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

ACADEMIC PRESS

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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 (

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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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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

GLYCtOGEN

GLUCOSE-1-PHOSPHATETE-~UDP-GLUCOSE ZNAD

n

Is

UTP

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PPi

4

GLUCOSE-6-PHoSPHATE FRUCTOSE-6 -PHOSPHATE

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GLUCOSE

-

ASCORB,C AC,D

L \ UDP-GLUCURONATE t t

NOT IN HUMANSAND PIG

D-GLUCURONATE L-GULONOLACTONE

IPENTOSE PHOSPHATE '\ PATHWAY)

NADP

L-GULONATE NAD

'\\ '\

*

D-XYLULOSE-5-PHOSPHATE

D-XYLULOSE

3-KETO-L-GULONOLACTONE

L-XYLULOSE

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 +

co2

-

-

FRUCTOSE-6-PHOSPHATE

t

I

GLUCOSE

(PENTOSE PKXPHATE PATHWAY)

I

i I

I

D -XYLULOSE -5-PHOSPHATE 4

I

? D-XYLULOSE -XYLJTOL-L-XYLULOSE

n

NADH

FIG. 2.

NAD

Metabolism of exogenous xylitol

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

REIN0 YLIKAHRI

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

METABOLIC AND NUTRITIONAL ASPECTS OF XYLITOL

165

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

166

REIN0 YLlKAHRl

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

METABOLIC AND NUTRITIONAL ASPECTS OF XYLITOL

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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

METABOLIC AND NUTRITIONAL ASPECTS OF XYLITOL

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

..

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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189

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

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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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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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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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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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20 1

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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21 I

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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MILFORD S. BROWN IE SEED

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.

217

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