ADVANCES IN FOOD RESEARCH VOLUME 22
Contributors to This Volume
N. T. Crosby G. Hobbs Gary E. Petrowski Y. Pomeranz ...
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ADVANCES IN FOOD RESEARCH VOLUME 22
Contributors to This Volume
N. T. Crosby G. Hobbs Gary E. Petrowski Y. Pomeranz R. Sawyer Samuel M. Weisberg John R. Whitaker
ADVANCES IN FOOD RESEARCH VOLUME 22
Edited by C. 0. CHICHESTER The Nutrition Foundation, Inc. New York, New York and University of Rhode Island Kingston, Rhode Island
G . F. STEWART University of California Davis, California
E. M. MRAK University of California Davis, California
Editorial Board J. F. KEFFORD S. LEPKOVSKY EDWARD SELTZER W. M. URBAIN
E. C. BATE-SMITH W. H. COOK J. HAWTHORN M. A, JOSLYN J. R. VICKERY
1976
ACADEMIC PRESS
New York
San Francisco
A Subsidiary of Harcourt Brace Jovanovich, Publishers
London
COPYRIGHT 0 1976, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC. 111 Fifth Avenue, New York,
New York 10003
Uiiited Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWI
LIBRARY OF CONGRESS CATALOG CARD NUMBER: 48-7808 ISBN 0- 12-016422-1 PRINTED IN THE UNITED STATES O F AMERICA
CONTENTS Contributors to Volume 22
............................................
vii
N-Nitrosamines: A Review of Chemical and Biological Properties and Their Estimation in Foodstuffs
..
.
N T Crosby and R Sawyex
I . Introduction .................................................. I1 Chemistry of Nitrosamines ........................................ I11 Biological Properties of Nitroso Compounds .......................... IV AnalyticalAspects .............................................. V Conclusions ................................................... References ....................................................
. . . .
1 8 27 33 55 56
Development of Flavor. Odor. and Pungency in Onion and Garlic
John R .Whitaker
.
I Introduction .................................................. I1 Sulfur Compounds of Intact Allium ................................. I11 Biosynthesis of Sulfur Compounds of AIliums ......................... Iv Sulfur Constituents of Crushed ANium .............................. V Methods of Measuring Allium Pungency. Flavor. and Aroma .............. VI Enzymes in Flavor. Aroma. and Pungency Development in ANium ......... VII Summary ..................................................... VIII Research Needs ................................................ References ....................................................
. . . . . . .
73 74 83 90 114 116 124 125 127
Clostridium botulinum and I t s Importance in Fishery Products
.
G Hobbs
. .
I Introduction .................................................. I1 Incidence and Distribution ........................................ 111 Classification and Nomenclature
.
...................................
135 137 142 V
CONTENTS
vi
IV. Methods for Isolation and Identification
. . . . .
.............................
V Properties of the Toxins .......................................... VI Factors Affecting Germination. Growth. Sporulation. and Toxin Production VII Inhibition of Clostridium botulinurn ................................ VIII Reservation of Fishery Products IX Summary and Conclusions ........................................ References
.
................................... ....................................................
146 151 154 157 160 168 169
Food Products Intended to Improve Nutrition in the Developing World
. I. Introduction .................................................. I1. Review and Discussion ........................................... 111. Conclusions ................................................... References .................................................... Samuel M Weisberg
187 189 201 203
Scanning Electron Microscopy in Food Science and Technology
Y .Pomeranz
. . . . . . .
..................................................
I Introduction I1. Principles and Applications ....................................... 111 Elemental Analyses ............................................. N Methods of Specimen Preparation and Ancillary Techniques .............. V Applications in Microbiology VI Applications in Plant Investigations VII Miscellaneous Biological Applications VIII . Studies of Foods and Food Products Ix Some Reflections on Scanning Electron Microscopy References ....................................................
...................................... ................................. ............................... ................................ ....................
206 208 222 231 239 250 252 258 292 293
Emulsion Stability and I t s Relation to Foods
. Emulsions ..................................................... Emulsifiers .................................................... Fatsand Oils .................................................. Other Emulsion Factors .......................................... Directions .................................................... References ....................................................
310 318 327 330 346 347
Subject Index .................................................. Errata ........................................................
361 366
Gary E Petrowski
I. I1 I11
.
. V.
IV.
CONTRIBUTORS TO VOLUME 22 Numbers in parentheses indicate the pages on which the authors’ contributions begin.
N. T. CROSBY, Department of Industry, Laboratory of the Government Chemist, London, England (1)
G. HOBBS, Ministry of Agriculture, Fisheries and Food, Torry Research Station, Aberdeen, Scotland (135) GARY E. PETROWSKI, Carnation Research Laboratories, Van Nuys, California (309) Y . POMERANZ, U. S. Grain Marketing Research Center, Agricultural Research Service, U. S. Department of Agriculture, Manhattan, Kansas (205) R. SAWYER, Depurtment of Industry, Laboratory of the Government Chemist, London, England (1) SAMUEL M. WEISBERG, League for International Food Education, Washington, D. C. (1 87)
JOHN R. WHITAKER, Department of Food Science and Technology, University of California,Davis, California (73)
vii
This Page Intentionally Left Blank
N-NITROSAMINES: A REVIEW OF CHEMICAL AND BIOLOGICAL PROPERTIES AND THEIR ESTIMATION I N FOODSTUFFS BY N . T. CROSBY AND R.SAWYER Department of Industry Laboratory of the Government Chemisi London. England
I . Introduction .................................................... A Nitrites and Meat Preservation B The Chemistry of the Curing Process C Botulism .................................................... D Toxicity of Nitrites E Historical Development of the Nitrosamine Problem I1 Chemistry of Nitrosamines A Nitrosation and Rate of Reaction B General Synthetic Routes to N-Nitrosamines C Properties and Structure D. ChemicalReactions 111. Biological Properties of Nitroso Compounds A . Carcinogenicity B. Biochemical Changes IV. Analytical Aspects A Nitrosamines in Biological Materials-The Analytical Problem B . Nonvolatile Nitroso Compounds .................................. C. Occurrence of Nitrosamines in Foods D Nitrosamines in Tobacco V Conclusions References
. . . . . . . . .
.
.
.
.................................... ............................... ............................................ ................... ......................................... ................................. ........................ ........................................ ............................................ ........................... ............................................... ........................................... ............................................... ........... .............................. ........................................ .................................................... .....................................................
1 2 4
5 6 7 8 8 10
13 17 27 27 32 33 33 46 47 53 55 56
I. INTRODUCTION The use of common salt along with varying amounts of nitrates and nitrites for the curing and preservation of meat is long established. In recent years. 1
2
N. T.CROSBY AND R. SAWYER
following the discovery of the hepatotoxic (Barnes and Magee, 1954) and later the carcinogenic properties (Magee and Barnes, 1956) of N-nitrosodimethylamine, the advisability of using curing salt containing nitrates and nitrites has been reviewed in several countries. This worldwide concern stems from the possibility that nitrites could react during curing, storage, or cooking, with amines occurring naturally in certain foods, to form nitroso compounds, thereby constituting a toxicological hazard to man. Equally, concern has been expressed following the realization that the nitrosation reaction could also occur in the stomach, and at a more rapid rate, during digestion. Present research is, therefore, directed toward further study of the biological properties of the nitrosamines and related compounds, together with investigations into the distributions of nitrates, nitrites, amines, and nitroso compounds in the human environment. Although the qualitative behavior of many of these compounds is well known, it has not been possible to establish a dose-response relationship, and hence a no-response level, even in animal studies. Further problems arise in extrapolation of the results to the human situation, and in the large numbers of animals in both study and control groups which are necessary when operating at the low dosage levels required for the results to be statistically significant. Some of these problems have been discussed recently by Grasso (1973). A complementary approach employs epidemiological studies of human cancer and its relation to environmental factors. This approach has been successful in the past, but with the modern trend to increasing movement and mixing of populations, it is less likely to provide reliable quantitative data in the future. More attention has been given in recent years to the development of reliable methods of analysis for the detection and determination of nitroso compounds, and considerable success has been achieved. Such methods are a prerequisite not only in determining the distribution of nitroso compounds in the environment but also in monitoring the diets used in experimental studies on animals. This review summarizes the chemical and biological properties of nitroso compounds, and discusses in detail work which has been proceeding in the analytical area and the results obtained recently with these methods on a wide range of foods. The formation of nitroso compounds both in model systems and in vivo is considered with reference to kinetic data and toxicological techniques, and present and future research programs are examined.
A. NITRITES AND MEAT PRESERVATION From ancient times Man has had a need to conserve and preserve his food. Fresh meat is one of the more perishable of Man’s foods; after the slaughter of an animal putrefaction sets in rapidly, particularly in hot climates, unless preventive measures are taken immediately. Smoking and salting have been used
A’-NITROSAMINES: A REVIEW
3
for preservation from the earliest times; refrigeration and canning are examples of relatively modern processing techniques. Spoilage of food by microorganisms can be prevented in two ways: 1. By destroying the causative organisms through heat (sterilization) or irradiation, and subsequent protection against reinfection. 2. By addition of preserving agents to inhibit the development of the microorganisms. This can be achieved in several ways-for example, by reduction of the available moisture content through the addition of salts or sugars having an osmotic effect, by refrigeration and freezing, or by packaging under vacuum or in the presence of an inert gas.
Smoking, in addition to reducing the moisture content of the food, may also have a bacteriostatic action by virtue of substances such as phenols and aldehydes present in the smoke. Changes of flavor are also observed. In addition to its curative action, salting imparts a desirable flavor to the final product. On cooking, the color of fresh meat changes to brown, but with preserved (salted) meat it was noticed many years ago that red patches formed on the surface. These patches were subsequently thought to be caused by nitrates present in the curing salt as impurities, and as a result nitrates were deliberately added in an attempt to produce a greater uniformity of color. Further work showed that, in fact, nitrites were the agent responsible for the development of the red color in cured meat products, the nitrites being produced from nitrate by bacterial or enzymatic reduction. Nitrates and nitrites are now widely used for curing many varieties of meat products, and modern methods such as direct multineedle injection into the vascular system of the carcass have speeded up the curing process from several weeks to a few days. The most rapid curing method, in which slices of meat are passed through a suitable curing solution, is complete in only a few minutes. In addition to the fixation of the characteristic pink color of cured meats, nitrites also impart a characteristic flavor to the product. Thus, ham and bacon possess a flavor quite distinct from that of salted pork. Furthermore, nitrites confer resistance to the growth of botulinum organisms and possibly some protection against other food-poisoning organisms-for example, Clostridiurn welchii. In uncured meats, putrefactive organisms multiply rapidly and the odors produced afford some degree of protection, since consumers would automatically reject the product on esthetic grounds alone, In cured products, however, the growth of such “indicator” organisms is retarded, thus presenting a greater hazard to the consumer where other food-poisoning organisms are present. Ingram (1973) and Wasserman (1973) have reviewed the microbiological effects of nitrite and the flavor effects of nitrite on cured meats. Mottram and Rhodes (1973) were able to establish that for curing of bacon the flavor effect
4
N.T.CROSBY AND R. SAWYER
FIG. 1. Diagrammatic representation of the structure of myoglobin.
due to nitrite in the curing solution reached a maximum of about 1500 mg of nitrite per liter, but that the effect was also dependent on salt concentration.
B. THE CHEMISTRY OF THE CURING PROCESS Nitrous acid can act both as an oxidizing and as a reducing agent. Thus, under suitable conditions, it can oxidize ferrous salts to the ferric form, and evidence of reducing properties can be found in the conversion of bromate to bromide and in its ready oxidation to nitric acid. Sodium nitrite in solution at the pH values normally found in fresh meat exists very largely in the dissociated form. However, in curing brines some decomposition to nitric oxide can occur. The normal dark red color of fresh meat is due to the muscle protein myoglobin, although the surface layer may be oxygenated and will be somewhat brighter red in appearance owing to the presence of oxymyoglobin. The basic structure of myoglobin is shown diagrammatically in Fig. 1. The nonpeptide portion of the molecule, referred to as the “heme,” consists of an atom of iron surrounded by a large planar ring (the porphyrin) made up of pyrrole units linked by methylene bridges. Myoglobin and nitric oxide react under reducing conditions to form nitrosyl myoglobin, the red pigments seen in preserved meat. The reaction can be written as follows: globin
N, I,N Fe
N’
1 ‘N
I
H*O Myoglobin
globin ~
N, N ,l
Fe
N’
j
‘N
I
NO
Nitrosomyoglobin
Under reducing conditions the iron remains in the ferrous form. Where the meat pigments have been oxidized, metmyoglobin is formed. Here, the iron is in the
N-NITROSAMINES: A REVIEW
Myoglobin : purplish red ferrous form
IN0 ferrous form
oxygenation L
5
OxymYOglobin : bright red ferrous form
, \ation
nitrous oxide
i
oxidation nitrite
brown
Denatured forms.
FIG. 2. Relationship between some oxidized and reduced forms of myoglobin.
ferric form and the pigment is brown in color. On heating, a denatured form of the pigment is produced. Thus, although the curing process is undoubtedly complex, in essence it involves the interaction of nitrite with myoglobin under reducing conditions to form the nitrosyl product. Satisfactory color development requires the reduction of both the nitrite and the pigment itself. The relationship between the various processes is shown in simplified form in Fig. 2. The chemistry of the curing process has been reviewed by Mohler (1973) and by Atkinson and Follett (1973). C. BOTULISM
As well as for its action in the formation and fixation of a satisfactory color in cooked meats, nitrite is added to control the growth of CZostridium botulinum and consequential toxin production where subsequent heat treatment does not effect total sterilization and destruction of the toxin. The precise mode of the inhibition is not fully understood, but a number of interdependent factors are involved, such as concentration of nitrite and of salt, pH, degree of heat treatment, and number of spores present initially. Roberts and Ingram (1973) have studied the relationships between temperature, pH, sodium chloride, nitrite, and nitrate on the inhibition of various types of CZ. botulinum in model systems. Baird-Parker and Baillie (1973) carried out studies of a similar kind but took into account the effects of ascorbic acid and meat extracts in their studies. They showed that, since individual strains of CL botulinum varied markedly in their resistance to sodium nitrite, they could be divided into two main groups on the basis of nitrite tolerance. The most resistant strains, generally growing in at least 150 to 200 mg of sodium nitrite per liter at 25"C, occur among the heat-resistant types. The most sensitive strains (inhibited by 100 to 150 mg of
6
N.T.CROSBY AND R. SAWYER
nitrite per liter) are the heat-sensitive, nonproteolytic, and psychrotrophic types. Shank et al. (1962) have shown that undissociated nitrous acid is the molecular species responsible for the inhibition of Cl. botulinum spores and therefore maximum protection is afforded at low pH values. The pH of fresh meat is around 5 to 7, and it cannot easily be lowered owing to its high buffering capacity. Botulism (derived from the latin botulus, meaning a sausage) is one of the most serious forms of food poisoning, and fatalities have been reported. In less serious attacks the victim suffers from vomiting, abdominal pain, and difficulty of vision (W. R. Thomson, 1958). While the toxin is relatively heat-labile, the organism itself produces spores which are considerably more resistant to heat. To avoid excessive shrinkage in certain canned meat products-for example, large hams-heat treatment during canning is unlikely to be sufficient to destroy all the spores throughout the whole volume of the meat. Nevertheless, outbreaks of botulinum poisoning have been relatively rare. Certainly other factors in addition to nitrite cures have played their part in safeguarding meat supplies: systematic slaughter control and meat inspection procedures, refrigeration, and hygiene, for example. Still, it would be foolhardy to abandon long-established techniques of meat preservation without a thorough examination of the evidence, and moreover the establishment of the safety of alternative measures. Further information on all aspects of the botulism problem can be found in the Proceedings of the 1st U.S./Japan Conference on Toxic Microorganisms (Herzberg, 1970).
D. TOXICITY OF NITRITES Before we consider the chemical and biological properties of nitroso compounds, a note on the reactions of nitrite itself is not out of place. Nitrite acts as a vasodilator and hypotensive agent (Rubin et al., 1963); it can reduce the storage of vitamin A in the liver, and it may also disturb thyroid function (Emerick et ul., 1963). The oxidation of hemoglobin to the ferric form, methemoglobin, occurs in an analogous way to the myoglobin reaction which has been discussed. In this form the pigment is incapable of carrying oxygen in the bloodstream. This is particularly serious for infants, who have a high liquid intake per body weight. Cases (some fatal) of methemoglobinemia in babies given feeds prepared from well waters containing high levels of nitrate have been described (Wood, 1961). Certain baby foods may contain relatively large amounts of naturally occurring nitrate (Phillips, 1971). These aspects of nitrite toxicity have resulted in similar regulations in different countries to control the nitrite intake in the diet. However, following the discovery of the hepatotoxic properties of nitrosamines (formed by the interaction of nitrite with amines) the problem has had t o be reexamined.
A'-NITROSAMINES: A REVIEW
I
E. HISTORICAL DEVELOPMENT OF THE NITROSAMINE PROBLEM The hepatotoxic properties of N-nitrosodimethylamine were first described by Barnes and Magee in 1954. Further tests for possible carcinogenic action showed that malignant tumors developed in nearly all the rats fed on a diet containing 50 mg of this compound per kilogram (Magee and Barnes, 1956). These initial findings catalyzed a new field of research so that today the biological activity of a large number of nitroso compounds is well documented. However, at this stage interest in nitroso compounds was limited to an academic study of carcinogenesis. Fresh impetus to these studies resulted from the outbreak of a rare liver disease in ruminants and mink in Norway during the period 1957 to 1962. Subsequent work showed that the disease was related to feeding the animals with a herring meal that had been preserved with sodium nitrite. The toxic substance present in the feed was isolated and identified as N-nitrosodimethylamine (Ender et al., 1964, 1967). N-Nitrosodimethylamine (30 to 100 mg/kg) was detected in six toxic batches of the meal but not in six nontoxic samples. Model tests using various methylamines occurring naturally in fish were carried out, and it was established that several of these could react with nitrite to form nitrosamines. N-Nitrosodimethylamine was formed in the presence of dimethylamine and nitrite on prolonged storage even at pH 6.5 and 0°C. This finding led to the suggestion that nitrosamines might also occur in human food through the interaction between naturally occurring (or added) precursor materials-namely, amines and nitrites. Several authors (for example, Mirvish, 1970) who studied the kinetics of the reaction in model systems were able to show that secondary amines and nitrite react only slowly to form nitroso compounds at ambient temperatures and neutral pH values, but that the reaction proceeds rapidly at a pH value close to that of the human stomach. It has, therefore, been suggested that the formation of nitroso compounds in vivo may well represent a greater hazard to man than the ingestion of the same compounds formed in minute quantities during the processing of foods containing nitrite. Tannenbaum et al. (1974) have recently discussed the importance of salivary nitrite to the in situ formation of nitrosamines. An essential prerequisite to research in this field is the development of analytical methods capable of detecting, identifying, and measuring minute quantities of nitroso compounds in a food matrix, or in biological tissues where the available sample weight is limited. Methods have recently been described which it is claimed will detect quantities of certain nitrosamines at levels as low as 1 part in lo9, and some positive identification of nitrosamines in foods have been reported (see Part IV,C). It remains to assess the significance of such low levels as a hazard to humans, and further biological testing is now directed toward this end.
N. T.CROSBY AND R. SAWYER
8
II. CHEMISTRY OF NITROSAMINES N-Nitrosodimethylamine is the first member of the class of aliphatic nitrosamines in which the nitroso group NO- is attached to nitrogen. This compound has been used as an industrial solvent and as an intermediate in the synthesis of the rocket fuel 1,l-dimethylhydrazine. There are patents covering the use of various nitrosamines as solvents in the fiber and plastics industry, as antioxidants in fuels, as softeners for copolymers, as nitrogen-sparing additives to fertilizers (Goring, 1966), as synergists to selfextinguishing agents used in expanded polymer materials (Ingram, 1966), and as insect repellants, insecticides, fungicides, and bactericides (Goodhue, 1966). Nitrosamines are also employed as intermediates in the manufacture of medicinal preparations (Maitlen, 1961; Levering and Maury, 1963; Getz, 1960). N-Nitrosodimethylamine has been suggested for use as a nematocide in the form of aqueous solutions or as powders. The application rates employed for tomatoes and carrots vary from 56 to 112 kg/hectare. At the rates quoted, Maitlen (1961) has shown that it is not phytotoxic for carrots, tobacco, cabbage, and the cotton plant; however, it does inhibit the growth of lima beans and peas. A. NITROSATION AND RATE OF REACTION
The study of the chemistry of aliphatic nitrosamines began in 1863 when Geuther obtained N-nitrosodiethylamine by the reaction of diethylamine hydrochloride with sodium nitrite. Taylor and Price (1929) undertook a systematic study of the kinetics of the reaction between nitrous acid and a variety of amines including dimethylamine in aqueous solution. They postulated that the reaction involved the amine cation, the nitrite ion, and an undissociated molecule of nitrous acid; that is, velocity varies as the product (RzNH,’) (NO,-) (HNO,). In this work the amount of dimethylamine lost was measured rather than the amount of N-nitrosodimethylamine formed, and the influence of pH on the rate of reaction was cursorily examined. Modern work summarized by Ridd (1961), Mirvish (1970), and Fridman et al. (1971) suggests that the N-nitrosation reaction may be represented generally by the following scheme: R,
:R
NH
+
NO-X
+
R,+ H N:
R:LNO + x-
where X may be 0-alkyl, NO,, halogen, O H : ,
-
R, -k
R:N-No
OH+-alkyl or 8
The amine participates in the reaction in a nonprotonated form, and therefore it is desirable to carry out nitrosation of highly basic amines in weakly acidic media. Although other mechanisms are known (Fridman et al., 1971; Austin, 1961), consideration of the reaction in aqueous solution is relevant to the
9
N-NITROSAMINES: A REVIEW
situation which occurs in foodstuffs. In aqueous solutions of nitrite and secondary amines the suggested reactions are: NO,
R,
,NH
+
H,O + HNO,
+
H,O
R,
2 HNO,
R,
,NH R,
+
N,O,-
-
+
OH-
R, ,NH, R,
+
+
N,O,
+
H,O
R, ,N-NO R,
+
(1)
OH-
(3)
HNO,
(4)
The rate of nitrosation reaction is given by the product k l x (RRINH) (HN02)’; in this form k is independent of pH. If expressed in terms of total nitrite content and total amine content the rate is given by the product kzx (total amine) (total nitrite)’; in this case kz is pH-dependent. Mrvish (1970) showed that, for dimethylamine and nitrite under standard conditions of temperature and total species concentration, a plot of the reaction yield against pH had a maximum at pH 3.4. The duplex effect of acidity on the amine/nitrous acid reaction was explained in the following manner: over a pH range 9 to 5, for each decrease of 1 unit in pH the concentration of non-ionized amine decreases approximately tenfold and that of the non-ionized HNOz is similarly increased. As the reaction rate is proportional to (HNO2)’ and to (R’NH), the rate increases tenfold for each unit drop in pH. Below the pK of HNOz at pH 3.36, nitrite becomes almost completely non-ionized, so that the main effect of a further drop in pH is a continuing decrease in (RzNH). At a constant pH of about 3, the relative nitrosation rate, k z , has been shown to be dependent on the basicity of the amine (Mirvish, 1971a). For four basespiperidine, dimethylamine, morpholine, and piperazine-kl vaned only fourfold at pH 3, indicating that the non-ionized species of the four amines have relatively similar reactivities, but kz was increased 1.85 X 10’ times on proceeding from piperidine (pKb ‘2.8) to piperazine ( p K b 8.4). Mirvish suggested that below pH 3 another mechanism is involved in the nitrosation reaction. In systems that approximate those in foodstuffs the basicity of the amine is a dominant factor in determining the rate and extent of nitrosation. Mrvish (1971a) studied also the rates of nitrosation of amino acids. He showed that the optimum pH is in the region of 2.2 to 2.5. The shift of pH is due to the effect of the four molecular species RNH;COOH, RNH;COO-, RRINH*COOH, and RRINHCOO-;of these, only the latter two can be nitro-
10
N. T. CROSBY AND R . SAWYER
sated. The k2 values obtained showed that at the optimum pH hydroxyproline and sarcosine are nitrosated at rates similar to that for morpholine (-200 times the rate for dimethylamine). Proline was shown to be nitrosated at about forty times the rate for dimethylamine. Similar studies on the kinetics of nitrosation of sarcosine in vivo and in vitro were carried out by Friedman (1972). First-order kinetics for nitrosation reactions with respect to nitrite concentration at pH 2.0 were demonstrated for sarcosine and dimethylamine. In this work nitrite is expressed in terms of sodium nitrite concentration, and the pK of nitrous acid is ignored. Sander et al. (1971) showed that nitrosation of alkylureas and N-alkylurethans proceeds rapidly and between pH 3 and pH 1 follows the kinetics:
or Rate = k4 (RNJ3-C0.Rl)(total nitrite)(H’) The stoichiometric constant k4 depends on the ionization of nitrite and hence on pH, but not on the ionization of the alkylureas, since they are almost completely non-ionized at pH above 2 (Perrin, 1965). Rates are quoted for methylurea, ethylurea, N-methylurethan, N-ethylurethan, citrulline, and hydantoin. The simple alkyl nitrosoureas and N-alkyl-N-nitrosourethansare known to be powerful carcinogens. Mirvish (1971b) showed also that nitrosation of methylguanidine proceeds slowly to yield N-nitrosomethylurea, N-nitrosomethylcyanamide, and 1-methyl-1-nitrosoguanidine.The nitrosation of amides and the chemistry of the resulting nitrosamides have been reviewed extensively by Challis and Challis (1970). The catalytic effect of various ions (acetate, bromide, and iodide) on the nitrosation of secondary amines has been studied by Ridd (1961). Boyland et ul. (1971) showed that thiocyanate ions present in saliva have a catalytic effect similar to that of the iodide ion; they used morpholine and N-methylaniline in these studies.
B. GENERAL SYNTHETIC ROUTES TO N-NITROSAMINES
The methods of preparation of nitrosamines fall into three main classes: (1) nitrosation of secondary amines in the acidic medium; this includes reactions of metal nitrites with acid salts of secondary amines; (2) nitrosation with oxides of nitrogen; and (3) cleavage and nitrosation of tertiary amines.
N-NITROSAMINES: A REVIEW
11
1. Reactions of Alkali Metal Nitrites with Solutions of Amines in Acids
Following the work of Geuther (1863), Fischer (1875) found that, when dimethylamine hydrochloride is acidified with sulfuric acid and treated with sodium nitrite in an aqueous medium, N-nitrosodimethylamine is formed in high yield. Other workers have used acetic acid and nitric acid, but hydrochloric acid has found widest application as the source of hydrogen ions (Fridman et al., 1971). Various methods have been employed to separate the reaction products; distillation, steam distillation, and solvent extraction have been favored most, the solvent extraction systems used depending on the nature of substituent in the alkyl side chains. The effect of temperature on the reaction is complex. Since the denitrosation reaction is acid-catalyzed, under conditions of high acidity lower temperatures are favored; however, under conditions of low acidity increase of temperature promotes the nitrosation reaction and improves the yield of nitrosamine. As indicated earlier, a detailed assessment of appropriate reaction conditions also requires knowledge of the basicity of the amine. Many workers have reported reactions in which the alkylamine hydrochloride is reacted at elevated temperature with excess sodium or potassium nitrite. Vogel (1948) quotes two such methods using separation by simple distillation for the preparation of Ndimethyl- and N-diethylnitrosamines with high yield. Instructions for laboratory preparation of approximately 44 g of the pure compounds are given in this paper. 2. Reactions of Oxides ofNitrogen with Secondaiy Amines Piria (1848) observed that “oxides of nitrogen in impure nitric acid lead to a deamination of amino compounds.” Paulman (1894) in studies on sarcosine found that a reaction with gaseous nitrous acid occurred in which a nitroso derivative was formed. Nitrogen trioxide has been obtained by the reaction between sodium nitrite and sulfuric acid (Brookes and Walker, 1957) and by the reaction of nitric acid with arsenic oxides (Curtius, 1917); because of the relatively complicated requirements of the method, this latter route has found only limited application in the synthesis of substituted N-nitrosodiamines such as the dinitrile and dimethyl esters of nitrosoiminodiacetic acid. The reagent has been used in the synthesis of the class of compounds known as sydnones; these compounds are also the products of the reaction of dehydrating agents on N-nitro derivatives of N-substituted a-amino acids (Brookes and Walker, 1957;. Stewart, 1964). Nitrosamines are formed when N-chlorodiakylamines are acted upon by nitric oxide in the presence of Few or Cuz+ (Minisci and Galli, 1964). Processes have
N.T.CROSBY AND R. SAWYER
12
been patented involving the use of pressure on reaction mixtures of nitric oxide and secondary amines in the presence of transition metal salts (Reilly, 1964). An oxidation by gaseous oxygen of the adduct of nitric oxide and diethylamine to yield N-nitrosodiethylamine has been reported (Ragsdale el al., 1965). Nitrosation of amines and amides with nitrogen tetroxide and nitrosyl chloride has been reported; nitrosyl chloride may be passed into ethereal solutions of secondary amines or their hydrochlorides to give the appropriate N-nitroso derivatives in good yield (White, 1955; Grimes et al., 1964; Addison et al., 1951).
3. Cleavage Reactions Involving Tertiary Amines Many textbooks contain statements to the effect that tertiary amines show no reaction with nitrous acid, and the nitrosation-denitrosation sequence has long been used as a technique for separation of secondary amine from mixtures with primary and tertiary amines. Geuther (1 864) obtained N-nitrosodiethylamine from triethylamine, but the experiment was later discredited and the possibility of reaction was discounted for over a hundred years after Geuther’s work. The reaction was rediscovered by Crowley et al. (1940) and confirmed by Smith and Loeppky (1967). The mechanism has been discussed in detail; typically, conversion reached 50 t o 70%. Reactions proposed are: +
HNO, R,R,N-CHR,R,
+
+
R,R,N=CR,R,
2 HNO
~
Hzo
=
H
,
RAN:
CHR,R, NO
+
+ Rl~-NNH,
RIR,C=O
N
,
O
,
+
RIR,N=CRIR,
~
H
HNO, C
+
HNO
R,R,N-NO
,+ O N,O
Conversion of triarylamines to N-nitrosodiarylamine plus aldehyde by refluxing an alcoholic solution with nitrous acid was reported by Addison et al. (1951). Fiddler et al. (1972a) studied the reactions of a number of naturally occurring quaternary ammonium compounds and tertiary amines with sodium nitrite at pH 5.6. The conditions chosen were said to simulate those occurring in the processing of comminuted meat products. They showed that N-nitrosodimethylamine is formed from a range of compounds of biological significance; these included neurine, carnitine, betaine, choline, and acetylcholine. Higher yields were obtained from 2-dimethylaminoethylacetate and dimethylglycine. Lijinsky and co-workers (1972), Lijinsky and Greenblatt, (1972) studied the reactions between nitrite and various drug materials under conditions approximating those in the human stomach. These authors report the formation of
N-NITROSAMINES: A REVIEW
13
N-nitrosodimethylamine from aminopyrine and oxytetracycline, and of N-nitrosodiethylamine from disulfiram and nikethamide. Nitroso compounds were also obtained from tolazamide and piperine. Tetranitromethane has been used as reactant for tertiary amines in the presence of pyridine (Schmidt and Fischer, 1920). R, R, NCH, R + C, H, N + C(N0, ).,
--t
R, R, N-NO + RCHO + C, H, N*HC(NO,) 3
A similar cleavage was achieved in the presence of acetic acid (Schmidt and Schumacher, 1921). 4. Other Methods Meyer and Forster (1876) and Meyer et at. (1877) studied the reaction of silver nitrite on the hydrochlorides of n-propyl- and n-butylamines and noticed that low yields of N-nitrosodipropyl- and N-nitrosodibutylamines were obtained in the process. Fusion of nitrite or nitrate salts of a dialkylamine is reported to yield the appropriate N-nitrosamine (van Zande, 1889); alkyl nitrites have also been employed as nitrosating agents (Titov, 1946), and methyl nitrite produces an efficient means of nitrosating dimethylamine in solution with alcohol (Tindall, 1960). Catalysis by zinc or copper in nitrosation reactions has also been studied (Chute et al., 1948; Brackman and Smit, 1965). 5. Biochemical Formation
Numerous workers have studied the reaction of acidified sodium nitrite and a variety of amines in conditions approaching those found in the mammalian stomach. The effects of gastric juices, enteric bacteria, and saliva1 secretions have been studied in vitro. In vivo testing in a range of animals has included the following amines: dimethylamine, piperidine, and morpholine. In many cases the evidence for positive reaction has been based on the induction of tumors. Work in this field has been summarized by Alam et al. (1971) in a report on the formation and chemical identification of N-nitrosopiperidine in the intestinal loop of a rat. C. PROPERTIES AND STRUCTURE 1, General Properties
The simple aliphatic nitrosamines are yellow or yellow-green nonhydroscopic liquids which boil without decomposition, the boiling points lying between 150°C (N-nitrosodimethylamine)and 220°C. Spectroscopic evidence (Tarte,
14
N.T.CROSBY A N D R. SAWYER
1955; Haszeldine and Jander, 1955) was interpreted to indicate hydrogen bonding or association, which the high boiling points suggest. The compounds are partially soluble in water, the degree of solubility varying according to molecular weight, and they are readily soluble in organic solvents. Nitrosamines may be salted out of solution in water with potassium carbonate. They show only weakly basic properties; a hydrochloride of N-nitrosodimethylamine is formed by passing hydrogen chlorine into an ethereal solution, and the salt is completely decomposed in the presence of ethanol or water. The simple aromatic nitrosamines, such as N-nitrosomethylphenylamine,are low-melting solids or yellowish oils of a characteristic, somewhat nutty odor. They are insoluble in water and can be distilled under reduced pressure, although at atmospheric pressure they undergo decomposition. The densities of the majority of nitrosamines lie in the range 0.9 to 1.Z g ~ m - increasing ~, with molecular weight. Dipole moments of N-nitrosodialkylamines indicate considerable polarity in the molecule, and the value is reduced by introduction of phenyl groups (Cowley and Partington, 1933; George and Wright, 1958; Lutskii and Kondratenko, 1959). Thermochemical parameters for N-nitrosodimethylamine have been reported by Korsunskii et al. (1967) as follows: heat of combustion (382 kcal mole-'), heat of formation in standard state (-10.7 kcal mole-'), heat of evaporation (9.9 kcal mole-'), heat of formation in the gas phase (-0.8 kcal mole-'). The dissociation energy of the N-N bond in N-nitrosodimethylamine has been variously quoted as 32 kcal mole-' (Bamford, 1939), 43 kcal mole-' (Gowenlock et al., 1961), and 55 kcal mole-' (Korsunskii et al., 1967). The higher measurements are consistent with the hypothesis of u-7r conjugation in the nitrosamine group and higher N-N multiple bond character when the appropriate values are compared with those for N-nitrodimethylamine (Gould, 1959; Chow and Lee, 1967; Rademacher et al., 1968). 2. Spectroscopic Evidence and Structure
The disposition of the atoms in the nitrosamine group in one plane facilitates the overlapping of the 0-71 orbitals. The resultant restricted rotation around the N-N bond leads to the appearance of s-cis-trans isomerism; multiplicity of the N-0 bond in nitrosamines is between 1 and 1.5 (Goubeau, 1961). Evidence that unsymmetrical nitrosamines exist in nonclassical geometrically isomeric forms has been demonstrated in nuclear magnetic resonance (NMR) studies (Karabatsos and Taller, 1964; Suhr, 1963). In the case of N-nitrosobenzylmethylamine the cistrans ratio is 3: 1;the forms proposed are shown below: PhH;C,+ /NHSC
N
,O-
N-NITROSAMNES: A REVIEW
15
Only the cis-methyl isomer has been detected for N-nitrosomethylphenylamine. Both isomers have been found for N-nitrosoethylphenylamineand N-nitrosoisopropylphenylamine. Cistrans isomers of benzyl-2,6-dimethyl-N-nitrosoanilines were separated by thin-layer chromatography (TLC), both substances being stable in the crystalhe state (Karabatsos and Taller, 1964). N-Nitrosodimethylamine shows two peaks in its Nh4R spectrum, since the two methyl groups do not lie in the same field. Bond length and bond angle studies in N-nitrosodimethylamine indicate that both nitrogen atoms are sp2 hybrids. The N-N bond is shorter than the equivalent bond in N-nitrodimethylamine, whch agrees with the enhanced multiplicity and strength of the bond in the nitroso group (Rademacher et al., 1968). The planar disposition of the nitrosamine group has been established by x-ray diffraction studies on the adduct with copper chloride (Klement and Schmidpeter, 1968). A characteristic intense Cotton effect in the range 250 to 400 nm has been observed in optical rotary dispersion studies on a range of nitrosamines (C. Djerassi et ul., 1961). A review monograph of optical circular dichroism includes work on the nitrosamines (Velluz et ul., 1965). The bond structure of the nitroso group in the nitrosamines is usually given as >N-N=O. It has been pointed out that the compounds do not show an infrared absorption band at 1520 cm-' (that assigned to the N:O stretching mode). Thermochemical evidence quoted above indicates a significfnt multiple bond character to the N-N bond and the extreme situation N - N a has been represented, but this view has not found wide acceptance (Could, 1959). Williams et ul. (1964) postulated that the actual structure may be mesomeric between these two. Haszeldine and Jander (1954) observed three relatively intense bands in the infrared spectra of the nitrosamines at 1410 to 1350 cm-' , 1320 to 1160 cm-' , and 1095 to 1045 cm-' . The first two bands were assigned to vibrations of N=O and the last to vibrations of the N-N bond, the middle band being attributed to the resonance between the two limiting canonical structures cited above; confirmatory studies were carried out with a variety of solvents in addition to the observations on pure liquids (Haszeldine and Jander, 1954; Haszeldine and Mattinson, 1955). It was found that the spectra of nitrosamines in the liquid state differ from those recorded in carbon tetrachloride and chloroform solutions, particularly in the frequencies characteristic of the N=O bond. Haszeldine and co-workers attributed the changes to three causes: (a) dipoie-dipole interaction, (b) intramolecular binding of hydrogen, and (c) dimerization.
16
N. T. CROSBY AND R. SAWYER
The predominance of one state over the other was said to depend on the physical conditions under which the nitrosamine exists. The dimerization hypothesis has been rejected by Williams et at. (1964) on the basis of their observations of the infrared spectra in polar solvents and on interpretation of the work of Earlet al. (1951) and Bellamy et al. (1959). The ultraviolet spectrum of a nitrosamine resembles that of an alkyl nitrite, with a low-intensity maximum at ca. 365 nm which shows fine structure and a high-intensity maximum at ca. 235 nm. The marked shift to the longer wavelength of the former maximum, with loss of fine structure, which occurs when the solvent is changed from light petroleum to ethanol distinguishes a nitrosamine from a nitrite (Haszeldine and Jander, 1954, 1955). The solvent dependency of the low-intensity nitrosamine band at 365 nm was investigated in more detail by Haszeldine and Mattinson (1955); they suggested that the effects due to changes in solvent supported the idea that the band at ca. 235 nm is characteristic of the N-N bond and that the absorption at the higher wavelength is due to -N=O. The large shift of the main peak due to a change from nonpolar solvents to water (30 nm) is further confirmation of this conclusion. The evidence was supported by Grimes et al. (1964) from studies onN-nitrosodi(2,2,2trinitroethy1)amine. Druckrey et al. (1967) included ultraviolet absorbance data in their review of physical properties of N-nitrosamines.
3. Mass Spectrometry Collin (1954) reported the mass spectra of four symmetrical dialkyl nitrosamines ranging from dimethyl to dibutyl. Schroll et al. (1967) investigated these compounds together with several substituted N-nitrosamines and alicyclic nitrosamines. Pensabene et al. (1972) extended the work on mass spectrometry to include a number of commercially unavailable compounds; they also included physical data and other spectroscopic data-principally infrared absorbances on the range of compounds studied. Saxby (1972) reported mass spectral fragmentation patterns for twenty-four alkyl nitrosamines with straight- and branched-chain substituents of both symmetrical and unsymmetrical conformation; deuterated compounds were also studied. The mass spectra of dialkyl nitrosamines show major fragments corresponding to a-cleavage of the side chain, and subsequent loss of NOH. For asymmetric dialkyl chains, cleavage takes place predominantly on the longer chain. Relatively large parent ions and fragments due to loss of OH are characteristic. For the low members of the series the parent ion is the base peak. The intensity of the parent ion diminishes as the series is ascended, and for most nitrosamines the base peak is mfe 42 or 43. Accurate mass measurement has shown that mfe 42 is predominantly C2H4N with a small contribution of C3H6, and m/e 43 is C2HSN and C3H7. In the case of N-nitrosodimethylamine large peaks at 42 and 43 arise by loss of H2NO and HNO, respectively.
17
N-NITROSAMINES: A REVIEW
A suggested pathway is: +
+ CH2=N=CH,
m/e 4 2
The phenyl nitrosamines all have an appreciable parent peak and a strong M-30 peak. Benzyl nitrosamines also have a strong parent peak and m/e 91, which is often the base peak. Other nitrosamines, in general, undergo thermal degradation to form the corresponding amine by abstraction of H and the loss of NO.
D. CHEMICAL REACTIONS In general, the lower alkyl nitrosamines are stable and they can be distilled at atmospheric pressure. They are, however, reactive under certain conditions. The occurrence of 0-71 conjugation with a residual charge on the oxygen is responsible for a number of reactions. The reactions undergone by the nitrosamines include complex formation, reaction with inorganic acids, reduction to hydrazines and to secondary amines, oxidation and nitration, cyclization to sydnones, and photochemical reactions.
I . Hydrolysis Geuther and Schiele continued the pioneer work on nitroso compounds by studies of the reactions of dialkyl nitrosamines with mineral acid to form amine salts. They showed that, when hydrogen chloride is passed into an aqueous emulsion of N-nitrosodiethylamine,the nitroso derivative dissolves. Similar treatment of an ethereal solution results in the formation of a salt, (C2H5)2N*NO* HCl, which is readily decomposed on addition of alcohol or water (Renouf, 1880). N-Nitrosodimethylamine decomposes on heating with hydrochloric acid, a reversal of the method of formation; similar reactions occur with the other simple nitrosamines. The method of nitrosation and denitrosation has been used as a ready means of preparing pure specimens of secondary amines in ether or toluene solution. In these cases gaseous hydrogen chloride has been used as the more effective denitrosating agent (LGffler, 1910; Forlander and Wallis, 1906). Apart from hydrochloric acid gas, bromine and sulfuric acid (Rohde, 1869) and hydrogen bromide in glacial acetic acid (Eisenbrand and Preussmann, 1970) have been used as denitrosating reagents. Studies of the kinetics of hydrolytic
N. T. CROSBY AND R.SAWYER
18
decomposition, at temperatures varying from 70°C to llO"C, of four nitroso compounds which represented four different structural characteristics were reported by Fan and Tannenbaum (1972). The compounds examined were N-nitrosodimethylamine, N-nitrosopyrrolidine, N-nitrososarcosine, and N-nitrosoproline. The authors showed that, in a solution with acidic pH (2.2 to 4), the compounds containing carboxyl groups adjacent to the amino nitrogen had high decomposition rates; at alkaline pHs (11 to 12.5) the compounds with cyclic structures had high decomposition rates. No evidence was obtained of decarboxylation of N-nitrosoproline and N-nitrososarcosine to yield N-nitrosopyrrolidine and N-nitrosodimethylamine. The decomposition studies carried out by these workers were extended to include the effects of many food constituents. No evidence of acceleration of decomposition was obtained with sugars, amino acids, reducing agents, or nucleic acids. Nitrosamines were found to decompose at ambient temperatures in the presence of Fe2+ and ascorbate; this is known as Udenfriend's hydroxylation system (Malling, 1966). Evaluation of the kinetics of the acid cleavage of alphatic nitrosamines has led to the hypothesis that the splitting off of the nitroso group is preceded by a protonation and subsequent formation and splitting off of the nitrosonium cation (Zahradnik, 1957,1958). R~RN-N=O
+
R,R--~;H-N=o
H+-
-
R,RNH
+ io
(slow)
The hydrolysis is easier, the more electrophilic the R or R1 group, and denitrosation takes place more readily in hydrochloric acid than in sulfuric or perchloric acid. The kinetics of the decomposition of N-alkyl derivatives of nitrosoguanidine has been studied at pH 7.5 and pH 9 by Haga et al. (1972). A further denitrosation reaction occurs when N-nitrosodimethylamine is heated with methyl iodide (Schmidpeter, 1963b). 2(CH,),N*NO+4CH31+2[(CH,),fiI~]+ 2NO+I,
2. Hydrogen Bonding and Adduct Formation The importance of hydrogen bonding in the denitrosation reaction is advanced by a number of workers; there is evidence that the oxygen atom of the nitroso group is involved in adduct formation. For example, Layne e t al. (1963a, b) have proposed that the reaction of nitrosamines with trichloroacetic acid in cyclohexane yields compounds with the following structures:
R1- NI R
/?*'"\ N
!* and
o...m
: / /
Ri-N-N
I
R
19
N-NITROSAMINES : A REVIEW
Layne el al. (1963b) have suggested that the W spectra of aqueous sulfuric acid solutions of N-nitrosodimethylamine indicate that acid-base reactions of the type R, R, NNO . . . (H, O)-+R, R, NNO
..
*
(H, O),HSO, == R, R, NNO (H, O),(H, SO, )2
account for the observed changes in spectra. Attempts to isolate sulfuric acid salts were unsuccessful; however, unstable white crystalline adducts of 72% perchloric acid with a number of N-heptyl- and N-octylnitrosamines were obtained. Hydrogen bond formation also takes place with formic acid, acetic acid, trifluoroacetic acid, phenols, alcohols, and amines (Meister and Nikolaeva, 1962; Bhowmik and Basu, 1963,1964). NMR spectroscopy has been used in studies of the protonation of lower alkyl nitrosamines and N-nitrosopiperidine. The protonation was found to occur in fluorosulfuric acid and other acids at temperatures below 0°C (Kuhn and McIntyre, 1966). The spectra indicate protonation of the oxygen rather than the amino nitrogen. The basic nature of the oxygen atom is indicated by the capacity of nitrosamines to form stable colored complexes with bromoplatinic acid. The composition corresponds to (R2N.NOV2 PtBr6 (Gutbier and Rausch, 1913). Spectroscopic analysis indicates that the oxygen is the electron donor in other adduct reactions (Goubeau, 1961). NMR spectra of the adducts of dimethyl nitrosamines with BF3, Els,SbC15, AlC13, and ZnBr2 showed that the methyl groups are nonequivalent, indicating that the amino nitrogen remains in the sp2 hydridized state. Consequently the acceptor is linked to the oxygen; the structure is also confirmed by high dipole moments (Schmidpeter, 1963a). Oxygen adducts are also formed with triethyloxonium antimonates, triethyl oxoniumfluoroborate, or a mixture of alkyl iodide and silver perchlorate (Huenig et al., 1963; Huenig and Geldern, 1964). Dimethyl sulfate forms an oxygenalkylated product with dialkyl nitrosamines; Klamann and Koser (1963) have indicated that boron trifluoride adducts R1 R2N-N-O-BF3 are formed readily in dry ether. The stability of the complexes increases with electron-donating capacity of the substituents linked to nitrogen; rearrangement reactions occur : with N-methyl-N-nitrosoaniline FH3
Ph-N-N-0-BF,
+
ON-Ph-NHCH,
+ BF,
The above reaction parallels the Fischer-Hepp transformation which occurs when the nitrosoaniline is dissolved in a mixture of alcohol and ether and undergoes reaction with gaseous hydrogen chloride. Stable complexes are also formed with
20
N. T. CROSBY AND R. SAWYER
di(trinitromethy1) mercury in water; the compounds may be readily purified (Fridman et al., 1968). Formation of adducts of the nitrosamines may prove to be useful analytically, since this renders the lower alkyl nitrosamines involatile. Such a step is important in the concentration of extracts from natural materials. Alternatively, adduct formation has been used in an attempt to enhance the sensitivity of detection methods (Brooks et al., 1972). Brooks et al. attempted to produce a fluorinated derivative of N-nitrosodimethylamine with the intention of applying gas chromatography with electron capture detection. They reported that a reaction occurred between chloroform solutions of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, A'-nitrosopiperidine, and heptafluorobutyric anhydride in the presence of pyridine. The authors showed that pyridine was essential to the reaction and that the adduct was not a 1,l-dimethylhydrazine derivative (that is, preliminary reduction did not occur). Mass spectrometry of the adducts was carried out, but no parent peaks were obtained.
3. Dansnitrosation Challis and Osborne (1972) studied the transfer of the nitroso group from N-nitrosodiphenylamine to other amines in the absence of nitrous acid. They point out that transnitrosation reactions may play a part in metabolic pathways and may give rise to carcinogenic nitrosamines, thus acting as proximate carcinogens; this has led to considerable speculation on the potential of a variety of food chemical adjuncts and contaminants to act as carriers of the nitrosating function. Transnitrosation has been reported under two sets of conditions, the most common method being the heating of a nitroso acceptor in an organic solvent with an aromatic nitrosamine (Bumgardner et ul., 1961; Sieper, 1967). Secondly, Morgan and Williams (1972) suggest that transnitrosation occurs in aqueous acid in connection with the Fischer-Hepp rearrangement. Challis and Osborne (1972) studied the reaction between N-nitrosodiphenylamine and N-methylaniline: Ph, NNO + PhNHMe + PhMeNNO + Ph, NH
The reaction proceeds to equilibrium in dilute acid (0.12 M). The evidence presented suggests that transnitrosation takes place without intermediate formation of free nitrous acid, a protonation mechanism being proposed. Transnitrosation reactions between aliphatic amines involving the release of nitrous acid are observed under more rigorous conditions-that is, 4M HCl at 80°C. Yoneda et al. (1972) report the use of N-nitrosodimethylamine to carry out simultaneous dimethylamination and nitrosation of 4-amino-6-chloro-2-methylpyrimidine and 6-amino-l,3-dimethyl uracil, the products being 4amino-6-
N-NITROSAMINES: A REVIEW
21
dimethylamino-2-methylpyrimidine and 1,3,6,8-tetramethyl-2,4,5,7 pyrimido [5,4-g] pteridinetetrone, respectively. The presence of both reactants was found to be necessary for the formation of the pteridinetetrone, although the dialkylmethylpyrimidine could be formed by reacting the appropriate reagent pairs.
4. Reduction The nitroso group undergoes reduction reactions to yield either the appropriate N,N-substituted hydrazine or the corresponding secondary mine. Fischer (1875) discovered the hydrazine reaction when investigating synthetic routes to dimethylhydrazine; in this case the reductant was zinc dust in acetic acid. Wieland and Fressel(l912) isolated ammonia and dimethylamine as by-products of the reaction; tetraethyltetrazene and diethylhydrazine were found following the reduction of N-nitrosodiethylamine with the same reducing agents. Paal and Yao (1930) obtained tetraethyltetrazene by the use of hydrogen in the presence of palladium on calcium carbonate: 2(C,Hs),NN0 + H,
+
(C,H,),N-N=N-N(C,H,),
Zinc in acetic acid, sodium amalgam, and tin in hydrochloric acid have been employed as reducing agents. In the latter case the nitroso group is eliminated and the product is the appropriate dialkylamine. Evidence on yields and products of reduction is somewhat contradictory, but this is probably due to a lack of control of pH during the reaction; the mechanism is discussed in detail later in the light of electrochemical evidence. The use of zinc and acetic acid to produce hydrazines is said to be effective only for methyl-, ethyl- and propyl-substituted nitrosamines (Leicester and Vogel, 1950); secondary amines are the main products for higher molecular-weight nitrosamines. A general method using lithium aluminum hydride has been shown to give products the nature of which depends on the molar ratio of the two reactants. Thus for reduction ofhr-nitrosodimethylamine to dimethylhydrazine (Schueler and Hanna, 1951) the ratio must be 1:2, and in the case of N-nitrosodiphenylamine (Poirier and Benington, 1951) the ratio must be 1:1. The yield of product is affected by the order of mixing the reactants. In the first stage of the reaction colored complexes are formed; these are converted to hydrazine by reaction with water (Hanna and Schueler, 1952). 2R, R,N-NO + ZLiAlH,
(R, R, N-N),
-t
(R, R,N-N),AlLi
AlLi + 2H, 0 +2R, R, N-NH,
+ LiA10, + 2H,
+ LiAlO,
Conjugation with the benzene ring which reduces the polarity of the NO bond
N. T. CROSBY AND R. SAWYER
22
is taken as an explanation of the appearance of side products in the case of reduction of aromatic nitrosamines. A number of hydrogenation methods are described in the patent literature. These include reactions based on catalysis by palladium, under pressure in the presence of iron salts (Tuemmler and Winkler, 1961), and reduction with zinc in hydrochloric acid (Derr, 1960). The reaction of N-nitrosodialkylamines with zinc dust in formic acid in the presence of mercury(I1) results in the formation of N-isocyanodialkylamines (Bredereck et al., 1965). Modifications of the hydrogenation method also occur in the patent literature; compared with the catalytic methods, the use of sodium amalgam, or of sodium in liquid ammonia or in alcohol, gives a lower yield of the hydrazine (Zimmer et al., 1955). Hydroxylammonium salts may be formed when the alkyl nitrosamines are hydrogenated in sulfuric, hydrochloric, nitric, acetic, or oxalic acid in the presence of Gp VIII elements on charcoal or silica gel under pressure (Mador and Rekers, 1960). Metalation reactions have been used in the production of chain-lengthened derivatives of the nitrosamines (Seebach and Enders, 1972). The reagent was diisopropylamide, and subsequent reaction with carbon dioxide, benzylbromide, acetaldehyde, or benzophenone gave a range of products with carbon addition at the CH3 group of alkyl-N-nitrosomethylamine.The general reaction is R
R, ,N-NO
>-NO -HC
H A H
‘Li
E = COOH, C,H,CH,
-
R, ,N-NO Hac.E
, C&CH(OH),
(C,H&C(OH)
Reduction of the nitroso group to give the appropriate secondary amine may follow. Total loss of nitrogen occurs when nitrosamines react with sodium dithionite in alkaline medium; a similar reaction occurs in liquid ammonia in the presence of lithium. R, RN-NO
OH-
Na,
R,RN-NHOH
s, 0,
-
-H2 0
R, R N - N ~ R R ,
The reaction is known as the Overburger-Lombardine reaction; extensive reviews have been published by the discoverers of the reaction (1966) and by Smith (1966). Reduction with lithium aluminum hydride to give high yields of hydrocarbon has been reported by Gleason and Paulin (1973). Controlled reduction may be obtained by electrochemical methods; much of the early work on electrochemical reduction relates to the application of polarographic techniques for analytical purposes. For N-nitrosodimethylamine, Smales and Wilson (1 948) found a half-wave potential in 0.2 N HC1 in the region of -0.9 volt versus the standard calomel electrode (SCE); other workers found that many of the
N-NITROSAMINES: A REVIEW
23
common alkyl nitrosamines are reduced within a narrow range of potential against a standard electrode (English, 1951; Heath and Jarvis, 1955). Few of the earlier workers considered the nature of the products of the reduction. The most complete investigation was carried out by Lund (1957). He studied the reduction of N-nitrosodimethylamine, N-nitrosomorpholine, N-nitroso-Nmethylaniline, and N-nitrosodiphenylamine over a pH range 0.2 to 12.5. Each nitrosamine produces a single wave in both acid and alkaline solution. The diffusion currents are reasonably constant from pH 0 to 5; around pH 7 the currents drop to a value which is half that in acid solution. At pH 9 and above, the diffusion current is again independent of pH. By studying the effect of the height of the mercury column on limiting current it was shown that waves are diffusion-controlled and hence that the ratio of electrons used in the two conditions is 2 : l . AU waves are irreversible, and in acid solution the half-wave potentials become more negative as the pH is raised. In alkaline solution they are independent of pH. Preparative use of a macro mercury electrode was used to demonstrate the course of the reduction. It was found that the N-nitrosamines at pH 1 to 5 yielded the unsymmetrical hydrazines by a four-electron reduction, yields being about 80%.No other products were isolated. RR1N-N-OH'+4e
+ 4 H + + R R l N - N H , + + 2H,O
In alkaline solution the reduction consumes two electrons per molecule and secondary amine is produced along with nitrous oxide. 2RR, N-N=O + 4e + 3H, 0 +. 2RR1NH + N, 0 + 4 0 H -
Two independent studies confirmed the above conclusions (Holleck and Schindler, 1958), bulr they did not identify products. However, Zahradnik and co-workers (1959) identified 1,l-diethylhydrazine as the reduction product of N-nitrosodiethylamine in acid solution. The identity of the reduction product in alkaline solution was not established, but it was shown to be neither a hydrazine nor a hydroxylamine. Half-wave potentials in acid solution varied between -0.47 and -0.94 volt according to the electrolyte and the nitrosamine; the values obtained in alkaline solution were in the range of -1.03 to -1.170 volt. Thus for controlled potential electrolysis of an aqueous alkaline solution of nitrosamines to the corresponding amines a voltage of -1.7 to 1.8 against the SCE is necessary to complete the reduction. Pulidori e l ai. (1970) and Borghesani et al. (1971) studied the reduction mechanism at the dropping mercury electrode for a range of nitrosamines and also the effect of the molecular skeleton on the reducibility. As far as the symmetrical straight-chain alkyl nitrosamines are concerned, these workers concluded that the relative reducibility in alkaline medium is the reverse of that observed for the same series of compounds in acidic medium. The
24
N. T. CROSBY AND R. SAWYER
isobutyl and isopropyl homologs showed analogous behavior. Alkaline medium favors the reduction of N-nitrosodimethylamine relative to N-nitrosodibutylmine; the sequence of reducibilities in acid medium was confirmed by Iversen (1970, 1971) in his studies on the electrosynthesis of the unsymmetrical hydrazines. Nikulin and Klochkova (1972) studied the electrochemical reduction of N-nitrosodimethylamine on a lead electrode in 0.2 N HzSO4 and found that the sole reduction product under a range of experimental conditions was 1,I-dimethylhydrazine. 5. Oxidation Nitrosamines are the starting materials for the synthesis of secondary N-nitramines which may be regarded as derivatives of nitramine, HZN.NOz. A variety of oxidizing agents have been described; hydrogen peroxide with nitric acid gave low yields (Brockman et ul., 1949), but better results were obtained with nitric acid and ammonium persulfate (Chute et al., 1948) and trifluoroperacetic acid (Emmons and Ferris, 1953). Emmons (1954) described a reaction system based on hydrogen peroxide and trifluoroacetic anhydrate in methylene chloride; reaction yields of pure nitramines in excess of 90%of the theoretical amounts were obtained. Addison and Conduit (1952) showed that a vigorous reaction occurred between N-nitrosodiethylamine and nitrogen pentoxide, the reaction being controlled only by dilution of the reactants. Z(C,H,),NNO + N,O,
+.
2C,H,NO, + 2CH,COOH + HNO, + HNO,
C,H,NO+HNO,
+.
CH,-C(NOH)NO, + H , O
Reactions of nitrosamines with nitric acid have been studied (van Romburgh, 1896; Paal and Deybeck, 1897;White and Grisley, 1961), but these reactions are not considered further here. Much of the recent work in this field relates to reactions of N-nitrosopolynitroalkylamines (Klager, 1958; Grimes et al., 1964; Frankel and Klager, 1963).
6. Cyclization The dehydrating activity of a number of reagents, notably acetic anhydride, on N-nitroso derivatives of N-substituted amino acids has been known and studied for a number of years; the products of the reaction are known as sydnones. Earl The and Mackney (1935) observed the reaction with N-nitroso-N-phenylglycine. genera1 reaction is often effected by heating the nitroso compounds with excess acetic anhydride on a water bath for several hours, and this procedure is generally satisfactory with simple alkyl or aryl derivatives (Eade and Earl, 1946).
N-NITROSAMINES: A REVIEW
25
However, in many cases it is better to allow the reaction to take place at room temperature over several days. The formation of the sydnones relies on the nucleophilic nature of the oxygen in the nitroso group and takes place via a mixed anhydride (Baker and Ollis, 1957).
-
RNCH2COOH I
N=O
0 0 II II RNCH2COCCHs-I N=O
CH-C=O R% N- 0
HC-C-O-RN\O
/
I
N-0
Other dehydrating agents have been described including trifluoracetic anhydride, thionyl chloride, and Nfl-diisopropylcarbodiimide W t t e r and Wolfrum, 1959a, b; Baker and Ollis, 1955). In view of the novelty of their electronic structure and the possibility of biological activity, interest in the class has been widespread. Extensive reviews by Baker and Ollis (1955) and by Stewart (1964) have been published on the chemistry, properties, and biological activity of the class. The fact that sydnones are derived from a-amino acids via the appropriate nitrosamine provides a reasonable basis for the assumption that biological activity might be observed. Widely different types of biological activity have been observed for sydnones and sydnone imines.
7. Photochemistry The fact that nitrosamines undergo photolytic decomposition was recognized some years ago. Chow (1964) and Burgess and Lavanish (1964) were concerned with identifying the products of the decomposition reactions and the mechanism of the reaction. These studies were carried out on aqueous or methanolic solutions of alkyl nitrosamines in the presence of hydrochloric acid. Reaction pathways to explain the formation of amidoximes and alkylidenimines were postulated; subsequently Chow (1967) recognized the formation of aldehydes and ketones. Photochemical reactions have found extensive application in the development of analytical methods. Concurrently with the fundamental studies a number of workers were investigating methods for the identification and analysis of N-nitrosoalkylamines. Preussmann et al, (1964a) and Daiber and Preussmann (1964) reported a thin-layer chromatographic method for the separation of N-nitrosoalkylamines in which the method for locating, identifying, and quantifying the nitrosamines was based on the photolytic cleavage of the NO groups. Subsequently, they used Griess reagent to estimate the amount of nitrite formed as a result of the reaction. Under the conditions of their experiments the photolytic decomposition reaction took place either on the silica gel support material or in aqueous alkaline solution.
26
N. T. CROSBY AND R. SAWYER
A thin-layer method was described by Krijller (1967) in which he claimed that nitrosamines could be positively identified by the detection of nitrous acid and secondary amine following photolytic cleavage of the nitrosamines. Griess reagent was used to identify nitrous acid, and ninhydrin was used for location of the secondary amines. Further evidence on the degradation products following exposure to sunlight was advanced by Althorpe er al. (1970); these workers claimed that, when standard solutions of N-nitrosodialkylamines in hexane were exposed to sunlight and subsequently analyzed by gas chromatography and mass spectrometry, an increase in molecular weight by sixteen mass units occurred. In later work they utilized peroxytrifluoroacetic acid as a reagent for production of nitramines. Thorburn Burns and Alliston (1971) reported studies on the kinetics of photolysis of a number of nitrosamines in which they observed the loss of individual nitrosamines by the reduction in absorbance at the peak wavelength in the region of 230 nm. No attempts were made to identify reaction products in this work, because of the apparently conflicting views expressed in the literature cited above and additionally by Chow and Lee (1967) and by Sander (1967a, b). Thorburn Burns and Alliston (1971) studied the decomposition kinetics at differing pH values, and in view of the interest in extraction of nitrosamines from foodstuffs they examined the effects of supporting electrolyte and of solvent on the decomposition. Irradiation was carried out in two systems-one a low-intensity U V source and the other a Hanovia photochemical reactor. They concluded that the photolysis of the nitrosamines studied-N-nitrosodimethylamine, N-nitrosodibutylamine, and N-nitrosopiperidine-followed first-order kinetics. The reaction half-lives of the nitrosamines proved to be characteristic of the individual nitrosamine but varied with the solution and irradiation conditions. Decomposition was faster at low pH and in methanol, ethanol, and dichloromethane except that N-nitrosodimethylamine appeared to be more stable in methanol. The results showed that the effects of acidity were less significant than hitherto suggested and that the nitrosamines are not stable (Chow, 1964), nor are the decomposition reactions hindered in organic solvents (Mohler and Mayrhofer, 1968a, b). Other photochemical reactions which have been reported include photolytic addition to olefms (Chow et al., 1967; Chow, 1965a, b). The photoaddition reaction takes place via the dissociation of the N-N bond with subsequent addition of nitroso and amino groups to the C=C bond; C-nitroso compounds are thus the primary reaction products which may then undergo secondary reactions. Photochemical transnitrosation is said to take place between nitrosamines and diphenylamine in the presence of ethanol and palladium(I1) chloride (Yoe and Overholser, 1939; Overholser and Yoe, 1941). This reagent system has been used as*a spray reagent in thin-layer chromatography (Preussmann et al., 1964a, b).
N-NITROSAMINES: A REVIEW
21
Chow (1965b) has described a photoaddition reaction to yield oximes by the following route:
(CH,(CH,),),
+ N-NO +
Chow et al. (1971) have carried out extensive studies on photoreduction in the presence of methanol to yield amine and formamide derivatives: 280 nm
o,/N
I
I
H
I
NHCHO
Dissociation of C - C bonds has also been achieved (Chow, 1965a). Numerous other reactions involving .reduction, addition, rupture, and elimination have been reported by Chow et al. (1967, 1972). Suryanarayanan and Bulusu (1 972) reported the formation of N-nitrosodimethylamine by the photolysis of the dimethylnitramine in the solid state. By the use of ”N-labeled dimethylnitramine, the authors showed that the reaction takes place by N - O bond cleavage. Further evidence that nitrosamines undergo light-induced rearrangement reactions was also afforded.
111.
BIOLOGICAL PROPERTIES
OF NITROSO COMPOUNDS A. CARCINOGENICITY 1. Epidemiological Studies
Approximately one-fifth of the deaths recorded annually in Great Britain are attributed to the various cancerous diseases. Similar statistics have been issued by medical officers in other countries. Environmental factors are thought to be a major cause of human cancer, and it is natural to suspect chemicals as being among the prime agents responsible. However, relatively few compounds have been implicated positively on a “cause-and-effect” basis as inducers of cancer in humans. For example, despite all the extensive investigations into the relationships between lung cancer and smoking, some pieces of the jigsaw remain to be identified and fitted into place. Where evidence has been obtained, frequently it has been the result of high accidental exposure or, in the case of chronic dosage,
28
N. T.CROSBY AND R. SAWYER
through occupational and epidemiological studies. Thus, scrota1 cancer was found to be predominant among the now defunct occupation of infant chimney sweeps; cancer of the bladder occurred among workers in the dyestuff and rubber industries owing to contamination with aromatic amines; skin cancer can be caused by contact with certain distillation fractions of the coal and oil industries; ultraviolet and ionizing radiations have also been identified as causative agents under certain conditions, although, paradoxically, they are also employed in the treatment of cancer, high doses being used on a localized area for the total destruction of the tumor. It is obviously not possible to mount direct studies of the effects of potentially dangerous chemicals on humans. The problem is, therefore, normally approached indirectly, and two different routes are available. First, the suspect chemicals are added to the diets of experimental animals whose development is then followed; on death further investigations of organs, etc., are carried out, and the results are compared with those obtained from a control group not exposed to the suspect chemical. Valuable as animal studies are-and little progress in medical research would have been achieved without them-there is a danger and considerable uncertainty in extrapolating any results to man. Some of the work in this area will be described later. An alternative approach is the study of cancer patterns with respect to human populations throughout the world. This encompasses ratios of one type of cancer to another and the influence of dietary patterns, sex, race, and age, as well as global variations. Where an unusual or distinctive pattern is found, an attempt is made to correlate the findings with an environmental factor. When this effort is successful, preventive measures can then be instituted. However, for cancers that are widespread throughout the population it is far more difficult to isolate the causative agents, particularly as there is normally a lengthy lag period between exposure and appearance of the tumor. The International Agency for Research on Cancer (IARC) has sponsored a number of field studies into the epidemiology of cancer and associated factors. One such study, in which the Laboratory of the Government Chemist was also involved, concerned the investigation of esophageal cancer. This cancer shows a dramatic variation in frequency throughout East Africa. The frequency changes abruptly from relatively high to very low over a few miles in a country where the people live in isolated villages. Previous studies had suggested that alcoholic drinks might be implicated but that the quantity of alcohol consumed is not the sole factor responsible. Many of the brews are locally produced, often illicitly, and previously McGlashan et al. (1968) had reported the presence of nitrosamine-like substances in samples of alcoholic drinks produced in Zambia. Accordingly, a number of samples of homemade spirits distilled from maize, millet, banana, and honey beers were collected from both high- and low-incidence areas. The samples were subjected to a preliminary screening by polarography, and
N-NITROSAMINES: A REVIEW
29
nitrosamine-like substances in concentrations as high as 21 mg/kg were recorded. Since polarography is not specific for nitroso compounds, the samples giving high results were subjected to further analysis by gas chromatography (GLC) and high-resolution mass spectrometry. As only low concentrations of nitroso compounds were likely to be present, the samples were first concentrated by a factor of 50 on a commercial spinningband distillation column. In this way the alcohol fraction was removed and the concentrates were then subjected to GLC with a nitrogen-selective detector. Any samples giving a positive response were further characterized by mass spectrometry with parent-ion monitoring. None of the samples was found to contain nitrosamines at a level in excess of 0.005 mg/kg of original sample. There was, therefore, no association between the nitrosamine content and the incidence of cancer in the areas studied. Fuller details of this work have been published by Collis et al. (1971). An example of a positive association between an environmental factor and dietary patterns was reported by Dungal (1959). He observed a correlation between those sections of the Icelandic population consuming smoked foods and an increased incidence of gastric carcinoma. The practice of preserving fish and mutton by heavy smoking appears to result in the deposition of significant amounts of the carcinogenic 3,4-benzpyrene in the food. Rats fed this smoked food also showed an increase in malignant tumors. Other associations between stomach cancer and the consumption of specific foods have been noted (Haenszel et al., 1972). The highest risks were attributed to pickled vegetables and salt-dried fish eaten by Japanese migrants in Hawaii. Although there are distinct differences between Japanese and Western foods, changes in dietary habits over a period of time do serve to complicate the picture. However, some experimental evidence in support of the epidemiological findings was reported in this study.
2. Animal Studies Magee and Barnes (1967) have published a comprehensive review of the chemical and biological properties of the nitroso compounds. Their review covers both the acute and the chronic effects produced in the cellular structure of a number of affected sites together with an extensive discussion of work on the metabolism and biochemical reactions induced by these compounds. Current theories of carcinogenic mechanisms are also presented and discussed. The main points are summarized below, particularly those of interest to the food chemist. a. Acute Toxicity. In general, large doses of the dialkyl nitrosamines produce severe liver necrosis accompanied by extensive hemorrhage both in the liver and at other sites. The single, oral LDs0 for rats has been determined for a number of these compounds (Magee and Barnes, 1967), and in general the required dose increases with the length of the chain in the alkyl group. Thus, the
30
N. T. CROSBY AND R. SAWYER
LDs0 value for N-nitrosodimethylamine is 27 to 41 mg/kg, for N-nitrosodiethylamine 216 mgfkg, and for N-nitrosodibutylamine 1200 mgfkg. Acute poisoning by N-nitrosodimethylamine in humans has been reported following an industrial accident (Barnes and Magee, 1954), and in animals, particularly mink (Section I, E). However, foodstuffs are likely to contain only minute (if any) quantities of these compounds (Section N,B), so the acute toxicity is of interest principally to medical and biochemical workers. b. Subacute Effects. At levels below the toxic dose, nitroso compounds have been shown to display carcinogenic activity (Magee and Barnes, 1956; Ma, 1971), and the LDs0 value is not necessarily a reliable guide to the power of this activity. For example, at lower dose levels the compounds N-nitrosodimethylamhe and N-nitrosodiethylamine show approximately equal activity in rats despite a large difference in the acute toxicity of the two compounds reported above. Indeed, there is some evidence that the diethyl compound is the more active liver carcinogen (Druckrey et al., 1963). The study of nitrosamine carcinogenesis has been extended so that nearly one hundred compounds have now been tested; of these, approximately three-quarters have shown carcinogenic activity to a greater or lesser extent (Druckrey et al., 1967; Preussmann, 1971). Activity is not always restricted to the liver, and other sites which have been shown to be susceptible include the kidney, esophagus, bladder, lung, and alimentary system (Magee and Barnes, 1967). The target organ varies with the dose and chemical structure of the compound, the animal under test, the method of dosing and route of administration, diet, and probably other factors as well. Thus nitrosodimethylamine has produced tumors under varying conditions in the liver, kidney, lung, and nasal sinus of the rat, and nitrosodiethylamine has been shown to be active in the liver, kidney, and esophagus of the rat. N-Nitroso-di-n-butylaminegives rise to bladder tumors only when injected subcutaneously, but causes liver, esophageal, and bladder tumors almost equally when administered orally (Druckrey et a2.; 1964, 1966). A further complication is that in some experiments evidence of repair of the liver cells was noticed in animals that survived, although tumors subsequently developed in the kidney (Magee and Barnes, 1962). Magee and Barnes (1956) also reported a shorter feeding trial using rabbits in which no tumors developed. Although many of the experimental studies have been carried out with rats, other animals have also been tested, since variations in response frequently occur from species to species. Schm’ihl and Osswald (1967) tested the carcinogenic activity of nitrosodiethylamine in rats, mice, guinea pigs, rabbits, dogs, monkeys, grass parakeets, and pigs. Cancer of the liver was detected in all these species at a daily dose of 3 mg/kg administered in the drinking water. The frequency of liver tumors was very high even in the guinea pig, an animal that is normally resistant to chemical carcinogenesis. No tumors were produced in the monkey, although severe liver damage was observed. Other workers, however, have detected tumors
N-NITROSAMINES: A REVIEW
31
in this animal after observations over a longer life span (Kelly et al., 1966). Hence, nitrosodiethylamine is now known to produce a carcinogenic response in twelve species, including primates, and no animals are known to be resistant to this chemical. Nitrosodimethylamine has been less extensively investigated but has been shown to be active in the rat, mouse, hamster, guinea pig, rabbit, and rainbow trout. Therefore, the overall conclusion which can be drawn from a large number of these and similar experiments with animals is that nitrosamines are active at low dosage levels and in a number of different species and that they can affect several organs in the bodies of animals depending on the particular conditions of the experiment. The limiting dose required to produce a carcinogenic response is not known with any degree of certainty. Terracini et al. (1967), working with nitrosodimethylamine, found that the incidence of liver tumors among surviving animals (rats) after 60 weeks was 1 out of 26 at a dietary concentration of 2 mg/kg, and 8 out of 74 at a level of 5 mg/kg. The incidence of tumor formation was even greater at high levels of nitrosodimethylamine in the diet. A similar study for nitrosodiethylamine has been reported by Druckrey et al. (1 963). Doses higher than 0.1 5 mg/kg of body weight per day produced a 100% tumor yield. At the lower end of the scale (0.075 mg/kg per day) 20 animals survived for more than 600 days. Of this group 11 had benign or malignant tumors of the liver and of the esophagus. All 4 animals that survived more than 940 days developed tumors. These authors concluded that the marginal effect dose was around 0.5 mg/kg. There are many difficulties in assessing these data and extrapolating the findings to man. Concern arises from the overall picture which has emerged following a number of experimental studies carried out in several countries, rather than from any individual finding. The principal features of interest can be summarized as follows: 1. Nitroso derivatives of dimethylamine and diethylamine are known to cause a carcinogenic response in a wide range of animal species-no known animal has been shown to be totally resistant to this class of compound. 2. Nitroso derivatives of dimethylamine and diethylamine produce tumors at several different sites in the animal and are active at low dosage levels. Other compounds in this series are also known to be active, although the relationships between compound, dose, animal, diet, and so on are very complex. 3. Although no direct evidence of the carcinogencity of nitrosamines in humans is available, it has been shown as a result of an industrial accident that similar toxic symptoms arise in the human liver as with experimental studies on animals; furthermore, metabolic studies in vitro using human and rat liver slices have shown that the pathway of biochemical change is very similar (see below), as regards the alkylation of cellular components and the rate of production of
32
N. T. CROSBY AND R. SAWYER
carbon dioxide. The significance of such cellular changes in the induction of cancer has been discussed by Swann and Magee (1968).
B. BIOCHEMICAL CHANGES As was indicated above, nitrosamines generally produce tumors at sites distant from the point of application. Nitrosamides, on the other hand, are less chemically stable, especially in neutral or alkaline media, and consequently display a local irritant action as well as selected cytopathic activity. The early work on a wide range of nitroso compounds showed that the liver was the organ most frequently affected, and, as this organ is the center of metabolic activity in the animal, it was suggested that a metabolite of nitrosarnines was the true active species and not the compounds themselves. Consequently, studies were made of the metabolic changes in vivo following administration of nitrosodimethylamine to the animal in an effort to isolate the active metabolite. Subsequent changes in normal biochemical processes were also investigated. Magee (1956) showed that nitrosodimethylamine was uniformly distributed in the body tissues of the rat following oral administration, there being no selective concentration in the liver. None of the original dose was recovered after 4 hours in mice, or after 24 hours in rats. Only a very smdl percentage of the dose was excreted in the urine. Dutton and Heath (1956) used nitrosodimethylamine labeled with 14C and found that the principal radioactive product was expired as carbon dioxide during the first few hours following injection. These experiments were repeated (Heath and Dutton, 1958) with nitrosodimethylamine labeled with 14C, "NO, and "NC. Again much of the I4C was expired as 14C02; smaller quantities were detected in the liver and in the urine, with traces in a number of other tissues, Most of the "N compounds were located in the urea fraction, the nitrogenous liver fractions being only lightly and evenly labeled. The position of labeling had little effect on the distribution of "N. It was concluded that nitrosodimethylamine is oxidized to one carbon atom intermediate and that the nitroso group is partly reduced to ammonia. In a later paper Heath (1962) extended this work to other nitroso compounds, and the results suggested that the toxic metabolite for the dialkylnitrosamines was a diazoalkane, or a monoalkylnitrosarnine or carbonium ions formed from it. Early microscopical studies had established that nitrosodimethylamine attacked the liver microsome material which is believed to play an important role in protein synthesis. The influence of this compound on protein synthesis was, therefore, investigated (Magee, 1958). These experiments showed that the incorporation of labeled amino acids into liver protein was reduced by about 50% at 3 hours following a dose of 50 mg of nitrosodimethylamine per kilogram of body weight in the rat. Incorporation into kidney and spleen proteins was unimpaired. These experiments were repeated in vitro with liver slices (Hultin et al., 1960).
N-NITROSAMINES:A REVIEW
33
This work showed that the incorporation in vitro of 14C-valineinto proteins of rat liver slices was inhibited after preincubation of the slices with nitrosodimethylamine. Similar effects were noted in the incorporation of 14C-adenine into ribonucleic acid fractions. Confirmation of these ideas can be found in the work of Brouwers and Emmelot (1960), who showed in addition that certain other enzymatic functions remained normal. No similar effects were observed with rat kidney slices. Further work on liver tissue preparations established the fact that the ability to metabolize nitrosodimethylamine was located only in the microsome + cell sap fraction. Neither fraction was significantly active alone, but activity was restored on mixing. Certain coenzymes-diphosphopyridine nucleotide and the tri- compound-appeared to restore activity to dialyzed fractions (Magee and Vandekar, 1958). Further work on the methylation of cell constituents was reported by Magee and Hultin (1962) and by Magee and Farber (1962). The first paper showed that oxygen was necessary for the reaction to occur and that the methylating agent released attacks the imidazole group of histidine at the 1 and 3 positions, and possibly free carboxyl groups of the protein molecules as well. The second paper established that nitrosodimethylamine can methylate RNA, and possibly DNA, in vivo, producing 7-methylguanine. Experiments with both whole animals and liver slices established that the radioactivity of the liver RNA was significantly higher than that of the liver protein. The ratio of the two activities was approximately 5:l. Kidney RNA is similarly methylated but to a smaller extent. Montesano and Magee (1970) reported experiments in which human liver slices were incubated with labeled nitrosodimethylamine. The rate of production of carbon dioxide and the rate of methylation of nucleic acids was only slightly slower than the rates found for rat liver slices in vitro. It was concluded in this paper that Man is likely to be approximately as sensitive to this compound as the rat on the basis of these findings.
IV. ANALYTICAL ASPECTS A. NITROSAMINES IN BIOLOGICAL MATERIALSTHE ANALYTICAL PROBLEM The reasons for the analytical interest in the levels of nitrosamines present in foodstuffs and biological materials have been discussed in the earlier sections of this review. Since the work of Magee and Barnes (1956), there has been an exponential growth in the number of publications on analytical evidence and methodology for nitrosamines in a variety of contexts. Much of the concern over the levels of nitrosamines in the diet is related to the widespread occurrence of nitrosamine precursors (nitrates, nitrites, secondary and tertiary amines) in
34
N. T. CROSBY AND R. SAWYER
foodstuffs and food adjuncts (Lijinsky and Epstein, 1970; BIBRA, 1968;Lancet editorial, 1968). This concern extends to the possible interaction of the precursors during processing and cooking (Ender and Ceh, 1968; 1971) and to the possibility that reactions can take place in the mammalian stomach after ingestion of combinations of foodstuffs containing the necessary precursors (Sander et al., 1968; Sander, 1967a, b; Sen et al., 1969a). Nitrates are widely distributed in the human environment (Wilson, 1949; Bodiphala and Ormrod, 1971) and are easily reduced to nitrite. A review of the occurrence of nitrates and nitrites in foods has been published by Ashton (1970). Information on the distribution of precursor amines is meager (Lijinsky and Epstein, 1970). Conditions and reaction mechanisms under which nitrosamines may be formed from naturally occurring amines have been studied by Ender and co-workers (1964; Ender and Ceh, 1971); they demonstrated the formation of large quantities of N-nitrosodimethylamine by the use of high levels of nitrite and processing conditions which were not typical of industrial practice. The presence of nitrosamines at low levels (microgram per kilogram) in various foodstuffs has now been established; the results and validity of the experimental evidence are summarized in a later section. The question as to whether the nitrosation reactions occur after ingestion of a combination of foods containing precursor amines and nitrites in natural amounts has not yet been satisfactorily resolved by the analytical methods. Apart from a few reports which are concerned with unusually high levels of nitrite and secondary amine as precursors, substantive analytical evidence for the in vivo formation of nitrosamines is rare. Many of the results of biological experiments are based on the evidence of tumor formation and tissue damage. Work of this type has been summarized by Shank and Newberne (1972), Magee and Bames (1967), Magee and Schoental(1964), and Magee (1969,1972).
I. Methods of Isolation of the Volatile Nitrosmnines A general method of isolation which is applicable to a variety of foodstuffs is not available. Many of the methods are tedious and inefficient in operation, and the production of artifacts during sample processing is not unknown. A survey of analytical techniques employed in the isolation and detection of nitrosamines has been published by Wasserman (1971). More recent developments have been summarized by Eisenbrand (1973a, b). Evaluation of isolation techniques is dependent on the use of one or more end methods for the measurement of their efficiency. In many cases the end methods have been inadequate in that they have been demonstrably inaccurate or lacking in specificity and sensitivity. As a result many of the findings reported in early papers must be regarded with some suspicion.
N-NITROSAMINES: A REVIEW
35
Many of the extraction procedures employed by different investigators have comprised a number of stages; there are examples of solvent extraction, distillation in steam or in vucuo, and a variety of chromatographic methods. These steps have been employed in various combinations, but the primary stages of extraction have generally consisted in a direct solvent extraction step or a distillation from an aqueous slurry of the foodstuff with pH adjustment varying from acid to alkaline. Of the organic solvents which have been used as primary extractants, dichloromethane has pride of place as the most favored solvent (Neurath et al., 1965; Sander et ul., 1968; Sander and Sief, 1969); however, ether and boiling water are sometimes used as alternative extractants. Solvents, other than dichloromethane, which have been employed as secondary extractants include heptane and acetonitrile-heptane mixtures (Eisenbrand et ul., 1969). Use of the traditional Soxhlet process directly on a range of foodstuffs with dichloromethane as extractant has been described by Marquardt and Hedler (1966), Thewlis (1967), Hedler and Marquardt (1968), Schuller (1969), and Eisenbrand et ul. (1970a). However, Mohler and Mayrhofer (1968a) reported that ether and dichloromethane were ineffective solvents for the direct removal of nitrosamines from meats and cheeses; recoveries of 20 to 40%were obtained. They preferred steam distillation from an acidified medium as the best means of separation. Contrary evidence was provided by Sen et ul. (1969b), who claimed that efficiencies of 65 to 90% were obtained with ether as solvent for 10 to 4000 mg of N-nitrosodimethylamine per kilogram added to wheat flour. The same authors claimed recoveries from 30 to 80% for additions of 5 to 25 mg/kg to a variety ofmeats, fish, and cheese (Sen et ul., 1969b). In this latter work solvent extraction was carried out by homogenization of the foodstuff with dichloromethane in the presence of alkali; variations of the same procedure were used on nitrite-treated fish and meat products (Sen, 1972; Sen et ul., 1970, 1972). Van Ginkel(l970) reported on ether as primary extractant for the determination of nitrosamines in cheese, but his results were inconclusive. Ender and Ceh (1967, 1968) used hot distilled water to extract fish preparations; the extracts were cooled to remove fats and proteins, and basic materials were precipitated with phosphotungstic acid. While recording findings of Nnitrosodimethylamine in the microgram-per-kilogram range in smoked fish, smoked meats, and mushrooms, these authors state that a recovery of 70% was obtained for N-nitrosodimethylamine added at “low levels” to the various foodstuffs studied. Solvent extraction techniques have been employed as secondary cleanup processes. In the majority of cases solvent partition is used after a distillation stage; Eisenbrand et ul. (1969,1970b) employed a liquid-liquid extraction system to separate lipids and nitrosamines. Primary extracts of wheat flour were prepared by direct solvent extraction with dichloromethane; a liquid-liquid
36
N. T.CROSBY AND R. SAWYER
extraction stage was performed with acetonitrile saturated with n-heptane. Three extractions of the n-heptane phase with acetonitrile were sufficient to concentrate more than 95% of ten nitrosamines into the polar phase. N-Nitrosodimethylamine was recovered in 85% yield when 200 ml of acetonitrile containing 75 pg of nitrosamine was concentrated to 3 ml. The acetonitrile was concentrated in contact with alumina, the nitrosamines being desorbed with methanol. The results obtained from application of the method to wheat flour were inconclusive. Distillation has been widely utilized as the primary separation stage by a number of workers. Steam distillation from acid, neutral, or alkaline media has often been used in the examination of foodstuffs. Heath and Jarvis (1955) effected quantitative separation of N-nitrosodimethylamine from animal tissues to which had been added varying quantities of the compound. In this work an alkaline homogenate was used. Molder and Mayrhofer (1968b) reported that steam distillation of acidified food samples was an inefficient method for the isolation of nitrosamines from the food matrix. Du Plessis and Nunn (1971) reported that a combination of steam distillation and solvent extraction with dichloromethane gave inconsistent results. Eisenbrand et ul. (1970b) found that steam distillation at atmospheric or reduced pressure of aqueous solutions of volatile nitrosamines gave good recovery from alkaline, neutral, and acid media. Bogovski ef ul. (1971) employed distillation from an acid medium in the presence of tannins, while Crosby et al. (1971) reported that steam distillation from alkaline solutions (pH 10) of foodstuffs gave extracts that contained more interfering materials than was obtained when the same foodstuffs were distilled under neutral or mildly acid (pH 5) conditions. Recovery of nitrosamines present in the microgram-per-kilogram range in a number of foods varied according to the molecular weight of the nitrosamines. Vacuum distillation was fust applied in nitrosamine analysis by Lyderson and Nagy (1967); they quoted recoveries of N-nitrosodimethylamine and N-nitrosodiethylamine of 95% and 100% from aqueous alkaline solutions, but with fish samples recoveries varied from 40 to 80%.Devik (1967), Heyns and Koch (1970, 1971), Telling et al. (1971), and Scanlan and Libbey (1971) reported on the vacuum distillation method for the examination of biological materials. Du Plessis and Nunn (1971) described freeze-drying techniques employed as a means of isolating volatile nitrosamines. Dichloromethane was used to extract the distillate; recoveries in the range 80 to 100% were obtained for nitrosamines at 125 to 0.25 mglkg in cellulose, beans, marrow, and fish. Vacuum distillation of an aqueous slurry of foodstuff (250 g) together with sodium chloride (50 g) and potassium carbonate (10 g) in a total of 250 ml of water was the preferred method of Telling ef al. (1971). These workers failed to find any nitrosamines in a range of meat products at concentrations greater than the detection limit of the method, which was quoted as 25 to 65 pg/kg
N-NITROSAMINES: A REVIEW
31
(according to type of nitrosamine and experimental recovery factors). Stage-bystage recovery data are recorded in this latter publication. Modified limits of detection in line with those of other workers were later claimed by Bryce and Telling (1972). Distillation at atmospheric pressure from solutions of sodium chloride and potassium carbonate was preferred by Essigmann and Issenberg (1972) in studies on a range of meats and fish, but no results were reported. Newell and Sisken (1972) also favored this distillation procedure for apples and milk, but no positive findings at a quoted detection limit of 3 pg/kg were reported by these workers. Alliston el aZ. (1972) favored distillation with steam from a mixture of the foodstuff with sodium chloride. Recoveries varying from 40 to 100% according to food matrix and nitrosamine were claimed, and positive findings to a limit of 1 pg/kg were reported. A method favored widely in the United States for application to meats and fish was developed in the FDA laboratories (Howard et ul., 1970; Fiddler et ul., 1971). The proteinaceous food is first digested by refluxing in methanolic KOH. The dispersion is diluted with water and then distilled. Further cleanup by partition in solvent and by chromatographic procedures follow. Positive findings of 1 to 5 pg/kg were reported by these workers for a range of meat and fish products. Recoveries quoted for N-nitrosodimethylamine at 10 pg/kg were generally in the range 70 to 90%. However, in work on spinach, Keybets el al. (1970) reported that a method based on digestion of the spinach with sodium hydroxide followed by steam distillation and extraction with dichloromethane was inefficient; samples spiked with N-nitrosodiethylamine and N-nitrosodimethylamine gave recoveries of 60% and 20%, respectively. Hedler et al. (1971) used a similar method for the analysis of soya beans. Walters et al. (1971) reported variable recoveries for the separation of a range of nitrosamines (including cyclic and high-molecular-weight species) from a variety of foodstuffs. They used steam distillation, distillation under reduced pressure, or repeated extraction with dichloromethane. Recovery data obtained by distillation of solutions of nitrosamines in 20% aqueous saline solution were also reported to be in the range 70 to 100%. Issenberg and Tannenbaum (1971) reported a simultaneous distillation and extraction method, based on the apparatus of Likens and Nickerson (1964). Recoveries of N-nitrosodimethylamine and N-nitrosodiethylamine were 60% and 70% at 100 pglkg. Fractional distillation procedures have been used on aqueous solutions of nitrosamines as a means of increasing the concentration of the nitrosamines. Casselden et al. (1969) used fractional distillation from methanol and salt solutions, the nitrosamine-rich fraction being collected between the alcohol and aqueous phases. Concentrations of low-molecular-weight nitrosamines increased thirtyfold, whereas those of the higher homologs were increased by fifteen- to twentyfold. Walters et al. (197 1) confirmed that N-nitrosodimethylamine could be concentrated fortyfold when fractionally distilled from an aqueous solution
38
N. T.CROSBY AND R. SAWYER
containing 5% methanol; 50% total recovery was obtained. Williams etal. (1971) combined vacuum distillation, solvent extraction, and fractional distillation to examine a potable spirit. Recoveries of the two lowest homologs of the symmetrical nitrosamine series at 1 &liter and 2 &liter were 20% and 30% respectively. According to the work of Du Plessis and Nunn (1971), the greatest loss of nitrosamine would have occurred during fractional distillation. Most of the published methods have utilized distillation and solvent extraction procedures in one combination or another as the primary or secondary extraction stages. In addition, there are reports of other methods of cleanup-for example, ion-exchange resins used on steam distillates prepared after solvent extraction of fish,cheese, meats, cereal, etc. (Sen et al., 1970; Sen, 1971). In these methods a polyamide column was used in conjunction with the ion-exchange resin, and a dichloromethane extract of the eluate was subsequently dried with sodium sulfate and fractionated on alumina. A number of variants of this procedure have been adopted by Sen and his co-workers, each variation being adapted to deal with particular problems arising from a change of food matrix under study. Variations of the method with omission of the ion-exchange step were reported by Friemuth and Glaser (1970); recovery of added nitrosamine from samples of cheese, anchovy, cured meat, and rye bread was 60 to 70%. Ender and his co-workers (1964) used cellulose column chromatography h t o clean up ethanolic extracts obtained from cod skins and herring meal. J Plessis et al. (1969) employed a dry column chromatographic procedure with alumina columns to clean up a methanolic extract of the fruit of Sotanurn incanurn; the nitrosamines were stripped from the column with ether. Eisenbrand et al. (1 969) utilized alumina for the cleanup of their extracts from wheat flour, using the liquid-liquid partition method with acetonitrile-heptane. It did not effectively purify the extract, and significant losses of nitrosamines occurred in the full procedure. Howard et al. (1970) employed a column of Celite 545 as the final stage in the purification of dichloromethane extracts obtained in the method favored by the American workers. The nitrosamines adsorbed on Celite columns were purified with pentane and reextracted with dichloromethane. Other workers (Fiddler et al., 1971; Wasserman et al., 1972) have chosen the same procedures for examination of ham and other meat products, but the former group omitted the column chromatography stages without any apparent adverse effects on the results of the analysis. Ender and Ceh (1967) found that N-nitrosodimethylamine at concentrations of 1 pg/liter in water was quantitatively adsorbed by activated charcoal. The adsorbed nitrosamine could be removed by steam distillation or by extraction with dichloromethane. No details were given of the optimum conditions required for successful application of t h i s technique. Walters et al. (1970) studied the adsorption-desorption conditions for aqueous solutions of nitrosamines (10
N-NITROSAMINES: A REVIEW
39
pgjliter) onto activated carbon. They reported that the adsorption was effective but that quantitative desorption was less readily achieved; the method was not applied to biological materials. In most of the methods described the removal of solvent from extracts is an essential stage of the analytical process; the low-molecular-weight nitrosamines, despite having relatively high boiling points, are extremely volatile. The wide variation in recoveries of added nitrosamines indicated in the papers quoted may reflect not only inefficient extractive processes but losses incurred by inadequate precautions on evaporation. A Kuderna-Danish evaporator with a microconcentrator tube is advocated in most of the more recent publications, and a number of workers recommend n-hexane as a replacement solvent, as dichloromethane is removed by evaporation. Typical details of such methods are described by Telling et al. (1971), Howard et al. (1970), and Crosby et al. (1972).
2. Methods of Detection and Estimation
As was indicated earlier, many of the methods used for the evaluation of extractive procedures have proved to be of doubtful value. Such methods have included polarography, W absorption, thin-layer chromatography, and gas chromatography. This is not to say that all the evidence obtained by these methods is valueless, but, as more investigators have taken part in analytical studies, an increasing number of contradictory observations have been made with respect to certain classes of foodstuffs. Investigation of the contradictions generally shows up the inadequacies of analytical methods and reagent systems hitherto regarded as selective for the species under study. Although there has been a desire for rapid methods of analysis for screening a range of foodstuffs, there has also been a natural gravitation on the one hand toward individual species methods and on the other hand toward “total nitroso group” systems. In the latter group belong methods such as polarography and UV absorption; to a lesser extent other spectroscopic methods have been used in special cases. Methods employed in the former (that is, compound-selective) category have included thin-layer and gas-liquid chromatography. a. Polarography. The N-nitrosamine group is reducible at the dropping mercury cathode (Heath and Jarvis, 1955). As a result of further work with cathode-ray instruments, N-nitrosamines in solution in water, aqueous methanol, or ethanol can be estimated at a concentration of about 0.05 pg/ml (Walters, 1971). The well-formed half-waves disappear at pH values in excess of 2 (English, 1951), but the peak potentials of a range of N-nitrosamines in 0.2 N hydrochloric acid showed a range of about 0.3 volt (Walters e t al., 1970). This precludes the adoption of the method for the determination of individual nitrosamines in a mixture. The fact that the half-waves disappear in alkaline solution has been utilized as a means of differentiating responses for nitro-
40
N. T.CROSBY AND R. SAWYER
samines from those of other electroreducible species which may occur in food extracts. Many substances of biological origin are likely to be reduced within the voltage range of the nitrosamines, but numerous workers have attempted to improve the specificity of the method. Lydersen and Nagy (1967) removed polarographic contaminants from extracts of fish products by passing nitrogen through the sample at 5OoC for 30 minutes. Ammonium sulfatesulfuric acid was the supporting electrolyte. Devik (1967) employed the same method to examine the distillate from roasted potato starch spiked with glucose and amino acids in attempts to assess the likelihood of nitrosamine formation in the Maillard reaction in natural products. He attributed two half-waves to the presence of nitrosamines in the reaction mixture. The reaction was further studied by Kadar and Devik (1970), who noted the existence of pyrazines as interfering species in this work. These findings were also corroborated by Heyns and Koch (1970, 1971), who found that the half-wave potentials of pyrazines and nitrosarnines overlapped. Walters (1971) proposed that the method of observation in acid and alkaline condition would avoid this complication. Walters et al. (1970) also proposed a system based on differential polarography and the use of a W-irradiated reference cell as a means of improving the sensitivity of the polarographic method, the photosensitive nitrosamines being degraded to products with half-waves at more negative potentials while photostable polarographic contaminants were not affected. This method is still not specific, since the photosensitivity of compounds other than nitrosamines-for example, unsaturated aldehydes and ketones-would affect the result. Casselden et al. (1969) utilized a polarographic method to detect nitrosamines in distillates from native distilled spirits examined in a study on esophageal cancer, but in view of the possible interferences and nonspecificity of polarographic methods the findings must be of dubious value. Williams et al. (1971) also chose polarography as a screening method in the examination of potable spirits; under the conditions used the half-wave potential of furfural was similar to that of the range of nitrosamines under study. b. Spectroscopic Methods. Ultraviolet spectra of nitrosamines are characterized by the low-intensity absorption band at about 340 nm and the highintensity band at about 230 nm. Solvent effects on the spectra have been discussed in the chemistry section. Many workers have utilized measurement of the intensity of UV absorption as a means of quantitative estimation. Preussrnann (1 964) estimated N-nitrosodimethylamine, N-nitrosomethylethylamine, and the other alkyl nitrosamines in aqueous solution by this means. Ender et al. (1967) used the absorption band at 230 nm as a means of quantifying the nitrosarnine formed in model studies simulating conditions relevant to the nitrite preservation of fish meal. Eisenbrand et al. (1970a) selected the same band to estimate the partition of nitrosamines in the n-heptane acetonitrile system. The extinction coefficients for N-nitrosodiethylamine are such that concentrations of
A’-NITROSAMINES: A REVIEW
41
2 to 3 pg/ml and 200 to 2500 pg/ml may be measured by use of the bands at 235 nm and 341 nm, respectively, if 1-cm quartz cells are employed. Mohler and Mayrhofer (1968b) chose water, pentane, and n-hexane as solvents for the nitrosamines in their studies, while Eisenbrand et al. (1970a, b) used solutions in 50% aqueous methanol, dichloromethane, or dichloromethane n-pentane mixtures. Ender et al. (1964, 1967) utilized W absorption and IR absorption measurements as a means of confirming the presence of N-nitrosodimethylamine in herring meal which had been subjected to processing in the presence of large quantities of sodium nitrite (that is, concentrations exceeding those found in human foodstuffs by factors of lo4 to lo’). Application of this method to extracts from normal foodstuffs is limited by coextracted material which also absorbs in the ultraviolet region. Such measurements have been used in studies on the kinetics of nitrosation of a variety of amines; again the question of interfering substances did not arise in these studies. c. Colorimetric Methods. The formation of colored derivatives has been used as a means of quantifying nitrosamines in extracts from a number of materials. Neurath et al. (1964) described a method in which the nitrosamines were reduced with lithium aluminum hydride and the hydrazine obtained was then condensed with 5-nitro-2-hydroxybenaldehyde to form a benzalhydrazine. The method was used on cigarette smoke condensate and on extracts from flour and cheese, but in the latter two cases the amounts of benzalhydrazine formed were so small that TLC methods had to be used to demonstrate the presence of diethylbenzalhydrazine. Hedler and Marquardt (1968) claimed to have demonstrated the presence of N-diethylnitrosamine in wheat flour (heated and unheated), pasteurized milk, and cheese, but the results were described in terms of concentration factors of the extract from a 500-g sample to give a positive response to the test. The results could be interpreted to give values of 50 mg/kg for heated wheat meal. The results of the analysis have since been questioned. Ender and Ceh (1967, 1968) employed zinc and hydrochloric acid as reducing agent and condensed the product with p-dimethylaminobenzaldehyde; the absorption of the reaction product was measured at 458 nm. In addition these workers utilized a reduction method based on that of Neurath and Doerk (1964) by which the N-nitrosodimethylamine was reduced to dimethylamine and reacted with 4-nitro-4-azobenzoyl chloride to give the corresponding amide which has an absorption maximum at 336 nm; the detection limit was 0.3 to 0.4 pg/ml. Daiber and Preussmann (1964) also utilized the release of nitrite by irradiation of nitrosamines with W light. The resultant solution was made alkaline with sodium carbonate and treated with Griess reagent, and the absorbance of the reaction product was measured at 525 nm. Recovery of nitrite was 98% of the theoretical value for the alkyl nitrosamines; the yield was reduced if an aryl group was present in the nitrosamine. Methanol also acted as an inhibitor in the nitrite release reaction. This method has been used by a number of
42
N.T.CROSBY AND R. SAWYER
workers for the estimation of nitrosamines (Neunhoeffer et al., 1970; Ender and Ceh, 1967; Walters, 1971), and it was also proposed as a screening technique in automated systems (Fan and Tannenbaum, 1971; Issenberg and Tannenbaum, 1971). Its sensitivity was claimed to be of the order 0.1 pg/ml for N-nitrosodimethylamine. Sander (1967a) described a method similar to that of Daiber and Preussmann, with acetone as solvent; sensitivity was of the order of 1 pg of nitrosamine per milliliter. Inorganic nitrite and esters of nitrous acid also gave positive reactions. Thus there is evidence that the presence of coextracted material will affect the sensitivity of the nitrite-release method. Eisenbrand and Preussmann (1970) described a reagent system based on hydrogen bromide in glacial acetic acid; the products of reaction were amine and nitrosylbromide. The nitrosylbromide was then reacted with sulfanilic acid; the diazo ion coupled with N-( 1-naphthy1)ethylenediamine , and the absorbance was measured at 550 nm. The sensitivity of the reaction was equivalent to approximately 1 pg/ml for a number of nitrosamines, but the presence of water in the solvent containing nitrosamine adversely affected the reaction. Johnson and Walters (197 1) examined the application of the method to nitrosamides, nitrous esters, and other related compounds. They concluded that the technique of Eisenbrand and Preussmann (1970) is applicable only to N-nitrosamines and N-nitrosamides; among the range of compounds tested the method can differentiate between N-nitroso-N-methylurea and N-nitro-N-methylurea. A cross-check on the method of Eisenbrand and Preussmann was possible by detection of the amine produced in the reaction by the methods of Seiler and Wiechmann (1966,1967), Neurath and Doerk (1964), and Walle and Ehrsson (1970). d. Gas-Liquid Chromatography. Nitroso compounds have been separated successfully on gas chromatographic columns, and a variety of operating parameters and stationary phases have been described in the literature. The diversity of conditions selected for analysis indicates that there is little difficulty in effecting a satisfactory separation of individual nitrosamines from a standard mixture, despite their relatively high boiling points and moderate to strong polarity. Papers describing practical conditions for chromatography can be found in the reports of the two latest Working Conferences on N-nitroso compounds sponsored by the International Agency for Research on Cancer in 1971 and 1973. Wasserman (1971) has prepared a survey of analytical procedures, including detailed references to gas chromatographic methods. Examples of successful separations serve to illustrate the wide-ranging approach to the problem. Foreman et al. (1970), using microporous polymer beads in a 6-foot column, 1/8 inch in diameter, at 200°C reported a separation of six dialkyl nitrosamines. In subsequent studies it was found that, although these conditions worked well for standard solutions of nitrosamines, insufficient separation from coextractives was achieved with foodstuffs, and therefore stationary phases such as FFAP or Carbowax 20M on Chromosorb W were preferred (Crosby et al.,
N-NITROSAMINES: A REVIEW
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1971). Heyns et al. (1971) selected alkali-treated polypropylene glycol capillary columns and were able to separate nitroso derivatives of eighteen different secondary amines. However, capillary columns have the disadvantage that only very small loadings can be applied and frequently a stream splitter is required, so that at the low levels occurring in foods this approach limits the concentrations that can be detected. Published information indicates that column temperatures have been in the range 100°C (Serfontein and Hurter, 1966) to 260°C (Kroller, 1967) when columns are operating isothermally; improved separations are achieved by temperature programming. Column lengths have varied from 3 feet (Du Plessis et al., 1969) to 150 feet (Serfontein and Hurter, 1966), normally with a diameter of 1/8 inch, although capillary columns and those up to 1/2 inch in diameter have been used. Stainless steel, glass, and Teflon are the preferred column materials, although Saxby has reported that nitrosamines undergo decomposition on stainless steel. Stationary-phase materials recommended include Carbowax 1540 (Rhoades and Johnson, 1970), Reoplex 400 (Sen et al., 1969a), polyethylene glycol 4000 (Serfontein and Hurter, 1966), and SE 30 (Kroller, 1967) at loadings varying from 1% to 20%, with 10% being a typical value. Two columns in series have been advocated by KrGller (1967) and by Saxby (1970). The second column used by Saxby consisted of a mixture of copper(1) thiocyanate and acid-washed, silanized Chromosorb W to absorb any pyrazines present in the extract. i. Detectors. Early experiments with flame ionization detectors showed that at the sensitivities required coextractives interfered so that positive identification could not be made from retention times of chromatographic peaks alone, despite extensive cleanup procedures prior to chromatography. A further step forward occurred with the introduction of nitrogen-selective detectors. Howard e t al. (1970) described a homemade thermionic detector consisting of a platinum coil coated with potassium chloride for the determination of nitrosodimethylamine in various foods, and subsequently by the modification of the cleanup technique this procedure was extended to the detection of other nitroso compounds. It has also been used successfully by other workers-for example, Fiddler et al. (1971) and Wasserman et al. (1972). On the other hand, Crosby et al. (1972) used a commercially available detector fitted with a rubidium sulfate salt tip. Potassium chloride detectors are less subject to interference from chlorinated solvents, but the former are more sensitive, although for both types the response is critically dependent on operating parameters. Another type of nitrogen-selective detector depends on the thermal degradation andfor catalytic reduction of organic compounds to ammonia and subsequent detection conductimetrically in solution (Coulson, 1965). This detector was first used in the pyrolytic mode to detect nitroso compounds in cigarette smoke (Rhoades and Johnson, 1970) and subsequently by Sen (1971) and
44
N.T. CROSBY AND R. SAWYER
Eisenbrand (1973b) for food extracts. Crosby et al. (1972) used the detector in the reductive mode for the estimation of nitrosamines in bacon, fsh, cheese, and meat products. In the reductive mode, the detector is more sensitive but also more subject to interfering compounds. At the higher furnace temperatures (-800°C) used in the reductive mode, all nitrogen-containing organic compounds will be thermally degraded and subsequently catalytically reduced to ammonia, whereas the lower operating temperatures (-400°C) of the pyrolytic mode are chosen so that only the nitroso group, and any less thermally stable components, are degraded. The difference in sensitivity between the two modes suggests that conversion to ammonia is only partially complete at the lower temperature. A comparative evaluation of all types of nitrogen-selective detectors is urgently required. Palframan et al. (1973) published the results of a study in which the rubidium salt tip detector was compared with the Coulson conductivity detector. The influence of a number of operating parameters is discussed, and it is concluded that, although there is little significant difference in sensitivity between the two detectors, the Coulson model is less disturbed by small changes in operating conditions and is therefore more suitable for routine use in the field of nitrosamine analysis. This paper also describes a flow-diverting system which enables the thermionic detector to be used with chlorinated solvents. Cough and Webb (1973) further examined the performance of the thermionic detector, and Gough and Sugden (1973) have studied the stability of the Pye model. ii, Collaborative studies of GLC methods. Two collaborative studies of methods for the trace analysis of volatile nitrosamines organized by the International Agency for Research on Cancer have been reported. Participants were allowed to choose their own method for the analysis and, although techniques varied widely, gas chromatography was almost exclusively the end method preferred. In the first study solutions of diethylnitrosamine and N-nitrosopyrrolidine in water and in methylene chloride were distributed. An overall interlaboratory precision of around +lo% was achieved among the fifteen laboratories, and there were no significant differences between results obtained by different detectors. In the second study samples of luncheon meat to which additions of four nitrosamines (N-nitroso derivatives of dimethylamine, diethylamine, dibutylamine, and pyrrolidine) had been made at the 20-mg/kg level were distributed along with a blank. Initial results indicate, as expected, that extraction and cleanup techniques are a greater source of error than the final measurement, particularly in the case of nitrosopyrrolidine. In the second study, the coefficient of variation was as high as *50% in some cases, confirming the known difficulties in analysis at this level (IARC, 1973). iii. Derivative methods. To improve the sensitivity and selectivity of gas chromatographic methods using flame ionization detection, attempts have been made to prepare derivatives of nitrosamines which can then be estimated by
N-NITROSAMINES: A REVIEW
45
means of an alternative detector. The electron capture detector is highly sensitive to compounds containing halogen, and therefore several proposals have been put forward in which fluorinated derivatives are prepared. The derivitization reaction introduces a further element of specificity, although at the same time it also increases the length and complexity of the analytical method, thereby reducing the overall recovery. Eisenbrand and Preussmann (1970) introduced the acid-catalyzed denitrosation of nitrosamines to the corresponding amines which were then converted into heptafluorobutyryl derivatives and estimated by using an electron capture detector or by monitoring the C3F7+fragment in a mass spectrometer. Unfortunately this technique is not applicable to aqueous systems. Alliston et al. (1972) employed a similar approach but carried out the reduction electrochemically at alkaline pH values. They claim that controlled-potential electrochemical reduction is more reproducible than reduction by chemical means. After formation of the heptafluorobutyryl derivatives the solutions were chromatographed. One special feature of this method is that the chromatogram obtained is compared with another obtained from an equal portion of the original extract that has not been subjected to the reduction process. In this way the reliability of the method is increased by eliminating peaks due to the presence of adventitious amines and other interfering compounds. Reduction to the corresponding hydrazine compounds has also been suggested. Exploratory work in the authors' laboratories showed that it was possible to couple hydrazines with 1,l ,l-trifluoro-2,4-pentanedioneto produce derivatives that are sensitive to the electron capture detector. However, difficulty was experienced in obtaining consistent results. Hoffmann and Vais (197 1) used diboran for the reduction step and condensed with 3,s-dinitrobenzaldehyde.The resulting hydrazones were again estimated by electron capture detection. An alternative approach to reduction was proposed independently by Althorpe et al. (1970) and by Sen (1970). In this method nitrosamines are oxidized to the corresponding nitramine compounds with peroxytrifluoroacetic acid. Further studies of the technique have been published by Telling (1972). The cleanup procedure involves column chromatography on alumina before and after the oxidation stage. The final extracts are very clean, and the method can be used at levels as low as 0.1 pg/kg in the original food. e. Mass Spectrometry. Many of the methods described above do not provide conclusive identification of nitrosamines. They serve as a screening technique, but there is still a need for a method that can provide confirmatory evidence of the identity of the extracted compound. Mass spectrometry is now universally regarded as the most reliable procedure for this purpose. However, there are dangers in an uncritical acceptance of mass spectroscopic data, and it is necessary to state the conditions (particularly resolving power) employed in the analysis, since 29 Si(CH3)3, present in some antifoaming agents, may be mis-
46
N.T.CROSBY AND R . SAWYER
taken for nitrosodimethylamine even at a resolving power of 10,000 (Dooley et al., 1973); Gough and Sawyer, 1973). A necessary condition for the use of a mass spectrometer as a detection system following gas chromatography is an efficient separator system. A number of variants have been described, and the efficiencies of various forms of separator, especially in relation to the nitrosamine problem, have been discussed (Gough and Webb, 1972; Gough and Sawyer, 1973). Recent advances in instrumental techniques have overcome many of the problems inherent in such a system to provide potentially the most powerful of a l l analytical tools for the analysis of mixtures of organic compounds. In low-resolution mass spectrometry the identification is based on the inspection of a complete spectrum, but this is possible only for extracts that contain few interfering compounds. This is seldom the case for extracts obtained from foodstuffs even after extensive cleanup treatments. In high-resolution analysis, a characteristic ion of the compound is monitored with respect to an internal standard of similar mass. Where peak matching facilities are available, this technique is highly definitive. In the early papers (for example, Telling et ul., 1971) NO' ion monitoring was adopted, but in later studies (Bryce and Telling, 1972; Gough and Webb, 1972) the more sensitive molecular ion monitoring approach was preferred. This latter technique gives a limit of detection about one order better than that obtained with NO' ion monitoring. Further problems encountered are the adjustment of the chromatographic conditions to ensure the minimum analysis time for a single run consistent with an adequate separation of the components in the eluate. Gough and Sawyer (1973) have described the advantages of pressure programming, column switching, and the use of a recently developed interfacial system adapted for the analysis of food extracts containing nitrosamines. Undoubtedly combined gas chromatography and mass spectrometry is expensive in terms of both capital equipment and running costs and as such will be outside the scope of all but the largest analytical laboratories. Clearly reliable screening methods are needed that will limit the analytical effort required from such expensive sophisticated systems as gas chromatography combined with mass spectrometry.
B. NONVOLATILE NITROSO COMPOUNDS The division of nitrosamines into two separate groups-those that are volatile in steam and those that are essentially nonvolatile-is arbitrary but nevertheless serves to highlight differences in the analytical approach required, since initial separation by distillation cannot be applied. This distinction was approved by the working group of experts at the First International Conference on Nitrosamines held in London during 1969. Since that time most of the research effort to develop analytical methods for nitrosamines has been directed toward the
N-NITROSAMINES: A REVIEW
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volatile group of compounds, since the problems encountered, although formidable, were likely to be less than those found with the nonvolatile type. One further difficulty has been that there is no agreement as to which nonvolatile nitroso compounds are most likely to occur in foods. One of the earliest attempts to separate nonvolatile nitroso compounds from food extracts was reported by Walters et al. (1970). They showed that these compounds were readily adsorbed onto activated carbon columns; unfortunately the efficiency of the desorption process was rather low. Walters and co-workers then adopted the denitrosation technique of Eisenbrand and Preussmann (1970) developed for volatile nitrosamines and nitrosamides in which the compounds are hydrolyzed with hydrobromic acid in glacial acetic acid to produce nitrosyl bromide. They obtained similar results and showed that the reaction was specific to the nitroso group (Johnson and Walters, 1971). A later publication (Walters et al., 1973) indicated that there were advantages in using thionyl chloride for the denitrosation reaction liberating the more volatile nitrosyl chloride which could then be trapped in alkali. Determination of the nitrite produced was effected colorimetrically, and yields approached the theoretical values obtained with the HBracetic acid system. A number of compounds containing NO were tested, and again the reaction was found to be specific for nitrosamines. In working with foods fewer interferences were encountered in the final colorimetric determination. C. OCCURRENCE OF NITROSAMINES IN FOODS Following the realization that nitrosamine compounds were not merely useful for an academic study of carcinogenesis but also presented a potentially serious hazard in human food technology, a wide range of foodstuffs has been examined for the presence of nitroso compounds. The immensity of the analytical problem has been discussed in a previous section; in the light of these comments the results reported in the literature may be reviewed. Apart from the industrial accident reported by Barnes and Magee (1954), the first indication of the presence of nitrosamines in the environment came from studies of a disease of mink in Norway characterized by extensive liver damage. Injurious effects in the animals were first noticed about 1957, and similar symptoms were later reported in ruminants (Koppang, 1964). Herring meal had been used as an animal feed for many years, but following investigation the disease was connected with certain batches of the meal that had been processed with high levels of nitrite. The toxic factor was later isolated from the nitritepreserved meal and identified as nitrosodimethylamine (Ender et al., 1964). Further studies were made of the conversion of dimethylamine and trimethylamine oxide, both present in herring, to nitrosodimethylamine by reaction with nitrite under controlled conditions. It was found that the reaction could proceed
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N. T.CROSBY AND R. SAWYER
even at 0°C and a pH of 6.5. In a recent paper Sen et al. (1972) reported a similar incident in Canada in which fishmeal was again implicated in the liver disease of mink. Examination of the suspect product with TLC, GLC, and mass spectrometry showed that nitrosodimethylamine was present at levels in the range 0.12 to 0.45 mg/kg. In 1961, Herrrnann claimed that cultures of the edible mushroom Clitocybe suaveolens contain the compound p-methylaminobenzaldehyde.In a search for nitrosamines in Transkeian Bantu foodstuffs, Du Plessis et ul. (1969) had identified nitrosodimethylamine in the fruit of Solanum incanum which is used by herdboys for the curdling of milk. Techniques used for the identification included TLC, IR, GLC, and NMR. In a later study this finding could not be repeated (Roach, 1971), but minute quantities of nitrosodiethylamine were identified by means of mass spectrometry in samples of maize and beans from the same area. Cycad meal, prepared from Cycm circinalis nuts, has been shown to induce malignant tumors in rats, and the presence of cycasin, a glucoside structurally similar to nitrosodimethylamine, has been reported (Laqueur et al., 1963). These preliminary findings stimulated the search for nitroso compounds in a number of raw, processed, and cooked products, particularly where nitrite is added or is likely to be present naturally along with amines. Some examples of the earlier studies will now be given. Marquardt and Hedler (1966) examined wheat plant, grain, and flour and claimed to have detected nitrosodiethylamine. In some samples they reported levels as high as 50 mg/kg after prolonged heating, although the method was only semiquantitative. Further work by Hedler and Marquardt on wheat and on samples of milk and cheese was reported in 1968. Nitrosodiethylamine was again detected in the samples of wheat, and in one sample of pasteurized milk and both samples of cheese. Ender and Ceh (1968) analyzed samples of smoked fish, mushrooms, and meat products and found nitrosamines at concentrations in the range 0.5 to 40 pg/kg, using the hydrazine method; results for cheese products were negative. These results were substantially confirmed by the Daiber and Preussmann technique (1964) and qualitatively by the methods of Neurath and Doerk (1964) and Neurath et ul. (1964). Freimuth and Gl'aser (1970) examined commercial samples of cheese, anchovy paste, cured pork (Kasseler), and pumpernickel. Nitrosodimethylamine (120 pg/kg) and traces of nitrosodiethylamine were found in one sample of Tollense cheese; nitrosodiethylamine (40 pg/kg) was found in one sample of raw Kasseler. Separation was effected by TLC followed by nitrite release and reaction with Griess reagent for quantitative estimation. Experiments on Cantonese salt-dried fish were carried out by Fong and Walsh (1971), who found both nitrosodimethylamine (0.6 to 9 mg/kg) and nitrosodiethylamine (1.2 to 21 mglkg). Identification was made by TLC and GLC procedures. Fong and Chan (1973) repeated the study and, using mass spectroscopy, confirmed the presence of nitrosodimethylamine in some samples.
N-NITROSAMINES : A REVIEW
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Devik (1967), using polarography, TLC, and GLC, claimed that nitrosamines could be found as by-products of the Maillard reaction-that is, during normal browning reactions occurring on heating of foods through the interaction of aldoses with amino acids. McGlashan et aJ. (1968) reported the presence of nitrosodimethylamine in locally distilled spirits (Kachasu) at levels up to 3 mg/kg; these results correlated with a high incidence of esophageal cancer prevalant in areas in Zambia where these spirits are consumed. The main technique used was polarography, and it was shown that the active ingredient was destroyed on exposure to W irradiation and the expected color reaction was obtained by using Preussmann’s reagents after TLC. Some negative findings have also been reported. Thus, Thewlis (1968) was unable to detect nitrosamines in his samples of wheat flour, and Keybets e f al. (1970) failed to detect such compounds in spinach-a vegetable with a very high nitrate content. Limits of detection with this technique were 0.1 to 0.5 mg/kg. Kroller (1967) worked with thirty samples of wheat flours and was unable to detect nitrosodiethylamine at levels greater than 0.01 mg/kg in more than one of these samples. Negative findings were reported for a number of samples of cheese. These findings by TLC were confirmed by GLC. There was an indication that very low levels of nitrosodi-n-propylamine or nitrosodiisopropylamine might have been present in the samples of cheese. Investigations of British alcoholic spirits were undertaken by McGlashan et al. (1970). Polarographic results indicated that between 2.5 and 8.5 mg of nitroso compounds per might be present in these products, but unlike the samples of African spirits the extracts of British spirits did not break down to release nitrite on irradiation with W light. Subsequent gas chromatographic-mass spectrometric analysis gave no indication of the presence of nitroso compounds at levels above 5 X lo-* mg/kg. ColIis et al. (1971), working on samples of alcoholic drinks collected in western Kenya and Southern Uganda, reported that nitrosamines were not present at levels greater than 0.1 mg/kg despite the fact that results obtained with an initial polarographic screening technique had suggested quantities as high as 21 mg/kg. Sen et al. (1969b) found little evidence for the presence of the nitroso derivatives of dimethylamine, diethylamine, or dipropylamine in samples of flour, cheese, pickled herring, or spinach using TLC and GLC methods for identification at levels above 50 to 150 pg/kg, depending on the particular nitrosamine. Undoubtedly some of these early results were unreliable, as the methods of detection used-for example, polarography, gas chromatography with flame ionization detectors, and color reactions-were nonspecific for nitroso compounds even after extensive predetection cleanup procedures such as distillation, solvent extraction, and column chromatography. Furthermore, many of the early methods had a limit of detection above the levels of nitrosamines now thought to be present in food. In 1971 Williams et al. showed that the half-wave potential of furfural is in the
N. T. CROSBY AND R. SAWYER
50
range of that of many of the nitrosamines. Pyrazines are also likely to interfere with both polarographic and gas chromatographic techniques. Figure 3 is a gas chromatogram of a concentrated food extract prepared in our laboratories. The multitude of peaks indicates the degree of interference encountered when the system is operating at such low limits of detection and consequently the unreliability of this technique alone for the identification of nitrosamines in food samples. Further work on the Maillard reaction has shown that nitrosamines are not produced in any great quantity and that the main products of the reaction which have been identified are pyrazines together with certain higher alcohols and ketones (Kadar and Devik, 1970; Heyns et al., 1971;Scanlan and Libbey, 1971). Hence, the conclusions to be drawn from the earlier studies are that, although techniques such as color reactions, polarography, thin-layer, and gas-liquid chromatography are useful for screening purposes, positive identifications of nitroso compounds in foods should be made only by using high-resolution mass spectrometry (IARC, Heidelberg 1971). Preferably, the resolving power should be 10,000 or greater. A number of studies have been reported in which the combined gas chromatographic and mass spectroscopic techniques had been used. These represent the best information to date of the distribution of nitrosamines in foodstuffs.
t W
u)
2
P
u)
w K
K
2u W
Iw -
a
I
4
DPN
DEN
I
1
DMN 8
TIME
FIG. 3. Chromatogram of a meat extract using a flame-ionization detector. The retention times of nitrosodimethylamine (DMN), nitrosodiethylamine (DEN), nitrosodipropylamine (DPN), and nitrosodibutylamine (DBN) are shown.
N-NITROSAMINES: A REVIEW
51
Howard et ul. (1970) reported a procedure for the determination of nitrosodimethylamine only in smoked chub using a modified flame ionization detector. Their results indicated that this compound was not produced in the flesh during the processing of fresh water species, but in raw and smoked sable, salmon, and shad trace quantities in the range 4 to 26 pg/kg were detected and confirmed. In a subsequent paper (Fazio et uZ., 1971) this technique was applied to certain meat products but in only 1 out of 51 samples was nitrosodimethylamine found and confirmed at the 5-pglkg level. Other samples might have contained lower amounts of this compound, but the identity could not be confirmed by the techniques available at that time. These workers have now modified their procedure so that other volatile nitrosamines can be detected. With this “multidetection” procedure some 50 samples of meat products together with the original samples of fish were re-examined for other nitrosamines. Few, if any, indications were obtained for the presence of other nitrosamines at levels greater than 10 pg/kg (Fazio et ul,, 1971). Recovery of added nitrosamines was found to be generally in the range 70 to 100%at the lO-pg/kg level. Sen and his team (1971, 1972) have analyzed a large number of food samples for nitrosamines using thin-layer and gas-liquid chromatography with the Coulson electrolytic conductivity detector in the pyrolytic mode. The latest results of this group (Panalaks et ul., 1973a) show that 57 out of 197 samples of cured meat products contained detectable amounts of nitrosodimethylamine in the range 2 to 12 pgtglkg. Recovery tests gave results of 70 to 8% at the 10-pg/kg level, but the tentative identifications made were not subjected to confirmation by mass spectrometry, although in a previous study by Sen et al. (1972) this was done. One sample of salami examined was found to contain 60 to 80 pg/kg of nitrosodimethylamine; this result was confirmed by gas chromatography of the prepared nitramine derivative and by gas chromatographic-mass spectrometric analysis. Telling et al. (1971) described a method based on vacuum distillation and combined GLC and MS in which detection was effected by monitoring the common fragment ion NO* in the extract following separation on a gas chromatographic column. The limit of detection was 25 to 65 pglkg, depending on the particular nitrosamine, and no positive results were reported in the meat products examined. They also showed that the limit of detection for certain nitrosamines could be lowered to 10 pg/kg by monitoring parent ions, but no results obtained with this modification were presented. Osborne (197 1) reported the results of more recent studies with this technique. He found that, although nitrosodimethylamine, nitrosodiethylamine, and nitrosopyrrolidine had been detected at times in samples of raw and cooked foods including meat, fish, cheese, spinach, bread, potato, and mushroom, the quantities present were usually below 10 pg/kg. The present authors (Crosby et d., 1971, 1972) carried out a preliminary
52
N.T.CROSBY AND R. SAWYER
survey of certain foods employing gas chromatography with a nitrogen-selective detector followed by m a s spectrometry, using a technique sensitive down to the 1-pg/kg level. Initial findings on varieties of food including bacon, fish, and cheese show that minute quantities of certain nitroso compounds (normally below 10 pg/kg) do occur in some foods. Further studies are under way. Fiddler et al. (1972b) have analyzed samples of frankfurters produced commercially. Samples of five out of eight lots contained no detectable quantity of nitrosodimethylamine. In other samples quantities in the range of 11 to 84 pg/kg were detected and confirmed by mass spectrometry. A further study (Fiddler et al., 1973a), in which the effect of nitrite concentration on the formation of nitrosodimethylamine under controlled processing was followed, showed that approximately one-half of the nitrite added was destroyed during processing and that more of the compound was formed at higher nitrite levels and with longer heating times. Levels of nitrosodimethylamine in the range of 3 to 19 pg/kg were identified. These levels were reduced in the presence of sodium ascorbate, the reduction being more marked at higher levels of ascorbate. Sodium erythorbate is similarly effective (Fiddler et ul., 1973b). Bacon has been extensively studied over the last few years following the discovery that nitrosopyrrolidine was formed during frying (Crosby et al., 1972). Levels of up to 40 pg/kg were found in some samples, and these initial findings have since been confirmed by several other groups of workers. Thus Fazio et al. (1973) examined eight commercial brands of bacon and were able to detect nitrosopyrrolidine at levels varying from 10 to 108 pg/kg in the fried product; nothing was detected in uncooked bacon. Nitrosopyrroline, being fat-soluble, was found at a higher concentration in the fat than the meat portion. Sen et al. (1973a) also analyzed a number of samples of fried bacon and detected amounts of nitrosopyrrolidine as high as 250 pg/kg in some samples. Smaller amounts (up to 30 pg/kg) of nitrosodimethylamine were also thought to be present. In one experiment they were able to show that 70% of the nitrosopyrrolidine was concentrated into the fatty phase. Telling et ul. (1973) studied the effect of cooking temperature on the yield of nitrosamines during grilling. They found that levels of nitrosodimethylamine remain fairly constant, but levels of nitrosopyrrolidine increase significantly as the cooking temperature is raised. However, 200'C was the highest temperature tested in this study. Fiddler et al. (1973b) working with model systems showed that 365°C was the optimum temperature of frying to produce the highest yields of nitrosopyrrolidine. These findings were confirmed in samples of bacon, and it was also shown that the duration of frying was unimportant. Different methods of cooking were tested, and it appears that microwave heating produces the smallest quantity of nitrosopyrrolidine. Additions of sodium ascorbate again reduce or inhibit nitrosamine formation. The presence of nitrosopyrrolidine and nitrosopiperidine in a meat curing mix
N-NITROSAMINES: A REVIEW
53
was reported by Sen et al. (1973a). The use of this curing agent in one particular brand of Mettwurst sausage resulted in the formation of nitrosodimethylamine, nitrosopyrrolidine, and nitrosopiperidine. It is thought that nitroso compounds are formed from amines present in the spices by interaction with nitrite on prolonged storage. Recommended codes of practice for these premixes have now been established.
D. NITROSAMINES IN TOBACCO The possibility that nitrosamines could be formed during the burning of tobacco was first discussed by Druckrey and Preussmann (1962). A large number of nitrogenous compounds are known to be present in tobacco smoke condensate, either naturally or produced in situ during the smoking process by the pyrolysis of more complex molecules. Most of these compounds occur at very low levels so that large numbers of cigarettes are required to provide a suitable concentration for the analytical determination. Smoking machines are now available in which many cigarettes are smoked simultaneously under controlled conditions designed to simulate, as far as is possible, the normal process. Norman and Keith (1965) examined the smoke from several brands of cigarettes to determine the ratio of the various oxides of nitrogen present. They found that the main constituent of fresh smoke is nitric oxide (NO) with only traces of the dioxide (NO,). Concentration of NO varied between 400 and 1000 ppm, depending on the nitrate content of the tobacco. Factors affecting the level of nitrate in tobacco leaves have been discussed by Broaddus et al. (‘1965). Among the types used for American cigarettes the highest levels of nitrate were recorded in Burley varieties. The availability of fertilizer nitrogen has a significant effect on the nitrate content of the resulting crop but there is no simple relationship between nitrates and smoking quality. The general dependence of nitric oxide content of the smoke on the original nitrate present in the tobacco was confirmed. Oxidation of nitric oxide can occur according to the reaction 2 N 0 + 0, + 2N0,
but under the conditions pertaining in cigarette smoke the rate of conversion is very slow, being half complete in 500 seconds (Neurath, 1971). An equimolar mixture of nitric oxide and nitrogen dioxide favors the formation of nitrosamines from secondary amines in the gaseous phase (Neurath et al., 1965). Hence, the above oxidation reaction is likely to be the limiting step in the formation of nitrosamines in tobacco smoke. Less information as to the amine content of cigarette smoke is available, and the picture is further complicated by the pyrolytic reactions taking place which
54
N.T.CROSBY AND R. SAWYER
in turn will depend on the particular conditions of smoking employed. The principal secondary amines which have been identified are anabasine, nomicotine, dimethylamine, pyrrolidine, methylethylamine , diethylamine, piperidine , and proline (Boyland et al., 1964; Neurath, 1971). The first two have relatively low vapor pressures and are likely to remain in the particulate phase (Neurath, 1971). Hence, N-nitroso derivatives of dimethylamine, pyrrolidine, and methylethylamine are the most likely compounds to be detected in the smoke of tobacco products. In addition to the control of smoking parameters, the collection system should be properly designed to preclude both the formation of artifacts and the loss of any nitroso compounds formed. The most usual device consists of a solvent trap cooled to a defined temperature. Interfering compounds are just as great a problem as in the analysis of foods, and therefore extensive cleanup procedures are required. Although some of the early methods of detection were nonspecific for nitroso compounds, the results have in general been confirmed by more recent studies using mass spectrometry. Neurath et d. (1965) first reported the presence of nitrosodimethylamine and nitrosopyrrolidine at a combined level of 4 ng per cigarette. In a follow-up study Krijller (1967) trapped the smoke in acetone cooled in an icesalt mixture. Purification of the condensate was achieved through steam distillation, precipitation first with lead acetate and then with strontium hydroxide, followed by extraction into methylene chloride. This solvent was then acid-washed followed by an alkali wash and concentrated. Chalk was added, and the mixture was extracted with diethyl ether. This procedure was repeated with n-hexane before thin-layer chromatography on silica gel plates. After development, the chromatogram was exposed to U V radiation and Griess and ninhydrin reagents. With this procedure nitrosodi-nbutylamine and nitrosopiperidine were thought to be present in the smoke condensate, but, despite such an extensive cleanup technique, it was not possible to confirm the presence of either of these compounds by gas-liquid chromatography owing to the continued presence of other interfering substances. Rhoades and Johnson (1970) used the Coulson electrolytic conductivity detector in the pyrolytic mode for the analysis of smoke condensates. They claimed that under these conditions the detector was more specific for nitroso compounds than operation in the normal mode at higher furnace temperatures. They reported the presence of nitrosodimethylamine and nitrosomethylethylamine in tobacco smoke condensate from experimental and branded cigarettes using GLC with two stationary phases for identification. The presence of nitrosodimethylamine in the range 0 to 140 ng per cigarette was confirmed by mass spectrometry. A more recent study of cigarette smoke condensate has been reported by . These workers used experimental cigarettes prepared McCormick et ~ l (1973). from tobacco grown under conditions of a high rate of nitrogen fertilization.
N-NITROSAMINES: A REVIEW
55
The smoke was condensed in methanol in a cold trap at -78°C. The methanolic condensate was steam-distilled first in the presence of potassium carbonate and then in a saturated sodium chloride solution before extraction into methylene chloride. Detection was effected by gas chromatography followed by molecular ion scanning with a high-resolution instrument. Values for nitrosodimethylamine ( 5 to 180 ng per cigarette), nitrosomethylethylamine (up to 40 ng), nitrosodiethylamine (up to 28 ng), nitrosopyrrolidine (1 to 110 ng) and nitrosopiperidine (up to 9 ng) are reported. The authors conclude that the levels of nitrosamines found are dependent on the level of total volatile bases in the tobacco and on its nitrate content. Hoffmann er al. (1973) examined the smoke from standard American brands of cigarette. Smoke from the automatic machine was collected in traps containing (1) acid and (2) alkali. Nitroso compounds were separated by distillation and ether extraction. The extracted compounds were then reduced with diborane to the corresponding unsymmetrical hydrazines and condensed with 3,5-dinitrobenzaldehyde. The resulting hydrazones were characterized by gas chromatogaphy and mass spectrometry. The authors were able to identify dimethylamine (84 ng per cigarette), methylethylamine (30 ng), and traces of diethylamine in the smoke. Filter tips reduced the quantities found in the extract.
V. CONCLUSIONS International concern over the presence and formation of nitroso compounds in foods and in other areas of the environment will undoubtedly continue for several years ahead. The principal analytical task of devising methods capable of detecting, identifying, and measuring nitroso compounds at concentrations as low as 1 part in lo9 has now been accomplished. Further progress in analytical methodology is likely to center around the modification of existing techniques to improve the speed or specificity of the method and to reduce losses encountered during the analysis. Further collaborative and comparative testing of a few of the most promising procedures is considered worthwhile. New methods of cleanup and techniques for detection will be welcomed, but they will require evaluation over a period of years before they can replace the procedures that have now been tested in a number of different laboratories in several different countries. Surveillance programs for the estimation of levels of nitroso compounds in the environment are costly in terms both of the equipment and of the quality of manpower required to carry out the analysis. This process is never likely to become a routine procedure, although a simple screening test might prove useful in pinpointing areas for further study by the more critical and sophisticated techniques available in the larger analytical laboratories. It seems unlikely that
56
N. T. CROSBY AND R . SAWYER
the outlines of the present picture will be changed significantly by these further studies, but, because of its importance in human terms, this work should continue at present until the information is complete. Nonvolatile nitroso compounds present additional difficulties which remain to be solved. Further development of the high-pressure liquid chromatography technique, as described by Cox (1973), may prove useful in this respect. At the present time the significance to Man of the carcinogenic properties of nitroso compounds is uncertain, although some experimental evidence has justifiably aroused concern and highlighted the need for further work. Further studies on experimental animals, particularly at low levels of dosing, are now under way. Statistically, the results will be difficult to interpret because of the natural variations in population studies at such low levels, where the effects are observed only near the end of the animal’s life span. Further problems arising from the combined effects of one or more carcinogens occurring together, leading to a possible synergistic effect, remain to be evaluated. The influence of cocarcinogens, and of inhibitors, may well prove to be equally important factors requiring careful examination. Other areas for study include the role of microorganisms in the formation of nitroso compounds in vivo, and more detailed knowledge on the distribution of precursor materials in the food chain and in the environment. The restriction of nitrate and nitrite levels in cured products, the separation of reactants in certain premix spiced cures, and the use of alternative drugs where possible are examples of arrangements which can be made to reduce the possible risk. More attention to the safety aspects of laboratory work involving nitroso compounds is desirable, particularly in educational establishments. The hazardous nature of these compounds, and the precautions to be taken in their use, deserve the widest publicity. Since this review was prepared, an international symposium on Nitrite in Meat Products was held at the Central Institute for Nutrition and Food Research TNO, Zeist, The Netherlands, on September 10-14, 1973. The proceedings of this symposium have now been published (B. Krol, B. J. Tinbergen eds., Wageningen, 1974). ACKNOWLEDGMENTS We wish to thank the Government Chemist, Dr. H. Egan, for permission to publish this review, and Mr. H. Baxter, for his help with the literature survey.
REFERENCES Addison, C. C, and Conduit, C. P. 1952. The liquid dinitrogen tetroxide solvent system. Part XI. Compound formation and ionic species in solutions of diethylnitrosamine in liquid dinitrogen tetroxide. J. Chem Soc., p. 1390.
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Addison, C. C., Conduit, C. P., and Thompson, R. 1951. The liquid dinitrogen tetroxide solvent system. Part V. Reactions involving some alkylammonium salts with particular reference to solvolysis and the formation of diethylnitrosamine. J. Chem. SOC.,p. 1298. Alam, B. S., Saporoschetz, I. B., and Epstein, S. S. 1971. Formation of N-nitrosopiperidine from piperidine and sodium nitrite in the stomach and the isolated intestinal loop of the rat. Nature (London) 232,116. U s t o n , T. G., Cox, G. B., and Kirk, R S. 1972. The determination of steam-volatile N-nitrosamines in foodstuffs by formation of electron capturing derivatives from electrochemically derived mines. Analyst 97,915. Althorpe, J., Goddard, D. A., Sissons, D. J., and Telling, G. M. 1970. The gas chromatographic determination of nitrosamines at the picogram level by their conversion to their corresponding nitramines. J. Chromatogr. 53, 371. Ashton, M. R. 1970. The occurrence of nitrates and nitrites in foods. B.F.M.I.R.A. Lit. Sum. No. 7. Atkinson, J. L., and Follett, M. J. 1973. Biochemical studies on the discoloration of fresh meat. J. Food Technol. 8,51. Austin, A. T. 1961. Nitrosation in organic chemistry. Sci. Progr. 49,619. Baird-Parker, A. C., and Baillie, M. A. H. 1973. The inhibition of Cl. botulinum by nitrite and sodium chloride. In “Proceedings of the International Symposium on Nitrite in Meat Products” (B. Krol, and B. J. Tinbergen, eds). Zeist, Wageningen. Baker, W., and OIIis, W. D. 1955. Mesc-ionic compounds. Chem. Ind. (London), p. 910. Baker, W., and Ollis, W. D. 1957.Meso-ionic compounds. Quart. Rev. Chem. SOC.,London, 1 1 , 15. Bamford, C. H. 1939. A study of the photolysis of organic nitrogen compounds. Part I. Dimethyl- and diethylnitrosamines. J. Chem. SOC.,p. 12. Barnes, J. M., and Magee, P. N. 1954. Some toxic properties of dimethylnitrosamine. Brit. J. Znd. Med. 11, 167. Bellamy, L. J., Conduit, C. P., Pace, R. J., and Williams, R. L. 1959. Infra-red spectra and solvent effects. Part 5. Solvent effects on X = 0 dipoles and on rotational isomers. Truns. Faraday SOC.55,1167. Bhowmik, B. B., and Basu, S. 1963. Studies on hydrogen bond. Part 3. Trans. Faraday SOC.
59,813. Bhowmik, B. B., and Basu, S. 1964. Studies on hydrogen bond. Part 4. Trans. Faraday SOC. 60, 1038. B.LB.RA. 1968. Articles of general interest-Nitrosamines: A jig-saw puzzle with missing pieces. Brit. Znd. Biol. Rex. Ass. 7, 223. Bodiphala, T., and Ormrod, D. P. 1971. Factors affecting the nitrate content of vegetables and fruit foods. J. Can. Znst. Food Technol. 4,6. Bogovski, P., Castegnaro, M., Pignatelli, B., and Walker, E. A. 1971. The inhibiting effect of tannins on the formation of nitrosamines. Zn “N-Nitrosamines Analysis and Formation” (P. Bogovski, R. Preussmann, and E. A. Walker, eds.), p. 127. I.A.R.C., Lyon. Borghesani, G., Pulidori, F., Pedriali, R., and Bighi, C. 1971, Reduction mechanism of nitrogen compounds at the dropping mercury electrode. 11. Polarographic reduction of aliphatic N-nitrosamines. J. Electroanal. Chem. Interfacial Electrochem. 32, 303. Boyland, E., Roe, F. J. C., and Gorrod, J. W. 1964. Induction of pulmonary tumors in mice by nitroso-nornicotine, a possible constituent of tobacco smoke. Nature (London) 202,
11 26. Boyland, E., Nice, E., and Williams, K. 1971. Catalysis of nitrosation by thiocyanate from saliva Food Comet. Toxicol. 9,639. Brackman, W., and S i t , P. J. 1965. Homogenous catalysis 11: Competitive formation of alkyl nitrite and nitrosamine in the reaction of NO with diethylamine-alcohol mixtures. Rec. Trav. Chim Pays-Bas 84,312.
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Bredereck, M., Foehlisch, B., and Walz, K. 1965. N-Isocyanodialkylamines.Justus Liebigs Ann. Chem. 92,686. Broaddus, G. M., York, J. E., and Moseley, J. M. 1965. Factors affecting the levels of nitrate nitrogen in cured tobacco leaves. Tobacco Sci. 9, 149. Brockman, F. J., Downing, D. C., and Wright, G. F. 1949. Nitrolysis of hexamethylenetetramine. Can. J. Res. 278,469. Brookes, P., and Walker, J. 1957. Formation and properties of sydnone imines, a new class of meso-ionic compound, and some sydnones related to natural *amino acids. J. Chem. Soc., p. 4409. Brooks, J. B., Alley, C. C., and Jones, R. 1972. Reaction of nitramine with fluorinated anhydrides and pyridine to form electron capturing derivatives. Anal. Chem. 44,1881. Brouwers, J. A. J., and Emmelot, P. 1960. Microsomal N-demethylation and the effect of the hepatic carcinogen dimethylnitrosamine on amino acid incorporation into the proteins of rat livers and hepatomes. Exp. CeN Res. 19,467. Bryce, T. A., and Telling, G. M. 1972. Semiquantitative analysis of low levels of volatile nitrosamines by gas chomatography-mass spectrometry. J. Agri. Food Chem. 20, 910. Bumgardner, C. L,McCallum, K. S., and Freeman, J. P. 1961. Small ring heterocyclic mtrasamines. J. Amer. Chem. SOC.83,4417. Burgess, E. M., and Lavanish, J. M. 1964. Photochemical decomposition of N-nitrosamines. Tetrahedron Lett. 19-20,1221. Casselden, R J., Johnson, E. M., Ray, N., and Walters, C. L. 1969. Concentration of volatile N-nitrosodimethylamine by fractional distillation. Proc. I.A.R. C. Meet. Nitrosamine Analysis, London. Challis, B. C., and Challis, J. A. 1970. Reactions of the carboxamide group. In “The Chemistry of Amides” (Zabicky Jacob, ed.), p. 731. Interscience, London. Challis, B. C., and Osborne, M. R. 1972. Chemistry of nitroso compounds. Reaction of N-nitrosodiphenylamine with N-methylaniline. Dnect transnitrosation. Chem. Commun., p. 5 18. Chow, Y. L. 1964. Photolysis of N-nitrosamines. Tetrahedron Lett. 34,2333. Chow, Y. L. 1965a. Carbon-carbon double bond cleavage by photoaddition of N-nitrosodialkylamine to olefms. J. Amer. Chem. Soc. 87,4642. Chow, Y. L. 1965b. Photoaddition of N-nitrosodialkylamines to cyclohexene. Can. J. Chem. 43,2711. Chow, Y . L 1967. Photochemistry of nitroso compounds in solution. V. photolysis of N-nitrosodialkylamines. Can J. Chem. 45,53. Chow, Y . L., and Lee, A. C. M. 1967. Photochemistry of nitroso compounds in solution. VI. Photolysis of N-nitrosoamides in acidic media. Can. J. Chem. 45,311. Chow, Y. L., Colon, C., and Chen, S. C. 1967. Photochemistry of nitroso compounds in solution. VII. Photoaddition of nitrosamines to various olefins. J. Org. Chem. 32,2109. Chow, Y . L, Lau, M. P., Cessna, A. J., and Yip, R. W. 1971. Flash photolysis of N-nitrosopipendine. The reactive transient. J. Amer. Chem. SOC.93, 3808. Chow, Y. L., Lau, M. P., Perry, R. A., and Tam, J. N. S. 1972. Photoreactions of nitroso compounds in solution. XX. Photoreduction, photoelimination and photoaddition of nitrosamines. Can. J. Chem. 50,1044. Chute, W. J., Herring, K. G., Toombs, L. E, and Wright, G. F. 1948. Catalysed nitration of amines. 1. Dinitroxydiethylnitramine. Can. J. Rex 26B, 89. Collin, J., 1954. Mass spectrometry of nitrogen derivatives. Dialkylnitrosaminesand nitroso derivatives. Bull Soc. Roy. Sci. Liege 23, 201. Collis, C. H., Cook, P. J., Foreman, J. K., and Palframan, J. F. 1971. A search for nitrosamines in E. African spirit samples from areas of varying oesophageal cancer frequency. Gut 12,1015.
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Mottram, D. S., and Rhodes, D. N. 1973. Nitrite and the flavour of cured meat. In “Proceedings of the International Symposium on Nitrite in Meat Products” (B. Krol and B. J. Tinbergen, eds.). T. N. 0. Zeist, Wageningen. Neunhoeffer, O., Wilhelm, G., and Lehmann, G. 1970. Enzymic rearrangement of carcinogenic nitrosamines. Z. Naturforsch. 25, 302. Neurath, G. 1971. Nitrosamine formation from precursors in tobacco smoke, In “NNitroso Compounds Analysis and Formation” (P. Bogovski, R. Preussmann, and E. A. Walker, eds.). I.A.R.C., Lyon. Neurath, G., and Doerk, E. 1964. Identifizierung und quantitative Bestimmung einzelner priminer und sekundirer Amine aus Gemischen als 4‘-Nitro-azobenzol-Carbons~ure(4)-amide. Chem. Ber 97,172. Neurath, G., Pirmann, B., and Dunger, M. 1964. Identifiiierung von N-Nitrosoverbindungund Anwenden und asymmetrischen Hydrazinen als 5-Nitro-2-hydroxy-benzalderivate ung in Mikromassstab. Chem. Ber. 97,1631. Neurath, G., Pirmann, B., Liittich, W., and Wichern, H. 1965. Zur Frage der N-Nitrosoverbindungen im Tabakrauch 11. Beitr. Tobakforsch. 3, 251. Newell, J. E., and Sisken, H. R. 1972.Determination of nitrosodimethylamine in the low parts per billion. J. Agr. Food Chem. 20,711. Nikulm, V. N., and Klochkova, V. N. 1972.Mechanism of the electrochemical reduction of dimethylnitrosamine on a lead electrode. Elektrokhimiya 8,499. Norman, V., and Keith, C. 1965. Nitrogen oxides in tobacco smoke. Nature (London) 205,
915. Osborne, D. R. 1971.Use of vacuum distillation and gas chromatograph-mass spectrometry for detection of low levels of volatile nitrosamines in food. In “N-Nitrosamines Analysis and Formation” (P. Bogovski, R. Preussmann, and E. A. Walker, eds.), p. 43. I.A.R.C., Lyon Overburger, C., hselme, J., and Lombardine, J. 1966. “Organic Compounds with Nitrogen-Nitrogen Double Bonds.” Ronald Press, New York. Overholser, L. G., and Yoe, J. H. 1941. The colorimetric detection and determination of palladium with compounds containing the p-nitroso-phenylamino group. J. Amer. Chem. SOC.63,3224. P A ,C., and Deybeck, S. 1897.Ueber Derivate der p-Tolylsulf-nitrosaminsaure.Ber 30,880. Pad, C., and Yao, W. N. 1930.Catalytic reduction of some nitrosamines. Ber 63B,57. Palframan, J. F., Macnab, J., and Crosby, N. T. 1973. An evaluation of the alkali flame ionisation detector and the Coulson electrolytic conductivity detector in the analysis of N-nitrosamines in foods. J. Chromatogr. 76,307. Panalaks, T., Iyengar, J. R., and Sen, N. P. 1973a.Nitrate, nitrite, and dimethylnitrosamine in cured meat products. J. Assoc. Offic.And. Chem. 56,621. Panalaks, T., Iyengar, J. R., Donaldson, B. A., Miles, W. F., and Sen, N. P. 1973b.Further survey of cured meat products for volatile N-nitrosamines. J. Assoc. Offic.Anal. Chem. 56, in press. Paulman, W. 1894. Beitrage zur kenntris des sarkosins. Arch. Pharm. (Weinheim) 232,601. Pensabene, J. W., Fiddler, W., Dooley, C. J., Doerr, R., and Wasserman, A. E. 1972.Spectral and gas chromatographic characteristics of some N-nitrosamines. J. Agr. Food Chem. 20,
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N-NITROSAMINES: A REVIEW
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Piria, R. 1848. Recherches sur la constitution chimique de L’asparagine et de l’acid aspartique. Ann. Chim. (Paris) 22, 160. Poirier, R. H., and Befigton, F. 1952. Reduction of N-nitrosodiphenylamine to unsymdiphenylhydrazine by lithium aluminium hydride. J. Amer. Chem. SOC. 74,3192. Preussmann, R. 1964. The oxidative breakdown of nitrosamines by enzyme-free model systems. Arzneim. Forsch. 14,769. Preussmann, R. 1971. In “N-Nitroso Compounds Analysis and Formation” (P. Bogovski, R. Preussmann, and E. A. Walker, eds.). I.A.R.C., Lyon. Preussmann, R., Daiber, D., and Hengy, H. 1964a. A sensitive colour reaction for nitrosamines in thin layer chromatograms. Nature (London) 201,502. Preussmann, R., Neurath, G., Wolf-Loretzen, G., Daiber, D., and Hengy, H. 1964b. Anfirbemethoden und Dunschicht-Chromatographie von organischen N-Nitrosoverbindungen. 2. Anal. Chem. 202,187. fitter, R., and Wolfrum, G. 1959a. Ascaricidal sydnones. Brit. Patent 823,001. fitter, R., and Wolfrum, G. 1959b. Sydnones. Ger. Patent 1,057,124. Pulidori, F., Borghesani, G., Bighi, C., and Pedriali, R. 1970. Reduction mechanism of nitrogen compounds at the dropping mercury electrode 1. Dipropyl N-nitrosamine. J. Electroanal. Chem. 27,385. Rademacher, P., Stoelevik, R., and Luettke, W. 1968. Structure of nitrosodimethylamine and nitromethylamine. Angew. Chem. Znt. Ed. Engl. 7,806. Ragsdale, R. O., Karstetter, B. R., and Drago, R. S. 1965. Decomposition of the adducts of diethylamine and isopropylamine with nitrogen (11) oxide. Znorg. Chem. 4,420. Reilly, E. L. 1964. Nitrosamine manufacture. U.S. Patent 3,153,094. Renouf, E. 1880. Ueber das Dimethylhydrazin. Ber 13,2169. Rhoades, J. W., and Johnson, D. W. 1970. Gas chromatography and selective detection of N-nitrosamines. J. Chromatogr. Sci 8,616. Ridd, J. H. 1961. Nitrosation, diazotisation and deamination. Quart. Rev. Chem. Soc., London 15,418. Roach, W. A. 1971. Zn “N-Nitroso Compounds Analysis and Formation” (P. Bogovski, R. Preussmann, and E. A. Walker, eds.). I.A.R.C., Lyon. Roberts, T. A., and Ingram, M. 1973. Inhibition of growth of CI. botulinurn at different pH values by sodium chloride and sodium nitrite. J. Food Technol. 8,467. Rohde, W. 1869. Ueber einige Zersetz ungen deg Tribenzylamino. Justus Liebigs Ann. Chem. 151,366. Rubin, A. A., Zitowitz, L., and Hawker, L. 1963. Acute circulatory effects of diazoxide and sodium nitrite. J. Pharmacol. Exp. Ther. 140,46. Sander, J. 1967a. Detection of nitrosamines. Hoppe Seyler’s 2. Physiol. Chem. 348,852. Sander, J. 1967b. Kann Nitrit in der menschlichen Nahrung Ursache einer Krebsentstehung durch Nitrosaminbildung sein. Arch. Hyg. Bakteriol. 151,23. Sander, J., and Seif, F. 199. Bakterielle Reduktion von Nitrat in Magen des Menschen als Ursache einer Nitrosaminbildung. Arzneim. Forsch. 19,1091. Sander, J., Schweinsberg, F., and Menz, H. P. 1968. Formation of carcinogenic nitrosamines in the stomach. Hoppe Seylers 2. Physiol. Chem. 349,1691. Sander, J., Buerkle, G., Flohe, L., and Aeckens, B. 1971. In vitro studies on the possible formation of carcinogenic nitrosamides. Arzneim. Forsch. 21,414. Saxby, M. J. 1970. The separation of volatile N-nitrosamines and pyrazines. Anal. Lett. 3, 397. Saxby, M. J. 1972. Mass spectrometry of volatile derivatives. V. N-Dialkynitrosamines. J. Ass. Offic. Anal. Chem. 55,9.
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DEVELOPMENT OF FLAVOR. ODOR. AND PUNGENCY I N ONION AND GARLIC
BY JOHN R .WHITAKER Department of Food Science and Technology University of California. Davis. California
.
I Introduction .................................................... 73 I1. Sulfur Compounds of Intact Alliurn .................................. 74 A Alkylcysteines ................................................ 77 3. Akylcysteine Sdfoxides ........................................ 77 C y-Glutamyl Peptides ........................................... 79 I11 Biosynthesis of Sulfur Compounds of AIliurns .......................... 83 A Cysteine .................................................... 85 B Alkylcysteines and Alkylcysteine Sulfoxides ......................... 85 C y-Glutamyl Peptides ........................................... 90 Iv Sulfur Constituents of Crushed Alliurn ................................ 90 A Initial Compounds Formed from Enzymatic Activity .................. 91 B Secondary Products in Crushed Alliurn ............................. 100 V . Methods of Measuring Alliurn Pungency. Flavor. and Aroma ............... 114 VI Enzymes in Flavor. Aroma. and Pungency Development in Allium .......... 116 A. Alliinase .................................................... 118 B y-Glutamyl Peptidase and y-Glutamyl Transpeptidase ..................122 VII Summary ...................................................... 124 125 VIII ResearchNeeds .................................................. References ..................................................... 127
. . . . . . . . . . . . .
I . INTRODUCTION Onion and garlic have had a long and interesting history of use as vegetables and spices and as home remedies in the treatment of illnesses Use of garlic in treatment of certain diseases probably dates back several hundred years; it was interest in this that led Lehmann (1930) and Stoll and Seebeck (1951) to investigate the substances present in garlic responsible for its antibiotic effects.
.
13
14
JOHN R. WHITAKER
Not only did they isolate the major antibiotic compound in garlic, allicin (Cavallito and Bailey, 1944), they also showed that it is produced from a precursor following damage to garlic tissue through the action of an enzyme, alliinase (aWn alkyl-sulfenate-lyase; EC 4.4.1.4) (Stoll and Seebeck, 1947). Interest in the flavor components of onion and garlic oils led to isolation of some sulfides from garlic oil (Wertheim, 1844, 1845) and onion oil (Semmler, 1892) by the middle of the nineteenth century. However, the major effort in elucidation of the flavor precursors of intact onion and garlic tissue came through the efforts of Virtanen and co-workers in Finland and the synthesis work of Carson and co-workers in the United States during the period 19551970. During this same period Schwimmer, Mazelis, and others worked extensively on the enzyme(s) involved in converting the flavor and lachrymatory precursors to the active compounds. At this time, the invention of gas chromatography made it relatively easy to study the changes in composition of the head space above crushed onion and garlic tissues and the composition of onion and garlic oils. Although there is still some disagreement among workers because of the complexity of the secondary products formed, the general features of the biosynthesis of the flavor constituents, their enzymatic conversion to primary products, and the rearrangement, decomposition, and interaction of these primary products t o give a large number of secondary products responsible for the flavor and odor of onions and garlic are generally understood. Now appears to be a good time to correlate and integrate the information in this field and to point up the areas of deficiency in our knowledge of flavor, odor, and pungency development in onion and garlic.
II. SULFUR COMPOUNDS OF INTACTALLIUM One of the outstanding features of the chemical composition of Allium plants is the large amount of organic bound sulfur. The number ofmlfur compounds is much larger than that usually found in most organisms. These sulfur compounds in onion and garlic have received a lot of attention because of their potential antibiotic (Lehmann, 1930; Cavallito and Bailey, 1944; Stoll and Seebeck, 1947) and flavor properties, as we shall see. A number of sulfur-containing compounds have been isolated from onions. When onions are injected with 35S-labeled inorganic sulfate, the label soon appears in a number of organic sulfur compounds (Ettala and Virtanen, 1962; Matikkala and Virtanen, 1967; Granroth, 1968, 1970). For example, 7 days after injection of onions, twenty-one different labeled spots were found on two-dimensional chromatograms (Ettala and Virtanen, 1962). Subsequent studies using separation by an amino acid analyzer (Matikkala and Virtanen, 1967)
TABLE I SULFUR COMPOUNDS O F INTACT ONION Compound
la
2b
3c
4d
&g/g onion fresh weight)
I. L-Valinee 2. L-Serinee 3. LCystineGf 4. L-Methioninee 5. L-Methionine sulfoxidee 6. S-Methyl-L-cysteineepxg 7. S-Prowl-L-cysteind 8. trans-S-(l-Propenyl)-L-cysteineeBf 9. S-(Carboxymethyl)-L-cysteinJ 10. S-(2-Carboxyethyl)-L-cysteinef 11. S-(2Carboxypropyl)-~-cysteine"f 12. S-(Carboxy-isopropy1)-L-cysteind 13. S-Allylcysteineeh 14. S-~2Carboxylpropyl)glutathione~~f 15. (+)S-Methyl-L-cysteine sulfoxidedsGf;k 16. (+)S-Propyl-L-cysteine sulfoxideaelf;hi 17. trans-(+)S-(l-Propenyl)-l-cysteine sulfoxidea exi 18. (+)S-Allyl-L-cysteine sulfoxidef 19. cycloalliind.e*f 20. yLGlutamyl-L-methioninedfe 2 1. y-LGlutamyl-S-methyl-L-cysteined*e 22. y-LGlutapylS-methyl-Lcysteine sulfoxidd 23. y-LGlutamyl-trans-(+)a-(1-propeny1)L-cysteine sulfoxidedae 24. yGlutamylS-(2arboxypropyl)-~cysteinyklyeei 25. Glutathionef 26. Glutathione cysteine disulfidei 27. Glutathione-y-glutamylcysteine disulfidd 28. S-Sulfoglutathiond
65.4 166 4.7
59.2 178 3.6 4.2
16.7
15.8
9.2
7.8
-
5 23
-
5.1
-
341
345 -
330 200 50
1927
1922
-
40
127 190
-
103 159
2500 50 50
-
-
1965
1300
aEthanol extract (Matikkala and Virtanen, 1967). bNeutral and basic extract (Matikkala and Virtanen, 1967). CAcidextract (Matikkala and Virtanen, 1967). dEttala and Virtanen (1962); amino acid analyzer data. eMatikkak %,id Virtanen (1967); separated by twodimensional electrophoresis and chromatography. fGranroth (1968). gS-Methylcysteine content of four varieties of onions ranged from 9.3 to 19.3 pg/g of onions wet weight (Kuon and Bernhard, 1963). k a r s o n and Wong (1961a). !Belo (1972). fVirtanen (1969).
76
JOHN R. WHITAKER
5
46 41
FIG. 1. A chromatogram from an amino acid analyzer of an ethanol extract of onion 2321. From Matikkala and Virtanen (1967). Identity of the peaks is as follows: (1) Sugars. (2) Unknown. (3) Isomers of S-methyl-L-cysteine sulfoxide. (4) 7-Glutamyl-S(l-propeny1)cysteine sulfoxide. (4a) Unknown. ( 5 ) S-(2Carboxypropyl)-glutathione. (6) Cycloalliin. (7) Isomers of S-propyl-L-cysteine sulfoxide. (8) Isomers of L-methionine sulfoxide. (9) S-(l-Propenyl)-L-cysteine sulfoxide. (10) 76lutamyl-S-methylcysteine. (11) Aspartic acid. (12) 7-Glutarnylvaline. (13) Asparaghe. (14) Threonine. (15) Serine. (16) 7-Glutamylmethionine. (17) y-Glutamylisoleucine. (18) Glutamic acid. (19) S-(2-Carboxypropy1)cysteine. (20) Citrulline. (21) Unknown. (22) Proline. (23) S-Methylcysteine. (24) Glycine and unknown. (25) Alanine. (26) 7-Glutamylphenylalanine.(27) S-Allylcysteine. (28) Valine. (29) Pipecolic acid. (30) Methionine. (31) S-(1-Propeny1)cysteine (fruns or cis). (32) S-(1-Propeny1)cysteine (cis or trans). (33) Unknown. (34) Isoleucine. (35) Leucine. (36) Tyrosine. (37) PAlanine. (38) Phenylalanine. (39) 7-Aminobutyric acid. (40) Ethanolamine. (41) Ammonia. (42) Lysine. (43) Histidine. (44) Unknown. (45) Tryptophan (?). (46) Arginine. Reproduced by permission of the Scandinavian Chemical Society.
and by two-dimensional electrophoresis and chromatography (Granroth, 1968, 1970) have confirmed the initial observation. Identified organic sulfur compounds from onions are listed in Table I along with an indication of the amounts of the major compounds. It must be kept in mind that the amounts will vary with variety, maturity, and cultural and climatic conditions. For example, Freeman and Mossadeghi (1970) found that flavor strength, lachrymatory potency, total sulfur content, and dipropyl disulfide content of crushed onions
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
I1
showed a significant correlation with sulfate concentration of the nutrient medium. On the other hand, the relative proportions of the various sulfur compounds may not be greatly influenced by habitat, stage of growth, or plant part examined (Saghlr el al., 1965) and can probably be used for taxonomic classification of the Alliums. More limited studies have been done on the incorporation of 3 5 S from inorganic sulfate into organic compounds of chives (Granroth, 1970) and garlic (Suzuki et al., 1961a,b; Granroth, 1970). Suzuki and co-workers (Suzuki et aL, 1961a,b) found y-L-glutamyl methyl-L-cysteine, y-L-glutamyl methyl-L-cysteine sulfoxide, S-(2-carboxypropyl)glutathione, L-cysteine, L-methionine, allyl-Lcysteine sulfoxide, and methyl-L-cysteine sulfoxide together with many unidentified sulfur compounds. Figure 1 shows the amino acid-containing compounds of an ethanol extract of onions as determined on an amino acid analyzer (Matikkala and Virtanen, 1967). Note particularly the relatively large amounts of y-L-glutamyl-S-(lpropeny1)cysteine sulfoxide (peak 4) and S-(l-propenyl)L-cysteine sulfoxide (peak 9; see also Table I). A. ALKYLCYSTEINES
The nonsulfur-containing amino acids valine and serine are listed in Table I, as they will become important in our discussion of the biosynthesis of the sulfurcontaining compounds. L-Cysteine, L-cystine, and L-methionine ocCur in relatively low amounts in onions, indicating that they are not terminal metabolic products, as we shall see later. On the other hand, L-methionine sulfoxide does appear to be a terminal compound, since it does not seem to be involved in the formation of other sulfur-containing compounds (Granroth, 1970). In part, it may be formed from L-methionine during the isolation procedures. Low amounts of seven alkyl-, alkenyl-, and carboxyalkyl-L-cysteinesare found in onion. They apparently are intermediates in the biosynthesis of the corresponding sulfoxides, as injection of 35 S-labeled cysteine derivatives leads to rapid formation of the corresponding sulfoxides (Granroth, 1970). Substantial are found in onion. amounts of S-(2-~arboxypropyl)glutathione
B. ALKYLCYSTEINE SULFOXDES From the standpoint of flavor development in the Alliums the most interesting compounds in Table I are the four alkyl- and alkenyl-L-cysteine sulfoxides. 0
t
These sulfoxides are: (t)-S-methyl-L-cysteine sulfoxide [CH3-S-CH2 CH(NH2)COOH; methylalliin] ; (+)S-propyl-L-cysteine sulfoxide [CH3-CHz -
I8
JOHN R. WHITAKER
0 t CH2-S-CH2 -CH(NH2 )-COOH; dihydroalliin or propylalliin] ; trans-(t)-S-(l0 t propeny1)-L-cysteine sulfoxide [CH3-CH=CH-SXH2 -CH(NH2)-COOH] ;
P
and (t)S-allyl-L-cysteine sulfoxide [CHZ =CH-CH2-SXH2 -CH(NH2)COOH; alliin] . (tv-Allyl-L-cysteine sulfoxide was the first of these compounds isolated. In their fundamental work on the origin of the antibiotic action of garlic, Stoll and Seebeck (1947, 1948) isolated this compound from Allium sativum (garlic) and Allium ursinum. It occurs in amounts up to 2.4 g/kg in garlic (Stoll and Seebeck, 1951) and in low amounts in other Alliums including onion (Table I). The corresponding thiolether, S-allyl-L-cysteine, has also been isolated from garlic (Suzuki et al., 1961b). (t)S-Methyl-L-cysteine sulfoxide and (t)-S-propyl-L-cysteine sulfoxide were isolated from onion in 1959 (Virtanen and Matikkala, 1959) and in 1961 (Carson and Wong, 1961a). Methylcysteine sulfoxide appears to have the widest distribution of all the a w l sulfoxides (Fujiwara et al., 1958). It is probably found in all Allium species, and it occurs in several members of the Liliaceae and Cruciferae families as well as sporadically in the families Compositae, Umbelliferae, and Leguminosae. Propylcysteine sulfoxide occurs in rather low amounts; it appears to be restricted to a number of Allium species and to the closely related plant Brodiaea uniflora Engl. (synonymous Ipheion uniflorum Raf., 7Yiteleia uniflora, Distagma uniflora). trans-(t)S-( 1-Propeny1)-L-cysteinesulfoxide was isolated from onion in 1961 (Virtanen and Spare, 1961) during investigation of the lachrymatory properties of onions. This compound is the precursor of the tear-producing factor (lachrymatory factor) released from crushed onion. The amount of trans-(t)S-( l-propeny1)-L-cysteine sulfoxide is about 2 g/kg of onion (Matikkala and Virtanen, 1967), approximately equivalent to the allylcysteine sulfoxide content of garlic. The corresponding thiolether, S-(1-propenyl)-L-cysteine, has been isolated from garlic (Sugii et al., 1963b), but not the sulfoxide. The naturally occurring S-(1-propeny1)cysteine sulfoxide is a derivative of L-cysteine, is the (t)isomer with respect to the sulfoxide oxygen, and has the trans configuration at the double bond (Carson et al., 1966). (+)-S-Ethyl-L-cysteine sulfoxide does not appear to occur in the genus Allium, although it is found in the closely related plant Brodiaea uniflora Engl. (syn. Zpheion uniflorum) (Fujiwara et al., 1958) and possibly also in Tulbaghia violacia (Jacobsen et al., 1968). The report of Sethylcysteine sulfoxide and S-butylcysteine sulfoxide in garlic (H6rhammer et al., 1968) has not been confirmed.
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
79
The cyclic sulfoxide, cycloalliin (.5-methyl-l,4-thiazan-3-carboxylic acid 1oxide), and its homologs have been reported in AZZiurn as well as in other plants. There is good evidence that cycloalliin is an original substance of onion (Virtanen and Matikkala, 1961), although it may be formed to some extent by the alkaline conditions used in chromatographic separation of the amino acid constituents (Ettala and Virtanen, 1962). President evidence indicates that it is one of the three major sulfur-containing compounds in onions, being present to an extent of 0.2% on a fresh-weight basis (Table I). It would seem that this point merits more research work, particularly as to effect of variety, maturity, and cultural conditions, since this derivative does not contribute to flavor, odor, or pungency. Cycloalliin was first isolated in its reduced thiolether form (Virtanen and Matikkala, 1956). Norcycloalliin (1,4-thiazan-3-carboxylicacid 1-oxide) is not present in Allium species but has been isolated from a red algae (Chondria crussicuulis) (Kuriyama et ul., 1960) and a brown algae (Unduria pinnatifida) (Tominaga and Oka, 1963). The higher homolog of cycloalliin, Sethyl-l,4thiazan-3-carboxylic acid 1-oxide, has not been found in nature; it is slowly formed in alkaline solution from synthetic trams-(1-buteny1)cysteinesulfoxide (Carson et uZ., 1969). In vitru, alkaline conditions are sufficient to convert trans-(+)-S-(l-propenyl) L-cysteine sulfoxide to cycloalliin (Virtanen and Spfire, 1961;Carson and Boggs, 1966) [and S-vinylcysteine sulfoxide to norcycloalliin (Dgbritz and Virtanen, 19691, as shown in Fq. (1). Carson and Boggs (1966) obtained 29% cycloalliin sulfoxide after 10 days in 1 N N H 4 0 H at from cis-(+)S(l-propenyl)-L-cysteine room temperature, and Schwimmer (1969) found 28% of the sulfoxide remaining after 10 hours in 1 N NaOH. This does not appear to be the pathway of biosynthesis as discussed below, however, since I4C-labeled truns-(t)-S-(l-propeny1)L-cysteine sulfoxide injected into an onion bulb produced no labeled cycloalliin (Milller and Virtanen, 1965). The structure of cycloalliin has been established by x-ray crystallography (Palmer and Lee, 1966). 0
t
HC II / ~ \ F H , H,C/i
N/;\COOH H2
-
trans-(+)- S- (1-propenyl)L-
cy steine sulfoxide
OH-
0
t
H p ? ,
(1)
H,C+N+COOH H Cycloalliin
C. y-GLUTAMYL PEPTIDES At least fourteen y-glutamyl peptides have been isolated from onion, of which nine contain sulfur (Table I). The other five peptides are y-glutamyl derivatives of valine, isoleucine, leucine, phenylalanine, and tyrosine (Virtanen, 1969). In
JOHN R. WHITAKER
80
TABLE I1 7-GLUTAMYL PEPTIDES ISOLATED FROM ONION, GARLIC, AND C H I V P
1. HOOC-CH~H, )+cH,
)
x
-+
COOH CH, ~ ~
~
~
CH3 y-glutamylvaline (onion)
2. HOOC
HCH,), 4 0 - N H
y-Glu-isoleucine (onion, chive) COOH CH, 3. HOOCXH(NH, HCH, ) , X O - N H ~ H - C H , ~ H LH3 y-Glu-leucine (onion) COOH 4. HOOC-CH(NH, H C H , ), 4 0 - N H J H - C H , - C H , -S-CH3 y-Glu-methionine(onion, chive)
5. HOOC-CH(NH, )-(CH,
),-CO-NH
y-GluS-methylcysteine (onion, chive)
6 . HOOC-CH(NH,)-(CH,),-CO-NH y-GluS-methylcysteine sulfoxide (onion)
7. HOOC-CH(NH,)+CH,),-CO-NH
XH,
yGluSpropylcysteine (garlic, chive)
8. HOOC-CH(NH, )-(CH,),-CO-NH
-CH=CH,
yG1u-S-allylcysteine (garlic, chive)
9. HOOC-CH(NH, )+CH,
),
4O-NH
yGluS-( 1-propeny1)cysteine (chive)
-S-CH=CH
d
~
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
81
TABLE I1 (continued)
10. HOOC-CH(NH, H C H , ), -CO-NH
ry
H-CH, -S-CH=CH-CH,
y-Glu-S-(I-propeny1)cysteine sulfoxide (onion, chive) -S-CH=CH-CH,
11. HOOC-CH(NH, )-(CH, ), -CO-NH I
0
NH
H-CH, JOOH
-LCH=CHXH,
y-GluS-( 1-propeny1)cysteinyl-S-(1-propeny1)cysteinesulfoxide (chive)
12. HOOC-CH(NH, )-(cH,
)2
COOH - C O - N H ~ H- CH, C, H,
y-glutamylphenylalanine (onion) COOH 13. HOOC-CH(NH,)+CH,
Q 4~
),xo-NH-LH-cH,-
y-glutamyltyrosine (onion) COOH 14. HOOC-CH(NH, HCH, ),-CO-NH-LH-CH,-SH y-glutamylcysteine (chive) COOH 15. HOOC-CH(NH, HCH,
1,-
),-~o-NH
CH(NH, )--COOH N,K-bis(y-glutamy1)cysteine (chive) FOOH 16. HOOC-CH(NH, H C H , )2-C0-NH-CH-CHz-S-CH2 (CH,),-CH(NH, )-COOH N~-bis(y-glutamy1)-3,3'-(2-methylethylene-l,Zdithio)dialanine (chive)
H, S-S-CH,
17. HOOC-CH(NH, )-(CH,),-CO-NH
TH-NH-CO-(CH, ),-€H(NH, CO-NH-CH, X O O H 0-NH-CH, -COOH
WOOH
glutathione (onion)
(continued)
82
JOHN R. WHITAKER TABLE I1 (continued)
18. HOOC-CH(NH, )-(CH),-CO-NH
~-glutamyl-S-(2-carboxy-1-propyl)cysteinylglycine (onion) H2-S-S-CH,4H(NH, 19. HOOC-CH(NH, )-(CH,
),
NOOH
40-NH CO-NH-CH, 4 O O H
glutathione cysteine disulfide (onion)
20. HOOC-CH(NH, &(CH,),-CO-NH-
CH, -S-S-CH,-CH-NH-CO-(CH, ),,I CH LOOH CH(NH, W O O H
LO--NH-CH,-COOH
glutathioney-glutamylcysteine disulfide (omon) CH,-S-SO,
H
21. HOOC-CH(NH, H C H , ) , 4 0 - N H 0-NH-CH, 4 O O H S-sulfoglutathione(onion)
=From Virtanen (1969).
addition to these peptides from onions, y-L-glutamyl-S-(1-propeny1)-L-cysteine, 7-1,-glutam ylS-propyl-L-cysteke, y-~-glutamyl-S-allyI-~ -cysteine, y-L-glutamylS-( 1-propenyl)-L-cysteinyl-S-(l-propenyl)-L-cysteine sulfoxide, T-L -glutamyl-~ -
cysteine, N,N‘-bis(y-L-glutamyl)-L-cysteine, and N,N’-bis-(y-~-glutamyl)-3,3’-(2 methylethylene-1 ,2-dithio)dialanine have been isolated from chives (Virtanen, 1969). Garlic has been reported to contain y-L-glutamyl-S-propylcysteine and y-L-glutamyl-S-allylcysteine(Virtanen, 1969). A quantitative method for determination of C-S-lyase-susceptibley-glutamyl S-substituted cysteines (and sulfoxides) in onion and in garlic has been reported (Schwimmer and Austin, 1971b). The structures of some of these more complex peptides are shown in Table 11. Y’L-Glutamyl-trmzs-(+)S-(1-propeny1)-L-cysteinesulfoxide, found in about 0.2% in the onion (Table I), is the most important of these compounds as far as potential flavor precursors are concerned. The significance of the y-glutamyl peptides in the nitrogen metabolism of these plants is not clear, although they apparently serve as a storage reserve. The peptides disappear from sprouting onion bulbs when green leaves appear from the bulb (Mattikkala and Virtanen, 1965b). In summary, at least twenty-six sulfur-containing compounds have been iso-
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
83
lated from intact onion (Allium cepa). Eight of these account for more than 90% of the total sulfur of the onion (compounds 14 through 21 and compound 23 of Table I) (Ettala and Virtanen, 1962). trans-(+)-S-(1-Propenyl)-L-cysteine sulfoxide , y-L-glutamyl-trans-(t)-S-(1-propeny1)-L -cysteine sulfoxide , and cy cl oalliin account for approximately 75% of this total; each is present in about 0.2% of the wet weight of the onion. A typical onion contains about 11% soluble carbohydrate (Pant et al., 1962), on a comparative basis.
111.
BIOSYNTHESIS OF SULFUR COMPOUNDS OF ALLIUMS
Studies on the biosynthesis of the sulfur compounds in Allium are rather limited. Suzuki and co-workers (Suzuki et al., 1962; Sugii et al., 1963a, 1964) found that I4C from uniformly labeled valine injected into garlic bulbs was incorporated into S-(2-~arboxypropyl)-~-cysteineand S-(2-carboxypropyl)glutathione. On this basis, they suggested that these compounds are synthesized from methacrylic acid and cysteine or glutathione, respectively (Suzuki et al., 1962). Following administration of 35 S-methionine into garlic bulbs, 35 S was found in methylcysteine and methylcysteine sulfoxide, suggesting that a thiomethyl transfer mechanism (see below) is involved in their biosynthesis (Sugii et al., 1963a). An extensive investigation of the biosynthesis of sulfur compounds in onion (Allium cepa), garlic (Allium sativum), and chives (Allium schoenoprasurn) has been reported by Granroth (1970), although it has left many questions unanswered. The following information on biosynthesis is from that report unless otherwise indicated. In most cases the tips of green leaves were used because of their vigorous uptake of labeled sulfate, although in some cases slices of bulb tissue were used with the same results. On the basis of this evidence, Granroth proposed the pathway in Fig. 2 leading to trans-(+)$-( 1-propeny1)-L-cysteine sulfoxide (trans-PrenCySO), (+)-S-methyl-L-cysteine sulfoxide (MeCySO), (+)-Spropyl-L-cysteine sulfoxide (PrCySO), and (+)-S-allyl-L-cysteine sulfoxide (AllCySO). The most surprising thing about the proposed pathway is that tmns-(+)S-(l-propeny1)-L-cysteine sulfoxide arises from cysteine, while the other three sulfoxides arise from serine. When "S-labeled sulfate is assimilated by roots of sprouting onion, there is synthesis of various low-molecular-weight sulfur compounds (Granroth, 1970). After 3 days about 56% of the inorganic sulfate was assimilated and 75% of the label showed up in trans-(+)&( 1-propeny1)-L-cysteine sulfoxide, with some labeled cycloalliin (-4%), (+)-S-methyl-L-cysteine sulfoxide 0 2 % ) , S-(2carboxypropy1)-L-cysteine,methionine, and S-methylmethionine sulfonium ion. The same general pattern was found when green leaves of onion were used. With
JOHN R. WHITAKER
a4
-so;-
so;-
Cystathimine
~r j2cH3 +AIISH
CPG-C.
Met hionine
+
i
I
y-Glutamdtrans- PrenCys - - m - P r e n C y s
i
1
Y-Glutamyltrans-PrenCy S O - m - P r e n C y
SO
1 I 1
MeCySO
PrCySO AIICySO
FIG. 2. Metabolic relationships of some sulfur compounds in Allium species (Granroth, 1970). GSH, glutathione; CPG, 4(2-carboxypropyl)glutathione; CPC, 9(2-~arboxypropyl)L-cysteine; MMS, S-methylmethionine sulfonium ion. Reproduced by permission of the Finnish Academy of Sciences.
garlic the pattern was similar, with trans-(+)-,!-( 1-propeny1)L-cysteine sulfoxide being labeled in the greatest amount. (t)S-Methyl-L-cysteine sulfoxide was higher in garlic tissue and was about one-half that of trans-(+)-,!-( 1-propeny1)-Lcysteine sulfoxide. No labeled (t)-S-allyl-L-cysteine sulfoxide or cycloalliin was found. Therefore, one may conclude from these studies that sulfate is reduced in roots, bulbs, and leaves of sprouting onion and garlic. Many short-lived intermediate sulfur compounds are synthesized which do not accumulate but react further to give a large amount of trans-(+),!-( 1-propeny1)-L-cysteinesulfoxide and smaller amounts of (t)-S-methyl-L-cysteine sulfoxide. No rapid formation of (t)-S-allyl-L-cysteine sulfoxide is found, even in garlic (most surprising). Cycloalliin is not formed in developing green leaves, although small amounts are formed in bulbs. Let us look at each of the steps involved in Fig. 2, at least in summary fashion.
FLAVOR, ODOR,AND PUNGENCY IN ONION AND GARLIC
85
A. CYSTEINE The synthesis of cysteine in higher plants (Thompson, 1967) from sulfide and serine is experimentally well documented and probably is also the same for yeast, bacteria, mold, and some animal tissues. Inorganic sulfate is reduced to sulfide by an enzyme in the tissue (Eq. 2): so$- --Ky-so:+2 H
7
+6 -H
2
-
3 H,O
H2O
A sulfite reductase [hydrogen sulfide :(acceptor) oxidoreductase; EC 1.8.99.11 has been purified from Allium odorum by Tamura (1965). The enzyme reduces sulfite to sulfide. Whether a separate enzyme is involved in reduction of S042to S032-is not known. More attention to this step(s) in Allium is needed. Cysteine is then probably synthesized in the following way from serine and sulfide (Eqs. 3 and 4): AcetylCoA + serine + 0-acetylserine + CoA
0-Acetylserine + HS-
-t
L-cysteine + acetate
(3) (4)
The enzymes catalyzing these reactions have been isolated in pure form from Salmonella and been shown to exist partly as a complex of weight 309,000 (Kredich et al., 1969). Pyridoxal phosphate is the activating cofactor of this enzyme system. The enzyme serine acetyltransferase (acetyl-CoA:L-serine 0acetyltransferase; EC 2.3.1.30) catalyzing the reaction in Eq. (3) has been found in extracts of a number of higher plants (Smith and Thompson, 1969), while the enzyme cysteine synthase [0-acetyl-L-serine acetate-lyase (adding hydrogen sulfide); EC 4.2.99.8; also called 0-acetylserine sulkydrylase J catalyzing reaction (4) has been found in spinach leaf (Giovanelli and Mudd, 1968), turnip leaf (Thompson and Moore, 1968), and onion (Granroth, 1974). Granroth (1974) recently reported the partial purification of the onion enzyme which is present in low amounts compared to the amount found in Salmonella (Kredich et al., 1969).
B. ALKYLCYSTEINES AND ALKYLCYSTEINE SULFOXIDES It is proposed (Granroth, 1970) that S-methylcysteine may arise via three routes-from the incorporation of a methyl group into cysteine (Eq. 5), from the reaction of serine with methionine via a thiomethyl transfer (Eq. 6), and from a transulfuration via cystathionine (Eq. 7).
JOHN R. WHITAKER
86
[Methyl-'*C] methionine
'
L-Cysteine
2
-
Homocysteine
[14CJS-methyl- L- cysteine
The reaction in Eq. ( 5 ) is well known in plant and animal tissue. It is necessary to postulate the formation of S-methyl-L-cysteinefrom free cysteine in order to explain the relatively large amounts of S-methyl-L-cysteine formed when [35 S] S042- or [35 S] L-cysteine is fed to A12ium. rS5S1 Methionine
-
Homoserine
\spss*
(6) :[ 3SS]S-methyl-~-cysteine
L- Serine
When [35 S] methionine is fed to leaves of onions, garlic, and chives, as well as onion slices, S-methyl-L-cysteine, S-methylmethionine sulfonium ion, (+)-Smethyl-L-cysteine sulfoxide, and truns-(t)-s-( 1-propenyl)-L-cysteine sulfoxide are rapidly labeled with 35 S, while there is no activity in (+)-S-propyl-L-cysteine sulfoxide or (t)-S-allyl-L-cysteine sulfoxide. Incorporation of 35 S into the first three products above requires a thiomethyl transfer as shown in Eq. (6); 14C was incorporated into these products when [14C] serine was used. In contradiction to the proposed pathway in Eq. (6), in radish leaves doubly labeled [14CH335 S] methionine showed separate transfer of the two labels (Thompson and Gering, 1966). Thompson and Gering (1966) concluded that S-methylcysteine is formed in radishes by methylation of cysteine. Equation (6) does not explain the incorporation of 3s S into trans-(t)-S-( l-propeny1)-L-cysteine sulfoxide, however, when [ 35 S] methionine is fed. It seems necessary (Granroth, 1970), therefore, to postulate the following transulfuration reaction (Eq. 7). In Eq. (7), cysteine is formed from cystathionine by a 7-cleavage, a reaction reported not to occur in higher plants. Only the &cleavage of cystathionine, which gives homocysteine, pyruvate, and ammonia, has been demonstrated in higher plants (Giovanelli and Mudd, 1971). Obviously, more research on this point of major importance is needed. Methionine
- CH,
[%I
Homocysteine
["Sl Cysteine
+c/ fS5S]S-methyl- L- cysteine
+
serine
NH,
[%] Cystathionine
I
t + a-Ketobutyrate
methacrylate
\
[s3S]trans - (+) - S-(1-propenyl)~ - c y s t e i n esuIfoxide
(7)
FLAVOR, ODOR,AND PUNGENCY IN ONION AND GARLIC
81
Even though the biosynthesis of S-methyl-L-cysteine from methionine has been demonstrated in Allium via Eqs. (6) and (7), it appears that under normal circumstances the route via Eq. (5) is more important for t h s compound. Methionine sulfoxide, S-methylmethionine sulfonium ion, S-adenosylmethionine, and S-methylthioadenosine were found not to serve as methyl or thiomethyl donors in these reactions (Eqs. 5-7). Enzymatic splitting of S-methylmethionine sulfonium ion to dimethyl sulfide and homoserine has been demonstrated in cabbage leaf (Lewis et ul., 1971) and with certain bacteria (Mazelis et ul., 1965). While the same enzymatic activity, termed "methionine sulfonium lyase" (Mazelis et ul., 1965), has been found in low amounts in onion seedlings and dormant onion bulbs Wattula and Granroth, 1974), it does not appear to be of primary importance in onion flavor development or in S-methylmethionine metabolism in onions. The available evidence (Granroth, 1970, 1974) indicates that S-propyl-Lcysteine and S-allyl-L-cysteineare synthesized via Eqs. (8) and (9). L-Serine + propyl mercaptan L-Serine + ally1 mercaptan
+ S-propyl-L-cysteine + S-allyl-L-cysteine
The evidence for this is as follows: (1) No radioactivity was incorporated into the isolated (+)-S-propyl-L-cysteine sulfoxide or (+)S-allyl-L -cysteine sulfoxide when ['4C]cysteine was fed, thus ruling out alkyl or alkenyl transfer to cysteine. (2) Radioactivity was incorporated into the isolated sulfoxides when [I4C] serine was fed. (3) Feeding of several mercaptans (methyl, ethyl, propyl, allyl, benzyl) along with [14C]serine to Allium species led to isolation of the corresponding labeled alkyl- or alkenylcysteine sulfoxides. While these alkylcysteines can be synthesized by this route in plants (Giovanelli and Mudd, 1968), it may not be the major route, since methylcysteine appears to be synthesized in radishes from transfer of a methyl group of methionine to cysteine (Eq. 5). There is no information to indicate the source of the propyl and allyl groups (as mercaptans?) in vivo. Feeding of [35 S] S-propylmethionine sulfonium ion did not result in labeled (+)-S-propyl-L-cysteine sulfoxide. Feeding of [2-14C] acetate gave no labeling of (+)-S-allyl-L-cysteine sulfoxide in garlic. Further work is needed in which [35 S ] alkyl mercaptans are fed. The work with [ 14C]cysteine is complicated, since cysteine can go to cystine. The propenyl residue of truns-(+)S-( 1-propenyl)-L-cysteine sulfoxide clearly comes from valine, probably via the mechanism shown in Fig. 3. It is well known that methacrylyl-CoA is an intermediate in the catabolism of valine, and when ['4C]valine or ['4C]cu-ketoisovaleric acid is fed t o Allium species there is rapid labeling of trans(+)-S-(1 -propenyl)-L-cysteine sulfoxide. It is not known whether methacrylyl-CoA or methacrylic acid couples with cysteine to give S-(2carboxypropy1)-L-cysteine. trans-S-(1-Propeny1)-L-cysteine is formed from S-(2-~arboxypropyl)-~-cysteine via an oxidative decarboxylation reaction. The
JOHN R. WHITAKER
88
Isobutyryl-CoA
Methacrylyl-CoA +C~ASH *CH3-C-"CH2
I
Methacrylic acid Cysteine
*cH,-cH-~cH,-
I
COOH
S-CH~CH-COOH
I
I
NH2
t-"l
*CH,-CH =*CH - S - C H ~ ~ H - C O O H
CPC
trans -PrenCys
\ -
*C H c H -*c H- S- C H ~ C H co OH I
m-(+)-PrenCySO
NHz
FIG. 3. Proposed pathway of biosynthesis of trans-(+)S(l-propeny1)-L-cysteine sulfoxide(trans-(+>PrenCySO) from valine and cysteine. From Granroth (1970). *, *.C label; CPC, S-(2-carboxy-propyl)-L-cysteine;trans-PrenCyS, trans-&( 1-propeny1)-L-cysteine.
nature of the one-carbon fragment released is not known, but it is probably C02. Neither is anything known about the enzyme involved. The alkyl- and alkenylcysteines are rapidly oxidized to the corresponding sulfoxides via an oxidizing enzyme in Allium species as may be shown by feeding the various alkyl- and alkenylcysteines to these species. The role of S-(2-~arboxypropyl)glutathionein the biosynthesis of trans-(+)-S(I-propeny1)-L-cysteine sulfoxide is not clear at the present time, although some suggested reactions are shown in Fig. 2. The origin and role of the other carboxyalkylcysteine derivatives found in Allium species (Table I) are not known. The pathway of biosynthesis of cycloalliin in onion is not clear. in vitro in alkaline solution, trans-(+)S-(1-propeny1)-L-cysteine sulfoxide is cyclized to cycloalliin (Eq. 1). However, when the 14C-labeled sulfoxide is fed to onion,
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
89
there is no incorporation of label into cycloalliin (Mliller and Virtanen, 1965). There is some evidence that S-(2-~arboxypropyl)-~-cysteine is the precursor of cycloalliin (Granroth and Virtanen, 1967). When radioactive S(2-carboxypropy1)-L-cysteine was injected into onion bulb, much of the radioactivity appeared in cycloalliin after 1 day. With the corresponding sulfoxide, appreciable incorporation occurred only after 7 days. It appears from these studies as well as from others that the alkylcysteine sulfoxides, including cycloalliin, are metabolically rather inert; the sulfoxides appear not to be active members of a main metabolic chain, but rather are end products, storage products, or waste products of the Allium species. As such they occur in large amounts in Allium species. The sulfoxides might possibly perform a protective role in the plant. When the tissue is bruised or otherwise damaged, the sulfoxides and enzymes are permitted to interact to form thiolsulfinates which are fungistatic and bacteriostatic. A proposed biosynthetic pathway for cycloalliin (Granroth and Virtanen, 1967) is shown in Eq. (10) starting with S-(2-~arboxypropyl)-~-cysteine synthesized as shown in Fig. 3.
H,c~'\~H, H,C\'
-
HOOCc, H N /c, H COOH
-
co,
HCI'\~H~
II
H,&
N+COOH H,
H2
S- (2- carboxypropy1)~-cysteine
-
H,CH'\~H,
I
H,C+N+COOH H
-
trans S - (1-propenyl)-
Thio le the r of cycloalliin
~-cysteine
/ 0 /LO1
t
Cycloalliin
In apparent contradiction to this proposal, Granroth (1970) found very little incorporation of ["S] SO4'- into cycloalliin, particularly with growing leaves of Allium species. Therc was somewhat more label incorporation into cycloalliin in growing bulb slices (-4%) as compared with 76% label incorporation into truns-(+)-S-(1-propeny1)-L-cysteine sulfoxide. Wilting leaves of Allium species incorporated more label into cycloalliin. Could it be that more cycloalhin is formed in dormant tissue, resulting in the high amounts reported in Table I? Clearly, more information is needed on this point, since, once cycloalliin is formed, there is no evidence that it can be converted to flavor in the crushed onion.
90
JOHN R. WHITAKER
C. y-GLUTAMYL PEPTIDES
The y-glutamyl peptides are undoubtedly formed through transpeptidation reactions involving the various alkylcysteines and alkylcysteine sulfoxides. In the growing tissue the level of these y-glutamyl peptides is low, indicating that they can be mobilized, probably via y-glutamyl peptidase (see below). Whether the level of y-glutamyl peptides in dormant bulbs is determined by the relative levels of y-glutamyl residues and amino acids and amino acid derivatives present is not known. Since approximately 5% of the important flavor precursor, trans-(t)-S(1-propeny1)-L-cysteine sulfoxide, is present as the y-glutamyl peptide in onion, it is essential to have more information about these reactions and how they are controlled in vivo. Onion leaves and sprouting bulbs, but not dormant bulbs, contain a hydrolase which hydrolyzes y-glutamyl peptides (Matikkala and Virtanen, 1965b; Austin and Schwimmer, 1971). y-Glutamyl transpeptidase [y-glutamyltransferase; (y-glutamy1)-peptide:amino acid y-glutamyltransferase; EC 2.3.2.21 occurs in many plants including sprouted onion bulbs (Schwimmer and Austin, 1971a). A y-glutamyl peptidase has been partially purified from germinating seeds of chives (Allium schoenoprasum) (Matikkala and Virtanen, 1965a). We shall have more to say about these enzymes later.
IV. SULFUR CONSTITUENTS OF CRUSHEDALLIUM Intact samples of leaves and bulbs of the AZZium species have no distinctive flavor or odor characteristics, nor does intact onion have lachrymatory properties. Immediately on cutting or crushing a sample, however, there is a rapid development of the flavor and odor characteristics of each species and a development of the lachrymatory factor in onion. This has been known for hundreds of years. Just when it was recognized that the strong-smelling, volatile oils from the different species of Allium are formed as a result of an initial enzymatic cleavage of nonvolatile precursors is not clear. Wertheim (1844, 1845) and later Semmler (1892) isolated some volatile constituents from steamdistilled garlic oil including mainly diallyl disulfide and smaller amounts of diallyl trisulfide and diallyl polysulfide. Rundqvist (1909) was the first to attempt to isolate the basic principle in garlic which gives rise to diallyl disulfide when garlic is crushed. His isolated product contained considerable amounts of carbohydrate, leading him to the conclusion that the basic principle was a glycoside. This he named “alliin,” a name that has remained. The continuing interest in isolating the basic principle in garlic was due more to the medicinal properties of garlic than to its flavoring properties in foods. In
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
91
1944, Cavallito and Bailey (1944) isolated a compound from crushed garlic by aqueous ethanol extraction followed by steam distillation. This compound possessed strong antibacterial action against numerous microorganisms. Cavallito et al. (1944) showed the compound, which they named allicin, to have the following structure. 0 CH, = C H - C H ~ - & - S ~ H , - C H = C H , diallyl thiosulfinate (allicin)
Ln some classic work summarized in a review (Stoll and Seebeck, 1951), Stoll and Seebeck investigated the conversion of alliin to allicin and studied a number of the properties of the enzyme involved in this conversion. They named the enzyme alliinase. Various other names have been used for the enzymes in Allium which are involved in this conversion. These names include alkylcysteine sulfoxide lyase, cysteine sulfoxide lyase, and C-S-lyase. According to the Commission on Enzymes its specific name is alliin alkyl-sulfenate-lyase (EC 4.4.1.4) with a trivial name of alliin lyase or alliiiase. The official name is a misnomer in at least two respects. Alliin is only one of several natural substrates of the enzyme, and a sulfenic acid does not appear to be the initial product of action of the enzyme on (t)-S-allyl-L-cysteine sulfoxide. This enzyme, which we shall call alliinase, will be described more fully later. A. INITIAL COMPOUNDS FORMED FROM ENZYMATIC ACTIVITY The key compounds in the intact Allium which serve as precursors of antibacterial and flavor and odor compounds are the alkyl- and alkenylcysteine sulfoxides (Table I). In garlic, the major compound is (t)-S-allyl-L-cysteine sulfoxide, with smaller amounts of (+)-S-methyl-L-cysteine sulfoxide and (+)-S-propyI-Lcysteine sulfoxide. in onion, the major compound is trans-(t)S-( 1-propenyl)-Lcysteine sulfoxide, with smaller amounts of (t)-S-methyl-L-cysteine sulfoxide and (t)S-propyl-L-cysteine sulfoxide (Table I). The enzymatic conversion of (t)-S-allyl-L-cysteine sulfoxide (alliin) to diallyl thiosulfinate (allicin), pyruvic acid, and ammonia as suggested by Stoll and Seebeck (1951) is shown in Fig. 4. The proposed intermediate products are allylsulfenic acid and aminoacrylic acid. Careful examination of this reaction by mass spectral analysis did not confirm allylsulfenic acid as an intermediate in the enzymatic conversion of (t)S-allyl-L-cysteine sulfoxide to diallyl thiosulfinate (Virtanen, 1964). An alternative mechanism shown in Fig. 5 has been proposed (Dlibritz and Virtanen, 1965) which does not involve allylsulfenic acid as an intermediate. This proposal involves a bimolecular reaction between two mole-
JOHN R. WHITAKER
92
2 I
CH
1
-
7%
CH, II CH
- H E 01
CH, I1 CH I
7th
7%
OtS-S
H-S+O
CH, I
Allylsulfenic acid
s+o
2 ~
Allicin
I
7%
7%
H,N-CH I COOH
EYN-C I COOH
AllCySO
+2Hc?-
Aminoocrylic acid
2C-0+2NH3 I COOH Pyruvic acid
FIG. 4. A proposed mechanism for the action of alliinase on (+)-S-aUylcysteine sulfoxide (AUCySO). From StolI and Seebeck (1951).
cules of (+)-S-allyl-L-cysteine sulfoxide leading to diallyl thiosulfinate and aminoacrylic acid (which spontaneously hydrolyzes to ammonia and pyruvic acid). The reactions involving (+)S-methyl-L-cysteine sulfoxide and (+)-S-propyl-Lcysteine sulfoxide appear t o follow a course identical to that for (+)S-dylL-cysteine sulfoxide. The initial products formed are methyl methanethiosulfinate and propyl propanethiosulfmate. Interestingly, the mixed alkyl and alkenyl thiosulfinates have also been reported (Table 111) (Fujiwara et aL. 1955). The mixed thiosulfinates can readily arise from the action of catalytic amounts of thiol compounds on the thiosulfinates as demonstrated for methyl methanethiosulfmate and propyl propanethiosulfmate and cysteine in Eqs. (1 1) and (12). 0 CH,-
0
A-S-CH,
f
HS-Cys
t
CH,-S-S-CyS
+
CH,-SH
(11)
0 Cb-SH
f
t
C~CH~-CH,-S-S-CH,CH,CH,
P CH,-CH,-
I
CH,-S-S-CH,
Il
(12)
. .
+
CH3-CH,-CH,-SH
The failure to detect allyl-, propyl-, and methylsulfenic acid intermediates (Dgbritz and Virtanen, 1965) does not necessarily rule out their being the first product formed by alliinase. Unlike 1-propenylsulfenic acid, which can be stabilized by isomerization to thiopropanal S-oxide, they may rapidly dimerize to the respective thiosulfinates.
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
93
FIG. 5. An alternative proposed mechanism for the action of alliinase on (+)a-allylcysteine sulfoxide. From Dabritz and Virtanen (1965).
The thiosulfinates are rather unstable compounds. They have not been shown to be present by gas chromatographic procedures. Rather, one takes advantage of their reactivity with thiols (Cavallito e l aL, 1944) to form stable compounds which may be isolated (Eq. 13). Fujiwara et a/. (1955) have used this reaction to determine the nature of the initial products following action of alliinase. These workers report the presence of mixed thiosulfinates in Allium species. They (Fujiwara e l al., 1955, 1958) also showed that diallyl thiosulfinate reacts with the sulfur of thiamine to give allihamine. Thls reaction may be of importance when animals eat food containing Alliurn species. Allithiamine possesses vitamin B1 activity but is not susceptible to the action of thiaminase (thiamine hydrolase; EC 3.5.99.2) present in raw fish, which is part of the Japanese diet. In Japan, vitamin formulations contain allithiamine instead of thiamine. More recently, Lukes (1971) has used this reaction to study the formation of these compounds as well as the lachrymatory factor in onions. The reaction between cysteine and thiosulfinates or 1-propenylsulfenic acid occurs over the range of pH 2 to 7 (Lukes, 1971). The reaction is faster, the higher the pH; however, this is offset by instability of 1-propenylsulfenic acid and cysteine (oxidation to cystine) at the higher pH values. 2 HS-CH, -CH(NH,)-COOH cysteine
+ R-S-
x
-R’
thiosumnate
-+
R-S-S-CH,CH(NH,)-COOH R‘-S-S-CH,
+
(NH, )-COOH + H,O
(13)
The action of alliinase on trans-(+)-S-( 1-propeny1)-L-cysteine sulfoxide, the major precursor in onion, leads to the formation of 1-propenylsulfenic acid (or thiopropanal S-oxide; see below), pyruvic acid, and ammonia, as shown in Eq. (14). Evidence that trans-(+)-S(1-propeny1)L-cysteine sulfoxide is the precursor of
TABLE 111 COMPONENTS OF CRUSHED GARLIC (ALLIUM SATIVUM) Compound
Compositiona
b 1. 2. 3. 4.
AUyl alcohok Methyl mercaptan” Dimethyl sulfide” Allyl methyl sulfide (tentative)” 5. DiaUyl sulfidehpi
c
d
Reference
e
5.4
Bernhard etal. (1964); Brodnitz et af. (1971a) Oaks et al. (1964) Oaks et al. (1964); Schultz and Mohrmann (1965)
P
Oaks et al. (1964)
6. Dimethyl disulfide”.’
<1
3
3
4
1
3
7. AUyl methyl disulfid&ph#i
1.2
31
22
33
8. Diallyl disulfidC h#i
5.7
55
74
61
3 <1 5
<1 <1 <1
<1
9. 10. 11. 12.
Methyl propyl disulfide Dipropyl disulfide Allyl propyl disulfide Dimethyl trisulfid&~h~i
f
2.4
<1 <1
Wertheim (1844, 1845); Semmler (1892); Oaks el al. (1964); Saghir et al. (1964); Bemhard et al. (1964); Schultz and Mohrmann (1965) Oaks et al. (1964); Saghir et al. (1964); Schultz and Mohrmann (1965) Oaks et al. (1964); Saghir et al. (1964); Brodnitz et al. (1971a) Wertheim (1844, 1845); Semmler (1892); Oaks et al. (1964); Saghir ef al. (1964); Schultz and Mohrmann (1965); Brodnitz et al. (1971a) Saghir et al. (1964) Saghir er al. (1964) Saghir et al. (1964) Oakset al. (1964); Brodnitz etal. (1971a)
e
H
Methyl 1-propyl trisulfideh Allyl methyl trisulfid&*h*i Diallyl trisulfid&li Diallyl thiosulfinatd (allicin) 17. Methyl methanethiosulfmate
13. 14. 15. 16.
1.5
1.0 t t t k
i
18. Propyl propanethiosulfmate
i
19. 1-Propyl methanethiosulfmate 20. Allyl methanethiosulfinate 21. Propyl 2-propenethiosulfinate
i
++ ++
Oaks ef al. (1964) Oaks ef al. (1964); Brodnitz et al. (1971a) Oaks el al. (1964); Brodnitz ef al. (1971a) Cavallito and Bailey (1944); Fujiwara ef al. (1955) Matsukawa et al. (1953); Fujiwara ef al. (1955) Matsukawa ef al. (1953); Fujiwara ef al. (1955) Fujiwara et al. (1955) Fujiwara ef al. (1955) Fujiwara el al. (1955)
aBased on percentage total flavor components. bBrodnitz e l al. (1971a). See footnoteg. 'Great headed garlic (A. ampeloprasum L.). Saghir el al. (1964). From head space above freshly chopped sample. d"California Early" garlic. Saghir ef al. (1964). From head space above freshly chopped sample. e"California Late" garlic. Saghir ef al. (1964). From head space above fully chopped sample. fFujiwara et al. (1955). BDomestic garlic of unknown origin extracted with trichlorofluoromethane (Brodnitz et al., 197 la). Seventy-nine percent of the total peak areas was due to two compounds tentatively identified as 3-vinyl-l,2-dithid-ene and 3-vinyl-l,2dithi4ene postulated to arise from dehydration of diallyl thiosulfmate. hComponents from garlic head space (Oaks ef al., 1964). Seven gas chromatographic peaks not identified. !Components from garlic hexane extract (Oaks ef al., 1964). Three gas chromatographic peaks not identified. JA major flavor component of fresh garlic. The workers (Cavallito and Bailey, 1944) isolated 1.5 g/kg of garlic. However, the thiosulfmates are quite upstable and give rise to the secondary products-sulfides, disulfides, and trisulfides.
?i
r
> c
0
P
0
b
0
P
JOHN R. WHITAKER
96
truns-(+)-S-(l-
propeny I-) - L - cy steine sulfoxide
1 -Propenylsulfenic acid
or thiopropanal S-oxide
the lachrymatory factor, 1-propenylsulfenic acid (or thiopropanal S-oxide; see below), has been carefully examined over the years (Virtanen and Spare, 1961; Spare and Virtanen, 1961; Moisio et al., 1962; Carson and Wong, 1963; Carson and Bogs, 1966; Schwimmer, 1969). Recently, Schwimmer (1968, 1969) has reexamined this question. He concludes (Schwimmer, 1968) that it is the major precursor involved not only in eye tearing (lachrymatory factor) but also in development of the sensory factors of odor, bitter taste, and tongue-biting sensation in fresh onion, and odor of cooked onion (Schwimmer, 1969). It has also been implicated in the development of pink color in onion homogenates (Shannon et al., 1967; Bandyopadhyay and Tewari, 1973), although it appears to this writer that other possibilities have not been excluded. Methyl- and propylcysteine sulfoxides, however, may be the major producers of fresh onion flavor. Schwimmer’s evidence (Schwimmer, 1969) that trans-(t)-S-(1-propeny1)-r,cysteine sulfoxide is the primary substrate for alliinase is based on the following: 1. The relative amounts of sulfoxides in onions [10.9 pmoleslg of onion for 1-propenyl, 2.3 pmoles/g of onion for methyl, and 0.3 pmolelg onion for propyl derivatives (Matikkala and Virtanen, 1967; Table I) 1. 2. The K , values for these compounds with onion alliinase ( 6 , l l ,and 34 mM for 1-propenyl, propyl, and methyl derivatives, respectively). 3. The V,, values of 2.9, 0.9, and 0.9 pmole of pyruvate per milliliter per 5 minutes for 1-propenyl, propyl, and methyl derivatives, respectively.
Based on these data, rough calculations show that the relative rates of hydrolysis expected would be 1, 0.03, and 0.01 for the 1-propenyl-, methyl-, and propylcysteine sulfoxides, respectively. The recent report (Bandyopadhyay and Tewari, 1973) that there are probably three thioalkanal S-oxide-like compounds in white onion purdes may indicate that all three compounds contribute to the process. Evidence for the structure of the lachrymatory factor was obtained from mass spectral data on this compound (Moisio et al., 1962; Spare and Virtanen, 1963). From the parent mass of 90 and the fragmentation pattern it was concluded the compound was 1-propenylsulfenic acid (Eq. 14). Prior to this work, Niegisch and Stahl (1956) suggested the structure 0-hydroxypropanthial, and Wilkens (1961)
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
91
proposed thiopropanal S-oxide as the possible structure. These compounds all have a mass of 90. H I
H I
CH,-C-C=S I
CH,-CH,-
CH=S=O
CH,-
CH= CH S
P
H
OH p-Hydroxypropanthial
Thiopropanal S-oxide
1-Propenylsulfenic acid
Brodnitz and Pascale (1971) have shown the structure of the isolatable compound to be that of thiopropanal S-oxide by chemical synthesis of the lachrymatory factor. However, l-propenylsulfenic acid may be the initial compound produced which then rearranges to the more stable thiopropanal S-oxide (Schwimmer and Friedman, 1972). There is no evidence from any of the workers in this field that any di-(I-propenyl) thiosulfinate is formed from trans-(+)-S-(l-propeny1)-L-cysteine sulfoxide when the latter compound is acted on by alliinase. Mixed alkenyl alkyl disulfides are found, however. It should also be mentioned here, as is shown later, that thiosulfinates apparently do not survive gas chromatography. It has been reported that pH has a marked influence on the development of the initial enzyme products in onion (Lukes, 1971). At pH 5.4 to 5.6 and room temperature the concentration of lachrymatory factor (thiopropanal S-oxide) reached a maximum within 2 minutes of start of grinding. This was followed by a rapid decrease in concentration of this factor. After 5, 15, and 40 minutes, there was 50%, SO%, and 97% reduction in the amount of the compound. Propyl propanethiosulfmate was formed rapidly and was relative1)- stable. Methyl methanethiosulfinate was formed more slowly over a period of 10 to 15 minutes. At pH 3.8 to 4.0 (adjusted with acetic acid prior to blending), there were higher yields of lachrymatory factor, and decomposition was slow (Lukes, 1971). Methyl methanethiosulfinate and propyl propanethiosulfmate did not develop to any extent at this pH. The role of the alkyl- and alkenylcysteine sulfoxides and/or alliinase in the development of a bitter substance in onion juice is not clear. In 1969, Schwimmer (1969) concluded that the bitter taste is probably due to products formed from the action of alliinase on trans-(+)-S-(1-propeny1)-L-cysteine sulfoxide. Earlier, Schwimmer (1967) had considered whether the bitter substance might be due to the presence of a flavonoid such as quercetin or its glycoside, quercetrin. However, he found neither quercetin nor quercetrin to be bitter. The reaction does involve an enzyme, since onions treated with acid or heated did not possess or develop bitterness until a crude onion protein fraction was added to the heated onion. It does appear that onion odor and pungency develop at a faster rate than the bitter taste, indicating either that a different enzyme and
JOHN R. WHITAKER
98
CH,=C-C<
I
/o ..’..o-
HOCH,-C-COO-+
,N.... HC’ 0’ -HO,POH
PC&oI
PALP
+ CH,=
t
H HS-CHpCH,-F-C, ’N., HC/
PAP
d
ADDITION TO B-CARB
:-COONH,+
Hno_ CH,-C-COO-+ II
NH:
0
R~ ....0-
p+
B.y ELIMINATION
cni
CH,-C-
8
coo-+
NH,+
+ H,S + P A L P FIG. 6. Schematic representation of reactions catalyzed by pyridoxal phosphate. A common Schiff base intermediate between serine and pyridoxal phosphate is shown in upper center. This, then, may break down to give five types of products. The lower scheme shows 0,~ elimination involving the -SH group of homocysteine. The intermediate is also a Schiff base. B+ is an acidic group on the enzyme. PALP and PAP are pyridoxal phosphate and pyridoxamine phosphate, respectively. From Whitaker (1972); reproduced by permission of Marcel Dekker, Inc.
reaction are involved or that the bitter taste arises from secondary reactions following alliinase action. In this connection, it is interesting that, of the four cysteine sulfoxides found in onions and garlic, only trans(t)S(l -propenyl)-Lcysteine sulfoxide, when treated with alliinase devoid of substrate, gave rise to all the sensory factors associated with crushed onions (Schwimmer, 1968). Prevention of the bitter taste can be achieved by acidification of fresh extract to pH 3.9 followed by readjustment of pH to the original value (5.5 to 6.0) or by addition of cysteine (Schwimmer and Guadagni, 1967). In the above proposed reactions of alkyl- and alkenylcysteine sulfoxides with alliinase, it is essential to consider the reaction in relation to the known
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
99
requirement for pyridoxal phosphate. Types of reactions involving pyridoxal phosphate-containing enzymes are summarized in Fig. 6 (Whitaker, 1972). The type of reaction catalyzed by aUiinase appears to be typical a,@ elimination reaction. It is easy to see how the lachrymatory factor (1-propenylsulfenic acid or thiopropanal S-oxide) arises from this reaction (Eq. 15). The kinetics of stimulation of alliinase activity by added pyridoxal phosphate and its mechanism of action have been investigated (Schwimmer, 1964; Schwimmer and Friedman, 1972), and a scheme consistent with that of Fig. 6 has been proposed. 0 -
0 CH,- CH=CH-
t
s-H,c,H,c
0
11
c
‘ Hc’/”..B‘;
\ -
P - H o p o H z c ~ -eliminationa, P +/
N H
CH,
/ Aminoacrylate
NH:
+
CH,-
+
CH,-
0
CH,:CH=S-CO
c-cooII
0 Pyruvate
Reactions involving (t)-S-allyl-L-cysteine sulfoxide, (t)S-methyl-L-cysteine sulfoxide, and (t)-S-propyl-L-cysteine sulfoxide are more complex and obviously need more detailed investigation of the mechanism involving formation of the thiosulfmates. This is even more true when one considers the presence of mixed thiosulfinates reported in garlic (Fujiwara et al., 1955). In summary, the primary reactions in crushed garlic involve the action of alliinase on (t)-S-allyl-, (+)-S-methyl-, and (t)-S-propyl-L-cysteine sulfoxides to produce the corresponding thiosulfinates, pyruvate, and ammonia. Diallyl thiosulfmate is the predominant thiosulfmate produced. In crushed onion, alliinase acts on trans-(t)S-(1-propeny1)-L-cysteine sulfoxide to produce thiopropanal S-oxide, pyruvate, and ammonia, and on (+)S-methyl- and (t)S-propyl-Lcysteine sulfoxides t o produce the corresponding methyl methanethiosulfinate and propyl propanethiosulfinate. These latter compounds may be largely responsible for the desirable flavor characteristics of fresh onion.
JOHN R. WHITAKER
100
B. SECONDARY PRODUCTS IN CRUSHED ALLIUM The primary products, thiopropanal 5’-oxide, diallyl thiosulfinate, methyl methanethiosulfmate, and propyl propanethiosulfmate are quite unstable and rapidly undergo spontaneous reactions to form the compounds isolated from the head space of crushed onion and garlic or from distillates of the crushed products. As shown by Tables I11 and IV, an impressive number of compounds are formed by these secondary reactions. 1. Alcohols and Aldehydes A much greater number of alcohols and aldehydes are isolated from crushed onions than from crushed garlic. This appears to be a direct result of the formation of large amounts of thiopropanal S-oxide in onion, not formed in garlic. The following spontaneous reactions (Eqs. 16-20) have been proposed in the decomposition of thiopropanal S-oxide (SpBre and Virtanen, 1963). 0 CH,-CH,-
CH=S=O
CH,-CHZCH-SH
t
(16)
t
+
0
0 CHs-CH=CH-SH
+
H,O-
CHS-CH=CHOH
+
[HSH]
(17)
Vinyl alcohols are known to readily undergo such reactions to aldehydes. The small amounts of 2-methyl-2-pentenal may result from aldol condensation of
propanal. Hydrogen sulfoxide has never been isolated, so its existence is hypothetical. However, Schwimmer (1969) noted that solutions in which alliinase hydrolyzed trans-(t)-S
FLAVOR, ODOR, AND PUNGENCY IN ONlON AND GARLIC
= CHSCH,CHCOOH * I
HC,HC,
0
J
101
or CH,CH,CH=SO
1 (CH,CH=CHOH)
I
CH,CH,CHO
I
CH,CH,CH,CHCHO I CH,
+
NH, CH,COCOOH
+ NH,
I
t
+S
\
CH.CH.CH.CH0
t
CH,CH,CHCHO I CH3
FIG.7. Formation of carbonyl compounds in onion from trans-(+)-S-(1-propeny1)cysteine sulfoxide as proposed by Boelens et ul. (1971).
cysteine sulfoxide (Fig. 7). Their scheme involves the conversio., of pyruvate to aldehydes also. It appears that enzymes must be involved in some of these steps. Such enzymes are known to be present in higher plants. The observed alcohols could arise from the action of alcohol dehydrogenase (alcoho1:NAD' oxidoreductase; EC 1.1.1.1) on the corresponding aldehydes, provided enough NADH or a NADH regenerating system survived the crushing process. This point needs to be investigated. Propanal is stated to be one of the more important flavor compounds in raw onions (Boelens et al., 1971). It must be pointed out that the flavor of fresh onions is modulated by the sugars present. The aldehydes appear to be formed much more rapidly in cut onion than are the sulfide compounds, as shown in Fig. 8. Fifteen minutes after chopping, the head space gas consisted primarily of ethanal, propanal, 2-methylbutanal, and 2-methylpentanal. Very little dipropyl disulfide was present. After 60 minutes there was substantially more dipropyl disulfide, and after 120 minutes the dipropyl disulfide was the major volatile component present. Methyl propyl disulfide was in the next highest amount after 120 minutes. The same workers (Boelens e t d., 1971) found that the rate of formation of these constituents depends on, among other things, the degree of homogenization of the onions. This has also been noted by Kohman (1952). Presumably the maximum rate would be reached only when every cell of the tissue is disrupted so that all enzyme and a l l substrate are brought together. Gas chromatography could give a
JOHN R. WHITAKER
102 2
1
I
I
I
2
3 AFTER 60 MINUTES
12
I I
1
I
AFTER 120 MINUTES
FIG. 8. Compounds in the head space of cut onion. From Boelens et aL (1971). Identity of peaks is as follows : (1) Ethanal. (2) Propanal. (3) 2-Methylbutanal. (4) 2-Methylpentanal. (5) Dimethyl disulfide. (6) 2-Methyl-2-pentenal. (7) 2,4-Dimethylthiophene. (8) Methyl propyl disulfide. (9) 3,4-Dimethylthiophene. (10) Methyl cis-propenyl disulfide. (1 1) Methyl Duns-propenyl disulfide. (12) Dipropyl disulfide. (13) cis-Propenyl propyl disulfide. (14) trans-Propenyl propyl disulfide. Reproduced by permission of the American Chemical Society.
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
103
distorted picture of the actual sequence of events, since the compounds found are those that are thermostable, thus surviving the separation process, and those that arise as a result of the heat. 2. Sulfur Compounds The amounts of these compounds formed depend on variety, maturity, cultural and environmental conditions, and sample preparation methods, while the relative amounts of the components may depend only on variety or species (Saghir et d.,1965). Of more immediate attention for our consideration here is the variation of these compounds depending on sample preparation methods. Three general procedures have been used in studying the compounds of crushed Allium species. One method involves analysis of the head space above the sample. Another method involves the extraction (either cold or at room temperature) of the crushed sample with low-boiling organic compounds followed by evaporation of the solvent. The third method involves steam distillation of the sample (homogenate or oil) at 25" to 40°C. The results obtained depend on the method employed. When the head space method is used, the major sulfur compounds of freshly chopped onions are mainly disulfides (Bernhard et ul., 1964; Saghir et ul., 1964, 1966; Bernhard, 1968; Boelens et ul., 1971), as shown in Table N.Dipropyl disulfide is present in the largest amount (44 to 93%), followed by methyl propyl disulfide, dimethyl disulfide, allyl propyl disulfide, and smaller amounts of allyl methyl disulfide, diallyl disulfide, and perhaps diallyl sulfide. (t)S-AllylL-cysteine sulfoxide, originally thought not to be present in onion, has been found in small amounts (Granroth, 1968), thus accounting for these allyl disulfides. Tri- and higher polysulfides are not reported in the head space of freshly chopped onion, although they apparently are present in small amounts in the head space of crushed garlic (Oaks et ul., 1964) (see Table 111). Diallyl disulfide and allyl methyl disulfide are the major components of the head space of freshly crushed garlic (Oaks et ul., 1964; Brodnitz et ul., 1971a) (see Table 111). The relative amounts of disulfides in the head space of freshly cut onion and garlic are a function of time and temperature of holding prior to sampling (Saghir et al., 1964; Boelens et ul., 1971). Dipropyl disulfide (Fig. 8) is much higher in a sample of chopped onions held at room temperature for 120 minutes than in a sample held for 60 minutes. Saghx et ul. (1964) reported an increase in the disulfides in the head space above chopped garlic held at 40°C up to 30 minutes (Fig. 9). After this time, methyl propyl disulfide decreased to zero at 1 hour, allyl propyl disulfide decreased to zero after 2 hours, and the other components appeared to remain constant through 4 hours at 40°C. Granroth (1970) showed that a sample of diallyl disulfide stored at 100°C for 1 day gave
TABLE IV COMPONENTS OF CRUSHED ONION (ALLILIM CEPA) ~
b 1. Methyl alcohoB 2. Ethyl alcoholh 3. n-Propyl alcohoFsh 4. Isopropyl alcoholh 5. Ethanalg.h*i 6. propanmi 7. n-Butanalh 8. 2-Methylbutanali. 9. 2-Methylpentanal' 10. 2-Methyl-2-pentenali*i 11. 12. 13. 14.
Acetone Methyl ethyl ketoneh Sulfur dioxidg Hydrogen sulfidCh
k 15. PropanethioF*hpj9
Reference
Compositiona
Compound
c
d
e
f Niegisch and Stahl(l956) Carson and Wong (1961b) Niegisch and Stahl(1956); Carson and Wong (1961b) Carson and Wong (1961b) Niegisch and Stahl(1956);Carson and Wong (1961b); Boelens et al. (1971) Challenger and Greenwood (1949); Niegisch and Stahl(l956); Boelens ef al. (1971); Kawakishi et al. (1971); Belo (1972) Carson and Wong (1961b) Boelens et nl. (1971) Sp&e and Virtanen (1961); Boelens ef al. (1971) Stoll and Seebeck (1951); Boelens ef al. (1971); Belo (1972) Carson and Wong (1961b) Carson and Wong (1961b) Niegisch and Stahl(l956) Niegisch and Stahl(l956); Carson and Wong (1961b) Challenger and Greenwood (1949); Niegisch and Stahl(l956); Carson and Wong (1961b); Kawakishi ef al. (1971); Belo (1972)
16. DiaUyl sulfidezpm
0
0
0
0
4
17. Dipropyl sulfide 18. Dimethyl disulfideh*i~m~n~o
2
0
4
4
18
19. Methyl propyl disulfideh~i~m*n*o
4
2
9
25
34
20. Methyl c!s-1-propenyl disulfideZpn
Brodnitz ef 41. (1969); Brodnitz and Pollock (1970); Boelens et al. (1971); Belo (1972)
21. Methyl trans-1-propenyl disulfidejt"
22. Ally1 methyl disulfidei*m~fl~o 23. Dipropyl disulfid&~~m*n~o
Bernhardef al. (1964);Saghiref nl. (1966); Bemhard (1969) Belo (1972) Carsonand Wong(1961b);Saghiretal. (1964, 1966); Bernhard (1968, 1969); Brodnitz ef al. (1969); Boelens et al. (1971); Belo (1972) Carson and Wong (1961b); Saghiretal. (1964, 1966); Bernhard (1968,1969); Brodnitz ef aZ. (1969); Brodnitz and Pollock (1970); Boelens ef al. (1971); Belo (1972)
<1 86
<1
93
1
80
2 60
0
44
Brodnitz et al. (1969); Brodnitz and Pollock (1970); Boelens ef al. (1971); Belo (1972) Saghir ef al. (1964, 1966); Bernhard (1968, 1969); Brodnitz ef 4Z. (1969) Niegisch and Stahl(l956); Carson and Wong (1961b); Saghir ef al. (1964, 1966); Bernhard (1968, 1969); Brodnitz etal. (1969); Brodnitz and Pollock (1970); Boelens ef al. (1971); Kawakishi et al. (1971); Belo (1972)
0 U 0
P 2-
z
U
0
z
E;
2
>
z
t 3 0
> w
t: n
~ _ _ _ _ _
(continued) c
fn 0
c1
TABLE IV (continued) Compound
s?
Compositiona b
c
d
Reference e
f
24. cis-l.-Propenyl propyl disulfidel*"
26. Ally1 propyl disulfide~m~n*o 27. Diallyl disulfidei~m~o 28. Dimethyl trisulfidehPn 29. Methyl propyl trisulfidehJ
30. Dipropyl trisulfideh'" 31. Methyl 1-propenyl trisulfide"BP 32. Methyl 1-propenyl trisulfiden*9 33. Propyl trisulfide" 34. 1-Propenylpropyl trisulfide"9P 35. 1-Propenylpropyl trisulfide"*q 36. Methyl rnethanethiosulfiiate'
Brodnitz et QZ. (1969); Brodnitz and Pollock (1970); Boelens el nl. (1971); Belo (1972)
6
4
5
9
0
<1
<1
<1
<1
0
Brodnitz er ul. (1969); Brodnitz and Pollock (1970); Boelens et 01. (1971); Belo (1972) SaghireraZ. (1964,1966);Bemhard (1968, 1969); Brodnitz et 01. (1969) Saghir er al. (1964, 1966); Bernhard (1968, 1969) Carson and Wong (1961b); Brodnitz et QZ. (1969) Carson and Wong (196 lb); Brodnitz et QI. (1969); Brodnitz and Pollock (1970); Belo (1972) Carson and Wong (1961b); Brodnitz and Pollock (1970) Brodnitz el QZ. (1969) Brodnitz et QI. (1969) Brodnitz etaZ. (1969); Belo (1972) Brodnitz et QI. (1969) Brodnitz et QI. (1969) Fujiwara el QZ. (1955); Lukes (1971); Belo (1972)
L o
0
x z
P
e
3 1 >
? i7J
37. Propyl propanethiowlfinate‘ 38. 39. 40. 41. 42. 43.
1-Propenyl-1-propenethiosulfmate Methyl methanethiosulfonateS Propyl methanethiosulfonat$ Propyl propane thiosulfonateS 2,4-Dimethylthiophene: 3,4-Dimethylthiophene1p
Fujiwara et nl. (1955); Lukes (1971); Belo (1972) Belo (1972) Boelens et al. (1971) Boelens et al. (1971) Boelens et al. (1971) Boelens et al. (1971) Brodnitz et al. (1969); Brodnitz and Pollock (1970); Boelens et al. (1971)
=Percentage of total volatile constituents from head space of freshly cut onions. bFrom head space of freshly cut “Australian Brown 5” onions (Saghir et al., 1964). CFrom head space of freshly cut “Crystal Grano” onions (Saghir et al., 1964). dFrom head space of freshly cut “potato” onions ( S a m et aL, 1964). eFrom head space of freshly cut “top-set” onions (Saghir et QI., 1964). fFrom head space of freshly cut “Southport White Globe” onions (Bernhard, 1969). gNiegisch and Stahl(1956); common yellow onion, minced while frozen, distillation at room temperature under vacuo. hCarson and Wong (1961b); “Sunspice” onions. Vacuum and steam distillation (at -25°C) of freshly chopped onions, volatiles trapped on carbon, extracted with ether, and ether evaporated to give oil; alternative procedure: freshly chopped onions pressed to obtain juice, juice steam distilled (not over 4 2 ” Q isopentene extraction, and concentration. jFrom head space of Dutch or Egyptian cut onions (Boelens et aL, 1971). jKawakishi et al. (1971); “Sensyu Yellow” onions, from head space of blended onions. kFrom aspiration of chopped onions (Challenger and Greenwood, 1949). ‘From head space of some freshly cut cultivars (Bernhard et al., 1964). m“Southport White Globe,” from head space analysis (Saghir et al., 1966). nFrom onion oil produced commercially and representing mixtures of varieties, maturity, and sizes (Brodnitz el aZ., 1969). OFrom head space of 53 lots of fresh and dried onions (Bernhard, 1968). PProbably cis (Brodnitz eta/., 1969). 4Probably trans (Brodnitz et al., 1969). ‘Following reaction with thiamine. SDutch or Egyptian onions extracted with dichloromethane (Boelens et al., 1971).
108
JOHN R. WHITAKER
HOURS AT 4OoC
FIG. 9. Effect of time of incubation on head space composition of a sample of chopped tissue of California Late garlic (Saghir et al., 1964). Note the semilogarithmicplot. Al, allyl; Me, methyl; PI, propyl. Reproduced by permission of the American Society of Horticulturists.
rise to at least twenty peaks on gas chromatography. Some of the peaks are quite large (Fig. 10). More work is needed on this point, and it is obvious that for a complete analysis of the volatiles of onions samples must be examined periodically, perhaps every 10 minutes. The head space composition of samples of rehydrated dried onions is not the same as that of freshly chopped onions (Bernhard, 1968). Bernhard found the principal disulfides present in the head space of fresHy cut onions to be dipropyl disulfide, followed in descending order of concentration by allyl propyl disulfide, methyl propyl disulfide, ally1 methyl disulfide, dimethyl disulfide, and diallyl disulfide. In rehydrated dried onions, methyl propyl disulfide was found to be the principal disulfide followed by dimethyl disulfide, allyl methyl disulfide, dipropyl disulfide, allyl propyl disulfide, and diallyl disulfide. He reported an average loss of 98% of the disulfides as compared with fresh onions. This point should be examined carefully by those producing dehydrated onions and/or garlic. If substantiated, this decrease represents a tremendous economic
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
109
I:
6
20
5
10
15
minutes
FIG. 10. Gas chromatogram of diallyl disulfide after incubation at 100°C for 1 day (Granroth, 1970). Peak 3, diallyl sulfide; peak 6, diallyl disulfide; the other peaks were not identified. Reproduced by permission of Finnish Academy of Sciences.
loss, and the flavor and odor of rehydrated onions would not be expected to be the same as that of fresh onions. The relative contributions of these compounds to the flavor and odor of onion has not been established. The relatively large amounts of allyl disulfides reported by Bernhard (1968) to be present in the head space of chopped fresh and dried onion samples have not been confirmed by other workers. In onion oil, Boelens et al. (1971) found a trace of allyl propyl disulfide (Fig. 1 1 and Table V, peak 27) between two large peaks of dipropyl disulfide (peak 26) and cis-propenyl propyl disulfide (peak
FIG. 11. Gas chromatogram of steamdistilled onion oil on a UCOH-coated SCOT column, programmed 80" to 180", 1" per minute (Boelens et al., 1971). For identification of the peaks see Table V. For clarity of the drawing some peaks are not numbered. Reproduced by permission of the American Chemical Society.
JOHN R. WHITAKER
110
TABLE V COMPOUNDS IDENTIFIED IN STEAM-DISTILLED ONION OILa
Compound Oxygen compounds Propanal Dimethylfurad 2-Methylpentma@ 2-Methyl-2-pentenal Tridecan-2-oneb
5-Methyl-2-n-hexyl-2,3-dihydrofuran-3-oneb Disulfides Dimethyl disulfide Methyl propyl disulfide Allyl methyl disulfide Methyl cis-propenyl disulfide Methyl trans-propenyl disulfide Isopropyl propyl disulfideb Dipropyl disulfide Allyl propyl disulfide cis-Propenyl propyl disulfide transPropeny1 propyl disulfide Diallyl disulfide Allyl propenyl disulfide (two isomers), tentativeb Dipropenyl disulfide (three isomers), tentativeb Monosulfides Dimethyl sulfideb Allyl methyl sulfideb Methyl propenyl sulfideb (two isomers) Allyl propyl sulfideb Propenyl propyl sulfideb (two isomers) Dipropenyl sulfideb (three isomers) Trisulfides Dimethyl trisulfide Methyl propyl trisulfide Allyl methyl trisulfide Methyl cis-propenyl trisulfide Methyl trans-propenyl trisulfide Diisopropyl trisulfideb Isopropyl propyl trisulfide* Dipropyl trisulfide Allyl propyl trisulfideb Diallyl trisulfideb cis-Propenyl propyl trisulfide trans-Propenyl propyl trisulfide Thiophene derivatives 2,5-Dimethylthi~phene~ 2,4-Dimethylthi~phene~ 3,4-Dimethylthiophene
Peak No.'
5 9
11 13 47 50
12 20 21 22 23 25 26 21
28 30 29 31 32
1 8 10 14
16 18 24
33 34 35 36 37 38 41 42 43 44 45
15 17 19
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
111
TABLE V (continued) Compound
NO.^
Peak
3,4-Dimethyl-2,5-dihydro thiophen-2-oneb Thiols Hydrogen sulfide Methane thiolb Propane thiol Allylthiolb m/e 154 C,HPS,SHb m/e 180 C, H,, S, SHb m/e 182 C,H,,S,SHb Tetrasulfide Dimethyl tetrasulfideb Hydrocarbon Pr openeb Additional compounds identified in an onion extract Methyl methanethiosulfonateb Propyl methanethiosulfonateb Propyl propanethiosulfonate*
49 3 4 6 7 40 46
48 39 2
OFrom Boelens e f al. (197 1). bNot reported earlier as a constituent of onion (Allium cepa). CAsnumbered on the chromatogram in Fig. 11.
28), and even a smaller amount of diallyl disulfide (peak 29) between large peaks of cis- and trans-propenyl propyl disulfides (peaks 28 and 30). Similarly, the peak for allyl methyl disulfide, present in onion oil in trace amount (peak 21), is very close to that of methyl cis-propenyl disulfide (peak 22). Although different sampling techniques were used in these two studies, the results indicate the need for further investigation using well-characterized standard compounds, since the elution positions of the homologous propenyl and allyl derivatives are so similar. The head space of freshly chopped onions is reported to contain very small amounts of 2,4-dimethylthiophene and 3,4-dimethylthiophene (Boelens e t ul., 1971). Heated samples contain substantially more of these compounds. It is thought that these arise, along with mono- and trisulfides, from the decomposition of alkyl and alkenyl disulfides, as shown in Eqs. (21) and (22) (R = methyl or propyl) where the two are competing reactions.
CH,-CH=CH-
S-S-R
CH3-CH=CH-S-S-R-
-H3cuCH3 f R-S-S-R
i
CH,-
+ H,S
(21)
CH=CH-S-R f
CH,-CHICH-
S-CH=CH-CH,
+
R- S- S- S- R
(22)
112
JOHN R.WHITAKER
It has been reported that UV photolysis of cis-S-( 1-propeny1)-L-cysteinegives rise to thiophenes (Nishimura et al., 1973a). For example, a 20 mM oxygen-free neutral solution of cis-S-(1-propeny1)-L-cysteine irradiated for 20 hours with a 50-watt low-pressure mercury lamp gave 2,4-dimethylthiophene (ca. 15%), 3,4dimethylthiophene (7%), 2,5-dimethylthiophene (<1%), and 3-methylthiophene (5%), as well as 1-propenyl mercaptan (40%), di-(1-propenyl) sulfide (<1%), and 1-propenyl 1-propyl sulfide (
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
113
thiosulfinate was observed to undergo dehydration to form major amounts of 3-vinyl-l,2-dithi-S-eneand 3-vinyl-I,2-dithi4-ene. Synthetic diallyl thiosulfinate held at 20°C for 20 hours underwent nearly complete decomposition to give diallyl disulfide (66%), diallyl sulfide (14%), diallyl trisulfide (9%), and sulfur dioxide. Only traces of the above 3-viny1-1,2-dithieneswere formed. H,C=HC
““=“‘Q 3-Vinyl-l,2-
3-Vinyl-1, 2dithi-4-ene
dithi-5-ene
Methyl methanethiosulfonate, propyl methanethiosulfonate, and propyl propanethiosulfonate (R-CH2 -SOz S-CH2 R) have been found in dichloromethane extracts of freshly chopped Dutch and Egyptian onions (Boelens et al., 1971). Propyl propanethiosulfonate was estimated to be present in about 0.5 ppm, while the sensory threshold value of t h s compound is 1.5 ppb (see Table VI). The workers (Boelens et al., 1971) reported that thiosulfonates with four or more carbon atoms have a distinct odor of freshly cut onions, that propyl- and propenyl-containing di- and trisulfides have more the flavor of cooked onions, and that the flavor character of 2,4- and 3,Cdimethylthiophenes is distinctly that of fried onions. Dimethyl disulfide has a “cabbage”-like odor and flavor (Lukes, 1971). It is of interest that recent patents have been taken out for preparation of synthetic thiosulfonates (Brodnitz et al., 1971b, 1972) and unsaturated disulfides (Brodnitz, 1969) for use in simulating onion flavors. The mechanisms by which the di- and trisulfides and thiosulfonates arise from TABLE V1 THRESHOLD VALUES OF ONION COMPOUNDS‘
Substance
Concentration @g/liter of water)
Onion oil Dipropyl disulfide Methyl propenyl disulfide Propenyl propyl disulfide 3,4-Dimethylthiophene Propyl methanethiosulfonate Propyl propanethiosulfonate
0.8 (0.1-2.7)b 3.2 (2.2-4.0) 6.3 (2.7-8.1) 2.2 (0.3-2.7) 1.3 (0.2-2.7) 1.7 (0.3-2.7) 1.5 (0.3-2.7)
‘From Boelens el d.(1971). bRange of responses from the panel.
JOHN R. WHITAKER
114
Is
RCH,SSSCH,R
R=
hydrogen or ethyl
FIG. 12. A proposed scheme for formation of primary and secondary sulfur compounds from alkyl- and alkenylcysteine sulfoxides in onion. From Boelens et al. (1971).
the thiosulfinates and from thiopropanal S-oxide is not well understood. Barnard (1957) has shown that the disproportionation of aryl thiosulfinates to thiosulfonates and disulfides occurs spontaneously at room temperature (Eq. 23). The disulfides can then undergo further reaction to m o n e and trisulfides as shown in Eq. (22). These compounds may also arise from alkyl- and alkenylsulfenic acids, as proposed by Boelens er QZ. (1971) in Fig. 12. It would appear that a great deal of fundamental work remains to be done in this field. 0
4
2 RS-S-R
-
0 I1
R-S-S-R
II
+
R-S-S-R
0
In summary, the secondary products from thiosulfinates and thiopropanal S-oxide do not form at the same rates in crushed onion or garlic, nor do they have the same stabilities in stored products. The carbonyl compounds appear first, followed by the disulfides and later by the monosulfides, trisulfides, and even polysulfides. Thiosulfonates appear to be important flavor components produced in these secondary reactions.
V. METHODS OF MEASURINGALLIUM PUNGENCY, FLAVOR, AND AROMA Any investigation designed to determine the effect of variety, genetic manipulation, maturity, cultural conditions, environmental conditions, storage, or
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
115
processing conditions on the quality attributes of Allium must depend on the method selected to measure these quality attributes. This is not easy as one might expect from reading to this point. Pungency, flavor, and aroma in Allium have no standardized definitions. To most workers pungency is the eye-tearing effect onion gives in the raw state when it is being disintegrated. Flavor and aroma are probably a reflection of the quantitative and qualitative alkyl and alkenyl di- and trisulfide and thiosulfonate composition of Allium, modified in part by soluble sugar composition, aldehydes, and alcohols. Various methods have been advocated for measuring these parameters. If pungency, which is related to the lachrymatory factor (thiopropanal S-oxide or 1-propenylsulfenic acid), is to be measured, then a method of measuring this lachrymatory factor is needed. Spare and Virtanen (1963) developed a semiquantitative method in which 4 to 6 drops of plant extract adjusted to pH 6 to 7 was placed in a small weighmg vial. Five milligrams of alliinase powder was added, the suspension stirred for 10 seconds, and the vial held tightly to the eye. Depending on concentration, the lachrymatory effect was perceived after 15 to 45 seconds. This method cannot be used with garlic or other Allium species that do not produce the lachrymatory factor. Both Currier (1945) and Platenius (1935; Platenius and Knott, 1941) advocated the use of volatile sulfur as a criterion of pungency. Although they found a good correlation between total sulfur in the distillate and pungency, they were not measuring the lachrymatory factor. Others have also suggested the use of total volatile sulfur as an index of pungency and/or flavor (Kohman, 1952; Foley, 1955; Farber, 1957). Allicin is the major primary product from the action of alliinase on (+)S-allylL-cysteine sulfoxide; it gives rise through secondary reactions to the typical garlic flavor and aroma. Allicin is also responsible for the major antibacterial effect of garlic extracts (Stoll and Seebeck, 1951). Presumably, bacteriological assays could be used to measure the allicin content. Conditions would have to be carefully standardized, since the secondary products are much less inhibitory than is allicin. Saguy et ul. (1970) have suggested the use of the Chemical Oxygen Demand (COD) method of McNary et al. (1957) as modified by Dougherty (1968) for estimation of the aroma and flavor components of onions. The procedure consists in oxidizing a distillate of the product with chromic acid and estimating the total volatile compounds colorimetrically. They reported a correlation coefficient of 0.98 between odor threshold and COD values for fresh onion; correlation was poor for dehydrated onion samples. The olfactory threshold and the pyruvate content of onions developed enzymatically have a good correlation (r = 0.97), making this a suitable method for both fresh and dried onion samples when used under carefully controlled conditions (Schwimmer and Weston, 1961; Schwimmer and Guadagni, 1962; Schwimmer et al., 1964a). The pyruvate content is estimated colorimetrically
116
JOHN R. WHITAKER
after the 2,4dinitrophenylhydrazonederivative has formed. Saghir et al. (1964) did not find a correlation between the aliphatic disulfide content and pyruvate, however, which is not surprising in terms of the continued reactivity of the primary products of the alliinase reaction. More recently, the correlation between rate of pyruvate and odor formation has been reexamined (Schwimmer and Guadagni, 1968). The results suggest that pyruvate and odor are probably developed by the same enzyme; however, it appeared that the odor was formed somewhat later than the pyruvate. This is not entirely unexpected, since disulfides, particularly dipropyl disulfide, contribute to the characteristic odor of onion, and these disulfides arise through secondary reactions from products of the enzyme-catalyzed reaction. Lukes (1971) has used the reaction of cysteine with thiosulfmates and thiopropanal S-oxide to estimate these initial products of allinase action in crushed onion. The derivatives are separated on thin-layer plates. This method measures directly the primary products of enzyme action on the pungency, flavor, and aroma precursors. The method, while slow and laborious, could be quite useful for analysis of pungency, flavor, and odor potential. Freeman and McBreen (1973) have developed a rapid spectrophotometric method for determining thiosulfmates in hexane extracts of fresh, freeze-dried, and oven-dried onions. There was a good correlation between measured thiosulfmates and pyruvate concentration (linked above to the olfactory threshold level). It might appear that the best method for measuring flavor and aroma is garliquid chromatography under carefully controlled conditions, perhaps as described by Bernhard (1968), Boelens et al. (1971), and others. The individual components can be rapidly separated and identified by mass spectrometry, and their quantitative amounts used to correlate with flavor and aroma properties. The method is imperfect in that it measures secondary compounds of the enzymatic action, and the relative contribution of these compounds to overall flavor and aroma is not known. The method also suffers in that the compounds and amounts found may be a result of the temperature and other conditions used in their separation. For these reasons, liquid chromatography, particularly by means of high pressure, may be the best method for separating the primary compounds formed from enzymatic action for correlation with sensory determination of flavor and odor.
VI. ENZYMES IN FLAVOR, AROMA, AND PUNGENCY DEVELOPMENT IN ALLIUM Numerous enzymes are involved in the biosynthesis of the flavor, aroma, and pungency precursors, in their breakdown to primary products, and perhaps also in the conversion of some of the primary products to sensory constituents or
FLAVOR, ODOR, AND PUNGENCY IN ONION AND GARLIC
117
modifiers of sensory perception. It is an indication of the sad state of our knowledge of the enzymes of Allium that only two of these have been investigated to the point where they can be described as entities. We refer here to alliinase and y-glutamyl transpeptidase. The proposed pathway for the biosynthesis of the alkyl- and alkenylcysteine sulfoxides from inorganic sulfate is presented in Fig. 2 . If one assumes that coupling of methyl mercaptan, propyl mercaptan, and ally1 mercaptan with serine to give the corresponding alkyl- and alkenylcysteines involves a single enzyme (cysteine synthase; Granroth and Samesto, 1974) and that oxidation of the four alkyl- and alkenylcysteines to the sulfoxides involves another single enzyme, there is a minimum of eleven enzymes in the biosynthesis of the sulfoxides. This includes the three enzymes found in the conversion of valine to methacrylyl-CoA (Fig. 3), which is then coupled to cysteine to give 2-carboxypropylcysteine. In the biosynthesis of trans-(+)a-(1-propenyl)-L-cysteine sulfoxide alone there is a minimum of nine enzymes. Additional enzymes would be required in the side route involving glutathione (Fig. 2), and at least one enzyme, y-glutamyl transpeptidase, in the conversion of the alkyl- and alkenylcysteines and sulfoxides to the corresponding y-glutamyl peptides, probably as a storage form in the dormant bulb. Tamura (1965) has purified a sulfite reductase from Allium odorum which reduces sulfite to sulfide. This enzyme should be studied in onions (Allium cepa) and garlic (Allium sativum), as it is a key enzyme in conversion of inorganic sulfate to organic sulfur. Whether a separate enzyme is involved in the reduction of sulfate to sulfite is not known. The reactions involved in the coupling of sulfide with serine to give cysteine have been studied in higher plants (Thompson, 1967; Eqs. 3 and 4), and the enzyme complex from Salmonella has been purified and studied in some detail. The enzyme catalyzing the reaction of acetyl-CoA with serine to give O-acetylserine (and CoA) is serine acetyltransferase, which has been found in the extracts of a number of higher plants (Smith and Thompson, 1969). The condensation of 0-acetylserine with sulfide (and other mercaptans) to give L-cysteine (and alkyl-L-cysteines) is catalyzed by the enzyme cysteine synthase which has been found in spinach leaf (Giovanelli and Mudd, 1968), in turnip leaf (Thompson and Moore, 1968), and in low amounts in onion bulbs (Granroth, 1974). Pyruvate is one of the primary products from the action of alliinase on the alkyl- and alkenylcysteine sulfoxides. Thiopropanal S-oxide from the action of alliinase on trans-(t)S-(1-propeny1)-L-cysteine sulfoxide undergoes nonenzymatic decomposition to propanal and sulfur. Both propanal and pyruvate probably undergo additional reactions as proposed in Fig. 7 leading to the formation of several aldehydes and on reduction to alcohols which are observed in the head space of crushed onions, particularly. While the formation of some
118
JOHN R.WHITAKER
of the compounds can be rationalized on the basis of aldol condensations, such condensations are not very likely at pH 6 in the environment of the crushed onion. It is more likely that enzymes known to be present in highei plants are involved. Alcohol dehydrogenase may be involved in conversion of aldehydes to alcohols.
A. ALLIINASE The enzyme alliinase [alliin alkyl-sulfenate-lyase (EC 4.4.1.4), synonyms alliin lyase, alkylcysteine sulfoxide lyase, cysteine sulfoxide lyase, C-S-lyase] has been studied extensively, particularly in garlic, onions, and mimosa beans. Alliinase is probably found in most if not all of the Allium species as well as being present in some related genera of the Liliaceae such as Ipheion and Thulbaghia (Jacobsen et al., 1968). Mazelis (1963) has studied an alliinase from Brassica species (broccoli), which, like the Allium enzymes, acts rapidly on alkyl- and alkenylcysteine sulfoxides but not on the alkyl- or alkenylcysteines. De Lima (1974) has obtained evidence for possibly three alliinase-like enzymes in cauliflower (Brassica oleraca var. botrytis L.). The three partially purified enzymes had different relative activities on L-cysteine S-sulfate, L-cystine, and S-methyl-L-cysteine sulfoxide. Each enzyme was purified five- to tenfold, and some of its enzymatic and physical properties were studied. Alliinase-like enzymes have also been reported in Pseudomoms cruciviae (Nomura et al., 1963) and Bacillus subtilis (Murakami, 1960). The seeds of Albizzia lophantha contain an enzyme which acts rapidly on alkyl- and alkenylcysteines and much more slowly on alkyl- and alkenylcysteine sulfoxides (Gmelin et al., 1957;Schwimmer and Kjaer, 1960). Enzymes in bovine liver (Anderson and Schultze, 1965) and in Escherichia coli (Saari and Schultze, 1965) have been reported to cleave S-(1,2-dichlorovinyl)-~cysteine. The alliinase of garlic was studied by Stoll and Seebeck in 1947 (Stoll and Seebeck, 1947, 1948, 1951). Purification consisted in extraction from garlic, and purification by two pH adjustments to pH 4.0 and suspension of the precipitate in buffer at pH 6.4.No data are given on the degree of purification attained; however, it is unlikely to have been very high. As prepared, garlic alliinase lost its activity in 14 days at 3°C. Activity was determined by the ammonia produced, according to Folin’s method, or by precipitation of pyruvic acid with 2,4-dinitrophenylhydrazine.The reaction catalyzed by garlic alliinase is shown in Fig. 4 and Fig. 5 . Garlic alliinase prepared by Stoll and Seebeck (1948)had a broad pH optimum of 5 to 8 and a temperature optimum under the assay conditions of 37°C. In an extensive study of garlic alliinase specificity, Stoll and Seebeck (1948) found that, to be acted upon, a compound must have the following properties. 1. The compound must be derived from (-)-L-cysteine. Derivatives of (t)-Dcysteine, homocysteine, or penicillamine are not substrates.
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2. The sulfur atom of cysteine must be linked to an aliphatic group such as ethyl, propyl, isopropyl, allyl, or butyl. Derivatives in which an aromatic residue is linked to sulfur are not substrates. 3. The amino group of the cysteine portion of the compound must not be substituted. N-Anilinoformylal.hn is not a substrate. 4. The sulfur atom of the cysteine derivative must be present in the sulfoxide form. Deoxyalliin is not a substrate. The sulfur oxygen may be in either the (t) or the (-) configuration; however, the (+) isomer is split much more rapidly. More recent work (Mazelis and Crews, 1968) on garlic alliinase has confirmed much of the data of Stoll and Seebeck. A sevenfold purified enzyme had a pH optimum of 6.5 on S-methyl-L-cysteine sulfoxide, and the best activity on S-allyl-L-cysteine sulfoxide [relative specificities (V,,, / K , ; units mgl mM-') for the S-alkyl-L-cysteine sulfoxides were: S-allyl-, 20; Sethyl-, 14; S-propyl-, 10; S-butyl-, 10; S-methyl-, 51. The thiolether analogs of the substrates were competitive inhibitors of the enzyme. The enzyme was stimulated by added pyridoxal phosphate and inhibited by hydroxylamine and cysteine. In summary, the best substrate for garlic alliinase was found to be (+)S-allylL-cysteine sulfoxide. No physical and chemical properties of garlic alliinase, other than stability, have been reported. Needed is definitive purification work leading to knowledge of how many alliinase-like enzymes are present (isoenzymes), and the physical and chemical properties of the molecule. Also needed is information on the amount of enzyme in various cultivars of garlic and the effect of cultural, climatic, and drying conditions on the enzyme. To our knowledge such information is not available for either garlic or onion alliinase. There have been considerably more publications on onion alliinase; however, our knowledge of this enzyme is only slightly further advanced than that for garlic alliinase. In large part, this is due to lack of sufficient purification of the enzyme to permit definitive chemical and physical studies. Most workers have achieved only some five- to sixfold purification of the enzyme (Kupiecki and Virtanen, 1960; Schwimmer, 1971a). Purification (Kupiecki and Virtanen, 1960; Schwimmer, 1971a) usually consisted in extraction of blended onions with 0.2 M potassium phosphate buffer, pH 6.8, centrifugation and filtration through Celite, and precipitation with 0.75 saturated ammonium sulfate. The precipitate was dissolved in water and used without dialysis, as this was found to result in a large decrease in activity (Schwimmer and Mazelis, 1963). Brosin (1969), using a modified procedure including chromatography on carboxymethylcellulose, achieved a 30- to 35-fold purification of the enzyme from Sunspiced variety of white bulb onions, the highest purification reported. The purified enzyme had a pH optimum of 7.5 on S-methyl-L-cysteine sulfoxide in 5 mM phosphate and maximum activity on S-allyl-L-cysteinesulfoxide (Table VII). The enzyme was completely inhibited by 50 f l hydroxylamine and was competitively inhibited by thiolether analogs of the substrates and by methionine sulfoxide and methionine sulfoxime.
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TABLE VII SUBSTRATE SPECIFICITY OF ONION ALLIINASE~ ~~
Krn Substrate
(mM)
vmax &moles pyruvate/min)
S-Allyl-L-cysteinesulfoxide S-Butyl-L-cysteinesulfoxide S-Ethyl-L-cysteinesulfoxide S-Methyl-L-cysteinesulfoxide S-Propyl-L-cysteine sulfoxide
7.13 5.1 12.5 9.1 6.25
1.47b 0.62 0.20 1.28 1.32
uFrom Brosin (1969); 5 mM phosphate, pH 7.5, in 20%glycerol at -20°C. bCalculated from K, and activity values at a given substrate concentration.
Very little information is available on the protein purity of such preparations (disc gel electrophoresis, for example), the possibility of more than one protein with alliinase activity, or the physical and chemical properties of the enzyme. Kupiecki and Virtanen (1960) did report their preparation to be homogeneous by free boundary electrophoresis (not a very definitive criterion of purity), although some earlier work indicated the possibility of more than one enzyme (Spgre and Virtanen, 1964). Be10 (1972) reported that onion alliinase (Spartan Banner onions) prepared by the method of Schwimmer and Mazelis (1963) contained sixteen protein bands and three activity bands by disc gel electrophoresis. Lyophilized preparations of the enzyme containing ammonium sulfate are stable at -13°C. In solution, the enzyme is very unstable below pH 4.5, in contrast to garlic alliinase. Partial heat inactivation followed by inhibition with S-propyl-L-cysteine suggests the presence of more than one enzyme (Schwimmer et al., 1964b). The enzyme is destroyed by freezing onions (Schwimmer and Guadagni, 1968). The reason for this is not known and should be investigated further. Onion alliinase has the following enzymatic properties. 1. The pH optimum is around 8 and depends on the buffer used. The reported optima are 8.6, 8.1, and 7.4 in sodium pyrophosphate, Tris-HC1, and potassium orthophosphate buffers, respectively (Schwimmer and Mazelis, 1963). The relative activities, compared at their pH maxima, are 1.0, 0.74, and 0.50 in the buffers listed in the order above. 2. Onion alliinase appears to be a pyridoxal phosphate-requiring enzyme (Kupiecki and Virtanen, 1960; Schwimmer and Mazelis, 1963), since it is inhibited by hydroxylamine and is stimulated by addition of pyridoxal phosphate (Theodoropoulos, 1959).
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3. The enzyme has specificity for S-substituted L-cysteine sulfoxides; S-substituted L-cysteine and derivatives of cystine are not substrates. 4. The Ssubstituent may be alkyl, alkenyl, or aryl (Kupiecki and Virtanen, 1960), in contrast to garlic alliinase (Stoll and Seebeck, 1948). The unsaturated derivatives (ally], 1-propenyl) are better substrates than the corresponding saturated alkyl derivatives (Schwimmer, 1969). The V,, values, in micromoles of pyruvate per milliliter in 5 minutes, for 1-propenyl, propyl, and methyl derivatives are 2.9, 0.9, and 0.9, respectively, while the corresponding K , values are 6,11, and 34 mM. 5. Both (t) and (-) diastereomers, with respect to oxygen on sulfur, are substrates. In general the K , for the (t)diastereomer is lower than that for the (-) diastereomer, but the Vmaxis the same. 6. While S-substituted cysteines are not substrates for onion alliinase, they are inhibitors of the enzyme (Schwimmer et al., 1964b). 7 . The naturally occurring substrate of onion alliinase is predominately trans(+)-S-(1-propeny1)-L-cysteine sulfoxide. Action of alliinase on this compound leads to formation of thiopropanal S-oxide (or 1-propenylsulfenic acid), pyruvate, and ammonia (Eq. 12). The rate of pyruvate production is used to follow the reaction (Kupiecki and Virtanen, 1960; Schwimmer and Mazelis, 1963). The products of action of this enzyme on other alkyl- or alkenyl-L-cysteine sulfoxides are dialkyl or dialkenyl thiosulfinates, pyruvate, and ammonia (Figs. 4 or 5). 8. The enzyme is destroyed in onions by freezing (Schwimmer and Guadagni, 1968). The reason for this interesting reaction needs further investigation.
An alliinase-like enzyme has been purified some 200-fold from Albizzia lophanta (Schwimmer and Kjaer, 1960). The enzyme differs from onion and garlic alliinase in that it splits alkyl- and alkenylcysteines more rapidly than it does alkyl- or alkenylcysteine sulfoxides. The enzyme has a pH optimum near 8.5 like onion alliinase (Gmelin el al., 1957). Both the cis- and trans-(t)S-(1-propeny1)L-cysteine sulfoxides are split by the enzyme (Carson and Bogs, 1966). An alliinase-like enzyme has been purified sixfold from Tulbaghia violacea (Jacobsen et al., 1968), a genus closely related morphologically to Allium. On crushing, this plant gives a garlic-like odor. The enzyme has the same specificity requirements described above for garlic alliinase including a pH optimum at pH 6.6. In summary, onion and garlic both contain enzymes that split (t)S-alkyl- and (t)S-alkenyl-L-cysteine sulfoxides to tluosulfinates (thiopropanal S-oxide in the case of trans-(+)&( 1-propeny1)-L-cysteine sulfoxide), pyruvate, and ammonia. The primary product from trans-(t)S-( 1-propenyl)-L-cysteine sulfoxide, thiopropanal S-oxide, is the lachrymatory factor in onions, whereas the major flavor and odor components of onions and garlic arise from the breakdown of the thiosulfmates and thiopropanal S-oxide. The substrate specificities of these
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alliinases have been described in some detail, but little is known about their chemical and physical properties because of insufficient purification.
B. 7-GLUTAMYL PEPTIDASE AND 7-GLUTAMYL TRANSPEPTIDASE
As was pointed out in Table I, onion and garlic contain substantial amounts of y glutamyl peptides of which some seventeen isolated from onion, garlic, and chives contain sulfur. y-L-Glutamyl-trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide is present in the largest amounts in onion (-0.2%) and as such represents about 50% of the potential flavor and odor precursors of onion. Therefore, considerable attention has been given to the role of y-glutamyl peptidases and y-glutamyl transpeptidases in the metabolism of these peptides and in possible enhancement of the flavor and odor of onions. The reactions catalyzed by y-L-glutamyl peptidase (a hydrolase) and YLglutamyl transpeptidase [y-glutamyltransferase; (y-glutamy1)-peptide:amino acid y-glutamyltransferase; EC 2.3.2.21 are shown in Eqs. (24) and (25), respectively, where propenylCyS0 is trans-(+)&( 1-propeny1)L-cysteine sulfoxide, which is taken as an example of substrate. Y-L-Glutamylpropenyl- CySO
+
Y-glutamY1 peptidase
-
L- Glutamic
+
acid
(24)
propenyl- CySO
H*O
R
u-L-Glutarnylpropenyl-CySO
+
R-
7
c -COX I
y-glutamyl transpeptidase
-
Y-L-Glutamyl- NH- C -COX I
+
H
(25)
propeny 1- CySO
NH*
Amino acid or peptide
Both enzymes are widely distributed in nature, and y-glutamyl transpeptidase is present in large amounts particularly in mammalian kidney (Orlowski and Meister, 1970). However, both enzymes are absent or occur in very low amounts in dormant onions. In 1965, Matikkala and Virtanen (1965b) reported the presence of a y-glutamyl peptidase in sprouting onion bulbs and in the seeds of germinating chive (Matikkala and Virtanen, 1965a). The ability of a buffer extract of onions to hydrolyze y-DL-ghtamyl-p-nitroanilide was taken as evidence for the presence of a glutamyl peptidase. The data presented do not rule out the possibility of a y-glutamyl transpeptidase, however, as there would be sufficient amino acids present in undialyzed onion extract (one preparation was
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dialyzed, but the results were not conclusive owing to turbidity) to permit transpeptidation. Evidence that the enzyme from germinating seeds of chive is a peptidase and not a transpeptidase is reasonably good (Matikkala and Virtanen, 1965a). The enzyme was purified some 200-fold, it had a pH optimum of 8, there was no detectable transfer of the y-glutamyl group of y-glutamylisoleucine to methionine (or from glutathone to methionine), and the products of the action of the enzyme on y-glutamylisoleucine and glutathione were glutamic acid and isoleucine and glutamic acid and cysteinylglycine, respectively. Austin and Schwimmer (1971) extracted an enzyme from sprouting onion bulbs which catalyzed the release of p-nitroaniline from y-L-glutamyl-p-nitroanilide. Although they described the enzyme as a peptidase with an optimum pH of 8, they present no evidence that would discriminate between hydrolase and a transpeptidase. In a subsequent paper Schwimmer and Austin (1 97 1a) purified an enzyme from sprouting onion bulbs some 800-fold and showed that it was a y-glutamyl transpeptidase. Evidence for transpeptidase activity is based on the substantial enhancement in rate of release of p-nitroaniline from y-L-glutamyl-pnitroanilide by amino acids, including S-methylcysteine and the competitive inhibition of the enzyme by y-glutamyl derivatives. The pH optimum was 9.0. While the data are indicative that the purified enzyme is a y-glutamyl transpeptidase (transferase), the authors suggest that a y-glutamyl peptidase (hydrolase) may be present in crude extracts from sprouting onions. Their reasoning is based on the following. The course of hydrolysis of y-glutamyl-p-nitroanilide by crude enzyme is bimodal in that it consists in an initial rapid conversion of 15 to 20% of the substrate, followed by a slower and steady rate of conversion of more than 50% of the substrate. On the other hand, with purified enzyme there was a rapid conversion of about 10%of the substrate, after which the reaction stopped in the absence of added amino acids (to function in transfer reactions). The evidence for the presence of a hydrolase in the crude enzyme is not conclusive, since amino acids may have been present. (We find no evidence that the crude enzyme was dialyzed before use.) The behavior of the purified enzyme in conversion of -10% of the substrate in the absence of added amino acid is stronger evidence for a peptidase in the purified preparation. More data are needed on this point. Preparations from onion (Schwimmer and Austin, 1971b) and from kidney (Schwimmer, 1971b) containing y-glutamyl transpeptidase are useful in enhancing the quality of dehydrated onion and garlic. A patent on this process has been granted (Schwimmer, 1973). It is of interest that, since the alliinase from AZbizziu Zophantu can split alkyl- and alkenylcysteines producing the corresponding mercaptans, it can produce more flavor compounds from onion and garlic than can the native enzymes. In combination with a y-glutamyl peptidase, maximum flavor can be released (Schwimmer and Austin, 1971a). In summary, many plants contain y-glutamyl transpeptidase activity (Thomp-
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son et ul., 1964), although dormant onion bulbs apparently do not. Sprouting onion bulbs contain y-glutamyl transpeptidase activity and perhaps y-glutamyl peptidase activity. The germinating seeds of chive appear to contain a y-glutamyl peptidase. These enzymes may be used to enhance the flavor and aroma of onion and onion products.
VII. SUMMARY Onions and garlic contain large amounts of organic sulfur compounds. In garlic the flavor and odor precursors are (+)-S-allyl-L-cysteinesulfoxide (major), (+)amethyl-L-cysteine sulfoxide, and (+)-S-propyl-L-cysteine sulfoxide. In onion the flavor, odor, and lachrymatory precursors are trans-(+)a-(1-propenyl)-L-cysteine sulfoxide (major), (+)S-methyl-L-cysteine sulfoxide, and (+)-S-propyl-L-cysteine sulfoxide. As much as 50% of the total @um-(+)-S-(1-propeny1)-L-cysteine sulfoxide is present as the y-glutamyl derivative in onion and is not converted to flavor, odor, and lachrymatory factor in crushed dormant onions. As much as 30% of the total sulfur of dormant onion bulbs may be in the form of cycloalhn and not capable of being converted to flavor, odor, and lachrymatory factor in crushed onion. The pathways of biosynthesis of the flavor precursor compounds in Allium are only partially understood. trans-(t)S-(l-Propeny1)-L-cysteine sulfoxide is probably synthesized via reaction of sulfide (from inorganic sulfate) with serine to give cysteine followed by coupling with methacrylyl-CoA produced from degradation of valine. Most of the (+)-5'-methyl-L-cysteine sulfoxide is probably synthesized via methylation of cysteine. (+)-S-Allyl-L-cysteine sulfoxide (major compound of garlic) and (+)a-propyl-L-cysteine sulfoxide are probably synthesized from serine and the appropriate mercaptan via a thiolalkyl transfer reaction. Alternative pathways have not been conclusively ruled out. The flavor precursor compounds are acted on by an enzyme in Allium called alliinase to give several primary products. trans-(+)&( 1-Propenyl)L-cysteine sulfoxide in onion gives 1-propenylsulfenic acid (or thiopropanal S-oxide), which is the lachrymatory factor, pyruvic acid, and ammonia. (+)-S-Allyl-L-cysteine sulfoxide, (+)S-methyl-t-cysteine sulfoxide, and (+)-S-propyl-L-cysteine sulfoxide are split by alliinase to give the corresponding thiosulfinates, pyruvate, and ammonia. The primary sulfur-containing products of alliinase action are not stable but are rapidly converted, probably nonenzymatically, to disulfides and thiosulfonates which are primarily responsible for the odor and flavor characteristics of Allium. The decomposition of 1-propenylsulfenic acid leads to formation of several aldehydes and alcohols which may contribute to the flavor of onions.
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Distillation and/or heat treatment of crushed Allium or oils as well as storage leads to formation of substantial amounts of alkyl and alkenyl tri- and polysulfides and thiophenes from further reaction of the disulfides and thiosulfonates. The alhinases from onion and garlic have specificity for S-alkyl- and S-alkenylL-cysteine sulfoxides. Nothing is known about the physical and chemical properties of these enzymes because of inadequate purification. Sprouting onion bulbs, but not dormant ones, contain a y-glutamyl transpeptidase which removes the y-glutamyl group from y-L-glutamyl-trans-(+)-S-( 1propeny1)-L-cysteine sulfoxide to give the flavor, odor, and lachrymatory precursor. This enzyme, as well as one from kidney, has potential use, in virro, for enhancing the quality of dehydrated onions in food usage. The flavor and odor characteristics of freshly crushed onions, rehydrated dried onions, and onion oils are expected to be different because of different compositions of the components leading to these properties. Gas chromatographic methods are thought to be superior to several other described objective methods for assessing the quality of Allium for food purposes.
VIII. RESEARCH NEEDS Several deficiencies in our knowledge of how to maximize the flavor and odor of onions and garlic have already been indicated. It is well to summarize some of these again. Experimental methods have been developed for separation and determination of the quantities of the four alkyl- and alkenyl-L-cysteine sulfoxides in Allium. It would appear useful to apply these methods, along with others, in selection, and genetic breeding, of better Allium and in processing and storage for food uses. The major steps in the biosynthesis of the alkyl- and alkenyl-L-cysteine sulfoxides appear to have been elucidated. However, major work remains to be done on the enzymes involved so as to maximize production of the sulfoxides from inorganic sulfate. Of great importance is the need to understand the regulatory mechanism determining whether the reaction goes through cysteine to form trans-(+)-S-(1-propeny1)-L-cysteine sulfoxide and (+)S-methyl-Lcysteine sulfoxide, or through serine to form (+)-S-allyl-L-cysteine sulfoxide and (+)S-propyl-L-cysteine sulfoxide. Factors controlling the conversion of rrans-(+)-S-(l-propenyl)L-cysteine sulfoxide to the y-glutamyl derivative should be thoroughly investigated, since this appears to happen in the dormant onion bulb and may lead to loss of as much as 50% of the potential flavor and odor content. Some transformations of trans-(+)-
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JOHN R. WHITAKER
S-( 1-propenyl)-L-cysteine sulfoxide to flavor constituents may occur while it or
its precursors are still attached to glutamic acid. These investigations will be aided by a new synthesis of y-glutamyl dipeptides of sulfur-containing amino acids (Carson and Wong, 1974). Major effort should be given to understanding and control of the formation of cycloalliin in the dormant onion bulb, since some 30% of the total sulfur goes into this inert compound. While specificity of onion and garlic alliinases has been elucidated, nothing has been done on determining the amounts of this enzyme in various cultivars grown under various conditions, and treated in various ways in processing and storage. Chemical and physical properties of this important enzyme(s) should be elucidated. Modern protein purification methods should make this possible. Better understanding of the mechanism of conversion of 1-propenylsulfenic acid to secondary products is needed. How much is decomposed to sulfur and carbonyl compounds compared with that which goes into formation of alkenyl disulfides and thiosulfonates which are more important flavor and odor components? Can this decomposition be controlled in favor of the latter process? Since very small amounts of 1-propenyl disulfides are found in the head space gases of onions, the first route must be the major one. It is expected that alkyl- and alkenylsulfenic acids would be the primary products from the action of alliinase on allyl-, propyl-, and methylcysteine sulfoxides, although these have not been detected. Further research is needed in this area. More information is needed on the mechanism of formation of the alkyl and alkenyl di-, tri-, and polysulfides and the thiosulfonates so as to optimize conditions for conversion to most flavorful compounds. The research needs described in this, and previous, paragraphs should be greatly facilitated by the fundamental work of Nishimura and co-workers (Nishimura ef QZ., 1972,1973b) on the mass spectra of volatile sulfur compounds of foods. The large relative concentration of dipropyl disulfide found in the head space of crushed onions (Table IV) does not appear to correlate with the relatively small amounts of (+)-S-propyl-L-cysteinesulfoxide found in onions. An area outside this report but an important one for the food processor and the supplier is the effect of other food components on the onion flavor components before and after cooking.
ACKNOWLEDGMENTS I wish to express my appreciation to Basic Vegetables, Inc., Vacaville, California, for a grant-in-aid for preparing the review, and to Dr. Hank Grill of Basic Vegetables, Inc., to Dr. Mendel Mazelis, University of California, Davis, and to Dr. Sigmund Schwimmer, USDA, Berkeley, for valuable discussions.
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REFERENCES Anderson, P. M., and Schultze, M. 0. 1965. Cleavage of S-(l,2-dichlorovinyl)-L-cysteineby an enzyme of bovine origin. Arch. Biochem. Biophys. 111,593402. Austin, S . J., and Schwimmer, S. 1971, L-y-Glutamyl peptidase activity in sprouted onion. Enzymologia 4 4 273-285. Bandyopadhyay, C., and Tewari, G. M. 1973. Thin layer chromatographic investigation of color developer involved in pinking of white onion purees. J. Agr. Food Chem. 21, 95 2-954. Barnard, D. 1957. The spontaneous decomposition of aryl thiosulfinates. J. Chem. Soc., pp. 46754676. Belo, P. S., Jr. 1972. Enzymatic development of volatile components of onions. Ph.D. Thesis, Michigan State University, Lansing, Michigan. Bernhard, R. A. 1968. Comparative distribution of volatile aliphatic disulfides derived from fresh and dehydrated onions. J. Food Sci, 33, 298-304. Bernhard, R. A. 1969. Sulfur components ofAllium species as flavoring matter. Qual. Plant Mater. Veg. 18, 72-84. Bernhard, R. A., Saghir, A. R., Jacobsen, J. V., and Mann, L. K. 1964. Isolation and identification of allyl monosulfide and allyl alcohol from Allium. Arch. Biochem. Biophys. 107, 137-140. Boelens, M., de Valois, P. J., Wobben, H. J., and van der Gen, A. 1971. Volatile flavor compounds from onions. J. Agr. Food Chem. 19,984-991. Brodnitz, M. H. 1969. Unsaturated disulfides as seasoning mixtures simulating cooked onion taste and odor. Ger. Patent 1,918,056. Brodnitz, M. H., and Pascale, J. V. 1971. Thiopropanal S-oxide: A lachrymatory factor in onions. J. Agr. Food Chem. 19, 269-272. Brodnitz, M. H., and Pollock, C. L. 1970. Gas chromatographic analysis of distilled onion oil. Food Technol. (Chicago) 24,78-80. Brodnitz, M. H., Pollock, C. L., and Vallon, P. P. 1969. Flavor components of onion oil. J. Agr. Food Chem. 17,760-763. Brodnitz, M. H., Pascale, J. V., and Van Derslice, L. 1971a. Flavor components of garlic extract. J. Agr. Food Chem. 19, 273-275. Brodnitz, M. H., Pascale, J. V., and Vock, M. H. 1971b. Onion surrogating thioalkand oxides and S-propenyl propanethiosulfonate and/or S-propenyl-1-propenethiosulfonate. Ger. Patent 2,108,727. Brodnitz, M. H., Pascale, J. V., and Vock, M. H. 1972. Flavoring propyl propenylthiosulfonate. Ger. Patent 2,166,074. Brosin, J. M. 1969. A purification and partial characterization of alliinase from Allium cepa. M.S. Thesis, Univ. of Calif., Davis, California. Carson, J. F., and Boggs, L. E. 1966. The synthesis and base-catalyzed cyclization of (+)and (-)-cis-S-(1-propeny1)-L-cysteine sulfoxides. J. Org. Chem. 31, 2862-2864. Carson, J. F., and Wong, F. F. 1961a. Isolation of (+)-S-methyl-L-cysteine sulfoxide and of (+>S-n-propyl-L-cysteine sulfoxide from onions as their N-2,4-dinitrophenyl derivatives. J. Org. Chem. 26,4997-5000. Carson, J. F., and Wong, F. F. 1961b. The volatile flavor components of onions. J. Agr. Food Chem. 9,140-143. Chem. Znd. Carson, J. F., and Wong, F. F.. 1963. Synthesis of cis-S-(prop-l-enyl)-L-cysteine. (London), pp. 1764-1765. Carson, J. F., and Wong, F. F. 1974. Properties of y-glutamyl dipeptides of sulfur-containing amino acids. J. Chem. Sac., Perkins Trans. 1, 685-687.
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Carson, J. F., Lundin, R. E., and Lukes, T. M. 1966. The configuration of (+)-S-(l-propenylkl-cysteine S-oxide from Allium cepa. J. Org. Chem. 31, 1634-1635. Carson, J. F., Lundin, R. E., and Boggs, L. M. 1969.Base cyclization of frand-(1-buteny1)L-cysteine S-oxide. J. Org. Chem. 34, 1996-1998. Cavallito, C. J., and Bailey, J. H. 1944.Allicin, the antibacterial principle of AIlium sativurn. I. Isolation, physical properties and antibacterial action. J. Amer. Chem. SOC.
66,1950-1951. Cavallito, C. J., Buck, J., and Suter, C. 1944. Allicin, the antibacterial principle of Allium sativurn. 11. Determination of the chemical structure. J. Amer. Chem. Soc. 66, 1952-
1954. Challenger, F., and Greenwood, D. 1949. Sulfur compounds of the genus Allium. Detection of n-propylthiol in the onion. The fission and methylation of diallyl disulfides in cultures of Scopulariopsis brevimulis. Biochem. J. 44, 87-91. Currier, H. B. 1945. Photometric estimation of volatile sulfur in onions as a criterion of pungency. Food Res. 10,177-186. Dilbritz, E., and Virtanen, A. I. 1965.S-Vinylcysteine S-oxide, a homolog of the precursor of the lachrymatory substance in onion. Chem. Ber. 98,781-788. De Lima, D. C. 1974. Isoenzymes cleaving S-cysteine derivatives from cauliflower. M.S. Thesis, Univ. of Calif., Davis, California. Dougherty, M. H. 1968. A method for measuring the water-soluble volatile constituents of citrus juices and products. Food Technol. (Chicago) 22, 1455-1456. Ettala, T., and Virtanen, A. I. 1962. Labeling of sulfur-containing amino acids and 7glutamyl peptides after injection of labeled sulfate into onion. Acfa Chem. Scand. 16,
2061-2063. Farber, L. 1957. The chemical evaluation of the pungency of onion and garlic by the content of volatile reducing substances. Food Technol. (Chicago) 11,621-624. Foley, R. F. 1955. Factors affecting the volatile sulfur content of the onion, Allium cepa. Ph.D. Thesis, Cornell Univ., Ithaca, New York. Freeman, G. G., and McBreen, F. 1973. Rapid spectrophotometric method of determination of thiosulfinate in onion (Allium cepa) and its significance in flavor studies. Biochem. SOC.Trans. 1, 1150-1152. Freeman, G. G., and Mossadeghi, N. 1970. Effect of sulfate nutrition on flavor components of onion (Altium cepa). J. Sci. Food Agri 21, 610-615. Fujiwara, M., Yoshimura, M., and Tsuno, S. 1955. “Allithiamine,” a newly found derivative of vitamin B , . 111. On the allicin homologues in the plants of the AZZium species. J. Biochem. (Tokyo) 42,591-601. Fujiwara, M., Yoshimura, M., Tsuno, S., and Murakami, F. 1958. “Allithiamine,” a newly found derivative of vitamin B,. IV. On the alliin homologues in the vegetables. J. Biochem. (Tokyo) 45,141-149. Giovanelli, J., and Mudd, S. H. 1968. Sulfuration of Qacetylhomoserine and 0-acetylserine by two enzyme fractions from spinach. Biochem. Biophys. Res. Commun. 31, 275-280. Giovanelli, J., and Mudd, S. H. 1971.Transsulfuration in higher plants. Partial purification and properties of P-cystathionase of spinach. Biochim. Biophys. Acfa 227,654-670. Gmelin, R., Hasenmaier, G., and Strauss, G. 1957. Occurrence of djenkolic acid and C-Slyase in the seed of Albizzia lophantha. Z. Naturforsch. 12B, 687-697. Granroth, B. 1968. Separation of Allium sulfur amino acids and peptides by thin-layer electrophoresis and thin-layer chromatography. Acta Chem. Scand. 22, 3333-3335. Granroth, B. 1970. Biosynthesis and decomposition of cysteine derivatives in onion and other Allium species. Ann. Acad. Sci Fenn. Ser. A 2 154, 1-71. Granroth, B. 1974. Partial purification of cysteine synthase (0-acetylserine sulfhydrylase) from onion (Allium cepa). Acta Chem. Scand. B28,813-814.
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Granroth, B., and Sarnesto, k 1974. Synthesis of S-substituted cysteine derivatives by the cysteine synthase (Oacetylserine sulfhydrylase) of onion (Allium cepa) and Escherichia coli. Acta Chem. Scand. B28,814-815. Granroth, B., and Virtanen, A. I. 1967. S-(2-Carboxypropyl)cysteine and its sulfoxide as precursors in the biosynthesis of cycloalliin. Acta Chem. Scand. 21, 1654-1656. Hattula, T., and Granroth, B. 1974. Formation of dimethyl sulfide from S-methylmethionine in onion seedlings (Allium cepa). J. Sci. FoodAgr. 25, 1517-1521. HBrhammer, L., Wagner, H., Seitz, M., and Vejdelek, Z. J. 1968. Evaluation of garlic preparations. I. Chromatographic studies of the actual components of AIlium sativum. Pharmazie 23,462467. Jacobsen, J. V., Yamaguchi, M., Mann, L. K., Howard, F. D., and Bernhard, R. A. 1968. An alkylcysteine sulfoxide lyase in Tulbaghia violacea and its relation to other alliinase-like enzymes. Phytochemistry 7 , 1099-1108. Kawakishi, S., Namiki, K., Nishimura, H., and Namiki, M. 1971. Effectsofy-irradiation on the enzyme relating to the development of characteristic odor of onions. J. Agr. Food Chem. 19, 166-169. Kohman, E. F. 1952. Onion pungency and onion flavor: their chemical determination. Food Technol. (Chicago) 6, 288-290. Kredich, N. M., Becker, M. A., and Tomkins, G. M. 1969. Purification and characterization of cysteine synthetase, a bifunctional protein complex, from Salmonella typhimurium. J. Biol. Chem. 244,2428-2439. Kuon, J., and Bernhard, R. A. 1963. An examination of the free amino acids of the common onion (Allium cepa). J. Food Sci 28, 298-304. Kupiecki, F. P., and Virtanen, A. I. 1960. Cleavage of alkylcysteine sulfoxides by an enzyme in onion (Allium cepa). Acta Chem. Scand. 14, 1913-1918. Kuriyama, M., Takagi, M., and Murata, K. 1960. A new amino acid, chondrine, isolated from red alga. Nippon Suisan Gakkaishi 26,627;Chem. Abstr. 55, 13559 (1961). Lehmann, F. A. 1930. Investigation of the pharmacology of Allium sativum (garlic). Arch. Exp. Pathol. Pharmakol. 147, 245-264. Lewis, B. G., Johnson, C. M., and Broyer, T. C. 1971. Cleavage of Se-methylselenomethionine selenonium salt by a cabbage leaf enzyme fraction. Biochim. Biophys. Acta 237,
603-605. Lukes, T. M. 1971. Thin-layer chromatography of cysteine derivatives of onion flavor compounds and the lacrimatory factor. J. Food Sci. 36,662-664. McNary, R. R., Dougherty, M. H., and Wolford, R. W. 1957. Determination of the chemical oxygen demand of citrus waste waters. Sewage Ind. Wastes 29, 894-900. Matikkala, E. J., and Virtanen, A. I. 1965a yGlutamyl peptidase (glutaminase) in germinating seeds of chive (Allium schoenoprasurn). Acta Ckem. Scand. 19, 1258-1261. Matikkala, E. J., and Virtanen, A. I. 1965b.y-Glutamyl peptidase in sprouting onion bulbs. Acta Chem. Scand. 19,1261-1262. Matikkala, E. J., and Virtanen, A. I. 1967. On the quantitative determination of the amino acids and 7-glutamyl peptides of onions. Acta Chem. Scand. 21, 2891-2893. Matsukawa, T., Yurugi, S., and Matsuoka, T. 1953. Products of the reactions between thiamine and ingredients of Allium species. Detection of allithiamine and its homologues. Science 118, 325-327. Mazelis, M. 1963. Demonstration and characterization of cysteine sulfoxide lyase in the Cruciferae. Phytochemistry 2, 15-22. Mazelis, M., and Crews, L. 1968. Purification of the a l h lyase of garlic, AIlium sativum L. Biochem. J. 108, 725-730. MazeIis, M., Levin, B., and Mallinson, N. 1965. Decomposition of methylmethionine sulfonium salts by a bacterial enzyme. Biochim. Biophys. Acta 105, 106-1 14.
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Moisio, T., Spare, C.-G., and Virtanen, A. 1. 1962. Mass spectral studies of the chemical nature of the lachrymatory factor formed enzymatically from S-(1-propeny1)cysteine sulfoxide isolated from onion (Allium cepa). S u m . Kemistilehti B35, 29, MUller, A. L., and Virtanen, A. I. 1965. On the bicsynthesis of cycloatliii. Acta Chem. Scand. 19,2257-2258. Murakami, F. 1960. The nutritional value of allium plants. XXXVI. Decomposition of alliin homologs by microorganisms and formation of substance with thiamidemasking activity. Bitamin 20, 126-131; Chem. Abstr. 61,15094d (1964). Niegisch, W. D., and Stahl, W. H. 1956. The onion: gaseous emanation products. Food Res. 21,657-665. Nishimura, H., and Mizutani, J. 1973a. cis-trans Isomerization products from cis-S-(l-propeny1)-L-cysteine and its sulfoxide irradiated in aqueous solutions. Agr. Biol. Chem. 37, 21 3-21 7. Nishimura, H., and Mizutani, J. 1973b. The SC cleavage of cis-S-(1-propeny1)-L-cysteine sulfoxide by OH radicals from irradiated aqueous solutions. Agr, Biol. Chem. 37, 1749-1750. Nishimura, H., Kawakishi, S., and Namiki, M. 1970. Studies on y-radiolysis of sulfur containing amino acids. I. 7-Radiolysis of S-n-propyl-L-cysteine sulfoxide and S-allyl-Lcysteine sulfoxide in aqueous solution. Agr. Biol. Chem. 34,609-616. Nishimura, H., Mizutani, J., and Obata, Y. 1971a. Studies on y-radiolysis of sulfur containing amino acids. 11. Volatile products from S-n-propyl-L-cysteine sulfoxide and s-allyl-Lcysteine sulfoxide irradiated in oxygen-free aqueous solutions. Tefrahedron 27, 307313. Nishimura, H., Asahi, A., Fujiwara, K.,Mizutani, J., and Obata, Y. 1971b. Changes in flavor components of onion by 7-irradiation. Agr. Biol. Chem. 35, 1831-1835. Nishimura, H., Tahara, S., Okuyama, H., and Mitzutani, J. 1972. Mass spectra of sulfurcontaining amino acids and peptides. Tetrahedron 2 8 , 4 5 0 3 4 5 1 3. Nishimura, H., Hanzawa, T., and Mizutani, J. 1973a. Photochemistry of sulfur-containing amino acids. Formation of thiophene derivatives. Tetrahedron Lett. 4, 343-344. Nishimura, H., Koike, S., and Mizutani, J. 1973b. Mass spectra of volatile sulfur compounds in foods. Agr. Biol. Chem 37,1219-1 220. Nomura, J., Nishizuka, Y.,and Hayaishi, 0. 1963. S-Alkylcysteinase; enzymic cleavage of S-methyl-L-cysteine and its sulfoxide. J. Biol. Chem. 238, 1441-1446. Oaks, D. M., Hartmann, H., and Dimick, K. P. 1964. Analysis of sulfur compounds with electron capture hydrogen flame dual channel chromatography. And. Chem. 36, 156& 1565. Orlowski, M., and Meister, A. 1970. The y-Glutamyl cycle: a possible transport system for amino acids.Proc. Nut. Acad. Sci. US.67, 1248-1255. Palmer, R. J., and Lee, K. S. 1966. The structure of cycloalliin hydrochloride monohydrate. Acta Crystallogr. 20, 790-795. Pant, R., Agrawal, H. C., and Kapur, A. S. 1962. The water-soluble sugars and total carbohydrate contents of onion (Allium cepa), garlic (Allium sativum) and turnip (Brassica rapa). Flora (Jena) 152,530-533. Platenius, H. 1935. A method for estimating the volatile sulfur content and pungency of onions. J. Agr. Res. 51, 847-853. Platenius, H., and Knott, J. E. 1941. Factors affecting onion pungency. J. Agr. Res 62, 371-379. Rundqvist, C. 1909. Pharmacological investigation of Allium bulbs. Pharm. Notisblad 18, 323-3 3 3. Saari, J. C., and Schultze, M. 0. 1965. Cleavage of S-(1,2-dichlorovinyl)-~-cysteine by Escherichia coli B. Arch. Biochem. Biophys. 109, 595-602.
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Saghir, A. R., Mann, L. K., Bernhard, R. A., and Jacobsen, J. V. 1964. Determination of aliphatic mono- and disulfides in Allium by gas chromatography and their distribution in the common food species. Proc. Amer. Soc. Hort. Sci, 84, 386-398. Saghir, A. R., Mann, L. K., and Yamaguchi, M. 1965. Composition of volatiles in Allium as related to habitat, stage of growth, and plant part. Plant Physiol. 40, 6 8 1 4 8 5 . Saghir, A. R. B., Mann, L. K., Ownbey, M., and Berg, R. Y. 1966. Composition of volatiles in relation to taxonomy of American Alliums. Amer. J. Bot. 53,477-484. Saguy, M., Mannheim, C. H., and Peleg, Y. 1970. Estimation of volatile onion aroma and flavor compounds. J. Food Technol. 5,165-171. Schultz, 0 . E., and Mohrmann, H. L. 1965. Analysis of constituents of garlic, Allium sntivum. 11. Gas chromatography of garlic oil. Pharmazie 20,441447. Schwimmer, S . 1964. L-Cysteine sulfoxide lyase. Competition between enzyme and substrate for added pyridoxal phosphate. Biochim. Biophys. Acta 81,377-385. Schwimmer, S . 1967. Development of a bitter substance in onion juice. Food Technol. (Chicago) 21, 292. Schwimmer, S. 1968. Enzymic conversion of trans-(+)S-1-propenyl-L-cysteineS-oxide t o the bitter and odor-bearing components of onion. Phytochemistv 7 , 4 0 1 4 0 4 . Schwimmer, S . 1969. Characterization of S-propenyl-L-cysteine sulfoxide as the principal endogenous substrate of L-cysteine sulfoxide lyase of onion. Arch. Biochem. Biophys. 130,312-320. Schwimmer, S . 1971a. S-Alkyl-L-cysteine sulfoxide lyase [ANium cepa (onion)]. Methods Enzymol. 17B, 4 1 5 4 7 8 . Schwimmer, S. 1971b. Enzymatic conversion of y-L-glutamyl cysteine peptides to pyruvic acid, a coupled reaction for enhancement of onion flavor. J. Agr. Food Chem. 19, 980-983. Schwimmer, S. 1973. Flavor enhancement of Allium products. U. S . Patent 3,725,085. Schwimmer, S., and Austin, S. J. 1971a. y-Glutamyl transpeptidase of sprouted onion. J. Food Sci. 36,807-811. Schwimmer, S., and Austin, S. J. 1971b. Enhancement of pyruvic acid release and flavor in dehydrated Allium powders by gamma glutamyl transpeptidases. J. Food Sci 36, 1081-1 085. Schwimmer, S., and Friedman, M. 1972. Genesis of volatile sulfur-containing food flavors. Flavor Ind. 137-145 (March). Schwimmer, S., and Guadagni, D. G. 1962. Relation between olfactory threshold concentration and pyruvic acid content of onion juice. J. Food Sci 27, 94-97. Schwimmer, S., and Guadagni, D. G. 1967. Cysteine-induced odor intensification in onions and other foods. J. Food Sci. 32,405408. Schwimmer, S., and Guadagni, D. G. 1968. Kinetics of the enzymatic development of pyruvic acid and odor in frozen onions treated with cysteine C-S-lyase. J. Food Sci 33, 193-196. Schwimmer, S., and Kjaer, A. 1960. Purification and specificity of the C-S-lyase of Albizzia lophantha. Biochim. Biophys. Acta 42, 3 16-324. Schwimmer, S., and Mazelis, M, 1963. Characterization of alliinase ofAllium cepa (onion). Arch. Biochem. Biophys. 100,66-73. Schwimmer, S., and Weston, W. K. 1961. Enzymatic development of pyruvic acid in onion as a measure of pungency. J. Agr. Food Chem. 9,301-304. Schwimmer, S . , Venstrom, D. W., and Guadagni, D. G. 1964a. Relation between pyruvate content and odor strength of reconstituted onion powder. Food Technol. (Chicaw) 18, 121-124. Schwimmer, S., Ryan, C. A., and Wong, F. F. 1964b. Specificity of L-cysteine sulfoxide lyase and partially competitive inhibition by S-alkyl-L-cysteines. J. Biol. Chem. 239, 777-782.
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Semmler, F. W. 1892. The essential oil of onion (Allium cepa L ) . Arch. Pharm. 230, 434443. Shannon, S., Yamaguchi, M., and Howard, F. D. 1967. Precursors involved in the formation of pink pigments in onion purees. J. Agr. Food Chem. 15,423-426. Smith, I. K., and Thompson, J. F. 1969. The synthesis of 0-acetylserine by extracts prepared from higher plants. Biochem. Biophys. R e s Commun 35, 939-945. Spire, C.-G., and Virtanen, A. I. 1961. The volatile carbonyls and alcohols in the flavor substances of onion (Allium cepa).Acta Chem. Scand. 15, 1280-1284. Spbre, C.-G., and Virtanen, A. I. 1963. On the lachrymatory factor in onion (Allium cepa) vapors and its precursor. Acta Chem. Scand. 17,641-650. Spare, C.-G., and Virtanen, A. I. 1964. On the lachrymatory factor in onion (Allium cepa) vapors and its precursor. In “Primary Plant Substances and Decomposition Reactions in Crushed Plants Exemplified Mainly by Studies on Organic Sulfur Compounds in Vegetables and Fodder Plants” (A. 1. Virtanen, ed.), pp. 120-132. Biochemical Inst., Helsinki. Stoll, A., and Seebeck, E. 1947. Alliin, the pure mother substance of garlic oil. Experienta 3,114-115. Stoll, A., and Seebeck, E. 1948. Allium compounds. I. Alliin, the true mother compound of garlic oil. Helv. Chim. Acta 31, 189-210. Stoll, A., and Seebeck, E. 1951. Chemical investigations on alliin, the specific principle of garlic. Advan. Enzymol. 11,377-400. Sugii, M., Nagasawa, S., and Suzuki, T. 1963a. Biosynthesis of S-methyl-L-cysteine and S-methyl-L-cysteine sulfoxide from methionine in garlic. Chem. Pharm. Bull. 11, 135136; Chem. Abstr. 58,1447b (1963). Sugii, M., Suzuki, T., and Nagasawa, S. 1963b. Isolation of (-)S-propenyl-L-cysteine from garlic. Chem. Pharm. Bull. 11,548-549; Chem. Abstr. 59,6509b (1963). Sugii, M., Suzuki, T., Kakimoto, T., and Kato, J. 1964. Sulfur-containing amino acids and related compounds in garlic. I. Assimilation of sulfate35S in garlic. Bull. Znst. Chem. Res. Kyoto Univ. 42, 246-251; Chem. Abstr. 62,5582g (1965). Suzuki, T., Sugii, M., and Kakimoto, T. 1961a. New 7-glutamyl peptides in garlic. Chem. Pharm. Bull. 9,77-78; Chem. Abstr. 55,21258i (1961). Suzuki, T., Sugii, M., Kakimoto, T., and Tsuboi, N. 1961b. Isolation of (-)S-allyl-L-cystehe from garlic. Chem. Pharm. Bull 9, 251-252; Chem. Abstr. 55,237028. (1961). Suzuki, T., Sugii, M., and Kakimoto, T. 1962. Metabolic incorporation of L-valine-C’4 into S-(2-carboxypropyl)glutathione and S-(2-~arboxypropyl)cysteine in garlic. Chem. Pharm Bull. 10,328-331; Chem. Abstr. 57, 11561b (1962). Tamura, G. 1965. Sulfite-reducing systems of higher plants. 11. Purification and properties of sulfite reductase from Allium odorum. J. Biochem. (Tokyo) 57, 207-214. Theodoropoulos, D. 1959. Synthesis of certain S-substituted L-cysteines. Acta Chem. Scand. 13,383-384. Thompson, J. F. 1967. Sulfur metabolism in plants. Annu. Rev. Plant Physiol. 18,59-84. Thompson, J. F., and Gering, R. K. 1966. Biosynthesis of S-methylcysteine in radish leaves. PlantPhysioI. 41,1301-1307. Thompson, J. F., and Moore, D. P. 1968. Enzymatic synthesis of cysteine and S-methylcysteine in plant extracts. Biochem. Biophys. Res. Commun. 31, 281-286. Thompson, J. F., Turner, D. H., and Gering, R. K. 1964. yGlutamy1 transpeptidase in plants. Phytochemistv 3, 33-46. Tominaga, F., and Oka, K. 1963. The isolation and identification of 1,4-thiazan-3carboxylic acid S-oxide from the brown algae, Undaria pinnafijida. J. Biochem. (Tokyo) 54, 222-224. Virtanen, A. I. 1964. Pharmacologically significant organic sulfur compounds in foods of humans and domestic animals. Suom KemisfilehfiA37, 108-125.
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Virtanen, A. I. 1969. Antimicrobial and antithyroid compounds in some edible vegetables. @ul. Plant. Muter. Veg. 18, 8-28. Virtanen, A. I., and Matikkala, E. J. 1956. A new sulfur-containing amino acid in the onion. Suom. Kemistilehti B29, 134-135. Virtanen, A. I., and Matikkala, E. J. 1959. The isolation of S-methylcysteine sulfoxide and S-n-propylcysteine sulfoxide from onion (Allium cepa) and the antibiotic activity of crushed onion. Actu Chem. Scund. 13, 1898-1900. Virtanen, A. I., and Matikkala, E. J. 1961. Evidence for the presence of yglutamyl-S-(1-propeny1)cysteine sulfoxide and cycloalliin as original compounds in onion (AIZium cepu). Suom. Kemistilehti B34, 114. Virtanen, A. I., and Sphre, C.-G. 1961. Isolation of the precursor of the lachrimatory factor in onion (Allium cepa). Suom. Kemistilehti 34,12. Wertheim, T . 1845. On the relationship between mustard and garlic oils. Ann. 55,297-364. Wertheim, T. 1884. Investigation of garlic oil. Ann. 51, 289-315. Whitaker, J. R. 1972. “Principles of Enzymology for The Food Sciences,” p. 393. Dekker, New York. Wilkens, W. F. 1961. The isolation and identification of the lachrymogenic compounds of onion. Cornell Univ., Ph.D. Thesis. University Microfilms, Inc., Ann Arbor, Michigan. Yamanishi, T., and Orioka, K. 1955. Chemical studies on the change of flavor and taste of onion by boiling. . I . Home Econ. 6 , 4 5 4 7 .
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CLOSTRIDIUM BOTULINUM AND ITS IMPORTANCE IN FISHERY PRODUCTS BY G. HOBBS Ministry of Agti’nrlture, Fisheries and Food Torry Research Station, Aberdeen, Scotland
I. 11. 111. N. V. VI. VII. VIII. IX.
Introduction .................................................... Incidence and Distribution ......................................... Classification and Nomenclature ..................................... Methods for Isolation and Identification .............................. Properties of the Toxins ........................................... Factors Affecting Germination, Growth, Sporulation, and Toxin Production Inhibition of Clostridium botulinum ................................. Preservation of Fishery Products .................................... SummaryandConclusions ......................................... References .....................................................
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I. INTRODUCTION Botulism is a neuroparalytic disease affecting man and animals which generally results from the consumption of food cmtaining toxins produced by one or other of a group of anaerobic bacteria classified as Closrridium botulinum. The historical documentation of botulism has been summarized by Dolman (1964a). As a food-borne disease it has been recognized for more than 1000 years; it was certainly well known as a clinical disease in Germany during the eighteenth century where it was particularly associated with the consumption of a popular type of smoked blood sausage. In 1820 and 1822 Justinus Kerner attributed the disease to a toxic fatty acid developing within the sausage during storage. The term “botulism” (botulus-latin for sausage) was first used by Miller in 1870, but it was not until 1897 that the causative organism was first isolated and described by Van Ermengen from an outbreak in Holland resulting from the consumption of raw salted ham. He noted the similarities of this 135
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outbreak with sausage poisoning and named the causative organism Bacillus botulinus. It was soon realized that at least two kinds of anaerobic bacteria, proteolytic and nonproteolytic, producing at least three pharmacologically identical but serologically distinct toxins, were involved in outbreaks of botulism (Bengtson, 1924). The strain isolated by Van Ermengen was nonproteolytic and produced type B toxin; proteolytic organisms producing types A and B toxins were recognized, and Bengtson (1922) and Seddon (1922) both described nonproteolytic strains producing type C toxin which caused botulism in animals. A fourth toxin, type D, was then described by Thieler et al. (1927), also produced by strains causing botulism in animals. In the mid-1930's a further type was discovered as a result of a survey of samples from fish carried out in Russia. The survey was prompted by a history of fish-borne botulism in Russia and by the largest epidemic recorded there in 1933 where there were 230 cases of botulism and 94 deaths resulting from the consumption of food containing stuffed eggplant (Dolman, 1964a). Although type A was shown to be responsible for the epidemic, the fish samples yielded ten strains of an organism which was not particularly heat-resistant, was nonproteolytic, and was not neutralized by antitoxins to types A through D. These strains were examined and described by Gunnison et al. (1935), who proposed that they should be designated type E. Two earlier outbreaks, in 1932 from smoked salmon and in 1934 from canned sprats, were subsequently shown to be caused by type E also (Hazen, 1937, 1938). It was not until 1960 that a proteolytic organism producing type F toxin was isolated (Mglller and Scheibel, 1960) from an outbreak on the Danish island of Langeland resulting from the consumption of liver paste. Subsequently it was shown that nonproteolytic strains were also able to produce type F toxin (Eklund and Poysky, 1967). More recently still a proteolytic organism producing yet another toxin, type G, has been isolated from soil (Gimenez and Ciccarelli, 1970a). Type G toxin has not yet been found in an outbreak of botulism. Botulism resulting from the consumption of fish has been recorded in the Russian literature since 1818, and in fact an anaerobic organism responsible was isolated from smoked sturgeon in 1914 and named Bacillus ichthyismi (Konstansov, 1914). It was conceded, however, that it was probably only a variety of B. botulinus. The history of fish-borne botulism has been well documented by Dolman and his colleagues (see Dolman, 1964a), who were also responsible for the thorough documentation of botulism caused by the consumption of uncooked marine mammals and fish among the Eskimos in Alaska, the Northwest Territories, and Labrador. From about 1950 onward it became apparent that type E botulism resulting from the consumption of fish was also common in Japan (Sakaguchi, 1969). Before the early 1960's it was generally accepted that commercial processing of foods had reasonably well taken care of the botulism hazard. The properties of the organism and its toxin were well known, and the commercial canning
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industry had long since taken account of the heat-resistant spores of this organism. Botulism outbreaks were generally a result of poor home processing or the consumption of raw products in certain parts of the world. In 1963, however, there was a sudden increase in cases of botulism in the United States; 46 were recorded, of which 22 were from commercially processed and distributed foods including smoked fish, canned tuna, and canned liver paste. The outbreaks from fish caused an immediate upsurge of investigations into Cl. botulinum and its relationships with fishery products. The U. S. Department of Health called a conference on botulism in 1963, and there was an international conference on botulism in 1966 in Moscow. The proceedings of these two meetings effectively summarized the state of knowledge on this subject at that time (Lewis and Cassel, 1964; Ingram and Roberts, 1967). The main properties of the organisms and their toxins which are of importance to food processors were known at the time of Van Ermengen’s classical report in 1897. He recognized that botulism could be caused by the consumption of an assortment of sausages and preserved meats, that it was probably identical with the “ichthyism” in the Russian literature, and that it was distinguishable from paralytic shellfish poisoning. He claimed that the organism did not cause an infection and it did not produce a toxin in the living animals. He also calculated that the toxin was the most potent produced by bacteria and noted that there were marked differences in the susceptibility of different animals to the toxin. He demonstrated that the toxin was inactivated by light, air, alkalinity, and relatively mild heat treatment. He described the spores and their heat resistance and the effects of salt and temperature on growth. The optimum temperature for growth was 20” to 30”C, sodium carbonate improved growth in most media, and cultures had a characteristic butyric acid odor. Detailed descriptions of the morphology and biochemical reactions of his nonproteolytic strain were also given. Botulism has therefore for many years resulted from failure to observe one or other of the known factors which will control this organism and its toxin. This has added significance in more recent years with the increase in large-scale food processing and catering. It was not possible to consider all of the very large amount of information accumulated in the literature on botulism and CI. botulinum in the space available. Those aspects important to the processing and consumption of fish will be considered, and other aspects such as the pharmacology of the toxin and immunity to and treatment of botulism will be referred t o only where necessary.
II. INCIDENCE AND DISTRIBUTION All the early work on Cl. botulinum indicated that it was primarily a soil organism and that soil was the main source of food contamination. Although Van Ermengen suspected this, it was not until the intensive surveys carried out
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in the early 1920’s that evidence accumulated to support the idea (Meyer and Dubovsky, 1922a, b, c, d). Investigations of fish botulism from the 1930’s onward have demonstrated a peculiar relationship of type E with fish and fishery products. This led to the suggestion that fish intestines were a potential reservoir of type E. In reviewing this subject Dolman (1957a, b, 1964a) described the accumulated data on the incidence of type E in the marine environment and suggested that, although the intestinal tract of fish may be a reservoir, the primary source of type E spores was probably soil. He suggested that the main cause of regional variations in the incidence of botulism was the variable incidence of spores in local soils or off-shore waters and the extent to which local food preservation methods render them subject to contamination by viable spores, whch could then germinate and grow to produce toxin. At about the same time, Johannsen (1962,1963, 1965a) reported widespread dissemination of type E spores in the soil and catchment area of the Baltic Sea. His evidence supported the view that the primary source of type E was the soil and that the highest concentrations of spores occurred where reservoirs of water were fed by run off from a large land mass. Such a situation is well illustrated by Johannsen’s figures for the stretch of water between Sweden and Denmark, where 100% of sediment samples yielded type E. The unusually high incidence in this area was confirmed by Cann e t QZ. (1965a, 1967) with some additional data which showed a much lower incidence in the Skagerrak and in the North Sea. Johannsen (1963) had also reported a low incidence of type B from sediments taken off the Norwegian coast in the North Sea. Similarly Cann et al. (1968) found a low incidence of type B strains in sediments collected around the coast of the United Kingdom. An occasional type E and one type F were also found in this study. Although there is no recent evidence, the earlier surveys of Meyer and Dubovsky (1922a, b, c, d) demonstrated a low incidence of only type B in soil samples from England. Further studies of marine sediments and fish from around the United Kingdom have consistently shown a very low incidence which contrasts markedly with that reported by Johannsen in Sweden. Following the outbreaks from Great Lakes fish in the United States, a number of surveys of sediments and samples of aquatic animals and fish demonstrated areas with an incidence similar to that in Scandinavia. Bott et al. (1965) described an extremely variable incidence in the intestines of fish from different parts of the Great Lakes. The incidence varied from 0.7% to well over 50%, all type E. Pace et al. (1967a, b) found an incidence of 8 to 30%, and Fantasia and Duran (1969) found 6 to 50% also in Great Lakes fish. Subsequent studies by Bott et al. (1968) showed that, in Green Bay on Lake Michigan, the high incidence in the sediments could not be explained by fresh-water runoff from contaminated soil. They suggested that in this case growth of type E occurred in the sediment. Further evidence for this was provided by Sugiyama (1971), who showed in addition that spores acquired in the fishes’ food did not establish themselves in their intestines.
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On the Pacific Coast of the United States, Eklund and Poysky (1965, 1967) examined fish, shellfish, and marine sediments. A high incidence of type E was found, particularly in the estuary of the Columbia River. Further from the mouth of the river the incidence was less until in their samples furthest south, off the coast of California, type E was virtually absent, although some types A and B were found. The type B strains were shown to be nonproteolytic with properties very similar to those of type E (Eklund and Poysky, 1967). In addition, Eklund and Poysky (1965) and Eklund et al. (1967) described two strains from their marine sediments which produced type F toxin. Unlike the Langeland strain of type F, however, these were nonproteolytic and had properties similar to those of type E. These studies were complemented by those of Craig and Pilcher (1967) and Craig et al. (1968), who examined fish intestines and sediments from the Pacific Northwest. These authors (Craig and Pilcher, 1966) also described a nonproteolytic strain of type F, this time isolated from the intestine of a salmon caught in the Columbia River. In a series of surveys around the Gulf Coast of the United States and the southeastern Atlantic coast, varying numbers of types A through F were found, although not the high incidence reported in other areas (Carroll et at!, 1966; Ward et al., 1967a, b, c, d). These results indicated that a rather higher incidence could be found in summer than in winter and that type E tended to be more common in the eastern part of the Gulf Coast. In the same area a few percent of samples of sediments and oysters were found to contain types C, D, and E (Presnell et al., 1967). Surveys along the eastern coast of the United States and Canada showed that type E was also present in fish and sediments (Nickerson et al., 1967; Cabelli and Richards, 1966; Laycock and Loving, 1972; Laycock and Longard, 1972). Occasional samples yielded types B and C, and two proteolytic strains of type F were isolated from crabs (Williams-Walls, 1968). The data of Laycock and Loving (1972) and Laycock and Longard (1972) provides further convincing evidence for a relationship between fresh-water runoff and concentration of type E spores in sediments. The distribution was correlated with patterns of terrigenous sedimentation. Other studies in the United States showed type E to be present in inland lakes other than the Great Lakes (Chapman and Naylor, 1966), in commercially caught salmon off the coast of Alaska (Houghtby and Kaysner 1969), and in marine sediments in the same area (Miller and Clark, 1971; Miller et al., 1972. In the U.S.S.R. similar reports of the incidence of CI. botulinum in soils have appeared (Kravchenko and Shishulina, 1967); types A through E were found with an incidence ranging from 2.1 to 14% depending on area. In Japan Yamamoto et af. (1970) described an incidence of 2.8 to 17% in fresh-water sediments and fish. In Indonesia and Java about 10% of marine sediments and animals yielded C1. botulinum types A through F (Mortojudo et al., 1973), and 135 sediment
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samples from the Caspian coastal waters of Iran contained twenty-two type E and two type B (Rouhbakhsh-Khaleghdoust, 1973). Since 1963 few surveys have been carried out in Europe and Scandinavia. Baumgart (1972), reported that 18 out of 252 samples of marine fish landed in Germany by commercial fishing boats yielded type E. However, renewed interest resulted from an outbreak of botulism in Germany (Baumgart, 1970) following the consumption of smoked trout fillets. An investigation of trout farms in Germany showed that a high incidence of type E occurred in some instances (Bach and MUller-Prasuhn, 1971; Bach et al., 1971; Wenzel et al., 1971a, b). The high incidence in trout was related to a relatively heavy contamination of the pond sediments. It has since become evident that a very high incidence of type E can occur in farmed trout in Denmark (Huss et al., 1974a, b) and that contamination can be high in some farms in the United Kingdom (Torry Research Station, unpublished data). In t h i s latter case nonproteolytic strains of types B and F have been found in addition to type E. An examination of farmed trout imported into the United Kingdom has shown that the incidence here varies from 10 to 60% (Jarvis and Corbett, 1972). The accumulated evidence now strongly suggests that Cl. botulinum type E, and probably the other types occurring in the aquatic environment, are essentially of terrestrial origin and that they enter the aquatic environment and hence fish by terrigenous sedimentation. The more recent evidence bears out the conclusions drawn by Dolman (1957a). Although there is no direct evidence that C1. botulinum will grow in marine sediments, there is no reason to suppose it would not if the temperature were high enough. Growth would be slow, however, since a salt concentration of 2.5 to 3% will slow down growth appreciably, although not inhibiting it completely. In fresh-water sediments there is evidence from the work carried out in the Great Lakes, and more recently from the work on trout farms in Europe, that growth can occur under suitable climatic conditions. There is no evidence that C1. botulinum can establish itself in the intestines of fish; indeed such evidence that is available suggests the contrary (Buttiaux, 1968a, b; Sugiyama, 1971; Bach and MUller-Prasuhn, 1971; Bach et al., 1971; Wenzel et al., 1971a, b; Huss et aL, 1974a, b). This is in accordance with the long-held view that the type of intestinal flora found in fish is governed by their feed (Shewan and Hobbs, 1967). Along with studies on the incidence of Cl. botulinum in the aquatic environment there has been some interest in the incidence on fish as it reaches the consumer. In the United Kingdom 5 out of a total of 646 vacuum-packed fish products were found to contain type E (Hobbs et al.. 1965b; Cann et al., 1966a). In Sweden Johannsen (1965b) found type E in 18 out of 144 samples of smoked herring and one sample of smoked trout, and in Denmark an incidence of 1.68% of type E was reported in smoked salmon purchased from retail outlets
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(Nielsen and Pedersen, 1967). In an examination of the smoking of Great Lakes chub in the United States, Pace et al. (196713) showed that although the smokmg process reduced the contamination level, appreciable numbers of fish were still contaminated after processing. An examination of 240 retail samples of smoked fish in the Pacific Northwest of the United States showed that 11 were contaminated with type E (Hayes et al., 1969, 1970). In contrast, none of 100 samples of commercially prepared vacuum-packed frozen raw flounder contained Cl. botulinum (Insalata et al., 1967), and only one type B was found in a survey of a variety of convenience foods in the United States (Insalata et al., 1969, 1970). Love11 and Barkate (1969) failed to demonstrate this organism in commercially prepared fresh-water crayfish, and Lerke (1973) failed to demonstrate it in 35 retail samples of seafood cocktail. A further examination of convenience foods, however, demonstrated one type E and one type B from 113 samples (Taclindo et al., 1967). In Iran 22 out of 124 samples of smoked and salted fish contained type E and one sample of fish eggs contained type B (Rouhbakhsh-Khaleghdoust, 1973). The more recent information confirms the earlier findings that the incidence of C1. botulinum in freshly caught fish is extremely variable; there are geographical areas where a high incidence can be found, and other areas where there are few, if any. Dolman’s earlier view that the primary source of this organism was soil has largely been substantiated, although in addition aquatic sediments, at least those in fresh water, can support multiplication of the nonproteolytic varieties under suitable climatic conditions, and thus can act as a primary source of contamination. A smaller amount of data is available on the incidence in fishery products reaching the consumer. It is apparent, however, that a sufficiently high level of contamination remains on such products to indicate that appropriate precautions should be employed throughout processing and distribution. The incidence is too low in general, however, to permit much useful information to be obtained by routine sampling. Botulism is a rare disease, and to maintain an indication of consumer risk by routine survey work would require the testing of an inordinately large number of samples. Control of botulism must therefore be achieved by adequate process and storage control, and this requires a thorough understanding of all the factors affecting survival, growth, and toxin production. In the Introduction it was stated that many of these factors were known a long time ago. Nevertheless outbreaks of botulism still occur, usually as a result of mishandling at some stage. Dolman (1964a) summarized the situation up to 1963 as regards outbreaks of botulism from fishery products. Since then outbreaks still occur; for instance, in 1970 two outbreaks were reported in Japan (Craig et al., 1970; Fukuda et al., 1970), and in Norway four outbreaks have been reported recently from “rakefisk”-two type E and two type B (Hauge, 1970). A type E outbreak occurred in Poland following the consumption of
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canned fish (Meisel et ul., 1964) and commercially canned tuna was suspected of causing an outbreak in Australia (Bennett et al., 1968). In the U.S.S.R. 24 individuals contracted type E botulism after eating salted fish from the Sea of Azov; in this case prompt treatment prevented any deaths (Pak et ul., 1971). Several outbreaks are believed to occur annually in Iran as a result of consumption of fish. Smoked trout was responsible for a type E outbreak in Germany in 1970 (Baumgart, 1970), and another type E outbreak occurred in the United States from home-prepared gefdte fish made from Great Lakes whitefish chub (Armstrong et al., 1969). In Poland it has been reported that out of some few hundred cases of botulism occurring annually in recent years (mostly types A and B) an appreciable proportion (6.8%) resulted from consumption of fish (Anusz and Przybyszewski, 1972).
I I I. CLASS1F ICATION AN D NOM ENCLATU R E There has been disagreement concerning the taxonomy of the anaerobic spore-forming bacilli for as long as they have been known. The two most comprehensive works classifying these organisms which are currently in use are Bergey’s Manual of Determinative Bacteriology (1957) by Breed et al. and Les Bacteries Anaerobies (1967) by Prkvot et al. In the classification of Prkvot et al. the anaerobic spore-forming bacteria are placed in two orders, four families, and nine genera. In Bergey’s Manual of Determinative Bacteriology, which is more popular probably because of its relative simplicity, they are placed in one genus, Clostridium, along with the genus Bacillus in the family Bacillaceae. The eighth edition of Bergey’s Manual (Buchanan and Gibbons, 1974) retains this classification for most species; however, a small number of sulfate-reducing species are placed in a new genus, Desulphotomaculum (Campbell and Postgate, 1965). Classification of species within the genus Clostridium remains based primarily on convenience rather than any fundamental taxonomic principles. When first described, all those clostridia producing a neurotoxin causing the typical symptoms of botulism were included in one species, Clostridium botulinum, despite the fact that strains differed widely in their metabolic activities. In addition, strains were known that could be distinguished from Cl. botulinum only by their lack of ability to produce a neurotoxin; these were excluded from the genus. Bengtson (1924) proposed that, since proteolytic activity, or lack of it, was a more stable characteristic than toxin production, the various strains should be differentiated into two species on the basis of this property. Since Van Ermengen’s original strain was nonproteolytic, this and similar strains should be retained within the species Cl. botulinum. The proteolytic strains should then be placed in a new species, C1. parabotulinum. The different types of Cl. botulinum
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and Cl. parabotulinurn were distinguished by the serological specificity of their neurotoxins. Bengtson’s proposals were accepted and maintained up to the seventh edition of Bergey’s Manual (1957), where Cl. botulinum included the nonproteolytic strains of types B, C, D, and E, and Cl. parabotulinum the proteolytic strains of types A and B. During more recent years the use of the specific epithet parabotulinum dropped out of common usage, and when both proteolytic and nonproteolytic strains of type F and the proteolytic type G were described they were all called Cl. botuZinum. This common usage has been recognized in the eighth edition of Bergey’s Manual (Buchanan and Gibbons, 1974) where all strains are included in the one species. In the classification of PrCvot et al. they are all included in a subgenus CL botulinum in three species. With some additions the present descriptions of the proteolytic and nonproteolytic strains are essentially the same as when they were first described. Both are gram-positive, anaerobic rods, motile with peritrichous flagella, and producing oval subterminal spores. The spores of some strains have characteristic appendages. The G t C ratio of the DNA is 26 to 28%, in common with that of many other clostridia. All strains produce a lipase and hence give a characteristic reaction on egg-yolk media. The nonproteolytic strains generally do not break down proteins, although most strains slowly digest gelatin, and they ferment a wide range of carbohydrates. The products of fermentation are chiefly butyric acid with a small amount of acetic acid. The proteolytic strains break down a range of proteins with the production of hydrogen sulfide and generally ferment only one or two carbohydrates. The products of fermentation are acetic, butyric, propionic, isobutyric, and isocaproic acids, and propyl, isobutyl, butyl, and isoamyl alcohols. Colony variation has always been recognized as a common occurrence among clostridia, sometimes due t o differences in medium composition, sometimes due to the relative abundance of spores in the colony, and doubtless sometimes due to genetic mutations, although there is no direct evidence of this in the literature. Loss of toxicity in laboratory cultures is also a common enough experience, and toxin production by a particular strain can often be improved or reduced by colony selection. There has, however, been little to substantiate the occurrence of genetic mutations such as those described by Dolman ( 1 9 5 7 ~ )and by Dolman and Murakami (1961). These mutations were reported to involve the loss of toxicity, changes in colony morphology, and gross changes in a range of biochemical activities. Hobbs et al. (1965a) studied some of these mutants supplied by Dolman along with typical toxic strains and other nontoxic strains isolated by themselves. They employed additional biochemical and serological comparisons and concluded that the so-called mutants differed from toxic cultures in too many features to be accounted for by mutation. They suggested that the mutants isolated by Dolman were in fact commensal organisms belonging to the CL bifermentans-sordellii group. Walker and Batty (1967a) and
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Hodgkiss et ul. (1966, 1967) provided further evidence based on spore morphology. In addition, Lee and Riemann (1970a, b) and Wu et ul. (1972) showed that, on the basis of DNA homology studies, Dolman’s 0s cultures were not related to toxic strains of Cl. botulinum, whereas other nontoxic cultures such as those described by Hobbs et ul. (1965a) were. The more recent studies indicating phage control of toxin production (see later) also substantiate this conclusion. Deliberate attempts to obtain mutants of C1 botulinum by treatment with N-methyl-Nhitrosoguanidine yielded asporogenous mutants which had also lost the ability to produce toxin (Emeruwa and Hawirko, 1972; Hawirko e t ul., 1973). These mutants were morphologically and culturally similar to the parent toxic strain. In recent years an important contribution was made to the understanding of relationships between toxic strains and at least some nontoxic strains with the discovery that, in some cases at least, toxin production was controlled by temperate phages (Iida and Inoue, 1968; Inoue and Iida, 1968,1970; Eklund et ul., 1969, 1971, 1972; Eklund and Poysky, 1972; Dolman and Chang, 1972; Sugiyama and King, 1972). These findings were summarized in a review by Riemann (1973). Iida and Inoue did not isolate the phages, apparently because of difficulties in producing cell lawns. Eklund and his colleagues, however, carried out extensive studies on methods for isolation, host range, morphology, and role of the phages in toxigenicity. They showed that (1) phages of C1. botulinum have a narrow host range; (2) toxigenicity of types C and D is controlled by phage; (3) several phages exist which do not impart toxigenicity; and (4) double infection is possible and may be a cause of production of different toxins by the same strain. While it is clear, therefore, that some nontoxic strains are related to C1. botulinum in that they only lack a phage infection, it is equally clear that others differ in other ways. Some related nontoxic strains produce substances, boticins, which are sporostatic and bacteriocidal to toxic strains (Kautter et el., 1966; Ellison and Kautter, 1970; Anastasio et al., 1971). Boticins produced by nonproteolytic type E-like strains are active against all nonproteolytic strains of types B, E, and F but are not active against proteolytic strains of types A, B, and F. Although differentiation of the types of neurotoxins produced by Cl. botulinum is clearly important on medical and epidemiological grounds, its significance on taxonomic grounds is becoming less clear. That type C strains did not all produce serologicdy identical toxins has been known for a long time, and a relationship between Ca, Cp, and D strains was shown by Bulatova and Matveev (1965) to be due to the fact that different strains produced three different toxic components in varying proportions. This was clarified by Jansen (1971a, b) and by Jansen and Knoetze (1971), who showed that Ca strains produced three toxic factors-C1 ,C2, and D; Cp strains produced C2 ;and D strains produced C1
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and D. This was confirmed by the phage control work of Eklund and Poysky, who showed that the C1 and D toxins were phage-controlled but C2 was not. In addition, they showed that with trypsin activation Cz could also be demonstrated in some strains of type D. If cured of their phages the Ca, Cp, and D strains all became indistinguishable. In addition, since type F was first described it has been recognized that some degree of cross-neutralization occurred with type E toxin. A strain producing both type A and small amounts of type F toxin was recently described (Gimenez and Ciccarelli, 1970b; Ciccarelli and Gimenez, 197 1). This phenomenon was further studied and confirmed by Sugiyama et al. (1973). Gimenez and Ciccarelli had previously (1968) described a type F isolate which appeared to be somewhat different from the original Langeland strain in that it required more antitoxin (against the Langeland strain) to neutralize a given dose of toxin. These authors reviewed the serological typing of botulinum toxins (Gimenez and Ciccarelli, 1972) and proposed a revised system including subtypes as a taxonomic category. Immunodiffusion tests with toxin preparations and antitoxins have shown the presence of other soluble antigens besides the toxins themselves. Common soluble antigens have been demonstrated between Cl. sporogenes and proteolytic strains of C1 botulinum types A and B, although they were not identical (Zachaczewrki and Komorowski, 1968). Similar results were obtained by Vermilyea et al. (1968), who also showed that the A and B strains had no common soluble antigens with type E. Boiled extracts of vegetative cells have also been used as antigens to show cross-reactions between C, D, and E strains (Shevelev et al., 1964). Similar relationships have been shown by means of hemagglutination tests with crude toxin or toxoid preparations (Johnson eta]., 1966, 1967). Investigations of agglutinating antigens of vegetative cells and spores have shown that flagella or H-antigens show a high degree of strain specificity, whereas somatic or 0-antigens and spore antigens differentiate between proteolytic and nonproteolytic strains but not between toxin types. Some crossagglutination with other clostridia was also found (Lynt er aL, 1967; Solomon et al., 1969a, b, 1971). Agar diffusion tests using an antiserum made against type E spores and disintegrated spores as antigen have shown that the spores contain several soluble antigens of varying specificity but without cross-reactions with types A or B or Cl. bifermentans (Law and Hawirko, 1969). Also using agar diffusion tests, Rymkiewicz (1970) showed that crude toxin preparations of types E and F strains had common antigens. This author also showed, unlike other workers, that type F (Langeland strain) had agglutinating antigens in common with types BandE. Characterization of the metabolic products of anaerobic bacteria, particularly
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the volatile alcohols and acids, has been recognized for some time as a useful taxonomic aid (Moore, 1970). Among the CI. botulinum group the proteolytic strains generally produce a range of acids and alcohols, whereas the nonproteolytic strains produce chiefly butyric acid with smaller amounts of acetic and occasionally propionic acids (Moore et al., 1966; Moss et al., 1970). In recent years chemical analyses of bacterial cells have increasingly been examined for potential taxonomic value. Cummins (1970) in an examination of cell wall sugars and amino acids showed that all Cl. botulinum strains possessed alanine, glutamic acid, and meso-diaminopimelic acid but that the sugars separated them into three groups. The proteolytic types A, B, and F possessed only glucose, types C and D possessed no sugars or a trace of glucose only, whereas the nonproteolytic types E and F possessed glucose and galactose. Several workers have examined the lipids and fatty acids of the C1. botulinum group, and, although sufficient data are available to indicate that differences exist which could be of taxonomic value, more data are needed before any conclusions can be drawn (Moss and Lewis, 1967; Paterno, 1968; Kimble et al., 1969; Farsby and Moss, 1970; Ellender et af., 1970; Fugate et al., 1971; Hobbs et aL, 1971a; Hardy et al., 1971). The use of pyrolysis combined with gas chromatography has also been examined (Cone and Lechowich, 1970; Hardy et al.. 1971). Again there are differentiating features, but more data are required to assess the value of this analysis.
IV. METHODS FOR ISOLATION AND IDENTIFICATION Botulism normally results from the consumption of food containing neurotoxins; hence detection and identification of the toxin rather than isolation of the organism is of prime importance where an outbreak is investigated. The toxins produced by CI. botulinum cause characteristic symptoms in experimental animals and are serologically highly specific; it is therefore seldom necessary to isolate the organism in diagnostic work or in survey work where the aim is to find the incidence of the organism in a particular environment. In the diagnosis of botulism it is often possible to demonstrate toxin directly in the blood serum of the patient (Sebald and Saimot, 1973) or in remains of the food; otherwise toxin is usually sought in enrichment cultures of suspected materials. A review of enrichment and isolation methods for CI. botulinum was presented by McClung (1967) in which he pointed out that the methods originally used in the early part of this century were still by and large the most successful. More recently Hobbs et al. (1971b) reviewed methods for isolation of clostridia; hence only those aspects of recent work which seem to offer some benefit will be discussed.
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In earlier studies it was normal practice to heat enrichment cultures to 80°C for 10 to 30 minutes or to 70°C for 2 hours. The less heat-resistant types of Cl. botulinum, not described at the time of the early studies, would not have been isolated. In addition, studies with type E have shown that incubation at 37°C for relatively long periods was not desirable owing to destruction of the toxin after it had been produced, and to the fact that toxin was produced more readily at 25" to 30°C. Beef infusion media, usually with meat particles present, were most often used for enrichment and Robertson's Cooked Meat Broth in some form or other is probably still the most widely used enrichment medium for most clostridia. Few comparative studies have been published in recent years; however, Bott et al. (1968) showed that the levels of toxin obtained with type E with beef-heart infusion cooked meat broth generally were ten times as high as those obtained with trypticase-peptone-glucose, glucose-peptone, proteose-peptone-trypticase, fish infusion broth, Reinforced Clostridial Medium, or brain-heart infusion broth. Similarly, Rymkiewicz (1968) found that for a proteolytic type F strain a papain broth with added minced meat and glucose was the best of 48 media tested for toxin production. On the other hand, Tsaraphin (1971) found that a corn extract medium with added carbohydrate gave best toxin production with type F. Trypticase-peptone-glucose broth (Schmidt et al., 1962) has been a popular enrichment medium in survey work, especially in the United States. This medium was first employed to induce sporulation of type E, and there is therefore no good reason to suppose that it would be a good medium for enrichment where the requirement is for germination and toxin production. This is borne out by the findings of Bott et aZ. (1968) and of Ward et al. (1967a). Other workers have also generally found that some form of cooked meat medium is superior for growth and toxin production (Abrahamsson et al., 1966; Mikhailova, 1965; Matras, 1971); however, in an investigation of type C strains Segner et al. (1971a, b) found that, of the various media tested, only a fortified egg medium gave good growth. In looking for a nonmeat medium for growth and toxin production with type E, Perova et al. (1970) found that a casein and fish hydrolyzate gave a high yield of toxin. A significant increase in success with enrichment for type E was reported by Kautter and LiUy (1970) with the addition of trypsin to the enrichment medium. This had been suggested earlier (Kautter et al., 1966) to overcome the effect of boticins produced by similar nontoxic clostridia often present in the same samples. Another factor which may contribute to the success of adding trypsin is that germination of type E spores can be stimulated by proteolytic enzymes such as trypsin (Sebald and Ionesco, 1972). As was already pointed out, heat treatment of samples to reduce the numbers of commensal organisms present is not appropriate where the less heat-resistant
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types are sought. It is, however, possible to achieve the same end by chemical treatment. Johnston et al. (1964) described the treatment of samples with ethyl alcohol to kill vegetative organisms and leave behind spores. This proved successful in the isolation of type E. Either heat or alcohol treatment, however, must be used judiciously, since in both cases the assumption is made that Cl. botulinum is present in the sample as spores, and this may not always be the case. It is preferable, therefore, t o use an enrichment method which permits the detection of both spores and vegetative cells and to reserve heat and chemical treatment for samples where isolation of the organism proves difficult. A medium such as Robertson's Cooked Meat Broth incubated at 25" to 30°C will give the highest probability of detecting any of the types of C1. botulinum at present known; however, the addition of trypsin to the enrichment medium as suggested by Kautter and Lilly (1970) deserves further investigation with other media. The use of an enrichment medium in this way does, however, place restrictions on the size of sample examined. In the case of fish it has been found preferable to incubate the whole fish anaerobically (for example, in a vacuum pack) rather than place a sample such as the intestines into an enrichment medium. (Cann et ul.. 1965a). There is ample evidence in our own work that C1. botulinum may be present in the intestines, on the gills, or on the skin of fish, and this finding has been supported by other workers (Foster et al., 1965). The advantages of incubating the whole fish for enrichment are obvious, and most fish have proved to be excellent enrichment media. After incubation the fish is macerated with a suitable diluent and then handled like any other enrichment medium in tests for toxin or for isolation of the organism. The next step with either a contaminated food or an enrichment culture is to test for the presence of toxin. This is normally done by injecting the sample into mice; if symptoms and death occur, specific protection tests with suitable antitoxins are performed to determine the toxin type. The fact that different animals are not equally sensitive to the different types of toxin has been known since Van Ermengen first described the organism. Despite this, mice are still the most convenient and economical test animal. Other test animals such as guinea pigs and goldfish can be used (Crisley, 1960), but they offer no material advantages. One difficulty that arises with test animals is that they often die from causes other than botulism after injection with a suspect sample or enrichment culture. In some cases death is due to other known toxins such as tetanus toxin, but many deaths are due to unknown factors and are called nonspecific deaths. Experience in our own laboratory has shown that dilution of the sample by two to ten times will usually eliminate the nonspecific factors yet still leave sufficient toxicity; this finding has been confirmed by other workers (Insalata et al., 1970). Where death is due to infection caused by commensal organisms, treatment of the mice with antibiotics can be successful (Segner and Schmidt, 1968).
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Freezing and thawing of the sample has also been reported t o overcome some nonspecific deaths (Pivnick and Bird, 1965; Warnecke et al., 1967; Riemann, 1973). Suspected samples are usually administered to mice by intraperitoneal injection where death occurs in 1 to 48 hours if toxin is present. Antitoxin is either administered to the mice prior to injection of the toxic sample or mixed with the sample prior to injection. Both methods appear to be equally effective, although in our own experience the latter is more convenient. This test is sensitive, specific, and relatively simple to perform and interpret; it has some disadvantages, however. In addition to the problem of nonspecific deaths, it can be made quantitative only by the use of many mice, and even then it is not entirely satisfactory. The time required for this test can be shortened and better quantification can be achieved by intravenous injection of the mice (Boroff and Fleck, 1966; Sakaguchi et al., 1968). It would be highly desirable to have an in vitro test for the detection of Cl. botulinum toxins. It is apparent from the serological work already discussed that traditional serological tests with vegetative cells and spores do not have the same specificity as the toxins. For the same reasons the use of fluorescent antisera prepared against vegetative cells and spores has not provided the required specificity despite many attempts to absorb out the cross-reactions (Boothroyd and Georgala, 1964; Georgala and Boothroyd, 1967; Walker and Batty, 1967b; Ward et al., 1967d; Hunter and Rosen, 1967; Midura et al., 1967; Lynt et al., 1969, 1971; Emeruwa et al., 1970). Even if this were possible the available evidence suggests that the use of fluorescent antibodies to examine foods for the presence of cells or spores would be somewhat less sensitive than the mouse test for toxin (Midura et al., 1968). Attempts to use complement fixation, tube precipitation, immunodiffusion, passive hemagglutination, and bentonite flocculation have all shown some promise but lacked specificity because either crude toxin preparations or antisera prepared against such toxins were used and antigens other than the toxins interfered with the tests (Boroff and DasGupta, 1971; Johnson et al., 1966; Brantner and Dohmen, 1969). Recent work in Russia, however, suggests that nontoxic antigens of the required specificity for identification do exist (Ivanova et al., 1968; Bulatova et al., 1971). These authors describe some success using antiserum prepared against a purified somatic antigen. More promising results in the development of in vitro tests have come from investigations employing antisera prepared against highly purified toxins. Sakaguchi (1 97 1) demonstrated that reverse passive hemagglutination was satisfactory for type E if this type of antitoxin was used, and Aalvik et al. (1973) demonstrated the effectiveness of fluorescence microscopy using the same antitoxin. Miller and Anderson (1971) showed that, by immunoelectrophoresis with such highly specific antitoxin, type A toxin present at a level of 14 mouse LD50
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per 0.1 ml of food extract could be positively identified and quantified. An elegant radio-immunoassay technique for type A toxin has recently been described by Boroff and Shu-Chen (1973), the success of which depends on the use of highly purified toxin. There does therefore seem to be some hope that in vitro testing for Cl. botulinum toxins may well be possible and practicable in the foreseeable future. For isolation of the organism, toxic enrichment cultures must be plated out on agar media. Direct plating out of contaminated samples without enrichment is seldom successful, since Cl. botulinum is normally outnumbered by other facultative and strictly anaerobic bacteria. The two agar media most commonly used for the isolation of CZ. botulinum are fresh blood agar and egg-yolk agar. The first of these has no particular selective value, although it does provide a good growth medium for many clostridia. There are a number of egg-yolk media in use, and their value lies in the fact that CZ. botulinum, along with a limited number of other clostridia, produce active lipolytic enzymes and hence a characteristic reaction on these media consisting of a precipitate in the medium underneath the colony along with a coextensive iridescent film (or pearly layer) that covers the colony (McClung and Toabe, 1947; Willis and Hobbs, 1958, 1959). The lactose-eggyolk agar described by Willis and Hobbs is more useful for clostridia generally, since it provides information on lactose fermentation as well as lipase and lecithinase activity. There is, however, no particular virtue in this with clostridia such as Cl. botulinum with only lipase activity, since none ferment lactose. The choice of a basal medium for preparation of egg-yolk agar is very important. Liver-veal agar was at one time a popular base until it was pointed out that nonproteolytic strains of Ci.botulinum did not uniformly give a typical reaction with this medium (Hobbs et al., 1967). These authors found that on media containing high levels of fermentable carbohydrate production of the pearly layer was often inhibited. They also found that under these conditions the vegetative cells contained large numbers of iodinophilic granules. Subsequent workers have shown that the intracellular polysaccharide produced under these conditions is an a-D-glucan or amylopectin-like substance (Strasdine, 1968, 1972; Whyte and Strasdine, 1972). It was suggested that there was a relationship between the intracellular polysaccaride and sporulation. The results on egg-yolk agar suggest also a possible connection with fatty acid production. Interference by fermentable carbohydrate with the egg-yolk reactions of other clostridia has also been reported (Hobbs et al. 1971b). Both fresh blood agar and egg-yolk agar can be made selective by the addition of antibiotics or other inhibitors, but in general the nonproteolytic strains of C1. botulinum tend to be more or less sensitive to those tried. Such inhibitory media have therefore not found favor with most workers in isolating Cl. botulinum. An
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exception to this is the report by Mossel and de Waart (1968) that enrichment in sulfite-polymyxin iron broth at 27°C is preferable to nonselective enrichment for Cl. botulinum. Various sulfite media are popular for the isolation and enumeration of clostridia, and they have occasionally been recommended for C1. botulinum (Takacs, 1971; Greenberg et al., 1966). However, some sulfite media are inhibitory to C1. botulinum, and the nonproteolytic strains do not consistently give rise t o blackening of the medium. There is evidence to show that carbon dioxide will improve the growth of clostridia, probably by improving the germination rate of spores (Wynne and Foster, 1948; h d e r s e n , 1951; Treadwell et al., 1958; Roberts and Hobbs, 1968). This in fact has been known for many years; for instance, Van Ermengen in his original description of CI. botulinum noted that “1.43% of NazC03 afforded a good growth with toxin production.” Carbon dioxide can be supplied either in the atmosphere in an anaerobic jar as a mixture of hydrogen with 5% carbon dioxide, or by the inclusion of 0.1% (w/v) of sodium bicarbonate to culture media. There are various methods for achieving anaerobic conditions for the growth of clostridia. In considering these Hobbs et al. (1971b) concluded that an anaerobic jar equipped with a cold catalyst and provided with a hydrogencarbon dioxide mixture was most generally suitable. Other methods tend to be less effective or are more cumbersome to use. Whichever method is adopted, it is also essential to use freshly prepared and adequately reduced media. Following growth on agar media, typical colonies must be selected and tested in liquid media for their ability to produce toxin. Once growth of separate colonies has been achieved, it should not be assumed that these are necessarily pure; further subculture, directly onto more agar media, is desirable to exclude the possibility of mixed cultures. Even so, in our own laboratory we have found on occasions that small numbers of Cl. tetani, for instance, can persist in CZ. botulinum colonies for several subcultures without readily being detected. Once a strain has been isolated in pure culture further tests can be carried out as previously described to establish the general properties of the particular strain.
V. PROPERTIES OF T H E T O X I N S The neurotoxins produced by strains of Cl. botulinum are proteins, produced inside the vegetative cells during growth and released into the surrounding medium following autolysis. The enzyme responsible for autolysis of a type A strain has recently been partially purified and studied (Kawata and Takumi, 1971; Takumi et al., 1971). There has been considerable controversy in the
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literature concerning the properties of C1. botulinum toxins, and it is becoming apparent that much of this has resulted from the use of inadequate criteria for establishing the purity of toxin preparations. Not all the toxins have been prepared with the same degree of purity. However, recent work on the toxins of types A, B, and E has shown some measure of agreement (Boroff et al., 1966, 1967, 1968; DasGupta and Boroff, 1967, 1968; DasCupta e l al., 1968, 1970; Boroff and Meluche, 1970; Beers and Reich, 1969; Kitamura et d ,1967, 1968, 1969; Kitamura and Sakaguchi, 1969; Kitamura, 1970). The various conflicting data were discussed by Lamana and Sakaguchi (1971), who also proposed some rationalization of nomenclature for bacterial toxins. The toxins of Cl. botulinum are produced as aggregates with other proteins. However, the best available purified preparations all have a molecular weight of about 150,000, and this is the size of molecule of type E toxin that appears in the blood of animals whether they are given purified or crude toxin preparations (btamura et al., 1968, 1969). The earlier claims by Gerwing and her colleagues (Gerwing et al., 1964, 1965a, b, 1966) for the isolation of toxic material with a molecular weight of 9000 to 10,000 have not been confirmed by other workers. Moreover, their purified type B toxin was found to be heterogeneous and did in fact contain a toxic fraction with a molecular weight of about 150,000 (Boroff et al., 1968). Further evidence for the heterogeneity of some type E toxin and prototoxin preparations was provided by studies of their thermal inactivation curves. Although toxin and prototoxin behaved similarly, there were two distinct inactivation rates-an initial fast rate followed by a slower rate (Licciardello et al., 1967a, b). Because of the varying degrees of purity of toxin preparations, it is doubtful if the data available from earlier studies of amino acid composition are reliable. Recent data are available, however, for purified type A toxin (Boroff and Meluche, 1970) and for type B toxin (Beers and Reich, 1969). In addition, DasGupta and Sugiyama (1972a) have shown that a common subunit structure exists in the toxins of types A, B, and E. There is general agreement that the final stages of synthesis of toxin by C1. botulinum involves the activation of a nontoxic prototoxin by proteolytic enzymes. In the case of proteolytic strains this process is virtually complete by the time extracellular toxin appears. With nonproteolytic strains, however, the process is incomplete, and considerable activation can still be achieved in old cultures or toxic foods. As Lamana and Sakaguchi (1971) pointed out, there are several possible mechanisms for the observed activation, and only with type E is there evidence of an increase in specific activity (Kitamura et al., 1968; Sakaguchi and Sakaguchi, 1967). In laboratory experiments activation is achieved when necessary by treatment of the toxic material with trypsin. Within the growing organism the responsible
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enzymes are synthesized by the organism itself. Inukai (1963) described three proteolytic enzymes produced by type A, one of which would activate the prototoxin. Similar proteolytic enzymes have been found in other types (DasGupta, 1971; DasCupta and Sugiyama, 1972a, b). In a contaminated food, activation can also occur with enzymes produced by other bacteria and in the human or animal intestine. As was already mentioned, there are eight serologically distinct toxins produced by strains of CI. botulinum, and some strains produce more than one toxin type. Although not all have yet been purified, there is no evidence to suggest chemical differences which would account for their differing serological activities, and the differing susceptibilities of various animals to them. These are presumably a result of differences in the tertiary structure of the protein molecules. On the other hand, the fact that they all have the same highly specific pharmacological activity suggests a basic similarity in the relevant parts of the molecule. Few data are available on the composition of the active site of the toxin molecules. Boroff and his colleagues (Boroff et al.. 1967; Boroff and DasGupta, 1966) have presented evidence that tryptophan residues are essential to toxicity. When a proportion of these residues in the toxin are modified by photoxidation or chemical treatment with 2-hydroxy-5-nitrobenzyl bromide, the toxicity is lost, as is the ability to produce protective antibodies in rabbits. Toxicity is also lost when toxin is treated with formalin, a standard procedure for preparing toxoid. In this case, however, the toxoid is still able to stimulate production of protective antibodies. In this reaction formalin is believed to cause condensation of two tryptophan residues in two molecules of toxin with concomitant loss of toxicity but not antigenicity (Fraenkel-Conrat et al., 1947). It has also been suggested that sulfhydryl groups such as those found in cysteine residues are essential to toxicity. The available evidence suggests that the conformational stability of the toxin may be affected by modifications to sulfhydryl groups but that these groups are not an integral part of the active site (Beers and Reich, 1969; b o x et al., 1970). The suggestion by Lamka and Sakaguchi (1971) that toxicity depends more on the whole protein fitting the configuration of a substrate is more easily reconciled with the specificity of its activity and the high degree of specificity of antitoxins. This does not exclude the involvement of tryptophan, or other amino acids, in establishing the essential configuration, nor does it exclude the existence of specific active sites within the molecule. There is evidence that the specific configuration required for toxicity is not identical with that required for combination with antitoxin. Trypsin activation of type E toxin, for instance, results in a marked increase in toxicity but no increase in antitoxin-combining power (Kondo et al., 1969).
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VI.
FACTORS AFFECTING GERMINATION, GROWTH, SPORULATION, AND TOXIN PRODUCTION
It is well established that germination and outgrowth of bacterial spores and subsequent growth of vegetative cells are separate events and do not necessarily respond in the same way to changes in the environment. It is also apparent that factors affecting one bacterial species do not necessarily affect another in the same way. In many studies the effects of environmental changes on the different stages of germination and outgrowth have not been distinguished and germination has been judged by the occurrence of growth or toxin production. Several general reviews are available on the germination process in bacterial spores; only those aspects that are pertinent to Cl. botulinum will be discussed here (Schmidt, 1955; Murrell, 1961; Riemann, 1963; Dawes and Hansen, 1972; Hurst and Gould, 1969). The advantages of adding bicarbonate to media for isolation of CI.botu~jnum have long been known and have already been discussed. The fact that the success of using bicarbonate is probably due to its activity in germinating spores has recently been confirmed by Rowley and Feeherry (1970), who showed that with a type A strain in a chemically defined medium both the rate and extent of spore germination were increased as the concentration of bicarbonate was increased up to 8.3 mM. In addition, Ando and Iida (1970) and Ando (1971) found that in some chemically defined media bicarbonate was essential for germination. It has been known for some time that a relatively mild heat treatment or heat shock will enhance the germination rate of many spores, and heating at 80°C for 30 to 60 minutes is standard practice with suspensions of proteolytic strains of Cl. botulinum. The nonproteolytic strains are much more sensitive to heat, however, and it is generally found that a mild heat treatment which does not kill these spores, such as 60°C for 10 minutes, has little or no effect on their germination (Roberts and Ingram, 1965, 1967; Ando and Iida, 1970). With type A spores in a chemically defined medium cysteine increased germination up to a concentration of 32 mM and thioglycolate up to 2.2 mM (Rowley and Feehery, 1970). It is not clear if these results were due to some specific chemical effects or to a lowering of the Eh of the medium. In studies with type E, however, germination but not outgrowth was shown to be independent of Eh, although it was affected by a number of chemicals (Ando and Iida, 1970; Ando, 1971). In a casamino acid medium germination was markedly stimulated by lactate and the minimum requirements for germination were met with various combinations of Z-alanine, glucose, lactate, and bicarbonate. These results suggest that there are several possible pathways for germination and that no one unique set of conditions is essential.
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Proteolytic enzymes have also been shown to stimulate germination of type E spores (Sebald and Ionesco, 1972). Both temperature and pH are known to affect germination, and Ando and Iida (1970) have determined the optimum conditions for germination of type E spores in a complex medium. These authors also reported that the germination rate increased after storage of the spore suspension in an aqueous medium during 20 days at 4°C. Strasdine and Kelly (1967) studied the effects of aqueous extracts of various fish on the germination of type E spores and found that of those tested cod extract gave the highest germination rate; extracts of salmon, sole, and herring gave a lesser but still good rate of germination, whereas extracts of shrimp, oysters, and crab were relatively inactive. The active agent was found to be dialyzable; it was destroyed on ashing and contained a wide spectrum of amino acids and peptides. There was no correlation with any amino acids, although there was some relationship between germination and the ratio of peptide to amino acid concentration. This is consistent with observations on trypticasepeptone-glucose medium-if the proportion of amino acids to peptone is increased, then the germination rate is decreased. More attention has been paid to the factors affecting growth and toxin production where the effects on the various stages of germination and growth cannot be distinguished. Conditions favoring growth and toxin production were reviewed by Dolman (1964b) and Schmidt (1964), and several papers on this subject were presented at the Moscow symposium (Ingram and Roberts, 1967). In addition, various aspects have been reviewed by Foster and Sugiyama (1967), Gordon and Murrell (1967), Baird-Parker (1971), and Riemann (1973). The suitability of various bacteriological media for growth and toxin production has already been discussed. AU strains of Cl. botulinum grow best in rich organic media, and growth of the nonproteolytic strains is improved by the addition of fermentable carbohydrate. The optimum growth temperature is 35" to 37"C, whereas the optimum temperature for toxin production is 25" to 30°C. Most investigators now employ a temperature of 30°C which gives reliable toxin production and nearly maximum growth rate. The proteolytic strains have a minimum growth temperature of 10" to 15"C, whereas the nonproteolytic strains have a minimum of 3" to 5°C. The maximum growth temperature is between 40°C and 50°C. Growth occurs at a wide range of pH, the optimum being 6.8 to 7.0. The proteolytic strains will not grow at pH below 4.5, whereas the minimum for nonproteolytic strains is somewhat higher, about pH 5.0 to 5.2. The maximum pH permitting growth is about 8.5. Although growth occurs over this range of pH, the toxin is stable only at acid pH, and becomes increasingly unstable with increasing pH over 7.0. It is not possible to specify these various parameters more precisely, since
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there is variation between strains, and in any event they do not operate independently of each other. Since C1. botulinum is a strict anaerobe, the Eh of the medium is important for growth and toxin production. Unlike germination, outgrowth is markedly affected by Eh, although this again is dependent on other factors such as pH. In general, a properly reduced medium is necessary for good growth. Few data are available for clostridia where Eh measurements have been made. A discussion of the available information can be found in Smith and Holdeman (1968). One recent study on type E C1. botulinum showed that, if the Eh was sufficiently low to initiate growth, then the metabolism of the organism would further reduce the medium (Ando and Iida, 1970). Factors affecting sporulation of C1. botulinum were reviewed by Schmidt (1964). Spore suspensions had previously been prepared in complex media, often with meat particles present, and incubated for long periods. Schmidt described a trypticase medium without meat particles which gave high yields of spores with type A strains. The total spore yield was approximately proportional to the concentration of trypticase from 2 to 10%;addition of 1% ammonium sulfate increased the total cell population and hence the spore yield. Thiamine resulted in a higher percentage of sporulation with only a slight increase in the population. Tsuji and Perkins (1962) described a similar medium which gave good sporulation and showed that prolonged incubation could in fact reduce the spore count, suggesting that earlier procedures resulted in a loss of a proportion of the original spore crop. Perkins and Tsuji (1962) found that in a synthetic medium sporulation was increased by increasing the concentration of arginine. The later stage of arginine degradation from citrulline to ornithine appeared to be essential for sporulation. Schmidt et al. (1962) showed that type E strains also gave good spore yields in a 5% trypticase medium, and this or similar media have become widely used for the preparation of spores of most strains of C1. botulinum. The medium used by Schmidt contained 5% trypticase, 0.5% peptone, and 0.4% glucose. Other workers have added varying amounts of yeast extract with advantage for some strains. Further work by Roberts (1965) indicated that there was considerable strain variation in the degree of sporulation in a given medium and that sporulation varied markedly with different casein digests and peptones. Increased sporulation has been achieved by placing heavy suspensions of vegetative cells in saline inside a dialysis bag which was then submerged in a nutrient medium (Schneider et al., 1963). The use of a biphasic culture system has also been reported to give increased spore yields with types A and B (Anellis et al., 1972) and with type E (Brtfch et al., 1968). In this method the organism is inoculated into an aqueous phase overlaying a suitable nutrient agar. A recent study (Strasdine and Melville, 1971; Strasdine, 1972) offered evi-
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dence that sporulation of type E might be associated with depletion of fermentable carbohydrate in a medium. In a glucose broth medium an intracellular glucan is rapidly accumulated, and when the glucose is depleted the glucan reserve is utilized. It was suggested that sporulation was in some way associated with utilization of the glucan. The growth requirements of most clostridia are complex, and the requirements for germination, growth, sporulation, and toxin production are not all the same. A number of chemically defined media have been devised for Cl. bofulinum. Mager et al. (1954), Kindler et al. (1956), and Perkins and Tsuji (1962) described suitable media for growth, toxin production, and sporulation of type A. Gullmar and Molin (1967a, b, c), Ward and Carroll (1966, 1967), and Snudden and Lechowich (1 967) discussed media for type E, and Holdeman and Smith (1965) used a modification of the medium suggested by Mager et aZ. to investigate the growth requirements of a proteolytic type F strain. All these media contained a wide range of amino acids and growth factors. In the medium reported by Ward and Carroll the addition of citric or lactic acid was necessary before toxin was produced, whereas in all the other media growth and toxin production occurred together. Atypical morphology was commonly found in minimal media; for instance, Gullmar and Molin (1967a, b, c) found that addition of choline was necessary with their medium to obtain normal cell division and morphology of type E. The choline could be replaced by acetylcholine, N,N-dimethylethanolamine, or lecithin,but not by ethanolamine, N-methylethanolamine, or betaine. These authors also showed that the requirements for growth and toxin production were not necessarily the same. With type E, 0.1 mg per 100 ml of tryptophan was optimal for growth, whereas 0.6 mg per 100 ml was optimal for toxin production. With glucose 1% was optimal for growth, whereas either 0.1% or 6% was optimal for toxin production. Stimulation of toxin production by type A with tryptophan had previously been reported by Kindler et al. (1956).
VII. INHIBITION OF CLOSTRIDIUM BOTULINUM Since the discovery of the organism responsible for botulism, there has been considerable interest in inhibiting its growth and toxin production and in destruction of its spores. The inhibitory effects of temperature, pH, and salt have been recognized for many years, although more recently there has been a fuller appreciation of the interactions of these inhibitory factors. Inhibition of C1. botulinum in foods has been reviewed on a number of occasions (Riemann, 1969,1973; Riemann ef a)., 1972; Lechowich, 1970). The proteolytic strains of CI. botulinum do not grow and produce toxin at
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temperatures below 10" to 12°C. However, the minimum temperature for nonproteolytic strains is 3" to 5°C. All strains are inhibited at temperatures above about 45OC. The inhibitory pH levels for proteolytic strains are 4.5 and 9.0, and the nonproteolytic strains are somewhat more sensitive to extremes of pH. The proteolytic strains are inhibited to 8 to 10% of sodium chloride and the nonproteolytic strains by 4.5 to 5%. These various inhibitory conditions are generally true when other factors are optimal. As was stated earlier, however, they seldom act independently of each other. Studies in recent years have elucidated more clearly some of the interactions of inhibitory factors. Ohye and Christian (1967) studied the combined effects of temperature, pH, and water activity on types A, B, and E grown in trypticase-peptone-glucose. Their results clearly show that, at temperatures less than optimum, less salt was required to inhibit growth (that is, a higher water activity). Similarly, at pH values away from optimum less salt was required for inhibition. Similar results have been reported by other workers (Roberts and Ingram, 1973; Emodi and Lechowich, 1969a, b; Baird-Parker and Fream, 1967; Lubenieki and Schelhorn, 1972; Riemann, 1969). While most work generally supports the concept that inhibition by salts and other solutes is accounted for by water activity (aw), other factors are sometimes involved. Baird-Parker and Freame (1967) found results consistent with this concept except when glycerol was used to reduce the water activity, when a lower aw was required to inhibit growth than with other solutes. This was confirmed by Lechowich (1970), who also described abnormal morphology at limiting levels of water activity when salt was used which was not apparent when glucose or sucrose was used. Under optimum conditions of pH and temperature the inhibitory aw is 0.940 to 0.950 for proteolytic strains of types A and B and 0.970 to 0.975 for type E. At 20°C types A and B were inhibited by an aw of 0.970, and at 10°C type E was inhibited by an aw of 0.990. A further factor which determines whether or not growth will occur under inhibitory conditions is the number of organisms present. A small number of cells are more readily inhibited than a large number (Segner et ul., 1966a). Experiments on the inhibition of CI. botulinum in foods are often difficult to interpret because of the inherent problems of measuring precisely the salt concentration, pH, and other factors in foods where they are frequently not uniform throughout a particular sample. Other unknown inhibitors may also be present. Fish do not all support the growth of CI.bo~u~inum equally, and indeed a substance inhibitory for type E has recently been described in the flesh of haddock (Nickerson and Zak, 1973). Nevertheless, such data as are available suggest that the limiting parameters found in culture media are generally not very different from those operating in foods, and in any event any error would
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be on the side of safety. Boyd and Southcott (1971) showed that in cod homogenates at 30°C some type E strains did not produce toxin with 3.76% salt in the aqueous phase, whereas other strains required 4.4%. Somewhat higher salt concentrations were necessary to prevent vegetative growth. These authors also showed that, at 2.5 to 2.9% salt, toxin production was about 98% less than in control samples, and at 3.4% salt, some toxin was detected after 4 days at 30°C but none was present after 6 and 12 days. The use of other chemical preservatives in foods is governed by the current legal situation which differs in different countries. However, there is no doubt that some are effective against CI. botulinum. Sodium nitrite and nitrate have been widely used as preservatives in cured meats for many years. Segner et at. (1966b) showed that type E could be inhibited by sodium nitrite in homogenates of haddock. Rather more than 500 ppm of sodium nitrite was required to prevent growth and toxin production completely. Other workers have shown that about 100 ppm of sodium nitrite inhibited type E in smoked whitefish chubs containing about 3% salt in the aqueous phase (Graikoski, quoted by Lechowich, 1970; Weckel and Chien, 1969). Again there is a synergistic action between salt and sodium nitrite. Sodium benzoate and sodium parahydroxybenzoate have both been reported to be inhibitory to type E at low temperatures (Segner et al., 1966b). These authors also reported inhibition of type E to EDTA (ethylenediaminetetraacetate) at a concentration of 150 to 200 ppm, and similar results were found by Winarno et aZ. (1971). Other, less effective chelating agents such as sodium tripolyphosphate were found to be effective at concentrations of 1000 to 5000 ppm. The chelating agents are thought to bind cations essential to the germination of spores. Ethylene oxide has been found to be effective in foods, and it has been suggested that its use may be commercially feasible (Kuzminski et al., 1969; Winarno and Stumbo, 1971). Relatively little interest has been shown in the inhibition of Cl. botulinum by antibiotics. Kaufmann et al. (1954) found that a number of antibiotics, including neomycin, celiomycin, streptin, and circulin, were without effect on type B, although other work (Spencer, 1969) has shown neomycin to be inhibitory to type E in culture media. Subtilin has been reported to be inhibitory to type A, although resistant mutants can arise (Campbell and Winiarski, 1959). Tylosin and tylosin lactate have been shown to be effective against clostridia including CI. botulinum in canned foods (Denny et aZ., 1961; Greenberg and Silliker, 1964). As little as 5 ppm was found to inhibit outgrowth but not germination of spores. Tylosin lactate was also shown to be effective in smoked whitefish chubs (Sheneman, 1965). A concentration of 100 ppm incorporated into the brine left a residue of 0.5 to 5.0 ppm in the smoked fish,and these fish did not support growth and toxin production by type E after 14 days at ambient temperature.
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Abrahamsson (1964) reported that in a beef-heart infusion medium 50 pg/ml of tylosin and 0.5 p g / d of oxytetracycline inhibited type E for 4 days at 30°C. Vierling (1962) found that penicillin had some inhibitory action against type A, whereas erythromycin, tetracycline, chloramphenicol, streptomycin, and kanamycin had little or no effect. On the other hand, both oxytetracycline and chlortetracycline have been shown to be effective against type E in both media and fish (Kawabata and Sakaguchi, 1963). It has recently been suggested that the addition of everinomicin to foods will prevent the growth and toxin production of CL botulinum (Hass and Insalata, 1971). The sporostatic and bactericidal boticins produced by some other clostridia have already been discussed; other than this there is little evidence for inhibition of Cl. botulinum by other organisms. Escherichia coli and Streptococcus faecalis have been reported to be inhibitory, and it was suggested that this could explain why Cl. botulinum does not appear to establish itself in the human intestine (Crisley and Helz, 1961). There is ample evidence, however, that other bacteria can have a strong inhibitory effect by reducing the pH of food or media containing fermentable carbohydrate (Riemann, 1967,1973).
VIII. PRESERVATION OF FISHERY PRODUCTS In the preceding sections the incidence of C1. botulinum in fishery products and the environment has been considered, and the properties of the organism and its toxin have been discussed. A thorough understanding of all these factors is essential when one is considering the preservation of foods such as fish, especially where new or modified preservation methods must be evaluated. Even traditional preservation methods are continually changing, owing partly to changes in peoples' tastes and partly to the development of new technology. Although the effects of preservation methods on vegetative cells and toxins are important, it is their effects on spores that have received most attention, and a large proportion of the relevant literature is devoted to the destruction of spores or the inhibition of germination and outgrowth. The role of spore-forming bacteria in food spoilage and the resistance of spores have recently been the subject of detailed reviews (Ingram, 1969; Roberts and Hitchins, 1969), and the methods used for the control of C1. botulinum in foods were discussed by Riemann (1973). The only practical way of destroying the spores of (3'. botulinum in foods at present is by heat treatment. Prevention of growth can be achieved, however, by refrigeration, freezing, reduction of pH, reduction of the water activity by drying or the addition of salts or other solutes, and in some cases by the addition of chemical preservatives. Irradiation can be used to kill spores, but the adverse effects of the doses required to do this on the organoleptic quality of foods generally precludes its use for sterilization. On the other hand, there has been
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considerable interest in the use of lower irradiation doses to extend the shelf life of foods such as fish, but such processes cannot be considered to control CI. botulinum. Although the necessary conditions for the control of CI. botulinum using any of the above methods are well known, the conditions where two or more controlling factors are acting together are less well defined. Interest in these interactions has increased in recent years, since it may be possible to reduce the undesirable organoleptic effects of any of the above treatments by the judicious application of two or more preservative methods together. Experiments designed to investigate the interactions of two or more inhibitory factors tend to be complex and time-consuming even when laboratory media are used. The conditions prevailing in a food such as fish are more complex still and can vary considerably. While it therefore may be convenient to devise a preservation process on the basis of work in laboratory media, the final definition of the process must be determined in the food itself. In fish the conditions of pH, salt concentration, and previous history up to processing are frequently not amenable to control. There are, for instance, seasonal variations which can result in gross changes in the chemical composition of fish; for example, a tenfold variation in the fat content of herring is normal. There are other variations associated with the spawning cycle of the fish and with its feed. Finally, there are postmortem changes which can occur between catching and processing. AU these variations are compounded by differences between species which can be large. There is evidence that the conditions of temperature, pH, and water activity, the presence or absence of lipids and other organic compounds, and the ionic environment can all affect the heat resistance of spores produced under these conditions (Roberts and Hitchins, 1969; Upadhayay and W i k , 1969). There are, however, enormous difficulties involved in producing spores in foods under different conditions, and the final stages of process development normally involve the use of samples of the food inoculated with spores produced under laboratory conditions. Much more information is available on the effects of conditions during treatment of the spores (Roberts and Hitchins, 1969). Even here, however, much of the available data has been obtained under laboratory conditions, and the final stages must be testing of the prescribed process with spores treated in the food concerned. Ingram (1969), for instance, points out that, if heat resistance tests are carried out with inoculated packs, the estimated D values commonly increase with the time of heating. He also suggested that the satisfactory outcome of process calculations against CL botulinum results from the fact that they include large margins of safety. Other examples are also given where laboratory experiments give a misleading impression of resistance under practical conditions. A thorough review of the prevention of botulism by thermal processing was
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presented by Perkins (1964), and further detailed information was presented by Ingram (1969) and Roberts and Hitchins (1969). With low-acid foods (pH > 4.5) a process equivalent to Fo 2 2.8, combined with good process control, is effective against the most resistant Cl. botulinum spores. A milder heat treatment is possible with high-acid foods, and in this type of product other factors such as salt concentration (or water activity), pH, and possibly preservatives such as sodium nitrite are usually the factors controlling spoilage by organisms other than Cl. botulinum. Heat sterilization of low-acid foods has been found to be satisfactory for a long time. Reduction of processing requirements could have considerable organoleptic as well as economic advantages; hence studies on the effects of environmental conditions on processing are assuming increasing importance. Spores are generally more sensitive to heat at extremes of pH than at values near neutrality. A recent study with type A spores showed that the D value decreased with pH from pH 7 to pH 4 by a factor of about 5 (Xezones and Hutchings, 1965). Dry spores are much more resistant to heat than moist spores, and it has been shown that reducing the water activity with salt increases heat resistance. A decrease in water activity to 0.8 was found to increase the heat resistance of type E spores by about 30,000 times (Murrell and Scott, 1957, 1966). Simiarly, spores suspended in lipids have a high heat resistance, although it is not clear whether this is due entirely to a low water activity. Carbohydrates can also increase heat resistance, owing partly to reduced water activity, but not all sugars have the same effect (Roberts and Hitchins, 1969). Little is known about the basis of heat resistance of spores, but recent work indicates that their unique properties are related in some way to the hydrophobic nature of the spore coat (Grecz et al., 1970). Attempts to find substances that will reduce the heat resistance of spores have generally been unsuccessful. However, a recent observation by Dallyn and Everton (1971) merits further investigation. They described a water-soluble component of some olive and ground nut oils which significantly reduced the heat resistance of spores of a putrefactive anaerobe. It was suggested that the responsible component may have been a carbonyl compound. Another means of reducing the effects of heat processing on the food yet maintaining safety is to increase the temperature of the canning process when a shorter processing time is required. The effects of such a process have been discussed by Charm (1965). Where heat processing short of sterilization is applied to fishery products, the process must be considered to affect the normal spoilage flora rather than to control C1. botulinum. Such processes can, in certain circumstances, increase the botulism hazard in that spoilage can be delayed sufficiently for toxin to accumulate before the product is organoleptically spoiled. With such products, control of C1. botulinum must be achieved by other means such as refrigeration,
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salt curing, or reduction of pH. Nevertheless under practical conditions heat pasteurization can reduce the number of Cl. botulinum spores in fishery products. The information available shows that contamination of fish is normally with nonproteolytic strains, mostly type E, and only occasional contamination with proteolytic strains has been reported. The nonproteolytic strains are much more sensitive to heat; type E spores, for instance, have Dsooc values of 0.6 to 3.3. It is conceivable, therefore, that a pasteurization process would kill off small numbers of such spores. In studies on the heat resistance of type E spores in whitefish chubs it was found that 17 minutes at 82.2OC would kill an inoculum of lo6 type E spores (Crisley et al., 1968). In a study of commercial heat pasteurization of crab and shrimp, however, survivors were found after inoculation with lo3 type E spores. This would be expected from the published D values and the time and temperature of pasteurization-1 minute at 82.2"C (Lerke and Farber, 1971). An example of the deliberate use of heat pasteurization to control Cl. botuh u m type E is embodied in the regulations and codes of practice for the production of smoked fish operating in parts of the United States. These include a requirement for heating the fish during smoking to 82.2"C for a minimum of 30 minutes. A summary of the United States regulations and a discussion of experience with them over the past 10 years has recently been published (Pace and Krumbiegel, 1973). It is clear that in fish smoked in this way the numbers of type E spores present are drastically reduced. However, they are not eliminated, and further controls by temperature and salt concentration are still necessary (Christiansen et al., 1968). It has recently been suggested that the efficiency of heat pasteurization of smoked whitefish chubs can be increased by the proper control of relative humidity during the smoking process (Alderman et al., 1972; Pace et al., 1972). In doing this, however, due consideration should be given to product quality, which depends to some extent on drying. A code of practice for the production of smoked trout was introduced in Germany following a type E outbreak there (Wenzel et al., 197 1b). This code is essentially the same as the United States regulations and includes a similar heating process. The levels of heating required by these regulations and codes are much higher than those traditionally employed in the manufacture of smoked fish products. An important consideration in changing a process in this way is that the resultant product is not the same as the original. This means that all the problems of handling distribution and consumer acceptability have to be dealt with. The reduction of contamination levels in this way cannot be considered in isolation. In addition to the problems mentioned it is even more important to avoid recontamination after processing, and in any event the proteolytic strains are not affected. Mishandling of this type of product could therefore rapidly result in toxin developing before the product is overtly spoiled.
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With pasteurized products it is apparent then that general hygiene precautions are even more important than with raw products, and the general principle that raw materials should have as low a level of contamination as possible is crucial. In some recent work on contaminated farmed trout in Denmark (Huss et al., 1974a, b) it has been shown that efficient evisceration and washing of the trout can drastically reduce the level of contamination and possibly achieve the same ends as the increased heat treatment during smoking without introducing the additional problem of altering the nature of the product. Heat pasteurization will generally kill the vegetative cells of Cl. botulinum and will inactivate any toxin already present in the food. Most domestic cooking procedures will in fact do this also (Licciardello et al., 1967a, b; Hobbs et al., 1969). Cooking therefore provides a final safeguard against botulism, and it is only with products that are not eaten immediately after cooking, or are eaten without cooking, that the real problems exist. There is evidence that toxin present inside spores is more resistant than free toxin, although on release from the spores it is no different from that derived from vegetative cells (Grecz et al., 1967a). This finding suggests that any increased heat resistance is probably due to protective factors in the spore, an observation borne out by the demonstration that spore toxin is serologically identical to that from vegetative cells (Rymkiewicz, 1971). Heat-resistant toxin inside spores does have some significance, however, since it is conceivable that after processing toxin could be released from surviving spores without growth occurring. As was discussed previously, germination of spores can occur under conditions that do not permit growth and toxin production. Whether sufficient toxin could accumulate in food in this way to cause botulism will depend on the number of spores surviving processing. Control of botulism by refrigeration or freezing is perhaps the most important preservation procedure after heat sterilization. Freezing prevents germination, growth, and toxin production, and, although it has some slight lethal action on vegetative cells, it does not affect spores or toxin. Commercial freezing and cold storage processes are therefore governed by their effect on the organoleptic quality aspects of fish rather than their effect on food-poisoning bacteria. The minimum growth temperature of some strains of C1. botulinum is 3" to 4°C; hence refrigeration below this temperature will effectively prevent growth and toxin production. Again, toxin is stable and spores can germinate at such temperatures. Pace and Krumbiegel (1973) have stated that some smoked fish processors in the United States are able to maintain refrigeration temperatures of less than 3°C. Other authors, however, have stated that this cannot be expected in commercial channels (Lerke and Farber, 1971). Our own experience bears out this latter point of view-that with existing refrigeration equipment and practices a maximum temperature of 3OC throughout distribution and sale cannot be
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guaranteed. There are, however, additional safeguards in practice. With unprocessed fish the rate of toxin production at temperatures below 10°C is such that putrefaction will generally be evident before detectable amounts of toxin are produced. With processed fish such as smoked fish the rate of toxin production is slowed down even further, primarily because of the salt concentration. Nevertheless, since spoilage is also slowed down, toxin can appear in such products before they become spoiled even at 10°C. The imposition of a maximum shelf life of about one week as required by the United States regulations is an additional safeguard in this situation. In general there is an increased margin between spoilage and toxin production as the temperature is lowered below 10°C, whereas at higher temperatures toxin can be produced before the fish is spoiled (Hobbs, 1968). This is, however, a generalization, and there are exceptions, depending on other factors such as salt concentration. For instance, smoked trout stored at 10°C can become toxic before they are spoiled (Torry Research Station, unpublished data). Salt was used for the preservation of fish long before the basis for its preservative action was known. Salt fish was an important part of the diet of poor people in ancient Egypt (Wilkinson, 1878); they used brine pickling methods for the preservation of fatty fish such as the “scromboids,” and such fish became a relatively common commodity in the Mediterranean area. The prosperity of many cities, like those in the Baltic area at a later period, depended on their trade in dried, salted, and pickled fish (Smidth, 1876). The use of salt for preservation of herring in Northern Europe has been practiced for some twelve or thirteen centuries (Cutting, 1955). The early world-wide use of salt has resulted in a very large number of fish products which rely on salt for their preservation. A detailed review of the many methods of preserving fish with salt, alone or in combination with various fermentation processes, has recently been prepared by Mackie et al. (1971). The vast majority of products have a high salt content, and no botulism hazard exists if the processes are carried out properly. The records of botulism outbreaks show, however, that a hazard does exist with some salt-preserved products (Mackie et al.. 1971). As has already been pointed out, the sensitivity of Cl. botulinum to salt cannot be considered in isolation from pH, temperature, the presence of other curing salts such as sodium nitrite, and the numbers of organisms present. The interactions of salt, pH, and other related conditions in controlling CZ. botulinum in foods have been reviewed by Baird-Parker (1969), Riemann et al. (1972), and Riemann (1973). The precise combinations of these various conditions which will guarantee the inhibition of C1. botulinum in the many types of products available are not as yet worked out, and in general process descriptions tend to err on the side of safety. As these inhibitory conditions become better known it may be possible to reduce salt levels-for instance, where this would be desirable for organoleptic
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reasons. There is good evidence that this has occurred in the past-with smoked fish, for example-without the full implications being realized, or indeed without the basic data being available. It has been suggested that practical use could be made of the combined effects of salt, pH, and temperature in cured meats (Riemann, 1967; Riemann et al., 1972). If 1% glucose is added to commercially prepared semipreserved meat products, the other organisms present would usually ferment the glucose, thus lowering the pH. A salt concentration which on its own permitted growth would then be inhibitory. In view of the prevalence of saccharolytic strains of CZ. botulinum in fish products, the application of this suggestion to fish products might present problems. One further consideration is the prevailing legal situation regarding chemical additives. While, for instance, effective inhibition could be achieved in products such as smoked fish by the combined use of salt and sodium nitrite, it seems hardly profitable to discuss this in the light of pressures to reduce the existing use of nitrite in cured meats because of the possible significance of nitrosamine formation. Since C1. botulinum toxin is readily destroyed by most normal cooking procedures, the chief hazards exist with those products eaten without further cooking such as the "izushi" products of Japan and the smoked or marinated products of Northern Europe and North America (Hobbs et al., 1969). Even with these products botulism will occur only if the foods are not produced properly or mishandled. As was shown by Engleson et al. (1962), toxin can be produced during the preparation of pickled herring if salt penetration during preparation is not efficient. In the case of hot smoked fish products in Europe and North America the brining and smoking are more often done for organoleptic and cosmetic reasons rather than for preservation, a trend that has been encouraged by the development of cold storage and refrigeration chains. One other difficulty is the fact that present methods of brining result in a wide variation of salt concentration between different fish or even between different parts of the same fish. If, therefore, an inhibitory level of salt is to be attained in all fish, some will be too salty from the taste point of view. In any event it is not possible to make an acceptable product that will inhibit the proteolytic strains. It has been claimed that the smoke constituents have some inhibitory effect (Spencer, 1967; Nielsen and Pedersen, 1967); however, there is ample evidence that growth and toxin production occur at the concentration of smoke constituents present in most commercially available smoked fish and that salt is the main inhibitory agent (Baird-Parker, 1969). Although preservation of fish by irradiation is not permitted, a great deal of effort has been expended during the past twenty years on devising and evaluating irradiation processes. Several detailed reviews of this work are available,
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and only the main points will be discussed here (Hobbs, 1967, 1968; Kreuzer, 1969; Ingram, 1969; Roberts and Hitchins, 1969; Anon., 1973). The eventual acceptance of a radiation process will depend largely on whether the criteria laid down by health authorities governing the wholesomeness of irradiated foods can be satisfied. In the case of radiation sterilization it will also depend on the successful control of undesirable organoleptic changes induced in foods by the levels of irradiation required. Some partial success has been achieved by carrying out the irradiation at low temperatures (Grecz et aL, 1965, 1967b, c, 1971; El-Bisi e l al., 1967). With raw foods a successful process would still require some heat processing to inactivate autolytic enzymes. Although there is evidence for some synergistic action between heat and irradiation and lower levels may be required than with either process alone, the product would no longer be raw. The success of such a process would therefore depend to some extent on whether the products were an improvement on conventionally heatsterilized products. Synergism between y-radiation and other agents such as ultraviolet radiation and chemicals such as sodium chloride, nitrite, and nitrate has also been reported (Krabbenhoft et al., 1964; Grecz el aZ., 1967b; Durban and Grecz, 1969; Durban ef al., 1970). It is conceivable, therefore, that the combined use of one or more of these agents along with irradiation could result in the development of a process that would reduce the undesirable effects of heat sterilization. This would be of particular advantage with many fish products. Pasteurization by irradiation or radurization has received considerable attention, and fish has been considered one of the more promising foods for this application. In the earlier work a dose of about 0.3 Mrad was used, since this was the maximum dose that did not produce undesirable organoleptic effects in the fish. Apart from any considerations of wholesomeness, radurized fish products~ suffer from most of the disadvantages of heat-pasteurized fish. As far as Cl. botulinum is concerned, the process has little or no effect on the spores or on the subsequent rate of toxin production, whereas the shelf life is extended by two and one-half to three times. There is, in fact, some evidence that toxin production can be somewhat more rapid in irradiated fish than in untreated fish (Cann et al., 1966b; Ando et al., 1969; Ando and Karashimida, 1972; WilliamsWalls, 1969). In any event there is ample evidence that toxin production can occur before the fish is spoiled (Hobbs, 1968). More recently attempts have been made to overcome this problem by using lower levels of irradiation such as 0.1 Mrad. This gives a reduced shelf life extension but reduces the effect on spoilage bacteria to such an extent that spoilage w ill still occur first. As with heat pasteurization, therefore, other factors such as refrigerated storage or salt concentration would probably have to be used to control Cl. botulinum in radurized products. Therefore, while it is feasible to subject fish to a radurization process, possibly
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with some commercial benefits, the criteria for wholesomeness laid down by health authorities must be satisfied before further developments are possible.
IX. SUMMARY AND CONCLUSIONS For botulism to occur after the consumption of a fishery product, the organism must be present, it must survive processing, the product must support growth and toxin production, it must be stored under conditions that permit growth and toxin production, and it must be eaten without adequate cooking. Botulism outbreaks have historically been largely sporadic and have involved relatively few people. With the increase of large-scale processing and catering, however, the risks of more extensive outbreaks are increased accordingly. Although low levels of contamination occur with most types of fresh foods, there are obviously instances where high levels of contamination can occur in fishery products. In addition, the strains of CI. botulinum that occur in freshly caught fish are generally nonproteolytic and are able to grow at relatively low temperatures. Although it is unlikely that further research will lead to the total elimination of Cl. botulinum from fresh foods, much remains to be understood concerning the ecological conditions which permit the development of high levels of contamination such as occur in some fish farms. The occurrence of other clostridia which produce boticins is of obvious importance in preventing the establishment of C1. botulinum in some ecological situations, and there are also less well-described instances of inhibition by other microorganisms. In addition to the boticin-producing clostridia, there is a whole group of nontoxic clostridia which are otherwise indistinguishable from CZ. botulinum, and these are of fairly common occurrence. The precise relationship of these strains of toxic organisms is not clear. Although the recent work on bacteriophage has explained to some extent the loss of toxicity by Cl. botulinum, it has not yet given much indication of the degree to which the reverse process of acquiring the ability to produce toxin exists. In addition, there are still some toxins which do not appear to be controlled by phage. The recognition of Cl. botulinum still depends finally on the identification of one or other of the toxins by its effects in experimental animals and by the inhibition of these effects with specific antitoxins. There would be obvious advantages in using an in vitro test for the toxin, and the recent work using highly purified toxins for the production of antitoxins suggests that this might soon be possible. There would also be considerable advantages in the development of more rapid and sensitive methods for identification of both the organism and the toxin. The use of radioisotope-labeled antitoxin and the work on physicochemical methods of identification indicate that this may be feasible. In the handling and processing of fishery products there is still room for more
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effective application of existing knowledge. The statement by Dolman (1964a) is even more true now: “Though there is much to be learned about botulism, there is still more to be taught about it.” Any preventive program must first be aimed at reducing to a minimum the level of contamination. High standards of hygiene and cleanliness are effective in this respect. Handling and processing conditions which will prevent contamination and growth of the organism can in general be specified, although refinements are possible here which could improve the quality of the product and yet maintain its safety. A good example of this is the development of more reliable and controllable brining processes for fish. A better understanding of the interactions of pH, aw, temperature, and preservatives would lead to a more effective definition of preservation procedures, although this in turn would require the development of more precise methods of controlling these factors. In most parts of the world the incidence of botulism is not sufficiently high to merit continuous monitoring of fish for the presence of the causative organism. Routine testing of this kind is expensive and inefficient, and except where a high level of incidence is known or suspected to occur it is unlikely to be effective in preventing an outbreak. It is generally much more efficient and effective to control processing and storage conditions so that growth and toxin production can be prevented. It is unlikely that botulism will ever be totally eliminated, since it most frequently occurs as a result of faulty processing or handling. However, a thorough understanding of the properties of this group of organisms and their toxins will help to minimize the risks and will help to avoid the development of new products or processes which might permit a further predisposition to the occurrence of botulism.
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Ando, Y. 1971. The germination requirements of spores of Clostridium botulinum type E. Jap. J. Microbiol. 15, 515. Ando, Y., and lida, H. 1970. Factors affecting the germination of spores of Clostridium botulinum type E. Jap. J. Microbiol. 14, 361. Ando, Y., and Karashimida, T. 1972. Radiation resistance of spores of Clostridium botulinum type E. 111. Effect of exposure of spores to a lowdose level of yirradiation on subsequent growth and toxin production. Food Irradiat. 7, 1. Ando, Y., Karashimida, T., Ono, T., and Iida, H. 1969. Toxin production by Clostridium botulinum type E in radiation pasteurized fish. Food Zrradiat. 4, 11. Anellis, A., Berkowitz, D., Kemper, D., and Rowley, D. B. 1972. Production of types A & B spores of Clostridium botulinum by the biphasic method. Effect on spore population, radiation resistance and toxigenicity. Appl. Microbiol. 23, 734. Anon. 1973. Radiation Preservation Food, Proc. Syrnp. IAEA, Vienna. Anusz, Z., and Przybyszewski, S. 1972. A case of botulinum poisoning caused by Clostridium botulinum type E from fish. Epidemiol. Rev. 26,417. Armstrong, R. W., Stenn, F., Dowell, V. R., Ammerman, G., and Sommers, H. M. 1969. Type E botulism from home canned Gefilte fish. J. Amer. Med. Ass. 210, 303. Bach, R., and MUller-Prasuhn, G. 1971. Farmed trout as a carrier of Clostridium botulinum and a cause of botulism. I. Literature survey of Clostridium botulinum type E and fish botulism. Arch. Lebensmittelhyg. 22, 64. Bach, R., Wenzel, S., MUller-Prasuhn, G., and Glasker, M. 1971. Farmed trout as a carrier of Clostridium botulinum and a cause of botulism. 111. Evidence of Clostridium botulinum type E on a fish farm with processing station and in fresh and smoked trout from different sources. Arch. Lebensmittelhyg. 22, 107. Baird-Parker, A. C. 1969. Medical and veterinary significance of sporeforming bacteria and their spores. In “The Bacterial Spore” (G. W. Could and A. Hurst, eds.), p 517. Academic Press, New York. Baird-Parker, A. C. 1971. Factors affecting the production of bacterial food poisoning toxins. J. Appl. Bacteriol. 34, 181. Baird-Parker, A. C., and Freame, B. 1967. Combined effect of water activity, pH and temperature on the growth of Clostridium botulinum from spore and vegetative cell inocula. J. Appl. Bacteriol. 30, 420. Baumgart, J. 1970. Detection of Clostridium botulinum type E in trout prepared for sale. Reischwirtschaft 50, 1545. Baumgart, J. 1972. Occurrence and demonstration of Clostridium botulinum type E in salt water fish. Arch. Lebensmittelhyg. 23, 34. Beers, W. H., and Reich, E. 1969. Isolation and characterization of Clostridium botulinum type B toxin. J. Biol. Chem. 244,4437. Bengtson, I. A. 1922. Preliminary note on a toxin producing anaerobe isolated from the larvae of Lucidia caesar. Pub. Health Rep. 37, 164. Bengtson, I. A. 1924. Studies on organisms concerned as causative factors in botulism. US. Pub. Health Sew. Hyg. Lab. Bull. No. 136. Bennett, N. McK., Forbes, J. A., Lucas. C. R., and Kucers, A. 1968. Botulism: A report of two cases. Med. J. Aust. 1, 804. Boothroyd, M., and Georgala, D. L. 1964. Immunofluorescence identification of Clostridium botulinum. Nature (London) 202,515. Boroff, D. A., and DasGupta, B. R 1966. Study of the toxin of Clostridium botulinum. Effects of 2-hydroxy-5-nitrobenzyl bromide on the biological activity of botulinum toxin. Biochim. Biophys. Acta 117,289.
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Boroff, D. A., and DasGupta, B. R. 1971. Botulinum Toxin. In “Microbial Toxins” (S. Kadis, T. C. Montie, and S . J. Ajl, eds.), Vol. IIA, p. 1. Academic Press, New York. Boroff, D. A., and Fleck, U. S. 1966. Statistical analysis of a rapid in vivo method for the titration of the toxin of Clostridium botulinum. J. Bacteriol. 92, 1580. Boroff, D. A., and Meluche, H. P. 1970. Amino acid analysis of the isolated and purified components from crystalline toxin of Clostridium botulinum type A. Infec. Immunity 2, 679. Boroff, D. A., and ShuChen, G. 1973. Radioimmunoassay for type A toxin of Clostridium botulinum. Appl. Microbiol. 25, 545. Boroff, D. A., Townend, R., Fleck, U. S., and DasGupta, B. R. 1966. Ultracentrifugal analysis of the crystalline toxin and isolated fractions of Clostridium botulinum type A. J. Biol. Chem. 241,5165. Boroff, D. A., DasGupta, B. R., and Fleck, U. S. 1967. Study of the toxin of Clostridium botulinum. Jap. J. Microbiol. 11, 371. Boroff, D. A., Das Gupta, B. R., and Fleck, U. S. 1968. Homogeneity and molecular weight of toxin of Clostridium botulinum type B. J. Bacteriol. 95, 1738. Bott, T. L., Deffner, J. S., McCoy, E., and Foster, E. M. 1965. Occurrence of Clostridiurn botulinum in fish of the Great Lakes. Bacteriolfioc., p. 1. Bott, T. L., Johnson, J., Foster, E. M., and Sugiyama, H. 1968. Possible origin of the high incidence of Clostridiurn botulinum type E in an inland bay (Green Bay of Lake Michigan). J. Bacteriol. 95, 1542. Boyd, J. W., and Southcott, B. A, 1971. Effects of sodium chloride on outgrowth and toxin production of Clostridium botulinum type E in cod homogenates. J. Fish. Res. Bd. Can. 28, 1071. Brantner, H., and Dohmen, H. 1969. Investigation into the detection of Clostridium botulinum in soil medium by immuno-diffusion. Arch. Hyg. Bakteriol. 153,239. Breed, R. S., Murray, E. G. D., and Smith, N. R. 1957. “Bergey’s Manual of Determinative Bacteriology,” 7th ed. Baillibre, Tmdall& Cox, London. BrUch, M. K., Bohrer, C. W., and Denny, C. B. 1968. Adaptation of biphasic culture technique to the sporulation of Clostridium botulinum type E. J. Food Sci. 33, 108. Buchanan, R. E., and Gibbons, N. E. 1974. “Bergey’s Manual of Determinative Bacteriology,’’ 8th ed. Williams & Wilkins, Baltimore, Maryland. Bulatova, T. I., and Matveev, K. I. 1965. An antigenic structure of Clostridium botulinum type C strains isolated in the U.S.S.R. Zh. Mikrobiol. Epidemiol. Imrnunobiol. 8, 79. Bulatova, T. I., Ivanova, L. G., and Matveev, K. T. 1971. The use of highly specific antibotulinum sera for the detection of Clostridium botulinum types A & B by the fluorescence serological method. Zh. Mikrobiol. Epidemiol. Immunobiol. 48, 101. Buttiaux, R. 1968a. Marine pollution and Public Health. Rev. Int. Oceanogr. Med. 11, 157. Buttiaux, R. 1968b. Modes of contamination of food products by Clostridium botulinum. Ann. Inst. Pasteur Lille 19, 139. Cabelli, V. J., and Richards, N. L. 1966. Ecological studies on Clostridium botulinum and related organisms. Bacteriol. Proc.. p. 11. Campbell, L. L., and Postgate, J. R. 1965. Classification of the sporeforming sulphatereducing bacteria. Bacteriol. Rev. 29, 359. Campbell, L. L., and Winiarski, W. 1959. Isolation and properties of a subtilin resistant strain of Clostridium botulinum. Appl. Microbiol. 7, 285. Cam, D. C., Wilson, B. B., Hobbs, G., Shewan, J. M., and Johannsen, A. 1965a. The incidence of Clostridium botulinurn type E in fish and bottom deposits in the North Sea and off the coast of Scandinavia. J. Appl. Bacteriol. 28,426.
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Wilkinson, J. G. 1878. “The Manners and Customs of Ancient Egyptians.” Revised by S. Birch. Williams-Walls, N. J. 1968. Clostridium botulinum type F: Isolation from crabs. Science 162,375-376. Williams-Walls, N. J. 1969. Effects on growth and toxin production of exposure of spores of Clostridium botulinum type F to sublethal doses of gamma irradiation. Appl. Microbiol. 17,128-134. Willis, A. T., and Hobbs, G. 1958. A medium for the identification of clostridia producing opalescence in egg yolk emulsions. J. Pathol. Bacteriol. 75, 299. Willis, A. T., and Hobbs, G. 1959. Some new media for the isolation and identification of clostridia. J. Pathol. Bacteriol, 77, 51 1. Winamo, F. G., and Stumbo, C. R. 1971. Mode of action of ethylene oxide on spores of Clostridium bohrlinum. J. Food Sci. 36, 892. Winamo, F. G., Stumbo, C. R., and Hayes, K. M. 1971. Effect of E.D.T.A. on the germination of and outgrowth from spores of Clostridium botulinum. J. Food Sci. 36, 781. Wu, J. I. J., Riemann, H., and Lee, W. H. 1972. Thermal stability of the DNA hybrids between the proteolytic strains of Clostridium botulinum and Clostridium sporogenes. Can. J. Microbiol. 18, 97. Wynne, E. S., and Foster, J. W. 1948. Physiological studies on spore germination with special reference to Clostridium botulinurn. 111. Carbon dioxide and germination with a note on carbon dioxide and aerobic spores. J. Bacteriol. 5 5 , 331. Xezones, H., and Hutchings, 1. J. 1965. Thermal resistance of Clostridium botulinum (62A) spores as affected by fundamental food constituents. 1 . Effect of pH. Food Technol. (Champaim) 19, 1003. Yamamoto, K., Kudo, H.,Asano, H., Seito, Y.,Nakya, S., Horiuchi, Y.,Awasa, K., Sasaki, T., and Kinura, K. 1970. Examination of CZostridium botulinum in samples taken from Lake Tourada. Hirosaki Med. J. 22,92: (Biol.Abstr. 52, 139138.) Zachaczewrki, J., and Komorowski, A. 1968. Differentiation of Clostridium botulinum A and B and of Clostridium sporogenes by agar gel precipitation. Exp. Med. Microbiol. 20. 43.
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FOOD PRODUCTS INTENDED TO IMPROVE NUTRITION IN THE DEVELOPING WORLD
BY SAMUEL M.WEISBERG* League for International Food Education Washington, D. C.
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I. Introduction
11. Review and Discussion
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B. Some Experiences with Nutritious Low-Cost Foods in Developing Countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 C. Intermediate Food Technology for Developing Countries . . . . . . . . . . . . . . . 199 111. Conclusions References
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I. INTRODUCTION In developing countries, the urgent need for nutritious, low-cost foods has long been recognized. Along with meeting daily calorie requirements, such foods should contain substantial amounts of good quality protein as low as possible in price to match the low per capita income. Numerous efforts have been made over the years to supply this need through government intervention and private enterprise. However, the long term impact of such efforts has been quite limited, and the successful private enterprises have been very few. A careful examination of this area indicates that, to achieve progress within a reasonable time span, inputs from private food industry, government, food science institutions, and external agencies are all necessary. Governments can establish advisory boards to define needs and set standards for low-cost nutritious foods (LCNF). They can also provide direct and indirect economic support along the following lines: *Executive Director Emeritus. 187
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1 . Classify the LCNF industry as an essential one. 2. Grant maximum tax incentives to such an industry. 3. Establish price controls for essential raw materials. 4. Encourage farmers to grow needed crops to ensure a reliable ingredient supply. 5. Buy such foods for institutions such as motherchild welfare centers. 6 . Promote low-cost nutritious foods on a nonbrand, conceptual basis.
In general, they can provide a favorable climate for encouraging private enterprise to produce LCNF. Private industry can and should be of central importance in providing low-cost nutritious foods. Its role includes the following types of support: 1. Persuade by demonstration to sources of capita1 that such foods can be profitable. 2. Help stimulate through communications media a desire for people to buy the essential foods. 3. Organize to provide farmers volume purchase and realistic prices for the needed crops. 4. Help to establish centralized warehousing and suitable storage for food ingredients so that the supply and prices of needed crops can be stabilized.
Up to the present, the experience of private enterprise with low-cost nutritious foods has been rather frustrating. The fact remains, however, that such enterprises can: 1. Create markets for desirable crops, such as legumes and oilseeds. 2. Provide employment and income for many people. 3. Create markets for equipment and supplies. 4. Provide tax money for government functions. 5. Maintain continuity of supply of nutritious foods at reasonable cost, once established. This is perhaps the most important role of all.
External agencies that are resident in developed countries, such as the Agency for International Development and organs of the United Nations, also make important contributions toward developing low-cost nutritious foods in many countries. They have made possible the channeling of technology, capital, equipment, and food ingredients from developed to developing countries. They have also played an important role in the creation and development of local science and technology institutions. Nonprofit private voluntary organizations also have made valuable contributions in designing and providing low-cost nutritious foods and in distributing donated foods. Educational institutions that specialize in science and technology can offer major assistance to local private industries in the development of low-cost
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nutritious foods (Hulse and Daniels, 1974). Such private industries often cannot afford even simple research and development facilities or special personnel. By turning to properly attuned technology institutions in their own country, they can obtain the needed technology and continued technical support for new low-cost nutritious foods. Here the food technology institutions must be strongly oriented toward providing extension services for fledgling local food industries. The foregoing general statements sketch in broad outline the need for low-cost nutritious foods and the necessary inputs for their achievement. Let us now examine the current state-of-the-art in applied food technology and nutrition, especially in developed countries. This examination will clearly indicate that a basic understanding of nutritional needs and adequate food technology techniques are necessary in providing the foundation for low-cost nutritious foods. Such technology, however, must be accompanied by a thorough understanding of complex marketing problems and how to overcome them.
II. REVIEW A N D DISCUSSION The scope and complexity of world food problems are so immense that all available applied technology in food and nutrition would not cure the ills. Just as we must be selective in utilizing our resources merely to cope with the crises, this review and discussion will focus on only a few cases of success, failure, and promise. The grouping of examples by food category, experience, and intermediate solutions is not intended to imply any cause-andeffect relationships. The degree of detail is directed more toward possible reader interest than relative importance.
A. FOOD CATEGORIES By training and experience, the food technologist is inclined to think about food problems largely in terms of the products themselves. Therefore, the first and major portion of the discussion will address these interests.
1. Textured Protein Products One of the most exciting developments in applied food technology in the past decade is the emergence of textured vegetable protein products. The early foundations for such products (made from plant proteins) were created by Dr. John Harvey Kellogg about 1879 (Robinson, 1972). But, until very recently, their use was accepted only by vegetarians. Essentially two types of such
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products have now moved into the marketplace in the United States. (Newsletter, 1971). a Expanded Vegetable Proteins. These products are made by cookingextruding a mixture of soy flour with flavoring and coloring so as to produce expanded bite-size pieces (generally). The equipment is similar to that used for producing thermoplastic resins. The pieces will absorb about twice their weight of water. They are essentially sterile when manufactured and when reconstituted may have about twice the shelf life of natural meats. Depending on the flavoring and coloring additives, it is possible to simulate beef, chicken, ham, pork, bacon, and many other food products. b. Spun Vegetable Proteins. These products are usually made from isolated protein (mostly soy at present). The protein is dispersed with alkali and extruded through spinnerettes into a coagulation bath. The resultant fibers are then stretched to provide chewiness in the finished product. The fibers are combined into bundles and cut into suitable lengths. When the fibers are mixed randomly with a heat-coagulable binder and then baked, the texture becomes meatlike. When the fibers are well-oriented and supplemented with certain binders and emulsifiers, they can simulate a seafood texture. Spun products require highly sophisticated technology and a major capital investment. They should not be considered near term for developing countries. The extruded products will have first development. It must be remembered, however, that the closest simulation to animal products is that achieved through the spun fiber route. About five years ago, only six or so companies were producing vegetable protein products in the United States. At present, at least fifteen are in this business. Rapid improvements in function, nutritive value, flavor, and organoleptic properties have opened the way for increased public acceptance. The protein efficiency ratio (PER) of these products is stated to be in the range of 2.34 to 3.10, compared to a casein standard of 2.5. In parallel with development in the United States, there has been worldwide interest in textured protein products. Research and development are underway in Mexico, Thailand, Uganda, South Africa, Taiwan, India, Hong Kong, and Venezuela. Such products are already in commercial production in Israel, Japan, and Hong Kong, with strong indications of commercialization in other countries. In the United States, the primary protein source for such products has been soy. In developing countries, peanuts, cottonseed, legumes, and other native sources are also likely candidate proteins. In many developing countries, meat products of any kind are often very scarce and costly. Therefore, it seems possible that textured protein products may move into the marketplace at even faster rates than in the United States, where a variety of animal proteins are still relatively abundant. The present annual level of usage of vegetable protein products in the meat industry in the United States
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is 145 million pounds. It has been predicted that within about 15 years this usage may go to approximately 2.45 billion pounds per annum. Attention has been focused largely on making meat analogs from textured vegetable protein. However, the production of a great variety of snacks of nutritional merit can also be visualized. Nutlike products simulating the texture and flavor of peanuts, cashews, almonds, and other nuts are feasible. One can also visualize crisp confections made by thermal extrusion. Obviously, a great variety of snacks of many shapes and densities can also be made with built-in nutrition. In summarizing the prospects for textured protein products in developing countries, the following observations seem pertinent. Favorable aspects include the following:
I . They will be less expensive than their animal equivalents. 2. They can be made from in-country vegetable protein sources. 3. They can be flavored and colored so as to match almost any desired flavor and color pattern. 4. Since they are dehydrated products, they will have a long shelf life in contrast to their animal meat equivalents. 5. They can be packaged relatively cheaply and simply because of their stability. 6 . They can be shipped long distances because of long shelf life and will occupy minimum space until reconstituted with water for serving. 7. They can be readily adapted to institutional feeding needs as for industrial canteens or school feeding. 8. When they are made properly, the protein nutritive value is good. The constraints to their rapid acceptance in developing countries appear to be a need for:
1. Substantial capital investment in equipment. 2. Sophisticated engineering, quality control, and food technology inputs. 3. Marketing studies to determine price requirements, consumer acceptance, etc. 4. Research and development to fit indigenous vegetable protein products into the processing system to be adopted. 5. Consumer education respecting the nature of products and motivation to use it. 2. Baked Goods
The concept of using baked goods as a convenient vehicle for improving nutrition for people in developing countries has received increasing research emphasis in the past decade. This has come about for several reasons. New
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research technology has indicated the prospect of replacing an appreciable percentage of wheat with in-country oilseed or legume flours. This can result in a reduction in the amount of wheat imported, thus saving scarce currency. A second benefit from the use of these native crops can be their contribution of valuable protein, vitamins, and minerals to the bread. It is now possible to provide a respectable portion of the daily nutrition requirement by means of such baked goods. Consumer acceptance for baked goods has been growing even where, for centuries, rice has been the traditional food and the major source of energy. Baked goods seem to enjoy a certain prestige which helps their acceptance. in Bangkok, for example, one of the wedding gifts is a bun of special shape. Thus, baked goods can constitute a very acceptable means for providing improved nutrition in many developing countries. Until fairly recently, however, it was not possible to maintain a good bread structure when a substantial reduction was made in the wheat content. The advent of bread improvers, such as glyceryl monostearate, the stearoyl lactylates, and sucrose fatty esters, does permit the development of satisfactory bread structures even when substantial percentages of oilseed flours are used. However, most often oilseed flours in developing countries are not of edible quality. They may have an undesirable color, and off-flavors may be present. Those oilseed flours containing appreciable oil content tend rapidly to become rancid. Therefore, the in-country production of better grades of oilseed or other protein flours compatible with wheat flour is essential. There are, for example, research indications that peanut flour and sesame flour may function better in permitting a satisfactory bread structure than cottonseed or sunflower. Carefully controlled heat treatment of the oilseed flour will be needed. Each type differs in cornpatability with wheat flour and will need its own optimum heat treatment to make it function best. At the same time, if the goal is improved protein nutrition by means of baked goods, attention must be paid to the effect on protein quality of any oilseed processing system. If wheat flour is fortified with oilseed or legume flour, it should not be assumed that this automatically results in improved protein quality. The ultimate indispensable test is a bioassay for protein quality of the finished baked goods. Such an assay integrates the supplementary value of the added protein source with the overall effects of the baking process. The protein of all oilseed or legume flours is not equally stable with respect to processing or baking conditions. For the long term, there is a well-defined need for upgrading the technology of oil extraction in developing countries, so that the residual oilcake is not nutritionally damaged, discolored, or otherwise impaired in its potential as a bread ingredient. However, such new technology will require a minimum capital investment in equipment. On an interim basis, therefore, it may be desirable to import from developed
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countries such oilseed products as edible soy flour or blends of wheat and soy flour. These permit composite flour breads of acceptable nutritional value and desired consumer appeal-always provided the cost of such ingredients can be accepted. By adding appropriate oilseed or legume flours to wheat, it becomes possible to achieve a protein quality level equal to that of casein. The structure and flavor of the bread can often be made (if necessary) to closely resemble that of all-wheat bread. There seems little doubt that this promising approach to improving nutrition in developing countries will receive increasing attention and eventually result in progress. Some additional constraints, however, must still be overcome. Flour millers must be persuaded that native crops, such as rice, peanuts, or legumes, can be processed with their regular equipment. Otherwise, special milling equipment must be provided. Provisions must also be made for blending the composite flours. Bakers must be persuaded that the necessary flour blends will handle as well as all-wheat flours. Consumers must be persuaded that breads from composite flours meet their requirements for appearance, flavor, and eating qualities. The government must also be willing to redefine what ingredients go into bread (generally, wheat is the only permissible cereal at present). The level of worldwide interest in more nutritive baked goods is high. Research on baked goods from composite flours is being conducted in Belgium, Canada, Colombia, Czechoslovakia, England, Fiji, Guatemala, Holland, India, Italy, Nigeria, Paraguay, Peru, Philippines, Portugal, Senegal, Trinidad, Turkey, the United States, West Germany, and Yugoslavia. The nonwheat crops being tested in these countries include breadfruit, cassava, coconut flour, corn, cottonseed flour, fish protein concentrate, guinea corn flour, millet, mungbean, nonfat dry milk, peanut flour, pumpkin meal, rapeseed, rice, sodium caseinate, sorghum, sunflower seed flour, and sweet potatoes. Fortification of flour with amino acids, minerals, and vitamins constitutes another valid approach to the use of baked goods as a nutrition vehicle. Bread products made in this way closely resemble in texture, color, and eating qualities the customary bread of commerce. Therefore, expensive persuasion campaigns should not be necessary to gain organoleptic acceptance of such bread products. The primary amino acid used in fortification is lysine, made in Japan by fermentation processes. It has been stated that only large volume requirements are needed to bring the price of lysine into the range of sodium glutamate. The required minerals and vitamins are quite reasonable in cost and do not cause unmanageable increases in the price of bread products.
3. Biscuits, Cookies, and Crackers In many countries, “biscuits” are equivalent to cookies or crackers in the United States. Products rich in protein can be made from completely indigenous
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ingredients, as the textural problems associated with breadmaking do not arise. Since they are relatively dense in structure and low in moisture, packaging requirements per unit weight are reduced. The useful shelf life is generally quite long, on the order of 6 months to 1%years, depending on packaging. This long shelf life allows bisuits to be safely shipped long distances and marketed in villages as well as in urban areas. Package sizes can also be varied over wide limits, according to the needs of the marketplace. It cannot be taken for granted, however, that merely putting together highprotein ingredients in a biscuit will ensure retention of protein quality during baking. Careful selection of the ingredients, their quality, and baking temperature and time are also important. Nutritional worth must be demonstrated by bioassay tests. Moreover, the texture, color, and flavor of the biscuit must be acceptable to the consumer. Of necessity, the price must be low if such biscuits are to reach the least affluent people. In India, widespread marketing of protein enriched biscuits has been achieved by the private sector. The Brittania Biscuit Company markets Provite and Probisk. These biscuits are made of wheat flour, peanut oil, skim milk powder, minerals, and vitamins. They contain 15%protein and, in the case of Probisk, are fortified with vitamins A and D, thiamine, riboflavin, calcium, phosphorus, iron, and iodine. The Parle Biscuit Company and the Shalimar Biscuit Company also market protein-enriched biscuits which have achieved extensive distribution in India. 4. Fried Noodles
Fried noodles do not fit the usual pattern of baked goods. Yet, as a basic wheat product made from dough, they deserve our attention. Fried noodles are widely eaten in many developing countries, and especially in Asia. In Korea, fried noodles are a major component of the daily diet. The Sam Yang Noodle Company in Seoul has expanded its operations year after year to keep up with the demand for this popular food. One can visualize making these noodles from composite flours, such as wheat plus soy, sunflower, peanut, or whatever oilseed or legume flour may be available for improving the protein content and quality of the fried noodle. This company is conducting research and development in the area. Similar studies are being made by CARE in India. One problem is the high oil pickup in frying which reduces the net protein content and increases the cost of the food. Bioassays for protein quality indicate that no impairment results with frying temperatures not exceeding 180" to 200°C for 1 minute. This study was made with noodles composed of CSM (corn-soy-nonfat milk solids) or Balahar (bulgur wheat, peanut flour, and Bengal gram flour.)
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5. Pasta Products Pasta products are highly acceptable foods throughout Latin America as well as in other parts of the world. Pasta is traditionally made from wheat flour. Recently, fortified pasta products have been developed using wheat flour, corn flour, and defatted food-grade soy flour with added minerals and vitamins. Macaroni of this type is being successfully marketed in Brazil. Fortified pasta has many merits. The price is low, the nutritional value is equal to that of milk casein, the cooking time is short, and the organoleptic properties closely resemble those of traditional pasta.
6. Protein Beverages Lowcost protein beverages based primarily on soybeans have been growing in sales volume in Hong Kong, Singapore, Malaysia, and Thailand. So far, commercially successful products have been milky beverages made largely from the dehulled soybean. The sterilized products are generally marketed in glass bottles. Profitable commercialization of milky soy beverages was not an easy task. Only after eight years did a Hong Kong enterprise become profitable. At first, the product was not sterilized, which led to spoilage and numerous returns. Beverage makers initially believed that the product should have the organoleptic properties of cow’s milk-namely, a rather rich unctuous taste. It soon became evident that consumers wanted a thin, low-viscosity profile like conventional soft drinks which had already achieved great popularity in Hong Kong. This constitutes a pertinent example of the need to meet consumer preferences if marketing success is to be achieved. Recently efforts have been made to introduce clear protein beverages with fruitlike flavors. Such beverages require proteins that can withstand at least pasteurization and preferably heat sterilization without coagulation. Thus, the development of insoluble protein particles can be avoided. The fractionation of whey by reverse osmosis techniques is believed to produce protein fractions suitable for this purpose. Some of the whey protein fractions can withstand beverage carbonation without coagulating. Some soy protein isolates are also believed to be suitable. The technology may soon be in hand for the production of carbonated protein beverages. To the extent that such protein beverages displace soft drinks of no nutritional value, they do make a contribution to nutrition.
7. Donated Foods Donated foods have had a major and continuing impact on alleviating hunger in the developing world. Under Public Law 480 Title 11, the United States has so
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far supplied approximately 7.8 billion pounds of nonfat milk solids, nearly 2 blllion pounds of CSM (corn, soy, nonfat milk solids blend) and 200 million pounds of WSB (wheat, bulgur, soy). CSM and WSB are man-made cereal blends fortified with minerals and vitamins. The ingredients are heat-processed to improve the nutritive value, dispersibility, and digestibility. More recently, these blends have been instantized which renders them quickly reconstitutable and thus better suited for such purposes as weaning foods. These products are made in the United States by private cereal companies and provided to the government at prices nearly always substantially lower than would be possible within the developing countries. The ready availability of donated products, widely distributed in quantity and accompanied by detailed information with respect to recipes in the languages of the developing countries, has created a widespread awareness of the suitability of appropriate blended cereals for improving nutrition at low cost. Donation of food products by government obviously cannot continue indefinitely at such a massive rate. The established processing technology, along with product design and development of recipes, should eventually serve as the basis for indigenous blends produced and marketed through the food system of the recipient country. In fact, this has been happening in many countries. In some instances, development of indigenous low-cost optimum-nutrition blends antedated products supplied under PL 480 Title 11. However, such products have experienced much difficulty in achieving sizable volume and making substantial impact. These products will be discussed in the following section on product experiences.
8. Snack Foods The acceptance of snack foods has been increasing in both developed and developing countries. Such foods have great appeal, especially to children. Until recently, such snacks were not seriously considered as candidates for meeting nutritional needs. However, attention is now being directed to both taste acceptance and nutritional input. A recent example of such a product is Pro-Teen, based on corn-soy-sodium caseinate and a modified starch. The protein is 17% and the protein quality is 80%that of Animal Nutrition Research Council reference protein. The snack is deep-fried in vegetable oil and seasoned with a spice blend containing a vitamin premix adequate to provide 20% of the National Academy of Science-National Research Council-Recommended Daily Allowance levels of seven key nutrients and iron (per ounce of product) for children over age four or adults. Pro-Teen is an extruded dough product which leaves the extruder as a flat ribbon. The ribbon is cut to size and dried down to 10% moisture content. In this form, the product is hard, glazed, and free of spices, frying oil, or nutritional
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additives. It is relatively uninviting to insects or rodents and can be shipped to the final processing and packaging site. Frying is done and flavor and vitamin additives are incorporated prior to packaging. Producers believe that the unfried form of this product could be supplied to developing countries for frying, adding locally acceptable flavors, and incorporating the vitamin premix. Perhaps only a modest investment in processing equipment and packaging would be required.
B. SOME EXPERIENCES WITH NUTRITIOUS LOW-COST FOODS IN DEVELOPING COUNTRIES 1. Incaparina
One of the best publicized attempts at providing a low-cost nutritious food for developing countries has been the experience with Incaparina. This product derives its name from the Institute of Nutrition for Central America and Panama (INCAP), located in Guatemala. Basically, Incaparina is a cereal-blend flour, consisting generally of corn or corn equivalent, edible cottonseed flour, torula yeast, vitamins, and minerals. The protein content is approximately 27%, and the protein quality is good. The product is generally consumed as a gruel or beverage. It is well suited for a weaning food. The experience with this product in several countries in proximity to each other and with populations of more or less similar ethnic background will illuminate the formidable types of problems involved in processing and marketing such products. a. Guatemala. Incaparina achieved a market of some 2.5 million pounds per annum. It was fairly successful because it had the support of public health officers, was low in cost, and had good nutritive value. It was handled by private industry, with its own sales organization. The lowest income group were substantial users of the product, and the sales were largely in the urban areas. The product generally had acceptable organoleptic properties. Fairly extensive advertising and continuing consumer evaluation led to product and packaging improvements. Obviously, these factors are important. However, it must be noted that a volume of 2.5 million pounds per annum cannot make a large impact in improving nutrition. b. Colombia. Incaparina was produced by a private company at the rate of some 5 million pounds per annum. It consisted of cornmeal, sesame flour, cottonseed flour, torula yeast, dehydrated kikuyu flour, vitamins, and minerals. The protein content was approximately 28%,and the protein quality was good. The product had some success because it had the support of health centers and doctors, excellent industrial management, and low packaging costs. The flavor and other organoleptic properties were improved as a result of continuing
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consumer evaluation efforts. The reconstituted Incaparina resembled a local thin gruel drink (colada) which is consumed by nearly all population groups. A white Incaparina (Incaparina Blanca) based on rice rather than corn also experienced fairly good acceptance in Colombia. It was marketed at a relatively low price, compared to similar products intended as nutritionally important foods, such as weaning products. c. El Salvador. In 1961-1962, a private company made a vigorous effort to market an Incaparina of attributes similar to those found in the Guatemala product. It was to be consumed warm, as a thin gruel-type beverage. However, the Salvadorans are not accustomed to this type of food, and they would not accept it. The packaging was also unsatisfactory, and the promotion of the product with a pharmaceutical orientation proved to be a bad decision. It is important to note that there was country-wide promotion, support of the medical profession, and government backing. But the product failed even after major modifications were made in packaging and promotion. It must be assumed that persuading consumers to accept the product was too formidable a task. d. Panama. Production was begun in 1966 by a private company with the marketing advantage of its own retail outlets. The product also had the enthusiastic support of a key member of the ownership family. During the first trial marketing, the product became rancid after a short shelf life and developed a bitter, unpleasant taste. This gave the product a serious marketing setback as, of course, consumers would not purchase the product and its favorable image had been hurt. The defect was overcome by toasting the cornmeal component, which also improved the flavor of the product. Packaging problems were also encountered and resolved. A sizable consumer market never developed beyond the local area around David, Panama. It was felt that a country-wide market could have been achieved by more promotion and government support. However, company management decided that more profitable use could be made of company funds, and the project was terminated in 1970. Currently, there is renewed interest by the company in marketing a product Like Incaparina that might fill the need for a nutritious low-cost food in Panama. This time, direct government participation in the project will be sought.
2. Duryea A product recently marketed in Colombia merits special consideration because a special effort was made to avoid the marketing pitfalls of cereal blends intended to provide balanced nutrition at reasonable cost. Duryea was derived entirely from indigenous ingredients: defatted soy flours, Opaque-2 (high-lysine) corn flour, and nonfat milk solids. The product was fortified with vitamins A, B1 , B2, and C, plus iron and calcium. Before cooking,
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a little sugar and water were added. The price of the product was approximately 25 cents per pound. The marketing company made corn the primary flavor component, using a level of 35% in accord with the established corn-consuming habit of the urban populations of Colombia. A continuous effort was made, however, to upgrade the corn component by encouraging the growing of Opaque-2 corn in Colombia. This variety contains protein of excellent quality, compared to the customary corn varieties. Careful attention was paid to destroying insect and microbiological life in the raw ingredients. This, in turn, permitted the use of indigenous packaging materials relatively low in cost. Major expenditures were made for research to determine market size, distribution and consumption patterns, product acceptance, package development, and nutritional adequacy. There were also sizable advertising and promotion budgets. Yet, ten years were required for the company to move from product concept to the current market position. It took four years to market the successful prototype. By 1971, Duryea had achieved a respectable volume, showing some indications of being a success. This experience with Duryea indicates that an integrated approach encompassing many essential components is needed for success. Additional ingredients include long-suffering patience, a serious commitment of much lead time, and a considerable sum of money.
C. INTERMEDIATE FOOD TECHNOLOGY FOR DEVELOPING COUNTRIES The application of major advances in food technology seems best suited to urban areas where a mass market for new food products exists which warrants the substantial capital investment required. In many developing countries, large numbers of people still live in villages and rural communities. Mass markets do not exist, and business enterprises are small, often at the single family level. For such areas, grass roots or intermediate food technology systems are needed. An example that contrasts the two types of food technology is related to manufactured fish products. Edible fish protein concentrate is made by a quite sophisticated process involving solvent extraction, solvent recovery, and solvent recycling. The plant investment for such a process is very considerable and the sophisticated processing operations require a high level of professional skill. The resultant fish product will have a long shelf life. However, as a new food ingredient, it has required extensive market development to achieve consumer acceptance. On the other hand, the production of a salted fish block can be carried out as a family enterprise, using relatively simple equipment and processes. The fish flesh
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is minced, simultaneously salted, and then compressed into a block. The block may be sun-dried. It can be reconstituted with hot water, still retaining many of the organoleptic properties of the original fish. Presumably, therefore, a major marketing effort to achieve consumer acceptance may not be needed. The shelf life will not be so long as for FPC, but it may be long enough for the intended market. Osmotic dehydration presents an excellent example of intermediate food technology that is well adapted to needs of developing countries. It can be readily applied to such fruits as mangoes, plantains, and bananas. In this process, a 67% sugar solution is prepared in unheated water. Ripe bananas or plantains are cross-cut into segments about 2 to 4 cm thick, placed in this sugar solution (four times weight), and held for 18 hours with occasional gentle mixing. Next, the slices are transferred to a 60% sugar solution containing 1% SO2 supplied from potassium metabisulfite. After 1 hour, the slices are removed and drained thoroughly. Then they are given a brief dip (1 minute) in clear, cold water to reduce stickiness. If extended storage is needed, the partially dehydrated slices can be sun-dried or even shade-dried. If available, a cabinet dryer will accelerate the drying which can be safely completed in about 18 hours at 48"C, 50% relative humidity, and linear airflow of 300 meters per minute. One can visualize an array of tropical fruit confections made by variations of this osmotic dehydration process. Fermentation technology is also readily adaptable to developing country needs. Such grass roots or intermediate technology has been extensively applied in Asian village and family enterprises. An example of fermented products is Tempeh, a major Indonesian food. Whole soybeans are washed and soaked overnight in tap water. The seed coats are removed and the beans are boiled in water for 30 minutes. After draining and cooling, the beans are inoculated with the mold Rhizopus oligosporis. Small patties of the beans are tightly packed in banana leaves and incubated for 20 to 24 hours at 31' to 32°C. At that time, the patties are completely bound together with mold fdaments and can be sliced and fried in oil. The texture and flavor are remarkably good, even for the palate of the western world. The mold culture is transferred from batch to batch of the fermented beans, and the environmental conditions of humidity and temperature are well suited to its growth. While mold cultures have been extensively applied in Asia for preparing valuable foods from soybeans, fermentations based on milk-souring types of bacteria have not received equal attention. Yet, it is clearly indicated that food products similar to buttermilk and yoghurt can be made by fermenting soy milks with such cultures as Lactobacillus acidopholus,Streptococcus lactis, and Leuconostoc citmvontm. Cheeselike products can also be made from soy milk with the help of Streptococcus thermopilus.
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The art of fermenting vegetables has been practiced for more than 4000 years (Prescott and Dunn, 1937). Cabbages, cucumbers, tomatoes, brussels sprouts, cauliflower, peppers, olives, carrots, and other horticultural products have long been successfully preserved by fermentation in many parts of the world. However, improved technology can surely be applied to such fermentation procedures by the use of pure mixed cultures and/or the use of a hot-water blanch to pasteurize the vegetables prior to inoculation with suitable cultures of microorganisms. Such improvements can greatly speed fermentation, prevent spoilage, and produce products of better flavor. There is also the excellent possibility of extending suitably controlled fermentation technology to a great variety of perishable products, such as animal protein foods (fish, meat, eggs). Pickling procedures involving the use of such acids as acetic and/or lactic also still offer unexplored possibilities for preserving many perishable foods or food ingredients. The use of such organic acids can bypass the need for fermentation procedures and the extended time involved. However, one cannot expect the same organoleptic properties to be developed.
I I I. CONCLUSIONS Attempts to provide lowcost foods of optimum nutrition in developing countries have often led to frustration and relatively little impact in alleviating malnutrition problems. Examination of constraints that inhibit progress may lead to useful concepts for doing better. There is often a lack of elementary understanding of nutrition on the part of the populations to be reached. There is new evidence that mass media (radio, television, movie theaters, and newspaper advertising) can be effectively used in urban areas for consumer motivation to purchase nutritious foods low in cost. In developing countries, however, the use of mass media for this purpose must be assisted by the government. Formal nutrition education is simply not accessible to rural people. The people must be reached where they are-at home, in the public food market, etc. A second constraint is the lack of suitable food crops, especially protein crops, such as oilseeds or legumes. Often where such crops are available, as in the case of oilseeds, processing the seeds to remove oil leaves a protein cake unsuited for edible use. Improved processing systems are becoming available which can lead to a residual cake in better condition. With respect to food crops, unacceptable losses result from insect pests and rodents. Urgent improvement is needed in grain storage at all levels. Sometimes there are indigenous crops that might be suited for producing nutritive foods if the nutrient composition were known. This problem can best
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be alleviated by establishing laboratories in technical institutions or in government agencies where an ongoing program of nutrient analysis can be conducted. Once data are obtained, it may become possible also to make regional arrangements to exchange suitable crops between countries. Based on such analysis, technology can be developed for removing toxic components and concentrating desirable fractions. A third constraint to developing low-cost nutritious foods is the shortage of capital available at a reasonable rate of interest. Several years are usually required to achieve profitability, and capital is essential during this gestation period. Such high-risk capital would need to be underwritten through government or international funding resources. Very often private entrepreneurs cannot or will not take the necessary business risk. A fourth constraint often encountered is the need for suitable low-cost packaging materials. Packaging preserves the product, presents it in a favorable format to the consumer, and tells what the product is and how to use it. Regional centers might be set up to produce food packages in volume, thus reducing the cost. At present, a suitable package often represents a prohibitive portion of the total cost of a processed food. A fifth and very important constraint is the lack of coordination and communication between government agencies, universities, and the private sector. This is a serious matter where the food industry lacks personnel to do research and development. It is, therefore, essential that businessmen be able to turn to government and university resources for technical assistance. University people need help in planning and funding projects relevant to needs in the private sector. This problem could be alleviated by setting up organizations where representatives of industry, government, and universities can meet on a regular basis and establish teaching programs that provide grass roots approaches, extension efforts, and assignment of students to field problems as part of their training. Clearly of utmost importance is the lack of consumer marketing research and development. Desired foods have succeeded with proper packaging and good keeping qualities. The organoleptic properties were designed to meet precisely what the consumer wanted. The process and product were continually improved and made available to the targetted consumer area. Careful attention was paid to the distribution chain whereby the product reached the consumer. Milky types of beverages and pasta products have met with some success to date. Products believed to have a promising future are: 1. Relatively low-cost weaning foods. 2. Textured vegetable protein foods. 3. Vegetable protein spreads. 4. High-protein snack foods.
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5. Baked goods made from composite or fortified flours using oilseed protein products and/or amino acids. 6. Protein beverages (both milky types and clear ones). 7. Modifications of fully accepted foods. It seems clear that, to succeed in marketing low-cost nutritious foods in the developing world, an inventory must be made in advance of all the applicable constraints, and appropriate resources must be marshaled to overcome them. Where the constraints are too many and too forbidding, the enterprise should not be launched. In the long run the emphasis must be placed on use of’indigenous crops, local enterprises, and in-country technology resources. This is the best way to keep costs down and to identify the local needs that marketing must always encounter and adapt to. “Outside” food enterprises imposed in this effort have had very limited success.
REFERENCES Hulse, J. H., and Daniels, W. D. 1974. The economics of transfer. “Newsletter, League for International Food Education,” September, 1974, pp. 1-4. Washington, D.C. “Newsletter, League for International Food Education.” Meatlike products: textured vegetable proteins, April, 197 1, pp. 1-3. Prescott, S. C., and Dunn, C . G . 1937. “Food Technology,” 1st ed., pp. 515-519. McGraw-Hill, New York. Robinson, R. F. 1972. What is the future of textured protein products? J. Food Technol. 26(5), 59-60,62-63.
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SCANNING ELECTRON MICROSCOPY I N FOOD SCIENCE AND TECHNOLOGY* BY Y. POMERANZ US. Grain Marketing Research Center, Agricultural Research Service U.S.Department of Agriculture, Manhattan, Kansas
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 A. Historical Background . . . . . . . . . . . . . . . . . 206 B. Sources of Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 It. Principles and Applications . . . . . . . . . . . . . . . . . . 208 A. The Basic Instrument . . . . . . . . . . . . . . . . 209 B. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 111. Elemental Analyses . . . . . . . . . . . . . . . . . . . . . 222 A. Electron Probe Microanalyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 B. Recent and Potential Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 IV. Methods of Specimen Preparation and Ancillary Techniques . . . . . . . . . . . . . . . 231 A. General Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 B. Freeze-Drying and Freeze-Etching . . . . . . . . . . . . . . . . . . . . . . . 233 C. Miscellaneous Ancillary Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 D. Specimen Manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 V. Applications in Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 A. Sample Preparation ...................................... 239 B. Morphology of Micr rns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 C. Storage of Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 D. Microbial Growth ...................................... 245 E. Effects of Antibiotics . . . . . . . . . . . . . . . . . . . ...... ... 249 VI. Applications in Plant Investigations . . . . . . . . . . . ...... ... 250 VII. Miscellaneous Biological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 VIII. Studies of Foods and Food Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 A. CerealGrains ..................... . . . . . . . . . . 259 €3. Processing of Cereal Grains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 C. MaltingandBrewing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 *Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
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D. Starches ................................ ..................... E. Oilseeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Milkproducts ........................... . . . . . . . . . . . . . . . . . . . . . G. Miscellaneous Foods ........................................... H. Sanitation ................................................... IX. Some Reflections on Scanning Electron Microscopy ..................... References .....................................................
281 286 287 288 292 292 293
I. INTRODUCTION A. HISTORICAL BACKGROUND
In the mid-1930’s several German physicists (notably M. Knoll and M. von Ardenne) made major contributions to the science of electron optics. Those contributions led in subsequent years to the development of several types of electron optical instruments. The most important one was the conventional transmission electron microscope (TEM)) The early scanning electron microscope @EM),$ constructed in the 1930’s in Germany and improved in the early 1940’s by Zworykin and co-workers at the RCA laboratories in the United States, did not have the resolution and did not show the three-dimensional images of modern SEM instruments (Everhart and Hayes, 1972). World War I1 delayed work on SEM, but after the war development of the SEM began in France and England. In France, Davoine reported the construction of an SEM with a resolution of about 2 pm. In England, Oatley and co-workers introduced the photomultiplier tube and improved the obtainable image quality (Kimoto and Russ, 1969; Black, 1970). The first commercial scanning electron microscope evoled from the design by Oatley’s group (Wells, 1957) and appeared in prototype form about 1963 (Everhart and Hayes, 1972). Commercial instruments with a guaranteed resolution of about 250 A became available in England and Japan in 1965. Today, at least ten manufacturers are building SEM’s. Since the commercial availability of the SEM there has been an explosive growth in its application. This growth in the number of instrument users, new techniques and applications, and volume of literature has been attributed (Black, 1970) to several factors: excellent depth of field (300 to 500 times that of an optical light microscope), good resolution, easy interpretation of three-dimeosional images, wide range of magnification (from 20X to lOO,OOOX), minimal specimen preparation, simple operation, and the capacity to interphase with various instruments (that is, electron microprobe to determine chemical composition of selected micro-areas). As was pointed out by Hollenberg and Erickson, (1973), it is possible to use the SEM to study the surface of fractured, tTEM is used also as an abbreviation for transmission electron microscopy in this report. $SEM is used also as an abbreviation for scanning electron microscopy in this report.
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quick-frozen biological specimens in the cold stage of the SEM, determine the elemental composition of sectioned material by x-ray spectroscopy, and map the distribution of enzymes and antigens on the surfaces of cells and tissues prepared by cytochemical and immunological techniques. The growth of SEM in the last few years has been as astounding and, it is hoped, as enlightening as the growth of valuable information from studies by TEM. There are good indications that SEM has an even greater potential than TEM to contribute to the solution of many elusive and controversial problems in the area of food science and technology. B. SOURCES OF INFORMATION It is difficult to list the many sources of information on the theory and applications of such a rapidly expanding field as SEM. The most important sources are research publications scattered in the numerous scientific journals in physics, optics, instrumentation, chemistry, biochemistry, biology, and technology. The more “concentrated” sources are technical literature supplied by manufacturers of instruments and companies which make the instruments available for use on a fee or contract basis. Other sources include books and chapters in books, review articles, annotated bibliographical lists, and proceedings of symposia. Proceedings of three Stereoscan Symposia were published by the Engis Equipment Co., Chicago, Illinois (1968, 1969), and by Kent Cambridge Scientific, Inc., Morton Grove, Illinois (1970). 0. Johari (Illinois Institute of Technology, Research Institute, Chicago) has edited proceedings of annual SEM symposia since 1968. 1. Books
Following the book by Thornton (1968) on scanning electron microscopy, Heywood (1971) edited a multiauthored volume on systematic and evolutionary applications of SEM. Heade et al. (1972) are the authors of a handbook for users of SEM. Oatley (1972) is the author of a multivolume series on SEM. Volume I deals with the instrument. It covers an introduction to the physics of the instrument and its construction principles, a description of design limitations, reasons for design choices, and detailed discussions of the major components. The objectives of the progenitor of the new research technique were to promote intelligent use of the instrument, to explain basic ideas, and to make use of the instrument as simple and as manageable as possible. Application of SEM in the study of polymers and coatings was discussed in a book edited by Princen (1970). Elucidation of plant structure by SEM is reviewed by Troughton and Donaldson (1972) and by Meylan and Butterfield (1972) in a study of the three-dimensional structure of wood. Fujita et al. (1971) produced an atlas of
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SEM applications in medicine. Gilmore (1972) is the author of a popular book on SEM; the book contains a collection of about 200 excellent half-tones of micrographs. Wells (1973) is the author of a book that deals with basic principles and practical applications of scanning electron microscopy; the book includes excellent micrographs and an extensive bibliography. A book on scanning electron microscopy of plants was published by Troughton and Sampson (1974). Hayat (1974) is the editor of two volumes on principles and techniques (biological applications) of scanning electron microscopy.
2. Chapters and Reviews Earlier work on SEM was reviewed by Oatley et al. (1965); Oatley (1966) discussed limiting factors and valuable features of the instrument. A comprehensive review on principles and applications of SEM in biology and medicine was published by Hayes and Pease (1968). A summary of the latter paper was presented by Pease and Hayes at the 1968 SEM Symposium of IIT, Chicago. Excellent reviews on the principles, characteristics, and applications of SEM were published by Kimoto and Russ (1969), Thomas and Hull (1969), Black (1970), Baker and Parsons (1971), Baker and Princen (1971), Nixon (1971), Everhart and Hayes (1972), Hollenberg and Erickson (1973), and Hayes (1973). Echlin (1968, 1971) is the author of two reviews. The first deals with the use of the SEM in the study of plant and microbial material. The second is a thoughtprovoking discussion of new developments and future applications of SEM in biological research. A more recent review (Echlin and Fendley, 1973) discusses the future of electron microscopy in biology.
3. Proceedings and Bibliography SEM has been the subject of annual symposia (since 1968) organized by the Illinois Institute of Technology, Research Institute. Proceedings of the symposia, including bibliographical lists, were published. Other sources of information on SEM include proceedings of annual meetings of microscopy societies, the biennial reviews on electron microscopy in the April issues of Analytical Chemistry, and comprehensive bibliographical lists by Rossi (1968) and Wells (1969, 1970).
11. PRINCIPLES AND APPLICATIONS The two most important parameters in the evaluation of any microscopy system are localization of information (resolution) and transformation and display of that information. The ideal system combines high resolution with high information content. The ultimate resolving power of a light microscope is
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between 2000 and 3000 A (half the wavelength of ordinary light). The greatest effective magnification of the light microscope is 1000 (the average resolving power of the human eye-about 0.2 mm-divided by the resolving power of the light microscope). To improve the resolution, it is necessary to change light to some other form of radiation for illuminating the specimen; this is done in the TEM. The wavelength of electrons is inversely proportional to the potential to which they are accelerated-that is, to their velocity. New high-voltage TEM’s give resolutions between 2 and 5 A. However, there are several characteristics that limit the type of material examined and information obtained from the TEM. The TEM can be used to examine thin sections (about 500 A); this requires tedious and careful specimen preparation which may complicate interpretation of results. The TEM can provide useful information on the three-dimensional spatial structure, chemical composition, and electrical properties of the specimen, or on relations that would exist in a living, physiologically intact specimen (Hayes and Pease, 1968). The TEM has a substantially higher depth of field (about 20X) than the optical light microscope. Thin sections are an excellent source of information when viewed in three dimensions. Excellent information can be obtained in the SEM, although, admittedly, at the expense of resolution (the resolving power of SEM is about 100 However, it would be incorrect to view the SEM as a replacement for other types of microscopes. Rather, it complements them. It is an extension of the reflected light microscope (which has rather complicated optics and low resolutions) and is a bridge between light and electron microscopy (Baker and Princen, 1971). If visible light is used as the source of radiation, we see a picture that is rich in information but limited in resolution. In the TEM, the reverse is true. To combine high resolution with high information one must separate these two functions so that they are not performed by the same radiation (Hayes and Pease, 1968). In SEM, electrons are used for localization, and visible light (cathodoluminescence), secondary electrons, backscattered electrons, or x-rays are used to transfer information. Instead of the lens system, SEM uses for image formation a time sequence of modulated points; this is, essentially, the system used in television. In SEM the correspondence between object and image is attained not by optical focusing but by time sequencing. Figure 1 compares image formation in the optical, transmission electron, and scanning electron microscopes (Baker and Princen, 1971). Important features of the three types of microscope are summarized in Table I (Black, 1970).
a).
A. THE BASIC INSTRUMENT
In the SEM, the electrons from the gun are accelerated toward the anode, where they pass through several electron lenses, and the image of the electron
TABLE I COMPARISON OF IMPORTANT FEATURES OF THE SCANNING ELECTRON MICROSCOPE (SEM), TRANSMISSION ELECTRON MICROSCOPE (TEM), AND OPTICAL LIGHT MICROSCOPE (0LM)n
Characteristic or feature
OLM
TEM
SEM with W filament
Lens system
Generally glass or quartz
Electromagnetic lenses
Electromagnetic lenses
Image
Displayed on eye retinas, frosted-glassviewing screen, or film
Displayed on fluorescent viewing plate or film
Energy
Radiation from the W through the visible spectrum to the infrared
Thermonic emission of electrons accelerated by 50-1000 kV
Displayed on CRT for viewing-visual and photographic screens available Thermonic emission of electrons accelerated by
Wavelengths of 2200 A (quartz optics) energy 7000 A Resolution, d Down to about 1000 A with U V radiation
1-30 kV
>1 A 5 A at 500,OOOX down to 250 A at 4000X
1 pm at l O O X up to loo$ at 10,OOOX
SEM with field emission gun Electromagnetic lens TV monitor or CRT
STEMb Electromagnetic lens Displayed in either TEM or SEM mode
Field emisField emission source, sion source 0.3-3.5 kV of electrons tip voltage; 30-100 kV oil-20 kV acceleration voltage >I A >1 A
20 A
25 AC
Depth of field, 250 pm at 15X down to 500 pm at 4000X down to Excellent at all magnifications; Almost 1000 pm at lOOX down to infinite 0.08 pm at 1200X. 0.2 pm at 500,OOOX. 10 pm at 10,OOOX Generally poor Generally good 20-100,000x 200-300,OOOX 20-50,OOOX or 100,OOOX Magnification 15-800X with dry objectives in visible light; with range oil immersion optical system up to 1500X in visible light; quartz optics and W radiation of 2200 A, up to 3000X Same limits No real size or thickness Thickness critical, 0.1 pm Sample require- Surfaces and thin sections as SEM and ments prepared by standard methods. or less for 50 kV, 1 pm for limits-rough 3-D surTEM faces accepted. Non500 kV. Surfaces must be Thickness important metals must be Au- or Pdreplicated-specimen 2-3 mm in diameter coated
D
‘Updated from Black (1970). bJEOL lOOB TEM modified to do STEM work. CResolved 5 A point to point on special carbon fibers.
Superior t o TEM Same limits as SEM and TEM
Same limits as SEM and TEM
Y. POMERANZ
212 liihl Microscope [Upside Downl
u r-tiihl
Transmission tlrclron
Scannine tletlron Microscage
Microscope Source
KVA
A
-Electron
Gun
&VlJ
-,-
~ t l c r l r o nGun Is1 Aperlurc
+
Condenser Lens -Condenser
Lens
-Specimen -Objective
-2nd
Lens
Aperture
I
Image Formalion
FIG. 1. Diagram of image formation in (a) the optical microscope, ( b ) the transmission electron microscope, and (c) the scanning electron microscope. From Baker and Princen (1971).
source is formed in the plane of the specimen, after several reductions, with a diameter of several tenths to several tens of nanometers. The guns used in electron microscopy are of two types: thermionic emission and field emission. For thermionic sources, tungsten and more recently lanthanum hexaboride are heated to electron emission temperatures. In field emission, a strong positive field pulls the electrons from the metal. The field emission gun operates at room temperature. It is fabricated of monocrystdine tungsten shaped to a point about 1000 A in diameter (Coates and Brenner, 1973). The field emission gun emits a beam that is about one thousand times as bright as conventional thermionic sources and has an energy spread of less than 0.3 eV. A thermionic tungsten hairpin has a typical energy spread of emitted electrons of about 5 eV; a lanthanum hexaboride rod gives an electron energy spread of 3 eV. Consequently, chromatic aberration in field emission is reduced and higher currents can be concentrated into smaller spot diameters, yielding higher resolution and/or higher signal-to-noiseratios. In most SEM’s the electron beam is deflected twice within the objective lens and scanned over the specimen in a predetermined manner-generally, a square television type of raster. The primary electrons produce at the specimen surface secondary electrons, light, x-rays, infrared, ultraviolet, and semiconductor effects; any of those signals can be measured. The specimen itself can be moved in any direction, rotated, tilted with respect
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to several axes, and subjected to mechanical or thermal treatments or otherwise treated in the microscope. An amplifier enhances the collected signal; it may also modify the contrast and process (that is, differentiate or integrate) the information that comes from the specimen as the electron beam scans over the surface. As the electron beam scans the specimen surface, the raster of a cathode-ray tube (CRT) is scanned in synchronization with the electron beam. A schematic diagram of the SEM and CRT display is given in Fig. 2 (Black, 1970). The intensity of a point on the CRT raster depends on the intensity of a signal (secondary electrons) generated by the impinging primary electrons on the surface of the specimen. For optimal image quality, enough lines should be scanned for good coverage, and the scan speed should allow an adequate count of electrons or protons for each point. Although the image formed in SEM is produced by a time-sequencing technique similar to that used in commercial television, there are two major differences (Everhart and Hayes, 1972). In commercial TV the picture is made up of 525 horizontal lines; the image in SEM can be varied (generally, from 500 to 1000 lines). The rate of scan that produces an SEM micrograph is often much lower than the scanning rate in TV. A time exposure of several minutes may be needed to obtain high-resolution SEM micrographs. Resolution decreases with working distances. (Degree of electron scattering, vibration, and contamination
Visual Disploy
Grid
Q
tube
Camera for recording image Scintillator Grid
Specimen
-
Photo mu It i p y e w
Video omplif ier
MICROSCOPE C O L U M N
CRT D I S P L A Y
FIG. 2. Schematic diagram of scanning electron microscope and cathode-ray tube (CRT) display. From Black (1970).
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Y.POMERANZ
also affect resolution.) The working distance is the distance from the last lens to the specimen; it ranges in SEM from 2 to 3 mm to 24 mm. Depth of field varies with magnification and resolution from about 300 pm at lOOX and 500-A resolution to 100 pm at lOOOX and 200-A resolution for a 25-kV beam and a 12-mm working distance (Black, 1970). In addition, depth of field can be controlled, within limits, by the final aperture. Increasing the acceleration potential increases the resolution, but this is accompanied by damage to the specimen and by contamination. Under most conditions, even at higher potential, the probe diameter is the primary factor controlling resolution. The probe can be demagnified to less than 100 A, but, in practice, for most work demagnification to 200 A is used. Resolution limits of the SEM were discussed by Catto and Smith (1973). In addition to a CRT used as a visual screen, there is a second CRT in front of which a camera can be placed. The resulting image can be observed visually, photographed, interfaced with a computer for direct analyses, videotaped, etc. One of the important features of SEM is that, once the beam has been properly focused at a particular magnification, there is no need to refocus for lower magnification. Magnification is given by the ratio of the display area on the CRT (constant) to the area scanned on the specimen surface (variable). Most SEM’s have a magnification range of 20X to lOO,OOOX, with maximum useful magnification of 20,000 to 50,000 in the secondary mode. Magnification is adjusted by operating a switch; on some instruments, a potentiometer is used to adjust the magnification. Specific areas on the specimen are located at low magnification. Subsequently, the image is enlarged to reveal finer detail. The advantages of the SEM are as follows (Nixon, 1971): (1) Solid specimens can be observed (and experimented with) at high resolution and good depth of field. The very small angular aperture of the probe-forming system permits a large depth of field. At a resolution of 1 pm the depth of field is 1 pm in the optical microscope and 700 pm in the SEM. (2) In addition to high resolution and depth of field, the SEM pictures show excellent contrast. If the observer looks at SEM pictures of specimens at an angle to the electron beam, it appears as if the eye were along the axis of the electron beam and as if the illumination fell on the specimen from the electron collector placed to one side (Nixon, 1971). This contributes to a three-dimensional effect even when only one micrograph is used. It is possible to tilt the specimen, or the beam, record two micrographs, and obtain a stereographic pair. The stereographic pair can be viewed in a stereoscope to achieve a true 3-D image. With additional equipment, contour maps of the specimen can be plotted (Thomas and Hull, 1969). (3) The sample can be inserted rapidly into a vacuum with an airlock, and the whole column can be maintained at the desirable vacuum. Under special circumstances one can study live specimens (insects). The very low beam current (lo-’’ to ampere in SEM, compared with lo-’ to lo-’ in TEM) produces negligible specimen heating or damage.
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Howden and Ling (1973) reported that good low-magnification (5X to 5OOX) SEM pictures of dry, uncoated zoological specimens may be obtained with a low accelerating voltage (1.5 to 3.0 kV) in conjunction with a short exposure to the scanning beam. As in TEM, the sample in SEM is bombarded with a fine probe of electrons from a filament, usually a tungsten wire loop that has been brought to a point. The electrons are accelerated by applying a voltage up to 30 kV. The electron beam is focused with a series of electromagnetic lenses. The electron beam should have a diameter of 100 A or less. If a tungsten hairpin cathode is used, the “crossover” of the electron gun has a diameter of about lo6 A. Consequently, a demagnification of about 2 X lo4 is required (Oatley, 1966). For such demagnification, three magnetic lenses are needed. Instruments can be made with two lenses, thus simplifying operation and alignment. This was accomplished by improving the gun design to reduce the “crossover” from over 100 pm to about 13 pm and by using electromagnetic lenses, which provide slightly higher demagnification with less aberration (Kimoto and Russ, 1969). As in any electron optical system, the electron beam can function only in a high vacuum, and the electron source, electron lenses, and specimen are all enclosed in an evacuated tube. So far as operation of the instrument is concerned, a pressure of about lo-’ torr is satisfactory. However, for many applications, a much lower pressure may be needed to prevent contamination of the specimen. In the TEM, the image is produced by nearly monochromatic electrons that illuminate a thin specimen which serves to diffract or scatter the electrons. The image in TEM is a measure of the scattering power of each point in the specimen. The advantage of the SEM is that it is not controlled by the physical constraints imposed on the TEM. In SEM there is a wide choice of contrast mechanisms and image formation. This results from the fact that in SEM there is a separation between resolution and transformation of information, and the latter does not require optical processing. Consequently, any short-term effect that results from the interaction of the beam with the specimen, and that can be quantitated, can be used to present information about the specimen (Hayes and Pease, 1968). The types of information generated by the impingement of the electron probe on the sample are shown in Fig. 3 (Kimoto and Russ, 1969). According to Hayes and Pease (1968), the contrast mechanisms in SEM can be divided as follows: 1. Secondary electron emission. For a constant primary electron current, the secondary current depends on the angle of incidence, the atomic number of the specimen, and the surface properties of the specimen. In practice, surface properties other than topography (or voltage) have little effect. The atomic number affects backscattering in polished samples, but on rough surfaces the effect of the angle of incidence is usually the important one.
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Y.POMERANZ Electron
Pro b e
FIG. 3. Types of information generated by impingement of electron probe on sample. Kimoto and Russ (1969).
2. X-ray microanalysis. The spectrum of the x-rays produced when an electron beam strikes a specimen is characteristic of the elements in the specimen. By monitoring the spectra of the x-rays produced as the specimen is scanned by the electron beam, it is possible to determine the elemental composition of the specimen (Hayes and Pease, 1968). 3. Cathodoluminescence. Although the x-ray spectra give information about the elemental content of the specimen, to learn about its molecular makeup it is necessary to collect the protons emitted by the action of electron irradiation of the target cathodoluminescence. An extension of this method uses organic stains to tag certain molecular species as in conventional fluorescence microscopy. Both in x-ray contrast and in cathodoluminescence the efficiency of production and collection of light quanta is low, although the generation of light quanta takes place in a relatively large volume. Another difficulty of cathodoluminescence is that the materials stop luminescing (are poisoned) after irradiation. 4. Induced-current mode. The electron beam generates free carriers in the specimen. This is the principle on which the induced-current mode is developed. If these carriers are in the region of the electric field, or are able to diffuse to
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such a region, an electric current will flow. This current can be used to modulate the brightness of the display cathode-ray tube. The induced-current mode has been employed primarily in the examination of semiconductors. It may be applied in the future in studies of biological systems mayes and Pease, 1968). 5. Deflection-modulation display. This display is most useful in situations where the contrast is low or when quantitative information of the video signal is required. The SEM is generally utilized on solid samples which are usually too thick to transmit electrons. Some electrons are transmitted if the specimen is not thicker than a few micrometers and is of low atomic density. A scanning transmission electron microscope (STEM) that operates on the same optical principles as the TEM in a transmission mode was developed by Crewe and co-workers (Crewe, 1971; Crewe and Wall, 1970). The STEM has more depth of field than the TEM, and the signal may be processed and modified as in the SEM.
B. APPLICATIONS The various operational modes of the stereoscan system of one of the major manufacturers of instruments for SEM are illustrated in the diagram shown in Fig. 4. The two most important modes for the food scientist and technologist are (1) secondary electron emission from a specimen caused by scanning it with an electron beam (used to reproduce topographical detail, the most convenient technique for direct examination of samples with rough surfaces), and (2) x-ray microanalysis in which x-rays emitted from the specimen as a result of excitation
I X-rays
11
Dispersive
Non-
dispersive
Specimen current
I
Beam-lnduced conductivity
Carhodoluminescence
1
Secondary electrons Electron channeling Selected-area diffraction
Transmission Voltage contrast
Electron-beam
FIG. 4. Uses and information from Cambridge Stereoscan, 54-10. Courtesy Cambridge Scientific Instruments, Ltd.
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Y . POMERANZ
by the electron probe are analyzed to determine the detailed chemical composition.
1. General Information The present status of SEM and future potential applications have been reviewed in two recent articles (Anon., 1972a, McAlear, 1972). According to McAlear (1972), there are about 400 SEM instruments in the United States and about twice that many in the world. Whereas, originally, most instruments were produced by two manufacturers, there are today about ten companies in the field. Several promising new developments have been indicated. Some of the recent instruments (HISCAN HHS-2R, Perkin-Elmer Corp.) have the following features: resolution routinely guaranteed less than 100 A, magnifications variable from 1OX to 300,00OX, two CRT’s for viewing of specimens at two magnifications at the same time, and inclusion of a light optical sample viewer. A relatively inexpensive (below $20,000, including auxiliary equipment) MiniSem (International Scientific Instruments) has a guaranteed resolution of 300 A and a magnification range of 30X to 40,OOOX. Lanthanum hexaboride electron guns with significantly improved beam intensity can be.adapted to most contemporary SEM’s. Ion pumped instruments with a field emission gun (Cwickscan) recently have been introduced (Coates and Welter Instrument Corp.); differentially pumped field emission sources seem to be assuming increased significance in improving the resolution of the SEM. The greatest activity and promise lie in improved methods of sample preparation and in x-ray microanalysis. Wet stages have been constructed for the SEM. Such stages should make examination of living cells possible, but many difficulties remain to be overcome-for example, cooling of the specimen from the rapid evaporation of water in the vacuum. Recording in most SEM’s is rather slow, and examination of frozen samples shows potentially greater promise than examination of wet samples. The fifth SEM symposium in Chicago centered around the analytical capabilities of the SEM and directions in which development of SEM is proceeding (Anon., 1972a). The following are some of the topics presented. High-resolution capabilities of field emission electron guns clearly demonstrated the potentialities of the new boride emitters which operate under less stringent vacuum conditions than the previously used guns. SEM’s working at high and low temperatures, and new high-contrast cylindrical secondary electron collectors are now available. A storage-display system shows great promise for examination of labile specimens. High-resolution x-ray microanalysis may now be carried out on a wide range of materials, and further improvements may be expected in the sensitivity of energy-dispersive x-ray detectors. New techniques of thin-film deposition of nonconducting surfaces permit more information to be obtained. New methods for measuring the height and depth of specimen surfaces and quantitative analyses are available w a r d , 1972).
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2. Cathodoluminescence As was mentioned previously, the efficiency of production and collection of photons in cathodoluminescence is low. The generation of light quanta takes place in a volume which is much larger than indicated by the cross-sectional parameters of the primary beam. If thin sections are used, the generation of light is lowered even more, and it is necessary to have long exposure times to obtain a satisfactory picture image. Cathodoluminescence resembles fluorescence optical microscopy. Specific fluorochromes may be added to living cells in low concentrations without causing toxic damage. Unfortunately, many types of fluorochromes which can be used in fluorescent light microscopy are quenched when used in a cathodoluminescent system. Although the cathodoluminescence system has a high contrast, the resolution is only about 80 to 100 nm and is not useful in quantitation (Echlin, 1971). According to Echlin (1971), the availability of appropriate dyes could extend the application to cathodoluminescence in two directions: (1) Observation could be made of naturally occurring luminescent materials in tissues, so that luminescent by-products of their metabolism could be localized. (2) Fluorescent stains with a specificity for particular regions or biochemical moieties could be added to cells. The stain could react with defined cellular entities, detect foreign substances, or be used in combination with labeled antibody methods. It would seem that progress in using cathodoluminescence in SEM hinges much on the availability of appropriate dyes. DeMets and Lagasse (1971) evaluated thirty-three organic chemicals for their cathodoluminescent properties. No consistent relationship was established between molecular structure and luminescent yield. More promising results were obtained in studies conducted by Falk and co-workers. Fak (1972) examined common biological dyes and herbicides for cathodoluminescence. Examination of leaf surfaces treated with herbicides showed cathodoluminescence in the herbicides contact area. Ong et al. (1973) reported that a wide variety of herbicidal compounds can be excited to fluoresce in the SEM; the compounds included variously substituted five- and sixmembered nitrogen heterocycles, anilines, and benzenes. Generally, the more highly substituted an amino group, the greater was the fluorescence intensity. The fluorescent properties of the herbicides could be used to spatially locate them on the surfaces of the leaves. Some insecticides show a similar fluorescence. The authors suggested using the method to estimate the persistence of such insecticides in treated fields. 3. X-Ray Microanalysis This is an area of great progress and promise; the status of development is summarized in Section 111.
220
Y.POMERANZ
4. Sample Preparation and Ancillary Techniques This is another area of great promise; it is reviewed separately in Section IV. 5. Useas TEM The regular SEM is a rather expensive instrument. It requires electron optical components of a quality comparable to that needed for regular TEM and more electronic equipment. Consequently, it costs rather more than the TEM and much more than high-quality light microscopes (Oatley, 1966). Use of the SEM in the transmission mode can be justified only if it offers unique advantages. There are many opaque specimens for which no satisfactory replica can be made; in such instances use of the SEM may be considered. Generally, resolution of the SEM in the transmission mode is about 100 A, compared with 5 A or below in conventional TEM. It is, however, possible to improve resolution to 20 A by reducing the probe diameter. Such a reduction does not improve resolution in regular SEM based on secondary electron emission because of spreading in the sample (Kimoto and Russ, 1969). The availability of very bright electron sources has made possible the development of SEM operating at high resolution in the transmissive mode (Echlin and Fendley, 1973). Such instruments can isolate and count the transmitted electrons of any particular category for each point of the specimen. That information can be used to measure the ratio of inelastically to elastically scattered electrons and to determine the average atomic number of the material. The high-resolution scanning transmission electron microscope (STEM) developed by Crewe and associates produced a beam spot of 0.5 nm (potentially as small as 0.1 nm) (Black, 1970). Wall (1973) reviewed the development of the high-resolution STEM, and Komoda e t al. (1972) described a STEM using a field emission electron gun. The instrument has the ability to resolve molecules such as DNA spread on a support fdm (Crewe and Wall, 1971). As it is possible to detect the distribution of atomic species to within about 1OZ numbers, different bases in the DNA could be labeled and a color-coded composite image could demonstrate the base sequence and read out the genetic code directly. Theoretically, at least, it should be possible to record additional signals and obtain x-ray, energy loss, and auger electron analyses in this improved STEM (auger electron analyses are explained in Section 111,B). A high-voltage STEM for study of living cells is being developed. In light of all these developments, one must concur with the optimistic prediction of McAlear (1972) that STEM may in the not-too-distant future replace TEM for many applications (see also Beer, 1974).
6. Moving the Laboratory into the Microscope In a SEM the distance between the specimen and the final lens need never be less than 3 to 5 mm; when the highest magnification is not needed, it can be
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much greater. The large working distance combined with the practically unlimited size of the specimen chamber make it possible to carry out experiments on an object under observation. Some of the earlier uses in this area were reviewed by Oatley (1966). They included sputtering of metals by positive-ion bombardment; activation, followed by poisoning, of thermoionic cathodes of the dispenser type; thermal decomposition of unstable chemical compounds, such as azides; and electron-beam machining of thin films in which the electron beam of the microscope is also used for machining the films. More recently, Nixon (1968) reviewed the possibilities of moving “the whole laboratory into the microscope.” The examples included observations of a small area of metal subjected to several cycles of heating or to various gaseous atmospheres and use of a tungsten point as a microhardness tester. A galvanometer was used in reverse, and a few microamperes flowing in the coil deflected the tungsten point by a known amount; thus a known force was applied to the surface of the specimen. In the latter case, the hardness indentations were considerably smaller than in normal testing (by a factor of 20 to 50) and could be positioned accurately on the sample. The volume affected by the hardness tester varied as the cube of the indentation size; decreasing the sample length ten times decreases the indentation volume a thousand times. Use of rheological measurements to follow thermal (or chemical) modifications in food products could provide important information on food texture. Similarly, rheological parameters could be theoretically applied to pinpoint the site of strains resulting from mechanical stresses in various plant and animal tissues. The minor modifications could be more easily evaluated in terms of basic rheological parameters than could major visual changes. Nixon (1968) pointed out that, as we decrease the size of an observed object, it is easier to follow the changes that take place. I should like to add that at the same time we can get information on the microstructure that cannot be obtained from examination of the gross macrostructure. This applies to the two processes mentioned previously (thermal and mechanical modification) and to corrosion studies. Minor, yet important, changes which may not be seen for months by the naked eye or for weeks in the optical microscope can often be seen within minutes in the SEM. Fulrath (1972) described the construction of a “hot stage” which enables the creation of temperatures to 1600°C without damage to the SEM chamber. The hot stage could be used to char foods for conductivity tests; to determine volatility, destruction, or heat stability of components; and to map the distribution and quantity of mineral components in a food or section of plant or animal tissue (Hollenberg and Erickson, 1973). Finally, SEM can be used as a tool in tissue preparation. Echlin ef d. (1969) described a method for removal of parts of the specimen surface inside the SEM column. The ion source was a cold cathode argon discharge unit, the beam from which was focused onto the specimen for controlled, accurate, and selective
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Y. POMERANZ
etching under direct observation. The power density from a controlled electron beam can theoretically equal the power of most lasers (Hollenberg and Erickson, 1973). The SEM primary beam can be used for specimen microdissection. The precision of the electron beam dissection is better than that obtained with the conventional laser beam.
7. Miscellaneous Recently some units have been equipped with a TV monitor which can follow rapid thermal or rheological modifications (Black, 1970). A conventional video tape recorder can be used for recording and reproducing the image; the image can be observed in a lighted room. The feasibility of a scanning ion microscope should be mentioned. Little thought has been given to the possibility of using beams of high-energy ions (Martin, 1973). Atomic collisions of high-energy heavy ions produce large yields of x-rays. Heavy ions have the theoretical advantage of having a smaller de Broglie wavelength than electrons. If the ratio of heavy ion yield to radiation damage is high enough to make the ions effective probing particles in a scanning microscope, the resolution of the microscope could be superior to that of the best electron microscopes. First pictures from a proton microscope showed great promise (Anon., 1974). The new proton microscope is based on technology similar to that developed by Crewe and co-workers in designing the STEM.The protons produced, by field ionization, use a tungsten tip at liquid hydrogen or helium temperatures. The microscope is designed to work at 100 kV, and the protons can penetrate to the depth of 1 pm of biological materials. The instrument is expected to provide information on cell composition at the molecular level. Wells, Broers, and Bremer (Anon., 1973b) have developed a new method capable of a finer resolution (by a factor of 5) than that obtained in the best SEM. By placing the specimen inside the electron lens, a short focal length may be used. In addition, an electron beam 5 to 10 A in diameter is employed. Preliminary experiments indicated point-to-point resolutions of 30 A; it is believed that resolutions of 10 A can be achieved.
111.
ELEMENTAL ANALYSES
It has been known for a long time that elements with a high atomic number backscatter more incident electrons than do elements with a low atomic number. This phenomenon can be used to obtain information on the approximate distribution of elements in a sample. However, while t h i s production of backscattered electrons depends on the element, it also depends on the binding
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energy of the various orbital electrons and varies with different chemical combinations of an element (Kimoto and Russ, 1969). Another way of examining the composition of specimens is cathodoluminescence. Its limitations were described in Section 11. The most precise way to determine composition is by identifying in an electron microprobe x-rays emitted from the specimen. There are two basic systems for elemental analysis and compositional determination by x-ray analysis. They are the wavelength-dispersivemethod (WDM) and the energy-dispersivemethod (EDM). In the early days of x-ray microanalysis the WDM was used exclusively as the x-ray detector system on electron beam microprobes. In this system, x-ray spectrometers are equipped with metal crystals. In the late sixties, the lithiumdrifted silicon detector brought x-ray analysis by the EDM into prominence. The EDM can be easily interphased with SEM and is less complicated to use than the WDM. The operation of the EDM is simple and rapid and is characterized by good detector resolution and high sensitivity. However, the resolution of spectra is poorer in the EDM than in the WDM. Important features of the two systems are as follows (Anon., 1973a): Wavelengthdispersive analysis. Better element separation (resolution), higher count rate on individual elements, better peak-to-background ratios, analysis of elements higher than sodium. Energydispersive analysis. Rapid, simultaneous multielement analysis of the full x-ray spectrum, display of entire spectrum in digital format, high collection efficiency, less sensitivity to geometric effects, lack of higher order lines which are generated in crystal diffraction, digitally produced outputs for elemental line scans and distribution maps. According to Black (1974), the advantages of the EDM outweigh its disadvantages. The use of both systems in x-ray analysis in combination with SEM has been described (Anon., 1973a; Dao, 1974). A. ELECTRON PROBE MICROANALYZER The technique of electron probe microanalysis was first introduced and applied to metallography by Castaing and Guinier in 1949 (Coslett, 1962). Concepts, principles, and uses of the electron probe for microanalysis were described by Castaing (1960), Coslett and Nixon (1960), Coslett (1967), Birks (1972), Hall and Coslett (1972), Andersen (1973), Hall et aZ. (1974), and Liebl (1974). The electron probe microanalyzer consists basically of an electron optical system, an x-ray detection system, an electron beam system, a light optical system, a mechanical sample stage, data recording and display systems, and several ancillary systems (Andersen, 1967). In addition to determining the elemental composition of a microvolume of material, the instrument can be used
224
Y. POMERANZ
FIG. 5. The electron column of an EMX-SM electron microprobe x-ray analyzer. A , tungsten filament; B, grid cap; C,anode plate; D, condenser lens; E, limiting apertures; F, focus lens; G, specimen; H , secondary electron detector;I, curved crystal;J, x-ray detector. From Rasmussen and Knezek (1971).
(1) in studies in situ of the nature of chemical bonding of many of the elements; (2) in studies of the structure of crystalline compounds within a specimen; and (3) in contact microradiography and x-ray projection microscopy. The electron column in a microprobe is shown schematically in Fig. 5 (Rasmussen and Knezek, 1971). The microanalyzer is an instrument which makes an essentially complete analysis on microstructures observed in their sections or in bulk. Elements of the periodic table above beryllium can be detected with good sensitivity in volumes of a few cubic micrometers (Andersen, 1967). Absolute detection limits are about g, and relative concentrations are 0.01 to 0.10% for most elements in biological specimens. According to Echlin and Fendley (1973), with probes formed from the tungsten filament electron emitter and the currently available detectors it is possible to determine by x-ray microanalysis lo-" g of an element in a biological tissue. The minimum weight-to-weight ratio in the volume exposed to the microprobe is about 100 ppm. The rare earth boride emitters, and especially the field emission gun, are several orders of magnitude higher than tungsten filaments and should make it possible to analyze a spot 2 nm in diameter. Echlin
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and Fendley (1973) visualize using x-ray microanalysis to determine the distribution and movement of ions and other species in cells and tissues; to measure ion concentrations within organelles, between different parts of the cytoplasm, and across cell boundaries; and even to measure concentration gradients across and within individual membranes. Detection limits in analysis of metals in biological materials by various microprobe techniques were reviewed by Treytl et al. (1972). Recently, radioisotope-excited fluorescence of x-ray-tubeexcited fluorescence for elemental analyses in such areas as pollution and production quality control has been reported (Duggan et al., 1971). X-ray fluorescence is claimed to have exceptional sensitivity and reproducibility. The area to be subjected to elemental analysis is selected either by using a conventional light microscope or by attaching the probe to an SEM. The analysis of a microvolume of specimen can be either qualitative or quantitative. For quantitative analysis, the intensity of seIected lines emitted from the specimen is compared to that emitted from a reference standard. The application of the electron probe x-ray microanalyzer in metallurgy and mineralogy has been extensive. Its usefulness in the biological field is increasing rapidly (Tousimis, 1969). Early biological studies indicated that the electron probe microanalyzer can be used to analyze small inclusions in tissues (precipitates in membranes from organs in pathological conditions), normal calcification during bone growth, hardening of blood vessels, degeneration of tumors, and localization of several elements (iron, zinc) in organometallic complexes in tissues (Tousimis, 1966). A is to An electron probe of about 1-pm diameter and of currents of placed in position either electrically (magnetic deflection scanning) or mechanically (specimen x-y movement under a stationary probe) on the desired part of sample that is to be analyzed. To position the specimen, a light microscope reflex or transmission objective, with a resolution of better than 1 pm, is used (Tousimis, 1963). The intensities of the characteristic lines are measured with appropriate detectors. The signals from the detectors are amplified, and recorded (strip chart) or counted. They can also modulate the intensity of an oscilloscope beam which is synchronized with the probe. It is thus possible to record the characteristic x-rays given off from small volumes of specimen or changes in intensity during the scanning of a selected line. When a single spot is analyzed, generally only x-ray signals are recorded by detectors. The detectors record the number of x-ray photons per unit of time, and the information can be used for quantitative analyses. The x-ray line scan and x-ray image from the CRT are only semiquantitative. If a quantitative analysis of a large area is desired, a static spot can be used with the specimen driven under the beam with specimen stage motors combined with data readout on a strip recorder (Rasmussen and Knezek, 1971). Hayward and Parry (1973) described the use of the three techniques in
226
Y.POMERANZ
electron probe microanalysis studies of silica distribution in barley. Electron microscopy and x-ray microanalysis in forensic science were described by Brown and Johnson (1973). Detection of diffusible ions in insect osmoregulatory systems by electron probe x-ray microanalysis using SEM and a cryscopic technique was the subject of an investigation conducted by Marshall and Wright (1973). Van Hofsten (1973) detected magnesium, calcium, and sulfur in the globoid region of the protein bodies in Crambe abyssinica by x-ray microanalysis. Linear scan profiles and multilinear scan images were made by a scanning electron microprobe. According to Lehrer and Berkley (1972), without appropriate standards only relative measurements can be made in biological materials with electron microprobes. The authors described a procedure for the preparation and application of gelatin standards in the quantitative elemental analysis of microscopic portions of tissue sections in the electron microprobe x-ray spectrometer. Gelatin suspensions containing known concentrations of the elements to be measured were quick-frozen, sectioned at the same thickness as the examined tissue, and freeze-dried. Standard sections were mounted with each tissue section and analyzed simultaneously. Concentrations of the elements in portions of the tissue as small as liter were then determined directly from a regression curve or equation derived from the standards. Several points should be considered in electron probe x-ray microanalysis (Tousimis, 1963). Penetration of the electrons in the specimen is especially important in thick specimens. Heterogeneous elemental distribution at the micrometer and at the submicrometer level may introduce errors in determining concentration of elements with atomic numbers above 26. The effects are reduced in elements with low atomic numbers and thin sections. Thin coating (about 200 A) with aluminum or carbon renders the specimen electrically conductive and eliminates excessive temperature rise. When the microprobe is used in combination with an SEM, the specimen is coated with gold-palladium (60%/40%). Contamination of the specimen surface at the point of electron bombardment by oil from vacuum pumps, gaskets, and greases may affect results. Echlin et al. (1973) reviewed preparative techniques for quantitative analyses of various elements in cells and tissues by a SEM fitted with diffracting and (or) energy-dispersive detectors and a transmitted electron detector for sections 0.1 to 0.2 pm thick. The review indicated a need to refine the experimental procedure for preparing and handling specimens, to reduce ice contamination, and to improve the instrumentation associated with signal acquisition and specimen localization. The high water content of plant and animal tissues must be removed without disrupting the microstructures of the tissues and without change in elemental distribution (Andersen, 1967). The dry tissues have a low density and are highly
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heterogeneous-both of these characteristics often complicate analyses. Biological materials, generally, consist primarily of carbon, nitrogen, and oxygen, which until recently were not detectable by the electron probe technique. Recent developments (described below) indicate, however, that despite those limitations there may be a bright future for the electron probe in biology. According to Ingram e l al. (1972), electron probe analysis of soft biological tissues requires a preparation technique that (1) presents a flat surface of the specimen to an electron beam in a vacuum, (2) can withstand the rigors of the vacuum, (3) can withstand the damage of the electron beam, and (4) retains the original distribution of elements under study. The authors prepared soft tissue, without use of aqueous solutions, for electron probe analysis. The tissue was quick-frozen at about -196"C, dried in a vacuum at -60" to -85"C, fixed with osmium tetroxide vapor, and embedded in low C1 epoxy. For thick sections, a low accelerating voltage of about 10 kV and a sample current of 50 mA were found
FIG. 6. Cross section through aleurone layer of barley (1800X). Arrows point to microareas in aleurone grain, aleurone cell wall, and starch granule (from the starchy endosperm) which were selected for x-ray microanalysis.
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Y.POMERANZ
to be reasonable working compromises between x-ray production, on the one hand, and spatial resolution and specimen damage, on the other. Some of the numerous areas of food investigation in which x-ray SEM may be used include distribution of elements in plant and animal tissues, homogeneity of milled or mixed foods, the presence of extraneous matter or enrichment trace elements, the purity or contamination of foods, corrosion of containers, and effects of heat treatment on redistribution and composition. Rasmussen (1968) used electron microprobe x-ray analysis to follow the mode of entry and distribution of aluminum in corn; Waisel et al. (1970) determined by x-ray microanalysis the site of aluminum in cortex cells of beans and barley. Spectroscopic microanalysis of the element-characteristic x-rays produced by a scanning electron microprobe was employed to detect calcium and carbon in both intact and thin-sectioned spores of Bacillus cereus and B. megaterium (Scherrer and Gerhardt, 1972). Calcium, similarly to carbon, was distributed throughout the
FIG. 7. X-ray map of the area in Fig. 6. The higher the concentration of light spots is, the higher the concentration of phosphorus. The area was scanned for 500 seconds.
SEM IN FOOD SCIENCE AND TECHNOLOGY
LARKER
o 0.751
CONQUEST
-
229
TOTAL
I
FIG. 8. Recording of x-ray spectra in selected microsections of aleurone grains, aleurone cell wali, and starchy endosperm of barley (see Fig. 6 ) ;and in the whole aleurone cell of two barley cultivars (Conquest and Larker). Elements present in relatively high concentrations are identified. The areas under the peaks represent counts X lo3 per 200 seconds. (The graphs of individual runs are shifted vertically for easier identification.)
spore but was concentrated mainly in a central region corresponding to the spore protoplast. Greaves (1974) used SEM in conjunction with energy dispersion analysis of x-rays to measure cellular elemental composition of bacterial cells. Using SEM, Kaufman et al. (1972) determined the kinds and distribution of epidermal cell types in Avena sativa inflorescence bracts (glume, lemma, and palea). Electron microprobe analysis of silica deposition in epidermal cells indicated that silica cells constituted one of the important deposition sites. Probe ratio data indicated that the deposited silica was 74% pure. Significant amounts of silica were deposited in the trichromes, small amounts in the walls of long epidermal cells, and none in the stomata. Figure 6 shows an SEM photomicrograph of a cross section through barley; in Fig. 7 location of phosphorus in the specimen (Fig. 6 ) is indicated; and Fig. 8 shows an example of spectra from the analysis of selected sections in the barley (Pomeranz, 1973).
B. RECENT AND POTENTIAL DEVELOPMENTS Since x-ray production is a bulk effect, resolution is limited to about 500 nm, which is within the range of optical light microscopy. Using a finer probe or thinner specimens has the disadvantage of reducing the rate of x-ray production. Echlin (1971) suggested that detection and localization of artifically produced
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specific heavy metal stains may be a useful companion to x-ray microanalysis. Such an extension of the conventional microanalytical assay could be based on the specificity of silver salts for halides, the stoichimetric binding of uranium to DNA, the affinity of lead salts for nucleoproteins containing RNA, and the availability of ferritin-coupled antibodies. X-ray microanalysis in the reflection mode is still limited in resolution; the analysis of sections in the transmission mode can produce much higher resolution (McAlear, 1972). The use of nondispersive detectors has improved the effective resolution. It is possible to display several images showing different elements in contrasting color, superimposed on the relatively high-resolution secondary electron image. The recently developed technique of auger electron microscopy has been used widely in the study of surface chemistry of solids; the technique can be applied in elemental analyses. Auger electron analysis differs from x-ray microanalysis in two respects: (1) X-ray analysis samples the chemical composition of a volume of the order of 1 pm3 , whereas the auger electron analysis samples the chemical composition of a surface layer of only a few angstroms in depth. (2) Auger spectroscopy is much more sensitive to elements having low atomic number (2 below 11) (MacDonald et aZ., 1970). For beam diameters of the order of tenths of micrometers and for auger electrons with energies of less than 1 keV, the spatial resolution is determined by the diameter of the electron beam; for x-ray analysis, the resolution is governed by a volume of the order of the range of the primary electrons. Auger electron analysis must be performed in a vacuum of at least 1 X lo-' torr. Resolution of auger electron analysis can be increased by using a smaller beam diameter, but this requires appreciable probe current. High-spatial-resolution auger electron analysis requires long recording time ; the upper limit on recording time depends on the cleanliness and pressure of the vacuum environment. When the electron microprobe is used for examining thin sections, the backscattered electron image may be an inadequate visual guide of the specimen features. To permit exploring the variation in element concentration within an individud cell, it may be necessary for the minimum probe size to be much smaller than 1 pm. To meet those requirements, Coslett and Hall (1971) constructed a combined electron microscope-microandyzer. The instrument is based on an electron microscope to which two fully focusing x-ray spectrometers have been added. A minilens allows one to obtain a probe down to 0.2 pm. As an electron microscope the instrument has a resolution of 10 A and provides two ranges of magnification: 150X to 1300X, and lOOOX to 160,OOOX. Sutfm et al. (1971) converted an SEM to an electron microprobe with high spatial resolution by adding a transmitted electron detector and a solid-state x-ray detector. This permitted microanalysis of individual mitochondria1 granules with diameters of less than 1000 A. The SEM was combined with a
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23 1
vacuum system and gas jet to reduce contamination to the point where qualitative and semiquantitative analyses of structures with diameters less than 1000 A could be performed (Sutfm, 1972). The ion microprobe mass analyzer (Andersen and Hinthorne, 1972) performs elemental analyses by bombarding microvolumes of surface with a high-energy beam of ions which causes the atoms to be sputtered away. Some of the sputtered atoms are charged, collected, and analyzed according to their mass-tocharge ratio. The instrument complements the electron probe x-ray analyzer. It provides information on concentration and distribution of elements on the surface, and isotope ratios can be measured. Theoretically, all elements can be analyzed (with the possible exception of inert gases), but detection and assay vary with the matrix, the element, and the polarity of the sputtered ion. The technique expands spatial microanalysis to trace elements. It is possible to determine analyses of deeper layers by successive eroding away of atomic layers by the bombarding ion beam. The electron probe microanalyzer is a promising powerful research tool. Combination of the electron probe microanalyzer technique with various classical techniques of histochemistry and chemical analysis promises to yield much new information in the area of biochemistry.
IV. METHODS OF SPECIMEN PREPARATION AND ANCl LLARY TECHNIQUES Preparation of specimens for SEM is relatively straightforward for most applications. The best specimen preparation is the least preparation (McAlear, 1972). However, for some specimens sample preparation is required, and the field is gaining sophistication as the types of specimens studied, applications of the instrument, and information sought continue to increase. Thus, the 1972 annual scanning electron microscopy symposium held in Chicago, Illinois, was followed by a one-day workshop sponsored by the National Institutes of Health and devoted to preparation of biological specimens for SEM. Preparation of biological materials for the SEM was discussed in excellent and comprehensive reviews by Boyde and Wood (1969) and by Hollenberg and Erickson (1973). Idle (1971) discussed preparation of plant material for the SEM. An excellent outline, detailed instructions, and a comprehensive bibliography on preparation of biological specimens for SEM were included in Jeol News (Anon., 1972~).The outline is reproduced in Fig. 9. Figure 9 does not show the use of ethanol, a frequently employed solvent, as a dehydrating agent. In addition, the figure does not include the critical point method, an increasingly popular drying procedure. The figure shows the double coating method utilizing carbon and gold and points to the possibility of using low voltages for nonconductive specimens.
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FIG. 9. Preparation of biological specimens for scanning electron microscopy. Courtesy Jeol Ltd.
Specimen holder
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SEM IN FOOD SCIENCE AND TECHNOLOGY
23 3
A. GENERAL PREPARATION The basic steps in tissue preparation and examination are: cleaning and fixation of a selected sample, dehydration, mounting on a stub, coating, and examination. Hard materials require little preparation other than cleaning, to remove extraneous material, and cutting or fracturing. Soft biological materials are washed and, frequently, fmed and embedded so that they can be dehydrated with minimum damage to the tissue. To accent various facets of the natural surface morphology, various methods can be employed. They include selective (chemical or enzymatic) digestion and extraction of interstitial material (removal of muscle collagen by collagenase, removal of mucopolysaccharides from dermal tissue with hyaluronidase, extraction of extraneous mineral components by chelating agents, or removal of extracellular organic materials with sodium hypochlorite). Some of those treatments can be used to modify the morphological appearance of smooth specimens. Typically, a biological specimen is mounted on a specimen stub with a 50% mixture of nitrocellulose cement and silver conducting paint (Black, 1970). The specimen is then covered, under vacuum, with gold or a combination of gold and palladium. The metal coating imparts to the sample excellent secondary emission characteristics which are important in studying surface topography. The specimen is rotated and tilted during coating. Boult and Brabazon (1968) described a rotating device for use in metallizing nonconducting SEM specimens. For most applications, a coating of 300 A is satisfactory; this does not cover up or mask surface detail. B. FREEZE-DRYING AND FREEZE-ETCHING Most biological specimens contain high concentrations of water and must be dried for examination in the SEM. The drying must be conducted under conditions that minimize alteration of structure and composition. It is in this area that most of the effort has been concentrated. One of the most common methods used for preparation of specimens for SEM is freeze-drying. Although some excellent preparations can be produced, the technique in general is rather disappointing for display of intracellular details because the gel matrix seems to disintegrate and leave deep cavities around the membrane structures (McAlear, 1972). Although it is possible to retain chemical activities and structural entities in frozen specimens, examination of such specimens at the electron optical level of resolution presents many technical difficulties. Several investigators have developed preparative procedures involving low-temperature microtomy combined with low-temperature electron microscopy of hydrated as well as dehydrated
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specimens. A temperature-controlled stage module has been developed for the SEM by Echlin et al. (1970). Labile tissue can be examined at temperatures ranging from t25"C to -180°C. Some plant materials (leaves, root hairs) were quench-frozen by plunging specimen stubs with the material mounted on the surface into liquid nitrogen at -196°C. The cellulose of the plant cells apparently was sufficiently robust to prevent or retard extensive damage by the small ice crystals. Micro-algae, protozoa, and animal tissues were drained or damp-dried, and either placed directly on aluminum specimen stubs or on platinum disks which were attached to cooled stubs. The stubs were plunged into liquid Freon 22 which was maintained at its melting point of -140°C by liquid nitrogen. The procedure yielded samples free of ice-crystal damage (Echlin et al., 1970). Specimens preserved by any of these methods could be examined at low temperatures provided condensation of water and removal of ice crystals were carefully controlled. The cold stage has been useful in retaining the threedimensional shape of algae and protozoa and in examining tissue culture cells. Electron microscopic observations of biological specimens in their native state by cryogenic techniques were described by Tokio et al. (1972) and Tokunaga and Tokunaga (1973). Frozen specimens of biological materials prepared without dehydration or metal coating were directly observed under a SEM equipped with specially designed cold devices (Tokio et al., 1973). The specimens included pistils, pollen, and petals of chrysanthemum, fruit fly larvae, and sections of hamster tongue. The internal structure of the specimens was also manifested on the newly formed surface which has been fractured in the frozen state with a cold knife. Bole and Parsons (1973) described a technique in which the SEM has been used to study the internal cellular organization of the plant organs and the interaction of the plant with the environment. Transverse surfaces were revealed by fracturing quick-frozen specimens and freeze-drying the fragments. The greater depth of focus of the SEM compared with the optical microscope enabled the production of clear, realistic micrographs with a three-dimensional appearance. Another method by which biological materials can be viewed in the frozen state is that of freeze-etching (Park, 1972). The procedure, originally proposed by Steere (1957), is based on the premise that sublimation of ice from a frozen specimen in vacua produces a relief of the sample. However, the method involves indirect visualization. It requires the use of replicas; the frozen cellular details are preserved by means of evaporated metallic films. On the other hand, the replication reveals three-dimensional details that cannot be seen in samples prepared by ordinary thin-section techniques. In addition, freezeetching circumvents chemical furation, dehydration, and embedding (Koehler, 1968). In the basic process, the sample is quick-frozen on a metal specimen holder and placed
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under vacuum. The sample is fractured by manipulation from outside the vacuum through a sealed actuator. After fracturing, the frozen specimen is held at about -100°C for a short time, while 100 A or more of the surface ice sublimes; this is followed by metal shadowing of the specimen and replication with carbon. The sequence of the freeze-etching process is shown in Fig. 10. According to McAlear (1972) freeze-etching may prove to be the most generally convenient means of treating tissues. Freeze-etched samples show excellent morphological correspondence to cellular structures characterized by other wellestablished methods. Unfortunately, it is not possible to combine freeze-etching with recently developed techniques of ultrastructural histo- and cytochemistry. However, as Koehler (1968) pointed out, freeze-etching offers some unique opportunities in several areas. For instance, one can observe in freeze-etched samples cellular and organelle membrane faces. The technique has revealed new classes of particulate substances associated with membranes. The identity of those particulates has yet to be ascertained by biochemical fractionation and analytical procedures. The critical point method and many of its modifications are becoming increasingly popular as means of avoiding artifacts. The method was developed by Anderson in 1952. When a liquid in equilibrium with its own vapor is heated in a confined space, a critical temperature is reached at which all liquid is converted to gas; at higher temperatures no additional compression will force the gas to condense back to the liquid phase. As the pressure increases, the density of the vapor phase increases until at the critical point (critical temperature and pressure) it is equal to that of the liquid. Surface tension approaches zero, and there is no boundary between the liquid and gas. Specimens immersed in an appropriate liquid and carried through the critical point are surrounded only by gas, and there is no distortion by surface tension. As the gas is bled off above the critical ( b)
(a) specimen
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specimen block/
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FIG. 10. Sequence of freezeetch system. Courtesy Denton Vacuum, Inc.
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temperature, the specimen remains dry and unchanged. Anderson (1952) first used carbon dioxide; the method has been employed since with many other liquefied gases. It is usually necessary to replace the water in the specimen with a fluid that is miscible with the liquefied gas. When carbon dioxide is used, the water is first replaced with absolute ethanol and the latter with amyl acetate. (See also Horridge and Tamm, 1969.) Smith and Finke (1972) described a simple apparatus for critical point drying. Worthen and Wickham (1972) compared conventional freeze-drying with the critical point method; the apparatus for the critical point method was less expensive, and the technique was faster and more consistent. The authors concluded that the critical point method yielded the most accurate preservation of tissue for SEM. Briarty (1971) reported that, to observe details of cell walls of living plant cells by SEM, digestion with a protease followed by freeze-drying gave the best results. Turner and Green (1973) found that nematodes and mildew-infected barley leaves when examined in the scanning electron microscope after critical point drying (CPD) from sulfur dioxide (critical temperature 157OC) showed no obvious physical damage, but the specimens had a surface deposit which were probably heat-damaged natural waxes. The nematode Caenorhabditis elegans and clover roots (Trifolium subterraneum) showed no physical or heat damage after CPD from monochlorodifluoromethane (Freon 22, critical temperature 96°C). The hyphae and conidia of unfvred mildew on barley were damaged after CPD from Freon 22, probably owing to the Freon's extracting lipids from the cell walls. Freon 22 is preferred for most specimens, as it is cheap, easy to obtain, and not very toxic. According to Humphreys et al. (1973), the main methods for stabilizing biological soft tissue for observation in the SEM include the critical point method, freeze-drying, freeze-fracturing followed by freeze-drying, observation of specimens frozen and fractured in the SEM specimen stage, applied glycerol substitution, and fractography of plastic-embedded cells. Viewing cells embedded and fractured in plastic permits studies of both extracellular and intracellular structures. Fractography of plastic-embedded cells also permits a direct comparison of structures seen by the SEM with structures viewed by the TEM in sections cut from the same embedment in the same block. Boyde and Vesely (1972) grew rat fibroblasts on glass cover slips, plastic dishes, or aluminum stubs, and prepared them for SEM by air drying, critical point drying, and freeze-drying after fixation with formaldehyde and glutaraldehyde or Os04,in various buffer systems. They reported that ethanol or acetone dehydration was responsible for noticeable shrinkage during critical point drying and that additional small shrinkage (cell flattening) may take place at subsequent stages. Whereas critical point drying caused less displacement of cells from the
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substrate than did freeze-drying, both techniques caused cytoplasmic tearing. Freeze-drying preserved the three-dimensional image of cell shape but produced artifacts (ice splitting and crystal hole puncturing). The authors concluded that the choice of method depended on the specimen and on “weighting” the significance of artifacts in interpretation of results. As Hollenberg and Erickson (1973) pointed out, many ingenious methods of sample preparation have been developed recently. However, no universally acceptable method for all biological materials is available. C. MISCELLANEOUS ANCILLARY TECHNIQUES Operating the primary electron beam at high accelerating voltages permits a reduction in probe diameter and improves resolution. This is achieved, however, at the expense of deeper penetration, uneven specimen charging, and uneven contrast. Limiting the voltage to 10 kV results in a resolution in the 20- to 30-nm range but decreases artifacts. Examination of uncoated fresh material at an accelerating voltage of 1 to 2 kV gives a resolution of between 80 and 90 nm. There is less beam penetration and a considerable reduction in the radiation flux impinging on the specimen. The advantage of low-voltage operation is that specimens can be examined at medium resolution with little, if any, preparation, coating, or danger of artifacts. Even relatively labile materials can be studied provided they are examined within a few minutes after they are introduced into the microscope column (Echlin, 197 1). The use of a beam of inert gas ions to etch surfaces has been applied in recent years to biological specimens; by this technique it is possible to remove parts of the specimen inside the microscope column. Echlin et al. (1969) used a demountable cold cathode argon discharge unit to etch the surface of complex resistant biological specimens (pollen grains). This etching can be also combined with examination of uncoated specimens at low accelerating voltages. By etching at two angles it is possible to learn about the crystallinity and structure of a specimen; crystalline materials are etched differentially at different crystal planes. Finally, differences in rates of etching may reveal differences in tissue texture and composition. Hodges et al. (1972) examined mammalian and avian cells in the SEM either after prior radiofrequency sputter ion etching with different ions (hydrogen, helium, argon, or oxygen) or after argon ion bombardment in the SEM. While the pattern of erosion was similar in the different specimens, the etching pattern varied with the different gases. The authors could not relate the etching pattern to characteristic subsurface structures. They concluded that, contrary to earlier optimistic opinions, the ion-etching technique is of little value in examining subsurface structures, especially of soft biological tissues.
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Y.POMERANZ D. SPECIMEN MANIPULATION
Observation of specimens by SEM is facilitated by availability of a large specimen chamber which can accommodate bulky items. Most commercial instruments are equipped with devices for simple manipulation of specimens mounted on stubs. However, those general features may be insufficient for some specimens or for certain types of manipulations (dynamic experiments). Sikorski ef al. (1969) described several types of specimen holders for examination of fiber periphery, for sections of stretched and deformed fibers, for thin blades and frictional experiments, and for tilting of fibers that are to be viewed as stereopairs. The devices were subsequently modified (Sikorski et al., 1970) to improve precision and reproducibility of settings, and to increase maximum flexibility of the specimen and of the required ancillary controls. Fiber rotation to obtain the cylindrical projection of the outer surface required intermittent opening of the specimen holder (Sikorski et al., 1969). This is a tedious and time-consuming process which often poses difficulties in locating the examined area, in relation to the area observed previously. By the use of suitable d.c. micromotors mounted on the specimen stage of an SEM, Bywater and Buckley (1970) eliminated some of the difficulties in locating the area. The micromotors were found to be so versatile that linear displacement in all directions and rotations around the tilt and beam axes could be actuated adequately. In a micromanipulator designed by Pawley and Hayes (1972), independent motion of two etched tungsten needles in three directions is obtained by a system of piezoelectric crystals, mounted on the end of an 18-inch shaft. The piezoelectric head can be positioned near the specimen during use or retracted into the microscope airlock for tip replacement. The shaft can be controlled by servo-driven differential screws in three directions with an accuracy of 5 pm. Dynamic changes in the specimen were observed and recorded by using the television scanning accessory with a video tape recorder. Dobbs (1972) described a device which can be used to cause mechanical strains in materials inside the SEM and to study the deformations as they are formed at high magnification. The devise employs a triangular wedge to produce a uniform strain. As the wedge moves forward, two sliding crossheads are forced apart. The crossheads apply the strain to the specimen attached with screws to the sliding crossheads. The optimum specimen dimensions are about 1.5 X 0.5 cm. Positioning and straining of the specimen are controlled from outside the chamber by universal joints and micrometer screws. Unsworth and Hepworth (1971) described an accurately calibrated goniometer stage (including a new specimen carnage and specimen holder) for use with the SEM to facilitate the preparation of accurate stereomicrographs. A detailed calibration procedure showed that the apparatus was reproducible and gave
SEM IN FOOD SCIENCE AND TECHNOLOGY
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errors of less than 2% in stereomicrographs. Theoretical aspects of stereoscopy of cylindrical objects in the scanning electron microscope were discussed by Hepworth and Sikorski (1972).
V. APPLICATIONS IN MICROBIOLOGY Some of the earlier investigations on the use of scanning electron microscopy in studies of microorganisms were reviewed by Oatley et al. (1965) and by Hayes and Pease (1968). Hawker, Williams and Veltkamp, Heim and Perreau, and Alvin are the authors of chapters (in a book edited by Heywood, 1971) on scanning electron microscopy of fungi and its bearing on classification, and the value of scanning electron microscopy for the examination of actinomycetes, studies of basidiospores, and investigation of fossil epiphyllous fungi. According to Greenhalgh and Evans (1971), the potential of SEM in mycology lies in investigations of surface characteristics of spores, hyphae, etc; in studies on the formation and distribution of exogenous spores; and in ecological studies designed to follow the presence of hyphae and spores on the surface of various substrates and in the air. Applications of SEM in studies of microorganisms were reviewed by Bulla e l al. (1973) and by Nickerson et al. (1974). A. SAMPLE PREPARATION Echlin (1968) reviewed the various methods of preparing plant and microbial material for study by SEM. He emphasized that the major problem is to remove water from the specimen under conditions that minimize damage to tissue. He divided the specimens into three main classes: robust dry, robust wet, and labile specimens. Robust dry specimens (spores) can be sprinkled on the tacky surface of a stub coated with a glue (Durafix or glue obtained from dissolving the adhesive from tape with chloroform). The robust specimens can either be air-dried in a dust-free atmosphere or dried over silica gel or phosphorus pentoxide. Most robust wet specimens can be placed onto the specimen stub directly and the liquid allowed to dry. Specimens rich in surface polysaccharides attach quite firmly to the stub. Bacteria and algae which have a thick sheath and capsules adhere firmly, dry slowly, and are well preserved. The specimens are dried slowly in air, and residual moisture is removed by using an appropriate desiccant. Cultures or samples of smaller algae and microbial material can be prepared by adding 1 or 2 drops of a 1% solution of osmium tetroxide and removing the osmium after a few minutes by repeated centrifugation and washing. The sample can be dehydrated through a graded acetone or ethanol series. However, in some
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cases freeze-drying is the best procedure. The material is applied to the stub in the form of an aerosol, as a thin layer, or sprinkled on a glue surface. As soon as the specimen has been applied to the surface it should be freeze-dried rapidly. According to Greenhalgh and Evans (1971), coating of specimens and their examination under vacuum may cause the hyphae to wrinkle and collapse and lead to shrinkage of soft-structured tissues around dense cores of internal substances. Distortion of delicate structures can be reduced, although probably not eliminated, by freeze-drying. Generally, distortion is smaller in thick-walled spores than in hyphae. The authors emphasized that, for meaningful and reliable interpretation, SEM observations should be evaluated in relation to parallel studies on the same structures with the light microscope. Separated conidia, ascospores, etc., can be collected on a small circle of frlter paper which can be attached to the specimen stub and coated. The spores are retained between the filter paper fibers. A small food particle can be attached to the stub, or the sticky surface of the stub can be attached to the food surface and removed with adherent particles. Williams and Davies (1967) used SEM to study intact sporing structures of actinomycetes. They developed a method for transferring material from petri dish cultures to the specimen stubs. They inserted, at an angle of 45", sterile circular glass cover slips (% inch in diameter) into the solidified agar medium, and inoculated the organism along the line of insertion. After incubation they removed the cover slips and the adherent mycelium, glued the cover slips to the specimen stubs, coated the stubs, and viewed the mycelium in the SEM. The technique is best for fungal cultures which produce very minimal aerial mycelium. It is also possible to remove from a growing culture a thin slice of agar and microorganism, cut away most of the agar, and either dry it carefully or place it directly in the coating chamber before examination. B. MORPHOLOGY OF MICROORGANISMS Potentials of scanning electron microscopy in studying the morphology of microorganisms were described by Barlett (1967). Hilbert (1967) used SEM in the study of carboniferous microspores, and Williams and Davies (1967) for the examination of Actinomycetes. The following examples illustrate some of the recent studies on applications of SEM in microbiology. Ito et al. (1970) examined several strains of Aspergillus (A. niger, A. oryzae), Pencillium chrysogenum. and Candida utilis with the SEM. They suggested on the basis of characteristic features of those strains that SEM could be used in morphological classification of fungi. Ascospores of four species of Schwanniomyces were examined by the SEM (Kurtzman et al., 1972). Spores of S. alluvius, S. castelli, and S. occidentalis, which were essentially identical, had abundant, long proturberances and wide, thin equatorial rings. Two strains of S. persoonii differed from the other
SEM IN FOOD SCIENCE AND TECHNOLOGY
24 1
species and from each other. Also examined were spores of Schwanniomyces hominis (= Saccharomyces rosei) which lacked a ring and were covered with short irregularly shaped protuberances, a finding consistent with the morphology of spores from other strains of S. rosei. Fennell et al. (1974) suggested that differences in conidiophore wall ornamentations in the genus Aspergillus, as observed by SEM, could be useful in delineating major groupings in the genus. Passmore (1973) used SEM to study colony structures of four yeast species associated with cider making. A glutaraldehyde fixation technique was compared with direct observation of untreated specimens. Passmore and Haggett (1973) demonstrated by SEM an in virro association between cells of Saccharomyces cerevisiae and Leuconostoc sp. These organisms are often naturally associated in fruit products and fermenting beverages such as apple juice and cider. A chemoautotrophic, thermophilic, and acidophilic microorganism capable of oxidizing reduced sulfur and iron compounds and leaching concentrates of molybdenite and chalcopyrite at 60°C was characterized by SEM and TEM (Brierley and Murr, 1973). This constitutes the first observations of microorganisms on ore fines. The isolate consisted of clustered (colony) structures. Observations of individual isolate clusters were made in order to study the isolate-mineral interface. Apparently, the isolate in many cases attaches to the mineral interface. Nondispersive x-ray analysis of isolated organisms showed them to contain trace amounts of copper. Readily identifiable groups of microorganisms present on nonliving particulate organic matter (detritus) in the upper waters of Lake Tahoe are attached in specific ways and appear to be responsible for detrital aggregation (Pearl, 1973). These microflora are associated with active heterotrophic metabolism, but deeper waters possess little detrital microflora and little heterotrophic activity. Todd and Kerr (1972) compared scanning electron micrographs of a Pseudomonas species, Staphylococcus aureus, and Bacillus subtilis on two membrane fdtration systems. The comparison was conducted to establish conditions for identification and calibration of microbial biomass. Grey (1972) used SEM to characterize soil microorganisms. Brandenburger and Schwinn (1971) studied by SEM the different types of surface fine structures (caves, ridges, spines, etc.) in thirteen representative species of the teleuto- and uredospores of rust fungi. They observed several new fine structures and reinterpreted previous findings based on light microscopy. The tissue structure and the development of the apothecium of the discomycete Ciboria acerina were studied by TEM and SEM (Elliot and Corlett, 1972). The two microscopic methods complement each other. SEM was particularly useful in demonstrating details of each ascus, paraphysis, interwoven hyphae, and cell tissue; TEM brought out details of wall thickness, color, smoothness, and septation. Fass (1973) used SEM (after critical point drying) in
24 2
Y.POMERANZ
parallel with light and TEM to study L colonies produced by a stable L-phase variant of Staphylococcus aureus.
C. STORAGE OF CEREALS Cereal grains are important as food because of their excellent keeping qualities. Moisture content is the major factor in determining the storage behavior of grain, which is also influenced by temperature, oxygen supply, inherent characteristics, history and condition of the grain, length of storage, and biological factors (molds and insects). In dry grain, the respiratory rate is low. As the moisture content is raised, the respiration increases gradually until a certain critical moisture is reached above which marked acceleration in the rate occurs and heating tendencies appear. This sharp increase in respiration is due to the germination and growth of certain molds (predominantly various species o f Aspergillus and Penicillium) commonly found in soil and in previously used storage bins. Molds are invariably found on the grain and within the seed coats, even though the grain is harvested under ideal conditions.
FIG. 11. Fungal mycelium on the inside of oat palea (630X).
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24 3
FIG. 12. Plaque with microbial growth on the inside of oat palea (650X).
The most xerophytic molds begin to grow at relative humidities of about 7056, and moisture contents of individual kinds of grain which are in equilibrium with this humidity have been found to be very close to the critical moistures. Critical moisture for most cereals lies around 14%. Heating of grain, chiefly attributable to microorganisms, usually commences in localized areas of high moisture content. Although cereal grains normally are considered safe for storage if the moisture content does not exceed 14%, prolonged storage in bulk (where temperature differences occur) may result in spoilage. Temperature gradients across a bulk of grain, such as at the surfaces due to a change in the outside temperature, cause a transfer of moisture by convection from warmer to cooler areas. If the temperature gradients are sufficiently steep and prolonged, the humidity of the interseed air in the cooler portions may become sufficiently high to promote mold growth and heating. Recent investigations have indicated that the palea (one of the grain husk layers) is the site of microorganisms in oats (Pomeranz and Sachs, 1972b). Oat diseases are a major factor in oat production in North America. The losses from oat diseases are generally higher than those from other small grains because most
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FIG. 13. Conidium (C) and conidiophores (CP) in the crease of an oat groat (34X).
of the crop is grown in warm and humid regions which are normally favorable to disease development. In addition to regular parasitic fungi of the growing plant, several saprophytes occur on the seed coat. Fungal mycelium on the interior surface of the palea of a seemingly healthy oat seed is shown in Fig. 11;a plaque with microbial growth (probably a slime-producing bacterial colony) can be seen against the background of hair-covered ridges in the inside of the palea in Fig. 12. The area between the palea and the crease seems to create a favorable microenvironment for growth of microorganisms and to harbor them in the mature and dried grain. The presence of fungi in the crease area, beneath the palea, is also indicated in Fig. 13,which shows a conidium and conidiophores. Buckwheat (Fagopyrum esculentum Monch), although the fruit of a dicotyledonous plant, is classed in agriculture and commerce with the cereals. Structurally and chemically the endosperm of buckwheat resembles the endosperms of cereals in that it has a nonstarchy aleurone layer and a starchy endosperm, but the structures of other parts and the external appearance of common buckwheat and cereals are dissimilar. To the inside of the buckwheat hull are attached fiber layers whch may
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FIG. 14. Fibrous layers in the inner surface of a buckwheat hull (470X).
harbor fungal growth (Fig. 14) (Pomeranz and Sachs, 1972~).The longitudinally elongated wavy-walled cells of the outer spermoderm surface, with some attached sections of the pericarp fiber layer, are shown in Fig. 15. A higher magnification of a section of the outer epiderm of the spermoderm is shown in Fig. 16. This layer is found to harbor conidia and hyphae, which is not surprising considering its large and rugged surface, and its fine texture.
D. MICROBIAL GROWTH When a colony of the fungus 7’richoderma viride Persoon ex Fries is exposed to light, a sudden ramification of aerial hyphae commences from the periphery. This leads to the production of a typical ring of conidiophores. SEM revealed (Galun, 1971) a clear distinction between hyphal types and enabled early detection of hyphal initiation, Recognition of morphological changes at early stages of photoinduced differentiation in 7’richoderma leading to conidiation could be made by SEM but not by light microscopy. A population of aseptate pycnidiospores of the fungus Botryodiplodia theo-
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FIG. 15. Spermoderm surface of a buckwheat groat (285x1.
bromae can be induced to germinate or to form septa delimiting two cells; this developmental process depends on nutritional and environmental factors (Wergin et d.,1973). Transmission electron microscope investigations indicate that, during germination of the aseptate spore, a new inner wall layer is synthesized de nouo at the site of germ tube emergence. Formation of the septum also involves the de nouo synthesis of an inner wall layer which comprises the majority of the septum and completely surrounds the spore. The wall of the germ tube emerging from the septate spore is a direct extension of this inner layer deposited during the formation of the septum. Although the early stages of spore germination may involve localized enzymatic degradation of the internal layers of the spore wall, transmission and scanning electron micrographs of germinating spores show that the outer wall layers are physically fractured by the emerging germ tube. It was suggested that spore germination and septum formation are initially similar processes with regard to cell wall genesis but that some mechanism responsive to environmental and nutritional conditions determines the course of development. The morphological aspects of the process of sporulation in Aspergillus group, as observed by scanning electron microscopy, were presented by Tokunaga et al. (1973). In the earliest stage of sporulation, the immature sterigmata arise almost
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FIG. 16. Conidia and fungal hyphae on the spermoderm surface of buckwheat (1300X).
synchronously from the entire surface of the vesicle, as spherical protrusions which mature into sterigma of about 500 in number. The sterigmata of each species of Aspergillus have a characteristic appearance. The sterigmata of A . niger form complicatedly branched secondary sterigmata at their distal ends. When a mature sterigmata is removed from the vesicle, a pore with a diameter of about 0.5 pm is seen, corresponding to the proximal end of the sterigma. It was suggested that the pores may transmit nutrients and an energy source to the cell tips. The first conidiospores to appear develop from the apices of the sterigmata as spherical swellings, covered by the outer walls of the parent sterigmata. The subsequent conidiospores first extend in rod shape and later swell in globules. In the formation of the sterigma from the vesicle and of the conidiospores from the sterigma, their new wall layer was shown, by transmission electron microscopy of sections, to be formed within the parent cell wall. It was surmised that the manner of cell wall formation in the forming sterigma and conidiospores of Aspergilli may correspond to the mechanism observed in the reproductive phase of some aerial hyphae. Tokunaga and Tokunaga (1973) described the use of cryoscanning SEM in studies of conidiospore formation in Aspergillus niger.
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S a l e (1973) examined the life cycle of Neurospora crassa by SEM. Buhse et al. (1973) synchronized cell division in the protozoan Tetrahymena pyriformis W by the application of seven 30-minute heat shocks, each interspersed 30 minutes at optimal temperature. Examination under the SEM pointed to three stages. In the “anarchic field stage” at the end of the heat treatment, the kinetosomes were in various stages of ciliation ranging from primitive cilia in the posterior margin of the field to the almost complete cilia in the anterior border of the field. In the second stage, ciliary growth within each of the membranelles began with two kinetosomal rows proximal to the cytosome, while the most distant row ciliated at a later time. By the beginning of the third stage (cytokinesis), oral regions of both the proter and the opisthe were in the same stage of development and continued to develop in a synchronized manner until the cell divided. Candida albicans can grow either as a budding yeast or as a pseudomycelium form; it may produce characteristic chlamydospores. In the presence of certain sera, C. albicans produces so-called germ tubes. Whitaker and Drucker (1970) showed by SEM that growth of C. albicans was mainly at the base rather than throughout the colony. Because of budding of the yeast, the size of the organisms was variable; the surfaces of the colonies were covered with a film. Barnes et al. (1971) presented a SEM study of selected morphological stages of C. albicans. Stages represented were budding yeast cells, mycelial-like forms, clamydospores, germ tube formation, and an unusual rough cell type. Rousseau et al. (1 972) examined single spores of Saccharomyces cerevisiae, during germination and outgrowth, by SEM and phase-contrast microscopy. The microscopic examinations were made in combination with determinations of changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. Development of the vegetative cell from a spore followed a definite sequence of physical and chemical modifications. The developmental stages were: rapid loss in cellular absorbance, followed by an abrupt gain in absorbance; reduction in cell weight and subsequent progressive increase; modification of the spore surface and concomitant decrease in refractility; elongation of the cell and augmentation of surface irregularities; rapid decrease in trehalose content of the cell and formation of KOH-soluble carbohydrates; and bud formation. Belin (1972) examined vegetative cells of Succhuromyces uvarum in the expoential growth phase. SEM studies confirmed the existence of two types of scars-birth scars and bud scars. Birth scars had larger diameters than bud scars; both remained visible on old cells. The distribution of the buds on the mother cell was selective and was related to cell polarity. Springer and Roth (1972) made a topographic study by SEM of the bacterial colonies of Diplococcus pneumoniae and Streptococcus pyogenes. The colonies of D. pneumoniae were found to have gradually sloping sides and an area of autolysis at the top of the colony. The area of autolysis had a convoluted, stippled surface, free of intact cells or cell remnants. Colonies of S. pyogenes
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were rounded, dome-shaped colonies with steep sides. An amorphous supporting matrix was observed under the top layer of cells in the colony of S. pyogenes. Colonization of soybean buds by bacteria was studied by Leben (1969). Bulla et al. (1969) studied use of SEM and phase-contrast microscopy in investigations of bacterial spores. In a subsequent study, they followed germination and outgrowth of spores from Bacillus thuringiensis and Bacillus alvei (St. Julian et al., 1971). The three-dimensional images produced by SEM showed distinctive changes in spore surface and overall anatomy during the developmental process. Scanning electron micrographs of ascospores of Neurospora crassa reveal two of the structures that develop during germination and outgrowth: (1) a germination pore and (2) the probably site of initiation of hyphal cell wall synthesis (Sullivan et al., 1972). George et al. (1972) used the SEM to follow spore germination in Dictyostelium discoideum. Bibel and Lawson (1972a,b) studied by SEM protoplasts and L-phase variants derived from Streptococcus faecium strain F 24. In the first investigation, effects on morphology of several osmotic stabilizers, the duration and kind of fixation, the type of specimen mounting, and the method of drying were examined. Optimized conditions for biological studies of the L-phase were determined. Whereas air drying of broth-grown variants was satisfactory, agar-grown colonies required the critical point method of drying. In the second investigation, the growth of the streptococci in broth and upon membrane filters was followed. Osmotic sensitivity, shape, size, and surface features of L-phase bodies, ratios of large bodies to granules, and numbers of units in clusters varied with the phase of growth in the broth. During the lag phase of growth, the L-phase elements seemed to have a weakened membrane. This was suggested by their increased susceptibility to osmotic lysis and by their flattened shape. Irregular protrusions, which may be associated with formation of elementary bodies or degeneration of membranes, were found on large bodies in stationary phase. Replication in broth appeared to occur by single budding of large bodies or binary fission of granules. SEM of streptococci grown on membrane filters pressed upon uniform agar medium differentiated well among various strains and groups of the microorganisms. Ewe11 et al. (1972) used SEM to correlate toxin production in Clostridiurn botulinum with morphology under varying conditions of cultivation. At 48 hours, cells grown in a basal medium began to sporulate. Cells grown in a basal medium with added glucose showed much autolysis and little sporulation. In a medium with EDTA, cellular sporulation was inhibited and many elongated rod forms were present. E. EFFECTS OF ANTIBIOTICS Several investigators used SEM to follow changes in bacterial morphology and structure induced by antibiotics (Greenwood and O’Grady, 1969, 1972; and
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Klainer and Perkins, 1970). Perkins and Klainer (1970) studied the effects of carbenicillin on the surface morphology of Proteus vulgaris and Pseudomonas aeruginosa. Exposure of P. vulgaris to carbenicillin produced a series of structural changes related to concentration of the antimicrobial agent. The equivalent of 1.O minimal inhibitory concentration (MIC) caused enlargement, elongation, and failure of cell division. Higher concentrations (10 and 100 MIC) caused formation of spherical protrusions at midcell and of isolated intact and collapsed spherical forms; still higher concentrations (1000 MIC) produced large spherical forms consistent with spheroplasts. But even 0.1 MIC caused minor, though consistent, irregularities in shape. In P. aemginosa, 10 MIC caused multiple saccular protrusions along the shaft in a susceptible strain of the bacilli. In a more resistant clinical isolate, large, single, spherical defects developed at the site of transverse septum formation. The authors concluded that the effect of carbenicillin on gram-negative bacilli may occur at multiple points along the shaft of the organism in addition to the transverse septum site. Nishino and Nakazawa (1972) found that exposure of Escherichia coli to minimum inhibitory concentrations of cephalexin resulted in the formulation of marked filamentous cells and spherical cells having multiple small saccular outpouchings, presumably spheroplasts. Prolonged exposure led to additional enlargement of cells and rupture. The morphology of cells of the H37Ra strain of Mycobacterium tuberculosis exposed to 0.5 pg of isonicotinic acid hydrazide (isoniazid) per milliliter was examined by scanning electron microscopy (Takayama et al., 1973). Cells that were exposed to isoniazid for 3 hours showed no detectable change, whereas cells exposed to the drug for 24 hours exhibited diverse morphological features. From examination of these SEM pictures, the authors reconstructed the probable sequence of morphological changes to be as follows: (1) the wrinkling of the cell surface was ascribed as the earliest observable change; (2) the cell surface then became very rough and ragged; (3) eventually the cytoplasmic material was extruded from the cell; (4) this event produced a collapsed cell; (5) the cells began to fragment; (6) the fragmented cells then coalesced to form an amorphous mass of cell debris.
VI. APPLICATIONS IN PLANT INVESTIGATIONS The extensive use of the SEM in plant investigations can be deduced from the fact that in the first half of 1972 two books were devoted to description of plants as seen under the scanning electron microscope. Troughton and Donaldson (1972) used pictures taken on the SEM to review several aspects of plant anatomy and physiology which are important to plant growth. They described plants as integrated organisms in which one structure is related to another, and structure is related to function. Meylan and Butterfield
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(1972) used SEM pictures to explain the three-dimensional structure of wood. Use of SEM in plant taxonomy has been reviewed by Heywood (1969). More recent taxonomic studies using the scanning electron microscope were reported by Baum (1971) for oats, by Whiffin and Tomb (1972) for the neotropical capsular-fruited melastomataceae, and by Chuang and Heckard (1972) for cordylanthus. Scurfield e f al. (1970) described the application of SEM in classification of hardwoods based on the architecture of the tracheary elements. However, Scurfield and Silva (1969) emphasized the fact that the greatest biological value of the SEM lies not so much in taxonomic surveys of the surface architecture of plant parts and tissues but rather in the examination and interpretation of the effects of applied physical and chemical treatments upon this architecture. They conducted two such studies. One concerned the reaction of wood tracheids formed on the undersides of gymnosperm stems grown out of the vertical. The second study Concerned the effects of chemical treatments of vessels in the wood of eucalyptus. The treatments included delignifying and pulping agents such as acetic acid/hydrogen peroxide, oxidants such as potassium permanganate, and mineral acids. Use of the SEM in studies of the structure of wood fibers was reviewed by Scurfield (1 973). SEM was utilized as an aid in the study of wood decay (Findlay and Levy, 1969), of weathered wood (Borgin, 1971), of weathered wood surfaces damaged by mold fungi (Kuhne et al., 1970), and of the mechanism of fracture and cohesive failure of the structure of wood (Borgin, 1971). Pollen in many species has been studied by electron microscopy and SEM. Some of the earlier work on pollen was reviewed by Echlin (1968). More recent work was reported by Walker (1971) and by Christensen et al. (1972). Striking pictures of the surface of diatom, algae, plant roots, and leaves were reported (Dart, 1971). Muir and Grant (1971) studied spores of two ferns by SEM and other techniques; his review contains many excellent photographs. Mia and Allison (1972) examined with the SEM the substructures of the surfaces of the capsules (sporophyte) and the tetrad spores (gametophyte) of Polyfrichum. The capsule revealed a variety of surface configurations ranging from elevated domes to undulating ridges. The spore wall displayed many wartlike structures or projections which were strikingly uniform in size and shape. Rogers (1971) used SEM to obtain a view of the separation surfaces exposed when the stem was pulled from the fruit of Valencia oranges. Separations were made at 0, 24, and 48 hours after ethylene treatment of the fruit. Ethylene treatment promotes abscission in citrus, a separation layer is formed, and the tensile strength of the junction falls considerably. The most noticeable differences were the lessened amount of tearing in the proximal vascular bundles at 48 hours and decreased rupture of distal parenchyma cells first observed at 24 hours. Blanchard (1972) used SEM to study ultrastructure of ascocarp development in Sporomia australis. SEM has had an interesting application in the evaluation of cotton textiles
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(Anon., 1970). It has been used to compare surface appearances of chemically modified and untreated, and abraded and unabraded fabrics to get a better understanding of wear abrasion in durable-press and easy-care fabrics. Backer (1972) described the use of SEM to characterize classical and modern fabric textures and to follow changes that take place when yarns are texturized to increase bulk, fuzziness, and stretchability. Finally, SEM has been used to follow penetration of stomata by liquids (Schonherr and Bukovac, 1972) and the nature of the waxes and cuticles on plant surfaces. The latter affords a mechanical protection to many leafy foods and seeds. Determining the structure of the protective tissue is, therefore, of significance in improving shelf life and consumer acceptance of some foods. As the surface waxes easily soften and melt during scanning, it was necessary to develop techniques to minimize beam damage (Parsons et al., 1972).
VII. MISCELLANEOUS BIOLOGICAL APPLICATIONS Applications of the SEM in biology and medicine have increased rapidly over the last few years. The extent of this increase can probably be judged best from the fact that, in addition to several books published in the last few years, an atlas of scanning electron microscopy in medicine was also published (Fijita et al., 1971). The atlas starts with an introductory chapter which discusses briefly the theoretical basis of SEM and methods of specimen preparation. The main body of the volume includes chapters on visceral organs, tissues of the joint, skin and sensory organs, free cells, cell organelles, gall and urinary stones, dental tissues, parasites, and fungi and bacteria. Most chapters include brief explanatory notes on specimen preparation; in some, the SEM photomicrographs are accompanied by explanatory drawings. Hayes and Pease (1968) reviewed miscellaneous biological applications of SEM under the following headings:
Cells. General configuration and fine detail of the surface structure of human cells, cytological studies of normal and pathological cells, surface properties of malignant cells, and automatic cell identification in hematological preparations. Whole mount tissues. The compound eye of the insect, human lung tissue, the endothelial lining of the human coronary artery, and leaf surfaces. Sectional tissue. A bridge between the light and transmission electron microscope. Particles. Protein crystals, virus paracrystals, serum lipoprotein macromolecules, polystyrene latex molecules. Human chromosomes. Microorganisms. Bacteria, fungal hyphae and spores, morphology of microorganisms (particularly protozoans).
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Pollen grains. Dental enamel and skeletal material. Fibers. Synthetic and natural fibers, wood fibers, and collagen fibers and relation of their structure to mechanical strength and repairing tissue in healing wounds. Insects. Among the first biological specimens looked at with SEM; some can be examined while alive (Pease et al., 1966). SEM has proved to be a useful, auxiliary tool in taxonomic, genetic, and physiological studies in entomology. Living specimens. The flour beetle, fibolium confusum, at all stages of development (egg, larva, pupa, and adult) can survive in a vacuum of torr for 30 minutes; no sectioning is needed; the electron current is relatively low and limited to the outer cm of the specimen surface. Humphreys et al. (1970) investigated mites, using a 20-kV accelerating voltage and a vacuum of 5 X torr . No attempt will be made here to list the numerous publications dealing with the various facets of SEM in biology and medicine. Many were reviewed by Oatley et al., (1965), Hayes and Pease (1968), Pease and Hayes (1968), and Hinton (1969), and in several books and additional reviews listed in the Introduction. In the following paragraphs some additional and/or novel applications of SEM will be mentioned. Schull (1973) examined by SEM meiotic cells in Lilium longiforurn. SEM of the outer surface of sea urchin eggs sampled at intervals during the first 3 minutes after insemination revealed the detailed structure of the vitelline layer during its transformation into the fertilization membrane (Tegner and Epel, 1973). A sperm attachment-detachment sequence was described for the large number of sperm which transistorily bind every egg during fertilization. Miller (1973) conducted a comprehensive SEM study on the auditory system and on sensory hair cells in reptiles. Kozlowski et al. (1973) demonstrated by SEM the functional significance of the regional differences in surface structure of the third ventricle of sheep in relation to cerebrospinal fluid movement, ependymoabsorption, and ependymosecretion. Scanning electron microscopy has been employed to study the central axis and laminae of the olfactory rosette in adult sea trout (Salmo trutta truttu L . ) caught in the River Umealven when they were homing from sea (Bertmar, 1972). Both flat sides of the primary laminae are secondarily folded all over their surface. In one organ there are about 200 secondary laminae usually arranged in longitudinal, parallel ridges crossing the surface of the primary laminae. Initially they are covered with sensory epithelium, but as the folds grow they become covered with an increasing area of indifferent ciliar epithelium with bushes of cilia separated by microvilli cells and goblet cells. Parts of the central axis and primary laminae have a nonciliar indifferent epithelium. The sensory epithelium has irregularly arranged cilia. Like those of the indifferent epithelium, they have uniform thickness and granulated surface.
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Investigations by SEM demonstrate the existence of several morphologically different types of olfactory receptors in Carassius auratus (Breipohl et al., 1973a). The structural differences, however, do not allow a definite classification of sensory cells into functionally different elements. The olfactory organ has a central axis with lamellae emerging at both sides of it. On these lamellae, sensory areas with densely packed receptor cells and with groups of ciliated cells exist. The terminals of the receptor cells show a great polymorphism of their surface. The morphologically different receptor cells are not equally distributed over the olfactory organ but differ from each other in quantity and density. The morphological results are discussed in relation to olfactory theories and in relation to the question of whether there are topographical projections between the peripheral olfactory organ and the bulbus olfactorius. The appearance of the fine structure of the surface of endoepithelial glands in the regio olfactoria of Carassius auratus was described by Breipohl et al. (197313). The phases of accumulation of secretory droplets, their excretion, and their significance for the production of the terminal mucous film were demonstrated and discussed. Two different kinds of endoepithelial gland formation were described. It is possible that these glands are only modifications of a single cell type. The first modification is referred to as goblet cells; the second, as endoepithelial glands with balloonlike projections over the surface of the olfactory epithelium. On the surface of the goblet cells can be seen two classes of microvilli, which differ in structure and pattern of distribution. In addition, membrane protrusions, which are interpreted as bundles of fused cilia, are sometimes found. External and internal surfaces of the compound eye of the flesh fly, Sarcophaga bulfata,were examined with the SEM (Carlson and hrsen, 1972a). A low, patterned corneal nipple-ridge array and sparse setiform interfacetal hairs were observed on the corneal lens surface. Particular cleavage planes revealed outlines of the Semper cells, their nuclei, and distal terminations of photoreceptor cells. The latter, with their axonal processes, were visualized and described. These axons were noted traversing the external chiasma and entering the lamina ganglionairs where suggestions of synaptic contact were pointed out. The descriptions were correlated with those taken from literature of the TEM. The superposition eye of the sphingid moth, Manduca sexta, was explored by means of the SEM (Carlson and Larsen, 1972b). Specifically examined were the corneal nipple array, corneal lens, crystalline cones and tracts, and photoreceptor cells and their axons. Descriptions of the external ultrastructure of the componenets were correlated, where possible, with previously published accounts of internal ultrastructure as obtained from TEM studies. A key finding was the demonstration of the axial rotation of the eccentrically situated retinular cell, its externally noted prominence, and the arrangement of the other photoreceptor cells composing the retinula. Because of the interest in superposi-
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tion eye theory, the functional significance of various preretinular optic components was reviewed where it specifically related to Manduca. Wolf et al. (1973) used a new method for coating sensitive biological specimens with gold. In their “sputtering” method the golden layer is thin and uniform, By examining the trombiculide mite, Ascoschongastia latyshevi, the authors described systematical characteristics like the scutum, the sensillae, the body hairs, and the eyes. Sensory structures of ticks have been studied with the SEM (Woolley, 1972a). Postulations of certain functions of the sensilla have been made with inferences from the literature. The species examined were Amblyomma americanum,Argas persicus, Boophilus annulatus, Dermacentor andersoni, D. variabilis, Haemaphysalis leporispalustris,Ixodes kingi, and I. ricinus. According to Woolley (1972b) the SEM reveals greater structural details of the respiratory apparatus of ticks than previously has been possible. SEM micrographs were used to elucidate the respiratory features of two soft ticks, Argus persicus and Otobius rnegnini. The hard ticks included Dermacentor andersoni, D. variabilis, Haemaphysalis leporispalustris, Ixodes kin@., I. ricinus, I. ochotonae, Amblyommu arnericanum, and A . cajenennse. Cutaway sections of spiracles in D. andersoni and D.variabilis observed by SEM particularly revealed aeropyles opening into primary atrial chambers with interatrial ostia and elongated, fenestrated secondary atrial chambers which connect with the tracheae. Ethanol sterilization of nitrocellulose (Millipore) filters did not alter the permeability for 3H-thymidine or bovine serum albumin, and the porosity was unchanged (Lehtonen et al., 1973). Ethanol pretreatment did not decrease the pore size as judged from scanning electron micrographs. This was also confirmed by freeze-etching electron microscopy. Thus, sterilization of nitrocellulose filters with 70% ethanol seems to be a safe procedure for their use-for example, in embryological and immunological experiments. LoBuglio et al. (1972) have shown that latex particles (0.23 /.fm in diameter) can be used as an immunological marker for SEM. The principle of this method lies in the specific binding of latex particles coated with antibody against a target cell to the surface of that cells. Latex particles coated with normal gamma globulin do not specifically attach to the cell surface. The technique involves three steps: (1) coating of latex particles with gamma globulin; ( 2 ) incubation of gamma globulin-coated latex particles with glutaraldehyde-fixed target cells; and (3) washing of cell preparation and examination by SEM for latex attachment to target cell surface. Major factors in the successful application of this technique relate to the pH of the suspending medium, the ratio of gamma globulin to latex particles, and the use of sedimentation rather than centrifugation for washing of cells with attached particles. This technique has been useful in demonstrating that the red cell membrane is intact antigenically over what appeared to be surface disruptions in malaria-infested red cells. A second application has been in
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the detection of antibody activity in antilymphocyte sera. Examples of the detection of antibody-antigen reaction on the surface of mouse red cells, mouse lymphocytes, and human lymphocytes were given. The cytochemical detection, by SEM, of concavalin-A complexed with peroxidase was described by Bretton et al. (1973). Knowledge of structural details of chromosomes is a prerequisite to understanding and correlating genetic and morphological changes. Light microscopy provides little detailed definition of chromosome morphology. Similarly, transmission electron microscopy cannot reveal the detailed external structure of the chromosome. Christenhuss et al. (1967) and Yu (1971) have shown that the SEM can be used to obtain information on the three-dimensional external architecture of the chromosome. Chromosomes can be visualized in the SEM only after the surface coating of nonchromosomal proteins has been removed; this can be done by tryptic digestion. However, if proteolytic enzymes are used to remove the surface proteins, some of the chromosomal proteins may be attacked. Pawlowitzki and Blaschke (1970) described a method of protein removal based on protein extraction under mild conditions, following mechanical injury to living cells. The search for chromosome damage as the common cause of tumors has spawned dozens of conflicting reports from leading medical centers. Recently, in several of those centers SEM and the scanning electron probe microanalyzer have been employed to establish whether and how cancer is induced by chemicals, viruses, or other means (Anon., 1968). Raphides are needle-shaped crystals of calcium oxalate, occurring within specialized cells of certain plants. Ingestion of raphide-containing plant tissue results in severe irritation of the mouth and the throat. Sakai et a!. (1972) found, by SEM, barbs and grooves on raphides and suggested that the barbs act as mechanical irritants and carry a chemical irritant into the wound produced by the crystal. Kohn e t al. (1972) have studied by SEM the hollow, harpoonlike radula tooth of the toxoglossan gastropod, Conus. The technique elucidated the structure and relationship of the component parts of the tooth. On the basis of this structure, the relationship of the component parts, and knowledge of the animal’s food, the authors proposed a scheme for the functional roles of the tooth components in prey capture. Nordbring-Hertz (1972) studied by SEM the nematode-trapping fungus, Arthrobows oligospora. A special technique was developed to visualize the capture organs in their erect position. The fungus was grown directly on agar, or on Millipore filters, or on glass cover slips placed on agar. Before freeze-drying and coating, the material was fixed in Os04 vapor to avoid shrinkage. Young capture organs, spontaneously formed and nematode-induced, remained standing better than old ones. An adhesive substance on the capture organs was observed; it was suggested that the substance aids in trapping nematodes.
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Boyde ef al. (1968) reported the use of SEM in the challenging and important area of spinal cord structure. In examination of cultures of chick spinal cord several cell types were identified and in a single case possibly a neuron was described. It was shown that what appeared under light microscopic examination to be single structures often possessed a multicomponent structure consisting of axon bundles rather than single axons. Scanning electron microscopy of cells in culture provides a technique for examining the cell division cycle and cellular interactions. It is useful in the study of malignant cell behavior, cellular immune reactions, and other aspects of cell interactions. Boyde ef aZ. (1972) described improved techniques for SEM of cells in culture. The surface morphology of several types of normal and malignant cells was described by SEM in correlation with cinematic and still light microscopy of the same cells in the living state. The authors have outlined some possibilities for further improvement and development of SEM analysis of cell cultures. They suggested that the most comprehensive picture of cellular organization can be obtained from correlative studies of scanning and transmission electron microscopy. In addition, one can utilize x-ray energy analysis in the SEM for study of cells that have been stained by reagents tagged with metals. Taylor (1971) reported that SEM is being evaluated as an aid in several areas of interest to forensic scientists. The technique has been used to identify timber and charcoal fragments without the need to cut sections. Spermatozoa were identified in seminal stains by direct examination of stained cloth. A number of fibers can be identified rapidly and nondestmctively. The comparison of such materials as laquered hair, machine tool swarf, and glazing putty can be achieved more easily by SEM than by other techniques. Additional applications of SEM to the medicolegal and forensic medical fields were described by Boehm (1971) and by Devaney and Bradford (1971). It would seem, however, that techniques of interpreting pictures from SEM and relating them to optical micrographs of the same object may require improvements before they can be used to solve forensic problems. The high cost of the equipment is a deterrent. As was mentioned before, smaller units at prices comparable to the cost of high-quality optical microscopes are available. Some of the smaller units can be used as electron microprobes by the addition of energy-dispersive x-ray analysis equipment. This combination seems particularly attractive to the forensic scientist for several reasons: The analysis is rapid, nondestructive, and semiquantitative or quantitive, and it can be performed either on single particles or on very small areas. In this case there is no problem of interpretation and the results can be related to conventional chemical analyses (Anon., 1972a). Forensic scientists might be interested in a method of determining, by electron microprobe, the chemical composition of microgram samples (Inman, 1972). The technique was adapted for assay of samples from space flights. It requires the dissolution of the specimen in a lithium tetraboratelithium carbonate (5-1) flux contained in gold crucible at 1025°C. The
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quenched melt is removed by shattering, and the glass fragments are polished. The low-atomic-weight flux simplifies the corrections to be applied to the x-ray intensity data obtained from the microprobe. Although the history of Jane Austen’s (1775-1817) hair may not be of great concern to many food scientists and technologists, the SEM study of her hair (exhibited in the museum at Chawton, Hampshire) provides some interesting insights into potentialities of the technique. Swift (1972) concluded that, during the last three years of her life, Jane Austen “did little to tend her hair and that brushing, combing, and handling were minimal.” Slight erosion of the hair (presumably after death) was attributed to the presence of two types of saprophytic yeasts. The examination of the hair provided for the first time direct association between the two types of microorganisms (Malassesia furfur and Pityrospoturn orbiculare). Another item of interest along similar lines was reported by Lynn and Benitez (1974). Perforated eardrums in the preserved temporal bones of a mummified Egyptian male, in otherwise normal middle ears, were shown by SEM as evidence of acute otitis media and probably defective hearing among ancient Egyptians. Watters and Buck (1972) used SEM to study fundamental differences between repair of mesothelial wounds and repair of skin or mucosal wounds. Price (1972) constructed three-dimensional models of ruffling membranes (a phenomenon associated with the moving boundary of many types of cells maintained on a solid surface) by assembling enlarged Styrofoam duplicates of serial sections. The sections were taken from a cultured monkey kidney cell. Various features of the models were corroborated by SEM. SEM may have great potential value as a diagnostic instrument for the pathologist, Aranyi et al. (1970) used SEM in an examination of virus-infected culture cells. SEM has also been used in the analysis of congenital hair defects (Brown et at., 1971), to determine the morphology of normal and diseased hair, nails, and skin lesions (Carteaud, 1969), to study the long-term effects of sodium glutamate on the rat retina (Hansson, 1970), and to study the morphology of emphysema (Nowell el al., 1971). And finally, I should like to describe the application of the SEM as a diagnostic tool in an entirely different, yet indirectly related to food processing, area. According to Scott and Mills (1973), the SEM in combination with microprobe analysis can be the basis of a powerful diagnostic tool to detect stress in critical roller bearing applications, to prevent material failure, and to allow better control of adequate maintenance.
V III. STUDIES OF FOODS AND FOOD PRODUCTS Microscopy of food is especially useful in detection of adulteration, determination of identity, control of food manufacture, development of new products,
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trouble-shooting during the investigation of problems arising in control of established processes, and determining the hygienic status of a food. Microstructure may provide useful indications of the best way of milling cereal grains, producing breakfast cereals, and drying such foods as milk and eggs. Particle size distribution is important in the rheology of chocolate and fondant, in the refining of sugar, and in starch manufacture (Lyne and Moss, 1971). Light microscopy is frequently used in studies of frozen fruits and vegetables. However, most specimens are seldom received in a form that is suitable for direct microscopic examination. The food materials require fairly lengthy preparation by the classical methods. In recent years it has become increasingly evident that direct examination of food surfaces can make important contributions to solving problems encountered by the food industry. Some of those applications of SEM are mentioned in practically all the other sections of this review. As in studies of other specimens of biological origin, the interior structure of the sectioned material can be revealed as surface structure for examination in the SEM.
A. CEREAL GRAINS Structures of cereal grains are important in relation to kernel function and to their processing and utilization as foods. A clear understanding of the structural details is therefore of interest to plant breeders, plant physiologists, plant pathologists, cereal chemists, and food processors. Sullins and Rooney (1974a) used SEM to illustrate differences in corn endosperm structure which account for differences in nutritive value of the grain. High-lysine corn has a reduced amount of protein bodies in the endosperm. Breeding efforts have developed high-lysine corn lines that contain a relatively normal endosperm, and the reduction of protein bodies can be readily observed with the SEM. With the reduction in protein bodies as a marker, screening of lines with normal endosperms for high lysine is practical with the SEM. It is also possible to remove only the cap of the kernel, examine the kernel with the microscope, and have the remainder of the kernel be viable and germinated. Distribution of the proteins in the peripheral endosperm area of waxy grain was different from that observed in nonwaxy grain. This difference, presumably, accounted for part of the improved digestibility of waxy grain over nonwaxy grain when fed to animals. Improved digestibility was due to starch granules in the peripheral endosperm area; those starch granules vaned in their susceptibility to enzymatic degradation. In vitro studies indicated that waxy starch was more rapidly solubilized than nonwaxy starch. Scanning electron micrographs of soft endosperms from normal, opaque-2, or modified opaque-2 corn showed loosely packed, nearly round starch granules associated with thin sheets of protein and many intergranular air spaces (Robutti et al., 1974). The hard endosperms had tightly packed, polygonal starch granules
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FIG. 17. Transverse section through the palea (PA), lemma (LE), pericarp (PE), and subaleurone layer, including starch granules (S) of barley (165X).
associated with a continuous protein matrix, and no intergranular air spaces. Normal hard endosperms had zein bodies embedded in the protein matrix; modified hard endosperms did not. Starch damage was greater in the hard endosperm than in the soft because of a stronger adhesion between starch and protein. The low density and opaqueness of soft endosperm were attributed to the intergranular air spaces. Interaction between protein matrix and starch granules during drying explains the shape of starch granules. Pomeranz and Sachs (1972a) studied by SEM the detailed structures of the immature and mature barley kernel. A cross section through the lemma and palea (husks) of barley is shown in Fig. 17; the differences in the appearance of the top of a cross section and of the side of the lemma (with several protruding tubes) and of the pericarp are shown in Fig. 18. A transverse section through the pericarp, seed coat, multilayered aleurone, and endosperm is shown in Fig. 19. The average length of the aleurone cell (in a transverse section at the germ end) was 28 m, and its average width was about 15 pm. The whole wall between adjacent cells was about 3 pm thick. Several compound middle lamellae can be seen between the cell walls of two aleurone
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FIG. 18. Section through lemma (LE) with protruding tubes, pericarp (PE), aleurone (AL), and starch containing endosperm of barley (17OX).
cells in Fig. 19. The section shows the multilayered structure of the pericarp, the two-cell-deep aleurone layer, and the subaleurone layers. The aleurone layers are in part open, showing the spherical aleurone grains (diameter of about 1.5 pm), and in part covered by a wrinkled cell wall material. The section through the aleurone cell wall seems to indicate in several places a multilayered fibrous structure. Some of the aleurone grains have a rugged surface, indicating the possible presence of spherosomes, structures which can be seen by TEM. The size of the aleurone grains determined by SEM on dry cells is substantially smaller, indicating considerable swelling from water imbibition. In the subaleurone layer are several lentil-shaped (6 to 8 pm long) starch granules, apparently embedded in a relatively thick protein matrix and forming cells separated by endosperm cell wall material. Mares and Stone (1973) described a method for the isolation of wheat endosperm cell walls free from nonendospermic cell walls. A technique was described for SEM of walls in endosperm sections. Sections were treated with boiling water followed by digestion with amylase. The appearance of the
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FIG. 19. Transverse section through pericarp and endosperm of barley (710X): PE, multilayered, cordlike pericarp; AW, aleurone cell walls; AG, aleurone grain; PM,protein matrix of subaleurone layer; S, starch granules; EW, cell wall of subaleurone layer; CL, compound lamelle.
endosperm cell walls in the intact kernel sections and in cell wall isolates was examined by light microscopy, SEM, and TEM. SEM micrographs showed folding of the walls on the cell wall contents and different patterns of prismatic and central walls. The cell walls have a microfibrillar skeleton embedded in the amorphous matrix components. Stevens (1973a) devised methods for the isolation from wheat bran of large fragments of aleurone layer, contents of aleurone cells, aleurone cell walls, and aleurone granules. Dimensions of the structures examined by SEM were generally smaller than those reported by workers who used conventional methods. The difference was attributed to swelling of sections mounted in water and examined by light microscopy. The polygonal prismatic aleurone cells examined
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FIG. 20. Longitudinal section through central endosperm of immature barley (1450X): S , starch granules.
by SEM varied in size and shape; an idealized cell was described as a 35-pm cube, having walls 2.5 pm thick. Aleurone grains ranged in diameter from 0.25 to 3.5 pm; the majority measured about 2 pm. The grains were densely packed in a cementing matrix within the cell walls. The cell wall appeared to have a fibrillar structure; the contents of the cells displayed a characteristic granular appearance. The samples of aleurone layer were subsequently extracted with water, 1 M NaCl, 0.04 M HCl, and 0.04 M NaOH (Stevens, 1973b). The extracted materials were examined by SEM. The results indicated that the walls of the aleurone cells were not impermeable to the protein of the cell contents, since substantial amounts were extracted by all the extractants. The results contradicted the widely held view that the aleurone grains are proteinaceous in nature. Os04 has been frequently used for electron microscopic futation of proteins, lipids, and lipoproteins of wheat grain. It is known to oxidize unsaturated fatty acids and to create diols on the site of ethylenic double bonds. It may also involve the formation of intermolecular linkages by breaking these double bonds and by forming an addition compound. Heavy metals increase the electron
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FIG. 21. Longitudinal section through central endosperm of mature barley (1450X): S, starch granules with attached protein (bottom) or with indentations (top); EW, cell wall.
density of the specimen, and hence the electron scattering efficiency. The furation increases the lipids’ contrast and makes them observable by electron microscopy. In the protein bodies of endosperm cells Buttrose (1963) and Morton et al. (1964) observed unidentified areas after osmium fixation. In wheat flour or endosperm fragments many authors found osmiophilic globules that they identified as lipid inclusions. These osmiophilic granules are also present in the gluten lipoprotein compounds in dough, and in the insoluble fraction in 4 M urea of glutenin. However, Hess (1960), using a replica surface technique, indicated that in flour a layer of lipids that are not directly observable will surround the starch granules more or less uniformly. In the same way, observations by Aranyi and Hawrylewicz (1969) and Evers (1969b) of flour surface using SEM did not show the presence of lipid zones or droplets; Aranyi and Hawrylewicz (1969) found a structural modification of proteins only after n-butanol delipidation.
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FIG. 2 2 Appearance of a dehulled oat kernel (groat); hairs on the dorsal side showing embryo (16X ).
Crozet and Guilbot (1974) attempted to determine whether OsO4 could aggregate unsaturated lipids observable in wheat flour, particularly on the starch granule surface. The results were inconclusive. A longitudinal section through the pericarp, aleurone, and starchy endosperm of immature barley showed no clear cellular differentiation of the aleurone layer. A higher magnification of material in the central endosperm of immature barley (Fig. 20) showed only isolated and small (diameter about 4 ym) starch granules. The pitted appearance of the starch granules in Fig. 21 from the longitudinal section of mature barley is an artifact that resulted from prolonged exposure to the electron beam in the scanning microscope. The high concentration of starch granules in the central endosperm of the mature endosperm is in sharp contrast to the picture in Fig. 20. This contrast would be expected, since the ratio of protein to starch may be as much as fifty times as high in the earliest stages of growth as it is in the mature barley grain (Pomeranz et d.,1971). According to Harris (1962), the starch granules of barley are first seen as small spheres in the cells of the endosperm a few days after the beginning of seed
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FIG. 23. Transverse section through oat groat ( I S O X ) showing crease (CR), with hairs (HI, pericarp (PE), aleurone layer (AL), and starchy endosperm (E).
development. These spheres later develop into the bean-shaped and lenticular forms characteristic of mature starch. The diameters of the large starch granules in Fig. 21 are as much as 16 pm; there were also many smaller granules (about 1.5 pm). The large granules seem to be relatively free; the smaller ones are embedded in a protein matrix. Some material, apparently proteinaceous, adheres to several of the larger starch granules. Starch granules are formed in amyloplasts, which probably accounts for adherent proteinaceous material. The presence of cell wall material is indicated in Fig. 21. In addition to the kidney-beanshaped starch granules, granules with surface indentations are present. The indentations indicate areas in which starch granules pressed against each other. Many oat cultivars are quite hairy, as can be seen from the dorsal view of the groat (dehulled kernel) (Fig. 22). A transverse section through the crease area (Fig. 23) shows the relatively large hairs, cordlike pericarp layers, the aleurone layer, and starchy endosperm (Pomeranz and Sachs, 1972b). The aleurone cells in the area adjacent to the crease area are almost elliptical in shape and covered by a cell wall with many fine protrusions. There were two lines of aleurone cells
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FIG. 24. Structure of the aleurone cell wall in oats (3750X).
in the distal side of the caryopsis; the cells in that area were almost hexagonal or rectangular. Aleurone cells in a transverse section at the germ end were generally elliptical and somewhat larger than the aleurone cells in the transverse section through the center of the kernel. The fine structure of the wall covering the aleurone cells in oat is shown in Fig. 24. The appearance of the center of the endosperm indicates, as in other cereal grains, a protein matrix in which starch granules are embedded. Tanaka et al. (1973) isolated subcellular particles from the phosphorus-rich portion of rice bran using a differential centrifugation in nonaqueous media. Isolated particles were characterized by high contents of phosphorus and metals and low contents of proteins. Most of the phosphorus was found as phytic acid. Chemical composition and SEM studies confirmed that isolated particles were derived from the aleurone layer and were distinguished from protein bodies. Long-grain rice was milled by nine different treatments to obtain samples with a wide range in degree of milling (Watson et aZ., 1975). Surface lipid, ash, and protein contents were determined, and examinations of the milled rice were made by SEM. The milling treatment, surface lipid and ash contents, and the
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FIG. 25. Cross section through area adjacent to the crease in triticale: AL, aleurone layer; PE, pericarp layers; EN, starchy endosperm (300X).
amount of pericarp and aleurone removed were related. However, there was a variation in the amount of pericarp and aleurone removed from different kernels and from various sites on the same kernel within a treatment. Triticale is a man-made cereal species derived from a cross between wheat (niticum) and rye (Secale) (Briggle, 1969). Objectives in making this cross included combining the grain quality, productivity, and disease resistance of Triticum with the vigor and hardness of Secale. Cross sections through the areas adjacent to the crease (midway between the brush and germ ends) in wheat, rye, and triticale were compared (Pomeranz, 1971). There is a general similarity in the three cereals in the arrangement, size, and structures of the pericarp and aleurone layers on both sides of the crease, and in the starchy endosperm in which starch granules are embedded in protein matrices (Fig. 25). Higher magnifications of transverse sections through the central endosperm and the aleurone layer of triticale indicated basic similarities to corresponding structures of wheat, rye, and barley. The starch granules in the central endosperm of triticale vary in size, but the larger granules (average
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diameter of about 20 pm) are much bigger than the more uniform starch granules in the subaleurone layer (average diameter of about 12 pm). The aleurone cell wall consists of several layers, and the aleurone grains in the exposed aleurone cell are small (average diameter of 2 pm) and embedded in a matrix. Dronzek (1972) examined by SEM the developing (from 3 to 42 days flowering, at 3-day intervals) endosperm of a hexaploid triticale, its rye and durum wheat parents, and a hard red spring wheat. At early stages of development, triticale starch granules developed at the same rate and in a pattern similar to that of starch granules in the durum parents. Enzymatically degraded starch granules could be seen in the triticale kernels about 21 days after flowering. At maturity, starch granules of triticale were similar in appearance to starch granules of the parents. The results indicated that the endosperm carries cell characteristics of both parents. Glutenins of one variety of triticale and its rye and durum parents were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and SEM (Orth et al., 1974). Results of all techniques showed that glutenin of triticale is simply inherited from its parents. All glutenin subunits of the triticale could be accounted for by corresponding subunits in one, or both, of its parents. The amino acid composition of the triticale glutenin was essentially intermediate between that of its rye and durum parents. Distinct differences in the ultrastructure of glutenin of spring rye (cv. Prolific) and amber durum (cv. Stewart) were noted. Both types of ultrastructure were evident in the micrographs of triticale glutenin. Orth et al. (1973a) analyzed by SEM glutenins of two hard red spring wheat cultivars, one durum cultivar, a synthetic hexaploid wheat and its tetraploid and diploid parents, and one variety of spring rye. The glutenins of hard red spring and the synthetic hexaploid were fibrous in structure; the glutenins of the durum wheat and the tetraploid were characterized by ribbonlike and film structures. The rye glutenins were rodlike. Reduction of disulfide bonds in the hard red spring glutenin destroyed the fibrous structure. Proteins from a bread wheat were separated by gel filtration (Orth et al., 1973b). The fractions were heterogeneous when separated by electrophoresis. The fractions also differed in size and shape when examined by SEM. Seckinger and Wolf (1973) studied the structure of grain sorghum endosperm protein of five commercial hybrids and eight experimental lines with both TEM and SEM. Vitreous endosperm showed a well-developed two-component structure consisting of protein bodies embedded in a matrix protein. On the basis of the solubility properties of the proteins, it was suggested that the globular protein was the site of prolamine (kafferin) deposition and that the matrix protein was the site of the glutelin fraction. The matrix protein was apparently amorphous; the globular bodies showed concentric rings and a core that indi-
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cated variations in composition. Protein bodies were fairly uniform in size-2 to 3 pm in diameter. However, one experimental line which had above-average lysine content had smaller protein bodies. This was postulated to confirm the well-known negative correlation between alcohol-soluble protein and lysine contents. Distribution of protein within the sorghum grain was similar to that of other cereal grains. The peripheral vitreous area of the kernel contained the highest protein content; interior areas had gradually decreasing amounts of protein. Protein bodies constituted, according to microscopic observations, 70 to 80%of sorghum protein. Wrigley (1972) used the SEM to show differences between wheat starches varying in amounts of adhering protein. The structure of grain sorghum samples representing a wide genetic base was examined by SEM (Hoseney et af., 1974). The soft or opaque endosperm was characterized by relatively large intergranular air spaces. The starch granules were polygonal in shape and covered with a thin protein matrix. Embedded in the protein matrix were protein bodies. It was suggested that in sorghum, as in wheat, the hard protein is caused by strong adhesion between protein and starch. When fractured, many starch granules broke, and little break occurred at the starch-protein interface. A dwarf variety from Sudan had relatively few protein bodies in the endosperm. Because the protein bodies were found to be kafferin, a prolamine presumably low in lysine, it was surmised that the sample would be high in lysine. Subsequent amino acid analyses confirmed that the sample contained 3.0 g of lysine per 100 g of protein, a value significantly higher than that found in regular sorghum. (See also Badi et al., 1974.) Sullins and Rooney (1973) conducted light and SEM studies of the peripheral endosperm of waxy and nonwaxy endosperm sorghum varieties. Sorghum varieties are known to differ widely in endosperm type-yellow, sugary, waxy, and nonwaxy. It has been observed in feeding trials that sorghum grains with waxy endosperm tend to have higher feed efficiencies than nonwaxy varieties. The peripheral endosperm area of sorghum is composed of starch granules embedded in an amorphous protein matrix containing relatively indigestible (alcoholsoluble) protein bodies. The waxy sorghum varieties contain fewer of these spherical protein bodies, and therefore the digestibility of the whole grain is higher. With the lower relative proportion of the protein bodies, the waxy grain apparently is more completely broken down during processing (steam-flaking, micronizing, pulverizing, popping, exploding, and reconstituting). This also may contribute to the difference in feed efficiency between waxy and nonwaxy sorghum grains. Sullins and Rooney (1974a) have subsequently shown that SEM can be used in screening sorghum grains for cultivars rich in lysine. SEM was found to be a useful tool in examining cereal grains for differences in structure and the relation
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FIG. 26. Cross section through a dehulled buckwheat achene showing spermoderm (SP), embryo (EM), and starchy endosperm (SE) (25X).
of that structure to differences in nutritive value (see also Rooney and Sullins, 1974; S u b s and Rooney, 1974b). As was mentioned previously, buckwheat is the fruit of a dicotelydenous plant but is classed in agriculture and commerce with cereals. Common buckwheat has a sharply three-angled or three-keeled pointed dry fruit (achene), with a relatively smooth hull (pericarp), and often part of the calyx attached at the base of the hull. The fruit in most varieties is 4 to 6 mm long; and in the Japanese type it is 6 to 9 mm long. A cross section through the achene (without hulls) shows at low magnification (Fig. 26) the spermoderm; the embryo with its two cotyledons, folded to form a double curve; and the starchy endosperm. The cotyledon cells are much smaller than the cells in the starchy endosperm (Pomeranz and Sachs, 1972~). A cross section through the spermoderm, the aleurone layer, and the subaleurone starchy endosperm is shown in Fig. 27. The section through the spermoderm shows the outer epiderm of longitudinally elongated wavy cells, the
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FIG. 27. Cross section through the spermoderm showing the outer epiderm (OE), spongy parenchyma (PP), inner epiderm (IE), aleurone layer (AE), and starchy endosperm (SE)of buckwheat (1750X).
spongy parenchyma, and the inner epiderm. The starch granules in the endosperm are round or polygonal and fill the endosperm cells. The structure of the aleurone layer (including the aleurone cell wall and aleurone grains) and subaleurone endosperm is shown in Fig. 28. The surface of the endosperm cell wall enclosing the starch granules is shown in Fig. 2 M . The aleurone grains are embedded in a matrix; the grains are much smaller than the starch granules. Figure 28B shows the outer and inner surfaces of the walls of two adjacent aleurone cells. The multilayered aleurone cell wall is apparently finely textured on the inside and has a relatively smooth outside surface. The previous sites of the small aleurone grains contrast clearly with the large starch granules. The starch granules in the center of the endosperm (Fig. 29.4) fill the contents of cells surrounded by relatively thin cell walls. Higher magnifications of the center of the endosperm (Fig. 298) indicate that the starch granules are not free but are surrounded by a matrix, presumably proteinaceous, which strengthens structural unity of the cell contents.
FIG. 28. A , Cross section through spermoderm (SP), aleurone layer (AL) with aleurone grains (AG), subaleurone endosperm with starch granules (S), and endosperm cell wall (EW) of buckwheat (1230X). B, Wall of two adjacent aleurone cells (AW) and starch granules (S) of subaleurone endosperm of buckwheat (3145X).
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FIG. 29. A , Starch granules in center of buckwheat endosperm (155x1. EW, cell wall of starchy endosperm. B, High magnification of starch granules from center of endosperm of buckwheat (line bar = 4.6 gm).
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B. PROCESSING OF CEREAL GRAINS Aranyi and Hawrylewicz (1968) compared wheat flour and dough samples at similar magnifications. At lower magnifications, flour particles could still be recognized as broken-up endosperm tissue. Surrounding the lentil-shaped and round starch grains was a protein matrix with rough, broken, and curled edges. In dough, the starch grains were distributed more evenly over the entire area, and the edgy, rough protein matrix was transformed into a veil-like structure w h c h appeared to cover the starch granules. This was in agreement with the general concept of dough formation, according to which, during hydration and mechanical dough mixing, the proteins and lipoproteins of dough form a viscoelastic network in which the starch granules are embedded. Sternberg (1973) showed by SEM that in partially developed dough the gluten film was imperfectly formed. A continuous gluten matrix enveloping starch granules was present in fully developed dough. SEM was used by Christianson et al. (1974) t o compare the surface structure and appearance of the internal crumb of wheat flour bread and starch-xanthan baked products enriched with soy proteins. Freeze-fractured bread crumb was used to examine the interior of gas cell walls in the bread. Comparison by SEM of endosperm areas, as seen in a wheat kernel section, and milled flour at corresponding magnifications demonstrated that flour was identical with fragmented endosperm tissue (Aranyi and Hawrylewicz, 1969). SEM revealed changes that take place during air classification of wheat flour into high- and lowprotein fractions and during extraction of flour lipids by butanol. Functional properties of wheat flour depend on the interaction of the protein, lipid, and carbohydrates of the flour (Pomeranz, 1968; Pomeranz et al., 1970). Simmonds (1971) reviewed recent work on the morphology of the intact wheat kernel, flour, and flour fractions and the relation of the structure to known biochemical and functional parameters of flour components. SEM indicated that the strength of adhesion between storage protein and starch reserves in wheat endosperm cells varies between hard and soft wheats and plays an important role in determining vitreousness of the grain and its milling behavior. Endosperm cells of soft wheat contain starch granules embedded in a friable matrix which is readily crushed by rollers in wheat milling. Consequently, the starch granules are released cleanly, with little damage. On the other hand, the endosperm cells of the hard wheat tend to shatter (rather than powder), and this enhances breakage of both starch granules and protein matrix. This results in flour from hard wheat showing high starch damage and protein adhering to the surface of starch granules. Wheat cultivars differ in the vitreousness and hardness of their mature air-dry endosperms. Micropenetration studies conducted by Barlow et al. (1973) showed little difference between either starch or proteins from different culti-
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vars. The authors suggested that there are large differences in the nature of the starch and storage protein interface of hard and soft cultivars. This conclusion was based on studies of the interphase by light microscopy combined with extraction of soluble proteins and protein staining, by TEM, SEM, and freezeetching, and by fluorescent antibody staining. It was shown that the area between starch granules is filled with materials that stain as protein. Watersoluble proteins are limited to an area immediately surrounding the starch granules. Hydration results in swelling of that area. The authors presented evidence that residues of the original amyloplast membranes, as well as of the endoplasmic reticulum, surround the starch granules. The soluble proteins associated with the starch granules form a heterogeneous, complex group. Associated with the soluble proteins are polysaccharides which yield on hydrolysis glucose. The authors concluded that the total water-soluble material acts as a cementing substance between starch granules and storage proteins. It was inferred that the genetic control of grain hardness is expressed through the amount and composition of the water-soluble material forming the interphase between starch granules and storage proteins (see also Simmonds, 1974). Hoseney and Seib (1974) concluded on the basis of SEM of wheats and wheat flours that hardness of wheat is governed by the strength of the proteinstarch bond. The opaque character of soft wheat and the yellow berry character of low-protein hard wheats is caused by light diffraction by air spaces in the endosperm. The air spaces, presumably, are formed after physiological maturity during the drying of the wheat kernel. During that drying, the protein matrix shrinks, ruptures, and creates air spaces. In harder, or more vitreous, wheat kernels the kernel becomes denser during drying, but the protein matrix remains intact. In hard wheats the protein matrix appeared to adhere tightly to the starch, with little of the starch surface exposed. In some instances the starch-protein adherence was strong enough to break the starch granule rather than to separate the adherence. In the endosperm of a soft red winter wheat kernel, the surface of the starch granules was exposed and the protein matrix was not as continuous as in hard red winter wheat. This was presumed to be conducive to the ease of separation of starch from protein in air classification. On the basis of SEM studies of wheat, Belderok (1973) suggested methods of selecting wheat cultivars of high bread-making quality. Bemardin and Kasarda (1973) have prepared a film which depicts what happens when thin sections or particles of wheat endosperm are moistened with a drop of water. There is an explosive reaction wherein protein is hydrated within 0.05 second. This hydration is demonstrated by the almost instantaneous appearance of the protein fibrils streaming out of broken endosperm cells. The fibrils exhibit viscous flow and elastic deformation. Starch granules can be seen adhering to the fibrils. If a fibril is stretched by the flow to a breaking point and
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recoils elastically, some starch granules may be dislodged and float free in the “solution” while other granules continue to adhere to the fibrils. Adjacent flour particles spreading similar protein networks interact to form a continuous interacting system that can be worked mechanically to form a dough. Fibrils with diameters of 50 to 300 A were observed. It was suggested that larger fibrils may be composed of lateral aggregates of the smaller fibrils. The observations indicated that endosperm protein acquires elastic properties immediately on hydration and before dough formation. Bernardin and Kasarda (1973) described results of SEM and TEM of the fibrils. Endosperm protein was present in sheet form when moistened. These sheets, presumably, resulted from a laminar deposition of storage protein in the protein bodies of the developing wheat kernel. The sheets of protein ruptured under stress (hydration), forming fibrilar webs of protein composed of fibrils ranging in diameter from 50 to several thousand angstroms. The streaming of fibrils was impeded by high flour concentrations. All wheat varieties exhibited the fibril formation phenomenon. Triticale and rye developed fibrils to a lesser extent; corn, rice, and barley showed no tendency to form fibrils. Once fibrils were formed, they dissolved slowly and persisted in solution for several hours. Protein fractions of gluten, obtained either by the Osborne separation method or by acetic acid solubilization followed by chromatography on Sephadex G-100, were observed by electron microscopy (Crozet et al., 1974). Gliadin and fractions with a high gliadin content have a smooth, compact, and nearly electron-lucent structure; glutenin and fractions with a high glutenin content present a granular and fibrillar structure. The fibrils have a diameter of I00 to 200 A and form compact networks. Albumins and globulins have a structure which is more like glutenin than gliadin. In gluten there is a smooth, compact matrix similar to gliadin, which, however, encloses zones of structure similar to those of glutenin, albumins, and globulins. Flour proteins also present two kinds of structure: fragments of relatively compact and nearly electron-lucent proteins, and granular zones and fibrillar felting which adhere to both starch granules and protein fragments. Wheat flour produced by conventional roller milling contains particles of different sizes (from 1 to 150 pm), such as large endosperm chunks, small particles of free protein, free starch granules, and also small chunks of protein still attached to starch granules. The regular flour can be ground relatively fine to free the high-protein material from the starch granules. The reground flour can then be passed through an air classifier. If the classifier is used, a fine fraction, made up of particles about 40 pm and smaller, is removed and passed through another air classifier, where particles about 20 pm and smaller are separated. They produce a fraction which has a high protein content (15 to 22%) and comprises from 5 to 15% of the weight of the original flour. Air classifica-
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tion is relatively inexpensive and its advantages are numerous, such as manufacture of more uniform flours from varying wheats, increase of protein content of bread flours and decrease of protein in cake and cookie flours, controlled particle size and composition, and production of special flours for specific uses. New high-lysine cultivars of corn which have improved levels and balance of amino acids, and some of which have inherent higher protein content, offer the opportunity to produce protein concentrates of high nutritional value. Christianson et al. (1971) found that, on treatment with isotonic buffer, the endosperm of corn fragmented into fine particles that gave improved protein shifting during air classification. Release of adhering protein from starch granules was demonstrated by optical and scanning electron microscopy. Before treatment, protein adhered to the starch granules in layers. During treatment, the protein slid off the starch granule in sheaths and left the starch granules free and smooth. (Protein sheaths are larger in size than starch granules and can be partially separated by sieving.) Munck (1972) reported that the SEM could be used to characterize endosperm tissues of high- and low-protein barley cultivars varying in concentration of lysine in the protein. Headley ef al. (1972) reported that tempering corn grits for 24 hours at 120°F and 30% moisture with bromelain (2 mg of enzyme per gram of dry solids), flaking them to 0.012-inch thickness, and freeze-drying them increased grinding capacity in a pin-mill approximately fourfold and reduced energy consumption by about 75%. At the same time, the proportion of total grit dry substance was reduced to a size below 32 pm and increased from 31 to 94%. Yet, despite the nearly complete mechanical disintegration of the corn grits, air classification produced (from original grits containing 8.5% protein) no fraction with less than 5.5% protein and no fraction with more than 31.4% protein. The reason for this failure to achieve a better protein shift was demonstrated by SEM; flour from finely ground enzyme-treated grits contained starch granules with attached pieces of protein. Walker et al. (1972) found the SEM useful in determining the cellular structure of popped milo, and the effects of processing (flaking in corrugated differential rolls or grinding in a hammer mill) on that structure. C.
MALTING AND BREWING
Dronzek et al. (1972) used scanning electron microscopy in combination with light microscopy to study the changes that occur in starch granules during germination of wheat. Enzymatically degraded starch granules were observed near the aleurone layer in grains germinated for 2 days. Most of the degradation was confined to the larger starch granules. Differences between the mode of enzymatic attack on the larger and smaller granules were interpreted to indicate differences in physical structure of the granules.
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The objective of malting barley for brewing is to modify grain into a product that can yield an aqueous extract containing (1) fermentable products, (2) available substrate for yeast nutrition, and (3) precursors for imparting the desirable organoleptic qualities to the beer (Preece, 1954). The sum total of physical and chemical changes that take place during malting is termed “modification.” According to MacLeod (1967), the term “modification” describes “a rather nebulous but nonetheless real condition which has resulted from the transformation of endospermic constituents to give the best possible material for mashing.” In practice, malting conditions are selected with the purpose of attaining optimum modification that will yield a maximum of extractable solids while minimizing malting losses and excessive degradation of the barley’s high-molecular-weight components. Modification results in transformation of tough barley into friable malt. That transformation can be assessed by physical methods ranging from the simple biting test to tests employing elaborate self-recording mechanical devices. Among chemical indices, the increase in soluble proteins is probably the most important single parameter. Another index which has gained acceptance in evaluating modification is the difference in extract of fine- and coarse-ground malt. Known chemical and physical tests for evaluating modification are of limited value (MacLeod, 1967). Studies of Palmer (197 1) have indicated that modification of the endosperm in germinated barley commences at the dorsal (nonfurrowed) surface of the grain. The rate of endosperm modification depended more on the effective dispersal of hydrolytic enzymes than on the total amounts of these enzymes in the grain. Microscopic analyses showed that starch grains and hemicellulosic materials of the cell walls were coated with proteinaceous materials. Proteases were found to play a more active role than carbohydrases in the conversion of hard barley into friable malt. Changes in the aleurone layer and in the starchy endosperm of steeped, malted, and kilned barley were followed by SEM (Pomeranz, 1972). The surface of aleurone cells in steeped barley was highly pitted. The walls of aleurone cells were progressively degraded during malting and kilning. An increase in diameter of the aleurone grains during steeping was followed by further distortion of the spherical granules during kilning. Partial breakdown of cell walls in the center of the starchy endosperm of malted barley was accompanied by extensive dissolution of the protein matrix and “freeing” of small starch granules that were previously embedded in that matrix; the effect on the appearance of the starch granules was s m d . In the central endosperm of kilned barley malt, the cell wall dissolution was extensive and was accompanied by mechanical breakdown of the large starch granules. Composition, use, and structure (by SEM) of malt sprouts (a by-product of malting) was described by Pomeranz and Robbins (1971). SEM was used to follow modification in malting of a low-protein and a high-protein barley cultivar (Pomeranz, 1974). In the low-protein cultivar, degradation of the protein matrix was extensive and some of the degraded protein
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was deposited in kilned malt on large starch granules. In the high-protein cultivar, much of the protein matrix was largely intact and some protein was retained in the form of a modified but coherent and continuous thick film covering the starch granules. It was suggested that the thick film might be responsible for difficulties in malting of high-protein barleys, reduction of wort extract, and persistence of undegraded proteins which enhance chill haze formation in beer. According to Palmer (1972a), malting of barley reduces the population of small starch granules which are slower to gelatinize than are large granules at the infusion mashing temperature of 65°C. Acidulation of selectively abraded barley results in a more rapid development of both carbohydrate and nitrogenous extract in response to gibberellic acid (Palmer, 1972b). The amount of abrasion required is related to the shape and size of the grain; preliminary trials to determine optimum treatment indicated that the amount of abrasion was not always simply related to variety. SEM showed that acidulation facilitated the removal of the hemicellulosic walls. The amount of 0-glucans recoverable from extracts of acidulated malt was lower than that from normal malts, and the p-glucans precipitable by 30% (NH4)2SO4 were of lower specific viscosity. The cause of the accelerated attack on endosperm hemicellulose was not established. However, the concomitant acceleration of degradation of proteins which bind endosperm cells together might be correlated, as early stimulation of proteolytic enzymes might facilitate the action of hemicellulases on the walls. The results indicated that adequate proteolytic activity was essential to complete and rapid modification during malting. Palmer (1974) suggested on the basis of SEM that, during malting, hydrolytic enzymes migrate into the endosperm to disrupt and solubilize mainly the cell walls, complex protein materials, and the small starch granules. Satisfactory modification in malting should result in degradation of cell wall material throughout the endosperm and release of starch and degraded protein during mashing. However, in nonabraded malt some areas of the endosperm (especially at the distal end) may contain undegraded endosperm cell walls in which starch extract can be trapped and which give rise to glucan (gum) materials during mashing. Bathgate and Palmer (1 973) investigated the susceptibilities to amylolytic hydrolysis of the two types of starch granules in barley and malt. The large and small granules from germinated and ungerminated grain were subjected to the action of malt a-amylase under conditions that simulated those of a conventional infusion mash. Large starch granules from malt are hydrolyzed faster than those from barley. The faster conversion is probably due to prior modification of the starch granule during malting. The small granules from barley are highly resistant to attack by a-amylase, and even precooking does not appreciably increase their susceptibility to amylase attack. Still, the small granules in malt
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can be hydrolyzed to a larger extent than the small granules in barley because of partial removal of a protein layer from the surface of the former. Although during malting the small granules of barley are hydrolyzed more rapidly than the large granules, the situation is reversed during mashing. Little decrease in extract can be attributed to the resistance to hydrolysis of small starch granules in all-malt mashes. If raw barley is used in the grist, substantial amounts of small starch granules may remain in the mash. Clutterbuck and Briggs (1973) prepared aleurone layers, with testa attached, from degermed, decorticated barley. The layers that appeared intact under the SEM were used in studies on the induction, formation, and release of &-amylase in the presence of gibberellic acid. Changes that take place during conversion on a laboratory scale of barley into beer were followed by scanning electron microscopy of lyophilized samples (Pomeranz and Sachs, 1973). The observations included studies of the raw materials (barley malt, corn grits, and hops); of changes in conversion of mash components into wort, hopped wort, and yeasted wort; of yeast and beer filtration sediments; and of differences between total beer and haze in beer. The results were interpreted in light of biochemical changes known to take place during the production of beer. Stage (1972) discussed the possible use of SEM in studying fitration efficiency in brewing. Rennie (Anon., 1972b) discussed various aspects of wort production in relation to recent developments in materials and methods which may affect economics of brewing. Mash tun adjuncts may reduce speed of runoff. Examination of the “fines” in the mash by SEM and other means indicated that the physical nature of this fraction affected performance of the adjunct. Once the cause of the slow runoff is understood, it should be possible to extend the use of economically valuable adjuncts. The contribution of small barley granules to the “fines” was discussed by Bathgate (cited in Anon., 1973~). Royle (1970) studied the relationship between the infective units, zoospores, of a fungus and hop leaf stomata. Mode of infection and penetration of stomata by zoospore germ tubes were followed by SEM.
D. STARCHES Several investigators have used SEM in studies of starch. Hall and Sayre (1972) emphasized the limitations of light microscopy and of SEM in studying structures of starch granules. Starch is a transparent crystal which often gives images that are difficult to define by light microscopy because of diffraction and internal structure which may appear as surface phenomena. On the other hand, examination by the SEM is limited to the surface. To provide more information on internal details, both microscopic techniques should be combined to determine details which are due to internal features of the starch granule.
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SEM has revealed considerable variation in shape and surface structure among starch granules from wheat endosperm (Evers, 1969a, b). Evers confirmed the presence of an equatorial groove in large granules and its absence in small granules. This provided evidence in support of the different types of starch suggested by transmission electron microscopy. Further evidence for the homology of granules of intermediate size in the subaleurone layer with larger granules in the inner starchy endosperm was presented. In a preliminary study on the structure of wheat starch granules, modifications resulting from a-amylolysis were examined in the SEM (Evers and McDermott, 1970). Much variation existed among the starch granules. Consequently, only tentative conclusions could be reached regarding the precise mode of a-amylase attack on a few individual granules. It was found, however, that, in starch granules with a groove, the groove was generally the site of amylolytic attack. Complete penetration either occurred exclusively at this site or accompanied penetration at apparently randomly located sites on the major surfaces. Starch granules attacked by fungal glucoamylase showed a pattern of erosion which was different from that resulting from wheat a-amylase attack (Evers e t al., 1971). The glucoamylase pattern was characterized by a relatively uniform erosion of the surface. Shetty et al. (1974) devised a method to measure gelatinization of starch. The method involves selective digestion of gelatinized starch with either of two glucoamylases followed by determining the released glucose. SEM of partially digested large wheat starch granules revealed different morphological modes of attack by the two glucoamylases. During the first 30 minutes of digestion, both enzymes exposed equatorial grooves on many starch granules; the equatorial groove was not seen in the control starch granules. At longer digestion times, glucoamylase from Rhizopus niveus attacked principally the surface from the large granules, giving their exterior a spongy appearance which was occasionally pierced by a sharply defined, small cylindrical hole. On the other hand, digestion by an industrial-grade glucoamylase from Aspergillus niger (Takamine Diazyme) was characterized by deep and wide penetrations into the interior and creation of scoop-shaped depressions on the surface of the granules. In addition, tunnels beginning chiefly at the equatorial groove, and widening as they penetrated, were often observed. Such tunnels were absent in granules exposed to R . niveus glucoamylase. It is possible, however, that the tunneling and higher degree of digestion by Takamine Diazyme could be from a-amylase in this commercial enzyme preparation. Baked products and alimentary pastes are affected adversely if made from flour milled from sprouted wheat. Jones and Bean (1972) found that amylases from sprouted wheat attacked wheat starch granules preferentially along the equatorial groove of the granules. When the central part of the granule was penetrated, the starch in that region was digested out rapidly. It was suggested that damaged starch granules may easily break up to yield a large number of
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fragments that would already be primed with amylase. The degraded granules would ultimately yield a dough that is unsuitable for production of bread or noodles. Bean et ul. (1974) found that enzymes from a malted wheat flour extract entered wheat starch granules at certain sites, often preferring an equatorial groove, degrading some of the surface layer, and then following a path toward the interior of the granules. Some granules were attacked at many surface sites. The enzyme then apparently follows a path of least resistance toward the interior layers of the granules. This mode of attack had been postulated previously by Sandstedt (1955) using the light microscope. Gallant et uf. (1973) subjected starch granules from potato, wheat, manioc, and corn (regular, high-amylose, and waxy) to hydrolysis by pig pancreatic juice. The type of starch degradation varied with the starch species. In some starches, only exocorrosion occurred; in others, exocorrosion was accompanied by endocorrosion at perferential sites. In some cases, randomly distributed pits appeared. At maturity, wheat endosperm contains two types of starch granules: large lenticular granules (A-type) ranging in size from 15 to 40 pm, and small spherical granules (B-type) ranging from about 1 to 10 pm. The A-type develops first, and each A-type granule develops within its own amyloplast double membrane. The B-type granules develop later in evaginations of the membrane which surrounds the A-type granule. As the B-type granules develop under conditions of tight packing, many adopt the shape of the limited space in which they develop. Study of A-type starch granules in maturing wheat with SEM suggested a developmental sequence (Evers, 1971). It has been shown that the initial minute spherical granule becomes a “nucleus7’ which is progressively surrounded in the equatorial plane by a continually enlarging structure resembling two lips with a furrow between them. When the lips have completely surrounded the ‘‘nucleus,” there is a continuous increase in the thickness and diameter of the granule. This increase results in the furrow’s becoming progressively shallower until it is present in the mature biconvex structure only as a shallow ridge. Banks e t af. (1974) examined the complex granules of wrinkled-seeded pea starch and related their structure to changes in the starch during growth. Only small, simple types of starch granules were found at early stages of development in the wrinkled-seeded variety of pea. In the mature pea, a variety of simple and compound granules existed. The formation of the compound granules may be explained by a mechanism in which spherulitic crystallization is initiated simultaneously at several nuclei. It was postulated that, in pea starch, different types of granule are laid down at various stages of growth. Mahlberg (1973) compared by SEM the unique morphology of starch grains isolated from the latex of two species of Eupkorbiu. In E. terrucinu they were elongated and greater in diameter at the midregion than at the tips, while in E. timcalfi they were osteoid. Starch granules varied in size in both species,
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although in E. tirucaZZi the largest grains, which measured 49 pm, were approximately twice the length of those in E. terracina. The occurrence of morphologically variable starch grains in these two species indicated that grain morphology was species-specific and could be used for morphological and taxonomic studies. For some time a controversy has existed regarding the internal structure of the starch granule. Whereas some investigators presented evidence for the existence of a fibrillar structure, others concluded that their work supported the concept that starch granules were organized basically as isodiametric granules. Sterling (1971) studied Lintnerized potato starch with the SEM. All fracture faces showed a radial fibrillar system as the structural foundation of the starch grain. Ringlike areas, corresponding to tangentially fractured concentric lamellae of the grain, were present. The fibrillar system, centered at the middle of the rings, radiated outward from that center and traversed the ring without obvious interruption. The longest fibrillar ridge had a length of 14 pm. Hall and Sayre (1969, 1970a, 1971a) reported in a series of three publications the shapes, sizes, and surface details of many starches. The first report covered nine root and tuber starches (potato, arrowroot, sweet potato, canna, dasheen, two types of tapioca, name, yucca, and malanga). Cereal starches included barley, five types of corn, oats, rice, two types of sorghum grain, and wheat. The series concluded with starches from peas (wrinkled, smooth-shell, chick, and blackeyed), cow cockles, acorns, sagos, beans (tender white, pinto, and lima), shoti, dieffenbachia, pineapples, peanuts, tamarinds, and chestnuts. In most cases, both SEM and light microscopy were used. To determine the internal architecture of potato and canna starches, crushing and swelling studies were conducted (Hall and Sayre, 1970b, 1971b). When the granules were crushed in such a way as to obtain a uniform fracture of the starch, growth appeared to be principally by intussusception, proceeding along a hilum canal toward the periphery of the granules. Some evidence of a membrane encasing potato and canna starches was cited. The internal details of wrinkled-pea starch were also studied. In this starch, the observations pointed to a shell-type layering in which the growth was apparently by apposition. As the crushing could introduce some artifacts, the authors (Hall and Sayre, 1971b) examined the potato and canna starches after treatment with cold dimethylsulfoxide (DMSO) and 2 M calcium nitrate. DMSO dissolves some starches without swelling: potato starch swells considerably, and canna starch swells little in 2 M calcium nitrate. Additional evidence of a pie-layered granule in potato and canna starch was obtained from the swelling studies. Some evidence for the presence of a membrane around potato starch was obtained by SEM observations of abraded starch granules. In canna, one can observe directly a relatively thick apparent membrane. The interior of potato starch is more homogeneous than the interior of canna starch. It was suggested that the presence of a Mum canal was respmsible for cavitation during gelation of potato starch in hot water.
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French (1 973) reviewed relation of starch composition, structure, and physical properties to utilization of starch as a food or feed. Hill and Dronzek (1972) followed gelatinization of a commercial wheat starch and two types of corn starch (high-amylose and high-amylopectin) by SEM. Loss of birefringence of the wheat and corn starches was followed by light microscopy. Swelling and deformation of the starch granules observed in the SEM were correlated with loss of birefringence. The SEM pictures provided pertinent information on the gelatinization stage of the starches. Swelling of the wheat starch granules was first observed in the larger, A-type granules. The two corn starches had widely different gelatinization properties, indicating their differing compositions. Those differences were reflected in SEM observations. Maximum viscosity of a wheat starch suspension heated in an excess of water takes place after most of the granule swelling ceases (Miller et al., 1973). Studies by light microscopy and SEM showed that the increase in viscosity was due mainly to an exudate (seen as a filamentous network after freeze-drying a fully heated starch suspension) released from the starch granules. In the absence of the network no viscosity developed. Similar effects appeared during testing of corn, waxy maize, and potato starch. Direct microscopic observation of starch in limited water systems was used to develop a visual reference series showing structures typical of those formed in any baked, cooked, or processed food product (Derby et af., 1974). In such products, the swelling of starch is controlled not only by temperature but also by the amount of moisture available to the granules. The amount of moisture available is influenced by the formula or recipe; by the presence of materials such as proteins, pentosans, and sugars which compete with the starch for water; and by the degree of protection given starch by fat. The data suggested that the recognized value of pin-milling cake flour may be due to the release of starch granules from the protein matrix so that water can contact the starch, causing the structure to change. Oxidized starches typically have low viscosity and free flowing characteristics; these properties make attractive their use for the surface size application to paper and the warp sizing of textiles. The oxidized starches are insoluble in cold water and retain the granular structure and general appearance of unmodified starches under the ordinary light and polarizing microscope. Hall et af. (1971) found that, for corn starches treated with up to 6% chlorine, there was little change in the appearance of the granule surface. At the 8%treatment level, SEM micrographs indicated some fusion at the surface. The surfaces of the starch grains at the higher chlorine treatment were somewhat smoother than those at the lower treatments. Hall and Brown (1972) explained the loss in swelling and solubility of yellow dent corn starches oxidized by high chlorine levels on the basis of surface fusion or redeposition, both of which were observed by scanning electron microscopy. A parallel study was made with a series of waxy corn starches at three chlorine concentrations and at pH 5 to 6 and pH 9 to 10. In
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each case, SEM was used to study surface changes resulting from oxidative chlorine treatment. Gallant et al. (1972) studied the relation of the nature of the rupture in potato starch grains induced by ultrasound, to the ambient atmosphere, the concentration of the suspension, and the frequency and time of irradiation. It has been tentatively concluded that principally mechanical effects have been induced under conditions that would promote vaporous cavitation (280 kHz, 15 watts/cm2). In an atmosphere of hydrogen, many deep, conical pits were produced. In an atmosphere of air or oxygen, deep pitting was somewhat less pronounced than in hydrogen but injury to other parts of the surface was greater. Virtually no effect was produced in vacuo, and under carbon dioxide the effect was very weak. The extent of damage increased with time of radiation and decreased with increase in concentration of starch in the suspension. It was suggested that damage produced by ultrasound indicated a primarily radial structure of submicroscopic units in the starch grain. Hood et al. (1974) evaluated the ultrastructure of hydroxypropyl distarch phosphate-skim milk gels with SEM, TEM, and light microscopy. SEM of ungelatinized granules showed that chemical modification affected neither the surface nor the size of the granule. Two types of gelatinized granules were observed with the TEM. One had a homogeneous granular texture throughout, while the other had a granular coat and a dispersed, less dense core. It was suggested that the coat-core type of granule may result from chemical modification. Casein micelle subunits were evident. Micelles were not aggregated together, and there was no evidence of a continuous network among the micelles or the micelles and the starch granules. The modified gels were stored for up to 60 days at -3" to -32°C (Hood and Seifried, 1974). The effect of cyclic freezing and thawing and automatic defrost freezers on gel structure was evaluated by- electron microscopy. The coat-core type of granules disappeared after several freeze-thaw cycles. Starch granules were ruptured by freezing and thawing, and the nongranular starch was dispersed throughout the continuous phase. Casein micelles were distorted and the subunits were almost completely disaggregated after 60 days. Syneresis increased with time, but the amount of increase varied with the conditions of frozen storage. E. OILSEEDS Wolf (1970) prepared protein bodies from defatted soybean flour. The protein bodies contained spherical particles 1 to 3 pm in diameter, plus amorphous material. Full-fat and defatted soy flour contained particles 1 to 10 ,um in diameter. The results pointed to disruption during isolation of the larger protein bodies. This and a subsequent study (Wolf and Baker, 1972) suggested that
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soybean protein bodies are of two types: those 1 to 3 pm in diameter, which are stable to isolation by sucrose gradient centrifugation, and those of larger diameter, which are not stable. The second study was extended to cover structural details of the soybean seed coat and cotyledon surfaces and interior. Vix el al. (1971) used SEM to study the site and structure of pigment glands in cottonseed. Van Hofsten (1972) studied the ultrastructure of rapeseeds both by SEM and by TEM. Rapeseeds are covered by a heavy seed coat of a lacelike appearance, containing numerous holes. Most cells in the seed contain lipid droplets; storage proteins are concentrated in aleurone grains. The aleurone grains contain varying numbers of globoids and are the site of toxic compounds. Whereas rapeseed feed meal containing 40% protein had a complex morphology, detoxified rapeseed flour with 70% protein had a homogeneous appearance.
F. MILK PRODUCTS Several investigators have studied by TEM the internal structure of spray-dried milk particles. However, the use of water in preparation of the samples may modify the structure of the particles and introduce artifacts. Saito (1973) presented electron micrographs of surface replica of casein micelles and scanning electron micrographs of frozen materials (whole milk and dispersion of casein micelles). Casein micelles were fractionated to three size groups by centrifugation (10, 47, 107 X lo3 g, 60 minutes, at 0" to 1"C), and some of their properties were compared. Compositional differences among them were slight. The content of P-casein, however, decreased slightly in entire micelles and definitely in the portion solubilized with 1 M NaC1, with decreasing micelle size. The solubilized portion of whole casein micelles showed a higher content of 6-casein and less precipitability at the pH range of 3 to 5. The temperature-dependent accumulation of 0-casein on casein micelles was discussed. Buma and Heustra (1971) found by SEM that fractured particles of whole dried milk had a rough cleavage structure, whereas dried slum milk particles had a smoother cleavage structure. Deep surface folds, presumably formed by casein, were observed. It was suggested (Buma, 1971) that the pores or cracks in whole milk powder might be related to the free-fat content of such powders. A high correlation (r = 0.94) was established between the free-fat content and the particle porosity of whole milk powders. The author concluded that particle porosity, amount of surface fat, and arrangement of the fat globules in the particles govern the extraction of milk fat from whole milk powders. Photomicrographs with the SEM showed cracks and pores in particles with high porosity; in less porous powder particles only surface folds and occasional cracks could be observed. Broken or cut particles showed vacuoles but no capillaries connecting the capillaries with the exterior of the particle. SEM photomicro-
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graphs, in combination with other data, were used to explain why powders from unhomogenized concentrated milk have a higher particle porosity than those from homogenized concentrated milk, and why smaller particles have a higher porosity than the larger ones from the same powder (Buma, 1971). Hall and Creamer (1972) have studied the submicroscopic structures of cheeses. The cheeses were etched with trypsin to remove surface proteins, freeze-dried, and examined. Fat globule distribution shown by SEM was even in Cheshire cheese, layered (onionskin) in Gouda, and nonuniform with some rupturing and aggregation in cheddar cheese. G. MISCELLANEOUS FOODS Schaller and Powrie (1971) used SEM to study the ultrastructure of skeletal muscle from rainbow trout, turkey, and beef immediately after death of the animals (prerigor) and at various postrigor intervals. Prior to fixation, the muscle was cryofractured. Samples of prerigor muscle showed intact fibrils with elevated, equidistant, transverse elements. These elements were presumed to overlay the Z discs. The surface of each sarcolemma in prerigor muscle had a criss-cross pattern of strands; those strands were about the same size as the transverse elements. On the surfaces of fibrils in all muscles were noted, between transverse elements, longitudinal strands and transverse central elevated bands (presumably sarcoplasmic reticulum). The transverse elements decreased in size and the sarcolemmae became perforated in trout and turkey muscles stored at 3°C for 6 days. Collapsed transverse elements were noted in commercially aged longissimus dorsi. In postrigor muscle, breaks across the fibrils were generally at the sites of the transverse elements. The effects of heating postrigor skeletal muscle from beef, chicken, and rainbow trout were also examined by SEM (Schaller and Powrie, 1972). With muscles heated to 97"C, the structural integrity of myofibrils was lost near, or at, the transverse elements. In addition, fibrils from trout muscle had transverse fissures at the level of the H zone. Granular matter was observed beneath the sarcolemma of fibers in heated beef and chicken muscles. When the sarcoplasm was removed from intact beef muscle, no granular matter was noted in the heated tissue. Heated (97"C)'beef sarcoplasm had a granular appearance; the diameters of the granules were similar to those in heated intact beef muscle. Endomysial fibers were not detected in the muscles heated to 97"C, but in the tissue heated to 60°C swollen fibers were apparent. Changes in muscles from beef and chicken heated to 60°C were small, although excessive damage was apparent in muscle from rainbow trout. Gotte et al. (1972) examined by SEM samples of bovine ligamentum nuchae elastin before and after purification and found that the elastic substance was composed of fibers about 6 pm in diameter and that the elastic substance was embedded in an amorphous matrix. The fibers were oriented chiefly along the
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main axis of the ligamentum and were often interwoven, branched, or twisted. In many cases smaller fibrillar components were observed. Each single fiber could be stretched up to three times its original length before failure, whereas the maximum elongation of macroscopic samples was less than twice the original length. It was concluded that elastin is not a homogeneous system at different levels of organization. The surface structure of porcine psoas muscle varying in degree of contraction was examined by SEM and compared with subjective and objective textural evaluations (Stanley and Geissinger, 1972). Examination by SEM distinguished and measured the major A and I bands of the sarcomere; the A band was raised and the I band was depressed. As the sarcomeres contracted below the length of the A bands, a progressive bulging of the sarcomeres was noted. The bulging was attributed to a buildup of thick filaments to the point where excess A band substance, unable to penetrate the Z discs, pushed outward in the area of the M band. Severely contracted muscle fibers had an irregular wavy appearance with a repeat pattern of 20 to 40 gm. Sarcomeres became more difficult to shear, break, or chew as they progressively contracted. SEM studies of Eino and Stanley (1973a, b) indicated that cathepsins are present in muscle and become active about the time when certain textural changes occur. Catheptic enzymes also may produce structural surface alternations which resemble changes in aging at the same pH and temperature conditions. Lamvik et al. (1973) studied the structures of rabbit tropomyosin-Mg-tactoids and isolated myofibrils by the STEM. Although the instrument can produce high-contrast images of unstained and unfvred biological materials, it was not clear which preparation methods might best maintain the native structure of the material. The fine banding pattern of tropomyosin was visualized in both conventional and scanning microscopes. Thin sections of deep-frozen unfvred muscle were studied in a STEM equipped with a cytostage for vacuum compatibility of hydrated tissue (Bacaner et al., 1973). With an energy-dispersive x-ray analysis system, intracellular atomic species in the scan beam path were identified by their fluorescent x-rays and spatially localized in correlation with the electron optical image of the microstructure. Marked differences were noted between the ultrastructure of deepfrozen hydrated muscle and that of fixed dehydrated muscle. In frozen muscle, myofibrils appeared to be composed of previously undescribed longitudinal structures 400 to 10,000A wide (macromyofilarnents). The usual myofilaments, mitochondria, and sarcoplasmic reticulum were not seen unless the tissue was fixed before examination. Fluorescent x-ray analyses of the spatial location constituent elements identified all elements heavier than sodium. Intracellular chlorine was relatively heavier than expected. The retention of organic volatiles in freeze-dried foods is important in ensuring a high-quality flavor. Samples of maltodextrin which had been freeze-dried from solutions containing organic volatiles were examined by SEM (Flink and Gejl-
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Hansen, 1972). In many cases the dry maltodextrin contained entrapped droplets of the liquid volatile. The structure of the freeze-dried maltodextrin cake was shown (by light microscopy and by SEM) to consist of numerous intersecting plates which had the retained volatile compounds within the plate. Droplets of similar appearance were observed within the solids of freeze-dried coffee. Subsequently, Flink et al. (1973) reported on microscopic investigations of the freezing-drying of flavor-containing model food solutions. Hodge et el. (1972) studied by chromatographic and microscopic methods crystals of maltose isolated from starch hydrolyzates which contained at least 90% maltose. SEM was useful in following properties of crystals grown in various media and in studying changes that take place during dehydration of &maltose H20 crystals. Mustakas (1974) isolated a lipid-protein concentrate, in 94% protein yield, from soybeans. The raw material (full-fat soy flour), the intermediate (freezedried acid curd), and a beverage base (spray-dried lipid-protein concentrate) were examined by SEM. The acid-precipitated curd produced a thin weblike structure, whereas the spray-dried beverage base particles assumed a rounded, irregular spherical shape and were indented as though the spheres collapsed during spray-drying. If colloid milling and homogenizing were omitted, the particles were more agglomerated; this resulted in more solid separation in the clarification step, poor mouth-feel, and some sedimentation. The colloid mill and homogenizer could not be eliminated, since they are important for achieving good dispersion of the added fat. The structure of shells and membranes of the hen’s egg was studied by SEM by Simmons and Wiertz (1970). King and Robinson (1972) made a SEM study of the radial fracture surfaces of strong and weak eggshells. Generally, in weak and thin eggshells the mammilae were of irregular shape, very porous, frequently not attached to the outer shell membrane fibers, and cleaved both radially and transversally during fracture. Mayer et al. (1973) studied the suitability of eggshell as a calcium source in layer diets by comparing pulverized and crushed sterilized eggshells against oyster shell, limestone grit, and pulverized limestone, as calcium supplements. Breaking strength of the eggs, determined at 90 and 150 days of feeding, showed that eggs from the hens fed the diets containing oyster shell, pulverized eggshell, and limestone grit were significantly stronger than eggs from hens fed diets containing pulverized limestone as the calcium source. Crushed eggshells as a dietary source of supplemental calcium promoted eggshell quality equivalent to that obtained with pulverized limestone. Scanning electron micrographs revealed that most of the changes responsible for differences in shell breaking strength apparently take place in the palisade layer, while the mammillary layer remains approximately constant. It appeared
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that the palisade layer contributes most of the strength in the shell. As the palisade layer decreased in thickness and in concentration, there was a closely predictable decrease in breaking strength of the eggs. Evidence was obtained indicating a poor correlation between breaking strength and total shell thickness. Breaking strength is a superior measure of eggshell quality because it takes into consideration both the thickness and the concentration of the palisade layer around the crystalline mammillary portion of the eggshell. Hasiak et al. (1972) followed by SEM and TEM structural changes induced in egg yolk by chemical and physical treatments. SEM indicated that the surface structure of the concentrated and frozen yolk samples was much more “open” than that of normal yolk. TEM showed changes in both low-density (LDF) and high-density (HDF) fractions. The background continuous phase (LDF) showed more irregularity, and the electron-dense particles (HDF) more changes in shape and organization in the concentrated and frozen than in the control samples. The photomicrographs suggested that the freezing process induced aggregation of the low- and high-density yolk lipoproteins. The aggregation presumably resulted in the formation of the type of three-dimensional structure which entraps large quantities of water and could result in increased yolk viscosity. Murray et al. (1972) have shown by electron probe analysis that potassium increased in the fruit apex of tomatoes ten times and decreased in the fruit base two times in blossom-end rot fruits. In diseased fruits, unlike in healthy ones, potassium was independent of magnesium and calcium, and magnesium showed inverse relations. Rockland and Jones (1973) used SEM to study effects of cooking on rehydrated large lima bean cotyledons. The scanning electron microscope was used to make a photographic comparison between the cellular characteristics of raw, partially cooked, and completely cooked, water-soaked as well as quick-cooking beans (Rockland and Jones, 1973). The cookmg process involved gelatinization of starch granules contained within integral cell units and concurrent dispersion of intercellular components of the middle lamella which facilitated separation of intact cells without rupture of cell walls. Mechanical stresses due to starch gelatinization, protein denaturation, swelling, and heat convection may have promoted cell separation. Except for differences in the rates at which these processes progressed, there were n o conspicuous differences between the structural characteristics of the watersoaked beans and the quick-cooking beans. Behnke et al. (1973) examined by SEM urea-containing protein feed supplements. Of eleven urea-containing extrusion processed protein supplements (corn, sorghum, barley) tested, five were toxic to cattle when administered at 50 g of urea per 100 kg of body weight via rumen cannula. All but one toxic composition was highly starch-damaged, contained urea in crystal form, and produced blood ammonia concentrations >0.8 mg of ammonia nitrogen per 100 ml of
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blood. All nontoxic components were also highly starch-damaged, but contained urea in a pocketed, amorphous, noncrystalline form. Thus, production of amorphous urea structures and extensive starch damage using moisture, temperature, and pressure in the extrusion-cooking process is essential in the manufacture of high-quality urea-containing protein supplements.
H. SANITATION Simmonds et al. (1970) used optical microscopy and SEM to determine the nature of the dust that arises during the handling of wheat and to determine means for its reduction or elimination. The fibrous nature of the pericarp suggested that surface abrasion during handling could explain the origin of some dust particles that arise when cereal grains are moved. In addition, there is the brush end of the kernel with bristles which shear off readily. The bristles provide particles that can be expected to have highly irritant properties when inhaled. Formation of dust particles depended on variety, seasonal harvest conditions, and commercial practices in grain handling. Benner (1971) examined by SEM air filters in a commercial brewery before and after coalescence of liquids and deposition of bacteria and dust particles. On the basis of the photomicrographs, he suggested means for increasing purifying efficiency of compressed air.
IX. SOME REFLECTIONS ON SCANNING ELECTRON MICROSCOPY The objective of this review has been to outline the principles and features of a relatively new instrument, the scanning electron microscope, which plays an increasingly important role in many areas of scientific research and technology. I hope this review has demonstrated that SEM can be a valuable instrument for the food scientist and technologist. The list of food researchers who cannot use the instrument in their work would seem much shorter than the list of those who can put it to good use. In most fields of biology, physics, and technology “the list of applications seems boundless, the results staggering, and the micrographs astounding” (Black, 1970). The excitement and enthusiasm of users of SEM results to a large extent, from the fact that SEM adds a new dimension-both literally and figuratively-to the capabilities of the available light and electron microscopes. As Everhart and Hayes (1972) stated, each type of microscope supplies different types of information, each type complements the others, and each type unravels more mysteries about the microstructure of biological systems. It is hoped that SEM will contribute as much to the expertise of the food researcher and technologist as it contributes to his pleasure and esthetic satisfaction.
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EMULSION STAB1 LlTY AND ITS RELATION TO FOODS BY GARY E.PETROWSKI Carnation Research Laboratories Van Nuys. California
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Emulsions A Introduction ................................................. B Terminology ................................................. C Types D Stability .................................................... I1 Emulsifiers ..................................................... A Theory B. Types ...................................................... C Emulsifier Characterization I11 Fatsandoils A Introduction B Characterization .............................................. IV Other Emulsion Factors .......................................... A Emulsifier Mixtures B ApplicationsofHLB C Limitations of HLB ............................................ D Emulsifier Concentration ....................................... E Effect of Emulsifier Structure .................................... F EmulsionType G Proteins H. Solids I Hydrocolloids ................................................ J Aerated Emulsions K Emulsifiers and the FDA L Emulsion Preparation M Emulsifier Identification ; V Directions References
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I.
EMULSIONS
A. INTRODUCTION Emulsions, and in particular food emulsions, have long been known to man. Although the natural occurrence of fat and water together in foods indicated the immiscibility of these two phases, Nature's chance mixing of these unlike materials was accepted. While progress was being made in unraveling the mysterious forces of emulsions in nonfood areas, understanding of food emulsions was lacking. Improvement in the stability of milk to creaming by homogenization did not come into general use until the late 1930's. Although the use. of emulsifiers greatly improved the stability of nonfood emulsions, research in the food area did not develop as rapidly, owing to the necessarily restricted use of emulsifiers. This review will outline the theories of emulsion stability, and the role of emulsifiers and their relation to the food industry, with references to current original literature when possible. B. TERMINOLOGY An emulsion can be viewed most simply as small droplets of one immiscible liquid dispersed in another liquid. The presence of a third component residing at the interface between the droplet and medium also must be considered in explaining the stability of such a system. For an understanding of emulsions, some terminology must be introduced. The suspended droplets are referred to as the dispersed or internal phase; the medium in which they are suspended is the external or continuous phase. The interfacial components are emulsifiers or surface-active agents (Mittal, 1971).
C. TYPES 1. Discussion
Most emulsions involve water and oil or fat as the two immiscible phases. Two types of emulsions are possible, depending on the composition of the phases. When water is the external phase and oil the internal phase, the emulsion is referred to as an oil-in-water emulsion, or O/W (Fig. 1). Common examples are milk and mayonnaise. This type is by far the most common, and the majority of the discussion will deal with this type. Conversely, when oil is the external or continuous phase and water the internal phase, a water-in-oil or W/O emulsion exists, such as butter or margarine. More complex multiple systems present in some food systems such as bakery products will be covered later (Section IV,J).
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WATER- IN-OIL (W/O)
OIL-IN-WATER (O/W)
FIG. 1. Emulsion types.
2. Testing The type of emulsion can be determined in several ways, all based on the fact that the emulsion will exhibit the properties more of the external phase than of the internal phase. A visual observation may provide initial clues. Oil-in-water emulsions are generally white and creamy, while W/O emulsions are darker and exhibit a greasy, oily texture. If the emulsion is of the O/W type, addition of a drop of the emulsion into water will result in dilution, with complete mixing into the water; if it is a W/O type, the drop will remain unchanged, usually floating on the surface, similar t o an oil droplet. Use of a water- or oil-soluble dye will differentiate similarly: Oil-soluble dyes stain W/O emulsions and not ON. Water-soluble dyes stain O/W emulsions and not W/O emulsions. These observations may be made with the naked eye or with a microscope. Filter paper can differentiate on the basis of capillarity. An O/W emulsion drop will spread on the paper, while W/O emulsions will not. Pretreatment of the paper with cobalt chloride makes the effect more visible. Finally, measurement of electrical resistance or conductivity can also be helpful: O/W emulsions exhibit lower resistance and higher conductivity than do W/O emulsions (Roehl, 1972a). D. STABILITY
1. Stabilization The balance between the attractive and repulsive forces of particles is the basis for theories of emulsion stability (Deryagin and Landau, 1941; Verwey and Overbeek, 1948; giving rise to the DLVO theory). The repulsive forces, electrostatic in nature, are stabilizing, since they tend to keep the droplets separated (attractive forces, on the other hand, would be destabilizing). However, once
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GARY E. PETROWSKI
aggregates of droplets have formed, other physical or mechanical properties prevent the next stage of destabilization-the merging of droplets, or coalescence. The latter phenomenon will be discussed in the section on emulsifiers. Electrical charges on droplets in emulsions can arise by ionization, absorption, or frictional contact (Alexander and Johnson, 1949). In the absence of emulsifiers, only frictional contact appears capable of charging droplets, if at all. An extension of a rule regarding solid dielectrics provides a possible explanation: A substance having a high dielectric constant is positively charged when in contact with another substance having a lower dielectric constant (Coehn, 1898). Since water has a higher dielectric constant than oil, it would be positively charged and the oil negatively charged. Like charges on the droplet surface would cause mutual repulsion between droplets, and thus a measure of stability would exist. Coalescence of two droplets of necessity involves contact between droplets. When this effect occurs with just oil and water, in the absence of emulsifiers, the stabilization is small. The presence of an electrical double layer around fhe droplet was proposed by Helmholtz (1879), who reasoned that, if ions of one charge lined the droplet surface, another oppositely charged layer would surround it, as in the two plates of a capacitor. The existence of such a sharp potential drop was questioned by Gouy (1909), who argued that the double layer was more diffuse, extending some distance into the medium but dropping off exponentially. Stern (1924) combined the two theories, providing for a single, immobile counter-ion layer at the interface, called the Stern layer, surrounded by a diffuse (Guoy) layer of mobile ions extending outward. Experimental investigations using electrophoretic mobility, sedimentation potential, etc., led to the quantitative determination of the experimentally obtainable term, the zeta potential, 5. (Although the value tells nothing about the structure of the double layer, the magnitude and variation of 5 must relate to the double layer.) Further discussion is beyond the scope of this article, but for details the reader is referred to Becher (1965), Garrett (1965), Lange (1965), Ottewill (1967), Riddick (1970), Robinson (1968), Sennett and Olivier (1965), and Woods and Burrill (1972). The generalization to be made is that the electrical charge surrounding the droplets provides a degree of stability by the repulsive interaction of droplets possessing like charge.
2. Destabilization a. Derivation. As was mentioned previously, emulsion stability depends on a balance between attractive and repulsive forces, which can be written
EMULSION STABILITY
313
where V i s the total potential energy of interaction, and V, and V A are the respective potential energies of repulsion (positive) and attraction (negative). It follows that, for maximum emulsion stability, V , should be maximized and VA minimized, resulting in a maximum value of Before discussion of V,, the factors affecting V, referred to in the previous section should be mentioned. The addition of inorganic electrolytes decreases the size and intensity of the droplets’ double layer. This would result in a decrease in V , and a net decrease in emulsion stability. The main source of attraction between droplets, V A , arises as a consequence of van der Waals forces or London dispersion forces. Although short range with respect to individual atoms, when summed up over all the atoms in the droplet the forces are long range. In its simplest form
v
VA = -Aa/6H0
(2)
where A is the Hamaker constant, a is droplet size, and Ifo is the distance between particle surfaces. Thus, the larger the droplet size, the greater the attractive force will be, and the less stable the emulsion. A plot of the potential energies may be helpful (Fig. 2). The shape of the curve in Fig. 2B will depend on the net difference of the forces of attraction and repulsion and will vary with droplet size and ionic strength of the medium. At high ionic strength, the curve approaches the attraction curve of Fig. 2A with little or no repulsion contribution. The larger B
A
+ II
.I< I
fI N C R E A S I N G H,
L 10 20
H,
100 200
fxlO*
CM)
FIG. 2. Potential energy (PE) plots versus droplet separation. ( A ) VR is PE of repulsion, VA is PE of attraction, and H, is distance between droplets. ( B )P is primary minimum, M is stability maximum, and S is secondary minimum.
GARY E. PETROWSKI
3 14
A
////I \t . .
F
E
FIG. 3. Emulsion destabilization scheme. Reprinted with permission from J. Amer. Oil Chem. SOC.51,110 (1974).
the particles, the greater the attractive forces will be, and the lower the stability maximum, M. When the value of M decreases to -5kT, the energy of Brownian motion is sufficient to overcome this stability barrier. An irreversible aggregation occurs at very small particle separations (P)owing to the reaching of the primary energy minimum. The depth of the secondary energy minimum, S, is affected by droplet size; that is, the larger the size, the deeper is the minimum and the greater the probability of reversible loosely associated aggregation (internal droplet distance may be as great as 200 A). b. Dpes. With this cursory presentation of factors affecting emulsion stability, attention may be directed toward its macroscopic physical manifestations. Destabilization involves a change from the original suspension of small droplets of one liquid to another. Several discrete steps are involved: Reversible aggregation or flocculation is the initial step, concurrent with or followed by creaming, and finally coalescence, as shown in Fig. 3. Flocculation or aggregation means literally a coming together. Small droplets are attracted to each other and form groups or clusters, with the individual droplets retaining their original shape. This process was just discussed.
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The phenomenon of creaming also brings droplets closer together, owing t o density differences. Since an oil-in-water emulsion is usually involved, oil droplets in clusters or individually tend to rise to the surface of the emulsion, since they are usually less dense than water. Because the droplets still retain their size and shape, the process is reversible, with only gentle stirring necessary to restore the original emulsion. The rate of destabilization by creaming is governed by Stokes’ law:
where V is the rate of rise or fall of the droplets, r is the radius of the droplets, S1 is the specific gravity of the aqueous phase, S2 is the specific gravity of the oil phase, g is the force of gravity, and 77 is the viscosity.
The final step in the destabilizing process-coalescence, or breaking of the emulsion-involves the merging of the droplets to form larger droplets and ultimately the complete destabilization of the emulsion (that is, the formation of two separate layers). Methods have been developed to accelerate the destabilization process for the purpose of comparing the stability of different, fairly stable emulsions. The determination of emulsion stability and all its ramifications can be carried out experimentally in many ways. After preparation, the condition of the emulsion can be observed visually and changes that occur with time noted. In a food product that contains an emulsion, a shelf-life expectancy of one to two years is not unreasonable. Storage of a new product for that length of time, followed by visual examination of the product to determine acceptability, would be the most foolproof. Seldom, however, is one afforded the luxury of such time limits in the development of new products, so other methods must be used. If the rate of destabilization under ordinary conditions is not sufficiently rapid, other methods may accelerate it. Caution must be used that the acceleration does not alter the mechanism of the destabilization (Sherman, 1971). By heating the emulsion, a more rapid destabilization can be affected. With reference to Stokes’ equation, V is increased probably by the decrease of q and the higher kinetic energy of the droplets resulting in more droplet collisions (more rapid Brownian movement). This method need not be confined to high isothermal temperatures. Cycling from ambient to high temperature as often as is necessary to achieve the desired effect or to simulate expected conditions may also be carried out. Another temperature variation involves freeze-thaw cycles, a very strong stress (Singleton et al., 1960). Centrifugation either by itself or in concert with one of the methods just described provides another stress on the emulsion (Vold and Mittal, 1972, 1973; Wachs and Reusche, 1960).
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GARY E. PETROWSKI
3. Determination Having described methods t o enhance emulsion destabilization to facilitate stability determination, we may now make the actual determination. Most simply, a visual observation may be all that is necessary to determine whether a particular emulsion is acceptably stable or not. Perhaps aggregation can be tolerated, but not coalescence. Perhaps slight coalescence is acceptable. Measurement of the amount of coalesced oil present before or after the various stress techniques described previously provides a quantitative value for comparing the stabilities of many emulsions. A method that measures the tendency of the fat droplets to rise to the surface of a partially destabilized emulsion as in C or D in Fig. 3 via microwave irradiation has recently appeared (Petrowski, 1974). Other methods determining essentially the same property-namely, the dielectric constant of the emulsionhave also been reported (Cyganska, 1968; Kaye and Seager, 1966; Mudgett et al., 1974; Noguchi and Maeda, 1973). Frequently the future stability of an emulsion can be determined by an examination of the particle size range of the emulsion produced, since droplet size affects the rate of aggregation and creaming. The droplet size range for normal emulsions will fall in the broad range 0.1 to 50 M. (With smaller droplets, below 0.1 p, the solutions are transparent and are termed microemulsions.) Determination of droplet size for such small sizes is difficult. The most direct method would be optical microscopy in which the particles are sized while viewed through a microscope equipped with a calibrated ocular grid. This is obviously tedious. Particle counters are available which, although not inexpensive, provide meaningful data. For a constant weight of internal phase, the more particles there are, the smaller they will be (Groves and Freshwater, 1968). Viscosity and reflectance are physical properties more easily measured which indirectly provide information regarding relative particle size. When the average droplet is smaller, the viscosity will be higher, and the reflectance greater. Roehl (1972a) observed an increase in electrical resistance when O/W emulsions destabilized, caused possibly by the increased suspended aggregates of the oil phase.
4. Improvement Up to this point, the methods used to improve emulsion stability are apparent. From the attractive energy equation for aggregation and the Stokes equation for creaming, we find that the critical factor is droplet size. Whatever can be done to decrease droplet size (and maintain it) will increase stability. The method chosen
EMULSION STABILITY
317
for emulsification will determine the initial particle size and thus the ultimate stability. If a homogenizer is used, higher pressures tend to result in smaller droplet size. Use of a blender or colloid mill should be investigated to determine the best operational parameters for minimum droplet size (Gopal, 1968). Differences in density between the oil and water phases which affect the rate of creaming generally cannot be changed; however, one interesting example is found in the beverage industry. Cloud agents have been used to impart an opalescence found desirable in colorless soft drinks. The cloud agent, usually flavoring oil, should ideally remain suspended in the beverage, neither sinking t o the bottom to form a sediment nor rising to the surface to form a ring over an expected shelf life of one to two years. Such stringent requirements were met until recently by the use of brominated vegetable oil (BVO). When BVO was mixed with the flavoring oils, the resultant mixture possessed a density similar to that of the aqueous medium; thus, there was little tendency toward rising or settling. After the Food and Drug Administration severely restricted the use of BVO, a similar increase in the specific gravity of the oil was brought about through the use of an approved additive, glycerol abietate, and through improved emulsion systems (Kesterson and Hendrickson, 1969; Oppenheimer, 1971; Simon, 1967). Another factor affecting stability is viscosity. With higher viscosity, Brownian motion is decreased, and the viscosity term in Stokes’ equation leads to a lower rate of creaming. Practically speaking, viscosity is increased most frequently, with increased emulsion stability, by the addition of gums and other stabilizers. More will be said about these later. As was already mentioned, the smaller the droplet, the greater will be the viscosity (Richardson, 1950). The purely random Brownian motion of the droplets in the medium is affected by temperature directly (the lower the temperature, the slower the motion and the less likely a collision between droplets) and indirectly (the lower the temperature, the higher the viscosity and the greater the resistance to particle collisions). In fact, the effect of temperature deserves further mention. In typical food products, ranging from salad dressing to ice cream, the formulating is carried out hot for better mixing, melting of fats and emulsifiers, etc. Normal processing requires heat to produce a commercially sterile product, and the heating benefits both sterilization and emulsification. However, once the emulsion has formed, heat becomes detrimental to the emulsion and should not be maintained any longer than necessary. The ionic strength of the aqueous medium is an important factor determining the repulsive energy between droplets. The lower the concentration of inorganic electrolytes, the higher will be the repulsive energy and the more stable the emulsion toward aggregation. This property often leads to compromises between flavor and stability.
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GARY E. PETROWSKI
O/W
EMULSION
W/O EMULSION
FIG. 4. Stabilized emulsion droplets.
II. EMULSIFIERS A. THEORY
In the search to further improve the stability of emulsions, it was discovered that the addition of certain compounds-for example, emulsifiers-greatly improved emulsion stability. Further investigation revealed that they were concentrated at the oil-water interface or generally on the oil droplet surface in O/W emulsions-hence the name surface-active agent or surfactant. The lowering of interfacial tension at the droplet surface was recognized quite early as a factor that increased emulsion stability (Donnan and Potts, 1910; Ford and Furmidge, 1966; Rosen, 1974). The effect, however, is not directly related simply to oil droplet coalescence rates (Ishida et al., 1968). Further stabilization comes as a result of a monomolecular film existing at the interface. Such a stabilization can be visualized as an oriented wedge (Harkins and Beeman, 1929). Cations or hydrophilic portions were drawn as circles and the hydrophobic portion was seen as a jagged line (Fig. 4). B. TYPES For emulsifiers to concentrate at the oil-water interface, they should be neither extremely water nor oil-soluble. Such solubility requirements are possible if the molecule contains both a polar and a nonpolar portion. Emulsifiers have been classified by Schwartz and Perry (1949). For purposes of this discussion, several types will be given, with examples of each. Anionic: (based on hydrophilic group) salts of long-chain fatty acids (soaps) Cationic: amine salts such as cetyl trimethyl ammonium bromide Nonionic: monoglycerides, polyoxyethylene adducts with alcohols, glycols, phenols, acids, sugars, etc. The hydroxyl groups and polyoxyethylene chain are the polar or hydrophilic
EMULSION STABILITY
319
portion, and the fatty acid chain and carbon skeleton are the nonpolar lipophilic portion of the nonionic emulsifiers. Because of toxicity considerations, nonionic emulsifiers have been of most interest in the food industry, and attention will be focused on them. Of special interest are the naturally occurring emulsifiers such as lecithin, mono- and diglycerides, lanolin, cholesterol, and natural gums. From the time of the discovery of emulsifiers to the present, the number of these surface-active agents available has increased logarithmically. Thus, today one is faced with the bewildering task of choosing the emulsifier or mixture of emulsifiers best suited for a particular application. C. EMULSIFIER CHARACTERIZATION
1. Hydrophile-Lipophile Balance The choice of emulsifier should be based on the evaluation of some characteristic of the emulsifier, and, on this basis, many exist. The idea that an emulsifier molecule should contain a balance between polar and nonpolar moieties was pointed out by Clayton (1943). Differences in soiubility provided an initial differentiation among the relative polarities of emulsifiers. The more polar emulsifiers tended to be more soluble in water and promoted the formation of oil-in-water emulsions. Less polar emulsifiers tended to be oil-soluble and promoted the formation of water-in-oil emulsions. This approach was further delineated in quantitative terms by Griffin (1949, 1954) with the introduction of the concept of the hydrophile-lipophile balance (HLB). Essentially, by a long experimental procedure, he was able to assign values to emulsifiers ranging from lipophilic to hydrophilic using a scale of 1 to 20, arbitrarily assigned to oleic acid TABLE I EMULSIFIER HLB RANGE AND APPLICATION‘
HLB range
Applications
4-6 7- 9 8-18 13-15 15-18
Water-in-oil emulsifier Wetting agent Oil-in-water emulsifier Detergent Solubilizer
“Reprinted with permission from J. Soe. Cosmet. Chem. 1, 311-326 (1949).
GARY E. PETROWSKI
320
TABLE I1 EMULSIFIER HLB RANGE AND SOLUBILITY‘
HLB range
Water solubility
1- 4 3- 6 6- 8 8-10 10-1 3 13-
No dispersion Poor dispersion Milky dispersion (vigorous agitation) Stable milky dispersion Translucent to clear dispersion Clear solution
Dig. Fed. ‘Reprinted with permission from Off: Paint Vam. Prod. Clubs 28,446 (1956).
and potassium oleate, respectively. Suitable applications were assigned to the ranges as shown in Table I. A theoretical basis for this empirical approach has been proposed by Beerbower and Hill (1971). It remained for several other workers to observe correlations between the HLB values of emulsifiers and various other physical properties and parameters. Griffin (1956) observed the relationship between water solubility and emulsifier HLB shown in Table 11. When the structure of a polysorbate type of emulsifier molecule is known, the HLB value can be calculated from the relation HLB = (E +p)/5
(4)
where E is the weight percentage of oxyethylene content, and P is the weight percentage of polyhydric alcohol content. This method works well for the series of sorbitan and polyoxyethylene sorbitan esters but suffers from its limitations-not applicable are noionic surfactants containing other hydrophilic oxide units, sulfur or nitrogen-containing surfact ants, or inonic surfactants. By examining the HLB values for a large number of emulsifiers, Davies (1957) was able to derive group values for the structural moieties of the molecule in the following equation HLB = 7 + B(hydrophilic group numbers) - s(lipophilic group numbers)
(5)
The last term is usually 0.475n, where n is the number of methylene groups in the lipophilic chain. Although good agreement with observed values for the sorbitan and polyoxyethylene-sorbitan esters was found, less satisfactory results were obtained with other emulsifiers.
EMULSION STABILITY
321
Griffin (1949, 1954) and Tashika et ~ l(1956) . also observed that HLB = 20(1 S / d )
(6)
where S is the saponification number of the ester, and A is the acid number of the acid. This method appears limited by its inability to give good values for S (Neuwald, 1964). HLB values for a series of poly oxyethylene polyoxypropylene block copolymers have been obtained experimentally. The values calculated by regression analysis based on the molecular weights of the two portions compared favorably with the experimentally obtained values (Lowenthal, 1968). By the use of thin-layer chromatography, HLB values of emulsifiers were correlated with Rf values (Hayano and Asahara, 1969; Nakagawa and Nakata, 1956). As another approach, Harva et QZ. (1959) used the emulsifier as the liquid phase on a gas chromatograph (GC). A linear relationship was obtained between the partition coefficients (water and diisobutylene) and the HLB numbers of the emulsifier used as the liquid substrate on the column packing. In an effort to further simplify the GC procedure, Becher and Birkmeier (1964) injected a hexane-methanol mixture onto columns coated with various emulsifiers. A linear relationship was obtained when HLB was plotted against p, defined as the ratio of the retention time of ethanol to the retention time for hexane. Scatter of as much as 3 HLB units was observed in the middle (8 to 12) of the HLB range. Recently, Titus and Mickle (1971) measured emulsifier HLB by a similar GC technique, injecting isoamyl alcohol on columns containing the various emulsifiers. A linear relation was again obtained by plotting retention time of the alcohol aginst known HLB values. In another HLB determination by GC, Petrowski and VanAtta (1973) obtained a linear relationship between the In p’ value for emulsifiers and published HLB values. In this case, refined values for the retention time ratios (p’) brought out the logarithmic nature of the relationship. A similar logarithmic relationship has been observed in correlating the dielectric constant of the emulsifier with the published HLB already mentioned (Gorman and Hall, 1963). The generally wide acceptance of HLB as a means of characterizing emulsifiers has resulted in the publication of many reviews in Enghsh (Griffin, 1955; Hackett, 1969; Hellsten, 1965; Morns, 1965; Riegelman, 1962; Rimlinger, 1969; Thalheimer and Rusch, 1970) and in foreign languages also (Angla, 1965; Bergwein, 1967; Hedrusiak, 1969; Kassem, 1963; Okada, 1960; Paquot, 1967; Racz, 1963; Rahm et d., 1969a,b; Rozhdestvenskii and Pugacheva, 1965; Sirovica, 1963).
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GARY E. PETROWSKI
2. CriticalMicelle Concentration Solution properties of surface-active agents are much different from those of other materials such as salts. For example, surface tension plotted against concentration is shown in Fig. 5. Other deviations from linearity at some point in the low concentration range also occurred when the concentration of the surface-active agent was plotted against specific conductance, ionic transport (related to conductivity), and osmotic pressure (Becher, 1965). McBain (1944) hypothesized that these phenomena are due to the formation of clusters of these molecules or micelles. Since the abrupt variations in linearity occurred over a very narrow concentration, this micellization must be critically dependent on concentration. Thus, the concentration at which micelles become appreciable has been termed critical micelle concentration, or c.m.c. More detailed explanations have appeared (Becher, 1967b; Hall and Pethica, 1967). Critical micelle concentrations have been determined for many nonionic emulsifiers; in general, the c.m.c. increases with increasing polarity of the emulsifier as exemplified by the HLB value (Kanig and Desai, 1964; McDonald, 1970; Reinhardt and Wachs, 1968; Schott, 1969a; Wachs and Reinhardt, 1965). That the relationship of HLB to c.m.c. was logarithmic was observed by Baba and Bushita (1964), Lin (1972), Mankowich (1962, 1963), Wachs and Hayano (1962). The c.m.c. has also been correlated with the fatty acid chain length: log c.m.c. = 2.41 - 0 . 0 9 4 ~
/--
0.1
(7)
SALTS, SUGARS
0.2
0.3
0.4
WEIGHT ( % )
FIG. 5. Surface tension versus concentration.
EMULSION STABILITY
323
where x = the number of carbon atoms in the hydrophobic portion (Baba and Busluta, 1964) and logarithmically with the polyoxyethylene mole ratio (Mankowich, 1962). The c.m.c. values for a series of emulsifiers were calculated by Wachs and Reinhardt (1965) by determining the decrease in interfacial tension of four monoglycerides in decalin/water. Micellular behavior of lecithin, sodium cholate, and other naturally occurring emulsifiers was covered by Dervichian (1 968).
3. Nuclear Magnetic Resonance Examining emulsifiers by means of nuclear magnetic resonance spectroscopy, Greff and Flanagan (1963) and Ben-Et and Tatarsky (1972) obtained the ratio of hydrophilic protons to lipophilic protons. These values correlated well with the published HLB values.
4. Solubility Partitioning The solubility of the emulsifier was put on a more quantitative basis by Greenwald et al. (1 961), who measured the distribution of polyoxyethylene (POE) emulsifiers of various POE units between water and isooctane. They observed greater partitioning into water as the number of oxyethylene groups increased. Similar approaches were taken by Davies (1957), Heusch (1970), Kruglyakov and Koretskii (1971), McDonald (1970), Mickle (1971), and Nakagaki and Sone (1964), with the solubility results correlating well with HLB values of the emulsifiers studied. Solubility in a third solvent such as ethanol likewise provides meaningful data which also correlate well with HLB (Rimlinger, 1964, 1966, 1968). Partitioning between the phases and the interface has been related to HLB by Prince (1970). Partitioning of emulsifiers at a liquid-liquid interface has been the subject of a review by Cratin (1 968).
5. Water Number Quantitative emulsifier solubility data have also been obtained by dissolving the emulsifier in a suitable solvent and then titrating with water to a turbid end point (Greenwald et al., 1956). In general, the more water-soluble the emulsifier, the more water will be required to reach the turbidity end point. This method, resulting in the determination of the water index, water number, or cloud number, as they are called for the emulsifiers, has also been utilized by others for similar determinations (Angelescu and Barbulescu, 1966; Mendes et al., 1964; Middleton, 1968; Nakamura et al., 1960; Szatlmayer, 1970; Tagawa et al., 1962; Tanaka, 1957). In general, emulsifiers with increasing water numbers also
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exhibited increasing HLB values in a linear manner. An exception was reported. At low HLB values, the trend was reversed in compounds containing four or fewer carbon atoms (hgelescu and Barbulescu, 1966). A normal but nonlinear relation of water number with HLB was reported (Szatlmayer, 1970). A logarithmic relationship: HLB = 16.04 log A
- 7.24
(8)
where A = cloud number in milliliters, was also reported (Hayashi and Fukushima, 1967). An inverse relationship (increasing water number with decreasing HLB) between water number and HLB was reported by Mendes et al. (1964). In the latter case, the emulsifier was added to petrolatum, and the end point was recorded when no more water was absorbed. Such a trend would be reasonable if W/O emulsions were being made; the lower HLB emulsifier containing emulsions could absorb more water before breaking. It thus does not appear to be a water number in the same context as in the previous examples.
6. Cloud Point As the temperature of an aqueous solution of an emulsifier is changed, at some point cloudiness due to insolubility occurs. This is referred to as the cloud point, and it has been used to characterize emulsifiers. The polyoxyethylene portion of nonionic emulsifiers is less hydrated at higher temperature; thus, heating an aqueous solution of the emulsifier would cause it to associate, leading to insolubility. As the cloud point increases, the calculated HLB values based on structure also increase, although not quite linearly (Schott, 1969b). Other factors such as molecular weight, molecular weight range, branching, unsaturations, and functional groups appeared to play a larger role. Perhaps if experimentally determined HLB values had been used rather than calculated values, these factors would have been accounted for a more linear fit would have been obtained. Others have obtained a linear correlation of emulsifier cloud point with experimentally determined HLB values: HLB = 0.0980~+ 4.02
(9)
where x = cloud point ("C) (Hayashi and Fukushima, 1967). The effect of structural differences in emulsifiers on their cloud points was also reported (Schonfeldt and Almroth, 1961). A further extension of this approach involved the determination of the cloud point of the emulsifier in an emulsion of oil and water. These results were correlated with HLB and with a property of the emulsion known as the phase
EMULSION STABILITY
325
inversion temperature, which will be discussed later (Arai and Shinoda, 1965; Mizutani, 1966; Shinoda, 1967; Shinoda and Arai, 1964).
7. Calorimetly A method of determining the proportion of ethylene oxide in emulsifier molecules based on a measurement of the heat of hydration of such molecules with a sensitive calorimetric system has been published (Kruglyakov and Koretskii, 1971; Racz and Orban, 1965; Orban, 1970). Good linear correlations with HLB values were obtained. 8. Polarity Index The polarity index of an emulsifier is a term used to denote the relative polarity of the molecule. It can be determined by gas chromatography in which the emulsifier is used as the column packing and homologous hydrocarbon mixtures are injected onto the column. The polarity index, P, is derived from the equation P = l0010g (C-4.7) + 60
(10)
where C is the carbon number of methanol, obtained from a graph of the logarithms of the hydrocarbon retention times against the number of carbon atoms in the hydrocarbon. A linear correlation of polarity index with HLB values was observed by Broniarz et al. (1972), Endo et al. (1969), and Huebner (1962), while Krivich and Gluzman (1973) found a linear logarithmic relation. By extending this approach, polarity indices were obtained for portions of the emulsifier molecule, thus allowing predictions of the number of ethylene oxide units necessary for particular polarity index values (Fineman, 1967, 1969).
9. Dielectric Constant Another parameter closely associated with polarity is the dielectric constant. Measurement of the dielectric constants of a group of twenty emulsifiers revealed an increase in dielectric constant with increasing polarity of the molecule (Lo et al., 1972). A logarithmic relationship was observed with the HLB values with some scatter in the midrange (Gorman and Hall, 1963). 10. Surface and Interfacial Tension Since the lowering of the surface and interfacial tensions is an important contributory factor in emulsion stability, measurement of these properties for
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GARY E. PETROWSKI
aqueous solutions of emulsifiers against air (surface tension) and oils (interfacial tension) provides further characteristic information (Rosen, 1974). The surface tension of aqueous solutions of nonionic emulsifiers increases as the number of oxyethylene groups in the emulsifier molecule increases (Bashura et al., 1969; Kravchenko and Rybinskaya, 1966; Wachs and Hayano, 1962). A linear correlation of surface tension and HLB was also observed (Bashura et al., 1969; Mankowich, 1963). The interfacial tension of aqueous emulsifier solutions measured against Vaseline (Bashura et al., 1969) and toluene (Chun and Martin, 1961) likewise exhibited linear correlations with HLB and number of ethylene oxide units in the emulsifier molecule.
11. Phase Inversion Temperature The temperature at which the emulsion type changes from O/W to W/O, or vice versa, has been used as a method of determining emulsion stability. The phase inversion temperature (PIT) has also been used to characterize the emulsifiers present in the emulsions. The length of the polyoxyethylene chain of the emulsifier directly affects the phase inversion temperature of emulsions; the longer the chain length, the higher the PIT will be (Shinoda and Arai, 1967). A similar correlation was observed between the PIT and the cloud point of the emulsifier (Arai and Shinoda, 1965; Mizutani, 1966; and Shinoda and Arai, 1964). Emulsions exhibited lower PIT'S when the emulsifier was more oil-soluble (Shinoda and Arai, 1964). Conversely, emulsions containing a mixture of nonionic emulsifiers and sodium lauryl sulfate (a water-soluble ionic emulsifier) exhibited increasing PIT'S as the amount of sodium lauryl sulfate was increased (Arai and Shinoda, 1965). A linear correlation between PIT and the HLB value of the emulsifier was also reported (Aoki et al., 1963; Arai and Shinoda, 1967; Matsumoto and Sherman, 1970; Parkinson and Sherman, 1972; Shinoda, 1967). This method has been proposed as an alternative to the cumbersome original method of Griffin for determining HLB values (Ranny, 1969; Shinoda, 1968). This concept, in fact, has been offered as an alternative to HLB as the generalized best characterization of emulsifiers, since it resolves some of the ambiguities of the HLB approach (Boyd et al., 1972; Shinoda, 1968). It has not yet received the acceptance given the HLB. The fact that HLB has been known for fifteen years longer may be a factor. Methods to calculate PIT based on emulsifier structure have not appeared. In the methodology, emulsions have to be prepared, and standardized conditions-for example, type of oil used, phase volume, and manner of emulsification-have not been established. Thus interlaboratory comparisons of results would be difficult.
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12. Emulsifier Characterization Critique Of all the emulsifier characteristics mentioned, the hydrophile-lipophile balance concept appears to have won the greatest recognition. Most of the other characteristics have been correlated with the HLB. In fact, many were developed as a means of obtaining HLB values more easily than with the originally published method of Griffin (1 949, 1954). Manufacturers of emulsifiers are now frequently supplying HLB values obtained experimentally or by calculation as an aid in making the proper choice. The HLB approach as well as others possess shortcomings, as will be discussed. Therefore new approaches, such as the phase inversion temperature or combinations of the others, should be developed to alleviate the problems. The guidelines are quite obvious: relatively simple methodology, easy reproducibility from laboratory to laboratory, greater scope, universal acceptance by the technical community, and, most important, its utility as a predictive tool for determining emulsion stability.
111.
FATS AND OILS
A. INTRODUCTION Having characterized emulsifiers, we shall now examine the properties of fats and oils. The most common physical properties such as melting point, solid fat index, unsaturation, and saponification number are of limited value for purposes of discussing emulsion stability. Ideally, distinguishing characteristics could be found which would relate to a property of an emulsifier, and they could simply be matched to ensure maximum emulsion stability. B. CHARACTERIZATION
1. Required HLB Value Determination of the required HLB was attempted initially by Griffin (1949, 1954), who prepared many emulsions using water, oil, and emulsifiers (or mixtures of emulsifiers) possessing HLB values over the entire HLB range. After emulsification, the HLB of the emulsifier in the most stable emulsion of the series indicated the “required HLB” of that particular fat or oil. This method, or slight variations of it, were used to obtain required HLB values for many fats and oils (Anon., 1963; Atherton and Maxcy, 1967; Griffin et al., 1966; Hayashi, 1967; Lo et aZ., 1972; Mima, 1958; Ohba, 1962; Racz, 1964; Racz and Kohari, 1965; Robbers and Bhatia, 1961; Scheller, 1960; Seiller et al., 1968, 1970,
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GARY E. PETROWSKI
1972; Strianse and Lanzet, 1960; Sunkes and Sperandio, 1961; Suwalska and Kujkowa, 1965; Tober and Autian, 1958; Wachs and Reusche, 1960). This method of characterizing fats, while helpful, is not without its problems. Required HLB values are often expressed over a sometimes broad range. Reported values may differ, depending on investigator, method of determination, or application. Another more basic cause for these variations may be the sample itself: cottonseed oil or beeswax, for example, examined by different investigators may be sufficiently different due to variety, source, processing, etc., to give different HLB values; yet each different value will be reported as being for the same material. This problem will be discussed further in Section IV,C.
2. Dielectric Constant Once these “wet” methods had been used to determine required HLB values for fats and oils, measurement of other physical properties of fats and oils were made. The results of these measurements were then correlated with the required HLB values already obtained. Gorman and Hall (1963) correlated the log of the dielectric constants of melted fats and oils with the published required HLB values. Griffin et al. (1966), however, were unable to obtain a similar correlation by the same technique. 3. Gas Chromatography
Determination of the required HLB values of fats and oils by means of the gas chromatography technique described previously for emulsifiers resulted in little or no correlation with published required HLB values (Griffin et al., 1966; Petrowski and VanAtta, 1973). If, however, the required HLB values are determined by wet methods and the gas chromatographic determination is made from the same sample of fat or oil, reasonably good agreement is obtained for the values obtained (Petrowski and VanAtta, 1973).
4. Spreading Coefficient The stability of emulsions has been related to the spreading coefficients of the internal phase liquid on the surface of a solution of the emulsifier in the continuous phase (Becher, 1960; Flath, 1971; Padday, 1967; Ross etal., 1959). Essentially, the procedure involves preparing aqueous solutions of emulsifiers of various HLB values. A drop of the oil to be investigated is placed on the surface of each solution, and the condition of the droplet is observed. In the emulsifier solution of high HLB value the droplet spreads over a considerable area. Moving to the other solutions, as the HLB of the emulsifier decreases, the amount of spreading decreases until there is none. At even lower HLB values the same
EMULSION STABILITY
329
unchanged oil droplet still exists. The point at which spreading ceases is the required HLB value. Without going into a theoretical derivation, the analogy to actual emulsion conditions becomes clear. As the small oil droplet in the emulsion rises in the mixture and comes to the surface, if it coalesces and spreads out on the surface, it destabilizes. This process repeated many times results in complete two-phase separation. If the droplet does not spread out, but retains its integrity, it may move back into the bulk of the emulsion and the emulsion remains stable. 5. Phase Inversion Temperature
As we have described previously, in the discussion of emulsifier characteristics, the temperature at which an O/W becomes a W/O emulsion, or vice versa, depends to a large extent on the emulsifier used, and especially on its HLB value. It has likewise been shown that the nature of the fat will affect the PIT. Furthermore, the PIT is related to emulsion stability in that the higher the PIT, the lower the rate of droplet coalescence, and hence the more stable the emulsion will be. In the determination of the phase inversion temperatures for a series of emulsions containing different emulsifier mixtures, at the maximum PIT the HLB of the emulsifier mixture corresponded to the required HLB value (Parkinson and Sherman, 1972; Racz and Kohari, 1965; Racz, 1964). It has been proposed that the PIT may be a better identifying characteristic of fats and oils than the required HLB, since it overcomes some of the latter’s shortcomings in determining conditions for maximum emulsion stability (Boyd et al., 1972; Shinoda and Saito, 1969). Although this approach has merit, it still begs the question, since the PIT is dependent on the emulsifier used and thus, of necessity, will have meaning only when the oil and emulsifier are defined. Thus, the problem of how to characterize oil, emulsifier, and PIT becomes circular, since each is related to the other two. 6. Water Number In a manner similar to that previously described for emulsifiers, titration of fats and oils gives water numbers that correspond to required HLB values. Direct matching of water numbers of oils with emulsifiers possessing the same water numbers was not done (Middleton, 1968).
7. Critique The methods described to evaluate fats and oils with respect to their behavior in emulsions are general approaches. Many involve examining their behavior in emulsions. Since no property has evolved that can be universally used as a
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GARY E. PETROWSKI
predictive tool for emulsion stability, it remains the onus of food scientists to evaluate the fat in their particular system.
IV. OTHER EMULSION FACTORS A. EMULSIFIER MIXTURES Up to this point single emulsifiers and mixtures of emulsifiers have been implicitly assumed to exhibit similar characteristics. In fact, over the years, emulsifier mixtures have extended the versatility of individual emulsifier properties. The subject of emulsifier mixtures has not progressed from an art to a well-defmed science as rapidly as the subject of emulsifiers themselves. The desirability and even necessity for a mixture of emulsifiers rather than a single one appears to be based on early observations by Schulman and Cockbain (1940) prior to the introduction of the HLB concept. They observed that a mixed interfacial film formed by simultaneous adsorption of a water-soluble and an oil-soluble emulsifier resulted in an emulsion of greater stability than one from either component separately. Such an idea was reinforced through the years by published (Maso and Conrad, 1969), and probably unpublished, experiences of those “familiar with the art.” For instance, Barry (1969) published on the need of emulsifier mixtures necessary to form fairly viscous emulsions and the requirements of the oil- and water-soluble emulsifier components. A thermodynamic treatment of mixed surfactant interactions has recently appeared (Lucassen-Reynders, 1973). It has been generally assumed that the hydrophile-lipophile balance of a mixture of emulsifiers is the algebraic sum of the HLB values of the individual emulsifiers:
Becher and Birkmeier (1964), however, showed a curvature in what should have been a linear correlation of emulsifier mixture p values determined by the ratio of retention times of ethanol/hexane versus calculated HLB values of the mixture obtained from the formula just presented. In a later study of the In p’ values of mixtures of POE sorbitan monostearate (HLB 14.9) and sorbitan monostearate (HLB 4.7), a good linear correlation was obtained between gas chromatographic retention time data and calculated HLB values. Because such ideal behavior might not be expected with chemically dissimilar mixtures, emulsifier combinations of POE sorbitan monostearate and Atmos-150 (a mixture of mono- and diglycerides, HLB 3.2) were evaluated with similar
EMULSION STABILITY
331
linear results. Thus, in mixtures of chemically similar or dissimilar emulsifiers, the resultant HLB values can be determined by the algebraic summation method (Petrowski and VanAtta, 1973). Early reports indicated that emulsifier properties as described in Section II,C were indeed generally additive (Arai and Shinoda, 1967; Jain and Sharma, 1971; Jelescu et al., 1966; Mickle et al., 1971; Roehl, 1972b; Shinoda and Kunienda, 1973; Thalheimer and Rusch, 1970), with a few reports to the contrary (Chun and Martin, 1961; Endo et al., 1969; Kamada and Matsumoto, 1959). For the practicing food scientist, a good emulsifier mixture to begin with might be composed of a polysorbate type of emulsifier for the high-HLB component, and a sorbitan ester type or mono- and diglyceride type for the low-HLB component. With this mixture, a broad range of HLB values can be obtained, depending on the proportions of each in the mixture. Once the optimum HLB requirements have been determined for the particular system, further selection of the emulsifier system will be accomplished with the least amount of “hit-or-miss” philosophy. B. APPLICATIONS OF HLB The literature is replete with examples of applications in which the use of a particular emulsifier resulted in a more stable emulsion. The list is considerably attenuated when the emulsifier was chosen as a result of a systematic study employing predictive techniques based on some generally recognized principle rather than a hit-or-miss proposition. While much of the predictive work employed in determining proper emulsifiers for optimum stability in individual instances is probably locked in the vaults of corporate proprietary information, sufficient examples have been published to indicate the general approaches used; for example, see Kunieda and Shinoda (1972). Because the concept of HLB provides a generally accepted quantitative emulsifier characteristic, many manufacturers are including this information. For very precise research work, however, HLB values should probably be determined independently, since the published values have sometimes been arrived at by calculations based on structure rather than experimental determinations. A good source of emulsifiers commercially available is the annually updated McCutcheon’s Emulsifiers and Detergents. A listing of only food-approved emulsifiers has also been published (Petrowski, 1975). Besides being listed alphabetically, emulsifiers are listed by increasing HLB values when the data are available. Many published reports on the use of HLB to improve emulsion stability have been summarized in a comprehensive bibliography covering the years 1949-1969 (Becher and Griffin, 1970). Several representative examples bear mentioning.
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GARY E. PETROWSKI
In the field of pharmaceuticals, maximum stability of ointments and release of active ingredients has been achieved by considering the HLB values involved (Carro and Jato, 1961; Czetsch-Lindenwald, 1961; Fincher et al., 1966; Kata, 1969; Mima, 1958; Rhyne et ul., 1960; Spittle and Hartman, 1960; Yousef and Khawam, 1964). Maximum herbicidal activity has also been determined in a similar consideration of HLB values (Jansen, 1964; Matsumoto and Aoki, 1962; Smith et al., 1966). This increased herbicidal activity at optimum emulsifier HLB may be due to a consideration of the contact angle of the droplet on the surface-in this case, the leaf surface (Becher and Becher, 1969). Use of HLB values for the selection of food emulsifiers has been outlined by Knightly (1963) and by Thalheimer and Rusch (1970). Other reports on emulsions in foods do not comment on the use of HLB to determine the best emulsifier for a particular application (Brennan, 1970; Cyganska and Witwicka, 1967; Fujita et al., 1972; Lancrenon and Sirami, 1972; Nash and Brickman, 1972; Savostikova et al., 1970; Terada and Kono, 1969).
C. LIMITATIONS OF HLB Having discussed at some length the advantages of using the HLB concept as an aid for the characterization of an emulsifier and oil, let us now consider its limitations. Having established the required HLB value of the oil or fat to be emulsified, and knowing the HLB values of the emulsifiers, thus eliminating many from consideration, one is still left with a bewildering array of emulsifiers or mixtures of emulsifiers, all possessing the optimum HLB. The choice must now be based on other considerations. Furthermore, once an emulsifier is properly chosen, no indication of emulsifier level necessary to achieve stabilization can be gleaned; in fact, different HLB values (Titus et al., 1968) and different emulsion types were evident when the same emulsifier was used at different levels (Becher, 1958). Other workers have concluded that different oil phase levels require different HLB emulsifiers (Burt, 1965). These shortcomings have been pointed out (Rahm, 1969a, b; Riegelman and Pichon, 1962). The HLB serves as a guide to emulsifier selection. The final choice will still have to be made on an individual basis, depending on stability desiredagglomeration, coalescence, stability to freeze-thaw or heating, particular processing conditions, limitations of flavor, FDA acceptability, cost, etc.
D. EMULSIFIER CONCENTRATION The question of the optimum level of an emulsifier deserves some attention. It appears that such levels are usually arrived at by trial and error. With the proper
EMULSION STABILITY
333
emulsifier, less is required to form a stable emulsion than if a less suitable emulsifier is chosen. By combining several ideas already expressed, a model system may be examined to consider the various factors. As described earlier, emulsifiers effectively prevent coalescence by covering the oil droplet (in an O/W emulsion) surface with a coating of monomolecular thickness. Further emulsifier is unnecessary; in fact, it becomes difficult to visualize a reasonable alignment of multilayers in the region of the droplet surface. By simple calculations, the surface area of the droplets can be obtained if the oil phase volume, density, and particle size (assuming uniform size) are known. For example, the total surface area of the oil droplets per unit volume of oil is equal to the surface area per droplet times the number of droplets per unit volume: Total surface area -surface area -
ml Oil
drop
X
number of drops
ml
where A = angstrom units and r = droplet radius. Thus, for droplets 2 I.( in diameter, the total surface area of 1 ml of oil is 3 X lo2' A2. The surface coverage of emulsifier molecules is usually measured in similar units. If the molecular weight and concentration of the emulsifier are known, the total monolayer surface of the emulsifier can be calculated also: Weight of emulsifier molecular weight
X
6.02 X loz3= number of emulsifier molecules
(16)
The number of molecules times the coverage per molecule equals the total emulsifier coverage. The effective surface area covered by an emulsifier molecule depends on its structure, but a value of -50 A' per molecule would be a good approximation. For a further review, see Ottewill(l967). Thus, from a previous calculation, 1 ml of oil suspended as droplets of 2 p diameter would require a minimum of (3 X 1O2')/5O = 6 X 10" emulsifier molecules for complete monolayer coverage. Putting this in more tangible terms, ) = lo-' mole. If the we obtain 6 X lo'* molecules = (6 X lo'* )/(6 X molecular weight of the emulsifier is 1000, this corresponds to 0.01 g or -1%
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GARY E. PETROWSKI
+ O I L DROPLET
STAB1 L I 2 E D DROPLET
+ MlCEL L E
t STARCH, GUM,
wCOMPLEX
PROTEIN
FIG. 6. Emulsifier equilibria. No structural features are intended in the starch-emulsifier complex.
based on the oil. For 100 g of an emulsion containing 30% oil, the emulsifier concentration should be at least 0.3% for monolayer coverage. It has been observed that the amount of emulsifier generally employed exceeds the amount necessary for monomolecular coverage based on the type of calculations just discussed. This may be due to the fact that not all the emulsifier in the emulsion resides at the droplet surface. However, by examining the emulsion immediately after formation, it has been observed that the interfacial area decreases quite rapidly initially, followed by a decrease at a much slower, almost constant, rate (Fischer and Harkins, 1932). If the initial emulsion results in an interfacial surface area too large to be covered by a monolayer of emulsifier molecules, coalescence will occur until the surface area is decreased to the point that coverage by the emulsifier is complete. At this point, the rate of coalescence will decrease. A complex equilibrium which may exist in the emulsion is illustrated in Fig. 6 . Besides coating the oil droplet surface (0) via K 1 ,the emulsifier monomers may form clusters or micelles ( K , ) if their concentration exceeds the critical micelle concentration (c.m.c.), as explained previously. Alternatively, in the presence of some other component such as starch, protein, or gum (n), a
EMULSION STABILITY
335
complex may form via K 3 , further removing the monomers from solution. Stabilization of the oil droplet via emulsifier monomers will be favored if the equilibrium constant, K 1 , is much greater than K 2 or K 3 . Because these equilibrium constants ultimately affect emulsion stability, the fact that different emulsifiers of the same HLB value afford different stabilities appears reasonable, since the equilibrium constants may be quite different relative to each other. The presence of starch or gum may be destabilizing if K 3 is much greater than K 1 , since there would be few emulsifier monomers t o absorb on the droplet surface. Besides the thermodynamic considerations, relative reaction rates for these processes may also be instrumental in determining stability. If the emulsifier concentration is below the c.m.c., equilibrium 2 favors monomer exclusively; likewise, in the absence of other components, equilibrium 3 need not be considered. Let us consider situations where the c.m.c. is exceeded. Several studies of emulsifier concentration versus droplet size have been reported (Riegelman and Pichon, 1962). For 25% mineral oil emulsions, Rowe (1 965) found the following logarithmic relationships: log C = 2.68 - 0.174d,
for sodium dodecyl sulfate
log C = 2.01 - 0.237d, for POE sorbitan monooleate
(17) (18)
where C = emulsifier concentration, and d, = droplet median diameter. The plot was linear at concentrations greater than their respective c.m.c. values (Becher, 1967b). Thus, it appears that at concentrations above the c.m.c. the increased interfacial area (smaller droplet size) is due to increased emulsifier adsorption at the interface. Micellar adsorption on the droplet surface, although possible, does not appear probable, leaving monomer adsorption as the explanation. An apparent discontinuity between emulsifier concentration and emulsion stability at the c.m.c. was reported by Rehfield (1962). Preparation of an emulsion of 50% benzene in water with varying levels of sodium dodecyl sulfate (SDS) as the emulsifier resulted in a linear relationship between the volume fraction of emulsion remaining after ultracentrifugation and the SDS concentration up to 0.26%, and another different linear relationship (slope = 0) at higher levels. Since the c.m.c. of SDS is 0.18 to 0.25%, the discontinuity was ascribed to the c.m.c. However, it must be pointed out that, at the 0.25% emulsifier level, the volume fraction of emulsion remaining after centrifugation was 1; thus n o improvement could be expected because the emulsion was physically as good as it could get under the conditions-a break in linearity had to occur. In another case, (Friberg and Mandell, 1970; Friberg and Wilton, 1970), the stability of an emulsion was observed to decrease at emulsifier concentrations greater than the c.m.c. The behavior was left an open question.
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GARY E. PETROWSKI
From the examples presented, it would appear that no unambiguous generalizations can be made regarding the effect of emulsifier concentration passing through the c.m.c. and its resultant effect on emulsion stability. A realization that the c.m.c. may cause anomolous behavior in an emulsion system may explain some hitherto unexplained observations and should be kept in mind. E. EFFECT OF EMULSIFIER STRUCTURE
A choice of emulsifiers based on structural considerations over the polarity features already discussed is limited (Bierre, 1971). A general rule has emerged that, to stabilize a saturated fat, an emulsifier possessing a saturated hydrophobic chain is desirable. Likewise, an unsaturated oil is best stabilized with an emulsifier possessing an unsaturated alkyl chain. The effect of emulsifier structure on its properties was the subject of a paper by Rosen (1972). A distinction was made between efficiency (concentration of emulsifier required to produce some significant reduction in surface tension) and effectiveness (minimum value to which the surface tension is lowered) (Fig. 7). Wetting agents need effective reduction of surface tension (Grishina et al., 1971) and thus tend toward the branched shape. Shorter straight-chain molecules are also good wetting agents. Foaming agents function by possessing good mechanical strength caused by close packing at the interface; thus, long straightchain hydrophobic groups are effective. Boyd et al., (1972), disregarding the resultant HLB value of the emulsifier mixture and the required HLB of the oil, considered the increased stability of emulsions containing sorbitan monooleate and POE sorbitan monopalmitate to be a result of a convenient meshing of the molecules on the oil droplet surface due to steric considerations (Fig. 8). Lucassen-Reynders (1 964) observed that emulsifiers should possess long (60-A) hydrophobic chains for best stabilization of water-in-oil emulsions. Conversely, Hallworth and Carless (1972) observed that, when sodium lauryl sulfate is used, the longer the paraffinic chain of the oil phase, the more stable the emulsion will be. Emulsifier hydrophobic chain length had no effect. Frank and Denk (1963) determined that emulsifier properties of straight-chain alkylphenol polyglycol ethers were determined not only by HLB (length of alkyl EFFICIENT
EFF E C T I V E
FIG. 7. Emulsifier structure variations.
EMULSION STABILITY
331
POE SORBITAN MONOPALMITATE WATER OIL D R O P L E T SORBITAN MONOOLEATE
FIG. 8. Enlarged oil droplet stabilized by a mixed emulsifier system. Reprinted with permission from J. Colloid Interface Sci. 41(2), 359 (1972).
group and polyoxyethylene) but by the position of the alkyl group on the phenol ring-for example, ortho versus para. The interrelationship of dye retention, HLB, and emulsifier structure was observed by Sekido and Tanaka (1963), who commented on three aspects of the emulsifier: hydrophobic, hydrophilic, and bridge groups. The effects of substituting alicyclic groups for linear aliphatic groups were shown by Goette (1969). Atherton and Maxcy (1967) and Hallworth and Carless (1972) found that, in formulating a lotion at a constant HLB value using glycerol monoesters of pure fatty acids, or long-chain paraffins, the viscosity, and hence the stability, of the emulsions increases with increasing chain length. This may be due to either an increase in film strength or a decrease in the demulsification rate as the chain length increases. In a study of the stability of milk fat-water emulsions, similar stabilities were obtained regardless of whether single or binary emulsifier systems were used as long as they possessed the same HLB value (Titus and Mickle, 1971). In spite of the example just cited, as a general rule, use of different emulsifiers or mixtures of emulsifiers all with the same HLB will result in emulsions of varying degrees of stability (Roehl, 1972b). In a study of the effectiveness of single or mixed emulsifiers in baking, cakes possessing properties deemed desirable by the consumer resulted from the use of emulsifier mixtures (Buddemeyer et aZ., 1962; McDonald, 1970). No consideration was made for the HLB values of the emulsifiers involved. The correlation of emulsifier structure to economic expediency can be illustrated by the chemical genesis of some of the most common food emulsifierssorbitan esters and polyoxyethylene sorbitan esters, as related by Schgnfeldt (1969). During World War I, the use of mercury fulminate made by Atlas Powder Company as a detonator explosive was phased out because supplies of mercury were scarce and had to be imported. Mannitol hexanitrate, a suitable substitute, likewise had the drawback that mannitol also was extremely scarce. An electrolytic process which converted readily available corn sugar to 20% mannitol and 80% sorbitol was discovered at this time. Such a process was
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GARY E. PETROWSKI
economical only if a use could be found for the major product. Esterification of the sorbitol led to the class of surface-active agents known under the trade name of SPANS. Water solubility of the emulsifiers was improved by ethoxylation to yield polysorbates or TWEENS, manufactured by the same company, Atlas Chemical Company, now ICI United States.
F. EMULSIONTYF'E Up to this point, the type of emulsion that forms has been referred to only in terms of HLB-emulsifiers with lower values tend to promote W/O emulsions, while those with higher values tend to produce O/W emulsions (Davies, 1957; 1961a,b; Mitsui et al., 1967). In the emulsification process,initially droplets from both phases will be formed. Both droplets will tend to coalesce, and the final emulsion type will be determined on the basis of which phase droplets have the greater rate of coalescence; that is, the phase having the greater coalescence rate becomes the continuous phase. With emulsifiers present, the emulsion type will frequently be determined by the characteristics of the emulsifier. Bancroft (1913) generalized that, when solids present in the oil-water mixture act as emulsifiers, the external phase will be the one that more readily wets the solid. With normal surface-active agents, the external phase will generally be the one in which the emulsifier is more soluble. The manner of preparation and the phase volume also play important roles. If one phase is slowly added to the other, the added phase will initially be the internal phase, since it is present in such low relative proportions. The general rule, based on phase volume-that is, the phase present in the greater proportion will be the external phase-has a sufficient number of exceptions to warrant limiting its use to indicating possible types. If an emulsion is visualized as being composed of spherically rigid droplets of uniform diameter, a maximum of about 74% of the emulsion can be the internal phase because it is physically impossible to pack any more spheres in. The interstices remain. Thus, if more internal phase were added, and the emulsion did not break, inversion would have to occur. In actual practice, emulsions with as high as 99% internal phase have been obtained. No fault in the theory exists, only an incorrect model. Emulsions rarely exhibit uniform droplet size, but rather a range of sizes with varying degrees of broadness. Thus, the smaller droplets could begin to fill the spaces. The idealized spherical shape may be distorted to various polyhedra, thus allowing an even closer packing. Such behavior may be brought about by the choice of emulsifiers. Emulsion level has been held accountable for emulsion type by some (Becher, 1958; Benson et al., 1962; Mitsui and Machida, 1969). In a study of other factors affecting emulsion type, Davies (1961b) included type of emulsifying machinery in contact with the two phases. More specifically, the last point refers to the wettability of the phases on the surfaces and the texture of the surface.
EMULSION STABILITY
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Factors responsible for the formation of the less frequently formed W/O emulsions have been studied by Ford and Furmidge (1966). A strong interfacial film is required t o prevent the coalescence of the water droplets. Molecular attraction forces such as electrostatic or hydrogen bonding may cause the interfacial behavior. HLB values of the emulsifier should be low, but this approach is not definitive. The initial location of the emulsifier may also affect emulsion type (Lin, 1968) as well as viscosity and, related to this, particle size and emulsion stability. One practical consideration in the formulation of food products is to avoid the presence of two antagonistic emulsifying agents. A solid present for some purpose, such as a whitener, flowing agent, or filler, may promote the formation of an emulsion of opposite type to that promoted by the conventional emulsifier. Such a situation could be destabilizing to the emulsion, perhaps to the point of making it unsatisfactory. G. PROTEINS The proteins of interest as stabilizers of emulsions are derived from three major sources: dairy, meat, and vegetable. The classic example of emulsions stabilized by proteins is milk. Extensive work has been carried out on the mechanism of stabilization and the structures of the principal components (Jenness and Patton, 1959). In its simplest explanation, proteins behave in a manner similar to that of surface-active agents. They tend to form mechanically strong monolayer fims at the interface (Kitchener and Mussellwhite, 1968). As proteins, their structure, and hence their behavior, is more affected by such variables as salt concentration, pH, and temperature, than would be found for the normal emulsifiers (Sabharwal and Vakaleris, 1972). Casein and sodium caseinate, proteins derived from milk and other vegetable proteins, notably soy, have been used in various food products with varying degrees of concomitant emulsion stabilization (Vakaleris and Sabharwal, 1972). The use of conventional emulsifiers in addition to proteins results in a complex situation with all the individual attendant mechanisms operative (described in Section IV,D),plus a competition between the two entities for adsorption onto the oil droplet surface, plus binding of the emulsifier to the protein (Green, 1971; Prakash and Srivastava, 1969). Karel(l973) summarizes the area succinctly. Comparisons of the emulsifying capacity of milk and vegetable protein, mainly soy, have been made with varying results as to which is the best. The conditions employed and stability tests obviously account for the differences (Acton and Saffle, 1971; Inklaar and Fortuin, 1969;Pearson et at., 1965). Proteins appear to have the rigid film-forming capabilities needed to form stable foams and, because of this, find considerable use in whipped toppings. Meat proteins and their ability to form stable emulsions were the subject of a recent review by Saffle (1968). Since that time, the HLB values of muscle tissue
340
GARY E. PETROWSKI
has been determined to be -14 (vanEerd, 1971). The emulsifying capacity of purified muscle protein has also been determined (Tsai et al., 1972).
H. SOLIDS The ability of finely divided inorganic solids to act as emulsifying agents has been recognized for a long time (Bennister el al., 1940; Mizrahi and Barnea, 1970; Pickering, 1907). Particle size is important, with smaller solid particles stabilizing the smaller, more stable droplets of the dispersed phase. The method of stabilization probably involves the positioning of the solid particles on the surface of the droplets, thus forming a protective layer as shown in Fig. 9. Bancroft (1913) observed that the liquid that wets the solid more easily will become the external phase. The formation of W/Oemulsions with carbon black and O/W emulsions with silica, calcium carbonate, and other hydrated oxides (Jain and Srivastava, 1970; Mukerjee and Srivastava, 1956) strengthened Bancroft’s generalization. By comparison with present-day emulsifiers, solids do not exert sufficient stabilization to make them practical for use as the primary emulsifier. Their stabilizing influence need not be neglected, however, since the presence of a solid may reinforce the emulsifier, or antagonize it if it tends to form one type of emulsion while the emulsifier promotes the formation of the other type. A common example will illustrate this point along with others already discussed. Mayonnaise is an O/W emulsion. Since it is 70 to 80% oil, phase volume considerations would point to the opposite type, W/O. The stability is due to two natural emulsifying agents, egg yolk and ground mustard seed. Two ingredients in egg yolk, lecithin and cholesterol, are surface-active agents and promote the formation of O/W and W/O emulsions, respectively. The ratio of one to the other will determine the type. In natural egg, the ratio would probably favor the W/O type. The resultant type formed in mayonnaise is due to the action of mustard seed which forms O/W emulsions. The coarseness of the mustard is critical-the finer, the better (Anon., 1968; Corran, 1946).
S O L I D PARTICLES OIL DROPLET WATER
FIG. 9. Solid emulsifier stabilization.
EMULSION STABILITY
34 1
Present-day research in the area of solid emulsifiers has centered around the interaction of solid emulsifiers with conventional surface-active agents (Enal’ev e l al., 1969; Fukushima and Kumagai, 1973; Jain and Srivastava, 1970; Koretskii and Taubman, 1959, 1969; Moriyama et al., 1969; Motawi e l aZ., 1965; Ross, 1973; Taubman and Koretskii, 1961) and their effect on emulsion stability.
I. HYDROCOLLOIDS The use of gums and starches to stabilize emulsions in the food and cosmetics industry is widespread (Glicksman, 1969; Meer and Meer, 1962). The mechanism of stabilization is most generally assumed to occur through an increase in viscosity. Some evidence has been presented, however, to indicate surface activity such as a concentration at the oil-water interface to form mechanically strong interfacial films (Serrallach and Jones, 1931; Serrallach et al., 1933). Interactions do occur between these stabilizers and the various other ingredients, and, because of this, the choice of gums is important. Such processing conditions as pH (Mukerjee and Shukla, 1965), temperature, oil level, solids, and salts will also affect the choice. The HLB values of gums have been determined (Chun el al., 1958; Guess, 1960, 1961), but the utility of these values has still to be demonstrated.
J. AERATED EMULSIONS Up to this point the working definition of an emulsion-that is, one immiscible liquid suspended in another-has been adhered to, yet one is aware of the many examples involving the introduction of yet another interface, namely air-liquid. The simplest example would be air entrained in a solution such as meringue from whipping egg whites or soap suds. The ability of aqueous solutions of proteins and some emulsifiers to entrain air under agitation has been both a boon and a bane to processors. To pursue this idea one step further in complexity, an air interface can be introduced into an emulsion. While the dynamics operative in simple foams can be visualized in terms of a simple model, none has yet evolved for the threephase system. These aerated emulsions, more dense and stiffer than foams, are found throughout the food industry: bakery products, ice cream, whipped toppings, whipped margarines, etc. (Hanks, 1973; Knightly, 1968, 1973). The stability of simple foams appears to depend on the physical strength of the films separating the air bubbles and will not be discussed further (Cante el al., 1973; Ross, 1967). The entrainment of air in emulsions may be viewed as two separate problems: emulsion stability and foam stability, with both being important.
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GARY E.PETROWSKI
It had been thought that, for a foam or aerated product to form from an emulsion, the emulsion had to “break” first. If this were the case, however, phase separation would occur, and the water or oil phase would synerese. In studies of factors affecting foam stability of aerated emulsions, it was observed that the best foams formed from the best emulsions; however, not all good emulsions formed good foams. The foams were judged for texture or stiffness, overrun (ratio of whipped volume to original volume), and stability (lack of syneresis or separation of phases). Factors other than emulsion stability were equally important: Solid fats formed good foams, while liquid oils did not; emulsifiers containing saturated aliphatic chains (hard emulsifiers) performed better than their unsaturated counterparts, giving rise to the rule that like stabilizes like, since the solid fats were also saturated. No correlation existed between HLB and foam stability other than the stability of the initially formed emulsion. In a recent study of aerosol emulsions containing fatty alcohols, no relationship between emulsion and foam stability was found (Sanders, 1974). The mechanism of the aerated emulsion stabilization is not definitively understood but may involve the crystalline structure of the solid fat. In fact, microscopic examination of good and bad aerated emulsions prepared from similar systems with different emulsifiers reveals crystallinity of the fats in the good foams and spherical amorphous shapes in the poor examples. The generalization that the emulsion must destabilize may be put in perspective: coalescence does not occur to form two phases, but the initially formed spheres are altered. Examples may be found in work in the ice cream area (Arbuckle, 1960). Actually the term aerated emulsion is a misnomer, since the nonaqueous phase is a solid; hence the term dispersion would be more proper. In the emulsion’s preparation, however, the mixture is heated, thus melting the fat, and a true emulsion exists. Today consumer demand and technology have increased the complexity even one step further-drying of the emulsion prior to its use, as in a dried, reconstitutable whipped topping. The area of dried emulsions ranges beyond just the application mentioned, especially in terms of spray-dried flavors and coffee whiteners (Clarke and Love, 1974). Thus, the food products that delight the child’s eye-ice cream, cake, and whipped cream-represent some of the most complex examples of emulsions, especially in terms of describing a model system.
K. EMULSIFIERS AND THE FDA Although the choice of emulsifiers available appears to be practically limitless, for food applications the list narrows considerably. Toxicity studies of emulsifiers have been reviewed (Elworthy and Treon, 1967). Nine types of emulsifiers
EMULSION STABILITY
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are listed in the Code of Federal Regulations (Title 21, Chapter I, Food and Drug Administration, Department of Health, Education & Welfare, Sub-chapter B, Food and Food Products Part 121, Food Additives, Subpart B 0 121.101) under substances that are generally regarded as safe (hence the acronym GRAS). In addition to these, another group of emulsifiers approved as food additives is included in Subpart D 0 121.100(F121.1238. These have been declared safe additives when used in the quantities and applications mentioned. Approved emulsifiers are listed alphabetically according to their FDA name in Table I11 (Petrowski, 1975). Since trade names have come to be known synonymously in some cases, these and other names have also been included. Trade names have been listed as an aid for recognition and should not be construed as being complete, since some of the emulsifier classes, such as the mono- and diglycerides, are manufactured by many companies. The cited reference contains additional pertinent information. Since emphasis has been placed on HLB as a means of characterizing emulsifiers, these values, when available, have also been included. Estimated ranges have been added when literature values are missing. Petitioning for approval of an emulsifier not listed is expensive and timeconsuming for the food manufacturer. Manufacturers of the emulsifiers themselves may petition for approval if projected sales warrant the time and expense of the testing and petitioning process. Of peripheral interest to the food scientist would be Subpart F, 0 121.250(F121.2616, which lists approved emulsifiers for products in contact with food, such as in packaging, and Subpart C, 0 121.200-121.337 which lists emulsifiers approved for feed. Further restrictions on the use of emulsifiers may exist if the product is subject to a standard of identity that does not allow the inclusion of ingredients other than those specifically defined ingredients. Since the regulations involving emulsifiers and other food additives are constantly changing, the reader is referred to the Federal Register, a daily government publication in which the amendments and their proposals appear.
L. EMULSION PREPARATION From the controlled environment of the laboratory bench and model systems to the complex engineering processes required to form a finished food product, the basic principles discussed in this review must be recognized if emulsion stability is to be more than a hit-or-miss proposition. In the transition, compromise will arise, but the chances of success will be increased if the formulator is aware of the basic principles governing emulsion stability. Other reports yield additional information in formulating emulsions (Becher, 1967a; Bensqn e t al., 1962; Gopal, 1968; Saad and Shay, 1972; Vold and Mittal, 1973).
W
PP
TABLE Ill ALPHABETICAL LISTING OF FDA-APPROVED EMULSIFIERS AND PERTINENT INFORMATIONa
FDA name (other names in parentheses) Acetylated monoglycerides Calcium stearoyl-24actylate Diacetyl tartaric acid esters of mono- and diglycerides Dioctyl sodium sulfosuccinate Ethoxylated mono- and diglycerides Fatty acids Glyceryl-lacto esters of fatty acids Hydroxylated lecithin Lactylated fatty acid esters of glycerol and propylene glycol Lactylic esters of fatty acids Lecithin Mono- and diglycerides
CFRsection HLB
Trade name equivalents
121.1018 121.1047 121.101 (GRAS) 121.1137 121.1221
-3 2-3 8-9
CETODAN (series); MWACET (series) ARTODAN (series); VERV DREWLATE 30; PANODAN (series); WITCOTEM AA-43, AA-45, C43-21, D66-1
13.1
ALDOSPERSE MS20; SANTELLE EOM
121.1070 121.1004
1 7-12
121.1027 121.1122
Med. Med.
GLYCON (series) ALDO LS; DREWLATE-10; DURLAC 1OO,1O1, 200,300 CENTROLENE -
121.1048
8.8 Med.
121.101 (GRAS) 121.101 (GRAS)
-
-3
MARVIC ACID; SLA ACTIFLO; CENTROL; CENTROLEX; CENTROPHILE; LECIDAN ALDO (series); ATMOS (series); ATMUL (series); DREWMULSE (series); DUR-EM (series); EMULDAN (series); HI-Q; HODAG GML, GMO, GMP, GMS; MWAPLEX-600; MWEROL (series); SUPER G (series)
m
Monosodium phosphate derivatives of mono- and diglycerides Polyglycerol esters of fatty acids
121.101 (GRW
Med.
121.1120
Poly oxyethylene( 2O)sorbitan tristearate (polysorbate 65) Polysorbate 60 (polyoxyethylene(20)sorbitan monostearate) Polysorbate 80 (polyoxyethylene(20)sorbitan monooleate) Propylene glycol mono- and diesters of fats and fatty acids Sodium lauryl sulfate Sodium stearoyl-2-lactylate Sodium stearyl fumarate Sorbitan monostearate
121.1008
Broad DREWPOL (series); DURKREME 310; HODAG PGO, SVO-629, SVO-1037, SVO-1047; POLY ALDO (series); SANTONE (series); TRIODAN; 10.5 WITCONOL (series) DREWPONE 65; DURFAX 65; GLYCOSPERSE TS-20; HODAG PSTS-20; TWEEN 65 14.9 DREWPONE 60; DURFAX 60; GLYCOSPERSE S-20; HODAG PSMS-20; HODAG SVS-18; TWEEN 60 15.9 DREWPONE 80; DURFAX 80; GLYCOSPERSE 0-20; HODAG PSMO-20; HODAG SVO-9; TWEEN 80 2-5 ALDO PME; ALDO PS; DREWLENE 10; HODAG PGMS; MWEROL P-06; PROMODAN (series)
Stearyl monoglyceridyl citrate Succinylated monoglycerides
121.1030 121.1009
121.1113
High 2-3
EMPHOS D70-30C, D70-31, F27-85
ARTODAN; EMPLEX
121.1012 121.1211 121.1183 121.1029
-
121.1080
Low
-
121.1195
-3
MYVEROL SMG
4.7
SI
C CI
-
DREWSORB 60; DURTAN 60; GLYCOMUL S; HODAG SMS; SPAN 60
aReprinted with permission from Food Technol. 29 (7) 52-62 (1975). Copyright @by the Institute of Food Technologists. W VI P
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GARY E. PETROWSKI
M. EMULSIFIER IDENTIFICATION Up to this point, the approach has been one of formulating emulsions. Situations may arise in which an emulsion is to be investigated to determine the presence and identity of emulsifiers contributing to the stability. The presence of an emulsifier can be determined by investigation of the physical properties of emulsions as covered previously: lowering of the surface and interfacial tension, foaming, solubilization, etc. Dye methods and precipitation processes are useful procedures but often yield ambiguous results. The tests are frequently nonspecific and affected by interfering substances. Further complications arise from the low proportions of emulsifier generally present. To concentrate the emulsifier, solvent extraction methods have been developed to remove the emulsifier from the remainder of the mixture. If an emulsifier mixture exists, chromatographic procedures may be utilized: TLC, paper, gas-liquid, liquid-liquid. Once the emulsifiers have been separated, identification is accomplished by classical organic analysis, determination of physical properties, spectral analysis, and the use of group-specific reagents. Quantitative information may be obtained in conjunction with some of the above methods, or separate spectral, chromatographic, or gravimetric methods may be used. A comprehensive treatise on analysis of surface active agents has been published by Rosen and Goldsmith (1972).
V. DIRECTIONS The methods developed to characterize emulsifiers, fats, and oils have been presented, and yet no generally accepted principle has evolved which accurately predicts the stability of the emulsion to be formed. Do any additional characteristics of emulsifiers and lipids exist so that they can be matched for greatest emulsion stability? Can additional emulsifier structure and effect correlations be derived? What additional factors determine whether a mixture of emulsifiers is better than a single well-chosen emulsifier? Can the effect of proteins, gums, and starches on emulsion stability be better understood? What interactions exist between these stabilizers and emulsifiers? Can emulsifiers not presently known be developed which will stabilize emulsions to a much greater extent? Regarding the testing of emulsion stability, can accelerated methods be correlated to normal shelf life over a wide range of applications? The above are basic questions, but the need for answers certainly goes beyond these to more complex problems and applications: Can model systems be developed to explain factors operative in the air-solid-liquid interfaces encountered in aerated and dried emulsion stability? The result of this research will be the completion of a large circle-specific approaches to problems which result in
EMULSION STABlLITY
347
a generalized working body of knowledge enabling future researchers, guided by these principles, to answer questions regarding the stability of specific emulsion systems.
REFERENCES Acton, J. C., and Saffle, R L. 1971. Stability of oil-in-water emulsions. 2. Effects of oil phase volume, stability test, viscosity, type of oil and protein additive. J. Food Sci 36, 1118-1120. Alexander, A. E., and Johnson, P. 1949. “Colloid Science.” Clarendon Press, London. Angelescu, I. E, and Barbulescu, E. 1966. Determination of the hydrophilic-lyophilic character of colloidal systems in the presence of cationic surfaceactive substances. Rev. Roum. Chim. 11(5), 567-572 (in Fr.); Chem. Abstr. 65, 1774131 (1966). Angla, B. 1965. Water solubility of essential oils and aromatic products. France Purfum 8(45), 323-334 (in Fr.); Chem. Abstr. 64,4857f (1966). Anonymous, 1963. ‘‘The Atlas HLB System.” Atlas Chemical Ind., Wilmington, Delaware. Anonymous, 1968. Stabilization of various emulsions in food products. French patent 1,533,528 issued to Deutsche Gold- und Siber-Scheideanstalt vorm. Roessler (in Fr.); Chem. Abstr. 71, 12259811 (1969). Aoki, M., Kamada, A, and Matsuzaki, T. 1963. Application of surfaceactive agents to pharmaceutical preparations. XII. The temperature of phase inversion in systems emulsified with nonionic surfactants. 1. Electric resistancetemperature curves and HLB (hydrophile-lipophile balance) of the surfactants. Yakugaku Zusshi 83, 1132-11 36 (in Jap.); Chem. Abstr. 60, 1185231 (1964). Arai, H., and Shinoda, K 1965. The correlation between cloud point in solutions of emulsifiers of Pluronic or Tween type and temperature of phase inversion of emulsion. Nippon Kuguku Zasshi 86(3), 299-301 (in Jap.); Chem. Abstr. 63,17181e (1965). Arai, H., and Shinoda, K. 1967. The effect of mixing of oils and of nonionic surfactants on the phase inversion temperatures of emulsion. J. Colloid Interface Sci 25(3), 396-400. Arbuckle, W. S. 1960. The microscopial examination of the texture and structure of ice cream. Ice Cream n a d e J. 5 6 , 6 2 4 8 . Atherton, J. G., and Maxcy, W. J. 1967. The effect of composition variations of glycerol esters on the physical properties of cosmetic emulsions. Proc. Sci Sect. Toilet Goods Ass. 1966, NO.46, 39-43. Baba, T., and Bushita, T. 1964. Fatty acid esters of sucrose as surfactants. 1. Critical micelle concentrations and solubilization powers of fatty acid esters of sucrose. Kogyo Kagaku Zusshi 67(12), 2077-2081 (in Jap.); Chem. Abstr. 63, 11878c (1965). Bancroft, W. D. 1913. Theory of emulsification. V.J. Phys. Chem. 17,501-519. Barry, B. W. 1969. Control of oil-in-water emulsion consistency using mixed emulsifiers. J. Pharm Pharmucol. 21(8), 533-540. Bashura, G. S., Gluzman, M. Kh., Labunskii, E. V., Trunova, M. A., and Levitskaya, I. B. 1969. Surfaceactive and emulsifying properties of polyoxyethylenated wool wax alcohols studied for use in making emulsions and ointments. Farm Zh. (Kiev) 24(3), 53-57 (in Ukr.); Chem. Abstr. 7 1 , 5 3 5 4 4 ~(1969). Becher, P. 1958. The effect of the nature of the emulsifying agent on emulsion inversion. SOC.Cosmet. Chem. 9,141-148. Becher, P. 1960. Spreading, HLB, and emulsion stability. J. SOC. Cosmet. Chem. 11, 325-332.
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Becher, P. 1965. “Emulsions: Theory and Practice.” Reinhold, New York. Becher, P. 1967a Effect of preparation parameters on the initial size distribution function in oil-in-water emulsions. J. Colloid Interface Sci 24(1), 91-96. Becher, P. 1967b. Micelle formation in aqueous and nonaqueous solutions. In “Nonionic Surfactants” (M. J. Schick, ed), p. 478. Marcel Dekker, New York. Becher, P. 1967c. Nonionic surface-active compounds. M. Micellar properties of monodisperse nonionic surfaceactive compounds. Chem Phys Appl. Surface Active Substances, Proc Int. Congr., 4th, 1964, 2,621-629. Becher, P., and Becher, D. 1969. Effect of hydrophilelipophile balance on contact angle of solutions of nonionic surface-active agents. Relation to adjuvant effects. Advun. G e m . Ser. 1967, No. 86,15-23. Becher, P., and Birkmeier, R. L 1964. The determination of hydrophildipophile balance (HLB) by gas-liquid chromatography (GLC). J. Amer. Oil Chem SOC. 41(3), 169-172. Becher, P., and Griffii, W. C. 1970. “HLB: A Bibliography.” Atlas Chemical Industries, Wdmington, Delaware. Beerbower, k , and Hill, M. W. 1971. The cohesive energy ratio of emulsions-a fundamental basis for the HLB concept. In “McCutcheon’s 1971/Detergents & Emulsifiers,” p. 223, Allured, Ridgewood, New Jersey. Ben-Et, G., and Tatarsky, D. 1972. Application of NMR for the determination of HLB values of nonionic surfactants. J. Amer. Oil Chem. SOC.49,499-500. Bennister, H. L, King, A, and Thomas, R K. 1940. Stability of emulsions. 111. A general survey of solid emulsifying agents with special reference to the hydrous oxides and hydroxides. J. SOC.Chem. Ind. 59,226-232. Benson, F. R, Griffm, W. C, and Truax, H. M, 1962. Statistical approach to common variables in emulsion preparation J. Soc. Cosmet. Chem. 13,437448. Bergwein, K. 1967. Emulsion technology and HLB-value. Fette Seifen Anstrichm. 69(5), 353-355 (in Ger.); Chem. Abstr. 67,55338e (1967). Bierre, M. 1971. Choosing an emulsifying agent. Soup, Perfum. Cosmet. 44,623-628,646, 650,652,654. Boyd, J., Parkinson, C., and Sherman, P. 1972. Factors affecting emulsion stability, and the HLB concept. J. Colloid Interface Sci 41(2), 359. Brennan, J. G. 1970. Emulsions in food technology. Process Eiochem 5(7), 33-37. Broniarz, J., Szymanowski, J., and Wisniewski, M. 1972. The interdependence between HLB value and the polarization index of saccharose esters. Przem. Chem. (Chem. Ind.) 51(8), 517-519 (in Pol.); Chem. Abstr. 78, 5696b (1973). Buddemeyer, B. D., Moneymaker, J. R., and Meyer, M. C. 1962. A cake-baking study of single and multiple emulsifier systems in a fluid shortening. CereuZ Sci Toduy 7 , 266. Burt, B. W. 1965. An approach to emulsion formulation. J. SOC. Comet. Chem. 16(8), 465475, discussion 475477. Cante, C. J., Frost, J. R, Hornyak, J., and Rosano, H. L 1973. Foamability and foam stability of aminohydroxy salts of long chain sulfates and carboxylates. J. CoZloid Interface Sci. 45(2), 242-251. Carro, R. C., and Jato, J. L. V. 1961. Diffusion of medicaments in ointment excipients. I. Free diffusion. Iodine ointment. A n Real Acud. Farm 27(2), 141-148. Chun, A. H. C., and Martin, A. N. 1961. Measurement of hydrophile-lipophile balance of surface-active agents. J. Phurm Sci 50, 732-736. Chun, A. H. C, J o s h , R. S., and Martin, A. N. 1958. The hydrophile-lipophile balance of gums, Part 1. Drug Cosmet. Ind. 82,164-165, 258-260, 312-313, 393-394. Clarke, R J., and Love, G. 1974. Convenience foods based on spray-dried emulsions. Chem. Ind. Feb., pp. 151-15s. Clayton, W. 1943. “The Theory of Emulsions.” Blakeston, Philadelphia, Pennsylvania.
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Coehn, k 1898.Uber das Ladungsgesetz fur Dielectrica Ann. Phys. (Leipzig) 66, 1191. Corran, J. W. 1946. “Emulsion Technology,” pp. 176-192. Chemical Publ. Co., Brooklyn, New York. Cratin, P. D. 1968. Partitioning at the liquid-liquid interface. Ind. Eng. Chem 60(9),
14-1 9. Cyganska, J . 1968. Application of the dielectric measuring test in the analysis of emulsified cosmetic preparations. Parfuem Kosmet. 49,149-152. Cyganska, J., and Witwicka, J. 1967. “Emulsifiers and Emulsions in the Food Industry,’’ 106 pp. WPLS, Warsaw, Poland (in POL). Czetsch-Lindenwald, H. V. 1961. The colloidality of ointments. Parfuem. Kosmet. 42,
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SUBJECT INDEX
A Alcohols, in Allium, 100-103 Aldehydes, in Allium, 100-103 Akylcysteine sulfoxides in Allium, 77-79 biosynthesis, 85-89 Alkylcys teines in Alliurn, 77 biosynthesis, 85-89 Allicin isolation of, from garlic, 74 as major flavor component in garlic, 94, 95 structure of, 91 AUiin, discovery of, 90 AUiinase discovery of, 74,91 enzymatic activity, 92,93,99, 121122 in flavor development, 118-122 substrates for, 120 AIlium alcohols and aldehydes in, 100-103 alkylcysteine sulfoxides in, 77-79 biosynthesis, 90 alkylcysteines in, 77 alliinase in, role in flavor development, 118-122 glutamyl peptidases in, role in flavor development, 122-123 ydutamyl peptides in, 79-83 pungency, flavor, and aroma of, 73-133 enzyme role in, 116-124 measurement, 114-1 16 research needs on, 125-1 26 sulfur compounds in, 74-83 biosynthesis, 83-90 in crushed Alliurn, 90-1 14
5'-Allyl-L-cysteine sulfoxide, as odor-principle in garlic, 91 Amines, as nitrosamine precursors, 11-12 Antibiotics, scanning electron microscopic studies of effects of, 249-250
B Baked goods, for improved world nutrition, 191-1 93 Beverages, high-protein type, for developing countries, i95 Biscuits, nutritious, for developing countries, 193-194 Botulism from fishery products, 135-185 occurrence and control of, 5-6 Brewing, scanning electron microscopy of, 278-281 C
Carbonyl compounds, formation in onion, 101 Carcinogens, nitrosamines as, 27-32 Cathodoluminescence, in scanning electron microscopy, 216, 219 Cereal grains scanning electron microscopy of, 259-274 processing studies, 275-278 Cereals, storage of, scanning electron microscopic studies of, 242-245 Chives. (See also Allium) yglutamyl peptides in, 80-82 Cigarettes, nitrosamines in smoke of, 53-55 Clostridium botulinum, 135-185 classification and nomenclature of, 142146 in fishery products, 135-185 36 1
362
SUBJECT INDEX
Clostridium botulinurn, continued germination and growth of, factors affecting, 154-157 incidence and distribution of, 137-142 inhibition of, 157-160 isolation and identification of, 146-15 1 toxins from factors affecting, 154-157 properties, 15 1-153 Cloud point, of emulsifiers, 324-325 Colombia, Incaparina use in, 197-198 Cookies, nutritious, for developing countries, 193-194 Crackers, nutritious, for developing countries, 193-194 Critical micelle concentration, of emulsifiers, 322-323 Curing of meat, chemistry of, 4-5 Cycloalliin, biosynthesis of, 88-89 Cysteine, in Allium, biosynthesis of, 85
D Deflection-modulation display, in scanning electron microscopy, 21 7 Dielectric constant, of emulsifiers, 325 Donated foods, for developing countries, 195-196 Duryea, use in developing S. American countries, 198-199
E Eggs, scanning electron microscopy of,
290-291 El Salvador, Incaparina use in, 198 Electron microscopy, scanning type, see Scanning electron microscopy Electron probe microanalysis, by scanning electron microscopy, 223-229 Emulsifiers, 318-327 calorimetric studies on, 325 characterization of, 319-321 critique of, 327 cloud point Of, 324-325 concentrations of, 332-336 critical micelle concentration of, 322-323 dielectric constant of, 325 equilibria of, 334 FDA-approved, 444-445 FDA regulation of, 442-445
Emulsifiers, continued hydrocolloids as, 341 hydrophile-lipophile balance (HLB)of, 31 9-321 identification of, 446 mixtures of, 330-331 nuclear magnetic resonance spectroscopy of, 323 phase inversion temperature (PIT) of, 326 polarity index of, 325 proteins as, 339-340 solids as, 340-341 solubility partitioning of, 323 structure effects of, 436-438 surface and interfacial tension of, 326 types of, 318-319 water number of, 323-324 Emulsions, 309-359 aerated, 341-342 destabilization of, 3 12-3 15 emulsifiers for, 318-327 of fats and oils, 327-330 preparation of, 443 proteins as stabilizers of, 339-340 stability of, 309-359 determination, 316 improvement, 316-31 7 stabilization of, 311-312 terminology of, 310 types of, 310-311 emulsifier effects on, 338-339 tests for, 311
F Fats and oils emulsion stability of, 327-330 dielectric constant, 328 gas chromatography, 328 HLB value, 327-328 phase-inversion temperature spreading coefficient, 328-329 water number, 329 Fermented foods, for developing countries, 201-202 Fishery products Ci. botulinum in, 135-185 preservation of, 160-168 Flavor development of, in onion and garlic, 7 3133
SUBJECT INDEX Flavor, continued enzyme role, 116-1 18 Food products, for improved world nutrition, 187-203 Food technology, intermediate, for developing countries, 199-201 Foods emulsion stability and, 309-359 nitrosamines in, 47-53 scanning electron microscopy of, 258-292 Freeze-drying, for scanning electron miCIOSCOPY, 2 3 3-237 Freeze-etching, for scanning electron microSCOPY, 233-237
G Garlic. (See also Allium) flavor and odor development in, 73-133 7-Glutamyl peptidase, in onion and garlic flavor development, 122-124 7-Glutamyl peptides, in Allium,79-83 biosynthesis, 90 7Glutamyl transpeptidase, in onion and garlic flavor development, 122-124 Guatemala, Incaparina use in, 197
H Hamaker constant, 31 3 Hydrocolloids, as emulsifiers, 341 Hydrophile-lipophile balance (HLB),of emulsifiers, 319-321 applications of, 331-332 limitations of, 332 pHydroxypropanthial, structure of, 97
I Incaparina, use in developing S. American countries, 197-198 Induced-current mode, in scanning electron ~ ~ C I O S C O P Y216-217 ,
L Lachrymatory factor, in onion, 96-97 Low-cost nutritious foods (LCNF), for improved world nutrition, 187-203
363
M Malting, scanning electron microscopy of, 278-281 Mass spectrometry, of nitrosamines, 16-17 Meat, curing of chemistry, 4-5 Microbial growth, scanning electron microscopic studies of, 245-249 Microbiology, scanning electron microscopy applied to, 239-250 Microorganisms, morphology of, using scanning electron microscopy, 240-242 Milk products, scanning electron microsCOPY of, 287-288 Muscle, scanning electron microscopy of, 288-289 Myoglobin, structure of, 4 N Nitrites as CI.botulinum inhibitor, 159 as meat preservants, 2-3 nitrosation of, 8-10, 11 toxicity of, 6 Nitrogen oxides, as nitrosamine precursors, 11-12 N-Nitrosamines, 1-71 adduct formation of, 18-20 analysis of, 33-55 methods, 3 9 4 6 animal studies on, 29-32 biochemical changes caused by, 32-33 biological properties of, 27-33 biosynthesis of, 1 3 as carcinogens, 27-32 chemistry of, 8-27 reactions, 17-27 colorimetric analysis, 4 1 4 2 cyclization of, 24-25 detectors for, 4 3 4 5 epidemiological studies on, 27-29 in foods, 47-5 3 gas-liquid chromatography of, 4 2 4 3 hydrogen bonding of, 18-20 hydrolysis of, 17-18 isolation of, 34-39 mass spectrometry of, 16-17,4546 from nitrosation, 8-10 nonvolatile, 46-47 oxidation of, 24
364
SUBJECT INDEX
N-Nitrosamines, continued photochemistry of, 25-27 polarographic analysis of, 3 9 4 0 properties of, 13-14 reduction of, 21-24 spectroscopy of, 14-16,4041 structure of, 14-17 synthetic routes for, 10-13 in tobacco, 53-55 toxicity of acute, 29-30 historical aspects, subacute, 30-32 transnitrosation of, 20-21 Nitrosomyoglobin, structure of, 4 Noodles, fried, nutritious, for developing countries, 194 Nutrition, in world, food products to improve, 181-203
S-(1-Propeny1)-L-cysteinesulfoxide as alliinase substrate, 96-97 as odor principle in onion, 91 structure of, 96 1-Propenylsulfenic acid, structure of, 97 Pro-Teen, use in developing countries, 196191 Protein beverages, for developing countries, 195 Protein food products, textured, for improved world nutrition, 187-203 Proteins, as emulsion stabilizers, 339-340 Pungency development of, in onion and garlic, 73133 enzyme role, 116-1 18 Pyridoxal phosphate, reactions catalyzed by, 98-99
R 0 Odor development of, in onion and garlic, 73133 enzyme role, 116-1 18 Oilseeds, scanning electron microscopy of, 286-287 Onion. (See also Allium) flavor and odor development in, 73-133 lachrymatory factor in, 96-97 threshold values of compounds of, 113Onion oil, components of, 1 10-1 1 1 Osmotic dehydration, as food technological method, for developing countries, 200
P Panama, Incaparina use in, 198 Pasta products, nutritious, for developing countries, 195 Phase inversion temperature (PIT), of emulsifiers, 326 Pickling, use for developing countries, 201 Plants, scanning electron microscopic studies on, 250-252 Polarity index, of emulsifiers, 325 Propanal, as flavor component in onions, 101
Radiation preservation, of fishery products, 166-167
S Salt preservation, botulism and, 165 Sanitation, scanning electron microscopic studies on, 292 Scanning electron microscopy (SEM), 205-307 ancillary techniques for, 237 of antibiotic effects, 249-250 applications of, 21 7-222 in microbiology, 239-250 miscellaneous biological, 252--258 in plant studies, 250-252 of cereal grains, 259-274 processing studies, 275-278 of cereal storage, 242-245 contrast mechanisms in, 215-217 elemental analyses by, 222-231 electron probe microanalysis by, 223-229 of foods and food products, 258-292 historical aspects of, 206 information sources on, 207-208 instrumentation for, 209-217 of malting and brewing, 278-281 of milk products, 281-288
SUBJECT INDEX SEM, continued of oilseeds, 286-287 principles of, 208-21 7 sample preparation for, 200, 231-240 in sanitation studies, 292 specimen manipulation in, 238-239 of starches, 281-286 transmission electron microscopy (TEM) compared to, 210-21 1 use as transmission electron microscopy. 220 Secondary electron emission, in scanning electron microscopy, 215 Smoked food products, botulism from, 164-165,166 Smoking, nitrosamines and, 53-55 Snack foods, of high nutritive quality, for developing countries, 196-197 Spectroscopy, of nitrosamines, 14-16 Starches, scanning electron microscopy of, 281-286
365
Stokes’ law, 315 Sulfur compounds in Allium, 74-83 biosynthesis, 83-90 crushed, 103-114
T Tempeh, as fermented food, 200 Thiopropanal S-oxide, structure of, 97 Tobacco, nitrosarnines in, 53-55 Toxins, from CI. botulinum, 151-153 Transmission electron microscopy (TEM), 206 comparison to scanning electron micxoscopy, 210-211
X X-ray microanalysis in scanning electron microscopy, 216, 21 9
E R RATA Volume 20 Page 237: Equation (17) should read:
Page 241, line 11: tJ =
o'2 5 3 tf= 0.0964tf (1/12)' X 72 X 220
Page 241, line 15: tf= 0.30/0.0964 = 3.11 hours (3 hours, 7 minutes) Page 246: Last parenthetical expression in Eq. (35) should read:
Page 247, line 4: Q = 130.5 Btu/lb Page 247, line 6: t f = [ 1.00445 X (70 - 27)] X
6: y23
x 1 x-+-0.0833 1 0.0833* 3.2
8
(
0.8
)]
= 3.08 hI
Page 247, line 13: D = 1/12 foot, not 1.12 ft Page 247, line 18: Should read: Eqs. (25) and (26) Page251,line2: w=O.5/12=O.O417ft Page 251, line 3: B = 3.2 X 0.0417 = o.0834 2 X 0.8 Page 251, line 5: G = (B + 1)/S = (0.0834 + 1) /1 = 1.0834 Page 251, line 10: 120.1 + (120.2 - 113.5) = 126.8 Btu/lb Page 2.51, line 13: Should read: From Eq. (40) we have Page 251, line 15:
A 6
- 1.0834 X 66 X 98.0 X 0.0417 3.2 X [ 21 - (-20)] = 2.23 hr
366
8 7 C 8
D 9 E O
F 6 H 1 J
1 2 3 4 5