero upon removal of stress; also referred to as viscous material behaviour. Viscosity or dynamic viscosity. Coefficient of dynamic viscosity, 77, is the ratio between shear stress and shear rate. = rl~ where r is shear stress and ~ is shear rate. Units: Pa.s or N.s/m 2 = 10 poise. Viscous modulus. See Loss modulus. Young's modulus. The ratio between normal stress and engineering strain (E = ole). =
Growth and Survival of Microbial Pathogens in Cheese C.W. Donnelly, Department of Nutrition and Food Science The University of Vermont, Burlington, USA
Introduction Cheesemaking evolved centuries ago as a means of preserving raw milk via fermentation. Selection of the beneficial natural flora in milk, such as lactobacilli, streptococci and lactococci, or direct addition of these as starter cultures, preserves products and in many instances allows competition with bacterial pathogens. However, cheeses can become contaminated with pathogens as a result of their presence in the raw milk used for cheesemaking and subsequent survival during the cheesemaking process. Alternatively, bacterial pathogens can contaminate cheese via post-processing contamination if sanitation and other measures in the processing plant are not sufficient to prevent re-contamination (Linnan et al., 1988; Johnson et al., 1990a). The characteristics of the specific cheese variety will dictate the potential for growth and survival of microbial pathogens, with ripened soft cheeses presenting a higher risk for growth and survival of pathogens than aged hard cheeses where a combination of factors, including pH, salt content and aw, interact to render cheeses microbiologically safe. Although cheeses have been linked with documented outbreaks of food-borne illness, epidemiological evidence collected from around the world confirms that this occurs infrequently (Johnson et al., 1990a; Ahekruse et al., 1998; De Buyser et al., 2001). This chapter will provide an overview of factors which affect growth and survival of microbial pathogens in cheese.
Factors that Influence the Safety of Cheese The pathogens, Salmonella enterica, listeria monocytogenes, Staphylococcus aureus and enteropathogenic E. coli (ETEC) pose the greatest risk to the safety of cheese 0ohnson et al., 1990a; De Buyser et al., 2001; Leuschner and Boughtflower, 2002). If active lactic acid starter cultures are used, Staph. aureus is considered to be a low-risk pathogen (Johnson et al., 1990a). However, in traditional cheeses where active starter cultures are not used, Staph. aureus may pose a significant risk for toxin production in cheese if numbers are sufficiently high. The factors that contribute to
the safety of cheese with respect to pathogenic bacteria include milk quality, starter culture or native lactic acid bacterial growth during cheesemaking, pH, salt, control of aging conditions and chemical changes that occur in cheese during aging (Johnson et al., 1990c). Other technologies (e.g., use of starter cultures that produce substances inhibitory to pathogens) may provide opportunities to add additional barriers to the growth of bacterial pathogens. It is particularly important for the producers of raw milk cheeses to have a documented and systematic approach to ensure product safety. Pathogens in raw milk
S. enterica, L. monocytogenes, Staph. aureus and ETEC are associated with raw milk. E. coli 0157:H7 can readily contaminate raw milk on the farm with contamination levels of 4.2-10% and 2% reported in the US and Canada, respectively (D'Aoust, 1989; Padhye and Doyle, 1991). Over 70 cases of E. coli infection, characterized by bloody diarrhea, haemolytic uremic syndrome (HUS) and kidney failure, have been traced to the consumption of raw milk (Martin et al., 1986; Borczyk et al., 1987; Bleem, 1994) with a few additional cases in England linked to yoghurt (Morgan et al., 1993). E. coli 0157:H7 was first characterized in 1982 during epidemiological investigations of two outbreaks which occurred in North America. Cattle are thought to be the principal reservoir for this important human pathogen, and in investigations where food has been identified as the vehicle of transmission, ground beef is the product most frequently linked to human illness. Shere et al. (1998), in a longitudinal study of E. coli dissemination on four Wisconsin dairy farms, identified contaminated animal drinking water as the most probable vehicle for infection of animals and a potential intervention point for on-farm control of dissemination of this pathogen. Since shedding of this pathogen by cattle is intermittent, re-inoculation from an environmental source rather than colonization of the pathogen is the more likely explanation than intermittent shedding.
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
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Growth and Survival of Microbial Pathogens in Cheese
S. enterica serovars Enteritidis, Typhimurium and Dublin have been associated with food-borne disease outbreaks involving raw milk and milk products (Maguire et al., 1992; Cody et al., 1999; Villar et al., 1999; De Valk etal., 2000). A 1987 FDA survey revealed the presence of salmonella in 32 of 678 (4.7%) samples of raw milk obtained from bulk-tank trucks in Wisconsin, Michigan and Illinois, with 10 of 16 (62.5%) collection sites also testing positive (McManus and Lanier, 1987). Salmonella spp. were isolated from 26 of 292 (8.9%) of farm bulk tank samples collected in eastern Tennessee and southwest Virginia (Rohrbach et al., 1992). Wells et al. (2001) examined recovery of salmonella from faecal samples obtained from dairy cows in 91 herds from 19 US states. Salmonella spp. were recovered from 5.4% of the samples. Recovery levels from cows on farms with less than 100 animals were much lower (0.6%) than those from farms with over 100 cows, where recovery levels were 8.8%. The incidence of Salmonella spp. in milk samples would be expected to occur at a much lower frequency than in faecal samples. Most farmstead cheesemakers maintain small dairy herds, where the lower incidence data would apply. S. enterica serotype Typhimurium definitive type (DT) 104 emerged in the UK as an important source of human infection in the late 1980s (Threlfall et al., 1996). Subsequent outbreaks of human illness traced to dairy sources were reported in Vermont (Friedman et al., 1998), Nebraska, California (Cody et al., 1999) and Washington State (Villar et al., 1999). This organism is notable because it is resistant to multiple antibiotics. Two outbreaks of S. enterica subsp, enterica serotype Typhimurium DT104 infection were recently linked to the consumption of Mexican-style soft cheese manufactured from raw milk (Cody et al., 1999; Villar et al., 1999). Aceto et al. (2000) conducted a survey to assess the herd prevalence of 5. enterica subsp, enterica serotype typhimurium DT 104 in Pennsylvania dairy herds. Of 51 farms surveyed, 11 were positive for salmonella species and 4 for 5. typhimuriurn, 2 of which were DT-104 positive. S. enterica serovar Dublin is present in dairy cattle and was identified as the most invasive of the salmonella bacteria for humans in studies conducted in Denmark (Lester et al., 1995). Beckers et al. (1987) and Lovett et al. (1987) estimate that extremely low levels of L. monocytogenes (0.5-1.0 ml) exist in commercial bulk-tank raw milk. Listeria is inactivated by pasteurization, and contamination of processed dairy products is therefore most likely a function of post-pasteurization contamination from the dairy plant environment. In fact, numerous surveys document the presence of listeria within the dairy plant environment, including floors in coolers, freezers, processing rooms, particularly entrances, cases
and case washers, floor mats and foot baths and the beds of paper fillers (Charlton et al., 1990; Klausner and Donnelly, 1991). Pritchard et al. (1994), in a study of dairy processing facilities, found that processing plants near a farm had a significantly higher incidence of listeria contamination than those without an on-site dairy farm. Arimi et al. (1997) demonstrated the link between on-farm sources of listeria contamination (dairy cattle, raw milk and silage) and subsequent contamination of dairy-processing environments. These investigators subjected listeria strains collected from farms and dairy processing environments over a 10-year period to strain-specific ribotyping using the automated Riboprinter T M microbial characterization system. A total of 388 listeria isolates from 20 different dairy processing facilities were examined along with 44 silage, 14 raw-milk bulk tank and 29 dairy cattle isolates. These 475 isolates included 93 L. monocytogenes, 362 L. innocua, 11 L. welshimeri, 6 L. seeligeri, 2 L. grayii and 1 L. ivanovii strains. Thirty-seven different listeria ribotypes (RTs) comprising 16 L. monocytogenes (including five known clinical RTs responsible for food-borne listeriosis), 12 L. innocua, 5 L. welshimeri, 2 L. seeligeri, 1 L. ivanovii and 1 L. grayii were identified. Greatest diversity was seen among the isolates from dairy-processing facilities with 14 of 16 (87.5%) L. monocytogenes RTs (including 5 clinical RTs), and 19 out of 21 (90.5%) non-L, monocytogenes RTs detected. Sixty-five of the ninety-three L. monocytogenes isolates belonged to the group of the five clinical RTs, which included one RT unique to dairy-processing environments, two RTs common to dairy-processing environments and silage, and one RT common to dairyprocessing environments, silage, raw milk and dairy cattle with the last RT appearing in dairy-processing environments, silage, raw-milk bulk tanks and dairy cattle. The finding of eight L. rnonocytogenes and twelve non-L, monocytogenes RTs common to both dairyprocessing and farm environments clearly implicates the farm as a natural reservoir for listeria RTs capable of entering dairy-processing facilities. These findings, which support the link between on-farm sources of listeria contamination (dairy cattle, raw milk and silage) and subsequent contamination of dairy-processing environments, stress the importance of farm-based Hazard Analysis and Critical Control Points (HACCP) programmes for controlling listeria. This work also showed that two important clinical L. monocytogenes ribotypes which were previously identified as RT 19092 and RT 19161 and epidemiologically linked to listeriosis cases involving pasteurized milk and turkey frankfurters were recovered from dairy-processing facilities A and B for 12 and 3 months, respectively, with L. innocua RT 19094 also present in these same two facilities for at least five years.
Growth and Survival of Microbial Pathogens in Cheese
Abou-Eleinin et al. (2000) analysed 450 goats' milk samples obtained from the bulk tanks of 39 goat farms for listeria spp. over a 1-year period. Modified versions of the USDA-FSIS (McClain and Lee,1989) and FDA (Lovett et al., 1987) protocols were used for recovery of listeria. Overall, 35 (7.8%) samples yielded listeria, with L. monocytogenes identified in 17 of the 35 (3.8%) listeria-positive samples and L. innocua in 26 (5.8%) of samples. Eight milk samples contained both L. monocytogenes and L. innocua. Milk samples from 18 of the 39 (46.2%) farms were positive for listeria at least once during the year-long study. Five different listeria RTs were identified from 34 selected L. rnonocytogenes isolates, 2 of which were deemed to be of clinical importance. Isolation rates of listeria were markedly higher during the winter (14.3%) and spring (10.4%), compared to autumn (5.3%) and summer (0.9%). Similar trends have been previously reported for cows' milk (Reaet al., 1992; Ryser, 1999). Milk quality
Raw-milk quality is important in producing all cheeses, but particularly for those made from raw milk. Low bacterial counts and low somatic cell counts are the key indicators of milk quality, and as their numbers increase, there is a higher risk for contamination of milk and cheese with pathogens. Monitoring and controlling bacteria and somatic cell counts in milk should be components of a HACCP programme to ensure product safety. As rapid, cost-effective methods become available for detection of bacterial pathogens in raw milk, the use of specific pathogen testing could become part of a HACCP programme. In general, when rawmilk bacteria and somatic cell counts are high, there will be other negative impacts on cheese quality that may reduce consumer acceptability and cheese yield. In most artisanal cheesemaking, the time from milking to cheesemaking is very short and in some cases the milk is made into cheese immediately on the farm without cooling. Minimizing the time from milk collection to the initiation of cheesemaking reduces the opportunity for the growth of undesirable bacteria in raw milk. Conversely, when milk is cooled and held in transport, the opportunity for pathogen growth, particularly growth of psychrotrophic pathogens, is increased. The European Community Directives 92/46 and 92/47 (Anonymous, 1992) contain regulations for the hygienic production and placing on the market of raw milk, heat-treated milk and milk-based products. These regulations establish hygienic standards for raw-milk collection and transport that focus on issues such as temperature, sanitation and microbiological standards, enabling the production of raw milk of the highest
543
possible quality. Raw cows' milk must meet quality standards, e.g., a standard plate count at 30 ~ of < 100 000 cfu/ml and somatic cell counts of -<400 000 per ml of milk. To meet these and other established standards, countries employ HACCP principles in the production of fluid dairy products. This involves identification of sites to be monitored and evaluated to ensure that products are produced under the correct conditions, as well as the development of critical limits established by valid and verifiable parameters. In the case of fluid milk products, many processors have identified length of shelf-life as a critical limit. Shelf-life is influenced by a number of factors, including cleaning and sanitizing of pipelines and milking equipment, condition of raw milk used to produce product and storage temperature. Pasteurization will eliminate some of the indigenous microflora in the raw milk, including pathogenic bacteria; however, thermoduric organisms survive pasteurization. Post-pasteurization contamination of milk is problematic if the processing/packaging environment is not maintained. Moreover, many contaminants, including listeria, are able to form biofilms which protect them from cleaning and sanitizing agents. Some regulations, such as those of the EU, have established microbiological limits at the sell-by-date for products such as cheeses. With respect to regulations which govern the use of raw milk for cheesemaking, limits have been established for Staph. aureus in raw milk. Finished cheeses must meet specific hygienic standards, in which case the presence of Staph. aureus and E. coli indicates poor hygiene. Heat treatment of milk
Milk contains heat-labile compounds (e.g., lactoferrin, lysozyme and lactoperoxidase) that are inhibitory to the growth of some pathogens. Recent work by Pitt et al. (2000) has demonstrated that the growth of Staph. aureus, S. enteritidis and L. monocytogenes was slower in raw milk held at 37 ~ for 72 h than in pasteurized milk held under the same conditions. During the first 16 h of incubation, the number of organisms increased in both raw and pasteurized milks, but after 16 h, the number of recoverable viable pathogenic organisms in the raw milk began to decrease; an overall decrease of 2-5 logs was found. The inhibitory effect of raw milk on the survival of the above three pathogens in milk, reported by Pitt et al. (2000), is probably of great importance for cheesemaking from raw milk, and additional research needs to be undertaken. The lactoperoxidase system (LPS) is a naturally occurring inhibitory system in raw milk and comprises three components, lactoperoxidase, thiocyanate and hydrogen peroxide. All three components are required to exhibit maximum antimicrobial effects.
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Growth and Survival of Microbial Pathogens in Cheese
Gram-negative psychrotrophs, such as pseudomonads, are extremely sensitive to this system. Approximately 0.5-1.0 I~g/ml lactoperoxidase is needed for effective inhibition, and bovine milk typically contains 30 ~g/ml (Bj0rck, 1978). Quantities of thiocyanate and hydrogen peroxide in milk are variable. For a full inhibitory effect, about 10 b~g/ml of hydrogen peroxide is required, and bovine milk normally contains 1-2 b~g/ml hydrogen peroxide. The thiocyanate levels in milk range from 0.02 to 0.25 mM but 8-25 mM is needed for optimum activity. Pitt et al. (2000) hypothesized that the inhibitory effect of raw milk in their study was due to activation of the lactoperoxidase system by hydrogen peroxide-producing lactic acid bacteria naturally present in the raw milk, which grew at 37 ~ The authors postulated that the reduction in growth of these organisms in the raw milk could result from inhibitory products produced by activation of the LPS. However, it is highly unlikely that pasteurization inactivates lactoperoxidase in milk, and so there must be an alternative explanation for the much greater inhibition of 5taph. aureus, S. enteritidis and L. rnonocytogenes by the raw milk. Some cheeses are made from milk that has been given a sub-pasteurization heat treatment at the farm, but are technically classified as raw-milk cheeses. This process can be beneficial when milk has to be transported and stored at refrigeration temperature at a cheesemaking facility and when there will be a time delay before cheese manufacture. These near-pasteurization thermal treatments are often called thermization and they help to reduce the gowth of psychrotrophic bacteria that cause quality defects in cheese. However, the thermization process may partially inactivate some indigenous antimicrobial milk components that were mentioned previously. Comprehensive studies conducted by the US Food and Drug Administration (FDA) and the US Department of Agriculture (USDA; Bunning et al., 1986, 1988) and by Health and Welfare Canada (Farber et al., 1992) have shown that listeria are unable to survive normal pasteurization conditions. Knabel et al. (1990) found that growing L. rnonocytogenes at 43 ~ prior to heat-inactivation caused an increase in thermotolerance, but a study conducted by Farber et al. (1992) demonstrated that even under worst-case scenario conditions, which included cultivation of L. monocytogenes populations at 43 ~ prior to inactivation, pasteurization would render a 4.5-6.2-D process. Therefore, while populations of L. rnonocytogenes have been shown to survive minimum pasteurization (71.1 ~ in various laboratory studies, survival under actual conditions of commercial milk pasteurization and processing is unlikely. Studies which define the impact of commercial heat treatment of raw milk,
either naturally or artificially contaminated with bacterial pathogens, are still relatively scarce. However, L. monoc.ytogenes is generally regarded as being more heat resistant than salmonella or E. coli 0157:H7 (D'Aoust et al., 1987; Line et al., 1991). Using raw milk inoculated to contain various pathogens at a level of 105 cfu/ml, D'Aoust et al. (1987) concluded that salmonella were inactivated in milk after heating to 64.5 ~ (148.2 ~ or above for 16.2 sec, except for S. senftenberg which survived until the treatment exceeded 67.5 ~ (153.5 ~ for 16.2 sec. Heating at 63 ~ (145.4 ~ for 16.2 sec reduced populations of S. senftenberg by 3 orders of magnitude. Thermal inactivation of E. coli 0157:H7 was complete at temperatures -->64.5 ~ (148.2 ~ for 16.2 sec (Line et al., 1991) which is similar to that required for most salmonella except S. sen ftenberg. Much of the aged raw milk cheese produced in the US is subjected to some form of heat treatment, generally thermization. This treatment generally consists of heat treatment at 55 ~ for a period ranging from 2 to 16 sec. The specific impact of this heat treatment combined with the interactive effects of salt and pH during subsequent ripening on pathogens such as listeria, salmonella and E. coli has not been well explored.
Extrinsic and intrinsic parameters in cheese which dictate microbial growth
Growth of microbial pathogens in cheese is dictated by extrinsic and intrinsic parameters. The important intrinsic parameters include moisture content, pH and acidity, nutrient content, redox potential, presence of antimicrobial compounds, either those occurring naturally or those which are added as food preservatives, e.g., NOs, and the presence of competitive microflora (ICMSE 1986). All of these factors dictate the potential for bacterial pathogens to grow, persist or decline in cheeses. Extrinsic parameters include factors such as type of packaging/packaging atmosphere, time and temperature of storage and holding conditions, processing steps, product history and traditional use. The interaction of these factors dictates the potential for microbial growth in cheese. Depending on the cheese variety, intrinsic parameters such as pH may serve to enhance or inhibit the growth of bacterial pathogens. Ryser and Marth (1987a) studied the behaviour of L. monocytogenes in Camembert cheese. The high moisture content and the neutral pH of this surface-ripened cheese facilitate growth and survival of pathogens such as listeria. Growth of listeria in Camembert cheese was found to parallel the increase in cheese pH during ripening and reached a final population of 106-108 per g. This contrasts with Blue cheese, where listeria failed to grow and decreased in number during
Growth and Survival of Microbial Pathogens in Cheese
56 days of storage (Papageorgiou and Marth, 1989). These authors suggested that Penicilliurn roqueforti may produce bacteriocins against L. monocytogenes. In hard cheese varieties like Colby and Cheddar, L. monocytogenes populations decline during aging, with survival strongly influenced by the moisture content and the pH (Ryser and Marth, 1987b; Yousef and Marth, 1990). Cheeses such as Camembert and Feta have nearly identical composition in terms of moisture content, water activity, % salt-in-water and ripening temperature. However, fully ripened Camembert has a pH of 7.5 versus Feta which has a pH of 4.4 that prevents the growth of listeria. Cheeses made from raw milk
In the US and other parts of the world, the manufacture of cheese from raw milk is a topic which is being revisited from the perspective of microbiological safety. Pasteurization of milk prior to cheesemaking is but one step that may reduce the risk of the presence of pathogenic bacteria in cheese. Current US regulations which govern the use of raw, heat-treated and pasteurized milk for cheesemaking were promulgated in 1949 (Anonymous, 1950; 21 CFR Part 133). One of the two options can be selected by cheesemakers to assure the safety of cheesepasteurize milk destined for cheesemaking or hold cheese at a temperature of not less than 1.7 ~ (35 ~ for a minimum of 60 days. Recent research has shown that S. typhimurium, E. coli 0157:H7 and L. monocytogenes can survive well beyond the mandatory 60-day holding period in Cheddar cheese prepared from pasteurized milk (Reitsma and Henning, 1996; Ryser, 1998). In a referral to the National Advisory Committee on Microbiological Criteria for Foods in April 1997, the FDA asked if a revision of policy requiring a minimum 60-day aging period for raw-milk hard cheeses was necessary. The FDA, in its communication, noted that such a duration may be insufficient to provide an adequate level of public health protection. The FDA cited numerous studies and outbreak investigations documenting the presence of listeria, salmonella and E. coli 0157:H7 in raw milk. Of particular concern was the report by Reitsma and Henning (1996) detailing the survival of E. coli 0157:H7 in aged Cheddar cheese. The FDA did note, however, that there was 'limited epidemiological evidence that food-borne illness results from consumption of raw-milk hard cheeses that have been aged for 60 days', citing work by Fabian (1947), D'Aoust et al. (1985) and Johnson et al. (1990b) in support of this claim. Groups outside of the US have recently expressed concern about the safety of raw-milk cheeses. The Institute of Food Science and Technology (IFST, 2000) in the UK issued a position statement drawing attention to the
545
potential public health hazards posed by pathogenic bacteria in cheeses made from raw milk. The IFST indicates that these hazards apply particularly to soft and semi-soft cheeses (IFST, 2000). Codex Alimentarious is presently recommending a 'combination of control measures' (including pasteurization) to achieve the appropriate level of public health protection (Groves, 1998). In a comprehensive review of all outbreaks of human illness associated with the consumption of aged rawmilk cheese, in the majority of instances, confounding parameters other than use of raw milk contributed to pathogens being present in the product at the time of consumption (Donnelly, 2001). Further, in challenge studies which examine the fate of pathogens in aged cheese, confounding factors can also explain the appearance of pathogens following 60 days of aging. Such confounding parameters in actual outbreaks or challenge studies involve the use of pasteurized versus raw milk in cheesemaking trials, inadequate development of acidity during cheesemaking, a low salt level, contamination by ill employees during manufacture, temperature abuse of milk designed for cheesemaking and environmental contamination during cheesemaking.
P r e v i o u s R e v i e w s on t h e S a f e t y of R a w Milk C h e e s e s
Two comprehensive reviews have been published regarding outbreaks of human illness linked to consumption of cheese. Johnson et al. (1990b) conducted a comprehensive review of the epidemiological literature during the 40-year period, 1948-1988. These authors identified only six outbreaks of illness transmitted by cheese produced in the US during this period. Post-pasteurization contamination was the most frequent causative factor in these outbreaks. Improper pasteurization equipment and/or procedures were implicated in only one outbreak each in the US and Canada, and use of raw milk was a factor in one outbreak in each of these countries. No outbreaks were linked to hard Italian cheese varieties such as Parmesan, Romano and Provolone. In rare instances, Swiss and Cheddar cheeses were linked to food-poisoning outbreaks. Factors other than pasteurization cited by Johnson et al. (1990b) as contributors to cheese safety include milk quality and management, lactic starter management, pH, salt, controlled aging conditions and natural inhibitory substances in the raw milk. These authors proposed three actions to improve the safety of raw milk cheeses: (1) Establish a guideline for minimum heat-treatment of milk for cheesemaking, e.g., 64.4 ~ (148~ for 16sec or equivalent with adequate process control, (2) Evaluate current safety
546
Growth and Survival of Microbial Pathogens in Cheese
technology and practices used for cheese manufacture and (3) Evaluate technologies not currently used in cheese manufacture for safety potential (Johnson et al., 1990c). Altekruse et al. (1998) reviewed all cheese-associated outbreaks reported to the Centers for Disease Control and Prevention (CDC) during the period 1973-1992. These authors noted the infrequency of large, cheeseassociated outbreaks reported during this period and suggested that improvement of cheesemaking methods and process control have resulted in cheese being a safer product. There were 32 cheese-associated outbreaks, 11 of which could be attributed to contamination at the farm, during manufacturing or during processing. Of the 11 outbreaks attributed to contamination prior to distribution, 5 were associated with the consumption of Mexican-style soft cheese versus only one outbreak linked to Cheddar cheese. It is notable that no outbreaks reported to the CDC during 1973-1992 were associated with raw milk cheese that was aged for a minimum of 60 days. The authors indicated that salmonella, E. coli 0157:H7 and L. monocytogenes may survive the aging process. However, the literature reference for survival of listeria points to Camembert cheese (Ryser and Marth, 1987a), and the authors failed to note the rapid decline of listeria populations in aged Cheddar cheese as documented by Ryser and Marth (1987b). Altekruse et al. (1998) suggest that aging alone may not be a sufficient pathogen control step to eliminate salmonella, listeria and E. coli 0157:H7 from cheese. Outbreaks involving Cheddar cheese
In 1976, seven lots of Cheddar cheese manufactured from pasteurized milk were contaminated with S. heidelberg and were responsible for 339 confirmed cases of illness and an additional 28 000-36 000 cases of illness (Fontaine et al., 1980). The cheese involved was aged for less than 60 days, and improper pasteurization was cited as the cause of the outbreak. Follow-up with the first few patients led epidemiologists to suspect cheese eaten in Mexican-style restaurants as the vehicle of infection. Seven lots of Cheddar cheese produced from pasteurized milk by a Kansas manufacturer and purchased from a single Denver distributor were identified as the potential sources of contamination. The epidemic began in July in two widely separated Colorado cities, Denver and Pueblo. Levels of S. heidelberg in these cheeses were estimated to be 0.36-1.8 per 100 g. The pH of contaminated cheese was 5.6, which may have been a factor in this outbreak. Poor manufacturing practices coupled with inadequate control programmes at the cheese plant were cited as causative factors in this outbreak. The Kansas State Health Department had
recorded 25 instances of non-compliance with good manufacturing practices by that particular food-processing plant. The Kansas Board of Agriculture required that raw milk contain <3 000 000 organisms/ml. Routine microbial analysis of the grade B or surplus grade A milk used at the plant revealed that counts greatly exceeded this standard. In the production of cheese, raw milk was stored for 1-3 days in an insulated but unrefrigerated holding tank prior to pasteurization at 71.6 ~ for 15 sec. The milk was filtered after pasteurization, which is a violation of FDA guidelines for pasteurization. Salmonella outbreaks in Ontario, Canada, during the period 1980-1982 occurred in raw-milk Cheddar cheese. 5. muenster was identified in the cheese and traced to a single farm where one cow was shedding the organism (Wood et al., 1984). Subsequent trials using milk from this infected cow were conducted to determine potential for survival during commercial preparation of raw milk cheese. Curd tested positive in 11 of 181 vats. During curing, one lot was negative after 30 days but one lot was positive after 125 days. It would be unlikely for this scenario to be repeated as cheese is rarely manufactured from milk from a single cow. Milk is co-mingled, and the dilution effect with milk from other animals and other farms reduces the level of pathogens, if present. A large Canadian outbreak of salmonellosis linked to the consumption of Cheddar cheese was reported in four Canadian Atlantic provinces (Newfoundland, New Brunswick, Prince Edward Island and Nova Scotia) between January and July 1984. This outbreak proved to be the largest single epidemic of salmonellosis ever to occur in Canada, ultimately involving more than 2700 cases of illness (Bezanson et al., 1985; Johnson et al., 1990b). Production of the cheese, which was manufactured from either pasteurized (73.8 ~ (165 ~ for 16 sec) or heat-treated (66.7 ~ (152 ~ for 16 sec) milk, was traced to a single plant on Prince Edward Island. Testing of the raw milk supply identified two cows in separate herds, one which shed 5. typhimurium and one which shed 5. heidelberg. D'Aoust et al. (1985) reported on the distribution and survival of 5. typhimurium phage type 10 isolated from Cheddar cheese in this outbreak. Levels of salmonella ranged from 0.36 to 9.3 per 100g. The pH of the cheese ranged from 4.97 to 5.40, consistent with normal Cheddar, which has a pH range of 5.0-5.5. S. typhimurium phage type 10 was found to survive in Cheddar cheese for up to 8 months at 4 ~ The data provided by D'Aoust et al. (1985) is very interesting. The authors compare salmonella recovery as a function of whether mild Cheddar cheese was manufactured from heat-treated (16 s at 66.7 ~ not pasteurized) or pasteurized (16 sec at 73.8 ~ milk.
Growth and Survival of Microbial Pathogens in Cheese
Tested samples of mild Cheddar manufactured from heattreated milk were found to contain 0.36-9.3 salmonella/100g. However, four lots of mild Cheddar manufactured from pasteurized milk were also found to contain 0.36-4.3 salmonella/100 g. Certain lots of cheese contained Staph. aureus at high levels (>105 per g), which may indicate poor starter activity (Johnson et al., 1990b) or contamination through handling. It is difficult to understand how D'Aoust et al. (1985) could support their concluding statement in this article 'Although pasteurization of milk used in cheesemaking increases the safety of the finished product, use of heat-treated (unpasteurized) milk in the manufacture of medium and old Cheddar cheese and survival of salmonella during prolonged periods of refrigerated storage raises legitimate doubts of the safety of current manufacturing practices.' In the data presented, pasteurization did not result in the unequivocal safety of mild Cheddar cheese. An evaluation of the pasteurization process, described by Johnson et al. (1990b), indicated that the employee in charge of the process manually overrode the electronic controls, which shut down the pasteurizer while milk continued to flow through the unit and into the vat. The pasteurizer was shut down after filling three vats and later restarted to fill the next three vat series. The first and the third vats of each three vat sequence tested positive for salmonella, except for the first vat of the day and the middle vat of each three vat series which consistently tested negative. This pattern only occurred when raw milk which included milk from the cow shedding S. typhirnurium was used. Bezanson et al. (1985) subsequently subjected outbreak strains to molecular analysis by biotyping, antibiotic resistance patterns, plasmid restriction and endonuclease analyses and revealed that two genetically distinct organisms were the aetiologic agents in this outbreak. These studies revealed the existence of a double infection, indicating that the incriminated cheese likely had two sources of contamination. S. typhimurium phage type 10 subgroup I strains were identified among cultures from raw milk and cattle associated with the incriminated dairy. S. typhimurium phage type 10 subgroup I and II strains were recovered from individuals employed at the dairy along with their family members. S. typhirnuriurn subgroup I and II strains were present in cheese curd samples obtained from the plant as well as from a consumer pack obtained from a distributor. Cheese plant workers from whom both subgroup I and II strains were cultured were involved in the production and/or packaging of Cheddar cheese, raising questions about the possibility of contamination of the cheese by ill workers. Salmonella were confirmed in a cheese-trim bucket. Plant inspections revealed that employees used their bare hands to transfer cheese to a forming machine, and an employee tested
547
positive for S. typhimurium. It is likely that this incriminated cheese was also responsible for an outbreak of illness reported at the same time in Ontario linked to S. typhimurium phage type 10 biotype 4 (D'Aoust et al., 1985). Hedberg et al. (1992) reported on a multi-state outbreak of S. javiana and S. oranienburg linked to the consumption of contaminated Mozzarella cheese and shredded cheese products. Cases were more likely to have consumed cheese manufactured at a single cheese plant or cheese shredded at processing plants that also shredded cheese from the single plant, than matched controls. The outbreak strains were isolated from 2 of 68 unopened 16-oz blocks of Mozzarella cheese. Inspections revealed deficiencies in plant sanitation and cleaning, and equipment was not routinely cleaned and sanitized between shredding different types of cheese from different manufacturers. However, no deficiencies in pasteurization were identified. Cheese-manufacturing equipment was found to be susceptible to environmental contamination and contamination by aerosols. Investigators believed that the contaminated Mozzarella cheese sent to four processing plants for shredding, crosscontaminated other cheese products at those plants. It is most likely that the cheese was contaminated from environmental sources or from infected production workers. Four outbreaks occurring in the late 1990s were reported in the UK, although detailed epidemiologic data on these outbreaks is lacking. An outbreak of E. coli 0157:H7 (phage type 8, Verotoxin gene 2) infection involving 22 cases was reported in Scotland in 1994. This outbreak was associated with the consumption of raw-milk cheese (Anonymous, 1997a). A December 1996 outbreak of salmonella gold-coast which occurred in England and Wales was linked to the consumption of a brand of mild, coloured, Cheddar cheese produced in August and September 1996 in Somerset, England. Phosphatase tests and examination of recording chart records from the pasteurizer indicated that pasteurization had failed at the plant on several occasions (Anonymous, 1997b). An outbreak of infection caused by E. coli 0157:H7 (phage type 21/28 VT2) was reported in 1999 in north-east England (Anonymous, 1999a,b). The vehicle of infection was Cotherstone cheese, a rawmilk cheese, manufactured in small quantities and distributed to specialty cheese shops in England. Samples from the dairy herd, slurry and environmental samples from the cheese manufacturing facilities were negative for E. coli 0157:H7. In March of 1999, a large outbreak of infection was reported in England and Wales due to consumption of contaminated milk from a single dairy. An outbreak of E. coli 0157:H7 infection was reported which was linked to the consumption of fresh cheese
548
Growth and Survival of Microbial Pathogens in Cheese
curd, which was held for < 60 days, from a dairy plant in Wisconsin (Durch et al., 2000). Nineteen of 55 laboratory-confirmed patients had purchased cheese curds from an unrefrigerated display at the cheese plant. To be legal, cheese curds must be manufactured from pasteurized milk. Vats of raw-milk Cheddar cheese were inadvertently used to make fresh curds, which were incorrectly labelled as 'pasteurized' Cheddar cheese curd. A comprehensive risk assessment would consider, among other factors, the degree to which the consuming population is exposed to risks associated with the consumption of aged raw-milk cheeses. Cheddar cheese is produced worldwide and is therefore considered an important variety of hard cheese. The USDA, National Agricultural Statistics Service, reports that Cheddar cheese was the most popular variety of cheese produced and consumed in the US in 1999, with a production level of 2.8 billion pounds (1.2 million tonnes) or 35.4% of the total cheese produced (Anonymous, 1999c). Given that a large amount of this cheese is produced from raw or heat-treated milk, the high degree of exposure (consumption) of this product coupled with the low incidence of disease outbreaks attests to the safety of aged cheese made from raw and heat-treated milk. Table 1 summarizes outbreaks involving Cheddar cheese which have occurred since 1976. Listed in this table are confounding parameters which contributed to the presence of pathogens in the finished product and the subsequent onset of human illness.
Challenge Studies Reitsma and Henning (1996) examined the survival of E. coli 0157:H7 during the manufacture and ripening of Cheddar cheese. E. coli 0157:H7 was inoculated at two levels into pasteurized milk, 1 x 103 cfu/ml and 1 cfu/ml. The organism showed a sharp decrease in numbers over the 158-day testing period. Treatment 1 (1000 cfu/ml) showed a 2-log CFU/g reduction after 60 days of ripening; however, E. coli 0157:H7 was still present even after 158 days of ripening when viable cells were detected in four of five replicates. Treatment 2 (1 cfu/g) showed a reduction to < 1 cfu/g in 60 days, with no viable E. coli 0157:H7 detected at 158 days. As the authors state, 'the results of this study cannot predict the behaviour of heat-injured cells which could result from the pasteurization of naturally contaminating E. coli.' Further, the low salt-in-moisture content (SM) and absence of natural inhibitors present in raw milk create an artificially protective environment for E. coli 0157:H7 in pasteurized milk. The SM determines the water activity, which, in turn, dictates the potential for growth of a micro-organism in the cheese environment. The SM in that study ranged from 2.75 to
3.76% with a mean of 3.25%, whereas in normal Cheddar, the SM ranges from 4 to 6%. The low SM could have affected the results in the study of Reitsma and Henning (1996) and the authors recommend further research with Cheddar containing a higher SM to determine if similar results would be obtained with an SM more commonly encountered in Cheddar cheese. NaC1 is an important inhibitor of microbial growth in cheese. The major roles of NaC1 in Cheddar cheese are to check lactic acid fermentation after an optimum peak has been attained, reduce moisture through syneresis of the curd, suppress the growth of spoilage micro-organisms and create physical changes in cheese proteins which influence cheese texture, protein solubility and protein conformation (Fox et al., 2000; 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1). While there are no state or federal standards for the amount of salt added to Cheddar cheese, variations in salt content from 0.8 to 2% are common. The minimum aw (adjusted with NaC1) for the growth of E. coli is 0.950 (Fennema, 1985). Further, most raw milk receives some form of heat treatment, albeit subpasteurization. The combination of heat, salt and natural inhibitors could provide barriers to the survival of E. coli 0157:H7. The experimental design used by Reitsma and Henning (1996) failed to consider these potential safeguards. It is plausible that the use of pasteurized milk for cheesemaking provides E. coli 0157:H7 with a more protective environment than raw milk, thus heat treatment could create more of a problem to food safety. The authors state 'The low number of outbreaks seem to indicate that pathogens in cheese are not a major problem.' The authors further state 'treatment 1 (1000 cfu/ml) would not likely be encountered in industry because of co-mingling of milk from several or many farms, thus creating a dilution effect.' Concern is expressed about the authors' concluding statement 'The current requirement for ripening of Cheddar cheese will not assure consumers of a safe product if the cheese is made from raw milk and a pathogen such as E. coli 0157:H7 is present in the cheese at the beginning of ripening.' This statement is contradicted by the authors' own data which show that E. coli 0157:H7 present at 60 cfu/g in curd after salting was reduced to < 1 cfu/g after 60 days, even in the artificially low SM of cheeses in the study. A subsequent study by Zhang and Henning (1999) described mathematically the decline of E. coli populations during cheese ripening. The authors inoculated pasteurized whole milk with E. coli biotype 1 at populations of 100-1000/ml. The authors used a complete factorial design to investigate the effects of high- and low-level environmental factors such as moisture (34-40%), pH (5.1-5.6), temperature (4-13 ~ and salt
Growth and Survival of Microbial Pathogens in Cheese
549
Table 1 Data from outbreak investigations involving aged raw milk cheese and confounding parameters which contribute to the presence of pathogens
Number of cases
Cheese type
Confounding parameter
339 confirmed; 28 00036 000 suspected
Cheddar made from pasteurized milk
1. Raw milk did not meet standards 2. Raw milk stored 1-3 days in holding tank - no refrigeration 3. Milk filtered after pasteurization 4. Cheese pH, 5.6 5.25 instances of noncompliance with GMP Milk traced to single farm; lack of co-mingling 1. Employee manually shut down pasteurizer 2. Group II type shed by workers 1. Deficiencies in cleaning and sanitation 2. Equipment not routinely cleaned and sanitized between shredding of different cheese types from different makers 3. Cheese equipment susceptible to contamination from environment/aerosols 4. Cheese contaminated by infected workers 5. No deficiencies in pasteurization Incorrectly labelled as a pasteurized product
Date
Location
Isolate
1976
Colorado
Salmonella heidelberg
1980-1982
Ontario
Salmonella muenster
1984
4 Canadian Atlantic Provinces and Ontario
Salmonella typhimurium phage type 10, group I and II
>2700 confirmed cases
Cheddar made from pasteurized and/or heattreated milk
1989
Multistate (Minnesota, Wisconsin, Michigan, New York)
Salmonella javiana and Salmonella oranienburg
164
Shredded cheese
1999
Raw-milk Cheddar
E. coil 0157:H7
concentration (0.8-1.7%) on survival of E. coli. Temperature and pH were found to have the most significant impact on survival, and there was no significant interaction among the four parameters studied. Salt concentration within the ranges used in this study (0.8-1.7%) was found to have no impact on survival of E. coll. Teo and Schlesser (2000) examined the survival of three groups of bacteria in raw-milk Cheddar cheese during cheesemaking and ripening; naturally occurring
Fresh cheese curd held for <60 days
Reference Fontaine et al., 1980
Wood et aL, 1984 Bezanson et aL, 1985; D'Aoust et aL, 1985
Hedberg et aL, 1992
Durch et aL, 2000
coliforms, a streptomycin-resistant strain of E. coli K12 (ATCC 35695) and E. coli 0157:H7. Populations of naturally occurring coliforms present at levels of ---105 cfu/ml experienced a l-log reduction after 60 days of aging at 7 ~ and a further 3-4-1og reduction after 180 days (Teo and Schlesser, 2000). In contrast, E. coli K12 populations exhibited a less than l-log reduction during 60 days of aging, and only a 1-2-1og reduction by 90 days. Similar results were recorded with a five-strain
550
Growth and Survival of Microbial Pathogens in Cheese
cocktail of E. coli 0157:H7 where populations declined by 1 log following 60 days at 7 ~ and by 1-2-logs following 90 days at the same temperature. A number of questions are raised by the data presented by Teo and Schlesser (2000). The coliform levels used were extremely high, and, in practice, such levels would raise concerns about raw milk quality. The FDA has set standards for ETEC and E. coli in cheese at levels of 103 and 104 cells/g, respectively (Anonymous, 1998). Thus the cheese produced by Teo and colleagues did not comply with these standards, and further, exceeded these standards by 103-104/ml as shown in their Figure 6 where initial populations of E. coli 0157:H7 exist at approximately 5 • 107/ml. This study has a biased objective, to 'confirm prior work that suggests 60-day aging inadequate to protect public health.' Figure 2 presented in the paper documents a decline in populations over time, thus if a reasonable starting population of 1-10 E. coli 0157:H7 were used, no viable cells should be present following aging. Studies by Ryser and Marth (1987a,b,c) examined the fate of L. monocytogenes during the manufacture of Cheddar, Camembert and Brick cheeses. Rapid growth to populations of 5 • 107 cfu/ml is observed in Camembert cheese, in which the pH normally increases during ripening, thereby creating a favourable growth environment for listeria (Ryser and Marth, 1987a). In contrast, listeria populations show a marked decline in viable population levels during the ripening of Cheddar cheese. However, population levels do not decline to undetectable levels. Current US regulations call for cheese made from raw or sub-pasteurized milk to be ripened at 1.7 ~ (35 ~ for at least 60 days prior to sale. Ryser and Marth (1987b) have shown that aging alone will not ensure the production of listeria-free Cheddar cheese. This stated, it is clear, that the greatest threat posed to the safety of cheese is due to post-process environmental contamination with listeria. While outbreaks of illness have resulted from the presence of L. monocytogenes in softripened and Hispanic-style cheeses (Linnan et al., 1988), no outbreaks of listeriosis have been reported as a result of survival of listeria in cheese aged for a minimum of 60 days. Genigeorgis et al. (1991) evaluated the ability of 24 types of market cheeses to support the growth of L. monocytogenes. Cheeses able to support growth included soft Hispanic-type cheeses, Ricotta, Teleme, Brie, Camembert and Cottage cheeses (pH range, 4.9-7.7). Cheeses which did not support growth, and which resulted in the gradual death of L. monocytogenes, included Cotija, Cream, Blue, Monterey Jack, Swiss, Cheddar, Colby, String, Provolone, MOnster, Feta and Kasseri (pH range, 4.3-5.6). A correlation was observed between the growth of listeria in cheeses having a pH of greater than 5.5, and in cheeses which were manufactured without a starter culture.
Approximately 80% of the cheeses made in Switzerland are manufactured from raw milk. However, the term 'raw milk cheese' as applied to Swiss cheese is a misnomer because Swiss cheese receives an extensive heat treatment during manufacture. Bachman and Spahr (1995) assessed the safety of Swiss hard and semi-hard cheeses made from raw milk. These authors inoculated Aeromonas hydrophila, Campylobacter jejuni, E. coli, L. rnonocytogenes, Pseudomonas aeruginosa, Salmonella spp., Staph. aureus and Yersinia enterocolitica into raw milk at levels ranging between 104 and 106 cfu/ml for the manufacture of hard (Swiss-type) and semi-hard (Tilsit-type) cheese. In the hard cheese, no pathogens were detected beyond 1 day. This was attributed to the curd-cooking temperature of 53 ~ (127.4 ~ for 45 min and 42 ~ (107.6 ~ for 15 min for Swiss hard and semi-hard cheeses. Further, the rapid decrease of the redox potential of Swiss cheese is likely to impart additional inhibitory effects. Pathogens were found to survive longer in the semi-hard than in the hard cheese. After 90 days of aging at 11-13 ~ when ripening was complete, all pathogens except L. monocytogenes were below detectable limits. Growth of L. monocytogenes was not observed in the interior of the cheese, but they grew well on the cheese surface. Thus, manufacturing parameters used in the production of semihard cheese are bacteriostatic, not bacteriocidal, for L. monocytogenes. Based upon these studies, the Swiss dairy industry has adopted a listeria-monitoring programme for cheese and other dairy products. The synergistic effects of active antimicrobial enzyme systems in raw milk coupled with antagonistic effects of starter cultures, fast acidification, inhibitory effects of lactic acid and high curd-cooking temperatures render a microbiologically safe hard cheese when produced under good manufacturing practices. Spahr and Schafroth (2001), in studies which examined the fate of Mycobacteriurn avium subsp, paratuberculosis, recorded pH values associated with Swiss hard and semi-hard cheese manufacture. After 24 h, cheeses manufactured under these curd-cooking conditions reached a pH value of 5.3 in hard cheese and 5.2 in semi-hard cheese, and these pH conditions remain for 10 days for hard cheese and 25 days for semi-hard cheese. Further, the rapid decrease of the redox potential of Swiss cheese likely imparts additional inhibitory effects. The synergistic effects of active antimicrobial enzyme systems in raw milk coupled with antagonistic effects of starter cultures, fast acidification, inhibitory effects of lactic acid and high curd-cooking temperatures render a microbiologically safe hard cheese when produced under good manufacturing practices. Pellegrino and Resmini (2001) examined the safety of the Italian hard cheeses, Grana Padano and Parmigiano Reggiano. The authors noted several parameters
Growth and Survival of Microbial Pathogens in Cheese
associated with these cheeses which contribute to their microbiological safety; (1) cooking of cheese curd to a temperature between 53 and 56 ~ for 15-20 min, with a total holding time of up to 85 min at these temperatures, (2) moulding of the cheese, whereby it is held at temperatures of 52 ~ (126 ~ and 56 ~ (133 ~ for at least 10 h at pH 5.0, (3) brine-salting of the cheese which lowers the aw to 0.9 and (4) extended ripening for periods of 9 months (Grana Padano) to 12 months (Parmigiano Reggiano) which promotes a further decrease in the aw to levels inhibitory for growth of bacterial pathogens. Resmini and Pellegrino (1996) demonstrated that the high-temperature-low-pH conditions occurring within Grana cheeses, which they describe as self-pasteurization, result in the inactivation of alkaline phosphatase, except within the outermost 3-4-cm layer. However, in this outer layer the SM ranges between 8 and 24% in the ripened cheese and the aw is close to 0.8. Staph. aureus, which is more tolerant of low aw, cannot survive below an ave of 0.86 and can produce toxins only above an aw of 0.90 (Sperber, 1983). Pecorari et al. (2001) examined the fate of pathogens during the production and ripening of Parmigiano Reggiano cheese. E. coli, S. typhirnurium, Staph. aureus and L. rnonocytogenes were inoculated into raw milk at levels ranging between 104 and 106 cfu/ml. None of the inoculated pathogens were detected 24 h after cheesemaking, confirming that the cheesemaking conditions of Grana cheeses do not support pathogen growth or survival. These results are consistent with those obtained by Yousef and Marth (1990) who reported a rapid decline of L. monocytogenes from an initial level of ---104g of Parmesan cheese to undetectable levels within 14-112 days of ripening. These authors attributed the decline of L. monocytogenes viability in Parmesan cheese to the following p a r a m e t e r s - addition of lipase (for US Parmesan) for flavour development, heat treatment of the curd and reduction in moisture content (aw) during ripening. Battistotti (1995), in an analysis of more than 100 samples of mature Italian Grana cheeses, failed to detect salmonella, Staph. aureus, L. monocytogenes, coliforms or enterococci, further confirming the microbiological safety of hard Italian cheeses. The results of the aforementioned challenge studies are summarized in Table 2. Most studies, which show the survival of pathogens, have been based on the use of pasteurized milk rather than raw milk in the experimental design. The growth rate of listeria (and presumably other pathogens) in milk is a function of the degree and extent of heat treatment. The fastest rate of growth is observed in UHT milk, followed in turn by HTST, heat-treated and raw milks (Northolt et al., 1988; Rajikowski et al., 1994). Therefore, challenge studies, which assess the survival of pathogens when
551
inoculated into pasteurized milk, may overestimate survival during 60 days of aging. The UK Institute of Food Science and Technology (IFST) has stated that the total health risk to the consumer is less from cheese made from pasteurized milk than from cheese of similar composition made from unpasteurized milk (IFST, 2000). Alternative hypotheses could be offered, including consideration that the use of raw milk provides protective effects from pathogens in milk and that post-pasteurization environmental contamination poses a far greater threat to the safety of cheese. As a result, the use of pasteurized milk in cheesemaking may provide an environment, which provides for optimal growth of pathogens whereas, in raw milk, the normal flora and natural inhibitors provide a margin of control over pathogen growth. In fact, a study conducted by Rudolf and Sherer (2001) showed a higher incidence of L. rnonocytogenes in cheeses made from pasteurized milk (8%) than in cheese made from raw milk (4.8%). Phage typing of isolates revealed persistent listeria contamination within dairy plant environments for periods of weeks to several months and documented cross-contamination within the plant environment as a significant factor associated with the contamination of cheese. The recommendation for mandatory pasteurization may ultimately lead to use of milk of inferior quality for cheesemaking. Pathogens harboured in this inferior quality milk can be transported to a processing facility and become established as environmental pathogens. A wiser strategy may involve routine testing of incoming lots of raw milk and working with producers when infected animals are identified to allow treatment and confinement of animals to control infectious disease. There is no evidence in the literature to support the view that cheese made from raw milk where pathogens are not present is a dangerous food. Thus, raw milk screening coupled with the use of good manufacturing practices to control environmental contamination during cheesemaking may be the most effective control strategy to improve the safety of aged cheese. The US FDA has recently stated 'a review of the literature relating to the potential for growth of pathogens in hard cheeses that are aged for at least 60 days shows that such growth is not likely to occur because of the combined effect of decreased pH, decreased water activity, and possibly other factors inherent to these cheeses' (Anonymous, 1999d). Although survival during aging is possible, the FDA cited a considerable body of evidence which showed that certain cheeses do not support the growth of pathogens during the aging process and subsequent storage. Both facultative and obligate heterofermentative lactobacilli have been isolated from Cheddar cheese, such as Lactobacillus casei, Lb. paracasei, Lb. plantarum, Lb.
552
Growth and Survival of Microbial Pathogens in Cheese
Table 2
Results of selected challenge studies which examine the fate of pathogens in raw milk cheeses and parameters which promote survival/decline of pathogens
Reference
Cheese type~pathogen
Milk inoculation levels
Reitsma and Henning, 1996
Cheddar/E. coil 0157"H7
1 cfu/ml and 1000 cfu/ml
No survival at 1 cfu/ml" Survival at 1000 cfu/ml
Ryser and Marth, 1987b
Cheddar/ L. monocytogenes
5 • 102 cfu/ml
Survival during aging at 6 or 13 ~
leo et aL, 2000
Cheddar/E. coil 0157:H7
105/ml
l-log decrease
Bachman and Spahr, 1995
Swiss hard/ semihard Aeromonas, Campylobacter, E. coil, L. monocytogenes, P. aeruginosa, Salmonella, Staphylococcus, Yersinia Italian Grana
104-106 cfu/ml
No detection of pathogens beyond 1 day
Parmesan/ L. monocytogenes
104-105 cfu/ml
Pellegrino and Resmini, 2001
Yousef and Marth, 1990
60 days of aging
Undetectable
Factors promoting survival~decline Cheese manufactured from pasteurized milk; low salt levels Decline in populations after 35 days of storage E. coil populations in cheese exceeded FDA standards Cook at 53 ~ redox potential
Curd cooked at 53-56 ~ brine-salting, extended ripening to lower aw Addition of lipase, heat treatment of curd, reduction of aw
SMP, Skim Milk Powder.
casei subsp, pseudoplantarurn, Lb. curvatus, Lb. brevis, Lb. rhamnosus and unclassified strains (Broome et al., 1990; Jordan and Cogan, 1993; McSweeney et al., 1993; Fitzsimons et al., 1999; Fox et al., 2000; Tammam et al., 2000). These are usually termed the non-starter lactic acid bacteria (NSLAB; see 'The Microbiology of Cheese Ripening', Volume 1). Lactobacilli are usually present at low numbers (<50/g) in cheese immediately after manufacture, but grow at a temperature-dependent rate during ripening and eventually become the dominant viable micro-organisms in cheese, reaching a population of 107 cfu/g in 10-60 days. The NSLAB population decreases with storage and usually approach 5 • 106 cfu/g after one year (Prentice and Brown, 1983). Conditions, which dictate the rate and extent of growth of NSLAB in cheese, include pH, moisture content, salt concentration and ripening temperature (Martley and Crow, 1993). In commercially produced cheese, NSLAB may originate from raw milk, postpasteurization environmental contamination and/or ingredients. These organisms may well offer a protective effect against the growth of pathogens and this role
should be studied as this may be a positive contribution of raw milk to the safety of raw-milk Cheddar cheese, consistent with competitive exclusion theories which have helped to advance the safety of products such as poultry. A lower total bacterial load in raw milk entering the pasteurizer results in a lower total bacterial count in pasteurized milk, but after 10-16 h, an increase in the number of NSLAB still occurs.
Growth and Survival of Bacterial Pathogens in Soft and Semi-Soft Cheeses Legitimate concerns can be raised regarding the safety of soft and semi-soft cheeses manufactured from raw milk, as well as high-moisture, low-sah aged cheeses. An outbreak of food-borne listeriosis linked to cheese was reported by Bille et al. (1992). This outbreak occurred in Vaud, Switzerland, and was linked to the consumption of Vacherin Mont D'Or cheese. A total of 122 cases during the period 1983-1987 were reported. The normal endemic rate of listeriosis in Switzerland is 5-10 cases/million persons. During the outbreak
Growth and Survival of Microbial Pathogens in Cheese 553 period, the rate of listeriosis rose to 50 cases/million persons. Sixteen cases were reported in 1983, 24 in 1984, 13 in 1985, 28 in 1986 and 41 in 1987. A mortality rate of 28% was associated with these cases. Of the clinical isolates available from the epidemic period, 111 of 120 (93%) were serotype 4b of two unique phage types, and 85% of these strains matched the epidemic phage types isolated from Vacherin Mont D'Or cheese. L. monocytogenes in Mexican-style cheese has been responsible for two major outbreaks of food-borne disease in the US. Mexican-style cheeses comprise a range of cheese products which include Queso Blanco, Quesco Fresco, Panela Ranchero, Queso de Hoja and soft Hispanic cheese (Bohon and Frank, 1999). These cheeses do not have a standard of identity, and most are coagulated and using rennet, may have added organic acids (citric, acetic and lactic); usually a lactic starter culture is not used (Bohon and Frank, 1999). The first documented link between cheese consumption and an outbreak of listeriosis was reported in California in 1985. Jalisco brand Mexican-style cheese was implicated as the vehicle of infection (Linnan et al., 1988). A total of 142 cases involving 93 pregnant women or their offspring and 49 non-pregnant, immuno-compromised adults were documented in Los Angeles County, CA. Forty-eight deaths were recorded, giving a mortality rate of 33.8%. The majority of afflicted individuals (62%) were pregnant Hispanic women. Although an additional 160 cases occurred in other parts of California, for logistical reasons, the study reported by Linnan et al. (1988) was limited to Los Angeles County. In this outbreak, the cheese was most likely manufactured from a combination of raw and pasteurized milks, and the cheese plant that manufactured the incriminated cheese was found to harbour listeria as an environmental contaminant. The epidemic strain in this outbreak was a serotype 4b, and this serotype was recovered from unopened packages of Queso Fresco and Cotija Mexican-style cheese. An outbreak of listeriosis associated with homemade, Mexican-style, fresh, soft cheese occurred in North Carolina between October 2000 and January 2001 (Boggs et al., 2001). The outbreak involved 12 cases, consisting of 10 pregnant women, 1 post-partum female and a 70- yea>old immuno-compromised male. The 11 women, upon hospital admission, reported symptoms of fever, chills, headache, abdominal cramps and vomiting. The cheese implicated in the outbreak was purchased from door-to-door vendors. L. monocytogenes isolates obtained from nine patients, three cheese samples from two stores, one cheese sample from a patient's home and one raw milk sample from a dairy all had indistinguishable PFGE patterns, indicating a common link. It is important to note that the manufacturing con-
ditions in this outbreak would not be those encountered in a licensed, inspected commercial cheese-processing facility. Microbiological surveys of raw milk conducted in the US have shown the presence of L. monocytogenes in 1.6-7% of samples. This incidence is similar to that in Canadian (1.3-5.4%) and Western-European (2.5-6.0%) raw milks. In the recently released Health and Human Services (HHS) and USDA listeria Risk Assessment and listeria Action Plan, USDA and FDA advise pregnant women, older adults and people with weakened immune systems that 'Cheeses that may be eaten include hard cheeses, semi-soft cheeses such as Mozzarella, pasteurized processed cheeses such as slices and spreads, Cream cheese and Cottage cheese.' However, persons in these risk groups are advised 'do not drink raw (unpasteurized) milk or eat foods that contain unpasteurized milk.' This advice may be ambiguous with respect to aged rawmilk cheeses (Anonymous, 2001).
Stress Adaptation of Pathogens and Impact upon Cheese Safety Over the past several years, microbiologists who study stress adaptation in bacterial pathogens are aware of genetic mechanisms which allow a number of Grampositive and-negative bacteria to adapt to hostile environments. Rpos is a sigma factor which is thought to allow induction of specific stress-related components in tolerant isolates. The rpos-regulated proteins enhance acid tolerance and cross-protect E. coli 0157:H7 against subsequent heat and salt challenges. The acid-tolerance response (ATR) gene encodes for the ability to withstand lethal pH conditions following adaptation to sublethal pH in L. rnonocytogenes, S. typhimuriurn, E. coli and A. hydrophila. These mechanisms play a role in predicting the fate of pathogens in acidic foods. Acid adaptation increases the general resistance, including acid tolerance, of L. monocytogenes, S. typhirnuriurn and E. coli, so that they survive better in both acidic and fermented foods than unadapted cultures. These findings have important implications for the safety of hard cheeses that are aged for at least 60 days where the combined effects of pH, salt and decreased aw dictate potential for pathogen survival. Leyer and Johnson (1992) inoculated the surfaces of commercially produced cheeses with adapted and nonadapted S. typhimuriurn at an initial level of 104/ml. Acid-adapted salmonella survived in Cheddar cheese through 74 days of storage at 5 ~ under aerobic storage compared with non-adapted salmonella, which were not detected after 14 days. In Swiss cheese, end products such as propionate and acetate produced by propionic acid bacteria were found to inhibit salmonella.
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Growth and Survival of Microbial Pathogens in Cheese
Dineen etal. (1998) examined the persistence of E. coli 0157:H7 in fermented dairy products (yoghurt). The authors concluded that post-processing contamination of fermented dairy products with E. coli 0157:H7 represents the greatest potential health hazard to humans. Those strains of E. coli 0157:H7 which possessed the rpos system appeared to contribute most effectively to bacterial survival under moderately lethal conditions, but did not appear to play much of a role in survival under sub-lethal conditions. The authors offered the following recommendations: (i) coliforms, including E. coli 0157:H7, may be present in raw milk, (ii) coliforms are destroyed by pasteurization, (iii) the primary objective of a comprehensive sanitation programme should be to prevent recontamination of pasteurized products and (iv) the presence of active starter cultures may help minimize the presence of bacterial pathogens in finished products. L. monocytogenes is able to withstand low pH following sub-lethal exposure to acidic conditions (O'Driscoll et al., 1997). According to Chawla et al. (1996) and Chen et al. (1997), temperature and acidity had a significant effect on the fate of acid-injured L. monocytogenes, with complete repair occurring at pH >6.6. At pH values <5.6 (which are typically found in Cheddar cheese), refrigeration temperatures were bacteriostatic, whereas higher temperatures were bacteriocidal. These findings are consistent with those reported by Ryser and Marth (1987b) where L. monocytogenes was inactivated faster in Cheddar cheese ripened at 13 ~ versus 6 ~ Since repair of sub-lethal injury requires optimal conditions, decreased survival of sub-lethally injured bacteria in Cheddar cheese would be expected due to low pH and high salt conditions. Mathew and Ryser (2002) assessed the ability of sublethally heat-injured L. monocytogenes cells to compete with a commercial mesophilic lactic acid starter culture during fermentation of UHT milk. L. monocytogenes strains were heat-injured by two treatments (low heatinured (LHI) and high heat-injured (HHI)) to yield greater than 99% injury. The UHT milk was inoculated to contain 104-106 LHI, HHI or untreated L. monocytogenes together with 0, 0.5 or 2% of a commercial Lactococcus lactis subsp, lactis/Lc, lactis subsp, cremoris starter. While listeria populations grew to levels of approximately 109 cfu/ml after 8 h of fermentation, after 24 h, 93% of the non-iNured control population became injured. In starter-free controls, >80% of both HHI and LHI cells were repaired within 10 h of incubation. These findings document the suppression of listeria growth by the starter culture which causes microbial injury, resulting in cells which are unable to grow or express pathogenicity. The potential of listeria and other pathogens to become inactivated and/or sub-lethally
injured during cheesemaking should be investigated. The combined effects of acid production by starter cultures, salt and mild heat alone or in combination all have the potential to injure bacterial pathogens such as L. monocytogenes, E. coli and salmonella. These interactive effects could provide an explanation for the remarkable safety record of aged raw-milk cheese.
Improvement in Cheese Safety Utilization of more sensitive methods for the detection of pathogens existing at low levels in Cheddar and aged raw-milk cheeses could do much to assure cheese safety. Baylis et al. (2000) compared the Oxoid Ltd SPRINT salmonella system (Oxoid, Ltd) against the ISO 6579:1993, Qualicon BAX PCR (Wilmington, DE, USA), bioMerieux VIDAS (Hazelwood, MO, USA) and Tecra Unique methods (Willoughby, NSW, Australia). The SPRINT system was developed for the rapid detection of low levels of injured salmonella in foods. This system utilizes an enrichment broth that contains a specifically developed peptone that allows consistent and rapid recovery of injured salmonella cells, coupled with a Recovery Supplement which contains an Oxyrase | Enzyme System that assists recovery through reduction in oxidative stress of the medium. After 5 h of incubation, selective agents are added to the medium. When tested with ice cream and skimmed milk powder containing low levels of heat-injured S. typhirnurium, the SPRINT method was superior (61% confirmed positive samples) to the ISO (37% positive), BAX (36% positive), VIDAS (30% positive) and Tecra (25% positive) methods. Similar improvements have been advanced by Pritchard and Donnelly (1999) for recovery of injured listeria in dairy products, where continuing work on enrichment of dairy environmental samples at the University of Vermont (UVM) and listeria Repair Broth (LRB) has shown that combining these two primary enrichment media into a single tube of Fraser broth for secondary enrichment yields a significantly higher (p<0.05) percentage of listeria-positive samples than when either LRB or UVM is used alone. Altekruse et al. (1998) stated that 'Because of inherent problems of statistical sampling of foods for microbial pathogens (ICMSE 1986), end-point testing may not assure the safety of cheese. These problems are amplified when organisms are present in small numbers below the sensitivity of the test or when there is intermittent contamination and the tested specimens do not contain pathogens.' Raw ingredient testing, i.e., screening of the raw milk supply, may overcome these shortcomings. In recent years, cheese and cheese products have been recalled due to the presence of pathogenic bacteria
Growth and Survival of Microbial Pathogens in Cheese
such as salmonella, L. monocytogenes and E. coli. In some instances, cheeses, both domestic and imported, have been linked to outbreaks of human illness. In November 1998, the FDA issued the Domestic and Imported Cheese and Cheese Products Food Compliance Program (Anonymous, 1998). The objectives of this programme are for the FDA to conduct inspections of domestic cheese firms, to examine samples of imported and domestic cheese for microbiological contamination, the presence of phosphatase, and filth and to take appropriate regulatory action when violations are encountered. Target pathogens for analysis include L. monocytogenes, salmonella, E. coli (ETEC), enterohemorrhagic E. coli 0157:H7 and Staph. aureus. Under this initiative, direct reference seizure or detention of cheese based on the presence of L. monocytogenes is authorized. It should be noted that ETEC analysis is performed only if E. coli is present at > 104 cfu/g. A review of the FDALs Product Recalls, Alerts and Warnings Archive (http://www.fda.gov/oc/po/firmrecalls/archive.html) for the calendar years 1999, 2000 and 2001 revealed several recalls due to the presence of L. rnonocytogenes in cheese, one recall involving E. coli contamination of Blue and Gorgonzola cheeses, and one recall involving salmonella contamination of Mexican White cheese. The strain of E. coli identified was not 0157:H7. Listeriacontamination appears to be a function of post-process contamination. In no instance during this period were aged cheeses made from raw milk the subject of a recall.
Future Research and Conclusions A number of gaps in the scientific literature have been identified as a result of this review. Future research is suggested in a number of areas: 1. Fully explore the impact of pasteurization of milk on the microbial ecology of cheeses aged for more than 60 days. Does pasteurization increase the susceptibility of cheese to the growth of pathogens introduced via post-processing contamination? 2. Evaluate the potential for survival of S. typhimurium DT 104 intentionally added at low levels (10 and 100 cfu/ml) to raw milk destined for aged raw-milk cheesemaking. 3. Evaluate improved microbiological methods for rawmilk screening and aged raw-milk cheese analysis. 4. Understand the contributions of microbial injury to the interactive effects of salt, pH and mild heat in the suppression of growth of listeria, E. coli and salmonella. Do acid-adapted cultures of these microbial species show enhanced ability to persist in aged raw-milk cheese by withstanding salt, aw
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and mild heat conditions encountered during aged raw-milk cheesemaking? 5. Develop microbiological criteria for raw milk destined for aged cheesemaking by setting tolerance limits for coliforms, E. coli, ETEC, enterohemorrhagic E. coli 0157:H7, salmonella, L. monocytogenes and Staph. aureus. Explore the impact of utilization of raw milk with stringent microbiological standards on the safety of raw-milk cheese. 6. Explore the development of risk reduction procedures and practices at both the primary production level (milk screening) and the cheese production level to improve the safety of aged raw-milk cheese. Aged cheeses made from raw milk are microbiologically safe when manufactured under conditions that use milk-screening procedures, GMPs and HACCP. The raw and heat-treated aged cheese issue is not unlike the listeria and potential for survival during pasteurization issue, which confronted the dairy industry in the 1980s. There was a body of scientific evidence which indicated that listeria was able to survive the pasteurization process. Through careful research and analysis over a period of years, it was established that pasteurization, in fact, offered adequate public health protection and that the greatest risk from listeria posed to dairy products was the threat of post-processing contamination. Careful investigation of the safety of aged, raw-milk cheeses may indicate that raw milk provides protective effects from pathogens in milk and that environmental contamination poses a far greater threat to the safety of cheese. This issue deserves the benefit of full study, careful evaluation of published research information and new research to fully assess all potential risks and benefits.
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unpasteurized cows' milk cheese. Epidemiol. Infect. 109, 389-396. Martin, M.L., Shipman, L.D., Wells, J.G., Potter, M.E., Hedberg, K., Wachsmuth, I.K., Tauxe, R.V, Davis, J.P., Arnoldi, J. and Tilleli, J. (1986). Isolation of Escherichia coli from dairy cattle associated with cases of hemolytic uremic syndrome. Lancet ii, 1043-1044. Martley, EG. and Crow, V.L. (1993). Interactions between non-starter microorganisms during cheese manufacture and ripening. Int. Dairy J. 3, 461-483. Mathew, E and Ryser, E.T. (2002). Competition of thermally injured Listeria monocytogenes with a mesophilic lactic acid starter culture in milk of various heat treatments. J. Food Prot. 65,643-650. McClain, D. and Lee, W.H. (1989). Development of a USDAFSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71, 660-664. McManus, C. and Lanier, J.M. (1987). Salmonella, Campylobacter jejuni, and Yersinia enterocolitica in raw milk. J. Food Prot. 50, 51-54. McSweeney, P.L.H., Fox, P.E, Lucey, J.A., Jordan, K.N. and Cogan, T.M. (1993). Contribution of the indigenous microflora to the maturation of Cheddar cheese. Int. DairyJ. 3,613-634. Morgan, D., Newman, C.P., Hutchinson, D.N., Walker, A.M., Rowe, B. and Majod, E (1993). Verotoxin-producing Escherichia coli 0157 associated with consumption of yoghurt. Epidemiol. Infect. 111,181-183. Northolt, M.D., Beckers, H.J., Vecht, U., Toepoel, L., Soentoro, P.S.S. and Wisselink, H.J. (1988). Listeria monocytogenes: heat resistance and behavior during storage of milk and whey and making of Dutch types of cheeses. Neth. Milk Dairy J. 42,207-219. O'Driscoll, B., Gahan, C.G.M. and Hill, C. (1997). Twodimensional polyacrylamide gel electrophoresis analysis of the acid tolerance response in Listeria monocytogenes LO28. Appl. Environ. Microbiol. 57, 2693-2697. Padhye, N.V. and Doyle, M.P. (1991). Rapid procedure for detecting enterohemmorrhagic Escherichia coli in food. Appl. Environ. Microbiol. 57, 2673-2697. Papageorgiou, D.K. and Marth, E.H. (1989). Fate of Listeria monocytogenes during the manufacture and ripening of blue cheese. J. Food Prot. 52,459-465. Pecorari, M., Guidetti, R., Panari, G., Perini, S., Merialdi, G. and Albertini, A. (2001). Indagine sul comportamento di germi potenzialmente patogeni nella tecnologia del formaggio Parmigiano Reggiano). Sci. Tec. Latt.-Cas. 52(1), 13-22. Pellegrino, L. and Resmini, P. (2001). Cheesemaking conditions and compositional characteristics supporting the safety of raw milk cheese Italian Grana. Sci. Tec. Latt.Cas. 52(2), 105-114. Pitt, W.M., Harden, T.J. and Hull, R.R. (2000). Investigation of the antimicrobial activity of raw milk against several foodborne pathogens. Milchwissenschaft 55,249-252. Prentice, G.A. and Brown, J.V. (1983). The microbiology of Cheddar cheese manufacture. Dairy Industry Int. 48, 23-26.
Pritchard, T.J. and Donnelly, C.W. (1999). Combined secondary enrichment of primary enrichment broth increases Listeria detection. J. Food Prot. 62,532-535. Pritchard, T.J., Beliveau, C.M., Flanders, K.J. and Donnelly, C.W. (1994). Increased incidence of Listeria species in dairy processing plants having adjacent farm facilities. J. Food Prot. 57,770-775. Rajikowski, K.T., Calderone, S.M. and Jones, E. (1994). Effect of polyphosphate and sodium chloride on the growth of Listeria monocytogenes and Staphylococcus aureus in ultra-high temperature milk. J. Dairy Sci. 77, 1503-1508. Rea, M.C., Cogan, T.M. and Tobin, S. (1992). Incidence of pathogenic bacteria in raw milk in Ireland. J. Appl. Bacteriol. 73,331-336. Reitsma, C.J. and Henning, D.R. (1996). Survival of enterohemorrhagic Escherichia coli 0157:H7 during the manufacture and curing of Cheddar cheese. J. Food Prot. 59, 460-464. Resmini, P. and Pellegrino, L. (1996). Evaluation and interpretation of alkaline phosphatase activity in hard cheeses made with raw milk. Food Australia 48,452-454. Rohrbach, B.W., Draughon, EA., Davidson, M.P. and Oliver, S.P. (1992). Prevalence of Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica and Salmonella in bulk tank milk: risk factors and risk of human exposure. J. Food Prot. 55, 93-97. Rudolf, M. and Sherer, S. (2001). High incidence of Listeria monocytogenes in European red smear cheese. Int. J. Food Microbiol. 63, 91-98. Ryser, E.T. (1998). Public health concerns, in, Applied Dairy Microbiology, Marth, E.H. and Steele, J.L., eds, Marcel Dekker, Inc., New York. Ryser, E.T. (1999). Incidence and behavior of Listeria monocytogenes in unfermented dairy products, in, Listeria, Listeriosis and Food Safety, 2nd edn, Ryser, E.T. and Marth, E.H., eds, Marcel Dekker, Inc., New York. pp. 331-404. Ryser, E.T. and Marth, E.H. (1987a). Fate of Listeria monocytogenes during manufacture and ripening of Camembert cheese. J. Food Prot. 50,372-378. Ryser, E.T. and Marth, E.H. (1987b). Behavior of Listeria monocytogenes during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50, 7-13. Ryser, E.T. and Marth, E.H. (1987c). Behavior of Listeria monocytogenes during manufacture and ripening of brick cheese. J. Dairy Sci. 72,838-853. Shere, J.A., Bartlett, K.J. and Kaspar, C.W. (1998). Longitudinal study of Escherichia coli 0157:H7 dissemination on four dairy farms in Wisconsin. AppI. Environ. Microbiol. 64, 1390-1399. Spahr, U. and Schafroth, K. (2001). Fate of Mycobacterium avium subsp, paratuberculosis in Swiss hard arid semihard cheese manufactured from raw milk. Appl. Environ. Microbiol. 67, 4199-4205. Sperber, W.H. (1983). Influence of water activity on foodborne bacteria. J. Food Prot. 46, 142-150. Tammam, J.D., Williams, A.G., Noble, J. and Lloyd, D. (2000). Amino acid fermentation in non-starter Lactobacillus spp.
Growth and Survival of Microbial Pathogens in Cheese
isolated from Cheddar cheese. Lett. Appl. Microbiol. 30, 370-374. Teo, A. and Schlesser, J. (2000). Survival of naturally occurring Escherichia coli and a streptomycin-resistant strain of E. coli K12 (ATCC 35695) during the aging period of hard cheeses made from raw milk. J. Dairy Sci. 83 (Suppl. 1), 109 (abstr.). Threlfall, EJ., Frost, A.J., Ward, L.R. and Rowe, B. (1996). Increasing spectrum of resistance in multiresistant Salmonella typhimurium. Lancet 347, 1053-1054. Villar, R.G., Macek, M.D., Simons, S., Hayes, PS., Goldoft, M.J., Lewis, J.H., Rowan, L.L., Hursh, D., Patnode, M. and Mead, P.S. (1999). Investigation of multidrug-resistant Salmonella serotype Typhimurium DT104 infections linked to raw milk cheese in Washington State. JAMA 281, 1811-1816.
559
Wells, SJ., Fedorka-Cray, P.J., Dargatz, D.A., Ferris, K. and Green, A. (2001). Fecal shedding of Salmonella spp. by dairy cows on farm and at cull cow markets. J. Food Prot. 64, 3-11. Wood, D.S., Collins-Thompson, D.L., Irvine, D.M. and Myhr, A.N. (1984). Source and persistence of Salmonella muenster in naturally contaminated Cheddar cheese. J. Food Prot. 47, 20-22. Yousef, A.E. and Marth, E.H. (1990). Fate of Listeria monocytogenes during the manufacture and ripening of Parmesan cheese. J. Dairy Sci. 73, 3351-3356. Zhang, Y. and Henning, D.R. (1999). Development of predictive modeling for reduction of Escherichia coli during the curing time of American type cheese. J. Dairy Sci. 82 (Suppl. 1), 8 (abstr.).
Toxins in Cheese N.M. O'Brien and T.P. O'Connor, Department of Food and Nutritional Sciences,
University College, Cork, Ireland J. O'Callaghan and A.D.W. Dobson, Department of Microbiology, University College, Cork, Ireland
Biogenic A m i n e s and M y c o t o x i n s In this chapter, we will discuss the formation of toxic compounds, such as biogenic amines and mycotoxins, in cheese. Both these classes of compounds have been reported to be present in cheese and are produced as a result of the activity of micro-organisms, both fungi and bacteria, either in the raw materials used in cheese manufacture or during the production and storage/ripening process. The factors that affect the formation of biogenic amines and mycotoxins will be discussed, together with their occurrence and potential toxic effects.
Biogenic A m i n e s Biogenic amines are non-volatile, low molecular mass aliphatic, alicyclic or heterocyclic organic bases which cause physiological effects (Davidek and Davidek, 1995). Typically, they originate in foods from the decarboxylation of specific amino acids. Decarboxylation can occur due to indigenous decarboxylases in foods or to decarboxylases produced by microorganisms in the food. Biogenic amines are found in a variety of foodstuffs, most commonly fish of the families Scombridae and Scombereoscidae, but also in cheese (Maga, 1978; Smith, 1981; Chang et al., 1985; McCabe, 1986;Joosten, 1988; kopez-Glaria et al., 2001; Innocente and D'Agostin, 2002). In cheese, biogenic amines are produced by decarboxylation of amino acids during ripening (see 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1). Levels produced vary as a function of ripening period and microflora (Renner, 1987; Leuschner et al., 1998). High levels of biogenic amines are most likely to be detected in cheeses heavily contaminated with spoilage micro-organisms (Joosten, 1987). The principal biogenic amines detected in cheese are histamine, tyramine, tryptamine, putrescine, cadaverine and phenylethylamine (El Sayed, 1996; Roig Sagues et al., 1998; Vale and Gloria, 1998; Novella-Rodriguez et al., 2000; Finoli et al., 2001a). Roig Sagues et al. (1998) reviewed the literature on the concentrations of histamine and
tyramine and other biogenic amines reported in cheese (Table 1). The ingestion of biogenic amine-containing foods may cause adverse toxic reactions (Stratton etal., 1991). Some of the biogenic amines have vasoactive properties (e.g., histamine, tyramine, phenylethylamine, tryptamine) while others act primarily by inhibiting histamine-detoxifying enzymes, e.g., the putrefactive amines, putrescine and cadaverine (Hui and Taylor, 1985).
Histamine Histamine has been reported to exert a wide range of effects in the body (Taylor et al., 1984). It stimulates both the sensory and the motor nerves, modulates gastric secretion and stimulates both vascular and extravascular smooth muscle. Histamine toxicity can result in a wide variety of symptoms such as rash, urticaria, inflammation, nausea, vomiting, diarrhoea, abdominal cramping, hypotension, tingling sensations, flushing, palpitations and headache (Taylor, 1986; Bartholomew et al., 1987). In general, toxic symptoms are relatively mild and many patients may not need medical attention. Thus, the exact prevalence of histamine toxicity worldwide is unclear. The prevalence of cheese-related toxicity is also unclear although, as discussed below, several incidences have been reported in the literature. Taylor (1986) comprehensively reviewed the toxicological and clinical aspects of histamine toxicity. He noted that the most common effects of histamine are on the cardiovascular system, causing dilation of peripheral blood vessels, capillaries and arteries with resultant hypotension, headache and flushing. Abdominal cramping, vomiting and diarrhoea may be related to histamine effects on H1 receptors. Urticaria may also be related to the interaction of histamine with H1 receptors, resulting in sensory and motor neuron stimulation. However, for most individuals, ingestion of even large concentrations of biogenic amines, such as histamine, does not elicit symptoms of toxicity since they are rapidly converted to aldehydes by monoamine
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
562
Toxins in Cheese
Table 1
Concentration (mg/kg) of biogenic amines in different cheeses*
Cheese
Histamine
Tyramine
Tryptamine
Em mental Blue cheese Camembert Dutch (Edam and Gouda) Cheddar Parmesan
69-650 3-910 nd-480 nd-450
0-917 40-1100 < 10-210 <0.1-670
nd-1100 nd-60 nd-200
nd-2120 nd-293
nd-1530 85-280
nd-300
Phenylethylamine
Putrescine
Cadaverine
<0.1 10
<0.5 44
16 42
<0.1
7-20
17-48
nd-300
* Adapted from Roig Sagues et aL (1998). nd, not detected.
oxidase (MAO) and diamine oxidase (DAO) and then to carboxylic acids by oxidative deamination (Edwards and Sandine, 1981). These enzymes, present in the gastrointestinal tract, may prevent/reduce the absorption of unmetabolised histamine into the bloodstream (Taylor and kieber, 1979; Lyons et al., 1983; Hui and Taylor, 1985). However, if the activities of MAO and DAO are impaired due to a genetic defect or the presence of potentiators such as food-borne putrefactive amines (e.g., putrescine, cadaverine) or pharmacologic agents (e.g., isoniazid), adverse reactions may occur on ingestion of biogenic amines (Rice et al., 1976; Diamond et al., 1987; Joosten, 1988). Putrescine and cadaverine have been reported to inhibit two histamine-detoxifying enzymes, DAO and histamine N-methyhransferase (HMT) (Hui and Taylor, 1985). Taylor and Sumner (1986) noted that many bacteria, especially Enterobacteriaceae, are capable of producing putrescine and cadaverine as they possess ornithine decarboxylase and lysine decarboxylase. Stratton et al. (1991) noted that putrefactive amine potentiators of histamine toxicity are usually formed by bacteria other than those responsible for histamine production since only relatively few bacteria possess histidine decarboxylase. Tyramine, tryptamine and phenylethylamine can also act as potentiators. Tyramine inhibits MAO, tryptamine inhibits DAO and phenylethylamine inhibits both DAO and HMT. Joosten (1988) reported that tyramine is the only inhibitor, of MAO and DAO, present in significant quantities in cheese. The anti-tuberculosis drug, isoniazid, inhibits histamine-metabolising enzymes and has been reported to result in histamine poisoning in conjunction with cheese consumption (Smith and Durack, 1978; kejonc etal., 197'9; Uragoda and kodha, 197'9). Other drugs administered as antidepressants, antihistamines or antimalarials can sometimes inhibit histamine-metabolising enzymes (Stratton et al., 1991).
Histamine is a normal constituent of the body; it is formed from histidine by a pyridoxal phosphatedependent decarboxylase and modulates several important bodily functions (Douglas, 1980). The concentration of histamine in the blood is strictly regulated. Orally administered histamine causes poisoning only when regulatory mechanisms fail to counteract the ingested dose, i.e., caused by consumption of a very high dose or inhibition of histamine-metabolising enzymes. Oral ingestion of up to 1 mmol ('--100 mg) of histamine does not elicit toxic symptoms in normal individuals (Motil and Scrimshaw, 1979). However, vasodilation and increased heart rate result following intravenous administration of 0.07 ~mol histamine, demonstrate the important detoxifying role of intestinal histaminemetabolising enzymes. Factors influencing formation of histamine and other biogenic amines
The presence of histamine-producing bacteria in foods such as cheese is a key factor in histamine formation. Enterobacteriaceae is the main family implicated in histamine production. However, Clostridium, Lactobacillus and some strains of Klebsiella, Morganella and Hafnia have also been reported to possess histidine decarboxylase, and hence are potential histamine producers (Sakabe, 1973; Taylor et al., 1978, 1979; Taylor, 1986; Marino et al., 2000). Low concentrations of free histidine are present in milk. However, proteolysis during cheese ripening can liberate large amounts of histidine (Hinz et al., 1956). Histamine formation can be controlled primarily by good hygienic practices and by low storage temperatures. Joosten (1988) reported that lactobacilli play a significant role in histamine formation in Gouda cheese; he reported that ripening temperature, pH and salt concentration influence the ability of Lactobacillus to produce histamine in cheese. Ripening for one year at 21 ~ resulted in 6.8 mmol histamine/kg cheese compared to 2.2 mmol/kg cheese after 1 year at 9 ~
Toxins in Cheese
6.5 mmol histamine/kg cheese was detected after 2 weeks of ripening when the pH was 5.39 whereas only 3.4 mmol/kg was detected when the pH was 5.19. A high salt concentration in the Gouda (salt-in-moisture, 4.8%) resulted in 3.5 mmol histamine/kg cheese while a salt-in-moisture of 2.6% resulted in 2.1 mmol histamine/kg cheese. Chambers and Staruszkiewicz (1978) reported that higher levels of biogenic amines are formed in cheese made from pasteurised milk than in raw milk cheese. It appears that bacteria responsible for biogenic amine formation are present in milk prior to processing rather than as post-processing contaminants. Thus, adherence to high standards of cleanliness during milk production can play a role in reducing the formation of biogenic amines in cheese. Storage temperature also appears to play a role in histamine formation in cheese. Elevated storage temperature increases the potential for histamine formation in cheese, particularly if significant numbers of bacteria with decarboxylase activity are present (Sumner et al., 1985). As noted earlier, increasing the storage temperature for Gouda cheese from 9 to 21 ~ results in higher histamine levels (Joosten, 1988). Enhancing proteolysis during cheese ripening by addition of proteolytic enzymes has been reported to increase the concentration of biogenic amines in cheese (Leuschner et al., 1998; Fernandez-Garcia et al., 2000). Biogenic amines in cheese
Only a few cases of histamine poisoning due to cheese consumption have been reported in the literature. Gouda containing 85 mg histamineYl00 g cheese was implicated in an outbreak in Holland (Doeglas et al., 1967). Salt-tolerant lactobacilli, which contaminated the rennet, were considered the most likely factor responsible for the high levels of histamine (Stadhouders and Veringa, 1967). Cheese-related histamine poisoning has also been reported in the United States. In 1978, 38 people exhibited symptoms of toxicity following consumption of Swiss cheese containing more than 9 mmol/kg of histamine (Chambers and Staruszkiewicz, 1978), and in 1980, 6 people aboard a naval ship were poisoned by Swiss cheese containing 16.8 mmol/kg of histamine (Taylor et al., 1982). An individual in Canada being treated with isoniazid exhibited toxicity after consuming Cheddar containing 4 0 m g histamine/100 g (Kahana and Todd, 1981). Similar reactions to histamine-containing cheese by individuals taking isoniazid have been reported by Uragoda and Lodha (1979) and Taylor
(1986).
563
Swiss cheese was implicated in an outbreak of histamine poisoning reported by Sumner et al. (1985). A strain of Lactobacillus buchneri, which possessed histidine decarboxylase activity, was isolated from the cheese. However, other strains of Lb. buchneri did not possess this enzyme activity and were incapable of producing histamine. Recsei and Snell (1982) reported that a strain known as Lactobacillus 30a, which closely resembled Lactobacillus delbruechii, is capable of producing large amounts of histamine. Sumner et al. (1985) reported that the ability to produce histamine appears to be limited to a few strains of lactobacilli, making them difficult to characterise. Other organisms such as Enterococcus faecium, Streptococcus mitis, Lb. delbruechii subsp, bulgaricus, Lb. plantarum, Lb. casei, Lb. acidophilus and Lb. arabinose have been shown to possess histidine decarboxylase activity (Stratton et al., 1991). Joosten and Northolt (1989) isolated five histamineproducing strains similar to Lb. buchneri from Gouda cheese. Tham (1988) reported that enterococci are probably irrelevant in cheese-related histamine toxicity. However, Gardin et al. (2001) reported that Enterococcus faecalis produced 2-phenylethylamine and also substantial amounts of tyramine in skim milk. They noted that the main biological feature influencing the formation of biogenic amines was the extent of growth of micro-organisms, such as Ec. faecalis, characterised by decarboxylase activity. In traditional and artisanal cheeses produced from raw milk, enterococci often reach levels of 107 cfu/g. Gardin et al. (2001) cautioned that it is important that the presence of biogenic amines due to the activity of these micro-organisms is maintained within safe levels, without affecting the positive effects of enterococci on the final organoleptic characteristics of the cheese. In addition to histamine, tyramine in cheese has also been reported to induce adverse reactions, such as headache and hypertension, in patients taking MAO inhibitors (Blackwell, 1963; Smith and Durack, 1978; Lejonc et al., 1979). Tyramine is found at levels ranging from non-detectable to 70 mg/100 g in cheese (Voigt et al., 1974). These workers detected tyramine in 81 of 85 samples of Cheddar cheese tested. Ingles et al. (1985) reported high levels of tyramine (625 Ixg/g) as well as histamine (490 Ixg/g) in Danish Blue cheese. Voigt and Eitenmiller (1978) concluded that many organisms may be responsible for generating biogenic amines in cheese but that most are adventitious rather than part of the starter culture population. The build-up of amines is influenced by the availability of substrate, pH, salt concentration and temperature (Joosten and van Boekel, 1988).
564
Toxins in Cheese
Mycotoxins
Mycotoxins are a group of secondary metabolites produced by various filamentous fungi which can cause a toxic response, termed a mycotoxicosis, when ingested at low concentrations by higher vertebrates and other animals (Fig. 1). The biosynthetic pathways for many of these mycotoxins have been extensively characterised, particularly the aflatoxin biosynthetic pathway (Fig. 2). Ingestion of mycotoxins can lead to the deterioration of liver or kidney function. Some mycotoxins are neurotoxins, while others produce effects ranging from skin sensitivity or necrosis to extreme immunodeficiency. This, coupled with the fact that aflatoxin B1 (AFB1) is regarded as the most potent liver carcinogen known for a wide variety of animal species, makes contamination of the human food chain, including dairy produce, with mycotoxins a significant problem in global food safety. The presence of mycotoxins in cheese is fundamentally due to three main reasons: (1) the presence of aflatoxin M1 (AFM1) in fresh or reconstituted milk (Blanco et al., 1998) used in cheese production, as a consequence of feed contaminated with AFB1 eaten by dairy cattle (Lund et al., 1995), which is often termed indirect contamination, (2) synthesis of mycotoxins by fungi such as Penicillium and Aspergillus species, which grow on cheese, termed direct contamination and (3) the production of mycotoxins by fungi which are used in the manufacture of mould-ripened cheeses. Indirect contamination
It is now well-established that the intake by dairy cows of feedstuffs contaminated with either aflatoxin B1 (AFB1) or aflatoxin B2 (AFB2) results in the excretion of the monohydroxylated AFM1 and AFM2 (Fig. 1) derivatives in their milk within a few hours (Allcroft and Carnaghan, 1963). It has been calculated that if cows ingest AFB1 in their diet at a level of 300 ng/g feed, they will produce milk containing 1-3 ng/ml AFM1 24 h later (Smith et al., 1994). According to two other studies (Veldman et al., 1992; Chopra et al., 1999), normal carry-over is about 0.4-0.6% and a daily intake of AFB1 -->70 Ixg by cows results in greater than the regulatory limit (0.05 txg/1 of AFM1) in milk accepted in most countries. The amount of AFM1 formed depends on the individual cow, with the excretion of AFM1 in the milk decreasing markedly about 1 day after the feeding of AFB1 had ceased, although small amounts are found for a further 2-3 days. The conversion ratio of AFB1 to AFM1 varies from 1:100 to 1:300. There is some evidence to suggest seasonal variations in the level of AFM1 in milk, with higher levels being observed in Albanian farm milk in winter than
in summer (Panariti, 2001), while in a Greek study, no seasonal effects were observed on AFM1 levels in milk (Markaki and Melissari, 1997). While AFM1 is much less toxic, less mutagenic and less carcinogenic than AFB1, it is nonetheless classified as a possible human carcinogen (Group 2B) and as such its presence in milk-derived products, such as cheese, must be a cause for concern. In addition, it is important to note that the consumption of AFM1contaminated infant formula and other milk products by infants is to be avoided and very low limits have been set (0.01-0.05 txg/kg) for infant foods, owing to the relatively high consumption level of these products by infants, their low body weight and the possibly greater susceptibility of younger children to aflatoxins (Aksit et al., 1997). The indirect contamination of milk with other mycotoxins, such as sterigmatocystin, T-2 toxin (van Egmond and Paulsch, 1986), fumonisins (Maragos and Richard, 1994) or cyclopiazonic acid (CPA; Dorner et al., 1994), has been reported. However, it is widely believed that these toxins do not represent a significant public health risk (Prelusky et al., 1990; Charmley et al., 1993), even though CPA can potentially be carried over into processed milk products (Prasongsidh et al., 1997). There is some evidence that ochratoxin A (OTA) can be present in cows' milk. A Swedish study showed OTA in 14% of 36 cows' milk samples at a level ranging from 10 to 40 ng/ml (Breithohz-Emanuelsson et al. , 1993). The results of quantitative surveys of the level of AFM1 in milk and milk products carried out in the late 1960s and 1970s in a number of countries were summarised by Smith et al. (1994). When compared with the results of surveys undertaken in the 1980s, it appears that the incidence of AFMl-contaminated milk in general decreased but this trend did not occur in all countries surveyed. This lowering of the incidence of AFM1 contamination may be as a result of the effect of legislation implemented in many countries on the contamination of feedstuffs with aflatoxins. In a recent review by Pittet (1998), including data from the Czech Republic, Slovakia, Czechoslovakia, France, Greece, Germany, Iran, Japan, Switzerland, Syria, the USA and the Netherlands, the incidence of AFM1 in cheese appears to be very varied. In another study in the south of Spain in which 35 samples of local cheese were analysed, AFM1 was detected in 16 samples (44.7%) at a concentration between 20 and 200 Ixg/g cheese (Barrios et al., 1996). In a survey in the Bursa Province in Turkey, the level of AFM1 in 7 of 57 samples of full-fat white cheese analysed exceeded 250 ng/kg (Oruc and Sonal, 2001). In other studies, the level of AFM1 was low, with only 4 of 204 samples of pasteurised milk, powdered
T o x i n s in C h e e s e
COOH
O
OH
OH2 O
O
i~
~ OH3
, i
N
H
H
Cl
N
Ochratoxin A
~ H~~N
Roquefortine C O
O O
O
u,/ O
O
OCH3
Aflatoxin B2
Aflatoxin B 1
O O
I~,~o o
O
O
o~0.3
O
O
OCH3
Aflatoxin G 2
Aflatoxin G1
OH 3
oJ~~L-OH
O H~176176176176
H
O
,,~
CH3
NH Sterigmatocystin
Cyclopiazonic acid
OH
...CH3
& ~'H
~
0
"
CHO CH3CO ' ~ OH
OH 3
OH 3 Citrinin Figure 1
~"
O
COOH
~ O"
0
O
Structure of some toxins produced by fungi.
Patulin
PR Toxin
OH 3
OH 3
565
566
Toxins in Cheese
(a) ~ ~ -
Acetate
+
(b)
HO O HO O
Hexanoyl-X
~
MalonyI-CoA
(c)
O Norsolorinic acid
OH O OH
HO O HO OH
HO
QH
HO~,~
H O ~ ~ o H 0
OHMe
O
Averufanin
5-Hydroxyaverantin
OH O OH
OH O OH
O
O HO OH
Averantin
HO O OH
'~
H
H O @ ~ M e O HO O OH
V(e)
O
HO O OH
HO~oP-oJ
OH
(fl) ( f 2 ) , , , ~
"----
O Versicolorin A
OH~
Versicolorin B
ocOH o LoA,o
e
Versiconal acetate
l'-Hydroxyversicolorane
Averufin
O O
O
"~ l@OH ~o~o~S~4o'~~
(i~~/Demethylsterigmatocystin
~
HI~O
H
O Versiconal
~)
Dihydromethlysterigmatocystin
~~OH
O'LO
H
~o~o~/~o';~~ Dihydrosterigmatocystin
Sterigmatocystin
(J)
(J)
~~_~OMe
Me
%AO N/~ OM~e
~o~,o~o~ ~ O-methylsterigmatocystin
"
0
Dihydro-O-methylsterigmatocystin
• 0
0
~
Q'o Aflatoxin BI/G 1
0
o
Logo Aflatoxin B2/G2
Figure 2 Aflatoxin biosynthetic pathway. Enzymes involved: (a) fatty acid synthase, (b) polyketide synthase, (c) norsolorinic acid reductase, (d) versiconal hemiacetal reductase, (e) esterase, (fl) versicolorin B synthase, (f2) versiconal cyclase, (g) desaturase, (h) O-methyltransferase (MT-II), (i) O-methyl-transferase, (j) O-methyltransferase (MT-I) (compiled from Trail et aL, 1995; Bennet et aL, 1997; Minto and Townsend, 1997).
Toxins in Cheese
milk and cheese analysed in Campinas, Brazil, being positive (De Sylos et al., 1996) and only 2 of 50 cheese samples tested in Argentina being positive, with levels of 0.33 and 0.20 tzg/l (Lopez et al., 1998). Fate of AFM1 in cheese during manufacture and ripening
Initially, it was believed that the processing of milk reduced the level of AFM1 present. However, later it became clear that the AFM1 content of milk is not reduced by heat treatments such as pasteurisation or sterilisation (Yuosef and Marth, 1989). The fate of AFMz in milk during cheese manufacture is affected by the principal manufacturing steps. Contradictory data have been reported for the recovery of AFM1 after cheese production. Some early studies showed variable losses of AFM1 during cheese manufacture, e.g., Purchase et al. (1972) reported that Cottage cheese made by acid coagulation of naturally contaminated milk contained no AFM1, which was present in the whey. However, a number of other studies have indicated that AFMz in milk partitions between the curds and the whey in both acid-coagulated and rennetcoagulated cheeses. A number of studies have shown that AFMz is stable during cheesemaking, and that 40-57% of total AFM1 is found in the curd (Stubblefield and Shannon, 1974; Stubblefield et al., 1980). Considering the partition coefficient of AFM1 in water, it would be expected that most of the toxin should partition into the whey. However, a greater than expected proportion of the toxin ends up in the curd, possibly due to the fact that AFM1 binds to casein (Brackett and Marth, 1982). Thus, the presence of AFM1 in cheese may be due to the fact that, on the one hand, this toxin binds to casein and, on the other hand, that a part of the whey remains in the curd. An examination of different types of cheese showed high stability of AFM1 during maturation and storage (Applebaum and Marth, 1982; Yuosef and Marth, 1989) and that while fluctuations in the level can occur during cheese maturation and storage, it appears that little if any of the AFM1 is lost during the cheesemaking process. Therefore, the presence of AFM1 in cheese and indeed in other casein-containing products is to be expected if contaminated milk is used as the starting material. The best way to control the presence of AFM1 in milk and cheese is to restrict its presence in the feed. With this in mind, the European Union has established an acceptable limit for AFB1 in animal feed of 10 tzg/kg (Moss, 1998). Production of toxic metabolites in cheese
The moulds, Penicillium camemberti and P. roqueforti, have long been used in the manufacture of mould-
567
ripened cheeses which are eaten throughout the world. P. roqueforti is an essential component of the microflora of a number of cheeses such as Roquefort (France), Stilton (UK), Tulum (Turkey), Gorgonzola (Italy), Blauschimmelkase (Switzerland) and Danish Blue (Denmark). P. camemberti produces cyclopiazonic acid while P. roqueforti produces at least three toxins, PR toxin, roquefortine and patulin (Fig. 1), and some strains can also produce mycophenolic acid, penicillic acid, cyclopiazonic acid, penitrem A, isofumigaclavine A and B, festuclavine and chaetoglobosin A. Cyclopiazonic acid (CPA) is produced by all strains of P. camemberti, and screening of P. camemberti isolates has failed to identify an isolate incapable of producing toxin (Leistner and Eckardt, 1979; Fig. 1). Cyclopiazonic acid has been reported in samples of commercial Blue cheese, at a level ranging from 0.05 to 1.5 Ixg/g (Le Bars, 1979). It appears to be found predominantly in the rind of the cheese but has also been reported to migrate to the core in Taleggio-type cheese (Finoli et al., 1999). Cyclopiazonic acid is produced by P. camemberti in cheese usually after 5 days at 25 ~ but not during normal storage at refrigeration temperatures (Still et al., 1978). The low level of CPA found in cheese (Le Bars, 1979), coupled with the relative instability of the toxin (Noroozian et al., 1998) and its low toxicity make consumption of these cheeses safe for the consumer. Finoli et al. (2001b) reported the presence of roqueforine C in cheese ranging from 0.05 to 1.47 mg&g, but PR toxin was not found. In any case, PR toxin has been reported to be unstable in Blue cheese (Scott and Kanhere, 1979). Roquefortine C has also been reported to be present in Valdeon, a naturally ripened Blue cheese from Spain (Lopez-Diaz et al., 1996), while mycophenolic acid has been reported in Manchego cheese (Lopez-Diaz et al., 1996). Thus, while there are reports of mycotoxin contamination of mould-ripened cheese, the low levels present, coupled with the fact that large quantities are seldom eaten, suggest that they are not a hazard to human health. In any case, there is no evidence to date of human toxicity resulting from the consumption of mould-ripened cheeses. Direct contamination of cheese with mycotoxins
Cheese is very susceptible to mould growth and is normally kept under refrigeration conditions; many retail packs are either vacuum-packed or flushed with an inert gas. Therefore, spoilage generally results from psychrotolerant moulds that can grow at low oxygen tensions. Mould growth during ripening and storage often necessitates trimming. Moulds have been reported to cause spoilage of vacuum-packaged Cheddar cheese during maturation (Hocking and Faedo, 1992). This
568
Toxins in Cheese
defect occurs sporadically in Cheddar blocks which are matured for up to 9-12 months at 8-12 ~ and is caused by the growth of fungi in folds and wrinkles of the plastic film in which the Cheddar is packaged. In one study, 195 fungi were isolated from vacuumpackaged Cheddar, about 27% of which were Penicillium species, with P. commune and P. glabrum being dominant (Hocking and Faedo, 1992). Given that P. commune can produce CPA, the presence of this fungus must be a concern. Indeed, the potential for mycotoxin production by mycotoxigenic fungi which contaminate cheese is a constant concern for both the manufacturer and the consumer. The most important spoilage organisms in hard, semi-hard and semi-soft cheeses from several countries, made without added preservatives, are P. commune and P nalgiovense (Lund etal., 1995). Less-important species include P. verrucosum, P. solitum, P. roqueforti, P. discolor, P. crustosum, P. palitans and Aspergillus versicolor (Fihenborg et al., 1996; Kure et al., 2001). Cheese has been reported to contain mycotoxins that are teratogenic (OTA, AFB1), nephrotoxic (OTA, citrinin), neurotoxic (penitrem A, CPA) or carcinogenic (AFB1, AFG1, OTA, sterigmatocystin; Filtenborg et al., 1996; Creppy, 2002). Others, including patulin, penicillic acid and PR toxin have also been reported but are known not to persist in cheese (Stott and Bullerman, 1976). A number of other secondary metabolites produced by different Penicillium species have also been reported to be present in cheese. These include novel metabolites such as cyclopeptin, viridicatol, rugulovasine A, meleagrin, chaetoglobosin A, compactin, viridic acid, PC-2, verrucolone, diportinic acid, anacine, verrucine A and sclerotigenin, which had not previously been reported from cheese (Larsen etal., 2002). A known metabolite of Penicillium associated with blue-veined cheese is mycophenolic acid, which exhibits antibiotic properties. Since mycotoxin-producing moulds are obligate aerobes, the appropriate packaging of cheese is important. For example, growth of potential mycotoxigenic moulds in cheese can be prevented by modified atmosphere packaging (Taniwaki et al., 2001). In addition, it appears that the production of roquefortine C and CPA by E roqueforti and E commune can be prevented or at least reduced considerably in cheese if adequate modified atmosphere packaging is used; atmospheres of 20-40% CO2 and 1% 02 reduce CPA production to very low levels (Taniwaki et al., 2001). Work has also been undertaken on the incidence of mycotoxins in cheese contaminated with Aspergillus spp. The ability of toxigenic aspergilli to produce ariatoxin during growth on Cheddar cheese was first demonstrated by Lie and Marth (1967), who demonstrated that aflatoxin can penetrate into cheese to a
depth of up to 4 cm from the surface. Aflatoxin has also been shown to be produced in Manchego-type cheese, at a level up to 130 Ixg/g cheese, following ripening at 15 ~ for 60 days (Blanco et al., 1988). Aflatoxin was also detected in both the outer 10-mm layer and in the second 10-mm layer following incubation at 28 ~ for 30 days. Sterigmatocystin was found in Ras cheese inoculated with A. versicolor; toxin was detected after 45 days of ripening and reached a maxim u m after 90 days (Abde Alia et al., 1996). The presence of mould growth on the surface of the cheese does not automatically imply that mycotoxins are present in cheese, as the minimum water activity (aw) for growth and toxin production can be quite different in mycotoxigenic fungi (Moss, 1991; Sweeney and Dobson, 1998). Therefore, even if mould growth does occur on cheese, the level of mycotoxin contamination is likely to be low, based on research findings to date. In any case, it is recommended that if cheese is visually contaminated with mould growth, the contaminated portion of the cheese be removed to a depth of a least 2.5 cm. As previously stated, there is no direct evidence of human toxicity resulting from the consumption of cheese contaminated with mycotoxins. However, this might simply reflect a lack of suitable human-related assay techniques rather than the actual absence of toxins.
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Nutritional Aspects of Cheese N.M. O'Brien and T.P. O'Connor, Department of Food and Nutritional Sciences, University College, Cork, Ireland
Introduction
Milk and dairy products are important components of our food supply. On average, these foods contribute 4% of total energy intake worldwide and approximately 10% of total energy intake in Europe, North America and Australia (FAO Food Balance Sheets, 1995-1999). Cheese is a nutritious, versatile dairy food. A wide variety of cheese types are available to meet specific consumer requirements and allow convenience of use. Per caput consumption of cheese in the European Union has been reported to be 15.2 kg/year. Greece has the highest per caput consumption of 23.5 kg/year (Burrell, 1996). Cheese contains a high concentration of essential nutrients relative to its energy level. Its precise nutrient content is influenced by the type of milk used (species, stage of lactation, full-fat, low-fat, skim), the manner of manufacture and, to a lesser extent, the degree of ripening. As outlined in detail elsewhere in this book, the water-insoluble nutrients of milk (coagulated casein, colloidal minerals, fat, fat-soluble vitamins) are retained in the cheese curd whereas the water-soluble milk constituents (whey proteins, lactose, water-soluble vitamins and minerals) partition into the whey. However, loss of water-soluble B vitamins in the whey may be compensated to a certain extent by microbial synthesis during ripening. Milk and dairy products, including cheese, contain components which may increase the risk of certain chronic diseases but reduce the risk of others (Norat and Riboli, 2003). Cholesterol and saturated fat are potential risk factors for atherosclerosis. A recent paper (Moss and Freed, 2003) has suggested that non-fat constituents of milk, particularly the calciummagnesium ratio, lactose and milk fat globule membrane antigens, have specific coronary atherogenic effects. However, other components may reduce risks, e.g., conjugated linoleic acid (CLA) which may have antioxidant and anticancer properties, calcium which may protect against hypertension and osteoporosis, and folic acid, vitamin B6 and vitamin B12 which may exert beneficial effects on plasma homocysteine levels (an independent risk factor for atherosclerosis). The
epidemiological evidence for an association between dairy products, including cheese, and colorectal cancer has been reviewed by Norat and Riboli (2003); no significant association between cheese consumption and colorectal cancer was noted. Epidemiological studies which attempt to investigate the effect of a specific food item (e.g., cheese) on disease risk are fraught with difficulty in interpretation as it is more likely that it is the overall dietary profile, made up of a balance of a wide variety of different foods, that may influence risk of chronic disease. Protein
Cheese contains a high content of biologically valuable protein. As shown in Table 1 (Holland et al., 1989), the protein content of cheese ranges from approximately 4-40%, depending upon the variety. The protein content of different cheese varieties tends to vary inversely with the fat content. During traditional cheese manufacture, most of the whey proteins pass into the whey. Whey proteins represent only 2-3% of the total protein in cheese, the remainder being casein, which is slightly deficient in sulphur amino acids. Thus, the biological value of cheese protein is slightly less than that of total milk protein. If the essential amino acid index of total milk protein is given a value of 100, then the corresponding value of the proteins in cheese varieties ranges from 91 to 97 (Renner, 1987). Cheese protein is almost 100% digestible, as the ripening phase of cheese manufacture involves a progressive breakdown of casein to water-soluble peptides and free amino acids. Hence, a significant degree of breakdown of cheese protein has occurred before it is consumed and subjected to the effects of gastrointestinal proteolytic activity. Milk proteins are a key source of a range of bioactive peptides (BP) which can exert hormone-like regulatory effects in the human body (Meisel, 1998; Gobbetti et al., 2002; Pihlanto-Leppala, 2002; Fitzgerald and Meisel, 2003). These peptides may be released from their parent protein by proteolysis in products such as cheese. The production of BP is influenced by
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
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the starter culture and ripening conditions. An important class of BP are peptides that inhibit the activity of angiotensin I-converting enzyme (ACE), inhibition of which mainly gives rise to antihypertensive effects but may also modulate immuno-defense and nervous system activity (Meisel, 1993). Angiotensin I-converting enzyme-inhibitory peptides have been reported in several ripened cheeses (Stepaniak et al., 1995; Meisel et al., 1997; Smacchi and Gobbetti, 1998). It appears the BP liberated by starter proteolytic enzymes during cheese ripening can be degraded further to inactive fragments, as the ripening progresses. For example, an antihypertensive peptide derived from Otsl-casein was observed in 6-month-old Parmesan cheese but was not detected in 15-month-old cheese (Meisel et al., 1997). Anticancer effects have been reported for peptides derived from a slurry of cheese made using Lc. lactis subsp, lactis as a starter culture (Kim et al., 1995). Bioactive peptides have potential as ingredients in functional foods and pharmaceuticals.
Carbohydrate Most of the lactose, the principal carbohydrate in milk, is lost in whey during cheese manufacture and hence most cheeses contain only trace amounts of carbohydrate (Table 1). Furthermore, the residual lactose in cheese curd is usually fermented to lactic acid by the starter bacteria. Thus, cheeses can be consumed without ill-effects by lactose-intolerant individuals who are deficient in the intestinal enzyme, [~-galactosidase.
Fat and Cholesterol The fat content of cheese varies considerably, depending on the milk used and the method of manufacture (Table 1). Fat affects firmness, adhesiveness, mouthfeel and flavour of cheese (see 'Rheology and Texture of Cheese', "volume 1). In some varieties of cheese, free fatty acids and their catabolites are important flavour constituents (see 'Lipolysis and Catabolism of Fatty Acids in Cheese', Volume 1). From a nutritional point of view, the digestibility of the fat in different varieties of cheese is in the range 88-94% (Renner, 1987). Most cheeses are potentially significant dietary sources of fat. For example, a 50-g serving of Cheddar provides 17 g fat (Table 1) which is a significant amount when compared with typical intakes of fat in affluent Western societies. A typical Western diet providing 2000 kcal with 40% energy derived from fat contains approximately 88 g fat. Cheese fat generally contains - 6 6 % saturated, 30% monounsaturated and 4% polyunsaturated fatty acids. Thus, cheese represents a significant dietary source of
575
both total fat and saturated fatty acids. Of the many saturated fatty acids in milk, only C12:0, C14:0 and C16:0 have the property of raising blood cholesterol and palmitic acid, C16:0, is relatively ineffective (Hayes et al., 1991). Many sets of dietary guidelines issued by expert panels worldwide have recommended reductions in the intake of both total and saturated fat in Western societies. In large measure, these recommendations are based on evidence that increased intakes of some saturated fatty acids elevate both total and low-density lipoprotein cholesterol in blood, which is associated with an increased risk of coronary heart disease. While some nutritionists dispute this hypothesis, the overwhelming body of medical opinion worldwide supports the concept of dietary guidelines. Market forces and consumers have responded to these guidelines and the market for food products low in fat, cholesterol and sodium has expanded significantly. The cheese industry has responded by developing 'light' cheese products with a reduced fat content (Olson and Johnson, 1990). The cholesterol content of cheese is a function of its fat content (Table 1) and ranges from approximately 10-100 mg/100 g, depending on the variety. Despite considerable consumer confusion and the widespread prevalence of misinformation, dietary cholesterol has much less influence on blood cholesterol level than dietary saturated fat (Keys, 1984). Thus, the cholesterol content of cheese is of lesser importance than its saturated fat content. The majority of individuals show little or no response in blood cholesterol level to increased dietary cholesterol intake in the range 250-800 mg/day. However, a minority (approximately 20%) of adults do exhibit an increased level of blood cholesterol in response to increased dietary intake (McNamara, 1987). In recent years, there has been widespread interest in the potential role of oxidation products of cholesterol on the aetiology of atherosclerosis (Brown and Jessup, 1999; Leonarduzzi et al., 2002). However, cholesterol oxides are formed to a negligible extent in cheese under normal conditions of manufacture, ripening and storage (Stanton and Devery, 2002). Conjugated linoleic acid (CLA) is a potentially beneficial component of milk products, including cheese (MacDonald, 2000). Conjugated linoleic acid is a mixture of positional and geometric isomers of linoleic acid (C18:2) that contain conjugated unsaturated double bonds. The principal isomer is cis-9, trans-11-octadecadienoic acid which accounts for more than 82% of total CLA in dairy products (Chin etal., 1992). Conjugated linoleic acid has been reported to have antioxidant and anticarcinogenic
576
Nutritional Aspects of Cheese
properties in vitro and in animal models (Ha et al., 1987, 1990; Ip et al., 1991). However, these suggested anticarcinogenic properties of CLA could not be confirmed in a recently published epidemiological study on humans (see Voorrips et al., 2002). These authors noted that cheese contributed approximately 21% of total CLA intake in their study group. Conjugated linoleic acid may also be anticholesterolaemic and antiathrogenic (Lee et al., I994; Mougios et al., 2001). On average, the concentration of CLA in milk and dairy products ranges from 0.2 to 1.6 g/100 g fat (Lin et al., i995; Fritsche and Steinhart, i998). Fritsche and Steinhart (1998) have estimated that the intake of CLA in Germany is 0.35 g/d for women and 0.43 g/d for men. Zlatanos et al. ( 2 0 0 2 ) reported that Greek Feta and hard cheeses contain 1.9 (average of 0.8) g CLA/100 g fat. These authors reported higher levels of CLA in Greek cheese derived from sheep's and goats' milk than the level of CLA reported by others (Lin et al., 1995; Jiang et al., 1997; Ma et al., 1999) in cheese derived from cows' milk.
Vitamins The concentration of fat-soluble vitamins in cheese is influenced by the same factors that affect its fat content. Most of the fat-soluble vitamins in milk are retained in the cheese fat. The concentration of water-soluble vitamins in cheese is generally lower than in milk due to losses in the whey. The loss of some of the B vitamins is offset, to a certain extent, by microbial synthesis during cheese ripening. In particular, propionic acid bacteria synthesize significant levels of vitamin B12 in hard cheeses such as Emmental (Renner, 1987). In general, most cheeses are good sources of vitamin A, riboflavin, vitamin B12 and, to a lesser extent, folate. The vitamin content of a range of cheeses is shown in Table 2 (Holland et al., 1989). Cheese contains negligible levels of vitamin C.
Minerals Cheese is an important dietary source of several minerals, in particular calcium, phosphorus and magnesium (Table 3). A 100-g serving of hard cheese provides approximately 800mg calcium. However, acid-coagulated cheeses, e.g., Cottage, contain considerably less calcium than rennet-coagulated varieties (Renner, 1987). Bioavailability of the calcium from cheese is equivalent to that from milk. Recker et al. (1988) reported that 22.9, 26.7 and 25.4% of total calcium was absorbed from cream cheese, whole milk and yoghurt, respectively.
While the aetiology of osteoporosis is very complex, it appears that adequate calcium intake during childhood and in the teenage years is important in assuring the development of high-peak bone mass. Maximizing bone mass early in life is considered to be a major preventative factor against the development of osteoporosis in later years (Heaney, 1991). Cheese has a potential role in supplying extra, highly bioavailable, calcium. Dairy products, including cheese, contribute little dietary iron (Table 3). Iron deficiency is commonly observed in both developing and developed countries. Hence, there has been interest in fortifying dairy products with iron to enhance their nutritional value. Cheddar and processed cheese have been successfully fortified with iron (Zhang and Mahoney, 1989a,b, 1990, 1991). As discussed elsewhere in this book, NaC1 serves several important functions in natural and processed cheeses (see 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1). A wide range of sodium levels are found in cheese due to different amounts of salt added during cheesemaking (Table 3). In general, the salt content of natural cheeses tends to be lower than that of many processed cheeses. There is considerable evidence that high sodium intake contributes to hypertension in a susceptible minority (20%) of individuals who are genetically saltsensitive. Unfortunately, there is no simple diagnostic test to identify salt-sensitive individuals. Hence, dietary guidelines for the general public usually recommend that salt intake be restricted. However, it is important to note that even in countries with a high consumption, cheese contributes only about 5-8% of total sodium intake (Renner, 1987).
Cheese and Dental Caries The aetiology of dental caries involves metabolism of sugars by oral micro-organisms to acids which gradually dissolve tooth enamel. However, it is now recognized that a number of dietary factors and nutrient interactions can modify the expression of dental caries (Herod, 1991; Kashket and DePaola, 2002). The cariogenic potential of food is influenced by its composition, texture, solubility, retentiveness and ability to stimulate saliva flow (Morrissey et al., 1984). Dental caries has been acknowledged as a 'silent epidemic' that represents a serious threat to children and adults (Surgeon General, 2000). A considerable body of research has been published on the cariostatic effects of cheese (see reviews by O'Brien and O'Connor, 1993; Kashket and DePaola, 2002). Early work (Shaw et al., 1959; Dreizen et al.,
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Table 3
Mineral content of selected cheeses, mg/100 g (Holland et aL, 1989)
Cheese type Brie Caerphilly Camembert Cheddar (normal) Cheddar (reduced-fat) Cheshire Cottage cheese Cream cheese Danish blue Edam Emmental Feta Fromage frais Gouda Gruyere Mozzarella Parmesan Processed cheese* Ricotta Roquefort Stilton
Na
K
Ca
Mg
P
Fe
Zn
700 480 650 670 670 550 380 300 1260 1020 450 1440 31 910 670 610 1090 1320 100 1670 930
100 91 100 77 110 87 89 160 89 97 89 95 110 91 99 75 110 130 110 91 130
540 550 350 720 840 560 73 98 500 770 970 360 89 740 950 590 1200 600 240 530 320
27 20 21 25 39 19 9 10 27 39 35 20
390 400 310 490 620 400 160 100 370 530 590 280 110 490 610 420 810 800 170 400 310
0.8 0.7 0.2 0.3 0.2 0.3 0.1 0.1 0.2 0.4 0.3 0.2 0.1 0.1 0.3 0.3 1.1 0.5 0.4 0.4 0.3
2.2 3.3 2.7 2.3 2.8 3.3 0.6 0.5 2.0 2.2 4.4 0.9 0.3 1.8 2.3 1.4 5.3 3.2 1.3 1.6 2.5
8 38 37 27 45 22 13 33 20
* Variety not specified.
1961) showed that the incorporation of dairy products in the diet greatly reduced the development of dental caries in rats. Reynolds and Johnson (1981) confirmed these findings. Later work (Jenkins and Ferguson, 1966; Weiss and Bibby, 1966) indicated that if enamel is treated with milk in vitro and subsequently washed, the solubility of the enamel is greatly reduced. This effect was attributed to the high levels of calcium and phosphate in milk (Jenkins and Ferguson, 1966) or to casein adsorption onto enamel surfaces (Weiss and Bibby, 1966). Reynolds and del Rio (1984) reported that both casein and whey proteins significantly reduced the extent of caries, with the former exerting the greater effect. Further evidence for the protective effect of casein was provided in a study on rats fed with caseinenriched chocolate (Reynolds and Black, 1987). Calcium and phosphate appear to become available under the acidic conditions of the plaque and reduce demineralization of enamel (Reynolds, 1997; Reynolds et al., 1999). Concentrates containing various levels of whey protein, calcium and phosphate but negligible amounts of casein, significantly reduced the incidence of dental caries in rats (Harper et al., 1987). Thus, there is evidence that milk proteins, calcium and phosphate all exert an anticariogenic effect. Guggenheim et al. (1999) reported that micellar casein inhibits oral colonization by the cariogenic Streptococcus sobrinus and dental caries in rats. Vacca-Smith et al. (1994) demonstrated that K-casein can reduce the adherence
of the cariogenic Sc. mutans to hydroxyapatite (the mineral of enamel). Rugg-Gunn et al. (1975) provided the first evidence that the consumption of cheese had an anticariogenic effect in humans. They showed that the consumption of Cheddar cheese after sweetened coffee or a sausage roll increased plaque pH, possibly due to increased salivary output. Similar effects were reported by Imfeld et al. (1978) who used a more sophisticated continuous wire telemetry procedure to monitor variations in plaque pH. The effect of eating patterns on dental caries in rats was investigated by Edgar et al. (1982). Rats fed additional meals of cheese while on a high-sucrose diet, developed fewer smooth surface carious lesions and exhibited increased salivary output (which buffers acid formed in plaque) and a reduction in the number of Sc. mutans. Harper et al. ( 1 9 8 3 ) suggested that the cariostatic effect of cheese in rats is due to its calcium phosphate and/or casein; the fat or lactose appeared to exert little influence. Further work by Rosen et al. (1984) on the effect of cheese, with or without sucrose, on dental caries and the recovery of inoculated Sc. mutans in rats indicated beneficial cariostatic effects of cheese consumption but little effect on Sc. mutans numbers. These data suggest that the cariostatic effects of cheese may not be directly related to effects on Sc. mutans. Work on the protective effects of four cheese varieties in an in vitro demineralization system suggested that most, but not all, of the protective
Nutritional Aspects of Cheese
effects of cheese could be explained by mass action effects of soluble ions, particularly calcium and phosphate (Jenkins and Harper, 1983). The effect of Cheddar cheese on experimental caries in humans was investigated by Silva et al. (1986) using an 'intraoral cariogenicity test' (ICT). Demineralization and consequent reduction in the hardness of enamel monitored in this test is assumed to be typical of the early stage of the development of caries. Enamel slabs were polished and their initial micro-hardness determined using a Knoop Indenter. The slabs were then wrapped in Dacron and fastened on a prosthetic applicance made specifically for each subject to replace a missing lower first permanent molar. The subjects chewed 5 g of cheese immediately after rinsing their mouths with 10% (w/v) sucrose. Chewing cheese immediately after sucrose rinses resulted in a 71% reduction in demineralization of the enamel slabs, raised plaque pH but caused no significant change in the microflora of plaque compared with controls. Silva et al. (1987) investigated the effects of the water-soluble components of cheese on human caries using the I CT procedure and an experimental protocol which avoided salivary stimulus caused by chewing cheese. An average reduction of 55.7% in the cariogenicity of sucrose was reported, indicating the presence of one or more water-soluble anticariogenic components in cheese. Further evidence that cheese may inhibit dental caries in the absence of saliva was provided by Krobicka et al. (1987); rats that had their saliva-secreting glands surgically removed developed fewer and less-severe lesions when fed with cheese in addition to a cariogenic diet when compared to appropriate controls. Trials on human subjects have confirmed that the consumption of hard cheese leads to significant rehardening of softened enamel surfaces (Jenkins and Hargreaves, 1989; Gedalia etal., 1991). Jensen and Wefel (1990) showed that processed cheese was both antiacidogenic and enamel-protective in human subjects fed with processed cheese four times a day for one month. Saliva flow is greatly reduced in individuals who receive head and neck irradiation for malignancies. These individuals are at high risk of developing dental caries. Sela et al. (1994) reported that hard cheese consumption by these individuals was effective in controlling caries. Moynihan et al. (1999) noted that the concentration of calcium in plaque was significantly higher in human subjects fed with cheese-containing meals than in control subjects fed with meals without cheese. The beneficial effects of cheese were observed even when it was incorporated into other foods, e.g., pasta with cheese sauce. Epidemiological studies (Pappas et al., 1995a,b) indi-
579
cate that high intake of cheese is negatively associated with root caries in elderly populations, many of whom are at high risk for such lesions. While more research is needed to define the precise mechanism(s) involved in the cariostatic effects of cheese, there is ample evidence to support the consumption of cheese as an anticaries measure (Herod, 1991; Kashket and DePaola, 2002). The most plausible mechanisms for the protective effect of cheese appear to be related to the mineralization potential of the casein-calcium phosphate of cheese, to the stimulation of saliva flow induced by its texture and/or flavour, the buffering effects of cheese proteins on acid formation in dental plaque and the inhibition of cariogenic bacteria.
References Brown, A.J. and Jessup, W. (1999). Oxysterols and atherosclerosis (review). Atherosclerosis 142, 1-28. Burrell, A. (1996). Economic Aspects of Milk Production in the EU. Eurostat Statistical Document, Eurostat, Luxembourg. Chin, S.E, Liu, W., Storkson, J.M., Ha, Y.L. and Pariza, M.W. (1992). Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognised class of anticarcinogens. J. Food Cornp. Anal. 5,185-197. Dreizen, S., Dreizen, J.G. and Stone, R.E. (1961). The effect of cow's milk on dental caries in the rat. J. Dent. Res. 40, 1025-1028. Edgar, W.M., Bowen, W.H., Amsbaugh, S., Monell-Torrens, E. and Brunelle, J. (1982). Effects of different eating patterns on dental caries in the rat. Caries Res. 16, 384-389. FAO Food Balance Sheets (1995-1999). http://apps.fao.org. Fitzgerald, R.J. and Meisel, H. (2003). Milk protein hydrolysates and bioactive peptides, in, Advanced Dairy Chemistry, Vol. 1, Proteins, Fox, RE and McSweeney, P.L.H., eds., Kluwer Academic/Plenum Publishers, New York. pp. 675-697. Fritsche, J. and Steinhart, H. (1998). Amounts of conjugated linoleic acid (CLA) in German foods and evaluation of daily intake. Z. Lebensm. Unters. Forsch. 206, 77-82. Gedalia, I., Ionat-Bendat, D., Ben-Mosheh, S. and Shapira, L. (1991). Tooth enamel softening with a cola type drink and rehardening with hard cheese or stimulated saliva. J. Oral. Rehabil. 18, 501-506. Gobbetti, M., Stepaniak, L., DeAngelis, M., Corsetti, A. and Di Cagno, R. (2002). Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing. Crit. Rev. Food Sci. Nutr. 42,223-239. Guggenheim, B., Schmid, R. and Aeschlimann, J.M. (1999). Powdered milk micellar casein prevents oral colonization by Streptococcus sobrinus and dental caries in rats: a basis for the caries-protective effect of dairy products. Caries Res. 33,446-454. Ha, Y.L., Grimm, N.K. and Pariza, M.W. (1987). Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis 8, 1881-1887.
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Nutritional Aspects of Cheese
Ha, Y.L., Storkson, J. and Pariza, M.W. (1990). Inhibition of benzo[alpyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res. 50, 1097-1101. Harper, D.S., Osborn, J.C. and Clayton, R. (1983). Cariostatic potential of four cheeses evaluated in a programmedfed rat model. J. Dent. Res. 62, 283 (abstr.). Harper, D.S., Osborn, J.C., Clayton, R. and Hefferren, J.J. (1987). Modification of food cariogenicity in rats by mineral-rich concentrates from milk. J. Dent. Res. 66, 42-45. Hayes, K.C., Pronczuk, A., Lindsey, S. and Diersen-Schade, D. (1991). Dietary saturated fatty acids differ in their impact on plasma cholesterol and lipoproteins in non-human primates. Am. J. Clin. Nutr. 53, 491-498. Heaney, R.P. (1991). Evaluation of Publically Available Scientific Evidence Regarding Nutrient-Disease Relationships. 3. Calcium and Osteoporosis. Life Sciences Research Office, Federation of American Societies for Experimental Biology, Rockville Pike, MD. Herod, E.L. (1991). The effect of cheese on dental caries. Aust. DentalJ. 36, 120-125. Holland, B., Unwin, I.D. and Buss, D.H. (1989). Milk Products and Eggs: The Fourth Supplement to McCance and Widdowson's The Composition of Foods, 4th edn, Royal Society of Chemistry/Ministry of Agriculture, Fisheries and Food, Cambridge, UK. Imfeld, T.H., Hirsch, R.S. and Muhlmann, H.R. (1978). Telemetric recordings of interdental plaque pH during different meal patterns. Br. Dent. J. 139,351-356. Ip, C., Chin, S.E, Scimeca, J.A. and Pariza, M.W. (1991). Mammary cancer prevention by conjugated dienoic derivatives of linoleic acid. Cancer Res. 51, 6118-6124. Jenkins, G.N. and Ferguson, D.B. (1966). Milk and dental caries. Br. Dent. J. 120, 472-477. Jenkins, G.N. and Hargreaves, J.A. (1989). Effect of eating cheese on Ca and P concentrations of whole mouth saliva and plaque. Caries Res. 23, 159-164. Jenkins, G.N. and Harper, D.S. (1983). Protective effect of different cheeses in an in vitro demineralization system. J. Dent. Res. 62,284 (abstr.). Jensen, M.E. and Wefel, J.S. (1990). Effects of processed cheese on human plaque pH and demineralization and remineralization. Am. J. Dent. 3,217-223. Jiang, J., Bjorck, L. and Fonden, R. (1997). Conjugated linoleic acid in Swedish dairy products with special reference to manufacture of hard cheeses. Int. Dairy J. 7, 863-867. Kashket, S. and DePaola, D.P. (2002). Cheese consumption and the development and progression of dental caries. Nutr. Rev. 60, 97-103. Keys, A. (1984). Serum cholesterol response to dietary cholesterol. Am. J. Clin. Nutr. 40,351-359. Kim, H.D., Lee, J.H., Shin, Z.I., Man, H.S. and Woo, H.J. (1995). Anticancer effects of hydrophobic peptides derived from a cheese slurry. Food Biotechnol. 4, 268-272. Krobicka, A., Bowen, W.H., Pearson, S. and Young, D.A. (1987). The effects of cheese snacks on caries in desalivated rats.J. Dent. Res. 66, 1116-1119.
Lee, K.N., Kritschevsky, D. and Pariza, M.W. (1994). Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 118, 19-25. Leonarduzzi, G., Sottero, B. and Poli, G. (2002). Oxidized products of cholesterol: dietary and metabolic origin and proatherosclerotic effects (review). J. Nutr. Biochem. 13, 700-710. Lin, H., Boylston, T.D., Chang, M.L., Luedecke, L.O. and Shultz, T.D. (1995). Survey of the conjugated linoleic acid contents of dairy products. J. Dairy Sci. 78, 2358-2365. Ma, D.W.L., Wierzbicki, A.A., Field, C.J. and Clandinin, M.T. (1999). Conjugated linoleic acid in Canadian dairy and beef products. J. Agric. Food Chem. 47, 1956-1960. MacDonald, H.B. (2000). Conjugated linoleic acid and disease prevention: a review of current knowledge. J. Am. Coll. Nutr. 19, 111S-118S. McNamara, D.J. (1987). Effects of fat-modified diets on cholesterol and lipoprotein metabolism. Ann. Rev. Nutr. 7, 273-290. Meisel, H. (1993). Casokinins as inhibitors of angiotensinconverting-enzyme, in, New Perspectives in Infant Nutrition, Sawatzki, G. and Renner, B., eds., Thieme, Stuttgart. pp. 153-159. Meisel, H. (1998). Overview on milk protein-derived peptides. Int. Dairy J. 8,363-373. Meisel, H., Goepfert, A. and Gunther, S. (1997). ACE inhibitory activities in milk products. Milchwissenschaft 52,307-311. Morrissey, R.B., Burkholder, B.D. and Tarka, S.M. (1984). The cariogenic potential of several snack foods. J. Am. Dent. Assoc. 109,589-591. Moss, M. and Freed, D. (2003). The cow and the coronary: epidemiology, biochemistry and immunology. Int. J. Cardiol. 87,203-216. Mougios, V., Matsakas, A., Petridou, A., Ring, S., Sagredos, A., Melissopoulou, A., Tsigilis, N. and Nicolaidis, M. (2001). Effect of supplementation with conjugated linoleic acid on human serum lipids and body fat. J. Nutr. Biochem. I2, 585-594. Moynihan, P.J., Ferrier, S. and Jenkins, G.N. (1999). Carlostatic potential of cheese: cooked cheese-containing meals increase plaque calcium concentration. Br. Dent. J. 187, 664-667. Norat, T. and Riboli, E. (2003). Dairy products and colorectal cancer: a review of possible mechanism and epidemiological evidence. Eur. J. Clin. Nutr. 57, 1-17. O'Brierl, N.M. and O'Connor, T.P. (1993). Milk, cheese and dental caries. J. Soc. Dairy Technol. 46, 46-49. Olson, N.E and Johnson, M.E. (1990). Light cheese products: characteristics and economics. Food Technol. 44 (10), 93-96. Pappas, A.S., Joshi, A. and Palmer, C.A. (1995a). Relationship of diet to root caries. Am. J. Clin. Nutr. 61,423S-429S. Pappas, A.S., Joshi, A. and Belanger, A.J. (1995b). Dietary models for root caries. Am. J. Clin. Nutr. 61,417S-422S. Pihlanto-Leppala, A. (2002). Bioactive peptides, in, Encyclopedia of Dairy Sciences, Roginski, H., Fuquay, J.W. and Fox, P.F., eds., Academic Press, London. pp. 1960-1967.
Nutritional Aspects of Cheese
Recker, R.R., Bammi, A., Barger-Lux, M.J. and Heaney, R.R (1988). Calcium absorbability from milk products, an imitation milk and calcium carbonate. Am. J. Clin. Nutr. 47, 93-95. Renner, E. (1987). Nutritional aspects of cheese, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, General Aspects, Fox, RE, ed., Elsevier Applied Science, London. pp. 345-363. Reynolds, E.C. (1997). Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J. Dent. Res. 76, 1587-1595. Reynolds, E.C. and Black, C.L. (1987). Reduction of chocolate's cariogenicity by supplementation with sodium caseinate. Caries Res. 21,445-451. Reynolds, E.C. and del Rio, A. (1984). Effect of casein and whey protein solutions on caries experience and feeding patterns of the rat. Arch. Oral Biol. 29,927-933. Reynolds, E.C. and Johnson, I.H. (1981). Effect of milk on caries incidence and bacterial composition of dental plaque in the rat. Arch. Oral Biol. 26,445-451. Reynolds, E.C., Black, C.L. and Cai, E (1999). Advances in enamel remineralization: casein phosphopeptide-amorphous calcium phosphate. J. Clin. Dent. 10, 86-88. Rosen, S., Min, D.B., Harper, D.S., Harper, W.J., Beck, E.X. and Beck, EM. (1984). Effect of cheese, with or without sucrose, on dental caries and recovery of Streptococcus mutans in rats. J. Dent. Res. 63,894-896. Rugg-Gunn, A.J., Edgar, W.M., Geddes, D.A.M. and Jenkins, G.N. (1975). The effect of different meal patterns upon plaque pH in human subjects. Br. Dent. J. 139, 351-356. Sela, M., Gedalia, I. and Shah, L. (1994). Enamel rehardening with cheese in irradiated patients. Am. J. Dent. 7, 134-136. Shaw, J.H., Ensfield, B.J. and Vv~ollman, D.H. (1959). Studies on the relation of dairy products to dental caries in caries-susceptible rats. J. Nutr. 67, 253-273. Silva, M.D. de A., Jenkins, G.N., Burgess, R.C. and Sandham, H.J. (1986). Effect of cheese on experimental caries in human subjects. Caries Res. 20, 263-269. Silva, M.E de A., Burgess, R.C., Sandham, H.J. and Jenkins, G.N. (1987). Effects of water-soluble components of cheese on experimental caries in humans. J. Dent. Res. 66, 38-41. Smacchi, E. and Gobbetti, M. (1998). Peptides from several Italian cheeses inhibitory to proteolytic enzymes of lactic
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acid bacteria, Pseudomonas fluorescenes ATCC948 and to the angiotensin I-converting enzyme. Enzyme Microbiol. Technol. 22,687-694. Stanton, C. and Devery, R. (2002). Formation and content of cholesterol oxidation products in milk and dairy products, in, Cholesterol and Phytosterol Oxidation Products: Analysis, Occurrence and Biological Effects, Guardiola, E, Dutta, R C., Codony, R. and Savage, G.R, eds., AOCS Press, Champaign, IL. pp. 147-161. Stepaniak, L., Fox, RE, Sorhaug, T. and Grabska, J.J. (1995). Effect of peptides from the sequence 58-72 of [3-casein on the activity of endopeptidase, aminopeptidase and X-proplyl-dipeptidyl aminopeptidase from Lactococcus. J. Agric. Food Chem. 43,849-853. Surgeon General (2000). Oral Health in America: A Report of the Surgeon General. US Department of Health and Human Services, Rockville, MD. Vacca-Smith, A.M., Wuyckhuyse, B.C., Tabak, L.A. and Bowen, W.H. (1994). The effect of milk and casein proteins on the adherence of Streptococcus mutans to saliva-coated hydroxyapatite. Arch. Oral Biol. 39, 1063-1068. Voorrips, L.E., Brants, H.A.M., Kardinaal, A.EM., Hiddink, G.J., van den Brandt, RA. and Goldbohm, R.A. (2002). Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands Cohort Study on Diet and Cancer. Am. J. Clin. Nutr. 76, 873-882. Weiss, M.E. and Bibby, B.G. (1966). Effects of milk on enamel solubility. Arch. Oral Biol. 11, 49-57. Zhang, D. arid Mahoney, A.W. (1989a). Effect of iron fortification on quality of Cheddar cheese. J. Dairy Sci. 72, 322-332. Zhang, D. and Mahoney, A.W. (1989b). Bioavailability of iron-milk protein complexes and fortified Cheddar cheese. J. Dairy Sci. 72, 2845-2855. Zhang, D. and Mahoney, A.W. (1990). Effect of iron fortification on Cheddar cheese. J. Dairy Sci. 73, 2252-2258. Zhang, D. and Mahoney, A.W. (1991). Iron fortification of process Cheddar cheese. J. Dairy Sci. 74,353-358. Zlatanos, S., Laskaridis, K., Feist, C. and Zagredos, A. (2002). CLA content and fatty acid composition of Greek Feta and hard cheeses. Food Chem. 78,471-477.
Factors that Affect the Quality of Cheese P.F. Fox, Department of Food Science, Food Technology and Nutrition, University College, Ireland T.M. Cogan, Dairy Products Research Centre, Teagasc, Fermoy, Ireland
Introduction As discussed in 'Cheese: An Overview', Volume 1 and 'Diversity of Cheese Varieties: an Overview', Volume 2, the manufacture of cheese exploits either of two properties of the casein system: precipitation/coagulation at the isoelectric pH (4.6), which is exploited in the production of fresh, acid-coagulated cheeses ( - 2 5 % of total cheese production), or by limited proteolysis using rennets which specifically hydrolyse the micellestabilizing protein, K-casein, following which the rennetaltered micelles coagulate in the presence of Ca 2+ at a temperature > 2 0 ~ usually 3 0 - 3 5 ~ ( ' 7 5 % of cheese production). Most acid-coagulated cheeses are consumed fresh (unripened) whereas the vast majority of rennet-coagulated cheeses are ripened for a period ranging from - 3 weeks to > 2 years. Although there are recognizable differences between the unripened curds for different cheeses, mainly with respect to moisture content and texture, the characteristic differences between the 1000 or so varieties of cheese develop during ripening. The quality of acid-coagulated cheeses is subject to some variation but the fact that they are consumed fresh and that no modifications are required after manufacture, makes them relatively easy to produce with consistent quality (see 'Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels', Volume 1 and 'Acid- and Acid-Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties', Volume 2). In contrast, the characteristic quality of rennet-coagulated cheeses develops mainly during ripening and frequently depends on the growth of a secondary microflora, which are not readily reproducible. During ripening, a complex array of microbiological, biochemical and chemical reactions occur and therefore there are many opportunities for problems to develop. In this chapter, the quality aspects of rennet-coagulated cheeses will be considered. Some of the principal areas of cheese science through which cheese quality can be improved through research will be discussed. Cheese is the quintessential convenience f o o d - it can be consumed as it is without preparation, can be
used as a sandwich filler, grated or diced and used as a condiment or as a component of several cooked dishes. At least 50% of cheese is used, at home or in the factory, as an ingredient or component of other foods. The principal applications of cheese as an ingredient are discussed in 'Cheese as an Ingredient', Volume 2. The use of cheese as a food ingredient is increasing and for many of these applications, the principal criterion of quality depends on the functionality of the cheese, which depends mainly on the physico-chemical properties of the proteins. When used as an ingredient in food applications, cheese is expected to perform one or more functions and there is considerable commercial interest in producing cheese products with functional properties tailor-made for particular applications. Cheese may be used as an ingredient in several forms: 9 natural cheese: sliced, diced or grated; 9 processed cheese products; 9 dried cheese products: - dried, grated natural cheese (a traditional product); - cheese powders. 9 enzyme-modified cheese products, which may be produced from mild cheese or fresh cheese curd or from blends of enzyme-treated casein and fats and are used mainly as high-intensity cheese flavours. There are several aspects to the quality of cheese; some are applicable to all cheese products and applications, others are significant for specific cheeses. Probably the most important aspects of cheese are: 9 9 9 9 9 9
safety from a public health viewpoint nutritional flavour texture functionality appearance, e.g., - Cheese must conform to the expected characteristics of the variety. There are obvious differences between the principal families of cheese but it may not be so easy to differentiate between cheeses claimed to
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
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Factors that Affect the Quality of Cheese
belong to similar varieties, e.g., Gouda and Edam, Cheddar and Gloucester, Brie and Camembert. The borderlines are blurred and within each variety, a certain degree of variability is tolerated and acceptable. - Reproducibility/reliability - consumers expect that a product will be reproducible and consistent between batches and over time with respect to flavour, texture, appearance and functionality, especially for the principal, 'mass-produced' varieties; some variability is tolerated, perhaps even expected, in artisanal cheeses.
Milk Selection Pre-treatment Standardization
Cheesemilk Addition of: starter culture (acidification) colour (optional) CaCI2 (optional) Coagulation [rennet or acid (produced in situ or pre-formed) or heat/acid]
Production of Rennet-Coagulated Cheese The production of rennet-coagulated cheese can be divided into two phases:
Coagulum (gel) Cut coagulum Stir Heat Acidification (rennet-coagulated cheeses) Separation of curds from whey
9 Conversion of milk to curd 9 Conversion of curd to cheese The key operations are summarized in Fig. 1. The production of good-quality cheese depends on a good milk supply from the chemical and microbiological viewpoints. Raw milk is a rather variable commodity and is subjected to a range of processes aimed at modifying, standardizing and optimizing the cheesemaking properties of milk. Given a good milk supply, the first objective is to produce curd of the desired chemical composition with the desired microflora. Unless these criteria are met, the curd will not evolve into cheese with the characteristic flavour, texture and functionality during ripening. The ripening of cheese, and hence its quality, is due to the activity of micro-organisms and enzymes from four or five sources: 9 9 9 9 9
Milk Rennet Primary starter Secondary cultures Adventitious (non-starter) bacteria
It is reasonable to expect that it should be possible to produce premium-quality cheese consistently by controlling these agents; however, in spite of considerable research and quality control efforts, it is not yet possible to do so. A very wide and diverse range of factors interact to affect the composition of cheese curd and hence the quality of the final cheese; an attempt to summarize these is shown in Fig. 2, which follows the same sequence as Fig. 1. Some of these factors/agents can be manipulated easily and precisely while others are more difficult, or perhaps impossible, to control. Indeed, the precise influence of many of the factors included
Curds Acidification Special operations (e.g., cheddaring, stretching) Salting (some varieties) Moulding Pressing (some varieties)
Fresh cheese
~
Salting (most varieties) Ripening (most rennet-coagulated cheeses)
Mature cheese Figure 1 General protocol for cheese manufacture (from Fox et al., 2000).
in Fig. 2 on cheese ripening and quality are not known precisely and many of the factors are interactive. It is possible to apply the principles of Hazard Analysis Critical Control Point (HACCP) analysis to cheese production and it is hoped that this article may stimulate efforts to apply HACCP principles to cheesemaking. The principal items in Fig. 1 and 2 will be discussed individually in the following sections.
Milk Supply It is well recognized that the quality of the milk supply has a major impact on the quality and consistency of the resultant cheese. Three aspects of quality must be considered: microbiological, enzymatic and chemical.
Species Breed Stage of lactation Plane of nutrition Animal health
Composition cell c o u n t
\ RAW MILK I--~-[ Public health and safety ', ' ,
On-farm jr hygiene Transport
1temperature
Thermization I Standardization-~[-~Pasteurization
I CREESE MILK I
In-factory L time
natural creaming centrifugal milk powder UF
Selection criteria Acid production Phage sensitivity Ripening properties Rate of lysis
Colour CaCI2 GDL Starter culture Secondary/adjunct culture
Gel strength subjective/objective assessment I - cheese yield - curd ~omposition
I COA~~~~jn;t I ~Cut ~
~
I C U R D S /IW H_E Y
Cheese quality
-
Acidification-
~
~ - cheese yield - curd composition
. -
{- curd syneresis curd composition - acidification retention of coagulant
Cook~ - syneresis 1 curd composition I curd structure ~, retention of coagulantI solubilization of CCP .J
Composition - casein - fat - calcium - pH - enzymes
Agitate
~ c u r d syneresis curd composiiton
Whey I CUI I D S I - Acidification Dehydration Texturization? Salting? Moulding Pressing? i
I s ~ ~--Rennet Milk enzyme StarterenzYmes Secondary culture Adventitious ~__
I
I- Salting ~- Special secondary cultures -
Coating/packaging~
, ~RIPENING I ~ //~ I - Composition - Temperature - Humidity - Time
Proteolysis Lipolysis j - Glycolysis - Secondary changes J MATURE CHEESE I -
pH ~Fat
~
Cl
)
Flavour
Texture ~ "
Functional properties
Figure 2 Interaction of compositional and technological factors that affect the quality of cheese. 585
586
Factors that Affect the Quality of Cheese
Microbiological
Three aspects of the microbiology of cheese milk are of interest/concern: 9 Public health 9 Off-flavours and spoilage 9 Desirable bacteria
Public health aspects As a product of animal origin, milk may become contaminated with pathogenic micro-organisms, and this, clearly, is of great concern. Previously, the principal pathogens of concern in milk were Mycobacterium boris and Brucella abortus, but in developed dairying countries today, these pathogens have been largely or entirely eliminated from the dairy herd. Today, a wide range of pathogens are of concern, mainly Listeria monocytogenes, enterotoxigenic strains of E. coli, e.g., E. coli O157 H7, Shigella, Erwinia, Campylobacter, Staphlyococcus, Salmonella spp. and M. paratuberculosis. Many of these micro-organisms do not grow in milk which simply acts as a vector. In cheese, these pathogens die off under the rather hostile conditions in well-made cheese which has a relatively low pH (5.3), a relatively high salt content (5-10% sah-inmoisture; S/M) and perhaps bacteriocins. For this reason, Public Health Authorities in many counties require that cheese made from raw milk be aged for 60 days, although this practice may not be fully effective. Alternatively, cheese must be made from pasteurized milk or the cheese itself must be pasteurized, as in processed cheese. Cheese, the pH of which does not decrease at the desired rate or to the desired extent during manufacture (e.g., due to bacteriophage infection or contamination with antibiotics) or if the pH increases substantially during ripening, e.g., surface mould- or smear-ripened cheese, are most at risk. High-moisture, fast-ripening cheeses are at a greater risk of harbouring pathogens than low-moisture, slowripening varieties. A considerable amount of cheese is made from raw milk, especially in France, Germany and southern European countries, but there is increasing pressure in northern Europe and North America to produce all cheese from pasteurized milk. There have been very few reported outbreaks of food poisoning from the consumption of raw milk hard, long-ripened cheese varieties. In all outbreaks for which adequate data are available, mitigating circumstances, e.g., lack of a starter culture or poor starter activity, have also been involved. Clearly, raw milk cheese is safe if adequate precautions are taken. However, it should be remembered that most raw milk cheeses are high-cooked, hard
or extra-hard v a r i e t i e s - many of these cheeses, e.g., Parmigiano Reggiano, Grana Padano, Swiss Emmental and Gruyere de Comte, are cooked at --~55 ~ the cooked curds are transferred while hot to moulds with a capacity of 20-60 kg curd and consequently, the curds cool s l o w l y - most of these 'raw milk' cheeses are in fact pasteurized, as indicated by a negative alkaline phosphatase test. High-moisture, raw-milk cheeses are of more concern but most of these have a low initial pH (4.6) and appear to be safe. It is probably significant that raw-milk cheeses are made on a small/very small scale from very fresh milk from healthy cows. For further discussion on pathogens in cheese, see 'Growth and Survival of Microbial Pathogens in Cheese', Volume 1. It is unlikely that it will be possible to produce raw milk guaranteed free of pathogenic bacteria. However, milk with very low numbers of pathogens can be produced from healthy animals and any pathogens that do enter milk can be: 9 killed (pasteurization or novel alternatives); 9 removed (bactofugation or microfihration (MF); see 'Application of Membrane Separation Technology to Cheese Production', Volume 1); 9 prevented from growing or killed, e.g., by use of low pH, selected bacteriocin-producing starters. To date, efforts to remove pathogens from cheese milk have concentrated on adequate pasteurization. There is ongoing research on alternative methods and it is likely that work in this area will continue and probably expand. Off-flavours and spoilage A second beneficial effect of pasteurization is the killing of spoilage micro-organisms, e.g., coliforms, pseudomonads and yeasts. In countries with a developed dairy industry, the quality of the milk supply has improved markedly during the past 30 y e a r s - total bacterial counts (TBC) are now usually <20 000 cfu/ml ex-farm. The TBC probably increases during transport to and storage at the factory, but growth can be minimized by thermization (65 ~ 3< 15 s) of the milk on reception at the factory, which is a standard practice in some countries. The presence of Clostridium tyrobutyricum poses special problems. Although many cheeses are made from raw milk, quantitatively, most cheese is made from milk pasteurized at or close to 72 ~ X 15 s. If produced from goodquality raw milk and if subsequently handled under hygienic conditions, pasteurized milk should have a very low TBC (a few hundred cfu/ml) and therefore
Factors that Affect the Quality of Cheese
represents a very uniform raw material from a microbiological viewpoint. Some alternatives to pasteurization are likely to become industrially significant.
Desirable hTdigenous bacteria Some of the adventitious micro-organisms in raw milk, especially the non-starter lactic acid bacteria (NSLAB), probably contribute positively to cheese f l a v o u r - it is generally accepted that the flavour of raw-milk cheese is more intense, although more variable, than that of pasteurized milk cheese. Although the reason(s) for the differences in flavour between raw and pasteurized milk cheese has not been explained to everybody's satisfaction and is still under investigation, there is broad support for the view that adventitious NSLAB are mainly responsible. These indigenous LAB are killed by pasteurization; attempts are being made to replace them through the use of adjunct cultures (see also 'Secondary and Adjunct Cultures', 'The Microbiology of Cheese Ripening' and 'Raw Mild Cheeses', Volume 1). While raw milk may be acceptable for cheesemaking in small-scale, manually operated factories, the cheesemaking quality of even good-quality raw milk is too variable to be used successfully in very large, highly automated factories such as those involved in the manufacture of Cheddar, Gouda or Mozzarella (Pizza) cheese. In these cases, it is highly probable that pasteurized milk will continue to be used, with selected adjunct cultures added to simulate the superior quality of raw-milk cheese. Alternatives to pasteurization
There are a number of alternatives to pasteurization for the decontamination of cheese milk: 9 Thermization- heat treatment at a sub-pasteurization temperature, e.g., 65 ~ • 15 s; thermization is intended to reduce the microflora of raw milk and extend the period for which it may be held at the factory without a risk of spoilage. Although thermization does not meet the requirements for pasteurization from the public health viewpoint, it is fairly widely used for cheese milk and in combination with other hurdles, e.g., cooking of the cheese curd, low pH, high S/M, is probably adequate to render good-quality milk free of pathogens and foodpoisoning bacteria; 9 Microfiltration; 9 Bactofugation, which may be used as a general, quite efficient method for the removal of bacteria and spores from milk but which is not widely used;
587
9 The LPO-H202-SCN- system, which is not used commercially for cheese milk; 9 Addition of H202, which is not used in developed dairying countries; 9 Pre-ripening milk with bacteriocin-producing cultures. Microfiltration is a very efficient method for the removal of bacteria from m i l k - >99.9% of the bacteria can be removed, i.e., it is more efficient than pasteurization. Microfiltration has the added advantage over pasteurization that no heat damage is caused to the whey proteins. In addition to killing bacteria, MF removes somatic cells, which are significant sources of enzymes and are generally believed to have a negative effect on cheese quality. However, the quality of Cheddar (McSweeney et al., 1993) and Gruyere de Comte (Beuvier et al., 1997) cheese made from pasteurized or MF milk was similar and different from that of raw-milk cheese, suggesting that NSLAB, rather than other factors, the distinguishing factors. At present, MF is not used commercially for the general removal of bacteria from cheese milk and it is not acceptable as an alternative to pasteurization from a public health viewpoint because it cannot guarantee milk free from pathogens. However, MF is provisionally accepted in France as a suitable alternative to HTST pasteurization for the decontamination of beverage milk (see 'Application of Membrane Separation Technology to Cheese Production', Volume 1). If no problems are encountered, it is likely that MF will become an acceptable alternative to pasteurization of cheese milk. A serious microbiological problem in many/most cheeses arises from the growth of Clostridium tyrobutyricum in the cheese during ripening which catabolizes lactic acid to butyric acid and H2, with the production of off-flavours and late gas blowing. Cheddar-type cheese is an exception owing to the rapid decrease in pH and the rapid increase in S/M to an inhibitory level. The principal sources of CI. tyrobutyricurn are soil and silage; the feeding of silage to cows, the milk of which is to be used for cheesemaking, is prohibited in Switzerland and parts of France. However, in most countries, the outgrowth of Cl. tyrobutyricurn is prevented by the use of NaNO3 or lysozyme or the spores are removed by bactofugation or ME Indigenous enzymes
Milk contains about 60 indigenous enzymes (see Fox et al., 2003), the significance of which for cheese quality has not yet been researched adequately. Several indigenous enzymes have the potential to affect cheese quality, especially lipoprotein lipase (LPL), proteinase(s), acid
588
Factors that Affect the Quality of Cheese
and alkaline phosphatase, xanthine oxidase (XO) and perhaps sulphydryl oxidase (SO), lactoperoxidase and y-glutamyl transpeptidase. Some of these enzymes are active in milk prior to cheesemaking and adversely affect the yield and/or quality of cheese. Many of the indigenous milk enzymes survive HTST pasteurization (72 ~ X 15 s) and at least some, e.g., plasmin, acid phosphatase and XO, are active during cheese ripening. Lipoprotein lipase has the potential to cause significant lipolysis in milk and the resulting fatty acids are concentrated in the cheese curd where they may cause hydrolytic rancidity, especially in mild-flavoured cheese. Normally, LPL has low activity in milk where it is separated from its triglygeride substrates by the milk fat globule membrane (MFGM). However, the MFGM is quite susceptible to damage due to rough handling of milk, leading to activation of the LPL, and rancidity. Plasmin, the principal indigenous proteinase in milk, hydrolyses O t s l - , Ors2- and, especially, [3-casein, producing y- and k-caseins, some of the proteose peptones (PP) and other peptides. Plasmin activity reduces cheese yield because the PPs are not incorporated into the cheese curd and is reported to damage the quality of the rennet-induced coagulum. Plasmin activity increases with advancing lactation, age of cow and mastitis and its action may result in a weak coagulum with poor syneresis properties- the consequences are reduced yield of cheese and a high moisture content. The formation of y-caseins in cheese during ripening clearly indicates that plasmin is active in cheese - it is mainly responsible for the hydrolysis of [3-casein in lowcooked cheeses and for total primary proteolysis in highcooked varieties in which the rennet is extensively or totally inactivated (see 'Proteolysis in Cheese during Ripening', Volume 1,). Studies on the effect of the plasmin inhibitor, 6-amino hexanoic acid, have shown that plasmin makes a significant, but not essential, contribution to proteolysis in Cheddar cheese; addition of exogenous plasmin accelerates proteolysis. A study of the effect of plasmin inhibitors on the ripening of high-cooked cheeses should be interesting. Milk contains at least four times as much plasminogen as plasmin. The indigenous plasminogen may be activated by added plasminogen activators (there are some indigenous plasminogen activators in milk), which accelerate proteolysis in cheese (Barrett et al., 1999). Dephosphorylation by acid phosphatase may be responsible for some of the variability in the level of phosphorylation exhibited by caseins but incomplete phosphorylation may also be responsible. The significance of the variability in the level of phosphorylation in cheese quality is unknown but dephosphorylation of casein-derived peptides in cheese may be significant. It is claimed that alkaline phosphatase is active in Grana
Padano cheese during ripening and is responsible for the dephosphorylation of casein phosphopeptides, which is significant for proteolysis. Alkaline phosphatase is inactivated by HTST pasteurization. Acid phosphatase survives pasteurization and since it is concentrated in the MFGM, it is concentrated in cheese curd. Many of the small water-soluble peptides produced by primary proteolysis are phosphopeptides and are partially dephosphorylated during ripening, either by milk acid phosphatase or by bacterial phosphatases. Since phosphopeptides are resistant to the action of proteinases and peptidases, dephosphorylation by phosphatase action is an important pre-requisite for secondary proteolysis in cheese. However, objective studies on the significance of phosphatase activity in cheese ripening and quality have not been reported. Xanthine oxidase reduces nitrate to nitrite which is needed for anti-clostridial activity Eventually, all nitrate and nitrite are decomposed to N2, probably by XO. Degradation of nitrate is important since it may react with amino acids to form carcinogenic nitrosamines. Sulphydryl oxidase oxidizes sulphydryl groups to disulphides: 2RSH ~
RmSmS--R
Several small sulphydryl compounds, e.g., H2S, methanethiol, dimethyl sulphide and dimethyl disulphide, are important for cheese flavour. Sulphydryl oxidase oxidizes and protects the sulphydryl groups of proteins and this may affect the redox potential (Eh) of cheese and the stability of thiol compounds and hence the quality and stability of cheese. Somatic cells are an important source of enzymes, especially proteinases, in milk. Somatic cell count (SCC) is negatively correlated with cheese yield and quality; an SCC < 300 000 per ml is recommended. As discussed above, the somatic cells in milk can be removed by ME which should, therefore, improve cheese quality and reduce variability. Although precise information is lacking, it is not likely that indigenous enzymes in milk are a major cause of variability in cheese quality; some of these enzymes contribute to cheese ripening and may contribute to the superior quality of raw milk cheese, a possibility that warrants investigation. Chemical composition
The chemical composition of milk, especially the concentrations of casein, fat, calcium and pH, has a major influence on several aspects of cheese manufacture, especially rennet coagulability, gel strength, curd syneresis, and hence cheese composition and cheese yield. When
Factors that Affect the Quality of Cheese
seasonal milk production is practised, as in New Zealand, Ireland and Australia, milk composition varies widely but there is some variability even with random calving patterns mainly due to nutritional factors. The constituents of milk are influenced by several factors, including species, breed, individuality, nutritional status, health and stage of lactation of the producing animal. Owing to major compositional abnormalities, milk from cows in the very early or late stages of lactation and those suffering from mastitis should not be used for cheesemaking. Somatic cell (leucocyte) count is a useful index of quality. Some genetic polymorphs of the milk proteins improve cheese yield and quality and there is an increasing interest in breeding for these. The milk should be free of chemical taints and free fatty acids, which cause offflavours in the cheese, and antibiotics which inhibit bacterial cultures. There is considerable information on the effects of protein content, [Ca] and pH on the various renneting parameters of milk in model systems and quite an amount of information on their effects in cheesemaking experiments. However, there is less information on the effects on the simultaneous change in two or more of these factors, especially in actual cheesemaking experiments. Studies on the interactive effects of these and other compositional factors on the cheesemaking properties of milk and on the quality of the resulting cheese are warranted. It is possible to reduce, but not eliminate, the variability in the principal milk constituents by standardizing the concentrations of fat and casein, not just the ratio (protein content can be standardized by adding UF retentate), the pH (using gluconic acid-8-1actone) and calcium content (by adding CaC12). Standardization of milk composition
589
9 adding cream; 9 adding milk powder or uhrafihration retentate; such additions also increase the total solids content of the milk and hence cheese yield. Calcium
Calcium plays a critical role in the coagulation of milk and in the subsequent processing of the coagulum; it is common practice to add Cat12 (e.g., 0.01%) to cheese milk, i.e., 40 mg Ca/1 milk. This is small in comparison with the indigenous concentration of Ca in milk, 1200 mg/l. Addition of 40 mg/l Ca to milk increases the concentrations of soluble, colloidal and ionized Ca and reduces the pH of milk, all of which have positive effects on the various renneting parameters (see 'Rennet-induced Coagulation of Milk' and 'The Syneresis of Rennet-coagulated Curd', Volume 1).
pH The pH of milk is a critical factor in cheesemaking (see 'Rennet-induced Coagulation of Milk' and 'The Syneresis of Rennet-coagulated Curd', Volume 1). The addition of 1-2% starter culture to milk reduces the pH of the milk immediately by about 0.1 unit. Starter concentrates (direct-to-vat starters; DVS), which are now used widely, especially for small and medium factories, have no immediate, direct acidifying effect. Previously, it was standard practice to add the starter to the cheese milk 30-60 min before rennet addition. During this period, the starter began to grow and produce acid, a process, referred to as 'ripening', which served a number of functions: 9 It allowed the starter bacteria to enter the exponential phase of growth and hence to be highly active during cheesemaking; this is not necessary with modern high-quality starters. 9 The lower pH favoured rennet action and gel formation.
Fat and casein
Different cheese varieties have a defined fat-in-dry matter (FDM) content, in effect, a certain fat-to-protein ratio, and this situation has legal status in the 'Standards of Identity' for many varieties. While the moisture content of cheese, and hence the levels of fat and protein, is determined mainly by the manufacturing protocol (including size of curd particles, pH, cook temperature, agitation, pressing), the fat to protein ratio is determined mainly by the fat to casein ratio in the cheese milk. Depending on the ratio required, it can be modified by: 9 removing some fat by gravity creaming, as practised in the manufacture of Parmigiano Reggiano, or by centrifugation; 9 adding skim milk;
However, the practice increases the risk of bacteriophage infection of the starter; phage become distributed throughout liquid milk but after it has coagulated, the phage cannot move through the coagulum and hence can infect only those cells in the immediate vicinity of an infected cell. This practice has been discontinued for most cheese varieties. The pH of milk on reception at the dairy is higher today than it was previously, owing to improved hygiene during milking and the widespread use of refrigeration at the farm and factory. In the absence of acid production by contaminating bacteria, the pH of milk increases slightly during storage due to the loss of CO2 to the atmosphere. The natural pH of milk is ---6.7 but varies somewhat (e.g., it increases in late lactation and during mastitic infection).
590
Factors that Affect the Quality of Cheese
To off-set these variations in pH and to reduce it as an alternative to ripening, the pre-acidification of milk by 0.1-0.2 pH units is recommended, either through the use of the acidogen, gluconic acid-8-1actone, or by limited growth of a lactic acid starter, followed by pasteurization (referred to as pre-maturation). Pre-acidification improves the uniformity of rennet-coagulated milk gels, which is reflected in the production of cheese of more uniform quality. Pre-acidification through the growth of a starter culture, which is fairly widespread in France, would appear to pre-dispose the system to the growth of phage, which are not killed by pasteurization, and undesirable bacteria. O
O
ii
C
I
il
C~OH
~
I
HC---OH
HC~OH
I
HO--C--H
H20
I
HO~C~H
HC--OH
I H~OH
HC
HC--OH
I
I I
CH2OH Gluconic acid-&lactone
I
CH2OH Gluconic acid
In addition to variations in gross composition, there are numerous minor differences and variations which are not easily removed or standardized. Some of the more significant of these are due to inter-species differences. Although the vast majority of cheese, worldwide, is produced from cows' milk, sheep's and goats' milk are very significant for cheese in southern Europe and in the Middle E a s t - many world-famous cheeses are made from sheep's milk, e.g., Manchego, Feta, Roquefort and the Italian Pecorino varieties. Sheep's milk is used mainly for the production of cheese and yoghurt. Goats' milk or mixtures of sheep's and goats' milk are also used widely for cheese production (see 'Cheeses Made from Ewes' and Goats' Milk', Volume 2). Buffalo milk is used for the production of cheese in southern Italy (Mozzarella di Buffala) and especially in Egypt. Bovine, ovine, caprine and buffalo milk differ from each other in many respects: concentrations of fat, protein, many inorganic salts, enzymes, fatty acid profile, types and proportions of caseins. These differences cannot be changed and are reflected in the quality of the cheese produced from these milks. The most obvious difference arises from differences in fatty a c i d s - ovine and caprine milk-fat have considerably higher concentrations of hexanoic, octanoic and decanoic acids and branched, medium-chain fatty acids than bovine milkfat and these are readily apparent as differences in the
flavour of the cheese. Ovine and caprine caseins are considerably different from the bovine caseins. It is likely that there are differences in the peptide and amino acid profiles of cheese produced from bovine, ovine, caprine or buffalo milk and that these affect the flavour of the cheeses. A notable example of this is that the rennet from the thistle, Cynara cardunculus, produces very satisfactory cheese from sheep's milk, e.g., Sera de Estrala (see 'Cheeses Made from Ewes' and Goats' Milk', Volume 2), but this rennet produces very bitter cheese from cows' milk. Cows transfer high levels of carotenoids from their feed to their milk or meat whereas sheep, goats and buffalo do not. Consequently cheese, butter and other dairy products produced from cows' milk are much more yellow than those made from milk of the other species and may be unattractive to certain consumers. The yellow colour can be destroyed by bleaching (H202 or benzoyl peroxide) or masked (by chlorophyll or TiO2). The milk of all species contains the same range of enzymes but at different levels; the significance of these differences is not known. Several sapid compounds are transferred from the animal's feed to its milk and affect the flavour of cheese made therefrom. There is a widely held view that the milk of cows fed on unimproved pasture yields better and more distinctive cheese than that from cows fed a more homogeneous diet. Further work in this area is warranted.
Coagulant
(rennet)
The key and characteristic step in the manufacture of rennet-coagulated cheeses is the coagulation of milk through the limited proteolytic action of certain proteinases, called rennets. Several proteinases can coagulate milk but only a few are suitable for cheese production. Traditionally, rennets were extracts of the gastric tissue of calves, kids or lambs, in which the principal enzyme is chymosin. Owing to increased production of cheese, concomitant with a reduced supply of calfs' stomachs, the supply of calf rennet has been inadequate for many years. This led to a search for 'rennet substitutes', four of which are commercially successful: bovine pepsin and proteinases from the fungi, R. meihei, R. pusillus and C. parasitica (porcine pepsin was used previously to a limited extent). All successful rennet substitutes are aspartyl (acid) proteinases. The gene for calf chymosin has been cloned in several micro-organisms and the product (referred to as fermentation-produced chymosin; FPC) is now widely used for cheesemaking in many, but not all, countries. The extract of the thistle, Cynara cardunculus,
Factors that Affect the Quality of Cheese
is used in the manufacture of certain cheeses in Portugal and Spain. The active enzyme is cardosin, which is an acid proteinase (which are rare in plants). Thistle rennet is unsuitable for cheesemaking in general. The mechanism by which chymosin coagulates milk is well established at the molecular level (see Fox and McSweeney, 1997; Fox et al., 2000; Hyslop, 2003; 'Rennetinduced Coagulation of Milk', Volume 1). Chymosin specifically hydrolyses K-casein, the protein responsible for the stability of the casein micelles, at Phel05--Metl06, releasing the hydrophilic C-terminal peptide (referred to as the glyco- or caseino-macropeptide) and destabilizing the micelles. All commercial rennet substitutes hydrolyse the Phel05--Metl06 bond except C. parasitica proteinase, which hydrolyses Serl04--Phel05. The rennetaltered micelles coagulate in the presence of Ca 2+ at a temperature >20 ~ (in cheesemaking, at 30-35 ~ It has been proposed (Andreeva et al., 1992; Gustchina et al., 1996) that chymosin normally exists in an inactive conformation but is activated when the substrate binds in the active site cleft of the enzyme. It has been suggested that the sequence--H.P.H.P.HB (residues 98-102 of K-casein) is responsible for this conformational change. This sequence occurs in the K-casein of cow, goat, sheep and buffalo but not in the K-casein of the mare, camel, pig, rat or human, in which the corresponding sequence is ..HPRPH.., ..RPRPR.., ..RPRPH.., ..HPINR. and ..RPNLH.., respectively (Iametti et al., 2001; Martin etal., 2003). Therefore, one would expect that calf chymosin would not coagulate the milk of the mare, camel, pig, human or rat. There have been few studies on the coagulation of non-bovine milk by calf chymosin. The commercial use of calf rennet in cheesemaking from sheep, goat or buffalo milk indicates that calf chymosin can hydrolyse the K-casein in these milks, as expected from the above hypothesis. Calf chymosin can also coagulate porcine milk (Fox, 1975b); in fact, porcine milk is coagulated by calf rennet at 4 ~ whereas bovine milk is not, due to the nature of the non-enzymatic secondary phase. Some investigators have reported that camel milk is not coagulated by calf rennet but Farah (1993) reported that it is coagulated slowly to a weak gel. The status of mares' milk with respect to K-casein remained unclear until very recently. Ochirkhuyag et al. (2000) reported that equine milk does not contain K-casein and that the micelle-stabilizing function is played by [3-casein; however, Malacarne et al. (2002) reported that it contains a low level (<7%) of K-casein which has been isolated and sequenced (Egito et al., 2001, 2002; Iametti et al., 2001). Equine K-casein is hydrolysed at Phe97--Ile98 (Egito et al., 2001) (which correspond to Phel05--Metl06 of bovine K-casein). However, equine milk does not appear (Fox, unpublished) to
591
be coagulated by calf chymosin or Fromase (R. miehei proteinase) at pH 6.6 (normal pH --~7.0 or higher). Studies on the coagulability of equine milk by different rennets and under different conditions appears to be warranted. We are not aware of studies on the coagulation of human or rat milk by calf chymosin. Studies on the action of chymosins from various species on the caseins from a range of species would be interesting. For a proteinase to be successful as a rennet substitute, two characteristics are important: 9 Specific hydrolysis of K-casein at or close to Phel05BMetl06; if other bonds in any of the caseins are hydrolysed, the resulting peptides may be lost in the whey, causing a reduction in cheese yield. 9 Its general proteolytic specificity during cheese ripening must be low and similar to that of chymosin (see below). It is generally accepted that calf chymosin produces the best quality cheese. An adequate supply of chymosin from genetically engineered micro-organisms is now available (although its use is not permitted in all countries) and therefore rennet quality should not be a cause of variability in cheese quality. In the presence of Ca 2+, the rennet-altered micelles in bovine milk coagulate to form a gel at a temperature >20 ~ this is referred to as the secondary phase of rennet coagulation. Renneted bovine milk does not coagulate <---18 ~ above which coagulation has a Q10 of 16. The very high temperature dependence of the secondary phase of coagulation has not been explained fully. Presumably, hydrophobic interactions are involved; perhaps the temperature-dependent dissociation of [3-casein from the casein micelles is a contributory factor. The temperature dependence of the coagulation of rennet-altered micelles is reduced by reducing the pH and increasing the [Ca 2+ ] or casein concentration, e.g., by UE As mentioned above, porcine milk is coagulated by rennet at 4 ~ The reason(s) for the difference between bovine and porcine milk in this regard has not been explained. In spite of many studies on the mechanism of coagulation of rennet-altered casein micelles and kinetics thereof, a generally applicable model of the phenomenon has not been developed. The subject is comprehensively reviewed in 'Rennet-induced Coagulation of Milk', Volume 1. Further research on various aspects of the secondary phase of rennet coagulation of, and the effect of low temperatures on, bovine milk and that of other species appear warranted. Some of the added rennet is retained in cheese curd. The amount retained varies with rennet type, cook temperature and pH at draining; these variables
592
Factors that Affect the Quality of Cheese
should be standardized if cheese of consistent quality is to be produced. The proportion of rennet retained in the curd is proportional to its moisture content, reflecting the presence of rennet mainly in the aqueous phase of cheese. However, everything else being equal, more chymosin than other rennets is retained in the curd, suggesting that chymosin is adsorbed more strongly on the caseins. The amount of chymosin and pepsin retained in low-cooked cheeses increases strongly as the pH of the curds-whey is reduced. In the case of chymosin, this is due to increased adsorption, for unknown reasons, onto the casein; for pepsin, which is very pH-sensitive (irreversibly denatured at pH 7), greater stability at a lower pH is also a major factor. Surprisingly, pH has no effect on the retention of fungal rennets, a lower proportion of which is retained in the curd than chymosin (see Fox and McSweeney, 1997). Obviously, cook temperature has a major effect on the level of residual rennet in the c u r d - chymosin and bovine pepsin are extensively or totally denatured in high-cooked cheese, e.g., Parmigiano Reggiano or Emmental; porcine pepsin is extensively denatured even in low-cooked cheeses due to its sensitivity to pH > 6.5. Low-cooked, low-pH, highmoisture cheese, e.g., Camembert, retains ---30% of the added chymosin activity; Cheddar retains "--6% and Emmental "--0%. Everything else being equal, increased retention of the coagulant in cheese curd results in greater initial hydrolysis of C~sl-casein; however, the significance of this variable on the flavour and texture of cheese has not been studied thoroughly. It has been suggested that the activity of chymosin in cheese curd is the limiting factor in cheese ripening; however, excessive rennet activity leads to bitterness. Proteolysis in cheese during ripening is discussed later; there have been relatively few studies on the significance of chymosin activity to cheese quality, an aspect which appears to warrant further research. Considering the importance of proteolysis in the ripening and quality of cheese and the significance of the coagulant thereto, studies on various factors that affect the retention of the coagulant in cheese curd appear warranted, e.g., 9 the adsorption of chymosin on casein micelles and the apparent lack of adsorption of fungal proteinases; 9 stability of various rennets under various conditions of temperature, pH and other factors.
Starter The second key reaction in cheesemaking is acidificat i o n - the pH of all rennet-coagulated cheeses should decrease to a value in the range 4.6-5.2 within a few
days of manufacture, or in some varieties, at the end of curd manufacture (5-6 h). Acidification at the appropriate rate and time is an essential and characteristic feature of cheesemaking- it is, in fact, a sine qua non. Among the important consequences of acidification are: 9 activity of the coagulant; 9 survival and retention of coagulant in the curd; 9 firmness of the coagulum, which affects the loss of fat and protein in the whey on cutting and hence reduces the yield of cheese; 9 syneresis of the curds and hence the composition of the cheese; 9 solubilization of colloidal calcium phosphate (CCP), which has a major effect on the texture, meltability and stretchability of the cheese; 9 inhibition of the growth of undesirable bacteria, most importantly pathogenic and food poisoning bacteria; 9 the activity of various enzymes in the cheese during ripening and consequently the rate of ripening and the quality of the cheese. Originally, acidification was due to the production of lactic acid from lactose by adventitious LAB. Acidification of some cheese varieties still depends on the activity of the adventitious microflora but most cheeses now are acidified using selected LAB added to the cheesemilk as a culture (starter). The idea of using starter cultures was introduced in ---1870 in Denmark. The cultures used today in cheesemaking can be divided into two groups: 1. Mesophilic - with an optimum growth temperature of "- 28 ~ 2. Thermophilic- which grow optimally at --~42 ~ Mesophilic cultures are used for cheese curds which are cooked at a temperature <40 ~ while cheeses in which thermophilic cultures are used are cooked at 50-55 ~ Mesophilic cultures contain strains of Lactococcus lactis subsp, lactis and/or Lc. lactis subsp, cremoris. Starters used for some cheeses, e.g., Gouda, Edam, Danbo, also include citrate-utilising strains of Lc. lactis subsp, lactis and/or Leuconostoc subsp., the principal function of which is the production of CO2 and certain flavour compounds. Thermophilic cultures contain species of thermophilic lactobacilli, e.g., Lb. helveticus and Lb. delbreuckii subsp, bulgaricus or Lb. delbreuckii subsp, lactis, alone or with Streptococcus thermophilus. It is possible to simulate the acid-producing function of the starter LAB by using acid or acidogen (usually GDL). Fresh acid-curd cheese (e.g., Cottage,
Factors that Affect the Quality of Cheese
Quarg, Cream) of satisfactory quality may be produced by direct acidification and some is produced commercially. Some rennet-coagulated cheeses are also produced by direct acidification, usually when the flavour is very mild or masked by other components or is less important than physico-chemical functional properties; examples include Feta-type and Mozzarella-type cheeses. Chemically acidified Mozzarella has better, more consistent and stable functionality than the biologically acidified product. However, most ripened rennet-coagulated, chemically acidified cheese does not develop a flavour typical of the variety. Possible explanations for this situation are: 9 the high redox potential (Eh) of chemically acidified cheese (c. 150 mV compared with c. - 4 5 0 mV for biologically acidified cheese); 9 a high concentration of lactose which may result in a high count of NSLAB. Both mesophilic and thermophilic cultures may by mixed-strain or defined-strain. Mixed-strain cultures contain an unknown number of strains of the same or different species. Several such systems were in use 20-50 years ago - the cultures were selected based on cheesemaking characteristics and various propagation procedures were used. These cultures were capable of producing very good quality cheese but they were susceptible to phage infection because many of the strains in the culture were sensitive to the same phage. The maintenance and propogation of mixedstrain cultures is laborious and not very reproducible. With the objective of improving resistance to phage, work commenced in New Zealand in the 1930s on the development of defined-strain cultures. The principal criterion for selecting strains for these cultures was phage unrelatedness, i.e., phage which infects one strain does not affect other strains in the culture; while a phage infection may kill off one strain, the other strains grow normally. Other essential criteria are acid-producing ability and strain compatibility; the ability to produce good quality cheese was assessed from experience and undesirable strains were removed from the culture. Originally, blends of 5-6 strains were usually used but blends of 2-3 strains are more common today. The definedstrain approach was introduced initially for Cheddar cheese and is now very widely used in New Zealand, Australia, Ireland and the USA. Although a different approach was used in the Netherlands to select definedstrain cultures for Gouda cheese, the essential outcome was similar. Defined-strain thermophilic cultures are now used also but less widely than mesophilic cultures. The use of defined-strain, phage-unrelated cultures has greatly reduced the risk of phage infection but to
593
reduce the risk even further, various techniques have been introduced to improve starter activity and ensure cheese quality. For discussions on starter technology, see Cogan and Hill (1993), Cogan and Accolas (1996) and 'Starter Cultures: General Aspects', Volume 1. There have been very considerable advances in the genetics of lactococci during the last 10 years and the complete sequence of the genome is known (see 'Starter Cultures: General Aspects' and 'Starter Cultures: Genetics', Volume 1). The genes for many of the important cheesemaking characteristics of lactococci, e.g., lactose metabolism, proteolysis and phage resistance, are carried on plasmids and hence are easily manipulated. Many genetically engineered strains of Lactococcus have been constructed but are not used in practice. However, lactococci can be genetically modified by natural mating (conjugation) and such genetically modified strains are being used commercially. The principal limitation with engineering superior starters is the lack of knowledge on the key enzymes in cheese ripening. The availability of the complete chromosome sequence of Lc. lactis (see 'Starter Cultures: General Aspects' and 'Starter Cultures: Genetics', Volume 1) opens up new avenues for research on cheese starter cultures. For example, five potential or rudimentary prophages were identified, suggesting that starter cultures are the ultimate source of phage. Examination of the chromosome for putitive proteinases, peptidases and lipases, especially those with unique activities, should be useful. Since 1999, the chromosome of 20 other LAB and other cheese-related bacteria have been or are being sequenced. These include other strains of Lactococcus, Lb. delbruckii, Lb. helveticus, Sc. thermophilus and B. linens. Comparative genomics of these different bacteria should be useful in delineating the differences that occur between them. However, the gene sequences are of little value unless the protein products (enzymes) are produced. Identifying how to turn on those genes which encode enzymes with potential cheese ripening properties, but which are normally not expressed, could be rewarding in studies on flavour development in cheese. Defined-strain cultures give very reproducible results in terms of acid production and overall cheese quality. However, the flavour of the cheese is considered to be rather bland, probably because of a lack of microbial diversity, both in the starter and also in the modern milk supply, which, if pasteurized, is essentially sterile. Attempts to overcome the lack of flavour will be discussed later. Traditionally, cheese starters were produced at the cheese factory from mother-cultures obtained from a culture supplier; this is still the usual practice in larger factories. However, the proper maintenance of starters is
594
Factors that Affect the Quality of Cheese
technically demanding and expensive. Consequently, starter concentrates, referred to as direct-to-vat starter (DVS) or direct vat inoculum (DVI), produced by culture suppliers have become quite widespread among small to medium cheese factories or as a back-up starter system for larger manufacturers. Another starter system warrants mention, i.e., artisanal or natural cultures. These cultures are produced in-house by the cheesemaker, who incubates some warm whey under conditions that select bacteria with desirable cheesemaking characteristics. Today, such cultures are usually used for high-cooked cheeses- hot whey, perhaps at ---55 ~ is transferred to insulated containers in which it cools slowly; these conditions are selective for thermophilic bacteria and by the time the whey has cooled sufficiently to allow mesophilic bacteria to grow, the pH has become inhibitory. These cultures are very complex and their composition is not known, certainly at the strain level, and probably not at the species level. Although the primary function of the starter culture is to produce acid at the appropriate rate and time, the starter bacteria or their enzymes also play an essential role during cheese r i p e n i n g - a typical and desirable flavour does not develop in starter-free cheese and many flavour defects, e.g., bitterness and fruitiness, are related to characteristics of the starter. The microbiology and biochemistry of cheese ripening will be discussed in a later section. In view of the significance of the starter LAB, both for the acidification and ripening of cheese curd, it is not surprising that the starter LAB have been the subject of extensive research since the very early days of microbiology and consequently, LAB are now very well characterized at the cellular, molecular and genetic Cultures: genetics levels (see 'Starter Cultures: General Aspects', 'Starter Cultures: Genetics' and 'Starter Cultures: Bacteriophage', Volume 1). Research on these very industrially important bacteria will very probably continue for the immediate future. Among the areas likely to attract attention are: 9 Characterization of Lactobacillus and Streptococcus, which at present are less well characterized than
Lactococcus; 9 Further work on the protection of LAB against phage; 9 Selection and improvement of starter strains, through genetic engineering techniques with respect to acid production and especially ripening attributes; 9 Selection of LAB strains with probiotic properties for cheese production; 9 The existing trend towards DVI starters will probably continue; success will depend on economic factors;
It is likely that the lack of diversity caused by the use of such highly defined starter systems will be offset by the use of adjunct starters, mainly Lactobacillus spp., but possibly also Streptococcus spp., and perhaps Enterococcus, for some varieties.
Post-Coagulation Operations A rennet-coagulated milk gel is quite stable if left undisturbed but if cut or broken, it synereses strongly, thereby creating the possibility of removing water and concentrating fat and protein. When the coagulum has reached the desired degree of firmness, usually 30-60 min after the addition of rennet, the gel is ready for further processing. The firmness of the gel at cutting should be optimized so as to reduce the loss of fat and protein from the curd particles into the whey. If the coagulum is too soft, extensive shattering will occur with high losses of fat and protein in the whey. If the coagulum is too firm, the coagulum may be difficult to cut using the usual equipment; it may run before the cutting knives and shattering may occur. Excessively firm curd is particularly problematic when using UF retentate. A uniform gel firmness at cutting also results in curd particles of more uniform size, leading to a cheese curd of more uniform composition and ultimately in cheese of more uniform quality. Traditionally, the point at which the gel was considered to be ready for cutting was determined subjectively by the cheese-maker but several devices are now available which permit objective assessment of gel firmness (see Fox et al., 2000; 'General Aspects of Cheese Technology', Volume 2). Some of these devices can be used to activate the cutting knives. The size of the curd particles affects the extent of syneresis- the coagulum for low-moisture cheeses is cut into small pieces while the gel for high-moisture varieties is cut into large pieces or is not cut at all but is scooped directly into moulds. Fat globules are lost from the cut surfaces; hence, finely cutting the coagulum increases the fat loss. If it were possible to achieve the same degree of syneresis by other means, it may be possible to increase cheese yield by using a larger cut. Contrary to what one might first think, the fat lost from the cut surfaces is mainly in large globules because large globules are more exposed on the cut surface on cutting. Would a very low degree of homogenization improve fat retention? The curd can be sedimented from a rennet-induced milk gel by a low gravitational force. Would it be technically feasible to coagulate milk by some form of centrifuge and to prepare curd of the desired moisture content by centrifuging the coagulum at an appropriate centrifugal force? It might be possible to eliminate
Factors that Affect the Quality of Cheese
cooking of the curds and all variability associated with syneresis of the curds. Once the gel has been cut, the pieces begin to synerese and expel whey. In addition to the size of the curd pieces, the rate and extent of syneresis are affected by several factors through which the cheesemaker can control the composition of cheese. The syneresis of rennet-induced milk gels is discussed in 'The Syneresis of Rennet-coagulated Curd', Volume 1. As discussed below, the composition of cheese has a major influence on the microbiology and biochemistry of cheese ripening and ultimately on the quality of the cheese. Syneresis is promoted by: 9 9 9 9
increasing the temperature (cooking); reducing the pH; vigorous stirring during cooking; removing some or most of the whey and continuing to stir.
The relative importance of these factors depends on the v a r i e t y - syneresis of some varieties depends mainly on cook temperature, e.g., Emmental, Parmigiano Reggiano and Cheddar, while others depend mainly on pH, e.g., Camembert. The cook temperature ranges from ---30 ~ (no cooking) to 55 ~ the extent of syneresis is related directly to the cook temperature. For reproducible cheese composition and quality, it is critical to control the rate and extent of syneresis. The cook temperature, pH, curd particle size and the extent of agitation are characteristic of the variety and were probably introduced/applied emperically long ago by artisanal cheesemakers. It seems reasonable to suggest that the manufacture of many varieties could be improved by changing the combination of the above parameters; research in this area appears to be warranted. The composition of the curd affects syneresis and this is a major reason for standardizing the composition of the cheese milk. The most important compositional factors are: 9 Fat, which has a negative effect, i.e., a high fat content causes poor syneresis, because fat is essentially an inert filler in the gel. 9 Protein, increasing the protein content, up to a certain value, promotes syneresis, but if the protein content is too high, the gel is too firm and synereses poorly. 9 Calcium, like other aspects of renneting, the extent of syneresis is directly affected by the concentration of calcium. 9 pH, syneresis is promoted by decreasing pH. The syneresis of rennet-induced milk gels has not yet been fully described at the molecular level. While
595
the effects of processing parameters (cutting, cooking, stirring, etc.) on syneresis are fairly well described, the effects of compositional factors, such as amount and type of casein, genetic polymorphs, casein micelle structure and size, effect of plasmin and other proteinases, fat globule size and integrity, milk salts, pre-treatments of cheese milk (e.g., heat treatment, high pressure treatment), have not been studied in detail. The deficiency is explained partly by a lack of good and appropriate analytical methods for the measurement of syneresis (see 'The Syneresis of Rennetcoagulated Curd', Volume 1). When the moisture content (as assessed subjectively by the cheesemaker based on evaluation of the texture of the curd) and the pH of the curd have reached desired values, the curds are separated from the whey and subjected to one of several treatments designed to regulate the composition, and in some cases the texture, of the curd. These include: 9 washing of the curd to reduce its lactose content (and thereby control its final pH) and perhaps to increase its moisture content, which occurs on washing with cold water, e.g., Monterey Jack, washedcurd Cheddar, low-fat cheeses; 9 replacing some of the whey by warm water, e.g., Gouda; this practice was probably used initially as a means of cooking the curds on farms lacking jacketed cheese vats and the ability to generate steam, but it has become a standard method for reducing the lactose content and acidity of the curd for some varieties; 9 cheddaring the curd, e.g., Cheddar, Mozzarella (Pizza cheese), which affects the texture of the curd but its main effect is probably to allow the pH of the curds to fall and the CCP to dissolve, thereby affecting texture; 9 kneading and stretching, as used for pasta-filata varieties, mainly to give the cheese a fibrous texture which affects the meltability and stretchability, i.e., the functionality, of the cheese; 9 moulding, applied to all varieties to give a characteristic shape and size, which are not simply cosmetic but are significant for the ripening of the cheese, e.g., if smear-ripened cheeses are too large, the surface will become over-ripe while the core is still immature; on the other hand, Swiss-type cheeses must be large so as to retain a substantial portion of the CO2 necessary for eye development; some cheeses have a characteristic open texture, with many mechanical openings, e.g., Samsoe, Havarti and blue varieties; in the latter, the openings are necessary for good mould development throughout the cheese; 9 pressing, applied to semi-hard and hard cheeses with the objective of removing some moisture but mainly
596 Factorsthat Affect the Quality of Cheese to consolidate the cheese mass which is important for texture and the retention of gas for eye development; 9 salting, which is discussed further below. In large-scale factories, all the above operations are mechanized and/or automated which improves the consistency of the product if executed properly; however, it is not possible for the cheesemaker to make ad hoc adjustments if the process is not progressing as planned. Very large cheese vats (e.g., 30 000 1 capacity) are used in large modern factories producing Cheddar, Gouda or Mozzarella, and possibly other varieties. The use of such large vats eliminates certain causes of variation in cheese but introduces others. About 30 min is required to empty these large vats and separate the curds from the whey; as a result, the curds at the start of draining differ from those at the end in several respects, e.g., moisture content (due to extra syneresis), pH and calcium content. These differences are probably reflected in the quality of the cheese but definitive studies have not been reported.
Salting Salting, one of the classical methods for food preservation, operates by reducing the water activity, aw, of the product. Most, probably all, cheeses are salted by one of four methods: 9 mixing dry salt witti milled or chipped curd, e.g., for Cheddar-type cheese; 9 brine salting of the moulded/pressed cheese; NaC1 diffuses into the cheese in response to the difference in osmotic pressure between the brine and the aqueous phase of the cheese; 9 surface application of dry salt to the surface of pressed cheese, e.g., Blue cheeses; 9 salting of cheese m i l k - for a few varieties, e.g., Domiati, a substantial amount of salt is added to the milk before renneting, traditionally, to control the microflora of the milk. There is a substantial literature on the technology, physics and significance of salting, which has been reviewed by Guinee and Fox (1993), Fox et al. (2000) and in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. The principal effects of salt in cheese are: 9 A major inhibitory and selective effect on the microflora. 9 A significant effect on the activity of many enzymes. 9 Through its effects on the microflora and enzymes, salt has a major indirect effect on the ripening, flavour and quality of cheese.
9 A direct effect on flavour. 9 An excessive dietary intake of NaC1 has several undesirable effects, e.g., hypertension and osteoporosis. Although cheese makes a relatively small contribution to dietary NaC1 intake, there is an economic incentive to reduce the NaC1 content of cheese, which may adversely affect its quality, or partially replace it by KC1. The significance of salt, and especially the uniformity of salt concentration, on the quality of cheese is well recognized. The physics of salt diffusion in cheese, the effect on various micro-organisms and the technology of salting are well known. Thus, it should be possible to achieve a very reproducible level of salt in cheese. However, this is not always achieved in practice and variations in salt concentration are probably a significant but avoidable cause of variations in cheese quality.
Use of UF in Cheese Production Ultrafiltration (UF) has many applications in cheese technology, as discussed in 'Application of Membrane Separation Technology to Cheese Production', Volume 1. Some problems with cheese quality persist but the potential for UF in cheese technology is great and research in its application will continue.
Ripening Fresh rennet-coagulated cheese curd is suitable for consumption and a little is consumed, e.g., Burgos cheese, but most is ripened (matured) for a period ranging from "--3 weeks (e.g., Mozzarella/Pizza cheese) to 2 or more years (e.g., Parmigiano-Reggiano, extramature Cheddar). During ripening, the characteristic flavour, texture, appearance and functionality develop along lines pre-determined by the microbiology and composition of the curd, as established during the manufacturing stage. However, the cheesemaker can influence the rate and, to some extent, the pattern of ripening by controlling the temperature and, for some varieties, the humidity of the environment. Many cheeses develop a characteristic microflora (bacteria, yeasts, moulds) during ripening and this microflora has a major effect on the sensory qualities of the cheese. Traditionally, this secondary microflora was adventitious, acquired from the milk and/or environment, and the growth of certain, desirable, contaminating micro-organisms was promoted by selecting certain environmental conditions such as pH, temperature, humidity, oxygen concentration, salt concentration and moisture level. However, the adventitious microflora was likely to be variable, leading to inconsistencies in
Factors that Affect the Quality of Cheese
cheese quality. In modern cheese technology, the adventitious microflora is replaced by selected secondary cultures, although adventitious micro-organisms may still grow, and even dominate in some cases. The principal secondary micro-organisms are: 9 Mesophilic lactobacilli, probably in all varieties but especially in internal bacterially ripened varieties, e.g., Cheddar and Gouda. Traditionally, these NSLAB were adventitious, probably variable and uncontrolled; today, it is becoming increasingly common to inoculate cheese milk with selected NSLAB (see below and 'Secondary and Adjunct Cultures' and 'The Microbiology of Cheese Ripening', Volume 1). 9 Propionic acid bacteria, characteristic of Swiss-type cheese (see 'Metabolism of Residual Lactose and of Lactate and Citrate', Volume 1 and 'Cheese With Propionic Acid Fermentation', Volume 2). 9 Penicillium carnemberti and P. roqueforti in surfacemould and blue-mould varieties, respectively. The inoculation of some mould-ripened cheeses with mould spores is adventitious but increasingly, the cheeses are inoculated with selected strains. The metabolic activity of the mould dominates the ripening, and hence the quality, of these cheeses (see 'Surface Mould-ripened Cheeses' and 'Blue Cheese', Volume 2). Therefore, ensuring the optimum growth of the mould is paramount. 9 Coryneform bacteria, e.g., Brevibacterium, Arthrobacter and Corynebacterium spp. are the characteristic microflora of surface smear-ripened cheeses and are responsible for their characteristic appearance, aroma and taste. Traditionally, surface smear-ripened cheeses acquired their secondary microflora from the environment and from older cheeses via smearing. However, for hygienic and consistency reasons, it is becoming increasingly common to inoculate the surface of the cheeses with selected coryneform bacteria (see 'Secondary and Adjunct Cultures', Volume 1 and 'Bacterial Surface-ripened Cheeses', Volume 2). Several species of yeast, e.g., Debaryomycos hansenii and Yarrowia lipolytica, have been isolated from cheese. These yeasts are adventitious contaminants on many varieties; since they are aerobic and acid-tolerant, they grow mainly on the surface of all cheeses but their growth on many varieties is prevented through packaging or rind formation. The growth of yeasts is essential on surface smear-ripened cheeses because they catabolize lactic acid, increase the pH of the curd and enable the corynebacteria, which cannot grow at < p H 5.8, to grow. Apart from their significance in the deacidification of smear-ripened cheese, their precise contribution to ripening has not been quantified. However, since yeasts are metabolically active, it is likely that their
597
contribution is considerable. With the objective of improving the consistency of cheeses in which they are a significant part of the microflora, the inoculation of such cheeses with selected strains of yeasts is becoming increasingly common. Geotricurn candidum is part of the adventitious surface microflora of many cheeses. A further variable through which the cheesemaker can influence the pattern of ripening and the quality of the final cheese is by preventing the loss of moisture from the cheese surface by appropriate packaging (rindless cheese) or by controlling its loss to form a rind. Scientific work on the significance of cheese packaging on cheese quality is lacking. The main focus has been on the prevention of mould growth on the surface and the loss of cheese yield. Undoubtedly, the changing composition of cheese (through evaporation of moisture) and the loss of gases and probably other volatile compounds affect cheese microflora and enzyme activity and consequently cheese quality. Although the technological advantages accruing from the packaging of cheese are great and perhaps cannot be off-set by other factors, a scientific comparison of various aspects of rindless and rinded cheese, e.g., Cheddar, may be interesting. Cheese ripening involves a very complex set of biological, biochemical and chemical reactions which can be classified into four groups: 9 Glycolysis and the catabolism of lactic and citric acids. 9 Lipolysis and the catabolism and modification of fatty acids. 9 Proteolysis and catabolism of amino acids. 9 Interactions between the products of the previous reactions. These reactions are catalysed by living micro-organisms or enzymes derived from four or five sources: 9 9 9 9 9
Milk Coagulant Primary starter Secondary starter (for most varieties) Adventitious microflora
The biochemistry of cheese ripening has been studied quite intensely in recent years and an extensive literature has accumulated, which has been the subject of several reviews, including Fox and McSweeney (1997), Fox etal. (1996a, 2000), Fox and Wallace (1997), and 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1. The principal features of glycolysis, lipolysis and proteolysis are summarized in Figs 3, 4 and 5, respectively. The principal
598
Factors that Affect the Quality of Cheese
Lactose
phflus
Glu. + Gal
Lc. lactis Lc. cremoris Sc. thermop~il(Sct~ate Q.~wiss L-Lactate Mould and ~ Surface smear
/
Propionibacterium
Propionate, acetate, CO 2
Cheddar Dutch
002, H20 NSLAB D,L-Lactate
Pediococcus
= Acetate
(lactobacilli) Figure 3 Summaryof lactose metabolism in cheese.
products of the various reactions have been characterized and include numerous large, medium and small peptides, amino acids, amines, ammonia, fatty acids and partial glycerides, other organic acids, aldehydes and ketones, thiol compounds, CO2, H2, hydrocarbons, pyrazines and furanones. Several hundred sapid and aromatic compounds have been isolated from several cheese varieties and identified (see 'Sensory Character of Cheese and its Evaluation' and 'Instrumental Techniques', Volume 1). The spectrum of these compounds is generally similar between the varieties that have been studied in detail but the varieties differ with respect to their concentration and proportions. There are four aspects to the quality of cheese; the relative importance of these depends on the variety and application of the cheese: 9 9 9 9
Appearance Flavour (aroma and taste) Texture Functionality The appearance of cheese includes such features as"
9 Depth and uniformity of colour 9 Presence or absence of mechanical opening or eyes due to gas production 9 Presence or absence of mould
Triglycerides~
f
8-Hyaroxyacios
ct3S%cohos
n-Fatty acid~
kactones
Methylketones Thioesters ~ Esters Figure 4 Summaryof lipolysis in cheese.
Usually, the appearance of cheese is the only attribute by which the purchaser can assess the quality of cheese and hence is of the utmost importance. Today, it is unlikely that cheese produced by large manufacturers and sold through reputable outlets will be defective in appearance although the appearance of artisanal cheese may vary. For cheese consumed as a table cheese, flavour is probably its most important quality attribute although flavour and texture are strongly interactive. The flavour of cheese is due to a subtle balance between several hundred compounds. It has been the subject of research since the beginning of the twentieth century, especially since the development of gas chromatography (GC) in the 1950s and the interfacing
Factors that Affect the Quality of Cheese
Casein Rennet=
Ca-pard-casein
Rennet, Plasmin
599
= Large p e p t i d e s ~ Lactococcal CEP \
~Oligopeptidases
/ Aminopeptidases
Small p e p t i d e s / / [Aminopeptidases~
} Dipeptidases
Deaminases j Amino acids Carbonyls ~ / ] ~C--C and C--S '~ ~..~J | ~ Lyases Esters~ Acid~" .~.~/~ CO.~ Decarb~ Alcohols /k
~.,~ Amines "s ~o~-Ketoacid
Various products, including sulphur compounds
F Aminoacid o~-Ketoacid
Figure 5 Summary of proteolysisand amino acid catabolism in cheese. of GC with mass spectrometry (MS). Considerable progress has been made on the characterization of cheese flavour by instrumental methods (see 'Instrumental Techniques', Volume 1). However, commercially, cheese quality is usually assessed by subjective sensory evaluation (see 'Sensory Character of Cheese and its Evaluation', Volume 1). It is still not possible to describe completely the flavour of cheese, especially more highly flavoured varieties. More research is needed in this area and it is very likely that it will continue. An objective method for grading cheese would be very useful. Analysis of cheese volatiles by GC-MS is probably the best approach at present but the present instruments are not capable of handling large numbers of samples and are too expensive. The electronic nose appears promising but considerably more work is required. The texture of cheese is important, both directly and indirectly. It is important directly because such important functional attributes as sliceability, grateability, crumbliness and eye development are, in fact, related to texture. To the consumer, texture is an indirect index of cheese flavour and general quality. In spite of its undeniable importance, the texture of cheese has received much less research attention than cheese flavour although as described in 'Rheology and Texture of Cheese', Volume 1, the texture of cheese can be described quite accurately by certain rheological parameters. The influence of various compositional parameters and the changes that occur during ripening have been described in theological terms. It should be possible to develop some of the present rheometers based on the principle of compression, penetration or cutting to take whole cheese
samples and hence could be used in a factory enviro n m e n t - it would appear that research in this area is warranted. Further work on the molecular basis of cheese texture and rheological properties is also required. All cheeses are expected to exhibit certain physicochemical or functional properties when cold, e.g., sliceability or crumbliness or grateability or when heated, e.g., meltability or stretchability. Most cheese (~70% in the USA) is used as an ingredient in other foods, either domestically or in a factory context. The flexibility and ease of use of cheese as a food component or ingredient are among its main attributes and are discussed in detail in 'Cheese as an Ingredient', Volume 2. Being able to provide the user, especially the industrial user, of cheese with a product with the correct functionality is a challenge to the cheese manufacturer. Considerable progress has been made but further work is required. The quality of cheese is determined in the first instance by the composition of the curd both directly and indirectly by its influence on the various ripening agents. The significance of principal ripening agents is described in the following sections.
Indigenous Enzymes Milk contains 60 indigenous enzymes (see Fox et al., 2003). Some of these enzymes, including LPL, acid phosphatase, alkaline phosphatase and XO, are located on the fat globule membrane, some, including plasmin, are adsorbed on the casein micelles while others are in the serum (whey) phase. Since the fat globules and casein micelles are concentrated in the cheese
600
Factors that Affect the Quality of Cheese
curd, cheese is enriched with many enzymes. Several enzymes in milk are quite heat stable and survive HTST pasteurization. The most significant of the indigenous enzymes as far as cheese ripening is concerned are: 9 Plasmin, which fully survives HTST pasteurization (in fact, its activity is increased due to inactivation of inhibitors of plasmin and plasminogen activators) and makes a significant contribution to primary proteolysis, especially in high-cooked cheeses in which the coagulant is extensively or totally inactivated; it is mainly responsible for the hydrolysis of [3-casein in most cheese varieties. Although the level of plasmin activity in milk is variable, it is unlikely that this variability causes significant variations in cheese quality although it may affect cheese yield and composition (through retarded syneresis) and functionality which is strongly influenced by the integrity of the casein network. 9 Cathepsin D, an acid lysozomal proteinase with a specificity similar to chymosin, occurs mainly in the serum phase of milk and therefore most of it is lost in the whey; furthermore, it is relatively heat labile. Therefore, there is probably little cathepsin D activity in cheese and, in any case, it is probably overshadowed by the much greater activity of chymosin. 9 Lipoprotein lipase, which is extensively or totally inactivated by HTST pasteurization, has little or no impact on the quality of cheese made from pasteurized milk although it probably contributes to lipolysis in raw-milk cheese; it is associated with the casein micelles and is incorporated into cheese curd. 9 Acid phosphatase is probably active in cheese but its significance has not been established. Many of the small peptides produced from casein by chymosin or plasmin are phosphopeptides and are partially dephosphorylated during ripening, indicating the action of an acid phosphatase. Work is needed to establish the contributions and significance of the indigenous and bacterial acid phosphatases in cheeses. One of the important nutritional features of cheese is its anticariogenic property due to the Ca-binding properties of the caseins and casein phosphopeptides (see 'Nutritional Aspects of Cheese', Volume 1). In this regard, the activity of acid phosphatase may be negative but dephosphorylation is necessary to enable the further degradation of phosphopeptides which may be important for flavour and texture development. 9 Xanthine oxidase reduces nitrate to nitrite, which is the active agent against clostridia and coliforms in cheese and contributes to the eventual disappearance of NO3- and NO2-: this is important from a toxicological viewpoint since nitrate may lead to the formation of nitrosamines.
It is possible that other indigenous milk enzymes are active in cheese during ripening and affect its quality; their activity may contribute to the differences in flavour between raw and pasteurized milk cheese but definitive studies have not been reported. Research on the significance of indigenous milk enzymes in cheese quality is warranted. Coagulant
The coagulant is mainly responsible for primary proteolysis in low-cooked cheese and for the desirable textural changes during the early stages of ripening. The peptides normally produced by rennet are too large to affect flavour but they serve as substrates for microbial proteinases and peptidases which produce small peptides and amino acids which contribute to background flavour. Amino acids serve as substrates for various catabolic reactions, the products of which (amines, NH3, acids, carbonyls, sulphur compounds) are major sapid compounds in cheese (see 'Proteolysis in Cheese during Ripening' and 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1). However, excessive rennet action or incorrect specificity may lead to bitterness. The C-terminal region of [3-casein is very hydrophobic and peptides released from this region, which contains the primary chymosin cleavage sites on [3-casein, are very bitter. The hydrolysis of [3-casein by chymosin is inhibited by NaC1 and it is not normally hydrolysed in Cheddar but is hydrolysed to some extent in surface-salted cheese, in which the concentration of NaC1 is sub-inhibitory for a considerable period. Chymosin is generally regarded as the best rennet; since there is now an unlimited supply of fermentationproduced chymosin, there is no excuse for rennetrelated problems in cheese. The natural function of chymosin is to coagulate milk in the stomach of the neonate, delay its discharge into the small intestine and thereby increase the efficiency of digestion. Chymosin is the most efficient milk coagulant known but it was not intended for cheesemaking although it is the best for this task also. However, it is likely that through genetic engineering, chymosin could be modified to improve its cheese-ripening properties, i.e., to increase its action on certain peptide bonds to yield desirable peptides or reduce it on other bonds to avoid defects such as bitterness. Some mutant chymosins have been produced (see 'Rennets: General and Molecular Aspects', Volume 1) but we are not aware of their use in cheesemaking trials. C. parasitica proteinase is much more active on [3-casein in cheese than chymosin, pepsins or Rhizomucor proteinases but it does not cause bitterness. Probably, it
Factors that Affect the Quality of Cheese
preferentially hydrolyses in the N-terminal region of [3-casein, which is hydrophilic. Characterization of the specificity of C. parasitica proteinase on caseins in cheese and in model systems is warranted. Perhaps a combination of chymosin and C. parasitica proteinase might produce cheese with interesting characteristics. The texture and functionality of cheese are affected strongly by even a low level of proteolysis, e.g., the stretchability of biologically acidified Mozzarella deteriorates after --~2 weeks at 4 ~ due to proteolysis. Thus, it is important to control the level and activity of rennet in cheese. As discussed earlier, the amount of rennet retained in cheese curd is affected by the moisture content of the cheese, the pH and temperature of cooking; activity during ripening is affected by pH, moisture, S/M and temperature. Starter
In addition to its essential role in the production of acid in the manufacture of cheese curd, the starter LAB also play a key role in cheese ripening. Experiments with chemically acidified Cheddar and Gouda cheese have shown that the starter is essential for normal flavour development. Even inoculation of chemically acidified cheese with NSLAB, which reached 108 cfu/g, did not produce good-flavoured cheese (Lynch et al., 1997). The starter LAB reduce the redox potential (Eh) of cheese curd to about - 2 5 0 mV and this may be of major significance in flavour development. There is very little information on the development and significance of Eh in cheese, possibly because it is very difficult to measure the Eh of cheese accurately; research in this area would appear to be highly desirable. The precise route and mechanism for flavour generation by the starter have not been elucidated fully but considerable progress has been made (see Fox and Wallace, 1997; McSweeney and Sousa, 2000; Yvon and Rijen, 2001; 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening' and 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1, and Volume 2, on the principal families of cheese). The growth of the starter ceases at the end of the curd manufacturing stage and the cells die at a rate characteristic of the strain. Therefore, it seems reasonable to conclude that starter enzymes rather than viable cells are involved in ripening and that differences in the enzyme profile of starter strains affect cheese quality. Modern defined-strain starters produce acid very reproducibly and, if properly selected and managed, show good resistance to phage. Lactococcus strains
601
have been selected mainly on the basis of acid-producing ability, phage resistance and compatibility. Based on pilot-scale studies and commercial experience, strains that produce unsatisfactory, especially bitter, cheese have been identified and excluded from commercial usage. However, there are substantial and recognizable differences in flavour quality and intensity between cheeses made using different defined-strain cultures, which presumably reflect differences in the enzyme profile of the component strains; systematic studies on strains are lacking. This probably reflects the lack of information on precisely what attributes of a starter are desirable from a flavour-generating viewpoint. Studies on genetically engineered strains that superproduce proteinase and/or the general aminopeptidase, PepN, showed that, although proteolysis was accelerated, cheese quality was not improved. Lactococcus strains lacking one or more peptidases in various combinations are available; mutants lacking any one or two peptidases can grow in milk but strains lacking three or more peptidases cannot. Published studies on the use of these peptidase-deficient mutants in cheese are lacking. Since all lactococcal enzymes, except the cell wallassociated proteinase, are intracellular and since the cells do not grow in cheese, the cells must lyse before these enzymes can participate in ripening; therefore, the rate of lysis of Lactococcus strains is being studied with the objective of selecting strains with improved cheesemaking properties. A bacteriocin-producing strain of Lactococcus has been isolated which accelerates lysis of the starter and consequently accelerates cheese ripening (Morgan et al., 1995). The enzymes of the glycolytic and proteolytic systems of Lactococcus are very well studied at the molecular, biochemical and genetic levels (see 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening' and 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1). Although less thoroughly studied, the phosphatase and lipase/esterase of a few strains have been isolated and characterized. During the past few years, there has been increasing awareness of the significance of the amino acid-catabolizing enzymes of the starter and a number of reports on lactococcal deaminases, transaminases, decarboxylases and lyases have been published (see McSweeney and Sousa, 2000; Yvon and Rijnen 2001; 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1). It is very likely that work on the lipolytic and amino acid-catabolizing enzymes of Lactococcus and their significance in cheese ripening and quality will be intensified in the immediate future.
602
Factors that Affect the Quality of Cheese
The enzymes of Lactobacillus and Streptococcus strains have been studied less thoroughly than those of Lactococcus but the systems of the three genera appear to be generally similar. Thermophilic lactobacilli lyse very rapidly and are more proteolytic than lactococci. Although considerable information is available on the individual enzymes of Lactococcus and to lesser extent of Lactobacillus, especially on the glycolytic and proteolytic systems, there have been few comparative studies on the different enzyme activities in starter strains. There have been even fewer studies on the relationship between different starter enzyme profiles and cheese quality. It would appear to be highly desirable that studies should be undertaken to relate cheese quality to the enzyme profile of natural starter strains or genetically engineered cultures. The availability of starter strains deficient in or over-producing one or more enzymes will facilitate such studies. It is very likely that the desirable cheesemaking properties of starters are due to a balance between certain, perhaps secondary, enzymatic activities, which have not yet been identified.
Non-Starter Lactic Acid Bacteria In addition to starter bacteria, cheese curd contains adventitious bacteria acquired from the milk and environment. When raw milk was used widely, it was probably the principal source of bacteria in cheese curd, especially since it was heavily contaminated during milking and was not cooled; counts >106 cfu/ml were common and 90% of the bacteria in milk are retained in the cheese curd. However, in the modern dairy industry, the microbial quality of the raw milk is very high and the milk is usually pasteurized; typically, the milk entering the cheese vat contains < 1000 cfu/ml. In large factories, the cheese is made in enclosed vats, with very little contamination from the environment. In all ripened cheeses, a NSLAB flora, which varies within and between cheeses made in the same plant, develops (see 'The Microbiology of Cheese Ripening', Volume 1). Cheese is quite a hostile environment due to: 9
9 9 9 9
a low pH a relatively high S/M anaerobic conditions lack of a fermentable carbohydrate the presence of bacteriocins and other inhibitory substances produced by the starter.
Hence, relatively few species of bacteria can grow, or even survive, in the centre of a well-made cheese. Recent studies have shown that the non-starter
microflora of Cheddar cheese is dominated by mesophilic lactobacilli, especially Lb. casei, Lb. paracasei, Lb. plantarum and Lb. curvatus. In cheese produced from good quality pasteurized milk in a modern plant these NSLAB typically grow from a few hundred per gram at the end of manufacture to 107-108/g within 2-3 months. Thus, while the population of starter LAB declines, the number of NSLAB increases and dominates the viable microflora of long-ripened cheese after 2-3 months (see 'The Microbiology of Cheese Ripening', Volume 1). Although less well studied that Cheddar, the NSLAB in Gouda, Emmental and Grana-type cheeses are also predominantly mesophilic lactobacilli and this is probably the normal situation in long-ripened cheeses. The significance of NSLAB for Cheddar and Dutch cheese quality is controversial. Many researchers consider their contribution to be negative (in the Netherlands, a maximum of 2 x 106 NSLAB/g is specified for Gouda). Although there are several studies on cheese with a controlled microflora, the ripening and quality of NSLAB-free cheese do not appear to have been compared with 'control' cheeses containing 'wild' NSLAB. Several comparative studies on cheese made under aseptic or non-aseptic conditions using Lactococcus starter alone or with selected Lactobacillus adjuncts indicate that inoculation of cheesemilk with selected strains of Lactobacillus improves cheese flavour and possibly accelerates ripening. However, a typical but mild flavour develops in Cheddar, Gouda and Emmental free of NSLAB, i.e., NSLAB do not appear to be essential for cheese ripening although they do affect the ripening pattern and cheese quality. Since the numbers and strains of NSLAB in cheese are uncontrolled, it is likely that they contribute to variability in cheese quality. Therefore, it appears worthwhile to determine what factors affect their growth. The number of NSLAB in Cheddar is strongly influenced by the rate at which the curd is cooled and subsequently ripened. Rapid cooling of the curd after moulding and pressing is the most effective way of retarding the growth of NSLAB, although they will grow eventually to ---107 cfu/g in cheese ripened at 4 ~ The growth of NSLAB can be prevented by ripening at ---1 ~ but all ripening reactions are retarded to an unacceptably slow rate. The growth of NSLAB does not appear to be influenced by the composition of cheese (moisture, salt or pH) within the ranges normally found in commercial cheese. Non-starter lactic acid bacteria grow mainly after the lactose in the cheese curd has been metabolized by residual starter activity. Although the growth substrates in cheese for Lactobacillus are not known, they
Factors that Affect the Quality of Cheese
can derive energy from the sugars of glycoproteins of the MFGM (Diggin, 1999). It is likely that available, suitable substrates are limiting (NSLAB normally plateau at ---107-108 cfu/g) and hence it might be possible to out-compete wild NSLAB by adding selected strains of Lactobacillus to cheese milk, thereby offering better control of the ripening process. Non-starter lactic acid bacteria may also be controlled by including a broad spectrum bacteriocin-producing strain in the starter culture (Fenlon et al., 1999).
Lactobacillus Species as Adjunct Cultures Cheddar and Cheddar-type cheeses do not have an intentional secondary microflora but there has been considerable interest in recent years in the use of an adjunct secondary culture (usually mesophilic lactobacilli) for the following reasons: 9 to intensify cheese flavour which is considered to have become too mild owing to the improved microbial quality of the cheese milk, pasteurization of the milk, the use of enclosed vats and other equipment (which reduce contamination from the environment) and the use of defined-strain starters, i.e., the cheese microflora have become too narrow; 9 to accelerate cheese ripening; the ripening of cheese, especially low-moisture, highly flavoured varieties, is a slow, and consequently an expensive, process. Various approaches to accelerate ripening have been assessed, including the use of mesophilic lactobacilli (see Fox et al., 1996b); 9 to give identifiable flavour characteristics to cheese produced by a particular manufacturer or sold by a particular retailer; 9 to improve the flavour of reduced-fat cheese, which generally lacks flavour; 9 inoculation of cheese with mesophilic lactobacilli which suppresses the growth of adventitious lacto-
pH 4.85-5.20
603
bacilli (NSLAB). Since the NSLAB are uncontrolled (they are the only really uncontrolled component of cheese), they probably contribute at least to some extent to variability. In this case, the adjunct lactobacilli need not contribute to the biochemistry of ripening, just suppress the growth of adventitious NSLAB. A considerable amount of research on the significance of mesophilic lactobacilli in cheese has been reported during the past 10 years and the results seem promising. Further research in this area is warranted. Thermophilic Lactobacillus spp. are more effective as adjuncts than mesophilic lactobacilli (Tobin, 1999), probably because they die rapidly in cheese, lyse and release intracellular enzymes. Both mesophilic and thermophilic lactobacilli and Sc. thermophilus are being used commercially as adjunct cultures for Cheddar cheese, and possibly for other varieties. Sc. thermophilus is used mainly to improve the phage resistance of the culture (since it is resistant to lactococcal phage) and to permit the use of a higher cook temperature, facilitating better control over cheese composition and hence ripening and quality.
Cheese Composition The quality of cheese is influenced by its composition, especially moisture content, NaC1 concentration (preferably expressed as % S/M), pH, moisture-in-non-fat substances (MNFS; essentially the ratio of protein to moisture) and % fat-in-dry matter. At least five studies (O'Connor, 1971; Gilles and Lawrence, 1973; Fox, 1975a; Pearce and Gilles, 1979; Lelievre and Gilles, 1982) have attempted to relate the quality of Cheddar cheese to its composition. These authors agree that moisture content, % S/M and pH are the key determinants of cheese quality but they disagree as to the relative importance of these parameters (see Fig. 6).
FDM 52-55% pH 4.95-5.10 FDM 52-55%
pH 4.95-5.15
Salt > 1.4%
Premium quality
MNFS 52-56% MNFS 50-57%
S/M 4.0-6.0% S/M 4.0--6.0%
Gilles and Lawrence (1973) Composition of cheeses was determined at 14 days and related to quality of mature Cheddar cheese.
Moisture < 38%
pH < 5.4
l Fox (1975a) Relationship between the quality and composition ._o!10-week-old Cheddar cheese.
MNFS 52-54%
S/M 4.2-5.2%
Pearce and Gilles (1979) Composition of cheeses was determined at 14 days and related to quality of Cheddar cheese.
Figure 6 Relationships between composition (determined at various stages during ripening) and the quality of mature Cheddar cheese (moisture-in-non-fat substances (MNFS) fat-in-dry-matter (FDM), and salt-in-moisture (S/M)).
604
Factors that Affect the Quality of Cheese
O'Connor (1971) found that the flavour, texture and total score of Cheddar were significantly correlated with % NaC1 and particularly with pH; moisture content had less effect on cheese quality. Salt content and pH were strongly correlated with each other, as were salt and moisture. Based on the results of a study on experimental and commercial cheeses in New Zealand, Gilles and Lawrence (1973) proposed a grading (selection) scheme which has since been applied commercially in New Zealand to young (14 day) Cheddar cheese. The standards prescribed for Premium grade cheese were: pH: 4.95-5.10; % S/M: 4.0-6.0%; MNFS: 52-56%; FDM: 52-55%. The corresponding values for First Grade cheeses were: 4.85-5.20%, 2.5-6%, 50-57% and 50-56%; young cheeses with a composition outside these ranges were considered unlikely to yield goodquality mature cheese. Quite wide ranges of FDM are acceptable; Lawrence and Gilles (1980) suggested that since relatively little lipolysis occurs in Cheddar cheese, fat content plays a minor role in determining cheese quality but if FDM is below about 48%, the cheese is noticeably more firm and less attractive in flavour. Pearce and Gilles (1979) reported that the grade of young (14-day-old) cheeses produced at the New Zealand Dairy Research Institute was most highly correlated with moisture content; the optimum compositional ranges were: MNFS: 52-54%; S/M: 4.2-5.2%; pH: 4.95-5.15. Fox (1975a,b) reported a weak correlation between grade and moisture, salt and pH for Irish Cheddar cheeses but a high percentage of cheeses with compositional extremes was downgraded, especially those with low salt (<1.4%), high moisture (>38%) or high pH (>5.4). Salt concentration seemed to exercise the strongest influence on cheese quality and the lowest percentage of down-graded cheeses can be expected in the salt range 1.6-1.8% (S/M: 4.0-4.9%); apart from the upper extremes, pH and moisture had little influence on quality in the sample studied. High salt levels tend to cause a curdy texture, probably due to insufficient proteolysis; a pasty body, often accompanied by off-flavours, is associated with low salt and high moisture levels. In the same study, the composition of extra-mature Cheddar cheeses was found to vary less and the mean moisture content was 1% lower than that of regular cheeses. A very extensive study of the relationship between the composition and quality of nearly 10 000 cheeses produced at five commercial New Zealand factories was reported by Lelievre and Gilles (1982). As in previous studies, considerable compositional variation was evident but was less for some factories than others. While the precise relationship between quality
and composition varied between plants, certain generalizations emerged: 9 within the compositional range suggested by Gilles and Lawrence (1973) for 'premium' quality cheese, composition does not have a decisive influence on grade, which decreases outside this range; 9 composition alone does not provide an exclusive basis for grading; 9 MNFS was again found to be the principal factor affecting quality; 9 within the recommended compositional bands, grades declined marginally as MNFS increased from 51 to 55% and increased slightly as S/M decreased from 6 to 4; pH had no consistent effect within the range 4.9-5.2 and FDM had no influence in the range 50-57%. 9 there were specific intra-plant relationships between grade and composition; therefore, each plant should determine the optimum compositional parameters pertinent to it. The results of the foregoing investigations indicate that high values for moisture and pH and a low salt content lead to flavour and textural defects. The desired ranges suggested by Gilles and Lawrence (1973) appear to be reasonable, at least for New Zealand conditions, but within the prescribed zones, composition is not a good predictor of Cheddar cheese quality. Presumably, several other factors, e.g., starter, NSLAB, activity of indigenous milk enzymes, relatively small variations in cheese composition and probably other unknown factors, influence cheese quality but become dominant only under conditions where the principal determinants, moisture, salt and pH, are within appropriate limits. Although the role of calcium concentration in cheese quality has received occasional mention, its significance has been largely overlooked. Lawrence and Gilles (1980) pointed out that the concentration of calcium in cheese curd determines the cheese matrix and, together with pH, indicates whether proper procedures were used to manufacture a specific cheese variety. As the pH decreases during cheese manufacture, CCP dissolves and is removed in the whey. The whey removed after cooking comprises 90-95% of the total whey expressed during cheesemaking and under normal conditions contains ---85% of the calcium and "--90% of the phosphorus lost from the cheese curd. Thus, the calcium content of cheese reflects the pH of the curd at whey drainage; there are strong correlations between the calcium content of cheese and the pH at 1 or 14 days and the amount of starter used (see Lawrence et al., 1984). Since the pH of cheese increases during ripening, the pH of mature
Factors that Affect the Quality of Cheese 605 cheese may be a poor index of the pH of the young cheese. Therefore, calcium concentration is probably a better record of the history of a cheese with respect to the rate of acidification than the final pH. Reduction in calcium phosphate concentration by excessively rapid acid development also reduces the buffering capacity of cheese and hence the pH of the curd will fall to a lower value for any particular level of acid production. No recent work on the level and significance of calcium in Cheddar cheese appears to be available. The calcium content of cheese has a major effect on its meltability and stretchability, e.g., pasta-filata cheese does not stretch well, or not at all, until the pH falls below ---5.4. Biologically acidified Mozzarella has poor stretchability and meltability immediately after manufacture but these properties improve during the early stages of ripening and are optimal after about 2-3 weeks; functionality deteriorates on continued ripening due to proteolysis. In contrast, directly acidified cheese is functional immediately after manufacture. The difference in behaviour is due to the lower calcium concentration in the directly acidified cheese owing to the faster decline in pH to ---5.6. Under such conditions, much of the CCP dissolves and is removed in the whey at drainage; the concentration of calcium per unit of protein, which is very important for cheese functionality, in biologically and chemically acidified Mozzarella cheese was 27.7 and 21.8 mg/g, respectively (Guinee et al., 2002). There is little published information on the relationships between composition and quality for other cheese varieties. However, it is very likely that similar factors affect the quality of all cheeses more or less to the same extent.
Ripening T e m p e r a t u r e Ripening temperature has a major influence on the rate of ripening and quality of cheese. Traditionally, cheese was ripened in caves or cellars at a relatively constant temperature. This practice is still widespread for some varieties but artificially refrigerated rooms are now used by large-scale manufacture. The ripening temperature is fairly characteristic of the variety, e.g., Cheddar, 6-8 ~ Gouda, 12-14 ~ ParmigianoReggiano, 18-20 ~ Emmental, 6 ~ for ---2 weeks, then at 22 ~ for 4-6 weeks to allow the propionic acid bacteria to grow rapidly and produce adequate CO2 for good eye development, then at ---4 ~ for several months to complete ripening; Camembert, 14 ~ for --~2 weeks to induce the growth of P. camemberti, then at 4 ~ for 2-4 weeks.
Ripening can be accelerated by increasing the ripening temperature but all reactions, desirable and undesirable, are accelerated and an unbalanced flavour or off-flavour may develop. Ripening at an elevated temperature is normally considered with the objective of accelerating ripening (see Fox et al., 1996b). Cheese flavour can probably be modified by manipulating temperature; however, this is rarely practised except for Swiss-type cheeses. The rate at which the curd is cooled after moulding has a major effect on the growth of starter LAB and NSLAB. The curds for most cheeses are moulded immediately after cooking and acidification occurs mainly in the moulds. Hence, the rate at which the curd cools in the moulds has a major effect on starter growth and rate of acid development, and is strongly affected by the size of the cheese and ambient temperature. The effect of cooling on starter growth is particularly noticeable for high-cooked cheeses, e.g., Swiss and Grana types. The thermophilic starters used for these cheeses do not grow at the cook temperature but begin to grow as the curd cools in the moulds. For consistency, it is important to control the ambient temperature. For Cheddar-type cheeses, acidification is almost complete at moulding. Traditionally, the moulded cheeses were pressed overnight at ambient temperature and the cheeses cooled close to ambient during this period, although ambient temperature probably varied significantly with season. In modern practice, the cheeses exit the Wincanton tower at ---36 ~ and are packaged and stacked on pallets (5 • 10 cheeses ---1 tonne) and transferred to ripening rooms. The cheeses at the centre of the pallet do not decrease to ambient (store) temperature for about 4 weeks and this causes considerable variation in the number and probably the type of NSLAB, and hence in the quality of the cheese. Many factories now cool the packaged cheese in a cooling tunnel overnight before stacking on pallets. If the cheese is cooled to <1 ~ and ripened at this temperature, the cheese will be free of NSLAB but the rate of ripening will be very slow. The humidity of the environment must be controlled, at 85-90% RH, for the ripening of many varieties, mainly those with a surface microflora, which will not grow if the cheese develops a rind. Traditionally, rind development was encouraged on internal bacterially ripened cheeses by reducing the RH slowly. The rind serves to protect the cheese against undesirable surface growth and the loss of moisture (weight). Today, many varieties, e.g., Cheddar and Gouda, are coated or wrapped in plastic, i.e., rindless cheese, to prevent weight loss and to protect the surface of the cheese against undesirable bacterial growth.
606
Factors that Affect the Quality of Cheese
Conclusions Through increased knowledge of the chemistry, biochemistry and microbiology of cheese, it should be possible to produce cheese of a very high quality consistently, although this is not always achieved owing to failure to control one or more of the key parameters that affect cheese composition and ripening. Milk is a variable raw material and although it is possible to eliminate major variations in the principal milk constituents, some variations persist. Variability in milk composition can also be compensated by manipulating some process parameters in the cheesemaking process. Most large factories operate on a strict time schedule and hence subtle manipulation of the process on an individual vat basis may not be possible. Therefore, strict control of milk composition and starter activity is critical. From a microbiological viewpoint, the milk supply to modern cheese factories is of very high quality and after pasteurization contains only a few hundred bacteria per ml. In modern factories where enclosed vats and other equipment is used, the level of contamination from the environment is very low; cheese curd containing <103 NSLAB/g at 1 day is normal. However, these adventitious NSLAB grow to c. 107-108 cfu/g and dominate the microflora of cheese after about 3 months. Since the adventitious NSLAB grow slowly in cheese, they are most significant in long-ripened cheese. Although the significance of the adventitious NSLAB in long-ripened cheese is unclear, it would appear to be desirable to control them, either by eliminating them or standardizing their number and type. In industrial-scale manufacture of cheese, it is not possible to eliminate NSLAB. It is possible to prevent their growth by ripening at ---1 ~ but the overall ripening process is also reduced to an unacceptable rate. Outcompeting indigenous NSLAB by an adjunct Lactobacillus culture, which does not have to contribute to ripening, is a possibility but this approach has not been investigated. Although it is now possible to avoid major defects in cheese produced using modern technology, further research on the biochemistry of cheese ripening is required to enable the process of cheese manufacture and ripening to be refined to an extent that will allow the consistent production of premium quality cheese. The key to successful cheesemaking is a good reliable starter, both from the viewpoint of reproducible acid production and subsequent ripening. If properly managed, modern starters are generally satisfactory and their performance is being improved progressively.
The use of starter adjuncts, usually mesophilic lactobacilli, for some varieties, especially Cheddar, is increasing, with the objective of intensifying and modifying flavour, accelerating ripening and perhaps controlling adventitious NSLAB and thus standardizing quality. Basically, cheesemaking is a relatively simple process, consisting of two phases: conversion of milk to cheese curd and transformation of the curd to mature cheese; both phases involve a number of steps. The key steps in curd manufacture are: acidification, coagulation, syneresis/dehydration and salting. With the knowledge currently available on the mechanism of these processes and the scale and quality of the cheesemaking equipment, it should be possible to produce cheese curd of consistently premium quality from chemical and microbiological viewpoints. Unfortunately, this is not the case in practice. Undoubtedly, variability in the composition and microflora of the milk contribute to the variability of cheese curd but there is variability in curd produced during the course of a single day from a single large batch of bulked milk using the same rennet and starter. One factor likely to be responsible for this variability is the time-lag in performing certain cheesemaking operations, e.g., it requires ---30 min to separate the curds and whey in the very large (->30 000 1) vats now used for Cheddar, Gouda or Mozzarella. This time-lag continues during later operations, e.g., cheddaring, milling, salting and pressing. The solution to this problem is the development of a continuous curd production system, such as the ALPMA system, but this is not used for hard cheeses. Work in this area appears warranted. The objective of cheesemaking is to consistently manufacture cheese with the desired, characteristic flavour, texture and functionality in the highest yield possible, as cheaply and as quickly possible. The closest we have come to achieving that objective is the production of enzyme-modified cheeses, which do not resemble closely the flavour, texture or functionality of any natural cheese but are used successfully to replace natural cheese in some applications (see 'Sensory Character of Cheese and its Evaluation', Volume 2). With improved knowledge of the biochemistry of cheese ripening, it may be possible to produce some of the milder, less complex cheese using the EMC a p p r o a c h - research in this area is warranted. Cheese ripening is a very complex biological, biochemical and chemical process which is determined and directed by the composition and microflora of the cheese curd; if these are reproducible and consistent, it should be possible to produce cheese of excellent quality consistently.
Factors that Affect the Quality of Cheese
References Andreeva, N., Dill, J. and Gilliland, G.L. (1992). Can enzymes adopt a self-inhibited form? Results of X-ray crystallographic studies on chymosin. Biochem. Biophys. Res. Commun. 184, 1074-1081. Barrett, EE, Kelly, A.L., McSweeney, P.L.H. and Fox, P.E (1999). Use of exogenous urokinase to accelerate proteolysis in Cheddar cheese during ripening. Int. Dairy J. 9, 421-427. Beuvier, E., Berthaud, K., Cegarra, S., Dasen, A., Pochet, S., Buchin, S. and Duboz, G. (1997). Ripening and quality of Swiss-type cheese made from raw, pasteurized or microfiltered milk. Int. Dairy J. 7, 311-323. Cogan, T.M. and Accolas, J.-P. (1996). Dairy Starter Cultures, VCH Publishers, Cambridge. Cogan, T.M. and Hill, C. (1993). Cheese starter cultures, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 193-255. Diggin, M.B. (1999). Potential Growth Substrates for Mesophilic and Thermophilic Lactobacillus spp. in Cheddar Cheese During Ripening. MSc Thesis, National University of Ireland, Cork. Egito, A.S., Giradet, J.-M., Miclo, L., Moele, D., Humbert, G. and Gaillard, J.-L. (2001). Susceptibility of equine K- and ~-caseins to hydrolysis by chymosin. Int. Dairy J. 11, 885-893. Egito, A.S., Miclo, L., Lopez, C., Adam, A., Giradet and Gafflard, J.-L. (2002). Separation and characterization of mares' milk of Otsl- , ~ - and K-caseins, ~/-casein-like, and proteose peptone component 5-like peptides. J. Dairy Sci. 85,697-706. Fenlon, M.A., Ryan, M.P., Guinee, T.P., Ross, R.P., Rea, M.C., Hill, C. and Harrington, D. (1999). Elevated temperature ripening of reduced fat Cheddar, made with or without lacticin 3147-producing starter culture.J. Dairy Sci., 82, 10-22. Farah, Z. (1993). Composition and characteristics of camel milk. J. Dairy Res. 60,603-626. Fox, P.E (1975a). Influence of cheese composition on quality. It. J. Agric. Res. 14, 33-42. Fox, P.E (1975b). Some physico-chemical properties of porcine milk. J. Dairy Res. 42, 43-56. Fox, P.E and McSweeney, RL.H. (1997). Rennets: their role in milk coagulation and cheese ripening, in, Microbiology and Biochemistry of Cheese and Fermented Milk, 2nd edn, B.A. Law, ed., Blackie Academic and Professional, London. pp. 1-49. Fox, P.E and Wallace, J.M. (1997). Formation of flavour compounds in cheese. Adv. AppI. Microbiol. 45, 17-85. Fox, P.E, O'Connor, T.P., McSweeney, P.L.H., Guinee, T.P. and O'Brien, N.M. (1996a). Cheese: physical, biochemical and nutritional aspects. Adv. Food Nutr. Res. 39, 163-328. Fox, P.E, Wallace, J.M., Morgan, S., Lynch, C.M., Niland, E.J. and Tobin, J. (1996b). Acceleration of cheese ripening. Antonie van Leeuwenhoek 70, 271-297. Fox, P.E, Guinee, T.P., Cogan, T.M. and McSweeney, P.L.H. (2000). Fundamentals of Cheese Science, Aspen Publishers, Inc., Gaithersburg, MD.
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Fox, RE, Olivecrona, T., Vilaro, S., Bengtsson-Olivecrona, G., Kelly, A.L., McSweeney, PL.H., Shakeel-Ur-Rehman, Fleming, C.M., Stepaniak, L., Gobbetti, M., Corsetti, A., Pruitt, K.N. and Farkye, N.Y. (2003). Indigenous enzymes in milk, in, Advanced Dairy Chemistry, Vol. 1, Proteins, 3rd edn, P.E Fox and P.L.H. McSweeney, eds., Kluwer Academic/Plenum Publishers, New York. pp. 465-601. Gilles, J. and Lawrence, R.C. (1973). The assessment of cheese quality by compositional analysis. NZ J. Dairy Sci. Technol. 8, 148-151. Guinee, T.P. and Fox, P.E (1993). Salt in cheese; physical, chemical and biological aspects, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, General Aspects, 2nd edn, P. Fox, ed., Chapman & Hall, London. pp. 257-302. Guinee, T.P, Auty, M.A.E., Feeney, E.P. and Fox, P.E (2002). Effect of pH and calcium concentration on some textural and functional properties of Mozzarella cheese. J. Dairy Sci. 85, 1665-1669. Gustchina, E., Rumsh, L., Ginodinan, L., Majer, P. and Andreeva, N. (1996). Post X-ray crystallographic studies on chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine-proline cluster of K-casein. FEBS Lett. 379, 60-62. Hyslop, D.B. (2003). Enzymatic coagulation of milk, in, Advanced Dairy Chemistry - Vol. 1 - Proteins, 3rd edn, P.E Fox and P.L.H. McSweeney, eds., Kluwer Academic/Plenum Publishers, New York. pp. 839-878. Iametti, S., Tedeshi, G., Oungre, E. and Bonomi, E (2001). Primary structure of K-casein isolated from mares' milk. J. Dairy Res. 68, 53-61. Lawrence, R.C. and Gilles, J. (1980). The assessment of the potential quality of young Cheddar cheese. NZ J. Dairy Sci. Technol. 15, 1-12. Lawrence, R.C., Heap, H.A. and Gilles, J. (1984). A controlled approach to cheese technology. J. Dairy Sci. 67, 1632-1645. Lelievre, J. and Gilles, J. (1982). The relationship between the grade (product value) and composition of young commercial Cheddar cheese. NZ J. Dairy Sci. Technol. 49, 1098-1101. Lynch, C.M., McSweeney, P.L.H., Fox, P.E, Cogan, T.M. and Drinan, ED. (1997). Contribution of starter lactococci and non-starter lactobacilli to proteolysis in Cheddar cheese with a controlled microflora. Lair 77,441-459. Malacarne, M., Martuzzi, E, Summer, A. and Mariani, P. (2002). Protein and fat composition of mare's milk: some nutritional remarks with reference to human and cows' milk. Int. Dairy J. 12,869-877. Martin, P., Ferranti, P., Leroux, C. and Addeo, E (2003). Non-bovine caseins: quantitation variability and molecular diversity, in, Advanced Dairy Chemistry - Vol. 1 - Proteins, 3rd edn, P.E Fox and P.L.H. McSweeney, eds., Kluwer Academic/Plenum Publishers, New York. pp. 277-317. McSweeney, P.L.H. and Sousa, M.J. (2000). Biochemical pathways for the production of flavour compounds in cheese during ripening. Lait 80,293-324.
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McSweeney, P.L.H., Fox, RE, Lucey, J.A., Jordan, K.N. and Cogan, T.M. (1993). Contribution of the indigenous microflora to the maturation of Cheddar cheese. Int. Dairy J. 3,613-634. Morgan, S.M., Ross, R.P. and Hill, C. (1995). Bacteriolytic activity due to the presence of novel plasmid-encoded lactococcins, A, B and M. Appl. Environ. Microbiol. 61, 2995-3001. Ochirkhuyag, B., Chobert, J.-M., Dalgalarrondo, M. and Haertle, T. (2000). Characterization of mare caseins. Identification of %1- and Ots2-caseins. Lait 80, 223-235.
O'Connor, C.B. (1971). Composition and quality of some commercial Cheddar cheese. It. Agric. Creamery Rev. 26 (10), 5-6. Pearce, K.N. and Gilles, J. (i979). Composition and grade of Cheddar cheese manufactured over three seasons. NZ J. Dairy Sci. Technol. 14, 63-71. Tobin, J. (1999). Effects of Adjunct Cultures and Starter Blends on the Quality of Cheddar Cheese. PhD Thesis, National University of Ireland, Cork. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11,185-201.
Index
Abondance, 199 Acid-coagulated milk gels, 6, 77-8, 105 casein micelles in, 105-106 effect of compositional/processing parameters on textural properties: fat content/homogenization, 118 heat treatment, 116-17 inoculation/gelation temperature, 115-16 pH/calcium content, 118-19 rennet addition, 117 solids non-fat (SNF) content, 117-18 mechanisms: physico-chemical aspects, 106-109 theoretical models, 106 physical properties: appearance, 113-14 microstructure, 112 permeability, 112-13 rheological, 109-11 texture/sensory, 111-12 whey separation/syneresis, 114-15 study by electron microscopy (EM)/confocal scanning laser microscopy (CSL), 112 syneresis, 114-15 see also Coagulation; Rennets Adjunct cultures see Secondary/adjunct cultures Amino acids, catabolism of see Catabolism of amino acids Appenzell, 199 Aroma, compounds: extraction methods, 491 dialysis, 492 headspace, 493 high-performance size-exclusion chromatography, 492-3 high-vacuum distillation, 491-2 solid-phase microextraction, 493 solvent, 492 steam distillation, 491 water-soluble extract (WSE), 493-4 identification using hyphenated GC techniques, 494-6 representativeness, 494 sample treatment, 491 see also Sensory characteristics of cheese; Flavour Arzua, 323 Aspartic proteinases see Chymosin/aspartic proteinases Autolysis, 126,289 Avenato, 323
Bacteriocins, 136, 153 Bacteriophage, 154, 163-70, 287,351,375 classification, 165 DNA homology, 167 host range, 165 morphology, 165 serology, 165-6 structural protein profiles, 166-7
control of, 164 engineered phage resistance systems, 179 antisense mRNA, 180 bacteriophage-triggered defence, 181-2 current status/future perspectives, 182 gene replacement/insertional mutagenesis, 180-1 phage-encoded resistance (Per), 179-80 recombinant superinfection exclusion/ immunity, 181 epidemiology, 167 Lactobacillus phage, 170-1 Sc thermophilus phage, 170 genome organisation/evolution, 171-6 lysogenic life cycle, 174 maintenance of lysogeny, 175-6 site-specific recombination, 174-5 superinfection exclusion, 176 lytic life cycle: bacteriophage lysis, 174 DNA packaging, 173 DNA replication, 172-3 lysogenic/lytic switch, 172 phage adsorption/DNA injection, 171-2 structural proteins, 173-4 multiplication, 163 natural resistance systems in lactic acid bacteria, 176-7 abortive infection, 178-9 adsorption inhibition, 177 injection blocking, 177 restriction/modification, 177-8 proteolytic enzymes, 131 raw milk cheeses, 321 resistance systems: engineered phage, 179-82 natural bacteriophage, 176-9 starter cultures, 129-38 Bavarian Blue, 193 Beaufort, 197 Bel Paese, 193, 195 Biogenic amines, 201,561-3 Bleu d'Auvergne, 193 Blue cheese, 123 contamination, 550 fatty acids, 378 mesophilic starters, 149 ultrafiltration, 269 use of salt, 211-12, 219 B r e v i b a c t e r i u m linens, 149, 192 Brie: flavour, 502 lipolytic agents, 376 moulds, 193 rheology/texture, 535 Brine-salted cheese see Salt Buffalo milk cheese, 5
610
Index
Cabrales, i93,306 Camembert, i23 contamination, 550 coryneform bacteria, 195,197 fatty acids, 377 flavour, 492,498,499, 500 lipolytic agents, 376 Listeria growth, 544 mesophilic starters, 149 microbial growth, 544-5 moulds, 193 raw milk, 321 rheology/texture, 528, 535 starter cultures, 126 uhrafiltration (UF), 271 use o f salt, 219,240 yeast flora, 306 Canestrato, 5,323 Casein, 7, 48-50, 55, 60, 71-2, 105-106,351,413, 415,417, 589,591 activity of cell envelope proteinase, 132 chemistry, 48 micelle assembly, 49 micelle properties, 49-50, 105-106 micelle stability, 50 self-assembly, 49 structures, 48-9 chymosin activity on, 52, 392-3 effect of NaC1, 212-13,214, 220-3 enrichment of cheese milk by ME 277-8 gel formation/properties of, 71-4, 78-81, 91, 94, 106-109, 111,115,116 hydration in cheese, 220-5 hydrolysis by cathepsin D, 396 mares' milk, 591 quality of cheese, 591-2 raw milk cheese, 324, 336, 339 use of capillary electrophoresis for analysis of casein, 420 Catabolism of amino acids, 152-3,201,302-303, 350-2,435,451 aromatic: phenylalanine, 444-7 tryptophan, 443 tyrosine, 443-4 branched-chain: deaminases, 449 decarboxylases, 449-51 production of volatile sulphur compounds: lyases involved in methionine catabolism, 442-3 transamination, 435-9 Catabolism of fatty acids, 376-80 Catabolism of lactate, 348-9 Cathepsins, 396, 399 Cauchy strain, 539 Cheddar, 5, 10, 12, 14 contamination, 545,546-9 dry-salted varieties, 245-6 fatty acids, 377 flavour, 335,337,492, 502, 605 lipolytic agents, 374,375 lysis, 136 mesophilic starters, 149 microbiology, 289,290, 296, 297, 301 moulds, 193 propionic acid bacteria, 200 quality, 595,596,604, 605 raw milk, 323, 327,330
reduced sodium, 226-7 rheology/texture, 516,517, 518-19,520, 527, 529, 530, 535 ripening, 348 sensory characteristics, 478-9 starter cultures, 126, 128 use of salt, 208-209,211,216-19,240 Cheddar curd: salt uptake: curd depth during holding, 238 degree of mixing, 238 level of salting, 237-8 mellowing period, 238 method, 237 moisture content of curd, 238-9 other factors, 239 temperature, 238 Cheese:
composition, 7-8, 603-605 fat-in-dry matter (FDM), 7, 589 moisture in non-fat substances (MNFS), 7 pH, 8 history of, 1-5,261,605 Cheshire, 5,208, 527 Chymosin/aspartic proteinases, 19-33,351,392, 600-601 active site, 23-7 catalytic mechanisms, 27-9 inhibitors, 33 natural sources, 19-20 physical properties/stability, 20-1 structure, 21-4 substrate-binding pockets/specificity, 30-3 zymogen activation, 29-30 crystals, 23 fermentation-produced chymosin, 51-2 milk clotting mechanism, 33-4 mutant chymosins, 32-3 physical properties/stability: enzyme solubility, 20-1 enzyme stability, 20 molecular weight/isoelectric point, 20 recombinant calf chymosin: eukaryotic expression, 35-6 prokaryotic expression, 34-5 ripening process, 351,356 stability (solubility), 20 structure: active site, 24-7 catalytic mechanisms, 27-9 gene sequences, 2i inhibitors, 33 primary, 21 secondary, 21, 23 substrate-binding pockets/specificity, 30-3 tertiary, 23-4 zymogen activation, 29-30 substrate specificity, 32 yield, 34-5 see also Pepsin Citrate metabolism, 130-1,151-2,367-8 Clostridia, 7, 153,202, 327, 328, 365-6, 562, 600 Clotting see Acid-coagulated milk gels; Coagulation; Rennets
Coagulation, 10-11, 47 Cynara cardunculus, enzymes from, 52 enzymes, 47, 391,392-3 influence of NaC1, 212-13
Index
physico-chemical mechanisms involved in gel formation from unheated milk, 106-109 post-coagulation operations, 11-12, 594-6 theoretical models, 106 visual, 11 see also Acid-coagulated milk gels; Rennets Comte cheese, 127 Conjugated linoleic acid, 575-6 Contamination see Pathogens; Safety; Toxins Cooking, 224-5 health/safety, 551 Coryneform bacteria, 195, 197, 597 antimicrobial activities, 198 form/use of adjunct culture, 198 lipolysis, 198 proteolysis, peptidolysis, amino acid catabolism, 198 selection as adjuncts, 197-8 useful properties in selecting surface bacteria, 197 Cottage cheese, 2, 9 acid milk gels, 105, 112, 116 addition of rennet, 117 contamination, 550 heat treatment, 116 reduced sodium, 227 ultrafiltration, 268 Cream cheese, 2 acid milk gels, 105, 112 compliance, 539 modulus, 539 whey removal, 114, 115 Cultures see Secondary/adjunct cultures; Starter cultures Curd, 12, 14 behaviour during processing: axial drainage, 95 compaction of curd column, 94-6 curd fusion, 93-4, 97 syneresis under pressure, 93 water content of cheese, 96-8 cooking temperature, 11 manufacturing process, 11-12 salt absorption/diffusion: direct mixing with milled curd, 229, 232 dry surface-salting of moulded pressed curd, 232 factors affecting uptake in Cheddar, 237-9 initial moisture content, 236-7 pH of curd/brine, 237 salt-in-moisture level/pre-salting, 208-11,235 temperature of curd/brine, 235 syneresis during curdmaking: effects of grain size, 86-7 methods of estimation, 85-8 rate equations, 85-6 stirring, 87-8 washing, 90 C y n a r a cardunculus proteinase, 3, 52 Danablu, Danish Blue, 193, 194, 306 Danbo: mould, 193 ultrafiltration, 268 use of salt, 220 yeast flora, 306 D e b a r y o m y c e s hansenii, 191,192, 196 Deformability modulus, 539 Dental caries, 576, 578-9 Domiati, 10, 219 Dry-sahed cheese see Salt
Dutch cheese, 128 lipolytic agents, 374 ripening, 348 see also Gouda; Edam Eastern European cheeses, 299 Edam:
fatty acids, 378 mesophilic starters, 149 starter cultures, 126 ultrafiltration, 268 Elastic material behaviour, 539 Elastoplastic material behaviour, 539 Emmental:
fatty acids, 378 flavour, 502 propionic acid bacteria, 200 starter cultures, 123, 126, 127 use of salt, 215 whey cultures, 127 see also Swiss cheese Enterococci, 290,295 Enzymes, 19, 133,288, 378, 448 coagulation, 47, 50-2, 391,392-3 indigenous, 587-8,600 microbial, 214-15 proteolytic, 19,413-15 ripening process, 352-7 secondary starter microorganisms, 195,413-15 sources: lyosomes, 19 micro-organisms, 19-20 plants, 19 stomach, 19 tissues, 19 see also Chymosin/aspartic proteinases; Lactic acid bacteria; Catabolism of amino acids; Catabolism of fatty acids; Lipolysis; Peptidases; Proteolysis; Ripening of cheese Ewe's milk cheese, 199,340 Exopolysaccharides, 137, 158 Fatty acids, 349,373-80 Feta, 5,268-9 contamination, 550 microbial growth, 545 microbiology, 290 ultrafiltration (UF), 271 use of salt, 219 Flavour, 3-4, 129, 289,306,332, 347, 466,489-91 acid milk, 112 contribution of lipolysis/catabolism of FFA, 379-80 dynamic methods for characterisation: model mouth systems, 501-502 release of non-volatiles in vivo, 501 release of volatiles in vivo, 499-501 fatty acids, 349 global/fast assessment, 502 electronic nose, 502-503 mass spectrometry-based systems, 503-504 peptides, 352 quality, 586-7, 599,603 raw milk, 335-8 sapid (non-volatile) compounds: extraction, separation, identification in relation to sensory properties, 496-9 water-soluble extracts (WSE), 496 smell/aroma, 466
611
612
Index
Flavour - contd. taste, 466 see also Sensory characteristics of cheese; Aroma, compounds Free-Choice Profiling, 477 Fungi, 36 Gammelost, 306 Gaziantep, 219-20 Gel formation see Acid-coagulated milk gels; Coagulation; Rennets; Syneresis of rennet-coagulated curd Genetic engineering, 155, 601 Geotrichum candidum, 191
Goat cheese, 1, 5, 14, 193,325,498 Gorgonzola, 193 Gouda, 14 fatty acids, 378 lysis, 136 mesophilic starters, 149 propionic acid bacteria, 199 quality, 595, 596 starter cultures, 126 use of salt, 223,237 Grana, 8, 126 microbiology, 289 whey cultures, 127 Gruy/~re: catabolism of fatty acids, 378 Coryneform bacteria in, 197 flavour, 492 rheology/texture, 529 starter cultures, 126, 127 whey cultures, 127 HACCP see Hazard Analysis and Critical Control Points
Havarti, 535 Hazard Analysis and Critical Control Points, 542, 543,584 see also Safety Health see Nutrition; Pathogens; Safety; Toxins Heterofermentative lactobacilli, 200 form/use of adjunct cultures, 202 species found in cheeses, 200-201, 291 useful properties to select as adjuncts: antagonistic activities, 201-202 formation of biogenic amines, 201 lipolytic activities, 201 probiotic properties, 202 proteolysis/amino acid catabolism, 201 Histamine, 561-3 Hygiene, 321-3 Illness see Pathogens; Safety; Toxins Italian cheeses, 298-9,338 Jarlsberg, 5 Kelvin element, 539 Kinematic viscosity, 539 Kluyveromyces, 195 Lactate: catabolism, 348-9 changes during ripening: metabolism by Clostridium tyrobutyricum, 365-6 metabolism by Propionibacterium, 366-7
oxidation, 364 oxidative metabolism in surface mould-ripened varieties, 364 racemization, 362-4 see also Lactose metabolism Lactic acid bacteria, 399-400 aminopeptidases, 411-12 carboxypeptidases, 403, 411 di- and tri-peptidases, 403 endopeptidases, 403 Lactobacillus delbrueckii, 290 Lactobacillus helveticus, 123 Lactococcus lactis, 123 peptidases, 400-403 proline-specific peptidases, 412-13 proteinases, 400 see also Starter cultures, Non-starter lactic acid bacteria, Bacteriophage Lactobacillus spp, 123,290 see also Non-starter lactic acid bacteria Lactococcus lactis, 123 chromosome, 149-50 genetic manipulation, 155 genetics of industrially important traits: bacteriocins, 153-4 bacteriophage, 154-5 lactose/citrate metabolism, 151-2 proteolysis/amino acid catabolism, 152-3 Lactose metabolism, 130, 151-2, 361-2 Leuconostoc, 155,290 Limburger, 195 Linear viscoelastic deformation, 539 Lipolysis, 198, 199-200, 201,303,349 agents, 373-6 contribution of FFA to flavour, 379-80 measurement of, 380,384-5 patterns of, 380 raw milk cheese, 325,327 Liquid pre-cheese (LPC), 269-73 Listeria monocytognes, 542-4, 550, 551,553-5 Livarot, 195 Loss modulus, 540 Low-concentrated retentates, 267-8 Lysis, 126, 289 see also Autolysis Lysogeny, 174 Maasdam, 5, 199,378 Mahon, 220 Manchego, 5,335 Membrane processing, 261 applications of: liquid pre-cheeses, 269-75 medium/intermediate concentrated retentates, 268-9 microfiltration, 276-8 milk protein concentrates, 278-9 on-farm concentration, 275-6 properties of UF retentates, 265-7 protein-standardized milk, 267-8 reverse osmosis, 275 UF in cheesemaking, 267-75 APV-sirocurd process: definitions, 262-5 design/configuration, 261 hollow fibre, 262-3 microfiltration (MF), 262 nanofiltration (NF), 262 plate and frame, 263
Index
reverse osmosis (RO), 262 spiral-wound, 263-4 tubular, 262 ultrafiltration (UF), 262 vibrating membrane system, 264-5 Mesophilic starters, 149-52, 597 Leuconostoc, 155-6 plasmids, 150-1 Mexican-style cheese, 553 Microbial pathogens see Pathogens Micrococcus, 197,304 Microfiltration: applications: casein enrichment of cheese milk, 277-8 microbial epuration of raw milk, 276-7 modifications, 278 selective fractionation of globular fat, 278 Milk: antiobiotics in, 7 casein chemistry, 48-50 chemical composition, 7, 588-9 clotting mechanism, 33-4, 47 composition, 91 fat, 47 gel formation, 106-109 heat treatment, 88 homogenization, 88 indigenous enzymes, 587-8 indigenous proteinases: others, 396-9 plasmin, 213-14, 393-6, 600 microbiology: desirable indigenous bacteria, 587 off-flavours/spoilage, 586-7 public health aspects, 586 pasteurized, 8,355 alternatives to pasteurization, 587 protein, 47-8, 573,575 protein-standardization, 267-8 quality, 584 rennet-induced coagulation, 50-65 safety: heat treatment, 543-4 pathogens, 541-3 quality, 543 standardization: calcium, 589 fat/casein, 589 pH, 589-90 syneresis of renneted-milk gel, 80-1 toxins in, 564 various additions to, 88-9 Milk gels see Acid-coagulated milk gels; Rennet coagulation of milk Milk protein concentrates, 278-9 Modulus of deformability, 540 Morbier, 195, 199,330 Moulds, 304-306, 395,597 contamination with mycotoxins, 567-8 form/use of adjunct culture, 194-5 species found in cheese, 193--4 useful properties in selecting as adjuncts: appearance on/in cheese, 194 de-acidification activity, 194 interactions with other microorganisms, 194 lipolytic activity, 194 production of aroma, 194 production of mycotoxins, 194
613
proteolytic activity, 194 Mozzarella, 9, 12 contamination, 553 flavour, 498 quality, 595,596 raw milk, 321 rheology/texture, 529,530 starter cultures, 123, 126 ultrafiltration (UF), 268, 272-3 use of salt, 222,240 MUnster, 195 Mycotoxins, 564, 567-8 Nitrogen metabolism in lactic acid bacteria, 131-2 amino acid degradation, 133-4 role of proteinase, 132 transport systems/peptidases, 132-3 see also Proteolysis; Peptidases; Lactic acid bacteria Non-starter lactic acid bacteria (NSLAB), 7, 289, 291,353 biochemical activities: amino acid catabolism, 302-303 citrate utilisation, 302 lipolysis, 303 proteolysis, 302 enterococci, 290, 295 growth/survival: environmental conditions, 296 interactions, 297 nutrient availability, 296-7 non-starter lactobacilli, 289-90 pediococci, 290 population dynamics: Cheddar, 297 Greek/eastern European cheeses, 299 Italian cheese varieties, 298-9 Portuguese cheese varieties, 298 Spanish artisanal cheeses, 297-8 Swiss cheeses, 297 quality, 602-603 ripening process, 356-7 significance: adjunct as probiotics, 301-302 influence on quality, 299, 301 use of other adjunct cultures, 301 source of, 295-6 see also Lactobacillus NSLAB see Non-starter lactic acid bacteria Nutrition, 573 carbohydrate, 575 cheese and dental caries, 576, 578-9 fat/cholestrol, 575-6 minerals, 576 protein, 573,575 vitamins, 576 Parmigiano-Reggiano, Parmesan: contamination, 545,550 fatty acids, 377-8 flavour, 492 raw milk, 322 sensory characteristics, 474 starter cultures, 123 whey cultures, 127 Pathogens, 7, 541 challenge studies, 549-52 Escherichia coli, 541,544, 545,548, 549-50, 551,555 growth/survival in soft/semi-soft cheeses, 552-3 reviews on safety of raw milk, 545-6
614
Index
Port Salut, 193
Quality of cheese, factors affecting: cheese composition, 603-605 coagulant (rennet), 590-2, 600-601 cultures, 592-4, 601-602 indigenous enzymes, 600 Lactobacillus species as adjunct cultures, 603 milk supply: alternatives to pasteurization, 584, 587 chemical composition, 588-9 indigenous enzymes, 587-8 microbiology, 586-7 standardization of composition, 589-90 non-starter lactic acid bacteria, 602-603 packaging, 597 post-coagulation operations, 594-6 production parameters, 584 ripening, 596-9,605-606 salting, 216-20, 596 starter, 592-4, 601-602 use of ultrafiltration (UF), 596 Quarg, 2, 5 acid milk gels, 105,112 addition of rennet, 117 ultrafiltration, 270 whey removal, 115 see also Acid-coagulated milk gels Queso Blanco, 5
Portuguese cheese, 298, 306 Processed cheese, 227 Prochymosin, 20, 30, 34-6 Propionibacterium, 303,449,597 see also Emmental; Swiss cheese Propionic acid bacteria, 198, 303,449,597 as adjunct cultures, 200 characteristics of species found in cheeses, 198-9 useful properties for selecting as adjuncts: lactate metabolism, 199 lipolysis, 199-200 probiotic properties, 200 proteolytic activities/amino acid catabolism, 199 Proteolysis, 152-3,156, 302,350-2 amino acids, 350-2 lactocepins, 400 monitoring: amino acid analysis, 421 capillary electrophoresis (CE), 420 chromatographic techniques, 420-1 fluorescent spectroscopy, 421 Fourier transform infrared spectroscopy (FTIR), 421 tryptophan, 421 ultrasonics, 421 urea-PAGE, 420 patterns, 415-19 primary, 323-4 raw milk cheese, 323-5 ripening, 391 coagulant, 391,392-3 exogenous proteinases/peptidases, 391-2 indigenous proteinases, 391,393-9 non-starter lactic acid bacteria, 200,391 secondary starter, 191,391,413-15 starter lactic acid bacteria, 391,399-413 secondary, 198, 199,201,324 water-soluble peptides, 416-19 Provolone, 545
Raclette, 323, 328, 335, 338 Raw milk cheese, 8, 319-20, 545-6 biochemical aspects: lipolysis, 325, 327 proteolysis, 323-5 volatile compounds, 327-35 safety aspects: diversity of microorganisms, 320-1 hygiene, 321-3 numbers of microorganisms, 320 sensory aspects: flavour/odour, 335-8 texture, 338-40 Reconstituted skim milk, 64, 139,208 Reduced-sodium cheese, 207,225-6 Cheddar, 226-7 Cottage cheese, 227 other cheeses, 227 Rennet coagulation of milk, 2, 3, 6, 8, 10-11, 19, 584 adhesive sphere models/viscosity, 55-6 development of theological properties, 56-7 effect of acidification, 117 fractal models/rearrangements, 62-3 heat treatment, 64-5 high pressure treatment, 64 kinetic models, 53-5 measurement of clotting time/curd-cutting time, 53 mechanisms of milk-clotting, 33-4 milk processing/gel formation, 63-5 modelling gel-firming kinetics, 60-2 post-coagulation operations, 594-6 preparation, 52 primary enzymatic phase, 11, 50-2 production, 584 quality, 590-2, 600-601 rennet, 354-5 ripening process, 354-5 secondary non-enzymatic phase, 11 substitutes, 10 theoretical basis of viscoelasticity, 57-60
Pathogens - contd. safety of cheese, 541 extrinsic/intrinsic parameters affecting microbial growth, 544-5 heat treatment of milk, 543-4 milk quality, 543 raw milk, 541-3,545 Salmonella, 542, 544, 546-8, 551,553,555 Salmonella enterica, 542,546, 548, 553 stress adaptation and impact on safety, 553-4 Pecorino, 5,124, 126, 127 Pediococcus, 290, 292 Penicillium camemberti, 193 Penicillium roqueforti, 193 Pepsin, 29, 31,354,393 Peptidases, 132-3,400,403,417 aminopeptidases, 411-12 carboxypeptidases, 403, 411 di- and tri-peptidases, 403 endopeptidases, 403 proline-specific, 412-13 Phage see Bacteriophage Pichia spp., 196 outbreaks involving Cheddar, 546-9 Poisson effect, 540 Pont l'Eveque, 535
Index
UF retentates, 266-7 see also Acid-coagulated milk gels; Coagulation; Chymosin/aspartic proteinases Rennets see Chymosin/aspartic proteinases; Pepsin Rheology, 511-33 cheese structure, 516-18 compliance, 539 creep/stress relaxation, 518-19 development of properties during rennet coagulation, 56-7 effect of NaC1, 223-4 empirical instrumental methods of measurement, 523 exopolysaccharides, use of, 158 gel formation, 75-7, 109-10, 113-14, 117-18 large strain deformation: bending tests, 530-1 definition/terminology, 520 effect of sample temperature, 532 fracture/work to fracture, 521 measurement using texture analyser, 520-1 shear measurements, 530 uniaxial compression, 527-30 wire-cutting, 532 mechanical models, 519-20 oscillatory rheometry for linear viscoelastic measurements: complex viscosity, 526-7 elastic shear modulus, 524-6 loss modulus, 524-6 overview, 511-12 sensoric methods, 521-3 terminology: bulk modulus/compressibility, 515-16 deformation and strain, 512 relationship between stress/strain, 515 shear/normal modes of stress/strain, 513-15 stress, 512-13 viscous deformation, 516 time-dependent measurement, 532 viscosity measurement, 532-3 see also Texture Rhizomucorprotease, 20 Ripening of cheese, 12, 14, 347,375,395 acceleration, 357 agents: cathepsin D, 355-6 NSLAB, 356-7 other indigenous enzymes, 356 plasmin, 355 rennet, 354-5 starter enzymes, 356 biochemical activities of NSLAB, 302-303 catabolism of amino acids, 350-2,435 aromatic, 443-7 branched-chain, 447-9 deaminases, 449 decarboxylases, 449-51 other, 451 transamination, 435-9 volatile sulphur compounds, 439-43 glycolysis of residual lactose/catabolism of lactate, 347-9 lipolysis/metabolism of fatty acids, 349 proteolysis, 350-2, 391-2 quality, 596-9,605-606 see also Proteolysis, Lipolysis; Catabolism of lactate; Catabolism of amino acids; Catabolism of fatty acids
Romano, 535,545 Roquefort, 5 lipolytic agents, 376 mould, 193, 194 rheology/texture, 535 yeast flora, 306 S a c c h a r o m y c e s cerevisiae, 196
Safety, 541 Cheddar, 546-9 improvements, 554-5 microbial growth, 544-5 milk: heat treatment, 543-4 quality, 543 raw, 541-3,545-9 soft/semi-soft cheeses, 552-3 stress adaptation and impact of pathogens, 553-4 St Nectaire, 193-4, 306 St Paulin, 136, 273,323 Salt, 10, 207-208,348, 576, 596 absorption/diffusion: brine- and surface dry-salted cheeses, 244-5 brine concentration/concentration gradient in brine-salted cheese, 232-3 brine-salted cheese, 228-9 Cheddar/dry-salted varieties, 245-6 cheese geometry, 233-4, 244 concentration gradient in dry-salted cheeses, 239-40 concentration of calcium in brine, 240 direct mixing of salt/milled curd, 229,232 dry surface-salting of moulded pressed cheese curd, 232 fat content of cheese, 243-4 initial moisture content of curd, 236-7 initial salt-in-moisture level of curd/pre-salting, 235 mechanisms, 228-32 method of brine-salting, 233 methods of salting, 228 moisture content of cheese, 98,241-3 pH of curd/brine, 237 salting time, 234-5 temperature of brine/cheese, 240 temperature of curd/brine, 235 uptake in Cheddar curd, 237-9 casein hydration/physical properties of cheese: cooking properties, 224-5 microstructure, 223 model systems, 220-3 rheology, 223-4 control of microbial growth, 208-12 effect on cheese composition: lactose content/pH, 249 moisture level, 247-9 salt content, 249 enzyme activity: coagulant, 212-13 microbial enzymes, 214-15 milk proteinase, 213-14 gel formation, 83-4 quality, 596 reduced-sodium cheese: Cheddar, 226-7 Cottage cheese, 227 other cheeses, 227
615
616
Index
Salt - contd. ripening/quality: Blue cheese, 219 Camembert, 219 Cheddar, 216-19 other cheeses, 219-20 salt/moisture equilibria in brine-salted cheese after salting, 244-7 salt/moisture equilibrium in Cheddar cheese, 246-7 water activity (aw), 215-16 Sbrinz, 127 Secondary/adjunct cultures: coryneform bacteria and staphylococci, 195,197-8 effect on quality, 603 heterofermentative lactobacilli, 200-202 moulds, 193-5 non-starter lactic acid bacteria, 7,289,291,353 propionic acid bacteria, 198-200 yeasts, 191-3 see also Starter cultures Semi-hard cheese, 273 Sensory characteristics of cheese, 455,463-5 cheese preferences, 455-6 consumer preferences, 14,480-1 definition, 455 evaluation methods, 467-8 consumer acceptability testing, 478 descriptive analysis, 475-7 discrimination tests, 475 grading/quality scoring, 468-75 time-intensity analyses, 477-8 human senses/sensory properties: cheese appearance, 462,466 cross-modal interactions, 466-7 flavour, 466 texture, 466 universal language, 480 variety, 456-62 see also Aroma, compounds; Flavour Shear modulus, 540 Sheep milk cheese, 1, 5, 14 Soft cheese, 270-3,552-3 Spanish artisanal cheeses, 297-8, 306 Staphylococcus, 195,197,304 antimicrobial activities, 198 form/use of adjunct culture, 198 lipolysis, 198 proteolysis, peptidolysis, amino acid catabolism, 198 selecting surface bacteria as adjuncts: effect on colour of cheese surface, 197-8 growth, 197 Starter cultures, 123, 149,287, 288-9,348 defined-strain, 164-5 genomics, 129-30, 158-9 mesophilic starter genetics: bacteriocins, 153-4 bacteriophage, 154-5 chromosome, 149-50 industrially important traits, 151-5 lactose/citrate metabolism, 151-2 Leuconostoc, 155-6 manipulation, 155 plasmids, 150-1 proteolysis/amino acid catabolism, 152-3 metabolism: autolysis, 136 bacteriocins, 136-7 citrate, 130-1
exopolysaccharide production, 137-8 growth, 134-5 lipases/esterases, 134 metabolic engineering, 135-6 nitrogen, 131-4 stress responses, 137 sugar, 130 mixed-strain mesophilic, 9-10, 164-5 pH control, 139-40 phage infection, 127, i39 preparation: preservation/distribution, 140-2 propagation, 138-40 time/temperature combination, 140 quality, 592-4 taxonomy, 123-4 types, 126, 128, 164 new sources, 129 primary, 123,124, 126, 191 secondary, 123, 191,195 see also Bacteriophage Stilton, 193, 194, 208 Streptococcus thermophilus, 123,157 Stress: relaxation modulus, 540 relaxation test, 540 Swiss cheese, 128 contamination, 545,552 flavour, 335,338,492 microbiology, 289,296,297 raw milk, 323,327,328 Syneresis of rennet-coagulated curd, 71, 114 during curdmaking, 84-5 effects of other process variables: acidity, 90 coagulation, 89 heat treatment of milk, 88 high-pressure treatment, 91 homogenization of milk, 88 temperature, 89-90 ultrafiltration, 90-1 various additions to milk, 88-9 washing of curd, 90 mechanisms, 78-9 methods for estimating: effects of curd grain size, 86-7 modelling process, 81-4 rate equations, 85-6 stirring, 87 renneted milk, 80-1 review of, 91-2 unified approaches to gel formation/syneresis: acid gels, 77-8 behaviour during processing, 92-3 compaction of curd column, 94-5 curd fusion, 93-4 effect of milk composition, 91 gel formation, 73-5 renneting, 72-3 rheological characteristics, 75-7 under pressure, 93 water content of cheese, 95-8 Texture: terminology, 533 evaluation: instrumental shear deformation, 535-6 texture profile analysis (TPA), 534 see also Rheology
Index
Thermophilic starters, 56, 126 Lactobacillus spp.: genetic manipulation, 157 important traits, 156-7 Streptococcus thermophilus: genetic manipulation, 158 important traits, 157-8 Tilsit, 306 Tomme, 193, 199 Torulospora delbrueckii, 196 Toughness, 540 Toxins: biogenic amines: formation, 562-3 histamine, 561-2 in cheese, 563 mycotoxins: direct contamination of cheese, 567-8 fate in cheese during manufacture/ripening, 567 indirect contamination, 564, 567 production of toxic metabolites in cheese, 567 Ultrafiltration (UF), 8-9, 90-1,265-75,596 cheese quality: functionality, 274-5 proteolysis/ripening characteristics, 274 texture, 273-4 liquid pre-cheeses: fresh unripened cheeses, 269-70 other applications, 273 semi-hard cheese, 273 soft cheese, 270-3 medium/intermediate concentrated retentates: APV-sirocurd process, 268 general considerations, 269 other cheeses, 269 structured Feta-like cheese, 268-9 properties of UF retentates: buffering capacity, 265-6 rennet coagulation, 266-7 rheological behaviour, 266
protein-standardized milk, 267-8 see also Membrane processing Uniaxal compression, 527-8 compressive strength, 540 effect of deformation rate, 529-30 effect of pre-test strain history, 529 effect of sample-machine interface conditions/sample dimensions, 529 influence of shape, 530 relationship between shear/normal stresses, 528 Viscoplastic material behaviour, 540 Viscosity/dynamic viscosity, 540 Volatile compounds, 327-8 alcohols, 331-2 carbonyl compounds, 330-1 esters, 332 lactones, hydrocarbons, 334-5 sulphur compounds, 332-4 volatile fatty acids (VFA), 328-30 Water-soluble extract (WSE), 493-4, 496 Whey, 105 heat treatment, 9 incubation, 9 preparation, 127 separation/syneresis, 114-15 Yeast, 36, 191,306-307, 597 forms/use as adjunct culture, 193 interactions with other microorganisms, 192-3 species found in cheeses, 191-2 useful properties in selecting adjuncts: effect on appearance of cheese surface, 192 lipolytic activity, 192 production of aroma, 192 proteolytic activity, 192 utilisation of residual sugars/lactate de-acidification activity, 192 Young's modulus, 540
617
Foreword
The art of cheesemaking has been augmented steadily by greater knowledge on the science of cheesemaking. This evolution has resulted from basic and applied research and from the increased need to understand and control the characteristics of milk, the microorganisms used in the manufacture and maturation of cheese, the manufacturing technologies, and the physical properties and flavour of cheese. Traditional methods of cheese manufacture have been modified by the need for greater efficiencies in the manufacture and maturation of cheese and by changes in the marketing channels for cheese. Accommodating these changes while maintaining the characteristics of a given cheese variety has been accomplished by the application of scientific principles. The need for greater understanding of the characteristics of cheese has also been driven by the increased use of cheese as an ingredient in other foods. This has required specific control of selected properties of cheese to impart the desired properties to the food, and to retain characteristics of the cheese during various food processing technologies. The successive editions of Cheese: Chemistry, Physics and Microbiology have documented the application of science to the art of cheesemaking. Certain characteristics are common in all editions: a thorough description and evaluation of scientific and technological advances, prodigious referencing to direct readers to more in-depth discussion of topics, and careful editing to impart consistency of discussion and a smooth transition between chapters. However, each edition has been revised to incorporate new information and to reflect recent trends in describing the science of cheesemaking and maturation and in the use of cheese as a food ingredient. Scientific principles emphasised in Volume 1 cover microbiological, chemical and physical attributes of cheese as in previous editions. Greater emphasis is given to the genetics and metabolic activity of lactic starters and on the secondary microflora in the third edition. Conversion of components (lactose, lactate, citrate, lipids, proteins) by microbial metabolism and enzymatic action is discussed in several chapters. Inclusion of modern sensory evaluation techniques and instrumental identification of flavour compounds recognises the relationship between these areas. A new chapter on acid gels provides the basic background for discussion in Volume 2 on cheese varieties made by acid or heat plus acid coagulation that are becoming more important as food ingredients. Volume 2, as in previous editions, focuses on various types of cheese, but the cheeses have been grouped into more logical categories based upon characteristics rather than geographical regions of production. The first chapter of Volume 2 provides an overview of the diversity of cheese varieties and systems of categorising varieties. A similar approach in the second chapter familiarises the reader with the general aspects of cheese technology to emphasise that there are common elements in cheesemaking and maturation and that cheese varieties result from specific deviations from or additions to these common elements. The last chapter is appropriately a discussion of cheese as an ingredient, which recognises recent trends in the science of cheese. A substantial bank of knowledge has been accumulated on cheese and this has been rigorously incorporated into the two volumes. It is inevitable that this bank of knowledge will be revised and expanded. The third edition of Cheese: Chemistry, Physics and Microbiology provides the base upon which these revisions and expansions can be undertaken objectively. N.E Olson Department of Food Science, University of Wisconsin, Madison
List of Contributors
Professor M.H. Abd E1-Salam
Dairy Department National Research Centre Dokki Cairo Egypt
Professor M. Carie University of Novi Sad Faculty of Technology Bulevar Cara Lazara 1 Novi Sad Serbia and Montenegro
Professor E. Alichanidis
Professor T.M. Cogan
Laboratory of Dairy Technology School of Agriculture Aristotle University of Thessaloniki 541 24 Thessaloniki Greece
Dairy Products Research Center Teagasc, Moorepark Fermoy Co. Cork Ireland
Professor Y. ArdO
The Royal Veterinary and Agricultural University Department of Dairy and Food Science Rolighedsvej 30 1958 Frederiksberg C Denmark
Dr L.K. Creamer
Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand
Dr H.P. Bachmann
Agroscope kiebefeld-Posieux Swiss Federal Institute for Animal Production and Dairy Products Schwarzenburgstrasse 161 CH-3003 Bern Switzerland
Dr u L. Crow
Mr R.J. Bennett
Dr E.-M. D~sterh6ft
Institute of Food Nutrition and Human Health Massey University Palmerston North New Zealand
Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand
NIZO Food Research PO Box 20 6710 BA Ede The Netherlands
Dr N.M. Brennan
Dairy Products Research Centre Teagasc, Moorepark Fermoy Co. Cork Ireland
Professor N.Y. Farkye
Dairy Produce Technology Center California Polytechnic State University San Luis Obispo CA 93407 USA
Dr M.D. Cantor
Danisco A/S Innovation Langebrogade 1 1001 Copenhagen K Denmark
Professor RE Fox
Department of Food and Nutritional Sciences University College Cork Ireland
x
List of Contributors
Dr M.T. Frbhlich-Wyder Agroscope Liebefeld-Posieux Swiss Federal Institute for Animal Production and Dairy Products Schwarzenburgstrasse 161 CH-3003 Bern Switzerland Dr J. Gilles Deceased 19 January 2003 (Retired from the New Zealand Dairy Research Institute.) Professor M. Gobbetti
Dipartimento di Protezione delIe Piante e Microbiologia Applicata Universita di Bari Via G. Amendola 165/a 70126 Bari Italy Dr J.-C. Gripon Unit4 de Biochimie et Structure des Prot4ines Instituto National de La Recherche Agronomique 78350 Jouy-en-Josas France Dr T.P. Guinee Dairy Products Research Centre Teagasc, Moorepark Fermoy Co. Cork Ireland Dr T.K. Hansen
The Royal Veterinary and Agricultural University Department of Dairy and Food Science Rolighedsvej 30 1958 Frederiksberg C Denmark Dr H.A. Heap Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand Dr C.G. Honor4 Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand
Dr M. Kalab Agriculture and Agri-Food Canada Food Research Program Guelph Ontario, K1A OC5 Canada Dr K.N. Kilcawley Dairy Products Research Centre Teagasc, Moorepark Fermoy Co. Cork Ireland Dr R Kindstedt Department of Nutrition and Food Sciences University of Vermont Burlington VT 05405-0044 USA Dr R.C. Lawrence 23 Pahiatua Street PaImerston North New Zealand (Retired from the New Zealand Dairy Research Institute.) Dr M. Loessner Technical University of Munich 21 EL, Abtilung Microbiologia Weihenstephan D-85354, Freising Germany Dr P.L.H. McSweeney Department of Food and Nutritional Sciences University College Cork Ireland Dr M. Medina
Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA) Crta. de la Corufia km. 7,5 28040 Madrid Spain Dr W.C. Meijer NIZO Food Research PO Box 20 6710 BA Ede The Netherlands
Mr K.A. Johnston
Professor S. Milanovi¢
Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand
University of Novi Sad Faculty of Technology Bulevar Cara Lazava 1 Novi Sad Serbia and Montenegro
List of Contributors
Dr M. Nufiez Instituto NacionaI de Investigacion y Tecnologia Agraria y Alimentaria (INIA) Crta. de la Corufia, km. 7,5 28040 Madrid Spain Professor G. Ottogalli Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche Sezione di Microbiologia Agraria Alimentare Ecologica Via G. Celoria 2 20133, Milano Italy
Dr B. Senge Technische Universit~t Berlin Faculty of Process Sciences Department of Food Rheology K~3nigin-.Luise-Str. 22 Sekr. KL-H 1 D- 14195 Berlin Germany Professor G. Smit NIZO Food Research PO Box 20 6710 BA Ede The Netherlands
Dr RK. Samal
Dr H.-E. Spinnler
Britannia Industries Limited Britannia Gardens Airport Road Bangalore 560 017 India (Formerly of Fonterra Research Centre, Private Bag 11 029, Dairy Form Road, Palmerston North, New Zealand.)
Laboratoire de Genie et Microbiologie des Procedes Alimentaires Instituto National de La Recherche Agronomique 78850 Thiverval-Grignon France
Professor S. Scherer Technical University of Munich 21EL, Abtilung Microbiologia Weihenstephan D-85354, Freising Germany Dr D. Schulz-Collins Arrabawn Co-Op. Nenagh Co. Tipperary Ireland
Dr G. van den Berg NIZO Food Research PO Box 20 6710 BA Ede The Netherlands Dr T. van den Tempel Chr. Hansen MS Cheese Culture Technology Boge Alle 10-12 2970 H~rshotm Denmark
xi
Preface to the First Edition
Cheese manufacture is one of the classical examples of food preservation, dating from 6000-7000 BC. Preservation of the most important constituents of milk (i.e. fat and protein) as cheese exploits two of the classical principles of food preservation, i.e.: lactic acid fermentation, and reduction of water activity through removal of water and addition of NaC1. Establishment of a low redox potential and secretion of antibiotics by starter microorganisms contribute to the storage stability of cheese. About 500 varieties of cheese are now produced throughout the world; present production is ---107 tonnes per annum and is increasing at a rate of ---4% per annum. Cheese manufacture essentially involves gelation of the casein via iso-electric (acid) or enzymatic (rennet) coagulation; a few cheeses are produced by a combination of heat and acid and still fewer by thermal evaporation. Developments in ultrafiltration facilitate the production of a new family of cheeses. Cheeses produced by acid or heat/acid coagulation are usually consumed flesh, and hence their production is relatively simple and they are not particularly interesting from the biochemical viewpoint although they may have interesting physico-chemical features. Rennet cheeses are almost always ripened (matured) before consumption through the action of a complex battery of enzymes. Consequently they are in a dynamic state and provide fascinating subjects for enzymologists and microbiologists, as well as physical chemists. Researchers on cheese have created a very substantial literature, including several texts dealing mainly with the technological aspects of cheese production. Although certain chemical, physical and microbiological aspects of cheese have been reviewed extensively, this is probably the first attempt to review comprehensively the scientific aspects of cheese manufacture and ripening. The topics applicable to most cheese varieties, i.e. rennets, starters, primary and secondary phases of rennet coagulation, gel formation, gel syneresis, salting, proteolysis, theology and nutrition, are reviewed in Volume 1. Volume 2 is devoted to the more specific aspects of the nine major cheese families: Cheddar, Dutch, Swiss, Iberian, Italian, Balkan, Middle Eastern, Mould-ripened and Smear-ripened. A chapter is devoted to non-European cheeses, many of which are ill-defined; it is hoped that the review will stimulate scientific interest in these minor, but locally important, varieties. The final chapter is devoted to processed cheeses. It is hoped that the book will provide an up-to-date reference on the scientific aspects of this fascinating group of ancient, yet ultramodern, foods; each chapter is extensively referenced. It will be clear that a considerably body of scientific knowledge on the manufacture and ripening of cheese is currently available but it will be apparent also that many major gaps exist in our knowledge; it is hoped that this book will serve to stimulate scientists to fill these gaps. I wish to thank sincerely the other 26 authors who contributed to the text and whose co-operation made my task as editor a pleasure. RE Fox
Preface to the Second Edition
The first edition of this book was very well received by the various groups (lecturers, students, researchers and industrialists) interested in the scientific and technological aspects of cheese. The initial printing was sold out faster than anticipated and created an opportunity to revise and extend the book. The second edition retains all 21 subjects from the first edition, generally revised by the same authors and in some cases expanded considerably. In addition, 10 new chapters have been added: Cheese: Methods of chemical analysis; Biochemistry of cheese ripening; Water activity and the composition of cheese; Growth and survival of pathogenic and other undesirable microorganisms in cheese; Membrane processes in cheese technology, in Volume 1 and North-European varieties; Cheeses of the former USSR; Mozzarella and Pizza cheese; Acid-coagulated cheeses and Cheeses from sheep's and goats' milk in Volume 2. These new chapters were included mainly to fill perceived deficiencies in the first edition. The book provides an in-depth coverage of the principal scientific and technological aspects of cheese. While it is intended primarily for lecturers, senior students and researchers, production management and quality control personnel should find it to be a very valuable reference book. Although cheese production has become increasingly scientific in recent years, the quality of the final product is still not totally predictable. It is not claimed that this book will provide all the answers for the cheese scientist/technologist but it does provide the most comprehensive compendium of scientific knowledge on cheese available. Each of the 31 chapters is extensively referenced to facilitate further exploration of the extensive literature on cheese. It will be apparent that while cheese manufacture is now firmly based on sound scientific principles, many questions remain unanswered. It is hoped that this book will serve to stimulate further scientific study on the chemical, physical and biological aspects of cheese. I wish to thank sincerely all the authors who contributed to the two volumes of this book and whose cooperation made my task as editor a pleasure.
RE Fox
Preface to the Third Edition
Very considerable progress has been made on the scientific aspects of cheese since the second edition of this book was published in 1993. This is especially true for the Microbiology of Cheese and the Biochemistry of Cheese Ripening; consequently those sections have been expanded very considerably. The general structure of the book is similar to that of the earlier editions, with the more general aspects being treated in Volume 1 and the more applied, variety-related aspects in Volume 2. The book contains 36 chapters. Reflecting the very extensive research on cheese starters in recent years, four chapters have been devoted to this topic in the third edition. Another new feature is the inclusion of two chapters on cheese flavour; one on sensory aspects, the other on instrumental methods. In Volume 2 of the second edition, cheese varieties were treated mainly on a geographical basis. While some elements of the geographical distribution remain, cheese varieties are now treated mainly based on the characteristic features of their ripening. Obviously, it is not possible to treat all 1000 or so cheese varieties, but the 10 variety-related chapters in Volume 2 cover at least 90% of world cheese production and it is very likely that your favourite cheese is included in one of those 10 chapters. Cheese is the quintessential convenience food and is widely used as an ingredient in other foods and in the USA approximately 70% of all cheese is used as a food ingredient. The use of cheese as a food ingredient is a major growth area; consequently, a ch~ipter has been devoted to the important features of cheese as an ingredient, including a section on Enzyme-modified Cheese. Each chapter is extensively referenced to facilitate further exploration of the extensive literature on cheese. While the book is intended for primarily lecturers, senior students and researchers, production management and quality control personnel should find it to be a very useful reference book. We wish to thank sincerely all authors who contributed to the two volumes of this book and whose co-operation made our task as editors a pleasure. Special thanks are due to Ms Anne Cahalane for very valuable assistance. P.E Fox P.L.H. McSweeney T.M. Cogan T.P. Guinee
Diversity of Cheese Varieties: An Overview P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland G. Ottogalli, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia Agraria, Alimentare, Ecologica, Milano, Italy P.F. Fox, Department of Food and Nutritional Sciences, University College, Cork, Ireland
A great diversity of cheeses are produced from the same raw materials (usually bovine, ovine, caprine or buffalo milks, lactic acid bacteria (LAB), coagulant and NaC1); indeed it has been said that 'there is a cheese for every taste preference and a taste preference for every cheese' (Olson, 1990). Although cheesemaking is an ancient art (see 'Cheese: An Overview', Volume 1), modern cheese production relies on the application of much science and technology, including the use of industrial enzymes, complex fermentations, sophisticated engineering and a dynamic biochemistry during ripening. Indeed, if cheese was developed today, it would be hailed as a triumph of biotechnology! Cheese production has a long history (see 'Cheese: An Overview', Volume 1) which is reflected in the wide range of technologies used for their manufacture. The idea of protecting and preserving the traditional diversity of foods, including cheese, commenced at the Paris Convention of 1883 where the term Appellation d'Origine ContrOlee (AOC) was introduced to recognize the specific heritage of food products from particular regions, while guaranteeing product authenticity (Bertozzi and Panari, 1993). This concept became widespread in Europe and was replaced by the EU scheme, Protected Designations of Origin (PDO), which applies to foodstuffs which are produced, processed and prepared in a given geographical area using recognized technology. Foods with the designation 'Protected Geographical Indication' (PGI) have a geographical link with a particular region during at least one stage of production, processing or preparation while 'Foods with Tradition Speciality Guaranteed' (TSG) status have a traditional character, either in their composition or means of production. A number of cheeses have PDO status (e.g., Roquefort, Stilton, Manchego, Grana, Padano, Parmigiano Reggiano, Gruyere de Comt6). Unlike commercial trademarks, PDO denomination reflects a collective heritage and
may be used by all producers of a particular variety in a defined geographical area. PDO cheeses are protected by the European Union under various international agreements (Bertozzi and Panari, 1993). A list of cheeses with PDO status is shown in Table 1. Other varieties may be produced outside the country or region of origin, e.g., Cheddar, Emmental, Gouda, Gruyere and Camembert, but the name of the producing country is often included. The FAO/WHO has published standards for several major cheese varieties in various editions of Code of Quality Standards for Cheese forming part of the Joint FAO/WHO Codex Alimentarius (see www.codexalimentarius.net). The concept of a Codex Alimentarius evolved from a meeting of European Governments at the Italian city of Stresa in 1951 but the idea for such a Codex Alimentarius dates from the end of the nineteenth century; in the Austro-Hungarian Empire between 1897 and 1911, a collection of standards and product descriptions for a wide variety of foods was developed as the Codex Alimentarius Austriacus.
A considerable international trade exists in the principal varieties of cheese, many of which are produced in several countries but which may not be identical. To assist international trade, to provide nutritional information and perhaps for other reasons, e.g., research, a number of attempts have been made to develop classification schemes for cheeses. There is no definitive list of cheese varieties. Sandine and Elliker (1970) suggest that there are more than 1000 varieties. Jim Path (University of Wisconsin) has compiled a list of 1400 varieties of cheese (available at ww.cdr.wisc.edu). Walter and Hargrove (1972) described more than 400 varieties and listed the names of a further 400 varieties, while Burkhalter (1981) classified 510 varieties (although some are listed more than once).
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
2
Diversity of Cheese Varieties: An Overview
Cheeses with protected designations of origin (PDO) or protected geographical indication (PGI)
Country
Variety
PDO
Belgium
Fromage de herve
X
Denmark
Danablu Esrom
Germany
AIIg&uerBergk&se AIIg&uer Emmentaler Altenburger Ziegenk&se Odenw&lder Fr0hst(~cksk&se
X X X X
Anevato
X X
Greece
Batzos Feta Formaella Arachovas Parnassou Galotyri Graviera Agrafon Graviera Kritis Kalathakai Limnou Kasseri Katiki Domokou Kefalograviera Kopanisti Ladotyri Mytilinis Manouri Metsovone Pichtogalo Chanion San Michali Sfela Xynomyzithra Kritis
Spain
France
Cabrales Idiaz~bal Mah6n Pic6n Bejes-Tresviso Queso de Cantabria Queso de I'AIt Urgell y la Cerdanya Queso de La Serena Pic6n Bejes-Tresviso Queso de Murcia Queso de Murcia al vino Queso Majorero Queso Manchego Queso Palmero o Queso de la Palma Queso Tetilla Queso Zamorano Quesucos de Li~bana Roncal Abondance Beaufort Bleu d'Auvergne Bleu des Causses Bleu du Haut-jura,de Gex, de Septmoncel Bleu du Vercors Brie de Meaux Brie de Melun Brocciu Corse ou brocciu Cantal ou Forme de Cantal ou Cantalet Camembert de Normandie Chabichou du Poitou
PGI
Country
Variety
PDO
France
Chaource Comt~ Crottin de Chavignol ou Chavignol Emmental de Savoie Emmental fran~ais est-central Epoisses de Bourgogne Fourme d'Ambert ou fourme de montbrison Laguiole Langres Livarot Maroilles ou Marolles Mont d'or ou vacherin du Haut-Doubs Morbier Munster ou Munster-G~rom~ Neufch&tel Ossau-lraty P~lardon Picodon de I'Ardbche ou Picodon de la Dr6me Pont-I'Ev~que Pouligny-Saint-Pierre Reblochon ou reblochon de Savoie Rocamadour Roquefort Saint-Nectaire Sainte-Maure de Touraine Salers Selles-sur-Cher Tomme de Savoie Tomme des Pyrenees
X X X
Ireland
Imokilly Regato
X
Italy
Asiago Bitto Bra Caciocavallo Silano Canestrato Pugliese Casciotta d'Urbino Castelmagno Fiore Sardo Fontina Formai de Mut Dell'alta Valle Brembana Gorgonzola Grana Padano Montasio Monte Veronese Mozzarella di Bufala Campana Murazzano Parmigiano Reggiano Pecorino Romano Pecorino Sardo Pecorino Siciliano Pecorino Toscano Prouolone Valpadana Quartirolo Lombardo Ragusano
X X X X X X X X X X
X X X X
X X X X X
X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X
PGI
X X X X X X X X X X X X X X X
X X X X X X X X X X X
X X X X X X X X X X X X X X
Diversity of Cheese Varieties: An Overview
3
continued
Country
Variety
Italy
Raschera Robiola di Roccaverano Taleggio Toma Piemontese Valle d'Aosta Fromadzo Valtellina Casera
The Netherlands Boeren-Leidse met sleutels Kanterkaas,Kanternagelkaas, Kanterkimijnekaas Noord-Hollandse Edammer Noord-Hollandse Gouda Austria
Portugal
PDO
PGI
X X X X X X X X
X X X X
Queijo de Azeit&o Queijo de Cabra Transmontano Queijo de I~vora Queijo de Nisa Queijo do Pico
X X X X X
Variety
Portugal
Queijo Mestis de Tolosa Queijo Rabas Queijo S~.o Jorge Queijo Serpa Queijo Serra da Estrela Queijo Terrincho Queijos da Beira Baixa
Sweden
Svecia
United Kingdom
Beacon Fell Traditional Lancashire cheese Bonchester cheese Buxton Blue Dorset Blue cheese Dovedale cheese Exmoor Blue cheese Single Gloucester Swaledale cheese, Swaledale ewe's cheese Teviotdale cheese West Country Farmhouse Cheddar cheese White Stilton cheese, Blue Stilton cheese
X X
Gailtaler AlmkAse Tiroler Almk~ise/Tiroler Grauk&se Tiroler Bergk&se Tiroler Grauk&se Vorarlberger AlpkAse Vorarlberger Bergk&se
Country
X X
PDO
PGI
X X X X X X
X X X X X X
X X
Source: http://europa.eu.int/comm/agriculture/qual/en/pgi_01 en.html
However, many of these varieties are very similar and should be regarded as variants rather than varieties. Walter and Hargrove (1972) suggested that there are probably only about 18 distinct types of natural cheese, no two of which are made by the same method, i.e., they differ with respect to: setting the milk, cutting the coagulum, stirring, heating, draining, pressing and salting of the curds or ripening of the cheese. They listed the following varieties as typical examples of the 18 types: Brick, Camembert, Cheddar, Cottage, Cream, Edam, Gouda, Hand, Limburger, Neufchatel, Parmesan, Provolone, Romano, Roquefort, Sapsago, Swiss, Trappist and whey cheeses. The authors acknowledged the imperfection and incompleteness of such a classification scheme and indeed a cursory glance at the list of the examples highlights this, e.g., listing Edam and Gouda as clearly distinct families appears highly questionable while exclusion of Feta and Domiati and all heat-acid coagulated cheeses appears to be major omissions. Attempts to classify cheese varieties exploit a number of characteristics of the cheese: 9 texture, which is dependent mainly on moisture content; 9 method of coagulation as the primary criterion, coupled with other criteria; 9 ripening indices.
Classification schemes based on texture
The difficulties in classifying cheese varieties were discussed by Schulz (1952) who reviewed earlier attempts to do so. Schulz (1952) was critical of these earlier schemes because they relied excessively on knowledge of the manufacturing process. He proposed a modified scheme consisting primarily of five groups based essentially on moisture content (moisture in fat-free cheese, MFFC): dried (<40% MFFC), grated (40-49.9% MFFC), hard (50-59.9% MFFC), soft (60-69.9% MFFC) and fresh (70-82% MFFC). The fresh, soft, hard and grated groups were each sub-divided into two sub-groups (i.e., eight sub-groups) based on whether or not the cheeses were cooked and/or pressed. An interesting development was the sub-division of each of the eight sub-groups into six sub-sets (a-g) on the basis of the concentration of calcium in the fat-free, NaCl-free solids, which reflects the rate and extent of acidification: >2.5%, 2.1-2.5%, 1.6-2.0%, 1.1-1.5%, 0.6-1.0% and <0.6%. Davis (1965) discussed the problems encountered in attempting to classify cheese and suggested a number of possible schemes. One scheme (Table 2) was based on the rheological properties, or, more precisely, on the moisture content of the cheese. In fact, most schemes include a similar criterion. In a second scheme (Davis, 1965), cheeses were classified primarily into hard,
4
Diversity of Cheese Varieties: An Overview
Suggested classification of cheeses based on rheological propertiesa
Type
Moisture, %b
pV
pM
pS
Very hard Hard Semi-hard Soft
<25 25-36 36-40 >40
>9 8-9 7.4-8 <7.4
>6.3 5.8-6.3 <5.8 <5.8
>2.3 2-2.3 1.8-2 > 1.8
pV, viscosity factor, logarithmic scale; pM, elasticity factor, logarithmic scale; pS springiness factor, logarithmic scale. a From Davis (1965). b Suggested moisture levels appear to be very low.
semi-hard and soft (Table 3); varieties were listed within each category according to type of milk, method of coagulation, cutting of the coagulum, scalding of the curds, drainage of whey and method of salting and moulding. Walter and Hargrove (1972) classified cheese into eight families (Table 4). However, this scheme has a number of inconsistencies, e.g., traditionally, Brick and M~inster cheeses are smear-ripened varieties but are listed in category 3.1 and are thus separated from the other smear cheeses in category 3.2. Likewise, although Mysost and Primost are unripened (category 4.2), they are quite hard. The species from which the milk is obtained was not included. Burkhaher (1981) classified 510 varieties based essentially on three criteria (Table 5): species of dairy animal (cow, sheep, goat, buffalo), moisture content and characteristic ripening agent. Scott (1986) also classified cheeses primarily on the basis of moisture content, i.e., hard, semi-hard and soft, and sub-divided these groups on the basis of cooking (scalding) temperature and/or secondary microflora (Table 6). The mechanism of coagulation was not considered by Scott (1986) and rennet-, acid- or acid/heatcoagulated cheeses are included in some groups. An alternative classification scheme suggested by Prof P. Walstra is shown in Table VIII in Fox (1993). Innovations were the use of the water:protein ratio rather than moisture content as the primary criterion for classification and replacement of cooking temperature by starter type, i.e., mesophilic, thermophilic. Classification schemes based on method of coagulation
The fundamental event in cheese manufacture is the conversion of liquid milk to a visco-elastic gel (coagulum). In fact one of the three coagulating agents may be used: rennet, acid and acid/heat, which suggests a clear primary criterion for classification. Rather surprisingly, the mechanism of coagulation was not used as a
classification criterion until Fox (1993) suggested the classification of cheeses into super-families based on the coagulating agent: 9 Rennet cheeses: most major international varieties. 9 Acid cheeses: e.g., Cottage, Quarg, Queso Blanco, Cream cheese. 9 Heat/acid: e.g., Ricotta, Manouri, Sapsago, Ziger, Schottenziger, some forms of Queso Blanco. Rennet-coagulated cheeses represent ---75% of total cheese production and almost all ripened cheeses. Acid-curd cheeses ('Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels', Volume 1; 'Acid- and Acid/Rennet-Curd Cheeses', Volume 2) represent ---25% of total cheese production and are generally consumed fresh. Coagulation by a combination of heat and acid is used for a limited number of varieties, including Ricotta and Manouri. Traditionally, they were by-products produced from the whey obtained from rennet-coagulated cheeses although today they are also produced from mixtures of milk and whey or even milk alone (see 'Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acid-heat Coagulated Cheeses', Volume 2). A minor group of cheeses are produced in Norway by the concentration of whey and crystallization of lactose, e.g., Mysost. Fox (1993) suggested that the classification schemes of Davis (1965), Walter and Hargrove (1972) and Burkhaher (1981) can be applied to rennet-coagulated cheeses, which form the most complex family, but are not really applicable to the other two super-families since most are high-moisture, soft cheeses and most, normally, are not ripened. The classification scheme of Fox (1993) was expanded and modified by Fox et al. (2000). Rennetcoagulated varieties were subdivided into relatively homogeneous groups based on the characteristic ripening agent(s) or manufacturing technology. The most diverse family of rennet-coagulated cheeses are the internal bacterially ripened varieties which include most hard and semi-hard cheeses. The term 'internal bacterially ripened' is somewhat misleading since indigenous milk enzymes and residual coagulant also play important roles in the ripening of these cheese varieties. This group may be subdivided based on moisture content (extra-hard, hard or semi-hard), the presence of eyes or a characteristic technology (e.g., cooking/stretching of pasta-filata varieties or ripening under brine). Many varieties in large-scale industrial production are included in this group. Grana-type cheeses (extra-hard), which are often used in grated form, are characterized by a high cooking temperature during their manufacture ('Extra-Hard Varieties',
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6
Diversity of Cheese Varieties: An Overview
Classification scheme for cheeses according to Walter and Hargrove (1972)
1.
Very hard (grating) 1.1 Ripened by bacteria: Asiago (old), Parmesan, Romano, Sapsago, Spalen
2.
Hard
2.1 2.2 3.
Ripened by bacteria, without eyes: Cheddar, Granular, Caciocavallo Ripened by bacteria, with eyes: Emmental, Gruyere
Semi-soft
3.1 3.2
Ripened principally by bacteria: Brick, MC~nster Ripened by bacteria and surface micro-organisms: Limburger, Port du Salut, Trappist 3.3 Ripenedprincipally by blue mould in the interior: Roquefort, Gorgonzola, Danablu, Stilton, Blue Wensleydale
4.
Soft
4.1 Ripened:Bel Paese, Brie, Camembert, Hand, Neufchatel 4.2 Unripened: Cottage, Pot, Baker's, Cream, Ricotta, Mysost, Primost
Volume 2). Cheddar and British territorial varieties (for which the curds are often textured and dry-salted) are classified as hard or semi-hard internal bacterially ripened cheeses ('Cheddar Cheese and Related Drysalted Cheese Varieties', Volume 2). Internal bacterially ripened cheeses with eyes are further sub-divided on the basis of moisture content into hard varieties (e.g., Emmental; 'Cheese with Propionic Acid Fermentation', Volume 2) in which the eyes are formed by CO2 produced on fermentation of lactate by Propionibacterium freudenreichii subsp, shermanii or semi-hard (e.g.,
Edam and Gouda; 'Gouda and Related Cheeses', Volume 2) in which a few small eyes develop due to the formation of CO2 by fermentation of citrate by the LAB. Pastafilata cheeses (e.g., Mozzarella; see 'Pasta-Filata Cheeses', Volume 2) are characterized by stretching in hot water which texturizes the curd. White-brined cheeses, including Feta and Domiati ('Cheese Varieties Ripened in Brine', Volume 2), are ripened under brine and have a high salt content and, consequently, they are grouped together as a separate category within the group of internal bacterially ripened cheeses. Soft cheese varieties are usually not included in the group of internal bacterially ripened cheeses because they have a characteristic secondary microflora which has a major effect on the characteristics of these cheeses. Mould-ripened cheeses are subdivided into surface mould-ripened varieties (e.g., Camembert or Brie; 'Surface Mould-ripened Cheeses', Volume 2) in which ripening is characterized by the growth of Penicillium camemberti on the surface, and internal mould-ripened cheeses ('Blue Cheese', Volume 2) in which P. roqueforti grows throughout the cheese. Smear-ripened cheeses ('Bacterial Surface-ripened Cheeses', Volume 2) are characterized by the development of a complex microflora consisting of yeasts and, later, bacteria (particularly coryneforms) on the cheese surface during ripening. The classification scheme of Fox et al. (2000) is not without inconsistencies. For example, cheeses made from the milk of different species are grouped together (e.g., Roquefort and Gorgonzola are both Blue cheeses
Classification of cheese according to source of milk, moisture content, texture and ripening agents* 1.
Cow's milk
1.1
Hard (<42% H20)
1.1.1 Grating cheese (extra-hard) 1.1.2 Large round openings 1.1.3 Medium round openings 1.1.4 Small round openings 1.1.5 Irregular openings 1.1.6 No openings 2.
Sheep's milk Hard; semi-hard; soft; blue-veined; fresh
3.
Goat's milk
4.
Buffalo's milk
1.2
Semi-hard/ semi-soft (43-55% H20) 1.2.1 Small round openings 1.2.2 Irregular openings 1.2.3 No openings 1.2.4 Blue veined
1.3
Soft (>55% H20)
1.4 Fresh, rennet
1.3.1 Blue veined 1.3.2 White surface mould 1.3.3 Bacterial surface smear 1.3.4 No rind
* Modified from (Burkhalter, 1981); unless otherwise stated, the cheeses are internal bacterially ripened.
1.5 Fresh, acid
1.6 Fresh
Diversity of Cheese Varieties: An Overview
7
Classification of cheese according to moisture content, cooking temperature and secondary microflora a Hard cheese (moisture content 20-42%) Low-scald Ns
Medium-scald Ns
High-scald Ns or Pr
Plastic curds Ns or Pr
Edam (NL) Gouda (NL) Cantal (F) Fontina (I) Cheshire (UK)
Cheddar (UK) Glouchester (UK) Derby (UK) Leicester (UK) Svecia (S) Dunlop (UK) Turunmaa (SF)
Grana (Parmesan; I) Emmental (CH) Gruyere (CH) Beaufort (F) Herrgardsost (S) Asiago (I) Sbrinz (CH)
Scamorza (!) Provolone (I) Caciocavallo (I) Mozzarella (I) Kaaseri (Gr) Kashkaval (YU) Perenica (Cz)
Semi-hard cheese (moisture content 44-55%; low-scald) Ns
Bs
Bv
St Paulin (F) Caerphilly (UK) Lancashire (UK) Trappist (Bill) Providence (F)
Herve (B) Limburg (B) Romadur (G) MOnster (F) Tilsit (G) Vacherin-Mont d'Or (S) Remoudou (B) Srainbuskerkase (G) Brick (USA)
Stilton (UK) Roquefort (F) Gorgonzola (i) Danablu (D) Mycella (D) Wensleydale (UK) Blue Vinny (UK) Gammelost (N) Adelost (S) Tiroler-GraukAse (D) EdelpitzkAse (A) Aura (ice) Cabrales (E)
Soft cheeses (moisture content >55%; very low or no scald) Bs or Sm
Bel Paese (I) Maroilles (F)
Sm
Ns
Un, Ac
Brie (F) Camembert (F) Carre d'est (F) Neufchatel (F) Chaource (F)
Colwich (UK) Lactic (UK) Bondon (F)
Coulommier (F) York (UK) Cambridge (UK) Cottage (UK) Quarg Petit Suisse (F) Cream (UK)
Pr, propionic acid bacteria; Ns, normal lactic acid starter of milk flora; Bs, smear coat (Brevibacterium linens and other organisms) Sm, surface mould (R camemberti); Bv, blue-veined internal mould (R roqueforti); Ac, acid-coagulated; Un, normally unripened, fresh cheese. a Modified from Scott (1986).
but the former is made from sheep's milk and the latter from cows' milk). Of course, the scheme can be readily modified by subdividing relevant categories to indicate the type of milk used. The subdivision between hard and semi-hard cheeses is somewhat arbitrary and overlaps. Most varieties lose moisture during ripening by evaporation from the surface, i.e., develop a rind. Several varieties, e.g., Pecorino Romano and Montasio, are consumed after various lengths of ripening and hence may be classified as semi-hard, hard or extra hard, depending on age of cheese at consumption. There is also some cross-over between categories. Gruyere is classified as an internal bacterially ripened
variety with eyes but it is also characterized by the growth of a surface microflora, while some cheeses classified as surface-ripened (e.g., Havarti and Port du Salut) are often produced without a surface microflora and thus are, in effect, soft, internal bacterially ripened varieties. Fox et al. (2000) considered pasta-filata and high-salt varieties as separate families because of their unique technologies (stretching and ripening under brine, respectively) but they are actually ripened by the same agents as other internal bacterially ripened cheeses. However, the scheme of Fox et al. (2000) is a useful basis for classification; the arrangement of topics within this volume largely follows this scheme.
8
D i v e r s i t y of C h e e s e Varieties: A n O v e r v i e w
Major omissions from the scheme of Fox et al. (2000) are processed cheeses, cheese-based products (cheese powders, enzyme-modified cheese), cheese analogues and cheese substitutes. Processed cheese products represent ---14% of world cheese production and thus surpass the production of most natural varieties except Cheddar, Gouda, Mozzarella and Camembert. None of the classification schemes referred to above includes processed cheeses - it would seem reasonable to include them as a separate category. From the discussion in 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2, it will be apparent that this is a very diverse group of products with respect to raw material, process technology and composition. One could also argue that each class of the other cheese-based products, which are described in 'Cheese as an Ingredient', Volume 2, warrants inclusion and of course this can be accommodated readily. It must be remembered that the dried and enzyme-modified cheeses are very heterogeneous groups. Although cheese analogues may not be considered to be authentic cheese products, there seems to be no
valid reason for their exclusion. They are usually based on dry rennet casein into which lipids and water are emulsified or absorbed, respectively. Their production involves many of the operations used for other types of cheese, e.g., rennet coagulation, cooking, syneresis (as for natural rennet-coagulated cheeses), heating and emulsification, packaging (as for processed cheese). Since they are not ripened, it seems reasonable to classify cheese analogues as 'processed unripened cheese'. The principal among such cheeses at present is analogue pizza cheese. A modified version of the classification scheme of Fox et al. (2000) is shown in Fig. 1, incorporating processed cheese, cheese-derived products and cheese analogues. Probably the most comprehensive classification scheme for cheese developed to date is that of Ottogalli (1998, 2000a,b, 2001) which organizes cheeses into three main groups (indicated by the Latin words: 'Lacticinia' (milk-like), 'Formatica' (shaped), 'Miscellanea' (miscellaneous; Table 7). The Lacticinia group includes products which are produced from milk, cream, whey or
Cheese Analogues
tt
"l
Enzyme-ModifiedCheese
~
~.
Acid-Coagulated
Ricotta
I
Cottage, Cream, Quarg
i
Dried Cheeses
Heat/Acid Coagulation
Cheese
Processed Cheese
Rennet-Coagulated
9
Concentration/CrystallizatiOnMysost
Most varieties of cheese may be processed
Natural Cheese
I Surface-ripened
Mould-ripened
Internal bacterially ripened
Havarti Limburger MOnster Port du Salut Trappist Taleggio Tilsit
I Surface mould
(usually P. camemberti ) Brie Camembert
Internal mould (P. roqueforti) Roquefort Danablu Stilton
Cheeses with eyes Hard Grana Padano Parmesan Asiago Sbrinz
Cheddar Cheshire Graviera Ras
Caerphilly Mahon Monterey Jack
Swiss-type (Lactate metabolism by Propionibacterium spp.) Emmental Gruyere Maasdam
High-salt varieties Domiati Feta
Dutch-type (Eyes caused by citrate metabolism)
Pasta-filata varieties Mozzarella Kashkaval Provolone
Edam Gouda
The diversity of cheese. Cheese varieties are classified into super-families based on the method of coagulation and further sub-divided based on the principal ripening agents and/or characteristic technology (modified from Fox et al., 2000).
Diversity of Cheese Varieties: An Overview
9
Classification of cheeses according to Ottogalli (1998, 2000a,b, 2001)
~5
Farn~
Class
Description
Examples
Yoghurt-like product, but with loss of some whey
Lebneh (Middle East); Fromage Blanc (Switzerland, France); Sauer-milchk&se, Quarg (Germany) Queso Blanco (Latin America); Cottage (UK, USA); Quarg (Germany); Tvorog (Poland) Whey cheese (UK); Ricotta (Italy); Manouri (Former Yugoslavia); Brunost, Getost (Norway) Whey cheese (UK); Ricotta (Italy); Ziger (Germany); Mysost (Norway) Mascarpone (Italy) Skyr (Iceland); Karish (Egypt); Buttermilk Quark (Germany); Aoules (Algeria); Kolostrumkase (Germany); Sa Casada (Italy), Armada (Spain)
Milk coagulated by addition of organic acid
c O. .m
Acid addition and heating of whey (goat or ewe) Acid addition and heating of whey (cow) Acid addition and heating of cream Acid addition and heating of buttermilk
o rL. LL
Acid addition and heating of colostrum or beestings Acid-rennet coagulation I
Rennet-acid coagulation c .m
c O.
I
I
~ c o II
Goat or sheep
~~Fresh-kneaded or plastic or stretched cheeses
r -g .~ gg O.
Coagulum cut into cubes and/or flakes cooked, drained, washed and water cooled Rindless, very short ripening phase Thin rind, short ripening (<1 month)
O c or) -~_ I
t-
(~
O
~r'-
c ~ II
m
Same as C1 or C2 but from goats' or ewe's milk
~ c Kneaded curds
oN "o c
gl i.. 0 .C or)
~ iI ::::3 ,,-0 .~,0 4-, X 9
-~
White-brined
Petit Suisse, Pates fraiches (France); Frischkase Quargel (Germany); Cream cheese (USA) Gervais T M (France); Jonchee, Caillebotte (France); Primo sale (Italy). Caprino (Italy); Goat cheese (UK); Cadiz, Soria, Villeria (Spain); Bruscion (Switzerland) Mozzarella di bufala, Fiordilatte (Italy); Oaxaca (Mexico); Pizza cheese (America) Cottage (UK, USA); Huttenkase (Germany); Farkost (Sweden) Crescenza (Italy); Butterkase (Austria); Cremoso (Argentina) Caciotta, Italico, Bel Paese T M (Italy); St. Paulin, Port Salut T M (France); Tetilla (Spain); Passendale (Belgium); Caerphilly (UK); Richelieu (Canada) Burgos, Azeitao, Puzol, Villalon (Spain); Capricorn goat (UK); Robiola di Roccaverano (Italy) Scamorza (Italy); Cascaval (Romania); Ostiepok (Czech Republic) Feta (Greece); Telemes (Romania); Domiati (Egypt); Brinza (Israel); Peynaz peynir (Turkey); Surati panir (India); Halloumi (Cyprus); Lightvan (Iran) continued
10
Diversity of Cheese Varieties: An Overview
continued
(5
Class
Family
Description
Examples
White-moulded rind
Camembert, Caprice de Dieux, Brie, Coulommiers, Chource, Carre de I'Est (France); Bouchester (UK); Tomme de Vadois (Switzerland); Casanova (Denmark); Scimudin (Italy) Romadour (Belgium); Brick, Liederkranz (USA); Havarti, Esrom (Denmark); Epoisses, Langres, Livarot, Maroilles, MOnster (France); Kernhem (The Netherlands); Ridder (Norway); Vacherin Mont d'Or (Switzerland); Limburger (Germany) Crottin, Chabichou, Bouche de Chevre, Pouligny, Saint Maure, Rocamadour (France); Altenburger (Germany); Capricorn goat (UK) Taleggio, Quartirolo, Robiola (Italy); Chaumes, Pont I'Eveque, Reblochon (France)
0 I c~ 0
II
Smear surface ~ Eu~
~=~_ "~-5 ~-
0
II ._I
E--
0.,~_ "~- 0
Same as D1 or D2 or D4 but goats' or ewes' milk
0
o~
Mould-ripened (white or blue) and smeary surface
I
u')
~
x: II
Cows' milk
L U ~ -o "r-
r"
m
~
White moulded rind
( D o 0 > ~'~ "~- L_"
Ewes' or goats'
~o~ N
9"~
m ~
~, ..=
~9 E I-r I
E
Untextured, usually semi-cooked and pressed
C 0 (I) "O
~ c(D • II c- ~
Washed curd (eyes caused by citrate metabolism or by heterolactic bacteria) Same as F1 but from goats' or ewes' milk
u~ E - ~ ~ 0 ._0 C~ ~.C_
c
c
9
0
Kneaded curds ('pasta filata') Propionic cheeses. Big round eyes
Textured (and dry salted) curd c- ~ .& E II
(0--
Smeared rind
Buxton Blue, Stilton, Dovedale (UK); Gorgonzola (Italy); Danablu, Mycella (Denmark); Bergader (Germany); Gammelost (Norway); Adelost (Sweden); Bleu d'Auvergne, Bleu de Causses, Bleu de Gex, Bleu de Laqueille, Fourme d'Aubert (France); Cashel Blue (Ireland) Bleu de Bresse (France); Cambozola (Germany) Roquefort (France); Cabrales (Spain); Kopanisti (Greece); Castelmagno, Murianengo (Italy) Montasio, Raschera, Bettelmatt (Italy); Pinzgauer (Austria); Beaumont, Laguiole, Murol (France); Raclette (Switzerland); Trappisten (Germany) Edam, Gouda (The Netherlands); Fontal (Italy); Mimolette (France); Blarney (Ireland) Serra (PR); Orduna, Mahon (Spain); Ossau-lraty (France); Pecorini: Pecorino Toscano, Canestrato (Italy); Altemburger (Germany) Caciocavallo (Italy); Ostwepock, Kasseri (Greece); Oaxaca (Mexico) Maasdamer (The Netherlands); Fol Epi (France); Jarlsberg (Norway); Samsoe (Denmark); Pategras, Colonia (Argentina) Lancashire, Colby (UK). Leiden (The Netherlands), Monterey (USA) Fontina (Italy); Tilsit (Germany); Appenzeller (Swtzerland); Stinking Bishop (UK)
Diversity of Cheese Varieties: An Overview
11
continued
Class
Fam~ c 0 if)
~ c-
c 9
I
9
0
.=
9r-- II E ~- -b- o
~~ -8=8 =o.
Description
Examples
Untextured, usually cooked and pressed
Asiago d'Allevo, Grana (Italy); Reggianito (Latin America); Sbrinz (Switzerland) Edam, Gouda (The Netherlands) Pecorino Romano, Pecorino Sardo (Italy); Kefalotiri (Greece); Manchego, Idiazabal (Spain); Ras (Egypt) Provolone (Italy); Parenica (Russia); Kashkaval (Bulgaria); Kasar peyniri (Turchia) Emmental (Switzerland, France); Svembo, Danbo (Denmark); Kefalograviera (Greece) Cantal (France); Cheddar, Cheshire, Derby, Single Gloucester, Double Gloucester (UK); Monterey (USA) Gruyere (Switzerland, France); Puzzone di Moena (Italy); Tete de Moine (Switzerland)
Washed curd, long ripened Same as G1 but goats' or ewes' milk Kneaded curds ('pasta filata')
0
~_o ii
Cheeses with eyes
e-, -d_J .~ x
-r--
m
I
I-
o x o
(D O') O 0 c cO 0 .@.., O) 0 ::t: "~
~-~
~. .-=
Textured (and dry salted) curd ('Cheddaring') Smeared rind The microbial coat causes the development of strong aroma Melted Smoked Grated or fractionated Mixed with other ingredients (fruit, vegetables, spices)
Ripened or kept under particular conditions, i.e., 'Pickled cheeses'
"0O
E 0 0 e-
o
Obtained using special technologies (i.e., ultrafiltration, sterilization or finished cheese) Products similar to cheese and with non dairy ingredients
Processed cheese, Spread cheese, Sottilette TM Oak-smoked Cheddar (United Kingdom) 'Grating cheeses' Friesan Clove cheese (NL); Sage Derby (UK); Kummelkas~, K&se mit Champignons (Germany); Sapsago (Switzerland); Ciboulette (France) Devon Garland (United Kingdom); Bruss (Italy); Kopanisti (Greece); Tupi (Spain); Fromage fort (France) PhiladelphiaTM (USA); BelgioiosoTM (Italy)
'Imitation cheeses', Filled cheeses
1Index of maturation (IM) = soluble N • 100/total N. 2Index of lipolysis (IL) = free fatty acids • 100/total fat.
buttermilk by coagulation with acid (lactic or citric), with or without a heating step. However, a small amount of rennet is often used to increase the firmness of the coagulum (e.g., Quarg and Cottage cheese). The Lacticinia group contains one class (A) comprised of seven families. Family A1 includes yoghurt-like products from which some whey is removed. Family A2 contains somewhat similar products but from which a large volume of whey is removed and acid is added. Families A3 and A4 are whey cheeses produced by the combination of heat and acid (e.g., Ricotta) while cheeses in Families A5, A6 and A7 are similar to other products in the Lacticinia group except that they are made from cream, buttermilk or colostrum, respectively.
The second group, Formatica (Table 7), contains most cheese varieties, all of which are coagulated by rennet. This is a large heterogeneous collection of varieties which are divided into 6 Classes (B-G), based essentially on the moisture content and the extent of ripening, and 31 families. Classes B and C include fresh cheeses and varieties with a short ripening period, respectively. The cheeses in Class D are soft surface-ripened varieties with a surface growth of moulds or smear bacteria. Blue cheeses are grouped in Class E while Classes F and G contain semi-hard and hard/extra-hard varieties, respectively. The third group of cheeses, Miscellanea (Table 7), is a heterogeneous collection of varieties and includes
12
Diversity of Cheese Varieties" An Overview
processed, smoked, grated and pickled cheeses, cheeses containing non-dairy ingredients (fruit, vegetables, spices), cheese analogues and cheeses made using ultrafiltration technology. The scheme of Ottogalli (1998, 2000a,b, 2001) takes into consideration the technological, chemical, microbiological and organoleptic characteristics of different cheese varieties, with the objective of a better classification of cheeses and related fermented dairy products into distinct categories. Chemical indices, which were given particular importance in the development of this classification scheme, included index of maturation (IM = soluble N • 100/total N, which can range from 1-2 to 60-70% although data for many cheeses are not available), lipolytic index (LI = free fatty acids • 100/total fat, which can range from 1-2 to 15-20%, although data for many cheeses are lacking) and fat:protein ratio (high fat = 2-5, medium fat = 1.2-1.5, low fat = <0.8). The organoleptic and microbiological characteristics of the families of cheeses in Table 7 are summarized in Table 8. According to this classification, cheeses and related products can be presented as in Table 7 or as in Fig. 2. An advantage of the system of Ottogalli (1998, 2000a,b, 2001) is that it allows the comparison of
cheeses from all over the world and the classification of products with similar characteristics. A disadvantage stems from the detailed and sharp sub-division of cheeses which necessitates exact knowledge of their technology. In addition, some products may move from one category to another during ripening (e.g., varieties which are consumed as semi-hard cheeses early in ripening but later become extra-hard varieties), and some varieties which are in fact quite different (e.g., white-mould cheeses and smear-ripened cheeses) are in the same class (D), although in different families. In addition, Quarg and Queso Blanco are placed in different families whether they are made with (B2) or without (A1) rennet. Finally, cheeses made from ultrafiltration retentate are grouped together in Family H6 although they may in fact be quite similar to cheeses in other families made using traditional technology. Classification based on ripening indices
Davis (1965) suggested the possibility of classifying cheese according to the extent of chemical breakdown during ripening and expressed the view that it might be possible within a few years (from 1965) to classify cheese on the basis of chemical fingerprints; nearly 40
Organoleptic and microbiological characteristics of the families of cheese described in Table 7 (Ottogalli, 1998, 2000a,b, 2001). See Table 7 for descriptions of the classes and families of cheese Cheeses character
Soft
Rind
Body Semi-hard and hard
Rind
Microflora
Rindless Rind with white surface mould Rind with smeared surface Rind with mould and smeared surface
No openings Blue-veined Brushed and cleaned during ripening Absence of cleaning operations
Body
No openings
Small openings
Large openings
Penicillium camemberti Geotrichum candidum Red-orange bacteria Penicillium spp. (or other moulds) and red-orange bacteria Lactic acid bacteria Penicillium roqueforti Microflora usually irrelevant
Relevant microflora (mainly moulds) Relevant microflora (mainly bacteria) Homofermentative lactococci and lactobacilli Heterofermentative lactococci and lactobacilli Propionic acid bacteria
Family~class
A,B D1, D3 D2, D3 D4
B-G El, E2, E3 F1, F2, F3, F5, F6 G1, G2, G3, G5, G6 F4, G4 F7, G7 F1, F3, F4, F6 G1, G3, G4, G6 F2, G2
F5, G5
Diversity of Cheese Varieties: An Overview
13
Examples of cheese from the principal groups of Ottogalli (1998, 2000a,b, 2001); see Table 7 for further details. (See
Colour plate 1.)
years later it is still not possible to do so reliably although some progress has been made in this area. An obvious problem encountered when attempting to fingerprint cheeses chemically arises from the fact that ripening cheese is a dynamic system and therefore the age at which the cheeses are fingerprinted creates a major problem of definition. Within any particular variety there is considerable variability with respect to any particular characteristic for several reasons, including the type (specificity) of the rennet, the activity and specificity of several enzymes from the primary starter, secondary starter or adventitious bacteria, the differences in composition, including zonal differences due to salt diffusion and/or the evaporation of water. At present, there is insufficient information, even on the major varieties, to permit such a chemical fingerprinting. However, it seems worthwhile to speculate on some possible methods and criteria that might be useful for the classification of cheese. The most effective analytical methods are:
9 Urea-polyacrylamide gel electrophoresis (PAGE) for resolving and identifying the large, water (or pH 4.6)-insoluble peptides. Sodium dodecyl sulphate (SDS)-PAGE or capillary electrophoresis should also be effective but to date have been used much less widely than urea-PAGE. 9 Reverse-phase (RP)-HPLC for resolving and perhaps identifying small, water (pH 4.6)-soluble peptides. Interfacing RP-HPLC and mass spectrometry (MS) should greatly facilitate the identification of small peptides. However, LC/MS is rarely used, possibly owing to cost. 9 The free amino acid profile of cheese may be a useful criterion for classification. While there is a considerable amount of information on the concentration of amino acids in a number of cheeses (see Fox and Wallace, 1997), we are not aware of its use as a criterion for cheese classification. 9 Profile of volatile compounds as determined by GC or GC-MS; attempts to classify cheeses based on their volatile flavour compounds are discussed in more
14
Diversity of Cheese Varieties: An Overview
detail in 'Cheese Flavor: Instrumental Techniques', Volume 1. Since many cheese varieties contain the same volatile compounds and many of the same proteolytic products, albeit at different levels (i.e., different varieties do not possess unique compounds), multivariate statistical approaches to data handling seem the most-promising. Most cheese classification schemes are based on, or include, an item for texture (and thus on moisture and fat content). Texture is usually assessed subjectively or indirectly by determination of moisture content. Classification schemes based on rheological measurements would be precise and sensitive. Davis (1965) recommended such a scheme (Table 2). A number of chemical or physico-chemical studies have been performed to compare different cheese varieties (e.g., Smith and Nakai, 1990; Martin-Hernandez et al., 1992; Fox, 1993; McGoldrick and Fox, 1995; Dewettinck et al., 1997; Dirinck and De Winne, 1999; Dufour et al., 2001; Manca et al., 2001) or to distinguish between cheeses of the same variety differing in age or quality attributes (e.g., Fritsch etal., 1992; Rohm, 1992; O'Shea et al., 1996; Garcia-Palmer et al., 1997; Frau et al., 1998; Contarini et al., 2001; Peres et al., 2002).
The objectives of Volume 2 of this book are to discuss the chemistry, physics and microbiology of the manufacture and the ripening of the major groups of cheese. Discussion of different cheese varieties generally follows the modified classification scheme of Fox et al. (2000; Fig. 1). In addition chapters are included on general aspects of cheese technology, processed cheese products, cheeses made from sheep's and/or goats' milk and uses of cheese as a food ingredient, including a brief discussion of enzyme-modified cheese. The remainder of this chapter will serve as an introduction to this volume by providing brief outlines of the science and technology of major groups of cheese. Most of these groups were reviewed in chapters in the second edition of this book, usually by authors from the same institution. Some groups of cheeses reviewed in the second edition have been omitted, e.g., Iberian cheeses, Italian cheeses, North European varieties, varieties produced in the Balkans and former USSR and non-European cheeses. However, these changes are due more to rearrangements than to omissions; the principal varieties from the above regions are covered under other headings, hopefully in a more objective way.
Extra-hard varieties
Extra-hard cheeses ('Extra-Hard Varieties', Volume 2) include a number of varieties which are ripened for a long period (usually 6-24 months). They are characterized by a hard granular texture, an aromatic flavour which can range from delicate to strong, very suitable for grating and are usually used as condiments for other foods, like pasta, as a topping or as a seasoning. Granatype cheeses, which have a brittle, grainy texture when mature, are made from raw cows' milk which is partially skimmed; the starters used are thermophilic lactobacilli (often as a whey culture) and the curds are scalded in the vat at 50-55 ~ for 20-30 min. During the long ripening period (c. 2 years), the temperature must not exceed 20 ~ (to avoid fat liquefaction or 'sweating' and a propionic acid fermentation) and the rind is brushed and oiled frequently. The best known extra-hard cheeses are the Italian 'Grana' types (Grana Padano, Granone Lodigiano, Parmigiano Reggiano), Asiago, Bagozzo, Bra, Formai de Mut; in addition, the 'Pecorino' cheeses (Pecorino Romano, Pecorino Sardo, Pecorino Siciliano, Pecorino Toscano, Pecorino Pepato, Fiore Sardo), which are made from ewes' milk, are included in this group, as are the Swiss varieties, "fete de Moine, Sbrinz, Sapsago, the Spanish cheeses, Cebrero, Pedroches and Manchego, the Greek cheeses, Kefalotiri and Gravera and Reggianito from South America. It must be emphasized that many of these cheeses may be consumed as hard or semi-hard cheeses at an earlier stage of ripening. Cheddar and related varieties
Cheddar cheese originated in England and is one of the most important cheese varieties made worldwide (see 'Cheddar Cheese and Related Dry-salted Cheese Varieties', Volume 2). It is a hard cheese, usually made from pasteurized, standardized cows' milk which is coagulated using calf rennet or a rennet substitute. A mesophilic starter (usually defined strains of Lactococcus) is used to acidify the milk, and the coagulum is cut and cooked to 37-39~ The drained curds are 'cheddared', which traditionally involves forming beds of drained curds along the sides of the vat, cutting the beds into blocks and inverting and piling the blocks of matted curds at regular intervals. The cheddaring process allows time for acidity to develop in the curds (pH decreases from c. 6.1 to 5.4) and places the curds under gentle pressure, which assists in whey drainage. The curd granules fuse during cheddaring and the texture of the curd mass becomes rubbery and pliable. When the pH has reached c. 5.4, the blocks of curd are milled into small chips and dry-salted. The salted curds are moulded and pressed overnight. Traditionally, Cheddar cheese was ripened in
Diversity of Cheese Varieties: An Overview
insulated rooms without temperature control. However, more recently, Cheddar is matured at 4-8 ~ (although a higher temperature, up to 14 ~ is used occasionally) for a period ranging from "-3 months to ->2 years, depending on the maturity desired. Although the traditional manufacturing procedure is still practised on a farmhouse level and in small factories, most Cheddar cheese is now manufactured in highly automated factories using multiple vats which provide a semi-continuous supply of cheese curd. Cheddaring is mechanized using a large tower in which the curds at the bottom are pressed gently by the weight of that above or using a belt system. Milling and salting are also mechanized. Pressing and moulding are done automatically using a 'block former' (a large tower in which the salted curds are compressed by their own weight and a close texture is ensured by applying a vacuum). Most Cheddar is now produced in block form, although traditional Cheddar cheeses were cylindrical, weighing 10-20 kg. Annatto or similar colorant may be added to the milk for Cheddar cheese; the resulting product is known as 'Red' Cheddar. The British Territorial varieties, Cheshire, Derby, Gloucester and Leicester, are dry-salted cheeses manufactured by a protocol similar to that for Cheddar cheese. Cheese with propionic acid fermentation
Cheeses with a propionic acid fermentation (see Volume 2) are characterized by the presence of many large (up to ---2 cm in diameter) round openings, called 'eyes', due to the metabolic activity of propionic acid bacteria which metabolize lactate, produced by LAB from lactose, to propionic acid, acetic acid, CO2 and H20; they also contribute to the development of the typical mild, nutty flavour of these varieties. For proper eye development, at least three conditions are necessary: 9 ripening of the cheese at 20-24 ~ for a period to permit the rapid growth of propionic acid bacteria and to soften the cheese for eye development; 9 a relatively low level of salt to which the propionic acid bacteria are very sensitive; 9 the physical properties of the curd, which must be sufficiently elastic and flexible to contain the gas and form the eyes. Emmental, which was first manufactured in the Emm valley in Switzerland, is traditionally made from raw milk acidified by thermophilic LAB but this cheese is now produced in Switzerland, France, Germany, USA, Finland and elsewhere. The curds are cooked at c. 54 ~ which denatures most of the rennet. Immediately after cooking, the curds are moulded and acidification
15
occurs mainly after whey drainage, leading to a high level of calcium in the cheese which, together with the low rennet activity caused by the high cooking temperature (which results in a low level of proteolysis), gives the cheese an elastic texture. Emmental cheese is ripened for at least 4 months, including "--3-6 weeks at c. 22 ~ for eye formation. Semi-hard cheeses with a propionic acid fermentation include Maasdamer, Leerdamer and Jarlsberg. Some other cheeses, e.g., Gruyere, may have eyes but they are not essential. Gouda and related varieties
Gouda cheese originated in The Netherlands but it, and similar varieties, is now produced worldwide from pasteurized cows' milk acidified by a mesophilic starter containing citrate-positive bacteria (see 'Gouda and Related Cheeses', Volume 2). The milk is coagulated using calf rennet or a rennet substitute and, after the coagulum has been cut, the curds and whey mixture is stirred for 20-30 min. Some (c. 30%) of the whey is then removed and replaced by hot water which has the effect of cooking the curds and removing some lactose (which helps to control the development of acidity after the curds are moulded). After cooking at 36-38 ~ and whey drainage, the curds are pressed under whey before being moulded, pressed and brine-salted. Traditionally, Gouda is coated with yellow wax and matured for 2-3 months at c. 15 ~ (although some are ripened much longer, e.g., up to 2 years). Gouda is an internal, bacterially ripened cheese, the ripening of which is also characterized by the catabolism of citrate to diacetyl, other volatile flavour compounds and CO2. The CO2 produced causes a few small eyes in the cheese. Edam is a Dutch variety similar to Gouda but is made from semi-skimmed milk (c. 2.5% fat). It has a characteristic spherical shape and, traditionally, is covered with red wax. Other Dutch-type varieties include Maribo and Danbo (Denmark), Colonia and Hollanda (Argentina), Norvegia (Norway) and Svecia (Sweden). Pasta-filata cheeses
Pasta-filata varieties (see Volume 2) are also known as 'kneaded' or 'plastic curd' cheeses, the curds for which are heated to c. 55-60 ~ kneaded and stretched. Pasta-filata cheeses are characterized by a unique texture which is malleable, smooth, fibrous and sliceable. These qualities arise mainly from the cooking/stretching step which is common to all these varieties, whether they are soft, semi-hard or hard. By far the most important pasta-filata cheese is Mozzarella which originated in southern Italy and was made originally from buffalo milk (Mozzarella di bufala).
16
Diversity of Cheese Varieties: An Overview
Mozzarella di bufala is still made on a small scale but most Mozzarella is made from pasteurized, partly skimmed cows' milk and is often referred to as Pizza cheese or, in the United States, low-moisture, partskimmed Mozzarella. The milk for this cheese is coagulated with calf rennet (or suitable substitute), acidified using Streptococcus thermophilus and a thermophilic Lactobacillus as starter; the coagulum is cut and the curds/whey mixture cooked to c. 41 ~ The whey is drained off and the curds are held to allow acidification (and may be cheddared). When the curd pH reaches 5.1-5.3, the curds are heated, kneaded and stretched in hot water or dilute brine (c. 78 ~ to a curd temperature of c. 58-60 ~ by hand, in the same fashion as a baker might knead dough, or mechanically, which is usually used in industry. Mozzarella cheese may be brine- or dry-salted and is usually consumed within a few weeks of manufacture; traditional Mozzarella is consumed as soon as possible after manufacture. Pasta-filata cheeses can be consumed fresh (often as a topping on pizzas) or ripened (semi-hard or hard) or smoked. Mozzarella is used mainly as a pizza topping for which its principal characteristics are its physicochemical functional properties, especially meltability and stretchability. The functionality of biologically acidified Mozzarella improves to a maximum after ripening for ---2 weeks at 6-8 ~ and then deteriorates due to excessive proteolysis. Chemically acidified Mozzarella is very functional immediately after manufacture. In addition to the use of Mozzarella cheese as a pizza topping, pasta-filata varieties include Mozzarella di Bufala (buffalo milk), Mozzarella di vacca (cows' milk; also called Fiordilatte, Scamorza or Provola), Caciocavallo, Cascaval, Kashkaval, Provolone, Kasseri and Kasar peyniri.
without cooking, into moulds to drain. When the curd is sufficiently cohesive, the moulds are removed and the cheese is cut into pieces and salted. The cheese pieces are then transferred to barrels or tin-plated cans, covered by a brine solution (c. 14% NaC1) and ripened at 14-16 ~ for c. 7 days until the pH has decreased to c. pH 4.5. The cheese-containing cans are then transferred to rooms at 3-4 ~ and stored for at least 2 months. Surface mould-ripened varieties
Surface mould-ripened varieties (e.g., Camembert and Brie) are soft cheeses characterized by the growth of Penicillium camemberti on the cheese surface. Mould spores may be added to the cheesemilk or sprayed onto the cheese after manufacture. Cheese milk is acidified using a mesophilic starter and coagulated using rennet extract. After the coagulum has formed, it is usually ladled directly, without cutting, into moulds, where drainage occurs. The cheeses are usually brine-salted and ripened at c. 12 ~ for 10-12 days for mould development. As discussed in 'Metabolism of Residual Lactose and of Lactate and Citrate', Volume 1 and 'Surface Mould-ripened Cheeses', Volume 2, the ripening of white-mould cheese is characterized by the extensive catabolism of lactate at the surface of the cheese by the mould, causing an increase in the pH of the surface zone (and thus creating a pH gradient from the surface to the core of the cheese) and the migration of lactate from the core. Calcium phosphate precipitates at the elevated pH of the surface and soluble calcium phosphate migrates through the cheese towards the surface. These changes, together with proteolysis, cause considerable softening of the cheese and mature whitemould varieties may flow under their own weight. Blue cheese
Cheeses ripened under brine
Feta, Domiati and related cheeses (e.g., Brinza, Beli Sir, Telemes, Kareish, Beyaz Peiniri; see 'Cheese Varieties Ripened in Brine', Volume 2), evolved in the eastern Mediterranean and Balkan regions; they are also known as 'pickled cheeses', so-called because they are ripened under brine. Feta is a Greek cheese made from sheep's milk with PDO status. However, similar whitebrined cheeses are also made from pasteurized cows' milk on a large industrial scale outside Greece, often using uhrafiltration technology (see 'Pasta-Filata Cheeses', Volume 1). Milk for Feta cheese is coagulated using rennet (which may also have lipase activity) and acidified using a thermophilic or mesophilic lactic starter. The coagulum is cut into small cubes and scooped,
Blue cheese varieties ('Blue Cheese', Volume 2) are characterized by blue/green veins throughout the cheese caused by the growth of Penicillium roqueforti. The milk for these varieties is coagulated by rennet extract; the curds are acidified using a mesophilic lactic culture and are cooked at a low temperature before being transferred to moulds. Some varieties of blue cheese are salted by repeated surface application of dry NaC1 while others are brine-sahed. The salted cheeses are ripened at a temperature and relative humidity which favour mould growth. Since P. roqueforti requires 02 for growth, the texture of Blue cheese must be open to allow the fungal spores and hyphae to germinate and grow. This open texture is achieved by encouraging mechanical openings during manufacture (by not pressing the curds after moulding) and by piercing the
Diversity of Cheese Varieties: An Overview
cheeses with needles (by hand or a special machine). The ripening of Blue cheese is characterized by extensive lipolysis. Blue cheeses have a soft texture and a strong flavour dominated by n-methyl ketones which are produced by the mould from fatty acids. Blue cheese varieties include Bleu d'Auvergne, Cabrales, Gorgonzola, Danablu (Danish Blue) and Stilton, all of which are made from cows' milk, and Roquefort which is made using sheep's milk. Bacterial surface-ripened cheese
Bacterial surface-ripened ('smear-ripened') cheeses are a diverse group of varieties characterized by the growth of a complex Gram-positive bacterial flora on the surface during ripening ('Bacterial Surface-ripened Cheeses', Volume 2). Soft smear cheeses usually are acidified using a mesophilic culture, are not cooked to a high temperature and are brine-salted. These cheeses have a high moisture content and are typically moulded as small cylinders (---200 g), with a high surface area:volume ratio which allows the surface smear to have an important influence on the characteristics of the mature cheese. During manufacture, the surface of the cheeses is washed periodically with a brine solution, a process referred to as 'smearing'. In many factories, old cheeses (which have fully developed surface microflora) are smeared first and the same smear liquid is used to smear young cheeses, which are thus inoculated. This practice, called 'old-young' smearing, assists in the development of the surface microflora, but has been criticized on the grounds of hygiene. Soon after manufacture, the surface microflora of smear cheeses is dominated by yeasts (e.g., Debaroymces hansenii) and Geotrichum candidum. Growth of these micro-organisms deacidifies the cheese surface and encourages the growth of coryneform bacteria (e.g.,
Corynebacterium, Arthrobacter, Brevibacterium), Micrococcacae and Staphylococcus. These bacteria gain access to the cheese from the milk (particularly for raw milk cheeses) or through post-pasteurization contamination. These cheeses are characterized by a strong aroma and high levels of proteolysis and lipolysis, mainly at their surface. Sheep's and goats' milk cheeses
Although sheep and goats are minor dairying species (each produces ~-2% of total world milk production compared with ---11% and ---85% for buffalo and cattle, respectively), they are quite significant in Mediterranean and Balkan countries, where most of their milk is used for cheese production. Many famous and popular cheeses are produced from sheep's milk, e.g., Roquefort, Feta, the various Pecorino varieties, Kashkaval and
17
Manchego. A more complete list of cheeses produced from sheepg and goats' milks is given by Kalantzopoulos (1993), and 'Cheeses Made from Ewes' and Goats Milk', Volume 2 is devoted to goats' and ewes' milk cheeses. These cheeses are considered together because the conditions of management of these relatively small animals are very similar and in many countries they are farmed in mixed flocks. In many cases, due also to the limited lactation period, cheeses are prepared from the mixed milk of these two species. Goats' and ewes' milk cheeses are produced mainly around the Mediterranean basin and in the Balkans. The gross composition of ewes' milk is markedly different from that of goats' and cows' milks, which have generally similar gross composition, although goats' and cows' milks differ in many respects, including their proteins and fatty acid profiles. These differences influence the characteristics of cheeses made from sheep's or goats' milk. Goats' milk cheeses are usually consumed fresh or ripened for a short period of time. Sheep's milk contains high levels of fat and protein, which are the main cheesemaking constituents. The coagulum is firm, the syneresis is rapid and the NaC1 diffusion is slow due to the low moisture content of these cheeses. By modifying the cheesemaking technology, it is possible to obtain a range of cheeses (fresh, short-, medium- and long-ripened) from ewes' milk. Acid-curd cheeses
Acid-coagulated cheeses are varieties for which milk or cream is coagulated on acidification to c. pH 4.6. Acidcurd cheeses were perhaps the first type of cheese produced since such products may arise from the souring of milk by the adventitious microflora. These cheese varieties are distinguished from yoghurt because their manufacture involves dehydration by removal of at least some whey. Acidification is usually achieved by the action of a mesophilic starter culture but direct acidification is also practised. A small amount of rennet may be used in certain varieties (e.g., Cottage or Quarg) to increase the firmness of the coagulum and to minimize casein loss in the whey but its use is not essential. The coagulum may or may not be cut or cooked during manufacture but it is not pressed. Acid-coagulated cheeses (e.g., Cream, Cottage, Quarg, some Queso Blanco) are characterized by a high moisture content and are usually consumed soon after manufacture. Acid-coagulated varieties represent ---25% of total cheese production (considerably higher in some countries, 'Cheese: An Overview', Volume 1). They are usually consumed when fresh although there are some minor varieties of ripened acid-curd cheese. The acid coagulation of milk is described in 'Formation, Structural Properties and Rheology of Acid-coagulated
18
Diversity of Cheese Varieties: An Overview
Milk Gels', Volume 1, and acid-coagulated cheeses are discussed in detail in 'Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese' Part C Acid-heat Coagulated Cheeses', Volume 2. Cheeses coagulated by a combination of heat and acid
A small group of cheeses are produced by a combination of heat and acid. The best-known and perhaps the most important member of this group is Ricotta, an Italian cheese variety (the name derives from ricottura, 'reheating') which is produced from rennet cheese whey, perhaps with some milk added, by heatinduced coagulation (85-90 ~ and some acidifying agent (e.g., lemon juice or vinegar). Ricotta curd is transferred to moulds surrounded by ice where drainage occurred. Mascarpone is made by a process similar to that for Ricotta except that it is made from cream and a slightly higher cooking temperature is used. The resulting cheese is creamier than Ricotta and is usually salted at a low level and whipped and formed into a cylindrical shape. Other heat-/acid-coagulated varieties and their country of origin include Ricotta Forte (Italy), Brocciu (Corsica), Cacio-ricotta (Italy, Malta), Mizthra and Manouri (Greece) and Ziger (former Yugoslavia). Some of these varieties were described by Kalantzopoulos (1993) and some aspects are discussed in 'Acid- and Acid~ennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acid-heat Coagulated Cheeses', Volume 2. Processed cheese products
Processed cheeses differ from natural cheese by not being made directly from milk but from various ingredients such as natural cheese (usually), emulsifying salts, milk solids, butter oil, other dairy ingredients, vegetable oils or other ingredients (Fox et al., 2000; see 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). Processed cheese is produced by blending shredded natural cheeses, varying in maturity, with emulsifying salts and often other ingredients, and heating the blend under vacuum with constant agitation until a homogeneous blend is obtained. Although connoisseurs of cheese often regard processed cheese as inferior to natural cheese, the former has a number of advantages, including stability and consistency and they provide an outlet for inferior quality cheese which might otherwise be difficult to sell. The nutritional value of processed cheese is generally similar to that of natural cheese; although it has usually a higher sodium
content than the latter, this can be reduced (see 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). Reducing the fat content of processed cheese has less undesirable consequences than for natural cheese. Since processed cheese can be produced in a wide range of flavours, shapes and consistencies, it is particularly popular for ingredient applications. About 2 • 106 tonnes of processed cheese are produced annually, i.e., ---14% of natural cheese. Cheeses made for use as food ingredients
In addition to processed cheese and cheese analogues (see below), most of which are used in ingredient applications, an increasing proportion of natural cheeses produced worldwide is consumed as an ingredient in other food products (see Guinee, 2003; 'Cheese as an Ingredient', Volume 2). While many traditional varieties have flavour or functional properties amenable to their use as ingredients (e.g., low-moisture Mozzarella for use as a pizza topping), it is likely that new 'varieties' of natural cheese will evolve in the future to meet requirements for cheese with tailor-made functional properties. Enzyme-modified cheeses (EMCs) are products with concentrated cheese flavours formed by the enzyme-catalysed hydrolysis of cheese curd or other ingredients by the action of exogenous proteinase, peptidase and/or lipase preparations (see Kilcawley et al., 1998; Wilkinson and Kilcawley, 2003; 'Cheese as an Ingredient', Volume 2). The advantages of EMCs over other sources of cheese flavours are their flavour intensity, range of flavours available, reduced production costs and shelf-life. Because of their high flavour intensity, EMCs are typically added as flavourings to foods at a very low level (c. 0.1%, w/w). Enzyme-modified cheeses are relatively new products, especially on a commercial scale. It seems very likely that their production will increase, made possible by the availability of purer and more specific enzymes and selected cultures. At present, Cheddarlike EMCs are the principal products but it is likely that the production of other varieties will increase. At present, EMCs are used only as ingredients; however, with increased knowledge of the biochemistry of cheese ripening in general, and of EMCs in particular, and of the flavour impact compounds in various cheese varieties, it seems conceivable that EMCs may evolve into table cheeses. Norwegian whey 'cheese'
A unique type of 'cheese' is produced in Norway by evaporation of water from whey by concentration to ---80% total solids and crystallization of the lactose. This
Diversity of Cheese Varieties: An Overview
type of cheese originated in Norway. Strictly speaking, it could be argued that such varieties are not cheeses in sensu stricto. These cheeses ('Brunost', brown cheese) are characterized by having a smooth but firm body, a sweet, caramel-like flavour and a long shelf-life. Sweet whey is the usual starting material although acid whey may be used for some brands. Sometimes, skim milk or cream is added to the whey to give a whiter, smoother product. Types of Brunost include Primost, Gjetost, Mysost, Niesost, Flmemyost and Gudbrandsdalost. The manufacture of these cheeses involves concentration of whey (or whey/cream mixture) by evaporation to high total solids to form a plastic mass. The Maillard reaction is encouraged and is important for the final colour and
19
flavour of the product. The concentrate is then cooled, kneaded and packaged. Crystallization of lactose is controlled so as to avoid sandiness in the product. Non-European cheeses
The majority of commercially important cheese varieties originated in Europe, and Europe and North America remain the most important regions for cheese production (see 'Cheese: An Overview', Volume 1). However, numerous minor cheeses are produced in Asia, Africa and Latin America, some of which are listed in Table 9 and were discussed by Phelan et al. (1993).
Some non-European cheese varieties (modified from Phelan et aL, 1993). Varieties discussed elsewhere in this volume are not listed
Country Asia Afghanistan
Bangladesh Bhutan
China India
Indonesia Iran Iraq Jordan Lebanon
Nepal
Pakistan Philippines Qatar Saudi Arabia Syria
Cheese
Remarks
Karut Kimish Panier Chhana Ponir Chhana Churtsi Durukhowa
Very hard cheese made from skim milk Semi-hard unripened cheese obtained by acid coagulation Acid coagulation of boiled milk Semi-hard ripened cheese (as above) Hard cheese made from yak and chauri milk Hard, rubbery cheese made from yaks' and chauris' milk (known as Chugga or Chhurpi in Nepal) Similar to Edam Acid-curd cheese from inner Mongolia Sour milk cheese made from cows' milk Soft heat-acid coagulated variety Fresh rennet cheese made from heated milk Sort cheese made from cows' and buffaloes' milk coagulated with vegetable rennet (bromelain) Variety ripened under brine similar to Feta Similar to Liqvan Hard brittle cheese with a sharp flavour Hard sun/air dried variety made from sheep's or goats' buttermilk Hard cheese made from sheep's and goats' milk Soft fresh cheese made from whole milk White cheese varieties
Long Giang Hurood Chhanna Paneer Panir Tahu Susu Atau Dadih Liqvan 'White cheese' Awshari Djamid Shankalish Akawieh Baladi/Baida/ Hamwi Chelal Karichee 'Fresh cheese' Umbris Chhana Chhurpi Shosim Langtrang Panir Peshawari Kesong Puti 'White cheese' Ekt Mesanarah Medaffarah Shankalish
Cheese in the form of strings or ropes Soft whey cheese Soft rennet-coagulated cheese Soft spreadable cheese made from raw goats' milk (as above) Similar to Durkhowa Soft cheese made from yaks' and chauris' milk Semi-hard cheese made from yaks' and chauris' milk Soft cheese variety Semi-hard cheese made from whole or partly skimmed cows' milk Soft fresh cheese made from carabao and cows' milk (as above) Sun-dried cheese made from sheep's buttermilk Sun-dried rennet-coagulated cheese made from sheep's milk Pasta-filata variety made from sheep's milk Rennet coagulated cheese made from partially skimmed milk continued
20
Diversity of Cheese Varieties: An Overview
continued
Country
Cheese
Remarks
Asia Turkey
Beyaz Peyneri
Yemen
Kasar Peyneri Mihalic Peyneri Tulum Peyneri Aomma Taizz
Semi-hard cheese ripened under brine made from sheep's milk or mixtures of milks Hard, pasta-filata variety Hard cheese made from raw sheep's milk and ripened under brine Hard cheese made from sheep's milk or mixtures of milks Pasta-filata variety No details available
Africa Algeria
Benin
Chad Dem.Rep.Congo Egypt
Ethiopia Kenya Madagascar Mali Niger Nigeria
Sudan
Latin America Argentina
Bolivia
Brazil
Chile Colombia Costa Rica Cuba Dominican Republic Ecuador Honduras
Takammart Aoules Takamart Woagachi/Wagashi Wagassirou/ Gassigue Pont Belie Mashanza Ras Karish/Kareish Daani Mish Ayib Mboreki Ya Iria 'Fromage' 'Fromage blanc' Wagashi Tchoukou Wara/Awara Chukumara Dakashi 'Country cheese' Karish Braided cheese Gibbna Mudafera 'White cheese' Goya Tafi Altiplano Quesillo Queso Benianco/ Quieso Chaqueno Queijo de Coalho Queiso de Manteiga Queijo Minas Queijo Prato Chanco 'Queso Blanco' Palmito Patagras Queso de Freir Queso Andino Quesillo de Honduras
Rennet-coagulated cheese made from goats' milk and air/sun-dried Heat/acid coagulated cheese made from buttermilk and air/sun-dried Rennet-coagulated cheese made from goats' milk and sun-dried Soft fresh cheese made from cows' milk coagulated using the sap of Calotropis procera Fresh cheese made from goats' or sheep's milk Soft fresh cheese made from cows' milk Hard, bacterially ripened variety Fresh, low salt acid-coagulated cheese variety Soft cheese made from sheep's or sheep's/goats' milk Karish cheese ripened in Mish (pickling solution) Heat/acid coagulated variety made from buttermilk Fresh soft cheese made from cows' or goats' milk Semi-hard cheese made from cows' milk Fresh soft cheese made from skimmed cows' milk (see above) Hard sun-dried cheese made from various milks Soft unripened variety similar to Wagashi Tough-textured cheese Heat-coagulated colostrum Hard cheese variety (see above) Semi-hard, braided cheese variety Similar to Feta or Domiati Semi-hard cheese made from cows' milk Soft white cheese ripened under brine Hard, ripened cheese made from cows' milk Semi-hard ripened cheese made from raw whole milk Soft fresh cheese made from raw whole cows' and sheep's milk Fresh, unripened soft cheese. Also known as Banela in Mexico, Paraguay cheese in Paraguay and Queso Blanco in Nicaragua Semi-hard ripened cheese made from whole cows' milk Semi-hard ripened cheese Processed cheese Semi-hard cheese made from raw cows' milk Semi-hard ripened cheese Semi-hard ripened cheese made from whole cow's milk Generic name for rennet- and acid-coagulated cheeses Pasta-filata cheese made from raw whole cows' milk Semi-hard, ripened cheese Type of Queso Blanco consumed after frying Soft, ripened cheese Pasta-filata cheese made from cows' milk
Diversity of Cheese Varieties: An Overview
21
continued
Country
Cheese
Remarks
Chihuahua Cotija Oaxaca Panela Queso Andino Requeson Colonia Yamandu De Mano Guayanes Llanero/Americano
Semi-hard, ripened cheese Hard, ripened cheese made from cows' or goats' milk Pasta-filata cheese variety Fresh, unripened cheese (see above) Heat/acid coagulated cheese similar to Ricotta Semi-hard, ripened cheese made from cows' milk Semi-hard, ripened cheese made from cows' milk Semi-hard unripened pasta-filata cheese Semi-hard unripened cheese Similar to Queso Blanco
Latin America
Mexico
Peru Uruguay Venezuela
Imitation and substitute cheese products
A wide range of imitation and substitute cheese products are produced worldwide, which may be classified into three broad categories: analogue cheese, filled cheeses and tofu-based products. Cheese analogues are cheese-like products produced by blending various oils/fats, proteins (usually rennet casein), flavours and other ingredients with water into a smooth homogeneous cheese-like blend with the aid of heat, shearing forces and emulsifying salts (Guinee, 2003). Analogues of low-moisture Mozzarella, Cheddar, Monterey Jack and processed (Cheddar) cheeses are produced and have the advantages of being cheaper and more easily manufactured than natural cheese; their functional properties may be tailor-made for specific applications. Developments in EMC technology should make it possible to improve and diversify the flavour of analogue cheese products. Filled cheeses differ from natural cheeses in that the milkfat is partially or totally replaced by vegetable oil which is dispersed using high-speed mixing and homogenization in skim milk or skim milk reconstituted from various dairy ingredients such as skim milk powder, whey and total milk protein dispersed in water. The filled milk is then used as the starting material for conventional in-vat cheesemaking (Fox et al., 2000). Tofu is a cheese-like product produced from soybeans which has been a staple food in the Orient for many centuries. Although the appearance resembles that of fresh cheese, and has similar culinary applications, its physico-chemical properties are clearly different from all the classes described in this chapter.
Bertozzi, L. and Panari, G. (1993). Cheeses with Appellation d'Origine ContrOl~e (AOC): factors that affect quality. Int. Dairy J. 3,297-312.
Burkhaher, G. (1981). Catalogue of Cheese. Document 141, International Dairy Federation, Brussels, Belgium. Contarini, G., Povolo, M., Toppino, P.M., Radovic, B., Lipp, M. and Anklam, E. (2001). Comparison of three different techniques for the discrimination of cheese: application to the ewe's cheese. Milchwissenshaft 56, 136-140. Davis, J.G. (1965). Cheese, Vol. 1, Basic Technology, Churchill Livingstone, London. Dewettinck, K., Dierckx, S., Eichwalder, P. and Huyghebaert, A. (1997). Comparison of SDS-PAGE profiles of four Belgian cheeses by multivariate statistics. Lait 77, 77-89. Dirinck, P. and De Winne, A. (1999). Flavour characterisation and classification of cheeses by gas chromatographic-mass spectrometric profiling. J. Chromatogr. 847, 203-208. Dufour, E., Devaux, M.E, Fortier, P. and Herbert, S. (2001). Delineation of the structure of soft cheeses at the molecular level by fluorescence spectroscopy- relationship with texture. Int. Dairy J. 11,465-473. Fox, P.E (1993). Cheese: an overview, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 1-36. Fox, P.E and Wallace, J.M. (1997). Formation of flavour compounds in cheese. Adv. Appl. Microbiol. 45, 17-85. Fox, P.E, Guinee, T.P., Cogan, T.M. and McSweeney, P.L.H. (2000). Fundamentals of Cheese Science, Aspen Publishers, Gaithersburg, MD. Frau, M., Simal, S., Femenia, A. and Rossello, C. (1998). Differentiation and grouping of chemical characteristics of Mahon cheese. Z. Lebensm. Unters. Forsch. 207, 164-169. Fritsch, R.J., Martens, E and Belitz, H.D. (1992). Monitoring Cheddar cheese ripening by chemical indexes of proteolysis. 1. Determination of free glutamic acid soluble nitrogen and liberated amino groups. Z. Lebensm. Unters. Forsch. 194, 330-336. Garcia-Palmer, EJ., Serra, N., Palou, A. and Gianotti, M. (1997). Free amino acids as indices of Mahon cheese ripening. J. Dairy Sci. 80, 1908-1917. Guinee, T.P. (2003). Cheese as a food ingredient, in, Encyclopedia of Dairy Sciences, Vol. 1, H. Rogenski, J.W. Fuquay and P.E Fox, eds, Academic Press, London. pp. 418-427. Kalantzopoulos, G.C. (1993). Cheese from ewes' and goats' milk, in, Cheese: Chemistry, Physics and Microbiology,
22
Diversity of Cheese Varieties: An Overview
Vol. 2, 2nd edn, RE Fox, ed., Chapman & Hall, London. pp. 507-553. Kilcawley, K.N., Wilkinson, M.G. and Fox, P.E (1998). Enzyme-modified cheese. Int. Dairy J. 8, 1-10. Manca, G., Camin, E, Coloru, G.C., Del Caro, A., Depentori, D., Franco, M.A. and Versini, G. (2001). Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope (C-13/C-12 and N-15/N-14) ratios and free amino acid ratios. J. Agric. Food Chem. 49, 1404-1409. Martin-Hernandez, C., Amigo, L., Martinalvarez, P.J. and Juarez, M. (1992). Differentiation of milks and cheeses according to species based on the mineral content. Z. Lebensm. Unters. Forsch. 194, 541-544. McGoldrick, M. and Fox, RE (1995). Intervarietal comparison of proteolysis in commercial cheese. Z. Lebensm. Unters. Forsch. 208, 90-99. Olson, N.E (1990). The impact of lactic acid bacteria on cheese flavor. FEMS Microbiol. Lett. 87, 131-147. O'Shea, B.A., Uniacke Lowe, T. and Fox, RE (1996). Objective assessment of Cheddar cheese quality. Int. Dairy J. 6, 1135-1147. Ottogalli, G. (1998). A global comparative method for the classification of world cheeses (with special reference to microbiological criteria). Ann. Microbiol. Enzimol. 48, 31-58. Ottogalli, G. (2000a). A global comparative method for the classification of world cheeses (with special reference to microbiological criteria). Revised edition. Ann. Microbiol. 50, 151-155.
Ottogalli, G. (2000b). Proposta di aggiornamento nella classificazione dei formaggi con particolare riferimento agli aspetti microbiologici. Alimenta 8, 147-165. Ottogalli, G. (2001). Atlante dei Formaggi, Hoepli, Milan. Peres, C., Viallon, C. and Berdague, J.L. (2002). Curie point pyrolysis-mass spectrometry applied to rapid characterisation of cheeses.J. Anal. Appl. Pyrol. 62, 161-171. Phelan, J.A., Renaud, J. and Fox, RE (1993). Some nonEuropean cheese varieties, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, Major Cheese Groups, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 421-466. Rohm, H. (1992). Regional classification of Swiss cheese based on its chemical composition. Z. Lebensm. Unters. Forsch. 194, 527-530. Sandine, W.E. and Elliker, P.R. (1970). Microbiologically induced flavors and fermented foods. Flavor in fermented dairy products. J. Agric. Food Chem. 18, 557-562. Schulz, M.E. (1952). Klassifizierung von Kase. Milchwissenshaft 7,292-299. Scott, R. (1986). Cheesemaking Practice, Elsevier Applied Science Publishers, London. Smith, A.M. and Nakai, S. (1990). Classification of cheese varieties by multivariate analysis of HPLC profiles. Can. Inst. Food Sci. Technol. J. 23, 53-58. Walter, H.E. and Hargrove, R.C. (1972). Cheeses of the World, Dover Publications, Inc., New York. Wilkinson, M.G. and Kilcawley, K.N. (2003). Enzyme modified cheese, in, Encyclopedia of Dairy Science, Vol. 2, H. Roginski, J.W. Fuguay and P.E Fox, eds, Academic Press, London. pp. 434-437.
Plate 1
Examples of cheese from the principal groups of Ottogalli (1998, 2000a,b, 2001)" see Table 7 for further details. (See page 13.)
Plate 2 APV Cheddarmaster belt system. Courtesy of NZMP Whareroa, New Zealand. (See page 33.)
General Aspects of Cheese Technology R.J. Bennett, Senior Lecturer in Dairy Technology, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand K.A. Johnston, Principal Research Technologist, Fonterra Research Centre, Palmerston North, New Zealand
Cheesemaking involves the conversion of liquid milk (an unstable, bulky but highly nutritious raw material) into cheese (a stable, flavoursome, concentrated product that provides eating pleasure and has an extended shelf-life). Cheesemaking has been practised for many thousands of years, for most of the time as a cottage industry. Towards the end of the nineteenth century, as industrialisation progressed, cheese manufacture moved to the factory; since then, there has been a progressive development of the technology, especially equipment, to the situation today with large, highly automated, modern factories employing minimal staff. This move has been driven by several f a c t o r s - scale, cost and availability of labour, increased hygiene and need for product uniformity and consistency. This development has been at the cost of some individuality and variety; therefore, in parallel with the increased mechanisation of manufacture, there has been a resurgence of many small boutique cheesemakers. The impact of computers and automation on the cheesemaking process has been dramatic, with many of the previously manual-controlling, programming, analysis and data-logging operations being replaced by computers. Thus, greater uniformity of production has been made possible. This chapter aims to introduce the steps involved in the cheesemaking process, explaining their purpose and then describing the equipment and the processes that have been developed to facilitate large-scale manufacture. Not all equipment types are included in detail but rather the major types to illustrate their purpose. Cheeses may be classified in various ways. The diversity of cheese types arises from composition (the manufacturing process) and from the cultures or microflora involved (Johnson and Law, 1999). This chapter focuses on the manufacturing process. A useful primary classification from a manufacturing technology viewpoint is based on cheese firmness (effectively, moisture content) and the salting technology involved. This is illustrated in Table 1 and forms the basis of the discussion of the manufacturing processes for the major cheese varieties
outlined in Fig. 1. The initial focus is on common steps to the end of the vat stage of manufacture. This is followed by discussion of the technology used for hard, dry-salted varieties such as Cheddar, with that used for other types being discussed in later sections.
Milk preparation The milk used for cheesemaking comes from cows, sheep, goats and buffaloes. As the key ingredient, its quality and preparation are of vital importance. As the equipment and processes used are standard dairy operations, they are not described in detail. Excellent explanations are provided by Bylund (1995a) and Muir and Tamime (2001). Hygienic milk harvesting, refrigeration and gentle handling are essential features of milk harvesting and transport to the factory. The absence of inhibitory substances such as antibiotics is also necessary for satisfactory cheese manufacture. Removal of foreign matter is a necessary first step in factory processing, and this is achieved by filtration through an appropriate mesh or by centrifugal clarification. Compositional adjustment of the milk is often required to achieve the desired final product specifications. This commonly involves centrifugal separation of part of the milk stream into skim milk and cream, followed by blending of the skim milk with the whole milk to achieve the desired fat content. For some products, a higher fat level may be necessary and this is achieved by incorporating additional cream. More recently, it has become feasible to also adjust the protein content of the milk. This is normally achieved through the use of ultrafiltration technology (discussed in 'Application of Membrane Separation Technology to Cheese Production', Volume 1). Skim milk is concentrated and then blended with other components to achieve the desired final composition. Advantages include a more uniform starting material, more profitable use of a lactose stream and greater throughput of milk solids through the cheese vat, as the milk is effectively partially concentrated.
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
24
General Aspects of Cheese Technology
Classification of cheese based on hardness and salting technology
Hardness
Salting technology
Examples
Hard/semi-hard Hard/semi-hard Soft/semi-soft
Dry Brine Brine
Cheddar, Cheshire Emmental, Gouda Camembert, Blue vein
Control of the microbiology of the cheese milk is a vital issue affecting the final product, and there is an ongoing vociferous debate on the merits of raw milk cheese versus cheese for which heat treatment, normally pasteurisation, has been used (Johnson and Law, 1999). Pasteurisation, through the use of a plate heat exchanger and holding tubes with typical time/temperature relations of 72 ~ s, is standard practice to
Milk preparation
VAT STAGE 9 Setting 9 Cutting 9 Cooking 9 Washing (some types)
Starter culture and coagulant addition
POST-VAT STAGES
Hard, dry- I~t" salted types
~
1
Hard~semihard types
Soft mouldripened types
T Dewheying
T Dewheying
Pre-pressing (some types)
Moulding
, Wheyto further ' processing ,
Dewheying
i
!
Drying Texturing (cheddaring) or stirring
Pasta-fila
Salting
Pressing
I
Coo ino stretc in I I
Milling
Brine-salted types
I
II
Moulding
I
rin,oo
I
Pressing
Acid development
Brining
Brining
Ripening
Ripening
-I Basic steps in cheese manufacture.
Despatch
I
I
I
Ripening
General Aspects of Cheese Technology
kill pathogenic organisms. If the raw milk is to be stored refrigerated for a long period before pasteurisation, thermisation (66 ~ s) is recommended to prevent the growth of psychrotrophic organisms and their associated production of lipases and proteinases. Alternative processes for the reduction of bacterial load include the use of specially designed centrifuges (bactofuges) and microfihration (Maubois, 2002). These avoid some of the perceived detrimental effects of thermal processes and are especially useful for the removal of spores, such as Clostridia, that survive pasteurisation and can cause problems in the final product. Following thermal or other treatments, the milk enters the cheese vat, typically at 32 ~ Starter culture preparation and addition
The use of cultures of micro-organisms, including bacteria, yeasts and moulds, is an integral component of cheese manufacture. 'Starter Cultures: Genetics', 'Starter Cultures: Bacteriophage', 'Secondary and Adjunct Cultures', of Volume 1 are devoted to detailed discussion of these cultures. The micro-organisms have two primary r o l e s - the reduction of the pH during manufacture due to the production of lactic acid from lactose, and the biochemical and physical changes during the curing or ripening phase after manufacture of the initial cheese curd. The cultures responsible for acid development are typically lactic acid bacteria and are commonly referred to as cheese starters, although they are also involved during ripening. Organisms, the primary role of which is post-initial manufacture, are known as starter adjuncts. Both groups of cultures are commonly incorporated into the milk in the cheese vat, although for some varieties, such as smear-ripened cheeses, the formed cheese may be inoculated with the culture. Cheese starters used for hard varieties such as Cheddar are commonly composed of Lactococcus lactis subsp, cremoris. The quantity of culture required for controlled, rapid acid development in the vat means that a substantial inoculation is necessary. This may be provided in a variety of ways, such as the direct addition of a powdered concentrated culture provided by a culture manufacturer. This may be frozen or freezedried and may need to be reconstituted before addition to the vat. The very successful system used in New Zealand, described by Heap (1998), depends on the use of frozen single-strain cultures, which are then grown in a heat-treated reconstituted skim milk in a pH-controlled environment, to produce a concentrated culture that is then metered into the milk at a level such as 0.3%, v/v, as the vat is being filled with milk. A simple fermentation vessel is used for bulk culture
25
production. The greatest hazard to the production of starter and satisfactory acid development in the vat is the presence of bacteriophage. Multiple vat filling throughout the day in large plants increases the potential for phage build-up. Stringent hygiene precautions, the use of several carefully selected phage-unrelated strains and the use of a phage-inhibitory growth medium for starter preparation are the techniques designed to minimise this risk. Starter adjunct cultures can be added directly to the vat, usually from 'potties' or suspensions of culture especially prepared by a culture manufacturer. As they are adjuncts, the quantities required are much smaller than the quantity of acid-producing starter. Coagulant addition
The most fundamental step in the cheesemaking process involves the conversion of the liquid milk into a semi-solid gel. Subsequent syneresis, or shrinkage and loss of whey from this gel, results in the formation of cheese curd. Detailed discussion of coagulants and syneresis is provided in 'Rennets: General and Molecular Aspects', 'Rennet-induced Coagulation of Milk', 'The Syneresis of Rennet-coagulated Curd', 'Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels', Volume 1. Coagulation involves the aggregation of the casein and is normally achieved by the addition of a coagulant to the milk in the vat stage of manufacture, although it can also be accomplished by pH reduction through acidification for some varieties, such as Cottage cheese. Traditionally, the coagulant of choice has been rennet, derived from the abomasum of young milk-fed calves, in which the principal active ingredient is chymosin. For reasons of supply, economy and ethics, alternatives are now also used frequently, derived from fungal sources such as Rhizomucor meihei or a natureidentical chymosin produced by genetic engineering technology. The coagulants are normally supplied by the manufacturer as stable liquid concentrates, which can be metered directly into the cheese vat at the appropriate stage via a distribution system. As the coagulant is a highly concentrated enzyme system, the quantity required is much lower than that of cheese starter, typically 0.01%, v/v, for calf rennet. As the enzymes are also involved in the ripening process, the level of addition and the enzyme characteristics are of vital importance to the cheese being produced. Vat stage
The cheese vat or cheese tank is the part of the cheesemaking equipment in which milk is converted from
26
General Aspects of Cheese Technology
a standardised liquid to a semi-solid gel. This part of the process concentrates the casein and the fat of the milk by removing moisture (whey). The first part of the process involves the addition of the coagulant to the milk, this being known as setting the vat. The coagulant is added and mixed in, as already described, and the vat contents are then left undisturbed. Determination of the appropriate coagulum strength for the next stage can be made by an experienced operator observing the curd or by using instruments such as the Stoelting Optiset | probe and others, discussed by Law (2001). Once a satisfactory coagulum has been formed, usually after about 40 min, the gel is cut into cubes of 6-10 mm size, to encourage moisture expulsion (syneresis). In most cheesemaking processes, the curds/whey mixture is then cooked to a higher temperature while lactose is fermented by the starter bacteria and acid is produced. Acid development is an important step in most cheesemaking processes and controls the rate and extent of syneresis, the composition, the final cheese pH and, perhaps of most importance, the degree of mineral solubilisation that occurs during the process. The cooking process has a fundamental role in controlling syneresis by influencing curd shrinkage and acid development. Following cooking, the curds/whey mixture is stirred until the drain pH target is reached and curds/whey separation (draining) or dewheying is initiated. For some varieties, a reduction in the lactose content of the curd and whey in the vat may be accomplished by partial removal of the whey followed by addition of water, which may be heated to also assist with cooking. This operation can be described as washing. Historically, cheese curd was produced in large, open, jacketed, square-ended, stainless steel vats. The cutting and stirring mechanisms were mounted above the vat and often both curd processing (e.g., cutting, cooking and stirring) and curd conditioning (e.g., cheddaring) were carried out in the vat. Labour costs were high and quality was often variable. Although this system is still used successfully in some small plants, more exacting hygiene standards, coupled with the demand for higher throughputs at reduced cost, resulted in the introduction of enclosed vat systems in the late 1960s. Since then, enclosed vat systems have been further refined to meet the needs of an increasingly mechanised and automated industry, an industry that in some countries is also having to deal with processing increasingly larger milk volumes because of extensive and rapid amalgamation of a number of smaller plants. This vat stage of cheese production is a batch process, and, for continuous throughput, a factory must have a number of vats, usually at least 6-8, to enable production to be sequenced to ensure a continuous output.
The majority of the enclosed vat systems available contain: 9 one or two revolving knife panels of various designs, which are used for both cutting and stirring operations, depending on their direction of rotation; 9 a fully or partially surrounding (steam or hot water) heating jacket; 9 whey removal systems for predraw and in-vat washing; 9 automated rennet addition, cleaning-in-place (CIP) and computer-controlled options for cutting/stirring speeds and cooking recipes (later models only). The choice of equipment for the vat stage of the cheesemaking process depends on many external factors, including the type of cheese to be made, downstream curd processing, flexibility, cost and throughput. Internal vat factors are also important. For example, the configuration of the vat and its cutting and stirring mechanisms, how the vat is heated and emptied, rennet addition and CIP configurations are also important. How the coagulum is cut is of particular significance. The cutting operation, together with the speed of stirring following cutting, influences how large the particles will be at draining and how much of the milk components (fat and casein) are lost to the whey. Johnston et al. (1991) showed that the speed and the duration of cutting in Damrow vats determined the curd particle size at draining and hence the moisture content in the final cheese, and that whey fat losses could be minimised depending on the cutting programme used. They also proposed a model for cutting that explains how variation in cutting speed and duration of cutting, followed by a constant stirring speed, determines the curd particle size distribution in a Damrow cheese vat. A similar study (Johnston et al., 1998) using Ost vats (30 000 1) gave similar trends. However, the study on Ost vats also showed that, although similar, the trends were sufficiently different from those for Damrow vats, to warrant characterisation of each vat type as to the effect of the speed and the duration of cutting on cheesemaking efficiency,before implementing a specific cutting regime. A number of vat types are available, including OST, Damrow, Scherping and APV CurdMaster. These are discussed in turn. There is a similar discussion of vats and their design in Law (2001). The O S T vat
One of the first and the most popular choices of enclosed cheesemaking vat was the Tetra Tebel OST (Ost Sanitary Tank) vat. To date, five models have been produced (OST I, II, III, IV and V) and there are two versions for each m o d e l - with or without predraw capability.
General Aspects of Cheese Technology
Both the OST I and the OST II vats were upright, single silo-shaped tanks with one (OST I) or two or more (OST II) vertically mounted knife panels. The tank volume ranged from 2000 to 20 000 L and these two models were first made in 1969. Manual, semi-automatic and fully automatic versions were available; however, in all cases, an operator was still required to add the coagulant. The last delivery of these models was made in 1977. The OST III vat was the first horizontally mounted vat of the OST series and its design was driven by a need to process larger (>20 000 L) volumes of milk. The operating principles of the design are illustrated in Fig. 2. Switching from the vertical to the horizontally mounted vats simplified the construction required to process the larger milk volumes. The essential difference between the three horizontal OST models (III, IV and V) is in the design of the cutting/stirring mechanisms. The knife in the OST III vat is thicker and its cutting/stirring speed is
27
limited to 6 rev/min. In comparison, the knife in the OST IV vat is thinner and has 'stay-sharp' qualities that reputedly reduce fat and fines losses to the whey. The construction and design of the OST V knife frames was revised to meet the latest hygiene requirements and to improve cheesemaking performance. In early 2002, Tetra Tebel delivered the thousandth vat of the series (OST III-OST V). OST vats have been installed in 35 countries and this vat type is used to make a range of cheese types, including semi-hard (Edam, Gouda, St Paulin, Havarti), hard (Cheddar, Emmental, Romano, Monterey Jack, Egmont, etc.) and low-moisture Mozzarella (Pizza type). The Damrow double-O vat
The vertical Damrow vat was developed in 1972 and has had two updates (Fig. 3). This vertical design was to become Damrow's 'proven standard', and to date
OST IV cheese vat. 1. Combined cutting and stirring tools, 2. Strainer for whey drainage, 3. Frequency-controlled motor drive, 4. Jacket for heating, 5. Manhole, 6. CIP nozzle. Courtesy of Tetra Pak, Sweden.
28
General Aspects of Cheese Technology
Damrow Double-O cheese vat. Courtesy of Damrow Inc., USA.
900 are in use worldwide. Although used to make a range of cheese types, the vertical Damrow vats were used almost exclusively in the New Zealand cheese industry in the early days of mechanisation to produce Cheddar and other dry-salt cheeses. Easily recognised with its 'double OO' configuration, the vertical Damrow vat has two vertical knife arrangements that were used both to cut and stir the curd. Capacity ranges between ~ 1000 and 22 700 1. The Damrow horizontal vat
The horizontal double OO Damrow (DOH) was Damrow's second-generation vat. The design was patented in 1994 and improved upon in 1997, 1999 and 2000 (Fig. 4). Superior draining capability, improved yield and a hot water or steam dimple jacket are characteristics of this vat type. To date, 49 DOH vats are in service in Canada, USA and New Zealand. Three vat sizes are available: ~ 1 6 000 1, ~ 1 8 000 1 and ~ 3 0 000 1. The Scherping horizontal cheese vat
(HCV)
The first dual-barrelled horizontal cheese vat was developed by Scherping Systems in 1988. Of interest are the unique design of the vat's 'counter-rotation', dual agitator, the cutting and stirring system and the
staggered design of the knife arrangement of the thirdgeneration model (see Fig. 5). The unique 'interlocking' action and the lower speed required by the two counter-rotating agitators in both cutting and stirring modes are claimed to reduce losses and to give a more uniform curd particle size distribution. A study on cutting similar to that of Johnston et al. (1991) was undertaken on the Scherping HCV by McLeavey (1995). Since 1998, 328 of the patented HCVs have been built mainly for US customers; HCVs have been installed in one plant in New Zealand. The most popular capacities are 25 000 and 30 000 1. As would be expected in a mostly American market, consumer cheeses made using HCVs are Americanstyle Cheddar, Colby, Swiss, Co-jack and Monterey Jack cheeses and the Italian-style Mozzarella, Asiago and Parmesan cheeses. Cheeses for further processing, such as the fat-free, reduced-fat or low-moisture barrel Cheddar and Swiss barrel cheeses are also made in HCVs. Scherping Systems, now a Carlisle company, has now developed and is producing the fully automated thirdgeneration HCV incorporating new counter-rotating agitators, dual curd outlets for more effective emptying and changes to the knife configuration of previous HCVs.
General Aspects of Cheese Technology
29
Damrow DOH horizontal cheese vat. Courtesy of Damrow Inc., USA.
The A P V CurdMaster
The first APV CurdMaster was produced in 1993 and its design is based on the Protech CurdMaster and the Damrow Double-O vat design, as shown in Fig. 6. As with the Damrow Double-O vat, each of the two knife panels of the APV CurdMaster is hung-off centrally located axes within each 'barrel'. However, the light stainless steel knives are mounted vertically in a stag-
gered formation across each panel, and the stirring blades are made of polypropylene. APV Denmark decided to concentrate on the DoubleO design because there were several advantages. The Double-O design allows: 9 for variable degrees of filling from 40 to 100%; 9 all shaft seals to be located above product level;
Scherping horizontal cheese vat. Courtesy of Scherping Systems, USA.
30
General Aspects of Cheese Technology
APV CurdMaster cheese vat. Courtesy of Invensys APV, UK.
9 efficient horizontal and vertical mixing; 9 minimal air entrapment after predraw or reduced fill levels. In addition, APV modified the attachment of the bottom of the vat to its support frame (floating bottom) to avoid welds cracking during heating and cooling. A 5 ~ incline and two outlets instead of one for more rapid and efficient emptying, staggered stay-sharp knives, polypropylene agitators and whey predraw during agitation are other modifications made by APV. Since 1993, APV Denmark, now part of the Invensys APV group of companies, has sold 146 APV CurdMaster vats to 56 customers throughout Europe and Latin America. The capacity ranges from 6000 to 30 000 1. Cheese types made using the APV CurdMaster include Danbo, Raclette, Mozzarella, Gouda, Edam,
Emmental, Tilsit, Blue, Feta, Maasdam, Cagliata, Provolone, Norvegia, Manchego, Camembert, Pecorino, Grana, Cheddar, Havarti, Port Salut and Parmesan. It is interesting to note that many of the cheeses listed are curd-washed varieties. Continuous processes
There have been various attempts to replace the batch vat process by continuous systems. Two systems warrant brief mention. An innovative system using uhrafihration technology and a sequential coagulation system was developed jointly by the CSIRO in Australia and APV, the process being named Sirocurd. Two commercial plants were developed and these successfully produced Cheddar-types cheese, with the benefits of increased yield from the uhrafihration stage (Jameson, 1987); however, the Sirocurd equipment is not now in operation.
General Aspects of Cheese Technology
The other system, which is still widely used, is the Alpma continuous coagulator. A diagram of this equipment is shown in Fig. 7. The system incorporates the use of a continuous belt, which is formed into a trough to hold the milk. This trough is then subdivided by a series of plates to effectively form mini-vats. As the belt moves, the vats also move along and the same processes that occur in a batch vessel are carried out on the belt, via the use of cutting tools, stirrers and other tools that are incorporated along the length of the belt. Partial whey drainage and water addition can also be incorporated, with the main curd/whey separation occurring at the end of the belt. Cooking is difficult with this system, which is therefore more suitable for the production of soft to semi-hard cheese types. Gentle treatment of the curd and evenness of particle size result in uniformity and continuity of output. These coagulators are in use worldwide, producing a wide range of cheese varieties from fresh curd to Havarti. Post vat stages - dry-salted types
Processing options here depend largely on whether the curd undergoes further development and handling as curd particles, followed by dry-salting and block formation, or whether the final cheese block is formed immediately, followed by subsequent brining for salt uptake. As shown in Fig. 1, distinctive processes are involved. The processes described here apply to hard cheese varieties such as Cheddar, Colby, Egmont and stirredcurd cheeses.
Oewheying The vats are emptied by pumping out their contents of curds and whey. This process is commonly described as running or draining the vat. Correct pump selection
31
is of vital importance as the curd can potentially be damaged, generating large quantities of fine particles that are lost into the whey stream. Large, slowly revolving, positive rotary lobe pumps are a common option, with the Sine | pump, which uses a specially formed impeller, becoming increasingly popular because of its gentle operation and low curd damage. During emptying of the vats, the stirrers remain in operation to ensure mixing of the vat contents. For the whole cheesemaking process to be effectively continuous, despite the batch vat stage, it is necessary for there to be a number of vats, e.g., eight vats operating and emptying in sequence to provide a continuous flow of curd. Even with this system, there is variation in acidity and composition between the curds that first leave the vat and those that leave towards the end. This effect can be minimised on multi-vat plants by overlapping vat emptying using dual pumps. The ratio of curd to whey also varies as the vat is emptied, with a higher proportion of curd at the start. The pump speed is controlled to increase during vat emptying to provide a uniform flow of curd to the next stage of the process. Primary separation of the curds and whey is achieved by pumping the curds/whey mixture from the cheese vat over a specially designed dewheying screen. This is normally parabolic in shape, fitted with horizontally oriented wedge wires, to maximise the efficiency of the separation process with minimal curd damage. The whey passes through the screen and the curd is transported to the next stage. The feed to the screen is designed to provide an even, gentle flow across its width; this is often achieved by the use of a weir feed arrangement. An example of the system used is illustrated at the top of Fig. 8, the Alfomatic cheesemaker. The whey that is removed through the screen is
Alpma coagulator. 1. Belt infeed, 2. Spacing plate insertion station, 3. Milk infeed, 4. Spacing plate in the coagulator, 5. Spacing plate transport, 6. Spacing plate extraction, 7. Curd-releasing station, 8. Curd cutter, longitudinal, 9. Curd cutter, crosswise, 10. Open syneresis sector, 11. Belt discharge, 12. Spacer plate cleaning. Courtesy of Alpma, Germany.
32
General Aspects of Cheese Technology
Alfomatic cheesemaker. 1. Whey screen, 2. Whey sump, 3. Agitator, 4. Conveyors (variable speed), 5. Agitators (optional) for stirred curd, 6. Chip mill. 7. Dry-salting system. Courtesy of Tetra Pak, Sweden.
collected and pumped to a tank prior to separate processing operations to produce a wide range of products. Initial processing operations include clarification to remove casein fines, centrifugal separation to recover fat and pasteurisation or thermisation to reduce the microbiological activity.
Drying (draining) the curd Commercial plants almost universally use a belt system for this next part of the process. Specially designed slotted plastic or stainless steel conveyor belts are used. These are usually fitted with peg-stirring devices mounted above the belts to agitate the curds in order to facilitate whey drainage and to prevent clumping of the curds. Residence times of 10 min are common. This belt often forms the first part of a cheese-texturing belt system. An example of these is the Alfomatic shown in Fig. 8.
Texturing (cheddaring) or stirring For varieties such as Cheddar, a traditional step in manufacturing protocol is the cheddaring stage, during which the curd is allowed to knit together, to flow and stretch and to develop a cooked chicken meat-type of structure. In the small open-vat process, cheddaring is achieved by heaping the drained curd along the sides of the vat and allowing it to fuse together. The fused mass is then cut into blocks of 10-20 cm and these are turned every 15-40 min over a period of 90-120 min to encourage flow and stretch to develop the desired
structure. There have been numerous attempts to replace this highly manual, labour-intensive process by a fully mechanised system. One such system is the cheddaring tower, a version of which was developed in New Zealand and is still available from Invensys APV. An example of this system is shown in Fig. 9. Essentially, the towers are cylindrical holding tubes, changing to a rectangular discharge section. Incorporated into their structure is a whey drainage system. Holding times of 1-2 h can be achieved with a capacity of up to 5000 kg curd/h. Large blocks of curd are guillotined from the column of curd as it exits from the base of the tower and fed into a curd mill. In the newer plants, a belt system has become very popular, typically with two belts running at different speeds to provide stretch, flow and inversion of the curd mass, and also to provide the desired holding time. Capacities of 12 000 kg of curd/h are possible. Examples of such equipments are the Alfomatic (Fig. 8), the Cheddarmaster (Fig. 10) and the Scherping draining conveyor (Fig. 11). These belt systems are totally enclosed in stainless steel housings. This provides a hygienic environment, and also the facility for in-place cleaning and maintenance of temperature. The belts are made of plastic or stainless steel and are generally not perforated, unlike the draining belts described earlier. The belts that are available for the cheddaring/holding stage can also be fitted with peg stirrers mounted
General Aspects of Cheese Technology
33
above the belt to facilitate the manufacture of stirred curd varieties, e.g., Cheshire and Egmont, on the same equipment. Similarly, the speed of the conveyors can be adjusted to provide the desired residence times.
Milling (size reduction) Following the texturing or cheddaring stage, the curd mass has fused into a solid structure. For the incorporation of salt in the next stage, it is necessary to reduce the solid mass to curd fingers (chips) of approximately 1.5 • 1.5 • 8 cm. This is achieved by the use of curd mills, of which there are a number of types. Most operate by using a rotating cutting tool, which cuts the curd mass in two directions using a blade and a comb. Prevention of fine particle generation is an important feature of the design. For stirred curd varieties, where little curd fusion has occurred, the mill still operates to break up any lumps that have formed. The mill is located at the base of the tower in a cheddaring tower system, or at the end of a conveyor belt in the more common belt systems.
Dry-salting and mellowing
APV cheddaring tower, with guillotine and mill at base. Courtesy of Invensys APV, UK.
Salting the curds is a vital part of the cheesemaking process. Salt has very important roles in flavour enhancement and in the control of microbiology, final cheese pH and moisture content. A detailed discussion on salting is given in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. Critical factors include the application of the correct ratio of salt to
APV Cheddarmaster belt system. Courtesy of NZMP Whareroa, New Zealand. (See Colour plate 2.)
34
General Aspects of Cheese Technology
Scherping cheese curd draining conveyor. Courtesy of Scherping Systems, USA.
curd, even uptake of the salt and controlled loss of moisture. The level of salt required will vary according to the type of cheese being manufactured. There are two components to the salting p r o c e s s - the application of the salt (salting) and the subsequent mixing, uptake and associated moisture loss (mellowing). There has been a range of equipment designs to achieve satisfactory salting, with variable success. Simpler styles have included belt systems in which the quantity of curd being conveyed is measured by means of a fork sensing curd depth, with dry salt then being air-conveyed and distributed across the belt by a reciprocating boom. The quantity of salt is varied in proportion to the curd flow and is metered by a funnel and salt wheel device in a dry area of the plant. Better control can be achieved by using load cells on the belt to weigh the curd flow. Twin-salting booms are another alternative, each applying a proportion of the salt. A widely used system is the trommel or drum salter, in which the curd flow is directed over a weighing belt and then into a rotating drum into which the salt stream is directed. This provides accurate measurement and good mixing. However, if this system is to be used in conjunction with a belt plant, the curds must be conveyed from the belt to the salter and returned to the next belt. An example of such a system is shown in Fig. 12. A variation on this concept involves the use of an auger conveyor instead of the rotating drum to provide mixing of the salt and curds, as they are conveyed back onto the mellowing belt.
The mellowing belt provides a holding time of 10-20 min to allow the applied dry salt to be mixed, dissolved and absorbed by the curd, at the same time as moisture is expelled. The belts are equipped with peg stirrers to encourage mixing and moisture loss, and they are also enclosed to maintain temperature. An alternative to the belt system is the use of finishing/sahing vats or tables, which are suitable for stirredcurd varieties. In these, the curds/whey mixture is pumped from the vat into these batch tanks, which allow whey drainage, holding time and pH drop, salt addition and mellowing, all in one vessel. An example is the Damrow enclosed finishing vat shown in Fig. 13.
Pressing~block formation- general discussion This process is common to most cheese varieties, exceptions being particulate cheeses such as Cottage cheese. Block formation involves the conversion of granular, particulate curd into a solid block of cheese. The degree of compression required and the techniques used vary according to the cheese type. For example, close-textured hard cheeses such as Cheddar require the application of considerable pressure and air removal to form appropriate blocks. Other varieties, such as Blue cheese, require little compression and pressure in order to produce an open texture enabling air penetration and mould growth. Varieties such as Gouda and Edam require preliminary block formation while submerged in the whey prior to further compression. A vital component of block formation during the history of cheesemaking has been the cheese hoop or
General Aspects of Cheese Technology 35
Figure 12 Trommel salting system. Courtesy of NZMP Edendale, New Zealand. (See Colour plate 3.) mould. Although its use has been superseded by blockformers in the large-scale production of dry-salted cheese, it is still a vital component of many other plants and also small-scale dry-salt plants. The cheese hoop or mould is a specialised container designed to hold and form the curd into the desired shape, permitting the further loss of whey and the application of pressure and vacuum, if so desired. The moulds were made originally of wood, with the inner shape being that of the final cheese. They were cylindrical or rectangular and had holes drilled through the sides, base and lid to permit whey drainage. They were often lined with cloth (hence the term cheesecloth) to provide a porous barrier between the curd and the walls to allow whey drainage. An early option was the use of metal, especially for rectangular blocks, and the use of telescopic lids and bases to permit compression of the blocks under applied external pressure. This system is still in use for small-scale operations, with stainless-steel moulds and synthetic cloths providing improved hygiene. A major technological development has been the introduction of plastic moulds. These may range from a simple plastic or metal tube with appropriate perforations, for a variety such as Camembert, to which no external pressure is applied, to a highly sophisticated micro-perforated, grooved, muhi-mould for Gouda. This technology has eliminated the need for cheesecloths, as drainage is via the grooves and the micro-porous holes. Hygiene is
maintained through an appropriate cleaning process, which may include ultrasonics. The desired cheese surface effect may be achieved by selecting an appropriate surface grooving. A major recent advance has been the introduction of welded plastic moulds, eliminating the use of metal screws as in earlier types. The Dutch company, Laude bv, has been at the forefront of developments in this field, and examples of its products are shown in Fig. 14. The appropriate pressing regime to be applied to the curd contained in the mould depends on the cheese type and is discussed separately. There is a risk in the application of too much pressure initially, which results in surface closure and poor subsequent whey removal.
Pressing/blockforming of dry-salted cheese For dry-salted cheeses, the next stage of the process is the conversion of the salted chips of curd into a solid block. The traditional process involved the use of hoops or moulds into which the curd was weighed and then compressed, often overnight, by externally applied pressure using hydraulic rams, commonly in horizontal gang presses. This system is still in use in small-scale plants, and developments in this area are discussed in more detail under brine-salted cheeses. The universal system adopted in large-scale dry-sah plants involves the use of blockformers, of which there are a number of varieties. Wincanton Engineering in the UK patented the original development over 25 years ago.
36
General Aspects of Cheese Technology
Damrow enclosed finishing vat. Courtesy of Damrow Inc., USA.
Plant capacity requirements usually mean that several blockformers are necessary and it is therefore important for reasons of product uniformity that an even feed is supplied to each blockformer. This may be achieved by using devices such as curd distribution tanks, which provide mixing of the curd from the mellowing belt and even distribution of the curd to the suction tubes feeding the blockformers. An example of these is shown in Fig. 15. All blockforming towers operate on a similar principle of using vacuum to draw curd into the top of the tower. The curd column is then subjected to further vacuum as it progresses down the tower. The internal side walls are perforated to facilitate whey and air removal, and the height of the towers (6-9.5 m) provides compression by gravity. As the curd travels down the tower, it is converted from individual curd par-
ticles into a fused column. This is discharged at the base via a guillotine arrangement, which produces blocks of cheese of a uniform shape and weight, typically 18-20 kg. The operation of the tower is illustrated in Fig. 16. The typical residence time in the towers is 30 min. Weight control is effected by adjustments to the platform height in the guillotine section. All the major equipment suppliers produce blockformers with variations in detail. Some of the more recent developments include extending the height to increase capacity and the provision of two different vacuum stages, as in the Tetra TwinVac Blockformer | This permits the use of a higher vacuum in the lower column, which is effectively separated from the upper column by a plug of curd, permiting the use of a lower transport vacuum in the upper section and a higher throughput.
General Aspects of Cheese Technology
37
Laude block mould. Courtesy of Laude by, The Netherlands. (See Colour plate 4.)
There are a number of variations of blockformers, producing differently shaped and sized blocks from 10-kg cylinders to 290-kg blocks. The type used depends on the product's end-use. A recent innovation by Cryovac | has been the introduction of bag loaders at the base of the towers, which automatically fit cheese bags to the discharge channels to receive the cheese blocks from the tower. The same company also supplies gusset stretchers to help present the bagged cheese in the appropriate form to the vacuum-sealing device. This equipment has removed another repetitive manual operation from the process. An example of blockformers fitted with bag presenters is shown in Fig. 17. The packing of the cheese is important as it plays a role during curing and storage, in the final cheese shape and appearance and in protection from the environment. The formed cheese blocks are discharged from the pressing towers into muhi-layered plastic bags. These are conveyed to a vacuum-sealing chamber where air is removed from the bag which is heatsealed. The gas and water permeability properties of the bag and the level of vacuum applied vary according to the cheese type. Prevention of moisture loss and prevention of mould growth are key factors for Cheddartype cheeses. The curd is still warm (typically 33 ~ as it exits the blockformers and is quite plastic. Therefore, the vacuum-sealed block requires the support of a carton
while cooling to maintain its desired shape and finish. Cartoning operations are normally fully automated with a variety of carton styles in use, ranging from a shoebox style with a separate base and lid to a wraparound one-piece type.
Ripening and storage This is a highly complex topic, which is the subject of several other chapters in this book (see 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese during Ripening', 'Sensory Character of Cheese and its Evaluation' and 'Instrumental Techniques', Volume 1). Cheese is essentially a complex matrix of protein, fat and carbohydrate, containing a range of enzymes and microorganisms. Their activities produce the changes that convert the young or green cheese into the desired final product, primarily through proteolysis, lipolysis and glycolysis. The primary objective of the cheesemaking process is to produce a material with the desired characteristics for ongoing changes during curing and storage. Factors such as salt content, pH and moisture content are of critical importance. The primary controllable factors after the young cheese has been made are the time and the temperature of storage. During ripening, changes in flavour and texture
38
General Aspects of Cheese Technology
which 40 or more blocks are stacked and shrinkwrapped. This format is suitable if the cheese is to be used for manufacture into processed cheese. Alternatively, the cheeses in cartons may be stacked on a pallet, or the cheeses in carton bases may be placed in bulk bins. These are strapped and tension is applied to help maintain shape and finish. This format is suitable for cheese intended for the precutting trade, where the large blocks are cut and repacked into consumer packs. Robots are normally used for these assembly operations. A typical assembly is shown in Fig. 19.
Ripening (curing). This involves the transfer of the palletised product to controlled-temperature storage rooms where the pallets are assembled onto racks. Typical temperatures are 8-10 ~ for a period of 35 days or so. Temperature and time after this stage will depend on the desired end-use for the product. For example, if a more rapid maturation is required, the temperature may be elevated to 15 ~ for 1 month. If a slower rate is required, a temperature of 2 ~ may be used. Once the desired degree of ripening has been achieved, the product is transferred to reducedtemperature storage to reduce the rate of further change. Storage. In this stage, the objective is for minimal change in product characteristics with time. This is achieved primarily through controlling the temperature. Freezing of the product is an option if the enduse for the product is processed cheese. Curd distribution tank. Courtesy of NZMP Stirling, New Zealand. (See Colour plate 5.)
occur. From a technological point of view, several stages can be identified- initial cooling, curing or ripening and controlled storage. The particular regime used depends on the cheese type and its intended use. Initial cooling of dry-salted cheese. This serves two
purposes. Firstly, a reduction in the temperature of the cheese curd causes the fat to solidify and the cheese to become firm and maintain its shape. Secondly, a sharp drop in temperature prevents the rapid growth of undesirable non-starter lactic acid bacteria, which could otherwise use residual lactose and produce undesirable gas and flavour defects. A reduction in temperature to 16 ~ within 12-16 h of manufacture is achieved by the use of open-rack stacking of the cheese blocks, which are then conveyed into a blast chiller, using air at 2-8 ~ Openrack stacking is necessary to permit good air flow and heat transfer. The rapid chillers operate on a first-in/firstout basis. An example is shown in Fig. 18. Following the rapid cooling operation, the cheeses are stacked into the form required for their long-term curing and storage. This may be a cartonless pallet on
Despatch The process described thus far is for the production of bulk blocks of cheese, typically weighing 20 kg. This product has many end-uses, such as an ingredient for many food products that contain cheese, conversion to grated cheese or processed cheese or cutting as natural cheese into consumer-size blocks. The uses of cheese as a food ingredient and as processed cheese are the subject of separate chapters ('Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', 'Cheese as an Ingredient', Volume 2). The preparation and packaging of cheese for domestic consumers involves the use of a wide range of sophisticated equipment and packaging technologies, the detail of which is beyond the scope of this chapter. Typical steps involve cutting the cheese blocks into the appropriately sized smaller blocks, followed by packaging in appropriate laminated material, under either vacuum or a modified atmosphere. Post vat stages - hard/semi-hard brine-salted types
Post vat processing of the cheese curd differs considerably for cheeses that are essentially formed into their final block shape on leaving the vat, as these generally require
General Aspects of Cheese Technology
Blockformer operating principles. Courtesy of Tetra Pak, Sweden.
Blockformers with bag presenters. Courtesy of NZMP Edendale, New Zealand. (See Colour plate 6.)
39
40
General Aspects of Cheese Technology
Dewheying For many varieties, partial whey removal occurs during the vat stage of processing, when the agitators are stopped for a period, allowing the curds to sink, and a whey-removal screen is lowered into the vat and the required amount of whey is drawn off. This is replaced by hot water, which serves to cook the vat contents and also to dilute the lactose and lactic acid content of the remaining whey. Further whey may be removed in the same way before the curds/whey mixture is pumped from the vat.
Pre-pressing
Rapid cooling tunnel. Courtesy of NZMP Hautapu, New Zealand. (See Colour plate 7.)
immersion in brine to achieve salt uptake. There are also processing differences depending on whether the cheeses are hard/semi-hard or soft and possibly mould-ripened. These differences are summarised in Fig. 1.
The presence of eyes or holes in the cheese is an important characteristic of several major cheese types, such as Gouda, Edam and Emmental. An important feature of the curd block formed for such cheese is the absence of air from within the block, and instead the presence of microscopic wheyfilled cavities in which micro-organisms can grow and produce gas, in particular CO2, which can ultimately form the characteristic round eyes (Martley and Crow, 1996; Kosikowski and Mistry, 1997). For the appropriate curd characteristics, the curds are formed into blocks below the surface of the whey prior to curds/whey separation, in contrast to the procedure with dry-sahed cheeses such as Cheddar. This process is known as pre-pressing. As block formation occurs prior to salting, an alternative salting technique, brine salting, also becomes necessary. To reduce the volume of material to be handled during block formation, some whey is removed using the vat sieve or strainer prior to pumping out the curds/whey
Robot stacking of cheese blocks. Courtesy of NZMP Hautapu, New Zealand. (See Colour plate 8.)
General Aspects of Cheese Technology
mixture to the pressing stage. An early development of a mechanised system to achieve the objective of pressing under the whey involved the use of prepressing vats, as illustrated in Fig. 20. The curds/whey mixture is pumped into a rectangular vat, and perforated metal or plastic plates are placed above the vat contents, and then lowered below the whey to the curd layer, which is supported by a woven plastic belt at the base of the vat. This layer is then compressed by the application of hydraulic pressure to the plates and a solid curd mass is formed. The whey is then removed, and the curd layer is conveyed from the base of the vat through the now-open end and is cut into appropriately sized curd blocks by cutting tools prior to being placed in moulds for further pressing and formation. More advanced systems use a semi-continuous prepressing blockforming system of which the Casomatic | equipment produced by Tetra Pak Tebel is a widely used example. A diagram illustrating the working principles is shown in Fig. 21. Buffer tanks are used to store the curds/whey mixture pumped from the cheese vat; they are essential to provide an evenly mixed feed to the pressing system. The curds/whey mixture in the ratio of about 1:4 is then pumped to the top of the column, which is about 3 m in height, with a total unit height of 5.5 m. The column is filled and the curds settle below the whey to a height of about 2 m. Whey is removed from the column via three whey drainage bands; a controlled rate of removal is
41
critical for the formation of a block of the correct density at the base of the column. The curd block is formed in a dosing chamber and is cut from the column above by means of a guillotine. The dosing chamber then moves forward and discharges the formed block into a mould or hoop for further pressing and formation. Several variations using the same operating principle are available to produce blocks of various shapes and sizes from 1 to 20 kg, with discharge of multiple blocks from one column being possible. Exchangeable perforated drainage columns within a common jacket can be used, as in the Casomatic | MC model. Cheese types with irregular holes or eyes, also known as granular, e.g., Parmesan, can also be handled using equipment such as the Casomatic | Pressing under the whey is not required, and curds/whey separation can be achieved by the use of rotating sieves or strainers placed above the columns, discharging curd into the column for initial block formation.
Pressing Having formed the curd into the final cheese block by moulding in the pre-pressing stage, further pressing of the block is necessary. This provides a further period for ongoing acid development and pH and texture change, and assists final whey expulsion, shape formation and also surface texture for subsequent rind formation, where appropriate. Simple vertical pressing systems are suitable for small-scale operation, where the cheese moulds are loaded into
Pre-pressing vat. 1. Pre-pressing vat, 2. Curd distributors or CIP nozzle (2a), 3. Unloading device, 4. Conveyor. Courtesy of Tetra Pak, Sweden.
42
General Aspects of Cheese Technology
programme. Again, simultaneous loading and unloading of the pressing bays are practised. An example of a conveyor pressing system is shown in Fig. 23. Pressing times and pressures vary with the cheese variety and block size. It is important that there is a gradual increase in pressure, as the application of too much pressure at the start can cause closure of the surface and prevent whey removal. A typical programme for 10 kg Gouda cheese is 1 bar (0.1 MPa) for 20 min, followed by 2 bar for 40 min. For cheeses such as Emmental where blocks of 30-100 kg are common, a specialised system has been developed by Tetra Pak Tebel; it incorporates a specialised mould-filling system that can also incorporate pressing, with a further external press equipped with inverting facilities to help improve cheese quality and uniformity. Another automated system for blocks up to 700 kg is available. Once the required pressing operation has been completed and the desired pH drop has been achieved, the cheese blocks are removed from the moulds and are conveyed to the next stage of brining. The used moulds and lids are returned to the system via a cleaning process.
Brining
Casomatic operating principles. 1. Curd/whey mixture inlet, 2. Column with sight glass, 3. Perforated whey discharge, 4. Interceptor, 5. Whey balance tank, 6. Cutting and discharge system, 7. Mould, 8. Pawl conveyor, 9. Whey collecting chute. Courtesy of Tetra Pak, Sweden.
the press and the appropriate pressure regime is applied by lowering hydraulic rams. For larger-scale operations, trolley presses, tunnel presses or conveyor presses are used. With trolley presses, the cheese moulds are placed on a trolley, which is then fed into a tunnel equipped with a series of individual vertical rams. These are subsequently lowered to apply the appropriate pressure to the batch of cheese. Automatically fed tunnel presses operate by automatically loading cheese into the tunnel, followed by the pressing programme for the whole batch. Simultaneous loading and unloading is possible. An example is the APV SaniPress system shown in Fig. 22. The conveyor press is another option, with the cheese moulds being loaded onto a conveyor system, where the blocks are assembled into groups. Each block or pair of blocks has an individual hydraulic ram and each group has its own individual pressing
Cheeses that have been formed into blocks under the whey cannot be salted prior to moulding and pressing. The application of dry salt to the cheese surface is one technique that is used for some cheeses, such as Blue, but for many cheeses brine-salting is simpler, provides greater uniformity and is less labour-intensive. Many cheeses that have traditionally been made using brinesalting can in fact be made using the simpler and cheaper dry-salting technology described already for Cheddar-type cheeses. However, eye development is not usually attempted, with the major objective being to produce the appropriate typical flavour and texture. As already mentioned, there is a detailed discussion of salting in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. Brine-salting basically involves the immersion of the cheese block into a brine bath. The brine is a solution about 19-21%, w/w, of NaC1. It should also contain an appropriate level of CaC12, e.g., 0.2%, w/w, to prevent leaching of calcium from the cheese. Its pH should be close to the cheese pH (typically 5.2-5.3) and its temperature should be 10-14 ~ As the brine is used, its salt concentration must be maintained as salt moves into the cheese and water/whey moves out, causing dilution. Also, the brine will become contaminated with cheese particles, whey proteins and undesirable bacteria. Filtration (including membrane filtration), centrifugal clarification and pasteurisation can be used to maintain brine quality. If properly cared
General Aspects of Cheese Technology
43
APV SaniPress tunnel pressing system. Courtesy of Invensys APV, UK. (See Colour plate 9.)
for, the same brine can be used for many years (Bylund, 1995b; Kristensen, 1999). The time required for adequate salt uptake in the brine depends on the size of the cheese block and the
desired final salt level. For example, a small 250 g Camembert may require only a few hours, whereas a 10 kg Gouda may require 2 days. Brining systems can be a simple tank in which the cheese is placed once it has
Conveyor pressing system, with Casomatics in foreground. Courtesy of NZMP Lichfield, New Zealand. (See Colour plate 10.)
44
General Aspects of Cheese Technology
been removed from its mould. Alternatively, a more continuous system, known as the serpentine or surface brining system, may be used, where the cheeses are floated in brine channels to holding pens for the required period. As the surface of the cheese is above the brine, periodic spraying of the surface with brine or forced dipping of the cheese below the surface is required to achieve even salt uptake. Another option for brine application is the TrayBrine System from APV (Fig. 24). Here, the cheeses are placed on plastic trays, which are stacked and connected to a brine distribution system. The brine flows down over the cheese surface, is recirculated for the required period and is then recovered. A common method of brining for large-scale operations is the deep brining technique, where the cheeses are floated onto shelves on racks which are then progressively submerged below the brine surface. Ideally, the racks should be emptied and the loading sequence reversed midway through the brining process to ensure the first-in/firstout principle for consistent salt uptake. An example of a deep brining system is shown in Fig. 25. In addition to the vital effect of providing salt uptake for control of the microbiology and flavour of the cheese, brining also provides a rapid cooling effect,
reducing the cheese temperature to a value close to that of the brine within several hours. This helps control the growth of undesirable bacteria in a similar fashion to the rapid cooling step used in Cheddar production.
Ripening Once the cheese has been brined for the required period, it is floated to the discharge point and removed from the brine via a conveyor. Its surface may be rinsed with a brine solution to remove any foreign matter and is then air-dried with a blower or air knife. Thereafter, packing and curing depend on the intended market. Rindless cheeses, which are very commonly produced for bulk markets, especially if they are to be used subsequently as ingredients, are packed into appropriate laminated plastics bags under vacuum. They are then put into cartons and are stacked on pallets and transported to the appropriate curing and storage conditions. If eye development is required, several stages of temperature change will be used, e.g., for Emmental, 3-4 weeks at 10 ~ followed by 6-7 weeks at 22-25 ~ for eye development, and storage/curing at 8 ~ for several months. For Gouda, conditions may be several weeks at 10-12 ~ followed
APV tray brining system. Courtesy of Invensys APV, UK. (See Colour plate 11 .)
General Aspects of Cheese Technology
45
Deep brining system. Courtesy of NZMP Lichfield, New Zealand. (See Colour plate 12.)
by 3-4 weeks at 12-18 ~ followed by several months at 10-12 ~ (Bylund, 1995b). If eye development is desired, as gas production is necessary, appropriately permeable laminated bags must be used to permit gas transport. If rinded cheeses are being produced, control of the humidity in the curing rooms is important (usually about 85-90%) to prevent undue moisture loss. Coloured wax coatings may also be applied to provide protection for the cheese. Some varieties, such as Parmesan and Emmental, require frequent turning during curing to maintain the desired shape. Mechanised systems, such as revolving shelf rails, are available for all the material-handling operations such as inversion of the final cheeses. As already discussed under Cheddar types, curing and maturation are a combination of time and temperature conditions, with the additional influence of humidity for cheeses that are not packed in plastic film.
Despatch The cheeses have the same multiple end-uses as already described for dry-salted varieties. However, as the brinesalting system tends to be more expensive, these products are more typically directed at the retail consumer market, requiring appropriate cutting and packaging. Post vat stages - brine-salted, soft mould-ripened
Cheeses such as Camembert and Blue fall into this category. Technological advances and automation have been applied to these varieties and ultrafiltration has had a major impact, as numerous advantages, includ-
ing yield improvement, can be obtained. The use of ultrafiltration is discussed in detail in 'Application of Membrane Separation Technology to Cheese Production', Volume 1. Discussion of these cheeses commences at the vat stage in Fig. 1. Uniformity of milk, starter and coagulant activity are of critical importance for the uniformity of syneresis, which is essential for these varieties (Pointurier and Law, 2001). The normal operations of coagulation, cutting, stirring and acid development occur in the vat. The milk entering the vat may have been pre-ripened with starter culture and is likely to include the mould spores for later development. However, because of the high moisture content, which changes rapidly with time due to syneresis, it is not practical or desirable to use large vats for the production of Camembert types, in particular, as the curd composition of the material first being discharged would be very different from that discharged 30 min later. Hence, curd formation in small vats of up to 300 1 is necessary, so that the contents may be discharged rapidly into multi-moulds where curds/whey separation (dewheying) occurs. This is combined with moulding and may be done by tipping the vats directly into the moulds or by using a specialised portioning system such as the APV Contifiller, illustrated in Fig. 26. Also illustrated here is the use of multiple small vats on a semi-automated line and handling systems for the filled moulds. The batch-continuous production system is necessary to obtain a uniform fill of curds/whey mixture into the moulds, as this is the determinant of the final cheese size and weight.
46
General Aspects of Cheese Technology
1. Curdmaking 2. Curd draining and filling 3. Stacking of mould batteries (A) and trays (B) 4. Turning of mould stacks
5. Acidification lines 6. Destacking 7. Transfer/turning of cheese from mould batteries to trays
8. Transport to climate room (A) and from brining (B) 9. Turning/emptying 10. Washing of mould batteries (A) and trays (B)
Process line for soft cheese with Contifiller. Courtesy of Invensys APV, UK.
The development of various systems, such as the ~11Pril'l,.., ,.,,..~.l~ nar"s ruP' 'c' ~c l~ i~ APcrrihPA i n m n r ~ , ,..,,..,.,.,.rlPtailhyu R p r l r a n r - ] (1987) and Pointurier and Law (2001). Some earlier systems include the use of micro-pans, which produce just enough curd for one mould. The Alpma continuous coagulator, already described in the vat stage section under continuous processes, has special application for these soft cheeses, being effectively a continuous series of small vats. The multi-moulds used to form the cheese may be in two sections to provide sufficient volume for the initial fill. The upper layer can be removed once initial block formation has occurred. The filled moulds can be stacked automatically and conveyed to the initial ripening rooms for further acid development, followed by brining in tanks for about 30 min, and then ripening for about 10 days in high humidity rooms for mould development. Frequent turning of the cheese is necessary during the first few days to ensure even block formation. This can be automated in larger plants. Final wrapping is done in air-permeable material and despatch follows. Variations such as dry-salting the cheese by surface application, may be used for Blue cheese. A feature of these mould-ripened cheeses is that a very open texture may be necessary to allow oxygen penetration for mould growth. Hence the cheeses are
not pressed by the application of any external pressurei11~t
J ""
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ir
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internal mould growth is desired, the passage of air is facilitated by spiking holes through the cheese with special needles. Smear-ripened cheeses are another type within both the semi-hard and the soft categories. The key process is the application and growth of a smear culture, predominantly Brevibacterium linens, on the surface of the cheese during ripening. Various mechanised brushing systems are available for smear application, which is usually repeated several times during ripening, where control of humidity and temperature is critical. Post vat stages - fresh cheeses
Cottage cheese falls into the soft/fresh category but is unusual in that the final product consists of curd particles packed in the final container with the appropriate dressing. Specialised equipment has been developed to mechanise and automate the production of this highly popular product. An example of this equipment is the O-vat by Tetra Pak Tebel. Quark, cream cheese and similar products also fit here but their manufacture is very different and is not described in detail (see 'Acid- and Acid/Rennet
General Aspects of Cheese Technology
Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acidheat Coagulated Cheeses', Volume 2). Following the formation of the coagulum in special ripening vats, the whey is separated using a specially designed centrifugal separator. The product is then blended with appropriate additional components, e.g., cream, and then filled directly into the final container. Post vat stages- pasta filata
Pasta-filata
cheeses are those varieties for which the curd has been worked or stretched and moulded at an elevated temperature before cooling. This process imparts a unique and characteristic fibrous structure that influences both the ripening and the functional profiles of the final cheese. Mozzarella is probably the best known of the Pastafilata cheeses, which are mainly Italian in origin. However, the category also includes cheeses such as Provolone, Scamorza, Caciocavallo, Kashkaval and Pizza cheese. Composition, particularly moisture level, and fresh versus ripened textures are characteristics that define the various varieties. The increase in popularity of the pizza in its various forms (from the thin-based traditional Italian pizza, with few or no toppings except Mozzarella and cooked in a wood-fired oven, to the American-style thick pan-based pizza, with a myriad of toppings and cooked rapidly in an impinge> type oven) has focussed attention on low-moisture Mozzarella or Pizza cheese (see 'Pasta-Filata Cheeses', Volume 2). del Prato (2001) discusses the various varieties of Pasta-filata cheeses and the traditional processes and purpose-built equipment to make them. However, another manufacturing option is to use existing equipment and to add on a cooker/stretcher and a cooling operation at the end of the curdmaking stage of the existing process. This has been the case in the development of New Zealand's Mozzarella industry. New Zealand produces only low-moisture part-skim (LMPS) Mozzarella and has adapted its Mozzarella-make procedure so that the existing Cheddar vats and curd-handling and cheddaring systems can be used to produce Mozzarella curd for stretching and subsequent cooling. Hence, the Pasta-filata process is included as a branch of the dry-salt Cheddar-type process in Fig. 1. Dry-salting can also partially or completely replace brining. Equipment designed to perform the stretching operation incorporates two essential components: cooking and stretching (the mechanical treatment of the curd following cooking). The cooking phase is where the Pasta-filata curd is transferred to the hot water section of a cooker/stretcher.
47
At this point, the curd is immersed, heated and worked by single- or twin-screw augers. The temperature of the water is determined by the temperature of the curd entering the stretcher, the curd flow rate and the target temperature of the cooked curd. Typical water temperature varies between 60 and 75 ~ with the cooked curd temperature varying between 55 and 65 ~ The mechanical treatment of the cooked curd influences the final cheese structure, composition and functionality. Moisture can be expelled or further incorporated. Salt and other ingredients can also be added at this point. Mechanical treatment or mechanical conditioning of the cooked curd is usually achieved by further working by single- or twin-screw augers or by 'dipping' arms in a relatively moisturefree environment. Following mechanical working, the curd may be extruded into a mould and immersed in chilled brine for cooling and salt uptake. Packaging and despatch follow, with shredding being a common option for pizza use. Almac s.r.l., Modena, Italy, Stainless Steel Fabricating, Wisconsin, USA and Construzioni Meccaniche E Technologia S.p.A (CMT), Italy, are examples of companies that manufacture a range of Pasta-filata processing equipment, including cooker/stretchers. Their equipment is described in the following sections. A l m a c s.r.L
Almac s.r.1, has been producing systems for making Pasta-filata cheese since the 1980s. They manufacture essentially three standard systems: for the production of high-moisture Mozzarella, for the production of Pizza cheese (low-moisture Mozzarella) and for the production of the ripened Pasta-filata cheeses (Provolone, Kashkaval and Kasseri). Turnkey design starts at curd draining and each system includes cheddaring (curd ripening), cooking/stretching, moulding and cooling (including pre-hardening and hardening), brining and packaging. Almac s.r.1, has an extensive range of cooker/stretchers with various capacities, built to handle a range of curd textures, depending on the type of Pasta-filata cheese to be made. An example is shown in Fig. 27. All the larger capacity cooker/stretchers use twin screws to convey the cut curd through the cooking section and all use the 'dipping arm' technology to condition the curd following cooking. All product contact surfaces are coated with a non-stick agent. A minimum quantity of water is used during the cooking phase to ensure high yields. Almac s.r.1, supplies Mozzarella cooker/stretchers to customers throughout Italy, other European countries and to Australia, Canada, Iran, Ecuador, Argentina, Brazil, the USA, Venezuela and Eygpt.
48
General Aspects of Cheese Technology
Almac cooker/stretcher. Courtesy of Almac, Italy. (See Colour plate 13.)
Stainless Steel Fabricating, Inc. qt.inloqq
qtool Fnhrirntino
(qqF)
is characterised by the fibrous nature of its texture and ,lqn
rn~Inllf~IrtllrOq
equipment for producing mainly low-moisture Mozzarella (American Pizza cheese) and Provolone. Stainless Steel Fabricating can provide cooker/stretchers, moulders and chilling-brining systems. It is a family-owned business, operating for the last 35 years and supplying Mozzarella equipment to Mozzarella manufacturers in North America, South America, Europe, Asia, Africa, Australia and New Zealand. Five models, ranging in capacity from <113 k g ~ (<250 lbs/h) to 9080 kg/h (20000 lbs/h) make up SSF's SUPREME cooker/stretcher range. An example is shown in Fig. 28. In contrast to the Almac design, SSF cooker/stretcher models use inclined twin augers to cook and condition the Pasta-filata curd. At the base of the incline, curd is cooked in circulating hot water. Curd conditioning takes place at the top of the incline, where the cooking water is encouraged to drain back to the base of the incline. Stainless Steel Fabricating also manufactures a range of moulders with capacities up to 1816 kg/h (4000 lbs/h) depending on the size of the mould. In addition, SSF produces an extruder for String cheese production. String cheese, which is essentially a thin stick of Mozzarella, is a popular snack food in the US and is used by some pizza makers, such as Pizza Hut, to fill the crusts of their 'stuffed-crust' pizzas. Each stick
m,~ro .......
r
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)'
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fibres. Using the SSF extruder, a ropes of Mozzarella is formed. The varied by adjusting the location which is activated by an electronic models of the automatic string ropes up to 3 m in length.
nf
thP nrntt~in t
series of continuous length of stick can be of the string cutter, sensing device. Other cutters can produce
Construzioni Meccaniche E Technologia S.p.A. (CMT) Construzioni Meccaniche E Technologia S.p.A., like Almac, is an Italian-based company and also produces a range of Mozzarella equipment including cooker/stretchers for customers similar to those supplied by Almac. As with SSF, CMT also produces equipment to make String cheese (Fig. 29). However, in the CMT machine, the String cheese is moulded rather than extruded. Construzioni Meccaniche E Technologia S.p.A. claims certain advantages, including the same fibrous structures as those obtained by extrusion but also more consistent weight and dimension control.
Cheesemaking is a centuries-old process that has developed from an art to a science as the demand for the product and the scale of production have increased. Conversion from a cottage industry to the
General Aspects of Cheese Technology
49
SSF cooker/stretcher. Courtesy of Stainless Steel Fabricating, Inc., USA. (See Colour plate 14.)
highly complex automated factories in use today has demanded major developments in technology. There have been many ingenious approaches to the technology requirements and the consumer has benefited from having very consistent, safe, nutritious and palat-
able products. Further technological developments will occur as our understanding of cheese increases and our ability to fractionate milk to its various components and reassemble them into desired products increases.
CMT String cheese moulder. Courtesy of Construzioni Meccaniche E Technologia, Italy. (See Colour plate 15.)
50
General Aspects of Cheese Technology
The authors are grateful to Tetra Pak AB, Sweden, for permission to use illustrations from the Dairy Processing Handbook and other sales literature. The following are also gratefully thanked for the supply and the use of technical sales information: Almac s.r.l., Italy; Alpma GmbH, Germany; Construzioni Meccaniche E Technologia S.p.A., Italy; Damrow Inc., USA; Hivolt Services Ltd, New Zealand; Invensys APV Ltd, Denmark, UK and New Zealand; Laude bv, The Netherlands; Scherping Systems, USA; Stainless Steel Fabricating, Inc., USA; Stoelting Inc., USA; Tetra Pak (New Zealand) Ltd; Tetra Pak Tebel bv, The Netherlands. Permission to use photographs from numerous sites of NZMP Ltd, New Zealand, is gratefully acknowledged, as is the assistance given by E Jeffery from Massey University, Palmeston North, New Zealand, with the preparation of the figures.
Bertrand, F. (1987). The main steps in manufacture, in, Cheesemaking, Science and Technology, 2nd edn, Eck, A., ed., Technique et Documentation-Lavoisier, France. pp. 413-443. Bylund, G. (1995a). Collection and reception of milk, in, Dairy Processing Handbook, Tetra Pak Processing Systems, Sweden. pp. 65-71. Byiund, G. (1995b). Cheese, in, Dairy Processing Handbook, Tetra Pak Processing Systems, Sweden. pp. 287-329. del Prato, O.S. (2001). Pasta Filata cheeses, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 266-295. Heap, H.A. (1998). Optimising starter culture performance in New Zealand cheese plants. Aust. J. Dairy Technol. 53, 74-78. Jameson, G.W. (1987). Manufacture of Cheddar cheese from milk concentrated by ultrafiltration: the develop-
ment and evaluation of a process. Food Technol. Aust. 39, 560-564. Johnson, M. and Law, B.A. (1999). The origins, development and basic operations of cheesemaking technology, in, Technology of Cheesemaking, Law, B.A., ed., Sheffield Academic Press, Sheffield. pp. 1-32. Johnston, K.A., Dunlop, EE and Lawson, M.E (1991). Effects of speed and duration of cutting in mechanized Cheddar cheesemaking on curd particle size and yield. J. Dairy Res. 58, 345-354. Johnston, K.A., Luckman, M.S., Lilley, H.G. and Smale, B.M. (1998). Effect of various cutting and stirring conditions on curd particle size and losses of fat to the whey during Cheddar cheese manufacture in Ost vats. Int. Dairy J. 8, 281-288. Kosikowski, EV. and Mistry, V.V. (1997). Cheese with eyes, in, Cheese and Fermented Milk Foods, 3rd edn, EV. Kosikowski, LLC, Westport, CT. pp. 226-251. Kristensen, J.M.B. (1999). Salting of the cheese, in, Cheese Technology- A Northern European Approach, International Dairy Books, Aarhus, Denmark. pp. 137-139. Law, B.A. (2001). Cheddar cheese production, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 204-224. Martley, EG. and Crow, V.L. (1996). Open texture in cheese: the contributions of gas production by microorganisms and cheese manufacturing practices. J. Dairy Res. 63, 489-507. Maubois, J.L. (2002). Membrane microfiltration: a tool for a new approach in dairy technology. Aust. J. Dairy Technol. 57, 92-96. McLeavey, L.J. (1995). Setting and Cutting of Curd in Scherping Cheese Vats. Diploma in Dairy Science Technology Thesis, Massey University, Palmerston North, New Zealand. Muir, D.D. and Tamime, A.Y. (2001). Liquid milk, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 53-93. Pointurier, H. and Law, B.A. (2001). Soft fresh cheese and soft ripened cheese, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 250-265.
Plate 1
Examples of cheese from the principal groups of Ottogalli (1998, 2000a,b, 2001)" see Table 7 for further details. (See page 13.)
Plate 2 APV Cheddarmaster belt system. Courtesy of NZMP Whareroa, New Zealand. (See page 33.)
Plate 3 Trommel salting system. Courtesy of NZMP Edendale, New Zealand. (See page 35.)
Plate 4
Laude block mould. Courtesy of Laude bv, The Netherlands. (See page 37.)
Plate 5
Curd distribution tank. Courtesy of NZMP Stirling, New Zealand. (See page 38.)
Plate 6
Blockformers with bag presenters. Courtesy of NZMP Edendale, New Zealand. (See page 39.)
Plate 7
Rapid cooling tunnel. Courtesy of NZMP Hautapu, New Zealand. (See page 40.)
Plate 8
Robot stacking of cheese blocks. Courtesy of NZMP Hautapu, New Zealand. (See page 40.)
Plate 10 Conveyor pressing system, with Casomatics in foreground. Courtesy of NZMP Lichfield, New Zealand. (See page 43.)
Plate 11 APV tray brining system. Courtesy of Invensys APV, UK. (See page 44.)
Plate 12 Deep brining system. Courtesy of NZMP Lichfield, New Zealand. (See page 45.)
Plate 13 Almac cooker/stretcher. Courtesy of Almac, Italy. (See page 48.)
Plate 14 SSF cooker/stretcher. Courtesy of Stainless Steel Fabricating, Inc., USA. (See page 49.)
Plate 15
CMT String cheese moulder. Courtesy of Construzioni Meccaniche E Technologia, Italy. (See page 49.)
Extra-Hard Varieties M. Gobbetti, Dipartimento di Protezione delle Piante e Microbiologia Applicata, Universit& di Bari, Bari, Italy
The various schemes proposed for the classification of cheeses (see 'Diversity of Cheese Varieties: an Overview', Volume 2) indicate that the description of extra-hard varieties is not always unequivocal. The FAO/WHO Codex Alimentarius defines as hard and extra-hard, those cheeses having values of moisture on fat-free basis (MFFB) and fat in dry matter (FDM) lower than 56% and higher than 45%, respectively. Davis (1965) proposed a classification of cheeses based primarily on moisture content and assigned values of 25-36% and <25% to hard and very-hard cheeses, respectively. Burkhalter (1981) used the same primary criterion but did not separate hard and extra-hard varieties, and characterised as hard cheeses those with a moisture content lower than 42%, further dividing them in subgroups based on the source of milk (e.g., cows', sheep's or goats' milk), texture and ripening agents. The temperature of cooking, low to high scald, the type of secondary microflora and the extent of chemical breakdown during ripening are other standards which have been used to differentiate cheeses within the hard and the extra-hard group (Walter and Hargrove, 1972). Davis (1965) proposed values for the classification of cheeses as hard, semi-hard and soft, based on viscosity, elasticity and springiness. A few preliminary considerations may, therefore, emerge: (i) the moisture content is probably the primary criterion by which extra-hard cheeses are differentiated; (ii) although based on the use of several standards, the distinction between hard and extra-hard varieties is not always well defined; (iii) extra-hard varieties are manufactured from cows', sheep's or goats' milk or their mixtures; and (iv) different names for the same or very similar cheeses are used in countries which are large producers of these types of cheese. The selection of extra-hard varieties is further complicated since a particular cheese may be consumed as an extra-hard variety but also after a shorter period of ripening, when the cheese is soft. This chapter will describe cheeses which, although sometimes consumed as a different category, are manufactured mainly as extra-hard varieties.
Most of the extra-hard varieties are produced in Italy. Some of them, like Parmigiano Reggiano, Grana Padano and Pecorino Romano, rank amongst the most famous international cheeses and have maintained their traditional features over time in spite of great changes in cheesemaking technology. Parmigiano Reggiano, Grana Padano, Asiago and Pecorino Romano are used traditionally as grated cheeses as flavouring for Italian 'pasta'. Swiss, Spanish, Russian, Balkan and nonEuropean extra-hard cheese varieties are also well known. Most of the European extra-hard varieties are produced under Protected Denominations of Origin (PDO). For instance, of the 979 060 tonnes of cheese produced in Italy in 2001, 441 360 tonnes were of cheeses which are legally designed by a PDO. Of the latter, 343 838 tonnes (c. 78%) were hard or extra-hard cheeses (Industria Lattiero-Casearia Italiana, 2002). Table 1 s h o w s the production of the more important hard and extra-hard Italian cheeses. Except for Fossa (pit) cheese, all other extra-hard Italian cheeses have PDO status. All these cheeses, with the exception of Grana Padano, Parmigiano Reggiano, Asiago, Montasio, Provolone and Ragusano, are, or may be, produced from ewes' milk alone or mixed with cows' milk. Most of the Italian cheeses made from ewes' milk are identified by the name 'Pecorino'.
The main chemical and technological features of the more representative extra-hard cheeses are shown in Tables 2 and 3, respectively. Nevertheless, the characterisation of some varieties is very poor and the related technological features are incomplete. Long-ripened pasta-filata cheeses, like Provolone and Ragusano, are described in 'Pasta-Filata Cheeses', Volume 2. The use of raw milk and natural thermophilic starters, cooking of the curd to a high temperature, long ripening, a very low moisture content and, generally, an ancient tradition are features common to most of the extrahard cheeses. Some of the main relevant technological traits of the more famous extra-hard varieties are described below.
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
52
Extra-Hard Varieties
Production of the principal extra-hard Italian cheeses, 2001
Milk quantity,
Cheese production,
Cheese yield,
Cheese variety
Animal species
tonnes
tonnes
kg milk/kg cheese
Grana Padano Parmigiano Reggiano Asiago Montasio Pecorino Romano Pecorino Siciliano Pecorino Sardo Fiore Sardo Canestrato Pugliese Castelmagno
Cow Cow
2 057 054 1 554 793
138 080 108 425
14.90 14.34
Provolone Valpadana Ragusano Fossa
Cow Cow Sheep Sheep Sheep Sheep Sheep Cow/sheep and Goat Cow Cow Sheep/cow
2 16 051 8 66 865 2 22 458 38 340 3 708 5 040 1 001 690
22 91 35 7
611 578 310 100 600 700 180 65
9.56 9.47 6.30 5.40 6.18 7.20 5.56 10.55
2 23 000 90 975
21 400 10 150
10.42 9.0 6.50
Source: Industria Lattiero-Casearia Italiana 2002.
Italian cheeses
Parmigiano Reggiano Parmigiano Reggiano, also internationally known as Parmesan, is, together with Grana Padano, a 'Grana' cheese due to the granular texture of the ripened cheese. In addition to these, there are Grana Bagozzo and Grana Lodigiano which, because of their limited production, have practically disappeared from the market. Parmigiano Reggiano cheese is produced according
to a traditional and well-defined technology in a restricted area of the Pianura Padana. For the manufacture of Parmigiano Reggiano, feeding of the cows is regulated carefully: (i) the ratio between forage and other feeds must be -> 1 to limit the dry matter (DM) derived from feeds which are rich in starch and proteins" (ii) ->25% of the DM of the forage used must be produced on the same farm where the cheese is manufactured; ->75% of the DM of the forage used must be produced
Gross chemical composition of the principal extra-hard cheese varieties (average data)
Cheese
Moisture (%)
Total protein (Nx6.38) (%)
Fat(%)
Ash(%)
Soluble N/Total N (%)
Grana Padano Parmigiano Reggiano Asiago Montasio Pecorino Romano Pecorino Siciliano Pecorino Sardo Fiore Sardo Canestrato Pugliese Castel mag no Fossa Sbrinz Mah6n Manchego Roncal Idiazabal Kefalotyri
32.0 30.8 34.0 32.0 31.0 31.5 31.0 26.5 34.5 35.0 32.0 31.0 31.7 35.5 29.4 33.2 35.0
33.0 33.0 29.0 26.0 28.5 32.5 27.2 30.0 26.5 26.0 27.0 31.0 26.9 24.0 24.7 23.3 26.6
27.0 28.4 31.0 34.0 29.0 28.0 35.0 32.5 30.0 33.0 35.0 32.0 32.6 33.6 38.8 37.8 28.7
4.9 4.6 5.0 n.a. 8.5 n.a. n.a. n.a. n.a. 5.0 n.a. 5.0 6.8 4.6 4.8 4.0 3.9
34.0 32.0 28.5 26.5 22.5 26.5 24.0 25.5 30.0 26.5 32.0 31.5 31.1 25.9 26.2 29.0 24.5
n.a., data not available; From various sources.
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Extra-Hard Varieties
within the district where Parmigiano Reggiano is legally produced; -<25% of the DM of the forage used may be produced in territories adjacent to the district; (iii) the feeding of silage as fodder is not allowed to minimise the number of spore-forming, gas-producing, bacteria in the milk; also, the storage of silage on the same farm is prohibited. The use of additives, other than rennet and NaC1, for cheesemaking is prohibited. A mixture of milk from two consecutive milkings is used; evening milk is partially skimmed after overnight creaming at c. 20 ~ in special tanks, 'bacinelle' (capacity, 10-50 hi), which contain a shallow body of milk. A slight microbial acidification occurs during creaming. After that, the partially skimmed milk is mixed in a ratio 1:1 with the whole milk from the following morning's milking. The fat content of the milk for Parmigiano Reggiano is c. 2.4-2.5%. The natural whey cultures used as starters for Parmigiano Reggiano and Grana Padano are prepared from the whey from the previous cheesemaking, which is held in a temperature gradient (from c. 50 to c. 35 ~ for 24 h. The microbial composition of the natural starter is very complex, subject to environmental factors and dominated by thermophilic lactic acid bacteria (c. 109 cfu/ml) such as Lactobacillus helveticus, Lb. delbrueckii subsp, lactis, Lb. delbrueckii subsp, bulgaricus and Lb. fermentum. The ratio of obligately homofermentative to heterofermentative species is c. 10:1 or higher. A large amount of the natural whey culture, c. 3% (v/v), is added to the milk. The calf rennet used for Parmigiano Reggiano contains less than 3-4% pepsin, based on clotting activity. The curd cooking temperature ranges between 53 and 55 ~ and the time from rennet addition at 32-34 ~ to the end of cooking is 22-23 min. The vats used for the manufacture of Parmigiano Reggiano and Grana Padano cheeses have a capacity of 10-12 hl and, traditionally, have the shape of an inverted bell. From each vat, two cheeses, each weighing 35-37 kg after ripening, are produced. Parmigiano Reggiano is ripened for 20-24 months at c. 18 ~ and an environmental humidity of c. 85%. Parmigiano Reggiano and Grana Padano have a cylindrical shape with a diameter of 33-45 cm and a height of 18-25 cm. The cheeses have a very low moisture content (c. 30%), a typical compact texture, with or without many very small eyes, and melt in the mouth with a sweet flavour, which is the result of very slow ripening, during which proteolysis is the main biochemical event (Bottazzi, 1962; Consorzio del Formaggio Parmigiano Reggiano, 1989; Gobbetti and Di Cagno, 2002). Grana Padano Grana Padano cheese is manufactured in several provinces of the Pianura Padana. Several major features distinguish it from Parmigiano Reggiano. For
55
Grana Padano, the feeding of high-quality silage fodder is allowed, and the cheese is produced from two consecutive milkings which are stored at 8 ~ on the farm. The milk is skimmed by creaming in 'bacinelle' or very large tanks (300-500 hi) for c. 12 h at 12-15 ~ The total microbial count of the milk after holding in the 'bacinelle' is low, c. 103-104 cfu/ml compared to c. 106 cfu/ml for milk for Parmigiano Reggiano, also due to the lower temperature of creaming (Bottazzi, 1979). The fat content of the milk for Grana Padano is c. 2.1-2.2% and ripening lasts 14-16 months (Bottazzi, 1962; Consorzio per la Tutela del Formaggio Grana Padano, 1990; Gobbetti and Di Cagno, 2002). Asiago Several types of Asiago cheese are manufactured which differ mainly in the duration of ripening. Asiago d'Allevo is a hard or extra-hard cheese variety, ripened for c. 12 months, and typically produced in the Veneto region. Previously, the cheese was manufactured from ewes' milk, but only cows' milk is used now. Raw milk from one or two consecutive milkings is partly skimmed by a creaming protocol similar to that described for Parmigiano Reggiano cheese. The natural whey culture used as starter is dominated by thermophilic lactic acid bacteria. The cooking of the curds is generally for 20-30 min and is divided into two steps. After cutting, the curds are heated to 40-42 ~ and held for 5-7 min; then, the temperature is increased to 46 ~ and held for 15-25 min. After moulding, the curds are pressed for c. 12 h. Generally, the cheeses are ripened for 1 year, exceptionally for 2 years. The cheeses are cylindrical in shape, 9-12 cm high and 30-35 cm in diameter and weigh 8-12 kg. The texture is rather compact and the flavour is slightly sweet (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002; Innocente et al., 2000). Montasio The cheese derives its name from the homonymous place located in the Julian Alps. Currently, its manufacture has been extended to the Friuli region and to several provinces of the Veneto region. A mixture of cows' milk from two consecutive milkings is used; the milk from the evening milking is partially skimmed after overnight creaming. The natural whey culture used as starter is added to the milk at 31-35 ~ and coagulation by calf rennet takes place in 30-40 min. After cutting to the dimensions of rice grains, the curds are cooked at 48-50 ~ for several minutes, pressed for 24 h and dry salted or immersed in saturated brine. Ripening of extra-hard Montasio cheese lasts 12 months at c. 18 ~ and an environmental
56
Extra-Hard Varieties
humidity of c. 80%. Cheeses have a cylindrical shape with a diameter of 30-40 cm, height of 8-10 cm and weigh 5-9 kg. The mature cheese has a brown rind, a granular texture with very small eyes and a pronounced and moderately piquant flavour (Battistotti et al., 1983; Ottogalli, 2001). Pecorino Romano
Pecorino Romano cheese is manufactured in the regions around Rome and in Sardinia. It is the best-known extrahard ewes' milk cheese. Pecorino Romano is usually made from raw or thermised milk which is inoculated with a natural culture, 'scotta fermento', which is produced by acidifying the 'scotta', the whey obtained from the manufacture of Ricotta. Thermophilic lactic acid bacteria, such as Streptococcus thermophilus, Lb. delbrueckii subsp, lactis and Lb. helveticus, dominate the microflora of this natural starter. The milk is coagulated at 37-39 ~ using lamb rennet paste and after cutting, the curds are cooked at 45-46 ~ After removal from the vat, the curds are placed in moulds, pressed manually and pierced with the fingers or a stick to increase whey drainage. The cheese is ripened for 8-12 months at 10-14 ~ to develop the characteristic flavour. The cheese is cylindrical in shape, 25-32 cm high and 25-30 cm in diameter and weighs 22-32 kg. The sensory characteristics of Pecorino Romano cheese depend mainly on lipolysis by enzymes (pre-gastric esterase) in the lamb rennet paste, and flavour intensity is relater-] tn t h e
cnntpnt
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octanoic acids. Proteolysis may show wide variations but the soluble nitrogen is always less than 30% of the total nitrogen (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Pecorino Siciliano
This variety of Pecorino cheese is manufactured only in Sicily, between October and June, when whole ewes' milk is available. Only natural whey cultures, containing mainly thermophilic lactic acid bacteria, are used as starter. Lamb paste rennet is used for coagulation, which occurs within 40-60 min. The coagulum is broken into pieces the dimensions of a pea using a traditional wooden tool, known as a 'rotella'. The curds are partially cooked at 40 ~ for c. 10 min by adding hot water (c. 70-80 ~ and moulded in a circular vessel, traditionally called 'canestro', where the curds are pressed slightly. The cheeses are ripened for at least 6-8 months to develop the moderate piquant flavour. The cheese has a cylindrical shape, 12-18 cm high and 35 cm in diameter, and weighs 4-15 kg. Pepato (peppery) is a variant of Pecorino Siciliano cheese which differs by the addition of black pepper to the curds during moulding (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002).
Pecorino Sardo
This is a variety of Pecorino cheese, the manufacture of which is limited to several provinces of Sardinia. Raw or thermised whole ewes' milk, natural whey or milk cultures and calf rennet paste are used in cheesemaking, which does not differ substantially from that of Pecorino Siciliano cheese. The ripening of this hard variety may last for 12 months. The shape of the cheeses is cylindrical, 10-13 cm high, 15-20 cm in diameter and weigh 1.7-4 kg. The straw-yellow rind is smooth and springy initially, but later it becomes darker and harder. The mature cheese has a pleasant pungent flavour and a firm, hard, fairly granular texture (Battistotti etal., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Fiore Sardo
The production of Fiore Sardo cheese is strictly limited to some provinces of Sardinia. Traditionally, it was produced by the shepherds in their cottages. Raw whole ewes' milk from a single milking is used. A large part of the milk is produced by an indigenous breed of sheep. Starters are not deliberately added and lamb rennet paste is used to coagulate the milk. The curds are not cooked and are pressed slightly during moulding. Treatment of the curds with hot water is necessary to make the rind thick and resistant. The cheeses are ripened for c. 6 months or more, and during the early phase of ripening, they may be smoked slightly by ex-nnqina them
tn ~ mnl c e f r n r n t h e w n n c ] n f M e c ] i t e r -
ranean scrub trees. During ripening, the cheeses are often rubbed with a mixture of olive oil and sheep fat. The cheeses have a cylindrical or wheel shape with curved sides, are 13-15 cm high and weigh 1.5-5 kg. The flavour of Fiore Sardo is pronounced, aromatic, moderately spicy and the rind varies from deep gold to dark brown with a sour smell (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Canestrato Pugliese
Canestrato Pugliese is manufactured only in the Apulia region. The cheese derived its name and traditional shape from the rush basket, 'canestro', in which the curds are ripened. Raw, whole ewes' milk of one or two daily milkings is generally used, but thermised or pasteurised milk may be processed also. A natural whey culture, composed mainly of thermophilic lactic acid bacteria, may be added, and liquid or powdered calf rennet, or, exceptionally, lamb paste rennet, is used. After cutting, the curds-whey mixture is heated to 45 ~ and held for 5-10 min. This treatment is generally not considered as 'cooking'. The cheeses are drysalted for c. 2 days and, during ripening (4-12 months) in the 'canestro', are turned regularly and rubbed with a mixture of oil and vinegar. Ripening in the 'canestro'
Extra-Hard Varieties
is limited to a few days for industrial production. Colonisation of the surface by moulds from the environment frequently becomes evident during ripening, which are removed by brushing after few months. The cheeses have a cylindrical shape, 10-14 cm high, 25-34 cm in diameter and weigh 7-14 kg. The rind is brown to pale yellow, and the interior is compact with small eyes. The flavour is pronounced and tends to be moderately piquant (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Castelmagno This cheese has been manufactured in the Grana valley, near Cuneo (Piedmont), since the twelfth century. Raw cows' milk is partly skimmed according to a protocol similar to that described for Parmigiano Reggiano cheese. Rarely, a mixture of cows', ewes' and goats' milks is used. The traditional technology does not involve the use of a natural starter, and acidification is due to the indigenous lactic acid bacteria. Liquid or powdered calf rennet, which may be combined with a small amount of lamb rennet paste, is used for coagulation. After cutting the coagulum and removal of most of the whey, the curds are traditionally harvested in cloth bags which are hung for 10-12 h at room temperature, allowing the removal of further whey The cheese is ripened in natural caves at 10-12 ~ and 85-90% relative humidity for more than 6 months. The cheeses have a cylindrical shape, are c. 20 cm high, 20-25 cm in diameter and weigh 4-6 kg. Penicillium spp. from the environment colonise the cheese surface, and occasionally the interior of the cheese. Castelmagno cheese may be considered as a hard Blue cheese variety with a compact but friable texture and a moderately piquant flavour (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Fossa The tradition of Fossa (pit) cheese originated in the Emilia-Romagna region (north of Italy) in the Middle Ages. The typical feature of this cheese involves ripening in flask-shaped pits which are dug in the tufa ground of this region. Cheesemaking is typically from raw ewes' milk but in some cases, mixed ovine-bovine milk is used. Traditionally, the cheese is produced only during a defined period of the year. Natural thermophilic starters in milk, comprised of indigenous lactic acid bacteria, are added to the cheese milk. The curds are not cooked and after moulding are held at c. 28 ~ for 4-8 h. Curds are generally ready for ripening in pits after a period (up to about 3 months) of maturation in rooms, which is necessary to achieve a certain degree of consistency and to eliminate the risk of whey losses when the cheeses are pressed into the pits.
57
Before they are placed and pressed in the pits, the cheeses are wrapped individually in cloths. The sides of the flask-shaped pits are covered with straw which is fixed by canes, horizontally linked by wooden rings. The pits are open during August and when completely filled with cheeses, they are hermetically sealed. The humidity inside the pits is close to saturation and the temperature ranges from 17 to 25 ~ Traditionally, the pits are opened on 28th November; at this time, the cheeses have been ripened for at least 6 months, including maturation in rooms. Due to the pressure inside the pits, the shape of the cheeses varies from cylindrical to very irregular and the weight ranges from 1.0 to 1.5 kg. The flavour is generally full, sharp, balanced and moderately piquant (Gobbetti and Di Cagno, 2002). Extra-hard Swiss cheeses
Most of the Swiss cheeses classified as hard or extrahard varieties are discussed in 'Cheese With Propionic Acid Fermentation', Volume 2, which deals with cheeses with the propionic acid fermentation. A few others are described below. Sbrinz The cheese derived its name from the locality of Brienz in Switzerland but now its manufacture has been extended to France, Germany and Italy. Raw, whole cows' milk of one day's milking is used. The milk is warmed to 34-38 ~ and a natural whey culture, containing mainly thermophilic lactic acid bacteria, is added. Liquid or powdered calf rennet is used to give coagulation in 15-20 min. During heating and mixing at 45-48 ~ the coagulum is cut to the dimensions of wheat grains. Cooking is at 54-56 ~ for a few minutes. After harvesting, the curds are pressed for 24 h, salted in brine for 18-22 days and ripened for 6-12 months at c. 18 ~ and an environmental humidity of c. 80%. The cheeses have a cylindrical shape, are 1 0 - 1 4 c m high, 4 0 - 5 0 c m in diameter and weigh 20-25 kg. The rind is yellow to brown at the end of ripening, and the interior is compact with a Grana-like texture. The moisture content is less than 35% and the flavour is pronounced (Battistotti et al., 1983; Fessler et al. , 1999). Saanenkiise This cheese is made from cows' milk of two consecutive milkings which is coagulated at 32 ~ by addition of calf rennet. After cutting, the curds are cooked at 50-52 ~ and pressed. Ripening lasts from 2 to 5 years and the moisture content is c. 25%. The cheeses have a cylindrical shape, are 10-14 cm high, 40-60 cm in diameter and weigh 20-40 kg. The interior and taste
58
Extra-Hard Varieties
are similar to those of Parmigiano Reggiano and Sbrinz cheeses (Battistotti et al., 1983).
compact with small eyes. The extra-hard variety has a moisture content of c. 35% and its flavour is pronounced (Marcos and Esteban, 1993).
Spanish extra-hard cheeses Roncal
All the Spanish cheeses listed below have a PDO status as established by national and European rules. Mahdn The cheese takes its name from the capital of Minorca (Balearic Islands), where it is produced. Raw or pasteurised cows' milk, containing 5% of indigenous ewes' milk, is used for cheesemaking. Natural whey cultures may be used as starters; the milk is coagulated at 30 ~ and, after cutting, the curds are pressed and salted in brine. Several variants are produced, including an extrahard cheese which is ripened for at least 10 months. The cheeses have a parallelepipedal shape, weigh 2-4 kg, the rind is white to yellow, oily due to treatment with olive oil, and the interior is compact with small eyes. The extra-hard variant has a moisture content less than 32% and its flavour is pronounced (Alcal~i et al., 1982; Esteban et al., 1982; Marcos et al., 1983). Manchego Manchego takes its name from the La Mancha region where the cheese was traditionally made from raw sheep's milk by shepherds. Because of increasing market popularity, its manufacture has spread throughout Spain. u s e s ewes' ,-,-,i11. bllli....lt~.,...~....IlLOthlll8 at a n lected over two consecutive days from herds in a demarcated area. The milk is pasteurised and a mesophilic starter culture (Lactococcus lactis subsp, lactis and Lc. lactis subsp, cremoris, mainly) and calf rennet or microbial rennet from Rhizornucor miehei are added. After c. 35 min at 30 ~ the coagulum is cut into pea-sized grains. The curd particles are heated to 37 ~ for 20 min and then stirred for another 30 min. After removal of the whey, the grains are transferred to a curd strainer and the beds of drained curd are cut into cube-shaped blocks, each of which is placed in a cylindrical PVC hoop, lined with a smooth cloth, in which the curds are moulded and pressed pneumatically at 0.3 MPa for 5 h. The cloths are removed and the curd pressed again at the same pressure for 17 h, after which it is immersed in a circulating brine bath at 14 ~ for 36 h. The blocks of curd are then placed in a drying room at c. 14 ~ and 85% environmental humidity where they are stored, with periodic turning, for 10 days, after which they are transferred to a curing chamber at c. 9 ~ and 95% environmental humidity. After 12 months, the cured cheeses are brushed and, in some cases, coated with a polyvinyl acetate emulsion containing an anti-fungal agent. The cheeses have a cylindrical shape, c. 20 cm in diameter and weigh 2.5-3 kg. The rind is green to black and the interior is ILLI,..Li..tLOLLL~ILL
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This cheese takes the name from the Navarrese valley where it is produced from December to July. It is manufactured from ewes' milk and the main technological traits are similar to those for Manchego cheese, except for the smaller dimensions of the ripened cheeses. A typical microflora composed of deliberately added mesophilic starter lactococci and adventitious lactobacilli persists during ripening (Ord6emz et al., 1980; Marcos and Esteban, 1993). Idiazabal This is another extra-hard variety made from whole ewes' milk in mountain caves of the Basque country. Raw milk from the 'latxa' breed of sheep is coagulated at 25-35 ~ in 30-45 min by addition of lamb rennet. The coagulum is cut to rice-sized grains, heated to and maintained at 40-45 ~ for several minutes, before being placed in moulds where the curds may be seasoned before pressing. Salting is performed by rubbing the rind with dry salt or by immersion of the cheese in brine for 24 h. The cheese is ripened for several months until a moisture content of c. 33% is reached. The cheeses have a cylindrical shape and weigh 1-2 kg. The rind of artisanal cheeses is engraved with drawings or symbols characteristic of the Basque culture. An optional smoking may be performed at the end of ripening by using smoke of wood from beech, birch, cherry or white pine trees. The taste is strong and pronounced, slightly acidic and piquant with a characteristic sheep milk flavour (Marcos and Esteban, 1993; Arizcun et al., 1997a,b). Other extra-hard cheeses
Russian cheeses SovetskiT, AltaiskiT and BriskiT are hard or extra-hard varieties made from cows' milk and are similar to Swiss-type extra-hard cheeses. The use of a mixed starter culture composed of 5c. thermophilus and Lb. helveticus, cooking of the curds at 50-55 ~ pressing for 4-8 h and ripening for at least 6 months are the main technological traits. Generally, cheeses have a moisture content of 32-36%, weigh 10-18 kg and have a rectangular shape (Gudkov, 1993). Balkan cheeses Kefalotyri is an extra-hard, salty Greek cheese, made exclusively from ewes' or goats' milk with the use of thermophilic and propionic starters. After coagulation by calf rennet at 35 ~ the coagulum is broken to dimensions of c. 6 mm and pressed for 5-10 h. Salting
Extra-Hard Varieties
is performed by rubbing the rind with dry sah and the cheeses are ripened for more than 3 months. The cheeses have a flat cylindrical shape, are c. 30 cm in diameter and weigh 5-10 kg. The flavour is strong, piquant and salty (Peji4, 1956; Scott, 1981). Manura is a Greek traditional farmhouse hard cheese variety manufactured on Sifnos island in the Aegean sea from raw ewes' milk or from a mixture of raw ewes' and goats' milks of local herds. Typically, after 3--4 months of ripening in straw beds, the cheeses are held for several days in red wine to soften them and then for a few days in wine residues. Cheeses are small and weigh c. 600 g. Par cheese means cheese from Pag, which is the name of the Adriatic island (Greece) where it is produced. It is a very-hard cheese made from ewes' milk which is ripened for at least 6 months. The cheeses have a high DM content, a firm compact texture, with no holes, and the flavour tends to be strong and piquant (PejiC 1956). Other extra-hard cheeses
Very-hard and hard varieties are produced in several non-European countries. Most of them are manufactured from ewes' and/or goats' milk, a starter culture is not always used, pressing of the curds is a very common feature and the cheeses are ripened for at least 6 months. Typical examples are Djamid from Jordan (Phelan et al., 1993), Ras from Egypt (Hofi et al., 1970) and Paphitico and Graviera from Cyprus (Phelan et al., 1993).
Although most of the extra-hard varieties considered above have high market popularity and are of great economic relevance, only a few of them have been characterised extensively. In addition, since the same cheese may be produced in a number of hard or extra-hard variants which differ with respect to the type of milk, season of milking, technology and ripening, the results available on cheese characterisation may differ markedly. Owing to the large size and the prolonged brine and/or dry salting, most extra-hard cheeses are commonly characterised by a decreasing NaC1 gradient from the surface to the centre and by an opposite moisture gradient, which is reflected in the water activity (aw) values. These gradients persist for a very considerable period after salting, and consequently, ripening in these cheeses shows variations which depend on the cheese zone. Changes in microflora during ripening
The lack of fermentable carbohydrates, low pH, aw (mainly due to NaC1) and temperature, and the presence of bacteriocins make the environmental conditions in extra-hard cheeses very hostile during ripening.
59
Generally, this favours a sharp decline of the number of thermophilic starter bacteria which are gradually replaced by mesophilic bacteria. For the extra-hard varieties, mesophilic bacteria are derived mainly from the raw milk used but environmental contamination is not excluded, as well as survival of bacteria following sub-pasteurisation or thermisation for those cheeses for which heat-treated milk is used. The composition of this population may vary but non-starter lactic acid bacteria (NSLAB) constitute the major part. Pediococcus spp., Lb. casei, Lb. casei subsp, pseudoplantarurn and Lb. rharnnosus are the predominant bacteria in Parmigiano Reggiano and Grana Padano cheeses (Bottazzi, 1979, 1993; Gobbetti and Di Cagno, 2002). Pediococci seem to be fundamental for maintaining the equilibrium within the cheese-related microbial community, probably also interfering negatively with the growth of clostridia, while Lb. casei, as the major part of the NSLAB, is very important for its peptidase activity (Gobbetti et al., 1999a,b). In Parimigiano Reggiano cheese, NSLAB decrease from c. 108 cfu/g at 5 months to 104 cfu/g after 24 months of ripening (Coppola et al., 1997). Lb. plantarum, Lb. casei and Enterococcus faecium prevail in Manchego cheese after 1 month of ripening (Nt~fiez et al., 1989). Lb. plantarurn and Lb. curvatus were the species isolated most frequently from Fossa cheese, with fewer numbers of Lb. paracasei subsp, paracasei (Gobbetti et al., 1999c). A more heterogeneous microflora, consisting of Lb. plantarum, Lb. pentosus, Lb. curvatus, Lb. brevis, Lb. paracasei subsp, paracasei and Leuconostoc spp., was found in Canestrato Pugliese cheese (Albenzio et al., 2001; Corbo et al., 2001). Lb. curvatus and Lb. paracasei subsp, paracasei were also found in Fiore Sardo at the end of ripening (Mannu et al., 2000). Together with components of the NSLAB microflora such as Lb. curvatus, Lb. plantarum and Lb. fermentum, and with a heterogeneous population of enterococci, the thermophilic Lb. dekbrueckii subsp, lactis was found occasionally in ripened Pecorino Romano (Di Cagno et al., 2003). Lb. casei, Lb. plantarum and Ln. mesenteroides subsp, mesenteorides and Ln. mesenteroides subsp, dextranicum were the bacteria found in aged Roncal and Idiazabal cheeses (Arizcun et al., 1997a). In most extrahard and hard cheeses, NSLAB reach c. 107-108 cfu/g after few months, which is generally maintained for a long time during ripening (McSweeney et al., 1993; Gobbetti et al., 1999c; Mannu et al., 2000; Albenzio et al., 2001). All these differences and changes in the microbial population are considered relevant factors which affect the cheese during subsequent ripening, especially regarding the extensive secondary proteolysis, which leads to an elevated concentration of small peptides
60
Extra-Hard Varieties
and amino acids, which is undoubtedly related to the peptidase activity of mesophilic bacteria.
-6.5
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Lactose metabolism
f
The lactic acid fermentation has been studied extensively in Parmigiano Reggiano cheese during the first 48 h after manufacture (Mora et al., 1984) (Figs 1 and 2). There are no comparable data for other extra-hard cheeses but the fermentation is generally similar in those varieties which are cooked to a high temperature and have a rather large size. The growth of the starter thermophilic lactic acid bacteria and the hydrolysis of lactose depend mainly on the rate at which the curds cool after removal from the cheese vat. Depending on the weight of the cheese, the temperature at the centre of the curd remains relatively high, e.g., >50 ~ for 12-16 h for Parmigiano Reggiano, while the exterior of the cheese cools rather suddenly (c. 2 h) to c. 42 ~ Consequently, bacterial growth starts earlier and is more intense in the external zone. While the residual lactose is consumed throughout the cheese within 8-10 h, bacterial numbers, pH and lactic acid concentration do not attain equal values in the centre and exterior of the cheese for a longer time. The concentration of lactic acid also may vary during ripening. For Manchego cheese, the concentration of lactic acid was c. 1.2 and 1.0% in the exterior and interior of the cheese during 2 weeks, then decreased to 1.0 and 0.8% ~tlttEt
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11
12
Time (hours) Changes in the concentrations of lactose (0), glucose (aa) and galactose (A)in the external (- -) and internal (--) zones of Parmigiano Reggiano cheese (Mora et al., 1984).
g g
~11 ...41
~ f"'t-Ji
....
r
10-
T -5.5 O_
/ /
0 0 -..~
o
-6.0
5-
_J
-5.0 r 2r , 4, r 6r , ~
r 10 , , 12 , . . . . . 2 r4
418
Time (hours) Changes in pH (O) and lactic acid concentration (aa) in the external (- -) and internal (--) parts of Parmigiano Reggiano cheese (Mora et aL, 1984).
Lipolysis
The ripening of most cheeses is accompanied by a low level of lipolysis but extensive lipolysis occurs in several extra-hard varieties. The length of ripening strongly influences lipolysis and since ripening varies markedly within the same variety, cheeses ready for the market may differ greatly. Lipolysis may be due to the action of the indigenous lipase in cheese made from raw milk, to the action of microbial lipases, PvPn
thn+lah
thP
lactic
~,'-irt h a c t P r i c i
in
~t~rt~,r ,"111_
tures have only weak lipolytic activity, or to the action of the lipases present in rennet paste used in cheesemaking for certain varieties. Several extra-hard Italian cheeses are probably unique in that rennet paste is used commonly. The desirable flavour which characterises the Pecorino cheeses (Romano, Siciliano and Sardo) and Fiore Sardo is due mainly to the action of pre-gastric esterase (PGE) in rennet paste, which is used as the source of both coagulant and lipolytic agents in cheese manufacture. Rennet pastes are prepared by grinding the engorged stomachs, including curdled milk, of young calves, kid goats or lambs which are slaughtered immediately after suckling or pail-feeding. The stomachs and contents are generally held for c. 60 days prior to grinding. Pregastric esterase, the physiological role of which is to aid in the digestion of fat by the young animals which have limited pancreatic lipase activity, is secreted during suckling and is carried into the stomach with ingested milk. The strong, balanced piquant flavour which characterises Pecorino cheeses and Fiore Sardo is due primarily to the relatively high levels of short-chain free fatty acids (FFAs), especially butanoic, hexanoic and octanoic acids. Although there are some inter-species differences, lamb,
Extra-HardVarieties
calf and kid PGEs preferentially hydrolyse fatty acids esterified at the sn-3 position of glycerol (Woo and Lindsay, 1984), which explains the relatively high rate of release of butanoic acid from milk fat, in which 90% of the butanoic acid is attached at the sn-3 position. Calf PGE does not hydrolyse monobutyrin and hydrolyses dibutyrin very slowly compared to tributyrin (Richardson and Nelson, 1967). The moderate accumulation of short-chain FFAs characterises the ripening of Parmigiano Reggiano, Canestrato Pugliese and Fossa cheeses, for which rennet paste is not used (Woo and Lindsay, 1984; Carboni et al., 1988; Gobbetti et al., 1999c; Albenzio et al., 2001). Table 4 shows the FFA profile of some extra-hard cheeses. The average values which are reported refer to ripened cheeses, with a high popularity on the market, but in general there is no standard flavour for such extra-hard Italian cheeses which is acceptable to all segments of the population. For Pecorino Romano cheese, there is a direct relationship between the flavour intensity and the concentration of butanoic acid (Richardson and Nelson, 1967) but the relationship between flavour desirability and butanoic acid concentration is more variable. Flavour desirability is influenced mainly by the relative proportions of the various FFAs. A strong, balanced, piquant Pecorino Romano cheese may be characterised by c. 10 500mg/kg total FFAs, principally butanoic (Car:0), together with hexanoic (C6:0), tetradecanoic (C14:0), hexadecanoic (C16:0) and octadecenoic (C18:1) acids (Table 4). It was shown that among these compounds, butanoic and hexanoic acids are the most important components of the aroma quality of Pecorino Romano cheese. The total FFA content of Parmigiano Reggiano approaches 20% of that generally found in Pecorino
61
cheeses, with variations in the proportions of FFAs. Congeners of C18 fatty acids dominate the FFA profile at the end of ripening (Carboni et al., 1988). The crude vacuum distillate of Grana Padano cheese contains large amounts of butanoic and hexanoic acids, which represent 50 and 35% of the total FFAs, respectively. These two FFAs may be important for the background aroma of Grana Padano cheese. A small change in the relative proportions of butanoic and hexanoic acids was found between 12 and 24 months of ripening (Moio and Addeo, 1998). Canestrato Pugliese and Fossa cheeses show very similar FFA profiles, although the former has a higher total concentration of FFAs (Gobbetti et al., 1999c; Albenzio et al., 2001). Butanoic acid, which occurs at the highest concentration, hexanoic, decanoic (C10:0), hexadecanoic and congeners of C18 acids dominate. Probably due to the lipolytic activity of moulds which colonise the cheese surface during the early period of ripening, Canestrato Pugliese also shows a rather high proportion of octadecenoic (C18:1) and octadecadienoic (C18:2) acids. A qualitative and semi-quantitative comparison of the FFA profiles of other extra-hard varieties produced from ewes' milk showed that butanoic, hexanoic, octanoic (C8:0) and decanoic acids were the dominant FFAs in Roncal, Pecorino Sardo and Fiore Sardo; levels were highest in the last cheese (Larr~iyoz et al., 2001; Di Cagno et al., 2003). Of 14 samples of Manchego cheese analysed, all contained high levels of short-chain FFAs, butanoic acid being the most abundant (Villasel~or et al., 2000). Extra-hard cheeses produced without the use of rennet paste may vary greatly in the concentration of FFAs depending on whether raw or pasteurised milk is used. Several studies have shown a higher level of
Concentration of individual and total free fatty acids (mg/kg cheese) in Parmigiano Reggiano, Pecorino Romano, Canestrato Pugliese and Fossa cheeses
Fatty acid
Parmigiano Reggiano
Pecorino Romano
Canestrato Pugliese
Fossa
Butanoic (C4:0) Hexanoic (C6:0) Octanoic (C8:0) Decanoic (C10:0) Dodecanoic (C12:0) Tetradecanoic (C14:0) Hexadecanoic (C16:0) C 18 congeners Total free fatty acids
172 48 44 107 107 225 565 1033 2301
3 043 1 428 429 1 009 690 778 1 306 1 843 10 526
425 178 42 98 46 85 172 322 1368
247 123 55 84 35 62 137 251 994
C18 congeners refer to octadecanoic (C18:0), octadecenoic (C18:1), octadecdienoic (C18:2) and octadectrienoic (C18:3) acids. The values indicated represent the average of several determinations made by different authors on cheeses which had a slightly different ripening time: Parmigiano Reggiano, 16-18 months; Pecorino Romano, 10-12 months; Canestrato Pugliese, 6-10 months; Fossa, 6-8 months. Source: Woo and Lindsay (1984); Carboni et aL (1988); Gobbetti et aL (1999c); Albenzio et aL (2001).
62
Extra-Hard Varieties
FFAs in cheese made from raw milk than in that made from pasteurised or thermised milk. Such differences are attributed mainly to heat-induced changes to the indigenous lipoprotein lipase of milk and to the lipase and esterase activities of the milk microflora, especially NSLAB, and become greater as the time of ripening increases. Studies on NSLAB (Gobbetti et al., 1996, 1997) showed that Lb. plantarum contains lipase and esterase which show a substrate specificity comparable to PGE and pancreatic lipase and since there is a very large population of NSLAB in cheese during ripening, their contribution to lipolysis has been suggested. Proteolysis
Proteolysis in extra-hard varieties does not differ substantially from that in other hard/semi-hard internal bacterially ripened cheeses. The low moisture and high salt content, which cause the persistence of gradients of moisture and NaC1 in the cheese, and the absence of a fungal microflora, which is evident only on the surface of Canestrato Pugliese and Castelmagno, are all factors which influence proteolysis during ripening. The principal proteolytic agents in the curd are the coagulant, depending on the intensity of the cooking treatment, starter and NSLAB proteinases and peptidases and indigenous milk proteinases (particularly plasmin) (Fox et al., 1996). Proteolysis in Parmigiano Reggiano and Grana
during the first month of ripening. The latter, together with the very low levels of 6-CN f1-192 and 6-CN f1-189, the primary products of [3-CN hydrolysis by chymosin, indicates considerable plasmin activity. Hydrolysis of [3-CN by chymosin during ripening is inhibited by 5% NaC1 and, in general, during curd cooking most of the chymosin activity is destroyed. The same pattern for [3-CN hydrolysis was found in Pecorino Romano cheese. Overall, [3-CN is rapidly and almost totally hydrolysed during the ripening of Parmigiano Reggiano, Grana Padano and Pecorino Romano cheeses, while Otsl-CN undergoes relatively less proteolysis (Fig. 3). One-year-old cheeses generally do not contain [3-CN, while at the end of ripening Parmigiano Reggiano cheese still contains unhydrolysed Otsl-CN. These findings confirm that chymosin, which is the primary proteolytic agent acting on Otsl-CN, is not very active in these cheeses. Addeo et al. (1988) proposed the ratio y-CNs/[3-CN as an index of proteolysis in Parmigiano Reggiano during ripening. During the first year, the y-CNs represent c. 20% of the oligopeptides, yl-CN being c. 30% of the total y-CNs. After this period, the percentage of yl-CN decreased, while that of Y2- and y3-CNs increased due to hydrolysis of the former by plasmin. SDS-PAGE and a specific anti-[3-CN monoclonal antibody identified ~/1- and "Y2-CNs in Grana Padano cheese during ripening which showed a correlation with the extent of
Paclano . . . . cheeqeq . . . . haq . . h e. e n. q. t l l.c l i.p d. 11qing . . . rnnnv,, ctiff~,r~,nt
rinPnino
analytical methods. Polyacrylamide gel electrophoresis and isolectric focusing in a polyacrylamide gel (Addeo et al., 1988) showed the rapid hydrolysis of C~sl-casein (CN) to the primary degradation product, OLsl-CN (f24-199) and the formation of y-CNs from [3-CN
same authors found that the area of cheesemaking, season of production, length of ripening and type of dairy are all factors which may influence proteolysis. The urea-PAGE profiles of pH 4.6-insoluble fraction of ewes' milk Fossa cheeses vary (Fig. 4). Nevertheless,
(l~.ni~r
~,t al
9DDI)
Nl~,,zorthol~,r162 t h o
Urea-polyacrylamide gel electrophoretograms of pH 4.6-insoluble nitrogen fraction of Parmigiano Reggiano cheeses at different times of ripening. C, whole casein (Addeo et al., 1988). PL, refers to Otsl-casein fragments with different electrophoretic mobility.
Extra-Hard
Urea-polyacrylamide gel electrophoretograms of pH 4.6-insoluble nitrogen fraction of Fossa cheeses. Lanes: Sb, bovine Na-caseinate; So, ovine Na-caseinate standard; 1-7 Fossa cheeses (Gobbetti et aL, 1999c).
the profiles are commonly characterised by the complete degradation of Otsl-CN after 6 months of ripening, while much of the [3-CN persists unhydrolysed (Gobbetti et al., 1999c). Fossa cheese is produced without cooking the curd and chymosin may remain active during ripening. The same was found for Manchego (Ordd~ez et al., 1978) and Canestrato Pugliese (Albenzio et al., 2001; Corbo et al., 2001) cheeses. In the last case, since the cheese may be produced from raw, thermised or pasteurised ewes' milk, RP-FPLC analysis of the water-soluble fraction showed a more complex peptide pattern in raw milk cheese which was positively linked to more intense proteolysis. The hydrolysis of the caseins leads to an increased proportion of water-soluble N which has been used as a ripening index for Parmigiano Reggiano (Panari et al., 1988). Fig. 5 shows the changes in the percentage ratio, soluble N/total N for Parmigiano Reggiano during ripening. The increase is very fast during the first 8-10 months, after which hydrolysis proceeds very slowly. At the end of ripening, the water-soluble N is c. 34% of the total N. Similar values (c. 32%) are found in Grana Padano cheese (Addeo and Chianese, 1990; Toppino et al., 1990). Since the pH of many extra-hard cheeses is in the range 5.0-5.5, the values of water-soluble and pH 4.6-soluble N do not differ significantly. Values of pH 4.6-soluble N/total N ranging from 19 to 29% were found in Romano-type cheese which
Varieties
63
coincided approximately with those for the 12% TCAsoluble N (Guinee and Fox, 1984; Guinee, 1985). Since pH 4.6-soluble N is produced principally by rennet, while starter and non-starter bacterial enzymes are principally responsible for the formation of 12% TCAsoluble N, these data support the view that rennet is not very active in this cheese and that once it produces soluble peptides, bacterial peptidases hydrolyse them relatively rapidly. Contradictory results were reported for proteolysis in Pecorino Romano which varied with the zones of the cheese. At the beginning of ripening, some authors found greater proteolysis in the interior of the cheese, which from 40 days onward was more extensive in the surface zone due to the inward diffusion of NaC1. Other authors (Guinee and Fox, 1984; Guinee, 1985) did not find differences in the level of water- and pH 4.6-soluble N at various locations in the Romano-type cheese throughout ripening. The level of pH 4.6-soluble N is very high also in Fossa cheese, ranging from 30 to 39% of the total N. The water-soluble N may range from 13 to 30% of the total N in Canestrato Pugliese cheese, depending on several factors including NSLAB activity. The extrahard Spanish varieties may be divided into two groups: Mahon and Idiazabal, with a content of soluble N of c. 30%, and Manchego and Roncal with slightly lower values of c. 25% of total N (Ord6fiez et al., 1980; Marcos and Esteban, 1993). Variations in the concentration of free amino acids during ripening may be considered as another index by which some extra-hard varieties can be compared (Table 5). Free amino acids accumulate in
40-
30-
OI
z
z
20-
10-
0
t
0
6
t
12 Months
!
t
18
24
Increase (%) in the level of water-soluble nitrogen (SN)/total nitrogen (TN) in Parmigiano Reggiano cheese during ripening. Open circles are the average of several cheeses, of the same age, at the end of ripening (Panari et aL, 1988).
64
Extra-Hard Varieties
Concentration individual free amino acids (mg/g cheese) in Parmigiano Reggiano, Canestrato Pugliese and Fossa cheeses
Amino acids Histidine Arginine Serine Aspartic acid + asparagine Glutamic acid + glutamine Threonine Glycine Alanine Tyrosine Proline Methionine Valine Phenylalanine Isoleucine Leucine Cysteine Ornithine Lysine Tryptophan Total free amino acids
Parmigiano Reggiano 8.20 2.50 13.60
Canestrato Pugfiese 3.82 5.01 8.85
25o4 .,,_, o 2013_ .,,._, 0
Fossa
~_ 1 5 < < 10-
2.44 0.25 3.09
~_ 5 -
18.60
2.99
4.09
45.50 12.30 6.40 6.90 6.30 n.d. 7.20 18.40 13.20 15.90 22.20 n.d. 3.80 30.80 n.d. 231.80
15.34 3.23 2.55 2.87 1.66 8.65 3.25 8.33 5.88 6.54 10.99 1.57 n.d. 13.31 0.03 104.87
19.19 2.07 1.8 5.83 2.02 5.6 3.97 9.56 5.42 6.24 13.83 5.00 n.d. 13.09 n.d. 103.49
The values indicated represent the average of several determinations made by different authors in cheeses which had a slightly different ripening time: Parmigiano Reggiano, 16-18 months; Canestrato Pugliese, 6-10 months; Fossa, 6-8 months. n.d., not determined. Source: Resmini et aL (1988); Gobbetti et aL (1999c); Albenzio et al. (2001); Corbo et al. (2001).
Parmigiano Reggiano until i5 months of ripening, after which their concentration remains relatively constant (Fig. 6) (Resmini et al., 1988). At the end of ripening, the average concentration of total free amino acids is c. 230 mg/g protein, which corresponds to c. 23% of the total protein content; therefore, Parmigiano Reggiano is one of the richest cheese in free amino acids. The same trend, with similar values, was found for Grana Padano, showing that the extension of ripening to more than 18 months did not produce a significant increase in free amino acids (Resmini et al., 1990). Nevertheless, large variability was found for the amino acid profile of cheeses of the same age. This variability is reduced by expressing the amino acid content as a relative percentage. A chemometric model was proposed to estimate the age and the organoleptic quality of Parmigiano Reggiano based on the level of serine, glutamine, arginine and ornitine which were used as markers (Resmini et al., 1988).
080
(D
r2 = 0 . 9 2 2
DSt = 1.231 I
0'
0
4 6 8 ,'0 ,'2 ;4,6 Months
Changes in the total concentration of free amino acids in Parmigiano Reggiano cheese during ripening (Resmini et al., 1988).
Long-ripened Mahon cheese may be differentiated from young cheese by the content of glutamic acid, glycine, serine and threonine, while cheese produced from raw or pasteurised milk can be differentiated by the concentration of asparagine and glutamine (Frau et al., 1997). The total concentration of free amino acids in Fossa cheese varies greatly between samples (Gobbetti et al., 1999c), which is relatively high compared to Cheddar cheese (Lynch et al., 1996) and even to internally mould-ripened cheeses such as Gorgonzola (Gobbetti et al., 1998). Canestrato Pugilese cheese manufactured from raw ewes' milk also has a high level of free amino acids (Albenzio et al., 2001). Apart from the high concentrations of threonine, isoleucine and phenylalanine in Parmigiano Reggiano cheese, glutamic acid, proline, valine, leucine and lysine are the amino acids commonly present at high concentrations in Parmigiano Reggiano, Pecorino Romano, Canestrato Pugliese, Fossa, Mahon and Manchego cheeses (Ordol~ez et al., 1980; Resmini et al., 1988; Frau et al., 1997; Gobbetti et al., 1999c; Albenzio et al., 2001; Di Cagno et al., 2003). Volatile compounds
Cheese flavour is the result of several non-enzymatic and many enzymatic reactions. Decarboxylation, deamination, transamination, desulphuration and cleavage of side chains convert amino acid to aldehydes, alcohols and acids which together with other compounds, derived by other routes (e.g., lipolysis and catabolism of fatty acids), compose the volatile profile of extra-hard cheeses. Based on High Resolution Gas Chromatography (HRGC)-Mass Spectrometry (MS) and different methods of extraction, volatile compounds in some longripened cheeses have been characterised (Moio and Addeo, 1998; Izco and Torre, 2000; Villasel~or et al., 2000; Larrayoz et al., 2001; Di Cagno et al., 2003) (Table 6). Overall, large variations for the same cheese
Extra-Hard Varieties
65
Several volatile compounds (~g/kg) found in Grana Padano, Canestrato Pugliese, Fiore Sardo, Pecorino Romano and Manchego cheeses
Compounds
Grana Padano
Canestrato Pugliese
Fiore Sardo
Pecorino Romano
Manchego
Esters Methyl butanoate Ethyl butanoate Methyl hexanoate Ethyl hexanoate 3-Methylbutyl butanoate Methyl octanoate Ethyl octanoate Ethyl decanoate
2 223 4.5 737 8 5 229 24
n.d. n.d. n.d. 4.17 n.d. n.d. 3.53 3.47
n.d. n.d. n.d. 3.68 n.d. n.d. 3.30 3.28
n.d. n.d. n.d. 3.69 n.d. n.d. 3.29 3.12
41 289 37 115 21 4 64 n.d.
Ketones 2-Pentanone 3-Hydroxy-2-butanone 2-H exanone 2-H eptanone 8- N onen-2-on e 2-Nonanone 2-U ndecanon e 2-Tridecanone
143 70 4 320 20 172 38 3
3.64 7.49 1.65 4.95 0.54 4.31 3.45 3.68
3.58 8.88 2.31 5.67 1.93 4.46 3.54 3.87
3.69 6.02 1.55 4.63 0.0 4.17 3.61 3.79
737 n.d. n.d. 368 n.d. 44 n.d. n.d.
37 47 430 290 8.6 20 94 25 6 n.d. n.d. n.d.
7.88 3.83 2.55 3.09 2.67 4.80 3.58 3.08 2.92 2.34 2.55 1.82
2.17 3.36 2.27 2.49 2.08 2.19 2.74 1.87 1.57 1.62 2.24 2.03
4.12 2.24 2.34 3.07 2.24 2.87 2.37 2.03 2.23 1.81 2.38 1.98
167 307 n.d. 180 n.d. n.d. 25 n.d. n.d. n.d. n.d. n.d.
3-Methyl-butanal 3-Methyl-thiopropanal Benzaldehyde Nonanal Hexadecanal Octadecanal 2- Fu rancarboxaldehyde Benzaldehyde
8 3 7 n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. 3.43 3.43 3.15 2.17 1.65
n.d. n.d. n.d. 2.90 3.32 3.13 2.27 1.71
n.d. n.d. n.d. 2.83 3.62 3.10 2.03 1.85
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Lactones ,,/-Hexanolactone $-Octanolactone ,y-Decalactone 5-Decalactone ~/-Dodecalactone 5- Dodecanolactone "y-Dodecenolactone ~-Tetradecanolactone 5- Hexad eca n ol acton e
n.d. n.d. 2 5 n.d. n.d. n.d. n.d. n.d.
1.99 2.11 2.70 4.15 3.95 4.02 2.99 3.20 2.29
2.78 1.94 2.44 4.07 3.63 4.02 2.62 3.10 2.21
2.39 3.74 4.43 5.53 4.15 3.12 3.30 2.24
n .d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Alcohols 1-Butanol 2-Pentanol 3- M ethyl-3-buten- 1-ol 3-Methyl- 1 -butanol 3- Met hyl- 2- bute n- 1-ol 1-Hexanol 2-Heptanol 1-Octa no I 2-Nonanol 1-Decanol Furanmethanol Phenethyl alcohol
Aldehydes
continued
66
Extra-Hard Varieties
continued
Compounds
Grana Padano
Canestrato Pugliese
Fiore Sardo
Pecorino Romano
Manchego
Miscellaneous Phytene A Phytene B P hytad ie n e Phytanol Phytol 4-Methyl phenol 3-Methyl phenol Dimethyl disulphide Dimethyl trisulphide Methional Limonene
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
2.87 3.22 1.43 1.96 3.00 2.37 2.10 1.55 1.67 4.17 1.08
4.16 2.99 3.24 2.34 2.33 1.62 0.61 1.51 1.70 4.19 0.55
2.89 3.19 1.89 2.09 1.99 1.48 1.44 1.57 1.71 3.76 1.38
n.d n.d n.d n.d n.d n.d n.d 4 n.d. n.d. 11
The values indicated represent the average of several determinations made by different authors in cheeses of different manufacture. n.d., not determined. Source: Moio and Addeo (1998); VillaseSor et al. (2000) Di Cagno et al. (2003).
variety were found to be related to cheesemaking practices, season of manufacture, duration of ripening and type of secondary microflora. Esters were the main neutral constituents in the aqueous distillate of Grana Padano cheese, constituting c. 41% of the total neutral volatiles (Moio and Addeo, 1998). Esters with a few carbon atoms have a perception threshold 10-fold lower than their alcohol precursors. Ethyl esters of butanoic, hexanoic, octanoic and decanoic acids represent c. 95% k}l
till
tO t~tl
E~LEL
D.
LtllJl
llCAdllOdtg,
WLLLI
a
~IID tlll~,
t
aroma of unripe apples, is present in the greatest quantity, c. 60% of the total esters. In 12-month-old Grana Padano cheese, this odorant is 10-fold the level found in fresh bovine milk (Moio et al., 1993). Ethyl butanoate is the second most important ester. Esters are the main volatile components of Canestrato Pugliese cheese (Di Cagno et al., 2003) and ethyl esters are the predominant esters in Manchego, Roncal, Mahon, Fiore Sardo and Pecorino Romano cheeses (Martinez-Castro et al., 1991 Moio et al., 1993; Izco and Torre 2000; Villasefior et al., 2000; Larr~iyozet al., 2001 Di Cagno et al., 2003). Ketones represent the second largest class of volatile compounds in Grana Padano cheese, accounting for c. 33% of neutral volatiles, similar to the amount found in Parmigiano Reggiano cheese, where they are the most abundant volatiles, representing c. 26% of total headspace chromatographic area (Barbieri et al., 1994; Moio and Addeo, 1998). The total concentration of methyl ketones in Parmigiano Reggiano (0.075 ~mol/g fat) is quite low compared to Blue cheese (5.18 b~mol/g fat for Roquefort) (Arnold et al., 1975; Piergiovanni and Volonterio, 1977; Gallois and Langlois, 1990). Ketones were also found to be the dominant volatile flavour compounds in Fiore Sardo (Di Cagno et al., 2003) and the second most abundant group of volatiles
in Roncal cheese (Izco and Torre, 2000) and were considered to be one of the major classes of volatiles which varied in Mahon cheese during ripening (Mulet et al., 1999). The major representatives of the 2-alkanones with odd numbers of carbon atoms in Grana Padano cheese were 2-pentanone, 2-heptanone, 2-nonanone and 2-undecanone. 2-Pentanone and 2-heptanone are the most abundant methyl ketones in aged Manchego cheese (Villasefior et al., 2000), while 2-heptanone and ,I.IIK 1 . . . . LWU l l l ~ t l i y l 2-tlVllaLLUttC ketones r. . . . a at .-,..~ highest level in Canestrato Pugliese, Fiore Sardo and Pecorino Romano cheeses (Di Cagno et al., 2003). All the methyl ketones with an odd number of carbons (C3mC9) were detected in Roncal, Pecorino Sardo, Manchego and Fiore Sardo cheeses at higher levels than those with an even number of carbons (C4mC12) (Izco and Torre, 2000; Villasefior et al., 2000; Larr~iyoz et al., 2001 Di Cagno et al., 2003). In Pecorino Sardo cheese, the concentration of methyl ketones generally increases during ripening. It was also presumed that the FFAs liberated through lipolysis are catabolised to methyl ketones by microbial activity (Izco and Torre, 2000). Alcohols represent the third class of volatiles in Grana Padano cheese, accounting for c. 23% of the total neutral volatiles. Those present in greatest quantity are 2-pentanol, 3-methyl-3-buten-l-ol, 3-methyl-l-butanol and 2-heptanol. 1-Octen-3-ol is a key aroma compound of mushrooms and has long been recognised as an important flavour compound produced by Penicillium roqueforti in Blue cheeses (Shimp and Kinsella, 1977). Alcohols are the predominant group of volatile compounds in Roncal and Pecorino Romano cheeses (Izco and Torte, 2000; Di Cagno et al., 2003). Butan-2-ol and propan-l-ol have been detected in the largest quantities VVCLC
,- . . . . . .
,-1....1
lOLIllg,
tll~
Extra-Hard Varieties
in the Spanish cheese, while 1-butanol and 1-hexanol characterised the Italian variety. Parmigiano Reggiano cheese contains at least 16 different chiral alcohols, the most abundant secondary alcohols found being 2butanol, 2-pentanol, 2-heptanol, 2-nonanol and 1octen-3-ol (Mariaca et al., 2001). Aldehydes and lactones contribute c. 0.6 and 0.1%, respectively, of the total neutral volatiles of Grana Padano cheese (Moio and Addeo, 1998). Low levels of aldehydes indicated a normal maturation; at higher levels, they were found to cause off-flavour. Lactones are the second largest class of volatiles in several Italian ewes' milk cheeses like Canestrato Pugliese, Fiore Sardo and Pecorino Romano, 8-dodecalactone and 8-dodecanolactone being found at the highest levels (Di Cagno et al., 2003). Eleven lactones were detected in the Parmigiano Reggiano cheese; 8-decalactone and 8-dodecalactone were found most commonly (Mariaca et al., 2001).
Some of the most famous Italian extra-hard varieties have also been characterised from the point of view of nutrition (Table 7). Compared to other varieties, Parmigiano Reggiano and Grana Padano are described as those cheeses with the highest content of protein and a low content of lipids and cholesterol (Turchetto, 1988; Berra and Ottina, 1990; Califfi and Mazzali, 2000). These cheeses have an energy value of c. 374-384 kcal/100 g which is similar to that of Pecorino Romano. One hundred grams of Parmigiano Reggiano cheese add c. 33 g
67
of protein to the diet. The protein content of these Italian varieties is of high quality since a great part is already digested to peptides of various size and amino acids which either facilitate digestion or stimulate the gastric secretions. A large part of the total free amino acids are essential amino acids (e.g., leucine, lysine, isoleucine and valine) and also the level of non-essential amino acids is very high which effectively reduces the metabolic energy expended on biosynthetic reactions. Except for cysteine + methionine, c. 50 g of Parmigiano Reggiano and Grana Padano cheeses are enough to meet the daily requirements of the other essential amino acids. Parmigiano Reggiano and Grana Padano cheeses contain c. 28% lipids, triacylglycerols being the main component. Cholesterol is present at a concentration less than 80-85 mg/100 g of cheese (Marchetti, 1988). The high content of calcium, c. 1.2%, and, especially, the optimum calcium/phosphorus ratio, is another important nutritional feature of these cheeses. Besides, the ratio of calcium/lipids is very high compared to other cheeses, which means that ingestion of an optimum intake of calcium (e.g., 800 mg/day) is not negatively correlated with an energetic surplus due to an elevated intake of lipids. Parmigiano Reggiano and Grana Padano cheeses are also very rich in other mineral constituents, e.g., 63 mg of potassium and 18 mg of iodine per 50 g of cheese which represent c. 20% of the human daily requirement (Ferri, 1990; Marchetti, 1990). Parmigiano Reggiano and Grana Padano cheeses have considerable levels of fat-soluble vitamins, A and D, and especially are highly appreciated for the elevated amount of vitamin B12 (Marchetti, 1990).
Average value of nutritional compounds for 100 g of Grana Padano cheese
Compound
Concentration
Moisture Protein Soluble peptides Free amino acids Lipids Carbohydrates Calcium Phosphorus Ratio calcium/phosphorus NaCI K+, Mg2+, Zn 2+, Fe2+, Cu 2+, Se 2+, IVitamin A, D3 and E Vitamin B1, B2, B6 Vitamin B12 Pantothenic acid Choline Biotin
32 g 33 g 1.5 g 6g 28 g absent 1165 mg 692 mg 1.7 1.4 g 881.5 #g 227.5 i~g 494 I~g 3 i~g 246 #g 20 i~g 6 i~g
The energy value of 100 g of Grana Padano cheese is 384 kcal (252 kcal from lipids and 132 kcal from proteins). Source: Califfi and Mazzali (2000).
Mazco Gobbetti wishes to thank Prof. Bruno Battistotti for the friendly and skilled revision of this chapter.
Addeo, E and Chianese, L. (1990). Cinetica di degradazione delle frazioni caseiniche nel formaggio Grana Padano, in, Grana Padano un Formaggio di Qualita: Studi e Ricerche Progetto di Qualitd, Consorzio per la Tutela del Formaggio
Grana Padano, Italy. pp. 97-130. Addeo, E, Moio, L. and Stingo, C. (1988). Caratteri tipici della proteolisi nel formaggio Parmigiano Reggiano. Composizione della frazione caseinica, in, Atti Giornata di Studio, Consorzio del Formaggio Parmigiano Reggiano, ed., Reggio Emilia. pp. 21-40. Albenzio, M., Corbo, M.R., Shekeel-Ur-Rehman, Fox, RE, De Angelis, M., Corsetti, A., Sevi, A. and Gobbetti, M. (2001). Microbiological and biochemical characteristics of Canestrato Pugliese cheese made from raw milk, pasteurised milk or by heating the curd in hot whey. Int. J. Food Microbiol. 67, 35-48.
68
Extra-Hard Varieties
Alcala, M., Beltr~in de Hredia, EH., Esteban, M.A. and Marcos, A. (1982). Distribucion del nitrogeno soluble del queso de Mahon. Arch. Zootecn. 31,257-267. Arizcun, C., Barcina, Y. and Torre, P. (1997a). Identification of lactic acid bacteria isolated from Roncal and Idiazabal cheese. Lait 77, 729-732. Arizcun, C., Barcina, Y. and Torre, P. (1997b). Identification and characterization of proteolytic activity of Enterococcus species isolated from milk and Roncal and Idiazabal cheese. J. Food Microbiol. 38, 17-24. Arnold, R.G., Shahani, K.M. and Dwivedi, B.K. (1975). Application of lipolytic enzymes to flavor development in dairy products.J. Dairy Sci. 58, 1127-1143. Barbieri, G., Bolzoni, L., Careri, M., Mangia, A., Parolai, G., Spagnoli, S. and Virgili, R. (1994). Study of the volatile fraction of Parmesan cheese. J. Agric. Food Chem. 42, 1170-1176. Battistotti, B., Bottazzi, V., Piccinardi, A. and Volpato, G. (1983). Formaggi nel Mondo, Arnoldo Mondatori Editore, Milano. Berra, B. and Ottina, V. (1990). Analisi degli acidi grassi trans della frazione lipidica totale in formaggio Grana Padano, in, Grana Padano un Forrnaggio di Qualita: Studi e Ricerche Progetto di Qualita, Consorzio per la Tutela del Formaggio Grana Padano, Italy. pp. 367-395. Bottazzi, V. (1962). Ricerche sulla microbiologia del formaggio grana. Nota III: Studio della microflora del sierofermento usato nella fabbricazione del formaggio grana tipico. Ann. Microbiol. Enzimol. 12, 59-72. Bottazzi, V. (1979). Aspetti microbiologici della produzione del formaggio grana, in, II Formaggio Grana Tomo 1, Latteria Didattica P. Marconi, Thiene. pp. 31-47. Bottazzi, V. (1993). Biotecnologia Lattiero-casearia, Edagricole, Bologna. Burkhaher, G. (1981). Catalogue of Cheese. Bulletin 141, International Dairy Federation, Brussels. pp. 15-33. Califfi, A. and Mazzali, E. (2000). Accadde rnolti secoli fa ... Grana Padano, Editoriale Sometti, Mantova. Carboni, M.E, Zannoni, M. and Lercker, G. (1988). Lipolisi del grasso del Parmigiano Reggiano, in, Atti Giornata di Studio, Consorzio del Formaggio Parmigiano Reggiano, Reggio Emilia. pp. 113-121. Consorzio del Formaggio Parmigiano Reggiano (1989). Regolamento per la Produzione del Latte, AGE Graficoeditoriale, Reggio Emilia. Consorzio per la Tutela del Formaggio Grana Padano (1990). Grana Padano un Formaggio di Qualitd: Studi e Ricerche Progctto di Qualita, Consorzio per la Tutela del Formaggio Grana Padano, Italy. Coppola, R., Nanni, M., Iorizzo, M., Sorrentino, A., Sorrentino, E. and Grazia, L. (1997). Survey of lactic acid bacteria isolated during the advanced stages of the ripening of Parmigiano Reggiano cheese. J. Dairy Res. 64, 305-310. Corbo, M.R., Albenzio, M., De Angelis, M., Sevi, A. and Gobbetti, M. (2001). Microbiological and biochemical properties of Canestrato Pugliese hard cheese supplemented with bifidobacteria. J. Dairy Sci. 84, 551-560. Davis, J.C. (1965). Cheese, Churchill Livingstone, London. Di Cagno, R., Banks, J., Sheehan, L., Fox, P.E, Corsetti, A. and Gobbetti, M. (2003). Comparison of the microbio-
logical, compositional, biochemical, volatile profile and sensory characteristics of three Italian PDO ewes' milk cheeses. Int. DairyJ. 13,961-972. Esteban, M.A., Marcos, A., Alcal~i, M. and Beltr~in de Hredia, EH. (1982). Caseinas y polipeptidos insolubles del queso de Mahon. Arch. Zootecn. 31,305-315. Ferri, G. (1990). La composizione minerale del formaggio Grana Padano, in, Grana Padano un Formaggio di Qualita: Studi e Ricerche Progetto di Qualita, Consorzio per la Tutela del Formaggio Grana Padano, Italy. pp. 413-432. Fessler, D., Casey, M.G. and Puhan, Z. (1999). Identification of propionibacteria isolated from brown spots of Swiss hard and semi-hard cheeses. Lait 79,211-216. Fox, P.E, Wallace, J.M., Morgan, S., Lynch, C.M., Niland, E.G. and Tobin, J. (1996). Acceleration of cheese ripening. Antonie van Leeuwenhoek 70,271-297. Frau, M., Massanet, J., Rossello, C., Simal, S. and Ca~ellas, J. (1997). Evolution of free amino acid content during ripening of Mahon cheese. Food Chem. 60, 651-657. Gaiaschi, A., Beretta, B., Ponesi, C., Conti, A., Giuffrida, M.G., Galli, C.L. and Restani, P. (2001). Proteolysis of [3-casein as a marker of Grana Padano cheese ripening. J. Dairy Sci. 84, 60-65. Gallois, A. and Langlois, D. (1990). New results in the volatile odorous compounds of French blue cheeses. Lait 70, 89-106. Gobbetti, M. and Di Cagno, R. (2002). Hard Italian cheeses, in, Encyclopedia of Dairy Sciences, Vol. 2, Roginski, H., Fox, P.E and Fuquay, J.W., eds, Academic Press, London. pp. 378-385. Gobbetti, M., Fox, P.E, Smacchi, E., Stepaniak, L. and Damiani, P. (1996). Purification and characterization of a lipase from Lactobacillus plantarum 2739.J. Food Biochem. 220, 227-246. Gobbetti, M., Fox, P.E and Stepaniak, L. (1997). Isolation and characterization of a tributyrin esterase from Lactobacillus plantarurn 2739.J. Dairy Sci. 80, 1110-1117. Gobbetti, M., Burzigotti, R., Smacchi, E., Corsetti, A. and De Angelis, M. (1998). Microbiology and biochemistry of Gorgonzola cheese during ripening. Int. Dairy J. 7, 519-529. Gobbetti, M., Lanciotti, R., De Angelis, M., Corbo, M.R., Massini, R. and Fox, P.E (1999a). Study of the effects of temperature, pH, NaC1 and aw on the proteolytic and lipolytic activities of cheese-related lactic acid bacteria by quadratic response surface methodology. Enzyme Microbiol. Technol. 25,795-809. Gobbetti, M., Lanciotti, R., De Angelis, M., Corbo, M.R., Massini, R. and Fox, P.E (1999b). Study of the effects of temperature, pH and NaC1 on the peptidase activities of non-starter lactic acid bacteria (NSLAB) by quadratic response surface methodology. Int. Dairy J. 9,865-875. Gobbetti, M., Folkerstema, B., Fox, P.E, Corsetti, A., Smacchi, E., De Angelis, M., Rossi, J., Kilcawley, K. and Cortini, M. (1999c). Microbiology and biochemistry of Fossa (pit) cheese. Int. Dairy J. 9,763-773. Gudkov, A.V. (1993). Cheeses of the former USSR, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, 2nd edn, Fox, P.E, ed., Chapman & Hall, London. pp. 281-299.
Extra-Hard Varieties
Guinee, T.P. (1985). Studies on the Movements of Sodium Chloride and Water in Cheese and the Effects thereof on Cheese Ripening. PhD Thesis, National University of Ireland, Cork. Guinee, T.R arid Fox, RE (1984). Studies on Romano-type cheese, general proteolysis. Ir. J. Food Sci. Technol. 8, 105-114. Hofi, A.A., Youssef, E.H., Ghoneim, M.A. and Tawab, G.A. (1970). Ripening changes in Cephalotyre 'RAS' cheese manufactured from raw and pasteurized milk with special reference to flavor. J. Dairy Sci. 53, 1207-1211. Industria Lattiero-Casearia Italiana (2002). Rapporto 2001, Milano 18 June 2002, Editoriale il Mondo del Latte. Innocente, N., Pittia, P., Stefanuto, O. and Corradini, C. (2000). Texture profile of Montasio cheese. Milchwissenschaft 55,507-510. Izco, J.M. arid Torte, P. (2000). Characterisatiorl of volatile flavour compounds in Roncal cheese extracted by the 'purge and trap' method and analysed by GC-MS. Food Chem. 70,409-417. Larr~iyoz, P., Addis, M., Gauch, R. and Bosset, J.O. (2001). Comparison of dynamic headspace and simultaneous distillation extraction techniques used for the analysis of the volatile components in three European PDO ewes' milk cheeses. Int. DairyJ. 11,911-926. Lynch, C.M., McSweeney, RL.H., Fox, RE, Cogan, T.M. and Drinan, EB. (1996). Manufacture of Cheddar cheese with and without adjunct lactobacilli under controlled microbiological conditions. Int. DairyJ. 6,851-867. Mannu, L., Comunian, R. and Scintu, M.F. (2000). Mesophilic lactobacilli in Fiore Sardo cheese: PCR-identification and evolution during cheese ripening. Int. Dairy J. 10, 383-389. Marchetti, M. (1988). Le vitamine nel formaggio Parmigiano Reggiano, in, Atti Giornata di Studio, Consorzio del Formaggio Parmigiano Reggiano, Reggio Emilia. pp. 107-112. Marchetti, M. (1990). Composizione e valore nutritivo del Grana Padano: le vitamine e lo Iodio, in, Grana Padano un Formaggio di Qualita: Studi e Ricerche Progetto di Qualita, Consorzio per la Tutela del Formaggio Grana Padano, Italy. pp. 433-444. Marcos, A. and Esteban, M.A. (1993). Iberian cheeses, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, 2nd edn, Fox, RE, ed., Chapman & Hall, London. pp. 173-219. Marcos, M., Esteban, M.A., Alcal~i, M. and Beltr~in de Hredia, EH. (1983). Actividad del agua, pH y principales minerales del queso de Mahon. Arch. Zootecn. 32, 1731. Mariaca, R.G., Imhof, M.I. and Bosset, J.O. (2001). Occurrence of volatile chiral compounds in dairy products, especially cheese - a review. Eur. Food Res. Technol. 212, 253-261. Martinez-Castro, I., Sanz, J., Amigo, L., Ramos, M. and Martin Alvarez, P. (1991). Volatile components of Manchego cheese. J. Dairy Res. 58, 239. McSweeney, P.L.H., Fox, RE, Lucey, J.A., Jordan, K.N. and Cogan, T.M. (1993). Contribution of the indigenous microflora to the maturation of Cheddar cheese. Int. Dairy J. 3, 613-634.
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Moio, L. and Addeo, E (1998). Grana Padano cheese aroma. J. Dairy Res. 65, 317-333. Moio, L., Dekimpe, J., Etievant, P.X. and Addeo, E (1993). Neutral volatile compounds in the raw milks from different species. J. Dairy Res. 60, 199-213. Mora, R., Nanni, M. and Panari, G. (1984). Physical, microbiological and chemical changes in Parmigiano Reggiano cheese during the first 48 hours. Scienza e Tecnica Lattiero-Casearia 35, 20-32. Mulet, A., Escriche, I., Rossello, C. and Tarrazo, J. (1999). Changes in the volatile fraction during ripening of Mahon cheese. Food Chem. 65,219-225. Nlifiez, M., Medina, M. and Gaya, P. (1989). Ewes' milk cheese: technology, microbiology and chemistry. J. Dairy Res. 56,303-321. Ordofiez, J.A., Barneto, R. and Ramos, M. (1978). Studies on Manchego cheese ripened in olive oil. Milchwissenschaft 33, 609-613. Ordofiez, J.A., Masso, J.A., M~irmol, M.P. and Ramos, M. (1980). Contribution /t l'etude du fromage ~Roncal~. Lait 60, 283-294. Ottogalli, G. (2001). Atlante dei Formaggi, Hoepli, Milan. Panari, G., Mongardi, M. and Nanni, M. (1988). Determinazione con metodi chimici delle frazioni azotate del formaggio Parmigiano Reggiano, in, Atti Giornata di Studio, Consorzio del Formaggio Parmigiano Reggiano, Reggio Emilia. pp. 85-96. Peji6, O.M. (1956). Technology of Milk Products, Nau~naknjiga, Beograd. Phelan, J.A., Renaud, J. and Fox, RE (1993). Some non-European cheese varieties, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, 2nd edn, Fox, RE, ed., Chapman & Hall, London. pp. 421-465. Piergiovanni, L. and Volonterio, G. (1977). Studio delle sostanze responsabili della formazione dell'aroma nel formaggio 'Grana'. 12Industria del Latte 13, 31-46. Resmini, R, Pellegrino, L., Hogenboom, J. and Bertuccioli, M. (1988). Gli aminoacidi liberi nel formaggio Parmigiano Reggiano stagionato, in, Atti Giornata di Studio, Consorzio del Formaggio Parmigiano Reggiano, Reggio Emilia. pp. 41-58. Resmini, P., Hogenboom, J., Pellegrino, L. and Pazzaglia, C. (1990). Evoluzione del contenuto quali-quantitativo di aminoacidi liberi nel formaggio Grana Padano, in, Grana Padano un Formaggio di Qualita: Studi e Ricerche Progetto di Qualita, Consorzio per la Tutela del Formaggio Grana Padano, Italy. pp. 193-213. Richardson, G.H. and Nelson, J.H. (1967). Assay and characterization of pregastric esterase. J. Dairy Res. 50, 1061-1065. Scott, R. (1981). Cheesemaking Practice, Elsevier Applied Science Publishers, London. Shimp, J.L. and Kinsella, J.E. (1977). Lipids of Penicillium roqueforti. Influence of culture temperature and age on unsaturated fatty acids. J. Agric. Food Chem. 25, 793-799. Toppino, P.M., Rampilli, M., Francani, R. and Pellegrini, N. (1990). Valutazione quali-quantitativa dei macrocomponenti, delle frazioni proteiche e degli acidi organici in formaggio Grana Padano, in, Grana Padano un Formaggio di Qualit& Studi e Ricerche Progetto di Qualitil,
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Extra-Hard Varieties
Consorzio per la Tutela del Formaggio Grana Padano, Italy. pp. 71-95. Turchetto, E. (1988). I1 Parmigiano Reggiano: aspetti nutrizionali, in, Atti Giornata di Studio, Consorzio del Formaggio Parmigiano Reggiano, Reggio Emilia. pp. 123-132. Villasel~or, M.J., Valero, E., Sanz, J. and Mart*nez Castro, I. (2000). Analysis of volatile components of Manchego
cheese by dynamic headspace followed by automatic thermal desorption-GC-MS. Milchwissenschafi 55, 378-382. Walter, H.E. and Hargrove, R.C. (1972). Cheeses of the World, Dover Publications, Inc, New York. Woo, A.H. and Lindsay, R.C. (1984). Concentration of major free fatty acids and flavour development in Italian cheese varieties. J. Dairy Sci. 67,960-968.
Cheddar Cheese and Related Dry-salted Cheese Varieties R.C. Lawrence, J. Gilles* L.K. Creamer, V.L. Crow, H.A. Heap, C.G. HonorS, K.A. Johnston and P.K. Samal, Fonterra Research Centre, Palmerston North, New Zealand
In the warm climates in which cheesemaking was first practised, cheeses would have tended to have a low pH as a result of the acid-producing activity of the lactic acid bacteria and coliforms in the raw milk. In colder climates, it would have been logical either to add warm water to the curds and whey to encourage acid production (the prototype of Gouda-type cheeses) or to drain off the whey and pile the curds into heaps to prevent the temperature falling. In the latter case, the piles became known as 'Cheddars', after the village in Somerset, England, where the technique is said to have been first used about the middle of the nineteenth century. The concept of cheddaring was quickly adopted elsewhere. The first Cheddar cheese factory, as opposed to farmhouse cheesemaking, was in operation in the United States (NY State) in 1861, followed by Canada (Ontario) in 1864 and by New Zealand and England in 1871. Development of cheddaring Cheddar cheese was apparently made originally by a stirred curd process without matting, but poor sanitary conditions led to many gassy cheeses with unclean flavours (Kosikowski and Mistry, 1997). Cheddaring was found to improve the quality of the cheese, presumably as a result of the faster and greater extent of acid production. As the pH fell below about 5.4, the growth of undesirable, gas-forming organisms, such as coliforms, would have been increasingly inhibited. The piling and repiling of blocks of warm curd in the cheese vat for about 2 h also squeezed out any pockets of gas that formed during manufacture. Cheesemakers came to believe that the characteristic texture of Cheddar cheese was a direct result of the cheddaring process. It is now clear that recently developed methods of manufacturing Cheddar cheese do not involve a traditional cheddaring step but the cheese obtained has a texture identical to that of traditionally made Cheddar.
* Deceased 19 January 2003.
The development of the fibrous structure in the curd of traditionally made Cheddar does not commence until the curd has reached a pH of 5.8 or less (Czulak, 1959). The changes that occur are a consequence of the development of acid in the curd and the consequent loss of calcium and phosphate from the protein matrix. Therefore, it is important to recognize that 'cheddaring' is not confined only to Cheddar cheese. All cheeses are 'cheddared' in the sense that all go through this same process of chemical change. The only difference is one of degree, i.e., the extent of flow varies due to differences in calcium level, pH and moisture (Lawrence et al., 1983, 1984). In addition, with brine-salted cheeses, flow is normally restricted at an early stage in manufacture by placing the curd in a hoop. However, if Gouda curd is removed from a hoop, it flows in the same way as Cheddar curd. Similarly, the stretching induced in Mozzarella by kneading in hot water is best viewed as a very exaggerated form of 'cheddaring'. All young cheese, regardless of the presence of salt, can be stretched in the same way as Mozzarella, provided that the calcium content and pH are within the required range (Lawrence et al., 1993). Development of dry-salting In the early days of cheesemaking, the surface of the curd mass was presumably covered with dry salt in an attempt to preserve the cheese curd for a longer period. In localities where the salt was obtained by the evaporation of seawater, it would have been a rational step to consider using the concentrated brine rather than wait for all the liquid to evaporate. The technique of dry-salting, i.e., salting relatively small pieces of curd before pressing, appears to have evolved in England, probably in the county of Cheshire, where rock salt is abundant. Cheshire has been manufactured for at least 1000 years and is thus a more ancient cheese than Cheddar. Variants of Cheshire and Cheddar were developed in specific localities of Britain and have come to be known as British Territorial cheeses. Blueveined cheeses such as Stilton, Wensleydale and Dorset are also dry-salted.
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
72
Cheddar Cheese and Related Dry-salted Cheese Varieties
Dry-salting overcomes the major disadvantage of brine-salting, i.e., the 'blowing' of the cheese due to the growth of such bacteria as coliforms and clostridia, but introduces new difficulties because the starter organisms and lactic acid formation are also inhibited by the salt. This inhibition is not a problem when the pH of the curd granules is allowed to reach a relatively low value prior to the application of salt, as in Cheshire and Stilton manufacture. However, the manufacture of a dry-salted cheese in the medium pH range (5.0-5.4), such as Cheddar, is more difficult than that of the Gouda-type cheeses in which the pH is controlled by limiting the lactose content of the curd by the addition of water to the curds/whey mixture in the vat. At the time of salt addition, a relatively large amount of lactose is still present in Cheddar curd (Turner and Thomas, 1980). However, this is not detrimental to the quality of the cheese provided that the salt-in-moisture (S/M) level is greater than 4.5% and the cheese is allowed to cool after pressing (Fryer, 1982). Differences obviously exist in the procedures used for the manufacture of dry- and brine-salted cheeses but these have relatively little effect on the finished cheeses; the production of dry-salted cheeses is similar in principle to that of brine-salted cheeses. Clearly, the rate of solubilization of the casein micelles and the activity of the residual rennet and plasmin in the curd 9. ,. , .i .l l.
ku~~,-
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uttvvtku
,-n . . . . .- u. l J - ~i ,. t. l- ,j ,v -,-,,.,-k
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brining but only during the first few weeks of ripening. There is no evidence to suggest that the mechanisms by which the protein is degraded are affected by the changes in salt concentration as the salt diffuses into the curd. Any differences between dry- and brinesalted cheeses of the same overall chemical composition will therefore decrease as the cheeses age. Traditional Cheddar cheese is visually different from the common brine-salted cheeses such as the Goudaand Swiss-type cheeses, which are more plastic in texture and have 'eyes'. However, both these characteristics are a result of the relatively high pH and moisture of these cheeses and not of brine-salting itself. The texture of a brine-salted cheese is less open than that of traditionally-made Cheddar cheese because the curd is pressed under the whey to remove pockets of air before brining. As a close texture is a pre-requisite for the formation of 'eyes', it has come to be generally believed that 'eyes' can be obtained only in brine-salted cheese. The technique of vacuum pressing allows the removal of air from between the particles of dry-salted curd. This can result in a closeness of texture similar to that of Gouda-type cheeses. Therefore, it is now possible to manufacture dry-salted cheese with 'eyes' provided that the chemical composition is similar to that of tradi-
tional brine-salted cheeses and if the starter contains gas-producing strains (Lawrence et al., 1993). Present and future role of Cheddar-like cheeses
Traditionally, Cheddar was a so-called 'table cheese' and was purchased by the consumer shortly before consumption. In line with the global changes in the dairy and food industries (Creamer etal., 2002), cheese, Cheddar in particular, is commonly purchased from the manufacturer, repackaged, often in vacuum packs, and sold on to supermarkets or food wholesalers. It is also used as the base material for a range of processed cheeses ('Pasteurized Processed Cheese and Substitute/ Imitation Cheese Products', Volume 2) and 'cheesefood' products ('Cheese as an Ingredient', Volume 2). Because of our understanding of the factors controlling the development of Cheddar cheese flavour and texture during maturation, it is possible to produce cheeses with a range of pre-determined characteristics using semi-automated mechanized manufacture ('General Aspects of Cheese Technology', Volume 2). Cheese, as a major ingredient in a food, needs to fulfil certain requirements, such as retention of the flavour and textural characteristics it confers on the food over a substantial storage period. This is coupled with strict composition and price criteria. A good example of meeting this challenge is outlined in detail by Chen and Johnson (2001) in producing a dry-salted cheese using a mesophilic starter suitable for hot-melt products, such as Pizza pies, without using the pastafilata (Mozzarella) process.
During the latter half of the twentieth century, there were a number of significant changes to the way in which Cheddar cheese is manufactured. The single most important factor supporting those changes has been the availability of reliable starter cultures. The successful development of continuous mechanized systems for Cheddar manufacture has depended upon the ability of the cheesemaker to control precisely both the expulsion of moisture and the increase in acidity required in a given time. This in turn has led to the recognition that the quality of cheese, now being made on a very large scale in modern cheese plants, can be guaranteed only if its chemical composition falls within pre-determined ranges. Nevertheless, Cheddar cheese is still a relatively difficult variety to manufacture because the long ripening period necessary for the development of the required mature flavour can also be conducive to the formation of off-flavours. In addition, its texture can vary considerably. The intermediate position of
C h e d d a r C h e e s e and Related Dry-salted C h e e s e Varieties
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73
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---
Classification of traditionally manufactured cheese varieties by their characteristic ranges of the ratio of calcium to solids-not-fat and pH.
Cheddar cheese in the total cheese spectrum (Lawrence et al., 1984) (Fig. 1) is particularly exemplified by its textural properties, which lie between the crumbly nature of Cheshire and the plastic texture of Gouda. The traditional manufacture of Cheddar cheese consists of: (a) coagulating milk, containing a starter culture, with rennet; (b) cutting the resulting coagulum into small cubes; (c) heating and stirring the cubes with the concomitant production of a required amount of acid; ( d ) w h e y removal; (e) fusing the cubes of curd into slabs by cheddaring; (f) cutting (milling) the cheddared curd; (g) salting; (h) pressing; (i) packaging and ripening (Fig. 2). Although it is impossible to separate the combined effects of some of these operations on the final quality of the cheese, they will, as far as possible, be considered individually. Effect of milk composition and starter culture
Cheesemaking basically involves the removal of moisture from a rennet-induced coagulum (Fig. 3). The four major factors involved are the proportion of fat in the curd, the curd particle size, the cooking (scalding) temperature and the rate and extent of acid production
o
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A typical manufacturing schedule for Cheddar cheese.
_
(Whitehead and Harkness, 1954; Lawrence et al., 1983; Johnston et al., 1991). In order to achieve uniform cheese quality in large commercial plants, the manufacturing procedures must be as consistent as possible. The first requirement is uniformity of the raw milk. This is achieved by bulking the milk in a silo to even out differences in milk composition from the various districts supplying milk to the cheese plant. Preferably, the milk should be bulked before use so that its fat content can be standardized accurately. For Cheddar cheese varieties, the milk is normally standardized to a casein/fat ratio between 0.67 and 0.72. The more fat present in the cheese milk, and therefore in the rennet coagulum, the more difficult it is to remove moisture under the same manufacturing conditions because the presence of fat interferes mechanically with the syneresis process. Standardization has traditionally involved manipulation of the fat content of the cheese milk to give a specific casein/fat ratio. This is usually achieved either by partially removing the fat from the whole milk stream or by removing all the fat from the whole milk and adding back a portion to the skim milk stream. However, recent developments in membrane
35-40 min 2 h 20 min-2 h 45 min
__
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The main factors in the expulsion of moisture from a rennet-induced milk coagulum.
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Dimensions of curd
<5
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74
Cheddar Cheese and Related Dry-salted Cheese Varieties
processing technologies have meant that the protein component of the whole milk can now be standardized also. There are a number of options by which the protein content of cheese milk can be standardized. An example is concentrating the level of protein in a skim milk stream by uhrafihration and adding the retentate back to the whole milk stream to boost the protein concentration in the whole milk to the target level, which is typically between 3.5 and 4%. The manufacture of Cheddar cheese is more dependent on uniform starter activity than that of washed curd cheeses, such as Gouda. The proper rate of acid development, particularly before the whey is drained from the curd, is essential if the required chemical composition of the cheese is to be obtained (Whitehead and Harkness, 1954; Lawrence etal., 1984). However, the curd is 'cooked' to expel moisture at a temperature that normally adversely affects the starter bacteria. The cheesemaker must therefore exert judgement to ensure that the desired acid development in the curd is reached at about the same time as the required moisture content. The starter system used in New Zealand cheese plants is based on the continuous use of a single triplet starter comprising three defined strains of Lactococcus lactis subsp, cremoris selected primarily on the basis of their acid production, phage resistance and flavour development (Heap, 1998). Defined starter systems are now widely used in the United States (Richardson 9-,~- ,-,1
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and Australia (Heap and Lawrence, 1988" Limsowtin et al., 1996) and have replaced the undefined commercial mixed-strain cultures of the type still used exclusively for the manufacture of Gouda-type cheeses in The Netherlands (Stadhouders and Leenders, 1984). If the cooking temperature is kept constant (for instance at 38 ~ throughout the cheesemaking year and standardized milk is used, by far the most important factor in producing Cheddar cheese of uniform quality is the extent of acid production in the vats. In New Zealand, this is managed successfully in two ways: (a) the use of reconstituted skim milk or suppliers' milk of good quality for the preparation of bulk cheese starter; (b) the ability of the cheese industry to produce neutralized bulk cheese starter and to control the ratios of the starter strains added to the cheese milk (Heap, 1998). To compensate for seasonal changes in milk composition, it is normally necessary only to vary the percentage inoculum of starter to achieve the required acidity at draining. Effect of coagulant
The amount of rennet added should be the minimum necessary to give a firm coagulum in the set-to-cut time
(time between rennet addition and cutting) required. In Cheddar cheese manufacture, the set-to-cut time is usually in the range 35-45 min. There is a range of animal, microbial and recombinant rennets to choose from and their advantages and disadvantages are discussed in 'Rennets: General and Molecular Aspects', Volume 1. Calf rennet, high in chymosin, has been used traditionally for Cheddar cheese production. The advantage of using a high chymosin content calf rennet is that the flavour and the texture of aged Cheddar are more predictable, with less bitterness. The same could be said for the recombinant chymosins. However, some customers have strong aversions to the use of genetically engineered ingredients in cheese. Some cheese manufacturers are now investigating the use of microbial rennets, which provide the added advantage of being suitable for Kosher, Halal and some vegetarian products. In addition, use of microbial rennets in Cheddar cheese production opens up the options for downstream whey products (whey protein concentrates, milk protein concentrates, etc.). Changes in the volume of rennet added, an increase or decrease in the setting temperature, addition of calcium chloride and/or pH adjustment may be required to avoid any seasonal changes in milk composition and functionality. The rennet-induced coagulum consists of a continuous network of protein that entraps both water and fat ~LUUULL~...~.
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units of protein held together by various forces. Several reports (Eino et al., 1976; Green et al., 1981, 1983) have concluded that the microstructure of the coagulum produced by different types of milk coagulant is a major factor determining the structure and texture of Cheddar cheese. It has been suggested (Green et al., 1981) that 'the structure of the protein network is laid down during the initial curd-forming process and is not fundamentally altered during the later stages of cheesemaking and that the fibrous and more open framework of curd formed by bovine and porcine pepsins might be a reason for the softer curd associated with their use' (Eino et al., 1976). This implies that different milk coagulants significantly affect the initial arrangement of the network of protein structural units. However, it is more likely that the proportion of minerals lost from the coagulum, as a result of the change in pH throughout the entire process, largely determines the texture of a cheese. As one would expect, the type of rennet used and the amount retained in the cheese curd affect the degree of proteolysis as the cheese ripens (Stanley and Emmons, 1977; Creamer etal., 1985) (cf. 'Rennetinduced Coagulation of Milk', 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis
Cheddar Cheese and Related Dry-salted Cheese Varieties
and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', and 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1). The early stages of Cheddar cheese manufacture, specifically gel assembly and curd syneresis, have been reviewed (Fox, 1984; Green, 1984) ('Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels', Volume 1). Electron microscopy studies (Kimber et al., 1974; Kalab, 1977; Stanley and Emmons, 1977) have shown that the casein micelles, which are separate initially, aggregate, coalesce and finally form a multi-branched casein network. The fat globules, also separate at first, are gradually forced together as a result of shrinkage of the casein network. After the coagulum is cut, the surface fat globules are exposed and washed away as the curd is stirred. This leaves a thin layer depleted of fat at the curd granule surface. During matting, the layers of adjacent curd granules fuse, leading to the formation of fat-depleted junctions (Lowrie et al., 1982). Starter bacteria are trapped in the casein network near the fat-casein interface, which has been shown to be the region of highest water content in the mature cheese (Kimber et al., 1974). In all cheese varieties, the outline of the original particles of curd formed when the rennet-induced coagulum is cut can be readily distinguished by scanning electron microscopy (Kalab et al., 1982). In addition, in traditionally-made Cheddar cheese, the boundaries of the milled curd pieces can be seen (Lowrie et al., 1982). These curd granules and milled curd junctions in Cheddar cheese are permanent features, which can still be distinguished in aged cheese. Effect of cutting
The objective of cutting the coagulum, and indeed the objective of the heating and stirring stages that follow cutting, is to facilitate syneresis ('The Syneresis of Rennet-coagulated Curd', Volume 1). However, the cutting operation, together with the speed of stirring following cutting, also influence how large the particles will be at draining and how much of the original milk components (fat and protein) are lost to the whey. The size distribution of the particles at draining is one of the key factors for controlling the moisture content of cheese. The larger the particles, the more moisture that is retained (Whitehead and Harkness, 1954). Maximizing moisture (or moisture in the nonfat substance (MNFS)) and minimizing losses (fat and cheese fines) to the whey will ensure the highest possible yield and profitability (Lawrence and Johnston, 1993). Therefore, cutting is a key operation in cheesemaking and influences not only the composition but also the yield of the finished cheese.
75
Johnston et al. (1991) showed that the speed and duration of cutting in 20 000 1 Damrow cheese vats during commercial Cheddar cheese production determines the curd particle size distribution at draining and hence the moisture content of the final cheese. The whey fat losses could be minimized by the choice of the cutting protocol used. They concluded that, as cutting proceeded, the particle size distribution increasingly favoured smaller particles and that there were two different effects (Fig. 4). In region I, where the cutting cycle is too short, large curd particles remaining after cutting will be reduced in size by smashing during the subsequent stirring phase. Smashing results in small curd particles and fines at draining and high whey fat losses. Between regions I and II, the curd particle size following cutting is small enough to avoid smashing during subsequent stirring and therefore the curd particle size is at a maximum and whey fat losses are at a minimum. In region II, continued cutting gives rise to a greater proportion of smaller curd particles and, in the absence of smashing, whey fat losses remain low. Based on this explanation, Johnston et al. (1991) proposed a model (Fig. 5) for cutting that explains how variations in cutting speed and duration of cutting, followed by a constant stirring speed, determine the curd particle size distribution in a Damrow cheese vat. Each of the five curves (Fig. 5) represents the variations in curd particle size distribution with the duration of cutting, for a constant speed of cutting. Each curve depends on the duration of cutting and is characterized by a specific duration of cutting at which the curd particle size is at a maximum. As the cutting speed is reduced and the duration of cutting is increased to avoid shattering during stirring, the maximum curd particle size increases. Cutting beyond a certain duration, irrespective of the speed of cutting, does not further reduce the curd particle size. A similar study (Johnston et al., 1998) using Ost vats (30 000 1) showed similar trends. However, the Ost vat study also showed that, although similar, the trends were sufficiently different to warrant the characterization of each vat type as to the effect of the speed and duration of cutting, before implementing a specific cutting regime. Effect of heating (cooking) the curd
During cooking, the curds are heated to facilitate syneresis and aid in the control of acid development. The moisture content of the curds is normally reduced from approximately 87% in the initial gel to below 39% in the finished Cheddar cheese. The expulsion of whey is aided by the continued action of rennet as
76
Cheddar
Cheese
and Related
Dry-salted
Cheese
Varieties
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I
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Effect of the speed and duration of cutting on the proportion of curd particles <7.5 mm at draining and the fat content of the whey at running.
well as the combined influence of heat and acid. The temperature should be raised to 38-39 ~ over a period of about 35 min. The curds shrink in size and become firmer during cooking.
801 o4 E 70
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,
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Curves showing the effect of the speed and duration of cutting on the proportion of particles <7.5 mm. Cutting speeds were: (9 2 rev/min; (A), 4 rev/min; (A), 5 rev/min; (I-1), 6 rev/min; ( i ) , 8 rev/min. The continuous lines show the data of Johnston et al. (1991) and the dotted lines are the anticipated trends.
at t h e v a t s t a g e
The single most important factor in the control of Cheddar cheese quality is the extent of acid production in the vat (Fig. 6) because this largely determines its final pH (Lawrence and Gilles, 1982; Creamer et al., 1988) and the basic structure of the cheese (Lawrence et al., 1983). As the pH of the curds decreases, there is a concomitant loss of colloidal calcium phosphate from the casein sub-micelles and, below about pH 5.5, the sub-micelles dissociate into smaller aggregates (Roefs et al., 1985). As the amount of rennet added and the temperature profile are normally constant in the manufacture of Cheddar cheese, the pH change in the curd becomes the important factor in regulating the rate of whey expulsion (Van Slyke and Price, 1952; Lawrence et al., 1984). In mechanized cheesemaking systems, the cheese is usually made to a fixed time schedule. In New Zealand, the time between 'setting' (addition of milk coagulant) and 'running' (draining of whey from the curds; also called 'pump out') is normally 2 h 40 min +_ 10 min. The percentage of starter added determines the increase in titratable acidity or
Cheddar Cheese and Related Dry-salted Cheese Varieties
Acid production in vat
%>
Decrease in pH of curd
Loss of calcium and phosphate
pH and mineral content of curd at draining
Basic structure of cheese
%> Breakdown of casein network during ripening
Cheddar texture and flavour Relationship between the extent of acid production up to the draining stage and the production of Cheddar cheese flavour and texture.
decrease in pH between 'cutting' and 'running'. The extent of acidity increase at this stage is particularly important because it also controls the increase in acidity from 'drying' (when most of the whey has been removed) onwards (Dolby, 1941). The actual increase in acidity may need to be adjusted at intervals throughout the year to achieve the required pH in the cheese at 1 day. This depends upon changes in the chemical composition of the milk, which, in turn, are determined by both the feed of the cow and the lactational cycle. The pH at draining also determines the proportions of residual chymosin (calf rennet) and plasmin in the cheese (Holmes et al., 1977; Lawrence et al., 1984; Creamer et al., 1985). Chymosin plays a major role in the degradation of the caseins during ripening and in the consequent development of characteristic cheese flavour and texture. While curds remain in the whey, there is a continual transfer of lactose to the curds. The whey thus provides a reserve of lactose which prevents any great decrease in lactose concentration in the curd. After the whey has been removed, this reserve is no longer available and the lactose content of the curd falls rapidly as the fermentation proceeds. Curd that has been left in contact with the whey for a longer period has a
77
higher lactose content than curd of the same pH value from which the whey has been removed earlier (Dolby, 1941; Czulak et al., 1969). Acid production can be under complete control only if defined starter systems, such as the single triplet starter, where the individual strains have been selected based on their sensitivity to the manufacturing temperature profile, are used (Lawrence and Heap, 1986; Heap, 1998). Use of this culture has allowed New Zealand cheesemakers to reduce the time from 'set' to 'salt' to about 4 h 30 min. Even shorter times are potentially possible but these are limited by the rate at which moisture can be expelled from the curds in the traditional Cheddar process. Experience has shown that it is preferable to produce lactic acid relatively slowly during the early stages of curd formation and cooking, followed by an increasing rate after draining the whey from the curds. This procedure retains more of the calcium and phosphate in the curd. A recent trend in Europe has been to include a thermophilic strain in starter blends comprising mesophilic strains used for making pressed cheeses (Beresford and Cogan, 1997), as well as soft-ripened cheeses. The rationale for the inclusion of this strain would appear to be in terms of providing a relatively slow rate of acid production at a low temperature of manufacture (high-pH, white-mould cheeses). However, phage attack of the mesophilic strains in these starter blends has led to variable rates of acid production in the cheese vats and problems controlling the final moisture content of the cheese (Heap, personal observation). Effect of cheddaring
The series of operations consisting of packing, turning, piling and re-piling the slabs of matted curd is known as cheddaring. The curd granules fuse under gravity into solid blocks. Under the combined effect of heat and acid, matting of the curd particles proceeds rapidly. The original rubber-like texture gradually changes into a close-knit texture, with the matted curd particles becoming fibrous. The importance attached to flow in the past varied markedly from country to country. In Britain, it was common for each Cheddar block to be made to spread into a thin, hide-like sheet covering an area of about a square metre, whereas in New Zealand only moderate flow was induced, the final Cheddar block being little different in dimensions from when first cut. Czulak and his colleagues (Czulak and Hammond, 1956; Czulak, 1958, 1959; King and Czulak, 1958) initially concluded that extensive deformation and flow were essential in Cheddar cheesemaking. However, further research in Australia
78
Cheddar Cheese and Related Dry-salted Cheese Varieties
(Czulak, 1962), New Zealand (Harkness et al., 1968) and Canada (Lowrie et al., 1982) slowly led to the view that 'cheddaring' is not an essential step and serves no purpose other than to provide a holding period during which the necessary acidity develops and further whey can be released from the curds. This loss of whey is controlled by the acidity and temperature of the curd. The temperature is important, both directly and indirectly, because the rate of acid development is also influenced by temperature. In general, a higher temperature during cheddaring increases the expulsion of whey from the curds. In the traditional process, manipulations of the curd, i.e., the cutting of the matted curd into different sized blocks, the height of piling and the frequency of turning the curd blocks, also aid in moisture control. Mechanical forces- pressure and f l o w - have been shown (Czulak and Hammond, 1956; Czulak, 1959) to be an important factor in the development of the fibrous structure in the curd. This is clearly seen in the arrangement of the fibres, which follow the direction of the flow. However, a fibrous structure cannot be brought about by pressure and deformation unless the curds have reached a pH of 5.8 or less (Czulak, 1959). This suggested that pressure and flow serve to knit, join, stretch and orientate the network of casein fibres already partly formed in response to rising acidity. The readiness to flow, the type of fibres and the density of
resulting in a change in the conformation of the caseins. The concomitant loss of moisture from the casein micelles may also possibly contribute to the conformational change. Czulak (1962) concluded that the characteristic close texture of Cheddar cheese could be obtained without cheddaring. However, he suggested that in mechanizing the cheesemaking process it was probably most convenient, while holding the curd for acidity to develop, to allow the particles to mat together 'but to apply no labour or equipment for its fusing beyond that necessary for ready handling'. Almost all modern mechanized Cheddar cheesemaking systems are based upon these conclusions and involve little or no flow of the curd mass. This development was supported by the success achieved in the manufacture of cheese of normal Cheddar characteristics, particularly in the United States, by 'the stirred curd' process. This strongly indicates that flow and the cheddaring process itself are of little or no significance in the Cheddar cheesemaking process. Similar conclusions were also reached by research workers (Harkness et al., 1968) in New Zealand.
tL .hL pl ~ i4 r .L t
(,~'~ ~4[~]
nl .L~,-p t ~LxYzVn~_P r bl
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and
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moisture. The warmer the curd and the higher its moisture content, the more readily it flows and the finer, longer and denser are the fibres. Czulak (1959) also concluded that it is possible to influence curd structure by manipulating pH, pressure and temperature and that a direct relationship exists between the structure and the water-holding capacity of the curd. This was confirmed by Olson and Price (1970), who showed that extension and rapid flow of curd during cheddaring produced a higher moisture content in the resulting cheese. Fluorescence microscopy has demonstrated the change of the casein from spherical granular particles to a fibrous network (King and Czulak, 1958). Whereas some granular structure was evident in curd grains, the conversion to the fibrous form was complete in cheddared curd. The fibrous shreds of cheddared curd consist of flattened, elongated curd particles that overlap each other, forming a networktype structure with the protein as a continuous phase. The exact mechanism responsible for these observed changes in cheddared curd is not known with certainty but the loss of minerals from the casein micelles in the curd is likely to be the major factor. The loss of calcium phosphate will destabilize the casein micelles,
Effect of milling
The milling operation consists of mechanically cutting the cheddared curd into small pieces in order to: iL .L .t.1 ~. ... .t . q ~ . . G A O %.. tL Lh t%... . . . .O
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more uniform salt distribution into the curd; (b) encourage whey drainage from the curd; (c) assemble the curd in a convenient form for hooping or block-forming. There is a practical upper limit to the cross-section of milled curd before salting for two reasons: (a) there is inadequate whey drainage after salting with large particles; (b) the larger the curd particles, the smaller is the surface/volume ratio. With larger particles, a higher salting rate is therefore required to achieve a given final level of S/M in the cheese. This increases the chance of seaminess (Conochie and Sutherland, 1965a) and gives higher salt losses in the whey (Gilles, 1976). The longer time required for salt penetration allows a greater development of acid in the centre of large curd particles than in smaller particles and this may result in a 'mottled' appearance of the final cheese. Gilbert (1979) pointed out that ideally the curd should be cut into spheres to obtain a uniform mass/surface area profile. However, the best that can be achieved (Breene et al., 1965; Gilbert, 1979) is to use a curd mill that produces a shredded curd, flakes of curd or tinge>like pieces of curd. The curd mill speed can be increased or decreased to change the curd particle size and shape, which in turn affect the
Cheddar Cheese and Related Dry-salted Cheese Varieties
cheese S/M ratio (Samal, personal observation). The more uniform the ratio of surface area to curd mass after milling, the more uniform will be the rate of salt diffusion into the milled curd particles and more consistent will be the amount of salt retained. It is worth noting that these conditions are more closely satisfied if the curd is not cheddared but is kept in the granular state prior to salting. Milling has little role in granular curd cheesemaking; see 'Stirred curd or granular cheese'.
79
Salt (and more specifically S/M) plays a number of roles in the quality of Cheddar cheese by controlling: (a) the final pH of the cheese (Thomas and Pearce, 1981; Lawrence and Gilles, 1982), (b) the growth of microorganisms, specifically starter bacteria and undesirable species such as coliforms, staphylococci and clostridia, and (c) the overall flavour and texture of the cheese. The S/M level controls the rate of proteolysis of the caseins by the rennet, plasmin and bacterial proteases. Proteolysis, and thus the incidence of bitterness and other off-flavours, decreases with an increase in salt concentration (Thomas and Pearce, 1981; Pearce, 1982). At S/M levels >5.0%, bitter flavours are rarely encountered (Lawrence and Gilles, 1969); below this level there is more or less an inverse linear relationship between S/M and the incidence of bitterness. General aspects of salt in cheese are considered in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1; some specific aspects in relation to Cheddar are considered below.
thereby released to dissolve more salt. The proportion of moisture in the curd and the amount of salt added both affect the rate of solution of the salt. The high salt content of the surface of the milled curd particles reduces the tendency of the particles to fuse together. The difference between dry-salting and brine-salting is, in effect, the availability of water at the surface of the curd. With brine-salting, salt absorption begins immediately; release of whey occurs, as in dry-salting, but is not a pre-requisite for salt absorption. In modern cheese plants, it is essential that the curd particles prior to salting are consistent from day to day with respect to moisture content, particle size and shape, acidity level and temperature, and that the application of salt is uniform. This gives the cheesemaker control over both the mean salt content and, equally important, variations (standard deviation) within a day's manufacture. Cheese specifications normally require both moisture and S/M to be within specified ranges. This means that in practice variations in the moisture content of the curd prior to salting must not be greater than ___1%. It has been suggested (Sutherland, 1974; Gilbert, 1979) that the size of the salt crystals used is important for both salt uptake and moisture control. In practice, however, the major requirement in mechanized cheese plants is that the size range of the salt crystals should be narrow. If the range is variable, the delivery of salt from the equipment is erratic. The presence of large amounts of very fine crystals also results in excessive salt dust within the plant environment. Although salt promotes syneresis, it should not be used in mechanized Cheddar cheesemaking as a means of making a significant adjustment to the moisture content of the curd. However, in practice, because of variations in milk buffering capacity, starter activity and plant breakdowns, day-to-day variations in the curd pH and moisture are not uncommon. Therefore, slight adjustments are made to the quantity of added salt to attain consistency in the salt and moisture contents in the curd. The salting techniques commonly used in mechanized cheesemaking are: boom-salting, in one or two stages, and trommel salting. The former uses salt addition to the curd on a mass/volume ratio whereas the latter uses a mass/mass ratio. More details on salting systems are included in 'General Aspects of Cheese Technology', Volume 2.
Salting of milled curd The salt crystals dissolve on the moist surfaces of the milled curd particles and form a brine. This diffuses into the curd matrix through the aqueous phase, causing the curd to shrink in volume, and more whey is
Mellowing after salting Sufficient time must be allowed after salting (the mellowing time) to ensure the required absorption of salt on the curd surface and continued free drainage of whey. It was suggested earlier that the curd could be
Mellowing prior to salting In the traditional procedure for Cheddar cheese manufacture, the milled 'chips' were left until the newly cut surfaces glistened as a mixture of whey and fat exuded from them. The mellowing period provided time to produce sufficient surface moisture to dissolve the salt crystals when they were applied and gave rise to better salt retention. The purpose of the traditional mellowing period ('dwell time') was to allow for further moisture release and acidity increase. In modern mechanized Cheddar cheese plants, salt is added to the curd pieces immediately after milling, and continuous agitation of the milled particles is used to encourage whey flow and salt absorption. Effect of salting
80
Cheddar Cheese and Related Dry-salted Cheese Varieties
hooped as soon as it had been salted. However, this led to problems in cheese made by these shorter processes (Czulak, 1963), specifically to the entrapment of whey and consequently to excessive moisture and uneven colour in the cheese. As a result, a number of investigations have been carried out to determine the factors that influence the amount of salt absorbed and the speed of its absorption (Breene et al., 1965" Sutherland, 1974; Gilles, 1976; Gilbert, 1979). The amount of salt absorbed by the curd and the rate of subsequent whey drainage are related to the availability of dissolved salt on the curd particle surfaces, and to the physical characteristics of the curd, e.g., fat-free curd allows faster diffusion (Sutherland, 1977). Even when a mellowing time of more than 30 min is maintained and the level of salt addition is uniform, large variations may still occur in the salt content of cheeses because other conditions that affect salt absorption are not controlled. For instance, the curd temperature, the depth of curd, the extent of stirring after salt addition and the degree of structure development in the curd are also significant factors in the control of salt absorption and subsequent whey drainage (Sutherland, 1974; Gilles, 1976). Therefore, it is not surprising that there have been conflicting reports as to how long the mellowing time after salting should be. It is clear that holding for at least 15 rain is necessary to minimize the loss of salt during pressing (Rreene at a l 1065"1 Other renort.~ ,~lltJ~e.qt that the pressing of the salted curd should be delayed for at least 30 min (Gilles, 1976) and preferably for 45-60 min (Breene et al., 1965). Some loss of salt occurs even when the mellowing time is extended to 60 min. However, an increase in the mellowing time substantially reduces the proportion of whey expelled during pressing and greatly improves the degree of salt absorption (Sutherland, 1974). Mechanized cheese plants nowadays have a mellowing time of 20-40 min, which is usually adequate for satisfactory salt uptake and whey removal (C.G. Honor~ and P.K. Samal, unpublished results). The irregular effect of curd temperature on the extent of salt absorption was thought (Breene et al., 1965) to be caused by a protective layer of fat exuding from the surface of curd particles. Less fat was present on curd surfaces at 26 ~ than at 32 ~ Above 38 ~ such fat was melted and dispersed in the brine solution that was present on the surface. In general, however, a decrease in the curd temperature at salting increases the S/M of the final cheese (Sutherland, 1974). Curd salted at a high pH retains more salt (Dolby, 1941) and is more plastic than curd salted at a low pH. Similarly, for a given salting level, the S/M is high when the titratable acidity is low (Gilles, 1976). x
. . . . . .
,
.
o
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-
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Salting the curd under the most favourable conditions for salt absorption reduces the proportion of salt required and reduces salt losses (Gilles, 1976) and also helps to overcome the defect of seaminess (Czulak, 1963; Czulak et al., 1964). Equilibration of salt within a cheese
The rate of penetration of salt into cheese curd is very slow (McDowall and Dolby, 1936" Guerts et al., 1974; Sutherland, 1977; Morris et al., 1985) and a mean diffusion rate of 0.126 cm2/day for salt in the water of Cheddar cheese has been reported (Sutherland, 1977). This corresponds well with salt migration values for Gouda cheese of the same moisture content (Guerts et al., 1974), suggesting that the matrix structures of the two cheese types are similar. Despite the low rate of salt diffusion, it was nevertheless believed that the S/M concentration in Cheddar cheese was essentially uniform within a few days (McDowall and Dolby, 1936). Reports (Morris, 1961" Fox, 1974; Sutherland, 1977; Thomas and Pearce, 1981) suggested that wide variations occurred in salt content between blocks from the same vat and even within a block. Also, there was appreciable variation in the salt and moisture contents of small plug samples taken from different cheeses from the same vat (Sutherland, 1977; Thomas and Pearce, 1981). With increased plant mechanization and automation, better designed cheese vats and improved salting devices, the S/M variation within a block of cheese and between blocks of cheese from the same vat is reasonably well controlled. As the consistency of cheese flavour is directly related to the extent of variability in S/M, the need to produce a curd mass consisting of particles of uniform cross-section at the time of salting cannot be over-emphasized (Fig. 7).
Acidity development Curd particle Curd acidity/pH cross-section at salting at salting
Moisture
,,'
content of curd
. . . . . 1~> Salt uptake <~
Salt-in-moisture
Salting rate
I
The main factors that affect the salt uptake and S/M level in Cheddar cheese.
Cheddar Cheese and Related Dry-salted Cheese Varieties
Seaminess and fusion
When curd particles are dry-salted, discrete boundaries are set up between the individual particles, in contrast to brine-salted cheeses where there is only one boundary, i.e., the cheese rind or exterior. The addition of dry salt causes shrinkage of the curd and a rapid rate of release of whey containing calcium and phosphate, particularly in the first few minutes of pressing. It has been suggested that the salted surface of the curd particle acts as a selective permeable membrane, thereby concentrating calcium and phosphate at the surface of the curd particle (McDowall and Dolby, 1936). It is possible that this calcium gradient is also accentuated, under some circumstances, by the variations in pH between the surface and the interior of the salted curd particle owing to inhibition of starter activity by the high salt concentration at each curd boundary. The establishment of a pH gradient leads, in turn, to a shallow calcium gradient (Le Graet et al., 1983), the magnitude of which will depend on the size of the curd particle and the proportion of salt added. In its most extreme form, the deposition of calcium phosphate crystals results in the phenomenon of seaminess in Cheddar cheese (Czulak, 1963; Conochie and Sutherland, 1965a; A1-Dahhan and Crawford, 1982), a condition in which the junctions of the milled curd particles are visible after pressing. Seaminess is more frequent and more marked with cheese of low moisture and high salt content and in some cases persists after the cheese has matured (Czulak et al., 1964). The binding between curd particles is usually weak, due to incomplete fusion. This often leads to crumbling when the cheese is sliced or cut into small blocks for packing. Photomicrographs show that, in both seamy and non-seamy Cheddar cheese, crystals of calcium orthophosphate dihydrate are dispersed throughout the cheese mass (Conochie and Sutherland, 1965a), but in seamy cheese they are concentrated in the vicinity of the surfaces of the milled curd particles to which salt was applied. To a depth of about 20 b~m below these surfaces, the protein appears to be denser than elsewhere, suggesting that severe dehydration of the surface occurs on contact with dry salt. The observation (Van Slyke and Price, 1952; Czulak, 1963) that seaminess is reduced by washing the curd after milling and before salting can be explained by the removal of calcium and/or phosphate from the surface layer. In addition, the provision of more water will lessen the dehydrating and contracting effect of salt on the surface layer. Seaminess and poor bonding between the curd particles occur together and treatment with warm water corrects both defects. Poor fusion of the curd as a con-
81
sequence of heavy salting results from changes in the protein at the surface, from poor contact between the hardened surfaces, from the physical separation brought about by the presence of salt crystals and, when these have disappeared, from the growth of the calcium ortho-phosphate crystals (Conochie and Sutherland, 1965a). Fusion of the particles is improved by an increase in the pH, temperature or moisture content of the curd. Effect of pressing
Traditionally, Cheddar cheese was pressed overnight using a batch method. The development of the 'blockformer' system (Wegner, 1979; Brockwell, 1981; Tamime and Law, 2001; 'General Aspects of Cheese Technology', Volume 2) offered two major advantages for modern cheesemaking plants: firstly it is a continuous process and secondly the residence time is reduced to about 30-45 min. The curd is fed continuously into an extended hoop (tower) under a partial vacuum, and mechanical pressure is applied at the base of the tower for a very short period, usually for about 1 min. In traditionally made Cheddar, the two common types of textural defect are mechanical- and slitopenness. Mechanical-openness (occurrence of irregularly shaped holes) is evident in very young cheese but decreases markedly during the first or second week after manufacture and changes little thereafter (Czulak et al., 1962; Irvine and Burnett, 1962; Price et al., 1963). However, in Cheddar cheese blocks from block-formers, mechanical-openness is barely visible immediately after manufacture and becomes prominent at about 4 weeks after manufacture (Samal, personal observation). Slitopenness is usually absent in freshly made cheese (Robertson, 1965a) but develops during maturation (Hoglund et al., 1972a). The extreme expression of this defect, known as fractured texture, is found only in mature cheese. A comprehensive survey of commercial Cheddar cheese on the United Kingdom market carried out during 1958-1961 showed that mechanically open cheese was usually almost free of fractures and conversely that badly fractured cheese usually had few mechanical openings (Robertson, 1965b). The term 'fracture' is normally used to describe long slits, i.e., slits longer than about 3.5 cm. As a result of the growth of the cheese pre-packaging trade, the importance attached to fractures in cheese has greatly increased because fractures can result in the break-up of cheese during prepackaging. The basic mechanisms for the formation of openness in Cheddar cheese depend firstly on mechanical-openness, i.e., microscopic nuclei or larger air spaces in the cheese structure, and secondly on gas production by microorganisms (Martley and Crow, 1996).
82
Cheddar Cheese and Related Dry-salted Cheese Varieties
From the observation (Walter etal., 1953) that cheese hooped under whey had a completely close texture and from their own studies of curd behaviour, Czulak and Hammond (1956) concluded that air entrapped during compression of curd was responsible for mechanically open texture. They considered that during compression of the salted, granular curd, the spaces between the granules diminish until they form a complex of narrow channels filled with air. Under further pressure, some of the air is forced out, the escape of the remainder being blocked by closure of the channels at various points and the high surface tension developed by traces of whey in the remaining narrowed outlets. The isolated pockets of trapped air form numerous small irregular holes in the cheese. Conventionally-made Cheddar cheese has a significantly closer texture than Granular cheese. The effect of cheddaring on texture appears to be due to the presence of milled strips of curd (fingers) compared with the relatively small granules of uncheddared curd present in Granular cheese. The larger the fingers of curd, the fewer are the pockets of trapped air and the closer is the texture of the cheese. As mentioned previously, however, there is a practical limit to the size of milled curd because large curd fingers may result in inadequate whey drainage after salting. During the last 50 years, there has been a marked reduction in the incidence of both texture defects by: (,-,'~
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,,c,.
,-,f
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(Whitehead and Jones, 1946)" (b) the change from the manufacture of 36 kg rinded cheese to smaller 20 kg rindless cheese" (c) the introduction of vacuum pressing; (d) the use of defined single-strain cultures from which gas-producing strains have been omitted. The beneficial effects of these modifications are undoubtedly associated with a reduction in the gas content of the cheese. The production of carbon dioxide during ripening by non-starter bacteria has been associated with the development of slit-openness (Hoglund et al., 1972a) but gas production is considered to be of secondary importance compared with manufacturing conditions (Hoglund et al., 1972b). It is the relatively insoluble and biologically inactive nitrogen in the entrapped air that contributes to the ultimate openness of the cheese because the oxygen is rapidly metabolized during ripening.
Vacuum pressing It was a logical step to prevent the entrapment of air between the curd particles by pressing the curd under vacuum, a procedure first patented in Canada by Smith etal. (1959). A moderately high vacuum, approximately 33 kPa pressure, is required. Vacuumtreated cheese is free, or almost so, of mechanical-
openness when 2 weeks old and remains free throughout maturation (Robertson, 1965a). There was some disagreement among the various research groups as to the optimum conditions for vacuum pressing (Robertson, 1965b). Initially, the cheddared and salted curd was pre-pressed under vacuum for 30 min before d r e s s i n g - or trimming, followed by normal pressing (Czulak et al., 1962). Later work suggested that pressures greater than 180 kPa appeared to be required during and after vacuum pressing to achieve a close texture (Robertson, 1965b). An important development in Australia was the hooping of granular, salted curd and pressing under vacuum (Czulak, 1962). It was found that the use of vacuum pressing ensured the characteristic close texture of Cheddar cheese and thus eliminated the need for cheddaring. This observation was particularly significant for the complete mechanization of Cheddar cheese manufacture. Trials in New Zealand (Robertson, 1965a) quickly confirmed the Australian conclusions. Maximum reduction in openness was achieved with the combined use of vacuum pressing of granular curd and a homofermentative starter (Hoglund etal., 1972a). Presumably, air can be removed more readily by vacuum from granular curd than from the closer textured cheddared curd. The technique used in the 'block-former' system of filling hoops under a partial vacuum is particularly effective in achieving a close tL1~.x~ o v t ,Ll ~rto-
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Mechanical-openness is rarely found in cheese made by the 'block-former' system, if the recommended operating procedures are practised in the blockformer, e.g., filling time, residence time, pressing time and appropriate vacuum. However, slit-openness does develop if gas-producing organisms are present. A factor that formerly restricted the size of Cheddar cheese blocks was the tendency for large cheeses to show severe mechanical-openness. With the aid of vacuum pressing, it has been found quite practicable to form curd into very large blocks (Robertson, 1967) which by extrusion into cutting equipment can be subdivided into 20 kg blocks.
Rapid cooling An unwelcome side effect of large blocks is that a temperature gradient is set up within the block because of the relatively slow cooling of the block interior. This, and the demands of the marketplace for evenly ripened blocks of cheese, has necessitated the introduction of rapid cooling of blocks by placing them in open stacks in well-ventilated areas of the cool room or in especially designed cooling devices. Generally, the core temperature of each block is cooled to below 18 ~ in 24 h to keep the growth of the non-starter
Cheddar Cheese and Related Dry-salted Cheese Varieties
lactic acid bacteria to a minimum (Fryer, 1982). Further details on the rapid cooling process and equipment are provided in 'General Aspects of Cheese Technology', Volume 2.
Developments in cheese marketing, coupled with increasing consumer standards, have resulted in a demand for cheese of greater uniformity of composition than in the past. Such uniformity is best achieved by a grading system based on compositional analysis, because the relationship between the composition and the quality of Cheddar cheese is now well established (Robertson, 1966; Lyall, 1968; O'Connor, 1971; Gilles and Lawrence, 1973; Fox, 1975; Pearce and Gilles, 1979). Lyall (1968) briefly reported on a procedure for evaluating chemical analyses of cheese, points being assigned on the basis of composition. However, the only scheme in commercial use for assessing Cheddar cheese quality by compositional analysis appears to be that proposed by Gilles and Lawrence (1973). Suggested ranges of MNFS, S/M, fat-in-dry matter (FDM) and pH for both first and second grade cheeses are given in Fig. 8. All New Zealand export Cheddar cheese is subject to compositional grading to ensure that it meets the appropriate specification. In addition, a sensory flavour assessment is carried out to ensure that the cheese is free from flavour defects (Lawrence and Gilles, 1980). Burton (1989) concluded that grad-
83
ing on the basis of composition may be a satisfactory method for deciding which cheese should be allowed to mature for the British market and which should be sold more quickly. Any grading system based on compositional analysis will be relatively complex because a further factor, the rate and extent of acid production at the vat stage, must also be considered (Lawrence etal., 1984; Lawrence and Gilles, 1986). The point in the process at which the curd is drained from the whey is the key stage in the manufacture of Cheddar cheese because it controls to a large extent its mineral content, the amount of residual chymosin in the cheese, the final pH and the moisture/casein ratio (Lawrence et al., 1984). All these factors influence the rate of proteolysis in the cheese. A relationship has also been found between the calcium content of the cheese, the concentration of residual chymosin and protein breakdown during ripening (Lawrence et al., 1983) and between the rate of acid development in the early stages of manufacture and proteolysis in the cheese (O'Keeffe et al., 1975). The calcium level is therefore an index of the extent of acid production up to the draining stage and also offers a rough indication of the rate of proteolysis that is likely to occur during ripening. Significant differences in the calcium content of Cheddar cheese would suggest differences in the proportions of residual chymosin in the cheese and thus differences in the rate of proteolysis and the development of flavour. However, variations in calcium content have a much smaller effect on Cheddar cheese quality than MNFS, S/M and pH. It is important to recognize that these three parameters are interrelated (Lawrence and Gilles, 1986) and must be controlled as a group to ensure first-grade cheese. Nevertheless, the effect of each of these factors will, as far as possible, be examined separately.
Effect of MNFS
Suggested ranges of salt-in-moisture (S/M), moisture in the non-fat substance (MNFS), fat-in-dry matter (FDM) and pH for first grade (shaded) and second grade Cheddar cheese. Analyses 14 days after manufacture.
There is considerable circumstantial evidence that the main factor in the production of the characteristic flavour of hard and semi-hard cheese varieties is the breakdown of casein. This is supported by the finding that the ratios of moisture to casein and of salt to moisture are critical factors in cheese quality (Gilles and Lawrence, 1973; Lawrence and Gilles, 1986) because both parameters affect the rate of proteolysis in cheese (Thomas and Pearce, 1981). Traditionally, cheesemakers describe cheese in terms of its absolute moisture content but the ratio of moisture to casein is much more important because it is the relative hydration of the casein in the cheese that influences the
84
Cheddar Cheese and Related Dry-salted Cheese Varieties
course of the ripening process (Lawrence and Gilles, 1980). However, it is difficult to measure the casein content of cheese accurately and most commercial plants analyse for only fat and moisture. Therefore, a practical compromise is to determine the ratio of moisture to non-fat substance rather than measure the moisture/casein ratio. The non-fat substance is not the same as the casein in the cheese but is equal to the moisture plus the solids-not-fat. Approximately 85% of the solids-not-fat consist of casein. Therefore, there is a relationship between the moisture/casein ratio and the MNFS. The level of MNFS in cheese gives a much better indication of potential cheese quality than the moisture content of the cheese in the same way as the S/M ratio is a more reliable guide to potential cheese quality than is the salt content of the cheese per se (Lawrence and Gilles, 1980). In large mechanized cheese plants, a significant relationship exists between the FDM and MNFS values for a cheese (Lawrence and Gilles, 1986), probably as a result of the relative inflexibility of the procedures available for the control of moisture. This is of commercial interest because changing the FDM is an effective way of controlling the MNFS in the cheese as the composition of the milk changes throughout the season. The actual MNFS percentage for which a cheesemaker should aim depends on the storage temperature used and
a key factor in determining the pH of dry-salted cheese (Fig. 9). However, the salting pH/titratable acidity is to a large extent controlled, in turn, by the pH/titratable acidity developed at draining (Lawrence and Gilles, 1982). The potential for a further decrease in pH after salting depends upon the residual lactose in the curd and its buffering capacity. The residual lactose will be determined by the rate at which an inhibitory level of NaC1 is absorbed by the cheese curd and the salt tolerance of the starter strains used. The buffering capacity is largely determined by the concentrations of protein and phosphate present, and to a much lesser extent by ions such as calcium. The concentrations of phosphate and calcium retained in the cheese are influenced mainly by the rate of acidification prior to the separation of the whey from the curd. The buffering capacity is also influenced by seasonal, regional and lactational factors. Given reliable starter activity at the vat stage, the actual pH reached in dry-salted cheeses is determined by the S/M value because this controls the extent of starter activity after salting, the rate of lactose utilization in the salted curd and thus the pH reached. An S/M concentration of 6% will inhibit the activity of all Lactococcus lactis subsp, crernoris strains, the starter organisms of choice for Cheddar manufacture (Lawrence and Heap, 1986). The proportion of residual lactose that remains unmetabolized in such
Experience has shown that if C h e d d a r cheese is to be stored at 10 ~ and the cheese is to be consumed after 6-7 months, then the MNFS of the cheese should be about 53%. The higher the MNFS percentage, the faster is the rate of breakdown. Thus, if one anticipates that the cheese will be consumed after 3-4 months, the MNFS percentage can be increased to about 56%. However, the higher the MNFS, the more rapidly Cheddar cheese will deteriorate in quality after reaching its optimum. The same is true for a Cheddar cheese with a relatively low S/M, i.e., less than 4%, or with a high acid content. Such cheeses tend to develop gas and sulphide-type off-flavours after they have reached maturity.
Thomas, 1980). However, in a cheese with S/M of 4.5%, the starter will not be inhibited completely and the lactose will be metabolized rapidly. This explains why the pH of 1-day Cheddar cheese may range from 5.3 (which is about the pH of the curd at salting) to pH 4.9. In general, the higher the pH, the greater
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Casein and mineral content of curd
Effect of pH
Every cheese variety has a characteristic pH range (Lawrence et al., 1984), within which the quality of the cheese is dependent upon both its composition and the way in which it is manufactured (Lawrence et al., 1983). The pH value is important in that it provides an indication of the extent of acid production throughout the cheesemaking process. In normal manufacture, the curd pH/titratable acidity at salting is
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Curd acidity/pH at draining
,,,. . . . . . . . . , ",[ . . . . . . . .
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---~
activity
Residual lactose in salted curd The main factors that d e t e r m i n e the pH of Cheddar
cheese.
Cheddar Cheese and Related Dry-salted Cheese Varieties
is the amount of lactose left unmetabolized. Under normal circumstances, this residual lactose does not affect the quality of Cheddar cheese at maturity (Gilles and Lawrence, unpublished results). The importance of measuring the pH at day 1 has been generally overlooked in the past, probably because it is relatively difficult to measure the pH of cheese accurately. This has led to a lack of appreciation of the significance of relatively small changes in pH. In addition, a pH value per se is sometimes difficult to interpret unless considered in conjunction with the calcium level in the cheese (Lawrence and Gilles, 1980), as well as the pH/titratable acidity at draining. Effect of S/M
The main factors that determine the S/M percentage of Cheddar cheese are summarized in Fig. 7. In young Cheddar cheese, the S/M ratio is the major influence controlling water activity. This in turn determines the rate of bacterial growth and enzyme activity in the cheese, specifically the proteolytic activity of chymosin (Fox and Walley, 1971; Pearce, 1982; Fox, 1987), plasmin (Richardson and Pearce, 1981) and starter proteinases (Martley and Lawrence, 1972). If the S/M value is low (<4.5%), the starter numbers will reach a high level in the cheese and the chance of off-flavours due to the starter bacteria is greatly increased (Lowrie and Lawrence, 1972; Breheny et al., 1975). For this reason, cheesemakers normally aim for an S/M value in Cheddar cheese between 4.5 and 5.5% (Lawrence and Gilles, 1980, 1982). Within this S/M range, the rate of metabolism of the lactose is controlled by a second factor, the temperature of the cheese during the first few days of ripening, because this controls the rate of growth of non-starter bacteria such as lactobacilli and pediococci (Fryer, 1982). Although nonstarter bacteria grow on energy sources other than lactose in cheese, undoubtedly the presence of lactose encourages their rapid growth. This tends to result in a more heterolactic metabolism of lactose, usually with the production of acetate, ethanol and carbon dioxide, and may lead to flavour and textural defects. Clearly, the initial number of non-starter bacteria in the salted curd should be controlled by hygiene during manufacture. Thereafter, their rate of growth, particularly after the first few days of ripening, should be kept to a minimum and this is largely controlled by the temperature of the cheese (Fryer, 1982). For this reason, large mechanized Cheddar cheese plants in New Zealand and Australia have incorporated rapid cooling systems, which reduce the core temperature of the 20 kg cheese blocks to less than 18 ~ within 24 h of manufacture.
85
Compositional ranges were introduced into the grading system to reduce variability within a day's manufacture, especially with respect to S/M. This measure has helped to reduce the previous variability. The rate of ripening will differ but all of the cheese is likely to be acceptable as long as its composition is within the required compositional range. For instance, variations in the moisture content and acidity of the curd before salting, in the accuracy of salt delivery by salting equipment and in the dimensions and structure of the milled curd will all result in considerable variation in salt uptake (Lawrence and Gilles, 1982). Despite improved understanding of salt diffusion (Baldwin and Wiles, 1996; Wiles and Baldwin, 1996) in large mechanized Cheddar cheese plants, as well as new improved ideas and equipment for salting cheese curds, inherent variation of S/M still exists. The effect of manufacturing Cheddar cheese with reduced sodium and S/M levels, on cheese quality, is discussed in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. Effect of FDM
The FDM in Cheddar cheese is less important than MNFS, S/M or pH, in that it normally influences cheese quality only indirectly through its effect on MNFS (Whitehead, 1948). Nevertheless, the FDM has more relevance to the cheesemaker than the fat content per se because moisture is volatile and legal limits for fat are usually specified in terms of FDM. Use of FDM has the further advantage that it can be controlled directly by altering the casein/fat ratio of the milk.
Most consumers of Cheddar cheese consider texture and flavour to be its most important attributes (McEwan et al., 1989; Jack et al., 1993). On the other hand, Cheddar purchased for repackaging also needs to withstand cutting, slicing and moulding. Hence the rheological properties are important (Gunasekaran and Ak, 2003). Cheddar destined to be used as a functional ingredient has the rather different requirements to give particular textural (and flavour) characteristics to the final product ('Rheology and Texture of Cheese', Volume 1). The desirable textural characteristics of a Cheddar cheese are different for different consumers, and this usually involves personal assessments of breakdown in the mouth, evenness of dissolution (melting), amount of chewing required, gritty remnants, etc. Traditionally, the textural properties of cheese sold for immediate
86
Cheddar Cheese and Related Dry-salted Cheese Varieties
human consumption are determined by trained graders. For texture research, such assessments are often made by consumer panels, and there have been many studies using either rheological parameters or the results from specific texture-measuring devices. Such studies have shown that perceived texture correlates moderately well with the indices investigated and developed by Szczesniak (1968, 1987) and discussed by Fox et al. (2000) and Gunasekaran and Ak (2003), and less well with the traditional rheological measures (Breuil and Meullenet, 2001). Nevertheless, there is still no clear method for discerning instrumentally which blocks of Cheddar cheese have acceptable textural properties. The complex interrelationships between the parameters that affect cheese texture make it almost impossible to design simple experiments in which the effect of a single parameter, such as fat content, can be examined in isolation. The wide-ranging experiments carried out by the New Zealand group in the 1970s and 1980s laid the basis for a good understanding of the factors underlying the production of Cheddar cheese of appropriate texture and flavour throughout the maturation cycle (Lawrence et al., 1993). Nevertheless, some recent studies demonstrate that fat content (Fenelon and Guinee, 2000) and pH (Pastorino et al., 2003) can affect the rheological properties of Cheddar cheese. However, by using modern statistical approaches, it is now possible to segregate the effects of several parameters, although each experiment needs to be very large and use many cheese samples (C.J. Coker, T.M. Dodds, S.P Gregory, K.A. Johnston and L.K. Creamer, unpublished results, 2000). Cheddar cheese has a texture that is intermediate (Fig. 10) between those of the relatively high pH cheeses, which flow readily when a force is applied, and the low pH cheeses which tend to deform, by shattering, only at their yield point. Scanning electron
microscopy has established that cheese consists of a continuous protein matrix but that this matrix is clearly different in the various cheese types (Hall and Creamer, 1972). The structural units in the protein matrix of Gouda are essentially in the same globular form (10-15 nm in diameter) as in the original milk. In contrast, the protein aggregates in Cheshire are much smaller (3-4 nm) and are apparently in the form of strands or chains, i.e., the original sub-micellar protein aggregates appear to have lost much of their identity. Cheddar is intermediate between Gouda and Cheshire, i.e., much of the protein in Cheddar is in the form of smaller particles than in Gouda (Fig. 10). As the pH decreases towards that of the isoelectric point of para-casein (approximately 4.5), the protein assumes an increasingly compact conformation and the cheese becomes shorter in texture and fractures at a smaller deformation (Creamer and Olson, 1982; Walstra and van Vliet, 1982). The texture of Cheddar cheese has a wider range of consumer acceptability than the texture of other varieties as a consequence of the intermediate position of Cheddar in the cheese spectrum. The high moisture and relatively high pH (5.2) of American Cheddar resulted traditionally in a more cohesive and waxy texture (Kosikowski and Mistry, 1997) than that of traditional English and New Zealand Cheddar. In North America, a relatively low 1.... 1 ,,r ~,.~A ..... developed ~,, ,~,. . . . . ~ up ,,- ,~,o ~1,_ ing stage (less than 0.65% titratable acidity). In contrast, English cheesemakers strove for a high salting acidity (about 0.85%) with consequently a low final pH (about 4.9). New Zealand cheesemakers aimed for a final pH of 5.0 and a moisture content of about 35%, in contrast to the 38-39% moisture level found in both American and English Cheddar. In recent times, however, most Cheddar-producing countries have tended
Diagrammatic representation of the effect of the pH on the microstructure and texture of cheese.
Cheddar Cheese and Related Dry-salted Cheese Varieties
towards the American style of 'sweet' Cheddar cheese with a final pH between 5.1 and 5.3 now being common. Effect of pH, calcium and salt
Although the mineral content plays an important role in establishing the characteristic structure (Lawrence et al., 1983, 1984), the texture of Cheddar cheese appears to be more dependent upon pH than on any other factor (Lawrence et al., 1987). For the same calcium content, the texture at 35 days can vary from curdy (pH >5.3) to waxy (pH 5.3-5.1) to mealy (pH <5.1). Trials in New Zealand have shown that, for any given pH value, the concentration of calcium in Cheddar can vary over a range of ___15 mmoles/kg with only a slight effect on the texture (Lawrence et al., 1993), although there is a general tendency for the cheese to become less firm as the calcium content decreases. However, the dominant effect of pH on texture can be modified by other compositional factors, particularly the levels of moisture, salt and calcium. Between pH 5.5 and 5.1, much of the colloidal calcium phosphate and a considerable part of the casein are dissociated from the sub-micelles (Roefs et al., 1985). These changes in the size and characteristics of the sub-micelles significantly increase their ability to absorb water (Tarodo de la Fuente and Alais, 1975; Snoeren et al., 1984; Creamer, 1985; Roefs et al., 1985), casein hydration reaching a m a x i m u m at about pH 5.35. More relevantly, Creamer (1985) found that casein hydration in renneted milk increased greatly in the presence of NaC1 between pH 5.0 and 5.4. Furthermore, at any given pH, the extent of solubilization of the micelles by the NaC1 decreased as the calcium concentration in the solution increased. This finding is in agreement with the effects of calcium in brine on the solubilization of the rind of Gouda-type cheese (Guerts et al., 1972). It also explains the observations that a higher Ca2+/Na + ratio results in a firmer cheese (Walstra and van Vliet, 1982), and that Cheddar cheese made from milk to which calcium has been added has a reduced protein breakdown and is of poorer quality (Babel, 1948; Ernstrom et al., 1958). The high level of calcium in buffalo milk (Rajput et al., 1983) may also account for the difficulty in manufacturing Cheddar cheese from buffalo milk. The extent of proteolysis is low (Neogi and Jude, 1978), presumably because the degree of solubilization of the casein micelles by the NaC1 is reduced. As a result, Cheddar cheese needs to be stored for a long period before its characteristic texture and flavour develop.
87
Therefore, it is not surprising that the texture of Cheddar cheese changes markedly as the pH varies between 5.4 and 4.9. A wide range of casein aggregates is present and differences in the sodium and calcium ion concentrations, as well as the proportion of water to casein, markedly affect the extent of swelling of the sub-micelles (Fig. 10). Salt also has a more direct effect on the texture of Cheddar cheese; excessive salting (i.e., an S/M > ~6%) produces a firm-textured cheese which is drier and ripens at a slow rate (Van Slyke and Price, 1952), whereas under-salting (i.e., an S/M < ~4%) results in a pasty cheese with abnormal ripening and flavour characteristics. Such factors as enzyme activity and the conformation of ors1- and [3-caseins in salt solutions (Fox and Walley, 1971), solubility of protein breakdown products, hydration of the protein network (Guerts et al., 1974) and interactions of calcium with the para-caseinate complex in cheese (Guerts et al., 1972) are all influenced by salt concentration. Effect of protein, fat and moisture
In dry-salted cheeses, water, fat and casein are present in roughly equal proportions by weight, together with small amounts of NaC1 and lactic acid. As protein is considerably more dense than either water or fat, it occupies only about one-sixth of the total volume. Nevertheless, the protein matrix is largely responsible for the rigid form of the cheese. Any modification of the nature or the amount of the protein in the cheese will modify its texture. Thus, reduced-fat Cheddar (17% fat) is considerably more firm and more elastic than full-fat Cheddar (35% fat), even when the level of MNFS in the cheese are the same (Emmons et al., 1980). This difference was explained by the presence in the reduced-fat cheese of about 30% more protein matrix, which must be cut or deformed in texture assessments, but such a large reduction in fat must also affect the texture of the cheese. Fat in cheese exists as physically distinct globules, dispersed in the aqueous protein matrix (Kimber et al., 1974). In general, increasing the fat content results in a slightly softer cheese (Bryant et al., 1995), as does an increase in moisture content, because the protein framework is weakened as the volume fraction of protein molecules decreases. However, relatively large variations in the fat content are necessary before the texture of the cheese is affected significantly (Lawrence and Gilles, 1980). Commercial cheese with a high FDM usually has a high MNFS (Lawrence and Gilles, 1986) and this causes a decrease in firmness. An inverse relationship between the fat content and cheese hardness has been reported (Whitehead, 1948; Baron, 1949; Fenelon and Guinee, 2000).
88
Cheddar Cheese and Related Dry-salted Cheese Varieties
Effect of ripening
Considerable changes in texture occur during ripening as a consequence of proteolysis (Hort and Le Grys, 2000, 2001). The rubbery texture of 'green' cheese changes relatively rapidly as the framework of Otsl-casein molecules is cleaved by the residual coagulant (Creamer and Olson, 1982; Johnston et al., 1994; Watkinson et al., 2001). A group of Cheddar cheeses examined over a period of nearly a year increased in hardness and decreased in elasticity with the age of the cheese, the greatest changes occurring during the first 30 days (Baron, 1949). Watkinson et al. (1997) measured proteolysis of ors1- and [3-caseins, and the strain at fracture (a measure of shortness (Gunasekaran and Ak, 2003)) as a function of ripening time. These results showed that the strain at fracture increased initially, probably as curd fusion continued, and then decreased continuously for the 400 days of the experiment. In part, this latter rheological (or textural) change is caused by the loss of structural elements, but another feature of proteolysis is probably important (Creamer and Olson, 1982): as each peptide bond is cleaved a molecule of water is incorporated into the resulting polypeptides and, in addition, two new ionic groups are generated and each of which will compete for the available water in the system. Thus, the water previously available for solvation of the protein chains becomes tied up by the new ionic groups, making the cheese more firm and less easily deformed. This change, in combination with the loss of an extensive protein network, gives the observed effect. Clearly, the change in texture during ripening depends upon the extent of proteolysis, which, for any individual cheese, is determined by the duration and temperature of maturation. The main factor that influences the rate of proteolysis appears to be S/M (Fox and Walley, 1971; Pearce, 1982; Fox, 1987). A direct relationship between S/M and residual protein was established whereas the correlation between moisture and residual protein was relatively weak. A cheese with a low S/M value has a higher rate of proteolysis and is correspondingly softer in texture than a cheese with a high S/M. The concentrations of residual rennet and plasmin in the cheese, together with the starter and non-starter proteinases present, are the important factors that determine the rate of proteolysis (Lawrence etal., 1983; C.J. Coker, T.M. Dodds, S.P. Gregory, K.A. Johnston and L.K. Creamer, unpublished results, 2000).
Cheese ripening is essentially the slow controlled decomposition of a rennet-induced coagulum of the constituents of milk to produce flavour (taste and aroma)
and textural changes. The final targeted flavour profiles and textures of ripened Cheddar and related dry-salted cheese varieties are variable as defined by different endcustomer requirements and traditional cultural flavour expectations. At the young end of the age range is cheese used solely as a source of intact casein for processed cheese, which has minimal flavour and textural change from the fresh curd. A low coagulant concentration, a low storage temperature, high S/M, short storage time or combinations of these are the main parameters used to achieve this end-use. At the other extreme are the strong flavoured Cheddar cheeses ripened for 12-24 months or more. During ripening, there are many changes and the ripening processes responsible are understood in general terms but many of the details are still being investigated. A vocabulary of sensory attributes has been developed to describe Cheddar (Muir and Hunter, 1992), and has been modified to include five odour, ten flavour and five textural attributes (Muir et al., 1995). Using this vocabulary with an experienced panel in combination with data analysis, the similarities and differences between Cheddar and 13 other hard cheeses popular in the United Kingdom have been described (Muir et al., 1995). The medium and vintage Cheddars stand out in a number of respects. In a similar analysis of 34 different Cheddars, a diversity of flavours was shown (Muir e t a l . , 1997). Cheddars made from raw milk were more intensely flavoured ,,,,,a ~,,,4 ,,t,,~,,,~l n . . . . . . . . . . ., ~,~ farmhouse cheeses It showing wide variations in composition and being associated with atypical flavour and texture. There is a significant correlation between the levels of proteolysis products and the extent of flavour development. Hydrolysis of the casein network, specifically e~sl-casein, by the coagulant appears to be responsible for the initial changes in the coagulum matrix (Creamer and Olson, 1982). The level of chymosin retained in the curd is pH dependent (Lawrence et al., 1983; Creamer et al., 1985). In fresh milk, plasmin, the indigenous alkaline milk proteinase, is associated with the casein micelles but it dissociates at low pH (Richardson and Pearce, 1981" Farkye and Fox, 1990). The activity of plasmin in cheese is reported to be dependent on cooking temperature (Farkye and Fox, 1990) as well as on pH and the salt and moisture contents of the cheese (Richardson and Pearce, 1981" Farkye and Fox, 1990). The role of plasmin in Cheddar cheese flavour has yet to be elucidated but it has been reported that the rate and extent of characteristic flavour development in Cheddar cheese slurries appeared to be related directly only to the degradation of [g-casein (Harper et al., 1971). Therefore, plasmin may well prove to be an enzyme of considerable importance in the development of cheese flavour. . . . . . .
Cheddar Cheese and Related Dry-salted Cheese Varieties
As the original casein network is broken down, ideally a desired balance of flavour and aroma compounds is formed. However, the precise nature of the reactions that produce flavour compounds and the way in which their relative rates are controlled are poorly understood. This has been due firstly to the lack of knowledge of compounds that impart typical flavour to Cheddar cheese, and secondly to the complexity of the cheese microflora as the potential producers of flavour compounds. Any organism that grows in the cheese, whether starter, adventitious non-starter lactic acid bacteria (NSLAB) or adjunct culture and any active enzyme that may be present, such as chymosin or plasmin, will have an influence on the subsequent cheese flavour (Fig. 11). Research in New Zealand has shown that if the growth of starter and NSLAB is limited (Fryer, 1982; Lawrence et al., 1983) and if as little chymosin as possible is used (Lawrence and Gilles, 1971; Lawrence et al., 1972), the flavour that develops in Cheddar cheese is likely to be acceptable to most consumers. This section is an attempt by the present authors to summarize what they consider to be relevant to flavour development in Cheddar. Since the last version of this section (Lawrence et al., 1993), more details have been published; however, the last word on the flavour of Cheddar cheese is still to come. For more
Basic structure for Cheddar (pH and mineral content)
Ripening conditions within cheese (Moisture-in-casein; salt-in-moisture; lactose; temperature)
Residual rennet, plasmin and starter activity
Non-starter activity " " " " " ,,
~ SSSSS
s
89
details on the biochemistry of cheese ripening, refer to 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1. Effect of milk-fat
It is well accepted that Cheddar cheese made from skim milk does not develop a characteristic flavour. Cheese with an FDM greater than 50% developed a typical flavour whereas cheese with an FDM less than 50% did not (Ohren and Tuckey, 1969). In this study, when a series of batches of cheese were made from milk of increasing fat content (from 0 to 4.5%), the quality of the flavour improved as the fat content increased. However, if the fat content was increased above a certain limit, the flavour was not further improved. Substituting vegetable or mineral oil for milk-fat still resulted in a degree of Cheddar flavour (Foda et al., 1974). This suggests that the water-fat interface in cheese is important and that the flavour components are dissolved and retained in the fat. Clearly, although milk proteins and lactose are the most likely sources of many of the flavour precursors in Cheddar cheese, the fat plays an important but not yet defined role; in part, the lack of understanding is due to the more limited fat modifications. The extent of lipolysis has been calculated to vary between 0.5 and 1.6% over time in good quality Cheddar (Perret, 1978). A number of fatty acids, keto acids, methyl ketones, esters and lactones in Cheddar are likely to have been derived from milk-fat; some are at concentrations to impact on flavour, but others contribute only to a background flavour (Urbach, 1995; McSweeney and Sousa, 2000). The residual activity after pasteurization of the indigenous milk lipase and the relatively low lipase/esterase activities of the starter and NSLAB are likely to be important in the hydrolysis of milk-fat to free fatty acids because of their flavour potency. The quality of the milk is probably a factor in excessive lipolysis in off-flavoured Cheddar (Perret, 1978). The catabolism of free fatty acids to other flavour compounds, by implication of their presence, occurs but the mechanisms are ill-defined.
i
Effect of proteolysis Acceptable Cheddar flavour
Off-flavours
The main factors that determine the development of flavour in Cheddar cheese.
As described earlier, the consequence of proteolysis of casein represents the most important biochemical ripening event in Cheddar, causing major texture changes and in addition making important contributions to both aroma and taste (Fox, 1989; Fox and
90
Cheddar Cheese and Related Dry-salted Cheese Varieties
McSweeney, 1996). A further consequence of proteolysis may be the release of flavour components that were previously bound to the protein (McGugan et al., 1979). The products of proteolysis include small- and intermediate-sized peptides and free amino acids and contribute at least to a background flavour (McSweeney and Sousa, 2000), or make a significant contribution to flavour intensity. It has been suggested (McGugan et al., 1979; Aston and Creamer, 1986) that the importance of low levels of such non-volatile compounds as peptides, amino acids and salts has been under-rated in the past. This view is supported by the highly significant correlations found between the levels of proteolysis products and the extent of flavour development (Aston et al., 1983). The level of phosphotungstic acid-soluble amino nitrogen was found to be a reliable indicator of flavour development. Above certain limits, however, the level of peptides results in bitterness. Cheddar cheeses made using temperatureinsensitive starter strains were found to become bitter because large numbers of starter cells contributed excessive levels of proteinases. These released bittertasting peptides from high molecular weight peptides that had been produced mainly as a result of chymosin action (Lowrie and Lawrence, 1972). The subject of bitterness, the single most common defect in Cheddar cheese, has been extensively reviewed (Crawford, 1977; Fox, 1989).
quality of the cheese decreased. Some amino acids such as phenylalanine and the branched amino acids yield Strecker degradation products, which in excess cause unclean flavour defects in Cheddar (Dunn and Lindsay, 1985).
for reactions that produce a range of flavour compounds (McSweeney and Sousa, 2000). Recent studies using gas chromatography-olfactometry and related techniques have identified key aroma components of Cheddar cheese (O'Riordan and Delahunty, 2001; Zehentbauer and Reineccius, 2002). Some of these (dimethyl sulphide, methional, dimethyl trisulphide and 3-methylbutanal) are likely to originate from amino acids (Urbach, 1995). Several reports strongly implicate the volatile sulphur compounds, specifically methanethiol, in Cheddar cheese flavour (Green and Manning, 1982; Lindsay and Rippe, 1986), but an Australian report (Aston and Douglas, 1983) concluded that none of these sulphur compounds is a reliable indicator of flavour development. However, it is conceivable that, although the volatiles do not make a measurable contribution to the intensity of Cheddar flavour, they may still be an essential factor in the quality of the flavour (McGugan et al., 1979). This is supported by the finding (Manning et al., 1983) that the quality of blocks of Cheddar cheese decreased, and off-flavours increased, with a decrease in block size. Headspace analysis showed that the concentrations of HzS and CH3SH, compounds that are extremely susceptible to oxidation, decreased as the
bitterness, which mask or detract from cheese flavour, are produced. A reduction in unpleasant flavour is associated with improved perception of the Cheddar flavour (Lowrie and Lawrence, 1972; Lowrie et al., 1974). The increase in the use of direct vat inoculum (DVI) cultures in Europe for the manufacture of cheese has led to greater usage of Lc. lactis subsp. lactis strains of starter. Because these strains have a greater tendency than Lc. lactis subsp, cremoris strains to produce bitterness in cheese, bitterness is more common with the use of DVI cultures than with bulk cheese starter (Heap, personal observation). During, or soon after, the manufacture of Cheddar curd, the starter viability decreases and is <1% by 3 months (Martley and Lawrence, 1972). The decrease in starter viability is generally an indication of starter autolysis, which is considered to have important consequences for the control of bitterness, as discussed in 'Effect of proteolysis', and for other ripening developments in Cheddar (Wilkinson et al., 1994a,b; Crow et al., 1995). Starter autolysis in Cheddar is influenced by the choice of starter strains (Martley and Lawrence, 1972), and the extent and the rapidity of autolysis are modified by manufacturing conditions, particularly
Role of starter
The absence of any Cheddar flavour in glucono lactoneacidified cheese and the development of typical, balanced Cheddar flavour in starter-only cheese (Reiter et al., 1966) established that Cheddar starter, normally Lactococcus lactis subsp, cremoris as discussed in 'Effect of milk composition and starter culture', has a role in the development of cheese flavour. However, the exact role has been much more difficult to determine. An important indirect role of the starter is considered to be providing a suitable environment that allows the development of characteristic cheese flavour (Lowrie et al., 1974). Starter activity results in the required redox potential, pH and moisture content in the cheese that allows enzyme activity to proceed favourably. In addition, the temperature during manufacture and the S/M must be controlled to ensure that the net metabolic activity of the starter organisms is low (Lawrence et al., 1972; Lowrie et al., 1974) but nevertheless adequate to allow the required pH at day 1 to be reached. Should the starter reach too high a | ~ t l . . . . . . . . . . i.ro L a U ~ V O t L ~ L L U I L U A ~tJtL V L V l , . .
. . . . .
tL~U ~ . . n lLr~,. .~P i,l ~~,
n . . . . . . . LLOtVIJL.~L
rlo~ont
. . . . .
h
~lc
q.at~.L~.~.1.00t.X'~Li CLO
Cheddar Cheese and Related Dry-salted Cheese Varieties
cook temperature, pH and salting, and the composition of the final cheese. A balance between intact and autolysed starter cells is important for good quality Cheddar flavour. Sufficient intact cells are needed for lactose removal and potentially for other physiological reactions such as oxygen removal (Crow etal., 1995). There is indirect evidence that starter autolysis results in higher concentrations of small peptides and free amino acids (Wilkinson et al., 1994a,b). The intracellular peptidases are released by autolysis before they can be very active in cheese as the peptide transport systems are probably inactive in the stressed starter cells (Crow et al., 1993). Role of s t a r t e r e n z y m e s
It is likely that a number of starter enzymes have a direct role in flavour development (Lawrence and Thomas, 1979; Law and Wigmore, 1983; Farkye et al., 1990). The starter proteinase is a cell-associated endopeptidase (lactocepin), which has been studied extensively, biochemically and genetically (Reid and Coolbear, 1998) and makes important contributions to proteolysis in cheese ripening (see reviews, Pritchard and Coolbear, 1993 and Kunji et al., 1996). Proteinasenegative starters have been used in different ratios with normal starter strains to demonstrate that a lower level of this enzyme during Cheddar ripening can reduce the development of bitter flavours (Mills and Thomas, 1980). Using proteinase-negative starters, Lane and Fox (1997) have shown that the absence of starter proteinase during cheese ripening gives rise to decreased levels of small peptides and amino acids, and a poorer quality Cheddar. Therefore, a balance of proteinase activity is often important to Cheddar flavour. There are also different starter proteinases (mainly types I and III), and their specificity differs (Broadbent et al., 1998) and/or their stability in cheese differs (Reid and Coolbear, 1999), factors that contribute to the starters' influence on proteolysis and bitterness. The other proteolytic activities of starters that are important for proteolysis in Cheddar are the peptidases (see reviews, Pritchard and Coolbear, 1993 and Kunji et al., 1996). The early appearance of free amino acids in Cheddar is due mainly to the starter peptidases (O'Keeffe et al., 1976). The mesophilic starters have about 15 peptidases with different specificities (McSweeney and Sousa, 2000); a number have been shown to be intracellularly located and the activities vary between strains (Crow et al., 1994). Although the collective importance of the peptidases in Cheddar proteolysis is reasonably well established, the individual roles of the peptidases are not clear. Genetic modi-
91
fication of the peptidase expression in the starter may clarify their roles (Kok and Venema, 1995). For example, Cheddar flavour was not accelerated using a starter that overproduced the general aminopeptidase, PepN (McGarry et al., 1994). This may not be surprising if the suggestion by Fox and Wallace (1997) is correct, that production of amino acids in cheese ripening is not rate limiting. To date, there is no strong evidence to suggest that the lactococcal starters have a true lipase that is capable of hydrolysing the milk triglycerides. However, they have esterase activity that acts on milk monoand di-glycerides, with the short chain fatty acids being released preferentially (Holland et al., 2002). There is evidence that the starter esterase can also produce short-chain fatty acid esters (Nardi etal., 2002). Starter strains have a range of esterase activity with a significant proportion located on the cell surface (Crow et al., 1994). Both the hydrolysis and ester synthesis reactions of the starter esterase probably play a role in Cheddar flavour but more investigations are needed to define the extent to which this impacts on Cheddar flavour. Role of other starter activities
In the young Cheddar curd, starters ferment the remaining lactose to lactic acid and possibly to other minor fermentation products. Some starters will also ferment citric acid to products including diacetyl, acetate and carbon dioxide. These products can contribute to flavour (Lawrence and Thomas, 1979) and the fermentations are dependent on intact viable cells (Crow et al., 1993). Other ripening reactions by starter that may be important in cheese are the modifications of amino acids and fatty acids (McSweeney and Sousa, 2000). It has been suggested that the enzymatic or chemical modification of amino acids is a rate-limiting factor in cheese ripening (Fox and McSweeney, 1996). Starters have a wide range of abilities to metabolize amino acids. This includes converting arginine to ornithine (Crow and Thomas, 1982), leucine, methionine and phenylalanine to their corresponding aldehydes (MacLeod and Morgan, 1958) and a range of amino acids to their corresponding organic acids (Nakae and Elliot, 1965). Many of the amino acid conversions by starters and other dairy bacteria rely on transamination reactions that are believed to be rate limited by the availability of the ot-ketoglutarate (Tanous et al., 2002). Enhancement of amino acid metabolism increased the aroma (Banks et al., 2001) and the flavour maturity (Shakeel-Ur-Rehman and Fox, 2002) in Cheddar made with added ot-ketoglutarate. Despite all this information, it is still not clear what constitutes a proper balance of amino acid transformations
92
Cheddar Cheese and Related Dry-salted Cheese Varieties
by starter with respect to a balanced Cheddar flavour. In mature commercial Cheddar, the availability of amino acids increases with time when the adventitious microflora are likely to contribute. In addition, at this later stage of ripening in good quality Cheddar, there is normally significant starter autolysis, which could affect the ability and the way the starters metabolize amino acids. It is clear that starters, because of the initial high biomass in young curd and associated ripening enzymes and fermentative abilities, will contribute to Cheddar flavour. In the past and currently, the choice of starter has been dictated more by its importance to commercial curd manufacture (particularly reliable acid production and associated phage resistance) than by its ripening properties, which have been focused mainly on minimizing flavour defects, such as bitterness. With the increasing understanding of Cheddar ripening and the role of the starter, it will be possible to use more lactococcal strains with specialized ripening attributes. Such strains can be used either as starters or as flavour adjuncts if their starter properties are compromised. Role of non-starter lactic acid bacteria
Cheddar contains a heterogeneous adventitious microflora originating from the milk and/or the manufacturing environment (Peterson and Marshall, 1990; Martiey and Crow, i993), in Cheddar, the main microflora identified are mesophilic lactobacilli and occasionally pediococci (Jordan and Cogan, 1993" Crow et al., 2001), commonly referred to as NSLAB. The most common species are Lactobacillus paracasei, Lb. casei, Lb. rhamnosus, Lb. plantarum and Lb. curvatus. Strains of heterofermentative lactobacilli (Lb. brevis and Lb. fermentum) are identified occasionally. The common species vary between countries (Fox et al., 1998) and the species and strains can vary between factories, within a factory and within a block of cheese during ripening (Crow et al., 2002). Cheddar cheese made under controlled bacteriological conditions and containing only starter streptococci develops balanced, typical flavour (Reiter et al., 1966) but it is intriguing that cheeses made in open vats develop such flavour more rapidly (Reiter et al., 1967 Law etal., 1976, 1979). This suggests that NSLAB present as a result of post-pasteurization contamination are beneficial. Nevertheless, there have been reports that conclude that NSLAB have little effect on normal Cheddar cheese flavour development (Law and Sharpe, 1977, 1978). The role of NSLAB in contributing positively to Cheddar cheese flavour has yet to be elucidated (Peterson and Marshall, 1990; Martley and Crow,
1993). In general terms, the numbers and types of dominant NSLAB are important (Crow et al., 2002). These factors are influenced by milk quality, factory hygiene, the rate of cooling of the cheese, the ripening temperature and the cheese composition (Lane et al., 1997; Fox et al., 1998). The inherent variability of the initial NSLAB strains makes consistent control of ripening by NSLAB a challenge. The rate of cooling of the cheese, after pressing the curd, appears to be a significant factor in controlling the cheese flora (Fryer, 1982) and appears to offer the easiest method of controlling cheese flavour (Miah et al., 1974). Recent evidence suggests that selected NSLAB can be used as adjunct cultures to provide an important additional tool in controlling Cheddar flavour (see 'Role of adjuncts'). The NSLAB have a diversity of metabolic and enzyme activities. Different NSLAB strains have a wide range of proteinase (Broome et al., 1991a), peptidase (Broome et al., 1991b) and esterase (Williams and Banks, 1997) activities and types, can catabolise a range of amino acids (Christensen et al., 1999) and can produce esters (Liu et al., 1998). Different NSLAB are likely to grow on different energy sources in cheese (Fox et al., 1998) and influence the redox potential in different ways (Thomas et al., 1985). The balance of all these activities is probably important to good quality Cheddar ripening. The prolonged presence of high numbers of some NSLAB species in Cheddar has been associated with lilJc,titt ~ ucL~+t~ ~u~+l, a~ un-LLavuuL~, ~.t~ atlu ~+ty~tal~ (Crow et al., 2001). Off-flavours can be produced by Lb. brevis and Lb. plantarum (Puchades et al., 1989). Slits have been attributed to heterofermentative lactobacilli (Laleye et al., 1987) and the formation of white spots of calcium lactate pentahydrate crystals has been associated with the racemizing activity of certain NSLAB (Thomas and Crow, 1983; Johnson et al., 1986, 1989; Dybing et al., 1988 Bhowmik et al., 1990). In Cheddar, the total lactate (usually the L(+) isomer) is at a concentration close to crystallizing out. Crystallization of calcium lactate on the surface of Cheddar cheese is a common and troublesome defect (Pearce et al., 1973). A number of lactobacilli isolates and all pediococci isolates can convert the L(+) isomer of lactate to the D(--) isomer in Cheddar such that an equilibrium is eventually reached where there is an equal mixture (a racemic mixture) of both isomers. As the racemic mixture of lactate is more insoluble than the separate isomers, there is a higher possibility of lactate crystallization in Cheddar containing a racemic mixture of lactate. Role of adjuncts
Cheddar-type varieties traditionally have starter cultures as the only dairy microorganisms deliberately added to the milk. Adjuncts, cultures that are added deliberately
Cheddar Cheese and Related Dry-salted Cheese Varieties
for features other than for acid production, and used in some other cheese types (e.g., propionibacteria in Swisstype cheese), have generally not been used for Cheddar. There has been an increasing interest in the use of adjuncts for Cheddar, usually for flavour acceleration but also for flavour consistency or for contributing to unique flavour profiles (Fox et al., 1998). A number of cultures with putative health attributes are also being investigated as adjuncts to produce probiotic cheese, including Cheddar (Ross et al., 2002). Much of the published work has concentrated on the use of NSLAB as adjuncts. Other adjuncts studied include attenuated thermophilic lactobacilli to accelerate ripening (Wilkinson, 1993). Addition of non-attenuated thermophilic lactobacilli, particularly Lb. helveticus, has been shown to accelerate ripening, reduce bitterness and provide a different flavour profile (Fox et al., 1998). Some other adjuncts studied in less detail include smear bacteria, Enterococcus, Pseudomonas and yeast (Crow et al., 2002). There is some commercial use of NSLAB adjuncts, but time and economics will determine if their use is sustained (Crow et al., 2002). Earlier work (Lane and Hammer, 1935; Reiter et al., 1967) has been followed by a recent increased effort in studying their use (Puchades et al., 1989; Broome et al., 1990; McSweeney et al., 1994; Lynch et al., 1996; Muir et al., 1996; Crow et al., 2001). In these studies, the NSLAB adjuncts often improved the flavour intensity. Although the desirable ripening mechanisms for suitable adjuncts are not known, some analysis shows that flavour improvement is associated with an increase in the concentration of amino acids and small peptides and that the volatiles are produced in a different ratio (Fox et al., 1998). Some strains tested produced flavour defects (e.g., Lee et al., 1990). The dynamics of the interactions between adventitious and adjunct NSLAB growing in Cheddar are not fully understood (Fox et al., 1998). For use in New Zealand Cheddar, successful NSLAB adjuncts are carefully selected from good quality cheese; to achieve competition against the range of adventitious NSLAB and to provide a balance of cheese ripening attributes, an adjunct is made up from more than one strain (Crow et al., 2002). Provided that the milk quality and the factory hygiene are high (i.e., a low level of adventitious NSLAB), the adjunct strains can overgrow the adventitious NSLAB and be the main population of NSLAB throughout ripening, thus providing consistency to mature Cheddar flavour development.
There is no one standard for measuring cheese quality. Young Cheddar cheese is judged on the basis of whether it has properties characteristic of its variety.
93
Compositional analysis provides an objective method for detecting atypical cheese and is to be preferred to subjective grading methods. In the case of mature cheese, quality assessment is largely a matter of specific market preference, with consumers in different countries differing considerably in their requirements with respect to cheese flavour. Cheddar cheese flavour requirements is specific to country, ethnicity and endapplication. Whereas sensory profiling of cheese provides a powerful tool for quality assurance and new product development, grading is a highly efficient method of identifying out-of-specification cheese early in the ripening period (Muir, 2002). Grading of cheese encompasses sensory evaluation and functionality tests on the finished product, and is carried out in tandem with chemical and microbiological analyses as part of the manufacturer's quality assurance programme. Grading is carried out as a series of checks during ripening to determine whether or not the manufacturer has achieved what he initially set out to achieve (S.P. Gregory, personal communication). Although the assignment of a grade to a consignment of cheese may be improperly influenced by the sample because differences may exist between blocks of cheese made from the same vat of milk, it has thus far been the most practical way of grading. Flavour defects, such as fruitiness and sulphide off-flavours, have sometimes been located in particular areas within a cheese (Gilles and Lawrence, unpublished results). Such lack of uniform flavour usually results from variations in S/M (Lawrence and Gilles, 1982). Differences between cheeses have also been attributed to an uneven cooling of cheese blocks stacked closely on pallets while the cheese is still warm (Conochie and Sutherland, 1965b). It is therefore possible that a grade score is highly biased if the assessment of a whole vat depends on a single randomly drawn sample (Sutherland, 1977). The texture of Cheddar cheese changes dramatically during the first few days of ripening. The simplest explanation for this observation is that the cheese microstructure consists of an extensive network of otsl-casein and that cleavage by chymosin (or rennet substitute) of just a few peptide bonds of otsl-casein greatly weakens this network (Creamer and Olson, 1982). This results in a relatively large change in the force necessary for deformation. It is differences in this force that a grader attempts to assess when he rubs down a plug of cheese between his thumb and forefinger. From this assessment of the texture, after the cheese has been allowed to ripen for about 30 days, the grader proceeds to predict what the quality of the cheese will be after it has matured (Lawrence et al., 1983).
94
Cheddar Cheese and Related Dry-salted Cheese Varieties
Based on the grade or quality attributes, the cheese is identified as one with potential for long-hold (mature Cheddar), medium-hold or short-hold (mild Cheddar). Bitterness is the most common flavour defect detected in Cheddar cheese. Therefore, the sensory method of prediction traditionally used by graders has some validity because the rate of change in cheese texture during the first few days of ripening is determined by the same factors, i.e., the pH at day 1, the salt/moisture ratio and the moisture/casein ratio, that also influence the quality of the cheese at maturity (Fig. 12). Experience has long shown that a Cheddar cheese with an atypical texture seldom, if ever, develops a characteristic flavour but unfortunately the reverse is not true. A good-textured cheese does not always develop an acceptable flavour, because off-flavours can still be produced if unsuitable manufacturing and ripening procedures are used (Lawrence et al., 1983). Detection of atypical cheese can be achieved more directly and objectively by compositional analysis.
Low-fat Cheddar cheese
Although a relatively minor product, low-fat Cheddar e]~ooco ~s
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i m n n r t ~ n t LILL~/UI tRILL
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tLU~.~L(,~k~ n , , - l , ' J , r ~ c~.~ hl l W o al , ]. ~t h( ~_k el t nJ knl -c- et .i. .nU,Ll lC~ J ~ . ~ l U
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ety, where consumers have become more concerned about the amount of fat in their diet. However, it has been difficult to produce a low-fat Cheddar cheese with the same flavour and texture characteristics as
Acid production at draining
those of a full-fat cheese (Guinee and Law, 2002). The flavour and acceptability at 3 months decrease with decreasing fat content (Banks et al., 1989). Cheddar cheeses containing only 15-30% fat are noticeably more firm and less smooth, when young, than full-fat cheese. The differences in texture, although marked in the early stages of ripening, apparently narrow after the cheese has matured for 1 or 2 months (Olson, 1984a). There has been some success at improving the quality of low-fat Cheddar cheese (Guinee and Law, 2002), but there is still a poor consumer perception of lower fat cheeses, judging by their relatively low consumption. The approaches taken to improve both the flavour and the texture have been reviewed (Guinee and Law, 2002). A novel idea for improving the body and texture of low-fat Cheddar cheese was through the use of sweet ultrafiltered buttermilk (Mistry et al., 1996). However, the differences in texture, seen at 4 weeks, between cheeses made with and without buttermilk, were smaller after 24 weeks. By using a combination of manufacturing changes and novel starter and starter adjuncts, Johnson et al. (1998) claim to have achieved a cheese with more acceptable texture, but with a flavour, although improved, that is not identical to that of Cheddar. Guinee et al. (1999) claim to have developed a halfity by modification of the cheesemaking procedure, including increasing the pasteurization temperature, and using selected starter cultures and bacterial culture adjuncts. The addition of fat mimetics to the milk is
pH change and mineral los
s~
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Proteinase activity (salt-in-moisture moisture-in-casein)
i
/" t____
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st
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An explanation for the general validity of traditional sensory testing of Cheddar cheese.
C h e d d a r Cheese and Related Dry-salted C h e e s e Varieties
also a method that is claimed to improve the quality of reduced fat cheese (Guinee and Law, 2002).
95
style and other washed-curd cheese types using a method similar to that outlined for traditional Colby and Monterey.
Stirred curd or granular cheese
As discussed earlier, Granular cheese preceded, historically, the manufacture of traditional Cheddar cheese. It is made as for normal Cheddar cheese except that the curd fusion or cheddaring step is omitted. Therefore, more acid has to be developed at the vat stage to compensate for the shorter total manufacturing time. Starter systems are available that allow very acceptable Granular cheese to be made. Maintaining curd in the granular form, without the need for milling prior to salting, has obvious attractions. However, there is a tendency for the curd to mat after drying unless it is agitated, and continued stirring may lead to higher fat losses. Moisture expulsion is also faster than during cheddaring. The salted curd particles take some time to fuse together, the rate of bonding depending largely upon the pH of the curds at salting. However, there are advantages in mechanized cheesemaking systems in having the curds in a granular form. The salt readily mixes with the curd, and the salted granules flow and can be hooped easily. Stirred curd cheesemaking is now widely used in the manufacture of 'barrel' (bulk pressed) cheese, although variations in moisture level may occur as a consequence of different temperatures within the block (Olson, 1984b; Reinbold and Ernstrom, 1988). The pressing of granular curd gives rise to opentextured cheese as a result of air being entrapped within the cheese (Czulak and Hammond, 1956). However, this has been overcome by the development and widespread use of methods of pressing the curd under vacuum (Brockwell, 1981; Tamime and Law, 2001). Granular cheese resembles Cheddar cheese in composition but it matures somewhat differently because of the relatively low acidity at which the curds are salted. Curds hooped in the granular form give a texture at 14 days which, although completely close, is just perceptibly different from that of normal Cheddar cheese (Czulak, 1962). However, this difference in texture diminishes as the cheese matures. Washed curd varieties
There has been a substantial increase in recent years in the consumption of washed-curd varieties of Cheddar cheese (Olson, 1981). Varieties such as Colby and Monterey are milder in flavour and have a more plastic texture than Cheddar. They are relatively high-moisture cheeses (39-40%) and ripen rapidly. It has also become popular in New Zealand and Australia to manufacture dry-salted Gouda-style, Edam-
Colby and Monterey The recent improvements in the production of granular Cheddar for processing are also indirectly responsible for the production of Colby and Monterey because these varieties are in fact washed-curd, granular cheeses. Traditionally, whey is drained off until the curds on the bottom of the vat are just breaking the surface and cold water is added to reduce the temperature of the curds/whey to about 27 ~ (Fig. 13). The moisture content of the cheese can be controlled by the temperature of the curds/whey/water mix. The moisture content decreases as the temperature is increased between 26 and 34 ~ The pH of the cheese is determined by the proportions of both whey removed and water added. The length of time the water is in contact with the curd is also important because this determines the level of residual lactose. As salt penetration into the interior of the granular curd particles is rapid, no pH gradient occurs and seaminess is not a problem.
Manufacture as for Cheddar up to running stage
Proportion of whey removed and water added
Contact time
Temperature of curd/whey/water mixture Residual lactose
pH
Moisture content
Typical texture of Colby
The main factors that determine the characteristic texture of Colby cheese.
96
Cheddar Cheese and Related Dry-salted Cheese Varieties
The calcium content of Colby tends to be slightly lower than that of Cheddar because of the higher percentage of starter used and a further small loss of calcium at the washing stage. However, as discussed previously, it is the pH that determines to a large extent the texture of a cheese. The addition of water results in a small increase (about 0.1-0.2 units) in the pH of the finished cheese but this is sufficient to give the cheese a more plastic texture than Cheddar. Recent trials (Creamer et al., 1988) have shown that the calcium content of Colby cheese can vary between 120 and 180 mmoles/kg cheese without influencing significantly the texture of the cheese as long as the pH is greater than about 5.2. The characteristic texture of Colby cheese is thus influenced almost entirely by its pH and moisture level (Fig. 13). Traditionally, Colby had a mechanically open texture but the use of shorttime pressing systems (Wegner, 1979; Brockwell, 1981; Tamime and Law, 2001), in which the curd is transported to be pressed under a partial vacuum, results in a texture that is as close as that of Goudatype cheese varieties. Monterey cheese has many similarities to Colby but is usually softer (Kosikowski and Mistry, 1997).
The editorial assistance of Ms Claire Woodhall and the advice of bar Philip X,u are gratefully acknowledged.
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Gouda and Related Cheeses G. van den Berg, W.C. Meijer, E.-M. DDsterh6ft and G. Smit, NIZO Food Research, Ede, The Netherlands
Gouda and related cheeses are the main representatives of the class of semi-hard cheeses and can be characterised by: (a) the use of fresh pasteurised cow's milk, the milk normally being partly skimmed (generally leading to at least 40% fat in the dry matter of the cheese); (b) milk clotting by means of rennet (usually extracted from calves' stomachs); (c) the use of, preferably, mixed-strain starters consisting of mesophilic lactococci and usually leuconostocs, that generally produce CO2; (d) a water content in the fat-free cheese below 63% (ratio of water to solids-not-fat < 1.70); (e) pressing the cheese to obtain a closed rind; (D acidification mainly in the curd block after separation of the whey during pressing, holding and the first hours of brining; (g) salting after pressing, usually in brine; (h) absence of an essential surface flora; (i) being at least somewhat matured (for 4 weeks) and thus having undergone significant proteolysis. Consequently, the cheese normally has a semi-hard consistency and a smooth texture, usually with small holes; the flavour intensity varies widely. After prolonged natural ripening, the consistency will be hard and short and the formation of amino acid crystals is common. Variation within cheese type and ripening time is considerable: (a) loaf size may be between 0.2 and 20 kg; (b) loaf shape may be a sphere (Edam), a flat cylinder with bulging sides (Gouda), a block, like a loaf of bread, etc.; (c) fat content in the dry matter ranges from 40 to over 50%; (d) water content in the fat-free cheese ranges from 53 to 63%; (e) salt content in the cheese water ranges from 2 to 7%; (f) pH may be anywhere from 4.9 to 5.6 (brittle Edam or old Gouda); (g) maturation may take from 2 weeks to 2 years under drying conditions, although foil-ripening of
block-form cheeses at a rather low temperature is applied now in large volumes. Recently, several companies have begun to produce cheese with a reduced fat content. In order to avoid too tough a consistency and too fiat a flavour, modified technology is used. The introduction of adjunct starters, often of thermophilic nature, to enhance flavour development, has also given more opportunities to achieve the desired texture. Besides calf rennet, the use of fermentation-produced chymosin and some microbial rennets is also practised nowadays in various countries. Generally, a larger loaf is likely to have a lower water content (initially) and is matured for a longer time than a smaller one. To this end, Baby Gouda (0.2-1.0 kg) is manufactured with a higher water content than the larger normal Gouda cheese. Smaller loaves, with a relatively large surface through which more moisture evaporates, are used for shorter ripening under natural conditions. Herbs or spices are sometimes added, particularly cumin (i.e., the seeds of Cuminum cyminum). Traditionally, two main types of cheese were made in The Netherlands: Gouda and Edam. Gouda cheese was made in fairly large loaves of flat cylindrical shape (mostly 4-14 kg) from flesh whole milk and was matured for a variable period (6-60 weeks) under natural conditions; it is still made on some farms from raw milk in much the same way ('Goudse boerenkaas'). Edam, a sphere of 1 or 2 kg, was made from a mixture of skimmed evening milk and flesh morning milk, leading to about 40% fat-in-dry-matter (FDM); the cheese had a somewhat shorter texture than Gouda and was usually matured for 6 months or more. Mimolette ('Commissiekaas'), a high-coloured sphere of 4 kg, is also related to this type. Later, a greater range of cheeses, differing in shape, body and taste, evolved from these types. Most modern types have a somewhat higher pH and moisture content than the cheeses had previously; one reason for this change was to obtain better sliceability of the matured cheese. It is good to be aware of the industrial scale on which production occurs. The past 45 years especially have witnessed drastic changes in the cheese industry. Refrigeration of the milk at the farm (--'4 ~ and collection of this milk every second or third day have
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
104
Gouda and Related Cheeses
become the accepted system in many countries. Rigorous control of the hygienic quality of the milk leads to far smaller variation in composition, thus facilitating the introduction of systems for process control. Cheese factories have been modernised and merged into plants with high capacity. Plants with an annual production of 30 000 tonnes of cheese, manufactured in a 6-day working week, are more or less standard now for Gouda-type cheesemaking. These plants are highly mechanised, automated and computerised, producing cheese of the desired quality at relatively low labour costs but with very costly equipment. Individual plants are often specialised in the manufacture of a single cheese variety. They process about 50 000 1 milk/h divided among three cheese vats (batches). Improved insights into cheese technology gained by much research made these developments possible.
Process principles
The milk for cheesemaking is stored and prepared by standardisation to produce a product of the desired composition. After pasteurisation, the milk is cooled to renneting temperature and starter, rennet and possibly other ingredients are added. The milk coagulates and forms a gel that is cut rather soon in order to be ahle to divide the r l l r d w i t h a l 1 / t a n m i l c h qtreqq on the still-fragile particles. The increased surface of the curd particles and gradually stirring more vigorously facilitates the expulsion of whey (syneresis). After some time, sufficient whey has been liberated and a significant part of this first (non-diluted whey) whey is removed to enhance the forces on the curd grains and to make room for the addition of curd-washing water. This water serves to dilute the whey to reduce the lactose content of the curd. Moreover, this water is heated to increase the temperature of the curds and whey mixture, which enhances syneresis to further reduce the moisture content of the curd. To this end, stirring is also intensified somewhat. This step takes approximately 30 min (>90% of lactose diffusion is reached) before the curd particles are allowed to settle. After 'matting', the whey is drained off at a limited speed, especially after the whey surface reaches the top of the curd mass. Particularly in this stage, inclusion of air between curd particles must be avoided carefully because it impairs later eye formation in the cheese. When the drainage is nearly complete, the curd particles have started to fuse and the bed is compacted, it is possible to cut the mass into blocks. These blocks are transferred to cheese moulds, with a liner, in which the mass still flows for a while to fill the mould entirely.
Then, the moulds with curd are transported to the press, where all cheeses from one batch are pressed at the same time. In the meantime, whey leaks from the blocks of curd, until pressing closes the surface. During pressing, pressure on the cheese surface is increased gradually to approximately 25 kPa in three or four steps. The cheese acquires its desired shape and a thin rind is formed, facilitated by the liner, to protect the loaf against the forces during subsequent process steps and greatly inhibits moisture loss until salting. After demoulding, the loaves are still held for further acidification upside down in a mould, without a liner when possible, for some time, depending on the cheese type. Then, the cheeses are salted, usually by immersion in brine. After brining, the cheeses are dried somewhat and coated, and curing starts. During ripening, the loaves are kept in an air-conditioned room to protect the integrity of the rind, control water evaporation from the cheese and prevent visible microbiological growth on the cheese surface. These essential process steps largely determine cheese composition and the efficiency of production. Important aims are to obtain maximum yield, to control cheese composition (and hence quality), and to keep the process as short as possible, while following a fixed time schedule. The main points to be considered in relation to cheese composition are: final fat and water content, pH of the cheese, quantity of calcium phosphate remaining in the cheese, and the quantity of rennet retained in the curd. To regulate the process, the rate of syneresis is of paramount importance. Several process parameters are important in this respect and have their impact on the water content of the cheese, its MNFS (moisture in the non-fat substances) in particular. An increase in the fat content of the milk, the heat load by pasteurisation and thermisation (see 'Milk quality'), of the curd cube size, the amount of curd wash water, and a decrease in scalding temperature, will diminish syneresis. Syneresis is enhanced by adding CaC12, adding more starter or using a more active starter, which causes a faster pH drop in the curd during the process. It is also enhanced by increasing the (scalding) temperature, the stirring intensity and time, the amount of whey removed before curd drainage and the time from filling the moulds until pressing. Increasing the moisture content by such process parameters also increases the inclusion of lactose, resulting in a lower pH of the cheese, unless there is a concomitant increase in the amount of curd wash water. Pre-acidification of cheese milk (which is not advisable), increasing the quantity of starter added, the rate of growth of starter bacteria and the time needed until the curd is separated from the whey, all reduce the pH of the curd before the rind is closed during pressing and,
Gouda and Related Cheeses
consequently, reduce the concentrations of calcium and inorganic phosphate retained in the cheese. Lower concentrations of these give a somewhat lower cheese yield, a lower buffering capacity and consequently a slightly lower pH, and may affect cheese texture somewhat, the consistency becoming slightly softer and shorter. During normal cheesemaking, acidification by lactose fermentation hardly occurs before filling the cheese moulds. Thereafter, pH should decrease considerably and acidification will be complete after approximately six hours in brine. A flow sheet for the production of Gouda cheese is shown in Fig. 1 to give an example of the steps in a practical process. The manufacturing process is discussed in more detail in the following sections. If more detailed information about the process equipment is desired, the reader is referred to van den Berg (2001). Milk treatment
The aim of treatment of the milk is to improve or maintain the quality of the milk for cheesemaking, with respect to cheese quality and composition, yield and ease of manufacture. Milk quafity Milk quality may be defined so as to include composition. The fat and the casein contents of the milk naturally affect cheese yield and fat content; lactose content affects cheese acidity (see 'Control of pH and water content'). Off-flavours, particularly if associated with the fat, may be carried over into the cheese. Although in a well-ripened Gouda-type cheese very limited lipolysis occurs, it is never enhanced to avoid any soapy off-flavour. Physical dirt should be absent as it shows up in the cheese. It can be removed easily by filtering or centrifugation. Such issues are among the official quality properties in the payment scheme for farm milk in The Netherlands, as mentioned in a survey on this subject by IDF (1995). The bacteriological quality of cheese milk is of great importance. Pathogenic organisms may survive in cheese, which may be a problem in raw milk cheese. Pathogenic Enterobacteriaceae and staphylococci might grow in cheese. However, proper manufacturing procedures, e.g., the use of an active lactic starter, ensure that throughout the cheese, either the sugar is fully and rapidly converted into acid by the starter organisms (van Schouwenburg-van Foeken et al., 1979), or that the salt content is already high enough to prevent the growth of pathogens. This is an important reason why there are no incidents with pathogens in well-made Gouda cheese. For more details on pathogens in cheese, the reader is referred to 'Growth and Survival of Microbial Pathogens in Cheese', Volume 1. Several bacteria present can cause
105
defects in the cheese: coliforms, Lactobacillus spp., Streptococcus thermophilus, faecal streptococci, propionic acid bacteria, Clostridium tyrobutyricurn (see 'Butyric acid fermentation'). The growth of psychrotrophic bacteria in the raw milk may lead to the production of sufficient thermostable lipolytic enzymes to cause undesired lipolysis in the cheese (Driessen, 1983); bacterial proteinases do not seem to cause undesirable effects but may break down casein prematurely and reduce cheese yield. Nowadays, milk is stored at the farm for some days so that the growth of psychrotrophs may have started already. Temperature control and good cleaning procedures are necessary and stimulated by quality demands on the total microbiological count of farm milk (IDE 1995). However, such milk is usually thermised (e.g., 10 s at 66 ~ upon reception at the cheese factory, sufficient to kill several types of bacteria, including most psychrotrophs, but not to greatly alter the milk otherwise (Stadhouders, 1982; van den Berg, 1984). Pasteurisation The milk is pasteurised, usually high temperature-short time (HTST) e.g., 15 s at 73-74 ~ just before cheesemaking. However, it is noteworthy that such an indication of pasteurisation intensity is not sufficient to indicate the total heat load to which the milk is exposed. Therefore, it is necessary to be aware of temperature changes with time when passing through the heat exchanger, including a large regeneration section (de Jong, 1996). Figure 2 gives time-temperature relations for effects that are important for cheesemaking; it should be realised that these are only examples since there is considerable variation in the thermolability of micro-organisms, etc. Pasteurisation serves the following functions:
(a) Killing of pathogens, including Listeria monocytogenes, which means that heating may be slightly more intensive than that needed to inactivate alkaline phosphatase only. (b) Killing of spoilage organisms. Spores of Clostridium tyrobutyricum survive but Enterobacteriaceae, propionic acid bacteria and most lactic acid bacteria are killed. Some species of Lactobacillus and Streptococcus may survive, but they are seldom present at high numbers in the milk. However, Streptococcus thermophilus may grow on the metal surface downstream of the large regeneration section of a modern heat exchanger (Bouman et al., 1982) and thus attain high numbers if pasteurisation is continued for a long time (e.g., 10 h). This may lead to undesirable flavour and texture in the cheese (see 'Thermo-resistant streptococci'). (c) Inactivation of milk enzymes. This is probably important only with regard to lipoprotein lipase (EC 3.1.1.34), but even the usefulness of this is variable.
106
G o u d a and R e l a t e d C h e e s e s
Thermisation 15s 66~ lr
Thermisation 15 s 66 ~
Separation Pasteurisation 10 min 95 ~
Standardisation e.g., 3.5% fat
Coating with latex
Jl,
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f
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Drying
Cream
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Mixing
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80 rain
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,
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Example of a modern flow sheet for the manufacture of 12 kg Gouda cheese (until curing). Time from start of cutting to start of moulding - 60 min. A curd-filling machine with vertical columns and plastic moulds with a nylon gauze lining is used. NaCI content of brine- 18%, brine temperature- 14 ~ brine pH -4.5. If bactofugation is omitted, about 0.015% NaNO3 is added.
Gouda and Related Cheeses
30-
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Effect of heat treatments of various severity of the of serum proteins, the inactivation of some enzymes and the killing of some micro-organisms, important for the cheese quality or safety. The figures indicate the fraction left unchanged in the milk after the treatment. Approximate average results from various sources (from Cheese: Chemistry, Physics and Microbiology, 2nd edn, Fox, P.F., ed., Volume 2, Chapter 2, Chapman & Hall, London, with kind permission from Kluwer Academic Publishers). denaturation
Several lipases and proteinases produced by psychrotrophic bacteria are not inactivated (Driessen, 1983). (d) Because the milk should be pasteurised shortly before renneting, it also serves to undo, more or less, the adverse effects of cold storage on the casein micelles and on the salt equilibrium (which in turn affects renneting properties; 'Rennet-induced Coagulation of Milk', Volume 1), to melt the fat in the globules and to bring the milk to renneting temperature. Heat treatment can also have undesirable effects, particularly if its intensity is distinctly higher than that needed to inactivate alkaline phosphatase: (a) Considerable denaturation of serum proteins by more intense pasteurisation leads to slow renneting, a weak curd and poor syneresis ('Rennet-induced Coagulation of Milk', 'The Syneresis of Rennet-coagulated Curd', Volume 1). It may also cause a cheese of poorer quality, particularly the development of bitterness and sulphide flavour (van den Berg et al., 1996b). The first defect is probably caused by stronger binding of chymosin and the second by breakdown of denatured (more heat-sensitive) sulphur-containing whey proteins. A small amount of denatured whey proteins, which is mainly precipitated on caseins, has little adverse effect on the renneting process or cheese quality, e.g., when denaturation occurs during sterilisation of small vol-
107
umes, such as bactofugate or starter milk which are then blended into a large volume of milk. Since heat denaturation also causes a profitable increase in cheese yield, fairly rigorous control is exerted in some countries, e.g., via the nitrogen content of the whey (which, for example, should be at least 95% of that of the whey made from raw milk). As sampling of the original raw milk has become increasingly difficult when (sometimes days later) the whey from the cheese vat is sampled for this goal, a new and more sensitive method has been introduced in The Netherlands. This is based on the different kinetics of denaturation of the individual whey proteins, e.g., bovine serum albumin and immunoglobulin G are denatured more rapidly between 70 and 100 ~ than [3-1actoglobulin. A small increase in pasteurisation temperature causes a distinct change in the relation between the contents of these native heat-sensitive proteins and that of native [3-1actoglobulin in the whey (van den Bedem and Leenheer, 1988). (b) Useful milk enzymes may be inactivated, especially xanthine oxidase (EC 1.2.3.2). This enzyme is needed to slowly convert (added) NO3 to NO2, which is essential for the desired action of nitrate against clostridia (see 'Butyric acid fermentation'). Although pasteurisation of cheese milk is widespread and has certainly helped to considerably improve average cheese quality, it is often held to be responsible for a certain lack of flavour, especially in well-matured varieties. This may be due to inactivation of lipoprotein lipase and killing of bacteria that may contribute to raw milk cheese flavour, which usually is more variable than the flavour of cheese made from pasteurised milk. Less-severe heat treatment may improve flavour. An old-fashioned way of improving milk quality is to let the fresh milk cream at a low temperature (5-10 ~ in this way, most bacteria accompany the cream because of agglutination (Stadhouders and Hup, 1970). By heat-treating the cream, but not the skimmilk, most bacteria are killed without greatly affecting milk enzymes. Bactofugation Another way to enhance bacteriological quality is bactofugation. This may be applied when cheese is made from raw milk. However, the main purpose is the removal of spores of Clostridium tyrobutyricum. As said before, these spores survive pasteurisation and will pass into the cheese, where they may cause butyric acid fermentation (BAF). This defect is often called 'late-blowing' and is a well-known risk for Gouda and related cheeses (see 'Butyric acid fermentation'). The addition of nitrate is effective against this defect but not desired in various markets. The use of a bactofuge, in particular the
108
Gouda and Related Cheeses
self-desludging type, is a successful answer to this question. The bactofuge is a kind of centrifuge for milk with special properties to remove the heavy sludge via nozzles mounted at the outside of the separator bowl. Bacterial spores have a rather high density in comparison with the bacteria themselves. The original idea of using a separation technique to improve the bacteriological quality of milk, in that case for liquid milk, came from Simonart and Debeer (1953). The bactofuge is mounted in line with the milk pasteuriser or the thermiser and is fed from the regeneration section because the best efficiency of the process is obtained at approximately 60 ~ This treatment reduces the number of spores drastically, even to about 2-3% of the number in raw milk (van den Berg et al., 1980, 1988). The sediment obtained contains the spores but also more casein than the original milk and its removal would cause a significant reduction in cheese yield (about 6%). Consequently, the sediment is commonly ultrahigh temperature (UHT) heated to kill the spores and added back to the milk; the concomitant denaturation of serum proteins is acceptable (see 'Pasteurisation'). Double bactofugation increases the efficacy of spore removal, but is more costly. Nevertheless, this treatment is necessary when no nitrate addition is allowed. In such a modern line, the continuously discharged heavy phase from the second separator is fed back into the milk supply to the first h~r-tnfllaP
hPr'a11cP thPrP
ir n n t
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spore load of the two liquids. On the other hand, the heavy phase from the first bactofuge, mixed with the intermittent discharges (by the self-desludging mechanism) from both machines, is the total volume to be sterilised. For further details of this technique, the reader is referred to van den Berg (2001). The technique of microfihration of the skimmed milk fraction is an alternative to bactofugation. The effect on the removal of spores might be similar to double bactofugation but the investments are higher. Moreover, the cream and the milk retentate must be sterilised, which is a considerably larger volume than in the case of bactofugation. Standardisation and additives In most cases, after thermisation, the milk is standardised so as to yield the desired FDM of the cheese (see 'Standardisation'). This generally implies some skimming of the milk. Usually, part of the milk is passed through a separator (which also removes dirt particles) and sufficient cream is removed. Skimmed milk, and often some sterilised whey cream, is added to whole milk shortly after reception. This standardised milk is stored until further processing. However, nowadays automatic in-line standardisation systems are receiving some interest in
large cheese factories because of possibilities to minimise storage tank volume. Substances added to the milk after pasteurisation and before renneting may include: (a) CaC12 to speed up, and particularly to reduce variability in, renneting and syneresis. (b) Nitrate, to prevent early blowing by coliforms and the growth of C. tyrobutyricum. Nowadays, nitrate is often added later, i.e., to the curds-whey mixture after the first whey has been removed. This serves both to save on nitrate and to avoid producing large quantities of whey that contains nitrate. (c) Colouring, either 13-carotene or annatto (an extract of the fruits of Bixa orellana), for obvious reasons. Its use appears to be waning and is often omitted, although some types are highly coloured, e.g., Mimolette. Spices, if any, are commonly added to the curds and whey mixture before draining, e.g., in the buffer tank. Cheesemaking
Curdmaking Renneting is usually done at 30-31 ~ and cutting starts 20-25 min afterwards. About 20 ml of rennet with a specific activity of 150 IMCU (International Milk Clotting Units) and 10-20 g CaC12 are usually arlrlpd
tn
l NN1 nf rhppep
milb
Thp
nil
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somewhat reduced by the addition of CaC12 and starter; 6.50-6.55 is the usual value. The aim is to produce a coagulum (gel) that can be cut easily and stirred without excessive loss of 'fines' in the whey and which shows rapid syneresis. If the milk is drawn from a very large quantity, and moreover if the calving pattern of the cows is fairly evenly spread throughout the year, milk composition is generally sufficiently constant to give good results with fixed quantities of rennet and CaC12 always. The coagulum is cut, usually into pieces of any given form, some 8-15 mm size, initially. When the coagulum pieces already have been cut sufficiently but still some time is required for syneresis, the mass is stirred (by turning the knives in the opposite direction) until the desired amount of whey can be drained off. This stirring should be carried out very gently to minimise loss of fines and fat. Curd fines are usually defined as the fraction < 1 mm size and can easily be lost in the separated whey. After a while, the curds are allowed to settle and part of the whey is removed so that stirring becomes more effective, i.e., the forces acting on the curd grains are higher and thus promote syneresis. After removing the first whey, stirring is immediately restarted to avoid too much curd fusion, as curd lumps may persist during
Gouda and Related Cheeses
the rest of the process. The temperature is increased (scalding), also to speed up syneresis, but not to a temperature high enough to harm the (mesophilic) starter organisms, which usually implies keeping below 38 ~ The curd wash water must be carefully spread over the cheese vat in order to prevent local overheating. During scalding, agitation should be more vigorous, again to avoid local curd fusion. Inside such curd lumps, whey expelled by individual curd particles cannot be removed and the washing of lactose is inhibited, resulting in acid, soft and 'nesty' spots (clusters of small irregular openings) in the cheese. Scalding can be done by indirect heating or by adding heated whey or hot water. The latter practice is the most common, since water usually has to be added anyway to regulate pH (see 'Control of the pH'). Initially, an increase in the size of the cheese vats became possible with the separation of the process steps of curdmaking, separation of curds and whey (curd drainage) and moulding. The time schedule for successive batches (vats) is programmed in such a way that (nearly) finished curds-whey mixtures can be pumped separately at constant time intervals into a buffer tank before being introduced into the continuously working curd drainage and moulding machine. The open cheese vats of the past have been developed into large enclosed tanks with integral cutting and stirring devices, built-in filling, emptying and dosing (of rennet and starter) pipework and metering devices (for sucking whey) and control and automation modules. Recently, the increased need for process control has also led to the successful introduction of a laser optical fibre device built into each cheese vat to control the renneting process. It indicates the beginning of aggregation, the increasing firmness of the coagulum and may warn when the desired gel firmness has been reached (ten Grotenhuis, 1999). All milk takes part in the renneting process and will contribute to cheese yield. However, during curdmaking and successive processing steps some loss of curd fines and fat is inevitable because of mechanical stress by the processing equipment. The cheesemaker has to perform the optimal process in relation to the equipment used and the curd properties desired at final drainage (curd size, water content, temperature, etc.). The losses in the whey of fat and, in particular, curd fines, should be minimal. Most of the fat in whey is already liberated when cutting the coagulum because cutting initiates the loss of the fat globules from the newly created surfaces. The amount of whey expelled during the first cuts has a very high fat content that decreases during syneresis and expulsion of whey at the end of cutting to <9% of the fat percentage of the cheese milk. Some further curd damage by mechanical stress during the process creates
109
new curd surfaces and also results in losses of fat and curd fines. This is particularly at risk when curd is allowed to settle before sucking the first whey and starting stirring and adding the washing water. Then, extra force is needed to avoid curd lumps during the next stage of curdmaking. The design of the cheese tank (vat) is important with respect to such losses and most types of vat have been thoroughly tested, as demonstrated by de Vries and van Ginkel (1984). The difference between the fat content of the whey at the end of curdmaking and after dilution with the wash water (calculated from the first whey) should be no more than 0.02. Curd fines, expressed as dry curd solids, in the first whey should be <150 mg/1 whey. This level may increase somewhat in the whey outside the curd particles during further processing, including the holding time in the buffer tank before drainage. However, it must be realised that during drainage, especially in the vertical draining cylinders, most of these fines are still entrapped between the larger curd particles.
Draining and moulding In the past, after the curds had lost enough moisture (the water content being ---68% in the case of Gouda cheese and the pH 6.45), stirring was stopped and the curd grains were allowed to settle. Partial fusion of deforming curd grains now occurs, whey is separated, and a continuous mass of curd is formed that can be cut into blocks. Considerable loss of whey from the curds occurs during these stages (Walstra et al., 1985). This was often promoted by applying some pressure (e.g., 400 Pa) by placing perforated metal plates on top of the bed of curd or later by the curd layer itself being deep enough; pressure also promotes curd fusion. The lower the fat content, the less easy curd fusion becomes, because of the lower scalding temperature and reduced ability of the curd grains to deform. As a consequence, more whey may be left between the curd grains after drainage, finally resulting in a more open texture. If a very low water content is desired, the drained curds may be stirred or worked; this causes considerable additional syneresis, but also loss of fines and fat and a cheese with an open texture (many irregular, small holes). The blocks of curd are transferred to cheese moulds. The traditional square block of curd is allowed to spread and fill the round mould after some time. Between the corners, it flows out and at the corners it may even be compressed, causing in the cheese locally a higher and a lower water content, respectively (Arentzen, 1972). So certain flow properties of the curd block are necessary. At these stages until brining, the temperature of the curd blocks should be maintained high enough for good curd flow, to continue acidification and achieve good rind formation during pressing.
110
Gouda and Related Cheeses
To this end, moulds are still warm after cleaning and the room temperature should be approximately 20 ~ and often the presses are practically closed. Forcing the flow of the curd block, e.g., by the premature start of pressing, should be avoided because it may locally destroy curd fusion and in such places more whey collects between the curd particles. This turns into 'nesty', soft and acid spots in the final cheese because this whey (with lactose) is resorbed, while the distance between these stronger acidifying curd grains remains too large and these spaces are filled later with gas. Essentially, the mechanism is similar to what may happen in the case of curd lumps (see 'Curdmaking'). The introduction of specific curd-sedimentation or pre-pressing vats, with a moving, perforated belt, was the first step in mechanisation of the Gouda cheesemaking process. At one end of this vat, the settled and drained curd layer is mechanically cut into pieces of the desired dimensions, which are transferred to moulds to be pressed. Such curd blocks are still square and require time to deform and fill the round Gouda cheese moulds. Furthermore, greater variation of water content in the cheese will occur. The water content of the curd blocks also deviates within a batch due to differences in drainage time before the transfer to the moulds. Although this system is still applied with improved equipment, in many cases it has been superseded by the following technique. The curds-whey mixture is fed from the buffer tank into continuously working machines that separate the whey from the curds, shape the curd blocks and fill them into moulds. The most common machine is the Casomatic | which operates with a downward curd stream. It has draining columns for cylindrical or rectangular cheeses. Technologically, these machines have the advantage that the weight of the loaves can be controlled accurately, the relative standard deviation being 0.5-1.5% this is especially important for small loaves, e.g., Edam cheese. These machines were less flexible than the pre-pressing vats for the production of cheeses of different shapes and sizes. However, modern models have been improved in this regard. During the mould-filling process, syneresis of the curd proceeds and would cause the water content of cheese to decrease if no precautions were taken. Therefore, syneresis during the moulding operation is slowed down by stirring the curds-whey mixture in the buffer tank only gently, but sufficiently to keep the mixture homogeneous, and by gradually reducing the temperature by, e.g., 1 ~ when the time between batches is more than 15-20 min. Because the first curd blocks of a batch contain more moisture and will loose more moisture before pressing than the others, the height of the curd block can be adapted during drainage of the batch. In this way, the moisture
content and the weight of the individual final cheeses can be controlled fairly accurately in combination with the time between filling the mould and start of pressing (de Vries and Staal, 1974). Nowadays, two buffer tanks are often used, alternately filled with the next batch, in order to let any fine air bubbles entrapped during stirring and pumping to escape. These could later disturb the eye pattern in the cheese, as they are entrapped during drainage. Consequently, the stirring time in the cheese vat is shortened. Figure 3 shows a section through a draining column of the Casomatic | The process in the draining columns was investigated thoroughly by Akkerman et al. (1996). During 'free' drainage, there is still considerable ongoing syneresis because whey is separated and the hydrostatic pressure has disappeared. At the same time, compaction, deformation of the curd particles and curd fusion take place, blocking the pores between the curd particles, and drainage decreases (Akkerman et al., 1994). This also slows down the compaction rate. In the draining column, the liquid pressure gradient between the centre and the wall of the drainage equipment is the important driving force. The column is divided into three sections in which this gradient increases in each section downwards in parallel with the compaction rate. To this end, the control of the outflow of the whey through the perforated wall has always been a very important feature of this machine. It is managed in relation to variables like the geometry of the drainage column, the softness (moisture content) of the curd grains and their size, especially the amount of curd fines. At the outlet, below the draining column, curd blocks are cut off (for round moulds, circular blocks), often pre-pressed slightly (without deformation of the block) to prevent loosening of curd during successive handling and transferred to the moulds. The filled moulds are moved to the press and on their way, a cover is put on top. The whey obtained at various stages may be collected separately, because it differs in pH, added water and added nitrate. The whey is usually separated and the cream obtained is pasteurised, e.g., 30 min at 95 ~ to destroy fully any bacteriophage that may be present. The whey cream is used to adjust the fat content of the next lot of cheese milk. Curd fines are usually separated from the whey by means of hydrocyclones or filters, but are not recycled to the curd because of the danger of contamination (bacteria and phage). They are pressed and often used for processed cheese.
Pressing The blocks of curd are pressed in moulds to obtain the typical shape of the cheese and a closed rind. The latter is necessary to avoid further loss of moisture until
Gouda and Related Cheeses
111
Cut-away view of the Casomatic CSC, the single column continuous whey drainage and portioning machine (reproduced with permission of Tetrapak Tebel B.V., Leeuwarden, The Netherlands). 1, Curds and whey supply; 2A-C, Whey drainage sections with pressure indicators; 3, Whey collection tube; 4, Whey collection vessel; 5, Pneumatically operated cylinder for opening and closing the curd knife; 6, Pneumatically operated cylinder for removing the curd block to fill the cheese mould.
112
G o u d a and Related C h e e s e s
brining, to achieve an even salt penetration through the cheese surface, to withstand the tension during brining and to serve as a barrier for micro-organisms during brining and ripening. Originally, wooden moulds were used, and the blocks of curd were wrapped in cloth; a pressure of 50-100 kPa was applied for several hours and the cheese developed a very distinct, firm rind. Nowadays, cheese loaves are formed in finely perforated metal or plastic moulds, or plastic moulds lined with a gauze or some kind of cloth to promote drainage and rind formation. The pressure is usually much lower and is applied for a shorter time. Consequently, only a weak rind is formed; although the rind should be fully 'closed', i.e., free of visible openings, it is not a fully effective barrier against the high numbers of micro-organisms during brining (Wilbrink e t a l . , 1981). In order to have an undisturbed process, sticking of the cheese to the liner of the mould must be avoided. This is achieved by the correct combination of a gradual increase of pressure over time and the firmness of the curds. Closing of the rind is due to complete fusion of the outermost layer of curd grains, promoted by local deformation of the surface around threads of the liner or corners of the perforations of the mould (Mulder e t a l . , 1966) and good drainage of the liberated whey. Essentially, it is a locally intensified syneresis process with a rearrangement of casein strands. Pressing, as it is applied nowadays, is generally 1.~, ,USlOnr " ,1., . . . . . 1.,,-,,,, +1.,,~ ;9ttau,,,~.,~.,tt . . . . m..;,..., to cause .... ,_,_,mp,~.te t,,,_,,.,~,,,_,,.,~ t,,,.. mass of the freshly pressed loaf. This implies that some moisture can still move fairly freely via small interstitial openings left between the curd particles through the cheese mass, possibly leading to uneven moisture distribution. During brining, the temperature decreases and the remaining whey is resorbed, while the interstitial openings practically disappear. Within 1-2 days, curd fusion is nearly complete and the cheese has a closed texture. The cheese looses considerable moisture during pressing, but moisture loss is slight once a closed rind has formed (Straatsma and Heijnekamp, 1988). This implies that starting the pressing earlier and applying a higher pressure lead to less moisture loss, and hence to a cheese with a higher water content. By varying the time between mould-filling and the start of pressing, water content can thus be regulated to some extent, especially to ensure the same water content in cheese loaves of the same batch. During stirring in the whey, syneresis proceeds. Consequently, the blocks from a batch formed last in a continuously operating draining and moulding machine have the lowest water content, and pressure should be applied to those loaves soon to 'keep in the moisture', while the blocks formed earlier should be left for a longer time before pressing (see
also 'Draining and moulding' and 'Control of water content'). In traditional cheesemaking, the freshly pressed loaves were turned in the moulds with a flat cover and left there until the next day for 'shaping' (Dutch: omlopen), i.e., to attain a symmetrical shape. After removing the cloths and before turning, the pressing rind created between the cover and the mould was trimmed off, which caused some risk of tearing open during brining. The main change occurring was, however, the complete conversion of lactose to lactic acid. This is important because during brining, fermentation by the starter bacteria is slowed down and even effectively stopped in the rind, owing to the combined effects of low temperature and high salt content. Nowadays, a somewhat greater quantity of a fastergrowing starter is usually added, which implies that much more lactic acid has already been produced a few hours after adding starter. The course of acidification in the core of the cheese is illustrated in Fig. 4. The short holding between pressing and brining is facilitated by a better design of the modern cheese moulds so that 'trimming' and 'shaping' are not really necessary any more. The shape of the curd block also approaches that of the mould better and the time necessary for curd flow before pressing diminishes. So the modern mould has made it possible to speed up the process to a great extent, thanks to improved starter ,~,.hnu,ugy. Instead of pressing cheese loaves in stacks, it has become common to press them in a single layer by individual pressing cylinders or one cushion for a great number of cheeses. Pressing a pile of cheeses in a column
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Pressing Time after adding starter (h) Wash water In brine Acid production during Gouda cheese manufacture (conditions comparable to those in Fig. 1). Quantity of lactose and lactic acid, and the pH of curd and cheese (core) as a function of the time after starter addition to the cheese milk.
G o u d a and Related C h e e s e s
under one pressing head, as in the past, started pressing of the first, more moist cheeses of the batch earlier instead of later (see 'Draining and moulding'). Nowadays, presses are filled and emptied continuously. Continuous pressing, however, is not applied because differences in water content still exist between loaves of a batch at the beginning and the end of moulding, due to differences in the extent of syneresis of the curd. When time between batches is not too long, say 20 min, the whole batch is pressed for the same length of time and pressuring programme. With respect to demoulding, mechanical systems have been designed which prevent damage to the relatively weak cheese rind. The lid is lifted perpendicularly, the mould is emptied after rolling on the side or the cheese is blown out of the linerless rectangular mould. When held for further acidification, the cheeses are kept in normal moulds. Then, the cheeses are weighed on their way to the brine as part of the system to control composition and yield. Brining
Nowadays, the cheese is put into brine within 1 h after pressing when some residual lactose is still present in the curd. (Incidentally, this means that even in a 2-week-old cheese, the outermost layer may contain about 0.2% lactose in the cheese moisture and also that the brine contains lactose.) The use of pasteurised milk, strict hygiene
113
and adequate rind treatment are needed to prevent the growth of undesirable micro-organisms, which might profit from the presence of lactose. Another requirement is a sufficient decrease in pH; after pressing, ---5.9 and, when it occurs, after 1.5 h holding, 5.4-5.5. Moreover, the modern curing room provides better drying conditions for the rind of the cheese. Otherwise, such cheeses will easily develop a slimy surface which is hard to repair. Brining is done primarily to provide the cheese with the necessary salt. Moreover, it serves to cool the loaves rapidly to below 15 ~ (to stop further syneresis, and prevent or slow down the growth of undesirable bacteria), and to give them a certain rigidity (due to the high salt content in the find) during the necessary handling shortly after brining. Brining causes a considerable loss of water (2-3 times the quantity of salt taken up) and loss of a little soluble matter (<0.2% of the cheese mass). Factors affecting these processes are discussed in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. Figure 5 indicates the effect of different salt concentrations in the brine on salt uptake and water loss during the process of manufacturing normal Gouda cheese. The brining process of Gouda and related cheeses was investigated extensively by Geurts et al. (1980). An increased fat content in the cheese hinders the process. Conversely, an increased water content before brining enhances salt uptake, relatively more than the
Salt content of dry matter (SDM)
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Changes (idealised) in water content (W) and salt content of dry matter (SDM) of Gouda cheese during brining for a normal process and for a longer brining time at a lower water content and weaker brine (dotted lines), according to van den Berg et ai. (1975).
114
Gouda and Related Cheeses
water loss. The shape and the size of the cheese have considerable impact on local diffusion processes. It may be expected that increased temperature and brine concentration speed up the process. However, in practice, strong dewatering of the rind zone hinders salt uptake and the net effect hardly increases above 20% salt or 18 ~ These phenomena are better described by the Maxwell-Stefan equation than by Fick's law, which Geurts et al. (1980) applied originally, as discussed by Payne and Morison (1999). The average salt content of the young Gouda cheese is generally 4-5 g/100 g water in the cheese; of course, it takes considerable time (up to 2 months in a large load for the salt to become more or less evenly distributed throughout the cheese. The brine usually contains 17-18% NaC1, but weaker brines might sometimes be used to allow brining the loaves for exactly 1 week. This poses the risk of growth of salt-tolerant lactobacilli in the brine that may even penetrate the cheese to some extent and cause flavour defects (Stadhouders et al., 1974; Wilbrink et al., 1981). Regular cleaning of the equipment and basin walls around and above the brine level is desirable (Stadhouders et al., 1985). Chemical disinfection of brine has negative effects on cheese flavour and, certainly in case of chlorine, forms toxic compounds. For similar reasons, irradiation of brine was not successful. Filtration techniques may be reason.~l,,
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application. The brine should contain enough Ca2+(0.2% at 17% NaC1) and acid (pH 4.5) to prevent dissolution of cheese protein, which would cause a slimy rind (Geurts et al., 1972). A weak brine has a similar effect but may contain more Ca 2+ (van den Berg et al., 1975). Brine is used for years, with daily removal of the surplus brine and adjusting the salt concentration. A certain balance exists in the non-sah components in the brine and the moisture of the cheese. These solids also serve to buffer the pH so that only a limited amount of HC1 is needed to control it. The lactose concentration in brine is higher when the cheese is salted at a higher pH, e.g., in case of rectangular blocks designated for foil-ripening. A consequence of brining is the loss of moisture, resulting in the dimensions of the cheese shrinking by a comparable percentage. Loaves with corners and edges shrink more at these places than elsewhere. The cheeses are transported by floating in the brine that is circulated continuously through the whole brining bath. The density of the cheese is approximately 1.07 while that of the brine is >1.10 g m1-1, depending on salt concentration. A bypass flow is used for cooling (the brine temperature is maintained at 13 ~
and for the uptake of salt from a silo. The daily amount of salt necessary to maintain a constant salt concentration is calculated from salt uptake by the cheese plus the increase of the brine volume by moisture from the cheese liberated by pseudo-osmosis. Two brining systems are in use. One uses stainless steel racks with horizontally tightened nylon nets to form several horizontal compartments; the racks can be moved up and down in a deep brine basin (2-3 m). Filling and, after brining, emptying of successive compartments is realised by opening gates, consisting of a frame with bars, at the front or the rear that do not hinder brine flow through the basin when closed. This circulation also serves to maintain an even salt concentration around all cheeses in order to control salt uptake by the cheese. Stronger agitation is, however, not necessary because salt uptake is, in effect, controlled by (slow) diffusion in the cheese when brine concentration is maintained. Moreover, increasing agitation by air injection through pipes with small openings at the bottom of the basin is not advised because precipitated calcium phosphates may block the openings. The air bubbles become smaller and yeast growth is enhanced (van den Berg et al., 1975). A high yeast count in the brine must be avoided because high counts on the cheese surface will inactivate natamycin (see 'Rind treatment and curing'). In the other brining system, the loaves are salted while floating in a shallow layer of circulating brine l~.,p,
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odically immerse the loaves take care of salting of the top of the loaves. The latter system is nowadays rarely installed in new factories because of the very large ground surface needed. The brine volume increases by about 2.5% of the cheese mass that is brined. The resulting surplus is often simply mixed with the effluent of the whole cheese plant. However, demands on the quality of the effluent and the (still threatening or already existing) financial consequences of discharging will make this solution less acceptable in many areas. Several techniques have been studied; concentration by evaporation under vacuum seems to be the most attractive one (Zoon et al., 1991). Rind treatment and curing
For Gouda and related cheese varieties, development of micro-organisms on the cheese surface is undesirable, because they may negatively affect cheese quality. In particular, the growth of moulds must be prevented, since they adversely affect the appearance of the cheese and some species may produce mycotoxins, e.g., sterigmatocystin by AspergiUus versicolor (Veringa et al., 1989). In former days, cheese was pressed in such a way as to obtain a thick and very tough rind; mould development
Gouda and Related Cheeses
was reduced by regular rubbing of the cheese rind with a dry cloth and unboiled linseed oil. After hardening, the coat formed also reduced water evaporation from the cheese rind, thus permitting it to remain relatively supple and springy. This practice was abandoned after the introduction of special plastic dispersions, which offer superior protection and permit the production of cheese with a weaker rind. This is one of the improvements that allowed mechanisation and a marked speeding up of cheesemaking. These plastic dispersions form, on drying, a coherent plastic film of hydrophilic nature that offers a protective coating against mechanical damage. When compared with oil treatment, it reduces the evaporation of water by ---50%. After the first week, water evaporation slows down steadily because diffusion inside the cheese is the limiting factor, while the relative humidity of the air is constant (Bouman, 1977). The film mechanically hinders mould growth, but it may also contain fungicides, e.g., natamycin (pimaricin), an antibiotic produced by Streptomyces natalensis, which is active only against moulds and yeasts, or calcium or sodium sorbate. In many countries, like The Netherlands, only natamycin is allowed while in some other countries only sorbates are permitted. Compared to sorbates, natamycin has the advantages that its migration into the cheese is generally limited to the outer 1 mm of the rind, it inhibits most of the microorganisms found on the surface of the cheese and it does not adversely affect the appearance, taste and flavour of the cheese (de Ruig and van den Berg, 1985). Moreover, natamycin is much more effective than sorbates; for comparable protection from mould growth, the amount of sorbate needed is about 200 times that of natamycin. Recently, growth of Penicillium discolor, which appeared to be less sensitive to natamycin, caused some trouble with Gouda cheese but adequate hygiene measures can prevent this when using this fungicide (van Rijn et al., 1997). With respect to public health, an acceptable daily intake of 0.3 mg natamycin~g body weight has been established. The EU cheese regulations limit the quantity to 1 mg/dm 2 of cheese surface when the cheese is sold. Recently, alternatives for plastic coatings, based on milk proteins, have been introduced. Generally, the cheese is cured at 12-15 ~ and 85-88% RH. The conditions must allow the coating to dry rather quickly, otherwise undesirable micro-organisms like yeasts, and successively coryneform bacteria or moulds, may develop and cause off-flavours and a sticky and dirty surface. However, if the coating layer dries too quickly, cracks may form in the plastic film, allowing mould growth in the cheese rind because natamycin penetrates into the cheese only to a very limited extent. Some residual moisture is necessary to keep this film sufficiently flexible. Particularly at the beginning of ripen-
115
ing, the cheese inevitably expels a little moisture, causing a high humidity between the loaf and the shelf, which favours bacterial growth. To prevent this and to allow the cheese to retain a good shape, loaves are turned frequently during this period. Upon prolonged ripening, this frequency is reduced. Other measures to maintain shape are sufficient evaporation of moisture to replace, to some extent, the 'framework' created during salting that wanes by salt diffusing to the core, and to maintain not too high a temperature. In this respect, 17 ~ is a practical hurdle. Above this temperature, most milk fat is liquid and the cheese exudes fat ('sweating'), while the consistency of the cheese may become greasy. If a higher temperature is desired to accelerate ripening, then a paper banderole is sometimes used around the cheese. A higher temperature increases the risk of 'late blowing' and more attention to the cheese surface is required to keep it clean. After brining, the cheeses are often dried slightly at room temperature, firstly removing the excess of brine. It is preferable to do this by means of a suction mouth just above the surface of the cheese. At this stage, the rind is under stress because of the brining process, and forced drying by heated air must be avoided. Cheese, just after removal from the brine and after application of the first coating, exudes extra moisture that will remain between the cheese and the shelf. The cheese will stick to the shelf, which hampers the following treatments of the cheese and destroys the plastic coating. (Even by pressing a finger on such quickly dried cheese, some moisture will appear immediately.) When the cheese is free of visible liquid, the first coating layer is applied on the top and most of the sides. Then, the cheese is conveyed in the same position to the store and automatically fed on to shelves to dry quietly for 1 or 2 days. The plastic coating is applied two or three times during the first 2 weeks and, during longer curing, repeated with gradually diminishing frequency. Care is taken that the cheese surface is sufficiently dry before each treatment. The cheese is turned and a coating machine with turning flexible flaps spreads the plastic dispersion evenly over the cheese. This treatment is combined with cleaning the upper surface of the shelf, and the cheeses are positioned on their dry and still clean other side. The drying of the cheese surface (and the shelves) is controlled by the climatic conditions of the curing room and, consequently, the decrease of the water content of the cheese or its weight loss. Treatments in curing rooms also have been highly mechanised: transport, coating and turning of cheese, cleaning of shelves, etc. Much progress has been made in controlling the temperature, relative humidity and velocity of air, in order to approximate the ideal situation in which each loaf is stored under
116
Gouda and Related Cheeses
identical conditions. Good insulation of the curing rooms is very important to prevent the condensation of water on the walls at the desired high humidity of the air, resulting in mould growth. Wooden shelves are normally used because they have the advantage of absorbing some moisture from the (young) cheese. However, they require special attention from a hygienic point of view. A strict maintenance programme of cleaning and drying combined with the treatment of the cheeses may guarantee sufficient safety. New wooden shelves need more attention because considerable quantities of fermentable sugars are still present in the wood. Thorough leaching in water, cleaning, disinfection and drying are necessary. Also, suitable glues must be used for the construction of these shelves (see 'Natural ripening'). Smaller cheeses, like baby types and Edam, are sometimes ripened hanging in coarse plastic nets or placed in perforated holes of a stainless steel plate where they can dry on all sides. The relative humidity of the air should be slightly higher to prevent excessive evaporation. Because such cheeses are coated on all sides, it is difficult to avoid the pattern of the net being imprinted on the surface. However, these cheeses are usually waxed before distribution. Just before they are put on the market, cheeses may be treated with paraffin (cheese wax), generally after they have been treated with latex. Before waxing, the loaves must have had, during the whole ripening pro-
also 'Foil-ripening'). A starter with low CO2-producing capacity is used to prevent loosening of the wrapping and too open a texture.
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vious bacterial growth on the cheese and a high humidity between the cheese and the wax layer favours bacterial growth, causing off-flavours and gas formation. In such cases, washing and drying of the cheese before waxing does not prevent these defects. Consequently, wax is applied predominantly to a matured cheese, mainly to prevent weight loss during transport. In some cases, especially for Baby Gouda or Baby Edam, often red wax is applied when the cheese has dried briefly after brining (so-called peel-off wax). Wax layers must be closed and cracks and pinholes must be avoided. Such cheese is stored under sufficiently cool conditions to maintain its shape in the cartons on pallets, consequently avoiding cracks due to bulging. Some cheeses are made in rectangular or square loaves for curing while wrapped in plastic foil. Treatment of the cheese, as mentioned above, is unnecessary. This type of cheese is particularly suitable for the processed cheese industry. However, it is also of increasing interest for customers who sell this type of cheese in prepacked portions or slices. Prolonged curing at the usual temperature, e.g., 14 ~ however, tends to produce cheese of poor flavour and consistency. Therefore, the cheese is kept at a low temperature ( < 8 ~ the resulting flavour is rather fiat (see
Milk standardisation and cheese yield
Cheese yield is usually defined as the mass of cheese obtained from a certain quantity of milk. When making cheese, individual milk components, including water, are converted to varying extents into the final cheese. During the process, salt is added. The conversion factors of the milk components are related to the process carried out and may differ slightly among cheese factories. The final cheese must have a certain composition because of legal and quality requirements. This primarily concerns water content and FDM but the actual parameter is the ratio of protein (para-casein) to water in the cheese. During the process, the conversion of protein from milk to cheese is of primary importance. This is why it has been advocated to calculate yield/kg para-casein in the milk (van den Berg et al., 1996a). This approach has also a close relation to the common practice of filling a cheese vat with such an amount of milk that a constant amount of cheese is obtained and that down-stream equipment is in full use. The cheese milk composition qtnndnrctiqPd
tn
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composition. To this end, the fat content is adjusted in a ratio to the protein content, as determined by infrared analysis at the plant. However, this protein content serves as a parameter for the para-casein content that is converted to cheese and may be obtained by the difference in protein content between milk and (undiluted) whey. It is important to calculate precisely the desired fat content of the cheese milk. The yield of cheese should also be predictable. Comparison with the ultimate analytical results may enhance understanding of the cheesemaking process. Usually, all cheese made from one vat of milk is weighed before brining, and again later. If this is always performed in much the same way it may be a valuable help, since it gives a first indication of cheese composition when milk composition and conversion factors of the main components are known. Standardisation
Under practical cheesemaking conditions, establishing the correct fat content of the milk causes specific problems. Firstly, the cheese mass is always inhomogeneous, causing difficulties in determining its real FDM. For this reason, borer samples may give considerable bias and therefore the whole loaf, a sector from it, or a quarter from a square-shaped cheese, is ground.
Gouda and Related Cheeses
was reduced by regular rubbing of the cheese rind with a dry cloth and unboiled linseed oil. After hardening, the coat formed also reduced water evaporation from the cheese rind, thus permitting it to remain relatively supple and springy. This practice was abandoned after the introduction of special plastic dispersions, which offer superior protection and permit the production of cheese with a weaker rind. This is one of the improvements that allowed mechanisation and a marked speeding up of cheesemaking. These plastic dispersions form, on drying, a coherent plastic film of hydrophilic nature that offers a protective coating against mechanical damage. When compared with oil treatment, it reduces the evaporation of water by ---50%. After the first week, water evaporation slows down steadily because diffusion inside the cheese is the limiting factor, while the relative humidity of the air is constant (Bouman, 1977). The film mechanically hinders mould growth, but it may also contain fungicides, e.g., natamycin (pimaricin), an antibiotic produced by Streptomyces natalensis, which is active only against moulds and yeasts, or calcium or sodium sorbate. In many countries, like The Netherlands, only natamycin is allowed while in some other countries only sorbates are permitted. Compared to sorbates, natamycin has the advantages that its migration into the cheese is generally limited to the outer 1 mm of the rind, it inhibits most of the microorganisms found on the surface of the cheese and it does not adversely affect the appearance, taste and flavour of the cheese (de Ruig and van den Berg, 1985). Moreover, natamycin is much more effective than sorbates; for comparable protection from mould growth, the amount of sorbate needed is about 200 times that of natamycin. Recently, growth of Penicillium discolor, which appeared to be less sensitive to natamycin, caused some trouble with Gouda cheese but adequate hygiene measures can prevent this when using this fungicide (van Rijn et al., 1997). With respect to public health, an acceptable daily intake of 0.3 mg natamycin~g body weight has been established. The EU cheese regulations limit the quantity to 1 mg/dm 2 of cheese surface when the cheese is sold. Recently, alternatives for plastic coatings, based on milk proteins, have been introduced. Generally, the cheese is cured at 12-15 ~ and 85-88% RH. The conditions must allow the coating to dry rather quickly, otherwise undesirable micro-organisms like yeasts, and successively coryneform bacteria or moulds, may develop and cause off-flavours and a sticky and dirty surface. However, if the coating layer dries too quickly, cracks may form in the plastic film, allowing mould growth in the cheese rind because natamycin penetrates into the cheese only to a very limited extent. Some residual moisture is necessary to keep this film sufficiently flexible. Particularly at the beginning of ripen-
115
ing, the cheese inevitably expels a little moisture, causing a high humidity between the loaf and the shelf, which favours bacterial growth. To prevent this and to allow the cheese to retain a good shape, loaves are turned frequently during this period. Upon prolonged ripening, this frequency is reduced. Other measures to maintain shape are sufficient evaporation of moisture to replace, to some extent, the 'framework' created during salting that wanes by salt diffusing to the core, and to maintain not too high a temperature. In this respect, 17 ~ is a practical hurdle. Above this temperature, most milk fat is liquid and the cheese exudes fat ('sweating'), while the consistency of the cheese may become greasy. If a higher temperature is desired to accelerate ripening, then a paper banderole is sometimes used around the cheese. A higher temperature increases the risk of 'late blowing' and more attention to the cheese surface is required to keep it clean. After brining, the cheeses are often dried slightly at room temperature, firstly removing the excess of brine. It is preferable to do this by means of a suction mouth just above the surface of the cheese. At this stage, the rind is under stress because of the brining process, and forced drying by heated air must be avoided. Cheese, just after removal from the brine and after application of the first coating, exudes extra moisture that will remain between the cheese and the shelf. The cheese will stick to the shelf, which hampers the following treatments of the cheese and destroys the plastic coating. (Even by pressing a finger on such quickly dried cheese, some moisture will appear immediately.) When the cheese is free of visible liquid, the first coating layer is applied on the top and most of the sides. Then, the cheese is conveyed in the same position to the store and automatically fed on to shelves to dry quietly for 1 or 2 days. The plastic coating is applied two or three times during the first 2 weeks and, during longer curing, repeated with gradually diminishing frequency. Care is taken that the cheese surface is sufficiently dry before each treatment. The cheese is turned and a coating machine with turning flexible flaps spreads the plastic dispersion evenly over the cheese. This treatment is combined with cleaning the upper surface of the shelf, and the cheeses are positioned on their dry and still clean other side. The drying of the cheese surface (and the shelves) is controlled by the climatic conditions of the curing room and, consequently, the decrease of the water content of the cheese or its weight loss. Treatments in curing rooms also have been highly mechanised: transport, coating and turning of cheese, cleaning of shelves, etc. Much progress has been made in controlling the temperature, relative humidity and velocity of air, in order to approximate the ideal situation in which each loaf is stored under
118
Gouda and Related Cheeses
composition and conversion of various components can be affected in various ways, determining yield. Milk protein content and composition are of primary importance because the conversion of many other SnF components is more or less related to para-casein. There are different factors that may affect protein composition, in particular para-casein content of the milk (Walstra, 2000). Natural variations in milk composition and processing affect the amount of protein converted to cheese but there is also the risk of premature proteolysis resulting in the loss of peptides in the whey. In practice, modern infrared techniques are used successfully for milk analysis in the cheese plant. Season. Under Dutch conditions, yield is relatively high in autumn and low in spring, the discrepancy amounting to over 10%. This is due to the variation of the protein content, more precisely the para-casein content, because the ratio of casein/total (or true) protein is also not constant. The variation in non-protein-nitrogen (NPN) content greatly contributes to this phenomenon. The different types of milk protein and their analysis, para-casein in particular, in relation to cheesemaking, are discussed by van den Berg et al. (1996a). However, other SnF components that are converted into cheese (calcium, phosphate, citrate) also do not have a constant level in milk; even their ratio to casein is variable. Genetic variants of milk proteins. These may affect cheese yield (vnn don Rpro et al 1QQ')"Jnl~nh nncl Pl,hnn 1995). The presence of the B variant of [3-1actoglobulin correlates with a higher ratio of casein to total protein (TP) than the A variant. The B variant of K-casein is correlated with a higher (K-)casein content and better coagulation properties than the A variant. This results in a somewhat better protein recovery in spite of the loss of more caseinomacropeptide. Bulk milk does not have the ideal composition as regards these genetic variants, but improvement will cost much time and much logistic effort.
Mastitis. Severe mastitis leads to the production of milk with a reduced casein content and a reduced casein/total N ratio (Barbano, 2000). Actually, since large quantities of bulk milk are used, real problems are seldom encountered but control of somatic cell count (SCC) is still important with respect to cheese yield. Cold storage of the milk. The indigeneous milk enzyme, plasmin, is continuously active, even at low storage temperature. It slowly decomposes [3-casein into y-caseins and proteose-peptones (PP) and also Ots2-casein is hydrolysed; some of the PPs are lost into the whey. van den Berg et al. (1998) found in good quality milk (low SCC) during 3 days storage at 4 ~ an increase of TP in whey by 0.018%. This increase
holds for raw as well as for thermised milk and the rate of proteolysis is constant with time. Such loss may increase when SCCs or the psychrotrophic counts are considerably increased. Pasteurisation of the milk. Increased intensity of pasteurisation will increase yield (see 'Pasteurisation').
Rennet type. Proteolysis, other than the hydrolysis of K-casein, during cheesemaking will reduce yield, but the loss is usually negligible, unless some older types of microbial rennets are used. Starter. A change in the amount of starter added introduces several other changes. Firstly, the incorporation into the curd of denatured serum proteins will increase with the quantity of starter. Banks and Muir (1985) reported such an effect for Cheddar cheese and such could be confirmed by the authors with Gouda cheese. The casein from the acid starter liquid behaves during renneting like normal milk casein but a very small amount of casein is decomposed during propagation of the starter bacteria. This amount is, however, practically negligible in comparison with the amount of denatured whey proteins. So, the higher yield is caused by the increased the retention of serum proteins from the starter, since it is prepared from severely-heated milk. Secondly, when more starter is used to increase the acidification it inevitably requires, in Gouda-type cheese manufacture, more curd wash water (see 'Control of pH and water control'), which increasingly dilutes the whey and hence reduces the yield. The net result of both factors may be almost nil. Increasing the rate of acidification, by more active starter in particular, reduces the pH of milk and curd, dissolving more calcium and inorganic phosphate. Probably, the subsequent loss in y is small, say 30 g for 10 kg cheese produced if the pH at the separation of curds from whey is as low as 6.25 instead of 6.5. Moreover, somewhat more PP will leave the micelles at a lower pH and will be lost in the whey. A lower yield (a higher protein content in the whey) might be expected but the findings of van den Berg and de Vries (1975) do not point to such an effect in normal cheesemaking. At an extremely high acidification rate, a slight increase in the protein content of the whey during pressing was observed. Bactofugation. The severe heat treatment of the sludge that is added back to the milk (see 'Bactofugation') means an increase in yield because of the denatured whey proteins. Practically all whey proteins are denatured, just as is the case with the starter milk. For the amounts used in practice, it does not harm cheese quality. By increasing the pasteurisation temperature of the cheese milk by only 4-8 ~ a similar increase in yield may be obtained. However, in that case the more
Gouda and Related Cheeses
heat-sensitive whey proteins are primarily involved and, as mentioned in 'Pasteurisation', they may even cause deterioration of cheese flavour (van den Berg et al., 1996b). CaCl2. Addition of CaC12 to the milk causes some increase of colloidal calcium phosphate in the micelles (Walstra and Jenness, 1984). When 1 mmolA CaC12 is added to enhance the clotting, it will presumably increase cheese yield by - 3 0 g/100 kg of milk. Inclusion of native whey proteins. An appreciable increase in cheese yield may be obtained by accumulation of serum proteins in the curd when ultrafihration and renneting of the milk is followed by no, or very short, curdmaking and quick pressing (Buijsse, 1999). However, this technique is hardly practised in Gouda cheesemaking. In standard Gouda cheese, no more than approximately 1% of the protein in the cheese consists of native whey proteins (de Koning et al., 1981). In view of the results of van Boekel and Walstra (1989), on steric exclusion of serum proteins with respect to (para)-casein micelles, it is likely that Goudatype (and many other types of) cheese produced in the usual manner (no ultrafihration) contains insignificant quantities of native serum proteins. Curd washing. The dilution of whey with water at scalding affects y. Increasing the quantity of added water, e.g., from 30 to 40% (expressed per mass of curd and whey after the first whey has been sucked off) reduces cheese yield by approximately 0.5%. The effect is illustrated in Fig. 6. In the calculation of cheese yield, the efficiency of reducing the concentration in the curd particles by the washing was taken to be 90% for lactose and other low molecular weight substances, and 50% for the serum proteins. Lolkema (1991) found a similar relation between wash water and cheese yield at a higher yield level. Salting. Absorption of NaC1 obviously causes a gain in weight of the cheese. Against this profit there is usually a greater loss of moisture (see 'Brining'), and hence a net loss of weight. This moisture also contains dissolved components from the cheese. The quantity of salt absorbed varies, e.g., from 1 to 3%, and the net weight loss may vary from, say, 0.02-0.06 kg/kg cheese produced. The loss of solids-not-sah ranges from 1 to 3 g/kg; this may include losses caused by mechanical damage during salting (such as slight amounts of curd pressed between the cover and the mould). Mechanical losses. During curdmaking and handling there are risks of mechanical damage to the curd that causes losses of fat and fines, as discussed in 'Curd-
119
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10.3
1213
10.2
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=
=
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0 2'0 4'0 6'0 ' 80 Water added to curds + whey (kg/100 kg) Yield of Gouda cheese (12 day-old, 41% water) as a function of the quantity of 'curd wash' water used. Water content and pH of the cheese are assumed to remain constant (recalculated from Posthumus et aL, 1963; from Cheese: Chemistry, Physics and Microbiology, 2nd edn, Fox, P.E, ed., Volume 2, Chapter 2, Chapman & Hall, London, with kind permission from Kluwer Academic Publishers).
making'. Moreover, during cutting of the drained and fused curd mass to be moulded, pieces of curd may be lost, but with modern draining and moulding machines such losses are normally rather limited. Strictly speaking, yield refers to the ultimate product, excluding curd remnants, fines and any rind trimmings which have to be discarded; y includes the (dried) latex coating. Clearly, yield cannot be predicted very precisely, the cheesemaking process being too complex. Even the random variation in water content makes exact prediction difficult. To predict the yield of cheese from a given vat of standardised milk one obviously will proceed on its protein content. When taking into account a standard process and the usual conversion factors, the weight of the cheese before brining already gives an indication of the water content at an early stage. Calculations of the conversion of many milk components into the final cheese are given by van den Berg et al. (1996a), including various practical complications of analytical methods and methods to set up a moisture control system. Control of pH and water content
Very few quantitative data have been published on this subject. Process control has made considerable progress during recent decades because most variables have been identified and can be controlled (Straatsma and Heijnekamp, 1988). Besides the control of the curdmaking
120
Gouda and Related Cheeses
process, a very important factor is the degree of acidification of the curd and the freshly made cheese. The control of starter activity is a key factor and will be discussed separately in 'Starters: composition and handling'. However, certain m i n i m u m variations will still exist because of practical analytical uncertainties and imperfections. For example, the use of a continuous draining and moulding machine makes the demarcation of batches somewhat arbitrary. When weighing batches of cheese to get an idea of the expected water content of the final cheese, one will only decide about adjustment of process parameters after a number of batches have passed into the brine. It is not easy to adjust the water content and pH of cheese independently of each other. In 'Interrelations', interrelationships under varying conditions are considered. On the other hand, nowadays modelling of such a complex process is desired in order to be able to adjust the process immediately, but reliably. This has been done for Gouda cheesemaking and maturation by de Jong et al. (2002). In addition, the need to have an in-line measuring method for the moisture content of the individual cheeses before brining still exists and the recent approach of this problem by Frankhuizen et al. (2002) is interesting. Control of water content
The basic information with respect to this subject is outlined in 'The Syneresis of Rennet-coagulated Curd', Volume 1. In fact, we have to deal here with MNFS, rather than with the absolute water content of the cheese, which, within one cheese type, decreases fairly proportionally with increasing fat content. Besides FDM, MNFS is characteristic of the type of cheese and affects textural properties. As a matter of fact, numerous factors affect the water content, as discussed in 'Cheesemaking'. How they turn out under practical conditions was investigated by Straatsma and Heijnekamp (1988). The effects of some process parameters are illustrated in Fig. 7. Under normal manufacturing conditions, however, the number of process parameters available in practice that can actually adjust MNFS turns out to be restricted. Important are: (a) Cutting of the coagulum. The smaller the grains, the higher the syneresis rate, causing a lower water content. Cutting the coagulum very finely, however, seems to increase MNFS, and it causes a greater loss of fines and fat into the whey. An inhomogeneous cheese mass may result if the initial size of the grains differs widely. (b) Stirring of the curds-whey mixture. This concerns the intensity of stirring, which increases with the stirring rate and on any removal of part of the whey, the
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The effect of some variables in the treatment of milk and in curdmaking on the water content of unsalted Gouda-type cheese, 5.5 h after renneting, other conditions being equal; time means time after cutting. The water content under standard conditions was about 46% (from Straatsma and Heijnekamp, 1988; from Cheese: Chemistry, Physics and Microbiology, 2nd edn, Fox, P.F., ed., Volume 1, Chapter 5, Chapman & Hall, London, with kind permission from Kluwer Academic Publishers).
duration of stirring and (scalding) temperature which is, in practice, the first factor used to adjust water content. Extended stirring causes a lower ultimate water content. If the temperature of the curd mass is kept constant after separation of the whey, then the time of separation should affect the final water content only slightly. This indicates that in practical cheesemaking, lowering of the temperature after whey drainage rapidly restrains syneresis. (c) The above process steps also play a part in the acidification rate, since the temperature affects the activity of the starter bacteria, and since stirring can be stopped earlier at a higher syneresis rate, hence at a higher pH. Other variables occurring at renneting, pressing, shaping and salting have effects also but they are less suitable process parameters through which to adjust the water content from one batch to another. However, it is necessary for optimising the process that all effects of different steps, in particular when starting a new production line, are analysed. In this respect, it is important to study the variability of the water content within the cheese, between the cheeses of one batch, between batches and between production days. This gives insight into possible systematic deviations caused in different stages of the process, so that related process parameters can be found that must be adjusted (Straatsma et al., 1984). To these measures also belongs the cooling programme in the buffer tank to slow down further syneresis (see 'Draining and moulding'). This provides a means to control moisture content during
Gouda and Related Cheeses
final drainage of the curd and filling the successive moulds of the batch by inhibiting syneresis until closing the rind during pressing. This should be done in relation to batch time, and time between the mould filling and the start of pressing (see 'Pressing'). Differences in water content, size and shape of the cheese types made on the same line have to be considered also because of differences in moisture loss before brining (Geurts, 1978). In this way, the most profitable cheesemaking process, with low losses, a good yield and a good quality cheese of the right composition, can be found. If the process has been established in this way, then the cheesemaker will adjust the water content primarily with small variations in the scalding temperature. To give an idea of the increase in the dry matter content (DM) at different stages of a modern process for normal Gouda cheese, the following values may be mentioned: milk, 12.8%; end of curd preparation, 32%; at mould filling, 42%; after pressing, 53%; after brining, 57%. Control of the pH Here, we deal with the pH at one or a few days after making the cheese; after this, the pH gradually but slowly increases as a result of citrate fermentation, loss of CO2 and proteolysis. The course of pH and lactose content in the core of normal Gouda cheese is illustrated in Fig. 4. During brining, the pH of the outer rind zone will decrease further because of the low pH of the brine. During ripening under well-controlled conditions, the pH of the outer rind soon increases to a level slightly higher than in the core. Any (undesired) visible microbial growth on the surface will increase the rind pH considerably. The lactose in the rind after brining (see 'Brining') will disappear within a few weeks. The pH of the cheese results mainly from the amount of lactic acid, on the one hand, and that of the buffering compounds on the other. The acid is produced by the starter bacteria metabolising the available lactose. The main buffeting substance in curd and cheese is the calcium para-casein-calcium phosphate complex, of which calcium phosphate contributes to roughly one-third of the buffering capacity. Lactic acid itself is a buffer in cheese at low pH (pK = 3.9). The other salts in the curd moisture presumably play a minor part; after about 2 weeks, citrate to a large extent has been fermented by starter organisms (DL-type). During cheesemaking, acidification should be under control. If Gouda cheese is brined shortly after pressing, some lactose is usually left in the outer rind portion, but it disappears under normal ripening conditions within a few weeks. In the core of Gouda cheese, all lactose is metabolised within 12 h, mainly to lactic acid. If we presume that the water content of the MNFS is adjusted to its desired value, important compositional characteristics of the milk in relation to the final pH are:
121
(a) The lactose content of the milk serum (rather than the lactose to casein ratio in the milk). (b) The quantity (and composition) of calcium phosphate in the casein micelles; a changed buffering capacity of the curd is due predominantly to a different calcium phosphate concentration. As soon as the cheese milk has been collected and bulked, these variables (a and b) are fixed. To make the desired type of cheese from this milk, the important process parameters involved are: (a) Factors affecting the water content of the cheese. The higher the water content, the more lactose, or its equivalent as lactic acid, that is present in the cheese, and the lower the pH will be. In other words, from the moment the cheese loaf is formed, the ratio between incorporated lactose and buffering substances controls the pH. It has been observed that, other things being equal, increasing the water content of Gouda cheese by 1% decreases the pH by 0.1 unit. (b) The decrease in pH during curdmaking, and the ensuing loss of calcium and phosphate into the whey, may play a role too. These phenomena depend on the buffering capacity of the curd, on the amount of added calcium chloride (which reduces the pH slightly), and on the degree of acid production, which is, in turn, affected by the amount and type of starter added, the temperature, any pre-acidification, infection with bacteriophage and the presence of inhibitory c o m p o n e n t s - antibiotics and disinfectants, agglutinins (active in milk but not in curd and cheese) and the peroxidase-H202-thiocyanate system. These factors should be under control. Stronger acidification will reduce the pH by only - 0 . 2 units at moulding; this causes dissolution of little calcium phosphate and hence it would have only a minor effect on the final pH, but it may affect texture. On the other hand, a decrease in pH increases the syneresis rate, which affects the water content and hence the pH (via point (a)). If the water content is kept constant by other means, a small effect still remains, since now a slightly smaller quantity of the buffering calcium phosphate is incorporated into the curd, causing a lower final pH and a lower cheese yield (see 'Yield'). So a well-controlled process is necessary to maintain a constant buffering capacity in the cheese. (c) If this is under control, then the best process parameter to adjust the pH is washing. After the addition of water to the curds-whey mixture, lactose diffuses from the grains into the whey to equalise the lactose concentrations inside and outside the particles, although equilibrium is rarely reached. When the size distribution of the particles is normal and the contact time with the wash water is 25 min, the efficiency of reducing the lactose concentration in
122
Gouda and Related Cheeses
the curd is at least 90% (van den Berg and de Vries, 1974). More water causes a lower yield (see 'Yield'), as well as a less-valuable whey. Traditionally, curd washing is a one-stage process but recently it has been proposed to reduce the total a m o u n t of washing water by a two-stage process. After removal of the first whey, a small a m o u n t of water is added and --~20 min later some whey is removed, followed by the addition of another small a m o u n t of water. This might reduce water usage by 10-20%, while still giving the same washing effect (Verschueren et al., 2002). Figure 8 illustrates the quantities of water to be added under conditions related to normal Gouda cheese manufacture. The apparent lactose content in the fat-free dry matter represents the ratio between lac-
35
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tose and buffering substances (see point (a)). The adjective 'apparent' means the lactose still present plus the lactose already fermented into lactic acid before salting; these values are combined in order to be able to make calculations. Note that increasing the water content of the cheese before salting from 45 to 47% increases the amount of water to be added by 15%. A 0.2% higher lactose content of the milk necessitates 5% more added water. Interrelations
If a cheese with a high water content is desired, e.g., 47% after salting, and a normal pH of 5.15, then in addition to gentle cutting and stirring, much water at a relatively low temperature must be added. To obtain a normal content and a high pH, whey drainage and water addition may be repeated. If the water content is to be normal and the pH low (4.9-5.0), addition of water should be omitted. Then, scalding can preferably be achieved by means of hot water in the vat jacket, or by adding heated whey (which formerly was a common practice in Edam cheese manufacture). If an extremely low pH is desired, giving a typical short consistency, the milk may be preacidified. When making a very brittle type of Edam cheese, even the addition of lactose to the milk has been practised. To obtain a low water content and a normal pH, the coagulum should be cut rather finely and, after removal of part of the whey, it should be stirred vigorously at a rather high scalding temperature. Heating should, again, be indirect and slow to prevent the formation of curd particles with a 'skin'. It will be obvious that such a process approaches that for hard cheese and it explains why in Emmental and Parmesan cheesemaking, the curd is never washed. Normally, the water content of the cheese is considered but concerning the control of pH, MNFS is primarily involved. Sometimes, FDM may be changed (see 'Standarisation'), which means that MNFS is changed also. It has been calculated for Gouda cheese that increasing FDM by 2% at a constant water content requires an increase of washing water by 7%. Starters-composition and handling
42
44
46
Water in cheese (%) Amount of curd wash water to be used in relation to the lactose content of the milk (A) and the water content of the cheese before brining (B). Figures near the curves indicate (apparent) lactose (%) in the fat-free dry matter of the finished cheese (A) and lactose (%) in the milk (B). In A, the water content of the cheese is 46%; in B, the (apparent) lactose content in the fat-free dry cheese is 4.85% (from van den Berg and de Vries, 1976).
Industrial cheese starters are usually composed of acidforming lactococci, Lactococcus lactis subsp, lactis and Lc. lactis subsp, cremoris, possibly in combination with citrate-positive strains of Lc. lactis subsp, lactis and/or Leuconostoc spp. Cheese starters are categorised into O, L and D type of starters. The L refers to the presence of Leuconostoc spp. in the starter, the D to citrate-positive strains in the starter. DL-Starters contain both types of citrate-utilising organisms, while o-starters lack both
Gouda and Related Cheeses
types of organisms. Selection of the type of starter is strongly dependent on the suitable properties of the starter, such as flavour formation, phage resistance and eye formation. Detailed information on these organisms, for example on their taxonomy, physiology, biochemical characteristics, phages and phage resistance, and on the composition of starters and their propagation can be found in 'Starter Cultures: General Aspects', Volume 1. Specific information on Dutch starters for cheesemaking was published by Stadhouders (1974) and Stadhouders and Leenders (1984). Industrial cheese starters can be divided into two groups, undefined and defined starters. Artisanal starter cultures, derived from raw milk production practice, are traditional undefined mixtures of strains. These spontaneously developed starters are still used in traditional, small-scale factories, located in various parts of the world, e.g., in Southern Europe. Their composition is complex, relatively variable and often poorly defined. The artisanal starters are phage-carrying and partly phage-resistant. Therefore, these starters can be used in cheese farms/factories without any controlled protection against air-borne bacteriophages. They do not suffer complete failure of acid production when they become contaminated with disturbing phages, but the strain composition of the starter is greatly affected and the rate of acidification may vary considerably (Cogan, 1996; Limsowtin et al., 1996; M~yr~-Makinen and Bigret, 1998). Modern large-scale cheese factories require the use of starters with consistent activity. Acid production in cheese must proceed fairly quickly and at a constant rate, the latter being essential for the control of syneresis and the water content of the cheese. Therefore, mixedstrain starters, originally derived from artisanal production practice, are propagated under controlled conditions. This ensures a more uniform bacterial composition of starters and controls their rate of acidification when they are propagated under complete protection from phage. In The Netherlands, the mixed-strain starters currently used were selected originally from artisanal practice, according to their taste and flavour formation properties, rate of acidification, capability to induce eye formation and phage resistance. They are kept as inoculated milk in a frozen condition. Sub-culturing is minimised, which preserves their functional properties, population and phage resistance. These so-called mother starters serve for the production of concentrates for bulk starter preparation. The concentrates are distributed to the cheese factories in a frozen state (Stadhouders and keenders, 1984). The most common procedure for the manufacture of bulk starter is as follows. Bulk starter milk is pasteurised, e.g., for 30 min at 95 ~ or 1 rain at 110 ~
123
The intensity of the heat treatment is aimed at the destruction of thermo-resistant phages in the milk. Specially designed bulk starter equipment offers an effective barrier against air-borne phages. Generally, the room above the milk in these tanks is provided with an over-pressure of phage-free air made up by passing a (high efficiency particulate air) HEPA filter. Moreover, a special device is mounted on top, enabling decontamination of the outer side of boxes of starter concentrate with hypochlorite solution before the starter is introduced into the tank (Stadhouders et al., 1976; Lankveld, 1984). Additional precautions should be taken to avoid accumulation of disturbing phages in the factory, which especially could affect the rate of acidification of the curd in the vat. These measures include: the manufacture of bulk starters in separate rooms, use of closed equipment, cheese vats in particular, frequent cleaning and disinfection of all installations. Cheese whey is a specially dangerous source of phage contamination, and its processing equipment, the self-desludging separators in particular, is also separated from the cheesemaking room. Starters are propagated for 18-24 h at 20 ~ In almost all modern factories, the starter is automatically metered and added to the cheese vat. Starters may be kept for a limited time (e.g., 24 h) below 5 ~ without loss of activity. The activity of the bulk starter should be the same on successive days of manufacture. Activity is usually assessed by an IDF-standardised activity test (Stadhouders and Hassing, 1980) performed with a standard, pasteurised, reconstituted, high-quality skim-milk powder, and also with the pasteurised cheese milk, which ought to be skimmed. The activity of the starter in either of these milks should be constant. Any change in activity can be an indication of (i) a contamination of the starter with disturbing bacteriophages, (ii) a reduced activity of the starter (e.g., if it had been kept too long at a low temperature), (iii) the presence of antibiotics and/or disinfecting agents in the cheese milk or (iv) variations in the composition of the milk. To a certain extent, variations in activity may be corrected by adjusting the quantity of starter added to the cheese milk, or by adjusting other conditions during curdmaking, e.g., the scalding temperature. It must be remarked that results of the activity test and the acidification rate of cheese are not always precisely related because of different conditions in milk and fresh cheese, notably phage concentration, but this should be under control. According to practical standards for the Dutch cheese industry, the pH of cheese should be 5.8-5.9 after 4 h from the start of manufacture, and 5.4-5.5 after 5.5 h (Northolt and Stadhouders, 1985), as indicated in Fig. 4.
124
G o u d a and Related C h e e s e s
Nowadays, most commercial suppliers market undefined mixed-strain starters for direct vat inoculation (DVI) as well. This requires a much higher concentration of the micro-organisms in the deep-frozen concentrate to obtain similar acidification rates compared with bulk starters. The technology of DVI eliminates unnecessary sub-culturing within the factory and reduces many difficulties associated with it (Sandine, 1996). Defined-strain starters are blends of two or more strains. They are frequently used nowadays instead of the former undefined mixed-strain starters. Since the risk of phage attack is greater here than with the use of undefined mixed-strain cultures, cultures with different phage-sensitivity profiles are used in rotation. Definedstrain starters are less common for Gouda-type cheese than, for example, for Cheddar cheese production (Pearce, 1969; Heap and Lawrence, 1976; Limsowtin et al., 1977; Heap, 1998). However, the use of single strains as an adjunct starter in combination with a DVI (undefined or defined) starter is increasingly popular in semi-hard cheeses, such as Gouda-type. The single-strain adjunct starters are highly flexible in generating various cheese features, such as eye size and cheese flavour.
the cheese is unequal, primarily concerning salt and water, as shown in Fig. 9. The more concentrated the brine and/or the greater the weight loss by evaporation in the curing room, the higher the water content before brining should be to obtain an equal water content after 2 weeks when cheese composition is officially checked in The Netherlands. This results in a higher water content in the core after brining and this will affect consistency and ripening processes (de Vries, 1978). The water content in the core of the cheese during the first weeks of ripening has practically already been established during pressing. As far as salt has diffused during brining, the water content has decreased by pseudo-osmosis and this reduction will continue during ripening. At the surface of the cheese, the salt concentration in the cheese moisture (Sw) is similar to that of the brine, and the stronger the brine, the lower the water content is in the outer rind zone. This is a totally different condition compared to cheese made from dry-salted curd, like Cheddar cheese. Further, the calcium and the Salt concentration in the cheese moisture (Sw) (%)
Water content (W) (%)
18
Maturation is the result of numerous changes occurring in the cheese. The structure and composition and organompuc properties of cneese . . . alter . .greatly. Development of cheese properties is due particularly to the conversion of lactose, protein, fat and, in Gouda-type cheeses, of citric acid. Cheese technology and composition greatly affect cheese consistency and performance, both directly and indirectly, as discussed by Lawrence and Gilles (1986). 1
16
W
..
Cheese composition during ripening
During ripening, enzymes such as residual chymosin or other clotting enzymes, residual plasmin and proteolytic enzymes from the starter bacteria are still active (see 'Proteolysis in Cheese during Ripening', Volume 1). Environmental factors in the cheese, e.g., available water (aw), salt concentration, lactate concentration, pH and lack of oxygen, as well as curing room conditions, are important for their activity. The degree of lysis of the starter bacteria, liberating their enzymes, is also affected by these conditions. In this respect, the composition of the cheese and the method of producing it are key factors for ripening, as discussed by van den Berg and Exterkate (1993).
Composition after brining After brining, when ripening has started, the cheese contains all components but the composition within
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Distance from surface (cm) Gradients (idealised) of water content (W) and salt concentration in cheese moisture (Sw) in a zone of 6 cm below the rind of Gouda cheese, just after brining, for a normal process (4 days) and for a longer brining time (7 days) at a lower water content and weaker brine (dotted lines), according to van den Berg et aL (1975).
G o u d a and Related C h e e s e s
phosphate contents have also been established before brining, mainly by the acidification rate. Control of the pH has been discussed (see 'Control of the pH'). During normal Gouda cheesemaking, "-7% of the chymosin added to the cheese milk is retained in the curd (Zoon et al., 1994). It is mainly adsorbed by the protein and not inactivated during processing because the scalding temperature is mild, in contrast to the high cooking temperature in hard cheesemaking. The amount of residual active chymosin will be increased by (for a-d see Stadhouders and Hup, 1975): (a) (b) (c) (d)
using more of this coagulant; lower pH at curd drainage; probably by low scalding temperature; probably by increased moisture content of the cheese (at least enhances activity); (e) more intensive milk pasteurisation (van den Berg et al., 1996b).
Some plasmin is present in the fresh Gouda cheese but not as much as can be expected in hard cheese from high-cooked curd (Fox and Stepaniak, 1993). Another essential variable is the bacterial population in the fresh cheese as it greatly affects ripening and the possible development of defects (see 'Possible microbial defects'). This concerns starter bacteria and contaminating organisms, respectively. Concerning the latter, strictly enforced hygienic measures must be taken to prevent the growth of, e.g., mesophilic lactobacilli (Stadhouders et al., 1983c). The viable count of lactic starter bacteria (LAB) in the standard process is practically constant, at 1 • 109/g cheese (Stadhouders, 1974). This number has been reached after 3-4 generations at normal inoculation; the pH is then 5.7, which is the case shortly after pressing. This count will soon start to decrease due to cell death, usually followed by lysis during ripening.
Natural ripening During ripening, normal Gouda cheese loses water by evaporation. The decrease in the overall water content of the cheese during the first 10 days is ---1.5% but the rate decreases steadily with time. Within a year, the water content will have decreased by - 1 0 % . The air humidity is the driving factor to maintain a water gradient in the cheese, and water diffusion within the cheese is the limiting factor in weight loss, except during the first 10 days, when increasing air velocity also enhances evaporation (Bouman, 1977). Certainly during the first month, ripening in the core of the cheese is practically not hindered by the increasing salt concentration or the decreasing water content. This and the higher curing temperature of at least 12 ~ favours the ripening process in comparison with, for example,
125
Cheddar cheese. So Gouda cheese is ready for consumption at 4 weeks after manufacture, when it has a mild flavour. However, salt diffusion into the core continues during further curing and in the meantime the water content decreases. The water content in the rind zone of 0.5 cm decreases to <30% within 3 months. It has been found that proteolysis has practically stopped in that area, as shown in Fig. 10. This drying and lack of proteolysis gives the rind of a mature cheese its tough and hard consistency and a horny appearance. It is clear that the lack of proteolysis in the cheese rind reduces the values for N fractions, e.g., SN, pH 4.6-SN, PTA-SN (or AN) of the usual representative cheese samples. In the outer rind zone, another phenomenon is worth mentioning, although all its consequences are not well understood. Inside the cheese, the redox potential (Eh) is approximately-140 mV, caused by lactic acid fermentation, which means that the core of the cheese is anaerobic. This implies that in the rind zone a certain redox potential gradient must be present because outside the cheese, normal oxygen pressure exists. This explains why slowly growing microaerophilic moulds can grow under the plastic coating of cheese contaminated by long-time contact with contaminated shelves (Stadhouders and Labots, 1962). Slight lipolytic activity from moulds and yeasts on the TN(%) 50
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126
Gouda and Related Cheeses
surface has been found in the rind zone of cheese (Stadhouders and Mulder, 1959). It may be expected that their growth, although practically invisible, cannot be fully prevented and the rind zone will acquire a slightly higher fat acidity (see also 'Proteolysis and lipolysis'). Further investigations are desirable on whether more micro-organisms can grow under such conditions. Moreover, it would not be surprising if other chemical or enzymatic reactions in this zone produce relevant flavour compounds (see also 'Foil-ripening'). It will be obvious that because of the direct contact between the cheese and the shelf, adequate hygienic measures must also be part of the treatment scheme for the shelves, but transfer of off-flavours of, e.g., chlorophenolic compounds into the cheese must be avoided also. In this respect, glues with phenolic components used for the construction of the shelves are dangerous, because disinfection by hypochlorite will easily result in the formation of these compounds.
Foil-ripening Nowadays, a considerable amount of Gouda or Edam cheese is packed after brining in a plastic foil with very low permeability for water and gases to avoid weight loss, mould growth and all the effort needed to keep the naturally ripening cheese in the curing room (see 'Rind treatment and curing'). Such cheeses are made in a rectangular shape and piled in palletised cases to keep their shape. After ripening, they can easily be cut into consumer-size packages without losses. However, there are clear differences when foil-ripening cheese is compared with naturally ripened cheese (see 'Yield'):
(a) A constant average water content during curing. (b) Before brining, the water content is lower by 2-3%; otherwise the cheese will be too soft after the usual (rather short) ripening time. (c) The ripening temperature is lower, namely 5-6 ~ (d) There is no oxygen available at the surface of the cheese. (e) Brining time is longer because of the lower water content. (t3 The cheeses are brined immediately after pressing, which means that the lactose content in the outer rind zone is still higher during the first weeks. At the start of brining, the pH is still so high that the final growth of the starter bacteria might be hindered by cooling in the brine. (g) Diffusion of salt and water after brining is still slow due to the lower curing temperature, the lower water content and the weaker water gradient in the cheese, because no water evaporation occurs. (h) Eventually, the physical rind zone disappears and is gradually subjected to normal proteolysis.
The majority of foil-ripened cheese is ripened for a short period of 1 month. After a longer time, when the proteolytic ripening parameters like the soluble nitrogen (SN) and the amino acid nitrogen fraction of total nitrogen (AN) are comparable with those of naturally ripened cheese, flavour formation is, however, generally poorer and consistency is softer and tends to be sticky. The differences in consistency may be caused to a large extent by the water content, at that time being higher than that of naturally ripened cheese. The influence of the absence of oxygen at the surface of the cheese is not well understood and warrants further investigation. An advantage seems to be that the lowripening temperature prevents the cheese from 'lateblowing'. However, if, after ripening, the temperature is not controlled during transport and storage, this risk still exists. Fermentation of lactose and citric acid
The formation of lactic acid by the starter bacteria is paramount for the preservation of cheese. By their action they: (a) ferment lactose quickly and almost completely; consequently, the cheese soon lacks available carbohydrate. (b) produce lactic (and a little acetic) acid and reduce the pH of the cheese to 5.1-5.2. At the end of fermentation (after about 10 h), the lactic acid concentration in the cheese moisture is about 3%. Part (usually 4-7%) of the lactic acid is present in its undissociated (i.e., bacteriostatic) form, the more so if the pH is lower. (c) reduce the redox potential of the cheese to about - 1 4 0 to - 1 5 0 mV at approximately pH 5.2, as measured with a normal hydrogen electrode (kangeveld and Galesloot, 1971; Northolt and Stadhouders, 1985). All these changes aid in inhibiting the growth of undesired micro-organisms; salt uptake by the cheese, the presence of a protective cheese rind and the adequate treatment of this rind also contribute (see 'Rind treatment and curing'). Microbial defects should always be prevented (see 'Possible microbial defects'). Individual strains in a mixed-strain starter may differ greatly as to growth rate, the maximum number to which they grow in cheese and the rate at which they lose viability and subsequently lyse during cheese ripening. Cheese milk is commonly inoculated at a level of 5 X 106-107 starter bacteria/ml of milk. Mechanical inclusion in the curd leads to 5 • 107-108 cfu/g of curd, where the bacteria grow to, at most, --~109 cfu/g; this implies that starter bacteria generate (divide) only a few times in the fresh cheese. After growth, fermentation
Gouda and Related Cheeses
is far from complete (pH of cheese - 5 . 7 ) , and during further conversion of lactose, growth and fermentation are uncoupled. Fermentation of citric acid is of particular importance to eye formation in Dutch-type cheese. The D L and L-starters used in the manufacture of Dutch-type varieties ferment citric acid, but DL-starters do so more rapidly and produce more CO2; they are, therefore, used if more extensive eye formation is desired. The rate of decrease of the citric acid content in the young cheese may be used as an indication of the capability to induce eye formation (Northoh and Stadhouders, 1985); the rate of citric acid fermentation is, however, not the only factor involved in eye formation (see 'Texture'). Proteolysis and lipolysis
Proteolysis Protein breakdown in Gouda-type cheese is due mainly to the remaining action of coagulating enzymes, enzymes of starter bacteria and, to a much lesser extent, milk proteinases. Basic information about these proteolytic systems is given in 'Proteolysis in Cheese during Ripening', Volume 1. The separate and combined action of these systems in Gouda cheese has been studied intensively by making use of aseptic milking and cheesemaking techniques (Kleter and de Vries, 1974; Kleter, 1975, 1976, 1977; Visser and de Groot-Mostert, 1977; Visser, 1977a-d). Effectively, the action of calf rennet is determined predominantly by the amount remaining in the curd. Gouda-type cheese contains approximately 0.2 ml rennet (strength 150 IMCU)/kg of cheese (see 'Composition after brining'). The action of the coagulant enzymes, predominantly chymosin, is characterised by the rapid degradation of Otsl-casein at the onset of maturation, about 70-80% being hydrolysed within 2 months in standard cheese. [3-Casein is degraded far more slowly, about 40-50% remaining even after 6 months (van den Berg and de Koning, 1990). When using more calf rennet and a lower scalding temperature, the degradation of Otsl-casein is even more rapid (Visser, 1977d). Rapid breakdown of Otsl-casein is particularly favoured by the pH of the cheese being near to the optimum (about 5) for rennet action, and a still low NaC1 content in the cheese moisture of the core (see 'Composition after brining'). [3-Casein degradation is slowed down considerably, even at this low NaC1 content (Noomen, 1978b). Calf rennet appears to be responsible for the formation of most of the SN and the liberation of high and low molecular weight (MW) peptides, but only very low amounts of amino acids. After the primary proteolysis of the casein, the caseinderived peptides are hydrolysed to small peptides and
127
amino acids in the secondary proteolysis due to the action of the complex proteolytic system of the starter bacteria. The proteolytic system of dairy LAB has been studied extensively (Pritchard and Coolbear, 1993; Poolman et al., 1995; Christensen et al., 1999). The proteolytic system can be divided roughly into three main components with specific modes of action, as follows: (a) The first step in the degradation of the caseinderived peptides is catalysed by an extracellular proteinase, which is a member of the serine proteases of the subtilisin family. The proteinase is anchored to the cell membrane and is located extracellularly. The proteinase hydrolyses the casein-derived peptides to oligopeptides (Hugenhohz et al., 1984; Exterkate et al., 1993; Kok, 1993). (b) Subsequent degradation of the oligopeptides is catalysed by intracellularly localised peptidases. Therefore, peptide transport systems are crucial. Various transport systems with different specificities have been characterised. Amino acid transport systems, two ditripeptide carriers and an oligopeptide transport system have been characterised in dairy lactococcal cells (Kunji et al., 1995). All these transport systems are active, energy-driven processes. (c) The oligopeptides transported into the cells by the transport systems are subsequently hydrolysed by various peptidases into small peptides and amino acids. Approximately 13 different peptidases have been characterised with different specificities (Kok and de Vos, 1994). During cheese manufacture, the carbon source is depleted, which de-energises the microbial starter. Active transport of the oligopeptides into the cell is, for this reason, reduced. Starvation of the starter during cheese ripening and, subsequently, permeabilisation of the starter cells is essential for the ongoing amino acid production in cheese. Starters may vary greatly in their sensitivity to lysis (W.C. Meijer, E Kingma, A. van Boven and J. Hugenhohz, unpublished results). Bacterial lysis is of great importance for the final cheese flavour, since amino acids are the main substrate for the final cheese flavour (Wilkinson et al., 1994; Crow et al., 1995; Morgan et al., 1995). When acting alone in cheese, milk proteinases may hydrolyse Ors1-, o%2- and [3-caseins to some extent during prolonged ripening, due to which small amounts of low-MW peptides and amino acids are liberated (Visser, 1977c). The pH and the NaC1 content sof mature Gouda-type varieties are not very favourable for the activity of most enzymes; in particular, plasmin activity is reduced greatly (Noomen, 1978a). In normal cheeses, where all enzyme systems act together, no clear mutual stimulation or inhibition of
128
Gouda and Related Cheeses
the systems in the formation of soluble N components is observed. The action of rennet clearly stimulates the starter bacteria to produce amino acids and low-MW peptides, which is most likely due to the progressive degradation by starter peptidases of the higher-MW products of rennet action. Contents of soluble N compounds (Visser, 1977c) reflect the 'width' of ripening. The 'depth' of ripening is defined as the ratio between the amount of degradation products of low MW, e.g., amino acids or peptides with MW <1400, and the total amount of soluble breakdown products. In that sense, the 'width' of ripening of Gouda-type cheese is predominantly determined by rennet action, and the 'depth' by the action of starter bacteria. Serum proteins seem to be hardly degraded in cheese; an exemption might be made for denatured, heat-sensitive whey proteins (see 'Pasteurisation'). Many selections of starter bacteria have been made on the basis of proteolytic activity and/or lysis sensitivity. Use of these bacterial starters has demonstrated the huge impact of these parameters on flavour intensity and flavour diversification. However, shortening the ripening time of Gouda cheese with organoleptic characteristics exactly corresponding to those of the normal cheese is still a challenge. The application of specific, highly proteolytic, thermophilic starter bacteria has resulted in cheese which ripens more quickly and
scarcely noticeable (Kleter, 1976, 1977). One may question to what extent variations in the earlier results were caused by differences in susceptibility of the milk to lipolysis, e.g., due to mechanical damage of the fat globules. Under well-controlled conditions during the making and the curing of cheese made from pasteurised milk, lipolysis in cheese will result predominantly from the action of starter bacteria, residual milk lipase and, possibly, heat-stable lipases of psychrotrophic organisms. Such conditions, in particular, imply a good bacteriological quality of the milk prior to thermisation or pasteurisation (especially, psychrotrophs should be virtually absent), and the absence of visible microbial growth on the cheese surface during maturation. Enzymes of the starter bacteria have very low activity on triglycerides, but are able to produce free fatty acids (FFAs) from mono- and di-glycerides formed by milk lipase and/or other microbial lipases (Stadhouders and Veringa, 1973). The latter might originate from NSLAB. The activity of milk lipase is reduced by NaC1 in the cheese. The action of milk lipase is also affected by the pH of cheese. Although this action has been found to decrease markedly with decreasing pH when assayed on substrates greatly different from cheese, the acidity of cheese fat has been reported to increase faster in cheese of low pH (Raadsveld and Mulder, 1949a). The explanation is unclear, though it should be noted that at a lower pH a
An example of this is the Proosdij-type cheese, which has been successfully introduced in The Netherlands under various brand names such as Parrano | In addition to mesophilic starter bacteria, a mixture of thermophilic lactobacilli and streptococci is used in its production, which otherwise follows the normal process for Gouda cheesemaking (van den Berg and Exterkate, 1993). These kind of adjunct starters are also used for low-fat cheeses (see 'Origin and characteristics').
will be in the fat phase (Veringa et al., 1976). Lipase activity in cheese increases markedly with temperature (Raadsveld and Mulder, 1949b). In cheese made from milk containing high numbers of psychrotrophic bacteria (or their heat-stable lipases), lipolysis may be increased to undesirable levels. Also, growth of organisms on the cheese surface, e.g., moulds, coryneform bacteria and yeasts, may contribute to increased acidity of the fat. Growth of such organisms, however, is usually minimised but cannot be fully prevented; consequently, the rind portion of the cheese generally acquires a somewhat higher fat acidity. Homogenisation of the cheese milk greatly enhances lipolysis in cheese, but is seldom practised. The FFA of cheese milk normally amounts to, say, "-~0.5 mmol/100 g of isolated fat. In a 6-week-old Gouda cheese made from HTST-pasteurised milk, this value averages about 1.3 mmol, which increases to about 1.6 after 6 months and to about 1.8 after 1 year. Cheese made from raw milk usually shows a higher FFA level.
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Lipolysis In Gouda-type varieties, some lipolysis usually occurs and is even desirable, but it should be limited, otherwise the cheese has a soapy flavour. Factors that affect lipolysis have been studied intensively by Stadhouders and Mulder (1960) and Stadhouders and Veringa (1973). Cheese made from raw milk shows the distinct action of milk lipase; if made from aseptically drawn milk containing a negligible number of lipolytic bacteria, fat acidity increases gradually (Stadhouders and Mulder, 1957). The HTST-treatment of milk, e.g., 15 s at 72 ~ largely, but not completely, inactivates milk lipase. Cheese made from aseptically drawn, low-temperature pasteurised milk still shows an increase in fat acidity during maturation, although this increase is slight or even
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During the last decade, it became clear that flavour development in cheese results from a series of
Gouda and Related Cheeses
(bio)chemical processes for which the starter cultures provide the enzymes. The balance between proteolysis and peptidolysis is very important in order to prevent a bitter off-flavour and also results in the release of FAAs. In particular, the enzymes which catabolise these FAAs play a major role. Various enzymatic and chemical reactions have been identified in the conversion of amino acids to volatile flavour compounds, and a good balance between all the flavour components is essential for the desired sensory quality of Gouda or related cheeses. Apart from flavour components derived from the caseins, in Gouda-type cheeses some flavour components derived from carbohydrate metabolism should also be mentioned. The main conversion of lactose obviously leads to the formation of lactate by LAB, which affects the flavour, giving a mild fresh acid taste. However, a fraction of the intermediate pyruvate can alternatively be converted to various flavour compounds such as small amounts of diacetyl, acetoin, acetaldehyde or acetic acid, some of which contribute to typical yoghurt flavours. Moreover, citrate serves as the main source for diacetyl due to the presence of citrate-fermenting strains in the starters used for Gouda preparation. In young cheese, citrate-fermenting strains in the starters used for Gouda cheese manufacture. In young cheese, diacetyl flavour can be easily distinguished but after 3 months this flavour has disappeared to a great extent because diacetyl is converted to acetoin during further ripening. Nevertheless, the conversion of caseins is undoubtedly the most important biochemical pathway for flavour formation in Gouda and related cheeses (van Kranenburg et al., 2002). Degradation of caseins by the activities of rennet enzymes and the cell-envelope proteinase and peptidases from LAB yields small peptides and FAAs. The balance between the formation of peptides and their subsequent degradation to FAAs is very important, since the accumulation of peptides might lead to a bitter off-flavour in cheese (Visser et al., 1983; Stadhouders et al., 1983b; Smit et al., 1996, 1998). Various bitter-tasting peptides have been identified in Gouda cheese and especially these peptides should be degraded rapidly in order to prevent bitterness (Visser etal., 1983; Stadhouders etal., 1983b; Smit etal., 1998). Specific cultures have been selected with good ability to degrade bitter-tasting peptides (Smit et al., 1998) and such cultures are nowadays used frequently in the preparation of various varieties of Gouda cheese. For specific flavour development, further conversion of amino acids is required to produce various alcohols, aldehydes, acids, esters and sulphur compounds involved in the flavour perception of Gouda cheese. Amino acids can be converted in many different ways by enzymes such as deaminases, decarboxylases, transamin-
129
ases (aminotransferases) and lyases. Transamination of amino acids results in the formation of ot-keto acids that can be converted to aldehydes by decarboxylation and, subsequently, to alcohols or carboxylic acids by dehydrogenation. Many of these components are odouractive and contribute to the overall flavour of the cheese (Fig. 11). Using biochemical and genetic tools, the various flavour-forming routes from amino acids and the enzymes involved have recently been identified, mostly in Lc. lactis (Ahing et al., 1995; Gao et al., 1997; Yvon et al., 1997, 1998, 2000; Engels et al., 2000; Smit et al., 2000; Yvon and Rijnen, 2001; van Kranenburg et al.,
2002). Aromatic amino acids, branched-chain amino acids and methionine are the most relevant substrates for flavour development in Gouda cheese. Volatile sulphur compounds derived from methionine, such as methanethiol, dimethyl sulphide and dimethyl trisulphide, are regarded as essential components in Gouda cheese varieties (Urbach, 1995). In fact, a Gouda cheeselike flavour can be generated by incubation of methionine with cell-free extracts of Lc. lactis (Engels and Visser, 1996). Conversion of methionine can occur via an aminotransferase-initiated pathway by branched-chain or aromatic aminotransferases, or via an or,y- elimination of methionine by the lyase activities of cystathionine [3lyase (CBL), cystathionine y-lyase (CGL) or methionine y-lyase (MGL) (Alting et al., 1995; Bruinenberg et al., 1997; Yvon et al., 1997; Gao and Steele, 1998; Rijnen et al., 1999; Engels et al., 2000; Fernandez et al., 2000, 2002; van Kranenburg et al., 2002). It has already been mentioned that various LAB strains differ in their amino acid-converting abilities and that these activities are in fact linked to the ability to synthesise amino acids. Ayad et al. (1999, 2000) focused on the ability of Lactococcus strains isolated from various natural sources, the so-called 'wild lactococci'. These strains originated from dairy and nondairy environments and had unique flavour-forming properties, when compared to commercially available starter strains. Using combinations of these strains makes it possible to develop tailor-made starter cultures for Gouda and related cheeses, a development with strong application possibilities. Texture Structure
Cheese consistency is discussed in 'Rheology and Texture of Cheese', Volume 1 of this book. For Gouda and Edam cheeses, a detailed study has been carried out by Luyten (1988). The main factors affecting the consistency in the core of these cheese varieties are moisture content, extent of proteolysis, pH, NaC1 and fat level,
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any inhomogeneity of these variables throughout the cheese mass and, of course, temperature. Levels of calcium and phosphate are fairly constant under normal cheesemaking conditions but they are somewhat lower than in Emmental and higher than in other cheese types (e.g., Cheddar and Camembert). The influence of most of these parameters in Gouda cheese has been discussed by Visser (1991). During natural ripening, several changes occur that may be important to texture: (a) Structure and composition become more uniform, particularly during the early stages, due to further fusion of curd grains and reduction of salt and pH gradients. The moisture gradient persists for a long time. (b) The cheese looses water by evaporation and ongoing syneresis, especially near the rind. (c) Maturation primarily implies breakdown of the para-caseinate network, OLs:-casein quickly, followed by [3-casein more slowly (remaining after 6 months, - 2 0 % and 40-50% respectively, as reported by Visser (1977b), van den Berg and de Koning (1990)); it also causes a slight increase in pH (formation of alkaline groups by proteolysis, degradation of lactic acid). (d) Gas is formed. The common result is that during maturation, the apparent elastic modulus of the cheese increases, the deformation at which fracture occurs decreases and the fracture stress at first decreases and subsequently increases again (Luyten, 1988; Zoon, 1993). This is shown in Fig. 12. During maturation, proteolysis dimin-
131
ishes the strain, and the stress increases mainly because of the lower water content and to a lesser extent by the increased salt concentration (salt/water). The modulus seems, for the same pH and NaC1 content, to depend on the water content in the fat-free matter only. The only rheological parameter that appears to correlate well with the degree of maturation of the cheese is the deformation at fracture, as was, for instance, found in a study of several, widely different cheeses ranging in age from 4 to 20 weeks (H. Oortwijn, unpublished results). The relative deformation (Hencky strain) at fracture is, say, 1.6 for unsyneresed curd, higher than 1 shortly after salting and about 0.5 after 3 months of maturation (Luyten, 1988). The complete fusion of the curd within few days, the still low salt concentration and the rapid start of casein degradation in the core soon give the cheese a smooth texture, while its 'longness' disappears gradually with ripening and tan 6 increases. These changing viscoelastic properties of curd and cheese are very important. During cheese formation, the curd deforms during pressing and fusion but, after a relatively short time, it remains deformed because casein bonds are broken and new ones are formed, within the curd particles as well as between caseins of neighbouring curd particles. After complete curd fusion, the cheese should still be able to flow slowly during the first month, which is a prerequisite for the creation of spherical holes. A cylindrical sample (obtained with a trier) of young Gouda cheese can be bent extensively before it breaks; during maturation of the cheese this flexibility is increasingly smaller. The rind zone of Gouda cheese shows a different development from the interior of the cheese. After
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132
Gouda and Related Cheeses
brining, this zone is white ('salted out'), short and brittle. Some weeks later, when the salt concentration declines below 7.5%, these properties have disappeared. In this zone, casein degradation is hindered by the high salt concentration and later even inhibited by the low moisture content. Then, the rind becomes gradually more tough and springy and is still distinguishable as a somewhat darker and translucent zone in the older cheese. This zone slowly increases in thickness; 3-4 mm after i year, depending on the degree of water evaporation (see also 'Natural ripening'). This is not the case with foil-ripening (see 'Foil-ripening') because no water evaporates. In the end, the high water content and proteolysis throughout the whole cheese give a soft and sticky consistent body without a rind zone.
In Gouda and related cheeses, the number, the size and the shape of holes are considered an important texture characteristic. Figure 13 shows a section of a typical Gouda cheese. Conditions allowing hole formation have been studied in some detail by Akkerman etal. (1989). Holes can be formed if gas pressure exceeds saturation and if sufficient nuclei are present. The gas is commonly N2 already present in the milk because this is saturated with air at 4 ~ when received at the cheese plant. During cheese manufacture, any 02 present is
tribute to the formation of the holes. The supersaturation needed for hole formation (by approximately 0.3 bar) can be achieved when the rate of CO2 production is relatively fast (which depends on temperature, type and number of bacteria and citrate content), its rate of diffusion (D = 3 • 10 -1~ m 2 s -1) out of the cheese is slow (mainly depending on loaf size and shape) and if the partial pressure of N2 is high (usually ---0.9 bar). Quantitative relations have been given by Akkerman et al. (1989). Nuclei are usually small air bubbles, either incorporated as such between the curd particles when the curd mass is 'worked' after draining off all whey, or already present in the milk and incorporated within the curd particles. The latter nuclei presumably exist as tiny air bubbles adhering to dirt particles and very small granules or partially coalesced fat globules; they can remain only if the milk is (almost) saturated with air. After normal pasteurisation of, usually, cold-stored milk, the time in the cheese vat before renneting allows sufficient deaeration by gentle stirring; otherwise, many pinholes will be formed in the cheese. Incomplete fusion of the curd, local inclusion of whey (curd lumps and disturbance of the curd b l o c k - see 'Curdmaking' and 'Draining and moulding') and inclusion of air at drainage may also serve as nuclei or may even disturb regular eye formation ('nesty' spots). Nucleation predominantly determines the number of holes, and their shape depends on cheese consistency, while
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next few weeks, CO2 is produced by starter organisms. This CO2, and possibly H2 produced by undesirable bacteria (see 'Possible microbial defects'), can also con-
Section through a normal Gouda cheese of 12 kg.
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production. If the latter is not too fast and the cheese consistency allows for viscous flow of the cheese body, eyes (i.e., spherical holes) develop. If the consistency is
Gouda and Related Cheeses
short, or, more precisely, if the fracture stress of the material at slow deformation is low, slits may develop because the cheese mass fractures in the vicinity of the holes. Such may be the case for a cheese of low pH, low calcium phosphate content and considerable proteolysis at the time of gas production, but quantitative relations cannot be given yet. The problem is that most variables causing the fracture stress to be low (implying easy slit formation) also cause the elongational viscosity of the cheese to be low (implying a low overpressure in the hole and thus less possibility of fracture; see Akkerman et al., 1989). Possible microbial defects
Butyric acid fermentation Butyric acid fermentation (BAF) is characterised by the catabolism of lactic acid principally to butyric acid, CO2 and H2: 2CH3CHOHCOOH --+ CH3CH2CH2COOH + 2CO2 + 2H2 Consequently, the growth of anaerobic, spore-forming, lactate-fermenting butyric acid bacteria (BAB), especially of Clostridium tyrobutyricum, may cause the 'late blowing' of cheese due to excessive production of CO2 and H2, and a very bad off-flavour. Grass silage, used as a feed in winter, but sometimes also as a part of the ration in summer, represents the main source of contamination of the milk with spores of BAB, especially when it is insufficiently preserved. Such silage contains large numbers of BAB spores, which survive passage through the digestive tract of the cow and are concentrated in dung. The degree of contamination of the milk with spores therefore strongly depends on hygienic conditions during milking (de Vries and Stadhouders, 1977), but even with modern methods of milking, slight contamination with faeces present on the udder cannot be prevented. This problem is more serious since the spores fully survive the HTST-treatment normally applied to cheese milk. The spores do not germinate in milk but in the cheese, where they can grow at > 7 ~ under anaerobic conditions with lactate as C-source. The redox potential will then be reduced from - - 1 3 0 mV to < - 2 0 0 mV, which may be used as an indicator for BAE Gouda cheese, as a brined cheese, is especially vulnerable to BAE the more so for larger cheese loaves. The normal eye formation is limited and too large holes are easily detected as a defect. Because of the serious nature of the defect, much research has been undertaken to find ways to reduce the number of spores in milk and to prevent their germination and growth in cheese. Factors studied include: bactofugation of the milk, addition to
133
the milk of nitrate, hydrogen peroxide or other oxidising substances, or lysozyme, the use of a nisin-producing starter, the salt content and pH of the cheese, cheese ripening temperature and amount of (undissociated) lactic acid (for relevant literature information, see van den Berg et al., 1980, 1988; Stadhouders et al., 1983c). The technique of microfihration is practically not used for Gouda cheese (see 'Bactofugation'). Nitrate may be used effectively to prevent BAF and has been used for this purpose for about 170 years. The mechanism of inhibition requires the presence of xanthine oxidase (EC 1.2.3.2), which reduces nitrate to nitrite (Galesloot, 1961). Nitrite is considered to delay the germination of spores for a certain period after brining (but the actual mechanism may well be more complicated, according to Stadhouders et al., 1983a). Later on, the inhibitory action is taken over by NaC1 when it has become evenly distributed throughout the cheese and if it is present at a sufficient concentration. If nitrite is the only factor involved in the initial inhibition, it must be very effective since it is present at only a very low concentration. The formation of nitrite was a reason to investigate the presence of nitrosamines in Gouda cheese. From the results of Goodhead et al. (1976), the existence of this danger appears to be not likely. Since xanthine oxidase is a milk enzyme, its inactivation by pasteurisation will increase between 72 and 82 ~ (see Fig. 2). In this way the effectiveness of nitrate declines. High numbers of coliform bacteria and some strains of mesophilic lactobacilli will degrade nitrate to nitrite during the first weeks, increasing the risk of BAE In cheese, nitrite is supposed to be degraded slowly eventually to NO and N2 that may diffuse outside the cheese. At a given curing temperature, usually about 14 ~ the combined effect of several factors determines whether growth of BAB is possible or not. Important factors promoting growth are a large number of spores in the cheese milk, a low content of undissociated lactic acid (hence usually a high pH), a low nitrate content in the cheese and a low level of NaC1 in the cheese moisture. The rate at which salt becomes homogeneously distributed throughout the cheese mass, its final concentration and the initial nitrate content of the cheese are, therefore, crucial. For example, a cheese with a high pH requires a higher than normal final salt concentration to inhibit growth. Since the pH of the cheese is increased by BAE growth conditions for the organism then become more favourable and consequently the rate of fermentation is accelerated. It is the experience of the authors that germination of the first spores may occur in cheese very soon. Therefore, the presence of nitrate on the first day is necessary and even a later start of brining by some
134
Gouda and Related Cheeses
hours will increase the incidence of BAE An L- or Otype starter makes the cheese somewhat less-sensitive to BAF in comparison with the use of the DL-type starter, probably because of the difference in production of acetate (Stadhouders, 1990). Low numbers of spores in the cheese milk, which also can be achieved by bactofugation, permit the amount of nitrate to be reduced considerably. If one wishes to produce cheese without nitrate addition, the critical number of spores in milk capable of causing the BAF is extremely low, ---5 spores/1 milk as found by van den Berg et al. (1988). To this end, effective double bactofugation of the milk is certainly necessary when making standard 12 kg Gouda cheese. Critical numbers of spores at different nitrate concentrations are also given in this study, e.g., ---250 spores/1 need 2.5 g nitrate/100 1 milk and ---10 000 spores/l need 15 g nitrate/100 1 milk. The latter number of spores may occur in the winter season. If then, only 2.5 g nitrate/100 1 milk may be used, single bactofugation with sludge sterilisation will be necessary under north-west European conditions. Lysozyme has often been proposed as an alternative to nitrate. Its use is somewhat more expensive than bactofugation for a similar protective effect. At the amount recommended (e.g., 500 IU/ml of cheese milk), it is usually less effective than nitrate, according to experience with Gouda cheese (Stadhouders et al., 1986). In comhinalion with qinale harlnfllg~fiem thiq amount may be used. Some spores are quite resistant to lysozyme, whereas others are readily inhibited or are even more sensitive to lysozyme than to nitrate (Lindblad, 1990). Nisin shows antimicrobial activity against a broad spectrum of Gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus spp. Currently, it is used in a wide range of foods and beverages, such as processed cheeses. However, for normal cheese manufacturing it cannot be used because it is lost in the whey and inhibits many starter bacteria. Recently, defined nisin-producing starter cultures were selected and have begun to be marketed; these could be used to manufacture good-quality Gouda-type cheese. Starters which produce nisin in situ during cheese manufacture give a very strong protection against spores of Clostridium tyrobutyricum and Staphylococcus aureus bacteria (Meijer et al., 1998). Lactobacilli Growth of mesophilic normal or salt-tolerant lactobacilli may cause flavour and texture defects, especially in mature cheese. Even when initially present at small numbers, e.g., 10/ml of cheese milk, some strains of common lactobacilli (Lb. plantarum, Lb. casei, Lb. brevis) may grow slowly in cheese to more than
2 • 107/g in 4-6 weeks (Stadhouders et al., 1983c), causing gassy and putrid flavours and an excessively open texture. Probably, amino acids are used as a carbon source. The organisms are killed by adequate pasteurisation of milk, e.g., 15 s at 72 ~ In industrial practice, continuously working curd-drainage machines were often an important source of contamination but improved designs, minimising 'dead spots', make longer standing times possible. However, growth on surfaces of tanks should also be considered. Especially when the salting of cheese is carried out in brine of reduced strength, there is a risk of defects caused by salt-tolerant lactobacilli, some strains being able to survive even in the presence of >15% NaC1. Furthermore, they differ from normal lactobacilli by their continuing growth in cheese and their active amino acid metabolism, causing phenolic, putrid, mealy and H2S-like flavours in 4-6-month-old cheeses. Some strains also produce excessive quantities of CO2, causing the formation of holes, either eyes or cracks according to the consistency of the cheese (Stadhouders et al., 1974). More than 103 of these gas-forming lactobacilli per ml of brine is considered to be dangerous. The lactobacilli may enter the cheese by penetrating the rind during brining, this being facilitated if the cheese is insufficiently pressed and the rind not wellclosed (Hup et al., 1982). Of course, contamination of the cheese milk with these bacteria must be prevented. If ...... 1. k ~ ; . . (e.g., . . . . . . . . . j . . . . ~,. . . . . . . . . . . . . y acid (pH <4.6) and cold (13 ~ growth of the organisms usually does not occur and they die gradually. However, increased numbers in brine originate from their growth in deposits, which are often present on the walls of basins just above the brine level, on racks and other equipment, and so contaminate the brine. Growth conditions for the lactobacilli are more favourable in these deposits as a result of the action of salt-tolerant yeasts increasing the pH, a lower NaC1 concentration (due to absorption of water) and a somewhat higher temperature than that of the brine. Measures to keep the number of lactobacilli low in brine include good hygiene in the brining room with removal of deposits, and adjustment of the NaC1 content of the brine to at least 16% and of its pH to <4.5 (Stadhouders et al., 1985). Thermo-resistant streptococci These bacteria are normally present in raw milk. In particular, strains of Sc. thermophilus may be responsible for cheese of inferior quality. In contrast to the mesophilic streptococci, they can grow at 45 ~ and survive thermisation (e.g., 10 s at 66 ~ and, to some extent, pasteurisation (e.g., 15 s at 72 ~ of milk. During such heat treatments, after some time a few organisms may become attached to the walls of the
Gouda and Related Cheeses
regeneration section of the heat exchanger and may then start to multiply very rapidly (minimum generation time, 15 min); this may depend on their initial number in the milk. Continuous use of heat exchangers for too long a period without cleaning may cause heavy contamination of the cheese milk (about 106/ml). Cleaning of the thermiser and the pasteuriser within 8 h is normally necessary. As a result of their high number in curd and growth during the early stage of cheesemaking, their number may increase to more than 108/g of cheese. They render the flavour of cheese 'unclean' and 'yeasty'. Moreover, CO2 production by these bacteria may yield cheese with an excessively open texture after approximately 5 weeks, especially if a starter with high CO2-producing capacity is used for cheesemaking (Hup et al., 1979; Bouman et al., 1982). Propionic acid bacteria Very considerable growth of these organisms in cheese results in the development of a sweet taste and a very open texture, due to excessive gas formation. Propionic acid bacteria can convert lactate into propionic acid, acetic acid, CO2 and H20 according to:
3CH3CHOHCOOH --~ 2CH3CH2COOH + CH3COOH + CO2 + H20
Consequently, the pH of the cheese does not change significantly. Because the bacteria develop very slowly in cheese at the commonly applied ripening temperature and salt content, any serious defects occur only after prolonged ripening. Several conditions determine their growth in cheese. The pH is decisive, significant growth starting only from 5.1 and increasing at higher values. Increasing the concentration of NaC1 retards their growth. So higher salt concentrations near the rind of the cheese may be inhibitory. A higher storage temperature favours the growth of propionic acid bacteria. Nitrate hinders their growth. When conditions allow growth of these bacteria in cheese, the development of BAB (if present) may also be expected, provided that growth of the latter is not otherwise prevented. Propionic acid bacteria are killed by normal pasteurisation of milk, e.g., 15 s at 73 ~ Therefore, they are predominantly of interest in the manufacture of cheese made from raw milk, farm-made cheese in particular. However, in factories also making Maasdam cheese, cross-contamination must be prevented. Yeasts and coryneform bacteria Abundant growth of yeasts and coryneform bacteria (and in extreme cases of B. linens) on the cheese surface may lead to a somewhat slimy rind and various
135
discolourations or pink appearance. Growth of these organisms is favoured by insufficient acidification of the cheese, leading to a significant lactose content in the rind, salting of cheese in brine with a low NaC1 content and a high pH, inadequate drying of the cheese rind after brining (this is the main factor in practice) and the use of insufficiently cleaned shelves. Inadequate drying is the main factor in practice because when the other items are under control, it still may happen. Consequences for cheeses waxed after maturation have been discussed (see 'Rind treatment and curing'). Growth of moulds causes discolouration and may under extreme conditions pose a health hazard because of mycotoxin formation. To prevent their development, special attention must be paid to the treatment of the cheese rind, the drying of the cheese surface and the hygienic conditions in curing rooms (see 'Rind treatment and curing').
As this text is an adapted version of the chapter on Dutch-type varieties by Walstra, Noomen and Geurts in the previous edition, the authors are grateful for their kind permission.
Akkerman, J.C., Walstra, P. and van Dijk, HJ.M. (1989). Holes in Dutch cheese. 1. Conditions allowing eye formation. Neth. Milk Dairy J. 43,453-476. Akkerman, J.C., Fox, EH.J. and XNalstra, P. (1994). Drainage of curd; expression of single curd grains. Neth. Milk Dairy J. 48, 1-17. Akkerman, J.C., Buijsse, C.A.P., Schenk, J. and Walstra, R (1996). Drainage of curd; role of drainage equipment in relation to curd properties. Neth. Milk Dairy J. 50, 371-406. Ahing, A.C., Engels, WJ.M., van Schalkwijk, S. and Exterkate, EA. (1995). Purification and characterization of cystathionine [~-lyase from Lactococcus lactis subsp. cremoris B78 and its possible role in flavour development in cheese. Appl. Environ. Microbiol. 61, 4037-4042. Arentzen, A.GJ. (1972). Gerichte bemonstering bij de vochtbepaling in kaas (Special sampling for the determination of water content in cheese). Off. Orgaan FNZ 64, I003-1006. Ayad, E.H.E., Verheul, A., de Jong, C., Wouters, J.T.M. and Smit, G. (1999). Flavour forming abilities and amino acid requirements of Lactococcus lactis strains isolated from artisanal and non-dairy origin. Int. Dairy J. 9,725-735. Ayad, E.H.E., Verheul, A., Wouters, J.T.M. and Smit, G. (2000). Application of wild starter cultures for flavour development in pilot plant cheese making. Int. Dairy J. 10, 169-179.
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Gouda and Related Cheeses
Banks, J.M. and Muir, D.D. (1985). Incorporation of the protein from starter growth medium in curd during manufacture of Cheddar cheese. Milchwissenschaft 40, 209-212. Barbano, D.M. (2000). Influence of mastitis on cheese manufacture, in, Practical Guide for Control of Cheese Yield, S.I. 0001, International Dairy Federation, Brussels, pp. 19-27. Bouman, S. (1977). Condities in kaaspakhuizen (conditions in cheese stores). Zuivelzicht 69, 1130-1133. Bouman, S., Lund, D.B., Driessen, EM. and Schmidt, D.G. (1982). Growth of thermoresistant streptococci and deposition of milk constituents on plates of heat exchangers during long operating times. J. Food Prot. 45,806-812. Bruinenberg, P.G., De Roo, G. and Limsowtin, G.K.Y. (1997). Purification and characterization of cystathionine y-lyase from Lactococcus lactis subsp, cremoris SKll: possible role in flavour compound formation during cheese maturation. Appl. Environ. Microbiol. 63,561-566. Buijsse, C.A.P. (1999). Cheese from Ultrafiltered Milk; Whey Proteins and Chymosin Activity. Doctoral Thesis, Agricultural University, Wageningen. Christensen, J.E., Dudley, E.G., Pederson, J.A. and Steele, J.L. (1999). Peptidases and amino acid catabolism in lactic acid bacteria. Antonie van Leeuwenhoek 76, 217-246. Cogan, T.M. (1996). History and taxonomy of starter cultures, in, Dairy Starter Cultures, Cogan, T.M. and Accolas, J.-P. eds, VCH Publishers, Inc., New York. pp. 1-20. Crow, V.L., Martley, EG., Coolbear, T. and Roundhill, S.J. (1995). The influence of phage-assisted lysis of Lactococcus lactis subsp, lactis ML8 on Cheddar cheese ripening. Int. DairyJ. 5,451-472. de Jong, P. (1996). Modelling and Optimization of Thermal Processes in the Dairy Industry. Doctoral Thesis, University of Technology Delft, Montfoort, The Netherlands. de Jong, P., Verschueren, M., Vissers, M.M.M., Straatsma, J. and Smit, E. (2002). Hybrid modelling for development and optimisation of food production chains including costs and food quality. Proceedings of the 2nd International Conference on Simulation and Modelling in Food and BioIndustry, SCS Europe/University College, Cork, Blarney, Ireland. pp. 13-17. de Koning, P.J., de Boer, R., Both, P. and Nooy, REC. (1981). Comparison of proteolysis in a low-fat semi-hard type of cheese manufactured by standard and by uhrafihration techniques. Neth. Milk Dairy J. 35, 35-46. de Ruig, W.G. and van den Berg, G. (1985). Influence of the fungicides sorbate and natamycin in cheese coatings on the quality of the cheese. Neth. Milk Dairy J. 39, 165-172. de Vries, E. (1978). Relatie tussen het vochtgehalte en de kwaliteit van kaas (relation between water content and quality of cheese). Zuivelzicht 70,554-557. de Vries, E. and Staal, H.J. (1974). Aspekte des kontinuierlichen Formens van Ktisebruch mit dem durch NIZO entwickelten Bruchportionierungsautomat (aspects of continuous shaping of cheese curd in an automatic curd portioning system developed by NIZO). Milchwissenschaft 29,651-655. de Vries, Tj. and Stadhouders, J. (1977). Boterzuurbacterien in melk. Zuivelzicht 69, 196-199.
de Vries, E. and van Ginkel, W. (1984). Test of a curd-making tank, Type Ost IV, with a capacity of 10 000 litres, manufactured by Tebel B.V. NIZO-report R120, Ede, The Netherlands. Driessen, EM. (1983). Lipases and Proteinases in Milk. Occurrence, Heat Inactivation, and their Importance for Keeping Quality of Milk Products. Doctoral Thesis, Agricultural University, Wageningen. Engels, W.J.M. and Visser, S. (1996). Development of cheese flavour from peptides and amino acids by cell-free extracts of Lactococcus lactis subsp, cremoris B78 and its possible role in flavour development in cheese. Neth. Milk Dairy J. 50, 3-17. Engels, W.J.M., Alting, A.C., Arntz, M.M.T.G., Gruppen, J., Voragen, A.G.J., Smit, G. and Visser, S. (2000). Partial purification and characterization of two aminotransferases from Lactococcus lactis subsp, cremoris B78 involved in catabolism of methionine and branched-chain amino acids. Intern. Dairy J. 10,443-452. Exterkate, EA., Alting, A.C. and Bruinenberg, P.G. (1993). Diversity of cell envelope proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region. Appl. Environ. Microbiol. 59, 3640-3647. Fern~indez, M., Van Doesburg, W., Rutten, G.A., Marugg, J.D., Ahing, A.C., van Kranenburg, R. and Kuipers, O.P. (2000). Molecular and functional analyses of the metC gene of Lactococcus lactis, encoding cystathionine [~lyase. Appl. Environ. Microbiol. 66, 42-48. Fernandez, M., Kleerebezem, M., Kuipers, O.P., Siezen, R.J. and van Kranenburg, R. (2002). Regulation of the metCcysK operon involved in sulfur metabolism in Lactococcus lactis. J. Bacteriol. 184, 82-90. Fox, P.E and Stepaniak, L. (1993). Enzymes in cheese technology. Int. Dairy J. 3, 509-530. Frankhuizen, R., Bastiaans, J.A.H.P., van Arem, E.J.E and Cruijsen, J.M.M. (2002). Eyes on cheese. Meetsysteem voor sturing van kaasbereiding. Voedingsmiddelentechnologie 35(23), 14-16. Galesloot, T.E. (1961). Concerning the action of nitrate in preventing butyric acid fermentation in cheese. Neth. Milk Dairy J. 15,395-410. Gao, S. and Steele, J.L. (1998). Purification and characterization of oligomeric species of an aromatic amino acid aminotransferase from Lactococcus lactis subsp, lactis $3. J. Food Biochem. 22, 197-211. Gao, S., Oh, D.H., Broadbent, J.R., Johnson, M.E., Weimer, B.C. and Steele, J.L. (1997). Aromatic amino acid catabolism by lactococci. Lait 77,371-181. Geurts, T.J. (1978). Some factors which affect the moisture content of cheese before brining. Neth. Milk Dairy J. 32, 112-124. Geurts, T.J., Walstra, P. and Mulder, H. (1972). Brine composition and the prevention of the defect "soft rind" in cheese. Neth. Milk DairyJ. 26, 168-179. Geurts, T.J., Walstra, P. and Mulder, H. (1980). Transport of salt and water during salting of cheese. 2. Quantities of salt taken up and of moisture lost. Neth. Milk Dairy J. 34, 229-254.
Gouda and Related Cheeses
Goodhead, K., Gough, T.A., Webb, K.S., Stadhouders, J. and Elgersma, R.H.C. (1976). The use of nitrate in the manufacture of Gouda cheese. Lack of evidence of nitrosamine formation. Neth. Milk Dairy J. 30, 207-221. Heap, H.A. (1998). Optimising starter culture performance in NZ cheese plants. Aust. J. Dairy Technol. 53, 74-78. Heap, H.A. and Lawrence, R.C. (1976). The selection of starter strains cheesemaking. NZ J. Dairy Sci. Technol. 11, 16-20. Hugenholtz, J., Exterkate, EA. and Konings, W.N. (1984). The proteolytic systems of Streptococcus cremoris: an immunological analysis. Appl. Environ. Microbiol. 48, 1105-1110. Hup, G., Bangma, A., Stadhouders, J. and Bouman, S. (1979). Groei van thermoresistente streptokokken in kaasmelkpasteurs. 1. Enkele waarnemingen in kaasbedrijven (Growth of thermoresistant streptococci in cheesemilk pasteurizers. 1. Some observations in cheese factories. Zuivelzicht. 71, 1014-1016. Hup, G., Stadhouders, J., de Vries, E. and van den Berg, G. (1982). Lactobacillen in "slappe" pekel en de kwaliteit van kaas (Lactobacilli in "weak" cheese brine and the quality of cheese). Zuivelzicht 74, 270-273. IDF (1995). Milk Payment Systems for Ex-farm Milk. Bulletin 305, International Dairy Federation, Brussels. pp. 2-17. Jakob, E. and Puhan, Z. (1995). Implications of Genetic Polymorphism of Milk Proteins on Production and Processing of Milk. Bulletin 304, International Dairy Federation, Brussels. pp. 2-25. Kleter, G. (1975). Apparatus for making cheese under strict aseptic conditions. Neth. Milk Dairy J. 29,295-302. Kleter, G. (1976). The ripening of Gouda cheese made under strict aseptic conditions. 1. Cheese with no other bacterial enzymes than those from a starter Streptococcus. Neth. Milk Dairy J. 30, 254-270. Kleter, G. (1977). The ripening of Gouda cheese made under strictly aseptic conditions. 2. The comparison of the activity of different starters and the influence of certain Lactobacillus strains. Neth. Milk DairyJ. 31,177-187. Kleter, G. and de Vries, Tj. (1974). Aseptic milking of cows. Neth. Milk Dairy J. 28,212-219. Kok, J. (1993). Genetics of proteolytic enzymes of lactococci and their role in cheese flavour development. J. Dairy Sci. 76, 2056-2064. Kok, J. and de Vos, W.M. (1994). The proteolytic system of lactic acid bacteria, in, Genetics and Biotechnology of Lactic Acid Bacteria, Gasson, M.J. and de Vos, W.M. eds, Chapman & Hall Ltd., London. pp. 169-210. Kunji, E.R.S., Hagting, A., de Vries, C.J., Juillard, V., Haandrikman, A.J., Poolman, B. and Konings, W.N. (1995). Transport of [3-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis. J. Biol. Chem. 270, 1569-1574. Langeveld, L.P.M. and Galesloot, T.E. (1971). Estimation of the oxidation-reduction potential as an aid in tracing the cause of excessive openness in cheese. Neth. Milk Dairy J. 25, 15-23. Lankveld, J.M.G. (1984). Hygienic processing- GMP in de zuivel. Voedingsmidellentechnologie 17(2), 33-36.
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Lawrence, R.C. and Gilles, J. (1986). Cheese composition and quality, in, Milk, the Vital Force: Proceedings of the XXII International Dairy Congress (The Hague), Vol. D, pp. 111-121. Limsowtin, G.K.Y., Heap, H.A. and Lawrence, R.C. (1977). A multiple starter concept for cheese making. NZ J. Dairy Sci. Technol. 12, 101-106. Limsowtin, G.K.Y., Powell, I.B. and Parente, E. (1996). Types of starters, in, Dairy Starter Cultures, Cogan, T.M. and Accolas, J.-p. eds, VCH Publishers, Inc., New York. pp. 101-129. Lindblad, O.T. (1990). Use of lysozyme for the prevention of butyric acid fermentation in Swedish round-eyed cheese. Brief Commun. Abstr. Posters, XXIII Int. Dairy Congr., Montreal, Canada. P. 483. Lolkema, H. (1991). Cheese yield used as an instrument for process c o n t r o l - Experience in Friesland, The Netherlands, in, Factors Affecting the Yield of Cheese, Special Issue No. 9301, International Dairy Federation, Brussels. pp. 156-197. Luyten, H. (1988). The Rheological and Fracture Properties of Gouda Cheese. Doctoral Thesis, Agricultural University, Wageningen. M~iyra-Makinen, A. and Bigret, M. (1998). Industrial use and production of lactic acid bacteria, in, Lactic Acid BacteriaMicrobiology and Functional Aspects, Salminen, S. and Wright, A. eds, Marcel Dekker, New York. pp. 73-102. Meijer, W.C., Verheul, A., Hugenholtz, J., Twigt, M. and Smit G. (1998). Prevention of spoilage and pathogenic microorganisms in raw milk cheeses, in, Proceedings of the Symposium on Quality and Microbiology of Traditional and Raw Milk Cheeses. p. 319. Morgan, S., Ross, R.P. and Hill, C. (1995). Bacteriolytic activity caused by the presence of a novel lactoccocal plasmid encoding lactococcins A, B and M. Appl. Environ. Microbiol. 61, 2995-3001. Mulder, H., de Graaf, J.J. and Walstra, P. (1966). Microscopical observations on the structure of curd and cheese. Proc. XII Int. Dairy Congr., Section D (Cheese), M~nchen. pp. 413-420. Noomen, A. (1978a). Activity of proteolytic enzymes in simulated soft cheeses (Meshanger type). 1. Activity of milk protease. Neth. Milk Dairy J. 32, 26-48. Noomen, A. (1978b). Activity of proteolytic enzymes in simulated soft cheeses (Meshanger type). 2. Activity of calf rennet. Neth. Milk Dairy J. 32, 49-68. Northolt, M.D. and Stadhouders, J. (1985). Microbiologische en biochemische normen voor de bereiding van Nederlandse kaassoorten (1 and 2). Zuivelzicht 77,324-327, 488-490. Payne, M.R. and Morison, K.R. (1999). A multi-component approach to salt and water diffusion in cheese. Int. Dairy J. 12,887-894. Pearce, L.E. (1969). Activity tests for cheese starter cultures. NZJ. Dairy Technol. 4, 246-247. Poolman, B., Kunji, E.R., Hagting, A., Juillard, V and Konings, W.N. (1995). The proteolytic pathway of Lactococcus lactis. J. Appl. Bact. Syrup. Suppl. 79, 65S-75S. Posthumus, G., Booy, C.J. and Klijn, C.J. (1963). Verband tussen eiwitgehalte van melk en kaasopbrengst. Off. Orgaan FNZ 55,986-990.
138
Gouda and Related Cheeses
Posthumus, G., Klijn, C.J. and Booy, CJ. (1967). Verband tussen eiwitgehalte van kaasmelk en opbrengst aan kaas en aan te houden vetgehalte in de kaasmelk. Off. Orgaan FNZ 59, 712-714, 740-742, 749, 769-772. Pritchard, G.G. and Coolbear, T. (1993). The physiology and biochemistry of proteolytic system in lactic acid bacteria. FEMS Microbiol. Rev. 12, 179-206. Raadsveld, C.W. and Mulder, H. (1949a). The influence of the temperature on the ripening of Edam cheese. Neth. Milk Dairy J. 3, 117-141. Raadsveld, C.W. and Mulder, H. (1949b). The influence of pH on the ripening of Edam cheese. Neth. Milk Dairy J. 3, 222-230. Rijnen, L., Bonneau, S. and Yvon, M. (1999). Genetic characterization of the major lactococcal aromatic aminotransferase and its involvement in conversion of amino acids to aroma compounds. Appl. Environ. Microbiol. 65, 4873-4880. Sandine, W.E. (1996). Commercial production of dairy starter cultures, in, Dairy Starter Cultures, Cogan, T.M. and Accolas, J.-P. eds, VCH Publishers, Inc., New York. pp. 191-204.. Simonart, P. and Debeer, G. (1953). Recherches en vue d'amr la qualitr microbiologique des laits par ultracentrifugation (Researches concerning improvement of the micribiological of milk by uhra-centrifugation). Neth. Milk DairyJ. 7, 117-128. Smit, G., Kruyswijk, Z., Weerkamp, A.H., de Jong, C. and Neeter, R. (1996). Screening for and control of debittering properties of cheese cultures, in, Flavour Science: Recent Developments, Taylor, A.J. and Mottram, D.S. eds, Royal Society of Chemistry, London. pp. 25-31. Smit, G., Kruyswijk, Z. and van Boven, A. (1998). Control of debittering activity of cheese starters. Aust. J. Dairy Technol. 53, 113 (1 page). Smit, G., Verheul, A., van Kranenburg, R., Ayad, E., Siezen, R. and Engels, W. (2000). Cheese flavour development by enzymatic conversions of peptides and amino acids. Food Res. Int. 33, 153-160. Stadhouders, J. (1974). Dairy starter cultures. Milchwissenschaft 29,329-337. Stadhouders, J. (1982). Cooling and thermization as a means of extending the keeping quality of raw milk. Kieler Milchwirtsch. Berichte 34, 19-28. Stadhouders, J. (1990). The Manufacturing Method for Cheese and the Sensitivity to Butyric Acid Fermentation. Bulletin 251, International Dairy Federation, Brussels. pp. 37-39. Stadhouders, J. and Hassing, E (1980). Description of a method for determining the activity of cheese starters, in, Starters in the Manufacture of Cheese. Bulletin 129, International Dairy Federation, Brussels. pp. 9-10. Stadhouders, J. and Hup, G. (1970). Complexity and specificity of euglobulin in relation to inhibition of bacteria and to cream rising. Neth. Milk Dairy J. 24, 79-95. Stadhouders, J. and Hup, G. (1975). Factors affecting bitter flavour in Gouda cheese. Neth. Milk Dairy J. 29, 335-353. Stadhouders, J. and Labots, H. (1962). Abnormale verkleuring van de kaaskorst bij gebruik van plastic bedekkingsmiddelen. I. Off. Orgaan FNZ 54, 35-37.
Stadhouders, J. and Leenders, G.J.M. (1984). Spontaneously developed mixed-strain cheese starters. Their behaviour towards phages and their use in the Dutch cheese industry. Neth. Milk DairyJ. 38, 157-181. Stadhouders, J. and Mulder, H. (1957). Fat hydrolysis and cheese flavour. I. The enzymes effecting the hydrolysis of fat in cheese. Neth. Milk Dairy J. 11,164-183. Stadhouders, J. and Mulder, H. (1959). Fat hydrolysis and cheese flavour. III. Surface organisms associated with fat hydrolysis in cheese. Neth. Milk Dairy J. 13, 291-299. Stadhouders, J. and Mulder, H. (1960). Fat hydrolysis and cheese flavour. IV. Fat hydrolysis in cheese from pasteurised milk. Neth. Milk Dairy J. 14, 141-148. Stadhouders, J. and Veringa, H.A. (1973). Fat hydrolysis by lactic acid bacteria in cheese. Neth. Milk Dairy J. 27, 77-91. Stadhouders, J., Hup, G. and Hassing, E (1974). Lactobacillen in "slappe" kaaspekel (Lactobacilli in brine of reduced strength). Off. Orgaan FNZ 49, 1170-1173. Stadhouders, J., Bangma, A. and Driessen, EM. (1976). Beheersing van de zuurvorming bij de kaasbereiding (2) Zuivelzicht 68, 180-184. Stadhouders, J., Hup, G. and Nieuwenhof, EEJ. (1983a). Silage and cheese quality. NIZO-mededeling M19A, Ede, The Netherlands. Stadhouders, J., Hup, G., Exterkate, EA. and Visser, S. (1983b). Bitter formation in cheese. 1. Mechanism of the formation of the bitter flavour defect in cheese. Neth. Milk DairyJ. 37, 157-167. Stadhouders, J., Kleter, G., Lammers, W.L. and Tuinte, J.H.M. (1983c). Groei van lactobacillen in Goudse kaas (growth of lactobacilli in Gouda cheese). Zuivelzicht 75, 1118-1121. Stadhouders, J., Leenders, G.J.M., Maessen-Damsma, G., de Vries, E. and Eilert, J.G. (1985). Vermindering van de besmetting van slappe pekel met zoutresistente lactobacillen door een betere hygiene in het pekellokaal (reduction in the contamination of weak brine with saltresistant lactobacilli by improving the hygienic conditions in the brining room). Zuivelzicht 77,892-894. Stadhouders, J., Stegink, H. and van den Berg, G. (1986). The use of lysozyme for the prevention of butyric acid fermentation in Gouda cheese. The limited effect of the enzyme. Meijeritieteellinen Aikakauskrja XLIV(1), 23-25. Straatsma, J. and Heijnekamp, A. (1988). Kwantitatieve invloed van diverse factoren op het kaasbereidingsproces (quantitative influence of several factors on the cheesemaking process). NIZO-mededeling M21, Ede, The Netherlands. Straatsma, J., de Vries, E., Heijnekamp, A. and Kloosterman, L. (1984). Beheersing van het vochtgehahe bij de kaasbereiding (control of the moisture content during cheese making). Zuivelzicht 76,956-959. ten Grotenhuis, E. (1999). Prediction of cutting time during cheese production. Eur. Dairy Mag., Feb., 40-41. Urbach, G. (1995). Contribution of lactic acid bacteria to flavour compound formation in dairy products. Int. Dairy J. 5,877-903. van Boekel, M.A.J.S. and Walstra, P. (1989). Steric exclusion of serum proteins with respect to (para)casein micelles. Neth. Milk Dairy J. 43,437-446.
Gouda and Related Cheeses
van den Bedem, J.W. and Leenheer, J. (1988). Heat treatment classification of low heat and extra low heat skimmilk powder by HPLC. Neth. Milk Dairy J. 43, 311-326. van den Berg, M.G. (1984). The Thermization of Milk. Bulletin 182, International Dairy Federation, Brussels. pp. 3-11. van den Berg, G. (2001). Semi-hard cheeses, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A. eds, Sheffield Academic Press Ltd., Sheffield. pp. 225-249. van den Berg, G. and de Koning, P.J. (1990). Gouda cheesemaking with purified calf chymosin and microbially produced chymosin. Neth. Milk Dairy J. 44, 189-205. van den Berg, G. and de Vries, E. (1974). Der Zusammenhang zwischen den Faktoren, die den pH van K~se beeinflussen (relation between factors influencing the pH of cheese). Milchwissenschaft 29,214-218. van den Berg, G. and de Vries, E. (1975). Whey composition during the course of cheese manufacture, as affected by the amount of starter and curd washing water. Neth. Milk DairyJ. 29, 181-197. van den Berg, G. and de Vries, E. (1976). Het gebruik van wrongelwaswater (the use of curd washing water). Zuivelzicht 68,878-879,924-926. van den Berg, G. and Exterkate, EA. (1993). Technological parameters involved in cheese ripening. Int. Dairy J. 3, 485-507. van den Berg, G., Stadhouders, J., Smale, E.J.W.L. and de Vries, E. (1975). Voor- en nadelen van "slappe" kaaspekel (merits and demerits of lower brine concentrations). Zuivelzicht 67, 984-988. van den Berg, G., Hup, G., Stadhouders, J. and de Vries, E. (1980). Application of the "Bactotherm" process (selfdesludging bactofuge, type MRPX 314 SGV, in combination with bactofugate sterilizer) in the manufacture of Gouda cheese. Technological effects on manufacture of Gouda cheese. NIZO-Report Rl12, Ede, The Netherlands. van den Berg, G., Daamen, C.B.G., de Vries, E., van Ginkel, W. and Stadhouders, J. (1988). Test of the bacteriaremoving separators, manufactured by Westfalia Separator AG, for the manufacture of Gouda cheese. NIZO-Report R127, Ede, The Netherlands. van den Berg, G., Escher, J.T.M., de Koning, P.J. and Bovenhuis, H. (1992). Genetic polymorphism of K-casein and [3-1actoglobulin in relation to milk composition and processing properties. Neth. Milk DairyJ. 46, 145-168. van den Berg, M.G., van den Berg, G. and van Boekel, M.J.S. (1996a). Mass transfer processes involved in Gouda cheese manufacture, in relation to casein and yield. Neth. Milk Dairy J. 50, 501-540. van den Berg, G., Neeter, R., Allersma, D. and de Jong, C. (1996b). Ripening of Cheese Made from Milk with Different Heat Treatments. Bulletin 317, International Dairy Federation, Brussels. p. 40. van den Berg, G., Boer, E and Allersma, D. (1998). Koel bewaren van melk van invloed op kaasopbrengst (consequences of coldstorage of milk for cheese yield). Voedingsmiddelentechnologie 31 (4), 101-104. van Kranenburg, R., Kleerebezem, M., Van Hylckama Vlieg, J.E.T., Ursing, B.M., Boekhorst, J., Smit, B.A., Ayad, E.H.E., Smit, G. and Siezen, R.J. (2002). Flavour forma-
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tion from amino acids: predictions from genome sequence analysis. Int. DairyJ. 12, 111-121. van Rijn, ET.J., Hoekstra, E.S., van der Horst, M.I., Samson, R.A. and Stark, J. (1997). Penicillium discolor in de Nederlandse kaasindustrie. Aanwezigheid leidt meestal niet tot problemen. Voedingsmiddelentechnologie 30(20), 19-23. van Schouwenburg-van Foeken, A.W.J., Stadhouders, J. and Witsenburg, W.W. (1979). The number of enterotoxigenic Staphylococcus aureus reached in Gouda cheese made under normal acidification conditions and the amount of enterotoxin produced. Neth. Milk Dairy J. 33, 49-59. Veringa, H.A., van den Berg, G. and Stadhouders, J. (1976). An alternative method for the production of cultured butter. Milchwissenschaft 31,658-662. Veringa, H.A., van den Berg, G. and Daamen, C.B.G. (1989). Factors affecting the growth of Aspergillus versicolor and the production of sterigmatocystin on cheese. Neth. Milk Dairy J. 43, 311-326. Verschueren, M., van den Hoven, G.A. and de Jong, P. (2002). Enhancement of curd washing efficiency: a twostage extraction process. Aust. J. Dairy Technol. 57, 144. Visser, EM.W. (1977a). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 1. Description of cheese and aseptic cheesemaking techniques. Neth. Milk Dairy J. 31,120-133. Visser, EM.W. (1977b). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 2. Development of bitterness and cheese flavour. Neth. Milk Dairy J. 31,188-209. Visser, EM.W. (1977c). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 4. Protein breakdown: a gel electrophoretical study. Neth. Milk Dairy J. 31, 247-264. Visser, EM.W. (1977d). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 5. Some observations on bitter extracts from aseptically made cheese. Neth. Milk DairyJ. 31,265-276. Visser, J. (1991). Factors affecting the rheological and fracture properties of hard and semi-hard cheese, in, Rheological and Fracture Properties of Cheese. Bulletin 268, International Dairy Federation, Brussels. pp. 49-61. Visser, EM.W. and de Groot-Mostert, A.E.A. (1977). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 3. Protein breakdown: analysis of the soluble nitrogen and amino acid nitrogen fractions. Neth. Milk Dairy J. 31, 210-239. Visser, S., Slangen, C.J., Hup, G. and Stadhouders, J. (1983). Bitter flavour in cheese. 3. Comparative gel-chromatographic analysis of hydrophobic peptide fractions from twelve Gouda-type cheeses and identification of bitter peptides isolated from a cheese made with Streptococcus cremoris HP. Neth. Milk DairyJ. 37, 181-192. Walstra, P. (2000). General principles, in, Practical Guide for Control of Cheese Yield, Special Issue 0001, International Dairy Federation, Brussels. pp. 6-13.
140
Gouda and Related Cheeses
Walstra, P. and Jenness, R. (1984). Dairy Chemistry and Physics, John Wiley, New York. Walstra, P., van Dijk, H.J.M. and Geurts, T.J. (1985). The syneresis of curd. 1. General considerations and literature review. Neth. Milk Dairy J. 39, 209-246. Wilbrink, A., Spoelstra, T. and Strampel, J. (1981). Scheurvorming in kaas bij gebruik van slappe kaaspekel. Zuivelzicht 73, 16-19. Wilkinson, M.G., Guinee, T.P., O'Callaghan, D.M. and Fox, P.E (1994). Autolysis and proteolysis in different strains of starter bacteria during cheese ripening. J. Dairy Res. 61,249-262. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11,185-201. Yvon, M., Thirouin, S., Rijnen, L., Fromentier, D. and Gripon, J.C. (1997). An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavour compounds. Appl. Environ. Microbiol. 63,414-419. Yvon, M., Berthelot, S. and Gripon, J.C. (1998). Adding ot-ketoglutarate to semi-hard cheese curd highly enhances
the conversion of amino acids to aroma compounds. Int. Dairy J. 8, 889-898. Yvon, M., Chambellon, E., Bolotin, A. and Roudot-Algaron, E (2000). Characterization and role of the branchedchain aminotransferase (BcaT) isolated from Lactococcus lactis subsp, cremoris NCDO 763. Appl. Environ. Microbiol. 66,571-577. Zoon, P. (1993). Physical properties of cheese, in, Proceedings of the Cheese Research & Technology Conference, April 13-14, 1993, Center of Dairy Research, Madison, WI. pp. 55-61. Zoon, R, Straatsma, J. and Allersma, D. (1991). Indampen van kaaspekel en de gevolgen voor de kaaskwaliteit (Concentration of cheese brine by evaporation and its effect on cheese quality). Voedingsmiddelentechnologie 24(11), 13-16. Zoon, P., Ansems, C. and Faber, E.J. (1994). Measurement procedure for the concentration of active rennet in cheese. Neth. Milk DairyJ. 48, 141-150.
Cheeses with Propionic Acid Fermentation M.T. Fr6hlich-Wyder and H.P. Bachmann, Agroscope Liebefeld-Posieux, Swiss Federal Institute for Animal Production and Dairy Products, Switzerland
The propionic acid fermentation leads to characteristic eyes and nutty flavour and can either occur spontaneously or can be achieved by a culture of selected propionibacteria. A spontaneous fermentation leads to irregular eye formation, because strain diversity of the natural propionibacterial flora is great. The number and size of eyes vary markedly, and cracks or splits are quite common. Comte and Beaufort are typical examples of cheese varieties with a spontaneous propionic acid fermentation. The application of a culture of selected propionibacteria allows a more regular eye formation as a result of a propionic acid fermentation which is under control. Such cheese varieties are often called Swiss-type cheeses. The body and texture correspond to those of hard or semi-hard cheeses. They were manufactured originally in the Emmental (Emmen valley) in Switzerland. Emmental is probably the best-known Swiss-type cheese and is frequently referred to simply as 'Swiss cheese'. There is no internationally recognised definition of Swiss-type cheeses that differentiates them from other varieties. Swiss-type cheeses have round regular eyes which vary in size from medium to large. The characteristics of Swiss Emmental are: 9 9 9 9
cylindrical shape; firm dry rind; w e i g h t - 60-130 kg; 1000-2000 round eyes per loaf of diameter 1-4 cm, caused by propionic acid fermentation; 9 f l a v o u r - mild, slightly sweet, becoming more aromatic with increasing age; 9 cheese b o d y - ivory to light-yellow, slightly elastic.
Today, Emmental-type cheese (Fig. 1) is produced in many countries and a great variety of other Swisstype cheeses is also available on the market, including Jarlsberg, Maasdamer, Leerdamer and generally many other products denoted as Swiss cheese. Their body and texture correspond to those of hard and semi-hard cheeses. They are manufactured by methods differing
from traditional Swiss procedures. Thus, the treatment of milk, the extent of mechanisation, the starters used, the weight, shape, ripening time and shelf life are often different from the original. Descriptions and analytical values presented in this chapter focus on Swiss Emmental cheese but most of the information is applicable to other cheese varieties with a propionic acid fermentation.
Lactic acid fermentation
Especially in the production of hard Swiss cheese varieties, mainly thermophilic lactic acid bacteria are used as starters, often as mixed cultures of lactobacilli (Lactobacillus helveticus, Lb. delbrueckii subsp, lactis) and streptococci (Streptococcus salivarius subsp, thermophilus). They guarantee the homofermentative catabolism of lactose to >90% lactate. The streptococci produce only L-lactic acid, whereas Lb. delbrueckii subsp. lactis converts lactose entirely to D-lactate. Both isomers are produced by Lb. helveticus. Lactose is fully hydrolysed within 4-6 h after addition of the lactic starters, and the lactic acid fermentation is completed after 24 h. Galactose from lactose breakdown is not utilised by the streptococci, but is metabolised by the lactobacilli. To avoid undesired fermentations, no residual galactose should remain after the lactic fermentation. During cheese ripening, the proteinases and peptidases of lactobacilli play a major role in the breakdown of casein. Some decades ago, Lb. helveticus was a major component of starter cultures in the manufacture of Swiss Emmental. Due to its intensive peptidolytic activity, which promotes late fermentation, it has been replaced by Lb. delbrueckii subsp, lactis. Streptococci play a minor role in proteolysis. In areas where the cheese milk is collected twice daily, it is quite common to add a mesophilic culture of lactococci (Lactococcus lactis) to the evening milk to pre-ripen it. In the production of semi-hard cheeses with a propionic acid fermentation, mesophilic species are also used.
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
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142
Cheeses with Propionic Acid Fermentation
Traditional Swiss Emmental cheese. (See Colour plate 16.)
Facultatively heterofermentative non-starter lactobacilli are purposely used in the Swiss artisanal cheese industry to slow down the propionic acid fermentation. They ferment hexoses almost exclusively to lactic acid. This group of micro-organisms contains, among others, Lb. casei and Lb. rhamnosus which are indigenous to raw milk. During cheese ripening, they grow by utilising citrate which is found in the fresh unripened cheese. Starting from 9 mmol/kg citrate in the cheese curd, native facultatively heterofermentative lactobacilli utilise approximately 3 mmol and those added as adjunct cultures metabolise all available citrate to formic acid, acetic acid and CO2 (Table 4).
about 1000 1 of milk). Propionic acid fermentation begins about 30 days after the start of manufacture at about 20-24 ~ for roughly 7 weeks and then continues at a slower rate at 10-13 ~ In cheeses ready for consumption, about 108-109 cfu/g of propionic acid bacteria are present. Propionibacteria are Gram-positive, non-motile, non-sporulating and appear under the microscope as short rods which grow at low oxygen concentrations only (anaerobic to aerotolerant), and occur naturally in the rumen and intestine of ruminants, in soil and in silage (Fig. 2). Strain diversity of the natural propionibacterium flora is great which, fortunately, has not been influenced by the wide use of commercially available cultures (Fessler, 1997). They are sensitive to salt and grow optimally at a pH between 6 and 7 (maximum 8.5, minimum 4.6). The optimal growth temperature is 30 ~ but growth occurs also at 14 ~ They develop well in cheese from low numbers, but do not grow in milk (Piveteau et al., 2000). The propionibacterial metabolism in cheese is rather complex and not yet fully understood (Crow et al., 1988" FrOhlich-Wyder et al., 2002). Three different metabolic pathways (Fig. 3) have been described for the utilisation of lactate as an energy source and aspartate as an electron acceptor, both of which are available in cheese (Brendehaug and Langsrud, 1985; Crow and Turner, 1986; Crow, 1986b). In the presence nf a~partale, the [ermentntinn of lnrtnto i~ rn,,plocl with the fermentation of aspartate to succinate and no propionate is produced. Consequently, more lactate is fermented to acetate and CO2 than to propionate. The role of pathway B (formation of succinate by fixation
Propionic acid fermentation
Nowadays, selected propionibacteria of the species P. freudenreichii are used in the manufacture of cheeses with propionic acid fermentation in order to achieve the characteristic eyes and nutty flavour. For Emmental cheeses, the inoculum size is very small (only a few hundred colony forming units (cfu) per vat containing
Scanning electron micrograph of a culture of Propionibacterium freudenreichfi (Source: Swiss Federal Dairy Research Station, CH-3003 Berne).
Cheeses with Propionic Acid Fermentation
143
(A) Classical propionic acid fermentation: 3 mol lactate ~
2 mol propionate + 1 mol acetate + 1 mol CO 2 + l m o l A T P
(B) Formation of succinate during propionic acid fermentation by CO2-fixation: 3 mol lactate ~
(2 - x) mol propionate + 1 mol acetate + (1 - x) mol CO 2 + x mol succinate
Wood-Werkman pathway
(C) Fermentation of aspartate to succinate during propionic acid fermentation: 3 mol lactate + 6 mol aspartate ~
3 mol acetate + 3 mol CO 2 + 6 mol succinate + 6 mol NH 3 + 3 mol A T P
Metabolic pathways for the utilisation of lactate by propionic acid bacteria according to Crow and Turner (1986) and Sebastiani and Tschager (1993).
of CO2) is certainly of minor importance, but it has not yet been clarified (Sebastiani and Tschager, 1993). Propionibacterial strains can differ markedly in their aspartase activity (Richoux and Kerjean, 1995). In the manufacture of Emmental cheese, the use of cultures with differing aspartase activity leads to different products (Wyder et al., 2001). Tables 1 and 2 show clearly the characteristics of Emmental cheeses made with propionibacteria with either strong or weak aspartase activity. Propionibacteria with weak aspartase activity are able to metabolise not more than 100 nmol aspartate per minute in vitro (Fr0hlich-Wyder et al., 2002). These strains metabolise lactate mainly by the classical pathway (A) and deaminate only little aspartate (Fig. 3). Strains with high aspartase activity are able to metabolise up to 8 0 0 n m o l aspartate per minute in vitro. During the ripening of Swiss-type cheese, aspartate is metabolised rapidly and L-lactate is used preferentially (Crow, 1986a; Piveteau et al., 1995). As an effect, usually all available aspartate is metabolised to succinate (Fig. 4) and lactate, preferentially the isomer L, is metabolised to propionate, acetate and CO2 (Tables 1 and 4). A comparison with other traditional cheese varieties from Switzerland, which do not undergo a propionic acid fermentation, reveals that the content of aspartate is always much lower and that of succinate much higher in Emmental cheeses (Sieber et al., 1988). A strong aspartase activity is generally coupled with a stronger growth rate of propionibacteria, leading to higher counts and higher concentrations of propionate, acetate and CO2 (Table 1). Piveteau et al. (1995) showed that the growth rate and yield of propionibacteria in whey can be enhanced by the addition of aspartate. Yet it is not possible to answer the question whether aspartase activity is the cause or just an indicator. The appearance of Emmental cheese is greatly affected by the aspartase activity of the propionibacteria used. Figure 5 shows clearly the outer appearance
of different Emmental cheeses. The number and size of eyes and the height of loaves are greater for cheeses made with a culture with strong aspartase activity (Table 2) as a result of increased CO2 release (Table 1). The storage time for the cheeses in the warm room may be shortened by up to 10 days (Fr0hlich-Wyder et al., 2002). Such cheeses are more prone to late fermentation which is not desired when the cheeses are ripened for a longer time (Bachmann, 1998a). Late fermentation is a resumption of the propionic acid fermentation during maturation. The intensity of taste, odour and aroma is also more pronounced compared to cheeses made with propionibacteria of low aspartase activity (Table 2). The main reason appears to be the higher concentrations of free short chain acids produced through fermentation as well as the free fatty acids, n-butyric and n-caproic acids, released by lipolytic activity of propionibacteria (Table 1). Thus, propionibacteria with strong aspartase activity accelerate the ripening process. This is a 15'I
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Asp + Asn (mmol/kg) Linear regression, with a 95% confidence interval, of succinate and sum of aspartate (Asp) and asparagine (Asn) in 6-month-old Emmental cheese (T, propionibacteria with high aspartase activity; A, propionibacteria with weak aspartase activity) (Wyder et al., 2001).
144
Cheeses with Propionic Acid Fermentation
Mean values of metabolites, proteolytic parameters and propionibacterial counts in Emmental cheese (6 and 12 months) made with propionibacteria with weak or strong a s p a r t a s e activity (Wyder et aL, 2001 )
Emmental cheeses at 6 months Parameter
Weak (N = 10)
Lactate a L(+)-Lactate a pH Free S C A a Acetate a Propionate a n-Butyrate a n-Caproate a Succinate a 002 a Propionibacteriab Total nitrogenc WSN a TCASN a Free a m i n o acids a Aspartate a Asparagine a
57.4 31.1 5.75 114.4 48.4 60.1 1.1 0.4 4.0 27.6
_ 10.5 _ 9.3 _ 0.02 +_ 5.2 ___ 1.3 ___4.4 _+ 0.2 +_ 0.1 _+ 0.6 _ 1.6 nd 3.17 _ 0.06 693.4 _ 33.5 469.2 _ 46.6 169.02 _ 23.72 2.219 +_ 0.861 2.863 +_ 1.100
Strong (N = 8) 45.3 17.0 5.79 126.0 53.1 67.1 1.2 0.5 11.9 33.6 3.20 720.4 470.4 165.58 0 0.125
_+ 17.4 _+ 8.9 +_ 0.02 _+ 5.2 _+ 5.1 _ 10.2 _+ 0.1 _+ 0.1 _+ 1.7 +_ 2.0 nd __ 0.07 _ 26.1 _+ 40.1 +_ 30.20 +_ 0 _ 0.237
Emmental cheeses at 12 months t-test
Weak (N = 10)
ns ** ** ns * ns ns * *** *** ns ns ns ns *** ***
47.0 25.4 5.63 117.4 47.6 63.2 1.7 0.5 5.1 6.7 3.16 901.0 682.6 266.92 4.834 1.886
+_ 8.5 _+ 8.1 _+ 0.06 +_ 5.9 _+ 0.6 _ 4.2 _+ 0.9 _+ 0.1 ___2.8 nd _ 0.9 _ 0.08 _ 28.3 _+ 50.3 +_ 34.51 +_ 0.585 _ 0.494
Strong (N = 8) 11.3 2.9 5.73 148.1 58.7 83.6 1.7 0.7 17.7 8.4 3.21 926.3 687.3 246.86 0.588 0.054
_+ 6.7 _+ 2.4 _ 0.02 _+ 5.0 _+ 1.7 _ 3.6 _+ 0.1 +_ 0.1 +_ 2.5 nd _ 0.3 __ 0.06 _ 28.7 _+ 46.8 +_ 22.95 _+ 0.097 _+ 0.154
t-test *** *** *** *** *** *** ns ** *** *** ns ns ns ns *** ***
a mmol/kg. b log CFU/g. c mol/kg. SCA, Short Chain Acids; WSN, Water-soluble N; T C A S N , 12% TCA-soluble N; nd, not determined; ns, not significant. *p < 0.05; **p < 0.01; ***p < 0.001.
combined effect of aspartate metabolism and of the lncreaseu numt)er of proplonlDacterla." - :' ....... " For the application of propionibacterial cultures in cheese production, their ability to utilise aspartate must be taken into consideration. Excessive aspartase activity has hidden dangers, such as late fermentation, as mentioned above; a moderate activity, however, may influence the quality of Emmental cheese positively, e.g., improving openness, increasing the intensity of flavour and reducing the maturation time. _9
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
1
.
.
.
Interactions
.
In Emmental cheese, interactions between propionibacteria and factors such as the type of lactic acid bacteria, season of milk production (feeding) and proteolysis have a major impact on the propionic acid fermentation. Nowadays, it is easy to control the propionic acid fermentation during the ripening of Emmental cheese. Since the introduction of starter lactic acid bacteria in the 1970s, of facultatively heterofermentative lactobacilli in 1989 and of propionibacteria cultures with weak aspartase activity in 1996, the defect of late fermentation has been practically eliminated in Switzerland. Nevertheless, it is still possible to produce Emmental cheese with eyes made to measure (Fig. 6) - large eyes are achievable with the use of Lb. helveticus together with a strongly aspartase-positive propionibacteria culture. Small eyes are obtained through the use of facultatively heterofermentative lactobacilli together with a weakly aspartase-positive propionibacteria culture. Feeding season
E m m e n t a l c h e e s e (12 m o n t h s old) m a d e with prop i o n i b a c t e r i a with strong (no. 2 6 - 2 7 ) or w e a k (no. 25) aspartase activity ( W y d e r et al., 2001).
Cheeses made from milk produced during the hayfeeding season (winter) are, from experience, more prone to the defect of late fermentation during ripening than cheeses made from milk produced during the grass-feeding season (summer). Cheese producers
Cheeses with Propionic Acid Fermentation
145
Sensory and quality parameters of Emmental cheese (6 and 12 months) made with propionibacteria with weak or strong aspartase activity (mean values and t-test)
Emmental cheeses of 12 months
Emmental cheeses of 6 months Parameter (Index)
Weak (N = 10)
Strong (N = 8)
t-test
Weak (N = 10)
Strong (N = 8)
t-test
Openness (1-6) Number of eyes (0-5) Size of eyes (1-5) Texture (1-6) Firmness (2-8) Maturity (2-8) Intensity of taste (1-6) Intensity of odour (0-7) Intensity of aroma (0-7) Sweetness (0-7) Saltiness (0-7) Sourness (0-7) Bitterness (0-7) Height of cheese (cm)
5.3 4.7 4.9 5.4 4.9 4.4 4.3 3.0 3.1 2.3 1.9 2.0 1.8 19.1
4.6 5.3 5.8 5.5 4.6 5.3 4.7 3.3 3.5 2.2 2.3 2.2 1.7 21.3
* * ** ns ns * ns ns *** ns ** ns ns *
4.6 4.4 4.5 5.3 4.6 6.5 4.5 3.6 3.7 2.5 2.3 2.6 1.8 18.1
4.6 5.4 5.8 5.0 4.7 6.8 4.4 3.5 3.8 2.4 2.5 2.8 1.9 20.6
ns *** *** ns ns ns ns ns ns ns ns ns ns **
+_ 0.6 _ 0.6 _ 0.3 +_ 0.4 _ 0.4 _+ 0.8 _ 0.5 _+ 0.3 _+ 0.2 _ 0.2 + 0.3 _+ 0.2 +_ 0.4 _ 1.5
+_ 0.6 _ 0.4 +_ 0.6 +_ 0.3 _ 0.5 _+ 0.6 _+ 0.3 ___0.3 _+ 0.2 _+ 0.1 _ 0.2 _ 0.2 + 0.4 +_ 1.7
_+ 0.6 _+ 0.6 _ 0.5 _+ 0.7 _+ 0.6 _+ 0.5 _+ 0.6 _ 0.3 _+ 0.4 _+ 0.3 _+ 0.2 _+ 0.3 _+ 0.4 _+ 1.8
_ 0.8 _ 0.3 _ 0.6 _+ 0.6 _+ 0.4 ___0.4 _+ 0.5 _+ 0.3 _ 0.3 _+ 0.2 _+ 0.3 _ 0.4 _+ 0.2 _+ 1.0
ns, not significant. *p < 0.05. **p < 0.01. ***p < 0.001: index indicate the range of appreciation (lowest number = lowest possible score; highest number = highest possible score).
generally observe a slightly slower rate of acidification of the winter milk, resulting in a higher content of water and thus a higher content of lactate in the cheese after 24 h and consequently a lower pH (Table 3). A low pH leads to a slower propionic acid fermentation, since the optimum pH range for propionibacteria is 6-7. Only with proteolysis, a change in pH can be anticipated. Thus, a higher number of propionibacteria is needed in order to start the propionic acid fermentation under this disadvantageous pH. This may be the cause for higher propionibacteria counts which lead to more lactate consumption and therefore more
propionic acid and CO2 production (Table 4). Due to a higher water content, proteolysis is also enhanced. As mentioned above, this is advantageous for the pH but also for the liberation of amino acids, such as asparagine and aspartate, which are substrates for the metabolism of aspartase-positive propionibacteria (Fr6hlich-Wyder et al., 2002). Facultatively heterofermentive lactic acid bacteria Facultatively heterofermentative non-starter lactobacilli are used in the Swiss artisanal cheese industry to slow down the propionic acid fermentation (Sollberger and
X-rays of 180-day-old Emmental cheese produced with strong aspartase-positive propionibacteria and Lb. helveticus (left) or with weak aspartase-positive propionibacteria and facultatively heterofermentative lactobacilli (right) (from Fr6hlich-Wyder et aL, 2002).
146
Cheeses with Propionic Acid Fermentation
Water, lactate, pH and proteolytic parameters for Emmental cheese (Fr6hlich-Wyder et al., 2002)
Factor
Feeding Grass Hay Propionibacteria Weak Strong
Water
Lactate
(g/kg)
(mmol/kg)
TN
WSN
NPN
Free AA
pH
(g/kg)
(% TN)
(% WSN)
(mmol/kg)
N
1d
180 d
1d
180 d
180 d
180 d
180 d
180 d
180 d
16 16
372.5 373.9
326.2 331.4
125.6 131.3
26.9 25.5
5.83 5.72
46.5 44.7
23.2 25.0
61.9 64.6
175.5 199.1
16 16
372.9 373.5
328.2 329.4
128.3 128.6
34.5 17.9
5.77 5.78
45.5 45.6
24.6 23.6
63.1 63.2
196.8 177.8
16 16
372.9 373.5
328.9 328.7
128.9 127.9
51.2 1.2
5.76 5.79
45.6 45.6
24.5 23.7
63.3 63.0
191.9 182.7
16 16
372.8 373.6
328.5 329.1
127.9 129.0
25.4 26.9
5.78 5.77
45.6 45.6
24.0 24.2
64.5 61.8
196.0 178.5
-
. . . . . . . . . *** *. . . . . -
Lb. casei
Added Not added Lb. helveticus
Added Not added ANOVA Feeding Propionibacteria
. -
Lb. casei Lb. helveticus
.
.
.
.
.
.
-
.
.
.
.
**
-
** * -
-, not significant. * p < 0.05. **p < 0.01. ***p < 0.001. TN, total N; WSN, water soluble N; NPN, non-protein N; AA, amino acids.
Free short-chain acids (FSCA), succinate and citrate in mmol/kg, as well as propionibacteria (PAB) and facultatively heterofermentative lactobacilli (FHL) in log cfu/g in 180-day-old Emmental cheese (n = 16 for each factor level) (Fr6hlich-Wyder et aL, 2002) Factor
Feeding Grass Hay Propionibacteria Weak Strong
C1
C2
C3
C4
C6
FSCA
Succinate
Citrate
FHL
PAB
1.7 2.4
41.5 51.6
75.8 88.7
0.87 1.17
0.32 0.36
120.4 144.4
9.8 10.4
4.2 3.3
7.24 7.23
8.12 8.03
2.3 1.8
43.3 49.8
76.6 87.9
1.03 1.02
0.32 0.36
123.8 141.0
4.2 15.9
3.8 3.7
7.30 7.17
7.56 8.59
3.5 0.6
47.3 45.8
68.6 95.9
1.05 0.99
0.33 0.35
121.1 143.7
9.3 10.8
0.2 7.4
7.53 6.94
7.95 8.20
2.2 1.9
47.3 45.9
82.0 82.5
1.02 1.02
0.34 0.34
133.0 131.8
10.2 9.9
3.6 3.9
7.30 7.17
7.97 8.18
Lb. casei
Added Not added Lb. helveticus
Added Not added ANOVA Feeding Propionibacteria
. .
Lb. casei Lb. helveticus
*** .
. . . . . . . . . . . .
. ***
.
.
m
-
.
.
-
.
-, not significant. *p < 0.05. **p < 0.01. ***p < 0.001. C1, formate; C2, acetate; C3, propionate; C4, butyrate; C6, caproate.
m
a
m
B
m
C h e e s e s with P r o p i o n i c A c i d F e r m e n t a t i o n
Wyder, 2000). Jimeno et al. (1995) found growth inhibition of propionibacteria in cheese of up to 80% compared to the control without facuhatively heterofermentative lactobacilli (Lb. casei and Lb. rhamnosus). As a consequence, less propionic acid is produced. The observed inhibition could not be reproduced in co-cultures, suggesting that bacteriocin production is not responsible for this effect. Citrate metabolism most probably plays the key role, since citrate-negative mutants were shown to inhibit propionibacteria much less than the corresponding citrate-positive strains (Jimeno, 1997). Lb. rhamnosus also produces small but appreciable amounts of diacetyl which has a lethal effect on propionibacteria. Acetate and formate seem to have an inhibitory effect on the growth of propionibacteria. In addition, the metabolism of citrate, which takes place before the propionic acid fermentation, leads to the release of the complexed copper. The ratio of citrate and copper plays an important role in the observed inhibition (Perez et al., 1987). However, the mechanism of inhibition is not yet conclusively clarified. Since the introduction of cultures of facuhatively heterofermentative non-starter lactobacilli in Switzerland in 1989, the defect of late fermentation has decreased considerably. Propionibacteria with differing aspartase activity are not inhibited in the same way, a fact already known by cheesemakers. A weak aspartase-positive culture together with Lb. casei requires a prolonged period in the warm room for Emmental cheese while a strong aspartase-positive culture without the addition of facuhatively heterofermentative lactobacilli leads to a shorter stay. Thus, propionibacteria with weak aspartase activity are inhibited much more than propionibacteria with strong aspartase activity. The question arises as to whether the weakly aspartase-positive propionibacteria are more sensitive to formate and acetate. The interaction in Fig. 7 shows that both cultures produce approximately the same amount of propionic acid after 180 days of maturation, but with the addition of Lb. casei, the propionibacteria with weak aspartase activity produce much less propionic acid. This is why propionibacteria with strong aspartase activity are generally more prone to provoke late fermentation. Lb. helveticus
Proteolysis is very important for the development of the texture and flavour characteristics of Emmental cheese. Intensified proteolysis generally leads to accelerated ripening of the product which is desired as long as no adverse effect on the storage quality is encountered. In Emmental cheese production, strong proteolysis, together with intense propionic acid fermentation, may,
147
100 -
Icn
90 -
m O
E 80E c-
70-
-"
Prop96
---1- Prop90
O .m O
a_ 6 0 50-
no
yes
I
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Addition of Lb. casei Two-way interaction between propionibacteria with different aspartase activity and Lb. casei for propionate in 180day-old Emmental cheese (Prop96, weak aspartase activity; Prop90, strong aspartase activity) (Fr6hlich-Wyder et al., 2002).
however, be the primary cause of late fermentation (Bachmann, 1998a; Baer and Ryba, 1999). The texture which becomes shorter and crumbly during proteolysis shows a loss of elasticity and the cheese can develop cracks because of excessive CO2 production. Several investigations have shown that thermophilic lactic acid bacteria, especially Lb. delbruechii and Lb. helveticus, can stimulate the growth of propionibacteria (Perez et al., 1987; Piveteau et al., 1995; Chamba, 2000; Kerjean et al., 2000). Baer (1995) found poor growth of propionibacteria in milk alone or with added rennet, but good growth in the presence of lactic acid bacteria alone or with added rennet. It was concluded that the growth of propionibacteria depends on the presence of free amino acids or small peptides. In later work, Baer and Ryba (1999) found that propionibacteria clearly prefer free amino acids to peptides. They concluded that the growth of propionibacteria, and thus the intensity of propionic acid fermentation and the risk of late fermentation, is correlated with the amount of free amino acids. In fact, Lb. helveticus is responsible for the liberation of a larger quantity of small peptides in Emmental cheese (Table 3, NPN, % of WSN). Piveteau et al. (1995) described the liberation of a heat-resistant stimulatory compound by Lb. helveticus which might be an aspartate or a peptide containing it. In contrast, the absence of nutrients is not the reason why propionibacteria fail to grow in milk when inoculated at <105 cfu/ml - i The same authors presented evidence for an inhibitory substance in milk, which is heat-stable and has a low molecular mass (Piveteau et al., 2000). It is removed by Lb. helveticus strains as a result of proteolysis, but not by Lb. delbrueckii or Lb. lactis strains. Consequently, the activation of propionibacterial growth may be the result of stimulation by the proteolytic activity of lactobacilli-liberating peptides and
148
Cheeses with Propionic Acid Fermentation
free amino acids and/or the removal of an inhibitory substance by the action of Lb. helveticus (Kerjean et al., 2000).
At this temperature, the curd dries and most of the undesirable micro-organisms are eliminated. This is not only important for the hygienic safety, but also for avoiding too intensive proteolysis in depth (peptidolysis), which leads to a 'shorter', i.e., more crumbly texture of the cheese, and increases the risk of splits and cracks during ripening. The high cooking temperature also causes the complete inactivation of the chymosin. A temperature above 54 ~ impairs the propionibacteria too strongly. Swiss-type cheeses are often manufactured in copper vats (Fig. 9) and pressed in cylindrical moulds (Fig. 10). The copper content should not be too high, because copper inhibits the formation of lactic and propionic acids. On the other hand, copper forms complexes with sulphur compounds originating from the catabolism of amino acids, and thus has a positive impact on the flavour and aroma of the cheese. A copper content between 120 and 200 Ixmol~g is optimal; it comes mainly from the vat surface. Because propionibacteria are sensitive to salt, brining is less intensive than for other cheese varieties. The average salt content in Swiss Emmental cheese is between 3 and 5 g/kg. Brining leads to a firm and dry rind, which reduces the loss of CO2 during the propionic acid fermentation and thus supports eye formation. The rind is also responsible for the sturdy shape of the cheeses during ripening.
The milk used for cheese manufacture should contain as few bacteria as possible so that the added starter cultures can have an optimum effect. If raw milk is used, the bacteriological requirements are particularly stringent. The microbial and hygienic state of farm milk, of course, also depends on the duration and temperature of storage, and secondary contamination before or after processing must be avoided. In Switzerland, Emmental cheese must be manufactured from raw milk from cows receiving no silage feeds. The whole technological sequence of operations is geared to creating optimum conditions for the propionic acid fermentation. A fundamental step is the addition of water (12-18%) to the milk or to the curd. This leads to a relatively high pH after the lactic fermentation (5.20-5.30), which consequently accelerates the propionic acid fermentation. Furthermore, this step leads to a soft and elastic texture and is also the explanation for the high calcium content of the cheese. A soft and elastic texture is crucial for regular eye formation. To avoid undesired fermentations, no residual sugar (galactose) should remain after the lactic fermentation. ]~/lnct q,~rlcc_t,rno
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ing or scalding temperature. Emmental curd is heated to 52-54 ~ after cutting. During pressing, the temperature remains at around 50 ~ for many hours (Fig. 8).
50
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42 40 38 36 34 32 30
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countries by technologies differing from the traditional Swiss procedure. Considering the techno]ogica| aspects,
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9 10 11 12 13 14 15 16 17 18 19 20 Pressing hours
Temperature profile of Emmental cheese during pressing (Steffen and Schnider, 1978).
Cheeses with Propionic Acid Fermentation
149
General aspects
Cutting of the curd. (See Colour plate 17.)
other hand, the treatment of milk, the extent of mechanisation, the weight and shape, the average composition (hard or semi-hard varieties both with different fat contents), ripening time and shelf life are frequently very different. Quite often, Swiss-type cheeses are manufactured in the form of blocks and ripened in plastic bags for large-scale production.
To initiate the typical propionic acid fermentation, the ripening temperature for the cheese must be raised to approximately 20-24 ~ for a certain period of time. As soon as the development of sufficient eyes has been accomplished, the propionic acid fermentation is retarded by storing the cheese at a lower temperature (10-13 ~ In the case of Swiss Emmental cheese, the period of the eye formation is between 40 and 60 days. By tapping the cheeses, the cheesemaker determines the right time for the transfer from the warm to the ripening room. The relative humidity in the ripening room is rather low (70-80%). This, and also the brining as mentioned above, leads to a firm and dry rind, which reduces the loss of CO2 and leads to the sturdy shape of the cheeses. Furthermore, the low humidity accelerates the sweating of the cheeses (secretion of fat) and reduces the growth of moulds on the surface of the cheeses and, therefore, the time needed for manual cleaning. The ripening period varies widely; some cheese varieties are sold directly after the propionic acid fermentation while others are ripened for more than i year. Lately in Switzerland, ripening under humid conditions (in rock caves) has gained more importance. By the action of specific moulds, the cheese surface becomes black and proteolysis is further affected, although the proteolytic enzymes of the surface flora do not penetrate into the cheese mass. An indirect, but strong proteolytic, effect arises from the accelerated increase in pH of the outer zones of the cheeses due to deacidification of the surface by the moulds. Moreover, soluble substances produced by the surface flora diffuse into the cheese mass. Flavour formation
Filling of the curd into the cheese moulds. (See Colour plate 18.)
Flavour development in cheese is very strongly dependent of the microbial flora of milk. Whilst the indigenous flora of milk is generally composed of unwanted microorganisms, which can influence the flavour directly by their fermentative activities or indirectly by other enzymatic reactions, the desired lactic acid bacteria must be added to the cheese milk as starter or adjunct cultures. The addition of rennet and the different operations involved in cheesemaking and cheese ripening influence flavour development. In Switzerland, Emmental cheese is made from raw milk. Certain flavour compounds in milk are in fact lost and others are produced when it is subjected to thermisation or pasteurisation before processing. The high temperatures applied during the early stages of manufacture and pressing of Emmental cheese are essential for flavour development. Other important factors are the fermentation and ripening processes.
150
Cheeses with Propionic Acid Fermentation
During ripening for 3-12 months, the intensity of odour, aroma, saltiness and sourness increase (Table 5). Sweetness and bitterness decrease slightly. Due to the propionic acid fermentation, the sweet taste is about 1-1.5 units higher than in other hard cheese varieties without the propionic acid fermentation. Warmke etal. (1996) evaluated the following substances as potent taste compounds: acetic, propionic, lactic, succinic and glutamic acids, each in free form and/or as ammonium, sodium, potassium, magnesium or calcium salts, as well as the corresponding chlorides and phosphates. Magnesium and calcium propionate mainly caused the sweetish note in the taste profile of Emmental. Although bitter-tasting amino acids and peptides occurred in the cheese, they were not detected in the taste profile. For analytical reasons, the flavour components are generally divided into two major groups: volatile and non-volatile compounds. The volatile compounds derive from glycolysis, proteolysis and lipolysis during ripening and include volatile short chain acids (Table 4), primary and secondary alcohols, methyl ketones, aldehydes, esters, lactones, alkanes, aromatic hydrocarbons and different sulphur- and nitrogen-containing compounds (Table 6). Methional and acetic and propionic acids are the most important volatile compounds for typical Emmental flavour. Ethyl butanoate, ethyl 3-methylbutanoate and ethyl hexanoate contribute to the fruity odour note. The two fllranone~ are responsible for the caramel-like flavour in Emmental cheese. Proteolysis is considered to be essential to the ripening process, contributing to flavour formation by the liberation of free amino acids. Recent results, however, showed that an increase of free amino acids in cheese could not be correlated with flavour formation. It is concluded that the rate-limiting factor in the formation of flavour is the degradation of amino acids by the micro-organisms present in cheese rather than their release (Yvon and Rijnen, 2001). Lactic acid bacteria are the main contributors, but propionibacteria
Flavour description of Emmental cheese (Mean values _+ standard deviation for n = 10) (taken from the work by Wyder et al., 2001)
Cheese age (months) Parameter
Scale
3
Odour intensity Aroma intensity Sweetness Saltiness Sourness Bitterness
0-7 0-7 0-7 0-7 0-7 0-7
3.0 2.5 2.5 1.9 2.1 2.0
6 -+ 0.3 _+ 0.3 _ 0.1 _ 0.2 +_ 0.3 _+ 0.3
3.1 3.1 2.3 1.9 2.0 1.8
12 -+ 0.2 _+ 0.2 _+ 0.2 _+ 0.3 +_ 0.2 +_ 0.4
3.6 3.7 2.5 2.3 2.6
_ 0.3 _+ 0.4 _ 0.3 ___0.2 ___0.3 1.8 _+ 0.4
Concentration of odorants (i~g/kg dry matter, except ammonia) in Emmental cheese (Mean values _+ standard deviation for n = 4) (unpublished results from the work by Wyder et aL, 2001)
Cheese age (months) Odorant
3
6
2,3-Butandione 2-Methylbutanal 3-Methylbutanal Ethylbutanoate Ethyl 3-methylbutanoate 2-Heptanone Dimethyltrisulfide Methional Ethylhexanoate 1-Octen-3-one 4-Hydroxy-2, 5-dimethyl3(2H)-furanone 5-Ethyl-4-hydroxy2-methyl-3(2H)furanone 2-sec-Butyl-3methoxy-pyrazine Skatole 8-Decalactone 3-Methylbutyric acid Ammonia, mg/kg
431 _ 147 181 _ 4 0 145 +__22 27-+ 17 0.40 _+ 0.16
605 _+ 354 251 _+43 167 +_ 16 73_+23 0.78 +_ 0.21
531 _+ 470 372 + 8 8 152 -+ 15 148_+72 2.48 _ 1.25
522 _+ 60 0.11 +_ 0.10 67__33 51 ___21 0.06 _+ 0.02 1186_+276
770 _+ 57 0.16 _+ 0.08 68_+22 164 _+ 63 0.05 _+ 0.02 658_297
2783 _+ 1517 0.21 _+ 0.16 50_+6 351 _+ 156 0.06_+ 0.02 1002_+387
253 _ 72
255 _ 86
547-_ + 232
0.07 _ 0.03
0.05 +_ 0.03
0.04_+ 0.01
4 7 _ 15 3751 _ 1216 2 0 _ 10
34_6 1 6 8 0 _ 97 3 0 _ 10
37_+ 10 1171 + 132 30+_ 10
560 -+ 150
12
720 _+ 160
970 _ 190
also have a high ability to convert branched-chain amino acids to acids. Isoleucine/Leucine is converted mainly to isovaleric acid (Thierry and Maillard, 2002). The cause of lipolysis in Emmental cheese can be bacterial lipase and the indigenous lipoprotein lipase in milk which is, however, thermolabile and therefore its activity is reduced by cooking to a temperature over 50 ~ Lactic acid bacteria have only limited lipolytic activity, with Sc. thermophilus having the highest (Gobbetti et al., 1996). Propionibacteria, in contrast, have high strain-dependent lipolytic activity, 10-100 times more than lactic acid bacteria (Dupuis, 1994). Lipolysis in Emmental is caused mainly by propionibacteria and is generally recognised as necessary to produce typical Emmental cheese flavour. The amount of free fatty acids present varies from 2 to 7 g/kg (Isolini et al., 2001). Nevertheless, higher contents give flavour defects (Bachmann, 1998b). The release of free fatty acids starts in the warm room simultaneously with the growth of propionibacteria (Chamba and Perreard, 2002). Long-chain free fatty acids, which have a minor influence on cheese flavour, accumulate during ripening, whereas short- and medium-chain free fatty acids are most probably transformed by ]3-oxidation and esterification to volatile flavour compounds (Bosset
Cheeses with Propionic Acid Fermentation
et al., 1995; Chamba and Perreard, 2002). Since propionibacteria have very strain-dependent lipolytic activity and also are the main lipolytic agents in Emmental, it is important to include the potential for lipolysis in the selection of new propionibacterial cultures. The non-volatile flavour compounds include peptides, free amino acids, amines (Table 1), free fatty acids, salt and minerals (Table 7). The peptides and free amino acids contribute to the background flavour. Free glutamic acid is mainly responsible for the umami taste. Salt (NaC1) and other minerals directly influence the saltiness and indirectly the total aroma intensity. Cheese off-flavour depends quite often on the properties of the cheese milk. Certain plants and feeds such as bulbous plants, leeks, vegetable wastes, herb mixtures and different mineral salt mixtures fed to dairy cows can influence the taste of milk and produce offflavours. Certain milk enzymes also can induce offflavours, e.g., rancidity induced by lipase. Today, Swiss-type cheese can be found on the market over a wide range of maturity from very young, elastic cheese with the typical sour lactic aroma and sweet taste up to cheese ripened for a very long period in humid caves (Fig. 11) with a more intensive flavour and a nutty and spicy note. Texture formation
Cheese body and texture are very important qualities for both traders and consumers. Variations from what is considered normal in body and texture within the same cheese variety are not tolerated because there is a close relationship between the body and texture and other qualities, such as eye formation, taste and shelf life. Texture denotes the structure and the consistency of the cheese body. A soft and elastic texture is crucial for regular eye formation. The high calcium content, which is the result of the high pH after the lactic fermentation (5.20-5.30), is very important for a 'long', elastic texture. Table 8 shows the development of the Concentration of non-volatile components (g/kg dry matter) in Emmental cheese (mean values _+ standard deviation for n = 4) (unpublished results from the work by Wyder et aL, 2001)
Cheese age (months) Taste component
3
Glutamic acid Sodiuma Potassium a Magnesiuma Calcium a Phosphate a
5.4 5.2 1.3 0.7 6.6 10.6
6 +_ 0.6 _+ 0.6 +_ 0.1 _+ 0.1 _+ 0.8 _+ 1.2
8.1 4.5 1.0 0.6 6.5 13.9
a Concentration in the aqueous extract.
Emmental, about 12-months-old, matured in a cave with a high relative humidity.
rheological parameters and penetrometry data during ripening from 3 to 12 months. The texture of Swiss Emmental changes during ripening from elastic and relatively soft to less elastic, more friable and more firm. Figure 12 shows the typical texture profile of 6-month-old Emmental cheese. Because of the low water content, Swiss-type cheese melts at a relatively high temperature. The average softening point measured with an automatic dropping point apparatus is 74 ~ The sum of free amino acids measured 1 day after manufacture by the Cadmium-ninhydrin method and the trichloroacetic acid-soluble nitrogen after 20 days allow an early prediction of flavour and texture development in Emmental cheese (Bachmann et al., 1999). Emmental cheese is characterised by a higher ratio of Otsl-:[3-casein than in cheeses which are not cooked at 50-55 ~ This is due to the specificity of primary proteolysis: Otsl-casein is hydrolysed at a slower rate and [3-casein at a faster rate. The slower proteolysis of Otsl-casein is due to inactivation of the coagulant, either chymosin or microbial enzymes, during cooking. The faster rate of primary proteolysis of [3-casein is due to the activity of plasmin. The high ratio of Otsl-:13-casein Results of penetrometry and uniaxial compression test on Emmental cheese during ripening (mean values __+standard deviation for n = 10) (adapted from Bachmann et aL, 1999)
12 _+ 0.3 _+ 0.2 __ 0.1 _+ 0.1 +_ 0.6 __+ 1.5
11.6 4.5 1.2 1.0 10.6 13.2
151
Cheese age (months) _ 1.2 _+ 0.7 _+ 0.1 _+ 0.1 _+ 0.7 __ 0.6
Parameter
Unit
3
Penetrometry Strain at fracture Stress at fracture Stress at 33% deformation
mm % kN/m2 kN/m 2
3.7 68.9 614 147
6 _+ 1.0 2.5 _+ 0.3 _+ 3.2 63.7 _ 2.5 +_ 121 437 +_ 58 _+ 16 157 +_ 20
12 4.6 46.5 319 244
___0.6 _+ 5.4 + 48 +_ 30
152
Cheeses with Propionic Acid Fermentation
Texture profile of 6-month-old Emmental cheese (adapted from Lavanchy and Betikofer, 1999).
is jointly responsible for the soft and elastic texture (Kerjean et al., 2001). The proteolytic activity of propionibacteria is insignificant. The course of proteolysis in Swiss Emmental cheese is shown in Table 9. Insufficient proteolysis may cause different cheese defects. If the level of protein breakdown is too low, the taste is fiat and the body consistency too 'long', i.e., rubbery. Sometimes, uneven openings also appear. Excessive proteolysis results in an overripe and sharp taste and a shorter body. Frequently, the extent of proteolysis in a cheese loaf varies from one zone to the other, a phenomenon that is due to a changing temperature profile in the cheese loaf during the lactic acid fermentation. Since the outer zone cools faster, it often develops a bacterial flora which is proteolytically more active than the flora of the centre of the loaf. This usually leads to cheese defects such as a short and firm body, or a sharp taste, or the development of white colour under the rind.
diversity of the indigenous propionibacterial flora is great. The number and size of eyes vary markedly, and cracks or splits are quite common. The application of a culture of selected propionibacteria allows a more regular eye formation as a result of a propionic acid fermentation which is under control. Small quantities of CO2 are already produced during the lactic acid fermentation and the degradation of citrate. The fermentation of citrate leads to a higher number of eyes in the initial stage of the propionic acid fermentation and to a lower number of eyes in the mature cheese (Fig. 13). To initiate the typical propionic acid fermentation, the ripening temperature for the cheese must be raised to approximately 20-24 ~ As soon as the development of sufficient eyes is accomplished, the propionic acid fermentation is retarded by storing the cheese at a lower temperature (10-13 ~ The different stages of CO2 production and eye formation may be summarised schematically as follows: CO2 production and CO2 diffusion (starts at the beginning of the lactic acid fermentation)
Accumulation of CO2 in the cheese body (degradation of citrate and propionic acid fermentation)
Over-saturation at the centres of future eye formation (propionic acid fermentation)
Onset of eye formation at these centres (after approximately 20-30 days) The propionic acid fermentation leads to the formation of characteristic eyes and a nutty flavour and can occur either spontaneously or be achieved by a culture of selected propionibacteria. A spontaneous fermentation leads to irregular eye formation, because strain
Increase in the number of eyes and their enlargement (propionic acid fermentation + decarboxylation of amino acids)
Proteolysis in Swiss Emmental cheese (mean values _+ standard deviation for n = 10) (adapted from Bachmann et aL, 1999)
Cheese age (months) Parameter
Unit
2/3
3
6
12
Water-soluble nitrogen 12% trichloroacetic acid-soluble nitrogen Sum of free amino acids
mmol/kg mmol/kg
218 +_ 17 90 _+ 9
610 _+ 31 386 _+ 39
693 +_ 33 469 _+ 47
901 _+ 28 683 _+ 60
mmol/kg
16 _+ 4
120 +_ 32
169 _ 24
267 _+ 35
Cheeses with Propionic Acid Fermentation
153
150
100 >,, 0 ..(3
E
z
sei
50
d,//
~
40 days
without Lb. casei
180 days Stage of ripening
Influence of the citrate metabolism on the number of eyes in Emmental cheese (adapted from the work by Fr6hlichWyder et al., 2002).
The development of eye formation depends mainly on: 9 9 9 9
time, quantity and intensity of CO2 production; number and size of the areas of future eye formation; CO2 pressure and diffusion rate; body texture and temperature.
At the beginning of eye formation, i.e., about 30 days after manufacture, only a few eyes appear; thereafter, the number of new holes increases progressively. The maximum rate is attained after about 50 days, which is also the time of rapid eye enlargement. The appearance of new eyes declines with decreasing CO2 production and simultaneous hardening of the cheese body. The quantity and distribution of the eyes also depend on other factors such as those mentioned above. The number of eyes is increased by the nonhomogeneity of the curd, physical openness and the content of gas. Centrifugation and heat treatment of the milk or application of vacuum after filling of the curds and during pressing of the cheese are performed in order to obtain a lower number of eyes. In cheeses produced from microfihered milk, the number of eyes is generally much lower, even when the retentate is added. This indicates that air bubbles are important areas of future eye formation. As regards eye formation, proper dip filling of the moulds is imperative since air inclusions can lead to undesirable openness. Figure 14 shows the deformation of the granules around eyes. In a cheese loaf of approximately 80 kg, total CO2 production is about 120 1 before the cheese is sufficiently aged for consumption. About 60 1 remain dissolved in the cheese body, - 2 0 1 are found in the eyes and - 4 0 1 diffuse out of the loaf (Flackiger et al., 1978).
The development of CO2 pressure shows two major phases (Fig. 15). The first covers the period of proper eye formation in the ripening room. During this period, the CO2 pressure remains relatively low, between 1500 and 2500 Pa, because of the low resistance of the soft cheese mass to gas compression at 22-24 ~ During storage, i.e., the second stage, the CO2 pressure increases to 4000-8000 Pa. The difference in pressure between various loaves is higher in the second stage than in the first. The pressure increase in the second stage is explained by the higher resistance to gas compression of the cheese mass, which is due to the decrease in temperature from 22 to 12 ~ and by continued gas production. During the first stage there is a marked pressure increase within the eyes. These observations go back to a period where it was common to use propionibacteria
Deformation of curd granules around eyes (L) in Emmentaler cheese (ROegg and Moor, 1987).
154
Cheeses with Propionic Acid Fermentation
40
:30 8..
~3 (D
(D
E 20 _=
or) or) (3. (D >
,-2
LU
o 10
!
i
1
5
Weeks:
!
7
|
i
11
9
Ripening room 4
,
13
i
15
,
17
~
19
i
21
23
Cold room ~.-,tl
Eye volume (IC]) and 002 overpressure (~)in Emmental cheese (adapted from FILickiger, 1980).
with high aspartase activity (Fl{ickiger, 1980). More recent results are not available. Excessive proteolysis becomes particularly evident when a large amount of casein is degraded to lowmolecular compounds (high non-protein nitrogen level). Additional CO2 production by decarboxylation of amino acids clearly reduces the keeping quality of the cheese and leads to oversized eye formation. In addition, the propionibacteria are stimulated which increases the production of CO2 (aspartate metabolism). The cheese body often cannot withstand the pressure of the gas and cracks or splits appear. This defect is referred to as late or secondary fermentation.
In Switzerland, Emmental cheese must be manufactured from raw milk. Despite intense hygienic efforts, contamination of raw milk by pathogenic microorganisms cannot be completely excluded. Infectious diseases in dairy cows or contamination of milk during milking, storage, transport or processing present potential hazards. This has led to discussions on their hygienic safety of raw-milk cheeses. Bachmann and Spahr (1995) and Spahr and Schafroth (2001) examined the ability of potentially pathogenic bacteria to grow and to survive during the manufacture and ripening of Swiss Emmental cheese made from raw milk (Figs 16 and 17). They concluded that the hygienic safety of Swiss Emmental cheese is comparable to cheese produced from pasteurised milk.
The decrease of the number of pathogens can be explained by the so-called hurdle technology, which implies the synergistic effects of a high milk quality, short milk storage (effect of active antimicrobial enzyme systems of fresh raw milk), antagonistic starter culture flora, rapid acidification, antimicrobial effect of lactic acid, high curd cooking temperature, brining, and ripening at an elevated temperature for at least 4 months. All these factors are also important determinants of flavour and texture quality.
8 7 6
"~ 4 g, 3 ~ 2 1
0
Milk
Curd after cooking
Cheese Cheese Cheese 1d 7d 30 d
Behaviour of Aeromonas hydrophila (~), CampyIobacter jejuni (~), Escherichia coil ( A), Listeria monocytogenes (A), Pseudomonas aeruginosa (0), Salmonella typhimurium (I), Staphylococcus aureus ([-7) and Yersinia enterocolitica (11) during manufacture and ripening of Swiss Emmental cheese made from raw milk (only data for batches with longest survival are shown); detection l i m i t , - - - - (Bachmann and Spahr, 1995).
Cheeses with Propionic Acid Fermentation 6~
i
i
i
0
i
i
i
30
60
90
i
o
0
\
., 120
150
Ripening days Inactivation curves for Mycobacterium avium subsp. paratuberculosis in Swiss Emmental cheese during 120 days of ripening (Spahr and Schafroth, 2001).
Bachmann, H.P. (1998a). Die Verg/irung von Aspartat durch Propions/mrebakterien steigert das Risiko von Nachg/irung beim Emmentaler K/ise, Agrarforschung 5, 161-164. Bachmann, H.P. (1998b). Lipolyse im K/ise: nicht zu viel, nicht zu wenig. Agrarforschung 5,293-295. Bachmann, H.P. and Spahr, U. (1995). The fate of potentially pathogenic bacteria in Swiss hard and semihard cheeses made from raw milk. J. Dairy Sci. 78,476-483. Bachmann, H.P., B1)tikofer, U. and Meyer, J. (1999). Prediction of flavour and texture development in Swiss-type cheeses. Food Sci. Technol. -Lebensm. -Wiss. Technol. 32, 284-289. Baer, A. (1995). Influence of casein proteolysis by starter bacteria, rennet and plasmin on the growth of propionibacteria in Swiss-type cheese. Lait 75,391-400. Baer, A. and Ryba, I. (1999). Interactions between propionic acid bacteria and thermophilic lactic acid bacteria. Lait 79, 79-92. Bosset, J.O., Gauch, R. and Mariaca, R. (1995). Comparison of various sample treatments for the analysis of volatile compounds by GC-MS. Application to the Swiss Emmental cheese. Mit. Gebiete Lebensm. Hyg. 86, 672-698. Brendehaug, J. and Langsrud, T. (1985). Amino acid metabolism in propionibacteria: resting cell experiments with four strains. J. Dairy Sci. 68, 281-289. Chamba, J.E (2000). Emmental cheese: a complex microbial ecosystem. Consequences on selection and use of starters. Sci. Alim. 20, 37-54. Chamba, J.E and Perreard, E. (2002). Contributrion of propionic acid bacteria to lipolysis of Emmental cheese. Lait 82, 33-44. Crow, V.L. (1986a). Utilization of lactate isomers by Propionibacterium freudenreichii subsp, shermanii: regulatory role for intracellular pyruvate. Appl. Environ. Microbiol. 52,352-358. Crow, V.L. (1986b). Metabolism of aspartate by Propionibacterium freudenreichii subsp, shermanii: effect on lactate fermentation. Appl. Environ. Microbiol. 52,359-365.
155
Crow, Vi. and Turner, K.W. (1986). The effect of succinate production on other fermentation products in Swiss-type cheese. NZ J. Dairy Sci. Technol. 21,217-227. Crow, V.L., Martley, EG. and Delacroix, A. (1988). Isolation and properties of aspartase-deficient variants of Propionibacterium freudenreichii subsp, shermanii and their use in the manufacture of Swiss-type cheese. NZ J. Dairy Sci. Technol. 23, 75-85. Dupuis, C. (1994). Activitr prot~olytiques et lipolytiques des bact~ries propioniques laiti~res. Th~se ENSA, Rennes. Fessler, D. (1997). Characterisation of Propionibacteria in Swiss Raw Milk by Biochemical and Molecular-biological Methods, Thesis No. 12328, ETH Zf~rich. Fh)ckiger, E. (1980). CO2- und Lochbildung im Emmentalerk/ise. Schweiz. Milchzeitung 106, 473-480. Fl(ickiger, E., Montagne, D.H. and Steffen, C. (1978). Beitrag zur Kenntnis der CO2-Bildung im Emmentalerk/ise vor Beginn der Propions/mreg/irung. Schweiz. Milchwirt. Forsch. 7, 73-78. Frohlich-Wyder, M.T., Bachmann, H.P. and Casey, M.G. (2002). Interaction between propionibacteria and starter/ non-starter lactic acid bacteria in Swiss-type cheeses. Lait 82, 1-15. Gobbetti, M., Fox, EE and Stepaniak, L. (1996). Esterolytic and lipolytic activities of mesophilic and thermophylic lactobacilli. Ital. J. Food Sci. 2, 127-135. Isolini, D., Casey, M.G., FrOhlich-Wyder, M.T., Meyer, J., Sollberger, H. and Collomb, M. (2001). Propions/mrebakterien und ihre lipolytische Aktivit/it: eine mOgliche Screening-Methode. FAM Intern. Bericht 35, 1-9. Jimeno, J. (1997). Lactobacillus casei et Lactobacillus rhamnosus citrate (+) et citrate ( - ) des MK 3007 et 3008: Croissance et antagonisme dans l'emmental module. FAM Intern. Ber. Biochem. 14, 1-18. Jimeno, J., Lazaro, M.J. and Sollberger, H. (1995). Antagonistic interactions between propionic acid bacteria and nonstarter lactic acid bacteria. Lait 75,401-413. Kerjean, J.R., Condon, S., Lodi, R., Kalantzopoulos, G., Chamba, J.E, Suomalainen, T., Cogan, T. and Moreau, D. (2000). Improving the quality of European hard-cheeses by controlling of interactions between lactic acid bacteria and propionibacteria. Food Res. Int. 33, 281-287. Kerjean, J.R., Bachmann, H.E and Cogan, T. (2001). Technical note: Cooking temperature of whey and curd during Emmental cheesemaking. Milchwissenschaft 56, 556-556. Lavanchy, E and Bfitikofer, U. (1999). Caract~risation sensorielle de fromages/t pate dure ou mi-dure fabriqu4s en Suisse. Mitt. Lebensm. Hyg. 90, 670-683. Perez Chaia, A., Pesce de Ruiz Holgado, A. and Oliver, G. (1987). Interaction between Lactobacillus helveticus and Propionibacterium freudenreichii subsp, shermanii. Microbiol. Aliment. Nutr. 5,325-331. Piveteau, P.G., Condon, S. and Cogan, T.M. (1995). Interactions between lactic and propionic acid bacteria. Lait 75, 331-343. Piveteau, P., Condon, S. and Cogan, T.M. (2000). Inability of dairy propionibacteria to grow in milk from low inocula. J. Dairy Res. 67, 65-71.
156
Cheeses with Propionic Acid Fermentation
Richoux, R. and Kerjean, J.R. (1995). Technological properties of pure propionibacteria strains: test in small scale Swiss-type cheese. Lait 75, 45-59. Ruegg, M. and Moor, U. (1987). The size distribution and shape of curd granules in traditional Swiss hard and semi-hard cheeses. Food Microstruct. 6, 35-46. Sebastiani, H. and Tschager, E. (1993). Succinatbildung durch Propions~urebakterien- Eine Ursache der Nachgarung yon Emmentaler? Dt. Molk.-Ztg. 114, 76-80. Sieber, R., Collomb, M., Lavanchy, P. and Steiger, G. (1988). Beitrag zur Kenntnis der Zusammensetzung schweizerischer konsumreifer Emmentaler, Greyerzer, Sbrinz, Appenzeller und Tilsiter. Schweiz. Milchwirt. Forsch. 17, 9-16. Sollberger, H. and Wyder, M.T. (2000). Propions~iurebakterien und fakultativ heterofermentative Laktobazillen. Schweiz. Milchzeitung 126, 5. Spahr, U. and Schafroth, K. (2001). Fate of Mycobacterium avium subsp, paratuberculosis in Swiss hard and semihard
cheese manufactured from raw milk. Appl. Environ. Microbiol. 67, 4199-4205. Steffen, C. and Schnider, J. (1978). Erhebungen t~ber den Temperaturverlauf im Emmentalerk~se. Schweiz. Milchzeitung 104, 383. Thierry, A. and Maillard, M.-B. (2002). Production of cheese flavour compounds derived from amino acid catabolism by Propionibacterium freudenreichii. Lait 82, 17-32. Warmke, R., Belitz, H.D. and Grosch, W. (1996). Evaluation of taste compounds of Swiss cheese (Emmentaler). Z. Lebensm. Untersuch.-Forsch. 203,230-235. Wyder, M.T., Bosset, J.O., Casey, M.G., Isolini, D. and Sollberger, H. (2001). Influence of two different propionibacterial cultures on the characteristics of Swisstype cheese with regard to aspartate metabolism. Milk Sci. Int. 56, 78-81. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11,185-201.
Plate 16 Traditional Swiss Emmental cheese. (See page 142.)
Plate 17
Cutting of the curd. (See page 149.)
Plate 18
Filling of the curd into the cheese moulds. (See page 149.)
Surface Mould-ripened Cheeses H.-E. Spinnler, Laboratoire de Genie et Microbiologie des Procedes Alimentaires, INA-PG, ThivervaI-Grignon, France J.-C. Gripon, Unite de Biochimie et Structure des Proteines, INRA, Jouy-en-Josas, France
Surface mould-ripened soft cheeses are characterised by the presence of a felt-like coating of white mycelia due to the growth of Penicillium camemberti on the surface. The presence of this mould gives these cheeses a characteristic appearance, as well as a typical aroma and taste. It also leads to more complex ripening patterns than in other varieties of cheese with more simple microflora. These cheeses are becoming increasingly popular with consumers, and the demand for them increases. In the present chapter, after reviewing briefly the different methods of production and technologies of these cheeses, the microflora, the various biochemical changes that occur during their ripening, their aroma and textural properties and the control of their ripening will be considered.
A typical example of surface mould-ripened cheeses is Camembert, which is a cheese with a soft consistency and a flat cylindrical form, approximately 11 cm in diameter and 2.5 cm thick. Camembert originated in the Normandy region of France. It is believed by some to date from about 1790 and has been attributed to a farmer's wife, called Marie Harel, from the small village of Camembert. Though first made on farms, Camembert has been made by industrial companies since the beginning of the twentieth century. The manufacture of surface mould-ripened cheeses became progressively widespread in France, followed by other European countries. Camembert manufactured in Normandy and meeting certain manufacturing norms benefits from a protected designation of origin (PDO; Camembert de Normandie). Conversely, the name 'Camembert' is not protected and is used for cheeses manufactured elsewhere in France or in other countries. The other principal cheeses with a surface mould are Brie, Coulommier and CaGe de l'Est (a mild fermented cheese). Products also exist which are marketed with a trade name. Brie from Meaux or from Melun are characterised by their large diameter (35 and 27 cm, respectively) and also
possess a PDO. France produced aproximately 300 000 tonnes of Camembert, Brie, Coulommier and Carre de l'Est in 2000 (CNIEL, 2002). Total production of surface mould-ripened cheeses is a little greater, as products marketed under trade names should be included. Germany produces 18 000 and 2000 tonnes of Camembert and Brie, respectively (again, these figures are under-estimates as they do not include products manufactured under other names). Denmark also produces significant quantities of surface mould-ripened cheeses. Many other countries around the world, including USA, Australia, Argentina, New Zealand and several European countries produce cheeses with a surface mould, but in limited amounts. In the absence of precise figures, it can reasonably be estimated that in 1999, surface mould-ripened cheeses represented 7-8% of the total production of cheese in the 15 countries of the European Union and 2-3% of the world production. (Reprinted from Encyclopedia of Dairy Sciences, Gripon, J.-C., pp. 401-403, 2 paragraphs only, with permission from Elsevier.)
Traditional Camembert is made from raw milk with the addition of a mesophilic starter. The pH at renneting is approximately 6.4 and the coagulation time is 30-45 min. Transfer of the coagulum to the moulds is by means of a ladle (5 ladles/mould), either manually or using an automated system. Draining takes place spontaneously through the perforated sides of the moulds during the first hours at 26-28 ~ then at a progressive reduction in temperature approaching approximately 20 ~ by the end of draining. A curd with a low mineral content is thus obtained, with a pH of 4.6-4.7 at the end of draining. The cheese is drysalted and maturated for a minimum period of 21 days in cellars at 11-13 ~ and 90% relative humidity. Camembert, without designation of origin, is manufactured from raw or pasteurised milk. Coagulation generally takes place continuously in an Alpma-type production system. The coagulum is cut into cubes of 2-2.5 cm/side and the curds are moulded (manually or automated, in multi-moulds) 30-50 min after cutting.
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
158
Surface Mould-ripened Cheeses
Curds are salted in brine. Distribution requirements, as well as market demands, have led to modifications in existing technology and have created new types of surface mould-ripened cheeses. The latter, called solubilised or stabilised cheeses, are often sold under trade names, Pasteurised milk is renneted after a very short maturation period. The coagulum is cut into cubes, 0.7-1 cm i n size, and the curds are stirred and washed. Part of the whey is drawn off before moulding. The starter used consists of thermophilic streptococci or a mixture of streptococci and lactococci. The curd obtained is much less acidic than that of Camembert. These cheeses rapidly acquire a soft texture, giving them a mature appearance; their taste is milder than that of traditional Camembert, and their storage properties are improved. (Reprinted from Encyclopedia of Dairy Sciences, Gripon, J.-C., pp. 401-403, 2 paragraphs only, with permission from Elsevier.)
Packaged 4 ~
14 ~ RH = 85%
~ o D ou_ ~ v_o to ~_ ~o e8 N o
10 2 ~ RH = 95t/, 9
8
[]
9 _T ~ { ~ i ~ ~
7 6 '
5 4 3 2
-8 1 .-~ > 0
D~
~7~
i
I
I
5
10
I
0
I I i l , l l l l l
I
I
I
,
l
l
l
l
l
l
15 20 25 30 Ripening time (days)
l
l
l
l
l
l
35
l
l
l
l
l
l
40
45
Changes in the number of D. hansen# (0), G. can(A) and B. linens (r-l) in Camembert ripening (Leclercq-Perlat et al., 2003). didum
The composition and the evolution of the flora of surface mould-ripened cheeses are complex, particularly when raw milk is used. Traditional Camembert is a good example of this complexity. In changing different parameters (salt, water activity, pH), cheese technologists hinder most of the microbial growth but the fungal and the bacterial flora of mould-ripened cheeses remain v e t ) / U I V C F b C I l l F~IW [ I l l l K C[ICCbC, LIIC pnyblco-clicnncal treatments select the 'technological' flora but in pasteurised milk cheese, most of the flora are nowadays added to the milk as starters. These micro-organisms (yeast, Geotrichum candidum, coryneform bacteria) generate different compounds responsible for different functions change in texture, taste, flavour, colour, antimicrobial activity and organoleptic qualities are tailormade by exploiting the properties of these different micro-organisms. The succession of micro-organisms is determined by the changes in the chemical environment. First, the lactic acid bacteria (mainly Lactococcus lactis subsp, lactis and Lc. lactis subsp, cremoris), by reducing the pH, will select acidophilic micro-organisms such as yeasts and filamentous fungi. On growing on the surface, stimulated by the oxygen, they will permit the development of ripening bacteria (mainly coryneform bacteria) which are adapted to the curd composition and ripening environment (Fig. 1). One can consider that the pH 5.8 is a barrier pH (Fig. 2) below which ripening bacteria cannot grow. _1- . . . . . . .
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Very soon after manufacture, yeasts grow on the surface (Leclercq-Perlat et al., 1999) forming a dense layer
about 200 ixm thick; Kluyveromyces lactis, Saccharomyces cerevisiae and Debaryomyces hansenii are the most common yeast species (Baroiller and Schmidt, 1990). The mould, Geotrichum candidum, appears at the same time as the yeasts but its growth is limited by salting. These fungi and Penicillium camemberti, by consuming lactate for their growth (Fig. 3), raise the pH (Fig. 2) and permit the growth of bacteria adapted to the water activity of the cheese such as staphylococci or coryneform bacteria. Debaryomyces hansenii and Kluyveromyces marxianus are usually added because their substrate consumption profiles are quite different. Indeed, Kluyverornyces consumes first lactose and only then lactate, but D. hansenii is able to consume both at the same time. In the past, Geotrichum candidum caused concern to cheese technologists because of its proteolytic activity and by causing a 'toad skin'-like surface on the cheese. But by its enzymatic properties, it plays a major role in taste and flavour formation. Now, the selection of strains which do not cause 'toad skin', and better control of their use, has led to the widespread use of this species. Nowadays, in order to improve the organoleptic quality of Camembert made from pasteurised milk, selected strains of Geotrichurn candidum, yeast and coryneform bacteria are generally added to the cheese milk, giving a product closer to traditional Camembert, and closer to the expectations of most consumers. Geotrichum is very sensitive to salt and therefore drysalting may stop its growth for a while. These yeasts, in starting to hydrolyse proteins and fat, will prepare the curd and help the growth of Penicilliurn. After 6 or 7 days of ripening, the growth
Surface Mould-ripened Cheeses
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Changes in pH at the surface (A) and in the core (A) in Camembert ripening (Leclercq-Perlat et al., 2003).
of E carnemberti is observed and a white felt covers the entire surface of the cheese. The growth of P. carnemberti is extremely fast compared to that of the other members of the ripening flora. In 2 or 3 days, its growth is completed and changes surface pH, exhausts lactate at the surface and produces a large amount of CO2 which may change the gaseous environment of the ripening cellar. It is clear that P. camemberti plays a major role and imparts its characteristics to the cheese. However, the secondary flora plays an essential complementary role in the development of the organoleptic quality of traditional products.
Initially, two species of Penicillum were distinguished, P. caseicolum and E camemberti. E caseicolum is now considered to be a white mutant of P. camemberti. Different forms of E camemberti can be distinguished (Moreau, 1979): 9 a form with a fluffy mycelium, white at first becoming grey-green; 9 a form with 'short hair', rapid growth, white, closenapped mycelium; 9 a form with 'long hair', rapid growth, white, loose, tall mycelium;
Changes in lactate (OO) and in residual lactose (AA) at the surface (closed symbols) and at the core (open symbols) during the ripening of Camembert cheese (Leclercq-Perlat et al., 2003).
160
Surface Mould-ripened Cheeses
'Neuchatel f o r m ' - vigorous, rapid growth, giving a thick white-yellow mycelium. Only the white forms are used for cheesemaking. Commercial strains differ mainly in the rapidity of their growth on cheese and the density of their mycelium. Penicilliurn spores are produced by specialised companies after culturing in a fermentor or in 'Roux flasks'. They can be added to the cheese milk, added to the surface in the form of a powder after curdmaking or mixed with the salt (when dry-salting is used). Bacteria
After 15-20 days, when the Penicillium has catabolised the lactic acid and deacidified the cheese, an aerophilic acid-sensitive bacterial flora becomes established on the surface. When the pH increases above 5.8, many bacteria grow again at the surface. Until recent work on taxonomy, most of the species involved in the ripening of white-mould cheese were described as belonging to Micrococcaceae and coryneform bacteria. Recent phylogenetic analysis of DNA changed the taxonomy of these groups (Irlinger etal., 1997; Stackebrandt et al., 1997; Irlinger and Bergere, 1999). Most of them belong to the huge coryneform bacteria group and others to Staphylococcus group and coliforms (e.g., Hafnia alvei). The most commonly found of these bacteria is Brevibacterium linens but a large diversity of coryneform bacteria such as Arthrobacter, Micrococcus, Corynebacterium and Brachybacterium, is also present on these cheeses. They play a major role in flavour generation and on the appearance of the cheese. Interactions between micro-organisms
Microbiologists are not at ease with complex ecosystems like that of cheese, but it is clear that the ripening flora should be considered as a whole. Many strains of the bacteria mentioned above are able to produce a specific flavour when alone in a medium (even a cheese curd medium), but when associated to the other microorganisms, the results are completely different and most of the time the interesting flavour detected in pure culture is, for several reasons, lost when the organism grows in the cheese ecosystem. The first reason is that bacteria may not grow because of competition or inhibition but it is also possible that the metabolic pathways are not expressed because of chemical changes to the environment. Inside the cheese, lactococci are clearly predominant; the yeast population remains much lower than on the surface (about 106 cells g-1 instead of 108 cells g-Z; Leclercq-Perlat et al., 2003). In the production of cheeses from pasteurised milk, the microflora
is less diverse, containing mostly micro-organisms added as starters, e.g., Lactococcus and P. camemberti. The populations of other micro-organisms are reduced and the cheese obtained has a more neutral aroma.
The lactic starters used to make Camembert are homofermentative mesophilic lactococci, and lactose breakdown leads essentially to the production of lactic acid by the hexose diphosphate pathway. For traditional Camembert, rennet is added to the milk after ripening when the pH is about 6.4. Intense acidification occurs mainly during draining, and the pH of the curd when taken from the mould is about 4.6. After the end of curdmaking, the surface fungal flora (i.e., yeast, Geotrichum and Penicillium in particular) use lactic acid for their growth. There is, as a result, a marked increase in the external pH and an internal migration of lactate towards the surface of the cheese. The surface pH increases steadily to about 7.0 at the end of maturation; the increase is slower in the interior, where the final pH is about 6.0 (Fig. 2). This neutralisation in cheese plays at least four different roles in the ripening process: As previously mentioned, acid-sensitive bacteria, including micrococci and coryneform bacteria ,..,1.,1i...h ,.,A on tl.,. . . . . r. . . . r moul d 1.. .
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ripened soft cheese and contribute to their traditional flavour qualities. Neutralisation also favours the activity of ripening enzymes, the pH optimum of which is often close to neutrality. Neutralisation also causes migration of minerals in the curd. Le Graet et al. (1983) showed considerable migration of calcium and phosphate towards the exterior of Camembert during mould growth on the surface. The rind of surface mould-ripened cheese attains high concentrations of calcium and inorganic phosphorus (17 and 9 g kg -1, respectively) while the concentrations of these decrease at the centre. Le Graet et al. (1983) observed that the high pH of the surface causes the formation of insoluble calcium phosphate, immobilising this salt at the rind. Electron microscopic studies of the rind showed the presence of crystals which were identified tentatively as calcium phosphate. This is of nutritional interest as far as the mineral supply in surface mould-ripened cheese is concerned, depending on whether the rind is eaten or not, since at the end of ripening, the rind contains about 80% of the calcium and 55% of the phosphorus of the cheese.
Surface Mould-ripened Cheeses
4. As discussed below, the increase in pH markedly modifies the rheological properties and gives rise to a softer curd.
Although less important than in Blue cheeses, proteolysis in surface mould-ripened cheeses is quite significant. In the outer part of a ripe raw-milk Camembert, pH 4.6-soluble nitrogen represents about 35% of total nitrogen; within the cheese, there is less breakdown and only 25% of the nitrogen is soluble at pH 4.6. The soluble nitrogen fraction contains mainly small peptides (nitrogen soluble in 12% trichloracetic acid is about 20% of total nitrogen). In ripe traditional Camembert cheese, ammonia, resulting from the deamination of amino acids, is also present. Electrophoretic studies reveal strong degradation of Otsl-casein in the whole cheese while [3-casein is highly degraded in the outer part but clearly less in the centre. This high level of proteolysis is due to the presence of three agents: rennet, plasmin and microbial proteinases, among which enzymes synthesised by P. camemberti are dominant. Camembert retains more rennet in the curd than other cheese varieties because acidification occurs during draining. It has been observed that about 50% of the rennet added remains in the curd while about 15% is retained in pressed cheese (Vassal and Gripon, 1984). A degradation product of Otsl-casein by rennet, %1-1 casein (%l-CN f24-199), is detected by electrophoresis in Camembert after 6 h of draining and the concentration of this peptide increases during ripening. However, the pH of the outer part of Camembert increases quickly, reaching 6 or more after 2 weeks and can reach 7.0 after 3-4 weeks (Fig. 2). Under these conditions, one may suppose that the action of rennet (the pH optimum of which on caseins is about 5.5) decreases at the end of ripening when the pH has increased. An increase in the level of the y-caseins, resulting from the degradation of [3-casein by plasmin, is observed at the end of ripening. This increased activity is not surprising since the pH of the outer region of Camembert at the end of ripening is not far from the optimum for plasmin (about 8.5). At the end of ripening, in the outer part of Camembert, this enzyme is probably more active than in semi-hard cheeses where the pH remains at about 5.2. Studies on aseptic curds (Desmazeaud et al., 1976), in which P. camemberti developed alone with no other micro-organism, have shown an extensive production of high and low molecular weight peptides, as well as of free amino acids. Thus, this mould has a high proteolytic potential due to the production of extracel-
161
lular endo- and exo-peptidases. It synthesises appreciable quantities of a metalloproteinase and an aspartate proteinase (Lenoir, 1984), which are optimally active at pH 5.5-6.0 and 4.0, respectively. Strains of P. camemberti have very similar enzyme profiles, with a variability of about 2-fold. The evolution of the proteolytic activity in curd has been studied in Camembert during ripening (Lenoir, 1970). At the centre of the cheese, this activity is very low. However, in the outer region it increases abruptly after 6-7 days of ripening, i.e., when the Penicillium begins to grow. Aspartyl proteinase and metalloproteinase are both synthesised in cheese and their concentrations are maximal after about 15 days and then decrease slowly. These two enzymes are thus fairly stable in cheese. Lenoir (1970) noted that the difference in the degree of proteolysis between the centre and the surface of Camembert was proportionally lower than the difference in proteolytic activity and suggested that the peptides migrate towards the centre of the cheese. Scanning electron microscopic studies of Camembert cheese show some lysis of the mycelium. However, electrophoregrams of cheese do not show the appearance of new hydrolytic products, indicating that intracellular proteinases play a much more limited role than the extracellular proteinases. P. camemberti produces large amounts of amino acids in cheese (Desmazeaud etal., 1976) due to the synthesis of extracellular exopeptidases. Ahiko et al. (1981) described an acid carboxypeptidase produced by P. camemberti, which is a serine enzyme with an optimum pH of 3.5, able to reduce the bitterness of a casein hydrolysate by releasing hydrophobic amino acids. An alkaline aminopeptidase, with a pH optimum of 8.5, has also been characterised (Matsuoka et al., 1991). Geotrichum candidum synthesises intra- and extracellular proteinases (pH optima near 6.0; Gueguen and Lenoir, 1976), but enzyme-production varies significantly from one strain to another (Gueguen and Lenoir, 1975). It is considered that its proteolytic action in cheese is clearly lower than that of P. camemberti since the proteolytic activity of the outer region of Camembert does not increase during the growth of Geotrichum but only during that of Penicillium (Lenoir, 1984). Also, Geotrichum alone seeded on the surface of the curds causes less proteolysis than P. camemberti alone (Vassal, personal communication). The proteolytic role of yeast is considered to be low. Schmidt (1982) observed an intracellular caseinolytic activity with an optimum pH of about 6.0 in 165 strains isolated from Camembert cheese. B. linens secretes extracellular proteolytic enzymes; several proteinases have been demonstrated (Foissy, 1974; Hayashi etal., 1990) and Rattray etal. (1995,
162
Surface Mould-ripened Cheeses
1996) purified and characterised an extracellular proteinase with pH and temperature optima of 8.5 and 50 ~ respectively. Extra- and intra-cellular aminopeptidases have been isolated and characterised (Foissy, 1978; Hayashi and Law, 1989; Rattray and Fox, 1997). These enzymes could participate in proteolysis of Camemberttype cheeses during late ripening but probably to a low extent. Rattray and Fox (1999) reviewed in detail the properties of the proteolytic system of B. linens. The presence and the action of lactic acid bacteria should not be forgotten. The cell-wall proteinase and the various peptidases contribute, as in other type of cheeses, to the hydrolysis of peptides produced by rennet, plasmin and microbial proteinases. The increase of pH in the outer part of Camembert could favour the action of the various peptidases since their optimum pH is generally near neutrality. Most of these enzymes have been isolated and characterised in the case of Lc. lactis, and their genes have been sequenced (see Christensen etal., 1999; Bolotin et al., 2001).
The intense degradation of fat is a common characteristic of mould-ripened cheeses. Moulds and yeasts are able to secrete a large diversity of lipases. These enzymes are active at the interface between fat globules and the continuous serum phase. Lipases (EC 3.1.1.3) L__..1__I
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cerides and free fatty acids. They are not very specific but can hydrolyse triglycerides more or less rapidly according to their molecular weight, with preferential liberation for fatty acids at positions Snl and Sn3. However, stricter specificities depending on the nature of the fatty acid can be observed. G. candidum synthesises two lipases, one of which preferentially liberates oleic acid and the other unsaturated C18 fatty acids at the Sn2 position of triglycerides (Veeraragavan et al., 1990; Bertolini et al., 1995). P. camemberti produces large quantities of an extracellular alkaline lipase (pH o p t i m u m - 9.0). At pH 6.0, this enzyme retains 50% of its maximal activity and remains very active at the temperature of ripening (Lamberet and Lenoir, 1976). It is the main lipolytic agent in Camembert cheese. It is more active on triglycerides composed of low molecular weight fatty acids. Other acids, such as C1-C4 or branched-chain C4 and C5 acids result from the action of micro-organisms on amino acids. The degradation of lactose by the same micro-organisms leads to acetic acid and propionic acid. For Camembert cheese, Kuzdzal-Savoie and Kuzdzal (1966) estimated that 5% of total free acids are not produced by lipolysis. From the organoleptic point of view, fat in cheese plays three main functions:
1. it is a component involved in the determination of the texture; 2. it is a solvent of the flavours molecules" 3. it constitutes a major precursor for the development of flavours. The role of fat in texture is as a lubricant, which gives the cheese a soft sensation in the mouth. The monoglycerides are very efficient emulsifiers and may reduce the size of fat globules in the cheese which may help to get a smoother mouth-feel and may also change flavour release (Wendin et al., 1999; Miettinen et al., 2002). Lipolysis is not homogeneous throughout the cheese and occurs mainly under the rind. Hassouna and Guizani (1995) reported that lipolysis is twice as intense just under the rind than in the interior of the cheese. The association of this phenomenon with proteolysis, and particularly the relatively high pH, gives the characteristic texture of the soft part of the cheese under a Camembert rind after a long ripening. As most of the flavour compounds are hydrophobic, most are more soluble in fat than in the serum. It is common to get ten times more flavour in the fat than in the water phase for flavour compounds with more than six carbon atoms (Overbosch et al., 1991). Free fatty acids are linear and have an even number of carbons. These compounds have quite high olfactive thresholds from a few mg kg -1 to several hundreds mg kg-1. But usually in this type of cheese the level of lipolysis is high. Blue cheeses undergo more intense lipolysis, reaching up to 50 meq/100 g of fat in a Danish Blue cheese while it is much less in surface mouldripened cheeses. In Camembert, lipolysis is usually less than 25 meq/100 g of fat and as low as 11 meq/100 g of fat in Brie (Vanbelle et al., 1978).
Flavours provided by fat catabolism
Fatty acids Long-chain free fatty acids (more than 12 carbon atoms) play a minor role in flavour, given their high perception thresholds. Short- and intermediate-chain even-numbered fatty acids (4-12 carbons) have much lower perception thresholds and each has a characteristic note (Molimard and Spinnler, 1996). Butyric acid has a rancid, cheesy odour. Octanoic, 4-methyloctanoic and, especially, 4-ethyloctanoic acids have odorous notes like that of goats. In goat cheeses, branched-chain fatty acids have much lower thresholds than the linear fatty acids. 4-Ethyl octanoic acid, has an olfactive threshold about 500 times lower than that of decanoic acid, which is linear with the same number of carbons. These fatty acids play a major role in the 'goaty' characteristics of these
Surface Mould-ripened Cheeses
cheeses. The young unlipolysed cheeses are much less goaty than the more ripened one. These branchedchain fatty acids are also present in ewes' milk cheeses (Ha and Lindsay, 1991) but not in cows' milk cheeses. According to their concentration and perception thresholds, volatile fatty acids can contribute to the aroma of the cheese or even, for some, give a rancidity defect. It is, in fact, the undissociated form of these acids which is aromatic. This form is found in the fat phase of the cheese, while the aqueous phase contains both forms, undissociated and ionised. Low pH reduces ionisation and increases volatility of the acids.
Compounds produced by partial/3-oxidation A homologous series of methyl ketones with an odd number of carbon atoms, from C3 to C15, is one of the most important aroma compounds in the aroma of Blue and surface mould-ripened cheeses (Gallois and Langlois, 1990). The longer chain ketones, probably, are much less important than the intermediate ones because of their lipophilicity, which probably limit their volatility in a fatty matrix, like cheese. Several studies have established the pathway for the formation of these products in cheese (Dartey and Kinsella, 1973; Okumura and Kinsella, 1985). In Camembert, methyl ketones are by far the most abundant volatile flavour compounds, in the order of 25-60mmol/100 g of fat, the two major compounds being nonan-2-one and heptan-2-one. All methyl ketones found in Camembert are also present in Blue cheese (Gallois and Langlois, 1990). Concentrations of heptan2-one and nonan-2-one in white-mould cheeses are very high and out of proportion considering the quantity of octanoic and decanoic acids present in milk fat, where the main fatty acid is palmitic acid (C16:0). We can therefore suppose that the corresponding fatty acids are not the only precursors of methyl ketones. Thus, the study of the oxidation of 14C-labelled palmitic and lauric acids by P. roqueforti spores has permitted elucidation of the formation of methyl ketones from long-chain fatty acids by successive cycles of [3-oxidation. Furthermore, addition of oleic acid (C18) in a milk-based medium causes an increase in the production of heptan-2-one and nonan-2one by P. camemberti, but the addition of lauric acid (C12) does not increase the production of undecan-2-one. Dumont etal. (1974a,b) isolated the aroma compounds of 11 samples of Norman Camembert by vacuum distillation and found 11 methyl ketones, all alkan-2-ones from C4 to C13, as well as octan-3-one (trace). The authors also identified 3-methylpentan-2one, 4-methylpentan-2-one, methylhexan-2-one (trace), nonan-2-one and undecan-2-one in larger amounts. The amounts of nonan-2-one, heptan-2-one and undecan-2one increased steadily during ripening.
163
In Camembert and Brie, most of the methyl ketones are present from the eighth day of ripening onwards but Moinas et al. (1973) identified butan-2-one and pentan2-one in young Camembert only (1-5% of the methyl ketones). These methyl ketones seemed to disappear during ripening. On the other hand, they observed an increase in the concentration of nonan-2-one during cheese ripening (1-5% of the methyl ketones in young Camembert compared with 20-40% of the methyl ketones in ripe Camembert) while the amount of heptan2-one remained constant (1-5% of the methyl ketones). From their odour notes typical of Camembert cheeses and from the amounts present in these cheeses, we are able to understand the important role played by ketones and methyl ketones in the aroma of these products. These volatile compounds are not only found in Camembert-type cheese, they are abundant in blue-veined cheeses in which heptan-2-one is the methyl ketone present in the largest quantity. Nonan-2-one, decan-2-one, undecan-2-one and tridecan-2-one are described as having fruity, floral and musty notes while heptan-2-one has a Blue Cheese note (Rothe et al., 1982). Oct-l-en-3-one has a mushroom note in aqueous media and a metallic note in lipid-rich media (Teranishi et al., 1981). Octa-l,5-dien-3-one is described as having a soil-like odour, octan-3-one a mushroom note and damascenone a woody note (Karahadian et al., 1985a). The principal agents in the formation of methyl ketones in mould-ripened cheeses are moulds, and the precursors are fatty acids. Methyl ketones are formed in a metabolic pathway which is connected to the [3-oxidation pathway. P. camemberti, P. roqueforti and Geotrichum candidum possess an enzymatic system which permits a diversion from the normal [3-oxidation pathway. The free fatty acid is oxidised to [3-ketoacylCoA. The action of a thiolase yields a [3-ketoacid which is rapidly decarboxylated by a [3-keto-acyl-decarboxylase to give a methyl ketone with one less carbon than the initial fatty acid; this metabolism has been extensively studied in yeasts (Fig. 4). For the micro-organism, this metabolic pathway represents a method for detoxifying fatty acids in the media. It needs only one molecule of coenzyme A, while complete degradation needs two. This mechanism allows faster recycling of the co-factor (Kinsella and Hwang, 1976). At low concentrations, fatty acids are oxidised completely to CO2 and H20, and very low amounts of methyl ketones are formed (Margalith, 1981). [3-Oxidation is a particularly important metabolic pathway since 60% of the carbonyl compounds produced by P. camemberti on a milk-based medium are methyl ketones (Okumura and Kinsella, 1985). The mycelium of P. camemberti
164 Surface Mould-ripened Cheeses
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Intra-cham oxidation of unsaturated fatty acids Linoleic and linolenic acid are precursors of 8C aroma compounds, particularly oct-l-en-3-ol, oct-2-en-l-ol, octa-l,5-dien-3-ol and octa- 1,5-dien- 1-ol and ketones such as octan-2-one, oct-l-en-3-one and octa-l,5dien-3-one. Oct-l-en-3-ol is well known for its raw mushroom odour. Considering its low perception threshold (0.01 mg kg-]), it gives Camembert cheese aroma a characteristic note. This compound is, without a doubt, one of the key compounds in the overall aroma UI
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work on the production of methyl ketones by moulds, especially E roqueforti (Creuly et al., 1992). The production of heptan-2-one continues to attract attention because it is preponderant in Blue-type cheeses. New flavourings with high aromatic power appeared on the market to flavour sauces, crackers, etc. Secondary alcohols found in mould-ripened cheeses are mainly heptan-2-ol and nonan-2-ol, which represent, together with the methyl ketones from which they are derived, 10-20% and 5-10%, respectively, of all aroma compounds found in Camembert (Moinas et al., 1973). Dumont et al. (1974b) also isolated significant quantities of pentan-2-ol from ripe Camembert. However, Moinas et al. (1973) did not report this alcohol in mature Camembert and have identified this alcohol in young samples only.
Decarboxylase j
O Fatty acid catabolism to methyl ketones by yeasts (from Ratledge, 1984). is more sensitive to inhibition by fatty acids than that of E roqueforti in spite of the fact that it uses fatty acids more rapidly. Mycelium and spores, but not germinating spores of Penicillium, are able to metabolise fatty acids to methyl ketones. The latter seem to be more sensitive to the inhibitory effect of fatty acids (Fan et al., 1976). G. candidum produces methyl ketones, including pentan-2-one, heptan-2one, nonan-2-one and undecan-2-one. It also produces pentan-3-one, which was found for the first time by Jollivet et al. (1994) in cultures of eight strains of G. candidum. The increasing demands for Blue cheese aroma compounds from the food industry gave rise to much
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(1975) on a neutral cheese base have shown that it is possible to mask partially the blue note of methyl ketones by oct-l-ene-3-ol. At concentrations of 5-10 mg kg -] in the cheese base, these authors obtained a flavour close to mature Camembert. If it is present in too large amounts, it is responsible for an aroma defect. Oct-l-en-3-ol represents 5-10% of the volatile compounds in Camembert. On the other hand, it is present in only very small amounts in young Camembert. Furthermore, its production is bound to P. camemberti metabolism, appears only late in cheese ripening and is a result of the secondary metabolism (Spinnler et al., 1992). Even-chain methyl ketones, except for butan-2-one, are probably produced by intra-chain oxidation. They are never present in large amounts, except in very ripe cheese. Camembert made from raw milk contains more even-chain methyl ketones and branched-chain methyl ketones. It was suspected by Karahadian et al. (1985a,b) that P. camemberti is capable of intra-chain oxidation of linoleic and linolenic acids, as are basidiomycetes (Chen and Wu, 1984). The principal enzymes believed to be involved are a lipoxygenase and a hydroperoxide-lyase, which are commonly present in moulds. Recently, Perraud and Kermasha (2000)
Surface Mould-ripened Cheeses
demonstrated that P. camemberti has lipoxygenase activity able to produce 9-, 10- and 13-hydroperoxy acids from polyunsaturated fatty acids. Perraud et al. (1999) also demonstrated lipoxygenase activity in G. candidum. Some aldehydes found in Camembert cheese, such as hexanal, heptanal and nonanal, are due to fat oxidation. Hexanal and (E)-hex-2-enal are known to give the green note of immature fruit. Their perception threshold in water is 9 and 24 txg kg-1, respectively (Ahmed et al., 1978). Octanal, nonanal, decanal and dodecanal are described by these authors as having an aromatic note, resembling orange. Their perception threshold in water is 1.4, 2.5, 2 and 0.5 Ixg kg-1, respectively.
Lactones Lactones found in Camembert are y-decalactone, 8-decalactone, y-dodecalactone and 8-dodecalactone. These compounds have also been identified in Blue cheeses (Gallois and Langlois, 1990). From an organoleptic point of view, lactones are generally characterised by very pronounced fruity notes (peach, apricot, coconut). 8-Lactones have a generally higher detection threshold than those of y-lactones. These thresholds are relatively low for y-octalactone, 7-decalactone and y-dodecalactone (7-11 Ixg kg -1 in water) and are lower for shorterchain lactones (Dufosse et al., 1994). Lactone precursors are hydroxylated fatty acids. The intra-molecular esterification happens under the action of pH and/or micro-organisms. The action of micro-organisms in the production of lactones has never been clearly demonstrated in cheese. Hydroxyacids, which are direct precursors of lactones, are present in triglycerides in milk. Lipases can liberate them and they are then cyclised to form lactones. Nevertheless, hydroxylated fatty acids can come from the normal catabolism of fatty acids and can be generated from unsaturated fatty acids by the action of lipoxygenases or hydratases. P. roqueforti spores can form 12-carbon lactones from long-chain saturated fatty acids (C18:1, C18:2). Chalier and Crouzet (1992) performed the bioconversion with spores of P. roqueforti using soya and copra oils as substrates. Flavour compounds produced from amino acid catabolism
The most common pathway, used by micro-organisms for amino acid breakdown, is Erhlich's pathway which leads to the production of branched-chain aldehydes, branched-chain alcohols or branched-chain acids from the branched-chain amino acids. Primary and secondary alcohols, along with ketones, are considered to be very important compounds in the
165
aroma of soft, mould-ripened cheeses. Regarding primary alcohols, 3-methylbutan-l-ol is present in relatively large quantities in Camembert and has an alcoholic, floral note. Phenyl-2-ethanol, with a perception threshold in a cheese base of 9 mg kg -1 and a characteristic rose floral note (Roger et al., 1988) and its ester, phenylethylacetate, play an important role in raw-milk Camembert where they are always present in important amounts (Dumont etal., 1974b). This alcohol is one of the major compounds in Camembert after 7 days of ripening, at a concentration of 1.15 mg kg -1. Its concentration stabilises at approximately 1 mg kg-1 at the end of ripening. It is lower than the detection threshold (9 mg kg -1) but is close to the detection threshold of the most sensitive panelist of the panel used in the study of Roger et al. (1988). In fact, these authors thought that phenylethanol and its esters have cumulative effects to give the perceptible floral note in certain Camembert cheeses. This alcohol is produced mainly during the first week of ripening, because it is, mainly, a metabolic product of yeasts (Lee and Richard, 1984). Ethanol, propan-2-ol, butan-2-ol, octan-2-ol and nonan-2-ol are also encountered in most soft cheeses. Eleven alcohols have been identified and quantified in two types of Brie. Ethanol and short-chain linear alcohols only have a limited aromatic role in cheese but are the precursors of several esters. By way of oxidative deamination or transamination, amino acids can be transformed to ot-ketoacids which can then be decarboxylated to aldehydes. The aldehydes can then be reduced to the corresponding primary alcohols or oxidised to acids. It has been shown in many models that the deamination/transamination step is very often a rate-limiting step in amino acid catabolism. Products arising from the reduction of aldehydes include 2-methylpropanol, 3-methylbutanol, 2-methylbutanol, 3-methylpropanol and phenylethanol. Production of phenylethanol from phenylalanine seems to be mainly performed by yeasts (Lee and Richard, 1984). In the same way, P. camernberti catabolises valine to 2-methylpropanol and leucine to 3-methylbutanol. The eight strains of G. candidum studied by Jollivet et al. (1994) produced isobutanol.
Aldehydes The main aldehydes found in Camembert are 2methylbutanal, 3-methylbutanal and benzaldehyde. These compounds, mostly at trace levels, are present as early as the first week of ripening in surface mouldripened cheeses like Brie and Camembert. Benzaldehyde is described as having an aromatic note reminicent bitter almond. Its detection threshold in water is 350 txg kg -1 (Buttery et al., 1988). With
166
Surface Mould-ripened Cheeses
detection thresholds in malt culture media of 0.1, 0.13 and 0.06 mg kg-1, respectively, 2-methylpropanal, 2-methylbutanal and 3-methylbutanal are also encountered in cheeses, including mould-ripened cheeses (Margalith, 1981). These compounds, can be oxidised to isobutyric, 2-methyl butyric and isovaleric acids. These acids are described as having a mild odour, reminicent of sweat. Aldehydes originate from amino acids either by transamination, leading to an cx-ketoacid which can be decarboxylated, or by chemical degradation. This last reaction is simple and can occur without enzymatic catalysis during ripening. Aldehydes are transitory compounds in cheese since they are transformed rapidly to alcohols or corresponding acids. Yeasts can contribute to the production of ethanal when alcohol dehydrogenase is less active than pyruvate decarboxylase. The biosynthesis pathway for benzaldehyde was determined recently by Nierop-Groot and de Bont (1999). It was shown that a chemical breakdown of phenylpyruvic acid is catalysed by divalent cations such as Mn 2+. This pathway seems also to be used for other amino acids, such as methionine, producing 2-methyl thioethanal which has a green-apple flavour (Yvon et al., 2001). Amines For some micro-organisms, the breakdown of amino acids starts by decarboxylation, with the production of (f.~LLIIIIL%.-O.
s
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in Camembert cheese, including methylamine, ethylamine, n-propylamine, isopropylamine, n-butylamine, 1-methylpropylamine, n-amylamine, iso-amylamine, anteiso-amylamine, n-hexylamine, ethanolamine, dimethylamine, diethylamine, dipropylamine and dibutylamine (Adda and Dumont, 1974). Dimethylamine has been detected in Camembert and in Blue cheeses at 0.811-1.623 mg kg-1 Nitrosamine have also been described in Camembert at a level of 25 nmol/10 g but has not been identified in Blue cheeses. We should keep in mind that ammonia, derived from amino acid deamination, is also an important element of Camembert aroma. P. camemberti, G. candidum and B. linens play major roles in ammonia production by deamination of amino acids (Karahadian and Lindsay, 1987). Many volatile amines are described as having fruity, alcoholic or varnish-like aroma notes. Ethylamine and butylamine have perception thresholds in water from 0.83 to 3.63 m g k g -1 and 0.24 to 13.9 m g k g -1 of free base, respectively (Laivg et al., 1978). Methylamine, dimethylamine and propylamine have perception thresholds in water of 182, 34.4 and 62.4 mg kg -1 of free base, respectively. Tertiary amines have much lower perception thresholds. Triethylamine,
with a fishy odour, is perceived at a concentration of 0.47 b~g kg -1 of free base in water. Some people have a specific anosmia for this amine which is a widespread pheromone in mammalian species. Amine biosynthesis Decarboxylation of amino acids leads to the production of CO2 and amines. This reaction needs the presence of pyridoxal-phosphate and co-enzyme. Decarboxylation of leucine gives isobutylamine, phenylalanine gives phenylethylamine and tyrosine gives tyramine. A low oxygen pressure favours these reactions. Amines are not final products but are subjected to oxidative deamination to form aldehydes. They can also be the starting point of compounds like N-isobutylacetamide encountered in Camembert, presumably by reaction with acetic acid. Catabolism of amino acid side chains
Indole ring Degradation of the side chains of tyrosine and of tryptophan by tyrosine-phenollyase and by tryptophan-indole lyase, respectively, leads to the formation of phenol and indole. Parliment etal. (1982) considered that phenol found in Limburger results from degradation of tyrosine by B. linens. The catabolism of tryptophan by B. linens has been recently studied by Ummadi and Weimer (2001). In model media, tryptophan was broken down to anthraniiic acid at a high rate. However, the physicochemical environment of ripening cheese is quite far from optimal conditions and these authors concluded that it is unlikely that B. linens could be responsible for faecal, putrid or meaty-brothy defects in Cheddar cheese. Sulfur compounds During their work on the identification of minor components present in aromatic extracts of Camembert, Dumont etal. (1976a,b) isolated four sulfur compounds from a fraction with a garlic flavour note: 2,4-dithiapentane, diethyldisulfide, 2,4,5-trithiahexane and 3-methylthio-2,4-dithiapentane. These authors also identified traces of a sulfur-containing alcohol, 3-methyhhiopropanol (or methionol), and ethyldisulfide. Other sulfur compounds are also found in Camembert cheese. Disulfides are generally absent from young cheeses. In these cheeses, a low level of proteolysis yields only a low level of sulfur amino acids, precursors of disulfides. In late ripening, sulfur compounds are quantitatively reduced and even disappear in some products. This can be explained by their high volatility. Nevertheless, in Brie cheese, Karaha(1985a) found sulfur compounds dian etal. (dimethyldisulfide, dimethyhrisulfide and methionol) only in aged Camembert cheeses with a growth of
Surface Mould-ripened Cheeses
Brevibacterium linens and other coryneform bacteria. Sulfur compounds found in cheeses are described as having a strong garlic or 'very ripe cheese' odour. Furthermore, these compounds have a very low detection threshold in water, from 0.02 Ixg kg -1 for methanethiol to 0.3 txg kg-1 for dimethylsulfide (Shankaranarayana et al., 1974). Sulfur compounds originate principally from methionine degradation, resulting from a carbon-sulfur bond cleavage by a methionine-y-demethiolase. This amino acid is a precursor of methanethiol which is itself the starting point for some other compounds, including dimethyldisulfide and dimethyltrisulfide. Many microorganisms are able to produce methanethiol from methionine. Among the ripening fungi many have this potential, such as P. camemberti, G. candidum and Y. lipolytica (Bonnarme etal., 2001; Spinnler etal., 2001). Molimard et al. (1997) have shown that some strains of G. candidum, although its growth was quite early in the ripening process, were able to change the characteristics of a Camembert cheese irrespective of which of the four strains of P. camemberti were used (Fig. 5). One strain of G. candidum caused the development of cabbage and cowshed notes. It was then shown that G. candidum growing in a curd medium enriched with methionine was able to accumulate a large variety of sulfur compounds, including various thioesters such as methyhhioacetate, methylthiopropionate, methyhhiobutyrate, methyhhioisobutyrate, methylthioisovalerate (MTIV) and methyhhiohexanoate (MTH; Berger et al., 1999a). These thioesters have various flavour notes, from cheesy (MTIV) to fruity (MTH; Berger etal., 1999b). Recently, the metabolism of G. candidum was explored and it was shown that this species, unlike B. linens, was able to accumulate 2-keto-4-methyhhiobutanoic acid as an intermediate in catabolism
167
(Bonnarme et al., 2001). The origins of different sulfurflavour compounds are summarised in Fig. 6. Smear bacteria have also been studied but mainly B. linens or Arthrobacter spp. (Bonnarme et al., 2000). Among these micro-organisms, coryneform bacteria, especially B. linens, are considered as the key agents in the production of sulfur compounds in cheeses in which they grow. The production of sulfur compounds by pure cultures of B. linens has been studied by Tokita and Hosono (1968) and Law and Sharpe (1978). Jollivet etal. (1994) studied the production of dimethyldisulfide by six out of eight strains of G. candidum in a cheese-based model system. It was recently shown that dimethylsulfide is produced by G. candidum using a separate pathway than methanethiol production from methionine (Demarigny et al., 2000).
Styrene Styrene has a very strong plastic-like odour. Its perception threshold in cream is 5 b~g kg -1. This compound has been described as a trace component in several cheeses, including Camembert (Dumont et al., 1974c). Adda et al. (1989) found an abnormally high quantity of styrene (5 m g k g -1) in Camembert with a pronounced celluloid taste. These authors demonstrated the role played by E camemberti in the production of this hydrocarbon. Spinnler et al. (1992) observed a correlation between the production of styrene and oct-l-en-3-ol in a minimal medium. These two compounds were produced after 15 days of culture, when there is no more glucose in the growth medium. Oct-l-en-3-ol is produced 2-3 days before styrene. 13C-styrene is produced from 13C-phenylalanine (Spinnler, unpublished data) suggesting that this amino acid is the precursor of styrene. Miscellaneous compounds
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Changes in the flavour profile of Camembert cheese made with a pure culture of R camemberti (strains P1-P4) or in association with G. candidum (strains G1-G3). Results where obtained with a trained panel (Molimard et aL, 1997).
Esters There is a great diversity of esters in cheese. Esters have been identified to the corresponding acids and alcohols present in Camembert. 2-Phenylethylacetate and 2-phenylethylpropanoate are qualitatively important in the flavour of Camembert cheese. On the seventh day of ripening, 2-phenylethylacetate is the principal compound in the aromatic profile, at a concentration of 4.6 mg kg -1 This concentration then decreases and stabilises around 1 mg kg -1 (Roger et al., 1988). Methylcinnamate, identified in Camembert by Moinas et al. (1975), seems to be particularly important in the aroma of this cheese. When varying the concentration of this compound in a neutral cheese base, to which heptan-2-one, heptan-2-ol, oct-l-en-3-ol,
168
Surface Mould-ripened Cheeses
L-Methionine (CH3S-CH2-CH2-CH(NH2)-COOH)
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Pathways for the catabolism of methionine by B. linens (left), in G. candidum the pathway using a transaminase and demethiolation of the KMBA (right) has been demonstrated but the existence of a methionine-y-lyase (left) cannot be excluded at the moment. ~, v~_ ~,_u,-,,,.,,-,~,,,.,...,,.,v~A,:,^ ~,_~.e,o_y_,~,,-.,~,y~thio butyric ~"~'~ H~,~^ o_~,,,4...... a_m..~,,,~,;,,_h,,~on,-,~,,o,-~..~_u'~a .~_u..t,, butyrate; AA, amino acids; FFA, free fatty acids; DMDS, dimethyl disulphide; DMTS, dimethyl trisulphide; GDH, glutamate dehydrogenase, ~I~-|~I~4~
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nonan-2-ol, phenol and butyric acid had been added, these authors developed a characteristic Camembert note. Most of the esters found in cheeses are described as having fruity, floral notes. The most-cited aromatic notes of these compounds are pineapple, banana, apricot, pear, floral, rose, honey and wine. Some of these esters have a very low perception threshold, e.g., isoamylacetate which is detectable in water to a concentration of 2 txg kg-1 (Piendl and Geiger, 1980). Low carbon number esters have a perception threshold approximately ten times lower than the corresponding alcohol. Esterification reactions occur between alcohols derived from lactose fermentation (ethanol) or from amino acid catabolism, and short- to medium-chain carboxylic acids. For example, acetates come from transesterification of alcohols with acetyl-CoA. These reactions are well-known detoxification reactions in media, enabling the elimination of toxic alcohols and carboxylic acids. A wide variety of enzymes are involved in esterification reactions, including carboxylesterases, which have a very wide range of substrates, and arylesterases, present in most of the micro-organisms which con-
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tribute to cheese ripening. Ester formation has been studied widely in fermented beverages in which they play an important aromatic role. Their production is due to yeast activity. In all cheeses, micro-organisms involved in ester production seem to be mainly yeasts. Production of esters occurs early during ripening. G. candidum is capable of producing numerous esters, some of which have a very pronounced melon odour. However, Latrasse et al. (1987) observed ester production by only one strain of G. candidum.
Terpenes Terpene alcohols such as 2-methylisoborneol (2-MIB) are produced by P. camemberti. 2-Methylisoborneol having a musty flavour but a very low detection threshold (0.1 lxg kg -1) is the reason for its role in the soft and the mould-ripened cheeses (Karahadian et al., 1985a,b).
Pyrazines Dimethylpyrazine and trimethylpyrazine were identified in Camembert by Dumont et al. (1976a,b). 2,5Dimethylpyrazine, which has a 'toasted hazel nut note', can be produced from threonine. 2-Methoxy-3-iso-
Surface Mould-ripened Cheeses
propylpyrazine comes from the degradation of L-valine, as demonstrated in Pseudomonas taetrolens (Gallois, 1984). This pyrazine is responsible for an aroma defect in Camembert, in which it causes a rotten soil, raw potato note. Its perception threshold is very low (0.002 Ixg kg -1 in a milk medium) and therefore it is important when present, even in very low amounts. Volatile contaminants
Many chlorinated compounds, present at trace levels, have been found in Camembert including chloroform, carbon tetrachloride, dichloroethane, trichloroethane, tetrachloroethylene, dichlorobenzene and trichlorobenzene. For those compounds, an external origin is likely: pesticide, cleaning agents, pollution or artefacts due to solvent extraction during analysis. Likewise, benzene and its derivatives have been identified, e.g., ethylbenzene, dimethylbenzene, trimethylbenzene, and the one derivative of toluene. Most of the studies demonstrating the presence of traces of these compounds have been done on extracts obtained by the use of solvents, where the origin of these compounds is likely to be impurities in the solvent used. But the recent intensive use of dynamic headspace confirms the occurrence of these compounds in cheeses, the fat of which is a good trap for these volatiles (Spinnler, 2003). Among the large number of compounds present in the volatile fraction of Camembert, methyl ketones and alcohols (oct-l-en-3-ol, 2-phenylethanol, etc.), as well as 2-phenylethanol acetate, are quantitatively the most important. These products, along with sulfur compounds, play an important role in the aromatic note of this cheese. On the other hand, we have very little knowledge at this time on the aromatic importance of most of the molecules often present at trace levels in this cheese. At present, it is not possible to say which compounds determine the organoleptic quality of these cheeses. The aroma of soft and mouldripened cheeses is, in fact, the result of a subtle and fragile equilibrium between all the numerous volatile compounds they contain.
The outer part of Camembert undergoes considerable modification of texture, and the curd which is firm and brittle at the beginning of ripening, later becomes soft. Softening is visible in a cross-section of the cheese and gradually extends towards the centre. The water content of Camembert is about 55% and, if it is too high, the outer part tends to flow when the ripe cheese is cut. These changes were previously attributed to the high level of proteolysis created by P. camemberti.
169
However, the diffusion of fungal proteases is limited and can affect only the outer few millimetres. The most important change caused by P camemberti and the surface flora is the establishment of a pH gradient from the surface to the centre due to the consumption of lactic acid and the production of ammonia (Fig. 7). This pH gradient can be simulated by incubating young Camembert (3 days of ripening without inoculation with Penicillium) in an ammoniacal atmosphere. The ammonia dissolves in the curd and, after equilibration, the pH gradient established is expressed by cheese softening; this process is more evident near the surface where the pH is highest (Vassal et al., 1984). Increasing the pH, therefore, plays an important role causing the cheese to soften. This may be explained by the fact that the increase in pH augments the net charge on caseins and modifies protein-protein and protein-water interactions. It also changes the water absorption capacity and the solubility of the caseins. According to Noomen (1983), the physico-chemical conditions (water content and pH) in Camembert alone cannot explain softening, which could also be related to rennet action. Indeed, experimental cheeses, containing no rennet and incubated in an ammoniacal atmosphere, do not soften but become hard and springy, while cheese with rennet activity softens. The softening of Camembert could thus be explained by three processes: breakdown of %l-casein by rennet, increase in pH caused by the surface flora and to the outward migration of Ca 2+ in response to the pH gradient (see Fig. 7).
The choice of the P. camemberti strain is important in the production of soft surface mould-ripened cheeses. However, the proteolytic activity of the different strains does not vary as much as their lipolytic and [3-oxidative activities (Lenoir and Choisy, 1970; Lamberet et al., 1982). The choice of a P. camemberti strain is also guided by the growth rate, colour, density and height of the mycelium, which contribute to the appearance and attractiveness of surface mould cheeses. Salting has a selective effect on the mould in soft cheeses. Too much salting limits the growth of G. candidum, while the growth of P. camemberti is much less affected. In whey culture, the growth of P. camemberti is slowed down when 10-15% salt is present. Conversely, too little salt, combined with insufficient draining, causes excessive growth of G. candidum and hinders the implantation of Penicillium, giving defective cheese; this defect is called 'toad skin'. Under-salting may also favour the surface implantation of Rhizomucor, altering
170
Surface Mould-ripened Cheeses
Inner cheese mass = 8-14 mm; sub-rind = 0-6 mm and cheese rind = 1-3 mm
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the appearance of the cheese; this defect is called 'cat hair'. Reducing the water activity by higher salting and using a Penicillium strain that implants quickly helps to correct this defect (Choisy et al., 1984). Salting also influences the activity of Penicillium enzymes, and at 4% it reduces the degree of proteolysis in Camembert V~
unsalted control). The effects of humidity and temperature in the ripening room on the growth of P. camemberti and the quality of Camembert-type cheese have been described by von Weissenfluh and Puhan (1987). The production of soft surface mould-ripened cheeses using milk highly contaminated with psychotrophic bacteria leads to organoleptic defects. The lipolytic activity of these bacteria is expressed by increased lipolysis and a rancid taste; bitterness has also been reported (Dumont et al., 1977). Listeria monocytogenes is able to survive the Camembert cheesemaking process and grow during ripening of the cheese. Control of L. monocytogenes (not detectable in 25 g of cheese) is obtained by the selection of good quality milk, adequate heat treatment and avoiding contamination during cheesemaking through good hygienic practices (good equipment design and appropriate cleaning and disinfection). Bacteriocin-producing lactic acid bacteria can also be used for cheesemaking. The number of L. monocytogenes in curd can be reduced very much by using strains of Lc. lactis that produce nisin (Maisnier-Patin et al., 1992). As mentioned above, uncontrolled development of G. candidum causes defects in the appearance and the taste of cheese, even though this mould probably contributes
significantly to the taste qualities of Camembert. Some strains of G. candidum clearly improve the taste and the aroma of Camembert cheese made from pasteurised milk. Their controlled growth results in a more typical Camembert flavour, close to that of traditional Camembert (Molimard et al., 1997). As in all cheeses, acidifi~..; ..,...,,~ . . . . f L~t, L L U L L UL
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by controlling syneresis and the degree of mineralisation. When acidification is too high, the Camembert curd is too dry and brittle and enzyme activities are limited; insufficient acidification results in a cheese, the moisture content of which is too high at the end of ripening. The last 20 years have witnessed an increasing interest in 'stabilised' cheeses. Washing the curd permits a higher pH to be obtained at the end of draining. This gives a less demineralised cheese that seems more ripe than a traditional cheese of the same age. These cheeses made from pasteurised milk have a milder taste and keep better than Camembert made using the traditional technology. This could be due to more limited activity of P. camemberti, perhaps because of the lower levels of available lactose and lactate. Due to their higher pH, these products are more sensitive to coliform bacteria. An investigation in France by Pelissier etal. (1974) showed that mould-ripened cheeses are more sensitive to bitterness than other varieties and the intensity of this defect may cause considerable damage to cheese quality. More recently, Molimard et al. (1994) have shown that G. candidum is able to ameliorate this defect, possibly due to its very efficient peptidase system compared to P. camemberti, which is more proteolytic than peptidolytic. P. camem-
Surface Mould-ripened Cheeses
berti plays a crucial role in the appearance of bitterness
in Camembert. Excessive growth of the mycelium can lead to the defect; if Penicillium growth is limited by the presence of G. candidum or by incubating the cheese in an ammoniacal atmosphere, proteolysis is reduced and the defect does not occur. Therefore, this defect could occur when there is too much proteolysis by Penicillium proteases (Vassal and Gripon, 1984). The level of rennet used and its augmentation does not seem to cause bitterness, perhaps because the pH of Camembert does not favour the action of rennet proteinases at the end of ripening. Lactic acid bacteria and their proteinases have also been reported to affect the occurrence of bitterness. The defect appears when a high population of lactic acid bacteria is present in the curd; on the other hand, if these populations are reduced (for example, by infection with bacteriophage), bitterness does not occur (Martley, 1975). This seems to be related to the degree of curd acidification, since the probability of bitterness is increased if the pH is low at the end of draining (Vassal, personal communication). Bitterness might not result directly from high amounts of lactic acid bacteria but could be related to Penicillium, the growth of which, and protease production, might be higher in very acid curds. In Camembert cheese, another very important point is mass transfer in the curd, from the core of the cheese to the surface or in reverse from the surface to the core. Due to different parameters such as relative humidity, lactate concentration gradient, moisture gradient, pH gradient and microbial activity, lactate migrates from the core to the surface leading to deacidification from the surface towards the core (Fig. 7). This deacidification changes the texture under the rind and even inside the curd. The faster the migration, the quicker is the ripening of Camembert. The stimulation of the microflora activity may lead to an exhaustion of their usual substrates, lactose and lactate. In that case, proteins and lipids are broken down, and is one of the possible reasons for the production, by P. camemberti, of styrene or 1-octen-3-ol. This risk increases when the ripening temperature is abnormally high or when the curds have been washed (stabilised curd technology; Spinnler et al., 1992).
The particular characteristics of P. camemberti are expressed in surface mould-ripened cheeses, giving the cheeses their characteristic appearance and contributing to the development of the rheological and sensory qualities. However, the secondary micro-flora contribute to the attainment of the traditional quality
171
of this variety. Great progress has been made during the last 20 years in our knowledge of the mechanisms of ripening in surface mould-ripened cheeses. However, the processes are very complex and no close relationship can yet be seen between the composition and the quality of mould-ripened cheese. While studies on traditional mould-ripened cheeses should not be abandoned, it should be remembered that more cheeses are now being produced in large, automated factories. The good quality of these products must be maintained, taking into account consumer taste, which often favours rather mild products. Improving the storage life of surface mould-ripened soft cheese should also make it easier to distribute and to expand its production.
Adda, J. and Dumont, J.P. (1974). Les substances responsables de l'arOme des fromages a pate molle. Lait 54, 1-21. Adda, J., Dekimpe, J., Vassal, L. and Spinnler, H.E. (1989). Production de styrene par Penicillium camemberti Thom. Lait 69, 115-120. Ahiko, K., Iwasawa, S., Ulda, M. and Nigata, N. (1981). Studies on acid carboxypeptidase from Penicilium caseicolum: II. Hydrolysis of bitter peptides by acid carboxypeptidases and large scale preparation of the enzyme. Report of Research Laboratory, Snow Brand Milk Products Co. 77, 135-140.
Ahmed, E.M, Dennison, R.A., Dougherty, R.H. and Shaw, P.E. (1978). Flavor and odor thresholds in water of selected orange juice components. J. Agric. Food Chem. 26, 187-191. Baroiller, C. and Schmidt, J.-L. (1990). Contribution a l'~tude de l'origine des levures du fromage de Camembert. Lait 70, 67-84. Berger, C., Khan, J.A., Molimard, P., Martin, N. and Spinnler, H.E. (1999a). Production of sulfur flavors by 10 strains of G. candidum. Appl. Environ. Microbiol. 65, 5510-5514. Berger, C., Martin, N., Collin, S., Gijs, L., Khan, J.A., Piraprez, G., Spinnler, H.E. and Vulfson, E.N. (1999b). Combinatorial approach to flavor analysis: II. Olfactory investigation of a library of S-methyl thioesters and sensory evaluation of selected components. J. Agric. Food Chem. 47, 3274-3279. Bertolini, M.C., Schrag, J.P., Cygler, M., Ziomek, E., Thomas, D.Y. and Vernet, T. (1995). Expression and characterization of Geotrichum candidum lipase I gene. Comparison of specificity profile with lipase II. Eur.J. Biochem. 228,863-869. Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, D. and Sorokin, A. (2001). The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res. 11, 731-753. Bonnarme, P., Psoni, L. and Spinnler, H.E. (2000). Diversity of L-methionine catabolism pathways in cheese-ripening bacteria. Appl. Environ. Microbiol. 66, 5514-5517.
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Surface Mould-ripened Cheeses
Bonnarme, P., Arfi, K., Dury, C., Helinck, S., Yvon, M. and Spinnler, H.E. (2001). Sulfur compound production by G. candidum from methionine: importance of the transamination step. FEMS Microbiol. Lett. 205,247-252. Buttery, R.G., Turnbaugh, J.G. and Ling, L.C. (1988). Contribution of volatiles to rice aroma. J. Agric. Food Chem. 36, 1006-1009. Chalier, P. and Crouzet, J. (1992). Production of lactones by P. roqueforti. Biotechnol. Lett. 14, 275-280. Chen, C.C. and Wu, C.M. (1984). Studies on the enzymatic reduction of 1-octen-3-one in mushroom (Agaricus bisporus). J. Agric. Food Chem. 32, 1342-1344. Choisy, C., Desmazeaud, M., Gueguen, M., Lenoir, J., Schmidt, J.-L. and Tourneur, C. (1984). Cheese ripening: microbial phenomena, in, Cheesemaking: From Science to Quality Assurance, A. Eck and J.C. Gillis, eds, Lavoisier, Paris. pp. 353-411. Christensen, J.E., Dudley, E.G., Pederson, J.A. and Steele, J. (1999). Peptidases and amino acid catabolism in lactic acid bacteria. Antonie van Leeuwenhoek 7,217-246. CNIEL (2002). EEconomie Laiti~.re en Chiffre, CNIEL Ed., Paris. Creuly, C., Larroche, C. and Gros, J.B. (1992). Bioconversion of fatty acids into methyl ketones by spores of Penicillium roqueforti in a water-organic solvent, two-phase system. Enzyme Microb. Technol. 14, 669-678. Dartey, C.K. and Kinsella, J.E. (1973). Metabolism of [U-14C]lauric acid to methyl ketones by the spores of Penicillium roqueforti. J. Agric. Food Chem. 21, 933-936. Demarigny, Y., Berger, C., Desmazures, N., Gueguen, M. and Spinnler, H.E. (2000). Flavour sulphides produced from methionine by two different pathways by G. candidum. J. Dairy Res. 67,371-380. Desmazeaud, M., Gripon, J.C., Le Bars, D. and Bergere, J.L. (1976). Etude du r~31e des microorganismes et des enzymes au cours de la maturation des fromages. III. Influence des microorganisms. Lait 56,379-396. Dufosse, L., Latrasse, A. and Spinnler, H.E. (1994). Importance des lactones dans les arOmes alimentaires: structures, distribution, propriet~s sensorielles et biosynthese. Sci. Alim. 14, 17-50. Dumont, J.P., Roger, S. and Adda, J. (1974a). Etude des composes volatils neutres presents dans les fromages /~ pate molle et crot?te lavee. Lait 54, 31-43. Dumont, J.P., Roger, S., Cerf, P. and Adda, J. (1974b). Etude des composes neutres volatils presents dans le Camembert. Lair 54, 501-516. Dumont, J.P., Roger, S., Cerf, P. and Adda, J. (1974c). Etude des composes volatils neutres presents dans le Vacherin. Lait 54, 243-251. Dumont, J.P., Degas, C. and Adda, J. (1976a). EAr~Sme du Pont l'Eveque. Mise en evidence de constituants volatils quantitativement mineurs. Lait 56, 177-180. Dumont, J.P., Roger, S. and Adda, J. (1976b). EArOme du camembert: autres composes mineurs mis en evidence. Lait 56, 595-599. Dumont, J.P., Delespaul, G. Miguot, B. and Adda, J. (1977). Influence des bacteries psychrotrophes sur les qualities organoleptiques des fromages /t pate molle. Lait 57, 619-630.
Fan, T.Y., Hwang, D.H. and Kinsella, J.E. (1976). Methyl ketone formation during germination of Penicillium roqueforti. J. Agric. Food Chem. 24, 443-448. Foissy, H. (1974). Examination of Brevibacterium linens by an electrophoretic zymogram technique. J. Gen. Microbiol. 80, 197-207. Foissy, H. (1978). Aminopeptidase from Brevibacterium linens. Production and purification. Milchwissenchaft 33, 221-223. Gallois, A. (1984). Biosynthi~se de la M~.thoxy-2-Isopropyl3-Pyrazine par Pseudomonas taetrolens. Doctoral Thesis, INRA. Paris-Grignon, France. Gallois, A. and Langlois, D. (1990). New results in the volatile odorous compounds of French cheese. Lait 70, 89-106. Gueguen, M. and Lenoir, J. (1975). Suitability of Geotrichurn candidum for production of proteolytic enzymes. Lait 55, 621-629. Gueguen, M. and Lenoir, J. (1976). Caracteres du systeme proteolytic de Geotrichum candidum. Lait 56,439-448. Ha, J.K. and Lindsay, R.C. (1991). Contribution of cow, sheep and goat milks to characterizing branched chain fatty acids and phenolic flavors to varietal cheeses. J. Dairy Sci. 74, 3267-3274. Hassouna, M. and Guizani, N. (1995). Evolution de la flore microbienne et des caracteristiques physico-chimiques au cours de la maturation du fromage Tunisien de type camembert fabrique avec du lait pasteurise. Microbiol. Hyg. Alim. 18, 14-23. Hayashi, K. and Law, B. (1989). Purification and characterization of two aminopeptidases produced by Brevibacterium linens. J. Gen. Microbiol. 135, 2027-2034. Hayashi, K., Cliffe, A.J. and Law, B. (1990). Purification and characterization of five serine proteinases produced by Brevibacterium linens. Int. J. Food Sci. Technol. 25,180-187. Irlinger, E and Bergere, J.L. (1999). Use of conventional biochemical tests and analyses of ribotype patterns for classification of micrococci isolated from dairy products. J. Dairy Res. 66, 91-103. Irlinger, E, Morvan, A., El Sohl, N., Bergere, J.L. (1997). Taxonomic characterization of coagulase-negative staphylococci in ripening flora from traditional French cheeses. Syst. Appl. Microbiol. 20, 319-328. Jollivet, N., Chateaud, J., Vayssier, Y., Bensoussan, M. and Belin, J.M. (1994). Production of volatile compounds in model milk and cheese media by eight strains of Geotrichum candidum Link. J. Dairy Res. 61,241-248. Karahadian, C. and Lindsay, R.C. (1987). Integrated roles of lactate, ammonia and calcium in texture development of mold surface-ripened cheese. J. Dairy Sci. 70, 909-918. Karahadian, C., Josephson, D.B. and Lindsay, R.C. (1985a). Volatile compounds from Penicillium sp. contribiting musty-earthy notes to Brie and Camembert cheese flavors. J. Agric. Food Chem. 33,339-343. Karahadian, C., Josephsorl, D.B. and Lindsay R.C. (1985b). Contribution of Penicillium sp. to the flavors of Brie and Camembert cheese. J. Dairy Sci. 68, 1865-1877. Kinsella, J.E. and Hwang, D.H. (1976). Biosynthesis of flavors by Penicilliurn roqueforti. Biotechnol. Bioeng. 18, 927-938. Kuzdzal-Savoie, S. arid Kuzdzal, N. (1966). Etude comparee des acides gras non volatils libres et esterifies dans les
Surface Mould-ripened Cheeses
fromages. Proc. XVII Int. Dairy Cong., Munich, Vol. D2. pp. 335-349. Laivg, D.G., Panhuber, H. and Baxter, R.I. (1978). Olfactory properties of amines and n-butanol. Chem. Sens. Flavour 3, 149-166. Lamberet, G. and Lenoir, J. (1976). Les caracteres du systeme lipolytique de l'espece Penicillium caseicolum: purification et propri4t4 de la lipase majeure. Lait 56, 622-644. Lamberet, G., Auberger, B., Canteri, C. and Lenoir, J. (1982). EAptitude de Penicillium caseicolum it la degradation oxidative des acides gras. Revue Laitiere Fran~aise 406, 13-19. Latrasse, A., Dameron, P., Hassani, H. and Staron, T. (1987). Production d'un arOme fruit~ par Geotrichum candidum (Staron). Sci. Alim. 7,637-645. Law, B.A. and Sharpe, M.E. (1978). Formation of methanethiol by bacteria isolated from raw milk and Cheddar cheese. J. Dairy Res. 45,267-275. Leclercq-Perlat, M.N., Oumer, A., Bergere, J.L., Spinnler, H.E. and Corrieu, G. (1999). Growth of Debaryomyces hansenii on a bacterial surface-ripened cheese. J. Dairy Res. 66, 271-281. Leclercq-Perlat, M.N., Buono, E, Lambert, D., Latrille, E., Spinnler, H.E. and Corrieu, G. (2003). Controlled production of Camembert-type cheeses. Part I: Microbiological and physicochemical evolutions. J. Dairy Res., submitted for publication. Lee, C.W. and Richard, J. (1984). Catabolism of L-phenyalanine by some microorganisms of cheese origin. J. Dairy Res. 51, 461-469. Le Graet, Y., Lepienne, A., Brul4, G. and Ducruet, P. (1983). Migration du calcium et des phosphates inorganiques dans les fromages/l pittes molles de type Camembert au cours de l'affinage. Lait 63,317-332. Lenoir, J. (1970). Eactivite proteasique dans les fromages it pitte molle de type Camembert. Revue Laitiere Fran~aise 275, 231-236. Lenoir, J. (1984). The Surface Flora and its Role in the Ripening of Cheese. Bulletin 171, International Dairy Federation, Brussels. pp. 3-20. Lenoir, J. and Choisy, C. (1970). Aptitude de l'espece Penicillium caseicolum it la production d'enzymes prot4olytiques. Lait 51,138-157. Maisnier-Patin, S., Deschamps, N., Tatini, S.R. and Richard, J. (1992). Inhibition de Listeria monocytogenes dans du Camembert fabriqu4 avec un levain producteur de nisine. Lait 72,249-263. Margalith, P.Z. (1981). Dairy products, in, Flavor Microbiology, R.A. Magalitz, ed., C.C. Thomas Publisher, Springfield, Illinois, USA. pp. 32-118. Martley, E (1975). Comportement et r01e des streptocoques lactiques du levain en fabrication de Camembert. Lait 55, 310-323. Matsuoka, H., Fuka, Y., Kaminogawa, S. and Yamauchi, K. (1991). Purification and debittering effect of aminopeptidase II from Penicillium caseicolum. J. Agric. Food Chem. 39, 1392-1395. Miettinen, S.M., Tuorila, H., Piironen, V., Vehkalahti, K., Hyvonen, L. (2002). Effect of emulsion characteristics on the release of aroma as detected by sensory evaluation,
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static headspace gas chromatography, and electronic nose. J. Agric. Food Chem. 50, 4232-4239. Moinas, M., Groux, M. and Horman, I. (1973). La flaveur des fromages. I.- Une methodologie nouvelle d'isolement de constituants volatils. Application au Roquefort et au Camembert. Lait 53,601-609. Moinas, M., Groux, M. and Horman, I. (1975). La flaveur des fromages. III.- Mise en evidence de quelques constituants mineurs de l'ar0me du Camembert. Lait 55, 414-417. Molimard, P. and Spinnler, H.E. (1996). Review: Compounds involved in the flavor surface mold ripened cheeses: Origins and properties. J. Dairy Sci. 79, 169-184. Molimard, P., Lesschaeve, I., Bouvier, I., Vassal, L., Schlich, P., Issanchou, S. and Spinnler, H.E. (1994). Amertume et fractions azot~es de fromages/~ pitte molle de type Camembert: r01e de l'association de Penicillium camemberti avec Geotrichum candidum. Lait 74, 361-374. Molimard, P., Lesschaeve, I., Issanchou, S., Brousse, M. and Spinnler, H.E. (1997). Effect of the association of surface flora on the sensory properties of mould ripened cheese. Lait 77, 181-187. Moreau, C. (1979). Nomenclature des Penicillium utiles/t la preparation du Camembert. Lait 59, 219-233. Nierop-Groot, M.N. and de BontJ.A.M. (1999). Involvement of manganese in conversion of phenylalanine to benzaldehyde by lactic acid bacteria. Appl. Environ. Microbiol. 65, 5590-5593. Noomen, A. (1983). The role of the surface flora in the softening of cheeses with a low initial pH. Neth. Milk Dairy J. 37, 229-232. Okumura, J. and Kinsella, J.E. (1985). Methyl ketone formation by Penicillium camemberti in model systems. J. Dairy Sci. 68, 11-15. Overbosch, P., Afterof, G.M. and Haring, P.G.M. (1991). Flavour release in the mouth. Food Rev. Int. 7, 137-184. Parliment, T.H., Kolo, M.J. and Rizzo, D.J. (1982). Volatile components of Limburger cheese. J. Agric. Food Chem. 30, 1006-1008. Pelissier, J.P., Mercier, J.C. and Ribadeau-Dumas, B. (1974). Problem of bitter flavor in cheese. Revue Laiti~re Fran~aise 325,817-821. Perraud, X. and Kermasha, S. (2000). Characterization of lipoxygenase extracts from Penicillium sp. J. Am. Oil. Chem. Soc. 77,335-342. Perraud, X., Kermasha, S. and Bisakowski, B. (1999). Characterization of lipoxygenase extract from G. candidum. Process Biochem. 34, 819-827. Piendl, A. and Geiger, E. (1980). Technological factors in the formation of esters during fermentation. Brewers' Digest 55, 26-38. Ratledge (1984). Microbial conversions of alkanes and fatty acids. J. Am. Oil Chem. Soc. 61,447-453. Rattray, EP. and Fox, P.E (1997). Purification and characterisation of an intracellular aminopeptidase from Brevibacterium linens ATCC 9174. Lait 77, 169-180. Rattray, EP. and Fox, P.E (1999). Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review. J. Dairy Sci. 82, 891-909.
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Rattray, EP., Bockelmann, W. and Fox, RE (1995). Purification and characterisation of an extracellular proteinase from Brevibacterium linens ATCC 9174. Appl. Environ. Microbiol. 61, 3454-3456. Rattray, RE, Fox, RE and Healy, A. (1996). Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine Otsl-casein. Appl. Environ. Microbiol. 62, 501-506. Roger, S., Degas, C. and Gripon, J.C. (1988). Production of phenyl ethyl alcohol and its esters during ripening of traditional Camembert. Food Chem. 28, 129-140. Rothe, M., Engst, W. and Erhardt, V. (1982). Studies on characterization of blue cheese flavour. Nahrung 26, 591-602. Schmidt, J.L. (1982). Proteolytic activity of yeast isolated from Camembert. Proc. XXI Intern. Dairy Congr. (Moscow), Vol. 1, p. 365. Shankaranarayana, M.L., Raghavan, B. Abraham, K.O. and Natarajan, C.P. (1974). Volatile sulfur compounds in food flavours. CRC Crit. Rev. Sci. Technol. 4,395-435. Spinnler, H.E. (2003). Off flavours due to interactions between food components, in, Taint and Off-flavours in Food, B. Baigrie, ed., Woodhead Publish. Ltd, Cambridge. pp. 176-186. Spinnler, H.E., Grosjean, O. and Bouvier, I. (1992). Effect of culture parameters on the production of styrene (vinyl benzene) and 1-octene-3-ol by Penicillium caseicolum. J. Dairy Res. 59,533-541. Spinnler, H.E., Berger, C., Lapadatescu, C. and Bonnarme, P. (2001). Production of sulfur compounds by several yeasts of technological interest for cheese ripening. Int. Dairy J. 11,245-252. Stackebrandt, E., Rainey, EA. and War-Rainey, N.L. (1997). Proposal for a new hierarchic classification system Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47, 479-491. Teranishi, R., Buttery, R.G. and Guadagni, D.G. (1981). Some properties of odoriferous molecules, in, Flavour 81,
P. Schreir, ed., Walter-de-Gruyter, Berlin, Germany. pp. 133-143. Tokita, E and Hosono, A. (1968). Studies on and behaviour of the amines produced by Brevibacterium linens. Milchwissenchaft 23, 690-693. Ummadi, M. and Weimer, B.C. (2001). Tryptophan catabolism in Brevibacterium linens as a potential cheese flavor adjunct. J. Dairy Sci. 84, 1773-1782. Vanbelle, M., Vervack, W. and Foulon, M. (1978). Composition en acides gras superieurs de quelques types de fromages consommes en Belgique. Lait 58,246-260. Vassal, L. and Gripon, J.-C. (1984). EAmertume des fromages a pate molle de type Camembert: r01e de la presure et de Penicillium caseicolum, moyens de la contr61er. Lait 64, 397-417. Vassal, L., Monnet, V., Roux, C., Le Bars, D. and Gripon, J.C. (1984). Relation entre le pH, la composition chimique et la texture des fromages de type Camembert. Lait 66, 341-351. Veeraragavan, K., Colpitts, T. and Gibbs, B.E (1990). Purification and characterisation of two distinct lipases from Geotrichum candidum. Biochim. Biophys. Acta 1044, 26-33. von Weissenfluh, A. and Puhan, Z. (1987). The effect of environmental conditions in the ripening room on the growth of Penicillium camemberti and the quality of Camembert cheese. Schweiz. Milchwitschaft Forsch. 16, 37-44. Wendin, K., Risberg Ellekjaer, M., Solheim, R. (1999). Fat content and homogeneisation effects on flavour and texture of Mayonnaise with added aroma. Lebensm. Wiss. Technol. 32,377-383. Yvon, M., Bonnarme, P., Chambellon, E., Semon, E., Spinnler, H.E. (2001). Transamination reactions initiates the methionine conversion to methylacetaldehyde by Lc. lactis. Proceedings of NIZO Dairy Conference on Food Microbes, Ede, p. 36.
Blue Cheese M.D. Cantor, Danisco A/S, Innovation, Denmark T. van den Tempel, Chr. Hansen A/S, Cheese Culture Technology, Denmark T.K. Hansen Leo Pharma A/S, Microbiological Research Laboratory, Denmark Y. Ard6, The Royal Veterinary and Agricultural University, Department of Dairy and Food Science, Frederiksberg, Denmark
Blue cheeses are characterised by the growth of the mould Penicillium roqueforti, giving them their typical appearance and flavour. Many countries have developed their own types of Blue cheese, each with different characteristics (Table 1) and involving different manufacturing methods (Fig. 1). The bestknown varieties today, worldwide, are considered to be Gorgonzola, Roquefort, Stilton and Danablu, all of which have been granted the status of Protected Designation of Origin/Protected Geographical Indication (PDO/PGI), together with a number of other European Blue cheeses. Blue cheeses have probably been produced for a long time, either deliberately or by accident, before they were described in writing. Gorgonzola was the first Blue-veined cheese to be mentioned in the literature, in 879, while Roquefort was described in customs papers in 1070; however, already in the eighth century chronicles from monasteries mention the transport of Roquefort across the Alps (Kloster, 1980). Stilton was not mentioned until the seventeenth century. In Denmark, the production of Danablu and Mycella, Blue cheeses from cow's milk, started in the 1870s. In 1916, a method for homogenising the cream was developed and used for the production of Danablu, making the cheese as white as the traditional Roquefort made from sheep's milk. Additionally, homogenisation influences ripening by accelerating lipolysis. As Blue cheeses are becoming more and more popular, there has been increased interest in the scientific characterisation of the various types. This chapter aims to review the present knowledge of different aspects of Blue cheese ripening, emphasising changes in the microenvironment, micro-organisms that contribute to ripening and various biochemical changes, i.e., lipolysis, proteolysis and aroma formation. Finally, recommendations for the selection of appropriate starter and mould cultures, as well as new, possible adjunct cultures, will be discussed.
The microenvironment in Blue cheese is, in general, heterogeneous with pronounced gradients of pH, salt, water activity (aw), etc. The ripening temperature is typically 8-15 ~ depending on the variety. Furthermore, there are considerable structural differences within these cheeses, which influence the level and distribution of 02 and CO2. These parameters and their changes during the course of ripening have a great impact on the growth and biochemical activity of the various micro-organisms present in the cheese and thereby the quality of the final product. Therefore, knowledge of the levels encountered at different ripening times is important in order to construct realistic model systems. The minimum pH of Blue cheese ranges from approximately 4.6-4.7 in Danablu (Hansen, 2001), Mycella (Hansen et al., 2001) and Stilton (Madkor et al., 1987a) to 5.15-5.30 in Gorgonzola (Gobbetti etal., 1997), Picon Bejes-Tresviso (Prieto et al., 1999, 2000) and Cabrales (Alonso et al., 1987). The conversion of lactose to lactic acid by the lactic acid bacteria (LAB) of the primary starter culture is facilitated by the manufacturing procedure; the curd is very moist when placed in hoops and no pressure is applied during whey drainage (2-3 days), giving the LAB access to large amounts of lactose. The amount of residual lactose decreases very quickly. In 15-day-old Gamonedo Blue cheese, the lactose content was only 0.15% of total solids (Gonzalez de Llano et al., 1992) whereas 0.9% lactose was found in 1-day-old Picon Bejes-Tresviso cheese, after which it was no longer detectable (Prieto et al., 2000). During ripening, the pH of Mycella increases to 6.5 in the core and to 5.9 in the surface layer (Hansen et al., 2001). Similar values were found in Danablu, as depicted in the partial least squares (PLS) plot in Fig. 2 (Hansen, 2001), and for other varieties of Blue cheeses (Zarmpoutis et al., 1997). However, higher pH values have been reported as well (Gonz/tlez de Llano et al., 1992; Zarmpoutis et al., 1996, 1997; Gobbetti et al., 1997). The pH of the interior rises more rapidly than
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
176
Blue Cheese
A few examples of Blue cheeses and the range in reported gross composition
Name
Origin
Cabrales x Chetwynd Danablu x Gamonedoa
Spain Ireland Denmark Spain
Gorgonzola x Kopanisti x
Italy Greece
Kvibille ,~,del Pic6n Bejes-Tresviso x
Sweden Spain
Roquefort x Stiltonx
France Great Britain
Type of milk used for production Raw cows' milk Pasteurised cows' milk Thermised cows' milk Raw cows', goats' and ewes' milk Pasteurised cows' milk Raw cows', goats' or ewes' milk or a mixture of these Pasteurised cows' milk Raw cows' milk or raw cows' and raw ewes' milk Raw ewes' milk Pasteurised cows' milk
% moisture
% fat
% protein
% NaCl
Reference
35.4-41.6 49.2-50.2 42.7-47.3 33-40.4
33.8-38.2 26 29-31 29.2-32.3
20.4-23.6 19.3-20.8 18.5-23.9 23.3-27.5
1.8-3.4 3.2-3.8 3-3.9 3.1-4.9
2 4 1,4,9,11 3
42.2-49.6 44.6-69.4
29.6-31 13-30
19-22.9 14.2-27
1.6-2.9 1-4.7
4,5,9 12
43 36.9-41.5 40.4-45.1 42-44 37-41.6
29 36.7-40.4 30.6-34.1 29 32-35.2
21 20.3-23.1 20.8-23.8 20 21-28.7
3-4 1.8-2.1 3.2-4.4 4.1 2.2-2.7
6 7,8 10,11 1,4,9
x Cheeses with PDO/PGI. a Gamonedo cheeses are smoked for 3-4 weeks. 1" Madkor et al. (1987a); 2: Marcos et aL (1983); 3: Gonz&lez de Llano et al. (1992); 4: Zarmpoutis et aL (1997)" 5" Gobbetti et al. (1997); 6: Palmquist and Brelin (1993)" 7: Prieto et al. (1999)" 8: Prieto et al. (2000); 9: Muir et al. (1995); 10: Matsui and Yamada (1996); 11" de Boer and Kuik (1987); 12" Kaminarides (1986).
that of the surface (Gobbetti et al., 1997; Hansen et al., cheeses (Marcos, 1993). Furthermore, the fat content 2001), as the level of NaC1 is lower, and therefore influences cheese structure and thereby the diffusion allows a faster and earlier growth of the mould cul- coefficient of NaC1 and the equilibration of aw throughtures. The increase in pH is due to the metabolism of out the cheese (cf. 'Salt in Cheese: Physical, Chemical lactic acid to CO2 by yeasts and moulds and the and Biological Aspects', Volume 1). In Danablu, the increased proteolysis, leading to production of NH3 highest aw, c. 0.98, is found in the interior after 1 week from amino acid~ (Godinho and Fox, l OR2; Zarmpollti~ nf rinenino while the vnl~e far the e:eterinr reoinn et al., 1996, 1997). ranges from 0.85 to 0.90 (Fig. 2). After 5 weeks, aw for Salting, done by immersing the cheeses in brine or both the interior and the exterior regions of Danablu is applying dry salt to the cheese surface, is an important usually in the range 0.91-0.94 (Fig. 2). Similar values step in the manufacture of most Blue cheeses. Both have been found for Mycella (Hansen et al., 2001) and methods create a NaC1 gradient from the surface of the Pic6n Bejes-Tresviso cheese (Prieto et al., 1999, 2000). cheese to the core, which equilibrates slowly during It is well known that the growth of fungi is affected ripening (Fig. 2) (Godinho and Fox, 1981b; Gobbetti by the gaseous composition in cheese, i.e., the conet al., 1997; Hansen et al., 2001). centrations of 02 and CO2. The level of 02 has been The overall NaC1 content in ripe Blue cheese ranges shown to decrease rapidly throughout the cheese; in from 2 to 5% (Madkor et al., 1987a; Zarmpoutis et al., Danablu, after 1 week of ripening, a 50% decrease was 1996, 1997; Gobbetti et al., 1997; Prieto et al., 2000). found 4 mm under the rind, whereas after 13 weeks, The high salt content is due to a fairly long salting 02 was completely absent, except in the outer 0.25 mm period for these cheeses (e.g., 2 days brine-salting for (van den Tempel et al., 2002). This anaerobic enviDanablu), the high moisture content and the loose ronment was evident already after 3 weeks of ripenstructure of the cheese matrix. The diffusion of NaC1 ing, except from small areas in the cheese, probably into the cheese core is faster in the piercing channels in fissures. The results are in accordance with obserand in areas with fissures creating an even more uneven vations in white-mould cheese (Boddy and Wimsalt distribution. The NaC1 concentration measured in penny, 1992), but are lower than values found in Danablu cheeses after 8 weeks of maturation was Roquefort (Thom and Currie, 1913), where oxygen approximately 2.0% (w/w) in the core and 4.0% (w/w) was measured in the gas phase of the cheese. P. roquein the surface layer, corresponding to a NaC1 in mois- forti is well adapted to growth inside Blue cheese ture of 7.5 and 10.0%, respectively (Hansen, 2001). where a low level of 02 is combined with a high level The concentration of NaC1, lipolysis and proteolysis, of CO2 (20-40%), as this does not significantly affect especially the increase in low molecular weight peptides, its growth (van den Tempel and Nielsen, 2000; Taniwaki influence the water activity, aw, significantly in Blue et al., 2001). l"
O
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9
Blue Cheese
177
Ax
Cows', goats' or ewes' milk or a mixture of 2 or 3 milk types $
$
Raw milk, thermisation (c. 62 ~ x 15 s) or pasteurisation (c. 72 ~
Pasteurised cows' milk (c. 73 ~ x 40 s)
15s)
Inoculation with starter (mesophilic + thermophilic starter culture, c. 106 cfu/ml of cheese milk)
4,
$
Addition of rennet
Inoculation with Penicillium roqueforti
Addition of starter (optional) Addition of Penicillium roqueforti (optional)
Addition of liquid calf rennet
4,
$
Coagulation, cutting and stirring
Coagulation at 30-34 ~
4,
4,
Moulding
Cutting of the coagulum (size of curd: c. 2.0-2.5 cm)
4,
4,
Whey drainage for c. 10-48 h. No pressure applied, but
Stirring
moulds are inverted frequently
$
4,
Moulding
Brine salting or dry salting for 24-48 h
$
4,
Whey drainage, no pressure applied, held at 18 ~ for 10 h
Piercing of the cheeses (optional)
with 4 turns
4, Ripening (in general at c. 10 ~
c. 85-95% relative
Salting (c. 200 g of salt spread over each cheese), 22 ~ for 40 h
humidity, for some varieties in caves) Ripening at 4-6 ~ for 83 days, 85-90% relative humidity
x Times, temperatures, etc. depend on the variety being produced.
Cheeses are pierced after 12 and 20 days
An outline of general steps in the manufacture of different varieties of Blue cheese (A) and the steps involved in the production of Gorgonzola (B).
Several micro-organisms make up the complex microbiota of Blue cheeses, contributing at different levels to ripening. The primary and secondary starter cultures, LAB and P. roqueJorti, respectively, are the most import-
ant, but yeast and non-starter lactic acid bacteria (NSLAB), even though they are not added deliberately to the cheese milk, most probably also influence ripening. It should also be noted that some varieties of Blue cheeses are ripened naturally, i.e., cultures are not
Partial least squares (PLS) contour plots of pH, NaCI and aw in Danablu 50+ after 1 and 5 weeks of ripening. The contour plots show the gradients from the core to the surface of the cheese, corresponding to the grey area on the cheese depicted. (Data were visualised by PLS regression using SIMCA-P, ver. 3.01 (UMETRI AB, Sweden)). 178
Blue Cheese
added during manufacturing. However, the abovementioned groups of micro-organisms are present in both naturally ripened Blue cheeses and Blue cheeses with added cultures, and these groups, and their characteristics, will be described in the following sections. Lactic acid bacteria
Mesophilic and thermophilic LAB are used as the primary starter culture for the production of different varieties of Blue cheese. A mesophilic, undefined mixed culture will typically contain lactic acid-producing Lactococcus lactis (Lc. lactis subsp, lactis and Lc. lactis subsp, cremoris) and sometimes also citrate-positive strains of Lc. lactis subsp, lactis and Leuconostoc species, which produce CO2 and open up the structure to facilitate the penetration of air and development of the mould. The thermophilic starters used in Blue cheese usually contain Streptococcus thermophilus and Lactobacillus delbrueckii subsp, bulgaricus. The most important role of the LAB starter culture is to acidify the milk by metabolising lactose to lactate. In general, the numbers of LAB (lactococci and lactobacilli) in the core decrease slowly from about 109 cfu/g after salting to 107-108 cfu/g at the end of maturation. The number on the surface after brining is 108-1010 cfu/g and remains almost stable to the end of maturation (Devoyod et al., 1968; Nufiez, 1978; Ordonez et al., 1980; GonzMez de Llano et al., 1992; Gobbetti et al., 1997; Hansen et al., 2001). Investigations of the LAB in the core and on the surface of Danablu, for which
179
only a mesophilic starter is used, immediately after brining to 4 weeks of ripening indicated that the number of lactococci decrease markedly in the surface layer during the first weeks of ripening. At the same time, an increase of a new microbiota dominated by Lactobacillus spp. was observed. For the whole period, the population in the core was dominated by lactococci (Hansen, unpublished results).
Penicillium roqueforti
Penicillium roqueforti has previously been known under other names, but several species, including P. stilton, P. italicum, P. gorgonzola, P. glaucum, P. bioruge, P. suavolens and P. aromaticum, were found to belong to the same species and collected under the taxon P. roqueforti (Pitt, 1979; Stolk et al., 1990). Taxonomically, P. roqueforti is classified under the genus Penicillium Link, the subgenus Penicillium and the species roqueforti Thom (Pitt and Hocking, 1997). The taxonomy of the fungi is based on phenotypic analysis though genotypic methods are becoming more and more common. Different methods.used to determine the taxonomy of P. roqueforti are shown in Table 2. Conidia of P. roqueforti may be added directly to the cheese milk, sprayed on the curd or colonise the cheeses naturally. The addition of conidia is crucial for the quality of Blue cheese varieties made from pasteurised milk. P. roqueforti can assimilate all the main carbohydrates that occur in cheese, i.e., lactose, glucose and galactose, utilise lactate and citrate and grow without
Methods used for the taxonomical classification of Penicillium roqueforti Methods
Analysis
Reference
Classical methods based on phenotypic classification
Micro- and macromorphology, growth rate on specific media, assimilation of carbohydrates and acids, growth on different nitrogen sources, resistance to preservatives and chemicals Production of secondary metabolites under specific and controlled conditions (assayed by TLC, HPLC and GC) Production of aroma compounds and their specific profile (assayed by GC and MS) RAPD ITS-PCR rDNA-RFLP AFLP Based on the same criteria as the classical methods, but instead of visual analysis of the macromorphology, digital image analysis and multivariate data analyses are used
Samson et aL (1977, 1995) Pitt (1979) Pitt and Hocking (1997)
Profiles of secondary metabolites Aroma profiles PCR-based methods
Image analysis
Frisvad (1982) Lund et al. (1995) Boysen et aL (1996) Larsen and Frisvad (1995a,b) Geisen et aL (2001) Boysen et aL (1996, 2000) Boysen (1999) D6rge et aL (2000)
TLC, Thin layer chromatography; HPLC, High performance liquid chromatography; GC, Gas chromatography; MS, Mass spectroscopy; RAPD, Random amplified polymorphic DNA; ITS-PCR, Internal transcribed spacer-Polymerase chain reaction; RFLP, Restriction fragment length polymorphism; AFLP, Amplified fragment length polymorphism.
180
Blue Cheese
difficulty at the pH and temperatures encountered during ripening of Blue cheese (Cerning et al., 1987; Vivier et al., 1992). P. roqueforti is the Penicillium species with the highest tolerance to low levels of 02 (Pitt and Hocking, 1997). It has been demonstrated that the rate of growth of P. roqueforti is not significantly affected in the range 4-21% 02 (Thom and Currie, 1913 van den Tempel and Nielsen, 2000; Taniwaki et al., 2001), but growth seems to be affected by interactions between the levels of 02 and CO2. P. roqueforti grows in the presence of 25% CO2 (van den Tempel and Nielsen, 2000) and 02 in the range 0.3-21%. Taniwaki (1995) found that growth and sporulation of P. roqueforti occur at 20% CO2 in an atmosphere with 0.5% 02. P. roqueforti grows in fissures and piercing channels in the cheese. The colour of the mould varies from white through several shades of green to brownish, depending on the strain and its age. The growth rate of P. roqueforti is strongly affected by increasing concentration of NaC1. The influence of aw on growth, sporulation and germination of four strains of P. roqueforti was investigated in laboratory media containing added NaC1 at concentrations corresponding to aw in the range 0.99-0.92 (0-13%, w/w, NaC1). The growth of most strains was stimulated by 3.5% NaC1, corresponding to aw 0.98 (Hansen and Jakobsen, 2003). Similar results have been reported by other authors (Godinho ~,,u~'a Fox, ,9ola; ,_.uv~-~,,,~ ~ . . ~ u u , ,,~,,,, et al., 1998). Higher concentrations of NaC1 cause a decrease in the growth rate, e.g., a 92% reduction at aw 0.92 compared to the optimum growth rate at aw 0.98 (Hansen and Jakobsen, 2003). Concerning sporulation, an optimum was observed at aw 0.98 for three of the four strains of P. roqueforti examined" the fourth strain showed an optimum at aw 0.96. Sporulation was strongly inhibited at aw 0.94 for the three salt-sensitive strains whereas the NaCl-tolerant strain still showed a pronounced sporulation at aw 0.94, but not at aw 0.92 (Hansen and Jakobsen, 2003). Germination of P roqueforti conidia is stimulated by 1-3% NaC1 for most strains, but differences in NaC1 tolerance have been observed (Godinho and Fox, 1981a Lopez-Diaz et al., 1996b). Below aw 0.96, the rate of germination decreases with decreasing aw (Hansen and Jakobsen, 2003) and it was observed that NaC1 inhibits the rate of swelling of the conidia as well as the further development of the germ tube. Germination rate was also influenced by the microenvironment in which the conidia were produced, i.e., conidia produced and harvested at aw 0.92 germinated faster at aw 0.99 than conidia produced at a higher aw (Hansen and Jakobsen, 2003).
The aw in the core of Blue cheeses after brining is optimal for germination and growth, and the concentration of NaC1 is in the range where P. roqueforti is stimulated (Godinho and Fox, 1981a). During the first 3 weeks of ripening, the NaC1 concentration in the core increases to a level that induces sporulation and reduces the germination rate and growth of mycelia. These changes influence the appearance of the cheese as the blue-green colour is due to the conidia and also prevents the growth of a thick mycelium in fissures and piercing channels. A thick mycelium feels like rubber in the mouth and is therefore undesirable in Blue cheese. Due to the NaC1 gradient, the development of P. roqueforti occurs from the interior to the exterior part of the cheese. The conidia in the exterior part of the cheese will germinate with a significantly prolonged lag-phase and a slow development of hyphae, compared to conidia in the interior. This difference in the rate of germination will persist only until the concentration of NaC1 in the exterior part is close to the concentration in the interior. Concerning the further growth of P. roqueforti, the aw values determined in the surface layer of, e.g., Danablu and Mycella, indicate that mycelial growth will not occur in the surface layer, which might be of importance with regard to the possible differences in enzymatic activity of conidia and mycelium. Yeast
It is not widely appreciated that yeasts can be an important component of the microbiota of many cheese varieties. However, yeasts form a substantial part of the microbiota in surface-ripened cheeses (Eliskases-Lechner and Ginzinger, 1995; Bockelmann and Hoppe-Seyler, 2001), white-mould cheeses (Schmidt and Lenoir, 1980a,b) and Blue cheeses (de Boer and Kuik, 1987; Gonzalez de Llano et al., 1992; Roostita and Fleet, 1996a; Gobbetti et al., 1997; van den Tempel and Jakobsen, 1998). Yeasts occur spontaneously in almost all types of cheese, and it is not unusual to find yeast counts as high as 107-108 cfu/g (Fleet, 1990; Viljoen and Greyling, 1995; Tzanetakis et al., 1998). Yeasts seem to originate from the raw milk and, for brine-salted cheeses, from the brine. Investigations have shown that yeasts can be found only at low numbers (<10 cfu/g) in Danablu cheese before brine-salting (van den Tempel, 2000). Changes in the sensory properties of cheese do not become apparent until the yeasts have grown to a population of 105-106 cfu/g (Fleet, 1992), but despite the frequent occurrence of yeasts in Blue cheeses, they seem
Blue Cheese
generally not to cause defects except brown spots (Weichhold et al., 1988; Nichol and Harden, 1993). Origin of yeasts in Blue cheese Raw milk. The predominant yeasts found in raw milk
from four Danablu dairies in Denmark included Debaryomyces hansenii (Candida famata), C. catenulata, C. lipolytica, C. krusei and Trichosporon cutaneum. Yeast populations exceeded 101-104 cfu/ml and a total of 37 isolates were identified (van den Tempel and Jakobsen, 1998). Other authors have also described the presence of D. hansenii (C. famata) in raw milk in Australia (Fleet and Mian, 1987; Fleet, 1990) and a German investigator (Engel, 1986) demonstrated the occurrence of other yeast species, including C. curvata and Saccharomyces spp. Pasteurisation (72 ~ • 15 s) or thermisation (61 ~ • 15 s) generally does not kill yeasts (Vadillo et al., 1987). Fleet and Mian (1987) reported 103 cfu/ml of pasteurised milk, with C. famata as the predominant species, followed by Kluyveromyces marxianus. Brine and the dairy environment. Salting, especially brine-salting, is a source of yeasts (Devoyod and Sponem, 1970; Kaminarides and Laskos, 1992; Eliskases-Lechner and Ginzinger, 1995; van den Tempel and Jakobsen, 1998). The composition and environmental conditions of the brine vary from country to country and from dairy to dairy. In France, for example, the brines used for Blue cheese production are typically 19-20% NaC1 (w/v), pH 4-6 and 13-16 ~ (Seiler and Busse, 1990), whereas brines used in Denmark have a higher NaC1 content (22-23%), a higher temperature (19 ~ and a pH of 4.5 (van den Tempel and Jakobsen, 1998). The environmental conditions in cheese brines select for salt-tolerant yeast species originating mainly from the dairy environment, the brine and the cheeses (Tudor and Board, 1993). Brines used for Danablu production may have a yeast population ranging from 104 to 106 cfu/ml, depending on the dairy (van den Tempel and Jakobsen, 1998). In spire of distinct differences in the composition of the yeast flora among the dairies, D. hansenii (C. famata) was the predominant species in the brines, except from one dairy, where C. globosa predominated. Several brines used for the production of soft surfaceripened cheeses have shown the occurrence of 104-105 cfu/ml, with C. famata as the predominant yeast species (Seller and Busse, 1990). The frequent occurrence of C. famata in brines used for cheesemaking is explained by its high tolerance to salt (Devoyod and Sponem, 1970; Kaminarides and Laskos, 1992; Eliskases-Lechner and Ginzinger, 1995). Other species
181
found were C. catenulata, C. lipolytica, Zygosaccharomyces spp., T. cutaneum and Cryptococcus laurentii. Occurrence and growth of yeasts in Blue cheeses
Yeasts develop spontaneously during the manufacturing, ripening and storage of Blue cheeses. Their occurrence is not unexpected because of their tolerance to low pH, elevated salt concentrations and low storage temperatures (Fleet, 1990). Furthermore, high concentrations of lactate, residual unfermented carbohydrates and small amounts of citric and acetic acids will assist the growth and prevalence of particular species of yeast. Blue cheese like Roquefort, made traditionally from raw milk, may reach a population of 107-108 and 105-106 cfu/g on the surface and in the interior, respectively, before brine-salting (Besancon et al., 1992). The same investigators showed that the yeast population on the surface decreases significantly (99%) after brinesalting, causing changes in the yeast population towards asporogenous yeast forms, C. famata in particular. These results confirm earlier investigations, which also showed a 90% reduction in the yeast flora and changes in yeast population, selecting for very salt-tolerant species, especially Candida spp. (Devoyod and Sponem, 1970; Galzin et al., 1970). The yeast flora in the interior of the cheese remains unaffected by salting during the early period of ripening, due to the slow diffusion of the salt from the surface to the interior of the cheese (Galzin et al., 1970; Hansen et al., 2001). Yeasts start to multiply on the surface of the cheese after a short adaptation period. There is an almost parallel development of the yeast population in the interior of the cheese, but with numbers 100-fold lower (Hansen et al., 2001). This can be explained by the low level of available oxygen and the high level of CO2, which reduce the growth of yeasts (van den Tempel and Nielsen, 2000). All investigators seem to show the predominance of D. hansenii (C. famata) in Blue cheese, except the Greek variety, Kopanisti, in which T. cutaneum seems to dominate over D. hansenii (C. famata) (Kaminarides and Anifantakis, 1989). A survey of the literature on yeasts isolated from Blue cheeses demonstrates the great diversity of the yeast flora (Table 3). In Blue cheeses such as Danablu, the yeast flora develops from a heterogeneous population towards a more homogeneous population as ripening progresses. On day 1 after salting, Danablu contains different species of yeasts, including C. famata, C. lipolytica, Zygosaccharomyces spp., C. rugosa and C. sphaerica. After 28 days of ripening at 10 ~ C. famata was the predominant yeast, reaching 6.2 • 106 and 1.4 • 108 cfu/g in the
182
Blue Cheese
Species of yeast isolated from Blue cheeses
Isolated species
Type of Blue cheese
Debaryomyces hansenii ( Candida famata)
Roquefort 1, 3, 4, Cabrales 2, Gorgonzola 3, 9, Danablu 3, 10, Bleu d'Auvergne 3, Bleu de Bresse 3, Gamonedo 5, Kopanisti 6, Valde6n 7, Australian Blue 8, unknown brand of Blue cheese 8 Roquefort 1, 3, 4, Cabrales 2, Gorgonzola 3, Danablu 3, 10, Bleu d'Auvergne 3, Bleu de Bresse 3, Kopanisti6, Valde6n 7, unknown brand of Blue cheese 8 Roquefort3, Gorgonzola 3, Danablu 3, 10, Bleu d'Auvergne 3, Bleu de Bresse 3, Valde6n 7, Australian Blue 8, unknown brand of Blue cheese 8 Roquefort 1, Cabrales 2, Gorgonzola 9 Danablu 10, Gamonedo 5, Valde6n 7 Kopanisti6, Valde6n 7 Australian Blue 8, unknown brand of Blue cheese8, DanablulO Valde6n7, unknown brand of Blue cheese8 Roquefort 3, Gorgonzola 3, Danablu 3, 10, Bleu d'Auvergne 3, Bleu de Bresse 3 Cabrales 2, Danablu 10 Roquefort 3, Gorgonzola 3, Danablu 3, Bleu d'Auvergne 3, Bleu de Bresse 3, Valde6n 7 Roquefort 3, Gorgonzola 3, Danablu 3, Bleu d'Auvergne 3, Bleu de Bresse 3, Valde6n 7 Roquefort 1, unknown brand of Blue cheese8 Roquefort 3, Gorgonzola 3, Danablu 3, Bleu d'Auvergne 3, Bleu de Bresse 3, Kopanisti6 Kopanisti6, Danablu 10 Australian Blue 8, unknown brand of Blue cheese8 Unknown brand of Blue cheese 8 DanablulO Valde6n 7 Unknown brand of Blue cheese 8 Danablu 10
Kluyveromyces marxianus ( Candida sphaerica) Yarrowia lipolytica ( Candida lipolytica) Pichia spp. Cryptococcus laurentii Rhodotorula spp. Candida catenulata Candida colliculosa Candida lambica Candida rugosa Candida zeylonoides Geotrichum candidum Kluyveromyces marxianus ( C. kefyr) Saccharomyces cerevisiae Trichosporon cutaneum Cryptococcus albidus Candida intermedia Candida norvegensis Candida parapsilosis Candida tropicalis Zygosaccharomyces spp.
1" Devoyod and Sponem (1970) 2 Nufiez et al. (1981) 3: de Boer and Kuik (1987); 4: Besancon et al. (1992); 5 Gonz&lez de Llano et al. (1992); 6: Kaminaride and Anifantakis (1989); 7: L6pez-Dfaz et al. (1995)" 8: Roostita and Fleet (1996a); 9: Gobbetti et al. (1997) 10: van den Tempel and Jakobsen (1998).
interior and on the surface of the cheese, respectively (van den Tempel and Jakobsen, 1998). Examination of Blue cheeses of different origin and age (> 12 weeks) showed that D. hansenii, or its asporogenous form C. famata, dominated in all cheeses examined (Table 4). Strong growth in the presence of salt, growth at a low temperature and the ability to utilise lactate and citrate are likely the key determinants that encourage the predominance of D. hansenii (C. farnata) in cheeses (van den Tempel and Jakobsen, 2000). Another yeast frequently found in Blue cheese produced from raw milk is Kluyverornyces rnarxianus (C. sphaerica). It assimilates and ferments lactose and, due to gas production, could play an important role in the formation of the characteristic open texture of Blue cheese (Lenoir, 1984; Fox and Law, 1991; Roostita and Fleet, 1996a). Furthermore, strains of K. marxianus (C. sphaerica) are able to assimilate lactic and citric acids, and have weak proteolytic and lipolytic properties (Lenoir, 1984; Fleet and Mian, 1987; Besancon et al., 1992; Roostita and Fleet, 1996b). They have, however, been shown to exhibit a pronounced inhibitory effect
on the growth of P. roqueforti (Kaminarides et al., 1992; Hansen and Jakobsen, 1998). A yeast species less frequently found in Blue cheese is Yarrowia lipolytica (C. lipolytica) which is characterised by the inability to ferment carbohydrates or assimilate nitrate and has strong lipolytic and proteolytic properties (Roostita and Fleet, 1996a; Freitas et al., 1999; van den Tempel and Jakobsen, 2000). Non-starter lactic acid bacteria
Non-starter lactic acid bacteria (NSLAB) are found in several cheese varieties during ripening, including Blue cheeses. As in many other cheeses, they are commonly facultatively heterofermentative strains of Lactobacillus, i.e., mainly of the Lb. paracaseilcasei complex and Lb. plantarum. Other NSLAB found in Blue cheese are Lb. fermentum, Lb. brevis, Pediococcus spp. and Leuconostoc spp. (Gonz/dez de Llano et al., 1992; Martley and Crow, 1993; Fox et al., 1996; Gobbetti et al., 1997; LOpez-Diaz et al., 2000). Nonstarter lactic acid bacteria grow in Blue cheese from
Blue Cheese
Occurrence of Debaryomyces hansenfi in selected Blue cheeses
Cheese
Surface, yeast/g cheese
Interior, yeast/g cheese
D. hansenii (%)
Roquefort Petite Fourme Bleu d'Auvergne Cambozola Saint Agur Gorgonzola Fourme d'Ambert
1.7 2.1 1.7 2.7 2.0 3.6 2.1
4.4 2.0 5.5 2.6 1.9 7.3 1.4
74 66 64 70 75 62 70
x • • • • • x
107 107 107 105 107 106 107
x • • • • • x
106 105 106 104 105 103 106
183
Another contaminant frequently found in Blue cheese is Geotrichum candidum (de Boer and Kuik, 1987; Lope> Diaz et al., 1995), which can cause considerable inhibition of the growth of Penicillium spp. (Nielsen et al., 1998; van den Tempel and Nielsen, 2000). G. candidurn has been isolated from Danablu at levels of 102-103 cfu/g, mainly from the interior of the cheese, as G. candidurn is sensitive to salt at concentrations above 1% (Philip, 1985; Lecocq and Gueguen, 1994; van den Tempel and Nielsen, 2000).
Modified from van den Tempel (2000).
10-100 cfu/g after brining to about 107 cfu/g at the end of maturation and are assumed to originate from the raw milk and the dairy plant environment (Nu~ez and Medina, 1979; Gonzalez de Llano et al., 1992; Gobbetti et al., 1997). In Blue cheeses made from raw milk, a high number of Enterococcus spp. have been isolated (Devoyod and Desmazeaud, 1971; Gonzalez de Llano et al., 1992; L6pez-D~az et al., 2000). However, it is not known how normal variations in the composition of the NSLAB flora influence ripening and flavour of Blue cheese. Contaminants
Micro-organisms other than P. roqueforti and LAB starter cultures can colonise and grow well on Blue cheeses, especially on the surface. Spoilage of cheese due to fungal growth is caused by the formation of off-flavours (Sensidoni et al., 1994), mycotoxins and possible discoloration of the cheese (Lund et al., 1998). The most important spoilage fungi of semi-soft cheeses are Penicillium spp., including P. commune and P. nalgiovense (Lund et al., 1995). Species of less importance are P. verrucosum, P. solitum and P. discolor, which were also found in Valdeon, an artisanal Spanish Blue cheese made from raw milk (L6pez-Diaz et al., 1995; Lund et al., 1995; Filtenborg et al., 1996). Of special interest for Blue cheese is the newly discovered species, Penicillium caseifulvum, which is frequently found on Blue cheeses (Lund et al., 1998). P. caseifulvum has to date been found in various Blue cheese dairies in Denmark and France (Lund et al., 1998), where it was isolated from cheese curd (102 conidia/g), brine (101-5 x 102 conidia/g) and from the surface of Danablu (102-103 conidia/g). P. caseifulvum is sensitive to CO2 (van den Tempel and Nielsen, 2000), and therefore grows only on the surface of the cheese, where it can cause discoloration in the form of brown spots.
During the production and ripening of Blue cheese, interactions between the primary starter culture, P. roqueforti, and the mould and yeast contaminants determine the maturation time, aroma, texture and appearance of the final cheese. The mechanisms behind these interactions can be broadly divided into two groups: Antagonism, representing negative interactions caused by antimicrobial metabolites, competition for nutrients or unfavourable changes to the microenvironment. Synergism, representing positive interactions as mutual use and production of nutrients, changes to a more favourable microenvironment and atmosphere composition, degradation of antimicrobial compounds, physical attachments between microorganisms and changes in microstructure. Microbial interactions have been deduced from examinations of Blue cheeses (Kaminarides et al., 1992; Hansen and Jakobsen, 1997; van den Tempel and Jakobsen, 2000; van den Tempel and Nielsen, 2000; Hansen et al., 2001), but there have been only a few detailed studies in this area. In the following, microbial interactions involving P. roqueforti will be described.
Penicillium roqueforti and lactic acid bacteria Positive and negative interactions between LAB and Penicillium spp. have been described (Salvadori et al., 1974; Gripon et al., 1977; Suzuki et al., 1991; Gourama and Bullerman, 1995; Roy etal., 1996; Gourama, 1997; Hansen and Jakobsen, 1997; Hansen, 2001). However, only a limited number of investigations have been carried out to examine how several strains of LAB, both starter and non-starter cultures, affect the growth and sporulation of different strains of P. roqueforti in model cheese systems. The results shown in Table 5 are based on screening of 20 strains of P. roqueforti and 15 strains of LAB in a model cheese system and a
184
Blue Cheese
laboratory substrate (Hansen and Jakobsen, 1997). As shown in Table 5, both negative and positive interactions were demonstrated. Furthermore, the type of interactions observed was related to the composition of the medium used. Positive interactions were stronger and found more frequently in the cheese agar compared to the laboratory medium. The interactions were found to be strain-specific for P roqueforti as well as for the LAB. The positive interactions were seen as faster growth and more pronounced sporulation of P. roqueforti. Further, P roqueforti developed a thicker and more velvet-like mycelium. Negative interactions were seen as a reduction in, or the absence of, the growth of P roqueforti. A synergistic effect on casein breakdown between P roqueforti and LAB has also been observed, indicated by the production of significantly higher amounts of non-protein nitrogen and phosphotungstic acid-soluble nitrogen when the enzymes of the two types of organisms were incubated together compared to the amounts produced by only one of them (Ottogalli et al., 1974). The benefits obtainable by selecting the right combination of cultures are emphasised by results showing that stimulation of the growth and sporulation of P roqueforti was stronger at higher levels of NaC1. The levels of NaC1 investigated corresponded to the concentration found in the surface layer of Blue cheese where the growth of P roqueforti can be limited or even absent. The stimulation 9~ f
.....
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,~,-,~,,,tb,
~,~A
th . . . . . .
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obviously enhance maturation, because both the conidia and the mycelium contribute to proteolysis and lipolysis in the cheese during ripening.
Penicillium roqueforti and yeasts
As for LAB and P roqueforti, selecting the right combinations of yeast and P. roqueforti cultures may stimulate the growth and sporulation of P. roqueforti and thereby enhance ripening and improve the appearance of the cheese in general.
Positive and negative interactions regarding the growth of Penicillium roqueforti in laboratory medium and cheese agar for 300 combinations of strains of P. roqueforti and lactic acid bacteria
Positive interaction Negative interaction No sign of interaction
Cheese agar
Laboratory media
136 49 115
22 195 83
Modified from Hansen and Jakobsen (1997).
Interaction experiments carried out under environmental conditions similar to those in Blue cheese production have demonstrated radial growth of P. roqueforti to be stimulated by selected strains of D. hansenii (van den Tempel, 2000). The mechanism behind the observed positive interactions might be explained by the stimulation of P. roqueforti by D. hansenii caused by the release of nutrients on autolysis due to low survival rates of yeasts at high levels of CO2 (Lumsden etal., 1986; Ison and Gutteridge, 1987; Dixon and Kell, 1989; van den Tempel and Nielsen, 2000). Positive interactions between a strain of Saccharomyces cerevisiae (FB7) and strains of P roqueforti have also been demonstrated (Hansen and Jakobsen, 2001" Hansen et al., 2001). Measurement of radial growth and visual observations of sporulation showed that whole cell inocula of S. cerevisiae promoted faster growth, thicker and more velvet-like mycelia and a more intense blue colour of the conidia. No interactions were seen when the supernatant or the disrupted cells of S. cerevisiae were examined. The mechanism behind the positive fungal-yeast interaction was found to be correlated to a synergistic effect in the breakdown of casein, as shown by capillary electrophoresis. S. cerevisiae FB7 degraded casein, and co-culturing with P roqueforti resulted in a higher number and different patterns of peptides. These findings have been confirmed in a large-scale production ,
oLxv
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FB7 to Mycella gave rise to faster growth and sporulation of P roqueforti, a softer cheese texture and a significantly higher relative concentration of aroma compounds (Hansen et al., 2001). Inhibition of P roqueforti by Y lipolytica has been observed in laboratory trials (van den Tempel and Jakobsen, 2000). The interactions were strain-specific for Y lipolytica as well as for P roqueforti. However, all strains of Y. lipolytica investigated were inhibitory to mycelial growth and sporulation of P roqueforti (van den Tempel, 2000). Competition for nutrients seems to be the most frequently occurring mechanism of interaction between yeasts and moulds in laboratory systems, but this does not exclude co-existence in more natural situations (Boddy and Wimpenny, 1992). The belief that competition for nutrients is the main interspecies mechanism between yeasts (e.g., Y. lipolytica and D. hansenii) and P. roqueforti is based mainly on investigations showing: (i) no inhibition of P roqueforti when using culture supernatant or disrupted cells, (ii) the quick colonisation of yeasts, e.g., Y lipolytica and D. hansenii, on cheese agar, (iii) the quantitative relationship between the numbers of yeasts and inhibition of P roqueforti, (iv) the inhibitory effect of Y lipolytica and D. hansenii being
Blue Cheese
185
Total concentration of fatty acids (FA) in different cheese
absent or reduced by addition of nutrients (van den Tempel, 2000; van den Tempel and Jakobsen, 2000).
varieties
Penicillium roqueforti and contaminants
Variety
FA, mg/kg
Variety
FA, mg/kg
Geotrichum candidum has shown a growth potential similar to P. roqueforti in the absence of salt, indicating a possible overlap between the two species in the interior of the cheese during the initial ripening stage. Contamination of Blue cheese by G. candidurn can cause inhibition of growth and sporulation of P. roqueforti resulting in 'blind spots', which affect the quality of the cheese significantly. This emphasises the importance of good manufacturing practice in the production of Blue cheese to prevent contamination by G. candidum. Studies by Tariq and Campell (1991) showed that G. candidum might compete by antibiosis, as volatile metabolites from arthrospore suspensions of G. candidum were found to inhibit conidial germination and reduce the rate of hyphal extension in different species of fungi, including P. roqueforti. Recent studies by Dieuleveux et al. (1998) demonstrated that G. candidum produces and excretes 2-hydroxy-3phenylpropanoic acid with a broad-spectrum antibacterial effect. Colonisation of the mould contaminant, P. caseifulvum, can occur on the surface of Blue cheese without major inhibition by any of the species investigated, thus causing colour defects on the cheese (Lund et al., 1998). The occurrence of P. caseifulvurn is unlikely to affect the growth and sporulation of P. roqueforti due to their different growth niches in Blue cheese.
Gamonedo c Blue (US) Cab rales b Danablu a Roquefort Parmesan
75685 35230 33153 32639 32453 4993
Provolone Gruyere Brie Cheddar Camembert Mozzarella
2118 1481 1314 1028 681 363
Lipolysis
Lipolysis in Blue cheeses, like proteolysis, is very intense compared to other cheeses. As seen from Table 6, high amounts of free fatty acids are found during the ripening of various kinds of Blue cheeses. In other varieties, this extensive lipolysis would cause a rancid taste, but in Blue cheeses, the free fatty acids are neutralised when the pH increases. In general, the total level of free fatty acids increases with ripening time, especially after the mould has sporulated (Alonso et al., 1987; Madkor et al., 1987b; Contarini and Toppino, 1995; Gobbetti et al., 1997), but a decrease at the end of ripening has also been observed (Prieto et al., 2000). This decrease could be caused by conversion of the fatty acids to methyl ketones. Due to the higher NaC1 concentration in the rind, which inhibits mould growth and thereby lipase production, a lower level of free fatty acids has been observed in the outer part
Adapted from Woo et aL (1984) except: a Unpublished results (Cantor). b Alonso et aL (1987). c Gonz&lez de Llano et aL (1992).
of the cheese compared to the core (Godinho and Fox, 1981c; Gobbetti et al., 1997). This effect can be altered to some degree by selecting more NaCl-tolerant strains of P. roqueforti. Generally, the levels of both saturated and unsaturated long-chained fatty acids (C12:0mC18:3) in Blue cheese is higher than the levels of shortchained fatty acids (C4:0--C10:0), which correspond to results obtained for P. roqueforti grown in butterfat emulsions (Larsen and Jensen, 1999). However, considerable differences in the levels of individual free fatty acids can be found between various types of Blue cheeses (Alonso et al., 1987; Madkor et al., 1987b; Prieto et al., 2000). Degradation of lipids in Blue cheeses is caused mainly by enzymes from P. roqueforti (Kinsella and Hwang, 1976; Coghill, 1979; Gobbetti et al., 1997). The lipolytic activity of commercial strains of P. roqueforti differs significantly, resulting in the release of different amounts of free fatty acids (Farahat et al., 1990; Larsen and Jensen, 1999) and thereby different flavour profiles of the cheeses produced (Farahat et al., 1990; Gallois and Langlois, 1990). P. roqueforti produces two extracellular lipases, an acidic and an alkaline lipase (Menassa and Lamberet, 1982; Lamberet and Menassa, 1983b; Mase et al., 1995). Intracellular lipase activity has also been reported (Niki et al., 1966; Stepaniak et al., 1980), but further research in this area is required. The acidic lipase has a pH optimum at 6.0 and a lesspronounced optimum at 2.8, with maximum stability between 3.7 and 6.0 (Lamberet and Menassa, 1983b). The optimum temperature is 35-40 ~ but it retains 37% of maximum activity at 5 ~ Optimum pH for the alkaline lipase is 8.8-9.0 at 30 ~ and 9.0-10.0 at 20 ~ but activity is retained between 4.5 and 11.0 (Lamberet and Menassa, 1983b), e.g., 15 and 20% of maximum activity is retained at pH 4.5 and 6.0, respectively. The relative importance of the acidic and the alkaline
186
Blue Cheese
lipases in cheese has not been determined fully. However, Lamberet and Menassa (1983a) investigated the lipolytic activity at pH 5.5 on tricaproin (acid lipase) and at pH 8.0 on tributyrin (alkaline lipase) in suspensions of seven French Blue cheeses. Activity at pH 5.5 dominated and only two samples showed measurable activity at pH 8.0. Even though the pH of Blue cheeses in general favours activity of the acid lipase, it should be noted that the alkaline lipase has the higher activity on milk fat (Eitenmiller etal., 1970; Lamberet and Menassa, 1983a,b). P.. roqueforti dominates the overall lipid degradation in Blue cheeses, but other lipolytic agents are also present. The native milk lipoprotein lipase contributes at the beginning of the ripening period, most significantly in Blue cheeses produced from homogenised milk, like Danablu and Stilton (Gripon, 1993). As lipoprotein lipase is almost completely inactivated by pasteurisation, its effect will be the most pronounced in cheeses produced from raw or thermised milk. The LAB, whether they are part of the starter culture or the non-starter microbiota, have very low lipolytic activity and are not likely to influence lipolysis in Blue cheese (El Soda et al., 1986; Meyers et al., 1996). Yeasts probably affect lipolysis, which could be positive (]akobsen and Narvhus, 1996), but this is very dependent upon the yeast species present. Almost all yqr.,..a3L,.~
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S. cerevisiae, Y. lipolytica, C. catenulata and Galactomyces geotrichum) have at least esterase activity, being able to hydrolyse short-chained fatty acids from triglycerides (Fleet and Mian, 1987; Roostita and Fleet, 1996a; Hansen and Jakobsen, 1998; van den Tempel and Jakobsen, 1998). Lipolysis of long-chained fatty acids has been demonstrated for Y. lipolytica, C. catenulata and G. geotrichum and the activity seems to be at the same level for these three yeasts (Roostita and Fleet, 1996b" van den Tempel, 2000). Regarding the release of free fatty acids, an increase has been observed when strains of S. cerevisiae grow in co-culture with P. roqueforti, whereas no effect was observed with strains of D. hansenii (Cantor, unpublished results). Strains of Y. lipolytica have strong lipolytic activity, which could be desirable in Blue cheese, but they also, in general, affect the growth of P. roqueforti negatively. However, these interactions are very strain-specific (Hansen and Jakobsen, 1998; van den Tempel and Nielsen, 2000). Recently, preliminary results indicated a positive effect of lipases from Y. lipolytica on the development of free fatty acids when interacting with P. roqueforti, both in Danablu and in milk (Cantor, unpublished results). This could be used to enhance the quality of Blue cheeses made from pasteurised milk, where lipolysis
and aroma formation are delayed and often weaker, unless the ripening period is prolonged. Proteolysis and amino acid catabolism
Several studies have revealed extensive proteolysis in Blue cheese compared to other cheeses (Marcos et al., 1979; Gonz~lez de Llano etal., 1995; Zarmpoutis et al., 1997). Casein is hydrolysed at more sites and at a considerably higher rate, and there are no intact caseins or primary breakdown products left in the ripened cheese (Marcos et al., 1979; Trieu-Cuot and Gripon, 1983; Fernandez-Salguero et al., 1989; Gonz~lez de Llano et al., 1992; Zarmpoutis et al., 1997). A larger number of different peptides are produced than in semi-hard cheeses (Fig. 3), and a high concentration of amino acids are released as a result of the peptidases, especially from mould and LAB working in concert (Ismail and Hansen, 1972; Gripon et al., 1977; Coghill, 1979; Zarmpoutis et al., 1997). The enzymes contributing to the complicated proteolysis in Blue cheese originate from the milk, rennet, starter and non-starter bacteria, moulds and yeasts, with the main contribution from the mould culture, P. roqueforti (Coghill, 1979). A significant increase in proteolysis has been observed when the mould has become visible in the cheese, typically after 2-5 weeks of maturation, depending on the cheese variety (Trieu-Cuot ~ZLIb.JL
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P. roqueforti is growing out, breakdown of the caseins is performed mainly by rennet (Hewedi and Fox, 1984). The main activity of rennet in cheese is on C~sl-casein to produce Otsl-CN (f24-199) and the peptide O~sl-CN (fl-23) (Table 7) whereas the milk protease, plasmin, hydrolyses [3-casein to ~/-caseins and proteose-peptones mainly during the first day. The cell envelope-associated proteinase of the Lactococcus or Lactobacillus starter culture hydrolyses the peptides produced from casein by rennet and plasmin. A limited release of amino acids by the starter aminopeptidases occurs during these first weeks of ripening. After a couple of weeks, P. roqueforti dominates proteolysis, liberating both peptides and amino acids using a variety of enzymes (Table 7) (Madkor et al., 1987a; Zarmpoutis etal., 1996, 1997). P. roqueforti expresses two extracellular proteases: a metalloprotease and an aspartic protease. The activity of these enzymes is maximal in Blue cheese at the stage when P. roqueforti has grown out and begins to sporulate. Both proteases are rather stable in cheese. The metalloprotease is active at pH 4.5-8.5 and it has an optimum for casein hydrolysis at 5.5, which corresponds to the pH often found in Blue cheese during ripening. The metalloprotease has a broad specificity
Blue Cheese
1.0
187
Otsl-CN (f1-13)
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Proteose-peptones
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55
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Retention time (min) Peptide profiles analysed by RP-HPLC of A: Danablu and B: Semi-hard yellow cheese of similar age (about 3 months). o~-LA: o~-Iactalbumin; 13-LG: 13-1actoglobulin; proteose-peptones: breakdown products from plasmin activity on 13-casein; O~sl-CN (f1-13): breakdown product from Lactococcus protease activity on the rennet-derived peptide otsl-CN (fl-23).
and hydrolyses both Ors1- and [3-caseins. Hydrolysis of Otsl-casein leads to eight peptides with molecular weights ranging from 7000 to 21 000 Da (Trieu-Cuot et al., 1982b). Hydrolysis of [3-casein in buffer gives nine peptides with molecular weights between 13 100 and 21 100 Da (Trieu-Cuot etal., 1982b), of which one has been shown to accumulate in Blue cheese (Le
Bars and Gripon, 1981 Trieu-Cuot and Gripon, 1983). Of special interest is that the metalloprotease cleaves [3-casein at Prog0--Glu91, which is not often hydrolysed by proteases because of the proline residue and that, like plasmin, it cleaves a bond close to Lys28 Lys29 (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982b; Trieu-Cuot and Gripon, 1983).
Main enzymes involved in proteolysis and amino acid release during ripening of Blue cheese with scrubbed surfaces to prevent the development of slime microflora (Gripon, 1993; Ard6, 2001) Enzyme
Specificity in cheese
Plasmin
13-CN and Ots2-CN after basic amino acids (Lys, Arg) Hydrolyses 13-CN to y-CN and proteose-peptones; preferred cleavage sites: 13-CN (28-29, 105-106, 107-108) Hydrolyses OLsl-CN to OLsl-CN (f24-199) and otsl-CN (fl-23)
Chymosin and other coagulants Lactococcal lactocepin Lactococcal peptidases NSLAB, peptidases R roqueforti aspartic protease R roqueforti metalloprotease R roqueforti serine carboxypeptidase (extracellular, acid) R roqueforti metalloaminopeptidase (extracellular, alkaline) Yeast CN, casein.
Hydrolyses peptides produced from casein by the action of plasmin, rennet or R roqueforti Produces different peptides from OLsl-CN (fl-23) depending on the lactococcal strain Releases amino acids from smaller peptides Broad-specificity aminopeptidase (e.g., PepN), PepC, PepX and dipeptidase specificity Contribute to the release of amino acid Hydrolyses 13-CN preferentially to produce 13-CN (98-209, 30-209, 1-29, 100-209, 1-97/99) Hydrolyses Otsl-CN Broad specificity Releases acidic, basic and hydrophobic amino acids Releases apolar amino acids (not next to Gly) Large variation between strains from no to excessive proteolytic activity
188
Blue Cheese
The aspartic protease is stable at pH 3.5-6.0 and has two optimal pH values for hydrolysis of casein, 3.5 and 5.5, which may be explained by conformation changes in the substrate. Casein is hydrolysed into mainly high molecular weight peptides and it does not hydrolyse di- or tri-peptides (Modler et al., 1974; Le Bars and Gripon, 1981). The first peptide released by the aspartic protease from %l-casein in solution corresponds in isoelectric point and molecular weight to Otsl-CN (f24-199), indicating specificity similar to chymosin (Trieu-Cuot etal., 1982a; Larsen etal., 1998). Later, this peptide is further hydrolysed to 4-5 new peptides. The aspartic protease hydrolyses [3-casein into five peptides; initially the peptides ~-CN (D8-209), (f30-209) and (fl-29) are released and then the peptides [3-CN (f100-209) and (fl-97/99) (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982a). In Blue cheese, [3-CN (f98-209) has been shown to accumulate (Houmard and Raymond, 1979" Le Bars and Gripon, 1981" Trieu-Cuot and Gripon, 1983). Le Bars and Gripon (1981), who compared electrophotograms of commercial Blue cheeses with those of sterile curds inoculated with either E roqueforti or purified aspartic protease, found that the patterns were very similar, indicating the great importance of P. roqueforti, and especially of its aspartic protease, for proteolysis in Blue cheese during ripening. A relationship has been established between the .-1 . . . . 1. . . . . U~V ElOplll~lll.
,.
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protease and the release of bitter peptides (Gripon, 1993). TO break down these bitter peptides, as we]] as other peptides, P roqueforti possesses several exopeptidases. An extrace]]u]ar acid carboxypeptidase, with a broad specificity, releases acidic, basic and hydrophobic amino acids and may be important in the debittering process. It is a serine enzyme with a pH optimum for hydrolysing an artificial substrate at pH 3.5, and it is stable at pH 5.0-5.5. P. roqueforti also produces an extracellular alkaline metalloaminopeptidase with a pH optimum of 8.0. It is specific for hydrophobic amino acids, and consequently, the debittering activity of P. roqueforti may increase with pH in Blue cheese. Several intracellular peptidases have also been detected, among them are alkaline carboxy- and amino-peptidases (Gripon, 1993), but their contribution to ripening is not known. The proteolytic activity, as well as the level of proteases and peptidases produced by P. roqueforti, varies greatly between strains (Larsen et al., 1998). The growth of mould within a pierced Blue cheese leads to an increase in pH that stimulates other proteolytic activities in the cheese, such as the LAB cell wall protease (lactocepin) and the milk protease, plasmin. At this later stage of ripening, hardly any [3-casein
remains in Blue cheese and plasmin activity is limited to any remaining peptides containing its specific cleavage sites. Salt inhibits the development P. roqueJorti and therefore also its proteolytic activity, which explains the hard and rather tasteless zone close to the rind of Blue cheese (Godinho and Fox, 1982). Yeasts in Blue cheeses belong mainly to the genus Candida (Table 3). Proteolytic activity, mainly intracellular, has been detected for a few Candida strains, but this property is poorly documented for larger number of strains within the same species (Pereira-Dias et al., 2000" Klein et al., 2002). Activity on casein at 10 ~ was not shown by any of six tested strains of D. hansenii originating from Blue cheese (van den Tempel and Jakobsen, 2000). However, in the same experiment, five of six strains of Y. lipolytica isolated from Blue cheese digested all the caseins at 10 ~ showing that yeast may contribute to proteolysis, e.g., a specific strain of S. cerevisiae used as an adjunct culture in Mycella was shown to enhance proteolysis and texture in the cheese (Hansen et al., 2001). However, an excessive contribution to proteolysis may cause a detrimental effect on the cheese. Aminopeptidase activity on branched-chain amino acids was shown for all strains of both D. hansenii and Y. lipolytica, and because yeasts grow to large numbers in many Blue cheeses, this activity may contribute to the release of amino acids during ripening (Klein et al., 2002). l~T
liglLttL
r
Uc:lLt[llr
LIIalVE
IL_J~LL
I ~ U I O . LI~.U
from Blue cheese and could, as in other cheeses, be expected to take advantage of the large amount of small peptides produced by the other micro-organisms present and produce mainly similar aroma compounds from amino acids as the starter bacteria. Amino acids, which are released at high amounts in Blue cheeses (Zarmpoutis et al., 1997), contribute to a background flavour, but further catabolism is needed to produce several aroma compounds characteristic of cheese (Hemme et aI., 1982" Yvon and Rijnen, 2001). However, the specific characteristic flavours of Blue cheese originate not from amino acids, but from lipolysis and a significant production of methyl ketones. Cheese flavour compounds are produced by LAB and moulds through amino acid catabolism (Hemme et al., 1982; Yvon and Rijnen, 2001). The free amino acids found most commonly in Blue cheese are glutamic acid, leucine, valine and lysine (Madkor et al., 1987b" Zarmpoutis et al., 1996, 1997). The metabolic pathways of LAB, starting with aminotransferase activity, dominate in hard and semi-hard cheeses, all of which have a low redox potential (ArdO et al., 2002). These activities are not very well studied in Blue cheese, but they are present. Oxidative deamination of amino acids may be performed by P. roqueforti within
Blue Cheese
the cheese and microbial flora on the cheese surfaces (cf. 'Bacterial Surface-Ripened Cheese', Volume 2). This activity produces ammonia in amounts that contributes to the flavour of Blue cheeses. Compounds resulting from different pathways of amino acid catabolism have been found in Blue cheese (as reviewed by Gripon, 1993). Glutamic acid is decarboxylated to y-aminobutyric acid (GABA) and CO2, and other amino acids are decarboxylated to amines and CO2 by P. roqueforti as well as by adventitious micro-organisms in and on Blue cheese. The concentrations of amines vary greatly and tyramine is usually observed in higher amounts than tryptamine and histamine (de Boer and Kuik, 1987). Catabolism of arginine to ornithine and citrulline has been shown in Blue cheese. The complex amino acid catabolism in Blue cheese varieties still needs much research to be understood fully. Formation of aroma c o m p o u n d s
A wide range of volatile and non-volatile aroma compounds are produced in Blue cheese during ripening, primarily by P. roqueforti, infuencing both the taste and the aroma of the final product. The varying proportions of these compounds determine the specific flavour profiles of the different Blue cheeses (Gallois and Langlois, 1990). A general overview of the different aroma compounds produced, their concentrations and characteristics will be given here. For more detailed information, excellent reviews have been published in recent years (Molimard and Spinnler, 1996; Sable and Cottenceau, 1999; McSweeney and Sousa, 2000). The characteristic favour and taste of Blue cheeses stems mainly from lipid degradation. Free fatty acids contribute both to the taste and the aroma, but even more important are the compounds produced from
189
them, e.g., the methyl ketones, which are essential for the sensory quality of Blue cheeses (Kinsella and Hwang, 1976; Rothe et al., 1994; Moio et al., 2000). As mentioned previously, the lipolytic activity of commercial strains of P. roqueforti differs significantly, resulting in different amounts of free fatty acids produced (Lopez-Diaz et al., 1996b; Larsen and Jensen, 1999) and thus leading to the different flavour profiles of the cheeses (Table 8) (Farahat et al., 1990; Gallois and Langlois, 1990). Most volatile fatty acids (C4:0-C12:0) have fairly low threshold values, rather pungent or rancid flavour notes, and are usually present at fairly high concentrations in Blue cheeses, but because of the high pH of Blue cheese, these acids are neutralised and hence contribute to the aroma of the cheese and not to a rancid defect (Molimard and Spinnler, 1996). Hexanoic and octanoic acids are especially important favour compounds (Rothe et al., 1994; Molimard and Spinnler, 1996; Sable and Cottenceau, 1999). Methyl ketones are the major aroma compounds in Blue cheeses (Table 8) (Day and Anderson, 1965; Dartey and Kinsella, 1971; Ney and Wirotama, 1972; Gallois and Langlois, 1990; Gonz/tlez de Llano et al., 1990; de Frutos et al., 1991; Moio et al., 2000). They have been reported to constitute 50-75% of the total volatile flavour compounds in Blue cheeses (Madkor et al., 1987b; Gallois and Langlois, 1990; de Frutos et al., 1991; Moio et al., 2000; Hansen et al., 2001), and their concentration in the cheese can be correlated to the intensity of a 'Blue cheese' note (Rothe et al., 1982, 1986, 1994). The methyl ketones found at the highest concentrations are 2-heptanone and 2-nonanone, but 2-pentanone and 2-undecanone are also important (Madkor etal., 1987b; Gallois and Langlois, 1990; Gonz/tlez de Llano et al., 1990; de Frutos et al., 1991; Contarini and Toppino, 1995). The total concentration
Total concentration (~g/kg cheese) of major groups of aroma compounds in Blue cheeses produced with different strains of R roqueforti Aroma
compounds Ketones Alcohols
Esters Lactones Aldehydes
Roquefort strain PF a
strain PO b
Roquefort
Roquefort strain PG c
Bleu de Causses d strain PG c
Bleu d'Auvergne e unknown strain
11 095 4 025 1 390 50 5
14 350 3 305 2 985 255 10
34 940 7 670 3 835 325 15
9345 3795 3155 425 0
9780 8110 2950 2230 250
Modified from Gallois and Langlois (1990). a b c d e
Low proteolytic/Iow lipolytic activity; 210 days ripening. High proteolytic/high lipolytic activity; 210 days ripening. Medium proteolytic/medium lipolytic activity; 210 days ripening. 100 days ripening. Approximately 45 days ripening.
190
Blue Cheese
of methyl ketones in Blue cheese depends on manufacturing procedure, ripening time and the strain of E roqueforti used (Table 8), whereas the proportions of the individual methyl ketones in the cheese depend mainly on the strain of P. roqueforti used (Gallois and Langlois, 1990). The odour impressions of the methyl ketones are, in general, fruity, floral and musty and, specifically for 2-heptanone, spicy and 'Blue cheese' (Molimard and Spinnler, 1996; Sable and Cottenceau, 1999). As increasing concentrations of free fatty acids have been shown to inhibit the growth of E roqueforti and thereby retard lipolysis, the formation of methyl ketones from free fatty acids has been proposed to be a detoxifying mechanism (Kinsella and Hwang, 1976). Methyl ketones with one less carbon atom are produced via part of the 6-oxidation pathway from the corresponding fatty acids. The first intermediate is a ]3-keto acyl-CoA which is converted to a [3-keto acid by a thiohydrolase and then decarboxylated to a methyl ketone and CO2. However, at low concentrations, the fatty acids enter the Kreb's cycle and are completely oxidised to CO2 (Molimard and Spinnler, 1996). Both conidia and mycelia are capable of producing methyl ketones (Fan etal., 1976). The majority of methyl ketones are derived directly from their fatty acid precursor, but the concentration of certain methyl ketones, e.g., 2-heptanone and 2-nonanone, is generO.11~
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precursors, C8:0 and C10:0 (Dartey and Kinsella, 1973a; Madkor et al., 1987b). Dartey and Kinsella (1973a,b) showed that these methyl ketones can also be produced from longer-chain fatty acids. Alcohols have been reported to represent between 15 and 30% of the total volatile flavour compounds in Blue cheese (Table 8) (Gallois and Langlois, 1990; Moio et al., 2000). Methyl ketones can be reduced to secondary alcohols, under anaerobic conditions, and these alcohols are generally more abundant in Blue cheese than primary alcohols. The main secondary alcohols are 2-heptanol, 2-nonanol and 2-pentanol, depending on the cheese type and strain of E roqueforti used (Gallois and Langlois, 1990; GonzMez de Llano et al., 1990). Their flavour is more or less similar to the corresponding methyl ketones, but at higher concentrations they can give a musty or mouldy impression (Kinsella and Hwang, 1976). Primary alcohols are also present, with 3-methyl-l-butanol being the most abundant (Gallois and Langlois, 1990; Moio et al., 2000). Other aroma compounds with fruity and floral notes are esters and lactones; the latter are usually found at low concetrations (Table 8) (Gallois and Langlois, 1990; Gonzalez de Llano etal., 1990). The higher
amount of lactones detected in Bleu d'Auvergne (Table 8) could be due to the use of pasteurised milk, as pasteurisation of milk has been shown to increase the level of lactones. Esters are believed to be formed in the cheese by microbial esterification of free fatty acids with alcohols. Apart from influencing the overall aroma profile of Blue cheese, the main contribution of esters is possibly by minimising the sharpness and bitterness arising from fatty acids and amines (Anderson and Day, 1966). Peptides and amino acids from proteolysis yield compounds which are important for the background flavour of the cheese and furthermore contribute with their own flavour, e.g., sweet, bitter, brothy (Nishimura and Kato, 1988; Yvon and Rijnen, 2001). Aldehydes are found at low levels (Anderson and Day, 1966; Ney and Wirotama, 1972; Gallois and Langlois, 1990; Table 8), but their impact on flavour is not known. The same is true for the volatile and non-volatile amines (Ney and Wirotama, 1972; Adda and Dumont, 1974), but it is believed that amines have an influence on the overall flavour sensation. The impact of sulphurcontaining compounds on Blue cheese flavour has not been investigated in detail, but they are presumed to make an important contribution (Kinsella and Hwang, 1976; Gallois and Langlois, 1990). Apart from P. roqueforti, yeasts also might contribute to the formation of aroma compounds, either directly, U~
1-/t U U LI ~.. l t l ~
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encing, e.g., growth, and thereby enzyme production of E roqueforti. The use of S. cerevisiae FB7 as an adjunct culture in Mycella cheese leads to an increase in the concentration of aroma compounds in the experimental cheeses compared to the reference cheeses without added yeast (Hansen et al., 2001). Positive effects on aroma production with certain strains of Y. lipolytica growing together with E roqueforti in a model cheese system has also been indicated, but this effect is very strain-specific regarding both the yeast and the mould (Cantor, unpublished results). Production and occurrence of mycotoxins
Penicillium roqueforti produces a range of secondary metabolites, or mycotoxins, like PR-toxin (Penicillium roqueforti-toxin) and its precursors, eremofortin A, B and C, the alkaloids roquefortine A, B and C and the isofumigaclavines, the marcfortines and mycophenolic acid (Scott, 1981; see 'Toxins in Cheese', Volume 1, for further discussion of mycotoxins). The occurrence of these metabolites in Blue cheese is shown in Table 9. The sesquiterpene PR-toxin is the most toxic of the secondary metabolites, inhibiting nucleic acid and protein syntheses, being cytotoxic in human and
Blue Cheese
191
Mycotoxins, produced by R roqueforti, detected in commercial Blue cheeses
Mycotoxin PR-toxin
PR-imine Roquefortine
Isofumigaclavine A Isofumigaclavine B Mycophenolic acid
No. of cheeses examined
No. of positive samples
13 60 30 60 10 16 12 13 30 16 16 32 100 10
0 0 0 50 1 16 12 13 30 13 6 4 38 0
Concentration ranges ( mg/kg)
0.019-0.042 nr a 0.05-6.8 0.16-0.65 0.2-2.29 0.05-1.47 traces-4.7 traces 0.25-5 nra-14.3
Detection limit reported ( mg/kg )
Reference
0.2 0.0015 nra 0.001 0.03 0.03-0.05 0.016 0.05 nr a 0.015-0.025 nra 0.075 0.02 0.02
1 2 9 2 3 4 5 6 9 4 4 7 8 3
a Not reported. 1: Engel and Prokopek (1979); 2: Siemens and Zawistowski (1993); 3: L6pez-Dfaz et aL (1996a); 4: Scott and Kennedy (1976); 5: Ware et aL (1980); 6: Schoch et al. (1984); 7: Engel et al. (1982); 8: Lafont et aL (1979); 9: Finoli et al. (2001).
porcine cell lines and in rat liver, in addition to being mutagenic (cited by Scott, 1981). Many strains of P. roqueforti used commercially as starter cultures or isolated from Blue cheeses have the ability to produce PR-toxin (Orth, 1976; Wei and Liu, 1978; Engel and Prokopek, 1979; Medina etal., 1985; Chang etal., 1991; Boysen et al., 1996; Geisen et al., 2001) or one or more of its precursors, eremofortin A, B or C (Moreau et al., 1980; Chang et al., 1991; Geisen et al., 2001) in synthetic media. Fortunately, PR-toxin is unstable in the cheese environment and is converted to the less-toxic PR-imine, which is also unstable, and PR-amide in the presence of basic and neutral amino acids (Scott and Kanhere, 1979; Chang et al., 1993). Furthermore, the optimum conditions for PR-toxin production, a high sugar content in the medium, a pH close to 4.5 and aeration, are far from the conditions prevailing in Blue cheese. PR-toxin has never been detected in commercial Blue cheeses or in experimental Blue cheeses made with known toxin-producing strains (Engel and Prokopek, 1979; Scott and Kanhere, 1979). Roquefortine C is a typical metabolite of P. roqueforti and P. carneum (Medina et al., 1985; Boysen et al., 1996; Lopez-Diaz et al., 1996a), found very often in Blue cheese. Isofumigaclavine A and its stereoisomer, fumigaclavine A, is also a characteristic secondary metabolite for P. roqueforti (Scott et al., 1976; Boysen et al., 1996; Geisen et al., 2001). Data on the biological activity of these alkaloids are scarce; the only reported toxicity values are a LDs0-value of 169-189 mg roquefortine/kg body weight and 340 mg isofumigaclavine A/kg body weight after intraperitoneal administration to mice (Ohmomo et al., 1975; Arnold et al., 1978).
Mycophenolic acid is not always produced by strains of P. roqueforti. Boysen et al. (1996) and Geisen et al. (2001) found that c. 50% of the strains investigated produced this metabolite, Engel etal. (1982) found it for 25% of the strains and LOpez-Diaz et al. (1996a) detected mycophenolic acid from only one strain out of nine. In contrast, Lafont et al. (1979) reported that all 16 strains of P. roqueforti investigated produced mycophenolic acid. The LDs0-values determined for mycophenolic acid is high, 2500 and 700 mg/kg for mouse and rat, respectively, but subacute toxic effects have been observed for monkeys and rats (Carter et al., 1969; Scott, 1981). However, taking into account the very low levels and the relatively low toxicity of the various mycotoxins present in the cheese, even large consumption of Blue cheese does not pose a risk to the health of the consumer.
Blue cheese is a very complex food ecosystem, with marked pH and NaC1 gradients and variable, but generally, low levels of 02 and CO2. This heterogeneous microenvironment creates different habitats on the surface and in the core of the cheese, which select for specific micropopulations. The technological characteristics of the LAB of the starter culture and the P. roqueforti culture have significant influences on the quality of the cheese. The primary LAB culture must be able to reduce the pH and survive phage attack, as acidification of the cheese milk is essential for the renneting of the milk and syneresis of the curd, and thereby the fundamental part of the
192
Blue Cheese
cheesemaking process. The secondary culture, P. roqueforti, is often chosen with regard to its proteolytic and lipolytic activities, depending on the type of Blue cheese, the targeted market and the desired shelf-life. The proteolytic activity of the strain of P. roqueforti used is extremely important for texture development, while the lipolytic activity determines the flavour profile. Next, the culture is chosen for its tolerance to NaC1, its growth rate and sporulation capacity. The right combination of these activities and characteristics is crucial for the development of a high quality product. It could, however, also be beneficial to include the interaction with the LAB as a factor and it should be considered whether to make use of the possible synergies in practice and to avoid antagonistic effects. Yeasts should also be considered as potential adjunct cultures as they are present in the cheese and have interesting technological characteristics. There are two major objectives in using yeasts as adjunct cultures in the production of Blue cheese: i) to secure the microenvironment by assimilating residual carbohydrates and organic acids, thereby promoting the growth of desired cultures and inhibiting the growth of spoilage and pathogenic micro-organisms, ii) to contribute directly to the desired cheese quality by their enzymatic activity and by stimulating P. roqueforti. But very careful selection is crucial to avoid undesirable antagonistic interactions between the different cmtuie~ anu to avotu tn~ p~oduction of pigm~ttt~, undesirable aroma compounds and uncontrolled enzymatic activity. A few selected yeasts will be described as potential adjunct cultures for Blue cheese. However, it is important to remember that several of their technological characteristics are strain-specific and cannot be seen as a general characteristic of the yeast species. Although D. hansenii is the yeast species most frequently isolated from Blue cheese (Tables 3 and 4), it is rarely used as an adjunct culture and only a few applications have been reported, e.g., as a surface culture in the production of Roquefort (Besancon et al., 1992). D. hansenii is very weakly proteolytic and has low lipolytic activity. The strains do not enhance proteolysis, but they might alter the aroma profile slightly without changing it significantly. The potential use of D. hansenii as an adjunct culture seems to be linked with its osmo-tolerance and good growth in Blue cheese. Furthermore, it can create a stable microenvironment which protects against undesired microbial growth by assimilation of residual carbohydrates and organic acids (van den Tempel and Jakobsen, 2000; van den Tempel and Nielsen, 2000). The potential of Y lipolytica as a ripening culture in cheese has been evaluated by Guerzoni etal. ___1
. . . . . . . . .
J
. . . . . .
" ..I
.L
(1998). It was demonstrated that Y lipolytica possesses some of the essential properties for use as an adjunct culture" i) ability to grow and compete with other naturally occurring yeasts, such as D. hansenii and S. cerevisiae, even though it assimilates only galactose and lactate, ii) compatibility with and possible stimulation of LAB when co-inoculated, and iii) its remarkable lipolytic and proteolytic activities. Y lipolytica is relatively salt-tolerant and its potential role as an adjunct culture in Blue cheese is linked mainly with early lipolysis at a time when the lipases from P. roqueforti are not present in significant amounts, but it may also contribute to proteolysis (van den Tempel and Jakobsen, 2000). Y lipolytica could be a potential adjunct culture, but should be controlled very carefully because of its strong enzymatic activity, its inhibitory effect towards P. roqueforti and its ability to discolour the cheese (Weichhold etal., 1988; Nichol and Harden, 1993). However, unpublished results (Cantor) indicate that the addition of lipase from Y lipolytica to the cheese milk could aid lipolysis in Blue cheeses made from pasteurised milk by assuming the role of the indigenous milk lipase. Strains of S. cerevisiae can stimulate the release of fatty acids by P. roqueforti, and a synergistic effect between P. roqueforti and S. cerevisiae has been demonstrated in the degradation of casein and the formation of aroma compounds (Hansen and Jakobsen, 2001; nm.t~,~c.t+t e~ uL., ~.uul . S. cerevisiae ~ . m L a~,,~,,~t~ ,csidual glucose, galactose and lactate. It has a relatively low tolerance to NaC1 and would be a suitable yeast culture only for the production of Blue cheese with a low level of NaC1, such as Gorgonzola or Mycella. The purpose of using S. cerevisiae as an adjunct culture would be to make a controlled contribution to aroma formation and proteolysis, as well as creating a stable microenvironment. For all cultures, whether they are already in use or under consideration as adjunct cultures, a thorough screening of their technological characteristics is extremely important. Only a few selected characteristics have been investigated and described to date, but with the new knowledge, especially on microbial interactions, new possibilities for applying and combining cultures in different ways have become available.
A number of aspects of Blue cheese ripening have been discussed in this chapter, but with the space available not all issues could be addressed. The importance of P. roqueforti for the quality of Blue cheese is indisputable, but newly gained knowledge on the microbial interactions and the importance of the adventitious
Blue Cheese
microbiota point at new possibilities for improving existing Blue cheeses or developing new varieties. Adding yeast as adjunct cultures would be an obvious opportunity for diversifying the product range, but there is a strong need for further study of interactions with the other cultures present, not only concerning growth, but also on enzymatic activity. Progress has also been made in understanding the complex mechanisms of ripening, but further research is required, especially concerning proteolysis, e.g., on patterns of casein breakdown caused by P. roqueforti proteases in vitro and in different Blue cheeses. Furthermore, the amino acid metabolism in Blue cheeses still needs research to be fully understood. This could be useful for clarifying the effect of individual cultures on proteolysis and thereby on structure development and taste. Research on texture development and its relationship with the proteolytic activity of P. roqueforti together with texture analysis of Blue cheese is very limited. It would, however, be a valuable tool for further characterisation of the cheese itself and the effect thereon of the microbiota, especially the strain of P. roqueforti.
Adda, J. and Dumont, J.P. (1974). Les substances responsables de l'ar6me des fromages /t p~te molle. Lait 54, 1-21. Alonso, L., Juarez, J., Ramos, M. and Martin-Alvarez, P.J. (1987). Overall composition, nitrogen fractions and fat characteristics of Cabrales cheese during ripening. Z. Lebensm. Unters. Forsch. 185,481-486. Anderson, D.E and Day, E.A. (1966). Quantitation, evaluation and effect of certain microorganisms on flavor components of blue cheese. J. Agric. Food Chem. 14, 241-245. Ard6, Y. (2001). Cheese Ripening: General Mechanisms and Specific Cheese Varieties. Bulletin 369. International Dairy Federation, Brussels. pp. 7-12. Ard6, Y., Thage, B.V. arid Madsen, J.S. (2002). Dynamics of free amino acid composition in cheese ripening. Aust. J. Dairy Technol. 57, 109-115. Arnold, D.L., Scott, RM., McGuire, RE, Harwig, J. and Nera, E.A. (1978). Acute toxicity studies on roquefortine and PR toxin, metabolites of Penicillium roqueforti, in the mouse. Food Cosrn. Toxicol. 16,369-371. Besancon, X., Smet, C., Chabalier, C., Rivemale, M., Reverbel, J.R, Ratomahenina, R. and Galzy, R (1992). Study of surface yeast flora of Roquefort cheese. Int. J. Food Microbiol. 17, 9-18. Bockelmann, W. and Hoppe-Seyler, T. (2001). The surface flora of bacterial smear-ripened cheeses from cow's and goat's milk. Int. Dairy J. 11,307-314. Boddy, L. and Wimpenny, J.W.T. (1992). Ecological concepts in food microbiology. J. AppI. Bacteriol. 73, 23S-38S.
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og Aspergillus). Licentiatafhandling. Laboratoriet for Levnedsmiddelindustri, DTH, Kobenhavn. Gallois, A. and Langlois, D. (1990). New results in the volatile odorous compounds of French cheeses. Lait 70, 89-106. Galzin, M., Galzy, P. and Bret, G. (1970). Etude de la flore de levure dans le fromage de Roquefort. Lait 50, 1-37. Geisen, R., Larsen, M.D., Hansen, T.K., Holzapfel, W.H. and Jakobsen, M. (2001). Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing. Int. J. Food Microbiol. 65, 183-191. Gobbetti, M., Burzigotti, R., Smacchi, E., Corsetti, A. and De Angelis, M. (1997). Microbiology and biochemistry of Gorgonzola cheese during ripening. Int. DairyJ. 7, 519-529. Godinho, M. and Fox, P.E (1981a). Effect of NaC1 on the germination and growth of Penicillium roqueforti. Milchwissenschaft 36, 205-208. Godinho, M. and Fox, P.E (1981b). Ripening of Blue cheese: salt diffusion rate and mould growth. Milchwissenschaft 36,329-333. Godinho, M. and Fox, P.E (1981c). Ripening of Blue cheese: influence of salting rate on lipolysis and carbonyl formation. Milchwissenschaft 36,476-478. Godinho, M. and Fox, P.E (1982). Ripening of Blue cheese: influence of salting rate on proteolysis. Milchwissenschaft 37, 72-75. Gonz/dez de Llano, D., Ramos, M., Polo, C., Sanz, J. and Martinez-Castro, I. (1990). Evolution of the volatile components of an artisanal blue cheese during ripening. J. Dairy Sci. 73, 1676-1683. Gonz/tlez de Llano, D., Ramos, M., Rodriguez, A., Montilla, A. and Juarez, M. (1992). Microbiological and physicochemical characteristics of Gamonedo blue cheese during ripening. Int. Dairy J. 2, 121-135. Gonz~lez de Llano, D., Polo, C.M. and Ramos, M. (1995). Study of proteolysis in artisanal cheeses: high performance liquid chromatography of peptides. J. Dairy Sci. 78, 1018-1024. Gourama, H. (1997). Inhibition of growth and mycotoxin production of Penicillium by Lactobacillus species. Food Sci. Technol. 30, 279-283. Gourama, H. and Bullerman, L.B. (1995). Antimycotic and antiaflatoxigenic effect of lactic acid bacteria: a review. J. Food Prot. 57, 1275-1280. Gripon, J.C. (1993). Mould-ripened cheeses, in, Cheese: Chemistry, Physics and Microbiology. Vol. 2. Major Cheese Groups, 2nd edn, Fox, P.E, ed., Chapman & Hall, London. pp. 111-136. Gripon, J.C., Desmazeaud, M.J., le Bars, D. and Bergere, J.L. (1977). Role of proteolytic enzymes of Streptococcus lactis, Penicillium roqueforti and Penicillium caseicolum during cheese ripening. J. Dairy Sci. 60, 1532-1538. Guerzoni, M.E., Gobbetti, M., Lanciotii, R., Vannini, L. and Chaves LOpez, C. (1998). Yarrowia lipolytica as potential ripening agent in milk products. Yeasts in the Dairy Industry: Positive and Negative Aspects, Proceedings of IDF Symposium, Copenhagen, 1996. pp. 23-33. Hansen, T.K. (2001). Microbial Interactions in Blue Veined Cheeses. PhD Thesis, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Blue Cheese
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Bacterial Surface-ripened Cheeses N.M. Brennan and T.M. Cogan, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland M. Loessner and S. Soberer, Abteilung Mikrobiologic, ZIEL, Technical University of Munich, Germany
Many cheeses are characterized by the development of microbial growth on their surfaces during ripening. These are called surface-ripened cheeses and are subdivided into mould-ripened and bacterial-ripened cheeses, depending on the major micro-organisms involved. Mould surface-ripened cheeses include the well-known varieties, Brie and Camembert. Bacterial surface-ripened cheeses are less well-known and include Beaufort, Brick, Butterk/~se, Comte, Epoisse, Esrom, Gruyere, Havarti, Italico, Limburger, Livarot, Mont d'Or, MOnster, Pont l'Eveque, Port du Salut, Reblochon, Serra da Estrela, Taleggio, Tetilla, Tilsit and Trappist. Bacterial surface-ripened cheeses are also called smear-ripened cheeses, because of the glistening appearance of the cheese surface, washed-rind cheeses, because their rind is washed several times with brine during ripening or red-smear cheeses, because of the red colour which characteristically develops on the surface of these cheeses. Bacterial-ripened cheeses are produced extensively in Austria, Belgium, Germany and France, but they are much less important in English-speaking countries. While these cheeses are made using lactic acid bacteria (LAB) as starters, their flavour is determined primarily by the growth of the surface microflora. The biochemical activity of these microflora results in the development of a cabbagy, garlicky or putrid flavour during ripening, due mainly to the production of sulphur compounds from methionine, particularly methanthiol (Adda etal., 1978; Hemme etal., 1982; Ferchichi et al., 1985; Manning and Nursten, 1985). Sulphur compounds have been identified in many cheese varieties and their importance in smear-ripened cheeses appears to be accentuated by their high concentration at the surface; interactions between the sulphur compounds generate the typical cheese flavour (Ferchichi et al., 1985; Gripon et al., 1991). The microbial composition of the smear of these cheeses is dominated by salt-tolerant yeast and Gram-positive bacteria, particularly coryneforms and staphylococci. Bacterial counts during ripening can exceed 109/cm 2 while those of yeasts are generally -107/cm 2. The pH of the surface also increases during ripening due to the catabolism of
lactate and the production of NH3 through deamination of amino acids by the surface micro-organisms. The last review of these cheeses was that of Reps (1993) who evaluated their chemistry, biochemistry and microbiology. Since then, there has been little additional information on the first two aspects, except for the studies of Leclercq-Perlat et al. (2000a,b) (see 'Brevibacterium linens'). The microbiology of the surface microflora of these cheeses is poorly understood and in this chapter we will review what is known about it, how it develops and is controlled during ripening, and the potential of the cheese surface to promote the growth of pathogens.
Manufacture
Typically, mesophilic mixed-strain cultures are used as starters for smear-ripened cheese, and the curds are cooked to a low temperature (<35 ~ are lightly pressed and are usually brine-salted after moulding. Consequently, these cheeses have high moisture contents and are either soft, e.g., Reblochon and Limburger, or semi-hard, e.g., Tilsit and Pont l'Eveque. Beaufort, Comte and Gruyere cheeses are exceptions to this general rule; they are made with thermophilic cultures, are heated to a high temperature, are pressed at a high pressure and, consequently, have low moisture contents. Smear cheeses are normally salted by brining for 4-18 h, depending on the size of the cheese, with smaller cheeses being brined for shorter periods, after which the cheeses are drained for some hours to remove excess brine. Again, Beaufort, Comte and Gruyere cheeses are an exception to this rule as their surfaces are rubbed with dry salt several times throughout ripening. After brining, smear cheeses are either deliberately inoculated with commercial preparations containing different combinations of Brevibacterium linens, Debaryomyces hansenii and/or Geotrichum candidum (Busse, 1989; Hahn and Hammer, 1990) or, in some countries, particularly Germany, young cheeses are smeared or washed with smears from older cheeses. This so-called
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
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Bacterial Surface-ripened Cheeses
'old-young' smearing ensures that all the organisms required for surface ripening are transferred to the young cheeses (Kammerlehner, 1995). The main disadvantage of this approach is that undesirable contaminants, such as Listeria, if they are present on the surface of the old cheeses, will contaminate all the young cheeses (Hahn and Hammer, 1990, 1993). In Austria, B. linens is the only micro-organism deliberately inoculated on the surface of the cheese; all other organisms on the surface of Austrian smear cheeses are adventitious contaminants. The cheeses are ripened at a high relative humidity (RH) and temperature in the range 10-15 ~ for a period ranging from 14 to 63 days (Table 1) and are washed frequently with a brine solution during the early stages of ripening. Sometimes, the cheese surface is inoculated a second time with the desired culture(s). Environmental factors
Environmental parameters like RH, ripening temperature, ripening time, microflora of the cheesemaking equipment, brine, etc. and the frequency of washing of the cheese all influence the development of the cheese microflora and dictate the surface characteristics of the final cheese. It is also likely that interactions occur between these parameters but this has not been studied. The high RH prevents the surface of the cheese from drying out while the relatively high temperature and the duration of ripening promote the growth of micro-organisms present while the wasmng' ensures a uniform distribution of micro-organisms on the cheese surface. Generally, one can see visible growth on the cheese surface within a few days of the beginning of ripening. The range of ripening temperature is relatively narrow and is part of the tradition used to make each cheese. A higher ripening temperature is used for
Gruyere and Comte, to promote the growth of propionic acid bacteria (PAB) in these cheeses. There does not appear to be an obvious correlation between the time, the temperature of ripening and the salt and moisture contents of the different cheeses (Table 1). It has been suggested that the microflora of the brine influence the microflora of the immature cheese (Siewert, 1986) but Eliskases-Lechner and Ginzinger (1995b) could not confirm this. Distribution of the smear is vital, as rapid spreading of the micro-organisms from cheese to cheese ensures uniform ripening and reduces the risk of unwanted contaminants, like moulds, colonizing the cheese surface. Washing the cheese disrupts the micro-habitat and the moulds are brought into direct competition with other microorganisms and are out-competed due to their slow growth rates. As a result, a uniform bacterial smear develops. After 2-3 weeks, the desired microflora has developed, and soft and semi-soft cheeses are then wrapped or transferred to another ripening room at a lower temperature for further maturation. The development of the surface microflora is also influenced by the presence or absence of oxygen. If the shelves on which the cheese is ripened are solid, the cheeses must be turned frequently; infrequent turning will limit the amount of oxygen that reaches the surface of the cheese in contact with the shelf, thereby limiting microbial activity to the upper surface and the ~,ori~notor
~ f t]~o r - h o o r
Physical and chemical characteristics of the cheese
pH, salt and moisture also affect the composition of the surface microflora. Variations in these factors, together with different treatments of the milk (pasteurized or raw), type of starter (indigeneous microflora or
Composition of, and ripening conditions for, some bacterial smear-ripened cheeses
Ripening conditions
Composition Cheese
Moisture %
MOnster Port du Salut Reblochon Taleggio Pont I'Ev~que Limburger
56 56 55 48-50 45-50 45-48 (50% max) 45-55 40-42 (44% max) 38-40 31
Tilsit Brick Beaufort Gruyere
From Robinson (1995).
Salt%
Typical aw
%RH
Temperature (~
Time (days)
-90 -90 -90
18-20 12-18 15 3-4 12-13 10-15 4-10 15 8-12
14-21 42-56 35-42 42-56 28-42 14-21 42-56 3O 14 56-89 120 14-21 21-90
1.1-1.3 2.0 1.8-3.0
0.98 0.98 0.97
80-85 -90
2.5 1.8-2.5
0.96 0.98
90 -90
1.1-1.3
-0.97
-92
8-12 10 15-18
Bacterial Surface-ripened Cheeses
commercial preparations), degree of cooking, pressing and salting of the curd before moulding and frequency of washing during ripening, ripening temperature and RH, and the length of the ripening period, have led to the development of the many different varieties of smear cheeses, few of which have been studied in detail. The pH of a young cheese, after acidification of the cheese curd by the LAB, is about 5.0. Yeasts and moulds can develop at this pH but it is generally felt that the salt-tolerant bacterial flora cannot grow at pH values less than 5.6, or even 6.0. Thus, the yeasts grow during the initial stages of ripening, de-acidifying the cheese surface. The consequent increase in pH allows the subsequent development of the sah-tolerant bacterial flora (see 'Staphylococci and micrococci' and 'Coryneforms'). However, recent data show that many bacterial isolates from the cheese surface can grow at pH 4.9, in the presence of 8% salt (Brennan et al., 2002). Generally, smear cheeses are brine-salted and the salt diffuses into the cheese relatively slowly, resulting in a salt gradient with the highest concentration on the surface (see 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1). The surfaces of some cheeses are dry-salted, e.g., Comte. The salt level, in turn, will reflect the method and the length of brining. The longer the cheese is salted, the higher will be the level of salt on the surface. Both the salt and the moisture levels directly affect the water activity (aw). The moisture content of surface-ripened cheeses varies from 38 to 56 g/100 g and the salt level from 1.1 to 2.5 g/100 g (Table 1). The contribution of other solutes besides salt (e.g., other ions, like Ca, phosphate, etc., and amino acids) to aw is very low, so the aw of cheese calculated from the salt and the moisture contents should be an accurate indicator of how quickly the surface flora will grow. The calculated aw values, based on salt and moisture levels (Table 1), vary from 0.95 for Comte, which is a hard cheese, containing <40% moisture, to 0.98 for M~inster, a soft cheese containing 56% moisture. Smear cheeses generally have a high surface area:volume (SA:V) ratio. The smaller the cheese, the higher will be the SA:V ratio and the larger will be the effect of biochemical changes produced by microorganisms present in the smear. The SA:V ratio also affects the salt level, as smaller cheeses will have more rapid salt diffusion.
201
content on the cheese surface. Little information is available on the initial number of yeast on the cheese surface. In an Austrian study (Eliskases-Lechner and Ginzinger, 1995b), where deliberate inoculation of the cheese surface with yeast is not practised, the number of yeast in cheese from 14 Tilsit cheese plants, 3 days after manufacture, ranged from <100 to >3 • 106/cm 2, with an average of 104/cm 2. There is some evidence that the initial number of yeast also determines the final level. For example, cheese with initial levels of < 10 or 1000 yeast/cm 2 reached final levels of 105 and 8 • 107/cm 2 after 2-3 weeks of ripening, respectively (Eliskases-Lechner and Ginzinger, 1995b). In contrast, in another plant in which an unspecified soft cheese was made, the maximum number of yeast was 107-108/cm 2 despite considerable variation in their initial number. The species of yeast found in several different redsmear cheeses are summarized in Table 2. Generally, D. hansenii and G. candidum are the most important species, with Trichosporon beigelii and Yarrowia lipolytica being important in some cheeses. Considerable variation occurred in the species present on Romadour, and to some extent on Limburger, produced in different factories. The significance of this is unclear. A succession of different species has also been noted at different stages of ripening (Wyder and Puhan, 1999). G. candidium has characteristics of both a yeast and a mould. In the past, it was often called the dairy mould but for the purpose of this review it is considered to be a yeast. During the past 50 years, it has gone through several name changes. In the older literature it is sometimes called Oospora lactis or Oidium lactis. Those strains isolated from soft cheeses grow more rapidly and have higher proteolytic activity than those isolated from hard cheeses. Therefore, two main biotypes can be distinguished based on growth rate and proteolytic activity: the first biotype has a rapid growth rate and a strong proteolytic activity, while the second biotype grows weakly and is only slightly proteolytic (Lenoir, 1984). There is considerable genetic diversity in G. candidum isolates from different French mould- and bacterial-ripened cheeses, including Chevre, Camembert, Livarot, Mont d'Or, Reblochon, Tomme de Savoie, Epoisses, St Nectaire, Pont l'Eveque, Brie, Morbier and some unknown cheeses (Marcellino et al., 2001; Gente et al., 2002). De-acidification
Yeast
The growth of yeast on the surface of cheese is not surprising because of the low pH, relatively low moisture content, low temperature of storage and high salt
The yeasts on the surface of smear-ripened cheese have two major functions: de-acidification and production of compounds which stimulate the growth of the smear bacteria. They metabolize the lactic acid produced by the starter bacteria to CO2 and H20, and deaminate amino acids, producing NH3, both of which
202
Bacterial Surface-ripened
Cheeses
Species of yeast found on the surface of different smear-ripened cheeses a
Teleomorph Candida catenulata Candida intermedia Candida rugosa Candida zeylanoides Cryptococcus laurentii Debaryomyces hansenfi Galactomyces geotrichum Saccharomyces dairenensis Torulaspora delbrueckii Trichosporon beigelii Trichosporon ovoides Willopsis california Yarrowia lipolytica Reference
Anamorph
Weinkase
Romadour
Limburger
German
German
German
Factory
Factory
Factory
A
C
A
B 2 2
Candida famata Geotrichum candidum
86 4
95 1
D
A
C
3
Swiss
Tilsit
Reblochon
Austrian
French
12 10
69 6
3
Candida colliculosa
55 21
64 17
85 2
22 52
79 5
2 59 6 28
24 3
22
Candida lipolytica
3
87
1
1
1
1
19
1
1
1
1 4 20
7
2
3
1" Valdes-Stauber et al., 1997; 2: Wyder and Puhan, 1999; 3: Eliskases-Lechner et al., 1995b" 4: B&rtschi et al., 1994. a Results from references 1 and 2 are given as a percentage of the surface yeast microflora; other results are given as a percentage of the number of strains isolated and/or identified.
result in an increase of the pH of the cheese surface from its initial value of---5.0 to >6.5. Thus, a pH gradient is set up with the core being more acid than the SRI l~LIC. Ill turn, tl,la Ill permits t i l t ; growth of the salt-tolerant bacterial flora. De-acidification not only enables the desirable bacteria to grow but also enhances the action of enzymes, important in ripening, the optimum pH of which is often close to neutrality (Gripon, 1997). The increase in pH modifies the rheological properties of the cheese, resulting in a soft body, which is typical of this type of cheese. Eliskases-Lechner and Ginzinger (1995b) examined the de-acidification properties of 305 strains of yeast in a medium containing 1% yeast-nitrogen-base solution and 3% lactate at pH 4.8. The strains differed in the final pH value attained and in the rate of pH increase: 23% of the strains neutralized (pH 7.0) the medium within 72 h, 33% within 94 h and 38% within 120 h. The inter-strain differences indicated that an unsuitable yeast flora can cause a delay in de-acidification. In such cases, the addition of rapidly de-acidifying yeast may be necessary. Differences in the rate of de-acidification by isolates of different origins were observed, especially in isolates from the brine, which showed very low de-acidification activity. This suggests that brine is not a major source of yeast. These workers also reported no correlation between the change in pH and the composition of the yeast flora on the cheese surface. In spite of low numbers of yeast, de-acidification on the cheese surface was
not delayed, indicating that de-acidification depends not only on the number of yeast present but also on the strain. Production of stimulatory compounds Yeasts on the surface of these cheeses also produce stimulatory growth substances, which appear to be necessary for the growth of B. linens. A study by Purko et al. (1951a) on the associative action between some yeast and B. linens showed that the latter did not grow on a vitamin-free agar medium but when the same medium was inoculated with yeast, B. linens grew around the yeast colonies. These workers also showed that strains of yeast isolated from the surface of Limburger cheese produced significant amounts of pantothenic acid, niacin and riboflavin. Purko et al. (1951b) demonstrated that B. linens grown on a semi-synthetic medium containing either pantothenic acid or p-aminobenzoic acid did not require biotin for growth and that the rate and the amount of growth of B. linens increased greatly in the presence of pantothenic or p-aminobenzoic acid. Lubert and Frazier (1955) demonstrated that the autolysates of yeast isolated from the smear of Brick cheese contained substances that stimulated the growth of Micrococcus caseolyticus, Mc. freudenreichii and Mc. varians. Two of these species, Mc. caseolyticus and Mc. varians have subsequently been reclassified as Staphylococcus caseolyticus (Schleifer et al., 1982), which has been reclassified again as Mc. caseolyticus (Kloos et al., 1998).
Bacterial Surface-ripened
Yeasts also contribute to the ripening process through their proteolytic and lipolytic activities which, although slight, would be active during several weeks of ripening. In addition, they prevent the surface of the cheese from drying out and influence flavour formation by producing volatile acids and carbonyl compounds (Siewert, 1986). Staphylococci
Cheeses
203
while Staphylococcus spp. have a low GC content and are included in the clostridial branch. Although they appear similar (clusters of cocci) under the microscope, they are easily separated from each other. Micrococci are resistant to furazolidone and lysostaphin and, when they produce acid from glucose, do so only under aerobic conditions, while staphylococci are sensitive to furazolidone and lysostaphin and produce acid from glucose anaerobically. Phylogenetic and chemotaxonomic analyses have shown that the genus Micrococcus is very heterogeneous and more closely related to Arthrobacter than to Staphylococcus. Recently, it has been separated into five genera, Micrococcus sensu stricto, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus, and these genera, together with others such as Arthrobacter and Stomatococcus, belong to the family Micrococcaceae (Stackebrandt et al., 1995, 1997). The species of Staphylococcus and Micrococcus found on the surface of several cheeses are summarized in Table 3. Whether Casar de Caceres should be considered a smear cheese is not clear. It is included because
and micrococci
Although coryneform bacteria play the most important role in the ripening of smear- cheese, recent studies have indicated that large numbers of micrococci and staphylococci are also found on the surface of these cheeses (Cacares etal., 1997; Irlinger etal., 1997; Vald~s-Stauber et al., 1997; Carnio et al., 1999; Irlinger and Berg~re, 1999; Bockelmann and Hoppe-Seyler, 2001; Brennan et al., 2002). Micrococcus and Staphylococcus have been placed in the same family, but they are not related phylogenetically. Micrococcus spp. have a high GC content and are included in the actinomycetes branch of the eubacteria,
Species of Staphylococcus and Micrococcus (number of isolates) isolated from different smear-ripened cheese
Pont I'Eveque
Livarot
Munster
Maroilles
Several French cheeses a
Casar de Caceres b
Staphylococci S. aureus S. capitis S. caprae S. caseolyticus S. cohnii S. epidermidis S. equorum S. gallinarum S. hominis S. intermedius S. lentus S. saprophyticus S. sciuri S. vitulis S. xylosus Unidentified Total number of strains
2
2
9 1
3
2 3
1
1
1
6 1 1 36 5
89 3
2
1
1 1 6
2
9 3 18
1 6 23
1 3 15
5 2 8
7 7 3 2O 16 1 145
1 3 10 3 115 183
Micrococci Micrococcus luteus Kocuria varians K. roseus K. kristinae Unidentified Total number of strains Raw milk Reference
22
3 3
7
+/1
5 5 +/1
1 1 +/1
1" Michaux (1983); 2: Irlinger et al. (1997); 3: C~.cares et aL (1997). a Mostly from the surface. b 1 cm below the surface. c Brachybacterium species.
1 1 8
3c +/1
10 2
+ 3
204
Bacterial Surface-ripened Cheeses
dry salt is spread on the surface a few times during ripening which may help it to develop a surface flora. Staphylococci are more important than micrococci with
Staph. equorum, Staph. saprophyticus, Staph. caseolyticus and Staph. xylosus being the most prominent species. Staph. caseolyticus has been reclassified as Macrococcus caseolyticus (Kloos et al., 1998). Staph. equorum was more or less the dominant bacterium on the surface of different French and German cheeses in the studies of Carnio et al. (1999) and Bockelmann and Hoppe-Seyler (2001). Recently, two new species of staphylococci, Staph. fleuretti and Staph. succinus subsp, casei, have also been isolated from smear-ripened cheeses (VernozyRozand et al., 2000; Place et al., 2002). Recent data show that a progression of bacteria occurs in the smear; staphylococci are the major organisms found early in ripening and are replaced by coryneform bacteria some days later (Brennan et al., 2002). In addition, low numbers of coagulase-positive pathogenic species, e.g., Staph. aureus, Staph. intermedius and Staph. hyicus have been isolated from a few cheeses. The cheeses from which they were isolated are all raw-milk cheeses which may imply that they originated in the raw milk. The dominant micrococci appear to be Kocuria varians and Mc. luteus. Most strains of staphylococci and micrococci can grow in the presence of 10% salt, which means that a general-purpose medium containing sufficient salt to inhibit the starter bacteria would be a very good selective medium. Staphylococci are also inhibited by furazolidone, implying that furazolidone-containing media should be selective for micrococci. Plate Count Agar containing 5% NaC1 was used by Eliskases-Lechner and Ginzinger (1995a) and Brennan et al. (2002) to isolate the bacteria from the smear of Tilsit and Gubbeen cheeses, respectively. At each stage of ripening (4, 16, 23 and 37 days) of Gubbeen, the predominant organisms were coryneforms, and very few staphylococci and no micrococci were found (the staphylococci were found mainly on day 4 of ripening). Significant numbers of non-pigmented micrococci (whether they were staphylococci or micrococci was not determined) have been found on the surface of Comte and Beaufort cheeses during ripening, and the flora was dominated by coryneforms (Piton-Malleret and Gorrieri, 1992). Coryneforms Coryneform bacteria (Arthrobacter, Brachybacterium, Brevibacterium, Corynebacterium, Microbacterium and Rhodococcus spp.) and related taxa occur almost everywhere on living and non-living matter in the environment. They are especially important on the surface of smeared cheeses, and numerous species have been
found in different cheeses, particularly Tilsit (Table 4). The classification of coryneform bacteria is very confusing. The methods used in recent years include chemotaxonomic techniques, e.g., polyamine patterns (Altenburger et al., 1997; Busse und Schumann, 1999), fatty acids (Kampfer und Kroppenstedt, 1996), numerical taxonomic analysis (Kampfer etal., 1993) and assessment of the heterogeneity of partial 16S rRNA sequences by temperature-gradient gel electrophoresis (TGGE; Felske et al., 1999). Other methods, which place more emphasis on the identification of coryneform genera, have also been reported, e.g., analysis of physiological characteristics by the BIOLOG Identification System (Lindenmann etal., 1995) or the API (RAPID) Coryne database (Funke et al., 1997) or the RapiD CB Plus system (Funke et al., 1998), comparative 16S rDNA sequence analysis (Bockelmann et al., 1997b), the use of genus-specific oligonucleotide probes (Koll6ffel etal., 1997), fluorescence in-situ hybridization (FISH) and colony hybridization (Koll6ffel et al., 1999), as well as Fourier-transform infrared spectroscopy (Oberreuter et al., 2002). The latter is receiving more attention, since an extensive database for the identification of bacteria from the two suborders Micrococcineae and Corynebacterineae, is now available (Oberreuter et al., 2002). Such studies have shown that many of the coryneform bacteria isolated from cheese have been misclassifled. For example, Microbacterium flavurn, which was isolated from an unspecified variety of cheese, has been redesignated as Corynebacterium flavescens (Barksdale et al., 1979), Caseobacter polymorphus, originally isolated from Limburger and Meshanger cheese, as C. variabilis (Collins et al., 1989), B. ammoniagenes as C. ammoniagenes (Collins, 1987) and methanethiol-producing coryneforms, isolated from Cheddar cheese and milk, as B. casei (Collins et al., 1983). In addition, the genus Aureobacterium has been amalgamated with Microbacterium (Takeuchi and Hatano, 1998). Such data suggest that many of the bacteria in Table 4 may be misidentified. New results from molecular bacterial taxonomy revealed that there is no taxon 'coryneform bacteria' (Stackebrandt etal., 1997). Today, the family names Micrococcaceae, Brevibacteriaceae and Corynebacteriaceae should be used with the corresponding genera (Table 5). For practical reasons, the expression, coryneform bacteria, is still widely used to group Arthrobacter, Brevibacterium, Corynebacterium and Microbacterium spp., and will be used in this paper, too. Brevibacterium linens
B. linens is a major micro-organism in the smear of surface-ripened cheeses. Its enzymes, especially its proteolytic and lipolytic ones, and biochemical characteristics
Bacterial Surface-ripened Cheeses
205
Different species (as number of isolates) of coryneforms found in smear-ripened cheeses
Limburger Species Arthrobacter citreus A. globiformis A. nicotianae A. variabilis Arthrobacter spp. Brachybacterium alimentarium Br. tyrofermentans Brevibacterium fermentans B. fuscum B. helvolum B. linens B. oxydans Brevibacterium sp. Corynebacterium ammoniagenes C. casei C. mooreparkense C. variabilis C. flavescens Corynebacterium sp. Curtobacterium insidiosum Curto. poinsettiae Curto. betae Microbacterium imperiale Microbacterium gubbeenense Total number Reference
a
b
Romadour
Weinkase
Harzer
Taleggio
1 5
1
5
2
5
1
2
7
2
Tilsit
Gubbeen
Gruyere
Beaufort
+ +
+ +
5
5
19 102 10 14 43
1 2
1 3
4
5
2
2
77 3
4 8
3
3
16
1
53 115 44
1
1 +
4
3
1
2
6
5 25
8 12 4
33 27
11
13
33
13
1
1
1
1
1
385 2
3
4
1" Valdes-Stauber et al. (1997); 2: Piantanida et al. (1996); 3: Eliskases-Lechner and Ginzinger (1995a); 4: Brennan et al. (2002); 5: Schubert et aL (1996). a Includes Weinkase. b Includes Romadour cheese.
influence the ripening and final characteristics of smear surface-ripened cheeses (for review, see Rattray and Fox, 1999). Because of its ubiquitous presence on the surface of a variety of cheeses, such as Limburger, Manster, Brick, Tilsiter and Appenzeller, it has long been recognized as an important dairy micro-organism. B. linens is strictly aerobic, halotolerant, with a rod-coccus growth cycle, temperature growth optimum of 20-30~ and optimum growth at pH 6.5-8.5. The growth of B. linens on the cheese surface is stimulated by vitamins produced by the yeasts (Purko et al., 1951). Extracellular, cell wall-associated and intracellular proteinases have been reported for B. linens, as well as
the ability to produce different bacteriocins and pigments for colour development on the cheeses (see 'Cheese pigmentation and colour development'). The early literature suggests that the only organism in the smear was Bacterium linens (Wolff, 1910), which was later reclassified as Brevibacterium linens (Breed, 1953). More recently, B. linens has been reported to represent only 30% of the bacteria on the surface of smear-ripened cheese (Brandl, 1980; Busse, 1989), 22% of the total microflora of Limburger cheese (E1-Erian, 1969) and 15% of the total microflora of Tilsit cheese (Bockelmann, 1997). Recent studies have indicated that several other coryneform bacteria, besides B. linens, including
206
Bacterial Surface-ripened Cheeses
Current hierarchic classification of bacterial families and genera with relevance for smear cheeses
Actinobacteria Micrococcineae Micrococcaceae
Arthrobacter, Micrococcus, Kocuria, Renibacterium Brevibacteriaceae
Brevibacterium Microbacteriaceae
Microbacterium/Aureobacterium, Clavibacter, Curtobacterium Dermabacteriaceae
Brachybacterium, Dermabacter Corynebacterinea Corynebacteriaceae
Corynebacterium, Turicella Nocardiaceae
Rhodococcus, Nocardia Firmicutes with low GC content of DNA
Staphylococcus, Enterococcus, Listeria, Bacillus From Bockelmann and Hoppe-Seyler (2001).
Arthrobacter citreus, A. globiformis, A. nicotianae, B. imperiale, B. fuscurn, B. oxidans, B. helvolum, Brachybacterium alimentariurn, Br. tyro-fermentans, Corynebacterium arnrnoniagenes, C. betae, C. casei, C. mooreparkense, C. insidiosum, C. variabilis, Curtobacterium pointsettiae, Microbacterium imperiale and Mc. gubbeenense, are also found on the surface of various cheeses (Table 4; Seiler, 1986; Busse, 1989; Eliskases-Lechner and Ginzinger, 1995a; Schubert et al., 1996; Irlinger et al., 1997; ValdesStauber etal., 1997; Carnio etal., 1999; Irlinger and Bergere, 1999; Bockelmann and Hoppe-Seyler, 2001; Brennan et al., 2002). The importance of each of the species listed in Tables 3 and 4, in the development of the flavour of smear-ripened cheese, has not been determined. However, Leclercq-Perlat et al. (2000a,b) carried out an extensive study of the growth of D. hansenii and B. linens, the utilization of lactate and the production of acid-soluble nitrogen and NPN, in aseptically made, model soft-cheese during ripening. D. hansenii grew rapidly during the first 2 days and then slowed down but it continued to grow exponentially (generation time - 7 0 h) until day 10 (pH of rind ---6), after which growth stopped. In contrast, B. linens did not begin to grow until about day 15 when the pH was --7 but it then grew exponentially until day 70 (generation time 70 h). Total cell numbers of each micro-organism were 10-100-fold higher than their respective viable counts. The pH of the rind increased linearly during the first 23 days at a rate of 0.12 pH units/day while the lactate level in the rind decreased linearly at a rate of 12.5 mmol/kg of dry matter per day up to day 25, then more slowly until day 35 when it was essentially zero. The lactate level in the inner mass of the cheese also
decreased linearly until day 41. Acid-soluble nitrogen increased exponentially throughout ripening, and the rate was faster when the cheese was stored at 4 ~ (day 43-day 76) than when the cheese was held at 13 ~ from day 2 to day 43. The most rapid increase in NPN was between day 22 and day 42 when the cheese was held at 13 ~ although it also increased during storage at 4 ~ The NH3 concentration in the rind remained low during the first 24 days of ripening after which it increased linearly to a level of 2 g/kg DM on day 76. The changes in NPN correlated positively with the growth of B. linens.
Corynebacterium Corynebacteriurn spp. are facultatively anaerobic, Grampositive chemo-organotrophs, which are widely distributed in soil, plants and waste water (Crombach, 1972) and may occur in various foods. Some species of the genus Corynebacterium are major components of the smear microflora of bacterial smear surface-ripened cheeses, e.g., C. ammoniagenes (Table 4). Many Corynebacterium and Arthrobacter isolates in Table 4 have not been identified to soecies level and recently two new species, C. casei and C. mooreparkense, were found in large numbers, especially at the later stages of ripening on the surface of an Irish smear-ripened cheese (Brennan etal., 2001a). Methanethiol is considered to be an important compound in flavour determination of these cheeses and all ten isolates of C. mooreparkense and three of ten isolates of C. casei were able to produce it. In addition, C. rnooreparkense and C. casei had esterase and leucine arylamidase activitities, and C. mooreparkense had lipase activity which probably play some role in cheese ripening (Brennan et al., 2001a). Other new species have also been isolated including Brachybacterium tyroferrnentans and Br. alimentariurn from the surface of Gruyere and Beaufort cheese (Schubert et al., 1996) and Microbacterium gubbeenense from Gubbeen cheese (Brennan et al., 2001b). It is probable that the surface of red-smear cheeses will be a source of further new species. Arthrobacteria
Arthrobacter spp. are Gram-positive, chemo-organotrophs which have a respiratory metabolism, never fermentative, are widely distributed among the bacterial population of soil, and are major components of the smear microflora of some surface-ripened cheeses (Mulder et al., 1966; E1Erian, 1969; Eliskases-Lechner and Ginzinger, 1995a; Valdes-Stauber et al., 1997, Table 4-). Mulder et al. (1966) isolated 22 strains of Arthrobacter, which formed grey-white colonies, from the surface of many types of cheese. In the presence of NaC1, they could develop at pH 5.5, i.e., considerably earlier than other coryneforms. E1-Erian (1969) isolated 173 strains
Bacterial Surface-ripened Cheeses
of Arthrobacter from Limburger cheese and classified them into four groups according to the colour of the colonies formed during growth. The studies of Seiler (1986) indicated the importance of yellow-pigmented Arthrobacter species for ripening of Tilsit cheeses. Coryneform bacteria on 21 brick cheeses were isolated and identified by Valdes-Stauber et al. (1997). Of 148 isolates differentiated in microtiter plates by comparison to reference strains, 21 Arthrobacter sp. and 14 A. nicotianae strains could be identified; A. citreus appeared only sporadically. Bockelmann et al. (1997a) found that the development of red colour could be promoted by mixed-cultures of yellow Arthrobacter strains and B. linens. The importance of yellowpigmented A. nicotianeae for the development of the typical reddish-brown colour of semi-hard cheese has been confirmed (Bockelmann et al., 1997a). Despite their presence at high cell numbers on the surface of several cheeses, only a few studies on the enzymology of dairy Arthrobacter spp. have been reported (see 'Flavour development'). Some Arthrobacter strains isolated from the surface of red-smear cheeses are known to produce bacteriocins or bacteriocin-like substances. Hug-Michel et al. (1989) isolated 80 bacterial strains from the surface of Vacherin Mont d'Or cheese, including seven A. protophormiae and one A. uratoxydans strains. A wide taxonomical distribution of the lin gene, responsible for production of Linocin M18 within coryneform bacteria, has been demonstrated by Valdes-Stauber and Scherer (1996); five out of six Arthrobacter strains tested possessed the gene necessary for the production of Linocin M18. From a total of 2613 individual colonies, eleven Arthrobacter strains were shown to produce clear zones of inhibition on solid media against six or more strains of Listeria monocytogenes (Carnio et al., 1999). The secondary flora Enterococci
Enterococci are ubiquitous bacteria which frequently occur in large numbers in dairy and other food products. Although they share a number of biotechnological traits (e.g., bacteriocin production, probiotic characteristics, usefulness in dairy technology) there is no consensus on whether enterococci pose a threat as food-borne pathogens (Giraffa et al., 1997). The presence of enterococci in dairy products has long been considered as an indication of poor hygiene during the production and processing of milk. On the contrary, many authors suggest that enterococci may have a potential desirable role in some cheeses because they occur in large numbers (up to 107-108 cfu/g) in the indigeneous microflora of many cheeses. For example, enterococci were found to be present in more than 96% of 48 Italian fresh, soft and semi-
207
hard cheeses examined, ranging from 101 to 106 cfu/g. In hard and semi-hard cheeses, the counts of Enterococcus spp. were greater, and the organisms persisted longer, than the other microflora (Gatti et al., 1993). Numerous strains of enterococci associated with food systems are capable of producing a variety of antibacterial proteins (enterococcins) with activity against food-borne pathogens, such as Listeria monocytogenes, pathogenic clostridia or Staphylococcus aureus (for review, see Giraffa, 1995). An overview of the potential of enterococcal bacteriocins in model cheese systems is given in 'Enterococcal bacteriocins'. Non-starter lactic acid bacteria
Non-starter lactic acid bacteria (NSLAB) are mesophilic lactobacilli and pediococci, which form a significant portion of the microbial flora of most cheese varieties during ripening (Beresford et al., 2001). They are not part of the normal starter flora and, generally, they do not grow well in milk (Cogan et al., 1997). Lactobacilli are either (i) obligatory homofermentative, (ii) facultatively heterofermentative or (iii) obligatory heterofermentative (Kandler and Weiss, 1986). Many species of mesophilic lactobacilli have been isolated from surface-ripened cheese, but the most frequently encountered are Lactobacillus caseilLb, paracasei, Lb. plantarum, Lb. rhamnosus and Lb. curvatus (Ennahar et al., 1996; Coppola et al., 1997). As NSLAB are found in cheeses made from raw or pasteurized milk, the major source of these microorganisms may be the raw milk itself, or in case of cheese made from pasteurized milk, either post-process contamination or failure of pasteurization to fully inactivate them (Beresford et al., 2001). Non-starter lactic acid bacteria may have some effect on the maturation of cheeses. Pediococci, which develop in Comte cheeses, have proteolytic, lipolytic and esterolytic activities (Bhowmik and Marth, 1990) and therefore could play a beneficial role during cheese ripening. In the same way, facultatively heterofermentative lactobacilli are potentially highly proteolytic (Khalid and Marth, 1990). Bouton etal. (1998) suggested that the high number of lactobacilli present at the end of ripening indicated that these bacteria could have an effect on flavour development. Only a few authors report significant numbers of NSLAB in red-smear cheeses, most of them in lowmoisture hard cheeses, whereas in other extensive studies (Carnio etal., 1999; Brennan etal., 2002), no information about the incidence of NSLAB is reported. Coppola etal. (1997) isolated lactobacilli (mainly Lb. paracasei subsp, paracasei and Lb. rhamnosus) and pediococci (Pediococcus acidilactici) from MRS agar during the later stages of Parmiggiano Reggiano cheesemaking. In the study of Bouton et al. (1998), the number of propionibacteria, facultative heterofermentative lactobacilli
208
Bacterial Surface-ripened Cheeses
and enterococci increased during the ripening of Comt~ cheese. On the other hand, Lb. plantarum WIlE 92 was found at high levels (108cfu/g cheese) among 1962 bacterial isolates from the surface of a soft smear cheese (Mfinster) and had anti-listerial activity (Ennahar et al., 1996; see 'Bacteriocins of lactic acid bacteria'). Propionic acid bacteria Propionic acid bacteria (PAB) grow in many cheese varieties and are the characteristic microflora associated with surface-ripened Swiss-type cheeses such as Gruyere, Appenzeller, or Comte. In cheese manufactured from raw milk, sufficient 'wild' PAB are present. However, with the advent of pasteurization, PAB are now added to the cheese milk at the beginning of manufacture to ensure that they are present in sufficiently high numbers (Vorobjeva, 1999). The cheeses undergo a propionic acid fermentation, and the propionic and acetic acids produced contribute to the development of characteristic flavours of the cheese. Flavour development
Sulphur amino acids Sulphur compounds are particularly important in the flavour of probably all cheeses, particularly smearripened varieties. Metabolism of sulphur amino acids by bacteria associated with cheese has been reviewed by Weimer et al. (1999) but was treated mainly from the involvement of lactococci and lactobacilli in Cheddar cheese. It was originally thought that methanethiol (CH3SH) was the major sulphur compound produced and that B. linens was the main organism responsible. Recent evidence suggests that other micro-organisms on the cheese surface, including Mc. luteus, S. equorurn, B. acetobutylicum (but not B. flavurn), C. glutamicum, Arthrobacter spp., several unidentified coryneforms, G. candidurn and the starters, Lc. lactis and Lb. helveticus, also produce CH3SH (Bloes-Breton and Bergere, 1997; Dias and Weimer, 1998a; Berger et al., 1999; Bonnarme et al., 2000). These studies have also shown that other sulphur compounds, including dimethyl sulphide (DMS), dimethyl disulphide (DMDS), dimethyltrisulphide (DMTS), thiols (2-propanethiol, 2methylpentanethiol), thioesters (methyhhioacetate, methylthiobutanoate, methyl thiopentanoate), are also involved. These are termed the volatile sulphur compounds (VSC) and quantitatively are produced in very small amounts but as they have very low flavour thresholds, small amounts (Ixg~g) are important in flavour perception. CH3SH is produced enzymatically from methionine and the other compounds, particularly DMS, DMDS and DMTS are formed from it by chemical (non-enzymatic) means (see 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1).
The most studied VSC is CH3SH, which is produced by two pathways. One involves its direct production from methionine by L-methionine-y-lyase in an oty elimination reaction in which NH3 and ot-ketobutarate are also produced (Fig. 1). The second mechanism involves a two-step reaction involving an amino transferase or transaminase, which produces ot-keto-y-methyl-thiobutyric acid (KMBA) and glutamate from methionine and ot-ketoglutarate, respectively, cx-Keto-y-methyl-thiobutyric acid is subsequently transformed to CH3SH. Both pathways appear to be present in many of the organisms examined but which is more important has not been examined. The enzyme activities are very low but so are the flavour thresholds of these compounds. In addition, these enzymes must be active at the relatively high salt (5-10%) concentrations in these cheeses. The k-methionine-3,-lyase (EC 4.4.4.11) from B. linens has been purified (Dias and Weimer, 1998b). It consists of four identical 43-kDa sub-units. Its optimum temperature is 25 ~ and its optimum pH 7.5. The enzyme is inhibited by carbonyl reagents and NaC1 but not by metal-chelating agents; 5% NaC1 reduces the activity to 10% implying that the enzyme may not be active in the cheese matrix. Dias and Weimer (1998a) found significant production of CH3SH by six of the seven strains of B. linens; however, a commercial strain, B. linens BLI, produced only insignificant amounts. B. acetylicurn produced almost as much as the best strain of B. linens, and B. flavum (seven strains) produced none. Micrococci (four strains) were poor producers, but Lc. crernoris S1 and Lb. helveticus CNRZ 32 produced significant amounts. Production of CH3SH from methionine and KMBA by growing cells by five strains of bacteria, commonly found on the surface of cheese, was compared by Bonnarme et al. (2000). Very small amounts of CH3SH were produced, but significant amounts of DMDS and DMTS were produced by B. linens ATCC 9175, Mc. luteus 790 and C. glutarnicurn D13. Resting cells of B. linens 9175 were also efficient, with Mc. luteus 790 and an Arthrobacter sp. producing 50% of that produced by B. linens, while Staph. equorurn 1265 and C. glutamicum D 13 only produced 35 and 27%, respectively. Cell-free extracts produced very little CH3SH from KMBA except for C. glutamicum, while B. linens, C. glutarnicum and the Arthrobacter sp. produced significant amounts of it from methionine; production by Staph. equorum and Mc. luteus was poor. Four of the ten strains of G. candidurn have also been shown to produce significant amounts of CH3SH, DMS DMTS, S-methyl thioacetate, S-methyl thiopropionate, S-methyl thioisobutanoate, S-methyl thioisovalerate and S-methyl thiohexanate (Berger et al., 1999). Except for CH3SH, the exact pathways involved in the production
Bacterial Surface-ripened Cheeses
209
Two pathways for the production of methanthiol. Reprinted from Bonnarme etal. (2000) with permission. Met ATase, methionine aminotransferase; KMBA, oL-keto-h,-methyl thio butyric acid DMDS, dimethyl disulphide; DMTS, dimethyl trisulphide; oL-KG, o~-ketoglutarate; MTL, methanethiol.
of these compounds have not been determined but the redox potential (Eh) is thought to be important. Proteinases and lipases The smear bacteria also contribute to the ripening process through their proteolytic and lipolytic activities. Hosono and Tokita (1970) studied the lipolytic properties of C. mycoderma and D. kloekeri isolated from the surface of Limburger cheese. Both yeasts produced extracellular lipases with a pH optimum of 4.5, and optimum temperatures of 35 and 30 ~ for the lipases of C. mycoderma and D. kloeckeri, respectively. These workers also reported that the lipase of C. mycoderma released considerably more lauric, myristic and palmitic acids and less stearic acid from milk fat than the lipases of D. kloekeri. Three esterases, which were active on aliphatic and nitrophenyl esters, were isolated from Brevibacteriurn strain R312 (Lambrechts et al., 1995); they had an optimum pH of 6-8, 7.6 and 8, and optimum temperatures of 43, 36 and 30 ~ respectively. Two of them appeared to be sulphydryl enzymes. An extracellular proteinese and an intracellular aminopeptidase have been purified from B. linens ATCC 9174 (Rattray etal., 1995; Rattray and Fox,
1997). The proteinase is a dimer of molecular mass 126 kDa. Its optimum pH is 8.5, its optimum temperature 50 ~ and it is activated by Mg 2+ and Ca2+; Hg 2+, Fe 2+ and Zna+ strongly inhibited the enzyme. NaC1 also stimulated the enzyme which appeared to be a serine proteinase. The aminopeptidase was a thiol enzyme with a mass of 59 kDa, had an optimum pH of 8.5, an optimum temperature of 35 ~ and was inhibited by Co 2+ and Zn 2+. Some of the putative extracellular enzymes from A. nicontianae, which may be important in cheese ripening, including two serine proteinases, a proline iminopeptidase and an esterase, have been purified by Smacchi et al. (1999a,b, 2000). The proteinases had molecular masses of 54 and 71 kDa and pH optima at 9-9.5; they also had considerable activity at pH 6 on Ors1- and [3-caseins. The proline iminopeptidase hydrolysed proline-containing peptides under the pH, temperature and salt concentration of surface-ripened cheese. An extracellular proline iminopeptidase has also been purified from C. variabilis (Gobbetti et al., 2001). It is a serine enzyme with a pH optimum of 8.5 however, significant activity also occurred at pH 6 and in the presence of 7.5% NaC1, implying that it would also be active on the cheese surface during ripening.
210
Bacterial Surface-ripened Cheeses
G. candidum produces several intracellular and extracellular proteinases (Lenoir, 1984; Hannan and Gueguen, 1985) and lipases (Alifax, 1979; Sidebottom et al., 1991). Cheese pigmentation and colour development
The smear bacteria are also thought to be responsible for the development of the surface pigmentation characteristic of red-smear cheese. Considering the low percentage of B. linens in the smear reported by E1-Erian (1969) and more recently by Bockelmann (1997), it is unlikely that this organism is solely responsible for the colour of the smear. Seiler (1986) demonstrated the importance of yellow-pigmented Arthrobacter species in the ripening of Tilsiter cheese, and Bockelmann et al. (1997a) reported that the development of the redorange colour in the cheese could be promoted by a combination of a yellow Arthrobacter spp. and B. linens. A more intense colour was observed on addition of casein hydrolysate to the growth medium, indicating that the strong proteolytic activity of B. linens may promote colour development. Lee et al. (1985) reported that the metabolism of phenylalanine and tyrosine is essential for the development of colour by coryneform bacteria isolated from cheese, while Ferchichi et al. (1986) reported that methionine is essential for the development of orange pigments by B. linens. These findings were confirmed by Bockelmann et al. (1997a), who observed that the development of the red-orange pigmentation was not achieved on addition of single amino acids to the growth medium. Tyrosine was reported to be the only amino acid which influenced colour, but the characteristic red-orange colour of smear-ripened cheese was not produced. Addition of combinations of tyrosine, phenylalanine and methionine, which have been reported to be responsible for colour development by coryneform bacteria, did not result in the development of pigmentation, indicating that the development of red-orange pigmentation is more complex. Piantanida et al. (1996) showed that the smear micro-organisms influence the surface colour of Taleggio cheese. A white surface indicated the presence of G. candidum, a yellow colour C. flavescens, yellow-green patches the presence of A. globiformis and A. citreus, while a uniform pink-red surface indicated the presence of B. linens.
Origin of the surface microflora
Bacterial smear-ripened cheeses have a long tradition. Without the knowledge of the bacterial nature of the surface flora, a large variety of smear cheeses was pro-
duced long before 1900 (Fox, 1993). Numerous species of yeast and bacteria have been isolated from the surface of smear cheese (Tables 2, 4 and 5). This raises the question 'where do these micro-organisms originate from?' Deliberate inoculation of the cheese surface is generally confined to commercially available cultures of D. hansenii, G. candidum and/or B. linens. Many cheese producers do not rely solely on these cultures (see 'Secondary and Adjunct Cultures', Volume 1). Traditionally, the surface flora of mature cheeses is used as the source of micro-organisms for smearing young cheeses; the necessary micro-organisms are transferred from the mature cheese to the brine before smearing of the young cheeses is begun. However, commercially available surface starters do not reflect the microbial composition of the cheese surface. Thus, cheese milk, brine, air, utensils and shelves are all possible sources of the surface microflora. Since the introduction of pasteurization, which has considerably improved food safety, the cheese milk flora has less influence on the surface microflora of cheeses (Holsinger et al., 1997), the dependance on an intact 'house microflora' and starter cultures has increased considerably. For cheeses made from raw milk, the evolution of the microflora of Comte cheeses made in duplicates from different sources was followed during ripening (Bouton et al., 1998). Comparison of the cheeses revealed no significant difference in the development of the microflora. Most of the micro-organisms found on the cheese surface are very salt tolerant, so brines could also be a source (Bockelmann and Hoppe-Seyler, 2001). However, Eliskases-Lechner and Ginzinger (1995b) found pure cultures of D. hansenii on the surface of Tilsiter cheese at all stages of ripening, even when other species, e.g., Kluyveromyces marxianus and Trichosporon beigelii, were the dominant organisms in the brine. In another study (Eliskases-Lechner and Ginzinger, 1995a), Austrian bacterial surface-ripened cheeses were examined for changes in microbial composition of the smear. The bacterial flora was a mixed population. B. linens, which had been deliberately smeared on the cheese surface, comprised 30% of the total bacterial count, and A. globiformis and C. ammoniagenes were also dominant, although they were not added to the smear. All other species found occurred only sporadically or were found in the smear from a single plant. Thus, one can conclude that all bacteria which grow on the surface and which have not been added deliberately are probably adventitious contaminants, which grow well in the presence of the high salt concentrations and relatively high pH of the cheese surface. It is likely that the major sources are the brine and the wooden shelves on which the cheese rests during ripening (Beresford et al., 2001).
Bacterial Surface-ripened Cheeses
Galli et al. (1996) investigated several Taleggio cheese samples at the end of ripening. The bacteria on the surface layer (109-10 l~ cfu/g smear) were either Gram-positive cocci (Mc. sedenterius, Staph. xylosus, Staph. sciuri) or were Gram-positive irregular asporogeneous rods (Microbacterium lacticum, B. linens, B. casei). Mb. lacticum and B. linens, together with some Micrococcus and Staphylococcus isolates were found to be mainly responsible for the typical orange pigmentation of the ripe cheeses. Bockelmann and Hoppe-Seyler (2001) analysed the surface flora of semi-hard Tilsit, soft Chaumes and semihard goat's milk cheese. D. hansenii was the predominant yeast at all stages of ripening, and 75-95% of the bacteria were coryneforms. Yellow-pigmented coryneform isolates (1-30%) were A. nicotianae, whereas B. linens was only 0-15%. Non-pathogenic staphylococci, mainly Staph. equorum, comprised 5-15% of the total flora. An extensive study of Danbo, a smear-ripened Danish cheese, showed that D. hansenii was the dominant yeast (Petersen et al., 2002). Using restriction analysis of mitochondrial DNA, these workers showed that none of the four strains in the brine were detected on the cheese surface when it was removed from the brine. Furthermore, there was a progression of strains, since another dominant strain of D. hansenii, which was different from those detected on the cheese early in ripening, developed later in ripening. This strain was able to grow better than the other strains at the pH and salt levels on the cheese surface. Recently, another extensive study was conducted (Brennan et al., 2002) of the bacteria on the surface of a farmhouse smear-ripened cheese at different stages of ripening. Of 400 isolates, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative staphylococci. The cheeses were dominated by single clones of novel species of C. casei (50.2%), C. mooreparkense (26%) and Mb. gubbeenense (12.8%). B. linens BL2 was not found in the surface flora of the cheese, even though it had been deliberately inoculated on to the cheese surface; in fact, it was inhibited by all the Staphylococcus and many of the coryneform isolates. The source of C. casei, C. mooreparkense and Mb. gubbeenense on the cheese surface was not determined. Defined cultures
Deliberate inoculation of the cheese surface is generally confined to commercially available cultures of D. hansenii, G. candidum and/or B. linens. However, these micro-organisms do not reflect the microbial composition of the cheese surface. So far, there is little knowledge about the influence of different strains on colour development of red-smear cheese. Because B. linens produces orange (or red)-coloured colonies, several
211
groups of workers have distinguished between the colour of the colonies in the smear cheese, e.g., orange, yellow or non-pigmented (Seiler, 1986; Piton-Malleret and Gorrieri, 1992; Eliskases-Lechner and Ginzinger, 1995a; Hoppe-Seyler et al., 2000). The orange-coloured colonies are considered to be B. linens and the yellow-coloured ones, Arthrobacter spp. (Hoppe-Seyler et al., 2000). However, in the study of Piton-Malleret and Gorrieri (1992), the orange- and yellow-coloured colonies comprised 82 and 68% Micrococcaceae (whether they were micrococci or staphylococci was not determined), respectively; the remainder were coryneforrns (genus also not designated). In the study of Eliskases-Lechner and Ginzinger (1995a), the cream- and yellow-coloured isolates were predominantly A. globiformis and C. ammoniagenes, respectively. Understanding the microbial ecology of the cheese surface is a prerequisite for the development of defined surface cultures for control of surface ripening (Bockelmann, 1997). Because of the numerous organisms found in smear cheeses, the development of defined-strain smear cultures is being investigated with promising results. Carminati et al. (1999) screened surface-smear organisms isolated from Taleggio cheese for their ability to inhibit different pathogens. Most of the isolates showing anti-listerial activity (19% of the total) were coryneform bacteria, mainly Mb. lacticum. Two bacterial mixtures (containing orange-pigmented strains and strains that inhibited Listeria) were studied as smear cultures for Taleggio cheese. However, the cheese did not undergo normal ripening, due to a delay in the growth of the smear bacteria. The successful use of a defined five-strain culture (D. hansenii, B. linens, A. nicotianae, C. ammoniagenes and Staph. sciuri) for Tilsit cheese was demonstrated by Bockelmann and Hoppe-Seyler (2001) on a pilot scale. No problems with yeast growth were observed, but ripening appeared to be slightly slower compared to another batch in which a commercial smear culture had been used. Nevertheless, after 6 weeks of ripening, cheese quality was good. However, commercially available surface starters do not reflect the diversity of the microbial composition of the cheese surface. Too much emphasis is put on B. linens because of the orange pigmentation. New results from Bockelmann and Hoppe-Seyler (2001) showed that the red-brown and orange pigments are most likely due to the growth of yellow-pigmented Arthrobacter spp. The mechanisms of developing the different shades of red are not yet understood. The presence of a rich and complex microflora on the surface of Taleggio cheese might help to control Listeria contamination. The role of the surface microflora, which develops during ripening, could exert a
212
Bacterial Surface-ripened Cheeses
'self-protecting activity' on the hygienic quality of the product (Carminati et al., 1999). Pathogens
Some cheeses, especially red-smear cheeses, may be risk products for bacterial contamination since they constitute a suitable medium for the growth of pathogens like Listeria monocytogenes (Terplan et al., 1986) and some other food-borne pathogens. This is partly due to the traditional 'old-young' smearing method, where unripe cheeses are inoculated with a complex, undefined microflora washed from the surface of ripened cheeses. A disadvantage of this approach is that undesirable contaminants will be transferred to the smear bath and will be spread throughout the factory. Smearing machines were found to be an important source of Listeria contamination (Hahn and Hammer, 1993).
Listeria monocytogenes Listeria monocytogenes is a food-borne pathogen, the control of which in food is made difficult by its ubiquity in the environment, its ability to grow at refrigeration temperature and its tolerance to low pH (
Schopfer, 1988; Pini and Gilbert, 1988; Weber et al., 1988; Eppert et al., 1995; Loncarevic et al., 1995, 1998; Rudolf and Scherer, 2000, 2001). While Listeria was the emerging food-borne pathogen of the 1980s, it has faded somewhat from public attention. Recent systematic investigations on the hygienic status of red-smear cheese are rarely available. It is therefore unknown whether the lack of outbreaks in recent years caused by red-smear cheese is due to an improved hygienic status of these cheeses. Investigations of Terplan et al. (1986), Comi et al. (1990), and Rudolf and Scherer (2001) have shown that L. rnonocytogenes was isolated from none of the 99 Italian hard cheeses (0%), 2 of the 48 (4.2%) German hard cheeses and 2 of the 45 (4.4%) other European hard cheeses. These results generally agree with those from other surveys in which L. monocytogenes was found more frequently in high-moisture than in lowmoisture cheese (Ryser, 1999). As a consequence of recontamination, L. monocytogenes and other species of Listeria may still appear frequently in red-smear cheese, even when pasteurized milk has been used for cheesemaking. Beckers et al. (1987) reported that 9 of the 14 mould-ripened cheeses made from raw milk, but none of the 36 soft cheeses made from pasteurized milk, were positive for L. rnonocytogenes. Eight years later, Eppert et al. (1995) and Loncarevic et al. (1995) also found a higher incidence of L. monocytogenes in soft and semi-soft cheese made from raw milk (33 and 42%, respectively) than in cheese made from pasteurized milk (9 and 2%, respectively). In contrast, Breer and Schopfer (1988) found that the incidence of L. rnonocytogenes in cheeses made from raw or pasteurized milk was similar (13.9 and 12.2%, respectively). However, more recently Rudolf and Scherer (2001) reported that more pasteurized milk cheeses (8%) were positive for L. monocytogenes than those made from raw milk (4.8%). Surface counts of Listeria seem to increase throughout cold storage because, generally, higher numbers were found close to the end of the shelf-life of the cheese (Rudolf and Scherer, 2001). Contamination with and multiplication of organisms may also occur in the retail chain and in the domestic refrigerator (Greenwood etal., 1991). Therefore, the consumer can be exposed to a potentially large number of pathogens even if the initial level of contamination is low. The ability of L. monocytogenes to grow at low temperatures, the length of time needed for maturation of ripened cheeses and the prolonged or unspecified shelf-life assigned to many products make the complete exclusion of L. monocytogenes of great importance.
Bacterial Surface-ripened Cheeses
The incidence of L. rnonocytogenes in soft and semisoft cheese in different investigations ranges from 1.1 to 22%, whereas other species of Listeria have been found in 0.5-24% of the cheese samples examined. The surveys published throughout the last 14 years (Table 6) involved quite different sample sizes and detection methods, as well as different cheese types. Therefore, a direct comparison of the incidence of L. monocytogenes in these studies is not possible. However, there is no indication that the incidence of L. monocytogenes in European red-smear cheeses has decreased sufficiently during this time period and, therefore, it still presents a considerable public health risk. Other species besides L. monocytogenes, predominantly L. innocua, are frequently found on cheese, sometimes in combination with the pathogen. Obviously, similar occurrences in the environment and the identical conditions of growth of both species may therefore be considered as an argument for considering L. innocua as a marker organism for contaminated dairy plants (Hahn and Hammer, 1990). In any case, the presence of non-pathogenic Listeria in a product indicates an unsatisfactory process, at risk of contamination with L. rnonocytogenes. It is therefore recommended that food samples in general should be tested for the occurrence of the genus Listeria. Staphylococcus aureus
Many of the food poisoning outbreaks of bacterial origin and of known aetiology are caused by the ingestion of foods containing Staph. aureus enterotoxins. Rawmilk cheeses have been repeatedly involved in staphylococcal outbreaks (Table 7; De Buyser et al.,
213
1985) and enterotoxin A (SEA) is commonly implicated (De Buyser etal., 1996). The incidence and growth of Staph. aureus and, in some cases, the production of enterotoxins during manufacture and ripening of red-smear cheeses, have been studied by several researchers (Gomez-Lucia et al., 1992; Otero et al., 1993; Bachmann and Spahr, 1995; De Luca et al., 1997). In Italy, De Luca et al. (1997) found 16.3% of all cheese samples made from pasteurized milk to be positive for S. aureus, with cell numbers ranging from 10 to 315 000 cfu/g. E. coli 0 1 5 7 : H 7
Since its identification as a human pathogen, E. coli O157:H7 has become a major concern for the food and dairy industries because of its ability to cause severe illness. Many outbreaks of E. coli O157:H7 have been linked with the consumption of contaminated meat and other foodstuffs, such as water, lettuce, alfalfa sprouts and apple juice (Buchanan and Doyle, 1997). The consumption of unpasteurized milk and dairy products manufactured from raw milk has also been associated with the transmission of E. coli O157:H7. In 1999, over 11% of the total number of reported cases of infection caused by E.coli O157:H7 in England and Wales were due to dairy products (CDSC, 2000). So far, little information is available about the incidence and behaviour of E. coli O157:H7 in surfaceripened cheeses (Table 8). Manufacturing cheese on a laboratory scale from milk inoculated with E. coli O157:H7, Maher etal. (2001) demonstrated that the manufacturing procedure promoted substantial
Incidence of Listeria spp. in red-smear cheeses from different countries since 1986 Origin of cheese
Type of cheese
No. of samples
L. monocytogenes (%)
Other Listeria spp.(%)
Authors~Year
D, other countries F A, DK, FIN, F, D, GR, NL, I, N, P, S, CH n.d. F F, UK, I, CY, D, DK, RL D, other countries I Other countries F, D A, DK, UK, F, D, GR, I, NL, N, RO, E, S A, DK, F, D, I, CH
Soft, semi-soft, hard Soft
420 69
3.3 14.5
3.3 n.d.
Terplan et aL (1986) Beckers et aL (1987)
Soft and semi-soft Red-smear cheese Soft and semi-soft Soft Soft, semi-soft, hard Soft Soft Soft
187 343 619 222 509 121 90 91
1.1 9.6 2.1 10.4 5.7 1.6 11.0 22.0
0.5 9.6 1.5 8.6 6.1 1.6 n.d. 24.2
Farber et aL (1987) Breer and Schopfer (1988) Gledel (1988) Pini and Gilbert (1988) Weber et aL (1988) Massa et aL (1990) Rorvik and Yndestad (1991 ) Eppert et aL (1995)
Soft and semi-soft Soft, semi-soft, hard
333 374
6.0 6.4
n.d. 11.8
Loncarevic et aL (1995) Rudolf and Scherer (2001)
A, Austria; CH, Switzerland; CY, Cyprus; D, Germany; DK, Denmark; E, Spain; F, France; FIN, Finland; GR, Greece; I, Italy; N, Norway; NL, The Netherlands; P, Portugal; RL, Lebanon; RO, Romania; S, Sweden; UK, England.
214
Bacterial Surface-ripened Cheeses
Examples of S. aureus outbreaks in cheese in different countries Country
Year
No. of cases
Food implicated
Type of milk
Reference
Canada USA France England Scotland England Brazil
1980 1981 1983 1983 1984 1988 1994
62 16 20 2 27 155 7
Cheese curd Cheese Farm ewe cheese Cheese Ewe cheese Stilton cheese Cheese
Unspecified Pasteurized Raw Pasteurized Raw Unpasteurized Unspecified
Todd et al. (1981 ) Altekruse et al. (1998) De Buyser et aL (1985) Barrett (1986) Bone et al. (1989) Maguire et aL (1991) Pereira et aL (1996)
growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. The authors concluded that the presence of low numbers of E.coli O157:H7 in milk, destined for manufacture of raw-milk cheese, could constitute a threat to the consumer.
of bacteriocin-producing cultures as biocontrol microorganisms, as starter cultures for fermented foods, as bacteriocin-containing microbial fermentates or as partially purified bacteriocins added directly to foods (Muriana, 1996). Bacteriocms of lactic acid bacteria
Bacteriocins
The most likely biological way to control pathogens in cheese is probably with bacteriocins. These are peptides, generally of low molecular mass, which are produced by many bacteria and inhibit the growth of other, generally closely related, species. They vary in their spectrum of activity, mode of action, molecular weight, genetic origin and biochemical properties. Klaenhammer (1993) defined four distinct classes of bacteriocins: class I, antibiotics; class II, small (<10 kDa), relatively heat-stable, non-lanthionine-containing membrane-active peptides, subdivided into Listeria-active peptides with the N-terminal consensus sequence,-Tyr-Gly-Asn-GlyVal-Xaa-Cys- (class IIa), poration complexes requiring two different peptides for activity (class lib) and thiolactivated peptides requiring reduced cysteine residues for activity (class IIc); class III, large (>30 kDa), heatlabile proteins and class IV, complex bacteriocins that contain essential lipid or carbohydrate moieties in addition to protein. Bacteriocin-producing strains can be used in numerous applications as protective cultures, including the use
A large number of bacteriocins produced by LAB have been characterized in recent years, most of them class II bacteriocins. While most bacteriocin producers synthesize only one bacteriocin, several LAB produce multiple bacteriocins (Quadri etal., 1994; Bhugaloo-Vial etal., 1996; Casaus et al., 1997). The classic example of a commercially successful naturally produced inhibitory agent is nisin. Known since the work of Rogers (1928), it is produced by some strains of kc. lactis and has been structurally characterized as a lanthionine-containing peptide by Gross and Morell (1971); nisin and nisinproducing strains have had a long history of application in food preservation, especially of dairy products (Molitor and Sahl, 1991). Lactic acid bacteria have been examined extensively for bacteriocin production because of their widespread use as food starter cultures (Nettles and Barefoot, 1993). Bacteriocins produced by LAB, however, are active mainly against Gram-positive bacteria and, therefore, many exploratory applications have been directed towards tisteria monocytogenes, which is likely to appear in red-smear cheese varieties.
Pathogenic E. coil outbreaks in cheese in different countries Country
Year
No. of cases
Food implicated
USA
1983
170
Denmark
1983
The Netherlands
1983
69
Sweden
1983
66
Scotland
1994
22
Brie and Camembert cheeses Brie from the same plant as for USA, 1983 Brie from the same plant as for USA, 1983 Brie from the same plant as for USA, 1983 Farm cheese
Type of milk
Reference
Pasteurized
MacDonald et al. (1985)
Pasteurized
Nooitgetagt and Hartog (1988)
Pasteurized
Nooitgetagt and Hartog (1988)
Pasteurized Raw
Nooitgetagt and Hartog (1988) Rampling (1996); Ammon (1997)
Bacterial Surface-ripened Cheeses
Sulzer and Busse (1991) examined 14 bacteriocinogenic strains of Enterococcus Jaecalis, Lb. paracasei and Lc. lactis isolated from cheese or raw milk for their ability to control L. monocytogenes during the manufacture of Camembert cheese. Complete inhibition occurred when the inhibitory strain was used as a starter culture and there was a low level of contamination with Listeria sp. during the early stage of ripening. Very little inhibition occurred if the inhibitory strain was added together with the starter culture. Of 1962 bacterial isolates from a bacterial surfaceripened soft cheese (M1)nster) screened for activity against L. monocytogenes, six produced anti-listerial compounds other than organic acids (Ennahar et al., 1996). The strain which displayed the strongest antilisterial effect was Lb. plantarum WHE 92, which produced pediocin AcH, which is normally produced by Pediococcus acidilactici (Henderson et al., 1992). The activity spectrum included all ten Listeria strains tested, Enterococcus, Micrococcus, Staphylococcus and Bacillus spp. Gram-negative bacteria tested were not affected. The anti-listerial effect of the strain was investigated in MOnster cheese (Ennahar et al., 1998). A cell suspension of Lb. plantarum WHE 92, sprayed on the cheese surface at the beginning of the ripening period, prevented the growth of L. monocytogenes. Moreover, the addition of Lb. plantarum WIlE 92 did not adversely affect the ripening process. The anti-listerial potential of Lb. plantarum WHE 92, which is commercially available as ALC 01 (anti-listerial culture, Danisco, Germany), was investigated in more detail by Loessner et al. (2003). Growth of L. monocytogenes WSLC 1364, which was isolated from a cheeseborne outbreak of listeriosis, on red-smear cheese was examined in the presence and the absence of the pediocin AcH-producing Lb. plantarum ALC01. An initial level of 102 cfu of L. monocytogenes/ml of brine was nearly completely inhibited (Fig. 2A), while a pediocin-resistant mutant of L. monocytogenes grew to high cell numbers on the cheese surface (Fig. 2B). The inhibition was due to the production of pediocin AcH by Lb. plantarum during growth before it was added to the brine solution, rather than bacteriocin production in situ on the cheese. Pediocin resistance developed in vitro at different frequencies in all L. monocytogenes strains investigated, and a resistant mutant remained stable and multiplied easily in smear cheese over a 4-month production period in the absence of selection pressure. Therefore, it was concluded that the addition of a pediocin AcH-producing Lb. plantarum culture is a potent measure for combating Listeria in a contaminated production line but, due to easy development of resistance, its use should be restricted to emergency situations.
215
Bacteriocins of coryneform bacteria Smear cheeses are characterized by the development of a relatively high pH (>6.5) at the surface during ripening. This, together with a relatively high ripening temperature, allows the growth of salt-tolerant pathogenic micro-organisms on the surface of the cheese, particularly L. monocytogenes. The undefined microflora from the surface of ripe cheeses which are used for the ripening of commercial red-smear cheeses were shown to have a strong impact on the growth of Listeria spp. (Eppert et al., 1997). Therefore, several workers (HugMichel etal., 1989; Valdes-Stauber etal., 1991; Ryser et al., 1994; Martin et al., 1995; Carnio et al., 1999, 2001; Motta and Brandelli, 2002) have evaluated smear bacteria for their ability to prevent the growth of Listeria and other pathogens. Hug-Michel et al. (1989) tested 80 strains of microorganisms which had been isolated from the rind of Vacherin Mont d'Or cheese for antagonistic activity against Listeria; 9 of the 80 strains (identified as Arthrobacter protophormiae, A. uratoxydans or Serratia marcescens) showed an inhibitory effect against all serotypes of Listeria monocytogenes tested; the identity of the inhibitor was not identified. Valdes-Stauber et al. (1991) showed that small numbers of B. linens (16 strains), A. nicotianae (4 strains) and A. nucleogenes (3 strains) inhibited 26-87% of the 91 strains of Listeria tested; B. linens M18 was particularly effective. The strain produces a substance (named linocin MIS) that inhibits the growth of Listeria spp. and several coryneforms and other Gram-positive bacteria (Valdes-Stauber and Scherer, 1994). The structural gene for linocin M18 has been detected in several strains of B. linens, five Arthrobacter spp. and four Corynebacterium spp. (Valdes-Stauber and Scherer, 1996), suggesting that the ability to produce this bacteriocin is widely distributed in coryneform bacteria. Ryser et al. (1994) found that less than 0.1% of 125 000 isolates from 105 French smear cheeses showed visible zones of inhibition against L. monocytogenes. Isolates possessed anti-listerial activity against various strains of Enterococcus faecalis, Staph. xylosus, Staph. warneri and coryneform bacteria; one strain, B. linens OC2, which was isolated from Comte cheese, was particularly effective. These data suggest that the number of smear isolates which can inhibit Listeria is low. In crude extracts of both organisms, the inhibitory properties were associated with molecules of high molecular mass but the purified bacteriocins, linenscin OC2 and linocin M18, have molecular masses of 1.2 and 31 kDa, respectively, and had broad spectra of activity inhibiting Staph. aureus, several Listeria, Bacillus, Arthrobacter, Brevibacterium and Corynebacterium spp. and LAB. Neither bacteriocin showed activity against
216
Bacterial
Surface-ripened
Cheeses
1.0E + 06 A A
1.0E + 05
v
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04
E o 1.0E + 03
I
O
1.0E + 02 plantarum ALC 01 1.0E + 01 I~I
ml~l Imlml
1.0E + 00" 1
3
5
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7
9
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I i
11
L'I P!la~talu~ ATCC 14917 l i i i i i 23 25 27 29 31 33 35
l i i i i
13
15
17 19 21 Days of ripening
37
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1.0E + 01 1.0E + 00i 1
3
5
7
9
11
13
17 19 21 Days of ripening
15
23
25
27
29
31
33
35
(A) Inhibition of growth of L. monocytogenes WSLC 1364 by Lb. plantarum ALC 01. Control cheeses were ripened with the non-pediocin AcH-producing type strain Lb. plantarum ATCC 14917. The cheeses were contaminated with 2 • 102 cfu/ml Listeria on day 1. (B) Inhibition of growth of L. monocytogenes WSLC 1364 by Lb. plantarum ALC 01 (open symbols). The contamination levels were 102 cfu/ml (squares) or 103 cfu/ml brine solution (circles). Control cheeses were contaminated with a bacteriocinresistant mutant of L. monocytogenes WSLC 1364 a (closed symbols). Ripening experiments were performed on soft cheese using a commercial, undefined multi-species microbial consortium (From Loessner et al., 2003).
Gram-negative bacteria. Linenscin OC2 lost no activity on heating at 100 ~ for 10 min, while linocin M18 was inactivated on heating to 80 ~ for 5 min but withstood heating to 50 ~ for 30 min (Valdes-Stauber and Scherer, 1994; Maisnier-Patin and Richard, 1995). The ability of linocin M18-producing strains of B. linens and undefined microbial consortia from the surface of smear cheeses to inhibit L. rnonocytogenes on deliberately inoculated cheese, was compared by Eppert etal. (1997). The results showed that the linocin-producing strains reduced the number of L. monocytogenes by only 1-2 log
cycles, while some of the microbial consortia caused complete inhibition of the Listeria. This finding implies that linocin M18 only partly inhibits the development of L. monocytogenes in smear cheeses and that other factors are also important. Martin et al. (1995) isolated three strains of B. linens from the brine used for a red-smear cheese that produced anti-listerial activity. The anti-listerial compound was stable between pH 4 and 9 and remained active after heating (80 ~ 30 min) at acid pH. No data of its use in cheese ripening were given.
Bacterial S u r f a c e - r i p e n e d C h e e s e s
The anti-listerial potential of 19 consortia of bacteria isolated from different French smear-cheese was analysed by Carnio et al. (1999). Forty-eight of 2613 isolates caused clear inhibition of one or more strains of L. monocytog,enes. In a study of 299 strains isolated from German dairy products, 30 strains inhibited at least one strain of L. monocytog,enes. Bacteria with antilisterial potential were members of the genera Arthrobacter, Brevibacterium, Corynebacterium, Enterococcus, Micrococcus, Microbacteriurn and Staphylococcus. The highly inhibitory Staph. equorurn WS 2733 was isolated from the surface of a French Raclette cheese (Carnio et al., 2000). This strain was shown to produce the antibiotic, micrococcin P1, which exhibited a bacteriostatic effect on a variety of Gram-positive bacteria. The anti-listerial potential of this strain as a protective starter culture was evaluated in in situ application in cheese-ripening experiments. A remarkable growth reduction of L. rnonocytog,enes was achieved compared to control cheese ripened with a non-bacteriocin producing strain (Fig. 3). Carminati et al. (1999) screened surface-smear organisms isolated from Taleggio cheese for their ability to inhibit L. rnonocytog,enes, Staph. aureus, E. coli and Hafnia alvei. Most of the isolates which showed anti-listerial activity (19% of the total) were coryneform bacteria, mainly Mb. lacticum. Two bacterial mixtures (containing orange-pigmented strains and strains inhibiting Listeria) were examined as surface-smear starter on commercial Taleggio cheese production; they partially but not c o r n -
217
pletely inhibited Listeria monocytogenes Ohio. However, the cheese did not ripen normally, due to a delay in growth of the surface-smear bacteria. Recently, Motta and Brandelli (2002) identified a bacteriocin produced by the red-smear cheese bacterium, B. linens ATCC 9175. A crude bacteriocin obtained from the culture supernatant fluid was inhibitory to some indicator strains, including L. monocytog,enes, but was inactive against the Gram-negative bacteria and yeasts tested. Enterococcal bacteriocms
Numerous strains of enterococci associated with food systems are capable of producing a variety of antibacterial proteins, called enterococcins, with activity against food-borne pathogens, such as L. monocytogenes, pathogenic clostridia or Staph. aureus (see Giraffa, 1995, for a review). In addition, several enterococcins have been assessed for their ability to inhibit Listeria spp. in dairy systems, where enterococci are often isolated as desirable microflora. Among nonlactic species, bacteriocins from Enterococcus spp., especially Ec. faeciurn and Ec. faecalis, are the most widely produced and characterized. Enterococcins share a number of common characteristics: (i) generally high heat stability; (ii) stability over a wide range of pH, but most often under acidic conditions; and (iii) broad spectra of activity on Gram-positive pathogenic bacteria, including L. monocytog, enes. Although enterococci are generally considered to be harmless,
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1.0E + 02
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Days of ripening Inhibition of growth of Listeria monocytogenes WSLC 1364 during ripening of cheese smeared with a bacteriocin-producing strain of S. equorum WS 2733 (open symbols) and a non-bacteriocin producing strain, DSMZ 20674 (closed symbols) after contamination at day 1 with 102 (circles) and 104 (squares) Listeria monocytogenes/ml brine (from Carnio et al., 2000).
218
Bacterial Surface-ripened Cheeses
the absence of haemolytic activity by enterococcinproducing strains should always be demonstrated. Only a few enterococcins have been assessed for their ability to prevent the growth of pathogens in model cheese systems. In preliminary experiments on Taleggio cheese, it was shown that Ec. faeciurn 7C5 bacteriocin was produced regularly in cheese during whey drainage and its activity was maintained at detectable levels until the end of ripening (Giraffa et al., 1995); an anti-listerial effect could also be observed on the surface of cheeses contaminated with 102 cfu/cm 2 L. rnonocytogenes Ohio (Giraffa and Carminati, 1997). Nu~ez et al. (1997) investigated the inhibitory effect of enterocin 4, a bacteriocin produced by Ec. faecalis INIA 4, on L. monocytogenes Ohio and Scott A during the manufacture and ripening of Manchego cheese. Counts of L. rnonocytogenes Ohio decreased significantly (3-6 log units during the first 7 days of ripening) in cheese made from milk inoculated with Ec. faecalis INIA 4, whereas L. monocytogenes Scott A was inhibited only slightly, when Ec. faecalis INIA 4 was used in combination with a commercial starter. In two different studies (Ennahar et al., 1998; Ennahar and Deschamps, 2000), the isolation of two bacteriocinproducing Ec. faecium strains from the cheese surface was reported. Both enterocin 81, produced by Ec. faeciurn WIlE 81, and enterocin A, produced by Ec. faeciurn EFM01, displayed a narrow spectrum of activity, which is directed mainly against Listeria sp. The activity of both bacteriocins was shown to be equally active at pH values ranging from 4.0 to 8.0, which is of considerable interest with regard to possible use in fermented foods. Some preliminary experiments on soft cheese have shown that enterocin 81 offers good protection against L. monocytogenes during cheese ripening (Ennahar et al., 1998).
Limitations of application of bacteriocins
The potential of bacteriocins as food preservatives is well-demonstrated. They are not only effective, but are also safe for use in food (Cleveland et al., 2001). Application studies have, however, shown that there are limitations to the usefulness of bacteriocins as antimicrobial agents. For example, nisin is unstable at pH > 5, and although bacteriocins of the pediocin family are able to extend the shelf-life of food products, full suppression of the spoilage microflora is rarely achieved. Moreover, one of the major concerns regarding the use of bacteriocins is the development of highly tolerant and/or resistant strains (Rasch and Knochel, 1998). It has been observed that Listeria develop tolerance to nisin and pediocin-like bacteriocins at a relatively high frequency in both the laboratory media and the model food systems (Ming and Daeschel, 1993; Rekhif et al., 1994; Gravesen et al., 2002; Loessner et al., 2003). Carnio et al. (1999) observed that the sensitivity of L. rnonocytogenes strains to the inhibitory activity of coryneform bacteria was quite different. On average, isolates from red-smear cheese were less sensitive to the inhibitory effects of red-smear bacteria than to animal or other food isolates. This observation indicates that selective pressure on the cheese surface might have resulted in the development of resistance mechanisms by micro-evolutionary adaption. From an applied point of view, the combined use of different bacteriocins is likely to be better than using one bacteriocin alone to prevent the growth of pathogenic bacteria. However, although bacteriocins in foods generally exhibit moderate anti-microbial activity, they are not suitable for use as a primary means of food preservation. However, they can be integrated into appropriate multi-hurdle preservation systems.
Anti-Listeria compounds of Geotrichum and Penicillium
Effects of complex smear cheese ripening consortia
As already outlined, yeasts contribute to the proper development and ripening of surface-ripened cheeses. It would be desirable if these organisms would also contribute to the inhibition of pathogenic micro-organisms during ripening. Only a few studies have discussed the possibility of using Penicillium spp. to inhibit the growth of undesirable micro-organisms in cheese (Larsen and Knochel, 1997). In that study, ten foodrelated strains of P. camemberti inhibited pathogens like L. monocytogenes. The inhibition was due to the production of acetaldehyde, benzaldehyde, 3-methylbutanal and 1-octen-3-ol. Dieuleveux and Gueguen (1998) showed that G. candidum produced compounds that inhibited L. monocytogenes, which were identified as D-3-phenyllactic acid and D-3-indollactic acid.
The undefined microbial flora derived from the surface of ripe red-smear cheeses show a strong impact on the growth of Listeria spp. This antagonistic behaviour is a stable feature of these microbial consortia, since the inhibitory effects could be reproduced with the smear of cheese produced over a period of several months to 1 year (Eppert et al., 1997). Ripening of cheeses with different undefined starters led to similar developments of pH and cell counts of yeast and bacteria, whereas development of deliberately inoculated Listeria on the cheese surface was dependent on the culture used for ripening (Eppert et al., 1997; Rudolf, 2001). In some cases, the microbial consortia inhibit Listeria almost completely (Fig. 4; Eppert et al., 1997; Maoz et al., 2002).
Bacterial Surface-ripened
Cheeses
219
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19 22 25 28 Days of ripening
MB KS WE Raclette
IIIII: III 31
34
37
40
: 43
Growth inhibition of Listeria monocytogenes by a complex consortium of soft red-smear micro-organisms from cheeses. Cheeses were artificially contaminated with 2-3 • 102 cfu Listeria monocytogenes WSLC 1364/ml brine solution at day 1 of ripening. Cheeses were obtained from German soft (MB, KS, WE) and semi-soft French (Raclette) cheese. The graph shows Listeria cells per cm 2 of cheese surface. Ripening experiments were repeated twice over a period of several months. Note that different cheese microbial consortia display very different inhibitory activity against Listeria monocytogenes (from Loessner et aL, 2003).
Nevertheless, the molecular basis of these effects is unknown. Bacteriocin production contributes to the inhibition of Listeria during the ripening of red-smear cheese, but the striking inhibitory effects observed with the industrial wash-off flora are not explained completely by bacteriocin production. Additional factors must be responsible for the inhibition of Listeria, e.g., the production of other inhibitory substances (Walstead et al., 1974) or u n k n o w n ecological interactions within the complex smear flora, such as competition and symbiotic relationships. A project partly funded by the EU is currently u n d e r w a y with two major objectives, viz., to obtain a clearer u n d e r s t a n d i n g of the surface microflora of five different cheeses, Limburger, Reblochon, Livarot, Tilsit and Gubbeeen, and to identify strains of yeast which have anti-listerial activity. Preliminary results show that such yeasts exist (http://www.teagasc.ie/ research/dprc/smearcheese.htm).
The surface of a smear-ripened cheese is a very complex microbial ecosystem and this review has discussed the ripening of such cheeses in terms of the microflora, paying particular attention to Gram-positive, catalase-positive, salt-tolerant bacteria and the problems associated with the development of pathogenic bacteria, particularly L. monocytogenes. It is likely that on all bacterial smear-ripened cheese, yeasts dominate during the early stages of
ripening, where they metabolize the lactic acid produced by the starter bacteria and produce an increase in the pH of the cheese surface. The most c o m m o n yeast is D. hansenii, followed by Kluyveromyces lactis, Geotrichum candidum and Yarrowia lipolytica. The bacteria on the surface of smear-ripened cheeses are Gram-positive, catalase-positive, salt-tolerant bacteria, which can be divided into two categories, coryneforms and staphylococci. The major difficulty in identifying these micro-organisms is that coryneform bacteria, as a whole, are not well defined taxonomically. Only after resolving their taxonomy can in-depth studies on the bacteria present in the smear and how these organisms interact with each other and their contribution to ripening be undertaken.
Financial support from the EU under contract QLK1CT-2001-02228 is gratefully acknowledged.
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220
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Motta, A.S. and Brandelli, A. (2002). Characterization of an antibacterial peptide produced by Brevibacterium linens. J. Appl. Microbiol. 92, 63-70. Mulder, E., Adamse, A., Autheunisse, J., Deinema, M., Woldendorp, J. and Zevenhuizen, L. (1966). The relationship between Brevibacterium linens and bacteria of the genus Arthrobacter. J. Appl. Bacteriol. 29, 44-71. Muriana, P.M. (1996). Bacteriocins for control of Listeria spp. in foods. J. Food Protect. Suppl. 54-63. Nettles, C.G. and Barefoot, S.E (1993). Biochemical and genetic characteristics of bacteriocins of food-associated lactic acid bacteria. J. Food Prot. 56,338-356. Nooitgetagt, A.J. and Hartog, B.J. (1988). A survey of the microbiological quality of Brie and Camembert cheese. Neth. Milk Dairy J. 42, 57-72. Nu hez, M., Rodriguez, J.L., Garcia, E., Gaya, P. and Medina, M. (1997). Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture and ripening of Manchego cheese. J. Appl. Microbiol. 83,671-677. Oberreuter, H., Seiler, H. and Scherer, S. (2002). Identification of coryneform bacteria and related taxa by Fouriertransform infrared (FT-IR) spectroscopy. Int. J. Syst. Evol. Microbiol. 52, 91-100. Otero, A., Garcia, M.C., Garcia, M.L., Santos, J.A. and Moreno, B. (1993). Behaviour of Staphylococcus aureus strains FRI 137 and FRI 361 during the manufacture and ripening of Manchego cheese. Int. Dairy J. 33, 85-86. Pereira, M.L., Do Carmo, L.S., Dos Santos, E.J., Pereira, J.L. and Bergdoll, M.S. (1996). Enterotoxin H in staphylococcal food poisoning. J. Food Prot. 59, 559-561. Petersen, K.M., Westhall, S. and Jespersen, L. (2002). Microbial succession of Debaryomyces hansenii strains during production of Danish surface-ripened cheeses. J. Dairy Sci. 85,478-486. Piantanida, L., Vallone, L. and Cantoni, C. (1996). Colorazioni superficiali del formaggio Taleggio. Industrie Alimentari 35,147-148. Pini, P.N. and Gilbert, R.J. (1988). The occurence in the UK of Listeria species in raw chickens and soft cheeses. Int. J. Food Microbiol. 6, 317-326. Piton-Malleret, C. and Gorrieri, M. (1992). Nature et variabilite de la flore microbienne dans la morge des fromages de Comte et de Beaufort. Lait 72, 143-164. Place, R.B., Hiestand, D., Burri, S. and Teuber, M. (2002). Staphyloccus succinus subsp, casei subsp, nov., a dominant isolate from a surface ripened cheese. Syst. Appl. Microbiol. 25,353-359. Purko, M., Nelson, W.O. and Wood, W.A. (1951a). The associative action between certain yeast and Bacterium linens. J. Dairy Sci. 34, 699-705. Purko, M., Nelson, W.O. and Wood, W.A. (1951b). The equivalence of pantothenic acid and p-aminobenzoic acid for growth of Bacterium linens. J. Dairy Sci. 34, 874-878. Quadri, L.E.N., Sailer, M., Roy, K.L., Vederas, J.C. and Stiles, M.E. (1994). Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B. J. Biol. Chem. 269, 12204-12211. Rampling, A. (1996). Raw milk cheese and Salmonella. Br. Med. J. 312, 67-68.
Rasch, M. and Knochel, S. (1998). Variations in tolerance of Listeria monocytogenes to nisin, pediocin PA-1 and bavaricin A. Lett. Appl. Microbiol. 27, 275-278. Rattray, EP. and Fox, P.E (1997). Purification and characterization of an intracellular aminopeptidase from Brevibacterium linens ATCC 9174. Lait 77, 169-180. Rattray, EP. and Fox, RE (1999). Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review. J. Dairy Sci. 82, 891-909. Rattray, EP., Bockelmann, W. and Fox, RE (1995). Purification and characterization of an extracellular proteinase from Brevibacterium linens ATCC 9174. Appl. Environ. Microbiol. 61, 3454-3456. Rekhif, N., Atrih, A. and Levebre, G. (1994). Selection and spontaneous mutants of Listeria monocytogenes ATCC 15313 resistant to different bacteriocins produced by lactic acid bacteria strains. Curt. Microbiol. 28,237-241. Reps, A. (1993). Bacterial surface-ripenend cheesese, in, Cheese: Chemistry, Physics and Microbiology, 2nd edn, Fox, P.E, ed., Vol. 2, Chapman & Hall, London. pp. 37-172. Robinson, R.K., ed. (1995). A Colour Guide to Cheese and Fermented Milks. Chapman & Hall, London. Rocourt, J. (1994). Listeria monocytogenes: the state of the science. Dairy, Food Environ. Sanit. 14, 70-82. Rogers, L.A. (1928). The inhibiting effect of Streptococcus lactis on Lactobacillus bulgaricus. J. Bacteriol. 16, 321-325. Rorvik, L.M. and Yndestad, M. (1991). Listeria monocytogenes in foods in Norway. Int. J. Food Microbiol. 13, 97-104. Rudolf, M. (2001). Listeria monocytogenes: Vorkommen in oberfldchengereiften Weichkdsen und Entwicklung antagonistischer Reifungskulturen. Thesis, Technical University, Munich. Rudolf, M. and Scherer, S. (2000). Incidence of Listeria and Listeria monocytogenes in acid curd cheese. Arch. Lebens. 51,118-120. Rudolf, M. and Scherer, S. (2001). High incidence of Listeria monocytogenes in European red smear cheese. Int. J. Food Microbiol. 63, 91-98. Ryser, E.T. (1999). Incidence and behaviour of Listeria monocytogenes in cheese and other fermented dairy products in, Listeria, Listeriosis, and Food Safety, Ryser, E.T. and Marth, E.H., eds, Vol. 2, Marcel Dekker, Inc., New York. pp. 411-503. Ryser, E.T., Maisnier-Patin, S., Gratadoux, J.J. and Richard, J. (1994). Isolation and identification of cheese-smear bacteria inhibitory to Listeria spp. Int. J. Food Microbiol. 21, 237-246. Schleifer, K.H., Kilpper-Baelz, R., Fischer, U., Faller, A. and Endl, J. (1982). Identification of Micrococcus candidus ATCC 14852 as a strain of Staphylococcus epidermidis and of Micrococcus caseolyticus ATCC 13548 and Micrococcus varians ATCC 29750 as Staphylococcus caseolyticus new species. Int. J. Syst. Bact. 32, 15-20. Schubert, K., Ludwig, W., Springer, N., Kroppenstedt, R.M., Accolas, J.P. and Fiedler, E (1996). Two coryneform bacteria isolated from the surface of French Gruyere and Beaufort cheese are new species of the genus Brachybacterium; Brachybacterium alimentarium sp. nov. and
Bacterial Surface-ripened Cheeses
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Todd, E., Szabo, R., Gardiner, M.A., Aktar, M., Delorme, L., Tourillon, P., Rochefort, J., Roy, D., Loit, T., Lamontagne, Y., Gosselin, L., Martineau, G. and Breton, J.P. (1981). Intoxication staphylococciqu liee a du caille de fromagerie- Quebec. Rapport Hebdomadaire des Maladies au Canada 7, 171-172. Valdes-Stauber, N. and Scherer, S. (1994). Isolation and characterization of Linocin M18, a bacteriocin produced by Brevibacterium linens. Appl. Environ. Microbiol. 60, 3809-3814. Valdes-Stauber, N. and Scherer, S. (1996). Nucleotide sequence and taxonomical distribution of the bacteriocine gene lin cloned from Brevibacterium linens M18. Appl. Environ. Microbiol. 62, 1283-1286. Valdes-Stauber, N., GOtz, H. and Busse, M. (1991). Antagonistic effects of coryneform bacteria from red smear cheese against Listeria. Int. J. Food. Microbiol. 13, 119-130. Valdes-Stauber, N., Scherer, S. and Seller, H. (1997). Identification of yeast and coryneform bacteria from the surface microflora of Brick cheese. Int. J. Food. Microbiol. 34, 115-129. Vernozy-Rozand, C., Mazuy, C., Meugnier, H., Bes, M., Lasne, Y., Fiedler, E, Etienne, J. and Freney, J. (2000). Staphylococcus fleuretti sp. nov., isolated from goat's milk cheeses. Int. J. Syst. Evol. Microbiol. 50, 1521-1527. Vorobjeva, L.I. (1999). Economic and medical applications, in, Propionibacteria, Vorobjewa, L.I., ed., Kluwer Academic Publishers, The Netherlands. pp. 209-243. Waisted, D.L., Reitz, R.C. and Sparling, RE (1974). Growth inhibition among strains of Neisseria gonorrhoeae due to production of inhibitory free fatty acids and lysophosphatidylethanolamine: absence of bacteriocin. Infect. Immunol. 10,481-488. Weber, A., Baumann, C., Potel, J. and Friess, H. (1988). Incidence of Listeria monocytogenes and Listeria innocua in cheese. Berl. MRnch. Tiergirztl. Wschr. 101,373-375. Weimer, B., Seefeldt, K. and Dias, B. (1999). Sulfur metabolism in bacteria associated with cheese. Antonie. van Leeuwenhoek 76,247-261. Wolff, A. (1910). Milchwirtschaftliche Bakteriologie. Cent. f. Bakt. Abt II 28,417. Wyder, M.T. and Puhan, Z. (1999). Investigation of the yeast flora in smear ripened cheeses. Milchwissenschaft 54, 330-333.
Cheese Varieties Ripened in Brine M.H. Abd EI-Salam, Dairy Department, National Research Centre, Dokki, Cairo, Egypt E. Alichanidis, Laboratory of Dairy Technology, School of Agriculture, Aristotle University of Thessaloniki, Greece
Cheeses ripened in brine are the oldest known group of cheeses (Scott, 1986). Traditionally, the manufacture of these cheeses was limited to the Mediterranean basin and the Balkans. However, their production has been extended to several parts of the world as a result of their popularity and increased demand in the international market (Mann, 1999). The manufacture of cheeses ripened in brine was carried out for centuries on a small scale, which is difficult to standardize. However, the last decades of the twentieth century have witnessed the development of large-scale, mechanized and standardized production of cheeses ripened in brine. Advanced technologies have been adopted for their production, including ultrafiltration (UF) techniques. It has been estimated that UF-Feta cheese represented ~56% of the total UF cheeses produced throughout the world (Jensen et al., 1988). These developments improved and better defined the characteristics of cheeses ripened in brine. Cheeses ripened in brine can be defined as those preserved in brine (pickle) from manufacture until they reach the consumer. They are characterized by the following: 9 Rindless, manufactured in various shapes and sizes but usually in pieces of less than 1 kg. 9 Clean, acid and salty taste when fresh; the ripened cheese has a sharp piquant flavour. 9 The cheese and brine have a high salt content which bestows good keeping quality in hot climates. 9 White colour arising from the use of sheep, goat or buffalo milk in their manufacture. When cows' milk is used to make cheese ripened in brine, methods are used to decolourize cows' milk fat in order to obtain the desired white colour. 9 Changes in the composition and properties of cheese during ripening and those of the brine used are interrelated. 9 Most varieties in this group are stored in sealed containers but some are stored in gas-permeable containers, which affect the biochemical changes which occur during ripening.
In this chapter, we will describe different cheeses ripened in brine, but special emphasis will be placed on Domiati and Feta cheeses, which are the most important members of this group and are recognized in international markets. Besides, more information about Domiati and Feta cheeses is available in the literature than other varieties ripened in brine.
Most cheeses ripened in brine are not well defined, which usually create problems in their classification. In many cases, cheese is described as 'white pickled cheese', a generic name that can apply to all cheeses ripened in brine. In addition, wide variations are found in the composition and texture of cheeses ripened in brine. Therefore, classification of these cheeses is necessary. The general systems used to classify cheese can be adapted for the classification of cheeses ripened in brine, as follows (country of origin is given in parentheses): 1. Soft cheeses (moisture content, 55-65%) 1.1. Acid-coagulated: Mish (Egypt). 1.2. Rennet-coagulated: 1.2.1. Salting of cheese curd (Feta type): Feta (Greece), Teleme (Romania), Brinza (Russia, Israel), Bli-sir-U-kriskama (Serbia), Bjalo or Belo Samureno sirene (Bulgaria), Chanakh (Russia), Beyaz peynir (Turkey), Akawi (Syria), Baida (Lebanon), Iranian white cheese (Iran). 1.2.2. Salting of cheese milk (Domiati type): Domiati (Egypt), Dani (Egypt; a variant of Domiati cheese made from sheep's milk), Gibna bayda (Sudan). 2. Semi-hard cheeses (moisture content, 45-55%): Halloumi (Cyprus), Braided Meddafara and Magdula (Syria, Sudan), Nabulsi (Jordan).
Domiati cheese can be considered as the most important cheese ripened in brine in the Middle East in terms of the quantity of cheese produced or available
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
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228
Cheese Varieties Ripened in Brine
information. It is unique among cheeses ripened in brine in the addition of a large quantity of NaC1 (8-15%) to the milk before renneting. This results in: 9 Partial solubilization of the colloidal calcium phosphate (Puri and Parkash, 1965). Addition of NaC1 up to 6% to cows' milk causes a significant increase in the level of soluble calcium (P < 0.001) (Elzeny, 1991). 9 An increase in the acidity of milk (Abd E1-Hamid et al., 1981) and a decrease in its pH (Elzeny, 1991). This has been attributed to a cation exchange reaction of Na + for H+. 9 The rennet coagulation time of cows', buffaloes', goats' and sheep's milk increases with the amount of NaC1 added up to 7.5-10% but decreases slightly at a higher level of added salt (Abd E1-Hamid et al., 1981). Aggregation of casein micelles is enhanced at a high NaC1 concentration, being dependent on the size of the micelles and the time of exposure to salt (Elzeny, 1991). 9 Addition of NaC1 (up to 1 M) to milk or casein micelles of variable sizes dispersed in simulated milk ultrafiltrate (SMUF) (Saito and Hirose, 1972; Abd E1-Salam et al., 1978) reduces the turbidity of the system due to a decrease in the average micellar size and increased non-preferential solubilization of casein fractions. 9 Casein micelles in salted milk have an irregular shape rather than the spherical shape characteristic of normal micelles (Elzeny, 1991). 9 The extent of hydrolysis of K-casein by rennet decreases by 43 and 61% on addition of 5 and 10% NaC1 to cows' milk, respectively (Elzeny, 1991). 9 Addition of NaC1 reduces the action of the coagulant; chymosin is affected less than Rhizomucor rneihei protease (Ibrahim et al., 1973). The overall effect of adding NaC1 to milk before renneting is the need for more rennet and a longer coagulation time for Domiati cheese compared to other cheeses. In addition, the coagulum formed is usually weak and therefore is ladled into moulds without cutting, and whey is permitted to drain for a long time (24 h). The cheese is consumed either fresh or after ripening in sealed tins under salted whey from the same cheese. Ripening usually occurs at room temperature (20 _+ 5 ~ Details of the manufacture of Domiati cheese have been described (Fahmi and Sharara, 1950; Abd E1-Salam et al., 1976; Abou Donia, 1991).
acterized by a relatively high pH (6.0-6.5), and high levels of moisture (60-65%) and NaC1 (5-8%). Changes in the gross composition of Domiati during storage are summarized in Table 1. The developed acidity strongly determines the changes in the gross composition of Domiati during storage. Acidification brings the pH of cheese close to the isoelectric point of casein and partially solubilizes colloidal calcium phosphate, which causes shrinkage of the cheese matrix and exudation of cheese serum into the brine (Hamed, 1955). The pH of ripened Domiati reaches as low as 3.3 as a result of two factors: the high lactose content of fresh cheese and the continuous supply of lactose from the salted whey used as brine for bacterial fermentation (Ahmed et al., 1972; Tawab et al., 1975). The use of salted whey diluted with aqueous NaC1 solution as brine reduces acid development in Domiati (E1-Abd et al., 1975). The available lactose (in cheese and brine) is more than that of which the cheese microflora can utilize, which explains the high level of residual lactose in ripened Domiati (Table 2). Lactose and galactose are found in Domiati even after 6 months of storage, but glucose is not detected (Abd E1-Salam, unpublished). It seems that the pathway for lactose fermentation by starter organisms in Domiati is similar to that used by yoghurt starters (Dellaglio, 1988). Several interacting variables affect the changes in the general composition and acid development in Domiati during storage. These can be summarized as follows. Type milk ever, goat, have et al.,
of milk. Domiati is made mainly from buffalo's or mixtures of buffalos' and cows' milks. Howthe use of reconstituted or recombined milks, sheep and even camel milk for Domiati cheese been described (Abou Donia, 1991; Kandeel 1991; Mehaia, 1993).
Summary of changes that occur in Domiati cheese during ripening
Constituent
Changes d u r i n g storage
Responsible factor(s)
Moisture
Decrease (about 2-3%)
Fat-in-drymatter
Increase (3-6%)
Changes during ripening
Acidity
Increase (1.0-1.5%)
General composition Extensive data are found in the literature concerning the moisture, fat, salt, pH and acidity of Domiati cheese during storage in brine. Fresh Domiati is char-
pH
Decrease (2-2.5)
Lactose
Decrease (1.5-2.0%)
Exudation of cheese serum Decrease in solidsnot-fat Lactic acid fermentation Lactic acid fermentation Lactic acid fermentation
C h e e s e Varieties R i p e n e d in Brine
Changes in the carbohydrate content of Domiati cheese during ripening (%, as lactose) Storage period (days) Fresh
15
30
120
180
Reference
3.5 ND
3.40 2.09
2.85 1.84
1.65 ND
0.54
Ahmed et al. (1972) Tawab et al. (1975)
ND, not determined.
Domiati from buffalo milk (unstandardized) contains more fat in dry matter (FDM), less moisture and lower developed acidity than cheese made from cow or goat milk. The use of reconstituted or recombined milk reduces the moisture content of Domiati (E1-Safty, 1969; Hagrass, 1971). However, raising the reconstitution ratio (i.e., higher total solids in cheese milk) increases the moisture content of the cheese (Moneib et al., 1981). Ripening temperature. Ripening at a low temperature reduces the rate of biochemical and microbiological changes in Domiati as apparent from the slow rate of acid development compared to cheese stored at room temperature (Abou Dawood, 1964; Teama, 1967). The moisture content of Domiati increases during early storage at a low temperature. This has been attributed to the relatively high pH of fresh cheese, which increases the hydration of the caseins at low temperature.
R i p e n i n g period. There is no standard period for the ripening of Domiati cheese. However, 3-4 months storage in brine at room temperature gives a good quality product. The composition of Domiati changes continuously during storage with the highest rate during the first month, which coincides with the growth of the cheese microflora (Abou Dawood, 1964; E1-Koussy, 1966; Ahmed et al., 1972; Naguib et al., 1974). Method of ripening. Storage in pouches or cans without brine has been suggested for Domiati, during which the acidity develops faster than cheese stored in brine (Abd E1-Salam et al., 1981; A1-Khamy, 1988; Gomaa, 1990).
229
Salt content. Variable levels of NaC1 are added to the milk for Domiati cheesemaking depending on season, quality of the milk and duration and temperature of ripening. The higher the percentage of salt added to the milk, the higher the moisture and the lower the extent of acid development in the cheese during ripening (Table 3). Heat treatment of milk.
Pasteurization of the cheese
milk has little effect on the gross composition of Domiati (Sharara, 1962; Teama, 1967; Naguib et al., 1974); a slight increase in moisture content and a slight decrease in acid development are apparent. Oltrafiltration. The manufacture of UF-Domiati has been described (Abd E1-Salam et al., 1981, 1982; Abd E1Salam, 1988; Gomaa, 1990; Hofi et al., 2001). The moisture content of UF-Domiati is usually higher and the fat content lower than those of traditional Domiati (Abd E1Salam et al., 1981; A1-Khamy, 1988; Gomaa, 1990) due to the poor syneresis and the high water-holding capacity of whey proteins retained in UF cheeses (Table 4).
Additives. Addition of lecithin to cheese milk increases the moisture content and acid development in Domiati (E1-Abbassy et al., 1991). The partial replacement of NaC1 by KC1 has no significant effect on the composition or pH of Domiati cheese (Ramadan, 1995). Oroteolysis
Domiati cheese undergoes continuous proteolysis during ripening in brine. Generally, the total nitrogen (TN) content of cheese decreases gradually due to the transfer of degradation products to the brine by diffusion, while the soluble nitrogen fractions of cheese increase continuously, indicating continuous proteolysis. Several factors affect proteolysis in Domiati cheese, as summarized in Table 5. The rennet contributes much to proteolysis in Domiati. This is due to the high concentration of rennet needed to coagulate salted milk (compared to most cheese varieties), the high level of rennet retained in cheese curd and to the storage in salted whey, which contains rennet. In Domiati, Otsl-casein is hydrolysed rapidly, while [3-casein is resistant to hydrolysis (Abd E1-Salam and E1-Shibiny, 1972; E1-Shibiny and Abd
Gross composition of Domiati cheese as affected by the level of NaCI in milk (Zaki et al., 1974) 8% NaCI
Moisture, % FDM, % Acidity, %
10% NaCI
Fresh
3 months
Fresh
58.6 34.6 0.27
51.4 49.7 2.24
59.2 35.0 0.24
FDM, fat in dry matter.
12% NaCI 3 months
52.2 48.2 2.02
15% NaCI
Fresh
3 months
Fresh
3 months
60.9 32.8 0.21
54.5 48.7 1.42
61.7 31.8 0.11
55.8 45.5 1.00
230
C h e e s e Varieties R i p e n e d in Brine
Gross composition of 30-day-old Domiati cheese made from cows' or buffaloes' milk by conventional or ultrafiltration techniques (Abd EI-Salam et aL, 1981) Conventional
Moisture, % FDM, % pH
Ultrafiltration
Cow
Buffalo
Cow
Buffalo
55.77 45.35 4.55
54.82 49.71 4.70
58.72 42.88 5.15
57.29 46.82 4.91
FDM, fat in dry matter.
E1-Salam, 1974; Abd E1-Salam et al., 1983; Mehanna et al., 1983). This pattern of changes arises from the action of rennet on cheese as affected by salt content (Fox and Walley, 1971). The high ionic strength and high ripening temperature seem to enhance the polymerization of [3-casein in Domiati via hydrophobic interactions and render it less susceptible to rennet action. The ]3/Otsl-casein ratio in Domiati increases continuously during ripening (Abd E1-Salam et al., 1983) and, after extended ripening, the water-insoluble proteins are mainly ]3-caseins which explain partially the soft body and texture of ripened Domiati, as reported for Cheddar cheese by Creamer and Olson (1982). A number of degradation products with high or low electrophoretic mobility are apparent in the electrophoretic pattern of the proteins and polypeptides of Domiati, including e~s]-casein (f24-199) (Ramos et al., 1988) produced from e~s]-casein by the action of chymosin, and the y-caseins produced from [3-casein by the action of plasmin (Eigel, 1977). The use of milk clotting enzymes other than calf rennet alters the pattern of proteolysis in Domiati (Abdou et al., 1976), but the use of different starters has only a slight effect (Abd E1-Salam et al., 1983). Analysis of the soluble nitrogenous compounds by gel permeation chromatography (Abd E1-Salam and E1-Shibiny, 1972)
Factors that affect proteolysis in Domiati cheese Increases proteolysis
Retards proteolysis
Cow milk > buffalo milk Homogenization Addition of denatured whey proteins, phosphate, citrate, capsicum tincture, cheese slurry Ultrafiltration
Low temperature storage Heat treatment of cheese milk H202-catalase treatment of milk
Direct acidification Salt-tolerant starters Storage in pouches
Use of reconstituted/recombined milk Increase in NaCI content
showed that this fraction consists mainly of low molecular weight components (amino acids and small peptides). Comparison of the free amino acid pattern in Domiati (E1-Erian et al., 1974) with the amino acid profile of cow and buffalo caseins reveals a marked reduction in the concentration of glutamic acid and the formation of y-amino butyric acid through deamination reactions. Also, arginine is almost absent, having been converted to ornithine. The ripened cheese has a high concentration of ammonia, which indicates the significance of deamination reactions in Domiati and which contributes to flavour development in this type of cheese. The concentration of biogenic amines in Domiati is very low (Mehanna et al., 1989). Tyramine is the principal biogenic amine found in Domiati, together with low concentrations of histamine, tryptamine, phenylethylamine and putrescine. Proteins of Domiati cheese seem to undergo three levels of proteolysis, as illustrated in Fig 1. The key point is that the soluble products diffuse into the brine to attain equilibrium with their concentration in the cheese. Lipolysis
Data on the volatile acids in Domiati cheese have been recalculated as acetic acid (Table 6) which is the principal volatile acid in Domiati (E1-Shibinyet al., 1974). Most of the changes in the volatile acids occur during the first month of ripening, which coincides with maximum bacterial growth (Naguib et al., 1974; Shehata et al., 1984). The pattern of free fatty acids in Domiati is comparable to the fatty acid profile of triglycerides in milk fat (Table 7), suggesting non-specific lipolysis. Analysis of glycerides from ripened Domiati cheese also indicates lipolysis (Precht and Abd E1-Salam, 1985). However, the origin of lipases responsible for fat hydrolysis in Domiati is not clear. The contribution of free fatty acids to flavour development in Domiati has been confirmed from the analysis of cheese of different fat contents (E1-Shibiny et al., 1974). Measurements of peroxide and thiobarbituric acid values indicate that fat oxidation occurs in Domiati cheese during storage (Hamed et al., 1987). Vitamin content
Almost all the vitamin A in milk is retained in Domiati and remains stable during ripening (Sabry and Guerrant, 1958). On the other hand, variable percentages of thiamine, riboflavin, niacin (Sabry and Guerrant, 1958), biotin, vitamin B12 and folic acid (Khattab and Zaki, 1986) are retained in fresh cheese. According to Sabry and Guerrant (1958), the level of biotin, vitamin B12 and folic acid remain unchanged
Cheese Varieties Ripened in Brine
First level
Fresh cheese proteins (%1, O~s2,~, par&K-casein)
Salted whey (brine)
231
Proteinases -mainly residual rennet -milk proteinase (plasmin)
Diffusion
Equilibrium =
Water-soluble components (mainly peptides) Insoluble cheese proteins (O~sl , O~s2, ~,
par&K-casein, large peptides)
Second level
Bacterial enzymes -peptidases -carboxypeptidases -aminopeptidases
Diffusion Equilibrium
Small peptides
Diffusion Equilibrium
Amino acids
Bacterial enzymes -deaminases (mainly) -decarboxylases
Diffusion
Third level
Ammonia, carboxylic acids, amines, C O 2
Equilibrium Schematic representation of proteolysis in Domiati cheese.
during storage while changes in riboflavin and niacin depend on storage conditions. Volatile flavour compounds In addition to the volatile fatty acids, the concentrations of acidic and neutral carbonyls increase in Domiati dur-
ing storage (Magdoub et al., 1983). Several aldehydes, ketones, alcohols, esters, sulphur compounds and hydrocarbons are found in the volatiles of Domiati cheese. Forty-four of these compounds have been identified using a dynamic headspace GC-MS technique (Collin et al., 1993). Most of these volatiles are formed
232
Cheese Varieties Ripened in Brine
Production of volatile fatty acids in Domiati cheese made from buffaloes' milk during ripening (expressed as acetic acid, %)
Ripening period (days) Coagulant
Fresh
15
30
60
90
Rennet a
0.073 0.073 0.034 0.044 0.052 0.029
0.150 0.121 0.089 0.092 0.084 0.078
0.158 0.142 0.107 0.097 0.105 0.107
0.173 0.184 0.109 0.107 0.108 0.118
0.179 0.219 0.111 0.109 0.111 0.120
R. pusillus protease a Calf rennet b Bovine pepsin b R. meihei protease b C. parasitica protease b
a EI-Safty and EI-Shibiny, 1980. b Abdou et aL, 1976.
after 2 months of maturation. Acrolein, butan-2-one, propan-l-ol, butan-2-ol, ethyl propionate, propyl acetate, ethyl butyrate, propyl propionate and propyl butyrate are found in good quality Domiati cheese. Various sulphur compounds are also found at low concentrations in good quality ripened Domiati cheese, but high concentrations of these compounds are associated with inferior quality cheese (Collin et al., 1993). Changes M the concentration of brine
The composition of salted whey used for the ripening of Domiati cheese changes continuously during maturation. This is attributed to the chemical and biochemical changes that occur in cheese and the diffusion of soluble constituents to attain equilibrium in their partition between cheese and brine. The following factors control the changes in the composition of brine: 9 Composition of the fresh cheese and brine. 9 Rate of the biochemical changes in Domiati, which are controlled by several factors. 9 Ratio of cheese to brine (usually 5-6:1). The volume of brine surrounding Domiati stored at room temperature increases (12.5%), and about 70% of the solids lost from the cheese during the first month of storage appear in the brine (Hamed, 1955). A further 5.9% of cheese solids are lost during the
Average proportion* of free fatty acids in Domiati cheese (Ramos et al., 1988)
C4:0
C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1
3.80
8.72
5.83
3.32
Total free fatty acids (mg/kg) * % of total free fatty acids.
4.03
12.55 31.53 2308
8.34
21.66
subsequent 2 months of ripening due to partial exudation of cheese serum into brine as a result of acid production and shrinkage of the cheese matrix. On the other hand, the volume of brine decreases during early ripening of Domiati cheese at a low temperature (Teama, 1967) through the hydration of the caseins. However, further ripening at low temperature is accompanied by an increase in the volume of brine and changes in its composition. The TN content of brine increases continuously during storage. However, the rate and extent of this increase is affected by the salt content and storage temperature (Teama, 1967) and homogenization (Ahmed et al., 1972). The concentrations of Ca and PO4 in the brine also increase initially but remain almost constant after 2 months of storage coinciding with changes in pH and acidity. About 25-30% of the Ca and PO4 in cheese are released into the brine (Ahmed et al., 1972). The salted whey used as a brine for Domiati cheese contains a small amount of fat, the level of which increases slightly with increasing salt content and heat treatment of the milk used for cheesemaking. Both factors weaken the cheese matrix and increase the loss of fat in the whey (Teama, 1967). However, the fat content of the brine changes very little during storage. The NaC1 content of the brine changes during early storage to attain an equilibrium between brine and cheese. Equilibration also occurs in the distribution of lactose and lactate. The brine used for Domiati usually contains a significant amount of lactose even after 4 months of storage (Ahmed et al., 1972). Texture and microstructure
The combined effect of decreasing pH and adding NaC1 to milk reduces significantly all textural parameters (elasticity, hardness, brittleness, adhesiveness, chewiness and gumminess) of Domiati cheese and increases its water retention (Elzeny, 1991). However, the textural parameters increase significantly with increasing rennet concentration, renneting temperature and addition of CaC12 to the milk (Elzeny, 1991). Maximum textural parameters (elasticity modulus, 1.1 x 10 N/m2; hardness, 0.9 kg; brittleness, 0.6 kg; elasticity, 30.9%; cohesiveness, 59.5; chewiness, 0.4 and gumminess, 1.2) were obtained at a milk pH of 6.6, a clotting temperature of 39 ~ a rennet concentration of 0.09% (15 000 SU) and a CaC12 level of 0.02% (Elzeny, 1991). The effect of composition and pH on textural parameters are in the following descending order: pH > NaC1 > protein > fat > moisture (Zaki, 1990). The textural characteristics of fresh Domiati (UF or traditional) cheese are significantly different. Ultrafiltration-Domiati is harder and more adhesive than the traditional cheese, while the latter is more chewy
Cheese Varieties Ripened in Brine
and gummy (Gomaa, 1990). Both types of cheese increase in hardness, adhesiveness and gumminess during the early stages of ripening, followed by a decrease in these parameters after 3 months of ripening in brine. However, traditional Domiati is more elastic than UFDomiati throughout ripening (Gomaa, 1990). It seems that the increase in the textural parameters during early ripening is related to the decrease in moisture and pH, leading to a firmer texture. During the latter stages, changes in texture are related more to changes in the protein matrix, due to proteolysis, particularly of Otsl-casein, and the loss of Ca. The textural parameters of Domiati are also related to the method of storage, i.e., in pouches without brine or in brine in cans. Cheese stored in pouches is significantly harder, more cohesive and gummy than cheese stored in brine (Gomaa, 1990). Also, the hardness of Domiati made from milk supplemented with whey protein concentrate (WPC) decreases as the level of WPC is increased (Gomaa, 1990). The hardness of flesh UF-Domiati can be controlled by changing the homogenization pressure, heat treatment and pH of the pre-cheese (A1-Khamy, 1988). Increasing the homogenization pressure and heat treatment, and reducing the pH of the pre-cheese increase the hardness of flesh UF-Domiati cheese (A1-Khamy, 1988). Electron microscopy of ultra-thin sections of Domiati (Abd E1-Salam and E1-Shibiny, 1973; Hofi et al., 2001) indicates that the internal structure of fresh cheese is a framework of spherical casein aggregates held together by bridges and occluding fat. On storage in brine, the casein aggregates dissociate into smaller spherical particles, forming looser structure. Differences have been observed in the microstructure of traditional and UF-Domiati (Hofi et al., 2001). The protein matrix of UF-Domiati is characterized by denser and bigger protein aggregates in which whey proteins are included with casein in the protein matrix. Additional proof that changes occur in the microstructure of Domiati cheese during storage was provided by scanning electron microscopy (Kerr et al., 1981; Zaki, 1990). The high salt content has little effect on the morphological characteristics of the surface of the cheese and fat globules per se are unlikely to be changed during storage. Most of the changes occur in the protein matrix. In fresh Domiati, hydrophobic interactions between casein molecules seem to be dominant and overcome the repulsive forces from the negatively charged protein matrix due to the relatively high pH (>5.8) of the cheese. The partial exchange of Na + for Ca 2+ weakens the strong interactions in the casein aggregates.
233
Microbiology Micro-organisms present Lactic acid bacteria are predominant in Domiati; lactococci grow during early storage and later lactobacilli (Naguib et al., 1974; Shehata et al., 1984). Salt-tolerant enterococci are the predominant cocci (94.5% of isolated cocci) in ripened Domiati (Hemati et al., 1998). Enterococcus faecalis, E. faecium, Lc. lactis subsp, lactis, Lc. lactis subsp, cremoris, Lb. casei, Lb. plantarum, Lb. brevis, Lb. fermentum, Lb. delbruekii subsp, lactis, Lb. alimentarius, Leuconostoc mesenteroides subsp, cremoris, Brevibacterium linens and Propionibacterium jensenii have been found in Domiati cheese (Naguib, 1965; Shehata et al., 1984; E1Zayat et al., 1995). Yeasts of the genera Trichosporon,
Saccharomyces, Pichia, Debaryomyces, Hansenula, Torulopsis, Endomycopsis and Cryptococcus are also found in Domiati (Ghoniem, 1968; Seham et al., 1982). Effect of manufacturing and ripening on cheese micoflora Raw milk Domiati generally has a higher bacterial count than cheese made from pasteurized milk during the first month of ripening, but cheeses made from both milks have similar counts thereafter (Naguib etal., 1974). The total microbial count increases rapidly to a maximum after a week of storage and then declines. Lactococci behave similarly, but disappear after 2-3 months of ripening. Lactobacilli reach a maximum after 2-4 weeks and then decrease gradually (Helmy, 1960; Naguib et al., 1974). The high salt content of the cheese milk reduces the total microbial and groups counts in Domiati (Shehata etal., 1984). Micrococci and lactobacilli are equally important in Domiati with a high salt content (Helmy, 1960). Starters Traditionally, starters are not used in the manufacture of Domiati cheese. Several attempts have been made to isolate salt-tolerant organisms from ripened Domiati for use as starters. These include Enterococcus faecalis, Pedicoccus spp., Lb. mesenteroides and Lb. casei (E1Gendy etal., 1983). The enterococci isolated from Domiati cheese have high esterolytic and autolytic activities and they can grow well in a medium with 9.5-10.0% NaC1 (Hemati et al., 1997). They are considered to be suitable starters for Domiati made from pasteurized milk. Survival of harmful organisms The presence of coliforms in Domiati is related to the level of salt added to the cheese milk. Not less than 9.5% NaC1 should be added to milk to suppress the growth of coliforms in Domiati made from raw milk (E1-Sadek and Eissa, 1956; Hegazi, 1972).
234
Cheese Varieties Ripened in Brine
Campylobacter spp. are present in Domiati, but C. jejuni has not been detected (E1-Nokrashy et al., 1998). However, added C. jejuni can survive for 21 days in Domiati made with or without Lb. casei as starter. Listeria monocytogenes remains viable in Domiati depending on the pH, NaC1 content and storage temperature. Storage in brine for 60 days at 20-25 ~ is recommended to ensure product safety (Tawfik, 1993). Aeromonas spp. (A. caviae, A. hydrophila, A. sobria) are found in Domiati (El-Prince, 1998). Clostridium spp. are found in Domiati made from pasteurized milk without the addition of starters. The species isolated are predominantly CI. tyrobutyricum and CI. perfringens (Naguib and Shauman, 1973). Bacillus cereus has been isolated from Domiati (E1Nawawy et al., 1981). Staphylococcus aureus can tolerate up to 15% NaC1 in Domiati but its enterotoxin has not been detected in this cheese (Ahmed et al., 1983). Salmonella typhi can survive for up to 16 days in Domiati made from milk containing 10% NaC1 (Naguib et al., 1979). Defects Early blowing is the principal defect in Domiati cheese, particularly that made from raw milk. It is characterized by the formation of gas holes in the cheese, a spongy texture and blowing of the tins. This defect arises from two factors: gas-forming yeasts or coliforms (Hegazi, 1972; E1-Shibiny etal., 1988) or electrolytic corrosion of tins by NaC1 and developed acidity (Abo Elnaga, 1971).
Introduction
One of the most famous cheeses ripened in brine is, undoubtedly, Feta, which has been produced in Greece since Homeric time (Anifantakis, 1991). Feta is the principal cheese produced in Greece and, in most cases, 'Feta' is synonymous with cheese in Greece. Feta represents over 50% of the total cheese consumed in Greece. The name Feta, which means 'slice' in Greek, has probably come from the original shape of the cheese or from the property which allowed it to be sliced without falling apart. Over the past 30-40 years, the name Feta has acquired an important trade value and, nowadays, it is used to designate many cheeses ripened in brine, which are made from different kinds of milk, using various technologies, even uhrafihrated cows' milk. Of course, the flavour and other sensory qualities of these cheeses does not equate to those of the original Feta cheese.
Manufacture
Milk The most suitable milk for the manufacture of Feta is sheep's milk, but also mixtures of sheep's milk with not more than 30% goats' milk are used. Milk is filtered and standardized to about 6% fat. The ratio of casein to fat is usually 0.7-0.8:1. The pH of the milk should be >-6.5. Heat treatment The majority of cheese milk for Feta is pasteurized (72 ~ 2 1 5 15-20s or 6 5 ~ However, in small enterprises and on farms, the cheese milk is either processed raw or receives a thermal treatment lower than pasteurization. Following heat treatment, the milk is cooled to 32-34 ~ and, if pasteurized, a 40% solution of CaC12 is added at a level of 200 ml/100 kg milk. Starter culture Starters used are a combination of lactic acid bacteria. A yoghurt culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp, bulgaricus, 1:1) or 24 h-old yoghurt was used traditionally, but have been gradually replaced partly by other commercial cultures capable of a higher acidification rate, e.g., Lactococcus lactis subsp, lactis and Lb. delbrueckii subsp, bulgaricus (1:3), Lc. lactis subsp, lactis and Lc. lactis subsp. cremoris. The culture is added to the cheese milk at a level of 0.5-1.0% (v/v) and incubated for 20-30 min before the addition of rennet. Coagulation Coagulation is performed at 32-34 ~ The quantity of the coagulant is regulated so that the coagulum is ready for cutting in 45-50 min. In large- and mediumsized factories, commercial calf rennet is used. In small enterprises and in mountainous areas, the traditional rennet (rennet paste) made from the abomasa of unweaned lambs and kids is used commonly alone or in combination with commercial calf rennet. Cutting and draining The coagulum is cut crossways into cubes of 2-3 cm and left for about 10 min for partial whey exudation. Then, the curds are ladled into perforated moulds, gradually in order to assist draining. The gradual transfer of the curds leads to the formation of small, almond-shaped openings in the cheese mass, which is a characteristic of the structure of Feta cheese. Moulds are cylindrical of various dimensions when the cheese is to be packed in barrels and rectangular (23 • 23 • 20-25 cm) when it is to be packed in tinplated cans (tin cans). The curds are left to drain in the moulds at 14-16 ~ without pressing for 2-3 h
Cheese Varieties Ripened in Brine
and the moulds are then inverted and left for another 2-3 h to complete draining.
Salting When the curd is firm enough, the mould is removed and the curd is cut into two (23 • 11.5 cm) or four (11.5 • 11.5 cm) pieces, which are placed close together on a salting table, the surface of which has already been sprinkled with coarse cooking salt (particles of the size of rice grains). The upper surface of the pieces is also sprinkled with salt which penetrates slowly into the curd mass. Every 12 h, the cheese pieces are inverted and the surface is dry-salted again. This procedure is repeated until the cheese contains about 3.0-3.5% salt. Following salting, the cheese blocks remain on the table for a few more days until a slime of bacteria, yeast and some moulds starts to develop on the surface. Dry salting and slime formation are essential for the development of characteristic Feta flavour during ripening. Before packaging, the slime is washed off from the surface of the cheese using a soft brush and water or brine. Nowadays, in large factories, moulding, draining and salting are performed mechanically. The curds are transferred by gravity to the moulds. The moulds on a belt conveyor pass under a special outlet of the cheese vat and are filled automatically by gravity (no pumps are used). After about 2 h, the palettes supporting the moulds are inverted to complete draining. Then, the curd is cut to the dimensions of the final cheese and dry-salted. Next morning, the cheese pieces are layered in tin-plated cans. The bottom of the can and the surface of each layer of cheese are sprinkled with coarse salt (rice grain size). After about 2 days, the cheese pieces are packed in the final container (tin-plated can).
Packaging Wooden barrels (kegs) were the traditional containers for Feta. However, handling a filled barrel (--~50 kg) is difficult. Nowadays, Feta is packaged mostly in tinplated cans weighting "--19 kg (net weight of cheese: ---16 kg), making the transportation easier and more economical. The cost of the barrels is also higher than that of tin-plated cans but the cheese develops a stronger and spicier flavour than when packed in tin-plated cans.
Ripening Cheese pieces are tightly packed in the tin-plated cans, allowing little space between them. Brine (6-8% NaC1 in water) is added to the container to fill the space between pieces and to cover the surface of the cheese. Usually, the ratio of brine to cheese is 1:8 (v/w). Cheeses are kept at 16-18 ~ until the pH reaches 4.4-4.6 and the moisture decreases to less than 56% (pre-ripening period, usually 2-3 weeks). From time to time, the lid of the container is untightened to per-
235
mit escape of the gases produced by fermentation and inspection of the level of brine, which must always cover the cheese surface. This is a usual practice with cheese ripened in barrels. If not covered by brine, the surface of the cheese becomes dry, its colour changes (from snow white to ivory or even light yellow) and the growth of yeasts and moulds is possible. After the pre-ripening period, the cans of cheese are transferred to a cold room (4-5 ~ to complete ripening. Feta is permitted to be sold at not less than 2 months postmanufacture (Greek Food Code, 1998). A good quality Feta cheese may be stored, always in brine, for up to 1 year at 2-4 ~ Yield and gross composition
The average dry matter of sheep's milk is 18-20% (Alichanidis and Polychroniadou, 1996) and a cheese yield of about 25% is expected for Feta cheese (Anifantakis, 1991; Mallatou etal., 1994). However, the yield varies with the percentage of goats' milk added to sheep's milk and, also, with the season, because the composition of the milk varies with season. The compositional provisions of the Greek Food Code (1998) for Feta cheese are: maximum moisture, 56% and minimum FDM, 43%. Analyses of 60 market samples (60-180-day-old) produced throughout the cheesemaking period in four major factories showed that the average composition (g/100 g) of Feta cheese is: moisture, 54.2; FDM, 50.82; protein, 17.23; salt-in-cheese moisture, 6.27. The average pH is 4.58 (Michaelidou, 1997). Biochemistry of Feta cheese ripening
The breakdown of the main cheese constituents (protein, fat and lactose) by the action of many enzymes involved in cheese ripening is of importance, since it greatly influences the texture and flavour of the mature cheese.
Proteolysis The most complicated event during cheese ripening is, undoubtedly, proteolysis. Proteolysis in cheese is mediated by the concerted action of many proteolytic enzymes, derived from various sources. The contribution of each enzyme depends, amongst other factors, on their relative concentration and on the environment of each cheese. One of the key points for the successful manufacture of Feta cheese is the high acidification rate exerted by starter cultures and the consequent significant drop in pH from about 6.5 to 5.0 in 6-8 h during coagulation and draining, and to about 4.8 after 18-20 h from the beginning of manufacture. This ensures that more rennet is retained in Feta than in some other cheeses
236
Cheese Varieties Ripened in Brine
(Samal et al., 1993; van den Berg and Exterkate, 1993). Furthermore, the pH of Feta (about 4.5) is favourable for the proteolytic activity of chymosin and, also, is close to the pH optimum of the indigenous milk acid proteinase, cathepsin D. However, only a small part ("-8%) of the activity of this enzyme survives pasteurization (Larsen et al., 2000), and its role is expected to be of some importance only in Feta made from raw milk. Even so, since this enzyme has many of the same cleavage sites in Ors1- and [~-caseins as chymosin (Larsen et al., 1996), its activity would be overshadowed by the far higher activity of chymosin in Feta cheese. The activity of the dominant indigenous milk proteinase, plasmin, differs substantially between cheese varieties (Sousa et al., 2001). No data are available for plasmin activity in Feta. The absence of a curd-cooking step during Feta cheese manufacture, the relatively low pH and the high salt content are conditions which do not favour either the conversion of plasminogen to plasmin or the activity of the enzyme itself. Bands in the y-casein region on the electrophoretograms of Feta cheese, indicating some plasmin activity, are in most cases not strong and their intensity does not change during ripening. Proteolysis is not very intense in Feta cheese. Only about 15-18% of the TN of the cheese is soluble in water (WSN) after 60 days of ripening, reaching a value of up to 20-25% in well-ripened cheese in 120-180 days post-manufacture (Fig. 2). The main reason for this relatively low proteolysis is the short ripening period (2-3 weeks) at 16-18 ~ after which the cheese is transferred to a cold room (---4 ~ where
all biochemical reactions, including proteolysis, are slowed down. Additionally, the activity of many proteolytic enzymes, other than chymosin, is not favoured by the low pH of Feta cheese. It is worth noting that the water-soluble fraction of Feta (and other similar cheeses) contains not only the hydrolytic products of caseins (peptides and amino acids), which are soluble in water, but also some whey proteins (mainly [3-1actoglobulin and ot-lactalbumin), which remain in the curd after draining. On the other hand, as the cheese matures in brine, some of the peptides and amino acids, as well as some of the whey proteins, diffuse into the brine. Consequently, the level of WSN measured (e.g., by the Kjeldahl method) is either overestimated at the beginning of the ripening period, due to the presence of the whey proteins, or underestimated later on, due to diffusion (Katsiari et al., 2000a). Because of the diffusion process, the TN of the cheese decreases continuously during ripening and storage (Alichanidis et al., 1984; Katsiari and Voutsinas, 1994; Katsiari et al., 2000a). The rate of proteolysis in Feta cheese is high during the first 15-20 days, when the cheese is in the warm room (Fig. 2) but slows down when the cheese is transferred to the cold room (4 ~ Large amounts of low molecular weight nitrogenous compounds are produced during ripening in the warm room; at the end of this period about 60% of the WSN is soluble in 12% trichloroacetic acid (TCA-SN). The composition of this fraction changes continuously; during further ripening, it is enriched in very small peptides (<600 Da) and free amino acids soluble in 5% phosphotungstic acid (PTA-SN).
25
__-------O
2O
g
15
10
5
0 0
20
40
60
80
100
120
Ripening time (days) Changes in the level of nitrogen soluble in water (9 12% (w/v) trichloroacetic acid (O) or 5% (w/v) phosphotungstic acid (A) as percentages of the total nitrogen during ripening of Feta cheese.
C h e e s e Varieties Ripened in Brine
The amino acid (AA) content of mature Feta varies from 2 to 7 g/kg cheese, depending on the culture used and on the age of the cheese. Leucine, valine, lysine and phenylalanine are the major AAs. Feta also contains significant amounts of non-casein AAs, such as y-aminobutyric acid and ornithine, while citrulline and ot-aminobutyric acid are present in very small amounts (Alichanidis et al., 1984; Valsamaki et al., 2000; Katsiari et al., 2000a). Biogenic amines are generated by the decarboxylation of amino acids, mainly by adventitious micro-organisms (Joosten and Stadhouders, 1987). Investigations have shown that the average amine content of Feta is 400 mg/kg cheese (Valsamaki et al., 2000). Market samples (15) analysed for their amine content had somewhat higher values (---480 mg/kg) (Alichanidis, unpublished results). In both investigations, tyramine constituted about 40% of the total amines, followed by putrescine, histamine and cadaverine. Electrophoresis in polyacrylamide gels containing urea has shown that the rates and extents of degradation of %l- and [3-caseins in Feta cheese are different. During the first 20 days, about 50% of the Otsl-casein is
237
hydrolysed, while more than 80% of the [3-casein remains intact. In well-ripened cheeses (120-240-day-old), only ---20% of the initial content of Otsl-casein remains intact compared to ---65% of [3-casein (Katsiari et al., 2000a). The extensive hydrolysis of Otsl-casein early during ripening is probably due to the residual rennet, which is much higher in Feta than in other cheese varieties (Samal et al., 1993), and due to the low pH, which favours chymosin activity. [3-Casein is also a good substrate for chymosin, but its degradation is strongly retarded by salt (Fox and Walley, 1971), the concentration of which is about 6% in Feta. Later in ripening, the degradation of large peptides and their parent caseins could be attributed to the synergistic action of chymosin, the cell-bound proteinases of starter bacteria, and, possibly, the proteolytic enzymes of the non-starter lactic acid bacteria (NSLAB). The important role of residual rennet in proteolysis and its high activity on Otsl-casein are confirmed from the identification of the major peptides found in the water-soluble fraction of Feta (Michaelidou etal., 1998). The majority of the peptides identified (Fig. 3)
100
0.4
80
0.3 7 60
E c
z Ckl
<
/ 6
0.2
1
2
3
1
4o 9
0.1
0 0
10
20
30
40
50
60
70
80
90
Elution time (min) Reversed-phase HPLC profile of the water-soluble fraction of 6-month-old Feta showing the peaks collected and identified. Eluent A was 1 ml of trifluoroacetic acid (TFA)/I of deionized water. Eluent B was 0.9 ml of TFA, 399.1 ml of deionized water and 600 ml of acetonitrile/I. Gradient: 0-10 min, eluent A; 10-90 min, 0-80% eluent B; 90-100 min, 100% eluent B. The flow rate was 0.8 ml/min. The absorbance of the eluate was monitored at 214 nm. Peak numbers correspond to the following compounds: 1, Tyr; 2, Phe; 3, OLslCN (f4-14) and o%I-CN (f40-49); 4, o%I-CN (f1-14); 5, 13-CN (f164-180), oLsl-CN (f102-109) and oLsl-CN (f24-30); 5A, K-ON (f96-105) and OLsl-CN (f91-98); 6, OLs~-CN (f24-32); 7, 13-CN (f191-205); 8, oL-LA; 9, 13-LG (f16-?); 10, 13-LG (from Michaelidou et aL, 1998, courtesy of the Journal of Dairy Science).
238
Cheese Varieties Ripened in Brine
originated from the N-terminal half of Otsl-casein. Cleavage of Phe23mVa124, Phe32~Arg33, Leu98mLeu99, Leul01~Lysl02 and LeUl09~Glull0 can be attributed to chymosin action. The two peptides identified as originating from the C-terminal half of [3-casein resulted from the cleavage of Leu190~Tyr191 and Ile205mLeu206 by chymosin. One peptide, Ala96~Phez05, originated from the C-terminal part of para-K-casein resulting from the cleavage of Met95mAla96 and Phelos~Metl06. The second cleavage site is a bond well-known to be cleaved by chymosin during renneting. The bond Met95~Ala96 could be a cleavage site of chymosin (Reid et al., 1997) or lactococcal proteinase (Reid et al., 1994).
Lipolysis The flavour of Feta cheese is critically affected by the high levels of short-chain carboxylic acids. All reports agree that acetic acid is the major free volatile acid (FVA) (Vafopoulou et al., 1989; Katsiari et al., 2000b; Kandarakis et al., 2001; Kondyli et al., 2002). It is worth noting that acetic acid is not produced from triglycerides by lipase activity. In Feta, acetic acid can be produced in high amounts during the early stage of ripening mainly from lactose and, perhaps, from the catabolism of citrate (Sarantinopoulos et al., 2002) and from amino acids (Kondyli et al., 2002). The concentration of free fatty acid (FFA) plus acetic acid in mature Feta usually ranges between 2 and 4 g/kg cheese. The accumulation of the FFAs varies with the heat treatment of the milk, the rennet used (home-made rennets from kid and lamb abomasa are rich in lipases), the starter culture, the kind of microbial contaminants and, also, the temperature at draining. Higher draining and warm room temperatures (21 ~ versus 15 ~ favour the formation of all short-chain (C2mC8) volatile acids. Most of the difference (90%) is due to the higher amount of acetic acid produced mainly during the early lactate fermentation (Kandarakis et al., 2001). Free volatile acid constitutes 30-50% of the total acids (except lactic) in mature cheese, acetic acid being the dominant (40-75%) FVA (Alichanidis etal., 1984; Georgala et al., 1999; Katsiari et al., 2000b; Kandarakis et al., 2001; Kondyli et al., 2002). Butyric and higher volatile fatty acids are vital for the development of the characteristic, slightly rancid, flavour of Feta cheese (Vafopoulou et al., 1989). Volatiles
While data on the concentration of acetic and other volatile acids have been provided by a number of studies, data on other volatile compounds are limited. From the data available (Horwood etal., 1981; Kondyli et al., 2002; Sarantinopoulos et al., 2002), it seems that ethanol, followed by butan-2-ol, are the
main volatiles in mature Feta cheese. Also, volatiles such as 3-hydroxy-butan-2-one, diethyl ether, 3methyl-butan-l-ol, acetone, pentan-2-one, propanal, ethyl acetate and acetaldehyde are present in appreciable amounts. Rheological and sensory properties
Data on the rheological properties of Feta cheese are limited, since this cheese easily breaks into pieces when compressed. From the data available, it seems that hardness (kg) varies from 2.7 to 7.0, force to fracture (kg) from 1.5 to 2.4 and compression to fracture (%) from 18.7 to 21.5 (Katsiari and Voutsinas, 1994; Katsiari et al., 1997; Kandarakis et al., 2001). Feta has a short, firm and smooth texture, snow-white colour, a moist surface without rind, and is sliceable. Irregular mechanical openings, distributed throughout the cheese mass, are desirable but the presence of small round holes is regarded as a defect. The flavour (taste and aroma) of Feta can be generally described as slightly acid, salty and mildly rancid. Although the flavour of Feta has not been studied extensively, from the data available it seems that the medium- and, especially, the short-chain volatile acids contribute to its flavour (Vafopoulou et al., 1989; Georgala et al., 1999). Besides these, several other volatile compounds are found in Feta cheese (Horwood et al., 1981; Kondyli et al., 2002; Sarantinopoulos et al., 2002), some at relatively high concentrations, which may contribute to the aroma of this cheese. Some of these compounds are produced solely from the catabolism of proteins or lipids or lactose and citrate, but several of them are common end products of metabolism of more than one of the above constituents. Experiments have shown that besides glycolysis and citrate catabolism, proteolysis should be accompanied by a certain amount of lipolysis and vice versa for a balanced flavour development (Vafopoulou et al., 1989; Katsiari et al., 2000b; Michaelidou et al., 2003). Microbiology
The microbiological quality of Feta cheese can be influenced by various factors, the most important of which are: the quality of the raw milk, the thermal treatment of the milk (no treatment, thermization, pasteurization) and the extent of microbial contamination during processing, especially during dry salting. As mentioned earlier (see 'Manufacture'), the early acidification of the curd is a crucial step in Feta cheese manufacture. This, together with the rate of salt absorption and its final concentration in the cheese moisture, are the most important features of the manufacture of high quality and safe cheese.
Cheese Varieties Ripened in Brine
Several starters are used for Feta cheese, which are mostly combinations of mesophilic cocci, thermophilic cocci and thermophilic rods (Vafopoulou et al., 1989; Litopoulou-Tzanetaki et al., 1993; Katsiari et al., 1997; Pappa and Anifantakis, 2001). Their counts increase rapidly during the first days, remain relatively high during ripening in the warm room and, later on, decline significantly, especially those of the mesophilic cocci. This is probably due to the low pH and high salt-inmoisture content of the cheese (Litopoulou-Tzanetaki et al., 1993; Sarantinopoulos et al., 2002; Manolopoulou et al., 2003). The enzymes of the starter culture are probably responsible for the high rate of production of small peptides and amino acids during the early stage of ripening (Butikofer and Fuchs, 1997). However, the environment of Feta seems to favour the growth of NSLAB, especially lactobacilli, which in a 30-day-old cheese represent about 90% of lactic acid bacterial isolates (Tzanetakis and Litopoulou-Tzanetaki, 1992). Among them, Lactobacillus plantarum is the predominant species, followed by Lb. paracasei subsp. paracasei and Lb. brevis. The dominant group of bacteria found in the brine throughout the maturation of Feta cheese are also NSLAB, and the principal species identified are Lb. paracasei subsp, paracasei and Lb. plantarum (Bintsis et al., 2000). Enterococci (Ec. faecalis and Ec. durans) and pediococci are also found in mature Feta cheese but at low numbers (Tzanetakis and Litopoulou-Tzanetaki, 1992). Investigations have shown that some strains of Ec. durans (LitopoulouTzanetaki et al., 1993) or Ec. faecium (Sarantinopoulos et al., 2002) improve the sensory properties of Feta when used as an adjunct culture. P. pentosaseus, used as adjunct in Feta cheese manufacture, also improves cheese quality and shortens the ripening time by 1 month (Vafopoulou-Mastrojiannaki et al., 1990). Salt-resistant yeasts grow to high numbers (6-8 log cfu/g) on the surface of Feta during dry salting but their number decreases significantly with ripening time (Tzanetakis etal., 1998). Among the species found, Saccharomyces cerevisiae is predominant, followed by Debaryomyces hansenii and Pichia farinosa. Defects
Early blowing This defect appears mainly during drainage but also during curd salting. It is characterized by the presence of small and/or large gas holes in the cheese mass, which gives it a spongy texture. This defect is due to the excessive growth of coliforms and/or yeasts. Coliforms can multiply very rapidly in the curd during the first few hours, when the pH and temperature are favourable. However, the problem is rare in modern dairies, provided that efficient pasteurization and good
239
manufacturing practices are followed. Furthermore, the activity of the starter culture is crucial for the control of coliforms by reducing the pH and the amount of lactose in the curd (Bintsis and Papademas, 2002). Late blowing This defect, which is not very common, causes blowing of the containers, and is attributed mostly to heterofermentative lactic acid bacteria and very rarely to some species of clostridia. Usually, this defect occurs when the containers are sealed before the completion of the intense fermentation in the warm room and before the pH drops to the normal values (<4.8). Mouldiness When the cheeses are not completely immersed in the brine, various species of mould grow on the cheese surface, causing visible defects and reduced quality. Softening of the cheese body In cheeses with normal pH and moisture, softening of the body is very rare; it occurs only when the concentration of salt in the brine in the final containers is lower than the salt-in-moisture content of the cheese. The problem is more frequent when cheeses with insufficient acidity are stored at a low temperature and when the salt concentration of the brine in the package is low. Ropiness of the brine This is actually a defect of the brine, which makes the appearance of the cheese unsightly; the cheese itself is edible and, in most cases, its flavour is normal but the brine is ropy. This defect is caused mainly by some strains of Lb. plantarum, but also by Lb. casei subsp. casei and Lb. casei subsp, rhamnosus and is due to the production of exopolysaccharides (Samaras, 1994).
Gibna bayda is the Arabic name for 'white cheese'. It is produced in Sudan following a method similar to that for Domiati cheese (Tannous, 1991; Abdelgadir et al., 1998) by adding salt to milk before renneting and the cheese is brined in salted whey for at least a month. It is made from cows' milk or variable mixtures of cows' and sheep's or goats' milks (Ahmed and Abdel-Razig, 1998). Gibna bayda can also be made from recombined milk (Ahmed and Khalifa, 1989).
Mish is one of the oldest known cheeses in Egypt (Abou Donia, 1991). It is made from naturally
240
Cheese Varieties Ripened in Brine
fermented, partially skimmed milk remaining after the separation of sour cream by gravity. Salt is sprinkled on the curd ladled onto cheese mats for whey drainage and then cut into suitable pieces. The flesh cheese (Kariesh) is either consumed directly or brined in concentrated salted buttermilk (laban zier) in earthenware containers for more than 1 year to obtain Mish (E1-Gendy, 1983; Abou Donia, 1991). Usually, morta (the heat-coagulated protein-rich precipitate left after the manufacture of butter oil (ghee/samna) by the boiling-off method), red pepper and paprika are added to buttermilk used as brine for Mish cheese. The Mish cheese and brine are served together for consumption. Ripened Mish has a yellowish-brown colour with a distinctly salty, sharp and pungent flavour. Commercially, Mish-like products are made from pieces of different Egyptian cheese varieties (Domiati/Ras) of variable degree of maturity. They are mixed in a barrel with water, NaC1 and emulsifying salts, spices and ripened Mish (about 2%) as a starter. The water and the different additives do not exceed 25% of the mixture. The container is kept sealed for 1 year and its contents are then heated to 70 ~ with agitation to give a homogeneous, spreadable mass, which is then packaged into retail packages (0.5-1 kg). No specifications have been issued for Mish. Therefore, wide variations are found in the composition of market samples of this cheese (Nassib and E1-Gendy, 1974; Abou Donia and ElSoda, 1986; Zaki and Shokry, 1988), as shown in Table 8. Marked proteolysis and lipolysis occur in Mish during ripening. Amino acid nitrogen represents a major part of the total N (E1-Erian et al., 1975). Also, the levels of total volatile fatty acids and butyric acid increase as Mish cheese ripens (Taha and Abdel-Samie, 1961). Bacillus spp., Clostridium mishii, CI. mishami, micrococci, arthrobacteria are found in Mish (Taha and Abdel-Samie, 1961; E1-Erian and E1-Gendy, 1975). Coliforms have not been detected in Mish.
Chemical composition of Mish cheese (Abou Donia and El-Soda, 1986; Zaki and Shokry, 1988) Constituent
Minimum %
Maximum %
Moisture Fat Protein Ash Calcium Phosphorus NaCI
54.76 0.5 6.95 11.13 0.229 0.180 10.0
75.68 4.60 13.13 19.79 0.403 0.215 15.20
Mudaffara cheese is a braided, semi-haM cheese originating in Middle Eastern Countries and in Sudan. In Syria, it is named 'Medafara' or 'Magdula'. Both Mudaffara and Magdula are Arabic names, which denote 'braided'. The technique of making Mudaffara cheese in Sudan (Ahmed, 1987) differs slightly from that for Magdula cheese made in Syria (Abou Donia and Abdel Kader, 1979). In Sudan, Mudaffara is usually made from raw cows' milk, but mixtures of cows' and sheep's or goats' milk are also used. Mudaffara has been made from cows', goats' and buffaloes' milk supplemented with skim milk powder or from reconstituted milk (Ahmed, 1987). Raw milk is renneted and left for 30-60 min after coagulation to ripen. After cutting, whey is partially removed and the curd is cut into small cubes. The curd cubes are left in whey for another 40-60 min to develop the proper acidity. The acidified curds are then cooked (5-10 min) in hot water (75 ~ Black cumin (Nigella sativa) is added to the hot paste, which is kneaded and pulled quickly while hot to form cords (2 m long). Three cords are braided to form a tress, cut into suitable pieces and immersed in brine or salted whey for 2 days. The cheese is packaged in brine or salted whey in sealed containers until consumed. Table 9 shows the composition of Mudaffara cheese made from different milks (Ahmed, 1987). Salt concentration and storage temperature affect the quality of Mudaffara cheese. The hardness of the cheese decreases significantly with continued storage and is more pronounced in cheese stored at 39 2 2 ~ than in cheese stored at a lower temperature (19 + 2 ~ Storage of Mudaffara in 10% salted whey gives a product of superior quality than cheese stored in whey containing 15 or 20% NaC1 (Abdel Razig et al., 2001). Syrian Magdula cheese differs from Mudaffara in that it is made from sheep's milk and the cheese is brined for 1 week, sun-dried for 2-3 days and then kept in tight containers until consumed. It is eaten after soaking in water for 24 h (Abou Donia and Abdel Kader, 1979). Composition of Mudaffara cheese from milk of different species (Ahmed, 1987)
Cows' milk
Reconstituted Goats' Buffaloes' cows' milk milk milk (20% TS)
Total solids, % 47.22 46.96 Fat, % 9.20 9.88 5.14 5.47 pH Total N (TN), % 3.76 3.83 0.448 0.27 Soluble N (SN), % SN/TN 11.9 7.05
49.0 10.6 5.1 4.1 0.48 11.8
48.0 5.8 5.4 4.2 0.3 7.2
Cheese Varieties Ripened in Brine
Nabulsi cheese is one of the most popular cheeses produced in Jordan. It is usually made in springtime when enough sheep's and goats' milk are available. However, the method of cheese preservation allows for its consumption throughout the year. Traditionally, Nabulsi is made without the intentional addition of a starter (Tannous, 1991). Fresh sheep's milk or mixture of sheep's and goats' milks is warmed to about 35 ~ and coagulated with rennet. The coagulum is pressed, cut into pieces (about 4 X 8 cm) and sprinkled with salt. The cheese pieces are then boiled in brine and flavoured with the spices mastic (Pistacia lentiscus) and mahlab (Prunus mahlab) during boiling. Boiling imparts the characteristic texture of the cheese and improves its shelf-life. Boiling usually continues for 5-15 min, after which the cheese pieces become soft and float to the surface of the brine. The cheese pieces are taken from the brine, re-shaped by slight pressing and packaged in tightly closed cans and covered with brine in which the cheese was boiled. Based on its moisture content, Nabulsi is considered a semi-hard cheese. Jordanian Standards (1991) stipulate a moisture content for Nabulsi of <50%. Typical market samples of Nabulsi have a moisture content in the range 36.1-51% (Humeid and Tukan, 1986, 1991). This variation has been attributed partly to the variable casein/fat (C/F) ratio of the cheese milk. The C/F ratio in the milk of Awassi sheep in Jordan varies from 0.7:1 to 1.1:1 with an average of 0.9:1 (Haddadin etal., 1995), which changes the chemical composition and sensory properties of the final cheese. Nabulsi cheese from milk with a C/F ratio of 0.7:1 is preferred, while cheese made from milk with a C/F of 0.5:1 or 1.0:1 is of poor quality. Attempts to manufacture Nabulsi cheese from pasteurized milk (Haddadin et al., 1995) with the use of commercial starter cultures yielded a cheese with strong, sharp flavour, which was not appreciated by the consumer. The use of salt-tolerant Lb. paracasei, Lb. rhamnosus, Lc. lactis subsp, lactis, Ec. faecalis, Ec. faecium and Ec. durans isolated from sheep's and cows' milks gives Nabulsi cheese made from pasteurized milk sensory properties as acceptable as the traditional product (Yamani et al., 1998). Staphylococcus aureus, E. coli, Bacillus cereus and Proteus vulgaris have been detected in retail Nabulsi cheese, but Listeria rnonocytogenes has not been detected (E1-Sukhon, 1993).
Akawi is a popular cheese in several middle eastern countries, especially in Lebanon and Syria. It is made from cows', sheep's or goats' milk. Traditionally, Akawi
241
is made from flash-heated (70-75 ~ milk, cooled to about 35 ~ and renneted. After 1 h, the coagulum is cut, the whey is drained and the curd pieces are removed and quickly wrapped in cheese cloth in small portions (12 • 12 x 3 cm), layered on the top of each other and pressed. Care is taken with respect to the temperature of the curd before pressing (24-26 ~ to avoid excessive fat losses or poor cheese texture. The curd is then brined (10% NaC1). The cheese is sometime used as a filler in traditional sweets after desalting for several hours. Akawi cheese has a close texture with no gas holes and can be sliced. The typical composition of Akawi is 49.1% total solids, 22.5% protein, 21.6% fat and 5% ash (Tannous, 1991). The manufacture of an Akawi-type cheese in Denmark has been described (Kristensen, 1983). The cheese was made from pasteurized cows' milk. A mixture of yoghurt starter and Cheddar cheese starter was used; 3 0 - 4 0 m l rennet, 10 g KNO3 and 10 g CaC12 were used per 100 1 milk. The cheese is sold in brine-filled casks or cans or, alternatively, wrapped in aluminium foil or plastic films. The manufacture of Akawi has also been mechanized (Olsansky etal., 1979), with no appreciable effects on the yield, composition or quality.
This cheese originated in Romania from where its manufacture spread to other Balkan countries and Turkey. Traditionally, it was made from sheep's milk, but for many years now it is made also from cows', buffaloes' or, even, goats' milk or mixtures thereof. In this case, milk with a high fat or total solids content (sheep's or buffaloes') is usually blended with cows' or goats' milk. The colour of the cheese is very white when made from sheep's, buffaloes' or goats' milk, and yellowish when cows' milk is used. It has no rind and its texture is smooth when sheep's milk is used but harder when goats' milk is used alone. Cows' milk sometimes gives a crumbly cheese. The flavour of Telemes can be described as slightly salty and acid; when this cheese is made from goats' milk (alone or mixed with sheep's milk) it has a piquant flavour, especially when it is very ripe. The manufacturing procedure (Table 10) varies from country to country to adapt to the local climate and to the type of milk used (Anifantakis, 1991; Mallatou et al., 2003). The technology has some similarities with that used for Feta, but differs considerably in the procedure of salting and of curd draining. In Feta, whey drains under gravity and by the action of coarse salt spread on the surface of the curd, while the moulded curd of Telemes is subjected to pressure to expel whey. Concerning salting,
242
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C h e e s e Varieties Ripened in Brine
Feta pieces are dry-salted for several days before being placed in the final container filled with brine containing 7-8% salt. Thus, the salt penetration into Feta blocks is slow. In contrast, after draining, the curd of Telemes is cut into pieces (usually 11 • 11 • 8-10 cm but sometimes smaller 7 • 7 • 7 cm), which are placed immediately in a brine containing ---18% salt for 20 h or even 22-24% salt for ---16 h. By this practice, salt quickly penetrates into the curd and, evidently, interrupts the biochemical activities in the ripening cheese and, primarily, the utilization of lactic acid, which accumulates in the curd following the initial fermentation of lactose by the starter culture. Consequently, the acidity of Telemes remains high for a long time, especially if it is refrigerated soon after manufacture (Efthymiou, 1967). The gross composition of Telemes cheese varies as, in different countries, milk of various species is used for cheesemaking. Additionally, in many cases, the C/F ratio is not standardized, a fact which, among others, affects the fat content of the final product. When Telemes is made from sheep's milk, its gross composition is comparable to that of Feta cheese. Electrophoresis of samples of Telemes made from sheep's milk (Alichanidis et al., 1981) or cows' milk (Kalogridou-Vassiliadou and Alichanidis, 1984) shows that the hydrolysis of %l-casein is much more extensive than that of [3-casein. From the analysis of 56 Feta and 120 Telemes samples obtained from the Greek market (Alichanidis, unpublished results), it was concluded that proteoly-
243
sis, as indicated by %WSN/TN, is lower in Telemes than in Feta cheese (13.8 and 19.9%, respectively). Also, other indices of proteolysis, e.g., %TCA-SN/TN and %PTA-SN/TN, show similar trends. Results from several experiments (Polychroniadou and Vlachos, 1979; Alichanidis et al., 1981; Kalogridou-Vassiliadou and Alichanidis, 1984; Zerfiridis et al., 1989) showed that all the above-mentioned indices and the FAA content were lower in Telemes aged for less than 90 days than in Feta cheese, but in older Telemes (> 120 days), their levels became comparable to those of Feta cheese. However, the rate of formation of products of casein degradation differs between the two cheeses. In Feta, the rate is high during the early ripening period, slowing down later (Fig. 2). In contrast, in Telemes, the initial rate is much slower but it continues nearly unchanged throughout ageing (Fig. 4), probably because of the differences in the cheesemaking procedure described above. The free fatty acid (FFA) content of Telemes is lower than that of Feta, ranging from 1 to 2.5 g/kg cheese. Although acetic acid is not a product of lipolysis it is, by far, the principal volatile acid, ranging from 55 to 86% of the total (Efthymiou, 1967; Alichanidis et al., 1981; Buruiana and E1-Senaity, 1986). In a recent study (Mallatou et al., 2003), batches of Telemes were produced using the same procedure but different kinds of milk, sheep's, cows', goats' or a 1:1 mixture of sheep's and goats' milk. The outcome of this study was that, irrespective of the milk used, acetic acid accounted for
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244
Cheese Varieties Ripened in Brine
more than 70% of the total volatile acids. In ripe cheese (60- and 180-day-old), the acid degree value was significantly higher in cows' milk cheese, as also was the sum of FFA produced by lipolysis.
Beyaz peynir is the most popular cheese made and consumed in Turkey. Originally, it was made from sheep's and/or goats' milk, but cows' milk is now widely used for its production. It is a typical cheese ripened in brine with a salty and acid taste. Its colour is very white when sheep's and/or goats' milk is used. It has no rind and its texture varies from soft to semi-hard, depending on the milk used and on the stage of maturity. The shape of the cheese is cubic, 7 • 7 • 7 cm, or rectangular, 7 • 7 • 10 cm. It can be consumed while fresh, but mostly is consumed after ripening in brine. In modern factories, the cheesemilk is pasteurized and starters are used for curd acidification. However, artisanal production is still very large and significant amounts of cheese are made from raw milk without starter addition (Erkmen, 2000). The technology of Beyaz peynir is given briefly in Table 10. Details of the manufacture and the microbiological and biochemical properties of this cheese are given in the review by Hayaloglu et al. (2002). It is difficult to give a reliable average gross composition for this cheese, because the reported data vary widely (Tekinsen, 1983; Turantas et al., 1989; Yildiz et al., 1989; Hayaloglu et al., 2002). These differences can be attributed to the lack of standardization of the C_Az ratio in the cheese milk and to the significant variations in the manufacturing procedure, concerning the heat treatment of the milk, the use of starters, the clotting time, the pressure applied to the curd during draining, the salting level, etc. (Tekinsen, 1983; Turantas et al., 1989; Yildiz et al., 1989). Besides composition, pH also varies substantially in mature cheese, ranging from 4.11 to 5.65 (Turantas et al., 1989) or even higher (Yildiz et al., 1989; Erkmen, 2000). For this type of cheese, a pH value higher than 5.0 could be detrimental for its keeping quality, when the salt-in-brine content is not very high. Proteolysis in Beyaz peynir has not been studied in detail. As with other cheeses ripened in brine, data from the electrophoresis of cheese samples show that Otslcasein is hydrolysed more extensively than [3-casein (Saldamli and Kaytanli, 1998). In mature cheese, the ripening index (WSN/TN) varies from 13 to 22.7%, with an average value of 19.5% (Hayaloglu et al., 2002). Free amino acid (FAA) development was studied by Oc~nc~ (1981), who found that mature cheese (120day-old) made from sheep's milk contained 853 mg FAA/100 g cheese, while the same cheese made from cows' milk contained 698 mg FAM100 g cheese.
The total biogenic amine content of commercial Beyaz peynir (22 samples) was found to be 179 mg/kg in cheeses made with starters and 442 mg/kg in cheeses made without the deliberate use of starter. In both groups, tyramine, putrescine and cadaverine were the predominant amines (Durlu-Ozkaya et al., 1999). The FFA content in cows' milk Beyaz peynir has been studied by Akin et al. (2002). It was found that in 30-day-old cheeses, the total FFA content was 638 mg/kg cheese and, as in other brine cheeses, acetic acid was the dominant volatile acid, accounting for 58.6% of the total volatile acids (C2-C8).
Bjalo salamureno sirene (Belo salamureno sirene or Bjalo sirene or, simply, Sirene) is the traditional and the most popular cheese in Bulgaria. It is a white, semi-hard sheep's milk cheese with a slightly salty and acid taste, and a smooth texture with no rind. Its shape is rectangular (12 • 12 • 10 cm) or cubic with a side of 10 cm. A variant of this cheese is made from pasteurized cows' milk or a mixture of cows' and sheep's milks. The manufacturing technology, in brief, is given in Table 10 (Dimov et al., 1975). As a result of the high fat content (7.5-8.5%) of sheep's milk, when cheese is made from unstandardized milk, it contains FDM ranging from 56 to 59% and protein from 14 to 16.2%. Usually, the C/F ratio in cheese milk is adjusted to 0.65-0.68:1 and the gross composition of the resulting mature cheese (>60 days) is 49-51% moisture, 50-53% FDM and > 17% total protein. The salt content varies between 3.5 and 4.5% and the pH between 4.35 and 4.7. Depending on the age of cheese and other factors, the ripening index (WSN/TN) ranges from 18 to 25% (Peichevski and Iliev, 1986; Mikov et al., 1996; Kafedjiev et al., 1998; Stankov et al., 1998).
Beli sir u kriskama means 'white cheese in pieces' in Serbo-Croatian. This type of cheese is produced in the countries of the former Yugoslavia under specific names, e.g., Bel Sprski, Travnin~ki, Sjeni&i, Sarplaninski, Homoljski, etc. (Zivkovie, 1963). Most of the cheeses were, traditionally, made from sheep's milk but nowadays some of them are also made from cows' milk or mixtures of milk. The manufacturing technology (Table 10) of all these cheeses is similar, with very few differences (Zivkovi~, 1971). All have the typical characteristics of white cheeses ripened in brine: sour-salty taste and tender but firm texture. Their surface is moist with no rind, although some of them have a very thin,
Cheese Varieties Ripened in Brine
greasy rind. Their shape is, usually, rectangular with typical dimensions of 10 x 10 x 10-12 cm or smaller.
Halloumi is the traditional cheese of Cyprus. For hundreds of years, it has been produced from raw sheep's milk or mixtures of raw sheep's and goats' milks; nowadays, large factories use also pasteurized cows' milk for its manufacture. Normally, the colour of the cheese is white, but that made from cows' milk is yellowish. It is a semi-hard cheese with no rind and no gas holes, and its texture is elastic and compact. The specific characteristic of its manufacturing procedure (Anifantakis and Kaminarides, 1983) is that the blocks of pressed curd (--~10 X 10 • 3 cm) are heated at 90-95 ~ in heat-deproteinated whey for at least 30 min. After cooling on a table, the curd blocks are salted with dry salt, often mixed with dry chopped leaves of mint (Mentha viriclis). Halloumi is sold fresh (immediately after production) or is stored at 4 ~ in the whey previously used to heat-treat the curd, containing ---120 g/1 salt. The severe heat treatment, together with the use of salted whey, makes this cheese suitable for storage without refrigeration for a short period of time. The average gross composition of Halloumi from the Cyprus market is: 42-53% moisture, 44.52% FDM, 24.46% protein (TN • 6.38), 3.54% NaC1 (Anifantakis and Kaminarides, 1983). The severe heating of the curd reduces substantially the microbial population in the cheese (Papademas and Robinson, 2000). Also, the residual rennet is probably inactivated under these conditions. Consequently, proteolysis is limited. In fresh ovine and ovine/caprine market samples, the WSN/TN is about 4.0% (Anifantakis and Kaminarides, 1983; Kaminarides et al., 2000). According to Papademas and Robinson (2000), in both ovine and bovine Halloumi, acetic acid is the dominant volatile acid in fresh and mature cheese, accounting for about 40% and over 80% of the total, respectively.
Prof. E. Alichanidis wishes to thank Prof. Ivan Stankov and Prof. Todor Dimitrov for providing literature on Bjalo Salamureno Sirene.
Abdelgadir, W.S., Ahmed, T.K. and Dirar, H.A. (1998). The traditional fermented milk products of the Sudan. Int. J. Food Microbiol. 44, 1-13.
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Teama, Z.Y.A. (1967). Studies on Some Factors Affecting the Yield and Ripening of Domiati Cheese. PhD Thesis, Ain-Shams University, Cairo. Tekinsen, O.C. (1983). Comparative studies on manufacturing methods of white pickled cheese. Ankara Universitesi Veteriner Facultesi Dergisi 30,449-466. Turantas, E, Onl(it(irk, A. and G6ktan, D. (1989). Microbiological and compositional status of Turkish white cheese. Int. J. Food Microbiol. 8, 19-24. Tzanetakis, N. and Litopoulou-Tzanetaki, E. (1992). Changes in numbers and kinds of lactic acid bacteria in Feta and Teleme, two Greek cheeses from ewes' milk. J. Dairy Sci. 75, 1389-1393. Tzanetakis, N., Hatzikamari, M. and Litopoulou-Tzanetaki, E. (1998). Yeasts of the surface microflora of Feta cheese, in, Yeasts in the Dairy Industry: Positive and Negative Aspects, Jacobsen, M., Narvhus, J. and Viljoen, B.C., eds, Special Issue 9801, International Dairy Federation, Brussels. pp. 34-43. Oc(inc(i, M. (1981). Untersuchungen fiber freie Aminostturen w/~rend des Reifens von t(irkischem Weisskttse (Feta-Kase) aus Kuh- und Schafmilch. Molkerei-Z. Welt der Milch 35, 634-638. Vafopoulou, A., Alichanidis, E. and Zerfiridis, G. (1989). Accelerated ripening of Feta cheese, with heat-shocked cultures or microbial proteinases. J. Dairy Res. 56, 285-296. Vafopoulou-Mastrojiannaki, A., Litopoulou-Tzanetaki, E. and Tzanetakis, N. (1990). Effect of Pediococcus pentosaceus on ripening changes of Feta cheese. Microbiol.Aliments-Nutr. 8, 53-62. Valsamaki, K., Michaelidou, A. and Polychroniadou, A. (2000). Biogenic amine production in Feta cheese. Food Chem. 71,259-266. van den Berg, G. and Exterkate, EA. (1993). Technological parameters involved in cheese ripening. Int. DairyJ. 3,485-507. Yamani, M.I., A1-Nabulsi, A.A., Haddadin, M.S. and Robinson, R.K. (1998). The isolation of salt-tolerant lactic acid bacteria from ovine and bovine milks for the production of Nabulsi cheese. J. Soc. Dairy Technol. 51, 86-89. Yildiz, E, Kocak, C., Karacabey, A. and G(irsel, A. (1989). Technological description of a standard quality pickled white cheese in Turkey. Doga Turk Veteriner ve Hayvancilik Dergisi 13,384-392. Zaki, N. (1990). Relationship between chemical composition, texture characteristics and microstructure of some soft cheese varieties. Egypt. J. Dairy Sci. 18, 293-302. Zaki, N. and Shokry, Y.M. (1988). Chemical and microbiological changes in Mish cheese and Mish during ripening. Egypt. J. Dairy Sci. 16, 119-129. Zaki, M.H., Metwally, N.H., Gewaily, E.M. and E1-Koussy, L.A. (1974). Domiati cheese stored at room temperature as affected by heat treatment of milk and different salting levels. Agric. Res. Rev. 52,217-231. Zerfiridis, G.K., Alichanidis, E. and Tzanetakis, N. (1989). Effect of processing parameters on the ripening of Teleme cheese. Lebensm.-Wiss. u.-Technol. 22, 169-174. Zivkovie, Z. (1963). Chemical changes in brine during the ripening of soft white cheese. J. Sci. Agric. Res. 16, 92-100. Zivkovie, Z.. (1971). Technology of White Serbian cheese manufacture. Mljekarstvo 21, 8-16.
Pasta-Filata Cheeses P. Kindstedt, Department of Nutrition and Food Sciences, University of Vermont, Burlington, VT, USA M. Carie and S. Milanovie University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, Serbia and Montenegro
The pasta-filata cheeses are a diverse group that originated primarily in the greater northern Mediterranean region, encompassing Italy, Greece, the Balkans, Turkey and eastern Europe. Traditionally, pasta-filata cheeses have been produced from the milks of the cow, goat, sheep or water buffalo. Some are soft or semi-soft cheeses that are, typically, consumed fresh or after only a brief period of ageing (e.g., fresh Mozzarella, low-moisture Mozzarella, Scamorza). Others are hard or semi-hard ripened cheeses that may undergo considerable ageing before being consumed (e.g., Caciocavallo, Kashkaval, Provolone, Ragusano). The term pasta-filata, which is derived from an Italian phrase that literally means 'spun paste' or 'stretched curd', refers to a unique plasticization and stretching process that is shared by all pasta-filata cheeses and which gives this diverse group their common identity. This chapter is divided into two major sections. The first section is dedicated specifically to low-moisture Mozzarella cheese (LMMC), often referred to as Pizza cheese, which is consumed fresh or after only a brief period of ageing, and which is the most economically important of the pasta-filata cheeses. The second section focuses on Kashkaval, a pasta-filata cheese that is aged extensively.
Global production of LMMC has experienced unprecedented growth during the last two decades and now exceeds that of all other pasta-filata cheeses because of its premier status as a pizza topping. Rapid market growth and keen competitive pressures have given rise to impressive increases in the production capacity of cheese plants that produce LMMC. Consequently, it is not unusual now to find cheese plants that routinely produce 100 000 kg or more of LMMC cheese per day. Cheesemaking on this scale requires precise control over all aspects of the manufacturing process, which has created a pressing need for a better scientific understanding of key manufacturing parameters and their influence on cheese composition, structure, function and yield. It is within this context that LMMC has
attracted considerable interest among cheese scientists. Indeed, the past decade has seen a remarkable increase in research aimed at understanding the effects of manufacturing parameters on LMMC and at elucidating the chemical, physico-chemical and microbiological factors that influence structure and functional properties. This section will focus primarily on the literature and scientific advances that have accrued during the past 10-15 years. Earlier research on LMMC was reviewed by Fox and Guinee (1987), Kindstedt (1991, 1993a,b) and McMahon et al. (1993). Overview of manufacturing technology
The basic manufacturing scheme and equipment lines that are commonly used in the industrial production of LMMC are very similar to those used for Cheddar cheese as far as the milling stage (Bylund, 1995; Kosikowski and Mistry, 1997a; Johnson and Law, 1999). The fat-in-dry matter (FDM) content of LMMC typically falls in the range of 30-45% (w/w). Therefore, the cheese milk is normally standardized to a higher casein to fat ratio by adding non-fat milk solids (e.g., non-fat dry milk, condensed skim milk or ultrafiltered milk protein concentrate) or, less often, by removal of cream (Barbano, 1996; Wendorff, 1996). The standardized milk is pasteurized and then inoculated with starter culture as the milk is pumped into horizontal or vertical enclosed vats. Low-moisture Mozzarella cheese can be manufactured using mesophilic (e.g., Lactococcus lactis subsp. lactis, cremoris) or thermophilic (e.g., Streptococcus thermophilus, Lactobacillus delbrueckii subsp, bulgaricus, Lb. helveticus) lactic acid bacteria. In either case, the principal role of the starter culture is to produce lactic acid in sufficient quantity to transform the curd into one that will plasticize and stretch in hot water. This is accomplished by attaining a suitable combination of pH and calcium content in the curd at the time of stretching. Furthermore, in order to obtain the correct moisture content in the final cheese, generally between 45 and 52% (w/w), the starter must produce acid much more rapidly than in the making of Cheddar cheese. Rapid acidification allows the total manufacturing time to be shortened, which reduces the total
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amount of syneresis during cheesemaking and enables a higher moisture content to be achieved in the final cheese (Barbano et al., 1994). Thus, the typical manufacturing time for LMMC is much shorter than that for Cheddar, averaging about 2.5 h or less from coagulant addition to the start of stretching (McCoy, 1997). In general, thermophilic starters are used much more widely throughout the world than mesophilic starters for LMMC cheese, although there are some noteworthy exceptions. The inoculated cheese milk is coagulated with rennet, the coagulum is cut and then cooked to a temperature of about 41 ~ if a thermophillic starter is used, after which part of the whey is drained off. The remaining whey and curds are then pumped to a large enclosed conveyor belt system where draining, matting and cheddaring of the curds proceed until the proper level of acidity is developed. By the time the cheddared curd reaches the end of conveyor belt, it should have attained an optimum pH value, usually between 5.3 and 5.1 if the curd is to be milled and then stretched immediately. Alternatively, the curd may be milled at a slightly higher pH and then dry-salted to incorporate a portion or all of the salt in the final cheese. The milled curds, salted or unsalted, are then plasticized and stretched mechanically in hot brine or hot water. The hot plastic curd is forced under pressure into a chilled mould which gives the cheese its shape and which precools the block sufficiently so that it will retain its shape when removed from the mould. The block may then undergo further cooling and salting by immersion in cold brine, although salting by brining is increasingly being replaced in part, and in some cases totally, by direct salting. Many variations of the basic process for making LMMC cheese are found in commercial practice but the underlying principles are common to all make procedures. A more detailed discussion of the manufacturing technology of LMMC can be found in a recent review by Kindstedt et al. (1999). Plasticization and stretching
In the industrial manufacture of LMMC, plasticization and stretching are usually performed using continuous single or twin screw mechanical mixers that recirculate hot water at a temperature that is precisely controlled by steam injection. An example of a commercial mixer for LMMC is shown in Fig. 1. Stretching is a two-stage process. During the first stage, the milled curd enters a reservoir of hot water at the front of the mixer, where it is warmed as it settles to the bottom of the mixer (1A). When the curd temperature increases to approximately 50-55 ~ the curd is transformed into a plastic and workable consistency. In the second stage, the
An example of a commercial mixer that is used to plasticize and stretch low-moisture Mozzarella cheese. (A) milled curd enters a reservoir of hot water at the front of the mixer; (B) plasticized curd is kneaded and stretched by counter-rotating augers; (C) hot (c. 60 ~ plastic curd exits the mixer under pressure and proceeds to the moulding machine.
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plasticized curd is kneaded and stretched by either a single pair of counter-rotating augers housed in an inclined barrel or a series of horizontal and inclined single or twin augers (1B). The action of the augers reorients the amorphous curd structure into a unidirectional fibrous ribbon of hot plastic curd that exits the mixer under pressure (1C). Physico-chemical characteristics of the curd
The ability of cheese curd to plasticize in hot water and reorganize into a unidirectional fibrous structure is presumed to be governed primarily by the amount of casein-associated calcium (more correctly calcium phosphate) that is available to crosslink the amorphous para-casein matrix at the time that heat is applied to the curd (Lawrence et al., 1987; Kimura et al., 1992; Lucey and Fox, 1993; Kosikowski and Mistry, 1997a). Furthermore, the hydration of paracasein increases as the level of casein-associated calcium decreases (Sood et al., 1979), which probably contributes strongly to the ability of the curd to plasticize. Curd that contains too much casein-associated calcium fails to attain a smooth, stretchable consistency upon heating and tears during stretching. Curd with too little casein-associated calcium becomes excessively soft and fluid-like during stretching. Two parameters determine the amount of casein-associated calcium in the curd at the time of stretching: (1) the total calcium content of the curd and (2) the distribution of total calcium between the soluble and the insoluble (i.e., casein-associated) states. The total calcium content of the curd is determined by the amount of calcium that is lost to the whey up to the time of stretching. It is well established that caseinbound calcium dissociates from the para-casein matrix to the water phase of cheese curd as the curd pH decreases, and is subsequently released from the curd along with the whey during syneresis (Lucey and Fox, 1993). Consequently, the ratio of total calcium to total protein in Mozzarella curd decreases progressively during cheesemaking as the pH decreases and syneresis progresses (Kindstedt, 1985; Kiely et al., 1992; Kimura et al., 1992). The amount of calcium lost depends on the timing of acidification relative to syneresis. When the pH decreases (and thus casein-associated calcium is solubilized) during the early stages of syneresis (i.e., up to draining), more calcium is lost than when the pH decreases during cheddaring, after most of the syneresis has already occurred (Kindstedt, 1985; Kiely et al., 1992; Kimura et al., 1992). Calcium losses are greatest when acidification occurs before the onset of syneresis (i.e., before coagulation and cutting), as in the making of preacidified and directly acidified Mozzarella. Therefore, directly acidified Mozzarella cheese characteristic-
258
ally contains a very low level of calcium relative to total protein (Kindstedt and Guo, 1997; Paulson et al., 1998; Metzger et al., 2000; Guinee et al., 2002). Thus, the total calcium content of Mozzarella curd may vary substantially at the time of stretching, depending on the conditions of acidification. Furthermore, the distribution of total calcium between the insoluble (i.e., casein-associated) and the soluble states may also vary substantially and, indeed, it is the combination of the total calcium content and its distribution that determine the level of casein-associated calcium and thus the capacity of the curd to plasticize and stretch. The distribution of calcium in Mozzarella curd at stretching is determined primarily by the pH of the curd, as evidenced by the strong pH-dependence of calcium distribution in the final cheese (Lucey and Fox, 1993; Guinee et al., 2000b; Kindstedt et al., 2001; Watkinson et al., 2001; Metzger et al., 2001b; Ge et al., 2002). Declining cheese pH favours a shift in calcium distribution from the casein-associated to the soluble state. Consequently, curd that contains a high total calcium content at stretching (e.g., c. 30 mg/g protein, as in the making of conventional cultured LMMC (Kiely et al., 1992)), must have a low pH in the range of c. 5.1-5.3, in order to attain a casein-associated calcium level that is low enough to allow the curd to plasticize and stretch. In contrast, curd with a low total calcium content (e.g., c. 22 mg/g protein, as in the making of directly acidified Mozzarella (Kindstedt and Guo, 1997)), is optimally stretched at a higher pH value (e.g., pH 5.6-5.7), the curd becoming excessively soft and fluid-like when stretched at a lower pH due to insufficient casein-associated calcium (Larson etal., 1967; Keller etal., 1974). Indeed, by demineralizing skim milk by electrodialysis before renneting, Kimura et al. (1992) demonstrated that curd with a very low calcium content (c. 19 mg/g protein) and very high pH (pH = 6.15) can be plasticized and stretched to produce string cheese. Mozzarella cheese analogues made from rennet casein, which combine very low calcium content (c. 18.5 mg/g protein) with a very high pH (c. 6.3), plus the addition of calciumsequestering salts, presumably are based on this same principle (Guinee et al., 2000b; O'Sullivan and Mulvihill, 2001). Thus, plasticization and stretching can be achieved over an extraordinarily broad range of combinations of total curd calcium content and pH. Thermo-mechanical treatment of the curd
Stretching is a thermo-mechanical treatment that involves the application of mechanical energy in the form of shear stress as the plasticized curd moves down the barrel of the stretcher and its temperature increases. Mulvaney et al. (1997) studied the effects of two key operating parameters of a twin-screw stretcher
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(i.e., the temperature of the stretching water and the speed of the augers (screw speed)) on the thermomechanical treatment of the curd and the chemical and functional characteristics of the final cheese (Yun et al., 1994a; Renda et al., 1997). Increasing the screw speed from 5 to 19 rpm at a constant and relatively low stretching water temperature of 57 ~ resulted in a shorter residence time in the stretcher and a lower curd temperature at the exit. Thus, the intensity of the heat treatment of the curd (in terms of both time and temperature) decreased with increasing screw speed. Furthermore, a higher screw speed resulted in a higher specific mechanical energy (a measure of the mechanical energy that was consumed during stretching), indicative of a more intense thermomechanical treatment. At the highest screw speed (i.e., 19 rpm), the curd temperature did not increase quickly enough in the 57 ~ stretching water to completely plasticize the curd before it was subjected to the shearing forces of the screws. Consequently, the curd underwent extensive tearing which resulted in higher fat loss in the stretching water and substantially lower fat and moisture levels in the final cheese (Mulvaney et al., 1997; Renda etal., 1997). The cheeses stretched at 19 rpm also had higher initial shear modulus (Go) in stress relaxation, which indicated a more extensive elastic network structure that probably resulted from their lower levels of moisture and fat. Increasing the stretching water temperature from 57 to 74 ~ at a constant mid-range screw speed of 12 rpm resulted in a shorter residence time in the stretcher, a higher curd temperature at the exit (ranging from 54.4 ~ (57 ~ water) to 66.5 ~ (74 ~ water)), a lower specific mechanical energy during stretching, a lower fat loss in the stretching water and cheese with higher FDM content and a higher Go immediately after manufacture. Also, during the 50 days of ageing at 4 ~ cheeses stretched at the highest temperature had much higher apparent viscosity and TPA-hardness and lower meltability values (Yun et al., 1994a). Thus, the temperature of the stretching water had a major impact on the time-temperature profile of the curd and the intensity of the mechanical treatment during stretching, and on the rheological and functional properties of the final cheese. The authors did not attempt to explain why a higher stretching water temperature resulted in the formation of a much more elastic network structure (in spite of a higher FDM in the cheese), as indicated by a substantially higher Go value in the final cheese immediately after manufacture. However, it seems reasonable to postulate that a higher stretching temperature may have favoured more extensive hydrophobic-mediated aggregation and contraction of para-casein into stronger, more elastic fibers. If this
were the case, then one might also expect to find greater phase separation of protein and water within the cheese structure (Kindstedt and Guo, 1998; Pastorino etal., 2002). The investigators did not specifically evaluate changes in the water phase of the cheese but they did observe that the highest stretching temperature produced cheese that released an unusually large amount of serum upon melting throughout the 51 days of ageing (Kindstedt et al., 1995b). In a different study, Kindstedt et al. (1995b) varied the screw speed from 5 to 19 rpm at a constant, relatively high, stretching water temperature of 74 ~ Stretching at a slow screw speed resulted in a longer residence time and a higher curd temperature at exit, ranging from 62 ~ at 19 rpm to 66 ~ at 5 rpm screw speed. Cheeses that were stretched at a slow screw speed and which, therefore, attained a higher temperature during stretching had a higher apparent viscosity and TPA hardness value throughout the 112 days of storage at 4 ~ indicative of a more elastic network structure. Furthermore, the amount of serum that was expressed from the cheeses upon centrifugation (12 500 • g for 45 min at 25~ during the first 12 days after manufacture increased, and the concentrations of intact caseins and calcium in the expressible serum decreased with increasing curd temperature during stretching. These results suggested that a higher stretching temperature may have favoured increased hydrophobically mediated aggregation of para-casein and a shift in calcium distribution to the casein-associated state, resulting in a more highly calcium-crosslinked, elastic, fibrous structure and a greater phase separation of protein and water. However, in this and the other studies cited, it is impossible to separate the effect of stretching temperature on the initial network structure that was established during stretching from other effects that occurred concurrently, such as on the rate of proteolysis and microbiological activity, as discussed below. Thus, further study is needed to differentiate and elucidate the multiple effects of stretching temperature on the structure and functional properties of the final cheese. Thermal effects on starter bacteria and coagulant
The heat treatment of the curd during stretching presumably influences the viability and activity of the starter culture bacteria in the final cheese. Yun et al. (1995a) reported that both Streptococcus thermophilus and Lactobacillus delbrueckii subsp, bulgaricus survived and remained metabolically active when stretching was performed at the low end of the stretching temperature range (e.g., 55 ~ curd temperature), as measured directly by plate count enumeration and indirectly by
Pasta-Filata Cheeses
increases in titratable acidity and in the level of N soluble in 12% (w/v) trichloroacetic acid in the cheese during ageing. Petersen etal. (2000) also enumerated starter Sc. thermophilus and Lb. helveticus in LMMC before and after stretching (water temperature, 83 ~ curd temperature unknown) and reported little or no change in the number of viable starter bacteria. However, starter activity appeared to be reduced substantially at higher stretching temperatures (e.g., 62-66 ~ exit temperature), as evidenced indirectly by a steep decline in titratable acidity and 12% TCA-soluble N in the final cheese (Yun et al., 1994a; Kindstedt et al., 1995b). Thus, the survival of metabolically active thermophilic starter bacteria appears to be highly temperature dependent within the normal range of stretching temperature employed in commercial manufacture. Residual coagulant activity in LMMC also may vary depending on the extent of heat inactivation during stretching (Gangopadhyay and Thakar, 1991). Indirect evidence of chymosin activity in LMMC during ageing, observed as either the breakdown of OLsl-casein by electrophoresis or the accumulation of N soluble in water at pH 4.6, has been reported in numerous pilot-scale cheesemaking experiments (Yun et al., 1993a,c,e, 1995a,b, 1998; Barbano et al., 1994; Kindstedt et al., 1995a,b; Renda et al., 1997; Guinee et al., 1998, 2000a, 2002; Hong et al., 1998; Walsh et al., 1998; Chaves et al., 1999; Feeney et al., 2002; Somerset al., 2002) In these studies, the curd temperature during stretching did not exceed c. 55-60 ~ i.e., the low end of the normal stretching temperature range. Microbial coagulants derived from Rhizomucor miehei (formerly Mucor miehei) and Cuophonectria parasitica (formerly Endothia parasitica) also remained active in LMMC stretched at 55 ~ (Yun et al., 1993c). However, chymosin activity appeared to be reduced substantially at higher stretching temperatures (e.g., 62-66 ~ exit temperature), as evidenced indirectly by a steep decline in the level of pH 4.6-soluble N in the final cheese (Yun et al., 1994a; Kindstedt et al., 1995b). More recently, Feeney et al. (2001) demonstrated conclusively that residual chymosin activity in LMMC decreased progressively when the curd temperature during stretching was increased from 55 to 66 ~ with the largest decrease occurring between 62 and 66 ~ From their results, one can conclude that stretching at a temperature higher than 66 ~ would have little effect on residual chymosin activity since very little activity remained after stretching at 66 ~ This was confirmed by Mayes and Sutherland (2002), who reported that the rate of proteolysis (measured as increases in pH 4.6- and 12% TCA-soluble N during ageing) decreased in LMMC when the stretching temperature was increased from 60 to 67 ~ However, increases from 67' to 75 ~ had little further effect
255
on proteolysis. The impact of curd pH at stretching has not been specifically investigated, but it is possible that a higher curd pH at a given stretching temperature may result in greater inactivation of chymosin and less residual chymosin activity in the final cheese (Guinee et al. 2002). Reorganization of curd structure
Several different microscopy techniques have been used to elucidate the changes in curd structure that occur during stretching. Using scanning electron microscopy (SEM), Oberg et al. (1993) and McMahon et al. (1999) observed that the amorphous curd structure before stretching was completely disrupted by the shearing forces of the screws, resulting in a reorganization of the aggregated para-casein matrix into roughly parallel aligned para-casein fibers. Furthermore, fat globules and starter bacteria became concentrated in longitudinal columns that separated the fibers. This striking reorganization is illustrated in Figs 2-4. Immediately after stretching and while the cheese was still hot, the para-casein had the appearance of smoothwalled fibers and there was little evidence of the fat, which had been removed during sample preparation for SEM (Fig. 3). However, after the cheese had cooled overnight, the walls of the fibers showed extensive spherical imprints left by the fat droplets as they solidified and acted as a template around which the pliable para-casein fibers moulded (Fig. 4). A similar transformation of curd structure during stretching was observed by Auty et al. (1998, 2001) using confocal scanning laser microscopy (CSLM). Furthermore, it was possible to directly observe both the protein and the fat phases of the curd, as well as non-fat, non-protein (presumably serum) regions. The CSLM images confirmed that fat globules became redistributed and concentrated in the channels that separated the para-casein fibers (Auty etal., 1998, 2001; Guinee et al., 1999). This striking reorganization of curd structure was also readily observed at much lower magnification by Taneya etal. (1992) using light microscopy and differential staining of the fat and protein phases of the cheese. The same researchers demonstrated, using cryo-SEM, that bulk phase water (serum) as well as fat occupied the columns that separated the protein fibers in newly stretched cheese. Functional properties
Cheese that is used as an ingredient in prepared foods must satisfy certain performance requirements that are determined by the function of the cheese in the particular food application in which it is used. As
256
Pasta-Filata Cheeses
Scanning electron micrograph of low-moisture, part-skim Mozzarella curd taken prior to dry salting and stretching. Scale bar equals 10 l~m (reproduced with permission from Oberg et al., 1993).
noted earlier, LMMC is used mostly as an ingredient in pizza and other prepared foods that contain melted cheese. Therefore, functional properties are essential determinants of the quality and acceptability of LMMC, and considerable research has been directed towards developing and applying new methods to measure functional characteristics and fundamental rheological
properties that determine and affect functional behaviour. A summary of recent developments relating to analytical testing for functional and rheological properties is presented here. For a more complete overview of the topic of LMMC functionality, the reader is directed to the recent comprehensive reviews by Rowney et al. (1999), Fox et al. (2000) and Guinee (2002b).
Scanning electron micrograph of low-moisture, part-skim Mozzarella curd taken immediately after stretching, longitudinal view. Scale bar equals 10 i~m (reproduced with permission from Oberg et al., 1993).
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257
Scanning electron micrograph of low-moisture, part-skim Mozzarella curd taken after stretching and following 1 day of storage, longitudinal view. Scale bar equals 10 #m (reproduced with permission from Oberg et aL, 1993).
Functional properties before heating LMMC is usually produced in block form, ranging in weight from c. 2.3 to 9.5 kg, and must therefore be comminuted (shredded or diced) before it can be used as an ingredient in prepared foods such as pizza. In the industry, the term 'shreddability' is used in reference to several important functional characteristics, including: the ease with which the cheese block is processed through a shredding machine (also referred to as 'machinability'); the geometry and integrity of the shreds (i.e., the extent to which shreds of uniform dimensions with cleanly cut edges are obtained); the susceptibility of the cheese to shatter and form fines during shredding; and the ability of the shreds to resist matting and remain free-flowing. Problems related to shreddability may occur when the body of the cheese is soft and pasty or wet, causing the shredding machine to become clogged with cheese and resulting in shreds with ragged edges and deformed geometry, along with the formation of fines and gummy balls of cheese. Such cheese is also likely to undergo excessive matting after shredding, which makes it difficult to handle, store and apply uniformly on the product in which it is used with portioncontrolled precision. At the opposite extreme, cheese that is excessively firm and dry, as is often the case with low-fat Mozzarella, may take longer to process through the shredding machine and fracture excessively to produce shattered shreds and fines, which also make handling and portion control more difficult (Kindstedt, 1995).
Only a few attempts to quantify aspects of shreddability directly by empirical and imitative tests have been reported. Apostolopoulos and Marshall (1994) used quantitative image analysis data to calculate a shreddability index as a function of the size, shape and number of shredded fragments obtained upon shredding. The shreddability index values obtained by image analysis were highly correlated with sensory panel visual assessments made on the basis of the length of the shreds, amount of fragments present and degree of stickiness between the shreds. An empirical test to measure matting behaviour was proposed based on the ability of cheese particles to penetrate down though a stack of vibrating sieves of decreasing mesh size (Kindstedt, 1995). Sticky cheese that mats excessively is retained by the larger sieves, whereas cheese that remains free-flowing may penetrate to the bottom of the stack. The aggregation index value of Mozzarella cheese, calculated as a weighted average of sieve size • mass of cheese retained by each sieve, increased during storage and with higher fat content, indicating increased susceptibility to matting (Kindstedt, 1995). Many researchers have used texture profile analysis (TPA) and similar uniaxial compression tests at a temperature ranging from c. 10 to 25 ~ to characterize the hardness or firmness of Mozzarella cheese. Generally, these studies showed that Mozzarella became significantly softer or showed a trend towards softening with increasing age and level of proteolysis (e.g., Tunick et al., 1993, 1995; Yun et al., 1993b,d,e, 1995a,b, 1998; Barbano et al., 1994; Kindstedt et al., 1995a,b; Renda
258
Pasta-Filata C h e e s e s
etal., 1997; Guinee et al., 1998, 2000a, 2001, 2002; Hong et al., 1998; Walsh et al., 1998), with increasing fat and/or moisture content (e.g., Tunick et al., 1991, 1993, 1995; Yun et al., 1993e; Rudan et al., 1999) and with decreasing calcium content (Yun etal., 1995b; Guinee et al., 2002) and pH (Guinee et al., 2002). Fundamental rheological testing methods, such as stress relaxation (Diefes et al., 1993; Yun et al., 1994b) and small amplitude oscillatory shear (dynamic) tests (Diefes et al., 1993; Tunick et al., 1993, 1995; Ak and Gunasekaran, 1996), have also been used to characterize the softening of LMMC during refrigerated storage. Testing has been performed in the temperature range of c. 10-26 ~ Of particular interest are the results of Tunick et al. (1995) and Ak and Gunasekaran (1996), who used dynamic testing to measure changes in G' (the elastic or storage modulus) and G" (the viscous or loss modulus) of Mozzarella cheese during storage. The elastic modulus (G') decreased during storage, as expected, indicating a softening of the cheese caused by a weakening of the para-casein matrix by proteolysis. However, the viscous modulus (G") did not increase as expected but, instead, decreased, indicating a reduction in viscous dissipation as the cheese aged. Ak and Gunasekaran (1996) suggested that the decrease in the viscous modulus (G") may have been caused by increased binding of water resulting from proteolysis. Changes in the state of water in Mozzarella cheese during ageing have been the focus of considerable study, as will be discussed later. The impact of such changes on rheological and functional properties as differentiated from the direct effect of proteolysis on the network structure of the para-casein fibers is not completely understood and warrants further investigation.
Heat-induced functional properties (melting) Heat-induced functional properties are essential determinants of the quality and acceptability of LMMC that is used as an ingredient in cooking applications such as for pizza. Important heat-induced characteristics include: meltability (more correctly, flowability, i.e., the extent to which melted cheese flows and spreads upon heating); stretchability (the ability of the molten cheese to stretch and form strings when extended); elasticity (the ability of the cheese strings to resist deformation during extension, which is related to chewiness); oiling-off (the release of free oil); and blistering and browning (the formation of dark-coloured patches of varying size and colour intensity). In general, LMMC for ingredient use in pizza should, upon melting, flow readily to form a continuous melt with complete loss of shred identity, possess a stretchable and slightly to moderately elastic, chewy consist-
ency, and display limited blister formation, limited intensity of browning and a glistening, but not greasy, surface.
Meltability. The meltability of LMMC has been evaluated extensively by empirical tests such as the Schreiber (Kosikowski and Mistry, 1997b) and Arnott (Arnott et al., 1957) tests, which measure the increase in diameter or decrease in height of a cylinder of cheese upon melting under standard conditions. Muthukumarappan et al. (1999a) proposed several modifications to improve the efficacy of the Schreiber test. More recently, Wang and Sun (2002a,b) applied computer vision image analysis to quantify the increase in the area of cheese samples upon melting as an index of meltability. Other widely used empirical approaches to measure flow properties or melted consistency include the method described by Olson and Price (1958), which measures the distance melted cheese flows in a horizontal glass tube, and helical viscometry (Kindstedt et al., 1989; Kindstedt and Kiely, 1992). The latter method measures the resistance on a rotating t-bar spindle as the spindle is drawn through a column of molten cheese, referred to as apparent viscosity. A high apparent viscosity generally corresponds to a fibrous, elastic, chewy melted consistency whereas a low apparent viscosity indicates a viscous fluid-like consistency. A more direct empirical method for measuring post-meh chewiness was described by Metzger and Barbano (1999). Post-meh chewiness was determined by blending melted cheese, that had partially cooled, with water in a stomacher, passing the contents through a series of sieves of decreasing mesh size, and determining the percentage of cheese solids retained in the largest mesh sieve (4.75 mm). Results of this empirical test were highly correlated with sensory ranking of chewiness. Guinee et al. (1998) described an empirical test for melt time, defined as the time required for a fixed weight of shredded cheese to melt and fuse into a molten mass, free of shred identity, on heating at 280 ~ Numerous researchers, who have used one or more of these empirical methods to study Mozzarella cheese, have generally reported that the melt time and apparent viscosity decreased and the meltability (flowability) increased with increasing age and extent of proteolysis (e.g., Oberg et al., I991, 1992; Tunick et al., 1993, 1995; Yun etal., 1993b,d,e, 1995a,b, 1998; Barbano etal., 1994; Kindstedt et al., 1995a,b; Fife et al., 1996; Renda et al., 1997; Guinee et al., 1998, 2000a, 2001, 2002; Hong et al., 1998; Madsen and Qvist, 1998; Walsh et al., 1998), with higher fat and moisture contents (Tunick et al., 1991, 1993, 1995; Yun et al., 1993e; Perry et al., 1997; Rudan et al., 1999; Petersen et al., 2000), with
Pasta-Filata Cheeses
lower calcium content (Yun et al., 1995b; Metzger et al., 200 la; Guinee et al., 2002) and with lower pH (Guinee et al., 2002). Reducing the calcium content of low-fat Mozzarella resulted in lower values for post-meh chewiness (Metzger et al., 2001b). Several different fundamental rheological tests have been used to study the melting process of LMMC. Guinee et al. (1999, 2002) used dynamic testing to characterize changes in the viscoelasticity of LMMC on heating from 20 to 80 ~ They reported that G' (the elastic or storage modulus) remained relatively constant from c. 20 to 25 ~ but then increased rapidly with increasing temperature to 45 ~ indicating a softening of the cheese which the authors have attributed, in part, to liquefaction of the fat phase. At temperatures above 45 ~ G' decreased more slowly because the fat is fully liquid at c. 40 ~ Diefes et al. (1993) also reported substantially lower G' values for LMMC at 60 ~ than at 20 ~ indicative of thermal softening. Furthermore, Guinee etal. (1999, 2002) showed that the phase angle (~) increased gradually when the temperature was raised from 20~ to between 45 and 60 ~ and then increased steeply with increasing temperature to c. 80 ~ These results indicated that the cheese underwent a phase transition from largely elastic to largely viscous in nature when the temperature exceeded the range of c. 45-60 ~ Consistent with these results, Taneya etal. (1992) reported that thermal softening occurred in string cheese over the temperature range 5-45 ~ as measured by compression and stress relaxation tests. The same investigators reported a strong temperature dependency of flow properties over the range of 45-75 ~ as measured by capillary rheometry. In summary, melting of LMMC is characterized by an initial thermal softening, resulting from the liquefaction of fat, followed by a phase change from solid-like to liquid-like as the para-casein matrix collapses, liquid fat globules coalesce and flow, and adjacent planes of para-casein are displaced (Guinee, 2002b). Much interest has developed around lubricated squeezing flow as an approach to profiling melting behaviour. Ak and Gunasekaran (1995b) used a lubricated squeezing flow test to characterize the thermal softening of LMMC over the temperature range 30-60 ~ and softening during ageing. Later, this group (Wang et al., 1998) developed a test device based on lubricated squeezing flow, named the UW Meltmeter, to objectively measure the melt/flow behaviour of cheese at different temperatures. They reported that the meltability of Mozzarella cheese, as profiled by several fundamental rheological parameters measured by the Meltmeter, increased with higher fat content in the cheese and higher melting temperature. Kuo et al.
259
(2001a) used the UW Mehmeter to characterize the flow behaviour of LMMC as a function of cheese age and holding time at 60 ~ for up to 20 min. They reported that the meltability of 1-week-old LMMC was not affected by holding at 60 ~ for up to 20 min. However, the meltability of older cheeses (6 and 12 weeks) decreased sharply with longer holding time at 60 ~ which they attributed to an apparent redistribution of moisture in the cheese caused by increased hydrophobic interactions in the para-casein structure. A different test device, called the UW Melt Profiler, which is also based on the principles of squeeze flow rheometry, was developed by Muthukumarappan et al. (1999b) and later modified by Gunasekaran etal. (2002). This device is used to measure the softening point of cheese, which is defined as the temperature at which cheese begins to flow under constant force. Using the UW Melt Profiler, Muthukumarappan et al. (1999b) demonstrated that the softening point of Mozzarella cheese decreased with increasing age and fat content of the cheese.
Stretchability. Both empirical and fundamental test methods have been proposed to evaluate the stretchability or elongational properties of Mozzarella cheese. Apostolopoulos (1994) and Guinee and O'Callaghan (1997) developed empirical tests to measure the distance to which the melted cheese could be stretched vertically or horizontally, respectively, before complete strand breakage. Authors who used the method of Guinee and O'Callaghan (1997) have generally found that the stretchability of molten LMMC increases with storage time at 4 ~ up to c. 15-20 days and thereafter remains relatively constant up to 50-75 days (Guinee et al., 1998, 2000a, 2001, 2002; Walsh et al., 1998). However, the stretchability deterioriates fairly rapidly as the level of pH 4.6-soluble N (as % of total N) exceeds c. 14% (Feeney etal., 2001; Guinee etal., 2001), as, for example, occurs on prolonged holding of cheese at 4 ~ (e.g., >130 days) or holding for a shorter time (e.g., 70 days) at higher storage temperature (e.g., 10-15 ~ (Guinee, 2002a). A more fundamental approach was described by Apostolopoulos (1994), who used lubricated squeezing flow to determine the elongational viscosity of melted LMMC at 65 ~ which can be used as a measure of the ability of the cheese to stretch and form strings. Cavella et al. (1992) used a spinning test method to objectively evaluate the stretchability of Mozzarella cheese. Horizontal (Ak etal., 1993) and vertical (Ak and Gunasekaran, 1995a) uniaxial extension methods have also been used to measure the elongational properties of LMMC. From the data presented in these reports, it appears that the horizontal method is more sensitive
260
Pasta-Filata Cheeses
than the vertical method to changes in the stretching behaviour of the cheese during 1 month of ageing.
Oiling-off. Oiling-off is caused by the release of free oil from the body of melted cheese. Excessive oilingoff results in pools of liquid fat at the surface and throughout the body of the melted cheese, giving the cheese a greasy appearance and mouthfeel that are generally regarded as undesirable. However, a moderate release of free oil contributes to desirable melting characteristics by creating a hydrophobic film on the cheese surface during baking, giving the surface a desirable sheen and, more importantly, slowing down evaporative loss of moisture. Excessive dehydration during melting, as occurs when insufficient free oil is released, results in the formation of a tough skin on the cheese surface that inhibits flow and scorches readily (Rudan and Barbano, 1998; Rudan et al., 1999). Free oil has been measured empirically by two different approaches: melting a disk of cheese on a filter paper and then measuring the area of the oil ring that diffuses into the filter paper; or melting and centrifuging the cheese to recover the free oil (Kindstedt and Rippe, 1990; Kindstedt and Fox, 1991). In general, oiling-off of Mozzarella cheese has been shown to increase with increasing fat content (Kindstedt and Rippe, 1990; Rudan et al., 1999), decreasing salt content (Rippe and Kindstedt, 1989; Kindstedt et al., 1992) and increasing time of storage and level of proteolysis (e.g., Tunick et al., 1993, 1995; Yun et al., 1993b,d,e, 1995a, 1998; Barbano et al., 1994; Renda et al., 1997; Hong et al., 1998; Poduval and Mistry, 1999). Furthermore, the release of free oil from Mozzarella cheese was reduced substantially when the milk or the cream fraction of the milk was homogenized before cheesemaking (Tunick, 1994; Rudan et al., 1998; Poduval and Mistry, 1999). Homogenization results in a much finer dispersion of fat within the cheese structure, as observed by SEM, which limits the ability of fat globules to coalesce and flow on melting. The use of a twin-screw extruder to stretch Mozzarella cheese also reduced oiling-off to a negligible level, presumably because the high shear mixing of the extruder produces a finer dispersion of the fat within the cheese structure (Apostolopoulos et al., 1994). Free oil was reduced by the addition of buttermilk solids to the cheese milk, presumably due to phospholipid-mediated enhancement of emulsification (Poduval and Mistry, 1999). Browning. Mozzarella cheese that contains both reducing sugars (i.e., lactose and galactose) and proteolysis products is susceptible to non-enzymatic (Maillard) browning reactions at high temperatures, such as that which occur during pizza baking. The browning potential of Mozzarella cheese has been evaluated
objectively by reflectance colourimetry after heating the cheese under various conditions (Johnson and Olson, 1985; Oberg et al., 1992; Barbano et al., 1994; Mukherjee and Hutkins, 1994). After heating and cooling, the cheese may be analysed for three colour indices, L '~ (light to dark), a ~ (red to green) and b '~ (yellow to blue), from which an evaluation of the intensity of browness can be made. Reduced browning potential in LMMC has been associated with lower galactose levels and the use of galactose-fermenting starter cultures (Johnson and Olson, 1985; Matzdorf etal., 1994; Mukherjee and Hutkins, 1994). Conversely, LMMC made from milk fortified with non-fat dry milk solids showed increased browning, presumably due to higher levels of lactose and galactose in the cheese (Yun et al., 1998). Directly acidified Mozzarella shows very little browning, presumably due to the absence of proteolysis products of starter culture origin (Oberg et al., 1992). Cultured Mozzarella cheese has generally been reported to increase in browning potential during ageing (Oberg et al., 1991; Barbano et al., 1994; Merrill et al., 1994; Yun et al., 1998). Presumably, increased browning is caused by the accumulation of proteolysis products and/or galactose released by non-galactose fermenting starter bacteria during ageing. Age-related changes in structure and function
Newly manufactured cultured LMMC generally melts to a tough, fibrous, chewy consistency that has limited ability to stretch and flow. Typically, it takes several weeks of storage at refrigerated temperatures before cultured LMMC attains its optimum melting characteristics (Kindstedt, 1995). Therefore, much research has been aimed at elucidating the age-related changes in the structure and function of Mozzarella cheese. However, it is important to recognize that the initial structure and functional properties of Mozzarella may vary substantially depending on the chemical composition of the cheese. Fat plays a particularly important role in the initial structure and function because the amount of fat determines the extent to which the paracasein fibers are interrupted by fat-serum columns (see Fig. 4). As the fat content of Mozzarella decreases, the volume fraction of the casein matrix increases and the para-casein strands become thicker with fewer inclusions of fat-serum channels between them (Merrill et al., 1996; McMahon et al., 1999). The abundance and size of the fat-serum channels influence the melting characteristics of the cheese because the channels act as a low viscosity lubricant which facilitates the displacement of adjacent planes of para-casein during heating (Guinee, 2002b). Consequently, cultured Mozzarella cheese with a reduced fat content initially
Pasta-Filata Cheeses
melts to a tougher, more chewy (higher apparent viscosity) and less flowable (lower meltability) consistency than Mozzarella made by the same process but with a higher fat content (Rudan et al., 1999). Furthermore, the distance separating the fat-serum channels from one another increases with decreasing fat content (Merrill et al., 1996), which restricts the ability of liquid fat globules in adjacent channels to flow and coalesce with one another to form pools of free oil. Consequently, the fat remains more finely dispersed on melting and the proportion of total fat that is released as free oil decreases with decreasing fat content (Rudan et al., 1999). The level of casein-associated calcium in the newly made cheese also plays a critical role in the initial structure and function of the cheese, as demonstrated by several recent studies in which different strategies to vary casein-associated calcium were used. Metzger et al. (2000, 2001a,b) used pre-acidification to vary the total calcium content of low-fat Mozzarella while holding other aspects of composition nearly constant. They reported that the level of water-insoluble (i.e., casein-associated) calcium decreased as the total calcium content decreased, which resulted in para-casein fibers that were less highly crosslinked with calcium and more highly solvated, the latter being evidenced by less serum expressed on centrifugation. Consequently, cheeses with less total calcium (and therefore less casein-associated calcium) had lower hardness, apparent viscosity and post-meh chewiness values immediately after manufacture, indicative of a softer cheese before heating and a less fibrous and chewy melted consistency. Several researchers (Kindstedt et al., 2001; Cortez et al., 2002; Ge et al., 2002) used a post-manufacture method to change the pH of cultured LMMC while holding other aspects of composition nearly constant. Increasing the cheese pH in the range of c. 5.0-6.5 caused a progressive increase in the amount of waterinsoluble (i.e., casein-associated) calcium and in the apparent viscosity of the cheese. Furthermore, changes in both calcium distribution and apparent viscosity were reversible when the pH of the cheese was reversed (Ge et al., 2002). These results, in combination with those reported by Metzger et al. (2001a,b), indicate that the initial cheese pH and the total calcium content independently affect the level of casein-associated calcium and, therefore, the initial structure and functional properties of Mozzarella cheese. Guinee et al. (2002) came to a similar conclusion by using direct acidification to simultaneously vary the pH and total calcium content of Mozzarella cheese. They observed that when the calcium level was typical, i.e., 28-30 mg/g protein, higher cheese pH, in the range 5.3-5.8, resulted in higher apparent viscosity, longer melt time,
261
and reduced flowability and stretchability. However, at a relatively low calcium level (e.g., 21 mg/g protein), LMMC with a high pH (i.e., 5.8) had functionality flow, stretch and apparent viscosity, at 1 day, similar to that of the control LMMC after storage at 4 ~ for 12-20 days. Furthermore, a lower total calcium content resulted in less serum expressed on centrifugation and a high degree of swelling of the para-casein fibers at the microstructural level immediately after manufacture, as observed by CSLM. From the results of the above studies, it may be concluded that initial cheese pH, in combination with the total calcium content, largely determines the amount of casein-associated calcium in the initial cheese structure. Casein-associated calcium, in turn, influences the amount of calcium crosslinking and solvation of the para-casein fibers and thus the initial cheese structure and functional characteristics. Less calcium crosslinking and greater solvation enable adjacent planes of para-casein to be displaced more readily during melting, resulting in greater meltability and stretchability and lower apparent viscosity and chewiness. Thus, the initial melting characteristics of Mozzarella cheese can vary widely, depending on the amount of caseinassociated calcium present in the cheese immediately after manufacture. During the first few weeks after the manufacture of cultured LMMC, it is well documented that meltability, stretchability and oiling-off increase, and the apparent viscosity, melt time and hardness decrease, as discussed earlier. These fairly dramatic functional changes are influenced by proteolysis that occurs concurrently during ageing, and proteolysis is clearly one of the driving forces behind the age-related changes in structure and function. For example, when proteolysis in LMMC was reduced by stretching at high temperature (i.e., cheese temperature at exit = 66 ~ the usual changes in hardness, meltabilty and apparent viscosity occurred more slowly (Yun et al., 1994a; Kindstedt et al., 1995b). Conversely, increasing the rate of proteolysis by using a more proteolytic coagulant or by storing LMMC at a higher temperature resulted in a faster decrease in the melt time and/or apparent viscosity and a faster increase in meltability (flowability) during ageing (Yun et al., 1993c,d; Guinee et al., 2002). However, proteolysis is not solely responsible for functional changes during ageing. Considerable interest has also been directed towards changes in the serum phase of Mozzarella cheese and elucidating their effects on structure and function (Kindstedt and Guo, 1998; McMahon etal., 1999). Several investigators have reported that the amount of serum expressed from cultured LMMC by centrifugation or pressing decreased from levels equivalent to c. 20-40% of the total cheese moisture immediately after manufacture
262
Pasta-Filata Cheeses
to no expressible serum after 2-3 weeks of ageing (Guo and Kindstedt, 1995; Kindstedt, 1995; Kindstedt et al., 1995b; Guo et al., 1997; Guinee et al., 2001, 2002; Kuo etal., 2001b). Thus, the water-holding capacity of cultured LMMC increases steeply during the first weeks after manufacture. Consistent with these results, data obtained using pulsed nuclear magnetic resonance suggest that a redistribution of water from a more- to less-mobile state occurs in cultured LMMC during the first 10 days of storage (Kuo et al., 2001b). McMahon et al. (1999) further demonstrated that the redistribution of water and the resulting increase in the water-holding capacity of Mozzarella cheese involved entrapped bulk water, whereas the amount of unfreezable (i.e., chemically bound) water did not change. The mechanism by which bulk water is redistributed has been elucidated using a couple of different approaches. Several studies have shown that intact caseins, especially [3-casein, and calcium are present in the expressible serum from cultured LMMC, and that their concentrations increase as the amount of serum decreases during storage (Guo and Kindstedt, 1995; Kindstedt et al., 1995b; Guo et al., 1997). These data suggested that a progressive dissociation of calcium and caseins from, and association of water with, the para-casein matrix occur over time. Guo et al. (1997) also observed that the solvation and solubilization of the para-casein matrix occurs much more slowly when
cultured LMMC contains no added salt (NaCI), as evidenced by higher amounts of expressible serum and lower concentrations of intact caseins in the serum obtained from the unsalted cheese. These investigators postulated that age-related changes in the water-holding capacity of cultured LMMC result in part from a NaCl-mediated process of swelling and solubilization of the para-casein matrix at the microstructural level. Furthermore, they suggested that the presumed microstructural swelling may be analogous to the swelling phenomenon known as 'soft rind defect' that occurs at the macrostructural level (Guo and Kindstedt, 1995; Guo et al., 1997). 'Soft rind defect' occurs when cheese is exposed to dilute salt brine (i.e., <6%, w/w, NaC1) with a low calcium content (Geurts et al., 1972). Further evidence of microstructural swelling was obtained using several different microscopy techniques. McMahon (1995) and McMahon et al. (1999) confirmed using SEM that the microstructure of LMMC changes substantially during the first weeks after manufacture, as seen in Figs 5-7. Initially, the fatserum channels appear to be very open, as evidenced by shallow imprints of fat globules on the walls of the para-casein fibers (Fig. 5). However, by day 7 the fat globules appear to be partly engulfed by the para-casein matrix (Fig. 6), and by day 21 they appear to be completely encased by swollen protein (Fig. 7). A similar process of microstructural swelling was observed by Auty et al. (1998, 2001) using CSLM. Also, Cooke et al.
Scanning electron micrograph of low-moisture, part-skim Mozzarella cheese 1 day after cooling and brining. Fat-serum channel walls show indentations formed by solidified fat globules of varying size and starter bacteria. Bacterial cells and residual fat globule membrane material adhere to the fat-serum channel walls. Scale bar equals 10 i~m (reproduced with permission from McMahon et aL, 1999).
Pasta-Filata Cheeses
263
Scanning electron micrograph of low-moisture, part-skim Mozzarella cheese after 7 days of storage at 4 ~ Fat globule indentations are more pronounced than at day 1, indicating that the cheese protein matrix has expanded into the fat-serum channels. Scale bar equals 10 l~m (reproduced with permission from McMahon et al., 1999).
(1995) reported that the average size and space between electron-dense clusters shown by transmission electron microscopy increased in Mozzarella cheese during ageing. The change in spacing of the electron-dense clusters, which corresponds to the sub-aggregates of the
para-casein matrix, is consistent with the swelling phenomenon that has been observed by SEM and CSLM (McMahon et al., 1999). Furthermore, Paulson et al. (1998) demonstrated that NaCI has a striking effect on the microstructure of non-fat directly acidified
Scanning electron micrograph of low-moisture, part-skim Mozzarella cheese after 21 days of storage at 4 ~ The hydrated cheese protein matrix fills the spaces between the solidified fat globules. Impressions of discrete fat globules attest to the completeness of the cheese protein matrix hydration and subsequent expansion into the fat-serum channels. Starter bacterial cells embedded in the matrix are evident. Scale bar equals 10 i~m (reproduced with permission from McMahon et aL, 1999).
264
Pasta-Filata Cheeses
Mozzarella cheese; salted cheese had a more homogeneous protein matrix that was free of serum pockets, whereas unsalted cheese contained pockets of free serum throughout the para-casein matrix and more widely spaced protein sub-aggregates. In summary, the above results strongly suggest that a NaCl-mediated redistribution of water, characterized at the microstructural level by swelling of the para-casein fibers, occurs in cultured Mozzarella cheese during the first few weeks after manufacture. The increase in solvation and decrease in calcium crosslinking of the swollen para-casein fibers during the first few weeks of ageing enable adjacent planes of para-casein to displace more readily and fat globules to coalesce and flow more freely upon melting. Consequently, meltability (flowability), stretchability and oiling-off characteristically increase, and the apparent viscosity, melt time and chewiness decrease as cultured LMMC ages. However, as noted earlier, it is possible to pre-empt many of the age-related changes in structure and functional properties by substantially reducing the initial level of casein-associated calcium in the cheese. This can be accomplished by directly acidifying the cheese milk, so as to attain a more highly solvated and less highly crosslinked para-casein matrix in the cheese immediately after manufacture (Kindstedt and Guo, 1997; Metzger et al., 2001a; Guinee et al., 2002). Such cheese may have functional properties immediately after manufacture that are largely similar to those attained by cultured LMMC after several weeks of ageing (Kindstedt and Guo, 1997; Guinee et al., 2002).
General characteristics
Kashkaval is one of the most popular hard cheeses in many Mediterranean countries, its production dating back to the eleventh and twelfth centuries. However, historical references suggest that Kashkaval has an even older tradition. According to the Roman writer Columella, a cheese named 'manurn pressurn' was produced in the Roman Empire by a method similar to that for Kashkaval. It has been assumed that Kashkaval was brought to the Mediterranean by nomadic tribes from the East during the second century BC through the seventh century. During Roman times, this technology was brought from Italy to Great Britain, adapted to the English conditions, modified, and resulted far later in a new type of cheese, named Cheddar, one of the most popular cheeses worldwide today. While in ancient times Kashkaval production was limited to the Greek and Roman empires, as well as their colonies, today it is produced in an area extending
from Crimea, South Ukraine, the Caucasus and Turkey, through Greece, Bulgaria, Romania, Yugoslavia, Albania and Hungary to Italy, Algeria, Tunisia, Egypt and Morocco. This entire area is characterized by a relatively hot and dry climate, a hilly terrain, with welldeveloped sheep breeding. International trade and globalization have led to the expansion of Kashkaval production to other parts of the world. The following Kashkaval-type cheese varieties, which differ in name and various aspects of manufacture, are produced in the Mediterranean region: Kashkaval Balkan, Kashkaval Preslav, Kashkaval Vitosha (Bulgaria), Kachkavalj (Yugoslavia), Kachkaval, Kachekavalo (USSR), Kasseri (Greece), Kasar (Turkey, Albania) and Cascaval Dobrogen (Romania). Kashkaval has also been given different commercial names according to the production district, e.g., Pirdop in Bulgaria, Epir in Greece, or Sarplaninski and Pirotski Kaskaval in Yugoslavia (Peji, 1956). The Italian version of Kashkaval is called Caciocavallo and in Egypt the name Romy is commonly used. Kashkaval is manufactured from cow, sheep, goat or mixed milk, which may be raw or pasteurized. For example, in Bulgaria, Kashkaval Preslav is produced from mixed milk, Kashkaval Balkan is produced from sheep's milk, while Kashkaval Vitosha is produced from cows' milk; in Romania, Cascaval Dobrogen is produced from sheep's milk only. The typical form of Kashkaval is flat, cylindrical, with a smooth, amber-coloured rind, 30 cm in diameter, 10-13 cm in height and 7-8 kg in weight. However, sausage-like or pear-like shapes are also produced (Carie, 1993; Carie and Milanovi~:, 1994). The typical composition of Kashkaval produced in several countries is shown in Table 1. The European Economic Community (1990) specified the characteristics of Kashkaval as follows: 'Kashkaval cheese of sheep's milk, matured for at least 2 months, of a minimum fat content of 45% by weight in the dry matter, and a dry matter of at least 58%, in whole cheeses of a net maximum weight of 10 kg, whether or not wrapped in plastic' (Carir 1993). The National Committee for Standardization in Yugoslavia has defined the National Standard for Kashkaval cheese (Yugoslav Standard, 1997). The Standard takes into account traditional manufacturing practices, as well as modern trends in the industrial production of the cheese. According to the Standard, 'Kashkaval is a semi-hard or hard, pastafilata cheese which is available in two types: Kashkaval (weigh 5-10 kg, minimum 56% dry matter, minimum 45% fat in dry matter and minimum 8 weeks ripening period) and Kashkaval Krstag (weigh up to 3 kg, minimum 54% dry matter, minimum 45% fat in dry matter and minimum 4 weeks ripening period)'. Both cheese
Pasta-Filata Cheeses
265
Composition of various types of Kashkaval cheese
Type of Kashkaval
Fat (%)
Dry matter (%)
Total protein (%)
Salt (%)
Ash (%)
pH
Author
Bulgaria
30.0
60.14
19.60
4.0
5.69
5.0
Greece
33.88
66.36
25.14
2.2
4.38
5.1
25-29 25.5-31.4 27-32 20.5-21.2 14.5 21.1-26.3
58-65 48.8-60.1 60-65 47.9-49.4 49.5-51.7 49.9-61.2
26.0 19.0-22.2 22.0-23.1 30.3-33.5 21.4-25.2
2.6-3.2 1.6-2.3 2.0-3.5 1.2-1.5 2.5-2.7 -
2.8-3.4 5.0-5.7 -
4.9-5.0 5.5-5.6 5.5-5.6 5.2-5.6
Kosikowski and Mistry (1997) Kosikowski and Mistry (1997) Robinson (1995) Kocak et aL(1996) Peji(; (1956) Alrubai (1979) Milanovi6 (1993) Omar and EI-Zayat (1986)
Turkey Yugoslavia
E gypt
types can be produced from cows', sheep's, goats' or mixed milk, which may be raw or pasteurized. Kashkaval, together with Mozzarella and Provolone, belongs to the pasta-filata group of cheeses. Kashkaval undergoes a texturization process that involves soaking the acidified curd in hot brine until a plastic consistency is achieved. The hot plastic curd is then kneaded and stretched to produce a homogeneous cheese with a fibre-like structure. The specific technology in the production of Kashkaval results in the characteristic structure of the final cheese: laminar, elastic, very close with visible layers and random slits, but no gas holes.
Manufacturing technology The manufacture of Kashkaval cheese consists of two independent stages: 1. production of the curd and its acidification (cheddaring); 2. texturizing of the acidified curd which involves heating and mechanical kneading and stretching by soaking in hot water or brine. The steps in the manufacturing process are shown in Fig. 8. Traditionally, Kashkaval cheese is produced from raw milk, which is generally of poor quality, without the addition of a starter culture. Heat treatment of the curd during texturizing has a preservative effect on the final cheese, enabling raw milk with a higher acidity, i.e., poor microbiological quality, to be processed. During the last two decades the use of pasteurized milk and starter cultures has been introduced gradually into commercial practice to standardize Kashkaval cheese quality (Carie, 1993; CariC and Milanovie, 1994; Milanovie and Carie, 1998; Pudja and Milanovie, 2000). Starter cultures for Kashkaval cheese usually consist Str. thermophilus, Lc. lactis subsp, diacetylactis (cit + Lc. lactis subsp lactis), Leuc. mesenteroides subsp, dextranicus, Lb. delbrueckii
subsp, bulgaricus, Lb. helveticus, Lb. casei, used in various combinations at a level ranging from 0.1 to 0.5%, w/w (Cari~, 1993; Cari~ and Milanovi~, 1994). Rennet is added in sufficient quantity to coagulate milk within 30-40 min. In addition to calf rennet, coagulating enzymes of various origins are now used. Recent studies comparing the effects of calf rennet, recombinant chymosin and protease from Rhizornucor miehei on the microstructure of Kashkaval curd demonstrated that recombinant chymosin as well as microbial coagulant may be used successfully in the manufacture of Kashkaval cheese from milk or UF retentate (Milanovi~, 1993, 1996; Milanovi~ and Cari~, 1998; Milanovi~ etal., 1998). The protein matrias of curds obtained by coagulating milk using calf rennet, recombinant chymosin or microbial protease are shown in Figs 9 and 10. Milk coagulated with standard calf rennet contained smaller casein particles compared to those obtained using recombinant chymosin or microbial rennet, the latter showing more advanced fusion of casein micelles into chains and clusters (Fig. 9). The microstructure of the Kashkaval curds obtained by coagulating UF retentate differs from those made from milk (Fig. 10). Chains of casein particles predominated over clusters in all UF curds, while clusters predominated in gels from non-ultrafiltered milk. The curd matrix obtained using recombinant chymosin or microbial rennet was considerably finer than the matrix obtained using standard calf rennet. The microstructure of conventional and UF Kashkaval curds differed from each other to an extent depending on their water and fat contents and the origin of enzymes used. The coagulum is usually cut finely into particles, 6-8 mm, stirred at 32 ~ for 5 min (Peji~, 1956; Scott, 1981), and then scalded at 42 ~ for 35 min in the making of Russian or Italian Kashkaval; in contrast, no scalding is used in the making of Balkan Kashkaval.
266
Pasta-Filata C h e e s e s
Milk
Pasterurization 75 ~
Starters Rennet
15-20 s
Cooling 32 ~ =~, Inoculation ~ Coagulation 32 ~ 30-40 min
Cutting 32 ~ 5 min Stirring 32 ~ 5 min Scalding 42 ~ 35 min = Whey tI Pressing 30-40 min I -~ Whey t "Fresh curd
Cutting in blocks Curd acidification 1-4 days, 20 ~
Salt
Ripe curd =~
Texturing 72-75 ~ 1 min Salt =~ Dry salting
1
Moulding
Cooling 20 ~ 24 h Pre-ripening 15-18 ~ RH 80-85~ 15 days Foils
=~ Packaging
10-12 ~
Ripening RH 80-85%, 2-3 months Storage 2-4 ~ 1 year Kashkaval
Manufacturing procedure for Kashkaval (Cari~ and Milanovi~, 1994).
cheese
The curds are then ladled into moulds and allowed to drain and self-press for 30 min. Alternatively, the whey may be drained from the vat and the curds allowed to fuse in the vat for 30--40 min. The bed of fused curds is then sliced into blocks and cheddared to allow the fermentation of lactose to continue. The length of the cheddaring process depends on the bacterial activity and curd acidity. During the warm season, cheddaring is usually completed in 1 day, while during winter, it may last 4 or 5 days, in both commercial and home-made procedures. The introduction of pasteurization and the use of starter cultures have improved the cheddaring process (Carie, 1993; Carie and Milanovik, 1994; Pudja and Milanovik, 2000). Proteolysis commences during cheddaring of the curd, which constitutes the first stage of Kashkaval ripening (Peji~, 1956; Djordjevi~, 1962; Cari~, 1993). Proteolysis as measured by the Van Slyke method (Djordjevid, 1962), during cheddaring is 25% of the total proteolysis during ripening in the Balkan procedure, 33% in the Russian and even 46% in the Italian procedure. During cheddaring, lactic acid fermentation takes place, with a concomitant increase in acidity to pH 5.4-5.5 when cows' milk is used, or pH 5.2-5.3 when sheep's milk is used. The difference in acidity can be attributed to the relative differences in the proportion of individual caseins between sheep's and cows' milk. Acidification causes an increase in the concentration of soluble calcium (the curd after cheddaring contains 53% more soluble calcium than the unacidified fresh curd) and results in the formation of monocalcium para-caseinate (Cari~, 1993). Acidification results in the characteristic fibre-like structure of the finished product. Additionally, the growth of lactic acid bacteria and the production of lactic acid inhibits the growth of many species of microorganisms (gas-forming, proteolytic, lipolytic) that can cause defects in the cheese. The ripened curd is then texturized, which is accomplished by soaking the blocks of curd (20-25 cm length; 5 - 1 0 c m width; 0.5-1 cm high) in a hot (72-75 ~ brine (5% NaC1) for 1 min (Peji~, 1956; Cari~ and Milanovie, 1994) or 12-18% NaC1 for 35-50 s (Cari~, 1993), in either the traditional or mechanized production technique. The traditional method for Kashkaval production required substantial manual labour. After forming and cheddaring the blocks by hand, the ripened blocks were placed into metal or wooden perforated baskets which were immersed in 5% NaC1 solution at 72-75 ~ The curd mass was then agitated with a strong, wooden stick in order to obtain a compact structure. The hot curd was then transferred to a table and hand-kneaded like a dough. At this stage, the curd has become plasticized and elastic.
Pasta-Filata Cheeses
Protein matrices (dark areas) of Kashkaval curds obtained by coagulating milk using: (a) rennet (Ha-Bo, Chr.Hansen's Lab. A/s, Denmark- CHR; (b) fermentation chymosin (Kluyveromyces lactis, Maxiren 15 I, Gist brocades, The Netherlands- GENC); (c) microbial protease (R. mieheL Rennilase 501, Novo Industri A/S, D e n m a r k - REN) (reprinted from Milanovi6 et aL, 1998, with permission from Elsevier Science).
267
Protein matrices of Kashkaval curds obtained by coagulating UF milk retentate using: (a) rennet (Ha-Bo, Chr.Hansen's Lab. A/s, Denmark- CHR; (b) recombinant chymosin (Kluyveromyces lactis, Maxiren 15 I, Gist brocades, The Netherlands - GENC); (c) microbial protease (R. mieheL Rennilase 501, Novo Industri A/S, Denmark- REN). Small arrows point to casein particle chains, large arrows point to clusters (reprinted from Milanovi6 etaL, 1998, with permission from Elsevier Science).
268
Pasta-Filata Cheeses
Texturizing has an additional advantage, i.e., a pasteurizing effect which suppresses undesirable microbial growth and encourages desirable fermentation and ripening, resulting in high-quality cheese. Microbiological evaluation of Kashkaval curd before and after texturizing showed that Escherichia coli and other coliform bacteria, which were present in the curd at the end of cheddaring, were absent from the final cheese due to heat inactivation during texturizing. Thus, texturizing enables the manufacture of Kashkaval in hot climates and the use of milk with high acidity in its production ( Carie, 1993). In traditional manufacture, Kashkaval cheese is partially salted with dry salt during kneading of the texturized curd. After moulding ( 2 4 h in metal or wooden hoops), a small amount of salt is applied 4-5 times to the cheese during the first 2-3 weeks of ripening. However, salting of Kashkaval is also performed by brining (18-20% NaC1, 10-12 ~ 5-6 days) (Pejie, 1956; Scott, 1981). In general, salt concentration depends on the specific variety of Kashkaval cheese (Table 1). Kashkaval is ripened at 15-18 ~ and at a relative humidity (RH) of 80-85% for 15 days and then at 12-16 ~ and 85% RH for 2-3 months. The shelf-life of Kashkaval is 10-18 months at 2-4 ~ During the past 20 years, the manufacturing process has been mechanized, replacing the manual labourintensive operations of slicing, heat treatment with agitation, salting and moulding (Carl(: and Milanovi~:, 1994; Tomatis, 1996). An example of processing equipment designed not only for Kashkaval but also for other pasta-
filata cheeses is shown in Fig. 11. The incorporation of mechanized texturing, milk pasteurization and starter cultures in the manufacture of Kashkaval has resulted in a more controlled process and better and more uniformquality cheeses. The development of Kashkaval structure at different stages of a mechanized process line is shown in Fig. 12 (Cari~:, 1993). It is evident that the para-casein curd is changed from an amorphous structure (Fig. 12a) to a fibrous material (Fig. 12b) after texturizing. At the end of the process, casein fibres form a compact, characteristic, laminar texture. Scanning electron microscopy micrographs of Kashkaval and other cheese varieties showed distinct differences in the microstructure of the cheeses, which helped to explain their textural properties (Hassan, 1988). Cheese hardness was highly correlated (R = 0.764) with its chemical composition: increased protein and NaC1 resulted in higher cheese hardness, while higher contents of water and fat and pH, as expected, reduced hardness. Kashkaval was ranked fourth according to its hardness, after Provolone, Ras and Gouda (Hassan, 1988). Although the manufacturing protocol for Kashkaval and Cheddar share many features, they differ in two fundamental aspects: 1 The main difference in the manufacture of these two hard cheeses is that Kashkaval has a specific operation: plasticization (heat treatment of cheddared curd with agitation in hot brine), resulting in a plasticized, homogeneous mass, which is afterwards formed into
Continuous texturing and moulding machine used in Kashkaval manufacture (Courtesy IMLEK Dairy, Sid, Yugoslavia).
Pasta-Filata Cheeses
269
Structure of Kashkaval cheese at different production steps: (a) acidified curd showing bacteria aggregated in nests; (b) textured curd with protein matrix having uniform orientation; (c) final product; (d) final product from another plant (Cari(~, 1993).
cheese, with no pressing. During the manufacture of Cheddar there is no plasticization, and moulding is followed by pressing (Milanovie and Tamime, 1990). 2 Traditionally, Kashkaval is produced from raw milk, without starter, while in Cheddar is usually produced from pasteurized milk, which is inoculated with a starter. The use of a starter increases the rate of acid production which affects the cheddaring time and, consequently, the time necessary for the complete manufacturing process. However, the use of pasteurized milk has been introduced recently for the manufacture of Kashkaval. Quality characteristics
The ripening process continues in the formed and salted cheese. During ripening, the distribution pattern of free amino acids (FAA) and free fatty acids (FFA) changes due to the complexity of the maturation process, resulting in the formation of the characteristic flavour of Kashkaval cheese (Omar and E1-Zayat, 1986). The concentrations of glutamic acid, serine, aspartic
acid, threonine, proline, alanine and lysine were higher in 4-month-old Kashkaval than in young cheese but the concentrations of valine, methionine, isoleucine, leucine and tyrosine were lower (Table 2). The increasing amount of total FAA in mature cheese, as well as the presence of particular FAAs, especially glutamic acid, leucine, valine and tyrosine at high concentrations, may be correlated with the typical flavour of Kashkaval. The concentration of FFAs increases c. 3-fold after 2 months and 6.5 times after 4 months of ripening. The main FFA are in the following order: C16, C18:1, C14 and C16. The volatile fatty acids, C4uC10, as well as C2 which contribute to cheese aroma, are present in low concentrations. Differences in the quality attributes of the numerous Kashkaval variants arise from the fact that its technology is subject to many variations with respect to curd composition, added cultures, degree of ripening and intensity of heat treatments. The chemical, rheological and sensory characteristics of Kashkaval cheese manufactured with different
270
Pasta-Filata Cheeses
Concentrations of amino acids in Kashkaval cheese (Omar and EI-Zayat, 1986)
Ripening period Young
Amino acid Lysi ne Histidine Arginine Aspartic acid Th reonine Serine Glutamic acid Proline Glycine Alanine Vali ne Methionine Isoleucine Leucine Tyrosi ne Phenylalanine Total
mg/100 g cheese 2.92 3.25 1.76 1.01 8.94 3.48 1.39 3.53 6.48 3.27 3.76 8.19 4.21 7.17 59.40
2 months Per cent of total free amino acids 4.92 5.48 2.96 1.70 15.10 5.86 2.34 5.94 10.92 5.51 6.33 13.80 7.11 12.10
mg/100 g cheese 5.91 1.79 2.12 8.68 4.93 2.73 26.4 9.16 3.07 6.53 9.92 4.53 6.36 13.20 8.15 16.20 130.00
coagulating enzymes and ripened for up to i year have been investigated in detail (Milanovie, 1993, 1995, 1996; Milanovie and Carir 1993, 1994a, 1995a,b, 1998). A progressive increase in non-protein nitrogen (NPN) is evident in all samples of Kashkaval during ripening, but the rate of increase varies depending on the type of coagulant used. The level of NPN in Kashkaval produced with fermentation chymosin is similar to that in control cheese made with calf rennet up to 150 days, but the values differ by about 25% in advanced ripening. Lower values for NPN were observed in samples made with R. miehei protease compared to cheese with standard rennet (i.e., 20% lower at the start and 10% after 1 year). These results are possibly related to the lower retention of microbial rennet in the curd and consequently in the cheese. In the same study, PAGE was used to evaluate protein degradation throughout ripening. Otsl-Casein was degraded more rapidly than [3-casein, which agrees with numerous earlier published data that residual rennet causes more intense hydrolysis of Otsl- than ]3-caseins, the latter being hydrolysed primarily by plasmin (Hassan and E1 Deeb, 1988; Carie and Milanovir 1994). Calf chymosin and fermentation chymosin caused a very similar pattern proteolysis of casein, with more intense degradation of ~XsX-than [3-casein, as has also been reported for Cheddar (Bines et al., 1989). Similar results were also obtained for Kashkaval made with Rennilase, which contrast with previous reports
4 months Per cent of total free amino acids 4.66 1.38 1.63 6.69 3.80 2.10 20.40 7.06 2.37 5.03 7.65 3.49 4.90 10.20 6.28 12.50
mg/100 g cheese 12.61 6.22 8.03 18.25 10.94 4.17 40.10 17.37 6.77 19.11 10.43 6.65 8.15 26.00 13.09 29.50
Per cent of total free amino acids 5.31 2.62 3.38 7.69 4.60 1.76 16.90 7.31 2.85 8.05 4.39 2.80 3.43 11.00 5.52 12.40
238.00
of higher breakdown of ]3-casein by Rennilase than by chymosin in Cheddar cheese (Creamer et al., 1988; Carie and Milanovie, 1994). The production of volatile aroma components in Kashkaval during ripening was evaluated by Milanovie (1993) and Carie and Milanovir (1994) using capillary gas chromatography of samples by a simultaneous distillation/extraction (SDE) method (De Frutos etal., 1988) The volatile components identified in 360-day-old Kashkaval are shown in Fig. 13. Based on retention times and mass spectrum, seven aroma components were identified: caproic, caprylic, capric and lauric acids and the ethyl esters of caproic, caprylic and capric acids. The presence of volatile fatty acids of the homologous series, CsmC12, and their role in Kashkaval aroma was reported by Hassan and E1 Deeb (1988) and is typical for semi-hard and hard cheese varieties (Scott, 1981). Caprylic and capric acids dominate the fatty acid profile in all Kashkaval samples throughout ripening (Milanovie, 1993; Cari~ and Milanovie, 1994), which agrees with the findings of Omar and E1-Zayat (1986). The distribution of volatile aroma components is influenced by the type of coagulant used in cheesemaking. The content of volatile fatty acids varied throughout the investigation, emphasizing the complexity of Kashkaval ripening. Differences in the formation of fatty acid ethyl esters show that they are formed in various ways during the degradation of cheese proteins and lipids (Gonz/dez de Llano et al., 1990;
Pasta-Filata C h e e s e s
CHR IS
271
GENC
4
IS
7
Time (min)
Time (min)
REN IS
5
6
7
I'L L
.4
Time (min) Chromatogram of the volatile aroma components of 1-year-old Kashkaval cheeses: (a) CHR - cheese produced with standard rennet; (b) GENC - cheese produced with fermentation chymosin; (c) REN - cheese produced with R. miehei protease. Volatile components are: 1-ethyl caproate; 2-ethyl caproate; 3-ethyl caproate; 4-caproic acid; 5-caprilic acid; 6-capric acid; 7-1auric acid (Milanovi6,1993).
Martinez-Castro et al., 1991). The use of fermentation chymosin (Maxiren) or microbial protease (Rennilase) resulted in different aroma profiles from that of control cheese made with standard calf rennet. Kashkaval cheeses produced from 1.8-fold concentrated retentate differed markedly from the conventional cheese in chemical composition, profile of proteolytic
degradation products, pattern of volatile aroma components and sensory characteristics (Milanovir and Carie, 1994b,c). In general, it may be concluded that conventional or UF Kashkaval made using fermentation chymosin is not significantly different from that made using calf rennet, confirming numerous published data that
272
Pasta-Filata Cheeses
recombinant chymosin is a satisfactory alternative to calf rennet in cheese manufacture. However, significant differences were noted in the proteolytic pattern of Kashkaval made using R. miehei protease compared to that made with standard calf rennet. Addition of an enzyme 'cocktail', containing protease (Acelase AHC 100) and lipase (Palatase M 1000 1), to the curd after 75% of the whey had been drained off to accelerate the ripening of conventional and UF Kashkaval cheeses has been assessed. Accelerated ripening by the added proteolytic and lipolytic enzymes was most evident during the early stages of ripening of both UF and traditional Kashkaval cheeses (Milanovie, 1993; Carie, and Milanovie, 1994; Milanovie and Carie, 1995c). Fungal lipase (R. miehei-Palatase M 200 1) added at a concentration of 5 or 15 ml/1001 milk before renneting, accelerated the ripening of Kasar cheese (a Kashkavallike cheese manufactured in Turkey) (Kocak et al., 1996). Titratable acidity, water soluble N and total volatile fatty acids level were significantly higher in lipase-treated cheese than in control cheese during 90 days of ripening. Cheeses with added enzymes had a stronger flavour during the first month of ripening, but developed excessive rancidity at higher amounts of added enzyme. The flavour of low-fat Kashkaval cheese (23% fat) can be enhanced by adding heat- or freeze-shocked Lb delbruecki var. helveticus cultures at a level of 2% to cheese milk prior to renneting. Incorporation of heat- or freezeshocked culture greatly enhanced proteolysis and slightly increased the content of FFAs in low-fat Kashkaval cheese during 6 months of ripening. Low-fat cheese without the added cultures did not develop typical Kashkaval flavour and had poor body and texture (Aly, 1995). The relationship between the level of biogenic amines and the level of hygiene during the production of Caciocavallo and other typical Sicilian cheeses were studied by Lanza et al. (1994). The content of biogenic amines, determined using HPLC, in Caciocavallo cheese was generally high and for histamine, tyramine and putrescine amounted to 2.8-119.1, 3.8-110.6 and 0.6-37.4 mg/kg, respectively. Differences were attributed to variations in the hygienic quality of the original milk and in the manufacturing procedure, including storage. Other relevant changes during Kashkaval cheese ripening were described by Carie (1993) and will not be discussed here. Many of the quality defects encountered by Kashkaval cheese are of microbial origin (see Carie, 1993).
Ak, M.M. and Gunasekaran, S. (1995a). Measuring elongational properties of Mozzarella cheese. J. Texture Stud. 26, 147-160.
Ak, M.M. and Gunasekaran, S. (1995b). Evaluating rheological properties of Mozzarella cheese by the squeezing flow method. J. Texture Stud. 26,695-711. Ak, M.M. and Gunasekaran, S. (1996). Dynamic and rheological properties of Mozzarella cheese during refrigerated storage. J. Food Sci. 61,566-568, 584. Ak, M.M., Bogenrief, D., Gunasekaran, S. and Olson, N.E (1993). Rheological evaluation of Mozzarella cheese by uniaxial horizontal extension. J. Texture Stud. 24, 437-453. Alrubai, A. (1979). Changes of Proteins During Ripening of Kashkaval Produced by Using Various Proteolytic Enzymes. PhD Thesis, Faculty of Agriculture, Beograd University, Beograd. p. 169. Aly, M . E . (1995). Flavour-enhancement of low-fat Kashkaval cheese using heat- or freeze-shocked Lactobacillus delbrueckii var. helveticus cultures. Nahrung 38, 504-510. Apostolopoulos, C. (1994). Simple empirical and fundamental methods to determine objectively the stretchability of Mozzarella cheese. J. Dairy Res. 61,405-413. Apostolopoulos, C. and Marshall, R.J. (1994). A quantitative method for the determination of shreddability of cheese. J. Food Qual. 17, 115-128. Apostolopoulos, C., Bines, V.E. and Marschall, R.J. (1994). Effect of post-cheddaring manufacturing parameters on the meltability and free oil of Mozzarella cheese. J. Soc. Dairy Technol. 47, 84-87. Arnott, D.R., Morris, H.A. and Combs, W.B. (1957). Effect of certain chemical factors on the melting quality of process cheese. J. Dairy Sci. 40,957-963. Auty, M.A.E., Guinee, T.P., Fenelon, M.A., Twomey, M. and Mulvihill, D.M. (1998). Confocal microscopy methods for studying the microstructure of dairy products. Scanning 20, 202-204. Auty, M.A.E., Twomey, M., Guinee, T.P. and Mulvihill, D.M. (2001). Development and application of confocal scanning laser microscopy methods for studying the distribution of fat and protein in selected dairy products. J. Dairy Res. 68, 417-427. Barbano, D.M. (1996). Mozzarella cheese yield: factors to consider. Proceedings of the Seminar on Maximizing Cheese Yield, Center for Dairy Research, Madison. pp. 29-38. Barbano, D.M., Yun, J.J. and Kindstedt, P.S. (1994). Mozzarella cheese making by a stirred-curd, no-brine procedure. J. Dairy Sci. 77, 2687-2694. Bines, V.E., Young, P. and Law, B.A. (1989). Comparison of Cheddar cheese made with a recombinant calf chymosin and with standard rennet. J. Dairy Res. 56,657-664. Bylund, G. (1995). Cheese, in, Dairy Processing Handbook, Tetra Pak Processing Systems AB, Lund. pp. 287-330. Cari~, M. (1993). Ripened cheese varieties native to the Balkan countries, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, 2nd edn, Fox, P.E, ed., Champan & Hall, London. pp. 263-279. Cari~, M. and Milanovi~, S. (1994). Recent advances in Kashkaval cheese technology, Proceedings of the Third California Cheese Symposium, University of California, Davis. pp. 1-17.
Pasta-Filata Cheeses
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on chemical composition, proteolysis, and functional characteristics. J. Dairy Sci. 78, 751-760. Yun, J.J., Barbano, D.M., Kindstedt, P.S. and Larose, K.L. (1995b). Mozzarella cheese: impact of whey pH at draining on chemical composition, proteolysis, and functional properties. J. Dairy Sci. 78, 1-7. Yun, J.J., Barbano, D.M., Larose, K.L. and Kindstedt, P.S. (1998). Mozzarella cheese: impact of nonfat dry milk fortification on composition, proteolysis, and functional properties. J. Dairy Sci. 81, 1-8.
Cheeses Made from Ewes' and Goats' Milk M. Medina and M. Nufiez, Instituto Nacional de Investigaci6n y Tecnologfa Agraria y Alimentaria (INIA) Madrid, Spain
Southern European countries account for most of the production of ewes' and goats' milk cheeses. Traditional cheesemaking procedures are strictly followed in some cases and there are also examples of cheese varieties in which they co-exist with modern industrial technology. The manufacture of many ewes' and goats' milk cheeses is regulated by a Protected Designation of Origin (PDO) at the national level, established mainly in Mediterranean countries to define and protect highquality traditional products against imitations (Nuftez et al., 1989). The European Union adopted this system in 1992 to promote and protect food products. A PDO (Europa, 2002) covers the term used to describe foodstuffs which are produced, processed and prepared in a given geographical area using recognized know-how. Most ewes' and goats' milk cheeses with PDO are named after the region in which they are produced. Ewes' and goats' milk cheeses have special tastes and flavours, very distinct from those of cheeses made from cows' milk. Compositional differences of ewes' and goats' milk with respect to cows' milk, mainly in proteins and fat, account for differences in the sensory characteristics of the cheeses. Genetic, physiological and environmental factors are responsible for variations in milk composition within a single species. Thus, the influence of breed, lactation stage, feeding regime, breeding conditions and milking system on the composition of ewes' and goats' milk has been dealt with in numerous studies. For reviews on the chemical composition of ewes' and goats' milk see Anifantakis (1986) and Juarez and Ramos (1986), respectively.
General aspects of ewes' milk cheeses
The production of ewes' milk in the European Union was 2 181 382 tonnes in 2001. Italy, Greece, Spain and France account for 95% of that production (FAOsTAT, 2002). The technology, microbiology and chemistry of ewes' milk cheeses were reviewed by Nufmz et al. (1989). Six main ewes' milk cheese families were con-
sidered: white or fresh, brined or pickled, hard and semi-hard, blue-veined, stretched curd and whey cheeses. The seasonal nature of ewes' milk production results in large variations in cheese production. Cheeses in European Mediterranean countries are produced mainly between December and June, from a milk production that increases sharply in the spring and decreases from July to November. Different procedures to regulate milk supply, including freezing of unconcentrated ewes' milk (Anifantakis et al., 1980), concentration of milk by ultrafiltration (Voutsinas et al., 1995), freezing of pressed curds (Fontecha et al., 1994; Sendra et al., 1999) and freezing of fully ripened cheeses (Tejada et al., 2002) have been investigated. Genetic polymorphism of ovine milk proteins and their relationships with the technological properties of milk have been reviewed by Amigo et al. (2000). Although there is considerable information on the effects of the genetic polymorphism of caseins in cows' or goats' milk on their composition and cheesemaking potential, the results obtained in ewes' milk are still preliminary. Technological consequences in the final product are not as important as for goats' milk, due to the higher casein content of ewes' milk. The effects of Otsl-casein CC, CD and DD genotypes on cheesemaking properties were investigated by Pirisi et al. (1999b). The CC milk had a higher casein content than CD or DD milk, a higher protein:fat ratio and a smaller casein micelle diameter. Cheesemaking trials with CC milk demonstrated its better renneting properties and cheesemaking characteristics than DD milk, while CD milk was intermediate. Three genetic variants of [3-1actoglobulin ([3-LG; A-C) have been described. The AA phenotype of [3-LG seems to be more efficient than other [3-LG phenotypes for cheese manufacture (Amigo et al., 2000). Some ewes' and goats' milk cheese varieties are manufactured with specific rennets. Pecorino Romano and Fiore Sardo are traditionally made with rennet paste prepared from macerated stomachs of lambs slaughtered immediately after suckling, which also contain pregastric esterases (PGEs). These cheese varieties
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
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C h e e s e s Made from Ewes' and Goats' Milk
have a sharp 'picante' flavour due to the high levels of short-chain fatty acids produced by PGE activity. Thus, these rennets play an important role in the development of cheese aroma and flavour. Vegetable rennet from Cynara cardunculus is used in the manufacture of many Portuguese and some Spanish cheeses made from ewes' milk. C. cardunculus is a thistle that grows wild and abundantly in dry, stony and uncultivated areas in Mediterranean regions. Aqueous extracts are prepared from flowers that are picked and dried in the shade in the open air. Proteinases present in the flowers of C. cardunculus were initially termed cynarases or cyprosins, and currently cardosins. Three acid proteinases have been partly characterized (Campos et al., 1990; Heimgartner et al., 1990; Faro et al., 1992). An acid proteinase was first isolated and shown to induce milk clotting via cleavage of the Phel05--Metl06 bond in bovine K-casein (Faro et al., 1992). Two new aspartic proteinases, cardosins A and B, isolated from fresh stigmas of C. cardunculus have been reported to be similar to chymosin and pepsin, respectively (Verissimo et al., 1995, 1996). Extracts from C. hurnilis are also used in the manufacture of ewes' milk cheeses (Fern~indez-Salguero and Sanjuan, 1999; Vioque et al., 2000). A powdered coagulant from crude aqueous extracts of C. cardunculus has been patented (Fernandez-Salguero etal., 2000) and used in cheese manufacture with similar results (Fern~indez-Salguero etal., 2002). Experiments on the use of C. cardunculus for cows' milk coagulation showed that cheeses tended to be bitter and to have textural defects (Vieira de S~i and Barbosa, 1972). Cardosin exhibits a preference for bonds between hydrophobic amino acids of bovine Otsl-casein (ala163--Va1167) and [3-casein (Ala189--Tyr193), which are less susceptible to attack by chymosin. Several bitter peptides were identified in the digests (QueirozMacedo et al., 1996).
Roquefort, the most important ewes' milk Blue cheese, is of very ancient origin and has been protected by a PDO since 1925. It is manufactured from raw ewes' milk in southern France and Corsica. Milk at 28-32 ~ is usually inoculated with a mesophilic lactic starter. Spores of Penicillium roqueforti are added to the milk or sprinkled, as a suspension, on to the curds when they are put into the moulds. After draining and salting, the cheeses are transported to the natural damp, aired caves of Roquefort-sur-Soulzon, where ripening must take place. Cheeses with a veined body are cylindrical in shape, around 10 cm high and weigh between 2.5 and 2.9 kg (see 'Blue Cheese', Volume 2). Ossau-Iraty is manufactured in south-western France from raw or pasteurized ewes' milk. After renneting, curds are heated at 36-44 ~ Cheeses are ripened for at least 3 months. Ossau-Iraty is similar to Roncal cheese made south of the Pyrenees, although produced in two different forms, 2-3 and 4-7 kg. The effects of lipolytic (L1, Lipomod) and proteolytic (PE Promod) enzyme preparations on proteolysis and the sensory characteristics of Ossau-Iraty cheese have been investigated (Izco et al., 2000). A bitter after-taste was detected in cheeses made with the PP preparation, with higher levels of free amino acids (FAAs) than in control cheese. Cheeses made with the L1 preparation had a more pungent flavour. Broccio is a whey cheese produced in Corsica and received a PDO status in 1988. Fresh whey is mixed with ewes' milk and heated to 80-90 ~ The resulting mass is placed into moulds and allowed to drain. The cheese, in the form of a ball of curd that has been flattened and drained, is presented in returnable wicker baskets known as 'canestres'. Broccio is usually eaten fresh, within 48 h of manufacture, and has a mild flavour. If drained and salted it can be ripened for at least 15 days. Greek ewe cheeses
French ewe cheeses
About 250 000 tonnes of ewes' milk were produced in France in 2001. Out of a total of 37 PDO cheeses made in France, only three varieties, Roquefort, Ossau-Iraty and Broccio, are made from ewes' milk. Their production in 2000 was 18 135, 2610 and 466 tonnes, respectively, representing approximately 40% of all cheeses produced in France from ewes' milk. In the same year, PDO cheeses made from goats' milk cheeses accounted for only 5%, and PDO cheeses made from cows' milk for 9.8%, of total cheese produced from milk of the respective species (CNIEL, 2002).
Greece has a very long tradition in breeding small ruminants, and consequently in cheese production. Of the total milk produced in Greece, 39% is cows' milk, 36% is ewes' milk and 25% is goats' milk (Zerfiridis, 1999). About 670 000 tonnes of ewes' milk were produced in Greece in 2001, and approximately 88% of Greek cheese production is from ewes' and goats' milk. Twenty-three of the twenty-six traditional PDO cheeses are made from ewes' milk mixed with goats' milk (http ://www.greece. org/hellas/cheese.html). Soft cheese production in 2000 was 84 240 tonnes, including 77 894 tonnes of Feta cheese. Semi-hard Kasseri cheese represented 4886 tonnes. Hard cheeses
Cheeses Made from Ewes' and Goats' Milk
accounted for 15 786 tonnes, including Graviera (8188 tonnes), Kefalograviera (3346 tonnes) and Kefalotyri (2753 tonnes). Whey cheese production was 13 245 tonnes (Greek National Dairy Committee, personal communication). Cheese consumption in Greece is 22 kg per caput, 8 kg of Feta and the remainder of Kasseri, Kefalograviera and different types of Graviera and Mizithra. Feta is the most famous traditional Greek cheese. It is a white soft cheese, ripened and kept in brine (10-12% NaC1) for at least 2 months. Feta is manufactured from pure ewes' milk or a mixture with up to 30% of goats' milk. Traditionally, Feta cheese was manufactured from raw milk in small family dairies. Nowadays, the greater part is produced from pasteurized milk with commercial mesophilic and thermophilic starter cultures. Feta has a salty, slightly acid taste, natural white colour, with a firm and smooth texture and pleasant organoleptic characteristics. It is marketed in barrels, in tinned boxes or as plastic-wrapped pieces. For further information on Feta and other cheese varieties ripened under brine see 'Cheese Varieties Ripened in Brine', Volume 2. Galotyri is a soft variety, with a creamy and spreadable texture, produced from ewes' or goats' milk or their mixture. The milk is heated to boiling point and left in containers at room temperature for 24 h. NaC1 (3-4%) is added and the milk is left for another 2-3 days at room temperature with occasional stirring. Rennet may be added to favour coagulation. The curds are put into containers which are held at 8 ~ for not less than 2 months when raw milk has been used. Kopanisti is a soft cheese produced from cows', ewes' or goats' milk or their mixtures. The milk is coagulated at 28-30 ~ in about 2 h. The coagulum is left to stand in the vat for 20-24 h, broken up and put into cloth sacks for draining. The drained curd is mixed with salt, put into containers and placed in a cool place with a high relative humidity to promote the growth of an abundant surface mould. Afterwards, it is mixed every 10 days for 30-40 days to facilitate even distribution of the mould. Kopanisti has a soft, spreadable texture and a salty and piquant flavour. High numbers of yeasts and moulds are found during ripening, with P. c o m m u n e identified in 90% samples (Tzanetakis et al., 1987). W h e n combinations of yeasts and Penicillium spp. were used to accelerate ripening, higher proteolysis was detected in cheeses containing Penicilliurn spp., (FFAs) and organoleptic characteristics were not affected by inoculation with selected cultures (Kaminarides et al., 1992). Pichtogalo Chanion is a soft cheese with a creamy texture, made from goats' or ewes' milk or their mixture. The milk is coagulated at 18-25 ~ within 2 h.
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The coagulum is left to acidify for 24 h and placed in cheese cloths to drain. Salt is added to 1%. Kasseri is a semi-hard cheese made from ewes' milk, to which up to 20% goats' milk may be added. Raw or pasteurized milk is used for manufacture (Moatsu et al., 2001). The coagulum is broken up and allowed to stand for 5-10 min. The curds are heated to 38-40 ~ under constant stirring and left to drain until the pH falls to about 5.2. The block of curd is cut into thin slices, which are stretched in water at 70-80~ for 15min, and put into moulds for 2-3 days. The cheese is dry-salted 12-14 times during ripening at 18 ~ for not less than 3 months. Formaella Arachovas Parnassou is a semi-hard variety made from ewes' or goats' milks or a mixture. The milk is coagulated at about 32 ~ for about 2 h. The coagulum is broken up and heated to 40 ~ for 10 min. The curds are left to settle and then divided into large pieces which fit in special moulds (hoops or wicker containers). The moulds are immersed in whey at 60 ~ for 1 h. Afterwards, the cheeses are inverted and re-immersed in whey at 75-80 ~ for 1 h. Cheeses are salted and left to dry for 24 h. Then, they are placed on shelves for 4 days to dry. Formaella cheese can be consumed fresh or ripened for not less than 3 months. Graviera Agrafon is a hard cheese produced traditionally from ewes' milk or from a mixture of ewes' and goats' milk. The milk is coagulated at 34-36 ~ with rennet. The coagulum is broken up after 25-35 min and the curds heated to 48-52 ~ Cheeses are pressed for several hours, left to dry on wooden shelves for up to 2 days and then placed in brine for 2-4 days. The cheeses are ripened initially at 12-15 ~ continued at 16-18 ~ and completed at 12-15 ~ The m i n i m u m ripening time is 3 months. Graviera Kritis is a hard cheese made in Crete from ewes' milk to which a low percentage of goats' milk may be added. It is a Gruyere-type cheese which undergoes a limited propionic acid fermentation that gives the cheese a slightly sweet taste. Milk is heated up to 68-70 ~ The coagulum is cut and the curds are scalded at 5 0 - 5 2 ~ Cheese wheels, with a diameter of 40 cm and a weight of 14-16 kg, are salted in 16-20% brine for 4 - 5 days and ripened for 90 days at 15-16 ~ Its composition and microbiological characteristics were described by Kandarakis et al. (1998). Inoculation of milk with starter cultures resulted in a faster increase of m e d i u m and small molecular mass nitrogeneous fractions (Moatsou et al., 1999). Kefalograviera is a traditional hard variety manufactured from ewes' milk or a mixture of ewes' and goats' milk, mainly in Western Macedonia. The coagulum is broken up, heated to about 48 ~ transferred to
282
Cheeses Made from Ewes' and Goats' Milk
moulds and pressed. The cheese is held at 14-16 ~ for 24 h, and then brine-salted for 2 days. Ripening begins at 14-16 ~ and during this period the surface of the cheese is dry-salted about 10 times. Afterwards, cheeses are held at less than 6 ~ for at least 3 months. Kefalograviera is circular in shape, with numerous holes, a pleasant salty flavour and a rich aroma. Lowfat Kefalograviera cheese, with the same body, texture and flavour as control cheeses made from full-fat milk (6%), was produced from milk containing 3% fat (Katsiari and Voutsinas, 1994). Cold storage of milk accelerated flavour development of full-fat cheese and enhanced flavour of low-fat cheese (Lalos and Roussis, 2000). Partial replacement of NaC1 by KC1 did not affect proteolysis or lipolysis of cheeses (Katsiari et al., 2001). Kefalotyri is a hard, heavily salted cheese, with a strong flavour and small irregular eyes. It is manufactured from ewes' milk, mixed ewes' and goats' milk or from cows' milk, without a starter culture. Kefalotyri is considered to be the ancestor of many hard Greek cheeses. It has a salty and piquant taste and a rich aroma after ripening for at least 3 months. Lactobacilli and enterococci counts are high, with Enterococcus faecium, Lactobacillus plantarum and Lb. casei as the predominant species. Leuconostocs and Streptococcus thermophilus disappear early in ripening (LitopoulouTzanetaki, 1990). Ladotyri Mytilinis is a hard cheese manufactured from ewes' milk or from a mixture of ewes' and goats' milk on the island of Lesvos. The coagulum is broken up and heated to 45 ~ The curds are pressed in the bottom of the vat, cut to their final cheese size and placed in special moulds. Cheeses are salted and ripened for not less than 3 months. After ripening, cheeses are immersed in olive oil or paraffined. Manouri is a soft whey cheese manufactured from whey obtained from ewes' or goats' milk or mixtures thereof. The mixture is heated to 88-90~ over 40-45 min with constant stirring. At 70-75 ~ salt is added together with 25% ewes' or goats' milk or cream. When the temperature reaches 88-90 ~ the curds are left for 15-30 min and then transferred to cloth sacks for draining for 4-5 h. After this, the cheese is kept at 4-5 ~ until consumed. Xynomyzithra Kritis is another soft whey cheese, with a sharp to sweetish taste and a granular to creamy texture, produced in Crete. The whey is filtered and heated to 90~ over 3 0 m i n while stirring. At 68-70 ~ a small quantity of full cream is usually added. The curds are left to stand for 30 min and then transferred to moulds for draining for 3-5 h. Salt is added and the cheese is put into cloth sacks. The
cheeses are pressed for 1 week before being placed at 10 ~ for at least 2 months. Italian ewe cheeses
Production of ewes' milk in Italy was 850 000 tonnes in 2001 and ewes' milk cheese varieties have great economic significance, particularly in central and southern Italy and in Sardinia. Varieties with PDO status are Pecorino Romano (33 650 tonnes in 2000), Pecorino Siciliano (735 tonnes), Pecorino Toscano (1328 tonnes), Pecorino Sardo (600 tonnes), Fiore Sardo (380 tonnes), Canestrato Pugliese (60 tonnes), Casciotta d'Urbino (230 tonnes) and Murazzano (62 tonnes). Cheese consumption per caput in Italy in 2001 was 19.8 kg. Pecorino Romano, the best-known Italian hard ewes' milk cheese, is produced in regions around Rome and in Sardinia. Milk, raw or thermized, at less than 68 ~ for not more than 15 s, is inoculated with a natural starter culture (scotta-fermento or scotta-innesto) obtained from the residual whey from Ricotta cheese manufacture. Starters consisting of Sc. thermophilus, Lactococcus lactis subsp, lactis and Lb. delbrueckii subsp, bulgaricus are also used for Pecorino Romano manufacture. Coagulation with lamb rennet paste takes place at 37-39 ~ in 14-16 min. The coagulum is cut, left to settle for 2-3 min and cooked at 45-46 ~ After 30 min, the curds are moulded, pressed and trenched to facilitate whey drainage. Cheeses are dry-salted periodically for 30-60 days and ripened at 10-14 ~ for 5-8 months (Battistotti and Corradini, 1993). Cheeses are cylindrical in shape, 25-32 cm high and 25-35 cm in diameter, and weigh 20-35 kg. Flavour is slightly piquant for cheeses ripened for 5 months, and piquant and very strong for older cheeses. The average composition is: dry matter (DM), 68-70%; fat, 28-30%; protein, 28.0-29.5%; NaC1, 3.2-4.5% (Battistotti and Corradini, 1993). Changes in the main microbial groups during ripening have been studied (Deiana et al., 1984). Staphylococcus, Micrococcus and yeasts are the main secondary microflora of traditional cheeses, with Debaryomyces hansenii and Kluyveromyces marxianus as the dominant yeasts (Deiana et al., 1997). Cheese pH 2 4 h after manufacture is 4.9-5.0 and DM, 58.6-59.6%; after 8 months, the pH is 5.6-5.9 and DM is 63.7-67.6%. Proteolysis proceeds slowly due to its high NaC1 content and its low moisture, with 23% pH 4.6-soluble N and 18% TCA-soluble N as a percentage of total N in 8-month-old cheeses. Sensorial characteristics of Pecorino Romano depend mainly on the lipolysis caused by the PGE in the lamb rennet paste. Free fatty acids range from 467 mg/kg (C8) to 1181 mg/kg (C6), distributed in the order C 6 > C 4 > C 1 0 > C 8 . Butanoic and hexanoic acids provide
Cheeses Made from Ewes' and Goats' Milk
intensity to the fatty acid flavour, 2-ethyl butanoic provides the butyric acid flavour notes and 3-methyl butanoic acid the sweat-like and fatty-acid like flavour notes. 4-Methyl octanoic and 4-ethyl octanoic acids, together with p-cresol, m-cresol and 3,3-dimethyl phenol, appear to be responsible for ewe notes in Pecorino Romano cheese (Ha and Lindsay, 1991b). Pecorino Sardo is a semi-cooked cheese produced in Sardinia from ewes' milk inoculated with a commercial or natural whey starter culture. Calf rennet is used to coagulate milk. The curds are heated to 41-42 ~ and held at this temperature for approximately 10min. Cheeses are brine-salted for 48 h. Mild Pecorino Sardo is ripened for 20-60 days, weighs from 1 to 2.3 kg and has an aromatic and slightly acid flavour. The mature type, which is ripened for at least 4 months, acquires a consistent structure and a strong and piquant flavour during ripening. Its weight varies from 1.7 to 4 kg. Thermophilic lactic acid bacteria (LAB) (Lb. delbrueckii subsp, bulgaricus, Lb. helveticus and Sc. thermophilus) predominate in industrial Pecorino Sardo manufactured from thermized milk with natural starter cultures (Mannu et al., 2002). Enterococci and lactococci, the main microbial groups in farm-made raw-milk Pecorino Sardo (Mannu et al., 1999), show a high genetic diversity (Mannu and Paba, 2002). The volatile components of Pecorino Sardo were described by Larrayoz et al. (2001). Pecorino Siciliano is a hard cheese, cylindrical in shape, with plane or slightly concave faces and a weight of 4-12 kg. It is manufactured from ewes' milk to which lamb rennet paste is added. The cheese surface is dry-salted twice in 10 days. Pecorino Siciliano is consumed at different stages of ripening, either fresh, dry-salted for 1 week or ripened for up to 4 months. The mature cheese has a pungent aroma and a characteristic sharp taste. The composition and physico-chemical properties of Pecorino Siciliano cheese were reported by Gattuso et al. (1995), and its microbiological characteristics by Giudici et al. (1997) and Migliorisi et al. (1997). Fiore Sardo is a hard cheese manufactured from raw ewes' milk in Sardinia. Coagulation takes place at 35-37 ~ in 20-30 min with lamb rennet paste. After 30 min, the coagulum is cut into rice-grain sized particles and left to settle. Cheeses are brined or dry-salted. After brining, the cheeses may also be slightly smoked and ripened for 6-12 months. Cheeses weigh around 1.5-4 kg. The flavour is flowery and fragrant, and the mature type is more piquant. Lc. lactis subsp, lactis predominates during most of the ripening period, but E..faecium and occasionally E..faecalis are the dominant species in 4-month-old
283
cheeses (Ledda, 1996). Lb. plantarum, Lb. casei and Lb. paracasei are the main mesophilic lactobacilli (Mannu et al., 2000). Cheeses produced from thermized milk and inoculated with Lc. lactis subsp, lactis and E. faecium had sensory characteristics similar to cheeses made from raw milk without starters (Ledda et al., 1994). Milk thermization did not influence secondary proteolysis, but resulted in changes in rheological characteristics (Pirisi et al., 1999a). Short- and medium-chain FFAs were significantly higher in rawmilk cheese than in cheese made from thermized milk, whereas long-chain FFAs, C18:1 and C18:2, were not affected by milk thermization (Pinna etal., 1999). Volatile components of 8-month-old Fiore Sardo were described by Larrayoz et al. (2001). Canestrato Pugliese is a hard cheese produced in Foggia and Bari. Its name derives from the reed baskets (canestri) used as moulds. It is cylindrical in shape, 25-34 cm in diameter, 10-14 cm high and weighs 7-14 kg. Milk is coagulated at 38-45 ~ with animal rennet for 15-25 min and placed in moulds. Cheeses are dry- or brine-salted and ripened for 2-10 months. Its flavour is strong, salty and piquant. Ripening has been characterized by Santoro and Faccia (1998). Raw-milk Canestrato Pugliese cheese has higher values of water-soluble and pH 4.6-soluble N, total FAAs (40mg/g), FFAs (1673mg/kg) and a greater diversity of NSLAB than pasteurized milk cheese (Albenzio et al., 2001). Pecorino Toscano is a soft or semi-hard cheese produced mainly in Toscany. Ewes' milk is usually inoculated with starter cultures and sometimes lipolytic enzymes are added. Coagulation with calf rennet takes place at 35-38 ~ in 20-25 min. The curds are heated to 40-42 ~ for 10-15 min. The cheeses are brine-salted for 24 h. The cheeses are cylindrical and weigh 1-3 kg. The m i n i m u m ripening period is 20 days for soft Pecorino Toscano and at least 4 months for the semi-hard variety. The taste is flagrant and slightly spicy. The technological and microbiological characteristics of pasteurized milk Pecorino Toscano cheese are described by Neviani et al. (1998). A wide diversity of LAB was described by Bizzarro et al. (2000) in raw and pasteurized milk Pecorino Toscano cheeses. Caciotta D'Urbino is a soft cheese manufactured from a mixture of 70-80% ewes' milk and 20-30% cows' milk. It is cylindrical in shape and weighs 0.8-1.5 kg. Milk is coagulated at 35-38 ~ with liquid or powdered lamb rennet. Cheeses are usually drysalted and ripened for 20-30 days at 10-14 ~ and 80-90% RH. The taste is sweet. Murazzano is a soft cheese variety manufactured from ewes' milk or mixtures of ewes' (60% minimum) and
284
Cheeses Made from Ewes' and Goats' Milk
cows' milk. Milk is coagulated at 37 ~ with animal rennet. The cheese, cylindrical in shape, is dry-salted and ripened for 4-10 days. Its weight varies from 0.3 to 0.4 kg. The body is soft and finely grained. Ricotta is a well-known whey cheese. The whey, usually from Pecorino cheese manufacture, is heated to 80 ~ The curd rises to the surface and is collected in shallow conical baskets where it drains for 12-14 h (Coni et al., 1999). Portuguese ewe cheeses
Most Portuguese cheeses are manufactured following traditional methods, the majority from raw milk without addition of a starter culture. The best-known Portuguese cheeses are Serra da Estrela, Serpa, Azeitao and Castelo Branco. These varieties, together with Evora, Nisa and Terrincho, have PDO status. The main characteristic that distinguishes them from other ewes' milk cheeses is their soft or semi-soft texture, due not only to the milk or the technology used, but also the highly proteolytic vegetable rennet used as coagulant. In 2001, ewes' milk production in Portugal was 98 000 tonnes, and 16 602 tonnes of ewes' milk cheeses were produced. Azeitao is a buttery, semi-soft cheese, with few or no holes, made near Lisbon. Coagulation of salted milk by vegetable rennet takes place at 30-35 ~ The cheese weighs 0.1-0.25 kg and is ripened in two stages. Initially, the cheese is held at room temperature for 10 days at 90-95% RH. During the second stage, the cheese is kept at 10-15 ~ and 85-90% RH for 2-3 weeks (Freitas et al., 2000). Castelo Branco is a semi-soft variety manufactured in central western Portugal (Freitas et al., 2000). It is related to Serra da Estrela cheese, with a stronger and slightly piquant flavour and a more compact rind. The cheese is obtained by slowly draining the curd after milk coagulation by vegetable rennet at 20-30 ~ Cheeses, 1 kg in weight, are ripened for 40-50 days at 8-14 ~ and 80-90% RH. Evora, a semi-hard to hard variety with a strong acidic and slightly piquant flavour is produced in the Alentejo region of southern Portugal from raw ewes' milk. Cheeses, 0.06-0.3 kg in weight, are ripened for 30 days in the case of the semi-hard type and a minimum of 90 days for the hard type (Freitas et al.,
2000). Nisa is a ripened cheese produced in the middle region of Alentejo. It is manufactured in two sizes, 0.2 and 1.3 kg, and has a semi-hard consistency, with a white-yellow colour and small holes. It is ripened for at least 45 days at 8-14 ~ and has an intense and slightly acidic flavour (Freitas et al., 2000).
Serpa is a buttery, semi-soft cheese with few or no holes, manufactured in the region of Serpa from Merino ewes' milk (Freitas et al., 2000). Cheeses are produced in four sizes, 0.2-2.5 kg in weight and have a strong, slightly hot and spicy flavour. Ripening takes place in two stages, at 6-10 ~ and 95-100% RH and at 7-11 ~ and 75-90% RH for 30-40 days. Serra da Estrela is the most traditional Portuguese cheese, produced in the Serra da Estrela mountains. It has a strong aroma, a slightly acidic flavour and a soft buttery texture with few eyes. Cheese is made twice daily from raw ewes' milk coagulated at 27-30 ~ for 1-2 h with vegetable rennet. The coagulum is cut manually and usually stirred by hand. The curds are poured into moulds and are slightly pressed, either by hand or mechanically. Cheeses are dry-salted and ripened for 30-45 days, at 6-12 ~ and 85-90% RH. They have a flat cylindrical shape, a diameter of 15-20 cm and weigh 1-1.7 kg. Changes in the microbiological and physico-chemical characteristics of the cheese have been reported by Macedo et al. (1993). A lower level of primary proteolysis occurs in cheeses made from milk coagulated with vegetable rather than animal rennet (Sousa and Malcata, 1997). [3-Casein is less susceptible to proteolysis than e~s-casein, and animal rennet is more proteolytic on both fractions than vegetable rennet. Higher levels of water-soluble N were observed in cheeses made with vegetable rennet, although levels of TCA- and PTAsoluble N were lower. Different patterns of peptides were detected at all stages of ripening (Sousa and Malcata, 1998). The main volatile compounds in Serra da Estrela cheese are derived from the degradation of sugars, FAAs and glycerides (Dahl etal., 2000). The short-chain carboxylic acids detected were acetic, propionic, iso-butyric and iso-valeric acids. High levels of 2,3-butanediol were attributed to the activity of spoilage bacteria, and high concentrations of ethyl octanoate and ethyl decanoate to yeast activity. Volatile short-chain fatty acids increased throughout ripening, with maximum levels of octanoic and decanoic acids detected at 150 days. The profile of volatiles in cheese was more complex if refrigerated milk was used instead of fresh milk, with higher concentrations of ethyl esters. Lactic acid bacteria and Enterobacteriaceae are the predominant microbial groups in Serra da Estrela cheese (Macedo et al., 1995; Tavaria and Malcata, 1998). Numbers of Enterobacteriaceae decrease whereas those of lactobacilli, lactococci and enteroccoci increase throughout ripening. Yeasts and staphylococci were 101-105 cfu/g and 104-107 cfu/g, respectively. Lc. lactis subsp, lactis, Lb. paracasei subsp.
Cheeses Made from Ewes' and Goats' Milk
paracasei, Leuc. lactis, Leuc. mesenteroides and Lb. plantarurn were the most frequently isolated LAB. A broad spectrum of yeasts are found in Serra da Estrela cheese. Terrincho is a semi-hard variety similar to other Portuguese ewes' milk cheeses, except that milk is coagulated with animal rennet (Freitas et al., 2000). Milk is coagulated at 35 ~ and the coagulum is cut to rice-grain size. Cheeses are pressed manually, drysalted and ripened at 5-12 ~ and 80-85% RH for 30 days. Terrincho cheese weighs 0.8-1.2 kg and has a slightly oily texture with small eyes. Spanish ewe cheeses
Production of ewes' milk in Spain was 306 000 tonnes in 2001. Six PDO ewes' milk cheeses are made: Manchego (6122 tonnes in 2000), Roncal (380 tonnes), Idiazabal (1131 tonnes) and Zamorano (448 tonnes) are hard, uncooked cheeses; La Serena (186 tonnes) and Torta del Casar are semi-hard cheeses manufactured from milk coagulated with vegetable rennet. Manchego cheese is made in La Mancha (central Spain) from raw or pasteurized milk of Manchega ewes. Cheeses weigh 2.5-3.5 kg and are cylindrical in shape. The rind is imprinted with a design recalling the woven esparto grass bands used traditionally as moulds. Rawmilk cheese is manufactured with or without addition of starter cultures, whereas mesophilic lactic cultures are used in the production of pasteurized milk cheese. Coagulation by animal rennet takes place at 30-32 ~ in 30-40 min. The coagulum is cut into 4-6 mm cubes, and scalded at 36-38 ~ for 15 min while stirring. Cheeses, 20-22 cm in diameter and 8-10 cm high, are pressed for 6-18 h, brine-salted for 24-48 h and held at 10-15 ~ for at least 2 months. Occasionally, Manchego cheese is immersed in olive oil during the later stages of ripening. Minimum pH (4.9-5.0) is generally reached in raw-milk Manchego cheese within 24 h of manufacture, after which the pH increases to 5.5-5.7 on day 90. In pasteurized milk Manchego cheese, the pH decreases to 5.1-5.3 during the first week and subsequently rises to 5.3-5.5 after 90 days. Dry matter is 40-50% in fresh curd and increases to 60-65% in 90day-old cheese. The salt-in-moisture is 5-7% on day 90. Greater degradation of Ors-casein than [3-casein occurs during ripening. Mean levels for pH 4.6-soluble N, TCA-soluble N and PTA-soluble N in 4-month-old raw-milk cheese were 23.6, 16.9 and 10.4% of total N, respectively, whereas levels of 21.1, 14.7 and 7.1% were found in pasteurized milk cheese (Gaya et al., 1990). Free amino acids accumulated during the l 1-month ripening period (Ordoflez and Burgos, 1980).
285
However, other workers (Marcos and Mora, 1982) reported a maximum level of FAAs after 4 months followed by a subsequent decrease. Lipolysis occurs only to a limited degree in Manchego cheese (Ramos and Martfnez-Castro, 1976). Lower levels of FFAs are found in pasteurized milk than in raw-milk cheese, due to the inactivation of indigenous milk lipase by pasteurization (Gaya et al., 1990). The majority of volatile compounds are more abundant in raw-milk Manchego cheese than in pasteurized milk cheese. Alcohols and esters predominate in raw-milk cheese, while methyl ketones and 2,3-butanedione (diacetyl) are quantitatively important in pasteurized milk cheese. Branched-chain alcohols were much more abundant in the raw-milk cheese. Aroma intensity is correlated with esters, branched-chain aldehydes and branched-chain alcohols in raw-milk cheese, and with esters, branched-chain aldehydes, 2-methyl ketones and 2-alkanols in pasteurized milk cheese. Diacetyl was positively correlated with the aroma attribute 'toasted' and negatively correlated with aroma quality in pasteurized milk cheeses (Fernandez-Garcfa et al., 2002). Lower concentrations of residual caseins were observed in pasteurized milk cheese made, with a commercial mixed-strain culture, than in pasteurized milk cheese made with a defined-strain culture made up of Lactococcus isolates from raw-milk Manchego cheese, or in raw-milk cheese made with any of the two cultures. The use of a commercial mixed-strain culture or a defined-strain culture did not affect flavour quality and intensity of raw- or pasteurized-milk Manchego cheeses (Gomez et al., 1999). Acceleration of the ripening of Manchego cheese by ripening at an elevated temperature (Gaya etal., 1990), addition of different enzymes to milk in liquid or solid form (Nufiez et al., 1991b; Fernandez-Garcfa et al., 1994) or encapsulation in liposomes (Picon et al., 1994, 1995, 1996) has been investigated, with increases in proteolysis and reduction of ripening period without impairment of cheese flavour. Flavour quality was improved and flavour intensity enhanced by the cysteine proteinase from Micrococcus INIA 528 (Mohedano et al., 1998). Increased proteolysis enhanced the formation of volatile compounds derived from amino acids, such as acetaldehyde, 2-methyl-2-butanal and 3-methyl-l-butanal (Mariaca et al., 2001). The microbial flora of Manchego cheese made from raw ewes' milk is well known (Nuflez et al., 1989). Lactococci, mainly Lc. lactis subsp, lactis, predominate during the first month of ripening. Thereafter, they are outnumbered by lactobacilli, mainly Lb. plantarum and Lb. casei. Enterococci, mainly E. faecium, leuconostocs, mainly Leuc. mesenteroides subsp, dextranicum
286
Cheeses Made from Ewes' and Goats' Milk
and Leuc. paramesenteroides, and pediococci, mainly Pediococcus pentosaceus, are found at lower numbers during ripening. Micrococci are commonly found in curd and 1-day-old cheese, and then decrease gradually during ripening. In pasteurized-milk Manchego cheese, starter lactococci predominate during the first month, but lactobacilli may reach 108 cfu/g in 30-day-old cheese. Idiazabal is a semi-hard or hard variety produced in the Basque country and Navarra from raw milk of Latxa ewes. Industrial production based on traditional methods and traditional production by small artisanal producers co-exist. Homofermentative starter cultures may be added to the milk before coagulation. Raw milk is coagulated with commercial bovine rennet or rennet paste at 30 ~ The coagulum is cut into 5-10-mm grains, stirred and heated to 37 ~ The curds are pressed in the vat, placed into moulds, pressed for 6 h and dry-salted or brined for 24-48 h. Cheeses are ripened at 10 ~ and 80% RH for 2-12 months. Smoking of cheeses during the third month of ripening by combustion of Alnus glutinosa wood for 24 h at 15 ~ is optional. Mean values of pH 4.6- and TCA-soluble N in Idiazabal cheeses were 11.5 and 14.4% of total N, respectively, indicating low levels of proteolysis. Glu, Leu, Val, Lys and Phe were the major FAAs detected (Orddflez et al., 1998). Butanoic acid was the main FFA, accounting for 33% of the total over the ripening period. The other common fatty acids (C6:0-C18:1) were also present (Najera et al., 1994). Differences in the amounts of short-chain FFAs were detected between winter and summer cheeses (Ch~ivarri et al., 1999). Effects of brining time and smoking on the microbiological (Perez-Elortondo et al., 1993), physicochemical (Iba~ez et al., 1995), FFA profile (Najera et al., 1994) and sensory characteristics (PerezElortondo et al., 2002) of Idiazabal cheese have been investigated. Smoking caused a decrease in the numbers of LAB and Micrococcaceae (P~rez-Elortondo et al., 1999a). Smoking lowered the aw and increased the pH, proteolysis and release of amino acids. Brining and smoking led to increases in FFAs (N~ijera etal., 1994). However, butanoic acid levels were lower in the smoked than in unsmoked cheeses. Brining had little effect on the sensory properties of Idiazabal cheese and smoking had a positive influence on the acceptability of rind, colour of the interior, aroma and texture. Cheeses made with rennet paste had higher contents of total and short-chain FFAs (Bustamante et al., 2000). Changes in LAB, Micrococcaceae, Enterobacteriaceae and psychrotrophic bacteria during ripening were
studied by Perez-Elortondo et al. (1998, 1999b), with citrate-utilizing (Cit +) strains of Lc. lactis and Lb. casei subsp, casei as the dominant LAB throughout ripening. Idiazabal cheese made from raw ewes' milk with the addition of a mesophilic starter culture had higher numbers of LAB during pressing, although no differences were observed during ripening. Flavour was typical of ripened ewes' milk cheese. Starter cultures also increased the levels of FAAs (Vicente et al., 2001), which reached higher levels in raw-milk cheese made with or without starter culture than in pasteurized milk cheese (Mendia et al., 2000). Roncal is a hard cheese manufactured in Navarra from raw ewes' milk. Its production is similar to that of Manchego, except for a higher renneting temperature (32-37 ~ and a smaller size (1.8-2.0 kg) and a ripening time of at least 4 months. The composition, biochemical and microbiological characteristics of Roncal cheese have been published (Orddfiez et al., 1980; Millan et al., 1992; Arizcun et al., 1997). The use of commercial calf rennet and aqueous extracts of dried, sliced and salted lambs' stomachs in Roncal cheese manufacture has been compared (Irigoyen et al., 2002). Physico-chemical parameters were not affected by the type of rennet, whereas cheese manufactured with lamb rennet showed higher levels of pH 4.6- and TCA-soluble-N and greater proteolytic activity on [3- but mainly on %~-casein. These changes did not result in any sensory differences. Alcohols are the predominant volatile compounds in 4-month-old Roncal cheese, with ethanol, 2-butanol and 1-propanol at high concentrations. Ketones were the next most prominent group, most of them being methyl ketones. 3-Hydroxybutan-2-one (acetoin) was present at high levels and influenced Roncal cheese aroma. Acetic and butanoic acids were the most abundant acids and ethyl esters were the predominant esters (Izco and Torre, 2000; Larrayoz et al., 2001). Lc. lactis subsp, lactis, Lb. casei, Lb. plantarum, Leuc. mesenteroides subsp, mesenteroides and Leuc. mesenteroides subsp, dextranicum were the predominant LAB in Roncal cheese (Arizcun et al., 1997). Starter cultures added to milk affected cheese flavour, with greater intensity for refreshing, astringent and sweet attributes and lower scores on bitterness. More homogeneous texture and higher elasticity were also observed in cheeses made with starter cultures (Ortigosa et al., 1999). Zamorano is a hard cheese produced in Zamora from raw milk of Churra and Castellana ewes. Milk is coagulated with rennet extract at 28-32 ~ for 30-45 min. The coagulum is cut by hand or mechanically into small pieces of 5-10 mm and the curds are heated to 38-40 ~ before moulding and pressing. Cheeses are dry-salted or
Cheeses Made from Ewes' and Goats' Milk
brined for 36 h and ripened for at least 100 days. Zamorano cheese has a firm texture and a dark grey rind, a cylindrical shape, 8-12 cm high and 18-22 cm in diameter, and weighs 3.5-4 kg. Castellano cheese, which is not protected by a PDO, is another ewes' milk hard variety made with similar technology (Roman-Blanco et al., 1999). La Serena is a semi-hard variety made from Merino ewes' milk in La Serena (Southern Extremadura). Raw milk at 25-32 ~ is coagulated in 50-75 min with vegetable rennet prepared by soaking dried flowers of C. cardunculus in tap water at room temperature for 24 h. The coagulum is cut into pieces of 10-20 m m in size and stirred for 2-3 min without scalding. After 10 min, the whey is removed. In traditional processing, the curds are transferred to woven esparto moulds where they are pressed by hand. Moulds are turned repeatedly for 1-2 h to assist whey drainage and the cheeses are dry-salted for 10-24 h or brined for 24 h. In modern dairies, plastic moulds, mechanical pressing and brine salting are used. Cheeses are ripened for at least 60 days at 8-12 ~ and 85-95% RH. La Serena is a flat round cheese, 18-20 cm in diameter and 4-8 cm high,-which weighs 0.8-1.5 kg. The rind is brownish in colour, with convex sides due to the softening of the cheese texture during ripening by the proteolytic action of the vegetable rennet. The cheese has a creamy to semi-hard texture with small eyes and a pronounced, sometimes slightly bitter, flavour. Chemical changes, pH and moisture content during the ripening of artisanal La Serena cheese were described by Fernandez del Pozo et al. (1988b). In fully ripened cheese, pH 4.6-soluble N was 38.5% of total N, and TCA-soluble N 10.3-14.6% (Fernandez-Salguero et al., 1978; Fernandez del Pozo et al., 1988b). Degradation of Ors-casein was faster than that of [~-casein. During the first month of ripening the texture of the cheese softens, due to hydrolysis of the casein, developing a more consistent body gradually as ripening progressed (Fernandez del Pozo et al., 1988b). Levels of FFAs increase gradually during ripening. Cheeses produced with animal rennet had a higher moisture content and a lower pH than cheeses made with vegetable rennet (Nufiez et al., 1991a). Proteolysis was more rapid in cheeses made with vegetable rennet, but there was less lipolysis. Softening of cheese texture was more pronounced in cheeses made with vegetable rennet, which received higher scores for flavour quality and intensity. Lc. lactis, atypical lactococci, Lb. casei, Lb. plantarum, Leuc. mesenteroides and E. faecium predominated in the interior of La Serena cheese (Fernandez del Pozo et al., 1988a). High yeast and mould counts
287
were found on the cheese surface from 30 days onwards. These were mainly lactic acid-utilizing species. Cheese flavour quality and intensity were significantly impaired in cheeses made with a strain of Lc. lactis subsp, lactis as a starter culture; the cheese had a firmer texture, which was attributed to retarded proteolysis and a lower pH (Medina et al., 1991). Alcohols were the major volatile compounds, with ethanol, propanol, 2-propenol, 2-propanol, 2-butanol, 2-pentanol and branched-chain 2-methyl propanol and 3-methyl butanol being the most abundant (Carbonell et al., 2002). Ethyl esters of acetic, butanoic, hexanoic and octanoic acids and 3-methyl-l-butanol acetate were the main esters. The concentrations of most esters increased dramatically during ripening and, because of their low perception thresholds, may be considered as key constituents of the aroma of this cheese variety. Torta del Casar is a semi-hard cheese made in Caceres (northern Extremadura) from raw milk of Merino ewes, which is coagulated with vegetable rennet at 25-30 ~ Manufacturing and ripening conditions are very similar to those for La Serena cheese. Torta del Casar has a PDO status since 2002. Chemical composition of cheese ripened for 60 days is 53.6% DM, 26.1% fat, 24.3% protein, 34.1% pH 4.6-soluble N and 13.0% TCA-soluble N as percentages of total N, and pH 5.21 (Mas Mayoral et al., 1991). The microbiology of Torta del Casar has been investigated (Poullet et al., 1991). Maximum counts of lactococci, lactobacilli, leuconostocs and enterococci are reached during the first 15 days of ripening. Coliform counts are 105-106 cfu/g during the first month and decrease to 102-104 cfu/g on day 60, whereas numbers of coagulasepositive staphylococci are 102-103 cfu/g during the first month and decrease to 10 cfu/g on day 60. The predominant LAB species during ripening were Lc. lactis subsp, lactis, Leuc. mesenteroides subsp, dextranicum, Leuc. mesenteroides subsp, mesenteroides, Lb. curvatus, Lb. plantarum and E. faecalis (Poullet et al., 1993). Another non-PDO Spanish ewes' milk cheese also made with vegetable rennet is Los Pedroches (Fernandez-Salguero and Sanjuan, 1999).
Technology and flavour of goats' milk cheeses
Goats' milk production in the European Union was 1 443 782 tonnes in 2001. Except for minor local production of goats' milk yoghurt or pasteurized goats' milk, all the milk is transformed into cheese, alone or mixed with cows' and/or ewes' milks. The textural characteristics of goats' milk curd are distinct from those of cows' milk curd produced under
288
Cheeses Made from Ewes' and Goats' Milk
the same conditions. A coagulum strength of 15.0-18.5 g was reported for goats' milk with a casein content of 29.5-30.8 g/kg versus a coagulum strength of 18.2-45.8 g for cows' milk with a casein content of 23.8-24.7 g/kg (Storry et al., 1983). The weaker mechanical properties of goats' milk curd constrain the manufacturing procedures used in goats' milk cheesemaking and limit the diversity of cheese types. Although there is some local production of goats' Blue cheeses, most cheeses made from goats' milk fall within the following groups: 9 Fresh or white unripened cheeses, with a low DM content, usually less than 25%. 9 Soft cheeses, traditionally made from predominantly lactic curd, of small size, cylindrical or pyramidal in shape, and generally with mould growth or ash on the surface. 9 Semi-hard or hard cheeses, made from predominantly rennet-coagulated curd, of larger size than the soft cheeses, fiat cylindrical shape (wheel) and dry rind. The composition of goats' milk casein is the main factor responsible for its technological limitations. Caprine casein contains a lower proportion of C~s-casein, especially OLsl-casein, and a higher proportion of [3-casein than bovine casein. The polymorphism of caprine Otsl-casein plays a role in these compositional differences. The A, B and C alleles are associated with high levels of O~sl-casein synthesis, while the E allele is with a medium level, the D and F alleles with a low level and the O allele with a null level of Otsl-casein synthesis (Grosclaude et al., 1994). Cheesemaking from goats' milk with a low Oql-casein content results in a less firm curd (Ambrosoli et al., 1988) and lower protein retention and cheese yield (Pirisi et al., 1994; Vassal et al., 1994) than when milk of a high OLsl-casein content is used. On the other hand, cheese made from A allele milk with high C~sl-casein content has a weaker goaty flavour than those from E or F allele milks, due to lower production of aroma compounds or due to the firmer texture of the A allele curd, which impairs the release and perception of volatile aroma compounds (Vassal etal., 1994). Pierre etal. (1998) found a weaker flavour in cheese made from A allele milk than in cheese from O allele milk, which was attributed to 50% lower levels of hexanoic, octanoic, nonanoic, decanoic, 4-methyl octanoic and 4-ethyl octanoic acids in the former cheese. The characteristic flavour of goats' milk cheeses is mostly due to volatile compounds. For a long time octanoic acid was considered to be the main 'goaty' compound in dairy products but, according to Ha and
Lindsay (1991a), its aroma and flavour lack the highly characteristic goatiness found in goats' milk cheese. Instead, 4-ethyl-octanoic acid was found by systematic assessment of the aroma of individual FA to be principally responsible for the goaty-type aromatic notes in goat-milk cheese (Ha and Lindsay, 1991a). Among medium-size FAs, the lowest perception threshold in water and in oil is that of 4-ethyl-octanoic acid (Brennand etal., 1989). Seven volatile compounds present in goat cheeses (hexanoic, octanoic, nonanoic, decanoic, 4-methyl-octanoic, 4-ethyl-octanoic, plus an unidentified compound co-eluting with decanoic acid) were characterized by olfactometry as having a specific goat cheese aroma (Le Qu~r~ et al., 1998). Salles et al. (2002) also concluded that 4-ethyl-octanoic acid is the most potent medium-chain FFA in typical goat cheese flavour. The concentration of 4-ethyl-octanoic acid in goat-milk cheese is 0.1 mg/kg versus 0.8 mg/kg for 4-methyl-octanoic acid and higher values for the other medium-chain FFAs. However, the threshold value for perception of 4-ethyl-octanoic acid in a cheese model was 0.0039 mg/kg versus 0.322 mg/kg for 4-methyloctanoic acid and considerably higher values for other medium-chain FFAs. Technological treatments influence the typical flavour of goats' milk (Morgan and Gaborit, 2001). Thus, skimmed goats' milk had a very mild flavour (0.78 points on a 10-point scale) compared with full-fat raw milk (4.14 points on the same scale). Heating milk at 65 ~ for 1 min led to a slight reduction in goaty flavour (3.06 points versus 4.06 points for control raw milk), accompanied by a decrease in the level of lipolysis. On the other hand, cold storage (4.68 points for milk stored 3 days at 6 ~ versus 2.96 points for control milk) and homogenization (4.90points versus 4.06 points for control milk) of milk increased goat flavour. The total content of FFAs was 40 p~g/ml fat in control milk, and 100 or 120 i~g/ml fat after cold storage or homogenization, respectively. Fresh cheeses made from goats' milk with high levels of lipolysis showed high levels of lipolysis and an increase in organoleptic defects (Morgan et al., 2001). However, no relationship was found between the level of lipolysis and the sensory characteristics of the ripened cheeses. The water-soluble extract of goats' milk cheese had an umami taste, although it was also regarded as salty, astringent and bitter, whereas the water-soluble extract from cows' milk cheese was described as showing mainly salty, umami and sour tastes, while that of ewes' milk cheese exhibited a strong umami taste, followed by a salty taste (Molina et al., 1999). Small peptides and FAAs in fractions of water-soluble extracts of goat cheeses had no direct impact on their taste, which was
Cheeses Made from Ewes' and Goats' Milk
influenced mainly by mineral salts and lactic acid (Salles et al., 2000). Also, when small peptides (MW < 1000 Da) or FAAs from the water-soluble extract of goat cheese were evaluated sensorially by incorporation or omission in a cheese model, no effects of those compounds on the taste descriptors, salty, bitter, sour, umami or astringent, were found (Salles et al., 2002). Saltiness of the water-soluble extract of goat cheese was explained by an additive contribution of sodium, potassium, calcium and magnesium ions, whereas sourness was mainly due to the synergistic effect of sodium chloride, phosphates and lactic acid, and bitterness to calcium and magnesium chlorides, partially masked by sodium chloride (Engel et al., 2000). The hydrophobic peptides present in the water-soluble extract of goat-milk cheese were not perceived as being bitter by panellists, probably due to their low concentrations (Sommerer et al., 2001). French goat cheeses
France produces more than 50 goats' cheese varieties. Goats' milk production was 460 000 tonnes in 2001. Total annual production of goat cheeses is close to 75 000 tonnes, of which 23% are farm-made cheeses. The nine varieties produced under a PDO are Chabichou du Poitou, Crottin de Chavignol, Picodon de la Dr0me or Picodon de l'Ardeche, Pelardon, Pouligny Saint-Pierre, Rocamadour, Sainte-Maure de Touraine, Selles-sur-Cher and Valengay, which account for a combined production of over 5000 tonnes per year. Detailed cheesemaking procedures for French goat cheeses have been reviewed by Le Jaouen and Mouillot (1990). Most French goat cheeses can be made from pasteurized or raw milks, but some PDO varieties, such as Pouligny Saint-Pierre, Rocamadour and Valencay must be made exclusively from raw milk. Many French goat-milk cheeses are soft varieties. In their manufacture, milk coagulation is due mostly to lactic acid production by the starter, with small doses of rennet, a coagulation temperature of 18-24 ~ and clotting times ranging from 16 to 48 h. One exception of considerable economic importance is Camemberttype goat cheese, made in the Poitou region. Pasteurized milk heated at 32-33 ~ undergoes a lactic-rennet type coagulation, with a rennet dose 2-3 times higher than that for soft cheeses, and a coagulation time of 45-50 min. Other goat cheeses with a lactic-rennet type coagulation are Blue des Aravis and artisanal Chevrotin, both traditionally made in the Alps. Some general microbiological studies have been carried out on varieties such as Crottin de Chavignol (Hosono and Shirota, 1994), Valenq.ay (Hosono and Sawada, 1995) and Sainte-Maure (Masuda et al., 2000).
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Nahabieh and Schmidt (1990) found that D. hansenii was the dominant yeast species in the main goat cheeses. However, detailed scientific and technical information on the microbiota of most French goat-milk cheeses is relatively scarce. The microbiology of a Camembert-type goat cheese made from raw milk was investigated by Sable et al. (1997b), who found similarities between its microbiota and the microbiota of Camembert cheese made from raw cows' milk (Lenoir, 1963). Lc. lactis was the dominant Gram-positive species, Hafnia alvei the dominant Gram-negative species and Staphylococcus the dominant salt-tolerant micro-organism throughout ripening. Chemical changes during ripening (31 days at 13 ~ and 90% RH) of raw-milk Sainte Maure-type goat cheese have been investigated by Le Quere et al. (1998). Cheese DM increased from 32.7% on day 2 to 53.6% on day 31. Cheese pH rose from 4.4 on day 2 to 5.1 on day 31 at the surface, but only from 4.3 on day 2 to 4.4 on day 31 in the centre, giving rise to a pH gradient. Proteolysis was slight, with 92% of the casein intact on day 2 and 87% on day 31, with none of the individual caseins being preferentially hydrolysed. Small peptides increased from 5% of total N on day 2 to 8.5% on day 31. This pattern of casein breakdown corresponded to the proteolytic activity of Geotrichum candidum, the surface mould. FFAs accounted for 1% of total FFAs on day 2, rose to 2% on day 18, and afterwards increased more rapidly, up to 6% on day 31. Lipolysis apparently occurred in a two-step process. A specific rise of saturated C6-C10 FFAs was observed in 2-day-old cheeses, which was attributed to the lipoprotein lipase of goat milk or the lipolytic activity of somatic cells. Afterwards, an increase in C18:1, and to a lesser extent in C16:1 and C18:2, was observed. Unsaturated FFAs were 35.7% of FFAs, and this specific activity was attributed to the Geotrichum lipase. The typical goat cheese aroma was attributed to mediumchain linear and branched-chain FFAs. From day 2 to day 31, 4-ethyl-octanoic acid increased from 0.001 to 0.027mg/kg, 4-methyl-octanoic acid from 0.012 to 0.139 mg/~g, hexanoic acid from 2.77 to 23.38 mg/kg, octanoic acid from 7.45 to 32.96 mg/kg and decanoic acid from 3.04 to 19.87 mg/kg. Those increases in FFAs might explain why fresh goat cheeses when submitted to sensory analysis are perceived as different from ripened goat cheeses. Fifty-one volatile compounds were identified and quantified during the ripening of Camembert-type goat cheese (Sabl~ et al., 1997a). Ethanol, 3-methyl butanol, 2-methyl butanol, ethyl acetate and 2-methyl propanol were the most abundant volatile compounds in 2-day-old cheese, whereas the major compounds in ripe cheese were ethanol, 2-heptanone, 2-nonanone,
290
Cheeses Made from Ewes' and Goats' Milk
2-heptanol, 3-methyl butanal, acetaldehyde, 2-propanol and acetone. The volatile profile of the cheese resembled that of Camembert cheese made from cows' milk and other surface mould-ripened cheeses (Molimard and Spinnler, 1996), but compounds such as limonene, 3-hexanone, 4-heptanone, 3-methyl 2-heptanone, 4-nonanone, 2-methyl butanol and acetaldehyde were considered by the authors to be characteristic of goat cheese. Yeast and mould strains used for ripening of French goat-milk cheeses have a remarkable influence on the flavour of both soft cheeses and Camembert-type cheeses (Gaborit et al., 2001). In soft cheeses, the use of G. candidum GC1, alone or associated with D. hansenii DH1 or Rhodosporidium infirmominiatum RI, resulted in the lowest levels of lipolysis and the highest scores for goat odour and goat flavour intensity. Goat flavour intensity increased when GC1 was used in conjunction with DH1 or RI. The use of Penicillium candidum PC1, alone or associated with DH1 or RI, was detrimental to the sensorial quality of the cheeses. In Camembert-type cheeses, lipolysis and scores obtained for most descriptors were lower than those found for soft cheeses. Lipolysis levels were higher for cheeses made using STRAIN GC1 with STRAINS PC1, or PC1 and DH1. The best sensorial quality cheeses were made with STRAIN GC1. Greek goat cheeses
Goats' milk production in Greece was 450 000 tonnes in 2001. There are no PDO cheeses in Greece made exclusively from goats' milk, which is generally added to ewes' milk for the production of ewes' milk cheese varieties. Feta cheese made from 100% goats' milk, mixtures of goats' and ewes' milks, or 100% ewes' milk have been compared (Mallatou et al., 1994). No clear relationship between milk used for manufacture and proteolysis was found, but significantly higher levels of lipolysis were found in cheese made from 100 or 75% goats' milk. The microbiology of white-brined cheese made from raw goats' milk has been studied (Litopoulou-Tzanetaki and Tzanetakis, 1992). Most microbial groups were at their highest levels in 15-day-old cheese, when the pH was 5.45. Coliforms, staphylococci and LAB declined by 4.0, 3.0 and 1.5 log cycles, respectively, from day 15 to day 90, when the pH was 4.50; in contrast, yeasts increased by 1.8 log cycles in the meantime and enterococci did not vary. Specific starters have been designed for the manufacture of white-brined cheese from pasteurized goats' milk (Tzanetakis et al., 1995). Anevato cheese is made from raw goats' milk which undergoes a lactic-rennet coagulation at 18-20 ~ for 12 h. The coagulum is cut, left to raise to the surface of the whey for 4-5 h and drained in cheese cloths for
24 h. Salt is added and, after thorough mixing, the curds are packed in plastic containers which are held at 4 ~ In Anevato cheese, the highest counts of most microbial groups were reached in curd after draining, when the pH was 4.28 (Hatzikamari et al., 1999). During cold storage for 60 days, the pH of the cheese did not change and counts of most microbial groups declined, but yeast counts increased by 1.2 log units. Anevato cheeses made from raw, pasteurized or thermized goats' milk inoculated with 0.5% of a Lc. lactis subsp, lactis culture were compared (Xanthopoulos et al., 2000). All microbial groups, except yeasts, were present at higher levels in raw-milk curd and declined during cold storage. Different patterns of hydrolysis of C~sl- and B-caseins during cheese ripening were recorded, with reduced proteolysis in cheeses made from pasteurized milk. Flavour scores were higher for raw-milk cheeses, but aroma and texture were not influenced by heat treatment of the milk. Italian goat cheeses
Production of goats' milk in Italy was 140 000 tonnes in 2001. There are no PDO goat cheeses in Italy, although some PDO cheeses (Bitto, Bra, Castelmagno, Raschera, Valle d'Aosta Fromadzo) are made from cows' milk to which small amounts of goats' milk are added. In the manufacture of PDO Robiola di Roccaverano cheese, a minimum of 15% goats' and/or ewes' milk must be included together with cows' milk, although Robiola 'classica' cheese is exclusively made from goats' milk. Technological aspects of Italian goatmilk cheeses have been reviewed (Emaldi, 1987). Studies on the microbiology and the biochemistry of Italian goat cheeses are scarce. Caprino tradizionale is a soft cheese made from raw goats' milk, to which a whey culture is added. Coagulation takes place after 18-24 h at 20-22 ~ The microbiology of this cheese variety has been studied, with maximum microbial counts in fresh curd, which had a mean pH of 4.35 (Foschino et al., 1999). After 10 days at 4 ~ the pH had declined to 4.32, and counts of lactococci, enterococci, S. aureus, coliforms and yeasts had decreased by 0.7, 3.7, 2.9, 5.3 and 1.5 log cycles, respectively. Bastelicaccia is a soft cheese made in Corsica by coagulating goats' or ewes' milks at 24-28 ~ for 60-90 min, cutting the coagulum to rice-grain size, draining part of the whey, stirring the curds, draining the rest of the whey, dry-salting the curds and ripening for 30 days. Ripe goats' milk cheese had a pH of 5.5-5.7, a high population of leuconostocs (>108 cfu/g) and a high coliform count (105 cfu/g). Soluble N of 30-day-old cheese was 18.2-28.4% of total N, and FAAs reached 3.7-4.5 g/kg (Casaha et al., 2001).
Cheeses Made from Ewes' and Goats' Milk
Cacioricotta cheese is made traditionally by heating goats' milk at 95 ~ cooling it to 40 ~ and adding a Sc. thermophilus culture. Use of lower doses of rennet and variable amounts of mesophilic lactic cultures increased the yield of 15-day-old cheese from 7.39 to 7.88% on a DM basis, probably due to reduced proteolysis (0.38% NPN instead of 0.47%; Caponio et al., 2001). Lipolysis was also retarded by the modified technology, which improved the palatability of the cheese. The use of thermized milk in the manufacture of farm-made goat-milk cheese has been studied in order to improve its microbiological quality (Clementi et al., 1998). Reduced proteolysis was found in thermized milk cheese compared with raw-milk cheese. Thermization of milk reduced the 'goaty' taste and led to a slightly more bitter and salty flavour, a harder texture and a more intense white colour. High-pressure homogenization (HPH) of goats' milk at 1000 bar (100 MPa) has been compared with pasteurization and thermization in the manufacture of soft cheese (Guerzoni et al., 1999). High-pressure homogenization of milk reduced counts of most microbial groups by at least 2 log cycles. Fresh curd yields were 16.0% for raw milk, 20.7% for thermized milk, 20.3% for pasteurized milk and 32.0% for HPH milk. Lipolysis was favoured in cheeses from HPH milk, with 6.89 mg FFAs/kg compared to 5.25 mg FFAs/kg in raw-milk cheese. Proteolysis was also enhanced in cheeses made from HPH milk, which received the highest overall score from panellists. Portuguese goat cheeses
Goats' milk production in Portugal was 35 000 tonnes in 2001. The only PDO cheese in Portugal made exclusively from goats' milk is Cabra Transmontano, although other PDO cheeses such as Picante da Beira Baixa, Amarelo da Beira Baixa and Rabacal are manufactured from a mixture of goats' and ewes' milks. The production of goats' milk cheese was 1295 tonnes in 2001, and the production of cheese from mixed ewes' and goats' milks, 4791 tonnes. Cabra Transmontano is a hard cheese made from raw Serrana goats' milk, which is coagulated at 35 ~ with animal rennet. The coagulum is cut manually into irregular pieces and pressed by hand. Cheeses (fiat cylinders) are dry-salted and ripened at 5-18 ~ and 70-85% RH for a minimum of 60 days. Ripe cheese weighs 0.6-0.9 kg. No scientific information is available on this cheese variety (Freitas et al., 2000). Raba~;al cheese is manufactured with variable proportions of ewes' and goats' raw milks, although a 2:1 ratio is considered to be optimal (Delgado, 1993). Milk is coagulated at 30 ~ with animal rennet in
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45-60 min and the coagulum is cut by hand to irregular grains. Cheeses (fiat cylinders) are pressed manually, dry-salted and ripened at 10-15 ~ and 7 0 - 8 5 % RH for 20 days. Ripe cheese weighs 0.3-0.5 kg. Sensory studies of this cheese variety describe its peculiar aroma and flavour as milky, floral and acid (Freitas et al., 2000). Picante da Beira Baixa may be manufactured from goats' or ewes' raw milk or their mixture, a 2:3 ratio being common. Milk at 28-30 ~ is coagulated with animal rennet in 40-50 min. The coagulum is cut into 1-1.5 cm cubes and pressed by hand. Cheeses (fiat cylinders) are dry-salted, stacked in groups of two or three and turned frequently. Ripening takes place at 10-18 ~ and 70-80% RH for 120-180 days. Ripe cheese weighs 0.4-1.0 kg. Picante cheese has high counts of staphylococci, up to 106 cfu/g, and coliforms, up to 108 cfu/g during the first week. Coliform counts decreased by 5-6 log cycles, and staphylococci counts by 3-4 log cycles, after 180 days in spite of an increase in pH from 4.5 to 5.2 in 9-day-old cheeses to 5.8-5.9 in ripe cheeses (Freitas et al., 1995). The predominant microbial species were identified by Freitas et al. (1996). Water-soluble N in ripe cheeses was 25-29% of total N, and NPN was 87-92% of soluble N. Residual Ors- and [~-caseins in ripe cheeses were 7-64% and 44-81%, respectively. The proportion of goats' to ewes' milk had no significant effect on cheese sensory characteristics (Freitas et al., 1997). Free amino acids of Picante cheese manufactured from different proportions of goats' and ewes' milks, animal or thistle rennets and salting once or twice were investigated by Freitas et al. (1999). The highest amount of FAAs was in cheese made using a mixture of goats' and ewes' milk (ratio of 1:4), animal rennet and salted once. Amarelo da Beira Baixa is a cheese variety similar to Picante, weighing 0.6-1.3 kg, with a straw to dark yellow rind (Freitas et al., 2000). Spanish goat cheeses
Spain is the third largest producer of goats' milk in the European Union, with 320 000 tonnes in 2001. Most of it is mixed with cows' and/or ewes' milks for the production o f - 2 0 non-PDO traditional cheese varieties, or new varieties such as Iberico cheese, manufactured from a mixture of milks of the three species with a minimum of 30% goats' milk. Technological aspects of Spanish goat cheeses have been reviewed (Franco et al., 2001). Twenty-eight varieties are made exclusively from goats' milk, although only four are PDO cheeses. In 2001, the production of PDO Majorero cheese was 352 tonnes, the production of PDO Ibores cheese began that year with 45 tonnes, and
292
Cheeses Made from Ewes' and Goats' Milk
the production of PDO Murcia and Palmero cheeses began in 2002. Majorero cheese is made in Fuerteventura, one of the Canary islands, from raw or pasteurized goats' milk. Coagulation with animal rennet takes place at 28-32 ~ in 60 min, after which the coagulum is cut to 1-cm-size grains and the whey is drained out. Cheeses are pressed, dry- or brine-salted, and ripened for 20-90 days at 12-18 ~ and a low RH. The surface is rubbed with oil, paprika or both during ripening. The shape is fiat cylindrical, and the weight 1-6 kg. In 90-day-old raw-milk cheese the DM was 83% and pH 5.44 (Fontecha et al., 1990). Two days after manufacture, coliforms and staphylococci reached 106-107 and 104-105 cfu/g, respectively, and after 90 days were less than 101 cfu/g. In 60-day-old cheese, residual Ors- and [3-caseins were 27% and 76%, respectively, and NPN was 19.0% of total N. Total FFAs reached 32.0 g/kg in 90-day-old cheese. Pasteurized milk cheese had a DM content of 61% and a pH of 5.46 on day 90 (Martin-Hern~indez et al., 1992). Residual %- and [3-caseins on day 60 were reduced to 47 and 81%, respectively, and NPN was 16.6% of total N. Total FFAs reached 6.11 g/kg in 90-day-old cheese, a much lower value than that of raw-milk cheese. Palmero, Tenerife and Conejero are traditional goat cheeses similar to Majorero made from raw milk, to which a Lc. lactis starter may be added, in different Canary islands. Tenerife cheese is a farm-house variety made from raw milk coagulated with animal rennet at 28-32 ~ in 30-60 min, of fiat cylindrical shape and weighing 0.9-1.2 kg, with an annual production close to 1500 tonnes. The DM increases slightly during ripening (46% after 2 days to 49% after 60 days) and the pH declines from 4.93 on day 2 to 4.64 on day 30, and remains constant during the second month of ripening. Coliform counts decreased from 107 cfu/g in 2-day-old cheese to 103-104 cfu/g in 60-day-old cheese, while S. aureus counts in 2-day-old cheese were 103 cfu/g and less than 10 cfu/g in 60-day-old cheese (Z~irate et al., 1997). Ibores cheese is made in Extremadura from raw milk, to which a Lc. lactis starter may be added. Milk is coagulated at 28-32 ~ in 60-90 min, generally with animal rennet. The coagulum is cut to medium-size (1-2 cm) grains. Cheeses of fiat cylindrical shape, weighing 0.7-1.2 kg, are pressed for 3-8 h, dry- or brine-salted and ripened for a minimum of 60 days. Seasonal differences have been recorded for pH, with higher values for cheeses made in winter than for those made in spring (Mas and Gonz~ilez Crespo, 1993). Cheese ripened for 60 days had a pH of 5.18, a DM of 59%, ---21% pH 4.6-soluble N as % of total N and ---10%
TCA-soluble N. In 60-day-old cheese, coliforms were 103-104 cfu/g, and coagulase-positive staphylococci less than 10 cfu/g. Lc. lactis subsp, lactis, E. faecium, Leuc. mesenteroides subsp, dextranicum and Lb. casei were the most abundant species within their respective genera (Mas et al., 2002). A total of 29 volatile compounds have been identified in Ibores cheese, including five ketones, five alcohols, two aromatic hydrocarbons, ten esters, four terpenes and one aldehyde (Sabio and Vidal AragOn, 1996). Murcia cheese is made from pasteurized milk. It may be fresh, ripened or 'al vino' (wine-cured). In fresh cheese manufacture, the milk is coagulated at 35-38 ~ in 30-60 min, the coagulum is cut and stirred, and cheeses are pressed for 2-4 h. After brinesalting, cheeses (fiat cylinders weighing 0.3-1.5 kg) are held at 4 ~ For ripened cheese manufacture, milk is coagulated at 32-33 ~ in 45-60 min with animal rennet. The coagulum is cut, stirred and heated to 35-37 ~ Cheeses (fiat cylinders weighing 1-2 kg) are pressed, brine-salted for 12h and ripened at 12-14~ and 75-85% RH for at least 21 days. Murcia cheese 'al vino' is made from washed curd. Cheeses are immersed in red wine for 30 min at the beginning of ripening, for 15-30 min on day 7, for 15-30 min on day 14, and on day 21 for a time depending on rind characteristics (Franco et al., 2001). There is no scientific information available on Murcia cheese. Gredos cheese, also called Tietar or La Vera, is farm-made from raw milk, coagulated with animal rennet at 25-30 ~ in 1.5-2.5 h. The coagulum is cut to rice-grain or smaller size, left to settle, scooped into moulds and pressed by hand. Cheeses, of flat cylindrical shape and weighing 0.8-1.2 kg, are dry-salted and ripened for 15 days at 8-10 ~ and 80-90% RH. If not consumed as fresh cheese, they are immersed in olive oil and held for 45-60 days at 8-10 ~ The pH declines from 6.27 on day 4 to 4.64 on day 45, while DM increases from 38% on day 4 to 45% on day 60. Most microbial groups reach maximum numbers after 15 days of ripening, with coliform counts of 105-106 cfu/g and coagulase-positive staphylococci counts of 102-103 cfu/g at that time. In 60-day-old cheese, coliform counts had decreased by 4 log cycles and coagulase-positive staphylococci by 2 log cycles. Residual ors- and [3-caseins were 22% and 40%, respectively, and NPN was 14.9% of total N in 60-day-old cheese (Medina et al., 1992). Cendrat del Montsec is made from raw milk inoculated with 3% Lc. lactis starter, coagulated at 15-20 ~ in 20 h using animal rennet. The coagulum is not cut, but scooped into cylindrical moulds where whey drains spontaneously for 6 - 7 h , and afterwards, cheeses weighing 1.5 kg are slightly pressed for 24 h.
Cheeses Made from Ewes' and Goats' Milk
Cheeses are dry-salted, and after 5 days are covered with oak ash. Ripening takes place at 10-15 ~ and 90-95% RH for 9 weeks. The pH increases from 4.02 in 1-day-old cheese to 4.40 in 63-day-old cheese, and the DM increases during this time from 46 to 53%. In ripe cheese, Ors-casein is almost completely degraded, but 50% [3-casein remains unaltered (Carretero et al., 1994). Due to the low pH value, coliforms and S. aureus were at low numbers (101-102 cfu/g) at the end of the ripening period (Mor-Mur et al., 1992). Valdeteja is farm-made from raw milk coagulated at 35 ~ with animal rennet in 105-120 min. The coagulum is cut to 1-cm-size grains, moulded and pressed for 12 h. Cheeses, fiat cylinders weighing 0.8-1.2 kg are dry-salted and ripened at 10-15 ~ and 70-80% RH for 30 days. During ripening, the pH declines to 5.1 on day 2 and 4.5 on day 10, and remains unchanged until day 30, while the DM is 48% on day 2 and increases to 62% on day 30. The acidity index of the fat increased from 0.89 on day 2 to 1.46 on day 30. Only 4-5% NPN of total N was found in 30-day-old cheese (Carballo et al., 1994). Armada cheese is farm-made from raw milk, to which a small amount of whey from the previous day is added, coagulated with animal rennet at 30 ~ in 60 min. The coagulum is cut, left to settle, cut again to a smaller size and scooped into cloths which are hung for 48 h. Afterwards, the curds are kneaded intensely by hand, transferred to new cloths, hung for a further 72 h and salt is added. The curds are kneaded again and moulded to cheeses, 20 cm in diameter and 20 cm high, which are wrapped in cloths and hung to ripen at 10-15 ~ and 70-85% RH for 60-120 days. During ripening, the pH declines to a minimum of 4.31-4.68 on day 7, increasing later to 4.89-5.25 on day 120, while the DM increases from 49-57% on day 7 to 75-82% on day 120 (Tornadijo et al., 1993). The NPN is 5.0% of total N on day 7 and increases to only 7.3% by day 120, whereas residual Ors- and [3-caseins were 93 and 98%, respectively, on day 120. Total FAAs increased from 2.1 g/kg on day 7 to 3.6 g/kg on day 120, and total FFAs increased in the meantime from 5.9 g/kg to 44.5 g/kg (Fresno et al., 1997). Cameros cheese is made from raw or pasteurized milk, coagulated at 32 ~ in 60 min with animal rennet. The coagulum is cut by hand, moulded in plastic baskets and slightly pressed for 8-12 h. Cheeses are dry-salted and ripened for up to 60 days at 12-14 ~ and 70-80% RH. Raw- and pasteurized-milk cheeses have been studied. A pH of 4.52-4.89 was reached on day 5, decreasing to 4.49-4.65 on day 30, followed by an increase to 4.70-4.98 on day 60. The DM was 51-56% on day 5 and increased to 79-83% on day 60. Proteolysis was slight, with only 5.0-7.7% NPN
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of total N on day 60. In raw-milk cheese, coliform counts were less than 10 cfu/g on day 60, but numbers of S. aureus were close to 106 cfu/g on days 5-15 and still over 103 cfu/g on day 30 (Olarte etal.,
2000). Recently, extensive studies on the effects of highpressure treatment on the microbiological (Capellas etal., 1996; Buffa etal., 2001b), physico-chemical (Trujillo et al., 1999; Capellas et al., 2001; Buffa et al., 2001a; Saldo et al., 2002) and textural (Saldo et al., 2000; Buffa et al., 2001c) characteristics of goats' milk cheeses have been carried out. High-pressure treatments of cheeses at 400-500 MPa improved microbiological quality, enhanced proteolysis and resulted in a more fluid-like texture. Lipolysis in cheeses made from high-pressure-treated milk was similar to that in raw-milk cheeses, and higher than lipolysis in pasteurized-milk cheeses. Cheeses made from high-pressure-treated milk were, like raw-milk cheeses, firmer and less fracturable than pasteurizedmilk cheeses.
More than 100 cheese varieties, many of them protected by a Designation of Origin, are made from ewes' or goats' milk in Europe. This rich heritage, dating in some cases from the Middle Ages, should be maintained for cultural and socio-economic reasons. Farming of ewes and goats and transformation of their milks into cheeses contribute to the sustainable development of many regions, mostly in Mediterranean countries. The peculiar flavour and texture typical of ewes' or goats' milk cheeses can be explained partly by compositional differences in caseins and fat, distinct from those of cows' milk. In raw-milk cheeses, a diverse microbiota composed of adventitious LAB (Cogan et al., 1997), but also of bacteria other than LAB, yeasts and moulds, contribute to their distinct sensory characteristics. In order to maintain the traditional characteristics of these cheese varieties, there is a need to preserve the biological diversity involved in the ripening process of ewes' and goats' milk cheeses, by the use of authoctonous lactic starters and mould cultures in their manufacture. Recent studies on ewes' and goats' milk cheeses have considerably enlarged our knowledge of their microbiology, chemistry and texture. However, current scientific information on many varieties, some of major economic importance, is still scarce and research for the better understanding and improving of their manufacture and ripening is needed.
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Cheeses Made from Ewes' and Goats' Milk
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Picon, A., Gaya, P., Medina, M. and Nufiez, M. (1995). The effect of liposome-encapsulated Bacillus subtilis neutral proteinase on Manchego cheese ripening. J. Dairy Sci. 78, 1238-1247. Picon, A., Serrano, C., Gaya, P., Medina, M. and Nuflez, M. (1996). The effect of liposome-encapsulated cyprosins on Manchego cheese ripening. J. Dairy Sci. 79, 1699-1705 Pierre, A., Le QuOre, J.-L., Famelart, M.-H., Riaublanc, A. and Rousseau, E (1998). Composition, yield, texture and aroma compounds of goat cheeses as related to the A and O variants of Otsl-casein in milk. Lait 78, 291-301. Pinna, G., Pirisi, A., Piredda, G., Addis, M. and Di Salvo, R. (1999). Effect of milk thermisation on Fiore Sardo DOP cheese. 2. The lipolysis progress during ripening. Sci. Tecn. Latt.-Cas. 50,366-377. Pirisi, A., Colin, O., Laurent, E, Scher, J. and Parmentier, M. (1994). Comparison of milk composition, cheesemaking properties and textural characteristics of the cheese from two groups of goats with a high or low rate of Otsl-casein synthesis. Int. Dairy J. 4, 329-345. Pirisi, G., Pinna, G. and Papoff, C.M. (1999a). Effect of milk thermisation on Fiore Sardo DOP cheese. 1. Physicochemical characteristics. Sci. Tecn. Latt.-Cas. 50,353-366. Pirisi, A., Piredda, G., Papoff, C.M., Di Salvo, R., Pintus, S., Garro, G., Ferranti, P. and Chianese, L. (1999b). Effects of sheep CCsl-casein CC, CD and DD genotypes on milk composition and cheesemaking properties. J. Dairy Res. 66,409-419. Poullet, B., Huertas, M., S~inchez, A., Caceres, P. and Larriba, G. (1991). Microbial study of Casar de C~iceres cheese throughout ripening. J. Dairy Res. 58, 231-238. Poullet, B., Huertas, M., S~inchez, A., C~iceres, P. and Larriba, G. (1993). Main lactic acid bacteria isolated during ripening of Casar de C~iceres cheese. J. Dairy Res. 60, 123-127. Queiroz-Macedo, I., Faro, C.J. and Pires, E. (1996). Caseinolytic specificity of cardosin, an aspartic protease from the cardoon Cynara cardunculus L.: action on bovine Ors- and [3-casein and comparison with chymosin. J. Agric. Food Chem. 44, 42-47. Ramos, M. and Martinez-Castro, I. (1976). Etude de la proteolyse du fromage type "Manchego" au cours de l'affinage. Lait 56, 164-176. Roman-Blanco, C., Santos-Buelga, J., Moreno-Garcia, B. and Garcia-Lopez, M.L. (1999). Composition and microbiology of Castellano cheese (Spanish hard cheese variety made from ewes' milk). Milchwissenschaft 54, 255-257. Sabio, E. and Vidal Aragon, M.C. (1996). Analysis of the volatile fraction of Ibores cheese. Alimentaria 278, 101-103. Sable, S., Letellier, E and Cottenceau, G. (1997a). An analysis of the volatile flavour compounds in a soft raw goat milk cheese. Biotechnol. Lett. 19, 143-145. Sable, S., Portrait, V., Gautier, V., Letellier, E and Cottenceau, G. (1997b). Microbiological changes in a soft raw goat's milk cheese during ripening. Enzyme Microb. Technol. 21, 212-220. Saldo, J., Sendra, E. and Guamis, B. (2000). High hydrostatic pressure for accelerating ripening of goat's milk cheese: proteolysis and texture. J. Food Sci. 65,636-640.
Saldo, J., McSweeney, P.L.H., Sen&a, E., Kelly, A.L. and Guamis, B. (2002). Proteolysis in caprine milk cheese treated by high pressure to accelerate cheese ripening. Int. Dairy J. 12, 35-44. Salles, C., HervO, C., Septier, C., Demaizieres, D., Lesschaeve, I., Issanchou, S. and Le QuOrO, J.-L. (2000). Evaluation of taste compounds in water-soluble extract of goat cheeses. Food Chem. 68,429-435. Salles, C., Sommerer, N., Septier, C., Issanchou, S., Chabanet, C., Garem, A. and Le Quere, J.-L. (2002). Goat cheese flavor: sensory evaluation of branched-chain fatty acids and small peptides. J. Food Sci. 67,835-841. Santoro, M. and Faccia, M. (1998). Influence of mould size and rennet on proteolysis and composition of Canestrato Pugliese cheese. Ital. J. Food Sci. 10,217-228. Sendra, E., Mor-Mur, M., Pla, R. and Guamis, B. (1999). Evaluation of freezing pressed curd for delayed ripening of semi-hard ovine cheese. Milchwissenschaft 54, 550-553. Sommerer, N., Salles, C., Prome, D., Prome, J.C. and Le Quere, J.L. (2001). Isolation of oligopeptides from the water-soluble extract of goat cheese and their identification by mass-spectrometry. J. Agric. Food Chem. 49,402-408. Sousa, M.J. and Malcata, EX. (1997). Comparison of plant and animal rennets in terms of microbiological, chemical, and proteolysis characteristics of ovine cheese. J. Agric. Food Chem. 45, 74-81. Sousa, M.J. and Malcata, EX. (1998). Identification of peptides from ovine milk cheese manufactured with animal rennet or extracts of Cynara cardunculus as coagulant. J. Agric. Food Chem. 46, 4034-4041. Storry, J.E., Grandison, A.S., Millard, D., Owen, A.J. and Ford, G.D. (1983). Chemical composition and coagulation properties of renneted milks from different breeds and species of ruminants. J. Dairy Res. 50, 215-229. Tavaria, EK. and Malcata, EX. (1998). Microbiological characterization of Serra da Estrela cheese throughout its Appellation d'Origine Protegee Region. J. Food Prot. 61,601-607. Tejada, L., Sanchez, E., Gomez, R., Vioque, M. and Fern~indezSalguero, J. (2002). Effect of freezing and frozen storage on chemical and microbiological characteristics in sheep milk cheese.J. Food Sci. 67, 126-129. The Greek Cheese page (1994). http://www.greece.org/hellas/cheese.html. Tornadijo, E., Fresno, J.M., Carballo, J. and Martfn-Sarmiento, R. (1993). Study of Enterobacteriaceae throughout the manufacturing and ripening of hard goats' cheese. J. Appl. Bacteriol. 75,240-246. Trujillo, A.J., Royo, C., Ferragut, V. and Guamis, B. (1999). Ripening profiles of goat cheese produced from milk treated with high pressure. J. Food Sci. 64,833-837. Tzanetakis, N., Litopoulou-Tzanetaki, E. and Manolkidis, K. (1987). Microbiology of Kopanisti, a traditional Greek cheese. Food Microbiol. 4, 251-256. Tzanetakis, N., Vafopoulou-Mastrojiannaki, A. and LitopoulouTzanetaki, E. (1995). The quality of white-brined cheese made with different starters. Food Microbiol. 12, 55-63. Vassal, L., Delacroix-Buchet, A. and Bouillon, J. (1994). Influence des variants AA, EE et FF de la caseine Otslcaprine sur le rendement fromager et les characteristiques
Cheeses Made from Ewes' and Goats' Milk
sensorielles de fromages traditionnels: premieres observations. Lait 74, 89-103. Ver~ssimo, P., Esteves, C., Faro, C. and Pires, E. (1995). The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specifities. Biotechnol. Lett. 17, 621-626. Verissimo, P., Faro, C., Moir, A.J.M., Lin, Y., Tang, J. and Pires, E. (1996). Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L. Eur. J. Biochem. 235, 762-768. Vicente, M.S., Ib~il~ez, E C., Barcina, Y. and Barr6n, L.J.R. (2001). Changes in the free amino acid content during ripening of Idiaz~ibal cheese: influence of starter and rennet type. Food Chem. 72,309-317. Vieira de Sa, E and Barbosa, M. (1972) Cheesemaking with a vegetable rennet from cardo (Cynara cardunculus L.). J. Dairy Res. 39,335-343. Vioque, M., Gomez, R., S~inchez, E., Mata, C., Tejada, L. and Fern~indez-Salguero, J. (2000). Chemical and microbio-
299
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Acid- and Acid/Rennet-curd Cheeses Part A: Quark, Cream Cheese and Related Varieties D. Schulz-Collins, Arrabawn Co-op, Nenagh, Co. Tipperary, Ireland B. Senge, Technische Universit&t Berlin, Faculty of Process Sciences, Department of Food Rheology, Berlin, Germany
Fresh cheeses are unripened cheeses, which are manufactured by the coagulation of milk, cream or whey using acid, a combination of acid and rennet or a combination of acid and heat. Fresh cheeses are ready for consumption immediately after production. In most countries and cultures, there is some traditional form of flesh cheese. With increased globalisation and tourism, the various regional types of fresh cheese have begun to spread outside their regions of origin. Cream cheese, Cottage cheese, Quark or Tvorog, Fromage frais and Ricotta are among the better-known types. Quark (in German-speaking countries) or Tvorog (in Eastern European countries) is essentially a milk protein paste. It is milky white to faintly yellowish in colour; smooth, homogeneously soft, mildly supple and elastic in body; mildly acidic and clean in flavour. Due to the high moisture content (--~82%, w/w), the shelf-life is limited to 2-4 weeks at < 8 ~ There should be no appearance of whey, dryness or graininess, bacteriological deterioration, over-acidification or bitter flavour during storage (Kroger, 1980; Siggelkow, 1984; Guinee et al., 1993). Hot-pack Cream cheese ('Soft Cheese' in the UK; 'Fresh Cheese' in Germany) is a creamy-white, slightly acid-tasting product with a mild diacetyl flavour; its consistency ranges from brittle, especially for double Cream cheese (DCC), to spreadable for single Cream cheese (SCC). Cream cheese, which is very popular in North America, has a shelf-life of "-3 months < 8 ~ (Guinee et al., 1993). Hard and brittle structures can be obtained only in high-fat Cream cheese (55-60%, w/w, fat-in-dry matter (FDM); Walenta et al., 1988). Quark and Cream cheeses can be consumed plain or in sweet or savoury dishes. Most fresh cheeses are very versatile and particularly suitable for processing into fresh cheese preparations or various dishes (e.g., cheesecakes, sauces, desserts).
Fresh cheeses can be divided into various categories, e.g., by the method of coagulation- acid, acidrennet, acid-heat, etc., their consistency- paste, grainy or gel-like, or raw m a t e r i a l - milk or whey (Fig. 1). In comparison to most ripened cheeses, fresh cheeses are generally low in dry matter (DM) and, hence, low in fat and protein and high in lactose/lactate (Table 1). As most of the calcium is solubilised during the acid coagulation and removed with the whey, fresh cheeses are much lower in calcium than rennet-curd cheeses. Classification and definition of cheeses are, in most countries, controlled by a codex or law, as done in Germany (Table 2) with the Kaseverordnung (Cheese order; Anon, 1986). German Quark is defined as containing at least 18%, w/w, DM, at least 12%, w/w, protein and a maximum 18.5%, w/w, whey protein in the total nitrogen content; products with a DM < 18%, w/w, are to be labelled as Frischktise (Fresh Cheese; Anon, 1986). In other countries, definitions can be less stringent or nonexistent. Often, only total moisture and protein contents are specified, as for, e.g., Kwark or Verse kaas (Quark or Fresh cheese) in The Netherlands, i.e., moisture maximum 87%, w/w, and protein minimum 60%, w/w, of non-fat DM (Anon, 1994b). American Cream cheese (>33%, w/w, fat, 45%, w/w, DM), Neufchatel (20-33%, w/w, fat, 35%, w/w, DM) and German Double Cream (fresh) cheese (26.4-38.3%, w/w, fat, 44%, w/w, DM) are similar in composition and comparable to Petit Suisse or Fromage frais/t la cr~me cheeses of France (Anon, 1986; Kosikowski and Mistry, 1997). World cheese production experienced a low of --~14 million tonnes in 1992/1993 due to the crisis in the former USSR. In 1995-1996 an upward trend started again and world production increased to 15.4 million tonnes in 1999. When cheese production was analysed for 26 countries that accounted for ---80% of the world production in 2001 (Table 3), the most distinct tendency is the remarkable upward trend for fresh cheese, which increased by 38% (from 2 660 000 tonnes in
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
302
Quark,
Cream
Cheese
and
Related
Varieties
and a c i d - c u r d
Acid/rennet-
cheese
varieties
I
I Fresh c h e e s e s
I I
Standard varieties
[
I
-- Paste-like consistency --Quark and quark-related
-
[
Mainly acid coagulated varieties
Baker's cheese - Topfen -Tvorog, Tvarog, Twarogow - Fromage Frais -- Labneh, Labaneh - Buttermilkquark - Petit Suisse - Neufchatel --Ymer -- Chakka, Shirkhand -- Skyr - - Queso Blanco - Cream cheese -Double Cream cheese -
Other varieties
Ripened
acid-curd
cheeses
Harzer Mainzer Olmuetzer Quargel Topfkaese
~ Cottage cheese Acid-heat coagulated t Ricotta, Ricottone Mascarpone Queso Blanco, Queso Fresco
Gel-like consistency
L Layered white cheese (Schichtkaese) Fresh cheese varieties.
1990 to 3 662 000 tonnes in 1999). In i999, 32% of the total cheese production was fresh cheese, compared to 30% 10 years ago (Sorensen, 2001). One reason for the steady increase in output of fresh cheese is that the ingredient sector is becoming more and more important. Major producers are the US (with a large ingredient sector) and the E U - in which Germany, France and Italy produce the highest levels, although Spain and Denmark have also experienced a large increase. Neither The Netherlands nor New Zealand and Australia have a fresh cheese output of importance on the world market (Anon, 1994a; Sorensen, 2001). Of the total production of fresh cheeses in the EU, approximately 47% is produced in Germany, 35% in France and 13% in Italy. In Germany and France, fresh cheese constitutes 47% and 33% of total cheese production, respectively. In Europe, the per capita consumption of fresh cheese is highest in Germany (8.7 kg/year in 1999), followed by France, Poland and Iceland. Almost half of the fresh cheese consumed in Germany is Quark (4.0 kg/year); the balance is Cream, Cottage and other fresh cheeses. Especially high growth rates have been observed for fresh cheese preparations con-
taining fruit or herbs (Richarts, 2001). Fresh cheese consumption is also very high in the Middle-East (e.g., in Israel, 12.3 kg/year in 1998). In Eastern European markets, particularly in Russia and Poland, Tvorog-type cheeses represent up to two-thirds of total cheese consumption (Rouyer, 1997). Poland and Russia are amongst the biggest Tvorog producers in Europe. Mann (1978a,b, 1982, 1984, 1987, 1994, 1997, 2000) has been following and reviewing the world literature on the manufacture, composition and utilisation of Quark and related products for almost the last three decades. The production of fresh acid- or acid/rennet-curd cheeses typically involves the addition of a starter culture and a relatively small amount of rennet to skim milk. Under these conditions, the milk undergoes slow quiescent acidification resulting in the formation of a gel at a pH value near the isoelectric pH of casein (typically 4.8-4.6). The gel is then stirred and concentrated by one of the several techniques, such as centrifugation or ultrafiltration (UF), which involve removal of whey or permeate. The resulting product might be cooled and packaged directly (e.g., Speisequark) or further processed (e.g., heat-treated Quark desserts, Fig. 2).
Quark, Cream C h e e s e and Related Varieties
303
Approximate composition (%, w/w) of various fresh cheeses
Variety (German) Skim Quark (German) Single Cream cheese (German) Double Cream cheese American Cream cheese Neufchatel Labneh Skyr Ymer Lactofil Buttermilk Quark Whole milk Ricotta Part skim milk Ricotta Mascarpone Cottage cheese Baker's cheese Cebreiro cheese
Dry matter
Fat
Protein
Lactose and lactate
pH
> 18
<1.8
> 12
3-4
4.6
39
19.5
n.a.
3.5
4.6
44
26.4-38.3
n.a.
2-3
4.6
>45 >35 22-26 18.5-20.5 14.5 16 15 28-41
>33 20-33 7-10 0.2-0.4 3.5 5 0.75-0.95 13-17
n.a. n.a. 7-10 12.5-16.0 5-6 5-6 9-10 11.3-1 8
2-3 2-3 --~4.2 3.6-3.8 n.a. n.a. 3.5-3.6 3.0
4.6 4.6 4.0-4.2 4.6 4.6 4.6 4.5-4.7 5.7-5.8
25
8
12
3.6
5.8
45-55 21 26 30-35
45-55 4.5 0.2 15-17
7-8 12.5 19 --~12
n.a. 2.6 3-4 n.a.
5.8 n.a. 4.6 4.55
Compiled from Anon, 1986; Tamime and Robinson, 1988; Jelen and Renz-Schauen, 1989; Modler and Emmons, 1989; Lehmann et aL, 1991; Guinee et aL, 1993; Kessler, 1996; Kosikowski and Mistry, 1997; Ozer et aL, 1999; Boone, 2001a,b; Fernandez-Albalat et aL, 2001.
Compositional specification of fresh cheeses according to German regulations
Fat category (Fettstufe)
German Quark Dry matter (%) Protein (%) Fat in dry matter (%) Fat, absolute (%)
Skim
Quarterfat a
Halffat
Threequarter fat
Fat
Fullfat
Cream
Double Cream
>18.0 b >12.0 c <10 <1.8
19.0 11.3 10.0 1.9
20.0 10.5 20.0 4.0
22.0 9.7 30.0 6.6
24.0 8.7 40.0 9.6
25.0 8.2 45.0 11.3
27.0 8.0 50.0 13.5
30.0 6.8 60-maximum 87 18.0-26.1
German Fresh Cream cheese d (Rahmfrischkiise) Dry matter (%) Fat in dry matter (%) Fat, absolute (%) German Fresh Double Cream cheese d (Doppelrahmfrischkiise) Dry matter (%) Fat in dry matter (%) Fat, absolute (%) a Layered cheese (SchichtkAse) is defined as quarter-fat cheese. b Products with DM <18% are to be labelled as Fresh cheese (Frischk&se). c German Quark: whey protein must not exceed 18.5% of total protein. d Compositional specification varies greatly with country. Data from K&severordnung (German Cheese regulations, Anon, 1986).
39.0 50.0 19.5 44.0 60-maximum 87 26.4-38.3
304
Quark, Cream Cheese and Related Varieties
Annual Fresh Cheese Production and Consumption (Approximate values based on data available for period 1990-1999)
Region~Country
Production 1990
Production 1999
Major types a
('000 tonnes)
('000 tonnes)
Per capita consumption 1999 (kg/head)
Mainly Quark
637in 1991 465 n.a. 307 in 1992 56in 1992
748 558 264 380 44.8 b
8.7 7.7 6.7 5.7 in 1992 3.6in 1992
424 48 31 37 32 24 12 n.a. 13 4.0 n.a. 1.6 1 n.a. n.a.
n.a. 85 36 52 24 42 14-20 d 18 in 1998 4.8 11 13.1 5.3 1.8 1 12.1 7.0
4.5in -1990 2.1 0.6 c 1.0 in 1996 n.a. 4.3 4.1 n.a. 2.5in 1996 2.2 0.2in 1998 <0.5 6.4 0.2 n.a. 4.7
60.2
75.3in 1998
12.3 in 1998
Europe Germany France Poland Italy Czech and Slovak Republics Russia Spain UK Denmark Austria Hungary Belgium Greece Finland The Netherlands Switzerland Ireland Iceland Norway Sweden Estonia
Asia Israel
Topfen (Quark) only
Quark only
Excluding whey cheese Fresh and soft cheese Quark only Including Quark, Cottage cheese and salted cheeses
Oceania Australia New Zealand America Canada USA
Includes Mozzarella, Ricotta, Cream and Cottage cheese
South Africa
17
48
1.2
n.a.
n.a.
n.a.
51 1069
54 1585
0.7 5.4in 1998
1.7
3.4
0.08
a If not otherwise stated, mainly Quark/'lvorog, Cream cheese, Cottage cheese and Mozzarella. b Czech Republic only. c Based on estimated production and population figures. d In 1999, output has been affected downwards due to contamination with dioxin. n.a., Data not available. Compiled from Guinee et al., 1993; Anon, 1994a; Serensen, 1995, 2001; Richarts, 2001.
Many manufacturers produce Cream cheese and similar products without rennet. In some acid-curd cheeses rennet may be added, but in much smaller quantities (2-20 ml of standard strength rennet/1000 1 of milk) than for rennet-curd cheeses ( - 2 0 0 m l of standard strength rennet/1000 1 of milk). The weaker acid gel in
comparison to an acid-rennet gel requires modifications of, for example, agitators and pumps. The process of acid gelation will be covered in 'Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels', Volume 1. The present chapter will focus on the combined acid-rennet gelation. Despite its importance in the technology of fresh cheeses, gel formation upon combined continuous
Quark, Cream Cheese and Related Varieties
305
~ Standardisedmilk) I Pasteurisationand homogenisationI ._1._1 Acidificationand pre-ripening I _~_ =', Renneting ~
"~ ~ Culture J [' Rennet
Acidification and coagulation Stirring _~_ Thermisation
)
_"-k,,. f Whey/permeate
Curd Cooling
. . . . . .Cooling-{. ........
.......
i .......
-I
.......
1- .......
.a
Mixing with cream, fruit, vegetables
M,x,n0 w,th cream, fruit, vegetables, stabilisers
) [
l
I M,x,ng w,th sa,t, I I M,x,n0 w,th sa't I stabilisers 1
I Pasteurisation I I Heattreatment I I
Cooling
1
Packing
@
I I .......
Cooting
I
HomogenisationI I Homogenisation I
ve0i'aes i ve0etaes
_~_ . . . . . . . . . . . . . . . .
_t ................
_! ........
,____Whippip_g.... 2 ', Mixingwith fruit,' ' Mixingwith fruit,' . . . . . . . . . . . . . . . . . . . . . .
I
I
Packing
-~- . . . . . . .
Cooling
I
l
Packing
II
Packing
I
uark, Topfen,~ aker's cheese,] (Quark dessert) f Hot-pack ~ f Cold-pack vorog, SkyrJ ~Cream cheeseJ LCream cheeseJ
Generalised flow-chart for the manufactureof fresh cheese. Optional processing steps (---) (from Guinee et aL, 1993; Ottosen, 1996). acidification and renneting has been relatively underresearched until recently. A few studies were dedicated to the subject around 10 years ago (kehembre, 1986; Noel, 1989; Dalgleish and Horne, 1991; Noel et al., 1991). Observations remotely related to acid-rennet coagulation or acid-rennet gels have been reported (Roefs, 1986; van Hooydonk et al., 1986a; Zoon et al., 1989; Roefs et al., 1990; Attia et al., 1993; Walstra, 1993; Gastaldi et al., 1996; Herbert et al., 1999; Kelly and O'Kennedy, 2001). Recently, there has been a renewed interest in the study of milk coagulation after
combined acidification and renneting using more sensitive rheological instruments (Schulz et al., 1999; Lucey et al., 2000; Schulz, 2000; Tranchant, 2000; Tranchant et al., 2001).
Physico-chemical changes Acidification promotes two major physico-chemical changes: a reduction of the negative surface charge on the casein micelles and solubilisation of micellar calcium phosphate. These changes (which influence other related
306
Quark,
Cream
Cheese
and
Related
Varieties
physico-chemical properties) confer metastability on the casein system, which, through structural rearrangements, reaches a new stable state in the form of a gel network. Physico-chemical changes induced by acidification (and limited renneting) have been discussed comprehensively by Guinee et al. (1993). Physico-chemical changes in combined acid-rennet gelation are to a large extent similar to acid gelation. However, there are differences that are discussed briefly below. The dissociation of calcium phosphate upon acidification is not altered by the addition of a small amount of rennet (van Hooydonk et al., 1986b). The dissociation of casein from the micelles (mainly [3-casein) is at a maximum at pH 5.6 and 30 ~ This maximum is less pronounced during the acidification of renneted milk and is caused more by non-specific rennet-induced proteolysis of [3-casein (van Hooydonk et al., 1986b). Both voluminosity and solvation are reduced slightly between pH 6.7 and 4.6 during combined acidification and renneting (Fig. 3). Maximum voluminosity and solvation are at pH 5.3 during acidification. This maximum is less pronounced for renneted milk and is shifted to pH 5.6. In contrast to pure acidification, renneting reduces the solvation and voluminosity by 27% and 37% at pH 6.7 and 5.3, respectively (van Hooydonk et al., 1986b). Creamer (1985) measured a weak maximum at pH 5.1 using different renneting conditions (e.g., renneting at 6 ~
.c_
Mechanism
Slow acidification leads to two 'adverse' reactions. On one hand, the casein micelles tend to aggregate due to the reduced negative surface charge and therefore reduced hydration and, hence, increased hydrophobic interactions; on the other hand, casein micelles disintegrate as a result of the solubilisation of colloidal calcium phosphate, which is completely in solution at pH 5.2 at 20 ~ (Walstra and Jennes, 1984; Heertje et al., 1985; Roefs, 1986; van Hooydonket al., 1986b; Gastaldi et al., 1996). As long as the pH is above the clotting pH (e.g., 5.3 at 30 ~ the disintegration processes dominate, i.e., no gel is formed. At a pH below 5.3, the aggregation forces are greater than the disaggregation forces (solubilisation of colloidal calcium phosphate), i.e., a gel is formed (Guinee et al., 1993). The mechanism during a combined acid-rennet gelation is even more complex. The effects of combined acidification and renneting are synergistic, with acidification potentiating the aggregating tendency of the renneted casein particles. The presence of renneted sites, i.e., supplementary reactive sites on the acid-modified casein particles, may mitigate the adverse effects of on-going casein demineralisation on gel cohesiveness and, ultimately, contribute to the structure of what may be regarded as a rennet-reinforced acid milk gel (Tranchant et al., 2001). A local maximum in complex viscosity (7/*) or storage modulus (G') around pH 5.6 has been reported by
4.0
4.0
3.0
3.0
E
O
~_
//../-"
"1"
---" ~ "-- -E..
. . ... .. . . . .-..-.. -o
_ ...--;or "
2.0
2.0 -
1.0
...-.E5
..
--
OS...
_ ..-IE]
I
I
I
I
I
I
4.6
5.0
5.4
5.8
6.2
6.6
pH
1.0
I
i
i
i
i
i
4.6
5.0
5.4
5.8
6.2
6.6
pH
Solvation (A) and voluminosity (B) of casein in three milk samples at 30 ~ during acidification (--) or combined renneting and acidification (---) (reproduced from van Hooydonk et aL, 1986a).
Quark, Cream Cheese and Related Varieties
several authors during combined acidification and renneting of milk, indicating a local maximum in gel strength or firmness (van Hooydonk et al., 1986a; Noel, 1989; Noel etal., 1991; Walstra, 1993; Schulz etal., 1999; Lucey et al., 2000; Schulz, 2000; Tranchant et al., 2001). On lowering the pH below 5.6, the firmness diminishes until pH 5.3-5.0 and, thereafter, increases again on further acidification towards the isoelectric pH (Fig. 4). Depending on experimental conditions, the local maximum and minimum have been found at different pH values; pH 6.0-5.5 and 5.5-5.0 at 40 ~ (Tranchant et al., 2001); pH 5.60-5.00 and 5.00-4.95 at 30 ~ (Schulz, 2000); pH 5.60 and 5.00 at 30 ~ (Noel, 1989); pH 5.6 and 5.3 at 25 ~ (van Hooydonk et al., 1986a), respectively. A local maximum has been reported at pH 5.3 at 30 ~ (Dalgleish and Horne, 1991) and a local minimum at pH 5.10-5.20 at 30 ~ (Lucey et al., 2000). The local maximum is most pronounced if the rennet concentration is high, the heat treatment is low and the pH at renneting is high. Addition of CaC12 does not markedly influence the presence of the local maximum (Luceyet al., 2000; Schulz, 2000; Tranchant et al., 2001). The local maximum occurs only if the acid coagulation sets in at an advanced stage of rennet-induced coagulation (Schulz, 2000). Similar to Noel (1989) and Tranchant et al. (2001), Schulz (2000) divided the coagulation process into several distinct stages, to which the following fermentation processes can be related: (i) adaptation of the starter and acidification until the desired pH of renneting;
307
(ii) primary phase of rennet coagulation (enzymatic hydrolysis of K-casein); (iii) onset of the secondary phase of rennet coagulation (aggregation and gel formation), i.e., complex viscosity increases; (iv) transition from rennet-type to acid-type gel and concominant microsyneresis (occurs if the gel is constrained and cannot shrink); segregation into dense and less dense gel network on a local scale, leading to wider pores on average (Walstra, 1993), i.e., complex viscosity decreases; (v) predomination of acid coagulation, formation of the final acid-rennet gel, i.e., a second increase in complex viscosity; (vi) syneresis; shrinking of casein strands, causes microsyneresis (no visible whey expression), followed by macrosyneresis (visible whey expression), i.e., complex viscosity decreases again. It is assumed that the decrease in firmness after the local maximum in the viscosity/time curve is due to demineralisation of the forming rennet gel, which is almost completed at pH 5.3-5.1 (Heertje et al., 1985; van Hooydonk et al., 1986b; Noel, 1989; Zoon et al., 1989; Gastaldi et al., 1996). Rheological properties of acid-rennet gels formed at pH values >---5.2 are essentially similar to those of rennet-induced gels, while at pH values <--5.2, the properties are similar to those of acid casein gels. At pH ---5.2, rennet-induced gels have a very high permeability coefficient and tan ~ (loss tangent) which indicates increased relaxation behaviour (shorter lifetime)
7.0 "1" Q.
6.0 5.0 4.0 180
"~" 160 a_ 140 E 120 O o
.-~ > x o_ E 0 o
Max1
IP1
IP3
Max2
4.0 3.5 3.0 2.5 2.0
100
1.5
80
1.0 0.5
60 40 20 ........................ ..........A 0 100 300
.n
8 "E" o FE ~ "~
x
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~g o ~ '~
000 ~ ~ -.5 ~ IP2
J I
500
Min I
I
I
700
900
1100
-1.0 rr -1.5 1300
Time (min)
Figure 4 Typical curves for complex viscosity (mA-) and rate of viscosity increase (m) during combined acid-rennet coagulation of skim milk. RA, rennet addition; A, aggregation; IP1, IP2 and IP3, inflection points 1, 2 and 3 of the complex viscosity/time curve (i.e., rate of complex viscosity increase is at a maximum or minimum); Max1 and Max2 donate the local (primary) and final maximum of viscosity/time curve (i.e., rate of complex viscosity increase is 0); Min donates a local minimum in the complex viscosity/time curve corresponding to a temporary reduction between the Max1 and Max2 (redrawn from Schulz et aL, 1999).
308
Quark, Cream Cheese and Related Varieties
of protein-protein bonds (Roefs et al., 1990; Lucey et al., 2000). In acid-induced gels, most bonds presumably are protein-protein salt bridges, and these are most abundant at pH 4.6, implying that the number of bonds keeping the gel together decreases on increasing the pH. At pH 6.7, the bonds may be mostly of the colloidal phosphate type, and lowering the pH leads to dissolution of colloidal calcium phosphate and, hence, to a reduced number (or strength) of bonds. At pH ---5.2, the resulting number of bonds presumably is at a minimum (Roefs, 1986; Roefs et al., 1990; Walstra, 1993; Lucey et al., 2000). The process of combined acidification and renneting marks a gradual transition from a rennet-type casein gel to an acid-rennet-type gel with a local minimum in complex viscosity or gel modulus due to microsyneresis which leads to a coarsening of the gel network (Lucey et al., 2000; Schulz, 2000). During microsyneresis, the gel network becomes more dense at some sites on a local scale and less dense at others. The surface-weighed average pore size and permeability increase (Walstra, 1993). Figure 5 shows that the permeability increases strongly with decreasing pH; the rate of change in the permeability over time is a measure of the tendency of a gel to exhibit syneresis (Walstra, 1993). The maximum in tan 8 at the local minimum during acid-rennet gelation (e.g., Noel, 1989; Noel et al., 1991; Lucey et al., 2000; Tranchant et al., 2001) is an indicator of the increased susceptibility of the gel to rearrangements and (micro-) syneresis (van Vliet et al., 1991; Lucey et al., 2000). Figure 6 shows the tendency of rennet gels to synerese as a function of pH and temperature (Roefs et al., 1990). There is a very sharp transition from the region of rapid syneresis to that of no syneresis around
4O
oG
Rapid syneresis
v
(D
3o
No
syneresis
!--
20 I
4.5
I
/
5.5 pH
6.5
Tendency of rennet-induced gels to synerese as a function of pH and temperature (redrawn from Roefs et aL, 1990).
pH 5.15, a transition that is hardly temperature dependent. Factors that influence the combined acidification and renneting process
The most important characteristics of acid gels (e.g., yoghurt) and acid-rennet gels (e.g., curd for Quark) are their gel strength and tendency to expel whey. Syneresis is desirable to a degree during Quark production at the separation step; however, spontaneous syneresis is undesirable in the finished product. Spontaneous syneresis is understood as the contraction of a gel without the application of any external force (e.g., separator or UF) and is related to instability of the gel network, i.e., large-scale rearrangements resulting in the loss of the ability to entrap all the serum phase (Walstra, 1993; Lucey et al., 2001). The acid (-rennet) gel structure determines quality aspects such as mouthfeel (i.e., smoothness and creaminess), appearance (coarseness) and physico-chemical stability (wheying-off) during processing and storage. Factors that influence the acidrennet gelation will be discussed below. Rennet concentration
pH 5.35 ~E
5.75
9~->' 1.0 c,
5.97
(1)
E
a_ 0.5
pH 6.33
0
~
p
I 0
1
H
I
6.68
I
2 3 Time (h)
Permeability of rennet-induced skim milk gels as a function of time after renneting at 30 ~ and various pH values (redrawn from Walstra, 1993).
German-type Quark and East European Tvorog are usually produced using small amounts of rennet in order to improve the draining characteristics of the curd, to reduce casein fines and increase curd firmness (Jelen and Renz-Schauen, 1989). Rennet is also known to produce bitter peptides by its proteolytic activity; therefore, a high rennet concentration can lead to bitterness in Quark (B~iurle et al., 1984; Sohal et al., 1988; Shah et al., 1990). The typical rennet concentration used in commercial Quark manufacture, depending on rennet type and strength, is 2-20 ml of standard strength rennet per 1000 1 of milk (Table 4). The rennet action enhances the destabilisation and aggregation of the casein micelles during acidification, i.e., the ratio between aggregation and disaggregation
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309
310
Quark, Cream Cheese and Related Varieties
forces during the early stages of acidification is increased (Guinee et al., 1993). If Quark is produced using acidification only, the coagulum is cut at pH 4.7-4.5 whereas the acid-rennet gel can be cut at a higher pH (4.8-4.9). The shift of the maximum storage modulus of a pure acid-induced gel from pH 4.5 to 4.7-4.8 for an acid-rennet gel is explained by the higher isoelectric point of the para-K-casein in comparison to the K-casein (Roefs et al., 1990). Therefore, over-acidification can be prevented by rennet addition. If the heat treatment is severe (i.e., 95 ~ for 5 min), there are no perceptible differences in UF Quark made with or without rennet (Sachdeva et al., 1993) as the casein is less susceptible to rennet hydrolysis when complexed with denatured whey protein. Ultrafiltration- or Thermoquarks are usually heated in the region 88-95 ~ for 3-6 min. If rennet is used in the process to obtain a firmer product, a lower heat treatment is used, i.e., 88 ~ for 3 min. Much firmer gels are produced when a small amount of rennet is added at the beginning of acidification as the enzymatic reaction is accelerated by lowering the pH (Fig. 7; Lehmann et al., 1991; Schkoda, 1998; Herbert et al., 1999; Schulz et al., 1999; Lucey et al., 2000; Schulz, 2000; Tranchant et al., 2001). The addition of rennet at the beginning of acidification induces a coarser network (Bishop et al., 1983; Roefs et al., 1990; Schkoda, 1998; Lucey et al., 2000; Schkoda et al., 2001a). Particle strands of stirred acid-rennet gels are thicker than in stirred acid gels, i.e., casein micelles partially fuse together (Roefs, 1986; Roefs et al., 1990; Schkoda, 1998). Casein particles in acidset curd are smaller than from enzyme-acid-set curd throughout various stages of Cottage cheese produc-
tion, i.e., after cutting, cooking and draining (Bishop et al., 1983). Rennet gels at pH 6.7 (tan 6 = 0.60) are more viscous-like than acid gels at pH 4.6 (tan ~ = 0.27) or acid-rennet gels at pH 4.6 (tan 6 = 0.26). As tan 6 is related to the relaxation of bonds in the gel during its deformation, rennet-induced gels are more prone to syneresis (Roefs, 1986; Roefs et al., 1990; Walstra, 1993). Therefore, syneresis of combined gels increases strongly with increasing rennet concentration (Roefs, 1986; Walstra, 1993; Schkoda, 1998; Schkoda et al., 2001a). However, Lucey et al. (2001) found more syneresis in low-rennet-GDL gels (10 ml of a double strength calf rennet/1000 1 of milk) than in high-rennet-GDL gels (150 ml/1000 1) for heated and unheated milks. Various parameters of rennet gels, acid gels and acid-rennet gels are compared in Table 5. In addition to the milk coagulation initiated by acidification alone, two other distinct types of coagulation profiles (Fig. 7) were identified, depending on the relative contribution of acidification versus renneting to initial gel formation (Dalgleish and Home, 1991; Schulz et al., 1999; Schulz, 2000; Tranchant, 2000; Kelly and O'Kennedy, 2001; Tranchant et al., 2001). Up to a certain, i.e., 'critical', rennet concentration, gel formation and firming due to acidification and renneting occur simultaneously, i.e., truly combined acidification and renneting (Schulz, 2000; Tranchant et al., 2001). This 'critical' rennet concentration was found to be 1.7-2.2 ml rennet per 1000 1 of milk (see Table 4 for rennet strength) at 30-40 ~ and a renneting pH of 6.4-6.6 (Dalgleish and Horne, 1991; Schulz et al., 1999; Tranchant et al., 2001). The highest gel firming rate and final complex viscosity of acid-rennet gels were found at this critical rennet concentration (Schulz etal.,
c
200
6.5
180 160 EL
6.0
E 140 v
120 g
~00
~"
80
~. 8
"r
5.5
6o
b
40 20 0 200
a 400
600
800
1000
5.0 4.5 1200
Time (min) Time-related change in complex viscosity (m) and pH ( ) of skim milk treated with a mesophilic starter culture and rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at different levels (ml rennet/1000 I milk: a, 0; b, 0.5; c, 2; d, 9; e, 20) during slow and quiescent acidification at 31 ~ (redrawn from Schulz, 2000).
Quark,
Properties of skim milk gels obtained by renneting (aged for about 1 h), by acidification (aged for 6-16 h) or by combined renneting and acidification. All gels were formed by slow quiescent acidification using GDL at 30 ~
pH Elastic modulus, G' at = 0.01 rad s -1 (Pa) Loss tangent, tan 8 at ~o = 0.01 rad s -1 Fracture stress (Pa) Fracture strain ( - ) Permeability B (l~m2)
Rennet gel
Acid gel
Combined gel
6.65 32
4.6 20
4.6 800
0.55
0.27
0.27
10 3.0 0.25
100 1.1-1.5 0.15
300 0.7 0.28
Data from Roefs et aL, 1990; Walstra, 1993; Lucey et al., 2000.
1999). Rennet concentrations higher than the 'critical' value lead to a distinct local maximum in gel consistency followed by a local minimum, i.e., sequential formation of rennet gel and acidification (Schulz, 2000; Tranchant et al., 2001). Rennet addition shortens the clotting time, and gelation occurs at a higher pH (Noel, 1989; Noel et al., 1991; Schkoda, 1998; Lucey et al., 2000; Schulz, 2000). There is a linear relationship between the inverse of rennet concentration and the clotting time as well as the time to reach the local viscosity maximum for the combined acidification-renneting method (Schulz, 2000). On increasing the rennet concentration in the range 0-20 ml/1000 1, the pH at aggregation and the local maximum of the complex viscosity increased
Cream
Cheese
and Related Varieties
311
from pH 5.44 to 6.31 and 5.05 to 5.38, respectively (Fig. 8; Schulz et al., 1999; Schulz, 2000; Fromase 220TL, DSM Food Specialties B.V., Dortmund). However, the local viscosity minimum and final viscosity maximum occured at the same pH, i.e., pH 4.95 and 4.45, respectively, when rennet was added at pH 6.45 and 30 ~ The complex viscosity of the local maximum is constant, i.e., does not change with rennet concentration. After the local maximum, the complex viscosity at all points, e.g., inflection points, local minimum and absolute (final) maximum in the viscosity curve (Fig. 4), decreases at rennet concentrations above the critical rennet concentration (Schulz, 2000). Noel et al. (1991) observed an initial increase of the storage modulus at the local maximum and then a slight decrease. Not only are acid-rennet gels firmer, but also the apparent viscosity of stirred products like Quark is higher than in those made by acidification alone. Syneresis of Quark produced by acid-rennet coagulation is also higher than in purely acid-fermented Quarks (Shah et al., 1990; Schkoda et al., 2001a). Heat treatment
Heat treatment of milk has very different effects on acid, rennet and acid-rennet coagulation and gels. Heat treatment of milk at > 70 ~ causes denaturation of the whey proteins, ot-lactalbumin and [3-1actoglobulin, some of which may complex with micellar K-casein by hydrophobic and disulphide intermolecular interactions (Smits and van Brouwershaven, 1980; Law et al., 1994; Lucey 1995; Singh, 1995). The yield of Quark can be increased by heat denaturation of the whey proteins (Puhan and Pltieler, 1974; RA
6.5 6.3
Enzymatic hydrolysis
.---------
6.1
A
5.9 5.7
Initial aggregation
IP1
-r{3. 5.5 5.3 5.1 4.9
Max1 /
~
4.7 4.5
I 2
Microsyneresis
Acid gel formation I I I 4 6 8 Rennet concentration (ml/1000 I)
IP3
I 10
Phases during combined acid-rennet coagulation of skim milk as a function of rennet concentration. The skim milk was heat-treated at 72 ~ for 30 s; gelation at 31 ~ was initiated by a mesophilic starter culture and rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at pH 6.45. The pH was measured at various points obtained from the complex viscosity/time curve (as in Fig. 4). RA, rennet addition; A, aggregation (initial increase of complex viscosity); IP1 and IP3, inflection points 1 and 3; Max1, local maximum (redrawn from Schulz, 2000).
312
Quark, Cream Cheese and Related Varieties
Sheth et al., 1988; Shah et al., 1990; Kelly and O'Donnell, 1998; Mara and Kelly, 1998) as exploited in the Thermoprocess. However, in the Thermoprocess, a higher level of rennet and a secondary heating prior to whey separation are necessary to enhance whey expulsion to give the desired DM content. The resulting Quark is softer and creamier than the traditional separator-type Quark (Dolle, 1977, 1981; Ott, 1977). In particular, when rennet is used in Quark production, the heat treatment must be lower than in yoghurt production. The degree of hydration and apparent viscosity of stirred acid-rennet gels (7%, w/w, protein) increase markedly up to a degree of whey protein denaturation of 30% when heated to 80 ~ with only a slight further increase at higher levels of whey protein denaturation (Schkoda, 1998). The optimum degree of whey protein denaturation for fresh cheese is 75-80%. If it is too low, the consistency of the product will be too soft, and not creamy; however, over-denaturation, i.e., at a temperature >120 ~ leads to protein aggregation which causes sandiness in the fresh cheese (B~urle et al., 1984; Schkoda and Kessler, 1997a,b). Depending on the manufacturing method used, the heat treatment is usually in the range 88-95 ~ for 3-6 min for Thermo- or UF Quarks (e.g., B/iurle et al., 1984; ROckseisen, 1987; Sachdeva et al., 1993; Rogenhofer et al., 1994). Several authors have observed spontaneous syneresis in combined gels made from unheated milk (Schulz, 2000; Lucey et al., 2001; Tranchant et al., 2001). Confocal scanning laser micrographs of acid-rennet gels made from unheated milk showed much larger pores than acid-rennet gels made from heated milk. This was
6.5
confirmed by permeability measurements (Lucey et al., 2001). Acid-rennet gels made from unheated milk are extremely prone to spontaneous whey separation, possibly due to considerable rearrangements of aggregated particles at an early stage of the gelation process (Schulz, 2000; Lucey et al., 2001). In pure acid coagulation, gelation occurs more rapidly and at a higher pH with increasing heat treatment (Heertje et al., 1985; Banon and Hardy, 1992). During combined acidification and renneting of low-heated milk (74 ~ for 30 s) or high-heated milk (86 ~ for 6 min), the pH at which aggregation begins remained constant at 6.3 but the pH for the local maximum (Maxl in Fig. 4) decreased from 5.6 to 5.0 (Fig. 9; Schulz, 2000). The initial increase in complex viscosity of acid-rennet gels is reduced by the heat treatment of milk (Lucey et al., 2000; Schulz, 2000). As this stage relates to the secondary phase of rennet coagulation (Schulz, 2000), this confirms the findings that this phase is more adversely affected by heat treatment than the enzymatic phase of rennet coagulation. Acid-rennet gels made from heated milk are firmer than those from unheated milks because the casein is crosslinked by denatured whey proteins and the local maximum/minimum are less pronounced due to reduced (micro-) syneresis (Lucey et al., 2000; Schulz, 2000). The maximum tan is smaller in gels from heated milks compared to unheated milks (---0.43 and 0.51, respectively), indicating that the proteins undergo fewer large-scale rearrangements (Lucey et al., 2000). Confocal micrographs indicate that the pores are much smaller and
RA A
Enzymatic hydrolysis
6.3 6.1
Initial aggregation
5.9 5.7 "T" T 5.5
IP1
~Maxl
Q.
5.3 5.1
Micro~ s y n e r e s i s ~
Rennet gel formation IP3
4.9 Acid gel formation
4.7 4.5
1:0
1 "1 Ratio Low-heatedhigh-heated skim milk
0:1
Phases during combined acid-rennet coagulation of skim milk as a function of the ratio of low-heat-treated skim milk (74 ~ for 30 s) and high-heat-treated skim milk (86 ~ for 6 min) in the milk blend used for gelation; gelation was initiated at 31 ~ by a mesophilic starter culture and 9 ml/1000 I rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at pH 6.45. The pH was measured at various points obtained from the complex viscosity/time curve (as in Fig. 4). RA, rennet addition; A, aggregation (initial increase of complex viscosity); IP1 and IP3, inflection points 1 and 3; Max1, local maximum (redrawn from Schulz, 2000).
Quark, Cream Cheese and Related Varieties
there appears to be more interconnectivity of the network in acid-rennet gels made from heated milk than those from unheated milk (Lucey et al., 2000).
313
The clotting time is also reduced at lower pH values during combined acidification and renneting (van Hooydonk et al., 1986a; Noel et al., 1991; Schulz et al., 1999; Schulz, 2000). There are discrepancies over the following stages of the coagulation process. Nod et al. (1991) investigated the effect of renneting pH in the range 5.98-6.62 up to the local minimum. At low rennet concentrations, the clotting time decreases markedly with decreasing pH whereas at high rennet concentrations the clotting time is independent of the renneting pH. The complex viscosity of acid-rennet gels at the local maximum (Maxl in Fig. 4) increases with decreasing pH for all rennet levels. Schulz et al. (1999) and Schulz (2000) found no difference in the final viscosity for renneting pH between 6.6 and 5.8. However, the initial aggregation reactions, i.e., due mainly to rennet, are affected by the renneting pH. If the rennet is added at a pH below 6.0, the typical local maximum and minimum are less pronounced as the two processes of acidification and renneting occur simultaneously. The pH values for clotting (pH 6.40-5.63), inflection point 1 (pH 5.65-5.17) and local maximum (pH 5.16-5.02) are directly related to the renneting pH (pH 6.6-5.8) whereas the pH values for the local minimum (pH 5.0), inflection point 3 (pH 4.80--4.85) and the final maximum (pH 4.45--4.50) are influenced solely by the acidification and not by the pH at renneting (Fig. 10). At pH values >5.9, the pH at which the rennet is added does not affect the magnitude of the complex viscosity ~/* of acid-rennet gels at the local maximum, local minimum and final maximum (Schulz, 2000). No information is
pH at renneting In Quark manufacture, rennet is rarely added simultaneously with the culture, but after 60-90 min when the pH is around 6.3. The correct moment of rennet addition and the effect on structural properties is based mainly on empirical experience. The pH value at which rennet is added (Table 4) varies from the natural pH to 6.00, and is mainly around 6.30-6.45. During rennet coagulation alone, the clotting time is markedly reduced at lower pH values as the pH is very important for the enzymatic activity of the rennet, with an optimum at pH 6.0 (Mehaia and Cheryan, 1983; van Hooydonk et al., 1986a; Zoon et al., 1989; Fox and Mulvihill, 1990; Dalgleish, 1992). With decreasing pH, the aggregation of micelles starts at a lower conversion of K-casein to para-K-casein (70% at pH 6.7 compared to 30% at pH 5.6) and the rate of aggregation and gel formation increases (van Hooydonk et al., 1986a). This is due mainly to the higher calcium ion activity at low pH values; the rate of aggregation is doubled by reducing the pH from 6.8 to 6.3 (Dalgleish, 1992). A lower pH possibly also leads to a faster rearrangement of strands and fusion of micelles, resulting in a faster increase in the storage modulus (G') directly after the onset of gelation and the earlier attainment of a plateau value of the storage modulus (Zoon et al., 1989).
RA 6.5
A
6.0 Initial aggregation -r
IP1
5.5
O.
~ 5,0
~
Rennet gel formation ~
Max1 Microsyneresis
-
IP3
Acid gel formation 4.5
5.8
, 6.0
I
I
I
6.2
6.4
6.6
pH at rennet addition Phases during combined acid-rennet coagulation of skim milk as a function of pH at rennet addition. The skim milk was heat-treated at 72 ~ for 30 s; gelation was initiated by a mesophilic starter culture and 9 ml/1000 I rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at 31 ~ The pH was measured at various points obtained from the complex viscosity/time curve (as in Fig. 4). RA, rennet addition; A, aggregation (initial increase of complex viscosity); IP1 and IP3, inflection points 1 and 3; Max1, local maximum (redrawn from Schulz, 2000).
314
Quark, Cream Cheese and Related Varieties
available on how the renneting pH affects the rheological and syneretic properties of the final product. Rate of gelation Culture addition and acidification profile are normally such that the milk has reached pH 6.3 after 1.5 h (pH for rennet addition) and pH 4.5-4.6 after about 16 h (German-type Quark). American-style Cream cheese or Quark is fermented in a shorter period of time, i.e., 5-6 h (Kosikowski and Mistry, 1997) or 8-9 h (Sohal et al., 1988). High rates of acid gelation lead to coarser networks with a greater tendency to syneresis. The rate of gelation increases with increasing rate of acidification, increasing temperature and increasing casein concentration (Heertje et al., 1985; Hammelehle, 1994). Incubation temperature For Quark-type products, either the cold method (22-24 ~ or warm method (28-31 ~ can be used. The amount of starter added is normally adjusted so that Quark can be separated the following morning, i.e., 16 h coagulation with an optional rennet addition after 60-90 min. The higher the temperature for acid gelation, the higher is the pH at which clotting and gelation begins during acidification (Heertje et al., 1985; Kim and Kinsella, 1989; Banon and Hardy, 1992). Increasing temperature also causes an increase in the maximum rate of coagulation due to an increase in the frequency of thermal collision between casein micelles (Kim and Kinsella, 1989). The coagulation rate of casein has a Q10 of 2-5 under various conditions (Walstra and Jennes, 1984). In acid gels, higher gelation temperatures result in a greater permeability coefficient, indicating the presence of larger pores and, therefore, increased susceptibility to syneresis (Lucey et al., 1997). Microscopic investigations show a coarser network at higher temperatures (Heertje et al., 1985; Rods, 1986). These effects at increased incubation temperatures may be attributed to a higher ratio of aggregation to dissaggregation forces during the early stages of acidification owing to decreased casein dissociation from the micelles, a reduction in repulsive forces due to increased hydrophobicity and a faster rate of acidification which is subject to the type of bacterial culture (Guinee et al., 1993). There is no information available on the effect of incubation temperature on the acid-rennet coagulation. Level and type of gel-forming protein Fermented milk gels and rennet curds are particle gels, networks built up of casein micelles or marginally modified micelles (Roefs, 1986; Home, 1998). The level and nature of proteins in the fresh cheese milk mainly determine the structure of the product. The manufacture of fresh cheeses involves a step to increase protein concentration (e.g., 12%, w/w, protein for Quark). Quark pro-
duced using the standard separator method incorporates a maximum of 15%, w/w, whey proteins; this type of Quark is generally described as firm, dry and sour. Thermoquark or UF Quark may contain all the whey proteins present in milk and is creamier, smoother, softer and often milder (Lehmann etal., 1991; Ottosen, 1996; Schkoda and Kessler, 1996; Hinrichs, 2001). The level of whey proteins in Quark is also regulated by law in Germany (> 12%, w/w, protein of which a maximum of 18.5%, w/w, is whey protein; Anon, 1986). For a gel with a given protein concentration, the final gel strength at 30 ~ and pH 4.6 increases up to a ratio of 1.5/10.5 whey protein/casein and decreases at a ratio 2.0/10 (Kelly and O'Kennedy, 2001). The proportion of pre-denatured whey protein required to give the desired synergism is substantially lower in the fresh cheese model compared to 2.5/10 in the model yoghurt system studied by O'Kennedy and Kelly (2000). The firmness of fresh cheeses increases with increasing total protein content (Korolczuk and Mahaut, 1991a; Mahaut and Korolczuk, 1992; Ozer et al., 1999). For a given protein type and degree of gel fineness, high levels of gel-forming protein result in a denser (i.e., greater number of strands of equal thickness per unit volume), more highly branched network which has a greater degree of overlapping of strands and a narrower pore size (Harwalker and Kalab, 1980; Modler and Kalab, 1983; Modler et al., 1983; Ozer et al., 1999). Increasing the protein concentration in skim milk by nanofihration from 3.5 to 7.0%, w/w, increases gel firmness, apparent viscosity, serum-holding capacity, solvation and fineness of the gel network; the rate of increase of the apparent viscosity over the protein range is slightly higher for acid-rennet gels than for acid gels (Schkoda, 1998; Schkoda et al., 2001a). Undenatured whey proteins do not participate in texture formation in acid-type fresh cheese (Korolczuk and Mahaut, 1991a,b; Mahaut and Korolczuk, 1992). For milk heated at 72 ~ for 15 s, increasing the whey protein content (from 19.6 to 25.6%, 32.9% and 41.4%, w/w, of total protein), by the addition of spraydried UF protein concentrate, reduced cheese viscosity substantially. However, as the heat treatment of the milk was increased to 92 ~ for 15 s or 92 ~ for 60 s, starting at a higher initial viscosity (i.e. at 19.6%, w/w, whey protein of total protein) increasing the whey protein content caused smaller decreases in cheese viscosity (Mahaut and Korolczuk, 1992). Factors which lead to an increase in the effective protein concentration include: (i) fortification with proteins, as often practised in the production of Fromage frais or Cream cheese by the addition of protein powders to either the milk or Cream cheese after separation;
Quark, Cream Cheese and Related Varieties
(ii) high heat treatment which causes the co-precipitation of denatured whey proteins onto the casein micelles and which therefore participate in gel formation; (iii) combining high-temperature heating and membrane technology to retain the denatured and aggregated whey proteins; (iv) homogenising of the fat-containing milk, as practised in Cream cheese production, which results in the incorporation of proteins in the fat globule membrane. Calcium chloride
Progressive solubilisation of salts bound to the casein leads to almost complete demineralisation at pH 5.00 (Heertje etal., 1985; van Hooydonk et al., 1986b; Dalgleish and Law, 1989). This suggests that the addition of CaCI2 to milk during flesh cheese production is not justified. If milk has been subjected to a high heat treatment, 500-800 ml of a liquid CaC12 solution (33%, ww) per 1000 1 milk can be added to improve its rennet coagulation properties (Spreer, 1998). The effect of CaC12 on the process of combined acidification and renneting is difficult to establish as it decreases the pH and, therefore, accelerates the rennet action (Walstra, 1993; Schkoda, 1998; Schulz, 2000). The viscosity of stirred acid-rennet gels is higher when CaCI2 is added (Schkoda, 1998). Schulz (2000) did not observe an effect of CaC12 on acid-rennet coagulation when the rennet was added at pH 6.45. Gastaldi et al. (1994) established the effect of calcium on combined acidification and renneting in the range of 10-30 ml rennet/1000 1 (for rennet specification see Table 4). No difference was found between calcium-free and calcium-enriched milk (6.25 mM) at 10 ml rennet/1000 1. At a rennet concentration >20 ml rennet/1000 1, the clotting time and pH were reduced by calcium, i.e., calcium affects the acid-rennet gelation only when >10 ml rennet/1000 1 are added and the gelation becomes more like rennet coagulation. Noel (1989) also found that the clotting time remained constant for various calcium concentrations (0-400 mg/hg). The storage modulus of the local maximum decreased with increasing calcium concentration (40-400 mg/hg), whereas storage modulus of the local minimum increased slightly (0-160 mg/~g) and then decreased (Noel, 1989).
The majority of acid- and acid/rennet-curd flesh cheeses are produced by acid (and rennet) coagulation, separation of the curd from the whey, various heating and homogenising steps. Fresh cheese preparations are blended with different ingredients (Fig. 2).
315
Q u a r k - traditional batch methods
Batch separation of curd from whey was done originally by draining and pressing the curd in filter bags. This process produces a granular textured Quark with a smooth mouthfeel and is still used for Farmhouse cheeses or Quarks with very high DM, up to 27-33%, w/w (Kroger, 1980; Dolle, 1991; Kessler, 1996). Semiautomated processes are the Berge-process (an oscillating suspended cloth method; Ramet, 1990; Kosikowski and Mistry, 1997) and the 'Schulenberg processor' (specially constructed double bottom Quark vat; Jelen and Renz-Schauen, 1989). Q u a r k - original (standard) separator process
Skim milk is pasteurised (72 ~ for 40 s), cooled to 28-30 ~ and coagulated with a mesophilic culture and a small amount of rennet within ---16 h. Rennet (--2-20 ml standard strength rennet/1000 1 of milk) is usually added approximately 90 min after culture addition at a pH around 6.3. The coagulated skim milk is then stirred for ---10-15 min and passed through a tubular strainer to remove larger particles. After separation (34-40 ~ the Quark is cooled, optionally blended with cream or other condiments and packed. The whey discharged from the separator still contains nearly all, i.e., -0.65%, w/w, whey proteins and 0.2%, w/w, NPN (Siggelkow, 1984; Ramet, 1990; Dolle, 1991; Lehmann et al., 1991; Senge, 2002a). Whey proteins in the native, undenatured state do not gel under the heating and acidification conditions used in standard separator Quark production. Various methods have been developed to increase the whey protein content of Quark and reduce losses in the whey. Early methods recovered the whey proteins from the whey and incorporated them either into the Quark or the following day's cheese milk (Centriwhey and Lactal processes, uhrafihration of whey). In the Centriwhey Process, the Quark whey is heated to 95 ~ to precipitate the whey proteins which are concentrated to 12%, w/w, DM by centrifugation and then added back to the cheese milk for the next batch of Quark (Dolle, 1977, 1981; Kroger, 1980; Jelen and RenzSchauen, 1989). In the Westfalia Lactal process, the heat-precipitated whey proteins are allowed to settle, and by partial decanting of the supernatant, a whey concentrate of 7-8%, w/w, solids is obtained. This is further concentrated in a Quark separator into whey Quark (17-18%, w/w, solids) which is added to regular Quark at a level of 20%, w/w (Dolle, 1977; Kroger, 1980; Jelen and Renz-Schauen, 1989). Uhrafiltration can also be used to concentrate whey instead of separators (Herbertz, 1982; Kn~pfer, 1982; Kreuder and Liebermann, 1983).
316
Quark, Cream Cheese and Related Varieties
Q u a r k - Thermo process (Westfalia)
The milk is pasteurised at 95-96 ~ for 2-3 min to denature and co-precipitate the whey proteins onto the caseins. The resulting finer milk coagulum after fermentation requires a further heat treatment at --~60 ~ for 3 min (so-called thermisation) in order to enhance aggregation and improve sedimentation characteristics. The stirred curd is then cooled to separation temperature (Dolle, 1977; Ott, 1977; Kroger, 1980; Siggelkow, 1984; Jelen and Renz-Schauen, 1989; Ramet, 1990; Lehmann et al., 1991). The majority of Quark in Germany is produced by this process. Q u a r k - filtration methods
Filtration technology can be used at different stages during the manufacture of Quark-type products, e.g., filtration of the acid whey, (partial) filtration of the sweet milk or filtration of (partially) acidified milk. The yield is higher than for Thermoquark as all whey proteins are incorporated. However, the structure is different from conventional Quark as UF Quark is generally softer, smoother and creamier. This can be an advantage if consumed as such; however, for cheese-cakes or desserts, the higher firmness of conventional Quark and Cream cheese is more desirable. When full filtration to final cheese solids was carried out before acidification, the sensory attributes of the resulting products were described as impaired due to bitterness contributed by the slower rate of acidification, failure to reach the desired pH and the high calcium content (Dolle, 1977; Kroger, 1980; Kreuder and Liebermann, 1983; Btturle et al., 1984; Mann, 1984; Patel et al., 1986). Labneh produced by culturing UF milk retentate was also not satisfactory (Tamime et al., 1989b). This problem has been overcome by UF of partially (pH 5.7-5.95) or fully (pH 4.8-4.6) acidified milk. Low-protein fresh cheeses, like Ymer and Lactofil (--6%, w/w, protein), are easily produced by ultrafiltering milk (Tamime and Robinson, 1988; Nakazawa et al., 1991; Kosikowski and Mistry, 1997). To produce UF Quark, acidified skim milk (pH 4.6) is heated to around 40 ~ and ultra- or micro-filtered to the desired DM content, cooled, optionally homogenised and packed (e.g., Btturle etal., 1984; Siggelkow, 1984; Dieu etal., 1990; Korolczuk and Mahaut, 1991a,b; Rogenhofer etal., 1994; Ottosen, 1996). The UF method gives complete recovery of whey proteins (native or denatured); however, NPN in the milk (-0.2%, w/w), is lost in the permeate. As native whey proteins are not retained during microfiltration, the curd is usually heat-treated (thermisation) before separating the curd form the whey (Dieu et al., 1990). Thermisation of the curd (60 ~ for 5 min)
before ultrafiltration also considerably reduces the development of stale, bitter and metallic flavours (Sachdeva et al., 1993; Rogenhofer et al., 1994). Ultrafiltration is carried out around 40-45 ~ in order to maintain good calcium solubility so as to remove calcium in the permeate (Ottosen, 1996). Quark and Labneh, ultrafiltered at higher temperatures, are described as gritty, granular and coarse (B~urle et al., 1984; Tamime et al., 1991a,b; Sachdeva et al., 1993). The viscosity of fresh cheeses produced by filtration is lower than of those manufactured by traditional technologies. In Germany, UF Quark is used only for Speisequarkzubereitungen (Quark preparations), as the possible slightly bitter flavour at the end of the shelf-life in plain Speisequark is not satisfactory. Several studies have been conducted to investigate the effect of the following during the manufacture of Quark using filtration methods: milk heat treatment, full (pH 4.6) or partial (pH 6.0) acidification of skim milk and type and configuration of membranes (Sachdeva etal., 1992a,b; Sharma etal., 1992a,b; Sharma and Reuter, 1993). Ultrafiltration using mineral membranes was found to be best for making Quark by UF from fully acidified skim milk (Sharma et al., 1992a; Sharma and Reuter, 1993). Recently, pilot-scale filtration methods have been developed by partially pre-concentrating the acidified milk in order to reduce the amount of acid whey. In the FML process (Forschungszentrum for Milch und Lebensmittel, Weihenstephan), skim milk is nanofiltered 2fold to 7%, w/w, protein and then fermented. The coagulum is stirred and concentrated by either ultrafiltration or separation. A separator needs to be adapted to the higher viscosity of the retentate coagulum in comparison to unconcentrated fermented skim milk. The texture of the final product is between that of conventional UF fresh cheese and of Thermoquark (Schkoda and Kessler, 1996, 1997a,b). Mucchetti et al. (2000) confirmed the findings of Schkoda and Kessler by nanofiltering milk 2.1-fold. In another method (Aubios process, Hannover), the skim milk is pre-concentrated 1.7-fold to 5.4%, w/w, protein (or up to 2.2-fold without causing bitterness) using microfiltration, producing a product which is similar to Thermoquark (Hulsen, 2002). A special combination of starter cultures is needed for the fermentation of retentates as more lactic acid must be formed than in unconcentrated milk. Pfalzer and Jelen (1994) enriched cheese milk with 25% sweet whey UF retentate containing 12%, w/w, DM and 4%, w/w, protein for an experimental Thermoquark-type fresh cheese produced using cheesecloth bags without significantly affecting the quality of the final product.
Quark, Cream Cheese and Related Varieties
Table 6 summarises the yield and whey protein recovery for the various methods. Q u a r k - recombination technology
Recombination technology is used to only a limited extent for the manufacture of Quark and related types. Fresh cheeses low in DM, like Frornage frais, can be produced by a method similar to yoghurt, i.e., skim milk is fortified with various milk proteins to approximately 14%, w/w, DM and then fermented. Labneh (a concentrated yoghurt with 23%, w/w, DM) can also be produced by direct recombination; fermentation at 23%, w/w, DM takes about 5-6 h in comparison with 3.5 h at 16%, w/w, DM (Ozer et al., 1999). Further treatments of the acid or acid/rennet gel
After fermentation, the gel is broken up by agitators and pumped through a sieve to the separators. Stirring the gel leads to breakage of the matrix strands, with the extent of breakage depending on the severity of the agitation. This non-Newtonian shear-thinning dispersion can be described rheologically by the Power-law model (Senge, 2002a). Increasing the temperature (25-50 ~ lowers the activation energy for aggregate interaction within the broken strands and facilitates the process of subsequent whey separation. A high pH (>4.6) at whey separation results in large losses of nitrogenous compounds in the whey (more casein fines) owing to greater physical damage to the softer gel. Any
317
factors which increase gel firmness at separation (e.g., rennet addition, higher level of gel-forming protein), will make it less susceptible to breakage for a given degree of shear and, therefore, reduce the amount of casein fines. Cooling of the gel to a temperature of <20 ~ to retard further acidification, may result in more destruction of the gel for a given degree of agitation (Guinee et al., 1993). Separation is generally carried out at a temperature between 34 and 40 ~ (Senge, 2002a). Whey separation causes concentration and aggregation of the broken gel pieces. Collision during concentration may be expected to result in the formation of large irregularly shaped conglomerates of varying thickness and length, which are forced into close proximity. The moisture content of the curd is closely related to the degree of aggregation. All factors which enhance aggregation (coarser gel structure, higher separation temperature) reduce the water content and increase the coarseness and firmness of the resulting curd (Guinee et al., 1993; Senge, 2002a). Increasing coarseness of the gel structure before separation results in a product with a coarser/rougher appearance and grainier mouthfeel (separator-produced Quark versus Thermoquark). This is particularly important for products that are packed at this stage (cold-pack Cream cheese, Fromagefrais, Quark). After leaving the separator, the Quark is subjected to further shearing (pumping, cooling, storing, optionally mixing with cream and condiments and packing). Such treatments will influence the structural, rheological
Yield of Quark and whey protein recovery using various production methods for Quark
Method
Principle
Westfalia Standard separator process Westfalia Thermoprocess Centriwhey/ Westfalia-Lactal/ Meggle-Aicor Ultrafiltration Ultrafiltration Weihenstephan (FMLa) process
Separation of acidified milk Separation of acidified milk Separation/Decanting/ Ultrafiltration (UF) of whey Full UF of milk Full UF of acidified milk Nanofiltration of milk to a volume concentration factor (VCR) of 2. UF or separation of acidified retentate Microfiltration of milk (VCR = 1.7-2.2). Separation of acidified retentate.
Hannover (Aubios) process
Typical yield (kg skim milk/kg Quark)
% Whey protein recovery in Quark
Flavour and texture
4.50-4.70
--~15
Firm and sour
4.08-4.30
50-70
Firm, smooth and mild
3.98
50-100
Whey taste possible
---3.8 3.60-3.98 -3.4
--- 100 --~100 <_100
Bitter taste Smooth Smooth, sweet mild flavour
4.00
<_60
As Thermoquark
a Forschungsinstitut fL~r Milch und Lebensmittel (Research institute for dairy and food), Weihenstephan, Germany. Data from Dolle, 1981" Anon, 1984; B&urle et aL, 1984; Lehmann et aL, 1991 Schkoda and Kessler, 1997a; Hinrichs, 2001" H(~lsen, 2002.
318
Quark, Cream Cheese and Related Varieties
and syneretic properties of the final product. The texture of Quark as measured by the yield value (r0, using the Bingham model) correlates with the extent of syneresis of the final product. The yield value (r0) of Quark leaving the separator decreases gradually during further processing (Senge, 2002a). Syneresis of the final product increases as the temperature at which the Quark is pumped increases. Below 15 ~ the Brownian motion of the serum is restricted; the high viscosity of the product also inhibits reincorporation of the serum (Senge, 2002a). After packing, the yield value increases during storage for 30-40 days (Hawel and Heikal, 1994; Senge et al., 1998; Senge, 2002a). In fresh cheese products where the stirred gel is concentrated to obtain the correct level of DM, the application of a relatively high pressure has already led to large-scale syneresis, which is necessary for whey separation. However, once the stirred gel has been concentrated to the correct DM, syneresis may continue due to delayed network arrangements, which in this situation is undesirable. The level of syneresis will depend on several conditions (i.e., composition and further treatments of the concentrated gel), which affect structure and porosity, and on the absence/presence of hydrocolloids that bind the moisture phase (Guinee et al., 1993).
(r0) and viscosity of the Quark decrease during this phase. During the second phase, the cream becomes gradually immobilised within the protein matrix and the yield value increases (Senge, 2002a). The addition of cream to Quark reduces the yield value and viscosity, especially in Quark with a high FDM (40%, w/w). This is due to a number of reasons: (i) the fat causes lubrication; (ii) adding cream up to 40%, w/w, FDM leads to an overall reduction in protein; (iii) the fat globules (0.5-10 Ixm) further interrupt gel particle interactions (Senge, 2002a). In Quark concentrated by filtration, the fat can be added either before or after fermentation and whey separation. When full-fat milk is homogenised at 15 MPa prior to fermentation, fat globules react with the protein matrix and the serum-holding capacity and apparent viscosity of stirred fermented milks (7%, w/w, protein) increase with increasing fat content. The addition of cream to the fermented milk, however, does not lead to an increase in serum-holding capacity and even results in a slight decrease in viscosity as the fat globules do not serve a structure-building function but are present in the fermented milk structure only as a filling substance (Schkoda, 1998; Schkoda et al., 2001b).
Addition of cream
A second heat treatment of either the curd before whey separation (60 ~ for 3 min) or the Quark-type product after separation (55-75 ~ for 30-60 s) is called thermisation. In the case of Thermoquark, thermisation of the curd is necessary to ensure sufficient whey separation from the finer curd structure due to the co-precipitated whey proteins. The thermisation of Quark after separation greatly increases shelf-life stability, particularly by preventing off-flavour developments such as bitterness, stale or acidic. The production of lactic acid is stopped and the proteolytic activity contributed by rennet and starter bacterial enzymes is reduced (B~iurle et al., 1984; Zakrzewski et al., 1991; Sachdeva et al., 1993; Rogenhofer et al., 1994; Mara and Kelly, 1998). The firmness of the rather soft UF fresh cheeses can be increased by a heat treatment (75 ~ for 2-3 min) after acid coagulation and whey removal if the heat treatment is carried out in the presence of undenatured whey protein. Native whey proteins are retained in the UF retentate, but not in microfihration or separatorproduced Quark (Bodor et al., 1996). The firmness of kabneh is higher when UF is carried out at 50-55 ~ instead of 35 ~ (Tamime et al., 1991a,b). For Quark containing no stabilisers with up to 40%, w/w, FDM, the limits for thermisation are pH <4.2 and a heating temperature <60 ~ otherwise the protein coagulum
Thermisation of curd or fresh cheese
In the manufacture of Quark using a separator, cream is added to the concentrated curd in order to reduce fat losses in the whey during separation. In some separator types, cream dosing takes place in the separator immediately after the Quark has left the separator bowl. Fat standardisation in Thermoquark, which is less sensitive to shearing, is usually done by continuous online mixing of the separated curd and cream using a dosing pump. Other additives, such as fruit, herbs, etc., can be incorporated simultaneously (Spreer, 1998; Senge, 2002a). Homogeneous mixing of Quark and cream is difficult due to a density difference of---40 kg/m 3. Sufficient mixing is necessary to fully incorporate the cream; however, if mixing takes too long, the fragile Quark structure can be damaged. Batch mixing of separator-produced Quark is obtained by a combination of a vertical screw pump and a scraped surface agitator. The latter ensures that all products are transported to the screw pump and cavities are created into which the cream can flow to achieve localised mixing. Two phases are described during batch mixing, which takes approximately 15 min: a dilution phase and a structuring phase. During the first phase, all ingredients are mixed homogeneously, so that the chemical composition is the same throughout the tank. The yield value
Quark, Cream Cheese and Related Varieties
would become too firm and sandiness/grittiness would develop (Spreer, 1998). This problem occurs particularly in low-fat cheeses, i.e., less than 10%, w/w, FDM (Bodor et al., 1996). Stabilisers can be added during thermisation. Thermisation can be followed by hot filling or aseptic cold filling. Starch is sometimes used in low-fat systems to build body and replace fat in Quark-based desserts. Instead of adding cream to skim milk Quark, microparticulated whey proteins (5-7.5%, w/w) can be used to obtain a creamier texture in flavoured Quark preparations (Hoffmann, 1994; Hoffmann and Buchheim, 1994). The milk can also be fermented with ropy culture strains, which produce exopolysaccharides (e.g., Desachy and Parmantier, 1998; Sebastiani et al., 1998). The combined use of acidifying strains and texturising strains is necessary to prevent the density of the acid gel being too close to that of the whey which would impair the separation process. The gel strength decreases significantly with increasing amount of texturising cultures (Sebastiani et al., 1998). Varieties directly related to Quark
Tvorog and Tvarog (Eastern Europe), Topfen (Austria) and Baker's cheese (Germany, US) Tvorog/Tvarog/Twarog are Slavic translations for Quark. Topfen and Baker's cheese with 22-24%, w/w, DM also belong to this group (> 18%, w/w, DM). For these high DM cheeses small amounts of rennet are necessary for good whey separation. Fromage Frais (France and UK) Fromage frais is very similar to Quark, but has a lower DM content (--~14%, w/w). Because of the lower DM, Fromage frais is produced increasingly by a recombination method similar to yoghurt fortification. Rennet is used only in a few cases. Buttermilkquark Because of its high lecithin content, Buttermilkquark is used as a baking emulsifier and for nutritional reasons. It can be produced from unacidified (sweet cream) buttermilk in the same way as normal Quark (Spreer, 1998; Boone, 2001b). If produced from acidified buttermilk, the buttermilk is heat treated (65-70 ~ for 40 s), held at 45-50 ~ for 1-3 h under agitation, to facilitate deaeration and protein clotting, and then separated (Spreer, 1998; Boone, 2001a). Approximately 20%, w/w, of the fat is phospholipids of which ---30%, w/w, is lecithin (Boone, 2001a).
Labneh (or Labaneh, Leben), tan (or than) and tulum (Middle East and Balkan regions) Labneh, a concentrated yoghurt (>22%, w/w, DM), is produced from full-fat milk acidified with a yoghurt
319
culture (optionally coagulated with rennet) at 42-45 ~ for 3.5-4.0 h. It is then concentrated. Similar to Quark, the traditional method (cheesecloth bags for straining the cold yoghurt) has been replaced by mechanised processes (mechanical separation or ultrafihration of the warm yoghurt immediately after fermentation) (Tamime et al., 1989a,b, 1991a,b; Lehmann et al., 1991; Ozer et al., 1998, 1999). If a Quark separator is used, cream is added to the concentrate after separation. Rennet addition increases the throughput of a separator by ---30% (Lehmann et al., 1991). Salt and other additives (e.g., dried herbs) are blended in after separation. Labneh can be stored in olive oil for several months. Traditionally, cows', sheep's and goats' milks have been used for the manufacture of Labneh. Labneh made from cows' milk has a more uniform structure and greater firmness than that made from goats' or sheep's milk. Homogenisation of Labneh markedly reduces the firmness of products from goats' or sheep's milk. The firmness of cows' milk Labneh is less affected by homogenisation (Tamime et al., 1991b,c). The milk for goats' milk Labneh can also be fermented with a mesophilic starter culture at 22 ~ for 16-18 h (Mehaia and E1-Khadragy, 1999). With increasing rennet level, the yield of Labneh and its DM content increase gradually. However, above a certain rennet concentration (depending on the milk type), an undesirable cheesy flavour was detected (E1-Tahra et al., 1999). The effect of different salt levels on Labneh has been investigated by Ammar et al. (1999) and Mehaia and E1-Khadragy (1999). Increasing the UF temperature from 35 to 55 ~ doubles the firmness of Labneh and halves the processing time (Tamime etal., 1991a,b). Labneh produced by UF at 5 0 - 5 5 ~ is very firm, similar to the traditional product. The increase in firmness has been attributed to enhanced formation of casein chains, as observed by transmission electron microscopy, at a temperature above 45 ~ similar to what happens to yoghurt when heated after fermentation (Tamime et al., 1991a,b). Uhrafihration Labneh (50-55 ~ which is not homogenised is very granular and has a rough texture. Chakka and Shirkhand (India) Chakka is produced from buffaloes' milk and is very similar to Labneh. Mixed cultures of mesophilic and thermophilic starter bacteria are used. Shirkhand is produced by blending Chakka with cream, sugar and cardamom (Tamime and Robinson, 1988).
Skyr (Iceland) Skyr is a concentrated fermented milk product (thermophilic bacteria in combination with lactosefermenting yeasts, optionally rennet) having a composition very similar to that of skim milk Quark. The
320
Quark, Cream Cheese and Related Varieties
concentrated Skyr normally undergoes a further heat treatment, i.e., thermisation (Tamime and Robinson, 1988).
Ymer (Denmark), Lactoffi (Sweden) The most common flesh cheese in Denmark, Ymer (14.5%, w/w, DM, 5-6%, w/w, protein, 3.5%, w/w, fat) is made from skim milk fermented with a mesophilic starter culture. As with Quark, cream is added after separation (Tamime and Robinson, 1988; Kosikowski and Mistry, 1997). As the protein is concentrated only to 5-6%, w/w, Ymer and Lactofil can easily be produced by fermenting a sweet UF retentate. Swedish Lactofil is similar to Ymer, the only evident difference being a higher fat content. Rheological and syneretic aspects of Quark-type cheeses
Rheological and microscopical investigations have shown that fresh cheeses can be described as dispersions or pastes of hydrated acid casein gel particles in whey (Korolczuk, 1993; Tscheuschner and Nimbs, 1993; Ozer et al., 1998; Senge et al., 1998; Senge, 2002a). Scanning electron microscopy (SEM) shows that acid-type cheeses and stirred yoghurts have similar structures. They are composed of irregular protein particles of varying dimensions, which consist of open, loose networks of casein micelles aggregated in branched chains and coarse clusters (Allan-Woitas and Kalab, 1984; Ozer et al., 1999). Traditional set-type Labnehs show a continuous structure with the voids and protein matrix evenly distributed, whereas stirred-type Labnehs have a disrupted discontinuous structure with thicker casein clusters. Small thread-like structures are visible between strands in UF Labneh (Ozer et al., 1999). Ozer et al. (1998) described Labneh (DM 22.5%, w/w, 6.4-9.2%, w/w, protein, 6.1-9.2%, w/w, fat) as a weak visco-elastic gel with the storage modulus higher than the loss modulus over the amplitude range measured (0.015-0.150 mNm at 25 Hz). Fromagefrais, as produced in France (DM 15-20%, w/w, protein 7-12%, w/w, FDM 0-58%, w/w), has a very soft consistency and is regarded as a visco-elastic liquid rather than a solid (Korolczuk and Mahaut, 1989; Korolczuk, 1996). Applying a frequency sweep, Senge et al. (1998) described Quark (DM 18%, w/w, protein 12%, w/w, FDM 0-40%, w/w) as a material with distinct solid-like properties. Fresh acid-curd cheeses of the Fromage frais and Quark type show shear thinning behaviour. The effect of shearing time shows that the material is thixotropic. The cheeses exhibit plastic flow which can be described very well by the Bingham model (Korolczuk and Mahaut, 1989;
Mahaut and Korolczuk, 1992a; Korolczuk, 1993; Hawel and Heikal, 1994; Senge et al., 1998; Senge, 2002a). With increasing shear rate, the structure progressively breaks down and at a sufficiently high shear, viscous flow can be observed. The stress decrease during shearing can be explained by a decrease in the extent of aggregation of the protein particles. The aggregates of casein gels are also capable of assuming some structural reformation by flocculation. The thixotropic behaviour suggests that under shear there is a continuous process of destruction and restoration, which is a function of the shearing time and shear rate (Korolczuk and Mahaut, 1989; Korolczuk, 1993; Senge, 2002a). Senge (2002a) described the rheological behaviour of Separator and Thermoquark during various stages of manufacturing. Of the various standard rheological models, the Bingham (linear, 2-dimensional) and Herschel-Bulkley (non-linear, 3-dimensional) are the most suitable models for separator-produced Quark and Thermoquark, respectively (Table 7). The rheological parameters, yield value and effective viscosity, are more temperature dependent for Thermoquark than separatorproduced Quark in the temperature range studied (5-30 ~ These differences are explained by the different microstructures and are reflected in different sensory properties of these two Quark types (Senge, 2002a). Proteolysis and bitterness in Quark
Bitterness may develop in Quark for a number of reasons and also depends on the production method. Schkoda and Kessler (1997a) attribute bitterness in UF Quark primarily to the composition of the retentate (mainly calcium) and the enzymes of the starter culture and rennet. The pH-sensitive solubility of calcium is the main cause of bitterness in Quark made from ultrafiltered sweet milk (Jelen and Renz-Schauen, 1989). In both UF and non-UF Quarks, enzymes responsible for proteolysis are mainly from rennet (Sohal et al., 1988; Mara and Kelly, 1998), but also enzymes from lactic acid starter bacteria, and milk enzymes like plasmin and the acid proteinase, cathepsin D (Mara and Kelly, 1998; Hurley et al., 2000). Significantly higher proteolysis during storage was observed in Quark produced with rennet (Sohal et al., 1988; Zakrzewski et al., 1991; Mara and Kelly, 1998). Starter proteinases contribute little to primary proteolysis and bitterness (Sohal et al., 1988; Mara and Kelly, 1998), probably due to the fact that lactic acid bacteria are generally weakly proteolytic (Fox et al., 1996). In Thermoquark, starter numbers are also considerably reduced by the thermisation step (Mara and Kelly, 1998).
Quark, Cream Cheese and Related Varieties
321
Rheological parameters of Thermo- and Separatorquark (Rheometer M C I temperature 10 ~ Profile: (i) pre-shear 100 s - l , 60 s; (ii) rest 0 s -1, 60 s (ii) shear rate sweep 0.1-100 s -1, 60 s; (iv) holding shear rate 100 s -1, 60 s; (v) shear rate sweep 100-0.1 s - 1, 60 s (used for regression)
Mode/
Equation
Separatorquark
Thermoquark
.g.
..,-... el.f)
r = f(4/)in Pa
E'
ffJ
n ~ n v
"T" v
"i" v
a_
Z
v
g
~_ m
v
-> Bingham (BH)
r-
1"0 4- 1 7 B H 5,
115
3.39
1.0
0.974
21.06
135
2.37
1.0
0.998
81
1.35
0.5
0.998
6.13
102
0.73
0.5
0.984
83
17.87
0.64
0.999
3.57
131
3.12
0.94
0.998
4.59
linear plastic, 2-dimensional Casson (CA)
Herschel-Bulkley
(HB)
r = ",Jroro+ ~/r/CA + 5'0 non-linear plastic, 2-dimensional r = 1"0 + K 5'n non-linear plastic, 3-dimensional
r, shear stress (Pa); r0, yield value (Pa) 5', shear rate s-l" r/, viscosity (Pa s)" K, consistency factor (Pa sion coefficient; s, standard deviation of shear stress (Pa) (Data from Senge, 2002a).
Cream cheese is produced from standardised (Double Cream Cheese (DCC), 8-12%, w/w, fat; Single Cream Cheese (SCC) 3.0-5.0%, w/w, fat), homogenised, pasteurised (72-75 ~ for 15-90s) milk or cream. Homogenisation is important for the following reasons: (i) it reduces fat loss on subsequent whey separation; (ii) it brings about, via coating of fat with casein and whey protein, the conversion of naturally emulsified fat globules into pseudo-protein particles which participate in gel formation on subsequent acidification. The incorporation of fat by this means into the gel structure gives a smoother and firmer curd (similar to yoghurt manufacture) and therefore is especially important for the quality of cold-pack Cream cheese for which the curd is not further treated (Guinee et al., 1993). Following pasteurisation, the milk is cooled (20-30 ~ inoculated at a level of 0.8-1.2%, with a D-type starter culture (Lactococcus lactis subsp, lactis, Lc. lactis subsp, cremoris and citrate-positive Lc. lactis subsp, lactis) and held at this temperature until the desired pH of 4.5-4.8 is reached. The resulting gel is agitated gently, optionally cooled to 10-12 ~ in order to prevent over-acidification, heated (80 ~ for up to 20 min) and deaerated. The curd is then concentrated by methods similar to those used for Quark, i.e., traditional method using bags, separator methods or UF methods. The acidified high-fat curd for DCC is obtained by centrifugal separ-
ation at 70-85 ~ 1996b).
12.7
5.11
sn); n, flow index; r, regres-
or UF at 50-55 ~
(Sanchez et al.,
Whey separation using separators
For separator-produced SCC, milk is standardised to 3.0-5.0%, w/w, fat. The specific weight of the cheese mass is greater than that of the whey and is separated outwards in the centrifuge during separation. The whey contains 0.2-0.5%, w/w, fat which can be reduced subsequently to ~-0.1%, w/w, by separation in a milk separator designed for this purpose (Lehmann et al., 1991). For separator-produced DCC, the milk is standardised to 8-12%, w/w, fat, giving a fat-protein mixture which has a lower specific density than that of the whey. At a fat content of 7%, w/w, the specific weights are too close to be separated by centrifugation (Dolle, 1991; Lehmann et al., 1991; Spreer, 1998). Whey separation using UF
Owing to the thick, viscous consistency of Cream cheese, concentration by UF necessitates a two-stage process (stage one: standard modules with centrifugal/positive displacement pumps; stage two: high-flow modules with positive displacement pumps) in order to maintain satisfactory flux rates and to obtain the correct DM level (Guinee etal., 1993). For fresh cheese, UF is normally carried out around 40-45 ~ for Cream cheese, 50-55 ~ can be used to improve throughput and reduce viscosity during concentration (Ottosen, 1996).
322
Quark, Cream Cheese and Related Varieties
Recombination technology
Recombination methods for experimental Cream cheesetype products include steps of combining a cheese base (e.g., dry Cottage cheese, Bakers cheese curd or fermented skim milk concentrate at pH 4.8-5.0) with emulsifying salts, bulking agents (e.g., buttermilk powder, corn syrup solids) and various gums (e.g., carrageenan or guar gum), followed by various heating, mixing and homogenisation steps (e.g., Baker, 198i; Crane, 1992). The advantage of these methods is that Cream cheese products can be formulated precisely to meet legal requirements without excess solids or butterfat. Another type of all-dairy Cream-type cheese is made by blending Ricotta cheese or Queso Blanco with a high-fat (58%, w/w) sour cream. The most critical aspect is proper dispersion of the large pieces of curd with the liquid components. The blend is standardised with cultured buttermilk, if necessary, pasteurised, homogenised and hot-packed. The final product has ---59%, w/w, moisture, 30%, w/w, fat and a pH of 5.29-5.55, depending on the cheese base used (Modler et al., 1985; Kakib and Modler, 1985a,b). Further treatments of curd after separation
In most cases, the curds are heat-treated (70-95 ~ mixed with salt (0.5-2.0%, w/w) and stabilisers (mainly hydrocolloids), homogenised (two-stage at 15-25 MPa at 65-85 ~ and either hot-packed or cold-packed after cooling to 10-20 ~ in a scraped-surface heat exchanger (Walenta etal., 1988; Sanchez etal., 1996b). Locust bean gum (0.30-0.35%, w/w), carrageenan (0.15%, w/w), xanthan gum, tara gum and sodium alginate are the most widely used stabilisers for hot-pack Cream cheese (Hunt and Maynes, 1997; Kosikowski and Mistry, 1997). Guar gum on its own gives a high processing viscosity, a soft body and undesirable texture. A synergistic effect of n-carrageenan on the gelling properties of tara gum was observed in Cream cheese (Hunt and Maynes, 1997). The extent of 'creaming' (emulsification and thickening) is influenced by the degree of heat and shear and the duration of cooking and has a major influence on the consistency of the final product. Increasing the holding time and shear during cooking generally results in a firmer product with an increasingly brittle texture (Walenta et al., 1988; Guinee et al., 1993). Static cooling of hot-pack cheeses gives a firmer texture than dynamic cooling (cold-pack cheeses; Jaupert and Vesperini, 1989; Mahaut, 1990; Sanchez et al., 1994b). The cheese firmness is already reduced by lowering the filling temperature from 85 to 75 ~ (Walenta et al., 1988). Cold-pack Cream cheese has a somewhat spongy, aerated consistency and a coarse appearance (Guinee et al., 1993).
The main particles structuring DCC, i.e., milk fat globules and milk proteins, undergo several thermal treatments, and therefore large temperature fluctuations and shear stresses during processing. Such technological treatments change the structure of particles (size, shape, state of aggregation) and physico-chemical properties (charge density and hydration of milk proteins, solid/liquid milk fat ratio and milk fat globule stability). The resulting micro- and macro-structural arrangements of particles, as well as the nature of interactions, mainly determine the texture and stability of DCC (Sanchez et al., 1996b). Jaupert and Vesperini (1989), Mahaut (1990), Sanchez et al. (1994a,b, 1996a,b,c), Sanchez and Hardy (1997) investigated the effects of processing parameters on the structure and stability of DCC. Cream cheese becomes firmer and more elastic after heating and homogenisation, and softer and more viscous after mixing and cooling. TEM and SEM show that the rheological changes during manufacture are correlated with aggregation (during heating and homogenisation) and disruption (during cooling) of milk fat globule/casein complexes. Dispersion of the fat globule clusters, formed on homogenisation, after cooling and aggregation of milk fat globules during storage causes structural instability to occur in Cream cheese. The following stages for structuring and destructuring processes occurred during the manufacture of experimental DCC (Sanchez et al., 1996c; Sanchez and Hardy, 1997): 9 Starting curd after centrifugal separation: Casein-fat
globule aggregates are first produced during curd formation. 9 Blending with water, salt and heat-denatured whey proteins: The casein-fat globule aggregates are destroyed
during blending of curd with ingredients. Fat globule destabilisation (e.g., coalescence) occurs during this stage. 9 Heat treatment: The broken aggregates reaggregate on heat treatment, but with extensive fat globule aggregation and coalescence. Leakage of oil and creaming can occur at the outlet of the heat exchanger. 9 Homogenisation: Emulsification of the system occurs during high-pressure homogenisation with strong clustering of fat globules, caused by a 'polymerbridging' mechanism. When homogenisation pressure is increased from 0 to 50 MPa, the firmness of DCC increases due to a stronger structural organisation within the cheese (Sanchez et al., 1994a). 9 Cooling: Homogenisation clusters are destroyed by high shear stresses developed in the heat exchanger during cooling to 20 ~ This effect is even more pronounced w h e n the final curd temperature is
Quark, Cream Cheese and Related Varieties
below 20 ~ (Sanchez et al., 1994b). The cooling rate and final temperature are the main factors for instability. An increase in curd cooling rate leads to a softer cheese with weaker structural organisation (reduced storage and loss moduli). Rheological and syneretic aspects of Cream cheese
TEM and cryo-SEM studies showed that DCC is structured mainly by compact casein/milk fat globule aggregates occluding large whey-containing areas, as well as partly coalesced milk fat globules. No rigid protein matrix was observed because of stirring and homogenisation of the curd during manufacture (Kahib et al., 1981; Kal~ib and Modler, 1985a,b; Sanchez et al., 1996b). The corpuscular structure seems to be responsible for good spreadability with a high moisture content facilitating the mobility of the corpuscular constituents during spreading (Kahib and Modler, 1985a,b). Double Cream cheese exhibits viscoelastic-plastic rheological behaviour with an unusual flow curve; the shear stress in the ascending shear rate curve shows, depending on the manufacturing stages, two or three peaks. The first peak is commonly referred to as the static yield value (Sanchez et al., 1994b, 1996c). The complex rheological behaviour of DCC indicates a 3-dimensional gel-like structure (Sanchez et al., 1994a). Double Cream cheese shows timedependent flow behaviour (partially thixotropic) and dynamic viscoelastic properties similar to those of non-chemically cross-linked polymers or pharmaceutical creams (Sanchez et al., 1994a,b, 1996c). Senge (2002b) also measured various cream cheese types in the oscillation and rotation mode.
Fresh cheese preparations (Frischk~isezubereitungen) are blends of Quark, Cream cheese or Cottage cheese with cream and up to 30%, w/w, fruit or vegetable preparations or up to 15%, w/w, of fruits, spices, herbs or other seasoning (KOseverordnung, Cheese order; Anon, 1986). A foamy consistency can be obtained by admixing nitrogen. Fresh cheese preparations can be heat-treated to increase shelf-life and may contain stabilisers. The components can be blended by either continuous in-line mixing or batch mixing (Lehmann et al., 1991; Kosikowski and Mistry, 1997; Spreer, 1998).
Acid-curd cheeses (Sauermilchkase), typical examples of which are Harzer, Mainzer or Olmatzer Quargel cheese, have a very strong flavour and odour, a white
323
to slightly yellow colour and a slightly brittle texture (Spreer, 1998). There are mould-ripened cheeses and yellow cheeses which have been treated with a 'smear' of red culture (Brevibacterium linens). Ripened acidcurd cheeses are generally produced in specialised plants which buy the acid Quark from dairies. The acid Quark used for these cheeses is produced by acid coagulation (cold: 1-2% mesophilic starter at 22-27 ~ in 15-20 h or warm: 2-5%, thermophilic culture at 40-45 ~ in 1.5-3.0 h). The coagulum is cut and cooked at 35-45 ~ while stirring until the granules are 2-4 cm in size. Whey is drained using filter bags or decanters. Acid Quark has a DM content >32%, w/w (pH --~4.6) and is granulated in a Quark mill and chilled below 10 ~ (Kessler, 1996; Bruckert, 1998; Spreer, 1998). In the manufacture of the cheese, 2-3%, w/w, of salt and caraway seeds are added, as well as NaHCO3 and CaCO3 (0.5-1.0%, w/w) to influence ripening (acceleration, neutralisation). The mass is mixed, milled, moulded into bars and spread on racks and then subjected to the following processes: drying at 18 ~ for 15 h; sweating at 20-25 ~ and 90% relative humidity for 2-3 days, ripening at 12-16 ~ and 90% relative humidity for 3 days and further ripening at 10-15 ~ After sweating, the cheeses are washed with salt water plus a culture of B. linens or sprayed with a culture consisting of Penicillium candidum and P. camemberti. The cheese ripens from the outside to the centre and is ready for distribution when up to 25% of the cheese mass is ripened, i.e., when the outside has a translucent, yellow appearance, even though the interior (75% of the cheese mass) is still white, dry, hard and crumbly and unripened. Cooked cheese (Kochk~ise, Topfk~ise), also manufactured from Quark, is a different type of cheese, usually with 20%, w/w, FDM. During ripening at 18-22 ~ for 3-6 days, the surface turns greasy and the pH increases to 5.5-6.0. Water, butterfat, salt, NaHCO3 and CaCO3 and caraway seeds are added to the cheese blend which is heated in a jacketed vessel at 70-110 ~ for 10-20 min. The hot cheese is filled into cups or pots (Kessler, 1996; Spreer, 1998).
Layered cheese (Schichtkiise)
German Layered cheese has a firm coagulated (gel-like) consistency with a middle layer which is higher in fat and therefore darker in colour. One-third of the cheese milk can be standardised to a higher fat level and/or colouring may be added. Standardisation of fat content must be carried out in the cheese milk, as it cannot be done in the drained curd without destroying the firm
324
Quark, Cream Cheese and Related Varieties
coagulated structure. Layered cheese is classified as a quarter-fat cheese (10%, w/w, FDM, Table 2), even though the middle layer has a higher fat content. The fermentation temperature is about 25 ~ the amount of rennet added is slightly higher than for Quark in order to shorten the gelation time by 3-4 h. The strong gel is cut into cubes (approximately 2 • 2 cm) and the curd then transferred into moulds starting with a lowfat white layer, followed by the yellow high-fat layer and finished with a low-fat white layer (Spreer, 1998).
Mascarpone Mascarpone, a high-fat ('--50%, w/w, fat) firm, spreadable fresh cheese with a mild buttery, slightly tangy flavour, is produced by direct acidification with an organic acid, instead of lactic acid bacteria, to a pH of about 5.0-5.8. Cream with 30%, w/w, fat is heated to 80-95 ~ organic acid added slowly and stirred for 10 min. Whey drainage takes about 16-24 h. To increase shelf-life, Mascarpone is usually heat-treated. No stabilisers are necessary due to the high fat content (Kessler, 1996).
The authors wish to thank Tim Guinee (Dairy Products Research Centre, Fermoy, Ireland) and Liam Gallagher (Dairygold Cooperative Society, Mitchelstown, Ireland) for useful comments on the manuscript.
Allan-Woitas, P. and Kal~ib, M. (1984). A simple procedure for the preparation of stirred yoghurt for scanning electron microscopy. Food Microstruct. 3, 197-198. Ammar, E.T.M.A., E1-Shazly, A.A., Nasr, M.M. and Omar, I.M.I. (1999). Comparative study of recombined Labneh, with buffalo and cow milk Labneh. II. Effect of different levels of rennet. Egypt. J. Dairy Sci. 27, 301-315. Anon (1984). Quark from uhrafiltration concentrates. Dtsch. Milchwirtschaft 35, 1000, 1002-1004. Anon (1986). German Cheese order (Kaseverordnung), 14 April 1986 BGB1. I S.412, in, Lebensmittelrecht, Vol. 1, Zipfel, W. and Rathke, K.-D. (2001). Verlag C.H. Beck, Munich. Anon (1994a). Consumption statistics for milk and milk products 1992. Bulletin 295. International Dairy Federation, Brussels. pp. 1-6. Anon (1994b). Dutch cheese order (Warenwet Decree on Dairy Products), 25 October 1994, Art. 13. Attia, H., Bennasar, M., Lagaude, A., Hugodot, B., Rouviere, J. and Tarodo de la Fuente, B. (1993). Ultrafiltration with a microfiltration membrane of acid skimmed and fatenriched milk coagula: hydrodynamic, microscopic and rheological approaches. J. Dairy Res. 60, 161-174.
Baker, D.B. (1981). Preparation of low fat imitation cream cheese. Pro Mark Companies, assignee. US Pat. No. 4, 244,983. Banon, S. and Hardy, J. (1992). A colloidal approach of milk acidification by glucono-delta-lactone. J. Dairy Sci. 75, 935-941. B~turle, H.W., Walenta, W. and Kessler, H.G. (1984). Herstellung von Magerquark mit Hilfe der Ultrafiltration. DMZ Deutsche Molkereizeitung 105,356-363. Bishop, J.R., Bodine, A.B. and Janzen, J.J. (1983). Electron microscopic comparison of curd microstructures of Cottage cheese coagulated with and without microbial rennin. Cult. Dairy Prod. J. 8 (1), 14-16. Bodor, J., Koning, M.M., Schmidt, M. and Stratmann, W. (1996). Process for preparing fresh cheese and fresh cheese obtainable thereby. Unilever NV, assignee. WO Pat. No. 9,637,114. Boone, M. (2001a). Cheese composition of the Quark type, and method for preparing fresh low-fat cheese. Marc Boone NV, assignee. WO Pat. No. 0,115,541. Boone, M. (2001b). New composition and method for preparing basic Quark and further processing of the basic Quark. Mark Boone NV, assignee. WO Pat. No. 0,178,518. Bruckert, W. (1998). Sauermilchk~se. Dtsch. Milchwirtschaft 49,493-494. Crane, L.A. (1992). Method of manufacture of a non-fat cream cheese product. Gen Foods, Inc., assignee. US Pat. No. 5,079,027. Creamer, L.K. (1985). Water absorption by renneted casein micelles. Milchwissenschaft 40, 589-591. Dalgleish, D.G. (1992). The enzymatic coagulation of milk, in, Advanced Dairy Chemistry - 1. Proteins, Fox, P.E, ed., Elsevier Applied Science Publishers, London. pp. 157-187. Dalgleish, D.G. and Law, A.J.R. (1989). pH-induced dissociation of bovine casein micelles, II. Mineral solubilization and its relation to casein release. J. Dairy Res. 56, 727-735. Dalgleish, D.G. and Horne, D.S. (1991). Studies of gelation of acidified and renneted milks using diffusing wave spectroscopy. Milchwissenschaft 46, 417-422. Desachy, P. and Parmantier, C. (1998). Fresh cheese. Nestl~ SA, assignee. WO Pat. No. 9,827,825. Dieu, B., Cuq, J., Tarodo de la Fuente, B., Bennesar, M. and Desroches, J.M. (1990). Method for producing cheese by means of microfiltration. Valmont SA, assignee. WO Pat. No. 8,804,141. Dolle, E. (1977). Technik des Therm~ Dtsch. Milchwirtschaft 22,709-712. Dolle, E. (1981). Erkenntnisse tiber das Westfalia-ThermoSpeisequark-Verfahren. Molkereizeitung-Welt der Milch 35,628-629,632-633. Dolle, E. (1991). Developments in Quarg processing. Dairy Ind. Int. 56 (9), 27-29. E1-Tahra, M.A.A., E1-Shazly, A.A., Nasr, M.M. and Omar, I.M.I. (1999). Comparative study on recombined Labneh, with buffalo and cow milk labneh. I. Effect of salt level on consumer acceptability. Egypt. J. Dairy Sci. 27, 127-139. Fem~indez-Albalat, M.P., Fernandez, M.A., Mendez, J. and Cobos,/it. (2001). Studies on the application of ultrafiltration for the manufacture of Cebreiro cheese. Milchwissenschaft 56, 392-394.
Quark, Cream Cheese and Related Varieties
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Acid- and Acid/Rennet-curd Cheeses Part B: Cottage Cheese N.Y. Farkye, Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, CA 93407
Cottage cheese is a soft, unripened, mild acid cheese with discrete curd particles of relatively uniform size. Creamed Cottage cheese is dry-curd Cottage cheese covered with a cream dressing. The specific origin of Cottage cheese is unknown. However, as the name implies, it was produced originally in homes (cottages) but industrial Cottage cheese production began in the USA in --1916 (Reidy and Hedrick, 1970). Cottage cheese is classified into different groups, subgroups, types, classes and styles (Table 1).
By definition, Cottage cheese and dry-curd Cottage cheese shall comply with the US Food and Drug Administration's Standards of Identity 21 CFR Part 122.128 for Cottage cheese or 21 CFR Part 133.129 for dry-curd Cottage cheese (see Table 2). Reducedfat, light and fat-free Cottage cheese or dry-curd Cottage cheese shall comply with 21 CFR Part 101.62 for nutrient claims for fat. Codex Alimentarius official standard (Codex Stan C-16) for Cottage cheese and creamed Cottage cheese (Codex Alimentarius, 1968) lists the raw material for manufacture as pasteurized bovine skim milk, and the following authorized ingredients: harmless lactic acid and aroma-producing bacteria, rennet or other suitable coagulating agent, CaC12 (maximum of 200mg/kg milk), NaC1 and water. Dairy ingredients allowed in cream dressing (creaming mixture) are: cream, skim milk, condensed milk, non-fat dry milk and dry milk protein. Other permitted ingredients in the cream dressing are: harmless lactic acid- or aroma-producing bacteria, chymosin or other suitable milk-clotting enzyme, NaC1, lactic acid, citric acid, phosphoric acid, hydrochloric acid, glucono-8-1actone (maximum level, 10 g/kg), sodium caseinate, ammonium caseinate, calcium caseinate, potassium caseinate. In addition, the following stabilizing agents are permitted: carob bean gum, guar gum, calcium sulphate, carrageenan or its salts, furcelleran or its salts, gelatine, lecithin, alginic acid or its salt, propylene glycol ester of alginic acid, sodium
carboxymethyl cellulose. Permitted carriers for stabilizers are sugar, dextrose, corn syrup solids, dextrine, glycerine and 1,2-propylene glycol. The limitations for ingredient use are as follows: (1) the weight of solids (including caseinates) added singly or in combination should not exceed 3% (w/w) of the cream dressing mixture and (2) the stabilizing solids, including carrier shall not exceed 0.5% (w/w) of creaming mixture.
Cottage cheese is produced by acid coagulation of pasteurized skimmilk or reconstituted extra low-heat skimmilk powder (RSM). The minimum heat treatment given to skimmilk or RSM for Cottage cheese manufacture is the minimum allowable pasteurization temperature • time of 62.8 ~ • 30 min or 71.7 ~ • 15 s. In a survey of seven Cottage cheese plants in California, Rosenberg et al. (1994) found that the average milk pasteurization temperature used for manufacture is 74-75 ~ Excessive heat treatment of milk (i.e., higher pasteurization temperature and/or longer time) results in a soft coagulum from which it is difficult to expel whey. The skimmilk or RSM used for Cottage cheese manufacture must be of good microbial quality and have a high dry matter (DM) content to ensure good quality and yield of cheese. The differences in DM content of milk from different breeds influence the yield and quality of Cottage cheese. Cottage cheese curds made from Friesian skimmilk (8.7% DM) are more fragile than curds made from Jersey skimmilk (9.8% DM) (Mutzelburg et al., 1982). According to Mutzelburg etal. (1982), when the DM content of Friesian skimmilk was increased to at least 9%, by the addition of Na citrate (0.1%, w/w) or Na caseinate (0.25-0.55%, w/w), curd formation improved during Cottage cheese manufacture. Reconstituted extra low-heat skimmilk powder can be used for Cottage cheese manufacture immediately after reconstitution without holding (Flanagan et al., 1978; White and Ryan, 1983). Increasing DM (8-20%) in RSM increases the moisture-adjusted (80%) Cottage cheese yield by 17.1-31.2%. However, using RSM containing >10.5% DM is not economical because the
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
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Cottage C h e e s e
Groups, types, classes and styles of Cottage cheese
Groups A B Sub-groups 1 2 Types I II III Classes 1 2 Style A B
Culture acidified Chemically acidified Normal shelf-life (14 days and over) Extended shelf-life (21 days and over) Dry-curd Cottage cheese Low-fat Cottage cheese Cottage cheese Unflavoured Flavoured (with nuts, fruit condiments) Small curd (0.635 cm) Large curd (0.953-1.27 cm)
additional yield advantage is offset by the extra cost of ingredients (White and Ryan, 1983), suggesting that 10.5% DM in skimmilk or RSM is optimal for Cottage cheesemaking. Also, cheese manufacturing time is increased when skimmilk containing >10.5% DM content is used because of its high buffering capacity. Emmons and Beckett (1984a) reported that it takes longer than 75 min (normal cooking time) to reduce the pH of skimmilk with a high DM content from 6.6 to 4.8 or lower when conventional bulk starter is used at a level of 5% (w/w). There are conflicting reports on the use of lactosehydrolysed skimmilk for Cottage cheese manufacture. Gyuricsek and Thompson (1976) reported that when >90% of the lactose in skimmilk is hydrolysed before Cottage cheesemaking, the manufacturing time is reduced by 135min because the starter bacteria ferment glucose better than lactose. The shortened manufacturing time results in reduced curd shattering and consequently increase in yields. However, Fedrick and Houliman (1981) found that the use of lactosehydrolysed skimmilk did not affect setting time, yield or quality of Cottage cheese.
The mode of setting (incubation) or acidifying milk for Cottage cheese manufacture depends on whether cultured or direct-acid Cottage cheese is being made. For cultured Cottage cheese, acidification is done by harmTable 2
Standards of Identity for Cottage cheese variants
Cheese type
Fat (%)
Moisture(%)
pH
Dry-curd Cottage cheese Cottage cheese
<0.5 <4.0
<80 <80
<5.2 <5.2
less mesophilic lactic acid bacteria while for direct-set (or direct acid) Cottage cheese, acidification is done using a combination of organic acid, mineral acid or acidogen. Cultured Cottage cheese may be manufactured by three methods that are based on the length of time it takes from the addition of starter to cutting the coagulum. The three methods are designated short-, long- or intermediate-set methods (Emmons, 1963a,b; Emmons and Tuckey, 1967; Kosikowski, i982; Scott, 1986). The temperature • time combinations for the method are given in Table 3. Regardless of the method used, the main principle behind Cottage cheesemaking is the coagulation of caseins at, or near, their isoelectric pH (4.6) and cooking of the curds obtained at a similar pH value. Also, the choice of method depends on the schedule of plant personnel. It is important to ensure that the incubation temperature remains constant and uniform throughout the entire mass for proper and uniform acid development. Sandine (1975) suggests that in the case of the long-set method, if the room temperature is above 22 ~ the incubation temperature should be 21 ~ and if room temperature is less than 21 ~ then the incubation temperature should be 22 ~ As a precautionary measure, and to ensure proper acid development, Reidy and Hedrick (1971) suggested that the titratable acidity of the skimmilk after incubation for 1.5 h should increase by 0.05--0.06% for the short-set method, and by 0.04-0.05% after 3.5 h for the long-set method. Another starter may be added if less acid has developed at that time.
Strains of Lactococcus lactis subsp, lactis or subsp. cremoris, which are least susceptible to agglutination, are used as cultures for acid production during Cottage cheese manufacture. The culture may be an active bulk culture or a frozen concentrated culture. The level of bulk culture used is usually in the range of 1-8%, depending on the method used for manufacture (Table 3), although 0.25-1.0% for the long-set method has also been recommended (Watrous, 1997) to prevent over acidification. Agglutination of starter bacteria in Cottage cheese vats has been reviewed (Salih and Sandine, 1980). When starter bacteria agglutinate, they form clumps and settle to the bottom of the vat during manufacture (Emmons et al., 1966). This results in localized production of lactic acid to give a pH difference of about 0.5 between skimmilk at the bottom and the top of the vat after approximately 4 h of incubation (Salih and Sandine, 1984; Milton et al., 1990). Consequently, precipitation of caseins occurs and sludge is formed at the bottom of the vat.
Cottage Cheese
331
Typical parameters used for short-, intermediate- and long-set methods for Cottage cheese
Method
Incubation temperature (~
Setting time (h)
Starter level (%)
Single strength calf rennet level (ml/454 kg skimmilk)
Short Intermediate Long
30-32.2 28.3 21.1-22.2
4-6 8-9 12-16
5-8 2-2.5 1-2
1.0-1.5 1.0-1.5 0.5-1.0
Brooker (1986) showed that starter agglutination causes minor sludge formation while major sludge, which results in total loss of the vat contents, is caused by slow acid production due to the destruction of starter bacteria by bacteriophage. This view is supported by electron micrographs of samples taken from the bottom of failed vats that show compacted casein micelles, starter bacteria at various stages of lysis and bacteriophage (Brooker, 1986). Minor sludge formation results in yield losses of 4-8% (Grandison et al., 1986). Agglutination can be prevented by homogenizing the skimmilk at 15.2 MPa (Emmons and Elliot, 1967) or bulk culture at 17.2 MPa (Milton et al., 1990) or the addition of de-oiled lecithin (0.5%) to the bulk culture (Milton et al., 1990). Homogenization of skimmilk destroys milk agglutinins while homogenization or addition of lecithin to the starter culture causes fragmentation of starter chains without affecting starter cell density or acid production. Low CO2-producing strains of citrate-positive Lc. lactis subsp, lactis (formerly Lactococcus lactis subsp. lactis biovar, diacetylactis) or Leuconostoc spp. are added for the production of diacetyl, which is important for flavour. However, most of the diacetyl produced (<3.2 mg/kg) is lost in the whey at cutting (Mather and Babel, 1959). Therefore, some processors prefer to add diacetyl to the creaming mixture or culture the creaming mixture with diacetyl-producing organisms (Gilliland, 1972). The production of diacetyl and other flavour compounds in Cottage cheese is discussed later in this review. The use of strains that produce excessive amounts of CO2 (through citrate metabolism) causes the curds to float during cooking. Also, the production of CO2 leads to erroneous determination of titratable acidity of the whey due to the formation of H2CO3 but this can be corrected by boiling whey before measuring its acidity (Emmons and Beckett, 1984b).
The use of food-grade acids or acidogen instead of starter bacteria is an alternate method for manufacturing Cottage cheese. The method of acidification is
based on patents issued to Hammond and Deane (1961), Corbin (1971) and Loter et al. (1975). Essentially, direct acidification is a two-step process in which a less-expensive acid (e.g., lactic or phosphoric acid) is added to cold (2-12 ~ skimmilk to give a pH of approximately 5.2. The cold skimmilk is warmed slowly to 32 ~ Then, glucono-8-1actone (GDL) is added to the warm skimmilk to slowly reduce the pH to about 4.6 in an hour. In solution, GDL hydrolyses slowly to gluconic acid, thereby reducing the pH of the skimmilk (Deane and Hammond, 1960). Skimmilk at pH 5.2 and 26.7 ~ requires between 4.8 and 5.4 g GDL per liter to reduce its pH to 4.7 in an hour (Satterness et al., 1978). The slow acidification of milk by GDL causes casein micelles to aggregate into a network with fewer links compared to clustered casein micelles observed (by electron microscopy) during rapid acidification with HC1 or lactic acid (Harwalkar and Kalab, 1980, 1981). During acidification of milk, casein micelle coagulation does not start until pH 5.1 (Bringe and Kinsella, 1990) when virtually all the colloidal calcium phosphate is solubilized (Gastaldi et al., 1996). The obvious advantage of direct acidification is efficiency of cheesemaking and the elimination of possible problems associated with starter performance in cheese vats (i.e., agglutination, bacteriophage and antibiotics (Sharma et al., 1980)). The total processing time from raw skimmilk to the end of washing during continuous Cottage cheese manufacture by direct acidification with HC1 is about 35 min (Ernstrom and Kale, 1975). However, Cottage cheese made by direct acidification using a continuous process has poor texture (Ernstrom and Kale, 1975), while direct-set Cottage cheese made by conventional methods usually has good texture (Geilman, 1981). Pre-culturing milk to pH 5.5 prior to direct acidification significantly improves the texture and body of the finished cheese (Ernstrom and Kale, 1975). A method for making Cottage cheese using a combination of starter bacteria and direct acidification has been patented (Reddy et al., 1990). Other patented methods for Cottage cheesemaking by direct acidification use HC1 (Ernstrom, 1963), an aliphatic dione (C2mC8 glyoxal) or H202 (Metz, 1980) as acidifying agents.
332
Cottage Cheese
A very low level of milk-clotting enzyme (e.g., chymosin) is required for Cottage cheese manufacture. Suggested levels of single-strength rennet (chymosin) used for short-, intermediate- or long-set cultured Cottage cheesemaking are given in Table 3. The milk-clotting enzyme is added to skimmilk within 1 h of starter addition for cultured Cottage cheese. Some manufacturers prefer to add the milk-clotting enzyme along with the starter. Because the level of chymosin added is small, adequate dilution and stirring is necessary to ensure proper distribution in the skimmilk. After the milk-clotting enzyme is stirred in, the skimmilk is left quiescent for gel formation. The pH of coagulation increases with the amount of rennet used. For directset Cottage cheese, rennet is added simultaneously with GDL. Emmons et al. (1959) recommended using 0.2 ml single strength rennet/454 kg skimmilk for cultured Cottage cheese manufacture in order to achieve a satisfactory coagulum ready to cut at pH 4.8. For direct-acid Cottage cheese, more rennet is necess a r y - about 2.9 ml single strength/454 kg skimmilk, and attempts to manufacture direct-acid Cottage cheese without rennet have been unsuccessful because the coagulum is very weak and the curds shatter extensively on agitation. This is consistent with findings by Kim and Kinsella (1989) who reported that acid-induced gelation with GDL did not occur for 3 h at 35 ~ Bishop et al. (1983) observed that the average size of casein micelles in cultured Cottage cheese curd made with microbial rennet is twice as large as that made without rennet, indicating that rennet promotes aggregation of casein micelles and shortens coagulation time. The United States standards permit the addition of <0.02% CaC12 to skimmilk to improve curd firmness at cutting during Cottage cheesemaking. However, Emmons et al. (1959) reported that the addition of CaC12 has no effect on curd strength or the quality of Cottage cheese, indicating that CaC12 does not play a significant role in the coagulation of casein micelles during Cottage cheesemaking because at pH values <5, most if not all, of the colloidal calcium phosphate in milk is dissolved from the casein micelles (Pyne and McGann, 1960) and the caseins are liberated into the serum phase (Roefs et al., 1985).
(Tuckey, 1964; Emmons and Beckett, 1984a). The size of wire knives used to cut the coagulum determines the size of Cottage cheese curd. Coagulum cut with 0.95 cm or larger wire knives produces large-curd Cottage cheese, while cutting with 0.64 cm knives gives small-curd Cottage cheese. At a cooking temperature in the range 46-60 ~ the firmness and cohesiveness of dry-curd Cottage cheese and its DM content increase with the pH at cutting in the range 4.6-4.9 (Perry and Carroad, 1980; Emmons and Beckett, 1984a). However, when the coagulum is cut at a high pH (e.g., 4.9), the curds have a tendency to matt during cooking (Emmons and Beckett, 1984a). Cutting at a pH lower than 4.6 results in curds that are soft and mushy, and that shatter during cooking. A soft coagulum (Emmons et al., 1959) that holds a high level of moisture after cutting and cooking (White and Ray, 1977) is obtained when heat treatment of milk prior to cheesemaking is more severe than normal pasteurization (72 ~ • 15 s). To obtain a firm coagulum that is easy to cut from milk that has been severely heated (e.g., 80 ~ • 30 min), more single strength rennet ( 1 5 - 2 0 m l / 4 5 4 k g ) is used (Emmons etal., 1959). Consequently, the pH at cutting is high (5.1-5.2) and the quality of the cheese is reduced (Emmons et al., 1959; Durrant et al., 1961). Some cheesemakers use time to indicate when the coagulum is ready to cut. However, this is not reliable. Other than pH measurement, titratable acidity measurement on the whey may be used. This is done by straining or centrifuging curd to obtain clear whey and titrating it with 0.1 N NaOH. The optimum titratable acidity at cutting ranges from 0.42 to 0.60% lactic acid or higher, depending on the DM content of the skimmilk (Table 4). Alternately, the A-C (acid coagulation) test (Emmons and Tuckey, 1967) is used to determine when the coagulum is ready for cutting. In the A-C test, a well-mixed sample of milk is taken from the vat after starter addition but before rennet or coagulant is added. The sample, contained in a stainless steel cup, is suspended in the vat so that it maintains the same Effect of dry matter in skimmilk on titratable acidity at cutting during Cottage cheese manufacture
Dry matter content of skimmilk (%)
7.8 8.7 The pH at which the coagulum is cut is perhaps the most critical step in Cottage cheese manufacture. The desired cutting and cooking pH during Cottage cheese manufacture are 4.75-4.8 and 4.55-4.6, respectively
9.6
10.5 12.4
14.3
Titratable acidity at cutting (%) 0.44 0.50 0.55 0.62 0.74 0.86
Cottage Cheese
temperature as the cultured and rennet-treated skimmilk in the vat. When whey appears in the sample as a knife is drawn through the coagulum in the A-C cup, then the coagulum in the vat is ready to cut. After cutting, the curds are left undisturbed in the whey for about 15 min to 'heal' (i.e., contract and expel some whey) before commencing agitation and heating (cooking) to 52-60 ~ over 1.5-2 h. Collins (1961) recommended cooking to a minimum temperature of 55 ~ and holding at this temperature for at least 18 min to kill coliforms and psychrotrophic bacteria since D-values for E. coli, Pseudomonas fragi, Lc. lactis subsp, cremoris and Lc. lactis subsp, lactis in whey (pH 4.6) at 55 ~ are 4.3, 1.88, 0.64 and 4.57 min, respectively. A high cooking temperature also increases the firmness and DM content of the curds (Chua and Dunkley, 1979; Emmons and Beckett, 1984a). However, at a constant cooking temperature in the range 46.1-60 ~ the DM in curd increases with cooking pH (4.5-4.8) (Emmons and Beckett, 1984a). When rennet is used to aid coagulation and the pH at cooking is <4.5, partial proteolysis of the caseins results in a soft curd even when cooked to a high temperature, e.g., 60~ (Tuckey, 1964; Emmons and Beckett, 1984a). Provided that agitation is not too rapid to shatter the curd, the optimum heating rate is the fastest rate at which no matting occurs (Emmons and Tuckey, 1967). An increase in heating rate from 0.11 ~ at the start to 0.3 ~ at the end of cooking, so that a temperature of 51.6-54.4 ~ is reached within 2 h, was suggested by Tuckey (1964) and Emmons and Tuckey (1967) for the production of high-quality Cottage cheese. The firmness and DM content of Cottage cheese curd increase with heating rate in the range 0.18-0.50 ~ (Chua and Dunkley, 1979). Tuckey (1964) proposed that when the heating rate is too rapid, a surface protein film, which acts as a semipermeable membrane surrounding each cube of Cottage cheese curd becomes denatured, causing whey to be trapped in the curd. However, scanning electron microscopy (SEM) of Cottage cheese (Glaser et al., 1979; Kalab, 1979) failed to demonstrate such a protein film but showed that casein particles on the surface of curds are densely packed and unevenly distributed with pores between them to allow for free outward movement of whey. Similarly, SEM of the interior of a dehydrated curd granule shows clustered and unevenly distributed casein particles. By transmission electron microscopy (TEM), Glaser et al. (1979) showed a distinct difference between the surface and interior of Cottage cheese curd and observed that a surface skin appeared only after severe heat treatment (70 ~ • 1 h), which is unusual for Cottage cheese manufacture.
333
After cooking, the whey is drained and the curds are washed 2-3 times with water to remove excess lactose and lactic acid, thereby stabilizing curd pH, and to cool the curd. It is important to drain hot water from the vat jacket during whey drainage so that curd temperature does not rise during washing. Washing also cools the curds and retards bacterial growth. To achieve a final curd temperature < 4 ~ wash water at 30, 16 and 7-4 ~ or 10 and < 4 ~ for 3 or 2 successive washings, respectively, is used. The volume of wash water varies (4.7-15.2 1/kg curd, i.e., 30-100% of the skimmilk volume, depending on the equipment used) among manufacturers (Dunkley and Patterson, 1977). A typical value is 80% of the original volume of skimmilk. The water is added slowly down the sides of the vat or by sprinkling, and the curds are stirred gently during washing. After adding the required volume of water, stirring is continued for 10 min before draining. In an industry survey, Rosenberg et al. (1994) reported that washing time in the vat varied from 20 to 45 min. Cottage cheese whey and waste wash water are acidic (pH, 4.5-4.6) and contain proteins, lactose and salts at levels that create disposal problems. Nilson and LaClair (1975) reported BOD levels ranging from 34 500 to 42 167 mg/1 and COD levels of 64 188- 67 213 mg/1 during Cottage cheese manufacture from 550 to 600 kg skimmilk. In addition to protein and lactose, most of the minerals in milk, both colloidal and soluble, are lost in the whey and wash water; 87, 72 and 87% of Ca, P and Mg, respectively, are lost (Wong et al., 1976, 1977). To reduce the pollution load of effluent from Cottage cheese plants, a reduction in the volume and number of washings has been suggested (Nilson and LaClair, 1975; Emmons et al., 1978). Cross et al. (1977) reported an average loss of 4% of the curd as fines in whey and wash water. Curd solids are lost into wash water by diffusion, the rate of which increases with temperature (Emmons etal., 1978; Bressan et al., 1981) according to the equation: Deft = (0.0658T + 1.72) • 10 - 6
where, Deft is the effective diffusion coefficient in cm2/s and T is in ~ (Bressan et al., 1981). Knowledge of Deft is useful for engineering calculations in designing equipment to optimize curd washing during Cottage cheese manufacture. The isothermal diffusion of whey components from dry small-curd Cottage cheese granules during washing is similar to that of a spherical geometric model (Bressan et al., 1981, 1982), suggesting that small-curd
334
Cottage Cheese
Cottage cheese can be modelled as spheres for mass transfer studies. The wash water obtained after the first 20 min of washing contains about 87 and 93%, respectively, of the DM and lactose present after 60 min of washing (Bressan et al., 1982). This suggests that diffusion of lactose from curds to wash water is more rapid than the diffusion of other soluble constituents like whey proteins, salts and lactic acid. Therefore, washing for <20 min may be economically and nutritionally advantageous. The quality of water used to wash curds affects the keeping quality of Cottage cheese. Washing curds with high pH water dissolves the casein on the surface of the curd particles, making them slick and slimy in appearance. Therefore, wash water is chlorinated (5-8 mg/kg), and then acidified (pH 5.5-6.0) before use (Angevine, 1959). Acid wash water would probably also cause less loss of protein due to solubilization. Wash water may also be pasteurized.
Cream dressing is added and mixed with dry curds after washing. A typical formula for cream dressing is 10.4% fat, 7.8% milk solids non-fat, 2.0% whey, 2.5% salt and 0.35% stabilizer (Morley, 1981); a typical curd to dressing ratio is 60/40 or 56/44 when some of the non-fat milk solids is replaced with whey solids (Morley, 1981). Methods for preparing cream dressing (Manus, 1957) and the ratio of dressing to curds needed to control the fat content of Cottage cheese have been described (Kemp and Schuhz, 1979; Lundstedt, 1980). Essentially, the dressing is prepared by blending desired quantities of the ingredients, pasteurization at 74 ~ • 30 min or at least 82.2 ~ • 30 s, homogenization (double stage - 13.8 MPa first stage plus 3.4 MPa second stage) and then cooling to 3.3-4.4 ~ Single-stage homogenization of the dressing at 17.2 MPa results in increased viscosity due to fat clumping but whether this treatment leads to improved absorption of the dressing is questionable (Morley, 1981). Alternately, after pasteurization and homogenization, the cream dressing is cooled to 22 ~ inoculated with 2% of a culture containing citrate-positive (cit +) Lc. lactis subsp, lactis, incubated for 6 h, and then rapidly cooled to 5 ~ before use (Sandine, 1975). Patented procedures have been developed (Sing, 1976) for adding concentrated cells of cit + Lc. lactis subsp, lactis to non-cultured chilled cream dressing just before mixing with dry-curd Cottage cheese for the purpose of flavour enhancement and to control the growth of psychrotrophic bacteria during storage. At the time of adding the cream dressing to Cottage cheese curds, flavour materials may be added. The desired final pH
of creamed Cottage cheese is ---5.1. This results from blending the dressing, which has pH of 6.4-6.6, with dry-curd Cottage cheese with a pH of 4.6. Whey separation and the appearance of whey in the package occur when the pH of creamed Cottage cheese is <5.0. On the other hand, creamed Cottage cheese with a pH >5.2 has a reduced shelf-life. Instead of adding cold cream dressing, Overcast and Mackens (1973) suggested the use of hot ( - 8 8 ~ cream dressing to improve shelf-life. Addition of ascorbic acid to a cream dressing as a preservative has been suggested (Custer, 1977). The cream dressing has several functions in finished Cottage cheese. Morley (1981) lists the functions of cream dressing as: 9 source of flavour in creamed Cottage cheese; 9 lubrication of curd to make the product easier to handle during pumping and packaging; 9 modifies perceived texture and improves palatability; 9 adds nutritive value to Cottage cheese; 9 helps to control the fat and moisture levels in creamed Cottage cheese to meet regulatory standards.
The microstructure of direct set and cultured Cottage cheese are similar except for slight differences observed during the early stages of coagulation (Glaser et al., 1980). During Cottage cheesemaking, the numberaverage diameter of the casein particles increases from ---88 nm in milk to ---182 nm during early gelation, ---185 nm at the end of healing, ---206 nm during cooking and finally to 207 nm at the end of cooking (Glaser et al., 1980). However, the average diameter of the micelles is larger in cheese made with rennet than in that made without rennet (Bishop et al., 1983), suggesting that rennet aids in the fusion of the micelles. Typical electron micrographs of Cottage cheese curds show a porous mass of casein particles in the form of clusters and short chains.
The typical yields (kg cheese at 80% moisture per 100 kg skim milk) of dry-curd (uncreamed) Cottage cheese made from HTST-pasteurized skimmilk is 15-17%, depending on the protein (casein) content of the skimmilk. Except for the results of White and Ray (1977), yields reported for direct-set Cottage cheese are generally slightly higher (Satterness et al., 1978;
Cottage Cheese
Sharma et al., 1980; Geilman, 1981) than for cultured Cottage cheese. This is attributable, in part, to loss of soluble peptides produced from casein on proteolysis by starter bacteria. The use of proteinase-negative ( p r t - ) starters minimizes yield losses resulting from the proteolytic activity of starter bacteria (Stoddard and Richardson, 1986). Other methods reported to increase the yield of Cottage cheese include: 1. heating of skimmilk to a temperature higher than minimum HTST (Emmons et al., 1959; Durrant et al., 1961; White and Ray, 1977); 2. thermization (74 ~ • 10 s), followed by storage at 3 ~ for 7 days, then HTST treatment (Dzurec and Zall, 1982); 3. addition of sodium hexametaphosphate (SHMP) (Dybing et al., 1982); 4. addition of iota-carrageenan + SHMP (Manning, 1985; Manning et al., 1985); 5. ultrafiltration (UF) of skimmilk to 6.4% (Mathews et al., 1976), 9% (Ocampo and Ernstrom, 1987) or 15% (Covacevich and Kosikowski, 1978) protein. Also, Kosikowski et al. (1985) described the use of retentate-supplemented skimmilk for Cottage cheese manufacture. It is not known whether the methods listed above have been adapted for the commercial production of Cottage cheese. The use of highly heated skimmilk, gums, polysaccharides and UF techniques is aimed at trapping more whey proteins in Cottage cheese.
While the commercial production of some high-moisture, acid-coagulated cheeses (e.g., Quarg) from UF milk is promising, commercial production of Cottage cheese from UF skimmilk (UF Cottage cheese) is currently unattractive partially because of the difficulty in making good UF Cottage cheese with 20% DM retentate (Ocampo-Garcia, 1987). Ultrafiltration Cottage
335
cheese contains higher DM (24%) than conventional cheese (20% DM), which results in a higher (2%) moisture-adjusted yield in the former. However, the high DM in the UF cheese is not accompanied by increased firmness, suggesting the need for research to make a better quality UF Cottage cheese containing 20% DM. The most acceptable firmness of conventional Cottage cheese by a consumer panel is 25-35 g/cm (Mackie et al., 1989), as measured by the resistance to the movement of a wire through the curds (Voisey and Emmons, 1966). Cottage cheese made from UF skimmilk (pH 6.6) absorbs cream dressing poorly, giving it a gelatinous appearance (Covacevich and Kosikowski, 1978). However, this problem can be corrected by acidifying the skimmilk to pH 5.8 prior to UF (Ocampo and Ernstrom, 1987). Another problem encountered during the manufacture of Cottage cheese from UF skimmilk is that the coagulum is tough to cut with conventional cheese knives. Heat treatment (71.7-82.2 ~ for 7 s) of the UF skimmilk (Raynes et al., 1988) or the addition of 0.3% sodium citrate (Geilman, 1988) to it prior to cheesemaking decreases coagulum firmness at cutting.
Nutritionally, Cottage cheese is a wholesome low-calorie food that supplies less than 110 kcal/100 g (Table 5). Cottage cheese contains less Ca (about 30 mg/100 g for dry-curd, 60-100mg/100 g for creamed curd and 68 mg/100 g for low-fat creamed curd) than rennetcoagulated cheeses (700-900mg/100 g). These values suggest that approximately 50% of the Ca in Cottage cheese comes from the cream dressing. The concentrations of Ca, Mg, K and Na in Cottage cheese vary seasonally (Bruhn and Franke, 1988), probably as a result of seasonal variations in their concentrations in milk. Increasing Ca and reducing Na content of Cottage cheese are major nutritional concerns for the dairy industry. Wong et al. (1976) reported a 5-fold increase in Ca and a nutritionally favourable P:Ca ratio of 1.5
Proximate composition of Cottage cheese varieties
Cottage cheese variety Component
Creamed (large- or small-curd)
Low-fat (2% milk fat)
Low-fat (1% milk fat)
Dry-curd (large or small curd)
Moisture (g/100 g) Protein (g/100 g) Fat (g/100 g) Ash (g/100 g) Carbohydrate (g/100 g) Energy (kcal/100 g)
79.0 12.5 4.5 1.4 2.6 103.0
79.0 14.0 1.9 1.4 3.6 90.0
82.5 12.4 1.0 1.4 2.7 72.0
79.5 17.3 0.4 0.7 1.85 85.0
336
Cottage Cheese
in Cottage cheese when 0.2% of either sodium pyrophosphate, sodium tripolyphosphate or SHMP was added to skimmilk prior to cheesemaking. Addition of CaC12 to milk does not affect the Ca content of Cottage cheese but the Ca content of large-curd Cottage cheese is about 60% higher than that of smallcurd Cottage cheese. The exposed surface area to water during washing for large-curd Cottage cheese is smaller than that of small-curd Cottage cheese. Consequently, more minerals leach out of the latter into the wash water during manufacture (Wong et al., 1976). Cottage cheese can be fortified with Ca (to 2 • normal levels) through the addition of calcium salts (chloride, lactate or phosphate) to the cream dressing without adverse effects on sensory and microbiological qualities (Shelef and Ryan, 1988). The use of UF skimmilk retentate or addition of carrageenan to skimmilk did not affect Ca content of Cottage cheese (Craddock and Morr, 1988). The concentration of Na in Cottage cheese is about 4 mg/g (Demott et al., 1984; Bruhn and Franke, 1988), most of which is added with the cream dressing because the recovery of milk Na (approximately 0.5 mg/g) in curd is only about 3% (approximately 0.11 mg/g) after three washings during Cottage cheese manufacture (Wong et al., 1976). A 25% reduction of the sodium content of Cottage cheese dressing (Wyatt, 1983) or replacement of up to 50% of the NaC1 by KC1 (Demott etal., 1984) results in low-sodium Cottage cheese with sensory qualities similar to regular Cottage cheese. Generally, dairy products are poor sources of dietary iron. The concentration of Fe in Cottage cheese is ---174.1 Ixg/100 g (Wong et al., 1977). Addition of Fe in the form of ferric ammonium citrate to skimmilk (to give 20 ixg Fe/ml milk) resulted in 58% Fe recovery in washed curds without adverse effects on cheese quality (Sadler et al., 1973). The concentrations of Zn, Cu and Mn in Cottage cheese are 482.6, 2.6 and 3.7 Ixg/100 g, respectively (Wong et al., 1977). Fortification of Cottage cheese with vitamins A and C has been reported to produce satisfactory results (Sweeney and Ashoor, 1989).
An earlier review on the microbial quality of Cottage cheese was published by Emmons (1963). Cottage cheese has limited shelf-life (time from manufacture to unacceptability). Surveys of the shelf-life of Cottage cheese (Table 6) in three countries show that it starts to deteriorate within 2 weeks of storage at 5-7 ~ and is dependent on temperature (Schmidt and Bouma, 1992).
Reported shelf-life of Cottage cheese stored at refrigerated temperatures in various countries Country
Shelf-life (days)
Reference
Canada USA UK
<16 17.8 9-15
Roth et aL (1971) Hankin et al. (1975) Brocklehurst and Lund (1988)
The most frequently found spoilage organisms in Cottage cheese are psychrotrophic bacteria (Pseudomonas, Achromobacter, Flavobacterium, Alcaligenes, Escherichia, Enterobacter), yeasts and moulds (Witter, 1961; Cousin, 1982). However, Pseudomonas fluorescens, Ps. putida and Enterobacter agglomerans are the principal cause of spoilage (Brocklehurst and Lund, 1985). Bishop and White (1985) found no correlation (r = -0.61) between bacterial numbers and Cottage cheese shelf-life but reported that proteolysis was inversely related to potential shelf-life. Psychrotrophic bacteria (Marth, 1970) and their proteases (White and Marshall, 1973) cause defects like surface discolouration, off-odours and off-flavours in Cottage cheese. Bitterness, resulting from increased proteolysis by psychrotrophic bacteria (Stone and Naff, 1967), occurs in Cottage cheese. The shelf-life of Cottage cheese can be extended by >75% by adding sorbic acid or potassium sorbate (0.075%, w/w) to the cream dressing to inhibit psychrotrophic bacteria (Bradley et al., 1962; Collins and Moustafa, 1969; Bodyfelt, 1979; Brocklehurst and Lund, 1985) and moulds without producing objectionable flavours. Sorbic acid is most effective as an anti-microbial agent in its undissociated form, which represents about 59-31% of the concentration used in Cottage cheese at pH 4.6-5.1 (Brocklehurst and Lund, 1985). Chen and Hotchkins (1991) found that dissolving CO2 in Cottage cheese prior to packaging in airtight containers inhibits the growth of gram-negative bacteria and extends shelf-life up to 60 days at 4 ~ Mannheim and Softer (1996) also reported that modified atmospheric packaging by headspace flushing with CO2 extends the shelf-life of Cottage cheese stored at 8 ~ by 150%. Other 'natural' ways of extending shelf-life are by the addition of bifidobacteria to inhibit Staphylococci (Brisolva, 1987) or a pre-cultured skim milk product, MicroGARD TM (Salih et al., 1990). The inhibitory effect of MicroGARD TM is due to a heat-stable, low-molecular weight (---700 daltons) peptide. It is claimed (Boudreaux et al., 1988) that the addition of 106-107 cfu/g Propionibacterium shermanii NRRL-B-18074 plus 106-107 cfu/g of either cit + Lc. lactis subsp, lactis (formerly, S. lactis
Cottage Cheese
subsp, diacetylactis) NRRL-B-15005, NRRL-B-15006, NRRL-B-15018 or ATCC 15346 to Cottage cheese dressing inhibits psychrotrophic bacteria and moulds by producing propionic and acetic acids which act as antimicrobial agents. Mather and Babel (1959) showed that the addition of acetic or propionic acid to Cottage cheese dressing retards slime formation. Some lactic acid bacteria produce anti-microbial substances that inhibit bacteria of other genera (Branen etal., 1975). The patented (Gonzalez, 1986) strains of citrate-positive (cit +) Lc. lactis subsp, lactis exert their preservative effect without metabolizing lactose or citrate because they are lac-, cit-; they lack a 41 MDa plasmid and a 5.5 MDa plasmid which control lactose and citrate utilization, respectively, in those organisms. The survival of Listeria monocytogenes (strains Scott A or V7) during the manufacture and storage of cultured Cottage cheese was studied by Ryser et al. (1985) who found that small numbers (<100 cfu/g) of the organism survived the cheesemaking process. However, Listeria-free direct-acid Cottage cheese was made using GDL but not HC1 because undissociated organic acids are more soluble in the bacterial cell membrane and are more bacteriostatic than dissociated acids (E1-Shenawy and Marth, 1990).
The acidic taste of Cottage cheese is due largely to lactic acid, which is present at a concentration in the range 124-452 mg/kg; other acids present include formic (23-306 mg/kg) and acetic (11-292 mg/kg), while the concentrations of propionic and butyric acids are <1 mg/kg (Brocklehurst and Lund, 1985). Formic, acetic, propionic and butyric acids are volatile, thus contributing to the aroma of Cottage cheese. The most distinct flavour compound in Cottage cheese is diacetyl which is produced by oxidative decarboxylation of oL-acetolactic acid, an intermediate compound formed during citrate metabolism by microorganisms that contain citrate permease and citritase (Seitz et al., 1963a), or by condensation of acetyl CoA (from acetic acid) and acetaldehyde, as a C2wthiamine pyrophosphate (TPP) complex (Collins, 1972). Diacetyl production is pH depedent, occurring at pH values <5.5 (Collins, 1972). Acceptable levels of diacetyl in Cottage cheese are estimated to be about 2 mg/kg (Hempenius etal., 1965) but a diacetyl:acetaldehyde ratio of 3-5 is desirable for good flavour (Lindsay et al., 1965). A ratio of diacetyl:acetaldehyde >5 or <3 results in harsh or green flavour defects, respectively. The lack of diacetyl flavour can be attributed partially to the oxidation
337
of diacetyl to acetoin by some starter or contaminating bacteria (e.g., coliforms, Pseudomanas and Alcaligenes) that contain diacetyl reductase (Seitz et al., 1963b). Since oxidation increases with temperature (Pack et al., 1968), storage of Cottage cheese at refrigeration temperatures is important for the retention of diacetyl flavour. The pathway for the conversion of citrate to diacetyl is very well understood. Further, the accumulating evidence on the central role of pyruvate in diacetyl synthesis, the diacetyl destructive enzymes, and procedures developed for selective isolation of mutants that favour the synthesis of the key intermediate, ot-acetolactate, have offered immense possibilities of 'engineering' high flavour-producing lactococci (Hugenholtz, 1993; Hugenholtz et al., 1994). Metabolism of citrate by citrate-fermenting lactic acid bacteria is dependent on the transport of citrate into the cell via a permease system, which functions optimally below pH 6.0. Genetic coding for the permease enzyme is found on a plasmid. Fermentation of citrate by citrate-fermenting lactococci starters also results in the generation of CO2. However, excessive gas production within Cottage cheese curds leads to floating curd cubes during cooking and causes improper, inadequate and non-uniform syneresis, resulting in defective texture and at times matting of individual curd particles, which is undesirable. The citrate-fermenting lactococci produce significant amounts of CO2 (some strains produce excessive amounts) and relatively high concentrations of acetaldehyde, which results in a 'yogurt-like green apple flavour'.
The following are common defects in Cottage cheese and their possible causes (Emmons and Tuckey, 1967; Sandine, 1975). 1. Mealy curd a. Cooling too fast b. Curd particles contacting hot surface during cooking (possibly due to inadequate stirring) 2. Matted curd a. pH too high, i.e., not enough acid at cutting 3. Shattered curd a. Excessive heat treatment of skimmilk b. Too much acid at cutting c. Rough handling of the curd, especially at cutting d. Too low solids e. Too much rennet 4. Rubbery curd a. Cooking temperature too high
338
Cottage Cheese
5. Weak pasty curd a. Excessive heat treatment of skimmilk b. Cooking temperature too low c. Low pH at cutting d. Too much acidity before and during cooking 6. Acid and unclean flavour a. Excess acid at cutting b. Cooking too rapid, especially during early stages c. Contamination during and after manufacture d. Poor quality skimmilk and/or starter 7. Gelatinous curd a. Spoilage bacteria b. Alkaline wash water 8. Poor shelf-life a. Unclean equipment b. Failure to keep cream, dry curd and creamed Cottage cheese cold ( < 5 ~ 9. Bitterness a. Contamination by psychrotrophic bacteria such as Ps. putrefaciens b. Cooking too fast 10. Gassiness a. Contaminated milk b. Use of starters containing high gas-producing cit + Lc. lactis subsp, lactis 11. Sediment in cheese vat at cutting a. Clumping of starter bacteria by agglutinins in milk; most c o m m o n with mastitic and early lactation milk 12. Medicinal flavour a. Use of chlorinated wash water high in organic matter.
Angevine, N.C. (1959). Keeping quality of Cottage cheese. J. Dairy Sci. 42, 2015-2019. Bishop, J.R. and White, C.H. (1985). Estimation of potential shelf life of Cottage cheese utilizing bacterial numbers and metabolites.J. Food Prot. 48, 1054-1057. Bishop, J.R., Bodine, A.B. and Janzen, J.J. (1983). Electron microscopic comparison of curd microstructures of Cottage cheese coagulated with and without microbial rennin. Cult. Dairy ProdJ. I8, 4-16. Bodyfeh, EW. (1979). Sensory, shelf life, microbial and chemical evaluation of creamed Cottage cheese treated with sorbates. J. Food Prot. 42,836 (abstr.). Boudreaux, D.P., Lingle, M.W., Vedamuthu, E.R. and Gonzalez, C.E (1988). Method for preservation of creamed Cottage cheese. US Patent 4,728,516. Bradley, R.L., Harmon, L.G. and Stine, C.M. (1962). Effect of potassium sorbate on some organisms associated with Cottage cheese spoilage. J. Milk Food Technol. 25, 318-323. Branen, A.L., Go, H.C. and Genske, R.P. (1975). Purification and properties of antimicrobial substances produced by
Streptococcus diacetylactis and Leuconostoc citrovorum. J. Food Sci. 40,446-450. Bressan, J.A., Carroad, RA., Merson, R.L. and Dunkley, W.L. (1981). Temperature dependence of effective diffusion coefficient for dry matter during washing of cheese curd. J. Food Sci. 46, 1958-1959. Bressan, J.A., Carroad, RA., Merson, R.L. and Dunkley, W.L. (1982). Modeling of isothermal diffusion of whey components from small curd Cottage cheese during washing. J. Food Sci. 41, 84-88. Bringe, N.A. and Kinsella, J.E. (1990). Acidic coagulation of casein micelles: mechanisms inferred from spectrophotometric studies. J. Dairy Res. 57,365-375. Brivosa, G.V. (1987). Izvestiya, Vyssnvikh Uchebnykh Zavedenii Pishchevaya Technologiya 3, 61-63 (cited from Dairy Sci. Abstr. 1988;50;584). Brocklehurst, T.E and Lund, B.M. (1985). Microbiological changes in Cottage cheese varieties during storage at 7 ~ Food Microbiol. 2,207-233. Brocklehurst, T.E and Lund, B.M. (1988). The effect of pH on the initiation of growth in Cottage cheese spoilage bacteria. Int. J. Food Microbiol. 6, 43-49. Brooker, B.E. (1986). Electron microscopy of normal and defective Cottage cheese curd. J. Soc. Dairy Technol. 39, 85-88. Bruhn, J.C. and Franke, A.A. (1988). Protein and major cations in California Cottage cheese and yogurt. J. Dairy Sci. 71, 2885-2890. Chen, J.H. and Hotchkins, J.H. (1991). Effect of dissolved carbon dioxide on the growth of psychrotrophic organisms in Cottage cheese. J. Dairy Sci. 74, 2941-2945. Chua, T.E.H. and Dunkley, W.L. (1979). Influence of cooking procedures on properties of Cottage cheese curd. J. Dairy Sci. 62, 1216-1226. Codex Alimentarius (1968). Codex international individual standard for Cottage cheese including creamed Cottage cheese. Codex Stan C- 16-1968. FAO/WHO, Rome, Italy. Collins, E.B. (1961). Resistance of certain bacteria to Cottage cheese cooking temperatures. J. Dairy Sci. 44, 1989-1995. Collins, E.B. (1972). Biosynthesis of flavour compounds by microorganisms. J. Dairy Sci. 55, 1022-1028. Collins, E.B. and Moustafa, H.H. (1969). Sensory and shelflife evaluation of Cottage cheese treated with potassium sorbate. J. Dairy Sci. 52,439-444. Corbin, E.A., Jr. (1971). Cheese manufacture. US Patent 3620768. Cousin, M.A. (1982). Presence and activity of psychrotrophic microorganisms in milk and dairy products: a review. J. Food Prot. 45, 172-207. Covacevich, H.R. and Kosikowski, EV. (1978). Cottage cheese by ultrafiltration. J. Dairy Sci. 61,529. Craddock, M.A. and Mort, C.V. (1988). Evaluation of alternative methods to increase calcium retention in Cottage cheese curd. J. Food Sci. 53, 1680-1683. Cross, S.D., Henderson, J.M. and Dunkley, W.L. (1977). Losses and recovery of curd fines in Cottage cheese manufacture. J. Dairy Sci. 60, 1820-1823. Custer, E.W. (1977). Manufacturing top-quality Cottage cheese. Cult. Dairy Prod. J. 12 (4), 18-20.
Cottage Cheese Deane, D.D. and Hammond, E.G. (1960). Coagulation of milk for cheesemaking by ester hydrolysis. J. Dairy Sci. 43, 1421-1429. Demott, B.J., Hitchcock, J.P. and Sanders, O.G. (1984). Sodium concentration of selected dairy products and acceptability of sodium substitute in Cottage cheese. J. Dairy Sci. 67, 1539-1543. Dunkley, W.L. and Patterson, D.R. (1977). Relations among manufacturing procedures and properties of Cottage cheese. J. Dairy Sci. 60, 1824-1840. Durrant, N.W., Stone, W.K. and Large, RM. (1961). The effect of increasing serum protein content of Cottage cheese curd on yield and quality. J. Dairy Sci. 44, 1171 (abstr.). Dybing, S.T., Parson, J.G., Martin, J.H. and Spurgeon, K.R. (1982). Effect of sodium hexametaphosphate on Cottage cheese yield. J. Dairy Sci. 65,544-551. Dzurec, D.J. and Zall, R.R. (1982). Effect of on-farm heating and storage of milk on Cottage cheese yield. J. Dairy Sci. 65, 2296-2300. E1-Shenawy, M.A. and Marth, E.H. (1990). Behavior of Listeria monocytogenes in the presence of gluconic acid and during preparation of Cottage cheese curd using gluconic acid.J. Dairy Sci. 73, 1429-1438. Emmons, D.B. (1963a). Recent research in the manufacture of Cottage cheese- Part I. Dairy Sci. Abstr. 25, 129-137. Emmons, D.B. (1963b). Recent research in the manufacture of Cottage cheese- Part II. Dairy Sci. Abstr. 25, 175-182. Emmons, D.B. and Beckett, D.C. (1984a). Effect of gas-producing cultures on titratable acidity and pH in making Cottage cheese. J. Dairy Sci. 67, 2192-2199. Emmons, D.B. and Beckett, D.C. (1984b). Effect of pH at cutting and during cooking on Cottage cheese. J. Dairy Sci. 67, 2200-2209. Emmons, D.B. and Elliot, J.A. (1967). Effect of homogenization of skimmilk on rate of acid development, sedimentation and quality of Cottage cheese made with agglutinating cultures. J. Dairy Sci. 50, 957 (abstr.). Emmons, D.B. and Tuckey, S.L. (1967). Cottage Cheese and Other Cultured Milk Products. Pfizer Cheese Monographs. Vol. 3. Chas. Pfizer and Co., Inc., New York. Emmons, D.B., Swanson, A.M. and Price, W.V. (1959). Effect of skimmilk heat treatment and methods of acidification on manufacture and properties of Cottage cheese. J. Dairy Sci. 42, 1020-1031. Emmons, D.B., Elliot, J.A. and Beckett, D.C. (1966). Effect of lactic streptococci agglutinins on curd formation and manufacture of Cottage cheese. J. Dairy Sci. 49, 1357. Emmons, D.B., Beckett, D.C., Campbell, N.J. and Humbert, E.S. (1978). Reducing washing of Cottage cheese and increased recovery of whey solids. Cult. Dairy Prod. J. 13 (2), 13-17, 22-24, 26-29. Ernstrom, C.A. (1963). Process for preparing cheese curd. US Patent 3089776. Ernstrom, C.A. and Kale, C.G. (1975). Continuous manufacture of Cottage cheese and other uncured cheese varieties. J. Dairy Sci. 58, 1008. Fedrick, I.A. and Houliman, D.B. (1981). The effect of lactose hydrolysis on the yield of Cottage cheese curd. Aust. J. Dairy Technol. 36 (3), 104-106.
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Flanagan, J.E, Thompson, M.P., Breower, D.P. and Gyuricsek, D.M. (1978). Manufacture of Mozzarella, Muenster and Cottage cheese from nonfat milk powder. Cult. Dairy Prod. J. 13 (4), 24-28. Gastaldi, E., Laguade, A. and Tarodo de la Fuente (1996). Micellar transition state in casein between pH 5.5 and 5.0. J. Food Sci. 61, 59-64. Geilman, W.G. (1981). Comparison of Skimilk Starter, Wheybased Starter and a Direct-set Method on Yield, Quality and Economics of Cottage Cheese Production. MS Thesis, Utah State University, Logan, UT. Geilman, W.G. (1988). Effect of milk quality and composition on the production of retentate made using ultrafiltration and a soft and semi-soft cheese made from retentate. Ph.D. Thesis. Mississippi State University, MS. Gilliland, S.E. (1972). Flavor intensification with concentrated cuhures.J. Dairy Sci. 55, 1028-1031. Glaser, J., Carroad, RA. and Dunkley, W.L. (1979). Surface structure of Cottage cheese curd by electron microscopy. J. Dairy Sci. 62, 1058-1068. Glaser, J., Carroad, P.A. and Dunkley, W.L. (1980). Electron microscopic studies of casein micelles and curd microstructure in Cottage cheese. J. Dairy Sci. 63, 37-48. Gonzalez, C.E (1986). Preservation of foods with rlonlactose fermenting Streptococcus lactis subspecies diacetilactis. US Patent 4,599,313. Grandison, A.S., Brooker, B.E., Young, P. and Wingmore, A.S. (1986). Yield loss of Cottage cheese curd due to the formation of minor sludge: the beneficial effect of homogenizationJ. Soc. Dairy Technol. 39, 123-126. Gyuricsek, D.M. and Thompson, M.P. (1976). Hydrolyzed lactose cultured dairy products. II. Manufacture of yogurt, buttermilk and Cottage cheese. Cult. Dairy Prod. J. 11 (3), 12-14. Hammond, E.G. and Dearie, D.D. (1961). Cheesemaking process. US Patent 2982654. Hankin, L., Stephens, G.R. and Dillman, W.E (1975). Quality control significance of special media for enumeration of microbial groups in Cottage-type cheese. J. Milk Food Technol. 38, 738-744. Harwalkar, "
340
Cottage Cheese
Kemp, A.R. and Schuhz, J.R. (1979). Increasing Cottage cheese profitability. Cult. Dairy Prod.J. 14 (1), 15-18. Kim, B.Y. and Kinsella, J.E. (1989). Effect of temperature and pH on the coagulation of casein. Milchwissenschaft. 44, 622-625. Kosikowski, EV. (1982). Cheese and Fermented Milk Foods. 2nd edn, Edward Bros, Inc., Ann Arbor, MI. Kosikowski, F.V., Masters, A.R. and Mistry, V.V. (1985). Cottage cheese from retentate-supplemented skimmilk. J. Dairy Sci. 68, 541-547. Lindsay, R.C., Day, E.A. and Sandine, W.E. (1965). Green flavor defect in lactic starter cultures. J. Dairy Sci. 48,863-869. Loter, I., Dissly, H.G. and Schafer, R.E. (1975). Preparation of cheese. US Patent 3,882,250. Lundstedt, E. (1980). The techniques involved in the creaming of Cottage cheese. Cult. Dairy Prod. J. 15 (2), 8-13. Mackie, D.A., Emmons, D.B., Beckett, D.C. and Elsaesser, J.L. (1989). Sensory and instrumental analyses of Cottage cheese firmness. Can. Inst. Food Sci. Technol. J. 22,456-459. Manning, D.W., Jr. (1985). Increasing cheese yields with carrageenan. Proc. 22nd Marschall Invitational Cheese Seminar, Madison, WI. pp. 49-54. Manning, D., Jr., Witt, H. and Ames, J. (1985). Increasing cheese yields with carrageenan, in, Gums and Stabilizers for the Food Industry 3, Phillips, G.O., Wedlock, D.J. and Williams, RA., eds, Elsevier Applied Science Publishers, London. pp. 379-385. Mannheim, C.H. and Softer, T. (1996). Shelf life extension of Cottage cheese by modified atmosphere packaging. Lebensm.-Wiss.u.-Technol. 29,767-771. Manus, L.J. (1957). High viscosity Cottage cheese dressing. Milk Prod. J. 48 (10), 56-58. Marth, E.H. (1970). Spoilage of Cottage cheese by psychrophilic organisms. Cult. Dairy Prod. J. 5 (1), 14-17. Mather, D.W. and Babel, EJ. (1959). Studies on the flavor of creamed Cottage cheese. J. Dairy Sci. 42,809-815. Mathews, M.E., So, S.E., Amundson, C.H. and Hill, C.G., Jr. (1976). Cottage cheese from uhrafiltered skimmilk. J. Food Sci. 41,619-623. Metz, EL. (1980). Method for preparing cheese. US Patent 4,199,609. Milton, K., Hicks, C.L., O'Leary, J. and Langlois, B.E. (1990). Effect of lecithin addition and homogenization of bulk starter on agglutination. J. Dairy Sci. 73, 2259-2268. Morley, R.G. (1981). Suggestions for the preparation of cream dressing for Cottage cheese, in, Proceedings of the 2nd Biennial Marschall International Cheese Conference. Dane County Exposition Center, Madison, WI. Mutzelburg, I.D., Dennien, G.J., Fedrick, I.A. and Deeth, H.C. (1982). An investigation of some factors involved in curd shattering during Cottage cheese manufacture. Aust. J. Dairy Technol 37, 107-112. Nilson, K.M. and LaClair, EA. (1975). Pollution load of Cottage cheese whey and wash waters. J. Milk Food Technol. 38, 532-536. Ocampo, J.R. and Ernstrom, C.A. (1987). Cottage cheese from ultrafiltered skimmilk. J. Dairy Sci. 70 (Suppl. 1), 67 (abstr).
Ocampo-Garcia, J.R. (1987). Cottage Cheese Made from Ultrafiltered Skimmilk by Direct Acidification. MS Thesis, Utah State University, Logan, UT. Overcast, W.W. and Mackens, D. (1973). The use of hot cream as dressing for Cottage cheese. Cult. Milk Prod. J. 8 (1), 6-7, 31. Pack, M.Y., Vedamuthu, E.R., Sandine, W.E., Elliker, P.R. and Lessment, H. (1968). Effect of temperature on growth and diacetyl production by aroma bacteria in single and mixed strain lactic cultures. J. Dairy Sci. 51,339-344. Perry, C.A. and Carroad, P.A. (1980). Influence of acid related manufacturing practices on the properties of Cottage cheese curd. J. Food Sci. 45,794-797. Pyne, G.T. and McGann, T.C.A. (1960). The colloidal phosphate of milk. II. Influence of citrate. J. Dairy Res. 27, 9-17. Raynes, R., Ernstrom, C.A. and Hicks, C.L. (1988). Sensory and curd characteristics of Cottage cheese manufactured from 16% dry matter retentate.J. Dairy Sci. 71 (Suppl. 1), 81 (abstr). Reddy, M.S., Mullen, J., Washman, C.J., Brown, C.G. and Hunt, C.C. (1990). Cheese manufacture. US Patent 4,959,229. Roefs, S.P.EM., Walstra, P., Dalgleish, D.G., and Horne, D.S. (1985). Preliminary note on the change in casein micelles caused by acidification. Neth. Milk Dairy J. 39, 119-122. Rosenberg, M., Tong, ES., Sulzer, G., Gendre, S. and Ferris, D. (1994). California Cottage cheese technology and product quality: an in-plant survey. 1. Manufacturing process. Cult. Dairy Prod. J. 29 (1), 4-11. Roth, L.A., Clegg, L.EL. and Stiles, M.E. (1971). Coliforms and shelf life of commercially produced Cottage cheese. Can. Inst. Food Sci. Technol. J. 4, 107-111. Reidy, G. and Hedrick, T.I. (1970). Highlights of Cottage cheese industry in the USA. Cult. Dairy Prod. J. 5 (3), 18-20. Reidy, G. and Hedrick, T.I. (1971). Highlights of Cottage cheese industry in the USA. Cult. Dairy Prod. J. 6 (1), 21-24. Ryser, E.T., Marth, E.H. and Doyle, M.P. (1985). Survival of Listeria monocytogenes during manufacture and storage of Cottage cheese. J. Food Prot. 48, 746-750. Sadler, A.M., Lacroix, D.E. and Alford, J.A. (1973). Iron content of Bakers and Cottage cheese made from fortified skimmilk. J. Dairy Sci. 56, 1267-1270. Salih, M.A. and Sandine, W.E. (1980). Lactic streptococci agglutinins: a review. J. Food Prot. 43,856-858. Salih, M.A. and Sandine, W.E. (1984). Rapid test for detecting lactic streptococci agglutinins in cheese milk. J. Dairy Sci. 67, 7-23. Salih, M.A., Sandine, W.E. and Ayres, J.W. (1990). Inhibitory effects of Microgard TM on yogurt and Cottage cheese spoilage organisms. J. Dairy Sci. 73,887-893. Sandine, W.E. (1975). Quality Cottage cheese. Cult. Dairy Prod.J. l0 (2), 12-16. Satterness, D.E., Parson, J.G., Martin, J.H. and Spurgeon, K.R. (1978). Yields of Cottage cheese made with cultures and direct acidification. Cult. Dairy Prod. J. 13 (1), 8-13.
Cottage Cheese Schmidt, K. and Bouma, J. (1992). Estimating shelf life of Cottage cheese using hazard analysis. J. Dairy Sci. 75, 2922-2927. Scott, R. (1986). Cheesemaking Practice, 2nd edn, Elsevier Applied Science Publishers, London. Seitz, E.W., Sandine, W.E., Elliker, P.R. and Day, E.A. (1963a). Studies on diacetyl biosynthesis by Streptococcus diacetilactis. Can. J. Microbiol. 9, 431-441. Seitz, E.W., Sandine, W.E., Elliker, P.R. and Day, E.A. (1963b). Distribution of diacetyl reductase among bacteria.J. Dairy Sci. 46, 186-189. Sharma, H.S., Bassette, R., Metha, R.S. and Dayton, D. (1980). Yield and curd characteristic of cottage cheese made by culture and direct-acid set methods. J. Food Prot. 43,441-446. Shelef, L.A. and Ryan, R.J. (1988). Calcium supplementation of Cottage cheese.J. Dairy Sci. 71, 2618-2621. Sing, E.L. (1976). Preparation of Cottage cheese. US Patent 3,968,256. Stoddard, G.W. and Richardson, G.H. (1986). Effect of proteolytic activity of Streptococcus cremoris in Cottage cheese yields. J. Dairy 5ci. 69, 9-14. Stone, W.K. and Naff, D.M. (1967). Increases in soluble nitrogen and bitter flavor development in Cottage cheese. J. Dairy 5ci. 15, 1497-1501. Sweeney, M.A. and Ashoor, S.H. (1989). Fortification of Cottage cheese with vitamins A and C.J. Dairy Sci. 72,587-590. Tuckey, S.L. (1964). Properties of casein important in making Cottage cheese. J. Dairy Sci. 47,324.
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United States Department of Agriculture (1978). Cheese Varieties and Descriptions. Agric. Handbook 54. Washington, DC. pp. 99-100. Voisey, EW. and Emmons, D.B. (1966). Modification of curd firmness test for Cottage cheese. J. Dairy Sci. 49, 93-96. Watrous, B. (1997). Lactic cultures in Cottage cheese, in, Cultures for the Manufacture of Dairy Products. Chr. Hansen, Inc., Milwaukee, WI. pp. 101-107. White, C.H. and Marshall, R.T. (1973). Reduction of shelf life of dairy products by a heat-stable protease from Pseudomonas fluorescence P26. J. Dairy 5ci. 56, 849-853. White, C.H. and Ray, B.W. (1977). Influence of heat treatments and methods of acidification of milk on manufacture and properties of Cottage cheese. J. Dairy Sci. 60, 1236-1243. White, C.H. and Ryan, J.M. (1983). Defining optimal conditions for making Cottage cheese from reconstituted milk powder. J. Food Prot. 46, 686-689. Witter, L.D. (1961). Psychrophilic bacteria - a review. J. Dairy 5ci. 44, 983-1015. Wong, N.P., LaCroix, D.E., Mattingly, W.A., Vestal, J.H. and Alford, J.A. (1976). The effect of manufacturing variables on the mineral content of Cottage cheese. J. Dairy Sci. 59, 41-44. Wong, N.P., LaCroix, D.E. and Vestal, J.H. (1977). Trace minerals in Cottage cheese. J. Dairy Sci. 60, 1650-1652. Wyatt, C.J. (1983). Acceptability of reduced sodium in breads, Cottage cheese and pickles. J. Food Sci. 48, 1300-1302.
Acid- and Acid/Rennet-curd Cheeses
Part C: Acid-heat Coagulated Cheeses N.Y. Farkye, Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, CA 93407
Several cheeses are manufactured throughout the world by a combination of acid and heat coagulation of milk. Notable are Queso Blanco (Central and South America), Paneer (India) and Ricotta (Italy), which are reviewed herein. Unlike Cottage cheese in which coagulation is induced by acidification, coagulation and curd formation in this group of cheeses is by a combination of acid and heat (Kosikowski, 1982). The high-heat treatment (>70 ~ • 20 min) given to the milk causes denaturation of whey proteins which form a complex with K-casein (Sawyer, 1969) via disulfide linkages and subsequently co-precipitate on acidification of the heated milk to pH <5.5. During acidification of the heated milk, there is a progressive removal of calcium phosphate from the casein micelles to form a soluble calcium salt (Rao et al., 1992) and at pH 5.2, most, if not all, of the calcium is solubilized (Pyne and McGann, 1960; Bringe and Kinsella, 1990; Gastaldi et al., 1996). Acid-heat coagulated cheeses are generally fresh, soft, unripened varieties manufactured from whole milk (e.g., Queso Blanco, Paneer), cream (e.g., Marscapone) or whey or whey blends (e.g., Ricotta, Ricottone).
Queso Blanco (White Cheese) is a soft mildly acid variety that is popular in Latin America and in Caribbean countries, where it is known by different names depending on the country of origin (USDA, 1978). Cheeses similar to Queso Blanco are also manufactured in other parts of the world, e.g., Paneer, a popular cheese in India and Pakistan. Queso Blanco (Queso del Pias or cheese of Puerto Rico) was first introduced in the USA by Weigold (1958). Since then, researchers in the USA and Canada have worked to standardize the manufacturing methods and elucidate the properties of Queso Blanco as made in North America.
Queso Blanco can be manufactured from whole, low-fat, skim or recombined milk by methods described by Chandan et al. (1979), Hill et al. (1982) and Kosikowski (1982). A continuous method for making Queso Blanco is described by Modler (1984, 1988) and Modler and Emmons (1989a,b). The main principle of Queso Blanco manufacture is heat-acid co-precipitation of milk proteins. When whole milk is used, there is a large variation (60-85%) in fat recovery (Hill et al., 1982; ParnellClunies et al., 1985a), resulting in a yield of 11.5-22% for milk containing 3-6% fat (Siapantas and Kosikowski, 1965, 1967; Hill et al., 1982). This suggests mechanical occlusion of fat in heat-acid coagulated milk protein and indicates an upper limit of 4.5% fat or a protein:fat ratio of 1:1.2 for the production of acceptable Queso Blanco with high yields (Hill et al., 1982). However, in India, Paneer is manufactured traditionally from buffalo milk standardized to 6% fat (see Torres and Chandan, 1981a,b; Kalab et al., 1988). Fat recovery increases to >93% when the cheese milk is homogenized (13.7 MPa) but a high-moisture (Parnell-Clunies etal., 1985a) soft cheese, lacking desirable slicing properties (Siapantas and Kosikowski, 1967), results.
In the traditional methods for making Queso Blanco or Paneer, milk is heated to boiling and acid (sour) whey is added, with continuous stirring, until coagulation is completed. In industrial processes, several heat treatments have been r e p o r t e d - mostly 82-90 ~ for 0-30 min (Weigold, 1958; Siapantas and Kosikowski, 1973; Chandan et al., 1979; Hill et al., 1982; Rao et al., 1992; Farkye et al., 1995). However, it appears that 85 ~ for 5 rain is the best heat treatment for the production of Queso Blanco with the most desirable qualities (Parnell-Clunies et al., 1985a).
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
344
Acid-heat Coagulated Cheeses
For the manufacture of Paneer, buffalo milk is heated to 90 ~ without holding (Kalab et al., 1988) or to 82 ~ for 5 min (Rao et al., 1992), then cooled to 70 ~ before acidification, to reduce curd firmness (see Kalab et al., 1988).
The hot milk is acidified using food-grade acids, e.g., HC1 (Chandan etal., 1979), H3PO4 (Siapantas and Kosikowski, 1973), lactic, tartaric, citric or glacial acetic acid (Siapantas and Kosikowski, 1973; Chandan et al., 1979; Farkye et al., 1995), fruit juices (Kosikowski, 1982) or acid whey concentrate (Hirschl and Kosikowski, 1975). However, citric or glacial acetic acid is used most frequently. The amount of acid required for coagulation depends on the buffering capacity of the milk (Siapantas and Kosikowski, 1973; Hill et al., 1982). To achieve a final pH of 5.2-5.3 in Queso Blanco, 120 ml glacial acetic acict/45.5 kg milk (Siapantas and Kosikowski, 1967) or 0.34% (w/w) citric acid monohydrate (Hill et al., 1982) is added to the milk. Prior to addition to the milk, the acid is diluted with nine parts of water (Siapantas and Kosikowski, 1967; Chandan et al., 1979) but Parnell-Clunies et al. (1985b) indicate that dilution of the acid to 1-2% before adding to milk produces more cohesive curd and cheese with improved body and texture. Rao et al. (1992) suggested that 1.5-3 g citric or lactic acid per kg milk is needed for the manufacture of Paneer.
After acidification, the curds are allowed to settle, then trenched in the vat to facilitate whey drainage. For Paneer, whey is drained off above 63 ~ (Mistry et al., 1992). Following whey removal, salt (2-2.5%, w/w) is added to Queso Blanco curd, which is then hooped and pressed for a few hours. After removal from the press, Queso Blanco is stored at < 8 ~ On the other hand, Paneer curd is not salted but hooped and pressed for 15-20 min. After removal from the press, Paneer is cut into small cubes that are immersed in chilled water (4-6 ~ for 2-3 h. Then, the cubes are removed from the water and allowed to drain or wiped with a dry cloth before cold storage and marketing (Vishweshwaraiah and Anantakrishnan, 1985; Rao et al., 1992). Fresh Queso Blanco has an average composition of 15-20% fat, 21-25% protein, 50-56% moisture, 2 - 2 . 5 % NaC1, 2.5-2.7% lactose and a pH in the range 5.2-5.5. It contains approximately 341,357 and 665 mg Ca, P and Na, respectively, per 100g (Torres and Chandan,
1981b). The chemical composition of Paneer varies depending on whether it is made from bovine, buffalo or mixed milk. The typical composition of Paneer from milk containing 3.5% fat is "-55% moisture, 19% fat, 21% protein, 2% lactose, 1.6% ash and pH 6.0 (Mistry et al., 1992). The typical composition of Paneer made from buffalo milk is ---51% moisture, 18% protein, 27% fat, 2% lactose and 1.8% ash (Chandan, 1991; Rao et al., 1992).
The microstructure of Queso Blanco is affected by the severity of heat treatment of the milk. Increasing heating/coagulation temperature in the range 62.8-98 ~ increases compactness of the curd and results in cheese with a smoother mouth-feel (Kalab and Modler, 1985). Unlike curd from rennet-treated milk, which consists of distinguishable casein particles fused together in chains and clusters, the microstructure of fresh Queso Blanco shows relatively large protein particles composed of transformed and indistinguishable casein particles (Kalab and Modler, 1985). According to Harwalkar and Kalab (1980, 1981), curds obtained by acidification of hot milk to pH --5.5 possess a so-called 'core-and-lining' structure in which a solid casein micelle core (>300 nm in diameter) is surrounded by an outer lining, 30-50 nm thick with a void space 50-80 nm wide that separates the lining and the core. Harwalkar and Kalab (1988) suggest that a pH of 5.2-5.5 is critical for the development of the core-and-lining structure because in this pH range, casein micelles have optimal voluminosity or hydrodynamic volume, a high percentage of non-sedimantable casein and little or no colloidal calcium phosphate. They suggest that the heat-induced interaction between [3-1actoglobulin and K-casein, enhanced by the presence of calcium ions, results in the development of filamentous appendages. Caseins, particularly [3-casein, which dissociate from the micelles during heat treatment, precipitate on the filamentous appendages to form a lining and leave an annular space between the casein core and the lining formed. Dissociation of caseins occurs on heating milk to high temperatures (Fox et al., 1967). The intensity of the core-and-lining structure in Queso Blanco increases with heat treatment of the cheese milk. The average diameter of casein particles in Queso Blanco made from milk coagulated at 62.8~ is 0.1 bLm compared to 0.5-5 b~m when coagulation is at 96-98 ~ (Kalab and Modler, 1985). Kalab et al. (1988) also observed coreand-lining structures in Paneer made from cow and buffalo milks.
Acid-heat Coagulated Cheeses
Queso Blanco made without rennet has a unique functionality- it has good slicing properties (Siapantas and Kosikowski, 1967) and resists melting when fried (Chandan et al., 1979). However, Queso Blanco made with rennet has excellent melting properties (Siapantas and Kosikowski, 1973). The small indistinguishable casein particles in Queso Blanco permits its use as an ingredient in the manufacture of cheese spreads free of grittiness (Modler et al., 1985, 1989). The texture, and hence the sliceability, of Queso Blanco is influenced by the moisture content of the cheese (Chandan etal., 1979) and the age of the cheese (Torres and Chandan, 1981b). Parnell-Clunies et al. (1985b,c) reported that the hardness of Queso Blanco increased linearly over time (17 days at 5 ~ but decreased with increasing moisture in the range 50-54%. Farkye et al. (1995) studied the textural properties of Queso Blanco made with acetic, citric or lactic acid and reported that texture profile analysis (TPA) hardness, fracturability, chewiness and gumminess were highest for cheese made with acetic acid and lowest for that made with lactic acid. They also found that TPA springiness and cohesiveness of Queso Blanco were independent of acid type, and that all the textural parameters except cohesiveness increased with age of cheese up to 7 weeks at 5 ~ Traditionally, Queso Blanco is consumed fresh because the nature of the processing conditions allows for very little biochemical changes during storage. However, Torres and Chandan (1981b) reported that lactobacilli or exogenous lipases can be added to the dry curd before salting and pressing to improve the flavour of the cheese during ripening (12 weeks at 10 ~ The rate of increase in non-protein nitrogen during the 12 weeks ripening period was slight (0.23%) but greater in cheese containing added lactobacilli than in cheese without (0.17%). Treatment with lipase increases the concentration of free fatty acids in cheese >300-fold. Major volatile compounds contributing to the flavour and aroma of Queso Blanco include acetaldehyde, acetone, ethyl, isopropyl and butyl alcohols and formic, acetic, propionic and butyric acids (Siapantas, 1967). Unlike most cheese varieties, the pH of Queso Blanco decreases from approximately 5.2 to 4.9 during ripening. The fermentation of residual lactose by heat-stable indigenous bacteria in milk that survive cheesemaking or by post-manufacture contaminating bacteria (Torres and Chandan, 1981b), or perhaps the dissociation of residual coagulating acid may account for the decrease in the pH of Queso Blanco during storage.
345
Information on the microbiological quality of Queso Blanco made in the US or Canada by methods described above is limited, even though poor keeping qualities of such cheeses made by different methods have been reported (Arispe and Westhoff, 1984a,b). In commercial Venezuelan Queso Blanco made without exogenous acids or starter bacteria, micro-organisms enumerated include Salmonella, Escherichia coli, Staphylococcus aureus, Bacillus cereus, Clostridium perfringes, Lactobacillus plantarum, Lb. casei, yeasts and moulds (Arispe and Westhoff, 1984b). Those cheeses were made under poor sanitary conditions and had a pH > 5.3. The high heat-acid treatment of milk, together with the low pH of the cheese and the presence of undissociated coagulating acid prevent the growth of spoilage organisms during refrigerated storage of Queso Blanco made in North America. Glass et al. (1995) reported differences in the efficacy of different organic acids and a bacteriocin-type product in the control of L. monocytogenes in Queso Blanco-type cheese. Siapantas (1967) reported that storage of Queso Blanco at a high temperature (>26 ~ results in butyric acid fermentation due to the growth of spore-forming bacteria in the milk used for manufacture.
Ricotta is an unripened soft cheese that originated from Italy. In Latin American and the Hispanic communities in North America, Ricotta is known as Requeson. The USDA specifies three types of Ricotta cheese: 1. Whole milk R i c o t t a - manufactured from whole milk, and the finished product shall contain not more than 80.0% moisture and not less than 11.0% milk fat. 2. Part-skim Ricotta - manufactured from milk with a reduced fat content, and the finished product shall contain not more 80.0% moisture and less than 11.0% but not less than 6.0% milk fat. 3. Ricotta (Ricottone) from whey or s k i m m i l k manufactured from skimmilk, whey or a blend of these products and the finished product shall contain not more than 82.5% moisture and less than 1.0% milk fat. Whole milk or part-skim Ricotta is a soft creamy cheese and has a pleasant and slightly sweet or caramel flavour whereas Ricottone has a slightly sweet, bland flavour. Typically, Ricotta is made from whey containing
346
Acid-heat Coagulated Cheeses
5-20% whole milk, skimmilk or non-fat dry milk (NFDM; Shahani, 1979). However, to produce Ricotta with desirable curd handling characteristics, it is necessary to add at least 5 parts of whole milk to 95 parts of whey, or 1 part NFDM to 99 parts of whey (Shahani, 1979). Traditionally, the starting material used for the manufacture of Ricotta cheese is whey resulting from Mozzarella cheese production. At present, Ricotta can be made from almost any type of sweet whey, provided the initial titratable acidity of the whey is -<0.16% lactic acid and its pH -->6.0. The best initial titratable acidity of whey for Ricotta cheese manufacture is 0.13-0.14% lactic acid (True, 1973). The use of whey concentrates containing up to 36% DM as starting material for Ricotta cheese manufacture has been reported (Nilson and Streiff, 1978). In the traditional method, whey or whey and milk blends are heated to 40-45 ~ and NaC1 is added. The mixture is heated continuously in large open kettles to 80-85 ~ A slow heating rate produces a better coagulum than rapid heating (True, 1973). Then, a suitable food-grade acid is added to reduce the pH to 6.0, thereby inducing coagulation. The coagulated curds float to the surface and are scooped off and placed in perforated hoops to drain and cool. In industrial methods, the whey is first neutralized to pH >6.5 (6.9-7.1) with a 25% (w/v) solution of NaOH. pH manipulation minimizes protein aggregation and produces a more cohesive coagulum (Modler and Emmons, 1989b). The neutralized whey is heated to 65-70 ~ then, whole milk or skimmilk equal to 5-25% of the whey volume is added and heating of the whey/milk mixture is continued to 75-80 ~ Cream may be added at this stage. Next, NaC1 (0.5%, w/v) is added and heating continued to 85-95 ~ Alternately, CaC12 may be added. NaC1 dehydrates the whey proteins and has a destabilizing effect on bovine serum albumin. Similarly, calcium destabilizes the whey proteins. Then, dilute food-grade acetic or citric acid is added for coagulation and curd formation. Typically, --1.5% (v/v) of dilute (---3.85%) acetic acid is needed to clot the whey/milk mixture. The curds are left in the hot whey for about an hour to increase in firmness and enhance whey drainage. The curds, which float on the surface of the whey, are ladled off. Alternately, the whey may be drained from the bottom, leaving the curds in the vat or kettle. Optimal coagulation occurs at pH 5.6-5.8 to give maximum yield (Weatherup, 1986). Approximately 5 kg of fresh Ricotta is obtained from 100 kg whey to which 5 kg of whole milk has been added. True (1973) obtained 30-39 g Ricotta cheese from 750 ml of whey; the highest yield was from whey heated to - 8 8 ~
Table 1 cheese
Proximate composition of different types of Ricotta
Ricotta cheese varieties
Component Moisture (g/100 g) Fat (g/100 g) Protein (g/100 g) Carbohydrate (g/100 g) Ash (g/100 g) Energy (kcal/100 g)
Part-skim
Whey (Ricottone)
72 13 11 3
74.5 8 11.5 5
77 2.5 16 3.5
1 174
1 138
1.0 100
Whole milk
Kosikowski (1967) describes the following procedure for the manufacture of whole milk Ricotta. Whole milk is adjusted to pH 6.0 or titratable acidity of 0.30-0.31% lactic acid, preferably with lactic starter, before heating. During heating, NaC1 (1.86 g/kg milk) is added. Also, stabilizer (0.23 g ~ g milk) is added to prevent foaming of the milk during heating. When the temperature of the milk reaches "-76 ~ a wideblade spatula is passed through the milk to observe the initiation of curd formation. Heating is continued to 80 ~ The floating curd is left undisturbed for about 10 min. Then, the curd is moved gently away from the wall of the vat or kettle towards the centre. This is continued for about 15 min and the curd is ladled from the top. The remaining whey is subjected to a second precipitation by heating to 85 ~ and adding granular citric acid (0.12 g&g milk) to give a pH of 5.4. The curd is ladled off. The curds from the primary and secondary precipitations are cooled and packaged. The use of ultrafiltration techniques to improve the yield of Ricotta cheese has been demonstrated (Maubois and Kosikowski, 1978). Also, a continuous manufacturing process for whole milk Ricotta cheese, with yields of 14.45-15.11kg/100kg milk, was reported (Modler, 1984, 1988; Modler and Emmons, 1989b). The typical composition of whole milk and partskim Ricotta, and Ricottone are given in Table 1.
Shelf-Life of Ricotta Ricotta has a relatively short shelf-life- about 3 weeks if properly packaged and stored at 4 ~ or lower (True, 1973), although Kosikowski (1967) reported a shelf-life of 70 days for whole milk. Ricotta cheese is packaged under vacuum, gas flushed and stored at "--4 ~
Acid-heat Coagulated Cheeses
Arispe, I. and Westhoff, D. (1984a). Manufacture and quality of Venezuelan white cheese. J. Food Sci. 49, 1005-1010. Arispe, I. and Westhoff, D. (1984b). Venezuelan white cheese: composition and quality. J. Food Protect. 47, 27-35. Bringe, N.A. and Kinsella, J.E. (1990). Acidic coagulation of casein micelles: mechanisms inferred from spectrophotometric studies. J. Dairy Res. 57,365-375. Chandan, R.C. (1991). Cheeses made by direct acidification, in, Feta and Related Cheeses, Robinson, R.K. and Tamine, A.Y., eds, Ellis Horwood, New York. pp. 229-252. Chandan, R.C., Marin, H., Nakrani, K.R. and Zehner, M.D. (1979). Production and consumer acceptance of Latin American white cheese. J. Dairy Sci. 62, 691-696. Farkye, N.Y., Prasad, B.B., Rossi, R. and Noyes, O.R. (1995). Sensory and textural properties of Queso Blanco-type cheese influenced by acid type. J. Dairy Sci. 78, 1649-1656. Fox, K.K., Harper, M.K., Holsinger, V.H. and Pallansch, M.J. (1967). Effect of high-heat treatment on stability of calcium casein aggregates in milk. J. Dairy Sci. 50,443-450. Gastaldi, E., Laguade, A. and Tarodo de la Fuente (1996). Micellar transition state in casein between pH 5.5 and 5.0. J. Food Sci. 61, 59-64. Glass, K.A., Bhanu Prasad, B., Schlyter, J.M., Uljas, H.E., Farkye, N.Y. and Luchansky, J.B. (1995). Effects of acid type and Aha TM 2341 on Listeria monocytogenes in Queso Blanco type of cheese. J. Food Prot. 58, 737-741. Harwalkar, V.R. and Kalab, M. (1980). Milk gel structure. XI. Electron microscopy of glucono-8-1actone-induced skim milk gels. J. Texture Stud. 11, 35-49. Harwalkar, VR. and Kalab, M. (1981). Effect of acidulants and temperature on microstructure, firmness, and susceptibility to syneresis of skimmilk gels. Scanning Electron Microsc. III, 503-513. Harwalkar, V.R. and Kalab, M. (1988). The role of [3-1actoglobulin in the development of the core-and-lining structure of casein particles in acid-heat induced milk gels. Food Microstruct. 7, 173-179. Hill, A.R., Bullock, D.H. and Irvine, D.M. (1982). Manufacturing parameters of Queso Blanco made from milk and recombined milk. Can. Inst. Food Sci. Technol. J. 15, 47-53. Hirschl, R. and Kosikowski, EV. (1975). Manufacture of Queso Blanco using whey concentrates. J. Dairy Sci. 58, 793 (abstr.). Kalab, M. and Modler, H.W. (1985). Development of microstructure in a cream cheese based on Queso Blanco cheese. Food Microstruct. 4, 89-98. Kalab, M., Gupta, S.K., Desai, H.K. and Patil, G.R. (1988). Development of microstructure in raw, fried, and fried and cooked Paneer made from buffalo, cow and mixed milks. Food Microstruct. 7, 83-91. Kosikowski, EV. (1967). The making of Ricotta cheese. Proc. 4th Annual Marschall Invitational Italian Cheese Seminar, Madison, WI. pp. 1-7. Kosikowski, EV. (1982). Cheese and Fermented Milk Foods, 2nd edn, Edward Bros, Inc., Ann Arbor, MI.
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Maubois, J.L. and Kosikowski, EV. (1978). Making Ricotta Cheese by ultrafiltration principles. J. Dairy Sci. 61, 881-884. Mistry, C.D., Singh, S. and Sharma, R.S. (1992). Physicochemical characteristics of Paneer from cow milk by altering salt balance. Aust. J. Dairy Technol. 47, 23-27. Modler, H.W. (1984). Continuous Ricotta manufacture. Mod. Dairy 63 (4), 10-12. Modler, H.W. (1988). Development of a continuous process for the production of Ricotta cheese. J. Dairy Sci. 71, 2003-2009. Modler, H.W. and Emmons, D.B. (1989a). Production and yield of whole milk Ricotta manufacture by a continuous process. I. Materials and methods. Milchwissenschaft 44, 673-676. Modler, H.W. and Emmons, D.B. (1989b). Production and yield of whole milk Ricotta manufactured by a continuous process. II. Results and discussion. Milchwissenschaft 44, 753-757. Modler, H.W., Poste, L.M. and Butler, G. (1985). Sensory evaluation of an all-dairy fermented cream-type cheese produced by a new method. J. Dairy Sci. 68, 2835-2839. Modler, H.W., Yiu, S.H., Bollinger, U.K. and Kalab, M. (1989). Grittiness in a pasteurized cheese spread: a microscopic study. Food Microstruct. 8, 201-210. Nilson, K.M. and Streiff, P. (1978). Comparison of whey Ricotta cheese manufactured from whey and whey concentrates. Proc. 15th Marschall Invitation Cheese Seminar, Madison, WI. pp. 1-12. Parnell-Clunies, E.M., Irvine, D.M. and Bullock, D.H. (1985a). Heat treatment and homogenization of milk for Queso Blanco (Latin American white cheese) manufacture. Can. Inst. Food Sci Technol. J. 18, 133-136. Parnell-Clunies, E.M., Irvine, D.M. and Bullock, D.H. (1985b). Composition and yield studies for Queso Blanco made in pilot plants and commercial trials with dilute acidulant solutions. J. Dairy Sci. 68, 3095. Parnell-Clunies, E.M., Irvine, D.M. and Bullock, D.H. (1985c). Textural characteristics of Queso Blanco. J. Dairy Sci. 68, 789-793. Pyne, G.T. and McGann, T.C.A. (1960). The colloidal phosphate of milk. II. Influence of citrate. J. Dairy Res. 27, 9-17. Rao, K.V.S.S., Zanjpad, P.N. and Mathur, B.N. (1992). Paneer technology- a review. Indian J. Dairy Sci. 45 (6), 281-291. Sawyer, W.H. (1969). Complex between [3-1actoglobulin and K-casein: a review. J. Dairy Sci. 52, 1347-1355. Shahani, K.M. (1979). Newer techniques for making and utilization of Ricotta cheese. Proc. 1st Biennial Marschall International Cheese Conference, Madison, WI. pp. 77-87. Siapantas, L.G. (1967). Biochemical Changes in "Queso Blanco" Cheese during Storage at High Temperatures. IDM Potential for Developing Countries. PhD Thesis, Cornell University Press, Ithaca, NY. Siapantas, L.A. and Kosikowski, EV. (1965). Acetic acid preparation phenomenon of whole milk for Queso Blanco cheese. J. Dairy Sci. 48, 764 (abstr.). Siapantas, L.G. and Kosikowski, EV. (1967). Properties of Latin American white cheese influenced by glacial acetic acid. J. Dairy Sci. 50, 1589.
348
Acid-heat Coagulated Cheeses
Siapantas, L.G. and Kosikowski, EV. (1973). The chemical mode of action of four acids and milk acidity in the manufacture of Queso Blanco. J. Dairy Sci. 56, 631. Torres, N. and Chandan, R.C. (1981a). Latin American white cheese: a review. J. Dairy Sci. 64, 552-559. Torres, N. and Chandan, R.C. (1981b). Flavor and texture development in Latin American white cheese. J. Dairy Sci. 64, 2161-2169. True, L.C. (1973). Effect of various processing conditions on the yield of whey Ricotta cheese. Proc. l Oth Marschall Invitational Cheese Seminar, Madison, WI. pp. 1-11.
United States Department of Agriculture (1978). Cheese Varieties and Descriptions. Agric. Handbook 54. Washington, DC. pp. 99-100. Vishweshwaraiah, L. and Anantakrishnan, C.P. (1985). A study on technological aspects of preparing Paneer from cow's milk. AsianJ. Dairy Res. 4 (3), 171-176. Weatherup, W. (1986). The effect of processing variables on the yield and quality of Ricotta. Dairy Ind. Int. 5 (8), 41-45. Weigold, G.W. (1958). Development of a factory method for the manufacture of Queso Del Pias. Milk Prod. J. 49 (10), 16-17, 25.
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products T.P. Guinee, Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland M. Cari~, University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, Serbia and Montenegro M. Kah~b, Agriculture and Agri-Food Canada, Food Research Program, Guelph, Ontario, Canada
The products in this group differ from natural cheeses in that they are not made directly from milk (or dehydrated milk), but rather from various ingredients such as skim milk, natural cheese, water, butter oil, casein, caseinates, other dairy ingredients, vegetable oils, vegetable proteins and/or minor ingredients. The two main categories, namely pasteurized processed cheese products (PCPs) and analogue cheese products (ACPs), may be subdivided further depending on the composition and the types and levels of ingredients used (Fig. 1). The individual categories will be discussed separately below.
Introduction
Pasteurized PCPs are cheese-based foods produced by comminuting, melting and emulsifying into a smooth homogeneous molten blend, one or more natural cheeses and optional ingredients using heat, mechanical shear and (usually) emulsifying salts (ES). Optional ingredients permitted depend on the product type, i.e., whether processed cheese, processed cheese food (PCF) or processed cheese spread (PCS), and include dairy ingredients, vegetables, meats, stabilisers, ES, flavours, colours, preservatives and water (Tables 1 and 2). Cheese, as an ingredient of PCPs, ranges from a minimum of 51% in pasteurized PCSs and PCFs to "-095% in pasteurized processed cheese (Code of Federal Regulations, 1986; Fox et al., 1996). Attempts to increase the shelf-life of cheese during the early twentieth century were inspired by the possibility of increased cheese trade, via the production of more stable transportable products, and by the existence of heated cheese dishes such as Swiss Fondue, Welsh Rarebit and Kochktise. Many of the early approaches
were unsuccessful; the heat-treated cheeses were unstable, undergoing oiling-off and moisture-exudation during cooling and storage. In 1911, Swiss workers, Walter Gerber and Fritz Stettler, produced a stable heat-treated Emmental cheese, known as Schachtelk~ise, by the addition of a 'melting salt', sodium citrate, to the comminuted cheese before processing (i.e., heating and shearing; Meyer, 1973). Subsequently, it was found that other cheeses (e.g., Cheddar) could be also processed to form stable products by the addition of other 'melting salts' (e.g., sodium phosphates) or blends of different ES. The 'melting salts' were gradually referred to as ES when their function became known, i.e., mediation of the processes of protein hydration and emulsification of free fat during processing. Initial successes were followed by numerous patents for different melting salt blends and later for the inclusion of food ingredients other than cheese. Processed cheese products are used in many applications, in both the raw and the heated forms. The suitability for particular applications depends primarily on the textural and the flavour characteristics of the unheated cheese and the cooking properties of the heated cheese. In the unheated form, it may be used as a table product with a spectrum of consistencies ranging from firm, elastic and sliceable to creamy, smooth and spreadable. The variations in consistency make it suitable for a range of uses, e.g., substitute for natural sliceable or shredded cheese (e.g., on bread, crackers or in sandwiches), table spread, sauces and dips. Processed cheese products are also used as an ingredient in several cookery applications, e.g., as slices in burgers, in toasted sandwiches, pasta dishes, au-gratin sauces or cordon-bleu poultry products. Processed cheese products may be also be dried, as cheese powders, which are then dry-blended with other ingredients in the preparation of formulated foods such as dry
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
350
P a s t e u r i z e d P r o c e s s e d C h e e s e and S u b s t i t u t e / I m i t a t i o n C h e e s e P r o d u c t s
Pasteurized processed and analogue cheese products
Pasteurized process cheese products
Analogue cheese products
I
9 manufactured by blending, heating and shearing mixtures of ingredients, mainly of dairy origin 9 natural cheese must be >51% (w/w) of the final product
Categories - Processed cheese - Processed cheese food - Processed cheese spread - Blended cheese -Blended cheese spread
9 manufactured by blending, heating and shearing mixtures of ingredients of dairy and/or vegetable origin 9 not necessary to include natural cheese 9 natural cheese may be added at a low level (e.g., 5% ) to impart cheesy flavour or to comply with a particular customer specification
Categories - Dairy analogue - Part-dairy analogue - Non-dairy analogue
Generalized classification scheme for pasteurized processed and analogue cheese products; the analogue cheeses may be either substituted or imitated depending on the nutritional equivalence compared to natural cheese (Analogue cheese products, ACPs).
soup or sauce mixes, ready-prepared meals, snack coatings (see 'Cheese as an Ingredient', Volume 2). The production of pasteurized processed cheese in different countries is shown in Table 3. Global production of PCPs, based on available information, is estimated to be "--2.0 million tonnes/annum, which is equivalent to ---13% of natural cheese production. Production in the EU15 increased steadily at a rate of ---1% per annum during the period 1996-2000, i.e., at a rate lower than that for natural cheese (1.6%) over the same period, but has increased by 2.7% per annum for the 1999-2000 period (ZMP, 2001). Factors contributing to the continued growth of PCPs include: 9 Their versatility as foods which offer wide variety in flavour, texture (e.g., elasticity, firmness, spreadability, sliceability), cooking attributes (e.g., degrees of flowability, browning, viscosity), size and shape of the final product and overall consumer appeal made possible by differences in formulation and processing conditions, condiment addition and packaging technology (Mann, 1970, 1972, 1974, 1975, 1978a,b, 1981, 1986, 1987, 1990, 1993, 1997; Price and Bush, 197ara,b; Abou-E1-Nour, 2001; Subak and Petranin, 2001).
9 Their popularity with children of different ages owing to their safe ingestable consistency (for infants), mild flavours and their packaging (colour, caricatures, strength, ease of opening, size) and shape (e.g., triangles, fingers, cartoon characters) which is generally attractive and convenient for lunch boxes. 9 Their nutritive value (e.g., especially as a source of calcium and protein) as a food for children. 9 Their ability to meet special dietary needs if fortified with vitamins and minerals (Zhang and Mahoney, 1991; Sukhinina et al., 1997), which is technologically easy in the manufacture of PCPs. 9 Their adaptability as an ingredient with properties customized to the needs of several sectors of the food formulation and assembly industries (e.g., manufacturers of cheese powders, cheese-flavoured coated snacks, soups, cheese-meat products, prepared meals). 9 Their convenience of use in the culinary and food service sectors, especially the fast food trade, and the home because of their excellent preservation (stability), consistent tailor-made functionality (e.g., cooking properties), convenient portion size and packaging (e.g., as slices for the beef burger and sandwiches trade).
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
351
Optional ingredients permitted in pasteurized processed cheese productsa,b
Ingredient type
Main function~effect
Examples
9 Standardization of composition 9 Contributes to flavour, texture and cooking characteristics 9 Standardization of composition 9 Assist in 'creaming' (thickening of blend during manufacture) and formation of product 9 Contribute to texture and rheological (e.g., fracturability, hardness) and cooking properties 9 Low-cost filler; may affect texture
Cream, anhydrous milk fat, dehydrated cream, butter
Dairy Ingredients Milk fat
Milk proteins
Lactose Cheese base
Stabilizers
Acidifying agents Flavourings
Flavour enhancers Condiments Sweetening agents Colours Preservatives
9 Substitute for young cheese 9 Similar in behaviour to milk proteins, it contributes to thickening during manufacture, texture and cooking properties 9 Assist the formation of a physico-chemically stable product 9 Impart desired texture and cooking characteristics 9 Assist control of the pH of final product 9 Impart flavour to processed cheese foods and spreads, especially where much young cheese, cheese base, or milk proteins are used 9 Accentuate flavour 9 Affect appearance, flavour and texture, and product differentiation 9 Increase sweetness, especially in products targeted to young children 9 Impart desired colour 9 Retard mould growth; prolong shelf-life
Casein, caseinates, whey proteins, milk protein concentrates (ultrafiltered milk and microfiltered milk preparations), co-precipitates, skim milk powder Whey powder, skim milk powder, whey permeate powder Typically, high dry-matter milk solids (=60%, w/w) prepared by evaporation of milk ultrafiltrates to which starter culture and rennet have been added Emulsifying salts: sodium phosphates and sodium citrates Hydrocolloids: carrob bean gum, guar gum, xanthan gum, sodium carboxymethylcellulose, carageenan Food-grade organic acids, e.g., lactic, acetic, citric, phosphoric Enzyme-modified cheese, starter distillate, wood smoke extracts, spices NaCI, yeast extract Sterile preparations of meat, fish, vegetables, nuts and/or fruits Sucrose, dextrose, corn syrup, hydrolysed lactose Annato, paprika, artificial colours Nisin, potassium sorbate, Ca- or Na- propionate
a The ingredients permitted depend on product type, category, regulations in the region of manufacture. b The effects of different ingredient types are discussed in detail in 'Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs' and 'Properties of ES important in cheese processing'.
9 Low cost relative to natural cheese due to the incorporation of low-grade natural cheese, off-cuts and cheaper non-cheese milk solids (e.g., skim milk powder, whey, casein and caseinates). Casein and fat in cheese are generally more expensive, on a weight basis, than casein and fat in the form of ingredients such as casein powders and butter oil. 9 Relatively long shelf-life, good physico-chemical stability (e.g., compared to natural cheeses in which fat and/or moisture separation sometimes occur on prolonged storage) and absence of waste (e.g., compared to natural cheeses with rind or surface mould or smear). This makes them easy to use in the food service and food formulation assembly sectors. 9 The developments in manufacturing technology, emulsifying salt blends and functional dairy ingredients which facilitate the manufacture of consistent quality products with customized quality attributes, shape, size and appearance (e.g., processed cheese
slices with holes similar to eye cheeses; Polkowski, 2002). Classification of PCPs
There are various types of PCPs, with standards of identity (relating to composition and levels and types of permitted ingredients) that vary somewhat from country to country. Hence, in the UK there are two categories of PCPs, namely processed cheese and cheese spread (as specified by the Cheese and Cream Regulations, 1995, SI 1995/3240, HMSO, London) whereas in Germany there are four categories, viz., Schmelzk/~se (processed cheese), Schmelzk~isezubereitung (processed cheese preparation), Kasezubereitung (cheese preparation) and K/isekomposition (cheese composition), as detailed in the Deutsche K/iseverordnung of 12 November 1990. Currently, the IDF, under the auspices of the Codex Alimentarius Commission, a
352
Pasteurized Processed-Cheese and Substitute/Imitation Cheese Products
Ingredients and composition specifications of different categories of pasteurized (processed) cheese productsa, b
Compositional specifications
Product category
Permitted ingredients
Pasteurized blended cheese
Cheese; cream, anhydrous milk fat, dehydrated cream (in quantities such that the fat derived from them is less than 5%, w/w, in finished product); water; salt, food-grade colours, spices and flavourings (other than those which simulate the flavour of cheese, and wood smoke extracts); mould inhibitors (sorbic acid, potassium/sodium sorbate at levels _<0.2%, w/w, of, and/or sodium propionates at levels -<0.3%, w/w, of, the finished product) when product is in the form of slices or cuts in consumer packs As for pasteurized blended cheese, but with the following extra optional ingredients: emulsifying salts (such as sodium phosphates and/or sodium citrates at a level of -<3%, w/w, of finished product), food-grade organic acids (e.g., lactic, acetic or citric) at levels such that the pH of the finished product is not less than 5.3 As for pasteurized processed cheese but with the following extra optional ingredients: dairy ingredients (milk, cream, skim milk, buttermilk, cheese whey, whey proteins - i n wet or dehydrated forms) As for pasteurized processed cheese but with the following extra optional food-grade hydrocolloids (e.g., carob bean gum, guar gum, xanthan gum, gelatin, carboxymethylcellulose and/or carageenan) at levels <0.8% (w/w) of finished products, and food-grade sweetening agents (e.g., sugar, dextrose, corn syrup, glucose syrup, hydrolysed lactose) As for pasteurized process cheese spread, except that emulsifying salts are not permitted
Pasteurized processed cheese
Pasteurized processed cheese foods Pasteurized processed cheese spread
Pasteurized cheese spread
Moisture (%, w/w)
Fat (%, w/w)
FDMc (%, w/w)
_<43
-
_>47
-<43
-
_>47
Not less than 5.3
_<44
_>23
-
Not less than 5.0
40-60
_>20
-
Not less than 4.0
40-60
_>20
-
Not less than 4.0
pH
a Data presented are summarized from the Code of Federal Regulations (CFR, 1986). b For each category, there may be product variations for which compositional specifications differ from those presented. c FDM, fat-in-dry matter.
subsidiary body of the FAO/WHO, is endeavouring to draft a single standard for pasteurized PCPs which will be accepted globally. It is expected that a Codex standard will assume increased importance because such a standard will be used by the World Trade Organization in the resolution of trade disputes. In the United States, the code of Federal Regulations defines four types of PCPs based on permitted ingredients and composition (Table 2). Under this system, which is detailed in the Code of Federal Regulations, Food and Drugs, Part 133 (Edition 4-1-93), four main categories of PCPs are identified, namely pasteurized processed cheese, pasteurized PCE pasteurized PCS and pasteurized blended cheese. The criteria for classification include permitted ingredients and compositional parameters; the main aspects of the different categories are summarized in Table 2. Pasteurized processed cheese is usually sold in the form of sliceable blocks (e.g., processed Cheddar) or slices; spreads and foods may be in the form
of blocks, slices, spreads or pastes (e.g., in tubes). Pasteurized blended cheese, which is the least common category, is usually sold in forms giving a natural cheese image. An arbitrary fifth category of 'non-standarized processed cheese-like products' may be considered as consisting of processed cheese-like, processed cheesebased products such as dips and sauces. Manufacturing protocol for PCPs
The manufacture of PCPs involves the following major steps (Fig. 2): 9 formulation and selection of the different types and levels of ingredients to be included; 9 cleaning and comminution of the cheese; 9 blending with water and other permitted ingredients; 9 processing (heating and shearing) of the blend; 9 homogenization of the molten blend (optional); 9 hot packing and cooling.
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
Production data for pasteurized processed cheese products in the period 1995-2000a, b
Region~Country EU 15 Belgium Denmark Germany Spain France Ireland Italy The Netherlands Austria Portugal Finland Sweden United Kingdom Norway Czech Republic Hungary Russian Federation USA Canada Australia New Zealand Japan Approximate Global
1996 ('000 tonnes)
1995 ('000 tonnes)
517 54 15 157 39 126 12 20 30 14 4 15 5 27 3 18 11 80 c 1081 75 c 50 12c 97 1,944
538 55 20 171 37 134 11 20 17 18 1 16 8 32
Mean annual change in period 1996-2000 (difference as % 1996 quantity) +1.0 +0.8 +8.1 +2.2 -1.3 +1.5 -2.1 +0.1 -10.9 +8.3 -18.4 +1.4 +13.0 +4.2
a Compiled from data by Hetzner and Richarts (1996), Serensen (1997), ZMP (Zentral Markt- und Preisberichtstelle GmbH, 2001). b For countries for which data are available. c Data for Russian Federation, Canada and New Zealand are for 1994.
353
PCP. However, age (and hence level of proteolysis) appears to have major effects, as reflected by the use of cheese age as a major selection criterion for blend formulation at production level. Block processed cheeses with good sliceability and elasticity require predominantly young cheese (70-90% intact casein) whereas predominantly medium ripe cheese (60-75% intact casein) is used for cheese spreads (Meyer, 1973). Hence, the meltability of processed Cheddar, in which 4-6-month old Cheddar was substituted by ultrafiltered milk retentate (UMR) at a level of 50%, w/w, on cooking increased significantly as the degree of proteolysis in the UMR was increased by the addition of a proteinase from Aspergillus oryzae (Sood and Kosikowski, 1979). Owing to inter-cheese variation in microstructure, composition and level of proteolysis (Fox et al., 2000), different types of cheese give processed products with different consistency characteristics. Hence, it is generally recognized that hard and semi-hard cheese varieties, such as Cheddar, Gouda and Emmental, give firmer, longer-bodied processed products than mouldripened varieties such as Camembert and Blue cheeses (Meyer, 1973). The latter cheeses undergo more extensive proteolysis during ripening and therefore, have a lower degree of intact casein than the former cheeses (Gripon, 1993; Lawrence et al., 1993; Walstra et al., 1993; Steffen et al., 1993). The effects of cheese characteristics (type, age) and the other ingredients used in the manufacture of PCPs on its textural and cooking properties are discussed in 'Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs'.
Formulation of blend
Cleaning and size reduction of cheese
Formulation involves selection of the correct type and quantity of natural cheeses, ES, water and optional ingredients to give a PCP with the desired composition, textural and functional properties. Cheese is the major constituent of PCPs, ranging from a minimum level of ---51%, w/w, in spreads and foods to --~98%, w/w, in processed cheeses. Consequently, the type, the blend and the degree of maturity of the cheeses selected for processing have a major influence on the consistency of the product. In some countries, pasteurized processed cheese made from only one type of cheese is very popular, e.g., Cheddar in the UK and Australia, Cheddar, Gruyere and Mozzarella in the USA and Canada and Emmental in France and Germany. However, PCPs, especially cheese foods and cheese spreads, are produced from a blend of various types of natural cheese. The mixed selection facilitates the procurement of the desired flavour and texture in the finished PCP. Little published information is available on how the attributes of the raw cheese impact on those of the
Cleaning generally involves the removal of surface contamination (e.g., adventitious mould growth) or rind using rapid motor-driven scrapers. The cheese is cut, using hydraulically operated blades, into segments, which are finely minced by passing through high-speed shredders or large mincing machines. The rind of the cheese may also be size-reduced, using counter-rotating stainless steel rollers, to particles sufficiently small (< 1 mm) to enable the adequate uptake of moisture during subsequent processing. Increasing the surface area of the cheese, by size reduction, increases the homogeneity of the formulated blend, maximizes the surface area of the cheese which facilitates heat transfer to the blend during subsequent processing and the interaction between the cheese and other ingredients (e.g., between ES). Pre-mixing of formulation materials
In batch cooking, the finely ground cheese is conveyed directly to the cooker where it is blended with ES, water and optional ingredients. Alternatively, the cheese may
354
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
Hard cheese
Semi-hard cheese
Derindering, cleaning
Rindlesscheese
Soft cheese
Removing, wrapping, cleaning
Washing in water, brushing, cleaning
Derinding, cleaning
I
Cutting machinery
Additives, other ingredients, water
I Rollers I t
1
I T
t Mixer I
Cutter
1
,
l
Processing I cooker
1
I Ho~o0en'zerI Filling machines I I [ ~ I
I Tro"e~s I J 1 Ventilator
Blocks
Portions
I coo, c
I
er I
I ~onve~or~e'It ~
I
Cans
Pallets
I
I
Icoo,,n0 tunne'l
1Cooling tunnel I
l VVar~roo~ I
Sterilizer I Sorting, labeling machines I"
I Slowly cooling I
L
I l Processed cheese
I-
Schematic overview of the manufacturing process for pasteurized processed cheese products.
Cooling
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
be pre-mixed at room temperature with ES (and some or all of the water and optional ingredients) using various types of pre-blenders (e.g., Drais, Blentech). Some cheese cookers, with interchangeable bowls, allow one batch to be filled and pre-mixed while another is simultaneously being cooked (processed). Pre-mixing has two main effects: 9 it allows the physico-chemical changes, which occur during processing (e.g., water uptake by the protein), to take place at a lower temperature prior to cooking; 9 it is conductive to a more uniform quality in the end product. For a given cheese variety, variations in cheese composition (e.g., pH, calcium-to-casein ratio and the level of intact casein) can occur owing to variations in milk composition, cheesemaking conditions and degree of maturation. In turn, such variations can affect its processability, e.g., how readily the para-casein in the cheese becomes hydrated and emulsifies free fat during subsequent processing. Pre-mixing evens out the effects of differences in composition and proteolysis, and thus processability, of the raw materials (e.g., cheese) on the consistency of the final product. The efficacy of pre-mixing depends on the type and the capacity of the pre-blender and the capacity of the cooker. However, the capacity of the pre-blender should not be so great as to cause a considerable difference in the premixing time between the first and the last sub-batches withdrawn for processing. Otherwise, time-related differences in the degree of physico-chemical changes (as discussed below) between the first and the last lots of a given pre-mix at processing could lead to differences in processability and ultimately in the consistency of the end product. The degree of physico-chemical change in a blend after a given time depends on the type of pre-blender which influences the level of shear applied, the shear rate and the degree of mixing and interaction between the different ingredients. Processing of the blend Following pre-mixing, the blend is tipped into bins and carried on wheels or rails to a hoist, and discharged into the cooker, where it is processed. When pre-mixing is not practised, the ingredients are added directly to the cooker. The order of addition of ingredients varies with plant practices, cooker type, overall plant design and duration of cooking. A typical order of addition is: ground cheese, a dry blend of ES and optional dairy ingredients (e.g., skim milk powder), water and flavours. When the cooking time is relatively short, the ES may be dispersed in a portion of
355
the water prior to addition and only a portion of the water added at the beginning of processing. This approach minimizes the time required for the ES to dissolve during cooking, increases the concentration of ES during early cooking and thereby enhances the effectiveness of the ES in promoting the desired physico-chemical changes in the blend. After a pre-set time, the remaining water may be added manually, delivered by metering pump, or drawn in by vacuum inside the vessel. Flavours may be added later in the process to minimize the loss of volatile flavour compounds. Processing refers to the heat treatment of the blend, by direct or indirect steam, with constant agitation. Application of a partial vacuum during cooking is optional, but may be used to regulate moisture when using direct steam injection, and is also beneficial in removing air and thus preventing the presence of air openings in the finished, set product. Processing has two main functions: 9 to kill any potential pathogenic and spoilage microorganisms, and thereby extend the shelf-life of the product; 9 to facilitate the physico-chemical and microstructural changes which transform the blend to an end product with the desired characteristics and physico-chemical stability. Processing may be performed in batch (e.g., Stephan, Damrow, Blentech, Scanima) or continuous cookers (e.g., Kombinator, Votator, Choc-Steriliser) connected to supplies of water, steam and vacuum. The temperature-time treatment in batch processing varies (e.g., 70-95 ~ for 4-15 min) depending on the formulation, the extent of agitation, the desired product texture, the body and the shelf-life characteristics. The heat treatments are generally sufficient to kill vegetative cells (Warburton et al., 1986); they are not adequate to eliminate microbial spores (see 'Characteristics of different ES in the manufacture of PCPs and ACPs'). However, a temperature > 130 ~ may be required to kill some spores. A temperature of 140 ~ can be achieved in continuous cookers by virtue of their design (Zehren and Nusbaum, 1992). For example, scraped surface heat exchangers which maximize the surface area of contact between the heating medium (e.g., stainless steel heated by steam, oil or hot water) and the blend, ensure sufficient agitation to prevent burn-on of the blend on the heat transfer surface. In continuous cookers, the blend is, typically, heated to and held at 140 ~ for 5-20 s and then cooled to 70-95 ~ by flash evaporation of moisture due to a pressure drop, or by passing through scrape surface tubular coolers. The product is then held at this temperature for 4-15 rain to allow adequate time for interaction of
356
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
the different blend ingredients, the desired physicochemical changes to occur and the development of the desired textural characteristics. The blend thickens progressively with holding time at 70-95 ~ Processing may also be performed continuously using extrusion whereby the blend of all ingredients is pumped directly to a twin-screw extruder and worked at a temperature of 70-90 ~ (Zuber et al., 1987; Blond et al., 1988; Tatsumi et al., 1989; Begueria, 1999). This form of cooking gives a high degree of protein hydration and emulsification very rapidly and may be used to produce ES-free PCPs. Extrusion cooking results in unidirectional alignment of the layers of the molten mass through die plates and is conducive to the formation of a fibrous texture. Hence, microscopic studies have shown that extrusion-cooked pasteurized processed Gouda and Cheddar (containing added whey protein concentrate (WPC)) have superior fibrousness compared to the corresponding kettle-cooked (control) PCPs (Ido et al., 1993).
tic film, e.g., saran-coated polyester, which is automatically flattened and crimped into a chain of individual wrapped slices using crimping conveyors and rotating crimping heads (Zehren and Nusbaum, 1992). The chain of slices is then passed through a water-cooling tank and cooled to <10 ~ dried by removing water using fans and/or scrapers, and finally cut into individual slices which are stacked and packed. Alternatively, the hot molten cheese may be pumped through a manifold with 8-12 nozzles which extrude it in the form of a continuous sheet onto the first of two or three counter-rotating refrigerated chill rolls which cool it rapidly from 80-70 ~ to <30 ~ The cooled sheet is then automatically cut into parallel ribbons, each of which is cut into slices which are individually wrapped in plastic films, stacked and packed. Principles of manufacture of PCPs
However, since homogenization requires capital investment, increased operating and maintenance costs, it is practised mainly for high-fat PCPs only (Carie and Kalab, 1993).
The principles of processed cheese manufacture have been reviewed extensively (e.g., Guinee, 1987; Zehren and Nusbaum, 1992; Carie and Kalab, 1993; Fox et al., 1996, 2000; Carie, 2000). Processing in the presence of ES, such as sodium citrates and sodium phosphates, results in a number of physico-chemical changes, which bring about a structural transformation from a 'coarse' oil-in-water (o/w) emulsion physically encased within a particulate cheese para-casein matrix, as in natural cheese, to a 'finer' o/w emulsion in a concentrated para-casein(ate) dispersion, in PCPs. Moreover, the composition of the emulsifier differs; in natural cheese, it is the native fat-globule membrane which consists mainly of protein and phospholipids (cf., Fox and McSweeney, 1998) and in PCPs it is a reformed membrane of re-hydrated para-casein(ate) which interacts, to a greater or lesser degree, with the casein in the bulk phase. Since the structural change is central to the formation of a physico-chemically stable PCP and its functionality (e.g., rheological and cooking properties), the microstructures of both the natural and the processed cheeses are discussed below.
Hot packing and cooling For most packaging formats, typically, the processed blend is conveyed (e.g., pumped directly or by gravity flow) from the cooker (e.g., by way of hopper) or homogenizer to the filling machine (sometimes via an intermediate buffer tank with gentle agitation). Numerous packaging formats are possible through the use of specialized filling machines (Meyer, 1973; Zehren and Nusbaum, 1992): individually wrapped portions (e.g., foil-wrapped triangles), blocks, sausageshapes, cans, tubes or slices. In the manufacture of slices, the hot molten cheese is pumped continuously into an endless 'tube' of plas-
Microstructure of rennet-curd cheese The rennet-induced coagulation of milk is characterized by casein micelles aggregating into interconnected clusters and forming a network in which fat globules are interspersed as loose inclusions (Gavarie etal., 1989). Electron microscopy (EM) is the method of choice to study this structure (Brooker, 1979; Kalab, 1983, 1995), particularly during the early stages of its development before the minute para-casein particles fuse into larger clusters during cheesemaking. Optical or light microscopy (LM) using specific staining to distinguish different components (Flint, 1994) is used to study the general structure of various cheeses
Homogenization The hot molten mass may be homogenized, with typical first and second stage pressures of 15 and 5 MPa, respectively. Homogenization has a number of effects (Meyer, 1973):
9 it assists in further mixing and size reduction of any coarse (e.g., rind) or undissolved particles (e.g., ES, dry ingredients), and thereby contributes to a more homogeneous and smooth end product; 9 it results in further shearing of the blend and interaction of blend ingredients; 9 promotes a finer dispersion of fat droplets (Walstra and Jenness, 1984); 9 generally promotes thickening.
Pasteurized Processed C h e e s e and Substitute/Imitation C h e e s e Products
(Awad et al., 2002). Confocal laser scanning microscopy (CLSM), which uses monochromatic laser light, is gaining popularity (Auty et al., 2001) for its ability to section the sample optically by focusing at predetermined levels below the surface of the sample. The resulting stack of images is then processed by a computer to produce a view of the three-dimensional structure. Both optical and electron microscopy have been used extensively to study the microstructures of milk gels and cheeses (Hall and Creamer, 1972; Knoop, 1972; R~egg and Blanc, 1972; Kimber et al., 1974; Kalab, 1977, 1979; de Jong, 1978; Green et al., 1981, 1983; Kiely etal., 1992, 1993; Mistry and Anderson, 1993; Bryant et al., 1995; Desai and Nolting, 1995; Guinee etal., 1998, 1999, 2000a; Auty et al., 2001). The protein matrices of both acid- and rennetinduced milk gels are particulate (Fig. 3; GavariC et al., 1989), being composed of entangled clusters of partially fused casein or para-casein aggregates. Ongoing aggregation of the casein, or para-casein, and whey expulsion lead to a gradual fusion of the para-casein network. Consequently, the matrix changes from being particulate to a highly fused aggregated structure (Fig. 4). The integrity of the matrix is maintained by various intra- and inter-aggregate electrostatic and
357
hydrophobic attractions between amino acid side groups on the para-casein molecules, and between calcium ions and organic serine phosphate groups or ionized carboxyl residues (calcium bridges; Walstra and van Vliet, 1986). The protein network is essentially continuous, extending in all directions, although some discontinuities exist at the macro- and micro-structural levels. Discontinuities at the macro-structural level exist in the form of curd granule junctions and, in Cheddar and related dry-salted cheese varieties, as curd chip junctions. Both kinds of junction are discernible by the naked eye in appropriately prepared sections (Kalab and Emmons, 1978; Brooker, 1979; Kalab, 1979; Lowrie et al., 1982; Ruegg et al., 1985; R(iegg and Blanc, 1987; Kalab et al., 1988). Unlike the interior of the curd particles, which consists of protein and fat at a ratio corresponding closely to that of the overall cheese, the junctions are comprised mainly of casein, being almost devoid of fat. The difference in cheese composition between the interior and the surface of curd particles arises as a result of the cutting or breaking of the coagulated milk (gel) into curd particles, which leads to the loss of fat globules from the freshly cut surfaces into the surrounding whey. As the protein matrix contracts and adjoining curd particles
Rennet gel from non-homogenized milk, showing fat globules (F; retained by post-fixing the gels with osmium tetraoxide) encased within a particulate para-casein matrix (P). Bar - 5 i~m (courtesy of M. Kalab).
358
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
in the interior and the exterior of curd particles and thus to differences in structure-function relationships. The protein matrix occludes fat globules (clumped or coalesced to varying degrees), moisture and its dissolved solutes (minerals, lactic acid, peptides and amino acids) and enzymes (e.g., residual rennet, proteinases and peptidases from starter and non-starter micro-organisms; Kimber etal., 1974; Laloy etal., 1996; Guinee et al., 2000a). The fat phase may be described as a concentrated o/w emulsion within a para-casein network. Evidence that clumping and coalescence of fat globules occur in stirred-curd cheeses is provided by both transmission (TEM) and scanning electron microscopy (SEM); in TEM micrographs (Fig. 5), the clumps retain their fat globule membranes. Scanning electron microscopy micrographs, obtained by examining cheese samples (e.g., Cheddar) from which the fat globules had been extracted during sample preparation, reveal irregularly shaped voids in the para-casein matrix (Mistry and Anderson, 1993; Bryant et al., 1995; Fig. 4). The occurrence of coalesced fat in the form of elongated pools between the para-casein fibres has been similarly demonstrated in Mozzarella and String cheeses by SEM (Taneya et al., 1992; Kalab, 1993, 1995; McMahon et al., 1993, 1999; Tunick et al., 1993) and CLSM (Fig. 6; Auty et al., 2001; Guinee et al., 2002). The presence of fat clumps and pools suggests partial coalescence of denuded liquid fat droplets, probably formed as a consequence of damage to the native fat globule membrane during curd manufacture, handling and plasticization, while the curd is still warm. Transmission electron microscopy micrographs
Scanning electron micrographs of full-fat (33.0%, w/w) Cheddar cheese at low (1800• a) or high (7000• b) magnification. The arrows correspond to the para-casein matrix and the arrowheads to the areas occupied by fat and free serum prior to their removal during sample preparation; bacteria (most likely starter lactococci) are visible in b, being concentrated mainly at the fat-para-casein interface ((a) reproduced with permission from the Society of Dairy Technology and adapted from Guinee et aL, 1998 and (b) adapted from Fenelon et aL, 1999).
mat through their fat-depleted surface layers, these fatdepleted areas become part of the internal cheese structure. Curd chips form another type of junction in Cheddar cheese and related dry-salted varieties (Lowrie etal., 1982). The difference in cheese composition between the interior and the surface of curd particles (or chips) probably leads to differences in the molecular attractions between contiguous para-casein layers
Transmission electron micrograph showing the protein matrix (M) of 1-day-old Cheddar cheese interspersed with fat globules (F) encased in fat globule membranes (arrows); B = bacterium (from Cari6 and Kalab, 1993).
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
359
Bar = 25 gm Confocal scanning laser micrographs showing protein (a) and fat (b) as light areas against a dark background in 1-dayold unheated Mozzarella cheeses. Bar corresponds to 25 i~m (from M.A.E. Auty and T.P. Guinee, unpublished results).
taken during the course of Cheddar manufacture clearly show the aggregation of fat globules, which is first notable at maximum scald, increases with the progression of cheesemaking as the protein network shrinks and forces the fat globules into closer proximity (Kimber etal., 1974; Kal~ib, 1995; Laloy etal., 1996). In the temperature range used for cheesemaking ( - 3 0 - 5 5 ~ most or all of the milk fat is liquid (Norris et al., 1973) and therefore flows on the application of stress. Some coalescence of the fat globules probably also occurs during the ripening of Cheddar, Mozzarella and other varieties, as reflected by the increases in level of fat that can be expressed from the cheese at room temperature when subjected to hydraulic pressure or centrifugation (Guinee et al., 1997, 2000a; Thierry et al., 1998) or by extraction of the melted cheese (at - 7 4 ~ with a water/methanol mixture, followed by centrifugation (Yun et al., 1993). The increase in the degree of aggregation of fat during ageing, as revealed by SEM (Tunick and Shieh, 1995), also supports this view. Coalescence is possible, as a large quantity of fat ( - 2 0 - 3 0 % of total) is still expected to be liquid at the ripening temperature ( - 4 - 7 ~ used for Cheddar or Mozzarella (Norris et al., 1973). The agerelated increase in free (expressible) fat may be accentuated by a possible increase in the permeability of the fat globule membrane during maturation due to storagerelated hydrolysis of membrane components by lipoprotein lipase activity (Sugimoto et al., 1983; Deeth, 1997) and by proteolysis of the casein matrix, which holds the fat globules in place (Tunick and Shieh, 1995).
In contrast to Cheddar, Mozzarella and String cheeses, relatively little clumping and coalescence of fat globules is evident in other cheese types such as Cheshire, Gouda (Hall and Creamer, 1972) and Meshanger cheese (de Jong, 1978). Heating natural cheeses in the absence of ES
At the microstructural level, CLSM indicates that heating of Cheddar to 95 ~ results in extensive clumping and coalescence of fat globules, and a less homogeneous distribution of the fat and the para-casein phase (Fig. 7; Guinee and Law, 2002). The ensuing partial phase separation increases with the fat content of the cheese and decreases with homogenization of the cheesemilk (Guinee et al., 2000b). Similarly, Paquet and Kal~ib (1988) observed, using SEM, that heating Mozzarella cheese in a conventional or microwave oven resulted in extensive coalescence of fat globules and shrinkage of the protein matrix. The heat-induced effects were the most severe in high-fat cheeses, less in low-fat cheeses and the least in processed cheese (Paquet and Kalab, 1988). Hence, application of heat (70-90 ~ and mechanical shear to natural cheese, as in processing, without the presence of stabilizers usually results in the formation of a heterogeneous, gummy, pudding-like mass which undergoes extensive oiling-off and moisture exudation during manufacture and, especially, on cooling. These defects arise from: (i) coalescence of the liquefied fat due to the shearing of the fat globule membrane and (ii) partial dehydration/aggregation and shrinkage of the
360
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
Bar = 25 #m Confocal scanning laser micrographs of 5-day-old unheated Cheddar cheese (a, b), and the same cheese after heating to 95 ~ and then allowed to cool to room temperature (c, d). The micrographs show protein (a, c - l o n g arrows) and fat (b, d - short arrows) as light areas against a dark background. Bar corresponds to 25 ~m (modified from Guinee et aL, 2000b).
para-casein matrix induced by the relatively low pH of cheese (i.e., for most cheeses, <5.7) and high temperatures applied during processing. The modified structure, consisting of a shrunken para-casein matrix with large pools of free oil and free moisture, has impaired ability to occlude fat and free moisture. Consequently, free moisture and de-emulsified liquefied fat seep through the more porous, modified structure. The role of ES in the formation of a physicochemically stable product
In the presence of ES, high heat and shear result in the formation of a smooth, homogeneous, stable product. This transition is facilitated by the ES-induced partial hydration and solubilization of para-casein which emulsifies the dispersed droplets of free fat (Templeton and Sommer, 1936; Meyer, 1973; Rayan et al., 1980; Lee etal., 1986; Cavalier-Salou and Cheftel, 1991; Marchesseau etal., 1997; Bowland and Foegeding, 2001). The fat, released by heating, is emulsified into
small globules (Fig. 8; Rayan et al., 1980) and new membranes develop on fat particle surfaces (Cari~ and Kal~ib, 1993). Extensive emulsification produces fat globules smaller than 1 ~m in diameter (Heertje et al., 1981; Tamime et al., 1990), particularly if the processed cheese is subsequently homogenized. The most commonly used ES include sodium citrates, sodium orthophosphates, sodium pyrophosphates, sodium tripolyphosphates, sodium polyphosphates (e.g., Calgon), basic sodium aluminium phosphates (e.g., Kasal) and phosphate blends (e.g.,JOHA and SOLVA blends from BK Giulini Chemie & Co., OHG, Ladenburg, Germany; Kasomel blends from Prayon S.A., Siege Social, B-4480 Engis, Belgium). These salts generally have a monovalent cation (i.e., sodium) and a polyvalent anion (e.g., phosphate). While the salts are not emulsifiers per se, they promote, with the aid of heat and shear, a series of physico-chemical changes within the cheese blend which result in rehydration of the insoluble aggregated para-casein (matrix) and its conversion to an
Pasteurized Processed C h e e s e and Substitute/Imitation
Scanning electron micrograph showing the protein matrix (M) of processed cheese interspersed with fat globules (asterisks). Insoluble calcium phosphate crystals (white arrow), in the lower left, differ in appearance from imprints of soluble emulsifying salts (arrow heads). Bar corresponds to 25 tzm (adapted from Kal~.b et aL, 1987).
active emulsifying agent. These changes include calcium sequestration, para-casein hydration and dispersal, upward pH adjustment and stabilization (buffering), emulsification and structure formation (Templeton and Sommer, 1936; Becker and Ney, 1965; Morr, 1967; Lee et al., 1986; Cavalier-Salou and Cheftel, 1991; Marchesseau et al., 1997), and are discussed briefly below. While ES are the main agents used to promote para-casein hydration and fat emulsification during the formation of PCPs and ACPs, other emulsifying agents, such as Tween 80, lecithin and protein hydrolysates, have been used experimentally (Marshall, 1990; Drake et al., 1999; Kwak et al., 2002). The ability to sequester calcium is one of the more important functions of the ES. The principal caseins in cheese (Ors1-, Ots2-, [3-) are amphilic in nature, having both apolar lipophilic segments and polar hydrophilic segments which contain most of the sepine phosphate (Fox and McSweeney, 1998). This structure allows the caseins to function as emulsifiers (Mulvihill, 1992). In cheese, a large proportion (i.e., 65% of total in Cheddar at pH --5.3) of the calcium is insoluble (Guinee et al., 2000c) and probably exists in the form of calcium-phosphate complexes precipitated onto the casein matrix, or in the form of calcium bridges which interlock and aggregate the para-casein molecules (Walstra and van Vliet, 1986). Precipitated calcium phosphate probably reduces the intermolecular repulsive effect of the negative charge on the casein molecules and, thereby, enhances the degree of casein aggregation (Horne, 1998). Consequently, the paracasein in cheese is essentially insoluble; it is estimated that only -15%, w/w, of the total moisture in cheese is
C h e e s e Products
361
bound to the protein (Geurts et al., 1974). Calcium sequestration involves the exchange of the Ca 2+ (attached to casein via the carboxyl groups of acidic amino acids and/or by phosphoseryl residues, or precipitated on the matrix) of the para-casein for the Na + of the ES. The sequestration of the calcium results in partial hydration of the insoluble para-casein and conversion to a sodium phosphate para-caseinate dispersion (sol; Morr, 1967; Nakajima et al., 1975; Sood etal., 1979; Wagner and Wagner-Hering, 1981; Lee et al., 1986; Marchesseau et al., 1997). The increase in the hydration of para-casein is paralleled by large increases in the levels of water-soluble N, and N that is non-sedimentable on ultracentrifugation. The degree of calcium sequestration and casein hydration (as determined by the proportion of total N that is nonsedimentable on ultracentrifugation of the processed cheese) is dependent on the processing conditions and the type and level of ES (calcium chelating strength, pH and buffering capacity; Irani and Callis, 1962; van Wazer, 1971; Thomas et al., 1980; Lee et al., 1986; Cavalier-Salou and Cheftel, 1991; Fig. 9). However, the mechanism by which added ES chelates calcium and increases para-casein hydration during manufacture is not entirely clear; this may differ with type and concentration of ES (cf. Nakajima et al., 1975). In the commercial manufacture of ACPs and PCPs, trisodium citrate, as the sole ES, is generally added at a much higher level (e.g., 3%, w/w) than orthophosphates (e.g., 1%, w/w). This suggests that added orthophosphates may largely sequester calcium bound 100
"
9
r
~o 90 80 70
o~ ~.
60 50
~o 40 0
0.5
1 1.5 2 Emulsifying salt, % w/w
I
!
2.5
3
Influence of concentration of emulsifying salt on the percentage of total N, which was non-sedimentable on ultracentrifugation (300 000 g x 1 h at 20 ~ of a dispersion prepared by blending a 5 g sample of analogue cheese in distilled water and different levels of trisodium citrate (O) or sodium tripolyphosphate (A; redrawn from Cavalier-Salou and Cheftel, 1991).
362
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
to phosphoserine residues (i.e., in the form of calcium bridges) whereas added citrate may additionally sequester calcium from precipitated calcium phosphate. Thus, it is noteworthy that the content of non-sedimetable soluble calcium in ACPs decreases with increasing level (0-3%, w/w) of added sodium phosphate ES (disodium monohydrogen phosphate, tetrasodium pyrophosphate and sodium tripolyphosphate) and increases marginally with trisodium citrate (CavalierSalou and Cheftel, 1991). The increase in para-casein hydration during processing is paralleled by a physical swelling of the caseinate dispersion (Nakajima et al., 1975) and an increase in the viscosity of the melting processed cheese mass (Wagner and Wagner-Hering, 1981). The increase in viscosity is frequently referred to as creaming in the processed cheese industry (see 'Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs'). The use of the correct blend of ES usually shifts the pH of cheese upwards (typically from---5.0-5.5 in the natural cheese to 5.6-5.9 in the PCP) and stabilizes it by virtue of their high buffering capacity (Gupta et al., 1984; Cavalier-Salou and Cheftel, 1991; Marchesseau et al., 1997). This change contributes to an enhanced calcium-sequestering ability of the sodium phosphate ES per se (Irani and Callis, 1962) and an increased negative charge on the para-caseinate. The latter changes lead to an increased casein hydration and a more open reactive sodium (phosphate) para-caseinate conformation with superior water-binding and emulsifying properties (Nakajima et al., 1975; Lamure et al., 1988; Marchesseau et al., 1997). Hence, the buffering capacity of the ES is a critical factor controlling the rheological, textural and melting attributes of PCPs and ACPs (Rayan et al., 1980; Thomas et al., 1980; Gupta et al., 1984; Cavalier-Salou and Cheftel, 1991; Savello et al., 1989; Guinee and Corcoran, 1994). The increase in hydration and dispersion of the paracasein, sometimes referred to as peptization, is enhanced by increasing the temperature and the shear within the normal limits applied during processing (see 'Processing of the blend'). Under the conditions of cheese processing, the dispersed hydrated para-caseinate contributes to emulsification by coating the surfaces of dispersed free fat droplets, and to emulsion stability by immobilization of a large amount of free water (Phillips, 1981; Mulvihill, 1992). During the cooling of PCPs, the homogeneous molten viscous mass sets to form its characteristic body, which, depending on blend formulation, processing conditions and cooling rate, may vary, from a firm sliceable product to a semi-soft spreadable consistency. Factors contributing to the setting probably
include fat crystallization, protein-protein interactions, and interactions between the dispersed emulsified fat globules and the bulk phase para-casein. The occurrence of rheological changes in PCPs during storage, to a degree dependent on storage time and temperature in the range 10-30 ~ (Tamime et al., 1990), lends support to the contribution of the latter interactions. Comparison of the microstructure of natural and processed cheeses indicates that the latter differs markedly from the former by the absence of curd granule and/or curd chip junctions and the more homogeneous distributions of the fat and protein phases. Micro-structure of PCPs and ACPs
Micro-structural studies on PCPs or ACPs indicate that the structure typically consists of a concentrated emulsion of discrete, rounded fat droplets of varying size (typically ---1-10 Ixm) in a hydrated protein matrix (Fig. 8; Kimura et al., 1978; Rayan et al., 1980; Taneya et al., 1980; Heertje et al., 1981; Lee et al., 1981; Kalab et al., 1987; Savello etal., 1989; Tamime etal., 1990; Kal~ib, 1995; Guinee et al., 1999; Auty et al., 2001). There is less clumping or coalescence of fat globules than in natural cheese. Consequently, the mean fat globule size tends to be generally smaller (Sutheerawattananonda and Bastian, 1995), although it varies depending on the type and level of ES, milk protein additions, processing time and extent of shear (Rayan etal., 1980; Kal~ib etal., 1987, 1991; Savello etal., 1989; Tamime etal., 1990). Generally, for most ES, the fat globule size decreases as the processing time at a high temperature increases, e.g., up to 40 min (Rayan et al., 1980; Kalab et al., 1987). The para-caseinate membranes of the emulsified fat globules appear to attach to the matrix strands, thereby contributing to the continuity of the matrix. The positive correlations between the degree of emulsification (DE) and firmness or elasticity, and the inverse relationship between DE and flowability of PCPs support this suggestion (Rayan et al., 1980; Carie et al., 1985; Savello et al., 1989). The incorporation of emulsified para-caseinate-coated fat globules, which can be considered as pseudo-protein particles, into the new structural matrix may be considered as increasing the effective protein concentration (van Vliet and Dentener-Kikkert, 1982; Marchesseau etal., 1997; Michalski et al., 2002). The fat globule size is important as it influences the firmness of the final PCP and the ability of the fat to become free and contribute to oiling-off when the PCP is subsequently cooked for consumption. When cheese is baked or grilled, some oiling-off is desirable, as it limits drying-out of the cheese and thus contributes to
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
the desired flowability, succulence and surface sheen of the melted product (see 'Cheese as an Ingredient', Volume 2). Generally, for a given formulation, a reduction in the mean diameter of the emulsified fat globules gives PCPs which are firmer and, which on cooking, exhibit a low tendency to oil-off and have poor flowability (Rayan etal., 1980; Savello etal., 1989). Comparative studies on the effect of different ES indicate that, for a given processing time, the mean fat globule diameter is generally smallest with tetrasodium pyrophosphate (TSPP) or sodium tripolyphosphates (STPP), largest with basic sodium aluminium phosphate (SALP) and intermediate with trisodium citrate (TSC) or disodium phosphate (DSP; Rayan et al., 1980; CavalierSalou and Cheftel, 1991). Hence, in practice, SALP is generally claimed to give PCPs and ACPs with good melting properties. Increasing the concentration of ES (1-4%, w/w) and processing temperature (80-140 ~ results in a progressive decrease in mean fat globule diameter and a concomitant increase in firmness. Generally, for most ES, the fat globule size decreases as the holding time at a high temperature increases, e.g., up to 40 min (Rayan et al., 1980; Kalab et al., 1987); the resultant products become firmer, more elastic and less flowable. High-resolution TEM (e.g., 60 000 x) has been used to study the structure of the protein matrix in PCPs (Kimura et al., 1978; Taneya et al., 1980; Heertje et al., 1981; Klostermeyer et al., 1984; Lee et al., 1996). The protein phase consists of varying proportions of individual para-caseinate particles and strands, which are probably formed through end-to-end association of para-caseinate particles. The individual particles (20-30 nm diameter) may correspond to casein submicelles released from the para-casein micelles in the matrix of the natural cheese as a result of calcium sequestration by the ES (Kimura et al., 1978; Taneya et al., 1980; Heertje et al., 1981). The proportions of strands to individual particles vary with the rheolgy and the texture characteristics of the processed cheese. Hard PCPs contain a high level of long protein strands (e.g., ---100 versus 25 b~m) which form a matrix (Fig. 10) that is finer than the protein matrix of natural cheese. In contrast, the protein matrix of soft PCPs usually consists predominantly of individual particles. However, abundant protein strands were also found in a soft processed cheese made with a mix of orthophosphate and polyphosphate when direct steam heating was used (Cari~ and Kal~ib, 1987). The presence of protein strands was apparently associated with the use of polyphosphates but no conclusive study has been reported on this subject. Apart from a few exceptions, including acid-heated coagulated Queso blanco-type cheese (Kakib and Modler,
363
The protein matrices of soft (A) and hard (B) processed cheeses. The black string-like protein strands (highlighted by the arrows) are longer and thicker in the hard product than in the soft product. F = fat globule. Bar corresponds to 0.2 #m (reproduced with permission from Kimura et aL, 1978).
1985) and Paneer (Kalab et al., 1988), it is not possible to identify the type of cheese used to make PCPs. The casein particles in the acid-heat type cheeses, above, have a characteristic core-and-shell uhrastructure (Harwalkar and Kalab, 1981; Kalab and Modler, 1985; Kalab et al., 1988), which is very stable and withstands the conditions of cheese processing. Microstructural analyses of processed cheeses frequently reveal the presence of various crystalline species such as calcium phosphate. Crystallization, if excessive, may be give rise to visual defects, and is discussed in 'Characteristics of different ES in the manufacture of PCPs and ACPs'. Properties of ES important in cheese processing
Differences in the functionality of ES offer the manufacturer of PCPs and ACPs a major lever with which to impart customized characteristics to the finished product, e.g., sliceability, spreadability and meltability (Thomas et al., 1980; Gupta et al., 1984; Abdel-Hamid et al., 2000a,b; Awad et al., 2002). The ES most commonly
364
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
used are sodium citrates, sodium hydrogen orthophosphates, sodium polyphosphates and sodium aluminium phosphates. Other potential emulsifying agents include gluconates, lactates, malates, ammonium salts, gluconic lactones and tartarates (Meyer, 1973; Price and Bush, 1974 a,b; Gupta et al., 1984). Tartaric acid from wine, added as an ingredient, acts as a calcium-sequestering agent in the manufacture of Swiss Cheese Fondue (Schar and Bosset, 2002). At the commercial level, ES are supplied increasingly as blends of phosphates (e.g., Joha C special, Solva 35S) or of phosphates and citrates (e.g., Solva NZ 10), tailormade to impart certain functionalities (e.g., different degrees of meltability, sliceability, spreadability) to different pasteurized products (e.g., blocks, slices, spreadable) manufactured under different conditions (e.g., from cheeses of varying degrees of maturity or using cookers with varying degrees of shear input).
Major types of E$ Salts consisting of a monovalent cation and a polyvalent anion have the best emulsifying characteristics. Of the many citrates available, trisodium citrate (Na3C6HsO7) is used most commonly. Monosodium citrate, when used alone, gives over-acid PCPs which are mealy, acid and crumbly and show a tendency to oiling-off due to poor emulsification (Gupta et al., 1984). The use of disodium citrate as the sole ES also leads to high acidity and to water separation during solidification of the molten PCP (CariC and Kalab, 1993). The dissociation constants (pKa) of citric acid at the ionic strength of milk are 3.0, 4.5 and 4.9 (Walstra and Jenness, 1984). Owing to their acidic properties, mono- and di-sodium citrates may be used to correct the pH of a processed cheese blend, for example, when using a high proportion of very mature, high-pH cheese or skim milk solids. The terminology and manufacture of phosphates has been reviewed by Bell (1971) and van Wazer (1971). The phosphates have a structure in which each phosphorus atom is surrounded tetrahedrally by four oxygen atoms. Neighbouring PO4 groups may react and share one or more oxygen atoms to form m P - - O m P m bonds and form condensed phosphates with a phosphorus content, expressed as percentage P205, >72.5 (Zehren and Nusbaum, 1992). The condensed phosphates are called linear condensed phosphates when one oxygen atom is shared by neighbouring PO4 groups, and metaphosphates (or cyclic phosphates) when three oxygens are shared. The generic structures of some of the sodium phosphates are given in Fig. 11. The phosphate-based ES used in cheese processing are mainly the sodium salts of orthophosphates (e.g., disodium monohydrogen phosphate (Na2HPO4), trisodium
Group Monomers, orthophosphates
Phosphoric acid Potassium dihydrogen orthophosphate Dipotassium hydrogen orthophosphate Tripotassium orthophosphate Sodium dihydrogen orthophosphate Disodium hydrogen orthophosphate Trisodium orthophosphate
Polymers, linearly condensed polyphosphates
Tetrapotassium diphosphate Disodium diphosphate Triosodium diphosphate Tertrasodium diphosphate
Pentapotassium triphosphate pentasodium triphosphate
Structure O II MO - P - OM I OM
0 0 II II M O - P - O - P - OM I I OM OM 0 0 0 II II II MO- P-O-P-O-P-OM I I I OM OM OM
Sodium tetrapolyphosphate 0 0 0 II II II MO - P - O - P - O - P - 0 I I I OM OM OM Sodium hexametaphosphate (Graham's salt) Soluble sodium polyphosphate
0 II P - OM I OM
oF ,o1 o .o- ,~1o- ~-io-~-o~ o. / o;
Insoluble sodium polyphosphate (Madrell's salt) M(n+2)PnO(3n+l)
Cyclical polyphosphates
Sodium trimetaphosphate
o
\\/ P
/
OM
\
O
o
p //\
p
I,o
7\
0
0 O
Sodium tetrametaphosphate
OM
\\/ P
o7
MO... /
OM
~0
\o
P
P
~\o \
/ \oM 70 p //\
0
OM
M: metal ion (Ca, K)
Structure of different sodium phosphates used in the manufacture of pasteurized processed and analogue cheese products (courtesy of M. Cari6).
monophosphate (Na3PO4)) which contain one PO4 group, and linear condensed phosphates such as pyrophosphates (two PO4 groups) and polyphosphates (3-25 PO4 groups, e.g., tripolyphosphate with three PO4 groups). Potassium and sodium aluminium phosphates may also be used in the manufacture of PCPs and ACPs (Karahadian and Lindsay, 1984), e.g., the latter ES is used widely in the manufacture of analogue pizza cheese
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
(APC). Of the orthophosphates, disodium hydrogen orthophosphate (Na2HPO4) is the most commonly used; when used alone, the mono- and trisodium salts tend to give over- or under-acid products, respectively (Templeton and Sommer, 1936; Scharf, 1971; Gupta et al., 1984). Comparative studies have shown that the potassium salts of orthophosphates, pyrophosphates and citrates give PCPs with textural properties similar to those made with the equivalent sodium salts at similar concentrations (Gupta etal., 1984; Karahadian and Lindsay, 1984); however, the use of the potassium salts reduced slightly the flowability of the melted PCs. Hence, while potassium ES may have potential in the preparation of reduced-sodium PCs, they are rarely used in practice as they impart a bitter taste to the finished product, which becomes more pronounced with storage (Templeton and Sommer, 1936), and are more expensive. Some of the characteristics of the commonly used ES in aqueous solution are presented in Table 4. Characteristics of different ES in the manufacture of PCPs and ACPs
The properties of different ES in both PePs and ACPs have been studied and reviewed extensively (Swiatek, 1964; Scharf, 1971; van Wazer, 1971; Meyer, 1973; Tanaka et al., 1979; Rayan et al., 1980; Lee et al., 1986;
365
Carie and KaDb, 1987; Savello et al., 1989; CavalierSalou and Cheftel, 1991; Fox et al., 1996, 2000; AbdelHamid et al., 2000a,b). Discrepancies between these studies vis-/t-vis the influence of ES on different physico-chemical changes exist, probably because of inter-study differences in product formulation (e.g., levels of total protein and intact protein, pH), levels and combinations of ES and processing conditions (e.g., cooker type, degree of shear, time-temperature treatment). However, these studies indicate definite trends that are summarized in Table 5 and discussed below. In practice, individual salts are rarely used, with blends of two or more ES being used widely to combine the best effects of the individual salts (Carie and KaDb, 1993; Abdel-Hamid et al., 2000a,b). Customized blends with specific functionality are prepared by suppliers of ES or developed in-house by manufacturers of PCPs and ACPs. Calcium sequestration. The ability to sequester calcium is closely related to the ability to hydrate and solubilize protein. Calcium sequestration involves the exchange of the divalent calcium cations, which aggregate and interlock the casein molecules in the paracasein network of the cheese or rennet casein, for the monovalent cations of the ES. It is best accomplished
Properties of emulsifying salts used in cheese processinga
Group
Emulsifying salt
Formula
Mol. mass (Da)
Citrates
Monosodium citrate monohydrate Trisodium citrate dihydrate Trisodium citrate undecahydrate Sodium dihydrogen phosphate (SDP) SDP monohydrate SDP dihydrate Disodium hydrogen phosphate (DSP) DSP dihydrate DSP heptahydrate DSP dodecahydrate Trisodium phosphate (TSP) TSP hemihydrate TSP-dodeca-hydrate Disodium pyrophosphate Trisodium pyrophosphate Tetrasodium pyrophosphate Pentasodium tripolyphosphate (PSTPP) PSTPP hexahydrate Sodium tetrapolyphosphate Sodium hexametaphosphate (Graham's salt) Sodium aluminium phosphate
NaH2C6H507.H20 NaH2C6H507.2H20 2NaH2C6H507.11H20 NaH2PO4 NaH2PO4. H20 NaH2PO4-2H20 Na2HPO4 Na2HPO4-2H20 Na2HPO4.7H20 Na2HPO4.12H20 Na3PO4 Na3PO4.0.5H20 Na3PO4.12H20 Na2H2P207 Na3HP2OF.9H20 Na4P207.10H20 Na5P3Olo
232 294 714 120 138 156 142 178 268 358 164 173 380 222 406 446 368
Na5P3010-6H20 Na6P4013 (NaPO3)n
476 470 (102)n
Orthophosphates
Pyrophosphates
Polyphosphates
Aluminium phosphates
NaH14AI3(PO4)8.4H20
a Compiled from Scharf and Kichline (1968), van Wazer (1971), Cari6 and Kal~b (1993).
t:'205 content (%) m
m
59 51 45 5O 40 26 20 44 41 19 64 35 32 58 45 60 70
Solubility at 20 ~ (%) 16.8 75 79.4 85.2
pH(1% solution)
13 32 10 14.6
3.75 8.55 7.95 4.5 4.5 4.5 9.1 9.1 9.1 9.1 11.9 11.9 11.9 4.1 6.7-7.5 10.2 9.7
170.0 157.0
9.7 8.5 6.6
m
39.9 9.3 80 2.0 11 m
m
8.0
366
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
Emulsifying salts commonly used in pasteurized processed cheese products and their properties during cheese processing
Physico-chemical changes during processing Group Citrates Orthophosphates Condensed phosphates Pyrophosphates
Polyphosphates
Commonly used forms
Calcium sequestration
Buffering action
Para-casein hydration
Fat emulsification
Trisodium citrate Disodium phosphate Trisodium phosphate
Low
High
Low
Low
Low
High
Low
Low
Medium
Medium
Very high
Very high
High to very high
Low to very low
High to low
Very high to low
Disodium pyrophosphates Trisodium pyrophosphates Tetrasodium pyrophosphates Pentasodium tripolyphosphate Sodium tetrapolyphosphate Long-chain polyphosphates
Compiled from various sources: Cark5 and Kalab (1993), Fox et al. (1996, 2000), Guinee (2002a).
by ES with a monovalent cation and a polyvalent anion, and effectiveness generally increases with valency of the anion. The general ranking of the calcium sequestration ability of the ES used in PCPs is in the following order: polyphosphates > pyrophosphates > orthophosphates > sodium aluminium phosphate = citrates (Nakajima et al., 1975; Wagner and Wagner-Hering, 1981; Lee etal., 1986; Cavalier-Salou and Cheftel, 1991; earle and Kal~ib, 1993; Chambre and Daurelles, 2000). However, the sequestering ability, especially of the shorter chain phosphates, is strongly influenced by pH. The increased ion-exchange function at higher pH values is attributed to more complete dissociation of the sodium phosphate molecules resulting in the formation of a higher valency anion (van Wazer, 1971). Thus, for the short-chain phosphates, calcium binding generally increases in the following order: NaH2PO4, Na2HPO4, Na2H2P2OT, Na3HP2OT, Na4P207, Na4P207 (Cari~ and Kal~ib, 1987).
pH adjustment and buffering. An appropriate pH value (e.g., 5.6-6.1) during processing is important for several reasons: it affects protein conformation and hydration, solubility of the ES, calcium sequestration by the ES and ultimately the DEE. It also affects the textural and melting characteristics of the final PCP or ACP. The effect of pH on the texture of processed cheese was clearly demonstrated by Karahadian and Lindsay (1984), using mono-, di- or trisodium phosphates for which the respective pH of a 1%, w/v, solution was 4.2, 9.5 or 13.0. Cheese made with NaH2PO4 (low pH) was dry and crumbly, whereas cheese made with Na3PO4 (high pH) was moist and plastic; the texture of cheese made with Na2HPO4 was intermediate. The buffering capacity of sodium phosphates, in the pH range normally encountered in PCPs (i.e., 5.5-6.0), decreases with increasing chain length and is effectively
zero for the long-chain phosphates (n > 10). This decrease in buffering capacity with chain length is due to the corresponding decrease in the number of acid groups per molecule; these occur singly at each end of the polyphosphate chain (van Wazer, 1971). The orthoand pyrophosphates have a high buffering capacity in the pH ranges 2-3, 5.5-7.5 and 10-12; thus, in cheese processing, they are not only very suitable as buffering agents but also as pH-correction agents. Within the citrate group, only the trisodium salt has buffering capacity in the pH range 5.3-6.0; the more acidic mono- and di-sodium citrates give over-acid, crumbly cheese with a propensity to oiling-off (Gupta et al., 1984). The pH of PCPs and ACPs is related to the pH of the ES (blend) used and to its buffering capacity. The pH of ACPs made with trisodium citrate or different sodium phosphate ES, at equal concentrations (3%, w/w), decreases in the following order: tetrasodium pyrophosphate ~ trisodium citrate ~ pentasodium tripolyphosphate > disodium hydrogen phosphate > sodium polyphosphate (Cavalier-Salou and Cheftel, 1991). The pH of cheese increases linearly with the concentration of ES in the range 0-3%, w/w, for trisodium citrate, tetrasodium pyrophosphate, sodium tripolyphosphate and disodium hydrogen phosphate (Cavalier-Salou and Cheftel, 1991). Similar observations have been made by others (Templeton and Sommer, 1936; Swiatek, 1964; Gupta et al., 1984) for PCPs. However, Swiatek (1964) reported that increasing the concentration of polyphosphate had little effect on the pH of PCPs.
Casein hydration and dispersion. The increase in para-casein hydration during the manufacture of PCPs and ACPs is supported by the large increases in the levels of water-soluble N and non-sedimentable N (when a dilute mixture of cheese and water is centrifuged; Templeton and Sommer, 1936; Ito et al., 1976;
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
Thomas et al., 1980; Lee et al., 1986; Cavalier-Salou, 1991). While all the commonly used ES increase casein hydration, there are large differences between the effects of the different salts at the level (-<3%, w/w) typically used in cheese processing. The results of some studies (Templeton and Sommer, 1936; Lee etal., 1986) indicate that para-casein hydration and dispersion decrease with the chain length of sodium phosphates while the results of Cavalier-Salou and Cheftel (1991) suggest the opposite trend. Similarly, Thomas et al. (1980) reported that trisodium citrate gives lower casein hydration than ortho- or pyrophosphates while others (Templeton and Sommer, 1936; Cavalier-Salou and Cheftel, 1991) found it gave levels that were comparable or higher. These discrepancies may be related to differences in the blend formulation and composition, concentration of ES, processing time (Csok, 1982) and pH. Casein hydration for sodium phosphates and trisodium citrate decreases as the pH falls in the range 7.5-5.5, with the decrease at pH <6.5 being particularly large for orthophosphates and trisodium citrate (Lee etal., 1986); the more highly condensed phosphates are the least susceptible to a decrease in pH.
Ability to promote emulsification. The effectiveness of different ES in promoting emulsification in PCPs, as determined from electron microscopy and oiling-off studies, is in the following general order: sodium tripolyphosphates > pyrophosphates > polyphosphates (P > 10) > orthophosphates ~ citrates (slightly) > basic sodium aluminium phosphates (Templeton and Sommer, 1936; Roesler, 1966; Rayan et al., 1980; Thomas et al., 1980; Cavalier-Salou and Cheftel, 1991). This trend generally coincides with that of calcium sequestration. Hydrolysis (stability). Linear condensed phosphates undergo varying degrees of hydrolysis and conversion to orthophosphates during processing and storage of PCPs (Glandorf, 1964; Roesler, 1966; Scharf, 1971; van Wazer, 1971; Meyer, 1973). Hydrolysis proceeds rapidly to tripolyphosphates and pyrophosphates and then more slowly to orthophosphates. The extent of degradation increases with processing time and temperature, product storage time and temperature, moisture level in the final product and phosphate chain length (Glandorf, 1964). Hydrolysis decreases as the concentration of added ES increases (Glandorf, 1964). In experiments with pasteurized processed Emmental, the level of polyphosphate breakdown (n > 4) during melting at 85 ~ varied from 7% of total for block product (processed for 4 min) to 45% for spreadable product (processed for 10 min; Roesler, 1966). While the breakdown of condensed phosphates to monophosphates was complete in the spreadable cheese after
367
7 weeks, low levels were detectable in block processed cheese even after 12 weeks. The greater extent of polyphosphate degradation in the spreadable processed cheeses is also expected due to their higher pH and moisture values (Scharf, 1971; van Wazer, 1971). The effect of temperature on the hydrolysis of phosphates and polyphosphates in dilute solution (1%, w/v) has been demonstrated clearly by Berger et al. (1989). The consequences of hydrolysis probably include variations in the functionality (e.g., buffering capacity, calcium sequestration) of the ES blend with processing conditions as the ratio of short-chain to long-chain phosphates increases; ultimately, this may affect product pH and degree of casein hydration/emulsification. Continued hydrolysis may lead to several problems during storage. The change in hardness that is frequently observed in PCPs during storage, and which is more pronounced as the storage temperature is raised from 10 to 30 ~ has been attributed to the hydrolysis of polyphosphates to orthophosphates (Ney, 1988; Tamime et al., 1990; Chambre and Daurelles, 2000). This is because the continued effects (calcium sequestration, protein hydration and emulsification) of ES differ as the proportions of phosphates of different chain length change. Other problems associated with the hydrolysis of polyphosphates is an increased propensity to crystallization of ES in the PCP, due to precipitation of dodecahydrate disodium orthophosphate (Na2HPO4"12H20) on product storage (Scharf and Kichline, 1969), and labelling difficulties in relation to declaration of the ES used.
Tendency to crystallize during storage Occurrence and types of crystals. A c o m m o n defect in processed cheese, known at a commercial level as crystallization, is the formation of crystalline deposits on the surface and in the interior of processed cheese. The deposits are visible to the naked eye as a delicate white powdery covering on the surface of the cheese, and sometimes are referred to as a haze or a bloom. A coating of crystals imparts a dull grainy, uneven appearance to the surface, especially in slices, which is otherwise normally smooth and shiny; hence, products containing crystal deposits are not aesthetically pleasing and can sometimes be rejected by the retailer/consumer who may confuse the white deposits with mould contamination. The crystalline deposits are frequently observed by SEM or TEM in PCPs (Fig. 12; Pommert et al., 1988; Carie and Kal~ib, 1993; Kal~ib, 1995). Using various techniques such as electron microscopy, X-ray diffraction analysis, infrared spectroscopy and energy dispersive X-ray spectrometry analysis (EDSA), several crystal types have been identified, including monoclinic calcium
368
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
9 The incomplete dissolution of the ES (especially when excess ES is added), the carryover of crystal inclusions from the natural cheese (e.g., insoluble tyrosine in Swiss cheese (Fluckiger and Schilt, 1963), or calcium lactate from Cheddar cheese (Brooker et al., 1975; Brooker, 1979)). 9 The use of excess lactose (e.g., in added skim milk powder or whey) which leads to the supersaturation and formation of lactose crystals which may then act as nuclei for the crystallization of mineral species (e.g., Ca) which are present at supersaturated levels. The tendency to lactose crystallization may, however, be reduced if the lactose is first hydrolysed enzymatically (Patocka and Jelen, 1988).
Light micrograph of processed cheese crystals of insoluble calcium phosphate (P) and sodium citrate (C), emulsified fat in globules (F) and bacteria (b) dispersed in the protein matrix (M). Bar corresponds to 5 i,m (modified from Cari6 and Kala.b, 1993).
pyrophosphate dihydrate, disodium phosphate dodecahydrate, unreacted melting salts, tyrosine, calcium citrate, lactose and complexes of various materials such as calcium, fatty acids, protein and lactose (Scharf and Michnick, 1967; Scharf and Kichline, 1968, 1969; Morris et al., 1969; Scharf, 1971; Uhlmann et al., 1983; Klostermeyer et al., 1984; Yiu, 1985; Bester and Venter, 1986; Pommert et al., 1988; Kondo et al., 1990). The composition of crystalline structures in natural cheeses and PCPs was studied by Washam et al. (1985) using SEM, EDSA and X-ray diffraction. Yiu (1985) identified calcium phosphate crystals by optical microscopy using Alizarin Red as a stain specific for calcium. Calcium phosphate aggregates were observed to grow beyond the size of 30 b~m in diameter in PCP made with sodium diphosphate (Rayan et al., 1980). Other crystalline inclusions may be calcium lactate in the form of randomly arranged aggregates up to 80 I~m in diameter (Brooker et al., 1975; Brooker, 1979) or crystalline amino acids such as tyrosine, e.g., in Swiss cheese (Fluckiger and Schilt, 1963). Careful standardization of the moisture content and the use of phosphates as ES were credited with the lower incidence of calcium lactate crystals in processed cheese than in natural cheese. Crystals of a tertiary sodium calcium citrate, NaCaC6H507, were identified in PCPs by Klostermeyer et al. (1984). Eliminating citrate from the ES blend may prevent their development. Major causes of crystallization.
The main causes of
crystallization in PCPs and ACPs probably include: 9 The formation of insoluble calcium phosphate crystals (as a result of the interaction between the anion of the ES and the Ca of para-casein).
Treatments which lead to dehydration of the cheese, such as smoking, are conducive to crystal growth on the processed cheese surface. Other factors (e.g., pH) may contribute to the formation of crystal deposits involving ES in PCPs and ACPs, as discussed below. Effects of calcium phosphate level and pH. Hard rennet-curd cheeses tend to be supersaturated with calcium phosphate (cf., Morris et al., 1988). In Cheddar cheese, only ---38% of total calcium (i.e., ---750 mg/100 g) is soluble at pH 5.0. This is equivalent to a calcium concentration of " - 7 2 0 m g / 1 0 0 m l cheese serum (Guinee et al., 2000c), which is ---&fold the total soluble Ca concentration in milk at pH 5.0 (van Hooydonk et al., 1986). Hence, it is not surprising that crystalline inclusions already noticeable in natural cheese (Brooker et al., 1975; Blanc et al., 1979; Bottazzi et al., 1982; Paquet and Kal~ib, 1988), particularly those composed of insoluble calcium phosphate, which are not affected by processing, are subsequently also found in processed cheese. For a given formulation, the risk of crystal formation in the resultant PCP probably decreases as the levels of calcium and phosphate in the natural cheese used decrease. Hence, it would be expected that the risk would be in the following order: Camembert-type cheese < Blue cheese < Cheshire < Cheddar ~ Mozzarella < Emmental. The addition of relatively large quantities of sodium phosphate ES (e.g., up to 3%, w/w) accentuates the risk of precipitating salts containing phosphate. Moreover, much of the water in PCPs is bound and, presumably, not available for solution of salts or other solutes such as lactose and amino acids. The levels of bound and free water in PCP made by heating at 95 ~ for 1-30 min varied from 1.4 to 1.6 g/g solidsnon-fat (SNF) and from 0.5 to 0.7 g/g SNE respectively (Csok, 1982). For a given formulation and processing conditions, the pH and the concentration of ES have major effects on the susceptibility to crystal formation, especially where sodium orthophosphates are used as ES. This is
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
because the pH determines the level of dissociation and hence the ratio of salt-to-acid forms of the salt, according to the Henderson-Hasselbalch Equation:
7.5
369
-
7
O .XE
pH-
salt(e.g. Na2HPO4) pKa + log acid(e.g" NaH2PO4)
-o 6.5
o o
Oo
cat}
o
Bacteriocidal effects. Cheese is the main ingredient used in PCPs. The occurrence of pathogens in cheese has been reviewed recently (Fox et al., 2000). While natural cheese is a relatively safe food, pathogenic bacteria may occur in cheese, especially in those made from raw milk (Fox et al., 2000). However, very few food-poisoning outbreaks have been attributed to cheese, e.g., a total of 32 outbreaks in western Europe, USA and Canada in the period 1970-1997 from an
O
O o
L C~
The salt and the acid forms differ markedly in their solubility in aqueous solution (Table 4) and hence their ratio determines the likelihood of crystallization at a given concentration of ES. At the pH of PCPs (pH 5.5-6.0), NaH2PO4 and Na2HPO4 are the major forms present, irrespective of the type of orthophosphate added, since the pKa values for H3PO4, are 2.14, 6.86 and 12.4 at 25 ~ The ratio of Na2HPO4 to NaH2PO4 varies from 0.1 to 0.15 in PCPs, depending on pH. At the temperatures used during processing (e.g., 75 ~ the disodium salt occurs mainly in the form of NaH2PO4"TH20 and is very soluble (80%, w/v; Scharf and Kichline, 1968). At temperatures <35 ~ the dodecahydrate disodium salt (Na2HPO4"12H20) is the main form of the disodium salt in PCPs (Scharf and Kichline, 1968), and its solubility (~1.5-2.5%, w/v, Na2HPO4) is much lower than that of the monosodium salts (Table 4). Hence, in experimental PC slices made using sodium phosphates, Na2HPO4" 12H20 is the predominant crystalline species (Scharf and Kichline, 1968), and its tendency to crystallize increases markedly with small increases in the pH of the PCP in the pH range 5.5-6.5. However, there is an inverse relationship between the pH required in the PCP to prevent the formation of crystals and the phosphate content of the PCP (Fig. 13). Trisodium citrate is normally used with phosphate in PCPs. However, at the pH of PCPs (pH 5.5-6.0), the forms of citrate present are Na2HC6H507 and NaH2C6HsO7 (e.g., at a molar ratio of --8:1 at pH 6.0). In contrast to Na2HPO4"12H20, the sodium citrates are highly soluble (Table 4). However, the resultant calcium citrates are relatively insoluble, e.g., the solubility of calcium citrate tetrahydrate, Ca(C6HsO7)2, at 18 ~ is 0.08%, w/v, and decreases on reducing the temperature (cf., Scharf and Kichline, 1969). Hence, refrigerated storage of PC increases the susceptibility to the formation of calcium citrate deposits.
O
6
ee ~ ~
_
o
9
O
9
.1o_ 5.5
9
9
5
1.4
Q
I
I
I
I
I
I
i
i
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
P205 content, %, w/w
Relationship between pH and phosphorus content in the development of crystals on processed cheese slices exposed to air. The cheese slices beneath the regression line (O) did not develop crystals whereas samples above (O) developed crystals (NaH2PO4.12H20), as detected using X-ray diffraction analysis; the regression line (-) was fitted to the experimental data points (9 O). (redrawn from Scharf and Kichline, 1968).
estimated cheese production of 235 million tonnes. The micro-organisms involved in these outbreaks of food poisoning included Listeria monocytogenes, Clostridium botulinum, Salmonella spp., Staphylococcus aureus and Escherichia coli O157. The main reasons for the occurrence of these bacteria, which apart from the spores of Cl. botulinum, are killed by pasteurization (72 ~ • 15 s), in cheese are poor starter activity, poor plant hygiene, gross environmental contamination and faulty pasteurization (Fox et al., 2000). Cheese processing normally involves the use of a temperature (70-95 ~ for 4-15 min) that kills the vegetative cells of most bacteria, yeasts and moulds, but not spores. Hence, PCPs may contain viable spores and their vegetative cells, especially of the genus Clostridium, which may originate in the cheese (especially if cows are fed poor-quality silage) or the formulation ingredients and condiments (Meyer, 1973; Thomas, 1977; Wagner and Wagner-Hering, 1981; Sinha and Sinha, 1988; Warburton et al., 1986; Carie and Kalab, 1987). Conditions favourable for the germination of spores (e.g., Cl. tyrobutyricum, Cl. sporogenes) in PCPs during storage include the heat activation of spores at the high processing temperatures, the anaerobic environment and the relatively high pH, water activity (ROegg and Blanc, 1981; Csok, 1982) and moisture content of PCs, compared to most natural cheeses (Russell and Gould, 1991). This may lead to problems such as blowing of cans, protein putrefaction and off-flavours. Perhaps, more importantly, contamination with Cl. botulinum spores could lead to the growth and formation of toxin on storage at a high temperature (e.g., 30-35 ~ to an extent dependent on ES type, moisture level and pH (Kautter et al.,
370
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
1979; Tanaka et al., 1979; Eckner et al., 1994). Bacteria may also gain access to PCPs following manufacture. Various studies have been undertaken to determine the ability of PCPs to host pathogens and thereby simulate the effects of post-processing contamination. These studies have monitored the changes in populations of various pathogenic bacteria inoculated into the cooled PCP, or the development of bacterial toxin during storage (Kautter etal., 1979; Jaskulka et al., 1995). Glass et al. (1998) inoculated PCP slices (water activity, 0.926-0.918; pH, 5.61-5.78; NaC1, 2.5%, w/w) with various strains of pathogenic bacteria and followed their growth over 96 h incubation at 30 ~ The populations of the Listeria monocytogenes, Escherchia coli 0157:H7 and Salmonella serotypes which were inoculated at levels of 1.3 • 103 to 4 • 103cfu/g slice decreased by 0.5-2.0 log counts. In contrast, the population of inoculated Staphylococcus aureus in PCP remained constant over the 96-h period. In a large predictive modelling study involving the inoculation of PCPs with CI. botulinum, Jaskulka et al. (1995) developed a prediction equation to relate the composition of the spreads to clostridial toxigenosis after 6 months' storage at 30 ~ Bacterial spoilage in PCPs is minimized by a number of factors:
Some ES also possess bactericidal properties. Polyphosphates and orthophosphates inhibit the growth of various Salmonella species and many Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes and CI. botulinum, with the effect increasing with the level added (van Wazer, 1971; Tanaka et al., 1979, 1986; Wagner, 1986; Ter Steeg et al., 1995; Loessner etal., 1997). The inhibitory effect of sodium orthophosphates depends on the moisture and the sodium salt levels and pH of the PCP (Tanaka et al., 1986). However, polyphosphates were found to be much more bacteriostatic than orthophosphates in laboratory media (Wagner, 1986) and than both orthophosphates and pyrophosphates in PCPs (Eckner et al., 1994). The general bacteriostatic effect of phosphates may reflect their interactions with bacterial proteins and sequestration of calcium, which generally serves as an important cellular cation and cofactor for some microbial enzymes (Stanier et al., 1981). Citrates appear to have no bacteriostatic effect (Chambre and Daurelles, 2000) and may even be subject to microbial degradation, thus reducing product keeping quality (Cari~ and Kalab, 1987). Tanaka et al. (1979) reported that the inhibitory effect of sodium orthophosphates on the growth of CI. botulinum in pasteurized PCSs with moisture levels in the range 52-58%, w/w, was superior to that of sodium citrates.
9 The addition of preservatives, such as nisin, ascorbic acid, sodium bisulphite, sodium sorbate, sodium propionate or sodium nitrite (Somers and Taylor, 1987; Gouda and E1-Zayat, 1988; Ryser and Marth, 1988; Delves-Broughton, 1990; Russell and Gould, 1991; Roberts and Zottola, 1993; Plockova etal., 1996) or the use of natural cheese made using nisinproducing lactococci (Zottola et al., 1994). 9 Formulating to keep the pH, water activity and moisture level as low as possible and to keep the NaC1 level moderately high (e.g., > 1.5%, w/w; cf., Briozzo et al., 1983; Tanaka et al., 1986; Leistner and Russell, 1991; Eckner et al., 1994; Rajkowski et al., 1994; Ter Steeg et al., 1995) without affecting the product quality otherwise. 9 Using ES with bacteriostatic properties. 9 Good manufacturing practice, minimization of manual handling of product, avoiding post-processing contamination and reducing storage temperature (Ter Steeg et al., 1995; Palmas et al., 1999).
Flavour effects. It is generally recognized that sodium citrates impart a 'clean' flavour while phosphates may impart off-flavours described as soapy (especially orthophosphates), chemical or salty (Meyer, 1973; Gupta et al., 1984). Pyrophosphates may cause bitterness if added at a level of 2%, w/w (Templeton and Sommer, 1936); potassium citrates also tend to cause bitterness (Templeton and Sommer, 1936; Meyer, 1973).
Bacterial spoilage can be effectively eliminated by the use of UHT processing (sterilization), which destroys heat-resistant spores such as Cl. butyricum, Cl. tyrobutyricum, Cl. sporogenes, in combination with hot filling at 85-95 ~ to eliminate post-pasteurization contamination (Sch~r and Bosset, 2002).
Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs
Processed cheese products are consumed directly as table products or as ingredients in certain cooking applications (Guinee, 2002a). As a table product, different PCPs offer a spectrum of consistencies ranging from firm, elastic and sliceable to creamy, smooth and spreadable. The variation in consistency makes PCPs suitable for a range of uses, e.g., substitute for natural sliceable or shredded cheese (e.g., on bread, crackers or sandwiches), table spread, sauces or dips. When consumed as table products, PCPs are subjected to various stresses and strains in the form of shearing (e.g., during spreading, mastication), cutting (e.g., during slicing and ingestion) or compression (e.g., during chewing). The rheological properties of the PCPs characterize its response (e.g., degree of spread,
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
fracture, crumbling, springiness) to the applied stresses or strains (see 'Rheology and Texture of Cheese', Volume 1) and have a major impact on the textural and sensory characteristics (see 'Sensory Character of Cheese and its Evaluation', Volume 1). Processed cheese products are also used as an ingredient in several cookery applications, e.g., as slices in burgers, toasted sandwiches, pasta dishes, au gratin sauces and cordon-bleu poultry. A key aspect of the cooking performance of cheese is its heat-induced functionality, which is a composite of different attributes, including softening (melting), stretchability, flowability, apparent viscosity and tendency to brown (see 'Cheese as an Ingredient', Volume 2). Hence, the textural properties of the unheated PCP and cooking characteristics of the heated product are major factors affecting quality. Consequently, numerous investigations have been undertaken on the effects of different factors on the rheological characteristics of PCPs. Although some discrepancies exist between studies, probably as a consequence of inter-study differences in factors other than that being investigated (e.g., formulation and processing conditions), definite trends are evident. The quality of PCPs is influenced by many factors, including: the type and level of ES, the composition and degree of maturity of the natural cheese used, the type and level of optional ingredients, the processing conditions and the interactions between the different factors. These are summarized in Table 6 and are discussed briefly below.
Processing time Processing conditions can vary markedly. As discussed in 'Principles of manufacture of PCPs', the heat and shear applied during processing contribute to hydration of the para-casein and other ingredients and to emulsification of free fat/oil. They do this by aiding: 9 the mixing and the uniform distribution of all ingredients throughout the blend; 9 the dissolution of the ES and their interaction with the para-casein (in the cheese) or casein aggregates (as in added milk-protein ingredients such as milk powders, caseinates, caseins); 9 destruction of the structure of the natural cheese being processed (by promoting aggregation and dehydration of the para-casein matrix and by destruction of the milk fat globule membrane in the natural cheese); 9 dispersion of free (non-globular) fat/oil and moisture; 9 transformation of the structure, e.g., from a paracasein gel with occluded fat globules and moisture (as in cheese), or from a protein aggregate/precipitate
371
in the case of added milk protein ingredients, to a concentrated o/w emulsion. Increasing processing time and shear (speed of mixing) is generally accompanied by an increase in the DE, as reflected by an increase in the number, and reduction in the mean diameter, of the emulsified fat globules (Rayan et al., 1980; Kimura et al., 1986; Tatsumi et al., 1989). The increases in casein hydration and DE result in a progressive thickening of PCPs with holding time at a temperature in the range 70-90 ~ (cf. Swiatek, 1964; Rayan et al., 1980; Kalab et al., 1987). The thickening, referred to as creaming or creaming effect in the industry, may be attributed to the ongoing interaction of the ES with the casein and the consequent increases in para-casein hydration and DEE. Creaming is desirable, especially in high-moisture PCS, as it imparts the desired viscous consistency to the molten blend for filling/packing (which prevents splashing) and gives a thick, creamy-bodied final product; in such products, the lack of an adequate creaming gives a thin runny consistency. However, extending the holding time (e.g., due to a delay or stoppage of packaging lines) of the molten product at 70-90 ~ can result in a defect known as over-creaming. In PCPs, over-creaming manifests itself as the development of a short, stiff, heavy, pudding-like consistency and dull appearance; this development may not become obvious until the product has cooled. In block PCPs and slices, it is reflected by the appearance of an 'orangepeel'-like surface and development of an over-firm and heavy pudding-like (coarse) structure which leaks free moisture and exudes beads of free oil (through the 'surface dimples'), especially on cooling. Over-creaming is highly undesirable in practice as it creates problems in pumping/filling (e.g., clogging of filling heads, excessive stand-up in packages) of the product and causes a deterioration in the end product quality, e.g., loss of spreadability, loss of surface sheen, non-uniform greasy appearance (in slices/blocks), loss of cooking properties. In experimental studies, increasing the processing time from 0 to 40 min at 70-82 ~ resulted in progressive increases in the elasticity and the firmness of the unheated PCPs and a decrease in the flowability of the melted PCPs, to an extent dependent on the ES type (Rayan et al., 1980; Harvey et al., 1982; Tatsumi et al., 1991). In contrast to these results, Swenson et al. (2000) found that increasing the processing time at 75 ~ from 0 to 20 min resulted in a decrease in firmness and an increase in the flowability of fat-free PCPs. These results may suggest the absence of a creaming process in the fat-free PCPs and highlight the importance of fat content and degree of fat emulsification to the creaming process.
372
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
General effects of various parameters on the textural characteristics and heat-induced flowability of pasteurized processed cheese productsa,b,c,d,e,f,g
Firmness
Elasticity
Spreadability
Heat-induced flowability
t 4,
f 4,
NA NA
f 4,
4, f
4, 1'
4, f
4, f
f f t
t t NA
4, 4, NA
4, 4, 4,
NA NA NA t NA NA
NA NA NA NA NA NA
NA NA NA NA NA t
4, 4, 4, 4, t NA
4, 4,
4, 4,
t t
NA t
t t
t t
~ ~
,~
Formulation Emulsifying salt concentration Increasing level in the range 0.0-0.5%, w/w Increasing level in the range 0.5-3.0%, w/w Cheese Increasing degree of proteolysis Increasing content of intact casein Substitution of rennet-curd cheese by: Reworked processed cheese Cheese base Acid-heat coagulated cheeses Dairy ingredients Whey proteins Total milk proteins Milk ultrafiltrates Calcium co-precipitate Calcium caseinate Skim milk powder
Composition of PCP Increasing moisture content Increasing pH
Processing conditions Increasing temperature Holding time at maximum temperature
a Modified from Guinee (2002a). b The general effects of the different parameters, as summarized from a review of the published literature, are presented. However, the precise effects of changing any parameter may depend on the particular formulation, processing conditions and the effects of their interaction. c NA, data not available, data limited, or conflicting data from which no general trends emerge. d Arrows, magnitude of factor (e.g., firmness) increases 1' or decreases ~. e Rework refers to pasteurized processed cheese product that is not packaged for sale; it is obtained from the 'left-overs' in cookers and filling machines, damaged packs and batches that have 'over-creamed' (thickened) and are too viscous to pump or fill. f Cheese base refers to milk ultrafiltrate which is diafiltered, inoculated with starter culture (and sometimes with rennet also) until the pH reaches ---5.2-5.8, pasteurized and concentrated to a dry matter content of - 6 0 % , w/w. g See text for more detail on effects ('Blend ingredients: cheese base (CB), ultrafiltered milk retentate (UFMR), cheeses from high heat-treated milks and whey proteins').
Several factors may contribute to over-creaming. Prolonged holding at a high temperature is conducive to aggregation and dehydration of para-casein. Hence, Csok (1982) reported that on holding a cooked processed cheese at 95 ~ the bound water increased to a maximum (e.g., at - 1 5 min) and decreased thereafter (Fig. 14). The initial increase may be attributed to increased solution of the ES (not fully solubilized at the end of the heating step) and calcium sequestration, while the eventual decrease may reflect aggregation of the paracaseinate on prolonged holding at the high temperature. In agreement with the above hypothesis of casein dehydration and aggregation, Bowland (1997), using image analysis of light micrographs, concluded that the level of protein incorporated into the matrix of PCP
increased with creaming time (holding time at the cooking temperature). Moreover, Tatsumi et al. (1991) reported that the level of water-insoluble N in the PCP increased with holding time at 80 ~ and that there was a significant inverse relationship between the holding time and the flowability of the cooked PCP. The increased degree of protein aggregation is consistent with the increase in firmness and elasticity that occurs with processing time (Rayanet al., 1980). Moreover, microstructural analyses of a processed cheese food (PCF) showed that the number and the area of electron-dense zones in a very firm product, cooked to 85 ~ and held for 5 h, was markedly higher than in the control, which was cooled after 3 min at 85 ~ (Kalab et al., 1987). The electron-dense zones may correspond to regions of
Pasteurized P r o c e s s e d C h e e s e and Substitute/Imitation C h e e s e P r o d u c t s
1.65 '7" c O c
.g1.35 m O u')
if) ID t-O
1.05
-O U) if) C) O
0.75 . cm (1) .4-.,
0.45 0
10 20 Holding time, min
30
Changes in the level free water (A) and total bound water (A) in pasteurized processed cheese as a function of processing time at 95 ~ (redrawn from Csok, 1982).
strand overlap and/or reflect areas with a relatively high degree of aggregation and fusion of the paracaseinate particles. The release of moisture and free oil during over-creaming of block PCPs also suggests that the process coincides with the onset of protein dehydration, emulsion destabilization and phase inversion. It is noteworthy that at a micro-structural level, clumping and coalescence of fat globules was evident in the PCF held for 5 h at 85 ~ but was absent, or markedly less, in fresh PCF held for 3 min at 82 ~ (Kal~ib et al., 1987). Another factor contributing to the over-creaming with time is the increase in the degree of fat emulsification (Rayan et al., 1980; Kalab et al., 1987). For a given protein-to-fat ratio in PCPs, increasing the DE leads to an increase in the surface area-to-volume ratio of the emulsified fat globules, which may be considered to behave as structure-building pseudo-protein particles. These particles are expected to increase the firmness of the PCP (see 'Micro-structure of PCPs and ACPs'). This hypothesis concurs with the positive correlation between the DEE and the firmness or elasticity, and the inverse relationship between the DE and the flowability of PCPs (Rayan et al., 1980; Cari~ et al., 1985; Savello et al., 1989). While increasing the DE beyond the critical emulsification point (where all the 'available' protein in the system is not sufficient to cover the available fat surface) maximizes the surface area of emulsified fat particles, it may also lead to free fat separation, especially in high-fat PCPs.
373
Processing temperature and shear According to Meyer (1973), processing at a temperature >95 ~ results in a decrease in product firmness. This coincides with observations in practice where UHT treatment, as in continuous processing, frequently gives PCPs which are more fluid than those processed at a lower temperature. The effect of temperatures >95 ~ may be attributable to thermal hydrolysis of polyphosphate ES, a consequent reduction in paracasein hydration and DE, and/or an increase in the rapidity of, and in the degree of, thermal-induced para-casein aggregation (which would reduce the extent of hydration and viscosity). However, tee et al. (1981) observed a positive relationship between the firmness of processed Emmental and the processing temperature in the range 80-140 ~ The effects of increased processing temperature are less clear when whey proteins are present in the PC blend. These undergo thermal denaturation and complex with para-K-casein at the high processing temperature (Jelen and Rattray, 1995; Singh, 1995). This denaturation may in turn lead to aggregation/pseudo-gelation on cooling the formed PCP (Doi et al., 1983a,b, 1985) to an extent which would be expected to increase with processing temperature. In this case, while a thin consistency may be observed in the kettle, the product may firm up more than usual on cooling. In contrast to Lee et al. (1981), Swenson et al. (2000) found that increasing the processing temperature from 70 to 90~ gave a significant increase in flowability and decrease in the spreadability of fatfree PCP; the firmness was highest at 70~ and lowest at 80 ~ Blend ingredients: ES Numerous studies have compared the effects of different ES blends on the texture and cooking properties of PCPs and ACPs (Templeton and Sommer, 1936; Swiatek, 1964; Thomas etal., 1980; Harvey etal., 1982; Gupta et al., 1984; Cavalier-Salou and Cheftel, 1991; Sutheerawattananonda and Bastian, 1998; Swenson et al., 2000; Abdel-Hamid et al., 2000a,b). Discrepancies between the various results may be due to inter-study differences in cheese (type, age and composition), blend pH, quantity of ES, processing conditions, moisture content and other compositional parameters and assessment methodology. However, general trends emerge showing that orthophosphates, citrates and sodium aluminium phosphates give relatively soft processed cheeses, which generally undergo a slight oiling-off ('sweating') on heating and have desirable melting properties (i.e., good flowability, moistness and surface sheen). In contrast, condensed phosphates generally give harder processed cheeses,
374
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
which show little, or no, oiling-off on heating and have poor melting properties (little or no flow, skin formation and crusting, dull and dry surface appearance). Overall, the flowability and oiling-off on cooking of PCPs or ACPs made with the different ES show the following general trend: sodium aluminium phosphate trisodium citrate (slightly) > disodium orthophosphate > >sodium tripolyphosphates ~ tetrasodium pyrophosphates > higher chain sodium polyphosphates. Generally, the opposite effect is observed with firmness. The above trends reflect the greater calcium sequestration and hydration effects of the condensed phosphates (Table 5) which affords them better emulsification and hence structural-forming properties. It is noteworthy that the DE is positively correlated with firmness and elasticity of the unheated PCP or ACP and inversely correlated with flowability of the heated products (Rayan et al., 1980). In contrast to the above, Lazaridis and Rosenau (1980) noted the following trend for the effect of ES on the flowability of a melted PCP made from a chemically-acidified curd (50%, w/w, moisture; pH 5.5): Na3PO4 > Na2HPO4 > trisodium citrate > sodium aluminium phosphate (kasal). This trend was probably due to the very low calcium sequestering ability of the latter two ES at pH 5.5 (see 'Characteristics of different ES in the manufacture of PCPs and ACPs'). Blend ingredients: cheese As cheese is a major blend constituent in PCPs, it is expected that both the cheese type and the degree of maturity would have major effects on the texture, flavour and cooking characteristics of the final product. The results of the few published studies, the authors' experience and the undocumented evidence from experienced manufacturers suggest that the following are important criteria: type (variety), composition (e.g., contents of moisture, fat, protein and Ca; pH; Thomas et al., 1980; Shimp, 1985; Salam, 1988; Marshall, 1990), age and level of proteolysis (Sood, and Kosikowski, 1979; Thomas et al., 1980; Lazaridis et al., 1981; Mahoney et al., 1982) and flavour. Proteolysis is inversely related to the level of intact casein (Fenelon and Guinee, 2000; Feeney et al., 2001; Guinee et a/.,2001). The pH, intact casein content and calcium-to-casein ratio are expected to influence the degree of casein hydration during processing, and in turn the DE, degree of casein aggregation and elasticity of the final product. However, there is very little direct experimental evidence to clearly demonstrate relationships between the various attributes of PCPs and the characteristics of the unheated cheese. Harvey et al. (1982) found that the flowability of heated processed Cheddar increased
markedly (from 0.5- to 2-fold) with age (from 3 to 6 months) of the Cheddar cheese used, the effect becoming more pronounced as the processing time of the PCP increased; no data on proteolysis were presented. Arnott etal. (1957) found no relationships between the levels of fat, moisture, pH or the level of proteolysis (measured by tyrosine content) in commercial Cheddar cheeses of different age (0-340 day) and the meltability (flowability) of the resultant PCPs. Variability in the flowability of the PCPs was attributed to the interactive effects of the different cheese characteristics. Surprisingly, Holsinger et al. (1987) reported that the melt index of processed Cheddar decreased as the proportion of mature (135-278 days) to young (90 days) Cheddar (stored at - 1 7 . 8 ~ increased from 100:0 through to 0:100. While few experimental details were given, the results of the latter study suggest an increased creaming reaction as the proportion of mature Cheddar increased. Lazaridis et al. (1981) investigated the effect of increasing the level of proteolysis in a pasteurized processed model chemically-acidified curd system by treating the processed curd (varying conditions: 40-55 ~ pH 5.5-9.0) with a proteinase from Aspergillus oryzae. In contrast to the studies cited earlier, there was a strong positive relationship ( r = 0.96) between the flowability and the extent of proteolysis (non-protein N). Excessive proteolysis was, however, associated with textural defects, including overshortness, faulty body and graininess. In a subsequent study (Mahoney et al., 1982), the same group found that optimal flowability of the processed chemicallyacidified curd was obtained when the proteolysis products were in the molecular mass range 10-25 kDa; smaller peptide sizes ( < 1 0 kDa) gave an excessively soft PCP which overflowed on cooking. In model experiments with processed Gouda, Ito et al. (1976) found an inverse relationship between the age (and hence level of proteolysis) and its emulsifying capacity (defined as ml of added oil absorbed per gram of cheese protein). A lower DE, due to greater proteolysis, would be expected to reduce the contribution of emulsified fat globules to structure building and the creaming effect, favour more oil-release during melting, and improve the flowability of the melted PCP (cf. Rudan and Barbano, 1998; Guinee et al., 2000b). Thus, in the studies of Lazaridis et al. (1981) and Mahoney et al. (1982), a decrease in the DE may explain the increase in flowability of the melted PCPs as the level of proteolysis in the raw cheese increased. Blend ingredients: rework Rework refers to a PCP which, for various reasons, is not packaged but instead is stored (refrigerated at a low
Pasteurized P r o c e s s e d C h e e s e and Substitute/Imitation C h e e s e Products
temperature or frozen) and reused (re-processed/ reworked) as a blend ingredient in later batches of PCP. It is obtained from left-overs in the cooking/filling machines, damaged packs and batches, which are overcreamed or are too viscous to pump. Meyer (1973) identified three types of rework: (A) that made from young cheese, quickly processed and long in structure; (B) that with a typical creamed character (i.e., processed cheese with texture characteristics considered normal for the product type) and (C) over-creamed product with a brittle structure. 'Hot melt', a North-American term, is a type of PC rework, which is the hot 'hardened' PCP that is removed from the packing pipelines following a plant breakdown, especially during continuous processing operations (Kal~ib et al., 1987). Microscopical examination of 'hot melt', which may be considered as an overcreamed rework, revealed the presence of dark areas (Fig. 15) that developed to a degree depending on the extent of heating and the melting salt used (Kal~ib et al., 1987). The dark areas represent regions where the protein absorbed an increased concentration of osmium during fixation. Klostermeyer and Buchheim (1988) reported that the protein matrix of processed cheese heated at 140 ~ contained areas of compacted protein as revealed by a freeze-fracturing technique followed by replication with platinum and carbon. It is probable that the areas of compacted protein observed in the study of Klostermeyer and Buchheim (1988) correspond to the osmiophilic dark areas reported by Kalab et al. (1987). Klostermeyer and Buchheim (1988) also observed that the dimensions of areas of relatively low protein concentration in the protein matrix of PCP decreased as the creaming effect increased: 1-2 b~m in diameter with no creaming (melting time, 4 min), - 0 . 5 p~m with mild creaming (melting time,
Scanning electron micrograph of a processed cheese food showing the presence of an electron-dense area (black area shown by arrow) that developed after holding the product for an extended time (5 h) at 82 ~ Bar corresponds to 0.2 i~m (adapted from Kalab et aL, 1987).
375
6 min) and completely absent at optimal creaming (melting time, 9 min), resulting in a uniform protein matrix. Rework that is free of crystals can sometimes be useful for initiating, or enhancing, the creaming effect in blends that are slow to thicken during processing. The recommended usage levels of rework types A, B and C are 1-2%, w/w, 2.0-30%, w/w and 0.0-1.0%, w/w (maximum), respectively (Meyer, 1973). Type A rework is particularly useful to impart creaming to PCS blends with a high proportion of mature (e.g., intact casein level, "--70% total) or very mature (e.g., low intact casein level, <65% total) cheese. Type B rework imparts firmness and elasticity to block processed cheese blends (Meyer, 1973). Unlike regular rework (type B), type C rework has a very strong creaming effect and can lead very quickly to over-creaming. Hence, the inclusion of type C rework should be avoided, except at very low levels (< 1%, w/w). Kalab et al. (1987) evaluated the effect of the following types of commercially produced rework in an experimental PCF (45%, w/w, moisture) made with trisodium citrate or trisodium orthophosphate: regular cheese food slice (RPCF), quickly frozen cheese food slice - prepared by cooling from 82 to 4 ~ in 10 min and then freezing (QFPCF), and hot melt cheese food slice (HMPCF), - prepared by slow cooling from 82 to 4 ~ over 5 h and then freezing. Added at a level of 20%, w/w, all types of rework increased the apparent viscosity of the hot PCF (immediately after processing) and the firmness of the stored PCE and reduced the flowability of the melted PCF (Fig. 16), with the apparent viscosity and flowability being the most affected. The effects were highest for the HMPCF and lowest for the QFPCF and increased with the level of HMPCE The mechanism by which rework exerts its effects on the physical properties of PC is not clear. However, tentative explanations include: 9 Further heating of precooked cheese may cause a higher degree of thermally-induced dehydration and aggregation of the para-casein (especially if hot melt is used), thereby increasing the degree of product elasticity. 9 A more effective dissolution of ES in the rework (due to the longer contact time) which leads to a more rapid hydration of the fresh para-casein introduced to the new blend. 9 A higher effective concentration of protein in the processed blend due to the high DE and, hence, high level of pseudo-protein particles in the rework (especially in hot melt). A high protein concentration would give a high viscosity, which in turn would give a more efficient fat dispersion and emulsification in the fresh blend.
376
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
.--. 1000 09 0_
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Type of processed cheese foods Apparent viscosity and flowability, on heating at 140 ~ for 6 min, of different types of processed cheese food: control, made with a regular formulation with no added rework, stored at 4 ~ after manufacture and tested at 24 h; QFPCF, as for the control except that it contained 20%, w/w, added rework which was a regular processed cheese food which was frozen/held at - 1 0 ~ immediately after manufacture; RPCF, as for the control except that it contained 20%, w/w, added rework which was a regular processed cheese food which was held at 4 ~ immediately after manufacture; HMPCF, as for the control except that it contained 20%, w/w, added rework which was a processed cheese food which was held for an extended time (5 h) at 82 ~ (drawn from data of Kal~.b et al., 1987).
Blend ingredients: cheese base (CB), ultraffitered milk retentate (UFMR), cheeses from high heattreated milks and whey proteins Attempts to reduce formulation costs of PePs and improve end-product consistency have led to extensive investigation on the development of, and study of the effects of, ingredients which are more cost-effective than cheese (Mann, 1970, 1981, 1984, 1990, 1997). In this regard it has been attempted to replace blend cheese by milk ultrafiltrate (Sood and Kosikowski, 1979; Anis and Ernstrom, 1984) or CB (Ernstrom et al., 1980; Park et al., 1992; Simbuerger et al., 1997). A major difference between these materials and rennet curd cheeses is that they contain whey proteins in addition to casein or para-casein (as in natural cheeses). Whey proteins may be also added to PePs and ACPs in the form of WPCs (Schulz, 1976; Savello et al., 1989; Nishiya et al., 1990; Hill and Smith, 1992; Kaminarides and Stachtiaris, 2000), total milk proteinates (Abou E1-Nour et al., 1996), co-precipitates (Thomas, 1970) and cheese with a high level of whey
protein, e.g., UF cheese or acid-heat coagulated curd (Kalab and Modler, 1985; Collinge and Ernstrom, 1988; Collinge et al., 1988; Kalab et al., 1991). Production of CB generally involves uhrafihration and diafihration of skim milk, inoculation of the retentate (typically 20-25%, w/w, dry matter) with a lactic culture, incubation to a set pH (5.2m5.8), pasteurization and scraped-surface evaporation to, typically, 60%, w/w, dry matter (Ernstrom et al., 1985; Ganguli, 1991; Sutherland, 1991). However, rennet may be added to the retentate to form a curd from which a small quantity of whey is removed (compared to that in natural cheese manufacture) and which is dry-salted and pressed, and stored as natural cheese. The retentate may also be treated with lipase to enhance the flavour of the final PeP; it was claimed (Aly et al., 1995) that up to 80%, w/w, of Ras cheese solids could be replaced by the lipase-treated retentate, with the resultant PePs having flavour and consistency considered to be superior to those of the control. A recent patent submission (Hyde et al., 2002) describes the preparation of CB by the acidification and cooling of a blend comprising of one or more powdered milk protein ingredients, milk fat, NaC1, edible acid and/or preservative. Increasing the level of substitution of natural cheese by CB, made in the conventional manner, or UFMR, normally results in a 'longer-bodied', firmer PeP which is less flowable on heating (Collinge and Ernstrom, 1988; Tamime et al., 1990; Younis et al., 1991). The lower flowability may be attributed to a number of factors, including: 9 a higher degree of intact casein in the CB; 9 the presence of whey proteins in the CB (---8.7%, w/w) which are denatured and complex with para-K-casein to form a pseudo-gel at the high processing temperature (85-90 ~ for 3 min; cf., Doi et al., 1983a,b, 1985). The adverse effect of whey proteins on the functionality of PePs is probably due to their ability to form thermally induced para-K-caseinlf3-1actoglobulin aggregates or gels at the high temperature (typically "-98 ~ reached during baking/grilling, when present in significant quantities (e.g., 3-7%, w/w) in the cheese. The tendency to aggregate and gel is probably accentuated by the high levels of protein and soluble calcium in the cheese (Doi et al., 1983a,b; Jelen and Rattray, 1995). On setting, the gels would impede the flow of the cheese as the fat phase melts and coalesces (Sood and Kosikowski, 1979; Savello et al., 1989). However, the effects on flowability vary depending on the method of preparation of the CB and UFMR and the subsequent heat treatment during processing: (i) Decreasing the pH of milk, from 6.6 to ->5.2, prior to UF resulted in CBs with lower calcium levels
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
and processed products with improved meltability (Anis and Ernstrom, 1984). (ii) Rennet treatment of the UF retentate results in poorer meltability (Anis and Ernstrom, 1984), an effect which may be attributed to the higher degree of interaction between [~-lactoglobulin and para-K-casein (than with native casein) during subsequent processing (Doi et al., 1983 a,b). (iii) Treatment of retentate with exogeneous proteinases (i.e., Savorase-A, and enzymes from Aspergillus oryzae and Candida cylindracea), which increase the level of proteolysis in the CB, yields PCPs which are softer and more meltable than those made with untreated CB (Sood and Kosikowski, 1979; Tamime et al., 1990, 1991). (iv) Increasing the processing temperature in the range 66-82 ~ results in processed products with reduced meltability, an effect attributed to the gelation of whey proteins at the higher temperatures, especially when rennet-treated CB is used (Collinge and Ernstrom, 1988). Similarly, the addition of calcium co-precipitate (to a level of 5%, w/w) to processed Cheddar reduces flowability, with the effect decreasing as the level of proteolysis in the Cheddar cheese increases (Thomas, 1970). The direct addition of whey proteins to PCPs and ACPs, as a substitute for cheese or casein, generally has been found to increase the fracture stress and firmness, and reduce the flowability of the heated cheese (Savello et al., 1989; Gupta and Reuter, 1993; Abou-E1-Nour et al., 1996; Gigante et al., 2001; Mleko and Foegeding, 2001). In this context, it is noteworthy that a PCP which is resistant to flow on cooking, can be prepared by adding a heat-coaguable protein (3-7%, w/w, lactalbumin or egg albumen), at a temperature <70 ~ to the PCP blend on completion of processing (Schulz, 1976). In contrast, Kaminarides and Stachtiaris (2000) reported that the hardness of PCPs with similar final composition decreased from 3- to 5-fold with the replacement of Kasseri cheese by added WPC (24%, w/w, protein, added at a level of 9-39%, w/w) and soybean oil. However, the quantity of ES used was added according to the quantity of added cheese in the blend and was, therefore, greatly reduced from 2.6%, w/w (control), to 1.5%, w/w, at the highest WPC level. French et al. (2002) investigated the effects of replacing sodium caseinate by a range of milk protein concentrates (MPCs, 72-82.5%, w/w, protein), whey protein concentrates (WPCs, 80 or 34%, w/w) or lactalbumin (80%, w/w, protein) on the hardness, cohesiveness and springiness of PCP. For each ingredient, the effects depended on the ratio of the different ES used, i.e., trisodium citrate and disodium orthophosphate. At both ES ratios, the
377
MPCs gave higher hardness than the control, while the lactalbumin and the WPCs gave lower hardness; however, experimental details are scarce. The use of WPCs as a replacement for cheese solids has also been found to accelerate storage-related flavour deterioration, which increased with the level of WPC added in the range 0-20%, w/w (Thapa and Gupta, 1992). In contrast to the above studies, the flowability of pasteurized processed Ras-Quark cheese was enhanced by the substitution of WPC for Quark, with the effect becoming more pronounced as the level of added whey protein was increased from - 3 to 6%, w/w (Abd E1-Salam et al., 1996). A similar trend was reported by A1-Khamy et al. (1997) who found that the magnitude of the effect varied with the type of ES and storage time of the PCP at room temperature. Similar observations were made by Kalab et al. (1991) who substituted a non-flowable acid/heat-coagulated white cheese (AHC) for Cheddar cheese in a PCP. The flowability of the PCP increased by - 4 0 % as the level of AHC was increased from 0 to 16%, w/w, and then decreased to a value slightly higher than the control as the level of AHC was further increased to 33%, w/w; the addition of 16 or 33%, w/w, AHC was equivalent to adding 0.64 or 1.28%, w/w, whey protein. Discrepancies between the foregoing studies vis-a-vis the effect of added whey protein on the functional characteristics of PCPs and ACPs may be due to differences in the characteristics (e.g., level of denaturation, pH, levels of Ca and protein, particle size) and format (e.g., as AHC, WPC, ~,TpI) of the whey protein added and in PCP formulations (i.e., type of casein, type of ES, cheese age) and processing conditions. These factors may determine the degree of aggregation of the whey proteins, aggregate size and interaction with the caseinlpara-casein; they may also influence the pH of the blend during processing which, as discussed below, has a major influence on casein hydration and the characteristics of the end-product. Blend ingredients: caseins Caseinates and caseins (acid and rennet) are used widely in PCPs and ACPs, the main attractions being lower cost (relative to cheese protein), a consistent level of intact casein, good emulsifying capacity of caseinates and good stretching properties of rennet casein which makes it ideal for APC. Caseinates (especially sodium) find most applications in processed cheese spreads (PCSs) where their high water-binding capacity and good emulsifying properties promote a desired creaming effect. Gouda et al. (1985) reported that full replacement of cheese solidsnon-fat by calcium caseinate caused deterioration in spreadability of Cheddar PCS, probably due to an excessive creaming effect. However, partial replacement in a
378
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
formulation (with skim milk powder, calcium caseinate, ripe Cheddar, butter oil and ES at respective levels of 6-8, 5-7, 15, 14 and 3%, w/w) improved the meltability of the PCS, suggesting a desirable level of creaming. Caseinates may be used in spreadable ACPs (Hokes et al., 1989; Marshall, 1990). Rennet casein, despite its insolubility, is generally preferred in the manufacture of APC, which is the major imitation cheese product (McCarthy, 1990; Fox etal., 2000; Guinee, 2002b; 'Analogue cheese products (ACPs)'). Recently, the use of casein hydrolysates has been found to reduce the quantity of ES required for the emulsification and formation of a stable product (Kwak et al., 2002). When added at a level of 3%, w/w, to replace the ES completely, the hydrolysate gave a PCP which on cooking had a high flowability but excessive oiling-off.
(Piergiovanni et al., 1989; Kombila-Moundounga and Lacroix, 1991). However, Hong (1990) found that replacement of experimental cheeses by lactose at levels of 5-20%, w/w, reduced the firmness of PCP. Excess lactose may also increase the propensity to crystallization in PCPs during storage, with the formation of mixed crystals containing various species, e.g., Ca, P, Mg, Na, tyrosine and/or citrate. Owing to the relatively high level of bound water in PCPs (a maximum of 1.6 g/g solidsnon-fat; Csok, 1982), the effective lactose concentration in the free moisture phase may easily exceed its solubility limit (---15 g/100 g H20 at 21 ~ This may result in the formation of lactose crystals which could serve as nuclei for the crystallization of mineral species which are supersaturated (Uhlmann et al., 1983; 'Characteristics of different ES in the manufacture of PCPs and ACPs').
Blend ingredients: co-precipitates Co-participates are protein products containing casein and whey proteins and are formed by heat treatment of the milk and subsequent precipitation of the protein complex by acidification and calcium addition (Mulvihill, 1992). Depending on the level of CaC12 added, three types may be obtained, namely, high-, medium- and low-calcium co-precipitates containing 2.5-3.0, 1.0-2.0 and 0.5-0.8%, w/w, calcium, respectively. The use of various co-precipitates at a level up to 5%, w/w, of the blend material resulted in PCPs with increased firmness and sliceability, lower meltability/flowability and higher emulsion stability (Thomas, 1970). Because of their excellent emulsifying capacity, Thomas and Hyde (1972) reported that the level of ES can be reduced from 3.0 to 2.0-2.5%, w/w, if calcium co-precipitate is used in the blend at a level of 2-3%, w/w. However, a high level (>3%, w/w) significantly reduced the flowability of the PC, especially when a high proportion of young cheese was used.
Compositional parameters Although the rheological attributes of PCPs with the same moisture content can differ significantly due to variations in blend composition and processing conditions, increasing moisture content yields products which are softer, less elastic and viscous, sticky and spreadable (Kairyukshtene and Zakharova, 1982; Salam, 1988; Gupta and Reuter, 1993). Marshall (1990) studied the effect of varying moisture-in-non-fat substances (MNFS; 50, 55 and 60%, w/w) and fat level, which was varied from 5.0 to 20%, w/w, at each MNFS level, on the rheological properties of model ACPs. Rheological measurements by uniaxial compression at large deformation included maximum stress, ~max, deformation at 8max, DMS; work to ~max, WMS; other analyses included stiffness, measured by low deformation compression, and work to fracture (WF), analysed by measuring cutting force. There was an inverse relationship between the levels of MNFS and protein; however, details on the actual levels of dry matter and protein were not presented. Linear regression analysis indicated that DMS was negatively related to the MNFS content and positively to the protein content. Multiple regression analysis showed that an increase in the levels of both fat and MNFS resulted in marked decreases in DMS, WMS, stiffness and WE However, as discussed earlier, the DE for a given fat content and protein-to-fat ratio has a major effect on the rheological and cooking properties of PCPs. Hence, as postulated by Shimp (1985), the protein-to-fat ratio is a major determinant controlling the rheological and cooking properties of PCPs, but only at levels of emulsification below the maximum, or the critical, DE. pH has a major effect on the texture of commercial and experimental PCPs (Scharf, 1971; Gupta etal., 1984; Shimp, 1985), in which the pH is varied by changing, among other factors, the type and level of ES.
Blend ingredients: skim milk powder Addition of skim milk powder to PCP blends at a level of 3-5%, w/w, results in softer, more spreadable products (Kairyukshtene and Zakhrova, 1982). However, higher levels (7-10%, w/w) lead to textural defects such as crumbliness and lack of cohesiveness (Thomas and Hyde, 1972; Kairyukshtene and Zakhrova, 1982) and may remain undissolved. However, a high level may be added if the skim-milk powder is first reconstituted and then precipitated by proteolytic enzymes or citric acid, and the curd added to the blend (Thomas, 1970). Blend ingredients: lactose Added lactose, in the range of 0-5%, w/w, results in lower spreadability, lower water activity and increased propensity to non-enzymatic browning in PCPs during processing (especially at a high temperature) and storage
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 379 Low pH (4.8-5.2), e.g., due to the use of monosodium citrate, monosodium phosphate or sodium hexametaphosphate alone, gives short, dry, crumbly cheese which shows a high propensity to oiling-off (Gupta et al., 1984). High pH values (>6.0) give PCPs that tend to be very soft and flow excessively on heating (Gupta et al., 1984). Similar trends were noted by Lee et al. (1981), who noted that increasing the pH of PCP from 5.75 to 6.05, by increasing the level of added sodium polyphosphate, was accompanied by a 2-fold decrease in hardness (as measured by penetrometry). Marchesseau et al. (1997) studied the effect of pH (5.7, 6.1, 6.7) in experimental PCPs made using a standard formulation with the same type (commercial polyphosphate blend) and level of ES, by adding NaOH or HC1 to the blend before cooking. Increasing the pH resulted in marked decreases in the elastic shear modulus (G' index of elasticity and firmness) and loss modulus (G"I index of viscous component of stress; 12-fold) and an increase in the loss tangent (tan & from 0.25 to 1.39). Scanning electron microscopy analysis of the PCPs showed that increasing the pH from 5.7 to 6.1 led to a decrease in the level of the para-casein aggregation and a finer para-casein matrix, and a further increase to pH 6.7 led to a decrease in the continuity of the matrix (Marchesseau etal., 1997). These structural changes coincided with increases in the hydration (moisture of pellet obtained on ultracentrifugation of the cheese at 86 000 g x 25 min) and solubilization (the ratio of supernatant N to total N on centrifugation of the cheese at 300 000 g for 45 min) of the para-casein. Since pH reduction in the region 6.1-5.7 (typical of commercial processed cheeses) markedly reduces the calciumcheating effects of ES ('Characteristics of different ES in the manufacture of PCPs and ACPs'), the study probably does not reflect the direct effect of pH, but rather the combined effects of pH and degree of calcium sequestration. Similar to the results of Marchesseau et al. (1997), Lee and Klostermeyer (2001) reported that increasing pH caused reductions in hardness and viscosity and an increase in tan 8 of ACPs prepared from sunflower oil and sodium caseinate. Cavalier-Salou and Cheftel (1991) reported that increases in the pH (---6.1-6.7) of ACPs, as affected by increases in the level of ES, caused a 1.5- to 2-fold increase in the flowability of the melted product when using NaH2PO4 and trisodium citrate as ES. pH had little, or no, effect when sodium phosphates with -> 2P were used as ES. The results of studies to date suggest that pH probably exerts its influence on the rheology and texture of PCPs and ACPs via its effects on protein-protein interactions and casein hydration, and on the calcium sequestering ability of the ES (Marchesseau et al., 1997; Cavalier-Salou, 1991; cf., 'The role of ES in the formation of a physico-
chemically stable product' and 'Characteristics of different ES in the manufacture of PCPs and ACPs'). However, further studies are required to elucidate the direct effect of pH.
Stabilizers (binding agents) and hydrocolloids Stabilizers, which include carob bean gum, guar gum, carageenan, sodium alginate, gum karaya, pectins and carboxy methylcellulose, are permitted in PCS at a maximum level of 0.8%, w/w (Code of Federal Regulations, 1986). These products stabilize by virtue of their waterbinding and gelation capacities (Phillips et al., 1985). In cheese processing, they are normally used at a level of 0.1-0.3%, w/w, to firm up the structure in instances of high water content or low creaming action (thin consistency) due to, for example, the use of over-ripe cheese or an unsuitable ES blend. More recently, they have found application in reducing firmness, and improving the spreadability and cooking properties (meltability and flowability) of reduced-fat PCPs (Brummel and Lee, 1990; Swenson et al., 2000). While it is difficult to determine the efficacy of the hydrocolloids in the latter studies due to the absence of low-fat controls, both firmness and flowability varied significantly with the type and the level used. Hydrocolloids (locust bean gum, guar gum, modified starch, xanthan gum, low methylated pectin) have recently been investigated as substitutes for sodium phosphate ES (Pluta etal., 2000); a mixture of locust bean gum (0.8%, w/w) and modified starch (2%, w/w) was claimed to give a stable ES-free product and was recommended as a substitute for sodium phosphate in the manufacture of PCPs. Various food-grade emulsifiers (e.g., lecithin, Tweens and Spans) have been used in PCPs, especially in reduced-fat products, to impart softness and improve flowability on melting (Drake et al., 1999). Lee et al. (1996) reported the effects of adding low molecular weight emulsifiers [(sodium dodecyl sulphate (SDS), Nacetyl-N,N,N-trimethylamonium bromide (CTAB), lecithin, mono- and diglycerides)] on the rheological properties of model PCPs. All emulsifiers led to finer dispersions compared to the controls, but their effect on the rheological properties was largely determined by protein-emulsifier interactions which depended on the emulsifier charge. The cationic CTAB increased hardness and elasticity while the anionic SDS gave a PCP which was softer and less elastic than the control; the neutral lecithins and glycerides had little effect.
A n a l o g u e cheese products (ACPs) Analogue cheese products may be classified as cheese substitutes or imitations, which partly or wholly substitute or imitate cheese and in which milk fat, milk protein or both are partially or wholly replaced by
380
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
non-milk-based components, principally of vegetable origin. However, their designations and labelling should, by law, clearly distinguish them from cheese or PCPs. The labelling requirement for imitation and substitute cheeses has been reviewed by McCarthy (1991). In the USA, an imitation cheese is defined as a product which is a substitute for, and resembles, another cheese but is nutritionally inferior, where nutritional inferiority implies a reduction in the content of an essential nutrient(s) present in a measurable amount but does not include a reduction in the caloric or fat content (Food and Drugs Administration Regulation 101.3, Identity Labelling of Food in Packaged Form (e)). A substitute cheese is defined as a product which is a substitute for, and resembles, another cheese and is not nutritionally inferior. Outside the USA, there is little specific legislation covering imitation or substitute cheeses. Few, if any, standards relating to permitted ingredients or manufacturing procedures
exist for imitation cheese products. For more pertinent information regarding designation and labelling, the reader is referred to IDF (1989), McCarthy (1991), current National Regulations and Codex Alimentarius. Other cheese-like products, which may be classified as imitation or substitute, are Tofu and Filled Cheeses; the latter products have been discussed briefly by Fox et al. (2000) and will not be reviewed here. The general aspects of ACPs have been reviewed recently (Ennis and Mulvihill, 1997; Fox et al., 2000; Guinee, 2002b). Analogue cheese products are cheeselike products manufactured by blending various edible oils/fats, proteins, other ingredients and water into a smooth homogeneous blend with the aid of heat, mechanical shear and ES. The array of ingredients used in ACPs and their functions are listed in Table 7. The effects of various ingredients, processing conditions and low temperature storage on the quality of imitation cheese products have been reported extensively
Ingredients used in the manufacture of cheese analoguesa,b,c, d
Ingredient
Main function~effect
Examples
Fat
Gives desired composition, texture and meltability characteristics; butter oil imparts dairy flavour Give desired composition, semi-hard texture with good shreddability, flow and stretch characteristics on heating Assist in the formation of physico-chemical stable product Gives required composition Low cost relative to casein Rarely, if ever, used commercially as sole protein owing to product defects; may be used at low levels (e.g., 2-3% w/w) Substitution for casein and cost reduction
Butter, anhydrous milk fat, native or partially hydrogenated soya bean oil, corn oil, palm kernel oil Casein, caseinates Whey protein
Assist in the formation of physico-chemically stable product; modify textural and functional properties Enhance product stability; modify texture and functional properties See Table 1 See Table 1 See Table 1 See Table 1 See Table 1 Improve nutritive value
Sodium phosphates and sodium citrates
Milk proteins
Vegetable proteins
Starches Stabilizers Emulsifying salts
Hydrocolloids Acidifying agents Flavours and flavour enhancers Sweetening agents Colours Preservatives Minerals and vitamin preparations
Soya bean protein Peanut protein, wheat gluten
Native and modified forms of maize, rice, potato starches
Hydrocolloids: guar gum, xanthan gum, carageenans See Table 1 See Table 1 See Table 1 See Table 1 See Table 1 Magnesium oxide, zinc oxide, iron, vitamin A palmitate, riboflavin, thiamine, folic acid
a Modified from Guinee (2002b). b The ingredients permitted are subject to the prevailing regulations in the region of manufacture. c Whey proteins mainly for products used in cooking applications where flow resistance is required. d See text for more details on effects of different ingredients (see 'Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs' and 'Formulation')
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
(Abou El-Ella, 1980; Lee and Marshall, 1981; Yang and Taranto, 1982; Marshall, 1990; Cavalier-Salou and Cheftel, 1991; Kiely et al., 1991; Suarez-Solis et al., 1995; Ennis and Mulvihill, 1997; Abou E1-Nour et al., 2001). Many of these have been discussed in Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs'. Similarities with PCPs include: 9 the use of many ingredients in common, including ES, stabilizers, non-cheese dairy ingredients, colours, flavours and flavour enhancers; 9 similar manufacturing technology, involving the application of heat and shear to the formulated blend, followed by hot filling, packing and cooling; 9 similar microstructures which may be generally described as an o/w emulsion, stabilized by hydrated (para) caseinate which occurs as a concentrated dispersion (e.g., high-moisture, low-protein ACPs) or as a weakly gelled (para) caseinate dispersion, depending on product composition and hardness (see 'The role of ES in the formation of a physico-chemically stable product' and 'Micro-structure of PCPs and ACPs'); 9 the absence of a ripening period (even though relatively minor changes can take place during cold storage of PCPs and ACPs (cf., Tamime et al., 1990; Guinee, 2002b) 9 the diverse range of textures, flavours, cooking properties and packaging formats; 9 the use of both as alternatives for natural cheese and in similar applications (cf., 'Cheese as an Ingredient', Volume 2). The major difference between ACPs and PCPs is in the permitted ingredients (as discussed in 'Formulation'), with most commercial analogues containing vegetable-derived fat, rather than milk fat, as in natural and processed cheeses. Analogue cheese products may be arbitrarily categorized as dairy, partial dairy or non-dairy depending on whether the fat and/or protein components are from dairy or vegetable sources (Shaw, 1984; Fox et al., 2000). Partial dairy analogues, in which the fat is mainly vegetable oil (e.g., soya oil, palm oil, rapeseed and their hydrogenated equivalents) and the protein is dairy-based (usually rennet casein and/or caseinate) are the most common. Non-dairy analogues, in which both fat and protein are vegetable-derived, have little or no commercial significance and, to the authors' knowledge, are not commercially available. Dairy analogues are not produced in large quantities because their cost is prohibitive. Partial dairy ACPs were introduced to the market in the USA in the early 1970s and constitute by far the largest group of imitation or substitute cheese products.
381
Since then, the commercial manufacture of analogues of a wide variety of natural cheeses (e.g., Cheddar, Monterey Jack, Mozzarella, Parmesan, Romano, Blue, Cream cheese) and PCPs have been reported in the trade literature (Dietz and Ziemba, 1972; Graf, 1981; Anonymous, 1982, 1986; Shaw, 1984; Morris, 1986). Based on feedback from the marketplace, current annual production of analogue cheese in the USA, the primary manufacturer, is - 3 0 0 000 tonnes (personal communication: Martin O'Donovan, BL Ingredients LLC, Chicago) with the major products being low-moisture Mozzarella, Cheddar and pasteurized processed Cheddar. These products have numerous applications: frozen pizza toppings, slices in beef burgers and ingredient in salads, sandwiches, cheese sauces, cheese dips and ready-prepared meals. Compared to the USA, European production is estimated to be relatively small (e.g., 20 000 tonnes/annum). This may be attributed to the lack of a common European effective legislation policy, the efforts of groups concerned with the protection of the designation of origin of milk and dairy products and/or the relatively low consumption of pizza and cheese as an ingredient in Europe (cf., Guinee, 2002c). Moreover, cheese flavour ingredients (e.g., EMCs) are still insufficiently developed to give analogue cheeses, which could be consumed as table cheeses (K.N. Kilcawley, personal communication), which is the major form of EU cheese consumption. The following have contributed to the success of (partial dairy) ACPs in the USA: (i) their lower cost relative to natural cheeses, coupled with the increase in overall cheese consumption; the low cost of analogues is due to the low cost of vegetable oils (compared to butterfat) and of price-subsidized casein imported from Europe, the absence of a maturation period, which for natural cheeses amounts to -US$1.6/tonne/day and the relatively low cost of manufacturing plant relative to that for natural cheese; (ii) the diversity they can offer by way of functionality (e.g., flowability, melt resistance, shreddability), made possible by tailor-making formulations, coupled with their relatively high functional stability during storage; (iii) the popularity of fast food and ready-prepared meals; (iv) their ability to meet special dietary needs and to act as a vehicle for health benefits/supplements, e.g., lactose-free, low in calories, low in saturated fat, vitaminenriched (Andreas, 1985; Anonymous, 1986; Morris, 1986; Keane and Glaeser, 1990); this is made possible by formulation changes. The following discussion relates to partial dairy analogues, especially analogue low-moisture Mozzarella cheese (LMMC), frequently referred to as analogue pizza cheese, APC.
382
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
APC" principles and manufacturing protocol
The principles of manufacture of APC from rennet casein are similar to those for PCPs involving: 9 the sequestration of Ca from the rennet casein by added ES at the high temperatures (typically ---80-84 ~ 9 upward pH adjustment of the blend by the added ES; 9 concomitant hydration of the casein by the ES, shear and heat; 9 dispersion of added fat by the shear and its emulsification by the hydrated para-caseinate; 9 structure formation during cooling. The manufacturing technology for ACPs is also very similar to that for PCPs (Ennis and Mulvihill, 1997; Fox et al., 2000; Guinee, 2002b), as described in 'Manufacturing protocol for PCPs'. While production methods vary, a typical manufacturing procedure (Fig. 17) involves the following sequence of events: simultaneous addition of required quantities of water and dry ingredients (e.g., casein, ES), addition of oil
I Formulation of blend i A
B
C
Cheese cooker Mix for -1-2 min
+
I
Process: heat to +85 ~ shear continuously
+ Homogeneous molten mass pH -8.5 Homogeneous molten mass I pH +6.0-6.4 I
+
i Mould and hot pack I
+
I Storeat4to-4~
and cooking to ---85 ~ (using direct steam injection) while continuously shearing until a uniform homogeneous molten mass is obtained (typically 5-8 min). Flavouring materials (e.g., EMC, starter distillate) and pH-regulator (e.g., citric acid) are then added and the mixture is blended for a further 1-2 min and hot-packed. Horizontal twin-screw cookers (e.g., Damrow, Blentech), operating at a typical screw speed of 40 rpm, are used in the manufacture of APC. This cooker design ensures adequate blending and a relatively low degree of mechanical shear (e.g., compared to the homogenizing effects of some processed cheese cookers). These process conditions, together with the correct formulation, promote a low degree of fat dispersion and hence a relatively large fat globule size (e.g., 5-25 I~m; Neville and Mulvihill, 1995; Ennis and Mulvihill, 1997; Neville, 1998; Guinee et al., 1999). The relatively large fat globule size ensures a sufficient degree of oiling-off from the APC topping when baked on pizza; this, in turn, limits dehydration of the cheese topping and is conducive to satisfactory flow and succulence characteristics (cf., Rudan and Barbano, 1998; Guinee et al., 2000b; 'Pasta-Filata Cheeses' and 'Cheese as an Ingredient', Volume 2). As for PCPs, there is generally an inverse relationship between the DE and the flowability of APCs (Neville, 1998; Mounsey, 2001). Addition of the acid at the end of manufacture, rather than at the beginning, ensures a high pH (--~8-9) in the blend during processing. This procedure is desirable in the manufacture of ACPs where insoluble rennet casein is the major protein ingredient. A high pH during processing leads to greater sequestration of calcium by the sodium phosphate ES, higher negative charge to the casein and higher degree of para-casein hydration. These changes enhance the conversion of the calcium para-casein to sodium para-caseinate, which binds water and emulsifies the vegetable oil (cf., 'The role of ES in the formation of a physico-chemically stable product' and 'Characteristics of different ES in the manufacture of PCPs and ACPs'). Thus, reducing the pH of the blend during processing increases the time required for the formation of the ACPs and probably affects its properties (e.g., firmness, meltability). The addition of flavouring ingredients, such as EMC, towards the end of processing minimizes the loss of flavour volatiles at the high temperature of processing.
I
Formulation Typical manufacturing procedures (A, B, C) for lowmoisture Mozzarella cheese analogue. The procedures differ with respect to the order in which the ingredients (1-5) are added, e.g., casein (1) followed by oil (4) and water (5) in procedure B.
A typical formulation (Table 8) shows that it differs from that for PCPs by the absence of cheese (though some cheese may be optionally introduced as a
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
Typical formulation of low-moisture analogue Mozzarella cheese a
Ingredient
Addition level (%, w/w)
Casein and caseinates Vegetable oil Starch Emulsifying salts Flavours and flavour enhancers Stabilizers Acidifying agent Colour Preservative Water and condensate
18-24 22-28 0.0-3 0.5-2 0.5-3 0.0-0.50 0.2-0.36 0.04 0.10 45-55
a Modified from Guinee (2002b).
flavouring agent) and the inclusion of vegetable oil and a relatively large level of casein(ate)s (cf., Table 1). The major protein source in dairy-based ACPs is caseinate or rennet casein (Nishiya et al., 1989; Ennis and Mulvihill, 1999), with the former being used mainly for spreadable products. Rennet casein is favoured for semi-hard block products and, especially, for APC where it generally imparts better stringiness and stretchability than acid casein or sodium or calcium caseinates. Rennet casein is formed by rennet coagulation of skim milk at normal pH, dehydration of the gel by cutting, stirring and heat treatment, washing of the curd to remove lactose, concentration of the curd by centrifugation and drying, grinding and separation of the dried casein into powders of different mean particle size (Mulvihill, 1992). At the micro-structural level, each powdered particle may be considered as a portion of dried skim milk cheese, with the casein in the form of an agglomerate of aggregates of paracasein. Similar to cheese, various types of attractions are expected to maintain the integrity of the paracasein aggregates (cf., Walstra and van Vliet, 1986), e.g., electrostatic bonds, hydrophobic bonds and calcium phosphate bridges. A further similarity between rennet casein and a young skim milk cheese (with a high level of intact casein) is insolubility in water (cf., Ennis et al., 1998; Fenelon and Guinee, 2000; Feeney et al., 2001). By choosing the appropriate blend of ES, the concentration of calcium cross-linking the paracasein molecules can be reduced to the desired level to give textural and cooking characteristics tailor-made to suit the envisaged application of the product (Fox et al., 2000). On cooking cheese, functional properties such as flow and stretch involve the partial displacement of contiguous layers of the para-casein on the application of stress (see 'Cheese as an Ingredient', Volume 2); a moderate displacement is desirable in cooked pizza cheese (Fox et al., 2000; 'Cheese as an
383
Ingredient', Volume 2). The level of displacement on cooking an ACP depends on the concentration of calcium cross-linking the casein molecules in the final product, which in turn is dependent on the type of casein ingredient used, its total calcium level, the colloidal calcium-to-casein ratio and the concentration and type of ES. For rennet casein which has a high calcium-to-casein ratio (--~36 mg/g casein), the degree of calcium sequestration and para-casein aggregation is easily controlled by using the correct blend of ES to give the desired degree of casein hydration/aggregation and fat emulsification in the ACP (Guinee, 2002b). This, in turn, gives the desired degree of flow and stretchability on cooking the APC. Compared to rennet casein, caseinates tend to over-hydrate, resulting in a degree of casein aggregation which yields good flowability but which is too low to achieve satisfactory stretchability. Owing to the relatively high cost of casein, much effort has been vested in its partial replacement by cheaper substitutes. Increasing the level of substitution of rennet casein by total milk protein, in the range 0-50%, resulted in a progressive increase in firmness and a decrease in the flowability of ACP (Abou-E1-Nour et al., 1996). In a subsequent study, Abou-E1-Nour et al. (2001) investigated the effects of replacing rennet casein by native phosphocasein (NPC) prepared by microfiltration and diafiltration with water (NPC-W) or ultrafiltered milk permeate (NPC-P) in block APC. At 20%, w/w, replacement, the addition of NPC resulted in an increased flowability of the melted APC, with the effect of the NPC-W being significantly greater than that of the NPC-P. In contrast, the NPC-W resulted in a slight decrease in firmness of the unheated ACP whereas the NCP-P gave a marked increase. A comparison between the NPC preparations and the MPC, prepared by UE indicated that the latter gave notably higher firmness in the unheated APC and lower flowability of the heated APC (Abou-E1-Nour et al., 2001). This trend concurs with that of previous studies showing that the addition of whey proteins to PCPs or ACPs, as a substitute for cheese or casein, impairs flowability and increases firmness (cf., 'Blend ingredients: cheese base (CB), ultrafiltered milk retentate (UFMR), cheeses from high heat-treated milks and whey proteins'). Hence, whey proteins are not used owing to the negative impact on flowability, except in applications where flow-resistant ACPs may be needed (e.g., cheese insets in burgers) when --~1-3%, w/w, whey protein is added. Studies have been undertaken on the effects of replacing casein in ACPs by various types of vegetable proteins, e.g., soybean (Lee and Marshall, 1981; Taranto and Yang, 198i; Yang and Taranto, 1982; Yang et al.,
384
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
1983; Kim etal., 1992; Ortega-Fleitas etal., 2001), peanut (Chen etal., 1979), pea protein (E1-Sayed, 1997) or wheat protein (Anonymous, 1981). These proteins gave varying results, depending on the ingredient preparation (e.g., soy flour or soy isolate, pH, fat content) and the type and level of other ingredients (e.g., hydrocolloids). However, the use of these protein substitutes, especially at a level > 10-20%, w/w, of the total protein, has, in general, been found to give ACPs which have a quality inferior to that made using casein only. Common defects include lack of elasticity, lower hardness, an adhesive/sticky body, impaired flow and stretchability and/or poor flavour. Hence, vegetable proteins are rarely used in the commercial manufacture of APCs. To date, starch has been the most effective low-cost casein substitute. Native maize starch appears to be the main type used commercially, with starches from other sources and with different types of modification (pre-gelatinized and/or chemically or enzymatically modified) being used less frequently (Ennis and Mulvihill, 1997). Native starches are used successfully commercially at a level of 2-4%, w/w, to replace ---10-15%, w/w, of total casein. At higher levels of substitution, product defects become noticeable- an increase in the firmness and brittleness of the unheated ACP and a decrease in the fluidity and flowability of the melted cheese, especially if the starch has a high amylose-to-amylopectin ratio (Mounsey and O'Riordan, 1999, 2001; Guinee, 2002b; Figs 18, 19). Moreover, on shredding, the unheated APC with added starch tends to fracture more easily to form curd fines and also tends to exude free moisture after a short period of cold storage, which often leads to sticking and bailing 70
during shredding operations. These defects, which occur to a degree dependent on the type and level of added starch (Mounsey and O'Riordan, 1999, 2001; Mounsey, 2001; Figs 18, 19), cooking temperature and time, degree of agitation and cooling rate, are probably related to storage-related retrogradation and gelation of the starch molecules (especially amylose). Starches (e.g., maize, ,#heat) with a high ratio of amylose to amylopectin tend to retrograde and undergo gelation more readily than those (e.g., waxy maize, rice, potato) with a lower level of amylose (cf., Miura et al., 1992) during storage of the ACP. The other factors above probably influence the degree of gelatinization of the starch during the manufacture of the ACPs and, thus, the concentration of free amylose molecules available for gelation. It is envisaged that a starch gel would impede the flow of the heated cheese when cooked on pizza. The adverse effects of starch may also be related to an increased degree of fat emulsification (Mounsey and O'Riordan, 2001), as a result of a higher apparent viscosity of the APC blend during manufacture when starch is added, especially at high levels. Composition and functionality
Analysis of commercial APC indicates large intra- and inter-factory variations in composition (Guinee et al., 2000c; Guinee, 2002), e.g., moisture, 40-52%, w/w; fat, 60
50
o
60
40
d C~ c-
o~
5o r
40 0 LL
30
13_
20
30 2O
10
10 0
0
I
I
I
I
I
I
30
60
90
120
150
180
Storage time at 4 ~
days
Changes in the flowability, after heating at 280 ~ for 4 min, of low-moisture Mozzarella (A) and low-moisture Mozzarella cheese analogues without (O) or with added native maize (A) or potato (I-1) starch during storage at 4 ~ (modified from Guinee, 2002b).
0 2 4 6 8 10 Level of added pre-gelatinized maize starch, %, w/w Effect of level of added pre-gelatinized maize starch on the fluidity of experimental analogue pizza cheese, after heating to 20 ~ or 95 ~ (drawn from data of Mounsey and O'Riordan, 1999).
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
22-30%, w/w; protein, 13-21%, w/w; 31-38 mg Ca/g protein. Such variations undoubtedly reflect differences in formulation, which suggest that formulation change is a key approach used by manufacturers in the production of APCs with customized nutritional, textural and/or functional (cooking) characteristics. Comparison with commercial Low Moisture Mozzarella Cheese (LMMC) shows that APC has a lower protein content, higher concentrations of moisture and fat, and higher ratios of Ca- and P-to-protein (Guinee et al., 2000c). The higher ratios of Ca- and P-to-protein reflect the use of rennet casein (which has higher concentrations of Ca and P on a protein basis than most natural cheeses) and the inclusion of sodium phosphate ES during formulation. Moreover, the mean value for the sum of moisture, fat, protein and ash in commercial APC is --96.5%, w/w, compared to ---99.5%, w/w, in the LMMC, indicating the addition of carbohydrate-based ingredients during formulation (Guinee et al., 2000c). The heat-induced functional properties of LMMC are discussed in detail in 'Pasta-Filata Cheeses', Volume 2. These generally change fairly markedly with storage time at ar ~ as reflected by reductions in apparent viscosity and an increase in the flowability of the heated cheese; depending on the cheese type, the stretchability of the melted cheese generally increases at first and decreases thereafter. The changes in these functional attributes are due to various factors including age-related physico-chemical changes in the cheese, including proteolysis, solubilization of casein-bound calcium and increases in para-casein hydration and in the level of non-globular fat (Kindstedt, 1995; Guinee 2002c). Similar to PCPs, the functionality of freshly manufactured APCs (e.g., after storage at 4~ for <7 days) is markedly influenced by formulation, processing conditions and product composition (cf., see 'Properties of ES important in cheese processing'; Savello et al., 1989; Cavalier-Salou and Cheftel, 1991; Abou E1-Nour et al., 1996, 2001; Ennis and Mulvihill, 1997; Bowland and Foegeding, 1999; Mleko and Foegeding, 2001). Few studies have considered the changes in casein-based APCs during ripening. Mulvihill and McCarthy (1994) reported a progressive increase in proteolysis (e.g., N soluble at pH 4.6 increased from --~3.5 at 0 days to 19.5% total N at 51 weeks) and reductions in elasticity and chewiness on storing at 4 ~ for 51 weeks. However, the changes during the first 6 weeks were relatively small; normally, analogues are used within 1 month after manufacture. In general, the functionality of APCs is much more stable than that of LMMC and other natural rennet-curd cheeses, as reflected by the relatively small changes in flowability, stretchability and apparent viscosity on storage (<4 ~ for 150 days (Kiely et al., 1991; Guinee, 2002b; Fig. 18).
385
Pasteurized processed cheese and analogue cheeses are a diverse groups of products manufactured by comminuting, heating and shearing into a homogeneous product, a blend of various ingredients which, depending on the product category, may include cheese, dairy ingredients, vegetable oils, flavours, colours and ES. Heating of cheese and other ingredients to a high temperature (e.g., 90~ while shearing, usually results in protein dehydration and formation of free fat which result in the formation of an unhomogeneous mass which exudes moisture and fat. The addition of ES to the blend prior to heating and shearing orchestrates a number of physico-chemical changes such as calcium sequestration, pH adjustment, protein hydration, emulsification of free fat and structural re-organization. The re-hydrated protein behaves as a water-binding and emulsifying agent and transforms the structure, e.g., from a gel in cheese or an aggregate/precipitate in ingredients such as rennet casein, to a concentrated para-caseinate-stabilized o/w emulsion. The degree of para-casein hydration, or aggregation, and the size distribution of the emulsified fat droplets have a major influence on the rheology and cooking properties of the resultant products. Many factors influence the degree of para-casein hydration and degree of fat emulsification, e.g., emulsifying salt type and level, degree of hydration of protein in the blend which in turn is affected by the level of proteolysis and bound calcium, pH of the blend and extent of heat treatment (i.e., temperature and holding time), and level of shear which determines the surface area of the fat phase. Exploitation of the factors affecting para-casein hydration and degree of fat emulsification facilitate the creation of an extensive array of pasteurized PCPs and ACPs which offer diversity in appearance, texture, flavour and cooking properties. Such diversity has contributed greatly to the increased consumption of these products.
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394
Pasteurized Processed Cheese and Substitute/Imitation Cheese Products
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C h e e s e as an Ingredient T.P. Guinee and K.N. Kilcawley, Dairy Products Research Centre, Teagasc Moorepark, Fermoy, Co. Cork, Ireland
There are a total of 500 (IDE 1981) to 800 (Hermann, 1993) varieties of cheese. These differ to varying degrees in nutritive value, appearance, flavour, texture and cooking properties. Consequently, cheese is capable of satisfying a diverse range of sensory and nutritional demands and, therefore, has very wide appeal. The diversity of cheese is extended when subjected to secondary processing to create an array of cheese-based products, as shown in Fig. 1. While it is generally assumed that cheese was originally eaten on its own or with bread, its use as an ingredient has been recorded since Roman times (Ridgway, 1986). Typical uses included the blending of hard cheese with oil, flour and eggs in the preparation of cakes and the mixing of soft cheeses with meat or fish, boiled eggs and herbs in the making of pies. Cheese has long been used in the home and in hostelries as an ingredient, along with other foods and condiments, to create an extensive array of dishes; typical applications include toasted sandwiches, omelettes, sauces and lasagna. Today, much of the cheese purchased through the food service/catering sector is to a large extent used in the preparation of fast foods (e.g., pizza pie, burgers) and culinary dishes (e.g., quiche, lasagna, cheesecake, toasted sandwiches, Mexican dishes; Anonymous, 1993; Fig. 2). Natural cheese is also used extensively by the industrial sector for the commercial manufacture of a vast array of assembled foods (e.g., pizza pie, sandwiches) or formulated foods (e.g., gratins, prepared meals, processed cheese products (PCPs), co-extruded products, cheese cake, dairy desserts). Moreover, cheese is also used by the industrial sector for the production cheese ingredients, including ready-to-use grated cheeses, shredded cheeses, cheese blends, dried grated cheeses, freeze-dried cheese pieces, cheese powders (CPs) and enzyme-modified cheeses (EMCs). Rapid cure cheeses (Fig. 1) are semi-hard cheeses (e.g., Cheddar type) used to impart intense cheese flavour to formulated foods. They are typically produced by the addition of exogeneous enzymes (proteinases, peptidases, lipases) and/or starter culture adjuncts to the cheesemilk and develop a high flavour intensity after a short ripening time (Fig. 2). These ingredients are, in turn, used by the manufacturers of formulated foods
(e.g., soups, dried cheese sauces, dehydrated potato mixes, infant meals, pasta dishes, snack coatings, bakery products; Duxbury, 1991; Lewin, 1996; Missel, 1996; King, 1999), and to a lesser extent by the catering/food service sector in the preparation of culinary dishes (Fig. 2). Additionally, non-standard cheese curd-like products, such as substitute cheeses and cheese bases, are also used as substitutes for natural or processed cheeses in a number of applications, e.g., imitation cheese products for pizza cheese, or cheese base for PCPs or EMCs ('see Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). The percentages of total cheese consumed via the retail, food service and industrial sectors are 70, 20 and 10, respectively, in the EU and --~33, 33 and 33, respectively, in the USA (Sorensen, 2001). This consumption pattern suggests that an estimated 35-45% of total cheese is consumed as an ingredient in other foods. Moreover, recent market analyses indicate that the consumption of cheese as an ingredient is growing rapidly, with a shift in the direction of the USA consumption pattern being evident, especially in Europe and Australia (Market Tracking International Ltd, 1998; Sutherland, 1998; Sorensen, 2001). Owing to the economic importance of cheese as a product per se and as an ingredient, research on different aspects of cheese, such as biochemistry and flavour, texture/rheology and cooking properties of individual varieties such as Mozzarella ('Pasta-Filata Cheeses', Volume 2), has been extensive. However, there have been relatively few reviews on cheese as a food ingredient (Fox et al., 2000; Guinee, 2002). In this chapter, we will review the functional requirements of cheese as an ingredient in assembled/formulated food, from the perspective of the rheological characteristics of the unheated cheese and the cooking performance of the heated cheese, and the properties of cheese ingredients, including EMCs and CPs.
When used as an ingredient in other foods or in the preparation of cheese ingredients, cheese is subjected to an array of treatments such as comminution (e.g.,
Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1
Copyright 9 2004 Elsevier Ltd All rights reserved
396
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Cheese, cheese ingredients and cheese-like products. Cheese may be used: (a) directly, as an ingredient in the home, food service and industrial sectors for the preparation of a variety of culinary dishes, formulated foods or assembled foods or (b) indirectly, in the preparation of various cheese ingredients, such as cheese powders and enzyme-modified cheeses, which are then used in an array of food applications. Various types of processed cheese products (PCPs), including processed cheese spreads (PCSs) and processed cheese foods (PCFs), may be formulated from natural cheese.
portioning, shredding, grating, grinding), cooling, freezing, thawing, heating and/or re-heating. In the home and the food service sector, the cheese-containing dish, once prepared, is generally cooked and consumed immediately In contrast, following assembly or formulation in the industrial sector, cheese-containing foods are frequently heated or pre-heated (pre-cooked) and frozen, and are then re-heated prior to consumption. The treatments of cheese during the manufacture of cheese-filled co-extruded products (e.g., cheese-filled croquette or meat-balls) are typical of those applied during the manufacture of many formulated foods; they include preparation of a cheese filling by dicing, grating, blending and/or processing (as in PCPs), co-extrusion of the cheese with the encasing material, deep-fat frying and freezing (prior to distribution and during retailing). Consideration of the various processes indicates that size reduction (comminution) and heating are the most common processes to which cheese is exposed when used as an ingredient. Hence, the behaviour of
the cheese during these processes is a major determinant of its functionality and its suitability as an ingredient. In the raw state, the cheese may be required to exhibit a number of certain rheological properties so as to facilitate its usage in the primary stages of preparation of various dishes, e.g., the ability to crumble easily, to slice or to shred cleanly, to bend when in sliced form. The rheological properties also determine the textural properties of cheese during mastication (cf. 'Rheology and Texture of Cheese', Volume 1). On grilling and baking, the cheese may be required to melt, flow, stretch, brown, blister, oil-off and/or stretch to varying degrees. The baked cheese may also be expected to be chewy (as in pizza pie) and contribute to certain mouth-coating characteristics (as in sauces and soups). In many dishes, e.g., sauces, the cheese is required to have the ability to interact with other food components such as water, carbohydrates, proteins and fats, during food preparation. The flavour of the unheated and the heated cheeses is important in
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Uses of cheese and cheese ingredients.
almost all applications, and it should be noted that flavour can be affected by various unit operations. Heating may adversely affect flavour due to the loss of key volatile compounds, or thermal processing may induce the Maillard reaction, resulting in the production of different flavouring substances. The development of rancid off-flavours can occur where interaction of fat and oxygen is promoted by any physical increase in the surface area of the fat during processing. The flavour of other ingredients (such as sauces, condiments, added flavours and flavour enhancers) used along with cheese in some formulated (e.g., soups) and assembled (e.g., pizza, sandwiches) foods and culinary dishes may somewhat mask or alter the flavour of the cheese. From a generic perspective, the functionality of cheese as an ingredient may be defined as its behaviour during all the stages in the preparation and consumption of the food in which it is incorporated. A functional cheese may be described as one which exhibits the required properties when raw or heated, and thereby contributes to the formation of, and/or
enhances the quality of, the food product in which it is used. Conversely, the absence of the correct functional attributes, or the occurrence of undesirable functional attributes at any stage of preparation, may impede the formation of a physico-chemically stable product and impair the usage performance and appeal. More specifically, the functionality of cheese may be defined as a composite property comprising different functional attributes, which may be classified into four main types: 9 rheology-related properties of the raw cheese, i.e., those properties exhibited when the cheese is subjected to a stress (such as cutting, sheafing, mastication) or strain (compression, extension). The rheological properties affect the textural attributes during consumption (see 'Sensory Character of Cheese and its Evaluation', Volume 1) and the deformation and fracture behaviour during formulation and processing, which in turn determines how the cheese grates, shreds, crumbles or slices (Table 1). 9 rheology-related properties of the heated cheese, i.e., those properties exhibited when the cheese is subjected to
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stresses throughout its mass as a result of the heatinduced physico-chemical and microstructural changes such as liquefaction of the fat, fat coalescence and changes in protein hydration and structural rearrangement of the matrix. These properties include the ability of the cheese to melt, flow and stretch. 9 physico-chemical- and microstructural-related properties induced by heating, including oiling-off, browning, blistering, fat coalescence and exudation/ separation, interaction of free amino groups with reducing sugars, moisture evaporation, para-casein aggregation and precipitation. 9 Flavour/aroma-related properties, which are characteristic of a given variety and may be positively or negatively altered during processing. Depending on the application, the combination of, and intensity of, individual attributes vary. Hence, a cheese which shreds well and melts and flows on heating, is ideal for the preparation of lasagna. However, a cheese which exhibits stringiness on heating is very unsuitable for the preparation of sauces, dips, gratins or cheese powders; a more suitable cheese would comminute to a sticky mass, blend easily with other ingredients, and, on heating, form a non-stringy, fluid, homogeneous mass.
as an Ingredient
399
In all applications, whether as a consumer product or as an ingredient, cheese is exposed to size-reduction operations involving a combination of shear and compressive stresses (e.g., >600 kPa) and strains (e.g., ->0.7) that are generally of a magnitude which results in large deformation and fracture (i.e., breakdown into smaller pieces): portioning of cheese into retail sizes, shredding into thin narrow cylindrical pieces (e.g., 2.5 cm long and 0.4 cm diameter), dicing into very small cubes (0.4 cm) and comminution by forcing precut cheese through die plates with narrow apertures. Similarly, when eaten, cheese is subjected to a number of strains which reduce it to a paste capable of being swallowed; first, the cheese is bitten (cut by the incisors), compressed (by the molars) on chewing and sheared (between the palate and the tongue, and between the teeth). The behaviour of the cheese when exposed to the different size-reduction methods constitutes a group of important functional properties, which are summarized in Table 1. In general, apart from shreddability, there is little information in the scientific literature on the functional properties of unheated cheese or how they
Rheological properties of unheated cheese which affect its functionality as an ingredient
Types of properties
Description
Measurements 1
Elasticity and related properties (springiness, toughness)
Tendency of cheese to recover to original dimensions following removal of the applied stress (o-, force per unit surface area)
Fracturability
Tendency of cheese to fracture into pieces when a stress (o-) is applied, e.g., during compression or extension
Recovery of sample after compression, obtained using Texture Profile Analysis Fracture stress (o-f) - force to fracture Fracture strain (sf) -displacement at fracture
(and related terms)
- brittleness
-Iongness
- crumbliness Firmness (and related terms) - Firm - Soft Adhesiveness
Tendency to fracture into pieces at a low deformation or displacement (strain; s, i.e., after a low-percentage compression). Low deformation at fracture, i.e., low sf Tendency to fracture at a large deformation, i.e., high 8 f The tendency to break down easily into small, irregular shaped particles (e.g., by rubbing) Resistance of a cheese to be deformed (e.g., compressed) when subjected to a stress (o-) High resistance to deformation, i.e., high O m a x Low resistance to deformation, i.e., low O m a x Tendency to be sticky and resist separation from a material it contacts
Firmness (O-max)- - stress (o-) required to achieve a given compression/extension
Texture Profile Analysis
References used in compilation: van Vliet (1991), Visser (1991), Fox etaL (2000), Guinee (2002). See 'Rheology and Texture of Cheese', Volume 1 for details of rheology tests. 1 Measurements obtained from large strain deformation tests, as in compression testing or Texture Profile Analysis using a Texture Analyzer; see 'Rheology and Texture of Cheese', Volume 1 for details of tests.
400
Cheese as an Ingredient
may be related to its rheological properties, which determine:
over the pizza base, preparation of sandwiches and use in salad bars. Mature Camembert or Chaumes, which are soft, short and adhesive, are very unsuitable for 9 the magnitude of the stress required to fracture shredded/diced cheese applications because of their (fracture stress, of); tendency to stick to the shredding equipment and of 9 the degree of strain (e.g., change in dimensions) the shredded cheese to bailing and clumping. Howrequired to fracture (fracture strain, ~f); ever, the ability of these cheeses to undergo plastic 9 the level of force or stress required to achieve a fracture and flow under shear (i.e., spread) makes given deformation (O'max); them ideal for spreading on crackers and for blending 9 the type of fracture (i.e., clean or jagged); with other materials such as butter, milk or flour in 9 the degree to which a piece of cheese recovers (in the preparation of fondues and sauces. The brittleness size dimensions) after being strained (e.g., comand tendency of hard cheeses, such as Parmesan and pressed or sheared). Romano, with low levels of moisture and fat-in-dry The various rheological terms (o'f, el, O'max) matter, to undergo elastic fracture (clean fracture withdescribed above are easily measured from the force (or out flow) endows them with excellent gratability stress, o-)/displacement (or strain, e) curve obtained (when crushed between rollers) and suitability as a during compression of a cheese sample, as described free-flow condiment for sprinkling, e.g., onto pasta in 'Rheology and Texture of Cheese', Volume 1. On dishes. However, these properties render the latter consideration of the forces operative during deforma- cheeses unsuitable for food applications that require tion, and the structure and the biochemistry of cheese, slices (e.g., filled sandwiches, cheeseburgers) or shredit can be inferred that relationships do exist between ded cheese. The crumbliness of Feta and Stilton makes the functional and the rheological characteristics of them very desirable for use in tossed salads and Greek unheated cheese. Similarly, cheese texture, which is a salads as the irregularly shaped, curd-like particles crecomposite sensory attribute resulting from a combin- ate an image of 'real' cheese and are more visually ation of physical properties that are perceived by the appealing to the consumer than cheese shreds. senses of touch (including kinaesthesis and mouthfeel), sight and hearing, has been found to be related Factors influencing the rheological (functional) to rheological (stress-strain) characteristics of cheese properties of unheated cheese (Szczesniak, 1963; Sherman, 1969; Brennan, 1988). Cheese rheology and the factors that affect it have The relationships between some common functional been studied (Culioli and Sherman, 1976; Vernon properties and the rheological parameters of the raw Carter and Sherman, 1978; Chen et al., 1979; Creamer cheese, as described below, are given in Table 1. and Olson, 1982; Green et al., 1985; Luyten, 1988; The rheological characteristics of the raw cheese Visser, 1991; Fenelon and Guinee, 2000) and reviewed have a major impact on how it behaves during com- extensively (van Vliet, 1991; Visser, 1991; Rao, 1992; minution and its usability as an ingredient (Table 2). Prentice et al., 1993; Fox et al., 2000; 'Rheology and Thus, it is difficult to cleanly portion hard cheeses Texture of Cheese', Volume 1). The rheology of cheese which have a relatively a low fracture strain (Parmesan) is a function of the combined effects of various factors, or which fracture in a jagged fashion (e.g., an over- including its composition, micro-structure (i.e., the acid Cheddar or Cheshire) owing to their tendency to spatial arrangement of its components and the break at the edges. Similarly, these cheeses are unsuit- strength of attractions between the structural elements) able for applications where shredded cheese is and the physico-chemical state of its components (e.g., required (e.g., pizza) because of their susceptibility to degree of casein hydrolysis). Moreover, it is difficult to fracture/shattering and the resultant formation of a quantify the direct effects of any of the gross composhigh level of curd fines/dust on the surface of the itional components (fat, protein or moisture) separately, uncooked pizza, which is aesthetically unappealing. owing to the fact that these tend to vary simultanConversely, other hard cheeses, such as Cheddar, low- eously, especially where large changes in the concenmoisture Mozzarella (LMMC) and Gouda-type, are tration of a particular component (e.g., fat) occur and unsuitable for grating owing to their lack of brittleness in the absence of process interventions. However, and to their elasticity and relatively high o'f and el, for convenience, the effects of individual factors are which enables a relatively high degree of recovery to discussed separately below. their original shape and dimensions following crushing. However, the latter cheeses generally shred very Protein level well to give pieces of uniform size which are relatively The concentration and the type of protein have a non-adhesive, which makes them ideal for distribution major influence on the rheological properties, as
Cheese
confirmed by the positive correlation between the volume fraction of the casein matrix and cheese firmness (O'max) and the o-f; de Jong, 1977; Guinee et al., 2000a; (Fig. 3), and by the effects of gel fineness or coarseness on the rheological characteristics of the matrix (Green etal., 1983; Green, 1990b; Guinee etal., 1993b). Hence, reduced-fat Cheddar, which has a high volume fraction of para-casein matrix relative to full-fat Cheddar, is firmer, and has a higher o-f, than the latter (Fenelon and Guinee, 2000). The large influence of protein becomes apparent when the effects of an applied stress to cheese structure are considered; the protein matrix provides the first resistance to deformation. The stress-bearing capacity of the matrix is dependent on its volume fraction and homogeneity, which determine the number of stress-bearing strands per unit area. Considering a gel to which a relatively small stress (i.e., much less than the fracture stress) is applied in the direction x, the elastic shear modulus (G', i.e., ratio of shear stress to shear strain, ofT), which is an index of elasticity or strength of the gel, can be related to the number of strands per unit area according to the equation (Walstra and van Vliet, 1986): d2A G' = C N ~ dx 2
where: N = number of strands per unit area of the gel in a cross section perpendicular to x, bearing the stress; C = coefficient related to the characteristic length
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Intact casein, %, w/w Relationship b e t w e e n the c o n t e n t of intact casein and (If]) and the fracture stress (m) in C h e d d a r c h e e s e s of v a r y i n g fat content in t h e r a n g e 6 - 3 1 % , w / w (reprinted from G u i n e e et aL, 2 0 0 0 a with permission from Elsevier).
firmness
as
an
Ingredient
401
determining the geometry of the network; dA = change in elastic energy when the aggregates in the strands are moved apart by a distance, dx, on application of the stress. The number of strands per unit area of a gel are determined by: 9 the concentration of gel-forming protein; 9 the fineness or coarseness of the gel, with a fine gel network having a greater number of stress-bearing strands than a coarse gel. As the concentration of casein in the matrix increases, the intra- and the inter-strand linkages become more numerous, and the matrix more elastic (Ma et al., 1997) and more difficult to deform (de Jong, 1976, 1978a; Chen et al., 1979; Prentice et al., 1993). At low temperatures ( < 5 ~ milk fat is predominantly solid and adds to the elasticity of the casein matrix. The solid fat globules limit the deformation of the casein matrix, as deformation of the latter would also require deformation of the fat globules enmeshed within its porous structure. However, the contribution of fat to the elasticity of cheese decreases rapidly as the ratio of solid-to-liquid fat decreases with increasing temperature and is very low at 40 ~ where all the milk fat is liquid (Guinee and Law, 2002). High heat treatment (HHT) of milk and denatured whey proteins High heat treatment of milk increases the level of in-situ denaturation of whey proteins and their complexation with K-CN at the micelle surfaces. The denatured whey proteins form appendages which protrude from the micelle surfaces and render the Phe]05mMetl06 bond of K-CN less susceptible to hydrolysis by rennet (van Hooydonk et al., 1987; McMahon et al., 1993b). These changes coincide with a reduction in the degree of casein aggregation/fusion during rennet-induced gel formation and the remaining post gel-cutting cheesemaking operations and an increased level of denatured whey proteins incorporated into the gel matrix (Pearse et al., 1985; Green, 1990a,b). Consequently, rennetinduced milk gels from HHT milk have a relatively fine structure, low porosity and an increased waterholding capacity. Cheese prepared from HHT milk (e.g., 82 ~ for 15 s) has lower o-f and Crmax than cheese made from milk pasteurized at a normal temperature (e.g., 72 ~ for 15 s; E1-Koussy et al., 1977; Marshall, 1986; Green et al., 1990a,b; Guinee et al., 1998). These effects are attributable to the reduced degree of para-casein aggregation, the increased level of denatured whey proteins in the protein network and the generally higher moisture level. Owing to its effect on cheese rheology, high levels of denatured whey proteins in cheese milk may
402
Cheese as an Ingredient
be exploited as a means of improving the texture (reducing the firmness and elasticity) of low-fat cheeses which tend to be excessively firm and rubbery (Guinee et al., 1998). For similar reasons, the inclusion of whey protein-based fat mimetics (e.g., Simplesse | 100 and Dairy Lo TM) in reduced-fat Cheddar reduces of, ef and O'ma x (Lucey and Gorry, 1994; Fenelon and Guinee, 1997). The whey proteins in these preparations, at least in the case of Dairy Lo TM, appear to interact with the casein to form a complex- type gel during Cheddar manufacture. Various studies have examined the effects of adding denatured whey proteins, in the form of partially de-natured whey protein concentrates (PDWPC; prepared by the Centriwhey, Lactal or UF processes), to cheese milk for the manufacture of hard or semi-hard cheeses, primarily as a means of enhancing cheese yield. The addition of WPC increases the moisture content, actual yield and moisture-adjusted yield, with the extent of the increase being correlated positively with the degree of denaturation of the added WPC (van den Berg, 1979; Brown and Ernstrom, 1982; Banks and Muir, 1985; Baldwin et al., 1986; Punidadas et al., 1999; Meade and Roupas, 2001). However, the addition of PDWPC has, generally, been found to cause defective body (greasy, soft) and flavour (unclean, astringent) characteristics in Gouda and Cheddar cheeses (van den Berg, 1979), with the intensity of the defects becoming more pronounced with increasing level of the PDWPC added. It has been suggested that these defects may be due to the large size of whey protein particles (aggregates) which do not fit compactly within the pores of the para-casein matrix, and thereby impede its shrinkage and syneretic potential (van den Berg, 1979). Fat c o n t e n t
reduction in the concentration of intact casein. Moreover, liquid fat confers viscosity and also acts as a lubricant on fracture surfaces of the casein matrix and thereby reduces the stress required to fracture the matrix (Marshall, 1990; Prentice et al., 1993). Similarly, reducing the fat content (e.g., from 21-25%, w/w, to "--9-11%, w/w) of low- (47.7-51.8%, w/w) or high(52.2-57.4%, w/w) moisture Mozzarella cheeses resulted in significant increases in hardness and springiness at 1 and 6 weeks, with the magnitude of the effect being the most pronounced for hardness (Tunick et al., 1993). There was a significant effect of the interaction between scald temperature and fat content on hardness, with the effect of fat reduction on hardness being more pronounced as the scald temperature was raised from 32.4 to 45.9 ~ This suggests a higher degree of para-casein aggregation at the higher temperature, an occurrence that would be expected to impede the level of displacement of contiguous casein layers obtained for a given stress. Owing to its effect on the ratio of solid-to-liquid fat in the cheese, temperature has a marked influence on cheese rheology, with the elastic shear modulus (G'), E, o'f and O'max decreasing as the temperature increased (Guinee and Law, 2002; 'Rheology and Texture of Cheese', Volume 1). The effect of the solid-to-liquid fat ratio, as affected by temperature, on the rheological properties of cheese and its use as an ingredient is evident in many instances. Hence, in pizza manufacture, cheese is tempered to, and maintained at, a low temperature prior to shredding (e.g., - 2 ~ so as to maximize the elastic contribution of fat and reduce the tendency of the cheese to stick or clump, and thereby facilitate free flow and distribution onto the pizza surface. Similarly, cheeses are maintained at refrigeration temperatures prior to portioning and slicing to get clean cutting and reduce the risk of surface smearing and greasiness by 'sweated' fat.
Alteration of the fat content has a major effect on the rheological properties of cheese varieties, including Cheddar, LMMC and Cottage cheeses (see Guinee and Law, 2002). Such effects are expected because of the differences in the viscoelastic contributions of fat and casein, as discussed above. However, the overall effects of the changing fat content may be attributed in large part to the interactive effects of changes in the levels of fat, moisture and protein. This is because a reduction in fat content (especially if large, e.g., >4%, w/w) is generally paralleled by increases in moisture, protein, intact casein and Ca. At temperatures of ~--4-20 ~ increasing the level of fat in Cheddar cheese results in decreases in elasticity (E), of, el, Crmax, cohesiveness, springiness, chewiness and gumminess and an increase in adhesiveness. The latter trends are expected because of the concomitant
Homogenization of cheesemilk and degree of fat emulsification Homogenization of milk is practised in the manufacture of some cheese varieties where lipolysis is important for flavour development, e.g., Blue cheese, to increase the accessibility of the fat to mould lipases and thereby increase the formation of fatty acids and their derivatives (e.g., methyl ketones; Fox et al., 1996). Moreover, homogenization is an essential step in the manufacture of cheeses from recombined milks and some acid curd varieties with a high fat content (e.g., Cream cheese; see 'Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acidheat Coagulated Cheeses', Volume 2). Homogenization reduces the mean fat globule size and increases the surface area of the fat by a factor of 5-6 (McPherson et al.,
C h e e s e as an Ingredient
1989). The newly formed fat globules are coated with a membrane consisting of casein micelles, sub-micelles, whey proteins and some of the original fat globule membrane (Walstra and Jenness, 1984; Keenan et al., 1988). The membrane enables the newly formed fat globules to behave as pseudo-protein particles which can interact with the casein micelles and become an integral part of the gel matrix formed during acid or rennet gelation of milk (van Vliet and Dentener-Kikkert, 1982; Green et al., 1983; Lelievre et al., 1990; Tunick et al., 1997; Michalski et al., 2002). Hence, the effective protein concentration of, and the overall level of protein-protein interactions in, the casein matrix are thereby increased. Homogenization of cheesemilk, e.g., at respective first and second stage pressures of 17.6 and 3.5 MPa, generally results in a higher moisture level and decreases in the magnitude of o-f and O'max o f reducedfat Cheddar (Emmons et al., 1980; Metzger and Mistry, 1994). Similarly, homogenization of milk for full-fat Mozzarella (---22%, w/w, fat) cheese, at combined first and second stage pressures of 250 or 500 kPa, resulted in significant decreases in hardness and springiness and an increase in cohesiveness; simultaneously, there were non-significant decreases in gumminess and chewiness (Jana and Upadhyay, 1991). The magnitude of these changes, which increased with homogenization pressure, coincided with a decrease in protein content and increases in the contents of moisture (i.e., - 5 % , w/w) and MNFS. In contrast, Tunick etal. (1993) reported that two-stage homogenization of milk at combined first and second stage pressures of 10.3 or 17.2 MPa resulted in a general increase in the hardness of low-fat ( - 9 % , w/w) or high-fat (~25%, w/w) Mozzarella cheese after storage for 1-6 weeks, the effect being more pronounced for low-fat cheese. Moreover, there was a significant effect of the interaction between homogenization pressure and scald temperature used in cheese manufacture, with the increase in hardness being more pronounced for the higher scald temperature cheeses. The higher hardness at the higher scald temperature probably reflects an increase in the degree of casein aggregation, an effect that would be enhanced as the effective casein concentration increases with homogenization of the milk. Rudan et al. (1998) reported that homogenization of cheesemilk or cream (first and second stage pressure, 13.8 and 3.45 MPa) did not significantly affect the hardness or springiness of reducedfat ( - 8 % , w/w) Mozzarella cheese at 30 days. The discrepancies between the latter two studies, in which the moisture content of the control and the homogenized milk cheeses were similar, may reflect differences in homogenization conditions, test conditions, age of cheese and fat content (see Fox et al., 2000; 'Rheology and Texture of Cheese', Volume 1).
403
Moisture content
Increasing the moisture content, while maintaining the ratios of the other compositional parameters relatively constant, reduces the concentration of protein and the volume fraction of the casein matrix (de Jong, 1978a). Hence, increasing the moisture content of Dutch-type Meshanger cheese from 40 to 60%, w/w, resulted in a marked reduction in O'max. Similarly, increasing the moisture content of 7.5-month-old Gouda cheese from --~32 to 46%, w/w, resulted in progressive decreases in E, crf and O'max (Luyten, 1988; Visser, 1991); the ef increased slightly with moisture content to an extent dependent on cheese pH and maturity. Similarly, Watkinson et al. (2002) reported that an increase in the moisture content of model Cheddar-like cheeses, from 40 to 48%, w/w, resulted in a large decrease in E and degree of cracking at fracture and large increases in ~f and adhesiveness (stickiness). Creamer and Olson (1982) reported a linear decrease in of as the moisture content of Cheddar was increased from 34.0 to 39.7%, w/w, with of at the lower moisture level being almost twice that at the higher moisture level. Salt (NaCI) content The effects of salt in the moisture phase (S/M) in the range 0.4-12%, w/w, on the rheology of model Goudatype cheeses, in which the levels of the other compositional parameters were relatively constant, were studied by Luyten (1988) and Visser (1991). The range of S/M investigated was inclusive of the values that span the spectrum of different varieties, e.g., from --~2.0%, w/w, in Emmental to --~12%, w/w, in Feta. Increasing the concentration of S/M in this range resulted in progressive increases in E, o-f (from --~28 kPa at 0.4%, w/w, S/M to --~83 kPa at 11.3%, w/w, S/M) and O-max (Visser, 1991). The fracture strain, el, increased slightly to a maximum at 4.5-5.0%, w/w, S/M, then decreased sharply to a value which was about half the maximum at 5.5%, w/w, S/M and thereafter remained relatively constant as the S/M was increased to 11.3%, w/w. The effects of salt are probably attributable to its effects on the degree of protein hydration. In low-concentration brines (i.e., -<6.5%, w/w, NaC1), para-casein in cheese absorbs water (Geurts et al., 1972; Guinee and Fox, 1986), an occurrence which is indicative of a salting-in effect on the protein matrix and a concomitant increase in casein hydration. Hence, the presence of NaC1, at a level of 5%, w/w, increased the degree of casein hydration in dilute suspensions of casein micelles, over the pH range 6.7-4.6, and especially at the pH of maximum hydration, i.e., "-'5.2-5.3 (Creamer, 1985). Conversely, when cheese (para-casein) is placed in a brine of higher concentration (e.g., >6.5%, w/w, NaC1), the loss of moisture, especially in the rind region, suggests a salting-out of the protein matrix and concomitant
404
C h e e s e as an Ingredient
casein dehydration/aggregation. An increase in dehydration would be expected to cause an increase in O'max,e.g., as seen by comparing the hardness of cheese rind to that of the cheese interior. The effect of salt, inter alia other factors, on the ef is apparent on comparison of the 'long' smooth body of low-to-medium salt cheeses (e.g., LMMC, Gouda or Swiss; 2-5%, w/w, S/M), with the 'short' crumbly body of high-sah varieties (e.g., Feta, Stilton, Blue; 8-12%, w/w, S/M; Table 1).
pH Small differences in cheese pH can have relatively large effects on its rheological properties which in turn may affect parameters (e.g., crumbliness, longness, shortness, softness, adhesiveness; see 'Rheology and Texture of Cheese', Volume 1) that are important in determining its suitability (e.g., portionability, sliceability, shreddability, gratability) for a particular ingredient application. The effects of pH on the rheological properties of cheese and its use as an ingredient are clearly manifest on comparing different varieties. Low-pH cheeses (e.g., Cheshire, Feta) generally tend to have low values of err and ef and to crumble into many pieces on fracturing, whereas relatively high-pH cheeses (e.g., pH 5.35-5.50; Emmental and Gouda) exhibit higher values of crf and ef and tend to fracture into larger pieces. Inter-varietal differences in pH occur mainly as a result of differences in make-procedure, composition and the type and level of biochemical changes during ripening. However, intra-varietal differences in cheese pH occur also due to a variety of other reasons (see Fox et al., 1996) including differences in: 9 lactate level resulting from differences in lactose level in the cheesemilk (Huffman and Kristoffersen, 1984; Fox and Wallace, 1997) for non-washed curd varieties and moisture level; 9 pH at whey drainage due to variations in the starter activity, pH at rennet addition, and time between starter addition and pitching; 9 buffering capacity, as a result of variations in phosphate content (Lucey and Fox, 1993) due to differences in pH at whey drainage (Lawrence et al., 1984); 9 extent of biochemical changes (e.g., as affected by type and level proteolysis, glycolysis, lipolysis deamination) as influenced by, among other factors, residual rennet activity, salt level, starter type and cell density, and ripening period. Creamer and Olson (1982) studied the effect of cheese pH (4.9, 5.15, 5.4) on age-related changes in ~f and o-f in Cheddar cheeses in which the pH was varied by altering the pH at whey drainage and in which the levels of fat, moisture-in-non-fat substances (MNFS) and salt were relatively similar. Increasing the pH from 4.9 to
5.4 resulted in a linear increase in el, an effect which became more pronounced with time over the 50-day investigation period (Creamer and Olson, 1982). The err changed little as the pH was raised from 4.9 to 5.15 but increased markedly on further increase of pH to 5.4. Similar trends were noted for model Gouda cheeses in which an increase in pH from 5.0 to 5.2 led to a reduction in E and a marked increase in ef but had little effect on err (Luyten, 1988; Visser, 1991). Moreover, increasing the pH from 5.2 to 5.6 led to a marked increase in err (to values much higher than those at pH <5.2), and an increase in E. The pH at which ef was maximal increased with ripening time, e.g., from - 5 . 2 in a i-week-old Gouda to ---5.4 in a 3-month-old Gouda cheese. The effect of pH probably ensues from its influences on: (i) the ratio of soluble-to-colloidal Ca (Guinee et al., 2000c), (ii) total calcium where pH is varied by reducing the pH at whey drainage (Lawrence et al., 1984), and, consequently, (iii) the degree of para-casein hydration or aggregation (Creamer, 1985). Model systems of rennettreated skim milk or casein suspensions have shown that the hydration of para-casein is maximal at pH ---5.2-5.3 (Creamer, 1985); the pH of maximum casein hydration may vary somewhat in different varieties due to differences in the degree of proteolysis and levels of calcium and NaC1. As aggregation of para-casein shows the opposite trend to para-casein hydration, it is expected that the degree of para-casein aggregation/fusion and the elasticity of the para-casein matrix would be minimal at pH 5.2-5.3. The effect of raising the pH above 5.2-5.3 is greater than that of reducing the pH below 5.2-5.3, an effect that may be attributed to the large increase in the calcium binding by the casein at the higher pH (van Hooydonk et al., 1986). The uptake of calcium reduces casein hydration (Sood etal., 1979) and greatly enhances cheese elasticity (Lawrence et al., 1987).
Ripening and para-casein hydrolysis For most varieties, the hydrolysis of Otsl-CN at the Phe23--Phe24 peptide bond, by residual chymosin early during ripening, results in a marked weakening of the para-casein matrix and decreases in ~rf and O-max (de Jong, 1976; Creamer and Olson, 1982; Fenelon and Guinee, 2000). The sequence of residues 14-24 of Otsl-CN is strongly hydrophobic and confers intact Otsl-CN with strong self-association and aggregation tendencies in the cheese environment (Creamer et al., 1982). It has been suggested that self-association of C~sl-CN in cheese, via these hydrophobic patches, leads to extensive cross-linking of para-casein molecules and thereby contributes to the overall continuity and integrity of the matrix (Creamer etal., 1982). Indeed, de Jong (1978b) reported a linear relationship between the content of intact Otsl-CN and the softness in Meshanger
Cheese as an Ingredient
cheese (a soft, internal bacterial-ripened Dutch variety) in which proteolysis was varied by altering the quantity of added coagulant. Moreover, at a microstructural level, proteolysis may result in discontinuities or 'breaks' in the para-casein matrix (de Jong, 1978a), a factor expected to reduce its stress-bearing capacity. Another factor contributing to the age-related weakening of the matrix structure is the increased hydration of the matrix, as reflected by the decrease in the level of expressible serum (on centrifugation or hydraulic pressing of the cheese) or, more appropriately, the increase in the level of non-expressible serum per gram of protein during maturation (Guinee et al., 2002). In contrast to the above, the firmness of some cheeses (e.g., brine-salted and/or surface dry-salted varieties that are not packaged for part of their ripening period) may increase initially even though proteolysis occurs during this period. The increase in firmness is a consequence of the loss of moisture and the concomitant increase in protein level. Other factors such as changes in pH and the increase in the salt content in the inner regions, as a result of inward diffusion from the surface rind zone, may also contribute to the initial increase in firmness. However, the softening associated with proteolysis becomes dominant when the composition has stabilized, and o-f and O'ma x decrease (de Jong, 1976, 1978b; Visser, 1991).
The heat-induced functional properties have been discussed in detail for Mozzarella cheese, and for pasteurized PCP and analogue cheese product (ACP) in 'Pasta-Filata Cheeses' and 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products' (Volume 2), respectively. Hence, this section will concentrate mainly on those of natural cheeses, other than Mozzarella. Types and definitions and principles of functional properties
Cheese is used extensively as an ingredient in cooking applications, e.g., grilled cheese sandwiches, pizza pie, cheeseburgers, pasta dishes and sauces; in these applications the cheese attains a temperature of---80-100 ~ A key aspect of the cooking performance of cheese is its heat-induced functionality, which is a composite of different attributes, including softening (melting), stretchability, flowability, apparent viscosity and tendency to brown. These attributes, which have been defined previously (Kindstedt, 1995; Fox et al., 2000; Guinee, 2002; 'Pasta-Filata Cheeses', Volume 2), are summarized in Table 3; the number and the intensity of the attributes required are determined by the application.
405
Heat-induced softening or melting involves liquefaction of the fat phase. Heat-induced flow or spread and stretchability may be defined as heat-induced rheological changes, involving strain displacement as a result of stresses on the para-casein matrix. These stresses may be of two types: 9 those which occur spontaneously during heating of the cheese under quiescent conditions (e.g., baking); 9 those applied externally to the hot molten cheese mass after cooking, e.g., manually during consumption or instrumentally during testing (e.g., shear during viscometric testing, compression during squeeze flow evaluation or extension during stretchability testing). The changes in viscoelasticity on heating cheese help to explain the mechanism of the melting process (Fig. 5). Spontaneous heat-induced stresses arise when the fat globules, which at low temperatures are solid and reinforce the para-casein matrix that surrounds them, melt and flow on heating. Consequently, the surrounding viscoelastic matrix deforms to a degree dependent on the ratio of the elastic-to-viscous character, which is in turn a function of the degree of casein aggregation or hydration. Additionally, the free oil (FO), arising from the coalescence of liquefied, nonglobular fat droplets, lubricates the displacement of adjoining layers of the deforming matrix and thereby contributes to flow. Clear evidence for heat-induced coalescence of fat globules/droplets in natural cheese is provided by dynamic microscopy of cheese during heating (Paquet and Kal~ib, 1988; Auty et al., 1999; Guinee et al., 1999; Fig. 4) and by the release of oil on baking (Rudan and Barbano, 1998; 'Pasta-Filata Cheeses', Volume 2). At the microstructural level, heating results in extensive clumping and coalescence of fat globules and a less homogeneous distribution of the fat and para-casein phase, at least in the case of Cheddar (Fig. 4) and Mozzarella (Paquet and Kal~ib, 1988). From the foregoing, it is clear that the measures (e.g., levels of flow, stretchability, apparent viscosity, elastic shear modulus, phase angle) of the functionality of heated cheese are to a large extent controlled mainly by the concentrations of fat and protein and their microstructural distributions, and the level of casein hydration. Factors that affect the functionality of heated cheese
The functionality of heated cheese is influenced by many factors (Kindstedt, 1993, 1995; Rowney et al., 1999; Guinee, 2002; 'Pasta-Filata Cheeses', Volume 2), including variations in: 9 milk pre-treatments, e.g., pasteurization conditions, homogenization;
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Bar = 25 #m Confocal scanning laser micrographs of 5-day-old unheated Cheddar cheese (a, b) and the same cheese after heating to 95 ~ and then allowed to cool to room temperature (c, d). The micrographs show protein (a, c - long arrows) and fat (b, d - short arrows) as light areas against a dark background. Bar corresponds to 25 i~m (Modified from Guinee et aL, 2000b).
9 make procedure, e.g., set pH, cooking temperature, pH at whey drainage, plasticization conditions; 9 compositional parameters, e.g., concentrations of fat, protein and moisture, calcium-to-casein ratio, pH; 9 degree of proteolysis; 9 other factors (e.g., absence or inclusion of fat replacers or whey proteins). Most of these factors exert their effects indirectly by influencing the microstructural distributions, and physico-chemical properties, of the protein (e.g., level of calcium binding, degree of aggregation or hydration, degree of casein hydrolysis) and the fat (e.g., degree of fat emulsification, level of fat coalescence) phases. Some of the major factors affecting functionality are discussed separately below. Comparison of different varieties Studies on the functional properties of retail samples of different varieties of natural cheese indicated that there
are considerable intra- and inter-varietal differences in melt time, flowability, stretchability and apparent viscosity (Park etal., 1984; Guinee etal., 2000a). These differences undoubtedly reflect inter- and intravarietal differences in the conditions of manufacture, composition, degree of maturity and/or formulation (e.g., levels and types of added ingredients and processing conditions) in the case of the PCPs and analogue pizza cheese (see 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). Studies (Guinee, 2002) on the storage-related changes in different functional parameters in different cheese varieties confirm the inter-varietal variation and show that functionality is dynamic, changing with storage time and proteolysis (Fig. 6). Compared to other varieties, pasta-filata cheeses (e.g., Mozzarella, Provolone and Kashkaval) are differentiated by their superior stretchability, relatively high apparent viscosity and moderate flowability. These functional attributes endow the pasta-filata cheeses with the characteristics that are
408
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typically associated with the melted cheese on pizza, i.e., sufficiently rapid melt and desirable levels of stringiness, chewiness and flow. In contrast to the pasta-filata cheeses, other types of cheese, including analogue pizza cheese and Cheddar and Emmental, have relatively low stretchability, low apparent viscosity and high (e.g., Cheddar) or low (some analogue cheese) flowability characteristics. If such cheeses were used exclusively, or as a substantial part (e.g., >30%, w/w) of a pizza topping, the melted cheese topping would lack the desired stringiness, would flow excessively and lack the desired chewiness. Conversely, stringiness, which is typical for LMMC and other pasta-filata cheeses, such as Kashkaval and Provolone, is an undesirable attribute for applications such as sauces, gratins, cordon bleu applications or fondues. Cheeses with a high flowability, such as mature Cheddar, Emmental, Raclette and Gouda (see Fox et al., 2000), are more satisfactory for the latter applications because of their relatively high flavour intensity and their lack of stringiness when heated. The flowability of retail Cheddar and other natural cheeses is generally
superior to that of cheese analogues, probably as a consequence, inter alia, of the higher degree of fat emulsification in the latter (see 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). The superior stretchability of pasta-filata varieties is largely attributable to plasticization (heating to ---58 ~ and kneading) of the fermented curd (pH typically ~-5.2) in hot water or dilute brine at "--80 ~ The relatively low curd pH and the high temperature are conducive to limited aggregation of the casein and the formation of para-casein fibres of high tensile strength (Taneya etal., 1992; Pagliarini and Beatrice, 1994; Guinee and O'Callaghan, 1997). Other factors that probably contribute to the high stretchability in these varieties are the low level of proteolysis, because of extensive inactivation of coagulant in the curd during plasticization (Feeney et al., 2001) and the relatively short storage time (at least for LMMC). A survey (Guinee et al., 2000b) of commercial cheeses indicated that the mean concentrations of pH 4.6 SN%TN in Cheddar and LMMC were 20.3 and 4.7, respectively). All other factors being equal, a low level of proteolysis in LMMC, compared to Cheddar (because LMMC is ripened for a short period), would be conducive to a more aggregated and intact casein matrix in the heated cheese, which when subjected to extension or shear stress would be less likely to fracture. The corollary to this is the general ability of unheated LMMC to withstand fracture when subjected to a high level (e.g., 75%) of compression. The rate of primary proteolysis in LMMC, as measured by urea-PAGE and the formation of pH 4.6-soluble N (pH4.6SN), is comparable to that in full-fat Cheddar stored at a similar temperature (7-10 ~ over 70 days (Fenelon and Guinee, 2000; Feeneyet al., 2001; Fig. 7). Undoubtedly, the higher moisture and the lower salt content in LMMC, compared to Cheddar, are more favourable for proteolysis by residual rennet than in Cheddar. Moreover, plasmin activity appears to make a greater contribution to proteolysis in LMMC than in Cheddar (Creamer, 1976; Yun et al., 1993b; Fenelon and Guinee, 2000; Feeney et al., 2001). Despite the increase in proteolysis and the concomitant decrease in the level of intact casein, the stretchability of LMMC is not significantly impaired until the concentration of pH4.6SN exceeds ---16% of total N, e.g., after storing at 4 ~ for >140 days or at 15 ~ for 17 days (Feeney et al., 2001; Guinee etal., 2001). Similarly, the stretchability of experimental Kashkaval does not decrease with increasing level of pH4.6SN in the range 2-16% of total N. In contrast, the stretchability of full-fat Cheddar cheese, which is only slightly inferior to that of LMMC when young (e.g., stored for 30 days at 4-7 ~ deteriorates rapidly on ageing as the level of pH4.6SN increases to a value >6% of total N (Fig. 7). The different stretchtime/pH4.6SN profiles of Cheddar and LMMC probably
C h e e s e as an I n g r e d i e n t
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Changes in levels of primary proteolysis (a) and heat-induced functional characteristics (b, c) of different cheese varieties during storage. Cheeses were: Kashakaval (A), low-moisture part-skim Mozzarella (11), half-fat low-moisture Mozzarella (10%, w/w, fat; F]), full-fat Cheddar (30.0%, w/w, fat; O), half-fat Cheddar (17.2%, w/w, fat; O) and Emmental-type cheese (A). All cheeses were experimental (produced on pilot-scale) apart from the Emmental-type cheese which was factory-produced. The data presented are the means of replicate treatments: 5, Kashkaval; 5, low-moisture part-skim Mozzarella; 3, full-fat Cheddar; 3, half-fat Cheddar; 2, Emmental; 3, half-fat low-moisture Mozzarella. Flowability was defined as the percentage increase in the diameter of a cheese disc on heating in a convection oven at 280 ~ for 4 min, and stretchability as the length of cheese strings at failure on uniaxial extension of molten cheese following heating in a convection oven at 280 ~ for 4 min, as described by Guinee et aL (2000a,c).
reflect differences in the state of aggregation of paracasein (as affected by the inclusion/absence of a plasticization stage), the ratio of soluble-to-colloidal Ca, the pH and the type of proteolysis (i.e., hydrolysis of e~slversus [3-CN; Guinee, 2003). Cheese varieties also differ markedly with respect to the change in flowability with ripening time and, consequently, the level of flow after any given storage time. Differences in flowability between different cheeses can result from differences in milk pre-treatment, make procedure, composition, proteolysis and ripening conditions (Kindstedt, 1993, 1995; McMahon et al., 1993a; Rowney et al., 1999; Guinee, 2002). The interactive
effects of these factors influence the degree of protein aggregation, or hydration, and the level of fat coalescence on heating, which in turn determine the level of heat-induced displacement. Varieties with low levels of fat and primary proteolysis, e.g., half-fat Cheddar, tend to have poor flowability. Differences in protein and fat contents
Increasing the protein content of Cheddar, by reducing the level of fat, impairs its functionality, as reflected by decreases in flowability, stretchability and an increase in the apparent viscosity of the melted cheese (Olson and Bogenrief, 1995; Guinee et al., 2000a,b). The extent of
410
C h e e s e as an Ingredient
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Changes in the levels of pH 4.6-soluble N (closed symbols) and stretchability (open symbols) in experimental low-moisture Mozzarella cheese samples ripened at 4 (A, A) or 10 (11, I-I) ~ and full-fat Cheddar (O, 9 ripened at 4 ~ for 30 days and at 8 ~ thereafter. Data presented are the means of triplicate trials for the Cheddar, and of duplicate trials for the Mozzarella cheeses.
the effect increases with the degree of fat reduction and protein increase. Hence, the level of intact casein in Cheddar and LMMC is correlated positively with the apparent viscosity of the molten cheese and negatively with its flowability (Guinee etal., 2001). Similarly, increasing the protein content of LMMC, by lowering the fat content, reduces the flowability and increases the apparent viscosity (see 'Pasta-Filata Cheeses', Volume 2). The adverse effects of increasing protein content is due to a number of concomitant changes which impede displacement of adjoining layers of the matrix. The changes include an increase in the volume fraction of the casein matrix, decreases in the levels of MNFS and proteolysis and the lower degree of heat-induced fat coalescence and FO (Rudan and Barbano, 1998; Rtiegg et al., 1991; McMahon et al., 1993a; Guinee and Law, 2002; 'Pasta-Filata Cheeses', Volume 2). Moreover, a reduction in the number of fat globules embedded in the casein matrix probably enhances the degree of fusion and aggregation of the rennet-altered micelles within the matrix during gel formation and post-cutting stages of cheese manufacture. Occluded fat globules limit the extent of contraction of the surrounding matrix and thereby physically impede casein aggregation. Storage time and proteolysis Numerous studies have shown that the various functional attributes of cheeses such as Mozzarella and Cheddar change during storage to a degree depending on the composition and functional attributes of the
cheese, e.g., whether stretch or flow (Fig. 6). Changes in proteolysis (e.g., level of pH4.6SN) and protein hydration are major factors contributing to age-related changes in functionality. For a given level of protein, the functionality of cheeses such as Cheddar and LMMC is markedly influenced by the extent of proteolysis (Arnott et al., 1957; Bogenrief and Olson, 1995; Guinee et al., 2001). This effect is reflected by the positive curvilinear relationship between the magnitude of primary proteolysis, as measured by the level of total N soluble at pH 4.6, and the flowability for different varieties (Fig. 8). Hence, elevation of primary proteolysis using different means, e.g., the addition of exogeneous proteinases, the use of coagulants more proteolytic than chymosin (Cryphonectria parasitica proteinase) or elevation of storage temperature, enhances the flowability of different cheese types (Lazaridis et al., 1981; Yun et al., 1993a,b; Madsen and Qvist, 1998; Feeney et al., 2001; Guinee et al., 2001). The positive effect of proteolysis may be associated with a number of concomitant changes, including the increased water-binding capacity (Kindstedt, 1995) and an increase in the number of discontinuities or 'breaks' in the casein matrix at the micro-structural level (de Jong, 1978a). The latter factors are expected to reduce the degree of casein aggregation, which should enhance heatqnduced displacement of adjoining layers of the casein matrix. The different relationships between flowability and primary proteolysis among varieties, as measured by
C h e e s e as an I n g r e d i e n t
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pH4.6SN (Fig. 8), clearly highlights inter-varietal differences in the contribution of other factors (e.g., curd treatment, pH, levels of fat and casein, calcium-to-casein ratio) to flowability. Hence, for a given level of proteolysis, the flowability of reduced-fat cheeses is much lower than that in their full-fat counterparts (Fig. 7). Degree of fat emulsification (DE), fat coalescence and milk homogenization Increasing the degree of fat emulsification (DE) in cheese by high-pressure homogenization of the cheesemilk (e.g., at first- and second-stage pressures of 25 and 5 MPa) impairs the flowability and stretchability of heated full-fat Cheddar (Guinee et al., 2000b), Halloumi (Lelievre et al., 1990) and full-fat Mozzarella (Jana and Upadhyay, 1991; Tunick etal., 1993). The effect of homogenization, which for Cheddar is similar to reducing its fat content from 30 to 1.3%, w/w (Guinee et al., 2000b), would be highly undesirable in applications such as pizza but highly desirable where a high degree of flow resistance is required, e.g., in frying. The adverse effects of milk homogenization coincide with marked reductions in the degree of fat coalescence in the unheated and the heated cheeses and in the release of FO on baking. Increasing the degree of emulsification in PCPs by the selective use of emulsifying salts and other blend ingredients and processing conditions has similar effects (see 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2).
In contrast to the above studies, Rudan et al. (1998) found that homogenization of milk or cream, at first- and second-stage pressures of 13.8 and 3.45 MPa, did not significantly affect the mean flowability of low-fat (---8.5%, w/w) LMMC over a 45-day ripening period. However, similar to the results of Jana and Upadhyay (1991), there was a significant reduction in the level of FO released on baking. The similar flowability of the control and the homogenized low-fat cheeses in the study of Rudan and Barbano (1998), despite the differences in FO, was probably due to the very low level of FO in all low-fat cheeses. The FO was --~0.25 or 0.6 and 3.9% of total fat for the homogenized-milk cheese, homogenized-cream cheese and the control low-fat cheeses at 40 days; the corresponding values for commercial LMMC ranged from - 1 0 to 40% of total fat, depending on FDM and age (Kindstedt and Rippe, 1990; Kindstedt, 1993). Thus, it is noteworthy that Tunick et al. (1993) reported that the interaction between fat content and homogenization pressure had a significant effect on the flowability of LMMC, ripened for 1 or 6 weeks. Homogenization of milk had little effect on the flowability of low-fat LMMC (--10%, w/w, fat) but impaired markedly that of regular LMMC with a higher fat level (----25%, w/w). The adverse effects of increasing the DE on the functional properties of heated cheese are due to the interaction of newly formed fat globules with the paracasein matrix. The effective protein concentration of, and the overall level of protein-protein interactions in,
412
C h e e s e as an Ingredient
the casein matrix are thereby increased (Guinee and Law, 2002). Consequently, it is expected that functional properties relying on displacement of contiguous layers of the casein matrix (e.g., flow and stretch) would be impaired by homogenization of the cheesemilk. Moreover, the recombined fat globule membrane stabilizes the newly formed fat globules to heat-induced coalescence (Guinee et al., 2000b). The consequent reduction in FO predisposes the cheese to dehydration during heating (Rudan and Barbano, 1998) and reduces the lubricating effect of oil on the surfaces of adjoining layers of the para-casein matrix during displacement. Thus, the adverse effects of homogenization on flowability and stretchability may be reduced (Lelievre et al., 1990) by: 9 lowering the homogenization pressure which has the effect of reducing the surface area of the fat phase and the number of newly formed fat globules; 9 preventing the casein micelles adsorbing at the fat-water interface by using a surface film of lecithin, which has the effect of making the newly formed globules more susceptible to heat-induced coalescence. Effect of whey proteins and casein-whey protein interaction on flowability In some cheese applications, softening or melt is essential but very limited flow, or a high degree of flow resistance, is required so as to preserve the shape and identity of the cheese. Examples of the latter include fried Paneer or Quesco Blanco, grilled or fried burgers containing cheese insets, deep-fried breaded cheese sticks, kebabs and casseroles in which the identity of cheese pieces following cooking is desirable (Chandan et al., 1979; Anonymous, 1999). Most other natural cheeses, especially when mature, are unsuitable for these applications owing to their excessive flow and oiling-off on cooking. In the case of cheese insets in deep-fried burgers, the latter attributes result in the melted cheese piece permeating the interstices of the coarse meat emulsion, and hence the inset looses its shape and visual effect in the cooked product (Guinee and Corcoran, 1994). Flow resistance in natural cheese is generally conferred by the presence of whey proteins, which may be included by several means: 9 in-situ denaturation of the whey proteins by HHT of
the cheese milk, e.g., ---65% of total whey proteins are denatured at 100 ~ • 120 s; 9 high concentration ultrafiltration (HCFUF) of milk, with or without H H T of the milk before UF or the retentate after UF, for cheese manufacture involving little or no whey expulsion (e.g., using the AL-curd coagulator);
9 addition of PDWPCs, prepared by HHT and acidification of whey, to the cheese curd (see 'High heat treatment of milk and denatured whey proteins'). High heat treatment of the milk (e.g., 95 ~ • 5 min) and the resultant incorporation of a high level of denatured whey proteins is a feature of the manufacturing process of acid-heat coagulated cheese types, e.g., Queso Blanco types and Paneer and some fresh cheeses such as Cream cheese (Guinee et al., 1993b; 'Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acid-heat Coagulated Cheeses', Volume 2). In contrast, H H T of milk is not usually practised in the manufacture of rennet-curd cheese varieties, because of the impaired curdforming properties of the heated milk, higher moisture content of the cheese and, generally, higher fat losses (see Fox et al., 2000). Guinee (2002) showed that increasing the level of whey protein denaturation (WPD) from 3 to 13% total in the milk ('--0.3-1.0%, w/w, denatured whey protein in the cheese) had little, or no, effect on the flowability of reduced-fat Cheddar. Even though the moisture content of the cheese increased with increasing level of WPD, there was a marked decrease in the flowability on further increasing the WPD to ~30% total ( - ~ 2 . 4 % , w/w, denatured whey protein in the cheese). The stretchability of the molten reduced-fat Cheddar was reduced at all levels of WPD, with the effect increasing with the degree of WPD. Similarly, the flowability of Mozzarella cheeses prepared from H H T milk (e.g., ---5-35% WPD; Schafer and Olson, 1975) or from HCFUF milk retentates (e.g., 42-48%, w/w, dry matter; Covacevich, 1981; Madsen and Qvist, 1998), was markedly lower than the corresponding cheeses from control milk. Some cheeses may be produced from HCFUF retentates (often referred to as liquid pre-cheeses) which are treated with starter culture and/or rennet, and then placed in containers, or moulded either before (as in cast Feta) or after curd coagulation and further curd treatments (e.g., as in Blue- and Brie types, Cream cheese, Cheddar, Mozzarella). This method of production involves little or no loss of whey following treatment of the retentate and retention of most of the whey proteins (see 'Application of Membrane Separation Technology to Cheese Production', Volume 1). Maubois and Kosikowski (1978) described a method whereby Mozzarella cheese with stretch properties similar to those of a control cheese could be manufactured by HCFUF (to --~43% dry matter) and diafiltration at pH 5.8 (to reduce the calcium content); however, little information was given on experimental details or flowability. Covacevich (1981) described the manufacture of Mozzarella curd, which plasticized
Cheese as an Ingredient
satisfactorily, from HCFUF milk retentate (---42%, w/w, dry matter) which had been pre-acidified and salted prior to diafihration to reduce its Ca content. However, the flowability of the UF Mozzarella at 1 week was less than half of that of commercial Mozzarella (Covacevich, 1981). The UF cheese had a markedly lower pH (---5.15 versus 5.9) and lower levels of moisture ("-460 versus 510 g per kg) and MNFS than commercial Mozzarella, changes which could affect flowability adversely (Metzger et al., 2000a,b; Guinee etal., 2002). Similarly, Madsen and Qvist (1998) reported that the flowability of Mozzarella produced from a pre-cheese (48% dry matter) prepared by HCFUF of milk and containing 7%, w/w, whey protein was significantly lower than that of the control Mozzarella throughout a 5-week ripening period. The adverse effect was partly counteracted by increasing the level of casein breakdown in the cheese by treating the curd with a proteinase from Bacillus licheniformis or Bacillus subtilis (Neutrase| The addition of denatured whey proteins (prepared by the Centriwhey process or by HHT of reconstituted WPC) at a level of 0.3-0.4%, w/w, to the cheese milk for LMMC, markedly reduced the flowability and/or the stretchability, and increased the apparent viscosity, of the melted cheese (Punidadas et al., 1999; Meade and Roupas, 2001). McMahon et al. (1996) reported different effects for added whey protein preparations, Simplesse | D100 and Dairy-Lo | on the flowability of low-fat (4-5%, w/w) Mozzarella cheese. At most times during a 28-day storage period, the flowability of the cheese containing Simplesse | was numerically, but not significantly, higher than that of the control while that of the cheese with added Dairy-Lo | was lower than that of the control. The latter trend is probably a consequence of: 9 a higher level of whey protein in the Dairy-Lo| containing cheese (estimated at 0.6%, w/w, versus 0.23%, w/w); 9 the HHT of Dairy-Lo| milk relative to the control and Simplesse| milks; 9 differences in the size of whey protein particles and their spatial distribution in the cheese matrix (McMahon et al., 1996); 9 the degree of interaction of the whey protein particles in the different preparations with the para-casein matrix, as affected by factors such as pH and calcium level (Jelen and Rattray, 1995). The adverse effects of whey proteins on the functionality of heated cheese are probably due to their ability to undergo self-aggregation or aggregation with the paraK-CN in the concentrated acid cheese environment to form aggregated protein structures (pseudo-gels) at the
413
high temperature (typically--~98~ reached during baking/grilling. The tendency to aggregate and gel is probably accentuated by the high content of soluble calcium in the cheese (Doi et al., 1983; Jelen and Rattray, 1995). On setting, these structures would impede displacement of adjacent layers of the para-casein matrix and thereby flow of the molten cheese mass. The formation of some type of aggregate or pseudo-gel is supported by the results of dynamic viscoelastic analysis on heating reduced-fat Cheddar cheese from 20 to 90 ~ At temperatures >61 ~ the phase angle, 6, decreased and G' increased uncharacteristically at a DWP level >1%, w/w, in the cheese (Guinee, 2002). A similar trend for 6 was observed by Horne et al. (1994) for Cheddar cheese prepared from HHT milk (110 ~ • 60 s). These trends suggest an abrupt increase in elasticity, and a decrease in fluidity, as a result of some aggregation/gelation at a temperature >61 ~ Browning of cheese as a consequence of the Maillard reaction
Maillard browning essentially involves reactions between an aldehyde group (e.g., of reducing sugars such as lactose and galactose) and a free amino group (e.g., or- or e-amino groups of amino acids, peptides and protein) and other reactive N-groups (O'Brien, 1995). Browning may occur sometimes during storage of the unheated cheese (e.g., Parmesan, Romano) or processed cheese (Piergiovanni etal., 1989; Younis etal., 1991; Abd E1-Salam et al., 1996; Gopal and Richardson, 1996) but more frequently on heating, e.g., Mozzarella and other cheeses made with a thermophilic culture and PCPs or ACPs. While slight browning of cheese may be desirable in some cooked applications (e.g., lasagna, pizza, crustinis), intense (dark) browning is unacceptable from aesthetic and nutritional viewpoints. Browning rarely occurs on cooking rennet-curd cheeses made with a mesophilic culture (e.g., Cheddar) since these have little or no residual sugars, even after a very short ripening time, e.g., > 14 days (Torres et al., 1995). However, these cheeses may be susceptible to browning on heating if residual lactose persists in the cheese as a result of excessive salt, which inhibits starter metabolism (Thomas and Pearce, 1981; Jordan and Cogan, 1993). Moreover, in the absence of reducing sugars, aldehyde groups, resulting from degradation of amino acids or FFAs (Fox et al., 1996; Fox and Wallace, 1997), may pre-dispose the cheese to heatinduced browning, especially if the cheese is mature and has a high concentration of amino acids. Cheeses made using galactose-negative thermophilic starters (e.g., most strains of Streptococcus thermophilus and Lacobacillus delbruechii subsp, bulgaricus) are very susceptible to Maillard browning, especially during
414
Cheese as an Ingredient
heating. Hence, Parmesan or Romano cheeses, which may contain residual galactose and free amino acids, are usually dried at a low temperature (e.g., <40 ~ to minimize the risk of brownish discolouration. Browning of Mozzarella cheese during cooking and its causes are discussed in 'Pasta-Filata Cheeses', Volume 2. Browning frequently occurs on cooking PCPs and ACPs, especially those with a high concentration of lactose or other reducing sugars added via ingredients such as maltodextrins, skim milk or whey powder, WPCs, milk protein or unfermented milk ultrafiltrates (Thomas, 1969; 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). Owing to their generally higher pH, high concentration of lactose and buffering capacity (associated with added sodium phosphates), PCPs are probably more prone to Maillard browning during storage and cooking than natural cheeses.
Cheese ingredients are prepared by subjecting cheese to either: 9 minimal primary processing involving a macrostructural change by the application of some physical form of comminution (e.g., as for diced, grated or shredded cheeses), or 9 more elaborate secondary processing involving processes (e.g., heating, shearing) and/or agents (e.g., enzymes, emulsifying salts) which lead to marked changes in microstructure, composition, levels of proteolysis and lipolysis, texture, flavour and/or physical form. The various types of cheese ingredients listed in Fig. 1 can be arbitrarily categorized as comminuted cheeses, dehydrated cheese ingredients (DCI) and concentrated cheese flavours (CCF) which comprise different types of enzyme-modified dairy ingredients (EMDI). Sometimes, EMDIs, such as EMC, may be dried for convenience of use (e.g., dry-blending with powdered formulations such as soups and cake mixes) and consequently fall into the category of DCI. The following sections focus mainly on DCIs and EMDIs. Little published information is available on the properties (e.g., free-flow, fines levels, shelf-stability) of comminuted cheese forms and how these are affected by the characteristics of the cheese. However, factors that affect the shreddability of Mozzarella cheese are discussed in 'Pasta-Filata Cheeses', Volume 2; moreover, much information on the physical properties of comminuted cheeses can be gleaned from an understanding of the fracture properties of cheese, which are discussed in 'Rheology and Texture of Cheese', Volume 1 and
'Rheology-Based Functional Properties of Unheated Cheese'. Dehydrated cheese ingredients (DCls): dried cheeses and cheese powders
The DCIs are industrially produced cheese-based ingredients which were developed during the Second World War as a means of preserving cheese solids under conditions to which natural cheese would not normally be subjected, e.g., temperature >21 ~ for a long time period. There are currently three main types of DCIs, namely, dried grated cheeses, cheese powders and dried EMCs. More recently (Anonymous, 1999; King, 1999), freeze-dried cheese pieces (e.g., cubes) have been developed as commercial products. Today, DCIs are of major economic importance owing to their ubiquitous use as flavouring agents and/or nutritional supplements in a wide range of foods (Duxbury, 1991; Lewin, 1996; Missel, 1996; King, 1999). These include bakery products, biscuits, dehydrated salad dressings, sauces, snack coatings, soups, pasta dishes, savoury baby meals, cheese dips, au gratin potatoes and readyprepared meals. Other uses are their inclusion in processed and analogue products as flavouring agents or as a functional ingredient in powdered instant cheese preparations, which can be reconstituted by the consumer for the preparation of instant functional cheeses (e.g., pizza type) for domestic use. Advantages over natural cheeses as an ingredient in the above applications areas include:
(i) convenience of use by fabricated food manufacturers. Dehydrated cheese ingredients can be applied easily to the surface of snack foods (e.g., popcorn, potato crisps, nachos) or incorporated into fabricated food formulations, e.g., by dry-mixing with other dry ingredients such as skim milk powder (e.g., as in dried soup, sauce or cake mixes) or blending into wet formulations. In contrast, natural cheeses require size-reduction prior to their use in these applications. (ii) their longer shelf-life, owing to their lower water activity (aw), than natural cheese. The aw for natural cheeses ranges from - 0 . 9 9 for Quarg to 0.917 for Parmesan (Ruegg and Blanc, 1981); from - 0 . 9 3 to 0.97 for PCPs (Kautter etal., 1979; Tanaka et al., 1979; Eckner et al., 1994), and from ---0.2 to 0.3 for various dairy powders (Spiess and Wolf, 1983). The relatively high stability of cheese powders allows them to be stored for a long period without alteration or deterioration in quality. In contrast, the changes which occur in natural cheese during storage may influence its processability (e.g., ability to be size-reduced, its inter-
C h e e s e as an Ingredient
action with other ingredients) and flavour profile and intensity. Hence, compared to natural cheese, cheese powders lend themselves to easier inventory management, set-manufacturing methods and end-products with consistent quality in large-scale manufacturing operations. (iii) the greater diversity of flavour and functional (e.g., mouth-feel) characteristics that can be obtained from a cheese powder, made possible by the use of different cheese types, EMCs and other ingredients in its preparation. Dehydrated cheese ingredients may be classified into three types, depending on the ingredients used: (i) Dried grated cheeses (e.g., Parmesan, Romano); (ii) Cheese powders, which may be natural (made using natural cheeses, emulsifying salts and, optionally, natural cheese flavours) or extended, which incorporate other ingredients, such as dairy ingredients (e.g., skim milk solids, whey, lactose), starches, mahodextrins, flavours, flavour enhancers and/or colours. Alternatively, cheese powders can be classified according to the proportion of cheese solids, as a % of total dry matter: high cheese solids (i.e., ---95%, w/w), medium cheese solids (>50%, w/w) or low cheese solids (<50%, w/w; Missel, 1996). (iii) dried EMCs. Dried cheeses
Dried grated cheeses are normally used as highly flavoured sprinklings (e.g., for pasta dishes) and in bakery products, e.g., biscuits. Essentially, the production of these products involves fine grinding of hard cheeses and conveyance of the ground cheese to a dryer (usually fluidized bed-type) where it is exposed to low humidity air (e.g., 15-20% relative humidity) at an air inlet temperature <30 ~ Under these conditions, the vibrated cheese is dehydrated rapidly and evaporatively cooled, thereby reducing the risk of fat exudation, the tendency to bailing/clumping and browning. The dried grated cheese (typically 17%, w/w, moisture) is generally pulverized and packaged under nitrogen to reduce the risk of oxidative rancidity during distribution and retailing. High moisture levels (e.g., >21%, w/w) are conducive to clumping and offflavour development, especially if the powder is exposed to air (Hermann, 1993). Certain properties are required for the production of dried grated cheeses, i.e., relatively low levels of moisture (e.g., 30-34%, w/w) and fat-in-dry matter (e.g., 39%, w/w), brittleness and elastic fracture characteristics. These properties lend themselves to efficient size reduction on grinding, minimize the susceptibility
415
to fat exudation and sticking of the cheese particles and contribute to efficient drying and a homogeneous product free of clumps. An intense cheese flavour is also a desirable characteristic. Generally, dried grated cheeses are used in small quantities, as sprinklings, with the objective of imparting a strong cheese note to pasta dishes, soups and casseroles. The cheeses which meet these criteria best are Parmesan and Pecorino cheeses, because of their composition, fracture properties and their strong, piquant, lipolysed flavour. In the Pecorino cheeses the latter characteristic ensues mainly from the addition of pre-gastric esterases (PGEs; from kid, goat or lamb) to the cheese milk, which preferentially hydrolyse the short chain fatty acids (C4-C8, especially butyric) from milk fat triglycerides during cheese maturation. High levels of butyric acid (i.e., 1500-2000mg per kg cheese) endow Romano cheese with its peppery piquant flavour (Fox and Guinee, 1987). Parmesan is produced from raw milk and ripened for a long time (--~2 years) and lipolysis is due mainly to the indigenous lipase. Owing to their generally lower firmness and high levels of moisture and FDM, cheeses such as mature Cheddar (moisture and F D M - "--37 and 52%, w/w, respectively) or Gouda (moisture and FDM "--41 and 48%, w/w, respectively) are unsuitable for drying. These characteristics render the cheese susceptible to fat exudation and clumping during grinding and drying. However, these cheeses also may be grated and dried provided that they are first shredded and blended with Parmesan or Roman-type cheeses before grinding. The moisture content of dehydrated grated cheese may be reduced further from 17%, w/w, by using the Sanders drying process (Kosikowski and Mistry, 1997). The grated cheese powder, placed on trays which are conveyed through a drying tunnel, is exposed to hot air which heats the cheese particles to 63 ~ and reduces the moisture content to --~3-4%, w/w. High-moisture (82%, w/w) cheeses such as Cottage cheese may also be dried directly to 3-4%, w/w, moisture, by first pulverizing and then subjecting them to specialized spray-drying operations (e.g., Silo spray drying using the Birs Dehydration Process; Kosikowski and Mistry, 1997). These low-moisture, dried natural cheeses are generally used for nutritional supplementation of foods, e.g., dried baby meals. Freeze-dried formats of a number of different cheeses such as mature Cheddar, Gloucester, Stilton and oaksmoked Cheddar are now produced commercially (Anonymous, 1999; King, 1999). The benefits of freezedrying, compared to air-drying or spray-drying, include: 9 the retention of volatile flavour compounds (e.g., degradation products of FFAs and amino acids),
416 Cheese as an Ingredient Cheese powders Manufacture. The manufacture of cheese powders essentially involves the production of a pasteurized processed cheese slurry (40-45%, w/w) which is then spray-dried (Fig. 9). The individual processing steps have been described (Hedrick, 1981; Guinee et al., 1993a, 1994; Missel, 1996; Darrington, 1999) and are discussed briefly below.
9 the ability to dry cheeses in the form of croutons, cubes or pellets, which convey an image of cheese pieces and greater naturalness compared to powder and are convenient to use (such pieces can be individually wrapped and added to soups and other products by the consumer), 9 the crunchy light texture of the dried cheese pieces which makes them easy to disperse (by rubbing between the fingers) as a topping and to re-hydrate allowing fast flavour release in the mouth.
(a) Formulation of the slurry blend. The blend usually consists of comminuted natural cheese(s), ,water, emulsifying salts, flavouring agents, flavour enhancers, colours, anti-oxidants such as propyl gallate, butylated hydrozyanisole (BHA) and/or filling materials such as
The production of freeze-dried cheese involves size reduction to the required format, layering onto trays, freeze-drying to - 3 % , w/w, moisture and packaging.
I Cheese I
so..~
i
whey / ~ buttermilk I ~ ~
Comminution I
skim milk J /
caseina~/~ ~
/Maltodextrin / I~ starches 2
\ -.. ~Formulation -.
/Flavours& ~ I ( flav~ enhancers~ I
/
/
~
(/~-Q Water ~
I
EMDIs I I NaCI I I yMeSGtextract/~j)
<
'~ salts /)
I I
I
I
Jf Dissolving tank (cooker)
~.__~fr Colours "'/ antioxidants | ee-flow agent~
Heat and shear
( Steam )
:> Jf
Hot molten slurry (35-45%, w/w, dry matter; 75-85 ~ Homogenization
~k
Atomization
',k
Spray-dry Cheese powder (>96%, w/w, dry matter) Outline of production processfor cheese powder.Abbreviations:EMDIs, enzyme-modifieddairy ingredients;MSG, monosodium glutamate.
C h e e s e as an Ingredient
whey or skim milk solids, starches, maltodextrins and butter-fat. In addition to antioxidants, fat encapsulation technology, which reduces the level of free fat in the powder, may be used to reduce the susceptibility to oxidative rancidity. The type and the level of ingredients used in the formulation depend on powder type (e.g., natural or extended), wettability and solubility characteristics and application (Anonymous, 1991). Typical formulations of the slurries required for natural and extended cheese powders with different levels of cheese solids are given in Table 4. The flavour profile and intensity of the final cheese powder is determined by the type(s) of cheese used and the type(s) and level(s) of other flavouring agents (such as EMC, hydrolysed butter-fat, starter distillates) and flavour enhancers (e.g., sodium chloride, monosodium glutamate, autolysed yeast extract). Generally, mature cheese with an intense flavour is used so as to impart a strong flavour to the final product. Apart from their lack of flavour-imparting properties, young cheeses with a high level of intact casein are unsuitable as they result in very viscous slurries, which are difficult to atomize and dry efficiently. Filling materials in extended cheese powders are usually added to replace cheese solids and thereby reduce the formulation costs. However, they may influence the flavour, wettability and mouth-coating characteristics of the product in which the cheese powder is used. (b) Processing of the blend and slurry formation. Processing principles and technology are similar to those used for the manufacture of PCPs. Processing involves heating the blend (using direct steam injec-
417
tion) to a temperature of---75-85 ~ in a processed cheese-type cooker, or in large (e.g., 5000 L) 'dissolving tanks' (e.g., Limitech) with shearing blades, while continuously shearing (e.g., at 1500-3000 rpm). Maintaining the temperature <85 ~ minimizes the loss of flavour volatiles and the risk of browning, especially in formulations containing high levels of lactose or high dextrose equivalent (DE) maltodextrins. The blend is worked until the hot fluid slurry is homogeneous in colour and consistency. The levels of fat and protein, pH and degree of hydration of the constituent ingredients are the main factors that control the viscosity of the cheese slurry. The viscosity in turn has a major influence on its tendency to foam and the levels of occluded and interstitial air in the resultant powder. High-viscosity slurries (e.g., 3.0 Pas) have less propensity to foam and thus give lower levels of air in the powder compared to low-viscosity (e.g., <0.3 Pas) slurries (Masters, 1976; Tetra Pak Processing Systems, 1995). Incorporation of air should be avoided as it affects the physical (bulk density) and instant (wettability, dispersability) characteristics of the powder and its susceptibility to oxidation on storage (Masters, 1976; Tetra Pak Processing Systems, 1995). The air content of the cheese powder is also influenced by the presence of ingredients which tend to promote (e.g., undenatured whey proteins) or depress (e.g., fat, food-grade antifoaming agents) foaming. However, for a given formulation, air incorporation may be minimized by de-aerating the slurry prior to spray-drying and by using the appropriate type of atomizer, e.g., a pressure nozzle rather than a rotary disc atomizer (J. Kelly, personal communication).
Typical formulations for cheese powders with low (25.5%, w/w, of total), medium (54.3%, w/w, of total) and high (95.4%, w/w, % of total) levels of cheese solids
Level of ingredients added during formulation (%, w/w, of total blend), prior to processing and drying
Ingredient
Low cheese solids powder
Medium cheese solids powder
High cheese solids powder
Mature Cheddar Extra-mature Cheddar Enzyme-modified cheese paste Enzyme-modified cheese powder Whey powder Skim milk powder Maltodextrin Emulsifying salt Sodium chloride Butylated hydroxyanisole Water and condensate
20.0 1.0 12.0 8.0 16.5 1.5 1.0 0.2 39.6
17.0 19.0 0.2 2.0 5.0 3.8 11.0 1.5 1.0 0.4 39.1
32.0 33.0 -
Based on data from Guinee et aL (1993a).
2.0 0.5 0.5 32.0
418
Cheese as an Ingredient
(c) Homogenization of the slurry. This step is optional but is commonly practised to ensure homogeneity of the slurry. The pressures applied (typically 15 and 5 MPa, first and second stages, respectively) have a major influence on the viscosity of the slurry, with higher pressures generally imparting higher viscosity for a given level of dry matter. (d) Spray-drying of the slurry. Several spray-drying processes (e.g., single stage or two stage) and dryer configurations (e.g., tall-form, filter mat, silo-form) may be used. The different processes and their operations (e.g., atomizer type and pressure, air-flow direction, air inlet and outlet temperatures, air humidity) influence the physical (e.g., bulk density, wettability and solubility) and flavour characteristics of the cheese powder. The wettability and solubility characteristics are important in applications requiring reconstitution of the cheese powder, e.g., ready-prepared soups, sauces and baby foods. Typical air inlet temperature ranges from 180 to 200 ~ and air outlet temperature from 85 to 90 ~ depending on the dryer type. The powder is cooled to <---20 ~ in an external fluidized bed, using dehumidified air before packaging. The moisture content of the dried powder is typically 3.0-4.0%, w/w, and generally decreases with increasing outlet air temperature. A high outlet temperature (>95 ~ for a tall form dryer) should be avoided to minimize Maillard browning, loss of flavour volatiles, loss of wettability characteristics, excessive oiling-off and free fat formation. Free fat in the cheese powder leads to lumpiness, flow problems and flavour deterioration. Commercially, cheese powders are normally manufactured using two-stage drying systems, e.g., filtermat (box) dryers are used frequently in the USA while tallform dryers with one or more integrated fluidized beds are commonly used in Europe. While the operating conditions of these dryers influence the quality of the final cheese powder, the tall-form dryer is generally considered to give better flavour retention, larger powder particles and better powder flowability. Conventional single-stage dryers are rarely used, except for experimental purposes, because of the high outlet air temperature (e.g., >95 ~ necessary to achieve the low moisture content (---4%, w/w) required. However, single-stage silo-dryers (with the drying tower ---70-m high compared to --~10 m for the tall-form dryer) may be used, as in the Birs Dehydration Process (Kosikowski and Mistry, 1997), where the drying air at 0-30 ~ is de-humidified. The moisture content of the powder emerging from the tower is ---10%, w/w, and is reduced to ---4%, w/w, in smaller drying chambers. The main advantages of this process over conventional
two-stage drying is that it results in improved colour stability and enhances flavour retention, especially in products with a high level of volatile flavour compounds (e.g., Cottage cheese, yoghurt). Applications of cheese powders. Cheese powders are generally used as flavouring ingredients in a wide variety of foods, especially snack coatings (e.g., pop corn, nachos, tortilla shells), extruded snacks, cheese sauces, soups, savoury dressings and savoury biscuits (Missel, 1996). The development of 'bake-stable' cheese powders is claimed to overcome problems (e.g., unpredictable rise during the baking of crackers, off-flavour development during the production of extruded pelleted snacks) encountered during high-temperature processing (Lewin, 1996). In snack foods, application involves dusting on the powder after the snack has been lightly sprayed with vegetable oil. In cheese sauces, the level of cheese powder is typically 5-10%, w/w, depending on the flavour intensity of the cheese powder and the types and levels of other flavouring ingredients in the formulation. Generally, the cheese powder, at these levels, has little influence on the rheological properties of the resultant sauces; the latter properties are controlled mainly by the types and the levels of starchy materials used (Guinee et al., 1994). Cheese powders may be also combined with various spices, such as cumin, garlic, chilli or onion, for the production of seasonings or savoury additives (Anonymous, 1993; Lewin, 1996; Mortensen, 1999). Enzyme-modified dairy ingredients: enzyme-modified cheese
Natural cheeses have certain limitations as a food ingredient: (i) low flavour stability due to ongoing biochemical/microbiological changes during storage, (ii) flavour inconsistency (e.g., due to changes in cheese composition), (iii) insufficient flavour strength to enable small quantities to impart a strong cheese flavour, (iv) the high cost due to the relatively long ripening time for most cheese varieties and the high usage level required to impart a given cheese flavour intensity to foods and (v) the necessity to comminute cheese prior to its application and the fact that cornminuted cheese is not suited to the bakery and the snack food industries which are large users of cheese. These deficiencies led to the development of EMC, by exploiting the natural biochemical cheese flavour development pathways through enzyme technology, which resulted in cheese flavour intensities of up to 30-fold that of the corresponding natural cheese (Kilcawley et al., 1998; Kilcawley, 2002). Enzyme-modified cheeses can be defined as concentrated cheese flavour ingred-
Cheese as an Ingredient
419
ients, which offer a cost-effective alternative to natural cheese as a source of cheese flavour. Enzyme-modified cheese variants of many natural cheeses, such as Cheddar, Blue, Romano, Parmesan, Colby, Gouda, Camembert, Mozzeralla, Gruyere, Brick and Emmental are available commercially (Freund, 1993; Holdt, 1996; Kilcawley et al., 1998). However, little scientific information is available on the types of enzymes and their specificities, enzyme dosage levels, substrate characteristics and process conditions. Most of the information relating to these parameters remain proprietary to individual manufacturers.
as: (i) chemical substances with flavouring properties obtained by either chemical synthesis or isolated by chemical processes and which are chemically identical to a substance naturally present in material of vegetable or animal origin and/or (ii) by chemical synthesis, but which are not chemically identical to a substance naturally present in material of vegetable or animal origin (European Union, 1998).
Applications Enzyme-modified cheeses are used principally as flavouring agents in industrial-based cheese products/ingredients such as PCPs, cheese substitutes/imitations, cheese powders, soups, sauces, dips, salad dressings, snack coatings, crackers and in prepared and semi-prepared foods. There has been a marked increase in the production of EMCs in recent years. This market continues to expand due to consumer demand, as modem life-styles have reduced the time available to prepare food in the home and increased the amount of food eaten outside the home (Cowan and Cronin, 1999). Legislation regarding the labelling of products containing EMCs varies between the USA and Europe. Enzyme-modified cheeses can be classified into two groups: EMCs per se which are produced by natural processes (e.g., enzymatic hydrolysis and fermentation) and EMC-WONF (with other natural flavours) which are similar to EMC but contain chemically synthesized nature-identical flavours, frequently referred to as 'top notes' in the industry. It is generally assumed that these substances are produced or isolated by chemical processes to mimic natural flavour compounds (Freund, 1995). In the USA, EMCs received GRAS (generally regarded as safe) status in 1969, and since 1970 have been added to specific categories of pasteurized process cheese, non-standard cheese products such a nontraditional reduced-fat or fat-free products and to various prepared foods. Non-standard cheeses include cheese products for which standards of identity do not exist (Code of Federal Regulations, 1986). There are no EU standards relating to the classification of EMDIs, including EMCs. However, a European Union Directive (European Union, 1998) classifies EMC as a 'flavouring preparation' which is defined as a substance obtained by physical, enzymatic or microbiological processes from material of vegetable or animal origin, either in the raw state or after processing for human consumption, by traditional food-preparation processes. Enzyme-modified cheeses with other natural flavours are classified as 'flavouring substances' which are defined
(i) Production of a cheese curd, as in conventional cheese manufacture; (ii) Formation of a paste substrate (typically, 400-600 g per kg dry matter) by blending the curd with water and emulsifying salts. Additional sources of fat and protein are often added as substrates for flavour generation or to enhance other characteristics, such as consistency and texture. Manipulation of compositional parameters is an integral feature of EMC production; (iii) The cheese paste substrate is usually pasteurized (e.g., 72-80 ~ for 10-20 min) to inactivate the existing cheese microflora and enzymes. Pasteurization reduces the risk of flavour inconsistencies due to variations in strain composition and populations of starter culture, NSLAB and residual enzyme activities (e.g., chymosin) in cheese curds; (iv) Often, the pasteurized cheese paste is homogenized to increase the surface area of fat available for optimal lipolysis; first and second stage pressures vary, e.g., 15-25 MPa and 5 MPa, respectively; (v) Treatment of the pasteurized cheese paste with the desired enzymes (blend of proteinases, peptidases, lipases) to give the required flavour profile and intensity in the final EMC. Starter cultures may be used to give more authentic natural cheese flavours but they are usually added in combinations with enzymes; (vi) Incubation of the pasteurized cheese paste. Process parameters are typically 25-45 ~ with constant slow agitation for --~1-4 days at pH 5-7; (vii) Pasteurization of the enzyme-treated paste to inactivate added enzymes and thereby to preserve the generated flavour characteristics with minimal change on storage; (viii) Homgenization of the hot pasteurized paste to reduce the tendency of phase separation on subsequent storage and thus to ensure product homogeneity. The homogenized cheese paste, known as EMC paste, may be then packaged, usually in opaque materials to minimize the risk of oxidative
Technology and manufacture The general manufacture of EMC typically involves the following steps (Fig. 10):
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rancidity and off-flavour development and stored at refrigeration temperatures; (ix) The EMC paste may be dried to give an EMC powder, which has a longer shelf-life than the paste and can be stored at room temperature. Powdered products are better suited for applications involving dry blending with other ingredients. There are several different industrial approaches to EMC manufacture, which are outlined in Fig. 10. The one-step process involves a single substrate which is hydrolysed simultaneously by proteolytic and lipolytic enzymes to the desired final flavour (Fig. 10a). Alternatively, a two-step process may be used in which a single substrate is hydrolysed initially by proteolytic enzymes and subsequently by lipolytic enzymes to create the final product (Fig. 10b). The two-step process offers more controlled flavour production by reducing the possibility of lipase degradation (by proteinases) and preventing the pH from falling below the optimum for proteolytic activity by neutral proteinases. A high level of lipolysis is an integral feature of EMC production, which increases the concentration of FFA, leading to a marked reduction in pH (Kilcawley, 2002). The component process involves the independent hydrolysis of individual substrates by either proteinases or lipases and their subsequent combination (Fig. 10c). This process enables the creation of a range of different product types from two substrates. A further alternative approach is often used for Blue mould-type EMC flavours only. This method involves the submerged aerobic fermentation of various milkfat substrates, such as high fat cream, with Penicillium roqueforti (Nelson, 1970; Dwivedi and Kinsella, 1974). This technology is based on the development of a highly lipolysed product, which directly provides flavour and aroma components associated with Blue cheese flavours, such as butyric acid and precursors of key methyl ketones known to be critically important in Blue cheese flavour. Commercially, blue mould cheese flavour is produced primarily for use in Blue cheese-flavoured dips and salad dressings.
Flavour generation M EMC production It is recommended that natural cheese of the same variety as the type of flavour desired in the EMC should be used as the substrate so as to ensure product consistency and quality (West, 1996). The age of the cheese used as the substrate is also important as young cheese is cheaper but lacks flavour compounds associated with the development of a more authentic natural cheese flavour. Emulsification of the substrate optimizes enzyme activity, in particular lipolysis, which is significantly more important in EMC production than in most natural
421
cheese varieties (Kilcawley et al., 2001; Kilcawley, 2002). Lipolysis generates key volatile flavour compounds, i.e., compounds with a low flavour threshold such as fatty acids, esters, alcohols, lactones and ketones (Fox et al., 1996; Wallace and Fox, 1998; Curioni and Bosset, 2002) which contribute significantly to both the aroma and the flavour of cheese and are therefore directly related to its perceived 'intensity' of flavour. A highly emulsified substrate is generally achieved by homogenization and increases the surface area available for lipolysis. The use of emulsifying salts is necessary to provide a stable emulsion and the salts typically used are those that bind calcium weakly, such as disodium phosphate, trisodium phosphate and trisodium citrate. These salts are not actually emulsifiers per se, but with the aid of heat and shear convert insoluble calcium para-casein to an active hydrated form capable of binding water. Emulsifying salts can also be used to adjust the pH of the substrate to a level optimal for the enzymes used in the process, thereby further accelerating flavour development. In EMC production, an excessive level of hydrolysis may adversely affect emulsion stability and thus influence rate of flavour development. To alleviate this problem, preliminary work is often necessary to optimize the level of emulsifying salts, enzymes and substrate composition. Many EMC products are produced with the inclusion of flavour additives, often called 'top notes' in the industry, to create a diverse product portfolio or to match a particular requirement. These EMCs are classified as EMC-WONE The most commonly used 'top notes' are monosodium glutamate and yeast extracts which are a source of free glutamic acid, which is known to mask bitterness and enhance the perception of flavour (Kilcawley, 2002). Organic acids such as acetic, butyric, propionic, valeric, lactic acids and diacetyl are also widely used to impart specific flavour notes to EMC products. These 'top notes' are used not only to impart specific flavours or enhance flavour, but also to harmonize the flavour of other ingredients, thus creating a more balanced overall flavour profile (Buhler, 1996). The widespread use of these compounds in EMC-WONFs, as evident from the work by Kilcawley et al. (2000, 2001), indicates that it may be difficult or uneconomical to match specific flavour requirements by enzyme technology alone. The relationship between the various commercially available EMC flavours and their corresponding natural cheese varieties is somewhat dubious. Hulin-Bertaud et al. (2000) carried out a detailed descriptive sensory analysis on 15 commercial Cheddar-type EMCs and were able to separate them into five separate clusters based on their sensory characteristics. This work highlighted major sensory differ-
422
C h e e s e as an Ingredient
ences between commercial EMC products marketed as having Cheddar flavour and also showed that none had a flavour identical to that of natural Cheddar cheese. The composition, flavour-forming reactions and flavour components of EMCs have been reviewed extensively (Talbott and McCord, 1981; Kilara, 1985; Moskowitz and Noelck, 1987; Grueb and Gatfield, 1989; Takafuji, 1993; Kilcawley et al., 1998). Enzymes used in EMC manufacture. The enzymes used in the EMC production process have major implications for the intensity and the type of flavour developed. These enzyme preparations contain proteinases, peptidases and lipases and act on casein and fat to produce flavour components and precursors of flavour components (peptides, free amino acids, amines, aldehydes, alcohols, ammonia, FFAs, ketones, lactones, esters and alcohols). A wide range of commercial enzymes are available, derived from plant, animal or microbial sources. Most of the enzymes used in EMC production are derived from microbial and/or animal sources. Plant enzymes are not generally used as they are comparatively impure and expensive, due to large volumes of plants required to produce sufficient amounts of enzyme. The choice of enzyme depends upon a number of factors, such as desired flavour, type and composition of substrate, cost and processing equipment available.
Proteinases/peptidases. Quite a number of commercial microbial proteinases are available for EMC production, most of which are derived from either Bacillus or Aspergillus species and are therefore neutral or acidic in nature. Bacillus proteinases have been implicated in the development of bitterness in cheese and cheese products, and this has precluded their use to some extent in EMC manufacture (West, 1996). Studies by Kilcawley et al. (2002a) have shown that some commercial proteinase preparations from Bacillus spp. lack general aminopeptidase and post-proline dipeptidyl aminopeptidase activities, which are critical in the degradation of bitter peptides. However, the use of proteinases derived from Bacillus spp. is not a problem if used in conjunction with commercial peptidase preparations that contain general aminopeptidase and proline-specific peptidases. Peptidase preparations from Lactococcus lactis have been shown to contain high levels of post-proline dipeptidyl aminopeptidase activity, and peptidases from Aspergillus spp. contain high levels of general aminopeptidase activity (Kilcawley et al., 2002b). Therefore, a cocktail of peptidases derived from both sources provides suitable peptidase activities to prevent the accumulation of bitter peptides.
Lipases. Lipolysis is very important in EMC production due to the production of volatile compounds, which are necessary in providing the perception of cheese flavour to a product to which EMC is added. Lipases are available from two main sources, animal and microbial. The most significant animal lipases are those isolated from bovine and porcine pancreatic tissues or the pre-gastric tissues of kid goat, lamb and calf (Nelson et al., 1977). Different-PGEs produce characteristic flavour profiles: calf PGE generates a 'buttery', slightly 'peppery' flavour, kid PGE generates a sharp 'peppery' flavour, often called 'piccante', whilst lamb PGE generates a 'dirty sock' flavour, often called 'pecorino' flavour (Birschbach, 1992). The use of animal lipases can often limit the application of products as they do not have vegetarian or Kosher status. Kosher status can be important, particularly in the USA, as these products are often perceived by consumers as being of higher quality than non-Kosher products. Microbial lipases are enzyme preparations derived from yeasts, moulds or bacteria, e.g., Rhizomucor miehei, Rhizopus arrhizus, Aspergillus niger, Aspergillus oryzae, Geotrichum candidum, Penicillium roqueforti, Achromobacter lipolyticum, Pseudomonas spp., Staphylococcus spp. and Candida cylindracea (Birschbach, 1994). Microbial lipases are generally cheaper than animal lipases due to lower production costs and have the added advantage of being suitable for use in vegetarian and Kosher foods and also do not contain amylases which may cause problems in foods to which EMCs are added (West, 1996). Commercial lipase preparations for potential use in EMC manufacture have been discussed by Kilcawley et al. (2002b). The selectivity of microbial lipases tends to vary with species and is an important facet in their choice in EMC flavour development, as differing ratios of FFAs impart characteristic flavours. However, it should be noted that extensive levels of lipolysis can lead to non-selective release of FFAs resulting in the development of similar lipolytic flavour profiles despite the use of lipases with different acyl selectivities. A major factor responsible for this is the migration of individual fatty acids on a glyceride, resulting in non-specific release of fatty acids. The effect of acyl migration can be markedly reduced by limiting the level of lipolysis or by using fat substrates of varying FA composition, e.g., the inclusion of vegetable fats with milk fat. It is now also possible to create structured lipids, i.e., lipids that contain specific fatty acids at precise positions on triglycerides. Interesterification catalysed by enzymes appears to offer the greatest potential and involves the exchange and redistribution of acyl groups among the lipid triacylglycerols. Using this approach, it is possible to enrich a fat substrate with specific fatty acids, known to impart flavour, nutritional
Cheese as an Ingredient
or functional characteristics (Balcao and Malcata, 1998; Bosley, 1999) and therefore possibly expand the use of EMC as an ingredient. Another problem associated with high levels of lipolysis is the production of surface active agents, e.g., mono- and diglycerides and soaps of FFAs, which inhibit lipolysis by blocking the interface between the lipase and the fat. This can reduce the rate of lipolysis, but can be alleviated by the addition of calcium salts which promote the formation of insoluble calcium soaps (Kilara, 1985). Starter cultures u s e d in EMC production.
Starter
cultures are used in the commercial production of EMCs but details on the type, concentration and pretreatments of cultures used are not available publicly. It can be assumed that the use of starter cultures in EMC production is analogous to the role of attenuated starter cultures to accelerate the ripening of natural cheese, i.e., they are added as an additional source of flavour-generating enzymes (Kilcawley et al., 1998). Lee et al. (2001) demonstrated the potential of producing strongly flavoured Cheddar-type EMC using exogeneous enzymes in combination with Lactobacillus helveticus DPC 4571 using a two-step process. Dried EMC
Enzyme-modified cheeses may be dried to extend shelf-life and/or for ease of use in specific ingredient formulations. However, the preparation of dried EMCs requires the addition of other ingredients to aid the drying process, which in turn has a diluting effect on the flavour intensity of the final product. This problem is exacerbated by the heat used in the drying process, which results in losses in key volatile flavour compounds. While it is difficult to quantify the negative effect of drying on the flavour of EMCs, as drying procedures vary depending upon the ingredients and process parameters used, it is probably significant. Recent advances in spray-drying technology offer the potential to alleviate flavour loss associated with traditional spray-drying (Fischer, 2000). The possibility of adding functional coating layers through fluidized bed technology has the potential to add further functional characteristics, thereby creating more applications. Enzyme-modified dairy ingredients: enzyme-modified fat
These ingredients, although not cheese per se, are discussed briefly as they are used widely to impart cheese-like flavours and are produced by technology similar to that used in EMC production. Their impor-
423
tance as ingredients is due to their very high flavour intensity, which enables small amounts to deliver sufficient flavour to a product. High levels of flavour intensity are achieved via lipolysis of fat, resulting in the production of volatile compounds, e.g., short chain fatty acids. These can be degraded and contribute to the formation of other volatile flavour/aroma compounds such as methyl ketones, lactones, esters, alcohols and aldehydes, which have low flavour thresholds and can be perceived at minute concentrations. The importance of FFAs and their catabolic products are known to be critical in many dairy flavours, e.g., cheese-, butter-, cream- and yoghurt-type products (Urbach, 1993; Fox and Wallace, 1997). Hence, enzyme-modified fats (EMFs) find application in the baking, dairy, confectionery and savoury food sectors. The manufacture of EMFs generally involves producing an emulsion containing predominately fat or an oil-based substrate. Typically, butte>fat or cream is used as the oil substrate since they contain high levels of lower-chain volatile fatty acids, which are the most important flavour-producing fatty acids. However, depending on the application, certain vegetable oils may be used for cost-effectiveness functionality, flavour or nutritional purposes. The substrate is hydrolysed by starter cultures and/or lipases chosen on the basis of their acyl specificity/selectivity and incubated under defined conditions until the desired flavour is attained. The reaction is usually terminated by heat to inactivate the cultures and/or enzymes used. Reviews of EMFs include Arnold et al. (1975), Kilara (1985) and De Greyt and Huyghebaert (1995).
Cheese is a highly versatile dairy ingredient, which is used directly or indirectly, in the form of cheese ingredients, in a vast array of culinary, formulated and prepared food products. The rheological, flavour and cooking properties are functional attributes, which have a major impact on the preparation and quality of these products. The interactive effects of various factors, including make procedure, cheese composition, degree of fat emulsification, proteolysis and lipolysis, affect cheese functionality. Many of the rheological and cooking functions may be viewed as displacement of adjoining layers of the casein matrix. Hence, these factors exert their effect mainly by affecting the microstructural distributions of fat and protein, and the degree of hydration (or aggregation) of the protein matrix in the raw and heated cheeses.
424
C h e e s e as an Ingredient
Abd E1-Salam, M.H., A1-Khamy, A.E, E1-Garawany, G.A., Hamed, A. and Khader, A. (1996). Composition and rheological properties of processed cheese spread as affected by the level of added whey protein concentrates and emulsifying salt. Egypt. J. Dairy Sci. 24,309-322. Anonymous (1991). Natural cheese powders. Food Process. 52 (11), 114, 116. Anonymous (1993). A savoury assortment. Dairy Ingredients Int. 58 (8) 30-31. Anonymous (1999). Flexible, functional, flavourful cheese. Food Eng. Int. (December) 30-31. Arnold, R.G., Shahani, K.M. and Dwivedi, B.K. (1975). Application of lipolytic enzymes to flavor development in dairy products.J. Dairy Sci. 58, 1127-1142. Arnott, D.R., Morris, H.A. and Combs, W.B. (1957). Effect of certain chemical factors on the melting quality of process cheese. J. Dairy Sci. 40,957-963. Auty, M.A.E., Fenelon, M.A., Guinee, T.P., Mullins, C. and Mulvihill, D.M. (1999). Dynamic confocal scanning laser microscopy methods for studying milk protein gelation and cheese melting. Scanning 21,299-304. Balcao, V.M. and Malcata, EX. (1998). Lipase-catalyzed modification of milkfat. Biotechnol. Adv. 16,309-341. Baldwin, K.A., Baer, R.J., Parsons, J.G., Seas, S.W., Spurgeon, K.R. and Torrey, G.S. (1986). Evaluation of yield and quality of Cheddar cheese manufactured from milk with added whey protein concentrate. J. Dairy Sci. 69, 2543-2550. Banks, J.M. and Muir, D.D. (1985). Effect of incorporation of denatured whey protein on the yield and quality of Cheddar cheese. J. Soc. Dairy Technol. 38, 27-32. Birschbach, R (1992). Pregastric lipases. Bulletin 269. International Dairy Federation, Brussels. pp. 36-39. Birschbach, R (1994). Origins of lipases and their action characteristics. Bulletin 294. International Dairy Federation, Brussels. pp. 7-10. Bogenrief, D.D. and Olson, N.E (1995). Hydrolysis of [3-casein increases Cheddar cheese meltability. Milchwissenschaft 50,678-682. Bosley, J.A. (1999). Enzymes used in oils and fats technology, in, Ingredients Handbook Enzymes, Rastall, R., ed., Leatherhead Food Research Association Publishing, Surrey, UK. pp 79-95. Brennan, J.G. (1988). Texture perception and measurement, in, Sensory Analysis of Foods, 2nd edn, Piggott, J.R., ed., Elsevier Applied Science, London. pp. 69-101. Brown, R.J. and Ernstrom, C.A. (1982). Incorporation of ultrafiltration concentrated whey solids into Cheddar cheese for increased yield. J. Dairy Sci. 65, 2391-2395. Buhler, T.J. (1996). Natural dairy concentrates going beyond flavour. Food Technol. Europe 3, 56-58. Chandan, R.C., Matin, H., Nakrani, K.R. and Zehner, M.D. (1979). Production and consumer acceptance of Latin American white cheese. J. Dairy Sci. 62,691-696. Chen, A.H., Larkin, J.W., Clark, C.J. and Irwin, W.E. (1979). Texture analysis of cheese. J. Dairy Sci. 62, 901-907. Code of Federal Regulations (1986). Part 133: Cheese and related products, in, Food and Drugs 21. Code of Federal
Regulations, Parts 100-169. US Government Printing Office, Washington, DC. Covacevich, H.R. (1981). Recent experiences in pasta-filata cheesemaking by ultrafiltration. Proc. Second Biennial Marschall International Cheese Conerence, Madison, WI. pp. 237-244. Cowan, C. and Cronin, T. (1999). Consumer attitudes to convenient foods, in, Process through Innovation. National Food Centre, Teagasc, Dublin. pp. 19-21. Creamer, L.K. (1976). Casein proteolysis in Mozzarella-type cheese. NZJ. Dairy Sci. Technol. 11,130-131. Creamer, L.K. (1985). Water absorption by renneted casein micelles. Milchwissenschaft 40, 589-591. Creamer, L.K. and Olson, N.E (1982). Rheological evaluation of maturing Cheddar cheese. J. Food Sci. 47, 631-636, 646. Creamer, L.K., Zoerb, H.E, Olson, N.E and Richardson, T. (1982). Surface hydrophobicity of Otsl-I, C~sl-caseinA and B and its implications in cheese structure. J. Dairy Sci. 65, 902-906. Culioli, J. and Sherman, P. (1976). Evaluation of Gouda cheese firmness by compression tests. J. Text. Stud. 7,353-372. Curioni, P.M.G. and Bosset, J.O. (2002). Key odorants in various cheese types as determined by gas chromatography-olfactometry. Int. Dairy J. 12,959-984. Darrington, H. (1999). The power of powder. Food Manuf. 74 (March), 39-40. De Greyt, W. and Huyghebaert, A. (1995). Liapase-catalysed modification of milk fat. Lipid Technol. 7, 10-12. de Jong, L. (1976). Protein breakdown in soft cheese and its relationship to consistency. I. Proteolysis and consistency of 'Noordhollandase Meshanger' cheese. Neth. Milk Dairy J. 30, 242-253. de Jong, L. (1977). Protein breakdown in soft cheese and its relation to consistency. 2. The influence of rennet concentration. Neth. Milk DairyJ. 31,314-327. de Jong, L. (1978a). The influence of moisture content on the consistency and protein breakdown of cheese. Neth. Milk Dairy J. 32, 1-14. de Jong, L. (1978b). Protein breakdown in soft cheese and its relation to consistency. 3. The micellar structure of Meshanger cheese. Neth. Milk Dairy J. 32, 15-25. Doi, H., Ideno, S., Huang Kuo, E, lbuki, E and Kanamori, M. (1983). Gelation of the complex between K-casein and [3lactoglobulin. J Nutr. Sci. Vitaminol. 29,679-689. Duxbury, D.D. (1991). Natural cheese powders. Food Process. 52 (ll), ll4, 116. Dwivedi, B.K. and Kinsella, J.E. (1974). Continuous production of Blue-type cheese flavor by submerged fermentation of Penicillium roqueforti. J. Food Sci. 39,620-622. Eckner, K.E, Dustman, W.A. and Rys-Rodriguez, A.A. (1994). Contribution of composition, physicochemical characteristics and polyphosphates to the microbial safety of pasteurized cheese spreads. J. Food Protect. 57, 295-300. E1-Koussy, L., Amer, S.N. and Ewais, S.M. (1977). Studies on making baby Edam cheese with low-fat content. II. Effect of milk heating. Egypt. J. Dairy Sci. 5,207-213. Emmons, D.B., Kal~ib, M., Larmond, E. and Lowrie, R.J. (1980). Milk gel structure. X. Texture and microstructure
C h e e s e as an Ingredient
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Index
Acid-and Acid/Rennet-curd cheese, 17-18, 301-304 acid-heat coagulated, 343 Queso Blanco and Paneer, 343-5 Ricotta, 345-6 combined acidification/renneting: calcium chloride, 315 concentration, 308-11 heat treatment, 311-13 incubation temperature, 314 level/type of gel-forming protein, 314-15 pH at renneting, 313-14 rate of gelation, 314 Cottage cheese, 329-38 Cream cheese, 321-3 fresh cheese preparations, 323 gel formation during combined acidification/renneting: factors influencing, 308-15 mechanism, 306-308 physico-chemical changes, 305-306 other varieties, 323-4 Quark, 315-21 theology: Cream cheese, 323 Quark, 320 unheated cheese, 399-405 ripened varieties, 323 see also Quark; Ricotta; Cottage cheese; Cream cheese Akawi, 241 Amarelo da Beira Baixa, 291 Analogue cheese products (ACPs), 21,349,379-85 composition/functionality, 384-5 formulation, 382-4 principles/manufacturing protocol, 382 see also Processed cheese products (PCPs) Anevato, 290 Appellation d'Origine ContrOl~e (AOC), 1 Armada, 293 Arthrobacteriaceae, 206 Asiago, 55 Austrian cheese, 319 Azeitao, 284 Bacteriocins: smear-ripened cheese: anti-Listeria compounds of Geotrichum and Penicillium, 218 coryneform bacteria, 215-17 enterococci, 217-18 lactic acid bacteria (LAB), 214-15 Baker's cheese, 319 Balkan, cheeses, 58-9, 319 Bastelicaccia, 290 Beli Sir U Kriskama, 244-5 Beyaz Peynir (Turkish White), 244
Bjalo Salamureno Sirene (White Brined), 244 Blue cheese, 6, 16-17, 175 micro-organisms contributing to ripening: contaminants, 183 lactic acid bacteria, 179 non-starter lactic acid bacteria, 182-3 Penicillium roqueforti, 179-80, 185 yeast, 180-2 microbial interactions, 183 microenvironment, 175-7 ripening: formation of aroma compounds, 189-90 lipolysis, 185-6 production/occurrence of mycotoxins, 190-1 proteolysis/amino acid catabolism, 186-9 selection of cultures, 191-2 Blue des Aravis, 289 Brevibacterium linens, 204 Brie, 199 Broccio, 280 Buttermilk Quark, 319 Cabra Transmontano, 291 Cacioricotta, 291 Caciotta D'Urbino, 283 Camembert, 157, 199 control of ripening, 169-71 flavours, 162-9 glycolysis, 160-1 goat-type, 289 lipolysis, 162 microbial flora, 158-60 proteolysis, 161-2 technology, 157-8 texture, 169 Cameros, 293 Canestrato Pugliese, 56-7, 283 Caprino tradizionale, 290 Castellano, 287 Castelmagno, 57 Castelo Branco, 284 Catabolism of amino acids: Blue cheese, 186-9 surface mould-ripened cheese, 165-7 Cendrat del Montsec, 292-3 Chakka/Shirkhand (India), 319 Cheddar, 14-15, 71 casein, 75 cheddaring, 71 chemical composition/quality: effect of FDM, 85 effect of MNFS, 83-4 effect of pH, 84-5 effect of S/M, 85
430
Index
Cheddar - contd. dry-salting, 71-2 flavour: milkfat, 89 proteolysis, 89-90 role of adjuncts, 92-3 role of non-starter lactic acid bacteria, 92 role of starter, 90-2 grading, 93-4 manufacture: acid production at vat stage, 76-7 cheddaring, 77-8 coagulant, 74-5 cutting, 75 heating/cooking curd, 75-6 milk composition/starter culture, 73-4 milling, 78-9 pressing, 81-3 salting, 79-81 texture: effect of pH, calcium, salt, 87 effect of protein, fat, moisture, 87 effect of ripening, 88 varieties: low-fat, 94-5 stirred-curd/granular cheese, 95 washed-curd, 95-6 Cheese analogues, 8, 379-85 Cheese as an ingredient, 18, 395-423 dehydrated cheese ingredients, 414-18 cheese powder application, 418 cheese powder manufacture, 415-18 dried cheeses, 414-15 enzyme-modified cheese (EMC): applications, 418-19 dried, 422 enzymes used in manufacture, 421-2 flavour generation in EMC production, 419-21 starter cultures, 422 technology/manufacture, 419 enzyme-modified milk fat, 422-3 functional properties of heated cheese: browning; Maillard reaction, 413 comparison of different varieties, 407-409 degree of fat emulsification, fat coalescence, milk homogenization, 411 differences in protein/fat contents, 409-10 storage time/proteolysis, 410 types, definitions, principles, 405 whey proteins/casein-whey protein interaction on flowability, 411-13 functional requirements, 396-9 rheology-based functional properties of unheated cheese: fat content, 402 high heat treatment of milk/denatured whey proteins, 401-402 homogenization of cheesemilk/fat emulsification, 402-403 moisture content, 403 pH, 404 protein level, 400-401 ripening/para-casein hydrolysis, 404-405 salt (NaC1) content, 403-404 Cheese categories, 1-22 Cheese technology: goats' milk cheese, 287-9 overview, 24
post-vat stages: brine-salted varieties, 38-46 dry-salted varieties, 31-8 fresh cheese, 46-9, 315-19,321-3,343 Pastafilata, 47-9, 251-3,265-6 processed cheese products, 353-6 Swiss cheese, 148-9 Vats, 25-31 Chevrotin, 289 Codex Alimentarius, 1
Colby, 95-6 Conjero, 292 Cooking: Cottage cheese, 332-3 curds, 75-6 Coryneforms, 204 Arthrobacteriaceae, 206-207 Brevibacterium linens, 204-206 Corynebacterium, 206 Cottage cheese: addition of CaC12/rennet, 332 common defects/possible causes, 337-8 cream dressing, 334 cutting/cooking curd, 332-3 direct acidification, 331 drainage, washing, cooling of curd, 333-4 flavour, 337 incubation of milk, 330 manufacture, 329-30 microbiological quality, 336-7 nutritional quality, 335-6 physical structure, 334 specifications, 329 starters, 330-1 use of UF skimmilk, 335 yield/quality, 334-5 Cream cheese, 321 further treatments of curd after separation, 322-3 recombination technology, 322 rheological/syneretic aspects, 323 whey separation: using separators, 321 using UE 321 Crottin de Chavignol, 289 Cultures see Starter cultures Cutting of milk gel: Cheddar, 75 Cottage cheese, 332-3 Feta, 234-5 Vats, 25-31 Danish cheese, 320 Dehydrated cheese ingredients (DCIs), 414-18 see also Cheese as an ingredient Domiati, 16, 227-8 microbiology, 233-4 ripening, 228-33 see also Ripened-in-brine cheese Dried cheeses, 414-15 Dry-salting, 71-2 Eastern European cheeses, 319 Emmental, 141 eye formation, 152-4 fermentations, 141-8 hygienic safety, 154 ripening, 149-52 technology, 148-9
Index
Emulsifying salts characteristics of: bactericidal effects, 369-7 calcium sequestration, 365-6 casein hydration, 367-8 crystallization, 367-9 emulsification, 367 flavour, 370 hydrolysis, 367 pH adjustment and buffering, 366-7 major types, 364 Enterococcus, 207 Enzyme-modified cheese (EMC), 418-22 applications, 418-19 dried, 422 enzymes used: lipases, 421-2 proteinases/peptidases, 421 flavour generation, 419-21 starter cultures, 422 technology/manufacture, 419 see also Cheese as an ingredient Equipment see Cheese technology Escherichia coli 0157:H7, 213-14 Evora, 284 Ewes' milk cheeses: French, 280 general aspects, 279-80 Greek, 280-2 Italian, 282-4 Portuguese, 284-5 Spanish, 285-6 Extra-hard varieties, 14, 51 Balkan, 58-9 Italian, 52-7 main chemical/technological features, 52-9 nutrition, 67 ripening: changes in microflora, 59-60 lactose metabolism, 60 lipolysis, 60-2 proteolysis, 62-4 volatile compounds, 64-7 Russian, 58 Spanish, 58 Swiss, 57-8 Eye formation, 132-3,152-4 Feta, 16,234, 290 biochemistry of ripening: lipolysis, 238 proteolysis, 235-8 volatiles, 238 defects, 133,239 manufacture, 234-5 microbiology, 238-9 rheological/sensory properties, 238 yield/gross composition, 235 see also Ripened-in-brine cheese Fiore Sardo, 56,283 Flavour, 149-51 amino acid catabolism: aldehydes, 165-6 amine biosynthesis, 166 amines, 166 catabolism of amino acid side chains: indole ring, 166 sulfur compounds, 166-7
Cheddar, 88-92, 88-93 Cottage cheese, 337 development, 128-9 Domiati, volatile compounds, 231-2 Enzyme-modified cheese (EMC), 419,421 Fatty acid catabolism, 162, 164, 165 Gouda, 128-9 miscellaneous compounds: esters, 167-8 pyrazines, 168-9 styrene, 167 terpenes, 168 volatile contaminants, 169 surface-ripened cheese: proteinases/lipases, 209-10 sulphur amino acids, 208 Formaella Arachovas Parnassou, 281 Fossa, 57 Fresh cheese, 323 Fromage frais, 319
Galotyri, 281 Gibna Bayda (Beida), 239 Glycolysis, surface mould-ripened cheese, 160-1 Goat's milk cheese: French, 289-90 Greek, 290 Italian, 290-1 Portuguese, 291 Spanish, 291-3 technology/flavour: Blue cheese, 288 composition, 288 Gouda, 15 manufacture: brining, 113-14 cheesemaking, 108-13 control of pH/water content, 119-22 curdmaking, 108-109 draining/moulding, 109-10 milk standardisation, 116-17 milk treatment, 105-108 pressing, 110, 112-13 process principles, 104-105 rind treatment/curing, 114-16 starters, 122-4 maturation: cheese composition during ripening, 124-6 fermentation of lactose/citric acid, 126-7 flavour, 128-9 lipolysis, 128 possible microbial defects, 133-5 proteolysis, 127-8 texture, 129-33 origin/characteristics, 103-104 yield: bactofugation, 118-19 CaC12, 119 cold storage of milk, 118 curd washing, 119 genetic variants of milk proteins, 118 inclusion of native whey proteins, 119 mastitis, 118 mechanical losses, 119 pasteurisation of milk, 118 rennet type, 118 salting, 119
431
432
Index
Gouda - contd. seasonal effects, 118 starter, 118 Grading, 93-4 Grana Padano, 55 Graviera Agrafon, 281 Graviera Kritis, 281 Gredos (Tier, La Vera), 292 Gruyere, 7, 199 Halloumi, 245 Havarti, 199 Hygiene, 154 Iberico, 291 Ibores, 291-2 Icelandic cheese, 319-20 Idiazabal, 58, 286 Imitation cheese see Analogue cheese products (ACPs) Indian cheese, 319 Ingredient, cheese as see Cheese as ingredient Italian-type cheese, 52-7,282, 290 Jarlsberg, 141 Kashkaval: general characteristics, 264-5 manufacturing technology, 265-9 quality characteristics, 269-72 Kasseri, 281 Kefalograviera, 281-2 Kefalotyri, 282 Kopanisti, 281 La Serena, 287 La Vera, 292 Labneh (Labaneh, leben), 319 Lactic acid bacteria (LAB), 92, 179, 183-4 Lactofil (Sweden), 320 Ladotyri Mytilinis, 282 Layered cheese (Schichtk~se), 323-4 Leerdamer, 141 Limburger, 199 Lipolysis, 61 Blue cheese, 185-6 Camembert, 162 Domiati, 230 extra-hard cheeses, 60-2 Feta, 238 Gouda, 128 surface mould-ripened cheese, 162 Listeria monocytogenes, 212-13
Low-moisture Mozzarella cheese (Pizza cheese), 251 age-related changes in structure/function, 260-4 functional properties: before heating, 257-8 browning, 260 heat-induced (melting), 258-60 meltability, 258-9 oiling-off, 260 stretchability, 259-60 manufacturing technology, 251-55 plasticization/stretching, 252-4 physico-chemical characteristics of curd, 253 reorganization of curd structure, 255 thermal effects on starter bacteria/coagulant, 254-5 thermo-mechanical treatment of curd, 253-4
Maasdamer, 141 Mahon, 58 Maillard reaction, 413 Majorero, 291,292 Manchego, 58,285-6 Manouri, 282 Mascarpone, 18,324 Microbial flora, 59, 158 bacteria, 160 interactions between micro-organisms, 160 Micrococcus spp., 203-204 Middle Eastern cheeses, 319 Milk: cold storage, 118 composition, 73-4 Domiati, 228-9 Feta, 234 genetic variants of proteins, 118 incubation for Cottage cheese, 330 preparation for cheese manufacture, 23-5 treatment: bactofugation, 107-108 pasteurisation, 105,107, 118 quality, 105 standardisation, 108, 116-17 Mish, 239-40 Montasio, 55-6 Monterey, 95-6 Mould-ripened cheese see Surface mould-ripened cheese Mozzarella di Bufala, 15-16 Mozzarella see Low-moisture Mozzarella cheese Mudaffara, 240 Monster, 199 Murazzano, 283-4 Murcia, 292 Nabulsi, 241 Nisa, 284 Non-European cheeses, 19-21 Norwegian whey cheese, 18-19 Ossau-Iraty, 280 Palmero, 292 Paneer, 343 acidification, 344 drainage/curd handling, 344 heat treatment of milk, 343-4 manufacturing methods, 343 Parmigiano Reggiano, 52, 55 Pasta-fi|ata varieties, 7, 15-16, 251 Kashkaval, 264-72 low-moisture Mozzarella (Pizza) cheese, 251-64 Pecorino Romano, 56,282-3 Pecorino Sardo, 56,283 Pecorino Siciliano, 56,283 Pecorino Toscano, 283 Penicillium roqueforti, 179-80 interactions with: contaminants, 185 lactic acid bacteria, 183-4 yeasts, 184-5 Picante da Beira Baixa, 291 Pichtogalo Chanion, 281 Pizza cheese see Low-moisture Mozzarella cheese Pont l'Eveque, 199 Port du Salut, 199
Index
Portuguese cheese: ewe, 284-5 goat, 291 Post-vat stages of cheese manufacture, 31-48 see also Vats Pressing of curd, 81-2 rapid cooling, 82-3 vacuum, 82 Processed cheese products (PCPs), 7, 18, 21,349-51 blend ingredients: caseins, 377 cheese, 374 cheese base/UF milk retentate, high heat-treated milk/whey proteins, 376-7 co-precipitates, 378 emulsifying salts, 373-4 lactose, 378 rework, 3 74-6 skim milk powder, 378 classification, 351-2 consistency/cooking characteristics: blend ingredients, 3 73-8 compositional parameters, 378-9 processing temperature/shear, 373 processing time, 3 71-3 stabilizers (binding agents)/hydrocolloids, 379 'Creaming' in processed cheese products: creaming effect, 371 over creaming, 372-3 emulsifying salts, 360-71 effect on consistency, 373-4 effect on cooking properties, 373-4 major types, 364 properties, 365-70 role in formation of physico-chemically stable product, 360-3 manufacturing principles: heating natural cheese in absence of emulsifying salts, 359-60 microstructure of rennet-curd, 356-9 manufacturing protocol: cleaning/size reduction, 353 formulation of blend, 353 homogenization, 356 hot packing/cooling, 356 pre-mixing of formulation materials, 353,355 processing of blend, 355-6 micro-structure, 362-3 see also Analogue cheese products Propionic acid fermentation, 135,141,142-4 effect of: facultatively heterofermentive lactic acid bacteria, 145,147 feeding season, 144-5 Lactobacillus helveticus, 147-8 eye formation, 152-4 hygienic safety, 154 lactic acid, 141-2 ripening: flavour formation, 149-51 general aspects, 149 texture formation, 151-2 technology, 148-9 Protected Designations of Origin (PDO), 1 Proteolysis: Blue cheese, 186-9 Camembert, 161-2 Cheddar, 89-90
Domiati, 229-30 extra-hard cheese, 62-4 Feta, 235-8 Gouda, 127-8 Quark, 320 surface mould-ripened cheese, 161 Quark, 315 manufacture: acid or acid/rennet gel treatment, 317-18 addition of cream, 318 filtration methods, 316-17 original (standard) separator process, 315 recombination technology, 317 thermisation of curd/fresh cheese, 318-19 thermo process (Westfalia), 316 tradition batch methods, 315 proteolysis/bitterness, 320 rheological/syneretic aspects, 320 varieties directly related, 319-20 see also Acid- and Acid/Rennet-curd cheese Queso Blanco, 343 manufacture: heat treatment of milk, 343-4 methods, 343 milk acidification, 344 whey drainage/curd handling, 344 microbial quality, 346 microstructure, 344 physico-chemical properties, 346 see also Acid- and Acid/Rennet-curd cheese Raba~al, 291 Ricotta, 18,284, 345-6 part-skim, 345 shelf-life, 346 whey/skimmilk, 345 whole milk, 345 see also Acid- and Acid/Rennet-curd cheese Ripened-in-brine cheese, 16, 227 Akawi, 241 Beli Sir U Kriskama, 244-5 Beyaz Peynir (Turkish White), 244 Bjalo Salamureno Sirene (White brined), 244 classification/nomenclature, 227 Domiati, 227-34 Feta, 234-9 Gibna Bayda (Beida), 239 Halloumi, 245 Mish, 239-40 Mudaffara, 240 Nabulsi, 241 technological features, 242 Telemea/Telemes, 241,243-4 see also Feta; Domiati Roncal, 58,286 Roquefort, 280 Russian cheese, 58 Saanenk~ise, 57 Sainte-Maure, 289 Salt, 119 Blue cheese, 176 Cheddar: equilibration, 80 mellowing, 79-80 milled curd, 79 seaminess/fusion, 81
433
434
Index
Salt - contd. Domiati, 229 effect on functional properties, 403-4 Feta, 235 surface-ripened cheese, 169-70, 201 Sbrinz, 57 Secondary/adjunct cultures, 92-3 Serpa, 284 Serra da Estrela, 284-5 Skyr (Iceland), 319-20 Smear cheeses: bacteriocins: anti-listerial compounds of Geotrichum and Penicillium, 218
Coryneform bacteria, 215-17 enterococci, 217-18 lactic acid bacteria, 214-15 defined cultures, 211-12 environmental factors, 200 manufacture, 199-200 micro-organisms: coryneforms, 204-207 flavour development, 208-10 pigmentation/colour development, 210 secondary flora, 207-208 staphylococci/micrococci, 203-204 yeast, 201-203 origin of surface microflora, 210-11 pathogens, 212 physical/chemical characteristics, 200-201 ripening, 199 ripening consortia, 218-19 Soft cheese, 6 Spanish cheese, 58 Staphylococcus, 203-204, 213 Starter cultures, 118 Blue cheese, 179, 191-2 Camembert, 157 Cheddar, 73-4 composition/handling, 122-4 Cottage cheese, 330-1 Domiati, 233 Emmental, 141 enzyme-modified cheese (EMC), 422 low-moisture Mozzarella (Pizza) cheese, 254-5 Mozzarella, 251 non-starter lactic acid bacteria (NSLAB), 92 Parmigiano Reggiano, 55 Pecorino Romano, 56 preparation, 25 role: enzymes, 91 other activities, 91-2 Sbrinz, 57 Smear, 199 Swiss, 141 Substitute cheese see Analogue cheese products; Processed cheese products (PCPs) Surface mould-ripened cheese, 6, 16, 157 control of ripening, 169-71 diversity, 157
flavour: catabolism of amino acid side chains, 166-7 fatty acid catabolism, 162-6 miscellaneous compounds, 167-9 glycolysis, 160-1 interactions between micro-organisms, 158-60 lipolysis, 162 proteolysis, 161-2 technology, 157-8 texture, 169 see also Camembert Swedish cheese, 320 Swiss cheese, 57-8 see also Emmental Tan (Than) cheese, 319 Technology see Cheese technology Telemea/Telemes, 241,243-4 Tenerife, 292 Terrincho, 285 Texture, 3-4, 151-2 Cheddar, 85-8 Domiati, 232-3 eye formation in Swiss cheese, 132-3 Gouda, 129-33 structure, 129, 131-2 surface mould-ripened cheese, 169 Tier, 292 Tilsit, 199 Tofu, 21 Topfen, 319 Torta del Casar, 287 Trappist, 199 Tulum, 319 Tvorog/Tvarog, 319 US cheese, 319 Valdetja, 293 Valencay, 289 Vats, 25-31 APV CurdMaster, 29-30 continuous processes, 30-1 Damrow Double-O, 27-8 Damrow horizontal, 28 OST vat, 26-7 Scherping horizontal cheese vat (HCV), 28 see also Post-vat stages of cheese manufacture
Yeast: Blue cheese, 180-1,184-5 brine/dairy environment, 181 occurrence/growth, 181-2 raw milk, 181 in smear cheese: de-acidification, 201-202 stimulatory compounds, 202-203 Ymer (Denmark), 320 Zamorano, 286-7
