Advances in Food Research, Volume 25

ADVANCES IN FOOD RESEARCH

VOLUME 25

Contributors to This Volume

R. B . Beelman Larry R. Beuchat Milford S. Brown J. F. Gallander D. Hadziyev Kauko K. Miikinen Stephen L. Rice L. Steele Reino Ylikahri

ADVANCES IN FOOD RESEARCH VOLUME 25

Edited by C. 0. CHICHESTER The Nutrition Foundation, Inc. New York, New York and University of Rhode Island Kingston, Rhode Island

E. M. MRAK

G . F. STEWART

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University of California Davis, Calqornia

Editorial Board E. C. BATE-SMITH J . HAWTHORN M. A. JOSLYN J . F. KEFFORD

S . LEPKOVSKY EDWARD SELTZER W. M. URBAIN J . R. VICKERY

1979

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9 8 7 6 5 4 3 2 1

CONTENTS Contributors to Volume 25

vii

Wlne Deacldlficatlon R. B. Beelman and J. F. Gallander

I. lntroduction . . . . . . . . . . . . . . . . . . . ... Acidic Components of Grapes and ... Acidity Changes During Normal Vinification .............. ... Physiochemical Methods of Wine Deacidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Methods of Wine Deacidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Ill. IV. V. VI.

I 3 10 13 25 42 44

Dehydrated Mashed Potatoes-Chemlcaland Blochemlcal Aspects D. Hadziyev and L. Steele 1. 11. 111. IV.

V. VI. VII. VIII. 1X.

lntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dehydrated Mashed Potato Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of Some Cell Constituents in Granule Processing. . . . . . . . . . . . . . . . . . Flavoring Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Sulfites as Additives . . . . . . ................... Microflora as Affected by Processing . . . ............................ Rancidity during Storage and Shipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Some Characteristics of Reconstituted Granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................................... Research Needs.. . . . ......................................... References . . . . . . . . .

55

56 61 102

I09 111 1 12 122 123 124

Xylitol and Oral Health Kauko K. Makinen

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Xylitol and Dental Caries. . . . . . . . . . . . . ............................... 111. Microbiological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V . ffa Vitro Plaque Studies. . . . . ...................................... V. Xylitol and the Exocrine Gla .................... V1. Xylitol and Periodontal Diseases . . . . . . . . . . . . . . . ....................

137 139 147

149 149 152 V

vi

CONTENTS

VI1 . Mechanism of Action of Xylitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153 156 156

Metabolic and Nutritional Aspects of Xylitol Reino Ylikahri

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of Endogenous Xylitol ........... ........... Metabolism and Metabolic Effects ylitol ...................... Use of Xylitol in Nutrition and Therapy . . . . . . . . . . . . . . . . . . . ............. Toxicological Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vl . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . ............. 11. I11 . IV . V.

159 160 162 167 170 174 176 176

Frozen Fruits and Vegetables: Their Chemistry. Physics. and Cryobioiogy Milford S. Brown

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Ice Formation in Biological Materials and Model Systems . . . . . . . . . . . . . . . . . . . . . 111. Survival of Plants at Low Temperatures ...................... IV . Refrigerated and Frozen Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Progress and Problems Remaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

181

183 193 211 229 230

.

Byssochlamys spp and Their importance In Processed Fruits Larry R . Beuchat and Stephen L . Rice 1. I1. 111. 1V . V. VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spoilage . . . . . . . . . ..... ................ Metabolic Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection and Enumeration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Subject Index

.............................................

237 238 242 252 277 280 281

289

CONTRIBUTORS TO VOLUME 25 Numbers in parentheses indicate the pages on which the authors' contributions begin.

R. B. Beelman, Department of Food Science, The Pennsylvania State University, University Park, Pennsylvania ( I ) Larry R. Beuchat, Department of Food Science, University of Georgia Agriculture Experiment Station, Experiment, Georgia 30212 (237) Milford S. Brown, Western Regional Research Center, Science and Education Administration, U.S.Department of Agriculture, Berkeley, California 94710 (181)

J . F. Gallander, Department of Horticulture, The Ohio Agricultural Research and Development Center, Wooster, Ohio ( I )

D . Hadziyev, Department of Food Science, University of Alberta, Edmonton, Alberta, Canada T6G 2N2 (55) Kauko M. Miikinen,* Department of Biochemistry, Institute of Dentistry, University of Turku, Turku, Finland (137) Stephen L. Rice,t Department of Food Science, University of Georgia Agriculture Experiment Station, Experiment, Georgia 30212 (237) L. Steele, Department of Food Science, University of Alberta, Edmonton, Alberta, Canada T6G 2N2 (55) Reino Ylikahri, Third Department of Medicine, University of Helsinki, Helsinki, Finland (159)

*Resent address: Department of Biochemistry and Biophysics, Texas A. & M. University, College Station, Texas 77843. thesent address: Department of Horticulture, Food Sciences Institute, Purdue University, West Layfayette, Indiana 47907. vii

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ADVANCES IN FOOD RESEARCH. VOL. 25

WINE DEACIDIFICATION* R. B. BEELMANT AND J. F. GALLANDERS

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Acidic Components of Grapes and W i n e s . . . . . . . . . . . . . . A. Wine Tartness . . ................................... B. Recommended Ac ines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Factors Affecting Acidity of Grapes. . . . . . . . . , , . , . . . . . . , , . . , . . . . . , 111. Acidity Changes During Normal Vinification . . . . . . . . . . . . . . . . . . . . . . . . . . A. Acids Produced During Fermentation . . . . . . . . . . . . . . B. Reduction in Wine Acidity . . . . . . . . . . . . . . . . . . . . . IV. Physiochemical Methods of Wine Deacidification . . . . . . . . . . . . . . . . . . . . . . A. Amelioration . . . . . . . . . . . . . . . . . . . . . . . B. Neutralization and Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Ion Exchange . . . . . . . . . . . . . . . . . . . . . . ..................... Deacidification . . . . . . . . . , . . . . . . . . , . . . . . . . V. Biological Methods of A. Malo-lactic Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fermentation of Malic Acid with Schizosacchnromyres pombe . . . . . . . . C. Carbonic Maceration , , , . . , , . . . . . . . . . . . . . . . . . , . . VI. Summary and Research N e e d s , , , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... ........................... References . . . . . . . . .

1.

i 3 3 4 5 10

10 11 13 13 16 23

25 25 32 38 42 44

INTRODUCTION

Maynard Amerine (1964b) once described wine as “a chemical symphony composed of ethyl alcohol, several other alcohols, sugars, other carbohydrates, polyphenols, aldehydes, ketones, enzymes, pigments, at least half a dozen vitamins, 15 to 20 minerals, more than 22 organic acids, and other grace notes that have not yet been identified. Since that time, many wine components have ”

*This paper is number 5597 i n the journal series of the Pennsylvania Agricultural Experiment Station and number 143-78 of the Ohio Agricultural Research and Development Center. ?Department of Food Science, The Pennsylvania State University, University Park, Pennsylvania. $Department of Horticulture, The Ohio Agricultural Research and Development Center, Wooster, Ohio. 1 Copyright @ 1979 by Academic Pie\\. Inc All rights of reproduction in any form reaerved ISBN 0-12-016425-6

2

R. B . BEELMAN AND J . F. GALLANDER

been identified, but his point that wines are immensely complex chemical solutions was well made. The factors which distinguish a great wine from just an ordinary wine are still not well understood. However, all good wines are at least properly balanced with respect to some of their major constituents. To produce good wine, the sugar, acid, and tannin content of the grapes should be properly balanced (Amerine and Joslyn, 1970). The proper sugar content is important to assure the development of the appropriate alcohol concentration for the particular type of wine. Tannin imparts astringency and is important in the maturation of wine. However, Milisavljevic (1971) stated that no component of the wine has such extensive and important functions as the acidity. The most important function is the tart taste imparted by the acids. Additionally, the acidity has an important influence on the color, clarity, and stability of the wine. Also, the acids in wine have important secondary effects on wine quality, e.g., functioning as substrates for microbial metabolism and hence increase sensory complexity of wine. However, the most readily apparent aspect of the acidity is its effect on taste. If too little acid is present in grapes, the resultant wines will taste flat or insipid. Too much acidity in wine will cause it to taste sour rather than pleasantly tart. Grapes are grown and made into wine in many areas of the world. Wines from grapes grown in warm climates such as countries in the Mediterranean basin, Australia, South Africa, and the interior valleys of California are generally somewhat bland, soft, high in alcohol, and low in acidity. On the other hand, wines from cooler climates like those of northern Europe and the coastal counties of California are often fruitier, lower in alcohol, higher in inherent acidity, and more delicate and subtle in aroma and flavor (Wagner, 1974). Unfortunately, grapes grown in the cooler areas often do not reach proper maturity before they are harvested. The sugar content of the grapes may be too low and/or the acidity may be too high to make well-balanced wines. The problem is intensified by unusually cold or wet growing seasons, poor vineyard sites, unfavorable variety selection, or poor cultural practices like overcropping. Grapes with high acidity are often used for winemaking even in famous viticultural areas such as the Burgundy, Champagne, and Alsace Districts of France, the Piedmont region of northern Italy, and the Rhine and Mosel River Valleys in Germany. The same problem exists in some of the lesser known areas of eastern Europe and in the midwestern, northeastern, and northwestern viticultural regions of the United States. The addition of sugar to the juice of immature grapes to correct for natural deficiencies, although not altogether desirable, is practiced in many cool viticultural areas (Amerine et ul., 1972). In itself, sugaring probably has no significant adverse effect on wine quality and it is easily accomplished in winery operations (Troost, 1972; Wagner, 1976). Reducing excess acidity in winemak-

WINE DEACIDIFICATION

3

ing is a different matter. Numerous alternative methods of reducing acidity are available, some of which require considerable technical skills. Also, some of these methods can have significant secondary effects on wine quality. The purpose of this report is to review the present knowledge of wine deacidification. Emphasis of the discussion will be on the technology relating to vinification of table wines

.’

II. ACIDIC COMPONENTS OF GRAPES AND WINES A.

WINE TARTNESS

Among the attributes of the acids found in wine, the acidic taste imparted by them is the prime factor in determining the acidity adjustments necessary during vinification. In most instances, the reduction of excess acidity to a level producing appropriate tartness does not cause problems with wine color or stability provided the method employed does not alter pH excessively. These defects are usually associated with wine lacking proper acidity with a correspondingly high pH values. Thus, winemakers working with high-acid wines implement deacidification with a special attention given to taste. Although tartness is important in all wine types, it is critical in table wines, since these wines are usually dry (lacking sweetness). In dry table wines proper acid balance is a major sensory characteristic. Enologists often describe wines high in acidity as “sharp,” “green,” “acidulus,” or “unripe.“ The term ‘‘tart” usually refers to a pleasing fresh taste. This characteristic is extremely important in table wines and receives considerable attention by winemakers. Wine tartness is influenced by the types and amounts of the various acids present, the buffering capacity of the wine, and the sugar and other components present. The major acids in wine are tartaric and malic acids but numerous other acids are usually present in varying concentrations. Amerine et al. (1965) employed a trained panel to rank the sourness of the different acids found in wine at the same total acidity and found malic > tartaric > citric > lactic. The ranking for sourness at equal pH was found as malic > lactic > citric > tartaric. The panel was able to detect differences of 0.05 pH units and 0.03 to 0.05% total acidity (expressed as tartrate). Berg et al. (1955) reported on both the threshold and minimum concentration differences for a number of acids found in wine. Ough (1963) found that citric acid was judged most sour, fumaric and tartaric acids about equal, and adipic least sour when they were added to dry white wines ‘For the purpose of this review, table wines are defined as wines made from grapes. still or sparkling, dry or sweet, containing less than 14% alcohol by volume.

4

R. B. BEELMAN AND J . F. GALLANDER

on a direct molar basis. Amerine (1964a) found that concentrations of acids were more distinguishable at lower sugar concentrations and that alcohol moderated the acidic taste of wine. Noordeloos and Nagel (1972) reported that added sugar reduced the apparent acid taste of wines. In this regard, Munz (1965) discussed the harmonizing influence of the residual sugar usually maintained in German wines on the acidity of these wines. Wine tartness is related both to total acidity and pH of wine (Amerine et al., 1965). Munz (1963a,b) stressed that the acid taste of wine is produced both by the hydrogen ion concentration as well as the fraction of the acids which are undissociated since most of the acids are partially neutralized at wine pH. Winemakers commonly balance the acidity of wine based only on the total acidity expressed as grams of tartaric acid/100 ml. It is usually determined by titration of a wine sample with a standard solution of sodium hydroxide to about pH 8.2 (Amerine and Ough, 1974). However, this is probably not sufficient, since Wejnar (1968) demonstrated that a poor correlation ( p = -0.046) existed between pH and total acidity of wine. He did demonstrate that a close positive correlation ( p = 0.785) existed between tartaric acid concentration and pH while a negative correlation ( p = -0.622) was found between malic acid concentration and pH. Wejnar (1968) also reported that pH was regulated mostly by tartaric acid content ( p = 0.789) and particularly by the ratio of tartaric acid to potassium content ( p = 0.914) and tartaric acid to alkalinity of the ash ( p = 0.933). Thus, winemakers wishing to control wine tartness must be concerned with both total acidity and pH. B.

RECOMMENDED ACIDITY FOR WINES

Numerous recommendations for optimum total acidity values in table wines are found in the literature. However, total acidity values in the range 0.55-0.85% are generally considered appropriate for table wines (Amerine et a / . , 1972; Amerine and Joslyn, 1970; Faber, 1970; Webb and Berg, 1955). Generally, values on the lower end of the above range are recommended for red wines and those on the higher end are considered best for white wines. In a study involving sensory evaluation of wines by numerical scoring, Amerine and Roessler (1964) stated that wines within the range 0.65-0.85% total acidity should receive the highest score. They also recommended that wines with total acidities in excess of 0.85% and between 0.50 and 0.65% should be given the next highest scores. Recommendation of a range and not a specific acidity seems reasonable since regional preferences vary and different styles of table wines undoubtedly require different degrees of tartness. The large variations in total acidity reported in commerical table wines from California (Ough, 1964), Ontario (Crowther and Clark, 1968), and Europe (Faber, 1970) support this assumption.

WINE DEACIDIFICATION

5

Large variations occur in pH of commercial wines. For example, Amerine et al. (1972) cited a range in the pH of California wines of 3.1 to 3.9 while Rebelein (1971a) reported a range of 2.8 to 4.0 in German wines. However, Amerine and Joslyn (1970) noted that table wines with pH values of less than 3.4 taste fresher and fruitier and have better color than wines of higher pH. Thus, winemakers attempt to deacidify wines (reduce total acidity) in a manner which does not increase pH excessively. Little is known concerning the optimum relationship between pH and total acidity in regard to sensory quality of wine. Nagel and McElwain (1977) attempted to determine this relationship for table wines based on sensory scores of wines where pH and total acidity data were known. They found in white table wines with pH values in the range 3.05-3.20, 3.2-3.3, and 3.3-3.5 that optimum range of total acidities were 0.60-0.65%, 0.6-0.85% and 0.85%, respectively. They also demonstrated that in wines of very low pH (<3.1) only minor differences in sensory scores were observed within the entire range of total acidity. They speculated that this might have been due to the generally better quality of the wines fermented at low pH. In this regard, Amerine and Joslyn (1970) related that musts with low pH often resulted in better flavored wines. C.

FACTORS AFFECTING ACIDITY OF GRAPES

Since grape acids are important to wine acidity, certain recommendations have been made regarding optimum acid levels in wine grapes (Amerine and Winkler, 1963; Amerine et al., 1972; Wagner, 1976). A general range for providing acceptable wine tartness is 0.70 to 0.90% total acidity. These limits are slightly higher than those established for wines, because a loss in acidity usually occurs during alcoholic fermentation. Cooke and Berg (1969) conducted a survey of 8 table wine wineries and reported the desirable Brix/acid ratios for 15 wine grapes. The overall average of the total acidities was 0.77%, but the white varieties contained slightly higher levels than the red varieties. The average minimum and maximum acidities were 0.68% and 0.86%, respectively. This lower limit is near the minimum value of 0.70% total acid suggested by Amerine and Winkler (1940) for grapes intended for production of table wines. The major organic acids in grapes are tartaric and malic acids. Malic acid is common to several fruits, but tartaric acid is rarely present in other fruits. These acids and their salts usually account for 90% or more of the total acidity in grapes (Amerine and Winkler, 1942). Several organic acids other than tartaric and malic acids have been identified in grapes. Kliewer (1966) found the following acids in Thompson Seedless grapes: tartaric, malic, citric, isocitric, ascorbic, cis-aconitic, oxalic, glycolic, glyoxylic, succinic, lactic, glutaric, fumaric, pyrrolidone carboxylic, a-ketoglutaric, pyruvic, oxaloacetic , galacturonic, glucuronic, shikimic,

6

R. B. BEELMAN AND J . F. GALLANDER

quinic, chlorogenic, and caffeic acids. Excellent reviews and listings of organic acids in grapes are reported by Amerine (1954), Amerine and Joslyn (1970), and Jaulmes and Flanzy (1971). 1 . Influence of Variety and Climate

Large variations in the acidity of grapes occur among different varieties and between the same varieties grown in different viticultural regions. This trend is illustrated in the data compiled in Table I. Comparisons between the data from California and Washington and the data from southern Ohio and northwestern Pennsylvania demonstrate that the grapes grown in the cooler areas (Washington TABLE 1 AVERAGE COMPOSITION O F MUSTS FROM SEVERAL WINE VARIETIES AT DIFFERENT LOCATIONS IN THE UNITED STATES

Variety

"Brix

pH

Titratable acidity (gm tartaric/ 100 ml)

"Brix

California" Chardonnay Gewiirtztraminer Semillon White Riesling Cabernet Sauvignon Gamay Beaujolais Pinot noir Zinfandel

24.3 22.9 22.3 22.0 22.0 23.1 22.9 21.3

3.22 3.46 3.38 3.21 3.33 3.27 3.30 3.16

pH

Washingtonb 0.79 0.65 0.68 0.80 0.81 0.82 0.76 0.94

23.4 20.4 20.8 21.4 21.4 22.6 22.3 21.7

Pennsylvania" Aurore Delaware Seyval White Riesling Catawba Villard Blanc De Chaunac Chancellor

17.5 19.5 17.9 17.3 18.2 16.2 17.7 17.3

3.32 3.38 3.16 3.29 3.22 3.17 3.28 3.21

Titratable acidity (gm tartaric/ 100 ml)

3.50 3.50 3.40 3.20 3.50 3.60 3.60 3.40

1.04 0.81 0.81 0.96 0.85 0.88 0.86 0.98

Ohio" 0.99 0.95 0.99 1.13 1.11

1.24 1.15

1.14

15.2 19.6 17.3 17.3 17.1 15.0 18.0 15.6

3.11 3.40 3.21 3.10 3.11 3.00 3.21 3.17

0.84 0.53 0.79 0.98 0.87 1.02 0.99 0.90

Values were taken from Region 1 for years 1946-1958. From Amerine and Winkler (1963).

* From Clore et a / . (1976).

"From Beelman (1971, 1972) and Beelman and McArdle (1973, 1974a). 'l From Gallander and Stetson (1977a).

7

WINE DEACIDIFICATION

and northwestern Pennsylvania) contained higher acidities with nearly equal sugar content ("Brix) compared to the same varieties grown in the warmer regions (California and Southern Ohio). In general, tartaric acid is the dominant acid in most grape varieties (Table 11). Amerine (1951) noted that European studies reported malic acid present in lower levels than tartaric acid in several wine grape varieties, ranging from 12.8 TABLE 11 CONCENTRATIONS OF TARTARIC ACID AND MALlC ACID OF SEVERAL WINE VARIETIES AT DIFFERENT LOCATIONS IN THE UNITED STATES

Variety

Species

"Brix

Tartaric (gm/IOO ml)

Malic (gm/lOO ml)

Tanratel malate ratio

0.70 0.65 0.60 0.58 0.68 0.66 0.74 0.67

0.41 0.31 0.34 0.27 0.28 0.19 0.51 0.29

I .71 2.10 1.76 2.15 2.43 3.47 1.45 2.31

0.70 0.64 0.85 0.83

0.63 0.57 0.55 0.66

1.11 1.12 1.55 1.26

0.50 0.85 0.65

0.51 0.66 0.32

0.98 1.29 2.03

0.52 0.64 0.42

0.45 0.44 0.25

1.16 1.45 1.68

0.28

0.52

0.72

California' 18.8 (early) 25.2 (late) 22.4 (early) 24.0 (late) 21 .O (early) 25.5 (late) 19.7 (early) 24.1 (late)

Chardonnay

V . vinifera

Semillon

V . viniferu

Cabernet Sauvignon

V . vinifera

Grenache

V . viniferu

Chardonnay Semillon Cabernet Sauvignon Grenache

V . viniferu V . viniferu V . vinijera V . vinifera

22.8 20.3 21.9 23.2

Aurore Catawha Niagara

French hybrid V . lahruscu V. lubrusca

15.5 17.5 13.6

Aurore Catawba Niagara

French hybrid V. lubrusca V. lahruscu

17.7 17.1 14. I

Scuppernong

V . roturidifoliu

Washingtonb

New York"

Ohio"

North Carolina' 13.4

From Kliewer et a / . (1967). 'From Nagel er a!. (1972). ' Values were calculated from data (1969-1972) of Rice (1974) From Gallander (1977a). ' From Carroll et al. (1971). (I

8

R. B. BEELMAN AND J . F. GALLANDER

to 46.7%.These results are in agreement with those reported by Amerine (1951) for 12 California varieties of Vitis vinijiera. At maturity, malic acid varied from 14 to 45% for Thompson Seedless and Sylvaner varieties, respectively. In an extensive study of 28 table and 50 wine varieties of V. vinijiera, Kliewer et al. (1967) found that the concentrations of tartrates were usually greater than total malates. Similar results were obtained for Washington-grown grapes of V. viaifera, Vitis labrusca, and American and French hybrids (Nagel ef al., 1972; Johnson and Nagel, 1976). As expected, most varieties contained higher amounts of tartrates than malates at full maturity. These findings were similar to the results reported for grape varieties commonly grown in the northeastern United States (Robinson et a/., 1959; Gallander, 1974a; Rice, 1974). It is noteworthy that tartaric acid is the major acid in most varieties of the two commercial species in the United States, V . viaifera and V . labrusca. This is in contrast to varieties of Vitis rotundifolia, which are commercially important in the southeastern United States. Carroll et al. (1971) analyzed the juice of 12 varieties of muscadine grapes (V. rotundifolia) in each of 3 years and found an average malate content of 0.50% while the tartrate content averaged 0.26%. The relative amounts of tartrates and malates in mature grapes am also dependent upon environment. Data from several studies of varieties grown at different locations in the United States are summarized in Table 11. While the amount of tartrates and malates vary among varieties, differences were also found between climatic regions. For the same varieties the percentage of malate in mature fruit was greater for the northern most regions (Washington and New York). The same trend was shown in Table I for total titratable acidities of grapes at four locations. Washington and Pennsylvania, being farther north than California and southern Ohio, have cooler temperatures and produce more acidic grapes. A further example of the influence of climate (temperature) on the total acidity of grapes is shown in Table 111. Amerine and Winkler (1963) found that fruit from TABLE 111 INFLUENCE OF CLIMATE ON TITRATABLE ACIDITY OF TWO VARIETIES OF GRAPES" Titratable acidity (gm tartarid100 ml) Climatic region

Average degree-days

I I1

2500 2501-3000 300 1 - 3500 350 1-4000 4001

111

IV V (I

From Amerine and Winkler (1963).

Carignane 0.73 0.69 0.67 0.59 0.58

Cabemet Sauvignon 0.72 0.61 0.59 0.65 0.54

9

WINE DEACIDIFICATION

Carignane and Cabernet Sauvignon contained less acid when grown in warmer regions of California. This temperature effect was supported by the results of several studies concerning the effects of temperature and light on the organic acid content of grapes (Kliewer, 1968, 1971; Kliewer and Lider, 1970). Kliewer (1971) found that acidities were higher in grapes ripened at 20°C than at 30°C. A general process during ripening is a simultaneous increase in sugars ("Brix) and decrease in titratable acidity. Amerine and Winkler (1 942) reported that malic acid decreased more rapidly than tartaric acid during maturation. Similar results were found by other investigators, all indicating a sharp decline in malic acid during ripening (Amerine, 1951, 1956; Johnson and Carroll, 1973; Johnson and Nagel, 1976). This phenomenon is also shown in the data of Table I1 and graphically in Fig. 1 taken from the results obtained by Kliewer et af. (1967) and Mattick et al. (1973), respectively. Kliewer (1971) suggested that respiration is the major cause for the decrease in malic and tartaric acids during ripening. Increased rate of respiration was suggested as the cause for increased acid loss at 30°C compared to 20°C. The lower percentage loss of tartrates during ripening may be due to accumulation of insoluble tartrate salts which are not readily degraded by enzymes (Saito and Kasai, 1968).

VARIETY-CONCORD YEAR- 1971

i

4.0

:i

- I6 - 14

28

-12

0 2.4 2

-10

2

m

-8 -6 TOTAL ACIDITY TARTARIC ACID

04t

30 9 19 29 F J U L Y +l-- AUG 10 20

8 18 SEPT

-

28

8

18

i k - OCT d

DATE OF HARVEST

FIG. I . Changes in "Brix and acid contents of Concord grapes during maturation. From Mattick et al. (1973).

10

R. B. BEELMAN AND I. F. GALLANDER

2 . Influence of Cultural Practices Titratable acidity can be influenced by thinning and pruning (Upshall and van Haarlen, 1934; Winkler, 1954, 1958; Weaver et al., 1961), rootstock (Ough et af., 1968), soil fertility (Clore et al., 1965), virus infections (Alley et al., 1963; Kliewer and Lider, 1976), and irrigation (Vaadia and Kasimatis, 1961). The first symptom of overcropping of grapevines is delayed maturation of fruit. Thus, the fruit needs a longer period for maturation. In warm regions, such as California, overcropping results in reduced acidity because of a progressive decline in acids during the prolonged ripening period, a consequence of more degree days (Winkler, 1958; Weaver et al., 1961). However, when overcropping grapes occurs in cool regions, the acid content may be higher at a given degree of ripeness, "Brix. This can be critical in extreme northern wine regions, such as the northeastern United States and northern France and Germany where normal maturity of certain varieties is not obtained in some short and cool growing seasons. Virus infection may also affect maturation. Kliewer and Lider (1976) reported that maturity of Burger grapes was delayed by leafroll virus infection. Higher titratable acidities were found in infected fruits on a given date when compared to healthy fruits. Soil moisture also influences titratable acidity of grapes. Grapes from vines exposed to high levels of moisture during fruit ripening tend to be higher in acidity (Vaadia and Kasimatis, 1961; Peynaud and Ribereau-Gayon, 1971). Other workers have found that fruit acidity is also related to vine vigor. Factors which produce vigorous vines tend to yield fruit with high titratable acidity. Ough et al. (1968) showed that the vigorous rootstock (St. George) yielded fruit with highest titratable acidity. Clore et al. (1965) found that fruit acidity is also affected by soil fertility. Vigorous vines on fertile soils produced fruit with higher acid content than from less vigorous vines on less fertile soils. Similar results were obtained from grapes grown in the eastern United States by Beattie and Baldauf (1960) and Upshall and van Haarlem (1934).

Ill. ACIDITY CHANGES DURING NORMAL VlNlFlCATlON A.

ACIDS PRODUCED DURING FERMENTATION

As already noted, the grape is the main contributor to wine acidity. Most of the acidity in wine is due to tartaric and malic acids. In addition yeasts produce minor amounts of organic acids, both nonvolatile and volatile, through the tricarboxylic acid cycle during aerobic metabolism (Kunkee and Amerine, 1970). In a comprehensive review, Amerine (1954) reported that the major acids formed during alcoholic fermentation included; lactic, succinic, citric, acetic, and formic.

WINE DEACIDIFICATION

11

The formation of lactic acid by yeasts is usually less than 0.1% (Amerine, 1950). Under anaerobic conditions, small amounts of D-( -)-lactic are formed through the metabolism of carbohydrates by wine yeasts and lactic acid bacteria. However, if malo-lactic fermentation occurs, larger amounts of L-( +)-lactic acid is formed through the conversion of L-malic acid. This reduction in malic acid leads to an increase of lactic acid content in the wines, but actually lowers the total acidity (see Section V,A). Succinic acid is the predominant nonvolatile organic acid formed during alcoholic fermentation. In commercial wines, Amerine (1954) and Schopfer (1971) reported that succinic acid concentrations are about 0.1%.

Small amounts of citric acid are found in both grape musts and wines. Amerine (1954) reported that wines contained 0.02 to 0.17% citric acid. Levels above

0.05% imply that citric acid was added to protect against ferric phosphate cloudiness or to improve flavor (tartness). More recently, Fong et al. (1974) analyzed 25 experimental wines to which no acid has been added and found approximately 0.20% citric acid. The volatile acidity of wines is one of the most important measurements for enologists. The amount is calculated as acetic acid which is the major volatile acid. Small quantities of acetic acid (about 0.03 gnd100 ml) are always produced during the normal fermentation process (Amerine and Ough, 1974). However, a high volatile acidity content is an indication of acetic acid spoilage, particularly by bacteria in the genus Acerobacter. The maximum volatile acidity permitted by the Federal regulations is 0.140 and 0.120 g d 1 0 0 ml for red and white table wines, respectively. B.

REDUCTION IN WINE ACIDITY

Even though several nonvolatile and volatile acids are produced during alcoholic fermentation, a net loss in titratable acidity is usually experienced when comparing the grape must to the finished wine (Kluba and Beelman, 1975). Grape variety is an important factor which influences the reduction in titratable acidity during vinification. Often, the greatest loss occurs with those varieties highest in initial titratable acidity. Kluba and Beelman (1975) reported on the changes in titratable acidity for 4 varieties (Table IV). The decrease in titratable acidity from the must to the wine ranged from 13.9% (Seyval) to 32.3% (Chancellor). The greatest reduction occumd in the variety Chancellor which exhibited the highest acidity, 1.21%. Similar results regarding acid reduction during vinification were reported by other investigators (Amerine and Winkler, 1963; Clore et al., 1976; Nagel et al., 1972; Rice, 1974; Gallander and Stetson, 1976). In addition to variety, these reports indicate that the degree of acid reduction for a given variety depends upon growing season, fruit maturity, and vineyard location.

12

R. B. BEELMAN AND J . F. GALLANDER

TABLE IV REDUCTION IN TITRATABLE ACIDITY, MALATE, AND TARTRATE CONCENTRATIONS FROM MUSTS TO FINISHED WINES" Finished wine

Must

Variety

Titratable acidity (gm tartaric/ 100 ml)

Malate (gd100 ml)

Tartrate (gm/100 rnl)

Titratable acidity (gm tartaric/ 100 ml)

Chelois Chancellor Seyval Villard Blanc

1.15 1.21 0.82 1.11

0.50 0.52 0.35 0.53

0.73 0.66 0.62 0.83

0.84 0.82 0.71 0.86

Tartrate (gdloo ml)

~

a

0.33 0.37 0.22 0.31

0.26 0.19 0.25 0.29

From Kluba and Beelrnan (1975).

The titratable acidity of the wine may exceed the acid level of the must, especially if the fruit is initially low in acidity. Several factors may be responsible for this apparent increase in acidity during vinification. Carter et al. (1972) showed the importance of sample preparation of grape berries for acid analyses and found that some methods are more representative of the must and wine than others. Also, total (titratable) acidity is expressed in tartrate equivalents. Certainly, other components such as inorganic acids, amino acids, and phenolic compounds contribute to titratable acidity. In addition, the production of acids during alcoholic fermentation (see Section III,A) may cause the wine acidity to exceed the titratable acidity of the must. 1 . Malic Acid Fermentation The reduction in titratable acidity during normal alcoholic fermentation is attributed to the loss in malic and tartaric acids. Kluba and Beelman (1975) demonstrated decreases in the malate and tartrate concentrations from must to wine (see Table IV). The reduction in malate content ranged from 28.8 to 41.5%. The prime explanation for the loss in malate content is the decomposition of L-malic acid by yeasts. Rankine (1966) reported that Saccharomyces yeasts utilized L-malic acid from 3 to 45% during fermentation. Examining several Bordeaux wines, Peynaud (1938), reported 10 to 14.5%reduction in L-malic acid during alcoholic fermentation. 2 , Tartrate Precipitation

Generally a net loss of tartrates which occurs during the vinification process lowers the total acidity to some degree. Kluba and Beelmen (1975) reported that

WINE DEACIDIFICATION

13

tartrates decreased more than malates during vinification (Table IV), apparently due to the precipitation of potassium bitartrate. As the alcohol content increases during fermentation, the solubility of potassium bitartrate decreases, and a portion is precipitated from the wine. Since crystallization of the excess potassium bitartrate is not immediate, wineries commonly employ low temperatures to accelerate the rate of precipitation. Cooling the wine to just above freezing for 2 to 3 weeks is usually sufficient to stabilize wines against tartrate deposition. However, the rate of bitartrate deposition during chilling varies greatly, not only among wine types but within each type (Berg et al., 1968; Berg and Akiyoshi, 1971). Tartrate-holding capacity of a particular wine depends upon its pH, alcohol, potassium, and tartrate contents (Berg and Keefer, 1958). Polyphenols and other wine components are also thought to increase the solubility of potassium bitartrate in wines. It is evident that potassium bitartrate precipitation from wines is a complex phenomenon and not a simple reaction between potassium and tartaric acid. Wejnar (1969a) studied the influence of the precipitation of potassium bitartrate from wine on the pH of the wine. He demonstrated that the pH may increase, remain the same, or even decrease as the result of the precipitation of tartrate during fermentation depending upon the buffering capacity of the wine which in turn is regulated by the solubility product. Thus, it is possible that both a decrease in total acidity and pH may occur in wine following tartrate precipitation.

IV. PHYSIOCHEMICAL METHODS OF WINE DEACIDIFICATION A.

AMELIORATION

In some winemaking regions where grapes commonly contain excess acidity and insufficient sugar to make balanced wines, a common practice is to add both sugar and water to the must or fermenting wine. This practice is referred to as amelioration in the eastern United States and Canada where it is a common method to reduce acidity. In Germany, where the addition of sugar solutions to musts has been a common practice to reduce acidity for many years, the process is called Nasszuckerung (wet sugaring) or Nassverhesserung (wet improvement). The legal definition (U.S. Internal Revenue Service, 1974) of amelioration in the United States is as follows: The addition to juice or wine, before, during. and after fermentation of either water or pure sugar, or a combination of water and pure sugar, or liquid sugar or invert sugar syrup, to adJust the acid content or develop alcohol by fernlentation.

14

R. B . BEELMAN AND J . F. GALLANDER

Thus, amelioration in the United States does not necessarily imply a process to reduce acidity, but can simply involve the addition of sugar to grapes to increase potential alcohol content of the resultant wine. In Germany, this is called Trockenzuckerung (dry sugaring). Such a practice is often necessary in cool winemaking regions or in cool years even in areas such as Burgundy. In France, the addition of sugar to musts is called chapitalisation. The addition of sugar increases wine volume and tartrate precipitation following fermentation and thus acidity is probably reduced slightly. However, amelioration is usually employed to simultaneously decrease acidity and increase sugar in the must. Even if the natural sugar content of a must is adequate for the desired alcohol level of the wine, sufficient sugar is added with the water to maintain the original level of sugar in the must. The degree of amelioration of wine is regulated in the United States (U.S. Internal Revenue Service, 1974) to allow the reduction of the total acidity of a grape must to 0.5 g d 1 0 0 ml as long as the added water and sugar does not exceed 35% of the total volume of the ameliorated material. Full amelioration is permitted in juice with a total acidity in excess of 0.769 g d 1 0 0 ml. The same regulations apply to all fruits and berry wines except those made from currants, gooseberries, and loganberries. With these fruits, the maximum level of amelioration is 60% of the resultant volume. Prior to the new German Wine Law of 1971, the addition of sugared water to German wines was permitted up to a maximum of 25%. The Common Market has authorized the use of amelioration until 1979 but limits the levels to 15% for ordinary table wines and 10% for quality wines (Faber, 1970). In Ontario, Canada, amelioration is limited only to the extent that the law permits the production of up to 250 gallons of wine per ton of grapes (Crowther and Clark, 1968). California regulations do not permit the addition of sugar or water to juice or wine (Amerine et al., 1972). The addition of water to facilitate crushing and for the addition of aqueous slurries of bentonite for clarification are exceptions to the California regulations. Rice (1974) discussed amelioration as practiced in commercial wineries in the eastern United States. The degree of amelioration used with a specific must depends upon the composition of the must and the type of wine to be made. Total acidity and sugar content, as well as the color and flavor intensity of the must, are considered by the winemaker as criteria for deciding the degree of amelioration to be employed. After pressing, the must is sampled and analyzed for total acidity. The amelioration credits allowable are determined in gallons of ameliorating material. The ameliorating material can be added before, during, or after fermentation, but must be used prior to finishing the new wine. The timing of this addition varies in practice. In musts low in sugar content, some sugar addition is usually necessary prior to fermentation to produce wines of sufficient alcohol to be stable. Thus, a portion of the amelioration credits is used at that time. Following fermentation the new wine is evaluated organoleptically to de-

15

WINE DEACIDIFICATION

termine whether remaining amelioration credits will be use or waived. Normally only a portion of the credits available for each wine are used, depending upon the winemaker and yearly variations in must quality. Kluba and Beelman (1975) studied the influence of various levels of amelioration on the acidity of musts and wines of several French-hybrid grape varieties used for winemaking in the eastern United States. Results with one variety (Table V) demonstrate the trends found in the study. The acidity of musts (total acidity, tartrate, and malate) was reduced by amelioration by about the amount expected from dilution. But in wines, the total acidity was reduced by about half the level expected from the dilution in comparison to unameliorated controls. For example, reductions of 4.9, 9.8, and 14.0%were observed when 10, 20, and 30% levels of amelioration were employed, respectively. The reductions were due mainly to decreases in malate. The tartrate content of the wine was not reduced significantly by any level of amelioration employed. The pH of both musts and wines was also unaffected by amelioration. This represents a definite advantage of this process since total acidity can be reduced without increasing the pH-a potential problem with other deacidification procedures like chemical neutralization (see Section IV,B). However, it was concluded that amelioration was an inefficient process for reducing wine acidity. They suggested that amelioration of wines with intense aromas and flavors (e.g., those made from native American varieties like Concord or Catawba) might benefit from appropriate levels of amelioration. On the other hand, they reasoned that dilution of aroma, flavor, body, and color intensity that results from excessive amelioration probably would not be appropriate with wines made from more neutral flavored varieties. In a study with German Riesling wines from the Mosel, Kielhofer (1959) demonstrated that amelioration (Nusszuckerung)did not adversely affect sensory quality. TABLE V INFLUENCE OF AMELIORATION ON THE ACID COMPOSITION OF MUSTS AND WINES OF THE FRENCH-HYBRID GRAPE VARIETY CHANCELLOR' Total acidity (gm tartrate equiv/ Tartrate 100 ml) ( g d 1 0 0 ml) Amelioration level (%) 0 10

20 30

Must

Wine

Must

Wine

1.21ab 0.82a 0.66a 0.19a 1.10b 0.76b 0.57b 0.20a 1 . 0 0 ~ 0 . 7 0 ~ 0 . 5 4 ~ 0.19a 0.89d 0.65d 0.47d 0.19a

Malate ( g d 1 0 0 rnl) Must 0.52a 0.43b 0.40~ 0.37d

Potassium (mg/100 ml)

PH

Wine

Must

Wine

0.37a 0.30b 0.26~ 0.24d

3.39a 3.39, 3.39a 3.40a

3.60a 3.60a 3.60a 3.60a

Must

Wine

195.3a 138.0a 167.5b 127.7a 147.0bc 108.6b 131.7~ 7 6 . 0 ~

" Adapted from Kluba and Beelman (1975). Means in a column followed by the same letter are not significantly different ( k = 100)

16

R . B . BEELMAN AND J . F. GALLANDER

Wines made from the same grapes with only sugar added (Trockenzuckerungdry sugaring) and the acidity adjusted to the same level (0.8%)with calcium carbonate were used for comparison. The wines made using amelioration (addition of a sugar-water solution) had slightly higher tartrate/malate ratios, lower mineral content, and lower pH values all of which were considered desirable and resulted because no calcium was added to the wine. Slightly higher dry extract values (0.1 gm/100 ml) were found in the wines made by dry sugaring mostly as a result of the increased mineral (calcium) content. Troost (1972) stated that 15 to 25% dilutions (amelioration) of acidic German musts, especially Rieslings, generally resulted in wines of superior sensory quality compared to musts that were dry sugared. He related that the dilution reduced the “green,” “unripe” taste often associated with the immature grapes. Rebelein (1971a) also suggested that the addition of sugar-water solutions to acidic German wines resulted in wines of improved sensory quality. He cited that an advantage of amelioration is that it can be employed in combination with other methods of deacidification to obtain desirable effects. In a study comparing various means of deacidification, Troost and Fetter (1 966) found that amelioration (25%) in combination with an addition of calcium carbonate to lower the total acidity to 0.9%, resulted in wines with the best sensory quality. Since amelioration mainly reduces malic acid (Kluba and Beelman, 1975) and neutralization with calcium carbonate reduces tartrate almost exclusively (see Section IV,B), a combination of these two methods would appear desirable from the standpoint of maintaining more natural tartrate to malate ratios in the wine. Rebelein (1971a) suggested the main disadvantage of amelioration is that it is considered an adulteration or falsification of wine. Increasing wine production for economic gain by unscrupulous winemakers through excessive amelioration is a potential problem. Watering of wine as well as other beverages (e.g., milk) has always been a problem in the food industry. The Code of Hammurabi, which dates back to 1792-1686 B.c., mentioned the problem of diluting wine with water (Amerine and Singleton, 1977). Amerine and Joslyn (1970) and Ribereau-Gayon et al. (1976a) reviewed numerous analytical procedures that have been suggested to detect dilution of wine with water. The procedures are based on determination of ratios of various wine components which only can provide presumptive evidence of dilution. In the United States, examination of winery production records is the main method used to detect excessive amelioration. B.

NEUTRALIZATION AND PRECIPITATION

1. Neutralization and Precipitation of Tartrate

Faber (1970) indicated that chemical deacidification (neutralization) was the most common method employed in Europe for reducing the acidity in high-acid

WINE DEACIDIFICATION

17

musts and wines. Acidity was neutralized by one of several mineral salts, with the main deacidification effect resulting from the precipitation of tartrate salts. Calcium carbonate is the most common chemical employed for this purpose. In the presence of excess tartrate (H,T), the reaction with calcium carbonate (CaCO,) is as follows: CaC03 + H 2 T e CaT

4 + H a + CO,

(1)

Potassium carbonate (K,CO,) and neutral potassium tartrate (K,T) both can be used for the same purpose. The reactions of the two chemicals in the presence of excess tartrate are as follows: K2COs + 2 H,T

e 2KHT 4 + H,O + CO,

K2T + 2H,T

* 2KHT

+ H2T

(2)

(3)

Treatment with calcium carbonate was cited by Faber (1970) to be the most practical alternative for use in wine, since potassium carbonate is not approved for use in most wine-producing countries and the expense of potassium tartrate is prohibitive. Jaulmes and Flanzy (1971) indicated a preference for the use of potassium carbonate for neutralization because of potentially fewer problems with calcium tartrate instability, but they also indicated that it was not approved for use in France. The problem of excessive pH increase following the use of potassium carbonate in wine has been reported (Nagel et al., 1975; Munyon and Nagel, 1977). Munyon and Nagel (1977) demonstrated that neutralization with calcium carbonate produced maximal reduction in total acidity with minimum shift in pH when compared to the use of potassium carbonate. They explained that this was due to the difference in the solubility products of the calcium and potassium salts of tartaric acid. The more soluble potassium bitartrate would result in a higher cation concentration in the wine which in turn would result in the formation of acid salts from acids and a greater increase in pH. They reported that this pH increase may be beneficial in wines with very low pH values. Faber (1970) reported that in Europe no consensus existed as appropriate stage of vinification for neutralization. Theoretically, the addition of 0.67 gm of calcium carbonate per liter of must o r wine would reduce the total acidity (expressed as tartrate) by 0.1%due to precipitation of 0.1% tartaric acid (Troost, 1972). Munyon and Nagel (1977) verified the quantitative deacidification using calcium carbonate as long as tartrate remained in the wines. Michod (1958) reported that the addition of the same quantity of calcium carbonate to must or young wine caused the same changes in the composition of the resultant wine. However, he recommended deacidification of wine rather than must because the pH of the must would be lower during the alcoholic fermentation and would favor improved sensory quality of the wine. Schopfer (1971) recommended neutralization

18

R . B. BEELMAN AND J . F. GALLANDER

with calcium carbonate immediately following alcoholic fermentation, since this also permits closer prediction of the final acidity of the wines and the amount of calcium carbonate required. Nagel et a / . (1975) observed that deacidification was more effective when calcium carbonate was added to wine following the natural precipitation of a large amount of potassium bitartrate. They found that deacidification was only about half as efficient when calcium carbonate was added to musts compared to wines. They reasoned that the calcium added to musts was competing for the same tartrate ions that normally would precipitate as potassium bitartrate after the alcoholic fermentation. This was demonstrated by the fact that very little change in the total anion content of the must was observed following the addition of calcium carbonate. They also observed that the potassium content of the must increased following addition of calcium carbonate-a phenomenon also observed by other researchers (Konlechner and Haushofer, 1956; Prillinger, 1958). On the other hand, Munz (1963a) suggested neutralization of musts rather than wines. A more rounded acid balance from a sensory quality standpoint was achieved in the wine due to soluble acid salts produced by the added cations. However, this was recommended only for moderately acidic musts in which very large acidity adjustments were not necessary. Opinions vary considerably regarding the optimum extent of deacidification by the neutralization. Faber (1970) reported that neutralization with calcium carbonate is limited in many countries by the requirement to maintain some tartrate in the wine. The minimum permissible tartrate concentrations allowable were reported to be 0.05,O. 10, and 0.15% in the wines of Germany, Luxembourg, and Switzerland, respectively. Schopfer (1971) stated that in Swiss wines calcium carbonate treatment was conducted in a manner to retain at least 0.15% tartrate and 0.75% total acidity in the wine following alcoholic fermentation. Benvegnin er al. (1951) related that if tartrate concentration was reduced below 0.15% in Swiss wines the quality of the wine was adversely affected. However, Prillinger (1958) reported that complete removal of tartrate using calcium carbonate resulted in no adverse effect on the wine unless the calcium concentration exceeded about 350 ppm, whereby sensory quality was harmed. Tanner and Sandoz (1974) suggested that the optimum acid reduction treatment of musts using calcium carbonate should be based on a treatment sufficient to increase the pH to 3.3 so that malo-lactic fermentation (see Section V,A) could further reduce acidity. They presented a simple method for empirically determining the desired amount of calcium carbonate required for this effect and related that prediction of resultant pH values was extremely difficult because of the variation in buffering capacity of different wines. Usseglio-Tomasset (1973) presented a mathematical formula (based on the Henderson-Hasselbach equation) to predict pH changes in wine following treatment with calcium carbonate and potassium carbonate. Munyon and Nagel (1977) reported excellent results with the prediction of pH and

WINE DEACIDIFICATION

19

total acidity changes in high-acid wines treated with calcium carbonate from empirically derived neutralization curves. The amount of neutralization required could be determined or the final pH and total acidity of treated wine could be predicted from the curves. Gnaegi (1975) presented a graphical method for calculating the proper amount of calcium carbonate to be used in wine deacidification based on the changes in total acidity and tartrate concentration. He preferred this graphical method over those involving measurement of pH changes because of inherent errors in pH determinations and since the method prevented the possibility of reducing the tartrate concentration below desirable levels. A potential problem associated with the use of calcium carbonate is the delayed precipitation of calcium &rate in the wine. Thus, wines treated in this manner are normally held for a sufficient time before bottling to assure stability. This delay was considered a disadvantage of the use of calcium carbonate by Haushofer (1971). For a discussion of factors affecting calcium tartrate stability in wine, see Berg and Keefer (1959). 2 . Double-Salt Deacidification

A major problem resulting with neutralization of musts or wines with calcium carbonate is that most of the deacidification is due to the precipitation of calcium tartrate. Most of the calcium malate formed remains in solution and can cause the wine to taste salty if the concentration is high enough (Wurdig, 1977). Neutralization often increases the pH of the wine excessively which can lead to problems with color and stability. Also, when tartrate concentration is reduced to a low level winemakers must be extremely careful to control malo-lactic fermentation (see Section V,A) since it could reduce the acidity to dangerously low levels and result in completely flat wine. Many of the problems associated with chemical neutralization are prevented with a process developed in Germany called double-salt deacidification. Several different enologists observed in the 1950s that the expected decreases in tartaric acid in musts and wines deacidified by the addition of calcium carbonate were never quite realized (Wurdig, 1977). The phenomenon was most noticeable when the calcium carbonate was added to a small portion of the must or wine to be treated. Kielhofer (1957) suggested that this observation was due to the simultaneous precipitation of tartrate and malate as an insoluble double salt as had been observed originally about 60 years earlier by a French enologist (Ordonneau, 1891). Munz (1960, 1961) recognized the potential of this phenomenon for wine deacidification and studied the conditions necessary for the precipitation of the double salt. He later demonstrated that calcium carbonate should be added to only a portion of the must until the pH was increased to 4.5, or above, whereby the double-salt was formed (Munz, 1961). The treated must was

R. B . BEELMAN AND J. F. GALLANDER

20

then settled overnight and filtered before blending it back with the untreated portion. Later work by Kielhofer and Wurdig (1963, 1964) led to development of an improved procedure for application of the process in winery operations now known as the Acidex procedure which was patented (Munz et af., 1968). The Acidex procedure received its name from the use of a special mixture of finely ground calcium carbonate with about 1% of the calcium double salt named Acidex by the manufacturer (C. H. Boehringer Sohn, 6507 Ingelheim Am Rhein, West Germany). The double-salt seed crystals are added to encourage nucleation and crystal growth for rapid and complete precipitation of the double salt. The double salt that forms as a result of this process is the racemate of an unequal pair of configuratively opposite molecules, namely calcium-D(+)-tartrateL-(-)-malate * 5 H20 (MW = 504). The following expression presented by Steele and Kunkee (1978) shows the reaction occurs: FOOH

YOOH

+

HCOH I HOF H

HOC H

COOH Tartaric acid

/

coo I

HcoH\ I

Ca

HTH / coo Calcium tartrate

+

+

fH2

"? + \HT OOC

Ca

ZCaCO,

,

COOH Malic acid

(4) COO -Ca- OOC I

2C02f

+

2H20-

Cale ium malate

HCOH I HOCH I

H27 HOC I

COO - Ca - OOC Calcium malate-tartrate (double salt)

The solubility product of the double salt was given by Wurdig (1977) in Eq. (5) (Cali)* x (malate2-) x (tartrate2-) = 2.1 x

at 20" C

(5)

Under normal conditions of neutralization with calcium carbonate and with equal molar concentrations of tartaric and malic acids, a mixture of calcium tartrate and double salt is formed. Because of higher pK, values of malic acid in comparison with tartaric acid (pK,'s for tartaric acid are 3.0 and 4.3 and for malic acid are 3.4 and 5.1), the malate ion concentration necessary for the maximum precipitation of the double salt is generally attained only about pH 4.5. For example, Wurdig (1977) reported that in a wine of 1.5% total acidity of which 45%was tartaric acid, the maximum double salt formation would be at pH

WINE DEACIDIFICATION

21

4.55. However, as malic acid concentration of the wine increases, the pH of maximum double salt formation becomes lower. If the above wine contained only 15% of the total acidity as tartaric acid, the maximum pH for double salt formation would be 3.9. Thus, it is only possible for the double salt to form at the pH of wine if the malic acid concentration is very high. The Acidex procedure for double-salt deacidification assures proper pH conditions for maximum double-salt formation. A portion of the must requiring deacidification is stirred into the calcium carbonate (Acidex) so that the initial pH of the treated portion is greater than 5.0 (Kielhofer and Wiirdig, 1963). In practice the calcium carbonate is suspended in a very small amount (about 10%) of the portion of must or wine to be treated. The remainder of the amount to be treated is then slowly pumped into the suspension with vigorous stirring. The double salt which forms is then separated by filtration, centrifugation, or settling. This treated portion is then blended back with the untreated portion of the original must or wine. It is recommended that wine treated by double-salt deacidification be allowed to stabilize for at least 3 months prior to bottling to prevent calcium tartrate precipitation in the bottled wine. Wiirdig (1977) presented formulas for calculating the portion of must or wine to be treated and the amount of calcium carbonate to be employed. Tables which provide the same information along with directions for practical application are available from the manufacturer of Acidex. The Acidex procedure is well suited to the deacidification of high acid musts and wines. Miinz (1961) demonstrated that the total acidity of musts could be reduced by as much as 1 .O-1.2% and wines with 1.2-2.2% total acidity could be deacidified as long as sufficient tartrate was present. Wurdig (1977) provided formulas for calculation of maximum deacidification of musts and wines based on tartrate content. This problem is also presented in the Acidex literature and a table is provided to determine the maximum deacidification possible with various percentages of tartrate in musts o r wines. However, determination of tartrate is only necessary when wine is made from frozen grapes or from musts with over 1.8% total acidity. It has been suggested that if the tartrate concentration is not sufficient for the required deacidification, it is possible to add tartaric acid at the end of the deacidification process (Rebelein, 1970, 1971b). An important advantage of double-salt deacidification is the nearly quantitative reductions in acidity and removal of nearly equilmolar quantities of both tartrate and malate. Deacidification of an acidic German wine by application of the double-salt process on both the must and wine is illustrated in Table VI. In this case the process was employed to reduce the total acidity of both the must and the wine to 0.8%. The data indicate that the procedure was almost equally effective in reducing the acidity when applied to either must or wine and resulted in only a moderate increase in pH of the final wine in both cases. Comparison of

TABLE V1 DEACIDIFICATION OF MUST AND WINE BY THE WUBLE-SALT PROCEDURE" ~~

Deacidification of mustb 2 3 4

1

Must before deacidification Time after deacidification (days) Total acidity @/lo0 mIj Tartaric acid (@lo0ml) Malic acid (dl03ml)

P"

Calcium (rndl) ~~

0 1.54 0.63 0.86 3.0 74

Deacidified fraction of I 0

0.23 0.03 0.18 4.7

United fraction of 1 + 2

0 0.81 0.31 0.49 3.4 340

Wine

6

7

Wine

Wine after fermentation of 1

5

Wine

41 90 0.77 0.77 0.150.14 0.49 0.52 3.3 3.3 125 Ilh

~

Adapted h i m Wurdlg (1977). 4.83 kg Acidex mixed with 540 liters of must separated from 980 liters of original must. 4.68 kg Acidex mixed with 590 liters of wine from 1018 liters of original wine.

203 0.76 0.14 0.49 3.2 125

0 1.49 0.45 0.76 3.0 I25

Deacidification of wine' 8 9

Deacidified fraction of 7

0 0.29 0.01 0.23

4.6

-

United fractions of 7 + 8 0 0.83 0.19 0.47 3.8 460

10

11

Wine

Wine

51

0.80 0.10 0.46 3.3 213

162 0.81 0.1 1 0.46 3.2 162

WINE DEACIDIFICATION

23

the tartaric and malic acid concentrations in columns 1 and 3 demonstrate that 0.37 g d 1 0 0 ml malic acid and 0.32 g d 1 0 0 ml tartaric acid were removed as double salt when the must was treated with Acidex. An additional 0.17 g/lOO ml tartaric acid apparently precipitated from the wine in the form of calcium tartrate and/or potassium bitartrate (compare columns 3 and 6 ) . These data demonstrate that 0.13 gd1OO ml tartaric acid was required to precipitate 0.10 gm/lOO mi malic acid. When wine was treated (columns 7,9, and 11) 0.12 gd1OO ml tartaric acid was required to precipitate 0.10 g d 1 0 0 ml malic acid. Since equilmolar losses of tartaric and malate would require a reduction of 1.12 gm/lOO ml tartrate for 1.00 g d 1 0 0 ml reduction in malate, these data indicate that equilmolar reductions were not quite achieved in either case. Kielhofer and Wiirdig (1 964) indicated that double-salt deacidification improved sensory quality when the process was applied to musts. They related that the reduction of insoluble grape solids prior to fermentation produced cleaner, more pleasant wines. On the other hand, Rebelein (1971b) suggested that double-salt deacidification was best applied to young wines rather than the corresponding musts. He related that it was not only more practical but also resulted in wines of superior quality. A number of studies have shown the double-salt process to be an effective process. Nagel et a/. (1975) found double-salt precipitation to be more effective in reducing acidity than neutralization with either calcium carbonate or potassium carbonate. Troost and Fetter (1 966) also reported that double-salt deacidification was preferable to neutralization with calcium carbonate. Munyon and Nagel (1977) found the double-salt process to be the most effective process in reducing acidity of high acid musts while having the least effect on wine quality. Steele and Kunkee (1 978) demonstrated that nearly quantitative deacidification was attained when the double-salt procedure was employed with high-acid grape musts from the western United States, despite the fact that they did not obtain the theoretical precipitation of equilmolar amounts of malic acid and tartaric acid. Also, essentially no problem with calcium tartrate instability was experienced using the process. C.

ION EXCHANGE

Ion-exchange resins were first used for treating wines in the late 1950s. Their use, including ion-exchange principles, resin types, and operating procedures has been reviewed by Percival et al. (1958), McGarvey et al. (1958), Fessler (1958), Mindler et al. (1958), Rankine ( 1 965), and Troost ( 1 972). If ion-exchange resins are used, certain federal regulations are imposed on the process and resultant wine (U.S. Internal Revenue Service, 1974). Other countries have similar type limitations and regulations on the use of ion-exchange systems and some may prohibit their use.

24

R. B. BEELMAN AND J . F. GALLANDER

I.

Cation Exchange

Most of the attention has been devoted to cation-exchangers for tartrate stabilization of wines. As noted earlier, wine is a supersaturated solution of potassium bitartrate. Unless excess salt is removed, crystallization of potassium bitartrate will occur after bottling. Although chilling is the common method of stabilizing wine for tartrate, cation-exchange systems are presently commercially important for this purpose. One such method makes use of a cation resin in the sodium form. Potassium ions are exchanged with sodium to form sodium salts which are soluble in wine. During ion-exchange a slight reduction in acidity occurs (Dickinson and Stoneman, 1958; Mindler er al., 1958). This small decrease in wine acidity does not warrant use of a sodium exchangers as a means of wine deacidification. In addition. the increase in sodium is not considered desirable.

2 . Anion Exchange The use of anion-exchange resins to treat wines is currently less popular than cation exchangers. Although tartrate stability may be obtained by replacing tartrate ions with hydroxyl ions, anion exchangers are primarily designed for reducing wine acidity. These resins are weakly basic and usually in the hydroxyl form. As the wine passes through the resin, the various anions are replaced with the hydroxyl ions thus reducing acidity. There is conflicting information concerning the merits of treating wines with anion exchange resins. Rankine (1965) stated that in Germany, wine deacidification by ion exchange was not comparable to the calcium carbonate treatment. In contrast, Moser (1956) reported that Austrian wines deacidified by anion exchangers were without noticeable defects in sensory quality. He further stated that the ion-exchanged wines tasted better than the calcium carbonate-treated wines. Other reports from Europe indicate that if the correct technology is applied, anion exchange can be used to produce an acceptable quality wine with lower acidity (Faber, 1970; Minarik, 1971; Haushofer, 1971). Although anion exchange can be successfully employed, there is concern that it is too drastic and may lead to the “fabrication” of wines. In addition, commercial anion exchange resins were evaluated in Canada for their influence on wine quality and degree of deacidification (Zubeckis, 1962). Results showed that the treated wines had less color and bouquet when compared to the untreated wines. Also, the degree of deacidification varied among resins used in the study. In another study, Zubeckis (1957) reported that the change in sensory quality of the treated wines was hardly detectable when the deacidified wines were mixed with the original wine to a desired acidity. Castino (1974) studied the deacidification of Italian wines using strong anion exchange resins in the carbonate form. Wine was added to the resin

WINE DEACIDIFICATION

25

in a batch operation. Ion exchange was reported to have less undesirable effect on wine composition and sensory quality than treatment with potassium carbonate.

V.

BIOLOGICAL METHODS OF WINE DEACIDIFICATION A.

MALO-LACTIC FERMENTATION

Malo-lactic fermentation is the bacterial conversion of L-malic acid to L-lactic acid and carbon dioxide that often occurs in new wine as a result of growth of certain strains of lactic acid bacteria. It occurs at least sporadically in wines of all viticultural regions (Kunkee, 1967a). The main effect of the fermentation is a decrease in acidity of the wine as a result of the decarboxylation reaction. Since malo-lactic fermentation serves as a natural means of reducing wine acidity, it is usually considered highly desirable and is often encouraged in regions that produce high-acid grapes. Malo-lactic fermentation is sometimes encouraged even in warm viticultural regions where most wines would not benefit from the deacidification which results (Kunkee, 1974). In such cases the bacteriological stability which results is its main value (Rankine, 1977). Many enologists feel that malo-lactic fermentation improves the sensory quality, especially in red wines, by giving the wine more complexity (Rankine, 1972). On the other hand, Castino et al. (1975) reported that little if any detectable differences in sensory quality, independent of acidity changes, were detected in Italian red wines following the fermentation. Radler ( 1968) suggested that malo-lactic fermentation had no significant effect on sensory quality of German red wines but adversely affected white wines. Radler (1972) stated that German wines made from Riesling grapes were adversely affected by malo-lactic fermentation. Faber (1970) stated that opinions vary as to the desirability of malo-lactic fermentation in European wines. He reported that it was almost always desirable in red wines, but not in white wines made in some countries where consumers were not familiar with the taste it imparts. The high level of lactic acid is thought to destroy the fresh fruity quality of such wines. 1 . Occurrence

The occurrence of malo-lactic fermentation in wine is often unpredictable. The fermentation takes place when a sufficient population of appropriate bacteria develop in the wine. The fermentation may take place immediately following the alcoholic fermentation or sometimes even years later, possibly even following bottling. When malo-lactic fermentation occurs following bottling, it is consid-

26

R . 9. BEELMAN AND J . F. GALLANDER

ered as spoilage, since the bacterial growth produces turbidity and the wine becomes inappropriately effervescent. A pattern of bacterial development and malo-lactic conversion in a high-acid wine inoculated with malo-lactic bacteria is illustrated in Fig. 2 (from R. M. Keen and R. B. Beelman, unpublished data, 1977). Malo-lactic conversion commenced in this wine after about 35 days (formation of L-lactic acid evident) coinciding with the middle of the log phase of bacterial growth. At the same time, a sharp decrease in malic acid was evident. The initial drop in malate was probably due its utilization by yeast during alcoholic fermentation (see Section 111,B). The bacterial fermentation was essentially complete by 85 days as illustrated by the utilization of L-malic acid and commencement of L-lactic acid formation. 2.

Malo-lactic Bacteria

Numerous species of bacteria in the genera Lactobacillus, Pediococcus, and Leuconostoc have been shown to cause malo-lactic fermentation in wine. Description of the malo-lactic organisms isolated from wines have been given by Radler (1962), Kunkee (1967a), and RibCreau-Gayon et al. (1975). Leuconostoc species have been reported to be the most prevalent bacterial type associated with high-acid (low pH) wines (Rice, 1974; Castino et al., 1975). Garvie (1974) presented a classification of the Leuconostocs in which all species of that genus isolated from wine would be named Leuconostoc oenos. A strain of this organism isolated from a California wine, L . oenos ML-34, has been studied extensively (Pilone and Kunkee, 1972). This same organism is called Leuconosroc gracile Cf 34 by French researchers (RiWreau-Gayon er al., 1975). 3 . Mechanism The unequivocal mechanism of malo-lactic fermentation has long eluded researchers. Morenzoni (1974) reviewed the literature concerning the many proposed mechanisms and presented a new concept, based on data concerning the malic acid utilization by L . oenos ML-34. He reported that most of the L-mafic acid is directly decarboxylated to L-lactic acid (reaction 6 ) while small amounts of pyruvic acid and NADH2 are formed as end-products (reaction 7) rather than as intermediates, as proposed earlier. Both enzymatic activities were shown to be on the same protein and catalyze both reactions 6 and 7 as follows: L-Malic acid L-Malic acid

+ NAD

NAD

Mn2+

+

L-Lactic acid

Pyruvic acid

+ CO:,

+ NADH, + COP

(7)

WINE DEACIDIFICATION

-

.8

BACTERIA A 0

L-MALICACID L-LACTIC ACID

- .7

-

-

I

.6

2 \

5

.5

; -; Y

- .4

4 A

-.3

s Y

-.2

3 A I

-.I

TIME (DAYS)

FIG. 2. Relationship between bacterial growth and malo-lactic fermentation in wine inoculated with a pure culture of Leuconosroc oenos ML-34 (from unpublished data of R . M . Keen and R . B . Beelman, 1977).

The first activity was referred to as the malo-lactic activity of the enzyme. Kunkee (1975) referred to the second activity of the enzyme as the NADH or pyruvic acid-forming activity and demonstrated that, proportionally, only a very small amount of L-malic acid is converted to pyruvic acid. However, Pilone and Kunkee (1976) demonstrated that this second activity of the enzyme was responsible for the stimulation of the early growth rate of the organism in the presence of malic acid. This stimulation was especially striking at low pH (below 4.0) like that found in wine. 4.

Acidity Changes

Theoretically, malo-lactic fermentation would result in a reduction in total acidity amounting to about one-half of the titratable acidity due to malic acid present in the wine at the initiation of the bacterial fermentation. Typical changes in the acidity of several commercial New York State wines as a result of malolactic fermentation are illustrated in Table VII. The reductions in total acidity and

28

R. B. BEELMAN AND J . F. GALLANDER TABLE VIl ACIDITY CHANGES IN COMMERCIAL MUSTS AND WINES FROM THE EASTERN UNITED STATES"

~~

Must

New wine

Wine following MLFb

Variety

pH

Total acidity

pH

Total acidity

pH

Total acidity

Aurore Delaware Niagara Catawba Concordd Concord' lves" Baco noir"

3.08 3.1 1 3.03 2.77 2.81 3.12 3.22 3.28

1.16 1.15 0.83 1.72 I .64 1.37 1.18 1.53

3.39 3.30 3.10 2.97 2.97 3.20 3.22 3.38

0.89' 0.95 0.71 1.31 1.26 0.91 0.89 1.13

3.46 3.30 3.13

0.65 0.71 0.69

3.35 3.37 3.50

0.72 0.69 0.82

a

(I

-b -

-

Adapted from Rice (1974) Malo-lactic fermentation did not occur. Decreases include the effects of partial amelioration (-10%) and deposition of tartrates Cold press. Hot press.

increases in pH between the musts and new wines were a result of normal changes during alcoholic fermentation (see Section III,B) and an approximate 10% amelioration treatment (see Section IV,A). The changes in total acidity and pH between the new wine and the wine following malo-lactic fermentation were due primarily to the conversion of rralic to lactic acid. Two of the wines with extremely low must pH did not undergo malo-lactic fermentation. The average total acidity of the 6 wines which did complete malo-lactic fermentation was reduced from 0.91 to 0.71%. The average pH increase of the same 6 wines was from 3.27 to 3.35 or 0.08 pH units. Bousbouras and Kunkee (1971) demonstrated the change in pH and total acidity in wine resulting from malo-lactic fermentation was dependent on the initial pH of the wine. Increases in pH were greater with higher initial pH values and ranged from 0.09 to 0.21 pH units with initial pH values of 3.15 to 3.83, respectively. Decreases in total acidity were greatest with an initial wine pH around 3.5. This was attributed to greater precipitation of potassium bitartrate at that pH, since pH 3.5 is about midway between the two pK, values of tartaric acid. The increase in pH which generally occurs following malo-lactic fermentation may have significant effects on wine quality in addition to the effect on wine tartness. If pH increases to about 3.5, the precipitation of potassium bitartrate will increase as mentioned above. This loss in tartrate would result in a slight additional deacidification (Bousbouras and Kunkee, 1971). Also, in red wines a loss in color intensity occurs as pH is increased. If the increase is great enough

WINE DEACIDIFICATION

29

the color hue may change from red to bluish-red (Kunkee, 1967a). Rankine (1977) stated that in Australian wines with initial pH values about 3.8, malolactic fermentation is undesirable and may be detrimental to quality. He suggested that the pH of such wines could be lowered by hydrogen-ion exchange or by the addition of tartaric acid. Nagel et al. (1975) made similar suggestions to decrease pH following malo-lactic fermentation. Wejnar (1969b, 1972) demonstrated that it is theoretically possible for the pH to drop following malo-lactic fermentation if it were to occur during the precipitation of tartrate; however, we have seen no report of this actually happening in wine. 5 . Factors Influencing Bacterial Growth

Wine is a hostile environment, even for malo-lactic bacteria. Lack of nutrients, low pH, the high concentrations of ethanol and sulfur dioxide present, and the normally low temperatures associated with storage all contribute to the inhibition of the bacteria to various degrees. The numerous factors associated with winemaking which influence the susceptability of wine to malo-lactic fermentation have been reviewed (Radler, 1966; Kunkee, 1967a, 1974; Amerine and Kunkee, 1968; Castino et al., 1975; Amachi, 1975; Yoshizumi, 1975). Low pH (Bousbouras and Kunkee, 1971) high concentrations of sulfur dioxide (Rankine et al., 1970; Vetsch, 1973; Lafon-Lafourcade and Peynaud, 1974) and low storage temperature (Bambalov, 1973; Rice, 1974) are the most important factors inhibiting malo-lactic bacteria and delaying the fermentation. Wines with pH values below 3.2, initial sulfur dioxide concentrations above 50 ppm or storage temperature below 10°C are not generally considered susceptable to the fermentation. White table wines are often made under these conditions; therefore, it is not surprising that the incidence of malo-lactic fermentation is much lower among white than red wines (Ingraham and Cooke, 1960; Rice, 1965; Radler, 1968; van Wyk, 1976). Winemakers wishing to stimulate the fermentation use minimal levels of sulfur dioxide and maintain cellar temperatures around 18 to 22°C. Rankine et a / . (1970) indicated that sulfur dioxide concentration was the most important factor and related that some Australian winemakers withhold the use of sulfur dioxide until the bacterial fermentation is complete. Rice (1974) demonstrated that if initial sulfur dioxide addition to New York State wines was less than 20 ppm, malo-lactic fermentation occurred at pH values as low as 3.0 even with low cellar temperatures. Van Wyk (1976) reported that delayed racking without sulfur dioxide addition greatly stimulated malo-lactic fermentation in South African table wines. Mayer et al. (1976) demonstrated that growth of malo-lactic bacteria was inhibited by sulfur dioxide producing yeast strains in experimental Swiss wines and suggested selecting yeast strains that produce little or no sulfur dioxide when encouraging malo-lactic fermentation.

30

R. B . BEELMAN AND J. F. GALLANDER

Kunkee (1967a) suggested the possibility that wines with low pH values could be deacidified partially by some other method to increase the pH, thereby improving the susceptability of the wine to malo-lactic fermentation. Blending acidic wines with wines with higher pH values was suggested as one possibility. The use of ion exchange or the addition of chemicals to increase pH was also suggested. It is common practice in some European countries to treat low pH musts with calcium carbonate to raise the pH and stimulate malo-lactic fermentation (Schopfer, 1971; Minarik, 1971; Rebelein, 1971a; Jaulmes and Flanzy, 1971). In Switzerland, calcium carbonate is often added to raise the pH of the musts to about 3.3 for this purpose (Schopfer, 1968; Tanner and Sandoz 1974). Calcium carbonate treatment may have a secondary effect, since Mayer (1974) observed that the carbon dioxide released into the wine subsequent to calcium carbonate addition was markedly stimulatory to the growth of malo-lactic bacteria. Other researchers have reported the stimulatory effect of carbon dioxide on malo-lactic fermentation (Vetsch, 1973; Brechot and Chauvet, 1971). In the eastern United States, partial amelioration has been used to deacidify wines which subsequently undergo malo-lactic fermentation (Rice, 1974; Wagner, 1976). Amelioration has little effect on wine pH (Kluba and Beelman, 1975) but it might possibly dilute the concentration of substances inhibitory to malo-lactic bacteria. Brechot et al. (1974) observed that initial concentration of malic acid in musts influenced the initiation of malo-lactic fermentation. They reported that concentrations of malate exceeding 40 mM (0.54%) delayed the initiation of the bacterial fermentation. Thus amelioration could possibly stimulate malo-lactic fermentation, since it would lower the malate concentration significantly (Kluba and Beelman, 1975). Other factors associated with vinification also influence malo-lactic fermentation. Leaving wine in contact with the yeast sediment following completion of the alcoholic fermentation (delayed racking) has been shown to increase the incidence of malo-lactic fermentation (Fornachon, 1957; van Wyk, 1976). Presumably nutrients stimulatory to the bacteria are released by yeast autolysis (Liithi and Vetsch, 1959). The presence of grape skins during alcoholic fermentation has been shown to be stimulatory to malo-lactic fermentation (Kunkee, 1967b; Beelman and Gallander, 1970). Most red table wines are made using the fermentation “on the skins” process to extract the anthocyanin pigments. However, hot-pressing (also called thermovinification) is currently being used more widely to extract color in red wine production. Some studies have found that wines made from hot-pressed musts were more resistant to malo-lactic fermentation (Beelman and Gallander, 1970; Beelman et ul., 1977). Martiniere et al. ( 1.975)found that wines made using thermovinification and sulfited resisted malolactic fermentation. Wines made from the same grapes that were made using thermovinification without sulfite treatment, completed malo-lactic fermentation soon after alcoholic fermentation. Rice (1974) indicated that red wines made by hot-pressing and with limited use of sulfur dioxide routinely completed malo-

WINE DEACIDIFICATION

31

lactic fermentation. It may be that an interaction between sulfur dioxide and heated musts produces something inhibitory to malo-lactic bacteria. 6 . Inoculation with Malo-lactic Bacteria

Since wines often do not undergo natural malo-lactic fermentation, winemakers sometimes induce the fermentation. Blending the resistant wine with a portion of wine which previously completed a desirable malo-lactic fermentation is one method used. Kunkee (1967a) indicated that large “inoculations” (15 to 50%) are employed. Castino et al. (1975) reported success in inducing malolactic fermentation in resistant wines by the addition of wine (about 5 % ) which was undergoing malo-lactic fermentation. However, they cautioned that the bacterial strain added in this manner must be one that is active at the pH of the wine to be fermented. Vetsch (1973) studied the fate of added bacteria ( L . oenos) transferred from wine to wine. He showed that the death rate was greater at lower pH and a large inoculum was necessary for successful stimulation at low pH. He suggested that large inoculations using a wine with high populations of bacteria (such that the resultant wine would contain more than 10 million viable Leuconostoc oenos celldml) would in most cases initiate the fermentation in the blended wine. Peynaud and Domercq ( 1959) demonstrated that malo-lactic fermentation could be successfully induced by inoculation of wine with small volumes (0.01%) of appropriate bacteria grown in pure culture. Sudraud and Cassignard (1959) employed larger amounts (0.1%) of bacterial inocula. Kunkee et al. (1964) demonstrated that the addition of a large inoculum (1 .O%) resulted in a rapid malo-lactic fermentation in California wines. Mixed cultures of yeast and malolactic bacteria have also been used with some success (Galzy and Plan, 1957; Lafon-Lafourcade et al., 1968). Recent review papers have remarked about the interest in the use of pure culture inoculation of wine with known strains of bacteria to induce malo-lactic fermentation (Kunkee, 1967a, 1974; Radler, 1968; Gandini, 1969; Rankine, 1977). Many of the recent studies concerning bacterial inoculation have been with strains of L . oenos. Kunkee (1974) discussed procedures for the preparation of pure cultures of malo-lactic bacteria and inoculation into wine and recommended the use of pure culture inoculation only if trained personnel and adequate microbiological facilities are available. L . oenos ML-34 was the organism recommended for inoculation. Pilone and Kunkee (1972) cited numerous applications of this strain for the induction of malolactic fermentation under laboratory conditions and in commercial wineries. Beelman et al. (1977) demonstrated the potential importance of selecting a strain of L . oenos adapted to local conditions for wine inoculation. Rankine (1977) suggested that it may be necessary to prepare bacterial cultures in a central laboratory. He also related that liquid cultures are not convenient since they are at maximum viability for only a short time. Commercially pre-

32

R. B . BEELMAN A N D J . F. GALLANDER

pared freeze-dried cultures of malo-lactic bacteria are available (Equilait, 38 Avenue de la Republique, 15-Aurillac, France) along with directions for their cultivation and inoculation into wine (Ardin, 1972). However, we have seen no published work on the use of commercially prepared cultures to induce malolactic fermentation. Even following inoculation with bacteria, stimulation of malo-lactic fermentation is sometimes difficult to control, especially under conditions unfavorable to bacterial growth. Lafon-Lafourcade (1970, 1975) demonstrated that malo-lactic fermentation could be induced more consistently by the addition of large amounts of nonproliferating (resting) bacterial cells. She demonstrated that the use of nonproliferating cells avoided the unpredictable multiplication of the bacteria from a small population into the log phase of the growth cycle. Malo-lactic fermentation was induced by this procedure under conditions (high alcohol, low pH, and high sulfur dioxide concentrations) where growth of the bacteria from small populations was extremely difficult. She has suggested that if large quantities of malo-lactic bacterial cells could be cultivated economically and the enzymatic capabilities ‘‘fixed’ ’, malo-lactic fermentation could be induced in wine more predictably. Several processes have been developed which involve “continuous” malo-lactic conversion (Flesch and Jerchel, 1966; Berdelle-Hilge, 1975; Kozub e f al., 1976). However, no significant application of any such process has yet been reported.

B. FERMENTATION OF MALIC ACID WITH SCHIZOSACCHAROMYCES POMBE The utilization of malic acid by Schizosaccharomyces spp. has been the basis of numerous enological studies dealing with wine deacidification. Many of these studies have been summarized in several excellent texts and review articles (Amerine et a l . , 1972; Amerine and Ough, 1972; Amerine and Kunkee, 1968; Kunkee and Amerine, 1970; Reed and Peppler, 1973). 1.

Species and Characteristics

Biochemical investigations have shown that Schizosaccharomyces spp. convert malic acid to ethanol and carbon dioxide under anaerobic conditions. These yeasts resemble the normal wine yeast, Saccharomyces cerevisiae, in producing ascospores and by being a strong alcoholic fermenter. Schizosaccharomyces belong to the same family as Saccharomyces, Saccharomycetaceae, but are classified into the subfamily, Schizosaccharomycoideae.Their vegetative propagation by fission is unlike Saccharomyces which reproduces by multilateral budding. Lodder (1970) comprised the genus Schizosaccharomyces into four species: Schizosacch. pombe, Schizosacch. malidevorans, Schizosacch. japonicus, and

WINE DEACIDIFICATION

33

Schizosacch. octosporus. Jn general, these species differ in their number of ascospores (four to eight) and ability to ferment sucrose, maltose, melibiose, and raffinose.

2 . Metabolsim One of the first studies to observe the degradation of malic acid through fermentation was conducted by Osterwalder (1924). He isolated a yeast from fermenting apple and grape juice and named it Schizosaccharomyces liquefuciens (now pombe). Malic degradation through Schizosaccharomyces metabolism has been studied by several workers (Mayer and Temperli, 1963; Peynaud et al., 1964; Dittrich, 1963b, 1964). Both aerobic and anaerobic metabolism of L-malate by Schizosaccaromycespombe, strain 196, was investigated by Mayer and Temperli ( 1 963). Under their oxidative conditions, L-malate was completely oxidized to COPand H,O. The anaerobic reaction (reaction 8) of malic acid by Schizosaccharomyces pombe is as follows: HOOC
---t

2C0,

+ CH,CH?OH

(8)

The same end-products with a discussion of their possible pathway formation was reported by Peynaud et al. (1964) and Peynaud and Lafon-Lafourcade (1965). 3. Reduction in Wine Acidity In many instances, the amount of deacidification through Schizosaccharomyces fermentation is quite variable. The ability to utilize malic acid greatly depends upon the selection of the S~.hizosaccharomyces species and strain. An extensive study to determine these differences was performed by Peynaud et a!. (1964). They reported that total acidity decreased as much as 45% among 18 strains of Schizosaccharomyces. These decreases were related to the decomposition of malic acid during alcoholic fermentation. Similar results were obtained by Peynaud and Sudraud (1964) for 10 strains of Schizosaccharomyces pombe. Under anaerobic conditions, the average malic acid decrease was 75% with the strain 206 reducing the malate concentration by 92%. Ribireau-Gayon and Peynaud (1962) tested the malic acid decomposition of five Schizosaccharomyces yeasts in fermenting grape musts. The level of malic acid was reduced for each yeast when compared to a normal wine yeast, Sacch. cerevisiae var. ellipsoideus. Schizosacch. liquefaciens (now pombe) was found to have the greatest ability to ferment malic acid, 84% decomposition. In an excellent review and study concerning Schizosaccharoniyces fermentation, Bidan et al. (1974) reported on malic acid reduction by three strains of

34

R. B. BEELMAN AND J . F. GALLANDER

Schizosacch. pornbe. Results of one phase of their research indicated that malic acid was completely metabolized in Chasselas musts. Yeast populations were also determined and showed that malic acid degradation occurred at about 150 X l o 5 cells per ml. This was considerably less than the Saccharomyces samples which showed no acid reduction. Bidan er al. (1974) also measured the reduction of total acidity in fermenting Chasselas musts which were pasteurized and inoculated with 2 strains of Schizosaccharomyces. Although the differences in wine acidities were slight, the length of malic acid fermentation by strain 105 was longer than 106. In comparing pasteurized and sulfited musts, total acidity of the Riesling wines was less for the pasteurized samples. This was expected for the naturally occurring yeasts probably outgrew Schizosaccharomyces in sulfur dioxide treated musts. As indicated previously, the intensity of Schizosacch. to utilize malic acid during fermentation is strongly related to yeast selection. Nonomura et al. (1968) selected an effective Schizosacch. pombe strain, 0-77, in fermenting malic acid in grape musts. A species resembling Schizosacch. pombe was isolated by Rankine and Fomachon (1964) and was identified as Schizosacch. malidevorans. This yeast decomposed L-malic acid completely but not the D-isomer in grape juice and synthetic media. However, during fermentation, Schizosacch. malidevorans produced noticeable amounts of hydrogen sulfide. The employment of Schizosacch. in reducing wine acidity of six grape varieties for two seasons was reported by Yang (1973a). The degree of wine deacidification depended upon malic acid content of the grape varieties and season. As much as 34% of the wine acidity was decreased by Schizosacch. fermentation when compared to Sacch. fermented wines. In one variety, the malic acid was not fully metabolized, and the total acidity was reduced by only 10.7%. This slight reduction was explained by the unusually high acidity (approximately 1.5%) of the Sauvignon blanc variety. For several high malate varieties in Ohio, Schizosacch. fermentation was found to reduce the malic acid to trace amounts (Gallander, 1974b). The average reduction in total acidity for all Schizosacch. fermented wines, in relation to the Sacch. fermented wines, was about 50%. The utilization of malic acid by Schizosacch. in six varietal wines was reported by Peynaud and Sudraud (1964). They compared the total acidity and malic acid content of wine fermented spontaneously to Schizosacch. fermented wines. In no wines was malic acid metabolized completely, and in many instances, the loss in wine acidity was not even significant. The degree of deacidification appeared to be related to the stronger growth of the natural yeasts in the Schizosacch. inoculated wines. 4.

Factors Influencing Malic Acid Degradation

Temperature is an important factor influencing the growth of Schizosacch., and its capacity to ferment malic acid. Dittrich (1963a) measured the growth of

WINE DEACIDIFICATION

35

Schizosacch. at four temperatures (10, 17,20, and 25°C) through the loss in wine acidity and sugar content. Within this temperature range, the length of fermentation varied inversely with wine temperature. The fermentation was completed in 15 days at 25°C while at 10°C malic acid disappeared in 39 days. In the same study, Dittrich (1963a) found that Schizosacch. possessed a higher optimum temperature for growth than Sacch. cerevisiae. Grape musts were inoculated with a mixture of Schizosacch. acidodevoratus (pombe) and Sacch. cerevisiae and fermented at several temperatures. The reduction in wine acidity was highest at 35°C. indicating predominance of Schizosacch. At lower temperatures, Sacch. outgrew the Schizosacch. yeasts as represented by higher wine acidities. Similar results were obtained by Peynaud and Sudraud (1964) in determining optimum temperature for Schizosacch. They measured the CO, evolution of fermenting Schizosacch. species and found maximum growth at approximately 35°C. This was 10 degrees higher than the optimum for the wine yeasts Sacch. cerevisiae and Sacch. oviformis. These results paralleled the findings of Nonomura et al. (1968), who established growth curves for Schizosacch. pomhe and Sacch . cerevisiae . The amount of L-malic acid decomposed by Schizosaccharomyces is inversely related to pH. Testing two strains of Schizosacch. pombe, Peynaud et al. (1964) found that the highest utilization of malic acid occurred at pH 2.8. These yeasts became less active as pH increased to 4.8. Complete degradation of L-malic acid was not observed in their study, but decreased from 27 to 16%. Later, Rankine (1966) was able to decompose all L-malic acid in fermenting grape juice with Schizosacch. malidevorans. The pH values vaned from 3.2 to 3.7; thus, malic acid utilization was pH-dependent. The same study also showed a strong relationship between pH and L-malic decomposition for Saccharomyces. The lower the pH values, the greater the amount of L-malic acid metabolized. Although these decreases were less than for Schizosaccharomyces, the trend was identical to the findings of Peynaud et ul. (1964). More recently, Yang (1973b) studied effects of pH on malic acid fermentation by Schizosaccharomyces pombe. Musts of Pinot blanc were adjusted to different pH values, sulfited and inoculated with Schizosacch. The yeast utilized all the malic acid present when the pH was above 3.0. This finding was in agreement with Rankine (1966). However, below pH 3.0, the percent utilization of malic acid decreased with decreasing pH. This was in contrast to results obtained by Peynaud et al. (1964) and the findings of Rankine (1966) with Succharomyces. The difference, in part, may be attributed to overgrowing of naturally occurring yeasts in the wines at low pH values. Additional information is needed to verify that the amount of L-malic acid decomposed is inversely related to pH. Sulfur dioxide is widely used in winemaking to inhibit undesirable microorganisms and oxidation. In general, Schizosaccharomyces are tolerant to normal levels of SO, (75 to 125 ppm) commonly used in treating musts prior to fermentation. Some studies have indicated that Schizosacch. spp. are more resistant to

36

R. B. BEELMAN AND J . F. GALLANDER

SO, than the established wine yeasts (Dittrich, 1963a; Yang, 1975). Most recently, Yang (1975) reported on the comparison of Schizosacch. pombe and Sacch. cerevisiae to initiation of alcoholic fermentation at several levels of SO,. At 100 ppm SO2, wines inoculated with Schizosucch. pombe started 1 day earlier than Sacch. cerevisiae. The difference was even greater at 200 ppm SO, more than 10 days sooner for Schizosacch. This conclusion was not reported by earlier studies (Minarik and Navara, 1967; Nonomura et al., 1968). Although certain Schizosacch. strains can tolerate usual SOp levels, their greater resistance to SOn than Sacch. has not been firmly established. According to Nonomura et al. (1968), the tolerance of Schizosacch. pombe to SOz was considered the same as Sacch. cerevisiae at 150 ppm. Both yeasts initiated alcoholic fermentation 5 days after inoculation. At 175 ppm SO, Schizosacch. and Sacch. musts did not ferment to any degree. Also, Minarik and Navara (1967) could not confirm the tolerance of Schizosacch. to high levels of SO,. They reported that Sacch. cerevisiae yeasts fermented twice as fast as Schizosaccharomyces species at 200 ppm so:!. Under similar conditions, certain Schizosacch. yeasts usually produce as much alcohol as regular wine yeasts. In addition to the alcohol produced through sugar fermentation, these yeasts metabolize malic acid to ethanol and carbon dioxide. Thus, alcohol production may be greater in Schizosacch. wines than in wines fermented by Sacch. cerevisiae. Results by Nonomura et al. (1968) indicate that Schizosacch. pombe, strain 0-77, produce as much alcohol as a typical wine yeast, Sacch. cerevisiae. Similar results were reported by Bidan et al. (1974) and Benda and Schmitt (1966) for strains of Schizosacch. pombe. In fact, Bidan et al. (1974) in many cases obtained slightly higher alcohol concentrations when grape musts were inoculated with Schizosacch. pombe. Bujak and Dabkowski (1961) found that ethanol has a profound effect upon malic acid fermentation. At 15% ethanol, the malic acid fermentation did not occur in wines inoculated with Schizosacch. acidodevoratus (pombe). The decomposition of malic acid was delayed 10 days in wines at 10% ethanol as compared to 5% ethanol. The conversion of malic acid to ethanol and carbon dioxide by Schizosaccharomyces species parallels alcoholic fermentation. The loss in malic acid proceeds until the sugar is utilized during alcoholic fermentation. Bidan et al. (1974) graphically illustrated this by relating the decline in wine acidity to the decrease in sugar during fermentation. Their results also indicate that metabolism of malic acid was highest near the beginning of alcoholic fermentation. This coincided with intense yeast activity. Comparable results were obtained by Dittrich (1963a) in regard to reduction of sugar and malic acid during fermentation. He reported that malic acid decomposition is coupled with sugar fermentation. Nonomura et al. (1968) compared the growth of Schizosacch. pombe, 0-77, to Sacch. cerevisiae at various sugar concentrations in a synthetic medium. Optimum growth for both yeasts was essentially the same at 20% sugar. However,

WINE DEACIDIFICATION

37

at 30% sugar, growth of the Schizosacch. strain was much higher than Saccharomyces. Bujak and Dabkowski (1961) found the rate of malic acid decomposition was highest at 20% sugar for four strains of Schizosacch. acidodevoratus (pornbe).The sugar percentages were varied between 10 to 35% with 35% sugar prolonging the fermentation the most. 5 . Sensory Qualiry and Mixed Cultures

Although Schizosaccharomyces spp. are effective in reducing malic acid, several researchers have found that Schizosacch. wines are inferior to wines fermented by Sacrharomyces. Benda and Schmitt (1966) reported wines fermented by Schizosacch. pombe were unacceptable when compared to Sacch. wines. The Schizosacch. wines did not possess the desirable flavor characteristics of the grape variety. The authors attributed this low quality to the high optimum fermentation temperature of the Schizosacch. species. Dittrich (1963a) found the same disadvantage for Schizosacch. species, i.e., high optimum fermentation temperature. Another undesirable characteristic of certain Schizosacch. spp. is production of hydrogen sulfide during fermentation (Rankine, 1966; Bidan et al., 1974). Nonomura et a!. (1968) reported better tasting wines from Schizosaccharomyces fermentation than from fermentation with true wine yeast. However, the Schizosacch. wines were scored inferior to the control wines in bouquet. In contrast, acceptable quality wines have been fermented with strains of Schizosaccharomyces by other workers (Minarik and Navara, 1967; Yang, 1973a; Bidan et al., 1974). However, their results lacked complete sensory evaluation data with statistical analysis. This has been the weakness of most studies concerning wine deacidification by Schizosaccharomyces. Interest in combining the benefits of both Schizosaccharomyces and wine yeasts has been shown by several investigators (Peynaud and Sudraud, 1964; Bujak and Dabkowski, 1961; Wienhaus, 1967; Rankine, 1966). The utility of this procedure would be attractive when excessively high malic acid grapes are used in making wines. The major disadvantage of mixed yeast culture is that one yeast usually dominates fermentation. This holds true in culturing grape musts with Schizosaccharomyces and Saccharomyces, where the true wine yeast outgrows the slow fermenter, Schizosaccharomyces. An illustration of the malic acid fermentation as influenced by mixed yeast cultures is shown in Table VIII (Rankine, 1966). The reduction of malic acid is directly related to the level of Schizosaccharomyces cells in the initial inoculum. The yeast counts during fermentation also indicate that the growth rate of Schizosaccharomyces was slower than that of the wine yeast. These results are in agreement with findings of other mixed yeast culture studies (Peynaud and Sudraud, 1964; Bujak and Dabkowski, 1961; Wienhaus, 1967). In a similar investigation, Gallander (1977b) studied the reduction of malic acid in wines fermented

38

R. B. BEELMAN AND J . F. GALLANDER TABLE Vlll PERCENTAGE OF L-MALIC ACID METABOLIZED IN GRAPE JUICE BY PAIRS OF YEASTS MIXED IN DIFFERENT PROPORTIONS" ~

% 422 in

Yeast pair

i noc u I um

Schizosacch. (422) and Sacch. (703)

0 10

50 90 100

Schizosucch. (442) and Succh. (723)

a

0 10 50 90 100

% 422 at middle of fermentation

% 422 at

end of fermentation

% L-Malic decomposed

0

0

2 8

2 11

18

47

25 35 79 96

I00

100

100

0

2 4 16

0 3 7 29

21 47 87 96

100

100

100

From Rankine (1966).

with Schizosacch. pombe and re-inoculated with a wine yeast at various time intervals. Loss in malic acid was greatest when Succh. inoculation was delayed 6 days after inoculation with the fission yeast. C.

CARBONIC MACERATION

Holding intact bunches of grapes in tanks filled with carbon dioxide for a period of time prior to crushing or pressing is generally referred to as carbonic maceration (maceration carbonique in France). Variations of this process have been used in winemaking for many years. Some are still used in the Rioja district of Spain, the Piedmont region of Italy, southern France, on the Rhone, and especially in Beaujolais (Amerine and Ough, 1968; Amerine and Joslyn, 1970). Detailed descriptions of the carbonic maceration process as given by Flanzy and Andre (1973) were reported by McCorkle (1974). 1.

Intracellular Fermentation

Enzymatically catalyzed intracellular reactions occur in intact berries in the anaerobic environment during carbonic maceration. These reactions, generally referred to as intracellular fermentation, occur when the oxygen concentration falls below about 5% or when the carbon dioxide concentration is increased about 50% (McCorkle, 1974). One of the main reactions to occur is the degradation of malic acid, mainly to ethanol. In addition, metabolites of malic acid are formed which influence wine quality (Jouret, 1971; Flanzy et a l . , 1974). The malate

WINE DEACIDIFICATION

39

degradation results in wines of significantly lower total acidity and higher pH. Thus, the process is of interest as a potential means of reducing acidity of wines from overly acidic grapes (Jaulmes and Flanzy, 1971). However, Andre (1971) emphasized that an important interest in carbonic maceration is its effect on the sensory quality of the wines. Nitrogenous substances and organic acids, such as quinic and shikimic, formed during intracellular fermentation are thought to be precursors of important bouquet components (Flanzy, 1971). According to Flanzy and Andre (1965), ethanol formed by intracellular fermentation results in the death of the grape cells. A selective diffusion of polyphenols from the grape skins to the pulp and juice also occurs during the anaerobic holding period (Bourzeix, 1971). Andre (1971) reported that under the proper conditions wines can be made with carbonic maceration that have equal color but less tannin than those produced by normal vinification. Wines made by carbonic maceration are claimed to have better bouquet and softer taste and to mature earlier than wines made by conventional methods (McCorkle, 1974). Flanzy (1935a,b) was the first to systematically study carbonic maceration in vinification and reported on the potential advantages of the process for making early maturing red wines. His work was initiated to confirm earlier claims of the desirable odors associated with grapes held under anaerobic conditions and to examine the potential of the process in reducing the amount of sulfur dioxide required in vinification. McCorkle (1974) described the details of the carbonic maceration process for making red table wines. The intact grapes are loaded into tanks previously filled with carbon dioxide. This is done without previous crushing and with as little damage to cellular integrety as possible. A large quantity of carbon dioxide dissolves in the juice of the berries [about 10-15% of the grape volume (Andre, 197 l)] thus, it is necessary to add additional carbon dioxide to the tanks early in the process. Brechot and Chauvet (1971) emphasized the importance of continued application of carbon dioxide throughout the holding period and even after pressing. They indicated that this was the major difference between carbonic maceration and traditional Beaujolais vinification. Some grapes become crushed during loading the tanks and some juice is released from grapes during the holding period. Therefore, the holding tank contains intact grapes in a gaseous carbon dioxide environment in the top of the tank, crushed or partially crushed grapes which yield juice to the bottom of the tank, and intact bunches submerged in juice. The reactions of the intracellular fermentation occur most readily in the intact grapes in the gaseous environment, and to a varying degree in those grapes in the liquid phase. The juice which develops during the holding period is subject to microbial attack and is usually partially fermented. After the holding period, the partially fermented free-run juice is drained from the tank. The remaining grapes, which still contain much of the original sugar, are pressed yielding a juice which ferments quickly to dryness. This pressed juice is considered

40

R. B . BEELMAN AND J . F. GALLANDER

superior in potential wine quality to the free-run, since it originated from the intact grapes where the intracellular fermentation occurred. This is opposite of normal vinification of crushed grapes where the free-run juice is usually thought to make superior wine. Following pressing, the alcoholic fermentation completes rapidly and malo-lactic fermentation usually occurs shortly thereafter. Both the yeast (see Section II1,B) and bacterial (see Section V,A) metabolism of malic acid reduces the acidity further. Brechot and Chauvet (1971) reported that carbonic maceration stimulates both the growth of yeast and malo-lactic bacteria. 2 . Mechanism of Malate Degradation Although the mechanism of malate degradation which occurs during intracellular fermentation has not been conclusively determined experimentally, RibCreauGayon et a f . (1976b) postulated the following reaction sequence: Malic Acid

+ NADP S

Pyruvic Acid

+ CO, + NADPH + H i

Pyruvic Acid S Acetaldehyde Acetaldehyde

+ Con

+ NADH + H+* Ethanol + NAD

(9) (10)

(11)

The initial reaction (reaction 9) is probably catalyzed by L-malate NADP oxidoreductase (EC 1.1.1. 40). The activity of this enzyme in grapes held under anaerobic conditions was studied by Garcia et al. (1974). Pyruvate is apparently decarboxylated to acetaldehyde (reaction 10) which is. subsequently reduced to ethanol (reaction 11). Andre (1971) demonstrated that between 0.44 and 2.2% ethanol was formed during 10 days of intracellular fermentation at 35°C which represented between 15 and 57% of the malate metabolized. Thus, the loss in total acidity occurs partly through an alcoholic fermentation of malic acid during intracellular fermentation. This degradation is independent of the degradation of malic acid by yeasts or the malo-lactic fermentation caused by bacteria which occurs in the liquid phase or following pressing. 3 . Acid Reduction

The transformation of malic acid to pyruvic acid during intracellular fermentation is not complete and depends upon the “malic enzyme” activity of the particular grape variety (Ribereau-Gayon et af., 1976b). Andre (1971) reported that the malate degradation during carbonic maceration at 35°C for 10 days vaned from 40 to 60% among several varieties. He also reported that some variation in malate degradation occurred within the same varieties from different locations, but little year-to-year variation occurred within the same varieties from

41

WINE DEACIDIFICATION

the same vineyard location. Earlier research revealed losses of malate ranging from up to 20% (Brechot et al., 1966), 25% (Chauvet el al., 1963) and 40% (Peynaud and Guimberteau, 1962). The research of Flanzy et al. (1967a,b), demonstrated that the limit and rate of malate degradation during intracellular fermentation was highly dependent on temperature and duration of the holding period. Flanzy (1971) also demonstrated the effect of temperature and duration of holding period on both malate degradation and total acidity reduction (see Fig. 3). After 10 days of intracellular fermentation the percentage loss of malate compared to the original concentration was 59, 26, and 14% at 35, 25, and 15°C respectively. Apparently most of the loss of total acidity that occurred was due to malate degradation. However, some reports have indicated reduced tartrate concentrations in wines made by carbonic I00

100

~

VARIETY - CARIGNAN NOlR YEAR - 1970

-

0 90

090

- 080

080

-

0 70

070

--

--

E

E 060 0

- 060

E

- 050

\

0 \

0

350

2

4" -

2

" -

0

c

s 040

I

-5

- 040 r-" I I

0 30

-ox)

020

-

010

0

1

1

I

5

10

15

I

20

L

25

I

'

020

010

O

TIME (Days)

FIG. 3. Effect o f temperature and duration o f intracellular fermentation of the carbonic maceration process on the reduction in total acidity and malate concentration (adapted from Flanzy, 1971).

42

R. B . BEELMAN AND J . F. GALLANDER

maceration (Benard and Jouret, 1963; Flanzy et al., 1967a,b; Beelman and McArdle, 1974b). Flanzy (1971) questioned whether the decrease in tartrate observed previously actually occurred during intracellular fermentation or was the result of other factors associated with vinification. Jaulmes and Flanzy (1971) remarked that carbonic maceration was one of the most advantageous biological processes for obtaining wine deacidification. However, much of the interest in carbonic maceration has concerned the influence it has on the sensory quality independent of acidity changes. Much interest in the process has been in warm areas where acidity losses might be detrimental, but the improved bouquet of the otherwise neutral wines would be of value (Andre, 1971). In such cases the addition of sulfur dioxide and acidification of the grapes with tartrate were recommended to prevent excessive intracellular fermentation and control possible spoilage (Andre, 1971). Several experiments conducted in California (Amerine and Ough, 1968, 1969; Amerine and Fong, 1974) indicated that the process had little practical value under California conditions. However, Beelman and McArdle (1974b) indicated that it had potential at least as a deacidification process in Pennsylvania wines. Andre (1971) related that the optimum conditions for vinification using carbonic maceration must be different for each variety, viticultural area, and the type of wine desired. Thus, the temperature and duration of the anaerobic holding period, the handling of the free-run and pressed juice, etc., must be determined for individual conditions. For a thorough discussion of the adaption of the process to different viticultural and enological conditions and wine types see Andre (1971).

VI. SUMMARY AND RESEARCH NEEDS In cool viticultural areas of the world some form of wine deacidification is often necessary to make well-balanced wines with the appropriate tartness. The goal of the enologist is to perform this deacidification, when necessary, in a manner which will result in stable wines with the best possible organoleptic quality. Thus, winemakers generally attempt to reduce the total acidity to a desired level without increasing the pH excessively. Presently, the winemaker must empirically determine the appropriate tartness for each wine based upon sensory examination. Basic guidelines are available concerning proper pH and total acidity levels for table wines. However, more information regarding the optimum relationship between pH and total acidity in regard to sensory quality would be helpful in the selection of a deacidification method and the employment of that method for specific wines. The problem of overly acidic grapes can be somewhat diminished by proper vineyard site and variety selection. Close attention to the cultural practices which can improve the optimum maturation of the fruit and delaying the harvest as long

WINE DEACIDIFICATION

43

as practical can also help to alleviate the problem. More emphasis by grape breeders regarding the development of new varieties that have more optimum acidity for winemaking when grown in cool climates might be helpful. Probably the most natural form of wine deacidification is blending the excessively tart wines with less acidic wines made in good years or from warmer climates. This is often done, but it is limited by trade restrictions and labeling regulations. A natural reduction in acidity occurs during normal alcoholic fermentation due to the partial utilization of malic acid by the yeast and precipitation of some tartaric acid as potassium bitratrate during and following fermentation. However, this reduction in acidity is often not sufficient and the winemaker must resort to some other form of deacidification. The neutralization of must or wine with calcium carbonate is a commonly used method to reduce acidity, but it has the serious drawback of removing only tartaric acid by precipitation. If used in excess, it may inordinately increase pH which can cause problems with chemical and biological stability of the wine. The double-salt precipitation procedure improves upon the ordinary use of calcium carbonate in that malic acid is also removed in addition to the tartaric acid. This results in less change in pH and wines with a more natural tartrate to malate ratio. This method seems particularly well suited to highly acidic, low pH white wines. Malo-lactic fermentation is thought to be a highly desirable, natural means to reduce acidity especially in red wines. The influence of this fermentation on the sensory quality of the wine other than the acidity change is still a moot point, Additional research is needed to determine the desirability of the fermentation in different wine types. The inability to consistently stimulate malo-lactic fermentation, even following pure culture inoculation, still remains as a problem to the enologist. A more basic understanding of the factors which control the growth of malo-lactic bacteria in wine is needed. The development of commercially prepared cultures to provide for convenient and reliable inoculation of wines with proven strains of bacteria would appear useful. Also, the potential of immobilizedcell or immobilized enzyme reactors for obtaining the malo-lactic conversion in wine more consistently might prove useful. Amelioration will probably continue to be used to reduce the acidity of wines, at least some of those made in the eastern United States and Canada. Despite the criticism of being an adulteration of wine, the method does have merit in that malic acid is reduced preferentially to tartaric acid and it has a negligible effect on pH. Amelioration appears to be a particularly desirable method for use with wines where a dilution of some inherent odor or taste as well as the acidity would be beneficial. A significant reduction in acidity occurs during the intracellular fermentation of the carbonic maceration process. The deacidification results mostly from the enzymatic degradation of malic acid. Carbonic maceration seems to have a limited potential as a deacidification treatment per se, since it requires special

44

R . B . BEELMAN AND J . F. GALLANDER

cellar techniques and equipment and results in a unique style of wine. However, continued study of the mechanism of malate degradation in intracellular fermentation may prove useful in the future. There appears to be a lack of information concerning the suitability of lowering high acid wines with anion exchange resins. Some literature indicates that anion exchangers are effective in reducing wine acidity, but with undesirable flavor and aroma changes. However, there is also evidence that wines can be deacidified by anion exchange are without noticeable defects. More research is needed to establish the correct technology for the successful treatment of wines by anion exchange. With the advent of new and more selective resins, the potential for ion-exchange treatment of wines could be improved. Much research has been conducted in recent years concerning wine deacidification through malic acid fermentation by Schizosaccharornyces pombe. However, there is still a need of screening strains of Schizosacch. for their effect on wine quality and intensity to metabolize malic acid. Special attention should be given to sensory evaluation with statistical analysis for such quality attributes as color, flavor, and bouquet. There is also a need to control the level of deacidification during fermentation. This will require more information on those factors which greatly affect the growth of Schizosacch., such as temperature, pH, and sulfur dioxide. In addition, research should be continued in determining the influence of mixed cultures and must treatments on the utilization of malic acid by Schizosaccharornyces. Combinations of one or more of the different deacidification processes are often complimentary and produce desirable results. Increasing the susceptibility of high-acid wines to malo-lactic fermentation by a controlled treatment of the must with calcium carbonate, sufficient to increase the pH to about 3.3, is commonly used in northern Europe. Carbonic maceration also has been shown to increase the susceptability of the resultant wine to malo-lactic fermentation. A moderate amelioration (about 15 to 25%) of must followed by addition of calcium carbonate to the wine appear to be complimentary deacidification practices in specific circumstances, since amelioration preferentially reduces malic acid while calcium carbonate preferentially removes tartrate from the wine. Other combinations undoubtedly exist and might serve as areas for additional investigation. Also, it is probable that different procedures or combinations would be more appropriate for use with certain grape varieties, or with different wine types. Many of these differences are yet to be determined experimentally.

REFERENCES Alley, C. J . , Goheen, A. C., Olmo, H. P . , and Koyama. A . T. 1963. The effect of virus infections on vines, fruit and wines of Ruby Cabernet. Am. J . Enol. Vitir. 14, 164-170.

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Peynaud, E. 1938. L’acide malique dans les moGts et les vins de Bordeaux. Ann. Falsc. Fraudes 31, 332-347. Peynaud, E., and Domercq, S. 1959. Possibilte de provoquer la fermentation malolactique en vinification a I’acid de batteries cultivees. C. R. Seances Acad. Agric. Fr. 45, 355-358. Peynaud, E., and Guimberteau, G. 1962. Modification de la composition des raisins au cours de leur fermentation propse en anaerobiose Ann. Physiol. Veg. 4, 161-167. Peynaud, E., and Lafon-Lafourcade, S. 1965. Etude dun dosage simple de I’acide malique applique aux vins a I’aide de Schizosarcharomyces pombe. A n n . Technol. Agric. 14, 49-59. Peynaud, E., and Ribkreau-Gayon, P. 1971. The grape. In “The Biochemistry of Fruits and Their Products” (A. C. H u h , ed.), Vol. 2, pp. 171-205. Academic Press, New York. Peynaud, E., and Sudraud, P. 1964. Utilisation de I’effect desacidifiant des Schizosaccharomyces in vinification de raisins acides. Ann. Technol. Agric. 13, 309-328. Peynaud, E., Domercq, S., Baidron, A. M., Lafon-Lafourcade, S., and Guimberteau, G. 1964. Etude des levures Schizosaccharomyces metabolisant I ’acide L-malique. Arch. Mikrobiol. 48, 150- 165. Pilone, G. J., and Kunkee, R. E. 1972. Characterization and energeticsof LeuconosIocoenos ML34. Am. J . Enol. Vilic. 23, 61-70. Pilone, G. J., and Kunkee, R. E. 1976. Stimulatory effect of malo-lactic fermentation on the growth rate of Leuconostoc oenos. Appl. Environ. Microbiol. 32, 405-408. Prillinger, F. 1958. Deacidification by means of calcium carbonate and inhibition of tartaric acid precipitation. Mitt. Hoeheren Bundeslehr- Versuchsanst. Wein- Obsrbau. Klosrerneuburg, Gartenbau, Schoenbrunn, Ser. A 8, 134-42; Chem. Absrr. 53, 8530 (1959). Radler, F. 1962. Uber die Milchsaurebakteriem des Weines und den biologischen Saureabbau. Vitis 3, 144-176 and 207-236. Radler, F. 1966. Die mikrobiologischen Grundlegen des Saureabbaus im Wein. Zentralbl. Bakteriol.9 Parasitenkd., Infektionskr. Hyg., Abt. 2 120, 237-287. Radler, F. 1968. Apfelsaureabbau in deutschen Spitzenweinen Z. Lebensm.- Unters. -Forsch. 138, 35-39. Radler, F. 1972. Problems in malo-lactic fermentation. Weinberg Keller 19, 357-370; Chem. Absir. 77, 162987 (1972). Rankine, B. C. 1965. Ion exchange treatment of wine. Ausr. Wine Brew. Spirit Rev. 85, 59-62. Rankine, B. C. 1966. Decomposition of L-malic acid by wine yeasts. J. Sci. Food Agric. 17, 312-316. Rankine, B. C. 1972. lnfluence of yeast strain and malo-lactic fermentation on composition and quality of table wines. Am. J. Enol. Viric. 23, 152-158. Rankine, B. C. 1977. Developments in malo-lactic fermentation of Australian red table wines. Am. J . Enol. Vitic. 28, 27-33. Rankine, B. C., and Fornachon, J. C. M. 1964. Schizosaccharomyces malideuorans sp. n., a yeast decomposing L-malic acid. Antonie van Leeuwenhoek 30, 73-75. Rankine, B. C., Fornachon, J. C. M., Bridson, D. A,, and Cellier, K . M. 1970. Malo-lactic fermentation in Australian dry red wines. J. Sci. Food Agric. 21, 471-476. Rebelein, H. 1970. Verfahren zur beliebig weitgehenden Entsauerung von Traubenmosten. Weinblatt 65, 283-287. Rebelein, H. 1971a. Correction de I’acidite des moats et des vins. Rapport Allemand. Bull OIV 480, 136- 141. Rebelein, H. 1971b. Ein Weg zur beliebig weitgehenden Entsauerung von Jungweinen. A&. Dtsch. Weinfachzrg. 107, 1208-121 1. Reed, G., and Peppler, H.J. 1973. “Yeast Technology.” Avi Publ. Co., Westport, Connecticut. RiMreau-Gayon, J . , and Peynaud, E. 1962. Application a la vinification de levures metabolisant I’acide malique. C. R . Seances Acad. Agric. Fr. 48, 558-560.

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R. B. BEELMAN AND J. F. GALLANDER

Riureau-Gayon, J., Peynaud, E . , RiMreau-Gayon, P. and Sudraud, P. 1975. “Trait6 d’oenologie, Sciences et Techniques du Vin,” Tome 2. Dunod, Paris. RiWreau-Gayon, I . , Peynaud, E., Sudraud, P., and RiEreau-Gayon, P. 1976a. “Trait6 d’Oenologie, Sciences et Techniques du Vin,” Tome 1. Dunod, Paris. RiWreau-Gayon, J . , Peynaud, E., RiWreau-Gayon, P., and Sudraud, P. 1976b. “Traite d’Oenologie, Sciences et Techniques du Vin,” Tome 3. Donod, Paris. Rice, A. C. 1965. The malo-lactic fermentation in New York State wines. Am. J. Enol. Viric. 16, 62-68.

Rice, A. C. 1974. Chemistry of winemaking from native American grape varieties. Adv. Chem. Ser. 137, 88-115. Robinson, W. B., Shaulis, N., Smith, G .C., and Tallman, D. F. 1959. Changes in the malic and tartaric acid content of Concord grapes. Food Res. 24, 176-180. Saito, K., and Kasai, 2 . 1968. Accumulation of tartaric acid in the ripening process of grapes. Plant Cell Physiol. 9, 529-537. Schopfer, 1. F. 1968. Vinification en blanc en Suisse. Fermenr. Vinifcafions, Symp. Inr. Oenol., 2nd. 1967 pp. 451-460. Schopfer, J. F. 1971. Correction de I’acidite des moDts et des vins. Rapport Suisse. Bull. OIV 479, 55-57.

Steele, J . T., and Kunkee, R. E. 1978. Deacidification of musts from the western United States by the calcium double salt precipitation process. Am. J. Enol. Vitic. 29, 153-160. Sudraud, P., and Cassignard, R. 1959. Travaux recents sur la fermentation malolactique. Vignes Vins 80, 10-13. Tanner, H., and Sandoz, M . 1974. Chemische Entsaurerungsmassnahmen im Hinblick auf die Einleitung eines einwandfreien biologischen Saureabbaues. Schweiz Z. Obsr- Weinbau 100, 356-367.

Troost, G. 1972. “Handbuch der Kellenvirtschaft,” 4th ed., Vol. 1. Ulmer, Stuttgart. Troost, G . , and Fetter, K. 1966. Erfahrungen mit der Saureminderung beim Jahrgang 1965. Drsch. Wein-Ztg. 102, 637-638 and 810-822. Upshall, W. H., and van Haarlem, J. R. 1934. Yield and quality of fruit from strongly vegetative Concord grape vines. Sci. Agric. 14, 438-440. U . S . Internal Revenue Service. 1974. “Wine,” Part 240 of Title 26, Code of Federal Regulations. ATF Publ. 5120.2, revised 12/74. US Gov. Printing Office, Washington, D.C. Usseglio-Tomasset, L. 1973. La correzione dell’acidita dei vini. Vini Iral. 85, 309-313. Vaadia, Y.,and Kasimatis, A. N . 1961. Vineyard imgation trials. Am. J. E n d . Vitic. 12, 88-98. van Wyk, C. J . 1976. Malo-lactic fermentation in South African table wines. Am. J. Enol. Viric. 27, 181- 185.

Vetsch, U. 1973. Untersuchung zur Vermehrung von Bacterium g r a d e (Leuconostocoenos) wahrend des biologischen Saureabbaus im Wein. Schweiz. Z. 0bsr.- Weinbau 109, 468-479. Wagner, P. M. 1974. Wines, grape vines and climate. Sci. Am. 230, 106-1 15. Wagner, P. M. 1976. “Grapes into Wine.” Alfred Knopf, New York. Weaver, R. J., McCune, S. B., and Amerine, M. A. 1961. Effect of level of crop on vine behavior and wine composition in Carignane and Grenache grapes. Am. 1. Enol. Vific. 12, 175-184. Webb, A. D., and Berg, H. W. 1955. Terms used in tasting. Wines Vines 7, 25-28. Wejnar, R. 1968. Untersuchungen zur Bedeutung der Weinsaure fur die Wasserstoffionenkonzentration des Traubenweines. I. Vergleichende Weinanalysen. 11. Weitere Komlationsuntersuchungen. Mitt. Hoeheren Bundeslehr- Versuchsansr. Wein- Obstbuu, Klosterneuburg, Gurtenbau. Schoenbrunn, Ser. A 18, 349-358. Wejnar, R. 1969a. Untersuchungen zur Bedeutung der Weinsaure fur die Wasserstoffionenkozentration des Traubenweines. 111. Einfluss des Weinsteinausfalls auf Gesamtsaure, Weinsaure und cH’ . IV. Die durch Weinsteinausfall verursachten Veranderungen des Kaliumgehaltes und der

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Aschenalkalitat. Mitt. Hoeheren Bundeslehr- Versuchsunst. Wein- Ohsthau, Klosterneuburg, Gartenbau, Schoenhrunn, Ser. A 19, 109-121. Wejnar, R. 1969b. Untersuchungen zur Bedeutung der Weinsaure fur die Wasserstoffionenkonzentration des Traubenweines. V. Apfelsaureabbau und cH’ des Weines. Mitt. Hoeheren Bundeslehr- Versuchsansr. Wein- Ohsthau, Klosrerneuburg. Gartenbau. Schoenhrunn. Ser. A . 19, 193-201. Wejnar, R. 1972. Untersuchungen zur Bedeutung der Weinsaure fur die Wasserstoffionenkonzentration des Traubenweines. VIII. Theoretische Eronerungen unter besonderer Berucksichtigung des Apfelsaureabbaus und der Weinsteinausfallung. Mitt. Hoeheren Bundeslehr- Versuchsanst. Wein- Obstbau. Klosrerneuburg, Gartenhau, Schoenhrunn. Ser. A 22, 19-37. Wienhaus, H. 1967. Untersuchungen uber Zucker- und Apfelsaurevergarung durch Schizosuccharomyces pombe var. liquefaciens. Wein-Wiss. 22, 25-39. Winkler, A. J. 1954. Effects of overcropping. Am. J. Enol. Vitic. 5, 4-12. Winkler, A. J. 1958. The relation of leaf area and climate to vine performance and grape quality. Am. J . Enol. Vitic. 9, 10-23. Wurdig, G. 1977. Die Doppelsalzentsauemng von Most und Wein Bekanntes und Neues-eine Ubersicht. Drsch. Weinbau Jahrbuch, 1977 pp. 183-190. Yang, H. Y. 1973a. Deacidification of grape musts with Schizosacchrrromvces pombe. Am. J. Enol. Vitic. 24, 1-4. Yang, H. Y. 1973b. Effect of pH on the activity of Schizosaccharomyces pombe. J. FoodSci. 38, 1156-1 157. Yang, H. Y. 1975. Effect of sulfur dioxide on the activity of Schizosaccharomyces pombe. Am. J . Enol. Viric. 26, 1-4. Yoshizumi, H. 1975. A malo-lactic bacterium and its growth factor. In “Lactic Acid Bacteria in Beverages and Food” (J. G . Cam, C. V . Cutting, and G. C. Whiting. eds.), pp. 87-102. Academic Press, New York. Zubeckis, E. 1957. Deacidification of wine by ion-exchange. Rep., Ont., Horric. Exp. Sta. Prod. Lab. pp. 88-89. Zubeckis, E. 1962. Studies on wine treatment with ion-exchange resins. Rep.. Ont., Hortic. Exp. Stn. Prod. Lab. pp. 117-119.

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ADVANCES IN FOOD RESEARCH. VOL. 25

DEHYDRATED MASHED POTATOES-CHEMICAL AND BIOCHEMICAL ASPECTS D. HADZIYEV AND L. STEELE Deparimeni of Food Science, University of Alberta, Edmonton, Alberia, Canada

55

A. Granules. . . . . . . . . . . . . . . . . . . . . . . B. Flakes ................................. Ill. Involvement of Some Cell Constituents in Granule P

IV.

V. VI. VII.

VIII.

1X.

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nces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Starch . . . . . . ...................................... E. Vitamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flavoring Constituents . . ...... ................ A. Nonvolatile Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Volatile Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Interaction of Flavor Volatiles with Major Potato Constituents . . . . . . . . The Role of Sulfites as Additives ............ Microflora as Affected by Processi Rancidity during Storage and Shipment . . . . . . . . . . . . . . . . . . . . . , . . . . , , . . A. Rancidity Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Determination and Use of Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . Some Characteristics of Reconstituted Granules . . . A. Rehydration Rates , , , . , . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Texture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ Research Needs.. . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

1.

56 56 59 61 61 73 79 85 96 102 102 103 108

109 111 112 112 117 122 122 122 123 124

INTRODUCTION

The potato (Solanum tuberosum) is already an important constituent in the diets of a large segment of the world's population, but there is still room for it to assume an even more significant role. Furthermore, development of higher yield55 Copyright 0 1979 h) Academic F'rerr. Inc. All righis of reproducuon in any lorn1 restrved

ISBN U-1?-0164?5-h

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D. HADZIYEV AND L. STEELE

ing, hardier, and even more nutritious varieties increases the need to provide potatoes in a form that is stable over a much longer period of time and requires less rigorous storage control than raw tubers. Processing of potatoes into dehydrated granules or flakes is perhaps the most satisfactory method of creating a product that is not only nutritionally and organoleptically adequate, but remains so over an extended period. However, such processing must be carried out in a controlled manner to ensure that most of the potato’s original value is retained. A complete understanding of the effect of each step in a potato dehydration process requires knowledge of the chemical and biochemical makeup of the raw tuber. The process can then be designed to minimize deleterious changes and maximize the quality of the product.

II. DEHYDRATED MASHED POTATO PROCESSES Early development of dehydralted mashed potato technology was reviewed by Olson and Harrington (1955). Recent patents were reviewed by Hanson (1975) and Torrey (1974). Basic information related to potato dehydration was given by Feustel et al. (1964). A.

GRANULES

It appears that the only commercially applied potato granule process is the add-back process. Its basic features are: peeling, slicing, precooking or waterblanching, and water-cooling; followed by steam-cooking, mashing-mixing (with about two parts of recycled dry granules), conditioning, remixing, air lift drying, fluid bed drying, and cooling and sieving. The rationale for the sequence of the steps is as follows: slicing ensures effective and uniform heat transfer in subsequent cooking. Precooking and cooling avoids sloughiness during cooking and imparts firmness to cell walls which is required in the mash-mixing step. Cooking brings about final softening of the tissue. Hot mash-mixing results in tissue separation into individual cells or their aggregates with minimum cell rupture. The conditioning step in a stream of cold air is needed to equilibrate the mash moisture and, by keeping the moisture content above -30%, forces the free starch to retrograde and thus increases the friability of the moist mash. Re-mixing is done in order to further granulate the moist mash into essentially single cell particles. This and the previous mash-mixing step are the prerequisites for the moist granules to remain separated and to be conveyed and dried in the subsequent air lift drying step. The latter step reduces the moisture content of the granules from 30 to -15%, while the following fluid bed drying decreases the moisture content from -15 to 7%. The cooled granules are then sieved. A small

DEHYDRATED MASHED POTATOES

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portion of granules of particle size 80 mesh or less is collected as end product, while the rest is recycled. Particles of 10 mesh or greater are removed as rejects. A modified add-back process with minimum recycling was that of Willard (1966). Cooked, mashed potatoes and dehydrated flakes were admixed with the aid of emulsifier to produce an equilibrated mash with 35-45% moisture. The end product, of high bulk density, was a mixture of fine flakes and single and aggregated cells. Kueneman and Conrad (1963) developed an improved process by treatment during or after the add-back step with a hot, moist atmosphere. The advantage of continuous cooking in a tunnel-type boiler was outlined by Griffon (1969). Vigerstrom and Strid (1974) preheated sliced potatoes in an aqueous bath by direct passage of current prior to other conventional add-back steps. Though the add-back process has been improved, its major disadvantage still remains: I/io to ' / 6 of the solid material handled is the end product, while the remainder stays in the system by recycling. This has a bearing on operating costs. In addition, granule quality can be reduced because undesirable characteristics, once developed, remain in the system in appreciable percentage after numerous cycles, even over a week of continuous production. Therefore, procedures were found to attain subdivision of cooked tissue without the shear action of the add-back granules. Willard (1967) introduced a process which involved a simultaneous cooking and mashing step. Peeled, precooked, and cooled potatoes were steam-cooked in a cylindrical mashing cage equipped with numerous elongated parallel rods. The exterior portion of the tuber and then the interior portion (with lower solid content) were mashed by sliding across the cage. No portion of the tuber was over- or undercooked so cell separation proceeded with much less damage. Hendel et al. (1962) designed a process in which hot-mashing and partial dehydration were followed by chilling down to subfreezing temperatures. The conditioned mash was then granulated and dried. A patent by Shatila and Terrell (1976) produced dehydrated granules without employing precooking and cooling steps. Partially mashed steam-cooked potatoes were riced in the presence of glycerol monostearate in order to coat the surfaces of the separated cells. The additional steps were granulation, predrying, and fluid bed drying. During the process amylose solubilized, but did not retrograde, giving an end product capable of high cold water absorption. In the process developed by Kodras (1962) mashed potatoes were cooled and aged at low temperatures. The aged mash was slurried in water, filtered, and dried. It was claimed that no objectionable cell rupture occurred and that the end product was of superior quality. A filter cake made from potato slurry was reslurried in the presence of additives and spray-dried (Sienkiewicz and Hollis, 1966). The product consisted of single cells that had high textural quality and were not grainy or pasty.

58

D. HADZIYEV AND L. STEELE

FIG. 1 . Scanning electron micrographs of potato granules obtained by the (a) freeze-thaw and (b) add-back process. The latter granules, as seen in a close-up view (c), are rounded (Moledina et al.. 1978a).

A freeze-thaw process which also omits precooking and cooling steps was recently disclosed by Ooraikul(1977a, 1978). Mashed potatoes were frozen in an air-blast freezer at -29°C. The frozen mash was thawed to 0-5°C before being subjected to predrying, granulation, and final drying steps. The end product contained at least 85% of granules smaller than 60 mesh, and the broken cell count was not more than 3%. The surface structure of add-back and freeze-thaw granules (Fig. 1) has been studied by scanning electron microscopy (Jericevic and Ooraikul, 1977; Moledina et a / . , 1978a). A similar process to the above was that of Shub and Bogdanova (1976). Sliced tubers were steam-cooked and mashed, and the moist mash was frozen to - 10 to -40°C for 2-5 minutes. Then the product was fluid bed dried to 8-12% moisture. The ruptured cell count was 3.6%. Granulation of potato mash in the frozen state is exemplified by the process

DEHYDRATED MASHED POTATOES

59

FIG. lb.

developed by Notter and Hendel (1960). Hot, cooked potatoes were mashed, partially dried, and spread in thin layers and frozen. The frozen mash was then fed into the nip between two rolls held at room temperature. Dislodged flakes of one cell thickness formed small platelets which, when subjected to subsequent dehydration steps, broke up into granules of single or agglomerated cells. It was claimed that granulation in the frozen state occurred without cell rupture. Agglomerated granules have been produced from individual potato cells (Hutchings and Stringham, 1971) while clusters have been created from agglomerates by using milk solids as binder (Mathias and Holland, 1974).

B. FLAKES Conventional drum drying, as developed by Cording and Willard (1956), has been widely accepted in practice. Most of the initial granule processing steps

60

D. HADZIYEV AND L. STEELE

FIG. lc.

See legend p. 58.

(peeling, slicing, precooking, cooling, and steam-cooking) are retained in flake processing (Knaack, 1976). However, in the flake process, mashing is done with higher shear and compression forces and is not followed by a conditioning step to retrograde the released starch. Hence, in order to avoid the pastiness imparted by free starch, the starch is complexed during mashing by adding monoglycerides or related emulsifiers. Also, in order to preserve the color and to improve the shelf life of the low bulk density end product, sulfites and antioxidants are added during mashing to assure their uniform distribution. (Since low bulk density product packaging in nitrogen is too costly, antioxidants must be added to protect the flakes against rancidity.) The mash is dried in the form of a thin sheet on an internally steam heated single or double drum dryer to 4-10% moisture content. The doctor knife strips off the dried layer which is then cut into flakes. The cell thickness of the flakes depends on the number of unheated spreader rolls spaced

DEHYDRATED MASHED POTATOES

61

close to the drum. Each spreader roll applies its single layer of cells to those already partially dried. In addition, applicator rolls may be used in order to squeeze the partially mashed chunks and thus to impart uniformity and a higher density to the sheet. Gutterson (1971) and Hanson (1975) reviewed modifications of this basic process such as a two-stage cooking process (Barnes and Nora, 1967), and bulk density increase with or without recycling fines (Eskew, 1962; Pader, 1962). Snack foods based on dehydrated mashed potato products, such as extruded French fries, enriched granules or flakes, or low calorie products, do not require any significantly unique additional steps, hence, they are not reviewed here. Pertinent data along these lines were presented by Hanson (1975) and Robbins (1976).

111.

INVOLVEMENT OF SOME CELL CONSTITUENTS IN GRANULE PROCESSING A.

LIPIDS

The amount of lipids in potatoes is too low to be of nutritional significance. However, the high unsaturation of fatty acids and the accumulated evidence that off-flavors in dehydrated potato products are partly due to fatty acid oxidation has prompted research into lipids in both raw and processed potatoes. 1 . Composition Related to Growth, Storage, and Variety

A detailed study of potato lipids was provided by Lepage (1968) for potatoes grown in Canada: “Netted Gem” (Russet Burbank) and Kennebec as representative of excellent processing and storage qualities, and Irish Cobbler and Sebago as fair-to-good storage quality. The results showed only a few varietal differences in the content of some lipid classes, and no difference in the fatty acid composition of the total lipids. However, marked differences were observed in fatty acid composition of some individual constituents of polar lipids. The involvement of lipids in off-flavor development prompted Galliard and co-workers (Galliard, 1973; Galliard et al., 1975; Berkeley and Galliard, 1974a,b) to analyze some European potato varieties in an attempt to find a variety with a reduced content of polyunsaturated fatty acids. The results showed that the content of total lipids (0.5% on a dry weight basis) was similar for all varieties. The relative amounts of each lipid class showed no varietal differences. Fatty acid analysis established that at least 90% of the total acids were a saturated acid, palmitic, and two polyunsaturated acid, linoleic and linolenic. Furthermore, it was found that the percentages of individual acids varied only

62

D. HADZIYEV AND L. STEELE

slightly among varieties, and that a significant inverse relationship existed between linoleic and linolenic acids, such that the content of polyunsaturated acids was close to the narrow range of 70-76% of the total acids for all varieties. Galliard (1973) emphasized the need for choosing correct lipid solvents and inactivating tuber enzymes responsible for lipid breakdown. Doubts were raised about results from earlier studies where this was not done. It was stressed that polar solvents must be used for efficient lipid extraction since the majority of potato lipids were polar, i.e., phospho- and glycolipids. In addition, potato tissue should initially be treated in boiling isopropanol to inactivate the high levels of two lipid degrading enzymes, lipolytic acyl hydrolase and lipoxygenase. The activities of these two enzymes in some varieties were such that homogenization of the tuber tissue at 0°C could result in immediate and nearly complete degradation of endogenous membrane-bound lipids (Galliard, 1970). Separation and detailed characterization of potato lipid hydrolases was reported by Hasson and Laties (1976). At pH 7.5 they found the phospholipase activity to be at least four orders of magnitude higher than the hydrolase activity on galacto- and neutral lipids. In order to avoid possible deleterious effects on polyunsaturated acids, contributing to an overall oxidative breakdown, Galliard and Matthew (1973) tried to find a physiological stage during potato growth or storage that would ensure low activity levels of lipid degrading enzymes. With the exception of the variety Desiree, the 22 European varieties tested had high levels of hydrolytic enzyme and lipoxygenase activity. The effect of storage at 5 and 20°C on lipid content was also investigated for some European varieties which differed widely in their sprouting behavior, i.e., varieties with short and long dormancy (Berkeley and Galliard, 1974a,b; Galliard et al., 1975). The results suggest that neither the choice of potato variety, nor the length or temperature of storage would be likely to reduce the polyunsaturated fatty acid content to an extent which would avoid fatty acid breakdown (giving rise to off-flavors), a problem experienced by many processors of dehydrated potato granules. The distribution of lipids and their fatty acid composition in potato peels and in potato flesh (usually considered as peeled potato) was reported by Fricker (1970) and Cherif and Ben Abdelkader (1970). The lipid content of the peel was 2%, and that of the flesh 0.61%, while the whole tuber gave 0.77% (dry weight basis). Linoleic and linolenic acids were more abundant in the flesh than in the peel. However, peel was rich in acids with a chain length greater than CZ2. Mondy and Mueller (1977a) found that the apical bud end had significantly higher total lipid and phospholipid fractions than the basal stem end of the tuber. This agreed with the histological development sequence outlined by Reeve et al. (1969).

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63

Though the compositional data for lipid in peel and flesh are satisfactory from the processing point of view, the data concerning lipid distribution within the tuber according to its anatomical makeup are also of interest. Data on the fatty acids of the total lipids in peel, the vascular bundle region (phloem and xylem), stem and bud ends, eye regions, and pith were obtained by Fricker (1970) and Warmbier and Muller (1973). Regardless of the anatomical region analyzed, the major tuber fatty acids were palmitic, stearic, linoleic, and linolenic. Most of the other acids were present in amounts less than 1% of the total fatty acids. Oleic acid was 1% in the bud and 3% in sprouts. The bud end was enriched with heneicosic acid (6%)and palmitic acid (30%), while the pith was relatively rich in behenic acid (1.6%). Moving from the peel toward the pith, linolenic acid decreased from 25 to 13.1%, while linoleic acid increased from 46.3 to 53.5%. A comparison of stem with bud ends and the eye region showed that the bud end contained the most palmitic acid and the least linolenic acid. Stearic acid was 3.7% in the stem end, 5% in the bud end, 4.9% in the eye region, and a low 1.7% in the sprout. Linoleic acid content differed only slightly between stem and bud ends, and sprouts, but it was significantly lower in eye regions. Some of these acid distribution results might be explained by metabolic changes induced by sprouting. 2. Lipid Distribution within Membranes and Organelles As outlined earlier, the major lipids in potato tuber are polar, i.e., phosphoand glycolipids, all of which are associated with lipoprotein membranes of the cell or cell organelles. Some data along these lines exist for the raw tuber; however, little is known about the lipid distribution within processed granules. There is interest not only in the nature of polar lipids and their composition and unsaturation ratios, but also in the spatial distribution of lipids in the newly acquired cell architecture. If membrane lipids were embedded between the fused starch of single cell granules, less of them would be left in the space between the gelled starch and the porous cell wall. As a consequence, less lipid would be exposed to oxygen diffusion and subsequent oxidative deterioration. The lipid composition of some of the membranes and organelles of the potato is given in Table I (Pun et al., 1979). The main lipids in membranes and organelles were phospholipids. However, striking exceptions were peroxisomes, and starch grain membranes in which the glyco- and/or neutral lipids were predominant. Phosphatidyl choline and -ethanolamine constituted nearly 80% of the phospholipids in plasmalemma, and about 78% in both mitochondria and endoplasmic reticulum. Diphosphatidyl glycerol was the third major phospholipid in mitochondria (1 1.4%). As reported by Mazliak et al. (1975), disphosphatidyl

TABLE I COMPOSITION OF LIPIDS IN SOME MEMBRANES FOR ORGANELLES @4 RAW "NElTED GEM" TUBERS STORED AT 20°C FOR 1 MONTH"

Lipid

Plasmalemma

Mitochondria

Peroxisomes

Endoplasmic reticulum (microsomes)

Phospholipids Phosphatidyl choline Phosphatidyl ethanolamine Diphosphatidyl glycerol Phosphatidyl inositol

34.90 21.1 8.0 2.2 3.6

79.2 36.0 25.6 11.2 6.4

13.Y 7.3 2.9 0.4 1.o

41.7d 19.5 13.0 1.o 5.9

16.3 11.9 2.5 1.9

Glycolipids Digalactosy I diglyceride Monogalactosyl diglyceride Steryl glucoside Esterified steryl glucoside

49.6 11.6 9.2 4.7 24.1

11.8 4.3

I .9 3.7

31.7 8.1 7.5 6.6 9.5

42.7 15.3 10.9 10.0 6.5

26.2 5.6 7.5 3.7 9.4

Neutral lipids Free fatty acids Triglyceride Free sterol Esterified sterol Oxidized fatty acids

15.5 3.2 1.4 0.8 8.3 1.8

9.0 2.1 1.2 0.7 4.0 1.o

54.4 8.3 6.4 2.1 32.4 4.6

15.6 4.7 3.2 2.0 3.0 2.7

57.4 12.5 11.9 18.0 8.8 6.2

1.9

During analysis, lipid hydrolase activity was suppressed by at least 70% by nupercaine-HCI (Pun et a/.,1979). Wt% of total lipids. Also contained phosphatidyl glycerol (2.3%). Other PL: 2.3%.

Starch grain membranes

FIG. 2. Transmission electron micrographs of potato starch grains (a) before,and (b) after acetone extraction of the parenchyma tissue (Chung et al., 1979). SGM,starch grain (SG) membrane.

66

D. HADZIYEV AND L. STEELE

FIG.3. Transmission electron micrographs of potato cell (a) before, and (b) after acetone extraction (Chung et al., 1979). CW, cell wall; LB, lipid body; Px, peroxisome; V , vacuole; VM, vacuolar membrane (tonoplast).

glycerol was concentrated in the inner mitochondrial membrane. The two mitochondria1 membranes, as found by these authors, did not have the same phospholipid composition, the outer having twice as much phosphatidylcholine as -ethanolamine, while the inner membrane had equal amounts of both compounds. Glycolipids were present in all membranes and organelles. The digalactosyl to monogalactosyl diglyceride ratio was 1.26 in plasmalemma, 2.26 in mitochondria, and much less in other membrane systems. A ratio close to 2.12 in purified potato mitochondria was reported by Schwertner and Biale (1973). The monogalactosyl diglyceride surpassed the amount of digalactosyl diglyceride in the cell wall.

DEHYDRATED MASHED POTATOES

67

FIG. 3b.

Steryl lipids were 15.7% of the total lipids in raw “Netted Gem” tubers (Pun and Hadziyev, 1978). Steryl glycolipids (esterified sterylglucoside and sterylglucoside) were present in all membranes and organelles. The esterified form was predominant, its content being 3 to 6 times higher than the nonesterified form in plasmalemma, mitochondria, or peroxisomes, while the free form was slightly higher in endoplasmic reticulum and starch grain membranes. Neutral lipids were the major lipids in peroxisomes and starch grains. However, results should be cautiously interpreted, since the presence of free fatty acids within the fraction suggested activity of hydrolyzing enzymes during isolation of membranes and organelles. In agreement with some data from Table I are transmission electron microscopy data of potato celIs obtained before and after extraction with selective lipid solvents (Chung et al., 1979). Potato samples extracted at room temperature with acetone, known to be a poor phospholipid solvent, showed a complete loss

68

D. HADZIYEV AND L. STEELE

of amyloplast (starch) membranes (Fig. 2), as well as those of peroxisomes, lipids in cell walls, and rare lipoid bodies distributed in the cytoplasm (Fig. 3). Similar results were obtained in potato samples washed with petroleum ether. Extraction with ch1oroform:methanol (2: 1 v/v) removed all the lipid membranes. n-Butanol washed out all the cell membranes, except that of tonoplast, which was left behind as broken disrupted fragments. A similar effect was found when methano1:ethanol (1:l v/v) was used as a solvent system. When the findings on raw potato tissue were compared with those obtained by a transmission electron microscopic study of steam-cooked tuber (Chung et al., 1979), then, aside from the observations such as lack of cell distension or cell rupture, a substantial change in spatial organization of the lipids was evident. In the cooked potato it was difficult to differentiate cellular membranes and organelle structure. Upon cooking, the denatured cytoplasmic membranes were sandwiched between gelled starch and cell wall (Fig. 4). It is probable’that lipids associated with the cytoplasmic membranes were present in this region. No embedded lipid was detected within the gelled starch. The exact spatial arrangement of lipids in dehydrated granules has not been fully established. The degree of unsaturation of fatty acids associated with membranes and/or organelles is of great importance from the processing point of view, since it can contribute to a rapid oxidation of lipids in processed granules. As shown in Table I1 (Pun et al., 1979), such a contribution would be least expected from starch grains since their membranes are rather saturated. Palmitic acid was 22.4% of all the acid present, and the unsaturation ratio (UR: a ratio of the sum of linoleic and linolenic acids to that of palmitic and stearic acids) was only 1.4. Peroxisomes (UR 1.8) should also be considered as predominantly saturated. The greatest contribution to rapid oxidation might be expected from mitochondria (UR 2.9), and the endoplasmic reticulum. Plasmalemma (UR 1.7) should be rated as only a moderate contributor to lipid oxidation. The percentage composition of the fatty acids of potato tuber membranes reported by Mazliak et al. (1975) might lead to the same conclusions, except for their peroxisome preparation, for which they reported linoleic plus linolenic acids to be 69.3%, and saturated acids 26.1%. This composition would raise the UR of peroxisomes to 2.7, a value which would surpass even that of the endoplasmic reticulum. 3. The Effect of Processing

Compared to the wealth of data on lipid changes in potatoes during growth and storage, there is relatively little information on lipids of processed potatoes. A recent report by Mondy and Mueller (1977b) was limited to the effect of household cooking methods on lipid composition, and only some of the total lipids and their fatty acids were dealt with. The results of Schwartz et al. (1968) were related to fatty acid contents in dehydrated potato dices and flakes. When dehy-

DEHYDRATED MASHED POTATOES

69

FIG. 4. Transmission electron micrograph of steam-cooked potato cells (Chung er a [ . , 1979). CW, cell wall; GS. gelled starch surrounded by a lipid layer.

drated dices were prepared commercially by cooking diced potatoes followed by drying in a forced draft tray dryer, the polyunsaturated fatty acid content matched that of the original tubers. Flakes made by the standard flake process contained somewhat less of these acids. In both products the degree of unsaturation of the fatty acids changed little, and no off-flavors developed when the samples were reconstituted. Studies on lipid changes in dehydrated granules obtained by a freeze-thaw process (Ooraikul, 1978) were recently completed in our laboratory (Pun and Hadziyev, 1978). The total lipid in peeled and sliced, raw “Netted Gem” tuber was 0.17% on a fresh weight basis, or 0.65% dry weight (Table Ill). Fifty-two percent of the total lipid was in the phospholipid fraction, which was mostly phosphatidyl choline and -ethanolamine. With the exception of phosphatidyl inositol, other phospholipids were found to be trace or minor constituents and were

TABLE 11 FATTY ACID COMPOSITION OF THE TOTAL LIPIDS OF SOME POTATO TUBER MEMBRANES AND ORGANELLESa

Fatty acid

Plasmalemma

Lauric + myristic (12:O + 14:O) Palmitic (16:O) Palmitoleic (16:l) Stearic (18:O) Oleic (18:l) Linoleic (18:2) Linolenic (I8:3) Eicosenoic (20:1) Unsaturation ratio" Pun et al. (1979) Percent of total fatty acid. I. (Linoleic + linolenic acid) / (palmitic

tr.

27.3 -

7.7 3.9 47.9 11.1 2.0 1.7

a

+ stearic acid).

Mitochondria

Peroxisomes

1 .ob

3.8 22.2 5.0 4.0 11.0 42.5 10.9

19.4 4.7 2.4 59.6 12.1 0.8 2.9

tr.

1.8

Endoplasmic reticulum (microsomes)

Starch grain membranes

tr. 24.7 6.8 2.0 49.6 15.4 1.4 2.1

1.5 22.4 4.3 14.1 4.0 37.8 14.3 1.6

1.4

71

DEHYDRATED MASHED POTATOES TABLE 111 COMPOSITION OF LIPIDS IN RAW AND PROCESSED POTATOESa Weight in mg per 100 gm dry weightb ~

~~

Lipid

Raw potatoes

Steam-cooked mashed potatoes

Dehydrated potato granules

Total phospholipid Phosphatidyl choline Phosphatidyl ethanolamine Phosphatidyl inositol

335.2 161.3 108.6 59.1

299.9 (10.5)' 151.9 ( 5.8) 89.3 (17.8) 50.5 (14.6)

282.3 (15.8) 145.1 (10.0) 82.4 (24.1) 46.7 (21.0)

Total galactolipid Monogalactosyl diglyceride Digalactosyl diglyceride Other galactolipid

128.6 37.3 79.9 11.4

127.1 ( 1.2) 42.8 (14.7+) 73.3 ( 8.3) 11.0( 3.5)

125.2 ( 2.6) 49.0 (31.4+) 65.8 (17.6) 10.4 ( 8.8)

Total steryl lipid Esterified steryl glucoside Steryl glucoside Free sterols Sterol esters

101.3 27.7 28.1 14.8 30.6

80.8 (20.2) 25.5 ( 7.9) 24.1 (14.2) 7.2 (51.3) 24.0 (21.6)

81.9 (19.2) 24.9 (10.1) 24.1 (14.2) 9.7 (34.5) 23.2 (24.2)

15.0 32.8 33.4

11.3 (24.7) 24.4 (25.6) 19.7 (41.0)

11.5 (23.3) 24.4 (25.6) 26.7 (20.1)

645.5

563.2 (12.9)

Free fatty acids Triglyceride Other neutral lipid Total lipids ~

~

551.7 (14.7) ~

~~

Pun and Hadziyev (1978). An average of three determinations which did not differ by more than 2 2.5%. Values in parentheses are losses (in percent) of lipids as compared to raw potatoes. A plus sign indicates an increase.

not separately analyzed. The galactolipid fraction was 20% by weight of the total lipids. More than half was digalactosyl diglyceride, while one-third was monogalactosyl diglyceride. The steryl lipids were 16% by weight of the total lipids. Free sterol was present in low amounts, while its esters (sterylglucoside and esterified sterylglucoside) were about 4% each. Cholesterol, stigmasterol, and /3 -sitosterol were identified by coupled gas-liquid chromatography-mass spectrometry. The content of triglycerides was a low 5%, while free fatty acids were about 2%. No lysophosphatidyl lipids, or mono- or diglycerides were detected. The distribution of major fatty acids of the total lipid and its constitutents in raw and processed potatoes is shown in Table IV. At least 96% of the total acids consisted of palmitic, linoleic, and linolenic acids. The unsaturation ratio (UR) of 3.22 found by Buttery er al. (1961) stressed the important role of lipid in

TABLE 1V FATTY ACID COMPOSlTION OF LIPIDS FROM POTATOES AT VARIOUS STAGES OF PROCESSINGa Nonpolar lipids Stageb

Total lipids

NL'

TG

Raw Cooked Granulated

2O.gd 20.1 17.7

21.6 22.7 28.0

Raw Cooked Granulated

2.7 4.8 8.7

6.4 6.7 12.1

5.2

Raw Cooked Granulated

0.9 0.9 1 .o

2.0 2.8 1.5

Linoleic

Raw Cooked Granulated

57.7 59.0 59.1

Linolenic

Raw Cooked Granulated

17.9 15.2 13.6

Fatty acid Palmitic

Stearic

Oleic

Phospholipids

Glycolipids

SE

ESG

PC

PE

PI

17.9 17.8 17.7

60.3 61.8 62.3

59.1 58.1 58.5

18.2 18.6 19.0

24.4 25.1 26.1

36.4 40.5 44.2

4.2 3.6 4.6

11.5

3.9

3.8 4.9 4.6

9.1 12.2 16.4

2.2 2.9 4.8

2.7 3.4 4.1

4.0 5.3 5.3

2.3 2.8 2.4

8.9 10.0 10.1

3.4 4.5 4.3

Trace Trace Trace

1.6 2.3 2.7

1.3 1.7 1.4

1.0 1.6 1.8

1.0

2.6 1.2

0.5 0.9 1 .o

2.0 1.8 3.1

48.7 44.3 41.4

47.1 46.2 46.2

25.9 24.9 24.3

25.9 23.9 19.1

62.3 61.2 60.2

62.8 60.8 58.2

46.3 40.6 39.0

54.6 54.8 53.1

54.6 54.7 54.0

21.4 23.5 17.1

27.6 26.3 26.5

9.9 8.5 8.8

4.2 3.5 3.2

16.0 15.6 14.5

9.0 9.1 9.8

12.3

38.3 37.9 38.9

23.1 22.1 21.2

5.5

11.0 10.3

MGDG

DGDG

11.4 11.6

Pun and Hadziyev (1978)and Pun (1979). bRaw = peeled, sliced, and washed raw potatoes. Cooked = steam-cooked and hot mashed potatoes. Granulated = dehydrated, freeze-thaw potato granules. CAbbreviations used: NL = neutral lipids; TG = triglycerides; SE = free and esterified sterol; ESG = esterified steryl glucoside; PC, PE, and PI = phosphatidyl choline, -ethanolarnine, and -inositol, respectively; MGDG and DGDG = mono- and digalactosyl diglycerides. Percentage of total fatty acid (for each stage).

DEHYDRATED MASHED POTATOES

73

rancidity assessment. The acid composition and URs varied greatly in individual lipids, the steryl lipids giving the lowest, and the polar lipids the highest. The UR was highest in monogalactosyl diglyceride (14.29), while the more abundant digalactosyl diglyceride was found to be more saturated, having a UR of 3.81, which was close to that of phosphatidyl choline but somewhat higher than that of phosphatidyl ethanolamine. Steam-cooking and hot mashing caused a 12.9%loss of the total lipid, with the greatest loss in neutral and phospholipid fractions, and the least in galactolipids. Phosphatidyl ethanolamine and -inositol were among the most affected phospholipids. The 27% decrease in the content of triglycerides and the losses of other lipids were not accompanied by an increase in the content of free fatty acids, which decreased by 25%from the initial amount in raw tubers (Table 111). The fatty acid composition of total and individual lipids in the cooking, mashing, and final dehydration-granulation steps did not change appreciably from raw potatoes. Cooking and mashing caused only a slight decrease in the sum of polyunsaturates, and, consequently, a small decrease of total lipid UR. The decrease could not be ascribed to any particular lipid constituent, since the reduction among lipid classes appeared to be nonpreferential. The lipid composition of dehydrated granules with 7% moisture, when compared to raw, sliced potatoes, showed a 14.7% loss of total lipids during the entire process, or an additional loss of 1.8% commencing with the freeze-thaw step up to the end of granule processing. The least affected lipids were once again glycolipids. As seen from Table IV, the highest amount of polyunsaturated acids was found in galactolipids. The UR of monogalactosyl diglyceride was 13.14, while that of digalactosyl diglyceride was 3.46. The URs of triglycerides, phosphatidyl choline, and -ethanolamine were similar to digalactosyl diglyceride . When these results were compared to URs found for raw or cooked and mashed potatoes, it appeared that granule processing brought about a very small overall decrease in URs. Thus, the URs of all lipids retained by freshly made granules were still very high, increasing the likelihood of off-flavor development in granules during storage or shipping. In light of these results. it might be concluded that phospho- and glycolipids should be considered as potential major off-flavor precursors. B.

I.

PECTIC SUBSTANCES

Occurrence and Distribution in the Tuber

Pectic substances in the cell wall are part of the hemicellulose-pectin gel which functions both as a structural element and as a membrane (Pilnik and Voragen, 1970). Pectin in the middle lamella (Fig. 5) acts as a cohesive agent, and so it is referred to as “intracellular cement. ”

74

D. HADZIYEV AND L. STEELE

FIG. 5 . Transmission electron micrograph of the middle lamella sandwiched between cell walls, as revealed after n-butanol treatment (Chung er al., 1979). CW, cell wall; ML, middle lamella.

Potter and McComb (1957) determined that the amount of pectic substances in potatoes was 0.7-1.5% on a dry weight basis, while Sharma et al. (1959) reported a range of 0.8-1.5% fresh weight. Bettelheim and Sterling (1955) found that the uronide content of 10 varieties was 1.1-2.1% dry weight, whereas Jaswal (1969) reported uronide contents as high as 4.5-4.8% dry weight. Hoff and Castro (1969) found that cell walls were 5-7.2% of the potato dry weight, and that pectic substances made up 47.5-62.5% of the dry cell wall. This corresponded to 2.4-3.9% pectic substances in the dry tuber. Keijbets and Pilnik (1974), using a copper ion exchange method, rather than analyzing colorimetrically for polyuronide content by a carbazole test, as above, reported the galacturonan content of the variety Bintje to be 2.62%dry weight for the fraction below a specific gravity of 1.060. The value decreased as the specific gravity increased.

DEHYDRATED MASHED POTATOES

75

It appears that the discrepancies in reported uronide contents were due mainly to differences between the samples studied and the methods of analysis employed. Pectic substances in raw tuber exist in the form of protopectin, the waterinsoluble parent substance, and pectin, which is composed of water-soluble pectinic acids. Protopectin can be subdivided into two fractions. The first can be rendered water-soluble by treatment with sequestering agents, while the second is strongly bound by enmeshing with other filamentous macromolecules in the cell wall and is not soluble in the presence of sequestering agents. It could, however, be extracted by strong acid at elevated temperature (Bettelheim and Sterling, 1955). The ratio of protopectin to pectin varied with variety, maturity, and cultural practice (Bettelheim and Sterling, 1955; Linehan and Hughes, 1969). This ratio changed on prolonged storage at low temperatures (4-5"C), or on storage at higher temperatures (up to 24°C) for shorter periods of time (Sharma et al., 1959). Only about one-half of the pectic substances of potato cell walls and middle lamella were composed of polyuronide, while the rest were sugars (Hoff and Castro, 1969). 2 . Changes Induced by Processing Pectic substances in the cell wall and middle lamella appear to exert a profound effect on the textural properties of processed potatoes. A shift from insoluble toward water-soluble pectin generally occurs upon cooking. There is a loss of cellular cohesion of tissue, implying a relationship between cohesion and solubilization of pectic substances during cooking. Woodman and Warren (1973) found that potato polyuronides generally had a low degree of esterification, and were present in higher concentration in the cortex and periderm than in the interior regions. Furthermore, the exterior portions of the tuber, which tended to disintegrate upon cooking, had a lower degree of salt formation with calcium and magnesium ions than the more cohesive central portion of the tuber. A statistically significant correlation between the amounts and characteristics of extracted pectin fractions, and the intercellular cohesion retained by cooked potato tissue was reported by Warren and Woodman (1974). Linehan and Hughes (1969) found a significant correlation between the intercellular cohesion and a pectin fraction that was soluble in a calcium sequestering agent and was thought to be responsible for the structure of the retained intercellular cement. However, the role of pectic substances in intercellular cohesion, reflecting more or less the texture of raw and processed potatoes, is still not clear. A few attempts to clarify the mechanisms of degradation and solubilization of pectic substances during cooking were reported by Keijbets et al. (1976). In model systems consisting of purified cell walls, they confirmed that pectic galac-

76

D. HADZIYEV AND L. STEELE

turonan, at the natural pH of potato tissue (5.5-6.5), was depolymerized by /3- or transelimination, and that the extent of depolymerization increased with increasing pH. The mechanism of the elimination reaction was clarified by Neukom and Deuel (1958) and Albersheim et al. (1960). The involvement of pectin degradation with the texture of cooked potato tissue prompted Keijbets and Pilnik (1974) and Keijbets et al. (1976) to investigate the effect of major potato cations, such as calcium, potassium, and magnesium, and anions, such as citrate, malate, phytate, and chloride, on the extent of p-elimination. It was found that they stimulated breakdown at 100°C and that the nature of the ions, rather than their concentration, was the predominant factor. During cooking, potato starch calcium is transfered from phosphate groups at C-6 of some glucose residues of amylopectin to carboxyl groups of galacturonan. In model systems of potato cell walls and starches, Keijbets et al. (1976) found that calcium starches consistently decreased galacturonan solubilization, while hydrogen starches had no influence. This implied that the amylose fraction, which leached from starch grains during cooking, did not stabilize galacturonan. Changes in pectic substances of the potato variety “Netted Gem,” as induced by a freeze-thaw potato granule process, were reported by Ooraikul et al. (1974). The content of pectic substances in raw tubers was low in both water- and calgon-soluble fractions. Steam-cooking brought about a 6-fold increase of the water-soluble fraction, and a 3-fold increase of the calgon-soluble fraction. The apparent total pectic substances at this stage of processing were 1.45%. Freezing-thawing, predrying, granulation, and final drying had little effect on the level of the fractions. The effect of precooking at 70°C for 20 minutes in potato granule production is shown in Table V (Moledina et al., 1978b). In processing without precooking most pectic substances were converted to soluble forms. A lower solubilization of pectic substances occurred in precooked and then cooked tubers. Precooking of potatoes and subsequent steam-cooking increased the firmness of unmashed potato tissue (Ooraikul, 1973). Precooking also induced hydration, swelling, and gelatinization of starch (Moledina et al., 1978b). The cells were loosely filled with gelled starch, while the cell walls appeared to be tightly bound, indicating that the intercellular pectic substance was essentially retained in unsolubilized form (Fig. 6). The cells in a steam-cooked but not precooked sample were well separated. When precooked samples were mashed against the recycled dried granules, as in the add-back process, cell separation was good, with cell damage of 2%. However, precooked samples subdivided by the method required in the freeze-thaw process showed excessive cell damage, resulting in a gluey mash that was more difficult to dry into granules. The interaction of calcium ions with pectic substances in the precooking step of a flake process and its beneficial effect on texture was the basis of a process patented by Nelson et al. (1962). Potato slices were precooked for 15-45 min-

TABLE V EFFECT OF PRECOOKING ON WATER-, CALGON-, AND HCI-SOLUBLE PECTlC SUBSTANCES OF POTATOES" Uronide content (mg per 100 gm dry matter)b

Treatment type

(i) Loss in the cooking liquor ~____

Raw Precooked Precooked and steamed Steamcooked

39.9

5

8.1

39.9 2 8.1 -

(i)+ (ii) + (iii) Apparent total

(ii) Water-soluble

(iii) Calgon-soluble

154.4 5 22.8, 120.7 5 14.2,

101.4-t 8.6, 87.9 -t 13.5,

255.9 248.5

633.3 -t 32.5, 1106.5 -t 106.6,

129.9 2 13.3, 201.0 f 22.9,

803.2 1307.5

(iv) HC1-soluble

(Totalapparent total)

(i)+(iv) Total

~~

~~

_

_

_

~

~

1157 2 79.6

5

17.3 6.3

-

-

-t 5

25.6 86.9

-

-

5

1311.7

~

-t

82.1

508.5 -

~~~~

Moledina er al. (1978b). All values, expressed as X -t S, are an average of eight determinations done in duplicate. Values followed by the same letter are not significantly different at P = 0.01. a

78

D. HADZIYEV AND L. STEELE

FIG. 6. Scanning electron micrograph of precooked and cooked potato tissue (Moledina er a / . , 1978b). The walls from adjacent cells are tightly bound to each other while gelled starch loosely fills the cells.

Utes at 63-77°C in demineralized water containing about 35 ppm calcium. After cooling in water free of all minerals but calcium, the potatoes were steamcooked, mashed, and rapidly drum dried. The reconstituted product had good texture, flavor, and color. Use of demineralized water without calcium in precooking resulted in a pasty product. It appears that precooking renders the pectic substances in middle lamella more resistant to further thermal degradation, thus decreasing the loss of intercellular cohesion and the rarefaction of cell wall brought about by subsequent cooking. During boiling, calcium but not magnesium ions had the ability to stabilize the pectic galacturonan in potato cell wall, even when it was progressively depolymerized (Keijbets ef d., 1976). The binding activity towards calcium ions increased as the de-esterification of pectic galacturonan increased, suggesting that precooking might involve an important de-esterification reaction.

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3 . Pectin-Methylesterase and Its Role in Tuber Firming Pectin-methylesterase (PME, EC 3.1.1.11) is present in potato tubers and is associated with demethylation of pectic substances. Since it is usually bound to water-insoluble cell constituents, particularly those of the cell wall, it is freed by solubilization with sodium chloride at pH 7.5. The PME activity of potato slurry was determined by Vas et al. (1968) and Bartolome and Hoff (1972a). An instant mashed potato process in which precooking was done in a phosphate buffer appeared to rely on a pH optimum activity of PME to provide dehydrated flakes of superior texture (Cole, 1965). Bartolome and Hoff (1972b) demonstrated that PME in potato was not active to an appreciable extent until the tissue was heated to temperatures above 5O"C, whereupon PME reacted with the pectins of the cell wall. At 60°C the enzyme activity was halved, while above 70°C it was found to be rapidly destroyed. This evidence was consistent with the effect of preheating treatment of not only cell wall preparations, but of the whole tuber in the region of 60-70°C followed by boiling for 30 minutes. This brought about a consistent firming of the potato tissue. The calcium and magnesium contents appeared to increase within the cell wall, but decreased in gelatinized starch during preheating. Bartolome and Hoff (1972b) gave their interpretation of the involvement of pectic substances in tissue firming induced by a precooking step. Heating above 50°C destroys the integrity of plasmalemma, permitting intracellular electrolytes to diffuse to the cell wall where they cause PME to desorb, thereby activating the enzyme, which interacts with methyl ester groups on the galacturonan chains to produce free carboxyl groups. This de-esterification is facilitated by the fact that potato tissue does not contain PME enzyme inhibitors. Finally, diffusion of calcium and magnesium ions develops cross linkages between chains and renders the tissue more resistant to further thermal degradation. Their interpretation suggests that problems with cooked potato texture are governed by factors that influence the cell wall and the middle lamella, and that starch content and its gelatinization in the precooking step of an add-back process would have little to do with texture. However, as stated by Hoff (19731, the truth probably lies somewhere between these two opposing views. C.

I.

PROTEIN In Raw Tuber

The total-N content in raw potato tuber, as determined by the Kjeldahl method, varies between 0.24 and 0.36% on a fresh, or I-2% on a dry weight basis. This corresponds to 6-12.5% crude protein (N X 6.25). However, since crude protein consists of a heterogeneous mixture of N-containing compounds, the true protein content is one-third to one-half of that calculated from total-N

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(Adler, 1971; Reiter, 1956; Mothes and Wagner, 1957). Protein-N varies in the range of 37.5-63.7% of total-N, the rest being present as: amide asparagine-N 11.8-25.5%, and glutamine-N 7.6-19.7%; a-amino acid-N 6.4-12.8%; and basic-N 20.4-27.7%. In addition a small percentage of the total-N in the tuber is present as inorganic-N. It was found that nonprotein-N was highest in early maturing potatoes (Vecher et al., 1974). Some 70-80% of the extractable true protein belongs to so-called “storage protein” (Fig. 7). According to Lindner et al. (1960), protein is present in the mature tuber as water-soluble albumin (4% of the total protein), slightly and readily soluble globulins (1.4 and 74.6%, respectively), ethanol-soluble prolamin (1.8%), dilute-alkali-soluble glutelin ( 5 . 5 % ) , and nonsoluble skeletal protein (11%). However, fractionation of potato proteins by Levitt (1951) gave

FIG. 7 . Scanning electron micrograph of numerous readily soluble globulins of a potato cell comprising close to 75% of the total cell protein (P.Fedec and D. Hadziyev, unpublished results, 1978). A few larger starch grains are also visible.

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almost equal quantities of water-soluble and -insoluble components. Also, the water-insoluble fraction, which could be solubilized in 1 M NaCl, was only 4-28% of the total protein. It was separable into acid-soluble and -insoluble fractions. Similar results were obtained by Nakasone et al. (1972), who found that potato proteins were separable by dialysis into equal amounts of albumin and water-insoluble globulins. Kapoor et al. (1975) got conflicting results using two different procedures to determine albumin and globulin fractions. Stegemann ( 1 975) used gel electrophoresis and isoelectrofocusing for identification of potato varieties based on tuber sap protein profiles, as well as for study of the effects of environmental conditions on protein composition. Stegemann et al. (1973) and Loeschcke and Stegemann (1966) found that the dormant tuber of a given variety had a well-defined pattern of proteins that was not influenced by climate, soil, fertilizers, virus infection, or growth regulators. The genetic background of storage proteins was due mainly to protein charge differences, probably as a result of variations in the extent of protein amidation. The molecular size distribution of tuber proteins appeared to be independent of variety. The similarity of the size distribution and uniformity among the almost 200 varieties analyzed suggested that many of the proteins in the tuber originated from one parent protein, and that the diversity was developed through charge specificity during tuber maturation. Kaiser and Belitz (1971) and Kaiser et al. (1974) used high-resolution isoelectrofocussing to distinguish up to 42 protein bands and 21 inhibitors of trypsin or chymotrypsin activities, both patterns providing means for variety identification. Hoff et al. (1972) isolated crystalline cubical bodies (Fig. 8) from cytoplasm, mostly in the outer cortex of the tuber, and identified them as proteins, predominantly of molecular weight of 77,000. They were relatively high in lysine, and low in cysteidcystine. It was suggested that these crystals in young tuber may be a form of storage protein which remains intact until dormancy is broken and sprouting begins. As found by Stegemann (1973, the crystalline proteins could be split into smaller units at elevated temperature by prolonged reduction of disulfide bridges using mercaptoethanol. Protein content and patterns during development and storage of potato tubers were reported by Zimmermann and Rosenstock (1976). It was found that the protein content of young tuber was twice that of mature organs, and that it decreased during tuber maturation to a constant level. When the fully matured tuber was stored at 7°C for up to 9 months, the protein pattern of 19 distinct bands was unchanged. 2 . Amino Acid Composition

The nutritional value and the amino acid composition of proteins are related. A number of analyses have been reported on the content of amino acids, either free or bound, in potato protein (Schuphan, 1959; Lindner et al., 1960; Mulder and

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FIG. 8. Scanning electron micrograph of a cubical crystalline protein body adhering to a starch grain (P. Fedec and D . Hadziyev, unpublished results, 1978). The cube edge is close to ] O F , as opposed to cubical peroxisome bodies with edges of only about Ipm.

Bakema, 1956; Hughes, 1958). The free amino acids in 31 varieties of potatoes were reported by Davies (1977). His results strongly indicated quantitative differences between varieties, but there was also evidence of amino acid composition being affected by location and year of growth. Finally, results for free amino acids set out according to potato variety were reviewed by Synge (1977). Potato protein quality has been attributed to the presence of essential amino acids, which, except for histidine, are present in substantially higher amounts than in some cereals. The high lysine content of potato proteins appears to make them potentially valuable as supplements to wheat proteins. Jaswal(l973) found a total of 108.1 mg amino acids per gram on a dry weight basis, of which 23 mg were in the free state, in “Netted Gem” potatoes of

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specific gravity 1.065- I .075. Approximately one-third of the protein consisted of the essential amino acids. All the bound essential amino acids, particularly lysine, valine, leucine, and phenylalanine, were present in substantial proportions. The free essential amino acid fraction was high in arginine and valine, but was low in leucine. Similar results were found in “Netted Gem” tubers of high specific gravity; however, the total amino acid content was lower (90.3 mg/gm). Talley et al. (1970) also found high contents of asparagine, glutamine, and y-butyric acid in the free amino acid fraction of the tuber variety Russet Burbank. The availability of automatic amino acid analyzers has made possible the critical examination, andor verification of some earlier results for composition of free or bound amino acids with regard to dependence on variety, specific gravity, location of growth, and storage time (Talley and Porter, 1970). However, even this method of analysis cannot avoid the errors due to destruction of some amino acids during acid hydrolysis of proteins. Tryptophane and cystine, which are markedly labile, are destroyed to a great extent during hydrolysis. Also, the presence of large quantities of starch enhances the damage to tyrosine, methionine, cystine, and cysteine (Schram et al., 1953; Blackburn, 1968). As demonstrated by Kaldy and Markakis (1972) in Russet Burbank potatoes and several clonal selections, an average of 52.3% methionine and 62.2% of cystinekysteine was destroyed during acid hydrolysis. These authors provided quantitative data for 18 amino acids using an analytical technique which prevented the loss of these acids and thus provided better estimates. They reported 2219 mg of total essential amino acids per gram of total-N. When the acid composition was compared to that of whole egg (protein score = loo), they got an average protein score of 69 for all potato samples. This was much better than that of wheat flour (50), peas (60), or even cow’s milk (60), and supported the earlier finding of Kofranyi and Jekat (1965) that the daily protein requirements for humans in grams per kilogram body weight were 0.50 for whole egg, 0.55 for potato, and 0.57 for milk. 3 . Changes during Processing

Variations in the content of apparent crude and true protein during the production of dehydrated table potatoes were studied by Kempf et al. (1976). There were marked differences during processing in the ratio of crude to true protein. In the variety Desiree the ratio was 2.5 for raw unpeeled and 2.9 for peeled tuber. After slicing and washing, a slight decrease occurred to 2.8, while waterblanching brought the ratio to 2.1, and cooling to 2.0. Drying steps had no effect. Other varieties showed similar changes. The increase during peeling indicated that more precipitable true protein was located in the peripheral cortex zone than in the rest of the tuber, while the decrease during water-blanching suggested more nonprotein-N was leached out than pure protein.

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In low specific gravity “Netted Gem” tubers processed to flakes, there was a 24.3% loss of essential and a 15.1% loss of nonessential amino acids, and an overall 18.5%amino acid loss, with nonpreferential destruction of both free and bound amino acids (Jaswal, 1973). When the flakes were processed with 0.2% glycerol monostearate, added as an emulsifier in the mashing step, the loss increased by 4% for essential and 5% for nonessential amino acids, and the overall loss rose to 22.8%. Effects of flake processing on amino acid contents of high specific gravity “Netted Gem” tubers followed the pattern of the low specific gravity tubers. However, the extent of damage to total, bound, and free amino acids was significantly lower. The loss was only 6% for essential and 3% for nonessential amino acids, while the overall loss was only 3.9%. Again, the extent of damage was more than doubled when the flakes were processed with emulsifier as an additive. In this case the free amino acid fraction was the most affected. Lysine is the amino acid which is most easily rendered nutritionally unavailable during processing. It is widely held that its Zamino group has to be free and not bound to other food constituents for lysine to be nutritionally available. The total lysine content of the high specific gravity “Netted Gem” was 4.2 mg/gm dry weight, of which 1.4 mg% was in the free state (Jaswal, 1973). Of this, 88% was present in the available form. The flake process adversely affected these values, with the total lysine content dropping to 3.9 mglgm dry weight, and the extent of its availability to 82%. The quality of protein in drum dried flakes was reported by Rios Iriarte er al. (1972). The chemical score was similar to data of two growth bioassays: protein efficiency ratio (PER), and gain in body weight. The lowest chemical score was found for methionine. Weight gains, PER, and the apparent absorbability of potato proteins were significantly increased on the methionine supplemented diets (0.66 gm L-methionine per 100 gm potato flakes), confirming a known fact that the nutritive value of a protein can be increased if it is enriched in a deficient amino acid and verifying the finding of Kaldy and Markakis (1972) that methionine was the limiting essential amino acid in potatoes. Kies and Fox (1972) also suggested that the protein value of dehydrated flakes could be improved by methionine supplementation. They found that there was an increase in nitrogen retention when methionine was used as a dietary supplement, either alone, or in combination with leucine or phenylalanine. Suggested methods of achieving an improved protein value were to process potato varieties having a higher methionine content; to improve the processing steps to avoid methionine destruction; or to enrich the end product with purified t-methionine. The latter suggestion was feasible since sensory panel evaluation indicated that up to 1% supplementation with methionine had no adverse effect on the palatibility of dehydrated mashed potatoes.

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85

STARCH

Submicroscopic Structure

Early comprehensive reviews of this subject were by Sterling (1965, 1968). Subsequently, several new more detailed structural models have been put forward. Since they aid in better understanding of starch grain behavior during mashed potato processing, they are briefly reviewed. Gruber et al. (1973) suggested that the potato starch grain consists of spherulites (crystalline, globular, isodiametric micelles of folded molecules), with a core consisting mostly of amylose micelles. Isodiametric, fringed micelles of folded amylopectins are organized in concentric, tangentially cohesive layers outside the core. The fibrillar structure concept of the starch grain advanced by Sterling (1965) was supported by observations on initial starch gelatinization (Sterling, 1974, 1976). This concept suggests that the microfibrils of the grain are radially organized, often appearing to be individualized as micelles against an amorphous background. Seven individual micelles are closely packed hexagonally in a microfibril, and cemented together with amorphous portions of the fibril. The microfibrils are coiled, with several of them occasionally joining to form a larger strand. This fibrillar structure implies the presence of pores on the grain surface. Sterling suggested that the pore diameter for native starch would be at least 2 nm. Taking into account some features of core-shell structure, radial microfibrillar concepts, and the X-ray data of Blackwell et al. (1969), Frey-Wyssling (1974) developed a submicroscopic starch grain model. Parallel amylopectin chains give a hexagonal cross-sectional pattern resembling a pitch-fork. Gaps formed in the unit cells are filled with amylose molecules. The recent findings by Kassenbeck (1978) essentially support the fibrillar structure of the starch grain and also attempt to clarify the periodicity in lamellar structure, based on the arrangements of amylopectin molecules in side and main chains. 2 . Composition and Distribution in the Tuber Native potato starch grains have a water content of 10-18% when in equilibrium with normal atmospheric humidity. Upon drying, the starch grain loses its sharp X-ray diffraction pattern. This supports the idea that water is an integral part of starch crystallites (Kainuma and French, 1972). Conventional analytical methods do not differentiate between the water in a crystallite region and that present in the amorphous gellike matrix of the grain. A recent study by pulse nuclear magnetic resonance (NMR) spectroscopy revealed separate splittings

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caused by water molecules of crystallite and amorphous regions as well as around cations (Hennig, 1977). An approximate estimate showed 90% of water in starch to be associated with the amorphous region. Raw Alberta “Netted Gem potatoes processed into dehydrated granules have an average specific gravity of 1.098, which corresponds to 25% total solids and 17.5% starch (21.2% amylose and the rest amylopectin; for starch determination, see Adler, 1971). Starch grains of different size are distributed within the tuber (Fedec er al., 1977). Cork cells, or periderm, usually designated as skin, do not contain starch, while parenchyma cells in the cortex contain numerous round and oval-shaped grains with diameters of 4.3-18.6 rn in outer, and 20 x 38-5Ox 86 pm oval-shaped grains in inner cells of the cortex (Fig. 9). Storage parenchyma cells adjacent to vascular tissue contain starch grains which are generally ”

FIG. 9. Scanning electron micrograph of parenchyma cells within the cortex showing oval and round starch grains (Fedec et ul., 1977).

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small and round. The internal phloem or perimedullary zone, occupying close to 75% of the total tuber volume, contains starch grains similar in size to those of the cortex. Grain sizes in the pith range from 4.5-18.2 pm for spherical, and 24 x 32-28 x 50 pm for oval-shaped grains. This abundance of various sizes of starch grains is in agreement with findings of Johnston et al. (1968). They isolated starch grains from the variety “Netted Gem” and fractionated them on a set of sieves. The distribution obtained was 31.9% over 44 pm (nearly half of which were above 52 pm), 39.1% from 31-44 pm, 22% from 23-31 p n , and 7% under 23 pm. Native starch grains isolated from whole Russet Burbank tubers have a nearly 100-fold variation in size, with about one third averaging 28.4 pm (Reeve, 1967a). Light microscopy measurement in situ in specific tissue zone sections gave average grain lengths in microns of 26.7 for pith, 7.3 for vascular ring, and 23.5 and 31.6 for outer and inner cortex, respectively. Putz et a f . (1978) studied the influence of environment, variety, and fertilization on the grain size of potato starch and found that the year and location of growth were of vital significance. Under the same cultural conditions, variety and fertilization were of lesser importance. The amylose/amylopectin ratio of potato starch grains that were ungraded as to size has been found to be about 1:4. However, amylose content depends on starch grain size (Johnston et al., 1968). Of the five grain sizes analyzed from Alberta “Netted Gems,” the largest (over 44 pm) consistently had a slightly higher percentage of amylose (21.2-21.7%) than the smallest of less than 31 pm (20.6-21.0%). Moreover, though the distribution of sizes during storage of tubers at 4°C up to 6 months remained fairly constant, there was a steady increase in the amylose content. The overall increase was 1.6%. Similar results were obtained for “Netted Gems” grown in New Brunswick, though there was a slight decrease during the initial 1.7 months of storage. Reeve (1967a) found a change in size distribution, which depended on temperature during storage. At higher temperatures smaller grains were digested more rapidly than larger ones. However, MiEa (1975) reported a degradation of larger grains to smaller ones in European potato varieties stored at 2 and 10°C for up to 6 months, Also, the total starch content (73.4% of the tuber on a dry weight basis) decreased to 70.7% at 2°C and to 71.7% at a 10°C storage temperature. Changes in starch phosphorus, potassium, and calcium contents during storage have also been reported (MiEa, 1976).

3 . Changes Induced by Processing Potato slices subjected to add-back granule processing are usually precooked at 6540°C for 10-20 minutes, and then water-cooled. This caused partial cell separation due to hydrolysis of the pectic middle lamella, and no cell rupture

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occurred (Fedec et al., 1977). Subsequent steam-cooking for 20 minutes brought about further dissolution of the middle lamella, and separation of cells (Fig. 10). Starch grains in cortex cells (18-20 per cell) became hydrated, swollen, and gelatinized during precooking and subsequent steam-cooking. The spheroidal, crystalline structure of the grain (Fig. Ila) was lost, and the hydrated gel occupied nearly the entire cell volume (Fig. 1 lb). The void space left between the gelled starch and the cell wall was probably due to protoplasmic material displaced to the cell wall during starch swelling which was then washed out during sample preparation for microscopy. The swollen and gelatinized starch grains from the pith zone are also presented (Fig. 12). The available cell volume was not filled in this case, probably owing to the smaller size and lower number of grains per cell, both characteristics of this tuber zone. Thus, the void space

FIG. 10. Scanning electron micrograph of steam-cooked potato showing dissolution of intercellular pectin and extensive cell separation (Fedec el a / . , 1977).

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between the cell wall and gelatinized starch was larger than that found in the cortex zone. The fact that starch grain size has a profound effect on swelling and gelatinization was illustrated by grains present in the vascular ring zone. Many were small in size and retained their spherical shape with little or no swelling after precooking and steam cooking. This observation agrees with gelatinization temperatures found for isolated and size-graded grains of the same tuber (Chung, 1979). Starch grains less than 20 pm had the highest gelatinization temperature range of 60-68"C, with other size and temperature ranges being 20-38 p n , 58-65°C; 38-53 pm, 57-61°C; 53-74 pm, 56-60°C; and 74-106 pm, 55-59°C. Extensive swelling of individual starch grains (Fig. 13) brought about a fusion in which minute strands of protoplasmic material were embedded between the gelled starch grains. This contributed to the appearance of the fused starch as an irregular reticulum, as has also been described using light microscopy by Reeve (1 954b). Starch gelation is a complex process. Apart from the breakdown of crystallites resulting in loss of the fine grain structure, there is swelling of the grain, leaching of the low-molecular-weight amylose, and, if gelation is followed by cooling to room temperature, additional setting of the system to a rigid gel. The breakdown of the grain structure followed by loss of birefringence is not instantaneous. The wide temperature range of gelation illustrated this fact. The changes which occur within the fine grain structure on a molecular level are well concealed when this optical method is used. However, pulse NMR can differentiate between starch molecules in crystallites and free states. The fixed molecules in crystallites have a wide resonance line, while those in amorphous regions show a narrow line (Jaska, 1971; Lelievre and Mitchel, 1975). If starch is treated with deuterium oxide to replace the exchangeable hydroxyl protons, and gelation is carried out in deuterium oxide, the resonance of CH-protons of the starch molecules offers an accurate way to follow the gelation mechanism. Hennig et al. (1976) found no increase in molecular mobility up to 55°C in native potato starch. The onset of crystalline melting was observed at 60"C, and this increase was low until 63°C; above this temperature a high increase was observed. At 66°C half of the molecules were already mobile, increasing to close to 80% at 68"C, while at 70°C no crystallite was left, i.e., all molecules were free. Potato starch gelation is accompanied by an extensive leaching of lowmolecular-weight amylose. Greater removal of amylose from gelled starch at 82.2"C (180°F) than at 65.6"C (150°F)was observed by Reeve (1963). Cooking a starch suspension at 100°C showed that starch dissolution was proportional to time (Potter et al., 1959). When the suspension was precooked at 65°C and then cooked at lOO"C, there was a decrease in dissolved starch content. The decrease was more pronounced as the preheating time was increased, suggesting that at

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FIG. 1 I . Scanning electron micrographs of starch grains in potato cells (a) before, and (b) after precook and steam-cook treatments (Fedec et a / . , 1977).

preheating temperatures starch retrogrades (realignment and association by hydrogen bonding of starch molecules causing a gradual decrease in their solubility). However, when precooking was followed by a cooling step, the extent of retrogradation was even more profound (Potter, 1954; Reeve, 1954a,b; Harrington et al., 1959). The leaching of amylose is observable in intact potato cells. When the cells are ruptured, extensive extrusion of the total gelled starch occurs. More gelatinized starch is extruded from such cells when the tuber is only cooked than from potato cells cooked after a preheating and cooling step. Many processes based on the above observations have been patented in which cooked and mashed potatoes are freed from stickiness, pastiness or glueyness, or gumminess. Cording and Willard (1957) heated potato slices in water at 60-83°C

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FIG. I l b

before being cooked. A cooling step using water of less than 22°C for at least 14 minutes was inserted between precooking and final cooling steps in order to decrease pastiness and increase mealiness (Sullivan et al., 1961). These patents actually promote retrogradation of both extracellular starch and starch within the unruptured cell, thereby increasing mealiness, a required feature for good quality mashed potatoes. The solubilized amylose content in mashed potatoes is reduced further in additional steps of processing. Retrogradation of amylose in predrying, low temperature conditioning, or tempering steps of an add-back process (Olson and Hamngton, 1955), or the freeze-thaw step of a straight-through process (Ooraikul, 1977a) are two such examples. The undesirable effect of amylose may be avoided by a mashing step which keeps the rupture of the potato cells low, and/or by tying up the soluble amylose by incorporating monoglycerides at this step. Most of these options are applied in practice.

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FIG. 12. Scanning electron micrograph of gelled starch grains within the pith zone (Fedec et

a/.,

1977).

The patent literature dealing with the use of monoglycerides in the production of dehydrated mashed potatoes was reviewed by Gutterson (1971). Bourne et al. ( 1960) demonstrated that sucrose monostearate complexed readily with solubilized starch, precipitating 78% of the amylose and only 11 % of the amylopectin of potato starch. The complex inhibited iodine absorption in the helix of the molecule, suggesting that the stearate-starch complex could be a host-guest nonstoichiometric clathrate compound in which the fatty acid chain is located within the amylose helix. The stickiness of gelled starch extruded from ruptured cells can also be suppressed by incorporating monoglycerides either during the process or in the dry granules (Harrington et al., 1960). Flake manufacture employs similar practices. It appears that the use of a blend of monoglycerides is superior to the use of a single monoglyceride. Addition of My vatex (a blend of glycerol monostearate

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FIG. 13. Scanning electron micrograph of starch reticulation showing minute strands of embedded cytoplasmic material (Fedec et a / ., 1977).

and propylene glycol monostearate) to a mash with an average broken cell count of 2% gave the best results at a concentration of 0.2%, against 0.3% when glycerol monostearate alone was used (Ooraikul and Hadziyev, 1974). Cell rupture (Fig. 14) is the most detrimental factor in a mashed potato process. It occurs primarily during mashing. The cells in potatoes mashed at a temperature close to that of cooking are easily separated with little cell damage (Ooraikul et al., 1974). However, as the mashing temperature is decreased, the percentage of broken cells increases. Therefore, mashing immediately after cooking at “high” temperature is desirable. The amount of cell rupture correlates with the pastiness of the end product. Rehydrated potato granules with 20% broken cells are designated as very pasty, while those with 10-12% broken cells are average, and those with 6% or less are good or superior (Greene et al., 1949; Reeve and Notter, 1959).

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FIG. 14. Scanning electron micrograph of ruptured potato cell (Fedec ei d.,1977). The escaping gelled starch, in the form of "icicles," is highly magnified.

The ruptured cell count also correlates with the released free amylose and its blue iodine complex (Mullins er al., 1955). Good quality dehydrated mashed potatoes have a BVI (Blue Value Index) of 92-182. Higher values correspond to a texture which is slightly pasty (BVI below 200), pasty (BVI 200-280), or very pasty (BVI 280 o r above). The fluctuations of BVI values during an add-back process were reported by Mullins er al. (1955). Freshly cooked potatoes, passed through the system seven times and each time returned in part as a portion of the add-back powder, had BVIs of 102-112. Additional cycles sharply increased the BVI. The BVI changes in a freeze-thaw process were given by Ooraikul el af. (1974). The freeze-thaw step brought about the lowest BVI (27) with subsequent steps giving increases. The final unsieved granules had a BVI of 118. These results strongly

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suggest that cell damage might occur even when granules are exposed to mild abrasion forces during predrying and granulation steps. Earlier explanations of the influence of cooking on cell rupture were reviewed by Reeve (1954b). As starch swells, the cell walls are distended and some rupture. More gelatinized starch is extruded from cells that were only cooked, than from those that were cooked and cooled. This opinion has often been cited in the literature, and it has been related to potato texture. The findings of Bretzloff (1970) dispute this view since intact potato tissue slices heated on a microscope stage showed no change in linear dimensions of cells and no swelling under any cooking conditions. Also, as stated by Hoff (1972), no experimental verification exists on starch swelling pressure, while the intermittent change in volume of the cells, if found, should be fully ascribable to their thermal expansion coefficient. 4 . Starch and Textural Quality

Softening of a potato during cooking is associated with cell separation. The readiness of cell separation (sogginess or mealiness) is related to the specific gravity of the tuber, which in turn can be related to the starch content. On the other hand, the cell size and the size of its starch grains are related to tuber size which also correlates with specific gravity and mealiness (Sterling, 1966). Reeve (1967b) reviewed some factors affecting texture qualities of processed potatoes. However, the relationship of the many different textural qualities of cooked potatoes to chemical and microscopic data has not been fully elucidated. Microscopic observations (Reeve, 1954a-c) of cooked potato tissue showed cell separation to be accompanied by “rounding off” of the cells so that the walls of adjacent cells were pushed apart. It was claimed that “rounding off” was the result of swelling of gelatinized starch. However, based on other observations, it was strongly suggested that the texture of cooked potatoes was only related to the factors that influence the strength of the cell wall and middle lamella. Besides the starch content, these factors would involve the amylose/amylopectin ratio of the starch, the extent of retrogradation, and the effects of specific gravity and total solids, cell size, cell surface, contents of calcium and organic acids, and the age and storage time of the tuber (Hoff, 1972). Cell wall strengthening by diffused amylose, as suggested by Linehan and Hughes (1969), would not be justified (Keijbets et a l . , 1976). The validity of the above view was supported by findings of Warren et al. (1975) that the swelling pressure of gelled starch was not sufficient to account for the texture of cooked potatoes. Instead, the results suggested that sloughing of cooked tissue was due to excessive hydration of cell wall. High levels of polyuronides and phytate, and low levels of polyvalent cations in the cell wall

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favored cell separation. Nevertheless, experimental data given are still inadequate in terms of a coherent concept for the texture of cooked potato. A recent review by Warren and Woodman (1974) relating the texture of cooked potatoes, particularly mealiness and sloughing, to chemical composition of the tuber, was aimed toward such a goal.

E. VITAMIN C 1 , In Raw Tuber

Potatoes are one of the most important natural dietary sources of vitamin C in many countries. A serving of 100 gm of boiled potatoes is able to provide about a third of the daily vitamin C intake suggested by Canadian and British nutritionists and one-half of the American Recommended Dietary Allowance (Voirol, 1974). Varietal studies in relation to vitamin C are quite comprehensive. Murphy et al. (1945) tested over 54 varieties and concluded that there were varietal differences in vitamin C content, differences that were strongly inherited. A recent study led to the same conclusion (Augustin, 1975). Hyde (1962) showed that the vitamin C content within a variety was relatively unaffected by location of growth. During the period between harvest and placing the tubers in storage, the vitamin C content remains relatively constant in many varieties. However, immediately after harvest some of the commercial varieties such as Katahdin, Dakota, and Sebago show a vitamin C decay of practically constant rate until approximately halfway through the storage period. A newly harvested “Netted Gem” potato containing some 30 mg of vitamin C per 100 gm fresh weight typically dropped to 22 mg after the first month, 15 mg after the second, 1 1 mg after the third, and about 8 mg after the fifth month of storage, while additional storage had only a slight influence (Hadziyev and Steele, 1976). Wokes and Nunn (1948) found a 50% drop of vitamin C content during the first 3 months in autumn, with little change in the next 3 months, followed by further decay. The loss of ascorbic acid in fresh potatoes, variety Russet Burbank, during storage has been well documented (Bring, 1966; Bring and Raab, 1964; Sweeney er al., 1969). Augustin er al. (1975) demonstrated that an early harvest provided high levels of ascorbic acid, while delayed harvests gave low levels. Also, during storage at 7°C and 95% relative humidity the most important factor affecting ascorbic acid values was time. A plot of mean values against the logarithm of time gave a straight-line relationship, and permitted reasonably acurate calculation of the ascorbic acid content at any postharvest time. This pattern of loss of ascorbic acid during storage did not coincide with European potato varieties, as given by Somogyi and Schiele (1966).

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2 . Increase in Aerated Slices Increasing the vitamin C content in peeled potato slices by moist aeration prior to processing was reported as a possible means of offsetting the losses during storage and subsequent processing (Feldheim, 1970).It was claimed that such conditions induced biosynthesis of vitamin C in high yields without appreciable enzymic browning reactions. When the potato slices were treated with enzyme inhibitors of carbohydrate metabolism, an inhibition of ascorbic acid synthesis was obtained (Trautner, 1968). Based on these and other results (Feldheim and Hjelm, 1963;Somogyi, 1964),it was concluded that the vitamin C increase in aerated potato slices was due to a true biosynthesis of ascorbic acid. The ability of the variety “Netted Gem” to gain vitamin C by aeration of slices under certain conditions was shown to be similar to Bintje and Lori varieties from Europe (Hadziyev and Steele, 1976).This suggested that such gains were not variety-dependent, but were a common property of the tuber in response to its injury by peeling and slicing. The gain in vitamin C was oxygen-dependent and was inhibited by bisulfite. The gain by aeration was proved to be storagedependent. Freshly harvested tubers could not provide a gain, but tubers stored for at least 3 months gave positive responses (Feldheim, 1970;Hadziyev and Steele, 1976). The above findings were verified by chemical and physical methods of analysis (Hadziyev and Steele, 1976). Electron spin resonance spectroscopy of potato extract, and of pure vitamin C solution confirmed the presence of the L-ascorbic acid-free radical. A strong signal obtained from potato peels was shown to be due to involvement of the unpaired electron of a free radical with an oxygen within a lignin matrix rather than to vitamin C. This agreed with the findings of Brieskorn and Binnemann ( 1 974)that potato peel contained up to 22% lignin that was located within the suberized tissue of the peel. Thus, the high apparent vitamin C content found in peel by chemical methods of analysis, such as the 2,6-dichlorophenolindophenoltitration or the 2,4-dinitrophenylhydrazine (DNPH) spectroscopic method, should be considered highly unreliable (Hadziyev and Steele, 1976).

3 . Losses during Processing Relatively little information is available on the vitamin C content of industrially processed potatoes. Cording et al. (1961)demonstrated with the Russet Burbank variety that there was a 71-73% retention of both natural and enriched vitamin C after employing a flake process with Tenox IV as antioxidant. In a similar study Bring et al. (1963)found an ascorbic acid retention of 36.5-43% in flakes reconstituted with water. The retention after the mashing step was about

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73-86%. Myers and Roehm (1963) found substantial differences in ascorbic acid content of uncooked and cooked dehydrated potato dices, flakes, and slices. The slices retained the most ascorbic acid, and the flakes the least. Vitamin C losses in dehydrated mashed potato granules obtained by either an add-back or a straight-through, freeze-thaw process were determined by Jadhav et al. (1975). Peeling, slicing, and washing steps brought about an 8.2-14.5% decrease in vitamin C. In the add-back process, a 25.1 % loss occurred during precooking or water-blanching at 60-80°C for 15-20 minutes, followed by cooling in water. The enzyme ascorbate oxidase was not present in potato tubers (L. Steele and D. Hadziyev, unpublished results, 1978), hence, any loss in the precooking step cannot be ascribed to enzymic destruction. The steam-cooking step showed an apparent increase in vitamin C content, while the subsequent mashing-mixing step in the add-back process showed the highest loss of vitamin C, a loss much higher than that of the freeze-thaw process (Jadhav et al., 1975). The next step in the add-back process, the conditioning or tempering step, brought the total loss of vitamin C close to 75%. Freezing-thawing and predrying steps of the freeze-thaw process brought about a vitamin C loss near 30%. The predrying and final drying steps of both processes appeared to result in gains of vitamin C (Steele et al., 1976). These gains ranged from 4.7-5.6%, depending on the method of analysis. It was demonstrated that most of the standard methods for vitamin C analysis, thought suitable for raw potatoes, were unreliable for dehydrated potatoes dried at 60°C or higher, due mainly to an amino acid-sugar interaction which yielded products that analytically simulated ascorbic acid. Use of modified methods to eliminate interferences showed that instead of the apparent vitamin C gains in the granule processes only losses in fact occurred. The overall loss in the freeze-thaw process was about 37%, while that in the add-back process was close to 74%. The influence of cooking on retention of vitamin C is a subject of many conflicting reports. Leichsenring er al. (1957) found as much as a 100% increase in ascorbic acid as a result of boiling and assumed that there was a release of a bound form of the acid. Virgin et al. (1967) reported a vitamin C gain on boiling of potatoes in water. When polarography was used to determine vitamin C, losses were only 6% (Domah er af., 1974). Somogyi (1975) found losses of 27% for potatoes cooked under pressure, and 16% for those boiled in water. Use of a modifed DNPH method showed that cooking gave losses of close to 20% (Steele et al., 1976). Vitamin C in hot mash is readily susceptible to oxidative degradation. The pronounced effect of temperature was illustrated in the presence of excess oxygen and a mashing temperature of 85"C, when a 12.7-fold higher autoxidation rate of ascorbic acid was observed than at temperatures of 20-25°C (Jadhav et al., 1975). The 2 ppm of copper present in the tuber, if available as a free, nonchelated ion, would further augment the oxidation by 5.1 to 6.7 times.

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However, vitamin C destruction in the mashing step was much less than theoretically expected, implying that the mash was a poor medium for oxygen dissolution and/or that the copper was not free but chelated by compounds within the tuber. The latter suggestion was substantiated by the fact that citric, oxalic, and tartaric acids, and some amino acids were able to substantially decrease the rate of copper catalyzed oxidation of ascorbic acid at pH 5.6 (L. Steele and D. Hadziyev, unpublished results, 1978). It was found that the shortest chain polyphosphate decreased ascorbic acid oxidation by 85.9% at pH 5.0 and by 73.8%at pH 6.0, while polyphosphates of greater chain length were even more effective. In order to compensate for the vitamin C losses in commercial production of dehydrated mashed potatoes, synthetic vitamin C is usually added in a final enrichment step of processing. However, when the granular form of vitamin C is added to potato flakes or larger agglomerates or clusters of agglomerates, segregation might occur, causing great disparities within the packaged product. When vitamin C is finely powdered to a particle size below 200 mesh, potato granule surfaces may be dusted uniformly with the adherence improving as the particle size decreases. However, such dusted products may turn pink or brown during storage. The addition of vitamin C along with other additives in the mashing step of processing is not recommended since it appears to precipitate emulsifiers (monoglycerides) and prevents their uniform distribution in the mash. Consequently, antioxidants such as BHT, which are added along with emulsifiers, would not be uniformly dispersed. The presence of sulfites fails to alleviate the problem. There is variability of flavor and texture, along with rapid decay of vitamin C during drying and storage. The problem has been alleviated by separate incorporation of a sulfite solution of vitamin C into the mash (Irmiter and Rubin, 1962). No interaction occurred among the additives, and vitamin C stability appeared to be satisfactory in a drum drying step. Pulley (1974) suggested fortifying dehydrated mashed potatoes to a vitamin C level of 80-160 mg/100 gm product with an aqueous solution consisting of ascorbic acid (14-20%), sulfites, and up to 40% by weight of a film-forming oxygen bamer. A light yellow corn maltodextrin film was found to be particularly effective against discoloration, and it also protected against oxidation. As outlined in the patent, many starch derivatives which do not retrograde and are not sticky, like thin-boiling waxy maize starch or hypochlorite-oxidized starch, were equally useful as film-forming agents. In a patent of Hammes and Boroshok (1973) vitamin C was given a waxlike coating that became sticky at 40-60°C. It was then dusted in discrete granular form on warm flakes as they were agitated and moved along by conveyor, adhering as the flakes cooled. To avoid the ready segregation of vitamin C in fortified flakes, Pedersen and Sautier (1974) introduced vitamin C flakes that, once incorporated into potato

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flakes in amounts from 0.1-5%, did not segregate and were invisible to the eye. The vitamin C flakes were made up of water-soluble minerals, vitamin C, and 50-70% fat, predominantly mono-, di-, and triglycerides with an iodine value less than 2 and Wiley melting points from 55-86°C. This melting point range was essential to prevent vitamin C flakes from becoming greasy and to ensure both proper encapsulation of vitamin C during storage and its release upon reconstitution. 4 . Losses during Storage of Dehydrated Mashed Potatoes

Flakes with 5% moisture and added antioxidants retained 70-76% of their vitamin C during storage for 7 months at 21°C when packed in air, while those packed in nitrogen retained practically all their vitamin C (Cording et al., 1961). Pedersen and Sautier (1974) incorporated a “protected” form of vitamin C in potato flakes (for details, see Section 111, E, 3). After 3 months of storage at 23”C, all the vitamin C added was recovered in undegraded form, while after 7.5 months the retention was not less than 78%. No systematic studies exist on the stability of “unprotected” vitamin C during storage of dehydrated potato products, however, numerous related investigations on dehydrated foods would appear to offer significant information. Most have established the fact that the moisture content of food has a decisive effect on the extent of ascorbic acid loss. The relationship of moisture content and relative humidity and the status of water in freshly processed add-back and freeze-thaw granules was illustrated by their water sorption isotherms (Jericevic, 1976). As seen from Fig. 15, the freeze-thaw adsorption isotherm had inflection points close to water activities (a,) of 0.1 and 0.87. At the first point (water content of 5.42%)the water was adsorbed in a monomolecular layer. From this point up to a , 0.87 (water content of 18.2%)water was adsorbed in a multimolecular layer. Above an a , of 0.88 the water in granules was condensed in bulk within capillaries and interstitial pores. Karel and Nickerson (1964) were among the first to show that the rates of ascorbic acid destruction in dehydrated foods increased with increasing moisture content. All the adsorbed water, even that in a monomolecular layer, contributed to losses of ascorbic acid. There was a linear relationship between vitamin C content and storage time at different relative humidities. Moreover, their findings demonstrated that the loss of ascorbic acid was implicated in nonenzymic browning reactions in dehydrated foods. Kirk et al. (1977) found that the rate of destruction of ascorbic acid increased as the total moisture content and water activity increased. A dehydrated model system enriched with 76.6% carbohydrates, containing 10.2% protein, 1% fat, salt, and 11.25 mg% ascorbic acid, wasequilibrated at a,’s below, at, and above the a , corresponding to the calculated monomolecular moisture content, and

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Water Activity (a,) FIG. 15. Water sorption isotherms of freshly processed add-back and freeze-thaw granules (Jericevic, 1976).

then sealed and stored. At all a,’s and storage temperatures the loss of vitamin C was found to conform to a first-order rate function. The rate of total ascorbic acid (TAA) destruction for the initial 25 days of storage appeared to be influenced by the rate of destruction of dehydroascorbic acid. Under all conditions the rate of loss of this reduced form of vitamin C was the determining factor in the total loss, since no buildup of the dehydro- form was observed. When the model food system was stored in the presence of a large excess of oxygen, the rate constants were found to be greater than in the absence of oxygen. The rate of ascorbic acid destruction increased dramatically in the presence of an excess of oxygen. The increased rate of loss as a function of the a, of the model system indicated that it was the dissolved oxygen in the mono- or multimolecular water layers, more than gaseous oxygen, which increased the rate of loss of TAA and reduced ascorbic acid. This suggestion was supported by the finding that the level of oxygen dissolved in a dehydrated model system may be a function of its a,. The importance of the effect of oxygen on the rate of ascorbic acid degradation in a low moisture, dehydrated model food system was recently emphasized by Dennison and Kirk (1978). Ascorbic acid degradation rates were first-order and were dependent on a, and temperature. Degradation was greater in the presence

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of excess oxygen. The data suggested that dissolved oxygen and the excess of gaseous oxygen were the primary factors governing the stability of ascorbic acid during storage of dehydrated foods. The extent of ascorbic acid destruction during storage in the intermediate moisture range (a, 0.32-0.93) of model food systems was reported by Lee and Labuza (1975). Although the value of u, 0.32 would correspond to a moisture content of 9-10% for dehydrated granules, which is well above the 5.5-7.0% moisture obtained in granule processing, the data given by these authors might be applied to understanding high vitamin C losses observed for reconstituted mashed potatoes given various heating regimens (Ang et al., 1975). Based on all these findings and the supposition that they might be equally valid for dehydrated mashed potatoes, high stability of vitamin C could be expected only at low storage temperatures and water activities. Also, implication of dissolved oxygen as a primary factor in the storage stability of vitamin C justifies the storage of dehydrated mashed potatoes in containers with little or no oxygen present.

IV. FLAVORING CONSTITUENTS For potatoes and potato products to be even more widely accepted they should possess a high organoleptic quality. From a consumer standpoint, flavor is an important property of instant mashed potatoes. The bland flavor of dehydrated granules and flakes suggests the need for a great deal more knowledge concerning the behavior of natural flavoring constituents during processing. Most studies have dealt only with qualitative aspects involved in the flavor of raw, steamcooked, boiled, or baked potatoes, as well as chips, granules, or flakes. Very little attention has been paid to quantitative evaluation, and there is a particular lack of information on changes during processing into the dehydrated product. None of the primary taste sensations of a salty, sour, sweet, or bitter nature are ordinarily perceptible in cooked potatoes. The term “flavor,” when applied to potatoes, has been used to denote a whole complex of sensations including not only taste, but aroma, mouth-feel, texture, and even appearance (Burr, 1966). While the flavor of potatoes and their products has been studied for some time (Ryder, 1966; Self, 1967), an interpretation of the significance of the findings has been difficult. Investigations have ranged from work on aroma of raw potatoes to a variety of studies with baked, steam-cooked, or boiled potatoes, as well as potato chips. A.

NONVOLATILE CONSTITUENTS

In general, the study of nonvolatile potato flavorings has not been extensive. Buri ef al. (1970) stressed the importance of free amino acids and 5’-nucleotides

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for the basic flavor note. Taste panel responses indicated a strong preference for a reconstituted aqueous flavor system containing both nucleotides and amino acids. Although 5’-nucleotides play important roles as flavor potentiators, they possess no taste of their own unless present in or added to potatoes (Dumelin and Solms, 1976). When free amino acids are present along with nucleotides, the flavor is synergistically increased (Solms, 1971). The nucleotide content of raw potatoes, though low, is comparable to that of other starch containing plant materials. Small amounts of adenosine and uridine nucleotides were found in raw tubers (Rees and Duncan, 1972a,b; Duncan and Rees, 1972). However, cooking resulted in the formation of relatively high levels of 5’-nucleotides. Dumelin and Solms (1976) showed that these compounds probably arose from a complex hydrolysis of tuber RNA by potato enzymes. The optimum release of 5’-nucleotides in potato homogenates was at 52°C and pH 6.0. Other nonvolatiles, such as phenolics and glycoalkaloids, have been considered significant in potato flavor development, although the majority of these compounds have been associated with a bitter off-flavor (Sinden et al., 1976). B.

VOLATILE CONSTITUENTS

The essence of potato is comprised of a variety of functionally different components, the major classes being sulfur-containing compounds-carbonyls, thiazoles, and, most notably, pyrazines. Some volatile constituents, especially those in the low boiling range, were identified and thoroughly discussed by Self (1967), Burr (1966), and Johnson et al. (1971); however, none had a characteristic potato odor. Buttery et al. (1970) characterized some higher boiling components of ptatoes. The medium and high boiling volatiles were 0.1 ppm in raw Russet Burbank potatoes, and had an aroma similar to that of raw potatoes. The major components were 1-octen-3-01, trans-2-octena1, trans-2-octeno1, and geraniol. The volatile oil obtained at atmospheric pressure, in an amount equivalent to 1 ppm in the potato, had an aroma similar to that of cooked potatoes. Among the major components identified were 2-pentylfuran, hexanal, phenylacetaldehyde, trans-2-furfuro1, and pyridine. Tentative evidence was given for the presence of 2-methoxy-3-ethylpyrazine,now known to be a potent odorant in raw potatoes. The pattern of a potato aroma profile is highly dependent upon the form of the potatoes, and extensive treatment has been given to the study of volatile constituents in raw, steam-cooked, fried, and baked potatoes, as well as potato chips. Much less has been reported for the aroma profile of dehydrated potato products. A comparative study of the medium and high boiling volatiles in raw and cooked potatoes and dehydrated granules, all from the same batch, was reported by Nursten and Sheen (1974). The yield of essence was about 1 ppm from raw and cooked potatoes, and 0.2 ppm for granules which had very little raw potato

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character. Although most of the same compounds were present, their relative proportion was different when compared to that of raw potatoes. The olfactory assessment of potato odor, described as green, raw, or “earthy” potato, was rather widely distributed between peaks of gas chromatographic runs. This odor was found not to be due to methional (3-methylmercaptopropanal) or 2S-dimethylpyrazine, but it did coincide in many respects with that of 2-methoxy-3ethylpyrazine. The odor assessments of potato granule essence gave no evidence for the presence of the off-flavor compounds that were expected to be produced during granule processing. Thus, as Nursten and Sheen (1974) concluded, any inferiority in the flavor of reconstituted potato granules, as compared to freshly mashed potatoes, should be attributed to a general lack of volatiles rather than to the presence of a specific off-flavor. However, as already mentioned, processing of granules may bring about an altered pattern of the remaining flavor constituents. The only new compounds found to be present in granules were hydrocarbons, methional, and 2-furaldehyde. The latter is known to be a heat degradation product of many foods and of vitamin C, so its presence in granules is to be expected. The fact that methional is found in readily detectable amounts in granules is not surprising, as it is a Strecker degradation product of methionine.

I.

Sulfur-Containing Compounds

The importance of sulfur-containing compounds to potato flavor lies in their extremely low odor thresholds. Gumbmann and Burr (1964), identified 14 volatile sulfur compounds in cooked potatoes with methyl mercaptan and methyl sulfide making up 90% of the total mixture. They further stated that it may, on first thought, seem anomalous that such an array of highly odoriferous compounds occurs in a food as bland as potatoes. This was clarified by the explanation that sulfur compounds are important in providing only secondary flavoring characteristics in cooked potatoes, since it was felt that they lose their characteristic odor at extremely low concentrations. The major source for the production of volatile sulfur compounds during potato cooking is assigned to breakdown of the sulfur amino acids, and to a lesser extent to breakdown of thiamine, biotin, coenzyme A, and glutathione. Degradation of methionine yields mostly methyl mercaptan, while sulfonium compounds such as S-methyl methionine yield dimethylsulfide. The variety of sulfur compounds obtained should also be assigned to secondary reactions of the strongly nucleophilic sulfur ion and to disproportionation among disulfides which occurs readily due to their ability to form an alkyl sulfide radical. Of interest is the finding of Gumbmann and Burr (1964) that sufficient precursors remain after processing of dehydrated potato granules to provide hydrogen sulfide upon reconstitution in amounts comparable to fresh potatoes.

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Methional is the most notable sulfur-containing constituent of potato aroma. Its importance in the nonbasic volatile fraction was established by Buttery and Ling (1972), Guadagni e f al. (1972), and Buttery (1973). It is thought to be one of the more significant volatile constituents of baked potatoes (Buttery er a l . , 1973a), potato chips (Buttery and Ling, 1972), and cooked and dehydrated granules (Nursten and Sheen, 1974). Guadagni et al. (1971) conducted experiments on the flavor enhancement of dehydrated mashed potatoes and found that methional did not improve the flavor of flakes unless its concentration was at least 3 ppm. 2 . Curbonyls A multitude of carbonyl constituents in potato aroma have been identified and reported by various workers. Johnson et af. (1971), in a review of volatile compounds identified in potatoes, reported a total of 34 carbonyls which had previously been characterized. Of these constituents, 24 were found in boiled potatoes and only 11 were present in fresh tubers. Mookherjee et ul. (1965) identified 18 monocarbonyls in fresh potato crisps. However, they felt that the majority of these compounds probably originated from the fat used during frying and not from the potato. A number of aldehydes have been characterized in potato chips (Buttery and Ling, 1972; Chang, 1967; Mookherjee et a / . , 1965). Buttery (1973) reported a total of 8 aldol condensation-type compounds, as well as 4 other unusual carbonyls.

3 . Thiazoles

Buttery et a f . (1971) found that most of the components of the basic fraction of the volatile oil of potato chips were alkylpyrazines; however, there were a few components with odd molecular weights, indicating compounds containing one nitrogen (or an odd number). Subsequent to this, both alkyl- and alkanoylthiazoles were found in potato chips and in the basic fraction of the volatile oil from boiled potatoes (Buttery and Ling, 1974). It was thought that, due to their potent and characteristic odor, some of these compounds could be important to the aroma and flavor of potato products, including dehydrated granules. 4 . Pyrazines A review of pyrazines in foods, including potatoes, was given by Maga and Sizer (1973). Deck and Chang (1965) reported that 2,5-dimethylpyrazine, which they isolated from potato chips, had a typical raw, earthy potato flavor at a concentration of 10 ppm in oil. Buttery et al. (1971) found 18 pyrazine compounds in the basic fraction of the steam volatile oil from potato chips. Eight of these had been

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previously reported by Deck and Chang (1963, and Chang (1967), and a number were found by Sapers et al. (1971) in explosion puffed dehydrated potatoes. Preliminary sensory evaluation by Buttery et al. (1971) indicated that 2ethyl-3,6-dimethylpyrazinewas one of the most potent odorants derived from chips, and suggested that, due to its low odor threshold, it could make a definite contribution to potato chip aroma. Its aroma, usually described as a “toasted flavor, could be detected in dehydrated granules when they were overheated in the final fluid bed drying steps of granule processing. It is considered an objectionable off-flavor in granules. As found by Sapers et al. (1971), specific pyrazine compounds for which increased amounts gave increases in the toasted off-flavor included methyl-, 2,5-dimethyl-, 2,3 and/or 2,6-dimethyl-, ethyl-, trimethyl-, and ethyldimethylpyrazine. Gas chromatographic scans of the volatiles from conventionally dried and explosion puffed potatoes were similar; however, the relative quantities of compounds were significantly greater in puffed samples. Pyrazines have recently been characterized in boiled potatoes (Nursten and Sheen, 1974) and baked potatoes (Buttery et al., 1973a; Pareles and Chang, 1974). Although only traces were found in boiled potatoes, relatively large amounts were found among the volatiles of baked potatoes. Two compounds which contributed significantly to the aroma were 2-ethyl-3,6-dimethyl- and 2-ethyl-3,5-dimethylpyrazine. However, though the pattern of pyrazines found in chips and baked or boiled potatoes might be similar, their threshold values would differ in oil- and waterbased potato products. The differences in odor thresholds of some pyrazines in oil and in water were determined by Koehler et al. (1971) and Guadagni et al. (1972). Guadagni et al. (1972) introduced the odor unit concept for better evaluation of volatiles from processed potatoes and used it as a qualitative tool to identify the major flavor contributors. The unit is a value calculated by dividing the compound concentration found in potatoes by the threshold concentration as determined in water or oil. These authors concluded that only two of the 11 pyrazines found in potato chips contributed significantly to potato chip aroma: 3-ethyl-2,5-dimethyl- and 2,6-diethylpyrazine. However, they found that methional was the major contributor to chip aroma. The earthy aroma of potatoes was assigned to the presence of 2-methoxy3-isopropylpyrazine by Buttery and Ling ( 1973). The authors also indicated the possible presence of 2-methoxy-3-ethylpyrazine. Guadagni et al. (197 l), in a sensory study on the effect of adding both compounds separately to reconstituted dehydrated potato granules, found that, in contrast to the isopropyl derivative, only ethylpyrazine was able to effectively improve the flavor of the potatoes. Seifert et al. (1970) showed that the methoxy group, more than any other alkoxy group, was the major contributor to flavor. The observation that ”

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substitution of the methoxy group by other alkoxy groups brings about a marked reduction in odor intensity was confirmed by Parliment and Epstein (1973). Seifert et al. (1970) proposed that one side of the pyrazine ring must be left unsubstituted in order to obtain a very potent odorant. Takken et af. (1975) confirmed this supposition. Seifert er al. (1972) demonstrated that most of the potent odors were obtained when the alkyl substituent was in a position ortho to the methoxy group. Elegant evidence for the presence in potatoes of 2-methoxy-3-ethylpyrazine and its relation to raw earthy odor was given by Nursten and Sheen (1974). Guadagni et al. (1971) confirmed the important role of 2-methoxy-3-ethylpyrazine in potato flavor by showing that the flavor of several samples of dehydrated granules and flakes was consistently and significantly improved by the addition of this compound in amounts of not more than 0.1-0.2 ppm. while other volatiles had no effect. Several patents have been developed for flavor enhancement of processed potatoes with synthetic pyrazines (Flament, 1974; Gaudagni et al., 1973; Mookherjee ef al., 1973; Nakel and Dirks, 1971; Roberts, 1968). The most notable is that of Guadagni et al. (1973), which suggests the addition of about 0.1-0.3 ppm of the hydrochloric acid salt of 2-methoxy-3-ethylpyrazine to potato products. Mookherjee et al. (1973) recommended the use of a potato flavoring containing 0.005-20 ppm of 2-acetyl-3-ethylpyrazine in a carrier. Flament (1974) suggested the use of combinations of pyrazines, cyclohexenones, and/or thiazolidines to enrich the flavor of French fried potatoes. A patent for the synthesis of 2-methoxy-3-isobutylpyrazine was given to Buttery et al. (1973b); however, the authors did not specifically recommend the addition of this compound to any particular foodstuff. The feasibility of these patents for implementation in potato granule processing was not well elaborated since exact procedures were not given for flavor augmentation at any particular stage in the dehydration process. However, several processes have been designed to retain natural flavors during processing. Hollis and Borders (1965) processed tubers along with the peel and adjacent cells since they contained the largest portion of the aroma and flavor notes. Reeves and Hollis (1964) claimed a high retention of natural flavors in a dehydrated mashed potato process in which the tuber was separated into inner core and peel fractions and each cooked separately. The peel fraction was then mashed, and particles of peel eyes and rot were removed by screening. The clarified slurry was partially dried and combined with the mashed core fraction before further dehydration. A short process, in which precook-cool steps were omitted and partial mashing was done by ricing with simultaneous incorporation of 1% by weight glycerol monostearate, was claimed to retain natural potato flavor more than other conventionally produced mashed potatoes (Shatila and Terrell, 1976). The coat of emulsifier on separated potato cells appeared to

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D. HADZIYEV AND L. STEELE

complex soluble starch and lubricate cell surfaces. This free liquid phase retained part of the potato flavoring constituents even during fluid bed drying in the granule process. Marce and Hadziyev (1977) determined that flavor enrichment during the mashing step of a freeze-thaw granule process was not suitable since great losses occurred in subsequent dehydration steps. The major loss occurred in the predrying step. In granulation completed at a moisture content of 20-36%, additional losses ranged from 14-18%, with the exception of methylpyrazine (30%). Final drying brought about an end product in which the loss, irrespective of the added pyrazine, was 99.9%. The extent of the pyrazine losses appeared to be dependent only on the amount of moisture removed. Granule processing in which pyrazines were mixed with' microcrystalline cellulose, pregelatinized starch, amylose, amylopectin, or pectin and then incorporated into a moist mash and dehydrated, showed losses of pyrazines as high as 99.9%. As a result of the retention studies described above, those patents suggesting pyrazine addition to potatoes prior to drying operations would be unsuitable. Hence, the only useful procedure for flavor improvement appears to be the addition of flavors to the dehydrated end products, analogous to the method now being employed for enrichment of granules with vitamin C. Furthermore, due to the volatile nature of pyrazines, the most practical method for pyrazine enrichment would be one in which pyrazines are incorporated into processed potatoes as immobilized compounds which would release the flavor upon reconstitution with hot water.

C. INTERACTION OF FLAVOR VOLATILES WITH MAJOR POTATO CONSTITUENTS The study of flavor compound binding in foods has not been extensive, since whole, intact foodstuffs are difficult substrates for sorption experiments because of their heterogeneous composition (Maier, 1972). As a result, the majority of investigations concerning this behavior of flavor volatiles have dealt with interactions between the flavor compounds and model food components, including macromolecular constituents such as lipids, carbohydrates, and proteins. The interaction of volatiles with model food components was recently discussed and reviewed by Solms et al. (1973). The authors stated that there seems to be no universal mechanism for flavor binding in foods. They did conclude, however, that there are at least three major but quite different mechanisms responsible for the interaction of volatiles with major food components. The first and simplest of these involves physical laws of partition (presumably the major effect controlling retention and release of flavor substances in foods with a high fat content). Second, flavor volatiles may associate with major food components such as potato starch if secondary and tertiary structures of the solid matrices are

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formed, thus giving the molecules new binding zones that can then interact with the ligands (flavor molecules). A third mechanism responsible for flavor binding is a hydrophobic one in which the food protein interacts with ligands, along with unfolding and destruction of tertiary structures. Thus, the reaction would depend on the “unfolding capacity” of the flavor molecules toward the food protein. The interaction of potato starch with flavor compounds has been investigated. Starch combines with a variety of compounds to form so-called inclusion or clathrate compounds which are insoluble at room temperature. According to Greenwood (1956), starch in aqueous solution exists in the following forms: aggregated linear chains; chains with linear conformation; chains with helical conformation; and aggregated helices. The helical arrangements of amylose are known to be responsible for inclusion compound formation, as is the amylopectin fraction. As proved by Osman-Ismail and Solms (1973), potato starch possesses the greatest complexing ability, surpassing that of rice and corn-and even pure amylose. These authors also found that a minimum concentration of flavor molecules is required to initiate the reaction, i.e., the formation of aggregated helices. At a high concentration of flavor compounds, the insoluble starch is filled up with entrapped molecules. Experiments with n-aliphatic alcohols with a chain length of 6-8 carbons showed a decreasing amount of these molecules within the starch as their molecular weight increased. In experiments with a larger set of model substances, such as aldehydes, fatty acids, and some terpenes, potato starch formed complexes under equilibrium conditions which then permitted the calculation of binding parameters and starch binding zones (Osman-Ismail and Solms, 1972). In addition to formation of inclusion compounds, several authors assumed that flavor ligands were also bound to preferential binding sites on the exterior of the helix or to other regions of the macromolecules (Simpson, 1970; Nicolson et al., 1966a,b). The binding ability of potato starch and other macromolecules for flavoring volatiles, covered by several patents, was reviewed by Maier (1972).

V. THE ROLE OF SULFITES AS ADDITIVES Nonenzymic browning phenomena occur frequently during processing and storage of dehydrated mashed potato products. In many cases they are a distinct sign of deterioration of the color, flavor, and nutritional value of the product. The Maillard reaction, a major cause of color and flavor deterioration, was reviewed by Hodge (1953). It is independent of oxygen, and does not occur if the initial stage (reducing sugar-amino acid condensation, followed by the Amadori rearrangement) is blocked by sulfite. In processing of instant mashed potatoes it has been the general practice to add sulfites to inhibit browning, as well as to act as reducing, bleaching, or sanitizing

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D. HADZIYEV AND L. STEELE

agents, and as preservatives. Sodium sulfite and/or bisulfite are added along with emulsifiers, antioxidants, and other additives in the mashing step of processing. The amount used corresponds to 350-500 ppm (as SO,) in the dehydrated end product. An SOp level of not more than 500 ppm is permitted in Canada and the U.S.A., while the United Kingdom specifies 550 ppm. The status of sulfites as food additives was reviewed by the Institute of Food Technologists (1975). Freshly harvested Alberta “Netted Gem” potatoes have a reducing sugar content of 0.5% on a dry weight basis. Storage at 4”C, which is associated with starch degradation and concomitant reducing sugar accumulation, necessitates a reconditioning step at 18°C for at least 2 weeks prior to processing to reduce the accumulated sugar. Reducing sugars were high in tubers stored at about 4”C, and, while conditioning decreased the content, they were still higher than in tubers stored commercially at 7°C (Zaehringer et al., 1966). At this temperature the reducing and total sugar contents fluctuated very little and were not influenced by sprout inhibitors. The major constituents of the sugar fraction of the tuber were fructose, glucose, sucrose, melibiose, and raffinose (Kimura er al., 1969; Shaw, 1969). Urbas (1968) found 15 major and 5 minor components. Seasonal variation of sugar content is proportionally matched with appropriate amounts of added sulfites in the mashing step of granule or flake production. In spite of this, only 37-40% of total sulfite incorporated was still in the form of measurable SO, shortly after processing and dehydration (Gilbert and McWeeny , 1976). Apart from a small amount present as sulfate, the remainder of sulfite was present as organic sulfur compounds, other than sugar-bisulfite adducts, which do not release SO2 on treatment with boiling acid in the Monier-Williams method (Association of Official Analytical Chemists, 1975). The effect of added sulfite on the quality of instant mashed potato flakes was studied by Sahasrabudhe el al. (1976). The flavor quality of the reconstituted product showed no correlation with the residual SOz. The same authors also analysed the optimum level of residual SOe in flakes. Five samples containing sulfite in the range of 0-848 ppm (as SO,) were prepared under commercial conditions, packaged in polylined paper cartons, and stored at 21°C for up to 1 year, while controls were stored at -17°C. An SO2 level above 162 ppm in dehydrated potato flakes gave no additional beneficial effect on the storage quality of the product. All samples, regardless of the initial content of SO,, showed deterioration after 6 months of storage. The samples with no sulfite added deteriorated within 3 months, while the control retained its quality for up to 8 months. All samples had apparent SOz losses of from 33-63%. The loss of vitamin C after storage at 21°C for 1 year was 70-82% regardless of the SO, level. The whiteness of the flakes decreased significantly only after 8 months of storage except for the sample with the highest content of SOz. The change was practically the same for other samples. The results demonstrated that SO, inhib-

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ited browning during processing, and, moreover, it had a bleaching effect during storage at room temperature. The bleaching effect was much less in samples stored at - 17°C. The authors concluded that a level of 250 ppm of residual SO2 in dehydrated mashed potatoes would suffice to ensure a good quality product even after storage for over 1 year. A similar storage stability of dehydrated granules with sulfite at a level of 400 ppm (as SO,) was reported by Lisberg and Chen (1973). The sulfite loss was 36% during 5 months of storage. It was practically the same in granules packaged in cans under nitrogen or those packed in air in polyethylene-coated and foillined cartons. During this time, SO, loss paralleled that of the phenolic antioxidant. Nevertheless, the Agtron color values remained essentially unchanged, and the differences in aroma and flavor against the control were not all significant. The frequent need to control the residual SO2 content in dehydrated potatoes prompted some flake processors to develop rapid methods of SO, determination by employing simple equipment that was satisfactory for routine use in a quality control laboratory. Such a method was that of Ross and Treadway (1972) in which titration with iodine followed careful and rapid extraction of SO2 to prevent cell rupture and subsequent release of starch.

V I.

MICROFLORA AS AFFECTED BY PROCESSING

The presence and growth of microflora during processing can have definite effects on the quality of the product. A very limited amount of data are available on the microbiological aspects of potato granule production. The microbiological standards for dehydrated mashed potatoes, as specified by Canadian processors, require that the maximum counts for each gram of product be standard plate count, 50,000; coliforms, 25; Escherichia coli, Staphylococci and Salmonella, 0; yeast and molds, 100. Some quantitative and qualitative information on the microflora of potatoes processed to dehydrated flakes were given by Gorun et al. (1973). The count decreased steadily during processing, becoming 0 after the 30- to 45-minute steam cooking step. However, as a result of contamination from the air, a low count was reestablished during mashing. Gorun et al. (1973) also found that up to the precooking step the raw potato microflora were non-spore-forming, aerobic bacteria of the type Pseudomonas fluorescens, cocci, and a small number of mesophilic spore forming Bacillus species. Fungi and molds, such as Aspergillus niger andflavus, and Alternuria, were also found, as were yeasts and yeastlike molds of the Oidium lactis type. No intestinal coliform bacteria were found in the end product, though the presence of Aerobacter aerogenes and “coli citrovorum” was confirmed in initial steps of processing up to the precooking step. When the flakes (no more than 12%

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moisture) were packed in cartons double-lined with polyethylene and cellophane and stored for 1 year at 18-20°C, their sterility was maintained. Pertinent data were given by Ovrutskaya ez al. (1977) for changes in potato microflora during washing, slicing, and blanching. The contaminants found in these steps of processing included Micrococcus, Pseudomonas, Erwinia, Bacillus, Clostridium, Lacrobacillus, the Coli-aerogenes group, spores of the molds Penicillium, Mucor, Aspergillus, Oidium, and Fusarium, green-spored molds of Trichoderma, and molds of the genus Monilia. In the add-back production of dehydrated mashed potato granules, an adequate washing of raw potatoes caused a reduction in the number of microorganisms, as did steam-peeling (Steele and Hadziyev, 1976). Precooking or water blanching at 70°C reduced the count by 99%. The surface of freshly mashed potatoes was practically free of microflora, however, when the mash had cooled to 60°C, contamination could occur from molds, yeasts, and bacteria in the air. The microflora count increased greatly during conditioning, a step in which the duration and temperature regime simulated a temperature gradient incubator. In subsequent air-lift drying and fluid bed drying steps, the heat applied produced a large reduction in the count. All yeasts and most bacteria were destroyed, but spores of bacteria, and molds usually survived, as did vegetative cells of a few species of heat-resistant bacteria. Therefore, processing equipment must be cleansed and sanitized often to avoid a buildup of spores of thermophiles. To reduce the microflora count of dehydrated mashed potatoes, gaseous “cold sterilization” with propylene oxide was suggested rather than heat sterilization in order to avoid browning reactions and a change in flavor and odor of the dehydrated product (Steele and Hadziyev, 1976). Treatment with an optimum concentration of 0.1% w/w sterilant for 6 days at 22°C resulted in a reduction in the bacteria count from 3.4 x l o 5 to less than 5 x lo3. The propylene glycol residue was 29 ppm, while propylene chlorohydrin was 12 ppm. Evidence was given that such sterilization did not result in detectable etherification of starch. The need for a prolonged exposure of granules did not adversely affect quality, as long as the sterilant concentration was kept below 0.5%w/w. The use ofethylene oxide in gaseous sterilization of dehydrated mashed potatoes was not recommended because of the probable accumulation of two toxic compounds, ethylene chlorohydrin and its hydrolytic by-product, ethylene glycol.

V II.

RANCIDITY DURING STORAGE AND SHIPMENT A.

RANCIDITY DEVELOPMENT

The contribution of potato lipids to the gradual development of an oxidative off-flavor in stored potato granules was studied by Buttery et al. (1961). Russet

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DEHYDRATED MASHED POTATOES

Burbank granules were prepared by an add-back process. Sulfite (300 ppm as SO,) was the only additive. The granules were sealed in cans in an atmosphere of air, oxygen, or nitrogen, and stored at room temperature, while control samples were kept under nitrogen at -35°C.Storage in air caused an intensive autoxidation of linoleic and linolenic acids. The amount of linoleic acid dropped from 53.6 to 46.0% of the total acids after 2.5 months, and to 42.7% after 4.5 months, while linolenic acid decreased from its initial 20.5 to 16.1 and 12.6%, respectively. Granules stored for 3 months in oxygen lost 22.5% of linoleic and 9.7% of linolenic acid. A convenient index of the extent of autoxidation was the unsaturation ratio (UR: a ratio of the sum of linoleic and linolenic acids to that of palmitic and stearic acids). Freshly dehydrated granules had a UR close to 3.0. This decreased to 1.2 after 4.5 months storage in air and to 0.7 after 3 months storage in oxygen. It was established that 2 moles of oxygen were taken up for each mole of linoleic and linolenic acids oxidized. Also, the oxidation rates of the two acids were similar. Off-flavor scores for the stored granules, as obtained by a sensory panel, increased with the decrease in UR. Storage for 29 days was required for a duo-trio test to reveal a difference between the stored and control (unstored) samples. This time was called the first “detectable difference. The second “detectable difference” was after 42 days and the third after an additional 40 days. A plot of oxygen absorbed from the headspace versus storage time gave a curve typical of lipid oxidation. There was an induction period followed by rapid oxidation, and a tailing-off period. ‘‘Detectable differences showed the same trend. The first was at the end of the induction period, when the granule UR was 2.6, the second at the end of rapid oxidation (UR 1.7), and the third was during a tailing-off period when an additional deterioration in flavor was detected and the UR was 1.2. Buttery et al. (1961) also analyzed the headspace vapor of the can or the vapor above the hot reconstituted granules for low- and high-boiling components. They detected aldehydes, including 2-methylpropanal and 2- and 3-methylbutanal, and hydrocarbons. Hexanal was the predominant volatile compound. Its concentration was four times that of any other major component from granule autoxidation and ten times that of the majority of other compounds. Higher boiling compounds found at low levels were octanal, 2-pentenal, 2-octenal, heptanal, nonanal, and 2-hexenal. The volatiles from autoxidized granules corresponded to theoretically expected degradation products of linoleic and linolenic acids. Evans et al. (1969) postulated thermal breakdown of hydroperoxides, yielding specific hydrocarbons. The oxidation of pure linoleic and linolenic acids produced mostly pentane and ethane which were more than 90% of the hydrocarbons released (Amaud and Wuhrmann, 1974). Measurement of the concentration of hydrocarbons (pentane ”

”

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D. HADZIYEV AND L. STEELE

in particular) was suggested for determination of the extent of oxidative deterioration of dehydrated mashed potatoes. Potato granules stored in air do not show a high level of headspace volatiles, but do show a large concentration upon steam distillation, or in the headspace of hot reconstituted granules. This suggests that the bulk of off-flavor constituents derived from oxidized lipids are produced through breakdown of precursors after hot reconstitution. The relation of hexanal in headspace to subjective flavor estimates was reported by Boggs et al. (1964). Commercially produced granules (7% moisture, 2.5 ppm BHT, and 250 ppm sulfite as SO,) were sealed in cans under air and stored at 22°C. A control sample was packed under nitrogen and stored at -34°C. Air-packed granules, when reconstituted, showed a hexanal increase proportional to storage time. The increase was slow during the first 2 months, suggesting an induction period. This was followed by a rapid change after 80 days, after which a regular hexanal increase occurred up to 4 months of storage. A taste panel, with 75% correct decisions in a duo-trio test, detected an offflavor after 101 days of storage when hexanal content was 4.2 ppm, and by the end of 4 months storage, with hexanal at 8.5 ppm, the percentage of correct decisions was 100. The study indicated that hexanal concentration was closely associated with flavor deterioration of dehydrated granules as judged subjectively, but this did not imply that it was responsible for rancidity of stored granules. Lisberg and Chen (1973) stressed the possible advantages of using cartons for storage of dehydrated mashed potatoes. Their study involved freshly processed granules (6% moisture, 11.O ppm BHT, and 382 ppm sulfite as SO,) stored at 24°C and 50% relative humidity under air or nitrogen in sealed cans or foil-lined and polyethylene-coated cartons. Hexanal was not detected until the third month of storage. At this time only traces were present in the nitrogen-packed cans, with no change after 6 months of storage. Hexanal in air-packed cartons was 0.26 ppm, increasing to only 0.5 ppm after 6 months of storage. Differences in aroma and flavor were significant only after the fourth and sixth months. The content of BHT declined steadily to 6.7 ppm in cans, and 1.7 ppm in cartons. Overseas shipment of granules in polyethylene-lined paper bags during Summer may result in extensive rancidification. Add-back granules (7% moisture, 550 pprn sulfite as SOn, and close to 10 ppm BHT) became rancid, some shipments grossly so (Domay Foods, 1976). Hexanal levels, determined by the procedure described by Buttery and Teranishi (1963) and Boggs et al. (1964), but expressed in arbitrary units as a peak height in millimeters corrected to a 250-mm peak height of isobutyl acetate as internal standard, resulted in peaks four to five times higher than commercially permitted. In these arbitrary units rancidity is normally noticeable organoleptically at hexanal levels of 100-150 mm. The levels of SO2 decreased at the same time, and tended to reflect the

DEHYDRATED MASHED POTATOES

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extent of rancidity. The samples with minimal hexanal levels retained about 4 ppm of antioxidant, the slightly rancid ones only traces (0.1-0.2 ppm), while the highly rancid granules completely lost all antioxidant protection. Granules shipped under controlled temperature (not exceeding 23°C) did not develop rancidity. Their hexanal level in arbitrary units was 10-15 mm, with sulfite retention close to 79%, while the residual level of BHT was 6-8 ppm. The behavior of granules in bags simulated that of those in air-packed cartons. Walter and Purcell (1974) found that dehydrated sweet potato flakes underwent rapid oxidative deterioration unless stored in an atmosphere low in oxygen. Of relevance to rancidity problems of dehydrated mashed white potatoes was their suggestion that autoxidation of flakes occurred in a bimodal fashion, with surface lipids being oxidized at a faster rate than internally located, bound lipids. The loss of bound fatty acids was so slow as to be nearly undetectable, and the surface acids showed an induction period of 18 days. The oxidation of unsaturated surface fatty acids was found to be independent of peroxide values. The initial UR of 1.60 for free surface lipids steadily declined during storage to a value of 0.96 after 77 days. The bound lipids’ UR of 1.75 declined only slightly. The fact that 76% of unsaturated lipids occurred in bound lipids (Walter et al., 1972) and that the bound lipids were oxidized at much lower rates than surface lipids, strongly suggested that environment was often more important for autooxidation than lipid composition. A plausible explanation for bimodal autoxidation in flakes was that processing brought about a trapping of up to 90% of the lipids into the gelled carbohydrate matrix. This lipid protection then retarded autoxidation. However, lipids on the flake surface were freely exposed to air, and consequently, were readily oxidized. Therefore, the rancidity responsible for short shelf life of dehydrated mashed potatoes might be attributed mostly to surface lipids. Electron microscopy studies (Chung et al., 1979; Fedec et al., 1977) on processed white potato cells showed fused starch grains occupying nearly the entire cell volume. No lipids were detected within this newly formed matrix, though traces of trapped cytoplasm were occasionally evident. However, a major lipid layer was present around the starch matrix, with an additional broad, weak layer within the cell wall and bordering the denatured cytoplasm. The lipids appeared to be spread over distinct starch, protein, and cellulose and pectin matrices. Thus, autooxidation in white potatoes, instead of being influenced mainly by a ratio of the amounts of free and trapped lipids, would be governed primarily or even exclusively by a matrix effect. Model systems, consisting of a solid surface over which lipid is spread, have often been used in rancidity studies with the aim of relating the findings to real foods. Labuza et al. (1969) found that egg albumin decreased linoleate oxidation by almost 100 times, suggesting a protein-lipid interaction. Soybean oil dispersed on protein oxidized slowly, while oxidation was most rapid on carbohy-

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drate surfaces (Roubal, 1971). A study of oxidation of pea lipids suggested that lipid polarity, rather than degree of unsaturation, was the predominant factor controlling the rate of oxidation on protein and carbohydrate matrices (Haydar and Hadziyev, 1973). Accelerated autooxidation of potato lipids in light at 45°C on dehydrated potato cell wall preparations, proteins, starch, and pectic substances was conducted by Khan and Hadziyev (1979). The oxidation rate on all matrices was fastest for glycolipids, with only a short 5-hour induction period. Oxidation during the propagation step was, in microliters oxygen uptake/hour: starch 10; pectin (DE=55%) 7; cell walls 6; and protein 1. During the same time, oxidation of neutral lipids was slow or negligible. Oxidation of phospholipids was slow in the initial 30 hour, followed thereafter by an exponential rise. The highest rate was on starch (8 pl oxygen uptake/hour), while pectin gave half that rate, and the potato protein matrix suppressed oxidation. Relating these findings to granules would suggest that the lipid layer around the gelled starch matrix would be the major site for rancidity development, while glycolipids present in granule cell walls would be of minor importance. No in situ proof yet exists for for such a conclusion, although results of Buttery el al. (1961), that the phospholipids were first to oxidize in granules might imply that the lipids around starch were involved, since they were richest in phospholipids. Potato flakes have a shelf life of only 6 months in air at 23°C even when stabilized by incorporation of sulfite and antioxidants. Volatile components associated with storage changes can arise from reducing sugar-amino acid interaction, and from lipid oxidation. 2-Methylpropanal and 2- and 3-methylbutanal were detected in headspace vapor. Higher boiling reaction products (furfural, benzaldehyde, and phenylacetaldehyde) were found in steam distilled volatiles. Storage for 6 months resulted in only small increases in low boiling aldehydes and in furfural. Phenylacetaldehyde, the major component of potato flake volatile concentrate, increased slowly during the first 3 months of storage, as did benzaldehyde. Differences between the level of furfural and the Strecker degradation aldehydes in air- and nitrogen-packed flakes were small and variable (Sapers et al., 1972). These findings suggested that dehydrated potatoes, even flakes packed in nitrogen, may undergo further nonenzymic browning reactions during storage, yielding volatiles detrimental to flavor. The initiation of these reactions occurred during processing, particularly in the drum drying step, as a consequence of overheating (Sapers, 1975). However, the shelf life of flakes was not normally limited by flavor defects due to sugar-amino acid interaction. The major objectionable flavor defect was derived from lipid oxidation, which could be controlled to a certain extent by BHA or BHT, and by nitrogen-packing. Flakes stored in air at 23°C for up to 6 months showed a substantial increase in compounds clearly indicative of lipid oxidation (Sapers et al., 1972). These included n-hexanal, 2-pentenal, n-heptanal, 2-hexenal, 2-heptanone, and

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2-pentylfuran. The sum of peak areas of either headspace or volatile concentrate has been suggested as an index of the extent of oxidation in flakes (Sapers et al., 1973). The protective effect of water on shelf life of dehydrated foods sensitive to lipid oxidation was reviewed by Labuza (1971). In a freeze-dried model system consisting of microcrystalline cellulose, water up to a water activity (a,) of 0.5 had an inhibitory effect on linoleate oxidation. The protective effect was pronounced in the initial stage of lipid oxidation and was attributed mostly to retardation of oxygen diffusion and hydroperoxide decomposition, loss of free radicals, and hydration of trace metal catalysts (Maloney er al., 1966; Labuza et al., 1970). The bonding of water molecules to food surfaces which excludes oxygen from the lipid would also inhibit oxidation (Salwin, 1959). The optimum moisture content would correspond to a monolayer coverage of water on dehydrated food. Lipid oxidation could most readily occur below this level. Thus, the higher the water content or a,, the slower the lipid oxidation rate. However, it was demonstrated that lipid oxidation was promoted in the intermediate moisture range ( a , 0.55-0.85). Jericevic (1976) found that the monolayer coverage of water in freshly processed add-back and freeze-thaw granules, as calculated by a graphical B.E.T. procedure, was close to 5.5%. The monolayer moisture content for flakes was found to be 5.51% by Sapers et al. (1974). The above findings were reflected by the effect of moisture content on stability of potato flakes in air at 23°C (Sapers et al., 1974). Underdried flakes, with 7% moisture, gave a slightly lower level of volatile oxidation products than those with the desired 4.7% moisture. Overdried flakes (moisture content 3.1%) contained 2.2 times more of the volatile oxidation products after 6 months, and 1.5 times more after 1 year of storage than normally dried samples. Also, the flavor scores of underdried and normally dried flakes, which were similar, were better than those for overdried flakes. B.

DETERMINATION AND USE OF ANTIOXIDANTS

Several antioxidants are permitted for use in dehydrated potatoes to prevent or minimize autoxidation. The legal status in various countries is summarized in Table VI. Widely used phenolic antioxidants are BHA (a mixture of 2-and 3-terbutyl4-hydroxyanisole), BHT (ditertbutyl-p-cresol), and PG (propyl gallate). These antioxidants break the free radical chain reaction through removal of alkylperoxy radicals from the lipid oxidation process, and, hence, are classified as primary (type I) antioxidants. They protect lipids by prolonging the induction period of autoxidation. The reaction kinetics were thoroughly reviewed by Labuza ( 1971) .

118

D. HADZIYEV AND L. STEELE TABLE V1 ANTIOXIDANTS IN DEHYDRATED POTATOES-LEGAL

Country

BHAb

Australia Belgium Canada Denmark France Germany Italy Japan Norway South Africa Spain Sweden U.K. U.S.A.

0.01 0.02 0.005 0.02

tion,

BHT

SITUATION (1974)O

Gallates

Tocopherol

-

0.01

0.01

0.02 0.005 0.02 0.01

0.01

0.01

0.02 0.02

0.02

0.0025 0.02 g 0.001Q f 0.005

0.0025 0.02 g 0.001 f 0.005

Ascorbic acid 0.01

+ +

0.005 0.01 0.01 0.01 0.003

Ascorbyl palmitate

+ +

+

+ +

+ 0.03

-

-

-

-

+

0.01

+ +

-

Figures are given in percent. “g” = granules. “f” - not permitted.

=

flakes.

“+

”

= permitted without limita-

‘6-3’

Amount given is that permitted for the antioxidant alone, or where allowed, for mixtures of BHA, BHT, and/or propyl gallate.

Synergism in mixtures between BHA and BHT, and between BHA and PG provides increased antioxidant potency, and lengthening of the induction period relative to that which any one antioxidant could provide. Therefore, BHA and BHT are usually added together in the mashing step of processing. In order to obtain a good distribution in the mash, they are added in a solvent such as ethanol. An example is a patent assigned to the American Potato Co. (1967) in which an ethanolic solution of BHT was added to mashed potato prior to dehydration. Synergistic ability has also been ascribed to ascorbic and citric acids. However, in dry systems these acids function more as chelating agents (secondary or type I1 antioxidants) which complex with trace metals, making them less available for the initiation step of lipid oxidation. In the presence of increased moisture ascorbic acid performs as a primary antioxidant. Ascorbic acid (or its palmitate) should also be considered as an antioxidant which controls environmental factors rather than lipid oxidation rates. It functions as an oxygen scavenger in closed systems (type 111 antioxidant), offering a distinct advantage for granules contained in cans packed in air. Bielski and Allen (1970) demonstrated that, in order to initiate oxygen scavenging, position 2 on ascorbic acid must be unsubstituted in order to allow free radical formation.

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Based upon theoretical calculations of the amount of air required to convert 1 mole of ascorbic acid to 1 mole of dehydroascorbic acid, 3 mg ascorbic acid would be required for each milliliter of headspace. The control of oxygen in headspace appears to be a promising way to suppress rancidity. Deobald and McLemore (1964) found that type I antioxidants were effective with sweet potato flakes only when the oxygen level was about 10%. Drazga et al. (1964) found that exclusion of oxygen and its replacement with nitrogen provided as good or better protection than addition of BHA, BHT, or tocopherols to white potato flakes. Lipids in freeze-dried whole foods, where tissue integrity has been preserved, were in general not well protected by synthetic antioxidants (Porter ef al., 1977). The same antioxidants functioned well when they could be incorporated into the lipid, as in vegetable oils or lard. Hence, the poor protection in dried whole tissue foods, including granules or flakes, might be due to the lack of ready access to sites of oxidation, i.e., polyunsaturated, polar lipids of the membrane systems. Rapid screening of antioxidant effectiveness in dried membranes was reported on porous and dry model systems (acid washed and activated silica) coated with a monolayer of linoleic acid, which was deposited by equilibrium adsorption from a nonpolar solvent in order to obtain maximal autoxidation rates (Porter et al., 1977). The effectiveness of an antioxidant was defined as the time taken to reach a headspace oxygen content of 20%, divided by the analogous time using Da-tocopherol. Though a-,p, or y-tocopherol were not found in appreciable amounts in the unsaponifiable portion of potato lipids (Lepage, 1968), the choice of tocopherol for reference was still justified since it is present in most of the cell membranes. BHA and BHT were 3.6 and 1.1 times, respectively, more effective than tocopherol, while caffeic acid (2.1 times) and PG (1.3 times) were also superior (Porter et al., 1977). The caffeic acid result was of interest, since it is one of the phenolic acids in potato tuber. Flavonol glycosides present in potato tubers, such as myrcetin, quercetin, and kaempferol, were not effective natural antioxidants. These experiments showed that BHA was by far the most effective antioxidant in a silica system that simulated dry membranes. Model systems of microcrystalline cellulose, glycerol, and methyl linoleate (Labuza er al., 197 1) showed PG to be more effective than BHA, but only at water activities above those typical for dried foods. Improvements in shelf life with antioxidants in model systems consisting of methyl linoleate, glycerol, and microcrystalline cellulose at a,. 0.11 and 0.75 were reported by Ragnarsson et al. (1977). It was found that the primary antioxidants, BHA and BHT, gave significant protection in a temperature range of 25-45°C when compared with PG and tocopherol. These authors also reviewed the value of procedures referred to as accelerated shelf life tests (ASLT), in which the acceleration parameter was a substantial increase in temperature. They

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D.HADZIYEV AND L. STEELE

stressed the fact that predictions of room temperature shelf life based upon a single high-temperature ASLT could not be done with any degree of confidence unless the reaction rate increase for a 10" temperature rise was the same for the sample containing the antioxidant as for the control in which antioxidants were omitted. For a low water activity system (a, 0.1 I), the normalized induction period (the time required for 3% of the linoleate to oxidize, divided by the time needed for a control) was 20 for BHA and 15 for BHT at 45°C. At room temperature the values were more than doubled. This illustrated that a single ASLT study at 45°C could lead to a significant underestimation of the shelf life extension at room temperature, and, consequently, to a very significant overuse of antioxidant. Activation energy (E,) values were obtained for model systems used by Ragnarsson et al. (1977). The E, for acontrol at an a, of 0.11 was 13 kcaVmole. In the presence of primary antioxidants, it increased to about 20. Ascorbic acid, instead of increasing the E , of lipid oxidation, appeared to decrease it. Nevenheless, it was concluded that, in the presence of effective primary antioxidants in the range of 25-45"C, at least part of the decreasing effect of the antioxidants on the oxidation rate occurred as a result of a rise in the E, for lipid oxidation. Additional relevant data about the effects of antioxidant treatments on flavor quality and stability of dehydrated mashed potatoes were reported by Sapers et al. (1975). Since efforts to stabilize the membrane lipids in a mashed potato system must contend with the problem of dispersing fat-soluble antioxidants in a medium containing close to 80% water, it was suggested that antioxidants in flake processing should be added as components of emulsions containing other ingredients or as alcoholic sprays. Levels of added BHA and BHT were 55-60 ppm (dry weight). Drying the mash on a single drum dryer reduced the levels to only 13-20 ppm, regardless of the method of antioxidant addition. This corresponded to a recovery of only 15-35%, which was still better than the 10% experienced in potato granule production in which a fluid bed dryer was used. The initial antioxidant losses were typical for dehydrated mashed potato processes, and resulted mostly from volatilization and steam distillation during the addition of antioxidants to the hot mash and during drying. However, additional losses of antioxidants were encountered during storage of flakes in air at 23°C for up to 1 year. Under these conditions the losses of BHA were negligible, while those of BHT amounted to 14-22%. The method of antioxidant addition had only a small effect on the storage stability of air-packed flakes. Mean flavor scores obtained by a taste panel complemented the results of gas chromatographic analysis of oxidation products in headspace and in volatiles isolated by steam distillation. These scores were the lowest in flakes to which BHA and BHT were applied by spraying and were the highest when BHA and BHT were added in an emulsion. After 6 months of storage, the oxidation product levels increased by the same amount regardless of

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method of addition, while after 1 year the levels were highest in the samples to which BHA and BHT were applied as an alcoholic spray and in those where they were applied in a corn oil solution using an aerosol sprayer. Neither the mean flavor scores nor the volatile oxidation product levels appeared to correlate with initial or final amounts of BHA and BHT. Indication that antioxidant concentration was inversely related to oxidation product levels was observed only during the six to twelfth months of storage. Sapers et al. (1975) also tested the potential antioxidant value of quercetin and caffeic acid, both of which are present in raw potato tuber. Their antioxidant activity might be due to an ability to complex trace metal ions in potato, and/or to action as true primary antioxidants. When quercetin was added to a hot mash in an ethanolic spray and then the mash was drum dried, the recovery was about 70%. Moreover, the amount in flakes did not change after 1 year of storage in air at 23°C. Stability data for potato flakes containing quercetin, caffeic acid, or their combination with BHA and BHT indicated that all samples were initially satisfactory with respect to flavor scores and levels of volatile oxidation products. However, flakes containing just quercetin or caffeic acid alone deteriorated during storage twice as much as the samples containing BHA and BHT. This poor performance might be due to low mobility in the dehydrated system, or to the use of an inadequate concentration. However, higher concentrations probably could not be used because of bitter flavor and the objectionable yellow color of quercetin. Flakes containing quercetin in combination with BHA and BHT, with application in an ethanolic spray, had a flavor score after 6 months storage that was higher than flakes with BHA and BHT alone, and there were lower levels of volatile oxidation products after 1 year. This finding alone warrants further research in order to assess the potential value of such combinations. Quantitative determination of phenolic antioxidants in dehydrated mashed potatoes, though being standardized, is still the subject of further improvement. The method of Filipic and Ogg (1 960) for determination of BHA and BHT was based on reconstitution of dehydrated samples and recovery of antioxidants by steam distillation. BHA was determined colorimetrically with 2,6dichloroquinonechloroimide according to Gibbs (1 927). The BHT component was determined by color development with 2,2'-dipyridyl and iron (11) chloride (Emmerie and Engel, 1938). Other colorimetric procedures were proposed by Anglin et al. (1956), Lazlo and Dugan (1961), and Sloman et af. (1962). However, as pointed out by the latter authors, most of the methods for BHA and BHT determination, except those based on gas-liquid chromatography (GLC), are suitable only at levels above 10 ppm. Buttery and Stuckey (1961) extracted dehydrated potato granules with petroleum ether. The extract was concentrated, and BHA and BHT were determined by GLC on columns packed with 20% Apiezon L on fire brick. This method, although sensitive, had its drawbacks-a long extraction time and need

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for a careful concentration step. Schwecke and Nelson (1964) described a method which was not only sensitive, but accurate and rapid. Dehydrated mashed potatoes were extracted by percolation with diethyl ether. Di-BHA (3,4-ditertbutyl-4-hydroxyanisole)was added as an internal standard then the eluate was concentrated and an aliquot was analyzed by GLC at 150°C on a column packed with 2% SE-30 and 1% Tween-80 coated on Chromosorb W (80 mesh). The average recovery of antioxidants from potato granules was 98.2%. Since contamination of the column by potato lipids may be a problem in analysis by GLC, a short precolumn of siliconized glass wool located in the sample injection port block was suggested as a solution (Hartman and Rose, 1969).

VIII.

SOME CHARACTERISTICS OF RECONSTITUTED GRANULES A.

REHYDRATION RATES

Rehydration rates of freshly processed add-back and freeze-thaw granules were found to be dependent on the initial moisture content of the granules (Jericevic and LeMaguer, 1975). A water content below 5.2% led to a marked increase in the rate of rehydration, while no variation was observed above this value. Homogeneous rates of rehydration were best achieved at 8% moisture, a level which would not modify the product quality upon storage. Ooraikul (1977b) observed that granules stored for at least 6 months at room temperature reconstituted much more slowly than freshly processed granules. It was suggested that this rehydration change involved a molecular rearrangement of the granule constituents, especially starch, protein, and pectin. Rehydration rates of granules also depend on the ratio of free to retrograded starch. The more solubilized free starch, the higher the rehydration rate. Pertinent data for commercial scale conversion of retrograded into free starch, and rehydration rate measurements were provided by Purves and Snively (1975).

B.

TEXTURE

The texture of reconstituted dehydrated mashed potatoes has a major influence on consumer acceptance. Mullins et al. (1957) attempted to evaluate consistency and pastiness by measuring the diameter of a mashed potato ball falling upon a smooth surface from a given height. Smith and Davis (1963) used a modified L.E.E.-Kramer shear press to record the textural changes of reconstituted flakes, while Voisey and deMan (1970) and Voisey and Dean (1971) measured torque and energy required to mix the reconstituted flakes. As stated by Ooraikul (1974), most of these attempts actually measure overall textural quality, the character of which is a complex combination of several attributes such as firm-

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ness, glueyness, and smoothness. The objective glueyness, as determined by texturometer, correlated strongly with both subjective glueyness and overall textural quality. Firmness did not appear to be an indicator of the textural quality of mashed potatoes. However, bulk density, which is not a direct textural parameter, correlated relatively well with overall textural quality scores. Density appeared to correlate positively with objective glueyness, and negatively with objective firmness. According to Stadler and Schaller (1971, 1972a,b), the major properties governing the texture or flow behavior of reconstituted potato granules are the particle size and its frequency distribution ( A ) , the number of ruptured cells and concentration of extracellular starch ( B ) , and the water sorption capacity ( C ) . Reconstituted granules did not exhibit a pseudoplastic flow behavior. The thixotropic property of the reconstituted mash was dependent on the amount of dry matter. Thixotropic breakdown, expressed as log of time vs dry matter content, was linear. Simple and partial correlation analysis of parameters A , B, and C with instrumentally measured consistency (D),sensory consistency ( E ) , and sensory adhesiveness revealed a wide and insignificant simple correlation between A and the textural parameters B, C , and D. However, a highly significant correlation was found between C and D. In addition, B was found to be an objective measure of adhesiveness. The finding of a highly significant correlation between D and E suggested a method for reliable and practical quality control of reconstituted mashed potatoes.

IX. RESEARCH NEEDS The production of dehydrated potato granules is a year-round, well-established industry. The commercially used add-back processes are challenged by many improved techniques for which some chemical and textural aspects are fairly well understood, but sound studies on commercial viability are lacking. Further growth of the industry is envisaged, since the use of granules in the form of reconstituted mashed potatoes has been diversified by such products as extruded French fries, balls, rings, food bars, “Pringles,” etc. Consequently, the snack food industry can be expected to demand tailoring of granules according to their needs: size and form of granules; cell wall characteristics; amount of soluble free starch; rehydration rate; and oil uptake. Nevertheless, aspects of granule processing per se need further research. Prevention of rancidity through more effective use and formulation of antioxidants is a major problem. Another is maintenance of consistent textural qualities, which necessitates further understanding of the factors governing the thickness and integrity of cell walls, as well as the role of intercellular matrix constituents such as the quantity and quality of pectic substances and hemicellulose. The effect of

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calcium on the texture of the products needs further clarification. Changes induced in protein by processing and subsequent possible participation in intercellular cohesion and flavor enhancement should be investigated. Also required are data on changes in protein and other constituents during granule aging, an important step in the production of some snack foods.

ACKNOWLEDGMENTS The authors are grateful to Miss I. Chung for assistance in the preparation of figures, and to Drs. B. Ooraikul and A. A. Khan for their constructive criticism of the manuscript.

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Haydar, M., and Hadziyev, D. 1973. Pea lipids and their oxidation on carbohydrate and protein matrices. J. Food Sci. 38, 772. Hendel, C. E., Notter, G. K., and Reeve, R. M. 1962. Preparation of dehydrated potatoes. U.S. Patent 3,031,314. Hennig, H. J. 1977. Kemmagnetische Resonanzuntersuchungen zur Rolle der Wasserbindung fur die Struktur des nativen Starkekorns. Staerke 29, 1. Henning, H. J., Lechert, H., and Goemann, W. 1976. Untersuchung des Quellverhaltens von Starke mit Hilfe der Kemresonanz-impuls-spektroskopie.Staerke 28, 10. Hodge, J. E. 1953. Chemistry of browning reactions in model systems. J. Agric. Food Chem. 1, 928. Hoff, J. E. 1972. Starch “swelling pressure” of cooked potatoes. J. Agric. Food Chem. 20, 1283. Hoff, J. E. 1973. Chemical and physical basis of texture in horticultural products. HorrScierice 8, 108. Hoff. J. E., and Castro, M. D. 1969. Chemical composition of potato cell wall. J. Agric. Food Chem. 17, 1328. Hoff, J . E., Jones, C. M., Sosa, M. P., and Rodis, P. 1972. Naturally occumng crystals in the potato: Isolation and identification as a protein. Eiochem. Biophys. Res. Commun. 49, 1525. Hollis, F . , Jr., and Borders, B. 1965. Process for preparing dehydrated potatoes. U.S. Patent 3,220,857. Hughes, B. P. 1958. Amino-acid-composition of potato protein and of cooked potato. E r . J. Nurr. 12, 188. Hutchings, R. W., and Stringham, C. H. 1971. Method of agglomerating dehydrated potatoes. U.S. Patent 3,565,636. Hyde, R. B. 1962. Variety and location effects on ascorbic acid in potatoes. Food Res. 27, 373. Institute of Food Technologists. 1975. A scientific status summary by the Expert Panel on Food Safety and Nutrition and Committee on Public Information: Sulfites as food additives. Food Technol. 29, 117. Irmiter, T. F., and Rubin, G. 1962. Preparation of dehydrated cooked mashed potato product. U.S. Patent 3,027,264. Jadhav, S., Steele, L., and Hadziyev, D. 1975. Vitamin C losses during production of dehydrated mashed potatoes. Lebensm.-Wiss. + Technol. 8, 225. Jaska, E. 1971. Starch gelatinization as detected by proton magnetic resonance. Cereal Chem. 48, 437. Jaswal, A. S . 1969. Pectic substances and the texture of French fried potatoes. Am. Potato J . 46, 168. Jaswal, A. S. 1973. Effects of various processing methods in free and bound amino acid contents of potatoes. Am. Potato J. 50, 86. Jericevic, D. 1976. Sorption properties of potato granules. M.Sc. Thesis, University of Alberta, Edmonton, Alberta, Canada. Jericevic, D.,and LeMaguer, M. 1975. Influence of the moisture content on the rate of rehydration of potato granules. Can. Insr. Food Sci. Techno/. J . 8, 88. Jericevic, D., and Ooraikul, B. 1977. Influence of the processing on the surface structure of potato granules as viewed by SEM. Staerke 29, 166. Johnson, A. E . , Nursten, H. E., and Williams, A. A. 1971. Vegetable volatiles. A survey of components identified. Part 11. Chem. Ind. (London) p. 1212. Johnston, F. B . , Urbas, B., and Khanzada, G. 1968. Effect of storage on the size distribution and amylose/amylopectin ratio in potato starch granules. Am. Potaro J. 45, 315. Kainuma, K . , and French, D. 1972. Naegeli amylodextrin and its relation to starch granule structure. 11. Role of water in crystallization of B-starch. Eiopolymers 11, 2241. Kaiser, K . P., and Belitz, H. D. 1971. Proteinaseinhibitoren in Lebensmitteln. IV. Vorkommen und

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Woodman, J. S., and Warren, D. S. 1973. Distribution of cell wall components in potato tubers. A new titrimetric procedure for the estimation of total polyuronide (pectic substances) and its degree of esterifxation. J . Sri. Food Agric. 24, 769. Zaehringer, M. V., Cunningham, H. H., and Sparks, W. C. 1966. Sugarcontent andcolor of Russet Burbank potatoes as related to storage temperature and sprout inhibitors. Am. Potatof. 43,305. Zirnrnermann, H. J., and Rosenstock, G. 1976. Proteingehalt, Proteinmuster, Peroxidase- und Malatdehydrogenase-isoenzymrnuster wahrend der Entwicklung und Lagerung der Knollen von Solunum tuberosum L. Biochem. Physiol. Pflanz. 169, 321.

ADVANCES

IN FOOD RESEARCH. VOL.

25

XYLITOL AND ORAL HEALTH KAUKO K. MAKINEN* Department of Biochemistry, Institute of Dentistry, University of Turku, Turku, Finland

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Xylitol and Dental Caries.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. IV. V.

VI. VII. VIII.

A. Properties of Xylitol and Cariogenic Sugars in Relation to Dental Caries B. Lowered Cariogenicity of Foods Containing Xylitol . . . . . . . . . . . . . . . . . Microbiological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . In Vitro Plaque Studies.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . Xylitol and the Exocrine Glands . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . A. Preliminary Findings in Feeding Studies . . . . . . . . . . . . . . .. . . . . . . . . . B. Sialic Acid and Sialoproteins . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . Xylitol and Periodontal Diseases . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . Mechanism of Action of Xylitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Research Needs.. . . . . . . . . . . . . . . . . . . . . . . . . . ............ .......... . . . . . . . . . . . .. . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

I37 139 139 139 147 149 149 149 150 152 153 156 156

INTRODUCTION

The health of the oral cavity is an inseparable part of the general health of man. Physicians, dieticians, and nutritionists should take a serious look at the effects of improper nutrition on oral tissues, viz, the teeth, periodontal tissues, oral mucosa, salivary glands, etc., because in all peroral nutrition the oral tissues are first to react. It has been customary to show sucrose to be a decisive etiological factor in dental caries, but it has also been shown to contribute indirectly to the development of other pathological processes in the mouth (for example, in periodontal diseases). Other dietary carbohydrates (glucose, fructose, starch, etc.) are also cariogenic but the consumption of any of these carbohydrates per se should not be considered detrimental to the oral health. A decisive factor is the *Present address: Department of Biochemistry and Biophysics, Texas A. & M. University, College Station, Texas 77843.

137 Copyright 0 1979 by Academic Press. Inc All nghb of reproduction in any form reserved ISBN 0-12-016425-6

138

KAUKO K. MAKINEN

manner in which they are consumed: the frequency and form of intake and the properties of other dietary ingredients. The periodontal diseases most likely result from the overgrowth of the bacterial plaque and no specific pathogens have yet been shown to cause these diseases, although plaque organisms such as Bacteroides melaninogenicus may be particularly damaging (Socransky, 1970). The periodontal tissues are under a continuous repair process and disease may be a simple accumulative process. In dental caries several pathogens have been demonstrated. They belong to the genera Streptococcus, Lactobacillus, and Actinomyces. A particular organism, S . mutans, has been the focus of active research and has been suggested to be associated with smooth surface caries. Each tooth site (interdental spaces, fissures, gingival margin, etc.) forms its own local ecosystem characterized by special microbial and biochemical determinants. Consequently, caries at these tooth sites are most likely initiated as a result of the action of different microorganisms and it is quite possible that the role of S. mutans has been exaggerated. In all oral diseases, however, man’s innate defense mechanisms play a very important role (MacFarlane and Mason, 1972). Impaired protective ability of these mechanisms, which are mostly related to salivary components, may lead to an unbalanced situation between the host and plaque bacteria with concomitant pathological changes in the former. The innate defense mechanisms may not be able to maintain the required balance if dietary habits are poor or if the oral hygiene methods are insufficient. Both anatomical and genetic factors are also involved and an insufficient supply of fluorine during fetal and early childhood development also contributes to the final resistance of the oral tissues. The preceding comments thus indicate that at least dental caries, but to a certain extent periodontal diseases as well, are multifactorial diseases. The causal factors include diet, oral hygiene, fluorine prophylaxis, dental plaque, saliva with its defense mechanisms, hereditary factors, and the oral tissues themselves. The current use of sucrose and certain other fermentable carbohydrates has made it necessary to search for substitutes that would not be cariogenic. As the main chemical properties of sucrose that make it damaging under certain conditions are indirectly related to bacterial adhesion and bacterial growth, any substitute should have properties that influence these phenomena. In addition, direct stimulatory effects on saliva should also be considered. Recent extensive clinicochemical human trials suggest that xylitol is a promising sucrose substitute. Sorbitol, mannitol, and hydrogenated hydrolysis products of starch have been used for some years for this purpose but they have not been totally effective. The present review deals with results obtained in recent clinicochemical xylitol studies on humans and experimental animals. The article also elucidates the physiological and chemical factors that underlie the encouraging results obtained with this sucrose substitute.

XYLITOL AND ORAL HEALTH

139

II. XYLITOL AND DENTAL CARIES A.

PROPERTIES OF XYLITOL AND CARIOGENIC SUGARS IN RELATION TO DENTAL CARIES

The biological processes of bacterial plaque that contribute to the incidence of dental caries include glycolysis, which produces, from suitable hexose-based sugars, lactic acid. Other bacterial fermentations can also be involved and the dietary carbohydrates may additionally yield, via bacterial syntheses, extracellular polysaccharides which contribute to the adhesion of bacteria to surfaces. This in turn leads to plaque growth. These factors are shown in Table I. The acids formed at the plaque interface may cause demineralization of the hard tissue. The chemical reactions shown in Table I chiefly require six-carbon skeletons as substrate. This fact stems from the evolutionary preference of human plaque microorganisms for this type of carbohydrate. Xylitol, being a sugar alcohol of the pentitol type, has quite different effects on the physiology of oral microorganisms, as presented in Table 11. The net effect of the bacteriophysiological properties of xylitol is that the time periods with very little or no risk of caries initiation are noticeably prolonged during and immediately after peroral administration of xylitol in a suitable form. Although pentitols are not effectively metabolized by plaque bacteria, xylitol, for example, is utilized by humans through normal preexisting pathways. This is an important difference between oral microorganisms and the animal organism. The latter benefits from the caloric value of xylitol while to most bacteria xylitol is an inert or inhibitory compound (Makinen, 1978a).

B.

LOWERED CARIOGENICITY OF FOODS CONTAINING XYLITOL

I.

Turku Sugar Studies

Perhaps the best proof of the extremely low or nil cariogenicity of xylitolcontaining foods so far obtained has been in the Turku sugar studies (Scheinin and Mikinen, 1975). This was a series of collaborative studies camed out at the University of Turku, Institute of Dentistry, Turku, Finland, in which human volunteers were placed on strict xylitol, fructose, or sucrose diets for 2 years. The caries incidence in the xylitol group was reduced very strongly, suggesting approximately 90% reduction compared with the sucrose group (Fig. 1). This same reduction in the incidence of dental caries, associated with xylitol, was, however, also obtained in a 1-year chewing gum trial (Fig. 2). In other words, these two studies showed that both the full substitution, involving consumption of approximately 70 gm of xylitol per day and partial substitution of sucrose with

140

KAUKO K. MAKINEN

TABLE 1 BIOCHEMICAL PROPERTIES OF DENTAL PLAQUE INVOLVED IN THE ETIOLOGY OF DENTAL CARIES Cariogenic traits of plaque and carbohydrates

Significance of the microbial products formed

I . Glycolysis C,H,zO,

1 . Dissolution of the hydroxyapatite structures

+ CH,CH(OH)COOH

Glucose or other n(CG)sugars ( n

3

1)

2. Other fermentations Propionic acid fermentation Acetic acid fermentation Mixed-acid fermentation Fermentations forming carboxylic acids capable of chelation 3 . Synthesis of extracellular polysaccharides CizHmOit Sucrose" GTb

I

O-W2

0-CH)

"

OH

oH

~OH

H

z

0-CH

OH

I

"-*"_

L ^.. " .

1. Adhesive factors (to enamel, cement, etc.,

and to each other) 2. Diffusion barriers (against the rinsing effects of saliva)

forn other organisms ~ 3. Nutrients ~ ~

4. Protective layers against oral defensive

O-CH2

factors DylHH W OH I

OH

1. As above 2. Chelation of hard-tissue metal ions

a'$oH

H ~ C H z H OHH H

I

of hard tissues 2. Providing an acid environment for organisms favoring low pH values 3. Action as a nutrient substrate for other plaque bacteria

W

%H

O

H

5 . Inflammatory to periodontal tissues

(C& @,)n dextrans

+

fructose or

(continued)

XYLITOL AND ORAL HEALTH

141

TABLE I-(cuntinued) C b o g e n i c traits of plaque and carbohydrates

Significance of the microbial products formed

4. Synthesis of intracellular polysaccharides

Takes place intracellularly following sugar transport

5 . Formation of microbial intracellular and

extracellular enzymes

Form reserve energy sources during periods of low supply of nutrients (sugars and amino acids). Typical at the interphases of olderdental plaque. The final products differ very much in molecular weight branching points, etc., depending on the specificity of the enzyme, bacterial strain, chemical and physical environment, acceptor molecule involved at the start of the synthesis, substrate concentration. etc. Some of the enzymes may be important in the breakdown of covalent chemical bonds in the organic structure of hard tissues during pathogenesis

Glucose, fructose, and other carbohydrates may partly replace sucrose. G T = glycosyltransferase.

xylitol (6-7 gm per day and subject, or 3 to 4 chicles per day) produced essentially the same result. The 2-year feeding study showed that the consumption of a fructose diet reduced the incidence of caries by approximately 30% (Scheinin and Mainen, 1975). Some scientists have been under the impression that this reduction was primarily on smooth surface caries comprising precarious lesions not recognized as such by the U.S. dental profession, although recognized as caries in several other countries. It has to be emphasized, however, that in the above studies the caries incidence was expressed in several quantitative and qualitative terms, including the conventional ADMSF-index (increment in the number of decayed, missing, and filled tooth surfaces). Irrespective of the way of expressing the

TABLE I1 COMPARISON O F PROPERTIES OF XYLITOL AND SUCROSE THAT ARE IMPORTANT IN DENTISTRY AND NUTRITION PropeflY

Sucrose 1.o

I . Relative sweetness 2. Organoleptic properties

Good

3. Solubility in water (20°C) 4. Combustion value

199.4 gm/100 gm H,O 4.06 kcaVgm

5 . Occurrence in nature

Widespread

6. Fermentation in dental plaque 7. Production of organic acids in plaque

Rapid and virtually total Variety of acids via bacterial fermentations

Xylitol I .o Good (the strongly endothermic reaction when crystalline xylitol dissolves in saliva, i.e., cooling effect, is considered an advantage) 168.8 gm/100 gm H,O 4.06 kcaVgm (The true value is most likely about 5% lower, which would mean an advantage) Widespread in the plant kingdom but at lower concentrations than sucrose: values up to 0.3-1.0 g d 1 0 0 gm dry matter. Also occurs in certain mammalian organs (e.g., liver) Very rare Nil or extremely low rate of formation

8. Effect on plaque pH values

9 . Effect on whole saliva (oral fluid) pH values 10. Stimulatory effect on flow of saliva 11. Relation to periodontal tissues

12. Effect on the levels of salivary lactoperoxidase 13. Effect on levels of salivary proteins 14. Effect on the levels of HCO, ions in saliva

Usually produces acidic plaque rapidly (pH below the critical point of 5.5 as regards hydroxyapatite dissolution) Small initial increase (0.2-0.3 units), often during 5-10 minutes, then leveling off to starting values Moderate amounts stimulate as xylitol

May be associated with overgrowth of bacteria at the gingival margin Affects as the regular diet Affects as the regular diet

No remarkable effect

The pH values virtually never fall below 5 . 5 , but rather stay above 6.0 Rather strong rise (even 0.4-0.8 units); the leveling off takes place more slowly Moderate amounts (10-30 g d d a y in unadapted subjects) stimulate as sucrose. In adapted subjects, up to 70 gm may be regarded as moderate D o e s not promote the overgrowth mentioned. No irritation of periodontal tissues found May, under certain conditions, be associated with elevated levels As above May be associated with higher levels, whichindicates better buffering capacity in saliva

144

KAUKO K. MAKINEN

-

l2 X W

n

z

~d

lo

t

8 -

2

6 -

k l-

t .SUCROSE FRUCTOSE XYLITOL

2 m

wa

5

2

0

6

4

8

10 12

14

16

18 20 22 24 MONTHS

FIG. 1. Turku sugar studies. Development of Canes Activity Index in man during consumption of sucrose, fructose, or xylitol diets for 2 years. The Index equals all clinical and radiographic quantitative and qualitative changes, including the cumulative development of DMF (decayed, missing, and filled) tooth surfaces, all the secondary caries reversals, and all qualitative changes in the size of the caries lesions. Xylitol was shown to be essentially noncariogenic. From Scheinin et al. (1975a).

canes increment rate, the reduction in the canes incidence in the xylitol group as compared to the sucrose group exceeded 85%, and a corresponding 30% in the fructose group. Furthermore, although smooth surface lesions would not be included in calculations of caries activity, the appearance and disappearance of such lesions as a function of the sweeteners used, viz sucrose, fructose, and xylitol, should be considered. A smooth surface lesion may lead to more irreversible tissue destruction. It was also shown in a recent article (Mikinen, 1978c) 5 4

-

W SUCROSE CHEWING GUM O - - O XYLITOL CHEWING GUM

3 -

2 -

_-_-_---------*

-0

1

2

3

4

5

6

7

8

9

101112 MONTHS

FIG. 2. Turku sugar studies. Total canes activity following 12-month use of either sucrose or xylitol chewing gum. The ordinate gives the increment of decayed, missing, and filled tooth surfaces, all new secondary caries reversals, and the increment in lesion size of primary and secondary caries reversals. The subjects consumed 4.0pieces of chewing gun1 per day in the sucrose group and 4.5 in the xylitol group. Other diet factors were maintained normal. Even partial substitution of the dietary sucrose with xylitol strongly reduced the incidence of dental caries. From Scheinin er n l . (1975b).

XYLITOL AND ORAL HEALTH

I45

that the strong reduction in the incidence of caries observed at Turku was not caused by the inclusion of precarious lesions in the calculations. The fact that an intermittent exposure of three to four times a day of xylitol chewing gum accomplished the same as a total replacement of sucrose by xylitol in the diet shows that a full substitution is unnecessary in the raajority of cases. These results also suggest that xylitol would act ideally when used in a slowly soluble form (in chewing gum) which simultaneously enables active mastication and salivary flow. In a full substitution much of xylitol would be swallowed without causing these long-lasting local effects. The two studies thus suggested that it was not the total amount of xylitol which was decisive, but rather the continuous, immediately after meal use of chewing gum at a constant frequency. Basing on the results of the 2-year feeding study and the 1-year chewing gum trial, the authors of these studies suggested that xylitol may under certain circumstances be considered therapeutic (Scheinin and Makinen, 1975). These views were supported by biochemical and microbiological results obtained in the 2-year trial: the consumption of a xylitol diet reduced the concentration of lactic acid in dental plaque and mixed saliva (oral fluid) and virtually removed from mixed saliva an invertase-like enzyme group which liberates reducing sugars from sucrose (Mikinen and Scheinin, 1975). The first of these findings indicated reduced chances for dissolution of hydroxyapatite, the main tooth mineral. The second suggests that during the course of the consumption of a xylitol diet, the plaque’s ability to attack sucrose was reduced. For example, dental plaque obtained from xylitol-consuming subjects was characterized by increased nitrogen metabolism compared with plaque obtained from sucrose-consuming subjects (Miikinen and Scheinin, 1975). In the latter case the metabolism of carbohydrates was more prevalent than in the former. The above chemical effects can be regarded as normal consequences in many bacterial cultures (mixed or pure) in which the microbial cells are deprived of their best source of energy. In the case of dental plaque one of the best energy sources is sucrose. Under such circumstances microorgani;ms search for other nutrients available, e.g., proteins, peptides, and amino acids of the medium, with a concomitant increase in bacterial transaminations, peptide bond hydrolysis, and related traits of the nitrogen metabolism. The microorganisms then also may increase the breakdown of salivary glycoproteins and mucins secreted from the salivary glands. It has been observed that the consumption of a xylitol diet is associated with slightly increasing levels of the activity of salivary glycosidases. The enzymes whose levels have been found to increase include a-fucosidase, a-glucosidase, and Pgalactosidase (Makinen et al., 1975b). This is in full accord with the fact that the salivary glycoproteins contain fucose, glucose, and galactose in the prosthetic groups of the glycoproteins. An important function of the salivary glycoproteins is to protect the enamel in the form of a thin (0.001 mm) layer which is adsorbed and/or precipitated onto the enamel surface. This film is called acquired pellicle (Dawes, 1968). For the

146

KAUKO K. MAKINEN

subsequent discussion it is important to note that the carbohydrate side chains in the salivary glycoproteins often bear sialic acid (N-acetylineuraminic acid) as the terminal carbohydrate. Although sucrose seems to be most effective in producing caries, glucose and fructose also cause some damage, although usually at a lower rate. The particular chemical properties which make sucrose especially cariogenic are in part related to the special energy of hydrolysis of this disaccharide. The enthalpy of the reaction, AH, is about -28 kJ per mole. Other common disaccharides yield lower values. The energy mentioned is built in the glycosidic bond of the sucrose molecule and is partly utilized by cariogenic microorganisms in their production of extracellular polysaccharides. This biosynthesis thus proceeds without phosphorylated energy-rich intermediates. Extracellular sucrase enzymes may in turn facilitate the formation of glucose and fructose, which enter glycolysis. Xylitol does not offer the above energetic and other advantages to cariogenic microorganisms. The strong reduction in the incidence of dental caries in the 2-year study is easy to understand in view of the bacteriophysiological aspects mentioned. Because the same clinical result was achieved in the chewing gum trial as well, additional explanations will be required. These will be dealt with in subsequent paragraphs, and the previously mentioned glycoproteins may play an important role in this context. Numerous in vitro plaque and microbiological studies and in vivo animal studies on xylitol have been carried out. The results of a single in vitro experiment of the above type should not be extrapolated to show any anticariogenic effects of xylitol in humans, but as the number of supporting in vitro and animal studies is rather high, the accumulated total evidence strongly indicates the noncariogenicity, nonacidogenicity, and even anticariogenicity of xylitol under certain fixed conditions. Virtually all published xylitol studies, along with a few unpublished ones, have been reviewed and listed elsewhere (Mikinen, 1976a, 1978a,b). A few pertinent investigations will be mentioned in the subsequent sections. A detailed description of the Turku studies has already appeared (Scheinin and Miikinen, 1975), supplemented by a few generalizing articles (Makinen, 1976b-d; Scheinin, 1976a,b). 2 . pH Telemetry Muhlemann and his co-workers have observed the nonacidogenic nature of xylitol in human use by pH telemetry, which involves pH registrations of interdental areas with a microelectrode placed on a desired tooth site. Xylitol has acted in these experiments as an inert carbohydrate that is not to any significant extent, or at all, attacked by plaque bacteria. Xylitol does not cause pH drops in this type of experiment (Hassel, 1971; Miihlemann et al., 1977).

147

XYLITOL AND ORAL HEALTH FISSURE

CONTROL

P

(BD)

B D + 2000 S ED + SUC.

CARIES

CHOCOLATE

ED + FRU. CHOCOLATE

BD + SOR. CHOCOLATE BD+ XYL. CHOCOLATE

YIG. 3. Effect of various sugars on rat caries. Caries incidence in the lower jaws of rats after Irogrammed feeding o f various chocolates for 6 weeks. The concentrations of sucrose and sucrose substitutes were 10-30% in the basal diet. The arithmetic means and the standard deviations are indicated. BD = Basal Diet; 2000 S = a cariogenic diet; SUC = sucrose; FRU = fructose; SOR = sorbitol; XYL = xylitol. From Gehring and Karle (1974).

3 . Studies on Rat Gehring and co-workers have clearly demonstrated the noncariogenicity of xylitol in rat experiments (Gehring and Karle, 1974). For example, rats fed a cariogenic diet (Fig. 3) developed the expected caries incidence. When xylitol chocolate was added to the basal diet, the caries incidence remained unchanged.

111.

MICROBIOLOGICAL ASPECTS

To date the only long-term human clinical trial involving an in vivo microbiological follow-up study during continuous consumption of xylitol was carried out in connection with the Turku sugar studies. The consumption of xylitol did not affect the major microbial categories occurring in dental plaque and mixed saliva (Lamas et al., 1975). On the other hand, the mean values of viable S. mutans in plaque were lower in the xylitol group than in the sucrose or fructose groups throughout the study (Gehring et al., 1975). Furthermore, the geometric and arithmetic means of the colony-forming units on selective Rogosa S.L. agar were significantly lower in the xylitol group than in the fructose and sucrose groups, A reduction of the acidogenic oral flora was observed particularly in the xylitol group. During the course of the study, no evidence was obtained of microbial adaptation or mutation enabling acidogenic decomposition of xylitol

148

KAUKO K. MAKINEN

(Scheinin and Makinen, 1975), nor have any phenomena been observed after 4.5 years of continuous consumption of xylitol (Mikinen and Virtanen, 1978). One of the working hypotheses of the Turku sugar studies was that the human oral microorganisms would gradually become adapted to xylitol use. However, no adaptation to xylitol use was detected. During consumption of higher amounts of xylitol in place of sucrose or other fermentable sugars, the oral micoorganisms started to use extracellular proteins, peptides, and amino acids as a source of energy, with concomitant changes in the nitrogen metabolism, as mentioned previously. As discussed elsewhere (Mikinen, 1976b, 1978a), adaptation, from the microorganisms' point of view, may not be necessary as long as easily available and water-soluble hexose-based sugars are available in the diet. The nonfermentability of xylitol in human dental plaque seems to be genetically fixed, for reasons of evolutionary expediency. An illustrative example of the strong diet-dependent changes in the incidence of certain microorganisms in the human oral cavity is shown in Fig. 4. The subject, who first consumed a fructose diet for 1 year, showed the colony5000 4000

3000

20

2000

; 1000

15

0

v

v

0

z

cn

LL

500 400 300

10

2 Q

200

5

100 0

-

0

5

10

15

20 MONTHS

0

FRUCTOSE -XYLITOL-

(---

)

(- - - --)

FIG. 4. Turku sugar studies. The relationship between diet and the incidence of salivary microorganisms and dental caries. A female subject was on a strict fructose diet for 1 year, followed by the consumption of a strict xylitol diet for another year. The figure gives the total salivary colonyforming unit values (CFU) on Rogosa S. L. agar on a logarithmic scale, and clinically and radiographically detected dentine caries lesions, as well as newly filled surfaces. The change of diet resulted in a steep decrease in the levels of the parameters investigated. From Lamas ef d.( I 975).

XYLITOL AND ORAL HEALTH

149

forming unit values indicated. When fructose was replaced with xylitol, there was a very sharp decrease in the incidence of microorganisms in saliva. It is interesting to observe that when the diet was changed, the incidence of dental caries also began to decrease. The DMFS values finally showed a clear tendency to decrease below the maximum previously attained. These types of changes led the authors of the Turku studies to ascribe therapeutic and anticariogenic claims to xylitol (Scheinin and Miikinen, 1975). Other microbiological studies have shown that Actinornyces viscosus, a typical oral microorganism which may be associated with root caries, does not ferment xylitol (Noguchi and Muhlemann, 1976). Another microbiological investigation showed that, of more than 200 oral bacterial strains, representing 10 oral genera, none used xylitol (Havenaar et al., 1978). Xylitol was not shown to promote the growth of oral Candida (Makinen et al., 1975~). After a series of mutations, microorganisms outside of the human oral ecosystem may use xylitol even as a novel carbon. In these cases xylitol often uses transport systems initially developed for the transport of other carbohydrates across cell membranes (Makinen, 1978a).

IV. IN VITRO PLAQUE STUDIES Labeled xylitol binds to human dental plaque only to a very limited extent (Mikinen, 1976a; Makinen and Rekola, 1976). This may be an important finding, as the sugar transport across cell membranes usually requires binding of the carbohydrate molecule to the specific recognition sites of a cell wall (Kaback, 1970). The human oral microorganisms possess such sites to a very small extent only. It can furthermore be assumed that the observed low binding of xylitol resulted from nonspecific reactions. The activity levels of plaque xylitol dehydrogenase are constantly very low or nil, whereas those of sorbitol dehydrogenase are understandably higher (Makinen and Scheinin, 1975; Makinen and Virtanen, 1978). These enzymes are often regarded as the first enzymes in the possible decomposition of the polyols in dental plaque. However, certain oral bacteria may, as the first step, phosphorylate mannitol and sorbitol (Stegmeier et al., 1971). Gulzov (1976) has also shown the low ability of model systems to produce acids from xylitol.

V. A.

XYLITOL AND THE EXOCRINE GLANDS

PRELIMINARY FINDINGS IN FEEDING STUDIES

The first finding that suggested that sugar alcohols, compared with sucrose, would cause different effects on the biochemistry and physiology of the exocrine glands was obtained in the Turku sugar studies: a xylitol diet was associated with

150

KAUKO K . MAKINEN

clearly higher levels of the salivary lactoperoxidase than the sucrose or fructose diets (Makinen et al., 1975a, 1976). Lactoperoxidase is an oxidative enzyme which is a part of the body’s innate defense mechanisms. This enzyme may be important for the antimicrobial properties of such secretions as saliva, lacrimal fluid, and milk (Morrison and Steele, 1968). The enzyme needs thiocyanate ions (SCN-) and hydrogen peroxide as cofactors. These compounds are present in saliva and plaque at low concentrations and the enzyme system thus formed is capable of inhibiting pathogenic and apathogenic oral bacteria. For example, several species of Lactobacillus, Streptococcus, and Corynebacterium are sensitive to the lactoperoxidase system. The above finding suggested that the xylitolinduced elevated lactoperoxidase levels in saliva and the anticariogenic properties of xylitol are partly interrelated phenomena. The above studies in humans were followed by other experiments on monkeys, cows, and rats. The consumption of moderate amounts of xylitol for 3 days significantly increased the activity levels of monkey (Macacu mufutta) parotid and submandibular lactoperoxidase (Mikinen et al., 1978). Simultaneously, the levels of protein and a-amylase were also increased (Bird et al., 1977). Stimulation of parotid saliva with xylitol fruit pastils caused a slightly higher lactoperoxidase activity in human parotid saliva than the sucking of corresponding hexose-based pastils (Harper et al., 1977). Cows fed 0.5 kg/day of a polyol mixture containing 10% xylitol (w/w) showed slightly higher milk lactoperoxidase activity compared with feeding a molasses or control ration, but a 2-day intake of xylitol (0.5 gm per kg body weight and day) did not cause this effect in lactating mothers (unpublished results from this laboratory). Homogenates of the submandibular and lacrimal glands of the rat showed slightly higher lactoperoxidase activity following drinking of xylitol-sweetened (4%) water for 2 months compared with drinking a correspon2ing glucose solution (unpublished results from this laboratory). Due to the preliminary nature of these studies, verifying data are clearly required before further conclusions are drawn about the relationship between polyol feeding and exocrine gland function. The above results clearly suggest that the relation of sugars, sugar alcohols, and dietary ingredients in general to exocrine gland function is interesting and deserves investigation. It is most likely that the above selective enzyme changes only represent normal and physiological responses to the various dietary ingredients. It is unlikely that this line of research would reveal any pathologically alarming findings. Perhaps the best evidence for this was obtained in the 2-year study previously mentioned, which clearly demonstrated that the safety of moderate amounts of oral xylitol is indisputable. B.

SIALIC ACID AND SIALOPROTEINS

Sialic acids are a group of N - and 0-acyl derivatives of the 9-carbon 3deoxy-5-amino sugar neuraminic acid. The sialic acids are ubiquitously dis-

0

/CH3

\CH I H2N - C - H

c=o

e

ooc

I

c ooe

I L

HO-C-H I H-C-OH I

H - C - OH I CH20H

D -MANNOSAMINE

H2

\

OOOC

/c\

C HO-C-H

//

I H2N-C-H

HO - C

/

-

I

H-C-OH I

HO’I

‘C-H

0 H,r-C

I-H

‘CL I H-C-OH H - C -OH

I

I

CH20H

CHZOH

FORM PYRANOSE NEURAMINIC ACID

(I)

N -ACETYLacetylation J

I

H-C-OH

KETO

2; ‘C’

FORM

NE UR AM1NIC ACID (SIALIC ACID)

1 GLYCOPROTEINS MUCOPOLYSACCHARIDES

152

KAUKO K. M K I N E N

tributed in tissues in the form of mucopolysaccharides and mucoproteins, including salivary macromolecules. The sialic acid from human plasma has the structure shown in (I). The biosynthesis of N-acetylneuraminic acid takes place as an aldol condensation of the N-acetylhexosamine and pyruvic acid. Certain microorganisms produce enzymes (neuraminidases) which can effect hydrolysis to these products. Sialoproteins and sialopolysaccharides play an important role in the host defense mechanisms in the oral cavity. Sialoproteins contribute to the formation of the acquired pellicle and some of them may be involved in the elimination of microorganisms from the mouth. The study of the role of sialomacromolecules in oral and other diseases has intensified, and, as to the oral conditions, it has been suggested that dietary carbohydrates exert a selective effect on the secretion of such compounds. For example, polyol-stimulated whole saliva contained more sialic acid than paraffin-stimulated whole saliva. Because this type of effect is not readily seen in parotid saliva, it can be assumed that the submandibular and perhaps sublingual glands were specifically affected (unpublished results from this laboratory). Such findings should also be interpreted as normal physiological responses to changes in the diet, not as pathological consequences.

VI. XYLITOL AND PERIODONTAL DISEASES The consumption of a xylitol diet was not shown to cause any periodontal problems in man (Paunio et al., 1975). On the contrary, it can be assumed that xylitol would have advantageous indirect effects on the periodontal tissues in the form of enhanced host defense mechanisms (lactoperoxidase) and its inability to promote plaque overgrowth. As a matter of fact, xylitol has been shown to decrease the growth of dental plaque by approximately 50% compared with sucrose and fructose (Mainen and Scheinin, 1975). Of particular importance in relation to periodontal diseases are the properties of the crevicular exudate which is nearly always present in small amounts in the gingival pockets. Increased inflammatory changes in the periodontal tissues also increase the flow of the exudate. The exudate contains typical inflammatory cells and mediators. The mediators are responsible for the inflammatory reactions on the macroscopic, microscopic, and biochemical levels, and they affect, among other things, the microcirculation in capillaries. Gingival exudate, collected from subjects who were on a xylitol diet, displayed clearly smaller microcirculation velocity values in the hamster cheek pouch microvasculature than exudates obtained from subjects consuming sucrose or fructose (Fig. 5 ) . This finding should be interpreted as showing the insignificant inflammatory qualities of xylitol in peroral administration. The consumption of xylitol seemed to reduce the enzyme content of

153

XYLITOL AND ORAL HEALTH

1400

- 1200

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-

1000

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>

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0

9

600

W

>

400 200

0 0

2 6 6 TIME (rnin) SUCROSE

2 4 6 TIME (rnin) FRUCTOSE

2 L 6 TIME (min) XYLITOL

FIG. 5 . Turku sugar studies. Effect of individual samples of gingival exudate on the microcirculation of hamster cheek pouch (vital microscopy). Gingival exudate was collected by a filter paper method from human subjects who were on a strict diet with respect to the sweetener used (sucrose, fructose, or xylitol). The experiment was performed 12-13 months after the onset of the diets. The paper strips were treated in a buffer, and 10-pl aliquots of the resulting extracts were applied on a suitable area of the microvasculature of the cheek pouch spread on a specimen holder plane of the microscope. The velocity of circulation was determined. Exudate samples obtained from xylitolconsuming subjects caused smaller velocity values than those obtained from other subjects. From Luostarinen er a / . (1975).

exudate compared with sucrose and fructose. The enzymes which were studied include peroxidase (Makinen el al., 1975a), glycosidases (Mikinen et al., 1975b), and aminopeptidase (Paunio et al., 1975). These enzyme findings should be interpreted as showing an increased clearance of inflammatory compounds and microbial enzymes (particularly glycosidases) from the exudate during xylitol consumption.

VII.

MECHANISM OF ACTION OF XYLITOL

The mechanism of action of xylitol in dental caries prevention is rather well known. While a number of details still require intensive studies, the very near future will certainly reveal the most important remaining aspects. The following list presents the cornerstones which, according to the available literature, should be considered in the description of the xylitol effect: (a) The xylitol molecule is shorter than the hexitol molecules that are regularly metabolized by oral microorganisms. The difference of this molecular parameter

154

KAUKO K . MAKINEN

between xylitol and sorbitol is not big (Fig. 6), but in the chemistry of the active site of the microbial enzymes it is decisive. No matter what type of mechanism is involved in the substrate specificity of enzymes involved in the initial breakdown of hexitols, the improper length of the xylitol molecule makes it a poor substrate for most such enzymes. So, for example, xylitol may not be able to bring into effect such specificity mechanisms as lock and key, productive binding, and induced fit. The above not only concerns polyols, but the arrangement of Cs compounds versus C5 compounds is valid in many other cases in the biochemistry of carbohydrates as well. Consequently, the pentitol nature of xylitol is an important ecological chemodeteminant in plaque metabolism. The configurations of the molecules, however, also contribute to their suitability as substrates for bacterial metabolism. It is necessary to indicate that, unlike virtually all oral microorganisms by which xylitol is not metabolized, xylitol is metabolized by the human body. (b) The corollary to the above is: As a consequence of the inability of oral bacteria to metabolize xylitol effectively, it is virtually never converted to acid in human dental plaque: Consequently, the pH values attained at the plaque interface will most likely be on the safe side (above 5.5-6.0; Fig. 7) compared with the situation involving consumption of fermentable carbohydrates. The critical pH value (approximately 5 . 5 ) with regard to hydroxyapatite dissolution will not be reached readily during xylitol consumption.

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(c) Xylitol causes, as do the other sweet carbohydrates so far studied, increased salivary flow rates. With xylitol, however, the pH values reached are more advantageous (approximately 7.2-7.8) than with fermentable sugars and the salivary defense mechanisms act effectively. One of these mechanisms comprises the remineralizing ability of saliva. Saliva is normally supersaturated with regard to enamel (hydroxyapatite, or Caw and phosphate). The calcium and inorganic phosphate concentrations of paraffin-stimulated mixed saliva is usually 10-15 mg/l and 75-210 mg/l, respectively. These concentrations are sufficient to account for the supersaturated state mentioned. The maintenance of higher and constant pH values induced by xylitol (approximately 7.2-7.8) gives the supersaturated state possibilities for the repair of initial demineralized areas. (d) Xylitol can cause, under specified conditions, an elevation of the levels of other salivary defensive factors such as lactoperoxidase or glycoproteins, of which the latter are required in the formation of the acquired pellicle, and possibly other factors. The present data also suggest that the buffering capacity of saliva is maintained at a more effective stage following stimulation with xylitol compared with stimulation with sucrose, i.e., more HCO, ions would be present in the former case.

In summary, the mechanism of the xylitol effect is dual. It partly comprises the nonfermentability of xylitol in the human dental plaque, and partly the stimulation of a number of salivary host defense factors. The latter ones are also dual: there is the possibility of a direct stimulatory effect (e.g., via nerve impulses) and of a systemic effect via the following route: stomach (with possible release of

156

KAUKO K. MAKINEN

gastric hormones), circulation, salivary glands. Indications of both types of phenomenon hhve been obtained.

VIII.

RESEARCH NEEDS

1. Intestinalflora. The effect of the consumption of higher amounts of xylitol on

the composition of the intestinal flora and the biochemistry (vitamin synthesis, etc.) of the microorganisms involved should be studied. 2. Secretion of glycoproteins. The effect of xylitol (and dietary ingredients in general) on the secretion of glycoproteins and mucopolysaccharides from exocrine glands should be better elucidated. 3 . Lowest effective dose. It would be of great value to determine the minimum amount of xylitol, in the presence of various other carbohydrates that still produces a clear protective effect on the oral tissues and particularly dental caries. Severe clinical cases. Particular lines of research should be planned and pursued to demonstrate the possible benefits of xylitol in rampant caries, severe periodontal diseases, xerostomia, and in the treatment of the teeth of diabetic subjects, and as a salivary stimulator in gerontology. Xylitol products. Food technology and clinical sciences should cooperate in the planning of new xylitol products for health care. Such products would be combinations of xylitol with fluorine, vitamins (chew tablets or tonics), other pharmaceutical products, prophylactic tooth pastes, preparations used in endodontics (xylitol plus penetrating detergents in the treatment of carious dentine, etc.), and products aimed at enhancing the salivary flow.

REFERENCES Bird, J. L., Baum, B. J . , Makinen, K . K . , Bowen, W. H . , and Longton, R . W. 1977. Xylitol associated changes in amylase and protein content of monkey parotid saliva. J. Nutr. 107, 1763- 1767. Dawes, C . 1968. The nature of dental plaque, films, and calcareous deposits. Ann. N.Y. Acad. Sci. 153, 102-119. Gehring, F., and Karle, E. 1974. Tierexperimentelle Untersuchungen ubex Zuckeraustauschstoffe und Zuckerzusatzstoffe. Sonderforschungsber. 92 Univ. Wurzburg, B i d . Mundhohle, 1973 p. 192. Gehring, F., Makinen, K . K., Larmas, M., and Scheinin, A. 1975. Turku sugar studies. X. Occurrence of polysaccharide-forming streptococci and ability of the mixed plaque microbiota to ferment various carbohydrates. Acta Odontol. Scand. 33, Suppl. 70, 223-237. Gulzov, H.-J. 1976. Comparative biochemical investigations on the degradation of sugars and sugar alcohols by microorganisms of the oral cavity. I n t . J. Viram. Nutr. Res., Suppl. 15, 348-357.

XYLITOL AND ORAL HEALTH

I57

Harper, L. R., Poole, A. E., and Wolf, S. I. 1977. Xylitol stimulation of lactoperoxidase in human parotid saliva. J . Dent. Res. 56, Spec. Issue A , A62. Hassel. T. M. 1971. pH-Telemetrie der interdentalen Plaque nach Genuss von Zucker und Zuckeraustauschstoffen. Dtsch. Zahnaerrzl. 2. 26, 1 145-1 154. Havenaar, R., Huis in’t Veld, J. H. J . , Backer Dirks, 0.. and de Stoppelaar, J . D. 1978. Microbiological aspects of sugar substitutes. Caries Res. 12, 118. Kaback, H. R. 1970. Transport. Annu. Rev. Biochem. 39, 561-598. Larmas, M., Makinen. K . K., and Scheinin, A. 1975. Turku sugar studies. VIII. Principal microbiological findings. Acru Odonfol. Scund. 33, Suppl. 70, 173-216. Luostarinen, V., Paunio, K., Varrela, J . , Rekola, M., Luoma, S . , Scheinin, A., and Makinen, K . K . 1975. Turku sugar studies. XV. Vascular reactions in the hamster cheek pouch to human gingival exudate. Acfa Odontol. Scand. 33, Suppl. 70, 287-291. MacFarlane, T. W., and Mason, D. K . 1972. Local environmental factors in the host resistance to the commensal microflora of the mouth. I n “Host Resistance to Commensal Bacteria” (T. MacPhee, ed.), p. 64. Churchill-Livingstone, London. Makinen, K . K. 1976a. Microbial growth and metabolism in plaque in the presence of sugar alcohols. Microbiol. Abstr. 2, Spec. Suppl., 521-538. Makinen, K. K . 1976b. Dental aspects of the consumption of xylitol and fructose diets. I n t . Dent. J . 26, 14-28. Makinen, K. K. 1 9 7 6 ~ Long-term . tolerance of healthy human subjects to high amounts of xylitol and fructose: General and biochemical findings. Int. J . Vitam. Nutr. Res., Suppl. 15, 92-104. Makinen, K. K. 1976d. Possible mechanisms for the cariostatic effect of xylitol. I n t . J . Vitam. Nurr. Res., Suppl. 15, 368-380. Makinen, K. K. 1978a. Biochemical principles of the use of xylitol in medicine and nutrition with special reference to dental caries. Experientia, Suppl. 30, 1 - 160. Makinen, K. K. 1978b. Approaches to food modification: Xylitol. Proc., Workshop Cariogenicity of Food, Beverages, Confections, Chewing Gum, pp. 99-1 13. Am. Dent. Assoc. 1977. Makinen, K. K . 1978c. The use of xylitol in nutritional and medical research with special reference to dental caries. Proc., Sweeteners Denf. Caries, 1977. Feeding, Weight & Obesity Absfr. Spec. Suppl., 193-224. Makinen, K . K., and Rekola, M. 1976. Xylitol binding in human dental plaque. J . Dent. Res. 55, 900-904. Makinen, K. K . , and Scheinin, A. 1975. Turku sugar studies. VII. Principal biochemical findings on whole saliva and plaque. Acfa Odontol. Scand. 33, Suppl. 70, 129-171. Makinen, K. K., and Virtanen, K . 1978. Effect of 4.5-year use of xylitol and sorbitol on plaque. J . Dent. Res. 57, 441-446. Makinen, K . K . , Tenovuo, J . , and Scheinin, A. 1975a. Turku sugar studies. XII. The effect of the diet on oral peroxidases, redox potential and the concentration of ionized fluorine, iodine and thiocyanate. Acra Odontol. Scand. 33, Suppl. 70, 247-263. Makinen, K. K., Laikko, I., Scheinin, A,, and Paunio, K. 1975b. Turku sugar studies. XVII. The activity of glycosidases in oral fluids and plaque. Acta Odontol. Scand. 33, Suppl. 70,297-306. . of xylitol on the growth of three oral Makinen, K . K., Ojanotko, A , , and Vidgren, H. 1 9 7 5 ~Effect strains of Candidn albicans. J . Dent. Res. 54, 1239. Miikinen, K. K., Tenovuo, J., and Scheinin, A. 1976. Xylitol-induced increase of lactoperoxidase activity. J . Dent. Res. 55, 652-660. Makinen, K. K., Bowen, W. H., Dalgard, D., and Fitzgerdld, G. 1978. Effect of peroral administration of xylitol on exocrine secretions of monkeys. J . Nutr. 108, 779-789. Morrison, M., and Steele, W. F. 1968. Lactoperoxidase, the peroxidase in the salivary gland. I n “Biology of the Mouth” (P. Person, ed.), p. 89. Am. Assoc. Adv. Sci., Washington, D.C.

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KAUKO K . MAKINEN

Miihlemann, H. R., Schmid, R., Noguchi, T . , Imfeld, T . , and Hitsch, R . S. 1977. Some dental effects of xylitol under laboratory and 0 7 vivo conditions. Caries Res. 11, 263-276. Noguchi, T., and Miihlemann, H. R . 1976. The effect of some carbohydrates on in vitro growth of Streptococcus rnutuns and Actiriornyces viscosus. Schweiz. Monatsschr. Zuhrrheilkd. 86, I 361 1370. Paunio, K., Makinen, K . K., Scheinin, A.. and Ylitalo, K. 1975. Turku sugar studies. IX. Principal periodontal findings. Acra Odonrol. Scund. 33, Suppl. 70, 217-222. Scheinin, A . 1976a. Xylitol in relation to the incidence of dental caries. Int. J . Vitam. Nutr. Res., SUPPI. 15, 358-367. Scheinin, A. 1976b. Caries control through the use of sugar substitutes. Int. Dent. J. 26, 4-13. Scheinin, A., and Makinen, K . !,. 1975. Turku sugar studies. I-XXI. Acta Odontol. Scand. 33, SUPPI. 70, 1-348. Scheinin, A , . Makinen, K . K., and Ylitalo, K. 1975a. Turku sugar studies. V. Final report on the effect of sucrose, fructose and xylitol diets on the caries incidence in man. Acta Odontol. Scund. 33, Suppl. 70, 67-104. Scheinin, A., Makinen, K. K., Tammisalo, E., and Rekola, M. 1975b. Turku sugar studies. XVIlI. Incidence of dental caries in relation to I-year consumption of xylitol chewing gum. Acta Odontol. Scand. 33, Suppl. 70, 307-316. Socransky, S . S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. Dent. Res. 49, 203-222. Stegmeier, K., Dallmeier. E., Bestman, H . - J . , and Kroncke, A. 1971. Untersuchungen iiber den Sorbitabbau unter Venvendung von ' 'C-markierten Substanzen und der Gaschromatographie. Dtsch. Zahnaerztl. Z. 26, 1129- 1134.

ADVANCES IN FOOD RESEARCH. VOL. 25

METABOLIC AND NUTRITIONAL ASPECTS OF XYLITOL REIN0 YLIKAHRI Third Deportment of Medicine, University of Helsinki, Helsinki, Finland

.................................. 111. Metabolism and Metabolic Effects of Exoge ................ A. Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Intermediary Metabolism . . . . . . . . . . . . . . . . . . . C. Effects on Hepatic Metabolism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Effects on the Carbohydrate, Lipid, and Ketone Body Metabolism of the Whole Body . . . . . . . . . . . . . . . ... ............. IV. Use of Xylitol in Nutrition and Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Parenteral Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Diets of Diabetics . . . .................... C. Therapy of Hemolytic -Phosphate Dehydrogenase Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Other Clinical Uses . . .................................. V. Toxicological Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Amounts without Adverse Effects ............................... B. Adverse Effects . . . . . . . . . . . . . ... .................... VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Research Needs ........................... References . . . . . . . . . . . . . ...........................

1.

159 160 162 162 163 164 165 I67 167 168 169 170 170 170 171 174

I76 176

INTRODUCTION

Xylitol is a five-carbon polyalcohol, pentitol, which is widely distributed in nature. Plants and fruits contain relatively large amounts of it but trace amounts of xylitol are also found in animals. The development of gas chromatography and enzymatic techniques during the last 20 to 30 years has markedly increased our knowledge of the physiological and metabolic role of xylitol in the human body as well. I59 Copyright @ 1979 hy AcnAemic Press. Inc. All nghir ot rcprcduclinn in any fnmi rrrerved ISBN 0-1?-0164?5-6

160

R E I N 0 YLIKAHRI

From a metabolic point of view xylitol has a double role in the mammalian organism. It is an endogenous metabolite of the liver but it can also be used as an exogenous nutrient. The physiological role of the endogenous metabolism of xylitol is not yet completely explained, but several grams of xylitol are produced by the liver daily. The liver also has an active enzyme system which is able to metabolize considerable amounts of exogenous xylitol for energy production. As a nutrient xylitol has two main uses. It has been recommended for parenteral nutrition, because it is claimed to have a better anticatabolic action than glucose in conditions where there is insulin resistance (e.g., postoperative and posttraumatic states). This assumption was mainly based on the hypothesis that xylitol is metabolized independently of insulin. Nowadays, however, we know that only the first steps of the xylitol metabolism are independent of insulin. Therefore, the benefits and disadvantages of xylitol in parenteral nutrition are still a matter of great debate. The second nutritional use of xylitol is as a sweetener in the normal diet and in the diet of diabetics. The use of xylitol as a general sweetener is based on its anticariogenic properties. The main argument for its use by diabetics is that it does not disturb the diabetic control. Severe metabolic side effects, even deaths, have been reported after the parenteral use of xylitol. However, due to the slow absorption of xylitol from the intestine, the metabolic effects of parenteral and oral xylitol are quite different. This review considers the metabolic pathways of endogenous and exogenous xylitol and the effects of both parenteral and oral xylitol on the metabolism of the human body. The tolerability and toxicity of the parenteral and oral xylitol are also discussed.

II. METABOLISM OF ENDOGENOUS XYLITOL The concentration of xylitol in the organs of different animals is negligible and only trace amounts of it have been found in urine under physiological conditions (Pitkinen and Sahlstrom, 1968). Therefore, it was not regarded as an intermediate of normal metabolism until the studies of Touster and his co-workers revealed it as a metabolite in the uronic acid cycle (see Touster, 1960, 1974). They studied the pathogenesis of a symptomless metabolic disorder, essential pentosuria, in which L-xylulose is excreted in urine (Touster, 1969). They found that L-xylulose could be reduced to xylitol by a specific NADP-linked dehydrogenase localized in both hepatic mitochondria and cytoplasm (Touster el al., 1956; Arsenis and Touster, 1969). Later it was confmed that this enzyme is defective in essential pentosuria (Asakura el al., 1967; Wang and van Eys, 1970). Touster’s group had also found that xylitol could be further converted to

161

METABOLIC AND NUTRITIONAL ASPECTS OF XYLITOL

D-xylulose by a NAD-linked polyol dehydrogenase (McCormick and Touster, 1975). This enzyme was rather nonspecific and it is located in the cytoplasm of hepatic cells (Smith, 1962). D-Xylulose is phosphorylated to Dxylulose-5-phosphate, which is a metabolite of the pentose phosphate cycle (Touster and Shaw, 1962). When these findings were combined with those concerning the ascorbic acid synthesis it was evident that xylitol was a physiological metabolite of the uronic acid cycle (Bums, 1959; Touster, 1969) (Fig. 1). The exact turnover rate of this cycle and thus the daily production of xylitol are not known, but the pentouric patients excrete 5-15 gm of L-xylulose per day. Thus the daily production of xylitol ought to be of the same order of magnitude (Hollmann and Touster, 1964). The enzymes involved in the uronic acid pathway are found only in the liver (Touster and Shaw, 1962), indicating that there is no endogenous metabolism of xylitol in other tissues. The function of the uronic acid pathway is not completely known. In all animals, excluding primates and the guinea pig, it produces ascorbic acid (Gupta et al., 1972; Touster, 1974). In man, the main function of the pathway is probably to produce glucuronic acid for synthetic processes and detoxification reactions (see Touster, 1974). Thus xylitol seems also to be an endogenous intermediate of a physiologically important metabolic pathway in the human liver.

p

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XYLITOL FIG. 1. Xylitol as an intermediate of the uronic acid cycle.

162

111.

REIN0 YLIKAHRI

METABOLISM AND METABOLIC EFFECTS OF EXOGENOUS XY LlTOL A.

INTESTINAL ABSORPTION

Unlike glucose and galactose, xylitol is not actively transported through the intestinal mucosa. Its absorption is either by passive or facilitated diffusion (Bassler e t a l . , 1966a; Bassler, 1969; Lang, 1971). Thus the absorption of xylitol is much slower than that of glucose, and its exact rate in man is not known. SmaIl doses of xylitol are probably completely absorbed (Bassler, 1969; Lang, 1971) but after large doses (over 50 gm) the absorption is incomplete and some xylitol reaches the colon, causing osmotic diarrhea (Bassler et al., 1962; Lang, 1971; Makinen, 1976; Ylikahri and Leino, 1979). Also, in rats, xylitol easily induces diarrhea (Bassler et al., 1966a), but in dogs the absorption seems to be faster and perhaps even more complete than in man (Kuzuya et al., 1969). Due to slow absorption and rapid metabolism in the liver the blood concentrations of xylitol are low after oral administration (Bassler et a f . , 1962; Ylikahri and Leino, 1979). When 1.0 gm of xylitol per kilogram of body weight was given in one dose the maximal concentrations in blood were less than 1 mMlliter (Ylikahri and Leino, 1979). In dogs some higher concentrations have been measured (Kuzua el al., 1969). A peculiar feature in the absorption of xylitol is an adaptive increase of absorption during prolonged administration (Bassler et a f . , 1966a; Lang, 1971). For a nonadapted subject who has not eaten xylitol previously the maximal LACTATE + PYRUVATE +

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METABOLIC AND NUTRITIONAL ASPECTS OF XYLITOL

163

tolerable dose of xylitol is 20-40 gdday; larger doses cause osmotic diarrhea. Continuous administration of xylitol in increasing doses enhances the maximal tolerable dose both in man and in animals (Bassler et al., 1966a; Dubach et al., 1969; Mikinen, 1976). Even doses greater than 400 gm per day have been tolerated by human subjects without side effects (Makinen, 1976). The adaptive enhancement of the intestinal absorption by continuous administration is unique to xylitol. It is somewhat unexpected, since the absorption of xylitol is a passive process. The mechanism of adaption is not completely understood. It has been suggested that regular intake enhances the metabolism of xylitol in the liver by decreasing the concentration of xylitol in peripheral blood and increasing the concentration gradient between the blood and the intestinal lumen (Bassler et al., 1966a; Lang, 1971). However, the concentration of xylitol in peripheral blood is, in any case, so low that its further decrease can hardly affect the rate of xylitol absorption significantly. Thus the exact mechanism of the adaption of xylitol remains to be elucidated. B.

INTERMEDIARY METABOLISM

Although xylitol can enter almost all cells of an organism (Bassler et al., 1962; Lang 1971) the liver cells are especially permeable (Froesch and Jakob, 1974). In addition, only hepatic cells contain remarkable amounts of enzymes for metabolizing xylitol (Bassler et al., 1962). Some enzyme activity is found also in the kidney and testes, but the liver is by far the most important site of xylitol metabolism (Bassler et al., 1962). Xylitol has theoretically two possible pathways of metabolism in the liver. It could be oxidized to L-xylulose by a specific NADP-linked polyol dehydrogenase or to D-xylulose by a nonspecific NAD-linked polyol dehydrogenase (Fig. 2). The K , value of the NADP-linked enzyme for xylitol is high (3.2 x lop2M ) (Wang and van Eys, 1970), while that of the NAD-linked enzyme of sheep liver is only about 2 x lop4M (Smith, 1962). Thus it is clear that the latter pathway is preferred in the metabolism of xylitol (Froesch and Jakob, 1974). This fact has been confirmed in several in vitro studies (Bassler et al., 1966a; Jakob et al., 1971; Williamson et al., 1971; Froesch and Jakob, 1974). After entering the liver, xylitol is oxidized to o-xylulose by the NAD-linked nonspecific polyol dehydrogenase (Fig. 2). Its metabolism is then linked to the pathway of endogenous xylitol. D-Xylulose is rapidly phosphorylated by D-xylulose kinase to ~-xylulose-5-phosphate(see Froesch and Jakob, 1974), which can be converted to fructose-6-phosphate by the reactions of the pentose phosphate pathway. Three molecules of xylitol yield two molecules of fructose-6-phosphate and one molecule of glyceraldehyde-3-phosphate.These intermediates of glycolysis and gluconeogenesis can be metabolized either to glucose and glycogen or to pyruvate and lactate. Normally the conversion to

164

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glucose and hepatic glycogen seems to predominate (Froesch et al., 1971;Jakob et al., 1971; Froesch and Jakob, 1974).

Only the first metabolic steps are unique to xylitol. Via these steps it is rapidly converted to fructose-6-phosphate, and thereafter the fate of its carbon atoms is very similar to that of glucose (Froesch et al., 1971; Froesch and Jakob, 1974). This is of considerable significance in the clinical use of xylitol. The exact mechanisms controlling the metabolism of exogenous xylitol are not known. The rate-limiting step in the metabolic pathway is the polyol dehydrogenase reaction (Bassler et al., 1966a) which is under no allosteric control. Therefore one important regulatory factor in xylitol metabolism is the activity of the polyol dehydrogenase. To a certain limit (up to 6 mMlliter), the concentration of xylitol entering the liver also influences the rate of elimination (Jakob et al., 1971). Also, the redox state of the hepatic cytosol may affect the rate of elimination, since ethanol, which is known to reduce the hepatic redox state, inhibits xylitol elimination (Ylikahri and Leino, 1979). C.

EFFECTS ON HEPATIC METABOLISM

The metabolism of glucose in the liver is well controlled in several steps in the glycolysis. Perhaps the most important regulatory steps are the inhibition of hexokinase by glucose-6-phosphate and the regulation of phosphofructokinase and pyruvate kinase by the energy state of the cell. The first specific steps of xylitol metabolism are under no allosteric control. Therefore, the rate of xylitol metabolism is mainly determined by the activity of the polyol dehydrogenase and by the concentration of xylitol entering the liver, but the general control mechanisms of energy metabolism do not regulate xylitol metabolism as they regulate glucose metabolism. This rapid and at least partially uncontrolled metabolism affects the whole intermediary metabolism of the liver. Xylitol has been found to cause great changes in the concentrations of several metabolites in perfused rat liver (Jakob et al., 1971; Williamson et al., 1971). Most of these changes can be attributed to the reduction of the hepatic redox state, i.e., the increase in the ratio of free NADH to free NAD during xylitol metabolism. The redox change can be regarded as a primary metabolic effect of xylitol and it is due to rapid production of NADH in the polyol dehydrogenase reaction (see Froesch and Jakob, 1974). The change is greatest in the cytoplasmic compartment of the liver cells because the polyol dehydrogenase is located there (Jakob et al., 1971). From a practical point of view, the decrease in pyruvate concentration and increase in a-glycerophosphate concentration seem to be the most interesting changes. The decrease in the pyruvate concentration may inhibit gluconeogenesis (see Williamson et al., 1969a) and an increase in a-glycerophosphate concentration could stimulate hepatic lipid synthesis (see Fritz, 1961). There are no data about the effects of xylitol on hepatic triglyceride synthesis, but liver perfusion

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studies have clearly shown that xylitol inhibits gluconeogenesis from lactate although the total glucose production is increased (Jakob et a [ . , 1971). This finding is indirectly supported by in vivo experiments (see Forster, 1974, 1976). Jakob et al. (1971) have studied the effect of xylitol on the activity of the citric acid cycle in perfused liver. They found that xylitol had no effect on oxygen consumption in the liver. This suggests that the activity of the citric acid cycle is decreased and the mitochondria1 respiratory chain uses NADH produced in the polyol dehydrogenase reaction for energy production. Also, the second step in xylitol metabolism, the phosphorylation of D-XylUbSe to ~-xylulose-5-phosphate,is rapid and consumes ATP. The rapid consumption of ATP can lead to a decrease in hepatic ATP content, as has been found after the administration of fructose (Maenpaa et al., 1968). Some decrease has been found also after the addition of xylitol in perfused livers (Woods and Krebs, 1973), but the changes are much smaller than those reported after fructose. Also, the effect of xylitol o n the total content of adenine nucleotides in the liver is small compared to that of fructose (Jakob et al., 1971). The effects of xylitol on hepatic intermediary metabolism in vitro are to some extent similar to those found after the administration of ethanol, which also effectively reduces the hepatic redox state (see Williamson et al., 1969b) and inhibits gluconeogenesis and the citric acid cycle. The effect of xylitol on hepatic metabolism is highly concentration dependent (Jakob et al., 1971). Jakob and co-workers (1971) and Williamson and co-workers (1971) used 10 mM xylitol in their experiments. This is a very high concentration and can hardly ever be reached under clinical conditions even after parenteral administration. The oral administration of xylitol causes so small an elevation in blood xylitol concentration that its effects on hepatic metabolism are probably small. Although xylitol has marked effects on hepatic metabolism in vitro, the clinical significance of these effects is questionable, since they are induced only by very high concentrations of xylitol. Parenteral administration of large doses may, however, induce marked metabolic alterations. D. EFFECTS ON THE CARBOHYDRATE, LIPID, AND KETONE BODY METABOLISM OF THE WHOLE BODY Only the first steps of xylitol metabolism are specific for it and most of xylitol is rapidly converted to glucose in the liver. Thus the metabolism of the carbon skeleton derived from xylitol is similar to glucose outside the liver (Froesch et al., 1971; Keller and Froesch, 1972). The effects of xylitol on the energy metabolism of the whole body, however, seem to be different from those of glucose (see Forster, 1974, 1976). After oral or intravenous administration of glucose there is a rapid increase in the glucose and insulin concentrations in the blood of healthy subjects. When xylitol is given either orally or intravenously the increase in blood glucose

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concentration is small or negligible (Lang, 1971; Forster, 1974). This is surprising, since xylitol is very rapidly converted to glucose in the liver. Xylitol seems, however, to increase the hepatic glycogen stores significantly more than glucose (Forster, 1974). Thus a considerable part of the glucose produced from xylitol is stored as glycogen, from which glucose is only gradually liberated into the bloodstream, and the changes in blood glucose concentration are small. The inhibition of hepatic gluconeogenesis from other substrates by xylitol may also diminish its effects on the blood glucose level (Forster, 1976). The effect of xylitol on insulin secretion is different in different species of animals (Kuzuya, 1969; Kuzuya et al., 1971). In the dog, xylitol is a very potent stimulator of insulin release (Kuzuya er al., 1969; Wilson and Martin, 1970) and it may cause hypoglycemia. In other animals the effect is smaller; no effect is found in the horse (Kuzuya et al., 1971). Data about the effect of xylitol on plasma insulin level in human subjects vary somewhat, depending on the experimental design. Under basal conditions the effect of xylitol on plasma insulin concentration is usually smaller than that of glucose (Geser et al., 1967; Kosaka, 1969; Amador and Eisenstein, referred by Brin and Miller, 1974), but xylitol may exaggerate the arginine-stimulated insulin release (Seino et a/., 1976). In contrast to insulin, the secretion of glucagon seems to be inhibited by xylitol (Seino et al., 1976). The concentration of free fatty acids in plasma is decreased after the administration of xylitol (see Lang, 1971; Forster, 1976). This effect seems to be as great as, or even greater than, that of glucose (Forster, 1976). Forster (1974) has suggested that this effect is mainly due to the increased esterification of free fatty acids in the liver. This may be one mechanism, but perhaps a minor one, since no significant changes in the triglyceride concentrations of the liver and plasma have been reported after xylitol infusions in man. In addition, the peripheral lipolysis is generally regarded as a more important regulator of plasma-free fatty acid concentration than the hepatic esterification. Thus xylitol must have some effect on the peripheral lipolysis. The mechanism by which xylitol inhibits lipolysis is not totally known. It cannot be mediated by glucose and insulin because the xylitol-induced changes in their concentrations in plasma are minimal. On the other hand, xylitol itself is not metabolized by the adipose tissue (Froesch and Jakob, 1974). Thus it must have a direct pharmacological effect on peripheral lipolysis. Xylitol has been found to inhibit the release of fatty acids from adipose tissue also in virro (Opitz, 1969), but the concentration needed was over 2 mMlliter. Therefore, it is not probable that this effect wholly explains the decrease of plasma-free fatty acid concentration which has been found after the administration of xylitol. Since the effect of xylitol on peripheral lipolysis is, from a clinical point of view, of importance, its mechanism ought to be clarified. In fasted and alloxan diabetic animals and in human subjects, xylitol has a clear antiketogenic effect (Bassler and Dreiss, 1963; see Lang, 1971). The pro6

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duction of ketone bodies (P-hydroxybutyrate and acetoacetate) is inhibited and their concentrations in the blood decrease. This effect is partly due to a decrease in the plasma-free fatty acid concentration, which is known to decrease ketogenesis and partly to direct effects of xylitol on the liver (Bassler et al., 1966b). The xylitol-induced decrease in glucagon secretion may also contribute to the antiketogenic effect. Xylitol increases the hepatic concentration of a-glycerophosphate at least in vitro (Jakob et al., 1971). Because a-glycerophosphate is an important regulator of hepatic triglyceride synthesis (Fritz, 1961), xylitol could increase hepatic triglyceride production and raise the concentration of triglycerides in plasma. Xylitol has been found to increase plasma triglyceride concentration in dogs after parenteral administration (Thomas et al., 1974), but this phenomenon has not been observed in human subjects. Even a long-term (2 year) oral utilization of large amounts of xylitol seems to have no effect on the plasma triglyceride concentration (Huttunen, 1976). In the same study no effect of xylitol on the plasma cholesterol level was found. The studies of nitrogen balance in animals and in human subjects have revealed a good anticatabolic effect of xylitol (see Lang, 1971; Forster, 1974). This effect has been claimed to be greater than that of equivalent doses of glucose (Forster, 1974, 1976). The reason is obscure. The effect is probably not mediated by insulin, but the possible decrease in glucagon secretion may play some role. The inhibition of hepatic gluconeogenesis by xylitol decreases the need of amino acids for the substrate of glucose production and this may be one reason. Also, the decrease in the plasma-free fatty acid concentration may improve the ability of the muscles to utilize glucose, and so inhibit the catabolism of muscle proteins.

IV. USE OF XYLITOL IN NUTRITION AND THERAPY At the present time it is believed that cost and gastrointestinal side effects prevent the use of xylitol as a major source of calories in oral nutrition. As a sweetener it has, however, certain advantages compared to sucrose. It is expected that xylitol may replace sucrose in the foods causing caries and in some special diets and diseases. In this review the latter aspects are dealt with.

A.

PARENTERAL NUTRITION

Xylitol has been recommended for parenteral nutrition for two reasons. First, amino acids do not react with sugar alcohols as they do with glucose. It is therefore easier to produce infusion solutions containing sugar alcohols and amino acids than those with glucose and amino acids. Second, it has been claimed that the tissues can use xylitol under postoperative and posttraumatic conditions in which considerable insulin resistance prevents the effective utilization of glucose

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(see Lang, 1971; Forster, 1974; Ahnefeld er al., 1976). The first argument is not very important since it is possible nowadays to produce infusion solutions containing amino acids and glucose. The second argument is extremely important, if it is valid. The advantages of xylitol as compared to those of glucose in parenteral nutrition have been argued, as seen in the proceedings of a recent symposium (Ritzel and Brubacher, 1976). It is true that oversecretion of the “stress hormones” (cortisol, catecholamines, glucagon, growth hormone, etc.) in postoperative and post-traumatic states cause insulin resistance and defective utilization of glucose in most tissues. This leads to the release of free fatty acids from adipose tissue, to the stimulation of gluconeogenesis, and to the degradation of muscle proteins, i.e., to a general catabolic state. There are many, mainly German, studies, which show that xylitol effectively corrects all those catabolic disorders and converts the metabolism to the direction of anabolism (see Lang, 1971; Wilkinson, 1972; Forster, 1974). Because the peripheral tissues are able to use xylitol only after its conversion to glucose, it is hard to explain the beneficial effect of xylitol. How can the glucose derived from xylitol be better than exogenous glucose together with insulin? Certainly the glucoses are not different, but xylitol may have direct effects, which could explain the anabolic effect. The inhibition of the hepatic gluconeogenesis from other substrates may save amino acids and correct the nitrogen balance. Also the possible direct inhibitory effect on the peripheral lipolysis may contribute to the anabolic effect of xylitol as well as the inhibition of the glucagon secretion. These direct effects of xylitol are, however, poorly documented and therefore the benefits of xylitol in parenteral nutrition are still a matter of dispute. The side effects of xylitol are discussed in detail later, but in this connection reference is made to some serious complications, even deaths, reported after xylitol infusions (Thomas et af., 1972a,b, 1974). The patients who died during xylitol infusion were very sick, and it is not clear which of the complications were specific to xylitol (Brin and Miller, 1974) but at least the lactic acidosis seems possible after the administration of xylitol (Forster, 1974). However, it is reported (Forster, 1974) that xylitol is widely used in infusion solutions in Germany without any major side effects. B.

DIETS OF DIABETICS

The use of rapidly absorbed carbohydrates such as sucrose is contraindicated for diabetics. Consequently, noncaloric sweeteners and different nonsucrose sugars and sugar alcohols have been recommended for diabetics. One of them is xylitol. Theoretically, xylitol is a good sweetener for diabetics. Because it is slowly absorbed it does not cause rapid changes in blood glucose concentration. In

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addition, all of the glucose formed from xylitol is not immediately liberated into the bloodstream, but is also stored as glycogen in the liver in diabetics (Bassler and Heesen, 1963; Forster, 1974). There are several studies in both alloxan diabetic animals and in diabetic patients showing that xylitol has smaller effects on blood glucose concentration than do glucose and sucrose (Lang, 197 I ; Mehnert, 1976). Also, our own experiments on seven insulin diabetic patients hospitalized in a metabolic ward showed that the addition of 60 gm of xylitol daily to the diet for 1 week had no significant effect on the fasting blood glucose concentration or on the excretion of glucose into the urine (Pelkonen and Ylikahri, 1980). Gastrointestinal complaints seem to be the only side effects of oral xylitol in diabetics. No significant changes in the concentrations of lipids, urate, or other components of blood have been found in diabetics during xylitol administration (Mertz el al., 1972; Pelkonen and Ylikahri, 1980). Thus xylitol seems to be a suitable sweetener for diabetics. However, one must remember that xylitol is a source of energy and it must be taken into account when calculating the total energy content of the diet. Xylitol infusion has sometimes been recommended for the treatment of diabetic ketoacidosis (Bassler and Dreiss, 1963; Goto et d., 1965; Toussaint et al., 1967; Lang, 1971). Although there are reports of good results, the rationale of this therapy is, however, questionable. The basic reason for diabetic ketoacidosis is lack of insulin and there is always considerable hyperglycemia and dehydration in this situation. It is therefore more reasonable to give the patient insulin and saline than infuse xylitol, which may further increase osmotic disturbances and hyperglycemia. C. THERAPY OF HEMOLYTIC ANEMIA DUE TO GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY A hemolytic anemia due to a deficiency of the glucose-6-phosphate dehydrogenase in red blood cells is a fairly common hereditary disease in some parts of the world (Marks and Banks, 1965). The activity of the pentose phosphate pathway is decreased in the cells with the defective enzyme and they 'cannot produce NADPH at a normal rate. Due to the lack of NADPH they are unable to keep glutathione in the reduced state. Glutathione is present in the red blood cells to reduce peroxide-generating compounds and if the cells do not contain enough reduced glutathione the peroxides disrupt them. Thus the intact pentose phosphate pathway and NADPH production in the red blood cells are necessary for cellular integrity. The rationale of the use of xylitol in the treatment of hemolytic anemia due to deficiency of glucosed-phosphate dehydrogenase is that it could produce additional NADPH for the reduction of glutathione (van Eys et af., 1974). It has been shown in vitro that the red blood cells take up xylitol and are able to oxidize

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it to L-xylulose by the NADPH-linked xylitol dehydrogenase (van Eys et al., 1974). However, the K, value of this enzyme for xylitol is high (3.2 X lo-* M ) (Wang and van Eys, 1970), which means that a relatively high concentration (about 10 mMlliter) of xylitol is needed in the blood for significant rates of NADPH production. In human subjects it is very difficult to reach such concentrations by oral administration. According to our own experience the maximal tolerable dose gave blood concentrations below 1 mMlliter in nonadapted subjects (Ylikahri and Leino, 1979). Therefore, the practical usefulness of oral xylitol in the therapy of glucose-6-phosphate deficiency is questionable. Large intravenous doses of xylitol have protected experimental animals against hemolysis induced by a hemolyzing agent, acetylphenylhydrazine (Wang et al., 1971), but to our knowledge no clinical trials in human patients have been carried out. D.

OTHER CLINICAL USES

Xylitol has been tested sporadically in the treatment of several diseases (see Horecker et al., 1969). It has been used during corticosteroid therapy to prevent the suppression of adrenal cortex activity (Ohnuki, 1969). The protection was thought to be due to the increased production of ribose-5-phosphate for RNA synthesis. After the first promising results no further support is found in the literature. Also, the trials on the use of xylitol in the treatment of other diseases have not led to clinical use. Thus the only clinically important use of xylitol (in addition to the prevention of dental caries) seems to be in the diet of diabetics and perhaps in parenteral nutrition of some selected patients. Further investigations are, however, needed to clarify the latter indication.

V. TOXICOLOGICAL EVALUATION A.

AMOUNTS WITHOUT ADVERSE EFFECTS I.

Oral Administration

The amount of xylitol which can be taken per os is limited by its slow absorption and the resulting osmotic diarrhea. The maximum dose that does not cause diarrhea depends on many factors. The individual variations are large (Mikinen, 1976). In addition, xylitol seems to cause diarrhea more easily in liquid than in solid foods (Makinen, 1976). The usual tolerated amount is 20-40 gm per dose or 30-70 gm per day in nonadapted persons. The adaption to xylitol is, however, considerable, and daily doses up to 400 gm without side effects have been reported (Lang, 1971; Makinen, 1976). Even this amount is easily

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metabolized by an adapted liver. Thus the hepatic metabolism is not a limiting factor in the oral administration of xylitol. 2. Intravenous Administration In parenteral administration the factors limiting the dose of xylitol are the capacity of the liver to metabolize it and the possible side effects, which will be dealt with later. The capacity of the liver to metabolize xylitol is high. The elimination rate of xylitol after,one intravenous dose (0.3-0.5 g d k g body weight) has been estimated to be 3.0-4.5% per minute, which is about the same as the elimination rate for glucose (Lang, 1971; Forster, 1974). Using the constant infusion technique, the maximal rate of xylitol elimination has been estimated to be about 480 mg/kg/hour in human subjects (see Lang, 1971; Bassler, 1976), but also much greater infusion rates (up to 2.0 gm/kg/hour) have been used (Thomas et af.. 1974). Then, however, the concentration of xylitol in the blood rises and some xylitol is excreted in the urine. In addition the frequency of the side effects seems to increase (Bassler, 1976). Therefore, it has been recommended that in parenteral nutrition the infusion rate of xylitol should not exceed 0.25 gdkglhour (Bassler, 1976). During long-term administration the rate of xylitol elimination is increased due to the adaption of the hepatic enzyme system (Bassler, 1976). Diabetics seem to eliminate xylitol at the same rate as healthy persons do (see Lang, 1971). B.

ADVERSE EFFECTS

The only significant side effects of the oral use of xylitol are the gastrointestinal symptoms, diarrhea, meteorism, and abdominal pain, which are dose dependent. Even very long-term administration of large amounts of xylitol per 0s does not cause any changes in blood chemistry (Mikinen, 1976; Huttunen, 1976), and no pathological changes in any organs have been reported in human subjects. In contrast to oral administration some serious side effects, even deaths, have been reported after intravenous infusion of xylitol (Thomas et al., 1972a,b). The exact cause of the deaths is not clear (Thomas er al., 1974; Forster, 1974), and it is not known which side effects were directly related to xylitol (Forster, 1974). Large intravenous doses of xylitol may induce profound metabolic alterations which may be the cause of the side effects. At least, the following adverse effects have been reported after the intravenous administration of xylitol.

I . Lactic Acidosis The redox state of the liver is changed to a reduced direction during xylitol metabolism. The change inhibits the utilization of lactate by the liver (Jakob er

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al., 1971). In addition, considerable amounts of lactate are produced from xylitol itself. Both these facts tend to raise the concentration of lactate in the blood. Normally, the body is able to compensate for this increased lactate production, but under certain conditions it may induce lactic acidosis (Thomas et al., 1974; Woods, 1976). This danger is actual, especially in hypoxic patients, whose muscles and other peripheral tissues produce lactate at high rates (Woods, 1976). Also simultaneous administration of xylitol and ethanol may be dangerous, because both change the redox state of the liver in the same direction and inhibit the utilization of lactate. Lactic acidosis is not specific to xylitol, because the rapid infusion of glucose, fructose, and sorbitol also can increase the blood concentration of lactate (Forster, 1974). With xylitol the danger of lactic acidosis is perhaps the greatest (Thomas et a l . , 1974). The effect of xylitol on the blood lactate concentration is dose dependent. Thus when slow infusion rates are used, the danger of lactic acidosis is small (Bassler, 1976). 2.

Deposition of Calcium Oxalate Crystals in Tissues

In Australia, calcium oxalate crystals were found in the renal, cerebral, and vascular tissues in patients who died after xylitol infusion (Thomas et al., 1972a). The deposition was regarded as an important contributing factor to the deaths of the patients and it was thought that xylitol somehow increased the production of oxalate, leading to crystallization (Thomas et a l . , 1972a, 1974). However, it was found in animal experiments that xylitol increased the oxalate excretion only in animals whose diet was deficient in vitamin B6 (Thomas e t a l . , 1976). Later studies with 4C-labeled xylitol also showed that xylitol could not have been the source of oxalate (Hauschildt and Watts, 1976), but contradictory results have been published recently (Rofe et al., 1977). According to this study, considerable amounts of xylitol are converted to oxalate in rat liver, the oxidized redox state of the liver favoring this conversion. However, calcium oxalate crystals have also been found in the tissues of severely ill patients who received infusion therapy other than xylitol (Pesch et al., 1976) and even in patients who received no infusion therapy, especially in uremic patients (Brin and Miller, 1974). Thus the relation between xylitol infusion and deposition of calcium oxalate in the tissues is still obscure (Forster, 1974; Thomas et al., 1974). 3 . Hyperuricemia

Increased serum uric acid concentrations have been measured during and after xylitol infusions (Forster er al., 1970; Forster, 1974). Theoretically, xylitol could increase the serum uric acid concentration by two mechanisms. First, like

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fructose, it can induce the degradation of the hepatic adenine nucleotides and so increase the production of uric acid. Second, xylitol may inhibit the excretion of uric acid. The first possibility is unlikely, because the effect of xylitol on the hepatic adenine nucleotides is much less than that of fructose (Jakob et a l . , 1971). The second mechanism, on the other hand, is quite possible, because xylitol increases blood lactate concentration and lactate is known to effectively inhibit the excretion of uric acid. The xylitol-induced hyperuricemia is mostly mild and it has hardly any clinical significance (Forster, 1974). In any case, it is not specific to xylitol and can be caused also by other carbohydrates (Forster, 1974). 4 . Liver Damage

Increased concentrations of bilirubin, transaminases, and alkaline phosphatase, which are indicators of liver damage, have been found in serum after parenteral administration of xylitol in man and in animals (Thomas et a l . , 1972a; Schumer, 1971; see Forster, 1974). These findings, however, have been made after very large doses of xylitol and it is probable that they are at least partially attributable to the infusion of hypertonic solution in general and not especially to xylitol. Also, other hypertonic solutions including glucose cause similar changes (Wang et a l . , 1973; Forster, 1974). Thus it seems probable that reasonable doses of xylitol do not cause liver damage. 5.

Changes in Renal and Cerebrul Function

Osmotic diuresis and anuria and azotemia as well have been found in some patients after xylitol infusion (Thomas et a l . , 1972a, 1974). Most probably, these disturbances are attributable more to the infusion of hypertonic solution than to xylitol (Thomas e f a l . . 1974), although the role of calcium oxalate crystals in renal tissues and their relation to xylitol have not been totally elucidated (Thomas e f a l . , 1976; Paulini, 1976). Confusion, somnolence, and other cerebral disturbances have been observed during xylitol infusion (Thomas et al., 1974). They are also probably due to the changes in the osmolality of the extracellular fluid after infusion of hypertonic solution (Thomas et a l . , 1974). A slow infusion rate protects against these complications. 6 . Carcinogenicity

Prolonged toxicological studies on xylitol carried out in the Huntington Research Laboratories in England raised a question about the carcinogenicity of

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xylitol. No published data from these studies are available at present, but some preliminary information about the results has been received by personal communication (F. Gey, personal communication, 1977). The studies showed that male mice receiving 10 or 20% of their diet as xylitol had both benign and malignant tumors in their urinary bladder and also frequently had bladder stones. This may be an important finding because it is known that foreign material in the bladder of mice very easily induces tumors in these animals. No bladder tumors were found in female mice or in rats and dogs. It is assumed that the bladder tumors of the male mice were induced by the bladder stones and not directly by xylitol. The stones consisted of calcium oxalate and calcium phosphate. The reason for frequent bladder stones in male mice is not known, but increased oxalate excretion is one possibility. The same toxicological studies showed that hyperplasia and tumors of the adrenal medulla were more common in rats receiving xylitol than in controls. These tumors were found neither in mice nor in dogs. The mechanism by which xylitol affects the adrenal medulla is not known. The above observations were made using experimental animals. The relevance of these findings to humans is not clear. As yet, increased frequency of stones in the urinary tract, bladder tumors, or pheochromocytomas has not been reported in human subjects consuming xylitol. But before one can be sure that xylitol is not carcinogenic in humans it has to be clarified whether the bladder tumors induced by xylitol are specific to mice. Xylitol has proved nonmutagenic in in vitro tests for mutagenicity (Batzinger et a / . , 1977). This supports the idea that xylitol is not, at least directly, carcinogenic.

VI. CONCLUSIONS In the human body xylitol is both an endogenous metabolite of the uronic acid cycle and an exogenous source of energy. The exact physiological role of the endogenous metabolism of xylitol is not known, but it is probably connected to the detoxification processes. The daily turnover of xylitol seems to be about 15 gm. Xylitol is absorbed from the intestine by passive or facilitated diffusion. Therefore its absorption is much slower than that of glucose. However, regular oral use of xylitol increases the rate of absorption. The exact mechanism of this adaptation is not completely explained. The exogenous xylitol is metabolized almost exclusively in the liver, because reasonable amounts of the key enzymes of the metabolism are found only in the liver. At first, xylitol is reduced to D-xylulose by a nonspecific polyol dehydrogenase using NAD as a coenzyme. D-Xylulose is rapidly phosphorylated to

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~-xylulose-5-phosphateby ~-xylulosekinase. ~-Xyh1lose-5-phosphateis a normal metabolite of the pentose phosphate pathway and thus it can be converted to glucose-6-phosphate via the reactions of this pathway. Glucose-6-phosphate can then be converted into either glucose and glycogen or into pyruvate and lactate. Normally, a major portion of xylitol is converted to glucose or glycogen in the liver. The main factors controlling the rate of the metabolism of xylitol are the activity of the polyol dehydrogenase and the concentration of xylitol entering the liver. The activity of the polyol dehydrogenase in normal human liver is high. Thus the concentration of the xylitol may very much regulate the rate of the metabolism of xylitol. After large parenteral doses, the concentrations of xylitol entering the liver may be high, and they can cause considerable changes in the metabolism of the liver. After oral administration, the absorption of xylitol is slow. Thus the concentrations entering the liver are small and they do not cause any major changes in the hepatic metabolism. The main argument for the use of oral xylitol is its anticariogenic property. Xylitol can perhaps also be used as a sweetener for diabetics because it does not cause rapid changes in blood glucose concentration. Oral xylitol has also been tested in the treatment of many other diseases, but the trials have not led to any clinical use. Xylitol infusions have been recommended for parenteral nutrition because they have been reported to have greater anticatabolic effect than glucose in conditions with insulin resistance. All investigators do not agree with this point. Therefore, the benefits of parenteral xylitol are still a matter of debate. Osmotic diarrhea is almost the only side effect of oral xylitol. In nonadapted subjects the maximal dose of xylitol that does not cause diarrhea is 20-40 gm. However, after an adaptation period of several weeks, doses of up to 400 g d d a y have been tolerated. Even the long-term oral utilization of xylitol does not induce any changes in the plasma concentrations of triglycerides and cholesterol or in the function of the liver and the kidneys. Serious complications have been reported after the parenteral infusion of large amounts of xylitol. Lactic acidosis is probably the most common and dangerous complication, but changes in the plasma concentration of urate and liver damage have also been reported during xylitol infusion. Calcium oxalate crystals have sometimes been found in the organs of patients who have received xylitol infusions. These changes are not specific to xylitol and some of them may be caused merely by the rapid infusion of hypertonic solution. However, the largest recommended rate of xylitol infusion is 0.25 gm/kg/hour, which ought to be safe. Recent long-term toxicological studies have revealed both benign and malignant bladder tumors in male mice who received 10 or 20% of their diet as xylitol for 2 years. In female mice, rats, and dogs, no malignancies have been

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found. In the tests for mutagenicity, xylitol has not proved mutagenic. Thus it is thought that xylitol itself is not carcinogenic and that the bladder tumors may be specific to mice and not caused directly by xylitol.

VII. RESEARCH NEEDS At the least, the following points about the metabolism and metabolic effects of xylitol need further study: 1. The exact mechanism by which the long-term oral use of xylitol enhances its absorption should be clarified. 2. The effect of xylitol on hepatic glycogen content as compared to that of glucose and fructose should be studied in human subjects also. 3. The direct effect of xylitol on the rate of peripheral lipolysis should be studied both in vitro and in vivo. 4. A large-scale (perhaps multicenter) controlled study should be carried out to determine whether xylitol really has a better anticatabolic effect than glucose in postoperative and posttraumatic states. 5 . The effect of xylitol on oxalate excretion in the urine should be studied in human subjects and in other animals. 6 . The effect of oral xylitol on diabetic control ought to be carefully studied in different types of diabetes. 7 . It should be clarified as soon as possible whether bladder tumors appearing after the utilization of xylitol are found only in mice. Also, the effect of xylitol on the function of the adrenal medulla in human subjects should be studied.

REFERENCES Ahnefeld, F. W., Halmagyi, M., and Milewski, P. 1976. Glukose and Glukoseaustauschstoffe in der Infusions-therapie des operativen Bereiches. I n “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” (G. Ritzel and G. Brubacher, eds.), p. 242. Huber, Bern. Arsenis, C., and Touster, 0. 1969. Nicotinamide adenine dinucleotide phosphate-linked xylitol dehydrogenase in guinea pig liver cytosol. J. Biol. Chem. 244, 3895-3899. Asakura, T., Adachi, K . , Minakami, S . , and Yoshikawa, H. 1967. Non-glycolytic sugar metabolism in human erythrocytes. 1. Xylitol metabolism. J . Biochern. (Tokyo) 62, 184-193. Bassler, K . H. 1969. Adaptive processes corcemed with absorption and metabolism of xylitol. In “International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols” ( B . L. Horecker, K . Lang, and Y. Takagi, eds.), p. 190. Springer-Verlag, Berlin and New York. Bassler, K . H. 1976. Quantitative aspects of the metabolism of glucose, fructose, sorbitol and

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xylitol. In “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” (G. Ritzel and G. Brubacher, eds.), p. 22. Huber, Bern. Bassler, K . H., and Dreiss, G . 1963. Antiketogene Wirkung von Xylit bei alloxandiabetischen Ratten. Klin. Wochensrhr. 41, 593-595. Bassler, K. H., and Heesen, D. 1963. Die Bildung von Leber- und Muskelglykogen aus Xylit, Sorbit und Glucose bei gesunden und alloxandiabetischen Ratten. Klin. Wochenschr. 41, 595-598. Bassler, K . H., Prellwitz, W., Unbehaun, V., and Lang, K. 1962. Xylitstoffwechsel beirn Menschen. Zur Frage der Eignung von Xylit als Zuckerersatz beim Diabetiker. Klin. Wochenschr. 40, 791-793. Bassler, K . H., Stein, G., and Belzer, W. 1966a. Xylitstoffwechsel und Xylitresorption. Stoffwechseladaptation als Ursache fur Resorptionsbeschleunigung. Biochern. 2. 346, 171185. Bissler, K. H.. Fingerhut, M., and Czok, G . 1966b. Hemmung der Fettsaureoxydation alsein Faktor bei der antiketogenen Wirkung von Zuckern und Polyalkoholen. Klin. Wochenschr. 44, 899900. Batzinger, R. P., Ou, S.-Y. L., and Bueding, E. 1977. Saccharin and other sweeteners: Mutagenic properties. Science 198, 944-946. Brin, M., and Miller, 0. N. 1974. The safety of oral xylitol. I n “Sugars in Nutrition” (H. L. Sipple and K . W. McNutt, eds.), p. 591. Academic Press, New York. Bums, J. J. 1959. Biosynthesisof L-ascorbic acid; basic defect in scurvy. Am. 3. M e d . 26, 740-748. Dubach, U. D., Feiner, E., and Forgo, I. 1969. Orale Vertraglichkeit von Xylit bei stoffwechselgesunden Probanden. Schweiz. Mrd Wochmschr. 99, 190- 194. Forster, H. 1974. Comparative metabolism of xylitol. sorbitol and fructose. In “Sugars in Nutrition” (H. L. Sipple and K. W. McNutt, eds.), p. 259. Academic Press. New York. Forster, H. 1976. Metabolism of glucose substitutes compared to that of glucose. /ti “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” ( G . Ritzel and G. Brubacher, eds.), p. 68. Huber, Bern. Forster, H . , Meyer. E., and Zilge, M. 1970. Erhohung von Serumhamslure und Serumbilirubin nach hochdosierten Infusionen von Sorbit, Xylit und Fructose. Klin. Wochenschr. 48, 878-879. Fritz, 1. B. 1961. Factors influencing the rates of long-chain fatty acid oxidation and synthesis in mammalian systems. Physiol. Rev. 41, 52- 129. Froesch, E. R . , and Jakob, A . 1974. The metabolism of xylitol. I n “Sugars in Nutrition” (H. L. Sipple and K . W. McNutt, eds.), p. 241. Academic Press, New York. Froesch, E. R., Zapf, J . , Keller, U . , and Oelz, 0. 1971. Comparative study of the metabolism of U- I 4C-fructose, U- 4C-sorbitol and U- ’ 4C-xylitol in the normal and in the streptozotocindiabetic rat. Eur. J. Clin. Invest. 2 , 8-14. Geser. K . A,, Forster, H., Prols, H., and Mehnert, H. 1967. Zur Frage einer Wirkung von Xylit auf die lnsulinsekretion des Menschen. Klin. Wochenschr. 45, 85 1-852. Goto, Y.,Anzai, M., Chiba, M., Ohneda, A., Kawashima, S . , and Maruhama, Y. 1965. Clinical effects of xylitol on carbohydrate and lipid metabolism in diabetics. Lancer 2 , 918-921. Gupta, S. D., Chaudhuri, C . R., and Chatterjee, I. B. 1972. Incapability of L-ascorbic acid synthesis by insects. Arch. Biochem. Biophys. 152, 899-890. Hauschildt. S., and Watts, R. W. E. 1976. Studies on the effect of xylitol on oxalate formation. Biochern. Phurmacol. 25, 27-29. Hollrnann, S., and Touster, 0. 1964. “Non-Glycolytic Pathways of Metabolism of Glucose,” p. 107. Academic Press, New York. Horecker, B. L., Lang, K., and Takagi, Y . , eds. 1969. “International Symposium on Metabolism, Physiology and Clinical Use of Pentoses and Pentitols. ’’ Springer-Verlag, Berlin and New York. Huttunen, J. K . 1976. Serum lipids, uric acid and glucose during chronic consumption of fructose

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and xylitol in healthy human subjects. In “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” (G. Ritzel and G . Brubacher, eds.), p. 105. Huber, Bern. Jakob, A,, Williamson, J. R., and Asakura, T. 1971. Xylitol metabolism in perfused rat liver. J . B i o l . Chem. 246, 7623-7631. Keller, U., and Froesch, E. R. 1972. Vergleichende Untersuchungen iiber den stoffwechsel von Xylit, Sorbit und Fructose beim Menschen. Schweiz. Med. Wochenschr. 102. 1017-1022. Kosaka, K. 1969. Stimulation of insulin secretion by xylitol administration. In “International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols” (B. L. Horecker, K . Lang, and Y. Takagi, eds.), p. 212. Springer-Verlag. Berlin and New York. Kuzuya, T. 1969. Some recent observations on xylitol-induced insulin secretion. In “International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols” (B. L. Horecker, K . Lang, and Y. Takagi, eds.), p. 230. Springer-Verlag. Berlin and New York. Kuzuya, T . , Kanazawa, Y., and Kosaka, K. 1969. Stimulation of insulin secretion by xylitol in dogs. Endocrinology 84, 200-207. Kuzuya, T., Kanazawa, Y., Hayashi, M., Kikuchi, M., and Ide, T. 1971. Species difference in plasma insulin responses to intravenous xylitol in man and several mammals. Endocrinol. Jpn. in, 309-320. Lang, K. 1971. Xylit, Stoffwechsel und klinische Venvendung. K l i n . Wochenschr. 49, 233-245. McCorrnick, D. B., and Touster, 0. 1957. The conversion in v i v o of xylitol to glycogen via the pentose phosphate pathway. J . B i o l . Chem. 229, 451-461. Maenpai, P. H., Raivio. K. O., and Kekomaki, M. P. 1968. Liver adenine nucleotides: Fructoseinduced depletion and its effect on protein synthesis. Science 161, 1253-1254. Makinen, K. K. 1976. Long-term tolerance of healthy human subjects to high amounts of xylitol and fructose: General and biochemical findings. In “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics“ (G. Ritzel and G. Brubacher, eds.), p. 92. Huber, Bern. Marks, P. A., and Banks, J . 1965. Drug-induced hemolytic anaemias associated with glucose-6phosphate dehydrogenase deficiency: A genetically heterogenous trait. A n n . N . Y. A c a d . Sci. 128, 198-206. Mehnert, H. 1976. Zuckeraustauschstoffe in der Diabetes-diaet. I n “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” (G. Ritzel and G . Brubacher, eds.), p. 295. Huber, Bern. Mertz, D. P., Kaiser, V., Klopfer-Zaar, M., and Beisbarth, H. 1972. Serumkonzentrationen verschiedener Lipide und von Harnsaure wahrend 2-wochiger Verabreichung von Xylit. Klin. Wochenschr. 50, 1107- 1 I 1 1 . Ohnuki, M. 1969. Preventing effect of xylitol on suppression of adrenocortical function by steroid therapy. f n “International Symposium on Metabolism, Physiology, and Clinical Use of Pent o w and Pentitols” (B. L. Horecker, K, Lang, and Y. Takagi, eds.), p. 334. Springer-Verlag, Berlin and New York. Opitz, K . 1969. The influence of xylitol and other polyols and sugars on fat mobilization. In “International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols” (B. L. Horecker, K. Lang, and Y . Takagi, eds.), p. 238. Springer-Verlag. Berlin and New York. Paulini, K. 1976. Kristallablagerungen im Gewebe nach Infusionen im Rahmen einer Intensivtherapie. In “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” (G. Ritzel and G. Brubacher, eds.), p. 204. Huber, Bern. Pelkonen, R., and Ylikahri, R. 1980. Effect of dietary xylitol on glucose balance in insulin dependent diabetes. (To be published.) Pesch, H.-J., Krampf, F.-D., Menzel, H., Weiland, H . , Eidam, U.-W ., Prestele, H., and Heid, H. 1976. Zur Wirkung von Kohlenhydratinfusionen auf die Bildung von Calciurnoxalat-

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Niederschlaegen in der Niere: Morphologische und biochemische Befunde bei Verstorbenen und im Tierversuch. I n “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” ( G . Ritzel and G . Brubacher, eds.), p. 193. Huber, Bern. Pitkanen, E., and Sahlstrom, K. 1968. Determination of urinary polyalcohols by means of gas-liquid chromatography. Ann. Med. Exp. Biol. Fenn. 46, 151-157. Ritzel, G., and Brubacher, G . , eds. 1976. “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics.” Huber, Bern. Rofe, A . M., Thomas, D. W., Edwards, R . G . , and Edwards, J . B . 1977. [ T ] oxalate synthesis from [U- ’ ‘C] xylitol. In vivo a d in v i m studies. Biochem. Med. 18, 440-451, Schumer, W . 1971. Adverse effects of xylitol in parenteral alimentation. Metab., Clin. Exp. 20, 345-347. Seino, Y . , Taminato, T., Inoue. Y., Goto, Y., Ikeda, M., and Imura, H . 1976. Xylitol: Stimulation of insulin and inhibition of glucagon responses to arginine in man. J. Clin. Endocrinol. Merab. 42, 736-743. Smith, M . G. 1962. Polyol dehydrogenase. 4 . Crystallization of the L-iditol dehydrogenase of sheep liver. Biochem. J. 83, 135-144. Thomas, D. W . , Edwards, J. B., Gilligan, J. E., Lawrence, J. R., and Edwards, R. G . 1972a. Complications following intravenous administration of solutions containing xylitol. Med. J. Aust. 1, 1238-1246. Thomas, D. W . , Gilligan, J. E . , Edwards, J . B., and Edwards, R. G. 1972b. Lactic acidosis and osmotic diuresis produced by xylitol infusion. Med. J. Ausr. 1, 1246-1248. Thomas, D. W . , Edwards, 1. B., and Edwards, R. G. 1974. Toxicity of parenteral xylitol. In “Sugars in Nutrition” (H. L. Sipple and K. W. McNutt, eds.), p. 567. Academic Press, New York. Thomas, D. W., Hannett, B., Chalmers, A., Rofe, A. M., Edwards, I. B., and Edwards, R. G . 1976. Oxalate excretion during carbohydrate infusions. In “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” (G. Ritzel and G . Brubacher, eds.), p. 181. Huber,Bern. Toussaint, W., Roggenkamp, K., and Bassler, K. H . 1967. Behandlung der Ketonamie im Kindersalter mit Xylit. Z . Kinderheilkd. 98, 146-154. Touster, 0. 1960. Essential pentosuria and the glucuronate-xylulose pathway. Fed. Proc., Fed. Am. SOC. Exp. B i d . 19, 977-983. Touster, 0. 1969. The uronic acid pathway and its defect in essential pentosuria. In “International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols” ( 9 , L. Horecker, K. Lang, and Y. Takagi. eds.), p. 79. Springer-Verlag, Berlin and New York. Touster, 0. 1974. The metabolism of polyols. In “Sugars in Nutrition” (H. L. Sipple and K. W. McNutt, eds.), p. 229. Academic Press, New York. Touster, 0.. and Shaw, D . R . 1962. Biochemistry of acyclic polyols. Physiol. Rev. 42, 181-225. Touster, 0 . .Reynolds, V . H . , and Hutcheson, R . M. 1956. The reduction of L-xylulose to xylitol by guinea pig liver mitochondria. J. B i d . Chem. 221, 697-702. van Eys, J . , Wang, Y . M., Chan, S., Tanphaichitr, V. S., and King, S. M . 1974. Xylitol as a therapeutic agent in glucose-6-phosphate dehydrogenase deficiency. In “Sugars in Nutrition” (H. L. Sipple and K. W. McNutt, eds.), p. 613. Academic Press, New York. Wang, Y. M., and van Eys, J. 1970. The enzymatic defect in essential pentosuria. N. Engl. J. Med. 282, 892-896. Wang, Y. M., Patterson, J. H., and van Eys, J . 1971. The potential use of xylitol in glucose-6phosphate dehydrogenase deficiency. J. Clin. Invest. 50, 1421-1428. Wang, Y. M . , King, S. M . , Patterson, J. H., and van Eys, J . 1973. Mechanism of xylitol toxicity in the rabbit. Merab., Clin. Exp. 22, 885-894. Wilkinson, A. W., ed. 1972. “Parenteral Nutrition.” Williams & Wilkins, Baltimore, Maryland.

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Williamson, J . R., Scholz, R . , and Browning, E. T. 1969a. Control mechanisms of gluconeogenesis and ketogenesis. 11. Interactions between fatty acid oxidation and the citric acid cycle in perfused rat liver. J . B i d . Chern. 244, 4617-4628. Williamson, J. R . , Scholz, R . , Browning, E. T.. Thurman, R . G . , and Fukami, M. H. 1969b. Metabolic effects of ethanol in perfused rat liver. J . B i d . Chem. 244, 5044-5054. Williamson, J. R . , Jakob, A,, and Refino, C. 1971. Control of the removal of reducing equivalents from the cytosol in perfused rat liver. J . B i d . Chern. 246, 7632-7641. Wilson, R. B., and Martin, J. M. 1970. Plasma insulin concentrations in dogs and monkeys after xylitol, glucose or tolbutamide infusion. Diuberes 19, 17-22. Woods, H. F. 1976. The metabolic complicationsof intravenous nutrition. I n “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics” ( G . Ritzel and G . Brubacher, eds.), p. 54. H u h , Bern. Woods, H. F., and Krebs, H . A . 1973. Xylitol metabolism in the isolated perfused rat liver. Biochem. J . 134, 437-443. Ylikahri, R . H . , and Leino, T. 1979. Metabolic interactions of xylitol and ethanol in healthy males. Metah., Clin. Exp. 28, 25-29.

ADVANCES IN FOOD RESEARCH. VOL. 25

FROZEN FRUITS AND VEGETABLES: THEIR CHEMISTRY, PHYSICS, AND CRYOBIOLOGY MILFORD S . BROWN Western Regional Research Center, Science and Education Administration, U . S . Departmenr of Agriculture, Berkeley, California

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Ice Formation in Biological Materials and Model Systems . . . . . . . . . . . . , . , A. Crystal Growth, Vitrification, and Recrystallization . . . . . . . . . . . . . , , . , B. Freezing in Solutions, Cells, and Tissues . , . . . . . . . . . . . . . . . . . . . . . . . C. Chemical Reactions at Low Temperatures . . . . . . . . . .

D. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Survival of Plants at Low Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chilling Sensitivity . . . . . . . . . . . , . , . . . . . . . . , . . . . . . , . . . . . . . . , , , . . B. Chilling Requirement. . . . . . . . . . . . . . . . . . C. Winter Hardiness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ............ ........... IV. Refrigerated and Frozen Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plants Used for Food . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Refrigerated Fruits and Vegetables . . ... ............. C. Frozen Fruits and Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . .

............................. ............................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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181 183 183 185 188 192 193 194 199 202 209 21 1 21 1 213 214 228 229 230

I. INTRODUCTION Freezing has become an important method of food preservation during the last 30 years. In the United States, 3 to 4 billion pounds of vegetables, 213 to 314 billion pounds of fruit, and 90 million gallons of juices and juice concentrates are now frozen annually. The techniques used for freezing these foods are quite vaned, and depend on the commodity being frozen and its final use. For example, whole strawberries to be used for dessert toppings must be handled much more carefully than berries 181 ISBN 0-12-016425-6

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that will eventually be made into strawberry jam. Asparagus that will become part of an expensive dinner in a high quality restaurant could be frozen with liquid nitrogen or other cryogens to provide the best preservation of its texture. On the other hand, peas that are to be priced competitively in the grocery store must be handled by the most economical means. This probably would involve freezing on a moving belt with air at about -35°C (-30°F), followed by bulk frozen storage. Some time after the harvest season, the frozen peas would be repacked in 8 to 10 ounce cartons or small plastic bags for retail sale. The procedures used for freezing these foods have been studied extensively. Raw materials, processing steps, and the stored products have been examined by a large number of chemical and physical methods, and by the final judges of all foods, the human eyes, nose, tongue, teeth, and brain. Food technologists have determined the direction and magnitude of changes in the product that result from altering the processing or storage conditions, but often they have not determined how or why these changes occurred. If the most desirable food quality is that of the freshly harvested fruit or vegetable, then the ultimate in frozen food would be perfect preservation of the living state. Current commercial processing and storage methods do not accomplish this. The interest in cryogenic and other new food freezing systems is an indication of the need or desire for improved product quality. If new processing methods are to be successful, they should be developed on the basis of a thorough understanding of the requirements of quality maintenance throughout the many steps associated with harvesting, processing, storage, distribution, and use. Possibly the best understanding is to be gained through examination of the accomplishments of a number of disciplines concerned with the chilling and freezing of water, aqueous solutions, plant tissues, and living plants under a wide variety of conditions. Early studies of the effects of freezing in plant tissues were concerned with the ability of temperate zone plants to survive winter conditions and thus produce a supply of food for the following year. Many plants accomplish this by forming hardy dormant buds that rest until favorable weather returns in the spring. Other plants whose vegetative structures are entirely unable to tolerate freezing must be grown from seed each year. Although some alpine plants may survive daily exposure to freezing temperatures, most plants are unable to tolerate freezing during their period of active growth. In food production, this is most important in the spring. If a period of warm weather that causes growth to begin is then followed by a freeze, portions of the plant may be killed. Many fruit trees bloom at the beginning of their annual growing season, and frost at that time can reduce or eliminate the crop for that year. Research into these aspects of plant hardiness has led to other studies of responses to cold and the ability to survive very low temperatures, including exposure to liquid nitrogen.

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Low temperatures above the freezing point also affect plant growth. Some plants are injured or killed by such temperatures during part or any of their life. On the other hand, some plants require a period of chilling to terminate dormancy of their seeds or buds. These and other effects of low temperatures and freezing on plants and foods of plant origin will be discussed in the following sections. I have tried to present basic principles and examples of some of the research in low temperature physics, chemistry, biology, and food preservation. None of these areas is reviewed exhaustively. Rather, 1 have tried to indicate for the reader the scope of research and knowledge, as well as some of the problems remaining. I hope this will help the understanding and improvement of fruit and vegetable handling and processing.

11.

ICE FORMATION IN BIOLOGICAL MATERIALS AND MODEL SYSTEMS

Water is the major component of plants and most plant parts (except seeds and woody stems) and the major component undergoing a phase change during freezing. An understanding of freezing in plants should therefore begin with some basic principles of the formation of ice and proceed to the study of the influence of this process on the tissues in which it occurs. A.

CRYSTAL GROWTH, VITRIFICATION, AND REC RY STA LLIZATION

During freezing molecules lose energy, reduce their motion, and become ordered in a particular pattern, or crystal structure. With a pure substance, this phase change takes place at a specific temperature (Fig. 1). Before this ordering of molecules begins, however, it is necessary for a pattern to be present. This pattern, or crystal nucleus, can be a small solid particle of the same substance, either formed spontaneously or introduced, or it can be a particle of another substance containing a surface similar to that of the crystals to be formed from the liquid phase. If the crystal nucleus is above a certain critical size, which is a function of the temperature of the system, other molecules will align themselves with it to form a larger crystal. Particles smaller than the critical size, on the other hand, lose molecules to the liquid phase. If the temperature of the liquid is lowered slowly and there are no crystal nuclei present, the liquid may be cooled below its freezing point, or supercooled. The extent of this supercooling determines the rate of freezing when a nucleus is finally formed or introduced. Energy released by the phase transition warms the liquid until the freezing point is reached. As more energy is removed from the mixture of liquid and solid, crystallization continues as molecules from the liquid align themselves with the crystal surface.

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TEMP

BEGINNING

OF FREEZING

FREEZING POINT

.~

~

TIME FIG. 1. Temperature change with time during freezing of a pure substance. Under equilibrium conditions, temperature remains a1 the freezing point until the entire mass is frozen.

If this happens rapidly in water, long needle-like crystals appear to move through the liquid. The “motion” is apparent rather than actual, as each molecule on the crystal remains stationary, but is joined by others that cause the crystal to enlarge (Fig. 2). The size of the individual crystals formed during freezing depends upon the freezing rate. Rapid removal of energy from the liquid leads to the simultaneous formation of many nuclei spontaneously as the temperature drops considerably below the freezing point. Each nucleus thus forms a crystal that can only grow a small amount before encountering a neighboring crystal. If, on the other hand, the liquid is cooled slowly, the formation of nuclei is the result of random motion of atoms or molecules. At temperatures not far below the freezing point, this does not happen often, so the crystals that form are able to grow large before incorporating all of the surrounding liquid.

A

B

FIG. 2. Formation of an ice “needle” as seen through the microscope. Sequence A shows apparent movement of the ice crystal. Sequence B shows that the crystal has actually grown by the addition of more water molecules (shaded areas) to the original crystal.

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Under certain conditions, cooling can produce an apparently solid substance without the subunit order that is found in crystals. This process is called vitrification. The substance formed may be considered a liquid in an extremely supercooled state. The most common vitreous solid is glass. Aqueous solutions of glycerol may also be vitrified, but vitrification of pure water is very difficult. Nonetheless, vitreous ice has been found by condensing water vapor on a surface cooled with liquid oxygen (- 183°C). At this temperature the rate of nucleation is low, and solidification occurs before crystal formation can take place. When vitreous ice is warmed, it changes to crystalline ice. Several experimenters (McMillan and Los, 1965; Ghormley and Hochandel, 1971) have tried to determine the temperature of this transition, but an exact figure has not been obtained. The value appears to be between -150 and -111°C. In one set of experiments, a solid containing some vitreous and some crystalline ice was formed. When it was warmed to -129°C it became completely crystalline (Ghormley and Hochandel, 1971). For many years it appeared that freezing biological materials very rapidly would cause vitrification instead of crystallization. This conclusion was reached by a number of researchers because they could not see ice crystals in their frozen specimens. However, X-ray analysis later indicated the existence of some ordered structures in this frozen material, and it was assumed that crystallization had been imperfect or imcomplete (Luyet, 1965). Polarized light has also been used to observe ice crystals that were not visible in ordinary light. If small ice crystals are formed, either by rapid freezing or by crystallization of vitreous ice, they remain stable only at a low temperature. As the ice is warmed, some of the crystals grow at the expense of others in a process called recrystallization. Vapor pressure is a function of the curvature of the crystal surface, with the smaller crystals less stable than large ones. Thus, water is transferred from the smaller to the larger crystals, making them even larger. This process continues until all crystals are of a size that is stable at the new temperature. Meryman (1957) observed the formation of very small crystals from vitreous ice after 3 minutes at -96°C. As the temperature was raised in steps of 3 to 10°C over a period of 10 minutes to -7O"C, the crystals grew to about 10 times their original size. This phenomenon is a critical one in the preservation of biological material by freezing.

B. FREEZING IN SOLUTIONS, CELLS, AND TISSUES When a solution freezes, it usually undergoes changes of composition as well as phase. If the solubility of the solute is reduced as the temperature drops, the solution will become saturated, and the solute will separate as a solid. It is also possible for some solutes, particularly those of dilute solutions, to remain in solution until the freezing point of the solution is reached. Because of the pres-

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ence of the solute, this temperature will be lower than the freezing point of the pure solvent (water in the case of biological systems). Further cooling of the system causes some of the liquid water to crystallize, thus increasing the solute concentration in the remaining solution and lowering its freezing point even more. It is customary to consider a food or plant frozen when it appears rigid or feels firm. Actually, freezing to the temperatures usually encountered in nature or in commercial food processing usually leaves a portion of the water unfrozen. Whether freezing begins within or between the cells, the formation of some ice crystals leaves a remaining liquid phase of higher concentration and lowered freezing point. As the temperature drops, this process continues with the growth of ice crystals, between which are small droplets of solution. Reactions have been observed in these droplets at temperatures as low as -80°C (Kiovsky and Pincock, 1966a). In some systems, crystallization forms a solid of eutectic composition, in which the components exist in a definite ratio. On heating, this solid melts at a fixed temperature to form a liquid of the same composition. The behavior during freezing of even relatively simple mixtures can be quite complex. Biological systems are usually of very complex composition, and thus a detailed analysis of their behavior during freezing is almost impossible. For example, van den Berg and Rose (1959) found 11 eutectic points in the system composed of Na+, K+, K,POI 3, and water. These included four eutectic mixtures of ice and one salt, five of ice and two salts, and two of ice and three salts. To further complicate the system, three of the phosphate salts crystallized as hydrates (NaH,P0,.2H20, K2HP04.6H20,and Na,HPO,. 12H20). If the temperature of a biological specimen is recorded during freezing, the resulting curve does not show a period of constant temperature, as is found with a pure substance, but rather a gradual decrease. The slope of the curve depends upon the temperature, composition, and geometry of the specimen, and the temperature of the environment and rate of heat transfer to it. The most common cooling and freezing curve is that obtained by temperature measurement at the center of the specimen. This curve usually has a “plateau” of very slight temperature change, which is assumed by many people to represent the time for crystallization to occur. Meryman (1966) found that this is not necessarily the case; the plateau may represent only the last stage of cooling prior to freezing. If the temperature is recorded at several points within the specimen, the plateau is observed only near the center. Near the surface, the temperature drops continuously, although at a decreasing rate. Inflections begin to appear in curves of temperature measured below the surface, but only very near the center is there a plateau. If these curves really indicated the rate of crystallization, a faster rate, and therefore smaller crystal size, would be expected at the surface. Meryman (1966)

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froze starch gel in cylindrical cans and observed smaller crystals at the center than near the surface. This result is not surprising, as the portion of the specimen slightly beneath the surface loses heat only to the nearest part of the surface. However, heat from the center of the cylinder is conducted radially in all directions, so that freezing occurs more rapidly there than near the surface. In plant tissues most of the water is within the cells, in either the cytoplasm or the vacuole. Many plant tissues have spaces between the cells, in which there is water vapor. In such tissues, freezing usually begins in this intercellular water, because its freezing point is higher than that of the solution within the cells. As the intercellular water freezes, the vapor pressure in the intercellular spaces is reduced, and more water diffuses out of the cells to the intercellular ice crystals. This water loss also increases the solute concentration of the cellular fluid, reducing its vapor pressure and freezing point. These changes tend to hinder further ice formation, the former by reducing the transfer of water to external crystals and the latter by maintaining the liquid state within the cell. The extent of intercellular freezing is also controlled by the rate of cooling. If the tissue is cooled so fast that the freezing point of the cellular liquid is reached before its concentration is increased by loss of water to the intercellular ice, then ice nucleation can occur within the cell. Here, as in pure water described in the preceding section, there is an effect of temperature on nucleation and crystal growth. Because there are many substances within the cell to act as nuclei, supercooling can occur only to a limited extent. Once crystallization begins, there is an interaction between rate of nucleation and rate of crystal growth that determines whether the cell will contain many small ice crystals or few large ones. In vegetable tissues damaged by blanching, it is possible to have an ice crystal larger than a single cell. At slow cooling rates, a single crystal may grow through cell wall pores from one cell to another. Thus, the ice crystal in the second cell is merely an extension of the crystal in the first cell. In normal living cells, however, this does not occur. Brown and Reuter (1974) observed ice propagation in thin slices of tissue at slow rates of cooling. Following the freezing of the water surrounding the tissue specimen (Fig. 3), the peripheral cells that had been cut open also froze. As the temperature was lowered, only a few isolated cells froze. Further freezing almost always occurred in cells adjacent to those already frozen, and always with a delay period before the second cell froze. Apparently the nucleus for crystallization of the second cell was a small portion of the ice crystal that grew through a cell membrane pore. The rapidity of freezing in the second cell suggests that it was indeed supercooled, as does the fact that all of the water froze immediately as soon as crystallization began. The freezing point of water in a small capillary, such as would be found in a cell membrane, is lower than the freezing point of a larger mass of water. Thus, for it to nucleate freezing in a second cell, it must be cooled to a temperature lower than freezing point of the

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31

(B)

6 Sec. P-4

FIG. 3. (A) Drawing of cucumber tissue and thermocouple as seen through the freezing microscope. (B) Temperature recording during freezing of the cells in (A). Recording trace above baseline indicates that thermocouple on tissue is warmer than the freezing stage when heat of fusion is liberated as each cell freezes. Initial hump corresponds to the lowering of the stage temperature that initiated the freezing. Numbers on the temperature recording peaks correspond to the upper (larger) numbers in each cell. Lower numbers in each cell are the number of videotape frames ( 1 frame = 1/60 second) from the beginning of cellular freezing to the completion of freezing of that cell.

cell contents. When the adjacent cell froze, energy was released to the cells around it, raising their temperature. Time for the removal of this heat added to the delay between freezing of adjacent cells.

C . CHEMICAL REACTIONS AT LOW TEMPERATURES Chemical reaction rates are usually retarded by a temperature decrease. This is expressed mathematically by the Arrhenius equation: d In k dT

-=

E,IRT'

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in which k is the reaction rate constant; T, the absolute temperature; E a , the energy of activation; and R, the gas constant (1.987 calorieddegree mole). The relationship between rate constants at two temperatures, T I and T,, is expressed by the integrated form of the equation:

E, is calculated from values of k (amount of reaction in a given time, or the reciprocal of time for a certain amount of reaction) measured at two temperatures, and this value is then used to determine the rate constants at other temperatures. Such reaction kinetics data are frequently presented graphically by plotting the logarithm of k against the reciprocal of the absolute temperature. A linear relationship usually exists for nonenzymatic reactions, and also for enzymatic reactions below the temperature at which the enzyme loses its catalytic properties as a result of thermal denaturation. As the temperature drops to the freezing point of a solution of reactants, many systems deviate from the linear relationship. The rate constant for enzymatic reactions often decreases more rapidly at temperatures below the freezing point (Fig. 4). In a number of systems studied, the line appears to have an abrupt change in slope at the freezing point. It has been suggested, however, that the transition is actually a gradual one (Kavanau, 1950) resulting from a gradual transformation of some of the enzyme molecules to an inactive form. The rate constant for the hydrolysis of sucrose by invertase has been shown to undergo such a gradual

I

I WARMER

1/T

\

' \ I

COLDER

FREEZING POINT OF WATER

FIG. 4. Change of enzymatic reaction rate constant with temperature near the freezing point of the reaction mixture. Broken line indicates the gradual transition observed in some systems.

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MILFORD S. BROWN

transition (Lund et al., 1969). This study also showed that the reaction rate decreased when the concentration of either sucrose or sodium citrate buffer was increased, even in unfrozen solutions. Another solute effect will be described later in the discussion of nonenzymatic reactions. The rate constants for two solutions containing invertase, sucrose, and buffer in the same ratio, but in different concentrations, were the same if both were at the freezing point of the more concentrated solution. The more dilute solution, of course, was “frozen” at this temperature, although there would have been liquid regions at the same concentration as that of the other solution that was just at its freezing point because of its higher original concentration. In contrast to most enzymatic reactions, nonenzymatic reactions may proceed faster in the frozen state than at temperatures slightly above freezing. The story of the discovery of one of these was told by Grant (1966). He and two of his co-workers had been studying the hydrolysis of an amide linkage in penicillin in the presence of hydroxylamine. On Friday afternoon, they left one portion of their reaction mixture in the refrigerator, while another, as a control, was stored in the freezer. On Monday they found the refrigerated sample as they had left it, while penicillin in the frozen “control” had been hydrolyzed almost completely. Further study by this group and others has shown that a number of nonenzymatic reactions take place faster in ice than in the liquid state. Study of such systems is complicated by the difficulty of determining the extent of freezing and the volume of the remaining liquid phase. In most cases, the reaction is assumed to take place in the regions of liquid that exist between ice crystals, as described previously. However, Kiovsky and Pincock (1966a) suggested (as a student experiment) the following reaction between arsenic acid and iodide ion: H,AsO,

+ 31- + 2H’

+

H,AsO,

+ 1,- + H,O

This reaction occurs within a few minutes at -8O”C, at which temperature the solution presumably is completely frozen. The reaction does not take place in supercooled liquid at -5 to -1O”C, however. Other changes in the kinetics of nonenzymatic reactions are also observed on ice. Although decreasing the temperature reduces the rate of a reaction, the formation of ice increases the concentration of the reactants in the remaining liquid, and may thus increase the reaction rate. As the temperature is lowered even further, the temperature effect overcomes the concentration effect, and the reaction again decreases (Fig. 5). This effect of freezing and the two following ones should be considered changes in the “observed kinetics” rather than the actual kinetics. Reaction kinetics are usually determined on the basis of the volume of solution prepared. When this solution is partly frozen, the liquid volume in which the reaction takes

FROZEN FRUITS AND VEGETABLES

LOG K

191

I

A

P WARMER

l/T

'

COLDER

I

FREEZING POINT OF WATER WARMER

FIG.5 . Change of nonenzymatic reaction rate constant with temperature. (A) Decrease during cooling. (B) Minimum at freezing point. (C) Increase due to concentrating of reaction mixture as water crystallizes. (D)Decrease as effect of declining temperature overcomes concentrating effect o f crystallization.

place is less than the original volume. If the freezing involves only the formation of pure water ice, rather than a eutectic mixture of ice and solute, and if the solubility limits of the solutes have not been exceeded, then the solution concentration will have been increased by the amount that the liquid volume has been decreased:

c,/c,

= v2/v,

Thus, the reaction actually follows the expected kinetics if the calculation is done on the basis of the actual volume of liquid in the partly frozen system. Since it is often difficult or impossible to determine this volume, the total system volume is used, and the reaction kinetics appear to have been altered by freezing. A third effect of freezing is a change in reaction order. A reaction is of zero order if its rate is not a function of the concentration of the reactants, first order if the rate is proportional to the concentration of one reactant, second order if it is proportional to the product of the concentrations of two reactants, etc. If one of two reactants is present in great excess, the reaction may appear to be of zero order with respect to that reactant, i.e., small variations in the concentration of that reactant do not influence the reaction rate. Varying the concentration of the other reactant changes the reaction rate, so the reaction is said to be of first order with respect to that component. Although two different molecules must react, the rate is limited by the supply of only one of them. During freezing, ice formation and the resulting increase in the concentration of the remaining solution may alter the apparent order of the reaction by increas-

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ing the concentration of one reactant beyond the range in which it is rate limiting. This is most apt to happen when the concentration of one reactant is much greater than the concentration of the other. An example of this is the mutarotation of glucose, studied by Kiovsky and Pincock (1966b). Both the spontaneous and the acid-catalyzed reactions were found to be first order with respect to glucose. The acid-catalyzed reaction is of variable order with respect to acid, from a value of one at low acid concentration to almost zero at high acid concentration. In the discussion of enzymatic reactions, the inhibitory effect of high concentrations of substrate or buffer has been mentioned. Nonenzymatic reactions also appear to be influenced by solutes that do not participate in the reaction. In some systems, addition of such a solute affects the reaction by increasing the volume of the liquid regions in the ice. The freezing point of a solution is determined by the total concentration of solutes, whether or not they are participants in a reaction under study. If more solutes are added, less water will freeze, so that the same total concentration of solutes is maintained in the liquid regions. The reactive components of the system are then distributed in a larger volume of liquid. If the reaction rate is dependent upon concentration, it will be reduced even though the additional solutes do not participate in the reaction. D.

SUMMARY

Water, the major component of plants, undergoes a phase change during freezing. This requires a temperature below the freezing point, and also either a pattern (for heterogeneous nucleation), or an ice crystal (for homogeneous nucleation), to guide the rearrangement of water molecules from their random distribution in the liquid state to the ordered form of the ice crystal. Removal of energy then allows crystal growth at constant temperature. Cooling very rapidly promotes the spontaneous formation of many nuclei, each of which forms only a small crystal before using all of the water available to it. At slower cooling rates, fewer nuclei form, and crystals are larger. Under certain conditions, usually where the condensation of water vapor takes place on an extremely cold surface, a noncrystalline (vitreous) ice is formed. In such a case, if the temperature rises sufficiently to permit the necessary movement of the water molecules, crystalline ice forms from the vitreous solid. Very small ice crystals are stable only at temperatures considerably below the freezing point. As the temperature rises, the larger crystals grow even larger at the expense of neighboring smaller ones. When solutions freeze, loss of water to the solid phase leaves the remaining solution more concentrated. Some solutions also form solid phases containing solutes. Study of freezing in biological materials becomes very complex because

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of these additional phases of altered composition. Rates of chemical reactions are altered by changes of solution concentration and composition as water and other components are lost from the liquid phase.

111.

SURVIVAL OF PLANTS AT LOW TEMPERATURES

Plants differ widely in their responses to low temperatures. Some plants tolerate or even require periods of low temperature at certain stages of their yearly growth cycle, while others are damaged or killed by a brief exposure to cool temperatures above the freezing point. Many temperate zone perennial plants and their seeds have a rest period during which there is no growth even though environmental conditions may be favorable. A period of chilling is often necessary to terminate this rest and enable the plant to resume growth. Lima bean and cotton plants grow during warm weather and are so cold sensitive during germination that they can be killed by chilling to 5°C. This extreme sensitivity is lost as the plants grow. Low temperatures above freezing affect other plants less seriously, even though the changes may be of commercial importance. For example, tomatoes grown in a cool climate ripen very slowly, and are inferior to those grown in warm weather. Peas, on the other hand, are usually planted very early in the growing season so that they can be harvested before the weather becomes hot. Many plants encounter freezing conditions at some time in their life. Predicting the outcome of this encounter may be more difficult than predicting the results of the chilling. Freezing during the period of active growth probably would be damaging or even fatal, except to plants native to polar regions or very high altitudes. In temperate regions, this is less frequent than freezing in the winter, but the consequences of a single spring freeze after the beginning of growth are usually much more serious than winter freezing. Most plants that grow where winter temperatures are below freezing undergo some chemical changes that increase their hardiness during that time. Survival thus depends upon the condition of the plant as well as the rate and duration of freezing and the conditions of thawing. Some of these effects and interactions are known for certain plants but the outcome of a single unusual freezing experience, in many cases, cannot be predicted with certainty. Some of the recent additions to our knowledge about the responses of plants to low temperatures will be discussed in this section. Although writers in other countries sometimes use the word “frost” for any freezing conditions, the common U . S . usage will be followed here, with its meaning being restricted to a natural condition of temperature slightly below

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freezing, often occumng at night during the seasons that have day temperatures appreciably above the freezing point. A.

CHILLING SENSITIVITY 1.

Seeds

Seeds are a means of reproducing and increasing the number of plants. Frequently they also allow the species to survive a period of weather conditions that are unfavorable for growth or even the existence of the parent plant. In the temperate zone these conditions may include low temperature or insufficient water. Tropical plants, which grow in an area of adequate moisture and heat all year, are not always able to survive chilling or drying. Cacao (Theobroma cacao) seeds are particularly sensitive to chilling. Exposure to a temperature of 40°C for 15 minutes is lethal but after only 10 minutes of chilling, the injury can be reversed by immediate warming to 37" for 10 minutes. Ibanez (1964) found that the cacao embryo was not damaged by as much as 2 hours of chilling, but the metabolism of the cotyledons is altered. He was able to remove the embryonic axis from the chilled seed and grow it on a nutrient culture medium. Oxygen uptake of the cotyledons increased for 4 to 5 hours after chilling and then decreased. This was not the cause of death, however, because chilling followed by warming, which maintained viability of the intact seed, also caused a temporary increase of oxygen uptake (Casas et al., 1965). Woodstock et al. (1967) were able to inhibit normal respiration by adding iodoacetate or malonic acid to the cotyledons, but found that this does not appreciably reduce the uptake of oxygen. Inhibitors of polyphenol oxidase, such as pnitrophenol or 1-phenyl-2-thiourea, reduced the oxygen uptake somewhat. Apparently neither the normal respiratory reactions nor the reactions catalyzed by polyphenol oxidase were responsible for all of the greater oxygen uptake by the chilled cotyledons. This alteration of the normal metabolism apparently damaged the cotyledons to the extent that the intact seed was unable to germinate. Another type of chilling sensitivity has been found in lima bean and cotton seeds. These are sensitive during the early stages of water imbibition prior to actual growth. Cotton seeds are also sensitive to chilling after 18 to 30 hours of germination. Lima beans were studied by Pollock and co-workers (Pollock, 1969; Woodstock and Pollock, 1965), who found that chilling to 5 to 15°C for as little as 10 minutes at the beginning of water uptake retards growth. During this time the embryonic axis absorbs water, beginning at the root end, and the chilling sensitivity is lost before the entire axis is fully hydrated. Unlike cacao seeds, germinating lima beans do not recover if they are heated shortly after the cold

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exposure. However, Pollock (1969) found that the cold injury could be avoided by allowing warm seeds to absorb sufficient water vapor to bring their moisture content to 20%. The seeds used in these experiments had part of the seed coat removed so that all of them would be able to absorb water readily. The intact seed contains a protective mechanism that helps it to resist chilling injury. The permeability of the seed coat to water decreases with temperature, so that the seed is less apt to absorb water and be injured by low temperature. Although the cause of this chilling injury is not known, Woodstock and Pollock (1965) suggested that at low temperatures, respiration might be too slow to supply the energy required for stretching of cell membranes during cell expansion in the initial stages of germination. Pollock (1969) later reported additional work that showed the effect of moisture on temperature sensitivity is reapeatedly reversible. Thus, it must involve some process occurring in the seed during each imbibition of water, and not just an irreversible reaction that takes place only once. Pollock suggested that temperature might affect alterations in the metabolic pattern of the seed as it changes from storage of reserve materials during maturation to the utilization of these reserves for growth during germination. Cotton seeds also are sensitive to chilling during germination, but there are several important differences between their responses to cold and those of lima beans. Christiansen (1967) found not only a period of cold sensitivity at the beginning of germination, similar to that of lima bean, but also a second period between 18 and 48 hours afterward. The second cold-sensitive stage was at the beginning of elongation of the hypocotyl, or shoot portion of the embryo. In both cases, the radicle, or embryonic root, was damaged. Chilling during the beginning of germination inhibited elongation of cells at the root tip. This delayed further growth until new lateral roots formed above the tip. The later chilling caused disintegration of the root cortex above the meristem. Such plants never recovered sufficiently to attain a normal growth rate, measured as a change in the percent of the total seedling dry weight remaining in the cotyledons, in the 5-day observation period. Leffler (1976) found that chilling reduced ribonuclease, suggesting alteration of protein synthesis. Dogras et af. (1977) found that chilling caused glycerol to be incorporated into phosphatidylethanolamine and phosphatidylglycerol. In broad beans (Vicicia fuba L.) and peas, which are not chilling-sensitive, the glycerol went preferentially into phosphatidylcholine. The major difference between chilling injury in cotton and lima bean seeds is that the former could be permanently protected by a single warm hydration for 4 hours, followed by drying. Lima beans, on the other hand, were cold-sensitive each time they began to absorb water. Although both Pollock and Christianses suggested that cold sensitivity involves alteration of some metabolic processes, different systems must be affected in these two plants.

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MILFORD S. BROWN

Duke et al. (1977) studied the mitochondria1enzymes of germinating soybean seeds. Chilling altered the relative amounts of the dehydrogenases, and therefore, the respiration. In addition, NADP-isocitrate dehydrogenase was extremely cold-sensitive . 2 . Plants

In plants beyond the young seedling stage, a number of responses to chilling have been observed. Of these, a few have been studied in detail. Many of the published reports, however, are less extensive, ranging from observation of survival or death after chilling to changes in the amount of a particular chemical component in response to chilling. In reports of the latter type, it is frequently impossible to determine whether the change described is a primary effect, i.e., the component directly altered by the low temperature, or merely the consequence of some other chemical or physical change that alters metabolic pathways in the plant. The energy of activation of invertase from winter wheat leaves has been shown to vary with growing temperature; however, there was no change in the activation energy with temperature in a spring wheat. In a wheat variety of intermediate growth habit, there was only a slight change (Roberts, 1967). The leaves of a number of cold-sensitive plants, including corn, beans, cucumbers, and cotton fail to develop their normal green color at low temperature. The synthesis of chlorophyll apparently proceeds as far as the formation of the porphyrin ring, but the rate of esterification with phytol is reduced at low temperature. In addition, newly synthesized chlorophyll that has not yet been incorporated into chloroplasts does not contribute to photosynthesis in the plant. Instead, the energy that it absorbs when exposed to light is used for its own photodestruction (McWilliam and Naylor, 1967). Unless the rate of synthesis is greater than the rate of destruction, there can be no accumulation of chlorophyll in the leaves. In some plants, chilling even increases the sensitivity to photodestruction of chlorophyll in chloroplasts (Kislyuk, 1964a,b; Margulies and Jagendorf, 1960; McWilliam and Naylor, 1967). Apparently the chloroplasts lose the ability to remove photosynthetically-produced photooxidants by oxygen evolution. Kislyuk (1964a,b) found that cucumber, corn, and zebrina leaves kept at 2°C lose chlorophyll if they are illuminated. When they are returned to a higher temperature, the rate of photosynthesis is inversely proportional to the light intensity during chilling. Margulies and Jagendorf (1960) found similar behavior in chloroplasts from bean leaves. Chloroplasts from spinach, which is not injured by low temperature, do not lose the ability to carry out the reactions of photosynthesis after they are chilled. Although chlorophyll in corn is normally somewhat light-sensitive at temperatures as high as 15 to 17°C (McWilliam and Naylor, 1967), a strain has been

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found in which virtually no chlorophyll accumulates below 17°C (Millerd and McWilliam, 1968; Millerd e t a / . , 1969). Under similar illumination, the temperatures for equal chlorophyll accumulation in this and normal corn differ by about 4 or 5°C. Stated another way, at low temperatures, the abnormal mutant is about as sensitive to low intensity light (300 foot candles) as the normal variety is to full sunlight. Millerd and McWilliam (1968) have suggested that this very sensitive variety, which is considered a mutant form of the present day corn, might actually be the original form. As the cultivation of corn moved from tropical to temperate regions, plants capable of growing at lower temperatures would have been selected. Genetic studies indicate that the ability to accumulate chlorophyll is controlled by at least three genes. The behavior of another cell organelle, the mitochondrion, is also different in plants sensitive or insensitive to chilling. Oxidative phosphorylation in mitochondria is accompanied by swelling and contraction. Lyons et al. (1964) have observed a correlation between the chilling sensitivity of eight plant tissues and the ability of their mitochondria to swell or contract in hypotonic or hypertonic solutions. In addition, the mitochondria of plants sensitive to chilling contained less unsaturated fatty acid than those from insensitive plants. Low temperatures would tend to reduce the flexibility of mitochondria containing a large amount of saturated fatty acid, thus also reducing their ability to carry out the necessary metabolic processes. Several deviations from the expected pattern were observed. The mitochondria from bean and corn seedlings, which are chilling sensitive, showed a brief immediate response to changes in the tonicity of their suspending medium, but then did not change further. Mitochondria of chilling-resistant plants adjusted slowly over a period of an hour. Mitochondria from both chilling-resistant pea seedlings and chilling-sensitive bean seedlings and green tomato fruits were of intermediate unsaturated fatty acid levels. In spite of these few deviations from the expected pattern, the results suggest a relationship between mitochrondrial structure and function and chilling injury. The above conclusion is supported also by the work of Stewart and Guinn (1969), in which the ATP concentration of cotton seedlings was found to be affected by temperature. When the seedlings are transferred from a normal growing temperature range of 20-30°C to a chilling temperature of 5"C, the ATP concentration in the leaves begins to decrease within a few hours. If the plants are returned to the normal temperature after 1 day, the ATP concentration returns to the original level. If they are chilled for 2 days, however, they do not recover. The cotton seedlings can be hardened, or conditioned to resist chilling, by exposing them to 15°C for 2 days. At this temperature, the ATP concentration in the leaves increases at the rate of about 10% per day, possibly because the ATPgenerating reactions are not retarded as much by the low temperature as are the ATP-utilizing reactions. During a second day at 15", the ATP concentration rises

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MILFORD S. BROWN

even faster, but if the plants are then returned to 20 to 30°, the ATP concentration returns to normal. For prevention of chilling injury, apparently respiration must decrease before the energy supply becomes limiting. Lewis and Workman (1964) found that tomato fruit cells become permeable to electrolytes after a period of chilling. Phosphate esterification also declines after chilling, although initially it exhibits a temperature coefficient similar to that of the same reaction in cabbage, a cold-tolerant plant that does not become permeable to electrolytes at low temperatures. Amin (1969) studied the interaction of temperature and respiration in cotton, using the respiration inhibitors picolinic acid, sodium malonate, and Dexon (p-dimethylaminobenzenediazosodium sulfonate). Picolinic acid and Dexon were effective, but sodium malonate did not provide protection. In addition, the interactions of the inhibitors used in combination suggested that their protective effects might have been on specific metabolic systems required for growth and development after chilling. Although the tomato is a tropical plant that has adapted to lower temperatures, not all varieties are equally well adapted at all stages of their growth. For example, Kemp (1968) found that varieties that germinated well at the low temperature of 8.5"C do not necessarily grow vigorously or set fruit well at low night temperatures. It would seem possible, though, to breed a variety of tomato that would tolerate low temperatures at all stages of its growth. In an earlier study, Kemp (1965) found that the ability to set fruit at low night temperature is a recessive characteristic apparently controlled by a single gene. This would have to be combined with the ability to germinate and to produce good top and root growth at all stages of its development. In addition, of course, the fruit would have to meet the necessary criteria of acceptability for fresh consumption or processing. Temperature affects early yielding of tomatoes not only by influencing fruit set, but also by regulating the amount of shoot growth prior to the formation of the first influorescence (Phatak and Wittwer, 1965; Phatak el al., 1966). Low top temperatures, short day length, and high light intensity reduce the number of stem nodes formed before flower development. In grafted plants composed of late and early flowering varieties, a flowering stimulator or inhibitor appeared to be formed only in stocks with leaves. This was then translocated to the scion, where it promoted or delayed flowering. The number of flowers in the inflorescence was increased by low root temperatures. Thus, unlike plant growth and fruit set, early production of many flowers is promoted by the temperatures and day lengths that occur naturally in early spring. From the studies described here, we can see that plants differ widely in their responses to temperatures that are low, but above the freezing point. For some plants, chilling is fatal, but others may be only temporarily retarded by such an

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experience. In some cases the damage can be reversed or outgrown, while in others the consequences are permanent.

B.

CHILLING REQUIREMENT

Some plants require chilling temperatures to terminate their rest period. The existence of such a rest period during which the plant remains dormant even though conditions are suitable for growth is very important. It allows the plant to survive a part of the year during which the environment is usually unsuitable for growth. For some plants, the unfavorable condition may be lack of water, but for most, low temperature (winter) survival is a much more serious problem. Dormancy of its buds or seeds permits the plant or its progeny to survive these periods of adversity. Although there is some metabolic activity during this time, the rate is extremely low in comparison to the state of active growth. Thus, extreme changes of environment are tolerated at this time because they do not appreciably influence metabolic processes. Termination of the rest period requires, in many plants and seeds, a definite period of exposure to low temperature, usually somewhat above the freezing point. Once this chilling requirement is fulfilled, the plant or seed remains dormant only until its environmental conditions are favorable for growth. It is interesting that the same temperature range that is injurious to some plants of tropical origin is essential at a certain time in the growth cycle for some temperate zone plants. The period of chilling required by seeds is often referred to as either “afterripening” (since it takes place after the seed has ripened), or “stratification” (from the practice of storing layers of seeds in a moist medium at a low temperature for a period of time before planting). In nature, of course, seeds usually fall to the ground when they are mature, and remain until their rest period is terminated by cold exposure during the winter and the temperature again becomes favorable for growth. For annual plants that cannot survive winter but must grow, flower, and produce seeds in one growing season, this property is essential for survival. For perennial plants, it prevents germination in late summer or autumn, which would not allow time for the development of the woody structure and mature buds that can tolerate the unfavorable conditions of winter. Dormancy of seeds has been the subject of many studies. In some cases, the inability to germinate is not a property of the embryo itself, but the result of a growth inhibitor in another part of the seed. This is usually leached out by water during autumn and winter. Irving (1968) found that the dormancy of box elder (Arer negundo) seeds can be broken by either leaching or chilling. In some cases, however, the seed embryos can be induced to grow, but unless they have been chilled, they produce an abnormal plant. An unchilled peach embryo will

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MILFORD S . BROWN

grow if portions of the seed coat and endosperm are removed, but it produces a dwarfed plant, with short internodes and deformed leaves developing from the apical bud. Branches from axillary buds are normal, however. The dwarfing that occurs at 25°C can be prevented by cooling the germinating seed to 19°C. The period of temperature sensitivity is quite brief, extending from the beginning of root growth to the beginning of shoot elongation. The behavior of peach seeds and seedlings suggests control of growth by a self-duplicating system within the apical meristem (Pollock, 1962). Composition and metabolism in seeds changes during chilling, with a gradual increase in respiration during dormancy, followed by a more rapid increase at the beginning of germination (Pollock and Olney, 1959). Enzyme content also changes (Sanz et al., 1969), but this is to be expected as the seed shifts from an energy-storing to an energy-utilizing state. Gibberellins also appear to be involved in the changes that take place during chilling (Frankland and Wareing, 1962, Westwood and Bjornstad, 1968), but Fine and Barton (1958) found that using gibberellic acid to break dormancy does not produce the same changes in amino acid content that chilling does. The relationship between climate and seed chilling requirement was investigated by Westwood and Bjorns’tad (1968). They determined the chilling time and temperature requirements of the seeds of 14 species of pear that originated in various parts of the world, from northern China to Morocco. Seeds from the coldest climates were found to require the greatest amount of chilling to break their dormancy. Crosses between species have chilling requirements between those of the two parents. Although the chilling requirement is a characteristic of the variety of plant, it can be modified somewhat. By grafting two pear varieties of quite different chilling requirements, Westwood and Chestnut (1964) determined that the major control of dormancy is the grafted buds themselves. There is, however, some influence of the stock variety also. In addition, the chilled buds that are grafted on a chilled stock grew more than similar buds grafted on a stock that have not received the required amount of chilling. Both of these observations indicate that some factor influencing dormancy is translocated from roots or stems to the buds. Another indication of the protective function of dormancy is the observation by Kester (1969) that almond and almond-peach hybrid trees that bloomed early produced seeds that require less chilling than those from late-blooming trees. Dormancy of flower buds serves to delay blooming beyond the time of frosts that would kill flowers. Commercially, this assures the production of a good crop, and in plants growing naturally it also assures the production of seeds for propagation. Premature germination of these seeds, which could occur if the chilling requirements were satisfied too early, might result in seedlings being killed by a late frost.

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Strawberry plants become dormant as the temperature drops in autumn, and they then require a period of chilling to break dormancy. The chilling requirement is not the same for all varieties; those from warmer climates require less than those from cooler areas. If the day length is prolonged with artificial illumination in autumn, plants continue vegetative growth instead of being dormant (Piringer and Scott, 1964). Where weather conditions are favorable for plant growth all year, there may may be no dormant period. In Colombia, for example, grapes grown at temperatures between 20 and 30°C produce two crops each year. They are pruned 2 to 3 months after the first harvest, and then the new growth produces a second crop of fruit. In temperate regions, grapes become dormant after the harvest season and resume growth the following spring (de Carrizosa, 1965). In some plants, the transition from vegetative to reproductive growth requires a period of chilling known as “vernalization. This term was coined by Lysenko (from the Latin vernurn, meaning spring) to indicate the transformation of a winter cereal into a spring variety (Chouard, 1960). Winter cereals must be planted in autumn so that the plants are chilled in the winter and are thus able to produce a crop during the following spring and summer. If the seeds of such varieties are planted in the spring when the weather has become warm, the plants produce three to four times as many leaves before flowering. Thus, there is not enough time for the crop to mature before autumn. Spring varieties do not have this chilling requirement, and therefore they flower at an early stage of growth. Some plants, including all cereals, can be vernalized during the first day of germination, when the seed has imbibed water but has not yet begun to grow. Others must grow to a certain stage, frequently a rosette formed by a number of leaves on a stem with extremely short internodes. A variety of interactions with photoperiod exist also, including requirements for long or short days, insensitivity to day length, and growth responses that vary with the extent of exposure to long days. The light and temperature requirements may even vary within a genus or species (Chouard, 1960). For example, the genus Dianrhus includes species with partial or total requirements for long days and a complete range of chilling requirements. Chrysanthemum, a short day plant, also has a wide range of chilling requirements, depending upon the variety. Control of flowering is thus a complex process, and temperature is only one of the regulatory factors. In some cases, vernalization can be reversed by high temperatures or short days. Very low temperatures interrupt the process, as does drying of the seeds that are capable of vernalization when they are moist. In summary, several factors appear to be common to all forms of vernalization. The plants must be at some particular stage of development, and they must be capable of carrying out the necessary metabolic processes. Because active metabolism is involved, many changes occur, making it difficult to select the one ”

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or more factors that are actually responsible for the change from vegetative to reproductive growth. C.

WINTER HARDINESS

Temperatures near but above the freezing point may be either beneficial or harmful to plants, as described in the previous section. Freezing, on the other hand, is always a disturbing phenomenon, even though it is tolerated by many plants. Those plants that are unable to survive freezing in the vegetative state must, unless they are assisted and protected by man, either remain in tropical climates or spend the winter in the seed form and complete their vegetative and reproductive growth in the warm seasons of 1 year. There are many degrees of freeze-hardiness. A number of herbaceous plants have stems and leaves that are killed by freezing, but their roots survive. New shoots grow from the roots each year. Other herbaceous plants are able to withstand freezing of their aerial parts only at some stage of their life cycle. Those that grow in very cold climates, or at high altitudes, on the other hand, may experience freezing temperatures at night even during the summer without damage. Woody plants also differ in their degree of freeze-hardiness. Citrus, which are semitropical evergreen plants, can tolerate only a small amount of ice formation in their leaves. Most survive occasional freezing temperatures of short duration by supercooling without freezing. The buds, wood, and bark of most deciduous plants tolerate freezing during dormant periods, although dormancy is not neccessarily a prerequisite for hardiness. Temperate zone evergreen trees and shrubs withstand winter freezing, and some even tolerate temperatures slightly below freezing in the summer. In general, the needle-leaved evergreens are hardier than the broad-leaved ones. Some of the recent studies are discussed in this section. These include regulatory mechanisms that initiate hardening, metabolic changes associated with winter hardiness, and the dehardening that follows warmer weather. 1.

Development of Cold Hardiness

Most plants that survive freezing temperatures during winter experience a frost-free season during which their major growth takes place. During the growing season, most are relatively intolerant of freezing. Survival at low temperatures in winter must be preceded by adaptive changes in the metabolic processes and chemical constituents of the plant. Initiation and control of these changes have been studied quite extensively. Several distinct phases of hardening are recognized. The first of these is the result of the decreasing day length during late summer and autumn. The second takes place only at temperatures slightly above

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freezing. In addition, plants that can survive very low temperatures undergo further hardening at temperatures somewhat below freezing. A prerequisite for the beginning of hardening in woody plants is the termination of growth. In some plants this is not an important factor, because the annual growth is limited to the stem and leaves already formed in the buds of the previous year. Other plants continue to produce new leaves and stem tissue as long as environmental conditions are appropriate for growth. In the latter case, it is important that vegetative growth ceases and buds for the next season’s growth form in time for hardening to be fimpleted before really cold weather sets in. In plants native to a cold area, this is usually not a problem. Abnormal conditions, however, such as a drought followed by a period of ample water, or pruning or heavy fertilization of cultivated plants, can bring about late growth that is not able to begin the hardening process at the usual time. Another problem can arise when plants from a warm climate, where cold hardiness is not necessary, are transplanted to a colder area. For example, Perry and Hellmers (1973) found that seedlings of Massachusetts maples (Acer rubrum L.) ceased growth when exposed to short days and cool temperatures, but seedlings of the same species from Florida grew continuously. Axillary buds of these trees also responded differently; those of the cool climate plants required chilling before growth would resume, while those from the warmer area grew whenever the adjacent leaves fell or were removed. In a cold climate, the latter plants are not able to develop the cold hardiness necessary for winter survival. Fuchigami et al. (1971b) studied hardiness development in red-osier dogwood (Cornus srolonifera Michx.) from two different climates at the same latitude, which differed by as much as 8 weeks in the development of cold hardening. Although both can eventually survive extremely low temperatures if allowed to harden properly, the warm-climate plants are injured by early autumn frosts that the others are already prepared to tolerate. The results of experiments in which plants of different hardening characteristics were grafted together suggest that there is some inherent controlling factor. In maples, the differences were so great that the southern-grown clone could not be induced to harden by anything translocated from a branch of the northern clone. On the other hand, both clones of red-osier dogwood were able to harden. In this case, the hardiness promoter was apparently formed in the leaves and translocated to the less hardy branch, if the latter had been defoliated. The requirement for defoliation may be an indication that a regulatory substance was translocated with the carboyhdrates formed by photosynthesis in the foliated branch. Fuchigami et al. (1971a) suggested that those plants that continue growth late in the season may not accumulate the compounds necessary for hardening because the products of photosynthesis are needed for growth. The accumulated substances must not be the common carbohydrates, however, because glucose or

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sucrose in a culture solution did not enhance hardening of defoliated stems (Fuchigami ef al., 1973). The ability to harden is not always preceded by development of the dormancy that is broken only by chilling. Irving and Lanphear (1967a) achieved considerable hardening of viburnum ( V . plicatum tomentosum) in which dormancy was prevented by exposure to long days. They also found that hardiness can be developed if growth is retarded by low temperatures, particularly at night (Irving and Lanphear, 1967a,b), or by the application of growth retardant chemicals (Irving and Lanphear, 1968). They suggested that a hardening inhibitor was formed in leaves exposed to long days, and Irving (1969) suggested that in box elder Acer negundo, this inhibitor was abscisic acid. Defoliation, either manually or by exposure to low temperature, was also effective in promoting hardening, possibly by removing the site of inhibitor synthesis. Howell and Weiser (1970b) also presented evidence of a hardiness promoter, which actually might be a growth inhibitor, that is translocated from apple leaves exposed to short days. In their experiments with the northern and southern red maples, Perry and Hellmers (1973) found that both clones accumulated abscisic acid to the same extent in response to reduced day length and temperature, even though abscisic acid, however, did not bring about the formation of normal buds, cold tolerance, or dormancy. Accumulation of abscisic acid appears to be one of the consequences of short days and low temperatures, but not the primary controlling factor in the development of winter hardiness in these plants. Other plants may respond differently to added abscisic acid. In studies of the interactions of various stresses and abscisic acid, Boussiba et al. (1975) reported protection of tobacco ( N . rustica) and several cereals (as measured by the leakage of ninhydrin-reacting substances) by the addition of abscisic acid. In tobacco, the beneficial effect is not obtained during the summer, an indication of the complexity of the various systems that interact to regulate plant growth. Waldman et al. (1975) suggest that the development of hardiness is a function of the ratio of abscisic acid to gibberellin. In alfalfa, unlike the red maple mentioned above, no gibberellin is formed after abscisic acid is supplied to the plants. In plants grown without addition of growth regulators, this condition is found only in cold-acclimated plants of a hardy variety. In contrast, gibberellin remains in a nonhardy variety of alfalfa exposed to hardening conditions. This suggests that growth or hardening may be determined by the relative amounts of gibberellin and abscisic acid, rather than the actual amount of only one or the other of these compounds. 2 . Factors Affecting Hardiness Carbohydrates. The hydrolysis of starch to glucose has long been known to be a consequence of the exposure of hardy plants to low temperatures. The occur-

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fence of starch in granules within plant cells limits interaction with the cellular water. Hydrolysis of these large, insoluble molecules to glucose permits distribution throughout the cell of carbohydrate that can reduce disruption of the cellular organization by interfering with the removal of water to ice crystals. Moisture stress can also induce this carbohydrate transformation, increasing the resistance to water loss by drought as well as by freezing (Chen et al., 1977; Chen and Li, 1977). This reaction to temperatures slightly above freezing has been observed in a wide variety of plants, from cabbage (Dear, 1973) to maple trees (Marvin and Morselli, 1971). Pomeroy and Siminovitch (1971) used the electron microscope to follow the disappearance of starch granules in black locust (Robinia pseudoacacia L.) phloem cells in autumn prior to the development of maximum hardiness. Beginning in the spring, new starch granules are formed, reaching a maximum in early October. Storage of carbohydrate as starch removes it from participation in growth processes, and provides the reserve needed for winter hardiness. Lasheen and Chaplin (1971) observed a similar transformation in peach leaves and shoots. Flower buds, on the other hand, do not accumulate starch in the spring prior to blooming time, but do contain a large amount of both reducing sugar and sucrose. The concentration of sugars is highest in the most hardy variety studied. The potato is not normally considered to be a cold-hardy plant, because the leaves and stems are killed by frost. The tubers, however, exhibit the starch-tosugar conversion in response to low storage temperatures. Presumably this reflects the tuber’s ability to withstand the low temperatures of the high altitudes to which they are native. This property is no longer of value to the cultivated potato, because tubers for planting are stored during the winter at temperatures above freezing. It is important in food preservation, though, because storage temperatures low enough to preserve the tubers also cause sugar formation from starch. When these potatoes are then fried, the high sugar content causes excessive browning. Processors of potato chips and French fried potatoes must reduce the amount of sugar, either by washing the cut surface with hot water or by holding the tubers at the higher temperature for a few weeks before processing so that the sugar is metabolized (Brown and Morales, 1970). This adverse effect of low temperature can also be corrected by an additional low temperature treatment. Weaver and Hautala (1971) used a brief freeze to damage the surface cells of the cut potatoes and thus facilitate subsequent removal of the sugar with water. The effect of chilling on reducing sugar buildup does not necessarily require an intact tuber. Pollock and ap Rees (1975) observed it in cultures of tuber cells that had been transferred from the normal 25 to 2°C. Both reducing sugars and sucrose increased within 3 to 5 days after the temperature reduction. Amino acids and proteins. The effect of low temperature on amino acids and proteins appears to be the opposite of the carbohydrate cycle. In a wide variety of

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woody plants studied recently, the concentration of protein (i.e., the polymer) increases in autumn, while the free amino acids (the monomers) decrease. In black locust (Robinia pseudoacacia L.), the increase consisted initially of water-soluble proteins, followed by the formation of a glycoprotein during the latter stages of hardening (Brown and Bixby, 1975). A similar increase in soluble protein has been observed in Korean boxwood (Buxus microphylla) (Gusta and Weiser, 1972), along with an increase in membrane-bound proteins. None of these studies provided proof of the actual participation of these proteins in the protection of the plant against damage by freezing. Heber (1970) fractionated the proteins from spinach leaves and other plant tissues. One of the proteins, present only in hardy tissues, was able to prevent the destruction during freezifig of the ATP-forming system associated with chloroplast membranes. Very low concentrations of this protein (about 0.1% w/v) provided as much protection against freezing damage as 2 or 3% of sucrose or glycerol. The mechanism of protection is unknown. Lipids. Lipids serve a number of functions in plants, structural, metabolic, and energy storage. As a major component of cellular and mitochondria1 membranes, lipids provide a degree of flexibility required for metabolic function and the transport of metabolites. In many cases, there is no specific requirement for a particular fatty acid, but rather a need for certain physical properties. Thus, plants growing in warm climates or warm seasons tend to have larger amounts of saturated fatty acids (i.e. those of higher melting point) while cold climate plants contain more of the unsaturated fatty acids (Lyons et al., 1964). Similarly, among those plants capable of adapting to winter conditions. exposure to hardening temperatures brings about an increase in the amount of unsaturated fatty acids (Grenier et al., 1975; Stoller and Weber, 1975; Willemot et al., 1977). Phospholipids and galactolipids, also important constituents of cell membranes, likewise undergo changes during hardening. In alfalfa a temperature decrease produces an increase in phosphatidyl choline and phosphatidyl ethanolamine, while two other phosphatides, phosphatidyl glycerol and phosphatidyl inositol, are decreased (Kuiper, 1970). In black locust bark, hardening was accompanied by a doubling of the phospholipid content, but very little change in the degree of unsaturation of the fatty acids (Siminovitch et al., 1975). In potato leaves, which are not freeze-hardy, freezing causes a decrease of all of the phospholipids present. Supercooling to the same temperature does not cause these changes (Rodionov et al., 1973). In the bark of trees that survive winter freezing, phosphatidyl choline and phosphatidyl ethanolamine increased greatly during hardening, while other lipid fractions either increased slightly (Siminovitch et al., 1968), or decreased (Yoshida, 1974). In the latter case, it was suggested that the triglycerides might have been reduced by conversion to phospholipid during hardening. E n z y n r s . Alterations in a number of enzyme systems have been observed in plants undergoing changes of hardiness. The invertase of wheat leaves has been

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found to have a lower energy of activation when the plants were grown at 6" than when they were grown at 20°C (Roberts, 1967). Subsequent study showed that there were three isozymes of invertase, and in winter wheat, the relative amounts of two of these depend on the growing temperature (Roberts, 1973). Low temperature (6°C) promotes the formation of a large amount of a high molecular weight enzyme in which the protein is associated with carbohydrate, and reduces the amount of a lower molecular weight form, in comparison to plants grown at 20". This change does not take place in a cold-sensitive spring wheat, however. The third invertase isozyme does not appear to be related to hardiness, but increased in both the winter and spring wheats at the low temperature (Roberts, 1975). A number of peroxidase isozymes are also present in plants. Growing wheat at 6°C causes an increase in one of these over the amount present in wheat grown at 20°C. Like the third invertase isozyme described above, this peroxidase change is found in both hardy and cold-sensitive varieties. Thus, although it is affected by temperature, it apparently does not contribute to the development of hardiness in wheat grown at low temperatures (Roberts, 1969). Alfalfa peroxidase, unlike that in wheat, increases during hardening and decreases during dehardening (Krasnuk er al., 1975). Oxidation of indoleacetic acid, a reaction thought to be catalyzed by peroxidases, also varies with the peroxidase activity and hardiness changes. A greater ability to oxidize indoleacetic acid would provide a mechanism for reduction of growth rate as winter approached. In most cases, cessation of active growth is a prerequisite for the development of hardiness. Another feature of hardy varieties of plants is the ability to maintain an effective metabolic system. In sensitive varieties of winter wheat (Karmanenko, 1972), and in pea and potato plants (which do not survive freezing) (Sycheva and Vasyukova, 1972), subzero temperatures cause uncoupling of oxidation and phosphorylation reactions. Thus, energy reserves of the plant are used without the formation of the phosphorylated intermediate compounds required for the normal metabolic processes of the plant. Observation of cells of sugar maple ( h e r saccharurn Marsh) roots chilled on a microscope stage demonstrated a pH decrease from about 6.5 to 5.5 as the freezing point was approached (Marvin and Morselli, 1971). This could be a factor in the alteration of carbohydrate metabolism at low temperatures, although in this study the particular reactions sensitive to pH were not determined. A study of the enzymes of sugar phosphate metabolism has shown seasonal variation in the activity of a number of enzymes. The changes in cold-sensitive plants are those that would be detrimental to energy utilization. Cold-resistant plants, on the other hand, retained their metabolic organization under chilling conditions (Sagisaka, 1974). Enzymes in subcellular organelles appear to be protected from some of the deleterious effects of freezing. Singh et al. (1977) isolated mitochondria from

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rye plants that had been frozen to various temperatures. Normal respiratory reactions were found in all of the mitochondria, even after the plants had been killed by freezing. Similarly, Senser and Beck (1977) froze spruce chloroplasts and, after thawing, found them capable of carrying out their normal photochemical reactions. They suggested that damage to these organelles after freezing may be the result of interactions with compounds or enzymes released from more labile portions of the cell.

3 . Dehardening and Spring Frost Injury Dehardening (loss of the ability to survive low temperatures) is the response of hardened plants to temperature increases. During the rest period, this is a reversible process; hardiness follows temperature with a lag time of a few days. Thus, a warm period in winter causes loss of hardiness. If this is followed immediately by extremely low temperatures, plants will be damaged before their hardiness increases again. On the other hand, a gradual temperature decline is tolerated, because there is time for hardiness to increase again. When the chilling requirement of the plant has been satisfied, thus completing the rest period, favorable temperatures will permit growth to begin. As this condition is approached, hardiness is lost 2 to 3 times as fast as it is gained in the autumn (Howell and Weiser, 1970a; Hamilton, 1973; Zehnder and Lanphear, 1966). If there is no serious temperature decline, the plant develops normally. Often, however, a warm period may be followed by freezing temperatures. Several studies have shown that during such a period, rehardening is limited to the degree of hardiness existing at or slightly before the beginning of the temperature decline. Thus, once dehardening has proceeded for a while, the plant is not able to return to the level of hardiness that existed during the winter (Howell and Weiser, 1970a; Hamilton, 1973). Although dehardening may lead to freezing damage during dormancy if extremely low temperatures follow warm weather, the more common occurrence among crop plants is damage by frost after growth has begun. The first growth process of many fruit trees in the spring is the opening of the flower buds that were formed during the previous growing season. Thus, there is a higher probability of frost damage to the flowers than to the vegetative growth that follows. Damage to the flowers, of course, can reduce or eliminate the crop for that year, or cause damage to the fruit that reduces its value (Simons and Doll, 1976). Unlike fruit trees, grape vines produce shoots with flower buds at the beginning of the bearing season. These shoots are subject to partial damage in the range of - 1--3"C, and complete killing below that. In varieties with secondary buds that can produce flowering shoots, a smaller crop may be produced if the first growth flowers are killed. In some varieties, however, this secondary growth is not productive.

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A number of methods of spring frost protection are now available. The most obvious, of course, is the use of fuel-burning heaters. However, the rising cost of fuel has made it more economical to use this energy to move other sources of heat. Thus, in areas that have temperature inversions, wind machines are used to move warm air from above the orchard or vineyard to the ground level. The air motion also counteracts cooling of the plants by radiation, which can lower plant temperature below that of the surrounding air (Proebsting, 1975). Another source of frost protection is water. By spraying the plants to cool them during warm weather before the normal growing season, it will delay early growth that would be susceptible to later frost injury (Anderson el al., 1975). During frosty nights, the same system can provide protection as the heat of fusion is liberated when the water freezes on the plants. As long as sufficient water is applied to maintain some of the liquid phase on all of the plants, plant temperatures will not drop below 0°C. This, of course, is above the freezing point of the cell contents, and therefore is not harmful. Protection by this method requires the capability of applying sufficient water to all of the plants simultaneously, as long as the temperature remains below the freezing point of the plants. Although cooling with water to delay growth also requires a complete distribution system, continuous application is not necessary, and a smaller water supply can be distributed intermittently to parts of the orchard or vineyard. Bauer et al. (1976) delayed blooming of peach trees 15 days by this method. Wood hardiness was not altered, but fruit buds on sprinkled trees were hardier for the first 1S months of sprinkling. Although there was some damage to the sprinkled flower buds, more of the live buds set fruit, probably because of better conditions at the later blooming date. Moisture in the soil increases its capacity to absorb solar energy during the day, and this will warm the air at night. Similarly, water applied to the ground will provide some heat to the air. A fog of fine water droplets has been used to reduce the loss of heat by radiation, but under certain climatic conditions, it is difficult to maintain the fog coverage (Proebsting, 1975). Delaying spring growth with growth regulating chemicals is another means of reducing susceptibility of spring frost. Proebsting and Mills (1976) delayed the growth of flower and vegetative buds of sweet cherry (Prunus avium L.) by applying ethephon during late summer. The winter hardiness of buds was increased by only 1 to 2°C by this treatment, but blooming was delayed several days. Later blooming could reduce the need for other means of frost protection during the spring.

D.

SUMMARY

Plants exhibit a wide range of responses to low temperatures. cool temperatures slightly above freezing are fatal to some plants at some stage of their life.

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Others require such temperatures during the dormant stage of their annual cycle to enable active growth to resume in the spring. In some cases, chilling injury is most harmful if the low temperatures occur during some stage of germination in the spring. In others, damage is more apt to occur if that part harvested for food is kept at too low a temperature in an effort to prolong its life in storage. Various metabolic changes or imbalances then lead to the development of chemical or physical abnormalities. Some metabolic changes are the result of inflexibility of membranes whose lipids contain saturated fatty acids. Chilling-tolerant plants, on the other hand, tend to have membrane lipids that contain more unsaturated fatty acids, thus allowing greater flexibility at cool temperatures. Temperatures of the same range as those responsible for chilling injury in some plants are required by other plants to terminate their winter rest period and allow growth to resume when the temperature becomes favorable in the spring. This prevents winter growth that would be killed by subsequent freezing weather. Low temperatures are also required by some plants to initiate the change from vegetative to reproductive growth. Plants that survive freezing in the winter exhibit a wide range of degrees of hardiness. Citrus leaves can tolerate only a slight amount of ice formation. Some of the deciduous and coniferous trees of the temperate zones, on the other hand, can survive winter temperatures even lower than those that occur naturally. In laboratory experiments some have even survived immersion in liquid nitrogen (- 196°C). With few exceptions, however, plants are not able to survive freezing during the seasons of active growth. Hardening, the process of acquiring resistance to freezing, is a response to photoperiod, temperature, dormancy, or a combination of any or all of these factors. Some of the controlling factors are translocatable within a plant, even across a graft, while others appear to be an inherent property of the tissue itself. Growth promoting and inhibiting substances appear to participate in some plants. These and other compositional changes are related, in some plants, to changes of enzymatic activity. In most plants, hardening is accompanied by the breakdown of large carbohydrate molecules to simpler sugars, increasing the water binding capability of the tissues. Protein content, on the other hand, tends to increase, although the changes involved are not well understood. Only one protein has definitely been proven to increase cold hardiness. The fatty acid composition of lipids may reflect both the degree of hardiness and the native habitat of a plant. Plants from cool climates possess unsaturated fatty acids, while those of tropical or semitropical origin have saturated ones. Flexibility of membranes is required for certain metabolic processes, and thus only those plants that contain or can produce unsaturated fatty acids are able to function at low temperatures.

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Certain metabolic functions that change with temperature are those catalyzed not by a single enzyme, but by two or more isozymes. Each of these may differ slightly in its activity and temperature sensitivity, and thus the predominating reaction varies with temperature. Two enzymes known to function this way are invertase, which affects carbohydrate metabolism, and peroxidase, which regulates growth by its effect on indoleacetic acid. In some plants, cold sensitivity is the result of the uncoupling of respiration and phosphorylation reactions. Photosynthetic products are used, but the resulting energy is not available for other essential reactions. Plants that alter their metabolism and composition to increase hardiness in winter are also able to reverse these changes, or deharden, in response to temperature increases. During the winter rest period, these changes are reversible if the temperature drops again. However, rapid cooling is dangerous because the rehardening process may require several days. After the rest period has been terminated by sufficient exposure to low temperatures, dehardening may exceed the capability to reharden in response to subsequent cooling. Damage to the flowers of deciduous fruit-bearing plants by early spring freezes is probably the most serious economic consequence of this type of injury. A number of methods have been used to protect plants from freezing damage, including artificial heating, circulated naturally warm air, artificial fog, and water sprinkling. Sprinkling has also been used to cool plants and thus maintain their dormant state or delay growth. Growth regulating chemicals have also been used to delay blooming.

IV. REFRIGERATED AND FROZEN FOODS A.

PLANTS USED FOR FOOD

Fruits and vegetables include a wide range of plant parts harvested and eaten at various stages of maturity (Fig. 6). Most leaves are eaten at a relatively early stage of their life. So are some stems, such as asparagus, which is harvested shortly after it emerges from the ground. Others, such as those of broccoli, are eaten only after the plant has begun the reproductive phase of its growth. Broccoli flower buds that terminate these stems, however, are quite immature. Roots or other underground organs are eaten only after they have matured. Foods commercially classified as fruits are usually eaten in the mature state or even whtan senescent; thus their storage life is limited. Many vegetables are actually the plant parts botanically classified as fruits, since they are the seedbearing organs of the plant. Some of these (e.g., green and wax beans, cucumbers, and summer squash) are eaten before they are mature. Tomatoes and

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FIG. 6 . Hypothetical plant illustrating the paas of plants used as food.

melons are harvested at a later stage and have a relatively short storage life. Winter squash is also harvested at maturity, but can be kept for a long time without using special storage conditions. For the plant, seeds are a means of propagation and of survival under conditions unfavorable for growth. Thus, many mature seeds can be stored dry at moderate temperatures for a long time. This property has made grains the most important food for much of the world’s population. With the exception of rice, most grains are prepared for use as flour. Some vegetable seeds (e.g., peas and beans) are dried and then eaten after rehydration and cooking. Between rehydration and consumption, they may be preserved by canning or freezing. Small amounts of rice and other grains are also used this way. Other seeds are consumed at an earlier stage of development. Lima beans are often eaten when they have enlarged fully, but are still “green.” Peas should be

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harvested before the seeds have attained their maximum size and starch accumulation. Sweet corn is best at an even earlier stage when the endosperm still contains a large amount of soluble carbohydrate and water. These examples illustrate the wide range of plant products consumed as food, from very young seedlings to completely mature seeds. Many of these are chilled or frozen for storage so that we can enjoy them at any time of the year, rather than only during their limited fresh life after harvest.

B.

REFRIGERATED FRUITS AND VEGETABLES

Fresh fruits and vegetables are living organs that continue their metabolic processes after they have been harvested. In mature seeds, the rates of these reactions are so slow that storage at ambient temperatures is satisfactory. In most other plant organs, however, the reaction rates are so fast that within a few hours after harvest, the product begins to deteriorate due to utilization of energy storage compounds, development of flowers or seeds, senescent breakdown reactions, and/or loss of water. Most of these changes can be retarded by decreasing the temperature of storage. Usually it is best to begin the cooling as soon as possible after harvest. This is particularly important for crops that are harvested during the summer, when the high temperature promotes both metabolic processes and water loss. The development of refrigerated transport and storage facilities has made it possible to produce large quantities of crops in areas best suited for their growth. For some products, the slightly longer storage life allows them to be shipped to markets in distant parts of this country. Others, the storage life of which is greatly lengthened by refrigeration, make possible a varied diet of fresh fruits and vegetables at times other than immediately after harvest. For some crops, the metabolic rate is further retarded and storage life is lengthened by providing an atmosphere of reduced oxygen and increased carbon dioxide content. An exceptionally successful product for this controlled atmosphere storage is the apple, which is now available throughout the year. Not all crops are adapted to low temperature storage. Fruits and vegetables of tropical or semitropical origin are subject to the chilling injury previously described for plants (Section 111) (Lyons, 1973). A number of these are grown as warm season annual crops in temperate climates, including tomatoes, peppers, sweet potatoes, cucumbers, melons, and green beans. In most of these, chilling injury develops after storage at or below 12°C (54°F). Products with surface lesions due to chilling injury are susceptible to additional injury by molds and fungi. Chilling injury in fruits and vegetables, as in growing plants, appears to be the result of metabolic imbalances and changes in membrane permeability. Some of

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these are reversible if the temperature is raised before the damage becomes excessive. Products in which the damage is not reversible must be stored above the damaging temperature. C. FROZEN FRUlTS AND VEGETABLES 1 . Consequences of Commercial Freezing and Associated Processing Steps

Most fruits are borne on perennial plants. Although the roots and stems of many of these endure winter cold, the leaves, flowers, and fruit are not normally required to survive freezing temperatures. Most vegetables, on the other hand, are grown as annual plants. Although a few are able to survive cold, most are not freeze-hardy, and many do not even tolerate prolonged chilling at temperatures slightly above zero. Freezing would be expected to be even more disruptive than nonfrozen cold storage, and for most fruits and vegetables this has indeed been observed in practice. The fundamental cause of the undesirable chemical reactions accompanying freezing appears to be the disruption of the normal compartmentalization of living cells. Substances that are kept apart in the living cell can come together after this disturbance, and the subsequent reactions that take place can lead to the loss of desirable flavor, odor, and color components, and the formation of undesirable ones. The structural polymers are also subjected to degradation, which results in softening of the tissues. The most serious chemical causes of quality deterioration are certain enzymatic reactions. In 1929, Kohman of the National Canners Association, and Joslyn and Cruess of the University of California found that blanching (a brief heating of the product) will inactivate these enzymes (Tressler and Evers, 1943). In the absence of these biological catalysts, the vegetables are stable during long periods of frozen storage. Some minor negative consequences of blanching have been reported. Using an electron microscope, Crivelli and co-workers (Bassi and Crivelli, 1968, 1969; Crivelli and Bassi, 1969; Crivelli et af., 1971; Monzini e f a l . , 1969) observed a number of ultrastructural modifications in blanched vegetables. These in turn make the vegetables more susceptible to damage during freezing. The additional freezing damage that results from prior blanching is much less than the amount of damage done by freezing of unblanched products. No sensory appraisals of these vegetables were reported, but presumably the consequences of the additional freezing damage would be very slight. Certainly they would be preferable to the enzymatic changes that take place in the absence of blanching. Because heating can cause noticeable flavor changes in foods that are not normally cooked before they are eaten, other preservative methods are often used

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for such products. Sugars andor antioxidants are added to fruits to inhibit browning and other oxidative reactions that begin when cells are damaged by cutting or freezing. The texture changes in frozen fruit are more serious than the chemical changes. The major contributor to the texture of fresh fruits is the turgor, or internal pressure, of their cells. When the fruit is eaten, this provides resistance to compression, followed by a sudden yielding as the cell walls burst. This behavior is the texture characteristic known as crispness. In some vegetables, structural tissues also exhibit this behavior of resistance and yielding, because of the thickness of their cell walls. Most fruits, on the other hand, are composed of very thin-walled cells. When the membranes responsible for the retention bf fluid and solutes are damaged by freezing, the texture is altered. The poor quality of wilted lettuce or celery provides an excellent demonstration of the importance of cell turgor. A frozen strawberry has the same deficiency, but unlike the living tissues of the wilted lettuce or celery, cannot be revived by the addition of water. An additional texture change occurs as a result of cell wall breakage during freezing (described in more detail later). Although its contribution to softening is not as great as that of the turgor loss, reducing this damage by rapid freezing brings about a significant improvement in the quality of the frozen fruit (Wolford et al., 1971). It is often recommended that frozen fruits be served before thawing is completed, so that the ice crystals will provide a degree of firmness to compensate for that lost during freezing. This necessity for advance planning and careful timing is probably a factor in the limited popularity of frozen fruits. Freezing does preserve the fresh flavor and aroma of fruits better than other processing methods. While this seems to be a positive factor, it may also cause them to suffer the comparison with fresh fruits, rather than being favored over other preserved ones. Unlike fruits, certain frozen vegetables have become very popular. They are subjected to the same loss of turgor that occurs in fruits, but so are their fresh counterparts when they are cooked. Some softening during cooking is expected or even required, and the changes that result from blanching and freezing may merely reduce the amount of cooking required before the vegetables are eaten (Table I). The convenience of rapid preparation and the good preservation of color and flavor have both contributed to the increased use of frozen vegetables at the expense of canned and fresh ones. For those cuisines that demand only brief cooking, and for individuals who prefer the crispness of vegetables that are cooked only slightly, the texture of some frozen vegetables are not acceptable. Migration of water from its normal locations within the cells to centers of crystallization imposes stresses that may be relieved either by breaking of thin cell walls, or by separation of thick-walled cells from each other.

216

MILFORD S . BROWN TABLE I EFFECT OF PROCESSING STEPS ON SHEAR RESISTANCE OF GREEN BEANS Treatment"

Shear resistanceb

Raw BI B2 B, F B2F B,FC BpFC

100 72

62 40

30 14

11

1' B , , Blanched 5 minutes at 190°F; B,, blanched 2 minutes at 210°F; F, frozen; C , cooked. Multiple treatments were applied in order listed. Total work (area under curve on shear press recorder chart) expressed as percent of that for raw beans.

The extent of cell wall breakage or separation is a function of the rate of freezing. Brown (1967) showed that good preservation was achieved by freezing blanched green beans in 10 minutes or less (see Figs. 7 and 8). Freezing in 20 minutes or longer produced damage, recognizable by sensory appraisal panels in comparisons with faster frozen beans. Even more obvious was the texture improvement that results from the elimination of the small amount of damage in the beans frozen in 10 minutes. Apparently small differences are more readily recognized in very well preserved vegetables than in those with extensive damage. With the commercial development of very rapid freezing methods (to be described later) that minimize freezing damage, the ability to recognize this improvement in the cooked vegetable was questioned. It was thought that the improved texture of rapidly frozen products might be negated by overcooking. Brown (1971) froze carrot and zucchini slices and green beans to obtain different amounts of freezing damage, and then served them to sensory appraisal panels after short, normal, and long cooking periods (Table 11, Fig. 9). In most comparisons, differences in texture became more apparent with longer cooking times, even if they were not so recognized in the undercooked vegetables. As a further indication of the distinction between softening by cooking and that caused by freezing, pairs of green bean samples were presented to the panel in which the better preserved one was cooked 50% longer than the poorer one (21 vs. 14 minutes). Here also the better texture of the rapidly frozen sample was very obvious (Table 111). In a similar comparison of shorter cooking times (14 vs. 7 minutes), however, the difference was not apparent. From these experiments, it can be seen that faster freezing produces an improvement that is not lost during cooking.

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FROZEN FRUITS AND VEGETABLES

A

B

FIG. 7 . Cross sections of bean pods illustrating condition of frozen beans in Fig. 8 . ( A ) Good condition (black lines). ( B ) Fair condition (white lines).

Another method of increasing the firmness of fruits and vegetables is by the addition of calcium ions. These form additional intermolecular linkages in the pectin, strengtheniiig the cell walls. This contribution to the texture, unlike the turgor effect, remains after the initial breakage of the cells during chewing. A very important factor in the texture of some vegetables is their starch content. It is a major factor in seeds, and in some underground storage organs,

2-

1

. .. f

i

\

i

.i: :.: . . .. .. ... .. .. f

G

C

F

#

0

FIG. 8 . Effect of freezing time on condition of frozen green beans

MILFORD S. BROWN

218

TABLE II EFFECT O F FREEZING RATE AND COOKING TIME ON TEXTURE OF GREEN BEANS Percent ofjudgments indicating that sample frozen faster was firmer or preferred Freezing methods compared“

Freezing damageb

Cooking time (minutes) I 14 21

“Immersion in R-12, air blast through unpackaged pieces, or 1 -kg package cooled by circulating air. A = least damage, C = most damage. Letters correspond to those of Fig. 9 . Top figure indicates the “firmer” slatistic. ‘I Bottom figure indicates the “preferred” statistic. *Significant at P S0.05; **significant at P <0.01; ***significant at P 6 0.005.

A

0

C

FIG. 9. Cross sections of green beans lrozen according to methods listed in Table 11

219

FROZEN FRUITS AND VEGETABLES TABLE 111 EFFECT OF DIFFERENT COOKING TIMES ON TEXTURE OF FROZEN GREEN BEANS

Freezing methods comparedn

Cooking time (minutes)

R-12 Air

$4 I

R-12 Air

21

Percent of judgments indicating that sample frozen faster was Firmer

’

Preferred

56

53

8Zb

83b

14

Immersion in R-12, or air blast through unpackaged pieces. Significant at P 6 0.005.

such as potatoes. It occurs in dense granules that are not soluble in water at normal temperatures. On heating, the glucose polymer chains of which it is composed separate and become hydrated or “gelled. This expansion may cause the starch to occupy most of the cell volume in potatoes and other starchy vegetables, thus increasing the resistance to deformation. However, the swelling may proceed beyond this stage, causing the cells to rupture or separate from adjacent cells, thus degrading the texture of the product. When the gelled starch is frozen, water is withdrawn from the hydrated polymer, leading to reassociation of the glucose units (Chan and Toledo, 1976). This “retrogradation” hardens the starchy tissue (Reeve, 1967). During thawing, the water may escape from the tissue. If the vegetable is then cooked in water, the starch is rehydrated. In the absence of water, as with potatoes fried in oil, there is no possibility of regaining the lost moisture. Burr (1971) found that it is better to cook french fries directly from the frozen state, because moisture from the centers of the strips does not have a chance to diffuse to the surface, where it would be lost rapidly during frying. ”

2. Processing Steps prior to Freezing If frozen fruits and vegetables are to retain as much of their fresh character as possible, they must be handled carefully and processed with the least possible delay. It is essential to realize that no processing step improves the product; correct processing only minimizes the change or deterioration. On the other hand, once a product is frozen, the rate of change will be less than that occurring in the normal retail distribution channels of handling “fresh” produce.

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MILFORD S. BROWN

Fruits, particularly those that are very soft when harvested, should be subjected to as little handling as possible. Many vegetables are more durable, but all ate, to some degree, susceptible to mechanical injury. Both are composed of living tissues that are constantly changing. Their metabolic rate is increased by high temperatures, and this in turn can cause a further elevation of their temperature. Therefore, cooling is beneficial if processing must be delayed. In addition to its protective effect on the product, cooling also retards the growth of spoilage organisms if they are present. This is usually a more serious problem with fruits than with vegetables. The first processing step for most fruits and vegetables is cleaning to remove soil, unwanted plant fragments, insects, and other debris. This may involve combinations of dry or wet cleaning with various forms of agitation or scrubbing to aid removal of dirt from the surface. The current trend is toward reduction of the amount of water used, because it is no longer considered acceptable to burden the local sewage system with large volumes of water, or to dispose of it without treatment into rivers or other bodies of water. Some fruits and vegetables require peeling, usually done by degrading the surface with lye and then rubbing and washing off the peel. Some products can have the peel loosened by heating or freezing the surface (Leonard and Winter, 1974; Thomas et al., 1976). Abrasion peelers have been used for potatoes, although they are somewhat more wasteful than other methods. Processes that allow the waste to be used for animal feed or soil improvement are preferred over those that generate a useless waste that is only a disposal problem. Many fruits and vegetables are cut during the preparation for freezing. The seeds of peaches and apricots are removed as the fruit is halved. The ends of green bean pods are snipped off, and then the pods are cut or sliced. Many other vegetables are cut into pieces, either to facilitate further handling, or to provide pieces of suitable serving size. All of these cut surfaces allow some loss of nutrients, and this also causes some problems of equipment cleaning and waste disposal. The extreme example of this loss is the making of potato chips, in which peeling, slicing, and washing may result in the loss of 1/3 of the solids or 240 million pounds per year (Reeve, 1971). At some point before they are frozen, many products are sorted for uniformity of size and quality, and are subjected to examination and manual removal of defective or damaged pieces. Sizing before blanching permits optimum blanching for all pieces, since heating time is a function of piece size. Almost all vegetables, and some fruits, are blanched to inactivate enzymes responsible for deterioration during frozen storage. This is usually accomplished by conveying the product through hot water or steam. The optimum blanching temperature is often determined by the effect of heating on texture; higher temperatures often produce a more tender vegetable. Brown and Morales (1970)

22 1

FROZEN FRUITS AND VEGETABLES

measured the rate of softening of potatoes, and found that the shear resistance decreased much more rapidly at 95 and 85°C than at 75°C (Fig. 10). The time must then be selected for the desired degree of enzyme inactivation. Altematively, a longer blanching time may be used to reduce the amount of heating needed prior to serving. For example, boil-in-the-bag vegetables cook very little, even though the recommended boiling time is appreciably longer than the cooking time used for vegetables that are cooked directly in water. Thus, the prefreezing “blanching” is actually a combination of enzyme inactivation and cooking. Following blanching, the product should be cooled. Although this has often been done with large volumes of water, such a process is very wasteful of the water-soluble components of the food, and consequently adds to the problem of waste water disposal. A fine spray of water, most of which evaporates as the product cools, is preferable. The individual quick blanch (IQB) process of Lazar et al. (1971) is probably the most efficient method of blanching and cooling, because it reduces losses to about 1/10 of that from conventional processing. This is accomplished by drying the surface of the pieces with warm air, spreading them in a single layer on a belt and heating with steam, and then holding in a multilayer chamber for thermal equilibration. After a heating time sufficient to inactivate enzymes, the product is cooled with a fine fog of water, most of which evaporates or is absorbed. Thus, the loss of soluble components is minimized.

0

10

20

30

40

50

60

TIME (MINUTES) FIG. 10. Effect of blanching time and temperature on the shear resistance o f french fries. The resistance of uncooked potatoes is 100%.

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MILFORD S. BROWN

3. Freezing

Many systems have been devised for freezing food. Originally foods were merely packed in barrels and placed in a room at about OOF, but quality and economic factors have led to better methods of handling and freezing. Freezing in the container in which the food is sold requires time during the processing season for the packaging to be done, and also advance knowledge of customers’ package requirements. The alternative, most commonly used for foods that are to be consumed as small pieces, is to collect the frozen product in a large container, from which it is later transferred to smaller packages for sale. Commodities amenable to this type of handling include the seed vegetables and whole berries. The freezing method, or course, should not allow the pieces to become frozen to each other if further handling is intended. These are referred to as IQF (individual quick freezing) systems. It is important to avoid thawing during the transfer from bulk containers to smaller packages. In addition to its convenience to the packer, this system allows the user to select the amount needed from the package. A number of products cannot be handled as individual pieces. Many fruits are frozen in a sugar syrup, which requires a container. A recent development by Winter and Leonard substitutes a brief dip in an acidified solution of sugar and ascorbic acid, after which IQF and bulk storage systems may be used (Anonymous, 1971). Some products, such as chopped spinach or fruit purees, lack the rigidity that is needed for freezing as individual pieces. A solution to this problem is a freezer that forms small “ice cubes” of a fluid product. After these are frozen, they can be handled much like individual pieces of solid food. Other vegetables are too fragile, either before or after they are frozen, to be subjected to much handling. These would include asparagus spears and broccoli. Such products are placed in a container immediately after they are blanched and cooled, and the package protects them from further damage during freezing and storage. In the closed package, the product will freeze in several hours. For those products not restricted to container freezing, a number of freezing methods are possible, differing primarily in the means of heat removal. Some of these are also used to freeze packaged products. A number of these systems have evolved as improvements on the original method of placing large containers into a cold room to freeze in several days. To increase the rate of freezing, air circulation was introduced, and package sizes were reduced. Cooling by mechanical refrigeration was retained, but the freezer was separated from the storage facility. Various systems were developed to convey the product through the freezer. In some of these, cold air is blown across racks or belts carrying the product. As the depth of product is increased, heat transfer becomes less efficient. To aid heat removal, a fluidized bed can be formed by blowing the cold air up from beneath the product at a velocity great enough to lift the pieces slightly.

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This method, of course, is only suitable for unpackaged products, but is satisfactory for items as large as whole green beans or french fried potatoes. Because the pieces are separated from each other by air, this method is also suitable for somewhat fragile items. In such cases, the fluidized bed may be preceded by a freezer with slower air flow, in which the surface is frozen for protection against damage in the following fluidized bed. Although a moving belt can be used to help transport the product through the freezer, fluidized particles will flow toward the outlet as more unfrozen product is added. Since the quality of a number of products is improved by freezing at rates faster than that obtainable with various forms of air blast freezers, other heat removal systems have been devised. Most of these might be described as "remote refrigeration" systems, in which a refrigerant is prepared away from the freezing plant and delivered to it. The substances used for this purpose undergo a phase change at a very low temperature, with the absorption of energy from the product being frozen. Probably the most common of these cryogenic substances is liquid nitrogen. In early experiments with this refrigerant, the product was immersed in the liquid in an insulated chamber. It was soon found, however, that once the product is frozen, i.e., its water has undergone its heat-liberating phase change from liquid to solid, it is then subject to very rapid, nonuniform cooling and contraction. This contraction, which is greatest at the surface, causes stresses that are relieved by random cracking (Fig. 11) of the product pieces (Wolford and Brown, 1965). To avoid such damage, freezers were designed in which the liquid nitrogen is sprayed onto the product as it moves through on a belt. In many of these, the cold nitrogen gas within the freezer is circulated by fans. The cold gas is vented at the product entrance. This is done to increase thermodynamic efficiency by allowing the exhaust gas, at a higher temperature than that in the freezing zone, to extract heat from the warm entering product. Rasmussen (1967) has pointed out the possibility of evaporation losses from warm products, which should be avoided by rapid cooling to lower the vapor pressure of surface moisture. Thus, doing the initial cooling slowly with a gas close to ambient temperature may be economical of refrigerant, but more costly in the weight loss of an expensive product. With a product of low value, on the other hand, this loss can be tolerated because it is not as costly as the refrigerant that might be lost through the product entrance. Another remotely generated refrigerant for food freezing is carbon dioxide. This ubiquitous compound has the rather unusual property of existing only as a solid or gas at atmospheric pressure. The liquid state exists only between the triple point, -56.6"C and 4.23 kg/sq. cm (-69.9"F and 60.4 lb/sq. in.) and the critical point, 31.1"C and 74.0 kg/sq. cm (88.4"F and 1057 lb/sq. in.). At temperatures below that range it solidifies, and above, it vaporizes. If the liquid under pressure is released to the atmosphere, it forms a cold mixture of gas and

224

MILFORD S . BROWN I

FIG. 1 1 . Cross section of a liquid nitrogen frozen green bean, showing cracks caused by prolonged immersion. Cracks extend from surface to center of pod through several tissues, indicating that a single continuous frozen mass existed at the time of cracking.

solid. This unique property permits distribution of carbon dioxide as either a solid or a liquid. The liquid is stored either at very high pressure and ambient temperature, or at very low temperatures and moderate pressure (15-20 kg/sq. cm or 200-300 lb/sq. in.). Solid carbon dioxide is distributed at low temperature, but is subject to loss by sublimation. This can be minimized after delivery by storing it in cylinders in which it changes to a mixture of gas and liquid subject to the conditions described above.

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A number of carbon dioxide freezers have been designed. In some, small pieces of solid carbon dioxide are mixed or tumbled with the food pieces and then separated after the product is frozen. This method requires a continuous supply of solid carbon dioxide at the freezer. When liquid carbon dioxide is used, it can be sprayed into a freezer similar to those used for liquid nitrogen. The cold solid and gas formed at atmospheric pressure both contribute to the freezing of the product. As with the liquid nitrogen freezers, various configurations are possible, depending upon the requirements of the product, space availability, and ease of manufacture, transport, and installation. Some freezers have been designed to use either refrigerant. There is no contact between the food and the liquid refrigerant in carbon dioxide freezers. The rate of heat transfer from the food to solid or gaseous carbon dioxide is considerably slower than the rate to a liquid. Thus, excessive cooling rates are not encountered, but because of the low temperature, freezing is fast enough to bring about a quality improvement over that obtained with most air blast freezing systems. Regardless of the cryogen used, and the design of the equipment that allows it to remove heat from the food product to be frozen, there is no magic about cryogenic freezing. It should be remembered that freezing preservation never improves the quality of food, but at best merely preserves the quality of the product entering the freezer. Within a certain range of freezing rates, better preservation is achieved with faster freezing. Too much warm product or too little refrigerant will retard the freezing rate, and the product will be no better than if it had been cooled with air at the same temperature. Compared to the time required for preparation prior to freezing, or to the duration of frozen storage before use, the freezing time is so short that the composition of the atmosphere then cannot have a significant effect on the product. Although the possibility of microbiological contamination is of constant concern in food processing, the temperature within any food freezer is too low for bacterial growth. Frozen fruits and vegetables, the only products considered in this volume, have in fact not been troubled with such problems because of the low temperatures involved. Also, the presence of oxygen during storage eliminates the possibility of growth of Clostridium botulinum, the most serious concern in certain canned products. On the other hand, cleanliness during preparation, such as cutting and blanching, is important to prevent contamination with organisms that might grow if the food is thawed and then held at a high temperature before use. The need for cleanliness is not reduced by any type of freezing equipment. Another claim often made for cryogenic freezers is a reduction in the loss of weight by evaporation of water. Unfortunately the “conventional” freezer with which this process is compared is not described in detail, but when weight losses of 5% or more are mentioned, it is certainly not one in proper operating condition

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(Astrom, 1969). Comparisons with poor old equipment are only valid if the purchase of new equipment is uncertain. Once the decision to buy a new freezer has been made, the only valid comparisons are between the various new alternatives. Cryogenic freezing systems, in which energy is absorbed by the vaporization of a cold liquid or solid, reduce the moisture transfer during freezing. Since the dry gases are produced from the refrigerant and leave the freezer after circulating around the product, there will be some removal of water vapor also, primarily from the warm incoming product. If this loss is less than that experienced in another type of freezer, then the saving must be considered along with other benefits, against the costs of cryogenic freezing. In one comparison (Astrom and Londahl, 1969), the weight loss during freezing with liquid nitrogen was similar to that observed in a fluidized bed freezer, about 1.5 to 2%, but less than that in a conventional blast freezing tunnel (2.5 to 2.7%). Other considerations than product quality and safety may occasionally influence the decision to use cryogenic freezing. For example, the lower temperature might make it possible to freeze a certain quantity of product in less space than would be required by a conventional freezer. The space available in an existing processing plant might thus dictate the equipment to be used. A cryogenic freezer would also provide a temporary or short-term freezing capability without the large capital investment for refrigeration equipment. Although crops are usually planted to assure a continuing supply throughout the expected harvest season, variations in climate or other conditions may occasionally require that more than the anticipated amount of a crop be harvested at one time. Increasing the rate of cryogen flow might accommodate the excess, thus reducing the delay before freezing that can occur with mechanical refrigeration systems operated near capacity at the normal rate of harvesting. An alternative to cryogenic freezing that provides fast heat removal without extremely low temperatures is immersion in dichlorodifluoromethane (R-12). This essentially inert liquid is used as a heat transfer medium between the food product and a standard mechanical refrigeration system. It operates near its boiling point, -30°C (-22"F), which, for most products, avoids the cracking problem of liquid nitrogen immersion freezing. Since the current R-12 freezers use a combination of immersion and spray, the freezing rate can be regulated by controlling the flow of R-12 to the spray section of the freezer. Because of the good heat transfer between the product and the liquid refrigerant, the freezing rate is similar to that obtained with cryogenic systems, and the benefits to product quality are similar. The major difference between R-12 systems and others is the necessity to reclaim the cooling medium. Because of its relatively high cost and the need to minimize atmospheric pollution, the R-12 vapor must be condensed and returned to the freezer. In addition, the product entrance and exit are raised so that the density of the vapor will assist its retention within the freezer. Nevertheless there

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is some loss, so that the process is more costly than air blast freezing. For a number of products, however, the improved quality makes it possible to recover the additional freezing cost. A further saving is achieved by the immersion freezing just inside the entrance of the freezer, which solidifies the surface water and eliminates evaporative weight loss (Astrom and Lmdahl, 1969). 4.

Changes during Frozen Storage

Chemical changes affecting texture during frozen storage have been studied very little. Since freezing involves the transfer of water from various cell organelles to ice crystals, it seems probable that there would be some alterations of the bonds by which water is bound to these hydrated elements. On thawing, not all of the water would return to the site at which it was previously bound. This is known to occur, for example, in the freezing of gelatinized starch, where it is responsible for the phenomenon known as retrogradation. Undoubtedly it takes place to some extent in cell walls and other hydrophilic components of the cell also. Another change that may alter texture is the deesterification of pectin that was observed in cherries (Guadagni et al., 1958; Gee and McCready, 1957). Although this reaction was observed in frozen and thawed fruit stored at 2OoF, it may occur to some extent at other temperatures also. In frozen products that have not been blanched adequately, enzymatic reactions take place slowly to cause color and flavor changes and vitamin losses within a few weeks or months. There have also been some investigations of these changes in unblanched vegetables which emphasize the need for adequate blanching of foods for commercial distribution. Even in properly prepared frozen fruits and vegetables, losses of pigments and nutrients do occur very slowly. The chlorophyll of green vegetables is converted to pheophytin when magnesium is replaced by hydrogen. Both pigments are also subject to further decomposition. Ascorbic acid is oxidized to dehydroascorbic acid and 2,3-diketogulonic acid (Guadagni and Kelly, 1958; Guadagni et al., 1957). Changes of pH have been observed in foods and mixed salt solutions during frozen storage of several months duration (van den Berg, 1966). Peroxidase activity is a commonly used indicator of adequacy of blanching, because it is easy to determine and is relatively heat-stable in comparison to other enzymes of plant tissue. In peas blanched just sufficiently to inactivate the enzyme, some peroxidase activity has been observed after several months of storage at 18°C (Pinsent, 1962). This did not affect the quality of the peas, possibly because those enzymes responsible for deterioration are not as heatstable as peroxidase or are incapable of regeneration. Rates of chemical reactions increase with temperature, so it is essential to maintain a low storage temperature. Brief warming can occur during removal from storage (Farquhar, 1977; Olson and Dietrich, 1971), but usually only for

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MILFORD S . BROWN

short periods. If the product is allowed to thaw, the quality deterioration from subsequent refreezing at a very slow rate in storage will probably be greater than that resulting from chemical action. The primary physical defect occurring during frozen storage is dehydration of the product with concomitant formation of free ice in the package. If this dehydration is extensive enough to produce areas that are noticeably dry and discolored, it is referred to as “freezer bum. ”This form of dehydration is caused by temperature fluctuations below the freezing point. During warming, the vapor pressure of water in the product rises. When the temperature drops again, the package cools before its contents, and water vapor inside the package crystallizes on the colder package surface. During the warming part of the temperature fluctuation, the temperature of the package surface becomes higher than that of the product. Water can then transfer to the product surface, but it does not return to its original location within the product. As the process repeats, more water is withdrawn from the product and added to the ice within the package. Keeping the package in contact with the product minimizes the temperature gradients that favor this migration. D.

SUMMARY

Fruits and vegetables are obtained from a variety of plants whose edible parts are harvested at times ranging from the extreme immaturity of germinating seedling to the senescence of mature fruit. Many are preserved to provide a varied diet throughout the year. Freezing is the favored method for a number of these. Disruption of the cellular structure by freezing leads to undesirable changes of flavor, odor, and color. These are prevented by protective treatments prior to freezing. Products that are cooked before consumption are usually blanched to inactivate the enzymes that catalyze unwanted reactions. Products to be eaten without cooking, primarily fruits, are often frozen with antioxidants or sugar to stabilize them during storage. Structural changes caused by freezing also modify texture. The softening that results from this supplements the softening of cooking, but may also be of noticeably different character. This texture change seems more objectionable in fruits that are not cooked before they are eaten. Other prefreezing treatments include cleaning, peeling, and cutting. Product quality is best preserved by careful handling, and by processing as soon as possible after harvest. Some products require the protection of a package during or after freezing. Many, however, are frozen as individual pieces and stored in bulk containers for later packaging in smaller containers for sale. Freezers for individual quick freezing (IQF) use a variety of conveying systems to expose the product to cold air, nitrogen, c a h n dioxide, or liquid refrigerant. These systems freeze faster

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than those used for packaged products, achieving better preservation of texture and retention of cellular fluid after thawing. Freezing does not improve the quality of a product, but merely maintains it. This is best achieved at a low, constant temperature to minimize undesirable chemical and physical changes.

V.

PROGRESS AND PROBLEMS REMAINING

Refrigerated and frozen foods have been investigated very extensively. Fruits and vegetables have been subjected to a variety of storage conditions and times. They have been evaluated by chemical and physical analysis, and by sensory appraisal. Much of the chemical analysis has been to determine the degree of nutrient retention or loss. Changes that might influence the integrity of tissues, and their possible effect on texture have been examined by light and electron microscopy. The application of the results of these studies has made possible a large variety of foods in all seasons through improved storage and distribution facilities. Many fresh fruits and vegetables are available well past their harvest time. Many of these are also frozen so that they can be enjoyed all year. In the past 25 years, consumption of frozen vegetables has tripled, while consumption of all processed vegetables increased only 50%. More recently, in the decade between 1963 and 1973, use of frozen potato products increased from less than 10% to almost 28% of all potatoes eaten. Convenience is an important factor in this increase. Economic and quality factors have caused frozen peas to replace both fresh and canned peas for many consumers. In spite of the great amount of research, certain dreams of the food technologist andor merchant are not yet possible. When lettuce freezes, it is not destined for the winter salad bowl, but for the garbage. Frozen tomatoes for the salad came closer to reality, but failed to achieve commercial success. Freezing strawberries is probably the best way to preserve them, but there can be no doubt that is has been done. Low temperature plant physiology has also advanced to include studies of regulatory compounds, enzyme systems, and properties of biological membranes. Annual changes in the composition and quantity of carbohydrates, proteins, and lipids have been measured. The significance of some of these changes is understood, but others remain a mystery. Improvement of foods is the constant goal of horticulturists and food technologists. Some of the past improvements are the result of engineering advances, which now provide the capability of changing or holding the required temperatures. It seems reasonable to assume that some of the improvements in

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MILFORD S. BROWN

the future will be based upon the knowledge and understanding gained in other areas of cryobiology, as well as through continued efforts in food techology.

REFERENCES Amin, J. V. 1969. Some aspects of respiration and respiration inhibitors in low temperature effectsof the cotton plant. Physiol. Plant. 22, 1184-1191. Anderson, J . L., Ashcroft, G. L., Richardson, E. A., Alfaro, J. F., Griffin, R. E., Hanson, G . R., and Keller, J. 1975. Effects of evaporative cooling on temperature and development of apple buds. J . Am. SOC. Hortic. Sci. 100, 299 and 231. Anonymous. 1971. Dip process for 1QF fruit eliminates syrup but retains flavor, color of fresh. Quick Frozen Foods 33(9), 109-1 10. Astrom, S. 1969. Discussion. Bull. Int. Inst. Refrig.. Annexe 6, 172. Astrom, S . , and Londahl, G. 1969. Air blast in-line freezing versus ultra rapid freezing. A comparison of freezing results with some various vegetables and prepared foods. Bull. Int. Inst. Refrig., Annexe 6 , 121-127. Bassi, M., and Crivelli, G. 1968. Etude des modifications ultrastructurales, dues a la congelation, dans les produits vegetaux. 1. Observations concernant le chou-fleur (Brussicu olerucea botrytis). Rev. Cen. Froid 59, 1211-1222. Bassi, M., and Crivelli, G. 1969. Etude des modifications ultrastructurales, dues a la congelation, dans les produits vegetaux. 111. Observations concernant le haricot vert (Phuseoulus vulgaris L . ) . Rev. Cen. Froid 60, 1239-1250. Bauer, M., Chaplin, C. E., Schneider, G . W., Barfield, B. J . , and White, G. M . 1976. Effects of evaporative cooling during dormancy on 'Redhaven' peach wood and fruit bud hardiness. J . A n . Soc. Hortic. Sci. 101, 452-454. Boussiba, S., Rikin, A., and Richmond, A. E. 1975. The role of abscisic acid in cross-adaptation of tobacco plants. Plant Physiol. 56, 337-339. Brown, G. N., and Bixby, J . A. 1975. Soluble and insoluble protein patterns during induction of freezing tolerance in black locust seedlings. Physid. PIunt. 34, 187-191. Brown, M. S. 1967. Texture of frozen vegetables: Effect of freezing rate on green beans. J. Sci. Food Agric. 18, 77-81. Brown, M. S. 1973. Texture of frozen vegetables: Effect of freezing rate on softening during cooking. Proc. Int. Congr. Refrig., 13th, 1971 Vol. 3. pp. 491-497. Brown, M. S . , and Morales, J. A. W. 1970. Determination of blanching conditions for frozen par-fried potatoes. Am. Potato J. 47, 321-325. Brown, M. S., and Reuter, F. W. 1974. Freezing of nonwoody plant tissues. 111. Videotype micrography and the correlation between individual cellular freezing events and temperature changes in the surrounding tissue. Cryobiology 11, 185-191. Burr, H. K. 1971. Frozen French fried potatoes. Effects of thawing and holding before finish frying and their nonrelation to starch retrogradation. J . Food Sci. 36, 392-394. Casas, I . A., Redshaw, E. S., and Ibanez, M . L. 1965. Respiratory changes in the cacao seed cotyledon coincident with seed death. Nature (London) 208, 1348-1 349. Chan, W. S . , and Toledo, R. T. 1976. Dynamics of freezing and their effects on the water-holding capacity of a gelatinized starch gel. J . Food Sci. 41, 301-303. Chen, P. M . , and Li, P. H. 1977. Induction of frost hardiness in stem cortical tissues of Cornus stoloniferii Michx. by water stress. 11. Plant Physiol. 59, 240-243. Chen, P. M., Li, P. H., and Burke, M. 1977. Induction of frost hardiness in stem cortical tissues of Cornus stolonifera Michx. by water stress. I . Plant Physiol. 59, 236-239.

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Chouard, P. 1960, Vernalization and its relations to dormancy. Annu. Rev. Plant Physiol. 11, 191-237. Christiansen, M. N. 1967. Periods of sensitivity to chilling in germinating cotton. Plant Physiol. 42, 43 1-433. Crivelli, G., and Bassi, M. 1969. Etude des modifications ultrastructurales, dues a la congelation, dans les produits vegetaux. 11. Observations relatives au poivron (Capsicum annuuni L.), Rev. Cen. Froid 60, 421-431. Crivelli, G., Bassi, M., and Buonocore, C. 1973. Influence of freezing rate and storage on some modifications in carrots and summer squashes. Proc. Int. Congr. Refrig., 13th. 1971 Vol. 3, pp. 327-334. Dear, J. 1973. A rapid degradation of starch at hardening temperatures. Cryobiology 10, 78-81, de Carrizosa, F. L. 1965. The budding wine industry in Colombia. Wines Vines 46, 18. Dogras. C. C . , Dilley, D. R., and Herner, R. C. 1977. Phospholipid biosynthesis and fatty acid content in relation to chilling injury during germination of seeds. Plant Physiol. 60, 897-902. Duke, S. H., Schrader, L. E., and Miller, M. G. 1977. Low temperature effects on soybean (Glycine max [L.] Merr. cv Wells) mitochondria1 respiration and several dehydrogenases during imbibition and germination. Plant Physiol. 60, 716-722. Farquhar, J. W . 1977. Time-temperature indicators in monitoring the distribution of frozen foods. J . Food Qual. 1, 1 19-123. Fine, J . M., and Barton, L. V. 1958. Biochemical studies of dormancy and after-ripening in seeds. Conrr. Boyce Thompson Inst. 19, 483-500. Frankland, B., and Wareing, P. F. 1962. Changes in endogenous gibberellins in relation to chilling of dormant seeds. Nature (London) 194, 313-314. Fuchigami, L. H . , Weiser, C. J., and Evert, D. R. 1971a. Induction of cold acclimation in Cornus stolonifera Michx. Plant Physiol. 48, 98- 103. Fuchigami, L. H., Evert, D. R., and Weiser, C. J. 1971b. A translocatable cold hardiness promoter. Plant Physiol. 47, 164 and 167. Fuchigami, L. H., Weiser, C. J., and Richardson, D. G. 1973. The influence of sugars on growth and cold acclimation of excised stems of red-osier dogwood. J . A m . SOC. Hortic. Sci. 98, 444-447. Gee, M., and McCready, R. M. 1957. Texture changes in frozen Montmorency cherries. Food Res. 22, 300-302. Ghormley, J. A., and Hochandel, C. J. 1971. Amorphous ice: Denlity and reflectivity. Science 171, 62-64. Grant, N . H. 1966. The biological role of ice. Discovery 27, 26-30. Grenier, G . , Hope, H. J., Willemot, C . , and Themen, H.-P. 1975. Sodium-1,2- I 4C acetate incorporation in roots of frost-hardy and less hardy alfalfa varieties under hardening conditions. P lant Physiol. 55, 906-9 12. Guadagni, D. G . , and Kelly, S . H. 1958. Time-temperature tolerance of frozen foods. XIV. Ascorbic acid and its oxidation products as a measure of temperature history in frozen strawberries. Food Technol. 12, 645-647. Guadagni, D. G . , Nimmo, C. C., and Jansen, E. F. 1957. Time-temperature tolerance of frozen foods. VI. Retail packages of frozen strawberries. Food Technol. 11, 389-397. Guadagni, D. G . , Nimmo, C. C . , and Jansen, E. F. 1958. Time-temperature tolerance of frozen foods. XI. Retail packs of frozen red sour pitted cherries. Food Techno/. 12, 36-40. Gusta, L. V . , and Weiser, C. J. 1972. Nucleic acid and protein changes in relation to cold acclimation and freezing injury of Korean boxwood leaves. Plarrt Physiol. 49, 91-96. Hamilton, D. F. 1973. Factors influencing dehardening and rehardening of Forsyrhitr X ititermedi(i stems. J . A m , Sor. Hortir. S r i . 98, 221-223. Heber, V. 1970. Proteins capable of protecting chloroplast membranes against freezing. Fro;en Cell. Ciha Found. Symp. pp. 175-188.

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Howell, G. S . , and Weiser, C. J . 1970a. Fluctuations in the cold resistance of apple twigs during spring dehardening. J. Am. SOC. Hortir. Sci. 95, 190-192. Howell, G. S., and Weiser, C. J. 1970b. The environmental control of cold acclimation in apple. Plant Physiol. 45, 390-394. Ibanez, M. L. 1964. Role of the cotyledon in sensitivity to cold of cacao seed (Theobromu cucao). Nature (London) 201, 414-415. Irving, R. M . 1968. Study of dormancy, germination, and growth of seeds and buds of Acer negundo. Plant Physiol. 43, S-49. Irving, R. M. 1969. Characterization and role of an endogenous inhibitor in the induction of cold hardiness in Acer negundo. Planr Physiol. 44, 801-805. Irving, R. M., and Lanphear, F. 0. 1967a. Environmental control ofcold hardiness in woody plants. Plant Physiol. 42, 1191-1196. Irving, R. M., and Lanphear, F. 0. 1967b. The long day leaf as a source of cold hardiness inhibitors. Plant Physiol. 42, 1384-1388. Irving, R. M., and Lanphear, F. 0. 1968. Regulation of cold hardiness in Acer negundo. Plant Physiol. 43, 9-13. Karmanenko, N. M . 1972. Energy efficiency of respiration and phosphorous metabolism in winter wheat varieties of different winter hardiness. Sov. PIanr Physiol. (Engl. Transl.) 19, 683-687. Kavanau, J. L. 1950. Enzyme kinetics and the rate of biological processes. J . Gen. Physiol. 34, 193-209. Kemp, G. A. 1965. Inheritance of fruit set at low temperature in tomatoes. Proc. Am. SOC. Hortic. Sci. 86, 565-568. Kemp, G. A. 1968. Low-temperature growth responses of the tomato. Can. J . Plant Sci. 48, 281-286. Kester, D. E. 1969. Pollen effects on chilling requirements of almond and almond-peach hybrid seeds. J . Am. SOC.Horric. Sci. 94, 318-321. Kiovsky, T. E., and Pincock, R. E. 1966a. Demonstration of a reaction in frozen aqueous solutions. J . Chem. Educ. 43, 361-362. Kiovsky, T. E., and Pincock, R. E. 1966b. The mutarotation of glucose in frozen aqueous solutions. J . Am. Chem. Soc. 88, 4704-4710. Kislyuk, I. M . 1964a. Functional and structural changes in leaf cells in thermophilic plants at temperatures slightly above 0°C in light and darkness. Biofizika 9, 463-468. Kislyuk, I. M. 1964b. Influence of light on injury of Citcumis sativus L. leaves. Dokl. Akad. Nauk SSSR 158, 1434-1436. Krasnuk, M., lung, G. A., and Witham, F. H. 1975. Electrophoretic studies of the relationship of peroxidases, plyphenol oxidase, and indoleactic acid oxidase to cold tolerance of alfalfa. Cryobiology 12, 62-80. Kuiper, P. J . C. 1970. Lipids in alfalfa leaves in relation to cold hardiness. Plant Physiol. 45, 684-686. Lasheen, A. M., and Chaplin, C. E. 1971. Biochemical comparison of seasonal variations in three peach cultivars differing in cold hardiness. J . Am. SOC.Hortic. Sci. 96, 154-159. Lazar, M. E., Lund, D. B., and Dietrich, W. C. 1971. A new concept in blanching, IQB. Food Technol. 25, 684-686. Leffler, H. R . 1976. Altered development of ribonuclease activity and formation of polyribosomes in chilled cotton cotyledons. Crop Sci. 16, 71-75. Leonard, S.,and Winter, F. 1974. Pilot application of freeze-heat peeling of tomatoes. J . Food Sci. 39, 162-165. Lewis, T. L., and Workman, M. 1964. The effect of low temperature on phosphate esterificstion and cell membrane permeability in tomato fruit and cabbage leaf tissue. Aust. J . Biol. Sci. 17, 147- 152. Lund, D. B., Fennema, O., and Powrie, W. D. 1969. Enzymic and acid hydrolysis of sucrose as influenced by freezing. J . Food Sci. 34, 378-382.

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Luyet, B. J . 1965. Phase transitionsencountered in the rapid freezing of aqueous solution. Ann. N . Y . Acad. Sci. 125, 502-521. Lyons, J. M. 1973. Chilling injury in plants. Annu. Rev. Phnr Physiol. 24, 445-466. Lyons, J . M., Wheaton, T. A,, and Pratt, H. K. 1964. Relationship between the physical nature of mitochondria1 membranes and chilling sensitivity in plants. Plant Physiol. 39, 262-268. McMillan, J . A,, and Los, S. C. 1965. Vitreous ice: Irreversible transformations during warm-up. Nature (London) 206, 806-807. MeWilliam, J . R., and Naylor, A. W. 1967. Temperature and plant adaptation. I. Interaction of temperature and light in the synthesis of chlorophyll in corn. Plunt Physiol. 42, 171 1-1715. Margulies, M. M., and Jagendorf, A. T. 1960. Effect of cold storage of bean leaves on photosynthetic reactions of isolated chloroplasts. Arch. Biochem. Biophys. 90, 176-1 83. Marvin, J . W., and Morselli, M. 1971. Rapid low temperature hydrolysis of starch to sugars in maple stems and in maple tissue cultures. Cryohiology 8, 339-344. Meryman, H . T. 1957. Physical limitations of the rapid freezing method. Proc. R . Soe. London, Ser. B 147, 542-549. Meryman, H . T. 1966. Review of biological freezing. I n “Crybiology” ( H . T. Meryman, ed.), pp. 27-40. Academic Press, New York. Millerd, A , , and McWilliam, J . R. 1968. Studies on a maize mutant sensitive to low temperature. 1. Influence of temperature and light on the production of chloroplast pigments. Plant Physiol. 43, 1967-1972. Millerd, A,, Goodchild, D. J . , and Spencer, D. 1969. Studies on a maize mutant sensitive to low temperature. 11. Chloroplast structure, development, and physiology. Plunt Physiol. 44, 567583. Monzini, A., Bassi, M., and Crivelli, G. 1969. Freezing rates and ultrastructural modifications in some vegetables. Bull. Inr. Inst. Refrig., Annexe 6 , 47-50. Olson, R. L., and Dietrich, W. C. 1973. Factors affecting permissible temperature change during transport of frozen food. Proc. I n t . Congr. Refrig., 13rh, 197l Vol. 4, pp. 461-464. Perry, T. 0.. and Hellmers, H. 1973. Effects of abscisic acid on growth and dormancy of two races of red maple. Bor. Caz. (Chicago) 134, 283-289. Phatak, S . C., and Wittwer, S. H. 1965. Regulation of tomato flowering through reciprocal top-root grafting. Proc. Am. SOC.Hortic. Sci. 87, 398-403. Phatak, S . C . , Wittwer, S . H . , and Teubner, F. G . 1966. Top and root temperature effects on tomato flowering. Proc. Am. Soc. Hortic. Sci. 88, 527-531. Pinsent, B. R. W. 1962. Peroxidase regeneration and its effect on quality in frozen peas and thawed peas. J. Food Sci. 27, 120-126. Piringer, A . A , , and Scott, D. H. 1964. Interrelation of photoperiod, chilling, and flower-cluster and runner production by strawberries. Proc. Am. Soc. Hortic. Sci. 84, 295-301. Pollock, 8 . M . 1962. Temperature control of physiological dwarfing in peach seedlings. Plant Physiol. 37, 190-197. Pollock, B . M. 1969. Imbibition temperature sensitivity of lima bean seeds controlled by initial seed moisture. Plant Physiol. 44, 907-91 1. Pollock, B. M., and Olney, H. 0. 1959. Studies of the rest period. I . Growth, translocation, and respiratory changes in the embryonic organs of the afterripening cherry seed. Planr Physiol. 34, 13 1- 142. Pollock, C. J . , and ap Rees, T. 1975. Cold-induced sweetening of tissue cultures of Solanum tuberosurn L. Plunru 122, 105- 107. Pomeroy, M. K . , and Siminovitch, D. 1971. Seasonal cytological changes in secondary phloem parenchyma cells in Robinia pseudoacacia in relation to cold hardiness. Can. J . Bor. 49, 787-795. Proebsting, E. L., Jr. 1975. Reducing energy consumption in cold protection. HortScience 10, 463-465.

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ADVANCES

IN FOOD RESEARCH. VOL. 25

BYSSOCHLAMYS SPP. AND THEIR IMPORTANCE IN PROCESSED FRUITS LARRY R. BEUCHAT AND STEPHEN L. RICE* Department of Food Science, University of Georgia Agriculture Experiment Station. Experiment, Georgia

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Classification and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Perfect State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .........................

........... ........................

..........

B. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Factors Affecting Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Control of Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

........... B. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Mycotoxins. . . ...... ... ........... D. Mannitol . . . . . ...... ... ........... V. Detection and Enum ........................................ VI. Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . ............

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I. INTRODUCTION Spoilage of canned and bottled fruits by Byssochlamys spp. was first recognized in Great Britain in the 1930s and has since been documented in several countries around the world. Two species of the mold, Byssochlamys fulva and B . nivea, are responsible for spoilage. Both form structures called asci (singular = ascus), each containing eight exceptionally heat-resistant ascospores. These spores are capable of withstanding thermal processing treatments given to many fruits *Present address: Food Sciences Institute, Purdue University, West Lafayette, Indiana 47907

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Copyrighl 0 1979 by Academi' Pre\\. Inc All right* at reproduction in any form r e w v e r l ISBN 0-12-016425-6

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and fruit products, and thus represent a potential problem to the preservation of products during subsequent storage. Fruits infected with Byssochlamys undergo deterioration and softening due to production of pectinases that break down structural tissues. The mold is capable of growing under greatly reduced oxygen tension and, for this reason, can cause substantial deterioration of canned fruit without causing a sufficient change under vacuum to result in recognizable spoilage before the can is opened. The fact that some strains of Byssochlamys are mycotoxigenic, producing byssochlamic acid, patulin, byssotoxin A, and perhaps other antimetabolites gives impetus to gaining further knowledge regarding the control of growth and detection of these organisms in processed fruit products.

II. CLASSIFICATION AND NOMENCLATURE A.

PERFECT STATE

The genus Byssochlamys was established from a single species, B . nivea, by Westling (1909). The mold was characterized by the production of eight-spored asci in clusters, phialospores born on phialides, aerial hyphae or conidiophores, and by the presence of chlamydospores. A second species, B. fulva, was described by Olliver and Smith (1933). All isolations at that time had been made from canned and bottled fruits. Naumoff and Kiryalova (1935) isolated a third species from must of grapes and named it B . musticola. From the description provided, Brown and Smith (1957) and Stolk and Samson (1971) surmised that this species was very close to, if not identical with, B. fulva. Additional species designated as B . trisporus (Cain, 1956), B . nivea var. lagunculariae, B . zollerniae (Ram, 1968), and B . verrucosa (Samson and Tansey, 1975) have been documented. With the exception of B . nivea and B . fulva, other Byssochlamys spp. have not been implicated as causative spoilage organisms in the food industry and will not be considered in detail here. A more complete description of morphological and physiological characteristics of B. nivea and B. fulva, however, is in order. Colonies of B . nivea cultured on Czapek agar at 30°C spread rapidly, consisting at first of a loosely floccose to funiculose mat, drying down with age and appearing almost granular, with a thin, arachnoid margin during the growing period (Brown and Smith, 1957). Colonies are white to off-white, with slightly pinkish centers upon aging; the reverse is white, becoming faintly yellow. Drops of liquid are lacking at first, then large and colorless; odor is nil, then faintly musty. Growth is similar on potato dextrose agar, but not as rapid initially as on malt agar. On the latter medium colonies are more deeply floccose when young,

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white at first, changing to slightly yellowish white; the reverse is white. Byssochfamys nivea thrives more readily on Sabouraud’s agar than on other media, attaining a colony diameter of 4.5 cm after 2 days (Ram, 1968). Cleistothecia are not produced by B . nivea, but hyaline, obovate (globose to subglobose) eight-spored asci, ranging in various strains from 5 to 12 x 6 to 16 p n , but mostly 7 to 9 x 10 to 13 pm, are formed in clusters with or without loosely aggregated white vegetative hyphae around them. Ascospores (2.8 to 4.0 x 3.4 to 5 . 6 pm) are hyaline, ovoid to elliptical, and smooth. Chlamydospores, borne singly and usually terminally, are ovoid to pyriform, thick-walled, hyaline to buff, smooth or slightly roughened, and measure 4 to 10 x 5 to 12 p ,but mostly 6.1 to 8 x 7 to 1 1 pm. Conidial structures are more prevalent when B . nivea is cultured on Czapek agar than on malt agar. One-celled conidia are borne in chains on phialides and are hyaline, smooth, often slightly flattened on one end, and measure 1.5 to 4.5 X 2.2 to 6.5 pm, but mostly 2.0 to 4.5 X 3.0 to 5.5 pn (Kuehn, 1958). Conidia germinate by one germ tube, but ascospores swell and then put forth one or two germ tubes, which are usually produced terminally but sometimes laterally (Brown and Smith, 1957). Byssochlamysfulva grows rapidly on Czapek agar, consisting at first of a very thin basal felt with fine funiculose overgrowth, becoming a densely matted floccose or funiculose felt with loose funiculose overgrowth, later developing into granular patches (asci) (Brown and Smith, 1957). Colonies are pale, yellowish buff or tawny to snuff brown with the reverse pale, dull yellowish or brownish; exudate is lacking or limited to a few colorless drops. Growth is slower in submerged media (Olliver and Smith, 1933), turning white when reaching the surface, then buff to pale brown in central areas, slightly floccose or funiculose, after 7 to 10 days showing clusters of asci visible to the naked eye as globose masses partially embedded in the mycelial mat. Colonies on potato dextrose and malt agars are similar in appearance to those on Czapek agar, except that they are more funiculose. Cleistothecia are not formed by B . fulva. Hyaline, globose to subglobose, eight-spored asci are formed in clusters with dimensions of 9 to 12 X 10 to 13 pn. Ascospores are hyaline, smooth, and regularly elliposoidal, with dimensions of 5.5 to 7 x 3.5 to 4.5 pm, mostly 6.5 pm long. This species does not produce chlamydospores. Conidia are hyaline, smooth, ovate to elongate, cylindrical, or barrel-shaped, and are formed in long twisted, divergent, or loosely tangled chains. Dimensions are 1.5 to 3 x 4 to 9 pm. Ultrastructures of conidia and ascospores of B . fulva were investigated by Partsch et a / . (1969) using electron microscopy. The conidial cell wall has a rough, fibrous coat which covers the cell wall membrane. These structures are separated by an intermediate space consisting of several layers. The intermediate space in ascospores is considerably thicker than that in conidia and, together with

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the cell wall, might represent a factor related to heat resistance. The cytoplasm of conidia contains one to three nuclei, all varying in size and shape. In some dormant conidia large numbers of mitochondria may be found. Structures are less discernible in dormant ascospores but become more visible upon germination. Herbert (1973) analyzed the lipid composition of ascospores, conidia, and mycelium of B . fulva. He found that 22.75% (dry weight) of the ascospores is comprised of lipid materials, 75% of which is located in the sporoplasm (cytoplasm) and 10% in the coat. The asci contained more saturated fatty acids with carbon chains of 19 or longer than did the conidia and hyphae. King et al. (1969) noted that ascospores of B . fulva contained less than 0.02% dipicolinic acid, a component often associated with heat resistance of bacterial spores. Thompson (1969) did not detect the acid in purified ascospore preparations of B . fulva. Both B . nivea and B . fulva grow more rapidly at 30°C than at 24°C. If subcultures of B . fulva are always grown at 24°C or at lower temperatures, they tend to become entirely conidial, and the ability to produce asci may be permanently lost (Brown and Smith, 1957). The formation of colonies identifiable as Byssochlamys depends greatly on the relative abundance of asci and conidial structures, and this depends on the incubation temperature and medium on which the organism is grown. Cell walls of asci thicken with age (Hatcher et a l . , 1979). When speciation of Byssochlamys isolates from spoiled foods is desired, three characteristics can be used as aids to distinguish B . nivea from B . fulva. Byssochlamys nivea generally forms white colonies; it is capable of producing chlamydospores and its asci and ascospores are of smaller dimensions than are those of B . fulva. Conversely, B . fulva produces buff, tan, or brownish colonies but does not produce chlamydospores. The shape of the conidia may also be useful for identification purposes. It should be recognized, however, that the temperature and length of incubation as well as plating medium affect the rate of growth, texture and color of colony surface, type of colony margin, and degree and type of sporulation. Habit sketches of various structures of B . fulva are shown in Fig. 1 . In addition to publications cited here on Byssochlamys, reports on developmental morphology (Emmons, 1935; Raper and Thom, 1949; Kuehn, 1957), nuclear cytology (Rosenbaum, 1944), synonomy of species (Benjamin, 1956), and identification (Prest, 1969; Splittstoesser and Prest, 1976; Beneke and Stevenson, 1978) are recommended. B.

IMPERFECT STATE

The imperfect state of the genus Byssochlamys is classified as Paecilomyces. Bainier (1907) erected the form-genus Paecilomyces from a single species, P . varioti. Distinguishing features included fulvus colored colonies with a suggestion of a green tone, diverse fruiting structures consisting of single sterigmata (i.e., phialides) scattered along aerial hyphae, or of small clusters of sterigmata,

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FIG. 1. Habit sketches of various structures of Byssochlumys fulva. (A) Conidial structure: (B) conjdia; (C) ascus; (D) ascospores.

or fairly complex penicillate systems, often whorled, of branches and sterigmata, along the length of the conidiophore. The genus Paecilomyces Bainier and its perfect state Byssochlamys Westling are extensively reviewed by Brown and Smith (1957). Paecilomyces is closely related to Penicillium but is sufficiently distinct from the majority of penicillia to justify its existence as a separate genus. The rediscovery of the perfect state, in B. fulva, and the fact that the conidial state of the latter is almost indistinguishable from P . varioti (Brown and Smith, 1957), however, raises some question of the status of the form-genus. A few species of Paecilomyces approach Gliocladium, in that conidia are not completely dry, tending to slime into irregular masses. Other species freely produce solitary phialides instead of penicillate fruiting structures, suggesting some link with Cephalosporium and Verticillium. A general description of Paecilomyces is given by Brown and Smith (1957): phialides which are short, tubular, or more-or-less swollen in the basal portion and tapering into long, slender, conidium-bearing tubes which are mostly curved or bent slightly away from the main axis of the cell; phialides are variously arranged, partly in verticils and partly branching, partly irregularly arranged upon short branchlets, partly sessile on and arising from the fertile hyphae; conidia are smooth, ellipsoid to cylindrical, 3 to 6 x 1.5 to 3 p (mostly 4 to 5 x 2 to 3 pm for P . varioti), and in chains, or tend to slip sideways, or to slime down into balls; colonies are white, or pale colored, or in dull, brownish shades, occasionally with a greenish tone but never true green. Colonies of P . varioti grow rapidly on malt agar, attaining a diameter of 6 to 7 cm within 4 days at 24°C. Growth is somewhat slower on Czapek agar; reverse is deeper and more orange on malt agar. Growth on potato dextrose agar is more rapid than on Czapek agar, colonies reaching 7 to 8 cm in 7 days. Colonies consist of a dense felt of conidiophores, giving a powdery appearance, some-

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times floccose, funiculose, or tufted. The color is variable depending on the strain, ranging from deep or dark olive buff to yellowish olive. Darker shades are sometimes noted in older colonies due to the formation of chlamydospores. Colony odor may be sweet and aromatic and is more pronounced with age. The genus Paecilomyces was redefined and monographed by Samson (1974). Based on morphological characteristics, it was proposed that Paecilomyces be subdivided into two sections, Paecilomyces and Isarioidea. The section Paecilomyces would include mesophilic to thermophilic species with yellowbrown to brown colonies, the perfect state of some of these species being Byssochlamys. Paecilomyces fulvus, P . niveus, and P . zollerniae were designated as imperfect states of B . fulva, B . nivea, and B . zollerniae, respectively. This classification scheme is not universally accepted, however, and P . varioti is still recognized by many researchers to be the imperfect state for B . fulva. Paecilomyces varioti is common in the air and soil, and has been isolated from a wide variety of foodstuffs, including sorghum brandy (Saito, 1921), butter (Bisby et al., 1933), cocoa beans (Maravalhas, 1966), and cassava bread (Goncalves de Lima, 1971). It is a causal agent of biodeterioration of palm oil (Cornelis et a l . , 1965) and has been isolated from fruits (King et al., 1969; Splittstoesser et al., 1971), fruit juices (Senser and Rehm, 1965; Senser et a l . , 1967; Eschmann, 1971), and canned fruits (Olliver and Rendle, 1934; Spurgin, 1964; Eckardt and Ahrens, 1977a). Environmental conditions necessary for inducing ascospore formation, and hence the development of the perfect state, Byssochlamys, have not been fully defined. Incubation temperature, substrate pH and moisture content, and nutrient availability are thought to influence this process significantly. A more detailed discussion of these factors is presented in a later section (IV,A,l).

111. A.

SPOILAGE

DISTRIBUTION AND SOURCES OF CONTAMINATION

The first documented fruit spoilage outbreaks attributed to Byssochlamys occurred in England and it was thought at that time the organism was confined to that country (Olliver and Rendle, 1934). It is now recognized that Byssochlamys spp. are responsible for spoilage of processed fruit and fruit products in several other countries, including the United States (Maunder, 1969), Denmark (Jensen, 1960), Australia (Spurgin, 1964; Richardson, 1965), Holland (Put and Kruiswijk, 1964), Canada (Yates et al., 1968), and Germany (Eckardt and Ahrens, 1977a). Researchers in these countries have systematically examined harvesting, handling, and processing procedures to determine sources of infection. The first extensive investigations of the incidence of Byssochlamys on fruits and associated environments were conducted by Olliver and Rendle (1934). Initial

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studies were unsuccessful in detecting the organism in damaged fruit or packing material, sugar and water used for processing, or air in sorting, filling, and storage departments throughout a processing factory in the winter months. Wooden trays and baskets, glass bottles, and jars were found to be carriers, although they were not in themselves the main source of infection by B . fulva. Efforts then turned to the examination of heat-shocked samples of fresh fruits taken in fields and orchards and immediately on their arrival at the factory. Field samples were taken from the trees or plants or, in the case of damaged and fallen fruit, from the ground. Results showed that fields and orchards were the initial source of contamination in processed fruits. Strawberries and plums appeared to be attacked more readily than were other fruit, although some samples of gooseberries, loganberries, blackberries, black currants, and apples were positive. In all instances the field samples of stone fruit positive for B . fulva were taken from the ground. Since strawberries, too, are easily contaminated with soil, it was suggested that B . fulva may be a soil fungus. Further tests on plant roots with some soil adhering also gave positive results. Hull (1939) directed extensive research to determine sources of infection and ways to control spoilage of processed fruits by Byssochlamys. In the course of investigation, leaves, fruit, and straw from strawbeny fields were examined and it was found that 33% of the samples were positive for B. fulva. Mummified plums, raspbeny refuse, and the remains of fruit present in the linings of wooden baskets used to collect fruits were contaminated with ascospores of B . fulva. These findings confirmed previous observations that Byssochlamys was saprophytic rather than parasitic. Asci produced on field and orchard refuse overwinter on this refuse, most of which is on the ground and in surface layers of soil, and are spread to fruits produced the following season before they are transported to processing plants. Selective cultivation of heat-resistant molds from 200 soil samples collected from eight different strawberry growing areas in Holland showed that of 27 isolates of Byssochlamys, 25 were B . nivea and two were B . fulva (Put, 1964). From this investigation it was learned that ascospores were present in each of the growing areas and that B. nivea was present in higher concentration (<0.1 to 1000 ascospores per gram of soil) than was B. fulva (<0.1 to 10 per gram). Eckardt and Ahrens (1977a,b) isolated B . fulva from soil in different regions of Germany, and from strawberry leaves and fruits. The incidence of Byssochlamys spp. in soils of vineyards and orchards in North America has been studied. Yates (1974) found little evidence of the mold in Ontario soil samples and apples, but 25 of 78 grape samples obtained from off-loading trucks at a winery were positive. Splittstoesser et al. (1970) and Splittstoesser (1971) reported that over 70% of the samples of fruit, vegetation, and soil obtained in surveys of New York orchards and vineyards were contaminated with heat-resistant molds. Byssochlamys fulva was the predominant mold detected on sound and decayed grapes, grape vegetation, apples, blackberries,

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cherries, peaches, raspberries, apricots, pears, and orchard and vineyard soils. Although B . nivea was isolated, it did not predominate on grapes of this region as had been reported for California grapes (King et al., 1969). Washing, fluming, blanching, peeling, and pressing are some of the processing operations that would be expected to remove ascospores of Byssochlamys from contaminated fruits.

B. OCCURRENCE The first extensive investigation of spoilage by Byssochlamys evolved from studies by Olliver and Rendle (1934) in the general area of softening of processed fruit. A sample of fruit received for examination was in good condition when packed, and after processing at normal times and temperatures was found to be well cooked, but still firm. Upon storing for several weeks, it was found that a small percentage of canned product disintegrated. There was no obvious gas production, no abnormal odor or flavor, and no evident color change. Apart from occasional slight growth of mycelium, no signs of microbiological activity were evident. Further investigation revealed that B . fulva was responsible for the softening process. The mold was observed to grow on both fruit pulps and syrups of cooked fruits, including strawberry, plum, raspberry, loganberry, apple, orange, and currant. Fruits such as peaches, pears, cherries, apricots, apples, and strawberries disintegrated rapidly. The first sign of fungal action is usually a slight pulpiness of the fruit which may be suggestive of overcooking or the use of overripe fruit. Later, total disintegration takes place until, finally, a broken mass intermixed with skins and seeds is formed. Off odors may be detectable and a slightly sour taste usually develops. Substantial softening of fruit may occur without sufficient gas production to bulge the ends of the container, so that affected cans may be unrecognized until they are opened. Gillespy (1 946) estimated the degree of infection in cans and bottles of packed fruit in England during the years 1937 to 1940. Initial infection ran as high as 97% for bottled gooseberries and as low as 18% for canned plums. These fruits were subjected to an old, slow-cooking process in which the maximum temperature reached was about 75"C, since this procedure was considered to give the highest quality packed fruits. Maximum temperatures attained in most fruit processing schedules today are somewhat higher than 75"C,but fill temperatures of 80 to 90°C for juices, for example, may not be severe enough to inactivate ascospores of Byssochlamys. Thermal treatment at higher temperatures causes a marked reduction in product quality and therefore cannot be used to prevent spoilage. It appears that the risk of spoilage by Byssochlamys spp. can best be controlled by reducing or eliminating Contamination of fruit prior to processing. Since these early reports on Byssochlamys in England, others have documented the problem. Processed strawberries and grapes, perhaps more than

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other fruits, appear to be infected with greatest frequency. Gillespy and Thorpe (1962) detected B. fulva on strawberries for canning during the 1961 growing season. The mold most commonly isolated by Put and Kruiswijk (1964) from jars and cans of disintegrated strawberries was B. nivea. It had been observed that strawberries in The Netherlands sometimes spoiled between 6 and 12 months after processing and that spoilage was always accompanied by unpleasant odor and flavor. Analysis of fresh strawberries during the 1961 and 1963 growing seasons showed that ascospores of Byssochlamys were present on 6 of 50 and 10 of 24 samples, respectively. Spurgin (1964) reported an incident of spoiled canned strawberries during the 1961 season in Australia. Attempts to isolate an ascospore-producing mold from infected product failed, but, instead, P. varioti was detected. Various attempts to induce the formation of the perfect state were unsuccessful; however, heat-resistant ascospores were demonstrated in the infected fruit. Later Richardson (1965) cultured the isolate on strawberry syrup and observed the production of structures which fitted the description of asci of B . fulva. Further studies on morphology and heat resistance described by Spurgin confirmed that B. fulva was the causative agent of spoilage of canned strawberries. Eckardt and Ahrens (1977a) more recently reported the contamination of canned strawberries by B. nivea. On fresh strawberries growth of B. fulva occurred only if the surface of the fruit was damaged. The incidence of Byssochlamys on raw and processed grapes has been documented (King et al., 1969; Splittstoesser et al., 1971; Yates, 1974). The presence of the Byssochlamys on certain fruits is undoubtedly influenced by the extent of exposure of fruits to soil as well as to the prevalence of the mold in soil in which fruits are grown. For these reasons, both strawberries and grapes would appear likely to harbor Byssochlamys. Maunder (1969) presented a history of spoilage outbreaks in the United States due to Byssochlamys spp. from the years 1964 to 1969. Spoilage involved fruit juices and drinks, grape and pineapple concentrates, fruit pudding, blackbeny and cherry pie fillings, and canned blackberries and figs. Paecilomyces was detected in three fruit drinks, grape concentrate, apple juice, canned figs, and blackberry pie filling. Spoilage rates ranged from a few cans within a production code to 100% of a code. Factors contributing to spoilage problems were attributed to ingredient contamination, ingredient storage and handling, and inadequate heat treatment of the products. At least one incident of spoilage of a vegetable was reported to have been caused by Byssochlamys. Yates and Ferguson (1963) isolated B. nivea from cucumber brine stock after noting that surface fungi secreting pecteolytic enzymes had been incriminated in the softened material. The crocks had been sealed with air traps, preventing the entry of atmospheric gases. Substantial growth had developed within 4 weeks, and at this time, the atmosphere above the brine was found to contain 47% carbon dioxide and 3% oxygen.

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C.

FACTORS AFFECTING GROWTH

Whether processed fruit and fruit products ultimately become spoiled by Byssochlamys spp. depends upon the level of contamination in raw products, the severity of heat treatment, the chemical nature of the processed product, headspace in the product container, and storage temperature. As suggested previously, good sanitation practices to reduce or eliminate ascospores in raw products should be considered as the most practical approach in controlling the risk of spoilage. Overcooking will destroy the ascospores but is also likely to adversely affect organoleptic and nutritional qualities of the finished product. Considering a given fruit, cultivar and degree of maturity may influence the rate at which Byssochlamys will develop. For example, Rice (1977) and Rice er al. (1977a) reported that certain metabolic activities of B. fulva were affected by composition of peaches. In later studies (Rice and Beuchat, 1978a,b) it was determined that the mold grew differently in media containing various acids and sugars found in fruits. Control of spoilage by choosing fruits with specific chemical components does not appear to be a practical approach. However, water activity ( a , ) , the sugar content of fruit syrup or concentrate, oxygen tension within the container, and the temperature at which products are stored can be manipulated to some extent to disfavor the growth of Byssochfamys, and deserve further discussion here. Like all microorganisms, Byssochfamys spp. have minimal a , below which certain metabolic activities will not occur. These a, values vary considerably and are dependent upon nutrient availability, pH, temperature of incubation, and strain. Olliver and Rendle (1934) studied the maximal concentration of sucrose tolerated (minimal a , tolerated) by B . fulva in potato dextrose agar, CzapekDox agar, and physiological saline. The maximal concentration was found to be between 60 and 65" Brix when cultures were incubated at 28°C. Hull (1939) observed that heated ascospores of B . fulva germinated on agar plates containing up to 70% sucrose. Germination was more rapid on 20% sucrose than on 0 or 40% plates. In agars containing higher concentrations of sugar, the germ tubes of the ascospores were numerous and fine. Although delayed by high concentrations of sucrose, B . fulva will produce asci in grape concentrate adjusted to 50" Brix (Maunder, 1969). On the other hand, Olliver and Rendle (1934) reported that B . fulva did not produce ascospores in laboratory media containing greater than 30% sucrose. Germination of ascospores and growth of B . nivea in fruit products were studied by Beuchat and Toledo (1977). The lowest a , for growth of the organism in fruit juices and nectars supplemented with sucrose was 0.90. This occurred in peach nectar and in prune juice inoculated with heat-shocked asci and incubated at either 21 or 30°C. Outgrowth was much more rapid at 30°C. Sucrose, added to grape juice agar at a rate of 10 gm per 100 ml, promoted rapid colony develop-

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ment by B . nivea, whereas agars with no added sucrose or with 20 to 40 gm per 100 ml retarded mycelial growth. The total number of colonies was essentially the same at the end of the 12-day test period on agars to which sucrose had been added at a level of 30 gm or less per 100 ml. Colony formation was reduced by 80%, however, when asci were plated on grape juice agar containing 40 gm per 100 ml. At 60 gm of sucrose per 100 ml, no colonies were detectable by the unaided eye after 12 days of incubation. It was suggested that ascospores either returned to a dormant state or failed to survive possible injury incurred during heat treatment when plated on agar containing high concentrations of sucrose. Orth (1976b) studied the influence of a, on germination of conidia of B. nivea and several other mycotoxin producers. A limiting a, of 0.84 for germination was observed for all species tested. No growth was noted at lower a, even after a 3-week incubation period. Since sucrose concentrations in cans of processed fruits are well within a range conducive to germination of ascospores and growth of Byssochlamys, low a, cannot be relied upon to control spoilage. The generally lower a, of fruit concentrates, however, would appear to enhance preservation, especially when products are stored at temperatures not optimal for growth of the fungus. Experiments have shown that B. fulva will not grow in an atmosphere from which oxygen has been removed by alkaline pyrogallol (Hull, 1939; King et a f . , 1969). However, Byssochlamys is capable of growing under greatly reduced oxygen tension. Cultures of B . fulva have been grown on synthetic and on natural media under a partial pressure of 20 in. (Olliver and Rendle, 1934) and in an air and nitrogen atmosphere containing as little as 0.27% oxygen (King et al., 1969). The rate and amount of mycelial growth that develops on infected canned products depends on the original amount of oxygen present. This, in turn, is dependent upon the size of headspace, type of fruit, and temperature of sealing. Hull (1939) studied changes in composition of the headspace gas in control and infected cans of plums stored at 30°C. In fruit infected with B. fulva, the carbon dioxide concentration increased to 52% over a 13-day test period and this was accompanied by a gradual reduction of oxygen concentration to zero. In some instances cans of infected fruit were blown due to production of carbon dioxide, indicating that a form of anaerobic respiration took place. The amount of mycelial growth in infected plum syrup increased with increasing headspace, and this was accompanied by more carbon dioxide production and greater gas pressure. It was suggested that a depletion of oxygen rather than the production of an inhibiting concentration of carbon dioxide normally limits growth of B . fulva in the can. Lack of oxygen is probably the cause of eventual death of mycelium. Investigations have been conducted to determine in more detail the effects of carbon dioxide on metabolic activities of Byssochlarnys. Yates and Ferguson (1963) reported that between pH 3.5 and 6.0, increasing carbon dioxide concentrations up to 34% had little effect on growth of a strain of B . nivea isolated from

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cucumber brine. Inoculated, buffered broth through which carbon dioxide was bubbled for the duration of a 2-week incubation resulted in 16.2 mg of mycelium per 100 ml. When nitrogen was substituted for carbon dioxide, the weight was reduced to 2.7 mg per 100 ml. In a later study, Yates et al. (1967) reported that B. nivea grew in a synthetic medium in which the gaseous atmosphere was 100% carbon dioxide. Pure nitrogen atmospheres resulted in no measurable growth. The effect of temperature on growth of Byssochlamys was one of the first parameters studied with regard to preventing spoilage of processed fruits. Olliver and Rendle (1934) reported that optimum temperatures for growth range between 30 and 37°C. Storage at temperatures between 0 and 30°C would theoretically, then, retard the rate of mold growth and yet preserve organoleptic quality of the canned fruit. Storage at -12 to -7°C inhibits growth but does not kill Byssochlamys. Hull (1939) observed that growth of B. fulva diminished progressively as incubation temperature was reduced from 35°C; at 15"C, growth was very slow and at 8°C growth ceased. Behavior of ascospores of B. nivea in fruit products supplemented with sucrose was examined by Beuchat and Toledo (1977). No growth was observed on fruit juices and nectars stored at 7°C. Storage of ascospores at 7 and -30°C had a lethal effect, but the addition of sucrose to fruit products tended to have a protective effect. Eckardt and Ahrens (1978) studied the survival of conidia and ascospores of Byssochlamys fulva in desalted water at -18°C over a 34-week period. Viable conidia declined gradually, but the number of surviving ascospores remained more or less constant. Only very low or slightly alkaline pH values strongly affected the resistance of ascospores. Growth of Byssochlamys can therefore be controlled by storing potentially contaminated products at reduced temperatures; however, inactivation of ascospores at these temperatures cannot be assured. D.

CONTROL OF GROWTH

Destruction of ascospores of Byssochlamys in fruits and fruit products by thermal processing alone is impractical, since the timehemperatwe requirements to ensure lethality also adversely affect the organoleptic qualities of most products. Researchers have, as a consequence, studied the chemical nature of fruits, environmental factors during handling, processing, and storage, and the use of antimycotics in attempts to find ways to control the growth of Byssochlamys. The widespread use of machines to harvest many fruits in recent years has raised the question as to whether changes in harvest procedures might have increased the opportunity for contamination by Byssochlamys (Splittstoesser er al., 1974). A study was therefore initiated which revealed that mechanically harvested tart chen!es and Concord grapes were not more heavily contaminated with ascospores of Byssochlamys than were hand-picked fruit. Data suggested, however, that both harvest procedures may contribute to contamination, proba-

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bly because of soiling of fruit surfaces. The practice of transporting cherries to the factory in chilled water to prevent scald was thought to serve as a partial cleansing procedure to remove ascospores. The cleaning of harvesters with a strong detergent sanitizer at the end of each shift and the use of plastic-lined lug boxes for transport of fruits may also aid in controlling potential contamination of fruits as they enter the processing line. Others have suggested that heat-resistant fungi can be controlled by thorough washing of fruit and careful cleaning of plant equipment (Ruyle et a/., 1946; Jensen, 1960). Since fruit juices often are filtered before being made into concentrates, King et al. (1969) investigated the possibility that diatomaceous earth filters could be used to entrap ascospores and asci of B . fulva in grape juice. A 5-log reduction in count by filtering did not depend on inoculum size. Since natural populations of Byssochlamys, if present, are usually small, i.e., in the order of
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germination of ascospores of B . fulva at pH 3, whereas 240 ppm were required for the same effect at pH 5. In an earlier study (Gillespy, 1940) it was shown that 500 ppm of the preservative were required to inhibit growth of B. fulva at pH 3. The D values at 88°C for ascospores of B . fulva in grape juice with concentrations of 250, 90, and 0 ppm of sulfur dioxide are 1.6, 4.5, and 8.8 minutes, respectively (King et al., 1969). These researchers suggested that sulfur dioxide, heat, and filtration could effectively reduce ascospore populations to insignificant levels. At equivalent concentrations, sulfur dioxide appears to be more effective in preventing growth of B. nivea than are potassium sorbate, sodium benzoate, and diethylpyrocarbonate-complete inhibition being observed at 200 ppm of sulfur dioxide in potato dextrose agar at pH 3.5 (Beuchat, 1976). Because of detrimental effects at such low concentrations, activity of sulfur dioxide has been ascribed to its antioxidant properties (Jensen, 1960). Gillespy (1946), on the other hand, attributed the lethal factor associated with sulfur dioxide to nonionized sulfurous acid. Potassium sorbate and sodium benzoate, added to grape juice at a concentration of 250 ppm, completely inhibited the growth of B . fulva for a 16-day incubation period at room temperature (King et a/., 1969). Maunder (1969) stated that 1000 ppm of sodium benzoate was fungistatic in fruit pudding and in fruit drink inoculated with B. futva and incubated at 29°C for 17 months. At 500 ppm, the compound was effective in fruit drink but not in pudding. The inhibitory effects of potassium sorbate and sodium benzoate against Byssochlamys are more pronounced as the pH of the suspending medium is decreased. This phenomenon is attributed to the degree of dissociation of the chemicals, and has been documented for other fungi for some time. Complete inhibition of outgrowth of ascospores of B. nivea in grape juice (pH 3.2) can be achieved for at least 60 days at 30°C by adding 400 ppm of potassium sorbate (Beuchat, 1976). Under the same conditions, growth, although delayed, was observed in juice containing as high as 1000 ppm sodium benzoate. Potassium sorbate at 50 and 100 pprn in grape juice may have a stimulatory effect on activation of ascospores of B. nivea during heating at 70"C, whereas higher concentrations are lethal (Beuchat, 1976). Concentrations of sodium benzoate between 50 and 1000 ppm tended to inactivate ascospores. The time elapsed between heat shock and exposure to 100 ppm of potassium sorbate had little effect on the number of ascospores forming colonies on potato dextrose agar (pH 3.5); however, exposure to the same concentration of sodium benzoate between 30 to 120 minutes after heat shock was more lethal than immediate exposure or exposure 4 to 6 hours after heat shock. It was suggested that there was an apparent recovery or a reduction in sensitivity associated with extended incubation before exposure to sodium benzoate. The period of high sensitivity was theorized to have been correlated with physiological and morphological changes leading from dormancy through germination and possibly germ tube

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development. This could have practical implications for the fruit processor. Addition of sodium benzoate at a particular point subsequent to heat processing may have greater effectiveness in reducing or controlling outgrowth of ascospores of Byssochlamys. Diethylpyrocarbonate (DEPC) has been investigated for its ability to control growth of microorganisms in fruit juices, wine, and beer. Splittstoesser (1972) and Splittstoesser and Wilkison (1973) examined the effects of DEPC on lactic acid bacteria, Saccharomyces cerevisiae, conidia of Aspergillus niger, and ascospores and conidia of B. fulva. Ascospores of B . fulva were more resistant to DEPC than were conidia. In 5" Brix grape juice containing 200 ppm DEPC, the viable ascospore count was reduced by about 90%after a 24-hour holding period. Beuchat (1976) noted that DEPC was more effective at pH 5.5 than at pH 3.5 with respect to percentage reduction in the number of colonies formed on potato dextrose agar by heat-shocked ascospores of B. nivea. Complete inhibition of 800 to 1000 ascospores was observed in grape juice containing 600 ppm DEPC; the compound was ineffective at 400 ppm. The presence of 50 ppm of DEPC in grape juice during heat treatment tended to inactivate ascospores of B. nivea, and exposure of ascospores to 100 ppm of DEPC between 60 to 120 minutes after heat treatment was more lethal than exposure immediately after heating or after 4 to 6 hours. An advantage to using DEPC as a preservative is that it quickly decomposes to form carbon dioxide and ethanol once added to food materials. However, there are indications that DEPC or reaction products may be carcinogenic and the preservative is therefore not legally used in many countries, including the United States. Other antimicrobials have been examined for their effectiveness in controlling growth of Byssochlamys. Hull (1939) immersed suspensions of ascospores of B. fulva in various antiseptics and then analyzed for viability. It was observed that growth was obtained after immersion in 10% formaldehyde for 10 minutes, in 10% Lysol for 30 minutes, in 0.5% sodium hypochlorite for 10 minutes, and in 0.5% mercuric chloride for 36 minutes. High concentrations of ammonia and acetaldehyde inhibit growth of B . fulva but are not lethal (Olliver and Rendle, 1934). Takano and Yabuki (1964) reported that several antimycotic agents had little effect on growth and ascopore germination by B . fulva. The destruction of asci of B. fulva by gaseous methyl bromide and by aqueous solutions of chlorine, an iodophor, and peracetic acid was reported by It0 et al., (1972). Results showed that asci were resistant to exposure to 200 ppm of chlorine (pH 6.5) and 446 ppm of iodophor (pH 2.2). Even 100 asci per ml were not rapidly destroyed by 1000 ppm of chlorine. A 4% solution of peracetic acid caused a 99.9%reduction of asci in 1.3 minutes. Asci dispersed in tapioca starch (a, 0.69) were completely destroyed in 30 days by adding 90 to 120 ppm of methyl bromide. Few asci persisted as long as 120 days in starch with an a, of 0.36. It was suggested that low concentrations of methyl bromide might be

25 2

LARRY R. BEUCHAT AND STEPHEN L. RICE

effective in eliminating fungi from foods and spices, provided that the proper a, is maintained during treatment, storage, and shipment. Although not completely anaerobic, as discussed earlier, Byssochlamys is capable of growing under greatly reduced oxygen tension, making control through manipulation of atmospheric content difficult. Olliver and Rendle (1934) noted that in jars of fruit where B . fulva spoilage had taken place subsequent to processing, the partial pressure usually was between 5 and 15 in. King et al. (1969) observed that B . fulva would grow in an atmosphere of nitrogen (99.999% pure) containing a trace of oxygen but not under strictly anaerobic conditions. Hull (1939) had noted earlier that B . fulva did not develop in an atmosphere free of oxygen and suggested that the depletion of oxygen rather than the production of an inhibiting concentration of carbon dioxide was responsible for stopping growth and eventually causing death of the organism in sealed cans of plum syrup. The temperature of storage of processed fruits can affect the rate at which Byssochlamys grows and should, therefore, be considered in procedures for controlling growth. Hull (1939) concluded that with a large headspace, maximum growth of B . fulva occurred at 20 to 38°C within 14 days, but within 28 days at 15°C. It was suggested that, in practice, if cans are stacked while still warm and if heat diffusion is slow, the development of mycelium will be greater than if the cans are cooled thoroughly before stacking.

IV. METABOLIC ACTIVITIES A.

ASCOSPORES I.

Production

The mold isolated from spoiled processed fruits in many instances is Paecilomyces (Spurgin, 1964; Maunder, 1969; King er al., 1969; Splittstoesser er a [ . , 1971). It is suspected that certain strains of Paecilomyces are observed because their perfect states are poor ascospore producers and because the number of asci formed is too low to be detected microscopically. King et al. (1969) suggested that, in cases where only the imperfect state is observed, ascospores were produced under natural conditions, i.e., on decomposed fruit before processing and/or during storage after processing, but this ability was lost when the mold was cultured on synthetic media for identification purposes. Conditions affecting ascospore formation by Byssochlamys spp. have been studied since the time the organism was first isolated and identified. Food microbiologists have been particularly interested in sporulation, since information gained from research in this area might lead to practical methods for preventing

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the process fmm occumng in raw and processed fruits. The effects of different variables on ascospore production by B. fulva were studied quantitatively by Splittstoesser et al. (1969). Tests on nine fruit and vegetable juices showed that eight supported good sporulation. Relatively high populations of asci were produced in commercial samples of prune, grape, and pineapple juices, whereas apple, orange, tomato, and sauerkraut juices and vegetable cocktail afforded lower counts. Cranberry juice, which supported the production of relatively few ascospores, also failed to support good vegetative growth. The presence of benzoic acid, a natural constituent of cranberries, was thought to have been responsible for these growth responses. Rice et al. (1977b) also reported that B. fulva grew poorly on cranberry cocktail. Ascospore production by B. nivea on fruit juices and nectars was investigated by Beuchat and Toledo (1977). Again, poor mycelial growth was noted on cranberry cocktail, but since the extent of ascospore development on peach, grape, pineapple, cranberry, and prune products held at 30°C over a 12-week test period was similar, it was concluded that a direct relationship did not exist between the extent of hyphal and ascospore production by B. nivea. An incubation temperature of 28 to 35°C is optimal for growth and ascospore production of most strains of Byssochfumys. If some isolates are incubated on synthetic media at temperatures below 30°C, however, they may irreversibly lose their capacity to form asci. Splittstoesser (1969, 1972) and Splittstoesser et al. (1969) reported that relatively good vegetative growth of B. fulva was obtained over a range of 20 to 40°C and that at 30 to 31°C ascospore production was highest, reaching a peak population after 7 days. Data showed that temperature was more critical for sporulation than for total growth. Beuchat and Toledo (1977) noted that, although B. nivea hyphae appeared within a shorter time after inoculation when fruit products were stored at 30°C as compared to 21"C, the extent of ascospore production during a 12-week storage period at these temperatures was similar. Slightly greater production at 21 or 30°C was dependent upon the type of fruit substrate. Byssochlamys spp. grow in nutrient substrates with pH ranging from 2 to 9, but the optimum for growth is about pH 3. Splittstoesser et ul. (1969) studied the effect of pH on ascospore production by B. fulva. The optimal value was found to be below pH 3, and similar yields of ascospores were obtained over the wide pH range of 4 to 8 in malt extract broth. The hydrogen ion concentration rather than the type of acid appeared to be the important factor affecting sporulation, since comparable results were obtained in media containing hydrochloric or tartaric acids, Rice and Beuchat (1978a), on the other hand, reported that at a given pH the concentration and type of organic acid in a synthetic growth medium affected the rate of ascospore formation as well as the total number of ascospores produced by B. fulva. Tartaric acid, which supported the least amount of biomass production, promoted the best ascospore production, whereas

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malic acid, which gave the best growth, supported only slightly less production of ascospores. The lowest numbers of ascospores were produced in media containing citric acid. Poor growth in tartaric acid media may have induced ascospore formation; conversely, good biomass production in malic acid media may have disfavored metabolic pathways associated with formation of ascospores. It is interesting to note that tartaric acid, the predominant acid found in grapes, appears to promote sporulation and that of the fruits in which B.fulva has been isolated or shown to cause spoilage, grapes are consistently a problem. It is possible that high numbers of ascospores found on grapes or in grape products may enhance the probability of the mold surviving thermal processing and, eventually, causing spoilage. Herbert and Larson (1972) investigated the interacting effects of pH and nutrients in a synthetic medium on production of asci by B . fulva. Of the different pH values employed in a complete synthetic medium with sucrose, only an initial pH of 4 gave consistent results. Erratic production of asci occurred if the initial pH was 2 or 5 or if glucose was substituted for sucrose. A more rapid utilization of glucose did not appear to be important, since there was no appreciable difference in the rates of dry weight increases between media containing glucose or sucrose. Others have studied the effects of fruit sugars on growth and sporulation. Although the optimum sucrose concentration for growth is 10 to 20%, B.fulva will tolerate 60 to 65% sucrose (Olliver and Rendle, 1934). Maltose, fructose, and glucose promoted growth at a rate comparable to sucrose. Asci were produced in large numbers when sucrose concentration in a solid synthetic medium was between 5 and 20%, but no asci were formed when the concentration exceeded 30%. Maunder (1969) observed that B.fulvu formed asci on potato dextrose agar containing 50% sugar after 2.5 months, and on grape concentrate at 50" Brix but not at 55". No inhibition in production of ascospores by B.fulva was noted when the organism was grown on peach halves containing 17 to 22% sugar (Rice et al., 1977a). The interacting effects of pH and fruit sugars on sporulation of B.fulva was reported by Rice and Beuchat (1978b). Greater ascospore production occurred in a defined liquid medium containing 5% sucrose, glucose, or fructose, as compared to 1% sugar media, when the initial pH was 4 or 5 . This effect was diminished in 5% sugar media having an initial pH of 3, indicating that B.f u h favors a higher pH for maximum ascospore production than for optimum biomass production, under certain culturing conditions. A variety of liquid and solid synthetic and fruit-based media have been employed with varying degrees of success by investigators as substrates for mass production of asci. These include potato dextrose, potato sucrose, Czapek-Dox, Sabouraud, malt extract, yeast extract, wort, and fruit juice agars and broths. Herbert and Larson (1972) and Herbert (1973) investigated the role of vitamins on sporulation. They concluded that riboflavin and nicotinamide were not essen-

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tial requirements for growth and ascospore formation, but rather ancillary additives to provide for enhanced growth and sporulation. It was suggested that the function of ascorbic acid is probably not nutritional but rather to suppress germination of ascospores which are produced, thereby promoting increased total numbers at any given time. Production of chlamydospores by B . nivea, but not by B . fulva, is a characteristic used to differentiate isolates. At least one report (LaRocca and Goos, 1975) exists, however, that documents the production of these spores by B . fulva. Chlamydospores were found on cultures grown on corn meal agar, soil extract agar, Czapek medium, and potato dextrose agar containing 0.07% Tween 80. Sizes varied from 6.3 to 9.9 X 4.5 to 8.1 p m . A thermal death time of less than 1 minute at 80°C was observed, indicating that chlamydospores are probably not significant as survival structures during canning processes, although they may function in this capacity in nature.

2 . Activation It has been long recognized that a large percentage of any given crop of ascospores of Byssochlamys possesses a dormancy that can be broken by heat treatment. Gillespy (1938) obtained the highest percentage germination when a suspension of ascospores of B. fulva was heated for 10 minutes at 75°C in a potato sucrose medium. Hull (1939) recognized that unheated ascospores either do not germinate or do so sporadically, and found that germination was most extensive when ascospores were heated for 10 minutes at 75 to 80°C. Splittstoesser er al. (1969) reported that a 20-minute treatment at 70°C completed activation whereas King et al. (1969) heat-activated ascospores of B . fufva by heating at 80°C for 4 minutes. The nature of the suspending medium can greatly affect the extent of activation at a given temperature of treatment. Splittstoesser et af. (1970) reported that activation was completed in 60 minutes or less when ascospores of B . fulva were heated at 60°C in Concord grape juice, whereas heating in water for as long as 3 hours produced no detectable changes in viable count. Activation also was enhanced by heating in grape juice at 70"C, although at this temperature a significant number of spores could also be activated in water. From studies using numerous strains it was concluded that 70°C was the optimal activation temperature and that grape juice could be diluted to as low as 2" Brix without reducing activation rates. Studies on the mechanism of stimulation revealed an interaction between pH and an active factor(s). Adjusting grape juice to pH 4 caused a marked reduction in the number of ascospores activated, whereas at pH 5 to 7 the viable counts were no higher than obtained when ascospores were heated in water. It was shown that pH per se was not responsible for the effect, since ascospores heated at pH 3.5 in solutions of malic and tartaric acids failed to

256

LARRY R. BEUCHAT AND STEPHEN L. RICE

enhance activation. Further investigation showed that a 5% solution of yeast extract gave results comparable to those using grape juice. Additional search for a substance(s) in grape juice that stimulates ascospore activation led to the discovery that activation was enhanced further when ascospores were treated with a solution of 0.1 N hydrochloric acid (Splittstoesser et al., 1972). At 60°C maximal activation occurred in both hydrochloric and nitric acid solutions. The concentration of hydrogen ions and the type of anion were critical, little activation being achieved above pH 1.6, and the addition of sodium or potassium chloride resulted in increased activation. Tests to determine the fate of activated ascospores exposed to an aqueous environment at 32°C revealed that about 50% reverted to a dormant state during a 7-day holding period.

3 . Germination Like bacterial spores, ungerminated ascospores are highly refractile when viewed under the phase microscope, but lose this characteristic on germination before the germ tube(s) emerges. King et al. (1969) studied sequential changes occurring in activated ascospores of B. fulva (Fig. 2). On emergence from the ascospore, the hypha commonly produces a basal swelling attached to the ascospore, from which one or two hyphae grow. The time required for germination may vary considerably so that a microscopic field may include ungerminated and germinated ascospores, as well as substantial amounts of mycelium. Germinating ascospores with emerging hyphae are less dense than are ungerminated spores. Determination of specific gravities using density gradient centrifugation showed that density of ascospores and asci vaned but was always in the range of 1.34 to 1.37 (King et al., 1969). Since the specific gravity of juice concentrates may be somewhat higher than that of ascospores, it would be impractical to separate ascospores by centrifugation, because they would float rather than settle. The temperature optimum for spore germination appears to be higher but the temperature range is narrower than for mycelial growth. Hull (1939) indicated that germination began after 10 and 15 hours, respectively, when heat-activated ascospores of B . fulva were incubated at 38 and 32°C. At 25 and 20"C, germination began within 24 and 43 hours, respectively, but ungerminated ascospores were observed long after this. At 15"C, there was no germination; however, it has been reported by several researchers that Byssochlamys will grow at this temperature. The effects of sucrose on germination of ascospores have been examined. Germination of B . fulva was observed within 18 hours on agar plates having sucrose concentrations up to 50% and within 37 hours on agar containing 70% sucrose. Germination was more rapid on 20% than either 0 or 40% sucrose agar plates. Beuchat and Toledo (1977) studied the rate of colony development and

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FIG. 2. Sequential changes occurring in the germination of ascospores of Byssochlarnys fulvu. Phase microscopy, 325 X . (A) Asci containing refractile ascospores, before germination; (B) ascospores darkening prior to emergence of germ tube; ( C . D) germinating ascospores. Note swelling of hyphae next to the ascospore from which they emerged. (E) Germinated ascospores and hyphae which they produce. Note refractile, ungerminated ascospores. From King e t a / . (1969).Reproduced with permission from the American Society for Microbiology.

growth of B . nivea on grape agar containing various levels of sucrose. Agars to which 20 gm of sucrose or more per 100 ml had been added retarded germination of heat-shocked ascospores as well as colony growth. Colony formation was reduced by 80% when asci were plated on grape juice agar containing 40 gm per 100 ml, and at 60 gm no colonies were detectable by the unaided eye. These studies indicate that the maximum sucrose concentration in which ascospores of Byssochfumys can germinate may be somewhat lower than that tolerated for growth.

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LARRY R . BEUCHAT AND STEPHEN L. RICE

Yates et al. (1968) investigated the requirements of ascospores for nutrients during germination. Soluble starch, ascorbic acid, alanine, thioglycollic acid, and dipotassium phosphate significantly depressed germination, whereas calcium chloride, potassium iodide, potassium acetate, threonine, glycine, methionine, leucine, aspartic acid, serine, phenylalanine, and potassium acetate promoted germination rates of B . nivea ascospores. Acetate resulted in the most significant improvement in germination as well as the highest germination rates, and was found to be the most effective germination stimulator of the compounds tested. Tricarboxylic acid cycle compounds were tested for their ability to replace acetate as a germination stimulant, but none was appropriate. Citrate was found to be inhibitory. Heat-activated ascospores are responsive to oxygen and carbon dioxide. Germination of ascospores of B . nivea is adversely affected by carbon dioxide at concentrations greater than 70% (Yates et a l . , 1968). Above 70% germination was rapidly reduced to 11% at 100% carbon dioxide. Aeration, on the other hand, promotes germination. Having observed that phosphate drastically reduced germination of ascospores of B. nivea, Yates (1973) deduced that cation binding may influence germination. Studies were conducted to determine the effects of cations on respiration of B . nivea. Calcium, magnesium, copper, manganese, potassium, aluminum, and sodium chlorides at 10 mM did not enhance respiration; however, cupric chloride resulted in a marked inhibition in respiration in every test. Noting that increased respiratory activity accompanied germination and that the site of action of Cu * appears to be the membrane, it was concluded that data provided further evidence that permeability of the ascospore is critical in the germination process. 4 . Inactivation Byssochlamys is a problem in fruit spoilage mainly because of the exceptionally high heat resistance exhibited by its ascospores. This characteristic was first recognized by Hirst and McMaster (1933) and has led to considerable research effort to determine environmental conditions affecting the degree of heat resistance. Numerous studies have also been conducted to determine other environmental conditions favorable or adverse to inactivation of ascospores. Nearly all investigations in this area have had as objectives the determination of processing and handling procedures which will prevent spoilage. Most studies on heat resistance of Byssochlamys have been carried out using asci, not free ascospores. Therefore, observed rates of inactivation for asci usually do not result in traditional straight-lined logarithmic thermal death rate curves, at least early in the inactivation process (Bayne and Michener, 1979; King et a l . , 1979). Even studies using 100% free ascospores, however, do not result in first-order death rate curves (Splittstoesser and Splittstoesser, 1977).

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Death of one to seven of the eight ascospores within an ascus cannot be detected by observing colony formation on laboratory plating media. Assuming, however, that lethality rates are uniform among ascospores in asci in a suspension subjected to heat, logarithmic death, as translated from plate counts, can be observed only after the eighth ascospore in each ascus fails to germinate and grow to produce a colony. Gillespy (1946) stated that when less than 10%of the asci survive, the probability is that only one ascospore per ascus survives. It was further theorized, based on observed thermal death rate curves for asci, that destruction curves for ascospores would be straight lines, as for bacteria. This would allow for decimal reduction times for ascospores to be calculated as approximately one-third of the time required to reach 1% survival of asci. Splittstoesser et al. (1969) also recognized problems inherent in enumerating thermally treated ascospores. Data suggested a single destruction rate for ascospores throughout heat treatment, and the authors indicated that more realistic counts for ascospores might be obtained by extrapolating from the straight-lined logarithmic inactivation portion of destruction curves. Even by using this procedure, however, an accurate count for viable ascospores may not be obtained, since some ascospores may remain dormant due to lack of activation or an improper nutrient environment in plating media. Procedures have been devised for freeing ascospores from asci and may be used to prepare uniform suspensions of spores for heat resistance studies. Michener and King (1974) accomplished this with the aid of a pressure cell from which a suspension of asci under high pressure was released to atmospheric pressure through a small orifice. Ascospores so treated had about the same heat resistance as untreated spores. Partsch (1969) used a series of filters to separate ascospores from mycelium. Ascospores of Byssochlamys vary in their capacity to withstand thermal treatment. Heat resistance is influenced by many factors, including species, nature of heating menstruum, and, most importantly, the temperature of treatment. A recent report (Hatcher e t a / . , 1979) indicates that thickness of ascus walls may be correlated with degree of heat resistance. Gillespy (1946) observed that ascospores of B. nivea were much less heat-resistant than those of B. fulva, thus indicating that spoilage of canned fruit by B. nivea may not be a problem. Liithi and Hochstrasser (1952), however, showed that B. nivea survives heating for 2 minutes at 90°C. Put and Kruiswijk (1964) found that the heat resistance of B . nivea ascospores in strawberries appeared to be about the same as that of B. fu/va. Long-chained fatty acids in spores may enhance heat resistance (Banner et al., 1979). The presence of antimycotic agents in juices tends to enhance the lethal effects of heat. Sulfur dioxide (Gillespy, 1940; King et a t . , 1969; Beuchat, 1976), potassium sorbate, sodium benzoate, and diethylpyrocarbonate (Beuchat, 1976), when added to grape juice, markedly increase the rate of inactivation of ascospores of Byssochlamys.

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Heat resistance of ascospores is influenced to some extent by the pH of the medium in which they are suspended (Hull, 1939). Tolerance of B. fulva was greatest at pH 5 . The effect of pH on heat resistance was studied by adjusting the reaction of grape juice with tartaric acid and sodium hydroxide (Splittstoesser and Splittstoesser, 1977). The highest percentage of ascospores of B. fulva surviving after 1 hour at 85°C was noted in suspensions adjusted to pH 3 and 4. The type of acid in heating media greatly affects the survival of ascospores. Splittstoesser et al. (1976) heated ascospores of B . fulva in tomato, apple, and Concord grape juices for 3 hours at 85°C and found surviving populations 1000fold higher than for spores heated in water, a pineapple-grapefruit blend, and a vinifera grape juice. Food constituents that protected ascospores during heating included glucose and malic, citric, and tartaric acids. Comparable survivals obtained with these acids over a pH range of 3 to 6 indicated that pH per se had little effect on resistance. When fumaric, lactic, succinic, and acetic acids were tested, lower numbers of survivors were obtained at pH 3.5 and below. Splittstoesser and Splittstoesser (1977) and Splittstoesser (1978) pointed out the practical application to using certain organic acids to control Byssochlamys in fruit juices. Thermal death times for B. fulva were determined in Concord grape juice that had been acidified to pH 3.0 with 0.5% citric, fumaric, or lactic acids. Samples containing 30,000 ascospores per ml were then heated at 90"C, incubated, and examined for growth. The thermal death time in juice acidified with citric acid was 25 minutes compared to less than 5 minutes in juice acidified with fumaric or lactic acids. These results showed that process requirements with respect to B. fulva could be reduced by using the proper organic acid. The protective action of sucrose against heat inactivation of ascospores has been documented by several researchers. Hull (1939) observed that percentage germination of ascospores of B. fulva heated at 84°C for 30 minutes was increased from 1% to 75%by increasing the concentration of sucrose in the heating menstruum. Gillespy (1938) noted that, at any given heat treatment, the greater the concentration of sucrose in the suspending medium, the greater the percentage survival of ascospores. This was true in fruit juices at pH 3 and in buffer solutions at pH 3 to 7. Later, Gillespy (1946) reported a Qlo value of approximately 70 for the destruction of ascospores of B. fulva heated at 85 to 90°C in water, buffer, or fruit juice with 6% solids. When the suspending medium was changed to syrup of approximately 30" Brix, the Qlo value decreased to 15. Thermal inactivation of ascospores of B. fulva in grape juice and grape concentrate was determined by King el al. (1969). The D values were slightly higher for the concentrate, the z value being 6.7"C. Baumgart and Stocksmeyer (1976) monitored the heat resistance of ascospores of Byssochlamys spp. in apple juice supplemented with sucrose. About the same resistance was noted for B . nivea heated at 90°C in juice containing I .7% sucrose and at 99°C in juice containing 4.7% sucrose.

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26 1

The interacting effects of a, and heat on inactivation of ascospores of B . nivea were studied by Beuchat and Toledo (1977). Ascospores were heated at 75°C in grape juice supplemented with various amounts of sucrose to reduce the a, to as low as 0.84. After 7 hours, the spore population was reduced from l o 5 per ml to zero in juice containing no added sucrose, whereas the number of viable ascospores held at 75°C in juice at a, 0.84 was reduced by only 70% after 8 hours. The reason for the protective effect of sucrose is not clear, but the osmotic pressure differential between the supporting medium and ascospores apparently influences resistance to heat inactivation. Hydrolysis of sucrose after extended heating at low pH may also occur, thus further influencing the rate of inactivation. Lubieniecki-von Schelhorn and Heiss (1975) studied the heat resistance of ascospores of B . fulva in atmospheres with reduced relative humidity. They reported a D value of 25 minutes at 0% relative humidity and 120°C. Although ascospores showed exceptional resistance to heat treatments in the dry state, they did not exhibit as high a degree of heat resistance as do spores of several Bacillus SPP. An interesting series of results arose from experiments to test the effect of intermittent heating on asci of B.fulva (Gillespy, 1938). Finding that two short treatments at 75°C with an interval of 6 hours at 37°C destroyed all asci in suspension, attempts were made to determine the best heating conditions and the shortest time possible between exposure to heat or necessary to kill all the asci. The optimum process required heating ascospores at pH 3 for 10 minutes at 77°C followed by an incubation period of 30 minutes at 46°C and a second 10-minute heat treatment at 77°C. Germination of ascospores apparently occurs during the intermediate holding period at 46"C, thereby making them more sensitive to heat during the second heat treatment. It is difficult to compare reports on the heat resistance of Byssochlamys spp. because of a lack of standardized testing conditions such as methods for preparing ascospores, concentration of ascospores in the heating medium, nature of the heating medium, and recovery media. There also appears to be some variation among species and strains. A summary of results from reports on the resistance of ascospores of Byssochlamys to adverse temperatures is given in Table I. The information presented attests to the fact that the organism is exceptionally heat resistant and would be expected to withstand processing conditions normally given to many fruit products. Ascospores are not easily killed by desiccation, as cultures of Byssochlamys kept in a dried condition for 4 years will grow on subculturing (Hull, 1939). Gamma irradiation of fruit juices infected with Byssochlamys, in addition to preservatives and heat, has been explored as a pasteurization or control technique. Partsch and Altmann (1970) examined the sensitivity of ascospores o f B. fulva in apple, grape, and orange juices and reported that outgrowth could be

TABLE I SENSITlVlTY OF ASCOSPORES OF Byssochlumys spp. TO ADVERSE TEMPERATURES

Species

Temperature ("C)

B . fulva

85

Grape juice, 5" Brix

85

Grape juice, 5" Brix

85

Grape juice, syrup

85

Cranberry juice, 5" Brix

85

Glucose solution

85

Organic acid solutions

86 87.7 87.8

Grape juice Grape drink Grape juice

88

Plum, strawberry, gooseberry, and raspberry syrups Distilled water

88

Suspending medium

Comments 10.001 to 50% survival after 60 minutes, depending on strain 8.4%survivalafter60minutes,2.l%after 75 minutes 0.21% survival in 5" Brix juice at pH 1.4, 4.7% at pH 2.3, 36% at pH 3 and 6; 0.36% survival in 50" Brix juice at pH 3, >84% at pH 4 to 6 1.3% survival after 60 minutes 21% survival in 40% solution after 75 minutes, 2.3% survival in 10% solution 78% survival in tartaric, 42% in malic, 1.9% in acetic, (1% in succinic, lactic, and fumaric acids, 14% in water after 120 minutes D value = 14 minutes D value = 10.32 minutes D value = 4.8-1 1.3 minutes, depending on strain Survived for 30 minutes Maximum time of resistance was 40 minutes

Reference Splittstoesser et ul. (1974) Splittstoesser and Splittstoesser ( 1977) Splittstoesser ef al. (1976)

Splittstoesser and Splittstoesser (1977) Splittstoesser and Splittstoesser (1977) Splittstoesser and Splittstoesser (1977)

Michener and King (1974) Denny and Brown (1969) King ef ul. (1969) Olliver and Rendle (1934) Hull (1939)

B. nivea

N W 01

90 90.4 90.4 90.4 92 93

Distilled water Fruit drink Punch Blackberries, fruit pudding Distilled water Fruit

94

Distilled water

95

Grape juice concentrate

95 95 98 101

Distilled water Strawbeny syrup, 34.8" Brix Plum syrup Apple juice

I20

75

Atmosphere, 0% relative humidity Fruit juices, syrups Grape juice, syrup

84 87.5 88 88 99

Distilled water Strawbeny juice Grape juice Sucrose solution, 30% Apple juice

- 30

Survived 3 minutes, but not 5 minutes lo4 asci per ml killed in 5 minutes 10' asci per ml killed in 10 minutes 10' asci per ml killed in 15 minutes D value = 27-37.5 minutes 1 % survival after 12,21, and 40minutes in gooseberries, plums, and strawberries, respectively Maximum time of resistance was 2 minutes 6.8 minutes required to destroy 3 X 10' asci Survived 5 minutes Survived 1 minute, but not 1.5 minutes Survived Survived in juicecontaining 4.7% sucrose, but not in 3.7% or lower D value = 25 minutes

Schumann and Werner (1973) Maunder (1969) Maunder ( 1969) Maunder ( 1969) Thompson (1969) Gillespy (1946)

Hull (1939)

King er a / . (1969)

D values of about 60 minutes in juice, 470

Peter (1964) Richardson (1965) Olliver and Rendle (1934) Baumgart and Stocksmeyer (1976) Lubieniecki-von Schelhorn and Heiss (1975) Beuchat and Toledo (1977) Beuchat and Toledo (1977)

minutes in juice with 66% sucrose Survived 3 minutes, but not 5 80% survival after 10 minutes Survived 60 minutes Survived 45 minutes Survived in juice containing 4.7% sucrose, but not in 3.7% or lower

Schumann and Werner (1973) Put and Kruiswijk (1964) King et a / . (1969) Yates and Ferguson (1963) Baumgart and Stocksmeyer (1976)

Slow loss in viability, protected by sucrose

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LARRY R. BEUCHAT AND STEPHEN L. RICE

retarded with irradiation doses as low as 100 krad. However, 100% of ten 10-ml samples of each juice initially containing 10 ascospores per ml showed growth after 15 days of incubation at 37°C when treated with 140 krad. At 150 krad, differences between grape and apple juices were noted. While all samples of grape juice and some samples of apple juice showed germination even after an extended period of storage, no growth occurred. Growth in orange juice was observed at doses above 200 but not at 240 b a d . Simultaneous treatment of infected juices with gamma irradiation and heat was investigated by Partsch et al. (1970) to see if there was a synergistic effect. It was found that heat (72°C) and simultaneous irradiation of 180 krad for grape and orange and 160 krad for apple juice guaranteed 3 months of storage without spoilage. At 50°C, with pretreated spores, the irradiation dose can be reduced by about 20 to 40 krad, but the treatment is technically more difficult to perform. Gamma irradiation may have a detrimental effect on certain enzymes involved in germination. Partsch et al. (1968) studied isoenzymes of conidia of B . fulva to determine radioresistance. Although 1500 krad had no significant effect in vivo, there were some changes in patterns of malate dehydrogenase and lactate dehydrogenase in vitro.

B . ENZYMES 1.

Pectinases

Olliver and Rendle (1934) first reported that B . fulva disintegrated fruit by attacking pectin. They showed that B . fulva breaks down citrus pectin and theorized that the destruction of “cementing pectinous substances” in fruits infected by the fungus caused rapid softening. The addition of inorganic salts and peptone to pectin solutions was shown to enhance the rate of decomposition by B . fulva. The presence or absence of naturally occurring substances favorable to the organism was offered as an explanation for the varying degree of rate of fruit disintegration observed in the food industry, and also for the lack of correlation between disintegration and rigidity of fruit structure. Concurrent with the report by Olliver and Rendle, Hull (1939) noted that growth of B . fulva was accompanied by the production of a “pectin-destroying’’ enzyme which caused the breakdown of fruit tissues. The enzyme was detected by the softening of potato disks in solutions in which B . fulva had been growing. In a more thorough study, Beaven and Brown (1949) reported that B . fulva produced protopectinase and a disaggregating enzyme which reduced the molecular size of citrus pectin without the production of galacturonic acid. The absence of polygalacturonase and pectin esterase was noted. Reid (1952) reported that B . fulva did produce both polygalacturonase and pectin esterase in addition to arabinase and galactanase. It was suggested that Beaven and Brown

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(1949) were unable to detect polygalacturonase due to low activities in their enzyme preparations. There was no evidence for the presence in B. fulva of “disaggregating enzymes. Yates and Mooney (1968) studied pectic enzyme production by B. nivea. They observed that the fungus produced endopolygalacturonase and endopolymethylgalacturonase but not pectin methylesterase, exopolygalacturonase, or pectate transeliminase. Endopolygalacturonase activity was greatest at pH 4.9 to 5 . 5 , dropped at pH 6.0, and returned to a high level at pH 6 . 5 . Maximal enzyme production was obtained in a glucose-pectin medium after 5 days of incubation at 35°C. Enzyme activities increased with increasing concentration of citrus pectin. A study of the production of pecteolytic enzymes by eight isolates of B. fulva revealed that there was considerable variability among strains (Chu and Foster, 1970). Only two isolates produced appreciable amounts of pectinases, among which were pectin transeliminase, polygalacturonate lyase, and polymethylgalacturonate lyase. A pectin transeliminase was partially purified and found to have an optimal pH for activity of about 4.9. The enzyme was activated by Mg * and Ca *, and to a lesser extent by Na+ . Later Chu and Chang (1973) examined some of the same isolates of B. fulva for their capacity to produce polygalacturonase, pectinesterase, and pectate lyase in a liquid medium consisting of glucose, yeast extract, pectin, and salts. Chu (1969) had indicated earlier that such a medium was best for supporting the production of pecteolytic enzymes. None of the eight isolates of B. fulva examined by Chu and Chang (1973) produced all three enzymes; however, all produced at least two of the enzymes. Byssochlamys fulva proved to be a potent organism for producing extracellular constitutive pectinase activity (Abdel-Fattah and El-Hawwary, 1973). The mycelial sporulation showed no correlation to pectinase activity and it was suggested that extracellular and intracellular pectinase activities were different. The growth of B. fulva in canned peaches of differing variety and maturity was studied by Rice et al. (1977a). The pectic enzymes secreted by B. fulva over a 16-day test period at 30°C resulted in an initial increase in water-soluble pectin followed by a decrease in total and water-soluble pectins. Very little growth was evident on inoculated peaches during the first phase of change in pectin composition. Textural changes were noted in inoculated peaches as compared to controls, and it was observed that variety and stage of maturity had an effect on the susceptibility of pectin to alterations by B. fulva. Growth rates were more rapid on mature peaches, which had lower acidity and higher initial levels of soluble solids. Rice and Beuchat (1978a,b) investigated the effects of acids and sugars in fruits on polygalacturonase production by four strains of B. fulva. Polygalacturonase activity was higher in a liquid medium supplemented with tartaric acid than in media containing malic or citric acids. Most activity was produced ”

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between 4 and 8 days of incubation; however, production was dependent upon the initial pH and acid concentration. Polygalacturonase activity was similar in media containing glucose and sucrose whereas it was slightly less in media supplemented with fructose. Most activity was produced between 4 and 8 days on media containing 1% sugar. On the basis of biomass accumulation, enzyme production in 1% sugar media was much greater than that in 5% sugar media. It was suggested that under conditions of low concentration of utilizable carbon sources, higher polygalacturonase activity would be required to break down pectin into a form that could be utilized. Higher concentrations of acids and sugars in fruits and fruit products may then retard polygalacturonase production somewhat, although the prolonged result of pectinase activity would probably be the same, i.e., disintegration of the fruit.

2, Proteinases The enzyme rennin is a specific proteinase which transforms soluble casein into insoluble p-casein without significant proteolysis. Knight (1966) examined 39 common filamentous fungi for their capacity to produce rennin-like enzymes. When the supernatant liquids from a whey medium in which the fungi were cultured were tested for rennin-like activity it was observed that B. fulva produced enzymes having good activity throughout the fourth to ninth days of growth. Reps et al. (1969, 1970) and Reps and Poznanski (1970) obtained a purified milk-coagulating protease by growing particular strains of B. fulva in a liquid medium and then characterized the optimal temperature of milk coagulation, changes in coagulation activity as related to milk acidity, the product of coagulation time as affected by enzyme activity, and kinetics of milk fermentation several hours after treating with the protease. Activity was investigated on the basis of nitrogen, calcium, and phosphorus content in whey. The optimal temperature of milk coagulation range between 64 and 66"C, and the enzyme was less susceptible, as compared with rennet, to changes in milk acidity. The contents of nitrogen, calcium, and phosphorus in whey after curd separation were almost the same for both rennet and the B. fulva protease preparation. It was suggested that the protease of B. fulva could be used for milk coagulation in cheese manufacture. The culture filtrate of B. fulva was characterized by Abdel-Fattah et al. (1972) as having high milk-coagulating activity and low proteolytic activity while the contrary was found with cell-free extracts. The results also showed that milkcoagulating and proteolytic enzymes were different in the culture filtrate as well as in the cell-free extract, and that intracellular and extracellular proteolytic activities seemed to be different. Rymaszewski et al. (1973) characterized the proteolytic activities of proteases of B. fulva. a,-Casein was hydrolyzed with the highest intensity; isoelectric casein and &fraction were hydrolyzed to a lesser extent and the hydrolysis of %-casein was the lowest. The highest proteolytic

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activity, several times higher than that of rennet, was shown at pH 6.0 and 6.6. Simultaneously, enzymes had the ability to produce considerable structural changes in the casein molecule and its fractions, suggesting that both endo- and exopeptidases were present in B . fulva preparations. The production of rennin-like enzymes by B . fulva varies considerably with the isolate (Chu et al., 1973). They reported that of seven isolates tested, three were good enzyme producers. A partially purified enzyme from one strain, given the name byssochlamyopeptidase A, had a pH optimum at 2.9, a temperature optimum around 60"C, and appeared to be relatively stable at 40°C between pH 3.0 and 6.85. The milk-coagulating activity of byssochlamyopeptidase A was dependent on pH and no extensive proteolysis was observed during coagulation. Later, Sun (1976) made an extensive study of production, purification and characterization, substrate specificity, and enzymatic activities of byssochlamyopeptidase A. Production of the enzyme was greatly influenced by variations in culture medium, carbon and nitrogen sources, and incubation period. Glucose was found to be the most efficient carbon source, and the addition of casein or sodium chloride to the basal medium resulted in marked improvement in enzyme production. Purified byssochlamyopeptidase A has a molecular weight of 34,000 and is stable for 24 hours between pH 3 to 6 at 4°C and between pH 4 to 5.5 at 37°C. The enzyme is less sensitive than pepsin to pH 4.6 to 6.6 for milkcoagulating activity. It was suggested that byssochlamyopeptidase A should be evaluated for its effects on cheese texture, flavor, and yield. Properties of rennin-like enzymes produced by P. varioti were examined by Sawada (1963, 1964). The pH and temperature optima were 2.5 to 3.0 and 60"C, respectively-very similar to those of milk-coagulating enzymes of B. fulva. 3 . Amylases

Surface-bound amylase was detected in B. fulva by Thompson (1969). Potato dextrose broth in which the fungus was grown was negative for the enzyme, a buffered extract was weakly active, and a mycelial residue extract was strongly active. Amylase was measured colorimetrically and viscometrically with soluble starch as substrate. C. I,

MYCOTOXINS Byssochlamic Acid

In the course of studying the chief metabolic products of B . fulva cultured on a synthetic medium containing glucose as a sole source of carbon, Raistrick and Smith (1933) noted that in preliminary tests an ether-soluble precipitate formed upon the addition of hydrochloric acid to 4-week-old Czapek-Dox culturing fluid. Further experiments showed that the amount of precipitate obtained in-

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LARRY R. BEUCHAT AND STEPHEN L. RICE

creased with continued incubation and reached a maximum when the glucose was entirely consumed. Yields varied with the temperature of incubation, being less at 30 than at 24°C and became negligible at 37°C. The acid precipitate was toxic to mice and was given the name byssochlamic acid. The compound titrates as a tetrabasic acid, giving salts of the type CI8H,,O8&, and is theorized to exist in the culture fluid as a mixture of salts of this general which immediately loses two formula, acidification giving free acid C1BH2408, molecules of water to give CI8H2,O6. Further characterization of byssochlamic acid revealed that it couId be obtained by crystallization from a mixture of ether and benzene in the form of colorless prisms, m.p. 163°C (Raistrick and Smith, 1933). There is no obvious decomposition at the melting point, and the melt is clear and colorless but does not resolidify on cooling, even when seeded. Its solubilities at room temperature in various organic solvents, expressed as grams per 100 ml, are approximately: acetone, 63; ethyl acetate, 33; chloroform, 20; benzene, 7.5; ether, 0.33; ethanol, 0.3;light petroleum,
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The molecular structure of byssochlamic acid was determined by X-ray analysis of the heavy atom derivative, byssochlamic acid bis-p-bromophenylhydrazide (Hamor et al., 1962; Paul et al., 1963). The crystals are tetragonal, of space group P4,2,2, with eight molecules of C30H3oBr2N404 in the unit cell dimensions a = b = 10.07, c = 57.61 8, (Paul et al., 1963).

CH2-CH2-

-CH,

0

Phase determination was based initially on the bromine atoms, and Fourier and least-squares refinement methods were employed in the determination of the atomic coordinates. Baldwin et al. (1965) later confirmed the constitution and stereochemistry of byssochlamic acid as determined by X-ray crystallography of its bis-p-bromophenylhydrazide derivative. The absolute configuration of byssochlamic acid was determined by fission of the nine-membered ring to give acidic fragments of known absolute configurations. Studies using laboratory animals indicate that byssochlamic acid is toxic when administered at relatively high levels. Intraperitoneal injection of 12 mice with 25 mg of the sodium salt of byssochlamic acid resulted in 100% mortality in 24 hours (Raistrick and Smith, 1933). A second group received 12.5 mg and, of these, one died in 24 hours, two after 2 days, and one after 3 days. King et al. (1972) studied the acute toxicity of byssochlamic acid to mice by intraperitoneal injection and oral administration. Injection of the acid as a solubilized sodium salt gave an LDSo of 94 mg/kg body weight, based on 7-day survival. The LDso was 80 to 195 mg/kg body weight when byssochlamic acid was suspended in aqueous 10% gum acacia before intraperitoneal injection, suggesting that, in spite of its low solubility, the acid has nearly the same LD5o as its solubilized form. The oral mouse LDSo of byssochlamic acid suspended in corn oil was greater than 2.5 g d k g of body weight but less than 4.0 g d k g . Thus the oral LD,,, was shown to be nearly 30 times greater than the intraperitoneal value. The toxin is weakly hepatotoxic to guinea pigs (Gedek, 1971). King et al. (1972) also studied the effects of feeding high dietary levels of whole dried mycelium of B. fulva to rats. Four groups of weanling male rats (eight per group) were fed 0, 2, 4, and 8% dietary levels of powdered culture of B.fulva that had been grown on 15" Brix white grape juice concentrate for 41 days at 25°C. The freeze-dried material contained 39 mg of byssochlamic acid per gram. All rats survived a 90-day feeding period; however, the mean body

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LARRY R. BEUCHAT A N D STEPHEN L. RICE

weight of the group of rats fed the diet containing 8% dried culture was significantly lower than that of the control group. A small but significant reduction in blood hemoglobin concentration was observed in the group of rats fed the highest intake of B . fulva mycelium. Microscopic examination of urine and tests for specific gravity, pH, occult blood, ketones, glucose, protein, and bilirubin were negative. No gross abnormalities were detected when test rats were autopsied but liver weights of animals fed all levels of dried mycelium of B . fulva were increased significantly over the control. Because of the low solubility of byssochlamic acid, it was suggested that most of the compound ingested in the diet would be eliminated in the feces, and fecal extraction data tended to support this latter premise. Assuming that the adverse effects in laboratory animals could be extrapolated to man, King et al. (1972) stated that a wide margin of safety would appear to exist between oral mycelium intake of rats and the relatively low levels of possible exposure in humans. In terms of spoiled canned fruit juice, it was estimated that comparable food levels in humans would be about 50 liters per day. The sodium salt of byssochlamic acid at a concentration of 0.002% was reported to greatly reduce the growth of hypocotyls and radiculae of light- or dark-germinated seed of Sinapsis alba (Meyer and Rehm, 1969). At a 0.1% level, germination was completely inhibited or greatly reduced. Sodium byssochlamate at 0.000 1 M delayed fermentation by Saccharomyces cerevisiae, while at 0.001 M fermentation was completely suppressed. Schmidt and Rehm (1970) investigated the effects of byssochlamic acid on several enzymes of primary metabolism. In vitro tests showed that adenosine deaminase was completely inhibited by 0.01 M byssochlamic acid, whereas at 0.001 M, the acid had a weak effect. Alcohol dehydrogenase and isocitrate hydrogenase were also inhibited at 0.01 M, but malate dehydrogenase, glutamate dehydrogenase, and glutamate-oxalacetic transaminase were not. Production of byssochlamic acid by P. varioti and Byssochlamys spp. cultured on laboratory media and on feeds, foods, and food components has been investigated. Meyer and Rehm (1967) studied production by B . fulva in Czapek-Dox solution supplemented with various carbon-containing compounds. The toxin was first detected after 8 to 10 days of incubation in solution containing 5 % glucose; however, the major portion was formed after 1 to 2 months after all of the glucose was utilized. It was theorized that byssochlamic acid was produced by an autolytic process. An increase in the concentration of glucose to 10% had little effect on the amount of byssochlamic acid formed. Sucrose, maltose, fructose, galactose, lactose, xylose, citric acid, gluconic acid, and glycerol promoted toxin production. Byssochlamic acid was produced in media adjusted between pH 2.5 to 8.0. Escoula (1975e) reported on the effects of formic and propionic acids on the production of byssochlamic acid by B . nivea. A strain was cultured for 5 months at 26°C on kernels of corn containing 56% moisture. Formic acid at

BYSSOCHLAMYS SPP.AND PROCESSED FRUITS

27 1

0.15 and 0.3% and propionic acid at 0.5 and 1.O% retarded growth and toxin production. In the presence of 0.6% formic acid and especially with 2.0% propionic acid, growth of B . nivea was inhibited. A maximal byssochlamic acid concentration of 390 ppm was obtained by growing a strain of B. nivea isolated from silage for 60 days at 26°C on kernels of corn containing 80% moisture (Escoula, 1975a). Of seven other liquid and solid substrates tested, the lowest amount (15.2 ppm) was formed on soybean meal, also containing 80% moisture. Maximal production of byssochlamic acid was obtained after 60 days of culture, i.e., during the autolysis stage. Growth of B. nivea in anaerobiosis and in pure culture on sterilized standing forage or in ecological conditions very similar to ensilaging is accompanied by production of byssochlamic acid (Escoula, 1975b). In pure culture and in anaerobiosis, toxin level reached 34.25 ppm after 82 days of incubation. The toxinogenesis of ten strains of B . nivea, four of B . fulva, and eight of P. varioti, all isolated from forages, was later studied in an enriched Czapek liquid medium at 26°C (Escoula, 1975d). Six filtrates from B . nivea cultures, all four from B. fulva, and three from P. varioti contained byssochlamic acid after 60 days. The concentrations observed varied from 40 to 540 ppm. The production of byssochlamic acid by B . fulva in fats and fat-containing foods was investigated by Schmidt and Rehm (1969). After 2 or 3 months of incubation at 28"C, the mold did not produce any byssochlamic acid when palmitic, stearic, linoleic, and oleic acids were the only carbohydrate sources in a synthetic culture solution. With glycerol as a carbohydrate source, 80 to 100 ppm of the toxin were detected in the culture fluid. In margarine, olive oil, bacon fat, and palm oil no byssochlamic acid was formed after growth of B. fulva for 2.5 or 7 months. A substance was formed in butter which was similar to byssochlamic acid. The concentration of this substance was calculated to be comparable to 75 ppm byssochlamic acid. In peanuts, hazelnuts (filberts), walnuts, poppy seeds, and coconut no byssochlamic acid was produced, although the mold developed well. From these studies it was concluded that in fats and fat-containing foods in which measurable amounts of free glycerol are absent, no intoxications by byssochlamic acid are to be expected. Health hazards associated with spoilage of these foods by Byssochlamys spp. and P . varioti have not been adequately assessed, however, and since Paecilomyces is responsible for spoilage of several animal and plant oils (Ponte and Tsen, 1978), continued research effort is needed. King et al. (1972) examined three strains of B . fulva for their capacity to produce byssochlamic acid in tomato and grape juices in sealea cans. Although the mold grew on these juices, the toxin was not detected in tomato juice after 1 year or in grape juice after 2 months at 25°C. A method for separation and quantitation of byssochlamic acid from nutrient solutions and fruit juices was published by Schmidt and Rehm (1968). The procedure involves extraction of acidified (pH 1.0) fluids with chloroform or ethyl acetate followed by concentrating the extract and spotting on silica gel

27 2

LARRY R. BEUCHAT AND STEPHEN L. RICE

thin-layer chromatography plates. Byssochlamic acid is visible after fluorescent quenching. The dark spots can be quantified following extraction by measurement of UV,, absorption by 224 nm. Fritz et al. (1976) reported that the recovery rate from fruit juice is 80% and the limit of detection is 0.5 ppm. They did not detect byssochlamic acid in commercially available fruit juice or in juices produced from moldy fruits. 2 . Patulin Several species of Penicillium, most notably P . urticae ( = patulum) and P . expansum, and Aspergillus have been demonstrated to produce patulin (4hydroxy-4H-furo[3,2-c[pyran-2(6H)-one).Karow and Foster (1944) reported that an unidentified species of Gymnoascus cultivated for 7 to 10 days on CzapekDox medium containing 3% corn steep liquor yielded a filtrate that inhibited Escherichia coli at 1: 100 and Staphylococcus aureus at 150. Through chemical and physical analyses, the inhibitory substance was identified as patulin. Kuehn (1957) later identified this specimen as Arachniotus rrisporus, which since then was recognized to be synonymous with B. nivea (Kuehn, 1958). During the 1940s and early 1950s patulin was described as an antibiotic and was thought to have therapeutic activity against the common cold. Indeed, patulin is a wide-spectrum biocide, having activity against many bacteria, protozoa. fungi, mammals, plants, and some viruses (Wilson, 1976). It was found to be too toxic for use as a therapeutic agent, however, and research efforts soon shifted to studies of its chemical properties, natural occurrence, and detection in feeds and foods. Patulin has been assigned a variety of names by researchers who have isolated the compound from mycelium or substrates on which molds have grown: clavicin, clavitan, claviformin, expansin, gigantic acid, leucopin, myocin c, penicidin, and tercinin. Patulin has an emperical formula of C71-&04,a molecular weight of 154, and a melting point of 110 to 112°C (Wilson, 1976; Davis and Diener, 1978). Patulin crystals are large monoclinic tables (001) and unit cell dimensions are: a = 12.42, b = 9.47, c = 7.78 A (6 = 46.7"); it absorbs in the UV at 276 nm. Patulin is soluble in water, ethanol, acetone, ethyl acetate, and chloroform, slightly soluble in ethyl ether and benzene, and insoluble in petroleum ether. The structure and synthesis of patulin were determined by Woodward and Singh (1949, 1950). The biosynthetic pathway for patulin from ['4C]glucose and acetate was elucidated by Bu'Lock et al. (1965) and later summarized by Stott and Bullerman (1975) as requiring the combining of one acetyl-CoA molecule and three malonyl CoA molecules followed by reduction and decarboxylation to produce 6-methylsalicylic acid. Upon further decarboxylation and oxidation, 6-methylsalicylic acid is converted to m-hydroxybenzaldehyde which then undergoes a rearrangement, resulting in one molecule of patulin.

BYSSOCHLAMYS SPP. AND PROCESSED FRUITS

27 3

The toxic effects of patulin are well documented. In a review of reports on toxicity, Ciegler et af. (1971) stated that more than 75 bacterial species have been tested and none was completely resistant to the effects of patulin. It exhibits strong inhibitory activity against some fungi, but is without effect against others, and its phytotoxic activity has been evaluated in a number of plant systems. Patulin isolated from B . nivea (Gymnoascus sp.) by Karow and Foster (1944) had an LDlooof 12.5 mg per 20-gm mouse. Other reports cited by Stott and Bullcrman (1975) indicated that intravenous injection of patulin into mice and rats gives LDsovalues varying from 0.3 to 0.7 mg per 20 gm of body weight. The lethal dose for mice by intraperitoneal injection was 0.1 to 0.25 mg of patulin per mouse. Cats, rabbits, chickens, quail, guppies, brine shrimp, and zebra fish larvae are also sensitive to patulin. Canguilhem et u f . (1976) studied toxicological effects in sheep. Patulin inhibits cell and/or nuclear division, but observations regarding mechanisms of action are not in agreement (Ciegler et al., 1971). It has been implicated in abnormal developments in the cell nucleus, including total or partial fragmentation of chromosomes. The mode of action of patulin has also been attributed to blockage of respiration as a result of its effect on cellular membrane permeability, and to its reaction with sulfhydryl groups in the active sites of enzymes. Conversely, an excess of sulfhydryl groups would detoxify patulin, supposedly binding all of the patulin molecules before they could react with a vital group (Stott and Bullerman, 1975). In addition to being overtly toxic, patulin exhibits carcinogenic activity when administered in sublethal doses to animals. Many methods have been reported for extraction, identification, quantitation, and bioassay of patulin from laboratory media as well as from some foods, and procedures are reviewed by Stott and Bulleman (1975), Wilson (1976), and Bullerman (1978). Extractants include acetonitrile-water, acetonitrile-hexane, and ethyl acetate. Patulin has been detected and quantitated by thin-layer, gas, and liquid chromatography. Bioassay procedures used for quantitation utilize chicken embryos, cockrels, ducklings, quail, mice, brine shrimp, and zebra fish larvae. Several bacterial systems have also been utilized to assay for patulin. Production of patulin by several strains of Byssochfamys isolated from silages and forages has been reported. Escoula (1975a,b) studied strains of B . nivea that produced both byssochlamic acid and patulin. Grown on a modified Czapek liquid medium, a maximal level of 816 ppm patulin was observed after 9 days of incubation at 26°C (Escoula, 1975a). Only 30 ppm were detected in soy meal containing 80% moisture. Patulin accumulation preceded maximal growth of B .

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LARRY R . BEUCHAT AND STEPHEN L. RICE

nivea and appeared to coincide with phialospore formation. In competition with other molds or in the presence of complex microflora of silages, patulin production was irregular; a maximum concentration was obtained after 45 and 82 days of culture, respectively (Escoula, 1975b). Irregularities were attributed to random competition between microorganisms or fixation of patulin on sulfhydryl radicals. Scurti et al. (1973) isolated an atypical strain of B . nivea from ensiled corn that they demonstrated to be capable of producing patulin on a synthetic medium. Kis et al. (1969) also reported that B. nivea produced patulin. The toxinogenesis of 18 strains of B . nivea, four of B . fulva, seven of P . varioti, two of Aspergillus clavatus, one of A . terreus, and one of P . urticae isolated from ensiled forages was tested at 26°C in enriched (0.8% glucose, 0.2% yeast extract) Czapek liquid medium (Escoula, 197%). All 18 strains of B. nivea, one strain of B. fulva, and one strain of P . varioti produced patulin after 9 days of incubation. Concentrations recovered ranged from 0.5 to 1120 ppm. Later Escoula (1975d) tested the same strains for byssochlamic acid production. There appeared to be no relation between the production of patulin and byssochlamic acid in the species tested. Formic and propionic acids, when added to wet corn, were found to decrease or prevent growth of B . nivea and patulin production (Escoula, 1975e). Percebois (1974) studied a strain of B . nivea isolated from heat-processed strawberries. Grown on a synthetic liquid medium, antibiotic activity toward Escherichia coli, Shigella sonnei, and Salmonella typhi appeared after 12 days. A later report from the same laboratory revealed that among 43 strains of gramnegative bacteria, none was resistant to the antibiotic substance (Percebois, 1975). With the exception of certain strains of Bacillus, the great bulk of gram-positive bacteria tested was little influenced, and the filtrate was ineffectual against mycobacteria and fungi. Isolation of the antibiotic metabolite from culture fluid (Colin and Percebois, 1975) revealed, upon purification and chromatographic characterization, that the substance was patulin (Percebois et al., 1975). The toxin was reported to be stable in strawberry juice. Over a period of 5 years, Frank et al. (1976, 1977) detected patulin in apples, pears, bananas, pineapples, grapes, peaches, and apricots which were infected with brown rot fungi. In a laboratory study, mold growth and patulin formation were then studied in various live tissues of fruits and vegetables experimentally inoculated with Penicillum expansum, P . urticae, and B . nivea. When stored at room temperature, patulin was detected in apples, pears, peaches, strawberries, bananas, tomatoes, and honeydew melons infected with penicillia, but only in apples and honeydew melons infected with B . nivea. Although the penicillia grew and produced patulin in inoculated red and green peppers, cucumbers, and carrots, B . nivea did not grow on any of these vegetables. Patulin production by ten strains of B . fulva and three strains of B. nivea cultured in enriched Czapek-Dox broth was tested by Rice et al. (1977b). Two strains of B . fulva and all three strains of B. nivea produced the mycotoxin.

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Thirteen commercially processed fruit juices and nectars free of added microbial preservatives were then tested as substrates for growth and production of patulin by one strain of B.fulva. The organism produced patulin in greatest amounts on blueberry, red raspberry, and boysenberry juices, while lower concentrations were observed in pear and peach nectars, grape, apple, Strawberry, pineapple, and black cherry juices, and cranberry cocktail. No patulin was detected in inoculated prune or tomato juices incubated for 14 days at 25°C. Excellent growth of B. fulva occurred in a?l juices except cranberry cocktail, probably because of the natural presence of benzoic acid, and there appeared to be no relation between soluble solids or pH and the amount of patulin formed in various juices. For example, 6.11 mg of patulin per 50 ml of blueberry juice (pH 3.16, 14.6% soluble solids) were detected as compared to 0.81 mg per 50 ml of strawberry juice (pH 3.33, 6.4% soluble solids) and 0.13 mg per 50 ml of peach nectar (pH 3.84, 14.5% soluble solids). The effects of temperature and time of incubation on biomass and patulin production by B. fulva cultured in Concord grape juice was studied by Rice et al. (1977b). Of the incubation temperatures studied (18, 25, 30, and 38"C), 18°C resulted in the greatest amount of patulin produced after approximately 25 days. Increasing the temperature markedly reduced the patulin accumulation. The next highest amount was obtained at 30°C after approximately 6 days; incubation at 38°C was unfavorable for patulin production. In all cases a fairly rapid decline in patulin concentration occurred after the maximum was reached. Sommer et al. (1974) found that P. expansum formed greater amounts of patulin at 5 to 20°C than at 30"C, although it is not known why patulin formation is favored by this low temperature. They observed a decline in patulin concentration in potato dextrose broth after reaching a maximum and suggested that metabolic destruction may be responsible. This may be the case, considering the highly reversible nature of rn-hydroxybenzyl alcohol dehydrogenase, a key enzyme in patulin biosynthesis (Forrester and Gaucher, 1972). Patulin loses its biological activity at alkaline conditions and reacts with sulfhydryl and amino groups. Thus changes in medium constituents during the course of growth and autolysis of B. fulva in fruit juices could affect the amount of patulin detectable by extraction and quantitation techniques. The initial rate of growth of B . fulva in grape juice is increased by elevating the temperature of incubation from 18 to 38°C (Rice et al., 1977b). However, the greatest amount of biomass produced occurs at 25 and 30"C, and at 38°C there were definite signs of autolysis after 11 days. There appeared to be little relation between maximum biomass and patulin production at various incubation temperatures. Noting that patulin occurs widely in commercial apple juice in North America (Stoloff, 1976) and that Byssochlamys has been isolated from grapes, Scott et al. ( 1 977) investigated the potential of grapes to support patulin production by molds and hence give rise to patulin in processed grape juice. It was concluded

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that P . expansum was likely to have been the mold responsible for the presence of patulin in grape juice made from Rauschling grapes. Byssochlamys was not detected in moldy grapes from which juice was prepared. The influence of one- and two-sided regulated atmospheres with lower oxygen and increased carbon dioxide concentrations in comparison with air on growth and patulin production by P. expansum, P . urticae, and B . nivea was studied by Orth (1976a). Low oxygen levels (0.5 to 2%) had little effect on development and toxin production in various nutrient media after 14 days at 25°C; B . nivea produced patulin in a nitrogen atmosphere containing 0.2%oxygen. Only higher carbon dioxide and lower oxygen concentrations than that of air (e.g., 40% carbon dioxide, 6% oxygen) reduced patulin production, whereas growth was not significantly retarded. Atmospheres with 90% carbon dioxide and 10% air permitted patulin production, but nitrogen atmospheres with 10% carbon dioxide markedly inhibited the growth of B . nivea and prevented toxin production. Patulin is moderately stable at 22°C in apple and grape juice but not in orange juice (Scott and Somers, 1968). Heating the juices at 80°C for 10 or 20 minutes did not completely destroy patulin. Stability in apple and grape juices was attributed to very low levels of sulfhydryl compounds. Pohland and Allen (1970) also reported that patulin was stable in apple juice for up to 12 days. Upon fermentation of apple juice with Saccharomyces, patulin disappeared (Harwig et al., 1973). In light of reports showing that Byssochlamys spp. can produce patulin on a wide variety of processed fruit juices under seemingly adverse conditions, it appears that the mold could produce patulin under conditions found in canned, bottled, or bulk-stored fruits and fruit products. In addition to the longrecognized esthetic problems associated with fruit products spoiled with Byssochlamys spp., the potential for patulin contamination of these products appears to be substantial. 3 . Byssotoxin A , Asymmetrin, and Variotin

Foods, feeds, and grains that become moist during storage may subsequently overheat and provide thermophilic fungi with a competitive growth advantage over other microorganisms. Since no thermophilic fungi had been isolated that produced mycotoxins, Davis et al. (1975) examined 23 strains of fungi representing 13 thermophilic and thermotolerant species for toxigenicity to brine shrimp, chicken embryos, and rats. Extract from an enriched shredded wheat substrate on which a Byssochlamys sp. was grown was lethal to 50% of the chicken embryos tested. Intraperitoneal injections of rats with extracts of Byssochlamys appeared to cause reduced fecal output. In later studies reported from the same laboratory, Kramer et al. (1976) found that chloroform-ethanol extracts from culture media of a strain of B . fulva isolated from corn were toxic to brine shrimp, chicken embryos, and rats. The

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extract was slightly inhibitory to the germination of pea seeds, but was nontoxic to 10 species of bacteria and 1 yeast. It did not contain byssochlamic acid, patulin, or variotin, previously described mycotoxins produced by Byssochlamys spp. One metabolite was isolated, partially characterized chemically and physically, and given the trivial name byssotoxin A. The UV spectrum of byssotoxin A showed AMeOWmax 247 nm ( E = 50,600), 285 nm ( E = 17,600), 356 nm ( E = 11,200), and 472 nm (E = 146). Through further chemical and physical characterizations using IR, NMR, and mass spectral analyses, a probable empirical formula for byssotoxin A was deduced to be C2,H,,N203. The structure was not determined, but data indicated that byssotoxin A was neither an indole nor an alkaloid. Tests were negative for primary tertiary amines, amino acids, and amides, but positive for a secondary arnine. Fragmentation analysis indicated that byssotoxin A consists of a large, stable ring structure plus a side chain of eight or nine carbons that terminates in an amine or nitrosamine group. Several other metabolites of varying toxicity have been reported to be produced by Byssochlamys and Paecilomyces spp. Zaroogian and Curtis (1963, 1964) isolated a plant growth inhibitor from the culture filtrate of B . nivea and named it asymmetrin. Paecilomyces spp. produce the antibiotic variotin which has been shown to be toxic to mice and to have antimycotic properties. D.

MANNITOL

A major metabolic by-product of B. fulva is mannitol (Raistrick and Smith, 1933) which may be formed in amounts up to 30% of the total sugar utilized. Several strains of B.fulva also form D[ -1-pantoic acid from added ketopantoic acid in unusually high yields and optical purity (Lanzilotta er af., 1974). D[-1Pantoic acid would be useful in the production of the nutritionally active D-isomer of pantothenic acid.

V. DETECTION AND ENUMERATION Several methods have been devised to detect and enumerate ascospores of Byssochlamys spp. in foods. The success of procedures depends largely on first activating dormant spores, usually by heat treatment. A variety of treatments, ranging from 5 minutes at 75°C to 35 minutes at 80°C,have been used (Olliver and Rendle, 1934; Gillespy, 1938; Yates et al., 1968; King et a l . , 1969). Differences in sensitivity to heat exist among strains and species, thus leaving any particular timehemperature treatment less than optimal for all situations. If only the imperfect state, Paecilomyces, is present, heat treatment would normally be lethal to vegetative cells, thereby resulting in false-negative data for the potential spoilage of foods by Byssochlamys at some later date in the storage period. The menstruum in which ascospores are heated may influence the extent

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of inactivation and, thus, the accuracy of enumeration. Also, since ascospores are produced in groups of eight in asci that often do not rupture during standard processing procedures, results from assays indicating the presence of Eyssochlamys may underestimate the total number of ascospores present in a food sample. On the other hand, clumping of asci hinders detection and estimation of ascospore populations, especially when most-probable-number techniques are used. If present, levels of ascospores in foods are usually low, in the order of 10 to 100 per 100 gm (Splittstoesser et al., 1971), thus requiring sensitive methods for detection. Approaches to quantitative estimation of Byssochlamys have been made with these problems in mind, and will be reviewed at this point. If spoiled, processed foods are not of the type that would support the growth of Byssochlamys spp. or if other microorganisms are more likely to have caused the spoilage, then time spent analyzing for the presence of ascospores of Byssochlamys may have been wasted. It is important, therefore, to recognize the possibility of contamination and spoilage by Byssochlamys. When contamination by heat-resistant fungi prior to processing is suspected, particularly in the case of acid foods such as fruit juice concentrates and fruits, the causative spoilage microorganism is usually E . fulva and related or similar species (Corlett, 1976). In the experience of Denny and Brown (1969), only mold filaments, not spores, have been found in spoiled acid canned foods. Spoilage is evidenced by a moldy taste and odor, fading color, and sometimes a loss under vacuum and slight swelling of the ends of the containers. Products may appear watery and, with advanced deterioration, soft and mushy. Centrifugation has been used to concentrate asci and ascospores before analysis. Rubin and Friedman (1969) reported a method for detection and enumeration of low levels of asci of B . fulva in fruit juice concentrates. Suspect material is diluted with an equal volume of water and then, for large volumes, centrifuged using a Westphalia-type continuous centrifuge which employs a desludging feature. A conventional batch centrifuge can be used for smaller samples. The sediment is then resuspended in a known volume of supemate or water, diluted, and plated on potato dextrose agar. Kuss (1969) separated asci from conidia by repeated spinning of water suspensions for 15- to 30-second intervals at low speed in a clinical centrifuge or by centrifugation through layers of different concentrations of sucrose at higher gravity for intervals of 5 to 10 minutes. Speed and length of centrifugation is influenced by sample size, viscosity, and specific gravity (Splittstoesser, 1976). Because of the low incidence of ascospores of Byssochlamys in many foods, detection often depends upon the use of relatively large samples (Splittstoesser, 1976). For example, 100 gm or more have been cultured for the enumeration of ascospores on sound grapes, apples, and other fruits (Splittstoesser et al., 1970). These workers outlined a procedure requiring a dilution of the fruit sample with an equal volume of sterile water in a screw-capped blender jar followed by homogenation and heat treatment at 70°C in a water bath for 2 hours. The jar is

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placed in a polyethylene bag before heating to safeguard against leakage of water. A 2-hour hold assures that the contents are at the equilibrium temperature for about 1 hour. After heating, the entire homogenate is distributed in 10-ml aliquots into petri dishes and an equal volume of potato dextrose agar acidified to pH 3.5 with 10% tartaric acid is added to plates and mixed. Solidification is assured by increasing the agar content of the medium to 3%. Colonies are counted after incubation for 4 to 5 days at 32°C. A comparison of plate count, 5% yeast extract, 5% malt extract, and Put (1964) agars, and concentrations of Concord grape juice from 1 to 15" Brix indicates that many heat-resistant molds are not particularly fastidious in that a number of strains gave comparable counts in all media. When differences are noted, potato dextrose agar usually provided the higher figures. An advantage in using acidified potato dextrose agar is that most bacteria will not form colonies. However, since partial hydrolysis of agar may occur, Put (1964) investigated the use of antibiotics in potato dextrose agar at pH 5.5 as inhibitors of spore-forming bacteria. It was observed that heat-resistant molds of several genera, including Byssochlamys, could be cultivated selectively in the presence of Bacillus spp. when at least 10 ppm of chloramphenicol or chlortetracycline were added to potato dextrose agar. Penicillin, sulfafurazole, streptomycin, oxytetracycline, and furazolidone did not give complete selectivity or optimal growth conditions. Denny and Brown (1969) describe a procedure for preliminary screening of juice concentrates for ascospores and asci of Byssochlamys. About 15 ml of concentrate is mixed with 100 ml of sterile water and shaken in a separatory funnel. The liquid is then drained and discarded, and the foam is settled by adding a second 100-ml portion of water, The foam liquid is filtered through a Millipore filter which is then examined microscopically (400X ) for ascospores and asci. Samples of concentrate can be heated (10 minutes at 77°C) and plated on potato dextrose agar if the presence of Byssochlamys is suspect. Two procedures were reported by Canada (1969) to aid in the detection of Byssochlamys in foods and food ingredients. The first requires the use of an enrichment broth in which the sample is heated at 80 to 85°C and incubated for 48 hours at 35°C. Serial dilutions are then surface-plated on Sabouraud dextrose agar or potato dextrose agar and incubated again for 48 hours at 35°C before colonies are counted. Although this method is highly sensitive, it does not permit quantitative estimation of the original ascospore load. A quantitative method was also reported in which samples were heated in the presence of bacterial amylase and then plated on potato dextrose agar containing hexachlorophene. The range in sensitivity of this method is 25 to 5000 ascospores per 10 gm of test material. A most-probable-number technique for detecting asci of Byssochlamys in fruit and fruit products was described by Kuss (1969) and Splittstoesser er al. (1970). Samples are diluted with an equal volume of water and heated for 60 minutes at 70°C. Portions (10 ml) are added to screw-capped bottles containing 50 ml of 5%

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malt broth acidified to pH 3.0 with tartaric acid. The bottles are incubated at 32°C and examined daily for macroscopic evidence of growth. Although one ascus per 20 gm of fruit homogenate could be accurately estimated, the procedure has a serious drawback in that an extended incubation, up to 22 days, is required before all potentially positive broths exhibit growth. Maunder (1969) described a method for enumerating Byssochlamys in grape concentrate. The procedure consisted of diluting 25 ml of concentrate with an equal volume of 0.5% peptone solution in a prescription bottle. The sample was then heated at 70°C for 20 minutes, cooled, and incubated at 30°C with the bottle placed on its side, cap loosened. This procedure was further modified by Murdock and Hatcher (1976) to specifically monitor grape, apple, and cherry concentrates and juice bases for the presence of heat-resistant molds. After heating 50 g of product and 50 ml of water at 77°C for 30 minutes, the sample is distributed in petri dishes and mixed with 2% agar. Plates are incubated at 30°C and examined for mold growth for up to 30 days, although outgrowth usually occurs within 3 to 5 days. The procedure is reported to be satisfactory for routine screening of incoming fruit products at processing plants. Enumeration of Byssochlamys may be hampered by the spreading of colonies, especially if more than 10 to 15 colonies are formed per plate. Splittstoesser (1976) suggests adding 8.3 pg of rose Bengal dye per ml of potato dextrose agar (pH 5.6) to restrict colony growth and thus permit as many as 200 colonies per plate to be readily counted. This concentration of dye does not reduce the recovery of viable ascospores subjected to thermal stress. It is sometimes difficult to determine the number of ascospores of Byssochlamys in fruit products because of clumping. This makes conventional plate count methods inaccurate. In an attempt to find a solution to this problem, Partsch (1969) devised a simple method for the separation of ascospores. The procedure consists of subjecting the sample to agitation in the presence of glass beads followed by separation of asci from mycelium using sintered glass plates and a suction device. After centrifugation, asci are again agitated, this time more vigorously to release ascospores, and filtered. Well-separated ascospores are obtained. Although this procedure would be a valuable research tool, it would appear to be impractical for use in quality control laboratories for routine monitoring of foods for the presence of Byssochlamys.

VI.

RESEARCH NEEDS

There are several deficiencies in our knowledge of how to control or at least minimize the incidence of spoilage caused by Byssochlamys. It is well to bring to the attention of researchers the need for further investigations in these areas. It does not seem feasible to eliminate Byssochlamys spp. from fruit refuse and soils which contaminate fruits as they arrive from the field. There may be room

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for improvement of handling and preprocessing procedures, however, which would lead to more effective sanitation of raw products. Physical and chemical treatments devised to reduce the ascospore loads on raw fruits would then decrease the risk of spoilage of material subsequent to processing. Considerable variation in tolerance of ascospores to heat is reported in the literature. This may be due in part to the chemical nature of the heating menstruum, concentration of ascospores, type of recovery medium, etc., but there is also evidence that the environment in which ascospores are produced may influence their capacity to withstand thermal treatment. Investigations need to be pursued to further define the effects of nutrients and physical parameters on sporulation per se as well as the chemical makeup and heat stability of ascospores. Knowledge gained from these studies may enable processors to more effectively design pasteurization techniques to inactivate Byssochlamys. Environmental conditions influencing the production of mycotoxins by Byssochlamys spp. have not been fully explored. It is more efficient and effective to control processing and storage conditions so that toxins will not be formed than it is to routinely analyze suspect materials for the presence of mycotoxins. To accurately assess the microbiological quality of foods, reliable and sensitive methods must be used. Because of the low numbers of ascospores generally found in products and because a heat shock is required to activate them, detection of Byssochlamys spp. requires special methodology. A substantial amount of research has been conducted in this area and yet a single procedure permitting accurate enumeration of Byssochlamys in a wide variety of fruits and fruit products has not been advanced. 'Further work is needed so that a method can be made available for reliably analyzing a wide range of products for the presence of Byssochlamys.

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Eckardt, C., and Ahrens, E. 1977a. Investigations about Byssochlumys fulvu Olliver & Smith as a potential spoilage agent in tinned strawberries. Part 1. Occurrence and growth of Eyssochlamys fulva. Chem. Mikrobiol., Technol. Lebensm. 5 , 71-75. Eckardt, C., and Ahrens, E. 1977b. Eyssochlamys fulva as a potential spoilage agent in tinned strawberries. Part 2. Heat resistance of ascospores of Eyssochlamys fulva. Chem. Mikrobiol. Technol. Lebensm. 5 , 76-80. Eckardt, C., and Ahrens, E. 1978. Resistance of Eyssochlamys fulva Olliver & Smith during freezing and pasteurization processes. Lebensm. Wiss. Technol. 11, 137-141. Emmons, C. W. 1935. The ascocarps in species of Penicillium. Mycologia 27, 128-150. Eschmann, K. H. 1971. Stoffwechselprodukte der schimmelpilize in fruchtsaften und konzentraten. Arch. Lebensmittelhyg. 6, 126-129. Escoula, L. 1975a. Toxinogenic moulds in silage. 11. In vitro kinetics of patulin and byssochlamic acid biosynthesis by Eyssochlamys nivea Westling in liquid medium. Ann. Rech. Vet. 6 , 155-163. Escoula, L. 1975b. Toxinogenic moulds of silage. 111. Patulin and byssochlamic acid production by Byssochlamys nivea Westling on a laboratory silage model. Ann. Rech. Vet. 6 , 219-226. Escoula, L. 1 9 7 5 ~ Toxigenic . moulds of silage. IV. Patulin production in liquid medium using fungus species isolated in silages. Ann. Rech. Vet. 6, 303-310. Escoula, L. 1975d. Toxigenic moulds in silage. V. Production of byssochlamic acid in liquid medium by Eyssochlamys niwea Westling, Eyssochlamys fulwa Olliver & Smith and Paecilomyces varioti Bainier isolated from forages. Ann. Rech. Vet. 6, 3 1 1-314. Escoula, L. 197%. Toxinogenic moulds in silage. V1. Effect of propionic and formic acids on the production of patulin and of byssochlamic acid by Eyssochlamys niwea Westling. Ann. Rech. Ver. 6, 315-323. Forrester, P. I., and Gaucher, G . M. 1972. m-Hydroxybenzyl alcohol dehydrogenase from Penicillium urricae. Biochemistry 11, 1108-1 114. Frank, H. K., Orth, R., and Hermann, R. 1976. Patulin in foods of vegetable origin. 1. Pomaceous fruit and products made out of it. Z . Lebensm.-Unrers. -Forsch. 162, 149-157. Frank, H. K., Orth, R., and Figge, A. 1977. Patulin in foods of plant origin. 2. Several kinds of fruits and vegetables and fruit and vegetable products. Z . Lebensm.-Unrers. -Forsch. 163, 1 1 1-1 14. Fritz, W., Engst, R., and Donath, R. 1976. Zur bedeutung der byssochlaminsaure im fruchtsaftschimmel. Nahrung 20, 539-542. Gedek, B. 1971. Mycotoxine in lebensmitteln und ihre bedeutung fur de gesundheit des menschen. Muter. Med. Normark 23, 130-141. Gillespy, T. G . 1938. Studies on the mould Eyssochlamys fulva. 11. Annu. Rep. Fruit Veg. Preserv. Res. Srn. pp. 67-78. Univ. of Bristol, Campden, England. Gillespy, T. G . 1940. Studieson the mould Eyssochlamys fulva. Ill. Annu. Rep. Fruit Veg. Preserv. Res. Stn. pp. 55-61. Univ. of Bristol, Campden, England. Gillespy, T. G . 1946. Studieson the mould Eyssochlamys fulva. IV. Annu. Rep. Fruit V e g . Preserv. Res. Stn. pp. 31-39. Univ. of Bristol, Campden, England. Gillespy, T. G . , and Thorpe, R. H. 1962. Survey of Eyssochlamys fulva ascospore infection on strawberries for canning - June and July, 1961. Tech. Memo Fruit V e g . Canning Quick Freez. Res. Assoc. No. 44. Univ. of Bristol, Campden, England. Goncalves de Lima, 0. 1971. Occurrence of P. varioti Bainier in moldy manioc loaves from the (Brazilian) state of Maranhao. Rev. Inst. Anribiot., Univ. Fed. Pernambuco, RecifP 9, 83-85. Hamor, T. A., Paul, I . C., Robertson, J . M., and Sim, G. A. 1962. The structure of byssochlamic acid. Experieriria 18, 352-354. Hanvig, J . , Scott, P. M., Kennedy, B. P. C., and Chen, Y. K. 1973. Disappearance of patulin from apple juice fermented by Saccharomyces spp. Can. Insf. Food Sci. Technol. J . 6, 45-46. Hatcher, W. S., Weihe, J. L., Murdock, D. I., FoIIinazzo, I. I., Hill, E.C., and Albrigo, L. G. ~

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1979. Growth requirements and thermal resistance of fungi belonging to the genus Bys. sochlamys. J. Food Sci. 44, 118-122. Herbert, R. J. 1973. Byssochlamys fulva. Nutrition of ascospore formation. Fatty acid profiles of ascospores, conidia, and mycelia. Electron microscopy of ascospores and conidia. Ph.D. Dissertation, Louisiana State University, Baton Rouge. Herbert, R. I., and Larson, A. D. 1972. Asci production by Eyssochlamys fulva on a synthetic medium. J . Food Sci. 37, 883-885. Hirst, F., and McMaster, N. B. 1933. Preliminary report on the heat-resistant mould Eyssochlamys fulvu. Annu. Rep. Fruit Veg. Preserv. Res. Stn. Univ. of Bristol, Campden, England. Hull, R. 1939. Study of Eyssochlamys fulva and control measures in processed fruits. Ann. Appl. Eiol. 26, 800-822. Ito, K. A., Seeger, M. L., and Lee, W. H. 1972. The destruction of Byssochlamys fulva asci by low concentrations of gaseous methyl bromide and by aqueous solutions of chlorine, an iodophor and peracetic acid. J . Appl. Eacteriol. 35, 479-483. Jensen, M. 1960. Experiments on the inhibition of some thermoresistant molds in fruit juices. Ann. Inst. Pasteur Lille 11, 179-182. Karow, E. O., and Foster, 1. W. 1944. An antibiotic substance from species of Gymnoascus and Penicillium. Science 99, 265-266. King, A. D,, Michener, H. D., and Ito, K. A. 1969. Control of Eyssochlamys and related heatresistant fungi in grape products. Appl. Microbiol. 18, 166-173. King, A . D., Jr., Booth, A. N., Stafford, A. E., and Waiss, A. C., Jr. 1972. Eyssochlamys fulva, metabolite toxicity in laboratory animals. J . Food Sci. 37, 86-89. King, A. D., Jr., Bayne, H. G., and Alderton, G. 1979. Nonlogarithmic death rate calculations for Byssochlamys fulva and other microorganisms. Appl. Environ. Microbiol. 37, 596- 600. Kis, 2.. Furger, P., and Sigg, H. P. 1969. Isolation of pyrenophorol. Experientia 25, 123-124. Knight, S. G. 1966. Production of a rennin-like enzyme by molds. Can.J . Microbiol. 12,420-422. Kramer, R. K., Davis, N. D., and Diener, U. L. 1976. Byssotoxin A, a secondary metabolite of Eyssochlamys fulva. Appl. Environ. Microbiol. 31, 249-253. Kuehn, H. H. 1957. Observations on Gymnoascaceae. IV. A new species of Arachniotus and a reconsideration of Arachniofus frisporus. Mycologia 49, 55-67. Kuehn, H. H. 1958. A preliminary survey of the Gymnoascaceae. 1. Mycologia 50, 417-439. Kuss, F. R. 1969. Approaches to a quantitative estimation of E . fulva in raw or processed fruit preparations. N . Y . State Agric. Exp. Stn., Geneva, Eyssochlamys Semin. Abstr., Res. Circ. 20, 9-11. Lanzilotta, R. P., Bradley. D. G., and McDonald, K. M. 1974. Microbial reduction of ketopantoyl lactone to pantoyl lactone. Appl. Microbiol. 27, 130-134. LaRocca, M. A. K., and Goos, R. D. 1975. Chlamydospore production by Eyssochlamys fulva. Dev. Ind. Microbiol. 16, 326-332. Lubieniecki-von Schelhorn, M., and Heiss, R. 1975. The influence of relative humidity on the thermal resistance of mould spores. In "Water Relations of Foods" (R. B. Duckworth, ed.), pp. 339-348. Academic Press, New York. Liithi, H., and Hochstrasser, R. 1952. Uber zwei neue, gegenwartig haufigei aftretende pilzinfekionen in der bauerlichen sussmosterei. Schweiz. Z. 0bsr.- Weinbau 61, 301-359. Marvalhas, N. 1966. Mycological deterioration of cocoa beans during fermentation and storage in Bahia. Rev. Int. Choc. 21, 375-378. Maunder, D. T. 1969. Spoilage problems caused by molds of the Eyssochlamys-Paecilomyces group. N . Y . State Agric. Exp. S m . , Geneva, Byssochlamys Semin. Abstr.. Res. Circ. 20, 12-16. Meyer, H., and Rehm, H. 1. 1967. Zur bildung der byssochlaminsaure. Natunvissenschaften 14, 370. Meyer, H., and Rehm, H. J . 1969. Germination-inhibiting and fermentation-inhibiting effects of byssochlamic acid. Nutunvissenschafren 56, 563.

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SUBJECT INDEX A

in processed fruits, 237-288 research needs on, 280-281 spoilage from, 242-252 Byssotoxin A, from Byssochlamys spp., 276277

Acidex procedure, for wine deacidification, 21 Acidity, of grapes and wines, 3-10 Amelioration, wine deacidification by, 13- I6 Amino acids, in plants, winter hardiness and, 205-206 Amylases, of Byssochlamys spp., 267 Anion exchange, wine deacidification by, 24 Antioxidants, for dehydrated mashed potatoes, 117-122 Arrhenius equation, 188 integrated form of, 189 Ascospores, of Byssochlamys spp., 252-264 Asymmetrin. from Byssochlamys spp., 276-277

Carbohydrixes, in plants, winter hardiness and, 204-205 Carbonyls, in potato products, 105 Carcinogen, xylitol as possible, 173-174 Cation exchange, wine deacidification by, 24 Cold hardiness, of plants, development, 202209

B

D

Bacteria, role in tooth decay, 138 Byssochlamic acid, from Byssochlamys spp, , 267-272 Byssochlamys spp., 237-288 ascospores of, 252-264 activation, 255-256 germination, 256-258 inactivitation, 258-264 production, 252-253 classification and nomenclature of, 238-243 detection and enumeration of, 277-280 distribution and sources of contamination from, 242-244 enzymes of, 264-267 growth of control, 248-252 factors affecting, 246-248 imperfect state of, 240-242 metabolic activities of, 252-277 mycotoxins of, 267-277 occurrence of, 244-245 perfect state of, 238-240

C

Deacidification of wine, 1-53 by amelioration, 13-16 biological methods for, 25-42 by carbonic maceration, 38-42 by ion exchange, 23-25 by malo-lactic fermentation, 25-32 by neutralization and precipitation, 16-23 double-salt type, 19-23 physiochemical methods of, 13-25 by Schizosaccharomyces pombe, 32-38 Dehydrated mashed potatoes, 55-136 antioxidants for, 117-122 cell constituent role in, 61-102 flavoring constituents in, 102-109 interaction processes, 108-109 lipids in, 61-73 distribution, 63-68 processing effects, 68-73 varietal effects, 61-63 microflora in, 111-112 pectic substances in, 73-79 distribution, 73-75 289

290

SUBJECT INDEX

Dehydrated mashed potatoes (continued) enzyme effects, 79 processing effects, 75-78 processes for, 56-61 flakes, 59-61 granules, 56-59 protein in, 79-84 amino acids, 81-83 processing effects, 83-84 in raw tuber, 79-81 rancidity in, 112-122 determination, I 1 7-122 development, 112- I I7 reconstituted, 122- 124 rehydration rate, 122-123 texture, 122-123 starch in, 85-96 composition in tuber, 85-87 processing effects, 87-95 submicroscopic structure, 85 texture effects, 95-96 sulfite additive role in, 109-1 1 1 vitamin C in, 96-102 in aerated slices, 97 processing effects, 97-101 in raw tuber, 96 storage effects, 100-102 Diabetes, xylitol use in, 168-169

E

G

Glucose-6-phosphate dehydrogenase, deficiency of, xylitol effects on, 169-170 Granules, potato, processes for, 56-59 Grapes acidic components of, 3- 10 factors affecting, 5-10 importance i n wine making, 2

H Hemolytic anemia, xylitol therapy of, 169- I70 Hyperuncemia, after xylitol infusion, 172-173

I Ice. formation of, in frozen fruits and vegetables, 183-193 Ion exchange, wine deacidification by, 23-25

L Lactic acidosis, in xylitol metabolism, 171-172 Lipids in dehydrated mashed potatoes, 61 -73 in plants, winter hardiness and, 206-208 Liver, xylitol effects on, 173 Low temperatures chemical reactions at, 188-192 plant survival at, 193-21 1

Enzymes, of Byssochlamys sp., 264-267 Exocrine glands, xylitol and, 149-152

F Flakes, potato, processes for, 59-61 Flavoring constituents, in dehydrated mashed potatoes, 102- 109 Frost. effects on plant, 208-209 Frozen fruits and vegetables, piocessing of, 181-235 Fruits (frozen), 181-235 freezing effects on, 214-218 ice formation in, 183-193 problems in, 229-230 steps prior to freezing, 219-221 storage changes in, 227-228 Fruits, Byssochlamys spp. in, 237-288

M Malic acid fermentation of during vinification, 12 by S. pombe, 32-38, 40 Malo-lactic fermentation wine deacidification by, 25-32 Mannitol, from Byssochlamys spp., 277 Methional, in potato products, 105 Microflora, in dehydrated mashed potatoes, 1 1 1-1 12

Mycotoxins, of Byssochlamys spp., 267-277

0 Oxalate, deposition of, from xylitol infusion, 172

29 1

SUBJECT INDEX

P Patulin, from Byssochlumys spp., 272-276 Pectic substances, in dehydrated mashed potatoes, 73-79 Pectin-methylesterase, role in potato tubers, 79 Pectinases, of Byssochlamys spp., 264-266 Periodontal disease, xylitol and, 152-153 Plants chilling requirement of, 199-202 chilling sensitivity of, 194-199 parts used as food, 212 survival of, at low temperatures, 193-21 1 winter hardiness of, 202-208 Plaque dental biochemistry of, 140-141 xylitol effects on, 149 Potatoes, dehydrated, mashed, 55-1 36 Protein(s) in dehydrated mashed potatoes, 79-84 in plants, winter hardiness and, 205-206 Proteinases, of Byssochlamys spp., 266-267 Pyrazines, in potato products, 105-108

R Rancidity, in dehydrated mashed potatoes, 112-122 Refrigerated foods, processing of, 21 1-228

S Schizosuccharomyces pombe, wine deacidification by, 32-38 Seeds, chilling sensitivity of, 194-196 Sialomacromolecules, xylitol effects on, 150152 Starch, in dehydrated mashed potatoes, 8596 Sucrose, dental and nutritional properties of, 142-143 Sugars, dental canes and, 139 Sulfites, as dehydrated mashed potato additives, 109-Ill Sulfur compounds, in dehydrated mashed potatoes, 104-105

T Table wines, acidic components of, 3-5 Tartness, of wine, 3-4 Tartrate, precipitation of, during vinification, 12-13, 16-23 Teeth, xylitol effects on, 137-180 Thiamles, in potato products, 105 Turku sugar studies, dental canes and, 139-147

U Uronic acid cycle, xylitol in, 161

V Variotin, from Byssochlumys spp., 276-277 Vegetables (frozen), 181-235 freezing effects on, 214-218 ice formation in, 183-193 problems in, 229-230 steps prior to freezing, 219-221 storage changes in, 227-228 Vitamin C, in dehydrated mashed potatoes, 96-I02

W Wine acidic components of, 3-10 changes during vinification, 10-13 recommended levels, 4-5 deacidification of, 1-53 biological methods for, 25-42 physicochemical methods, 13-25 fermentation of, acids produced during, 10- I I tartness of, 3-4

X Xylitol, 137-180 adverse effects of, 171-174 carcinogenicity studies on, 173-174 dental caries and, 139-147 dental and nutritional properties of, 142-143 effects on renal and cerebral function, 173 exocrine glands and, 149-152 foods containing lowered cariogenicity, I39 mechanism of action of, 153-156

292 Xylitol (continued) metabolism of, 160-161 hepatic, 164-165 intermediary, 163-164 intestinal absorption, 162-163 microbiological aspects of, 147 periodontal disease and, 152-153 plaque studies on, 149

SUBJECT INDEX research needs for, 176 sialomacromolecules and, 150- 152 therapeutic use of, 167-170 for diabetics, 168-169 for hemolytic anemia, 169-170 in parenteral nutrition, 167-168 toxicology of, 170- 174