Advances in Food Research, Volume 17

ADVANCES IN FOOD RESEARCH VOLUME 17

Contributors to This Volume

F. AYLWARD ITAMARBEN-GEM MIKHAILA. BOKUCHAVA D. R. HAISMAN N. CZYHFUNCIW AMIHUDKRAMER NINAI. SKOBELEVA

ADVANCES I N FOOD RESEARCH VOLUME 17

Edited by C. 0. CHICHESTER

E. M. MRAK

University of California Davis, California

University of California Davis, California

G. F. STEWART University of California D a v f s , California

Editorial Board

E. C. BATE-SMITH w.H. COOK M. A. JOSLYN

S. LEPKOVSKY EDWARDSELTZER W. M. URBAIN

J. F. VICKERY

1969 ACADEMIC PRESS, New York and London

COPYRIGHT @ 1969, BY ACADEMIC PRESS, INC. ALL FUGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

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CONTRIBUTORS TO VOLUME 17

F. AYLWARD," Fruit and Vegetable Preservation Research Association, Chipping Campden, England ITAMARBEN-GEM,! Department of Horticulture, College of Agriculture, University of Ma y l a n d , College Park, Maryland MIKHAIL A. BOKUCHAVA,Bakh Znstitute of Biochemistry, USSR Academy of Sciences, Moscow, USSR

D. R. HAISMAN,$Fruit and Vegetable Preservation Research Association, Chipping Campden, England N. CZYHRINCIW,"" School of Biology, Faculty of Sciences, Central University of Venezuela, Caracas AMIHUD KRAMER, Department of Horticulture, College of Agriculture, University of Maryland, College Park, Maryland

NINA I . SKOBELEVA, Bakh Znstitute of Biochemistry, USSR Academy of Sciences, Moscow, USSR

'Present address: Department of Food Science, The University of Reading, Berkshire, England. tPresent address: Food Institute, Centre for Industrial Research, Haifa, Israel. $Present address: Unilever Research Laboratory, Colworth House, Bedford, Beds., England. ''Present address: Department of Chemistry and Technology, Faculty of Agronomy, University Central of Venezuela, Maracay, Venezuela. V

Mortimer Louis Anson

Mortimer Louis Anson 1901-1 968

Those who professionally delve into the life sciences are singularly blessed when reflecting upon the death of a friend. The quiet and sustaining comfort of certain knowledge has long prepared them for the inevitable processes that lead all living things to common ground. Notwithstanding, the removal from the daily scene of a prized companionship and the intellectual stimulation that it brings creates an immediate and stark sense of loss. The audience to which this encomium is addressed is surely cognizant of the background, stature, and accomplishments of Tim Anson. It is the abstract qualities or the essence of a lifetime with which we are now concerned. Tim Anson was a stem taskmaster in matters of education and scientific research. His personal directness and approach to discipline and problem alike were often misunderstood, yet these were the roots of his excellence and accomplishment. But above all else there persisted in the man a gentle and dedicated concern for the fulfillment of man’s basic needs. Internationalist at heart and in action, he unselfishly devoted his considerable talents and energy to the food and nutritional needs of people everywhere. The worldwide appellation and accolade he justly earned as a distinguished protein chemist, were secondary to his main dedication in life. More importantly, he was a great scientist who early advanced the concept of the need to drastically increase food protein resources to overcome world malnutrition stemming from traditional agricultural practices and inadequate technology. In this, Tim Anson offered persuasion and inspiration through personal advice and counsel, authorship, and editorship. He was also a major force in organizing international meetings to speed the exchange of scientific and technological information toward alleviating the ravages of human starvation; the first two International Congresses on Food Science and Technology, held in London and in Warsaw, and the First International Symposium on Oilseed Protein Foods, held in Japan, where scientists from the West and East met to explore a common interest and the values of and means for extending the rise of plant proteins as human food. At the time of his death he vii

viii

MORTIMER LOUIS ANSON

had already made a substantial contribution to plans for the Third International Congress on Food Science and Technology to be held in Washington, D.C. in 1970, and was intimately involved in planning a symposium dealing with the better utilization of marine raw materials to produce protein-rich foods. Tim Anson was a man of many talents. This was the result of inborn ability, superior academic training, and the determination to make full use of his capabilities. In the present difficult job involved in this editing of a full and highly productive life, there consistently arises the memory of a dominant quality in Tim Anson that struck quickly at the heart of a matter. A practiced discipline became the analytical factor in the weeding out of the superficial and unfounded in the search for truth. A negative attitude was quickly replaced by positive thinking in reaching the means for accomplishment. Stand and opinion were based on the proven fundamentals of a liberal education and subsequent specialization in science. Motivation came from a deep affection for all mankind. This was the scientist, the humanitarian, and the gentle man; the man who used abundant talent in the cause of others. According to the ancient prophets, there is nothing better than that a man rejoice in his own works, for that is his portion, for who shall bring him to see what shall be after him. The Anson legacy will endure. The donor knew what he was about and rejoiced in the service of mankind. His vision and course gave credence to that which would follow. The solid foundation of his work indelibly inscribes his epitaph.

C. 0. CHICHESTER E. M. MRAK GEORGEF. STEWART

CONTENTS

.

CONTRIBUTORS TO VOLUME17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MORTIMERLOUISANSON . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

V

vii

Oxidation Systems in Fruits and Vegetables-Their Relation to the Quality of Preserved Products

F. AYLWARD AND D. R. HAISMAN I. 11. 111. IV. V. VI. VII.

.

.

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Introduction . . , . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidizing Enzyme Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Respiratory and Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative and Other Changes in Lipids . . . , . . . . . . . . . . . . . . . , . , . . . . . . . . . . . Thermal and Other Environmental Factors Modifying Enzyme Activity . . . . .~ Regeneration of Enzyme Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Research Needs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

2 7 19 23 34 53 57 61

The Utilization of Food Industries Wastes

ITAMAR BEN-GERAAND AMIHUD W

M E R

Introduction.. . . ...................................... Vegetable-Proces .................. .., .. .. . . .. Fruit-Processing Wastes . . . . . . . . . . . . . . . . . . . . . Total Utilization of Plant Residues . . . . . . Oilseeds and Grains . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . Starch-Production Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sugar-Manufacturing Wastes . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distillery, Brewery, and Winery Wastes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. .......... IX. Animal and Marine Product Wastes . . . References ..........................................................

I. 11. 111. IV. V. VI. VII. VIII.

78 80

.

. .

..

.

106 115 122 126 132 135

Tropical Fruit Technology

N. CZYHRINCIW I. 11. 111. IV.

Introduction ......................................................... The Significance of Fruits . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology and Anatomy of Fruits . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . Physical Properties of Fruits . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . .

.

.

.

.

.

..

.

153 157 163 165

ix

CONTENTS

X

V. Some Chemical Properties of Fruits .................................... VI . Technical Problems .................................................. VII Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ..........................................................

.

174 185 204 207

The Chemistry and Biochemistry of Tea and Tea Manufacture

MIKHAILA . BOKUCHAVA AND NINA1. SKOBELEVA I . Introduction ........................................................ I1 Chemical Constituents of Tea Leaf and Manufactured Tea ................ I11. Significance of Biochemistry .......................................... IV. Thermal Treatment to Enhance Quality and Vitamin P of Black Tea . . . . . . . V. Biological and Nutritional Value of Tea ................................ VI . Conclusion .......................................................... References ...........................................................

.

SUBJECTINDEX............................................................

215 219 260 272 278 279 280

293

OXIDATION SYSTEMS IN FRUITS AND VEGETABLESTHEIR RELATION TO THE QUALITY OF PRESERVED PRODUCTS BY F. AYLWARD*AND D. R. HAISMAN~ Fruit and Vegetable Preservation Research Association, Chipping Campden, England 2 I. Introduction Introduction.. . . . .. . . . . . . . 7 11. 11. Oxidizing Enzyme S Systems 8 A. Peroxidase Peroxidase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... 9 B. Pseudo-Peroxidases . . . 10 C. Catalase . . . . . . . . . . 10 D. Cytochrome Oxidase . 11 E. o-Diphenol Oxidase . 12 F. p-Diphenol Oxidase . 13 G . Ascorbate Oxidase . . 13 H. Amine Oxidases . . . . 13 I. Clycolate Glycolate Oxidase . . . . . . . . . ............. 14 J . Oxidation Mechanisms . 18 K. Correlation between Enzyme Activity and Food Deterioration 19 111. Respiratory and Other Enzymes 19 A. Respiration . . 20 B. Fermentation . . . . . . . . . . . . . . . . . . . . . . . .20 20 C. Respiratory Enzymes and Food Deterioration 21 D. Pectic Enzymes. . . . . . . . . . . . . . . . 22 E. Chlorophyllase . . 22 F. Enzymes of Amino Acid Metabolism 23 IV. Oxidative and Other Changes in Lipids . 24 A. Degradation of Lipids . 26 B. Lipoxygenase . ,27 C. Autoxidation of Lipids 28 D. Decomposition of Hydroperoxides 131 E. Antioxidants . . . . . . . . . . 31 F. Lipid Oxidation in Relation to Food Quality I

I

'Present address: Department of Food Science, The University, Reading, Berks. England. +Present address: Unilever Research Laboratory, Colworth House, Sharnbrook, Beds., England.

11

F. AYLWARD AND D. R. HAISMAN

2

V. Thermal and Other Environmental Factors Modifying Enzyme Activity . . . . . 34 A. Thermal Inactivation -General Principles . . . . . . . . . . . . . . . . . . . . . . . . . .35 B. Thermal Inactivation of Oxidizing Enzymes-Experimental Data . . . . . .39 C. Enzyme Action at Low Temperatures .............................. .47 D. Effects of pH and Ionic Strength ................................... .47 E. Effects of Water. . . . . . . . . . . : . . . . . . . . . . . . . . .................... . 4 8 F. Multiple Molecular Forms of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . .t.48 G. Adsorption of Enzymes on Natural Substrates ....................... .50 H. Specific Inhibitors ................................................ .53 VI. Regeneration of Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3 A. Test Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Heat Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54 C. Storage Conditions .......................... . 5 6

VII. Research Needs

..........

Biochemical ......................... . 5 8 Enzyme Systems: Substrates-Primary and Secondary Reactions . . . . . . 58 Plant Lipids: Oxidation Mechanisms and Reaction Products . . . . . . . . . . . 59 Plant Components Inhibiting or Modifying Enzyme Activity. . . . . . . . . .59 F. Heat Resistance of Enzymes . . . . . . . . . . ........................ .60 G. New Methods of Preservation ....................................... .60 References ......................................................... 61

B. C. D. E.

I. INTRODUCTION

The fruit and vegetable canning, quick-freezing, and dehydration industries can be considered from the economic standpoint in terms of a chain stretching from farm to consumer and including growers and processors and groups concerned with transport, storage, and distribution. The scientific and technological problems of the industry must be considered in the same context. During the growth of the canning industry, and later of the quickfreezing and dehydration industries, the importance of careful control of postharvest and processing conditions became well recognized, and scientific and technological investigations sponsored by industrial and other groups were directed to the production of preserved foods of good quality. Not so much attention was given to the changes in quality of food after leaving the factory, but it was gradually recognized that changes1 in quality could and did take place, especially with frozen and dehydrated foods. Realization of the extent of such changes has led to various investigations, notably those sponsored b y the Western Utilization Research Laboratory of the United States Department of Agriculture.

PLANT-TISSUE OXIDATION SYSTEMS

3

Quality changes may result from three main types of reaction within the foodstuffs or between the food and its environment: (a) microbiological; (b) enzymic, and (c) chemical nonenzymic changes. This review is concerned primarily with the effects of enzymes. It is well known that active enzyme systems can spoil fruits and vegetables even at subzero temperatures and low moisture levels. For this reason most vegetables and some fruits which are to be preserved by canning, freezing, or dehydration are given a preliminary heat treatment (for instance, blanching in boiling water) to inactivate the enzyme systems in the tissues. The blanching operation reduces the level of infection by microorganisms, and may improve color and flavor by expelling volatile degradation products formed during the postharvest interval (Adam et al., 1942). Blanching is omitted with certain products, such as onions and peppers and other strongly flavored materials, in which enzymic deterioration either does not occur or has no noticeable effect on quality (Makower, 1960). The literature on the effect of enzymic activity on the quality of foods has been comprehensively reviewed by several workers. Joslyn (1949) examined the evidence for the enzymic nature of flavor changes, the effectiveness of blanching, and the tests available for measuring its efficiency. He noted that the best indicator for blanching efficiency was peroxidase activity, and believed that until more was known about the enzymic systems involved in off-flavor formation, technological developments would be limited to improvements in techniques of peroxidase estimation. From a survey of the experimental evidence, he concluded that there was no doubt of the activity of enzymes in frozen foods. In a later publication Joslyn (1951) dealt with enzyme activity in the dried state and in concentrated solutions, drawing parallels with the situation in frozen tissues. In more recent reviews Joslyn (1961, 1966) has emphasized that procedures for the control of enzymic activity during the preparation and freezing of fruits and vegetables are largely empirical; further biochemical research is required to define postharvest changes and changes during processing and storage. Leeson (1957) reviewed the inactivation of enzymes by heat, the effect of residual enzyme activity on the quality of fruits and vegetables, and evidence for the regeneration of enzyme activity. He emphasized that in devising tests of blanching efficiency the possibility of enzyme regeneration should not be overlooked. McConnell (1956) tabulated data on the heat resistance of enzymes in fruits and vegetables, and showed that in high-temperature short-time process-

4

F. AYLWARD AND D. R. HAISMAN

ing, enzyme inactivation may take longer than the destruction of microorganisms. Board (1961) surveyed the effect of enzymes on the stability of canned foods; in describing the changes caused by enzymes he gave examples of nonenzymic catalysis such as the effect of copper and hemochromogens on the destruction of ascorbic acid and concluded that the mechanisms of the deteriorative changes were still obscure, but that both enzymic and nonenzymic reactions may take place. The investigations of the Western Regional Laboratory, to which reference has already been made, were initiated in 1948 and covered not only fruits and vegetables but also other frozen products. The research program covered the behavior of frozen foods within the extremes of time and temperature which might be encountered in commercial practice. The main aims of the investigation were to establish tolerable deviations from ideal conditions for different products, to identify and improve critical operations, to seek technological improvements ensuring greater tolerances, and to establish tests of product quality. The results were published from 1957 onward, starting with a definition of the problem (van Arsdel, 1957); surveying the quality of frozen soft fruits (Guadagni et al., 1957a,b,c, 1958, 1960; Guadagni, 1957; Guadagni and Nimmo, 1957a,b, 1958; Guadagni and Kelly, 1958), poultry products (Hanson and Fletcher, 1958; Hanson et al., 1959; Klose et al., 1959), vegetables (Dietrich et al., 1957, 1959a,b, 1960, 1962; Boggs et al., 1960), and liquid products (Hanson et al., 1957; McColloch et al., 1957), and covering relations between temperature history and product quality (van Arsdel and Guadagni, 1959) and bacterial population (Michener et al., 1960). In frozen fruits the degree of browning and percentage loss of ascorbic acid (based on content of ascorbic acid, dehydroascorbic acid, and diketogulonic acid) were found to be closely related to overall quality. In peaches, partial inactivation of the oxidizing enzymes, followed by vacuum-packing in sealed containers and freezing, was sufficient to retard browning for a reasonable time after opening and thawing; complete inactivation of the enzymes by conventional heat processing caused serious flavor changes and increased leaching losses (Guadagni and Nimmo, 1957a). In frozen vegetables, the loss of ascorbic acid was also found to be a useful index to quality, but the percentage retention of chlorophyll (considering chlorophyll-pheophytin ratios) was found to give the closest correlation with overall quality (Dietrich et al., 1959a,b).

PLANT-TISSUE OXIDATION SYSTEMS

5

Chlorophyll losses were affected by the initial heat treatment given the product: the greater the loss during blanching, the lower the stability during storage. Walker (1964a,b) also found that the rate of change of chlorophyll during frozen storage increased when green beans were overblanched. Chlorophyll degradation in green snap beans was minimized by the use of high-temperature short-time blanching (Dietrich et al., 1959b). Water-blanching of Brussels sprouts was more effective than steam-blanching in that enzyme inactivation was faster and chlorophyll degradation less (Dietrich and Neumann, 1965). The search for improved quality in preserved fruits and vegetables has led to proposals to reduce the period of heat processing to a minimum, and so retain to the maximum extent the “natural” characteristics of the food. It is extremely important to have precise estimates of the effectiveness of different types of heat treatments and process times on enzymes, as well as on microorganisms. Many authors have stressed that with high-temperature short-time processes enzymes, such as peroxidase, may be more difficult to destroy than microorganisms; enzyme inactivation may therefore be the deciding factor in assessing the efficiency of the process (e.g., McConnell, 1956; Leeson, 1957; Adams and Yawger, 1961; Yamomoto et al., 1962). Deterioration of foods through enzyme action can lead to the development of off-flavors and also to marked changes in color and texture. Despite the efforts of many investigators over the past thirty years the enzymes responsible for quality deterioration have not been positively identified except in a few cases, mostly concerned with changes in texture. There is general agreement that where flavor is concerned several enzymic systems may be involved, working in sequence or simultaneously. The problem is complicated by the fact that the substances responsible for off-flavors are also largely unknown. Many compounds which could be involved can be detected by taste at extremely low concentrations, of the order of 1 part in lo9(Lea and Swoboda, 1958), that is, at levels at which chemical isolation and identification are difficult. In some frozen vegetables a good correlation can be obtained between acetaldehyde content and off-flavor, so that acetaldehyde levels can be used as an index of quality deterioration (Gutterman et al., 1951; Lovejoy, 1952). Acetaldehyde is not the cause of the off-flavor, but appears as a reaction by-product, possibly from anaerobic glycolysis. Anaerobic conditions may exist in frozen tissues (Fuleki and David, 1963).Joslyn (1966) has noted that the odors and flavors formed during

6

F. AYLWARD AND D. R. HAISMAN

the storage of frozen unblanched vegetables resemble those of fresh vegetables held in oxygen-deficient atmospheres at room temperature, and that particular vegetables develop quite characteristic odors. Progress has been made in identifying both the precursors and volatile products responsible for flavor and odor changes in certain products. Falconer et al. (1964) found that the violetlike off-flavor in dehydrated carrot was closely related to the oxidation of p-carotene, and was derived from p-ionone and other oxidation products. The formation of pyrollidone carboxylic acid from glutamine has been shown to cause bitter phenolic off-flavors in beet products (Shallenberger and Moyer, 1958). Gas-liquid chromatographic examination of the volatile compounds from stored frozen vegetables has yielded interesting results. Bengtsson and Bosund (1964) evaluated the volatile substances from stored frozen peas, and found that the compounds formed slowly during frozen storage resembled those found in rapid postharvest changes at ordinary temperatures. The main components were acetaldehyde, ethanol, and hexanal; the hexanal content showed promise as an indicator for off-flavor development. It was shown later (Bengtsson et al., 1967) that the hexanal concentration in the vapor over cooked frozen peas correlated well with the postharvest deterioration which had occurred. Off-flavor development during the first months of storage at -5°C coincided with the formation of hexanal, but over longer periods the hexanal concentration decreased, suggesting that it may be unreliable as a single quantitative indicator of quality. Other workers have examined hexanal formation in different products. Thus, Whitfield and Shipton (1966), in their examination of the volatile carbonyls from frozen unblanched peas in storage, found that the major components were acetaldehyde (96%) and hexanal (3.5%).Hexanal was found to be a major component in the volatiles produced during low-temperature oxidation of sunflower oil (Swoboda and Lea, 1965)and oxidative degradation of potato granules (Buttery et al., 1961). In later experiments-during storage of potato granules for up to four months -the hexanal content of the vapor over the potato, after reconstitution at 93"C, followed subjective flavor scores closely (Boggs et al., 1964). The experiments on the production of carbonyl compounds provide support for the theory that the unsaturated lipids, although present in only trace amounts in some types of vegetable material may serve as substrates for oxidative degradative changes during storage.

7

PLANT-TISSUE OXIDATION SYSTEMS

The present review summarizes information available that is relevant to fruits and vegetables on: (1) the enzymes and, in particular, oxidizing systems which may be important in postharvest changes; (2) the possible relationship of some of these oxidizing systems to lipid oxidation by enzymic and nonenzymic mechanisms; (3) thermal and other factors modifying or inactivating the oxidizing enzyme systems; and (4)the relationship between enzymic activity and quality.

II. OXIDIZING ENZYME SYSTEMS There are many enzymes in plant tissues which possess an oxidizing function, and it is quite conceivable that most play some part in deteriorative processes, albeit a definite link has been established in only a very few cases. The enzymes which have been studied in this connection are listed in Table I, together with some of their distinguishing features. Their properties are discussed in more detail below. The catalytic activity of most of these enzymes depends on a prosthetic group containing copper or iron, and consequently the possibility cannot be ignored of nonspecific metallic catalysis of various

OXIDIZING

Enzyme

TABLE I ENZYMES IN

PLANTS

Nature of pH optimum prosthetic group(s) for activity

~~

Peroxidase

Heme

7.0

Catalase

Heme

5.3-8.0 -

o-Diphenol oxidase

Heme and copper Copper

5.5-7.0

p-Diphenol oxidase

Copper

-

Ascorbate oxidase

Copper

5.6

Amine oxidase Glycolate oxidase Lipoxygenase

Copper Flavin None

8.5

Cytochrome oxidase

Comments

~

8.3 5-5-10.0

Widespread, concentrated in root material In all plants, in conjunction with cytochrome systems Photodissociable carbon monoxide complex Inactivated by carbon monoxide Insensitive to carbon monoxide Insensitive to carbon monoxide Isolated from pea seedlings Isolated from spinach -

8

F. AYLWARD A N D D. R. HAISMAN

reactions before or after inactivation of the enzymes. This factor may be of some importance in the breakdown of lipid hydroperoxides, and examples of this, together with a detailed consideration of the action of lipoxygenase, are reserved for a later section, devoted to lipid oxidation.

A. PEROXIDASE The activity of peroxidase with reference to deteriorative changes in vegetable tissues has been studied more extensively than any other enzyme system. Because of its relatively high resistance to thermal inactivation, and its extensive distribution, peroxidase has been widely used as an index of enzyme activity in plant tissues. It has been generally accepted that if peroxidase is destroyed by a given heat treatment it is unlikely that any other enzyme system will have survived. As a practical test this has worked very well, its main disadvantage being lack of agreement between different techniques for detecting the active enzyme. Methods of estimating peroxidase activity have recently been reviewed (Wood and Lopez, 1963), and levels of activity in various vegetables have been determined (Bottcher, 1961,1962). A full account of the biochemistry of this enzyme has been given by Saunders et al. (1964). Various types of peroxidase exist, and the properties of the enzyme depend to some extent on its source. True peroxidases are hemoproteins, and have in common the prosthetic group protohematin IX in the proportion of one hematin residue per molecule of enzyme. Different peroxidases can be distinguished by differences in their absorption spectrum (Mehler, 1957) and in their behavior toward different reducing agents. Thus, milk peroxidase will oxidizes resorcinol but not nitrite (Elliott, 1932a,b). Even peroxidases isolated from the same plant source may exhibit different properties, and there is little doubt that multiple forms of the enzyme occur. Horseradish peroxidase, the best characterized of the peroxidases, consists of a colorless protein reversibly bound to protohematin IX (Maehly, 1955). The iron atom in protohematin has six coordination positions, four of which are taken up by porphyrin nitrogens and the fifth by a protein attachment. The sixth position can be occupied by water, cyanide, or another radical, and the enzyme appears to operate by the exchange of groups at this position. The protohematin can be easily and reversibly detached by acetone and hydrochloric acid below WC,a property not shared by other peroxidases.

PLANT-TISSUE OXIDATION SYSTEMS

9

Peroxidases are quite specific in their primary reaction with peroxides, but, by means of coupled reactions whereby the primary oxidation products react with secondary substrates, they can promote a variety of consequential reactions. They appear to be most stable at pH 7.0, where they also exhibit maximum catalytic activity. Maehly (1955) found horseradish peroxidase to be stable between pH 3.5and pH 12.0 in the absence of inhibitors, and Wilder (1962)confirmed this. Lenhoff and Kaplan (1955) found that at pH 7.0 cytochrome c peroxidase was most active and also stable. Axelrod and Jagendorf (1951) found peroxidase (and phosphatase and invertase) to be stable in autolyzing tobacco leaves. Even though the leaves lost 45% of the cytoplasmic protein nitrogen during storage, the levels of enzyme activity were unaltered. The enzyme is severely inhibited by azide, cyanide, fluorides, and other halides in acid solutions (Maehly, 1955; Lenhoff and Kaplan, 1955).

B. PSEUDO-PEROXIDASES There are many organic and inorganic substances which can catalyze certain typical peroxidase reactions involving peroxides (Saunders et al., 1964). These include hematin compounds, chelated iron salts, amorphous heavy-metal hydroxides, aldehydes, granite, charcoal, platinum, and palladium. More specialized cases are acetyl choline, which can catalyze the oxidation of benzidine and vitamin A, and the carotenes, which can catalyze the oxidation of potassium indigosulfonate. Generally speaking, these substances are much less effective catalysts than peroxidase itself, as shown by the comparison between horseradish peroxidase and hematin compounds in Table 11. TABLE I1 THECATALYTIC ACTIVITY OF HEMATINCOMPOUNDS TOWARD PURPUROGALLIN~ Relative catalytic activity Horseradish peroxidase Pyridine hemochrome Hemoglobin Denatured globin hemochrome Hematin "Bancroft and Elliott, 1934.

1,000,000 4.5 16 35 1.6

10

F. AYLWARD AND D. R. HAISMAN

C. CATALASE Catalase and peroxidase activities are often grouped together. Both are hemoproteins, use hydrogen peroxide as a substrate, and occur in several modifications according to source. Pure crystalline catalase can be obtained from blood, is red at a neutral pH, and contains 1.1% protohemin and 0.09% iron, which is equivalent to four hematin residues per molecule of enzyme (Bonnichsen, 1955).Plant catalase has been isolated from spinach, and contains 0.049% iron, approximately half the value for a pure four-hematin enzyme (Galston, 1955). Preparations of the plant enzyme are stable indefinitely at 1°C between pH 5.3 and 8.9, and the enzyme activity is greatest between pH 5.3 and 8.0, falling off quickly at more acid values, and slowly at more alkaline values (Galston, 1955). Sapers and Nickerson (1962a) prepared spinach catalase and found it to be quite stable below 36°F. Otherwise, its stability was greatly influenced by storage temperature and pH. At 80°F and pH 7.0 it became inactivated rapidly. Inactivation was more rapid in acid solutions. The enzyme was also susceptible to attack by microorganisms.

D. CYTOCHROME OXIDASE The characterization of cytochrome oxidase is still the subject of experiment, but the available evidence suggests that it is constituted of heme, copper, lipid, and protein (Wainio, 1961).In a recent review, Beinert (1966) tentatively concludes that the enzyme is a 1:1combination of cytochrome a and cytochrome a,. It is thought that the cytochrome a, reacts directly with oxygen (Smith and Conrad, 1961).The enzyme is readily distinguished by its absorption spectrum and by the spectral shift during the formation of its photo-dissociable carbon monoxide complex. It is specific to cytochrome c, which it converts to the oxidized form in the presence of molecular oxygen. The coupled system can oxidize many other substrates, such as phenols and amines, and sustains other oxidizing enzymes such as succinate dehydrogenase, a particularly important system in many microorganisms. The estimation of the enzyme may be difficult in the presence of phenol oxidases. Hare1 and Mayer (1963), working with lettuce seeds, found that the presence of oxidized phenolic compounds depressed cytochrome oxidase activity. The activity was restored when phenolase inhibitors were added. The final stages of respiration in potato tubers appear to be shared by cytochrome oxidase and o-diphenol oxidase, the relative impor-

PLANT-TISSUE OXIDATION SYSTEMS

11

tance of each depending on the maturity of the tuber (Mondy et al., 1960). Mapson and Burton (1962) found that 70% of the respiration of potato tubers passes over the cytochrome system. Other authors have found that cytochrome oxidase activity is high enough to account for the whole of the respiration of potato tubers (Goddard and Holden, 1950; Schade et al., 1949; Thimann et al., 1954). The substrate of cytochrome oxidase, cytochrome c, is known as a stable hemoprotein. Its chemistry has recently been reviewed by Margoliash and Schejter (1966). It is notable for its remarkable stability in conditions commonly deleterious to proteins, and its resistance to dilute acid and alkali and to boiling (although a small proportion may be denatured). It is easily reduced by molecules such as cysteine and ascorbic acid, and reoxidized by cytochrome oxidase, and also by peroxidase. Cytochromes found in plant tissues have been surveyed by Bonner (1961), and may be slightly different from those from other sources. The purification of cytochrome c from wheat germ has been described (Wasserman et al., 1963), and this compound was found to be very unstable in solutions of low ionic strength at pH 7.0.

E. O-DIPHENOL OXIDASE o-Diphenol oxidase is widespread in occurrence. Bonner (1957)reviewed the function of the enzyme in plant tissues, and Brooks and Dawson (1966) surveyed aspects of its chemistry. It is a coppercontaining enzyme, easily inactivated by carbon monoxide. o-Diphenol oxidase displays activity toward a great range of substrates, as is indicated by the various names by which it is commonly known: catecholase, tyrosinase, cresolase, polyphenoloxidase, phenolase, etc. The enzyme apparently occurs in various forms, which can be classified into two broad groups, both oxidizing o-dihydric phenols but not ascorbic acid, one of which possesses the added ability to catalyze the o-hydroxylation of monophenols (Robb et al., 1965). The ratio of the activities toward mono- and o-dihydric-phenols in an enzyme preparation varies according to the source and methods used in its isolation. Although catechol and tyrosine or cresol are commonly used for estimation of the activity, the natural substrates are probably more complex phenolic compounds, such as chlorogenic acid. Alberghina (1964) found that o-diphenol oxidase from potato tubers had an affinity much higher for chlorogenic acid and methyl catechol than for catechol or dihydroxyphenylalanine. One enzyme from eggplant oxidized chlorogenic acid much faster than any other substrate tried,

12

F. AYLWARD A N D D . R. HAISMAN

while one from avocado showed greatest affinity for nordihydroguaiaretic acid when compared with catechol and catechin (Knapp, 1965). Another eggplant phenol oxidase was active toward anthocyanins (Sakamura et aZ., 1966). Tate et aZ. (1964) characterized an o-diphenol oxidase from Bartlett pears, and found it was active only toward o-dihydric phenols. Other phenols were not attacked. Walker (1964c,d) isolated the enzyme from apples and pears, and found that it was not only very active toward o-dihydric phenols but over longer times catalyzed the o-hydroxylation of p-coumaric acid to cafFeic acid. The conversion of p-coumaric acid to caf€eic acid is efficiently catalyzed by mushroom o-diphenol oxidase in the presence of ascorbic acid, but the reaction can also be brought about nonenzymatically by dihydroxymaleate or an iron-ascorbate system (Embs and Markakis, 1966). The optimum pH for maximum activity of the enzyme appears to vary between preparations and according to substrate from about pH 4.0 to pH 7.0. It is difficult to measure the activity of the enzyme directly, owing to the large number of secondary reactions which follow the initial enzymic oxidation. Assay methods have been compared critically (Mayer et al., 1966); polarographic measurement of the initial oxygen uptake appears to give the most sensitive estimate of the enzyme activity. The level of o-diphenol oxidase activity and the concentration of various substrates in different fruits and vegetables have been determined by Herrmann (1957, 1958).The cellular location of the o-diphenol oxidase responsible for the darkening of cut red beet has been examined by Boscan et al. (1962). The interrelationship between cytochrome oxidase and o-diphenol oxidase activities in potato tubers, particularly during storage at different temperatures, has been examined by Mondy et al. (1966a,b). F. ~ D I P H E N OOXIDASE L

Commonly known as laccase, p-diphenol oxidase, another widespread copper-containing enzyme, has been reviewed by Bonner (1957) and Levine (1966). The pure enzyme is deep blue and is not appreciably inactivated by carbon monoxide. Alleged to be principally an extracellular enzyme, it catalyzes the oxidation of a large number of aryl dihydric phenols and diamines, where the functional groups have an o- or a p- relationship. Like o-diphenol oxidase, it indirectly oxidizes ascorbic acid through coupled oxidations with phenolic or amine substrates.

PLANT-TISSUE OXIDATION SYSTEMS

13

G. ASCORBATE OXIDASE Ascorbic acid is a powerful reducing agent, and its oxidation is catalyzed by metals as well as by several enzyme systems. Methods have been devised (Butt and Hallaway, 1958) for distinguishing between true ascorbate oxidase activity and the action of other less specific catalysts. Ascorbate oxidase is another copper-containing enzyme widespread in plants and microorganisms. Its chemistry has been reviewed by Bonner (1957) and Dawson (1966). The pure enzyme is blue and insensitive to carbon monoxide. Its natural substrate is assumed to be ascorbic acid, but it is also active toward ring analogs with a dienol grouping adjacent to a carbonyl group, and substituted polyhydric and amino phenols, including 2,6-dichloroindophenol, which is oxidized to a blue quinoid dye. The activity of the enzyme has been determined in a number of different fruits and vegetables (Huelin and Stephens, 1948; McCombs, 1957). H. AMINE OXIDASES

The characterization of these enzymes is still the subject of intensive investigation. Although a tentative classification into monoamine oxidases and diamine oxidases has been proposed, Nara and Yasunobu (1966) suggest in a recent review that a rigid classification is no longer tenable. The enzyme is of interest since it catalyzes the conversion of amines to aldehydes; hydrogen peroxide and ammonia are the other primary reaction products. The production of hydrogen peroxide leads to various secondary reactions, including inactivation of the enzyme. Most work has been carried out on the amine oxidase system in pea seedlings (Kenten and Mann, 1955; Mann, 1961; Hill and Mann, 1962, 1964). The enzyme contains copper, and is inhibited by various chelating agents. It is unusual in that solutions of purified enzyme are pink. The preparation from pea seedlings catalyzes the oxidation of aliphatic monoamines, diamines, phenylalkylamines, histamines, spermidine, agmatine, lysine, and ornithine. I. GLYCOLATE OXIDASE Glycolate oxidase, a flavoprotein, catalyzes the oxidation of ahydroxy acids by oxygen to the corresponding 0x0 acids, with the concomitant production of hydrogen peroxide. The most important

14

F. AYLWARD A N D D . R. HAISMAN

substrates have been found to be glycolic and L-lactic acid (Zelitch and Ochoa, 1953).Kolesnikov (1948a,b; 1949) noted that glycolic acid had a catalytic effect on the degradation of chlorophyll and ascorbic acid in barley leaf macerates. In the presence of glycolic acid, oxygen absorption by the macerate increased up to 15 times over the amount needed to oxidize the glycolic acid. The rate of chlorophyll degradation was dependent on the level of glycolic acid present. Tolbert and Burris (1950) also found that the oxidation of glycolic acid in green leaves was accompanied by bleaching of the chlorophyll. No11 and Burris (1954) detected glycolate oxidase activity in 17 species of plants. Recent work has shown that the enzyme also catalyzes the oxidation of glyoxylic and a-hydroxybutyric acids (Richardson and Tolbert, 1961)and aromatic a-hydroxy acids (Gamborg et d.,1962). J. OXIDATION MECHANISMS

Although quite distinct in their primary reactions, the oxidative enzymes are alike in their ability to utilize a wide range of secondary substrates. The main features of the oxidative reactions are shown in Table 111.

1. Peroxidase The combination of peroxidase with substrate can be followed spectrophotometrically, which facilitates study of the oxidation mechanism (Chance, 1949a,b). The cyclic reaction illustrated in Fig. 1 has been well established (Saunders et aZ., 1964). Methyl or

Oxidized donor

Peroxidase

H’ore donor Compound Il

Compound I

H’or e donor

Excess H,O,

Compounds If1 and N (enzymically inactive)

FIG. 1. The peroxidase oxidation cycle.

TABLE I11 OXIDATION MECHANISMS

Enzyme

Substrate(s)

Primary product

Peroxidase

HzOz (and some other peroxides)

“Oxidized” peroxidase

Catalase Cytochrome oxidase

HA“ Cytochrome c and oxygen

o-Diphenol oxidase

o-Dihydric phenol

Monophenols p-Diphenol oxidase Ascorbate oxidase

-

0-and

p-Dihydric phenols Ascorbic acid

2 HZO + 0 2 Oxidized cytochrome c Quinone

o-Dihydric phenol Quinones Dehydroascorbic acid

Secondary reaction Electron transfer to hydrogen donor such as m i n e or phenol Coupled oxidation of phenols, mines, etc. Quinone formation may lead to further coupled oxidations Oxidation of dihydric phenol ensues May lead to further coupled oxidations Spontaneous delactonization to diketogulonic acid

“At low concentrations of hydrogen peroxide, catalase can act as a “peroxidase,” using aliphatic alcohols and other compounds as hydrogen donors.

F.AYLWARD AND D. R. HAISMAN

16

ethyl hydroperoxides, peracids, or hypochlorite can be substituted to some extent for hydrogen peroxide in the first stage of the reaction. The commonest hydrogen donors are amines (such as aniline, p-toluidine, mesidine, etc.) or phenols. Side reactions are very common, and methyl, methoxy groups, or halogen atoms may be eliminated from the ring during the oxidation of aromatic amines. An alternative oxidative pathway involving peroxidase was discovered when Kenten and Mann (1950) showed that MnZf was oxidized by peroxidase systems. Since then, systems containing peroxidase, manganese, and mono- or dihydric phenols have been found to oxidize a variety of substrates including dicarboxylic acids (Kenten and Mann, 1953), phenylacetaldehyde (Kenten, 1953), NAD, and NADP (Akazawa and Conn, 1958). Mudd and Burris (1959) observed that plant peroxidases have broad substrate specificities, acting peroxidatively in the presence of hydrogen peroxide, or oxidatively in the presence of phenols and manganous or cerous ions. The peroxidase-manganese-phenol system also oxidizes indoleacetic acid and, although the point is still controversial, may account for activity formerly attributed to a specific enzyme, indoleacetic acid oxidase (Hare, 1964). This oxidation is inhibited by certain quercetin derivatives (Furuya et al., 1962). The oxidation of crocin in sugar-beet leaves may be mediated by a similar system. Dicks and Friend (1966a,b) found two enzyme systems associated with mitochondria from sugar-beet leaves which could accomplish crocin oxidation. One was attributed to a coupled oxidation involving lipoxygenase and unsaturated lipids, and the other, which was stimulated by 8-hydroxyquinoline and other phenols, probably involved peroxidase or another hemoprotein. The presence of free radicals during peroxidase oxidations was demonstrated by Yamazaki et al. (1960) with peroxidase from turnips on substrates of hydroquinone and ascorbic and dihydroxyfumaric acid. 2. Catalase

The characteristic reaction of catalase is the catalytic decomposition of hydrogen peroxide to water and oxygen. This is a two-stage reaction and can be followed spectrophotometrically. Catalase

+ 2 H,O,

-

Compound I

-

Catalase

+ 2 H,O + 0,

Keilin and Hartree (1955) discovered that, in the presence of the very low concentrations of peroxide generated by oxidase systems in

PLANT-TISSUE OXIDATION SYSTEMS

17

oitro (e.g., xanthine oxidase, glucose oxidase, etc.), catalase could bring about the coupled oxidation of alcohols and other donors, such as nitrites, carboxylates, and aldehydes. Compared with the decomposition of peroxide, the coupled oxidation is a slow reaction.

3. Diphenol Oxidases Characteristically, the phenol oxidases catalyze the aerial oxidation of dihydric phenols. Mayer studied the effect of various inhibitors on the activity of the o-diphenol oxidase (Mayer, 1962; Mayer et al., 1964) and also the effect of nuclear substituents on the rate of enzymic oxidation. It appears that the oxidation takes place by electrophilic attack. The inactivation of o-diphenol oxidase during the oxidative reactions which it catalyzes is well known and was investigated by Ingraham (1954). He concluded that no extensive damage occurred to the protein in this inactivation, which seems most likely to be due to an interaction between the enzyme and the quinone produced by the oxidation (Brooks and Dawson, 1966). 4 . Ascorbate Oxidase

The first product of oxidation of ascorbic acid is dehydroascorbic acid. Opening of the lactone ring, with the formation of diketogulonic acid, can follow spontaneously or by enzymic catalysis. Roe et al. (1948) devised a method of estimating all three compounds in the presence of one another which has been much used in subsequent investigations. The mechanisms of the destruction of ascorbic acid in cauliflower, bitter gourd, and tapioca leaves have been investigated in detail by Tewari and Krishnan (1960, 1961). They found that both steps from ascorbic acid to diketogulonic acid were enzymically catalyzed, and that tapioca leaves contained a further enzyme system which degraded diketogulonic acid. Tapioca leaves also contained a natural inhibitor of ascorbate oxidase which stabilized the natural ascorbic acid. Like o-diphenol oxidase it is reaction-inactivated, but in this case the inactivation is thought to be due to hydrogen peroxide produced nonenzymically by traces of free copper associated with the enzyme (Dawson, 1966). Lillehoj and Smith (1966) found that an ascorbate oxidase from Myrothecium uerucaria took up more than 0.5 mole of oxygen per mole of ascorbate oxidized, and that 10%of the oxidized product disappeared. They thought that either the ascorbate oxidase had peroxidative capacity for a reductant other than ascorbic acid, or free radicals were produced during the oxidation.

18

F. AYLWARD A N D D . R. HAISMAN

K. CORRELATION BETWEEN ENZYME ACTIVITY AND FOODDETERIORATION

1. Peroxidase Wagenknecht and Lee (1958) added various enzymes to blanched peas and found a good correlation between added peroxidase and offflavor production, but emphasized that the flavor changes were only minor. In a similar experiment Zoueil and Esselen (1959) added peroxidase to sterile packs of green beans and turnips and found that off-flavors and off-odors developed and the acetaldehyde content of the pack increased up to fivefold. Joslyn and Neumann (1963)used the decrease in ascorbic acid content in frozen vegetables as an index of peroxidase activity. Pinsent (1962) noted that when peroxidase was not completely inactivated during the blanching of green peas, offflavors developed during storage of the frozen product. Grommick and Markakis (1964) found that anthocyanin pigments could be discolorized by peroxidase.

2. Catalase Wagenknecht and Lee (1958) found that additions of catalase to blanched peas resulted in a mild off-flavor when the peas were stored frozen. A later experiment, with added endogenous catalase, produced a disagreeable off-flavor over 18 months of frozen storage.

3. Oxidases Although the function of the oxidases in the metabolic processes of plants is still obscure, their ability to catalyze direct oxidation by molecular oxygen makes them potential agents in quality deterioration. The browning of plant tissues, particularly after injury, due to the oxidation of polyphenolic constituents is a familiar problem. Joslyn and Ponting (1951) reviewed the enzymes responsible for the browning of fruit and pointed out that, although the phenol oxidases are the primary browning agents, other enzymes, such as cytochrome oxidase, which participate in coupled oxidations, may easily be involved. This applies to both oxidative and reductive changes. Makower (1964a,b) showed that adenosine triphosphate (ATP) inhibited the browning of potato slices although it was not itself a reducing agent. It appeared that reduced nicotinamide adenine dinucleotide (reduced NAD) was the effective reducing agent, and that the function of the ATP was to maintain the supply of reduced NAD.

PLANT-TISSUE OXIDATION SYSTEMS

19

Investigations into the effect of oxidase activity on quality have concentrated on color changes and oxidation of vitamin C. Phenolic oxidation products may also contribute to changes in flavor (Mapson and Swain, 1961), however, and the possibility should not be overlooked of apparently unrelated effects due to secondary reactions of the oxidation products. Ill. R E S P I R A T O R Y AND O T H E R E N Z Y M E S Although peroxidase and catalase may be involved in the respiratory process, their exact role is not clearly understood. There are, however, many enzyme systems operating along the respiratory pathways whose function is defined, and most of them have been detected at one time or another in the higher plants (e.g., Bonner and Varner, 1965).

A. RESPIRATION In essence, respiration is controlled oxidation of organic material to carbon dioxide and water, producing energy in a form which can be utilized in other cellular processes. It is carried out by a sequence of enzyme systems that transfer electrons from successive degradation products to molecular oxygen by a stepwise process. The energy produced is transported to other systems by means of the reduced forms of coenzymes such as nicotinamide-adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and energy-rich” organic phosphates such as adenosine triphosphate “

(ATP). The process can be divided into two stages. In the first instance, hexoses are oxidized to pyruvate ions under the action of phosphorylating, isomerizing, chain-splitting, and dehydrogenating enzymes. Next, the pyruvate is oxidized to carbon dioxide and water through operation of the Krebs cycle, again involving a variety of phosphorylating, isomerizing, dehydrogenating, and decarboxylating enzymes. Most of the oxidative reactions in the respiratory chain are coupled, often using common intermediates such as NAD or NADP, and the pathways of electron transport from the original substrates are obviously complex and probably involve cytochrome systems and flavoproteins. Although various oxidases have been proposed as “terminal” oxidases (Lee,catalyzing the final step in the transfer of electrons to oxygen) most have a comparatively low affinity for oxygen and are more likely to operate through some other acceptor (Bonner, 1957).

20

F. AYLWARD AND D. R. HAISMAN

Cytochrome oxidase has a high affinity for oxygen, and almost certainly catalyzes at least part of the oxygen uptake of all plant tissues.

B. FERMENTATION Under anaerobic conditions the terminal respiratory pathways are blocked. The breakdown of hexose continues, but the pyruvate produced is no longer oxidized via the Krebs cycle. Instead, it is either reduced to lactate through the agency of lactate dehydrogenase and reduced NAD (the predominant mechanism in muscle tissue) or decarboxylated under the action of pyruvate decarboxylase to acetaldehyde and carbon dioxide. The acetaldehyde can then be reduced to alcohol by alcohol dehydrogenase in the presence of reduced NAD. The latter mechanism predominates in plant tissues. Hatch and Turner (1958) showed that pea extracts could quantitatively convert starch, hexoses, and hexose phosphates to carbon dioxide and ethyl alcohol when cofactor levels of ATP, NAD, and magnesium were supplied.

c. RESPIRATORY ENZYMESAND FOODDETERIORATION Delay between vining and processing peas brings about rapid deterioration in quality, and the conditions are often such that respiratory by-products might be expected to accumulate. Off-flavors can develop in less than two hours (Talburt and Legault, 1950) and, even when ice-cooling is used, are noticeable after four hours (Lynch et al., 1959). The enzymes of the respiratory pathways have been suspected as factors in the development of off-flavors mainly because acetaldehyde and alcohol, typical products of anaerobic glycolysis, have been found in relatively large amounts in some deteriorated products (Gutterman et d.,1951; Joslyn and David, 1952). More extensive investigations with peas (David and Joslyn, 1953) and broccoli (Buck and Joslyn, 1953) showed that, in addition to acetaldehyde and alcohol, smaller amounts of acetoin and diacetyl were produced. In the presence of thiamine pyrophosphate, pyruvate decarboxylase is known to catalyze acyloin formation as well as a-keto acid decarboxylation (Singer, 1955): RCOCOOH + RCHO + CO, RCOCOOH RICH0 -+ RCHOHCOR' RCHO RICH0 + RCHOHCOR'

+ +

+ CO,

PLANT-TISSUE OXIDATION SYSTEMS

21

and the results indicated that a pyruvate decarboxylase system was still active in underblanched peas. David and Joslyn (1953)demonstrated that pyruvate decarboxylase was active in green peas but was inactivated within 2 min at 60°C. Buck and Joslyn (1956) followed the activity of the enzyme in broccoli, but found that the amounts of acetaldehyde, acetoin, and diacetyl produced could not be correlated with the intensity of undesirable flavors. Ralls (1959) reported that levels of acetoin are appreciable (up to 300 ppm) in many canned vegetables and can be induced in frozen peas through the nonenzymic thiamine-catalyzed conversion of pyruvic acid. Although acetoin is probably a flavor component, it cannot be classed as an off-flavor. Fuleki and David (1963) examined the production of alcohol, acetaldehyde, and off-flavors in frozen snap-beans, and concluded that neither acetaldehyde nor alcohol level gave an objective measure of off-flavor development. Controlled blanching experiments showed that the fermenting enzymes were more easily inactivated than the “off-flavor-producing” enzymes, and that most of the acetaldehyde and alcohol were produced under anaerobic conditions before and during freezing. In a study of immediate postharvest changes, Wager (1964) measured the respiration of peas in and out of pods. Although shelled peas deteriorated fairly rapidly there was little change in their respiration pattern. On the other hand, the respiration of the pod changed markedly after removal of the peas. Wager postulated that a translocation of hormone from pod to peas delayed senescence. Experimental evidence thus far indicates that the occurrence of acetaldehyde and related substances in deteriorated products may be coincidental and unrelated to the more objectionable flavor changes. Conditions which favor the development of off-flavors may also encourage fermentation, though not necessarily. Generally, the products of fermentative processes occur at quite high levels. The amounts of acetaldehyde reported in the literature are commonly of the order of 1 mmole/kg, and thus are far in excess of the putative off-flavor components, such as hexanal and TBA-reactive substances, reported at levels of between 0.01 and 0.05 mmole/kg.

D. PECTICENZYMES Among other plant enzymes, the pectic enzymes have been studied extensively because of their influence on the texture and appearance of plant products. The occurrence, effects, and applications of pectolytic activity, particularly in fruits and fruit juices, have been discussed

22

F. AYLWARD AND D. R. HAISMAN

in many reviews (e.g., Charley, 1961; Reid, 1950; Demain and PhafF, 1957). Pectinesterase, which catalyzes the deesterification of polygacturonates, brings about the gelation of soluble pectins and increases the potential cross-linking of structural pectins through divalent ions such as calcium and magnesium, leading to a firmer texture. The enzyme is structure-bound and insoluble in its natural state. It can be solubilized in the presence of salts in slightly alkaline solutions. The bound enzyme is active over a smaller pH range than its soluble form, but both show maximum activity at pH 7.5 (Jansen et al., 1960). The optimum pH for the associated enzyme, polygalacturonase, is 2.5 to 4.5, depending on the substrate (Pate1 and Phaff, 1960a,b). The innate pectinesterase activity of tomatoes has been used to improve the texture of the canned product; there is some evidence that short heat treatments (such as a 30-second blanch) activate the enzyme in situ (Hsu et al., 1965). Pectic enzymes are not inhibited by sulfur dioxide, and can show considerable activity at temperatures as low as -12°C (Doesburg, 1951). E. CHLOROPHYLLASE Chlorophyllase hydrolyzes chlorophyll to a chlorophyllide and phytol, converting it from a fat-soluble to a water-soluble pigment. Its influence on retention of the natural color of green vegetables during processing has not been investigated. The enzyme is localized in the chloroplasts, and is easily solubilized and activated by treatment with trypsin (Boger, 1965). Holden (1961) obtained a soluble preparation from sugar-beet leaves. She found it was most active in sugar beet, peas, beans, wheat, and barley. Brussels sprouts contained only a low level of activity.

F. ENZYMES OF AMINO ACID METABOLISM Eriksson and von Sydow (1964) examined the effect of postharvest treatments on the levels of glutamic, y-aminobutyric, and aspartic acid in green peas. When peas were damaged during harvesting, y-aminobutyric acid was formed through the action of glutamate decarboxylase. The concentration of this acid increased with the time the peas were held after vining, indicating its possible value as an index of quality. The glutamic acid concentration in the peas was maintained by proteolytic enzymes and aspartate aminotransferase

PLANT-TISSUE OXIDATION SYSTEMS

23

acting on aspartic and a-ketoglutaric acid. Enzyme activity was highest in the skins of the peas; it was postulated that, in damaged peas, changes in permeability brought enzymes and substrates into proximity.

IV. OXIDATIVE AND OTHER CHANGES IN LIPIDS There is now substantial evidence that changes in the lipids of food components may play an important, sometimes dominant, role in deterioration in the quality of foodstuffs. Our knowledge of lipid chemistry and biochemistry has advanced rapidly in recent years, both because of more workers in this field and because of the availability of new analytical techniques - in particular, chromatographic methods. Various standard textbooks and monographs (e.g., Witcoff, 1951; Eckey, 1954; Lovern, 1955; Deuel, 1951, 1955, 1957; Hilditch and Williams, 1964) give authoritative reviews of lipids in terms of their types, composition, and distribution in plants, animals, and microorganisms. It has long been recognized that two major types of lipids are widely distributed in plant and animal cells, namely the mono-, di-, and triglycerides, and the phospholipids (such as phosphatidyl choline), containing glycerol, fatty acids, phosphoric acids, and a nitrogenous base. Work over the past twenty years has established the existence of several other types of lipids of varying degrees of complexity. Authors frequently group within the term “lipids” the sterols and fat-soluble pigments and vitamins, which are often associated with glycerides and phospholipids in tissues, and are extracted from tissues by similar solvents. These sterols and pigments (including polyene pigments) of varying degrees of unsaturation may participate in oxidation processes in foodstuffs. The glycerides and phospholipids contain a range of fatty acids which differ in the number of carbon atoms and in the number of unsaturated links. In plant tissues the lipid content varies greatly from one type of material to another. Large quantities are present in oilseeds, and substantial amounts in cereals. Vegetables and fruits may contain only small amounts (perhaps less than 1% of the wet weight). The fact that the lipids are only minor components of the common fruits and vegetables in no way detracts from their potential importance in quality deterioration. There has been an increasing interest in the location and function

24

F. AYLWARD A N D D. R. HAISMAN

of lipids in the plant cell. In this connection the protein and carbohydrate complexes of lipids are important, both in the living plant and in plant foodstuffs. Much of the earlier work on lipids (cf. Hilditch and Williams, 1964) was concerned with the nature and distribution of fatty acid components. A second stage of investigations covered the isolation of individual lipids and their characterization in terms of both fatty acids and other components. Much work has been carried out on oilseeds (texts already cited, and Markley, 1950; Aylward and Nichols, 1961, 1962). An increasing amount of effort has been devoted to cereals (Aylward and Showler, 1962a,b; Fisher, 1962; Fisher et al., 1964). Relatively little systematic work has been carried out on the common fruits and vegetables, although mention should be made of investigations by Wagenknecht (1957a,b) into the lipids of peas. Reviews of the literature on plant phospholipids (e.g., Aylward, 1956) show that proper characterization of lipids is difficult and that reliable information is scarce for most plant materials, although many gaps in knowledge are being filled by the application of newer techniques (James, 1960). In the absence of detailed information about lipids in many plant foodstuffs, the role of lipids in deteriorative processes in such foodstuffs is necessarily obscure, although much can be learned by analogy with studies on animal products (such as milk and fish). A. DEGRADATION OF LIPIDS

Changes in lipids can be brought about by different methods (see Fig. 2), which can be summarized as follows: (1)Partial or complete hydrolysis of the lipid to fatty acids and other components, followed by oxidation of unsaturated fatty acids; and (2) direct oxidation of the unsaturated acids (or other unsaturated components) in the intact lipids, followed by hydrolytic degradations. The hydrolytic processes are brought about by enzymes normally classified as lipases; the oxidation processes may be catalyzed by enzymes (in particular the enzyme lipoxygenase) or by metals and their salts or organic complexes. Among the degradation products likely to be of special importance in relation to food quality are (1) short-chain ( <12C) volatile fatty acids present in some intact lipids and liberated by hydrolysis; (2) other volatile substances (such as acids and aldehydes) formed by oxidation of fatty acids or polyene components; and (3)nitrogenous or other nonfatty components (e.g., from phospholipids).

PLANT-TISSUE OXIDATION SYSTEMS

Action of lipases- stepwise hydrolysis

J

Mono- and diglycerides

t

Phosphatidic acids

25

Autoxidation TY--..L

Nitrogenous bases

Chain length reduced by one carbon atom

.. . .

ted and saturated

,

Fatty acids \

Chain length reduced by two carbon atoms

\

\ 1,4-Pentadiene structure attacked 1,3-Diene-5-hydroperoxide produced

FIG.2. T h e degradation of lipids.

Most of the work on lipases has been in connection with animals rather than vegetables. Studies on vegetable lipases include those of Hanahan and Chaikoff (1947a,b, 1948) on the degradation of phospholipids from carrots and cabbage, and of Long et al. (1962) on phospholipase D from cabbage. It is probable that several lipase systems exist, some of which are selective, preferentially attacking either the a or p fatty acid chains of triglycerides. There is some evidence that the enzymes in animal tissue are activated by freezing and can operate at moisture levels down to 1.5% (Lea, 1961a). The general outlines of the biological oxidation of lipid materials have been well defined (e.g., Mahler, 1964). Probably the most important degradative pathway is the @-oxidationspiral, which involves coenzyme A, NAD+, and magnesium ions and shortens the fatty acid chain by two carbon units in successive cycles to produce acetyl coenzyme A. The p-oxidative enzymes have been shown to be present in both the mitochondria and the soluble proteins of plant cell homogenates, and the acetyl coenzyme A which is produced is subsequently consumed in the tricarboxylic acid and glyoxylate cycles. The longer chain fatty acids (C13-C18)can also be degraded by a-oxidation through a two-enzyme sequence, reducing the chain length by one carbon atom per cycle. Both saturated and unsaturated acids are susceptible to this attack (Martin and Stumpf, 1959; Hitch-

26

F. AYLWARD A N D D. R. HAISMAN

cock and James, 1963).The reaction requires continuous generation of hydrogen peroxide, such as might be provided by the action of glycolate oxidase (widespread in plant tissues). The degradation proceeds through the action of a specific fatty acid peroxidase followed by a long-chain aldehyde dehydrogenase in the presence of NAD+. The enzymic oxidative degradation which has received most attention in relation to deterioration of the quality of foods is that due to the enzyme lipoxygenase.

B. LIPOXYGENASE Bergstrom and Holman (1948) reviewed the chemistry and properties of lipoxygenase, an enzyme which is distributed widely, particularly in legumes, potatoes, tomatoes, and various herbs. According to the earlier literature it displays maximum activity at pH 9.0. No known prosthetic group or cofactor is involved in lipoxygenase catalysis, but there have been reports of an activating agent in seeds, which may in fact be an emulsifier assisting close approach of enzyme and substrate. The enzyme is specific in attacking only 1,Cpentadiene structures, e.g., it attacks linoleic, linolenic, and arachidonic acids, but not oleic acid (Dillard et al., 1961). The product of the action of lipoxygenase is an optically active cis-trans-conjugated hydroperoxide (Privet? et al., 1955). At one time it was thought that the enzyme attacked free acids only, but now it is accepted that it will act also on triglycerides and other esters. Koch et al. (1958) isolated two groups of lipoxygenases from soybeans, one of which was active toward triglycerides and the other toward free acids. Dillard et al. (1960) showed both triglyceride and fatty acid types of activity in beans, peanuts, and peas, and found that the optimum pH varied with the substrate. Siddiqi and Tappel (1956) identified a lipoxygenase in peas which had maximum activity at pH 6.9 at temperatures between 0" and 15°C. Surrey (1964) introduced an improved spectrophotometric method for the estimation of lipoxygenase activity, and found pH optima for maximum activity ranging from pH 5.5 to 7.0, depending on the substrate. The discrepancies in the reported effects of pH on the activity of the enzyme are probably due to variations in the ionic strength of the reaction medium, Ames and King (1966) found that the pH profile of lipoxygenase activity varied between pH 5.5 and 10.0, depending on the type of medium used.

PLANT-TISSUE OXIDATION SYSTEMS

27

The enzymic reaction is inhibited by antioxidants such as a-tocopherol and dibutylhydroxytoluene and to some extent by surface-active agents such as Triton X-100 (Dillard et al., 1961). Blain and Shearer (1965) found that long-chain polyacetylenic acids were potent competitive inhibitors, in contrast to nordihydroguaiaretic acid, which they found prolonged the induction period. A natural inhibitor of lipoxygenase activity, found in peanut testa, has also been reported (Narayanan et al., 1963).

c. AUTOXIDATIONOF LIPIDS As anyone with experience in lipid studies will testify, constant and elaborate precautions are necessary to guard against autoxidation. The oxidation of unsaturated fats in the presence of molecular oxygen proceeds through free-radical chains and is thus autocatalytic (Lundberg, 1962).It is accelerated by heat and light and catalyzed by heavy metals and their salts and organometallic compounds; by all oxidative enzymes; by hemoglobin and all hematin compounds; and by photochemical pigments. Autoxidation reactions are readily distinguished from lipoxygenase activity because: (a) they are quite unspecific; (b) they are autocatalytic; and (c) they yield oxidation products which are not optically active. The catalytic action of metals and their compounds has been reviewed by Ingold (1962). Examples of simple hematin compounds acting as oxidative catalysts are quoted by Lemberg and Legge (1949); the hematin compound is destroyed, indicating that this is a peroxidative reaction. Tappel (1962)states that all naturally occurring hematin compounds catalyze the oxidation of unsaturated lipids and other olefins. The hematin compounds hemoglobin, myoglobin, hemin, and cytochrome c all have similar catalytic activities (Tappel, 1955). Hematin catalysis appears to be unspecific, requiring only an unsaturated compound which forms a peroxide. The rate of oxidation is proportional to the square root of the catalyst concentration. Hematin-catalyzed linoleate oxidation has a relatively low activation energy of 3 to 5 Kcal/mole, of the same order as lipoxygenase catalysis. The occurrence of cytochrome systems in vegetable tissue, similar to the cytochromes a, b, and c of animal tissue, was demonstrated by Lundegardh (1954),and Butler and Baker (1965)have found an active ferri-hematin compound in peanuts. Peroxidase and catalase, both containing hematin prosthetic groups, are widespread in vegetable tissue, so that this oxidative pathway could be of considerable im-

28

F. AYLWARD AND D. R. HAISMAN

portance. Blain and Styles (1961) attempted to elucidate the function of hematin compounds in soya extracts in p-carotene destruction. They concluded that carotene was destroyed by a coupled oxidation using linoleate hydroperoxide and catalyzed by hematin compounds. In the absence of p-carotene, cytochrome c promoted the formation of conjugated diene hydroperoxides. The relative concentrations of catalyst and fatty acid may be critical in this process. Lewis and Wills (1963) reported the inhibition of autoxidation by high concentrations of hematin proteins, which catalyzed peroxidation within a certain concentration range. D. DECOMPOSITION OF HYDROPEROXIDES As indicated above, hydroperoxides can be formed either through the action of lipoxygenase or by autoxidation. Lea (1962) outlined decomposition routes of hydroperoxides (Fig. 3), and Keeney (1962) showed how aldehydes, alcohols, ketones, epoxides, and esters are produced by free-radical chain reactions. Free radicals have been detected during the action of soybean lipoxygenase (Walker, 1963), and in lyophilized lipid-rich foods in the presence of oxygen or after heating to 100°C (Munday et al., 1962). The production of dec-1-yne, a compound with a potent off-flavor, together with dec-1-ene and n-decanol during the autoxidation of soybean and cottonseed oils has Lipid component

I

oxidation

Hydroperoxide

/

further oxidation, polymerization, or fission

Aldehydes, acids, ketones, hydroxy- and epoxycompounds, polymers causing "Off" odors and flavors and various secondary reactions

coupled oxidations Destruction of vitamins, pigments, and other constituents

FIG. 3. Formation and decomposition of hydroperoxides. Formation: catalyzed by lipoxygenase, oxidative enzymes, hemes, pigments, and metals and their salts. Decomposition: catalyzed by metals, hemes, and oxidative enzymes.

29

PLANT-TISSUE OXIDATION SYSTEMS

also been reported (Smouse et al., 1965). The decomposition can be accelerated by heat. Lea and Hobson-Frohock (1965), using autoxidized sunflower and linseed oils, showed that Iinolenate peroxides decomposed more readily than linoleate peroxides under the action of heat and produced a higher proportion of volatile carbonyl compounds. Apart from the fatty acids, many other substances, such as pigments, vitamins, and proteins, are susceptible to free-radical action once this is initiated. Thus oxygen-labile compounds such as vitamin A and p-carotene are readily cooxidized, and amino acids such as histidine, serine, cystine, and methionine can be destroyed. Glycosides and aglycones have been found to break the chain reaction in the hemecatalyzed oxidation of lipids (Pratt and Watts, 1964; Pratt, 1965). Mapson and Moustafa (1955) demonstrated the coupled oxidation of glutathione in the presence of linoleic acid by a lipoxygenase from pea seeds. The decomposition of hydroperoxides into free radicals is catalyzed by many substances, and, once more, heavy metals and hematin compounds constitute an important catalytic group. Mikhlin and Bronovitskaya (1948) observed that cytochrome c peroxidase could utilize the hydroperoxides produced by the action of lipoxygenase on linoleic acid. Maier and Tappel (1959a) measured the comparative catalytic activity of hematins and other metal catalysts in decomposing linoleate hydroperoxide, with results shown in TabIe IV. They also (1959b) examined the products of the heme-catalyzed oxidation of unsaturated fatty acids, and found the concentrations of catalyst and substrate to be critical.

TABLE IV CATALYSIS OF DECOMPOSITION OF LINOLEATEHYDROPEROXIDE Catalvst Catalase Hemoglobin Hematin Cytochrome c Peroxidase Ferric triethylenc tetramine Manganic protoporphyrin

Rate constant

36 29

4.8 1.7 0.22 0.45 0.17

30

F. AYLWARD A N D D . R. HAISMAN

Banks et al. (1961) studied inhibition of the decomposition of methyl linoleate hydroperoxide by different concentrations of cytochrome c. O’Brien (1966, 1967) examined the kinetics of the reaction between linoleic acid hydroperoxide and cytochrome c and other heme catalysts at various pH values, and postulated that the hemoprotein coordination bonds are loosened to expose the hematin ring. The mechanism of destruction of carotenoids is still obscure. It has been attributed to the action of free radicals produced during the chain oxidation of fats, but the oxidative bleaching of carotene in sugar-beet chloroplasts appears to be independent of the peroxidation of the unsaturated fats present (Lea, 1961b). Dahle (1965)suggested that lipoxygenase activity was a major factor in carotene destruction in the milling of semolina, but found that the destruction was dependent on the concentration of free fatty acids, which are the principal substrate for the lipoxygenase. Friend and Nakayama (1959) measured the destruction of carotenoids in the chloroplasts of different leaves and postulated the existence of two differently located enzyme systems. Friend and Mayer (1959) found that the action of chloroplasts on crocin (the glycoside of crocetin, a polyene dicarboxylic acid) was due to aerobic oxidation catalyzed by a metalloprotein. Reviewing the oxidation of carotenoids in green plant tissue, Friend (1961) noted the loss of p-carotene from frozen unblanched spinach and examined degradation mechanisms. The coupled oxidation of &carotene with linoleate was catalyzed by lipoxygenase and ferrous phthalocyanin; epoxides, hranoxides, and conjugated polyene aldehydes were produced. Friend concluded that two different carotenoid-destroying enzyme systems were operating, with pH optima of 4.8 and 7.5. Since one system was inhibited by cyanide, it probably included a hemoprotein. There is some evidence that hydroperoxides can be enzymically decomposed (e.g., Mikhlin and Bronovitskaya, 1948). Gini and Koch (1961) found that soya-flour extracts contained a heat-labile factor which accelerated the breakdown of lipoxygenase-formed hydroperoxides and had many of the properties of a peroxidase. It was less heat-stable than lipoxygenase, had an optimum pH of 8 to 9, and was activated by storage at 4°C. Frankel (1962) reviewed the properties of various hydroperoxides, including their isolation by molecular distillation, for example, and their characterization. Fat hydroperoxides can be relatively stable substances that, when formed, can persist for relatively long storage periods. This observation may provide an explanation of delayed changes in food quality during storage (see Section IV,F).

PLANT-TISSUE OXIDATION SYSTEMS

31

E . ANTIOXIDANTS Extensive studies have been made of the use of antioxidants in preserving edible oils and fats (Tappel, 1961), but their effect on deteriorative changes in preserved vegetables has not been fully explored. Mention has already been made of various inhibitors of lipoxygenase activity; similar compounds effectively inhibit the heme-catalyzed oxidation of unsaturated fatty acids (Tappel, 1954). The effect of the naturally occurring inhibitors, particularly cy-tocopherol, on oxidative changes during the storage of plant materials may have considerable significance, particularly since the level of vitamin E in different organs of the plant is known to fluctuate during growth and according to season (Sironval and El Tannir-Lomba, 1960). Rhee and Watts (1966b) investigated the effect of various additives on lipoxygenase activity in different systems and found that propyl gallate, turnip-green extract, and sodium tripolyphosphate retarded the oxidation of pea lipids. They also found evidence (1966~)of a certain amount of natural antioxidant activity in plant systems. Pratt (1965) attributed significant antioxidant activity to the flavone glycosides and cinnamic acid derivatives in onions, peppers, and potato skins. The presence of water inhibits oxidation in freeze-dried material, and this has been demonstrated in the autoxidation of methyl linoleate with and without metal catalysis. Amino acids were also effective in prolonging the induction period and reducing the rate of oxidation (Maloney et al., 1966; Labuza et al., 1966; Karel et al., 1966).

I?. LIPID OXIDATIONIN RELATION

TO

FOODQUALITY

Some instances of food deterioration during storage can be explained by assuming that hydroperoxides are formed before or during processing and then decompose by a free-radical path, gradually producing rancid odors and flavors. As already mentioned, some peroxides have been shown to be relatively stable compounds and may not decompose for some weeks or months (depending, perhaps, on the diffusion of a particular catalyst). Consequently, if this theory is correct, eventual deterioration of quality may be long delayed. Koch (1962) showed that flavor deterioration in precooked dehydrated beans was due to lipoxygenase activity while the beans were soaking, prior to cooking. It has been postulated that peroxy free radicals, either produced by lipoxygenase or generated by heat processing, propagate chain reactions that degrade chlorophyll and

32

F. AYLWARD A N D D. R. HAISMAN

pheophytin (C.S.I.R.O., 1962- 1964). This theory accounts for the deleterious effect of both over- and underblanching. The same report noted, however, that free fatty acid, peroxide, or diene content could not be correlated with off-flavor development. In fuller publications, Walker (1964a,b) showed that the oxidative degradation of chlorophyll and pheophytin in frozen French beans occurred only after 12 months storage and was coincident with an increase in fat peroxide. He thought that the time lag could be attributed to a natural antioxidant which slowly became exhausted. Wagenknecht and Lee (1958) have examined the part played by lipids in the deterioration of frozen peas. They showed that the addition of lipase and lipoxygenase preparations to blanched peas caused off-flavors and chlorophyll breakdown. Lipids extracted from frozenstored unblanched peas contained a high proportion of unsaturated carbonyl compounds not present in fresh or cold-stored blanched peas (Lee, 1958). They were able to isolate from peas a lipase which effectively degraded chlorophyll to pheophytin (Wagenknecht et al., 1958). Using enzymes isolated from fresh peas, they showed that the addition of these enzymes to blanched peas led to the degradation of chlorophyll and produced some flavor changes during storage (Lee and Wagenknecht, 1958). They concluded that the mechanism of offflavor production during frozen-storage was complex and that several enzymes were involved, including lipase and lipoxygenase, acting in sequence. In investigations of underblanched corn on the cob, Wagenknecht (1959) detected residual lipoxygenase activity which appeared to be partly responsible for off-flavor production. Lee and Mattick (1961) showed a considerable breakdown of phospholipids and losses of unsaturated fatty acids from the triglycerides in unblanched peas stored one year at -17.8"C. Pendlington (1962) presented evidence for lipid breakdown, with a corresponding increase in choline and phosphate, in unblanched peas, but concluded that the mechanism of off-flavor production remained obscure. In further studies of frozen unblanched peas, Bengtsson and Bosund (1966) found that, between -20" and -5"C, the temperature coefficients remained about the same for the rates of production of offflavors and formation of free fatty acids, and that both were much lower than would be expected if the degradative changes were nonenzymic. Holden (1964) reported that the chlorophyll-bleaching activity of legume seed extracts is correlated with the action of lipoxygenase and (1965) appears to arise from a chain reaction involving the

PLANT-TISSUE OXIDATION SYSTEMS

33

peroxidation of long-chain fatty acids which are subsequently decomposed by a heat-labile factor. Lea and Parr (1961) described the off-flavors which arise during oxidative deterioration of the lipids in crude leaf protein, dividing them into two categories: polyunsaturated acids of galactoglycerides, or phospholipids which give “fishy” flavors; and carotenoids and chlorophylls, which produce “violet” or “haylike” odors. Some aspects of quality deterioration attributed to lipids may be nonenzymic in origin. Thus, Burton and McWeeny (1963) implicated phosphatides, such as lecithin, in nonenzymic browning reactions. In contrast, experiments using the 2-thiobarbituric acid (TBA) test as a measure of lipid oxidation in frozen peas appeared to establish that rancidity was not a primary cause of flavor deterioration (Rhee and Watts, 1966~). The TBA test depends on the formation of a red pigment when thiobarbituric acid reacts with oxidized fat. In many cases the intensity of the pigment has been found to be proportional to the oxidative degradation which has taken place. The reactive material is malonaldehyde, which is produced from the hydroperoxides of unsaturated fatty acids containing three or more methylene-interrupted double bonds (Dahle et al., 1962). Oleic and linoleic acid do not react, although it is reported that malonaldehyde may be a secondary oxidation product in other lipid oxidations (Lillard and Day, 1964). It is unlikely that malonaldehyde exists in the free state in oxidized products; it is probably combined with other food components, particularly some proteins (Crawford et al., 1967). Most workers have agreed on the necessity of using acid extraction of the food material to obtain maximum color development. Like other reactive intermediates, malonaldehyde is itself destroyed during the later stages of oxidation, and, in view of this and the various side reactions which can occur (Tarladgis et al., 1962), the results obtained with the test must be treated cautiously. Rhee and Watts (1966a) used the TBA test on plant materials and suggested that a comparison of the TBA values of tissues blended with and without acid (to inactivate lipoxygenase) might provide a useful measure of the “lipid oxidation potential.” As already mentioned, when the test was applied to stored frozen blackeye peas, whether raw, blanched, or cooked, the TBA numbers were all less than the threshold of 1.0 established for rancid odors in animal products, even though the raw peas were judged unpalatable by sensory methods (Rhee and Watts, 1966~). Rancid peas were found to have a TBA number of 7.8.

34

F. AYLWARD AND D. R. HAISMAN

V. THERMAL AND OTHER ENVIRONMENTAL FACTORS MODIFYING ENZYME ACTIVITY The activity of an enzyme, whether considered in its natural environment in the living cell or in isolated tissues or extracts, is dependent on many factors, and much work has been carried out on activating and inhibiting agents in various enzyme systems, as well as on the effects of changes of pH, temperature, and other conditions. Some of the factors that have been studied are summarized in Table V. It is well known that many enzymes consist of two linked components, the protein moiety (or apoenzyme) and a prosthetic group (coenzyme). Destruction or modification of activity can be brought about by dissociation of this complex or by changes in either component. On this basis it is to be expected that enzyme activity will be lost through conditions that lead to the destruction of proteins. The prosthetic groups are all molecules of relatively low molecular weight; many of the oxidizing enzymes embody metallic (e.g., Fe or Cu) compounds in the prosthetic group, and in such cases reagents which will react with these metals would be expected to reduce activity. In view of the importance of thermal processes in food preservation, the effects of heating and cooling on plant enzymes will be considered in some detail before discussing the other factors outlined in Table V.

TABLE V hfODIFICATION OF ENZYMEACTIVITY 1. Methods of inactiuation

(a) Destruction or dissociation of protein-prosthetic group (apoenzyme/coenzyme) complex (b) Changes in protein (apoenzyme) leaving complex (c) Changes in prosthetic group (coenzyme) intact

1

2. Factors modifying actiuity (a) Temperature (b) PH (c) Water (d) Sugar and related compounds (e) Multiple forms (0 Adsorbents of different types (inert substrates, active substrates) (g) Action of specific inhibitors, e.g., agents affecting the prosthetic groups (coenzyme)

PLANT-TISSUE OXIDATION SYSTEMS

35

A. THERMAL INACTIVATION-GENERALPRINCIPLES 1 . Effect of Temperature on Enzyme Activity In any enzyme system, two independent processes are simultaneously accelerated by an increase in temperature: the catalyzed reaction; and thermal inactivation of the enzyme. The rate of the catalyzed reaction normally follows the general pattern of other chemical reactions; the temperature coefficient, Qlo, defined as the factor by which the velocity is increased on raising the temperature by 10"C, is normally between 1 and 2 (Dixon and Webb, 1958). The temperature coefficient for enzyme inactivation is usually very much higher (see Section V,B,7). As a consequence of these two different temperature coefficients, an optimum temperature for the reaction is observed. Below the optimum, changes in temperature have the greater effect on the catalyzed reaction, whereas above it the inactivation of the enzyme becomes the predominant factor. Both coefficients are highly dependent on the environment of the system (e.g., the ionic strength and pH). The optimum temperature for most enzyme reactions usually lies between 30" and 50°C.

2. Temperature Coefficients of Reactions As noted above, the temperature coefficients for enzyme reactions usually lie between 1 and 2, but exact measurements have been made on comparatively few systems. Sizer and Josephson (1942) measured the kinetics of three enzyme systems over the range -70" to +50"C, and found that the rates conformed to the Arrhenius equation:

d In k' - E dT RP (where k' is the specific rate constant, T is the temperature in degrees Kelvin, R is the gas constant, and E is the energy of activation) except for a discontinuity at the melting point of each system. Abnormal temperature coefficients were obtained below the melting point; lipase had a Q," of 1.5 in the range 0" to 50"C, changing to 26 in the range -70" to 0°C. Maier et al. (1955) studied phosphatase- and peroxidase-catalyzed reactions at subzero temperatures, and also found pronounced deviations from the Arrhenius equation. This point is taken up later (Section V,C).

36

F. AYLWARD AND D. R. HAISMAN

3. Znactivation of Enzymes by Heat Like all proteins, enzymes have a definite three-dimensional structure, maintained by a multitude of secondary bonds (hydrogen bonds and London dispersion forces), which is easily disrupted by thermal or chemical attack. The processes of denaturation of proteins and inactivation of enzymes appear to comprise a structural breakdown of this sort. Breaking bonds, even weak secondary bonds, needs energy, and only those molecules possessing energy much above the average can rearrange to the denatured or inactive conformation. At a fixed temperature, the Maxwell-Boltzmann distribution law shows that, for any random assembly of molecules, the number, ni,having an energy in excess of a certain value is proportional to the total number of molecules,

!!!= e-EIRT n

(2)

Hence, the rate of denaturation or inactivation is proportional to the concentration of the unaltered protein or enzyme. Let this be c ; then

-dc -dt

- k‘c

(3)

Integrating between times t , and tz In 5 = k’(t2- t , ) C2

(4)

If x is the fraction of activity remaining after heating for t min at temperature T

2.3 log x = k’t

(5) and k’, the specific rate constant, can be obtained from a plot of log x against t . 4 . Practical Units The thermal inactivation of enzymes is in many ways analogous to the thermal destruction of bacterial spores, and, when estimating degrees of enzyme inactivation, food technologists have found it convenient to adopt the conventions and mathematical treatments introduced by Ball (1923) for the evaluation of sterilizing processes. Ball (1943) applied these methods in a study of phosphatase inactivation during the pasteurization of milk.

PLANT-TISSUE OXIDATION SYSTEMS

37

The decimal reduction time, D, defined as the time taken for a 90 % decrease in the activity of the enzyme, can be obtained from the relationship

D

= 2.3/k'

D values provide a practical measure of the heat resistance of an enzyme system at a particular temperature. A related practical parameter that is used more widely is the FT value, the time required to reduce the activity to a desired level, again at a particular temperature. The chosen level of activity usually corresponds to the detection limit of the enzyme system in question (say 1 % or 0.1% of the original activity). A complete description of the heat resistance of an enzyme system requires that we know not only the rate of inactivation at one temperature, but also the way in which this rate varies as the temperature is changed. Experimentally, for both enzymes and bacterial spores it is found that, over short temperature ranges, plots of the logarithms of the rates of destruction against either the temperature or the reciprocal of the absolute temperature approximate to straight lines (Gillespy, 1948). log D

= a - (t°F/z)

(7)

or log D = (M/T°K)- b

(8)

where a, z, M , and b are constants. The former equation is very useful in practice, and defines the factor z as the temperature change in degrees Fahrenheit necessary to produce a tenfold change in the rate of inactivation. Thus, the parameters D or F , together with z, completely describe the heat inactivation of an enzyme system over a restricted range of temperature.

5. Thermodynamics of lnactivation Although Eq. (7) has practical value, it cannot be justified theoretically. On the other hand, Eq. (8), which is of the same form as can be derived from strict postulates the Arrhenius equation [Eq. (l)], and, properly treated, can yield thermodynamic constants which illumine the mechanism of inactivation. Unfortunately, in practice the full potential of this treatment is seldom realized, because of the difficulty of applying the necessary restrictions to the system. The earliest relationship between rate of inactivation and tempera-

38

F. AYLWARD A N D D. R. HAISMAN

ture was derived from the collision theory applied to the conversion of normal molecules to the activated transition form, which preceded the final structural rearrangement:

k' = C e - A / R T

(9) where A came to be identified with the energy required to activate the molecule, and C was a constant embodying frequency and probability factors. In practice, both A and C were found to vary with temperature, and various modifications have been suggested to produce a better fit between the theoretical equation and practical data. The advent of statistical mechanical treatment of the problem in terms of absolute reaction rates (Stearn, 1938; Eyring and Stearn, 1939), however, showed that the collision theory amounted to a special case of a more general equation:

k ' = K -kT e h

-F*IRT

where K is a transmission coefficient (usually taken as unity), k is the Boltzmann constant, h is Planck's constant, F' is the Gibbs free energy of activation, K" is the equilibrium constant between activated complex and reactant, and y" and y o are activity coefficients. Thus, discrepancies between experimental data and theory could always be attributed to neglect of the correct activity coefficients. Using the transformation

AF'

= AH'

-

T AS'

(11)

(with H" and S' denoting enthalpy and entropy of activation respectively) Eq. (10)can be written

from which the usual Arrhenius equation (1) can be derived. The free energy, AF', the enthalpy, AH', and the entropy, AS', of activation can be calculated from the activation energy, AE', and the rate constant, k'. The values obtained will depend on the standard states which are adopted. Provided the inactivation rates are measured with due regard to the activity coefficients of the reactants and the activated complex, they will vary only with respect to temperature and pressure. As already mentioned, these requirements are most often

PLANT-TISSUE OXIDATION SYSTEMS

39

experimentally unrealizable, so that the thermodynamic constants refer to the unspecified standard states under which the measurements happen to be made.

6. Znterconversion of Parameters In the literature, heat inactivation data have been expressed in a variety of forms. The following relationships have been used in comparing results from different sources:

D = 2.303Ik' Qlo= antilog

(6)

10E" 2.303 X 1.987 X T 2

where T is the average temperature in degrees Kelvin, and E" is in calories.

z=--

18 T2 - 8.237 log Qio E

B. THERMAL INACTIVATION OF OXIDIZING ENZYMES EXPERIMENTAL DATA

1 . General Considerations The earlier literature on the heat resistance of different enzymes has been surveyed by McConnell (1956) and Leeson (1957). Both workers concluded that many of the published observations were of limited value in that experiments were often confined to one temperature or were only semiquantitative, and the modifying action of pH and other environmental conditions were usually not taken into account. McConnell tabulated all the data from which thermal inactivation curves could be drawn, recalculating, where necessary, the constants in terms of F7. and z values to facilitate comparison. The enzymes studied included the pectic enzymes, peroxidase, o-diphenol oxidase, catalase, ascorbate oxidase, and phosphatase. In vegetables, peroxidase was found to be by far the most heat-resistant enzyme. In fruits, where conditions are more acid, peroxidase was less stable, but still the most difficult enzyme to inactivate; ascorbate oxidase displayed almost as great a resistance to heat under these conditions. Pectic enzymes required, on average, long periods for destruction at moderate temperatures, but the rate of inactivation increased rapidly as

40

F. AYLWARD A N D D. R. HAISMAN

the temperature was raised. Both McConnell and Leeson emphasized that at high temperatures, processes adequate for sterilization purposes may not wholly inactivate the more heat-resistant enzymes. A selection of results from more recent experimental work on oxidizing enzymes is presented below. The information is presented first in terms of individual enzymes, and secondly for different systems. The empirical parameters D,F , and z have been chosen to express the results. Thermodynamic data, which must be regarded with greater caution (for the reasons already outlined), are presented in a later section.

2. Peroxiduse Many workers have studied the thermal inactivation of peroxidase under conditions corresponding, on the one hand, to industrial processes and, on the other, to precise examinations of highly purified systems. Thus, Lopez et ul. (1959)investigated the effect of blanching time (at 100°C) on peroxidase and catalase activity in peas, and found that 70 to 100% of the activity was destroyed in 1to 3 minutes. Baker and Goldblith (1961) investigated the effect of both heat and ionizing radiations on peroxidase activity in green beans. Ionizing energy had a measurable effect on enzymic activity, but this was insignificant compared with the effect of heat. Kyzlink and Chytra (1959) determined the thermal death rates of peroxidase in fourteen kinds of fruit and vegetables, and found large variations in the heat resistance of the enzyme systems, particularly among fruits. They emphasized the importance of the pH of the system, which may in fact be the only factor determining the thermal resistance of enzymes during the ripening of fruits. They found that the heat resistance of the enzyme was greater when the enzyme concentration was high. Addition of salt often decreased the heat resistance of the system. Wilder (1962) also found that the ionic strength of the medium had a profound effect on the heat inactivation of purified peroxidase in buffered solutions. Joffe and Ball (1962) measured the thermal inactivation of pure horseradish peroxidase precisely, and summarized the results of previous workers in a comparative survey of heatinactivation data. This information is presented in Table VI, together with some later work, they did not include. F values refer to the heating necessary to reduce the enzyme activity to below the limits of detection. The exceptional heat resistance of peroxidase is evident from the fact that nearly all the measurements have been made at temperatures above the boiling point of water. The great variations in

TABLE VI THETHERMAL INACTIVATION OF

Medium HRP in PO, buffer HRP in PO, buffer Green beans, aqueous extract Turnips Yellow turnips White turnips Broccoli Green beans Peas Peas Sweet corn, wholekernel heat-labile fraction heat-stable fraction heat-stable fraction Sweet maize, aqueous extract Sweet maize, wholekernel

Highest experimental temperature

z

(OF)

(OF)

FZSO (min)

302 302 250 270 300 300 300 300 300 260

49.8 64.3 47.0 46.0 72.5 57.5 63.0 86.0 48.0 52.0

25.7 3.0 11.3 3.1 2.1 1.4 0.6 6.0 7.7

200 200 290 270 310 270 310

87.0 54.0 65.0 40.0 86.0

Fsoo

(min)

PEROXrDAsE

Dzoo (min)

Dzm (min)

Investigators

7.5

]

0.4 1.8

I

Joffeand Ball (1962)

Zoueil and Esselen (1959)

Esselen (unpubl.) quoted by Joffe and Ball (1962) Farkas et al. (1956) Guyer and Holmquist (1954)

Yamamoto et al. (1962)

0.2 11.0

ca 4.0 1.78 0.26 7.0 2.1

i

Vetter et al. (1958)

42

F. AYLWARD A N D D . R. HAISMAN

F and D values are almost certainly a consequence of differences in pH, ionic strength, cellular materials, and assay and heating techniques. The temperature coefficient, z, is affected less by such vicissitudes, so there is closer agreement about its value, which on average is about 60°F. An investigation by Yamamoto and co-workers (1962) is of great interest since they demonstrated the existence of a peroxidase fraction (about 5% of the total activity) with abnormal thermal stability. The rate of inactivation of this heat-resistant form was fifty times lower than that of the heat-labile fraction. 3. Catalase The thermal lability of catalase is well established, and there have been several semiquantitative determinations of its inactivation by different blanching treatments (e.g., Lopez et aZ., 1959), showing that its heat resistance is low. Basic studies of the inactivation process have been confined principally to catalases isolated from animal and microbiological sources, and there are indications that plant catalases may show some differences in behavior. Frazer and Kaplan (1955), studying yeast catalase, found marked differences in behavior between the native (Lea,in situ)enzyme and crystalline preparations or aqueous extracts. Unaltered catalase in the yeast cell was less active but more heat-resistant than extracted or altered catalase. Deutsch (1951) found that the heat inactivation of crystalline horse erythrocyte catalase did not follow first-order kinetics, a fact he attributed to the presence of various species of catalase of differing thermal lability. In a basic investigation of a plant catalase (from spinach), Sapers and Nickerson (1962b) found that the rate of inactivation by heat was not first-order. They postulated the presence of a heat-labile inhibitor. Kinetic constants derived from the data of Sapers and Nickerson are shown in Table VII. All investigators are agreed on the relative ease with which catalase is inactivated.

4 . Lipoxygenase There has been only one basic investigation of the inactivation of lipoxygenase by heat (Farkas and Goldblith, 1962). The enzyme, a purified soybean lipoxygenase, showed a typical protein-inactivation response to heat treatment, varying with the pH of the system. The enzyme was most stable between pH 5 and pH 7. Heat resistance was also enhanced up to tenfold by the addition of 20% pea solids to the system. Under the same conditions the enzyme was quite sensitive to

43

PLANT-TISSUE OXIDATION SYSTEMS

ionizing radiation. Kinetic constants calculated from the heat inactivation data are shown in Table VIII.

5. o-Dipheno2 Oxidase Leeson (1957) compiled miscellaneous data from numerous investigations on thermal inactivation of the phenol oxidases. Of particular interest were results of a comprehensive investigation by Dimick et uZ. (1951)on the effects of time and temperature on the activity of the enzyme in fruit purbes. A D value of 3.5 min at 75”C, and a z value of about 10, were obtained with pear puree. The thermal stability of the enzyme was dependent on pH, but, surprisingly, the pH for maximum thermal stability varied widely between different fruits (apricots 3.9, grapes 4.5, pears 6.0, and apples 6.2). Studying the properties of the oxidase from apples, Walker (1964a) found that in solution at pH 5.0, where it displayed maximum activity, the enzyme had a half-life of 12 min at 70°C. This corresponds to a decimal reduction time of 50 min, in reasonable agreement with previous work. TABLE VII THETHERMAL INACTIVATION OF CATALASE Highest experimental temperature

z

D

Temperature for determination of D

Medium

(OF)

(OF)

(min)

(“F)

Spinach catalase purified in buffer Spinach extract Spinach extract

140 140 149

-

ca 2 22-31 ca 1

140 140 149

37 15

TABLE VIII INACTIVATION OF LIPOXYGENASE THETHERMAL Highest experimental temperature Medium

(“F)

(“F)

D (min)

McIlvaine buffer, pH 7.0 20% pea solids 20% pea solids

167 158 171

9.2 15.7 6.1

44 85 12

+ +

z

Temperature for determination of D

(“F) 149 154 163

44

F. AYLWARD AND D. R. HAISMAN

6. Other Systems Some of the experimental work on nonoxidizing plant enzyme systems will be noted for comparative purposes. The heat resistance of the pectolytic enzymes has been investigated extensively, particularly in connection with the firmness of fruits and gels and the manufacture of fruit juices. Quantitative data on heat resistance are limited, but the pectolytic enzymes, on the whole, appear to be labile and readily inactivated at temperatures over 70°C. Some peculiarities in behavior have been reported, however. Kohn et al. (1953) examined the destruction of pectic enzymes used for the clarification of apple juice, and found that polygalacturonase was rather more heat-stable than pectinesterase. The F values were 7.2 and 255 min at 65.5"C, and associated z values were 11.3and 11.5. McColloch and Kertesz (1948) isolated a polygalacturonase from tomatoes which appeared unusually stable, retaining 5% of its original activity even after boiling for 1 hour. The polygalacturonase in papaya also appears to possess a higher degree of heat resistance (Aung and Ross, 1965).The decimal reduction time at 82.2"C was found to be 10 min (pH 4.2), and the z value 11. When heat processes based on these data were evaluated, however, residual activities were higher than expected, indicating the existence of a more heat-resistant form.

7 . Comparative Thermal Stability of Diferent Enzymes Comparative values for the heat-inactivation constants from the sources already quoted are shown in Table IX. D values have been extrapolated to 176°F to facilitate comparison. Since z values are constant over only short temperature ranges, the estimates of D must be viewed with caution. The results substantiate data tabulated by McConnell (1956) and, once more, illustrate the exceptional position of peroxidase.

8. Thermodynamic Constants for Thermal Znactivation As explained in the derivation of the thermodynamic properties relating to inactivation, proper restrictions are seldom applied to the experimental system. In consequence, although it is permissible to compare the values for any property taken from one experimental set (in which standard states, although undefined, are probably constant), a comparison of results obtained in different systems may be quite misleading. Nevertheless, inspection of data available in the

45

PLANT-TISSUE OXIDATION SYSTEMS TABLE IX COMPARATIVE THERMAL STABILITYOF DIFFERENT ENZYMES z

D,16

Enzyme

(OF)

(min)

Peroxidase (horseradish) Peroxidase (green beans) Peroxidase (turnips) Peroxidase (sweet corn) Catalase (spinach extract) Lipoxygenase (+pea solids) o-Diphenoloxidase (pear) Polygalacturonase (papaya)

49.8 47.0 46.0 54.0 15.0 15.7 10 11

232 15 73 30 0.02 0.09 0.82 23

literature does reveal certain salient features which are at least qualitatively significant. The changes in total and free energy, heat content, and entropy which are usually calculated refer to the transition of the enzyme molecule from the normal to the activated state. Thus they apply only to the first part of the denaturing transformation. The whole process has been studied only in a reversible system (e.g., the denaturation of trypsin), in which an equilibrium constant for the complete reaction can be calculated. A selection of calculated thermodynamic properties for the inactivation of various enzymes is shown in Table X. One of the most striking points about the inactivation of enzymes and the denaturation of proteins by heat is the very large activation energy involved. Chemical processes requiring the same apparent energy of activation proceed at an infinitesimal rate below 100°C; for instance, the rate of depolymerization of dianthracene in phenetole, activation energy 39.4 kcal, is about 3 x lo-'* (sec-I) (MoelwynHughes, 1933). The comparatively rapid rate of enzyme inactivation is explained by the large increase in entropy involved, which counterbalances the high activation energy. The entropy of activation is visualized as the result of the fission of many secondary bonds in the protein, so that the activated molecule has a greater freedom in its spatial configuration. As might be expected, peroxidase proves an anomaly in this concept of thermal inactivation. Results from two independent investigations corroborate in showing that, for this molecule, the entropy of activation is negative; other results (e.g., Zoueil and Esselen, 1959)indicate a low positive entropy when a negative value is not obtained. On balance, it appears that the peroxidase molecule is more rigid in the

TABLE X THERMODYNAMICS OF INACTIVATION Enzyme

Temperature ("C)

Inactivation rate (sec-' x 104)

A H* (cal/mole)

A Fo (caVmole)

A So (caVmole "K)

Peroxidase (horseradish)

85

2.69

24,300

27,000

-7.39

Peroxidase (sweet corn)

85

34.9

15,700

24,900

- 26.0

Catalase (spinach)

60

14.5

60,900

23,900

+ 111

Lipoxygenase (soybean)

65

101,000

22,900

+ 242

o-Diphenol oxidase (pear)

80

100,000

24300

+212

8.71

59.1

Investigators Joffe and Ball (1962) Yamamoto et az.(1962) Sapers and Nickerson (1962b) Farkas and Goldblith (1962) Dimick et nZ. (1951)

PLANT-TISSUE OXIDATION SYSTEMS

47

activated state, which would indicate an unusual mechanism of inactivation in this case.

ACTION A T LOW TEMPERATURES c . ENZYME Although it is normally assumed that the rate of enzyme action diminishes rapidly on cooling, there have been several reports of the activation of tissue enzymes by freezing. Kiermeier (1947) found that the activities of catalase and lipase were increased after freezing and thawing in the presence of substrate, although repeating the operation had an adverse effect. Lea (1961a) also mentions the activation of lipases by freezing. Peroxidase activity in kohlrabi was stimulated eightfold by freezing (Kiermeier, 1951). It has been suggested that the activation during freezing is due to the liberation of endoenzymes from associated inhibitory constituents (Kiermeier, 1951; Sukhorokov and Barkovskaya, 1953). Some enzymic reactions appear to be accelerated by freezing. Thus, the transfer reaction between amino acid esters and hydroxylamine, catalyzed by trypsin, was twice as rapid at -23°C as at +1"C (Grant and Alburn, 1966),although in one case the pathway of the reaction changed. Such behavior is exceptional, for enzyme activity in frozen media is generally drastically reduced, being comparable to that in very concentrated solutions or dried tissues (Joslyn, 1951; Tappel, 1966). The important effect of freezing may be that the decrease in activity is not the same for all enzyme systems, so that interrelated reactions get out of balance, allowing particular intermediates to accumulate. Tappel and others (1953) found that lipoxygenase near the freezing point displayed relatively higher activity than did other systems, although the activity was sharply reduced (to 1% of the liquid system) upon actual freezing. Some enzymes are partially inactivated at low temperatures. The inactivation observed with phosphatase and peroxidase is reversible and is thought to be due to the formation of internal hydrogen bonds and associated conformational changes (Maier et al., 1955).

D. EFFECTSO F PH

AND

IONICSTRENGTH

In solution, the secondary structure of enzymes is highly dependent on pH and ionic strength, and examples of the effect of these variables on heat inactivation have already been quoted (Farkas and Goldblith, 1962; Sapers and Nickerson, 196213). Nakayama and Kono (1957)

48

F. AYLWARD AND D. R. HAISMAN

found that the rate of inactivation of sweet potato P-amylase increased as the buffer concentration was raised from 0.01 to 0.1 M and as the enzyme concentration was lowered over a fivefold range. The heat inactivation of glucose dehydrogenase is affected markedly by dimermonomer conversion, which occurs in solutions of this enzyme. Since the equilibrium between dimer and monomer depends on pH and ionic strength, the stability of the enzyme can be increased by a factor of lo6 by adding sodium chloride and changing the pH from 7.5 to 6.5 (Sadoff et al., 1965).

E. EFFECTSOF WATER Enzymic activity may persist in foods at quite low moisture levels (Acker, 1962; Blain, 1962). Peroxidase activity in solvent mixtures has been measured down to 12.5%water, and lipase activity in oats has been measurable at moisture levels between 6 and 12 %. Amylases were found to be active in the hydrolysis of glycogen in freeze-dried model systems containing meat enzymes at moisture levels of 3% or lower (Matheson, 1962). It is established that enzymes, like other proteins, are much more stable (e.g., to heat treatment) when dry, but in practice much will depend on the amounts of residual moisture present. The techniques of food preservation include not only dehydration by heat but methods (e.g., sugar and salt preservation) in which the effect is due in large part to reduction of the activity of water in the system. Sugars and other substances (e.g., lyophilic colloids) have been shown to inhibit enzyme actions in some types of experiments, and to stabilize enzyme systems in others-for example, during heat treatment. Thus, Chang et al. (1965) found that sucrose inhibited pectin methylesterase activity in papaya, and Kiermeier and Koberlein (1957) found that mono- and disaccharides could reduce the effect of heat treatment on the activity of catalase. In the presence of sugar, heating times had to be increased by factors up to 100, or temperatures raised 15"C, to inactivate the enzyme to the same degree. It seems likely that the action of sugar, at least in some cases, can be considered the equivalent of dehydration.

F. MULTIPLE MOLECULAR FORMS OF ENZYMES The idea is not new that an enzyme may occur in different forms, and it has often been used to explain discrepancies in properties in

PLANT-TISSUE OXIDATION SYSTEMS

49

comparisons of enzyme preparations isolated from different sources. Interest in the concept was stimulated by the unequivocal demonstration, in 1957, of different forms of lactate dehydrogenase, and this has led to the subsequent discovery of physically distinct forms of many of the common enzymes. The subject has been reviewed in a recent monograph (Wilkinson, 1965). The occurrence of multiple molecular forms is of both theoretical and practical importance. The different forms may display differences in substrate specificity and react differently to changes in pH or ionic strength and to heat. The properties of the five isoenzymes of lactic dehydrogenase were studied in detail by Wilkinson (1965). He found differences not only in chemical composition and physical properties but also in thermal stability, substrate specificity, response to coenzyme analogs and susceptibility to inhibitors. Moreover, the relative proportions of the isoenzymes were found to vary between different tissues and between individuals. The differences in lability between the isoenzymes have been used to devise a chemical test (Wroblewski and Gregory, 1961). Comparisons of activities before and after heating to 57" and 65°C provide approximate values for the relative proportions of labile, stable, and intermediate fractions. Peroxidases have long been known to differ in properties according to source. Horseradish peroxidase, verdoperoxidase, lactoperoxidase, and cytochrome c peroxidase have all been obtained in crystalline form, and chromatographic and electrophoretic techniques have shown that many of the crystalline preparations comprise several peroxidases (Saunders et al., 1964). McCune (1961) obtained six active peroxidase fractions from corn. Siege1 and Galston (1966) separated iso-peroxidases in peas by starch gel electrophoresis, and found that they differed in their secondary reactions. Kon and whitaker (1965) separated three peroxidases from fig latex, all more heat-stable than horseradish peroxidase. The confusion surrounding the properties of various phenol oxidases can be attributed, in part at least, to the existence of many variant forms. Hare1 et al. (1965) isolated four distinct o-diphenol oxidases from the subcellular particles of apples, and postulated that each enzyme was bound to a specific site in the cell, where it fulfilled a particular physiological function. Heterogeneous forms of this enzyme have also been recently reported in broad beans (Robb et al., 1965), tea (Gregory and Bendall, 1966), and eggplant (Sakamura et al., 1966). There is some evidence for transmutation between different forms; Jolley and Mason (1965) observed changes in the

50

F. AYLWARD A N D D. R. HAISMAN

forms of phenol oxidase from mushrooms when the enzyme was incubated for a short time in dilute buffers within a pH range of 3.8 to 10.4. Hultin and Levine (1963) isolated from bananas three forms of pectin methyl esterase, which differed in pH activity curves, response to surface-active agents, and differential temperature inactivation. One fraction could be completely inactivated by freezing to -30°C for 3 days, but was comparatively stable at 0°C and above. It appeared that the forms could be interconverted to some extent by treatment with sodium dodecyl sulfate (Hultin et al., 1966).

G . ADSORPTION OF ENZYMES ON NATURALSUBSTRATES Some of the variants in multiform enzymes may be cases where the enzyme is combined with another compound such as a protein, lipid, carbohydrate, or inorganic ion. Such a combination often greatly modifies the properties, especially the stability, of the enzyme. Since enzyme inactivation is frequently due to disruption of the protein structure through the breaking and rearrangement of secondary bonds, any compound or process which stabilizes the structure of the molecule will, to some extent, protect it against inactivation. Okunuki (1961), examining the relationship between the denaturation and inactivation of enzyme proteins, showed that inactivation, like denaturation, rendered enzymes more susceptible to attack by proteolytic enzymes. Taka-a-amylase was activated by calcium ions, which were bound to the enzyme molecule. Over a certain level, the bound calcium was found to protect the enzyme against heat inactivation and proteinase attack. London et al. (1958) related the potency of inhibitors of prostatic acid phosphatase to their structural affinities to the principal substrate, P-glycerophosphate. They found that inhibitors enjoying a multipoint attachment to the enzyme stabilized it against denaturation. Inhibitors such as chloride and sulfate, which made only singlepoint contact, had no effect on the stability of the enzyme. Adsorption of enzymes also leads to increased stability. Lilly et al. (1965) observed that chymotrypsin, ficin, and ribonuclease had a greater heat resistance when chemically bound to modified celluloses. The stabilization of glucose oxidase-catalase preparations by mixing with methylcellulose has been patented (Scott, 1961). In its natural state, pectin methylesterase adsorbed on cell walls has been found to be very resistant to heat inactivation (McDonnell et al., 1945). In a like manner, the presence of excess substrate can protect an enzyme,

PLANT-TISSUE OXIDATION SYSTEMS

51

presumably because of the increased rigidity of the enzyme-substrate complex. Taka-a-amylase was more heat resistant in the presence of starch and its hydrolysis products (Tonita and Kim, 1965), and the heat resistance of almond P-glucosidase was doubled in the presence of 0.2% or more of amygdalin, the primary substrate (Haisman and Knight, 1967). Enzymes are denatured when adsorbed at interfaces at low surface pressures (i.e., when the molecule is completely unfolded). At higher concentrations, allosteric effects may be observed, with the activity and stability depending on the conformation of the enzyme molecule (James and Augenstein, 1966). Frazer et al. (1955) found that catalase adsorbed at an oil-water interface was stabilized by lipids. The general protective action of plant constituents associated with enzymes has often been demonstrated, although the responsible factors have not been identified. Examples have already been quoted of the protection of lipoxygenase by pea solids and of catalase by spinach extract. Frequent references have been made to the stabilization of pectin methylesterase by associated tissue components, e.g., in snap beans (van Buren et al., 1962) and in apple juice (Pollard and Kieser, 1951). Manolkides (1962) attributed the heat resistance of catalase and phosphatase in milk to their association with other milk proteins. A striking example of the effect of associated constituents on the stability of enzymes has been observed with adenylate kinase, which controls the equilibrium between the adenosine phosphates: ATP + AMP

2 ADP

Colowick and Kalckar (1943) observed that this enzyme possessed an unusual stability and survived boiling with mineral acids and precipitation with trichloroacetic acid. Bowen and Kenvin (1956), however, found that purification of the enzyme drastically reduced its heat resistance. The intrinsic stabilization of enzymes and metabolic systems against extremes of temperature is demonstrated in the vegetation of the tundra and the desert, and in thermophilic microorganisms. Langridge (1963) reviewed the proposed biochemical mechanisms to account for the thermal stability of proteins in living organisms subject to extreme temperatures. Enzymes isolated from cacti have been found to be more heat-resistant than their analogs in temperatezone plants. Theories involving adsorption on cellular surfaces and combination with other proteins and metal ions have also been ad-

52

F. AYLWARD A N D D. R. HAISMAN

vanced to account for cases of unusual stability. A special mechanism appears to operate in certain bacterial spores, where the heat resistance has been shown to depend on the presence of dipicolinic acid, a substance found in large quantities in resting spores of Bacillus species (Powell, 1953; Church and Halvorson, 1959).Dipicolinic acid also retards the denaturation of bovine serum albumin (Mishiro and Ochi, 1966). There is indirect evidence for the existence of natural inhibitors, which may or may not have a stabilizing function, in association with many native enzymes. It has often been observed that the enzymic activity in tissue extracts can be stimulated by the addition of surfaceactive or other agents, or by mild heat treatments. For example, the activity of NAD pyrophosphatase in trichloroacetic acid extracts of Proteus vulgaris was enormously enhanced by boiling the extract for two minutes in the presence of inorganic pyrophosphate (Swartz et al., 1956). Similarly, o-diphenol oxidases, isolated from lower forms of animals or from the leaves of broad beans, have been activated by treatment with detergents, acetone, or urea or by heating to 60"-70"C (Swartz et al., 1956; Bailey, 1961; Kenten, 1958; Robb et al., 1966). In other cases, oxidase activity has been stimulated by the action of unrelated enzymes; thus, pectic enzymes have been shown to induce increased phenol oxidase activity in injured or infected potato-tuber tissue (Tomiyama and Stahmann, 1964), and proteolytic enzymes produced a fourfold increase in the activity of o-diphenol oxidase from sugar-beet chloroplasts (Mayer, 1966). The activity of pectinmethylesterase in fruit has been found to increase after mild blanching treatments (Hsu et al., 1965) or irradiation (Somogyi and Romani, 1964), and a thennolabile inhibitor of pectolytic enzymes has been reported in pear juices (Weurman, 1953). Lipoxygenase in pea seeds has been activated by the addition of certain alcohols (Mapson and Moustafa, 1955). The explanation usually advanced for heat activation postulates some form of combination between a relatively stable enzyme and a labile inhibitor. The inhibitor has generally been assumed to be an associated protein. Swartz et al. (1956) suggested that some enzymes may complex with RNA, since RNA breakdown products have been found in activated P-galactosidase systems. Proteins and pigments have been identified 'as phenoloxidase inhibitors in broad beans (Bailey, 1961) and mushrooms (Karkhanis and Frieden, 1961). Tannins and other polyphenolic compounds have been identified as potent enzyme inhibitors in many systems, including mitochondria1 preparations from apples and flowers (Hulme et al., 1964), pectic

PLANT-TISSUE OXIDATION SYSTEMS

53

enzymes from grape leaves (Porter et al., 1961; Porter and Schwartz, 1962), and glucosidases (Goldstein and Swain, 1965).

H. SPECIFICINHIBITORS There is an extensive literature on enzyme inhibition (e.g., Dixon and Webb, 1958; Hochster and Quastel, 1!363), and it will be recognized that various groups of chemical substances can act as inhibitors for many different enzyme systems. In some cases the chemical inhibitors may act through combination with, or destruction of, the protein part of the enzyme system. In other cases, the effect may be through the coenzyme. As already noted, many oxidizing enzymes have a prosthetic group incorporating iron or copper, and reagents which can combine with, or alter, the valency states of these metals may be expected to produce inactivation (reversible or irreversible) of the enzyme. The suggestion has been made that such inhibitory mechanisms might have practical applications in food preservation, and, in fact, treatment with carbon monoxide gas has been used as a substitute for blanching in the production of dehydrated vegetables (Brooks, 1955). Carbon monoxide inactivates o-diphenol oxidase (and also, in the dark, cytochrome oxidase), and hence might be expected to reduce browning reactions. Workers in Louisiana have found that vegetables dried in a stream of air containing 0.3%carbon monoxide retain an acceptable color after dehydration (Anon., 1966).

VI. REGENERATION OF ENZYME ACTIVITY The reversibility of the heat inactivation of certain enzymes is well established and is best illustrated by the completely reversible inactivation of trypsin, in which the equilibrium between native and denatured forms has been measured (Anson and Mirsky, 1934). The partial reversibility of the inactivation of peroxidase is also well authenticated. Leeson (1957) quoted many examples of the revival of peroxidase activity after apparently complete inactivation, and some features of the earlier work are worth recalling. The regeneration of peroxidase activity has been found to depend on three main factors: the test used for detecting the activity; the severity of the heat treatment applied to the system; and the conditions under which the “inactivated” system has been kept.

54

F. AYLWARD A N D D . R. HAISMAN

A. TESTMETHODS Balls (1942)pointed out that some tests for peroxidase activity were unspecific, depending on the catalytic activity of the prosthetic groups alone; many instances of pseudo-peroxidase activity have been quoted in this review. Some inconsistencies in earlier reports of peroxidase regeneration may be attributed to this factor. Schwimmer (1944),studying the regeneration of peroxidase in turnip and cabbage juices, found that heating separated the enzyme system into soluble and insoluble components. Although the soluble fraction retained some catalytic activity, regeneration was observed only when both components were incubated together for some time. Some evidence was adduced that the precipitated protein characterized the specificity of the peroxidase while the prosthetic group remained in the supernatant liquid. Reddi et al. (1950) used three different substrates - guaiacol, ophenylenediamine, and pyrogallol- to detect the regeneration of peroxidase activity in an apple extract, and although all the results followed the same general pattern, the differences found between substrates showed that experiments limited to one aspect of peroxidase activity could give misleading results. Nebesky et al. (1950) obtained similar results in tests on other products, and concluded that the guaiacol test gave the most accurate indication of peroxidase activity. B. HEAT TREATMENT It has been found consistently that the regeneration of peroxidase activity can be prevented by prolonged or rigorous heat treatment (Kaplan et al., 1949; Reddi et al., 1950; Farkas et al., 1956), and that regeneration is most likely to occur when the enzyme has not been wholly inactivated, or has been heated just to the elimination point as judged immediately after the heat treatment. Figure 4 illustrates some examples of regeneration, based on data obtained by Schwimmer (1944) and Farkas et al. (1956), in which the regeneration of peroxidase activity, during the day following the heat treatment, is shown for both the longest heating periods used and the heating period just sufficient to inactivate the enzyme at various temperatures. Where the enzyme was not completely inactivated, the residual activity is also depicted. It can be seen that the extent of regeneration was

55

PLANT-TISSUE OXIDATION SYSTEMS

70 r A

::I,

0

,

~

,

10

0 70

80

90

100

110

L ~

I20

I30

Temperature of inactivation ('C)

FIG.4. The regeneration of peroxidase. Blank area of column, residual activity after heat treatment; striped area, regenerated activity 24 hr after heat treatment; solid area, regenerated activity 24 hr after maximum heat treatment. A: Data of Schwimmer, 1944. B: Data of Farkas et nl.. 19.56.

greater when there was residual activity after heat treatment or when no extra heating was given after inactivation appeared complete. Furthermore, the amount of regeneration varied with the temperature of the heat treatment given and was considerably greater after shorttime high-temperature processes. Esselen and Anderson (1956)tested 17 different vegetables after processing at temperatures up to 300"F, and calculated the heat treatment required, first, to inactivate peroxidase, and, second, to prevent regeneration. The F values at 212°F to prevent regeneration were about 2-4 times as great as those for inactivation based on testing immediately after heating, and the z values were lower.

56

F. AYLWARD A N D D. R. HAISMAN

C. STORAGECONDITIONS Other factors which have been found to affect the recovery of peroxidase after inactivating treatments include the concentration of neutral salts, the concentration of the enzyme itself, and pH (Herrlinger and Kiermeier, 1944). As more and more cases of regenerated activity have been examined, however, it has become apparent that conditions of time and temperature during the storage of the "inactivated" products are critical factors determining whether or not regeneration will be observed. Schwimmer (1944) found that the activity regenerated in an inactivated turnip peroxidase preparation stored for 20 hours was greater at 25°C than at 6°C. Studying canned sweet corn, Vetter et al. (1958) found that regeneration of peroxidase took place for two days after processing and was greater when the cans were stored at higher temperatures in the range of 2"-38"C. Zoueil and Esselen (1959) found that peroxidase activity in green beans and turnips stored at 22"-23"C regenerated within 24 hours of processing. Pinsent (1962) observed regeneration of peroxidase activity in peas within a few hours after blanching if the peas were kept at room temperature. At -18"C, regeneration took several months. Working with solutions of pure peroxidase, Wilder (1962) observed a gradual regeneration of activity over 9 days after heat inactivation, increasing most in the first day. In a comparison of three storage temperatures - lo,22", and 38°C-the middle temperature was found to be optimum for maximum regeneration. Joffe and Ball (1962) investigated the regeneration of pure peroxidase in greater detail. In solutions stored at 30°C after heat treatment, the enzyme activity was slowly regenerated over 2 to 10 days after an initial time lag of 20 hours. Thereafter, the activity slowly decreased to zero again. At higher temperatures, both regeneration and the subsequent decrease in activity were accelerated. The amount of enzyme activity regenerated varied from 4 to 24%. Thus, for any particular product the amount of regenerated peroxidase activity which can be detected will depend both on the time elapsed since heat treatment and on the ambient temperature. Neglect of these factors probably accounts for discrepancies in the earlier literature. There are few examples of the regeneration of other enzymes considered important in relation to food preservation, but mention should be made of the transitory reappearance of catalase activity after heat treatment, reported by Sapers and Nickerson (1962~).Significantly affected by pH, storage temperature, and heating conditions, up to 29% regeneration of the original activity was observed at pH 7 and

PLANT-TISSUE OXIDATION SYSTEMS

57

30°C within 2 hours of inactivation. The reactivated enzyme was evidently unstable, and the regenerated activity decayed within 24 hours. D. EFFECTON QUALITY It has been generally assumed that regenerated enzyme activity must have an undesirable effect on product quality, but, because the borderline between residual and regenerated activities is ill-defined and depends on the sensitivity of the test method used, unequivocal measurement of this effect is difficult. In one of the few published investigations of this aspect of regeneration, Guyer and Holmquist (1954) found that canned peas given a minimum process at 250" or 260°F showed a very low level of regenerated peroxidase activity, and after eight months of storage possessed a definite off-flavor.

VII. RESEARCH NEEDS This review has made many references to off-flavors and deterioration in the color and texture of foods. Any discussion on food quality, however, must focus attention also on the remarkable progress made by the food industries in the past half century in providing an everwidening range of products both for the domestic consumer and for catering and institutional use (Aylward, 1966, 1967).The impetus for advances in food technology comes not only from the food industries but also from increasing demands by the consumer for variety in foods and for rising standards of quality. The search for improved quality is therefore almost self-perpetuating. A. THE SCOPE OF RESEARCH In surveying research needs we must consider not only individual topics, such as the properties of some particular enzyme system, but also the place of this enzyme system in the chain of reactions from farm to consumer. Deterioration from enzyme action may result from changes which occur at different points in this chain. a. After Harvest and before Processing. Undesirable enzymic reactions may be stimulated by bruising and other injury, changes in temperature, and anaerobic or oxidative conditions, leading to proteolysis, foreign flavors, and the possible production of reactive intermediate products which may induce further changes at a later stage. b. During Processing. Enzymic reactions may be accelerated or

58

F. AYLWARD A N D D. R. HAISMAN

activated by a rise in temperature; autoxidation and textural changes may be promoted. c. During Storage. As a result of residual enzymic activity, offflavors will become more pronounced as storage time is prolonged. Moreover, chemical and physical reactions may be stimulated by reactive intermediate compounds produced at an earlier stage in storage or processing. Such reactions by free-radical or other pathways can cause the destruction of pigments, vitamins, proteins, and other compounds. Autoxidation in particular may lead to flavor and color changes, and phase separation and conformational changes may lead to changes in texture. Poor quality may arise from the operation of any or all of these factors, and it is seldom possible to deduce the mechanism from a consideration of quality defects alone. Investigations carried out at the Western Regional Laboratory and elsewhere have shown that the quality of frozen foods may deteriorate even when the possibility of continuing enzymic activity is minimal. Although present-day processing procedures go far toward stabilizing the raw material, it is still potentially reactive, particularly if mistreated during storage. How far this potential is created or modified during processing treatments poses one of the more interesting problems requiring further investigation. B.

BIOCHEMICALENGINEERING

There will clearly be differences in individual processes such as canning, dehydration (by various methods), and quick freezing, and further work is still required on the effects of these processes on the plant constituents. Moreover, modifications of processes require investigation so that a balance sheet of advantages and disadvantages can be obtained. This points to the need for collaboration between chemical and biochemical engineers, concerned with machinery design and process innovation, and the biochemist and microbiologist. There is ample scope for investigations in this field, which can be broadly described as biochemical engineering.

c. ENZYMESYSTEMS: SUBSTRATES - PRIMARY AND SECONDARYREACTIONS In surveying the enzymes which may be involved in the deterioration of fruit and vegetable products, it appears that only a few (for example, ascorbate oxidase and the pectolytic and proteolytic en-

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zymes) affect quality directly. The undesirable effects of the majority of oxidative enzymes arise almost entirely from secondary reaction products and the reaction chains they initiate. It has been observed that the off-flavors and off-odors produced during the storage of underblanched frozen vegetables are characteristic of the vegetable concerned, which suggests that although the initiation of degradation may be a common reaction, its development depends on secondary reactions involving indigenous components. Considerable effort has been applied to identifying and determining enzyme activity in various plant tissues; it is probable that there is now a greater need for more information about enzyme substrates and the nature of the secondary reactions. In this connection, work of recent years on reactions involving the unsaturated lipids of plant material is of great importance.

D.

PLANT

LIPIDS:OXIDATION MECHANISMSAND REACTION PRODUCTS

Unsaturated lipids can be oxidized under a wide range of conditions: enzymically by the action of lipoxygenase, and otherwise by heat, light, and a variety of metallic catalysts including hemoproteins such as peroxidase (not necessarily active) and the cytochromes. The peroxides, once formed, may be long-lived; their eventual breakdown may be spontaneous or catalyzed by metals, hemoproteins, and the like, and can initiate chain reactions promoting more extensive degradation. The oxidation and decomposition of lipids in foods has been followed through the assay of intermediate products such as peroxides, malondialdehyde, and hexanal. These products represent phases in a complex sequence of reactions, and in isolation may not give an accurate picture of either the extent or course of degradation. An extension of work on the degradation pathways in these systems would be of great value.

E. PLANTCOMPONENTS INHIBITING OR MODIFYING ENZYMEACTIVITY There are numerous examples of modification of the properties of an enzyme system by factors in its natural environment. In situ, enzymes usually show an enhancement in heat resistance, and various inhibitors may disguise the existence of certain enzymes altogether. Natural antioxidants, such as the E and K groups of vitamins and the

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ubiquinones, may delay or prevent the onset of oxidative changes; analysis of many of these components presents difficulties, but there are indications that some at least vary in concentration according to season and maturity. The effect of these and other components on the potential stability of vegetable products is worth investigation in greater detail.

F. HEATRESISTANCE OF ENZYMES Study of the heat resistance of enzymes has been confined to comparatively few systems and has centered on peroxidase because of its demonstrably superior stability and reversible inactivation. There is general agreement that peroxidase activity should be completely suppressed to ensure that quality is maintained in long-term storage, but the actual effect of regenerated peroxidase activity has not been firmly established. More data are undoubtedly needed on the heat resistance of other enzyme systems, such as ascorbate and cytochrome oxidase, particularly in relation to subcellular environment. At the same time it is worth recalling the point emphasized by Joslyn (1966) that critical evaluation of the lability of enzymes is useful as a criterion for the technical treatment of raw materials only when the processing conditions are constant and reproducible.

G. NEW METHODS OF PRESERVATION There are two approaches to food preservation: (1)to seek methods whereby the essential attributes of the fresh material are least changed; and (2) to produce new types of products which have their own special desirable characteristics as well as stability during storage. If the first is the objective, then the ideal process would arrest all natural changes in perpetuity. In practice, no method has been found of controlling the complex biochemical balance maintained in natural organisms, although modern freezing techniques go some distance toward this goal. The second objective is the basis of many traditional methods for preserving both plant and animal materials. Products such as cheese, kippers, and dried fruits are different in many respects from their “fresh” counterparts. They are accepted as foodstuffs in their own right (Aylward, 1967). Despite the very considerable advances in knowledge of food science and technology, the majority of the methods used have their

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origins in antiquity (Tilgner, 1965).The possibility of new approaches to the preservation of foods should not be ignored, and must increase as knowledge grows on the biochemical mechanisms underlying maturation, senescence, and dormancy. There is agreement in many countries on the role of biochemistry as the theoretical basis for food technology (Oparin, 1966).

REFERENCES Acker, L. 1962. Enzymic reaction in foods of low moisture content. Adoances in Food Research 11,263-330. Adams, H. W., and Yawger, E. S. 1961. Enzyme inactivation and color of processed (fresh) peas. Food Technol. 15,314-317. Adam, W. B., Horner, G., and Stanworth, J. 1942. Changes occurring during the blanching of vegetables. J. Soc. Chem. Ind. (London)61,96-99. Akazawa, T., and Conn, E. E. 1958. The oxidation of reduced pyridine nucleotides byperoxidase. J. Biol. Chem. 232,403-415. Alberghina, F. A. M. 1964. Chlorogenic acid oxidase from potato tuber slices: partial purification and properties. Phytochemistry 3,65-72. Ames, G . R., and King, T. A. 1966. The assay and pH profile of lipoxidase. J. Sci. Food Agr. 17,301-303. Anon. 1966. Drying vegetables with carbon monoxide. Food Technol. in Australia 18, 173. Anson, M. L., and Mirsky, A. E . 1934. Equilibrium between active native trypsin and inactive denatured trypsin. J. Gen. Physiol. 17,393-398. Aung, T., and Ross, E. 1965. Heat sensitivity of pectinesterase activity in papaya puree and of catalase-like activity in passion fruitjuice.J. Food Sci. 30,144-147. Axelrod, B., and Jagendorf, A. T. 1951. The fate of phosphatase, invertase and peroxidase in autolysing leaves. Plant Physiol. 26,406-410. Aylward, F. 1956. Phospholipids in foods: plant phospholipids. Chem. Znd. (London) 1956,1360-1366. Aylward, F. 1966. Food technology in relation to the catering industry. Proc. 2nd. Intern. Congr. Food Sci. and Technol. (D. J. Tilgner and A. Borys, eds.), pp. 397-401. Warsaw. Aylward, F. 1967. The impact of food technology on the cateringindustry. In “Catering management in the technological age.” (J. Fuller, ed.), pp. 34-52. Barrie and Rockliff, London. Aylward, F., and Nichols, B. W. 1961. Plant lipids. I. The carbohydrate content of groundnut phospholipids. J. Sci. Food Agr. 12,645-648. Aylward, F., and Nichols, B. W. 1962. Plant lipids. 11. Free and combined sterols (sterol esters and glycosides) in commercial oilseed phospholipids. J. Sci. Food Agr. 13,86-91. Aylward, F., and Showler, A. J. 1962a. Plant lipids. 111. Phosphatidic acid and phosphatidylinositol in cereal grains. J. Sci. Food Agr. 13,92-95. Aylward, F., and Showler, A. J. 1962b. Plant lipids. IV. The glycerides and phosphatides in cereal grains. J. Sci. Food Agr. 13,492-495.

62

F. AYLWARD AND D. R. HAISMAN

Bailey, J. L. 1961. Latent phenolase from broad bean leaves. Biochem. J . 79, 514-516. Baker, R. W., and Goldblith, S . A. 1961. Complementary effects of thermal energy and ionizing energy on peroxidase activity in green beans.J. Food Sci. 26,91-94. Ball, C. 0. 1923. Thermal process time for canned foods. Bull. Natl. Research Council US.7, Part 1, NO. 37, 1-76. Ball, C. 0.1943. Short-time pasteurization of milk. Ind. Eng. Chem. 35,71-84. Balls, A. K. 1942. The fate of enzymes in processed foods. Fruit Prods. J. 22.36-39. Bancroft, G., and Elliott, K. A. C. 1934. The distributionof peroxidase in animal tissues. B i o c h m . J . 28, 1911-1919. Banks, A,, Eddie, E., and Smith, J. G. M. 1961. Reactions of cytochrome-c with methyllinoleate hydroperoxide. Nuture 190, 908-909. Beinert, H. 1966. Cytochrome c oxidase: present knowledge of the state and function of its copper components. In “The Biochemistry of Copper” (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), pp. 213-234. Academic Press, New York. Bengtsson, B., and Bosund, I. 1964. Gas chromatographic evaluation of the formation of volatile substances in stored peas. Food Technol. 18, 773-776. Bengtsson, B., and Bosund, I. 1966. Lipid hydrolysis in unblanched frozen peas (Pisum satiuum). J. Food Sci. 31, 474-481. Bengtsson, B. L., Bosund, I., and Rasmussen, I. 1967. Hexanal and ethanol formation in peas in relation to off-flavour development. Food Technol. 21,478-482. Bergstrom, S., and Holman, R. T. 1948. Lipoxidase and the autoxidation of unsaturated fatty acids. Adoances in Enzymol. 8,425-457. Blain, J. A. 1962. Moisture level and enzyme activity. Recent Adoances in Food Sci. 2, 41-45. Blain, J. A., and Shearer, G. 1965. Inhibition of soya lipoxidase. J. Sci. Food Agr. 16, 373-378. Blain, J. A., and Styles, E. C. C. 1961. Unsaturated fat oxidase activity of plant extracts. Soc. Chem. Ind. Monogr. 11,150-159. Board, P. W. 1961. Problems of stability of canned foods-enzymic aspects. Proc. 4th Intern. Congr. Canned Foods, 39-56. Boger, P. 1965. Chlorophyllase of Chlorella oulgaris Beijerinck. Phtyochemistry 4, 435-443. Boggs, M. M., Dietrich, W. C., Nutting, M. D., Olsen, R. L., Linquist, F. E., Bohart, G. S., Neumann, H. J., and Morris, H. J. 1960. Time-temperature tolerance of frozen foods. XXI. Frozen peas. Food Technol. 14, 181-185. Boggs, M. M., Buttery, R. G., Venstrom, D. W., and Belote, M. L. 1964. Relation of hexanal in vapour above stored potato granules to subjective flavour estimates. J. Food Sci. 29, 487-489. Bonner, W. D. 1957. Soluble oxidases and their functions. Ann. Reo. Plant Physiol. 8, 427-452. Bonner, W. D. 1961. The cytochromes of plant tissues. In “Haematin Enzymes” (J. E. Falk, R. Lemberg, and R. K. Morton, eds.), pp. 479-497. Pergamon Press, Oxford. Bonner, J., and Varner, J. E. (eds.) 1965. “Plant Biochemistry.” Academic Press, New York and London. Bonnichsen, R. K. 1955. Blood Catalases. I n “Methods in Enzymology.” Vol. I1 (S. P. Colowick and N.0.Kaplan, eds.), p. 781. Academic Press, New York. Boscan, L., Powrie, W. D., and Fennema, 0. 1962. Darkening of red beets, Beta oulgaris. J . Food Sci. 27, 574-582.

PLANT-TISSUE OXIDATION SYSTEMS

63

Bottcher, H. 1961. Activity of peroxidase and polyphenoloxidase in fresh vegetables. Z. Lehensm.-Untersuch. u. Forsch. 115, 527-534. Bottcher, H. 1962. Elimination of fermentation in the conservation of vegetables by freezing. 111. Influence on the fermentation activity remaining after blanching on the quality of the frozen product. Nahrung 6,446-459. Bowen, W. J., and Kerwin, T. D. 1956. Kinetics of myokinase. 11. Studies of heat deactivation, effect of salts and state of equilibrium. Arch. Biochem. Biophys. 64, 278-284. Brooks, D. 1955. Aphydatosis- promising process for drying foods. Food in Canada Nov., 26-27; Dec., 18-20. Brooks, D. W., and Dawson, C. R. 1966. Aspects of tyrosinase chemistry. In “The Biochemistry of Copper” (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), pp. 343355. Academic Press, New York. Buck, P. A., and Joslyn, M. A. 1953. Accumulation of alcohol in underscalded frozen broccoli. J. Agr. Food Chem. 1, 309-312. Buck, P. A., and Joslyn, M. A. 1956. Formation of alcohol, acetaldehyde and acetoin in frozen broccoli tissue. J. Agr. Food Chem. 4, 548-552. Burton, H. S . , and McWeeny, D. J. 1963. Role of phosphatides in non-enzymatic browning. Nature 197, 1086-1087. Butler, W. L., and Baker, J. E. 1965. A haemoprotein from the lipid of peanuts. Nature 205, 1319-1321. Butt, V. S., and Hallaway, M. 1958. Ascorbic oxidase activity in extracts from higher plants. Biochem. J. 69, 20P. Buttery, R. G . , Hendel, C. E., and Boggs, M. 1961. Autoxidation of potato granules. J. Agr. Food Chem. 9, 245-252. Chance, B. 1949a. The properties of the enzyme-substrate compounds of peroxidase and peroxides. I. The spectra of the primary and secondary complexes. Arch. Biochem. 21,416-430. Chance, B. 1949b. The enzyme-substrate compounds of horseradish peroxidase and peroxides. 11. Kinetics of formation and decomposition of the primary and secondary complexes. Arch. Biochem. 22,224-252. Chang, L. W. S., Morita, L. L., and Yamamoto, H. Y. 1965. Papaya pectinesterase inhibition by sucrose. J. Food Sci. 30, 218-222. Charley, J. L. S. 1961. The use of enzymes in the processing and storage of juices and other fruit products. Soc. Chem. Znd. Monogr. 11, 107-117. Church, B. D., and Halvorson, H. 1959. Dependence of the heat resistance of bacterial endospores on their dipicolinic acid content. Nature 183, 124-125. Colowick, S. P., and Kalckar, H. M. 1943. Role of myokinase in transphosphorylations. 1. Enzymatic phosphorylation of hexoses by adenyl pyrophosphate. J. Biol. Chem. 148, 117-126. Crawford, D. L., Yu, T. C., and Sinnhuber, R. 0. 1967. Reaction of malonaldehyde with protein. J. Food Sci. 32, 332-335. C.S.I.R.O. 1962-1964. Colour changes in stored or processed foods. Diu. of Food Preseru. Ann. Rept. p. 11. Dahle, L. 1965. Factors affecting oxidative stability of carotenoid pigments of Durum milled products. J. Agr. Food Chem. 13, 12-15. Dahle, L. K., Hill, E. G . , and Holman, R. T. 1962. The TBA reaction and the autoxidation of polyunsaturated fatty acid methyl esters. Arch. Biochem. Biophys. 98, 253-261.

64

F. AYLWARD AND D. R. HAISMAN

David, J. J., and Joslyn, M. A. 1953. Acetaldehyde and related compounds in frozen green peas. Food Reseurch 18,390-398. Dawson, C. R. 1966. Ascorbate oxidase: a review. In “The Biochemistry of Copper” (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), pp. 305-334. Academic Press, New York. Demain, A. L., and PhafF, H . J. 1957. Recent advances in the enzymatic hydrolysis of pectic substances. Wullerstein Labs. Communs. 20, No. 69, 119-139. Deuel, H. J. 1951. “The Lipids.” Vol. I. Chemistry. Wiley (Interscience), New York. Deuel, H. J. 1955. “The Lipids.” Vol. 11. Biochemistry: Digestion, Absorption, Transport and Storage. Wiley (Interscience), New York. Deuel, H. J. 1957. “The Lipids.” Vol. 111. Biochemistry: Biosynthesis, Oxidation, Metabolism and Nutritional Value. Wiley (Interscience), New York. Deutsch, H. F. 1951. The effect of heat and urea treatment on crystalline horse erythrocyte catalase. Act4 Chem. Scund. 5 , 1074-1086. Dicks, J. W., and Friend, J. 1966a. Crocin oxidation by Triton extracts of sugar-beet leaf mitochondria. Biochem. J . 99, 38P. Dicks, J. W., and Friend, J . 1966b. The effect of phenolic compounds and peroxidase on crocin oxidation by sugar-beet leaf mitochondria. Biochem. J . 99, 38P. Dietrich, W. C . , and Neumann, H. J. 1965. Blanching Brussels sprouts. Food Technol. 19, 1174-1177. Dietrich, W. C . , Lindquist, F. E., Miens, J . C., Bohart, G. S., Neumann, H. J.. and Talburt, W. F. 1957. The time-temperature tolerance of frozen foods. IV. Objective tests to measure adverse changes in frozen vegetables. Food Technol. 11,109-113. Dietrich, W. C., Nutting, M.-D., Olsen, R. L., Lindquist, F. E., Boggs, M. M., Bohart, G . S., Neumann, H. J., and Morris, H. J. 1959a. Time-temperature tolerance of frozen foods. XVI. Quality retention of frozen green snap beans in retail packages. Food Technol. 13, 136-145. Dietrich, W. C., Olsen, R. L., Nutting, M.-D., Neumann, H. J., and Boggs, M. M. 1959b. Time-temperature tolerance of frozen foods. XVIII. Effect of blanching conditions on colour stability of frozen beans. Food Technol. 13,258-261. Dietrich, W. C., Boggs, M. M., Nutting, M.-D., and Weinstein, N. E. 1960. Timetemperature tolerance of frozen foods. XXIII. Quality changes in frozen spinach. Food Technol. 14, 522-527. Dietrich, W. C., Nutting, M.-D., Boggs, M. M., and Weinstein, N. E. 1962. Time-temperature tolerance of frozen foods. XXIV. Quality changes in cauliflower. Food Technol. 16, 123-128. Dillard, M. G . , Henick, .4. S., and Koch, R. B. 1960. Unsaturated triglyceride and fatty acid lipoxidase activities of navy beans, small red beans, peanuts, green peas, and lima beans. Food Research 25,544-553. Dillard, M. G., Henick, A. S., and Koch, R. B. 1961. Differences in reactivity of legume lipoxidases. J. Biol. Chem. 236, 37-40. Dimick, K. P., Ponting, J . D., and Makower, R. 1951. Heat inactivation of polyphenolase in fruit juices. Food Technol. 5 , 237-241. Dixon, M., and Webb, E. C. 1958. “Enzymes.” Longmans, London. Doesburg, J. J. 1951. Behaviour of pectins in stored frozen fruits. Proc. 7th Intern. Congr. Refrig. London 548-551. Eckey, E. W. 1954. “Vegetable Fats and Oils.” Reinhold, New York. Elliott, K. A. C. 1932a. Milk peroxidase. Biochem. J . 26, 10-24. Elliott, K. A. C . l932b. Oxidations catalysed by horseradish and milk peroxidases. Biochem. J . 26, 1281-1290.

PLANT-TISSUE OXIDATION SYSTEMS

65

Embs, R. J., and Markakis, P. 1966. Enzymatic conversion of p-coumaric acid to caffeic acid. Mich. State Uniu. Quart. Bull. 49,51-54. Eriksson, C., and von Sydow, E. 1964. Post-harvest metabolism of green peas (Pisum sutiuurn) with special reference to glutamic acid and related compounds. J. Food Sci. 29, 58-64. Esselen, W. B., and Anderson, E. E . 1956. Thermal destruction of peroxidase in vegetables at high temperatures. Food Reseurch 21, 322-325. Eyring, H., and Stearn, A. E. 1939. The application of the theory of absolute reaction rates to proteins. Chem. Reu. 24, 253-270. Falconer, M. E., Fishwick, M. J., Land, D. G., and Sayer, E. R. 1964. Carotene oxidation and off-flavour development in dehydrated carrot. J . Sci. Food Agr. 15, 897-901. Farkas, D. F., and Goldblith, S. A. 1962. Kinetics of lipoxidase inactivation using thermal and ionizing energy. J. Food Sci. 27, 262-276. Farkas, D. F., Goldblith, S. A., and Proctor, B. E. 1956. Stopping storage off-flavours by curbing peroxidase. Food Eng. 28 (Jan.), 52-154. Fisher, N. 1962. Wheat Lipids. I n “Recent Advances in Food Science,” (J. Hawthorn and J. M. Leitch, eds.), Vol. 1, pp. 226-245. Buttenvorths, London. Fisher, N., Broughton, M. E., Peel, D. J., and Bennett, R. 1964. The lipids of wheat. 11. Lipids of flours from single wheat varieties of widely varying baking quality. J. Sci. Food Agr. 15, 325-341. Frankel, E. N. 1962. Hydroperoxides. In “Symposium on foods: Lipids and their oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), pp. 51-78. Avi, Westport, Connecticut. Frazer, M. J., and Kaplan, J. G. 1955. The alteration of intracellular enzymes. 111. The effect of temperature on the kinetics of altered and unaltered yeast catalase. J. Gen. Physiol. 38,515-547. Frazer, M. J., Kaplan, J. G., and Schulmann, J. H. 1955. Activity of catalase-lipid complexes at oil/water interfaces. Discussions Faraday Sac. 20,44-54. Friend, J. 1961. The oxidation of carotenoids in green plant tissue. “Production and application of enzyme preparations in food manufacture.” Soc. Chem. Ind. Monogr. 11, 160-167. Friend, J., and Mayer, A. M. 1959. The destruction of crocin by isolated chloroplasts. Biochem. J. 73, 18P. Friend, J., and Nakayama, T. 0.M. 1959. Destruction of carotenoids in isolated chloroplasts. Nature 184, B.A.66. Fuleki, T., and David, J . J. 1963. Effect of blanching, storage temperature and container atmosphere on acetaldehyde and alcohol production and off-flavour formation in frozen snap-beans. Food Technol. 17, 1313-1316. Furuya, M., Galston, A. W., and Stowe, B. B. 1962. Isolation from peas of co-factors and inhibitors of indolyl-3-acetic acid oxidase. Nature 193, 456. Galston, A. W. 1955. Plant catalase. In “Methods in Enzymology” (S. P. Colowick and N. Kaplan, eds.) Vol. 11, p. 789, Academic Press, New York. Gamborg, 0. L., Wetter, L. R., and Neish, A. C. 1962. The oxidation of some aromatic a-hydroxy acids by glycollate; oxygen oxidoreductase. Phytochemistry 1,159-168. Gillespy, T. G. 1948. The heat resistance of the spores of thermophilic bacteria 111. Thermophilic anaerobes. Ann. Rept. Fruit and Vegetable Preseru. Research Sta., Campden, Glos. pp. 34-43. Gini, B., and Koch, R. B. 1961. Study of a lipohydroperoxide breakdown factor in soya extracts. J. Food Sci. 26, 359-364. Goddard, D. R., and Holden, C. 1950. Cytochrome oxidase in the potato tuber. Arch. Biochem. 27. 41-47.

66

F. AYLWARD AND D. R. HAISMAN

Goldstein, J. L., and Swain, T. 1965, The inhibition of enzymes by tannins. Phytochemistry 4, 185-192. Grant, N. H., and Alburn, H. E. 1966. Acceleration of enzyme reactions in ice. Nature 212,194. Gregory, R. P. F., and Bendall, D. S. 1966. Purification and some properties of the polyphenol oxidase from tea. Biochem. J. 101,569-581. Grommick, R., and Markakis, P. 1964. Effect of peroxidase on anthocyanin pigments. J . Food Sci. 29, 53-57. Guadagni, D. G. 1957. Time-temperature tolerance of frozen foods. VIII. Organoleptic evaluation of frozen strawberries, raspberries and peaches. Food Technol. 11, 471-472. Guadagni, D. G., and Kelly, S. H. 1958. Time-temperature tolerance of frozen foods. XIV. Ascorbic acid and its oxidation products as a measure of temperature history in frozen strawberries. Food Technol. 12,645-647. Guadagni, D. G., and Nimmo, C. C. 1957a. The time-temperature tolerance of frozen foods. 111. Effectiveness of vacuum, oxygen removal, and mild heat in controlling browning in frozen peaches. Food Technol. 11,43-47. Guadagni, D. G. and Nimmo, C. C. 1957b. The time-temperature tolerance of frozen foods. IX. Effect of time and temperature on colour distribution in retail packs of frozen raspberries. Food Technol. 11,606-608. Guadagni, D. G., and Nimmo, C. C. 1958. Time-temperature tolerance of frozen foods. XIII. Effect of regularly fluctuating temperatures in retail packages of frozen strawberries and raspberries. Food Technol. 12, 306-310. Guadagni, D. G., Nimmo, C. C., and Jansen, E. F. 1957a. The time-temperature tolerance of frozen foods. 11. Retail packages of frozen peaches. Food Technol. 11, 33-42. Guadagni, D. G . , Nimmo, C. C., and Jansen, E. F. 195%. The time-temperature tolerance of frozen foods. VI. Retail packages of frozen strawberries. Food Technol. 11,389-397. Guadagni, D. G., Nimmo, C. C., and Jansen, E. F. 1957c. Time-temperature tolerance of frozen foods. X. Retail packs of frozen red raspberries. Food Technol. 11,633637. Guadagni, D. G., Nimmo, C. C. and Jansen, E. F. 1958. Time-temperature tolerance of frozen foods. XI. Retail packs of frozen red sour pitted cherries. Food Technol. 12, 36-40. Guadagni, D. G., Eremia, K. M., Kelly, S. H., and Harris, J. 1960. Time-temperature tolerance of frozen foods. XX. Boysenberries. Food Technol. 14,148-150. Gutterman, B. M., Lovejoy, R. D., and Beacham, L. M. 1951. Preliminary report on correlation of acetaldehyde with off-flavour in frozen vegetables. J . Assoc. Ofic. Agr. Chemists 34,231. Guyer, R. B., and Holmquist, J. W. 1954. Enzyme regeneration in high-temperature short-time sterilized canned foods. Food Technol. 8,547-550. Haisman, D. R., and Knight, D. J. 1967. p-Glucosidase activity in canned plums. J . Food Technol. 2,241-248. Hanahan, D. J., and Chaikoff; I. L. 1947a. Phosphorus-containing lipids of the carrot. J . Biol. Chem. 168, 233-240. Hanahan, D. J., and Chaikoff, I. L. 194%. A new phospholipid-splitting enzyme specific for the ester linkage between nitrogenous base and the phosphoric acid grouping. J . Blol. Chem. 169, 699-705.

PLANT-TISSUE OXIDATION SYSTEMS

67

Hanahan, D. J., and Chaikoff, I . L. 1948. Phosphorus containing lipids of cabbage leaves and their relation to a phospholipid-splitting enzyme contained in these leaves. J. Biol. Chem. 172, 191-198. Hanson, H. L., and Fletcher, L. R. 1958. Time-temperature tolerance of frozen foods. XII. Turkey dinners and turkey pies. Food Technol. 12,40-43. Hanson, H. L., Fletcher, L.R., and Campbell, A. A. 1957. Time-temperature tolerance of frozen foods. V. Texture stability of thickened precooked frozen foods as influenced by composition and storage conditions. Food Technol. 11,339-343. Hanson, H. L., Fletcher, L. R., and Lineweaver, H. 1959. Time-temperature tolerance of frozen foods. XVII. Frozen fried chicken. Food Technol. 13,221-224. Hare, R . C. 1964. Indoleacetic acid oxidase. Botan. Reo. 30, 129-165. Harel, E., and Mayer, A. M. 1963. Interference of phenolase in the determination of cytochrome oxidase activity in lettuce seeds. Phytochernistry 2,417-420. Harel, E., Mayer, A. M., and Shain, Y. 1965. Purification and multiplicity of catechol oxidase from apple chloroplasts. Phytochemistry 4, 783-790. Hatch, M. D., and Turner, J. F. 1958. Glycolysis by an extract from pea seeds. Biochem. /. 69, 495-501. Herrlinger, F., and Kiermeier, F. 1944. Inactivation and regeneration of heated peroxidase solutions. Biochem. Z. 317, 1-12. Herrmann, K. 1957. Oxidative enzymes and phenolic substrates in vegetables and fruit. 11. Content of peroxidases and o-polyphenoloxidase in vegetables. Z. Lebensm.Untersuch. u. Forsch. 106,451-454. Henmann, K. 1958. Oxidative enzymes and phenolic substrates in vegetables and fruits. 111. Catechols, hydroxy-cinnamic acid and o-polyphenoloxidase in fruit juice. Z. L,ebensm.-Untersuch. u. Forsch. 108, 152-157. Hilditch, T. P., and Williams, P. W. 1964. “The Chemical Constitution of Natural Fats.” Chapman & Hall, London. Hill, J. M., and Mann, P. J. G. 1962. Inhibition of pea seedling diamine oxidase by chelating agents. Biochem. /. 85, 198-207. Hill, J. M., and Mann, P. J. G. 1964. Further properties of the diamine oxidase of pea seedlings. Biochem. 1.91, 171-182. Hitchcock, C., and James, A. T. 1963. Breakdown of unsaturated fatty acids by plant tissue. Biochem. J. 89, 22P. Hochster, R. M., and Quastel, J. H. 1963. Metabolic Inhibitors. Vol. I and 11. Academic Press, New York. Holden, M. 1961. Breakdown of chlorophyll by chlorophyllase. Biochem. /. 78, 359364. Holden, M. 1964. Enzymic breakdown of chlorophyll. Rothamsted Ann. Rept. p. 117. Holden, M. 1965. Chlorophyll bleaching by legume seeds. J. Sci. Food Agr. 16,312325. Hsu, C. P., Deshpande, S. N., and Desrosier, N. W. 1965. Role of pectin methylesterase in firmness of canned tomatoes. J. Food Sci. 30, 583-588. Huelin, F. E., and Stephens, I. M. 1948. The enzyme-catalysed oxidation of ascorbic acid in fruit and vegetable suspension. Australian J . Sci. Research (Series B ) 1, 58-64. Hulme, A. C., Jones, J. D., and Wooltorton, L. S. C. 1964. Mitochondria1 preparations from flowers. Nature 201, 795-797. Hultin, H. O., and Levine, A. S. 1963. On the occurrence of multiple molecular forms of pectinesterase. Arch. Biochem. Biophys. 101, 396-402.

68

F. AYLWARD AND D. R. HAISMAN

Hultin, H. O., Sun, B., and Bulger, J. 1966. Pectin methylesterases of the banana: purification and properties. J. Food Sci. 31, 320-327. Ingold, K. U. 1962. Metal catalysis. I n “Symposium on foods: Lipids and their oxidation.” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), pp. 93-121. Avi, Westport, Connecticut. Ingraham, L. L. 1954. Reaction-inactivation of polyphenolase: temperature dependence. J . Am. Chem. Soc. 76,3777-3780. James, A. T. 1960. Lipid analysis. In “Methods of Biochemical Analysis” (D. Glick, ed.) Vol 8, pp. 1-60. James, L. K., and Augenstein, L. G. 1966. Adsorption of enzymes at interfaces: film formation and the effect on activity. Advances in Enzymol. 28, 1-40. Jansen, E. F., Jang, R., and Bonner, J. 1960. Orange pectinesterase binding and activity. Food Research 25, 64-72. Joffe, F. M., and Ball, C. 0. 1962. Kinetics and energetics of thermal inactivation and the regeneration rates of a peroxidase system. J. Food Sci. 27,587-592. Jolley, R. L., and Mason, H. S. 1965. The multiple forms of mushroom tyrosinase. Interconversion. ]. Biol. Chem. 240, PC 1489-1491. Joslyn, M. A. 1949. Enzyme activity in frozen vegetable tissue. Advances in Enzymol. 9, 613-652. Joslyn, M. A. 1951. The action of enzymes in concentrated solution and in the dried state. J. Sci. Food Agr. 2, 289-294. Joslyn, M. A. 1961. The freezing preservation of vegetables. Econ. Botany 15,347-375. Joslyn, M. A. 1966. The freezing of fruits and vegetables. In “Cryobiology” (H. T. Meryman, ed.), pp. 565-607. Academic Press, London and New York. Joslyn, M. A., and David, J. J. 1952. Acetaldehyde and alcohol in raw or underblanched peas. Quick Frozen Foods 15,51-53. Joslyn, M. A., and Neumann, H. J. 1963. Peroxidase in frozen vegetables. J. Assoc. Ofic. Agr. Chemists 46, (4), 712-717. Joslyn, M. A., and Ponting, J. D. 1951. Enzyme-catalyzed oxidative browning of fruit products. Advances in Food Research 3, 1-44. Kaplan, A. M., Esselen, W. B., and Fellers, C. R. 1949. Enzyme systems of canned acid foods. Effect of processing conditions of time and temperature. Ind. Eng. Chem. 41, 2017-2020. Karel, M., Tannenbaum, S. R., Wallace, D. H., and Maloney, J. F. 1966. Autoxidation of methyl linoleate in freeze-dried model systems. 111. Effects of added amino acids. J. Food Sci. 31,892-896. Karkhanis, Y., and Frieden, E. 1961. On the induction period and a protein inhibitor of mushroom tyrosinase. J. B i d . Chem. 236, PC1-2. Keeney, M. 1962. Secondary degradation products. In “Symposium on foods: Lipids and their oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), pp. 79-89. Avi, Westport, Connecticut. Keilin, D., and Hartree, E. E. 1955. Catalase, peroxidase and metmyoglobin as catalysts of coupled peroxidative reactions. Biochem. J. 60,310-325. Kenten, R. H. 1953. The oxidation of phenylacetaldehyde by plant saps. Biochem. J. 55, 350-360. Kenten, R. H. 1958. Latent phenolase in extracts of broad bean leaves. Biochem. J. 68,244-251. Kenten, R. H., and Mann, P. J. G. 1950. The oxidation of manganese by peroxidase systems. Biochem. J. 46, 67-73.

PLANT-TISSUE OXIDATION SYSTEMS

69

Kenten, R. H., and Mann, P. J. G. 1953. The oxidation of certain dicarboxylic acids by peroxidase systems in the presence of manganese. Biochern. J. 53, 498-505. Kenten, R. H., and Mann, P. J. G. 1955. Purification and properties of the amine oxidase of pea seedlings. Biochern. J . 59,609-620. Kiermeier, F. 1947. Influence of freezing on the activity of enzymes. Biochern. 2.318, 275- 296. Kiermeier, F. 1951. Enzyme activity in frozen plant tissues. Proc. 7th. Intern. Congr. Refrig. London 344-346. Kiermeier, F., and Koberlein, W. 1957. The influence of mono- and disaccharides on the heat inactivation of crystalline catalase. Biochem. Z. 329, 247-254. Klose, A. A., Pool, M . F., Campbell, A. A,, and Hanson, H. L. 1959. Time-temperature tolerance of frozen foods. XIX. Ready-to-cook cut-up chicken. Food Technol. 13, 477-484. Knapp, F. W. 1965. Some characteristics of eggplant and avocado polyphenolases. /. Food Sci. 30,930-936. Koch, R. B. 1962. Dehydrated foods and model systems. In “Symposium on foods: Lipids and their oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), pp. 230-251. Avi, Westport, Connecticut. Koch, R. B., Stern, B., and Ferrari, C. G. 1958. Linoleic acid and trilinolein as substrates for soya bean lipoxidase. Arch. Biochern. B i o p h y s . 78,165-179. Kohn, G . G., Esselen, W. B., and Fellers, C. R. 1953. Thermal inactivation of pectolytic enzymes used in the clarification of apple juice. Food Research 18, 511-515. Kolesnikov, P. A. 1948a. The role of glycollic acid in the oxidation metabolism of green cells. Biokhimiya 13,370-377. Kolesnikov, P. A. 1948b. Catalytic action of glycollic acid on the oxidation of chlorophyll in ground leaves. Dok. Akad. Nauk S S S R . 60,1353-1355. Kolesnikov, P. A. 1949. Participation of organic peroxides and glyoxylic acid in the oxidation of chlorophyll. Biokhirniya 14, 124-129. Kon, S., and Whitaker, J. R. 1965. Separation and partial characterization of the peroxidases of Ficus glabrata latex.J. Food Sci. 30,977-985. Kyzlink, V., and Chytra, M . 1959. Conditions of thermal inactivation of redox enzymes that deteriorate the quality of canned fruit and vegetables. Scientific papers from Institute of Chemical Technology, Prague, pp. 9-42. Labuza, T. P., Maloney, J. F., and Karel, M. 1966. Autoxidation of methyl linoleate in freeze dried model systems. 11. Effect of water on cobalt-catalysed oxidations. J. Food Sci. 31,885-891. Langridge, J . 1963. Biochemical aspects of temperature responses in living organisms. Ann. Rev. Plant Physiol. 14,441-462. Lea, C. H. 1961a. Enzymic deterioration of lipids. I. Animal lipids. In “Production and application of enzyme preparations in food manufacture.” Soc. Chern. Ind. Monogr. 11, 169-177. Lea, C. H. 1961b. Biological aspects of fat deterioration. Food Technol. 15 (7), 33-40. Lea, C. H. 1962. Oxidative deterioration of food lipids. I n “Symposium on foods: Lipids and their oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), pp. 3-28. Avi, Westport, Connecticut. Lea, C. H., and Hobson-Frohock, A,, 1965. Flavour volatiles of fruits and fat-containing foods. I. Degradation of the peroxides of autoxidised sun flower and linseed oils. J. Sci. Food Agr. 16,18-27. Lea, C. H., and Pan, L. J . 1961. Some observations on the oxidative deterioration of the lipids of crude leaf protein. J. Sci. Food Agr. 12,785-790.

70

F. AYLWARD AND D. R. HAISMAN

Lea, C. H., and Swoboda, P. A. T. The flavour of aliphatic aldehydes. Chern. Znd. (London)1958,1289-1290. Lee, F. A. 1958.Effect of blanching on carbonyl content of the crude lipid during the storage of frozen peas. Food Research 23,85-86. Lee, F. A,, and Mattick, L. R. 1961. Fatty acids of the lipids of vegetables. I. Peas. J . Food Sci. 26,273-275. Lee, F. A., and Wagenknecht, A. C. 1958.Enzyme action and off-flavor in frozen peas. 11. The use of enzymes prepared from garden peas. Food Research 23,584-590. Leeson, J. A. 1957.The inactivation of enzymes by heat. Fruit and Vegetable Cunning and Quick Freezing Research Assoc. Sci. Bull. 2. Lemberg, R., and Legge, J. W., eds. 1949.“Haematin compounds and bile pigments,” p. 341.Wiley (Interscience) London and New York. Lenhoff, H. M., and Kaplan, N. 0. 1955. Cytochrome c and cytochrome c peroxidase from Pseudomonus fluorescens. I n “Methods in Enzymology” (S. P. Colowick and N. 0.Kaplan, eds.), Vol. 11, p. 758.Academic Press, New York. Levine, W. G. 1966.Laccase: a review. In “The Biochemistry of Copper” (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), pp. 371-387.Academic Press, New York. Lewis, S . E., and Wills, E. D. 1963.Inhibition of autoxidation ofunsaturated fatty acids by haematin proteins. Biochem. et Biophys. Acta. 70,336-338. Lillard, D. A., and Day, E. A. 1964.Degradation of monocarbonyls from autoxidising lipids. J . Am. Oil Chemists Soc. 41,549-552. Lillehoj, E. B., and Smith, E. G. 1966.Reaction properties of the ascorbic acid oxidase from Myrothecium oerrucaria. Plant Physiol. 41,1553-1560. Lilly, M., Money, C., Hornby, W., and Crook, E. M. 1965.Enzymes on solid supports. Biochem.J . 95,45P. London, M., McHugh, R., and Hudson, P. B. 1958.Unified theory of enzyme catalysis and denaturation. I. Specificity considerations of prostatic acid phosphatase. Arch. Biochem. Biophys. 73,7244. Long, C., Odavic, R., and Sargent, E. J. 1962.Action of cabbage-leaf phospholipase D on lysolecithin. Biochem. J . 85,33P. Lopez, A., Bocklet, M. F., and Wood, C. B. 1959. Catalase and peroxidase activity in raw and blanched southern peas. Food Research 24,548-551. Lovejoy, R. D. 1952.Acetaldehyde content as a measure of flavour in frozen peas and Agr. Chemists 35,179-181. frozen asparagus. J . Assoc. Ofic. Lovern, J. A. 1955.“The Chemistry of Lipids of Biochemical Significance.” Methuen, London. Lundberg, W. 0. 1962.Mechanisms of oxidation. In “Symposium on foods: Lipids and their oxidation” (H. W. Schultz, E. A. Day and R. 0. Sinnhuber, eds.), pp. 31-50. Avi, Westport, Connecticut. Lundegardh, H. 1954.A new cytochrome in living roots. Nature 173,939-941. Lynch, L. J., Mitchell, R. S. and Casimir, D. J. 1959.The chemistry and technology of the preservation of green peas. Adoarices in Food Research 9.61-151. McColloch, R. J., and Kertesz, Z. I. 1948.An unusually heat-resistant pectolytic factor from tomatoes. Arch. Biochern. 17,197-199. McColloch, R. J., Rice, R. G., Bandurski, M. B., and Gentili, B. 1957.The time-temperature tolerance of frozen foods. VII. Frozen concentrated orange juice. Food Technol. 11,444-449. McCombs, C. L. 1957.Ascorbic acid oxidase activity of certain vegetables and changes in the content of reduced and dehydroascorbic acid during shelf life. Food Research 22,448-454.

PLANT-TISSUE OXIDATION SYSTEMS

71

McConnell, J. E. W. 1956. Review of literature on thermal inactivation of enzymes in foods. Natl. Canners Assoc. Rept. No. 0-582 Berkeley, California. McCune, D. C. 1961. Multiple peroxidases in corn. Ann. New York Acad. Sci. 94, 723-730. McDonnell, L. R., Jansen, E. F., and Lineweaver, H. 1945. Properties of orange pectinesterase. Arch. Biochem. 6,389-401. Maehly, A. C. 1955. Experiments with peroxidase. 111. Recombination of hemin and protein to the active enzyme. Arch. Biochem. Biophys. 56,507-524. Mahler, H. R. 1964. Biological oxidation of fatty acids. In “Fatty Acids: Chemistry, properties, production and uses” (K. S. Markley, ed.), Part 3, pp. 1487-1550. Wiley (Interscience) New York. Maier, V. P., and Tappel, A. L. 1959a. Rate studies of unsaturated fatty acid oxidation catalysed by haematin compounds.J.Am. Oil Chemists Soc. 36,8-12. Maier, V. P., and Tappel, A. L. 1959b. Products of unsaturated fatty acid oxidation catalysed by haematin compounds.]. Am. Oil Chemists Soc. 36,12-15. Maier, V. P., Tappel, A. L., and Volman, D. H. 1955. Reversible inactivation of enzymes at low temperatures. Studies of the temperature dependence of phosphatase and peroxidase catalyzed reacti0ns.J. Am. Chem. Soc. 77,1278-1280. Makower, R. U. 1960. Chemical inactivation of enzymes in vegetables before dehydration. Food Technol. 14,160-164. Makower, R. U. 1964a. A.T.P.-induced inhibition of potato browning. Effect of ascorbic acid oxidase and of reducing substances. Plant Physiol. 39,520-522. Makower, R. U . 1964b. Effect of nucleotides on enzymic browning in potato slices. Plant Physiol. 39,956-959. Maloney, J. F., Labuza, J. P., Wallace, D. H., and Karel, M. 1966. Autoxidation of methyl linoleate in freeze-dried model systems. I. Effect of water on autocatalyzed 0xidation.J. Food Sci. 31,878-884. Mann, P. J. G. 1961. Further purification and properties of the amine oxidase of pea seedlings. Bi0chem.J. 79,623-631. Manolkides, K. 1962. Heat resistance of some enzymes. Milchwissenschaft 17,136-140. Mapson, L. W., and Burton, W. G. 1962. Terminal oxidases of the potato tuber. Biochem. ]. 82, 19-25. Mapson, L. W., and Moustafa, E. M. 1955. Oxidation of glutathione by a lipoxidase from pea seeds. Biochem.]. 60,71-80. Mapson, L. W., and Swain, T. 1961. Oxidation of ascorbic acid and phenolic constituents. In “Production and application of enzyme preparations in food manufacture.” Soc. Chem. Znd. Monogr. 11,121-133. Margoliash, E., and Schejter, A. 1966. Cytochrome c. Advances in Protein Chem. 21, 113-226. Markley, K. S. (ed.). 1950-1951. “Soybeans and Soybean Products,” Vol. I and 11. Wiley (Interscience) New York. Martin, R. O., and Stumpf, P. K. 1959. Fat metabolism in higher plants. XII. a-Oxidation of long chain fatty acids.J . Biol. Chem. 234,2548-2554. Matheson, N. A. 1962. Enzymic activity at low moisture levels and its relation to deterioration in freeze-dried foods.]. Sci. Food Agr. 13,248-254. Mayer, A. M. 1962. Inhibition and substrate specificity of lettuce phenolase. Phytochemistry 1,237-239. Mayer, A. M. 1966. Catechol oxidase: Enzymic liberation from sugar-beet chloroplasts. Phytochemistry 5,1297-1301.

72

F. AYLWARD AND D. R. HAISMAN

a specific competitive Mayer, A. M., Harel, E., and Shain, Y. 1964.2,3-Naphthalenediol, inhibitor of phenolase. Phytochemistry 3,447-452. Mayer, A. M., Harel, E., and Ben-Shaul, R. 1966. Assay of catechol oxidase-a critical comparison of methods. Phytochemistry 5,783-789. Mehler, A. H. 1957. “Introduction to enzymology.” Academic Press, New York. Michener, H. D., Thompson, P. A,, and Dietrich, W. C. 1960. Time-temperature tolerance of frozen foods. XXII. Relationship of bacterial population to temperature. Food Technol. 14,290-294. Mikhlin, D., and Bronovitskaya, Z. S. 1948. The connection between the cytochrome-c peroxidase and other oxidative systems. Dokl. Akad. Nauk. SSSR. 60, 1547-1549. Mishiro, Y., and Ochi, M. 1966. Effect of dipicolinate on the heat denaturation of proteins. Nature 211, 1190. Moelwyn-Hughes, E. A. 1933. The kinetics of enzyme reactions. Ergeb. Enzymforsch. 2,1-22. Mondy, N. I., Klein, E. P., and Smith, L. I. 1960. Effect of maturity and storage on phenolic content, enzymatic activity and discolouration of potatoes. Food Research 25,693-705. Mondy, N. I., Gedde-Dahl, S. B., and Mobley, E. 0.1966a. Effect of storage temperature on the cytochrome oxidase and polyphenol oxidase activities and phenolic content of potato.J . Food Sci. 31,32-37. Mondy, N. I., Gedde-Dahl, S. B., and Mobley, E. 0. 1966b. Relationship of specific gravity to the enzymatic activity and phenolic content of potatoes. J. Food Sci. 31, 157-160. Mudd, J. B., and Burris, R. H. 1959. Participation of metals in peroxidase-catalysed oxidations.]. Biol. Chem. 234,2774-2777. Munday, K. A., Edwards, M. L., and Kerkut, G. A. 1962. Free radicals in lyophilised food materials. J . Sci. Food Agr. 13,455-458. Nakayama, S., and Kono, Y. 1957. Effect of concentration on the rate of heat inactivation of enzymes. J. Biochem. (Tokyo)44,25-31. Nara, S., and Yasunobu, K.T. 1966. Some recent advances in the field of amine oxidases. In “The Biochemistry of Copper” (J. Peisach, P. Aisen, W. E. Blumberg, ed.), pp. 423-436. Academic Press, New York. Narayanan, K. M., Bains, G. S., and Bhatia, D. S. Lipoxidase inhibitor in groundnut testa. Chem. Ind. (London)1963,1588. Nebesky, E. A., Esselen, W. B., Kaplan, A. M., and Fellers, C. R. 1950. Thermal destruction and stability of peroxidase in acid foods. Food Research 15, 114-124. Noll, C. R., and Burris, R. H. 1954. Nature and distribution of glycollic acid oxidase in plants. Plant Physiol. 29,261-265. O’Brien, P. J. 1966. The effects of hydrogen peroxide or lipid peroxide on cytochrome-c. Biochem. J . 101,12P. O’Brien, P. J. 1967. Lipid peroxide in a peroxidase-type reaction. Biochem. J. 102,9P. Okunuki, K. 1961. Denaturation and inactivation of enzyme proteins. Aduances in Enzymol. 23,29-82. Oparin, A. J. 1966. Biochemistry as a theoretical basis for food technology. In Proc. 2nd Intern. Congr. Food Sci. and Technol. (D. J. Tilgner, and A. Borys, eds.), pp. 475-481. Warsaw. Patel, D. S., and PhafF, N. J. 196Oa. Purification of tomato polygalacturonase. Food Research 25,37-46. Patel, D. S., and PhafF, H. J. 1960b. Properties of tomato polygalacturonase. Food Research 25.47-57.

PLANT-TISSUE OXIDATION SYSTEMS

73

Pendlington, S . 1962. Chemical changes in unblanched peas after vining. 1st Intern. Congr. Food Sci. and Technol.Abstr. p. 120. Pinsent, B. R. W. 1962. Peroxidase regeneration and its effect on quality in frozen peas and thawed peas. J. Food Sci. 27,120-126. Pollard, A., and Kieser, M. E. 1951. The pectase activity of apples. J. Sci. Food Agr. 2,30-36. Porter, W. L., and Schwartz, J. H. 1962. Isolation and description of the pectinaseinhibiting tannins of grape leaves. J. Food Sci. 27,416-418. Porter, W. L., Schwartz, J. H., Bell, T. A,, and Etchells, J. L. 1961. Probable identity of the pectinase inhibitor in grape leaves. J. Food Sci. 26,600-605. Powell, J . F. 1953. Isolation of dipicolinic acid (pyridine-2: 6-dicarboxylic acid) from spores of Bacillus megatherium. Biochem. J. 54,210-211. Pratt, D. E. 1965. Lipid antioxidants in plant tissue. J. Food Sci. 30,737-741. Pratt, D. E., and Watts, B. M. 1964. The antioxidant activity of vegetable extracts. T . Flavone aglycones. J. Food Sci. 29,27-33. Privett, 0. S., Nickell, C., Lundberg, W. O., and Boyer, P. D. 1955. Products of the lipoxidase-catalysed oxidation of sodium linoleate. J . Am. Oil Chemists SOC. 32, 505-511. Ralls, J. W. 1959. Non-enzymatic formation of acetoin in canned vegetables. J. Agr. Food Chem. 7,505-507. Reddi, R. R., Esselen, W. B., and Fellers, C. R. 1950. The peroxidase activity of apple tissue. Food Technol. 4,63-68. Reid, W. W. 1950. Pectin degrading enzymes and their implication in the fruit products industry. In “Recent Advances in Fruit Juice Production” (V. S. Charley, ed.), pp. 145-161. Commonwealth Bureau of Horticulture and Plantation Crops. Rhee, K. S., and Watts, B. M. 1966a. Evaluation of lipid oxidation in plant tissues. J. Food Sci. 31,664-668. Rhee, K. S., and Watts, B. M. 196613. Effect of antioxidants on lipoxidase activity in model systems and pea slurries. J. Food Sci. 31,669-674. Rhee, K. S., and Watts, B. M. 1966c. Lipid oxidation in frozen vegetables in relation to flavour change. J. Food Sci.31,675-679. Richardson, V. E., and Tolbert, N. E. 1961. Oxidation of glyoxylic acid to oxalic acid by glycollic acid oxidase. J. Biol. Chem. 236,1280-1284. Robb, D. A., Mapson, L. W., and Swain, T. 1965. On the heterogeneity of the tyrosinase of broad bean (ViciafabaL.). Phytochemistry 4,731-740. Robb, D. A., Swain, T., and Mapson, L. W. 1966. Substrates and inhibitors of the activated tyrosinase of broad bean (Vicia faba L.). Phytochemistry 5, 665-675. Roe, J. H., Mills, M. B., Oesterling, M. J., and Damron, C. M. 1948. The determination of diketo-1-gulonic acid dehydro-I-ascorbic acid and I-ascorbic acid in the same tissue extract by the 2,4-dinitrophenyl hydrazine method. J. Biol. Chem. 174, 201-208. Sadoff, H. L., Bach, J. A,, and Kools, J . W. 1965. Significance of multiple forms of glucose dehydrogenase in relation to its heat resistance. Spores 3,97-110. Sakamura, S., Shibusa, S., and Obuta, V. 1966. Separation of a polyphenol oxidase for anthocyanin degradation in egg plant. J. Food Sci. 31,317-319. Sapers, G. M., and Nickerson, J. T. R. 1962a. Stability of spinach catalase. I. Purification and stability during storage. J. Food Sci. 27,277-281. Sapers, G. M., and Nickerson, J. T. R. 1962b. Stability of spinach catalase. 11. Inactivation by heat. J. Food Sci.27,282-286. Sapers, G. M., and Nickerson, J. T. R. 1962c. Stability of spinach catalase. 111. Regeneration. J. Food Sci. 27,287-290.

74

F. AYLWARD AND D. R. HAISMAN

Saunders, B. C., Holmes-Siedle, A. G., and Stark, B. P. 1964. “Peroxidase.” Butterworths, London. Schade, A. L., Levy, H., Bergmann, L., and Harris, S. 1949. Studies on the respiration of white potato tubers. 111. Changes in the terminal oxidase pattern of potato tissue associated with time of suspension in water. Arch. Biochem. 20,211-219. Schwimmer, S. 1944. The regeneration of heat-inactivated peroxidase. J . Biol. Chem. 154,487-495. Scott, D. 1961. Heat stabilised enzyme preparations. British Patent 918,056. Shallenberger, R. S., and Moyer, J. C. 1958. Relationship between pyrollidone carboxylic acid and an off-flavour in beet puree. J . Agr. Food Chem. 6, 604-606. Siddiqi, A. M., and Tappel, A. L. 1956. Catalysis of linoleate oxidation by pea lipoxidase. Arch. Biochem. Biophys. 60,91-99. Siegel, B. Z., and Galston, A. W. 1966. Isoperoxidases of Pisum satioum. Plant Physiol. 42,221-226. Singer, T. P. 1955. Plant carboxylases. In “Methods in Enzymology” (S. P. Colowick, and N. 0.Kaplan, eds.), Vol. 1, pp. 460-471. Academic Press, New York. Sironval, C., and El Tannir-Lomba, J . 1960. Vitamin E and flowering of Fragaria oesca L. Nature 85,855. Sizer, I. W., and Josephson, E. S. 1942. Kinetics as a function of temperature of lipase, trypsin and invertase activity from -70 to 50°C. Food Research 7,201-209. Smith, L., and Conrad, H. 1961. Studies on problems of cytochrome c oxidase assay. In “Haematin Enzymes” (J. E. Falk, R. Lemberg, and R. K. Morton, eds.), pp. 260-274. Pergamon Press, Oxford. Smouse, T. H., Mookherjee, B. D., and Chang, S. S. 1965. Identification of dec-1-yne in the initial autoxidation products of some vegetable oils. Chem. Znd. (London) 1965,1301-1303. Somogyi, L. P., and Romani, R. J. 1964. Irradiation-induced textural change in fruits and its relation to pectin metabo1ism.J. Food Sci. 29,366-371. Stearn, A. E. 1938. The theory of absolute reaction rates applied to enzyme catalysis. Ergeb. Enzymforsch. 7, 1-27. Sukhorokov, K. T., and Barkovskaya, G . E. 1953. Effect of low temperatures on plant enzymes. Byull. Glaon. Botan. Sada 16,55-60. Surrey, K. 1964. Spectrophotomeh.ic method for determination of lipoxidase activity. Plant Physiol. 39.65-70. Swartz, M. N., Kaplan, N. O., and Frech, M. E. 1956. The significance of “heat-activated” enzymes. Science 123,50-53. Swoboda, P. A. T., and Lea, C. H. 1965. Flavour volatiles of fats and fatcontaining foods. 11. A gas chromatographic investigation of volatile autoxidation products from sunflower oil.]. Sci. Food Agr. 16,680-689. Talburt, W. F., and Legault, R. R. 1950. Timelapse gets top blame for shelled-pea off-flavour.Food Inds. 22,1021. Tappel, A. L. 1954. The mechanism of vitamin E action. 11. Inhibition of unsaturated fatty acid oxidation catalysed by haematin compounds. Arch. Biochem. Biophys. 50,473-485. Tappel, A. L. 1955. Unsaturated lipid oxidation catalysed by haematin compounds. J . Biol. Chem. 217,721-733. Tappel, A. L. 1961. Autoxidation and antioxidants. In “NY Interscience” (W. 0. Lundberg, ed.), 1,325. Tappel, A. L. 1962. Haematin compounds and lipoxidase as biocatalysts. Zn “Symposium on foods: Lipids and their oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), pp. 122-138. Avi, Westport, Connecticut.

PLANT-TISSUE OXIDATION SYSTEMS

75

Tappel, A. L. 1966. Effects of low temperatures and freezing on enzymes and enzyme systems. In “Cryobiology” (H. T. Meryman, ed.), pp. 163-177. Academic Press, London. Tappel, A. L., Lundberg, W. D., and Boyer, P. D. 1953. Effect of temperature and antioxidants upon the lipoxidase-catalysed oxidation of sodium linoleate. Arch. Biochem. Biophys. 42,293-304. Tarladgis, B. G., Pearson, A., and Dugan, L. R. 1962. The chemistry of the 2-TBA test for the determination of oxidative rancidity in foods. I. Some important side reactions. J. Am. Oil Chemists SOC. 39,34-39. Tate, J. N., Luh, B. S., and York, G. K. 1964. Polyphenol oxidase in Bartlett pears. J. Food Sci. 29,829-836. Tewari, C . P., and Krishnan, P. S . 1960. Enzymatic transformation of dehydroascorbic acid to diketogulonic acid. Nature 188,144. Tewari, C . P., and Krishnan, P. S. 1960. Enzyme-catalyzed breakdown of dehydroascorbic acid in plant tissue.J. Food Sci. 26,416-421. Thimann, K. V., Yocum, C. S., and Hackett, D. R. 1954. Terminal oxidase and growth in plant tissues. 111. Terminal oxidation in potato tuber tissue. Arch. Biochem. Biophys. 53,239-257. Tilgner, D. J. 1965. Food Science and technology. Four principal tasks. Chem. Ind. (London)1965,711-718. Tolbert, N. E., and Burris, R. H. 1950. Light activation of the plant enzyme which oxidizes glycollic acid. J. Biol. Chem. 186,791-804. Tomiyama, K., and Stahmann, M. A. 1964. Alteration of oxidative enzymes in potato tuber tissue by infection with Phytophthora infestans. Plant Physiol. 39,483-490. Tonita, G., and Kim, S. S. 1965. Substrate effect on heat inactivation of Taka-amylase A. Nature 205, 46-48. van Arsdel, W. B. 1957. The time-temperature tolerance of frozen foods. I. Introduction-the problem and the attack. Food Technol. 11,28-33. van Arsdel, W. B., and Guadagni, D. G. 1959. Time-temperature tolerance of frozen foods. XV. Method of using temperature histories to estimate changes in frozen food quality. Food Technol. 13,14-19. van Buren, J. P., Moyer, J. C., and Robinson, W. B. 1962. Pectin methylesterase in snap beans. J. Food Sci. 27, 291-294. Vetter, J. L., Nelson, A. I., and Steinberg, M. P. 1958. Heat inactivation of sweet maize peroxidase in the temperature range 210-310°F. Food Technol. 12,244-247. Wagenknecht, A. C. 1957a. Occurrence of plasmalogens in lipids of green peas. Science 126,1288. Wagenknecht, A. C. 1957b. The lipids of green peas. J. Am. Oil Chemists SOC. 34, 509-513. Wagenknecht, A. C. 1959. Lipoxidase activity and off-flavor in underblanched frozen corn-on-thecob. Food Research 24,539-547. Wagenknecht, A. C., and Lee, F. A. 1958. Enzyme action and off-flavour in frozen peas. Food Research 2 3 , 2 5 3 1 . Wagenknecht, A. C., Lee, F.A., and Graham, R. J. 1958. Preparation of lipase from peas and the action of C. steapsin and pea lipase on crude pea lipid. Food Research 23, 439-445. Wager, H. G. 1964. Physiological studies of storage of green peas. J . Sci. Food Agr. 15, 245-252. Wainio, W. W. 1961.The composition of cytochrome c oxidase. In “Haematin Enzymes” ( J . E. Falk, R. Lemberg, and R. K. Morton, eds.), pp. 281-300. Pergamon Press, Oxford.

76

F. AYLWARD AND D. R. HAISMAN

Walker, G. C. 1963. Formation of free radicals during reaction of soya bean lipoxidase. Biochem. Biophys. Research Commun. 13,431-434. Walker, G. C. 1964a. Colour deterioration in frozen french beans. J . Food Sci. 29,383388. Walker, G. C. 1964b. Colour deterioration in frozen french beans. 2. The effect of blanching. J . Food Sci. 29, 389-392. Walker, J. R. L. 1964c. Studies on the enzymic browning of apples. AustralianJ. Biol. Sci. 17,360-371. Walker, J. R. L. 1964d. Polyphenoloxidase of pear fruit. Australian J . Biol. Sci. 17, 575-576. Wasserman, A. R., Gamer, J. C., and Burris, R. H. 1963. Purification of cytochrome c and other hemoproteins from wheat germ. Phytochemistry 2,7-14. Weurman, C. 1953. Pectinase inhibitors in pears. Acta. Botan. Neerl. 2, 107-121. Whitfield, F. B., and Shipton, J. 1966. Volatile carbonyls in stored unblanched frozen peas. J. Food Sci. 31,328-331. Wilder, C. J. 1962. Factors affecting heat inactivation and partial reactivation of peroxidase purified by ion exchange chromat0graphy.J. Food Sci. 27,567-573. Wilkinson, J. H. 1965. “Isoenzymes.” Spon, London. Witcoff, H. 1951. “The Phosphatides.” Reinhold, New York. Wood, C. B., and Lopez, A. 1963. Evaluation of quantitative methods of determining peroxidase in vegetables. Food Technol. 17,497-499. Wroblewski, F., and Gregory, V. F. 1961. Lactic dehydrogenase isozymes and their distribution in normal tissues and plasma and in diseased states. Ann. New York Acad. Sci. 94,912-932. Yamamoto, H. Y., Steinberg, M. P., and Nelson, A. I. 1962. Kinetic studies on the heat inactivation of peroxidase in sweet c0rn.J. Food Sci. 27, 113-119. Yamazaki, I., Mason, H. S., and Piette, L. 1960. Identification by electron paramagnetic resonance spectroscopy of free radicals generated from substrates by peroxidase. J . Biol. Chem. 235,2444-2449. Zelitch, I., and Ochoa, S . 1953. Oxidation and reduction of glycollic and glyoxylic acids in plants. I. Glycollic acid 0xidase.J. Biol. Chem. 201,707-718. Zoueil, M. E., and Esselen, W. B. 1959. Thermal destruction rates and regeneration of peroxidase in green beans and turnips. Food Research 24,119-133.

THE UTILIZATION OF FOOD INDUSTRIES WASTES BY

ITAMAR

BEN-GERAAND AMIHUDKRAMER

Department of Horticulture. College of Agriculture. University of Maryland. College Park. Maryland

I . Introduction.. ....................................................... I1. Vegetable-Processing Wastes ........................................... A . Introduction . . . . . . . . . ........................................... B. Tomato . . . . . . . . . . . . . ............................................ C . Asparagus ......................................................... D. Sweet Potato., .................................................... E. Potato ............................................................. 111. Fruit-Processing Wastes ............................................... A . Introduction ....................................................... B. Citrus By-products ................................................. C . Pear and Apple ..................................................... D . Pineapple. . . . . . . . . . . . . . . ................................ E . OtherFruits ....................................................... IV. Total Utilization of Plant Residues . .............................. A . Introduction . . . . . . . . . . . . . ................................ B. Vegetable Wastes as Leaf Meals .....................................

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

Wastes as Protein Sources ................................

E . Fruits .............................................................. V Oilseeds and Grains ................................................... A. Introduction ....................................................... B . Soybeans .......................................................... C. Cottonseed ......................................................... D. Coconut . . . . . . . . . . . . . . . ...................................... E . Safflower . . . . . . . . . . . . . . . ...................................... F. Grain Mill Feeds .................................................. VI . Starch-Production Wastes .............................................. A. Introduction., ...................................................... B. Wet Milling of Corn ................................................ C . Potato Starch. ............................................... VII . Sugar-Manufacturing Wastes ............................................ A . Introduction ........................................................ B . Bagasse and Beet Pulp .............................................. C. Molasses ...........................................................

.

78 80 80 81 85 86 87 88 88 89 94 95 96 96 96 98 99 100 104 106 106 108 108 110 112 114 115 115 117 122 122 123 124

77

78

ITAMAR BEN-GERA AND AMIHUD KRAMER

VIII. Distillery, Brewery, and Winery Wastes.. ............................... A. Introduction.. ..................................................... B. Distilleries.. ....................................................... C. Breweries.. ........................................................ D. Wineries ........................................................... IX. Animal and Marine Product Wastes. .................................... A. Dairy .............................................................. B. Meat and Poultry.. ................................................. C. Marine Products.. .................................................. References .............................................................

126 126 126 128 131 132 132 133 135 135

I. INTRODUCTION Waste utilization is both a necessity and a challenge. In the food industry the recovery and modification of wastes is becoming increasingly important. The aim is more complete utilization of the raw material, and minimization of the problems of pollution and waste treatment. Good examples of substantial and successful processing waste utilization already in effect are found in the meat-packing and citrus-processing industries. (Burch et al., 1963). With the increase in world population and the existing shortage of high-quality low-cost foods, recovery of nutrients from presently wasted sources and their utilization as foods or feeds will help curb the danger of increasing the gap between the world population and world food supplies. The transformation of a part of an agricultural crop or product from a state of discarded waste into a by-product, a coproduct, and finally into the main product, is a lengthy process, influenced and regulated by accumulated knowledge, needs, economics, and legislation on a worldwide or local basis. The relative value of different fractions may change from time to time, depending on the methods by which an agricultural commodity is broken down, as a result of harvesting, sorting, or processing procedures and techniques. The relative values of the different fractions of an agricultural commodity change as their importance and potential change. The prices of cotton and cotton by-products show this trend. Prices of cotton generally declined in the last ten years (U.S.D.A., 1966a). Cottonseed oil prices increased between 1955 and 1957 and have generally declined since then, while seed meal gained in price and importance as more knowledge was gained and more uses were developed. In less than a century, cottonseed grew from the stage of a waste and, later, a disposal problem in the cotton-producing areas, to a source of edible oil and animal feed and a promising source of

THE UTILIZATION OF FOOD INDUSTRIES WASTES

79

low-cost high-quality proteins to be used not only in animal feeding but also in feeding humans. The world shortage of proteins and food calories was recently discussed by Sen (1960), Altschul (1965a), Abbot (1966), and others. According to Brown (1965), food output per person in the future will increase in North America, western and eastern Europe, and Oceania, but the prospects for many of the less developed countries are not good, and in many of these countries it will be difficult to arrest the present downward trend in per capita food production. This will result in further deterioration of the present inadequate world food supplies. Channeling sources of energy and proteins from livestock feeding to human consumption, which involves both education and technological and scientific research and development, may help minimize the food shortage but will also create difficult problems. According to Szebiotko (1966), half or more of the potatoes produced in Poland and the USSR are used in animal feeding. More than 17,000,000 tons of oilseed meal and mill feeds of a high potential nutritive value for humans were used in the United States during 1964 for animal feeding (U.S.D.A., 1966a). Peru, the world's largest producer and exporter of high-protein fish meal (utilized mainly as animal feed), experiences the tragedy of food shortage and malnutrition. Overcoming this wastage of valuable nutrients is essential as part of a total effort to improve overall world food supplies. Thus, today's potential foods that are presently used as animal feeds must be diverted to human use. Then, one alternative would be to replace the animal feeds with synthetic mixtures free of proteins. This was demonstrated successfully by Virtanen (1966) in feeding trials with dairy cows. Utilization of urea as a nitrogen source was not found to have any adverse effect on the cow or on the milk produced, which opens the road to further developments leading to the release to human consumption of materials presently fed to livestock. Changes in agricultural and industrial practices have a direct influence on the types of wastes which become utilizable, as well as on established methods for waste utilization. The introduction of mechanical harvesting and automatic sorting of tomatoes in the United States will affect both the type and amount of tomato wastes. This new technique will make the tomato vine available for utilization while it is still of a potential nutritive value (Leffel, 1968). Changes in the industrial utilization of waste can be demonstrated with molasses. The use of molasses in production of ethanol in the

80

ITAMAR BEN-GERA A N D AMIHUD KRAMER

United States is constantly declining. Today, less than 2 %of the alcohol produced in the United States comes from molasses fermentation, whereas 30 years ago about 86% of U. S. alcohol production was from molasses. The present review mainly covers utilizable and potentially utilizable wastes originating from the production and processing of major foods of a plant origin. Only a brief account is given of animal and marine products. Emphasis is on the potential use of wasted agricultural commodities as feeds or foods, and attention is directed toward their protein fraction and its nutritive value in terms of the world protein shortage. Water is the biggest single wasted material in the food processing industry. According to Mercer (1965),an average of about 50 gallons of water is required per case of canned food. Recycling of waters, as well as renovation, is the key to the solution of problems related to local or seasonal water shortage and to pollution. This subject is not covered here. Important information has been provided by Hardenbergh and Rodie (1961),Isaac (1960),Gurnham (1955),Rhoads (1965, 1966),and Popper et al. (1967).

I I . VEGETABLE-PROCESSING WASTES A. INTRODUCTION

Utilization of vegetable-processing wastes has been reviewed by Wiegand (1937, 1944), Sanborn (1944, 1945, 1961), Morris (1946), Cruess (1958), Kelley (1958), Joslyn (1961), and Mercer (1965). Sorting, peeling, trimming, and coring are the major operations in the vegetable-processing industry that result in the formation of vegetable waste. Table I shows the amount of waste formed in the processing of different vegetable crops. The disagreements between the different reports in the data quoted reflect differences in defining what constitutes waste. Data in the table under Report 1 include only losses of edible material. The other data, in contrast, refer to total amounts of solid waste, including waste related to harvest, such as pea and bean vines, leaves, and corn husks. The United States Department of Agriculture (U.S.D.A., 1965) estimated for the period of 1951-1960 average annual losses during processing of more than 19,000,000 dollars in edible vegetable material, on the basis of the percent losses in Table I, the price of

THE UTILIZATION OF FOOD INDUSTRIES WASTES

81

the raw material, and the quantities processed. This includes loss of soluble nutrients through leaching during the different steps of processing, as well as losses from excessive peeling and trimming and the removal of culls. Some of the losses are uneconomical to recover. This is mainly the case with nutrients lost through leaching. Losses through blanching, reported by Dickinson (1960), are shown in Table 11. Kramer (1945) reported the distribution of proximate and mineral nutrients in the drained and liquid portions of canned vegetables (Table 111). Other types of waste are not yet fully utilized, mainly because of the seasonal nature of vegetable-canning operations and the perishable nature of the raw waste material (its high moisture content makes a drying treatment necessary if the material is to be utilized over a period of time). The high price of drying is still a major obstacle in the road to economical utilization of vegetable wastes (Dennison, 1967, private communication). According to Mercer (1965),utilization of dehydrated solid vegetable waste is largely confined to tomato wastes and, to a limited extent, asparagus and spinach wastes. Sanborn (1961) states that the only vegetable wastes utilized in quantity are pea vines and corn husks and cobs. These are utilized for animal feeding during winter time. Cruess (1958) agrees that pea and corn wastes are seldom wasted but are used as livestock feed. Cobs and husks are usually shredded and transported to dairies and livestock establishments, where they are ensiled or utilized fresh. Problems in utilization of vegetable and fruit processing or production wastes as foods or feeds may rise from the level of residue of chemicals found in them. The level of concentration of the types of residues which are under control are determined in the United States by the Food and Drug Administration and published as Official FDA Tolerances in the Federal Register, in the annual publication of the Association of American Feed Control Officials, and in the N.A.C. News and Pesticide Review of the National Agricultural Chemicals Association. This section discusses utilization of processing wastes. Waste related to harvest and preparation procedures are discussed later (Section IV).

B. TOMATO Rabak (1917)estimated the average annual quantity of wasted skins and seeds from tomato-processing plants in the United States at 16,000 tons on a wet basis (approximately 3,000 tons of dry material).

82

ITAMAR BEN-GERA AND AMIHUD KRAMER TABLE I VEGETABLE CROPS: LOSSESRELATED

TO

PROCESSINGa

Commodity

Report lb

Report 2c

Report 3d

Asparagus Beans, green Beans, lima Beets Broccoli Brussels sprouts Cabbage Carrots Cauliflower Corn Cucumber, pickling Peas Potatoes Spinach Sweet potatoes Tomatoes

3.2 5.0 6.0 7.0 20.0 10.0 5.0 18.0 8.0 20.0 5.0 6.0 5.0 10.0 15.0 5.0

41 12 85 25 25 33 86 79 40 25

30 38

40

-

52

Report 4'

20

-

-

-

-

-

-

72

-

-

8

8

-

-

-

40

-

"Data presented given in % of weight. bFrom U.S.D.A. (1965). 'From Sanborn (1945). dFrom Mercer (1965). eFrom Dickinson (1960).

TABLE I1 LOSSESOF SOLUBLES DURING BLANCHINGOF VEGETABLES' Percentage of total sugars lost

Vegetable Fresh peas* French beans Whole Sliced Broad beans Carrots Whole Sliced Diced Rutabagas, diced Dried peasb Dried beansb

1-min Water blanch

Percentage of protein lost

Water blanch

3-min Steam blanch

1-min Water blanch

3-min Water blanch

3-min Steam blanch

9-12

18-22

12-18

1-9

10-14

0-5

0 19 2

3

34 12

0 13 12

8 0

3 13 5

3 3 0

14 27 13 42 7- 10 14-16

11 26 17 15 1 2- 6

10 30 23 16 1 0-2

10 37 25 19 8- 12 2-6

10 26 7 11 4-10 0-2

20

-

35 2 9-1 1

"From Dickinson (1960). bAccording to variety.

3-min

T H E UTILIZATION O F F O O D INDUSTRIES WASTES

83

TABLE 111 PERCENT

OF

Product Asparagus max. min. av.' Beans, green max. min. av.c Beans, lima max. min. Beets max. min. av.d Carrots max. min. av.d Corn max. min. av.' Peas max . min. av.c Spinach max. min. avSd

TOTALNUTRIENTSOF ENTIRE CAN FOUND IN LIQUID PORTIOi@ Protein

Fat

Fiber

Ash

Carbohydratesb

Calcium

Phosphorus

Iron

20.4 10.2 14.3

6.0 2.6 4.4

8.4 2.0 3.9

38.3 27.9 33.1

30.8 25.0 27.6

37.2 22.6 30.3

25.3 15.4 19.5

34.8 20.8 27.0

P

e

6.6 2.2 4.4

38.6 35.1 37.2

25.3 19.2 22.7

19.4 10.4 16.6

31.4 23.0 26.7

29.1 18.4 24.5

12.4 7.7

2.0 0.0

1.8 0.6

39.5 31.4

10.9 8.6

32.3 23.1

30.7 25.3

38.1 26.6

35.1 27.2 31.2

e e

1.9 1.o 1.5

40.2 28.2 31.7

35.9 22.7 28.7

12.6 9.4 11.1

39.6 26.4 31.4

29.3 15.0 24.9

22.3 10.7 16.8

c c

1.9 1.6 2.6

40.2 21.7 26.9

35.9 24.2 28.9

12.6 13.5 17.2

39.6 16.1 23.5

29.3 20.6 33.4

9.6 6.6 7.8

4.2 0.6 1.5

2.4 1.1 1.6

37.2 27.1 32.6

17.8 10.6 14.9

26.1 16.5 22.7

37.9 24.6 30.1

35.1 20.6 25.5

18.6 11.1 14.3

5.7 3.6 4.6

1.4 0.3 0.9

40.3 34.8 38.1

27.4 13.2 17.4

23.0 14.8 17.5

29.5 23.2 25.8

35.7 20.4 27.7

P

2.7 0.8 1.3

29.0 21.4 23.8

27.9 19.5 22.1

1.1 0.3 0.7

32.9 23.3 27.6

14.3 9.3 11.5

17.7 10.4 15.4

7.6 5.5 6.3

c

F

P

e

"From Kramer (1945). By permission of.American Dietetic Association. bCarbohydrates (other than crude fiber) by difference. CAverageof 5 samples, each consisting of 4 cans. dAverage of 5 samples, each consisting of 6 cans. %significant quantity.

Edwards et al. (1952) reported that recoverable tomato waste, based on data for 1941 to 1950, amounts to about 19% of the total solids in the original tomatoes. For the period mentioned, this amounted annually to 33,131 tons of tomato solids. The 1965 commercial crop of tomatoes for processing was close to 5,000,000 tons (U.S.D.A.,

84

ITAMAR BEN-GERA AND AMIHUD KRAMER

1966).According to Ries and Stout (1962), about a third of the tomatoes delivered to processing plants end as processing waste. Thus, with 5,000,000 tons of tomatoes processed annually, more than 2,000,000 tons are wasted. The introduction of mechanical harvesting is estimated to have doubled the quantity of such waste material. These figures do not include material that remains in the field. The tomato seed cake, a by-product of the tomato oilseed recovery process, is a product of considerable interest. The seed cake contains, on a dry basis, 37% protein according to Carusi (1945), and over 39% according to Vecchiotti and Piva (1964). Table IV shows the amino acid composition of the seed cake protein. The amino acid composition of the tomato seed proteins compares favorably with that of soybean protein. Limited experimental evaluation of the nutritive value of the seed cake proteins was reported. Vecchiotti and Piva (1964) report that rnethionine was the limiting amino acid in feeding tests with pigs. Mainardi (1951) reported that heating decreased the nutritive value of the seed cake proteins as determined in feeding tests with rats. TABLE IV AMINO ACID COMPOSITION OF TOMATO SEED PROTEIN AS COMPARED WITH ESSENTIALAMINO ACID PATTERN OF COMMERCIAL SOYBEAN PROTEIN ISOLATE Amino acid

Tomato seed proteina (%)

Commercial in soy protein isolateb (%)

Lysine Histidine Arginine Threonine Tryptophan Valine Methionine I soleucine Phenylalanine Leucine Aspartic acid Glutamic acid Serine Proline Glycine A 1anin e Tyrosine Cystine

5.41 1.98 8.66 3.20 1.01 3.96 1.67 3.93 4.21 5.68 9.68 19.33 5.60 5.07 4.83 4.49 3.43 0.80

6.3 2.4 6.6 3.4! 1.3 4.8 1.1 4.6 5.4 8.4 n.v. n.v. n.v. n.v. n.v. n.v. n.v. n.v.

"From Vecchiotti and Piva (1964). "Supra 610, Ralston-Purina Company technical data sheet.

THE UTILIZATION OF FOOD INDUSTRIES WASTES

85

Most of the attention directed toward the recovery of tomatoprocessing wastes has been concentrated on utilization of the tomato pulp and pomace as animal feeds. Although Webster (1929) reported on recovery of the solid waste from tomato-processing plants and its use as a fertilizer, Rabak (1917) and Hays (1919) showed earlier that wastes from the manufacture of catsup (tomato skins, seeds, and cores) were used satisfactorily in swine feeding. Templeton (1947) and Edwards et al. (1952) studied the cost of dehydrating tomato-processing wastes. Dehydration of the wastes and their use as animal feeds was suggested b y those workers as the most economical method for disposing of these wastes. Tomato pomace, a dry product consisting of skins, pulp, and crushed seeds that remain after extraction of the tomato juice, was found b y Stewart (1931) to be useful in cattle feeding, by Tomhave (1931, 1932) to be suitable for dairy cow feeding when included in the diet at the 15% level, by Tange (1937) to be of low efficiency with swine but well accepted and utilized by sheep when included in the diet at a 30% level, and by Esselen and Fellers (1939) to be well accepted by chicks when included in the diet at a level of 11.6%. In addition to the nutritive value, several reports were published on the antidiarrheal effect of tomato pomace in humans and animals (McCay and Smith, 1940; Smith, 1941; Morrison, 1946). Dried tomato pulp is another product made of tomato wastes. It consists of whole cull fruit. Cull tomatoes have become a serious waste-disposal problem in the tomato-growing regions of the United States (Brooke and Capel, 1958). Dehydration of these culls not only solves a waste disposal problem but produces a product of commercial value. Limited experimental evaluation of the nutritive value of the tomato pulp was reported. Chapman et al. (1958) reported satisfactory gains by grazing steers which consumed 16 to 19 lb daily of a concentrate in which 10 to 30% of the dried citrus pulp was replaced by dry tomato pulp. Ammermann et al. (1963a, 1965) studied the nutritive value of dried tomato pulp for steers, lambs, and poultry. Some of their results are given in Table V. In lamb feeding tests Ammermann et al. (1963a) found that the Net Apparent Value of nitrogen was 2976, compared with 44% for soybean meal. C. ASPARAGUS The 1965 crop of asparagus for processing consisted of 119,150 tons (U.S.D.A., 1966a). Losses related to processing are rather high, as

86

ITAMAR BEN-GERA AND AMIHUD KRAMER TABLE V COMPOSITION AND DIGESTION COEFFICIENTS FOR TOMATO PULP IN STEERS

FEE DING"^ Crude

Composition Digestibility, tomato pulp fed with hay Digestibility, tomato pulp fed alone

Ash

Protein

fiber

Nitrogenfree extract

Ether extract

10.6

24.0

17.8

43.3

4.2

-

58.0

50.1

79.5

94.6

-

54.6

42.2

78.4

85.8

"From Ammermann et al. (1963a). By permission of American Chemical Society.

* 70.

can be seen in Table I. Some estimates run as high as 50% (Cruess, 1958). These losses result from discarding the butts of the stalks prior to canning or freezing. Some of these butts are used in preparing soup stock or lower-grade products. Asparagus wastes contain 93 to 97% moisture. If these wastes can be dried economically, they can be turned into stock feed (Cruess, 1958). Kline et al. (1944)reported the utilization of waste asparagus butts for recovery of bacterial proteinase. Asparagus-butt juice was also found to be an excellent medium for antibiotics production (Sanborn, 1945).Cruess (1958)and Mercer (1965) report that most asparagus waste is discarded, with only limited dehydration or use as animal feed. D. SWEETPOTATO About 900,000 tons of sweet potatoes were produced in the United States alone during 1965 (U.S.D.A., 1966a). Of this approximately 100,000 tons were canned (Almanac, 1966). Losses during processing are heavy (Scott, 1967, private communication) and may be as high as 52.5-53.5%.This includes losses due to trimming and cutting, as well as to the brushing and polishing of the peeled potato and irrecoverable losses from excessive lye peeling. The latter reaches 15%(U.S.D.A., 1965).Recoverable losses are estimated at 10 to 20%. Several workers (Grimes, 1941; Southwell and Blak, 1948) have established that sweet potato meal is a satisfactory complete or partial replacement for corn in finishing rations for beef cattle. Recently Bond and Putnam (1967) studied the nutritive value of dehydrated sweet potato trimmings fed to beef steers. Their findings indicate that this product, although somewhat less nutritious than corn, did not negatively affect the properties of the carcasses produced and

THE UTILIZATION OF FOOD INDUSTRIES WASTES

87

may have a place in cattle feeding, particularly under conditions when grain availability is limited. Another possible way to utilize sweet potato trimmings is suggested by Gray and Abou-El-Seound (1966a). Those workers utilized whole sweet potatoes as a substrate for Fungi impeIfecti. After an incubation period of 4 days, it was found possible to produce 81.2 lb of dried product from 100 lb of dry sweet potatoes. This dry product contained 31.6 lb protein, compared with 6.9 lb protein in 100 lb dry sweet potatoes. Kobayashi et al. (1964) reported 26% crude protein in dry residue of sweet potato alcohol distillers stillage. This approach was reviewed by Gray (1966). A number of new products are recently being produced from trimmings as well as from over- and undersize sweet potatoes. Among these are dehydrated flakes, granules, chips, prepared frozen whole, slices, mashed potatoes, and baby food purke (Dowskin et al., 1963). E. POTATO A large amount of information on potato waste utilization is found in the International Symposium on Utilization and Disposal of Potato Wastes (1966). According to preliminary data on world production of potatoes, the 1965 crop reached 230,000,000 tons (U.S.D.A., 1966). The Soviet Union is the largest potato producer, followed by Poland, West Germany, the United States, and France, in that order. According to Szebiotko (1966), 50% of the crop in the Soviet Union and about 55% of the Polish crop is fed to livestock. About 5 % of the United States potato crop is fed to animals (U.S.D.A., 1966a) and about 30% of the potato crop is processed into chips, french fries, and canned or dehydrated products (U.S.D.A., 1966a), with other estimates reaching as high as 50%. Losses of 5 % of edible material in potato processing were reported by the U. S. D. A. (1965). Table VI shows losses during the processing of potatoes for french fries. Some of the Iosses are recoverable and could be utiIized by the potato starch and alcohol industries. Such is reported by Szebiotko (1966) to be done in Poland. Attention was directed by Shaw (1966) toward recovery of edible wastes in potato-products processing plants, and he pointed out possible products from edible potato wastes. He suggested recovery of waste resulting from cutting or slicing, screening and sizing, off-grading in the manufacture of dehydrated potatoes as well as chips and french fries. Satisfactory utilization of potatoes as well as solid potato waste as a culture medium for yeast growth was reported by

88

ITAMAR BEN-GERA AND AMIHUD KRAMER TABLE VI LOSSESDURING PROCESSING OF POTATOES

FOR

FRENCHFRIESa’*

Plant A

Plant B

21.6 0.7 2.5

17.6 2.4 7.2

Peeling Trimming Grading OFrom Hesen (1966). % weight of raw material.

Janicki et al. (1966). Protein content reported in the dried yeast concentrate was 4 to 5 times as high as that of the raw material. Kramer et al. (1968) reported an increase in nitrogen content in potato discs stored under controlled atmosphere conditions. After several weeks of storage of discs in anaerobic conditions, nitrogen in the headspace of the container was depleted. They suggest that, in the total absence of oxygen, atmospheric nitrogen could be utilized through anaerobic fermentation for enriching the protein content of food materials of plant origin. It is thus possible that potatoes, a low-protein feed, could be enriched and utilization would become more efficient.

I II. FRU IT-PROCESSING WASTES

A. INTRODUCTION

Sanborn (1944, 1945, 1961), Cruess (1958), Joslyn (1961),and Mercer (1965) reviewed the literature in the field of fruit-processing wastes. Fruit wastes consist mainly of peels, cores, trimmings, pits and seeds, culls, and overripe and blemished fruit. As with vegetables (Table I), the percentage of processing waste reported varies with the source of information, depending largely on definition of the term “ waste.” Data on fruits are shown in Table VII. The data from Reports 2,3, and 4 are based on the amount of product produced from total raw material, whereas the data from Report 1 are related to the loss of edible material only. This loss is estimated at more than $45,000,000 annually, on the basis of average annual losses in processing between 1951 and 1960 (U.S.D.A., 1965). Among the fruits processed in the U. S. the most important processing wastes are derived from citrus fruits, apples, peaches, apri-

THE UTILIZATION OF FOOD INDUSTRIES WASTES

89

TABLE VII FRUIT-PROCESSING WASTES" ~

Apples Apricots Cranberries Cherries, sour Grapefruit Lemons Oranges Peaches Pears Strawberries

Report lb

Report 2b

Report 3b

Report 4b

35 25

47

55

-

-

15 58

-

-

40 42

11 46

12 8.0 2.0 5.0 3.0 3.0 3.0 20 12 10

-

-

-

-

10 -

-

10

"In %. bReports 1, 2, 3, and 4 are identified in Table I.

cots, pears, pineapples, and cherries. Wastes from tropical and other fruits marketed fresh are probably greater (Watkins, 1967).

B. CITRUSBY-PRODUCTS Citrus by-products, which represent between 45 and 58% of the original material before extraction of the juice, can be classified into 3 main groups: (1) animal feed; (2) raw material for the production or recovery of valuable materials; and (3) food products. This is shown in Fig. 1. The feeding value of citrus-processing waste was early recognized by Anon. (1922-1923), Mead and Guilbert (1926), and Reagan and Mead (1927). Scott (1926) used dried citrus pulp satisfactorily as a cattle feed. Dried citrus pulp has been produced in Florida on a commercial basis since 1932 (Hendrickson and Kesterson, 1965) and in California as early as 1927 (U.S.D.A., 1956). Dried citrus meal for animal feeds is still considered the principal waste-recovery product (Hendrickson and Kesterson, 1965; Sanborn, 1961). The procedure for the manufacture of dried citrus pulp is described in detail by Braverman (1949), U.S.D.A. (1956)and Hendrickson and Kesterson (1965). It consists essentially of a two-step water-removal operation. The first step is aimed toward destruction of the hydrophilic nature of the pectin in the peel, by liming, and then removal of water from the peel, by pressing. This step decreases peel moisture content from about 82 %to about 72 %. Further drying is accomplished in rotating dryers, where moisture content is reduced to 6 4 % .The composition of citrus feed products is shown in Table VIII.

90

ITAMAR BEN-GERA A N D A M I H U D KRAMER

FIG. 1. Origin and interrelationship of citrus by-products. From Hendrickson and Kesterson (1965).

Recently, Ammermann et al. (1966) discussed the factors affecting the physical and nutritional properties of dried citrus pulp. Dry citrus pulp is used mainly in feeding dairy and beef cattle. A review by the U.S.D.A. (1956) stated that citrus pulp is similar in feed value to beet pulp, being low in crude protein, fiber, and fat, but high in nitrogenfree extract or carbohydrate, which is 88 to 92% digestible. This is supported by studies of Becker and Arnold (1951), who found that dried citrus pulp was fully equal to dried beet pulp for milk production and that it is a desirable component of dairy and beef cattle feed. Kirk and Davis (1954)found dried citrus pulp a good source of energy in a feeding test for maintenance and fattening of beef, and Peacock and Kirk (1959) found it of the same value as corn feed meal and ground snapped corn in feeding trials with growing steers. Literature from previous studies is reviewed by von Loesecke (1950). Citrus molasses is the product of the concentration of citrus-peel press liquor. Table IX shows a typical analysis of Florida citrus molasses. Von Loesecke (1950), U.S.D.A. (1956), and Hendrickson and Kesterson (1964, 1965) reviewed the literature related to citrus molasses. Studies conducted by the Florida Agricultural Experiment Station demonstrated the good feeding value of citrus molasses for

THE UTILIZATION O F FOOD INDUSTRIES WASTES

91

TABLE VIII OF CITRUSFEEDPRODUCTSO COMPOSITION

Citrus product

Dry matter

Crude protein

(70)

(%I

(94

1.07 0.96 1.01 1.23 2.01 2.24

Whole fruit 13.64 Grapefruit, cullb Oranges, gratedb 14.84 Tangerinesb 17.39 Undried peel residue Citrus peelC 18.49 Citrus peel, pressedC 28.27 Grapefruit peel, pressedb 25.23 Dried citrus residue Citrus pulp (71 analyses)d 90.1 Citrus pulp (10 analyses 6465 productsj 92.0 Citrus pulp, sweetenedd 92.0 Grapefruit pulp (3 seasons)e 100 Grapefruit pulp (5 lots)’ 100 Dried meal Citrus mealb 88.27

Crude fiber

Nitrogenfree extract

Ash

( %)

Crude fat (%)

1.39 1.58 1.38

10.03 11.34 13.62

0.64 0.32 0.80

0.51 0.64 0.58

2.22 4.36 4.61

12.48 17.80 15.74

1.83 2.65 1.18

0.73 1.45 1.46

(%)

5.9

11.5

62.7

3.1

6.9

6.2

5.3 6.4 7.0

12.0 9.3 12.1 15.3

64.0 66.6 56.9 -

4.9 2.8 5.5 5.9

4.9 8.0

6.47

12.39

60.55

2.94

5.93

-

6.5

DFromHendrickson and Kesterson (1965). bKirk et al. (1949). cBecker et u1. (1946). dMorrison (1949). eArnold et al. (1941). Tulley and von Loesecke (1940).

cattle and swine. It has been shown to be palatable to all classes of beef cattle and used successfully to replace ground snapped corn in fattening rations. Citrus molasses is used mainly as feed, to a large extent mixed with dried citrus pulp. Dried citrus seed meal was the subject of several reports. The seedy varieties of citrus fruit contain 3.5% seed (U.S.D.A.,1956). Deriggers et al. (1951) reported that approximately one pound of seed can be secured from each box of processed oranges or seeded grapefruit. Ammerman et al. (1963b) examined 25 samples of dried citrus pulp and found them to contain 1.8 to 8.3% seeds. The composition of grapefruit seed press cake after partial removal of the oil is given in Table X. Ammermann et al. (1963b) found that citrus seeds, recovered from dried citrus pulp, contained (on a 90% dry-matter basis) 3.1% ash, 14.6% protein, 40.5% ether extract, 12.1%crude fiber, and 19.7%

92

ITAMAR BEN-GERA AND AMIHUD KRAMEK TABLE IX TYPICAL ANALYSISOF FLORIDA CITRUS MOLASSES' Constituent

%

"Brix Sucrose Reducing sugars Total sugars Moisture Protein (N X 6.25) Nitrogenfree extract Fat Fiber Ash Glucosides Pentosans Pectin Volatile acids Potassium (K) Calcium (Ca)

72.0 20.5 23.5 45.0 29.0 4.1 62.0 0.2 0.0 4.7 3.0 1.6 1.0 0.04 1.1 0.8

Constituent

70

Sodium (Na) Magnesium (Mg) Iron (Fe) Phosphorus (P) Manganese (Mn) Copper (Cu) Silica (SiO,) Sulfur (S) Boron (B)

0.3 0.1 0.04 0.06 0.002 0.003 0.04 0.17 0.0006

Niacin (ppm) Riboflavin (ppm) Pantothenic acid (ppm) Viscosity 25°C (centipoises) PH

35 11 10 2000 5.0

'From Hendrickson and Kesterson (1965).

TABLE X COMPOSITION OF GRAPEFRUIT SEED PRESS CAKE" Constituent Moisture Nitrogen as ammonia Nitrogen as protein Crude fat (ether extract) Crude fiber Ash Calcium Iron Magnesium Phosphates Silicone Sodium and potassium chlorides Sulfur

% 3.43 4.21 21.60 13.95 26.50 4.04 .35 .0014 .39 * 55 .081 2.48 .088

'From Nolte and von Loesecke (1940). nitrogen-free extract. A high-protein meal is obtained by removal of the ether solubles (fat). Glasscock et al. (1950), Deriggers e t al. (1951), and Ammermann et al. (196313) studied the nutritive value of citrus seed meal. When supplying 88% of the total protein in lamb rations, protein in dried citrus meal was found to be equal in digestibility and

THE UTILIZATION OF FOOD INDUSTRIES WASTES

93

biological value to protein from two samples of soybean meal (Ammerman et al., 196313).The toxic factor for swine and chickens was studied by Deriggers et al. (1951), and a method developed for its removal. According to Hendrickson and Kesterson (1965), citrus seed meal is presently blended with dried citrus pulp for cattle feed. Within the last decade there has been a substantial increase in the use of citrus in the manufacture of “comminuted juice.” The peel is finely comminuted and used as a fruit drink base (Charley, 1963; Braverman and Levi, 1960). This product has found a wide market in Europe, but has not yet gained a foothold in the United States. Several products of commercial value are recovered from citrus wastes. These are mainly the citrus peel oils, citrus pectin, flavonoids, and citrus seed oils. Detailed reviews describing recovery and utilization of these products were published by Braverman (1949), Joslyn (1961), Cruess (1958), U.S.D.A. (1956), Kefford (1966), Hendrickson and Kesterson (1954, 1965), Hull et al. (1953), and Kesterson and Hendrickson (1957). Several products from citrus-fruit residue are manufactured as food items, including brined and candied peels, marmalades, bland syrup, and peel seasoning. Detailed discussion of these products is found in the above-mentioned references. In addition to being concentrated into molasses, peel juice, or pressed liquor, they can be utilized as a carbohydrate source for fermentation for the production of feed yeast, industrial alcohol, vinegar, butylene, glycol, and lactic acid. Feed yeast, containing (on a dry basis) 50 to 55% protein, can be produced on a growth medium consisting of diluted citrus peel juice, enriched with minerals (U.S.D.A., 1956). This process was studied both in a batch-type (Nolte et al., 1942) and in a continuous operation (Veldhuis and Gordon, 1947). Yields as high as 60% (weight of yeast per weight of total sugar content of the medium) were reached. The protein in the product is low in methionine, and thus should not be considered as a possible single source for dietary protein. Industrial alcohol can be made from citrus peel juice or citrus molasses. Nolte et d. (1942) demonstrated the economic feasibility of this process. Long and Patrick (1961) investigated the conversion of peel juice to 2,3-butylene glycol through fermentation. Two Aerobacter species, A. aerogenes strains NRRL B-119 and A-101, were studied. Yields were 4.8 to 5.3%. McNary and Dougherty (1960) studied the possibilities of vinegar making from orange peel juice through acetic fermentation. Elimination of peel oil from the fermentation medium resulted in a satisfactory product. It appears, therefore, that the technology exists for manufacturing

94

ITAMAR BEN-GERA AND AMIHUD KRAMER

these fermentation products. Raw material for their production is available in the form of citrus wastes throughout the citrus-producing areas in the world. The efficiency of the processes, as well as availability of competitive raw materials, will determine whether citrus waste will be utilized for such purposes.

c. PEAR AND APPLE Potter et al. (1948) determined the composition of pear cannery waste for its by-product value. Brown et al. (1950)reviewed previous work on the utilization of pearcanning waste. They obtained 110 lb of pear pomace (8% moisture) and 270 lb of molasses with 50% sugar content from one ton of pear cannery waste. Graham et al. (1952) described the process and equipment used for recovery of pear-waste molasses and pomace in California. The plant described could process 150 tons of pear waste in a 24-hour day. Larger operations on a commercial basis were not found profitable (Mercer, 1965). Neubert et al. (1954) reported on a process of recovery of pear juice from waste to be utilized in canning pears. The process involves liming to facilitate separation of the juice from the solids, removal by ion exchange of the excess calcium, and decoloring. The possibility was suggested of replacing one-third of the refined sugar used in canning pears. This method of recovery of edible components of waste material, even if found to be economically feasible, might be questioned from the legal standpoint and dependent on high standards of hygiene. According to Mercer (1965) no commercial attempts to utilize fruit wastes by this method are known. Adams (1949) suggested the utilization of pear, apple, and cherry wastes for alcohol production through fermentation. This was done successfully (Anon., 1949). Stubbs et al. (1944) showed that Torula yeast (Torulopsis utilis) grows well on medium prepared from pear, apple, and peach press juice. Yields were 50%or higher, calculated on weight of sugar utilized for production of a given weight of yeast. Cruess (1958) reviewed the possibilities for the production of brandies from dried cull and surplus apples, grapes, oranges, and other fruits. Masuda et al. (1964) reported on the possible utilization of apple pomace and pulp for the manufacture of apple cider, and Amemiya et al. (1964)reported on the production of apple brandy of good quality from apple pomace through fermentation. The value of apple pomace as an animal feed has been reviewed by Smock and Neubert (1950).It can be fed fresh, dried, or as silage, and was generally found to be palatable to cows and sheep but of limited value in pigs’ diets.

THE UTILIZATION OF FOOD INDUSTRIES WASTES

95

Smock and Newbert (1950) and Tressler and Joslyn (1961) reviewed the production of apple pectin from apple pomace. Apple pomace and wastes from the processing of pears and other fruits or vegetables are possible sources for the manufacture of pectin (Baker and Goodwin, 1938; U.S.D.A., 1946; Tressler and Joslyn, 1961). At present the pectin industry requires that the raw material be rich in pectin and low in price, or offer a product of exceptional and special properties. Because of competition, the production of pectin from any source other than citrus, particularly lemon peels, is limited. In the United States there is no sizable production of pectin from apples or apple pomace. Apple pectin is produced in Europe to a greater extent.

D. PINEAPPLE Sugar-syrup for canning, alcohol, cattle feed, and organic acids are being produced on a commercial basis from pineapple processing wastes (Collins, 1960). The first by-product was the mill juice recovered from pineapple waste in order to facilitate its disposal. It contains about 11% soluble solids, of which 75-80%are sugars, 7-9% citric acid, 2 % malic acid, and 2.5-4% protein. Gould and Ash (1915) developed a clarification method consisting of partial removal of the citric acid (as a calcium salt), heat precipitation of the protein, and decolorization with charcoal. The clarified juice was used as a solvent for additional sugar for pineapple canning. According to Felton (1949) only one-third of the mill juice produced was utilized in this way. The rest was concentrated and fed to livestock or used as a syrup in pineapple canning. The use of concentrated mill juice in the canning of pineapple was found to be undesirable since it accelerated the development of off-flavors during storage of canned pineapple products. Treatment of clarified mill juice by ion exchange to remove almost all salts and organic acids prior to concentration was reported by Felton (1949). Syrup obtained by this process is used successfully in canning pineapples. Between 30 and 45 lb of sugar equivalent for human consumption is recovered by this process from each ton of fresh pineapple for processing. Henke (1931, 1945), Work and Henke (1940), and Henke et al. (1945) studied the feed value of pineapple wastes. Pineapple bran - a product obtained by drying pressed fruit shells and pressed pulpwas found by Henke (1931) to be a suitable feed for cattle, pigs, and chickens. One ton of processed pineapple yields 60 to 70 lb of dry pineapple bran. Collins (1960) discussed other possible feeds: alco-

96

ITAMAR BEN-GERA AND AMIHUD KRAMER

hol, feed yeasts, citric and malic acids from the mill juice; and starch, proteases, and fiber production from the plant residues. Utilization of waste pineapple juice for vinegar production was recently studied by Spurgin (1964)and Richardson (1967). E. OTHERFRUITS Fruit pits accumulate as waste from the canning of peaches, apricots, prunes, cherries, and other fruits. Recovery of oil and feed meal from the kernels, as well as other ways of utilization of the pits, was reviewed by Marshall (1954), Cruess (1958), Joslyn (1961)and Dang et al. (1964). As noted above, considerable progress has been made in the utilization of fruit-processing wastes in tropical and semitropical regions, particularly in the United States and Europe. Much less utilization of tropical fruits has been accomplished, even with highly commercial fruit crops such as bananas and cocoa. These are discussed in detail in the next section. IV. TOTAL UTILIZATION OF PLANT RESIDUES A. INTRODUCTION

Only 20 to 30%of the vegetable plant goes for human consumption in the United States (Willaman and Eskew, 1948). The rest is wasted. Considering the entire plant, most of the waste consists of leaves and stems. Efficient utilization of residual plant materials should be directed toward their nutritive value as food or feed. Such a procedure would not only solve a health and pollution problem but increase the value of the crop and the rate of food production per unit of cultivated farm land. The composite value of an agricultural crop was demonstrated by Ben-Sinai et a2. (1965a,b) in the case of peas. In that study, the food and fodder value of pea plant parts was related to harvest time and variety. The value of pea seeds is influenced by their tenderness, and the value of pods and vines reflects their chemical composition (Tables XI, XII). It was found that the stage of development of the pea plant has a turning point beyond which the crop would be utilized more economically as feed to animals. This turning point, differing with varieties of peas, represents poor acceptability for human consumption and, thus, a low commercial value. Similar data demonstrate

THE UTILIZATION O F FOOD INDUSTRIES WASTES TABLE XI TOTALVALUE OF AN ACRE

OF

Peas

A. Wet weight, lb/acre: Fancy Extra Standard B. Dry weight, lb/acre: Fancy Extra Standard C. Protein, lb/acre: Fancy Extra Standard D. Carbohydrates, lb/acre: Fancy E xtra Standard

97

PEAS"

Pods Vines

2452 3983 2925 3474 2530 2598

Total

6418 4964 3515

12,853 11,363 8,643

506 640 606

578 482 303

1211 967 826

2295 2089 1695

132 149 113

69 52 27

195 122 64

396 323 204

303 403 421

338 287 180

578 424 354

1219 1114 955

"From Ben-Sinai et al. (196513). Permission by Institute of Food Technologists.

TOTALRETURNSFROM PEA

TABLE XI1 ($/lo00 LB SEED), 1962 AND 1963"

CROPS

Total value of crop ($) Code Alaska peas 62-2 63- 1 62-4 63-2 63-4 62-6

Tenderometer Value of Seeds used value seeds ($) as food 92 99 120 152 199 208

60.00 60.00 58.00 12.50 12.50 12.50

78.00 78.17 67.70 27.53 23.07 21.91

Vined seeds Unvined plants used as fodder used as fodder 25.10 25.87 18.60 24.63 22.24 19.76

34.10 34.87 27.60 33.63 31.24 28.76

"From Ben-Sinai et nl. (1965a).

the additional nutritive value obtainable from lima beans and green beans (Kramer, 1964). Plant material has a high moisture content. To improve its keeping qualities and preserve it as a food or feed, removal of the water or the creation of conditions for desirable microbiological action is necessary. Practically, this may be achieved by dehydration or by ensilage.

98

ITAMAR BEN-GERA A N D A M I H U D KRAMER

The high protein content of leaves, which contributes to their nutritive value, has attracted the attention of many workers. In this respect leaves and stems, as vegetable residues, were considered as raw material for the preparation of protein concentrates or isolates, as animal feeds and human food. Other isolates of nutritional or economic importance were studied to a lesser extent.

B. VEGETABLEWASTESAS LEAF MEALS The utilization of vegetable leaf meals was reviewed by Kelley (1958) and Thompson (1958) and by Tressler and Joslyn (1961). As early as the forties, the United States Department of Agriculture, Eastern Regional Research and Development Division, was interested in the utilization of vegetable leaf and vegetable leaf waste for this purpose. They have reported on the availability and utilization of vegetable wastes (Morris, 1946), on their preparation and use (Willaman and Eskew, 1948), and on the production (Aceto et al., 1950) of leaf meals from vegetable wastes. Kelley (1948) reported on the chemical composition of beet, carrot, broccoli, kale, turnip, rutabaga, rhubarb, spinach, celery, cabbage, cauliflower, collard, and parsnip wastes, as well as pea and lima bean vines. Results were given on moisture, protein, crude fiber, ether extract, carotene, and riboflavin contents. Tomhave et al. (1948)reported on the use of vegetable leaf meals in poultry feeds. Some of the results are given in Table XIII. Broilers fed diets containing broccoli and turnip leaf meals at the 8% level were heavier and utilized the feed more efficiently than broilers fed alfalfa meal. Pigmentation was very good in birds fed broccoli and turnip leaf meals. Tomhave et al. (1948) found that even 1% of broccoli meal was adequate if supplemented with riboflavin, and was superior to 5% alfalfa meal. Kale leaf meal at a 1.5%level was equal to broccoli leaf meal at 1 % supplemented with riboflavin. Willaman and Eskew (1948) concluded that vegetable waste leaf meals have a value proportional to their vitamin content, or carotene content at least. Similar results were reported by Davis et a2. (1951) on the utilization of dehydrated celery tops in chicken rations. Evaluation of the feeding value of dried celery tops for steers was reported b y Haines et al. (1959). They found that dried celery tops are satisfactory in a steerfattening ration. A leaf meal containing 27% protein is used in SouthEast Asia primarily as an ingredient in poultry feed. This meal is

THE UTILIZATION O F FOOD INDUSTRIES WASTES

99

produced from leaflets of a leguminous tree known locally as Ipil; the pods are utilized to some extent as food (Kramer, 1967). The main economic problem in the utilization of vegetable waste, aside from their seasonal and scattered availability, is the price of dehydration. Willaman and Eskew (1948) and Aceto et al. (1950) discussed this problem. Isolation and purification of carotene, chlorophyll, tocopherol, and enzyme preparates are reviewed b y Tressler and Joslyn (1961) and Wall (1948). A potential exists for extracts in great profusion of many specific nutrients and biologically active material. Ordinarily the cost of isolation of a single component occurring in small quantities is prohibitive. Thus, for example, tomatine, a glycosidal alkaloid, was isolated from a11 parts of the tomato plant, with approximately 0.5 % in the leaves (on dry-weight basis). Certain therapeutic and fungistatic properties were ascertained, and laboratory, pilot-plant, and commercial-size procedures were developed for its recovery and isolation (Fontaine et al., 1955; Kalkstein, 1967, private communication). To this day, however, practically no tomatine is being manufactured, despite the availability of millions of tons of tomato residue. Only by developing a process in which other components of economic value can be produced simultaneously will it be possible to produce tomatine economically. Thus, work is now in progress at the University of Maryland to develop a process for total utilization of tomato plant material, with tomatine as one of the products to be recovered.

C . SILAGE A method which is relatively inexpensive and does not require heat treatment is the preparation of silage. As a method for preservation of grass it is not a new technique. Over the years, it has been employed in preservation and utilization of a large number of grasses, crop residues, and food-processing wastes. The applicability and usefulness of this method, as well as study of the process of ensilage and the techniques employed, have been the subject of many books and articles (Watson and Smith, 1951; Staniforth, 1966). As stated above, the use of a number of crop residues as feed is limited by drying costs. The tomato is one of many such crop residues. If tomato residues could be shown to ensile satisfactorily, their cost as feed would be reduced considerably below the cost of dehydrated products. Leffel(l968) fed ensiled tomato vines to growing lambs. He

100

ITAMAR BEN-CmERA AND AMIHUD KRAMER TABLE XI11

Analyses of diet Crude Pen No.

1 2 3 4 5

6 7

Diet

+ + + +

Basal 8% alfalfa meal Basal 8% pea vine meal Basal 8% lima bean vine Basal 8% turnip leaf meal Basal 8%broccoli leaf meal Basal 8% carrot leaf meal Controld

+ +

( %)

Riboflavin (mg/W

Carotene as vitamin A (IU/lb)

6.5 5.0 4.4 4.5

1,410 1,568 1,290 1,558

4,500 2,200 9,000 21,000

4.3 5.2

1.976 1,430 1,290

26,400 5,900 1,100e

Protein ( %)

fiber

20.8 20.0 19.8 20.4 21.6 19.4 20.2

3.8

“From Tomhave et al. (1948).By permission of U. S . Department of Agriculture. *An index of 100 would mean that broilers, given their choice, would eat that diet to the complete exclusion of a diet supplemented with alfalfa meal.

demonstrated that gains were satisfactory, particularly if the vines were chopped and mixed with cannery waste and corn cobs.

D.

VEGETABLE WASTES AS PROTEIN SOURCES

Vegetable wastes are high in protein content. Some data from Aceto et al. (1950) are shown in Table XIV. Bondi (1958) reviewed literature on the preparation of native protein from leaves. The essential amino acid composition of leaf proteins is comparable, with some exceptions, to that of casein. Proteins from different plant species, or from plants of different ages, manure treatment, or climate, do not show great differences in their amino acid composition. Vegetable leaf proteins resemble green forage proteins in composition. The amino acid compositions of leaf meals of vegetables or green forage source are reported b y Kelley and Baum (1953), Block and Bolling (1951),Baptist (1954), and others. Dean (1958), in his review on leaf proteins, discusses the role of leaves in the human diet. The greatest disadvantage of the leaf as a source of protein is its high fiber content. This may not be of critical importance in the feeding of ruminants, which may not require proteins, as such, in their diet. Pioneering work on the technology of recovering leaf proteins was done at the Rothamsted Experimental Station in England, by N. W. Pirie (1966a,b) and his associates. In the process, fresh leaves

101

T H E UTILIZATION O F FOOD INDUSTRIES WASTES

ANALYSESOF DIETS IN RUN 1 AND RESULTS OF FEEDING TESTSA T ENDOF 14 WEEKS"

Palatability indexbof diet

Mortality of birds

Av. wt. of birds

( %)

(W

Av. feed consumed per bird (Ibs)

-

11 15 7 11

2.55 2.10 2.51 2.59

9

2.73 2.65 1.94

62 78 87 96 93 -

6 60

Feed consumed perllb of gain of birds

(W

Pigmentation index of birdsc

11.78 10.71 11.76 11.46

4.6 5.1 4.6 4.4

48 48 59 66

11.31 11.87 9.11

4.3 4.5 4.7

95 53 6

cAn index of 100 would mean that every bird in the group had scored perfect in pigmentation. dThe control diet was the same as the basal diet except that it contained 35% ground yellow corn and 22% soybean oil meal instead of 30 and 20%, respectively. Calculated; none found by analysis.

TABLE XIV COMPOSITION OF VEGETABLE LEAF M E A L S ~ . ~ ~ ~ Leaf meal

Stems

Vegetable waste

Moisture

Protein

Fiber

Moisture

Protein

Fiber

Broccoli Spinach (field waste) Beet tops Lima bean vine Pea vine

5.75 4.23 3.21 6.05 9.00

39.5 31.9 20.8 15.2 16.9

8.5 9.15 7.55 20.5 22.5

52.9 37.5 34.4 38.2

23.2 27.6 16.3 12.0 11.6

16.7 15.3 12.9 51.8 25.3

"From Aceto et al. (1950). By permission of U. S. Department of Agriculture. Moisture-free basis. In 70.

are finely ground to liberate most of the protein from the cells, since much of the protein is located in the chloroplasts and their fragments, which are only 0.1 p in diameter (Pirie, 1959). Overheating during grinding should be avoided since excessive heat would result in in situ denaturation of the leaf proteins and a decrease in their extractability through loss of solubility. The juice is separated from the fibrous layers by pressing. If the pressed layer is too thick, the proteins may

102

ITAMAR BEN-GERA A N D A M I H U D KRAMER

not be fully recovered from the juice. A belt press was developed by Davys and Pirie (1965) for efficient separation of the juices from the fibrous pulp. The proteins can be recovered from the expressed juice in several ways (Bondi, 1958). Morrison and Pirie (1961) prefer heat. According to Pirie (1966b), bringing the leaf juice to at least 70°C results in the formation of a coagulum which can be recovered by filtration or centrifugation. Byers (1961), Singh (1964), Devi et al. (1965), and Byers and Sturrock (1965) studied the extraction of leaf proteins from legumes, cereals, and other grasses. Byers and Sturrock (1965) found that yields of extracted protein were greater from cereals than from legumes and other grasses. They concluded that a suitable succession of crops should make it possible to get 1000 kg of protein from a hectare in a year. Akeson and Stahman (1966) compared the average production of several essential amino acids from ten crops harvested for forage and fifteen crops harvested for seed, by determining both their protein contents and the amino acid composition of the proteins. Their results are shown in Fig. 2. These data demonstrate the potential of forages as protein sources. In corn, they show that the yield of leaf protein per acre is higher than the protein produced in the corn seed, and thus, several times as high as the protein produced by animals fed the grain. Pea-canning waste and sugar beet tops were found by Pirie (1966b) to be suitable raw materials for the recovery of proteins. Singh (1966) reported that plant residues remaining from several vegetable crops were satisfactory raw materials for the production of leaf protein concentrate. Amino acid analysis of leaf proteins shows methionine as a limiting amino acid, with adequate concentration of all other essential amino acids (Gerloff et al., 1965; Wilson and Tilley, 1965; Singh, 1966). Feeding tests conducted with infants, chicks, rats, and pigs, reported by Barber et al. (1959), Duckworth et al. (1961), Duckworth and Woodham (1961), and Waterlow (1962), showed that leaf protein concentrate has a nutritive value higher than those of soybean meal and white fish meal. Hartman et al. (1967) prepared leaf protein concentrates by spraydrying the juice expressed from fresh alfalfa and pea vines. Table XV shows the composition of the spray-dried juice, raw material, and residue. Extraction with 95 % ethanol removed the chlorophyll from the protein concentrate. Hartman et al. (1967)reported that the product obtained is a light-tan powder with a bland taste. Table XVI shows the biological value of the leaf protein concentrate.

--

THE UTILIZATION OF FOOD INDUSTRIES WASTES

3 L

300 -

250

g

-

a

Forage

Oil seed

103

.

0

Legume seed EZZZZd Cereal seed ~

200-

0 \

In 7J

50

150-

a

100

-

50

-

0-

FIG. 2. Yield of essential amino acids (lysine, methionine, tryptophan, phenylalanine, threonine, valine, leucine, isoleucine) per acre for various crops harvested as forage or as seed. Yields were calculated from crop yields compiled by the U. S. Department of Agriculture for the years 1953-1962. From Akeson and Stahman (1966).

Both Pirie (1966b) and Singh (1966) reported success in utilizing isolated plant proteins by incorporating them into existing foods. Singh (1966) incorporated leaf protein satisfactorily at a 10% level and higher. Acceptability was good, particularly in soup-powder mixes and chutneys. As mentioned previously, the amounts of tomato wastes are increasing considerably with the increased use of mechanical harvesting. A major change in the situation is the availability of green tomato vines, which represent a raw material of little economic value at present although a potential source of nutrients. According to Kwee (1967), tomato plants contain 14.1-15.1 % proteins (leaves and stems). With seeding for mechanical harvest there are 5,000 to 10,000 plants per acre yielding a dry weight per plant from 300 to 200 g, respectively. Tomato plants thus represent between about 255 and 300 kg protein per acre. Morrison and Pirie (1961) state that a recovery of 75% of the protein from a high-quality crop is possible. Chayen et al. (1961) separated up to 80% of the nitrogen from good-quality plant material. Even with less efficient recovery, tomato vines would represent a considerable amount of protein. The University of

104

ITAMAR B E N - G E M AND AMIHUD KRAMER TABLE XV COMPOSITION OF SPRAY-DRIED JUICE,SPRAY-DRIED JUICE EXTRACTED WITH 95 % ETHANOL,RESIDUE AFTER EXPRESSING JUICE,AND PLANT MATERIALBEFORE EXPRESSINGJUICE^'^'^ SamDle

Spray-dried juice Alfalfa (first cut) Alfalfa (second cut) Pea vine Spray-dried juice extracted with 95% ethanol Alfalfa (first cut) Alfalfa (second cut) Pea vine Residue after expressing juiceb Alfalfa (first cut) Alfalfa (second cut) Pea vine Plant material before expressing juice Alfalfa (first cut) Alfalfa (second cut) Pea vine

Moisture Protein

Fat Fiber

Nitrogenfree extract

Ash

4.9 4.2 4.8

34.9 31.3 18.5

6.6 5.8 2.5

0.7 0.9 1.4

40.2 45.7 60.0

12.7 12.1 12.8

2.6 2.3 3.1

42.8 37.0 26.2

0.6 0.6

0.5

0.8 1.2 1.6

38.8 44.8 47.0

14.4 14.1 21.6

7.4 5.8 6.3

15.9 15.5 9.7

3.1 32.6 2.8 31.0 3.3 28.5

34.6 38.2 41.2

6.4 6.7 11.0

5.5 5.1 5.5

21.7 19.5 12.2

4.5 23.0 3.6 24.1 3.4 21.4

37.5 40.6 44.6

7.8 7.1 12.9

"From Hartman et al. (1967).From the Journal of Agriculture and Food Chemistry. By permission of American Chemical Society. *Air-dried for 48 hours at 140°F. 'In 70.

Maryland is conducting a research program directed toward recovery and evaluation of tomato-vine proteins. Chayen and Ashworth (1953) studied the possibility of isolating protein-lipid complexes from plant and animal tissues by the Impulse Process. In this process, the membranes of fat-containing cells are ruptured by a series of high-speed impulses transmitted through the medium of a liquid. High-protein fat-containing fractions were separated and recovered by this technique from peanuts, coconut, cottonseed, alfalfa, and other materials of plant and animal origin. Smith (1966) reviewed the literature related to lipid-protein isolates.

E. FRUITS Bananas are a major article of international trade. Total world exports of bananas approximated 5,000,000 tons annually in recent years. It is generally estimated that less than one third of the bananas pro-

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105

TABLE XVI ESTIMATEDBIOLOGICAL VALUESOF SPRAY-DRIED LEAF PROTEINPREPARATIONS“

Sample Spray-dried juice Alfalfa (first cut) Alfalfa (second cut) Pea vine Spray-dried juice extracted with 95% ethanol Alfalfa (first cut) Alfalfa (second cut) Pea vine Other foodstuffs determined by same methodb Whole egg Egg white Lactal bumin Milk Leaf protein concentratec Casein Beef Yeast Soybeand Cottonseed meal‘ Wheat flour Gluten Zein Gelatin

Estimated biological value

80 81 68

83 70 97

87 84 83 83 76 75

71 65

64 50 45

26 17

“From Hartman et al. (1967); biological Values estimated by pancreative digest. bAkeson and Stahman (1966). cAverage of 14 samples prepared according to Morrison and Pirie (1961). dThe biological value range from 57 to 75, depending on preparation, according to Mitchell and Block (1946). “Biological value from rat feeding trials, according to Mitchell and Block (1946).

duced are exported from South America, Central America and African countries, with approximately one third utilized for food or feed locally, and the remainder wasted (Watkins, 1967). There have been some attempts at utilization of bananas other than as fresh fruit for export. An increasing market is canned or processed as frozen banana purke, used mostly in baby food, bakery goods, and ice cream mixes. Some effort has been directed toward the production of dehydrated products. Since the cost of the raw material can be as low as $0.15per 100 lb (Watkins, 1967), it is possible to produce a pound of banana flour for as little as $.02. This would provide a far cheaper source of

106

ITAMAR BEN-GERA AND AMIHUD KRAMER

food energy. A number of schemes have been proposed for the utilization of banana peels, stems, and leaves. Among other products that could be produced from these huge quantities of waste are furfural, pectin, wilted stock feed, paper, and wallboard. It has even been proposed to form the containers for bananas by compressing stems at high pressures. With the exception of some pilot plant operations for producing paper and pelletized feed, nothing has yet been accomplished in this direction. As with other wastes, the major obstacle in the utilization of banana stems is the cost of removing water. World exports of cocoa beans have been exceeding one million metric tons annually in recent years, with the majority of exports originating in central African countries. The usual practice is to remove the cocoa fruit from the tree when it reaches an appropriate stage of maturation, wrap a number of fruits in plantain palm or other suitable leaves, and allow the fruits to ferment until the mucilage surrounding the beans is sufficiently decomposed that the beans can be removed easily. The beans are then dried and bagged for shipment from the farm. This process wastes the entire fruit except for the beans. Forsyth and Quesnel (1963) developed an enzymatic process for separating the beans from the placental mucilage. Such a process would permit harvesting the entire fruit, including several million tons of nutritious fruit for feed and perhaps food uses. Many tropical regions have an abundance of papaya, avocado, and mango that are used to only a very small extent. High-quality oil resembling olive oil could be extracted from avocados if a method were available for removal of the bitter principle. Possibilities of utilizing coffee residues were discussed by Barbera

(1965). V. OILSEEDS AND GRAINS

A. INTRODUCTION The first part of this section discusses the transformation of a few selected oilseeds from a fertilizer or animal feed to a potential source of food for humans. New products from soybean and cottonseed, comprising together more than 20,000,000 tons of protein (more than enough to cover present world protein deficiencies), have now reached the stage of development where high-protein foods are becoming increasingly available for use by humans around the world.

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107

Peanuts, the third major oilseed, are consumed, either roasted or boiled, by humans in many parts of the world. Removal of the peanut oil results in a meal containing 50 to 55 % protein, deficient in lysine, methionine, and threonine. Peanut meal is used as food only to a limited extent, although its potential as an important tool in combating malnutrition is well recognized (Parpia and Subramanian, 1966).According to Milner (1966), it is estimated that 80% of the peanut meal produced in India, which produces more than a third of the world peanut crop, is used as manure. Rosen (1958) reviewed different aspects of the production, trade, composition, and utilization of peanuts and peanut meal. More than 14,000,000tons of peanuts, with 27% protein content and 48% fat, were produced in 1965, and used mainly for crushing for oil. The high-protein meal is potentially an important ingredient in mixes with other protein sources, where it can be supplemented and save valuable proteins of animal origin (Altschul, 1965a, 1967). Problems of high fiber content exist not only in coconut and safflower, which are discussed in this section, but also in sunflower and other meals, and present problems in using these meals as foods for humans. Future use of several meals as feed for livestock and humans will depend on the development of reliable procedures for detoxification. Examples are rapeseed meal, castor bean meal, and tung seed meal, with a combined annual production of more than 5,000,000 tons (compiled from U.S.D.A., 1966a).Sesame seed meal and linseed meal, produced from more than 4.5 million tons of raw material in 1966, are sources of good-quality protein, potentially of great importance, that are as yet almost untapped for human consumption. By-products of dry milling of grains are being used currently for animal feeding. The growing need for new sources of good-quality protein focuses attention on these foods. Pioneering work in the recovery of wheat-mill feed proteins, carried out at the Western Regional Research and Development Division of the U. S . Department of Agriculture, is mentioned in this section. Lipid content of rice bran varies considerably. According to Markely (1949) it is between 14 and 17%. Kumar et aZ. (1964) found the variation to be even bigger. Based only on the production of rice-mill feeds in the United States, about 60,000 tons of fat and wax could be recovered. With bran representing more than 8% of the weight of rough rice (Markely, 1949),the quantity of lipids potentially available for extraction around the world is much bigger. Solvent extraction of rice bran oil and wax was studied b y Reddi et aZ. (1948), Wellborn

108

ITAMAR BEN-GERA AND AMIHUD KRAMER

et al. (1951),Graci et al. (1953),Cousins et al. (1953),and Pominski et al. (1954,1955). Recently, Lynn and Lawler (1966)reported on a combined solventextraction and wet-milling process which results not only in decreased breakage of rice during milling but also in the recovery of high-grade food oils and a bland, light-colored, high-protein (17-20%) rice bran of food grade. Much information, most valuable in the preparation of this section and in reviewing the subject of oilseeds, can be found in Altschul (1958)and in Milner (1966).

B. SOYBEANS For the peoples of the Far East, soybeans were and still are a major component of the diet. Altschul(1965b) lists examples of soybean food products made in China, Japan, Korea, and Indonesia. When introduced into the Western World, soybeans were primarily a source of oil for human nutrition, and the oil meal was used first as a fertilizer and later as feed. Cravens and Sipos (1958)reviewed literature related to the production, composition, and utilization of soybean oil meal. Smith (1958)and Anson (1958)discussed opportunities for production and potential uses of isolated soybean and other legume seed proteins, and Circle and Johnson (1958)reviewed the early growth of the edible isolated soybean-protein industry. According to De et al. (1966),approximately 30,000 tons of soybean meal are utilized annually for food and industrial purposes in the United States, compared with 10,000,000tons of meal used as feed. Data on the production of soybean protein concentrates and soybean isolates are hard to come by. Meyer (1966)estimates that the soybean protein concentrates are selling at a level approaching 15,000,000lb per year, and the protein isolates at a level of 10,000,000lb per year. The development of meat analogs from soybean-isolated protein is discussed by Ode11 (1966)and Ziemba (1966);from dairy, bakery, meat, and other products by Meyer (1966)and Horan (1966);and from defatted and full-fat flours by Horan (1966) and Mustakas (1966). Such developments are an important step toward the successful use of the soybean for human consumption, a natural “protein concentrate” of high nutritive value. C. COTTONSEED More than 25,000,000tons of cottonseed were produced throughout the world in 1965 (U.S.D.A., 1966a). Delinted cottonseed contains

THE UTILIZATION OF FOOD INDUSTRIES WASTES

109

36% hulls and 65% meats (Thorp, 1948), of which about 39% is crude protein and about 30% is fat (Guthrie et al., 1949). Throughout the history of mankind and until recently, cottonseed, a by-product of cotton production, although known to contain valuable oil and to have certain feed value, received little attention and, if utilized at all, was used primarily as fertilizer, fuel, or cattle feed. Crushing of cottonseed for oil on a large scale is relatively new. Only 40% of the U. S. cottonseed crop was crushed for oil as late as 1897. The rest of the seed crop was considered waste. A condensed review of the history of cottonseed was published by Altschul et aZ. (1958). According to Harper (1965) the percentage of cottonseed crushed for oil in the United States has increased from about 81%, in 19361945, to 91 Yo, in 1957-1961, and to more than 95% in 1964 (U.S.D.A., 1966a). On a worldwide basis, however, the percentage of cottonseed crushed for oil must be lower since only 2,675,000 tons of cottonseed oil were produced during 1965 (U.S.D.A., 1966a). This oil is used primarily as food for humans. Altschul et al. (1958)reviewed the literature related to the uses of cottonseed meal in ruminant and nonruminant feeding. While cottonseed meal fed to ruminants offers a feed of good quality, high in protein and energy, the presence of gossypol (a toxic yellow-colored pigment located mainly in the dark glands visible when a cottonseed is cut open) provides a problem in nonruminant feeding because gossypol detoxification destroys cottonseed meal proteins. The physiological properties of gossypol, its relationship to feed value, and its role as a toxic agent were reviewed by Adams et .ul. (1960), Hale and Lyman (1948), Lyman (1964), and Altschul et d. (1958). A solution to the problems related to the presence of gossypol in cottonseed may be in sight with the development, through breeding work by the United States Department of Agriculture, of glandless (and thus gossypol-free) cottonseed. This is based on work of McMichael (1960). Lewis (1964) discussed the genetic aspects of this breeding program. The lipid and protein fractions of glanded and glandless cottonseed were compared by Watts (1964), who found that only minor differences in oil and amino acid composition of the proteins exist between the two types of cottonseed compared. Results reviewed by Watts (1964) indicate that the absence of gossypol in glandless cottonseed eliminates the problems of the color of crude cottonseed oil and makes it possible to use the meal in unrestricted amounts in feeding nonruminants, although some problems remain as to pigmentation of eggs produced by hens fed on the glandless cottonseed meal, and understanding is still incomplete as to the nutritional inferiority of

110

ITAMAR BEN-GERA AND AMIHUD KRAMER

glandless cottonseed meals produced by hexane extraction (Johnston and Watts, 1965). With the lint still the dominant economic factor in cottonseed production, the yield of lint by the newly developed varieties will, according to Altschul (1967), determine the future of the glandless cottonseed. The use of cottonseed protein in human food was reviewed by Altschul (1965a, b, 1967), Bressani (1965), Lambou et al. (1966), and Bressani et al. (1966). The amino acid composition of the cottonseed protein compares favorably with human requirements (Bressani, 1965). Table XVII shows the nutritive value of cottonseed protein for humans. The introduction of cottonseed protein-containing products, manufactured and distributed on a commercial basis after extensive research and development work, carried out mainly at the Institute of Central America and Panama (INCAP), in Guatemala, is discussed by Lambou et al. (1966), and Bressani et al. (1966). Incaparina, a product containing 38% cottonseed flour of 50% protein and 6% fat, was developed by INCAP. According to Lambou et al. (1966) it is being produced and marketed commercially or is under market study in Guatemala, Panama, Colombia, Brazil, Peru, Venezuela, and Mexico. Table XVIII shows the production of Incaparina (INCAP No. 9 and No. 15) in Latin America. The increased production of Incaparina and its distribution in areas where malnutrition prevails is an important step toward improvement of the nutritional state of the peoples of those areas. D. COCONUT

Curtin (1958) reviewed the literature on coconut-oil meal. Copra, the product obtained by dehydration of coconut meat, varies in TABLE XVII PROTEIN CONCENTRATE IN HUMAN SUBJECTS" NUTRITIVE VALUEOF COTTONSEED Protein source

Protein intake

Absorption (%)

Biological value

NPU

Subjects

Cottonseed protein Cow milk Cottonseed flour Cow milk

28.86 glday 30.55 glday 5 g/kg 5 glkg

81.97 90.73 71 85

62.12 82.15 57 79

50.83 74.50 41 67

Childrenb Children Prematuresc Prematures

"From Bressani (1965). By permission of Institute of Food Technologists. DeMaeyer and Vanderborght (1958, 1961). Snyderman et al. (1961).

THE UTILIZATION OF FOOD INDUSTRIES WASTES

PRODUCTION OF

111

TABLE XVIII POUNDS) IN LATINAMERICA^

INCAPARINA (IN

Trimester Year

First

Second

Third

Fourth

Total

1961 1962 1963 1964 1965

68,772 63,134 475,055 720,458

44,250 9,568 154,464 237,411 832,648

100,755 128,515 106,977 722,844

100,731

245,736 206,805 488,570 2163,344

b

163,995 728,034

“From Bressani et al. (1966). Not produced because of low availability of cottonseed flour.

composition but contains, according to Eckey (1954), between 63 and 68% fat. The protein content of this product is 6 to 9%,and water content is 6%. F A 0 (1965) recently estimated that world copra production for 1965 was well above 3,000,000 tons. So far, the main value of copra is its high oil content, which is recovered by compression or extraction, according to Curtin (1958).This process yields two products: coconut oil and coconut oil meal. Coconut oil is used widely in the chemical and food industries. Coconut oil meal, containing about 25% protein and 2 to 11% fat (dry basis) is used primarily as a feed or fertilizer (Curtin, 1958). The usefulness of coconutoil meal as a high-quality source of nutrients in feeding dairy and beef cattle, sheep, swine, and other animals was reported by Miller (1919), Guilbert (1927), Loosli et al. (1955), Warner et al. (1957), and Curtin (1958). Coconut-oil meal as fertilizer (U.S.D.A., 1951) was found by Tabayoyong (1947) and Andrion (1948) to improve both the chemical and biological properties of the soil. Until recently, according to Curtin (1958) and Rama Rao et a2. (1965), very little work, if any, was done on coconut meal as a potential protein source for human nutrition. A major obstacle to the use of coconut-oil meal or coconut protein by humans, poultry, and swine is its high fiber content, which can be as much as 13.3% (Morrison,

1950). Rama Rao et al. (1965) studied the effect of fiber on the utilization of protein in coconut-oil meals by children. A considerable interference of the fiber with digestibility of the meal and retention of the nitrogen was found. According to Chandrasekaran and King (1965), aqueous extraction of coconut proteins results in low extraction yield. Studies by those workers on enzymatic modification of the extractability of protein from coconuts improved the extractability of the protein from 50 to 85%, and destroyed about 50% of the crude fiber.

112

ITAMAR BEN-GERA A N D AMIHUD KRAMER

Another process for the recovery of coconut proteins, known as the impulse process (mentioned earlier in this text), was discussed by Smith (1966). In this process the raw material is fresh coconut meats. About 70% of the nitrogen of the original kernel is recovered in the lipid-protein complex, which contains 60% protein and 35 % lipid (on a dry basis). Coconut protein is of fairly good quality. Its first-limiting amino acid is lysine (Curtin, 1958). Smith (1966) reported that the NPU value for carefully prepared coconut protein concentrate was 63. Coconut-oil meal, with about 25% protein, could become an important source of protein for the peoples in areas where it is produced in quantity and where additional sources of protein are needed.

E. SAFFLOWER The production of safflower,once considered to be a minor oilseed, although known to mankind for centuries, is increasing rapidly. According to Kohler (1966), who recently discussed the potential of safflower as a source of protein for human food, 640,000 tons of safflower seed were produced throughout the world in 1964, mainly in the United States, India, Spain, and Mexico. Additional references to the production, processing, and utilization of safflower are in Knowles (1955,1965),Kneeland (1958),AOCS (1965). The composition of the safflower seed as reported by Kohler (1966) is: 55-65%kernel and 35-45%hull. The whole seed contains 35-40% oil and 13-17% protein. Fig. 3 shows the products theoretically obtainable from 1 ton of seeds of average composition. Kneeland (1958) and Kohler (1966) discuss the utilization of safflower oil b y the chemical and food industries, and the effects of its unique composition and oil content on the production of the crop, which had previously been used primarily as fertilizer and stock feed in India, and as feed for dairy and beef cattle, sheep, and poultry in the United States, Japan, and other countries. The amino acid composition of safflower meal was published by Lyman et al. (1956). Lysine is the first-limiting amino acid of safflower proteins. Kohler (1966) compares the amino acid composition of safflower and soybean with the original and adjusted F A 0 reference pattern, and with egg protein as a reference protein. A 3:2 combination of safflower and soybean has the high score of 93. Baliga et al. (1954) reported on the adequacy of supplementation of safflower by milk, fish, pulses, and other protein sources rich in lysine.

THE UTILIZATION OF FOOD INDUSTRIES WASTES

113

700 Ib oil

480 Ib flour

Safflower seed

containing 312 Ib protein

FIG. 3. Theoretical yields of products from one ton of safflower seed. From Kohler (1966).

The effect of supplementation of safflower flour with amino acids is shown in Table XIX. No evidence for the presence of antimetabolites or inhibitors in safflower was found by Kohler (1966), who reported that growth of chicks was faster on safflower than on soybean meal (at optimal level of lysine addition). Similar observations were made by Fisher et al.

(1962). According to Goss and Otagaki (1954), the fiber content of undecorticated safflower meal is 32.7%,and that of decorticated meal is 11.5%.This high fiber content is an obstacle to utilization of safflower meal as a protein source for humans. According to Kohler (1966), high-protein products should contain less than 5% fiber, at least for children. We are unaware of the availability of such a product from a commercial source, although an experimental laboratory process was reported by Kohler (1966) to produce a product containing 57-60 % protein and about 3 70fiber. The debittered product was incorporated successfully in meatlike products and in bread (5% level) on an -experimental basis. Dehulling and protein isolation, as reported by Van Etten et al. (1963), are important steps toward the development of a safflower product of high protein and low fiber content suitable for humans. TABLE XIX EFFECT ON PER OF SUPPLEMENTATION OF SAFFLOWER FLOUR WITH METHIONINE OR METHIONINE PLUS LYSINE"

Safflower flour (58%protein) Same plus methionine Same idus methionine and lysine "From Kohler (1966).

Cystine plus methionine, ( d 1 6 g N)

Lysine ( d 1 6 g N)

PER

3.34 4.20 4.20

3.09 3.07 5.00

1.39 1.59 2.09

ITAMAR BEN-GEM AND AMIHUD KRAMER

114

F. GRAINMILL FEEDS More than 4.7 million tons of wheat-mill feeds and about 400,000 tons of rice-mill feeds were used in the United States in 1964 (U.S.D.A., 1966a). The proteins of wheat, and other constituents as well, are not distributed evenly throughout the kernel. This was substantiated by Morris et al, (1946), Hinton (1947), Pomeranz and Shellenberger (1961), Normand et al. (1965), and others. The milling of wheat results in uneven distribution of the components between the different milling products. Table XX shows the composition of a Montana hard red spring wheat and its milling products. According to Morris et al. (1946) and Fellers et al. (1966a), the protein content of the extracted flour increases as extraction rate increases. Cave et al. (1965) studied the nutritional value for the growing chick of wheat shorts, bran, middlings, and wheat-germ meal. They concluded that wheat shorts, wheat middlings, and properly processed wheat-germ meal are good sources of protein, comparing favorably with soybean meal supplemented with methionine, if diets in which they are included are adequate in their caloric content. According to Hove et al. (1945), shorts and middlings, as well as germ meal, are of a higher biological value than other fractions of wheat.

TABLE XX PROXIMATE CHEMICAL COMPOSITION OF AN HRS WHEAT AND ITS MILLING PRODUCTS (DRYBAS IS)^

HRS wheat ( %)

Shorts (%)

Feed middlings ( %)

Straightrun flour ( %)

20.6 10.5 6.3 6.2 7.1 10.Ob 24.5 14.8

20.9 8.5 6.9 5.2 7.3 15.gb 19.7 15.6

19.5 4.4 4.4 3.3 5.7 37.7b 12.6 12.4

17.8 0.3 1.o 0.6 2.3 74.5 1.7 1.8

6.3

8.4

2.4

72.5

Coarse bran

Fine bran

(%I

Protein (N X 5.7) Fiber Fat Ash Total sugars Starch Pentosans Undetermined

18.3 2.5 1.9 1.9 2.9 58.8 6.6 7.1

(70) 19.2 12.6 5.5 6.9 6.1 5.8b 27.1 16.8

Whole wheat (9”)

100.0

10.4

From Fellers et al. (1966a). *Values may be somewhat low (1-270) because of levorotatory effect of high pentosan content. (I

THE UTILIZATION OF FOOD INDUSTRIES WASTES

115

The nutritional value of wheat-mill feeds compares favorably with that of other proteins (Marais and Smuts, 1940; Chick et al., 1947). Hove et al. (1945), McCance and Walsham (1948), Bolton (1954), and Fellers et al. (1966a,b) point to the potential of mill feeds as a source of good-quality protein for human food. Figure 4 shows a procedure for the manufacture of protein concentrate from milling products of wheat, developed by Fellers et al. (1966a, 1967) and based on milling and sifting of milling products of wheat. The yield and composition of the protein concentrate produced by this repetitive milling operation is influenced by the moisture content of the wheat shorts and can be adjusted by the sifting step. This is shown in Fig. 5. Fellers et al. (1967) studied the nutritive value of protein concentrate prepared from wheat shorts by determination of its Protein Efficiency Ratio (PER), which was found to be 1.9, compared with 0.7 for white flour and 1.2 for whole wheat. The manufacture, composition, and use of products of dry milling of rice as poultry, swine, and cattle feeds were reported and reviewed by Kik and Williams (1945) and Seeley (1958). Although the protein content of rice bran and rice polish can reach more than 11%, high fiber and ash contents limit its uses in nonruminant feeding. A process was developed by Palye (1968) for the release from the mill feed by mechanical methods (grinding and sifting) of a highprotein fraction representing 25-30 % by weight of the mill feed. The product, called “Aleurone Concentrate,” developed in Canada, is reported to have been incorporated successfully in several food products, to be of a high-protein low-fiber content, and of good flavor and acceptable color. VI. STARCH-PRODUCTION WASTES

A. INTRODUCTION

Hatfield (1961) and Greenfield (1965) have reviewed the field of starch-production wastes. According to the U. S. Department of Agriculture, 1,406,000 tons of gluten feed and meal were produced in the United States in 1964 from more than 200,000,000 bushels of wet-milled corn. In 1963, about 600,000 tons of potatoes were utilized for flour and starch production (U.S.D.A., 1966a).According to Dickey et al. (1966), a potato-starch factory produces about 8750 lb of wet potato pulp (80%moisture) for each ton of starch produced.

ITAMAR BEN-GERA A N D AMIHUD KRAMER

116

Moisture adjusted shorts

1st milling

1st Milling PC

Course residue 2nd milling

2nd Milling PC

-7XX

-100

+lo0

t140

Course residue

-140

FIG. 4. Production of protein concentrates from milling products of wheat. From Fellers et al. (1967).

70

27

60

26

50

25 24 .E e c

e

23 a

$

22

21

10

C 21

, , , , , , , , 3 5 7 9 I I 13 15 17

20

'/o Moisture of s h o r t s before milling

FIG. 5. Influence of moisture content and yield of flour on yield and composition of protein concentrate from wheat shorts (total of three millings). From Fellers et al. (1966a).

THE UTILIZATION OF FOOD INDUSTRIES WASTES

117

Wet milling of sorghum and the production of feeds and other by-products were reviewed by Seeley (1958) and Watson (1959,1967). The Texas Agricultural Experiment Station (1951) published a review of feeding tests with sorghum gluten meal and feed, which was summarized by Seeley (1958). The production of feeds from wet milling of sorghum is still small and limited. We know only of one plant, in Corpus Christi, Texas, which does it on a commercial basis. According to Watson (1968, private communication), sorghum gluten feed is utilized in cattle feeding. A high-protein product is used as a pet food.

B. WET MILLING OF CORN The main products of the wet milling of corn are starch, corn syrup, and corn oil. Corn gluten feed and gluten meal, concentrated cornsteep liquor, and corn oil meal are the principal by-products. The steeping of corn in water containing approximately 0.2 % sulfuric acid for 40 hours at 115"-130"Fsoftens the hulls and makes subsequent separation of the starch-protein complex easier. Soluble constituents of the corn move into the steeping liquid, which is concentrated later, usually to 50 % solids. Following removal, separation, and dehydration, oil is extracted from the germs, and the residue is the corn oil meal. Coarse fiber and fine fiber removed from the remainder of the grain after separation of the germ become part of the corn gluten feed and corn gluten meal. Corn gluten, removed by centrifugation from the gluten-starch mixture, is combined with the fine fiber to make up the corn gluten meal. The composition of several corn wet-milling by-products is given in Table XXI. Lyman et al. (1956) and others determined the amino acid composition of the major wet-milled corn by-products. Results are given in Table XXII. From amino acid analyses, Christensen et al. (1967) calculated the chemical score and essential amino acid index of proteins of the different wet-milled corn by-products. Results are presented in Table

XXIII. As shown, the gluten feeds are deficient primarily in tryptophan and lysine. The mineral and vitamin composition of corn by-products is reviewed by Seeley (1958). The high nutritive value of corn oil meal was reported b y Mitchell and Beadles (1944) and Schultz and Thomas (1949).Block and Bolling (1944) reported equal protein efficiency ratios (PER) for milk and corn oil meal. Beeson et al. (1947) reported PER for corn oil meal protein of 2.01, compared with 2.50 for egg protein. This is attributed

ITAMAR BEN-GERA AND AMIHUD KRAMER

118

TABLE XXI PROXIMATE ANALYSIS OF CORN WET-MILLINGBY-PRODUCTS~

By-product

Moisture

Protein Ether (N X 6.25) extract

-

Corn gluten feedb Corn steepwater solidsr Corn glutenC Corn fine branc Corn gluten mealb Corn oil mealb Corn oil mealc ZeinC Zein-extracted glutend

24.8 20.3 59.1 14.4 42.9 22.3 24.1 90.0 51.8

5.7 5.8 6.2

-

4.8 7.9 7.2

Ash

2.6 1.4 2.0 0.6 2.0 7.8 0.9 0.15

Crude fiber

6.4 7.3 1.1 0.5 2.5 2.3 2.5 0.1 2.8

Nitrogenfree extract

49.8 65.3 31.3 65.1 40.1 49.0 59.5 2.0 34.9

7.8

-

0.7 13.2 3.9 10.3 8.2 3.2

"In Yo. From Morrison (1948). From Christensen et al. (1967). dFrom Seeley (1958).

TABLE XXII AMINO ACID COMPOSITION OF CORN GLUTEN FEED, CORN GLUTEN MEAL,AND CORN OIL MEAL Total nitrogen in the protein (%)

Amino acid in the protein (%)"

Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

Corn gluten feed"

Corn gluten meal"

Corn oil meala

4.3 3.0 3.5 10.2 3.2 2.1 3.8 3.5 0.7 5.5

3.3 2.2 4.4 16.5 2.1 2.7 6.1 3.6 0.5 5.2

6.3 3.0 3.9 8.2 4.7 2.0 4.1 3.9 1.0 5.9

"From Lyman et al. (1956). From Seeley (1958). c g amino acid per 16 g N.

Zeinextracted glutin

5.0 3.0 13.7

-

4.0 4.2 6.0 4.0 0.55 5.7

Corn steepwater conc.b

8.2 6.8 3.4 5.9 4.0 1.1 2.0 3.4

-

3.9

THE UTILIZATION O F FOOD INDUSTRIES WASTES

119

TABLE XXIII CHEMICAL POTENTIAL OF THE PROTEIN OF CORN BY-PRODUCTS ACCORDING TO THEIR ESSENTIALAMINO ACID CONTENT" ~

Corn by-product

Chemical score

Limiting amino acid

Essential Amino Acid Index (EAAI) method

Corn germ oil meal Zein-extracted gluten Reconstituted starch free corn Corn gluten Corn steep-water solids Corn fine bran Zein

51 48 43 33 30 30 1

methionine lysine tryptophan tryptophan tryptophan lysine lysine

89 88 75 70 68 61 39

Chemical score method

"From Christensen et al. (1967). By permission ofJournal ofNutrition.

to the more desirable amino acid pattern of corn oil meal proteins than of the gluten feeds. Recently, Christensen et al. (1967) determined the nutritive value of corn-refining by-products. Their results are given in Table XXIV. Several workers have already reported a low biological value for corn oil meal. Schultz and Thomas (1949) reported a biological value of 83 for corn oil meal prepared by solvent extraction, versus 63 for meal prepared by the expeller process. A decrease in biological value

&SULTS

OF THE

Source of dietary protein Dried whole eggs Reconstituted starchfree corn Zein-extracted gluten Corn gluten Corn germ oil meal Corn steep-water solids Zein

TABLE XXIV GROWTHTRIAL USING WEANING Av . daily gain (g) 3.626 2.28C 1.82c 0.45d 0.OOd -0.16' -0.67d

RATS"

Av . daily feed intake

(€9 8.86

9.6b 8.86

5.5c 4.6bed 4.2bed 3.4d

OFrom Christensen et al. (1967). By permission ofJournal ofNutrition. b-eSame letter indicates no significant difference.

Protein efficiency ratio 2.42b 1.38" 1.21d 0.45e -

-

120

ITAMAR BEN-GERA A N D AMIHUD KRAMER

of corn oil meal proteins as a result of oil extraction was also reported

by Schiller (1957). Low digestibility of the protein in the study by Christensen et al. (1967) may at least partially explain its low biological value. As shown in Table XXIV, corn-steep water has a very low nutritive value. Its nitrogenous fraction consists mainly of free amino acids and short peptides. Its main utilization is in biotechnological processes where it is used as a growth medium (Underkoflar and Hickey, 1954). Zein-extracted gluten has a higher methionine and lysine content than gluten. Experiments with rats show a higher nutritive value for zein-extracted gluten than for gluten. Corn gluten feed is used widely in dairy cow feeding. Early reports (e.g., McCandlist and Weaver, 1922) indicate its value in dairy cow feeding to be equal to linseed meal, peanut meal, and soybean meal. Recently, Harshbarger and Schingoethe (1965) reported on the feeding value for dairy heifers of a product made by adding vegetable-oilrefinery lipids to corn gluten feed. The fatted corn gluten feed contains 22.8% protein, 7.3% fat, 7.2% fiber, 4.9% ash, and 48.8% nitrogen-free extract (on a 9%-moisture basis). In a feeding test in which fatted corn gluten feed at levels of 25 and 50% replaced part or all of soybean meal, oats, and corn, the energy value of the fatted corn gluten feed and that of the control group were equal when feed consumption and growth were compared. For nonruminants, corn gluten feed is a poor source of protein when unsupplemented with other proteins or amino acids. Corn gluten meal, with a higher protein content of a better nutritional quality and lower fiber content, is of wider utilization in ruminant feeding. In nonruminant feeding, corn gluten meal has to be supplemented with other proteins or amino acids because of its deficiency in lysine and tryptophan. The benefits of supplementation of the corn gluten meal proteins was demonstrated by Fritz (1955) in feeding broilers with a mixture of soybean and corn gluten meal. The mixture was superior in biological value to the separate values of the two meals. This confirmed results of several earlier studies summarized by Seeley (1958). Corn gluten meal was found by Huffman and Duncan (1950) and by Miller et al. (1937) to be a satisfactory cattle and lamb feed. Corn oil meal is used as feed in poultry and pig rations. Its use in poultry feeding is limited by its high fiber content, its poor palatability, and its low methionine content. The addition of fish meal or skim milk to feeds containing corn oil meal improved the protein nutritive value for feeding to poultry (Seeley, 1958). Geurin et al.

121

THE UTILIZATION OF FOOD INDUSTRIES WASTES

(1951) showed that in pig feeding, corn oil meal protein was equal to skim milk protein and better than soybean oil meal protein. Because of low palatability, its level in the diet has to be kept low. The proximate analysis and amino acid composition of zein-extracted gluten is given in Tables XXI and XXII. The product is very low in fat and high in protein content. Zein-extracted corn gluten has a higher chemical score and Essential Amino Acid Index (EAAI) than almost all other corn wet-milling by-products (Table XXIII). This is mainly attributable to its higher methionine and lysine content. Its nutritive value, as measured by PER with rats, is given in Table XXIV. According to Heiman (1968, private communication), production of zein-extracted gluten has been rather small, and this product is commercially unavailable at present. This situation may be changed if zein extraction (of which zein-extracted gluten is a by-product) would be undertaken on a large scale. Zein-extracted gluten was used in dairy cattle feeding as a successful substitute for soybean oil meal. Other applications are reviewed by Seeley (1958).

C. POTATOSTARCH Wet potato pulp is the residue of the potato starch recovery operation. This residue can be recirculated through the starch factory for more complete recovery of the starch, and later disposed of as waste. Its usefulness as feed material was reviewed by Dickey et al. (1966). The composition of wet potato pulp and commercial dry potato pulp is given in Table XXV. In feeding experiments with lactating dairy cows, dry potato pulp was found to be equal in value to yellow hominy feed when fed at 22.5%of the grain mixtures. TABLE XXV COMPOSITION OF POTATO PULP".*

Moisture Protein Fat Fiber Nitrogenfree extract Minerals =From Dickey et al. (1966). *In 70.

Wet potato pulp

Dried potato pulp

83.37 1.20 0.06 1.06 13.80 0.51

11.83 5.71 0.40 13.16 66.58 2.58

ITAMAR BEN-GERA AND AMIHUD KRAMER

122

Total digestible nutrients of about 79% were found by Dickey et al. (1966) in digestibility studies of dried potato pulp with dairy heifers. Digestible protein was close to 6 %. Dickey et al. (1966) showed good results in feeding dry potato pulp to steers. Enrichment of potato pulp with proteins recovered from waste water of potato flour factories by precipitation with an organic acid was reported by Dijkstra (1958a). The protein-enriched potato pulp was ensiled for 3 months and then fed to cows and heifers. Table XXVI shows the composition and percent digestion of the ensiled enriched pulp. The palatability of ensiled potato pulp and protein-enriched potato pulp was found to be good in experiments with heifers and cows (Dickey et al., 1966; Dijkstra 1958a). VII. SUGAR-MANUFACTURING WASTES

A. INTRODUCTION

The world’s total sugar production in 1964 exceeded 81,000,000 tons (U.S.D.A., 1966a). More than 72,000,000 tons of sugar were recovered by the centrifuga1 process. Of this, about 39,000,000 tons were recovered from beets and about 33,000,000 tons from cane. Sugar beet pulp, bagasse, and molasses are the main by-products of the sugar industry. About 1,286,000 tons of dried and molassed beet pulp was used as feed during 1964 in the United States (U.S.D.A., 1966a). The related literature was reviewed by Spencer and Meade (1945), Balch (1947), Long (1949), Scott (1950, 1953), West (1952), Hansen (1955), Gurnham (1955), Keller (1963), and Jensen (1965).

TABLE XXVI COMPOSITION AND DIGESTION COEFFICIENTS (%) OF ENSILED PROTEIN-ENRICHED POTATOPULP^ True protein Composition Average digestion coefficient

Ash

fiber

15.86 61.5

Crude

5.45 53.3

“From Dijkstra (1958b).

Nitrogen-free Crude extract protein

Organic matter

Dry matter

19.08

53.96

21.51

-

10.38

86.8

89.4

68.8

84.1

82.4

THE UTILIZATION OF FOOD INDUSTRIES WASTES

B. BAGASSE AND BEET

123

PULP

According to the definition of the International Society of Sugar Cane Technologists, bagasse is the fibrous residue obtained from the crushing of sugar cane and removal of the cane juice, in the cane sugar-manufacturing process. Litkenhous (1945),West (1952),Hansen (1955),and Keller (1963, 1965) reviewed the literature on the uses of bagasse. According to Keller (1965), 82,000,000tons of bagasse were produced during 1962-1963, of which about 37,000,000 tons consisted of fiber. It is used most widely as a fuel in sugar factories where it is produced, having, according to Chapman (1955), a fuel oil value of 12,000,000BTU’s, or approximately two A2-gallon barrels, to a ton of bone-dry bagasse. Bagasse is also a raw material for structural and acoustical wallboard and other building products, as well as for paper manufacture and other purposes (Long, 1949). Seventy-six percent of the dry weight of beet pulp is composed of the following carbohydrates in this proportion: 6%galactan, 20%araban, 25% cellulose, and 25% pectin. The remainder is mainly ash, proteins, and gums. According to Silin (1958) the dry matter of beet pulp is good feed for cattle, with its feed value placed between meadow hay and oats. Because of the high water content of the pulp (around 94%) it is very perishable. In most sugar beet plants today the pulp is pressed to remove as much water and sugar as possible, and is then dried to produce a livestock feed. Silage is prepared from sugar beet pulp by storage of the wet pulp in silos. These silos are equipped with drains for the removal of pulp drainage water. Sugar beet pulp is a potential raw material for the recovery of watersoluble gum, pressed board, pectin glue, and other products. In the Soviet Union a pectin glue is produced from beet pulp. Heating the pulp above 100°C makes the pulp pectin water-soluble. Separating it from the pulp (by pressing) and concentrating to 50% dry matter produces pectin glue in the form of a viscous mass. This product is used in the textile and printing industries. Whole sugar beets and pulp are considered a potential raw material for the production of fungal protein by Gray and Abou-El-Seound (1966b).The amount of protein produced in these experiments was too low to be of great importance when beet pulp was used as the growth medium. Beet roots, on the other hand, proved to be a better source of nutrients, and approximately 15.97 g of protein per lb of beet roots was harvested. A possibility exists that beet tops, at present discarded or fed to livestock, will offer a suitable nutrient source for the production of fungal protein.

124

ITAMAR BEN-GERA AND AMIHUD KRAMER

Gawecki et al. (1963),in feeding experiments with sheep and cows, determined that dry sugar beet pulp was well utilized as feed, with no negative or harmful effect on the animals or on the milk produced. C. MOLASSES With the recovery of sugar from the cane or beet juice, crystallization inhibitors are accumulated in the crystallization residue. Blackstrap molasses is the crystallization residue, in which the level of the inhibitor is high enough to render the continuation of sugar recovery by crystallization economically unfeasible. Several studies were carried out toward the feasible recovery of sucrose from molasses. Recently Schultz et al. (1967) reported on sucrose recovery from beet molasses by ion exclusion. Although patents for the recovery of sucrose from molasses were issued as early as 1913 (Binkley and Wolfrom, 1953) for the removal of reducing sugars and impurities by lime, invertase-free yeast, barium hydroxide, fuller’s earth, solvents, and ion-exchange resins, it does not seem that any of these have been accepted by the industry. The most important process for recovery (although incomplete) of molasses sucrose are the Steffen process and the Strontic process (McGinnis, 1951). Molasses -mixed with beet pulp, with other feeds, or direct-is fed successfully to livestock. Scott (1953) reviewed the use of molasses in feeding farm animals. Recently, Hamid Miah et al. (1965) concluded that ureamolasses replaces part of the protein requirements for growing dairy calves. Urea-molasses was substituted for green grass, and savings were achieved in feed expenditure. Utilization of molasses in mixed feeds and in silage preparation is well known (Bender, 1948). Molasses is utilized in several biotechnological processes. Some of these processes aim at maximum production of a fermentation product, whereas others seek maximum growth of the microorganisms utilized. The biotechnological utilization of molasses is reviewed by Underkoflar and Hickey (1954)and Silin (1958).Molasses is at present the main raw material for baker’s yeast production, according to Silin (1958). It is a suitable growth medium for production of yeasts for both food and fodder, according to Walter (1953), Wiley (1954), and Irwin (1954). Recently, Mannan and Ahmad (1965) studied the growth of Saccharomyces cereviseae and Torulopsis utilis in molasses and other carbohydrate-containing media. Yield, reported as weight of dry yeast recovered from weight of sugar utilized, was 42-47% with S. cereviseae, and 36-38% with T . utilis. When corn-steep liquor was

THE UTILIZATION OF FOOD INDUSTRIES WASTES

125

added to the growth medium, respective yields reached 60.0 and 52.6%. The S. cereviseae crop contained 50% protein, 2.5%fat, and 13% minerals, while that of T. utilis contained 42% protein, 5.6% fat, and 15.8%minerals. Shukla and Dutta (1967)reported the production of fungal protein from molasses. They used a strain of Rhizopus and were able to harvest a mycelium having 34.37%protein, with a yield of 28%. Fermentation processes for the production of butanol, acetone, and lactic, citric, and acetic acids were reviewed by McCutchan and Hickey (1954), Schopmeyer (1954), Johnson (1954), and Vaughn (1954). The utilization of molasses in fermentation production of 2,3-butanediol was reviewed by Ledingham and Neish (1954), and the production of riboflavin and other vitamins by Hickey (1954) and Van Lanen (1954). About 86% of all the 95% alcohol produced in the United States in 1935 was by fermentation from molasses. In 1945, when alcohol production was about 5.5 times as high, molasses accounted for only 20%. In 1955, with industrial-alcohol production reduced to a level of 250,000,000 gallons of alcohol, or only 2.5 times as high as in 1935, molasses was used for only 16%of the alcohol (Jackson, 1958). This is shown in Fig. 6. In 1966, with a production of close to 700,000,000 gallons of alcohol, only 1.59%of the total was produced from molasses (U. S. Treasury Dept., 1966).

600

500

Yeast, citric acid and vinegar Acetone -n-butyl alcohol Industrial alcohol

v)

6 400

-

Spirits and rum

0 0

m C

0

-

-

'Z 300 f 200

--

Livestock feed

3

! 00

0

FIG. 6.

Edible molasses and sirups

-

126

ITAMAR BEN-GERA AND AMIHUD KRAMER

Vlll. DISTILLERY, BREWERY, AND WINERY WASTES

A. INTRODUCTION

Potentially 17- 18 lb of dried feeds can be recovered from every 56 lb bushel of grain used by the grain-distilling industry (Boruff, 1961). According to Brinker (1967) a total of more than 427,000 tons of distillers’ feeds were produced in the United States in 1966. Of this, 379,000 tons were dried grains with solubles, and the rest was dried grain and dried solubles, an increase of about 20% over figures for 1960. The Distillers’ Feed Research Council, Cincinnati, Ohio, reports that only 1% of stillage in the United States is not recovered. About 85% of the stillage is dried by the distillers, and 14%is fed to livestock undried. About 12.5 lb of dried grains and 0.4 lb of dried yeast are recovered per barrel of beer produced (Quittenton, 1966). In addition, about a pound of press-liquor solids can be recovered per barrel of beer produced. According to Boruff (1961)0.3 to 0.5 lb of hops and residue are recovered that represent potentially utilizable material. Of the original grain entering the brewery, an average of about 25 % is recovered as dry of wet brewer’s grain. Total production of brewer’s dried grains in 1964 amounted to 295,000 tons. About 2,370,086 tons of grapes were crushed for wine, juice, etc., in 1965. About 15%of this quantity is converted into grape pomace (U.S.D.A., 1966a).

B. DISTILLERIES Figure 7 shows a basic distillery operation. The major product of this operation is alcohol, which is produced through fermentation by yeasts of milled, slurried, precooked, malt-saccharified grain. The beer produced, containing alcohol, spent grain, and solubles, goes through a distillation process for recovery of the alcohol. Several feeds are recovered from the stillage. These include light grains, the dried spent grain recovered from stillage by screening; dried solubles, produced by drum-drying of the thin stillage; and dark grains, which are a dried mixture of spent grain and concentrated thin stillage. Proximate analysis of typical distillers feeds from corn and rye mashes was published by Boruff (1961). It is possible, in the manufacture of beer, to use 100%grain malt, or a mixture of unmalted and malted grain. According to Jackson (1960)there is a difference in the composition of animal feeds recovered as by-products from completely and partially malted grains (Table XXVII).

127

THE UTILIZATION O F FOOD INDUSTRIES WASTES Groin

D I S T I L L E R Y OPERATIONS

Molt

J=-k

4 0 gal + / BU 5 - 7 % solids 17-19 Ibs /BU 25,000 ppm BOD 5 0 pop. eq / BU

I

Whole slilloge

I

grains

groins

FIG. 7. Simplified flow diagram of distilling and feed recovery processes. From Blaine and Van Lanen (1962).

TABLE XXVII ANALYSIS OF DISTILLERYBY-PRODUCTS".*

Substance Moisture Crude protein (nitrogen X 6.25) Ash Fat Fiber Calcium Carbohydrate (by difference) a From

Malt Malt distillery distillery dried dried concentratec solublesd

5.88 38.3 5.0 0.18 0.08 0.08 50.6

5.06 26.8 17.2 0.16 0.03 4.64 50.7

Grain distillery dried Dried drege concentrate

2.2 41.3 1.29 14.2 15.4 0.03 25.6

2.08 44.0 3.6 4.06 1.75 0.03 44.5

Jackson (1960).

%.

cDried solids consisting of dead yeast and organic debris from extracted barley malt, recovered by centrifugation. dDried stillage after removal of solids. eCrain remainings and insoluble solids that settle out in ponds (partially malted grain). Solids, mostly dead yeast, recovered from the stillage of partially malted grain after removal of coarse solids. f

128

ITAMAR BEN-GERA A N D AMIHUD KRAMER

Additional data on amino acid composition of the protein in distiller’s by-products, carotenoids, minerals, and vitamins were reported by Bauerfield et al. (1944a,b),Baumgarten et al. (1944, 1945), Boruff (1947, 1961), Anon, (1951),Williams (1955),Carpenter (1955),Lyman et al. (1956), Boruff and Van Lanen (1958), Jackson (1960), and Boruff et al. (1964). Successful utilization of distiller’s feed by-products in poultry feeding was reported by Synold et al. (1943),Hill et al. (1944),Couch (1956),and Jaffe and Wakelam (1956).Protein and amino acid requirements and availability in poultry-feed diets containing distillers feed by-products were recently discussed by Balloun (1966), Potter (1966), and Runnels (1966). Feeding of pigs was reported by Fairbanks et al. (1944), Krider and Terrill (1949), Conrad (1961), and Plumlee et al. (1966); of calves by Knodt and Williams (1950), Slack et al. (1951), Slack et al. (1958), and Jackson et al. (1965); of dairy cattle by Loosli (1960);and of beef cattle and sheep by Baker et al. (1957) and Little et al. (1965).General use of these by-products was reviewed by Boruff and Van Lanen (1958). The presence of known and unknown growth-promoting factor or factors was reported by Carrick (1948), Hauge (1948), Scott et al. (1957),Rasmussen et al. (1957),Couch and Stelzner (1961),Wakelam and Jaffee (1961),Dansi et al. (1966),Boruff et al. (1964),and Runnels (1966). Boruff et al. (1964) evaluated the use of scotch-whisky distiller’s feeds in poultry rations. Growth responses from dried distiller’s feeds in semipurified chick diets are shown in Table XXVIII. Analyses of scotch and U.S. bourbon feeds studied showed them as generally similar. The principal difference was that scotch light grains are relatively low, and scotch solubles relatively high, in B vitamin content as compared with bourbon light grain and bourbon solubles. Substantial growth and increased feed efficiency were secured with most of the distiller’s feed by-products when added to the diet at a 6%level, and even at a 3% level. C. BREWERIES Several by-products are recoverable from the brewing process. Those utilized most are brewer’s grains, brewer’s feed yeast, and hops residues. Proximate analysis was reported by Boruff (1961). Amino acid composition of brewer’s grains and malt sprouts was published by Boruff and Van Lanen (1958), and vitamin content was

129

THE UTILIZATION O F FOOD INDUSTRIES WASTES

TABLE XXVIII GROWTHRESPONSE FROM DRIEDDISTILLER'SFEEDSIN SEMIPURIFIED CHICKEN DIET^ Dried distiller's feeds

A

Av. wt. (g) after 4 weeks on test dietsb

Gain over basal ( Yo)

Feed efficiency (feed/gain)

466

-

2.39

496 487 494

6.4' 4.5d 6.0

2.09 2.01 2.01

48 1 475 472

3.2 1.9 1.3

2.10 2.09 2.16

496 474 481

6.4' 1.7 3.2

2.11 2.11 2.14

499 503 503

7.1" 7.9" 7.9'

2.02 2.00 2.02

508 483 478

9.0' 3.6 2.6

2.01 2.08 2.20

50 1 499 490

7.5' 7.1" 5.2d

2.06 2.19 2.10

A. Basal rationc B. 3% level of distillers' feed Bourbon Grains (light) Grains with solubles Solubles Scotch malt whiskey Grains (light) Grains with solubles Solubles Scotch grain whiskey Grains (light) Grains with solubles Solubles C. 6% level of distillers' feed Bourbon Grains (light) Grains with solubles Solubles Scotch malt whiskey Grains (light) Grains with solubles Solubles Scotch grain whiskey Grains (light) Grains with solubles Soh bles OFrom Boruff e t ul. (1964). *Equal numbers of males and females. Quadruplicate pens (64 chicks). dSignificant at the 5% level (t-test). Significant at the 1%level (t-test).

ITAMAR BEN-GERA AND AMIHUD KRAMER

130

reviewed by Norris (1948). According to Nissen (1946) 23 to 33% of the original grain entering the brewery is recovered as dry or wet brewer’s grains, at present mostly as dry products. Recovery of the spent grain and its utilization as animal feed are well established. The prime market is in beef and cattle feeds (Quittenton, 1966). Quittenton (1966) reported on addition of molasses to dry brewer’s grains. This mixture is being used successfully as cattle feed. Schneider (1947) determined the digestion coefficient of brewer’s dry grain by feeding experiments with wethers. Dijkstra (1955) ensiled brewer’s wet grain in a silo and in an earth pit for 7-8 months. The feeding value of the ensiled wet grain was determined by feeding tests with wethers. Results are in Table XXIX. In a similar feeding test, Dijkstra (1958a) compared the feed value of brewer’s wet grain and distiller‘s wet grain. The results (Table XXIX) show that the feed value of brewer’s wet grain, although good, is slightly inferior to that of distiller’s wet grain, both on starch equivalent and a digestible-protein basis. The utilization of dry brewer’s grains in poultry feed is similar to that in cattle, in spite of the fact that the poultry industry is in constant need of inexpensive sources of protein supplements. Thornton (1962),Thornton and McPhernon (1962),Kienholz (1964), and Quittenton (1966) reported on successful use of brewer’s dry grains in poultry feeding. They reported increased egg production during the pullet laying year, reduced layer body weight, and lower percent fat in liver during the laying period resulted from diets containing up to 40% brewer’s dry grains, as well as reduced mortality in caged hens. Success was also obtained in feeding trials with broilers,

FEEDVALUE

OF

TABLE XXIX DRYMATTER OF BREWERS’WET GRAINS AND DISTILLERS’ WET GRAIN Brewer’s wet grain“

Digestible crude protein (%) Digestible true protein (%) Starch equivalent Nutritive ratio ‘From Dijkstra (1955). *From Dijkstra (1958a).

From earth pit

From concrete silo

Distiller’s wet grainb

15.74

17.90

21.8

13.78 48.1 1:3,1

16.18 57.8 1:3.2

21.5 66.3 1:3.0

THE UTILIZATION OF FOOD INDUSTRIES WASTES

131

Hops represent a potential by-product much smaller in quantity than spent grain. Some breweries discard it whereas others mix it with spent grain and dehydrate them. Brown (1941)and Nissen (1946) reported that hops residues are palatable to dairy cattle when fed in mixtures with brewer’s dry grain. Recovery and dehydration of yeast to be used as food or feed can be a profitable operation if beer production is large enough. Weber (1943) stated that recovery of dry yeast is not profitable in breweries with an annual beer production of less than 500,000 barrels. Wet yeast from small breweries is collected for processing elsewhere or is disposed of as wet feed. Some of the yeast (a third of the dry yeast produced, according to Feustel and Thompson, 1946) is debittered for human consumption. Yeast extract, a product of plasmolysed yeast, is also manufactured for human consumption. Because of its high folic acid content and the high folic acid requirement of turkeys, yeasts are used extensively in turkey feeding (at a level of 2-3%). Stokes (1958) reviewed the distribution and composition of yeast nitrogenous compounds. Several other products can be recovered as by-products from the wastes of beer manufacture. The press effluent, or press liquor, contains about 2 % total solids, with about 50%protein, 25 % fat, and 20 % soluble carbohydrates. Recovery of these solids is desirable for pollution abatement, but is actually done in just a few breweries. Quittenton (1966) discussed different possible recovery methods.

D. WINERIES Grape pomace and winestone compose the bulk of the wastes from the production of grape juice and wine. The pomace, which consists mainly of grape skins, seeds, and pulp, represents 10 to 20 70of the weight of the original raw material. Analyses of 160 samples of pomace reported by Amerine and Cruess (1960) were as follows: moisture 5.1%, ash 6.42%,protein 13.45%,fat 7.35%, and fiber 26.94%. Pomace, with its high fiber content, is a potential livestock feed of relatively low value. Its protein and fat are due mainly to the grape seed. The dehydration of grape pomace and its use in animal feeding has been declining in recent years according to Amerine and Cruess

(1960). Grape seeds constitute about 25 % of the pomace weight. They contain, according to Cruess (1958) and Valebreque (1943), 7 to 19%fat. The oil recovered from grape seeds by solvent or expeller extraction

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around the world does not represent a substantial amount. The press cake can be used as animal feed if the amount of undesirable solvent residues is small. Decorticated seed meal has potentially good feed value. Counter-current water extraction from grape pomace of sugar or alcohol following fermentation was reported by Berg and Guyman (1951) and by Coffelt et al. (1965). The extent to which such methods are used today on a commercial basis for recovery of sugar or its fermentation products is unknown. Tressler and Joslyn (1961), Boruff (1961), and Amerine and Cruess (1960) reviewed the different measures taken to secure recovery of almost all of the tartrates and tartaric acid present in winery wastes. According to Boruff (1961) pomace is a good source of nitrogen, phosphate, and potash, and may be returned to the soil as one way of disposal. Jacob (1943) reported that a ton of pomace (50%moisture content) supplies about 20 lb of nitrogen, the same amount of potash, and 5 lb of phosphorus.

IX. ANIMAL AND MARINE PRODUCT WASTES A. DAIRY

By-products from dairy wastes were reviewed by Wheatland (1960), Eldridge (1961), and Watson (1965). Cheese whey, because of the large volume in which it is produced by the cheese-making industry, poses a vast disposal problem and offers great potentials for utilization, The average composition of whey, according to Eldridge (1961), is: protein 0.9%, fat 0.3%, lactose 4.9%, and total solids 6.9%. In cheese making, the use of 100 lb of milk results in 80 lb of whey. More than 2.7 billion lb of cheese were produced in the United States in 1966 (U.S.D.A., 1966b). This led to the production of about 23 billion lb of liquid sweet and acid whey, with 1.6 billion lb of whey solids containing 13% potentially utilizable protein, of which only about 300,000,000 lb are recovered at present. Additional data are presented by USDA (1966a) and Josephson (1966). Kosikowski (1967) summarized the nutritional potentials of whey for human consumption. Present utilization of whey is limited to the use of powdered whey in the manufacture of poultry and animal feeds (Eldridge, 1961). A fermentation process for the production of high-nitrogen cattle feed from whey was developed by Arnott et al. (1958).

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133

A method for the production of nonhygroscopic Cheddar cheese whey powder was developed recently.* The product is reported to be suitable for use in the food and baking industries. The cost of producing 3,000,000 lb of whey powder annually is estimated at about $.02 per lb. Utilization of a nonhygroscopic acid whey in the manufacturing of whole-milk Ricotta cheese was reported by Kosikowski (1967). Swanson and Ziemba (1967) reported on a process designed initially for commercial use of whey solids. In this process, which is based on separating molecules according to size, separate recovery is possible of products high in protein, lactose, or minerals. Products with tailored whipping, binding, bodying, and texturing properties can be manufactured by this process and are reported to be cheaper than currently used caseinates and egg and soy proteins. The use of whey in a commercially available product of food grade was also reported (Ben-Gera, 1967).An example is a dehydrated mixture of fluid whey, skim milk, and buttermilk, with a protein content of 21.5%, suggested for use in baked goods, emulsified meats, ice cream, and other products. f Utilization of whey through alcoholic fermentation of lactose in the development of a whey-based alcoholic beverage, is under study at the University of Maryland.

B. MEAT

AND POULTRY

The American Meat Institute Foundation (1960) described and defined the different types of meat by-products. Rudolfs (1961) and Johnson (1965) recently summarized waste-recovery operations in the meat-packing industry. Approximate percentage yields of meat products and by-products were given by Rudolfs (1961). Total meat production in the United States in 1964, as reported by the USDA (1966a), was above 16,000,000 tons. Tankage and meat meal disappearance for feed in the United States in 1964 approached 2,000,000 tons. Meat meals are used widely as sources of supplementary protein in livestock and poultry rations. Numerous papers have been published on the feed value of meat meal. The variability in nutritive value of the meal as a result of changing processing conditions is stressed in many of these reports and was recently investigated by 'Blaw-Knox Company, Buffalo, N e w York, Bull. 603. +Land O'Lakes, Minneapolis, Minnesota. Tech. Bull. 222, May 1967.

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ITAMAR BEN-GERA AND AMIHUD KRAMER

Gartner (1964), Gartner and Burton (1965), Summers et al. (1964), Sathe et al. (1964), and Sathe and McClymont (1966). Grace and Richards (1964) and Dahl and Persson (1966) studied the variation in nutritive value of meat by-products in relation to their amino acid content. Cooked blood of pig, chicken, and duck, according to Huang (1964), has been used as a human food for a long time. Huang (1964) studied the supplementation of rice or wheat diets with pig plasma protein or with pig blood meal, and reported remarkable increases in nutritive value of the cereal proteins as a result of supplementation with pig blood. Moran et al. (1967a,b) studied the nutritional value of hog hair as a protein and energy source for growing chicks. They reported an increase from 0.58 to 2.14 kcal/g of metabolized energy as a result of processing hog hair at 148°C for 30 minutes. Supplementation of hog hair with lysine, methionine, tryptophan, and glycine was reported to overcome the depression in nutritive value of a 207'protein cornsoybean diet when all the soybean was replaced by processed hog hair. Davis et al. (1961), Naber (1961), and Vigstedt (1965) thoroughly reviewed the processing, nutritional properties, and utilization of poultry wastes and by-products. Table XXX shows the possible yields of poultry-processing by-products. Davis et al. (1961) calculated the recovery of poultry by-products on the basis of data for 1955. According to his calculations, 90% of the offal, 75% of the feathers, and 44% of the blood were recovered by renderers, farmers, feather dealers, and others. He indicates that the continuing trend toward increased centralization of poultry processing will increase the recovery and utilization of poultry byproducts. According to Naber (1961), feather meal, which represents

TABLE XXX PROCESSED BY-PRODUCTS YIELD FROM PIUNCIPAL TYPESOF POULTRY" _ _ _ _ ~

Poultry type

Broiler Fowl Turkey

Feather meal (%)

By-product meal (%)

Blood meal (%)

Grease

5.16 4.27 4.17

0.78 0.67 0.78

0.64 3.17 0.83

5.50 5.50 5.9

"Lortscheret al. (1957).

( %)

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135

more than half the protein available from poultry by-products, contains about 85% protein, poultry meat scraps are 50-60% protein, and poultry blood meal is 65 and 70% protein. Recently, Summers et al. (1965), Barber et al. (1965), and Moran et al. (1966) studied the nutritive value of feather meal in feeding tests with poultry and pigs. According to Summers et al. (1965), feather meal as the sole source of protein in rations of growing chickens did not support weight gains. Supplementation of the meal with essential amino acids improved rate of gain. Barber et al. (1965) reported that partial substitution of dietary proteins by feather meal is practiced. A corn-soybean ration for growing chicks was not found by Moran et al. (1966) to degrade the nutritive value of the diet. Replacement of all soybean protein with corn and feather meal depressed weight gains severely. Supplementation with methionine, lysine, and tryptophan completely overcame the depression. C. MARINE PRODUCTS According to statistics published by the Food and Agriculture Organization, United Nations (FAO, 1963), the world catch of fish in 1963 was 46.4 million tons. Lovern (1966) estimates that about onequarter of the total world catch was converted into animal feeds. In view of the great world need for additional supplies of good-quality protein, the conversion of fish to animal feed is at least partly a waste. Solution of this wastage is offered through the conversion of fish to edible fish-protein concentrate (FPC) rather than to fish meal of feed grade. The current status of FPC was discussed and reviewed recently by Lovern (1966) and in a series of articles in Food Technology by Snyder (1967), Snyder et al. (1967), Knob1 (1967), and Yaney e t al. (1967). Brody (1965), Slavin and Peters (1965), and Lovern (1965) recently reviewed and discussed problems of waste and by-products recovery from fish and other marine products.

REFERENCES Abbot, J. C. 1966. Protein supplies and prospects: the problem. “World Protein Resources.” Advances in Chem. Ser. No. 57. Chap. 1. Am. Chem. Soc., Washington, D. C. Aceto, N. C., Edwards, P. W., Eskew, R. K., Eskew, C. S., Hurley, R. F., and Hoersch, A. 1950. Production of leaf meals from vegetable wastes in rotary alfalfa driers. U.S.Dept. Agr. Bur. Agr. lnd. Chem. AIC-286. Adams, S. L. 1949. Industrial alcohol for cannery wastes. Food Pucker 30 (9),26.

136

ITAMAR BEN-GERA AND AMIHUD KRAMER

Adams, R., Geissman, T. A., and Edwards, J. D. 1960. Gossypol, a pigment of cottonseed. Chem. Revs. 60,555. Akeson, W. R., and Stahman, M. A. 1966. Leaf protein concentrate: a comparison of protein production per acre of forage with that from seed and animal crops. Econ. Botany 20,244. Almanac. 1966. “The Almanac of the Canning, Freezing, Preserving Industries.” E. E. Judge, Westminster, Maryland. Altschul, A. M. (ed.) 1958. “Processed Plant Protein Feedstuffs.” Academic Press, New York. Altschul, A. M. 1965a. Edible seed protein concentrates: their role in control of ma]nutrition. Israel J. Med. Sci. 1,471. Altschul, A. M. 196513. “Proteins, Their Chemistry And Politics.” Basic Books, New York. Altschul, A. M. 1967. Food proteins: new sources from seeds. Science 158,221. Altschul, A. M., Lyman, C. M., and Thurber, F. H. 1958. Cottonseed meal. In “Processed Plant Protein Feedstuffs.” (A. M. Altschul, ed.) Chap. 17. Academic Press, New York. Arnemiya, S., Morozumi, S., Noritaka, S., and Hiroyuki, M. 1964. Studies on fermentative processing of apple fruit. VI. Production of brandies from apple pomace. J . Fermentation Technol. uapan) 42,388. American Oil Chemists SOC.1965.J . Am. Oil Chemists’ SOC. 42,457A. Ammermann, C. B., Arrington, L. R., Loggins, P. E., McCall, J. T., and Davis, G . K. 1963a. Nutritive value of dried tomato pulp for ruminants. j . Agr. Food Chem. 15,347. Ammermann, C. B., van Walleghen, P. A., Easley, J. F., Arrington, L. R., and Shirley, R. L. 1963b. Dried citrus seeds, nutrient composition and nutritive value of protein. Proc. Florida State Hort. SOC.76,245. Ammermann, C. B., Harms, R. H., Dennison, R. A., Arrington, R. L., and Loggins, P. E. 1965. Dried tomato pulp, its preparation and nutritive value for livestock and poultry. Florida Uniu. Agr. Expt. Sta., Bull. 691. Ammermann, C. B., Easley, J. F., Arrington, L. R., and Martin, F. G. 1966. Factors affecting the physical and nutrient composition of dried citrus pulp. Proc. Florida State Hort. SOC. 79,233. Andrion, V. C. 1948. Effects of copra meal on certain chemical and biological properties of Los Banos clay loam. Philippine J . Agr. 32,178. Anon. 1922-23. Nutritive value of orange pulp for dairy cows. Cali5 Univ. Agr. Expt. Sta. Ann. Rept. Anon. 1949. Western Canner and Packer 41( l), 33. Anson, M. L. 1958. Potential uses of isolated oilseed protein in foodstuffs. In “Processed Plant Protein Feedstuffs.” Chap. 11. (A. M. Altschul, ed.) Academic Press, New York. Arnold, P. T. Dix, Becker, R. B., and Neal, W. M. 1941. The feeding value and nutritive properties of citrus by-products. 11. Dried grapefruit pulp for milk production. Florida Uniu. Agr. Expt. Sta., Bull. 354. Arnott, D. R., Patton, S., and Kesler, E. M. 1958. A method for manufacturing a highnitrogen low lactose product from whey. ]. Dairy Sci. 41,931. Baker, G . L., and Goodwin, M. W. 1938. Pectin from apple thinnings. Fruit Prods. j . 18(2),36. Baker, F. H., Grainger, R. B., Long, R. A,, and Garrigus, W. P. 1957. Value of distillers feeds for ruminants. Proc. Distillers Feed ConJl2th Conf. Cincinnati, 1957,40.

T H E UTILIZATION O F FOOD INDUSTRIES WASTES

137

Balch, R. T. 1947. Wax and fatty by-products from sugarcane. Sugar Research Foundation, N.Y. Technol. Rept. Ser. No. 3. Baliga, B. R., Rajagopalan, J. L., and Shivaromiah, K. 1954. Nutritive value of safflowerseed-cake proteins. Zndiunl. Med. Sci. 8,704. Balloun, S. L. 1966. Protein and amino acid balance in poultry diets. Proc. Distillers Feed Res. Council Conf. 21st Con? Cincinnati, 1966,63. Baptist, N. G. 1954. Essential amino acids of some common tropical legumes and cereals. Brit.J.Nutrition 8,218. Barber, R. S., Braude, R. T., and Mitchell, K. G. 1959. Comparison of dried skim milk and white fish meal as protein supplements for fattening pigs. Proc. Nutr. Soc. Engl. Scof. 18, iii. Barber, R. S., Braude, R. T., Chamberlain, A. G., Mosking, Z. D., and Mitchell, K. G. 1965. The value of feather meal as a protein supplement for growing pigs. Animal Production 7 , 103. Barbera, C. E. 1965. L’utilization du marc de cafe. Cafe Cacao, 9,206. Bauernfield, J. C., Carey, J. C., Baumgarten, W., Stone, L., and Boruff, C. S. 1944a. Nutrient content of alcohol fermentation by-products. Znd. Eng. Chern. 36,76. Bauernfield, J. C., Smith, M. B., Gary, J . C., Baumgarten, W., Gustoff, F. H., and Stone, L. 1944b. Nutrient content of alcohol fermentation by-products from various grains. Cereal Chem. 21,421. Baumgarten, W., Bauernfield, J. C., and Boruff, C. S . 1944. Carotenoids in corn distillers by-products. Znd. Eng. Chem. 36,344. Baumgarten, W., Stone, L., and Boruff, C. S. 1945. Amino acid composition of grain alcohol fermentation by-products. Cereal Chem. 22,311. Becker, R. B., and Arnold, P. T. Dix. 1951. Citrus pulp in dairy rations. Florida Uniu. Agr. E x p t . Sta. Circ. S-40. Becker, R. B., Davis, G. K., Kirk, W. G., Arnold, P. T. Dix, and Hyman, W. P. 1946. Citrus pulp silage. Florida Unio. Agr. Expt. Sta. Bull. 423. Beeson, W. M., Lehrer, W. P., and Woods, E. 1947. Peas supplemented with wheat germ or corn germ as a source of protein for gr0wth.J. Nutrition 34,587. Bender, C. B. 1948. Use of molasses in grass silage preparation. Sugar Research Foundation, N.Y. Technol. Rept. Ser. No. 4. Ben-Gera, I. 1967. Proc Conf. Solid Waste Disposal. Engineering Foundation, New York. Milwaukee, Wisconsin. Ben-Sinai, I. M., Ben-Sinai, M., and Kramer, A. 1965a. University of Maryland. Unpublished. Ben-Sinai, I. M., Ben-Sinai, M., Ahmed, E. M., and Kramer, A. 196513. The food and fodder value of pea plant parts (Pisum satiourn L.) as related to harvest time and maturity. Food Technol. 19,856. Block, R. J., and Bolling, D. 1944. The amino acid yield from various animal and plant proteins after hydrolysis of the fat-free tissue. Arch. Biochem. 3, 217. Block, R. J., and Bolling, D. 1951. “Amino Acid Composition of Proteins and Foods.” Thomas, Springfield, Illinois. Bolton, W. 1954. The digestibility of the carbohydrate complex of bran and oats by adult cocks. Proc. World Poultry Congr. 94. Bond, J., and Putnam, P. A. 1967. Nutritive value of dehydrates sweet potato trimmings fed to beef steers.]. Agr. Food Chem. 15,726. Bondi, A. 1958. Plant proteins. In “Processed Plant Protein Feedstuffs,” (A. M. Altschul, ed.) Chap. 3. Academic Press, New York.

138

ITAMAR BEN-GERA AND AMIHUD KRAMER

Boruff, C. S. 1947. Industrial wastes-recovery of fermentation residues as feeds. Ind. Eng. Chem. 39,602. Boruff, C. S. 1961. The fermentation industries. In “Industrial Wastes -Their Disposal and Treatment,” (W. Rudolfs, ed.) Chap. 6. Library of Engineering Classics, Valley Stream, New York. B o ~ f f ,C. S., and Van Lanen, J. M. 1958. Fermentation feedstuffs. In “Processed Plant Protein Feedstuffs,” (A. M. Altschul, ed.) Chap. 27. Academic Press, New York. Boruff, C. S., Luthy, P. W., and Van Lanen, J. M. 1964. Evaluation of scotch whiskey distillers feeds in poultry rations. J. Sci. Food Agr. 15,364. Braverman, J. B. S. 1949. “Citrus Products.” Wiley (Interscience), New York. Braverman, J. B. S., and Levi, A. 1960. Comminuted orange, a novel process for its manufacture. Food Technol. 14,106. Bressani, R. 1965. The use of cottonseed protein in human foods. Food Technol. 19, 1655. Bressani, R., Elias, L. G., and Braham, E. 1966. Cottonseed protein in human foods. Advan. Chem. Ser. No. 57,p. 75. Brinker, G. M. 1967. President’s message to conference. Proc. Distillers Feed Res. Council Con5 Cincinnati 22,3. Brody, J . 1965. “Fishery by-products technology.” Avi, Westport, Connecticut. Brooke, D. L., and Capel, G. L. 1958. An economic analysis of alternative methods of cull tomato disposal in Dade County Florida. Florida Univ. Agr. Expt. Stas., Agr. Econ. Mimeo Rept. 59-2. Brown, B. M. 1941. Brewery by-products and their disposal. J. Inst. Brewing 47,176. Brown, L. R. 1965. Increasing world food output. U.S.Dept. Agr. Foreign Agr. Econ. Rept., No. 25. Brown, A. H., Ramage, W. D., and Owens, H. S. 1950. Progress in processing pear canning waste. Food Pucker 31(7) 30; (8),50. Burch, J. E., Lipinsky, E. S., and Litchfield, J . H. 1963. Technical and economic factors in the utilization of waste products. Food Technol. 17,1266. Byers, M. 1961. Extraction of protein from the leaves of some plants growing in Ghana. J. Sci. Food Agr. 12,20-30. Byers, M., and Sturrock, J. W. 1965. The yields of leaf protein extracted by large-scale processing of various crops. J. Sci. Food Agr. 16,341. Carpenter, L. E. 1955. The modern distilling industry and its feeds. Feedstuffs 27(9),30. Carrick, C. W. 1948. Known growth factors in distillers solubles. Proc. Distillers Feed Conf. 3rd Con5 Cincinnati, 7. Carusi, A. 1945. Composition and digestibility of tomato seed cake. Ann. Inst. Sper. Zootec. Roma 3,329. Cave, N. A. G., Summers, J . D., Slinger, S. J., and Ashton, G. C. 1965. The nutritional value of wheat milling by-products for the growing chick. 11. Evaluation of protein. Cereal Chem. 42,533. Chandrasekaran, A,, and King, K. W. 1965. Enzymatic modification of the extractibility of protein from coconuts. J. Agr. Food Chem. 15,305. Chapman, A. W. 1955. Purchasing, handling and storing bagasse. “Pulp and Paper Prospects in Latin America.” FAO, United Nations, New York. Chapman, H. L., Haines, C. E., Crockett, J. R., and Kidder, R. W. 1958. Dried tomato pulp for fattening steers on pasture. Florida Univ. Agr. Erpt. Stas. Everglades Sta. Mimeo Rept., 59-3. Charley, V. L. S . 1963. British comminuted drink production. Food Technol. 17, 987.

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139

Chayen, I. A., and Ashworth, D. R. 1953. The application of impulse rendering to the animal fat industry.]. A p p l . Chem. 3,529. Chayen, I. A., Smith, H. R., Tristram, G. R., Thirkell, D., and Webb, T. 1961. The isolation of 1eafcomponents.J. Sci. Food. Agr. 12,502. Christensen, D. A., Lloyd, L. E., and Crampton, E. W. 1967. Nutritive value for rats of certain by-products of the corn refining industry.]. Nutrition 91,137. Circle, S. J., and Johnson, D. W. 1958. Edible isolated soybean protein. In “Processed Plant Protein Feedstuffs,” (A. M. Altschul, ed.) Chap. 15, Academic Press, New York. Collins, J. L. 1960. “The Pineapple.” Wiley (Interscience), New York. Conrad, J. H. 1961. Recent research in the role of unidentified growth factors in 1961 in swine rations. Proc. Distillers Feed Conf. 16th Conf. Cincinnati, 41. Couch, J. R. 1956. Summary of information on unidentified growth factors -economic evaluation. Feedstufs 28 (17),30. Couch, J. R., and Stelzner, H. D. 1961. Vitamin-like, unidentified growth factors from corn distillers dried solubles. Proc. Distillers Feed Conf. 16th Conf. Cincinnati, 65. Cousins, E. R., Fore, S. P., Janssen, H. J., and Feuge, R. 0. 1953. Rice bran oil. VIII. Tank settlings from crude rice bran oil as a source of wax. J . Am. Oil Chemists SOC. 30.9. Cravens, W. W., and Sipos, E. 1958. Soybean oil meal. In “Processed Plant Protein Feedstuffs,” (A. M. Altschul, ed.) Chap. 14. Academic Press, New York. Cruess, W. V. 1958. “Commercial Fruit and Vegetable Products.” Chap. 23. McGrawHill, New York. Curtin, L. V. 1958. Coconut oil meal. In “Processed Plant Protein Feedstuffs,” (A. M. Altschul, ed.) Chap. 23. Academic Press, New York. Dahl, O., and Persson, K. A. 1966. Significance of constituents and processing on the amino acid pattern of meat meal. Acta. Chem. Scan. 20,911. Dang, R. L., Narayanon, R., and Rav, P. S. 1964. Apricot kernel oil: its composition and utilization. Indian oilseed]. 8 (2),10. Dansi, A., Pozzo, A. D., Zanini, G., Meneghini, E., and Craveri, A. 1966. Growth factors of distillers dried solubles. Ann. Chem. Liebigs 695,226. Davis, G . K., Mehrof, N. R., Driggers, J. C., and Dennison, R. A. 1951. Dehydrated celery tops in chickrations. Florida Univ.Agr. E x p t . Sta. Circ. S -37. Davis, J. G., Micchi, E. P., and Linewater, H. 1961. Processing of poultry by-products and their utilization in feeds. I. Processing of poultry by-products. U.S. Govt. Printing Office, Washington, D.C. Davys, N. M. G., and Pirie, N. W. 1965. A belt press for separating juices from fibrous pu1ps.J. Agr. Eng. Research 10,142. Dean, R. F. A. 1958. Use of processed plant proteins as human food. In “Processed Plant Protein Feedstuffs,” (A. M. Altschul, ed.) Chap. 9. Academic Press, New York. De Maeyer, E. M., and Vanderborght, H. 1961. Determination of the nutritive value of different protein foods in the feeding of African children. Natl. Acad. Sci., Natl. Research Counc., U.S. Publ. 843. Deriggers, J. C., Davis, G. J., and Mehrhof, N. R. 1951. Toxic factors in citrus seed meal. Florida Univ.Agr. E x p t . Stas. Bull. 476. De, S. S., Russell, J. S., and Andre, L. M. 1966. Soybean acceptability and consumer adoptability in relation to food habits in different parts of the world. Proc. Intern. Congr. Soybeon Protein Foods. U.S. Dept. Agr. ARS-71-35. Devi, A,, Rao, N. A. N., and Vijayaraghavan, P. K. 1965. Isolation and composition of leaf protein from certain species of India flora. J. Sci. Food Agr. 16,116.

140

ITAMAR BEN-GERA AND AMIHUD KRAMER

Dickey, H. C., Brugman, H. H., Plummer, B. E., and Highlands, M. E. 1966. The use of by-products from potato starch and potato processing. Proc. Intern. Symp. Utilization and Disposal of Potato Wastes. Fredericton, Canada. Dickinson, D. 1960. In “Waste Treatment” (P. C. G. Isaac, ed.) p. 385. Pergamon Press, New York. Dijkstra, N. D. 1955. Experiments about the preservation of brewers wet grains. Rijkslandbovwproef Station, Hoorn, Holland. Dijkstra, N. D. 1957. The digestibility and feed value of oat mill feed. Overdruk Vit Het Landbouwkundig Tijdschrqt 69,471. Dijkstra, N. D. 1958a. Digestibility and feeding value of Dutch distillers wet grains. Rijkslandbouwproef Station, Hoorn, Holland. Dijkstra, N. D. 195813. Digestibility and feeding value of protein enriched potato pulp. Overdruk Vit Het, Landbouwkundig Tijdschrift 70,821. Dowskin, P. B., Hester, 0. C., Kerr, H. W., and Bayton, J. A. 1963. Instant sweet potatoes. US.Dept. Agr., Market Research Rept. No. 580. Duckworth, J., and Woodham, A. A. 1961. Leaf protein concentrate. I. Effect of source of raw material and method of drying on protein value for chicks and rats. J , Sci. Food Agr. 12,5. Duckworth, J., Hepburn, W. R., and Woodham, A. A. 1961. Leaf protein concentrates. 11. The value of a commercially dried product for newly-weaned pigs. J . Sci. Food Agr. 12,16. Eckey, E. W. 1954.“Vegetable Fats and Oils.” Reinhold, New York. Edwards, P. W., Eskew, R. K., Hoersch, Jr., A., Aceto, N. C., and Redfield, C. S. 1952. Recovery of tomato processing wastes. Food Technol.6,383. Eldridge, E. F. 1961. Milk products waste. In “Industrial Wastes-Their Disposal and Treatment” (W. Rudolfs, ed.). Chap. 3. Library of Engineering Classics, Valley Stream, New York. Esselen, W. B., Jr., and Fellers, C. R. 1939. Nutritive value of dried tomato pomace. Poultry Sci. 18,45. Fairbanks, B. W., Krider, J. L., and Carroll, W. E. 1944. Distillers by-products in swine rations. J. Animal Sci. 3,29. Food and Agr. Organization, United Nations. FAO. 1963. Fishery Commodities, Yearbook of Fishery Statistics, 17. FAO. 1965. “FA0 Agricultural Studies.” Food and Agr. Organization, United Nations. Fellers, D. A., Shepard, A. D., Bellard, N. J., and Mossman, A. P. 1966a. Protein concentrates by dry milling of wheat mill feeds. Cereal Chem. 43,715. Fellers, D. A., Sinkey, V., Shepherd, A. D., and Pence, J. W. 1966b. Solubility and recovery of protein from wheat mill feeds. Cereal Chem. 43,l. Fellers, D. A., Shepherd, A. D., Bellard, N. J., and Mossman, A. P. 1967. Protein concentrates by dry milling of wheat mill feeds. 11. Compositional aspects. 52nd Ann. Meeting, American Assoc. Cereal Chemists, Los Angeles. Felton, G. E. 1949. Use of ion exchanges in by-product recovery from pineapple wastes. Food Technol. 3,40. Fisher, H., Summers, J. D., Wessels, J. P., and Shapiro, R. 1962. Further evaluation of proteins for the growing chicken by the carcass retention method. J. Sci. Food Agr. 13,658. Fontaine, T. D., SchafFer, P. S., Doukas, H. M., Scott, W. E., Ma, R. M., Turkot, V. A., DeEds, F., Wilson, R. H., and Doolittle, S. P. 1955. Tomato leaves, a potential alkaloid and sterol source. U.S. Dept. Agr. ARS-73-8. Forsyth, G . C., and Quesnel, V . C. 1963. The mechanism of cocoa curing. Adoan. Enzymol. 25 (1963),457.

THE UTILIZATION OF FOOD INDUSTRIES WASTES

141

Gartner, R. J. W. 1964. Evaluation of meat and bone meals in rations for growing chickens. Australian Poultry Sci. Convention, Surfers Paradise 30. Gartner, R.J. W., and Burton, H. W. 1965.Evaluation of meat-and-bone meals in rations for growing chickens. 3. Effect of cooking time in the preparation of meat-and-bone meal and effect of added tallow in poultry rations. Queensland J . Agr. Animal Sci. 22,281. Gawecki, K.,Frelich, A., and Ponikiewska, T. 1963.The use of dried sugar beet pulp, . molasses distillers residue added, in feeding of ruminants. Zeszyty P ~ o b lPostepow Nauk Rolniczych 41,121. Gerloff, E. D., Lima, I. H., and Stahmann, M. A. 1965.Amino acid composition of leaf protein c0ncentrate.J. Agr. Food Chem. 13,139. Geurin, H. B., Hoefer, J. A., and Beeson, W. M. 1951.Protein efficiency of corn byproducts and other protein feeds for swine gr0wth.J. Animal Sci. 10,607. Glasscock, R. S., Cunha, T. J., Pearson, A. M., Pace, J. E., and Busehman, D. M. 1950. Preliminary seed meal as a protein supplement for fattening steers and swine. Florida Univ. Agr. Expt. Stas. Circ. S-12. Goss, H., and Otagoki, K. K. 1954.Safflower meal digestion tests. Calif. Agr. 8(5), 15. Grace, N. D., and Richards, E. L. 1964.The nutritive value of meat by-products. J . Sci. FoodAgr. 15,711. Could, R. A., and Ash, C. S. 1915.Pineapple waste. U.S. Patent No. 1,166,674. Graci, A. V., Reuther, C. G., Eaves, P. H., Molaison, L. J., and Spadaro, J. J. 1953. Pilot-plant application of filtration extraction of rice bran. J . Am. Oil Chemists’ Soc. 30(4),139. Graham, R. P., Shepherd, A. D., Brown, A. H., and Ramage, W. D. 1952.Advanced fruit waste recovery. Food Eng. 24(2), 82,151. Gray, W. D. 1966.Fungi and world protein supply. Advan. Chem. Ser. No. 57, p. 261. Gray, W. D., and Abou-El-Seound, M. 0. 1966a.Fungal protein for food and feeds. 11. Whole sweet potato as a substrate. Econ. Botany 20,119. Gray, W. D., and Abou-El-Seound, M. 0. 1966b.Fungal protein for food and feeds. IV. Whole sugar beets or beet pulp as a substrate. Econ. Botany 20,372. Greenfield, R. E. 1965. Starch and starch products. In “Industrial Wastewater Control” (C. F. Gurnham, ed.) Chap. 7.Academic Press, New York. Grimes, J. C. 1941. Feeding value of sweet potatoes. Alabama Polytech. Inst. Agr. Expt. Sta. Rept. 52,15. Guilbert, H. R. 1927. A study of various rations for finishing range calves as baby beeves. Calif Unio. Agr. E x p t . Sta. Bull. 418. Gumham, C. F. 1955.“Principles of Industrial Waste Treatment.” Wiley, New York. Guthrie, J . D., Hoffpauir, C. L., Stansbury, M. F., and Reeves, W. A. 1949.The chemical composition of cotton fibers, cottonseed, peanuts and sweet potatoes. U.S.Dept. Agr. Bur.Agr. Ind. Chem. AIC-61. Haines, C. E., Chapman, H. L., and Kidder, R. W. 1959.The feeding value and digestibility of dried celery tops for steers. Florida Uniu. Agr. Expt. Sta., Eoergludes, Mimeo Rept. 59-13. Hale, F., and Lyman, C. M. 1948.Cottonseed as a source of Animal feedstuffs, In “Cottonseed and Cottonseed Products” (A. E. Bailey, ed.) Chap. 21.Wiley (Interscience) New York. Hamid Miah, M. A., Rahim, Q. M. F., and Ali, S. M. 1965.Utilization of molasses with and without o-phosphoric acid as livestock feed in East Pakistan. J. Biol. Agr. Sci. (Pakistan) 8,255. Hansen, R. B. 1955.Bagasse utilization. Sugar]. 18,32.

142

ITAMAR BEN-GERA AND AMIHUD KRAMER

Hardenbergh, W. A., and Rodie, E. B. 1961. “Water Supply and Waste Disposal.” International Textbook, Scranton, Pennsylvania. Harper, G. A. 1965. Trends in the cottonseed processing industry in the United States. Proc. Conf. Cottonseed Protein Concentrates. U.S. Dept. Agr. ARS-72-38. Harshbarger, K. E., and Schingoethe, D. J. 1965. The feeding value of fatted corn gluten feed for dairy heifers. 57th Ann. Meeting Am. SOC.Animal Sci. Hartman, G . H., Akeson, W. R., and Stahman, M. A. 1967. Leaf protein concentrate prapared by spray-drying.]. Agr. Food Chem. 15,74. Hatfield, W. D. 1961. Corn starch processes. In “Industrial Wastes-Their Disposal and Treatment” (W. Rudolfs, ed.) Chap. 7. Library of Engineering Classics, Valley Stream, New York. Hauge, S. M. 1948. Unidentified growth factors in distillers solubles. Proc. Distillers Feed Conf. 3rd Conf. p. 3. Cincinnati. Hays, F. A. 1919. Swine production in Delaware. Delaware Uniu. Agr. Expt. Sta. Bull. 124,28. Hendrickson, R., and Kesterson, J. W. 1954. Hesperidin, the principal glucoside of oranges. Florida Uniu. Agr. Expt. Stas. Bull. 545. Hendrickson, R., and Kesterson, J. W. 1964. Citrus molasses. Florida Uniu. Agr. Expt. Sta. Bull. 677. Hendrickson, R., and Kesterson, J. W. 1965. By-products of Florida citrus. Florida Uniu. Agr. Expt. Sta. Bull. 698. Henke, L. A. 1931. Pineapple bran as feed for livestock. Hawaii Agr. Expt. Sta. Circ. No. 2. Henke, L. A. 1945. Pineapple crowns as cattle feed. Hawaiian Acad. Sci. Ann. Rept. Abstr. Henke, L. A., Willet, E. L., and Maruyanea, C. 1945. Pineapple tops or crowns as a substitute for napier grass in cattle rations. Hawaii Agr. Expt. Sta. Progr. Note 4 5 , l . Hesen, J. C. 1966. The potato processing industry in the lowland area of Europe and the utilization and disposal of potato wastes. Proc. Intern. Symp. Utilization and Disposal of Potato Wastes. New Brunswick Research and Productivity Council, New Brunswick, Canada. Hickey, R. J. 1954. Production of riboflavin by fermentation. In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.) Vol. 2, Chap. 5. Chem. Publ. Co., New York. Hill, P. W., Scott, M. L., Norris, L. C., and Heuser, G. P. 1944. Deficiency of unidentified vitamins in practical chick rations. Poultry Sci. 23,253. Hinton, J. J. C. 1947. The distribution of vitamin B, and nitrogen in the wheat grain. Proc. Hoyal SOC.(London)B 134,418. Horan, F. E. 1966. Defatted and full-fat soy flours by conventional processes. Proc. Intern. Conf. Soy Protein Foods. U.S. Dept. Agr. ARS-71-35. Hove, E. L., Carpenter, L. E., and Harrel, C. G. 1945. The nutritive quality of some plant proteins and the supplemental effect of some protein concentrates on patent flour and whole wheat. Cereal Chem. 22,287. Huang, Po-Chao. 1964. Nutritive value of pig blood meal and the availability of lysine from its protein.]. Formosan Med. Assoc. 6 3 , l . H u h a n , C. F., and Duncan, C. W. 1950. The nutritive value of alfalfa hay. IV. Beet pulp, corn gluten meal and soybean oil as supplements to an all-alfalfa hay ration for milk production.]. Dairy Sci. 33,710. Hull, W. J., Lindsay, C. W., and Baier, W. E. 1953. Chemicals from oranges. Ind. Eng. Chem. 45,876.

THE UTILIZATION O F FOOD INDUSTRIES WASTES

143

International Symposium. 1966. Utilization and disposal of potato wastes. Proceedings, New Brunswick Research and Productivity Council, Canada. R. A. Gallop, Program Chairman. Irwin, R . 1954. “Food and feed yeast.” I n “Industrial Fermentation” (L. A. Underkoflar and L. J. Hickey, eds.) Vol. 1, Chap. 10. Chem. Publ. Co., New York. Isaac, P. C. G. (ed.) 1960. “Waste Treatment.” Pergamon Press, New York. Jackson, C. J. 1960. The treatment of distillery and antibiotics wastes. In “Waste Treatment” (P. C. G. Isaac, ed.). Pergamon Press, New York. Jackson, R. W. 1958. Potential utilization of agricultural commodities by fermentation. Econ. Botany 12,42. Jacobson, N. L., Bryant, J. M., Foreman, C. F., and McGilliard, A. D. 1965. Nutrient source and other factors affecting the utilization of protein and energy by the calf. Proc. 20th Distillers Feed Research Conf. 20th Conf. Cincinnati. 9. JafFe, W. P., and Wakalam, J. A. 1956. Unidentified chick growth factors in fermentation by-products. Nature (London)178,414. Jaky, M. 1947. By-products of tomato processing factories. Konzeruipar 2,21. Janicki, J., Szebiotko, K., and Stawicki, St. 1966. Potatoes and their industrial waste as media for yeast production. In Proc. Intern. Symp. Utilization und Disposal of P o t u t o Wustes. New Bninswick Research and Productivity Council, New Brunswick, Canada. 11. 135. Jensen, L. T. 1965. Sugar. In “Industrial Wastewater Control” (C. F. Gumham, ed.) Chap. 8. Academic Press, New York. Johnson, A. S. 1965. Meat. I n “Industrial Wastewater Control” (C. F. Gurnham, ed.) Chap. 2. Academic Press, New York. Johnson, M. J. 1954. The citric acid. In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.) Vol. 1, Chap. 13. Chem. Publ. Co., New York. Johnston, C., and Watts, A. B. 1965. The infrared spectra of glandless cottonseed meals of varying nutritional value. Poultry Sci. 44,302. Josephson, D. V. 1966. General outlook for milk proteins. In “World Protein Resources” Chap. 3. A d a Chem. Ser. No. 57. Joslyn, M. A. 1961. In “Fruit and Vegetable Juice” (D. K. Tressler and M. A. Joslyn, eds.) Chap. 13. Avi, Westport, Connecticut. Kefford, J. F. 1966. Citrus fruits and processed citrus products in human nutrition. In “World Review of Nutrition and Dietetics” Vol. 6. Krager, Basel, New York. Keller, A. G . 1963. Bagasse. “Encyclopedia of Polymer Science and Technology.” Vol. 2, p. 295. Wiley (Interscience) New York. Keller, A. G. 1965. “A Review of Methods for Depithing Sugar Cane Bagasse.” Proc. Congr. Pulp and Paper Development in Africa and Near East, Cairo. Kelley, E. G. 1948. In “Preparation and Use of Leaf Meals from Vegetable Wastes” (J. J. Willaman and R. K. Eskeu, eds.). Bureau Agr. Ind. Chem., U.S. Dept. Agr. Tech. Bull. No. 958. Kelley, E. G., and Baum, R. R. 1953. Protein amino acids contents of vegetable leaf proteins.]. Agr. Food Chem. 1,680. Kelley, E. G. 1958. Plant residues and pomaces. In “Processed Plant Protein Feedstuffs’’ (A. M. Altschul, ed.) Chap. 32. Academic Press, New York. Kesterson, J. W., and Hendrickson, R. 1957. Naringin, a bitter principle of grapefruit. Florida Uniu. Agr. Expt. Sta. Bull. 511A. Kienholz, E. W. 1964. Brewers dried grains as a protein supplement in chick starter, grower, layer and breeder diets. Feedstufs 36(20),34. Kik, M. C., and Williams, R. R. 1945. The nutritional improvement of white rice. Bull. Natl. Res. Counc. Reprint Circ. Ser. 112, 1.

144

ITAMAR BEN-GERA AND AMIHUD KRAMER

Kirk, W. G., and Davis, G. K. 1954. Citrus products for beef cattle. Florida Uniu. Agr. Expt. Sta. Bull. 538. Kirk, W. G., Felton, E. R., Fulford, H. J., and Hodges, E. M. 1949. Citrus products for fatteningcattle. Florida Unio.Agr. Expt. Sta. Bull. 454. Kline, L., MacDonnell, L. R., and Linewater, H. 1944. Bacterial proteinase from waste asparagus butts. Ind. Eng. Chem. 36,1152. Kneeland, J. A. 1958. Minor oilseed and tree nut meals. In “Processed Plant Protein Feedstuffs” (A, M. Altschul, ed.) Chap. 22. Academic Press, New York. Knobl, G. M. 1967. The fish protein concentrate story. 4. World efforts toward EPC. Food Technol. 21,1108. Knodt, C. B., and Williams, J. B. 1950. Milk replacement mixture corn distillers grain solubles for young dairy calves. Proc. Distillers Feed Conf. 5th Conf. Cincinnati, 1950 46. Knowles, P. F. 1955. Safflower production, processing znd utilization. Econ. Botany 9,273. Knowles, P. F. 1965. Safflower. Calif. Uniu.Agr. Erpt. Sta. Circ. 532. Kobayashi, K., Nagata, I., Nagata, M., and Iwata, H. 1964. Nutritional studies on the utilization of distillers stillage. ]. Agr. Chem. Soc.Japan38,317. Kohler, G . 0. 1966. Safflower, a potential source of protein for human food. In “World Protein Resources” Chap. 17.Aduan. Chem. Ser. No. 57. Kosikowski, F. V. 1967. Greater utilization of whey powder for human consumption and nutrition. J . Dairy Sci. 50,1343. Kramer, A. 1945. The nutritive value of canned foods.]. Am. Dietet. Assoc. 21,354. Kramer, A. 1964. University of Maryland. Unpublished. Kramer, A. 1967. Malaysia four report. The Ofice of Intern. Res. Natl. Inst. of Health, Bethesda, Maryland. Kramer, A., Palmer, S. J., and Kaffezakis, J. G. 1968. Preservation of raw food quality by gas exchange. Hort. Sci. 3 (In press). Krider, J. L., and Terrill, S. W. 1949. Present work at the Illinois Station on distillers grain solubles in swine rations. Proc. Distillers Feed Conf. 4th Conf Cincinnati, 1949. Kumar, D., Kumar, J. S., Mrithyunjaya Rao, D., Thirumala Rao, S. D., and Murti, K. S. 1964. Studies on Indian rice bran and rice bran oil. 11. Indian Oilseed]. 8,49. Kwee, W. H. 1967. University of Maryland. Unpublished. Lambou, M. G., Shaw, R. L., Decossas, K. M., and Vir, H. L. E. 1966. Cottonseed‘s role in a hungry world. Econ. Botany 20,256. Ledingham, G. A., and Neish, A. C. 1954. Fermentative production of 2,Sbutanediol. In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.) Vol. 2, Chap. 2. Chem. Publ. Co. New York. Leffel, E. C. 1968. University of Maryland. Unpublished. Lewis, C. F. 1964. Progress in breeding gland-free cottonseed. Proc. Conf. on Cottonseed Protein Concentrates. U S . Dept. of Agr., ARS-72-38. Litkenhous, E. E. 1945. Bagasse is a source ofpulp. Chemurgic Dig. 4,169. Little, 0. C., Bradley, N. W., and Mitchell, G. E., Jr., 1965. Corn distillers dried grains with solubles and urea in grain rations for beef cattle. Proc. Distillers Feed Res. Council Conf 20th Conf. Cincinnati 1965 35. Long, L. 1949. Sugar and sugar by-products in the plastics industry. Sugar Research Foundation N . Y . Technol. Rept. Ser. No. 5. Long, S . K., and Patrick, R. 1961. Production of 2,3-butylene glycol from citrus wastes. Appl. Microbiol. 9,244.

THE UTILIZATION O F FOOD INDUSTRIES WASTES

145

Loosli, J. K. 1960. The value of different protein supplements for milk production. Proc. Distillers Feed C o n J l 5 t h Conf. Cincinnati. 6. Loosli, J. K., Pena, J. D., Ynalvez, L. A,, and Villegas, V. 1955. The digestibility by swine of rice bean, copra meal, coconut meats, coconut residue and two concentrate mixtures. Philippine Agriculturist 38,181. Lortscher, L. L., Sachsel, G. F., Wilhelmy, O., and Filbert, R. B. 1957. Processing poultry by-products in poultry slaughtering plants. U.S. Dept. Agr. Market Res. Rept. 181. Lovern, J. A. 1965. Trash fish, is there money in it? World Fishing, June, July. Lovern, J. A. 1966. General outlook for edible fish protein concentrates. In “World Protein Resources.” Chap. 4. Advan. Chem. Ser. No. 57. Lyman, C. M. 1964. The role of gossypol. Proc. Conf. Cottonseed Protein Concentrates. U.S. Dept. of Agr. ARS-72-38. Lyman, C. M., Kuiken, K. A., and Hale, F. 1956. Essential amino acid content of farm feeds.J. Agr. Food Chem. 4, 1008. Lynn, L., and Lawler, F. K. 1966. Revolutionizes rice milling. Food Eng. 38 ( l l ) ,68. McCance, R. M., and Walsham, C. M. 1948. The digestibility and absorption of the calories, proteins, purines fat and calcium in whole meal wheaten bread. Brit. J. Nutrition 2,26. McCandlist, A. C., and Weaver, E. 1922. Coconut meal, gluten feed, peanut meal and soybean meal as protein supplements for dairy cows. J . Dairy Sci. 5,27. McCay, C. M., and Smith, S. E. 1940. Tomato pomace in the diet. Science 91,388. McCutchan, W. N., and Hickey, R. J. 1954. The butanol-acetone fermenting. In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.) Vol. 1, Chap. 11. Chem. Publ. Co., New York. McGinnis, R. A. 1951. “Beet Sugar Technology.” Reinhold, New York. McMichael, S. C. 1960. Combined effects of glandless genes gl, and gl, in pigment glands in the cotton plant. Agron.J.52,385. McNary, R. R., and Dougherty, M. H. 1960. Citrus vinegar. Florida Uniu. Agr. Expt. Sta. Bull. 622. Mainardi, B. 1951. Effect of heat treatment on the alimentary value of some oleaginous seeds. Atti. Soc. Ztal. Sci. Vet. 11,308. Mannan, A., and Ahmad, K. 1965. Production of food yeast on molasses, water hyacinth and juite stick hydrolysates and palm juice of East Pakistan 0rigin.J. B i d . Agr. Sci. (Pakistan)8,240. Marais, J. S. C., and Smuts, D. B. 1940. The biological value of the proteins of oats, barley, wheat bran and pollard. J. Vet. Sci. 15,205. Markely, K. S. 1949. Some aspects of processing and utilization of rice bran oil. Rice, October. Marshall, R. E. 1954. “Cherries and cherry products.” Wiley (Interscience) New York. Masuda, H., Noritaka, S., and Maraki, H. 1964. Studies on fermentative processing of apple fruit. IV. Fermentation of milled apple pulp in cider making. J . Ferment. Technol. 42,379. Mead, S . W., and Guilbert, H. R. 1926. The digestibility of certain fruit by-products as determined for ruminants. I. Dried orange pulp and raisin pulp. Calif. Univ. Agr. E x p t . Sta. Bull. 409. Mercer, W. A. 1965. Canned foods. In “Industrial Wastewater Control” (C. F. Gurnham, ed.) Chap. 6. Academic Press, New York. Meyer, E. W. 1966. Soy protein concentrates and isolates. Proc. Intern. Congr. Soybean Protein Foods. U.S. Dept. of Agr. ARS-71-35.

146

ITAMAR BEN-GERA AND AMIHUD KRAMER

Miller, J. I., Morrison, F. B., and Maynard, L. A. 1937. Relative efficiency for growing lambs of the protein in rations supplemented b y soybean oil meal, linseed meal or corn gluten meal.]. Agr. Res. 54,437. Miller, R. F. 1919. “Annual Report of the Director.” Calif. Univ. Agr. Expt. Sta. Milner, M. 1966. General outlook for seed protein concentrates. In “World Protein Resources” Chap. 5. Advan. Chem. Ser. No. 57. Mitchell, H. H., and Beadles, J. R. 1944. Corn germ: a valuable protein food. Science 99, 129. Mitchell, H. H., and Block, R. J. 1946. The relationship between the amino acid contents of proteins and their nutritive values for the rat.]. Biol. Chem. 163,599. Moran, E. T., Summers, J. D., and Slinger, S. J. 1966. Keratin as a source of protein for the growing chick. I. Poultry Sci. 45,1257. Moran, E. T., Summers, J. D., and Slinger, S. J. 1967a. Keratin as a source of protein for the growing chick. 11. Poultrg Sci. 46,456. Moran, E. T., Bayley, H. S., and Summers, J. D. 1967b. Keratin as a source of protein for the growing chick. 111. Poultry Sci. 46,548. Morris, 3rd, D. A., Colker, M. F., and Chemoff, M. F. 1944. Vegetable wastes, availability andutilization. Bureau ofAgr. lnd. Chem. U S . Dept. Agr. AIC-51. Morris, R. H. 1946. Vegetable wastes: their availability and utilization. Food Packer 27,43. Morris, V. H., Alexander, T. L., and Pascoe, E. D. 1946. Studies of the composition of the wheat kernel. 111. Distribution of ash and protein in central and peripheral zones of whole kernels. Cereal Chem. 23,540. Morrison, F. B. 1948. “Feeds and Feeding.” Morrison, Ithaca, New York. Morrison, F. B. 1949. “Feeds and Feeding.” Morrison, Ithaca, New York. Morrison, F. B. 1950.“Feeds and Feeding.” Morrison, Ithaca, New York. Morrison, J. E., and Pirie, N. W. 1961. The large scale production of proteins from leaf extract.]. Sci. FoodAgr. 12,l. Morrison, L. M. 1946. The control of diarrhea by tomato pomace. J. Digest. Diseases 13,196. Mustakas, G . C. 1966. Alternative processes for full fat soy flavors for use in developing countries. Proc. Intern. Con$ Soy Protein Foods. U.S. Dept. Agr. ARS-71-35. Naber, E. C. 1961. Processing of poultry by-products and their utilization in feeds. 11. Utilization of poultry by-products in feeds. U S . Gov. Printing Office. Washington, D.C. Neubert, A. M., Graham, D. W., Henry, J. L., Brekke, J. E., and Beardsley, C. L. 1954. Recovery of sugars from pear canning waste. j . Agr. Food Chem. 2,30. Nissen, B. H. 1946. Brewery waste utilization. Proc. 2nd Ind. Waste Conf., Purdue University. Nolte, A. J., and von Loesecke, H. W. 1940. Grapefruit seed oil manufacture and physical properties. Ind. Eng. Chem. 32, 1244. Nolte, A. J., von Loesecke, H. W., and Pulley, G. N. 1942. Feed yeast and industrial alcohol from citrus waste press juice. Ind. Eng. Chem. 34,670. Normand, F. L., Hogan, J. T., and Deobald, H. J. 1965. Protein content of successive peripheral layers milled from wheat, barley, grain sorghum and glutinous rice by tangential abrasion. Cereal Chem. 42,359. Norris, F. W. 1948. Vitamins in brewing materials and beer. Brewers Dig, 23 (8),45. Odell, A. D. 1966. Meat analogues from modified vegetable tissues. Proc. Intern. Con$ Soybean Protein Foods, U.S. Dept. Agr. ARS-71-35.

THE UTILIZATION O F FOOD INDUSTRIES WASTES

147

Parpia, H. A. B., and N. Subramanian. 1966. Plant protein foods in India. Adoan. Chem. Ser. No. 57. p. 112. Peacock, F. M., and Kirk, W. G. 1959. Comparative feeding value of dried citrus pulp, corn feed meal and ground snapped corn for fattening steers in dry lot. Florida Uniu.Agr. Expt. Sta. Bull. 616. Pirie, N. W. 1959. The large scale separation of fluid from fibrous pulp. J. Biochem. Microbiol. Technol. Eng. 1,13. Pirie, N. W. 1966a. Some suggestions on initiating work on leaf protein as a human food. Rothamsted Exptl. Sta. Harpenden, Herts, England. Pirie, N. W. 1966b. Leaf protein as a human food. Science 152,1701. Plumlee, M. P., Harrold, R. L., Conrad, J. H., and Beeson, W. M. 1966. Response of rats and young pigs to fractions of corn distillers dried solubles. Proc. Distillers Feed Conf. 21st. Conf: Cincinnati, 1966 25. Pomeranz, Y.,and Shellenberger, J. A. 1961. Histochemical characterization of wheat and wheat products. 11. Mapping of protein distribution in the wheat kernel. Cereal Chem. 24,109. Pominski, J . , Eaves, P. H., Vix, H. L. E., and Gastrock, E. A. 1954. Simultaneous recovery of wax and oil from rice bran by filtration extraction. ] . Am. Oil Chemists’ SOC.31,451. Pominski, J., Decossus, K. M., Eaves, P. H., Vix, H. L. E., and Pollard, E. F. 1955. Preliminary cost study of rice wax filtration extraction. lnd. Eng. Chem. 47, 2109. Popper, K., Camirand, W. M., Watters, G. G., Bouthilet, R. J., and Boyle, F. P. 1967. Recycles process brine; prevents pollution. Food Eng. 39 (4),78. Potter, E. F., Bevenue, A., and E. McComb. 1948. Test pear cannery waste for byproduct values. Western. Canner und Pucker, 40 (4), 35. Potter, L. M. 1966. Studies with distillers feeds in turkey rations. Proc. Distillers Feed Research ConJ21st Conf. Cincinnati, 1966 47. Pulley, G . N., and von Loesecke, H. W. 1940. Drying method changes composition of grapefruit by-product. Food Ind. 12 (6),62-63,100-101. Quittenton, R. C. 1966. An assessment of brewery by-products. Tech. Quart. 3 (2), 174. Rabak, F. 1917. The utilization of waste tomato seeds and skins. U.S. Dept. Agr. Bull. 632. Rama Rao, G., Doraiswamy, T. R., Indira, K., Mahadeviah, B., and Chandresehara, M. R. 1965. Effect of fibre on the utilization of protein in coconut cake: metabolism studies on children. Indian]. Exptl. Biol. 3,163. Rasmussen, R. A., Luthy, P. W., Van Lanen, J. M., and Boruff, C. S. 1957. Measurement and differentiation of unidentified chick growth factors using a new, semi-purified ration. Poultry Sci. 36,46. Reagan, W. M., and Mead, S. W. 1927. The value of orange pulp for milk production. Calif Uniu. Agr. Expt. Sta. Bull. 427. Reddi, P. V. B., Murti, V. S . and Feuge, R. 0. 1948. Rice bran oil. I. Oil obtained by solvent extraction. J . Am, Oil Chemists’ SOC. 25 (6),206. Rhoads, A. T. 1965. Disposal systems for food processors. Maryland Processors Rept. March. Rhoads, A. T. 1966. Food processing and canning plant operations. Presentation, Am. Chem. SOC., Dio. Water,Air and Waste Chem. Richardson, K. C. 1967. Submerged acetification of a vinegar base produced from waste pineapple juice. Biotech. G Bioeng. 9,171.

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Ries, S . K., and Stout, B. A. 1962. Bulk handling studies with mechanically harvested tomatoes. Proc. Am. Soc. Hort. Sci. 81,479. Rosen, G. D. 1958. Groundnuts and groundnut meal. In “Processed Plant Protein Feedstuffs” (A. M. Altschul, ed.) Chap. 16. Academic Press, New York. Rudolfs, W. 1961. Slaughter house and meat packing wastes. In “Industrial WastesTheir Disposal and Treatment” (W. Rudolfs, ed.) Chap. 5. Library of Engineering Classics, Valley Stream, New York. Runnels, T. D. 1966. The biological nutrient availability of corn distillers dried grains with solubles in broilers feeds. Proc. Distillers Feed Research Con$ 21st Conf. Cincinnati, 1966 11. Rutkowski, A., Grynberg, H., Szczepanska, H., and Beldowicz, M. 1960. Wheat g e m , rye germ, tomato and rose fruit seed oils, as a source of essential fatty acids. Nahrung4,1115. Sanborn, N. H. 1944. Food canning waste utilization. Proc. 1st Ind. Waste Utilization Conf., Purdue University. Sanborn, N. H. 1945. Canning wastes bring dollars. Food Packer 26(1),35. Sanborn, N. H. 1961. Canning, freezing and dehydration. In “Industrial Wastes -Their Disposal and Treatment” (W. Rudolfs, ed.) Chap. 4. Library of Engineering Classics, Valley Stream, New York. Sathe, B. S., and McClymont, G. L. 1966. Nutritive evaluation of meat meals for poultry. V. Effect of addition of antioxidant during and after processing on growth promoting value of high and low quality meat meals. AustralianJ. Agr. Res. 18,183. Sathe, B. S., Cumming, R. B., and McClymont, G. L. 1964. Nutritional evaluation of meat meals for poultry. 11. Effect of increasing protein concentration, removal of bone, and folic acid, pyridoxine and pantothenic acid supplementation of diets based on high and low quality meals. Australian J . Agr. Res. 15,698. Schiller, K. 1957. Effect of different protein sources in animal nutrition. IV. Biological value of corn protein by-products and some supplementary mixtures with dried skim milk, fish meal and soybean oil meal. Arch. Tlerernahr. 7,244. Schneider, B. H. 1947. “Feeds of the world.” Agricultural Expt. Sta., West Virginia University, Morgantown. Schopmeyer, H. H. 1954. Lactic acid. In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.) Vol. 1, Chap. 12. Chem. Publ. Co., New York. Schultz, J. A., and Thomas, B. H. 1949. The biological value of the proteins of corn germ and endosperm wet milled experimentally. Cereal Chem. 26,1967. Scott, J. J. 1926. Grapefruit refuse as a dairy feed. Florida Uniu. Expt. Sta. Ann. Rept. 25R-26R. Scott, M. L. 1953. Use of molasses in the feeding of farm animals. Sugar Research Foundation, N . Y. Technol. Rept. No. 9. Scott, M. L. 1957. Corn distillers dried solubles as a source of unidentified nutrients required by chickens and turkeys. Proc. Distillers Food Con$ 12th Con5 Cincinnati, 195761. Scott, W. 1950. “The Industrial Utilization of Sugarcane By-Products.” Kent House, Port of Spain, Trinidad. Seeley, R. D. 1958. Milling feeds. In “Processed Plant Protein Feedstuffs” (A. M. Altschul, ed.) Chap. 28. Academic Press, New York. Shaw, R. 1966. Potato specialties from potato waste. Proc. Intern. Symp. Utilization and Disposal of Potato Wastes. New Brunswick Research and Productivity Council, New Brunswick, Canada. Shukla, J. P., and Dutta, S. M. 1967. Production of fungal protein from waste molasses. Indian]. Technol. 5,27.

T H E UTILIZATION O F FOOD INDUSTRIES WASTES

149

Sen, B. R. 1960. The basic freedom-freedom from hunger. Food Agr. Organ., United Nations, Rome. Silin, P. M. 1958. “Technology of Beet Sugar Production and Refining.” U.S. Dept. Agr. and National Science Foundation, Washington, D.C. (Translated from Russian). Singh, N. 1964. Isolation of leaf proteins.]. Food Sci. Technol. 1,37. Singh, M. 1966. Studies on production of protein for food from green vegetation. A report on leaf protein work at the Central Food Research Inst., Mysore, India. Slack, S. T., Turk, K. L., Trimberger, G. W., and Reid, J. T. 1951. Distillers grain solubles-calves starters. Proc. Distillers Feed Con$ 16th Con$ Cincinnati, 1951 5. Slack, S. T., Holter, J. A., and Turk, K. L. 1958. Value of distillers feeds for dairy calves. Cornell Uniu. Agr. Expt. Sta. Bull. 932. Slavin, J. W., and Peters, J. A. 1965. Fish and fish products. In “Industrial Wastewater Control” (C. F. Gurnham, ed.) Chap. 3. Academic Press, New York. Smith, A. K. 1958. Vegetable protein isolates. I n “Processed Plant Protein Feedstuffs” (A. M. Altschul, ed.) Chap. 10. Academic Press, New York. Smith, R. H. 1966. Lipid-protein isolates. In “World Protein Resources” Chap. 10. Advances in Chem. Ser. 57. Smith, S. E. 1941. Tomato and tomato products for feeding fur animals. N. Y. State Agr. Expt. Sta. Rec. 81,93. Smock, R. M., and Neubert, A. M. 1950. “Apples and apple products” Wiley (Interscience) New York. Snyder, D. G . 1967. The fish protein concentrate story. 1. Bureau of Commercial Fisheries Program. Food Technol. 21, 1234. Snyder, D. G., Periser, E. R., and Chapman, W. M. 1967. The fish protein concentrate story. I. Enter: FPC. 11. The deep yam of FPC. 111. Fish catch for FPC. Food Technol. 21,1008. Snyderman, S. E., Boyer, A., and Holt, L. E . 1961. Evaluation of protein foods in premature infants. Natl. Acad. Sci., Natl. Res. Council, Publ. 843. Southwell, B. L., and Blak, W. H. 1948. Dehydrated sweetpotatoes for fattening steers. Georgia Agr. Expt. Sta. Bull. 45. Spencer, G. L., and Meade, G. P. 1945. “Cane Sugar Handbook.” Wiley, New York. Spurgin, M. M. 1964. Vinegar base production from waste pineapple juice. Queensland I . Agr. Sci. 21,213. Staniforth, A. R. 1966. An evaluation of extension methods in the early stages of a project on vacuum compression silage. Outlook on Agriculture 5,117. Stewart, E. D. 1931. Converting wastes into profits. Food Ind. 3, 112. Stokes, J. L. 1958. Microbial proteins. In “Processed Plant Protein Feedstuffs” (A. M. Altschul, ed.) Chap. 29. Academic Press, New York. Stubbs, J. J., Noble, W. M., and Lewis, J. C. 1944. Fruit juices yield food yeast. Food Ind. 16 (9), 694. Summers, J . D., Slinger, S. J., and Ashton, C . C. 1964. Evaluation of meat meal as a protein supplement for the chick. Can.]. Animal Sci. 44,228. Summers, J. D., Slinger, S . J., and Ashton, G. C. 1965. Evaluation of meat meal and feather meal for the growing chicken. Can.]. Animal Sci. 45,63. Swanson, E., and Ziemba, J. V. 1967. Seek more profits from plant wastes. Food Eng. 38 (7),110. Synold, R. E., Carrick, C. W., Roberts, R. E., and Hauge, S. M. 1943. Distillers dried solubles as a vitamin supplement in chick rations. Poultry Sci. 22,323. Szebiotko, K. 1966. Total utilization of potatoes, including the disposal of industrial wastes. Proc. Intern. Symp. Utilization and Disposal of Potato Wastes. New Brunswick Research and Productivity Council, New Brunswick, Canada.

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Tabayoyong, T. 1947. The effects of the additives of copra meal to Los Banos clay loam. Philippine J . Agr. 31,52. Tange, H. de. 1937. Feeding value of tomato pomace. Kiserletusyi Kozlemenyek 40, 125. Templeton, C. W. 1947. Cost studies on dehydrating tomato wastes. Food Packer 28, 53. Texas Agricultural Experiment Station. 1951. Grain sorghum by-products feeds. Texas Agr. Expt. Sta. Bull. 743. Thompson, C. Ray. 1958. Alfalfa and other leaf meals. In “Processed Plant Protein Feedstuffs” (A. M. Altschul, ed.) Chap. 25. Academic Press, New York. Thomton, P. A. 1962. An improvement in growth and egg production in chickens fed brewers dried grains. Feedstuffs 34 (15),50. Thomton, P. A., and McPhernon, T. A. 1962. Controlling body weight and liver lipid accumulation in the chicken with dietary brewers dried grains. Federation Proc. 21 (2),397f. Thorp, W. H. 1948. I n “Cottonseed and Cottonseed Products” (A. E. Bailey, ed.) Chap. 4. Wiley (Interscience) New York. Tomhave, A. E. 1931. Dried tomato pomace in the dairy ration. Delaware Uniu. Agr. Expt. Sta. Ann. Rept. 172,23. Tomhave, A. E. 1932. Dried tomato pomace in the dairy ration. Delaware Uniu. Agr. Expt. Sta. Ann. Rept. 179,23. Tomhave, A. E., Hoffman, E., Kelly, E. G., and Wall, M. E. 1948. In “Preparation and Use of Leaf Meals from Vegetable Wastes” (J. J. Willama and R. K. Eskew, eds.) U . S. Dept. Agr., Bureau Agr. Ind. Chem. Tech. Bull. No. 958. Tressler, D. K. and Joslyn, M. A. Fruit and Vegetable Processing Technology. 1040 pp. Avi. Westport, Connecticut. Underkoflar, L. A,, and Hickey, L. J. 1954. “Industrial Fermentation.” Chem. Publ. Co., New York. U.S.D.A. 1946. The preparation of a liquid apple pectin concentrate. U.S.Dept. Agr., Western Regional Research Lab. AIC-28. U.S.D.A. 1951. Foreign Agr. Service. Circ. FFO 17-51. U.S.D.A. 1956. Chemistry and technology of citrus, citrus products and by-products, U . S . Dept. Agr. Handbook No. 98. U.S.D.A. 1965. In “Losses in Agriculture.” U.S. Govt. Printing Office, Washington, D.C. U.S.D.A. 1966a. In “Agricultural Statistics 1966.” U. S. Govt. Printing Office, Washington. D.C. U.S.D.A. 196613. Annual Statistical Summary. Milk Production and Dairy Products. U. S . Govt. Printingoffice, Washington, D. C. U. S. Treasury Dept., Internal Revenue Service 1966. Alcohol and tobacco. Summary Statistics, Publication 67. U. S. Govt. Printing office, Washington, D.C. Valebreque, G. 1943. The production of oil from grape seeds. Wines and Vines 24 (1l),24. Van Etten, C. H., Rackis, J. J., Miller, R. W., and Smith, A. K. 1963. Amino acid composition of safflower kernels, kernel protein and hulls, and solubility of kernel protein. J . Agr. Food Chem. 11,137. Van Lanen, J. M. 1954. Production of vitamins other than riboflavin. In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.) Vol. 2, Chap. 6. Chem. Publ. Co., New York.

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Vaughn, R. H. 1954. Acetic acid-vinegar. In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.) Vol. 1, Chap. 17. Chem. Publ. Co., New York. Vecchiotti, G. G., and Piva, G. 1964. Amino acids level in tomato by-products. Proc. Congr. Soc. ltal. delle Sci. Vet. p. 279. Veldhuis, M. K., and Cordon, 0. W. 1947. Experiments on production of feed yeast from citrus press juice. Proc. Florida State Hort. Soc. 60,32. Vigstedt, C. R. 1965. Poultry and eggs. I n “Industrial Wastewater Control” (C. F. Gurnham, ed.) Chap. 4. Academic Press, New York. Virtanen, A. I. 1966. Milk production or cows on protein-free feed. Science 153, 1603. von Loesecke, H. W. 1950. Citrus cannery waste, its use and disposition. U . S. Dept. Agr. Bur. Agr. 2nd. Chem. AIC-290. Wakelam, J. A., and Jaffee, W. P. 1961. Unidentified chick growth factors in fermentation by-products. J . Sci. Food Agr. 12,335. Wall, M. E. 1948. I n Preparation and Use of Leaf Meals from Vegetable Wastes, (J. J. Willaman and R. K. Eskew, eds.) U. S. Dept. Agr., Agr. 2nd. Chem. Tech. Bull. No. 958. Walter, F. C. 1953. “Manufacture of Compressed Yeast.” Chapman & Hall, London. Warner, R. G., Loosli, J. K., and Davis, R. F. 1957. A study of the value of corn distilled dried grains, coconut oil meal and corn gluten feed for milk production. J. Dairy Sci. 40, 123. Waterlow, J. C. 1962. The absorption and retention of nitrogen from leaf protein by infants recovering from malnutrition. Brit. J . Nutrition 16,531. Watkins, R. J. 1967. Opportunities for expanding exports from Ecuador. 365 pp. Surveys and Research Corp., Washington, D.C. Watson, C. W. 1965. Dairy products. In “Industrial Wastewater Control” (C. F. Gurnham, ed.) Chap. 5. Academic Press, New York. Watson, S. A. 1959. Grain sorghum. Cereal Sci. Today 4,230. Watson, S. A. 1967. Manufacture of corn and silo starch. I n “Chemistry and Industry of Starch” (R. L. Whistler and E. F. Paschal], eds.) Vol. 11. Academic Press, New York. Watson, S. J., and Smith, A. M. 1951. “Silage.” Crosby Lockwood, London. Watts, A. B. 1964. Properties of glandless cottonseed. Proc. Conf. Cottonseed Protein Concentrates, U.S.D. Agr. ARS-72-38. Weber, P. J. F. 1943. Utilization of brewery by-products. Am. Brewer 76,60. Webster, R. 1929. Tomato cannery waste. Proc. 3rd Ann. Conf. Maryland Water and Sewage Assoc. 33. Wellborn, W. A., Parker, J. S., Molaison, L. J., and D’Aquin, E. L. 1951. Pilot-plant steam refining of high fatty acid rice bran oil. Rice]. 54 (8),6 . West, C. J. 1952. The utilization of sugar cane bagasse for paper, board, plastics and chemicals. Sugar Research Found., N.Y. Technol. Rept. Ser. No. 8. Wheatland, A. B. 1960. Treatment of waste water from milk products factories. In “Waste Treatment” (P. C. G. Isaac, ed.). Pergamon Press, New York. Wiegand, E. H. 1937. Agricultural and cannery waste utilization. Western Canner and Packer 29(8), 12. Wiley, A. J. 1954. In “Industrial Fermentation” (L. A. Underkoflar and L. J. Hickey, eds.) Vol. 1, Chap. 9. Chem. Publ. Co., New York. Willaman, J. J., and Eskew, R. L. 1948. Preparation and use of leaf meals from vegetable wastes. U . S. Dept. Agr., Bur. Agr. 2nd. Chem. Tech. Bull. No. 958.

152

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Williams, H. H. 1955. Essential amino acid content of animal feeds. Cornell Uniu. Agr. Expt. Sta. Memo No. 337. Wilson, R. F., and Tilley, S . M. A. 1965.Amino acid composition of alfalfa and of alfalfa and grass protein preparati0n.J. Sci. Food Agr. 16,173. Work, H.S., and Henke, L. A. 1940.Pineapple syrup and cane molasses for fattening pigs. Hnwctii Agr. Expt. Sta. Repts. 2 , 231. Yaney, E., Barja, I., Monckeberg, R., Maccioni, A., and Donoso, G. 1967. The fish protein concentrate story. G. Quintero fish protein concentrate: protein quality and use in foods. Food Technol. 21,1604. Ziemba, J. V. 1966.Let soy proteins work wonders for you. Food Eng. 38,82.

TROPICAL FRUIT TECHNOLOGY BY N. CZYHRINCIW Department of Chemistry and Technology, Faculty of Agronomy, Central Uniuersity, Maracay, Venezuela

I. Introduction .......................................................

11. The Significance of Fruits ........................................... ......................... 111. Morphology and Anatomy of Fruits . . . .

163 IV. Physical Properties of Fruits ............................................. 165 V. Some Chemical Properties of Fruits ...................................... 174 A. Color, Aroma, and Flavor. ............................................ 174 B. Vitamins and Mineral Salts ........................................... 179 C. Carbohydrates, Proteins, Fats, and Caloric Value ....................... 182 D. Enzymes ........................................................... 183 E. Other Substances . . ........................... 185 .................. 185 VI. Technical Problems ............................... A. Preservation of Fresh Fruits and Freezing.. ............................ 185 B. Separation of the Inedible Part (Skin). ................................. 188 C. Obtaining Juices, Pulps, and Concentrates ............................. 190 ................................... 194 D. Dehydrated Products . . . . . . . . E. Sweet Products. . . . . . . . . . . . . ................................... 195 F. Fermentation Products -Alcoholic and Acetic ......................... 196 G . Age of Elaborated Products ........................................... 199 H. Preservation by Irradiation ........................................... 204 VII. Conclusion . . . . . . . . . . . . ...................................... 204 References ............................................................. 207

I. INTRODUCTION With the expansion of the food industries at the beginning of the twentieth century, the science of food technology was established for study of the preservation of edible agricultural products in their natural state, and for the study of cheap and practical methods of processing foodstuffs, to preserve them and improve their quality. Processing is the series of manufacturing operations that transform the natural structure and change the proportions of the substances 153

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initially present in the raw materials. The science of food technology is concerned with study of the physical and chemical properties of the raw materials and the finished products, of the manufacturing processes, of the machinery used, and of the analyses used-organoleptic, physical, chemical, and bacteriological. Many modern industrial processes are based on ancient experience gained in primitive manufacturing and rural handicrafts. Desrosier (1961), for example, presented a historical survey of methods of cereal preservation known for thousands of years. H.: refers to the ancient civilizations of the Middle East, of India, of the Inca Empire in Peru, etc. Such products as salted and pickled fish, cured meats, fermented drinks, bakery goods, etc., were commonly sold in the markets of the Roman Empire. Storni (1942) reports that the aborigines of South America had developed processes to preserve certain food products, and that they could manufacture flour, sweets, beverages, and other things. Friedman (1963) states that “many of our established methods of food preservation and food processing come from prehistorical times, and their safety has been tested in the crucible of human experience, not always, however, definitively and with finality.” Studies in food technology cover various fields, characterized either by the nature of the raw materials (e.g., cereals, fruits, meats) or by the common processes of manufacturing and the resulting similarities in the finished products (e.g., bread baking, canning, bottling of soft drinks). As a part of food technology we study fruits, particularly the problems presented by tropical fruits. The development of the several fields of food technology is very important in the social and economic life of all countries, as may be seen from the history of Australia, where the great boom in the past century in farming and stock raising was brought about by the develop ment of frozen-meat technology and canning industry, which made it possible for high-quality products at satisfactory prices to be exported to the markets of distant and thickly populated Europe. Peterson and Tressler (1963) consider the social and economic development of Australia to be “almost an ideal case.” The industrialization of pineapple in Hawaii is another example of the significance of food technology. The growing and processing of pineapple in Hawaii has become a principal occupation of the people of the island. New, thoroughly mechanized factories produce 57% of the world’s canned pineapple and 82% of its pineapple juice (Cushing, 1960).

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In the United States 28% of industry is concerned with food processing, employing 13.29’0 of the national working force (Parker et al., 1952). It may be noted that during the last twenty years, the consumption of frozen foods has increased eight times, and that of baby foods 42 times (Proctor, 1960). In England, the consumption of canned juices has increased 400% in the last five years. The development of agriculture in distant areas would be impossible without modern technology to preserve and process farm products. Desrosier (1961) has estimated that the present world population will have doubled in the next forty years, from three billion to six billion; the demand for foodstuffs will grow proportionally. Areas in the temperate zone suitable for growing wheat or rye will soon be totally occupied; meanwhile, huge areas suitable for rice and corn lie untouched in the tropics. Also, land suitable for growing the white sugar beet (Beta uulgaris) soon may not be available. Although scientific research has increased the sugar content of this root from 6 to 18% within the last century, it does not seem probable that this can be carried any further. However, there is no shortage of space for growing sugar cane in the tropics. Equally vast possibilities exist in the tropics for the cultivation of other crops, especially for fruit plants. Thus, a great future may be expected in tropical farming, and inevitably, a parallel development in tropical food technology. This advance in tropical farming will occur sooner than the production of synthetic foods mentioned by Proctor (1960), despite the fact that the cost of many synthetic vitamins has decreased by almost half during the last six years, according to Fox (1963). Food technology must prepare in advance to respond to its future role as a principal factor in food production and processing. Desrosier (1961), in his absorbing book, has explained the possibilities of food production in the new tropical zones, and also its main technical difficulties. Wickiner (1960) has also discussed important aspects of farming in Asia, Africa, and Latin America. Food technology, particularly fruit technology, in the tropics is determined by certain specific conditions. (1) Natural fruit resources are potentially enormous. One recalls Humboldt’s calculation that the growing of 33 kg of wheat and 90 kg of potatoes requires the same ground area as the growing of 4000 kg ofbananas (Nicholls, 1901). There are many obscure fruit plants in the wild or semi-primitive state. The time necessary for growing plants to the first crop is relatively short in the tropics; this is an important factor in planning and

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forecasting needs for raw materials. For example, papaya and passion fruit trees start to bear at the end of the first year; cashew at the third year; the mango tree between the fourth and sixth years; and the coconut from five to eight years. Another advantage is that since fruit crops ripen at different times in the tropics the manufacturing processes can work on freshly harvested fruits almost all year long. (2) Transport of harvests and technological processes are usually carried out at high temperatures. Van't Hoff established that every increase in temperature of 10°C speeds chemical reactions two to three times. This necessitates special study on desirable or undesirable biochemical processes, autoxidation, and, particularly, on tinplate corrosion. Temperatures in production departments and storage houses always range between 22" and 32°C. Other negative factors, such as strong sunlight and high seasonal humidity, affect the manufacturing and storage of raw materials and elaborated products. (3) In the future development of tropical-fruit technology, export to far markets should be taken into account. Such factors as the conditions of manufacturing and packaging the semifinished products, as well as the stability of the exotic products, will be of great importance. (4) It is probable that the products of a majority of tropical countries will be less contaminated b y radioactive elements than the products of more northerly countries, as may be confirmed by comparative determinations of radioactive contamination made by Solanas et u1. (1964). (5) Since the larger and more experienced research institutions have been located in temperate and subtropical climatic zones, different aspects of the technology of tropical fruits have not yet been properly studied. Only botanical data and data on the principal chemical substances contained are known for tropical fruits (INCAPICNND, 1961; Popenoe, 1938, 1939; Landaverde, 1941; Nicholls, 1901; Chandler, 1958; Castaiieda, 1961; Pittier, 1926; Sturrock, 1959; Kennard and Winter, 1963; Aristeguieta, 1950; etc.). Among all tropical fruits, those most studied have been: the banana, by von Loesecke (1949) and Simmonds (1959); the pineapple, by Collins (1960); the mango, by Singh (1960); the passion fruit, by Pruthi (1963);and the coconut, by Child (1964). There is a serious lack of knowledge and technical data on tropical raw materials and on the most suitable methods for their processing. Some of the data and technical methods currently available, and lines for further research will be covered in the following chapters.

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II. THE SIGNIFICANCE OF FRUITS Fruits are edible products, borne on the perennial higher plants, having agreeable sweet-sour and semiastringent flavors. Their water content is high, and they deteriorate easily. The texture of the mature tissues is relatively soft. Fruits develop, according to their position on the plant, in contact with the air, not with the soil. The edible products of the fruit-bearing higher annual plants are called vegetables, e.g., tomatoes, red peppers, cucumbers. Melons and watermelons are a sweet class of vegetables that develop on the soil surface. Nuts, which are edible products of fruit-bearing plants, have an agreeable flavor, a high content of fat and protein, a reduced water content, and a hard texture when mature. The products of fruitbearing plants with a very high content of aromatic essences are spices (pepper, coriander with 2% oil, cardamon with 343% oil, etc.). The definition of fruit given by “Webster’s International Unabridged Dictionary” states: “There is no well-drawn distinction between vegetables and fruits in the popular sense; but it has been held b y the courts that all those which, like potatoes, cabbage, peas, carrots, celery, lettuce, tomatoes, etc., are eaten (whether cooked or raw) with the principal part of the meal are to be regarded as vegetables; while those used only for desserts are fruits.” This definition, quoted by Meyer (1960), deals more with the location of fruits within a dietetic dimension and in accord with popular experience than with the technical definition of these important products. Hughes (1962) gives the following definition for fruits: “the foods commonly designated as fruits, however, are pulpy in character, often juicy, and since they develop from the flowers of plants, they consist of the ripened seed or seeds with some edible tissues attached.” The famous Russian scientist F. W. Zerevitinoff stated 40 years ago that “fruits are poetical and vegetables are prose inspirations in the human nutrition.” Rietz (1961), in his “Gustametric Chart,” classifies fresh fruits as a separate group of foods, placing them within the scale of taste intensity (which ranges from zero, for water, to 940, for red pepper) between the numbers of 39 and 350. Most foods are included in this range, whole-wheat bread having a value of 12, and rum a value of 350. Thus, Rietz has confirmed one of the characteristics of fruits: “fruits are foods with the widest range of flavor factors.” Within the taste-intensity scale given above, the best-known tropical fruits have classification numbers shown in Table I. In comparison, nontropical fruits are shown in Table 11.

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Papaya Avocado Cherimoya Banana

39 40 44 48

Mango Guava Pineapple

75 83 100

TABLE I1 CLASSIFICATION NUMBERS OF NONTROPICAL FRUITS” Apples Grapes Oranges

52 72 122

Lemon Lime

260 350

aCalculated from “Gustametric Chart” of Rietz (1961).

Desrosier (1959) has classified foods according to their acidity, putting the fruits into two groups, one of pH 4.5-3.7, and one a highly acid group of pH 3.7-2.3. According to research of Ralls (1959), the specific flavor of cooked vegetables (green peas, red beets, spinach, canned asparagus) depends partially on acetoin and other products of its autoxidation. Acetoin is produced by glycol oxidation and is found in the fresh vegetables mentioned. It increases in content at the beginning of cooking. For instance, the content of acetoin in green peas cooked fifteen minutes may reach 340 ppm. The products of acetoin oxidation are also found in the baking of bread (75 ppm). Fresh and cooked fruits do not have such specific taste and aroma. Further studies of the above-mentioned substances would probably make possible more objective determinations of the characteristic differences between fruits and vegetables. Because of their organoleptic and chemical properties, fruits, with their pronounced flavors, are consumed b y man in relatively smaller quantities than are the simplest vegetable products, such as rice, potatoes, and beans. Fruits are eaten in the natural or the processed state; before meals to stimulate the appetite, or to slake the thirst; or as dessert after the main course, to cleanse the palate of the flavors of soup, fish, or meat. Although of relatively low caloric value, fruits are an important element of human nutrition throughout the world. Ruehle and Ledin (1955) report that the mango, for instance, “is probably of more

TROPICAL FRUIT TECHNOLOGY

159

importance to the people of the tropics than the apple or the peach to the people in temperate countries.” Fruit supplies abundant vitamins, mineral salts, and carbohydrates. The aromatic and gustatory substances, together with sugars, organic acids, and tannic substances, intensify secretion. A relatively rich cellular structure and the pectic substances contribute to the dynamics of digestion. For the importance of fruit in the popular diet, the calculations of Jaff6 et al. (1963) may be cited, to the effect that bananas supply the people of Venezuela with 7% of their calories, 26% of their vitamin ’ of their vitamin A in the form of carotene. C, and 48% Lassabliere et al. (1950) studying the development of human nutrition throughout the world, mentioned that fruits were consumed at an earlier date in hot climates. Bhutiani (1956), studying the importance of the development of horticulture, emphasized that in India, for instance, the population is mainly vegztarian. T h e medicinal properties of fresh and processed fruits, such as grapes, apples, and apple flour, have been studied intensely in many temperate countries, and it would seem profitable to study tropical fruits also, basing the studies on popular experience. Research by many workers has indicated a great variation in the chemical composition of fresh vegetable products. They reported that the chemical composition is influenced by genetics, fertilizers, degree of ripening, etc. It is enough to say, for example, that in 28 varieties of mango, the content of ascorbic acid may vary between 7 and 131 mg per 100 g Singh (1960) presented extensive data on the variation in chemical composition of different varieties of mangoes, and Cegarra (1964) presented data on some varieties of mango in Venezuela (Table 111). Riaz-ur-Rahman et al. (1956) studied 11 varieties of Indian guava, determining their shapes, sizes, color, and content of the principal chemical compounds. The ratio of sugar to acidity (citric acid) was found to fluctuate between 17.9 and 7.7. Rivas (1964) also determined the composition of varieties of guava (Table IV). All the above data demonstrate the difficulties presented to the analyst when he tries to determine the exact characteristics of fruits. According to research and common experience, the processing of fruit reduces its nutritive value to varying degrees, depending on the processes involved. In thermal treatment, such as cooking or sterilization, the finished product is still rich in nutritive value as compared with the raw material, but the enzymes are totally deactivated. Only with respect to enzymes do fresh fruits differ significantly from processed fruits, mostly canned or bottled.

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160

CONTENTS

OF

TABLE 111 SUGARS, ACIDS (AS CITRIC ACID), AND TANNINSOF 11 VARIETIES OF MANGOES"

Varieties Martinica Glenn Irwin Selection 80 Selection 85 Kent Zill Sensation Smith Lippens Blackman

Sugar (70) 11.68 15.15 15.24 12.26 17.00 16.37 16.11 15.95 13.65 16.58 16.75

Acids (Oh)

Tannins ( %)

0.12 0.13 0.17 0.19 0.25 0.31 0.34 0.29 0.26 0.20 0.28

0.026 0.026 0.018 0.046 0.039 0.026 0.018 0.016 0.021 0.014 0.023

"Cegarra (1964).

TABLE IV STUDYOF THREE INGRAFTEDVARIETIES Variety Weight Total solids (%) Soluble solids Total acidity (%) (citric) PH Ascorbic acid (mg/100 g) Pectins (70)

(a)

OF THE

GUAVA"

Periforme de Trujillo

Eloina

Dominica Roj a

60-95 g Av. 75 g 21.52 14.0

120-185 g Av. 170 g 19.10 9.0

90-150 g Av. 120 g 17.91 13.0

1.672 3.60 214.6 0.925

0.748 4.10 26.7 0.750

1.426 3.85 85.8 0.800

"Rivas (1964).

Depending on the area of origin and principal distribution of perennial plants, fruits may be classified as being tropical, subtropical, temperate, or subpolar. Popenoe (1939) defines tropical plants in the following way: "plants which will not grow where the temperature falls much below 4.4"C are here termed strictly tropical; by tropical plants are meant (following P. H. Rolfs) those of the zone in which coconut can be grown; and by subtropical plants, those of the zone of the orange." Dassler et al. (1957) classified bananas, pineapples, avocados, etc., together

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161

with citrus fruits, as “southern fruits,” which seems very inexact from the phytogeographic point of view. Among these four groups of fruit plants, the tropical group is the largest. Some 150 species are known, distributed among 40 different botanical families, and probably many more will be found. Such diversity does not exist in the temperate zones, and is far less in cold areas. However, only approximately a tenth of tropical species are cultivated on a large scale, and only this part is studied in its technical aspects. The origin of tropical fruit plants is very diverse, as is shown in Tables VA,B (Vavilov, 1950), which also show that the majority of tropical fruit plants are from Central and South America. Some fruits, generally harvested in a state of “pre-botanical” maturity, must either be processed immediately or else stored in suitable places; such harvesting may be done to intensify, retain, or slow their maturation for a longer or shorter period. In such conditions, the fruits reach the required grade of “commercial” or “industrial” maturity, which may or may not coincide with the condition of botanical maturity. The grade of commercial or industrial maturity is that in which the fruits show that they have the most adequate physical and che mica1 properties, by organoleptic tests, for consumption in the fresh state or for industrial processing to finished products. A study b y Krishnamurthy et al. (1960) on mango ripening may be taken as typical for demonstration of the principal dynamics of development of the physical and chemical properties for the majority of fruits (Table VI). During the ripening of fruits the content of protopectin is reduced and the content of pectin increases. Many fruits, such as bananas, the mango, and the papaya, are processed not only when they are ripe but also in the unripe state, which is not done with fruits of the temperate zones. Fruits are valued not only for their food value but also for their flavor and beauty. Fruits adorn the table, and fruit motifs are common in art. Fruits are symbols of feasting, both social and religious, in the poetry of many peoples. The Bible (Genesis I and 11) tells of Eden, the fruit garden of Paradise, created by God; Adam was tempted b y forbidden fruit. Simmonds (1959) reported many references to the banana in ancient Indian writings, and the fruit appears many times in ancient Indian art. Popenoe (1939) and Singh (1960) mentioned the role of the mango in the mythology and religious ritual of India. Alphonse de Candolle believed that the mango had been cultivated for 4000

162

N. CZYHRINCIW TABLE V.A SOME TROPICALFRUITS

English name

Spanish namea

Avocado Aguacate Banana Camblir Bullock‘s heart Chirimoya Cashew apple Merey Cherimoya Chirimorriiion Giant Granadilla Parcha Guava Guayaba Mango Mango Mammee apple Mamey Papaya Lechosa Passion fruit Parchita Plantain PIPtano Pineapple Piiia Pomerac Pomagis Sapodilla Nispero Sapote Sapote Soursop Guanibana Sugar apple Riiih Tamarind Tamarind0 Yellow mombin Job0 West Indian cherry Semeruco

Botanical name

Family

Persea americana, Mill. Musu paradisiacu sapientum, L. Annona reticulata, L. Anacardium occidentale Annona cherimoya, Mill. Passijbra quadrangularis Psidium guajava, L. Mangifera indica, L. Mammea americana, L. Carica papaya, L. Passijiora edulis, L. Musa paradisiaca, L. Ananas comosus, Merr. Syzygium malaccensis, L. Achras sapota, L. Colocurpum mammosum, L. Annona muricata, L. Annona squamosa, L. Tamarindus indica, L. Spondias lutea, L. Malpighia punicifolica

Laureaceae Musaceae Annonaceae Anacardiaceae An non ace ae Passifloraceae M yrtaceae Anacardiaceae Guttiferae Caricaceae Passifloraceae M usaceae Bromeliaceae M yrtaceae Sapotaceae Sapotaceae Annonaceae An nonace ae Cesalpinaceae Anacardiaceae Malpighiaceae

“The Spanish names given are those current in Venezuela.

years. Collins (1960) reports that the pineapple was not important in the religion of pre-Columbian Mexico, but this fruit came to be the symbol of lavish hospitality in Europe, especially among the upper classes. Dassler et a,!. (1957) reported that the fine flavor of the pineapple has given it the title of “the queen of all the Southern fruits.” Storni (1942) quoted a saying of the Indians of South America: “those who eat avocados live many years”; and Merory (1960) reported that the same people call papayas “the fruits of the angels.” We might mention many more examples of popular interest in fruits, as compared with other foods. However, we shall cite only the following: Among the many spoken languages, few names of things are similar in pronunciation except for words for common and important things, such as salt, water, and wine. English speakers say “fruits”; Spanish speakers say “frutas”; Germans say “friichte”; Russians say “frookty”; and the Arabs say “frote-fueke.”

163

TROPICAL FRUIT TECHNOLOGY TABLE V.B SOME TROPICAL FRUITS Fruit Avocado Banana Bullock's heart Cashew apple Cherimoya Giant Granadilla Guava Mango Mammee apple Papaya Passion fniit Plantain Pineapple Pomerac (Ohia) Sapodilla Sapote Soursop Sugar apple Tamarind Yellow mombin West Indian cherry

'bile Drupe Berry

-

Nut? Berry Berry Drupe Drupe Berry Berry Berry Multiple fruit Berry Berry Berry Aggregate fruit Legume Drupe Drupe

Color outside

Color inside

Origin"

Greenish Yellow Green Grey Green Green-yellow Yellow Yellow Brown Green to green-yellow Yellow Yellow

Yellow Yellow White Yellow or pink White Yellow Pink or yellow Yellow Ye1lowish

Ab Bb A A A A A Cb -

Yellow Yellow Yellow

A A B

Light brown Red Brown Yellow

Yellow White Yellow Yellowish

A A A

Green Green Brown Yellow Pink

White White Brown Yellow Yellow

A A C A A

"Extracted from Vavilov (1950). 'A = Central and South American Center; B = Indian Center; C = Asia. 'Adhering by a juicy pendiculum, which is the most attractive part of the fruit.

111. MORPHOLOGY AND ANATOMY OF FRUITS

A great number of different botanical families of the tropical fruit world have been investigated. As with phytoplankton or, indeed, almost any group of organisms, it has been found that, while the absolute numbers of a given species tend to increase poleward, the variety of species increases toward the equator. Thus, there are many more individual families and species of tropical fruits, very diverse in shape, size, and structure, classifiable as drupes, berries, aggregate fruits, etc. The digitoform contours of the banana (Fig. 1) and the rounded form of the guava (Fig. 2) are very different from the shape

164

N. CZYHRINCIW TABLE VI Fruits

Variety Badami

Raspuri

Tatapuri

Neelam

Fruit pulps

Stage of maturity

Pressure test in lbs

Brix at 20°C

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

25.85 12.66 6.30 3.90 15.75 7.37 2.80 1.15 20.06 14.25 2.41 30.00 18.44 10.08 3.47

8.30 13.30 18.31 19.32 7.29 15.31 16.31 18.31 12.80 14.31 17.31 12.80 14.31 16.31 18.31

WaterTotal insoluble solids solids (96) (9'0)

9.66 5.98 0.88 0.22 11.44 6.31 2.11 0.72

-

5.51 4.11 1.40 5.82 5.02 3.65 1.30

17.88 18.90 19.11 19.37 18.09 19.98 18.42 18.97 18.53 18.70 18.75 17.81 18.30 18.84 18.90

pH

2.68 2.85 3.68 4.04 2.64 2.94 3.22 3.82

-

3.07 3.11 4.24 3.06 3.10 3.46 4.74

Acidity (anhyd. citric acid) (%)

3.41 2.08 0.81 0.38 3.50 2.12 1.17 0.57 1.71 1.69 0.32 1.50 1.28 0.88 0.16

Xrishnamurthy et al. (1960).

of the pineapple, with its thorny crown. The forms of tropical fruits do not fit the geometry of classical pomology. Table V, A and B, gives an idea of the diversity of tropical fruits. The anatomy of fruits is interesting as an aid in identifying the origin of the pulp. Other points for study are the presence of stone cells; the micrograins of starch; the microcrystals present in some fruits, such as pineapple and pomegranate; and other elements of structure. For example, the micrograins of starch in the unripe mango are different from the micrograins of other plant products (Fig. 3). Lindorf (1966) studied the anatomy of different fruits, such as the sapodilla, pomegranate, and breadfruit (Figs. 4, 5, and 6). The microscopic structure of banana and pineapple tissues has been shown by Winton and Winton (1958). Weier and Stocking (1950) studied histological changes in fruits and vegetables during processing, particularly by heat. However, the first studies of the anatomy of tropical fruits, specifically those of India, probably were made by Griebel (1928).

TROPICAL FRUIT TECHNOLOGY

165

CHANCES IN PHYSICOCHEMICAL CHARACTERISTICS OF MANGOES DURING RIPENINGAFTER PICKINGO Fruit Pulps p-Carotene

Glucose

Pg (%)

( %)

Fructose ( %)

345 1413 4883 6607 1231 1498 3094 4952 513 566 1976 130 490 844 2381

0.22 0.70 1.91 2.07 0.25 0.84 2.13 2.06 0.74 1.85 0.55 1.79 1.91 2.46 3.07

1.06 1.79 2.95 4.04 0.75 1.45 3.11 3.44 2.52 4.69 3.23 2.71 2.66 2.68 7.75

Sucrose (70) 1.81 6.50 11.25 9.69 1.33 7.33 8.13 10.52 4.04 2.14 9.52 1.40 3.44 4.95 4.00

Total sugars (%)

Color

Flavor

3.09 8.99 16.11 15.80 2.33 9.62 13.37 16.02 7.30 8.68 13.30 5.90 8.41 10.09 14.82

Pale white Dull yellow Yellow Orange yellow Pale white Dull yellow Yellow Deep yellow

No flavor Mild Marked flavor Strong flavor No flavor Very mild Mild Marked No flavor Very mild Mild No flavor Very mild Mild Marked

-

Pale white Dull yellow Yellow Pale white Dull yellow Yellow Slight orange yellow

IV. PHYSICAL PROPERTIES O F FRUITS The chief physical properties of fruits to be considered are their weight, specific gravity, specific heat, porosity, juiciness, texture, and proportion of edible parts. Data are seldom given on the physical properties of tropical fruits. The average weight is not always given, and, though the average measurements may be offered, these are insufficient for technical purposes. The important properties of porosity, juiciness, and texture have not yet been studied. Only in recent years has there been published, in addition to data on general composition, information on the proportion of inedible parts of tropical fruits (INCAP-ICNND, 1961). The specific gravity of whole fruits may determine which type of washing machinery is to be used. It may be mentioned that passion fruit has a specific gravity of much less than one. The porosity of food products is generally related to their elasticity in determining their texture. Elimination of gas in the intercellular

166

N . CZYHRINCIW

FIG.1. Transverse and longitudinal sections of plantains, showing shape and size. (Czyhrinciw, 1952).

tissue spaces is important in the processing of raw vegetable tissues. Meyer (1960) states that this gas is mainly air, with a high content of COP,water vapor, and volatile substances. Oxygen must be reduced in the tissues to prolong the “life” of finished products, that is, to preserve their flavor and color and to reduce corrosion in cans. Many fruits are processed to the liquid state. This is commonly done by pressing or mechanical disintegration. It is necessary to know the juiciness coefficient of the raw material in order to select the best method. The texture of the uncut whole fruit and of the edible tissue is important. Skin hardness is decisive in resistance to phytopathological and entomological attack, and is important during transport. Finally,

TROPICAL FRUIT TECHNOLOGY

167

the texture of the skin and the edible tissue determines the best methods of cutting, peeling, etc. Table VII, based on experience gained during analysis and research (Czyhrinciw, 1955), gives data on the weight of the most important fruits. Data on the proportion of the inedible part are from INCAP-ICNND (1961). Tables VIII and IX give data on specific gravity, porosity, and juiciness (Mosqueda and Czyhrinciw, 1964). Tables X and XI (Czyhrinciw et al., 1967) give data on texture. Table VII shows the average weight of some tropical fruits that belong to different botanical families. It can be seen that the largest tropical fruits weigh a thousand times as much as the smallest, reaching weights expressed in kilograms in papayas and pineapples. Temperate-zone fruits and subtropical fruits -for example, grapes (2-8 g), oranges (120-150 g), and apples (120-350 g), show only about one-

Piriforme de Truli lo

FIG.2. Transverse and longitudinal sections of two varieties of guava (Rivas, 1964).

168

N. CZYHRINCIW

FIG. 3. Starch micrograins from unripe mango, 8 to 15 microns diameter. In polarized light (Chavez and Czyhrinciw, 1961).

tenth the variation in weight. The few berries of the subpolar regions show even less variation. A high waste index is typical of tropical fruits. Table VII shows that it reaches 58% in avocado and 67% in passion fruit. The waste index for temperate-zone fruits, belonging to the same families, such as the Rosaceae, is generally 2-15%, reaching 50% only in the citrus fruits. Porosity of edible tissues varies from 5.2 %, in the mango, to 24.9%, in the Pomerac (Table VIII). It is not easy to eliminate such porosity without prolonged precooking, which greatly softens edible tissues and complicates the manufacture of tropical fruit cocktails. For com-

TROPICAL FRUIT TECHNOLOGY

169

cel p

FIG.4. Ripe sapodilla (Achras sapote, L.) (c.) several layers of cork in skin; (ce1.p.) petrified cells; (cond.lat.)lactiferous ducts (Lindorf, 1966).

FIG. 5. Pomegranate (Punicu granntum, L.) Petrified cells (ce1.p.) surrounded by smaller parenchyma cells (Lindorf, 1966).

N. CZYHRINCIW

170

FIG. 6. Breadfruit (Artocarpus communis, Forst.) Parenchyma with intercellular spaces (i) (Lindorf, 1966).

TABLE VII AVERAGEWEIGHT AND PROPORTION OF INEDIBLE PART OF Weight in grams

SOME

TROPICAL FRUITS % Proportion of

Fruit

Min.

MU."

inedible pa&

Avocado Banana Cashew Guava Mammee apple Mango Papaya Passion fruit Pineapple Plantain Sapodilla Sapote Soursop West Indian cherry

500 60 32 30 500 50 2000 60 1000 220 130 460 1550 1

1000 150 80 100

46-58 34-40 18 4 54 47 25 67 41 31

800

450 4000 70 4000 450 170 900 2000 5

"Czyhrinciw, unpublished data. "Table of composition of aliments for South America (INCAP-ICNND).

44-53" 41 25

171

TROPICAL FRUIT TECHNOLOGY

TABLE VIII SPECIFICGRAVITY A ND PoRosrw OF THE EDIBLEPARTOF SOMETROPICAL FRUITS”

Fruit Avocado Banana “Manzana,” ripe Banana “Pineo,” ripe Bullock’s heart Guava Mango “Hilacha,” ripe Mango “La India” Papaya. ripe Papaya, unripe Passion fruit, ripe Passion fruit, unripe Pineapple, ripe Pineapple “Los Andes” Plantain, ripe Plantain, unripe Sapote Soursot,

Specific Porosity Reduction in porosity gravity in precooked Specific Porosity precooked fruits after precooking gravity (9’0) fruits ( %) ( %)

0.959 1.014 0.994 1.037 1.051 1.043 1.045 0.987 0.964 0.637 0.771 1.012 0.974 1.042 1.014 1.083 1.038

5.4 15.7 14.5 21.2 17.0 13.2 5.2 12.0 10.6 40.9 29.8 13.3 10.5 15.6 15.9 14.4 19.8

3.2 6.7 7.8 9.5 7.3 10.2 2.6 2.8 7.5

1.003 1.060 1.083 1.050 1.067 1.052 1.018 1.025 1.037 -

-

1.109 1.077 1.085 1.070

7.3 7.8 6.9 11.6

40.7 57.3 46.2 55.2 57.0 22.8 78.3 73.6 43.6 53.2 51.0 52.1 41.4

‘Mosqueda and Czyhrinciw (1964).

JUICINESSOF

THE

TABLE IX EDIBLEPART OF SOME ‘rROPICAL FRUITS“ ~~~

Fruit Banana “Pineo,” ripe Guava Mango Papaya Pineapple, ripe Pineapple, unripe Plantain, ripe Sapote Soursop Sugar apple

Juice at 9.6 atm for 5 min (%)

Juice at 14.2 atm for 5 min (Yo)

Total juice ( 70)

4.0

12.3

16.3

12.2 13.5 57.3 59.4

16.2 17.1 12.9 12.2

28.4 30.6 70.2 71.6

55.6

15.7

71.3

0.0

3.6

3.6

29.4 38.5 42.3

10.7 17.0 18.2

40.1 55.5 60.5

“Mosqueda and Czyhrinciw (1964).

N. CZYIHRINCIW

172

TEXTURE OF

Scale ~~

Relatively hard texture’

SOME

Scale

TABLE X TROPICAL FRUITSWITH Relatively semihard texture’

SKIN“

Scale

Relatively soft texture’

~

0-5

Pineapple Passion fruit

5-10

Mango “Hilacha,” unripe Soursop

11-25

Mango “Hilacha,” 101-300 Cashew (pendic-) ripe ulum) Avocado Guava Plantain, unripe Banana “Manzana” ripe Papaya, ripe Papaya, unripe 26-100 Plantain, ripe 301+ Sapodilla Banana “Pineo,” ripe

“Czyhrinciw et al. (1967). ”Determined by “Precision Penetrometer.”

TEXTURE OF

SOME

Relatively semihard texture”

A. 11-25 Papaya, unripe

B. 26-100 Mango, unripe Plantain, unripe

TABLE XI TROPICAL FRUITSWITHOUT

SKIN“

Relatively soft texture’

A. 101-300 Pineapple Mango “Hilacha,” ripe Plantain, ripe B. 301+ Banana “Pineo,” ripe Banana “Manzana,” ripe Papaya, ripe

“Czyhrinciw et al. (1967). ‘Determined by “Precision Penetrometer.”

parison, porosity is 25% in apples and 2% in potatoes (Smock and Neubert, 1950). Fruit beverages are now prepared in three ways: first, as transparent juices (apples and grapes); second, as semitransparentjuices, containing some homogenized tissue (oranges, grapefruit, and pineapple); and third, as nectars, in which the fruit is completely homogenized (pears, apricots, guava, mango, etc.). Transparent juices are usually

173

TROPICAL FRUIT TECHNOLOGY

extracted by pressing, requiring a pressure of 5 to 25 atm to separate the liquid phase. Smock and Neubert (1950) gave the juiciness of apples as 68.2-77.3%, and Reitersmann (1952)gave it as 55-75 96. At present, few tropical fruits are pressed for juice; of those mentioned in this article, only pineapple, papaya, and cashew may be considered as juicy fruits, with about a 70% yield. The tenacious retention of liquid phase by the edible tissue of many tropical fruits is of great theoretical interest, for the same phenomenon is observed in pears, apricots, and strawberries of the temperate zone. Table IX, on fruit juiciness, suggests that attempts to produce nectars from other fruits would be justified, as would studies directed toward the production of other transparent bottled juices. From Tables X and XI, which give data on the texture of the whole fruit (with skin) and edible tissue, respectively, it is possible to classify fruits into three groups - relatively hard, semihard, and soft - from readings of a “Precision Penetrometer” expressed as 0.1 mm penetration of the needle under a load of 50 g. The relatively hard group would be exemplified by apples and potatoes; the soft fruit by a ripe tomato (Table X). Physical properties vary not only with the maturity of the fruits but also with the type. Cegarra (1964) presented comparative data on 11 varieties of mango (Table XII). The specific heat of fruits must be known for calculations on refrigeration and freezing, in addition to the heat produced by respiration. The specific heat can be calculated from theoretical formulas. ManTABLE XI1 PHYSICAL PROPERTIES OF 1 1 VARIETIES OF MANGO” Average weight Variety

(td

Skin (%)

Martinica Glenn Irwin Selection 80 Selection 85 Kent Zill Sensation Smith Lippens Blackman

475 425 367 328 269 500 362 243 711 260 259

8.39 6.48 8.96 12 9.48 8.20 9 9 10.40 10.33 14.41

“Cegarra (1964).

(%)

Edible part (%)

Specific gravity edible

Porosity (%)

Total juiciness ( %)

8.62 13.39 9.18 19 9.23 12 12.56 15.13 10.32 12.33 16.60

83 80.13 81.86 69 81.29 79.80 78.44 75.87 79.28 77.34 69

1.026 1.053 1.053 1.031 1.053 1.042 1.064 1.050 1.053 1.042 1.064

13.85 7.80 7.68 12.37 10.53 8.85 6.38 8.16 6.32 8.85 5.85

20.4 46 48 30 30 30 33.4 34 26 43 32

Seed

174

N. CZYHRINCIW

zano (1964) determined the specific heat of the mango to be 0.889 cal; of the guava, 0.808 cal; and of the banana, 0.840 cal. These values are in very close agreement with the calculated values.

V. SOME CHEMICAL PROPERTIES OF FRUITS

A. COLOR, AROMA,AND FLAVOR Fruits show their type and maturity by their color, in the skin as well as in the edible tissues. Most skins of tropical fruits are green or yellow, rarely reddish (Table V). The edible tissue is usually yellow, sometimes white, with pinkish shades in some varieties. The green is from chlorophyll, which often disappears with maturity, leaving other pigments, such as xanthophyll and the carotenoids (Simmonds, 1959), to give the yellow color. Some fruits, such as the cherimoya and the soursop, retain the green color. Chlorophyll, when heated, darkens through the formation of pheophytin; this is another reason for the peeling of some fruits before further processing. Tropical fruits, which are deficient in anthocyanins, are not red or violet, except for a few like the West Indian cherry. Anthocyanins are red pigments, soluble in water, with the intensity of their color depending on pH. They appear mostly in the northern fruits, disappearing in fruits found toward the equator. Santini and Huyke (1956a) found malvin (an anthocyanin) in the West Indian cherry (Semeruco). Pruthi et al. (1961) found 1.4 mg pelargonin (belonging to the anthocyanin group) per 100 g of tissue in the skin of certain varieties of passion fruit. The color of the pomegranate is also due to anthocyanin. The edible yellow tissues of tropical fruits are very rich in a large group of carotenoid pigments. Morgan (1966) found, in the carotenoids of fresh pineapples, 50 Yo violaxanthins, 13 % luteoxanthins, 9%@-carotene,and 8%neoxanthins, in addition to smaller amounts of (-carotene, hydroxy-a-carotene, cryptoxanthins, lutein, auroxanthins, and neochromes. Pruthi (1963) reported eight forms of carotene in passion fruit, with @-carotenedominant. Carotenoids are fat-soluble and heat-resistant, but are easily destroyed by oxygen in the presence of sunlight in manufactured products. Some carotenoids are precursors of vitamin A in the human body. INCAP-ICNND (1961) gives data on “activity of vitamin A in micrograms” in the majority of the important fruits, calculated from the content of carotenoids by the use of a conversion factor (Table XIII).

TROPICAL FRUIT TECHNOLOGY

175

TABLE XI11 ACTIVITYOF VITAMINA" Fruit

Vitamin A activity in Pd100 g

Avocado Banana, ripe Banana, unripe Cashew Coconut Guava Mammee apple Mango Papaya Pineapple Plantain, ripe Plantain, unripe Soursop Tamarind West Indian cherry Yellow mombin

15-60 30-65 290 120 0 80 30 630 110 15 165- 175 380 5 20 10 70

"Extracted from INCAP-ICNND (1961).

The color of fruit tissue changes during storage in the fresh state and during processing. These changes, which may or may not be desirable, are the work of enzyme action or other processes. These processes include autoxidation of phenols during prolonged cooking, partial caramelization, the Maillard reaction, and reactions with iron utensils or with mineral impurities in the processing water. Special studies have been made on nonenzymatic browning in fruit products (Stadtman, 1948; Reynolds, 1963). Thus, it can be seen that color indicates not only the freshness and quality of the raw material but also the quality of the finished product. The flavor and aroma of fruits are due to many substances, volatile and nonvolatile, found in differing proportions in the skin and edible tissues of fruits. Not all these substances have been identified, either because they are present in very small amounts or because they cannot easily be isolated. The intensity of the flavor may depend partly on the variety of the fruit, on its growing conditions, and on its maturity. Some of these substances are specific for each species and even each variety; others are common to all fruits. The many specific substances which determine the flavor of some fruits are given in the following references, omitting common substances such as sugars, which give sweetness; organic acids, which give acidity; and tannins, which give astringency.

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N. CZYHRINCIW

1. Bananas Hultin and Proctor (1961) have found the following substances in aqueous distillate of the banana: acetic acid, methyl alcohol, ethyl alcohol, methyl acetate, ethyl acetate, 2’-hexenal, 2-pentanone, and possibly isoamyl alcohol, isoamyl acetate, and 2-octanone. McCarthy et al. (1963), from chromatographic analyses of bananas, found that the “bananalike” flavor is due to the ainyl esters of acetic, propionic, and butyric acids. The distinctive “fruity” and “estery” tones are attributed to butyl acetate, butyl butyrate, hexyl acetate, and amyl butyrate. Wick et ul. (1966) also studied the flavor and biochemistry of volatile banana components.

2. Pineapple Kirchner (1950) states that the summer pineapple has 190 mg of volatile oil per kg of tissue, and the winter pineapple has 15.6 mg/kg. The components of the volatile oil are given in Table XIV. Rodin et al. (1966) studied the sulfur-containing components of pineapple. The total volatile sulfur content of pineapple flavor concentrate was found to be 0.4-1.0% (13-32 ppb of whole pineapple). Silverstein et al. (1965), using mass spectrography and infrared and nuclear magnetic resonance spectroscopy, isolated and identified chavicol (p-allylphenol) and y-caprolactone from fresh pineapple.

3. Passion fruit Four components, n-hexyl caproate, n-hexyl butyrate, ethyl caproate, and ethyl butyrate, make up 95% of the oil of passion fruit. Of these four, n-hexyl caproate accounts for 70% of the volatile passionfruit essence (Hiu and Scheuer, 1961). The oil is present as 36 ppm of the juice. 4 . Papaya

Katague and Kirch (1965) analyzed the volatile components of papaya chromatographically. It is interesting that the content of volatile oil in whole oranges and lemons is 0.3 to 0.67%,according to Braverman (1949).This indicates that efforts to extract essential oils from tropical fruits are unlikely to be practical, for these oils are small in quantity.

TROPICAL FRUIT TECHNOLOGY

COMPONENTSOF

THE

TABLE XIV VOLATILEOIL OF

THE

177

PINEAPPLE"

Summer fruit

Winter fruit

Ethyl acetate Ethyl alcohol Acetaldehyde Ethyl isovalerate Methyl n-propyl ketone E thy1 acrylate Ethyl n-caproate

Ethyl acetate Acetaldehyde Methyl isocaproate Methyl isovalerate Methyl n-valerate Methyl caprylate Methyl ester of 5 carbon hydroxyacid Sulfur-containing compounds

"Kirchner (1950).

The basic sweet-acid and semiastringent flavor of fruit is due to sugars, organic acids, and tannic substances. Rapid determination of maturity for industrial purposes is done by refractometric analysis of the approximate content of sugars. Table XV gives data on the content of sugars, acids, and tannic substances (Czyhrinciw et d.,1967). Joslyn and Goldstein (1964) give data on fruit astringency. Fruits contain invert sugars (glucose and fructose) and saccharose. Some banana varieties have 20-50% of their sugar content as invert sugars; mango has 20-30% of its sugar as invert sugar. Sapodilla contains 3.7% glucose, 3.4 % fructose, and 7.02% saccharose, practically 50% invert sugar (Popenoe, 1939). Merory (1960) found 3.9%invert sugar and 7.5%saccharose in pineapple. Many organic acids, such as citric, tartaric, malic, and oxalic, are found in fruits, either free or as salts (e.g., oxalate crystals) and esters. Jansen et al. (1965) found that approximately 6 % of the citric acid in the avocado exists as an asym-monoethyl ester. Meyer (1960) mentioned citric and malic acid in plantains and pineapples; the avocado has traces of tartaric acid. Wolf (1958)mentioned citric and malic acids in bananas. In guava, citric acid prevails, and tartaric and levomalic acids are also present (Santini and Huyke, 1956b). Tartaric acid is dominant in the tamarind. Traces of oxalic, acetic, butyric, succinic, etc., acids are also found in fruits. Bananas, for example, contain 6.4 mg oxalic acid per 100 g pulp; Hawaiian canned pineapple contains 6.3mg/100 g (Anon., 1949). Gortner (1963) reported that the malic acid content of pineapples is quite sensitive to changes in sunlight or conditions favoring water evaporation, whereas citric acid does not change in response to cultivation factors.

N. CZYHRINCIW

178

TABLE XV CHEMICAL

DATAON

CERTAIN

FRUITS’

Fruit

Total sugarsb ( %)

Acidity, cibic ( %)

Tannins (CLgl100 g )

Avocado Banana “Pineo” Guava Mango “Hilacha” Papaya Passion fruit Pineapple Red cashew Sapodilla Sapote Soursop Tamarind Yellow cashew

1.84 20.00 6.15 11.38 6.36 12.00 10-13 9.12 10.96 11.69 11.52 34.50 10.89

0.18 0.30 1.28 0.50 0.07 4.64 0.50 0.64 0.07 0.12 1.04 11.53c 0.33

25 17 190 23 37 45 25 220 57 68 76 158 115

“Cryhrinciw et al. (1967). bAsdextrose after inversion. “As tartaric acid.

The pH of unripe banana varies from 5.02 to 5.6; in ripe banana, from 4.2 to 4.75 (von Loesecke, 1949). Garces (1967) stated that pH varies from 5.0 to 5.35 in the soursop, from 5.5 to 5.8 in the sapodilla, from 5.3 to 5.65 in the giant granadilla, and from 3.75 to 3.95 in the pineapple. Cegarra (1964) stated that pH varies from 4.5 to 5.35 in the mango, and Rivas (1964) reported a pH of 3.6 to 4.1 in the guava. The irregular distribution of tannic substances in cross sections of unripe and ripe plantain tissue has been demonstrated (Fig. 7). It is possible to characterize the flavors of fruits, to a certain degree, by a formula which involves determining the ratio acids-sugarstannins. Riaz-ur-Rahman et al. (1956) stated that the ratio of sugar content to acid content varies from 7.7 to 17.9 in 11varieties of guava. Smock and Neubert (1950) reported the basic flavor ratio (acids:sugars: tannins) in six varieties of apples to be 1:25:0.25. Mosqueda (1967) proposed classifying tropical fruits according to their basic flavors, which vary greatly. Such a classification would be of importance to fruit technology for it would permit classifying fruits, according to strength of flavor, for various uses. The first two groups, of milder flavor, would be used as table fruits. The third group would be processed, and the fourth group would be processed with some treatment to modify flavor, such as dilution with water or mixture with other fruits.

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179

It is proposed that each fruit be classified by an arbitrary number consisting of three digits, the first of which represents acidity, calculated as percent citric acid; the second, astringency, calculated as percent tannic acid; and the third, sweetness, calculated as percent dextrose. The possible range of each digit is from 1 to 9, with 9 being the maximum tolerable strength of flavor that does not fatigue the palate. By this scale, it is possible to classify fruits into four groups: accentuate (500-800); and simple (less than 300);moderate (300-500); penetrate (more than 800). According to past experience with tropical fruits, acidity is the most important criterion for the classification of flavor. Therefore, the names of the categories have been based on this quality. Table XVI shows a group of tropical fruits classified on this scale (Mosqueda, 1967).Included for comparison are two varieties of apples and one of pear. The acidity of the pear has been recalculated as percent of citric acid. It should be evident that this scheme of classification would be useful for characterizing different varieties within a species.

B.

VITAMINS AND

MINERAL SALTS

Some mention has already been made of the importance of fruits as sources of vitamins (see p. 159)and minerals. Table XVII gives further data.

FIG.7. Sections of unripe and ripe plantains. Reaction of ferric chloride solution showing distribution of phenolic substances (tannins) (Czyhrinciw, 1952).

180

N. CZYHRINCIW TABLE XVI CLASSIFICATION OF SOME TROPICAL FRUITSBY FLAVOR^ Flavor indexb

Fruit

Simple jlauor, 0-300 211 122 132 132 143

Avocado Papaya Sapodilla Sapote Pear

Moderate jlauor, 300-500 314 472 312 352 482 343

Banana Guava Mango “Hilacha” Yellow cashew Red cashew Apple

Accentuate ~ ~ U U O 500-800 T, Soursop Pineapple Apple Grape

732 512 582 654 Penetrate jlauor, ouer 800

Passion fruit Tamarind Cubarro

>922 >976 >923

“Mosqueda (1967). bNumbers of citric acid, tannins, and total sugar content.

Jaff6 et al. (1950) determined that Pomerac contains an average of 10 mg of vitamin C per 100 g, and sapodilla 4.5 mgllOO g. Ascorbic acid in the West Indian cherry varies from 1375 to 2259 mg/lOO g (Arostegui and Asenjo, 1954). Berries of the West Indian cherry from the state of Lara, Venezuela, average 600-800 mg/100 g (unpublished). Bukin (1963) stated that the dog-rose, with 700-4500 mg/lOO g, is the species richest in vitamin C. Bradfield and Roca Amalia (1964) found almost 3% vitamin C in the Camu-camu (Myrcieria paraensis Berg) from Peru. Success in synthesizing this vitamin, however, will probably limit interest in its natural sources. Vitamin C is not distributed uniformly in fruit tissues. For instance, Braverman (1963) stated that the ratio of the vitamin in the epicarp to that in the flesh and center of the guava of Israel is 9:4. Vitamin C, in addition to being important in the diet, is an antioxidant, sometimes being added to improve the flavor or color stability

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181

of fruit products. Bauernfeind (1953) reported that high pH, oxygen, and heating can easily destroy this substance, which is stable in media of low pH. Proper processing conserves 80-90% of vitamin C, so that analysis of the content of this vitamin might serve as a check on proper organization of a production line. Vitamin B is relatively low in fruits, compared with other foods. However, the large consumption of, for instance, bananas, which may contain thiamin and riboflavin in amounts ranging upward from 0.04 mg/100 g, introduces significant amounts of these substances into the diet. The total content of mineral salts in fruits varies from 0.3%, in pineapple, cashew, and mammee apple, to 1%, in coconut. According to INCAP-ICNND (1961), phosphorus in coconut is 83 mg/100 g; in avocado, 42 mg/100 g; in unripe plantain, 40 mg/100 g; in ripe plantain, 34 mg/100 g; in unripe banana, 35 mg/lOO g; and in mombin, 31 mg/lOO g. Calcium is abundant in mombin, with 26 mgllOO g; guava, with 22 mg/1OO g; in soursop, with 24 mg/lOO g; and in papaya, with 20 mg/1OO g. Maximum iron content is found in mombin, with 2.2 mg/ 100 g; in coconut, with 1.8 mg/lOO g; in cashew, with 1.0 mg/100 g; and in plantain and bananas, with 0.8-0.9 mgllOO g. Most fruits are very low in sodium (for example, 0.4-0.5 mg/100 g

TABLE XVII

VITAMIN

c CONTENT OF SOME TROPICALFRUITS~

Fruit Avocado Banana Cashew Cherimoya Giant granadilla Guava Mammee apple Mango, ripe Mango, unripe Mombin Papaya Pineapple Plantain Soursop Tamarind West Indian cherry ‘INCAP-ICNND (1961).

17 15 219 30 20 218 16 53 128 28 46 61 20 26 6 1790

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N . CZYHRINCIW

in the banana), but Wenkham et al. (1961)found that papaya has 3.676 mg/100 g. Papaya can have a salty flavor. Its chloride content is related to the distance from the sea at which it is grown. Gardner et al. (1939)gave data on the mineral content of fruits. It is interesting that those workers found banana and pineapple almost equal to apple in contents of sulfur, calcium, magnesium, and silicon.

c. CARBOHYDRATES, PROTEINS,

FATS, AND CALORIC VALUE

Among carbohydrates, the sugars, abundant in most fruits, get the most attention. The relation of sugars to the flavors of fruits is shown in Table XV. The starch content of fruits is of interest to technology, and tropical fruits can be divided into two groups on the basis of starch content. The first group is the fruits that contain starch when unripe, with starch decreasing with maturity. Such fruits are the plantains and bananas, with 15-25% starch; the mango, with 4-6%; and the tamarind, passion fruit, and cashew, with traces. The shape of the micrograins of starch in the unripe mango has been investigated by Chavez and Czyhrinciw (1961).Starch is found in unripe fruits of many of the Rosaceae of the temperate zone. The second group contains fruits which have no starch even when unripe, such as the pineapple, papaya, and guava. Starch in breadfruit (Artocarpus communis Forst) forms upon ripening. Cellulose, hemicellulose, and lignin form the cell walls and fibers of fruit tissue. The most fibrous of the fruits are guava (5.3%fiber) and coconut (3.8%).Other fruits range between 0.5 and 1.0%fiber. In 11 varieties of Venezuelan mango, fiber content varies between 0.41 and 0.77%(Carrillo, 1940). Pectic substances, found in the cell walls and juice as soluble pectin and insoluble protopectin, form gels under certain conditions (pH, sugar concentration, and cooking). The total content of pectic substance varies a great deal. Elwell (1939) stated that the guava and banana are rich in pectin, whereas pineapple and pomegranate are poor in pectin. Kertesz (1951) found 0.31-0.39% pectin in natural banana pulp. Unripe plantain pulp contains 0.53-0.77%pectin; ripe pulp contains 1.0%. The mammee apple has 0.14%; the ripe rose guava, 0.46%; and the yellow guava, 0.53%. Garces (1967)determined the pectin content of bananas (pineo) as 0.5-0.72%; soursop, 0.360.38%; sapodilla, 0.31-0.39%; giant granadilla, 0.37-0.44%; and cayena” pineapple, 0.01-0.06%. Bhatia et al. (1959) studied the possibility of extracting pectin from papaya, and Pruthi et al. (1960) studied guava pectin. ‘I

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183

Citrus fruits are richer in pectin. Braverman (1949) reported that the raw skin of such fruits has 1.5-3.0% pectin; the white lemon has 2.5-5.5 70. There is relatively little protein in fruits. The richest in protein among tropical fruits are coconuts, with 3.5% protein; avocado, with 1.5%; and plantain and bananas with 1.2%. Since most fruits have only about 0.5% protein, little work has been done on their amino acids, although Pruthi and Srivas (1964) studied free amino acids in passion-fruit juice, and Gawler (1962) studies those of pineapple juice. The chief point of interest in amino acids is their reaction with sugars, producing browning, during the preparation of concentrates. Little fat is found in fruits, the majority having only 0.1-0.2%. Those with the most are coconut, with 27.7%; avocado, with 10%; and mombin, with 2%. Chandler (1958) reported up to 26% in certain varieties of avocado. The above review indicates the essentially low caloric value of fruits, which in the majority is 30-70 ca1/100 g. Those with the highest values are, again, coconut (296 ca1/100 g), plantains and bananas (91132 ca1/100 g), and avocado (102-152 ca1/100 g).

D. ENZYMES Enzymes are organic catalysts; that is, they promote chemical reactions without becoming incorporated into the substances. Since they are part protein, they are very sensitive to oxygen, humidity, temperature, and changes of pH; high temperatures inactivate them. Thus, the preservation of fresh fruits or their processing may greatly affect enzyme equilibria. Enzymes are distributed irregularly in vegetable tissue (Czyhrinciw, 1951). The blanching or precooking process in industry is directed toward inactivating enzymes. Since peroxidase is relatively heat-stable, this process can be controlled by determination of the remaining peroxidase activity. According to our determinations (unpublished), the activity of the peroxidase in l-cm3 pieces of papaya is destroyed in 1 min at a water temperature of 7W-75"C. After 1 min at 60°C, only 20-25% of the activity remains. At 100°C, 6 minutes are needed to inactivate peroxidase in %-inch cubes of ripe pineapple. Peroxidase activity, expressed as PZ (the peroxidase index of Willstiitter) is 0.003-0.009 in ripe papaya, 0.05 in ripe pineapple tissue, and 0.084 in yellow orange skin. Sastry et al. (1961)found the PZ value, on a dry weight basis, for sugar apple (Annona squamosa) pulp to be 0.06. Garces (1963) gave data on the inactivation of enzymes by high temperature (Table XVIII). She further reported (1967)the following

184

N. CZYHRINCIW

INACTIVATION OF

TABLE XVIII ENZYMES IN FRUITPULP DILUTED 1:1“ Fruit

Enzyme

Mango

Guava

Papaya

Ascorbic acid oxidation

75°C 3 min 80°C 3 min 75°C 5 min 80°C 5 min

95°C 3 min 65°C 7.5 min 98°C 5 min 85°C 7.5 min

85°C 3 min 70°C 3 min 90°C 7.5 min -

Peroxidase Pectinesterase Phenolase

-

Avocado

-

85°C 10 min 85°C 7.5 min 85°C 5 min

“Garces (1963).

data for the inactivation of pectinesterase in other fruits (pulp diluted 1:l): “pineo” bananas, 95°C for 3 min; soursop, 85°C for 3 min; and giant granadilla, 85°C for 3 min. She stated that, for retention of ascorbic acid, soursop must be precooked at 80°C for 5 min, giant granadilla at 90°C for 5 min, and pineapple at 75°C for 5 min. Enzyme content varies with fruit maturity. Papain, from the papaya, is a proteolytic enzyme found abundantly only in the unripe fruit, while very similar bromelin from the pineapple is found in the ripe fruit. Phycin has lately been recommended as being far superior to those enzymes for tenderizing meat and clarifying beverages (Whitaker, 1957). Krishnamurty et al. (1960) studied the preparation of papain from papaya. Further studies on this general subject are those of Chang et al. (1965), on papaya pectinesterase inactivation by saccharose, and of Hultin et al. (1966), on the purification and properties of banana pectin methyl esterases. Rieckenhoff and Rios (1956) found pectin methyl esterase activity in guava, but very much less than in tomatoes or citrus flavedo. Reymond and Phaff (1965) studied the purification and properties of avocado polygalacturonase, and Knapp (1965) worked on avocado polyphenolases. Invertase in fruits may be important in alcoholic fermentation, since it differs from yeast invertase by being partially inactive during fermentation. Also, it must be inactivated by heat before invert sugar and saccharose in fruits can be determined (von Loesecke, 1949). Joslyn and Ponting (1951)demonstrated the importance of enzymes to fruit technology, with particular reference to color changes during storage and processing. Heid and Joslyn (1963)studied the desirable

TROPICAL FRUIT TECHNOLOGY

185

and undesirable effects of enzymes in food processing. Acker (1962) studied enzyme action in foods of low moisture content, particularly dried fruits.

E. OTHER SUBSTANCES Food chemistry identifies other substances in fruits that affect the flavor of the raw material and finished product. Fidler (1962) stated that many ripe fruits contain specific flavor substances; mango, for instance, can contain turpentine. Heating avocado pulp, in preparing sauce, causes it to form a very bitter substance, a technological problem not yet solved for that fruit. Also, the heating of papaya pulp produces a somewhat distasteful “not fruity” aroma, although this does not occur with the short process of heating used in preparing fruit cocktails. Anderson (1949) and Tatterfield and Petler (1940) investigated the distribution of a toxic alkaloid of the aporphine group in the Annonaceae (cherimoya, soursop, etc.). With these fruits it is necessary to select raw fruits of very good quality, and to separate the skin and seeds with great care. Small (1943) reported that the content of this alkaloid in plants varies with selection and growing conditions. The cashew nut is very nutritious, containing much protein, fat, and starch. Popenoe (1939) mentioned, however, that the nuts are roasted over a charcoal fire to decompose the toxic cardol and anacardic acid of the shell, making the nut safe to eat. The sapodilla contains a gumlike substance which renders the passage of the cooked pulp through a finisher somewhat difficult. Research is needed on all of the above problems. VI. TECHNICAL PROBLEMS

A. PRESERVATION O F FRESHFRUITS AND FREEZING Low temperatures (above OOC) are used to keep fruit in its natural condition for many days. Fruits are frozen only when the fruit is to be processed immediately after defrosting, because defrosted fruits lose their natural texture and begin to deteriorate. Low temperatures slow chemical and biological processes in fruits, particularly respiration. Fruits damaged mechanically, phytopathologically, or entomologically are subject to further deterioration. Microorganisms in general,

186

N. CZYHRINCIW

and particularly mold, easily penetrate into the edible interior tissues from wounds in the skin, thus stimulating decay. Evaporation of water content is regulated by the proper relative humidity and air temperature in the storage space. With intense evaporation, fruits wither; their natural resistance to superficial microorganisms (fruit immunity) is lowered; the equilibrium of the enzymatic systems is lost or shifts to an undesirable level; and the tissue elasticity is affected, which may cause distaste to the consumer of fresh fruits, or complications in technical processes, such as mechanical peeling. Fruit respiration is regulated by the temperature, within certain limits, and by the composition of the atmosphere in the storage space (increased COz, ethylene gas, etc.). Lowered temperatures, within certain limits, not only slow respiration, but help maintain the natural equilibrium of the enzymatic systems. Respiration is a natural process in all stored fresh vegetable products (cereals, fruits, and vegetables). Some solid substance is lost, and heat, COz, and ethylene are produced. Under too low a temperature, the enzymatic reactions involved may go over to an abnormal state, and the fruits will begin to deteriorate. Desrosier (1959) states that the minimum preservation temperature of bananas is 13.3"C; of avocados, 7.2"C;of the mango, 10.0"C;of the papaya, 7.2"C;and of the pineapple, 7.2"C.Fidler (1962) considers that the minimum temperature is 3°C for English apples, and lO"-ll"C for bananas, papaya, mango, avocado, and pineapple. Biale (1960) presented data on the respiration of some fruits at various temperatures (Table XIX). He reported that most tropical fruits have a higher degree of respiration than fruits from other climates, and he also presented data on ripening processes and on changes in certain fruit substances. Desrosier (1959) gave data on the storage temperature and humidity, preservation time, and freezing point of certain fruits (Table XX). All the conditions noted above vary somewhat with different varieties. Such conditions have been studied primarily in the preservation of bananas (von Loesecke, 1949). The development of the microflora of the fruit surface is controlled by proper humidity and lowered temperature of the air in the storage space. S h c h e z Nieva and Rodriguez (1958) determined that wild guavas may have 454 million microorganisms per pound on their surfaces. Washing the fruits reduces the microorganisms to 18 million per pound. Kapur et al. (1962) mentioned that mangoes of the Alphonso and Raspuri varieties, with added COz, can be stored for 35-45 days at

187

TROPICAL FRUIT TECHNOLOGY TABLE XIX RESPIRATION OF CERTAIN FRUITSIN RELATION

TO

TEMPERATURE" Respiration

Fruit

Temperature ("C)

(ml CO,/kg/hr)

Banana (Gros Michel)

12.5 15.0 20.0 25.0 31.0 2.0 4.5 9.0 10.5 20.0 2.0 11.0 30.0 7.5 10.0 15.0 20.0 25.0 30.0

23 38 64 79 130 7.7 10.0 18.7 35.4 44.5 2.6 4.2 34.0 27.0 41.0 76.0 165.0 200.0 120.0

Mango (Alphonse)

Pineapple (Cayenne)

Avocado (Fuerte)

"Extracted from Biale (1960).

TABLE XX STORAGE CONDITIONS FOR CERTAIN TROPICAL FRUITS" Storage temperature ("C)

Relative humidity ( %)

Banana

11.7- 15.6

85-90

Mango Pineapple, green Pineapple, ripe Papaya Pomegranate

10.0 10.0-15.6 4.4-7.2 7.2 -0.6-0.0

85-90 85-90 85-90 85-90 85-90

Fruit

"Extracted from Desrosier (1959).

Storage time

1-3 wk

15-20 days 3-4 wk 2-4 wk 15-20 days 2-4 mo

Freezing point ("C)

-1.0 (green) -3.3 (ripe) -1.2 -1.65 -1.15 -1.05 -2.2

188

N. CZYHRINCIW

between 5.5” and 10°C. Pruthi and Girdhari (1955) stated that the optimum conditions for passion fruit storage are temperature, 5.5”7.2”C, relative humidity, 85-90 %, and storage time, 4-5 weeks. Mathur and Srivastava (1956), Bogin and Wallace (1965), Hansen (1966), Scott and Roberts (1966), Burg and Burg (1966), and Dolendo et ul. (1966) have all studied chemical changes, respiration, ripening conditions, etc., in the storage of tropical fruits. Monvoisin (1953) indicated that the freezing point of unripe banana pulp is -O.PC, and that of ripe pulp is -3.3”C. Mathur et ul. (1958) showed that peeled fruits, cut up or divided into sections, can be preserved for 10%-12 months with excellent results if they are kept in syrup with added ascorbic acid (0.05% by weight of syrup) at -17.8%. The fruits were kept at -28.9”C for the first 48 hours. Monvoisin (1953) also reported on freezing pineapple at -15” to -18”C, the fruit being cut into l-cm thick slices and put into 40-50% saccharose syrup. Many other fruits will probably be frozen someday. Tressler and Evers (1957) presented a study on freezing food products, in particular fruits, dealing with the processing of bananas, pineapple, papaya, guava, avocado, and mango.

B. SEPARATION OF

THE

INEDIBLE PART(SKIN)

Removing the inedible part of the raw material is basic to food technology. Since fruits differ in size and shape, peeling is difficult, and is often done by hand. In the pineapple industry, mechanization has been carried to a high point, and machines are available that can peel and core 100 pineapples a minute, each machine requiring three attendants. In less industrialized countries, however, cost and complexity preclude machinery, and pineapples are peeled by hand. The hands of workers require protection from the bromelin in the pineapple, and from papain when papaya is processed (Fig. 8). The mango is tasty and nutritious, but preservation of this fruit remains at the “home-canning” stage because of difficulty in removing the skin and the seed. The seed is large, and the skin contains substances that fatigue the palate (Sherman et al., 1958; Singh, 1960). However, the ripe mango can be peeled by freezing it at -10” to -15°C until ice crystals form in the tissue; the fruits can then be peeled by a machine such as is used to peel potatoes (Fig. 9). These machines consist of a cylinder with an inside abrasive surface and a rotating bottom plate. The disintegrated skin is flushed away with water, and the weight loss is only 15-20%. The short and rapid freezing does not

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189

FIG.8. Peeling unripe papayas. (Courtesy Tiquire Flores Co., Venezuela.)

cause any change in the properties of the fruit. Unripe mangoes need no freezing, for their texture is like that of the potato. This same method can also be used for other fruits. Good results have been obtained with sapodilla and medium-sized soursop. Loss of weight did not exceed 10-20%.

FIG. 9. Ripe peeled mangoes after freezing (Czyhrinciw, 1967, unpublished).

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N. CZYHRINCIW

Guavas used in fruit cocktails can be peeled chemically by placing them in baskets and immersing them for 2-3 minutes in boiling 1.52.0% NaOH solution. The skin is disintegrated and separates from the fruit. The fruits are then thoroughly sprayed with water at room temperature, and any residual skin is cut off by hand. Loss of weight is 10-12%,compared with 15-22%from hand peeling. Mechanization of the peeling of other tropical fruits is still in the experimental stage. Some machines have been developed for paring coconuts for preparing shredded and dried coconut meat, but most countries that export such products (Ceylon, the Phillipines, etc.) rely on hand paring with a special knife. Bananas are also peeled by hand- a woman at a conveyor belt can peel 300 lb of fruit an hour. The problem of skin separation should be studied in relation to the aromatic, gustatory, and pectic substances present in the skins of some fruits. Ripe plantains baked in their own skins have a richer flavor, and guava paste has a better and stronger flavor when the fruits are cooked whole. Woodroof presented a complete survey of receiving and preparing fruit for processing (Joslyn and Heid, 1963).

c. OBTAINING JUICES,PULPS, AND

CONCENTRATES

Most fruit is bottled or canned as juices or nectars; this is particularly true in the thirsty tropics. The production of concentrates, flours, sauces, marmalades, baby foods, wines, cordials, etc., requires the preparation of edible tissue in the form of pulp. Juices and nectars can be classified into three groups according to content of the structural part of edible tissue: transparent (clarified), semitransparent, and nontransparent (see page 172).Table XXI gives an analysis of some tropical fruit beverages made in various factories. The “Gustametric Chart” of Rietz (1961)permits comparison of the flavors of some tropical fruits and of the juices and nectars made from them. It can be seen that the majority of tropical fruit juices and nectars fall between 65 and 91, values that probably reflect consumer preference. Flavor intensity is less in transparent and semitransparent juices than in the raw material, and is greater in the nectars (papaya: fruit = 39, nectar = 70; guava: fruit = 83, nectar = 146).This difference may be due to the disintegrated tissue, which is all present, serving as a “transporting agent” of flavor substances. Clarification of the juice is difficult. In addition, most tropical fruits have a large content of carotenoids which are retained in the structural tissue during pressing. Colloidal particles, which cause turbidity

191

TROPICAL FRUIT TECHNOLOGY TABLE XXI CHARACTERISTICS OF NATURAL FRUITBEVERAGES

Product Soluble solid substances, % Acidity as citric acid, % Average relation of soluble solid to acidity as citric acid Viscosity“ Structural phase by 5 min centrifugation, YO

Semitransparent juices

Nectars (mango, papaya, guava, tamarind)

14.5-16.5

15.0-16.5

0.4-0.6

0.25-0.6

34.4 3-5

37.0 10.0-40.0

2.5- 15.0

15.0-40.0

“In centipoises.

in the juice, carry flavor substances and natural antioxidant substances. Therefore, fully clarified juices from these fruits will lose a substantial part of their flavor, their attractive coloring, and vitamin A content, as seen in guava and pineapple. Tamarind and cashew juice have strong flavors, and tamarind is highly acid, so that they must be diluted with water. This makes it possible to prepare acceptable clarified juices from these fruits. Such juices may be bottled, for they do not form a sediment displeasing to the consumer. Bottled drinks are preferred to canned ones, which may explain the popularity of artificial drinks over natural juices. Mosqueda (1966) studied the production of clarified tamarind juice, and Jain et al. (1956) and Marvaldi (1966) worked on cashew juice. Disintegration is held to mean the reduction of edible fruit tissue to a semiliquid slurry or pulp. Fruits of the temperate zone are usually cooked in a little water before going to the “pulpers,” where they are disintegrated and the skin, seeds, and a large part of the fiber removed. S h c h e z Nieva et al. (1959) reported on the use of “Cowless”type disintegrators for the mango. The seed can then be separated by centrifugation (see Fig. 10). Such disintegration may be applied to other tropical fruits and vegetables, in which case, the cooking process can be omitted, with resulting conservation of the unstable flavor and aroma components. However, such a process must take into account the enzymatic reactions in the fruits, particularly those of the oxydase-phenolase group.

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FIG. 10. Mango seeds separated b y centrifugation (Czyhrinciw, 1967, unpublished).

Finishing of the pulp, before conditioning and pasteurization, is a most important process. Shnchez Nieva and Rodriguez (1958) explain the importance of removing “granular” particles from guava pulp. Such lignified, hard inclusions also occur in other fruits - pears, for example. The low pH of the majority of fruits, plus citric acid added during conditioning, permits pasteurization of the finished product, instead of sterilization. Tressler and Joslyn (1961) surveyed the production of juice and pulp from tropical fruits, particularly pineapple and passion fruits giving much data on juice concentration. Northcutt and Gemmill (1957) reported on the canning of banana purke, and Lawler (1967) gave information on technical progress in banana-purke canning in Honduras. Coussin and Ludin (1963) studied the manufacture of pomegranate juice and concentrate. Table XXII shows the organization of the production line for preparing juices and pulps from fruits. Factories may easily diversify their production to include sauces, creams, marmalades, etc. Passion

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193

TABLE XXII THE PREPARATION OF JUICES

FLOWCHART OF PROCESSES IN Ripe ya

pa

Ripe mango

Unripe ma !O

1

Freezing

Guava

Soitrsop

1

Cashew apple

AND

NECTARS

Sapodilla

1

I

PreCOC ing

'

Freezing

Mechanical peeling'

Seed removal b y hand

Soaking

I

Freezing

I

Mechanical peeling"

1

Tamarind

I gration Disintegration

I I

er-

I

Centrifugation

Beating0

Cutting Be ting

I

Centrifugation

I

t

Finishing (pulping)

I

Conditioning C

I

Pasteurization "Mechanical peeling in the type of machines used for peeling potatoes. bToaid in separation of seeds from edible tissue. cDilution with treated water to reduce acidity or viscosity; addition of citric acid; addition of sugar.

N. CZYHRINCIW

194

fruit is particularly interesting in that the high acidity of the pulp makes it a natural concentrate, which must be diluted with water in preparing the finished product (juices, wines, marmalades, etc.). The manufacture of guava and mango concentrates has already begun in different countries. Pruthi et al. (1963) have had success in manufacturing cashew concentrate. D. DEHYDRATED PRODUCTS High temperatures cause losses of flavor and color, particularly in pineapples and passion fruit, and darkening, particularly in bananas. Attempts to produce guava flour with the “L. M. Mitchell Drum Dryer” failed because the product was dark in color, and the flavor of the reconstituted pulp was inferior (Luna, 1963). Flour can be manufactured perfectly well from unripe mangoes by the same equipment, such flour being suitable for cattle feeding. Table XXIII shows the composition of two samples of the finished product. The product retained its good flavor over a period of two years. Czyhrinciw (1952) presented a quick-cooking method for producing flour from unripe plantains; the flour contains 2.6% protein, 1.5% ash, 70.0% starch, 2.2% sugars, and 6.9% water. Flour made from unripe bananas without precooking, unfortunately, loses its agreeable flavor within a few months. Rahman (1963) presented an “economical” method for making flour from unripe plantains. However, the plantains should be peeled first, if the product is intended for human consumption. Cancel et al. (1962) suggested preparing bananas in thin slices and frying them at temperatures from 176.7”188.2”C.

COMPOSITION

OF

TABLE XXIII FLOURMADE FROM UNRIPE MANGOES“

Fat, 70 Proteins, % Ash, 740 Fiber, % Glucosides, % Starch, % Ascorbic acid (mg1100g) Carotene (mg/100 g) “Chavez and Czyhrinciw (1961).

Sample 1

Sample 2

2.17 3.92 3.52 3.39 87.00 40.35 271.00 1.15

2.63 3.45 3.13 6.10 84.69 38.69 528.00 2.86

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195

Nother et al. (1958) studied the production of pineapple-juice powder; Siddappa and Nanjundaswamy (1960) studied the hygroscopic properties of tropical fruit flours; and Bates (1964) studied foaming and stabilization in these flours. Other studies on dehydrated bananas, mango, guava, etc., have been made by Balasubrahmanyam et al. (1960), Jain et al. (1962), Girdhari et al. (1960), Padmini et al. (1963), and Pruthi and Girdhari (1959). Child (1964) studied desiccated coconut. Vacuum-drying and freeze-drying have an important role to play in preserving tropical fruits. Joslyn reported on different drying methods (Heid and Joslyn, 1963); Burke and Decareau (1964) discussed problems of freeze-drying. The stability of flours made from tropical fruits is still a serious problem. The method of packing should be tailored to the product. In addition, the utmost care should be taken to guard any dehydrated product against insects of the genus Ephestia. This insect has a lifecycle of 9-10 weeks, and in the tropics may reproduce with explosive rapidity throughout the whole year. The larvae cause great loss in stored products by fouling them. E. SWEETPRODUCTS Good marmalades, jams, and jellies can be made from most tropical fruits. Although government regulation of sweet fruit products dates back to 1660 in France, there is still no widely accepted definition of any of these products (Rauch, 1952). Marmalades and jams are made by cooking fruit pulp with sugar (65-67%) and are nontransparent, whereas jellies are made by cooking clarified fruit juice with sugar, and are transparent or semitransparent. All have a ‘liellylike” texture, which comes from the formation of gels when a solution of pectin is heated and pectin polymolecules are formed, bonded together by sugar and acids. Citric or apple pectin must be added when they are prepared from pulps diluted with water. Rieckehoff and Rodriguez (1960) studied the addition of pectin in making guava products. Pectin need not be added to guava pulp with a final sugar content of 70-75%, but is needed with a sugar content of 65-67%. Industrial experience has shown that, to manufacture 1 kg of marmalade from pineapple, guava, mammee apple, or passion fruit, it is necessary to use 0.7-0.75 kg pulp, 0.55 kg sugar, and 10-15 g pectin (citric or apple at 100-150 index). Elwell (1939) presented comparative data on the manufacture of

106

N. CZYHRINCIW

jellied products. For example, when the temperature reaches 105°C in apple pulp, the desired concentration of 65 940 sugar, and the potential for gel formation, have been reached. Comparable temperatures are 106.lo-107.2"C for grape jellies and 107.8"C for guava jelly. Guava needs more sugar than does apple, since it is poorer in pectin. Marmalades made only from quinces or apples are not likely to need extra pectin. Consumer preference in tropical countries dictates a lower acidity in marmalades and jellies made there (0.3-0.6%) than in the temperate zone. Thus, there is between 110 and 225 times as much sugar as there is acid, in contrast to the usual 75 to 90 times of the northern countries. The low content of natural pectin and acidity requires the addition of pectin from other sources. Gomez (1963), in studying the quality of marmalades, found it to be necessary to control water dilution of the pulp; less dilution gives a more natural taste. He recommends ash determinations and alcohol precipitations in quality control of the finished product. For a better product, Luh et al. (1964) recommend raw material of good quality and use of the vacuum cooker. Manufacture of candied fruits has recently increased greatIy (Fig. 11). Unripe papaya and pineapple are promising raw materials. Fruits need no prior treatment with sulfurous acid if they are to be used immediately, but diced fruit for export must be treated for temporary preservation (Fig. 12). Chakraborty et al. (1962) studied the cashew apple in candy making. Methods are also being developed for canning fruits in syrup. Guava, mango, and, especially, unripe papaya are excellent for canning (Figs. 13, 14).Fruit cocktails, especially a mixture of pineapple and papaya with a little guava, have been manufactured and have found ready consumer acceptance.

F. FERMENTATION PRODUCTS- ALCOHOLIC AND ACETIC In the more northerly regions, alcoholic fermentation is usually applied to the production of beverages from grape juice or from cereals. In the tropics, rum is produced from sugar-cane molasses. In recent years wine has been produced from imported grape-juice concentrates, which, without going into detail, is regarded as a pseudoindustrial process since it deals with partially processed, denaturalized, raw materials. There are great possibilities for making wines in the tropics from local fruits. Sinchez Nieva (1951) draws attention to pineapple and

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197

FIG. 11. Manufacturing candied fruits froin unripe papayas. (Courtesy Selecta Co., Venezuela.)

tamarind wines; Dyal Singh (1956) refers to fruit wines in India; Czyhrinciw (1966) studied the making of wines from passion fruit and mango. Amerine and Cruess (1960) reported on the technology of wines made from pineapples, pomegranates, etc. As for countries in the temperate zone, Canada, in 1958, used more than 700 tons of loganberries and blackberries for winemaking. Wines made from apples and other fruits are well known in Europe. The Ukraine formerly had two research centers concerned with winemaking: one at Odessa, for grape wines, and one at Uman, for wines made from apples, strawberries, raspberries, etc. Making wine from fruits other than grapes differs from the traditional process mainly in the initial stage, preparation of the must for fermentation. This usually requires peeling and mechanical disintegration of the fruit. Also, must from fruits other than grapes is more

198

N . CZYHRINCIW

FIG. 12. Preparation of pineapples to be preserved in sulfurous acid (for export). (Courtesy Infrut Co., Venezuela.)

often conditioned by the addition of water, sugar, and citric or tannic acids. Conditioning may involve diluting the pulp, making up for a lack of natural sugar, or controlling the acidity or astringency of the final product. Sugar is usually added in two or three batches during the first few days of fermentation. Further study is warranted on the practicability of manufacturing wines, semidry and sweet, in a range of colors and having the specific flavors of fruits such as passion fruit and cashew apple. Various alcoholic fermentations, made with substantial amounts of added cane sugar, appear to indicate that many tropical fruits contain invertase (saccharase) of sufficient potential. The invertase content of the fruit, even though this enzyme is also present in the yeast, must be studied

TROPICAL FRUIT TECHNOLOGY

199

in relation to the addition of sugar. Satisfactory fermentation curves have been demonstrated in mango and passion-fruit musts; in both cases, Fleischmann’s active dry yeast was used (Fig. 15). Muller et al. (1964)investigated, by gas chromatography, numerous volatile substances in dry passion-fruit wine. Griinwald (1967) also investigated the fermentation dynamics of passion-fruit wine, and concluded that the flavor and aroma substances appear in like proportion in the raw material and the finished product. The esters, in particular, were determined by gas chromatography. Tables XXIV and XXV give data on passion fruit and mango wines. Production of vinegar from tropical fruits would be of interest in some countries. Arispe (1967) has prepared vinegar from several, giving analyses and technical data. The production of vinegar from pineapple waste has special interest (see Table XXVI).

G . AGE OF ELABORATED PRODUCTS Organoleptic criteria for determining the age of food products have been known for a long time, as witnessed by the Italian who said: “Today’s bread; last year’s wine.” It is true that fresh bread and cake are tastier, and probably more nourishing than when they are several days old. Staleness results from evaporation of water, the regrouping

FIG. 13. Cooking of fruits in syrup. (Courtesy of Tiquire Flores Co., Venezuela.)

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N. CZYHRINCIW

FIG. 14. Fruits in syrup (sterilized).(Courtesy of Tiquire Flores Co.,Venezuela.)

of certain chemical components, autoxidation, etc. On the other hand, liquors and wines become “smoother and mellower” when their chemical reactions have some time to proceed. Unfortunately, the majority of products made from tropical fruits do not improve with age. Ascorbic acid (vitamin C), being an active reducing agent, can serve as an index of the stability of products elaborated from raw material containing this substance. Stadman (1948) and others have regarded this substance as a natural indicator of autoxidation; the age of certain products might be correlated with the loss of ascorbic acid. Czyhrinciw (1954) studied the dynamics of vitamin C and the organo-

TROPICAL FRUIT TECHNOLOGY

20 1

leptic properties of papaya nectars stored 8 months at 29.4“ and 32.2”C finding the average loss of vitamin C to be 17.2%. He stated that “the tentative storage life under tropical conditions is proposed as being 2 years for papaya nectar and 1.5 years for bananas in heavy syrup.” Braverman ( 1963) mentioned that “extensive changes” in products of fruits, especially in color and flavor, run parallel to losses of ascorbic acid during storage. Most manufactured products are at their best when “young,” but a few days (a few hours for bread) are needed to stabilize the structure and reach uniformity. More study is needed on determining the age of food products, especially in tropical countries, where storage is difficult. Hearne (1964) discussed problems of storage, and Parpia (1956) reviewed food storage in India. The development of metal containers with protective linings of tin and enamel, of glass containers, and, lately, of plastic and of aluminum containers, is of great interest to food technology, for corrosion during storage is a serious problem. Internal corrosion of cans,

15 ..

- 14” 0

5 13.-

Days

FIG. 15. Rate of alcohol production during fermentation of passion fruit and mango wines. Passion fruit win-; mango wine-------(Czyhrinciw, 1966),

202

N. CZYHRINCIW

SENSORY

ANALYSIS OF THE

Wine Color Flavor Body Other indices Type of wine

TABLE XXIV WINES FROM PASSION FRUIT AND MANGO“

Passion fruit Typical white wine (slightly yellow) Pleasant, vinous special fruity touch Good Without defects Sweet dessert wine

Mango Typical white wine Pleasant, vinous Good Without defects Fine white table wine

“Czyhrinciw (1966).

going on a long time, may produce enough hydrogen gas to swell the cans. In humid places, external corrosion makes the cans unsightly. Britton (1952) reviewed the main criteria of tinplate quality: the thickness and porosity of the tin layer. For the tropics, we believe that 1.5 tinplate is to be preferred to 1.25. Cruess (1948) gave information on the rate of increase with age of the tin content of canned vegetables. Jakobson and Mathiesen (1946) consider that “the mean storing capacity of the products is more than doubled when the storing temperature is decreased by 10°C. The temperature coefficient is about the same for lacquered cans as for unlacquered ones (aluminum).” Various investigators, including Felin (1952), also dealt with lowtemperature storage of preserved products. Acidity or the concentration of the product is not always strictly correlated with corrosion. Especially active substances are the anthocyanins -certain amino acids, tannic substances, oxalic acid, etc. Numerous observations of the corrosive activity of various products show that because of their different origins or because of different processing methods they are not really comparable, although designed to be so. Corrosion can often alter the color or flavor of a product. Some acquire a “metallic” taste, which indicates how high the content of tin and/or iron is. Czyhrinciw (1954), working on the corrosion of tinplate, ranked fruit products in increasing order of corrosive ability as follows: tropical fruit cocktail, passion-fruit juice, tamarind juice, pineapple juice, papaya juice. Concentrated papaya pulp was the most corrosive. The products were stored 12-20 months at 25”-35”C. Boyle et al. (1957) recommended enameled cans (“citrus” or “T” enamel) for guava nectar. Sherman et al. (1958) state that mango may

TROPICAL FRUIT TECHNOLOGY

203

TABLE XXV PHYSICAL AND CHEMICAL MEASUREMENTSOF PASSIONFRUITAND MANGOWINES“

Wine Density Number of wines Alcohol at 15”C, % Brix scaleb Total acid as citric, Yo PH Volatile acid, % Tannic substances, % Esters as ethyl acetate, % Aldehydes as acetaldehyde, % Furfural, % Higher alcohols, %

Passion Fruit “Maracuya” (Age 8 mo)

Mango “Hilacha” (Age 15 mo)

1.0200 3 12.53 14.5 0.81 3.26 0.018 0.023

1.0002 3 13.68 5.0 0.79 3.20 0.027 0.008

0.038

0.040

0.0035 0.0004 0.072

0.0001 0.027

0.0050

“Czyhrinciw (1966). *Determination after distilling off two-thirds of the volume and diluting to original volume with distilled water.

PROPERTIES Properties

Specific gravity (at 15°C) Total acidity (acetic acid), g/100 cc Nonvolatile acidity (citric acid), g/l00 cc Volatile acidity, g/l00 cc Alcohol, % Reducing sugars (invert sugars), 7% Soluble solids, % Esters, mg/100 cc Aldehydes, mg/100 cc) Tannins, mg/100 cc Oxidation number “Arispe (1967).

TABLE XXVI SOME TROPICALFRUITS”

OF VINEGAR FROM

Vinegar from pineapple waste

Vinegar from passion fruit

1.014

1.011

5.18

5.1

0.35 4.83 0.30

2.0 3.1 0.2

0.15 4.00 6.30 20.60 35.00 178.00

3.0 6.0 12.0 32.0 320.00

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N. CZYHRINCIW

be canned with syrup in tin cans. Rodriguez (1962)suggested packing guava paste in cardboard boxes. H. PRESERVATION BY IRRADIATION Irradiation, the newest method of food preservation, is still in an early stage of development. Romani (1966), Maxie and Abdel -Kader (1966), and Sommer and Fortlage (1966) discussed theoretical problems and experimental results of this method. Fergason et al. (1966) studied the effects of y-radiation on bananas. Dharkar et al. (1966a,b) studied irradiation of mangoes, developing a heat-irradiation process for sterilizing mango and sapodilla slices in cans. These methods look promising.

VII. CONCLUSION During the last ten years a great development in tropical fruit technology has been carried out in research centers in California, Hawaii, Florida, Puerto Rico, Brazil, Venezuela, India (at Mysore), the Philippines, and other places. Much, however, remains to be done. There are certain characteristics of tropical fruits which can be correlated with latitude. For example, the anthocyanins in fruits decrease toward the equator. In addition, the number of botanical families, the diversity of fruit form, sensitivity to temperature, and rapid growth to the first fruit-bearing, all increase in fruits of lower latitudes. Tropical fruits have increased carotenoid content and a higher percentage of inedible part. The potential resources of tropical fruits are enormous. The great diversity in flavor and chemical composition of tropical fruits gives many possibilities for industrial enterprise, although it must be remembered that the parallel diversity in form and size will greatly complicate the technology. The giant granadilla has a flavor resembling that of pears. The passion fruit and the pineapple can be developed further. The papaya has little astringency, while the cashew and the yellow mombin are very astringent. Acid fruits, such as the lemon and the vinagrillo (Averrhou blimbi, Oxalidaceae) should be given attention. The high sugar content of ripe plantain (up to 21.1 %) and of the mango (up to 25%) merit their study as food products. The exotic flavors of these fruits are almost totally unknown in the north, and food products based upon them should find a ready market. In addition, the vitamin

TROPICAL FRUIT TECHNOLOGY

205

C and carotenoid content would be a point of interest to the public. The shortage of anthocyanins, which give the pleasing red color to fruits, suggests that such fruits as Piritu or Cubarro (genus Bactris, Palmaceae) should be cultivated. Excellent beverages may be made from mango, passion fruit, giant granadilla, cashew, tamarind, and soursop. Cupana (Paullinia cupana, Sapindacea) can be cultivated for beverages; “Guarana” types have been successful in Brazil. Schery (1956) mentions the high content of caffein and tannins in this fruit. Miller et al. (1957) submitted formulae for combined nectars of banana-guava, papaya-banana, and papaya-pineapple. Fruit sauces and condiments may be made from mango, tamarind, etc., to compete with tomato catsup. Guava and papaya catsup formulae have been submitted by Miller et al. (1957). Pruthi and Bedekar (1963) and Bedekar and Pruthi (1963)studied the salting of raw mango slices for pickling and for chutney, an Indian product popular the world over. The potential is great for producing marmalades, jams, and jellies; fruits in syrup; and particularly fruit cocktails. The exotic flavors of mango and other fruits should be appreciated in the north. Pomerac (Fig. 16) is a promising fruit. Wines and cordials can be made from many tropical fruits (mango, cashew, passion fruit, etc.) Passion fruit makes a good cordial, and an excellent red cordial can be made from Piritu or Cubarro. Wiistenfeld

FIG. 16. Pomerac (Ohia).

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N. CZYHRINCIW

(1953) reported on pineapple and banana liqueurs, and Reitersmann (1952) on banana liqueurs. Vinegar production has already been mentioned. Pulp concentrates, sliced semiproducts, and fruit flours may become important. Ross (1960), Seale and Sherman (1960), Sherman et aZ. (1958),Boyle et a2. (1957), Cruess (1961), Miller et a2. (1957), Kanhiro and Sherman (1956), and La1 (1956) have given formulae for many different products. Baby foods are also becoming important. The science of food technology should coordinate studies on the following:

1. Wild farming, and semiwild botanical species of industrial value. At the beginning, efforts should be made to eliminate the differences of opinion between growers and industrialists. Adam (1962) stated that “the processor is primarily interested in the qualities of color, flavor, and texture, whereas the grower is more concerned with yield, disease resistance, and ease of cultivation and harvesting; yet, the processor can no more ignore the grower’s requirements than the grower can affect indifference to the ultimate use of his crops.” 2. Influence of chemical composition on the technical aspect. The study of pectic substances, astringents (tannins), enzymatic systems, complete identification of sugars and acids, and the identification of gustatory and aromatic substances should be studied, in addition to the ordinary analyses of carbohydrates, proteins, fats, vitamins, mineral salts, and moisture. 3. Physical properties. Weight, specific gravity, porosity, juiciness, texture, freezing point, specific heat, etc., deserve study. 4. Bacteriological aspects. 5. Preservation in the fresh state at reduced temperatures and freezing. Temperatures, humidities, and storage times must be established for the various products. 6. Irradiation. 7. Mechanization of processes. 8. Retention of natural color and flavor in manufacturing processes. 9. Corrosive properties of tropical products. 10. Age of finished products, packing, and storage; temperaturehumidity. 11. Radioactive contamination. 12. Extension of available assortments. 13. Utilization of industrial wastes. 14. Historical aspects.

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207

15. International trade and export. It should be noted that few tropical fruits or fruit products are known outside their own areas. The “Fruit World Map” (1963) of the international fruit and vegetable trade, for example, shows only the important banana and pineapple centers. Other fruits are not even mentioned. 16. Quality standards for the raw material and finished products. 17. Production planning and cost accounting. 18. Bibliography. 19. Technical publicity. 20. Technical education. Work on any of the factors listed above would contribute enormously to the development of tropical fruit-producing countries. This applies with equal force to tropical vegetables.

ACKNOWLEDGMENTS I express my thanks to the following professors of the Faculty of Sciences of the Central University of Venezuela for their help and advice: Dr. Leandro Aristeguieta, Professor of Botany; and Dr. W. G. Jaffk, Professor of Biochemistry. I am also indebted to Dr. C. 0. Chichester, Department of Food Science and Technology, University of California, Davis, California, for his suggestions in preparing this text.

REFERENCES Acker, L. 1962. Enzymic reactions in foods of low moisture content. Adoances in Food Research. 11, 292, 298. Adam, W. B. 1962. Recent Adoances in Food Sci. 2,83. Butterworths. London. Amerine, M. A., and Cruess, W. V. 1960. “The Technology of Wine Making.” 462,482. Avi, Westport, Connecticut. Anderson, H. T. 1949. “The Plants Alkaloids.” McGraw-Hill (Blakiston). Anon. 1949. The canned food reference manual. Am. Can Co., New York, 477. Anon. 1963. Fruit World Map. 0. Bauer, Basel, Switzerland. Arispe, I. 1967. Tecnologia del Vinagre d e frutas tropicales. Dept. Tecnologia de Alimentos. Fac. Ciencias. Univ. Central d e Venezuela. Unpublished. Aristeguieta, L. 1950. Frutas comestibles d e Venezuela. Tip. La Nacibn. Caracas. Arostegui, F., and Asenjo, C. F. 1954. Studies on the West Indian Cherry Proc. Florida State Hort. S O C . 67, 251. Balasubrahmanyam, N., and Murthy, H. B. N., Sreenathan, V. R., Annandaswamy, B. and Iyengar, N. V. R. 1960. Studies on packaging of dehydrated bananas. Indian Food Packer 14,19,21. Bates, R. P. 1964. Factors affecting foam production and stabilization of tropical fruit products. Food Technol. 18, 93, 96. Bauernfeind, J . C. 1953. Use of ascorbic acid in processing foods. Adoances in Food Research 4, 381, 359, 409.

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Bedekar, S. V., and Pruthi, J. S. 1963. Some physico-chemical and technological aspects of preparation and curing of mango pickle and other commercially important pickles in oil. Punjab Hort. J . 3, 259, 263. Bhathia, B. S., Krishnamurthy, G. V., and Lal, G. 1959. Preparation of pectin from raw papaya. Food Technol. 13, 10. Bhutiani, R. C. 1956. The need for horticultural development for the benefit of preservation industry in India. Fruit and Vegetable Preservation Industry in India Central. Food Technol. Research Inst. Mysore 25. Biale, J . B. 1960. The postharvest biochemistry of tropical and sub-tropical fruits. Advances in Food Research 10, 300, 293-347. Bogin, E., and Wallace, A. 1965. Investigations of processes in avocado fruit ripening. Calif. Agr. 19 (lo), 11, 12. Boyle, F., Smith, H., Sakata, S., and Sherman G. 1957. “Guava Processing.” Univ. of Hawaii. Bradfield, R. B., and Amalia, Roca. 1964. Camu-camu-A fruit high in ascorbic acid. J. Am. Dietet. Assoc. 44 (l),28-30. Braverman, J . B. S. 1949. “Citrus Products.” (Interscience), New York, 211. Braverman, J. B. S. 1963. “Introduction to the Biochemistry of Foods.” Elsevier, Co., Amsterdam-London-New York, 21 1. Britton, S. C. 1952. The corrosion resistance of tin and tin alloys. Research Inst. Greenford, 18-62. Bukin, V. N. 1963. “Biochemical Principles of the Food Industry.” Pergamon Press, Oxford, 62. Burg, Stanley, P., and Burg, Ellen A. 1966. Fruit storage at subatmospheric pressures. Science 153, 314-315. Burke, R. F., and Decareau, V. 1964. Recent advances in the freeze-drying of food products. Advances in Food Research. 13,73-80. Canada Dominion Bureau of Statistics. 1959. The wine industry. Ottawa. Cancel, L. E., Gonzalez, M. A., and Sanchez Nieva, F. 1962. Elaboracibn del Platanutra. Univ. Puerto Rico. Ext. Expt. Agr. Carrillo, T. G. 1940. El Mango. SOC.Venezuela d e Quim. Caracas. Vol. 1. Castaiieda, R. 1961. Frutas silvestres de Colombia, BogotA. Vol. 1. Cegarra, J. R. 1964. Estudios comparativo de algunos indices quimicos y fisicos importantes en variedades d e mangos injertados. Fac. d e Agronomia. Univ. Central de Venezuela. Unpublished. Chakraborty, R. N., Sastry, L. V. L., Pruthi, J. S., and Siddappa, G. S. 1962. Changes in polyphenols and ascorbic acid during the candying of cashew apples. Indian Food Packer 7. Chandler, W. H. 1958. Evergreen Orchards. Lea & Febiger, Philadelphia. Chang, L. W. S., Morita, L. L., and Yamamoto, H. Y. 1965. Papaya pectin esterase inhibition by sacrose. J . Food Sci. 30 (2), 218-222. Chavez, J. F., and Czyhrinciw, N. 1961. Posibilidades tecnolbgicas en la industrializacibn del Mango Verde. Arch. venezolanos nutric. Caracas. 11,205-220. Child, B. 1964. “Coconuts.” Longmans, Green, London. Collins, J. L. 1960. “The Pineapple.” Wiley (Interscience) New York. Coussin, B. R., and Ludin, Asher. 1963. Utilisation of the pomegrarate. Food Ma& 38 (7), 376-378. Cruess, W. V. 1948. “Commercial Fruit and Vegetable Products.” McGraw-Hill, New York, 285. Cruess, W. V. 1961. Canning Hawaiian pineapple. Food Manuf. 36 (10).

TROPICAL FRUIT TECHNOLOGY

209

Cushing, R. L. 1960. Introduction to panel pineapple. Pacific Rim. Food. Conf. Honolulu. Czyhrinciw, N . 1951. Nota sobre las variaciones quimicas e n productos vegetales. Arch. Venezolanos nutric. 2. Czyhrinciw, N. 1952. Harinas precocidas d e plitanos. Arch. Venezolanos nutric. 3. Czyhrinciw, N. 1954. Calidad y estabilidad de algunos productos enlatados. Arch. Venezolanos nutric. 5, 134-135. Czyhrinciw, N. 1955. Aspect0 de la tecnologia de las frutas tropicales. Soc. Cienc. Nut. Caracas. 15, 245. Czyhrinciw, N. 1966. The technology of passion fruit and mango wines. Am. J. Enol. and Viticult. 17, 27-30. Czyhrinciw, N., Mosqueda, M., and GarcBs, M. 1967. Analisis industrial en la fabricaci6n de alimentos. Lit. y Top. Vargas, Caracas. Dassler, E., Lardschneider, R., and Fischer, P. 1957. Warenkunde fur den Fruchthandel. Paul Pareg in Berlin und Hamburg. 1-8. Desrosier, N. W. 1959. “The Technology of Food Preservation.” Avi, Westport, Connecticut. pp. 63, 69, 188. Desrosier, N. W. 1961. Attack on starvation. Avi, Westport, Connecticut. pp. 45-48, 59-87, 261. Dharkar, S. D., Savagaon, K. A,, Srirangarajan, A. N., and Sreenivasen, A. 1966a. Irradiation of mangoes: I and 11.J. Food Sci. 31, 863-877. Dharkar, S. D., Savagaon, K. A., Kumta, U. S., and Sreenivason, A. 1966b. Development of a radiation process for some Indian fruit: mangoes and sapodilla. J. Food Sci. 31, 22-28. Dolendo, A. L., Luh, B. S., and Pratt, H. K. 1966. Relation of pectic acid and fatty acid changes to respiration rate during ripening of avocado fruits. J. Food Sci. 31 (3), 332-336. Dyal Singh, J. 1956. Technology of fruit wines, ciders, bears and liquors in India. Fruit and vegetable preservation industry in India. Central Food Technol. Research Inst. Mysore. 86-93. Elwell, W. E. 1939. Pectin. Belle Reeves, Washington. 17-19. Felin, A. R. 1952. Retenci6n de vitamina C e n el Jug0 de Naranja, pasteurizado, durante el almacenaje a varias temperaturas. Inst. Nacional de Invest. Agron. Valencia. Espaiia. Fergason, W. E., Yates, A. R., MacQueen, K. F., and Robb, J. A. 1966. The effects of gamma radiation on bananas. Food Technol. 20. 105-107. Fidler, J . C. 1962. Recent Advances in Food Science. Butterworths, London 1, 269, 270, 277. Fox, S. W. 1963. The outlook for synthetic foods. Food Technol. 22, 388, 392. Friedman, L. 1963. Evaluation of wholesomeness in newer methods of food preservation. Exploration in future food processing techniques. Mass. Inst. of Technol. GarcBs Medina, Mary. 1963. Estudio de algunas enzimas en frutas tropicales. Dept. Tecn. de Alimentos. Fac. Cienc. Univ. Central d e Venezuela. Unpublished. GarcBs Medina, Mary. 1967. Algunas enzimas importantes en frutas tropicales en relacion con su procesamiento. Dept. de Tecn. de Alimentos. Fac. Cienc. Univ. Central de Venezuela. Unpublished. Gardner, V. R., Bradford, F. C., and Hooker, H. D. 1939. The fundamentals of fruit production. McGraw-Hill, New York, 159-172. Gawler, J . H. 1962. Constituents of canned malayan pineapple juice. J . Sci. Food Agr. 13, 57-61.

210

N. CZYHRINCIW

Girdhari, L., Krishnamurthy, G. V., Jain, N. L., and Bhatia, B. S. 1960. Stability of different varieties of mangoes for the preparation of mango cereal flakes. Food Sci. (Mysore)9, 121-123. Griebel, C. 1928. Uber den mikroskopischen Bau einiger tropischer Fruchte und ihren Nachweis in marmeladenartigen Zubereitungen wie “Lukutate-Mark.” Untersuch. Lebensmittel. 55,89-111. Gomez, L. J. R. 1963. Estudio de las posibilidades de determinar la desnaturalizacion de mermeladas. Dept. Tecn. de Alimentos, Fac. Cienc. Univ. Central de Venzuela. Unpublished. Gortner, W. A. 1963. A short-term effect of weather on malic acid in pineapple fruit. J . Food Sci. 28 (2), 191. Grunwald, Evelyn. 1967. Contribucibn a1 estudio del proceso de fermentacibn alcoholica del jug0 de parchita. Fac. Cienc. Univ. Central de Venezuela. Unpublished. Hansen, E. 1966. Postharvest physiology of fruit. Ann. Reo. Plant Physiol. 17,459-480. Hearne, J. F. 1964. Long-term storage of food. Food Technol. 18 (3), 60, 65. Heid, J. L., and Joslyn, M. A. 1963. “Food processing operations.” Vol. 2, AVI, Westport, Connecticut. Hiu, D. N., and Scheuer, P. J. 1961. The volatile constituents of passion fruit juice. J . Food Sci. 26,557-563. Hughes, 0. 1962. Introductory foods. Macmillan, New York, 23. Hultin, H. O., and Proctor, B. E. 1961. Change in some volatile constituents of the banana during ripening, storage, and processing. Food Technol. 15, 440-444. H u h , H. O., Bonnie, S., and Bulger, J. 1966. Pectin methyl esterase of banana. Purification and properties. J . Food Sci. 31,320-328. INCAP-ICNND. 1961. Tabla de composicibn de alimentos. Inst. de Nutrici6n de Centro America y Panaml, Guatemala. J&e, W. G., Budowski, P., and Gorra, C . 1950. Estudio sobre el contenido de acido ascorbico en las principales frutas de Venezuela. Arch. Venesol. de Nutricibn. Caracas. 1,83-107. J&e, W. G., Chavez, J, F., and Belkis de Koffman. 1963. Sobre el valor nutritivo de plltanos y cambures. Arch. Venezol. de Nutricibn, Caracas 13,9-23. Jain, N. L., Das, D. P., and Lal, G. 1956. Utilization of cashew apples. Central Food Technol. Inst., Mysore, 75-80. Jain, N. L., Nair, K. G., Siddappa, G. S., and Girdhari, L. 1962. Studies to improve the keeping quality of fried salted banana chips. Food Sci. (Mysore) 11, 335-338. Jakobson, F., and Mathiesen, E. 1946. Corrosion of containers for canned foods. Hos. Jakob Dybwad, Calc. 89. Jansen, E. F., Palmer, K. J., and Lundin, R. E. 1965. The isolation of Asym-monoethylcitrate from avocado fruit. J . Food Sci. 30,1021-1025. Joslyn, M. and Goldstein, J. 1964. Astringency of fruits and fruit products in relation to phenolic content. Adu. in Food Research 13,179-217. Joslyn, M. A., and Heid, J. L. 1963. “Food Processing Operations.” Vol. 2, AVI, Westport, Connecticut. Joslyn, M. A., and Ponting, J. D. 1951. Enzyme-catalyzed oxidative browning of fruit products. Advances in Food Research. 3,l-37. Kanchiro, Y., and Sherman, G. D. 1956. Polvo pectic0 con sabor a guayaba es rico en icido ascbrbico. Food Inds. 18. Kapur, N. S., Sarveshwarz, K., Rao., and Srivastawa, H. C. 1962. Refrigerated gas storage of mangoes. Food Sci. (Mysore)11,228-231.

TROPICAL FRUIT TECHNOLOGY

211

Katague, D. B., and Kirch, E. R. 1965. Cromatographic analysis of the volatile components of papaya fruit.J. Pharmacol. Sci. 54 (6),891-894. Kennard, W. C., and Winter, H. F. 1963. Frutas y nueces para el trbpico. A.I.D. Mexico. Kertesz, Z. L. 1951. “The Pectic Substances. Wiley (Interscience), New York. Kirchner, J . C. 1950. The chemistry of fruit and vegetable flavors. Adoances in Food Research 2,265-269. Knapp, 1965. Some characteristics of eggplant and avocado polyphenolases. Food Sci. 30,430-437. Krishnamurthy, G. V., Jain, H. L., and Bhatia, B. S. 1960. Changes in the physicochemical composition of mangoes during ripening after picking. Food Sci. (Mysore) 9,277-279. Krishnamurthy, G. V., Bhatia, B. S., Girdhari, L., and Subrahmanyan, V. 1960. Pilotplant studies on the preparation of crude papain from raw papaya. J. Sci. Food Agr. 8,433-436. Lal, G. 1956. Some important aspects of the researches in fruit and vegetable preservation at the Central Food Technological Research Institute. Fruit and vegetable preservation industry in India. C. Food Technol. Research Inst., Mysore, 67-74. 67-74. Landaverde, A. 1941.10 plantas tropicales. Ed. Agricolas. Mexico Lassabliere, P., Lesne, E., and Tanon, L. 1950. Encyclopedia de L’Alimentation. Maloine, J. A., Paris. Lawler, F. K. 1967. Banana challenges food formulators. Food Eng. 39 (5), 58-63. 39 (6),62-65. Lindorf, H. 1966. Desarrollo y anatomia d e varios frutos tropicales comestibles. Fac. Cienc. Univ. Central d e Venezuela. Unpublished. Luh, B. S., Chichester, C. O., Gudel, W., and PBrez, R. 1964. Evaluacibn d e la calidad de 10s alimentos enlatados venezolanos. Arch. Venezola-nos de Nutricibn 14, 151-157. Luna, C. 1963. Fabricacibn d e harina d e guayaba. Dept. Tecnol. d e Alim. Fac. Cienc. Univ. Central de Venezuela. Unpublished. McCarthy, A. I., Palmer, J. K., Shaw, C. P., and Anderson, E. E. 1963. Correlation of gas chromatographic data with flavor profiles of fresh banana fruit. J. Food Sci. 28 (4),379. Manzano Barrios, Diana. 1964. Determinacibn de calores especificos d e frutas tropicales. Dept. Ing. Quimica. Univ. Central de Venezuela. Unpublished. Marvaldi, Ely. 1966. Industrializaci6n del merey. Dept. Tecnol. de Alim. Fac. Cienc. Univ. Central de Venezuela. Unpublished. Mathur, P. B., and Srivastava, H. C. 1956. Cold Storage and freezing preservation of fruits and vegetables. Fruit and vegetable preservation industry in India. Food Technol. Research Inst. Mysore. 268-271. Mathur, P. B., Sing, K., and Srivastava, H. C. 1958. Studies in frozen storage of some fruits products. Food Sci. (Mysore)7 . Maxie, E. C., and Abdel-Kader, Adel. 1966. Food irradiation-physiology of fruits as related to feasibility of the technology. Aduances in Food Research 15, 105-138. Merory, J. 1960. “Food Flavorings.” AVI, Westport, Connecticut. Meyer, L. H. 1960. Food chemistry. Reinhold, New York 218, 274, 108, 254, 261. Miller, C., Bazore, K., and Batow, M. 1957. Fruit of Hawaii. Univ. of Hawaii. 18, 70, 115, 116.

212

N. CZYHRINCIW

Monvoisin, A. 1953. La conservation par le froid des denrkes pkrissables. “Dunod,” Paris. 489,495. Morgan, R. C. 1966. Chemical studies on concentrated pineapple juice. Carotenoid composition of fresh pineapple.]. Food Sci. 31,213-217. Mosqueda, M. B. 1966. Tecnologia del jug0 de tamarind0 clarificado. Soc. Cienc. Naturales, Caracas. 73,62-69. Mosqueda, M. 1967. Contribucibn a1 estudio del aroma y sabor de algunas frutas tropicales, y proyecto de clasificacibn s e g h dichas caracteristicas. Dept. Tecnol. de Alim. Fac. Cienc. Univ. Centr. de Venezuela. Unpublished. Mosqueda, M., and Czyhrinciw N. 1964. Propiedades fisicas principales de algunas frutas tropicales. Arch. Venezolanos de Nutricibn. Caracas. 14,91-102. Muller, C. J., Kepner, R. E., and Webb, A. D. 1964. Some volatile constituents of passion fruit wine.]. Food Sci. 29,569-575. Nicholls, H. A. A. 1901. Manual de Agricultura tropical. Tip. Nac. San Josk de Costa Rica. Central America. Northcutt, R. T., and Gemmill, A. V. 1957. New banana puree. Food Eng., April. Nother, C. K., Taylor, D. H., and Brekke, J. E. 1958. Pineapple juice powder. Food Technol. 12 (7). Padmini, T., Narayanaswamy, D., and Murthy, H. B. N. 1963. Packaging and storage studies on mango cereal flakes. Food Sci. (Mysore) 12,315-317. Parker, M. E., Harvey, E. H., and Statcler, E. S. 1952. Elements of food engineering. Reinhold, New York, 81. Parpia, H. A. B. 1956. Some aspects of canned food storage problem in India. Fruit and vegetable preservation industry in India. C. Food Technol. Research Inst., Mysore, 104- 116. Peterson, M. S., and Tressler, D. K. 1963. “Food Technology the World Over.” Avi, Westport, Connecticut, p. 473. Pittier, H. 1926. Manual de las plantas usuales en Venezuela. Caracas. Popenoe, W. 1938. Importantes frutas tropicales. Ofic. de Cooper. Agricola, Washington, D.C. Popenoe, W. 1939. Manual of Tropical and sub-tropical fruit. Macmillan, New York, 7. Proctor, B. E. 1960. “Food Enzymes.” Avi, 129-136. Pruthi, J. S., and Girdhari L. 1955. Refrigerated and common storage of purple passion fruits. ZndianJ. Hort. 12,205-211. Pruthi, J. S., and Girdhari, L. 1959. Studies on passion fruit juice concentrate and powder. Food Sci. (Mysore)8,5-6. Pruthi, J. S., Mookeii, K. K., and Girdhari, L. 1960. A study of factors affecting the recovery and quality of pectin from guava. Indian Food Packer 14,7- 13. Pruthi, J. S., Susheela, R., and Cirdhari, L. 1961. Anthocyanin pigment in passion fruit ring.]. Food Sci. 24,385-388. Pruthi, J. S. 1963. Physiology, chemistry and technology of passion fruit. Advances in Food Research. 8,203-282. Pruthi, J. S., Chakraborty, R. N., Sondhi, S. P., Sastry, L. V. L., and Siddappa, G. S. 1963. Studies on concentrating the juice of the cashew apple. Food Technol. (ll),95-98. Pruthi, J. S., and Bedekar, 1963. Studies on varietal trials in salting or brining of raw mango slices for subsequent use in chutney and pickle manufacture. Punjab Horticult.]. 3,264-271. Pruthi, J. S., and Srivas, S. R. 1964. Free amino acids in passion fruit juice. Sci. Cult. (Calcutta)30 (l),48-49.

TROPICAL FRUIT TECHNOLOGY

213

Rahman, A. R. 1963. Economical method for the production of flour from green plantains../. Agri. Univ. Puerto Rico 47 (1). Ralls, J. W. 1959. Nonenzymatic formation of acetoin in canned vegetables. J. Agr. Food Chem. (7),505. Rauch, G . H. 1952. Jam manufacture. L. Hill Ltd., London, 155-181. Reitersmann, R. 1952. Die frucht-Likijre. Carl Hnoppe Griiner Vez. Berlin Charlottenburg, 22. Reymond, D., and Phaff, H. J. 1965. Purification and certain properties of avocado polygalacturonase. J. Food Sci. 30 (2),266-273. Reynolds, T. M. 1963. Chemistry of nonenzymic browning. Advances in Food Research 8,1-52. Riaz-ur-Rahman, Saeed Ahmad, and Musahib-ud-Din Khan. 1956. Some studies on the quality of punjab guavas (Psidium guajava). Fruit and Vegetable Preservation Industry in India. Central Food Tech. Research Inst. Mysore, 34-41. Rieckehoff, I. G., and Rios, A. 1956. Occurrence of pectin methylesterase in guavas. J . Agr. Uniu. Puerto Rico Rieckehoff, I. G., and Rodriguez, A. I. 1960. Pasta de Guayaba No. 3. Jalea o cristal de guayaba. Univ. de Puerto Rico. Est. Experimental. Rietz, C. A. 1961. “A Guide to the Selection, Combination and Cooking of Foods.” Avi, Westport, Connecticut. Rivas, Nilo. 1964. Estudio de las variedades de guayaba. Fac. Agron. Univ. Central de Venezuela. Unpublished. Rodin, J. O., Coulson, D. M., and Silverstein, R. M. 1966. Volatile flavor and aroma components of pineapple. 111. The sulfur-containing components. J. Food Sci. 31, 721- 726. Rodriguez, A. J. 1962. Results of a consumer acceptance test for two of containers for guava paste. J . Agr. Uniu. Puerto Rico. 46 (2). Romani, R. J. 1966. Radiobiological parameters in the irradiation of fruits and vegetables. Advances in Food Research 15,57-93. Ross, E . 1960. Present and future of mango processing. Hawaii Farm Science, Univ. of Hawaii. Ruehle, G. D., and Bruce Ledin R. 1955. Mango growing in Florida. Univ. of Florida, 3. Sanchez Nieva, F. 1951. Estudio sobre la industrializacih d e frutas tropicales. Colegio de Quimicos. Puerto Rico. Sinchez Nieva, F., and Rodriguez, A. J. 1958. Pulpa d e guayaba. Univ. de Puerto Rico. 3,7,8. Sinchez Nieva, F., Rodriguez, A. J., and Benzo, J. R. 1959. Processing and canning mango nectars. Univ. of Puerto Rico. Santini, R., and Huyke, A. 1956a. Identification of the anthocyanin present in the Acerola. J . Agr. Uniu. Puerto Rico 40. Santini, R., and Huyke, A. J. 1956b. Identification of the polibasic organic acids. J. Agr. Uniu. Puerto Rico. Sastry, L. V. L., Bhatia, B. S., and Girdhari, L. 1961. Studies on some aspects of custard apple peroxidase. J. Food Sci.26,244-247. Schery, R. W. 1956. Plantas utiles a1 hombre. Salvat. Edit. Madrid 690-691. Scott, K. J., and Roberts, E. A. 1966. Polyethylene bags to delay ripening of bananas during transport and storage. AustralianJ. E x p t l . Agr. ANIM HWSB. 6 (21), 197199.

214

N. CZYHRINCIW

Seale, P. E. and Sherman, G. D. 1960. Passion fruit processes in Hawaii. Univ. of Hawaii. Sherman, H. C. 1958. Chemistry of Food and Nutrition. Macmillan, New York. Sherman, G . D., Sakata, S., and Hamilton, R. A. 1958. Mango canning. Univ. of Hawaii. 8. Siddappa, G. S., and Nanjundaswamy, A. M. 1960. Equilibrium relative humidity (E.R.H.). Relationships of fruit juice and custard powders. Food Technol. 14, 538-537. Silverstein, R. M., Rodin, J. O., and Himel, C. M. 1965. Volatile flavor and aroma components of pineapp1e.j. Food Sci. 30,668-672. Simmonds, N. W. 1959. Bananas. Longmans, Green, London, 310,229,226. Singh, L. B. 1960. “The Mango.” Wiley (Interscience), New York, p. 367, 360-363. Small, L. 1943. Alkaloids. Organic Chemistry. Adv. Treatise. New York. Smock, R. M., and Neubert, A. M. 1950. Apples and apple products. Wiley (Interscience), New York. 135,171,439. Solanas, S., Darder, B., Bertou, P., and Bertou, H. 1964. Contaminacibn radioactiva d e alimentos e n Venezuela. Inst. Venezolano de Investigaciones Cientificas. Caracas. Unpublished. Sommer, N. F., and Fortlage, 1966. Ionizing radiation for control of postharvest diseases of fruit and vegetables. Adoances in Food Research 15,147,184. Stadtman, E. R. 1948. Nonenzymatic browning in fruit products. Adoances in Food Research 1,325,369,350. Storni, S . J. 1942. Bromatologia Indigena. Tucuman, Argentina. Sturrock, P. 1959. Fruit for southern Florida. Southeastern Printing, Florida. Tatterfield, F., and Petler, 0. 1940. The insecticide properties of certain species of annona. Am. Appl. Biol. 27. Tressler, D. K., and Evers, C. F. 1957. The freezing preservation of foods. Avi, Westport, Connecticut, pp. 1,474,484,539,593,655.2,120,412. Tressler, D. K., and Joslyn, M. A. 1961. Fruit and vegetable juice. Avi, Westport, Connecticut. Vavilov, M. Y. 1950. The origin, variation, immunity and breeding of cultivated plants. Chronica Botanica. Waltham, Mass., 13, 1-6. von Loesecke, H. W. 1949. “Bananas.” Wiley (Interscience), 79,80,81,114. Weier, T. E., and Stocking, C. R. 1950. Histological changes induced in fruits and vegetables by processing. Adoances in Food Research. 2,298-340. Wenkham, W. S., Miller, C. D., and Kanehiro, Y. 1961. The sodium content of Hawaiigrown fruits and vegetables in relation to environment. j . Food Sci. 26 (l),31-37. Whitaker, F. R. 1957. Assay and properties of commercial ficin. Food Research 22. Wick, E. L., McCarthy, A. I., Myers, M., Murray, E., Nursten, H., and Isenberg, P. 1966. Flavor and biochemistry of volatile banana components. Adoances in Chem. Ser. 56,241-260. Wickiner, V. D. 1960. Some aspects of agricultural development in the Tropics. Trop. Agr. London 17 (l),163-176. Winton, A. L., and Winton, K. B. 1958. Analisis de Alimentos (esp.) H.A.S.A. BarcelonaBuenos Aires. Mexico. 693. Wolf, J. 1958. The organic acids of bananas. 2. Lebensm-Untersuch. u-Forsh. 107, 124-131. Wiistenfeld, H. 1953. Trinkbranntweine und Likore. Paul Pareg. Berlin, Hamburg, 334.

THE CHEMISTRY AND BIOCHEMISTRY OF TEA AND TEA MANUFACTURE BY MIKHAILA. BOKUCHAVAAND NINA I. SKOBELEVA Bokh lnstitute of Biochemistry, USSR Acudemy of Sciences, Moscow, USSR

I. Introduction.. .......................................................... 215 11. Chemical Constituents of Tea Leaf and Manufactured T e a . . . . . . . . . . . . . . . . 219 A. Phenolic Substances.. ............................................... 220 B. Nonphenolic Substances.. . . .............................. 231 C. Aromatic Substances.. ............................................... 248 D. Enzymes.. ..................................................... 255 111. Significance of Biochemistry ............................................. 260 A. Basic Principles of Manufacture of Different T e a s . . .................... 260 B. Biochemistry and Technology of Black T e a . ........................... 261 C. First and Second Stages of Fermentation.. ............................ 264 D. Molecular Basis of Fermentation.. .................................... 266 IV. Thermal Treatment to Enhance Quality and Vitamin P of Black T e a . . . . . . . . 272 A. Disadvantages of Classic Technology .................................. 272 B. Importance of Thermophysical Processes in Manufacture . . . . . . . . . . . . . . .273 V. Biological and Nutritional Value of T e a . .................................. 278 VI. Conclusion.. ............................................................ 279 References .............................................................. 280

I. INTRODUCTION

Tea is one of the most popular beverages in the world because of its excellent flavor and healthful effect on the human body. These qualities significantly distinguish tea from other products of plant origin which impart good taste and aroma to various foods. The tea shrub is a perennial evergreen plant. It belongs to the Camellia genus of the Theaceae family. Taxonomically speaking, two basic varieties of the tea plant are recognized: (1) the Northern (China) form, Thea sinensis;and ( 2 )the Southern (Assam) form, Thea assamica. 215

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Commercial teas are produced by the biochemical transformations to which tea leaves are subjected in processing. Since tea manufacturing is based on biochemical processes, tea-production technology can be reasonably regarded as biochemical technology. Tea manufacturing is one of the most ancient industries on our planet, In its native land-China-tea was brought into use a few centuries B.C. Tea leaves were used first as a drug. Later, however, an infusion of young tea shoots became widely used and recognized as a national Chinese beverage. As tea consumption grew, methods of manufacture were improved on the basis of practical observations. The development of tea production has been greatly promoted by achievements of related scientific branches. As is well known, the high quality of Indian and Ceylon teas of commerce is accounted for not only by favorable conditions for tea cultivation in those countries but also by successful application of scientific achievements in tea cultivation and manufacture. Of particular significance is the contribution made by the Toklai Experiment Station, in Assam. An important role in the development of the tea industry in Ceylon has been played by the Tea Research Institute (Ukers, 1935). The outstanding role of scientific research in progress in the tea industry is vividly illustrated by the development of tea manufacture in the USSR. In 1930 the USSR Research Institute of Tea and Subtropical Cultures was established. Later, the Technology Department of the Institute grew into the USSR Institute of Tea Industry, which has been actively elaborating improvements in procedures of tea-leaf processing. The rapid development of tea manufacture in Russia made it necessary to conduct a many-faceted study of raw tea and its processing methods. In 1933, regular investigations of the biochemistry of tea and tea manufacture began under the guidance of A. N. Bakh and A. I. Oparin. The studies were closely connected with the tea industry, and were usually performed in collaboration with the USSR Research Institute of Tea Industry at tea factories of the Georgian SSR. Since then, studies of tea biochemistry have been developing successfully. Results of this research have been published in a ten-volume work titled “The Biochemistry of Tea Manufacturing” (Biokhimiya Chainogo Proizvodstva), some monographs, and many papers in journals issued by the USSR Academy of Sciences and various specialized institutes. Extensive research work of A. N. Bakh, A. I. Oparin, A. L. Kursanov, their colleagues, and students helped reveal the essence of tea-leaf processing and elaborate the biochemical theory of tea manufacture.

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According to the theory, oxidative processes catalyzed by tea-leaf enzymes underlie the tea-manufacturing process. Oxido-reductive processes are well equilibrated in intact tissues of the tea leaf. Under the influence of oxidative enzymes, catechins are oxidized into corresponding quinones, to be immediately reduced at the expense of readily oxidized substances present in the tea leaf (amino acids, ascorbic acid, different phenolic compounds, etc.). Therefore, an undamaged tea leaf exhibits no colored products of catechin oxidation. Cell damage of plucked tea leaf during rolling leads to the predominance of oxidative processes over reductive. Products of catechin oxidation - quinones -oxidize various substances and gradually become denser, forming the red and brown pigments responsible for the color of tea liquor. It should be noted that the biochemical theory of tea manufacture has exerted a great effect on the development of tea industry in the USSR. On the basis of the theory, methods have been developed for biochemical control over manufacturing procedures. In connection with practical implementation of the theory, biochemical laboratories have been organized at all tea factories. Recent years have been marked by particularly great progress in the biochemistry of tea and tea manufacturing. During the last decade, ample data have been obtained on tea tannin and oxidative enzymes, as well as on oxidative processes underlying tea production. These data shed new light on many problems in the theory and practice of tea manufacture. They make it possible to introduce significant improvements into tea manufacturing and obtain products of higher quality. In addition, certain regularities discovered in tea biochemistry are of theoretical importance for general biochemistry. This can be exemplified by the fact that the formation of tannins in plants and their role in oxido-reductive processes have been studied in detail in the tea plant. Problems of the state of oxidative enzymes in the intact and damaged cell and of their high activity in the insoluble form also have been studied in the tea leaf and are important for complete understanding of the nature of enzymes and the mechanism of their effects in general. Results of studies of primary and secondary biochemical processes in tea manufacturing can also be used in other industries based on the processing of raw plant materials. The formation of aldehydes and other intermediates in the course of secondary oxidative processes which occurs during the interaction of tannin, amino acids, and sugars at normal temperatures in the presence of enzymes and

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at high temperatures in the absence of enzymes can be well applied to many branches of the food industry, e.g., in wine making, bread baking, coffee and cocoa manufacturing, brewing. In recent years some new properties of tea polyphenols have been uncovered. The following are major properties of tea-leaf polyphenols: ( 1)the capillary-strengthening property (vitamin P activity); (2) the antioxidative property responsible for the radioprotective effect; and (3)the antimicrobial property. In 1950 Kursanov et al. made an interesting discovery: they showed that tea catechins had a high vitamin P activity. Catechins with oxigroups located in the orthoposition [( -)-epicatechin and (-)epicatechin gallate] exhibited the highest activity. In addition to the capillary-strengthening effect, tea catechins were shown to promote an accumulation of ascorbic acid in the organism. The vitamin P activity of tea catechins was proved in laboratory experiments and clinical studies. This activity exceeded that of preparations commonly used for capillary strengthening- citrin, rutin, esculin. On the basis of this discovery, the production of vitamin P from tea leaves was organized in the USSR (Kursanov and Zapromyotov, 1954). It was later established that tea flavonols also had an appreciable vitamin P activity (Ulyanova, 1966). Interesting new data have recently been gathered on the therapeutic effect of the tea vitamin P (Kazantsev, 1966). One of the most important properties of tea polyphenols is their radioprotective effect. Japanese workers reported that tea catechins could almost completely eliminate the strontium-90 damaging effect on the human body. They absorb the radioactive isotope and remove it from the body before it reaches bone marrow (Ugai and Hayashi, 1959). Recent studies, particularly those by Baraboy, demonstrated that the radio-protective effect of tea catechins was associated with their antioxidative property. They were shown to have a favorable effect whether taken before or after irradiation (Baraboy, 1966). Thus, tea catechins may have both a preventive and therapeutic function. It should be stressed that consumption of catechin-rich tea varieties can be very significant for the prophylaxis of radiation disease. Another important property of tea polyphenols is their antimicrobial effect. Experiments carried out indicate that they have a high bactericidal activity, which accounts for the therapeutic effect of green tea infusion against dysentery (Bokuchava and Berdyeva, 1959; Kost and Bauer, 1966). A very wide distribution of tea throughout the world appears to be connected with its bactericidal effects. This refers primarily to green

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tea used in hot climates. Taking into consideration diverse positive effects of tea catechins, science and engineering experts make great efforts to increase their content in manufactured teas. Breeders try to select tea varieties with a higher catechin content (Bakhtadze, 1955), and agricultural technicians grow tea plants with a much higher catechin concentration in the leaves (Bziava, 1956). Biochemists and engineers develop manufacturing procedures that help reduce losses of valuable constituents during tea processing, producing manufactured teas which can be a rich source of vitamin P. Quite recently, scientists made another important contribution to the tea industry. Extensive experimental work has led to a method of producing instant green tea from fannings and crude leaves. This tea is of the same quality as green tea obtained from high-quality raw leaves (Bokuchava and Pruidze, 1964a,b; Pruidze, 1962). Also, a method has been elaborated for producing food dyes of yellow, brown, and green color with high vitamin P activity from poor-quality raw tea and fannings (Bokuchava and Pruidze, 1965, 1966). Food dyes obtained through a special process contain a large amount of vitamin P, caffeine, amino acids, soluble sugars, and other substances. An original method has been developed for stabilizing red pigments from table beet through applying tea dyes. The beet dye normally changes its color in the light, becoming violet or bleached, preventing its use in the confectionery industry. The treated dye has been used successfully in confections produced in Moscow (Bokuchava et al., 1966b). Thus, achievements in tea biochemistry have proved beneficial not only for tea manufacture but for medicine and the food industries as well.

II. CHEMICAL CONSTITUENTS OF TEA LEAF AND MANUFACTURED TEA

The chemical composition of the tea leaf and manufactured tea is very complex and not yet completely established, though the problem has been studied for more than a century. Our knowledge of the constituents of the leaf and of manufactured tea, however, has greatly increased in recent years through modern research techniques: paper chromatography, column chromatography, thin-layer chromatography, gas-liquid chromatography, spectrophotometry, radioisotope labeling, etc. New information has been accumulated on catechins, enzymes,

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volatile oils, aldehydes, organic acids, proteins, amino acids, and vitamins. Alkaloids, pectins, flavonols, leucoanthocyanins, carbohydrates, resinous substances, mineral substances, etc., have been well investigated. For convenience, material presented herein on the chemical constituents of tea leaf and manufactured tea is divided into four major groups: (A) phenolic substances; (B) nonphenolic substances; (C) aromatic substances; and (D) enzymes. These groups are discussed below, together with their significance to biochemical processes of tea manufacturing and the quality of manufactured tea.

A. PHENOLICSUBSTANCES 1 . Tannins Among the chemical constituents of tea leaf and manufactured tea, a complex of tanning substances, or tea tannin, is particularly important. Tannin is a complex mixture of organic compounds (known to be derivatives of multi-atom phenols) of a diverse chemical nature. In tea manufacturing, the tannin present in the leaf is subjected to various transformations that form the basis of tea manufacturing. Almost all characteristics of manufactured tea- its taste, color, and aroma- are associated directly or indirectly with conversions of the tea-leaf tannin. Therefore, scientists from many countries have given much attention to its study. The first paper dealing with this question was published in 1838 (Miilder, 1838). Previous investigations of tea-leaf tannins contained their total measurements only. However, tea manufacturers often failed to observe a direct correlation between the quality of manufactured tea and total tannins in the leaf. It was suggested that the quality of manufactured tea depended not upon the total content of soluble tannins but upon certain fractions. Early work by Oparin, Kursanov, Bokuchava, and others showed that tea-leaf tannins were heterogeneous in composition. Kursanov (1941) subdivided the mixture of tannins into two fractions: the so-called polyphenol-catechin fraction, of low molecular weight, which is soluble in sulfuric ether; and a tannin fraction of higher molecular weight, which is insoluble in ether and readily soluble in hot water, ethylacetate, and acetone. Bokuchava and Popov ( 1945) revealed the water-insoluble tannin fraction in tea leaf and tea, and developed a method for measuring

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its amount (1946). The tannin mixture of tea leaf and manufactured tea was separated into four fractions: (1)the fraction precipitated by mineral acid; (2) the fraction not precipitated by mineral acid; (3)that precipitated by ammonium sulfate; and (4) that not precipitated by ammonium sulfate (Bohmhava and Belinovich, 1946). Studies of the physical and chemical characteristics of the fractions established their different properties. Kursanov et al. (1947) fractionated the tannin mixture of tea leaf and manufactured tea, proceeding from their different affinity for solvents. Those experiments indicated that the major portion of tannin from fresh tea leaves (about 90%) is the ethyl acetate fraction which consists of catechins and their gallic esters, whereas the major portion of tannin from manufactured tea is the water-soluble fraction. Thus, studies have shown that the tea tannin is a mixture of many substances. Tsujimura isolated three constituents of tannin from green tea in the crystalline form: l-epicatechin, yielding 0.14% (1929); l-epigallocatechin, yielding 0.25 %; and l-epicatechin gallate, yielding 0.32 % (1934,1935) of tea dry weight. Using the hydrolysis of the tannin enzyme, Kursanov and Dzhemukhadze (1948) isolated gallic acid from tannin of Georgian tea. The isolation of crystalline components shed light on the chemical composition of the tea tannin, but the yield was too small to draw a convincing picture of the tannin mixture as a whole. It was the development of partition chromatography that assisted a deeper insight into the constituents of tea tannin. With partition chromatography on a gel column, Bradfield et al. (1947) and Bradfield and Penney (1948) isolated seven catechins from Ceylon green tea, their yield making 60 to 80% of the total amount of amorphous tannin. In addition to (-)-epicatechin, (&)-catechin, (-)-epigallocatechin, and (-)-epicatechin gallate, they first revealed (&)-gallocatechin, (-)-epigallocatechin gallate, and an unknown catechin later identified as (-)-gallocatechin gallate (Bradfield and BateSmith, 1950). Kursanov and Zapromyotov (1952) separated the tannin preparations isolated from fresh shoots of Georgian tea plants into eight crystalline components. They were mainly catechins and reached 98.57% of the total amount of the amorphous tea tannin, as can be seen in Table I. Studies of tea catechins by paper chromatography proved to be very valuable. Roberts and Wood (1951a,b) investigated the composition of phenolic compounds in the juice of young shoots of Assam tea by two-dimensional chromatography. This was followed by a number

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CHEMICAL CONSTITUENTS OF

TABLE I TANNIN FROM FRESHTEASHOOTS OF VARIETY

Catechin

THE

GEORGIAN Content ( % tannin)

Structure

0.4

(*)-Catechin

OH HO

(-)-Epicatechin

'

O

HO

W

O

H

1.3

H?

OH

(&)-Gallocatechin

2.0 \

HO

H*

OH

OH

12.0

of studies of tea catechins by paper chromatography (Zapromyotov, 1958a,b; Oshima et al., 1952; Dzhemukhadze and Shalneva, 1954, 1955; Dzhemukhadze et al., 1962; Bhatia and Ullah, 1962; Zapromyotov and Kolonkova, 1965). These studies demonstrated that the amount of specific catechins and their proportion in the tea plant vary

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TABLE I (Continued) Catechin

Structure

Content (To tannin)

(-)-Epicatechin gallate

18.1

(-)-Epigallocatechin gallate

58.1 HO OH ,OH

1.4

'OH

I

"""O \ &OH

Quercitrin

HO ~

0.27

0

~~

Concomitant pigments and gallic acid Total

1

5.0

98.57

in relation to plant species and age, and to environmental conditions. The qualitative composition of catechins is similar in various teas. It was established that (-)-epigallocatechin gallate is quantitatively the major constituent in all parts of the tea shoot (Kursanov and Brovchenko, 1950; Zapromyotov, 1952; Dzhemukhadze, 1966). The

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first and second leaves of the shoot are richest in catechins. As the leaf ages, the content of total catechins decreases with the amount of (-)-epigallocatechin gallate and (--)-epicatechin gallate falling sharply, and that of (-)-epigallocatechin and (-)-epicatechin rising. This affects the quality of black tea: two-leaf flushes give better tea than three-leaf flushes. The qualitative and quantitative composition of catechins also changes with the season of tea-leaf plucking. An increase of tannin content in raw tea in summer months is due mostly to an active synthesis of (-)-epigallocatechin gallate and (-)-epicatechin gallate (Dzhemukhadze, 1958,1959). The formation of tannin is one of the most interesting problems in the biochemistry and physiology of the tea plant. Therefore, it is not at all surprising that it attracts great attention from plant biochemists and physiologists. Nevertheless, the problem is still far from being entirely solved. The ideas on tannin origin presented in the literature can be summarized as follows:

(1) Tannin is a direct product of photosynthetic processes. (2) Tannin is a product of secondary (carbohydrate) origin. ( 3 ) Tannin is a product of protein origin. The authors of the first hypothesis (Westermaier, 1885; Moller, 1888; Biisgen, 1890) proceeded from the fact that plants are capable of accumulating tannin in the grean leaf in light. According to Gogiya (1950b), however, tea tannin can be accumulated in the dark as well. The second hypothesis, which presumes that its formation is independent of photosynthesis, is favored because of numerous observations indicating that tannin synthesis can occur in the dark (Gogiya, 1950; Kursanov, 1944). The hypothesis on the protein origin of tannin was put forth by Krauss in 1889. Later, it was shared by Michel-Durand (1929). This idea was not confirmed experimentally, though its author believed that there were two pathways for the formation of tannin: a primary formation in green organs in the light but not related directly to photosynthesis; and a secondary formation occurring in any organs in the light. It should be noted that the above hypotheses had many shortcomings. They were based chiefly on indirect findings, and their principal shortcoming was that they considered the tannin mixture as a homogeneous compound. It is well established today that the tea tannin is a mixture of

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many constituents, with the origin and formation of each requiring its own investigation. Realizing that tea tannin consists mostly of catechins, it appears desirable to discuss the biosynthesis of catechins in detail at this point. Much interesting information has been obtained quite recently. As early as 1935, Blagoveshchensky stated that formation of phenolic substances from sugars was most probable. He believed that glucose transforms first into inositol, and then into quinic acid, whose oxidation yields gallic acid, one of the most common tea-tannin constituents. He thought the tannin formation to be associated with plant aging. Studies by Bokuchava (1946), Dzhemukhadze (1946), and Kryukova (1946) on changes in tannin content with age demonstrated that young leaves of the tea shrub accumulated 2 or 3 times as much tannin as old leaves. Besides, the existence of tannins in the germ, shoots, and buds of the tea plant proves that tannin is formed in young cells, and consequently cannot be a product of plant aging. Investigations by Kryukova (1946, 1947) and Kursanov et al. (1948) dealt with the formation, via inositol, of polyphenolic rings from compounds having an open carbohydrate chain, i.e., sugars. Polyphenolic rings are supposed to be formed from hexose through aldole attachment to its terminal carbon atoms, resulting in the formation of inositol, which, in turn, losing three water molecules, forms phloroglucinol. This pathway appears to be very probable. It has been supported by numerous findings indicating the presence of phloroglucinol in many plants (Kursanov and Kryukova, 1941; Kursanov, 1943; Kryukova, 1946). Inositol, being in the free and bound state, has also been found in tea leaves (Kryukova, 1947). Sakato and Matumura (1955) discovered mesoinositol (0.01%) in aqueous extracts of dry tea leaves. Nizheradze (1946) found an inositol phosphorus derivative (phytin) in tea leaves. Kursanov (1952) has shown that phloroglucinol synthesis takes place during sugar infiltration into tea leaves. It is established that phloroglucinol is synthesized more readily from inositol than from sucrose. Nick (1953) demonstrated that immersion of leaf halves of the bistort plant in glucose and sucrose solutions causes an active formation of tannin. Using the radioisotope technique in photosynthetic administration of I4CO2,Zapromyotov (1958a,b) located the site of catechin synthesis in the tea plant. He showed that the main site is in young leaves and shoots; he also proved that the synthesis is not connected directly with

Eytrose phosphate

Pyruvate

/

CH&OOH

Quinic acid

HO Shikirnic acid

CH,-CO -COOH -

H

O

/

T

G

-

COOH

Prephenic acid

COOH

OH Subproduct hydroxylation, reduction, cyciization

Gallic acid

I

Epicatechin

Epigallocatechin

Epicatechin gallate

FIG. 1. Scheme of the biosynthesis of catechins. 226

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activity of the root system. The intensive formation of catechins in the youngest and most rapidly growing tissues is indicative of their active role. Studies of catechin synthesis (Zapromyotov, 1961,1962; Zapromyotov and Bukhlaeva, 1963) with labeled precursors helped to draw a basic scheme of catechin synthesis (Zapromyotov, 1964,1967) (Fig. 1). The pyruvate formed during the glycolytic decomposition of sugars can, on the one hand, undergo oxidative decarboxylation, forming acetate, and, on the other, condense with erythrosophosphate, producing shikimic acid. In the course of condensation, activated acetate in the form of acetyl-CoA yields phloroglucinol nucleus (A ring). Shikimic acid transforms into prephenic acid, serving as a source of pyrocatechin and pyrogallic nuclei (B ring). The gallic acid residue attached by the depside link to C, (C ring) appears to be produced by free gallic acid in tea leaves. Thus, the synthesis of tea catechins is approached today in a different manner. At present, ample data are available giving evidence that the tannin plays an active physiological part in the plant organism. As mentioned above, a high content of tannin in young growing organs, e.g., the germ, bud, and first leaf, indicates its vital significance for the plant metabolism. Applying the labeled I4C for the study of catechin location, Zapromyotov (1959) demonstrated that at least a portion of the radioactive label administered to the tea shoots could be detected in respiratory co,. Thus, in young shoots of the tea plant not only the formation of catechins but also their utilization as a respiratory material takes place. Zapromyotov established that an intensive decomposition of I4Ccatechins started only 20 to 30 hours following their introduction into the shoots. Thus, it can be assumed that respiration proceeds at the expense of carbohydrates until sufficient amounts of sugars and starch are present in tea leaves. As these energy sources are being exhausted, catechins become involved in the respiratory process (Zapromyotov, 1964). In the opinion of Oparin (1935),the tea-leaf tannin, like chlorogenic acid, plays the part of “respiratory chromogenes,” i.e., it serves as the hydrogen carrier in enzymic oxidative processes. This has found support in model experiments (Bokuchava, 1958). Tea-leaf catechins have certain antioxidative properties. Special experiments on the oxidative capability of specific fractions of tea tannin (Bokuchava, 1958) demonstrated that tea catechins absorbed

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?

d

Kaempferol (R,-OH;

HO* “

R,-%-H)

Quercetin HO

(R,-R,-OH;

0

%-H)

Myricetin

practically no oxygen from the atmosphere whereas tannin as such absorbed it significantly; in other words, tannin is a self-oxidizing compound. The presence of catechins, however, protects tannin from oxidation. This property of catechins was later used in developing the method of stabilizing red pigments isolated from beet.

2. Flavonols Flavonols of the tea plant represent a group of compounds which are very close to catechins in chemical composition but differ in a higher level of oxidation of the primary diphenylpropane nucleus. Flavonols of the tea plant include mono-, di-, and triglycosides of three aglycones: kaempferol, quercetin, and myricetin. As the sugar component they involve glucose, rhamnose, galactose, arabinose, rutinose, etc. Application of the chromatography technique allowed study of the flavonols of the tea plant in more detail. Oshima and Nakabayashi (1953), and Nakabayashi (1953) detected 23 flavonol-glycosides, 9 of which were later identified. Takino et al. (1953, 1954) identified the glycosides of quercetin and kaempferol. With paper chromatography, Roberts and Wood (1951b) and Roberts et al. (1956)demonstrated that flavonols from leaves of the Assam tea variety were derivatives of kaempferol, quercetin, and myricetin, whereas the sugar moieties were represented by glucose and rhamnose. Vuataz et al. (1959) confirmed the findings of Roberts for the green tea leaf. From green tea leaves growing in Japan, Oshima and Nakabayashi (1953) and Nakabayashi (1953)isolated 7 flavonol-glycosides: quercitrin, isoquercitrin, rutin, quercetin - 3- rhamnodiglucoside, astragalin, kaempferol-3-rhamnoglucoside and kaempferol-3-rhamnodiglucoside. Takino et al., (1962a,b) and Takino and Imagawa (1963a,b) isolated from tea leaves a new glucoside which contained galactose as a sugar moiety, the compound being identified as myricetin-3-galactoside.

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Dzhemukhadze and Nestyuk (1960, 1961) studied the content of flavonol-glycosides in the Georgian variety with relation to its diurnal changes and variations during rolling. The concentration of flavonoIglycosides was shown to be lower in the rolled leaf and fermented tea than in original leaves. Bokuchava and Skobeleva (1960) demonstrated that the content of flavonol-glycosides in manufactured black tea was higher if the tea leaf was given a shorter fermentation. Ulyanova (1962) performed a more detailed study of flavonols in the Georgian and Krasnodar tea varieties. Paper chromatography and polyamide column chromatography yielded two preparations -a mixture of flavonol-glycosides and a mixture of individual flavonolglycosides: astragalin, kaempferol-3-rhamnoglucoside, kaempferol3-rhamnodiglucoside, isoquercitrin, rutin, quercetin-3-rhamnodiglucoside, and myricetin-3-glycoside. These substances were identified, their functional groups assayed, and their ultraviolet spectra measured. Some of the flavonol-glycosides were studied with infrared spectroscopy. Ulyanova (1963) proved that flavonols occur in vegetative and reproductive organs of the tea plant. Young leaves and flower petals exhibited the highest content of flavonols. In the course of ontogenetic development the flavonol concentration in plant organs changes-it increases in shoots with age. If tea seeds contain 0.03% of flavonols, 30-day-old seedlings contain 10 times as much. As the plant ages, the flavonol content declines significantly. Ulyanova developed a method for quantitative estimation of the glycosides of kaempferol and quercetin in the tea plant. The ratio of the glycosides was shown to vary during ontogenetic development of the plant. Table I1 gives data on the content of glycosides of kaempferol and quercetin in different parts of the tea plant. These data indicate that different organs of the tea plant are characterized by different content of flavonols and their different ratio. As tea leaves age, the relative concentration of quercetin increases, especially that of rutin. From study of flavonol concentration during black-tea manufacture, Ulyanova (1966) revealed its gradual decrease beginning with the withering stage. The content of flavonols is 15 to 25% less in manufactured black tea than in fresh green leaves. The glycosides of myricetin undergo the greatest transformations. The author believes that manufacturing procedures of black tea provide more favorable

CONTENT O F

TABLE I1 GLYCOSIDES O F KAEMPFEROL AND QUERCETIN IN DIFFERENTPARTS O F THE TEA PLANT (% DRYMATTER) Glycosides of kaempferol

Glycosides of quercetin Ratio of glucosides Kaempferol-3Kaempferol-3Quercetinof kaempferol to Astragalin rhamnoglucoside rhamnodiglucoside Isoauerciirin Rutin 3-rhamnodirrlucoside those of auercetin 0.25 0.13 First leaf 0.16 3:1 0.18 0.44 0.49 0.25 0.28 Second leaf 0.23 1:l 0.21 0.32 0.36 0.15 0.21 Third leaf 0.17 1:l 0.12 0.05 0.14 Fourth to sixth leaves 0.32 0.20 0.45 1:3 0.05 Stalk 0.10 0.22 1.19 0.51 1:6 0.29 Buds (green) 0.38 0.54 0.10 0.20 0.05 3:l 0.17 0.48 Flowers 0.21 0.18 0.16 0.05 2:l 0.33 Petals 0.45 0.67 0.12 4:l 0.14 0.11

Plant Part

m

c

2-

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conditions for oxidative transformations than for hydrolytic transformations of flavonol-glycosides. According to data collected by Roberts and Wood (1951a,b) and Takino and Imagawa (1963a,b), in the presence of pyrocatechin or (+atechin, flavonol-glycosides are capable of being enzymically oxidized, forming dimer products - biflavonols. Takino and Imagawa (1963a,b) obtained the product of myricitrin oxidation- erycitrin. The oxidation occurred in the presence of pyrocatechin or (+atechin by means of tea polyphenol oxidase. Using peroxidase and hydrogen peroxide, Loth (1964) performed oxidation of astragalin, rutin, and myricitrin, and isolated oxidants which also were dimers. Ulyanova and Erofeyeva (1966) pointed out that the flavonolglycoside complex of black tea had vitamin P activity: it strengthens capillary walls and promotes an accumulation of ascorbic acid in the internal organs of animals.

B. NONPHENOLIC SUBSTANCES 1 . Carbohydrates

Similar to other plants, the tea plant contains various carbohydrates ranging from simple sugars to complex polysaccharides -cellulose and hemicellulose. Bamber (1893) found 0.2 to 0.5% sugars in the shoots of the Java tea variety. Harler (1933) detected a somewhat greater amount of soluble carbohydrates: 1 % in fresh tea leaf, and 1.5% in manufactured tea. Deuss (1937) and Sprecher (1936) found approximately 0.5% of soluble sugars in tea leaves. Roberts (1939) detected 1.4 % of soluble sugars in fermented tea. Kharebava (1946) established that the content of reducing sugars and sucrose in the tea leaf increased with age. According to Harler (1933), the starch concentration varies in different plant parts: 0.11% in the bud, 0.19 % in the first leaf, 0.30% in the second leaf, and 0.83% in the stalk. With leaf aging, the content of cellulose and hemicellulose increases sharply, which is indicative of the negative nature of raw tea (Shavishvili, 1940). Torii and Kanazawa (1954) used paper chromatography to, investigate the composition of carbohydrates in tea leaves. They established the presence of free glucose, fructose, sucrose, and two oligosaccharides. They also found glucose, rhamnose, galactose, and arabinose as glycoside components.

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232

Mizuno and Kimpyo (1955) reported free fructose, glucose, arabinose, sucrose, maltose, raffinose, and stachyose in black tea. In addition to the sugars detected by the Japanese researchers, ribose and rhamnose were identified by Cartwright and Roberts (1954)as free sugars of black tea. Bokuchava and Skobeleva (1961a) used paper chromatography and densitometry to investigate carbohydrates of the tea leaf. This technique was later used as the basis for a qualitative estimation of carbohydrates in fresh tea leaf and manufactured tea (Kharebava and Skobeleva, 1968). Table I11 presents the content of carbohydrates in the tea leaf. TABLE I11 SUCROSE, GLUCOSE, AND FRUCTOSE IN FRESHTEALEAF( % DRYWEIGHT) ~~

Sugars

July

August

September

Sucrose Glucose Fructose Total content of sugars

0.43 0.28 0.15

0.48 0.15 0.15 0.78

0.34 0.15 0.17

0.86

0.66

As can be seen from the table, the content of sucrose is higher than that of glucose or fructose. Table IV compares the carbohydrate concentrations of tea manufactured by classic procedures and by new technology based on thermal treatment of underfermented tea. TABLE IV IN TEA SUCROSE, GLUCOSE, AND FRUCTOSE MANUFACTURED BY CLASSIC AND NEWPROCEDURES ( % DRYMATTER) Manufactured black tea Sugars Sucrose Glucose Fructose Total content of sugars

New technology

Classic technology

0.17

0.09 0.78 0.48 1.35

0.46 0.40

1.03

The data show that the processing of tea leaf results in a decrease of sucrose content and increase of monosaccharide content in the manufactured tea. It should be noted that tea manufactured by the new technology contains 0.17% sucrose, against 0.09% in tea manufactured by classic procedures. The contribution of simple sugars to the liquoring characters of the tea once received insufficient attention. Kretovich and Tokareva

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(1948, 1949), Markh (1950), and Gerasimov (1955) reported that reducing sugars are of significance in production of the aroma of many food products (bread, vegetables, fruit, malt, tobacco, wine, etc.). In this connection, model experiments were carried out to study the interaction of reducing sugars with amino acids and tannin of the tea leaf at high temperatures (Bokuchava, 1958).The experiments showed that the interaction of glucose or fructose solutions, phenylalanine, and tannin of the tea leaf at higher temperatures leads to the formation of aldehydes, producing a whole spectrum of aromas-those of flowers, fruit, malt, honey, etc. Pure solutions of glucose and fructose exposed to high temperature give a caramel aroma like that often smelled during tea drying. Thus, experiments at the molecular level suggest that simple sugars play a certain part in forming the tea aroma.

2. The Pectic Substances The pectic substances are very important constituents of the tea leaf, though they were previously regarded as of no significance to tea quality. That is why so little research has been done on this aspect. Knowledge of the pectic substames in tea has been extended by experiments by Shaw (1934) and Lamb (1937) on the Assam variety; by Egorov (1940), Shavishvili (1940),and Gogiya (1950a) on the Georgian variety; and by Mizuno and Kimpyo (1955) and Nakabayashi (1956a, 1957)on the Japanese variety. It was ascertained that pectic substances affect some characters of tea leaf and manufactured tea: viscosity developing during leaf processing, the sweet taste of manufactured tea, hygroscopicity, etc. Gogiya (1950a) accumulated data showing that different parts of the tea flush contain different amounts of pectins (Table V). Besides the pectin content, the author investigated the composition of pectins and established that the tea leaf contained both hydropectin and protopectin. TABLE V DIFFERENT PARTS TEAFLUSH(70DRYMATTER)

PECTIC SUBSTANCES IN

Flush parts

OF THE

Pectic substances ~

1st leaf and buds 2nd leaf 3rd leaf 4th leaf Stalk

3.08 2.63 2.21 2.01 2.62

Shavishvili (1940) reported that pectin content is much higher in the young tender Ieaf than in the coarse leaf, whereas protopectin

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

content differs but slightly. Nakabayashi (1957) reported pectin content in different parts of the tea flush of the Japanese variety to be 4.9%in the bud, 6.1 % in the first leaf, 4.7% in the second leaf, and 7.6%in the stalk (all percent of dry matter). A comparison of the above figures indicates that tea plants of the Japanese variety contain more pectic substances than those of the Georgian variety (Gogiya, 1950a). Some research on the pectic substances was carried out by the Tocklai Experiment Station (Tocklai Ann. Reports, 1958), which reported the amount of total pectin in black tea to be 6.2%, of which 2.57%was water soluble. In 1934 Shaw put forward original ideas on the participation of pectic substances in forming tea aroma. In his opinion, the apple aroma developing in the withered leaf is related to pectin transformations. Unfortunately, his ideas were not developed further.

3. Alkaloids The great popularity of tea as a beverage can be accounted for mainly by the presence of alkaloids -caffeine, theobromine, and theoph ylline. For many years alkaloids were considered to be products of terminal metabolism, storage substances, or protection means. However, work by Ilyin (1948, 1966), Areshkina (1951), Lovkova (1964), Fairbairn and Paterson (1966), and others has demonstrated that alkaloids actively participate in plant metabolism. It should be mentioned that caffeine, theobromine, and theophylline are methylated derivatives of purine. Purine bases are well known as the most important components of the nucleoproteins, which make up the bulk of cellular nuclei and play an important role in the activity of the living organism. Hence, great biological significance is ascribed to caffeine. Caffeine or theine was discovered in tea by Oudry (1827). Caffeine content reaches 1.5% in coffee beans, and 3 to 5 % in tea leaf and manufactured tea. If calculated per cup of liquor, coffee and tea contain similar amounts of the alkaloid (Martinek and Wolman, 1955). Pure caffeine isolated from the tea leaf is a crystalline substance or of a bitter taste; its chemical formula can be written as C8HI0N4O2, CqN-CO I I NNC&

oc

I CbN-C,

7’

\cH

N

4

Caffeine has diverse effects on the human body: it affects central nervous activity, cardiac activity, and activity of muscular tissues and kidneys.

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Caffeine accelerates metabolism and oxygen intake by body tissues. Its effect on the central nervous system is confined to the cortical centers responsible for higher psychic functions. This results in a well-coordinated enhancement of the brain functions and, consequently, in greater vigilance and mental activity (Vorontsov, 1946). The caffeine effect on the muscle tissue implies a better blood supply to it, which in turn improves cardiac activity and increases the work capacity of skeletal muscles. Caffeine has another advantage: it is not accumulated in the body, thus ruling out the possibility of toxic effects from high tea consumption. Vorontsov (1946) presented the following figures on caffeine content in raw tea (in percent of dry matter): 1st leaf 2nd leaf 3rd leaf 4th leaf

3.39 4.20 3.40 2.10

5th leaf Old leaves Stalks

1.70 0.79 0.36

Indian research workers (Tea Board, India, 1958, 1959a,b) reported that the caffeine content of Indian teas ranged from 2.8 to 4.0%. Lo and Chu (1945) found the caffeine content of Chinese tea of the Yunnan variety varied from 2.64 to 3.62%. Schuen and Mei (1944) showed the caffeine content of Chinese teas varied from 2.9 to 4.0%. Michl and Haberlei (1954) gave 2.5% as the caffeine content of black tea. Caffeine content is not significantly reduced during tea processing, though it may decrease during firing. Caffeine is very important to tea quality. Shaw and Jones (1932) and Kevanishvili (1948) established that during tea processing caffeine reacts with tannin to form a compound termed caffeine tannate, which has a pleasant taste and aroma although separately each has an unpleasant, bitter taste. Roberts (1958a,b) showed that, during tea-leaf processing, caffeine is associated with the polymerized theaflavin to form a compound which imparts “briskness” to the tea infusion. Zoller and Libich (1871) discovered another alkaloid in tea, i.e., theobromine. HN-CO

I

OC

I,NCH,

$

‘CH N

At first, theobromine was found only in high-quality teas, but Fleischner (1956) detected it in teas of all grades.

236

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

The third specific compound of the tea plant is theophylline, which was found by Kossel (1889). CbN-CO I

OC CHSk-C,

I,NH

s

‘CH

4

N

The theophylline content of tea is lower than the theobromine content. According to Fleischner (1956), the theophylline content of black tea averages 0.33 mg 70. Tea leaves have been reported to contain also xanthine (Baginski, 1884), adenine (Kossel, 1889), hypoxanthine, and tetramethyl uric acid (Johnson, 1937). The above nitrogenous substances have been found in tea leaves and manufactured tea, but never in mature tea seeds (Serenkov, 1962). In addition to these compounds, another group of nitrogenous substances belonging to volatile amines has been found in fresh tea leaf, manufactured tea, and tea seeds (Serenkov, 1959; Serenkov and Proiser, 1961; Serenkov, 1962). The problem of caffeine synthesis in the tea plant has long attracted the attention of many scientists. Using the vacuum infiltration method, Blagoveshchensky (1935)proved that caffeine can be formed at the expense of arginine, histidine, and urea. An infiltration of some of these substances into a fresh tea leaf increased its caffeine content by 17%. Caffeine synthesis in the tea plant was studied in more detail by Serenkov (1962). An infiltration of methyl amine, ethyl amine, and butyl amine increased caffeine content significantly in tea leaves, whereas other secondary and tertiary amines found in the tea plant caused no rise in caffeine content. Using methyl-14C amine those workers demonstrated that the methyl amine molecule is completely incorporated into the caffeine synthesized. The other primary amines appear to be deaminated with a specific monoamine deaminase; nitrogen released is used for the caffeine synthesis. Of a good number of substances used for infiltration, only ammonium citrate, partially glycine, and probably RNA are involved in caffeine synthesis. Optimal conditions were found to be 25°C and pH 6.0.

4 . Proteins and Amino Acids Proteins are very significant constituents of the tea plant, and are of importance in tea manufacture as well. It should be-stressed, however,

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237

that a high protein content in the leaf at the expense of tannin can degrade the taste of manufactured tea, since the processing of black tea involves a protein-tannin reaction producing insoluble compounds. Thus, excess protein results in low amounts of tannin and extractives in the manufactured tea, lowering the quality of the beverage. Green tea quality seems to be affected less by a high content of proteins. It is known that Japanese tea breeders grow tea in shadowed plantations; this helps decrease tannin content and increase protein content. They claim that the method provides high-quality green teas. The tea leaf contains mostly alkali-soluble proteins, though small amounts of water-soluble, alcohol-soluble, and acid-soluble proteins can also be detected there. Vorontsov (1928) estimated the total protein at tea flush to be (in percent of dry matter) as shown in the following tabulation: buds 1st leaf

29.06 26.06

2nd leaf 3rd leaf

25.95 24.94

Thus, it can be stated that tea parts contain more proteins at flush than later on. According to the many reports of the Tea Institute of the Georgian SSR, the protein content in tea plants is significantly higher at the beginning of the vegetation period than at the end of it. Protein concentration varies with tea-leaf grade as well. Khocholava (1940) established that tea leaves of the first grade contained 19.31% proteins, leaves of the second grade 18.62%, and leaves of the third grade 16.06%. Of key importance is the study of amino acids of the tea leaf that has been made possible by the development of new research techniques. Krishnamurthy et al. (1952) detected the following amino acids in black tea: aspartic acid, leucine, glutamic acid, phenylalanine, valine, alanine, serine, asparagine, tyrosine, arginine, histidine, lysine, and proline. In investigations of green tea leaves, Roberts and Wood (1951a,b) additionally found isoleucine, threonine, glutamine, palanine, a-aminobutyric acid, and tryptophan. Bokuchava et al. (1954) detected 17 amino acids in tea leaves of the Georgian variety. Some Japanese workers (Jammamoto et al. 1954; Oshima et al., 1954) studied the pattern of changes of amino acids during the manufacture of black and green tea. They reported that amino acid concentration is greatest at the stage of withering, declining again at the stages of rolling, fermenting, and firing.

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Tsujimura and Osawa (1957) found 21 amino acids in green tea. Sakato (1950) detected an unknown compound, which he termed theanine. Roberts and Wood (1951a,b) isolated theanine from tea leaves of the Assam variety. According to data of Sakato, this compound is y-ethyl amide of L-glutamic acid with the formula H,C,~HN~OCCH,*CH,*CH.COOH.

I

NHz

Sakato synthesized theanine and isolated it in the pure state in the form of colorless needles. The melting point of the compound was 20 217"C, and (a)5 = 7.0. Cartwright et al. (1954) confirmed that the

+

compound synthesized by Sakato was identical to the amide from tea leaves. Theanine was also found in tea leaves of the Georgian variety (Popov, 1959). Popov (1959, 1966a) revealed that the germination of tea seeds is accompanied by an appreciable increase in theanine content: from 0.17% theanine of the dry weight of dormant seeds to 2.31% in seedlings after 60 days of germination. The accumulation in seedlings evidences the important physiological role of the compound. Theanine has been found only in the tea plant, in which, besides its specific effect, it appears to play a part similar to that of aspartine and glutamine in higher plants. Popov (1966a) estimated the content of theanine and free amino acids in different parts of the tea flush, and found theanine in greatest concentration in stalks. The younger the leaves were, the higher the content of free amino acids (Table VI). TABLE VI THEANINE AND FREEAMINO ACIDS IN 1)IFFERENT PARTS OF THE THREE-LEAF FLUSH ( % DRYWEIGHT) Part of the flush Bud 1st leaf 2nd leaf 3rd leaf Stalk

Theanine

Amino acids as calculated per glutamic acid

0.55 0.31 0.42 0.58 2.10

1.74 1.14 1.11 0.98 3.49

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239

Popov (1966a) also investigated changes in theanine and amino acid content in two-leaf flushes at the vegetation period, and at various stages of tea processing. Quite recently, Kit0 et al. (1966) used the tracer technique (ethylamine-l-14C) to demonstrate the possibility of theanine participation in catechin synthesis. Bokuchava and Popov (1954) claim that amino acids play a significant role in the formation of tea aroma. The tannin-amino acids reaction in the presence of polyphenol oxidase, or at high temperatures, results in the formation of aldehydes, which, as such or in the form of their transformation products, are responsible for the tea aroma. Experiments carried out have indicated that this is the process of oxidative deamination of amino acids by quinones present in tea catechins. Bokuchava and Popov (1954) advanced a scheme of oxidative deamination of amino acids by quinones resulting from tannin oxidation (Fig. 2). As a result of certain intermediate reactions, end products of amino acid oxidation are formed: COP,NH3, aldehyde, and monosubstituted quinone, which has the ability to deaminate the amino acid molecule further. Experiments carried out by Popov (1956), Skobeleva (1957), and Skobeleva and Popov (1962) established the formation of COP, NH3, and aldehydes. In model experiments, Popov (196613) observed the formation of aldehydes in the mixture of amino acids with ascorbic acid oxidized under the influence of p-quinone, Cu2+,or high temperature (SOo90OC). Dehydroascorbic acid that formed upon ascorbic acid oxidation performed oxidative deamination of amino acids, yielding aldehydes as one of the products. Oxidative deamination of amino acids, with the resulting formation of aldehydes, plays an important part in many food industries. This is well illustrated by the formation of tea aroma in the stages of fermentation and firing as well as during the thermal treatment involved in the new procedure of black tea manufacture developed at the Bakh Institute of Biochemistry, USSR Academy of Sciences, Moscow. Skobeleva (1967) studied changes in the composition of amino acids during this thermal treatment. By paper chromatography and spectrophotometry, she demonstrated that the thermal treatment of underfermented tea was accompanied by a decrease of alanine, phenylalanine, valine, and leucine isoleucine, and an increase of the corresponding aldehydes (acetic, phenylacetic, butyric, valeric).

+

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

240

H,O

+ H o @ i ' G ! k,CHOH l C O O H OH

NHR,CHCOOH

H.

NHR,CHCOOH OH

(11)

If

(111)

(IV)

FIG. 2. Scheme of the interaction of catechins and amino acids.

This led to a better tea aroma. Special experiments were conducted to establish the participation of amino acids in the formation of aldehydes. In these experiments, an addition of amino acids to the underfermented tea was followed by thermal treatment. The resulting data are presented in Table VII. As seen in the table, an addition of alanine to the underfermented tea with thermal treatment to follow increases the content of acetaldehyde by 13%.This is a convincing indication of the participation of amino acids in the formation of carbonyl compounds during thermal treatment of tea.

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TABLE VII EFFECTOFALANINEONACETALDEHYDECONTENTUPON THERMAL TREATMENT OF UNDERFERMENTED TEA (mg1100 g DRYWEIGHT)" Before thermal treatment

After thermal treatment Without alanine With alanine Increase (%)

15.7

21.5

24.3

13

"Experimental conditions: 200 g of underfermented tea + 1 g of alanine in 10 ml of water were kept at 70°C for 5 hours.

The data are in agreement with findings of Roberts (1962),who observed an increase in acetaldehyde content if alanine was added to the manufactured tea when it was brewed. 5. Chlorophyll and Concomitant Pigments Tea shrubs, like any other green plant, contain chlorophyll as well as carotene and xanthophyll. Information is scant on the content of chlorophyll and carotenoids in tea leaf and manufactured tea, and not much greater on the role of pigments in the manufacture of different teas and evaluation of their quality. Jammamoto and Muraoka (1932) assayed the content of chlorophyll and carotenoids in tea leaves and green tea. The resulting data are given in Table VIII. TABLE VIII AND CAROTENOIDS IN TEA LEAFAND CHLOROPHYLL GREENTEA( % DRYMATTER) Material

Chlorophyll

Carotene

Xanthophyll

Fresh tea leaf Steam-treated green tea Fired green tea

0.864 0.548-0.646 0.379-0.546

0.018 0.016 0.01-0.018

0.042 0.042 0.023-0.042

As seen in Table VIII, a major portion of the chlorophyll of the tea leaf is destroyed in the manufacturing process, especially during steam treatment fermentation and firing. Changes in the carotenoid composition are insignificant. Harler (1933) measured chlorophyll, carotene, and xanthophyll in the fresh tea leaf and in manufactured black tea, and discovered qualitative and quantitative changes of all the pigments during tea

242

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

manufacture. It should be pointed out that the reduction of pigments in manufacture is greater for black tea than for green tea. Bokuchava (1935) studied the changes of chlorophyll in tea leaves of the Georgian variety during the vegetation period. The changes found were considerable (% dry matter): May 31 June 3 June 18 June 26 June27 July 4

0.60 0.80 0.61 0.76 0.67 0.70

July 8 July 15 July 19 August 6 August 14

0.72 0.86 0.77 0.70 0.76

The tea plant contains less chlorophyll in spring than in summer. As the plant grows, the chlorophyll content in leaves increases, with crude old leaves being far richer in the substance than younger ones. Bokuchava (1955) found out that tea-leaf processing decreased chlorophyll content: the manufactured tea contains about 20 to 25 70 of the initial chlorophyll amount. It should be stressed that chlorophyll lowers the quality of manufactured black tea, imparting a green color, a grassy taste, and other negative properties. Recent investigations have shed new light on the role played by carotenoids in tea manufacture. Tirimanna and Wickremasinghe (1965) found in the fresh tea leaf 14 carotenoids: 9 carotenes and monooxyxanthophylls, 2 polyoxyxanthophylls, and 3 epoxides. The amount of carotenes was reported to increase during withering and to decrease during rolling, fermentation, and firing. Oxidation of carotenoids was reported to yield volatile substances (unsaturated aldehydes and ketones) which are responsible for the tea aroma. Nikolaishvili and Adeishvili (1966) made a detailed study of the pigments in tea leaf during processing. The data they furnished are presented in Table IX. The data given show that the content of chlorophylls a and b noticeably decreases during tea-leaf processing. Green pigments undergo most intensive destruction at the stages of withering, rolling, and firing. The amount of pheophytin a and b increases to a maximum during rolling, fermentation, and firing. It should be noted that the amount of pheophytin in manufactured tea makes up half of the destroyed chlorophyll. The authors conclude that chlorophyll is of importance for the biochemical processes occurring in tea manufacture. At the withering stage the amount of carotene and neoxanthin decreases whereas that of lutein and violaxanthin increases. During

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243

TABLE IX CHANGES OF TEA LEAFPIGMENTS DURING BLACK-TEAMANUFACTURE (mglkg DRYWEIGHT) Pigments Chlorophyll a Chlorophyll b Green pigments in total Pheophytin a + b Carotene Lutein Violaxanthin Neoxanthin Yellow pigments in total

Green-tea leaf

Withered leaf

Rolled leaf

Fermented leaf

Dried tea

2.57 1.47 4.04

2.265 1.19 3.46

2.15 1.17 3.32

1.85 0.95 2.8

1.0 0.76 1.76

0.22 0.183 0.0645 0.263 0.731

0.176 0.216 0.085 0.19 0.667

0.278 0.165 0.207 0.102 0.179 0.653

0.735 0.174 0.193 0.075 0.125 0.567

1.605 0.08 0.097 0.023 0.12 0.320

fermentation the content of carotene rises insignificantly, and that of the other pigments diminishes. Firing is accompanied by a noticeable reduction of yellow pigments. It seems likely that the yellow pigments take an active part in biochemical processes developing in the tea leaf during processing.

6. Organic Acids Organic acids play an important role in metabolic processes in living organisms. One of the primary functions of organic acids is participation in oxidoreductive processes involved in respiration. Organic acids are the stock material for the synthesis of carbohydrates, amino acids, and fats. For a long time organic acids of the tea plant and manufactured tea remained unknown. Bokuchava (1936) found malic acid in the fresh and fermented tea leaf. Later Khocholava (1940)and Kharebava (1946) detected oxalic, citric, malic, and succinic acids. Those workers showed that the content of succinic and malic acids increased during withering. Other investigators also reported citric, malic, oxalic, and succinic acids as well as some acids of phenolic origin in the tea plant (Tsujimura, 1963; Roberts and Wood, 1951a,b; Cartwright et d., 1955; Cartwright and Roberts, 1955; Sakato and Matumura, 1955; Sakato et al., 1955; Roberts and Russell, 1957). Research workers at the Tocklai Experiment Station (1958) reported that, of the ten organic acids involved in the Krebs tricarboxylic acid

244

MIKIIAILA. BOKUCHAVA AND NINA I. SKOBELEVA

cycle, only pyruvic, isocitric, and oxalsuccinic acids were not found in the tea plant (Stahl, 1962). A detailed study of the organic acid composition and changes in the ontogenesis of the tea plant was performed by Soboleva (1961, 1962, 1966). She also investigated the possibility of direct interaction occurring between organic acids and the polyphenoloxidase system. The basic organic acids of tea seeds were found to be citric, malic, oxalic, and pyruvic acids. The growth of seeds and development of seedlings are accompanied by an increase of these acids and the formation of succinic, oxalacetic, malonic, a-ketoglutaric, gallic, and quinic acids, and an acid similar in properties to phenylpyruvic acid. It has been suggested that the formation of malonic, gallic, quinic acids and of phenylpyruvic acid is associated with an active synthesis of catechins in the tea plant since these acids are known to be precursors of phenolic substances. It has also been demonstrated that the polyphenoloxidase system is capable of oxidizing citric and malic acids. This is indicative of the significant role of the organic acids in tea-plant respiration and tea manufacturing since quinone derivatives of catechins can be involved in hydrogen transfer from citric and malic acids to air oxygen, forming a single oxidoreductive system. Sanderson and Selvendran ( 1965) studied nonvolatile organic acids of the tea plant by silica-gel column fractionation.

7 . The Resinous Substances The odor or aroma of tea-one of its most important characteristics depends on essential oils and resins. These latter are formed in resin ducts, specific organs of the tea plant. Chemically, resins are very close to essential oils and terpenes, making up a mixture of different groups of organic compounds. Resins can be subdivided into several groups: (1) resin acids; (2) resin alcohols (resenols and phenols showing no tannin properties); (3) resin phenols showing certain tannin properties; and (4)alkaliinsoluble resenes. The tea aroma is contributed to by some of these compounds whose odor increases under the influence of high temperature. It seems probable that the aroma that develops when tea is brewed is stronger and more pleasant than the odor of dry tea because of the hightemperature effect on its essential oils and resins (Bokuchava, 1958). The content of resins in tea is quite large when compared with that of essential oils. Harler (1933) found 3% of resinous substances in the green-tea leaf and manufactured tea.

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245

Timoshenko (1936) performed original investigations with respect to the changes of resins in tea plants of different varieties throughout a year. On the basis of the data collected, he concluded that resin accumulation in the tea plant was related to its frost resistance, and that resin concentration during the autumn-winter period could be used as a reliable indication of the frost resistance of tea shrubs. Kharebava (1951) obtained peculiar results in determinations of the total content of resinous substances and their specific groups. As seen in Table X, the tea grade is correlated directly with the yield of resenes but not with the total content of resinous substances and the concentration of resin acids. TABLE X RESINOUS SUBSTANCES IN TEA LEAVESOF THE FIRSTAND SECONDGRADES( % DRYMATTER) Tea-leaf Total content of Resenes grade resinous substances content

Resin acid content

7.65 8.09

1.48 1.54

First Second

3.40 2.85

Unfortunately, the role of resins in the aroma of the tea leaf has received little attention. It is hoped that extensive research on tea resinous substances with up-to-date techniques will greatly promote the biochemistry of tea and tea manufacturing.

8. Vitamins The physiological efficiency of tea depends to a great extent on the vitamins in the tea leaf and manufactured tea. Tea containing a good number of catechins is a valuable source of vitamin P. Tea also contains some other vitamins, and therefore has certain nutritional and physiological benefits. Tsujimura and Masataro (1924-1926) were the first to report a high yield of vitamin C in the tea leaf and green tea. Golyanitsky and Bryushkova (1936) and Anufriyev (1940) found that the fresh tea leaf of the Georgian variety contained 3 to 5 times the vitamin C in lemon or orange juices. With regard to vitamin C content, the tea leaf is inferior only to the berries of some varieties of dog rose. Leaf processing, however, results in high losses of ascorbic acid. For instance, an appreciable portion of vitamin C is lost in fermentation and firing of black tea; thus, manufactured black tea has but small amounts of vitamin C - from 164 to 580-640 IU per kg of dry matter.

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

The manufacture of green tea involves lower losses of ascorbic acid because there is no fermentation. Green and yellow teas contain ten times the vitamin C of black teas (Bokuchava, 1958). According to Golyanitsky and Bryushkova (1936), vitamin C can be detected in different parts of the Georgian tea plant in the following concentrations (in mg/kg dry matter): Bud 1st leaf 2nd leaf

7.03 9.99 10.44

3rd leaf Coarse leaf

7.88 3.83

It should be noted that ascorbic acid is of biochemical significance in tea manufacture. Bokuchava et al. (1951)showed its important role in oxidoreductive processes occurring in the presence of the polyphenol oxidase catechin system. In model experiments Popov (1966b) demonstrated the role of ascorbic acid in oxidative deamination of amino acids, resulting in the formation of aldehydes responsible for tea aroma. Many other vitamins have also been discovered in the tea plant, including vitamin K (Umikov, 1947) and the vitamin B complexvitamin B, (thiamine), vitamin BB (riboflavin), nicotinic acid, and pantothenic acid (Egorov, 1950). Below are values of the vitamin B complex in the tea plant obtained by Egorov (in mg/kg dry tea):

+

Vitamin B, (thiamine) Vitamin B, (riboflavin) Vitamin PP (nicotinic acid) Pantothenic acid

0.3- 10 6-11 54-152 14-40

Wada and Sakurai (1952) found thiamine and riboflavin in green tea and Miyzawa (1953) detected folic acid. Indian researchers reported the vitamin B complex in black tea (Tea Board, 1957). They also elucidated the effects on vitamin B content of geographic location of tea plantations, seasonal variation, grades of tea, and stages of manufacturing (Tea Board, 1959a,b). As mentioned above, Kursanov et al. (1950) showed that tea catechins have a high vitamin P activity. Ulyanova (1966) demonstrated that this kind of activity can be attributed also to tea flavonols. It should be mentioned that rutin has been isolated from tea leaves (Nakabayashi, 1953; Ulyanova, 1966).

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The data presented show convincingly that tea is rich in various vitamins and has significant physiological benefits as a nutritional product. 9. The Mineral Substances

The important role played by mineral substances in plant life is widely recognized. Minerals are responsible for changes in the state of colloids and directly affect the cell metabolism. In many cases they function as catalysts of biochemical reactions. Minerals are also involved in changes in protoplasm turgor and permeability. They often become the centers of electrical and radioactive phenomena in the plant organism. All this can be referred to both micro- and macroelements accumulated in the plant. In the tea leaf and manufactured tea, unfortunately, study of the role of minerals has been scant. It is obvious that this matter deserves greater effort. Mineral elements make up 4-5% of dry matter of the fresh tea leaf and 5-6% of dry matter of manufactured tea (Vorontsov, 1946). The major constituent in the ash of young tea leaves is potassium, which reaches 50% of the total ash constituents (Zoller and Libich, 1871; Romburg and Lohmann, 1898; Nanninga, 1901). Potassium is necessary for plant metabolism in general, and for the formation of carbohydrates and proteins in particular. According to numerous data furnished by Godziashvili (1949), potassium deficiency can kill the tea plant. Tea-plant ash also has noticeable amounts of phosphorus, calcium, magnesium, and sulfur. Of particular importance are phosphorus and sulfur, which are necessary constituents of proteins, nucleoproteins, and other physiologically important compounds. In the tea leaf, phosphorus is involved in both organic and inorganic compounds. In the study of phosphorus metabolism in the Georgian tea plant, Nizheradze (1946) found the following organic compounds in the leaf: phytin, hexosomonophosphate, and hexosodiphosphate. Inorganic phosphorus could be detected in the form of orthophosphoric acid derivatives. Phosphoric acid is very important not only for vital processes in the tea leaf but also for tea processing. For instance, one of the factors causing a change of the medium reaction (pH) during tea manufacture is the release of phosphoric acid from the bound state. Among the ash elements, the third place (after potassium and phosphorus) belongs to magnesium, which forms a part of the chlorophyll molecule.

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

The content of iron in the tea plant is very limited though it plays a significant role in its vital activity: it is a constituent of certain physiologically important organic compounds and is a part of the enzyme peroxidase which is involved in oxidoreductive processes. An appreciable part in these processes is played also by manganese, which makes up 1 to 4 % of the total ash. In 1954 Japanese investigators (Matuura et al.) found fluor in the tea leaf. The occurrence of this element was confirmed by Zimmerman et al. (1957). Many workers attempted to correlate tea quality with total content of mineral elements in the leaf (Zoller and Libich, 1871; Kellner e t al., 1887; Carpenter and Harler, 1923; Frost and Elovsky, 1926). The mineral constituents of tea are usually subdivided into two groups -soluble and insoluble. Most important to the consumer are the soluble ones, which enter the liquor and, consequently, the human body. Some workers believe that higher grades of tea contain less total ash and more soluble elements than lower-grade teas. According to Vorontsov (1946), the average ash content of Georgian tea is (in percent of dry matter): Highest grade Second grade

4.97 5.33

Third grade Fourth grade

5.68 5.98

C. AROMATIC SUBSTANCES One of the most important characters of tea quality is aroma. Although the quality of different tea grades depends first on taste and aroma, study has been insufficient on the mechanisms of aroma formation and the chemical nature of constituents that contribute to tea aroma. Further, the data obtained are often at variance. Stahl (1962) wrote that the majority of scientists studied the substances responsible for the taste of tea and very few dealt with the aroma. This can be understood because experiments on aroma involve many technical difficulties, stemming mainly from the diverse composition of the aroma complex. The analytical difficulties are aggravated by negligible concentrations of the sum total (which makes up hundredths of percent) and the specific constituents of essential oils in tea. This naturally makes it very difficult to investigate tea aroma in detail. Nevertheless, the problem has long attracted the attention of many workers. Nanninga (1901) suggested that tea aroma has its source in glycoside, which, upon the manufacture of black tea, disintegrates into

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sugar and aromatic compounds under the influence of specific enzymes. The glycoside theory of aroma formation was held by Kozai (1890), who produced tea aroma by treating the glycoside with an enzyme which he did not specify. The theory was also shared by Katayama (1907). A different idea was advanced by Mann (1914), who pointed out that tea aroma, like tea pigments, developed from tannin oxidation by enzymes. This viewpoint was poorly supported experimentally, but it is to Mann’s credit that he was the first to become interested in changes in tea aroma during the black tea manufacture, and he revealed a relation between the oxidative process and aroma formation. Deuss (1915) put forward an original theory that was an attempt to correlate the glycoside theory with Mann’s standpoint. He supposed that tea aroma resulted from the decomposition of glycoside links of the tea tannin, with the production of simpler forms of tannins and carbohydrates which later transformed into odor-bearing esters. Unfortunately, this idea was not furthered. Similar to the ideas advanced by Deuss was a hypothesis voiced by Shaw (1934), who regarded the aroma formation of black tea as the decomposition of theotannins under the influence of hydrolytic enzymes. An interesting idea was put forward by Potapov in 1940. He believed that the essential oils of tea are of protein origin, synthesized upon direct participation of certain amino acids accumulated during tea-leaf withering. All the aforementioned theories had a similar disadvantage -they lacked experimental support. In 1936 Kursanov and Shubert conducted a detailed investigation of aroma formation at various stages of the manufacture of black tea. They introduced aqueous-distillate estimations of acid number, essential number, and saponification number, and evaluations of the volatile fraction. These methods encouraged a scientific approach to the determination of tea aroma. Vorontsov (1928, 1939, 1946) devoted many years to the study of tea aroma. From data in the literature and his own findings, he concluded that the black tea flavor included two odors: the odor of fresh leaf and the odor developed upon its processing. The chemical nature of the essential oil of tea also lacks clarity. One of the oldest definitions of the tea essential oil was given by Mulder (1838). He described the oil as a liquid of a lemon-yellow color which can readily solidify; the liquid is astringent and has a

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strong tealike odor. From analysis of numerous samples of manufactured teas, he found the essential-oil content to vary from 0.60 to 0.98%. These values are high as compared with the latest findings. Kolokolov (1906)also reported elevated values for tea essential oil. Groll (1897) indicated that the essential oil of black tea can be readily resinified in the air. Nevi11 (1928)reported that the tea essential oil is easily oxidized. The first attempt at detailed research on the chemical nature of the tea essential oil was made by Romburg and Lohmann (1898). They isolated 130 ml of essential oil from 2500 kg of freshly fermented old tea leaves (a yield of 0.006%).In the oil they detected methyl alcohol and about 0.33%of methyl salicylate. Gildemeister and H o b a n (1900) found an alcohol which was identified as p,y-hexenol. In their opinion, this compound bears the primary responsibility for tea aroma. Ampler information on the chemical composition of the essential oil of the fresh leaf and manufactured tea was furnished by Japanese scientists. “On the Aromatic Substances of the Green Tea Leaf,” by Takei and Sakato, was published in 1933. Those workers steam-distilled fresh tea leaves of the spring and summer crop, which yielded 0.014% and 0.007% of essential oil, respectively. From the oil they isolated large quantities of p,y-hexenol and hexylene aldehyde, which had a strong, fresh, grassy odor. They also found isobutyl and isovaleric aldehydes. Detected in the acid moiety were fatty acids (acetic, propionic, butyric, valeric, and caproic acids) and a higher acid (palmitic). Further studies of the essential oil of fresh tea leaves resulted in the isolation of methyl acetaldehyde, parahexyl alcohol, benzyl alcohol, phenylethyl alcohol, phenol, and cresol. Takei et al. (1935-1938) found considerable amounts of linalool and citronellol in the high-boiling neutral moiety of the essential oil of fresh tea leaves. They showed that during tea-leaf manufacture an appreciable portion of lower acids, P,y-hexenol, and hexylene aldehyde was lost, whereas benzaldehyde, butyric, isobutyric, and isovaleric aldehydes increased. On the basis of studies of the essential oil of black tea containing no hexylene aldehyde, they concluded that the aldehyde was entirely utilized in the formation of new odor-bearing substances upon tea manufacture. They stated that aromatic substances of manufactured black tea developed during the withering and fermentation processes. The essential-oil composition of fermented leaf and manufactured black tea was investigated by Jammamoto and co-workers (Jammamot0 and Muraoka, 1932; Jammamoto and Kato, 1935; Jammamoto

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and Ito, 1938). By steam distillation at 100°C they isolated 13 g of essential oil from 400 kg of fermented tea leaf (amounting to 0.03%). They pointed out that the oil isolated involved lower aldehydes whose odor developed during withering and fermentation, though they failed to identify the aldehydes because they were distilled within the ester boiling range. In 1937 they investigated the essential oil of black tea. From 1300 kg of manufactured black tea they isolated 250.4 g of essential oil (a yield of 0.02%). The oil was separated into groups of compounds illustrated in Table XI. The high content of the aldehyde fraction should be noted. Japanese scientists made a great contribution to the understanding of tea essential-oil constituents. TABLE XI CONSTITUENTS OF ESSENTIAL OIL OF TEA Classes of compounds Acids Phenols Bases Aldehydes Neutral essential oil

Total oil (%)

Yield (g)

32.0 8.3 0.5 36.4 173.2

12.78 3.31 0.20 14.54 69.17

Later, Georgian specialists (Gegele et al., 1941) studied the essential oil of Georgian tea. From 900 kg of fermented leaves they isolated 65 g of essential oil. The classes of compounds detected are listed in Table XII. TABLE XI1 CONSTITUENTS OF ESSENTIAL OIL OF TEA Classes of compounds Carboxylic acids Phenols Bases Aldehydes Neutral essential oil Losses

Content ( %)

3.20 2.52 0.49 0.35 73.2 20.24

Individual components of the essential oil were identified as quinoline (in the class of bases), salycilic acid (in the class of phenols), and palmitic, propionic, butyric, and isovaleric acids (in the class of acids).

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Goguadze (1940) tried to synthesize new compounds similar to tea odor-bearing substances. He obtained new derivatives of P-phenylethyl alcohol. Unfortunately, this line of research was not continued. Investigations by Japanese and Soviet workers have made it quite clear that essential oil is contained in both green leaf and manufactured tea, with tea-leaf processing resulting in the formation of new aromatic substances responsible for the specific odor of manufactured tea. The 1950’s were marked by publications giving impetus to new approaches to the problem of tea aroma. These papers ascribed an important role in aroma formation to aldehydes resulting from the reaction of amino acids with sugars, on the one hand, and with tannin, on the other. In model experiments Bokuchava and Popov (1954) observed a release of volatile compounds bearing different odors depending on the amino acid used. Their experiments were based on the oxidation of amino acids in the presence of tea tannin and polyphenol oxidase upon an addition of amino acids to the leaves. They also stated that broken leaves placed in vacuum produced no aroma. This is another proof of the involvement of oxidative processes in the production of tea aroma. In further studies those workers showed that the amino acid-tannin reaction at high temperature also yielded volatile substances of different odors. Addition of dimedone, an aldehyde-binding reagent, to the reaction mixture arrested the aroma formation. This is indicative of the aldehyde nature of the aroma constituents. Later, Popov (1956) and Skobeleva et al. (1958) demonstrated the production of aldehydes, C 0 2and NH3. Nakabayashi (1956b,c, 1958a,b,c) confirmed an important role of carbonyl substances in the formation of black-tea aroma. He showed that these substances changed qualitatively and quantitatively upon tea manufacture. Treatment of the distillates obtained with sodium bisulfite acid solution resulted in disappearance of the black-tea aroma, while subsequent alkali treatment restored it. Nakabayashi concluded that carbonyl group compounds are of significance in aroma production. The aroma is not directly dependent on the amount of carbonyl compounds, however, though related to their composition. Nakabayashi reported benzaldehyde, citral, perilla, and cinnamic aldehydes in black tea. Despite extensive research he could not give final determinations of the qualitative and quantitative composition of carbonyl compounds in black teas. Bokuchava and Skobeleva (1957) showed that an important role in

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the formation of black-tea aroma is played not only by aliphatic aldehydes but also by aromatic aldehydes. They identified vanillin, p hydroxybenzaldehyde, and cinnamic aldehyde in manufactured teas. These aldehydes underwent certain changes during tea manufacture. Mokhnachev and Kamenshchikova (1966) studied carbonyl compounds with paper chromatography and gas chromatography. They found at least 68 carbonyl compounds in manufactured black tea, plus others that they failed to identify. By paper chromatography, Gogiya and Motsonelidze (1966) detected acetaldehyde, acetone, isobutyric, isovaleric, and caproic aldehydes in the carbonyl compounds of tea-brew vapors and extract. They believe that the compounds mentioned take a direct part in production of the aroma peculiar to tea. By gas chromatography, Roberts (1961) found acetaldehyde and isobutanol in black tea. This technique facilitated greater insight into the compounds contributing to tea aroma. Graham et al. (1966) applied the method to study of the volatile constituents of green and black teas. They separated the aromatic compounds into 34 constituents, and identified 18: acrolein, n-butyric aldehyde, ethanol, nbutanol, isobutanol, hexanal, pentonal, 2-hexanol, 3 - hexen - 1-01, benzaldehyde, linalool, terpeniol, methyl salicylate, benzyl alcohol, P-phenyl ethanol, isobutyric aldehyde, geraniol, and acetophenone. The aromatic compounds of green teas were separated into 26 constituents, of which 5 were identified: 2-butanol, 2-hexenal, pentanol, 3-hexen-1-01, and benzaldehyde. Kozhin et aZ. (1966) attempted a comparative evaluation of different teas by gas chromatography. They used steam-distillation in a modified Klevenger apparatus and gas-extraction of brewed tea to isolate the total volatile constituents. Their experiments were carried out on samples of manufactured black tea of varied origin (Georgian, highest grade; Indian, highest grade; Chinese, second grade), on samples of green tea (Georgian variety), and on fixed green tea leaf (“Kimyn” variety). Comparison of the chromatograms led to the conclusion that samples of differing origin and variety did not differ qualitatively. The quantitative ratios of the volatile components, however, could be used for evaluating the tea samples. It is obvious, as the authors themselves pointed out, that these studies are only the first step in penetrating the chemistry of the volatile constituents responsible for the aroma of different teas. Yamanishi et aZ. (1966) applied gas chromatography to studying the changes in aromatic substances at different stages of tea manu-

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facture. They identified and estimated individual constituents of the tea essential oil at all stages, beginning with the green leaf and ending with manufactured black tea. They isolated essential oil from the green tea leaf and from withered, fermented, and manufactured tea, and fractionated the oil into 4 fractions: a carbonyl-free neutral fraction, a carboxylic fraction which was converted into methyl esters, a carbonyl fraction, and a phenolic fraction. Each fraction at various manufacturing stages was investigated by gas chromatography. The total alcohols decreased during the withering process, increased in the fermentation process, and decreased during the firing process (except that geraniol, benzyl alcohol, and phenyl ethyl alcohol increased during firing). Carbonyl fraction. Nine of the 30 peaks in the carbonyl fraction were identified. The total amount of carbonyl compounds increased during withering and fermentation, but decreased in firing. Isovaleric and phenylacetic aldehydes and trans-2-octana1, however, increased during firing. Carboxylic fraction (in the fomn of methyl esters). The total carboxylic fraction increased slightly during withering, increased significantly in fermentation, and decreased somewhat during firing. As to individual acids, acetic and propionic acids decreased sharply during withering, and increased noticeably in firing. trans-2-Hexenoic, salicylic, and n-caproic acids increased noticeably during withering and fermentation; in firing, the first two decreased and the last increased. Phenolic fraction. The total phenolic fraction was rather small. Mainly it contained methyl salicylate, which decreased appreciably in manufactured black tea. That occurred also with salicylic acid. The results indicate that different fractions of aromatic substances are subjected to considerable changes during tea manufacture. Most fractions increase in withering and fermentation, and decrease in firing. Some aromatic substances increase in firing, however: geraniol, benzyl alcohol, phenyl ethyl alcohol, phenyl acetic aldehyde, isovaleric aldehyde, trans-2-octana1, acetic, propionic, and n-caproic acids. Thus, the formation of the black-tea aroma proceeds during firing. Changes in the formation of essential oil during black-tea manufacture are being studied by gas chromatography in the Institute of Tea Industry of the Georgian SSR (Kharebava et al., 1967). Tirimanna and Wickremasinghe (1965) revealed that oxidation of carotenoids is one of the sources contributing to the formation of the

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volatile compounds (unsaturated aldehydes and ketones) which participate in the production of tea aroma. Thus, recent investigations have considerably extended our knowledge of the substances involved in formation of the essential oils and processes responsible for tea aroma.

D. ENZYMES

All basic transformations of substances in living organisms occur with the aid of enzymes, which gives enzymes exceptional significance in the vital activity of plants and animals. Modern concepts on enzymes and their role in living things are well presented by Kretovich (1967). It should be emphasized that enzymes play a very important part in the processing of raw materials of plant and animal origin. Enzymes of grain, flour, sugar beet, grape, malt, tobacco leaf, and tea shoot cause deep biochemical transformations, promoting the formation of specific taste and aroma substances that are peculiar to particular products. The conversion of fresh tea leaf into manufactured tea of different types and grades is based on the effect of enzymes in the raw material. Of particular importance to tea manufacture are oxidative enzymes. An application of enzymic oxidative processes in different ways results in teas of different types and grades. As shown by Glazunov (1935), Kharebava (1946), Nizheradze (1946), Kursanov and Brovchenko (1950), Gogiya (1950b), and others, the tea plant contains, besides oxidative enzymes, invertase, amylase, p-glucosidase, oxynitrilase, protease, and pectinase. These enzymes contribute to the conversion of appropriate substrates not only in the living tea leaf but also during processing. The exceptior 11 role played by oxidative enzymes in tea manufacture requires discussion of them in more detail. Although the physiological role of oxidative enzymes as catalysts of oxidoreductive reactions in the tea plant is as important as the technological one, their study has been concerned mainly with tea processing. The first studies on tea oxidative enzymes were carried out at the beginning of this century. Bamber and Wright (1902) put forward an idea that the conversion of tea leaf into black tea occurs under the influence of enzymes, not microorganisms, as thought previously. Those later investigators isolated an enzyme of the oxidase type from the tea leaf. Aso (1901) confirmed the idea of Bamber on the presence of oxidase

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in the tea leaf, and showed that the red and brown pigments of fermenting tea developed under the influence of oxidase. Newton (1901) classified the tea-leaf enzyme as an oxidase and termed it thease. Mann (1902, 1903, 1904) carried out research extensive for his time on oxidative enzymes of the tea leaf. Prostoserdov (1917)termed thease the enzyme causing fermentation of the Georgian tea leaf. Although those early studies of tea oxidative enzymes were hypothetical to a certain extent, they gave impetus to more extensive research in the field. There are numerous papers devoted to oxidative enzymes of the tea plant. They have established that polyphenol oxidase and peroxidase are the primary oxidative enzymes of the tea plant (Khocholava, 1940; Manskaya, 1935b; Bokuchava, 1935; Kharebava, 1946; Roberts,

1939,1940). Almost all investigators of tea-leaf peroxidase also studied oxidase. Nevertheless, their results remained rather poor for a long time. All workers unanimously reported an active peroxidase in the tea plant, but they were at variance with respect to oxidase. Bokuchava and Popov (1940) and Sreerangachar (1941, 1943) clarified the occurrence of an active polyphenol oxidase in fresh tea leaf. They demonstrated that polyphenol oxidase was present there mainly in the adsorbed state. It was later shown that the proportion of soluble and insoluble enzymes in the tea leaf varied in relation to the physiological state of the tea plant (Bokuchava and Popov, 1948; Oparin and Shubert, 1950). Oparin and Shubert (1950)made a detailed study of changes in the polyphenol oxidase state in the cell with regard to the age of the tea leaf. They failed to detect any amount of soluble polyphenol oxidase in an old leaf, and indicated that it made up about 7 % and 16.5% of the total activity in a mature and young leaf, respectively. From study of polyphenol oxidase and peroxidase, Bokuchava and Soboleva (1966)showed that the activity of the enzymes was higher in roots than in aerial organs. It should be pointed out that polyphenol oxidase occurs in the bound state in aerial parts of the plant whereas half of it in roots is in the soluble form. Peroxidase, however, exists in both states in both the aerial parts and roots of the tea plants, with the soluble form amounting to 35 % of the total enzyme activity. The localization of polyphenol oxidase in cellular structures of the tea plant has long been an issue. This was the subject of investigations performed by Li and Bonner (1947) and Oparin and Shubert

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(1950). Those workers ascertained that the enzyme was located in chloroplasts only. By the differential centrifugation method, Bokuchava and Soboleva (1966) established that polyphenol oxidase and peroxidase are located in the chloroplasts and mitochondria of the tea leaf, with higher activity in mitochondria. Japanese workers (Takeo, 1965; Takeo and Uritani, 1966) reported that polyphenol oxidase can be found in the cytoplasm, chloroplasts, and mitochondria of the cell of the tea plant, and indicated polyphenol oxidase localization to be dynamic and dependent on many factors. Wickremasinghe et al. (1967) demonstrated that the enzyme was localized in epidermal cells. This may explain the protective effect of the polyphenol oxidase system against pathogenic agents. Studying the substrate specificity of the tea polyphenol oxidase, Bokuchava (1958)concluded that, besides tea polyphenols and tannin, the enzyme oxidizes pyrocatechin, pyrogallol, and gallic acid, i.e., the compounds which are constituents of the tea tannin, being mixed and o-polyphenols. He also indicated that tea polyphenol oxidase cannot oxidize substrates belonging to mono- and p-phenols or tyrosine. Similar conclusions with respect to the polyphenol oxidase effect on hydroquinone and tyrosine were made by Roberts and Wood (1950). Takeo (1965) found that tea polyphenol oxidase first oxidizes (&)-catechin. Some new methods have been developed which help isolate and purify polyphenol oxidase from tea leaves. This is an important contribution to the study of tea enzymes since earlier investigators had to work with plant extracts, homogenates, and acetone preparations. Bendal and Gregory (1963) isolated and purified polyphenol oxidase from tea leaves by chromatography on diethylaminoethyl cellulose (DEAE) and carboxymethyl cellulose. In comparison with the acetone preparation, the polyphenol oxidase they obtained was purified by 2000 times. Using hydroxy apatite and amberlite, they subjected the fractions obtained to further purification, thus assuring a purification of 5000-fold over that of the acetone preparation. They isolated five active fractions of the enzyme and measured the molecular weight of the tea polyphenol oxidase at 144,000 k 16,000, and copper content at 0.32%, with each molecule containing 6-8 Cu atoms (Gregory and Bendal, 1966). Takeo and Uritani (1966) established that the soluble polyphenol

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oxidase was composed of three components, two representing basic proteins, and the third acidic protein. The components differed in substrate specificity. Roberts (1942) indicated the presence of cytochrome oxidase in the tea leaf. He attributed a very important role in tea fermentation to that enzyme because he believed that cytochrome oxidase formed hydrogen peroxide, to be later involved in peroxidase activity. Oparin and Shubert (1950), however, proved that the Georgian tea leaf contained no cytochrome oxidase. It is mentioned above that tea tannin transformations during plant vegetation and leaf processing are catalyzed by the oxidative enzymes polyphenol oxidase and peroxidase, but their functions in regard to tannin are different. Experiments by Bokuchava (1947) and Bokuchava et al. (1948) revealed that, under the influence of polyphenol oxidase, tannin undergoes oxidation involving consumption of large amounts of oxygen and the formation of colored products of tea infusion. Later, Roberts (1959, 1962) and Roberts and Smith (1961,1963) established that the color of the tea liquor depended mainly on the concentration of the products of tannin enzymic oxidation- theaflavins and thearubigens. The peroxidase effect resulted in the condensation of tannin to form leukocompounds. Changes in phloroglucinol concentration are suggestive of considerable variations of tannin induced by oxidative enzymes. According to Kursanov (1941) and Kursanov et al. (1947), the content of phloroglucinol decreases with tannin oxidation. Studying tannin condensation at the molecular level, Kursanov et a2. (1947) ascertained the condensation to take place mainly during fermentation. They found that the molecular weight of tannin of the unseparated preparation varied greatly in the fermentation process, the molecular weight of tannin from fermented tea (780-800) being twice that of raw tea catechins (358-414). These data allowed the conclusion that, during tea-leaf fermentation, catechins and catechin gallates can be condensed in pairs, forming double molecules (see Fig. 3). This was also suggested by Oshima (1936),who believed that amorphous tannin of the tea leaf consisted of paired catechin molecules. Thus, it has been established that the condensation of catechins is a result of oxidation, though the mechanism remains obscure. Kursanov et al. (1947) assumed that the elimination of the phloroglucinol reaction of tea tannin that results from the molecular weight increase indicated that the catechin condensation involved a phloro-

CHEMISTRY & BIOCHEMISTRY OF TEA &TEA MANUFACTURE

--n

I

259

0

0

on

FIG. 3. Scheme of the condensation of catechins.

glucinol ring of at least one of the two attached molecules. It seems more likely, however, that the attachment occurs not at the phenolic group of the phloroglucinol residue but at the hydrogen, located between two OH-groups in the metaposition (Fig. 3). The attachment of catechins becomes possible through their development of o-quinone or peroxide groups readily appearing under the influence of enzymic oxidation:

I

-C=O

I

-C=O

or

c-0 II I c-0

The experiments carried out suggested that the polyphenol oxidase and peroxidase of tea leaf actively affect tannins, causing significant changes involving oxidation, condensation, and the formation of insoluble tannins. Under natural conditions these enzymes can act on tannins of the tea plant simultaneously or one after another, depending on the plant requirements. The role of enzymes in tea manufacturing is discussed in Section 111, devoted to the fermentation mechanism.

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111. SIGNIFICANCE OF BIOCHEMISTRY A. BASIC PRINCIPLES OF MANUFACTUREOF DIFFERENTTEAS

Tea manufacture is based on biochemical processes that are of key importance. Oxidative processes make it possible for various teas of different quality to be obtained from the same raw tea. If processing procedures of the freshly plucked tea leaf assure proper oxidation processes through withering, rolling, fermentation, and firing, black tea results, with the widely known taste, aroma, and color. If, however, oxidation processes are stopped by high temperature at early stages of tea manufacture, green tea results, with a specific astringent taste and golden-colored infusion. The control of oxidative processes allows the production of red tea, oolong, and yellow tea, which occupy intermediate positions between black and green teas. Oxidative processes also underlie the manufacture of green brick tea, which is usually produced from old coarse tea leaves, and molding material. Thus, biochemistry plays an extremely significant part in tea manufacture. The basic principle underlying tea manufacture is the chemical transformations responsible for the formation of properties peculiar to a particular tea type - with its own taste, color, and aroma. The main task is to retain all the substances present in the original fresh leaves that have a positive effect on tea quality, and to destroy entirely those that have a negative effect. The quality of the final tea depends on the raw-tea quality and processing procedures. It is obvious that good tea cannot be manufactured from poor original leaves, whose quality, in turn, depends on many factors, the most important being biological, geographical, and agrotechnical. The biological factor, i.e., the variety of the tea plant, affects raw-tea quality markedly. It is widely recognized that the South-Assam variety of large-leaved tea plants can synthesize the greatest amounts of the most valuable compounds -tannins, catechins, and extractives. The significance of the geographic factor can be exemplified by Darjeeling teas. When cultivated in that area, North-China varieties of small-leaved tea plants yield tea of exclusive aroma. Cultivated in other areas of India, however, plants of the same variety do not yield such aromatic tea. Experiments at the USSR Research Institute of Tea and Subtropical Cultures have shown that specific agrotechnical measures for optimum cultivation not only increase tea yields but also improve quality.

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Nevertheless, the best raw material will yield only a mediocre product if manufacture is inferior. The necessary qualities of manufactured tea are finally developed during tea manufacture through biochemical transformations of the tea-leaf constituents induced by specific processing procedures.

B. BIOCHEMISTRYAND TECHNOLOGY OF BLACK TEA In all tea-producing countries, black tea is manufactured from freshly plucked tea leaves by withering, rolling, fermentation, and firing. This technique can be considered classic.

1. The Withering Process The withering process is the first step in black-tea manufacture. The physiological and biochemical processes in the living tissue before plucking continue afterward but become different. The withering changes the pattern and rate of these processes (metabolism, respiration, etc.), altering the chemical composition and physical properties of the tea leaf to prepare it for further processing procedures. The necessary biochemical changes develop at the highest rate under water-deficiency conditions, hence the withering. The loss of moisture in withering is accompanied by considerable biochemical changes of the tannin complex, amino acid composition, essential oils, carbohydrates, enzymic activity, etc. The physical and chemical changes involved are two aspects of the same process in the living leaf. The physical changes in the tea leaf involve reduced elasticity, turgor, size, weight, and volume. The rate of water losses varies with stage of withering. At first the cell juice loses water, which takes place rather rapidly, and then plasma colloids begin to lose water, which proceeds more slowly. The third period is characterized by a rapid water evaporation resulting from changes of the plasma colloids, which lose their hydrophilic nature (Manskaya, 1935a). Following this, the cell protoplasm irreversibly loses its hydrophilic properties and the leaf cannot restore its initial turgor (Oparin, 1935). This condition is the aim of the withering process. The physical changes thus induced are so obvious and profound that many experts have regarded the withering stage as mainly a physical process, attaching no importance to chemical changes. This approach cannot be scientifically justified, however, since the withering step has a very significant role in subsequent stages -rolling and fermentation - and the manufacture of high-quality teas. If the withering stage is omitted,

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the final product will be of lower quality than usual. Inadequate withering also results in inferior quality. Rational implementation of technological processes demands that the tea leaf should contain a definite amount of moisture. The freshly plucked tea leaf contains about 75-80% water, and the withered leaf usually 62-64 %. Certain tea-producing areas apply stronger (about 58%)or weaker (66-67%)withering. It is very important that all parts of the flush undergo uniform withering. The concentration of cell juice increases in the withered leaf, intensifying the reaction of its constituents. Thus, the withering process involves both physical and chemical changes. The biochemical changes resulting contribute to the formation of taste and aroma during fermentation. Proper withering also affects the aging of manufactured tea and the maintenance of its qualities. There are two methods of withering: natural and artificial. a. Natural Withering. This is carried out under natural air and temperature conditions in lofts at the tea factory. Tea leaves are spread out on racks located in tiers spaced 10-15 cm from each other. Leaves are distributed in a thin layer of 0.5 kg/m2. Depending on weather conditions and leaf quality, the tea leaf is usually withered for 12 to 18 hours. The process becomes longer if the weather is wet. In favorable atmospheric conditions the natural withering yields uniformly withered leaves that assure the manufacture of highquality tea. A significant disadvantage of natural withering is the complete dependence on weather conditions. That is why scientists and engineers have long studied the factors which could help eliminate the disadvantages of natural withering. Air and temperature can be controlled in special facilities to provide artificial withering of the tea leaf.

b. Artijkial Withering. Intensive research has been performed in the USSR to establish a theoretical basis for withering and to develop special installations for its implementation. Experiments at the Bakh Institute of Biochemistry indicated that withering time can be shortened to 6-8 hours. This period proved sufficient to allow physical and chemical changes in the tea leaf that produced a manufactured tea of high quality. In addition to biochemical investigations, much has been done to design and build special facilities for artificial withering. Outstanding among many machines built is the Mardaleishvili withering assembly, used at all tea factories of the Soviet Union.

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2. Rolling Rolling is the second important step in tea manufacture. This step changes the pattern of biochemical processes in the living tea leaf. Rolling provides the conditions necessary for rapid development of oxidative processes. This is done by rupture of tea-leaf tissues and damage of leaf cells by special rolling machines -rollers. The cells of the withered tea leaf are crushed and their components mixed up. The resultant mass is characterized by the destruction of biochemical processes, including respiration, which in the living organism are strictly coordinated. They are replaced by fermentation, which is the basic process in black tea manufacture. From the very initiation of the rolling stage, enzymic oxidative processes begin to lead to considerable transformations of all the constituents of the tea leaf. These chemical changes are manifested in a gradual change of leaf color from dark green to coppery red and brown, and the development of a specific pleasant odor. These changes are essentially chemical results of the rolling process. Thus, the physical process of rolling results in significant chemical changes of tea-leaf constituents. The main purpose of the rolling step is complete damage of tea-leaf tissues. This is vital to the best possible development of the raw tea for the manufacture of high-quality tea. Damage of any type is not acceptable, however. Tea-leaf tissues can be readily destroyed by freezing and thawing, for example, but this method involves undesirable chemical changes of tea proteins, giving the tea an unpleasant fishlike taste and odor. The cells of tea leaves can be thoroughly damaged by rollers, but the exposure and grinding of leaf fibers involved imparts a specific coarse taste and aroma to the tea liquor. This method of leaf bruising is also accompanied b y a very active tannin oxidation, with resultant high tannin losses. Tea leaves can be crushed by hot rolling, but this technique also causes a very rapid development of oxidative processes, promoting conversion of soluble substances into insoluble. Tea manufactured by this method contains only 6-7% soluble tannin, against 20-25% in the original fresh leaves. Thus, the method and conditions of rolling have an important place in the formation of tea constituents and quality. The operation conditions and the degree of fresh-leaf tenderness influence the number of rolls applied and their duration. Dajeeling and Assam tea factories use thrice-repeated rollings of

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30 min each. In Assam the so-called C.T.C. machine and a cutter based on the tobacco cutter are used for complete distortion and best utilization of fresh green leaves (Harler, 1963). In the USSR, rollers of single and double action, as well as differentiated rolling, are employed. At present great attention is paid to the development of continuous methods for the rolling process (Pomazanov et al., 1966a,b; Bokuchava, 1966). In use today are a line of the cascade type, designed at the USSR Research Institute of Tea Industry and Central Design Office, as well as a horizontal line designed at the Dagomys and Adler tea factories. C. FIRSTAND SECONDSTAGESOF FERMENTATION The most important stage in black-tea manufacture is fermentation. The chemical changes developed in the withering stage proceed at a most rapid rate in fermentation. These changes result in profound qualitative and quantitative changes in tea-leaf constituents, contributing to the formation of new taste and aroma products responsible for the character of manufactured black tea. The fermentation process starts with rolling but does not end there. Rolling is, in essence, the first stage of fermentation, usually requiring about 2 or 3 hours. The second stage of fermentation usually lasts for 2 to 5 hours, according to the classic procedure of tea manufacturing adopted in the USSR. Nevertheless, the fermentation time can be increased or decreased as dictated by fresh-leaf quality, seasonal variation, processing conditions, etc. In India the fermentation process takes approximately 3.5 to 4 hours by the classic procedure or 1 to 2 hours by the procedure involving the C.T.C. process or other cutting machines (Harler, 1963). Fermentation is performed as an independent technological process in a special room where boxes containing rolled leaves are kept at room temperature and relative humidity of 96-98%, and under constant oxygen supply; sometimes fermentation can be accomplished in the roller shop. During fermentation, rolled leaves lose their green color and grassy odor, acquiring a coppery-red shade and the aroma of fermented tea. The process also involves an accumulation of coppery-red and brown pigments (products of tannin oxidation) which are responsible for the specific color of the tea infusion. In addition, oxidation processes lead to disappearance of the bitter taste of unoxidized tannin and the development of a pleasant, less astringent taste. Thus, fermentation is the stage of formation of the taste and aroma characters specific

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for manufactured black tea, distinguishing it from other teas produced from similar fresh leaves. Fermentation proceeds at the expense of oxidative enzymes inherent in the tea leaf, mainly of polyphenol oxidase, consuming air oxygen. This distinguishes tea manufacture &om other food processes in which exogenous fermentation-inducing agents must be added to the raw material (breadmaking, brewing, champagne production, etc.). In addition to the positive processes occurring during fermentation, some negative ones also take place. The oxidation of catechins and tannins is accompanied by their protein precipitation and change of soluble tannins into insoluble compounds. The precipitation process increases as long as fermentation continues and the temperature of the fermenting leaf rises. This is accounted for by the fact that the enzyme, and other proteins of the tea leaf, form an insoluble compound with quinones present in the tea catechins and tannin. The process enhances with fermentation time because tannin oxidation results in its increased molecular weight and protein precipitability. Tannin oxidation and precipitation processes involved in fermentation lead to a twofold decrease of tannin and nearly complete disappearance of catechins in manufactured tea. This is obviously an undesirable phenomenon. Studies of enzymes, tannin, and other constituents of the tea leaf and of the effects of thermophysical processes make it possible to solve the problem in a new way. This is discussed in Section IV.

Firing The fermented tea leaf enters the firing room, where it undergoes The firing process, performed in special installafiring at 90°-95"C. tions, reduces tea moisture content to 3-4%. The main purpose of firing is to stop fermentation at the very moment when the amount of valuable substances accumulated in the tea leaf is highest. Firing arrests the activity of enzymes and, consequently, of biochemical processes. Hot air affects the fermented leaf, which, through evaporation, loses its coppery-red or brown color and transforms into black tea. During firing, the tea undergoes physical and chemical changes that impart to the manufactured dry tea specific taste, color, and odor. The tea quality depends to a large extent on the mode of firing, particularly on temperature, rate of air flow, depth of the tea layer, and exposure time. As is well known, firing is accompanied by a loss of tea aroma. It has been established that 75-80% of the essential oils formed in

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

fermentation volatilize during firing (Kursanov and Shubert, 1936; Vorontsov, 1939). Thus, firing at high temperatures is usually regarded as an unavoidable harm involved in tea manufacture. Studies have been carried out with the purpose of retaining essential oils formed in fermentation (Bokuchava et al., 1957a,b). Up-to-date methods and facilities in firing now successfully solve the problem. Lyophilization helps dry the material, without causing significant changes in chemical constituents or damage to native properties. Lyophilization of the fermented leaf helps maintain the strong aroma peculiar not to manufactured black tea but to fermented leaf. Tea dried by lyophilization is of a light-brown sandy color and has the grassy taste of the fresh leaf. It is interesting that an additional firing of lyophilized samples at high temperature does not restore the black color and other properties typical of manufactured black tea. If, however, the samples are wetted and dried again, this time at high temperature, the color and aroma characteristic of black tea are developed. Thus, water is important to the development of black tea characteristics in high temperature conditions. Firing at high temperatures in the presence of water involves necessary transformations which finally form the taste, color, and aroma of manufactured black tea. Thus, it can be stated that firing is an indispensable stage in tea manufacturing which, unfortunately, causes essential oil losses. In 1940 an attempt was made to perform tea drying by ultrahighfrequency current (Tsekhomskaya, 1940). Although the experiments gave very promising results, this line of research was not continued. Present investigations involve the use of radiant energy (infrared rays, visible light, and ultraviolet rays) for tea drying. These studies are still in the stage of laboratory experiments but have yielded interesting data (Nikolaishvili and Shipalov, 1967). Summing up the above, it should be said that, despite great losses of essential oils, firing is absolutely necessary because of biochemical changes that develop in the process. The essential oils are lost because of the effect of hot-air flow. Naturally, the question arises whether the tea aroma could be formed by firing, and it appears that the thermal treatment can be applied to the underfermented tea. That is described below.

D. MOLECULAR BASISOF FERMENTATION One of the most important problems of tea chemistry is the mechanisms underlying catechin oxidation, since oxidative transformations

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of catechins are the basis of the technological procedure in black-tea manufacture. In 1935 Oparin put forward a biochemical theory of tea manufacturing that considered the fermentation process as decompensated respiration. In the intact cell the normal respiration process can be represented by the following reactions:

-

+ +

catechin 0, o-quinone o-quinone respiration substrate catechin

+ CO,

In undamaged cells the two processes are very well equilibrated; thus, products of oxidation of catechins do not accumulate, since they are constantly reduced. During processing, however, an intensive oxidation of catechins induced by polyphenol oxidase starts in cells damaged by leaf rolling. This results in the formation of quinone forms of catechins. The quinones formed are not completely reduced at the expense of respiratory substrates, and they condense, forming colored products which primarily determine the quality of the manufactured tea. Thus, technological procedures of tea manufacture are based on the following reactions:

+

-

catechins 0, (o-quinones)

+

o-quinones H20 colored products

The formation of quinones as a result of catechin oxidation was first established by Bokuchava et al. (1951), in model experiments of ascorbic acid oxidation in the presence of catechins and polyphenol oxidase, and by Kursanov and Zapromyotov (1952), in experiments on the chemical and enzymic oxidation of (-)-epicatechin. Kursanov and Zapromyotov (1952) investigated the enzymic oxidation of different catechins and tea tannin. They found that the oxidation of (-)-epicatechin and (-)-epicatechin gallate proceeded at a considerable rate, involving the consumption of 1 mole of oxygen per 1 mole of catechin. In addition, the catechin oxidation was accompanied by COz release. Therefore, during the oxidation and condensation of catechins the initial molecules are partially split. Those workers also showed that the oxidation of (-)-epigallocatechin and (-)-epigallocatechin gallate proceeded at a slower rate, involving the consumption of smaller oxygen amounts than the oxidation of (-)-epicatechin and (-)-epicatechin gallate. The combined oxidation of (-)-epicatechin and (-)-epigallocatechin involved a more intensive oxygen consumption (by 45%) and a greater carbon dioxide release (by 300%) than separate oxidation of the catechins.

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

From these findings and data on the value of the reduction-oxidation potential of pyrocatechin and pyrogallol, Kursanov and Zapromyotov suggested that the combined oxidation of (-))-epicatechin and its ester would serve as an additional catalyst, accelerating the oxidation of gallocatechins and their galloyl esters. Those ideas found support in investigations by other workers (Roberts and Wood, 1951a,b; Zapromyotov and Soboleva, 1954; Dzhemukhadze et al., 1957, 1964; Buzun et al., 1966). Further development of classical studies by Palladin (1912) and Bakh (1912) on mechanisms of the oxidation of organic substances led to the conclusion that the biological oxidation should result in intermediates, i.e., free radicals. Using electron paramagnetic resonance to examine the mechanism of enzymic oxidation, Soboleva et al. (1966) found two stages in the formation of o-quinones, accompanied by the development of free radicals of the semiquinone type: catechin

= semiquinone

quinone

Greater difficulties surround the problem of the further fate of catechins, i.e., the mechanisms of their condensation. The problem has been considered by many scientists, who have advanced several schemes for the condensation. Freudenberg and Maitland (1934) formulated a scheme for the condensation of an unlimited number of molecules with the formation of the C2-C, bond and breaking-up of the pyrane ring. Freudenberg and Weinges (1962)defended that scheme but limited it to the dimer product. Roberts (1942) advanced a scheme in which the condensation process involved not catechins themselves but their quinone forms, to establish the C,-C, bond without breaking up the pyrane ring. This scheme also theoretically assumed the infinity of the catechin condensation. Kursanov et al. (1947) studied the molecular weight of the catechin condensation products formed in fermentation, and established that the weight of initial compounds only doubled. Bearing this in mind, they suggested a scheme of catechin dimerization that specified the preliminary oxidation of molecules and the formation of dimers via interaction of two quinone molecules. The scheme was later confirmed by experimental results (Zapromyotov, 1950). The composition of condensed products formed during tea fermentation was studied in detail by Roberts and co-workers and reported in a number of papers around a decade ago. From his investigations

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269

of the mechanism of catechin oxidation, Roberts concluded that (-))-epigallocatechin and (-)-epicatechin gallate were the principal substrates condensed during tea fermentation. These two catechins amount to 80% of the total catechin content of young shoots of the Ceylon and Georgian tea varieties. (-)-Epicatechin, ( 2)-catechin, and (-)-epicatechin gallate, found in tea leaves in lesser amounts, are oxidized by polyphenol oxidase at an even greater rate, but they cannot be involved in the condensation, because of their high reduction-oxidation potential. The oquinones of these catechins act as additional catalysts of the gallocatechin oxidation. They oxidize gallocatechins to the corresponding o-quinones, to be later restored to initial molecules of catechins. The quinones of gallocatechins and their esters thus formed show less stability than the quinones of (-)-epicatechin and (+)-catechin; that is why they undergo oxidation and condensation as soon as they are formed. In the opinion of Roberts (1958a,b), o-quinones of (-)-epigallocatechin and (-)-epigallocatechin gallate are condensed spontaneously to diphenoquinones. As a result of oxidoreductive reactions, two molecules of diphenoquinone form dicatechin and theaflavin (or theaflavin gallate). The combined oxidation of (-)-epigallocatechin and (-)-epigallocatechin gallate leads to the formation of theaflavin, whereas the oxidation of (-)-epigallocatechin gallate alone yields theaflavin gallate. Roberts and Mayers (1959) isolated theaflavin and theaflavin gallate from black tea, and measured their molecular weights and absorption maxima, theaflavin being obtained in the crystalline form. Further transformations of dicatechins and theaflavins yield the compounds which Roberts termed thearubigens. Roberts and Mayers (1959)and Roberts (1962) indicated that catechins with a pyrocatechin ring were necessary for the formation of thearubigens. Roberts thought the oxidation of theaflavins to thearubigens occurred as follows:

‘3 \

Hd

/

‘COOH

‘OH

Theaflavin

Thearubigen

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Proceeding from the data available, Roberts formulated a scheme of chemical transformations which take place in tea fermentation (Fig. 4). Therefore, theaflavins and thearubigens are the main products of the oxidation of tea catechins formed during tea-leaf fermentation. It should be noted that the content of theaflavins is far less than that of thearubigens, but they are of primary importance to tea quality sinck they impart the specific bright and vivid color to the liquor (Roberts, 1962; Roberts and Smith, 1963; Bokuchava et al., 1966a). Thearubigens are a duller brown but nevertheless important to the color of the tea liquor. The ratio of theaflavins to thearubigens is responsible for the strength of the tea liquor. The content of theaflavins and thearubigens chiefly depends on the duration and temperature of fermentation (Kharebava and Nikolaishvili, 1964; Bokuchava et al.,1966a). Vuataz and Branderberger (1961) isolated (from freshly fermented Ceylon tea and fired black tea) dicatechins in crystalline form (substances A, B, C, previously described by Roberts), and studied their properties in detail. They reported thearubigens to contain 0.55 % nitrogen. The acid hydrolysis of thearubigens revealed alanine, arginine, glycocoll, leucine, isoleucine, lysine, phenylalanine, proline, serine, threonine, tyrosine, valine, and aspartic and glutamic acids. Thus, thearubigens are a mixture of various compounds and seem to be the products of condensation of o-quinones present in the catechins (or dicatechins) with amino acids. Problems of the enzymic oxidation of catechins were covered by Takino and Imagava (1963a,b). They studied the products formed from enzymic oxidation of (k)catechin, epicatechin, and gallocatechin. In recent years, great progress has been made on the mechanisms of catechin oxidation and the nature of the products formed via catechin oxidation by polyphenol oxidase. It should be mentioned, however, that cells of the tea leaf also contain highly active peroxidase and catalase. The role played by each enzyme in fermentation has been discussed thoroughly by Bokuchava (1958). Therefore it is only necessary to say that the peroxidase effect on catechins yields uncolored products, contributing to a certain degree to the development of tea taste.

Gallic acid

A

t i I

I

-

(~ )-Epigallo-

catechin gallate

Qand Z

P

4 -, I + acid

Intermediate (1) I

I

I

; I

Theaflavin gallate

coupled

1

Theaflavin

oxidation

0-Quinone

Intermediate (1) (Monogallate)

I

~

Intermediate (1)

Thearubigena

1

I+

I + gallic I acid t R

=

Bisflavanol gallate (B)

+2 H

Bisflavanol (C)

cd

Y

FIG. 4. Scheme of oxidative transformations of catechins during tea fermentation (after Roberts, 1962).

CHEMISTRY & BIOCHEMISTRY OF TEA &TEA MANUFACTURE

Bisflavanol digallate (A)

0

271

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

IV. THERMAL TREATMENT TO ENHANCE QUALITY AND VITAMIN P OF BLACK TEA

A. DISADVANTAGES OF CI~ASSIC TECHNOLOGY Proper tea manufacture should assure 100% utilization of the raw tea, completely retaining its positive properties and eliminating the negative. Scientific analysis of processing procedures shows that the classic technology, involving withering, rolling, fermentation, and firing, does not meet this basic requirement because of certain disadvantages: (1) During withering, approximately 20-25 % of the tea-leaf tissues remain undamaged (Manskaya, 1935; Bokuchava, 1935). This portion cannot undergo biochemical transformation, and is therefore worthless in the production of high-quality tea (Oparin, 1936). (2) In fermentation, oxidative processes are rapid but not uniform in rate, resulting in high losses of catechins and tannin, valuable tea constituents. (3) During firing, about 70-80% of the essential oils are lost, weakening aroma. (4) Manufactured tea ages rapidly, in a few months, significantly losing its high quality. No ripening can occur to improve the tea taste and aroma. Early investigations and inventions have failed to provide a complete use of plucked leaves and assure high quality in manufactured tea. Greater promise seemed offered by thermal treatment of underfermented tea. (Fermented tea contains about 50% of the tannin of raw tea, overfennented tea contains 35-40%, and underfermented tea contains 65-75%). This thermal treatment, thus, is applied only to underfermented tea, which is very rich in tannin, catechins, and other valuable compounds. Underfermented tea has a coarse taste and grassy odor until the thermal treatment eliminates them, improving the flavor of the manufactured tea. In 1928, Nevill wrote that tea is heated and packed while still hot in much of China, and this method causes tea ripening and improvement of quality (Nevill, 1928). Carpenter and Harrison (1923) mentioned that in India also, particularly in Assam, tea is heated to 70°C and packed while hot to improve flavor. Thermal treatment of different sorts is also used in other tea-producing areas of India- Darjeeling and Kunur (Bokuchava and Popov, 1958).It should be indicated that in tropical climates underfermented tea is subjected naturally to a slow thermal treatment in factory or storehouse by ambient temperatures that reach 40°C.

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Nevertheless, it is only recently that thermal treatment as a scientifically grounded technological process has found wide application in the manufacture of black tea (Bokuchava, 1957). The procedure has yielded positive results in producing not only black tea but green brick tea as well, where this process, lasting for 8-10 hours, has been substituted for the fermentation stage which lasts for 10-15 days (Bokuchava, 1955). B. IMPORTANCEOF THERMOPHYSICAL PROCESSES IN MANUFACTURE 1. Mechanisms of Thermal Treatment As established by numerous investigations at the Bakh Institute of Biochemistry, USSR Academy of Sciences, thermophysical processes are of key importance in tea manufacture (Bokuchava and Popov, 1954; Bokuchava, 1957; Bokuchava and Skobeleva, 1957; Bokuchava et al., 1957a,b; 1966b; Skobeleva et al., 1958; Bokuchava, 1958, 1959, 1962, 1966; Bokuchava and Ulyanova, 1960; Fatali-Zade et al., 1960, 1962; Gulua, 1962; Ralyanov et al., 1962; Bagirov, 1964, 1966; Sokbeleva and Popov, 1962; Makhmudov, 1962; Kharebava and Melimonadze, 1966; Kharebava, 1966; Khavtasi, 1966; Oragvelidze and Bokuchava, 1966; Pomazanov et al., 1966a,b; Vepkhvadze et al., 1966; Bagirov et al., 1966). Ten to fifteen years ago it was believed that the formation of taste and aroma products during fermentation occurred chiefly under the influence of enzymes. Now it is widely recognized that their formation is also dependent on purely chemical nonenzymic processes of the thermochemical type. (1) It was previously understood that tannin transformations contributing to tea flavor during fermentation are produced by polyphenol oxidase and peroxidase. Today it has been proved experimentally that the bitter taste of tea tannin can be changed by thermal treatment. Under the influence of heating and moisture, tannin loses bitterness and acquires a pleasant astringent taste peculiar to black tea. This is accounted for by the epimerization and isomerization of catechins, constituents of tannin. Polyphenol oxidase oxidizes the latter to form quinones, yielding red and brown pigments. Simultaneously, tannin combines with proteins, sharply decreasing the tannin content of manufactured tea. The thermal treatment also causes tannin oxidation, resulting in the formation of quinones, red and brown pigments, without the production of insoluble tannin-protein complexes. This is due to the fact that proteins are denatured and enzymes inactivated, a great advantage for thermal treatment over that of enzymes.

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(2) Tea aroma is considered to develop as a result of tea fermentation. Experiments have indicated that the process also occurs during thermal treatment. Interactions of tannin, amino acids, and sugars at high temperature yield various aldehydes and esters responsible for tea aroma. Further improvement of aroma results from the firing performed on the maximal accumulation of essential oils 2 to 3 hours after withering has started. In addition, the thermal treatment is applied to a thick layer of tea, with air blowdown being excluded; therefore, tea adsorbs aromatic substances, and manufactured tea has a strong aroma. (3) The thermal treatment involves destruction and isomerization of certain substances that otherwise impair tea quality and impart a grassy odor (chlorophyll, P,y-hexenol, hexylene aldehyde). (4) The thermal treatment distributes moisture uniformly in the tea bulk. Because water acts as a heat carrier, this, in turn, promotes uniform development of oxidative processes in all cells. The raw material is thereby utilized more completely, and taste and aroma are improved. ( 5 ) The thermal treatment partially replaces mechanical effects, so not all the leaf tissues need be crushed in the rolling process. Thus, the formation of taste and aroma substances that takes place during fermentation can be accomplished better with the aid of a thermal treatment applied after firing. The manufactured tea, of course, must have contained the necessary initial compounds (soluble tannin, catechins, amino acids, sugars, etc.) that contribute to good tea flavor. This makes it possible to exclude the second stage of fermentation as an independent process and to shorten the withering-fermentation process by half. The above vividly demonstrates the importance of thermophysical processes in tea manufacture. Tea quality can be increased significantly, however, only on the basis of rational combination of the thermophysical processes with enzymic processes. 2 . Application of Thermal Treatment

The basis for a new technique of tea manufacture has been the result of many years of research on the biochemistry of tea and tea manufacture at the Bakh Institute of Biochemistry, data developed by Soviet and foreign workers in this and related fields, and intensive studies of tea manufacture in the Soviet Union and other countries.

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The technique includes thermal treatment to improve tea quality and its biological values and stability in storage. The basis is rational control of the biochemical processes that develop during tea-leaf processing. This is achieved through shortening fermentation and applying thermal treatment to the underfermented tea. In the new technology the withered tea leaf is subjected to rolling. Its quality and the season determine the specific rolling treatment used for producing well rolled tea containing 65-75% of the tannin of the original fresh leaves. After rolling, the tea is passed to the firing room. Firing is performed in a one-step process to assure a residual moisture content of 6-9% for fine fractions and 7-10% for large fractions, in contrast to the content of 3-4% achieved in classic technology. It should be mentioned that the new method increases tea-firing capacity by 30%. The tea obtained in such firing contains large amounts of tannin and catechins but has coarse and bitter taste and grassy odor. A thermal treatment eliminates these unfavorable characters and induces biochemical transformations necessary for the development of specific taste and aroma substances peculiar to black teas of high quality. Underfermented tea is treated thermally in a special chamber at 5Oo-65"C.Exposure time is 2-3 hours for fine fractions and 3-5 hours for large fractions, depending on season and moisture content after firing. The thermal treatment reduces moisture content to 4-670. After standard processing, the tea is subjected to sorting. The thermal treatment of underfermented tea significantly increases the concentration of extractives, tannin, catechins, and vitamin P, reducing their losses in tea processing. A study of catechins in tea manufactured with the aid of the thermal treatment showed that they contained 6 catechins: (-)-epigallocatechin, (-+)-gallocatechin, (-)-epicatechin, (2)-catechin, (-))-epigallocatechin gallate, and (-)-epicatechin gallate (Oragvelidze et al., 1966) (Table XIII). Sephadex gel filtration and paper chromatography were used to isolate four catechins in crystalline form from Georgian tea of the highest grade (Oragvelidze and Bokuchava, 1966) (Fig. 5). Black tea manufactured by the new technology contained 50-70 mg catechins per g (Bokuchava, 1967), in contrast to only traces in black tea manufactured by the classic procedure (Zapromyotov and Soboleva, 1954; Dzhemukhadze and Shalneva, 1955). Regular intake of black tea manufactured by the new technology can completely meet the requirements of the human body for vitamin

276

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA TABLE XI11 CATECHINS IN BLACKTEA MANUFACTURED BY ( % DRYWEIGHT) TECHNOLOGY Catechins (-(-Epigallocatechin (*)-Gallocatechin (-)-Epicatechin (&)-catechin (-)-Epigallocatechin gallate (-)-Epicatechin gallate Total

+

THE

NEW

Manufactured tea MI-BI (mg/g) ( %) 13.42 2.01 9.83 19.88 11.35 57.49

8.57 2.77 6.85 13.02 7.83 39.66

P since 3 g of the tea (the daily norm) contains approximately 150 mg of catechins. The vitamin P content of such black tea is thus comparable with that of green tea while retaining its specific character. It should be emphasized that the new method improves the tea taste and aroma, with tea quality increasing by 0.25-0.5 and in some cases by 0.75 tea taster ball (Bokuchava et al., 1966~).It thus increases the amount of teas of highest and first grade and decreases the amount of teas of lower grade. It also increases the content of tannin and extractives up to 6% in manufactured tea. Tea manufactured by this procedure shows greater stability in storage, retaining quality for a longer period (Bokuchava et aZ., 1966d). The new technology of black-tea manufacture makes it possible to mechanize all stages of the process, converting to production-line methods. Thermal treatment of underfermented tea is now in use at the Adler tea factory of the Krasnodar region and in many tea factories in Azerbaijan and Georgia. An improvement in quality and vitamin P content is reported for teas manufactured at some factories (Ralyanov et al., 1962; Bokuchava et al., 1966c; Pomazanov et al., 1966; Melimonadze, 1966; Bagirov et al., 1966). The Adler factory has been working by the new process for over 7 years. In this period the production of tea of highest and first grades has been increased appreciably, and the content of tannin and catechins enhanced. Average tannin content in tea manufactured at that factory was formerly 10-12%, reaching 14-15% in only the best teas. Now, however, it is 16-17%, reaching 19-20% in the best teas. Thus, the Adler tea factory is ahead of the many Indian factories which manufacture (besides high-tannin teas) teas with tannin con-

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277

tent of 8-10%. Data of the Central Research Food Institute in Maisur indicate for black tea a maximum tannin concentration of 20%, a mean of 13%, and a minimum of 8-9% (Bokuchava, 1958). It should be stressed that the new technology increases tannins and extractives in all tea grades, as illustrated in tea manufactured at the Adler factory.

FIG. 5. Crystalline catechins from black tea manufactured by the new technology: (a) (-)-epigallocatechin (X 170); (b) (-)-epicatechin (X 170); (c) (-)-epigallocatechin gallate (X 100); (d) (-)-epicatechin gallate (x 170).

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MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

V. BIOLOGICAL AND NUTRITIONAL VALUE OF TEA

As affirmed at the Second International Congress of Food Science and Technology, in Warsaw in 1966, the main purpose of the food industries is to increase the quality and nutritional value of various foods. Recent research has convincingly demonstrated that tea is very rich in vitamin P. As mentioned previously, tea catechins exhibit a strong vitamin P effect, restoring to normal an elevated permeability of capillaries. In vitamin P activity, tea catechins exceed all other known capillarystrengthening preparations: citrin, rutin, and esculin. The catechin content increases the physiological and nutritional value of tea. It is of particular importance since other foodstuffs contain very small amounts of catechins. The richest in catechins are green teas, with top-quality green teas containing 80 to 170 mg of catechins per g. An individual’s daily requirements for vitamin P can thereby be met completely through a normal tea intake. Black tea manufactured by classic technology contains very low amounts of catechins, i.e., about 3-4 mg per g, in contrast to 30-70 mg by new methods. Thus, a man can meet his vitamin P requirements by regularly drinking black tea. Black tea manufactured by the new method retains a third to half of the catechins of the fresh leaf (Table XIV). Quite recently the Bakh Institute of Biochemistry, in collaboration with the USSR Research Institute of the Tea Industry, developed a new technological procedure for the manufacture of green instant tea and vitamin P-rich food dyes of green, yellow, and brown. The catechin content amounts to 200-400 mg/g of dry concentrate in green

TABLE XIV WITH THERMAL CATECHINS IN BLACKTEAMANUFACTURED TREATMENT AT DIFFERENT TEA FACTORIES IN GEORGIA Catechins

Makharadze district

Tkibuli district

Gudauta district

(-)-Epigallocatechin (+)-Gallocatechin (-)-Epicatechin (&)-catechin (-)-Epigallocatechin gallate (-)-Epicatechin gallate Total

30.56 13.58 12.46 81.51 24.34 162.49

32.56 17.98 7.86 93.66 19.10 170.16

32.04 13.49 15.76 78.76 23.63 166.68

+

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279

instant tea and to 200 mg/g in food dyes. The instant tea and food dyes are produced from coarse inferior leaf and molding material left after trimming tea shrubs. The production of instant tea and food dyes from coarse tea leaves doubles vitamin P resources in the USSR, because yields of poor-quality leaves equal those of high-quality leaves. The antioxidative properties of catechins are used in a new method of stabilizing food dyes from beet, developed in Russia. A combination of tea dyes and beet dyes given special treatment imparts high stability to the latter and enriches them in vitamin P. Food dyes of plant origin can be used successfully in the confectionery industry and other food industries, entirely substituting for synthetic dyes, e.g., amaranth, which are not quite innocuous for human consumption. Taking into consideration the annual world volume of black tea (500,000 tons) and green and other teas, as well as of food dyes, it can help provide the world population with necessary amounts of vitamin P.

VI. CONCLUSION In conclusion, it is worthwhile to indicate the importance of biochemistry in tea manufacture. Biochemistry is in essence the theoretical basis of tea manufacture. It is responsible for the quality of raw and manufactured tea since biochemical processes underlie the transformations of substances during the growth and processing of the tea leaf. Tea quality, in its broad sense, is being formed during the growth and development of the tea leaf, when many of the compounds responsible for quality are synthesized. It should be borne in mind that the tea plant can synthesize constituents that may exert both positive and negative effects on manufactured tea quality. The task in tea manufacture is to control the transformations occurring in processing procedures so as to preserve all positive substances possible while destroying negative substances and producing catechin-rich teas. Biochemical analyses have furnished ample data establishing the chemical constituents and characters of raw and manufactured tea and providing insight on the manufacturing processes. The composition of tea-a content of rare and valuable compounds, such as caffeine, theobromine, theophylline, catechins, essential oils, aldehydes, various vitamins -makes it a nutritionally valuable product. The

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tonic effect of tea on the human body, with its outstanding ability to quench thirst, is very well known. The caffeine in tea makes it valuable from the pharmacological point of view, and its physiological significance is enhanced by its supply of vitamins P, C, B, PP, etc. According to current concepts, the physiological and nutritional value of tea is dependent primarily on catechins. Three main properties of catechins should be indicated: (1) the capillary-strengthening effect, vitamin P factor; (2) the antioxidizing effect, contributing to its radioprotective effect; and (3) the antimicrobial effect, related to the bactericidal and bacteriostatic effects of catechins. The favorable effect of tea was once attributed exclusively to the caffeine content. Today, biochemical studies have shown that it also depends on the content of catechins. They are responsible for the tea taste and vitamin P activity.

REFERENCES Anufriyev, M. F. 1940. Vitamin C in tea. Biokhim. Chain. Proiz. Akad. Nauk S S S R 4,73. Areshkina, L. Ya. 1951. On the role ofalkaloid N-oxides in plants. Biokhimiya 16,461. Aso, D. 1901. On the role of oxidase in the preparation of commercial tea. Bull. Coll. Agr. Imp. Unio. Tokyo 4,255; Chem.Abstr. 2,894. Baginski, A. 1884./. Physiol. Chem. 85,431. Bagirov, A. Yu. 1964. “Tea Manufacture in the Azerbaijanian SSR,” Baku. 128 pp. Bagirov, A. Yu. 1966. Efficiency of the introduction of new technology at tea factories of the Azerbaijanian SSR with respect to a decrease of tannin losses and an increase of soluble substances in tea. Biokhim. i Progr. Technol. Chain. Proizo. 281. Bagirov, A. Yu., Andreyev, I. F., and Makhmudov, K. I. 1966. On mechanization of the thermal treatment of underfermented tea and on the completion of the introduction of new technology at tea factories of the Azerbaijanian SSR. Biokhim. i Progr. Technof.Chain. Proizo. 388. Bakh, A. N. 1912. Chemical mechanisms of respiratory processes. Selected Works of Academician A. N. Bakh, 181. Bakhtadze, K. E. 1955. Results of tea selection for several years. Buff.USSR Research Inst. of Tea and Subtrop. Cultures 4, 14. Bamber, K. 1893. “A Text Book on the Chemistry and Agriculture of Tea.” Calcutta. Bamber, K., and Wright, H. 1902. Note prdiminaire sur I’enzyme du thi.. Ind. Plant. Card. Few. Baraboy, V. A. 1966. Radioprotective properties of tea catechins and other compounds having vitamin P activity. Biokhim. i Progr. Technof.Chain. Proizv. 357. Bendal, D., and Gregory, R. 1963. Enzyme Chemistry of Phenolic Compounds. Pergamon Press. Bhatia, J., and Ullah, R. 1962. Nature 193,658.

CHEMISTRY & BIOCHEMISTRY OF TEA & T E A MANUFACTURE

281

Blagoveshchensky, A. V. 1935. Caffeine and tannins of the tea leaf in relation to the variety. Biokhim. Chain. Proizv. Akad. Nuuk SSSR 1, 140. Bokuchava, M. A. 1935. Chlorophyll indicator as a method of the tea manufacture control. Biokhim. Chain. Proizv. Sb. 1,62. Bokuchava, M. A. 1936. Temperature conditions of tea fermentation. Biokhim. Chain. Proizv. Akad. Nauk S S S R Sb. 2,23. Bokuchava, M. A. 1946. Transformations of different tannin fractions in the course of tea leaf growth and its processing. Biokhimiya 11,3. Bokuchava, M. A. 1947. O n the enzymic oxidation of different fractions of tea tannins. Biokhimiya 12,59. Bokuchava, M. A. 1955. Biochemistry of the Manufacture of Green Brick Tea. Izd. Akad. Nauk SSSR (Moscow) 166 pp. Bokuchava, M. A. 1957. New technology of black tea manufacture and its theoretical basis. Bull. U S S R Research lnst. of Tea and Subtrop. Cultures, 3,92. Bokuchava, M. A. 1958. Biochemistry of tea and tea manufacture. Izd. Akad. Nauk S S S R (Moscow)586. Bokuchava, M. A. 1959. Theoretical basis of the new technology of black tea manufacture. Biokhim. Chain. Proizv. Akad. Nuuk SSSR Sb. 7,5. Bokuchava, M. A. 1962. O n the theory and practice of the new technology of black tea manufacture. Biokhim. Chuin. Proizv. Akad. Nauk SSSR Sb. 9,81. Bokuchava, M. A. 1966. Theoretical basis of mechanized-line production of tea. Biokhim. i Progr. Technol. Chain. Proizv. 254. Bokuchava, M. A. 1967. Biochemistry in the tea industry. Subtropicheskii Cultury, 5-6 (91-92), 105. Bokuchava, M. A,, and Belinovich, A. M. 1946. Fractionation of tannins. Biokhim. Chain. Proizo.Akud. Nauk S S S R 5, 15. Bokuchava, M. A., and Berdyeva, S. I. 1959. Bacteriostatic and bactericidal properties of tea tannins. Biokhim. Chain. Proizv. Akad. Nauk SSSR Sb. 7,209. Bokuchava, M. A., and Makhmudov, K. I. 1966. Study of the withering process. Biokhim. i Progr. Technol. Chain. Proizo. 250. Bokuchava, M. A., and Popov, V. R. 1940. Oxidation of different tannin fractions by insoluble tea enzymes. Rept. Lab. Tea Biocheni. lnst. Biochem. USSRAcad. Sci. Bokuchava, M. A., and Popov, V. R. 1945. An insoluble form of tea tannins. Biokhimiya

10,3. Bokuchava, M. A,, and Popov, V. R. 1946. Quantitative estimation of water-insoluble tannins. Biokhim. Chain. Proizo. Akad. Nauk SSSR Sb. 5,82. Bokuchava, M. A., and Popov, V. R. 1948. On the role ofpolyphenol oxidase and peroxidase in tea fermentation. Dokl. Akad. Nauk SSSR 60,620. Bokuchava, M. A., and Popov, V. R. 1954. The significance of amino acids in tea aroma formation upon their reaction with tannins at high temperature. Dokl. Akad. Nauk S S S R 99,145. Bokuchava, M. A,, and Popov, V. R. 1958. Modem state of the tea manufacture in India. Bull. USSR Research lnst. Teu and Subtrop. Cultures 4,25. Bokuchava, M. A,, and Pruidze, G. N. 1964a. T h e method of producing the tea dry concentrate. USSR Patent No. 166 228. Bull. 21. Bokuchava, M. A., and Pruidze, G . N. 1964b. T h e manufacture of green crystalline tea using freeze-drying. Bull. USSR Res. Inst. Tea Ind. 1 (16),3. Bokuchava, M. A., and Pruidze, G . N. 1965. Green instant dry concentrate of tea from the moulding material. Commun. Acad. Sci. Georgian S S R 3,587.

282

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Bokuchava, M. A., and Pruidze, G. N. 1966. Development of manufacturing procedures of instant tea and dyes having a high vitamin P activity. Biokhim. i Progr. Technol. Chain. Proizo. 328. Bokuchava, M. A., and Skobeleva, N. I. 1957. Study of aromatic aldehydes. Biokhimiya 22,3. Bokuchava, M. A., and Skobeleva, N. I. 1960. Transformations of flavones in the tea manufacture. Biokhimiya 25,3. Bokuchava, M. A., and Skobeleva, N. I. 1961a. Study of carbohydrates by paper chromatography and densitometry. Biokhimiya 26,2,361. Bokuchava, M. A., and Skobeleva, N. I. 1961b. Manual of the New Technology of Tea Manufacture. Izd. Akad. Nauk S S S R (Moscow)101 pp. Bokuchava, M. A., and Soboleva, G. A. 1966. On the state and localization of polyphenol oxidase and peroxidase in the tea plant. Biokhim. i Progr. Technol. Chain. Proizv. 21. Bokuchava, M. A., and Ulyanova, M. S. 1960. Conversions of catechins of the tea leaf under the influence of enzymes and thermal treatment. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 8,107. Bokuchava, M. A., Popov, V. R., and Shlipakova, L. Ya. 1966a. Study of theaflavins and thearubigens upon tea manufacturing. Biokhim. i Progr. Technol. Chain. Proizo. 42. Bokuchava, M. A., Shubert, T. A., Popov, V. R. 1951. The role of tannins in oxidativereductive processes occurring in plants. Dokl. Akad. Nauk S S S R 76,439. Bokuchava, M. A,, Popov, V. R., and Sidorov, V. S. 1954. Chromatographic fractionation of free amino acids from fresh and withered tea leaf. Dokl. Akad. Nauk S S S R 95,609. Bokuchava, M. A., Skobeleva, N. I., and Dmitriyev, A. F. l957a. Biochemical changes of aroma and taste substances of tea and its quality. Dokl. Akad. Nauk S S S R 115, 362. Bokuchava, M. A., Soboleva, G . A., and Knyazeva, A. M. 195713. Transformations of tea catechins under the influence of high temperature. Biokhimiya 23,266. Bokuchava, M. A., Knyazeva, A. M., Skobeleva, N. I., Dmitriyev, A. F., and Pruidze, V. G. 1959. Testing results of the new technology of the black tea manufacture. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 7,12. Bokuchava, M. A., Gryuner, V. S., Pruidze, G. N., Selezneva, G . D., and Podroikina, N. I. 196613. Application of dyes made of tea leaves in the confectionery industry, Biokhim. i Progr. Technol. Chain. Proizv. 335. Bokuchava, M. A., Skobeleva, N. I., and Knyazeva, A. M. 1966c. Results of application of the new technology of tea manufacture accumulated for several years. Biokhim. i Progr. Technol. Chain. Proizv. 299. Bokuchava, M. A., Skobeleva, N. I., and Knyazeva, A. M. 1966d. Changes in the composition and quality of tea manufactured by the new and classical technology upon long-term storage. Biokhim. i Progr. Technol. Chain. Proizo. 309. Bradfield, A., and Bate-Smith, E. 1950. Chromatographic behavior and chemical structure. 11. The teacatechins. Biochim. Biophys. Acta 4,441. Chem.Abstr. 44,5677. Bradfield, A., and Penney, M. 1948. The catechins of green tea. Part 2. J . Chem. S O C . 1948,2249, Chem. Abstr. 43,3394. Bradfield, A., Penney, M., and Wright, W. 1947. The catechins of green tea. Part 1. J . Chem. Soc. 1947,32. Busgen, M. 1890. Beobachtungen uber das Verhalten des Gerbstoffes in den PflanzenJenische L.F.Naturwiss. 24 (N.F.), 17.

CHEMISTRY & BIOCHEMISTRY OF TEA &TEA MANUFACTURE

283

Buzun, G . A., Dzhemukhadze, K. M., and Mileshko, L. F. 1966. Enzymic transformations of catechins in tea manufacturing. Proc. Second Intern. Congr. Food Sci. Technol. 192. Bziava, M. L. 1956. Effect of enhanced nutrition factors on the yield of tea plantations. Bull. USSR Res. Znst. Teu and Subtrop. Cultures 1 , 9 . Carpenter, P., and Harler, C. 1923. Variation in the composition of the tea leaf throughout the season. Znd. Tea Assoc. 1. Carpenter, P. G., and Harrison, S. D. 1923. “Tea Manufacture in North-East India.” Tiflis. Izd. “Chai-Gruzia.” Cartwright, R., and Roberts, E. 1954. The sugars of manufactured tea. 1. Sci. Food Agr. 5,593. Cartwright, R., and Roberts, E. 1955. Theogallin as a galloyl ester of quinic acid. Chem. G Ind. (London) 1955,230. Cartwright, R., Roberts, E., and Wood, J . 1954. Theanine-an amino acid N-ethyl amide present in tea. J. Sci.Food Agr. 5,597. Cartwright, R., Roberts, E., Flood, A., and Williams, A. 1955. Suspected presence of p-coumarylquinic acids in tea, apple and pear. Chem. 6 Znd. (London) 1955, 1062. D e w , J. 1915. Over enkele factoren in eventuell verband met de kwaliteit d e thee. Mededeel. Proefsta. Thee 4 2 , l . Deuss, J. 1937. Proc. K . Akad. 40,518. Dzhemukhadze, K. M. 1946. Results of the biochemical control of the tea manufacture. Biochim. Chain. Proizu. Akad. Nauk S S S R Sb. 6,116. Dzhemukhadze, K. M. 1958. Theoretical Basis of the Biochemical Control of the Tea Manufacture. Zwl. Akud. Nauk S S S R (Moscow). Dzhemukhadze, K. M. 1959. On the objective evaulation of raw tea. Biokhim. Chain. Proizv. Akud. Nauk S S S R Sb. 7,89. Dzhemukhadze, K. M. 1966. The significance of catechins in tea manufacture. Biokhim. i Progr. Technol. Chain. Proizv. 167. Dzhemukhadze, K. M., and Nestyuk, M. N. 1960. Changes of flavonoids in tea leaf withering. Dokl. Akud. Nuuk SSSR 133,469. Dzhemukhadze, K . M., and Shalneva, G. A. 1954. Transformations of catechins in the course ofthe tea leaf development. Dokl. Akad. Nauk SSSR 99,1069. Dzhemukhadze, K. M., and Shalneva, G. A. 1955. The method of quantitative evaluation of tea leaf catechins by paper chromatography. Biokhimya 20,336. Dzhemukhadze, K. M., Shalneva, G. A,, and Mileshko, L. F. 1957. Changes incatechins upon tea fermentation. Biokhimiya 22,5. Dzhemukhadze, K. M., Nestyuk, M. N., and Mileshko, L. F. 1961. Flavonoids of twoleaf flushes of the tea plant. Biokhimiya 26,694. Dzhemukhadze, K. M., Buzun, G . A., and Nestyuk, M. N. 1962. The diurnal changes of catechins and flavonols in the tea plant. Biokhini. Chain. Proizv. Akad. Nauk SSSR Sb. 9, 15. Dzhemukhadze, K. M., Buzun, G. A,, and Mileshko, L. F. 1964. Enzymic oxidation of catechins. Biokhimiym 29,882. Egorov, I. A. 1940. Pectic substances in tea. Biokhim. Chain. Proizu. Akad. Nauk SSSR 4,69 Egorov, I. A. 1950. On vitamins B complex in tea. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 6, 181. Fairbairn, I., and Paterson, A. 1966. Alkaloids as possible intermediaries in plant metabolism. Rept.f:Nature 210,5041, 1163.

284

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Fatali-Zade, F. A., Veisov, G. M., and Bagirov, A. Yu. 1960. Results of the testing and introduction of new technology of black baikhovi tea manufacture at Azerbaijanian factories in 1959. Biokhim. Chain. Proizu. Akad. Nauk SSSR Sb. 8,186. Fleischner, G. 1956. The theophylline content of tea leaves (with simultaneous consideration of theobromine). Pharmazie 2,248. Freudenberg, K., and Maitland, K. 1934. Der Quebracho-Gerbstoff Ann. Chem. 51, 193. Freudenberg, K., and Weinges, K. 1962. “The chemistry of flavonoid compounds.” 197 pp. Pergamon Press. Frost, V. A., and Elovsky, V. A. 1926. Tea. Leningrad, 200 pp. Gegele, V. G., Kikodze, G. G., and Rukhadze, I. S. 1941. Study of essential oil of the Georgian tea. Bull. USSR Res. lnst. Tea and Subtrop. Cultures4,43. Gerasimov, M. A. 1955. Conditions of the wine thermal treatment. Vinogradarstuo i Vinodelie SSSR 2,8. Gildemeister, R., and Hoffman, F. 1900. Les huiles essentielles. 606 pp. Leipzig-Paris. Glazunov, I. V. 1935. Dynamics of the enzymic effect. Biokhim. Chain. Proizu. Akad. Nauk SSSR Sb. 1. Godziashvili, G . S. 1949. Effect of potassium fertilizers on the yield of tea plantations cultivated on red-green soils. Bull. USSR Res. lnst. Tea and Subtrop. Cultures 2,194. Gogiya, V. T. 1950a. Pectic substances of the tea leaf. Biokhim. Chain. Proizu. Akad. Nauk SSSR Sb. 6,184. Gogiya, V. T. 1950b. The formation of tannins and changes in the oxidative enzymes upon the germination of tea seeds. Biokhim. Chain. Proizu. Akad. Nauk SSSR Sb. 6,70. Gogiya, V. T., and Motsonelidze, N. D. 1966. Study of aldehydes and ketones in black tea. Biokhim. i Progr. Technol. Chain. Proizu. 60. Goguadze, V. P. 1940. On the study of substances responsible for the tea aroma. Commun.Acad. Sci. Georgian SSR 1,513. Golyanitsky, I. A., and Bryushkova, K. A. 1936. On vitamin C in tea. Dokl. Akad. Nauk S S S R 4 (13),367. Graham, G., Moor, T., and Stein, R. 1966. Study of the tea aroma. Biokhim. i Progr. Technol. Chain. Proizu. 66. Gregory, R., and Bendal, D. 1966. The purification and some properties of polyphenol oxidase from tea. Bi0chem.J. 101,3,569. Groll, R. 1897. “Tea: A text book of tea planting and manufacture.” London. Gulua, K. P. 1962. Testing results of application of the new technology of black tea manufacture at the Anaseuli tea factory in 1960. Biokhim. Chain. Proizu. Akad. Nauk SSSR Sb. 9,88. Harler, C. 1933. Culture and Marketing of Tea. London. 125 pp. Haler, C. 1963. “Tea Manufacture.” 126 pp. Oxford University Press, New York, Toronto, London. Ilyin, G . S. 1948. On the relationship ofbasic tobacco alkaloids. Biokhimiya 13,2. Ilyin, G . S. 1966. 14C-nicotinedecomposition in relation to protein biosynthesis. Dokl. Akad. Nauk SSSR 169,l. Jammamoto, R., and Ito, K. 1938. Uber die Riechstoffe des schwarzen thees. Chem. Zentr. I, 200. Jammamoto, R., and Kato, J. 1935. Study on the essential oil of black tea. Sci. Papers lnst. Phys. Chem. Res. (Tokyo)27,582. Jammamoto,’R., and Muraoka, T. 1932.5.Agr. Chem. Soc.Japan8,749.

CHEMISTRY & BIOCHEMISTRY O F TEA &TEA MANUFACTURE

285

Jammamoto, R., Hayashi, I., and Takeo, K. 1954. Nutritional value of tea leaves during the process of green tea manufacture. I. Changes of free amino acids and sugars. Shig. Kenritsu Nagya. Tankidaigaku, Gakujutsu Hokoku Ser. 1,6,30.Chem. Abstr. 51,10789. Johnson, T. 1937. Science 85,431. Katayama, K. 1907. Sur I’arome du thC noir. Bull. Centr. Agr. E x p t . Sta.Japan (Tokyo) 1, 149. Kazantsev, A. P. 1966. Application of vitamin P in therapy. Biokhim. i Progr. Technol. Chain. Proizv. 369. Kellner, 0..Makino, K., and Ogasawazo, K. 1887. Die Zusammensetzung der Theeblatter in verschiedenen Vegatationsstadien. Zandw. Vers. St. 33,370. Kevanishvili, V. N. 1948. Tannate of tea caffeine. Bull. USSR Res. I n s t . Tea and Subtrop. Cultures 2.73. Kharebava, G. I . 1946. Enzymic processes in the living tea leaf. Biokhim. Chain. Proizu. Akad. Nauk S S S R Sb. 5,86. Kharebava, G. I. 1951. Resinous substances of tea. Bull. U S S R Res. Inst. Tea and Subtrop. cultures 1,10. Kharebava, G. I., and Nikolaishvili, D. N. 1964. Quantitative changes oftheaflavins and thearubigens in the tea fermentation process. Bull. USSR Res. Inst. of Tea Znd. 2 (17),29. Kharebava, L. G., Starodubtseva, V. P., and Surguladze, L. M. 1967. Study of volatile substances of tea. Tchai. Kultura i Proizu. (Tbilisi)1 (7),50. Kharebava, L. M. 1966. Effect of shortened withering on the composition and quality of tea upon the new manufacturing procedures. Biokhim. i Progr. Technol. Chain. Proizti. 268. Kharebava, L. M., and Melimonadze, S. N. 1966. Efficiency of the new technology application for the processing of raw tea from eastern and northern areas of the Georgian SSR. Biokhim. i Progr. Technol. Chain. Proizu. 263. Kharebava, L. M., and Skobeleva, N. I. 1968. Study of carbohydrates by the paper chromatography and densitometry technique. 11. Prikladnaya Biochim. i Mikrobiol. (In press). Khavtasi, V. A. 1966. A comparative study of temperature conditions at different stages of the tea thermal treatment. Biokhim. i Progr. Technol. Chain. Proizu. 277. Khocholava, I. A. 1940. Correlation of the quality of the tea leaf with its chemical constituents. B u l l . U S S R Res. Inst. Tea and Subtrop. Cultures 3 (7),13. Kito, M., Kokura, H., Izaki, J., and Sasao, K. 1966. Fate of the radioactive carbon of theanine labeled with ethylamine-l-’4C in tea seedlings. Agr. Biol. Chern. 30, 61,623. Kolokolov, V. 1906. “On the Tea on Russian Plantation.” Moscow. Kossel, A. l889.j. Physiol. Chem. 13,298. Kost, E. A., and Bailer, 0. E. 1966. Application of green tea brew for the treatment of dysentery patients. Biokhim. i Progr. Technol. Chain. Proizu. 365. Kozai, T. 1890. “Researches on the Manufacture of Various Kinds of Tea.” Tokyo. Kozhin, S. A., Chanturia, 0. D., Treiger, N. D., and Vasilevskaya, Yu. V. 1966. Use of gas chromatography for comparative evaluation of different teas. Biokhim. i Progr. Technol. Chain. Proizu. 76. Krauss, G. 1889. “Griindlinien zu einer Physiologie des Gerbstoffes.” Leipzig. Kretovich, W. L. 1967. “Introduction to Enzymology.” 350 pp. Izd. Nauka. Moscow. Kretovich, W. L., and Tokareva, R . R. 1948. Interaction of amino acids and sugars at high temperahlres. Biokhimiya 13,508.

286

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Kretovich, W. L., and Tokareva, R. R. 1949. Volatile aromatic substances in malt and bread. Dokl. Akad. Nauk S S S R 69,231. Krishnamurthy, K., Venkatasubramanian, T., and Giri, K. 1952. Circular paper chromatography of the amino acids of tea and coffee infusions. Current Sci. (India) 21, 133. Chem. Abstr. 49,9228. Kryukova, N. N. 1946. Age changes of tannins in the tea plant. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 5,41. Kryukova, N. N. 1947. Inositol in tea leaves. Biokhimiya 12,512. Kursanov, A. L. 1941. Assay of different tannins in plants. Biokhimiya 6,312. Kursanov, A. L. 1943. Transformations of different tannins upon tea leaf manufacture. Biokhimiya 8,188. Kursanov, A. L. 1944. Transformations of tannins in willows during spring growth. Biokhimiya 9,322. Kursanov, A. L. 1952. Synthesis and transformation of tannins in the tea plant. The 7th Bakh Lectures, Moscow. Izd. Akad. Nauk SSR. Kursanov, A. L., and Brovchenko, M. L. 1950. Tannins of different organs of the tea plant. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 6,53. Kursanov, A. L., and Dzhemukhadze, K. M. 1948. Gallic acid as a constituent of tea tannins. Biokhimiya 13,61. Kursanov, A. L., and Kryukova, N. N. 1941. Age changes of tannins in plants. Biokhimiya 6,321. Kursanov, A. L., and Shubert, T. A. 1936. The formation and changes of tea aroma upon black tea manufacturing. Biokhim. Chain. Proizo. Akad. Naut S S S R Sb. 2,45. Kursanov, A. L., and Zapromyotov, M. N. 1952. Oxidative transformations of tea catechins. Biokhimiya 17,230. Kursanov, A. L., and Zapromyotov, M. N. 1954. The method of producing vitamin P. Patent No. 97497. USSR Ministry Food lnd. 27,3. Kursanov, A. L., Dzhemukhadze, K. M., and Zapromyotov, M. N. 1947. Condensation of tea leaf catechins during their oxidation. Biokhimiya 12,421. Kursanov, A. L., Kryukova, N. N., and Vyskrebentseva, E. I. 1948. Inositol as an intermediate in the transformation of sugars into polyphenols. Biokhimiya 13,530. Kursanov, A. L., Bukin, V. N., Povolotskaya, K. P., and Zapromyotov, M. N. 1950. Biological effects of tea tannins. Biokhim. Chain. Proizu. Akad. Nauk S S S R Sb. 6, 170. Lamb, J. 1937. Research and the practical manufacture of tea. J . Tea Res. Inst. Ceylon 36. Li, L., and Bonner, J. 1947. Experiments on the localisation and nature of tea oxidase. Biochem.J . 41,105. Lo, Chein-Pen, and Chu, Lucy Ju-Yung. 1945. The caffeine content of Yunnan teas. J . Chem. Eng. China 12,15. Chem.Abstr.40,2905. Loth, H. 1964. Dimer flavonols produced by enzymatic oxidation of rutin and myricetin. Sixth Intern. Congr. Biochem. New York, Abstr. V., 443. Lovkova, M. Ia. 1964. Metabolism of nicotine in tobacco. Acta Biol. 14,273. Makhmudov, K. I. 1962. Single firing in the new technology of black tea manufacture. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 9,122. Mann, H. 1902. The ferment of tea leaf and its relation to quality of tea. Colombo. Mann, H. 1903. The ferment of tea leaf. Part 11. Calcutta. Mann, H. 1904. The ferment of tea leaf. Part 111. Calcutta. Mann, H. 1914. Fermentation of tea leaf. Russkii Subtropiki 2 , l .

CHEMISTRY & BIOCHEMISTRY O F TEA &TEA MANUFACTURE

287

Manskaya, S. M. 1935a. Biological state of the tea leaf during the withering process. Biokhim. Chain. Proizv. Akad. Nauk SSSR Sb. 1,18. Manskaya, S. M. 193513. Peroxidase in the tea leaf. Biokhim. Chain. Proizv. Akad. Nauk SSSR Sb. 1,96. Markh, A. T. 1950. On changes of fruit product color upon thermal treatment. Biokhimiya 15,2. Martinek, R., and Wolman, W. 1955. Xanthines, tannins and sodium in coffee, tea and cocoa. J . Am. Med.Assoc. 158,1030. Matuurd, S . , Kokubu, N., Wakimoto, S., and Tokimasa, M. 1954. Fluorine in tea. Met. Tac. Sci. Kyushu Univ. 2,1,34. Melimonadze, S. N. 1966. Biochemical characteristics of raw tea and manufactured tea of main typical areas of the Georgian SSR. Biokhim. i Progr. Technol. Chain. Proizv. 203. Michel-Durand, M. 1929. Recherches physiologiques sur les composes tanniques. Revue gen. botan. 41,772. Michl, H., and Haberlei, F. 1954. Determination of purines in caffeine containing drugs. Monatsh. 35,770. Chem.Abstr.49,4237. Miyzawa, S. 1953. Metabolism of microorganisms. VI. Determination of vitamins in foods by bioassay. Ann. Rept. Takamine Lab. 5.49. Chem. Abstr. 49,14211. Mizuno, T., and Kimpyo, T. 1955. Carbohydrates of tea. I. The kinds of carbohydrates in black tea.]. Agr. Chem. Soc.Japan29,11,847. Mokhnachev, I. G., and Kamenshchikova, S. V. 1966. Study of carbonyl compounds of tea. Biokhim. i Progr. Chain. Proizu. 87. Mdller, H. 1888. Ber. Deutsch. Botan. Ges. 6. Des gerbstoffes in den pflanzen.]ena. Z. Naturw. 24, (N.F.), 17. Miilder, C. J. 1838. Uber coffein und thein.]. Prakt. Chem. 15,280. Nakabayashi, T. 1953. Separation and estimation of rutin in tea leaves. J . Agr. Chem. Soc.Japan.27,274. Nakabayashi, T. 1956a. Pectic substances of tea. I. The determination of pectin in tea leaves. J. Agr. Chem. Soc.Japan 30,402. Nakabayashi, T. 1956b. The formation mechanism of black tea aroma. I. Chemical changes of essential oils in manufacture. J . Agr. Chem. Soc. Japan 30,451. Chem. Abstr. 52,610. Nakabayashi, T. 1956c. The formation mechanism of black tea aroma. 11. Determination ofthe volatile carbonyl compounds in black tea. J . Agr. Chem. Soc.Japan30,641. Nakabayashi, T. 1957. Pectic substances of tea. 111. Relation between the quality of green tea and pectin content. J . Agr. Chem. Soc. Japan 30,402. Nakabayashi, T. 1958a. Formation mechanism of black tea aroma. 111. Qualitative experiments on the volatile carbonyl compounds of black tea. J . Agr. Chem. Soc. Japan 32,110. Nakabayashi, T. 1958b. Formation mechanism of black tea aroma. IV. Effect of the condition of black tea manufacture on the formation of volatile carbonyl compounds. J . Agr. Chem. Soc. Japan 32,757. Nakabayashi, T. 1 9 5 8 ~Formation . mechanism of black tea aroma. V. The precursor of the volatile CdrbOnyl compounds.J. Agr. Chem. Soc. Japan 32,941. Nanninga, A. 1901. “Verslag onderzoekingen Java gecult theen.” Buitenzorg. Nevill, Y. 1928. “Tea Manufacture.” Tbilisi. Izd. Chai-Cruzia. Newton, C. 1901. Oxidising enzymes. Ind. Card. Plant 9,418. Nick, E. 1953. Beitrage zur Physiologie der Gerbstoffe und Flavonfarbstoffe Polygonum Bistorta. Pharmazie 8,940.

288

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Nikolaishvili, D. K., and Adeishvili, N. I. 1966. Chromatographic study of quantitative changes of pigments of the tea leaf upon its processing. Bull. USSR Res. Znst. of Tea Ind. 2 (21), 57. Nikolaishvili, D. K., and Shipalov, M. S. 1967. Study of the visible spectral characteristics of raw tea during its processing to manufacture black and green tea. Bull. USSR Res. Znst. of Tea Znd. 2, (3). Nizheradze, A. N. 1946. The role of phosphorus compounds in tea leaf metabolism. Biokhim. Chain. Proizv. Akad. Nauk S S S R Sb. 5,67. Oparin, A. I. 1935. Biochemical theory of tea manufacturing. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 1,6. Oparin, A. I. 1936. Technological control at tea factories. Biokhim. Chain. Proizv. Akad. Nauk. S S S R Sb. 2,73. Oparin, A. I. 1967. Achievements of Soviet biochemistry as the theoretical foundation of the food production. Prikl. Biochim. i Mikrobiol. 3,5,509. Oparin, A. I., and Shubert, T. A. 1950. On oxidative respiratory systems in the tea leaf. Biokhim. Chain. Proizv. Akad. Nauk S S S R Sb. 6,82. Oragvelidze, N. I., and Bokuchava, M. A. 1966. Isolation of crystalline catechins from high-quality black tea. Proc. Second Intern. Congr. Food Sci. Technol. 207. Oragvelidze, N. I., Bokuchava, M. A., and Knyazeva, A. M. 1966. The paper chromatography investigation of catechins from raw and manufactured tea. Biokhim. i Progr. Technol. Chain. Proizv. 98. Oshima, J. 1936. Chemical studies on the tannin substance of Formosa tea leaves. BulLAgr. Soc.japan 28,269. Oshima, J., and Nakabayashi, T. 1953. Studies on catechins and pigments in tea leaves. Part 5. Paper chromatography of tea flav0nes.J. Agr. Chem. SOC.japan. 27,754. Oshima, J ., Nakabayashi, T., and Nishida, S. 1952. Semiquantitative estimation of catechins in tea 1eaves.J. Agr. Chem. Soc. Japan. 26,367. Oshima, J., Nakabayashi, T., and Sakamoto, J. 1954. Studies on the black tea tannin. Part 1. Chemical changes on the polyphenol compounds and amino acids in the manufacture of black tea. J . Agr. Chem. Soc. Japan.27,264. Oudry, K. 1827. “Thein, eine organishe Salzbase im Thee.” Carlsruhe. Palladin, V. I. 1912. The role of respiratory pigments in oxidative processes of plants and animals. Zzvestia Akad. Nauk 6,5,437. Pomazanov, I. A., Gorbacheva, R. T., and Balukova, A. A. 1966a. Results of application of the new technology of tea manufacture at the Adler tea factory in 1960-1964. Biokhim. i Progr. Technol. Chain. Proizo. 297. Pomazanov, I. A., Shulga, V. G . , Balukova, A. A., and Kaplunova, C . A. 1966b. On biochemical control in the new technology application. Biokhim. i Progr. Technol. Chain. Proizv. 307. Popov, V. R. 1956. Oxidation of amino acids in the presence of tannins and polyphenol oxidase of tea. Biokhirniyu 21,380. Popov, V. R. 1959. On a new amide in tea. Biokhim. Chain. Proizu. Akad. Nauk S S S R Sb. 7, 163. Popov, V. R. 1966a. Quantitative evaluation of theanine and free amino acids during the development and processing of tea shoots. Biokhim. i Progr. Technol. Chain. Proizu. 110. Popov, V. R. 1966b. Participation of ascorbic acid in oxidative processes during the tea manufacture. Biokhim. i Progr. Technol. Chain. Proizu. 128. Potapov, A. I. 1940. The role of withering in the tea manufacture. Souet. Subtropiki 6.50.

CHEMISTRY & BIOCHEMISTRY O F TEA &TEA MANUFACTURE

289

Prostoserdov, N. N. 1917. “The Cultivation and Manufacture of Tea in the Transcaucasia.” Tiflis. Pruidze, G . N . 1962. Production of the dry tea concentrate by freeze-drying. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 9, 177. Ralyanov, A. P., Pomazanov, I. A,, Shulga, V. G . , and Balukova, A. A. 1962. Results of the application of the new technology of tea manufacture at the Adler tea factory in 1960-1961. Biokhim. Chain. Proizu. Akad. Nauk S S S R Sb. 9,96. Roberts, E. 1939. The fermentation process in tea manufacture. Biochem.]. 33,842. Roberts, E. 1940. The fermentation process in tea manufacture. Biochem.]. 34,500. Roberts, E. 1942. The chemistry of tea fermentation. Aduan. Enzymol. 2,113. Roberts, E. 1957. Oxidative condensation of flavonols in tea fermentation. Chem. 6 Znd. 13,55. Roberts, E. 1958a. The phenolic substances of manufactured tea. ]. Sci. Food Agr. 9, 212. Roberts, E. 1958b. The chemistry of tea manufacture.]. Sci. Food Agr. 9,381. Roberts, E. 1959. The nature of oxidized phenolic substances of black tea. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 7,133. Roberts, E. 1962. Economic importance of flavonoid substances in tea fermentation. I n “The chemistry of flavonoid compounds.” Pergamon Press. Roberts, E., and Mayers, M. 1959. The phenolic substances of manufactured tea. J . Sci. Food Agr. 10,167. Roberts, E., and Rastege, D. 1962. Volatile aldehydes in black tea extracts. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sh. 9, 182. Roberts, E., and Russell, G. 1957. Oxidation of gallic acid and gallic acid esters to aconitic acid. Chem. 6 Znd. (London)1957, 158. Roberts, E., and Smith, R. 1961. Spectrophotometric measurements of theaflavins and thearubigens in black tea liquors in assessments of quality in teas. Analyst 86,94. Roberts, E., and Smith, R. 1963. The phenolic substances of manufactured tea. IX. The spectrophotometric evaluation of tea liquors.]. Sci. Food Agr. 14,689. Roberts, E., and Williams, D. 1958. The phenolic substances of manufactured tea. 111. Ultraviolet and visible absorption spectra.]. Sci.Food Agr. 9,217. Roberts, E., and Wood, D. 1950. The fermentation process in tea manufacture. 11. Oxidation of substrates by tea oxidase. Biochem. J . 47, 175. Roberts, E., and Wood, D. 1951a. Oxidation of anthoxanthins by tea oxidase. Nature 167,608. Roberts, E., and Wood, D. 1951b. A study of the polyphenols in tea leaf b y paper chromatography. Biochem.]. 49,414. Roberts, E., Cartwright, R., and Wood, D. 1956. The flavonols of tea. J . Sci. Food Agr. 7,637. Roberts, E., Cartwright, R., and Oldschool, M. 1957. The phenolic substances of manufactured tea.]. Sci. Food 8,27. Romburg, V., and Lohmann, C. 1898. Onderzoekingen betreffende op Iava gecultiveerde theen. Verlag omtrent den stadt van s’hand Plantentuin te Buitenzorg. Batavia. Sakato, Y. 1950. Studies on the chemical constituents of tea. 111. On a new amidetheanine. Agr. Chem. Soc. Japan. 23,262. Sakato, Y., and Matumura, T. 1955. Studies on the chemical constituents of tea leaves. VII. Isolation of myoinositol from water extracts of dried tea leaves. Nippon Nogei-kagaku Kaishi 29,771.

290

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Sakato, Y., Ingar, T., and Matumura, T. 1955. Chemical constituents of tea leaves. hlippon NBgei-kagaku Kaishi 29,769. Sanderson, G., and Selvendran, K. 1965. The organic acids in tea plants. A study of the non-volatile organic acids separated on silica gel.]. Sci. Food Agr. 16,5,251. Serenkov, G. P. 1959. Nauch. dokl. oyshei shkoly, Ser. Biol. 2. Serenkov, G. P. 1962. Study of nitrogen metabolism in the tea plant. Biokhim. Chain. Proizo. Akad. Nauk SSSR Sb. 9,27. Serenkov, G . P., and Proiser, E. 1961. On the biosynthesis of caffeine in tea leaves. Dokl. Akad. Nauk S S S R 140,3,716. Shavishvili, M. N. 1940. Biochemical characters of green tea leaf and underfermented tea. Biokhim. Chain. Proizu. Akad. Nauk SSSR Sb. 4,53. Shaw, W. 1934. Studies on the pectic substances in tea. United Planters Assoc. South India, Tea Sci. Dept. Bull. (Madras)6,4. Shaw, W., and Jones, T. 1932. “Theotannin.” Madras. Shuen, T., and Mei, T. 1944. Caffeine and ascorbic acid contents of different varieties of tea from Kunming, China. Proc. Chinese Physiol. SOC. Chegty Br. 2,69. Skobeleva, N. I. 1967. Participation of free amino acids in flavour formation during tea thermal treatment.j. 7th Intern. Congr. Biochem. Abstr. V, 296, 1020. Skobeleva, N. I., and Popov, V. R. 1962. Interaction of tannins, amino acids and sugars under high temperature conditions. Biokhim. Chain. Proizo. Akad. Nauk S S S R Sb. 9, 185. Skobeleva, N. I., Bokuchava, M. A., and Knyazeva, A. M. 1958. Changes in the composition of volatile aldehydes during thermal treatment. Dokl. Akad. Nauk SSSR 118,6. Soboleva, G. A. 1961.The formation and transformation of organic acids in the tea plant. 5th Znternl. Congr. Biochem. Abstr. 1,383. Soboleva, G . A. 1962. Study of organic acids of the tea plant. Biokhim. Chain. Proizo. Akad. Nauk SSSR Sb. 9,20. Soboleva, G . A. 1966. Oxidation of citric and malic acids by the polyphenol-polypheno1 oxidase system. Biokhim. i Progr. Technol. Chain. Proizu. 37. Soboleva, G. A,, Tchetverikov, A. G., and Bokuchava, M. A. 1966. Study of the mechanism of enzymic oxidation of catechins by the electron paramagnetic resonance method. Biokhim. i Progr. Technol. Chain. Proizu. 116. Sprecher, A. 1936. “Der Teestrauch und der Thee.” Stuttgart. Sreerangacher, H. 1941. Studies on the fermentation of Ceylon tea. Biochem. J . 35, 1106. Sreerangachar, H. 1943. Studies on the fermentation of Ceylon tea. Bi0chem.J.37,653. Stahl, W. H. 1962. The chemistry of tea and tea manufacturing. Advances in Food Research 2,201. Takei, S., and Sakato, J. 1933. On the aromatic substances of green tea leaves. Bull. Znst. Phys. and Chem. Res. (Tokyo)12,1,13. Takei, S., Sakato, J., and Ono, M. 1935. The aroma of black tea. Bull. Znst. Phys. and Chem. Research (Tokyo)14,303. Takeo, T. 1965. Tea leaf polyphenol oxidase. I. Solubilization and properties of the structurally bound polyphenol oxidase in tea leaves. Agr. Biol. Chem. 29,6,558. Takeo, T., and Uritani, J. 1966. The leaf polyphenol oxidase. 11. Purification and properties of the solubilized polyphenol oxidase in tea leaves. Agr. Biol. Chem. 30,2, 155. Takino, Y.,and Imagawa, H. 1963a. Studies on the oxidation of myricetin-glycosides by tea oxidase. Formation of erycitrin, a new red-colored glycoside, having a benzotropolone nuc1eus.J. Agr. Biol. Chem. 27,9.

CHEMISTRY & BIOCHEMISTRY O F TEA &TEA MANUFACTURE

291

Takino, Y., and Imagawa, H. 1963b. Studies on the mechanism of the oxidation of tea leafcatechins.J . Agr. Chem. SOC.Japan 37,417. Takino, J., Imagawa, H., and Yoshida, H. 1953. Studies on the flavonoids in the leaves. J . Agr. Chem. SOC.Japan 27,150. Takino, J., Imagawa, H., and Yoshida, H. 1954. Studies on the flavonoids in the leaves. IV. On the quercetin-glycoside. J . Agr. Chem. Soc. Japan 28, 190. Takino, Y., Imagawa, H., and Yoshida, H. 1962a. Studies on the flavonoids in tea leaves. V. On the quercetin-glycoside, quertrin and the kemfetrin. J. Agr. Chem. SOC. Japan 36,943. Takino, Y., Imagawa, H., and Yoshida, H. 1962b. Studies on the flavonoids in tea leaves. VI. On the myricetin-glycoside. J . Agr. Biol. Chem. 26,lO. Tea Board, India, 1957. Nutritional aspects of tea. I. Studies on theamin, riboflavin, folic acid, pantothenic acid and nicotinic acid. Sci. Publ. Ser. 1. Tea Board, India, 1958. Chemical investigations of India tea. Sci. Publ. Ser. 2. Tea Board, India, 1959a. Chemical investigations of India tea. 11. Sci. Publ. Ser. 3. Tea Board, India, 1959b. Nutritional aspects of tea. 11. Sci. Publ. Ser. 4. Timoshenko, M. G. 1936. Tea selection for the chemical constituents. Souet. Subtropiki 1,25. Tirimanna, A., and Wickremasinghe, R. 1965. Studies on the quality and flavour of tea. I. The carotenoides. Tea Quart. 36,3,115. Tocklai Experiment Station. 1958. Tocklai Ann. Reports. Torii, H., and Kanazawa, J. 1954. Studies on the carbohydrates in the tea leaves. I. J . Agr. Chem. Soc.Japan 28,34. Tsekhomskaya, V. M. 1940. Biochemical studies of tea dried by ultrahigh frequency current. Biokhim. Chain. Proizv. Akad. Nauk SSSR Sb. 4,79. Tsujimura, M. 1929. On tea catechin isolated from green tea. Sci. Papers lnst. Phys. Chem. Research Tokyo 10,253. Tsujimura, M. 1934. Isolation of a new catechin - tea catechin I1 or gallocatechin from green tea. Sci. Papers lnst. Phys. Chem. Res. Tokyo 24,149. Tsujimura, M. 1935. Isolation of tea tannin in crystalline state from green tea. Sci. Papers Inst. Phys. Chem. Res. Tokyo 26,186. Tsujimura, M. 1963. Tannin in tea leaf. Proc. Japan Acad. 39,6,376. Tsujimura, M., and Masataro, M. 1924-1926. Vitamin C in green tea. Proc. Japanese Assoc. Agr. Chem. Tsujimura, M., and Osawa, N. 1957. Amino acids in green tea. JissenJoshi Daigaku Kigo, 4,5. Chem. Ahstr. 41,5924. Ugai, T., and Hayashi, I. 1959. Tea as an anti-strontium 90 agent. Moscow, Gosinti. Ukers, W. H. 1935. “All About Tea.” New York Vol. I. 559 pp; Vol. 11.568 pp. Ulyanova, M. S. 1962. Study of the qualitative and quantitative changes in flavones of the tea plant. Biokhim. Chain. Proizv. Akad. Nauk SSSR Sb. 9,7. Ulyanova, M. S. 1963. Flavones in tea leaves. 1st USSR Congr. Biochem. Abstr. 3, 31. Izd. Nauka, Moscow-Leningrad. Ulyanova, M. S. 1966. Transformations of flavonol glycosides in the course of the black tea manufacturing. Proc. 2nd Intern. Congr. Food Sci. Technol. 195. Ulyanova, M. S., and Erofeyeva, N. N. 1966. Isolation of flavonols from black tea and evaluation of their biological activity. Biokhim. i Progr. Technol. Chain. Proizu. 7. Umikov, N. Z. 1947. “Fruits, Berries, Vegetables, Cereals and Spices.” 120 pp. Tbilisi, Gruz. Medgiz. Vepkhvadze, V. S., Gulua, K. P., Khavtasi, V. A,, and Bokuchava, M. A. 1966. On the economic efficiency of the introduction of the new technology for the black baikhovi tea manufacture with the use of the thernial treatment of underfermented tea. Biokhim. i Progr. Technol. Chain. Proizv. 315.

292

MIKHAIL A. BOKUCHAVA AND NINA I. SKOBELEVA

Vorontsov, V. E. 1928. Peculiarities of the chemical composition of the Georgian tea. Trudy Priklad. Botan. Genet. i Selektsii 18, 106. Vorontsov, V. E. 1939. Tea aromatization. Proc. USSR Res. Inst. Tea and Subtropi. Cultures, (Tbilisi) 13. Vorontsov, V. E. 1946. “The Biochemistry of Tea.” 278 pp. Moscow, Pishchepromizdat. Vuataz, H., and Brandenberger, H. 1961. Plant phenols. I. Separation of fermented and black tea polyphenols by cellulose column chromatography. J. Chromatog. 5, 17. Vuataz, L., Brandenberger, H., and Egli, R. 1959. Plant phenols. I. Separation of the tea leaf polyphenols by cellulose column chromatography. J. Chromatog. 2, 173. Wada, T., and Sakurai, Y. 1952. The vitamin B, content of tea. J . Japan Soc. Food Nutrition 4, 217. Chem. Abstr. 46, 9228. Westermaier, M. 1885. B e d . Akad. Ber. 1115. Wickremasinghe, R., Roberts, G., and Perera, B. 1967. Localisation of the polyphenolase of tea. 7th Intern. Congr. Biochem. Abstr. V, 1-228, 1003. Yamanishi, T., Kobavachi, A., Sato, H., Nakamura, H., Osawa, K., Uchida, A., Mori, S., and Saijo, R. 1966. Flavor of black tea. IV. Changes in flavor constituents during the manufacture of black tea. Agr. Biol. Chem. 30, 8, 784. Zapromyotov, M. N. 1950. Participation of serial (1,2,3)hydroxyls in transformations of tea plant tannins. Biokhimiya 1.5, 137. Zapromyotov, M. N. 1952. Chromatographic fractionation of tea tannins. Biokhimiya 17, 97. Zapromyotov, M. N. 1958a. Quantitative evaluation of catechins upon their paper chromatography fractionation. Fiziologia Rastenii 5, 296. Zapromyotov, M. N. 195813. On the site of catechin formation in the tea plant. Fiziologia Rastenii 5, 51. Zapromyotov, M. N. 1959. On the ability of higher plants to split the benzene ring. Deep oxidation of I4C-catechins in tea shoots. Dokl. Akad. Nauk S.S.S.R.125,1359. Zapromyotov, M. N. 1961. Isolation of quinic and shikimic acids from tea shoots. Biokhimiya 26, 373. Zapromyotov, M. N. 1962. On the mechanism of catechin biosynthesis. Biokhimiya 27, 366. Zapromyotov, M. N. 1964. “Biochemistry of Catechins.” 294 pp. Moscow, Izd. Nauka. Zapromyotov, M. N. 1967. Biochemistry of phenol compounds. Uspekhi Sovremennoi B i d . 63, 3, 380. Zapromyotov, M. N., and Bukhlaeva, V. Ya. 1963. Gallic acid and catechin biosynthesis. Dokl. Akad. Nauk S.S.S.R., 151, 291. Zapromyotov, M. N. and Kolonkova, S. V. 1965. The diurnal dynamics of catechins and alicyclic precursors (quinic and shikimic acids) in the tea plant. Fiziologia Rastenii 12,646. Zapromyotov, M. N., and Soboleva, G. A. 1954. Study of the transformation of catechins upon the tea fermentation by paper chromatography. Dokl. Akad. Nauk S.S.S.R., 96, 1205. Zimmerman, P., Hitchock, A,, and Gwirtsman, J. 1957. Fluorine in food with special reference to tea. Contribs. Boyce Thompson Inst. 19, 1,49. Zoller, K., and Libich, L. 1871. Untersuchung eines Himalayathees. Ann. Chem. u. Pharm.

SUBJECT INDEX

A Acetaldehyde, 18 in anaerobic metabolism, 20 in flavor deterioration, 20-21 in frozen vegetables, 5, 6 role in off-flavor development, 5-6 Acetoin flavor of vegetables, 158 in food deterioration, 20-21 Acids, see also individual acids in fruits, 177 organic, in tea, 243-244 unsaturated, in off -flavor production, 33 Activation energy of, 38 enthalphy of, 38 entropy of, 4 5 Adenosine triphosphate, 18-20 Aerobacter, in fermentation, 93 Aglycones, 29 in flavonols, 227 Alcohol from fruits, 94 from molasses, 125 production of, 20-21, 93, 125, 126 Alcoholic fermentation, 196-199 Aldehydes formation of, 240-241 in tea aroma, 239 Aleurone concentrate, 115 Alkaloids, in tea, 234-236 Amino acids essential, 102-103 in corn by-products, 118 in fruits, 183 metabolism, enzymes of, 22-23

oxidation of, 29, 31 in tea, 236-241 Anaerobic glycolysis, 20 Animal feed, 79 Animal feeds apple pomace, 94 brewery wastes, 130 citrus molasses, 90-91, 124-125 citrus pulp, 90-93 corn, 81, 120 cottonseed, 109 from mangoes, 194 pineapple bran, 95-96 safflower meal, 112 Animal waste products, 132-135 Anthocyanins in corrosion, 202 decoloration by peroxidase, 18 in tropical fruit, 174 Antioxidants, 27, 31 natural, 59 Apoenzyme, of enzymes, 34 Apple wastes, 94-95 pomace, as animal feed, 94 Aroma development of, 274 of tea, 233, 248 Aromatic substances, in tea, 248-255 Arrhenius equation, 35 Ascorbic acid, see also Vitamin C accumulation of, 231 destruction of, 4-5,17 loss in frozen foods, 4-5 oxidation of, 13 as substrate, 16 in tea, 218 Asparagus, waste, 85-86 ATP, see Adenosine triphosphate 293

294

SUBJECT INDEX

B Bactericidal activity of tea polyphenols, 218 Bagasse, definition of, 123-124 Bananas flavor of, 176 usage of, 104-105 Beet pulp, composition of, 123-124 Biological value of tea, 278-279 Blanching effect on chlorophyll, 5 efficiency of, 3 4 of fruits and vegetables, 3 Boltzmann constant, 38 Brandy, production of, 94 Breadfruit, anatomy of, 170 Brewery wastes, 128-131 as animal feeds, 130 Bromelin, in pineapple, 188 Browning enzymic, 18-19 in frozen fruits, 4 C

Caffeine effects of, 234-235 structure, 234 synthesis, 236 in tea, 234 Capillary-strengthening effect, see Vitamin P Carbohydrates in fruits, 182-183 in tea, 231-233 Carbon monoxide, as inhibitor, 53 Carbonyls, see also individual carbonyls in peas, 6 production of, 20 unsaturated, in peas, 32 6-carotene, oxidation of, 6,28430 Carotenoids in fruits, 190 odors from, 33 pigments, in tropical fruits, 174 in tea, 241 Castor bean meal, 107 Catalase, 10 effect of temperature, 42-43 in food deterioration, 18

oxidation mechanisms, 16-17 regeneration, 56-57 Catalysis enzymic, 9 metallic, 7-8,27 nonenzymic, 4 Catechins condensation of, 268-271 in fermentation, 265 protection against Strontium-90, 218-219 reaction with amino acids, 240 structure of, 222-223 synthesis, 225-227, 244 in tea, 217, 221, 258, 276, 278 Vitamin P activity, 218 Catecholase, see Oxidase, o-diphenol Cellulose, in fruits, 182 Chlorogenic acid, reaction with o-diphenol oxidase, 11 Chlorophyll degradation of, 14, 3 1 3 2 effect of blanching, 5 in fruits, 174 odors from, 33 retention in frozen vegetables, 4-5 in tea, 241-243 Chlorophyllase, 22 Chromatography of aldehydes, 253 of amino acids, 239 of carbohydrates, 231 of flavonols, 228-229 gas-liquid, 6, 253-254 paper, of tea catechins, 221-223 partition, of tea tannins, 221 Citric acid, in fruits, 177-179 Citrus fruit by-products of, 89-94 composition, 91 origin, 90 Citrus pulp, nutritional properties, 90 Citrus seed meal, composition, 91-92 Cocoa beans, harvest, 106 Coconut extraction of, 111 impulse process, 112 oil and meal from, 111 proteins of, 111-112 usage, 110-112

SUBJECT INDEX Coefficients activity, 38 temperature, 35 Coenzyme A, 25 Coenzymes, 18-20, 34 Collision theory, 38 Constants Bokzmann, 38 equilibrium, 38 Planck's Copra, from coconuts, 110 Corn by-products, 117 gluten feed, 117-120 Corrosion, in cans, 201-212 Cottonseed glandless, 109-110 proteins, nutritive value, 110 products from, 108-110 usages, 78-79 Cresolase, see Oxidase, o-diphenol Crocin, oxidation of, 16, 30 Cytochromes, 19-20, 59 in electron transport, 19-20 Cyotchrome c, 10-11 inhibition of oxidation, 30 as oxidative catalyst, 27-28 of peroxidase, 29 Cytochrome oxidase, 10-11, 20 in tea, 258

D D value, see Decimal reduction time Dairy product wastes, 132-133 Deamination of amino acids, 239 oxidative, 246 Decimal reduction time ( D value), 37,43 in fruit purkes, 43 for oxidase, 43 Dehydrated products of tropical fruits, 194-195 Dehydration of citrus waste, 89-90 economics of, 99 Dextrose, in fruits, 179 Diacetyl, in food deterioration, 20-21 Dinucleotides, 19 Distillery wastes, 126-128

295

E Enzymes, see also individual enzymes absorption of, 50 activity, at low temperatures, 47 thermal and environmental factors, 34-53 of amino acid metabolism, 22-23 deterioration of foods, 5 effects of water, 48 in fruits, 183-186 heat resistance of, 3-4 inactivation of, 3-4, 34-46 inactivation, equation, 37 in fruits, 183-184 thermodynamics of, 37-39 inhibitors, 52-53 from plants, 59-60 molecular forms, 49-50 oxidative, 217 oxidizing, 4, 7-19 in plants, classification, 7 thermal inactivation, 3 9 4 7 pectic, 21-22 pectolytic, 44 regeneration of, 3, 53-57 respiratory, 19-23 iso, see Isoenzymes in tea, 255-259 effect of thermal treatment, 273 Essential oils, composition in tea, 250-251

F F, value, 37, 39 Fats in fruits, 183 unsaturated, 27 Fatty acids, losses of, 32 Fermentation, 20 of citrus by-products, 9 3 of molasses, 125 in tea manufacture, 264-272 Fermentation products, from tropical fruits, 196-199 Firing, in tea manufacture, 265-266, 272 Fish-protein concentrate (FPC), 135 Flavonols, in tea, 218, 227-231 Flavoproteins, 19

296

SUBJECT INDEX

Flavors changes due to pemxidase, 18 deterioration, 31-33 detection of, 5 “fishy,” 33 off-, 21 caused by oxidation, 33 production of, 32-33 in vegetables, 6 Folic acid, in turkey feeding, 31 Food processing definition, 153 importance of, 154-155 Food science, definition, 154 Food technology, definition, 153-154 Freezing of fruits, 185-188 Fruit beverages preparation of, 172-173 juice, 190-194 pulp, 191-194 Fruits acid content of, 177 aroma of, 174-179 carbohydrates in, 182-183 chemical properties of, 174-185 classification of, 158 color of, 174-179 definition, 157 dehydrated products of, 194-195 flavor, classification of, 174-179, 180 morphology of, 163-165 nutrients, 159 oxidative systems, 1-76 peeling of, 188-190 pH, 178 physical properties of, 165-172 preservation of, 185-188 processing wastes, 88-96 respiration of, 186-187 significance of, 157-163 technology, tropical, 155-156 tropical, classification, 162-163 wastes, 104-106

G Galactoglycerides, unsaturated acids of, 33 Glycollate oxidase, 13-14, 26 Glycollic acid, oxidation of, 14

Glycolysis, 225 anaerobic, 120 Glycosides ( flavonol), in tea, 228-230 Gossypol, in cottonseed, 109 Grains as feeds, 114-115 utilization of, 106-115 Guava, composition of, 160 H Hematin, catalytic activities, 27-29 Hexanal, in frozen vegetables, 6 Hop residue, 131 Hydrogen peroxide enzymic production of, 13-14, 17,26 as substrate, 16 Hydroperoxides decomposition of, 28-30 formation of, 31 of linolenate, 29-30

I Ionic strength, effect on enzymes, 47-48 j3-ionone, role in off flavors, 6 Impulse process, isolation of lipid-protein complexes, 104 Incaparina, 110 Inhibitors, enzyme, 51-53 Inositol, in tea, 225 Invertase, 198 in fruits, 184 Irradiation, in fruit preservation, 204 Iron, in tea, 248 Isoenzymes, 49-50 of lactic dehydrogenase, 49 of peroxidase, 49 of phenol oxidase, 49-50

J Juice, fruit, preparation of, 172-173, 190-194

K Krebs cycle, 19-20,25

SUBJECT INDEX

L Lactic dehydrogenase, 20, 49 isoenzymes of, 49 Lecithin, in non-enzymic browning, 33 Linseed meal, 107 Lipase, 24-25 deterioration of peas, 32 Lipids autoxidation of, 27-28 biological oxidation of, 25-26 degradation of, 24-26 oxidation in food, 31-33 oxidative changes, 23-34 plant-oxidation mechanisms, 59 unsaturated, in oxidation, 6 “Lipid oxidation potential,” 33 Lipid-protein isolates, 104 Lipoxidase, see Lipoxygenase Lipoxygenase, 52, 59 activity in beans, 31 in carotene destruction, 30 free radical formation, 28-29 heat inactivation of, 42 in off-flavor production, 32 specificity of, 26-27 Lysine, deficiency in seed, 107, 112, 117 M Malonaldehyde, 33 Mangoes composition of, 160 flour, 194 physical properties, 173 physiochemical changes, 164-165 starch micrograins from, 168 Manufacture of tea, 260 black tea, 261 Marine waste products, 132-135 Maxwell-Boltzmann, distribution law, 36 Meat, by-products, 133 Metal ions, influence on peroxidase, 16 Methionine, deficiency in leaf proteins, 102 Mineral salts, in fruits, 181 Minerals, in tea, 247-248 Molasses, 124-125 citrus, composition of, 92 production of, 90

297

production of, 94 alcohol from, 80

N NAD+, see Nicotinamide-adenine dinucleotide NADH, see Reduced nicotinamide dinucleo tide NADP, see Nicotinamide-adenine dinucleotide phosphate Nicotinamide-adenine dinucleotide, 19-20, 25 Nicotinamide-adenine dinucleotide phosphate, 16, 19 Nutrients, loss of, 81 Nutritional value, of tea, 278-279 0

Off-flavors, see Flavors, offOils, autoxidation, 28-29 Oilseeds, utilization of, 106-115 Oxidases amine, 13 ascorbate, 13, 17 cytochrome, 10-11, 20, 258 in food deterioration, 18 glycollate, 13-14 o-diphenol, 12, 17 reactions of, 11-12 thermal inactivation, 43 p-diphenol, 12 polyphenol, 239, 244 chromatography of, 257 in fermentation, 265 in oxidation, 267 in tea, 229, 256,258-259 in tea, 256 a-oxidation, 25-26 @-oxidation,25

P Papain, in papaya, 184, 188 Papaya, flavor of, 176 Passion fruit, flavor of, 176 Peanut meal, deficiencies of, 107 Pear juice preparation of, 94 wastes, 94-95

298

SUBJECT INDEX

Pectic substances in fruits, 182 in tea, 233-234 Pectin in gels, 195-196 manufacture of, 95 Pectinesterase, 22 in fruits, 184 Pectin methyl esterase, 52 in bananas, 50 Pectolytic enzymes, 52 Peroxidase, 59 in food deterioration, 18 oxidation mechanisms, 14-16 in papaya, 183 in processing, 5 properties of, 8-9 pseudo-, 9 regeneration of, 53-56 in tea, 231, 256 thermal inactivation of, 39-42 Peroxidase activity, as blanching index, 395 Peroxides formation of, 27 linolenate and linoleate, 29 pH, effect on enzymes, 47-48 Phenolase, see Oxidase, o-diphenol Phenolic substances, in tea, 220-231 Phenolic-(non) substances, in tea, 231-248 Pheophytin a and b, 242 formation of, 32 Phloroglucinol condensation of, 258-259 in tannin formation, 225 Phosphatase, inactivation of, 38-37 Phosphatides, in nonenzymic browning, 33 Phospholipids breakdown of, 25, 32 in off-flavor production, 33 in oxidation, 32-33 Phosphorus, in tea, 247 Phycin, 184 Pigments in fruits, 174 in tea, 241-243 Pineapple, bran as animal feed, 95-96

bromelin in, 188 flavor of, 178-177 juice, usage of, 95 wastes, 95-96 Planck's constant, 38 Plant residues, utilization of, 9&106 Polygalacturonase, 22 Polyphenols bactericidal activity of, 218 radioprotective effects of, 218 in tea, 218 Polyphenol oxidase, see Oxidase, polyphenol Pomegranate, anatomy of, 169 Potassium, deficiency of, 247 Potato pulp composition of, 121 protein enriched, 122 Potato starch, 121-122 Potato wastes, 87-88 Poultry, by-products, 133-134 Prephenic acid, in catechin synthesis, 225 Processing effect on chlorophyll, 5 high-temperature short-time, 3-4 Prosthetic group of enzymes, 7, 34 protohematin IX,properties of, 8, 10 Proteins in corn by-products, 119 denaturation of, 36,45 extraction of, 101-102 in fruits, 183 source of, 100-104 in tea, 236-241 in tomato seed, 84 from tomato wastes, 103-104 Protein concentrate, from soybeans, 108 Protein efficiency ratio, 115 Proteolytic enzymes, 22, 184 Protohematin IX,molecular properties of, 8 Purine, 234 Pyruvate decarboxylase, 20-21

Q Quinones formation of, 267-269, 273 in oxidative deamination, 239 in tea, 217

SUBJECT INDEX

R Radiation, ionizing, on peroxidase activity, 40 Radiation disease, prophylaxis of, 218 Radicals free, 17, 28-29 in peroxidase oxidation, 16 peroxy, 31 Radioisotope techniques, 225 Rapeseed meal, 107 Reduced nicotinamide dinucleotide, 18-20 Respiration, 19-20 in fruits, 186-187 Respiratory chmmogenes, 227 Ribonucleic acid, 52 Rice bran, 107-108 Rice-mill feeds, 114 RNA, see Ribonucleic acid Rolling, in tea manufacture, 263-264

S Saccharomyces cereoiseae, growth in molasses, 124 Safflower meal composition, 112-1 13 for human consumption, 113 Sapodilla, anatomy of, 169 Sesame seed meal, 107 Shikimic acid, in catechin synthesis, 225 Silage preparation, 99-100 Soybeans, 108 Starch in fruits, 182 potato, 121-122 production wastes, 115-122 in tea, 231 Starch-protein complex, in corn, 117 Strontium-90, 218 Substrate, enzyme absorption, 50 Sugars in fruits, 182-183 invert, of fruits, 177 manufacturing wastes, 122-126 reaction with amino acids, 232-233 reducing, 232-233 in tea, 231 Sweet potato, wastes, 86

299

Sweet products, from tropical fruit, 195-196

T Tannic acid, in fruits, 177-179 as enzyme inhibitors, 52 Tannins in fermentation, 265 formation of, 223-225 origin, 224 separation of, 220-223 structure of, 222-223 in tea, 217, 220-227, 258-259 TBA, see 2-Thiobarbituric acid Tea aldehydes, 239 alkaloids, 234-236 amino acids in, 236-241 aroma, 233 biochemistry of, 260-272 biological value, 278-279 caffeine in, 234 carbohydrates, 231-233 chemical constituents of, 219-260 chemistry of, 215-292 dyes from, 219 enzymes of, 217 essential oils in, 251 instant, 219, 278-279 manufacturing of, 216-219 resinous substances in, 244-245 varieties of, 215 Theaflavin, 269-271 Theanine, in tea, 238 Thearubigens, formation of, 269-271 Theobromine, in tea, 235 Theophylline, in tea, 236 Thermal treatment mechanism of, 273 of tea, 272-278 Thermodynamics of enzyme inactivation, 37-39 2-Thiobarbituric acid, 21 in rancidity tests, 33 a-tocopherol, 31 Tomatine, isolation of, 99 Tomato, wastes, 81-85, 103-104 Torulopsis utilis, growth in molasses, 124 Toxic substances, in fruits, 185

300

SUBJECT INDEX

Tropical fruit anthocyanins in, 174 origin of, 161-163 technology of, 153-214 Trypsin, regeneration of, 53 Tryptophan, deficiency of, 117 Tung seed meal, 107 Tyrosinase, see Oxidase, o-diphenol

U Ubiquinones, 60

V Vegetables, anaerobic deterioration of, 5-6 Vegetable-oil, autoxidation of, 28-29 Vegetable-oxidation systems, 1-76 Vegetable-processing wastes, 80-88 Vegetable wastes as leaf meals, 98-99 as source of protein, 100-104 Vinegar, production of, 93, 96 Vitamins, in tea, 245-247 Vitamin A oxidation of, 29 in tropical fruits, 174-175 Vitamin B, 246 in fruits, 181 Vitamin C, 200,246, see also Ascorbic acid content of tropical fruits, 180-181 oxidation of, 18 Vitamin E, as antioxidant, 59-60 Vitamin K, 59-60, 246 Vitamin P, 245-246 activity in tea, 218, 231, 278-279

content of black tea, 271-278 capillary-strengthening effect in tea, 218 therapeutic effects in tea, 218 Volatile carbonyl compounds, 29 Volatile flavor components, 6

W Waste asparagus, 85-86 potato, 87-88 sweet potato, 86-87 tomato, 81-85 utilization as food, 76-152 Water, effect on enzymes, 48 Wheat, source of protein, 114 Wheat-mill feeds, 114 Wheat milling products, composition, 114 Whey, uses, 132-133 Winery wastes, 131-132 Wines, 196-197 Withering process, in tea manufacture, 261-263, 272

Y Yeast, see also individual organisms Brewers, 130-132 recovery of, 131 Torula, growth of, 94

Z 2 value of enzymes, 44-45 thermal inactivation, 39, 42 Zein-extracted gluten, 121