Advances in Food Research, Volume 9

ADVANCES I N FOOD RESEARCH VOLUME 9

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ADVANCES IN FOOD RESEARCH VOLUME 9

Edited by

C. 0. CHICHESTER University of California Davis, California

E. M. MRAK University of California Davis, California

G. F. STEWART University of California Davis, California

Editorial Board E. C. BATE-SMITH B. E. PROCTOR EDWARD SELTZER W. H. COOK P. F. SHARP W. F. GEDDES W. M. URBAIN M. A. JOSLYN J. F. VICKERY S. LEPKOVSKY 0. B. WILLIAMS

1959 ACADEMlC PRESS, New York and London

Copyright

0, 1959, by

Academic Press Inc.

ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. I l l FIFTHAVENUE NEW YORK3, N. Y.

Uniied Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) LTD. 40 PALLMALL,LONDON S.W. 1

Library of Congress Catalog Card Number 48-7808

PRINTED I N THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 9

D. J. CASIMIR,Commonwealth Scientific and Industrial Research Organization, Division of Food Preservation and Transport, Homebush, New South Wales, Australia J. F. KEFFORD,Commonwealth Scientific and Industrial Research Organization, Division of Food Preservation and Transport, Homebush, New South Wales, Australia

AMIHUD KRAMER,University of Maryland, College Park, Maryland

HANSLUTHI,Swiss Federal Agricultural Experiment Station, Wadenswil, Switzerland

L. J. LYNCH,Commonwealth Scientific and Industrial Research Organization, Division of Food Preservation and Transport, Homebush, New South Wales, Australia R. S. MITCHELL, Commonwealth Scientific and Industrial Research Organization, Division of Food Preservation and Transport, Homebush, New South Wales, Australia B. A. TWIGG, University of Maryland, College Park, Maryland JOHN

R. WHITAKER, University of California, Davis, California

V

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FOREWORD With the enormous growth of the literature in all scientific fields, good reviews are needed now as never before. The multiplication of facts, the integration of previously discrete fields of knowledge, and the wealth of publications tax the ability of the individual in a broad field to keep abreast of the subfields within his area of interest. A good review should report on developments in the expert’s field, put the relevant facts into their proper perspective, and point out possible fruitful avenues of research within the field. We believe that the last point is particularly important since, from these suggestions, new advances within the field may be made. As in previous volumes of Advances in Food Research, the present volume is divided into commodity and functional areas. Four of the five chapters are oriented toward particular products, namely citrus fruit, juices, peas, and meats. The other deals with disciplines which have been brought to bear on a variety of products. A balance has been sought between the technological aspects of food processing and the application of basic sciences to research on food products. From this aspect, this volume constitutes reviews devoted to the application of chemistry, microbiology, biochemistry, engineering, and instrumental techniques to research in food science. The review by Whitaker covers the increasingly important topic of meat processing. The fundamental aspects of this subject certainly have not received the attention merited by the importance of the commodity. As the reviewer points out, the number of publications within the field of protein biochemistry is overwhelming. By considering the general protein field from the viewpoint of a food scientist, the reviewer brings the chemical, enzymatic, and microbiological aspects of meat aging to a more understandable perspective. The excellent review of the chemistry and technology of peas by Lynch, Mitchell, and Casimir is an intensive review of a particularly important commodity. The selection and preparation of raw materials are considered as a part of food processing, an approach which will become increasingly important in food science. The chemical and enzymatic changes in peas during and prior to processing are considered intensively, as are the yet unsolved research problems related to peas. Kramer and Twigg discuss the extremely difficult subject of analyzing food for quality. The application of objective measurements to vii

viii

FOREWORD

the appraisal of subjective quality factors is a field which is just now being developed. The individual working in this area must not only consider the instrumentation for the measurements of chemical and physical aspects of food material, but also must consider the physiological and psychological aspects of quality. The article analyzes and critically reviews the work which has been done on the appearance factors and the aesthetics of food materials. The authors also consider flavor factors, a subject which is particularly lacking in objective measurement. The consideration of microorganisms in fruit juices has become of great importance with the trend toward decreasing the use of chemicals for food preservation. The chemical changes induced by the presence of microorganisms in juices may lead to off-flavor or off-color and general degradation of product quality. Liithi’s review, the first on the subject, is extremely complete, considering the types of organisms found, the possible source of the microorganisms, their control, and the chemical changes which they may induce. In view of the tremendous volume of microbiological literature, abstracting the articles pertinent to juice products is an impressive accomplishment. In contrast to the review on non-citrus juices, the article on the organic constituents of citrus fruits, by Kefford, is a review in depth on a narrow range of commodities. The vast utilization of citrus products, both as food as well as raw materials for chemical processing, makes citrus fruit one of the world‘s most important crops. The composition of the fruit is particularly important in its relation to processing, nutrition, and acceptability. The reviewers consider the alteration of the composition of the fruit by such diverse factors as rootstocks, horticultural sprays, position on tree, and size of the fruit. The chemical composition of the fruit is discussed with respect to carbohydrates, acids, vitamins, nitrogen compounds, enzymes, pigments, lipids, and flavoring compounds. The extensive work upon the volatile and non-volatile flavoring constituents is reviewed in detail. One is amazed by the number and complexity of the compounds which have been identified in citrus fruits.

December, 1959

C. 0. CHICHESTER

E. M. MRAK G. F. STEWART

CONTENTS CONTRIBUTORS TO VOLUME 9 .

FOREWORD . .

. . .

. . . . . . . . . . v . . . . . . . . . . . vii

Chemical Changes Associated with Aging of Meal with Emphasis on the Proteins

JOHNR. WHITAKER Introduction . . . . . . . . . . . . . . . . Structure of Skeletal Muscle . . . . . . . . . . . Proteins of Muscle . . . . . . . . . . . . . . Chemical Changes Associated with Contraction and with Onset of Rigor Mortis . . . . . . . . . . . . . . . V . Chemical Changes Associated with Relaxation and with Resolution of Rigor Mortis . . . . . . . . . . . . . VI . Artificial “Aging” of Meat . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

I. I1. I11. IV.

1 3 5 10 26 37 46 47

The Chemistry and Technology of the Preservation of Green Peas

L. J. LYNCH.R. S. MITCHELL. AND Introduction . . . . . . . . . Chemistry . . . . . . . . . Maturity . . . . . . . . . . Unit Processes . . . . . . . .

I. 11. I11. IV. V. Future Research Requirements References . . . . . .

D. J . CASIMIR

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Principles and Instrumentation for the Physical Measurement of Food Quality with Special Reference to Fruit and Vegetable Products

AMIHUDKRAMERA N D B. A . TWIGG I . Introduction . . . . . . . . . . . . . . . . 153 I1. General Principles . . . . . . . . . . . . . . 154 I11. Appearance Factors . . . . . . . . . . . . . 157 IV . Kinesthetics . . . . . . . . . . . . . . . . 197 . . . . . . . 209 V. Flavor . . . . . . . . . VI . Summary and Conclusions . . . . . . . . . . . 213 References . . . . . . . . . . . . . . . . 214 ix

X

CONTENTS

Microorganisms in Noncitrus Juices

HANSLUTHI I . Introduction . . . . . . . . . . . . I1. Types of Microorganisms Found in Fruit Juice . I11. Occurrence of Microorganisms of Juice in Nature IV. Occurrence in Fruit Juice . . . . . . . . V. Changes in Appearance of Juice . . . . . . VI . Production of Alcohols by Microorganisms . . VII . Changes in the Organic Acid Content Induced organisms . . . . . . . . . . . . VIII . Other Changes in Juice Induced by Microorganisms IX. Additional Research Needs . . . . . . . References . . . . . . . . . . . .

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286 289 302 307 310 313 315 320 324 329 332 340 342 348 354 355

222 237 245 257 259

Micro-

. . . . 262 . . . . 271 . . . . 273

The Chemical Constituents of Citrus Fruits

J. F. KEFFORD I. Introduction . . . . . . . . . I1. General Composition of Citrus Fruits . I11. Carbohydrates . . . . . . . . IV. Acids . . . . . . . . . . . V. Vitamins . . . . . . . . . . VI . Inorganic Constituents . . . . . . VII. Nitrogen Compounds . . . . . . VIII . Enzymes . . . . . . . . . . IX . Pigments . . . . . . . . . . X . Lipids . . . . . . . . . . XI . Volatile Flavoring Constituents . . . XI1. Nonvolatile Constituents of Citrus Oils XI11. Flavonoids . . . . . . . . . XIV. Limonoid Bitter Principles . . . . XV . Research Needs . . . . . . . . References . . . . . . . . .

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. . . ERRATA: Volume VIII . . . . . . . AUTHORINDEX . . . . . . . . . SUBJECTINDEX .

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391

CHEMICAL CHANGES ASSOCIATED WITH AGING OF MEAT WITH EMPHASIS ON THE PROTEINS BY JOHN R. WHITAKER University of California, Davis, California

I. Introduction ...................................................... 11. Structure of Skeletal Muscle .............................. 111. Proteins of Muscle . . . . . . . .................... A. Muscle Fiber Proteins. . .................... B. Extracellular Proteins ................................ IV. Chemical Changes Associated with Contraction and with O n s Mortis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Theories of Muscle Contraction ............................... B. Experimental Models for Contraction and Relaxation . . . . . . . . . . . . . . C. Rigor Mortis ....................... ................... D. Thaw Rigor ........................ ................... V. Chemical Changes Associated with Relaxation and with Resolution of Rigor Mortis . . . . . . . . . . . . . . . . . A. ReIaxation of Muscle . . . . . . . . ...................... B. Resolution of Rigor Mortis . . . VI. Artificial ‘‘Aging” of Meat . . . . . . ............................... A. Historical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nature of the Enzymes Used . . ...................... C. Commercial Tenderizers . . . . . ...................... D. Evaluation of the Effect of Proteolytic Enzymes on Tenderness . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ...................................................

Page 1

10 10 13 14

23

26 37 37

38 39 44l 46 47

1. INTRODUCTION

While Bate-Smith (1948) was astonished at the bewildering rate of growth of the fundamental information concerning the biochemical properties, structure, and function of muscles at the time he wrote his review for Advances in Food Research, this reviewer is overwhelmed by the mass of information which has accumulated since that time. It is estimated that the number of papers published each year which deal with some phase of the biochemistry and physiology of the muscle is in excess of 500, and the number is increasing each year. This area is no longer the sole domain of the physiologists; the physicists, biophysicists, biochemists, and mathematicians have also contributed their share to our understanding of the muscle. The muscle has been stretched, relaxed, contracted, softened, extracted, photographed, and divided both 1

2

JOHN R. WHITAKER

physically and chemically. It would seem that no other single biological material has received so much attention as has the muscle. It was thought that, with the discovery that the contractile elements consist of two proteins, actin and myosin, which can combine with each other under the proper circumstances and which contract when ATP’ is added, a solution of the mechanism of contraction of muscle was close at hand. This was in 1939. Nineteen years and many thousands of papers later, man still must admit that he does not know how the muscle contracts, by what mechanism it is able to convert the chemical energy of the phosphate bond of ATP to useful work, or how it relaxes. The more the biochemist learns about the structure of the muscle the more confused the physiologist becomes about the workings of the muscle. Szent-Gyorgyi (1958) has suggested that what is needed is a new approach to the whole problem. Perhaps the four criteria most used by the consumer in evaluating a piece of meat are tenderness, flavor, juiciness, and color. In the case of beef, experience has shown that the first three of these criteria are usually improved by “aging” or “ripening” the meat at 0 to 1.5OC. for approximately 17 days. This is a very expensive process in the merchandising of beef. Watts (1954) has recently written an excellent article on the pigments of meat. The color of meat is influenced by an interaction between the pigment content of the meat and the transparency of the meat fibers. Usually the poorer the transparency of the fibers, which is associated with reduced water-binding capacity (also juiciness and perhaps tenderness), the paler the meat. Practically nothing is known at present about the changes in flavor which occur during aging and the compounds which are responsible for this flavor. It is anticipated that with the tremendous amount of work being done on irradiated meat flavor and with the new techniques of gas chromatography available to the flavor chemist significant advances will soon be forthcoming in this field. Juiciness is probably dependent upon a combination of factors including the fat and the water-binding capacity of the meat. Proteins and their ability to bind water play a prominent role in the tenderness of meat. So these criteria of meat quality are all interrelated, and it appears that they are all influenced in one way or another by the changes, primarily in the proteins, which take place in the muscle after death. *The following abbreviations are used throughout this review: ATP, ADP, AMP = adenosine tri-, di-, and monophosphates; ITP, IDP, IMP = inosine t+, di-, and monophosphates; creatine phosphate, CP; ethylenediaminetetraacetic acid, EDTA; inorganic phosphate, IP; inosine, IN; pyrophosphate, PP.

CHEMICAL CHANGES ASSOCIATED WITH AGING O F MEAT

3

The most prominent physical change which occurs soon after the death of an animal is a hardening of the muscles. The muscles become very hard, inflexible, contracted, and tough. If the animal is held or “aged” for a few days the muscles again become soft, pliable, relaxed, and tender. This review shall attempt to clarify the chemical changes which are responsible for this physical change in the muscle. II. STRUCTURE OF SKELETAL MUSCLE

All skeletal muscles are striated in appearance and make up more than 50% of the weight of the animal. The striations are due to the presence of alternating bands of material in the fibrils which possess different refractive indices. The nature of these striations will be discussed in more detail later. The color of the muscle is dependent on its myoglobin content which may be completely absent as in white muscle or as high as 5 to 7% of the fresh weight of seal and whale flesh. Red muscle is also richer than the white muscle in the particulate bodies or granules which contain the enzymes of the respiratory cycle. Lawrie (1952) has discussed many of the biochemical differences between red and white meat. The typical skeletal muscle is enclosed in a sheath called the perimysium and is permeated quite extensively by fat deposits and by the connective tissues (the endomysium) . The contractile tissue proper consists of fibers of diameter 10 to 100 p (microns) which eventually fuse with the tendon fibrils. The muscle fiber itself is a fusiform cell with many nuclei, and its contents are referred to as the intracellular material. The vascular system, nerve tissue, connective tissue, and the material of the interstitial space make up the extracellular components. All of the insoluble components of the extracellular tissue, together with the insoluble components of the sarcolemma (mainly reticulin) are called the stroma. Depending largely upon the function of the muscle in the body, the fibers may run parallel to the long axis of the muscle fiber, as in the psoas or sartorius, or may be inclined as in the gastrocnemius. Each fiber is enclosed in a membrane, the sarcolemma, which is considered to be a complex structure; one layer at least consists of fine interlacing reticular fibers. Besides the transverse striations, each fiber is split longitudinally into fibrils, often called myofibrils, of about 1 p in diameter, which are separated from each other by a sarcoplasmic gap of 0.5 p. The cross-striations so typical of skeletal muscIe are associated with the myofibrils. The pattern of the cross-striations is repeated regularly every few microns. The repeat unit is called a “sarcomere,” and the length changes in the whole muscle are brought about by length

4

JOHN R. WHITAKER

changes in the sarcomeres of its fibrils. The myofibrils consist almost entirely of protein (Perry, 1955). It has been shown that when myosin is extracted from washed glycerol-extracted fibrils, they lose about 68% of total protein; 50% of this is myosin, while 18% is another protein fraction called the X-protein (Szent-Gyorgyi et al., 1955). The other two main constituents of the sarcomeres are actin and the unextractable stroma. The sarcomere is 2.3 p in length and is bounded by the two narrow, dense Z lines (Fig. 1). In the middle is a dense anisotropic band, the A band, which is 1.5 p long. Alternating along the fibril with the A bands are the I bands, which are isotropic and much less dense than the A bands. Each I band is bisected by a Z line. In the middle of each 2.3

u

U

Z

H I

U

Z

I FIG. 1. Arrangement and dimensions of the bands in sarcomere of a myofibril, at rest length (Randall, 1957). A

A band is an H zone about 0.3 p long, which is less dense than the rest of the A band. By the use of the electron microscope, it has been shown that the myofibril itself is composed of thinner threads which are called “filaments.” There are apparently two types of filaments present (Huxley, 1953a,b; Huxley and Hanson, 1954, 1957). Figure 2 shows diagrammatically the apparent arrangement of these two types of filaments in the sarcomere. From the changes produced in the myofibrils by differential extraction of the proteins as measured by the electron microscope, it has been concluded that the thick filaments of the A band contain the myosin of the sarcomere; the thin filaments extending from the Z lines to the borders of the H zone contain the actin and tropomyosin of the sarcomere; and the third component, left behind when myosin, actin, and tropomyosin and other soluble materials have been removed, is the stroma (Hanson and Huxley, 1953,1955,1957; Corsi and Perry, 1958).

CHEMICAL CHANGES ASSOCIATED W I T H AGING O F MEAT

5

FIG.2. Arrangement of filaments in one sarcornere of a myofibril: top, stretched to 120% rest length while plasticized; middle, at rest length; bottom, contracted to

90% rest length (Randall, 1957).

Many excellent reviews have appeared recently on muscle physiology and structure (Szent-Gyorgyi, A, 1953; Gerard and Taylor, 1953; Bailey, 1954; Dubuisson, 1954; Mommaerts, 1954; SzentGyorgyi, A. G., 1955; Feigen, 1956; Weber, 1957; Gelfan, 1958). 111. PROTEINS OF MUSCLE

A. MUSCLEFIBERPROTEINS

I. Myogen Fraction If muscle is finely minced and then diluted with an equal volume of water or 0.9% sodium chloride solution, a viscous mass is obtained from which no juice can be pressed. After about 30 min. this suddenly contracts and undergoes syneresis. Actually the muscle mince is now in a state of rigor. The juice can be pressed out and contains all the components of the glycolytic cycle plus the nonprotein components of the muscle. These nonprotein components, the extractives, consist of carnosine, anserine, glutathione, carnithine, sarcosine, taurine, the common amino acids, purine bases, ATP, ADP, etc. These extractives of muscle were extensively studied in the early days of biochemistry. The press juice is often called “myogen” and was initially thought to be homogenous in nature. It now seems probable that all of the protein components of myogen are enzymatic in nature. Bailey (1954) has listed the more than fifty enzymes which have been found in myogen.

6

JOHN R. WHITAKER

Phosphoglyceraldehyde dehydrogenase ( 10%) , creatine phosphokinase (10%) , phosphorylase (2%), and the aldolase-isomerase system (5%) make up 27% of the myogen fraction. 2. Myosin

This protein has been known for almost a century. Kuhne (1859) discovered that a large amount of protein could be extracted from the muscle by strong salt solutions and that it precipitated when the solution was diluted. However, it was not until 1942 that it was shown that the “myosin molecule” as usually prepared consisted of two proteins, true myosin and actin (Schramm and Weber, 1942). Myosin is readily soluble in 0.5 M KCl to give a water-clear solution; its isoelectric point appears to be near 5.50 to 5.75 (MihAlyi, 1950), and it is easily denatured by freeze-drying, dehydration with organic solvents, or by heat. Its molecular weight (if myosin can be called a molecule) is 382,000 +- 20,000 (Mommaerts and Aldrich, 1957). It is 2200 A in length (Portzehl, 1950). Myosin is a fibrous protein with an a-structure, as its peptide chains are not fully elongated. It appears to be a cyclic protein as the number of N-terminal groups is smaller than 1 in 500,000 (Bailey, 1951) . Myosin is a sulfhydryl enzyme (Singer and Barron, 1944). One of its functions in the contraction process appears to be the splitting of ATP molecules as will be shown later. The myosin molecule can be broken down into units of smaller molecular weights by short treatment with trypsin (Gergely, 1950, 1951, 1953; Perry, 1951; MihAlyi and Szent-Gyorgyi, 1953a) or with chymotrypsin (Gergely et al.,1955). The action of these proteolytic enzymes is to fragment myosin into two distinct kinds of components called “heavy meromyosin” (mol. wt. 232,000) and Yight meromyosin” (mol. wt. 96,000) (Szent-Gyorgyi, A. G., 1953). These components possess all the properties of the original myosin except contractility (Gergely, 1953). The heavy meromyosin combines with actin and has all the ATPase activity of the intact myosin, while the light meromyosin has the peculiar solubility properties of myosin. The myosin molecule can also be broken down into smaller units by the action of urea (Weber and Stover, 1933) and more recently it has been shown that it is the light meromyosin that is depolymerized into approximately 20 units of molecular weight 4500 (Szent-Gyorgyi and Borbiro, 1956). These small units have been named protomyosin. They all appear to possess the same molecular weight but to differ in amino acid composition. Heavy meromyosin does not undergo similar splitting on treatment with urea.

C H E M I C A L C H A N G E S ASSOCIATED W I T H AGING OF M E A T

7

A schematic drawing of the myosin molecule as depicted by Laki (1957) is shown in Fig. 3. The bases for this model are the strong agreement between the amino acid analyses of the fractions and of the whole, agreement with labeling experiments, agreement between halflives of heavy meromyosin and actin (80 and 67 days, respectively) and light meromyosin and tropomyosin (20 and 27 days, respectively),

(,~{[.~ fl.)n~ j ~] +LMM

-11-

HMM

*I

t-LMM-1

FIQ. 3. Schematic representation of the myosin molecule. Weight: myosin, 420,000; actin, -80,000; tropomyosin, -50,000. KEY:LMM, L-meromyosin; HMM, H-meromyosin; TM, tropomyosin; A, actin (Laki, f 957).

as well as the fragmentation experiments mentioned above. [See also Laki et a2. (1958) .] 3. Actin

The discovery of actin as a major component of muscle followed from the observation that certain myosin-containing extracts of muscle lost their gel-like consistency on addition of ATP while others were much less affected (Szent-Gyorgyi, 1947). The molecular weight of the actin monomer is approximately 70,000. Actin exists in two forms, one of which is limpid in solution (globular actin or G-actin) and which can be converted by salt ions or acid into the second form, which is fibrillar (F-actin) and which is highly viscous and shows strong flow birefringence. The polymerized F-actin is considered to be more or less a linear aggregate of G-actin units, although there are certain discrepancies which cannot be explained by this view. The rate of polymerization of actin in sodium o r potassium salt solutions is increased by magnesium ions and inhibited by calcium ions. The transformation of G- to F-actin can be prevented by chloromercuribenzoate, Salyrgan (a mercury-containing arsenical), and copper glycinate (Turba and Kuschinsky, 1952). Inhibition by these reagents is explained on the basis that certain sulfhydryl groups are involved in the polymerization; only F-actin is capable of interacting with myosin to give a collodial system whose state is profoundly affected by ATP.

4. Tropomyosin

This protein was discovered in 1946 by Bailey and has been shown by Perry and Corsi (1958) to account for at least 10 to 12% of the total

8

JOHN R. WHITAKER

myofibrillar proteins. It has many properties in common with myosin and may be thought of as a prototype of myosin; it is almost identical in amino acid make-up but has a molecular weight of only 50,000. It is very viscous in water, but the addition of a small amount of salt very markedly decreases the viscosity. It is postulated by Laki (1957) that tropomyosin is an integral part of the myosin molecule, but the role of tropomyosin in the muscle machinery has not yet been elucidated.

5. Actomyosin To add to the confusion that exists in muscle physiology this protein is often called “myosin” or “myosin B.” Actomyosin is composed of a complex between actin and myosin which is formed at the moment of excitation.” It does not exist in the resting muscle. I t is the actomyosin complex which is contractile under the proper conditions. Actomyosin accounts for about 80% of the total structural protein extracted from contracted o r dead muscle. The values given for the combining ratio between actin and myosin vary from 1 : 3 to 1 :6 (Mihhlyi and SzentGyorgyi, 1953b; Snellman and Erdos, 1949; Spicer and Bowen, 1951). However, myosin solutions will contract in the presence of very small amounts of actin. Actomyosin does not appear to be nearly as labile as myosin since beef actomyosin was found to retain most of its ATPase activity and contractility after freeze-drying (Hunt and Matheson, 1958). Artificial threads of actomyosin which retain the property of contractility can be prepared by forcing solutions of this compound through narrow apertures into salt-free water. (
W

z

0

L

a t I

u) 3

3 000

loook 2000

-lZE

0

m

0 100

200

300

400

M A T U ROMETER READING

FIG. 6. Summation yields of size grades and corresponding maturities (Canners Perfection variety). Plotted from data of Lynch and Mitchell (1953).

in Fig. 7, which shows the frequency distribution of A.I.S. values and emphasizes the wide variation which can occur. When sweet peas are harvested at the O.H.T., size grades 2 and 7 are almost invariably immature and overmature, respectively. Size grade 3 is usually immature when the yield is high but is distributed between immature and first quality when the yield is low. Size grade 4 nearly always falls within the limits of first quality. In low yielding crops, size 5 is distributed on either side of the upper quality limit line, while in crops of higher yield it is entirely within desirable limits. Size

132

L. J. LYNCH, R. S. MITCHELL, A N D D. J. CASIMIR

grade 6 contrasts with size 3, being either totally overmature or containing a proportion of high quality peas. Except in the case of very large-seeded varieties, size 7 may always be regarded as overmature. For factory purposes, it is essential to determine the maturities of samples from individual size grades from all crops when using the size grader for quality segregation.

A.I.S.

%

FIG. 7. Frequency distribution of A.I.S. per cent in ungraded and size grade 5 peas from a cannery line (Canners Perfection variety). From Lynch and Mitchell (1950).

3 . Blanching Blanching is a mild heat treatment at a temperature close to that of boiling water for times of about 1 min. for freezing and dehydration and 3 to 6 min. for canning. Times adopted may vary widely, and Burton (1938) reported a blanching time of 20 min. Blanching is reputed to remove gases from tissues and to destroy enzymes. Removal of gas is essential for gravity separation and for adequate vacuum in the canned product, and enzyme destruction is a prerequisite to preservation by freezing and by drying. Hot water and steam are commonly used for blanching, though other methods, such as dielectric heating, have been

PRESERVATION OF GREEN PEAS

133

used experimentally. The blanching process should be terminated by cooling to insure a known time for the blanch treatment. Holmquist et al. (1954) found that steam-blanched peas had a grasslike flavor which was practically eliminated by a l-min. prewash in sodium hexametaphosphate (Calgon) solution at 75OF. Flavor, texture, nutritive value, and brine turbidity of the final product are influenced by the nature and severity of the blanch. Bitting ( 1937) stated that high-temperature blanches probably produce more splits than lower temperatures and cause cloudiness of the brine of canned peas. Legault et al. (1950) found that ruptured skins in a sample of peas which had been steam-blanched at 197OF. amounted to 15 to 20%, and TABLE IX MATUROMETER READINGSOF PEASBLANCHEDIN WATER AT 200°F. FOR DIFFERENT TIMES' Size grade composition Blanch tiinc (sec.)

(4-7)

(2-3)

0 15 30 45 60 90 120 150

347 255 22 1 216 212 198 180

180

151 145

151 126 110 I10 110 114 108 107 111 108

240 0

160

Data from Lynch et al. (1956-1957).

that this result was independent of time up to 5 min. Increase in temperatures up to 204OF. for periods just sufficient to give a negative peroxidase test did not give any substantial increase in ruptured skins. When times exceeded that required for negative peroxidase test, the numbers of ruptured skins increased if temperatures above 197OF. were used. At 204OF. for 150 sec., about 40% ruptured skins occurred and at 2 1 2 O F . , 30 to 40% ruptured skins were recorded after 38 sec. This work also provided evidence that large peas are more affected than small peas when blanching times or temperatures are excessive. Lynch et al. (1956-1957) measured the changes in texture of peas blanc,hed in water for different times, and the results set out in Table IX show a rapid change in the first 30 sec. Thereafter there was a

134

L. J. LYNCH, R. S. MITCHELL, A N D D. J. CASIMIR

steady decrease in hardness of larger peas, while little additional change occurred in smaller peas. Measurement by Mitchell and Casimir (1957-1958) of gas liberated from 100 g. of peas showed that during a 2-min. water blanch at 190OF. the volume was higher for the larger size grades. It amounted to 0.29 ml. for size 2 and 0.94 ml. for size 7. Evolution of gas was rapid over the first 20 to 30 sec. and slow thereafter. Peas of size grade 7, which were artificially injured by slitting, gave more gas than unslit peas when blanched for times up to 5 min. This result demonstrated that long blanches do not remove the whole of the gas from undamaged peas. Heberlein et al. (1950) , from extensive studies, concluded that even with extreme variation in blanch times no significant differences were detectable in the flavor of canned peas. Texture measurements were made by Lynch et al. (1956-1957) on canned peas which had been water-blanched at 200OF. for times between 0 and 240 sec. Maturometer values were higher for peas which had not been blanched, but after 15-sec.blanch little further change was recorded. Measurement of per cent transmission of light through can brine showed a decrease in turbidity as blanch time increased. Can vacua and drained weights were found to increase as blanch times were extended, but A.I.S. was apparently constant within the range of variation to be expected from can to can.

4 . Specific Gravity Grading Differences in S.G. permit the separation of quality grades by use of brine solutions, and this method of grading has been practiced commercially for many years. Burton (1938) reported that in 1919 Hugh Finderburg, of Keen Belvedere Canning Company, Belvedere, Illinois, used a tank of brine into which peas were poured continuously and the floaters were skimmed off. The floaters and sinkers were washed and canned separately. The basic requirements for segregation are appropriate brine strength, adequate time for separation, and absence of vertical brine movement. The peas must be added and removed from the separating tank with a minimum of disturbance to the system. There are several types of gravity separators, which may be grouped as those in which the brine is essentially still and the peas are added and removed mechanically, and those in which separation is effected by the movement of brine. The Olney grader is an example of the first group. It consists of a tank in the form of an inverted cone. The peas are fed over fingers mounted on a horizontally rotating arm to which is attached a skimmer plate which divides floaters from sinkers. The

PRESERVATION O F G R E E N PEAS

135

floaters flow in brine towards the center of the machine to the discharge point. The sinkers on reaching the bottom of the tank are ejected by a brine jet. Machines involving brine flow include the Lewis grader in which peas, added tangentially into a conical brine tank, flow in a circular path between a central cylinder and the outer side of the tank. Floaters pass over a weir at the discharge point, and sinkers are forced through an outlet in the bottom of the tank. In all graders, emerging peas are separated from the brine by screening, and the brine is recirculated. As the peas entering are wet with water and those leaving carry brine, the brine is continually diluted. To maintain correct brine density, automatic regulators are used to admit concentrated brine. Wagner et al. (1947b) produced data which indicated that the total solids in peas gravity-graded and then water-blanched was 21.0% for both floaters and sinkers. When peas were blanched before gravitygrading, the total solids content of the floaters was 20.5% and that of the sinkers was 25.2%. Since per cent total solids is related to maturity, it may be assumed that the gravity-grading of raw peas resulted in no maturity separation but that gravity-grading after blanching gave distinct maturity fractions. Peas must be washed and cooled after blanching to avoid contamination and temperature rise in the gravity grader. Link (1955) stressed the importance of the time of immersion in gravity separation, and published graphs showing the per cent sinkers in different brines with separation times of 6, 9, and 12 sec. Using brine of S.G. 1.06, 14% of peas in the samples sank in 6 sec., 30% in 9 sec., and 50% in 12 sec. The circular path of brine in a grader investigated by Lynch et al. (1956-1957) was found to give separation times of 18 sec. for peas moving near the periphery and 10 sec. for those closest to the center. Efficient grading demands the avoidance of continuous overloading by excessive feed rate or of temporary overloading arising from irregular input. With more than one layer of peas in the settling tank, free vertical movement is hindered, and heavy peas may be held up or light peas carried to the bottom of the grader. Link stated that maximum capacity is achieved when sinkers and floaters are present in equal proportions, Berth (1955) considered that a grader with a capacity of 8000 lb./hr. on a 50 to 50 basis would have a capacity of 6400 lb./hr. when the separation is 70 to 30, and only 4800 lb./hr. when the separation is 90 to 10 floaters to sinkers. Lynch et al. (1956-1957) studied the efficiency of a gravity grader by collecting the sinkers and floaters, separately size grading them, and

136

L. J. LYNCH, R . S . MITCHELL, A N D D. J. CASIMIR

testing the maturity of the size grades. As the peas were water-blanched before gravity separation, a special type of experimental size grader and a low range maturometer gauge were used. Data in Tables X and XI show that ungraded bIanched peas are TABLE X MATURITY OF PEASSIZEGRADED AFTER GRAVITY SEPARATION' Size grade Feed rate (lb./hr.) Fraction

Maturity measure

Floaters

A.I.S. (%)

2400

Sinkers 7800

Floaters Sinkers

Q

M.R. A.I.S. (a) M.R. A.I.S. (%) M.R. A.I.S. (%) M.R.

Ungraded

4

5

6

7

8

11.1 108 14.7 162 11.2 115 14.9 153

9.8 104 14.4 145 10.3 126 15.2 154

10.6 114 14.4 156 10.5 114 15.0 159

10.8 120 14.5 160 11.0 122 15.1 154

11.7 133 15.0 173 12.0 128 15.1 156

160 -

175 158 175

Data from Lynch et al. (1956-1957).

TABLE XI MATUROMETER VALUES OF SAMPLE^ OF BLANCHED AND GRAVITT-GRADED PEAS^ Size grade Fraction Floaters Sinkers Floaters Sinkers Floaters Sinkers Floaters Sinkers a

Ungraded

2

3

4

5

6

7

111 116 84 120 94 128 100 114

119 124 80 117 94 133 93 99

114 120 84 119 96 137 96 112

115 121 89 122 97 133 99 123

115 126 100 127 102 137 103 115

121 132 117 130 114 142

147 146 135 134 134 152 -

100

121

Data from Lynch et 02. (1950-1957).

separated into two maturity fractions. For perfect segregation, all peas in the floater fraction must be less mature than the sinkers, giving a division at a particular maturity level. Evaluation of grading efficiency would necessitate determining the maturity of each individual pea in representative samples of sinkers and floaters. This would be extremely difficult but an analysis may be obtained by evaluating maturity of size grades within the fractions. Tables X and XI demonstrate adequate separation in almost all cases. The separation was poor in the largest

PRESERVATION O F G R E E N P E A S

137

size grades of some crops, probably on account of incomplete liberation of internal gas during blanching. Holmquist et al. (1955) compared the effect of water-blanching at 205OF. with steam-blanching. Various blanch times between 30 sec. and 5 min. were used, and results showed that peas steam-blanched 1 min. or longer gave higher S.G. readings than water-blanched peas. This was attributed to differences in amount of leaching and in water uptake. They stated that steam-blanched peas required the grading brine to be 3 to 6* Salometer higher than for water-blanched peas for comparable separation. Lynch et a2. (1956-1957) observed that the time and temperature of water blanch influenced the ratio of sinkers to floaters. The results suggest that consistent quality separation can be obtained only if peas are evenly blanched under closely controlled conditions. Gravity grading may be used independently or in conjunction with size grading. The whole crop may be passed through one gravity grader or through two in tandem to give two or three quality grades, or single size grades or combinations of grades may be gravity-separated. Certain size grades considered as falling within fixed quality limits may not require further grading. Berth (1955), for instance, considered that sizes 2 and 3 in sweet peas did not require gravity grading. Large sizes in some crops are overmature and do not warrant grading. The gravity grader may be adjusted to give approximately equal amounts in each grade or to remove only a small amount of peas which are outside the quality range required. The gravity grader operates best when field control is efficient and crops are harvested at a constant maturity. Burton (1938) found that installation of quality graders in a factory stimulated field men to exercise careful control over harvest maturity. Physical measurements of maturity of raw peas serve as a guide to the operator in gravity-grading, but the selection of brine density is usually determined by the results of sinker tests. This test is simple and rapid but since it is based on factors which also govern separation in the gravity grader, it is affected by severity of blanch and other factors. Specific gravity determinations and measurements by mechanical instruments on blanched peas are similarly affected. A.I.S. is not affected by the blanch but, although useful as a check, is too time-consuming to be used for grader control. Walls and Hunter (1938) suggested that removal of air by vacuum treatment might replace the blanch prior to gravity separation, and Malrower (1957) as a preliminary to floater-sinker determination subjected peas immersed in the grading solution to a vacuum of 29 in. of mercury.

138

L. J. LYNCH, R. S. MITCHELL, AND D. J. CASIMIR

5. Final Processing a. Canning. Peas from the inspection belt pass to the hopper of the filler usually by means of a gooseneck elevator. From the hopper they “ f l o ~ into ~ ’ the pockets of a rotary filler, from which a measured volume is filled into each can. A machine of similar principle is used to add a measured quantity of brine, the latter consisting of salt and water with or without sugar. A short steam exhaust may be given to insure adequate can vacuum, though hot brine and steam flow closure is sufficient for this purpose. After closing, cans are processed in stationary retorts or in continuous cookers. Canned peas are typical solid-in-liquid packs which heat rapidly by convection. Processes recommended by National Canners Association (1955) are 36 min. at 240OF. or 16 min. at 250’F. for cans No. 2 size or smaller. No. I0 cans are used for institutional packs and are processed for 55 min. at 240OF. Special processes, as yet experimental, seek to lessen the overcooking which, from a culinary point of view, results from the usual processes. The rate of bacterial destruction increases more rapidly with rise in temperature than the rate of chemical change, and commercial sterilization can be rapidly achieved at temperatures in the range 265 to 270OF. with relatively small changes in texture, flavor, and color. Can rotation at these temperatures further reduces the time required for sterilization, but there is evidence that the heat treatment may be inadequate for enzyme destruction. b. Freezing. Peas may be frozen before or after packaging. Rapid freezing was thought to be an important factor in quality retention due partly to the formation of small ice crystals. However, Lee et al. (1946) from experiments including extreme rates of freezing, found that taste panels were unable to detect any difference in appearance and flavor of peas frozen at different rates. There are a number of €actors which favor fast freezing. It permits greater factory throughout and allows the use of smaller freezing units which occupy less floor space. Desiccation of the peas also tends to be less in fast freezing. Fast freezing is therefore an economic choice rather than a quality requirement. It is achieved by the use of low temperatures in the freezing medium, by efficient transfer of heat from the food to the medium through good contact, and by reduction to a minimum of the thickness of the peas on the belt and in the package. Peas are quick frozen commercially by contact with refrigerated metal plates or by a blast of cold air blown over the product. In platefreezing, heat is transferred to metal plates which in turn transfer it rapidly to the refrigerant reticulating internally through the plates.

PRESERVATION O F GREEN PEAS

139

Transfer of heat from packet to plate is assisted by intimate contact developed by mechanical compression. A series of plates is enclosed in an insulated cabinet, and batches of packets are loaded, frozen, and removed to store. Continuous plate freezers permit a constant flow of packages through the freezing stage. Blast freezers circulate air which is cooled by contact with refrigerated coils. Air has a low heat capacity, and the volume circulated must be high. Air-blast freezing is adaptable to continuous production in which packaged or loose material may be frozen during passage through a freezing tunnel. Packets of differing size and shape may be frozen in the same air-blast freezer. By contrast packages to be treated in plate freezers are limited in size and shape. Packaging materials must not taint the product and should resist damage to product and package. Moisture vapor transfer must be low to reduce desiccation. Paperboard containers treated to resist wetting provide strength and rigidity and may be improved when combined with a suitable flexible liner. A sealed ovenvrap may also be applied with advantage. Frozen packages are packed into shipping containers of corrugated fiberboard before they are placed in frozen storage. c. Dehydration. Peas intended for dehydration are treated with sulfur dioxide (usually combined as a salt) to minimize browning during drying and storage. They are treated by dipping or by spraying after blanching. Sodium and potassium sulfites and similar compounds are used to give an ultimate residual sulfur dioxide content of about 500 p.p.m. in the dried peas. Moyer et al. (1956a) found that slitting the seed coat of peas accelerated water transfer during drying and allowed more rapid and complete rehydration. The common method of drying is by spreading peas on trays at the rate of 1 lb./sq. ft. The trays are loaded on to trolleys which are moved through a dehydration tunnel. Hot air is blown over the peas at a velocity usually within the range of 600 to 1000 ft. per minute. Belt trough driers and rotary and vacuum-plate types are used to a limited extent. Dehydration is a two-stage process. The first stage removes the bulk of the moisture rapidly, and subsequent treatment reduces the moisture content to the desired level. In the initial stage, air temperatures of 180 to 200°F. are used in tunnel driers and about 300°F. in belt trough driers. The temperature of the peas during this stage is about 100 to 120°F., which is close to the wet bulb temperature of the circulating air. Air temperature is reduced to 140 to 160°F. when the product temperature tends to approximate that of the drying air. This

140

L. J. LYNCH, R. S. MITCHELL, A N D D. J. CASIMIR

stage is maintained until completion of the process. Peas are sometimes held for about 24 hr. at 100 to 120OF. in a bin drier to reduce moisture content from 8% to a final 5%, at which level the storage stability is improved. In-package desiccation by calcium oxide and other agents may be used to complete the drying. The desiccant in its own package is enclosed with the peas. The desiccant package must retain the solid but permit ready transfer of water vapor. A paper packet which is siftproof and allows moisture vapor transfer is surrounded by a cloth container which protects the paper from damage. The amount of desiccant is found by calculation from the moisture value of the sample and the desired final moisture content. In-package desiccation increases package volume by approximately 13%. Packages for dehydrated peas must be moisture-proof and must possess adequate tensile strength. Metal cans fulfill these requirements and are used to a limited extent. More commonly, flexible packaging materials with or without lamination are used. Moyer et al. (1957) found that brown discoloration which may occur in the final product decreased as maturity increased up to 12% A.I.S. and thereafter remained constant, Caldwell et al. (1946) stated that properly dehydrated peas are highly resistant to deterioration. Samples of large size grades showed little change when stored at 70°F. for 12 months, and small size grades had a slightly haylike odor but were acceptable when cooked. Moyer et al. (1957) found that rehydration capacity decreased with maturity due to chemical composition rather than to any change resulting from heat damage. Under similar conditions, older peas dried to a higher moisture content than peas less advanced in maturity. This was true for peas of all size grades and within any one grade. d . Dehydrofreezing. Howard and Campbell (1946) developed the method of partial drying followed by freezing. They considered that decrease in weight and volume resulted in savings in packaging materials, freezer capacity, and transport costs. Reconstitution was said to be rapid and quality was not impaired. Tressler and Evers (1957) dried peas to 50% of their blanched weight before freezing and found uniformity of size was an important factor in drying efficiency. Talburt and Legault (1 950b) considered that blanching procedures are critical and that long blanch times and high temperatures favor higher drained weights and higher volumes in cooked dehydrofrozen peas. Equivalent samples of peas preserved by freezing and by dehydrofreezing were examined by triangular taste tests and showed no signifi-

PRESERVATION

OF GREEN PEAS

141

cant differences between the two methods of preservation shortly after processing and after storage for 12 months. When dehydrofrozen peas were stored at -1OOF. for 6 months in atmospheres of air and of nitrogen no differences in quality were apparent. Talburt and Legault (1950b) found that cooking methods for frozen peas were found to be suitable f o r the dehydrofrozen product since no preliminary soak is required for reconstitution. V. FUTURE RESEARCH REQUIREMENTS

Scientific investigations into the chemistry and physiology of peas have contributed substantially to the development of the present high standard of technical efficiency in industry. Further study of the chemical and physical changes which occur during maturation and processing is likely to make useful contribution to the technology of pea preservation. There is a need to evaluate new and existing pea varieties by replicated field trials and a reliable method of maturity assessment. Results should be expressed in terms of yield, quality, and size grade distribution. The vining of peas provides scope for engineering research. Viner speed, beater angle, and rate of feed are important factors in recovery of peas and the extent to which they are damaged. The performance of stationary and mobile viners should be compared. Vining studies are complicated by the need for careful sampling and for sufficient replication to permit precise assessment of the results by statistical analysis. The rate of deterioration of vined peas transported in various ways needs further investigation. Observations should be made o n peas carried in small lugs and in bulk containers with and without ice. Changes in the amylose-to-amylopectin ratio in pea starch, the calcium-phytic acid relationship, and the changes in other components with maturity warrant additional investigation, particularly from the point of view of their influence on the acceptability of peas processed by different methods. It is important that the results of such work should be recorded in a way which makes their technological significance clear. In particular, the A.I.S. values, or some other reliable measure of maturity, should be quoted, and the methods of sampling raw material should be described in detail. The degradation of chlorophyll during processing and storage is probably the most important change which occurs in canned peas. The chemical mechanism is well known, but various methods for the control of this reaction have been effective only at the expense of undesirable flavor and texture changes. It would seem that the o n l y immediate

142

L. J. LYNCH, R. S. MITCHELL, AND D. J. CASIMIR

means of reducing the rate of loss during storage is by the use of temperatures in the 40 to 45OF.range. Present knowledge of the role of enzymes in flavor deterioration is incomplete. Emphasis has been given in the past to the presence or absence of catalase and peroxidase in the processed product, but the importance of enzymes associated with lipid breakdown has now been recognized. The destruction of these latter enzymes during blanching of peas for freezing may provide a better criterion than that which is at present accepted for control purposes. Precise data on temperature coefficients for enzyme destruction by heat are required in view of the problem of survival or regeneration in frozen and dehydrated foods and in those treated by high-short canning processes. The serious deteriorative changes which occur during blanching suggest that the reasons for the process should be clearly defined. Temperatures and times should be reduced to accord with the minimum requirement for canned, frozen, or dehydrated peas.

REFERENCES Adam, W. B. 1941. Factors affecting the vitamin C content of canned fruit and vegetables. Ann. Rept. Fruit Vegetable Preseru. Research Sta., Campden, Uniu. Bristol. p. 14. Adam, W. B. 1942. Factors affecting the vitamin C content of canned fruit and vegetables. Progress report 11. Ann. Rept. Fruit Vegetable Preserv. Research Sta., Campden. Univ. Bristol. p. 12. Adam, W. B. 1956. Experiments with the tenderometer and maturometer. Fruit Vegetable Canning Quick Freezing Research Assoc., Campden. Tech. Memo. No. 14. Adam, W. B., and Dickinson, D. 1945. Estimation of maturity of canned green peas. Ann. Rept. Fruit Vegetable Preseru. Research Sta., Campden. Univ. Bristol. p. 51. Adam, W. B., Homer, G., and Stanworth, J. 1942. Changes occurring during the blanching of vegetables. J . Soc. Chem. Ind. (London) 61, 96. Alexander, 0. R., and Feaster, J. F. 1947. Thiamin and ascorbic acid values of raw and canned peas. Food Research 12, 468. Allen Chlorophyll Co. Ltd. 1957. British Patent 779,560; Abstract Food Manuf. 32, 448. Anderson, A. J. 1949. The influence of plant nutrients on symbiotic nitrogen fixation. Proc. Specialist Conf. Plant and Animal Nutrition in Relation to Soil and Climatic Factors. Australia p. 190. Anonymous. 1947. “Canned Foods Reference Manual,” 3d ed. American Can Co. N.Y. Anonymous. 1952. “Refrigeration Data Book,” Applications Volume. 4th ed. pp. 2-02. Am. SOC.of Refrigerating Engineers. N.Y. Anonymous. 1953. Recommended dietary allowances. Natl. Acad. Sci. Natl. Research Council Publ. No. 302. Anonymous. 1957a. “Canned Food Tables.” Consumer Service Div. Natl. Canners’ Assoc. Washington, D.C.

PRESERVATION O F G R E E N P E A S

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Anonymous. 1957b. Bulk bins reduce field costs. Western Canner and Packer 49, 16. Armbruster, G., and Murray, H. C. 1951. The effect of canning procedures on the nutritive value of the protein in peas. J. Nutrition 44, 205. Association of Official Agricultural Chemists. 1950. “Official Methods of Analysis,” 7th ed. Washington, D.C. Bedford, C. L., and Hard, M. M. 1950. The effect of cooling method on the ascorbic acid and carotene content of spinach, peas, and snap beans preserved by freezing. Proc. Am. SOC.Hort. Sci. 55, 403. Bednarczyk, W. 1950. The choline content of some of the food products on the market in Poland. Roczniki Pahstwowego Zakladu Hig. 1, 225-238; Chem. Abstr. 46, 5737c. Bendix, G. H., Heberlein, D. G., Ptak, L. R., and Clifcorn, L. E. 1951. Factors influencing the stability of thiamine during heat sterilization. Food Research 16, 494. Bendix, G. H., Henry, R. E., and Strodtz, N. H. 1952. (To Continental Can Co., Inc.) Preservation of green color in canned vegetables. U.S. Patent 2,589,037; Chem. Abstr. 46, 5224b. Berth, L. 1955. Quality grading. Proc. Tech, Sessions, Ann. Conv. Natl. Canners’ Assoc. 48, 94 (Information Letter 1526). Bicknell, F., and Prescott, F. 1942. “The Vitamins in Medicine.” Heinemann, London. Bitting, A. W. 1937. “Appertizing or the Art of Canning; Its History and Development.” Trade Press, San Francisco, California. Blair, J. R., and Ayres, T. B. 1943. Protection of natural green pigment in the canning of peas. Ind. Enq. Chem. 35, 85. Boggs, M. M., and Hanson, H. L. 1949. Analysis of foods by sensory difference tests. Advances in Food Research 2, 219. Bohrer, C. W. 1955. Some spoilage and prevention aspects of washing and quality grading operations. Proc. Tech. Sessions, Ann. Conv. Natl. Canners’ Assoc. 48, 97 (Information Letter 1526). Bonney. V. B., and Palmore, J. I. 1934. The maturity of canned peas. Canner 78(18), 10. Bonney, V. B., and Rowe, S. C. 1936. Chemical studies on the maturity of canned peas. J. Assoc. Offic. Agr. Chemists 19, 604. Bonney, V. B., Clifford, P. A., and Lepper. H. A. 1931. An apparatus for determining the tenderness of certain canned fruits and vegetables. U.S. Dept. Agr. Circ. No. 164. Brenner, S., Wodicka, V. O., and Dunlop, S. G. 1948. Effect of high temperature storage on the retention of nutrients in canned foods. Food TPchnoZ. 2, 207. Briant, A. M., MacKenzie, V. E.. and Fenton, F. 1946a. Vitamin retention in frozen peas and frozen green beans in quantity food service. J . Am. Dietct. Assoc. 20, 507. Briant, A. M., MacKenzie, V. E.. and Fenton, P. 3946b. Vitamin content of frozen peas, preen heam and lima heans and market fresh yams prepared in a Navy mess hall. J. Am. Diptet. Assoc. 22, 605. Brush. M. I I-

W

a

a 0

a

SHEARING

FORCE

SHEARING

RATE

FIG.13. Viscosity of pseudoplastic-type flow in non-Newtonian systems.

FIG. 14. Geometry of pseudoplastic-type flow.

be catsup, cream-style corn, tomato juice, mayonnaise, and others. NonNewtonian materials have been classified as having three main types of flow; namely, pseudoplastic, plastic, and dilatant (Minard, 1954). Pseudoplastic materials are described in Fig. 13, which shows that the apparent viscosity decreases as the rate of shear at which the material is tested increases. Many emulsions show this effect. This phenomenon (of liquids reducing their consistency as the rate of shear increases) can be explained by the hypothesis illustrated in Fig. 14. The particles may be elongated or capsule in shape (as compared with the spherical particles in Newtonian liquids). The elongated particles have a tendency to stand up and obstruct the movement of the fluid. As the rate of shear is increased, the elongated particles tend to become more spherical, reducing the resistance to flow or apparent viscosity (Shaw, 1950).

178

AMIHUD KRAMER AND B. A. TWIGG

Plastic materials also show a decrease in apparent viscosity as the rate of shear increases, as do the pseudoplastic materials. They are further characterized, however, by a “yield value,” a n initial starting pressure required to initiate shear, or flow (Fig. 15). Tomato catsup is a good example of this type of material; it is because of its “yield value” that it may not flow from the bottle. After the bottle is struck, however, the initial pressure required to start flow (yield value) is exceeded, and the catsup will then pour (Brookfield Engineering Laboratory, b) .

SHEARING FORCE

FIG.15. Viscosity of plastic-type flow in non-Newtonian systems.

I

I

SHEARING

FORCE

SHEARING

RATE

-

FIG.16. Viscosity of dilatant-type flow in non-Newtonian systems.

Dilatant materials exhibit an increase of apparent viscosity (thickening) as the rate of shear increases (Fig. 16). This characteristic is very important in situations where materials of this nature need to be pumped, since they may become semisolid inside the pump, and thus increasingly difficult to move. Heavily-filled liquids, such as clay slurries, candies, milk chocolate filled with buttermilk powders, heavy starch suspensions, and some paints, are dilatant within a narrow range of concentration (Perry, 1950; Shaw, 1950). Most liquids of this nature return to their original consistency as soon as agitation stops. Thixotropy is another term often found in literature on rheology. Originally, the word was used to describe a reversible isothermal gel-solgel transformations, i.e., those gels that break up on being shaken and

PHYSICAL MEASUREMENT OF FOOD QUALITY

179

reset on standing (see Fig. 17). This curve demonstrates a hysteresis effect, in that the apparent viscosity at any particular rate of shear will depend on the amount of previous shearing (stirring, etc.) it has been subjected to (Brookfield Engineering Laboratory, b) . In common usage now, however, the term thixotropy (thixotropic flow) usually is applied to all systems that show reversible alteration in their flow characteristics when work is performed on them, the alteration being in the direction of greater fluidity with increased work or increased rate of shear (Anonymous, 1955). Thus thixotropic flow would include both plastic and pseudoplastic flow as already described.

SHEARING FORCE

FIG.17. Reversible gel-sol-gel transition in a non-Newtonian system.

The term rheopexy has been used to describe materials which increase in consistency with an increase in rate of shear (Wicker and Geddes, 1943). The rheological, non-Newtonian properties just discussed may change in some products as temperature and concentration are varied. A definite relationship exists between viscosity or consistency and temperature which must be considered. A rule of thumb is that the resistance to flow of a substance will vary 10% for a change of 1OC.Therefore, temperature, as well as other conditions under which the tests are made, must be held constant in order to obtain comparable results. A complete understanding of the properties of a product would be desirable before viscosity or consistency measurements are made in order to select the proper conditions and instruments for measurement.

2. Units of Measurement The measurement of the resistance to flow of a Newtonian fluid is termed viscosity. The unit of absolute viscosity is the “poise.” A mate-

180

A M I H U D KRAMER A N D B. A. TWIGG

rial requiring a shearing force of one dyne per square centimeter to produce a rate of shear of one inverse second has a viscosity of one poise. The centipoise is one-hundredth of a poise. Viscosity is expressed mathematically as follows (West, 1942) :

v = -7rPr4 t 8vl where: V = viscosity in poise; v = volume of liquid in cu. cm.flowing through a capillary tube in time t ; I = length of capillary tube in cm.; F = radius of capillary tube in cm.; P = pressure of system in dynes/ sq. cm.; t = time of flow through capillary tube in seconds. If the times of flow of equal volumes of two liquids through the same capillary are measured under the same head of liquid and at the same temperature (such as could be done in an Ostwald viscosimeter) the ratio of viscosity or relative viscosity is expressed as: n , - =&ti -1 d2tz

n nz

where: n = relative viscosity; d, = density of unknown or material being tested; tl = time of flow through the capillary of material being tested; d, = density of reference solution; tz = time of flow through the capillary of reference solution; n, = coefficient of viscosity of material being tested; n, = coefficient of viscosity of reference solution. This formula gives the viscosity of one liquid relative to that of the other. Water is commonly used as the reference solution. Absolute viscosity can easily be obtained by multiplying the relative viscosity, as obtained above, times the absolute viscosity of water at the temperature at which the relative viscosity was determined (West, 1942). The absolute viscosity of water at 2 O O C . is 1.0050 centipoises and 0.8937 centipoise at 25OC. Some instruments measure viscosity in absolute units; results from other instruments can be changed to absolute units by means of charts or graphs furnished by the manufacturer. The measurement of the resistance to flow of a non-Newtonian fluid is termed consistency. This term has been used to describe many food products which are essentially suspensions. Suspensions do not exhibit true viscosity like Newtonian liquids; however, consistency can be measured by methods similar to those used for viscosity. The results, however, are reported as apparent viscosity, which is a measure of resistance to shear or flow at a given rate of shear expressed in absolute units (Minard, 1954). It is measured at a given rate of shear since the apparent viscosity of non-Newtonian fluids would be different under different shearing rates. Thus, to fully characterize a non-Newtonian

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fluid, apparent viscosity values should be recorded for various rates of shear; however, values recorded at a constant rate of shear are applicable to quality control functions. The indices for obtaining apparent viscosity values vary with different instruments; for example, measurements made with the Stormer viscosimeter are expressed in time (seconds) required for the rotor to make 100 revolutions in the material; measurements made with the Bostwick consistometer are expressed in maximum distance of flow of the material in 30 seconds. These indices can be expressed as apparent viscosity in absolute units by conversion charts furnished by the manufacturer or by preparing a curve with standard sugar solutions. Nevertheless, apparent viscosity may be used to characterize any fluid and has been considered synonymous with consistency (Perry, 1950). Relatiue viscosity has also been used to express measurement of resistance to flow or shear in non-Newtonian fluid. For example, the relative viscosity of a fluid, using the Stormer viscosimeter can be obtained by dividing the time required for the rotor to make 100 revolutions in the fluid under test by the time required to make 100 revolutions in distilled water or other reference or standard fluids (Gould, 1953a). Fluidity expresses the tendency of a liquid to flow, while viscosity is a measure of the resistance to flow. Fluidity is the reciprocal of viscosity (Joslyn, 1950).

3 . Types of Measuring Systems Many instruments are in existence for the measurement of viscosity and consistency of food products. The purpose here is to illustrate various principles on which some objective instruments are based, a. Flow through capillary tube. One of the most widely used methods for determining the viscosity of pure fluids is still, in principle, that of Poiseville, though many modifications in experimental details have been made for different instruments (Hatschek, 1928). Thg Ostwald uiscosimeter is the best known instrument which measures viscosity by flow through a capillary tube. Results are calculated by the time required for the liquid to flow through a capillary a given distance while the instrument is immersed in a constant-temperature water bath. With the OstwaId viscosimeter, the determinations are made as relative viscosity, and the density of the fluids are used in the calculations. Such instruments which use density of the fluids in the calculations are known as kinematic viscosimeters. For the utmost accuracy, the rate of flow from the capillary should be reasonably slow. This can be accomplished by selecting the proper diameter and length of capillary and size of bulb (Joslyn, 1950). The objection to this type of viscosimeter

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is that the driving force, the hydrostatic head of the liquid, is not constant. The pressure decreases as the column of liquid decreases. This difficulty has been overcome in a modified viscosimeter by Ubbehlode, where the liquid is forced to flow through the capillary under pressure obtained with a manostat. Some recent applications of the Ostwald principle were utilized by Lipscomb (1956) on confections, Kulkavni and Dole (1956) on milk fat globules, Nutting (1952) on potato starch paste, and Whittenberger and Nutting (1957, 1958) on tomato products. The Jelrneter developed at the Delaware Agricultural Experiment Station (Baker, 1934) is a simplified version of an Ostwald-type viscosimeter. The Jelmeter is used to determine the correct proportions of sugar, pectin, and acid in making jelly, jams, and marmalade. A similar device is also used by some catsup manufactures. b. Flow through an orifice. Efflux time through an orifice of a specified diameter embodies the principle of several viscosimeters. This type of instrument is especially suitable for highly mobile materials, as is the Ostwald-type viscosimeter. Materials possessing a high yield value may require considerable hydrostatic head to produce suitable flow. An efflux viscosimeter based on this principle was used by Davis et aZ. (1954) to measure the consistency of tomato paste. An official ASTM instrument (Saybolt viscosirneter) for measuring the viscosity of viscous liquids also uses this principle. Results are reported in time of efflux of a definite volume of material through the orifice of a short capillary under constant temperature. The viscosity or consistency range of a system using the flow-through-an-orifice principle can be increased by using a pressurized system to produce flow through the orifice. With this type of viscosimeter, different rates of shear can be imposed. For example, time in seconds can be determined for an efflux of 10 g. at 5-lb. pressure, and the pressure can then be raised in steps for subsequent efflux of 10 g. each. c. Falling weight. Falling-weight viscosimeters depend on the measurement of the time required for a weight to fall through a tube of the material being tested. The weight may vary from spherical to discshaped. The range for this type of instrument may be varied simply by varying the size or the specific gravity of the weight, thereby increasing the driving force. The Gardner Mobilometer is one of several instruments operating on this principle. It measures the consistency by the time required for a plunger to fall between two reference points; the results can also be expressed as the product of the time and weight divided by the distance traveled by the plunger. The Mobilometer can also be equipped with a water jacket to insure more uniform temperature of the sample. It has been used to determine the consistency of oils,

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sirup, heavy cream products, mayonnaise, and tomato products (Gould, 1953a). The same principle is involved in the plastic bowl test by which the firmness of curd is measured by the depth to which a plastic bowl sinks (Baron, 1952). d. Rotation of spindle or cylinder in test material. The measurement of resistance to rotation of a spindle or cylinder immersed in the test material is the basis for several precise, industrial viscosimeters. Most instruments of this type can be classed also as torsion viscosimeters since the results are obtained by a measurement of the torque on the rotary part of the instrument. The measurement of torque, by a calibrated spring, on a spindle rotating at a constant speed in the test material is the principle of operation of the Brookfield Synchroletric-viscometer. The instrument’s dial is graduated so that readings are made directly in centipoises. The driving force for the motor is a low-speed, high torque synchronous motor. I t is geared for hfferent rates of shear, enabling a wide range of viscosity measurements to be made with a single instrument. Non-Newtonian materials can be measured at various rates of shear, provided that rapid means are available to determine the presence and extent of thixotropic, dilatant, and other rheological properties. The viscosimeter has been used on such food products as custards, pie fillings, starches, mustards, tomato products, mayonnaise, salad dressing, cream-style corn, and dairy products (Potter et al., 1949; Gould, 1953b; Hand ct al., 1955; Whittenburger and Nutting, 1957, 1958). The Stormer viscosimeter has also found wide use in the food industry. It measures viscosity or consistency by the time required for a definite number of revolutions of a rotating cylinder, or other type rotor, immersed in the sample. The test cup may be maintained at a desired temperature by means of a water or oil bath. The rotor is activated by the force of a falling weight acting through a series of gears. By increasing the weight, the rate of shear can be increased. Results can be reported as relative, apparent, or absolute viscosity. The absolute unit (centipoise) is obtained by means of a calibration table. The Stormer instrument is reported to have been used f o r determining the viscosity and consistency of starches, sugar solutions, cream-style corn, mayonnaise, pea slurries, catsup, and other tomato products (Kertesz and Loconti, 1944; Robinson et al., 1954; Davis et al., 1954; Luh et al., 1954; Hand et al., 1955; Elehwany and Kramer, 1956; Gardner Laboratory, 1958). e, Rotation of test material around a spindle or cylinder. Most viscosimeters of this type may also be classified as torsion instruments since the torque exerted on a stationary spindle by the rotating test material is a measure of the viscosity or consistency. The MacMichael

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uiscosimeter is a torsion-measuring instrument operating on this principle. A plunger of standard dimensions is suspended by a torsion wire of fixed length from the top of the instrument into a constant-speed revolving cup containing the test material. The sample is rotated by means of a motor. The amount of twist imparted to the wire, depending upon the viscosity or consistency of the material, is read on a graduated disc attached to the spindle. The readings are in arbitrary units; however, by standardizing against solutions of known viscosity in centipoise, the results can be interpreted in absolute units. A water or oil bath is available for uniform temperature conditions. The instrument is reported to have a range of viscosities from a little above that of water to that of stiff glue. The instrument has been used on food products such as gelatins, ice cream, starches, chocolate, cocoa solution, dairy products, and tomato products (Herschel, 1920; Stanley, 1941; Potter et al., 1949; Fisher Scientific Co., 1952). T h Fisher Electroviscometer is a torsion-type instrument which measures viscosity by means of a special torque-magnetic-electrical system. The instrument is based on the principle that fluids being measured have thin layers, each moving with a constant velocity in respect to adjacent layers. The sample exerts a torque on a stationary bobbin, about which it rotates. The bobbin is attached to a coil, which the bobbin’s torque tends to turn. The coil is a magnetic field which resists this tendency to turn.A restoring force from a voltage-regulated power supply tends to swing the coil back to its original position. The power of the restoring force necessary to keep the coil from turning registers on the meter, which is calibrated directly in centipoise. A range from 0 to 50,000 centipoise is possible by using different bobbin sizes. A heavily insulated, constant temperature bath surrounds the sample for more accurate results. The instrument is designed for testing a diversity of products (Fisher Scientific Co., 1952). f. Power consumption. The consistency or plasticity of a product can also be determined by recording total power necessary to drive a mixer or other type shearing instrument a certain number of revolutions. The power consumed can be recorded by a microwatt-hour meter (Bailey, 1930; Jacobs, 1951). The Brabender Farinograph operates on a similar principle in that the torque on the motor housing provides the measure of consistency. It is used widely in cereal chemistry and the baking industry. Dough character can be identified by its ability to absorb more or less water. The Farinograph measures and records this absorption consistency and also the stability and elasticity of the dough. The data obtained are useful in predicting baking qualities of flour and indicating proper fermentation kneading and baking time for highest

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quality (Pagenstedt, 1955). The slope of the extensograph (Grogg and Melms, 1956) was used similarly to describe the elastic and viscous properties of dough. g. Penetration into test material. Instruments of this type have found wide use in testing gelatin, glues, pectin, jellies, and some tomato products. They are particularly applicable for the testing of jelly strength, The Bloom Gelometer was originally developed for the determination of the stiffness of glues, but it also has been useful for measuring jelly strength (Fellers and Griffiths, 1928). The principle of the instrument is based on the lowering of a plunger a predetermined distance (usually 4 mm.) into the products being tested. The force (weight) applied to the plunger to drive it against the resistance of the material is a direct measure of the jelly strength or consistency of the material (Richardson, 1923). The penetrometer also measures the degree of penetration by a blunt instrument into materials, as produced by a given force applied over a given area for a measured length of time at a specified temperature. By the use of penetration cones, Underwood and Keller (1948) found this instrument applicable for the measurement of consistency of tomato paste. It has also been used in tomato paste by Underwood (1950) and McColloch et al. (1950). h. Spread or flow of material. The degree of spread or flow of a material in a given period of time is the principle involved in some widelyused consistometers for plasticlike products such as applesauce, catsup, tomato paste and puree, and cream-style corn. The Bostwick consistometer measures the consistency of viscous material by measurement of the distance over which the material flows on a level surface under its own weight during a given time interval. The test material is held at one end of the metal trough by a gate which can be opened instantaneously by release of a spring. When the gate is released, the product flows out because of the hydrostatic head produced by the weight and height of the product before it is released by the gate. This is the official instrument used by the U.S. Dept. of Agriculture in establishing the score points for tomato catsup (U.S. Dept. Agr., 1 9 5 3 ~ )I.n addition it has been used on other tomato products, jams, and preserves (Anonymous, 1939; Davis et al., 1954) and milk puddings (Rutgus, 1958). The Adams consistometer (Adams and Birdsall, 1946) measures the consistency of foods by the degree of spread or flow of the product in all directions in a given time. This instrument consists of a large metal disc upon which are engraved 20 concentric circles. A steel trucated cone, which holds the sample, fits tight against the center of the disc. When the cone is lifted vertically, the sample flows unrestrainedly over

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the disc. The force initiating the flow is due to the hydrostatic head from the sample in the cone. After 30 seconds, the extent of flow is ascertained by averaging the flow at four quadrant points. Less expensive instruments operating on the same principle are also available. This type of instrument has been used on tomato products, pumpkin, cream-style corn, and lima bean slurries (Davis et al., 1954; Lana and Tischer, 1951; Mason and Wiley, 1958). i. UZtrasonic uibration (Roth, 1957). An ultrasonic viscosimeter is now available, which measures the viscosity or consistency of a product electronically by means of a magnetostrictive sensing element which vibrates longitudinally. A signal generated by the vibrating reed in air is used as a standard. Subsequent immersion of the reed in any liquid causes damping of its vibrations, which in turn reduces the magnitude of the signal sent to the indicator. This reduction is in direct proportion to the viscosity of the sample. The instrument reads the solution viscosity in centipoise density units. The meter is calibrated for Newtonian liquids; however, it is said to give reproducible readings with many nonNewtonian liquids. j . Radioactive density gauges (Bossen, 1957). With the advent of the atomic age, radiation has been put to use as a measure of density of products. If gamma rays are permitted to pass through matter, the amount of absorption of these rays is primarily a function of density. Radioactive density gauges direct gamma rays from a nuclear source through the material being tested to a receiving cell containing an inert gas. The ionization of this gas by the rays passing through the test material produces a current which vanes with the radiation received by the cell. At present, these gauges are being used in industries other than food; however, it is likely they will become applicable for the measurement of consistency of food products in the future (Minneapolis-Honeywell Regulator Co., 1955). k. Continuous viscosity measurement. A continuous indication and record of the viscosity of a product under actual processing conditions may be desirable whenever product quality is directly or indirectly affected by this variable. Such continuous recording systems are available. The Viscometran measures viscosity or consistency by rotating a cylindrical spindle in the material; the torque required to maintain a constant rotation of the spindle is transmitted to an automatic recorder. The instrument is installed directIy on the process equipment, thereby eliminating the need of removing samples for manual testing (Minneapolis-Honeywell Regulator Co., 1954). Continuous and instantaneous viscosity or consistency measurement of many types of materials may be accomplished by ultrasonic vibra-

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tion. The UZtra-Viscoson measures viscosity by exciting a thin alloy steel blade on the end of a probe by a short current pulse. The oscillating blade is magnetostrictive, capable of transforming electrical energy to mechanical energy in the ultrasonic range. The ultrasonic waves produced in the material surrounding the blade cause layers of the material to slip back and forth over one another at a corresponding frequency. An electronic computer calculates the energy required to produce the sliding motion or shearing action, which is in turn proportional to the viscosity of the material. Value of viscosity density units in centipoisegram per cubic centimeter are indicated and fed into an automatic recorder (Minneapolis-Honeywell Regulator Co., 1957). The De Zurik continuous, automatic consistency controller for cream-style corn consists of a feeler-agitator suspended in a flow box or tank. The feeler agitator is rotated by a motor mounted on a ballbearing turntable. Every change in the density of the material makes a corresponding change in torque on the feeler-agitator. An arm from the ball-bearing motor turntable actuates a pilot-controlled dilution valve to increase or decrease the amount of dilution fluid being added (Food Machinery and Chemical Corp.) . The Plastometer consists of a flow bridge with an arrangement of tubing designed to produce a pressure differential between two reference points. The differential pressure in the flow bridge is reported to be a function of consistency (Eolkin, 1957). Rheological problems with different food commodities are discussed by Scott-Blair (1958). C. SIZE AND SHAPE Size and shape are such obvious factors of quality and ordinarily may be measured and controlled so easily, that they are occasionally overlooked entirely. Certainly their importance is frequently underestimated. A casual survey of a number of United States grades and standards, however, should suffice to impress the food technologist with the importance of the nature and, particularly, the uniformity of the size and shape factors (Kramer, 1957b). Grading into various size and shape categories is usually one of the first steps in food processing operations. This may be accomplished by hand o r by means of mechanical sorters, using screens, reels, slats, etc. Grading for size is done not only for the purpose of obtaining uniformity but also for providing each consumer with the size preferred, and at a specified price. Size grading may be used as a means of facilitating succeeding processing operations. Thus, for example, cutting, peeling, or blending operations may be facilitated, or accomplished more thoroughly and effi-

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ciently, if the field-run material is first separated in accordance with the size of the units. Size grading may also be an indirect means of grading for other quality characteristics, as for example small-sieve green beans, or peas are usually, though by no means inevitably, less mature and consequently more tender and desirable (Kramer and Twigg, 1957).

I . Weight Measurements Weight measurements are, of course, obtained by means of scales. Ordinarily spring scales are less precise than balance scales. With the latter the weight of the object to be measured is balanced by a calibrated counterweight. Weights may be recorded as total weight, average weight, weight per unit, percentage of units above or below a given weight limit, etc. The most common application of weight measurements is for fill-in weight, or drained weight of a container. In such cases, individual units are rarely measured, but rather a number of replicate containers such as cans, cartons, jars, etc. Such weight measurements lend themselves very readily to posting on control charts (Grant, 1946). Where uniformity of size is important, it is necessary to weigh out individual units of a product. Such measurements, however, may be accomplished more easily by other means, as described below. 2. Volume Measurements Measurement of volume is accomplished by a determination of the space occupied by the object being measured. The measurement may be one of apparent displacement, where no account is taken of the air spaces among the units. Absolute displacement involves the measurement of the space actually occupied by the unit and does not include any adjacent unoccupied voids. Apparent displacement is used very commonly in terms of units per container, such as number of oranges per box, o r number of apricots per can, or in recipe information that may appear on the label, such as number of pieces or units per can. Although at times it is described in terms of counts per unit of weight (for example, number of shrimp per pound), such a measurement is still one of volume rather than weight. To obtain a measure of absolute displacement, the unit or units may be immersed in a liquid medium, and the change in the level of the liquid noted. For example, water is poured into a graduated cylinder to the mark of 10 ml. A bean is then submerged in the water, and it is noted that the water level in the cylinder has risen to 12 ml. It may, therefore, be concluded that the volume, or absolute displacement, of

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that bean is 12 minus 10, or 2 ml. Particularly for such small units, samples consisting of many units are immersed simultaneously, and results reported as total volume per number of units, or as an average volume per unit. Thus, for example, if 100 beans raised the water level from 200 to 400 ml., the results could be reported as 200 ml. per 100 beans, or as an average of 2 ml. per bean. Exceptionally dense materials may be submerged in salt or sirup solutions of appropriate density. Similarly, buoyant materials may be completely submerged in water physically by the use of a screen or plate, or they may be submerged in lighter-than-water liquids, such as alcohol or xylene. In all cases, but particularly where rapidly penetrating liquids are used, the determination should be accomplished rapidly before any appreciable quantity of the liquid is absorbed into the product (Jenkins, 1954).

3 . Weight-Votume Ratio Although it utilizes two size measurements, this ratio is not strictly an expression of size, but rather that of the density of the product. Thus, absolute density is defined as mass (weight) per unit volume, and relatiue density as the relation of the density of a substance at a given temperature to the density of a standard (usually water) at the same temperature. When the relative density is corrected for the buoyancy of the atmosphere, specific gravity is obtained. An easy method of obtaining the relative density may be illustrated by continuing with the above example with beans whose volume was 200 ml. If the weight of these beans before immersion was 220, then their relative density is 2201200, or 1.1. For rapid in-plant control procedures, it may not be practical or desirable to take into consideration the air spaces among individual units and those between the product and the container walls. In such cases, a container of known volume is merely filled to capacity, and the net weight of the product noted. When this net weight is divided by the volume of the container, the result obtained cannot be properly called relative density or specific gravity. A more suitable term, which has been used by The United Company, Westminster, Maryland, would be apparent density. Such determinations are useful for establishing fill-in weights (Cover, 1948), 4 . Length, Width, and Diameter Measurements Length, width, and diameter measurements are used on numerous products, especially where uniformity of size is important, or when a restriction is stipulated on the minimum or maximum size. Many sim-

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ple devices are available for such measurement, the most common of which is the straight-edged ruler. For more accurate measurement a vernier caliper or a micrometer can be used. The U.S. Dept. of Agriculture raw-product inspectors use measuring devices designed for size grading of particular commodities (Batjer and Rogers, 1954). For rapid measurements in cases where more than one unit is to be classified, a series of screens or slits can be constructed, such as a laboratory pea size grader, or the procedure used by Kimball and Kertesz (1952) to determine the size distribution of suspended particles in macerated tomato products. Where very small particles are involved, which because of their size or condition are difficult to separate by means of screens, it may be necessary to resort to microscopic examination (Kazakov, 1958). Particles so small that they remain in suspension may be separated by sedimentation, so that after a period of standing time, distinct layers will form with the larger particles at the bottom. The speed of formation of the sediment may also be an indication of particle size (Toldby, 1958). Small pulp particles may be separated from the liquid phase (serum) by sedimentation, by gently washing in water over a screen, or by centrifuging. Ratio of length to width, or height to diameter may be used to characterize the shape of units of a product or its wholeness. Thus, for example, a whole, firm, canned tomato will have a higher height/diameter ratio than a broken-down, soft tomato. Wholeness of diced or sliced products may be reported as number or per cent of units that do not entirely conform to the proper shape.

5 . Symmetry Symmetry is defined as the mutual relationship of parts (as in size) arrangement and measurements. In reference to food products, uniformity of symmetry would mean the lack of mixed units of irregular sizes and shapes. To measure the conformity to a basic or average configuration, photographs, shadowgraphs, or models may be used as well as a visual observation for an estimate of symmetry. Simple tools and devices discussed under the other size and shape characteristics would also be useful.

6 . Curvature Curvature measurements may be needed for products which have a tendency to curve (such as snap beans and pickles). Figure 18 illustrates a procedure used by the US. Dept. of Agriculture for determining

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curvature of pickles. The results are reported in degrees of angle. The angle of a curved pickle means that angle formed by the intersection of lines projected from either end, approximately parallel to the sides of the pickle, adjoining the stem and blossom ends, respectively (U.S. Dept. Agr., 1954), The angle can be measured by a protractor.

FIG.18. Measurement of curvature in pickles. (Processed Products Standardization and Inspection Branch, A.M.S., U.S. Dept. Agr.) 7. Area

For a measurement of area of an irregular-shaped product, an outline of the produce is traced on a piece of paper to form an enclosed curve. After this has been done, several methods are available for determining its area. Weighing. Although there are obvious limitations in accuracy, it is possible to approximate the area within an enclosed curve by cutting out the area, weighing it, and comparing its weight with the weight of an area of readily-determined, known dimensions, assuming a proportionality to exist between weight and area. Planimeter. This is an accurate instrument widely used for determining the area included within a closed curve (Miller et al., 1956). It has an added advantage in that the determination can be made rapidly. The outline of the area is traced with a pointer while the base is held in a fixed position; the area is read direct from a vernier measuring roller. Mathematical. Mathematical approximations of area in a n enclosed curve are also available. By Simpson’s Rule, a base line is drawn at the bottom of the curve as shown in Fig. 19. Divide the base “OX” into an even number of equal parts, and measure the ordinate at each point of division. Add together the first and last ordinates, twice the sum of the other even ordinates; multiply the sum by one-third of the distance between consecutive ordinates. In other words, from Fig. 19, the approximate area = % a ( h o 4hl 2h2 4h3 2h4 4h5 2h6 4h7

+

+ + + + + + +

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hs). This technique permits the determination of the area included between a curve and a straight line. In order to find the area of the irregular-shaped figure in the diagram, it would be necessary to determine the area of the portion between the figure and the base line, and also the area between the top line of the figure and base line. The former area is then subtracted from the latter to give the area enclosed within the curve (University of Maryland, 1956). Auerage ordinate rule. Divide the base line (Fig. 19) into any number of equal parts, and at the center of each of these parts draw ordinate, e.g., mn, po, sr. Take the average length of these ordinates and multiply by the length of the base line. The result is the approximate area enclosed (University of Maryland, 1956). M

FIG.19. Determination of

area by mathematical approximations.

D. DEFECTS Most defects are another factor of quality still largely evaluated by the consumer’s eye, and thus are classified along with color and consistency as appearance factors. Many products of high quality in all other respects may be downgraded because of defects. Defects has been defined as “Imperfections, due to the absence of something necessary for perfection, or the presence of something that distracts from perfection” (Webster’s New International Dictionary, 1936). Since we may assume that absolute perfection is unattainable with any biological material, our problem is not to determine whether a particular unit is perfect or imperfect, but rather to determine whether a particular defect is of sufficient magnitude to be objectionable at a given level of acceptability. Thus in grading foods for defects, tolerances may be established in terms of maximum numbers of defective units allowable, such as number of discolored kernels in a No. 2 can of corn, or in terms of total quantity of a defect that may be present, such as area of unpeeled skin in peeled tomatoes (U.S. Dept. Agr., 1956a). Although defects may ordinarily be determined rather easily, they

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occasionally present difficult problems, usually quantitative rather than qualitative. Thus it is difficult to determine whether a particular discolored spot is dark enough or large enough to be counted as a defect, or whether two smaller spots are only as unacceptable as one darker, or larger spot. Attempting to establish tolerance limits for each of many such detailed criteria may result in unwieldy and lengthy procedures. 1. Classification of Defects Defects may be classified into the following categories: (1) geneticphysiological, (2) entomological, ( 3 ) pathological, (4) mechanical, and (5) extraneous or foreign matter. a. Genetic-physiological defects. Such defects occur as a result of hereditary abnormalities of the raw material or as the effects of unfavorable environmental conditions during the growth and maturation of the crop. Normal functions of metabolism of the plants may be disturbed by extremes of temperature, water supply, or nutrition, or genetic aberrations. These genetic-physiological defects may be further subdivided as follows: Structural defects are mostly identified as misshaped or misshappened pieces due to abnormal growth of tlie crop. Fasciation, a common malformation in plant parts resulting in enlargement and flattening, as if several parts were fused, is a common defect in asparagus and strawberries (Zielinski, 1945). Crooks and malformed cucumbers would constitute a defect in the packing of fancy whole pack pickles. Hollow stem is a defect of cauliflower (US. Dept. Agr., 1951). Off-color defects of a physiological nature are mostly caused by some genetic disturbance which would promote abnormal growth and coloration of tissue cells, as may be commonly observed in some seedtype crops, as off-colored edible seeds. A classical example would be the phenotypic phenomenon of xenia in white sweet corn. Xenia is the immediate effect of the sperm of the pollen parent .on the endosperm. It occurs, for example, when white sweet corn is accidentally pollinated by yellow corn. The result is the presence of some yellow kernels on what should be an entirely white-kerneled ear. These yellow kernels would be classified as defects in a white corn pack. Blond peas is another genetically induced off -coloring which can cause serious trouble for pea packers (Sinnott and Dunn, 1932). Character defects refer to the degree of development of certain tissues or plant parts, such as excessive development of fibrovascular bundles or floral or fruit organs. Such defects may best be described by definitions given in the U.S. Grades and Standards, such as: (A) Frozen asparagus: “The factor of character refers to the degree of development of the head and bracts, the tenderness and texture

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of the unit, and the degree of freedom from shriveling” (US. Dept. Agr., 1953a). (B) Canned sweet potatoes: “The factor of character refers to the texture and condition of the flesh, the degree of freedom from tough or coarse fibers, the tenderness of the canned sweet potatoes, and the tendency of sweet potatoes packed in a liquid packing medium or as vacuum-pack (without packing media) to retain their apparent original conformation and size without disintegration” (U.S. Dept. Agr., 1953b). b. Entomological defects. Insects cause injury to a wide variety of crops and thus are a major source of defects in fruits and vegetables, as well as other food materials. Each commodity may be attacked by few to many insect species, each causing a different type of damage. The damage (defects) may be direct, as a result of the insect’s own activities such as feeding, oviposition, and stings. I n other cases, the principal damage may be indirect, caused by a disease organism introduced into the crop by the insect (Ross, 1948). Holes and scars are defects of food products which are generally caused by insects with chewing mouthparts, where noticeable portions of the product are removed. Leafy-type crops may be seriously damaged by a class of insects called leaf miners as well as grasshoppers, Japanese beetles, etc. Stem- and pod-type crops may be damaged by the same type of insects and also by boring insects. Holes made by the chewing insects may be found in corn kernels, snap bean pods, and tomatoes. Roots and underground tubers are eaten by larvae of many beetles, flies, and moths. Lesions, 08-coloring, and curled leaves are defects caused by insects with piercing-sucking types of mouthparts, such as aphids and mites. I n this case, no gaping wounds are noticeable. When feeding on living tissue, sucking insects empty the plant cells, removing the green color and causing a whitening or etiolation followed by production of lesion o r scar tissue. Early feeding punctures often result in a tiny white spot, and when they are extremely numerous, the entire plant part may appear blanched. Discoloration of tissue may also result indirectly from damage inflicted by chewing-type insects, Curling of leaves is a common occurrence following heavy feeding from suckingtype insects. Egg laying and stings may also cause damage similar in appearance to defects caused by sucking. Insects often affect certain crops by disseminating pathological diseases. A few plant diseases, not actually carried by insects, gain entrance to the crop through insect feeding or oviposition punctures (Ross, 1948). c. Pathological defects. Failure of plantings to produce commercial products of satisfactory quality and quantity may result from the action

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of bacteria, fungi, mold, or virus. The crop may suffer only in quality, or in many cases there is lowering of quality along with a reduction in quantity (Heald, 1933). Structurally, the crop may be disfigured or deformed. The defects may appear externally only as surface phenomena in the form of lesions, scabs, or off-coloring. Hidden defects may occur as internal defects as off-coloring, corky tissue, mold, and rot. Regardless of the type damage on the crop, the lowering of keeping-quality due to the possibility of rot and mold is greatly enhanced. Pathological infections may do more than alter the appearance of the product; they may decrease wholesomeness. For example, Anthracnose disease of tomatoes not only causes a visual lesion to the tomato itself but also increases the mold count of tomato products. d. Mechanical defects. Mechanical defects arise from damage to the product of a physical nature. Although it may be impossible to eliminate such defects, the degree of severity of such damage can be regulated to a certain extent by care in handling and by proper adjustment of equipment. Bruising of tissue, which may result in discoloration, is undoubtedly the most commonly occurring defect of a mechanical nature. Damage inflicted on tissues by bruising, upset normal biochemical reactions. As a result, abnormal metabolism occurs causing discoloration and hastened deterioration. Care in handling is the best method of preventing the occurrence of these defects, as well as such things as broken pieces and loose skins. Ragged cuts and slices, crushed pieces, pulled kernels, etc., on the other hand, are examples of mechanical defects which are most readily controlled by proper adjustment of harvesting and processing equipment. e. Extraneous or foreign material defects. Defects in this classification refer to materials of a harmless nature which are not part of the edible portiop of the product, For example, with peas it would mean leaves, pea pods, stems, thistle buds, and buds and seeds from other harmless plants; with corn, it would mean pieces of cob, husk, and silk. Control of these defects starts in the field before harvest, continues at the washing and cleaning station, and ends with the sorting belt. 2. Instrumentation

Available instruments for measuring the defect factor may be considered largely as aids to visual examination rather than complete objective procedures in themselves. These may serve one or more of the following purposes: (1) improve visibility, (2) standardize conditions of examination, (3) serve as reference standards, (4) count or measure, and (5) eliminate defective material.

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a. Improving visibility. Examination for defects implies, at least in theory, 100% inspection of the sample. In practice this is rarely attained but may be more nearly approached as more care and time is taken to perform the visual inspection, especially when aids are used such as the following: dilution-with fluid products, the sample may be diluted with water, thus making it thinner, so that the defects may be seen more readily; white background-the sample may be spread out on a white surface to accentuate dark-colored defects. Color diflerence. If the defects are of a type that reflect light in a given manner, but differently from the normal material, the illumination can be adjusted in such a way that the color of the defects is maximized, and that of the normal material minimized, thus providing greater contrast between the defect color and the substrate. This may be done by illuminating the sample with reflected light composed of a spectral band limited as narrowly as practicable to the dominant wavelength of the defect. This in effect serves as a filter through which the defect may be seen to best advantage. For example, pieces of red apple skin may be seen in yellow-green applesauce more clearly if red light is used to illuminate the sample. Transmitted light may also be used for the same purpose. In this case, the light source would be placed under a transparent plate of glass or plastic on which the sample is spread. The hue of this light source should be complementary to, rather than the same as, the hue of the defect. b. Standardization of conditions. As stated above, accounting for 100% of the defects cannot be expected; however, comparable results may best be achieved if the conditions of the examination are standardized. Thus it is well to specify time limits for an examination, as well as sample size, container, and light qwlity and intensity. A sample with a plastic-type flow may be combed by the use of, some simple device which spreads out the material to a uniform depth so that defects may be noted more easily, or at least through a uniform depth of material. c. Reference standards. Illustrative material (from simple sketches to photographs, color chips, drawings, and painted plaster-of-Paris models) may serve as useful reference standards in identifying the nature of a specific defect as well as in determining its severity. Such visual aids are used extensively by the Standardization Section of the Fruit and Vegetable Division, A.M.S.,U.S. Dept. Agr. d. Counts and measures. In some instances, a defect or defective unit is merely counted. In other instances, it is first necessary to determine quantitatively the magnitude of the defect, Some aids are

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available or may be easily constructed for this purpose. A plastic disc with concentric rings of different diameters is useful to determine the size of a defective area. Squares or holes punched in metal or plastic may serve a similar purpose. Where counts are to be made, it may be helpful to superimpose grids over the sample surface, and count the defects systematically in each grid separately. Work is now in progress at the University of Maryland on the development of an instrument which will automatically tally defects in products such as applesauce and tomato catsup. With this instrument, the sample, combed to a predetermined thickness, is presented to an electronic scanning device which scans the entire surface of the sample in discreet 2-sq. mm. units. When such a unit transmits less than 50% of the light, as compared to the normal material, a photoelectric cell activates a counter. After the entire surface of the sample is exposed, the total number of such defective areas is tallied on the counter (University of Maryland, 1957). e. Isolation of defects. A number of devices and procedures are available by which the defects or defective units may be separated from the bulk of the sample, thereby simplifying the problem of their measurement. These include: Flotation. Some defects may be floated off by the use of water, oil, or gasoline. For example, loose skins and other light particles may be floated off merely by the use of liberal quantities of water. Defective units having an affinity to oil, such as weed seed, or to gasoline, such as insect fragments, will rise to the upper layer after mixing, and thus be separated from the bulk of the sample. Elution. This process is the reverse of flotation, whereby defective materials characterized by a high specific gravity may be separated by stirring with liberal quantities of water or other liquids. Thus particles of grit, or stone cells, will sink to the bottom of a container. The rest of the material is decanted from the defective material. Electronic sorting. Where defective units exhibit a color difference as compared to the normal units, the sample may be put through an instrument such as an electronic sorting eye, and the defective units separated out automatically. By the use of an instrument of this type, blond pea seeds may be separated from green; black beans separated from brown, etc. IV. KINESTHFTICS

Kinesthetic characteristics deal with the sense of feel, just as the characteristics of appearance have to do with the sense of sight. Our problem, therefore, is to find physical instruments which will simulate

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and measure the sensations which the consumer experiences through her sense of feel, with the fingers and more particularly in the mouth. A. CLASSIFICATION Some of these sensory characteristics are listed as follows: (A) Finger feel: (1) Firmness, as encountered by the consumer selecting a firm apple, measured physically by compression. (2) Softness, or yielding quality, as in selecting a peach, or plum, measured physically by compression. ( 3 ) Juiciness, as in immature sweet corn, where the thumbnail is used to test the ease and amount of juice squeezed out of a kernel, measured physically by puncturing, or juice extraction. (B) Mouth feel: (1) Chewiness, as sensed by the resistance of the product to compression and shearing action of the teeth. ( 2 ) Fibrousness, as sensed by the presence of an inedible residue remaining in the mouth after mastication, as well as resistance to cutting force of the teeth. ( 3 ) Grittiness, as sensed by the presence of small grit particles, such as sand, or stone cells. ( 4 ) Mealiness, as sensed by the coating of starch or other material with adhesive properties, over mouth tissues. ( 5 ) Stickiness, as sensed by the mouth while chewing foods with adhesive properties. (6) Oiliness, as sensed in the mouth, caused by oily or soapy products. In general, these factors lend themselves readily to objective measurement by the use of mechanical instruments. Thus a considerable array of tenderometers, texturemeters, puncturemeters, succulometers, fibrometers, and pressure testers are available. These instruments vary in their precision as well as in their accuracy. Considering the tremendous variability encountered in food materials because of differences in varieties, growing conditions, and soils, these instruments frequently require special calibration for use with different varieties, and perhaps in different geographical locations.

B. PRINCIPLES OF MEASUREMENT In spite of the large array of instruments that have been developed for the purpose of measuring these kinesthetic factors, actually there are only a few basic principles involved. Many of these sensations have in common the principle of resistance to force, so that in general, the

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unit of measurement may be in terms of pounds force. This force may be applied in a number of ways, or through a combination of two or more of these methods. Thus: (1 ) Compression. This refers to the squeezing together of the test material so that it still remains as a single undivided unit, but may

r r

FIG.20. Compression.

FORGE

fORCE

FIG. 21. Shearing.

FORGE

E

FIG.22. Cutting.

occupy less volume. Finger tests and pressure testers fall in this category (Fig. 20). (2) Sharing. This results &om the application of force where the test material is separated into two (or more) parts, with one part sliding beyond the other part (Fig. 21 ) .

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FIG.23. Tensile strength measurement.

FIG.24. Shear-pressure.

( 3 ) Cutting. This occws when force is applied in such a way that the test unit is divided, so that the portions remain in their original position in relation to each other (Fig. 22). (4) Tensib strength. This is the application of force away from the material rather than towards the material, when force is applied to pull the test material apart (Fig. 23). This type of test is more commonly used in the textile industry, although it does have some application to TABLE V RELATIONOF KINESTHETICCHARACTERISTICS TO PHYSICAL METHODS OF APPLICATION OF FORCE Sensory reaction

Physical test

Firmness Yielding quality

Compression Compression

Juiciness

Compression (juice extraction) Shear-pressure

Chewiness Fibrousn ess Grittiness Mealiness Stickiness

Cutting; comminuting -

-

Tensile strength

Instruments or procedures for measurement Pressure tester; shear-press Pressure tester; shear-press; ball compressor Puncture tester; succulometer; shearpress; moisture tests Tenderometer; texturemeter; shearpress; specific gravity; solids Fibrometer; shear-press; fiber analysis Comminution; elution; sedimentation Starch, and/or gum analysis Jelly strength, pectin, and/or gum analysis

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foods, such as the procedure used by the Food and Drug Administration (1954) to measure the toughness of strings in string beans. (5) Shear-pressure. This combined application of force simulates the action of teeth which first compress and then shear the food. Instruments such as the tenderometer, shear-press, and the texturemeter act essentially in this manner (Fig. 24). The relations of these consumer sensations (sensory) to the physical methods of application of force (physical) are summarized in Table V, together with some of the instruments and procedures that have been suggested as methods of measuring these characteristics.

C. INSTRUMENTATION The development of instruments for measuring kinesthetic characteristics may be said to have begun in 1917, when Professor 0. M. Morris (1925) attempted to simulate the “time honored custom of pressing the fruit with the ball of the thumb to determine its ripeness” by noting on a spring scale the pounds of pressure required to press a marble into the side of an apple. Since that time many ingenious devices have been constructed in which rods, bars, blades, wires, or needles are used to penetrate the test material. In the early studies, force was applied by the operator’s hand directly, or by means of a wheel or hand pump. Later, weights, liquid columns or electric motors provided the power source. Measurements of the force applied have been obtained by noting the contraction of calibrated springs, scales, hydraulic gauges, and dynamometers. More recently, instruments have been developed where the total work is recorded continuously on a chart in the form of a time-force curve (Decker et al., 1957; Proctor et al., 1955). The purpose of these measurements is not only to determine the resistance to force offered by the test material as a measure of the kinesthetic property, but also to predict such things as optimum harvest dates, soaking and cooking times and temperatures, fill weights, blending proportions, stage of maturity or ripeness, degree of fibrousness, and succulence. L

I . Compression Since the first pressure tester was introduced in 1917, many workers in state experiment stations and in the U.S. Dept. of Agriculture have added refinements to the fruit pressure tester and have developed empirical scales for use with specific commodities and varieties under given conditions (Haller, 1941; Magness and Taylor, 1925). The instrument in current use consists of a plunger (varying in diameter) attached to

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a calibrated spring encased in a sleeve which is graduated in terms of pounds. As the plunger is pressed into the fruit, the spring contracts, and an indicator shows the pounds pressure required to press the plunger into the fruit for a given distance. Although results with the pressure tester have been found to vary considerably because of difference in varieties, regions, and seasons, it is still widely used in the fruit industry. Other limitations of this method are that it is destructive, or damaging to the fruit, and that many individual readings must be obtained before an average value may be established for a lot of fruit. The puncture tester developed by Caldwell (1939) for use on products such as sweet corn, may be said t Q be a miniature pressure tester, in that a needle (approximately l/ls inch in diameter) is used instead of a plunger, and the scale is in grams instead of pounds. The method used by the Food and Drug Administration (1954) to measure the hardness of a pea cotyledon is quite similar to the puncture tester. Here the measure of the required force is not the compression of a spring but the volume of a liquid required to provide sufficient weight for the needle to plunge into the pea cotyledon. A ball compressor was proposed by Wearmouth (1952) to measure cheese body (texture). Briza (1955) used an adaptation on grapes which provides a measure of firmness of grapes in terms of g./sq. mm. The succulometer developed a t the University of Maryland (Kramer and Smith, 194613) makes use of the principle of compression indirectly, in that the volume of extractable juice under controlled conditions of time and pressure is the measure of quality, It is being used for measuring the maturity of sweet corn, the storage quality of apples, and the oil and water content of canned tuna (Food and Drug Administration, 1957). 2. Shearing The tenderometer developed by Martin (1937) at the American Can Company, the texturemeter developed by Christel in Wisconsin, and the maturometer developed by Lynch and Mitchell (1950) in Australia serve as examples of instruments measuring shearing force, although in actuality the force measured is a combination of shearing and compression, with compression preceeding the shearing action (Fig. 24). All three instruments were originally designed for measuring the maturity of raw peas for processing, and although their use was suggested for other commodities, by and large they are still utilized primarily for this one purpose. The tenderometer has become the recognized instrument for

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measuring raw pea quality because of its precision and high correlation with alcohol-insoluble solids of the processed product (Walls and Kemp, 1940). The grid assembly of the tenderometer simulates jaw action in the eating of peas, in that the lower and upper sets of grids, or bars, are hinged together. I n contrast to mouth parts, the lower set of bars remain stationary, while the upper bars rotate from the common hinge. These upper bars first engage the lower bars at the ends opposite from the hinge, with the sample compressed in between. As the upper bars continue to intermesh with the lower bars, they shear through the test sample. The test material is first compressed and then shorn with part of the material extruded ahead of the rotating bars. Power is provided by means of an electric motor and a hydraulic system. The force in pounds per square inch is shown by an indicating hand synchronized with counterweights. Although the tenderometer rates very high in precision, its use is limited because it is not easily portable and cannot be standardized easily in the field. Attempts to find some test material for calibrating the instrument have not thus far been successful (Kramer and Aamlid, 1953). The texturemeter, though highly portable and less expensive than the tenderometer, leaves much to be desired from the standpoint of precision. Here, a group of 25 rods (each 3/ls inch in diameter) travel through the mass of sample until they pass through matching holes in the bottom of the cylindrical cup. Power is applied by hand through the rotation of a handle and gears which force down the cylinder, to the bottom of which the rods are attached. Force in pounds per square inch is indicated on a hydraulic gauge which is attached to the top of the cylinder. Schneider (1955) described a homemade texturemeter, and Doesburg and Grevers ( 1952) have described a new type of tenderometer. The maturometer (Lynch and Mitchell, 1950) is similar to the texturemeter, except that each of its 143 rods shears through a single pea while it travels towards and through a matching hole on which the pea is positioned. Total force required to shear through the 143 peas is indicated on a single gauge.

3 . Cutting Wilder (1948) of the National Canners Association developed the fibrometer specifically for identifying fibrous asparagus stalks. The instrument consists of a channel shaped to contain an asparagus stalk. This channel is slit at each 0.5-inch interval to allow for the passage of a wire 0.035 inch in diameter. A 3-pound weight is attached to the wire in the form of a horseshoe. A stalk of canned asparagus is placed in the

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channel, and the wire is placed through the slits until it rests on the stalk. Any segment of the stalk which the wire does not cut through, is considered fibrous. The procedure is generally satisfactory and fairly well correlated with fiber content, except that it tends to be too severe for large size stalks, and/or too lenient for small diameter stalks (Kramer et al., 1949). The fiber-pressure tester was developed by Kramer et al., (1949) in an attempt to make the fibrometer principle more versatile, so that corrections could be made for differences in stalk diameter, and so that it would be used on raw as well as processed products. This instrument is essentially a modification of the pressure tester in which the plunger is replaced by a 1-inch square of stainless steel. 0.017 inch thick.

4 . Shear-Pressure As indicated above, tenderometer-type tests are actually combinations of compression and shearing forces. These tests simulate the action of the teeth, which first compress, and then shear the food being chewed. Proctor et al., (1955) developed a recording strain gauge denture tenderometer where the test cell actually consists of a set of plastic dentures. A mechanism is attached to provide a continuous chewing motion. Force required to chew the samples is transferred to a strain gauge, whose deflections are recorded as a curve. Since all the above methods require the application of force, it is logical to assume that all such kinesthetic measurements can be accomplished with one power unit in the same way that all color measurements may be made with one color instrument. One such multipurpose instrument is the shear-press (Kramer et al., 1951). Different test cell assemblies may be provided for use with the same power unit to accomplish the three different types of testing. Thus, one test cell similar in design to the pea tenderometer, may be used to measure hardness or firmness of such different products as peas, lima beans, sliced apples, chicken, beef, or spaghetti. Of course, the range of values for lima beans will be many times higher than for cooked spaghetti; however, both may be tested with the same instrument and test cell, and with the use of gauges of different ranges. Another test cell similar in design to the asparagus fiber-pressure tester, may be used for measuring the fibrousness of such commodities as asparagus and celery, while a third test cell, similar in design to the succulometer, may be used to test the succulence of sweet corn, or apples, or water content of canned tuna. The original shear-press model, while utilizing some of the principles of the tenderometer and texturemeter, was an attempt to develop

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an instrument more versatile, easily standardized, and portable than the tenderometer, and more versatile and accurate than the texturemeter (Kramer et al., 1951). The last model, released in 1956, has practically unlimited versatility accompanied with maximum precision (Kramer and Backinger, 1956). The basic unit consists of a hydraulic drive system for moving of a piston a t any predetermined rate of travel, adjustable from 15 to 100 seconds for full stroke. Automatic limiting pressure and fast return of drive piston are provided. For the standard unit primary hydraulic power is obtained from a gear pump driven by an electric motor. For field use where electric power is not available, the electric motor may be replaced by an air pressure accumulator. Measurement of force is provided by the compression of a proving ring dynamometer, similar to ones used by National Bureau of Standards for calibration of testing machinery (Wilson et al., 1946). Fixed point resolution of 0.5% may be obtained from individual calibration curves, Different rings are available capable of providing ranges from maxima of 100 pounds for relatively soft materials, to 6000 pounds for hard products. Readings may be obtained from gauges fitted into the proving ring, or electrically by the use of transducers. Where an electrical measuring device is used, a recorder may be attached to obtain a time-force curve for the entire stroke, instead of a mere maximum force reading (Decker et al., 1957). The test cell is attached directly to the proving ring, thus eliminating any possible frictional error, since the force developed by the resistance of the food material to shearing or compression is transferred directly to the measuring system. The standard test cell consists of parallel stainless steel blades which precisely mesh with the sample box, A unique design is provided for the positive meshing of the blades with the cell, thus obviating damage to the cell from improper meshing. The standard test cell has been used successfully for the measurement of maturity of raw peas, lima beans, southern peas, firmness or hardness of raw and canned apple slices, beets, chicken, beef, shrimp, and spaghetti. Additional cells have been developed for the measurement of fibrousness of asparagus and string beans, and succulence of sweet corn and apples.

METHODS D. PHYSICAL-CHEMICAL

In the absence of adequate devices for measuring directly kinesthetic properties, empirical, physical-chemical tests have been devised and used successfully. Some of these are highly accurate, particularly when

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B.

A. TWIGG

the details of the methods are adhered to rigidly. Although in comparison to the ordinary analytical chemical methods they may be considered rapid, they are still considerably more time-consuming than the instrumental methods just discussed.

I . Moisture Content Working with sweet corn and other vegetable crops, Caldwell (1939) demonstrated that as these crops approach maturity (and become more chewy and thus less desirable) they tend to accumulate solids. They therefore suggested the use of a moisture (solids) determination to measure this quality characteristic. The AOAC method (Association of Official Agricultural Chemists, 1950) of moisture analysis usually calls for the use of a sample of given size, spread over a given surface, to be dried to a constant weight in a vacuum oven, usually at 70OC. Although this procedure is excellent from the standpoint of precision, it requires a drying time of 6 hours or more. Thus many procedures have been developed in an attempt to reduce the time required and thus make the tests useful for quality control under commercial conditions. The toluene distillation and Brown Duuel methods involve the distillation of the water from a sample mixed with toluene or oil. The time requirement is reduced to less than 1 hour; however, a considerable amount of precision is lost (Geise et al., 1951). The Brabender moisture tester utilizes the rate of moisture evaporation as a means of indicating moisture content. A weighed sample of the product is evaporated at fairly high temperatures for a fixed time period, after which the sample is reweighed, and the moisture content is derived from a calibration curve. The drying and weighing is accomplished in a single compact unit which operates automatically and continuously. Cenco moisture balance is another automatic instrument operating on a similar principle, except that infrared radiation is used for drying. There are many electronic moisture testers which are satisfactory for relatively dry products whose moisture content does not exceed 20% or perhaps 30%. Most raw and canned or frozen products, however, contain moisture levels that are much too high for precise electronic moisture determination. The Steinlite Model No. 300 (Seedburo Equipment Co., 1957) overcomes this difficulty by first extracting the moisture from a small sample with a relatively large quantity of solvent of low conductivity. The conductivity of the resultant mixture is then measured in the instrument, and the moisture content of the original sample ascertained (Gamer, 1952).

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2. Alcohol-Insoluble Solids

For products to which soluble compounds such as sugar o r salt are added, a total solids determination is not valid as a measure of tenderness-maturity. For such products, Kertesz (1935) introduced the alcoholinsoluble solids determination, which consists of the extraction of the sample with 70 to 80% alcohol. The filtered, washed, and dried residue is a measure of the alcohol-insoluble starches, celluloses, fiber, pectins, and proteins which account for the chewiness and mealiness of the product. The method is particularly suitable for vegetables such as peas, sweet corn, and lima beans. It has been adopted as an official method by the Food and Drug Administration in the standards of quality for canned peas and sweet corn (Food and Drug Administration, 1954).

3. Fiber The AOAC method for fiber involves the isolation of that fraction of the food product which is not digested by boiling weak acid and alkali. A rapid modification of the procedure was developed by Bonney at the Food and Drug Administration, which consists of the boiling of the sample in water and 50% NaOH, stirring, filtering through a 30-mesh Monel metal screen, and drying (Food and Drug Administration, 1954). The dried residue consists mostly of strands of fiber and some gelatinous material. This is the official method included in the quality standards for canned green and wax beans, and it is being considered for asparagus. A more direct procedure, consisting of the physical separation of fibrous strands in beans and asparagus and skins in sweet corn, has been developed which is still more rapid and more directly related to fibrousness as sensed by the human taster (Kramer, 1951b). This method involves the maceration of a weighed sample in a Waring Blendor for 5 minutes, filtering on the 30-mesh screen, drying, and weighing. McArdle and Desrosier (1954) found that the drying time could be greatly reduced if the screen containing the washed residue would be immersed for 1 minute in acetone before drying. 4. Grit

Sand particles in a product like spinach, or stone cells in a product like pears, contribute to a sensation of grittiness. Such materials may be isolated and measured by comminuting the product in a Waring Blendor (or Osterizer) using a liberal quantity of water. The heavy grit particles sink to the bottom; the supernatant liquid containing the

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floating macerated tissues may then be eluted, and the grit particles collected, dried, and weighed (National Canners ASSOC.,1941). The “sand test” used by the Dried Fruit Association (1950) includes boiling the sample as a step in the procedure in order to facilitate the separation of the sand particles from the food. By the use of standard measuring tubes into which the sand is washed, the quantity of sand found in dried fruit is determined volumetrically. 5. Density

In general, denser units of a food product tend to offer more resistance to the grinding action of the teeth. It is common practice with such commodities as peas and lima beans to separate the lighter, more tender units from the heavier, more mature units, by the use of brine flotation or froth flotation equipment. Similarly, salad-type and lowstarch potatoes may be separated from heavier starchy potatoes that are more suited for baking (Kunkel et al., 1952). This same principle is utilized principally by U.S. Dept. of Agriculture inspectors to determine the tenderness-maturity factor for peas and other crops, where a specified number of units of the product are placed in salt solutions of varying concentration; the number of sinkers are counted (U.S. Dept. Agr., 1955). This procedure, though not as precise as the alcohol-insoluble solids method as a mesure of maturity, has the advantages of indicating the percentage of heavy units in a sample. It also has greater simplicity in the performance of the test and the equipment required. Where results are not satisfactory because of the occlusion of air particles under the skin, a preliminary scalding (blanching) treatment may be required, or the units may be skinned before floating. The use of salt solutions as the medium has also been criticized, and suggestions have been made that alcohol (Jenkins, 1954), xylose (Lee, 1941), or sugar (simp) solutions be used (U.S. Dept. Agr., 195613).

E. CORRELATED METHODS Certainly the best and most direct approach to the measurement of kinesthetic factors is by the use of instruments or procedures which measure directly the kinesthetic factor involved. Where such methods or instruments do not exist or are not suitable for the quality control operator, he may yet have available other procedures. These may not be specifically designed to measure that attribute with which he is concerned; however, they may happen to be sufficiently closely correlated, at least under specific conditions, with attributes in which he is interested, to justify the use of the method. It must be emphasized however, that any change in the conditions of the test or material may render such a procedure useless.

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1. Color as a Measure of Kinesthetic Properties Color has been used successfully for many years as a measure of tenderness of raw Lima beans for processing. Since all varieties of lima beans used f o r processing in the past tended to turn from green to white as they matured and hardened, the per cent of white lima beans in a sample was a good measure of their tenderness-maturity. However, with the development of “green seeded” varieties (i.e., varieties which retain the green pigmentation at all stages of maturity), this method of measuring the kinesthetic property of tenderness-maturity was no longer valid, and more direct methods must now be used (Kramer and Hart, 1954). Similarly the absence of green color in green asparagus is taken as an indication of fibrousness. Although there is a significant negative correlation between greenness of asparagus and its fiber content, there are all too many instances of white asparagus containing little fiber, and what is more important, of green asparagus that is fibrous to an objectionable degree (Kramer et al., 1949). Redness of apples and yellowness of peaches are other examples of inaccurate applications of color to the measurement of kinesthetic properties. On the other hand, the spectrophotometric measurement of the residual green pigment in peaches and apricots has proved to be the most satisfactory method to date of measuring the ripeness-tenderness of these fruits (Kramer and Smith, 1947). 2. Consistency as a Measure of Kinesthetic Properties Consistency is essentially a measure of shearing force, which in turn we defined as a kinesthetic property. On the assumption that hardness, density, and viscosity are all correlated, Elehwany and Kramer (1956) developed an apparent viscosity test to measure the tendernessmaturity factor in peas. The test consists of the blending of a sample of peas in a Waring blendor for 5 minutes, and the measurement of the consistency of the slurry in a Stormer viscosimeter. Results compared favorably with the alcohol-insoluble solids test. Additional work is now in progress at the University of Maryland in an attempt to utilize the same principle in the development of rapid tests of tenderness-maturity for peas, corn, and simiIar products. V. FLAVOR

Attributes of quality included in this group are largely those which the consumer evaluates with his senses of taste and smell, although the sense of feel in the form of touch, pain, warmth, and cold may also be

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involved (Beidler, 1957). I n contrast to appearance and kinesthetic factors, these flavor factors are difficult to evaluate instrumentally and are therefore still measured largely by subjective methods, such as taste panels or the profile method (Caul, 1957). This is undoubtedly due to the fact that there is no thorough understanding of the mechanism by which such sensations are stimulated in the human consumer of food. Until there is a thorough physical-chemical understanding of flavor perception, it is difficult to construct an instrument capable of measuring flavors qualitatively and quantitatively in a manner analogous to the evaluation of color by tristimulus colorimetry. In spite of these almost insurmountable difficulties, one substantial effort has been made by Hartman and Tolle (1957) to characterize flavors of vegetables quantitatively and qualitatively, by means of an apparatus which would present the volatile substances emanating from a food sample to a series of sensing electrodes, and the combined response to such sensing elements would be correlated with over-all flavor quality. Results obtained thus far with a platinum electrode showed characteristic, reproducible responses to many volatile flavoring constituents. The many other attempts at instrumental flavor measurement cannot be construed as over-all approaches, but rather measures of specific flavor or off-flavor constituents, and these may be conveniently divided into taste and odor categories.

A. TASTE There is general agreement that taste is a four-dimensional phenomenon, consisting of sweet, sour, salt, and bitter (Crocker, 1945). Sweetness can be determined very satisfactorily by the use of hydrometers or refractometers in relatively pure solutions of sirup in terms of degrees, Brix, or less accurately, but apparently to a commercially satisfactory extent, as measures of sweetness and general quality, for such products as cantaloupes (Stark and Matthews, 1951) and sweet corn (Scott and Mahoney, 1946). Sourness can be measured instrumentally by the use of a pH meter in relatively pure solutions, but the use of hydrogen ion concentration as a measure of sourness in a complex food medium is not generally as satisfactory as is a measurement of total titratable acid. In some instances, sugar or acid content has been found to be less indicative of flavor quality than a ratio of the two. Thus, for example, the flavor of oranges, applesauce, and prunes is indicated in this manner (Wiley and Worthington, 1955). Saltiness can be estimated by a chloride determination, or more

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rapidly by a sodium determination with the use of a flame photometer (Brown et al., 1952). As with sugar and acid, sugar/salt ratios are also useful in arriving at the desired taste evaluation. Bitterness is not estimated in any general way. It may be related to the bitterness of a given concentration of a substance such as quinine sulfate. B. ODOR The four-dimensional system of odor classification of Crocker and Henderson (Crocker, 1945) is not generally agreed upon. I n fact, the six-category system of Henning (Moncrieff, 1951) is gaining acceptance, and other proposed systems contain as many as 32 odor categories (Pilgrim and Schutz, 1957). With the single exception of the apparatus described by Hartman and Tolle (1957), the objective measurement of odor is thus far largely an attempt to identify, isolate, and measure quantitatively the specific substances responsible for certain odors. Since, by and large, these volatile substances which cause olfactory sensations occur in extremely minute quantities, their identification and quantitative estimation by the classic chemical methods is extremely difficult, and certainly impractical for use in routine quality evaluation. With newer methods, particularly chromatographic separation, and spectrophotometric charting, this approach has been given a tremendous impetus. Thus chromatographic methods have been used to study the flavor constituents of many foods, as for example, coffee (Mabrouk and Deatherage, 1956) and citrus (Stanley, 1957). A related method, gas chromatography, first proposed by Martin and Synge (1941), is gaining considerable popularity as a means of identifying and measuring flavoring components in foods. In this procedure, volatile constituents of the food sample are swept into chromatographic column by a carrier gas. Since the various components travel at different rates, they emerge from the column at different times, into a thermal conductivity detector which provides information on the concentration of each component. This information is presented as a graphic record (Fagerson, 1957). Such procedures have been utilized by Dimick and Course (1956, 1957) on strawberries. Much attention is being given currently by many workers to the possibilities of identifying and measuring flavoring substances by means of infrared or ultraviolet spectrophotometry (Savitsky, 1957; Hall and Clark, 1956). Radiant energy transmitted o r reflected from a food product or a preparation thereof is recorded in graphic form, in terms of per cent transmission, absorption, or reflection a t different wave lengths.

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Characteristic curves are related to the presence and concentration of specific substances which may be involved in flavor sensations. Direct determinations of volatile reducing substances, particularly nitrogen compounds, have been proposed for fish decomposition (Farber and Ferro, 1956), as was a determination of succinic acid (Hillig d al., 1950).

C. CORRELATED MEASUREMENTS I n the absence of direct methods of measuring flavor quality, advantage has been taken of the coincidental relationships between certain other measurements and flavor. If freshness is to be taken as flavor, then ascorbic acid retention may be used as a measure of flavor quality of vegetables (Kramer and Mahoney, 1940; Hartman and Tolle, 1957). Likewise, turbidity of washings has been suggested as a measure of freshness for cooked fish (Tomiyama et al., 1955). Change in pH after slaughter has been used as a test of freshness of meats (Yamakawa et al., 1956). Presence of enzyme activity is used widely for frozen vegetables as a measure of enzymatic off-flavor (Schmitt et al., 1954). With certain foods, the stage of development of the raw material provides information on the flavor quality of the product. This stage of development may be termed variously as age, maturity, or ripeness. Blanck (1955) suggested that the term age be limited to use with animal products, where the concept of aging of meat is well-established in the industry and is closely related to flavor quality as well as to other quality characteristics. Maturity is generally used for vegetables such as peas, where an immature stage is desirable since it is associated with sweetness and lack of mealiness. Ripeness is more frequently used with fruits such as peaches, where a fully-ripe condition is desirable, since it is only at the more advanced stage of development that these fruits attain their maximum potential of flavor quality. A direct measurement of stage of development would be in terms of time and temperature. Thus temperature summations (temperaturedegree-hours, or days) have been used to determine the optimal point in the stage of development of peas and other crops (Seaton, 1955) and for the purpose of planting and harvesting schedules. Indirectly, many of the methods described in the previous sections, provide information on the stage of development of the raw product, and thus coincidentally on flavor quality as well. Thus for example, a spectrophotometric determination of the presence of green pigment in peaches is not so much an indication of the color of the peach as it is a measure of ripeness and, consequently, of flavor quality (Kramer and Haut, 1948).

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VI. SUMMARY AND CONCLUSIONS

A. CURRENTSTATUS The basic principles and the instrumentation for the physical measurement of visual and rheological attributes of foods quality are generally available. Although a great deal of work has been accomplished, much still remains to be done in the adaptation of principles and equipment to specific commodities and conditions. On the other hand, there is still no basic understanding of the mechanism of flavor perception, which makes the development of instrumentation for measuring flavor quality per se extremely difficult. With few exceptions, therefore, work on flavor evaluation consists of the identification and quantitative measurement of specific flavoring components rather than a basic measurement of flavor as perceived by the human consumer. With the rapid development of radically new research techniques, such as ionizing radiation, gas chromatography, and nuclear magnetic resonance, there is the hope that a means for the direct measurement of flavor quality may be discovered in the not-too-distant future.

B. TERMINOLOGY STANDARDIZATION As with any rapidly evolving discipline, the area of food quality evaluation is replete with many conflicting and confusing terms, meaning different things to different workers. It is this situation which made it necessary for these authors to devote so much space to defining and classifying. This confusion may be attributed in part to the empirical nature of some of the contributions. Certainly the time has arrived when, by means of some official or voluntary group or committee, the terminology may be defined and clarified. Such a committee should represent not only food technologists, but physicists and instrument engineers as well.

C. DIRECT OR CORRELATED METHODS Where there is a choice of methods, it is advisable to use a direct method for the measurement of a specific quality characteristic, since a correlated method may lead to gross errors as a result of changed conditions. This point may be illustrated with the color measurement of tomato juice, where a quantitative estimation of the red pigment, lycopene, was used with apparently good results when only one variety was involved. However, gross errors in color quality evaluation were made by the use of this method when other varieties were tested. A

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direct color measurement by the use of a tristimulus colorimeter, on the other hand, provided accurate results for all varieties grown and processed under a wide variety of conditions. This does not mean that a correlated or abridged method cannot be used, if the procedure offers savings in time and equipment, particularly for routine quality control purposes, as long as its limitations are recognized. If an empirical procedure is used, some means should be provided by which these empirical data could be converted to basic data, so that results obtained by different procedures can be compared in terms of basic physical or chemical units.

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Twigg, B. A. (Ed.). 1958. The practical application of the shear-press in determining quality and grade of lima beans. Maryland Processors’ Rept. 4 (3), 1-15. Underwood, J. C. 1950. Factors influencing quality of tomato paste I, Chemical composition of California commercial tomato paste. Food Research 15, 1-7. Underwood, J. C., and Keller, G. J. 1948. A method for measuring the consistency of tomato paste. Fruit Prods. J . 28, 103-105. University of Maryland. 1956. Determination of area. Plant Biophysics Lab., Sheet No. 1-1, College Park, Maryland. University of Maryland. 1957. Maryland U., Agr. Expt. Sta. Ann. Rept. A87, 61. U.S.D.A. 1951. “US. Standards for Grades of Frozen Cauliflower.” Agr. Marketing Service, Washington, D.C. U.S.D.A. 1953a. “ U S . Standards for Grades of Frozen Asparagus.” Agr. Marketing Service, Washington, D.C. U.S.D.A. 1953b. “U.S. Standards for Grades of Sweet Potatoes.” Agr. Marketing Service, Washington, D.C. U.S.D.A. 1953c. “US. Standards for Grades of Tomato Catsup.” Agr. Marketing Service, Washington, D.C. U.S.D.A. 1954. “ U S . Standards for Grades of Cucumber Pickles.” Agr. Marketing Service, Washington, D.C. U.S.D.A. 1955. “U.S. Standards for Grades of Canned Peas.” Agr. Marketing Service, Washington, D.C. U.S.D.A. 1956a. “U.S. Standards for Grades of Canned Tomatoes.” Agr. Marketing Service, Washington, D.C. U.S.D.A. 1956b. Personal communication. U.S.D.A. 1957. “Color Evaluation of Canned Tomato Juice with Munsell Spinning Discs.” Agr. Marketing Service, Washington, D.C. Walls, E. P., and Kemp, W. B. 1940. Relationship between tenderometer readings and alcohol insoluble solids of Alaska peas. Proc. Am. SOC.Hort. Sci. 37, 279. Wearmouth, W. G. 1952. Some effects of variations in temperature on the firmness of chedder cheese. Dairy I d . 17, 994997. Webster’s New International Dictionary, 1936. G and C Merriam Co., Springfield, Mass. West, E. S. 1942. “Physical Chemistry,” pp. 276278. Macmillan Co., N.Y. Whittenberger, R. T., and Nutting, G. C. 1957. Effect of tomato cell structure on consistency of tomato juice. Food Technol. 11, 19-22. Whittenberger, R. T., and Nutting, G. C. 1958. High viscosity of cell wall suspensions prepared from tomato juice. Food Technol. 12, 4 2 M 2 4 . Wicker, C. R., and Geddes, J. A. 1943. A new recording viscometer for paint consistency measurements. ASTM Bull. No. 120, 11-18. Wilder, H. K. 1948. Instructions for use of the fibrometer in the measurement of fiber content in canned asparagus. Natl. Canners’ Assoc. Research Lab. Rept. No. 12313-C. San Francisco, California. Wiley, R. C., and Worthington, 0. S. 1955. The use of fresh fruit objective tests to predict the quality of canned Italian prunes. Food Technol. 9, 381-384. Wilson, B. L., Tate, D. R., and Borkowski, G. 1946. Proving rings for calibrating testing machines. Natl. Bur. Standards (U.S.) Circ. No. C495. Yamakawa, M., Kobayashi, T., Sato, H., Suzuki, M., and Makino, M. 1956. A practical and simple test for freshness of meats. I. Changes of p H and p H difference. Igaku to Seibutsugaku 38, 15-20. Zielinski, Q. 1945. Fasciation in horticulture plants with special reference to tomatoes. Proc. Am. SOC.Hort. Sci. 46, 263-268.

MICROORGANISMS IN NONCITRUS JUICES BY HANSLUTHI Swiss Federal Agricultural Experiment Station, Wadenswil, Switzerland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Types of Microorganisms Found in Fruit Juice.. . . . . . . . . . . . . . . . . . . . . . A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Yeasts ............... D. Molds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Other Organisms.. ................ .......... .... 111. Occurrence of Microorganisms of Juice ................... A. Occurrence in Soil.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Occurrence in Air and Water. ...... .......... C. Occurrence on the Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Factors Influencing Frequency of Occurrence. . . . . . . . . . . . . . . . . IV. Occurrence in Fruit Juice. .................................... A. Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reduction of Organisms by Treatment of Juice .......... V. Changes in Appearance of Juice. . . . . . . . . . . . . . . A. General . . . . . . . . . . . . ................... B. Mold Changes and Clarification.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Production of Alcohol sms.. . . . . . . . . . . . . . . . . . . . . . . . . A. Ethanol . . . . . . . . .... ........... B. Other Alcohols . . . .............................. VII. Changes in the Organic Acid Content Induced by Microorganisms. . . . . . A. Tartaric Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Malic Acid. .. ................................ C. Citric Acid . . . . . . . . . . . .............................. D. Other Acids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Other Changes in Juice Induced by Microorganisms.. . . . . . . . . . . . . . . . . . IX. Additional Research Needs.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... .... .......... References .

Page 221 222 222 223

234 236 237 237 238 239

945 247

258 259 259 261 26'2 262 269 270 271 273 273 273

I. INTRODUCTION

Fruit juices were first produced in the latter two decades of the nineteenth century. Production was on a small scale at first, and remained so until forty years ago when the fruit juice industry made its appearance. The development of this industry became very rapid in the period between the two World Wars, and because this accelerated growth has continued up to the present time, the industry has become 221

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an important factor in the economy of most of the fruit-growing countries of the West. It is to be expected that the importance of deciduous fruit juice production will increase in the future, particularly in European countries where an ever greater quantity of second-quality fruit is finding its way into the factories for conversion into juice or other products. The techniques of production and treatment of juices are continually improving. I n spite of these advances, however, the original methods (heat treatment, sterile filtration, concentration, cold storage, drying, and the addition of chemicals) remain in use today in preserving juice and food products from spoilage. The trend today is to reduce the use of chemicals as much as possible in preserving juice. Though a few countries do permit the use of sulfurous acid in raw juice destined for limited storage, the food and drug laws of most countries forbid the use of chemicals. When they are used, chemicals are usually added in order to inhibit undesirable chemical reactions and not in order to protect against bacterial spoilage, e.g., the addition of ascorbic acid to juice to prevent browning. Preservation methods are aimed a t eliminating two main causes of deterioration and spoilage of fruit juices: (1) microbial growth, and (2) chemical change, both of which occur during production and storage. Since control of microbial growth is the first prerequisite for successful fruit-juice production, it was the first problem to be solved. It is only recently that the study of the chemical changes which take place in the juice has become the object of greater interest. This paper deals with the many ways that microbial growth can modify fruit juice, the various problems involved in its preservation and storage, and the latest scientific developments in this field. II. TYPES OF MICROORGANISMS FOUND IN FRUIT JUICE

A. GENERAL The literature on microorganisms found in fruit juice is very scanty. The first studies concerning the occurrence of definite microorganisms in juice were carried out in the latter half of the nineteenth century and related to the alcoholic fermentation of various fruit juices, primarily grape juice and ciders. It is only with the development of the fruit juice industry within the last twenty years, however, that a large number of studies of the occurrence of these microorganisms in juices made their appearance. The studies are unevenly distributed throughout the literature on the various types of processed fruit. Best known is the microbiology of the citrus fruits, which is under-

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standable when one considers the great economic importance of these beverages, especially in the United States. These beverages, therefore, will not be considered in this paper, which will be limited to the relatively smaller literature on other fruits. With regard to the classic researches on the effects which various microorganisms have on fruit juices, we must place the studies of Pasteur (1866), which represent a part of his famous studies on wine, in first place. I n spite of the fact that they deal with fermented beverages, they offer valuable insights into the occurrence of certain organisms in fresh grape juice. The work of Hansen ( 1879), Miiller-Thurgau (1905), Osterwalder (1915a,b, 1924a), and Martinant and Rietsch (1891) must also be considered, since it was from these and other studies by their contemporaries that the first knowledge of the occurrence of the various microorganisms, above all the yeasts, in noncitrus fruits were obtained. The above-mentioned authors have provided us not only with information as to the occurrence of the various species of yeasts in these juices but also with their numbers and their dependence upon various factors such as vintage, degree of maturity, condition of the fruit, and season. Recent systematic investigations on the various species inhabiting juices are unfortunately rare, although we have the work of Marshall and Walkley (1951a,b, 1952a,b,c,d) to thank for excellent insight into the microbiology of apple juice. I n addition, Beech ( 1957), Carr ( 1956), Beech (1958a,b), and Clark et al. (1954) have intensively studied yeasts and bacteria in apple and pear juice with respect to cider production. The work of Castelli (1954), Peynaud and Domercq (1953), and Domercq (1956), who carried out studies on the occurrence of various yeast species in Italian and French grape musts and wines, should also be mentioned here. With the exception of these few complete studies, our knowledge of the microorganisms occurring in noncitrus juices forms a mosaic to which numerous authors have contributed. The studies have limited themselves mainly to the investigation of definite changes in fruit juices or to the effectiveness of various methods of preservation. Further systematic investigation would be most appreciated.

B. BACTERIA Those bacteria which are found in, and are capable of developing in noncitrus fruit juices, belong mainly to the groups of acetic acid and lactic acid bacteria. The pathogenic bacteria, which can also be found in these juices, are incapable of growth in this medium and generally die off rather rapidly. It is only under the special conditions of cold

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TABLEI BACTERIA OCCURRING IN FRUITJUICES Bacteria Acetic acid bacteria Acetobacter x y l i n u m

A . oxydans A. suboxydans A. melanogenum A . mesoxydans A. rancens A . aceti A . ascendens Lactic acid bacteria Lactobacillus delbriickii L . leichmannii L. plantarum

Source

Reference

Apples, pears, grapes

Many authors, Carr (1958), Marshall and Walkley (1952a) Marshall and Walkley (1952a) Carr (1958) Carr (1958) Carr (1958) Carr (1958), Luthi (1953,1954), Hochstrasser (1955) Vaughn (1955)

Apples Apples Apples Apples Apples California wines Wines

Marshall and Walkley (1952a) Marshall and Walkley (1952a) Marshall and Walkley (1952a), Carr (1956) L. pastorianus var. quinicus Apples, pears Carr (1956) L. brevis Carr (1956) Apples, pears Leuconostoe mesenteroides Carr (1956) Apples, pears Microbacterium Carr (1956) Apples, pears Leuconostoc Millis (19511, Apples, pears Streptococcus Apples, pears, gra,pes Luthi (1953), Vaughn (1955) Lactobacillus fermenti California wines Vaughn (1955) L. buchneri California wines Vaughn (1955) L. hilgardii California wines Vaughn (1955) L. trichodes California wines Vaughn (1955) Other bacteria Sporeforming soil bacteria Cool stored fruits Smart (1939a,b) , Fabian (1933a) Achromobacter butyri Smart (1939b) Cool stored fruits Bacillus mycoides Flugge Smart (1939b) Cool stored fruits Pseudomonas syncyanea Migula Cool stored fruits Smart (1939b) Spirillum volutans Cool stored fruits Smart (193913) Flavobacterium Cool stored fruits Berry (1933a) A erobacter Cool stored fruits Berry (1933a) Clostridium nigrificans Cameron and Esty (1940) Cool stored fruits Cameron and Esty (1940) and C. pasteurianum Pears, figs other authors C. botulinum Cool stored pears Meyer and Gunnison (1929) Staphylococcus aureus Fresh fruit Tanner (1944) Bacillus subtilis Fresh fruit Tanner (1944) B . thermophilus Fresh fruit Tanner (1944) Zymomonas anaerobia Cider Millis (1956) Apples Apples Apples

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225

storage of unpasteurized juices, that they can remain viable for prolonged periods of time. Table I shows those bacterial species which have been isolated from noncitrus juices. A discussion of the more detailed classification of the bacterial species has been omitted because of the many unanswered questions still existing on the taxonomy of the bacteria.

1. Acetic Acid Bacteria The acetic acid bacteria develop on overripe or damaged fruit in great numbers and are therefore present in most fruit juices. Further multiplication of these bacteria is, however, rare. An increased volatile acid content of the fruit juices, resulting from the activity of these bacteria, can in most cases be shown to be the result of using raw material of poor quality. Acetobacter xylinum Brown, as well as A . oxydans (Henneberg) Bergey, has been identified in grape musts and fruit juices by some authors. I n a study of the changes of the bacterial flora present during anaerobic and aerobic fermentations, Carr (1958) found A. suboxydans, A. melanogenum, A . mesoxydans, and A. rancens in addition to A . xylinum. He reports that he found the species A . suboxydans, A. melonagenum, and A . mesoxydans to be present in the fresh juice of the Yarlington Mill apples in the ratio of 70: 10 :20, respectively, and that the other two species could be found only during specific periods of the alcoholic fermentation. Acetobacter rancens (found also in Swiss wines by Liithi, 1953, 1957a and Hochstrasser, 1955), by acting as a symbiont, stimulated the growth of those bacteria which cause ropiness. Acetobacter aceti has been found in Californian wines by Vaughn (1955). It may well be assumed that those organisms, as well as A . ascendens, which are known to occur in wine, can also occur in fruit juices. 2. Lactic Acid Bacteria Of all the bacteria found in fruit juices, the lactic acid bacteria are the most important. As with the other bacteria which one finds in juice, some of them can be found on the fruit itself, while several, introduced during the preparation and storage of the juice, seem to appear typically as a secondary infection. The lactic acid bacteria of fruit juice are primarily heterofermentative. Vaughn (1955) describes Lactobacillus plantarum as being the sole homofermentative species which he and his co-workers could identify in California wine, although Carr (1956), on the other hand, was able to identify homofermentative strains other than L. plantarum in

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English ciders. These had a great similarity to L. arabinosus and L. leichmannii, as well as to some species of the genus Microbacterium. The taxonomy of the individual lactic acid bacteria is a difficult problem, the classification of individually described organisms being in many cases very controversial. One must keep this fact in mind when referring to the lactic acid bacteria in Table I. The growth of the lactic acid bacteria in fruit juices is dependent upon the chemical composition of these media; the pH of the juices is one of the most important factors influencing their development. Despite the normal variations in the chemical composition of a particular fruit juice, the growth limitations of individual lactic acid bacteria can be determined quite accurately with respect to pH. Liithi (1953, 1957a) has shown that the minimal p H a t which growth will occur can be significantly reduced by the presence of symbionts, and further, that some lactic acid bacteria are greatly dependent on the presence of particular nitrogen compounds, such as amino acids, peptides, and growth factors. Under unsterile conditions, various species of lactic acid bacteria can often be identified in fruit juice. As previously mentioned, it is not quite certain that they present an unmitigated danger to the juice. Such a danger is present only for acid-poor fruit, however, since the probability that a strain will develop is increased the higher the pH. For this reason it is advisable to blend those acid-poor juices immediately with juice of higher acid content. The number of lactic acid bacteria capable of developing in very acid fruit juice of pH 3.0 to 3.4 is very small. The strains which can are primarily those which are capable of attacking organic acids, such as malic acid, citric acid, and quinic acid, and converting them to lactic acid, succinic acid, and dehydroshikimic acid, respectively, as their main metabolic products, along with carbon dioxide. Biichi (1958) has recently shown that acetic acid, diacetyl, and acetoin can be formed in small quantities in apple juice. Most lactic acid bacteria grow at pH values above 3.5. Under these conditions, catabolism of the sugar and the organic acids produces chiefly lactic acid and carbon dioxide with considerable quantities of acetic acid, mannitol, and ethyl alcohol. A particularly interesting modification of fruit juice caused by the lactic acid bacteria is the formation of slime. Millis (1951) described slime formation in apple juice by certain species of Leuconostoc. The production of long-chain polysaccharides does not appear to be restricted to this genus alone, however, since it often appears to be a transient characteristic which has been observed in fermented ciders and in wines (Carr, 1956, in strains of Lactobacillus plantarum; Liithi,

MICROORGANISMS I N NONCITRUS JUICES

227

1953, in species of Streptococcus). The formation of slime is chiefly dependent upon the presence of particular sugars, such as sucrose, glucose, and fructose. The lactic acid bacteria can grow in fruit juice under conditions which would preclude the development of other organisms such as yeasts and molds. I n the European fruit juice industry, where large quantities of juice are stored for prolonged periods of time under carbon dioxide pressure by the Boehi process, there is often a considerable loss due to the development of certain lactic acid bacteria. Such cases have been reported by Schmitthenner (1949) and more recently by Koch et al. (1953), Liithi (1957a,b), and Mehlitz and Matzik (1955). The particularly frequent occurrence of such cases since the last war, when great storage facilities for fruit juice were first developed, was the main reason for the rapid introduction of cold storage in European plants. Lactic acid bacteria, as a rule, no longer develop at temperatures below 8OC.

3 . Other Bacteria As Table I indicates, bacteria other than those of the lactic acid and acetic acid groups have been shown by various authors to play a very important, though subordinate, role in infections of fruit juice. It is only in exceptional cases, however, that these bacteria can develop or remain viable in the juice. Some of these bacteria, Staphylococcus aureus, for example, can be found on fresh fruit in the market (Tanner, 1944). It has not yet been determined whether these bacteria are disseminated on the fruit by natural means or whether the fruit is contaminated after the harvest by bacterial carriers and other sources, such as the spore-forming soil bacteria Clostridia. The low pH of fruit juice does not permit growth of those bacteria which are not adapted to this medium. Luthi and Vetsch (1957) have shown that in extremely acid-poor juices, (pH > 4.0) a butyric acid fermentation occasionally occurs. Great care must be used in industrial installations to prevent the development of the butyric acid bacteria. Fruit juices can be deacidified considerably by careless cleansing of storage vessels or by cement. In such cases, the development of butyric acid bacteria can be observed again and again. Butyric acid bacteria can ruin the quality of juice stored in poorly treated concrete containers. Storage in such containers plays a n important role in Italy and France nowadays, where grape juice which is intended for export is so kept before it is cooled and shipped. As might be expected, at pH values below 4.0 those bacteria, in-

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cluding the spore-forming bacteria, which are not adapted to fruit juice, will not survive storage. Douglas and Edin (1930), as well as Scholz (1943) have shown that pathogenic bacteria die off in apple juice within anywhere from a few hours to a few days. The presence of pathogenic bacteria in frozen concentrates is a very serious problem since, under those conditions, we can expect the organisms to remain viable for long periods of time. The problem of the s ~ viva1 of pathogenic organisms in frozen citrus juice concentrates has been studied with particular thoroughness. Berry and his co-workers (1956) , as well as McFarlane, have made the most thorough studies in this field, and their work will be discussed later in this paper. The possibility of toxin formation in acid-poor fruit preserves by Clostridium botulinum has been considered by many authors. Meyer and Gunison (1929) described a case in homemade frozen pear preserves which resulted in two deaths. Wallace and Park (195313) have also described exceptional cases where the formation of toxin in strawberry and raspberry preserves took place. The same authors, in another paper (Wallace and Park, 1953a), report that while artificial infection of sour cherry preserves with colon typhoid-type bacteria showed that they could remain viable for 12 to 14 weeks at -17.8OC. Escherichia coli could remain viable for only 4 to 7 weeks at this temperature. No organisms could be found in frozen cherry juice at the end of the fourth week. The temperatures of -17.8O and -4OOC. showed no difference in their effect on the innoculated bacteria. Cases in which a frozen juice has been detrimentally modified by a pathogenic organism, or has been implicated as a causal factor in disease, appear to be very rare. Obviously, knowledge concerning the possible survival of pathogenic organisms in frozen preserves is of very great practical importance for the organization of industrial hygiene measures. C. YEASTS Numerous varieties of yeast may be found in all fruit juices; the number of these varieties is, however, too great to permit us to enumerate them here. It is difficult at the present time to make any generalizations concerning the occurrence of the various yeast genera or species in all fruit juices, nor can we generalize as to their occurrence with respect to geographic distribution. Since we do have detailed studies from a few countries on grape juice and apple juice, it would be best to discuss briefly the characteristics of these juices with respect to their yeast flora.

229

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1. Apple Juice

The yeast flora on apples and in apple juice (Table 11) have recently been the object of a detailed study for review by Marshall and Walkley (1951a,b, 1952a,b,c,d), Clark et al. (1954), Clark and Wallace ( 1954), Pollard ( 1956), and Beech (1958a,b). They isolated representatives of the genera Candida, Cryptococcus, Rhodotorula, and Torulopsis from healthy skin and core and from the freshly pressed juice of those apples. It is noteworthy that those strains found in the fermented SPECXESOF YEASTS ISOLATED

TABLE I1 CULTIVATED AND WILDAPPLES,AND

Source

Yeasts

Cultivated apples

Candida malicola Torulopsis jamata Rhodotorula glutinis var. rubescens Rhodotorula mucilaginosa Cryptococcus albidus Cryptococcus neojormans Candida malicola Cryptococcus albidus Cryptococcus laurentii Cryptococcus neojormans Candida scottii Pichia membranejaciPns Saccharomyces ovijormis Saccharomyces cerevisiae Saccharomyces steineri Dcbariomyces kloeckeri Torulopsis candida Candida mesenterica Pichia pohjmorpha

Wild apples

Apple cider

n

FROM

FROM

CIDER'

Number of cultures isolated 21 3 2 2

1 1 6

5 4 4

I 2 2 1 1 1 1

1

Data taken from Clark et al. (1954).

juice were chiefly sporogenous, and that those found on the fruits, or in the juice which was freshly pressed under laboratory conditions, were asporogenous. We must therefore conclude that the appearance of the sporogenous yeasts is the result of a secondary infection. Such infections have been found by various authors (Tressler and Pederson, 1936; Marshall and Walkley, 1952d; Ingram, 1949, 1954, 1958a; and Beech, 1958a). Clark et al. (1954) and Beech (1958a,b) have found that species of Cnndida are numerically most predominant in apples. In England, it is

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Candida pulchrrima, in Canada (Province of Quebec), it is the species Candidu malicola which has recently been described by Clark and Wallace (1954). It is of interest to note that the C. pulcherrima of Porchet ,1938) could also be identified in fruit and grape musts in west Switzerland. According to the above papers, species of apiculated yeasts similar to Kloeckera could be found on the fruit only in small numbers. Clark et al. (1954) have shown that all yeasts which were isolated from apples in the province of Quebec belonged to the family Cryptococcaceae. With the exception of the species of S a c c h m y c e s mentioned in Table 11, none of the other strains which were isolated fermented the usual sugars, More than 50% of the yeasts isolated from cider were nonfermenting types, and those strains which were isolated from the fruit were, without exception, nonfermenters. This finding supports the previous observation that those strains which are found on the fruit are not necessarily identical with those found in the juice. Beech (1958b), in his studies of yeasts in English apple juice and cider, has given the first description of the occurrence of a species of the genus Hansenula in juice. The presence of representatives of this species has been confirmed by Luthi and Howard (1959), who found such an organism in apple juice which had been stored under reduced carbon dioxide pressure. It forms a pellicle and is a strong ester-producer, thereby modifying the fruit aroma.

2. Fruit Concentrates At the present time, little is known of the yeasts which occur in fruit juice concentrates, and those yeasts which have been described have been classified under the “osmophilic” yeasts (von Schellhorn, 1950a, 1951a; Ingram, 1958a). According to Lodder and Kreger van Rij (1952), they belong to the species Saccharornyces mellis and S. romii, though earlier authors have considered these species to be of the Z ygosaccharomyces. These organisms develop in sugar-rich juice or on the surface of concentrates and produce a weak alcoholic fermentation. One such yeast has been observed to develop, given favorable temperatures, in apple juice and pear juice concentrates whose specific gravity was 1.349 ( 70° Brix) . The present author has found alcohol concentrations of between 0.35 to 1.25% per volume in rejected commercial apple juice concentrates of specific gravity 1.3337. The development of “osmophilic” yeasts is limited by the water content of the atmosphere with which the fruit juice concentrate is in equilibrium. For most yeasts, the limiting range of relative humidity permitting development lies between 95 and 85%. Some osmophilic

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23 1

yeasts, however, are capable of growing in sugar solutions of up to 80% (w/v) which are in equilibrium with the air which has a relative humidity of between 65 and 70%. Such solutions are usually very hygroscopic and have, therefore, a thin layer of less concentrated solution on their surface in which the yeasts develop. These yeasts also convert sugar to water, thus improving the concentration relationships of their environment. For these reasons, the fermentation of concentrates occurs in rather thin surface layers. An interesting attempt to explain this behavior has been made recently by Ingram (1958b). Until now the surface fermentation had been considered to be the result of the above-mentioned hygroscopicallycaused modification of the concentration relationships, coupled with the strong growth-stimulating influence of the atmospheric oxygen. Ingram and his co-workers were able to show, however, that even when the air was removed, fermentation could begin on the surface, though in this instance, it was somewhat inhibited. This proves that factors other than the redox potential must play a role in surface fermentation. The decisive basis for this reasoning was found in the specific gravity of the cells of the osmophilic yeast, S. rouzii. It could be shown that these sugar-tolerant yeasts had a specific gravity less than that of the medium in which they grew. Depending upon the method used, the concentrations in which the cells were found to be lighter than their media lay between 450 and 650 g. sugar per liter. This indicates that the cells of the osmophilic yeasts have a tendency to float on the surface of the norms1 concentrates. It is chiefly when the concentrates are exposed to air that the concentration relationships develop which permit growth. With the reduction of the specific gravity resulting from the conversion of the sugar, the cells sink to a region of higher concentration. Most rapid development takes place at sugar concentrations of 30% (w/v) . In this connection, we should especially note the detailed investigations of Kroemer and Krumbholz (1931, 1932) and Krumbholz (1931a,b) on the occurrence of osmophilic yeasts in late, or specially selected, highly concentrated grape musts used for late harvest wines, as well as the work of Lochhead and Farrel (1931a,b, 1936), which deals with the occurrence of osmophilic yeasts in honey.

3 . Grape Juice Existing studies indicate that there is a definite difference between the yeast flora of grape juice and that of apple juice. According to Mrak and McClung (1940), Peynaud and Domercq (1953), and Domercq (1956), the majority of yeast strains occurring in grape juice are sporogenous (see Table 111).

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The above-mentioned French workers, as well as Castelli (1954) and numerous earlier workers, consider the apiculated yeasts of the type Kloeckera apiculata to be of great importance. Rees, as early as 1870, had noted the frequent appearance of apiculated yeasts on fruit and in fruit juices. Martinant and Rietsch (1891) and others found that the apiculated yeasts in grape juice often accounted for 90% or more of the yeast flora and that the elliptical yeasts were, as a rule, less numerous. Great variations were noted, however, in comparisons of the frequency studies which covered a number of years. Cases were even found in TABLE I11 OCCURRENCEOF YEASTS IN GRAPES, MUSTS, AND NEW WINES' Yeast

Fresh grapes

Musts in new wines

Saccharomyces Zygosaccharomyces Hanseniaspora Pichia Debariomyces Hansenula Zygopichia Torulaspora Total number of cultures of sporogenous yeasts

43

58

7

6 2

Torulopsis Mycoderma Kloeckera Rhodotorula Candida Total numbers of cultures of asporogenous yeasts 0

10 2 3 2 I

1 1 1

68

69

17 2 15 6 20

4 3 1

60

11

3

Data taken from Mrak and MoClung (1940).

which the elliptical yeasts were more numerous. Niehaus (1932) has made a thorough study of the physiology and occurrence of the apiculated yeasts on grapes and in soil samples which confirms the above findings. It should be mentioned here that Niehaus never found these yeasts on unripe fruit. The frequent massive appearance of specific species of yeast (apiculated yeasts) in certain years is a well-known fact which has never been explained. Aside from studies on the influence of increased rainfall on the number of organisms found on fruit, the influence of external factors on the occurrence of yeasts has never been studied. A later work of Ciferri (1941) , on the distribution and cycle of the yeasts in nature, attempted to throw a gleam of light into the darkness

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of these relationships. This author pointed out that the sporogenous yeasts predominated in the soil which he investigated. He also noted the still unclear relationship between the yeasts which occur in the soils and those which occur in musts. These relationships were further illuminated in the work of Clark et arl. (1954) mentioned above. Mrak and McClung (1940) have described the occurrence of yeasts in grape juice and on grapes under the conditions prevailing in California. With respect to European conditions, we have the previously mentioned studies of Peynaud and Domercq (1953, 1956) on the occurrence of yeasts in the Bordeaux region. Attention should also be brought to Castelli’s (1954) valuable discussion on the distribution of several yeasts in various regions of Italy, while the yeast flora of the German and Swiss vineyards is somewhat better known through the earlier work of Behrens (1910) and Ostenvalder (1924a). It may be concluded from all of these works that Kloeckera apicutata and Saccharomyces ellipsoides represent the dominant yeast types found in grape juices, comprising some 80% of the yeast flora. Domercq (1956) isolated 28 different species from French grape musts, which could be divided into ten genera: Sporogenous

Asporogenous

Saccharomyces Tees Saccharomyces hansen Hansenula Pichia Torulaspora

Kloeckera Torulopsis Brettanomyces Rhodotorula Candida

Domercq found that among those strains isolated from red wine, 41 % were asporogenous yeasts and 59% sporogenous yeasts, while from the white wine of the Gironde region, his isolates were 70% sporogenous as against 30 % asporogenous. Saccharomycesouiformis plays a particularly important role in this region because of its excellent strong alcohol production and can be found in the majority of the local grape musts. In a few cases, Domercq isolated yeasts of the genus Brettanomyces. Earlier, Schanderl and Draczynski ( 1952) had also isolated representatives of these species in sparkling wine, Krwnbholz and Tauschanoff (1933) described a new species of yeast isolated from grape must which they had classified as Mycotorula intermedia n.sp. and which was later listed with the Brettanomyces. Recently, Peynaud and Domercq (1956) have compared the members of this genus which have been isolated from wine, and on the basis of their study proposed a classification for the group.

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There are, finally, two genera which have not as yet been mentioned but which were isolated from grape musts some time ago. In 1922, Kroemer and Heinrich isolated a species of Saccharomycodes ludwigii which was resistant to sulfur dioxide, and Ostenvalder (1924b) isolated a species, Schizosaccharomyces liquefaciens n.sp., which grew in a strongly SO,-treated grape must and which also showed an extraordinary resistance to sulfurous acid.

D. MOLDS The molds are very important causative agents of many secondary changes which take place in fruit juices, although compared with the usual organisms which are found in the juices, they are decidedly less frequent. Since cases of fungus growth resulting in loss of quality are numerous, however, they deserve our full attention. This applies particularly to the conditions which prevail in home and farm manufacture, where mold infection of stored fruit juice is the main problem facing the industry today. Elimination of mold infections in these small installations is particularly difficult. We have the above cited works of Marshall and Walkley (1951a,b, 1952a,b,c,d) to thank for the most complete study extant on the occurrence of molds in apple juice (see Table IV) . Only future investigation will show how far these results can be applied to other fruit juices. They were able to show that not all the molds which are found on the fruit can later be found in the juice. They further isolated 6 varieties of Mucorales, 10 aspergilli, 10 penicillia and related species, as well as 7 of the Fungi Imperfecti from the fresh juice of “Bramley’s Seedling.” A number of these mold genera had previously been noted in the investigations of earlier students of fruit juice. Of all the molds, the penicillia are responsible for the most frequent infections and quality damage. The very wide-spread distribution of infections of fruit juice by species of Mucor in home and farm production, noted by Baumann (1951), could not be substantiated by other workers, including the present author. An alterating relationship between Mucor and Penicitlium is not out of the question, however, in view of the interesting observation by Marshall and Walkley that the appearance of Mucor on fruit which had been infected with Penicillium was quite frequent. Some species of Penicillium appear to be quite harmless to fruit juice; those are the species which are inhibited in their growth by even very low concentrations of carbon dioxide. Several species, on the other hand, can achieve a very weak growth in apple juice, while still others, such as Penicillium expansum and P. crustosum, can develop extremely

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MICROORGANISMS IN N O N C I T R U S JUICES

TABLE IV Moms OCCURRINGIN FRUITJUICES Molds Mucorales Mucor mucedo Linn6 Mucor piriformis Fischer M U C Mracemosus Fresenius Mucor hiemalis Wehmer Zygorhynchus Moelleri Vuillemin Rhizopus nigricans Ehrenberg Aspergillaceae Aspergillus niger group A . ustus (Bain.) Thom et Church A. Sydowi (Bain. et Sart.) Thom et Church A . nidulans (Eidam) Wint. A . jtavipes (Bain. e t Sart.) Thom et Church Aspergillus wentii group Aspergillus ochraceus group Aspergillus fumigatus group A . Fischeri Wehmer A . versicolor (Vuillemin) Tiraboschi Penicillium and related species Penicillium glabrum series P . spinulosum Thom P . cyaneum (Bain. e t Sart.) Biourge Penicillium brevi compactum series P . lanosum Westl. Penicillium viridicatum aeries P . crustosum Thom P . expansurn (Link) Thom P . glaucum Link Paecilomyces varioti Bain. Scopulariopsis brevicaulis (Saccardo) Bain. Fungi Imperfecti (mainly Hyphomycetes) Fusarium Link Alfernaria tenuis (Dem.) pleosporia Cladosporium herbarum Link Botrytis cinerea Persoon Oospora laclis (Fresenius) Saccardo Oospora candida Wallr. Pullularia (Dematium) pullulans (Ue Bary et Low) Rerkhout illonilia candida (Bon.) Hansen Byssochlamis fulva Ollivier et Smith Bywxhlnnais niven Westl. Phialophora mustea Neergaard Monascus ruber 6

Reference"

Osterwalder (1940)

Lafar (1914) Lafar (1914) Olliver, Rendle (1934) Liithi (1952) Neergaard (1941) Luthi and Halter (1959)

If no indication, data are from Marahall and Walkley (1QSla.b;62a,b,c,d).

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rapidly. Buchi (1958) has related the well-known moldy taste to the production of diacetyl and/or acetoin by these molds. Marshall and Walkley have ranked the aspergilli second in frequency of occurrence in fruit juices. The spores of some varieties are extremely resistant and can remain viable for more than two years in pressure tanks. Several species occur as infections in bottled fruit juice. Unlike the Penicillium infections, the aspergilli appear to be somewhat less detrimental since they modify the juice quality less rapidly. One frequently meets with infections of Aspergillus niger, A. niduzans, and A. fumigatus, and occasionally Rhizopus. The above molds occur as primary infections in home and farm fruit juice production. Since this type of production is very important in Europe (Switzerland and Germany), we must give them careful attention. These molds are very rare in industrial fruit juice. In the last few years, infections by molds of the genus Paecilomyces have become an increasingly greater problem not only in the production of home- and farm-made juices, but also in juices produced by industrial plants. D a m (1951), Liithi and Hochstrasser (1952), Liithi and Vetsch (1955), and Liithi and Halter (1959) have noted the increasing frequency of infections by PaeciIomyces uarioti in apple and grape juices. The same authors have also noted an increasing frequency of infection by species of Byssochlamis, Monascus ruber, and Phialophora mustea. The latter had been isolated for the first time by Neergaard (1941) from Danish sweet cider. Raistrick and Smith (1933), Williams et al. (1941), and Olliver and Rendle (1934) had noted the frequency of infections by Byssochlamis fulva in the British preserves industry. Paecilomyces, Byssochlamis, Monascus ruber, and Phialophora mustea are thermoresistant fungi which withstand the temperatures usually used in fruit juice pasteurization. They are therefore extremely dangerous to the juice since a very minute quantity of mycelium growth suffices to impart an unpleasant moldy taste. Among the other fungus genera which occur and should be mentioned are Alternaria, Cladosporium, Botrytis, Oospora, and Fusarium, representatives of which can be found in juice immediately after pressing. They have not as yet been found in stored juice, however, and are therefore not of very great practical importance.

E. OTHERORGANISMS Most studies of the microorganisms of fruit juices include only the bacteria, the yeasts, and the molds. It is conceivable, however, that other, more highly developed organisms, such as protozoa, are

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237

capable of remaining viable in these juices for prolonged periods. Osterwalder (1915b) found amebae on the surface of a very acidpoor, fresh pear juice just prior to the onset of fermentation. These amebae often contained several yeasts in their cell plasma. I n the same work, he noted the occurrence of numerous other protozoa which formed a fine pellicle on the surface of the juice and which were very motile. The present author has examined the foam of freshly pressed juices, and he has also found numerous protozoa among which were primarily pear-shaped flagellates. Their unipolar flagella make them appear to belong to the genus Bodo. These organisms can be found only up to the time alcoholic fermentation sets in. Those very motile representatives of various species of flagellates which have been observed by Osterwalder and the present author have, at the time of this writing, not been studied more closely. They do not appear to be identical with the swarm cells of the ameba Physarium l e u c o p k u m which Chrzascz (1902) isolated from a pear juice which had been made from fruit heavily infected with Monilia fructigena, and which formed a delicate pellicle in a %day culture in fruit juice. I l l . OCCURRENCE OF MICROORGANISMS OF JUICE IN NATURE

A. OCCURRENCE IN SOIL Any discussion of the microorganisms which are responsible for changes in fruit juice must also include a consideration of the origin of these organisms and the most important sources of infection by them. As a matter of fact, the earliest studies of the beverage microorganism population also considered their origin. It is indeed a pleasure to read today the detailed and exact investigations which Hansen made in 1879 on those organisms which he found during various seasons in the air of the Carlsberg region, or his classic study of the life cycle of Saccharomyces apiculatus. These works were put out as an opus by Kloecker (1911). These and other later German and Swiss works indicate that the soil is the main source of microorganisms found on fruits and in fruit juices. Soil samples taken from orchards and vineyards have a higher number of those organisms which are dependent upon the respective local fruits than would be found in other areas. Spore-forming organisms have a wider distribution than those which are not spore-forming. The chemistry of the soil has a great influence on the viability of the organisms, and Ciferri (1941) has shown that acid soils are richer in yeasts. Miiller-Thurgau (l889) made the first study of the relationship be-

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tween species of Saccharomyces in vineyard soils and those found in grape musts. I n a work which was an extension of the work of Hansen, he showed that the superficial layer of soil is richer in yeast cells and that these organisms can be demonstrated to a depth of 30 cm. He demonstrated by means of cell counts that those grapes which grew close to the ground had a far greater number of microorganisms on them than those which hung high. With regard to the direct relationship between the microflora of the soil and that of the fruit, he has shown that when a vineyard is heavily infested with a particular species of yeast in the fall, those yeasts occur in the grapes in increased numbers the following year. A further orientation as to the numbers of yeasts in various soil samples is provided in the later publications of Lund (1958). Those counts may vary considerably. I n the same orchard, he found a maximal cell count of 245,000 yeasts per gram of soil at one time, and only 1350 in another sample. Studies by other researchers have shown that Sacchuromyces, Hansenula, Torulopsis, Candida, and Rhodotorula are those yeasts which are most commonly found in the soil. The differences in the various soil samples are easily understood. The frequency with which yeasts appear in the soil depends upon whether fruit or other plant material has been added to the soil, and, therefore, the average microorganism counts of the soil of an orchard can vary considerably, depending upon its condition and state of cultivation. New plantings of vines in areas where vine culture has not been undertaken for decades have shown that the cell count was considerably less than that in old vineyards. Those species which were present also caused unclean fermentations.

B. OCCURRENCE IN AIR AND WATER Hansen, in his 1879 publication, recognized the wind as the most important carrier for the microorganisms which are found on fruit. We have already noted, in discussing the work of Marshall and Walkley, the relationship between precipitation and the microorganism content of the fruit. It is to be expected that with prolonged periods of rain, the count of certain organisms would be reduced. Stalder (1953) has shown, however, that the frequency of infections occurring on grapes by Botrytis cinerea increases greatly immediately following a rainy period. The same is true for infections of grapes by acetic acid bacteria. Williams et al. (1956), who dealt with this problem as a secondary aspect of his work, came to the conclusion that no direct relationship between the number of organisms and the quantity of precipitation could be demonstrated.

MICROORGANISMS IN N O N C I T R U S J U I C E S

C. OCCURRENCEON

THE

239

FRUIT

I. Surface The number of microorganisms on the surface of fruit rises as the fruit ripens. That is the result both of increasing opportunity for reproduction offered the organisms by the ripening fruit and augmented infection resulting from increased visits by insect carriers. The fact that ripe fruit tends to ferment sooner or is ruined by molds more quickly than unripe fruit had early led some researchers to examine the microorganisms present. Some classic studies contain data on the microorganism content of fruits and freshly pressed juices. The investigations of Martinant and Rietsch (1891), in which, in addition to other data, the microorganism count per gram of Algerian grapes was 432,000, should be mentioned among other works of that period, as well as the detailed investigations of Miiller-Thurgau (1892-93) on the microorganism population of grapes. In the latter works, special consideration was given to the counts of bacteria, molds, and yeasts. These counts clearly illustrate the great differences which exist in the microorganism populations of healthy, damaged, and spoiled grapes. The classic works dealt exclusively with investigations of grapes in relation to wine production. Studies and counts on other fruits were lacking until a few years ago when newer, somewhat more thorough investigations were made which substantiated the results of the early researchers. Here again, it was Marshall and Walkley (1951a) who, in their systematic studies of the occurrence of microorganisms on apples, not only confirmed the enormous differences in the number of organisms present on healthy and damaged fruit found earlier (see Table V), but also pointed out the interesting fact that some varieties of fruit apparently contain fewer yeasts, molds, and organisms in general than others. Olliver and Rendle’s study (1934) of the occurrence of the mold Byssochlamis fulva on various fruits can be considered in the same light. They found that the mold occurs more frequently on some varieties of plums than on others. They were further able to show that while the mold occurred frequently on strawberries, it was practically never found on other berries such as raspberries, loganberries, or blackberries. A summary of the cell counts from the work of Marshall and Walkley on the distribution of yeasts among the healthy fruit of the “Bramley’s Seedling” variety is given in Table V. A comparison with

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other results in the same paper, shows that Bramley’s Seedling and “Newton Wonder” may be classed among those having the highest cell counts. I n this respect, it should be noted that Stalder (1953) has shown that the variety of grape known as “Pinot Noir” is considerably more susceptible to infection by the mold Botrytis cinerea than is the “Chasselas” strain. Despite the fact that the juice of the two varieties had TABLE V DISTRIBUTION OF YEASTS AND MOLDS IN BRAMLEY’S SEEDLING APPLES WITH AND WITHOUT INTERNAL ROT”

Count

No.

Total yeast count

1 2 1 2 1 2

Yeasts per gram Molds per gram a

Portion of pulp not showing Core and surrounding rot and apparently sound areas of rot (weight examined: 28.35 9.) (weight examined: 28.35 9.) 3000 8000 106 282 0 0

830,000 640,000 29,276 22,576 15,300 23,800

Data taken from Marshall and Walkley (1951a).

TABLE VI MAINYEASTSFROM WASHED KINGSTONBLACKFRUIT’ Yeast Hansenn la Candida F Candida pulcherrina C m d i d a p a r a p s i h i s var. intermedia Candida caterrulata Tordopsis A Toriilopsis C 0

b

Eyes

Cores

Stalks

20 40

206 75

-

-

-

4 -

60

-

-

9 90 -

Skin 40 6 50

-

-

Data taken from Beech (1958a). All figures per cent of total yeasts isolated.

practically the same sugar content, total acid content, and pH, the mold grew only one-fifth as strongly on Chasselas grapes than on the Pinot Noir variety. To date, no attempt has been made to study or explain this apparent ‘‘selection’’ of the organism by the fruit, a selection which was also observed by Romwalter and von Kiraly (1939). Romwalter and von Kiraly have found that in a limited geographic area, certain varieties of yeast occurred on certain fruits with a greater frequency than on others. They found species of Torda on Ribes

MICROORGANISMS I N NONCITRUS J U I C E S

24 1

grossularia which either never, or only rarely, could be found on Vitis vinifera. The results in Table VI have been extracted from the latest investigations of Beech (1958a) and offer an insight into the distribution of yeasts on fruit of the “Kingston Black” variety. It is apparent that the most frequent inhabitants in the region of the eye and core of the fruit are Torulopsis, and Candida, while the population of the skin consists primarily of Hamenula, Candida, and Debariomyces kloeckeri, which until now had not been found in either apples or apple juice. 2. Within the Fruit Since the work of Hansen (1879), there is no longer any doubt that the microorganisms which originate from the above-mentioned sources, end up on the fruit, and, under favorable conditions, reproduce there. Recently, several authors have claimed to have succeeded in isolating species of Sacchromyces from the healthy cells of fruit (RomWalter and von Kiraly, 1939). Schanderl (1950a, 1951, 1952a, 1953) has claimed to have found bacteria which had developed spontaneously from the mitochondria or chondriosomes of plant cells. TABLE VII DISTRIBUTION OF YEASTS IN SOUND BRAMLEY’S SEEDLINGAPPLES“ Section of fruit Whole apple Ep ider m Flesh Core a

Total yeast count

Yeast count per gram of sample

1,150,000 1,195,000 0 5507

13,988 12,892 0 790

Data from Marshall rtnd Walkley (195Ia).

That in a large percentage of cases, microorganisms can often be found within the fruit, is confirmed, not only by the data in Table VII, but also by the detailed investigations made by Marcus (1942), and Niethammer (1942), and others. Those authors examined a great quantity of plant material which included apples, pears, cherries, and gooseberries, and they found that the molds far outnumbered all other organisms in the healthy fruits and seeds. With varying frequency, bacteria and yeasts could also be found, and their numbers could be related to their presence in the region. It appears to be definitely proven that in those cases we are dealing with internal infection which probably occurred in the flower stage.

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Marcus (1942) isolated Torulopsis calbida from inside healthy gooseberries, and Rhodotorula glutinis from sour cherries. Niethammer ( 1942) isolated asporogenous yeasts from various plant tissues, and Burcik (1948) has shown experimentally that the number of organisms which could be found in tomatoes and beans can be related, at least in part, to natural infections which occurred during the blossoming period. Those fruits too, therefore, can contain bacteria, just as was found in the case of apples. The nature of the bacteria and the degree of infection are dependent upon the location of the plant. The means by which fruit is infected is known in some cases, and attempts at artificial infection have been successful, although the positive demonstration of organisms within the intact cell has been unsuccessful. On the basis of our present knowledge, we can therefore assume that infection as the result of spontaneous generation of microorganisms within the cells of the fruit has not been proven. The theory of the genesis of microorganisms from cell components of healthy fruit, as put forth by Schanderl (1950a) has only a limited number of adherents. Stapp (1951, 1952) and Woll (1956) have checked the theory experimentally and achieved only negative results. D. FACTORS INFLUENCING FREQUENCY OF OCCURRENCE

1. Season Early detailed investigations concerning the role of the soil as a source of infection during the various seasons were carried out by Wortmann (1897-98). He found that the number of organisms in the soil was highest in November and December, and dropped steadily until the grapes reached maturity, at which time the number of organisms again increased. Other workers found similar relationships in analyses of the air in which a sudden increase in the number of fermentative organisms could be noted at the onset of the ripening of the fruit (berries). The most recent studies of this phenomenon are those of Marshall and Walkley (1951a) in which (as shown in Fig. 1) the lactic acid bacteria are the most prevalent species at the onset of ripening; they give way to the acetic acid bacteria, which become most numerous towards the end of July. September, as might be expected, is the “season” for the yeasts. These findings run counter to the classic investigations and to those of Niehaus (1932), in which yeasts could never be found on unripe fruit.

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243

2. Climate Systematic studies on the influence of climate on the occurrence of certain yeasts were carried out by Castelli (1954). He found, in agreement with other authors, that Saccharomyces ellipsoides and Kloeckera apiculata were the yeasts most frequently found on grapes and in grape musts. Another interesting finding was that the numerical relationships between the two groups of yeast was dependent upon both latitude and altitude. The number of sporogenous yeasts and of S. -:

&&t

-.-: --I

Lactic acid bacteriq ,4cetic acid bacteria

FIG.I.The seasonal occurrence of yeasts, lactic acid, and acetic acid bacteria on apples. Drawn from the data of Marshall and Walkley (1951a).

ellipsoides increases, while those of K . apiculata and the usual asporogenous yeasts decreases as one moves south. With the decrease in the number of asporogenous there is an increase in that of the sporogenous, apiculated yeasts of the genus Hanseniaspora, which are normally not found in fruit juices in the more northerly parts of Europe. Mrak and McClung (1940) have found the latter yeasts on California grapes and during grape must production (see Table 111). These results should be compared with those of Beech (1958a) which are given in Table VI. The Hanseniaspora first make their appearance in mid-Italy and increase in frequency the farther south one goes below the 4 5 O parallel.

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Castelli (1954) has found that with increasing altitude, the number of sporogenous yeasts decreases and that of the asporogenous yeasts increases. When one compares these results with those of Peynaud and Domercq (1953) and Domercq (1956), one finds an excellent correlation. Even the average maximal alcohol-forming ability of the apiculated yeasts, which decreases from the south of Italy to the north, where it is on the order of 6 vol. %, agrees completely with that found for the apiculated yeasts at the mouth of the Gironde, which is at approximately the same northerly latitude. The data in Table I11 deserve consideration. They indicate that, under the conditions which prevail in California, both Saccharomyces species and the apiculated yeasts are most frequent, The data further indicate that the sporogenous yeasts are the most numerous types present in grape musts in southerly regions. Clark and Beech have stated that the predominant yeast on apples and in apple juice in England is Candida pulcherrima while that in the province of Quebec is C. malicola. Investigation of the influence of climate on microorganisms is still incomplete, and the work which is available deals almost entirely with the yeasts. Despite this, we can still unearth some very interesting relationships. It has been found, for example, that a genus of thermoresistant fungi Byssochlamis, which is relatively common in Europe, has not yet been found in the United States.

3 . Insect Vectors Insects have long been held responsible for the infection of fruits with microorganisms. It is known that yeasts are not only found in the intestines of many insects but are also capable of multiplying there. The relationship of species of Drosophila to the yeasts has been studied with particular thoroughness. Very specific species of yeasts have repeatedly been demonstrated in the digestive tracts of these and other insects. Various authors (Phaff et al., 1956; Shifrine 1956; Carson et d.,1956) have investigated these yeasts and have found them to be chiefly species of Saccharomyces, among which were 5'. montanus, and S . cereuisiae var. tetrasporus, and representatives of the genus Hansenula, Kloeckera, Torulopsis, and Cmdida. Further study is necessary, however, to fully explain the infection of fruits by certain microorganisms. The frequent occurrence of specific yeast strains on certain fruits or parts of fruits (see Table VI) could, as shown by the above-mentioned papers, be related to the visits of certain insects.

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4. Washing

The microorganism population on fruit can often be reduced considerably when the fruit is thoroughly washed, a fact which Marshall and Walkley’s (1951b) systematic studies have confirmed. In three experiments, they were able to show that the cell count per unit surface of healthy fruit can be reduced to a fraction by thorough cleansing. In the most extreme cases, this reduction was on the order of 140,000 down to 91 per fruit, while in the less extreme cases, the reduction was from 7000 down to 196 per fruit. However, the authors also called attention to the fact that the washing of spoiled fruit does not result in a very great reduction in microorganisms since the organisms are not limited to the surface. IV. OCCURRENCE IN FRUIT JUICE

A. SOURCES

It is understandable that those organisms which can be found on the fruit can also be found in the fruit juice. Innumerable authors have shown that fruit juice, as a result of unsanitary processing, insufficient cleaning, and unsuitable construction of equipment, can be extremely heavily infected. Fabian (1933a,b,c) investigated the influence of various manufacturing operations on the microorganism content of grape juice and found that the large infections were due primarily to the presses. Grape pulp, which as the result of heating, was practically germ-free, left the presses with counts of as much as 250,000 per milliliter, with yeasts being the chief organisms present. Infection with molds was insignificant. The work of Pederson (1936a,b) and Pederson et al. (1936) on the preservation of grape juice, confirms the results of Fabian. As one would expect, the frames and cloths are the greatest source of infection. This infection can be so heavy that fermentation occurs in a minimal time. Infection is particularly heavy in those installations which are in operation 24 hr. a day. A daily, thorough disinfection of all equipment is therefore necessary to avoid gross contamina tion. Ingram (1949) showed that a single reaming head used in the citrus industry contained about 1 g. of yeast. Similarly, one must consider that the pressing frames and cloths which are set out in the air offer ideal conditions for the development of certain strains of yeast. Beech (1958a) studied the yeast flora in the juice of English cider apples with respect to the presence of various genera. In further studies in which the juice was pressed under laboratory conditions and also in

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a well-organized industrial plant, he found great differences not only with respect to the number of yeasts in the pressings but also with respect to the occurrence of individual species. He found that after pressing, there was 100% more yeast in the large-plant pressings than in the pressings from the pilot plant (see Table VIII) . The yeast flora in the pilot-plant pressings, moreover, were the same as that found on the fruit, i.e., primarily asporogenous, while that in the commercial pressings were chiefly sporogenous. With such a majority of sporogenous yeasts present, it is understandable that the original asporogenous yeasts are lost in the usual isolation procedures and that special enrichment techniques must be used in order to determine their presence. Infection is primarily by Saccharomyces microellipsoides. TABLEVIII FLORA COUNTON FRUITJUICES PRESSED ON PILOT PLANT SCALE AND COMMERCIAL PLANTSCALE' Kingston Black Flora Yeasts Bacteria

Pilot plant

Comm. plant

300,000 1,055,000 -

Bramley 's Seedling Pilot plant

Comm. plant

477,000 17,000

665,000 139,000

ON

Sweet Scrocet Coppin Pilot plant

Cornm. plant

610,000 1,372,000 135,000 185,000

a Data taken from Beech (1Q58a). Yeast counts per milliliter on juices from eamples of same fruit pressed on pilot-plant scale and on commercial plant scale.

Liithi (1950) has shown that the tubes and containers used in the preparation of juice can also serve as a source of infection, though they play a subordinate role to that of the presses. Emch (1954) demonstrated that the corners and connections in the construction of the pumps and other apparatus used in fruit juice production permit the accumulation of microorganism-containing debris, which can often inhibit the effective disinfection of the installation and control of the microorganisms. This points up the necessity for closer cooperation between engineers and microbiologists in equipment design and in plant operations. Pederson (1936a) and Liithi (1949b) have pointed out the significance of the storage containers as a source of infection. In spite of careful treatment, one must reckon with a slight increase in the yeast and mold content of the juice. In Pederson's experiments, the microorganism count rose from 0 to 40 to 60 cells per milliliter in germ-free juice which had been stored in glass vessels and tanks. The significance of such small infections is dependent upon storage conditions.

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247

B. REDUCTIONOF ORGANISMS BY TREATMENT OF JUICE 1. Centrifugation Although introduced into the technology of the fruit juice industry at a very early date, centrifugation uses have been limited as the result of the development of the more efficient methods of filtration. Since the appearance in the mid-fifties of the automatically emptying centrifuges, however, centrifuges are again coming into more general use, mainly for the preclarification of the juice before storage in tanks. Usually only those components of the juice are separated which would sediment out within a short time. Microorganisms can be included in this category. Marshall and Walkley (1951b) have shown that by introduction of the centrifuge, the yeast population could be reduced to 3 to 4% of the original count. Brunner (1947) has shown that a reduction in the yeast population to 14 to 17% of the original could be effected by the use of a centrifuge with 4000 r.p.m. At 8000 r.p.m. the yeast population declined to 1 to 3%. With an appropriate reduction in the volume of the liquid flowing through the machine, essentially yeast-free fruit juice could be obtained. Beech (1958a) had similar experience when studying the yeast concentration in fermenting ciders before and after centrifugation. 2. Fining

The fining reaction as carried out with the addition of gelatin, enzymes, and bentonite results in the sedimentation of the microorganisms among the other suspended solids. Although, unfortunately, there have been no systematic investigations of the physicochemical reactions involved, the result of the treatment of fruit juices by such means has been the object of considerable study. Marshall and Walkley, in comparing the gelatin and enzymatic methods of fining, have favored the gelatin method, since it is usually carried out at much lower temperatures than the enzymatic method, thereby offering less favorable conditions for the development of microorganisms. The results of 34 experiments have shown the average reduction in the yeast population of apple juice to be 62%. It is interesting to note, however, that the mold population is not always reduced to this extent. Brunner (1947) appears to have obtained even better results in apple juice. He found that by the addition of an insufficient quantity of gelatin he was able to achieve a reduction in the yeast population of 94%, while the addition of the proper quantity resulted in a reduction of 99% Excessive addition of gelatin gave poorer results.

248

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It may therefore be assumed that the reduction in the microorganism content of juice is greatly dependent upon the nature of the fining agent as well as the chemical composition of the juice. Brunner (1947) could achieve a reduction in the microorganism population by 75% in a yeast-infected apple juice by the simple expedient of adding filtering cellulose. With the addition of 0.2% L‘Hyflocel,yy he could increase the efficiency of the reduction to 85%, while the same quantity of filtering asbestos resulted in juice with a cell count of less than 1% of that found in untreated juice.

3. Filtration According to the method used, filtration can reduce the microorganism population practically to zero. In order to obtain essentially germ-free juice, however, it must be filtered two or more times, depending upon the degree and nature of the turbidity. The Lcprecoat” method of filtration, which is very widely used today, is somewhat less adapted to the systematic study of the reduction of the microorganism population of juices. For this reason, more studies of this nature have been made with filter plates. The following data show that even filter plates of very great permeability (low numbers) can bring about very great reductions in the microorganism count. Marshall and Walkley (1951b) have found that an average reduction in the microorganism population to 3 to 4% of the original count can be achieved, while Brunner (1947), working with Seitz filters (No. 2, 3, 5, and 7), has been able not only to substantiate these results but has found that even with the higher-numbered plates a reduction could be obtained to only 1% of the original count. The accompanying tabulation has been taken from the work of Marshall and Walkley (1951a) and shows that not only are the filter plates effective on the first passage of the juice, but that multiple passages can reduce the microorganism population to practically zero. Microorganism count ~

Treatmcnt

Before filtration After first filtration After second filtration After third filtration

1

3270 34 26 2

Filter number 3 5 1147 265 323 1

3920 14 6 0

~~

7 82 3 0 0

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249

4 . Heat Treatment Fruit juice is heat-treated in order to accomplish two goals, first, the inactivation of the enzymes, which produce deleterious reactions immediately following pressing, and, second, the destruction of microorganisms. This treatment which is routine in industrial installations, is not generally used in home and farm production of fruit juice, which must therefore be given special consideration. In commercial practice, heat treatment is usually carried out with plate-type pasteurizers. The temperatures used are chosen primarily with respect to the enzymes to be inactivated, and not with respect to the microorganisms. The work of Dimick et al. ( 1951) on the inactivation of polyphenolases in fruit purees demonstrates the thermoresistance of these enzymes. As a result of this work, the fruit juice industry usually uses temperatures of 90°C. for about 30 sec. There are many basic studies available on the importance of the temperature-time relationship, e.g., Cruess and Irish (1933), Fabian and Marshall (1935), Pederson and Tressler (1938), Lund (1946), von Schelhorn (195 l a ) , and Tressler and Joslyn (1954), which have thoroughly covered the field so that there is no further need to discuss them in detail here. Treatment which inactivates the polyphenolases and pectinases suffices, as a rule, for the destruction of most microorganisms. Under the special conditions of grape juice manufacture, e.g., as described by Pederson (1936a,b) , lower temperatures can be used for enzyme treatment or color extraction. His experience has shown that the microorganism population is reduced rapidly at extraction temperatures of 62.2O to 68.8OC. The yeasts die off to a considerable extent at the lower temperatures, leaving only the molds which are also destroyed at 68.3OC. The yeast count naturally drops more quickly than that of the bacteria and molds. It is known that yeasts are destroyed in a few minutes by temperatures of 55O to 65OC. while the literature indicates that the mold spores are more resistant, normally requiring temperatures ranging between 75O to 80OC. for periods of up to 15 min. It should be kept in mind that juice is usually reinfected by the processing apparatus subsequent to heat treatment. Studies of this situation have been made by Pederson (1936a) and Fabian (1933a,b,c). The above-mentioned heat treatment must, therefore, be considered as a pretreatment prior to storage or bottling. In bottling, the juice must be kept at higher temperatures for longer periods. Under the conditions which prevail in home and farm production, maximum temperatures of 70° to 75OC. are the rule, the lower temperature being used in carboys, and the higher temperature in smaller

25 0

HANS LUTHI

bottles. The length of time at which the juice is kept at these temperatures depends upon the rate at which the vessels cool. In all cases, the cooling time is longer in small-scale production than in industrial installations and amounts to several hours at over 5OOC. using the common 25-liter carboys. Experience has shown that although these temperatures result in certain destruction for most of the microorganisms, the thermoresistant organisms, such as B y s s o c h h i s , Paecilomyces, and Phialophora, remain unaffected under these conditions as Fabian ( 1 9 3 3 ~ )Olliver ~ and Rendle (1934), Liithi and Hochstrasser (1952), and Liithi and Vetsch (1955) have shown. According to the latter authors, these organisms can remain viable in fruit juice for several minutes at temperatures of 90°C. In industrial practice, the fruit juice is rapidly heated to 90°C., cooled to 6 5 O to 7OoC., and bottled and held at that temperature for a time. This procedure has proven itself and results in germ-free juice. Under the conditions prevailing in Europe, where so much juice contains added carbon dioxide, pasteurization in undisturbed bottles is the method chiefly used. This technique, consists in heating the juice for about 20 min., at the end of which time a temperature of 6 8 O to 72OC. is reached, The juice is held at that temperature for another 20 to 25 min., after which time a cooling-off period follows which is of about equal duration. These conditions suffice for the pasteurization of beverages which contain carbon dioxide. In treating those beverages which contain little or no carbon dioxide, the possible appearance of thermoresistant organisms must be expected. Such considerations indicate that while heat treatment is insufficient protection against thermoresistant organisms in carbon dioxide-free juices, it is adequate for those juices which contain carbonic acid.

5 . Storage under Carbon Dioxide Pressure (Boehi Process) Most of the commercial fruit juice in Europe is prepared for storage by permeating it with carbon dioxide, a method introduced by the Swiss chemist Boehi (1912). In this process the juice is permeated with carbon dioxide at 15OC. until it contains 1.5% by weight of the gas, which is equivalent to a pressure of 7.7 atmospheres. Storage at 15OC. is considered unsuitable today and so, by cooling to 2OC., it has been possible to reduce the carbon dioxide saturation to 0.8% (wt.) which represents a pressure of only 3.5 atmospheres, and today not only fined, but also turbid juices are stored by this modified Boehi process. The combination of Boehi’s process and filtration, and also sterile

MICROORGANISMS IN N O N C I T R U S J U I C E S

25 1

filtration ( Boehi-Seitz) have nowadays been practically abandoned in favor of the plate-type pasteurizer. It must be noted, however, that some fruit juice is still stored by the Boehi process, though only after centrifugation or coarse filtration. In such cases the effect of carbonic acid on microorganisms is of special interest. Hofmann (1930), Osterwalder and Jenny (1939), Jenny (1940), and Schmitthenner (1949) have shown that yeasts are still capable of fermenting at the above-mentioned carbon dioxide concentrations, though they can no longer reproduce. Moreover, they can not only remain viable for long periods of time, but they can also reproduce when returned to more favorable conditions. The actual time during which they can remain viable depends upon the storage temperature. Lethal effects of carbonic acid on yeasts are observed only at much higher concentrations and pressures. According to Schmitthenner, the yeasts died after 40 days at 30 atmospheres, 30 days at 35 atmospheres, and 5 days at 40 atmospheres of carbon dioxide pressure. Kolkwitz (1921) and Lieske and Hofmann (1929) have shown that a carbon dioxide saturation of 1 mole/l00 ml. (38 to 40 atm.) was lethal within a very short time at room temperature. Few systematic investigations are available on the effect of carbon dioxide on the bacterial flora of fruit juices. There are, however, many more studies on the behavior of pathogenic organisms under carbon dioxide pressure or in carbonic acid-containing beverages. Schmitthenner (1949) has demonstrated that the quantity of carbon dioxide used in the Boehi process is insufficient to destroy lactic acid bacteria in apple juice. His experiments have further shown that the reduction in the total acid (malolactic fermentation), caused by the lactic acid bacteria, still occurred under the normal conditions of carbon dioxide impregnation and storage at 15O to 17OC. The speed at which the reduction occurred was determined primarily by the type of apple juice used. This insensitivity to the quantities of carbon dioxide used in normal practice frequently led to serious deterioration of the stored juice with commensurate losses. In the case of the pathogenic organisms which occasionally occur in fruit juice, carbon dioxide has a powerful lethal effect. The conditions under which the carbon dioxide is allowed to work are of prime importance in determining the degree of lethal effect. As Donald et al. (1924) were able to show in a study of the death rate of Bacillus typhosum and Escherichia coli, low pH greatly reinforces the lethal effects, causing the organisms to die out in a very few days in a commercial ginger ale which contained carbon dioxide in a concentration of 4.8 volumes. Eagon and Green (1957), using E . coli, Micrococcus

252

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pyogenes var. aureus, and Salmonella enteritidis as test organisms in the investigation of carbonic acid-containing commercial beverages, found that these organisms in four different, frequently consumed beverages, were destroyed in less than 24 hr. at 4O, 2 5 O , and 37OC. The inference that these effects are the result of the action of carbon dioxide alone, however, would be fallacious. The more common factors determining viability, such as the p H and the presence of organic acids, are just as important. Witter et al. (1958) found that the pH is more important in the destruction of the bacteria than is the carbon dioxide concentration. It is because of this sensitivity that in the pH range normally found in beverages (a pH which is too high to be of itself lethal) the addition of a small quantity of carbon dioxide can be critical and result in the destruction of microorganisms. The growth of E. coli and various strains of yeast could be completely inhibited by two volumes of carbon dioxide in beverages having a p H of 4.0 or less. He was also abIe to demonstrate that the survival of yeasts was chiefly a function of the carbon dioxide concentration, while that of the bacteria was more greatly dependent upon the pH of the medium. Forgacs et al. (1945) have shown that the antiseptic activity of a 0.02 N solution of lactic acid in apple juice was decidedly increased by the addition of 2.5% (vol.) of carbon dioxide, or 10% of sucrose. The influence of carbon dioxide on the growth of molds has been studied less in relationship to the storage of fruit juice than with respect to the preservation of other foodstuffs such as meat. Ruyle et al. (1946) found that Penicillium spores will no longer develop in an atmosphere which contains more than 90% carbon dioxide. 4. Cold Storage There are numerous works available which deal with the effects of low temperatures on the microorganisms which inhibit citrus juices and frozen concentrates. Studies of the microorganisms which can be found in the juice and concentrates of other fruits are, however, fewer. It had been very early noted that bacteria die off at a very rapid rate in ice (Keith, 1913) and at a considerably slower rate in foodstuffs such as eggs, milk, or juices. The mortality rate in sherbet ice was lower than that in solid ice. Such observations indicate that low temperatures slow the metabolism of the organisms, thereby tending to preserve them. It can be concluded that the greater survival rate of the organisms in foodstuffs over that of those found in water is the result of better physical conditions existing for the cells in the former medium. The favorable effects of large additions of sucrose and glycerin can be ex-

253

MICROORGANISMS IN N O N C I T R U S J U I C E S

plained in like manner. The systematic studies of McFarlane (1940a, b, 1941, 1942), Berry (1932a,b,c, 1933a,b, 1935), and Berry and Diehl (1934) on the behavior of microorganisms at subfreezing temperatures have provided us with a deeper knowledge of this field. There is generally a greater mortality rate at -1OOC. than at -2OOC. It is interesting that reduction of the concentration of sucrose in aqueous solution results in an increase in the mortality rate. These findings are in accord with those of Keith (1913). At pH 3.7 and -lO°C., 99% of the yeasts in a 1% solution were dead at the end of 2 weeks, while those in a 20% sucrose solution survived for 15 weeks. The acceleration in the death rate is also very greatly dependent upon pH, storage temperature, and species of microorganism. TABLE IX BEHAVIOR OF Saccharomyces ellipsoideus IN LOGANBERRY JUICE AND LOGANBERRY JUICE-SUCROSE SOLUTIONS'

IN

Wort agar plate counts (S. eZ1ipsoideu.s per ml.) After freezing Condition Juice J suc. t o J SIIC. t o J suc. to J SUC. to

+ + +

J

+ +

20%h 30% 40% 50% SUC. to 60%

Befort: freezing 10,800 10,600 6900 7600 10,100 lI,000

-

1 wk.

2 wk.

4 wk.

8 wk.

12 wk.

16wk.

650 2075 2150 2100 3900 4575

335 1000 1325 1470 2575 3525

54 373 628 805 1395 2290

7 84 360 445 775 1265

0 35 146 300 425 685

0 10 93 176 290 443

Data from V. H. McFarlrtne (1942). Juice solutions stored at - 17.8'C. ( O O F . ) . J auc. to 20 % means that sucrose has been added to juice in a quantity sufficient to give a total sahihle-solids content of 20 %. 0

b

+

Escherichia coli was much more sensitive in sucrose solutions of varying pH values and at temperatures of -loo and -2OOC. than species of Saccharomyces. Low p H values (between 3.2 and 3.8, as are usually found in fruit juices) have a particularly destructive effect on yeast and bacterial cells at higher sugar concentrations and lower storage temperatures. With increasing sugar concentration, the number of surviving organisms is greater at lower temperatures, as can be seen in Table IX. From this we may conclude that there is no great advantage in the addition of sugar to cold storage fruit juices as a means of protecting them from microorganisms. However, sugar is usually used to maintain the organoleptic characteristics of the juice. The work of McFarlane (1940b) on the distribution of microorganisms and dissolved substances in frozen fruit juice is of very great

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HANS LUTHI

practical importance. He has demonstrated that the microorganism population was considerably smaller in the outer regions than in the central zone of cans of fruit juice which had been frozen in a standing position. There was a similar shift in the sugar and ion concentrations. These variations are of great physiological importance, since pH differences between the various zones of the contents of the cans could be found which ranged from 3.28 to 6.11. In the case of a frozen sucrose solution, there were differences in concentration ranging between 9.5 and 28.0%, and an initial yeast cell count of 472,000 per milliliter resolved itself into a differential between the various zones of 544,000 to 1,062,00O/per milliliter. This observation explains why, under certain conditions, a reduction of temperature may result in an increased cell count. Recently, Harrison (1956) has shown that the death rate of microorganisms is, under certain conditions, directly dependent upon the concentration as well as the nature of the dissolved substances. Increase in concentration generally results in a decreased mortality rate. It can be demonstrated that glycerin has a specific favorable effect on survival, and that the addition of glycerin can, to some extent, counteract the deleterious effects of other substances such as sodium chloride. Glycerin also has a favorable effect in preventing heat-denaturization of the cell proteins.

7 . Treatment with Sulfur Dioxide In many countries a limited use of sulfur dioxide is permitted in the processing of fruit juice, Its use is desirable because the strong reducing activity inhibits undesirable chemical changes (such as browning) and because it has antiseptic characteristics. It also has very great practical importance because of its strong reactivity with substrates and its transiency resulting from its volatility. The use and nature of the chemical activity of sulfurous acid in fruit and vegetable production has been the subject of considerable discussion. Therefore, for those who would like to pursue the subject further, the reports of Cruess (1948) , von Schelhorn (1950b, 1951a,b), Borgstrom (1954), and particularly that of Joslyn and Braverman (1954) are recommended. Sulfurous acid has a selective action on microorganisms. It is known that yeasts are more resistant than acetic acid bacteria, lactic acid bacteria, or molds. There are great differences in the sensitivity of various strains to this agent, and resistance is greatly influenced by external factors such as pH, temperature, and the nature of the medium itself. Among the yeasts genera those which are primarily aerobic, such as Willia and Pichia appear to be considerably more resistant than the

MICROORGANISMS IN NONCITRUS JUICES

255

fermentative yeasts of the genus Sacchuromyces. That there are exceptions among the Saccharomyces can be seen from the work of Osterwalcler (1934) in which he reports finding a species (Saccharomyces ouijormis) which was particularly resistant to sulfur dioxide. This species was isolated from a grape must and was capable of withstanding a concentration of 225 to 235 mg. per liter of free sulfur dioxide in juice whose pH was not determined. It can be assumed that normally representatives of this genus would be destroyed by concentrations of 50 to 70 mg. per liter in such a medium. Unlike other species of this genus which can be adapted artificially to withstand higher concentrations of free sulfur dioxide, but which soon lose this resistance again, this species seems to be naturally resistant. Ostenvalder (1924b) has described an even more resistant yeast, Schizosaccharomyces liquefaciens, which was also isolated from an excessively sulfurated grape must. This strain is capable of fermenting musts containing 555 to 674 mg. per liter free sulfur dioxide and is much more resistant than Saccharomycodes ludwigii which Kroemer and Heinrich ( 1922) described. There also appear to be strains of molds which are resistant to sulfur dioxide. Schanderl (1952b) has described such a mold, Mucor racemosus, which had such an extraordinary resistance to sulfur dioxide that it required concentrations of as much as 600 mg. per liter to inhibit its growth in grape juice. von Schelhorn (1954) has, in a short review of Schanderl’s work, noted an error in the reasoning and calculations. Even taking this into consideration, however, Mucor still shows an exceptional resistance to sulfur dioxide. While an increase in temperature reinforces the antiseptic action of sulfur dioxide, the shift in pH towards the acid region is of special importance. Cruess et al. (1931) and von Schelhorn (1951b), who investigated the influence of pH on the effectiveness of sulfurous acid very thoroughly, have shown that in the very acid region of pH 3.5, two to four times the amount of sulfurous acid must be added in order to effect the same preservative action as at pH 2.5. At pH 7.0, 1000 p.p.m. were necessary in order to prevent the growth of bacteria in apple juice. In order to inhibit the multiplication of 2.5 x lo6 yeast cells, von Schelhorn (1951b) needed at least 50 times more sulfur dioxide at pH 4 than at pH 3. In order to effect a similar inhibition on the same number of Penicillium conidia, the quantities of sulfur dioxide used were in a ratio of 1: 10 between pH 3 and 4; I :100 between pH 3 and 5; and I :1000 between pH 3 and 6. The findings and conclusions of the earlier authors on the effects and mode of action of the hydrogen ion concentration has been con-

256

HANS

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firmed and enlarged by the work of Vas and Ingram (1949) , who were able to show that, in the natural pH region of the fruit juices (between pH 3 and 4), very small changes in the pH can have decided effects on preservation. Comparable results can be found in Table X. This effect can be related to the changes in the equilibrium proportions of HSO,-, SO,=, and molecular sulfur dioxide. The proportion of sulfur dioxide is practically zero at p H 4, and increases rapidly with decreasing pH. Bioletti and Cruess (1912) found that free sulfur dioxide has about 60 times more inhibitory action on fermentation than bound sulfur dioxide. Ingram (1948) also found that free sulfur dioxide was the chief active agent in the antiseptic activity of sulfurous acid, and that the sulfur dioxide is bound chiefly to the glucose. Braverman (1953) and Joslyn and Braverman (1954) have shown that the sulfur dioxide is also bound TABLE X EFFECTOF 150 P.P.M. SULFURDIOXIDE ON Saccharornyces uuarum IN STERILE APPLEJUICE' Time of contact Zero t i m e After 1 hr. After 2 hr. After 5 hr. After 24 hr. After 7 days a b

pH 3.4 3500

2000 1880 650

N. S . b 10,000,000

pH 3.7

pH 4.0

850 800 800 880 35,000

5300 5150 5150 6500 10,000

N.

N. S.b

S.b

Viable counts per milliliter. Data taken from Beech (1958s). N. 9. = Not sampled.

to other extract components of the juice and that the concentration of these substances must also be taken into consideration when referring to the bound sulfur dioxide. Low p H values reduce the velocity of the reaction, which binds the sulfur dioxide primarily to the glucose and secondarily to the other extract components. Vas and Ingram (1 949) have shown that this reduction causes the sulfur dioxide to remain in solution in its free form for a longer period of time with a commensurate prolongation and increase of its antiseptic activity. They point out that fruit juices can be preserved with a smaller addition of sulfur dioxide if the pH is first lowered. In most temperate countries, there is a tendency to cut down the use of sulfurous acid in fruit juice manufacture by substituting ascorbic acid as the reducing agent. W e can only decrease the concentration of the sulfur dioxide, and not eliminate it, since ascorbic acid cannot re-

MICROORGANISMS IN NONCITRUS J U I C E S

25 7

place its antiseptic activity. The technical installations and processing in use in temperate regions permit manufacture without the aid of sulfur dioxide. In a few countries such as France, the preservation of grape juice is still carried out exclusively with the aid of sulfur dioxide. T h e method suggested by Fabre (1947) which entails the addition of approximately 1000 p p m . H,SO, with the later removal of the same by heating in uacuo is used extensively since the strong dosages which this method permits results in the rapid destruction of the organisms present. From the above, we see that the valuable antiseptic activity of sulfur dioxide can be used to best advantage in low pH juices such as apple, grape, and berry, and for the destruction of thermoresistant molds which Gillespy (1946) found sensitive to small quantities of sulfur dioxide at higher temperatures. With the addition of sulfur dioxide, both pasteurization temperature and time can be reduced. The antiseptic activity of sulfurous acid can be also influenced by external factors. W e should keep in mind the capacity of the sulfur dioxide to combine with glucose, aldehydes, and other extracts. Sugar content and aldehydes (mainly acetaldehyde) , which are present in considerable amounts in some fruits at certain stages of ripening are important considerations when determining how much sulfur dioxide must be added to a juice. W e must also consider the characteristics of the large yeast and bacterial infections, since younger cultures are less sensitive than older cultures, and yeasts are known to have the ability to adapt to sulfur dioxide. The fruit juice industry is not alone in its high evaluation of sulfur dioxide as an antiseptic. It is used as a disinfecting agent for many purposes, usually in a 5% aqueous solution. I n the autumn, press cloths and frames are washed with this solution during the “rest” periods, and are kept in a 2 to 5% solution until needed. The same concentration is used for the disinfection of storage tanks and vessels of all types, and very recently, the firm of Seitz (Kreuznach, Germany) has introduced it for the disinfection of bottles used for juices and fermented beverages.

V.

CHANGES IN APPEARANCE OF JUICE

A. GENERAL Fruit juices should contain no developing organisms; should they be found in a juice, it can no longer be safely placed on the market. There is very little difficulty in checking clear beverages. Juice of apples, grapes, and berries have, until now, been sold on the European market primarily in clear form. The naturally turbid fruit juices are,

258

HANS LUTHI

however, coming into wider use, resulting in greater difficulty in checking for the growth of microorganisms. Fortunately, microorganisms in such turbid juices are rare. The more highly the fruit juice industry of a country is developed and the more technically equipped the installations, the rarer is the occurrence of such infections. In countries which are characterized by many small installations, however, difficulties of this nature are more frequent. Turbidity must be considered primarily among the superficial, easily marked modifications which can occur in fruit juice. It is more often of chemical than of biological nature. A m o n g the biologically induced turbidities those caused by the yeasts are the most frequent and are usually accompanied by an alcoholic fermentation. Torula species are very often implicated in yeast-caused turbidity and appear usually as an infection resulting from inadequately cleaned bottles. Those species of Torula are, in addition to the heat-resistant molds, usually the last survivors in the process of bottle cleansing. Since Torula are usually weak fermenters, fermentation by them usually runs its course before the containers are damaged; infections by these yeasts consequently result in less material damage than in the case of the more strongly fermenting yeasts, which usually burst the storage vessels. The cause of the infection is usually insufficient pasteurization. B. MOLDCHANGESAND CLARIFICATION MoId mycellia can grow to large size in beverages. Olliver and Rendle (1934) and Biichi (1958) have found that even a slight growth of Byssochlamis nivea and Paecilomyces varioti can cause great chemical and organoleptic modifications of juice, A moldy taste is frequently concomitant with this growth. As the result of the production of pectinsplitting enzymes, which many of the fruit juice molds have been found capable of producing, there is often a clarification of the juice. Some molds, e.g., Phialophora mustea, cause, as Neergaard (1941) found, a brown coloration in apple juice. Millis (1 951 ) has described a rare case of the production of slime in apple juice by Leuconostoc species. The frequent occurrence of molds in commercial fruit juices is the result of the greater heat resistance of these organisms. That the growth of these organisms is less in juices which contain carbon dioxide than in those essentially free of the gas may be considered to be primarily the result of its replacing the oxygen, and only secondarily the result of and preservative action of the carbonic acid. In this respect, we must again consider the conditions in home fruit

MICROORGANISMS I N N O N C I T R U S JUICES

259

juice production. In this case, unfortunately, mold infections are so frequent as to be very nearly the rule. However, with improved techniques and the use of air-tight vessels, it is to be hoped that the infection rate will very shortly be reduced. As Liithi and Hochstrasser (1952) and Liithi and Vetsch (1955) have shown, the thermoresistant molds play a very important role, since once they are brought into an installation, they are very difficult to eradicate. VI. PRODUCTION OF ALCOHOLS BY MICROORGANISMS

A. ETHANOL The presence of small quantities of alcohol in fruit juice cannot necessarily be considered to be the result of the activity of microorganisms. It is known that as the result of careless storage in the presence of increased carbon dioxide concentration (as is the case when stored in deep containers) fruit is capable of forming alcohol in the cells of the tissues by enzymatic means. Simultaneously with this alcohol production, the tissue becomes softer and begins slowly to disintegrate. Within a few days at higher temperatures, 0.5 to 0.7% of alcohol, which then acts as a cell poison and inhibits cell respiration, can be formed in the tissues. Ostenvalder and Kessler (1934) have studied the formation of alcohol in fruit tissues; more recent reports by Smock and Neubert (1950) and Ulrich (1952) are also available.

1. Yeasts The formation of alcohol in fruit juices is usually the result of the activity of yeasts. The danger of fermentation in grape or berry juice, even during the preparation of the raw material, is very great in warm climates, although normally the onset of fermentation is not a factor to be considered at this stage of the processing. With the direct bottling or canning of the juice, fermentation is limited to the rare case. Formation of alcohol in fruit juice subsequent to storage in tanks is possible. In spite of storage in sterile tanks after a sterile filtration (Seitz process) or with the commonly used flash-heating, there is still a very great risk that alcoholic fermentation will take place as the result of an infection by yeasts subsequent to processing. Therein lies the main reason why these processes are used with reluctance and are slowly being replaced. Storage under carbon dioxide pressure (as in the Boehi process) reduces the risk of a fermentation to practically zero. As Schmitthenner (1949) has shown, however, a slight production of alcohol can still be found as the result of fermentation by yeasts which remain in the

260

HANS

LUTHI

juice. That the formation of alcohol is the result of activity by these yeasts is indicated by the fact that with carbon dioxide concentrations as used industrially, i.e., 1.5% at 15OC. and 0.8% at 2OC., multiplication of the yeasts no longer takes place. Storage of the juice without a considerable prior reduction in the microorganism population by means of the usual methods, such as centrifugation and coarse filtration, can, at unsatisfactory storage temperatures, result in a n alcohol content of more than 0.5%. Lieske and Hofmann (1929), Osterwalder and Jenny ( 1939), and other authors have shown that the carbon dioxide concentration used in the Boehi process does not kill the yeasts. I n order for the compound to be lethal to these organisms, much higher concentrations (in the region of 22.4 liters carbon dioxide per liter of juice, which is equivalent to a carbon dioxide saturation of 4%) are necessary. As a consequence of this survival, the danger exists that the stepwise emptying of the tanks can lower the carbon dioxide concentration sufficiently to permit a resumption of cell multiplication and alcohol production. The chief possibilities for the production of alcohol by the yeasts have now been pointed out. The fact remains, that transient or prolonged storage in large containers always results in a clearly determinable alcohol content. The legal alcohol content for alcohol-free fruit juice in many countries is 0.5 to 0.7 vol.%, which is very high.

2. Bacteria Another, though rarer, possibility for alcohol production is as the result of bacterial activity. Some of the lactic acid bacteria which can be found in fruit juice are capable of producing ethyl alcohol, lactic acid, and acetic and carbonic acids from fructose, galactose, and sucrose. Miiller-Thurgau and Osterwalder ( 1913) have shown that certain strains of Bacterium mannitopoem can convert as much as 40% of the sugar (fructose) into alcohol. Hucker and Pederson (1930) have also shown that species of Leuconostoc are capable of converting glucose into alcohol, some strains being capable of forming up to 20%. The production of alcohol has also been demonstrated by members of the genera Betncoccus, Streptococcus, and Zymomonas.

3. Molds Production of alcohol in fruit juice by molds is as rare as that by bacteria. I n spite of this, it shouId be noted that molds have been known to produce considerable quantities of alcohol in some fruit juices. Thus, species of Mucor are known to be prime producers of alcohol. Fusarium

MICROORGANISMS IN N O N C I T R U S J U I C E S

261

and certain species of Aspergillus have also been known to produce alcohol in fruit juices under certain circumstances. Studies of alcohol production by these molds have been made by Kostytschew (1907) and Jacquot and Raveaux (1943).

B. OTHER ALCOHOLS The production of mannitol by some species of lactic acid bacteria has been observed in the fermentation of fructose. Spencer and Sallans (1956) have found that the osmophilic yeasts also form mannitol simultaneously with the production of ethyl alcohol. One of the most important metabolic products of these yeasts is glycerin; erythritol and D-arabitol are also produced. Mannitol can be found in considerable quantities. It is a reserve substance in the cells of Aspergillus niger. This mold, among others such as Botrytis cinereu, can produce glycerin in large quantities not only in the finished fruit juice but also in the raw material. Muhlberger and Grohmann (1956) found 2 to 7 g. per liter of glycerin in moldy grape juice. Their studies confirmed the ability of Aspergillus to produce glycerin and added certain species of Penicillium to the list of producers of this metabolic product. According to these authors, the high glycerin content of grapes and raisins which are used for the production of special wines can be shown to be produced primarily by the growth of molds on the berries, only a small quantity being produced by the yeasts during the alcoholic fermentation. Thus, as much as 20 g. per liter of glycerin has been found in grape juice. Charpenti6 (1954), in his detailed study of the growth of Botrytis cinerea in grape juice, confirmed not only the fact that this mold is capable of producing considerable quantities of glycerin but that there are also great differences in productive capacity between the various strains. He was further able to demonstrate that the production of glycerin by Botrytis reaches a maximum in grape juice and later declines with prolonged growth in this medium. It is known that glycerin is a normal metabolic product of yeast activity during alcoholic fermentation and that under normal conditions approximately 3 to 4% of the sugar is converted into glycerin. Grohmann and Miihlberger (1957) made a detailed study of the production of glycerin in grape musts and confirmed the known dependence of glycerin production on the strain of yeasts. A further stimulation of glycerin production in grape musts by sulfuration was noted, as well as a dependence of the production on the vitamin content of the juice. Thiamine had a particularly great effect, which is understandable when one considers its role in enzymatic reactions. Fining of

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the grape must and a low pH have an inhibitory effect on glycerin production. The production of glycerin is closely coupled to the production of ethanol; Grohmann and Miihlberger (1957) found that half of the glycerin was formed by the time approximately 35% of the total ethanol had been produced, and in addition 65% of the total glycerin content was produced by the time half of the alcohol was formed. Glycerin production in grape juice terminates before ethanol production is complete. VII. CHANGES IN THE ORGANIC ACID CONTENT INDUCED BY MICROORGANISMS

In recent years, the organic acids of grape, apple, pear, and cherry juice, and the juice of various berries have been the object of study by means of paper chromatography. Consequently, we now have a good knowledge of the modifications in organic acid content, including the appearance of new acids which result from the activity of microorganisms. The most important acids from a quantitative standpoint are tartaric, malic, and citric, of which the first two acids are of approximately equal importance in grape juice. In apples and pears, on the other hand, malic and citric acids are the most common, while in berries, citric acid can predominate. Other acids which can be found in fruit juices are: quinic acid, chlorogenic acid, galacturonic acid, shikimic acid, dehydroshikimic acid, succinic acid, and lactic acid, all of which are present in only milligram quantities and which are relatively unimportant quantitatively. The most recent complete studies on the acid content of apples and pears have been made by Hulme ( 1951) ,Rentschler and Tanner ( 1954, 1955), Tanner and Rentschler ( 1954), and Phillips et al. ( 1956). Modifications induced by microorganism growth in the most important organic acids of fruit juices will be briefly discussed.

A. TARTARIC ACID Tartaric acid and its salts are the most stable of the organic acids found in fruit juice with respect to the attacks by microorganisms. By mid-nineteenth century, however, the French students of the subject had already noted the ability of some bacteria to decompose tartaric acid and tartrates in wine. Muller-Thurgau and Ostenvalder (1919) and Osterwalder (1952) have shown, in their studies of the decomposition of tartrates in wine by pure cultures of the bacteria Bacterium tartarophorum, that only carbon dioxide and acetic acid are produced, the latter in large quantities.

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263

Berry and Vaughn (1952) were successful in isolating from spoiled red wine a bacteria which converted tartrate chiefly into lactic and carbonic acids. This bacterium, which they named Lactobacillus plantarum, has been the object of a detailed study by Krumperman et al. (1953). Other studies are available concerning the main metabolic products of the above-mentioned tartrate-catabolizing organisms of MullerThurgau and Ostenvalder (1919). Unfortunately, the exact classification of their “Bacterium tartarophthorum” cannot be made on the basis of their description. Vaughn (1955) is of the opinion that it very probably was not a representative of the lactic acid bacteria group. Vaughn and Marsh (1943a,b) and Vaughn et al. (1946) also studied the tartrate-decomposing ability of a number of species of Aerobacter and found that these bacteria could be differentiated into distinct groups according to the strength of gas production. They found that most of the strains did not decompose the Z-tartrates as well as the dl- o r d-forms. They showed that the ability to decompose tartrates is a characteristic typical of many strains of the genera Aerobacter and Escherichia. Bacterium succinicum, described by Sakaguchi and Tada (1940), produces large quantities of succinic acid during the decomposition of tartrate. Sakaguchi et al. (1952) were later able to work out the steps in the decomposition of d-tartrate. As a result of other investigations, Vaughn et al. (1946) have been led to believe that Bacterium succinicum is closely related to those representatives of the genera Aerobacter and Escherichia which they studied. In conclusion, we should note that Nomura (1953a,b) also described a tartrate decomposition for Pseudomonas incognita. By means of tracer techniques, Nomura was able to elucidate further the mechanism of tartrate catabolism. Small quantities of succinic acid, fumaric acid, and acetic acid were also found in the medium. Somewhat later, Nomura and Sakaguchi (1955) succeeded in working out the steps in the breakdown of the more resistant tartrates by Pseudomonas incognita, and found that this form of the tartrate partakes in the intermediate metabolism of the organism. The ability to metabolize tartrates does not appear to be as widely distributed among bacteria as it is among molds. Pasteur knew of the ability of certain penicillia to decompose tartrates, and used a PenicilZium to obtain the L-form of tartaric acid. The ability of the aspergilli to decompose tartrates is also well known. When growing anaerobically they also produce alcohol. Stadtman et a2. (1945) have shown that the ability to decompose tartrates is widely distributed among the molds. Studies of a large

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number of molds for their tartrate-metabolizing ability have shown that representatives of the genera Aspergillus, Penicillium, and Fusarium are very active in the decomposition of calcium tartrate, potassium tartrate, and tartaric acid. Her studies, especially those on Aspergillus niger, have shown that the decomposition of tartrates and tartaric acid in a synthetic medium begins only after a lag period of 5 days, by which time a heavy mold pad had developed, and that the decomposition of the tartrate proceeds in a regular manner at the rate of 2% per day in the case of calcium tartrate. The mold Botrytis cinerea, which occurs in grape juice, is of practical importance. The great chemical changes which this mold is capable of producing, even on ripe grapes, very early attracted the attention of enologists. One of the first thorough investigations was that of MullerThurgau (1888) which dealt with the reduction of the acid content by Botrytis. Stalder ( 1954) has approached these relationships from another standpoint, and corrected the long-standing misconception that the acid was the prime carbon source. He has shown that the mold is mainly dependent upon the sugar content of the nutritional medium, utilizing this sugar in large quantities and simultaneously metabolizing equal quantities of malic and tartaric acids. He found also, that though poor carbon sources in themselves, the tartrates do stimulate the mold growth. Charpentik (1954) has shown that there are great differences in the behavior of various strains of mold with respect to tartaric acid and tartrates. It can be seen from the above that the ability to decompose tartaric acid and tartrates is present in innumerable molds which are capable of living and multiplying in grape juice. This ability is limited in the case of the bacteria to a very few genera. W e are forced to admit, however, that our knowledge in this field is strictly limited and that further studies are necessary before generalizations can be made. Yeasts which occur in fruit juices, on the other hand, are incapable of utilizing either tartrates or tartaric acid.

B. MALE ACID I . Bacteria The breakdown of malic acid was first observed and closely studied in fermented fruit juice. Pasteur was one of the first to describe the reduction in the total acid content in wine which resulted from the growth of certain bacteria. More detailed studies of this phenomena Were made at the Swiss Federal Experimental Station in Wadenswil

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by Muller-Thurgau and Osterwalder (1913) after a number of lesser studies of some malic acid-metabolizing bacteria had been carried out. The reduction of total acid content in fermented beverages as a consequence of the decomposition of malic acid is of great practical importance in grape and fruit wine production. It is also of interest with respect to the naturally occurring reduction of the acid content of fruit juices or to undesirable changes which take place during storage. The importance of this reduction is best demonstrated by a study of the voluminous literature on malolactic fermentation. Since this fermentation is not as yet well known from a mechanistic standpoint, however, industry in many countries is now supporting a study of this type of fermentation in research laboratories. All the wineproducing countries have interested themselves in the study of the malolactic fermentation of wines and fruit juices with a view toward gaining a better knowledge of the responsible bacteria and thus gaining better control of the fermentation. It would therefore be of great advantage 10 find a biochemical method of inducing and controlling the decomposition of malic acid. This is the goal of many different lines of investigation. Newer systematic studies of the classification of malic acid-decomposing bacteria have been made by Vaughn (1955), Millis (1951), Carr (1952, 1956, 1957a), Lambion and Meskhi (1957), and Fornachon ( 1957). The Lactobacillus species which decompose malic acid are L. plantarum, L. fermenti, L. hilgardii, L. buchneri, and L. breuis. Among the cocci, Leuconostoc mesenteroides appears to play a special role. Carr (1956) and Lambion and Meskhi (1957) considered it as probably being identical with “Bacterium gracile” which had been made famous by Miiller-Thurgau. I n addition, they mentioned Leuconostoc deztranicum. Even today, it is not certain whether the other strains should be classified under the genus Streptococcus or Pediococcus, A few authors have described cocci which have very great similarities to representatives of the genus Pediococcus. It has been known since the time of the first thorough studies made by Miillet-Thurgau and Osterwalder (1913) that lactic and carbonic acids are the main metabolic products of the bacterial decomposition of malic acid. It must also be noted that acetic acid is produced in small quantities as a by-product of this fermentation. Vaughn and Tschelistcheff (1957) have speculated that minute quantities of acetoin and diacetyl can also be produced. Korkes and Ochoa (1948), Ochoa (1951), and Nossal (1951) also studied the biochemical reactions involved in the malolactic fermentation and succeeded in isolating an enzyme preparation from a malic

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acid-adapted strain of Lactobacillus arabinosus which converts malic acid into lactic acid and carbon dioxide in vitro. This enzyme was designated the “malic-enzyme.” They found that the reaction proceeded only in the presence of the manganese ion (Mn”) and diphosphopyridine nucleotide (DPN) . These workers have designated the previously generalized reaction as general Formula (I) and have detailed the reaction in Formulas (11) and (111) : COOH-CEI-CHOH-COOH l-malic acid

% CHj-CH(0H)-COOH

lactic acid

+

COz carbon dioxide

(1)

l-malic acid

+ DPN

pyruvic acid

Mn*+ ~

~~

pyruvic acid (11)

+ DPNHa

+ COZ + DPNHz

lactic acid

+ DPN

(111)

Jerchel et al. (1956) recapitulated the work on the decomposition of 1-malic acid with pure enzyme extracts using not only Lactobacillus arabinosus but also “Bacterium gracilae” (Leuconostoc mesenteroides) . They found that there were two ways in which malic acid could be decomposed, and that which pathway was operative depended upon the method used for the lysis of the bacterial cells. By careful treatment, (using the method of Ochoa), the breakdown proceeds directly from pyruvic acid to lactic acid, while by using other enzyme preparations a further intermediate product can be obtained (oxalacetic acid) which then is converted to pyruvic acid and lactic acid. This work, in addition to the theoretical considerations of Peynaud (1956), indicates that the malic acid breakdown reaction is in reality an endothermic one. Schanderl (1950b) was one of the first to note this. A satisfactory explanation of this reaction has not yet been made. Because of the fact that various workers have found an increase in ammonia nitrogen in the wine following malic acid breakdown, Schanderl believes that a concomitant breakdown in higher nitrogen compounds is possible. Peynaud (1956), who admits to this possibility on purely theoretical grounds, has pointed out the possibility that glycollides or mesoinositol can also serve as energy sources. This question has not been resolved. As the result of his own work the present author does not believe that there is an accumulation of ammonia during the acid breakdown. He is, on the other hand, convinced that higher nitrogen compounds do play an important role. In an interesting observation on this question, Carr (1956) found that two bacterial strains produced varying quantities of lactic and

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succinic acids during malic acid breakdown, and that in addition to the quantities of lactic acid produced, the production of succinic acid was dependent upon the p H of the medium. One of these strains produced small quantities of succinic acid in the normal pH range of fruit juice while the other produced only traces. In both cases the production of succinic acid increased with increasing pH. As Table XI shows, the primary product at higher pH values is succinic acid, with only traces of lactic acid being produced. Until now this possible pathway of malic acid breakdown had not been considered. The above studies indicate that the mechanism of the bacterial breakdown of malic acid in fruit juice has been considerably clarified. One would believe that as a result of this work, control of this breakdown could now be introduced. Unfortunately, this is not the case. Luthi (1952) in work which he carried out between 1949 and 1951 has shown that there were other factors besides pH which influenced the breakdown of malic acid in fruit juice (such as the presence of amino acids, Burroughs and Carr, 1956, and the presence of manganese, and magnesium) and that amino acids may have an inhibitory effect. In spite of the detailed clarification by Luthi and Vetsch (1953), Jerchel et al. (1956), Carr (1956), Liithi (1957b) and Flesch (1958), of the role of amino acids and vitamin requirements of the bacteria concerned, it has not as yet been possible to control the malic acid breakdown in natural media such as fruit juices and wines. This indicates that the reaction is not only dependent upon many factors but that there is most probably a decisive element which has not as yet been discovered. Luthi (1954, 1957b) pointed out that this effect should most probably be sought in the higher molecular nitrogen-containing compounds and that there is a probability that the effect is the result of streptogenin-like activity such as has been found in peptides. As Challinor and Rose (1954), Peynaud (1956), and Liithi (1957b) have pointed out, symbiotic and antibiotic effects must also be considered. 2. Yeasts Compared with the decomposition of malic acid carried out by bacteria in fruit juices, the attack upon this acid made by the yeasts is secondary in importance. It has long been known that small quantities of malic acid are decomposed by yeasts during alcoholic fermentation of fruit juices and that these organisms are capable of utilizing the acid as a carhon source (Ingram, 1955; Morris, 1958). Yeasts usually attack other carbon sources in the nutritionally rich and chemically complex fruit juices.

TABLE XI EFFECTOF PH ON

THE ENDPRODUCTS OF k

c ACIDBREAKDOWN" PH

Organism No.

Lactic acid Succinic acid Lactic acid Succinic acid

73

74

b

Acid

Data taken from Carr (1956). Key: T = trace = small amount -I-= moderate amount

+

+

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.2

+++ +++ ++ ++ + T T T + + + ++ ++ +++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++ ++ T T T T T ++ ++ ++ T ++fb

+ f + = fairly large amount

++f f = very

large amount

5

2: !!

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3 . Molds Prolonged growth of molds in fruit juice always results in a change in the total acid content. Malic acid and other acids can be decomposed to a very great extent, or even completely, as reported by Charpentie (1954). A strain of Botrytis cinereu which he used in his experiments was able to reduce the total acid content of a grape juice to one-half within a period of 20 days. After 38 days, the tartaric acid and malic acid completely disappeared. Innumerable molds which are capable of growing in fruit juices have the ability either to decompose or to produce malic acid. In this respect the considerable production of malic acid from sugar by Rhizopus nigricans (Rippel-Baldes, 1952) should be noted. C. CITRICACID Citric acid can be both produced and decomposed in fruit juices in a manner similar to that for malic acid. Most of the lactic bacteria in fruit juice are capable of decomposing citric acid, thereby producing acetic acid in considerable quantities in addition to lactic acid and carbon dioxide. I n those cases where citric acid comprises a considerable part of the total acid of the fruit juice, bacterial acid reduction can, according to Tanner and Rentschler ( 1954), result in serious deterioration in the quality of the juice as the result of the formation of acetic acid, Liithi (1949a) and Charpenti6 et al. (1951) have studied the bacterial decomposition of citric acid in wines. The French researcher found that in addition to the main production of acetic and lactic acids there was a secondary production of acetoin and butanediol. They therefore concluded that the breakdown proceeds with oxalacetic acid as an intermediate. The breakdown of citric acid by bacteria in fruit juice which has been stored under carbon dioxide pressure in the Boehi-process at unfavorable temperatures can be of practical importance since, as Liithi (1957a,b) has shown, the acetic acid which results from the bacterial reduction of the total acid and the sugar can rise in concentration to such an extent as to make the beverage worthless. The ability not only to decompose citric acid but also to produce it is widely distributed among microorganisms. This facility is known to be possessed by the following molds: Citromyces, Aspergillus, Penicillium, Mucor, Botrytis, Dematium, and Fusarium. Charpentik ( 1954) intensively investigated Botrytis in this respect and also noted the great differences between various strains. The growth of Botrytis results, as

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Rentschler and Tanner (1955) and Charpenti4 (1954) have found, in the production of gluconic acid. This acid also appears as a metabolic product of other molds such as aspergilli. In addition to the above acids, other organic acids, though not yet identified, can be produced as reaction end products in the development of these molds.

D. OTHER ACIDS Beech and Pollard (1955) have made detailed studies of changes which occur in the acid content of English ciders. They found that yeasts produce only limited acid modifications during fermentation, the most important of which is the production of smalI quantities of succinic acid. The greatest changes in the fruit juices result from bacterial activity which produces small quantities of succinic acid from malic acid in very acid-poor ciders. A reduction in the total acid content usually parallels acid production, resulting in the modifications of the acid content of the juice as mentioned above. In those juices which have an intermediate acidity, modifications in acid content resulting from bacterial metabolism can increase or decrease the total acid content. In the case of acid-poor ciders, changes in acid content are often accompanied by deleterious organoleptic changes in the beverage. Phillips et al. (1956) have shown that, in overripe raw material, the decomposition of malic acid can result in the production of significant quantities of succinic acid without any of the expected lactic acid being formed. The same worker, as well as Carr et at. (1954,1957) and Whiting and Carr (1957) demonstrated, for the first time, the decomposition of quinic acid in English ciders by lactic acid bacteria. They identified dehydroshikimic acid as the end product of this breakdown. A variety of Lactobacillus pastorianus, which is characterized by the ability to carry out the above-mentioned breakdown, has been named by them L. pastorianrcs var. quinicus. Cam et al. (1957)have been able to demonstrate an enzyme system in the resting and growing cells of certain lactic acid bacteria which can convert quinic acid and shikimic acid to dehydroshikimic acid. They were able, in addition, to detect the conversion of shikimic acid to quinic acid in resting cells, thus demonstrating the reversibility of this reaction. The question is therefore posed, whether the shikimic acid found in fruit juices can be considered to be only an intermediate product in the breakdown of the quinic acid to dehydroshikimic acid. Whiting and Carr (1957) have shown that chlorogenic acid, which very often occurs in English cider apples in concentrations of up to 0.25%, can also be decomposed by lactic acid bacteria. It is interesting

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to note that these changes occur only simultaneously with the conversion of quinic acid to dehydroshikimic acid. When present, caffeic acid was also decomposed. L. pastorianus var. quinicus is also capable of carrying out the above reactions. The first step in the decomposition of chlorogenic acid is the hydrolysis to caffeic acid and quinic acid. Both these products are further metabolized to dehydrocaffeic acid and ethyl catechol. VIII. OTHER CHANGES IN JUICE INDUCED BY MICROORGANISMS

Millis (1956) reported on a modification of fermented apple juice which is known particularly in the north of France (as “Framboisd”). She has shown that it is the result of the previously discussed and studied changes resulting from the activity of Zymomonas anaerobiae var. pomacea. This organism is capable of carrying out a practically quantitative conversion of glucose into ethanol and carbon dioxide. As is the case with all other bacteria capable of multiplying in fermented products, the ability of these bacteria to develop in unfermented fruit juice is greatly dependent upon pH. Zymomonas anaerobia var. pomacea develops only in those English ciders whose pH is higher than 3.5. It is interesting to note that in these experiments a considerable growth of these bacteria took place in a pH up to 8.0. The occurrence of butyric acid fermentation is relatively rare in unfermented juices, and at this writing has been found only by Luthi and Vetsch (1957). The bacteria causing this fermentation have not yet been described. Their development takes place only in juice whose pH is greater than 4. Bowen et al. (1953) described a butyric acid fermentation in canned pears and tomatoes and identified the causative organisms as Clostridium pasteurianum, whose growth takes place only when the pH is higher than 4. The addition of 0.2% citric acid can prevent this fermentation. Special consideration should be given to the production of diacetyl and acetoin in fruit juices. The production of these compounds was first studied in citrus juice and concentrates. T h e content of these substances is used as an index for the quality of the product. Buchi (1958) related the production of these substances in Swiss apple juice to the activity of species of molds of the genus Paecilomyces. Charpentik et a2. (1951) found that acetoin results from the bacterial decomposition of citric acid, while Hochstrasser (1955) found that acetoin results from the decomposition of malic acid. The production of acetoin and diacetyl in fruit juices as the result of other bacterial changes must also be considered. Hill et al. (1954) has worked out a simple determination

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method for these substances which is suitable for use in citrus juices. The production of acrolein is of very great practical importance in any consideration of modifications of fruit juice and fermented products brpught about by bacteria. Holz and Wilharm (1950) and Wilharm and Holz (1951) found acrolein in apple juice as well as in apple pulp and the resulting fermented products. According to those authors, sugar plays a very important role in acrolein production, and they were successful in improving its production with sugar additives. It appears possible that acrolein can also result from the decomposition of glycerin. Rentschler and Tanner (1951) showed that the bitter sickness of wine which was described by numerous earlier authors is the result of the production of acrolein from glycerol and the further reaction of the acrolein with the polyphenols of the wine. The result of this last reaction is a bitter substance which is the cause of the characteristic modification of the beverage, Unfortunately, no detailed study has yet been made of the characteristics of the bacteria which produce acrolein. The descriptions given by Wilharm and Holz (1951) are inadequate. In conclusion the production of symbiotically and antibiotically active substances by the microorganisms of fruit juices should be noted. To date, only very few studies are available although there is no doubt that there are many aspects to be discovered which would be of scientific and practical interest. We will here refer only to the work of the Institute in Bordeaux. RiGreau-Gayon et a2. (1952, 1955) found that the mold Botrytis cinerea, which grows on grapes, liberates substances into the berries which strongly inhibit fermentation. These substances are called “botryticin.” The addition of sulfur dioxide to fresh grape musts inactivates the botryticin. This antibiotic substance can be precipitated from the grape musts by the addition of 80% alcohol and then further purified and concentrated. The authors also found antibiotic effects and fermentation-inhibiting substances in culture media in which numerous Penicillium species had grown. They were able furthermore to isolate activators of alcoholic fermentation from dried cultures of Aspergillus niger, which strongly stimulated the growth of yeasts. A similarly acting preparation could also be isolated from dried Botrytis cinerea after previously boiling the mycelia in distilled water in order to first remove botryticin. Liithi (1953, 1957a) and Hochstrasser (1955) have found activators for bacterial growth in cultures of bacteria, yeasts, and molds. In this respect the above-mentioned strong stimulation of growth of certain lactic acid bacteria by Acetobacter rancens, Penicillium rogueforti, and Debariornyces kloeckeri should be remembered.

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IX. ADDITIONAL RESEARCH NEEDS

Pure research, as well as applied research, is necessary in the studies of microorganisms in fruit juices, concentrates, and fermented products. Our knowledge of the microorganisms which are capable of multiplying in fruit juices is far from complete. Systematic studies are still lacking in numerous areas of fruit juice production. I n this respect we have only to note our very scanty knowledge of the distribution of individual microorganisms with respect to geographic areas as well as our limited knowledge of the frequency of occurrence of most organisms over long periods of time. Most of the work which is available can be likened to snapshots. It can to some degree be assumed that certain microorganisms, e.g., the dangerous thermoresistant molds, occur in tremendous numbers in some years and in other years are quantitatively unimportant, as is the case with animal pests. A knowledge of these relationships would help to explain spontaneously occurring difficulties in preservation practice. There is also a very great need for a new systematic classification OI those bacteria which occur in fruit juice. Insecurity reigns supreme in this area today. A comparison of the numerous organisms which have been isolated to date and a general clarification of their taxonomic position would, without doubt, be very valuable. Another field for additional study is that of the biochemical changes which result from the growth of bacteria in the various fruit juices. The study of molds in fruit juices has, until now, been neglected. As producers of symbiotically and antibiotically active substances, they deserve more attention. In all countries, the importance of fruit juice concentrates is increasing. I n general, studies such as those which have been carried out by von Schelhorn (1956) and Ingram (1957) on the microorganisms in media of high sugar and salt concentrations are, unfortunately, very rare. This, therefore, is another region which needs further work.

ACKNOWLEDGMENTS The author wishes to thank Arthur M. Howard for the translation of this work and Paul Halter for checking the various references

REFERENCES Baumann, J. 1951. “Handbuch des Siissmosters,” 109-1 19. Ulmer, Stuttgart. Beech, F. W. 1957. The incidence and classification of cider yeasts. Ph.D. Thesis, University of Bristol, Bristol, England. Beech, F. W. 1958a. The yeast flora of apple juices and ciders. J. A p p l . Bncteriol. 21, 257-266.

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freie schweflige Saure widerstandsfahige Saccharomyces Garhefe. Landwirtsch. Jahrb. Schweiz pp. 1101-1132. Osterwalder, A. 1940. Nachteilige Veranderungen in alkoholfreien Trauben-und Obstsaften durch der grunen Pinselschimmel Penicillium glaucum. Landwirtsch. Jahrb. Schweiz. 54, 510-511. Osterwalder, A. 1952. Ueber die durch Bakterien verursachte Zersetzung von Weinsaure und Glycerin im Wein. Die Bakterien der Weinstein- und Glycerinzersetzung. Landwirtsch. Jahrb. Schweiz pp. 181-197. Osterwalder, A., and Kessler, H. 1934. Das Auftreten der Faulnis bei der Kuhllagerung des Obstes. Schweiz. 2. Obst u.Weinbau 43, 413-528. Osterwalder, A., and Jenny, J. 1939. Die wissenschaftlichen Grundlagen der Siissmosteinlagerung unter Kohlensauredruck. Das Verhalten der Garhefen gegenuber Kohlensaure und dem Kohlensauredruck. Landwirtsch. Jahrb. Schweiz pp. 371426. Pasteur, L. 1866. Etudes sur le vin, ses maladies, causes qui les provoquent, procPdCs nnuveaux pour le conserver et pour le vieillir. Imprimerie ImpCriale, Paris. Pederson, C. S. 1936a. The preservation of grape juice I. Pasteurization of Concord grape juice. Food Research 1, 9-27. Pederson, C. S. 1936b. The preservation of grape juice 111. Studies on the cool storage of grape juice. Food Research 1, 301-305. Pederson, C. S., and Tressler, D. K. 1938. Flash pasteurization of apple juice. Znd. Eng. Chem. 30, 954. Pederson, C. S., Beavens, E. A,, and Goresline, H. E. 1936. Preservation of grape juice. IV. Pasteurization of juices or musts prepared from several varieties of grapes. Food Research 1, 325-339. Peynaud, E. 1956. New information concerning biological degradation of acids. A m . J . Enol. 7 , 150-156. Peynaud, E., and Domercq, S. 1953. Etude des levures de la Girnnde. Ann. inst. natl. recherche agron. No. 4, 265-300. Peynaud, E., and Domercq, S. 1956. Sur les Brettanomyces isolCs de raisins et de vins. Arch. Mikrobiol. 24, 266-280. Phaff, H. J., Miller, M. W., Recca, J. A,, Shifrine, M., and Mrak, E. M. 1956. 11. Yeasts found in the alimentary canal of Drosophila. Ecology 37, 533-538. Phillips, J. D., Pollard, A., and Whiting, G. C. 1956. Organic acid metabolism in cider and Perry. I. A preliminary study. J . Sci. Food Agr. 7, 31-40. Pollard, A. 1956. The microbiology of apple juice. Vorbericht, IV. Intern. FruchtsnftKongress, Stuttgart. Bayley, Beuel-Bonn, 21 5-220. Porchet, B. 1938. Contrihution b 1’Ctude de la levure Torulopsis pulcherrina. Ann. fermentations 4, 1-20. Raistrick, H., and Smith, G. 1933. The metabolic products of Byssochlamis fulva Olliver and Smith. Biochem. J . 27, 1814-1819. Rees, M. 1870. “Botanische Untersuchungen iiher Alkoholgarungspilze.” Leipzig. Rentschler, H., and Tanner, H. 1951. Das Bitterwerden der Rotweine. Beitrag zur Kenntnis des Vorkommens von Acrolein in Getranken und seine Beziehung zum Bittenverden der Weine. Mitt. Gebiete Lebensm. u.Hyg. 42, 463-475. Rentschler, H., and Tanner, H. 1954. Ueber die Zusammensetzung der Fruchtsauren von schweizerischen Ohstsaften. I. Die Fruchtsauren der schweizerischen Mostapfelsafte. Mitt.Gebiete Lebensm. u. Hyg. 45, 142-158. Rentschler, H., and Tanner, H. 1955. Ueber den Nachweis von Gluconsaure in Weinen aus edelfaulen Trauben. Mitt. Gebiete Lebensm. u. Hyg. 46, 200-208.

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RibBreau-Gayon, J., Peynaud, E., and Lafourcade, S. 1952. Formation d’inhibiteurs et d’activateurs de la fermentation alcoolique par divers moisissures. Compt. rend. 234, 251-253. RibBreau-Gayon, J., Peynaud, E., Lafourcade, S., and Charpentih, Y. 1955. Recherches biochimiques sur les cultures de Botrytis cinerea. Bull. SOC. chirn. biol. 37, 10561076. Rippel-Baldes, A. 1952. “Grundriss der Mikrobiologie,” 2nd ed. Springer, Berlin. Romwalter, A., and von Kiraly, A. 1939. Hefearten und Bakterien in Friichten. Arch. Mikrobiol. 10, 87-91. Ruyle, E. H., Pearce, W. E., and Hays, G. L. 1946. Prevention of mold in kettled blueberries in Nr.10 cans. Food Research 11, 274-279. Sakaguchi, K., and Tada, S. 1940. On the formation of succinic acid by Bacterium succinicum. Zentr. Bakteriol. Parasitenk. Abt. I I . 101, 341-354. Sakaguchi, K., Takahashi, H., and Nomura, M. 1952. Decomposition of citrate and tartrate by bacteria. Ann. Rept. Res. Committee Appl. Art. Radioact. Isotopes Japan 2/I 54-61. Schanderl, H. 1950a. Ueber das Studium der Chondriosomen pflanzlicher Zellen intra vitam. Der Ziichter 20, 65-76. Schanderl, H. 1950b. “Die Mikrobiologie des Weines,” 110. Ulmer, Stuttgart. Schanderl, H. 1951. Ueber die natiirliche und kiinstIiche Verwandlung von Schimmelpilzen und Hefen in Bakterien. Mikroskopie 6, 146157. Schanderl, H. 1952a. Ueber die Isolierung von Bakterien aus normalem Pflanzengewebe und ihre vermutliche Herkunft. Ber. h u t . botan. Ges. 64, 35-36. Schanderl, H. 1952b. Ueber die Wirkung der schwefligen Saure auf Schimmelpilze, Hefen und Bakterien. Rhein. Weinztg. 2, 181-183. Schanderl, H. 1953. Methoden zur Auslljsung spontaner Bakterienentwicklung in normalen Pflanzengeweben bzw. Pflanzenzellen. Ber. deut. botan. Ges. 64, 79-87. Schanderl, H., and Draczynski, M. 1952. Brettanomyces, eine lastige Hefegattung im flaschenvergorenen Schaumweim. Dtsch. Wein-Ztg. Wein u. Rebe 20, 462-463. Schmitthenner, F. 1949. Die Wirkung der Kohlensaure auf Hefen und Bakterien. Wein-Wiss., Beih. Fachz. deut. Weinbau 3, 147-187. Scholz, U. 1943. Ueber die keimtotende Wirkung von Sussmosten. Obst- u. Gemiiseuerw. I d . B14, 106112. Shifrine, M. 1956. The association of yeasts with certain bark beetles. Mycologin 48, 41-55. Smart, H. F. 1939a. Further studies on behaviour of microorganisms in frozen cultivated blueberries. Food Research 4, 287-292. Smart, H. F. 1939b. Microbiological studies on commercial packs of frozen fruits and vegetables. Food Research 4, 293-298. Smock, R. M., and Neubert, A. M. 1950. “Apples and apple products.” Interscience, New York. Spencer, F. T., and Sallans, H. R. 1956. Production polyhydric alcohols by osmophilic yeasts. Can. J. Microbiol. 2, 7%79. Spiegelberg, C. H. 1940. Some factors in the spoilage of acid canned fruit. Food Research 5, 439-455. Stadtman, T. C., Vaughn, R. H., and Marsh, G. L. 1945. Decomposition of tartrates by some carnmon fungi. J. Bacteriol. 50, 692-700. Stalder, L. 1953. Untersuchungen iiber Graufaule (Botrytis cirierea Pers.) an Trauben. I. Mitteilung. Phytopathol. 2. 20, 315-344. Stalder, L. 1954. Untersuchungen iiber die Graufaule (Botrytis cinerea Pers.) an

MICROORGANISMS IN N O N C I T R U S J U I C E S

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Trauben. 11. Ueber den Zucker- und Saureverbrauch des Pilzes und die Wirkung einiger Nihrstoffe auf das Wachstum. Phytopathol. 2. 22, 345-380. Stapp, C. 1951. Zur Frage der Regeneration von Chondriosomen zu Bakterien. BioZ. Zentr. 70, 398417. Stapp, C. 1952. Konnen sich Chondriosomen (Zellbestandteile) in Bakterien umwandeln? Umschau 7. Tanner, F. W. 194.4.. “Microbiology of food,” 2nd ed. Garrard Press, Champaign, Illinois. Tanner, H., and Rentschler, H. 1954. Ueber die Zusammensetzung der Fruchtsauren von schweizerischen Obstsaften. 11. Die Fruchtsauren der schweizerischen Mostbirnensafte. Mitt. Gebiete Lebensm u. Hyg. 45, 305-311. Tressler, D. K., and Joslyn, M. A. 1954. The chemistry and technology of fruit and vegetable juice production. Avi, New York. Tressler, D. K., and Pederson, C. S. 1936. Preservation of grape juice. 11. Factors controlling the rate of deterioration of bottled Concord Juice. Food Research 1, 87-97. Ulrich, R. 1952. “La vie des fruits.” Masson, Paris. Vas, K., and Ingram, M. 1949. Preservation of fruit juices with less SOz. Food Manuf. 24, 41-16. Vaughn, R. H. 1955. Bacterial spoilage of wines with special reference to CaIifornia conditions. Advances in Food Research 6, 67-108. Vaughn, R. H., and Marsh, G. L. 1943a. Bacterial decomposition of crude calcium tartrate during the process of recovery from grape residues. J . Bacteriol. 45, 35-36. Vaughn, R. H., and Marsh, G. L. 1943b. Microbial decomposition of tartrates. Wines and Vines 24, 2 6 2 9 . Vaughn, R. H., and Tchelistcheff, A. 1957. Studies on the malolactic fermentation of California table wines. I. An introduction to the problem. Am. J . EnoZ. 8, 7479. Vaughn, R. H., Marsh, G. L., Stadtman, T. C., and Cantino, B. C. 1946. Decomposition of tartrates by the coliform bacteria. J . Bacteriol. 52, 311-325. von Schelhorn, M. 1950a. Untersuchungen uber den Verderb wasserarmer Lebensmittel durch osmophile Mikroorganismen. I. Verderb von Lebensmitteln durch osmophile Hefen. 2. Lebensm. Unters. u.- Forsch. 91, 117-124. von Schelhorn, M. 1950b. Ueberblick uber den Stand der Forschung auf dern Gebiet der Venvendung von SOz als Konservierungsmittel fur LebensmitteI. Ind. Obstu. Gemiiseuerw. 35, 181-182. von Schelhorn, M. 1951a. Control of microorganisms causing spoilage in fruit and vegetable products. Advances in Food Research 3,429-482. von Schelhorn, M. 1951b. Untersuchungen uber Konservierungsmittel. VIII. Wirksamkeit und Wirkungsbereich der schwefligen Saure. Deut. Lebensrn-Rundschau 47, 170-173. von Schelhorn, M. 1954. Wirkung von SO, auf den Hefepilz Mucor racemosus. Ind. Obst- u. Gemiiseverw. 39, 57. von Schelhorn, M. 1956. Untersuchungen iiber das Absterben von Hefen und anderen Mikroorganismen i n Substraten von hohem NaC1-bzw. Zuckergehalt. Arch. Mikrobiol. 25, 39-57. Wallace, G. I., and Park, S. E. 1953a. Microbiology of frozen Foods. IV. Longevity of certain pathogenic bacteria in frozen cherries and frozen cherry juice. J . Infectious Diseases 52, 146-149.

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LUTHI

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THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS BY J. F. KEFFORD Commonwealth Scientific and Industrial Research Organization, Division of Food Preseruation and Transport, Homebush, N e w Souih W a l e s , Australia Page I. Introduction . . . . . . . . . . . . . . .............................. 286 A. Taxonomical Considerati B. Morphological Considerations ............ 11. General Composition of Ci ..................... 289 A. Variability in Composition within Individual Fruits. . . . . . . . . . . . . . . 291 B. Effects of Genetic Factors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 C. Effects of Rootstock .............................. . . . . . . . . . . . 292 D. Effects of Maturity, ....... . . . . . . . . . . . . . 298

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

V. Vitamins

302

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

A. Ascorbic Acid.. . . . . . . . . . . . . . B. Other Vitamins. .... ........................................ .............

311 313

C. Horticultural Interest. . . . . . A. Factors Affecting the Nitrogen Content of Citrus Fruits. C. Nitrogen Compounds Containing Sulfur. . . . . . . . . . . . . . . . . . . . . . . . . . . D. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Analytical Applications. . ............. VIII. Enzymes . . . . . . . . . . . . . . . . . ............. A. Pectolyzing Enzymes. . . . . . . . . . . . . . . . . . . . . . .. ...... B. Acetylesterase . . . . . . . . . . . . ............................. C. Phosphatase . . . . . . . . . . . . ............. D. Glutamic Acid Decarboxylase. . . . . . ..................... E. Peroxidase . . . . . . . . . . . . ............. 285

3 18 319 319 320 320 322 322 323 323

286

J. F. KEFFORD

Page

F. Other Enzymes. . . . . . . . . . . . . . . . . .................. IX. Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... A. Oranges and Tangerines.. . . . . . . . . . . . . . . . . ................ B. Grapefruit .... ................ C. Citrus Fruits Containing Noncarotenoid Pigments. . . . . . . . . . . . . . . . . .

323 324 324 328 328

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

330

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

332

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

346

XIV. Limonoid Bitter Principles, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Some General Observations ................................ B. Chemistry of Limonin. . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other Bitter Principles.. . . . . . . . . . . . . . . . . . . . . . . . XV. Research Needs.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....................

348 349

B. Distribution of Citrus Flavonoids. .

355

I. INTRODUCTION

Citrus fruits may fairly be regarded as the most important fruit crop directly consumed as human food; the world crop, approaching 18 million tons in 1956-57 (Anonymous, 1957), is second only to that of grapes. The high acceptability of the citrus fruits is due to their attractive colors and distinctive flavors, and to the fact that they are the richest common sources of vitamin C. The large consumption as fresh fruit is now exceeded by the quantities of citrus fruits supplied as raw materials to branches of the food industry producing pasteurized, frozen, and preservatized juices and concentrates, cordials and soft drinks, canned and frozen citrus segments, marmalades, candied peels, and flavoring oils. By-products utilization in these industries has reached a high level of technical ingenuity and economic efficiency. T o serve the needs of the food processing and by-products industries for basic chemical information, an impressive body of knowledge on the composition of citrus fruits has been accumulated by workers in many countries. The literature on this subject up to 1947 has been comprehensively reviewed by Braverman (1949). The present review aims to

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

287

embrace the subsequent ten-year period--1948 to 1957; related reviews which partially cover this period were prepared by Kefford (1955) and the Agricultural Research Service (1956). Citrus products technology has been excluded from the review, but an attempt is made to indicate the technological relevance of the various chemical constituents of citrus fruits. CONSIDERATIONS A. TAXONOMICAL

In a review which discusses the chemical composition of natural plant products, it is desirable to identify as accurately as possible the particular fruit under discussion. Unfortunately, there is a lack of agreement among taxonomists about the botanical nomenclature of Citrus species, and the matter is further complicated by the existence of many mutants, hybrids, and horticultural varieties. A scheme is set out in Table I, following Swingle (1948) and Webber (1948), which attempts an orderly classification of all the citrus fruits specifically mentioned in the review; it may assist the reader to follow some genetic trends in chemical composition.

B. MORPHOLOGICAL CONSIDERATIONS When discussing the chemical composition of citrus fruits, it is frequently necessary to refer to component parts of the fruits, some of which are morphological structures while others are technological fractions. In order to avoid ambiguities, the main components to which reference will be made are defined as follows: (1) The flavedo, or outer peel, is a layer of tissue underlying the epidermis and containing chromoplasts and oil sacs. (2) The albedo, or inner peel, is a layer of spongy white tissue beneath the flavedo. The albedo and the core, or central axis, contain the vascular system which supplies the fruit with water and nutrients. ( 3 ) The peel is the flavedo and albedo together. The peel juice is the free fluid from the peel, expressed mechanically. (4) The endocarp within the peel is sometimes called the pulp and is the principal edible portion of citrus fruits. It consists of a series of segments, carpels, or locules, each of which contains a compact mass of elongated, thin-walled vesicles. ( 5 ) The juice is the cell contents from the vesicles, expressed by means of a variety of hand or mechanical devices, and usually screened. In addition to constituents in solution, the juice contains suspended chromoplasts and tissue fragments in amounts dependent on the fineness of screening. ( 6 ) The rag and pulp is the fraction screened from the juice and

TABLE I CLASSIFICATION OF CITRUSFRUITS” b

General name

Botanical name

Sweet orangesb

Citrus sinensis

Sour or bitter oranges

Citrus aurantium

Mandarins

Citrus reticulata (C. nobilis)

Grapefruits

Citrus paradisi

Pumelos

Citrus grandis (C. decumana, C. m a x i m a )

Lemons

Citrus limon

Limes

Citrus aurantifolia

Citrons

Citrus medica

Papeda Trifoliate orange

Citrus hystrix Poncirus trifoliata (C. trifoliata)

Tangors Tangelos Citranges

Varieties Normal group, e.g., Valencia Late, Shamouti (Palestine), Sathgudi (Madras, India), Malta (Punjab, India), Batavia (India), and many others Navel group Blood fruit group A m m a or Seville group Bittersweet group Bergamot group (C. bergamia) Kabusu (Japan) cyathifera (Japan) Kamala (India) Mandarin group Satsuma group (C. unshiu) Tangerine group, e.g., Dancy, Ponkan (China), Clementine (prob. hybrid) Sangtra (Nagpur, India) Coorg (India) Pale-fleshed group, e.g., Duncan, Marsh Pink-fleshed group, e.g., Ruby Red Thong Dee shaddock Pamparapana (Indian shaddock) Matheepala (India) Buntan (Japan) Eureka group Lisbon group Anomalous group (prob. hybrids), e.g., Meyer, Ponderosa, Rough Lemon (jambhiri) Mexican group Tahiti or Persian group Sweet Lime group (C. limetta) Sweet group Acid group Dabba (India) Naranja (India) abyssinica (Somaliland)

Hybrids C. reticulata X C. Temple orange sinensis Temporona(?) (Argentina) C . reticulata X C . Natsudaidai (Japan) paradisi or C. grandis Satsumelo P. trifoliata X C. sinensis Rusk, Morton, Savage

After Swingle (1948) and Wehher (1948). Throughout this review the name “orange,” used without qualification, should he understood t o mean sweet orange. a

b

288

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

289

consists of fragments of the core, the segment walls, and the juice vesicles. The rag and pulp fraction is generally combined with the peel for by-products manufacture, and together they amount to more than half the weight of the original fruit. The profitable utilization of these fractions is essential to the economic health of the citrus processing industry. Some information collected in Florida on the approximate quantitative relationships between the component parts of citrus fruits and also on the solids contents of the respective components is set out in Table 11. Analogous information, including determinations of additional constituents, has been tabulated by Money and Christian (1950) and Money et d.(1958). II. GENERAL COMPOSITION OF CITRUS FRUITS

The palatability of citrus fruits, in particular oranges, mandarins, and grapefruit, depends largely upon a balance of sweetness and acidity acceptable to the human palate (Sinclair and Bartholomew, 1947; Harding, 1954). Accordingly, the ratio of the soluble solids content (in degrees Brix) to the acidity (as anhydrous citric acid per cent), designated as the Brix/acid ratio, has come to be widely accepted as a useful index of palatability in citrus fruits (Baier, 1954). Thus the chemical determinations most frequently reported on citrus fruits are the soluble solids content and the acidity, as well as the ascorbic acid content because of its obvious nutritional significance. The large volume of information that has accumulated on these three characteristics of citrus fruits will be reviewed separately, before proceeding to discuss specific groups of constituents in detail. In Table 111 are presented ranges of values for the soluble solids content, the acidity, and the ascorbic acid content of six citrus fruits. This table has no statistical foundation; it has been compiled by inspection of many published tables in the light of the reviewer’s experience. The limits of the ranges tabulated are not the most extreme values ever recorded, but it is considered that values outside these ranges should rarely be encountered. The variability in composition between and within each kind of citrus fruit that is revealed in Table I11 is influenced by many factors: by genetic factors, by rootstock, by maturity, by the position of the fruit on the tree, by field factors, and by orchard practices. Moreover, there is even variability in composition within individual fruits. All of these factors which influence variability should be taken into account when a population of citrus fruits is sampled for analysis; conversely, the validity of published analyses of citrus fruits should be assessed in the light of the sampling procedures adopted.

RELATIVEPROP~RTIONS AND SOLIDS CONTENT^

Component part Whole fruit Flavedo Albedo Juice Rag and pulp Seeds 0

b

OF

TABLE I1 COMPONENTPARTSOF lMATuRB Cmus FRUITSIN FLORIDA

Orange0 (4 var.)

Tangerineo (Dancy)

Grapefruit* (5 var.)

Shaddock* (Thong Dee)

Lemona (Meyer)

Lime. (Tahiti)

Per cent Total of whole solids (%) fruit

Per cent Total of whole solids fruit (%)

Per cent Total of whole solids (%) fruit

Per cent Total of whole solids fruit (%)

Per cent Total of whole solids fruit (%)

Per cent Total of whole solids fruit (%)

100 9-10 12-20 35-50 25-30 0-3

14-18 22-27 20-25 9.5-13 14-18 40-50

100 7-11 30-40 45-55 2

From Hendrickson and Kesterson (1954a). From Kesterson and Hendrickson (19538).

15-20 25-30

-

1@-13 15-20 33-40

100 7-8 18-27 40-55 20-28 0.5-5

12-16 19-26 15-22 7-12 12-16 30-55

100 8-9 28-31 28-34 26-32 3-4

16-18 22-25 18-21 10-11.5 14-16 44-51

100 10 12-13 40 35-40 1-2

12 16-17 13-15 8-10 11-12 30-40

100 11-12

12.5 21

44-48 40-45 0

8 15-16 -

-

-

9 q

1 1 0

TI

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

29 1

TABLE I11 RANGEOF COMPOSITIONOF JUICES

Juice Sweet orange Sour orange Mandarin Grapefruit Lemon Lime

FROM

MATURE CITRUSFRUITS’

Soluble solids content (“Brix)

Acidity as citric acid (g./lOO ml.)

Ascorbic acid content (mg./lOO ml.)

6-14 9-14 6-14 6-13 6-12 8-14

0.4-2 1-5 0.3-1.5 0.5-2.7 4-9 4-8

25-80 20-40 10-50 25-50 30-60 20-40

0 Principal sources: Baier and Stevens (1954), Bartholomew and Sinclair (1951), Benk (1956, 1958), Burdick (1954), Money and Christian (lQ50).Von Loesecke (1954).

It is relevant to point out here that citrus varieties and horticultural treatments are frequently compared on the basis of the percentage concentrations ,of specific constituents in the juice or edible portion of the fruits. More significant, however, for the citrus grower and processor are comparisons based on: (1) The yield of a specific constituent, usually soluble solids, per ton of fruit. This yield depends upon the juice content of the fruit as well as upon the soluble solids content of the juice, or (2) The yield of a specific constituent per acre. This yield depends in addition upon the yield of fruit in tons per acre.

A. VARIABILITY IN COMPOSITION WITHIN INDIVIDUAL FRUITS It has long been known that gradients in the concentration of many chemical constituents exist within individual citrus fruits as they do in other fruits. Thus the juice from individual segments may differ considerably in composition, and the stylar or blossom half of mature fruits has a higher concentration of soluble constituents than the calyx or stem half (Haas and Klotz, 1935; Bartholomew and Sinclair, 1941). Further, the juice from the center of oranges and mandarins has a lower soluble solids content and a higher acidity than juice from the outer portions of the endocarp (Blondel, 1952; Hall, 1955).Accordingly, the minimum sugar content and the maximum acidity within an orange are found inwards from the stem end, and the maximum sugar content and minimum acidity in the outer endocarp at the stylar end. Again, the navel portion of Navel oranges has a higher soluble solids content and a lower acidity, and hence a higher Brix/acid ratio, than the rest of the fruit (Dupaigne, 1953). It is possible that the distribution of the

292

J . F. KEFFORD

main vascular system leads to a preferential supply of nutrients to the outer endocarp and the stylar end of citrus fruits. B. EFFECTS OF GENETICFACTORS The differences in appearance and flavor that distinguish the different kinds and varieties of citrus are, fundamentally, differences in chemical composition governed by genetic factors. However, although it is possible to define chemically the gross differences, such as those between oranges and lemons, the more subtle differences between horticultural varieties may not at all be reflected in the general chemical composition. Moreover, the effects of variety on composition are frequently overshadowed by the effects of the many field factors which are discussed below. It is not profitable, therefore, to attempt to summarize the copious information that is available on the composition of citrus varieties from different areas of production throughout the world. Sources of recent information on this aspect are listed in Table IV. As an example of a specific investigation of varietal differences, the work of Cohen (1956) on the ascorbic acid content of Palestine citrus fruits may be cited. This study embraced 29 varieties of oranges, 9 of grapefruit, 3 of lemons, and 21 of mandarins. The oranges were highest in ascorbic acid content and the mandarins lowest. The fact that taxonomically related varieties were in many cases similar in ascorbic acid content led Cohen to suggest that some taxonomic problems might be resolved on the basis of ascorbic acid content. Thus, the high ascorbic acid content (65 mg.%) of the Clementine mandarin provides evidence that it is probably a mandarin x orange hybrid (cf., Webber, 1948), while the low ascorbic acid content of the Meyer lemon (32 mg.%) (cf., Deaker, 1952) suggests that it is a lemon X mandarin hybrid (cf ., Swingle, 1948). C. EFFECTSOF ROOTSTOCK While genetic factors have a predominating effect in determining the chemical composition of citrus fruits, the rootstock on which a scion variety is grafted also exercises a profound influence. Sinclair and Bartholomew ( 1944) reviewed comprehensively the knowledge available at that time on the influence of rootstocks on the composition of citrus fruits, and they also reported the results of a wellplanned investigation under California conditions, involving 14 rootstocks for oranges and 13 for grapefruit. Although the statistical treatment of the results was inadequate, the principal conclusions are not disputed; indeed they have been repeatedly confirmed by other workers.

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

293

TABLE IV INFORMATION ON THE COMPOSITION OF CITRUSFRUITS Region Algeria Argentina Brazil California

China Costa Rica

El Salvador Florida

Varieties Oranges, mandarins Temporona orange Oranges (6 var.), limes (3 var.) Oranges, grapefruit Grapefruit Lemons Oranges, pumelos Tangerines, grapefruit, limes Sweet and sour oranges, mandarins, lemons, limes, pumelos Sweet and sour oranges, tangerines, grapefruit, limes Oranges (10 var.)

Tangerines Temple oranges Grapefruit Oranges, lemons, limes Mandarins, grapefruit, lemons, limes Oranges (2 var.) Israel Bitter oranges Oranges (21 var.) Italy Oranges (5 blood var.), lemons (2 var.) Oranges Bergamot oranges Mandarins (21 var.) Japan Natsudaidai Montenegro Oranges, tangerines, lemons Oranges, grapefruit Morocco New Zealand Lemons (2 var.) Oranges, tangerines, grapefruit, Nicaragua lemons, limes Tangerines, limes Peru Guatamala Hondiiras

Phillipine Is. Pumelos C . hystrix var. abyssinica Somaliland Oranges (9 var.), tangerines, Spain lemons, grapefruit (3 var.), bergamot oranges Oranges (23 var.) Oranges, grapefruit Surinam Imported oranges, grapefruit, Sweden mandarins

References Blonde1 (1952) Di Giacomo and Lo Presto (1956) Burger (1955) Sinclair and Bartholomew (1944) Rygg and Getty (1955) Bartholomew and Sinclair (1951) Lu and Chou (1955) Munsell et al. (1950d) Van der Laats (1954) Munsell et al. (1950b) Harding et al. (1945); Swift and Veldhuis (1957); Westbrook and Stenstrom (1957) Harding and Sunday (1949) Harding and Sunday (1953) Harding and Fisher (1945); Stenstrom and Westbrook (1956) Munsell et al. (1950a) Munsell et al. (1949) Samisch and Cohen (1949) Ephraim and Monselise (1955,1957) Fratoni and Spadoni (1951) Pennisi (1952) Lisanti and Catalano (1956) Cuzzocrea and Centonze (1951) Inagltki (1946, 1953) Nomura and Matsunaga (1952) GuguiSevi6-Ristid (1954) Patron and Swinzow (1956) Deaker (1952) Munsell et al. (1950~) Cordova (1953); Becerra de la Flor (1955) Francia (1954) Sacco (1957) Fuertes Polo and Roy0 Iranzo (1954) Garcia Alvarez et al. (1957) Spoon et al. (1951) Hellstrom (1955)

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J. F. KEFFORD

The highest concentrations of chemical constituents in the juice were, in general, found in fruit from trees grown on trifoliate orange and citrange rootstocks, and the lowest concentrations were found in fruit grown on rough lemon or sweet lime rootstocks. It is interesting to note that in another investigation (Haas, 1948), although trifoliate orange stock produced high contents of sugars in the endbcarp of Valencia oranges, it gave the lowest sugar content in the peel, among the stocks tested. In Florida experience also (Harding et al., 1945; Harding and Fisher, 1945; Harding and Wadley, 1945; Harding and Sunday, 1949, 1953), rough lemon as a stock for all varieties of citrus produces fruit which are low in soluble solids, acid, and ascorbic acid content, while citrange and sour orange stocks produce fruit which are consistently high in soluble constituents (Cook et al., 1952). Nevertheless, rough lemon rootstocks give the highest yields of soluble solids per acre because they promote vigorous growth and produce more fruit. Samisch and Cohen (1949) have stated that rootstock is the most important grove factor affecting the composition of citrus fruits under normal cultural conditions in Israel. Shamouti oranges on sour orange stocks contained 10% more sugars and 18% more citric acid than fruit grown on sweet lime stocks. In South Africa also, Valencia oranges and Marsh grapefruit on rough lemon rootstocks had lower soluble solids contents than fruit on the other stocks tested, but differences in acidity were slight and variable (Marloth, 1950, 1958). Among 6 rootstocks tested for lemons, trifoliate orange gave the highest soluble solids content, while rough lemon gave the lowest soluble solids content and acidity (Bartholomew and Sinclair, 1951; Batchelor and Bitters, 1954). The citric acid yields from lemons on 8 different rootstock-scion combinations were reported by Goodall and Bitters (1958). Marked differences in the palatability of juices from Washington Navel oranges grown on several rootstocks were observed by Marsh (1953), but he could find no differences in chemical composition that would account for the organoleptic differences. The most notable effect was that of rootstock on bitterness; this is discussed further in Section

XN.

D. EFFECTS OF MATURITY Investigations during the period under review have confirmed earlier knowledge of maturity trends in the chemical composition of citrus fruits.

THE C H E M I C A L C O N S T I T U E N T S O F CITRUS F R U I T S

295

1. Oranges, Mandarins, and Grapefruit During the maturation of oranges, mandarins, and grapefruit, the major changes in composition up to the optimal harvest time are slow increases in the concentrations of soluble solids, sucrose, and reducing sugars, and a steady fall in acidity. Hence the Brix/acid ratio increases with advancing maturity. The use of this ratio as an index of palatability has already been mentioned; it is also widely accepted as an index of maturity. A statistical study by Rebour (1951) indicated that the ratio of soluble solids content to acidity gave a more reliable measure of the maturity of oranges and mandarins than either value alone. Wedding and Horspool (1955) examined several chemical indices of maturity and found that the Brix/acid ratio was the only one which showed a relatively smooth trend with maturity. The cloud/acid ratio and the y-aminobutyric acid/acid ratio showed large sample-to-sample variations. The pattern of development described has been clearly demonstrated in several kinds of citrus fruits from a number of regions: e.g., California oranges and grapefruit (Sinclair and Bartholomew, 1944); Florida oranges (Harding et al.,1945; Westbrook and Stenstrom, 1957; Swift and Veldhuis, 1957) ; grapefruit (Harding and Fisher, 1945); tangerines (Harding and Sunday, 1949); and Temple oranges (Harding and Sunday, 1953); and Shamouti and Valencia oranges in Palestine (Samisch and Cohen, 1949). In some other studies of maturity trends, the changes in composition have taken a slightly different form. The decrease in acidity is still a definite and consistent trend, but the soluble solids content increases to a maximum and then levels out or declines slowly with advancing maturity. This pattern of change has been reported most frequently in grapefruit, e.g., in California and Arizona (Rygg and Getty, 1955), in Texas (Krezdorn and Cain, 1952; Burdick, 1954; Lime et al., 1954, 1956), and in Florida (Stenstrom and Westbrook, 1956). However, Marloth (1950) has traced a distinct maximum in soluble solids content during the maturation of Valencia oranges in South Africa. Bain (1958), working with Valencias in Australia, related trends in chemical composition to morphological and anatomical changes in the developing fruit throughout the whole period of its growth on the tree. During the later part of the cell enlargement period, the soluble solids and sugar contents increased and then remained steady through the maturation period. The acidity decreased during the later part of the cell enlargement stage and continued to decrease more slowly through the maturation period.

296

J. F. KEFFORD

2. Lemons and Limes Chemical changes during the maturation of lemons and limes are broadly the reverse of those in oranges and grapefruit. The soluble solids content in the juice remains almost constant from an early stage in the growth of the fruit until maturity, but the acidity increases greatly in total amount and in concentration, and there is a corresponding decrease in sugars and other soluble solids (Bartholomew and Sinclair, 1951). It is noteworthy that these changes in the composition of lemons may continue during storage (“curing”) after removal from the tree.

3 . Ascorbic Acid Content Trends in ascorbic acid content with advancing maturity are not so consistent between citrus varieties. A gradual decrease has been generally observed in grapefruit, tangerines, and Valencia oranges, but other orange varieties may maintain an approximately constant ascorbic acid content throughout the maturation period (Harding et al., 1945; Harding and Sunday, 1953). On the other hand, Lisanti and Catalan0 (1956) reported that the ascorbic acid content of oranges of the “Ovaletto di Calabria” variety increased as the fruit matured. Samisch and Cohen (1949) found no definite trend with maturity in the ascorbic acid content of Shamouti oranges in Palestine. Analyses of Australian orange juices (Anonymous, 1947) indicated a steady decline in the ascorbic acid content of Valencia juices throughout the season, but no decline in Navel juices.

E. EFFECTS OF POSITION ON

THE

TREE

A very thorough investigation of the variation in composition of individual oranges on one tree was undertaken by Sites and Reitz (1949, 1950a,b), who analyzed approximately 1800 fruits from a single Valencia orange tree, 28 years old, in Florida. All of the fruit was harvested within 6 days and analyzed within 15 days. As Table V shows, the individual fruits varied widely in composition, and the variation was related to the position of each fruit on the tree. The soluble solids content and ascorbic acid content varied with the height of the fruit on the tree, with the amount of shading by the foliage, and with the orientation of the fruit on the tree. Thus the highest contents of soluble solids and ascorbic acid were found in fruit towards the top of the tree outside the leaf canopy, and the lowest contents in fruit inside the canopy. In addition, fruit on the northeast sector of the tree, which received less incident sunlight, showed lower

T H E C H E M I C A L CONSTITUENTS O F CITRUS F R U I T S

29 7

values than fruit in the other sectors. In the Southern Hemisphere, fruit on the south side of the tree tends to be low in soluble constituents (Kefford, 1952). Throughout the tree, therefore, fruit developing under conditions of lower light intensity showed lower concentrations of soluble solids and ascorbic acid. The juice content in individual fruits was not related to position on the tree, nor was the acidity, except in the northeast sector where lower values were recorded. Similar observations by Winston and Miller (1948), extending over 6 varieties of oranges, Temple oranges, and Dancy tangerines, confirmed the fact that exposure to sunlight increases soluble solids content and ascorbic acid content, but has no consistent effect on acidity. On the other hand, Randhawa and Dinsa (1947), studying Valencia TABLE V RANGES OF COMPOSITION OF VALENCIA ORANGUFROM ONE TREE" Analysis

Soluhle solids content ("Brix, by refractometer) Acid content (as citric acid, g./100 9 . ) Brix/acid ratio Ascorbic acid content (mg./100 ml.) Juice content (g./lOO g . )

Minimum

Maxinium

Average

5.90 0.50 4.8 18.2 32.7

13.50 1.39 21 .o 59.6 65.8

10.24 0.885 11.56 37.1 49.15

From Sites and Reitz (1949, 1950a,b).

oranges in the Punjab, were unable to establish any significant effects of aspect, exposure, or height on soluble solids content. The Brix/acid ratio, however, was higher in exposed fruit and fruit on the upper half of the tree. An analogous study of the variation in mineral composition between individual fruits on a single Valencia tree was undertaken by Koo and Sites ( 1956) ; see Section VI. Some knowledge of the basic processes responsible for the effects of light on the composition of citrus fruits is provided by the studies of Cohen (1953) on Shamouti oranges in Israel. In conformity with earlier observations, Cohen found that the ascorbic acid content in both juice and peel was higher in exposed fruit, and was also higher on the scalded side of sun-scalded fruit than on the shaded side. In fruit permitted to develop in the absence of light, there was about 10% less ascorbic acid in the juice and 60% less in the peel. Further, exclusion of light from the adjacent leaves caused a marked decrease in the ascorbic acid content of the peel. Partial defoliation also decreased the ascorbic acid and sugar contents in the peel, and to a less extent in the juice.

298

J. F. KEFFORD

I n explanation of these observations, Cohen put forward the hypothesis that ascorbic acid is formed in the peel of citrus fruits by the action of light on assimilation products transferred from the leaves, and then passes from the peel to the endocarp. Most of the sugars in the fruit are derived from the leaves, but small amounts are synthesized in the peel. The ascorbic acid content in the fruit is therefore influenced by the amount of light received by both the fruit itself and the leaves which supply it with nutrients. Similar principles were applied by Sinclair and Bartholomew (1944) to account for some regional differences in the composition of citrus fruits. They attributed the higher contents of soluble solids and sugars in California inland fruit, compared with coastal fruit, to greater exposure to sunlight and higher mean temperatures, which promoted photosynthetic activity and led to increased accumulation of soluble carbohydrates.

F. EFFECTS OF FRUIT SIZE Another factor contributing to variability in composition between individual fruits is the effect of fruit size. Analyses reported by Harding and Lewis (1941) and Sites and Camp (1955) demonstrate a consistent decline in soluble solids, acid, and ascorbic acid contents with increasing fruit size in Florida oranges. Similarly in California oranges (Sinclair and Bartholomew, 1944) and Shamouti oranges (Samisch and Cohen, 1949) there was a n inverse relationship between fruit size and the concentration of sugars and acid. Again, Long et al. (1957) found that small fruit of Marsh and Duncan grapefruit had higher concentrations of ascorbic acid than larger fruit.

G. EFFECTS OF THE NUTRIENT STATUSOF THE TREE A large number of papers have appeared reporting investigations on the effects of fertilization practices on the general composition of citrus fruits. From the accumulated evidence, this general conclusion may fairly be drawn: For the production of fruit with desired chemical characteristics there are evidently optimal levels for the status of the tree, with respect to major and minor nutrients, but at present these levels are not clearly defined.

I, Nitrogen Status Although the supply of nitrogen to citrus trees has considerable influence on the yield of fruit, it appears to have only small effects on the composition of the fruit. For instance, Reuther et al. (1957) conducted trials with Valencia oranges in Florida extending over eight seasons

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

299

and involving two rates of nitrogen fertilization (applied according to three timing regimes and two N-P-K ratios). But they could establish no effects of commercial significance on soluble solids, acid, or ascorbic acid content. Similarly, Frith (1952) found no significant effects of nitrogen status on the soluble solids content of Australian oranges, while any effects on acidity were very complex. Evidence was reported by Sites et d. (1955) that the soluble solids content and the acidity of fruit from trees receiving nitrogen as ammonium sulfate or nitrate was higher than in those receiving nitrogen in other forms. An unfavorable effect of high nitrogen status on ascorbic acid content appears to be well established (Reuther and Smith, 1951; Sites et al., 1955), and an inverse correlation between ascorbic acid content and nitrogen content in the juice has been observed in grapefruit (Jones et al., 1944) and Navel oranges (Jones and Parker, 1947).

2. Phosphorus Status The effect of phosphorus fertilization on the chemical composition of citrus fruits depends upon the initial phosphorus status of the tree. In 1948, Reuther et d.reported that heavy applications of phosphate lowered the soluble solids, acid, and ascorbic acid content of Florida oranges. Similarly, Chapman and Rayner (1951) found that a high phosphorus status lowered the acidity and ascorbic acid content in Navel oranges, but phosphorus deficiency tended to produce higher acidity. Negative correlations between the phosphorus content and both the acidity and the ascorbic acid content in orange juice were established by Jones and Parker (1951). On the other hand, Bouma (1956) found significant positive correlations between leaf phosphorus content and both juice content and acidity in Navel oranges in Australia. With California Valencias of low phosphorus status, Embleton et at. (1956) observed that phosphate applications increased the soluble solids content of the juice but that as the phosphorus status of the trees improved, phosphate applications decreased the soluble solids content and tended to decrease acidity. The over-all effect of phosphate fertilization, however, was to increase the yield of soluble solids per ton of fruit since the juice content of the fruit was increased.

3 . Potassium Status In Florida experience, low potassium status leads to low soluble solids, acid, and ascorbic acid contents in citrus fruits (Sites and Camp, 1955; Harding et al., 1958), but potassium in excess also reduces the soluble solids content (Reuther and Smith, 1951; Sites and Camp,

300

J. F. KEFFORD

1955). With California Navel and Valencia oranges, Embleton et al. (1956) found that potassium applications tended to increase acidity and ascorbic acid content, and Jones and Parker (1951) established a positive correlation between the acidity and the potassium content of the juice. 4 . Minor Nutrients According to Sites and Camp (1955), an inadequate supply of the minor elements, magnesium, zinc, manganese, copper and iron, tends to lower the soluble solids, acid, and ascorbic acid contents of citrus fruits. Reuther and Smith (1951), however, reported that the rate of magnesium fertilization did not appreciably affect fruit quality. A high boron status consistently lowered the acidity and the ascorbic acid content in oranges and grapefruit without affecting the soluble solids content significantly (Smith, 1955).

H. EFFECTS OF HORTICULTURAL SPRAYS Pesticidal and hormonal sprays applied to citrus trees may significantly influence the composition of the fruit. 1. Oil Sprays

It is now well established that scalicidal oil sprays depress the soluble solids content, acidity, and ascorbic acid content of oranges and grapefruit (Stofberg and Anderson, 1949), but that the magnitude of the effect depends upon the timing of the oil treatment (Riehl et al., 1956). The detrimental effect on composition is greatest when the oil is applied during fruit development and least when it is applied as the fruit is approaching maturity. Riehl et al. (1957) were unable to establish any significant effects of an oil spray program on the acid, soluble solids, and juice content of lemons. There have been many comparative trials of oil sprays against the phosphatic scalicides, parathion and malathion, and it has been demonstrated conclusively that the latter substances do not adversely affect the composition of oranges (Harding, 1953; Taylor et aZ., 1956) or grapefruit (Bartholomew et d,, 1951; Thompson et al., 1951; Thompson and Deszyck, 1957). 2. Lead Arsenate and Copper Sprays Lead arsenate sprays, originally used for pesticidal purposes, are now applied in Florida because of a specific effect on the acidity of grapefruit. I n the ripe fruit, the total acidity is decreased about 25% as compared with that of untreated fruit. The soluble solids content

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

301

is not greatly affected (Harding and Fisher, 1945), but the ascorbic acid content may be increased (Deszyck and Sites, 1954, 1955). When applied to grapefruit in California and Arizona, lead arsenate had only a slight effect on composition (Rygg and Getty, 1955). I n contrast to lead arsenate sprays, copper fungicidal sprays increase the acidity of grapefruit and partly counteract the effects of lead arsenate sprays when both are applied together (Deszyck et d., 1952).

3 . Hormonal Sprays Applications of 2,4-D to prevent fruit drop of grapefruit and lemons had no important effect on the composition of the fruit (Stewart and Parker, 1954; Erickson and Haas, 1956), but lemons sprayed with 2,4,5-T showed a lower juice content, a lower acidity, and a higher reducing sugar content than untreated fruit. Gibberellin sprays applied to improve fruit set did not affect the content of juice, soluble solids, or acid in limes, lemons, and Navel oranges, but treatment of Navel oranges, when almost fully developed, increased the juice and ascorbic acid contents without affecting the soluble solids content and the Brix/acid ratio (Hield et al., 1958; Coggins and Hield, 1958).

I. EFFECTSOF MISCELLANEOUS FACTORS I . Moisture Status Differences in fruit composition which have been observed between trees grown under conditions of high- and low-soil-moisture status can reasonably be attributed to dilution effects. Thus, trees receiving ample soil moisture continuously tend to produce fruit with a high juice content but with low soluble solids and acid concentrations in the juice. On the other hand, moisture stress tends to increase soluble solids content and acidity, although the magnitude of the effect varies according to the stage in the development of the fruit at the time of stress (Sites et al., 1951; Hilgeman, 1951).

2. Frost Exposure of citrus trees to low temperatures may have major effects on the health of the trees and may also interfere with normal maturation processes so that the composition of the fruit is affected. Thus, frosted oranges and g r a p e h i t generally have a lower juice content than normal fruit, and the soluble solids, sugar, and acid contents of the juice are also lower (Bartholomew et aZ., 1950). Sugars are prob-

302

J. F. KEFFORD

ably translocated from the juice into the thickened peel of the frosted fruit (Samisch and Cohen, 1952). An unfavorable effect of low temperatures on the ascorbic acid content of citrus fruits, particularly lemons, was reported by Mirimanyan (1956). I II. CARBOHYDRATES

A. IN CITRUS JUICES

1. Sugars The soluble solids of citrus juices include reducing and nonreducing sugars. On the basis of chemical determinations by copper reduction and measurements of specific rotation, Curl and Veldhuis (1948) concluded that the principal sugars in Florida Valencia orange juice were sucrose, glucose, and fructose, which were present in the approximate proportions 2 : 1:1. These findings were in general agreement with the results of earlier work. Subsequently, McCready et al. (1950), using paper chromatography, found sucrose, glucose, and fructose to be the only sugars present in orange, grapefruit, and lemon juices. In grapefruit juice, the sucrose content was less than the reducing sugar content, while in lemon juice only a trace of sucrose was present (cf., Bartholomew and Sinclair, 1951). On the basis of the values shown in Table VI, Samisch and Cohen (1949) claimed that there was a difference in the relative proportions of monosaccharides and disaccharides in the juices of Valencia and Shamouti oranges in Palestine; this difference, however, was not tested for significance. Marsh (1953) found that the ratio of reducing sugar content to total sugar content in California Navel oranges grown on six rootstocks during two seasons was fairly consistent, but the range (0.39-0.43) which he reported does not embrace the values quoted in Table VI. Sucrose, glucose, and fructose were also the only sugars detected by Nomura and Matsubara (1952) and Ito and Sakasegawa (1952a) in mandarin and in natsudaidai juices, and by Siddappa and Bhatia (1954a,b) in Sathgudi and Coorg oranges. A report by Srivastava (1953) of the presence of maltose, as well as sucrose, glucose, and fructose, in oranges and mandarins must therefore be regarded with scepticism. During the processing and storage of citrus juices, inversion of the sucrose occurs, so that eventually only reducing sugars are found. For the rapid determination of total reducing sugars and fructose in citrus juices, Ting (1956) developed a method based on the differential rates of oxidation of glucose and fructose by ferricyanide at 55OC.

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

303

TABLE VI MONOSACCHARIDES AND DISACCHARIDES IN ORANGE JUICES“ Valericia oranges Determination Monosaccharides (%) Disaccharides (%) monosaccharides Ratio disaccharides monosaccharides Ratio total sugars 0

Shamouti oranges

March 1947

April 1948

January 1947

January 1948

4.2 4.3

3.8 4.7

4.6 3.5

3.7

0.98

0.81

1.31

1.35

0.49

0.45

0.52

0.57

5.0

From Bamisch and Cohen (1949).

2. Pectic Substances Citrus juices contain a cloud of suspended solids consisting of cell fragments and chromatophores, and this cloud is stabilized by soluble pectic substances, which also impart the consistency characteristics described by the term “body.” Recent knowledge of these components of citrus juices has been contributed largely by A. H. Rouse and C. D. Atkins working at the Florida Citrus Experiment Station, Lake Alfred, Florida. The quantity of free and suspended pulp is one of the quality characteristics included in the standards for grades of processed citrus juices (U.S. Department of Agriculture, 1954), where limits are specified in terms of the volume of compacted pulp following a standard centrifuging treatment. A more reliable measure, however, is the waterinsoluble solids content determined by filtration and drying; the alcohol-insoluble solids content may also be determined by a n analogous procedure (Rouse and Atkins, 1955a). The amount and composition of the suspended solids in citrus juices are greatly influenced by the method of juice extraction. Valencia orange juices extracted by four different methods (Atkins and Rouse, 1953) had pulp contents ranging from 8 to 12% by volume, while the water-insoluble solids content ranged from 0.060 to 0.136 g./100 g. of juice, and the total pectin content from 0.044 to 0.080 g. anhydrogalacturonic acid/100 g. juice in the same order. Rouse and Atkins (1955a) state that there is no relation between the pulp content by volume and the water-insoluble solids content, but their own results do show that, as might be expected, juices with high pulp contents tend to have high contents of water-insoluble and alcohol-

304

J. F. KEFFORD

insoluble solids, and vice versa. In orange and grapefruit juices, ranging in pulp content from 2 to 26% by volume, Rouse et d. (1954) found the range in water-insoluble solids content to be 0.52 to 3.55 g./100 g. of total solids. The juices from the seedy varieties-Pineapple oranges and Duncan grapefruit-always contained more water-insoluble solids than Valencia orange juice at the same pulp content. With increasing pulp content, the total pectin content in the same juices increased about 8-fold. The water-soluble pectin fraction, expressed as a percentage of the total pectin, decreased, and the sodium hydroxide-soluble pectin fraction increased with increasing pulp content. The water-soluble pectin fraction consists of pectic substances of relatively high methoxyl content, The pectic substances soluble in 0.05 N sodium hydroxide consist of protopectin and some calcium and magnesium pectates. By the use of an extractant with sequestering properties, e.g., sodium hexametaphosphate or ammonium oxalate, an intermediate fraction may be separated, consisting of low-methoxyl pectinates of polyvalent cations, chiefly calcium and magnesium. Methods for the fractionation of the pectic substances of citrus juices have been described by Atkins and Rouse (1953) and Rouse and Atkins (1955b). T o determine the content of pectic substances in the respective fractions, Dietz and Rouse (1953) devised a rapid colorimetric method based on the reaction of carbazole with hexuronic acids in the presence of sulfuric acid.

B. IN CITRUSPULPSAND PEELS 1. General The carbohydrates of the peel and pulp components of citrus fruits have been the subject of a series of painstaking biochemical studies by Sinclair and Crandall, working at the University of California Citrus Experiment Station, Riverside. Their findings are summarized in Table VII. The values shown may be regarded as indicating the composition of typical samples of the various components of citrus fruits, but the variability of the raw material samples analyzed was not examined statistically, and the significance of differences, e.g., between varieties, was not established. Moreover, the proportions of the constituents listed are greatly influenced by maturity, e.g., the amounts of water-soluble sugars and water-soluble pectin increase with increasing maturity. Sinclair and Crandall made a primary separation into alcoholsoluble and alcohol-insoluble solids by extracting the various component parts of the fruit with hot 80% ethanol. The alcohol-soluble solids (A.S.S.) consisted largely of sugars, mainly reducing sugars, to-

TABLE VII CARBOHYDRATE COMPOSITION OF PEELA N D PULP COMPONENTS OF CITRUSFRUITS ~

~

~

~

Soluble sugars as glucose (%

Component Whole peel Lemon, immatured Lemon, matured Grapefruit, fumigatede Grapefruit, oil-sprayede Valencia orangeAlbedo Lemon, immatured Lemon, matured Navel orangea Vesicles Valencia orange' Navel orange' Grapefruitg Pulp Navel orangef Lemon/

AlcoholAlcoholTotal soluble insoluble solids solids solids (%fresh ( % d r y (% dry wt.) wt.) A.SILb) wt.)

~

Pectin"

z z

M

Pentosans (%

Recovered (% dry

Calculated (% dry

A.I.S.)

wt.)

wt.)

5 LI

18.45 22.34 18.57 17.86

>

-

43.82 40.00 66.31 67.35 57.31

60.29 54.00 60.76 61.28 55.40

56.18 60.00 33.69 32.65 42.69

35.20 35.37 43.79 43.95 39.76

-

-

40.37 40.88 30.53

15.84 15.17 29.71

76.60 96.80 72.69

52.72 62.74 44.81

35.37 39.28 34.87

30.79 31.20 30.41

34.36 28.22 34.71

13.71 14.17

-

47.28 37.26 55.19

9.51 9.22

90.13 84.85

-

-

-

-

26.51 26.30 36.59

-

-

9.87 15.15 7.39

18.50 19.10

12.25 10.06

From Sinclair and Crandall (1953). A S S . = Alcohol-soluble solids. c A.I.S. = Alcohol-insoluble solids. From Sinclair and Crandall (1949a).

b

~

Pectin Cellulose total and hemi- Undeteras Ca pectate cellulose mined (% (% (% A.I.S.) A.1.S.c) A.I.S.)

84.44 86.37

-

-

15.56 13.63

33.48 36.47

-_

__

-

-

-

19. 78 21.22 14.75 14.34 16.97

27.31 27.31 20.24 22.90 20.12

-

18.65 24.64 15.63

24.44 31.97 19.84

10.58 12.i8 10.86

2.62 3.98 2.54

3.39 4.77 2.88

-

r o

5 2

2

2 w

9 2 c cn ~

0 ~.

-

From Sinclair and Crandall (1949b). From Sinclair and Crandall (1951). g From Sinclair and Crandall (1954).

13.62 10.64

5.21 4.07

6.04 6.61

5 ,?

6

I

w

0

m

306

J. F. KEFFORD

gether with small amounts of low-polymer galacturonides and such substances as essential oils, waxes, organic acids, and flavonoids. The alcohol-insoluble solids (A.I.S.) comprised the cell-wall components, principally pectic substances, hemicellulose, and cellulose. Starch and lignin were not present in significant amounts. Water-soluble and acidsoluble pectin fractions were determined (Table VII) , but the amount of pectin recovered was always considerably less than the pectin content computed from the yield of carbon dioxide from the A.I.S., following hydrolysis and decarboxylation in 12% hydrochloric acid. After removal of the pectin fractions, an insoluble residue remained which consisted of about one-third “hemicellulose” (extracted with 2% hydrochloric acid) and two-thirds cellulose (soluble in zinc chloridehydrochloric acid) (Sinclair and Crandall, 1949a). The undetermined fraction of the A.I.S. probably consisted of low-polymer uronides degraded from large hemicellulose molecules. Sinclair and Crandall also estimated a pentosan fraction by determining the yield of furfural from the A.I.S. and correcting for the furfural derived from the uronic acid anhydrides. By virtue of their carbohydrate content, the peel, pulp, and rag fractions of citrus fruits may be used as a bulky ration for cattle and pigs, and thus the principal by-product of the citrus processing industry in the United States is dried stock feed. Technologically, however, pectin is the most interesting of the insoluble carbohydrate constituents of citrus fruits, and it also has become a valuable by-product of citrus processing (Hull et al., 1953). 2. Pectin Among the common plant crops there are few richer sources of pectin than citrus fruits; the peels may contain 20 to 40% pectin on a dry basis. Accumulation of pectin in the cell walls of the peel is a characteristic anatomical feature in the development of citrus fruit (Bain, 1958). Orange, lemon, and grapefruit pectins, in general, contain 80 to 85% anhydrouronic acid and 8 to 14% methoxyl groups, and have an average specific rotation of +230° (McCready et al., 1951). The distribution of pectin in the component parts of several varieties of oranges, mandarins, and grapefruit grown in Florida was examined by Rouse (1953). In general, the pectin content was highest in the rag and peel fractions. Similar studies on lemons and limes (Rouse and Atkins, 1954) revealed that the pectin content in Villafranca lemons was highest in the rag, while in Persian limes the peel and rag fractions had the same pectin content on a dry solids basis. The effect of maturity in Pineapple oranges on the distribution of

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

307

pectic substances in the peel, pulp, and juice was studied by Rouse and Atkins (1953). During five months of the season, while the Brix/acid ratio increased from 10.5 to 18.8, the water-soluble and sodium hydroxide-soluble pectin contents tended to decrease and the polyphosphatesoluble pectin content to increase. The total pectin content did not change greatly until it declined in late maturity. Fuertes Polo and Roy0 Iranzo (1954) determined the pectin content of the residues from Spanish citrus fruits after juice extraction and reported the following pectin values: tangerines, 2.59%; oranges, 4.01-1 1.51%; lemons, 3.22%; grapefruit 2 . 5 4 1 % ; and bergamot oranges, 2.22% on a wet basis. Changes in the carbohydrate composition of the peel of the Japanese summer orange during maturation were studied by Shioiri et al. (1953). The total sugar content increased to 10.2% and the total pectin content to 5.6%, and then they declined. The peel of Shamouti oranges contains about 3.5% pectin, and the content of hydrolyzable polysaccharides tends to decrease as the season advances (Samisch and Cohen, 1949). Money and Christian (1950) and Money et al. (1958) have tabulated data on the pectin and total sugar contents of the peel and pulp of sweet and bitter oranges, grapefruit, and lemons. IV. ACIDS

A. IN CITRUSJUICES Organic acids are important constituents of the soluble solids of citrus juices, and in lemons and limes they become the principal soluble constituents. Citric acid, as its name implies, is the characteristic and predominant acid in citrus fruits, and it is accompanied by malic acid and some minor acids. Sinclair and his co-workers examined the titratable acids, and also the total acid radicals precipitated by lead acetate, in typical samples of California citrus fruits, and the ranges of concentrations which they observed are set out in Table VIII. Most of the acid radicals, up to 97% in lemon juice, occur as free acid. The remainder are combined with inorganic cations, probably mainly as potassium acid citrates since potassium provides 60 to 70% of the total cations (Sinclair and Eny, 1946a). Very recently, Wolf (1958) examined the acids present in alcoholic extracts of the endocarp of four kinds of citrus fruits by chromatography on ion exchange resins and silica gel, and obtained the results tabulated in Table IX. The proportions of malic acid are higher than previous workers have reported, and quinic acid is identified for the first time in citrus fruits.

308

J. F. KEFFORD

Moreover, an unidentified acid, which is evidently stronger than phosphoric acid, is claimed to be present in all four fruits, but in notably high concentration in oranges. I n addition to the acids listed, formic and acetic acids were detected in small amounts, and as many as seven fractions present in amounts less than 1% of the total acids were not identified; many of these fractions were probabIy acid esters of sugars. TABLE VIII CONCENTRATIONS OF ACIDSIN CITRUSJUICES Oranges* (wide range of maturity)

Determination

Grapefruith (mainly immature)

Lemonsc (green and yellow)

7.6-12.6 2.8-3.1 20.3-26.4 1.6-4.1 1.7-5.0 80-9 1

8.0-9.8 2.1-2.3 45.3-65.0 1.5-4.3 1.8-2.2 96-97

-

Soluble solids (%) PH Citric acid and citrates (mg./ml.) Malic acid and malates (mg./rnl.) Combined acids, as citric (mg./ml.) Free acid/total acid radicals (%) a 6 6

10 .6- 15.4 2.9-3.8 8.3-25.5 1.O-1.8 2.5-3.4 70-90

From Sinclair et al. (1945). From Sinclair and Eny (1948a) From Sinclair and Eny (1945).

TABLE IX RELATIVE PROPORTIONS OF ACIDSIN CITRUSFRUITS" Orange Acid Citric acid Malic acid Quinic acid Phosphoric acid} Unidentified acid Eapresscd

a.8

Mandarin (%)

Grapefruit

(%I

(%I

Lemon

(%I

65.6 16.2

86.1 7.6

87.2 2.1

91.8 4.9

4.1

-

1.7

0.5

12.2

2.8

7.0

2.5

per cent of total rtcids eluted from silica. gel column (Wolf, 1958).

A number of other acids have been identified as minor constituents of citrus fruits. Traces of oxalic and tartaric acids in grapefruit were reported in earlier work (Braverman, 1949). I n natsudaidai, Nomura and Takahashi (1952) found the composition of the total acids to be: citric, 72.5%; succinic, 15.0%; malic, 9.0%; tartaric, 1.0%; and oxalic, 0.5%, together with some acetic and formic acids. However, Ito and Sakasegawa (1952a) failed to find tartaric acid in Japanese mandarins, lemons, and natsudaidai. Mehlitz and Matzik (1956) found about 0.01% lactic acid in lemon, orange, and grapefruit juices and about

T H E CHEMICAL CONSTITUENTS O F CITRUS FRUITS

309

half this concentration of volatile acids, mainly acetic with some formic and two unidentified acids. Free galacturonic acid was detected in sound oranges (0.002% in whole fruit), but not in lemons, by Almendinger et al. (1954). Succinic acid was identified in lemons by Young and Biale (1956), and in bergamots by Calvarano (1958a). The bergamots also contained citric and malic acids and probably tartaric and malonic acids. Ting and Deszyck (1959) found Z-quinic acid in oranges, grapefruit, tangerines, lemons and limes, in amounts ranging from 161 to 233 mg./100 g. in the peel and from 27 to 146 mg./100 g. in the flesh. It has already been mentioned (Section II,D) that the concentrations of the acids in citrus fruits vary considerably with the maturity of the fruit. I n oranges and grapefruit, the amount of free acid per fruit increases in early growth then becomes approximately constant, but the concentration of free acid in the juice decreases by dilution as the fruit grows (Sinclair and Ramsey, 1944). The citric acid concentration shows the greatest change with advancing maturity; the concentrations of malic acid and combined acids remain more uniform. I n general, the pH of the juice increases as the fruit matures, but because citrus juices have a comparatively high buffering capacity, wide variations in titratable acidity may cause only small changes in pH (Sinclair and Bartholomew, 1947). Varma and Ramakrishnan (1956) followed the development of acidity in the fruits of Citrus acida (presumably a lime) by paper chromatographic techniques. At the earliest stages of growth, succinic acid was the principal acid, accompanied by two unidentified acids. When the fruit diameter increased to 1.5 cm., citric acid became the predominant acid with small amounts of malic acid, and the acidity increased as the fruits matured. In lemons also, the free acidity increases during maturation, and the pH decreases. Sinclair and Eny (1945) observed a drop in pH from 5.2 to 2.6 as the fruit diameter increased from 2 to 4 cm.; then the subsequent increase in free acidity caused a small additional fall in pH. As would be expected, lemon juice behaved as a citric acid-citrate buffer system (Sinclair and Eny, 1946b).

B. IN CITRUSPEELS The concentrations of organic acid radicals in citrus peels are much lower than in the juices, and the pH of the peel (5-5.5) indicates that they are present largely as salts and not as free acids. In orange, grapefruit, and lemon peel, Sinclair and Eny (1947) found about 0.03%

310

J. F. KEFFORD

citrate, 0.2% malate, and 0.1% oxalate, on a fresh weight basis. The relative concentrations in the juice are reversed, the concentrations of malate and oxalate in the peel being higher than that of citrate. Except for a trace of free acid in the peel juice, the oxalate is present as insoluble calcium oxalate, which occasionally appears as visible crystals. The low concentration of organic acids in the peel of citrus fruits has led Sinclair and Eny (1947) to suggest that the acids are not translocated from the leaves but are synthesized in the endocarp of the fruit, and this view is also held by Varma and Ramakrishnan (1956). It is further supported by the experiments of Erickson (1957, 1958), who successfully grafted developing lemon fruits from one variety to another. Sweet lemons (limes) grafted on a sour lemon tree remained low in acidity (0.52%) and high in reducing sugar content (5.2%), while sour lemons grafted on a sweet lemon tree remained high in acidity (5.2%) and low in reducing sugar content (1.5%). Evidently the site of acid synthesis is in the fruit itself and not in the leaves which nourish it. V. VITAMINS

A. ASCORBIC ACID The occurrence of ascorbic acid in citrus fruits and the many factors which influence ascorbic acid content have already been discussed in Section 11. One aspect which may be enlarged upon here is the distribution of ascorbic acid between the different parts of citrus fruits. Only about one-quarter of the ascorbic acid present in the whole fruit is contained in the juice, the principal portion entering human consumption. The remainder is present in the peel and rag fractions, and the concentration in the flavedo is considerably higher than in the albedo. Some comparative determinations of ascorbic acid in citrus fruits from various sources are tabulated in Table X. According to Iwasaki and Komatsu (1941), the peel on the stemhalf of a mandarin has a higher ascorbic acid content than the peel in the stylar-half. Naito et al. (1942), however, report exactly the oppos i t e t h a t the ascorbic acid content is higher at the stylar end. The matter is of minor practical importance, but it merits elucidation. If the peel is the main site of ascorbic acid synthesis (cf., Section II,E), then the stem-half may have a higher ascorbic acid concentration since it may receive more light radiation when the fruit is hanging on the tree. The seeds of citrus fruits contain only small amounts of ascorbic acid, e.g., 2.1 mg./lOO g. dry weight in the whole seeds of grapefruit

31 1

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

TABLEX ASCORBIC ACID CONTENTOF COMPONENT PARTS OF CITKUSFRUITS Ascorbic acid content Whole fruit (mg./ fruit)

Flavedo bg./ 100 9 . )

Albedo (mg./ 100 g.)

Rag (ing./ 100 9.)

Juice bg./ 100 g.)

Oranges (4 var.), Floridaa Oranges, Navel, Australiab Oranges, Valencia, Australiah Oranges, Shainouti, IsraeP

334 158-325 119-260 236

182 44-125 36-84 123

59 42

62 67-74 48-70 41

Oranges, Italy"

175-292

86-194

Rag and Juice: 45-73

Citrus fruits

Mandarins, Japane Mandarins, Punjab' Grapefruit (2 var.), Floridaa Lemons, New Zealandg Standard var. Meyctr

Flavedo and Albedo: 76-212 60-206 239 147

47

Flavedo and Albedo: 128 65

-

-

22-42 13-30 36

37 28

Atkins et al. (1945). Huelin and Stephens (1944), Anon. (1947) c Samisch and Cohen (1949). d Rauen et al. (1943). = From Insgaki (1946, 1853). I Prom Riaz-Ur-Rahman and Sadiq Ali (1955). 0 From Deaker (1952). 0

b

From From From From

(Miller and Jablonski, 1949). In conformity with general experience, the ascorbic acid content is increased about 10-fold when the seeds germinate.

B. OTHER VITAMINS In addition to ascorbic acid, other vitamins of considerable variety have been found in citrus fruits. However, the quantitative information that is consolidated in Table XI indicates that none of these factors is present in nutritionally significant amounts except i-inositol. In 1948, Davis and Kemmerer claimed that dried grapefruit peel contains a factor which stimulates milk production in dairy cows, but no other workers have confirmed this observation, and the factor has not been identified.

TABLEXI VITAMINS IN CITRUSFRUITS'

Factor i-Inositol Tocopherols Niacin Pantothenic acid Thiamine Pyridoxine Riboflavin p-Aminobenzoic acid Folic acid Biotin Vitamin Bla

Unitb (%)

Sweet orange

Sour orange

98-185 88-121 104-360 130-310 36-165 18-64 9-60 4-6 1.2-11 0.10-0.39 0.00114.0013

175-282 17-59

-

30

-

-

-

Mandarin

Grapefruit

135

67-112

-

-

-

198-269

29-196

-

80-252

67-160 23 21-36

67-370 520 15-57 8-30 10-28

4-125

9-66

1.2 0.45

0.8-1.8 0.364.97

-

4

Lemon 85

-

5-73

-

Lime

-

11-23 -

1

-

The values given are concentrations in the juice or edible portion, representing extremes of ranges from the following sources: Asenjo et al. (1946, 1948, 1950)-sweet orange, sour orange, grapefruit, lemon, sweet lime. Baier and Manchester (1949)--orsnge, grapefruit, lemon. Baier and Stevens (1954)-lemon. Burdick (1954)-grapefruit (canned juice). Inagaki (1946, 1953)-mandarin. Krehl and Cowgill (1950)-0range, grapefruit. tangerine. Munsell et al. (1949, 1950a,b,o,d)-aweet orange, sour orange, mandarin, grapefruit, lemon, lime, sweet lime. Penniai (1952)-orange. lime. Rakieten et al. (1951)-0range. In the present context it is not important t o distinguieh between values expressed on weight per volume aud weight per weight basis.

0

*

Sweet lime

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

313

VI. INORGANIC CONSTITUENTS

Information on the inorganic constituents of citrus fruits has accumulated mainly as a result of investigations in three different fields of interest.

INTEREST A. NUTRITIONAL Determinations of the inorganic constituents in citrus fruits have been made in order to assess their value as sources of mineral nutrients. Quantitative information, published during the period under review, on the ash content and the composition of the ash of citrus juices is summarized in Table XII. Analogous information on a number of the ash constituents of Central American citrus fruits (edible portions) has been collected by Munsell et al. (1949, 1950a,b,c,d). The high potasTABLE XI1 INORGANIC CONSTITUENTS OF CITRUSJUICES Constituent Total ash Alkalinity of ash (as KzCOd Potassium Sodium Calcium Magnesium Phosphorus Phosphorus pentoxideb-." Sulfur Chlorine Bromine Fluorine Iodine Iron Cobalt Copper Manganese Zinc

Unit (per 100 ml.)

Orange juicearb+

Grapefruit

Lemon juicebsa

0.29-0.63

0.25-0.56

0.15-0.56

0.24-0.53 197-350 12-17 4-15 4-16 14-20 19-72 3-8 1-4 0-240 38-94 0-2 78-350 8-80 45-101 0-28 0-9

0.26-0.45 170 2 5 5 11 27-43 2 1

0.13-0.42 99-128 1-5 6-28 9-1 1 5-17 20-42 2-8 2-4

-

140-690 -

Whole fruitf

Boron From From 0 From d From 0 From I From 0

b

230-401 Rakieten et al. (1951,1952), Stevens (1954). Morgan (1954). Stern (1954). Burdick (1954). Baier and Steven8 (1954). Bionda (1956).

-

20 20

-

-

-

-

-

314

J. F. KEFFORD

sium and low sodium content of citrus juices is noteworthy; potassium makes up about 40% of the ash, while sodium amounts to less than 3% (Sinclair and Bartholomew, 1944). The alkalinity of the ash of citrus juices (see below) has some nutritional significance (Sinclair and Eny, 1946a,c), since after metabolic oxidation of the organic anions, the cations are available to neutralize anions in the urine. Further, the alkalinity of the ash provides information on the state of combination of the cations in the original juice. Thus, Sinclair and Eny (1946a,c) calculated that in orange juice about 72% of the cations, and in grapefruit juice about 57%, are combined with organic acids, while the remainder are combined with inorganic anions such as phosphate, nitrate, chloride, and sulfate. Cations are also present, in the insoluble fractions of citrus fruits, in combination with organic groups, chiefly with the free carboxylic acid groups of pectin (Sinclair and Crandall, 1949b, 1951). The ash content of the peel and pulp fractions of citrus fruits is generally greater than that of the juice (Swift and Veldhuis, 1957; Ephraim and Monselise, 1957). The calcium content of the peel of oranges and grapefruit is about ten times that of the juice, on a dry basis, while the magnesium content is similar in the two fractions (Sinclair and Eny, 1947; Bartholomew and Sinclair, 1951). Most of the calcium is in a water-insoluble form, as calcium pectates and calcium oxalate. On the other hand, the phosphate content is higher in the juice and pulp than in the peel (Sinclair and Bartholomew, 1944).

B. ANALYTICAL INTEREST Interest in the inorganic constituents of citrus fruits has also arisen in connection with attempts to provide reliable methods for determining the content of citrus fruit in manufactured foods and beverages. Stern (1943) demonstrated that from determinations of the ash content, the alkalinity of the ash, and the phosphate in the ash of citrus beverages, it is possible to calculate approximately the citrus juice content. Determinations of the phosphate in the ash are particularly useful since this value is not affected by the addition of alkali metal salts to the beverage, e.g., as preservatives. Some values for the alkalinity of the ash and the phosphate in the ash of citrus juices, collected by Morgan (1954) and Stern (1954) are included in Table XII.

C . HORTICULTURAL INTEREST A third source of information on the inorganic constituents of citrus fruits is provided by horticultural studies concerned with the mineral

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

315

status of citrus trees. These studies have demonstrated that the amounts of inorganic constituents in citrus fruits are influenced by many field factors. Koo and Sites (1956) examined the variation in mineral composition between individual fruits on a single Valencia orange tree and found significant effects of height and amount of light received by the fruit. The potassium and phosphorus contents decreased with height, while the calcium content increased, and the magnesium content did not change significantly. The potassium and phosphorus contents were highest in fruits inside the canopy, but the calcium and magnesium contents were not affected by this factor. There was no significant variation due to the orientation of the fruit on the tree. Throughout the growth of oranges studied by Zidan and Wallace (1954) , the phosphorus, potassium, and magnesium contents, on a dry basis, decreased, but the calcium content showed a more variable trend. The absolute amounts of these elements per fruit increased while the fruit remained on the tree, even after horticultural maturity. The level of nitrogen nutrition influenced the levels of the other elements in the fruit. Jones and Parker (1951) found, as might be anticipated, that applications of phosphatic fertilizers to the trees increased the phosphorus content of citrus juices, and applications of potassium increased the potassium content, while both treatments decreased the calcium content. Wide ranges in the amounts of inorganic constituents were found by Haas (1948) in the peels of oranges, grapefruit, and lemons grown on different rootstocks under similar horticultural conditions. As an example of the results reported, the calcium, magnesium, potassium, and phosphorus contents tended to be high in fruit grown on trifoliate orange and citrange rootstocks, and low in fruit from rough lemon stocks. This was a fairly general but not universal observation, and it is of considerable interest in view of other observations of differences in composition between citrus fruits on the stocks mentioned (see Sections II,C and XIV). VII. NITROGEN COMPOUNDS

A. FACTORS AFFECTING THE NITROGEN CONTENT OF CITRUSFRUITS The total nitrogen content of citrus fruits is greatly influenced by horticultural factors. I n individual fruits on a single Valencia orange tree, the nitrogen content varied over the range 0.83 to 1.14 g./100 g. dry weight according to the position of the fruit on the tree (Koo and Sites, 1956). The nitrogen content decreased with height, and was

316

J. F. KEFFORD

higher in fruits inside the canopy than in those outside, but there was no significant variation with orientation on the tree. As might be expected, the application of nitrogen fertilizers to citrus trees increases the nitrogen content of the fruit (Jones and Parker, 1947, 1951). During the development of citrus fruits, the nitrogen content expressed as a percentage of the dry weight decreases, but the total amount of nitrogen per fruit increases up to and even beyond horticultural maturity (Zidan and Wallace, 1954). Bain (1958) followed changes in the distribution of nitrogen throughout the development of Valencia oranges. During the first 6 to 9 months of fruit development, while new cytoplasm was being synthesized, protein nitrogen predominated but gradually decreased in proportion to soluble nitrogen as the juice vesicles filled with juice. At maturity, the soluble nitrogen content per fruit was only slightly less than the protein nitrogen content. The amounts of protein nitrogen in peel and endocarp were practically equal, but the endocarp contained the greater part of the soluble nitrogen. The total nitrogen content of the pulp on a dry basis was higher in the stem-half of the fruit than in the stylar-half, while this relationship was reversed in the peel. The total nitrogen content of citrus juices normally lies in the range 50 to 200 mg./100 ml. and the amino nitrogen content in the range 10 to 60 mg./100 ml. (Rakieten et al., 1952; Munsell et al., 1949, 1950a,b,c,d). In California Navels, grown on six rootstocks, Marsh (1953) found that the ratio of amino nitrogen to total nitrogen was fairly consistent (0.32 to 0.38) during two seasons. Organic nitrogen compounds account for 5 to 10% of the total solids in citrus fruits. Knowledge of the soluble nitrogen compounds and proteins which make up tliis fraction has been greatly extended in recent years by application of chromatographic techniques. B. SOLUBLE NITROGEN COMPOUNDS

A considerable variety of amino acids and bases has been identified in citrus fruits. Present knowledge is summarized in Table XIII. Quantitative information is available mainly for orange juice together with one set of data for canned grapefruit juice. This information indicates a wide variability in the amounts of amino acids in citrus juices, but even the highest concentrations are not nutritionally important. There is evidence that the amino acids play a significant part in reactions leading to quality deterioration in processed citrus juices (Joslyn, 1957). The distribution of the alcohol-soluble nitrogen compounds in the tissues of citrus fruits was surveyed by Townsley et al. (1953); the alcohol-soluble nitrogen compounds were highest in the seeds of Va-

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

317

TABLE XI11 COMPOUNDSIN CITRUS FRUITS SOLUBLE NITROGEN ~~~

Conc. (mg./100 ml.) in juice Compound

Orange" Grapefruit* Lemonc

3-26 Alanint? y-Aminobutyric acid 4-73 Arginined 23-150 Asparagine 20-188 Aspartic acid 7-1 15 Glutaniic acid 6-71 Glutamine 3-63 Glycinc 5 Proline 6-295 Serine 4-37 Valine 10

&Ahnine or-Aminobutyric acid Citrulline Histidine

-

-

76

-

470 280

-

310 24

14

Notes on occurrence

1-31 This group of amino acids appears to be present generally in citrus 4-20 fruits; e.g., in American oranges, 25-106 grapefruit, mandarins, lemons, limes, and some hybrids;aoqme in 19-60 6-35 Italian oranges, mandarins and lemons;/ in Spanish oranges and lemons;g and in Japanese man27-53 darins and natsudaidaih,i.i 12-28

-

-

Hydroxyproline Leucines

-

24

-

Lysine

-

16

-

Ornithine Pheny lalanine

-

12

-

Threonine

-

10

-

-

In mandarins and natsudaidaiizjlk In Navel tissuese In Spanish oranges and lemonso In oranges,q grapefruit,b mandarins,j and lemonso In natsudaidaic I n oranges, lemons, mandarins! and grapefruit! In oranges, lemons and grapefr,iitb.d.d,r I n Spanish oranges and lemonsn In oranges,* grapefruit,b lemons, and natsudaidaii In oranges, grapefruit, and lemonsb3e3f*g.l

0 Extremes of ranges from Wedding and Sinclair (1954); Wedding and Horspool (1955); Rockland and Underwood (1956). b From Burdick (1954). c Rockland (1959). d Probably arginine and Iyaine, cf., Rockland and Underwood (1056). a Townsley et al. (1953). I Safina (1953). 0 Caabeiro (1956). Nomura and Munechika (1952). i Nomura (1953b). i Iseda. and Matsushita (1953). k Ito and Sakasegawa (1952b). Rakieten et at. (1952). Miller and Rockland (1952). n Herbst and Snell (1949). 0 Nelson et ol. (1933). P Hiwatrtri (1927). a Underwood and Rockland (1953).

318

J. F. KEFFORD

TABLE XI11 (Continued) Conc. (mg./100 ml.) in juice Compound

Orangen

Grapefruitb Lemonc

Tryptophan

-

4

-

Tyrosine

-

6

-

0.3-0.8

Cysteine Cystine Glutathione

-

Methionine

-

Betaine Choline Putrescine Stachydrine

39-63 7-16 -

-

0.18

-

0.35

-

2.8-7.8

-

-

-

-

-

-

Notes on occurrencc I n grapefruit,b mandarins and natsudaidai"." In oranges, grapefruit,, l e m o n s , b ~ e ~ ~ and mandarinsi Range of values for oranges, grapefruit, lemons, and limes" I n grapefruitb Range of values for oranges, grapefruit, lemons, and limesm I n grapefruit* Range of values for orange juicei Range of values for orange juicei In canned orange juice" I n orangeso and pumelosp

lencia oranges and grapefruit, in the navels of Navel oranges, and in the vascular tissue of lemons. The use of the ratio of the y-aminobutyric acid content to the citric acid content as an index of maturity in Valencia and Navel oranges was investigated by Wedding and Horspool (1955) but rejected because of large sample-to-sample variations which they attributed to transient environmental influences. Safina and Sara (1955) followed changes in the concentrations of aspartic acid, glutamic acid, asparagine, alanine, and serine in Italian oranges and lemons and found similar trends during two seasons.

C. NITROGENCOMPOUNDS CONTAINING SULFUR Glutathione was detected polarographically in immature oranges by Coulson et al. (1950), but they were unable to detect it in mature oranges, lemons, and grapefruit. Turrell ( 1950), however, estimated glutathione concentrations of 8.9 to 56.8 mg./100 g. in fresh lemon peel. Subsequently, Jansen and Jang (1952) isolated both glutathione and cysteine from mature Valencia and Navel oranges, and Miller and Rockland (1952) made the quantitative determinations shown in Table XIII. Citrus juices are thus among the few natural products known to contain free cysteine. Free hydrogen sulfide has also been detected in freshly expressed

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

319

orange juices (Kirchner et al. 1950). Micale and Sara (1955) were unable to detect hydrogen sulfide in juices distilled under vacuum at temperatures not higher than 35OC. and therefore considered that the hydrogen sulfide previously reported was a product of the decomposition of cysteine during analysis. Kirchner and Miller ( 1957), however, showed that hydrogen sulfide was present when nitrogen was passed through unheated, freshly reamed orange juice; consequently, they maintained that it was not an artifact produced by heating.

D. PROTEINS I n addition to soluble nitrogen compounds, the tissues of citrus fruits contain proteins, which are extractable with alkali. Paper chromatographic studies after hydrolysis of the proteins in juices (Rakieten et al., 1952), and in peels and chromatophores (Townsley et al., 1953) indicate that the component amino acids are mainly the same as those occurring in the free state in citrus fruits (Table XIII), with some differences in relative proportions. Wedding and Sinclair (1954) found some major differences in composition between the proteins of the eiidocarps of Navel and Valencia oranges which led them to suggest that the proteins were not identical; thus, although y-aminobutyric acid was relatively abundant in the Valencia protein, it was not detected in the Navel protein; and while ornithine was abundant in the Navel protein, it was not detected in the Valencia protein. Townsley et al. (1953), however, have reported the presence of ornithine in the chromatophore protein of Valencia oranges. Bartholomew and Sinclair (1951) found the protein of lemon endocarp to be similar to the orange proteins in general chemical properties, but they did not determine the constituent amino acids. The seeds of citrus fruits contain about 10% crude protein on a wet basis, while the seed meals prepared by removing the seed oil and the husks may contain 30 to 40% protein on a dry basis (Nolte and Von Loesecke, 1940; Driggers et al., 1951; Averna and Petronici, 1955). The component amino acids of the proteins of orange, lemon, and mandarin seeds were found by Safina (1953) to be mainly the same as those occurring in the juices (Table XIII) .

E. ANALYTICAL APPLICATIONS Attempts have been made to use the nitrogen compounds of citrus juices as a basis for the estimation of the juice content of citrus beverages. Siddappa and Rao (1955) used the “albuminoid ammonia nitrogen” values of Indian citrus juices to distinguish between genuine and

320

J. F. KEFFORD

artificial citrus beverages, but they found that the values were influenced by the amount of rag and peel in the juice. The application of the formol titration of amino acids for this purpose was examined by Benk (1954, 1956), but Safina and Sara (1955) could establish no relation between the formol titration and the concentrations of alanine, asparagine, aspartic acid, glutamic acid, and serine in Italian orange and lemon juices. Buffa (1954) found that the blue ninhydrin color was useful for the determination of citrus juices in carbonated beverages only when the citrus juice content was greater than 3%. VIII. ENZYMES

Among the proteins in citrus fruits, particular biochemical and technological interest is attached to the enzymes. The most important of these, technologically, are those which hydrolyze pectin. A. PECTOLYZING ENZYMES In processed citrus juices and citrus beverages, pectin performs the desirable function of suspending the cloud of chromatophores. If the pectin is hydrolyzed, the cloud settles out, and the appearance of the juice becomes unattractive. Further, in frozen concentrated citrus juices, and in juices preservatized in bulk for beverage manufacture, the hydrolyzed pectin may combine with calcium ions to form gels which do not reconstitute satisfactorily.

1. Pectinesterme The principal pectolyzing enzyme in citrus fruits is a highly specific pectinesterase which hydrolyzes the methyl ester groups of pectin about 1000-times as fast as it attacks the ester groups in nongalacturonide esters (MacDonnell et al., 1950). The general level of pectinesterase activity in citrus fruits is similar to that in other known plant sources such as tomatoes. The pectinesterase is associated with the insoluble components of citrus tissues and is not present in the filtered juice or the peel juice (MacDonnell et al., 1946). Thus, Rouse (1951) found that the pectinesterase activity in Valencia orange juice is proportional to the pulp content and the content of water-insoluble solids, while Atkins and Rouse (1953) reported a similar relationship in juices extracted by four different methods. The relative pectinesterase activity in the flavedo, albedo, and juice sacs of oranges, lemons, and grapefruit was found by MacDonnell et

THE C H E M I C A L C O N S T I T U E N T S O F CITRUS F R U I T S

321

al. (1945) to be approximately 1:0.8:0.5 on a wet basis. The distribution of pectinesterase in the component parts of citrus fruits was also examined by Rouse (1953) and Rouse and Atkins (1954). Their results, expressed on a dry-solids basis, showed the decreasing order of pectinesterase activity to be: for oranges and tangerines-juice sacs, rag, flavedo, albedo, seeds, and juice; for grapefruit-juice sacs, flavedo, albedo, rag, seeds, and juice; and for lemons and limes-peel, juice sacs, juice, rag, and seeds. The effect of maturity on pectinesterase activity was studied by Rouse and Atkins (1953) in Pineapple oranges harvested once a month for five months. While the Brix/acid ratio increased from 10.5 to 15.9 the pectinesterase activity on a dry basis also increased but thereafter declined in all parts of the fruit. Optimum extraction of pectinesterase from citrus tissues was obtained by MacDonnell et al. (1945, 1950) at pH values near 8 with a sodium chloride concentration about 0.25 M . By a purification procedure involving salt fractionation and adsorption, these workers were able to concentrate orange pectinesterase 100-fold, on a protein nitrogen basis. The enzyme is capable of de-esterifying pectin over a wide range of pH, but its activity and stability are considerably influenced by the concentration of cations. Pectinesterase shows a relatively high resistance to inactivation by heat; in 5 min. at 56OC. and pH 7.5, the loss of activity is about twothirds. The heat resistance of the enzyme decreases with decreasing pH (Rouse and Atkins, 1952). Methods for the determination of pectinesterase activity in citrus fruits, which depend upon continuous titration of the carboxylic acid groups liberated in pectin under standard conditions, have been reviewed by Rouse and Atkins (1955b).

2. Depolymerizing Enzymes The question as to whether pectinesterase is the only pectolyzing enzyme in citrus fruits or whether it is accompanied by enzymes capable of depolymerizing pectin remains unresolved. MacDonnell et al. (1945) were unable to demonstrate any change in the reducing value or molecular weight of pectin treated with an extract of orange flavedo, and they therefore concluded that polygalacturonase activity was absent. On the other hand, Pratt and Powers (1953) considered that enzymes which depdymerized pectin or pectic acid were present in 4 out of 14 batches of grapefruit juice which they examined. The activity, however, was slight and was demonstrated only by a decrease in viscosity.

322

J. F. REFFORD

B. ACETYLESTERASE ’ In addition to the highly specific pectinesterase, oranges, lemons, and grapefruit contain an esterase which Jansen et al. (1947, 1949) designated as acetylesterase since it showed its greatest hydrolytic activity on esters of acetic acid. All aliphatic esters of acetic acid were hydrolyzed, and also other simple esters such as methyl and ethyl butyrates, but at slower rates. With glycerides, even the water-soluble monoglycerides, the activity decreased markedly with increasing chain length of the fatty acid. The rate of hydrolysis of tributyrin was only 4% of that of triacetin. The enzyme is thus not a lipase; it was also shown not to be a cholinesterase. Acetylesterase has, in fact, novel properties among plant enzymes. Jansen et al. (1947) have postulated that its metabolic function is concerned with the synthesis of acetic esters found among the volatile flavoring substances of citrus fruits. Acetylesterase activity is highest in the flavedo, and it decreases in the albedo and pulp. Like pectinesterase, the acetylesterase activity is associated with the insoluble solids of citrus tissues, but there is significant activity in the juice from the flavedo. Since acetylesterase is unstable below pH 4 and is rapidly inactivated at 5OoC., it is not likely to be responsible for deteriorative reactions in processed citrus products. C. PHOSPHATASE An enzyme system having the properties of a phosphatase and generally similar in properties to other plant phosphatases was detected for the first time in oranges, lemons, and grapefruit by Axelrod (1947a). In contrast to pectinesterase and acetylesterase, some phosphatase activity is present in solution in the juice. Concentration of the enzyme was achieved by ammonium sulfate fractionation, followed by adsorption and dialysis. Orange phosphatase showed phosphomonoesterase activity but no diphenylphosphatase activity. It hydrolyzed a wide variety of phosphate compounds, including polyphosphates, but it was inactive towards glucose-I -phosphate and diphenylphosphate. The fact that the enzyme system also split off at least 90% of the phosphate of yeast nucleic acid was explained by the presence in the phosphatase preparation of ribonucleinase. Acetylphosphatase activity in orange juice was due entirely to the phosphatase and not to esterase activity. Citrus phosphatase is not very resistant to heat, but its stability increases with increasing pH. Axelrod (1947b) reported that its heat inactivation behavior was somewhat anomalous and did not follow the logarithmic course generally found for the heat denaturation of enzymes.

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

323

D. GLUTAMIC ACID DECARBOXYLASE The flavedo of lemons and oranges contains an enzyme system which decarboxylates glu tamic acid to yield y-aminobutyric acid. In fact, citrus flavedo is 10 times as rich in glutamic acid decarboxylase, on a wet basis, as the most active sources known in other plants (Axelrod et al., 1955). The highest specific activity on a protein-nitrogen basis was found in the flavedo of green lemons; the activity was lower in ripe lemons and in Navel and Valencia oranges. By water extraction of a flavedo suspension followed by ammonium sulfate fractionation, a 1O-fold concentration of the enzyme was achieved. The enzyme is specific and acts only upon L-glutamic acid. It shows a fairly broad p H optimum between 5.3 and 5.6, and it requires pyridoxal phosphate to prevent loss of activity under certain conditions of temperature and pH.

E. PEROXIDASE The occurrence of peroxidase in citrus fruits was first investigated quantitatively by Davis (1942). A survey of the tissues of oranges, lemons, grapefruit, and tangerines revealed that peroxidase activity was present in the flavedo, albedo, endocarp, and seeds, the highest activities being recorded in the inner seed coat and the outermost layer of the flavedo. Petronici and Averna (1955, 1957) also reported that peroxidase activity was high in the flavedo of lemons and oranges and lower in the albedo and membranes. Only limited activity was found in orange juice, while none could be detected in lemon juice, probably because of the low pH.

F. OTHERENZYMES

A cytochrome-oxidase system appears to be present in orange tissues, the activity being greatest in the flavedo (Hussein, 1944). Huelin and Stephens (1948) found a high level of ascorbic acidoxidase activity in orange peel but only negligible activity in the juice. These observations were confirmed by Avidor (1950), who also found that the ascorbic acid-oxidase activity was higher in the flavedo than the albedo. Slight proteolytic enzyme activity was found in lemon juice by Manchester (1942). Jansen et al. (1952) reported the presence in citrus peels of a di- and polyaminopeptidase in fairly high concentration. The peptidase preparations hydrolyzed a variety of peptides but catalyzed only slightly the hydrolysis of proteins. Axelrod and Jang (1954) reported the occurrence in orange flavedo

324

J. F. KEFFORD

of phosphoribo-isomerase, an enzyme which catalyzes the isomerization between ribose-5-phosphate and ribulose-5-phosphate. The possible presence in citrus peels of a flavanone synthease which converts chalcone glycosides to flavanone glycosides (Shimokoriyama, 1957) is mentioned in Section XIII. Some evidence of enzymic reduction of potassium permanganate led Miller (1946) to infer the presence of a reductase in lemon peel. In order to explain a flavor defect in frozen concentrated citrus juices, described as the “COF effect,” Blair et d.(1957) postulated the survival of an enzyme system capable of producing lipids, or lipid precursors such as unsaturated aldehydes, by the drastic reduction of sugars. The enzyme was considered to be more active in immature than in mature fruit. This hypothesis was based, however, on related model systems, and no direct evidence of the presence of such an enzyme in citrus fruits was presented. IX. PIGMENTS

Mature citrus fruits have distinctive and attractive colors, ranging from pale yellow through orange to red, which are due principally to carotenoid pigments located in chromoplasts in the flavedo and endocarp. Chlorophylls are also present in immature citrus fruits, but they generally disappear rapidly with advancing maturity. In oranges, the amounts of carotenoids increase with maturity, but in the lightercolored fruits, such as grapefruit and lemons, the carotenoid content tends to decrease (Miller et al., 1940). Early work had established that the pigments in citrus fruits were complex mixtures of carotenoids, mainly xanthophyll esters with small amounts of carotenes (Natarajan and Mackinney, 1952). Rabourn and Quackenbush (1953) found 8.3 pg/g. wet weight of phytoene in the edible portion of oranges but none in lemons. Both fruits, however, contained phytofluene. Knowledge of the pigments of citrus fruits has been greatly extended by the painstaking studies of A. L. Curl and his collaborators at the Western Regional Research Laboratory of the U.S. Department of Agriculture. This work provides an outstanding example of the usefulness of two modern techniques in the investigation of the chemistry of complex natural substances. A. ORANGESAND TANGERINES By application of countercurrent distribution, Curl (1953) was able to divide the carotenoids of Valencia orange juice into six fractions according to the number of hydroxyl and cyclic ether groups present.

T H E CHEMICAL CONSTITUENTS OF CITRUS FRUITS

325

Then, by column chromatography, these fractions were separated into individual components amounting to 25 in all, including stereoisomers (see Table XIV) , The major pigments were xanthophylls, and their epoxides and furanoxides (Curl and Bailey, 1954). Isomerization of xanthophyll 5,6-epoxides to the corresponding furanoxides (5,8-epoxides), which is catalyzed by acids, may have occurred in the fruit o r during the extraction process (Curl and Bailey, 1956a). It is possible that in the intact orange only the five parent epoxides occur: antheraxanthin, the monoepoxide of zeaxanthin; violaxanthin, the diepoxide of zeaxanthin ; trollixanthin, a polyhydroxy epoxide; and valenciaxanthin and sinensiaxanthin, two polyhydroxy epoxides not previously known. The polyhydroxy compounds are considered to be natural constituents of orange juice and not artifacts since these compounds were not found among the xanthophylls isolated from green leaves by similar techniques of extraction and fractionation (Curl and Bailey, 1957c). The majority of the xanthophylls occur in orange juice in the fully esterified form (Curl and Bailey, 1955). The acids combined in the xanthophyll esters were not identified, but they are probably the same fatty acids as occur in the lipids of orange juice (see Section X). A red carotenoid acid was also present but was not identified. An investigation of the effect of canning and storage a t room temperature for three years on the pigments of Valencia orange juice (Curl and Bailey, 1956b) revealed that the only major chemical change was the complete disappearance of the xanthophyll epoxides which had made up more than half of the total carotenoids in the fresh juice. These pigments were partially, but not completely, accounted for in the furanoxide fraction, having been isomerized at the acid pH of the juice. No significant hydrolysis of xanthophyll esters or changes in the composition of nonether carotenoids had occurred. The visual color of the juice was still good. The same techniques of countercurrent distribution and chromatographic separation were applied successfully to the identification of the pigments in orange peel and in tangerines. I n general, orange peel (Table XIV) contained the same pigments as orange juice, but the proportion of violaxanthin was much higher (Curl and Bailey, 1956a). I n earlier work, Zechmeister and Tuzson ( 1937) had found a polyenealdehyde, citraurin, among the pigments of orange peel. Tangerines yielded a complex mixture of carotenoids (Table XIV) generally similar to the pigments of oranges, but the redder color of these fruit may be attributed to the much higher concentrations of

TABLE XIV CAROTENOID CONSTITUENTSOF CITRUSFRUITS Approximate percentage of total carotenoids Valencia orange

Fractions and constituents

Fresho juice

Hydrocarbons Phytoene Phytofluene Phytofluene-like a-Carotene p-Carotene {-Carotene 7-Carotene-like Lycopene

Canned* juice EndocarPC

1 .o 0.8

0.7 2.1 3.2

4.0 13 0.5 1.1

5.4

Monols Cryptoxanthin Cryptoxantbin-like Cryptoxant hin epoxide Cryptoflavin Cryptochrome-like 3-Hydroxy-a-carotene' Hydroxy-a-carotene furanoxide-like Rubixanthin-like

Diols Lutein

Tangerined

Peelc

Endocarp

Peel

Endocarp

Peel

3.1

5.8 7.2 0.1 0.3 4.1 6.9 0.1 0.1

4.2 3.5 0.1 0.2 0.4 2.0

16 4.4

47 14

6.1

0.1 0.3 3.5

1.2 0.4 1.2 0.8 0.3

7

9.9

2.9

Ruby Red grapefruit"

1.2

-

27 3.5 0.8

0.02

40

33

24

0.7 0.4

0.9 1.0 -

1.4 3.4 0.1 0.6 0.4 0.2

0.2 0.5

2.9

3.3

0.3

0.8

-

0.4

4

0.1 7.2 7.2 0.4 11

r

1.4

-

-

-

1.3 0.2 0.1

-

0.3

0.9

B 2 0

s U

Zeaxanthin Hydroxycanthaxanthin-like Capsanthin-like Monoetherdiols Antheraxanthin Mutatoxanthins Flavoxanthin-like Dietherdiols Violaxanthin Luteoxanthins Auroxanthins Polyols Valenciaxanthin Valenciachromes Sinensiaxanthin Sinensiachrome-like Trollixanthin-like Trollichrome-like Trolleins From Curl 1953; Curl and Bailey (1954). a From Curl and Bailey (1956b). c From Curl and Bailey (1956s). d From Curl and Bailey (1957b).

3.3 0.1 -

3.5 2.7

5.8 6.2

-

6.3 1.7 -

9.7 2.2

6.2 2.8

0.7 0.4

7.4 17 12

44 16 2.3

14 3.5 0.4

24 9.1 1.9

0.9 0.4 0.3

2.8 1.0

2.2 0.7 3.5 0.2 0.5 0.8 -

0.2

0.4

0.2

0.2

1.1 2.6 2.7

14.2

4.5

-

-

-

-

20 6

-

-

30.9 0.6

16 5 P

-

P

-

P P

2.5 0.2

P P

-

7.2

-

7 .O 4.7

-

0.8 0.3

15

-

-

2.0

-

2.9 3.0

-

-

-

-

1.o 0.3 0.9

-

-

-

-

-

-

-

-

0.2

-

F

*From Curl and Bailey (1957a).

2

From Curl (1956). I P = present but percentage not given.

5

s

3

v)

328

J. F. KEFFORD

cryptoxanthin in the endocarp and peel and of p-carotene in the endocarp (Curl and Bailey, 1957b) than are found in oranges. The carotenoid composition of the edible portions and the peel of Japanese citrus fruits has been reported by Shioiri and Kimura (1955).

B. GRAPEFRUIT Khan and Mackinney (1953) investigated the pigments of white, pink, and red varieties of grapefruit. Marsh White grapefruit contained small amounts of phytofluene and 6-carotene, and a pale yellow pigment which was not identified. The mutation of Marsh White to Marsh Pink resulted in the appearance of p-carotene as the major pigment accompanied by lycopene; then in the further mutation to Ruby Red the concentrations of both p-carotene and lycopene greatly increased. The amounts of these carotenoids varied with the maturity of the grapefruit (Lime et al., 1954, 1956, 1957). Lycopene predominated at first, then declined; while p-carotene increased slowly, so that it exceeded the lycopene concentration about haIf-way through the season, then later declined slowly. Curl and Bailey (1957a) examined the pigments of Ruby Red grapefruit intensively and identified more than 20 carotenoid constituents (Table XIV) . Hydrocarbon pigments predominated, notably lycopene and p-carotene; and phytoene was a major constituent, particularly in the peel pigments. Xanthophylls occurred only in minor amounts, but most of those found in oranges and tangerines were represented, C. CITRUSFRUITSCONTAINING NONCAROTENOID PIGMENTS

1. Blood Oranges The sweet orange varieties known as “blood oranges” owe their distinctive colors not to red carotenoid pigments but to anthocyanins. In 1931, Matlack had observed aggregates of red anthocyanin crystals in the juice vesicles of blood oranges. Two pigments have now been isolated by Chandler (1958a) from the Moro variety of blood orange. One pigment, amounting to at least 95% of the total pigments, is cyanidin-3glucoside, and the other is probably delphinidin-3-glucoside. It is likely that these pigments are common to all blood oranges since the Tarocco, Florida and Ruby Red varieties contained the same principal pigment as the Moro variety. 2. Limes

In 1925, Hardy and Warneford stated that limes contained little carotenoid pigment, and they claimed that the principal pigment was a

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

329

phlobatannin, related to caffetannic acid. According to Bate-Smith (1954), this observation could not be confirmed in West Indian or West African limes. It appears that the pigments of limes might well be re-examined by means of modern techniques. X. LIPIDS

Early work by Matlack (1929, 1940) had shown that the peel and endocarp of oranges contain lipid material made up of oleic, linoleic, linolenic, palmitic, and stearic acids as glycerides and probably also as sterol esters. Sitosterol and sitosteryl-D-glucoside (see Section XII,B) were isolated; ceryl alcohol was found in the peel, and a hydrocarbon, probably pentacosane, was found in the endocarp. More recent work has extended our knowledge of the lipid constituents of citrus fruits. A. LIPIDSIN ORANGEJUICE By filtering the suspended solids from orange juice and extracting the residue with acetone and petroleum ether, it is possible to remove a lipid fraction which amounts to about 0.1% of the whole juice (Swift, 1946). Information on the composition of this fraction has been accumulated mainly by L. J. Swift and his collaborators at the U.S. Citrus Products Station, Winter Haven, Florida. The investigations were prompted by the possibility that chemical changes in the lipid fraction were responsible for flavor deterioration in processed orange juices. The lipid composition of some fresh and processed orange juices is set out in Table XV. One batch of fresh juice was compared with the same juice pasteurized (Huskins and Swift, 1953a) ; and another batch of fresh juice was compared with the same juice canned and stored for 2 years at temperatures above 73OF. (Swift and Veldhuis, 1951; Swift 1952a; Huskins et al., 1952). The experimental treatments were not replicated, and therefore the quantitative results should be interpreted with caution, but there appears to be no doubt about the main trends in the chemical changes. Pasteurization had only a small effect on lipid composition; there was evidence of slight hydrolysis of glycerides but not of phosphatides. During storage of the canned juice, however, extensive changes occurred. There were losses of phosphorus and nitrogen following the breakdown of phosphatides. These and other changes led to the disappearance of about 20% of the extractable lipid, presumably in the form of water-soluble fragments (Huskins et al., 1952). T h e changes during storage in the lipids of canned orange juice were studied more

330

J. F. KEFFORD

COMPOSITION OF

THE

TABLE XV LIPIDSFROM FRJLSH AND PROCESSED ORANGEJUICES Effect of storageb

Effect of pasteurization"

Constituent Unsaponifiable matter Fatty acids, free Fatty acids, total Saturated Oleic Linoleic Linolenic Arachidonic Conjugated dienes Conjugated trienes Conjugated tetraenes Resin acids Sterol glycosidesc Glyceryl radical Monoacid phosphate radical Total nitrogen Cholinyl radical Ethanolaminyl radical

Fresh juice (%)

Pasteurized juice (%)

Fresh juice

19.05 33.50 57.55 11.21 23.22 18.20 4.05 0.00 0.72 0.014 0.002 10.43 5.85 1.63 4.06 0.68 1.78 0.74

16.53 34.30 56.53 10.83 23.50 17.35 3.98 0.00 0.86 0.017 0.005 10.31 4.93 1.40 4.34 0.73 2.02 0.74

14.81

(%I

-

59.00 16.28 17.64 18.76 5.37 0.00 0.75 0.092 0.096 12.41 1.48 1.53 4.34 0.61 2.03 1.09

Stored juice (%)

14.86 63.81 21.42 19.90 16.01 3.20 0.00 3.03 0.025 0.00 12.54 1.oo

1.40 0.40 0.13 0.00 0.47

From Huskins and Swift (1953s). From Huskins el al. (1952). c Identified a8 ,9-sitosteryl-D-glucoside (see Section XI1,B).

a

closely by Huskins and Swift (1953b), and the losses of phosphorus and nitrogen, particularly choline nitrogen, pointing to the hydrolysis of phosphatides, were confirmed.

B. LIPIDSIN

THE

JUICE VESICLES

Deposits of a lipid substance occurring peripherally in the spaces between adjacent juice vesicles in citrus fruits were examined histologically by King (1947a,b). Suberin, a complex polyestolide of hydroxy fatty acids was identified as one constituent of these deposits, and phellonic acid, a major component of suberin, was isolated from an ether extract of the vesicles of oranges and grapefruit. Suberin may be responsible in part for the relative impermeability of the walls of the juice vesicles and segments of citrus fruits (cf., Bartholomew and Sinclair, 1941).

COMPOSITION OF Grapefruit' Trinidad Seed analysis

Oil contentf Saturated acids: Myristic Palmitic Stearic Arachidic Unidentified hydroxyacid Unsaturated acids: Oleic Linoleic Linolenic

TABLE XVI LIPIDSFROM CITRUSSEEDS

THE

Lime' Trinidad

-

Sweet orangeb Jamaica

-

Sweet orang& Calif. (Valencia)

Tangerine" Florida (Dancy)

Lemond Sicily -

Shaddock# India -

34

27

38 (max.)

39

+

20.7 15.3

(Foster)

(Marsh)

43

41

0.8 28.9 2.1 0.6

1.2 27.5 2.9 2.1

0.3 26.1 9.6 0.5

-

-

23.8 8.3 0.7

20.7 4.7 0.9

19.6 5.2 1.1

-

-

-

-

-

2.9

25.1 36.6 5.9

21.1 39.3 5.9

11.1 39.3 13.1

24.8 37.1 5.3

36.6 36.5 0.6

22.5 46.6 2.1

39 40 Component fatty acids'

-

+ ++

From Dunn et al. (1948). From Van Atta and Dietrich (1944). c From Swift (1949). d From Averna and Petronici (1955). e From Dasa Rao et al. (1940). i Per cent by weight of dried or air-dried see&. o Per cent by weight of total fatty acids, except for tangerine results which are per cent by weight of total mixed methyl eatera 6

-

55.5 8.1 0.5

332

J. F. KEFFORD

C. LIPIDSIN CITRUSSEEDS

In common with the seeds of many fruits, citrus seeds contain reserves of lipid material. By expression or extraction of the dried seeds, fatty oils may be recovered in yields up to about 40%. The most complete investigation of the composition of citrus seed oils was made by Dunn et al. (1948) on samples from West Indian grapefruit, orange, and lime seeds. Their results, together with those of some other workers, are collected in Table XVI. Citrus seed oils are semidrying oils which resemble cottonseed oil in the nature and distribution of the probable component glycerides, except for the presence of linolenic glycerides, which are absent from cottonseed oil. XI. VOLATILE FLAVORING CONSTITUENTS

A. IN CITRUSPEELS

In contrast to other common fruits, in which the volatile flavoring constituents are mainly aliphatic esters, citrus fruits owe their characteristic aromas and flavors to essential oils that are largely terpenoid in composition. 1. Distribution The citrus essential oils are contained in oil sacs located in the ffavedo. The amounts of essential oil in citrus peels are influenced by a number of factors, and particularly by variety and growing area. Valencia oranges, for instance, yield about twice as much essential oil as Navel oranges. Typical oil contents found by Bartholomew and Sinclair (1946) for California oranges from an inland area were: Valencias, 1.1 m1./100 sq. cm. and Navels 0.52 m1./100 sq. cm. area of peel; from an area nearer the coast: Valencias, 0.88 m1./100 sq. cm. and Navels 0.43 m1./100 sq. cm. area of peel. Large oranges yield more oil than small fruit when the yield is expressed per unit area of surface. The density of distribution of the oil sacs, and hence the oil content, increases progressively from the stem-end to the stylar-end of the fruit (Haas and Klotz, 1935; Bartholomew and Sinclair, 1951). Shioiri et al. (1953) followed trends in the oil content of natsudaidai peel during maturation and found that it increased to a level of 2.4% on a wet basis and then declined. Citrus peel oils, recovered by expression or steam distillation, are valuable by-products of the citrus processing industry and are widely used for flavoring foods, beverages, and confectionery.

THE CHEMICAL CONSTfTUgNTS OF CITRUS FRUITS

335

2. Composition The chemical composition of citrus peel oils has been comprehensively reviewed by Guenther (1949), and the information collected together by him has been summarized in Table XVII. The terpene hydrocarbon, d-limonene, is the major constituent in all citrus peel oils, but it contributes little to the flavor. However, “terpeney” off -flavors, appearing in citrus oils and citrus products during storage, may be due to oxidation of limonene to carveol and carvone (Proctor and Kenyon, 1949). The characteristic flavors of citrus oils are ascribed to the oxygenated constituents, principally aldehydes and esters; for instance, lemon oil owes its character to citral and isocitral, and orange and grapefruit oils to n-octanal and n-decanal. Kesterson and Hendrickson (1953b, 1958) have made a thorough study of the chemical and physical properties of orange, grapefruit, tangerine, lime, shaddock, and lemon oils from Florida fruit. Many factors affected the chemical composition as represented by the aldehyde and ester content, e.g., seasonal conditions, variety, maturity, the storage history of the fruit, the method of oil recovery (whether expression or distillation), and the yield (the proportion of the total oil expressed from the peel). Further, the quantity of aqueous phase coming in contact with the oil during processing largely determined the aldehyde content. A method for determining the citral content of lemon oil devised by Stanley et d.(1958) has been used by Stanley and Vannier (1958) to examine the citral content of the oil from individual fruits. Wide and random variations over the surface of the fruit were observed. No correlation of citral content with maturity was established. The citral content of lemon oils from the coastal areas of California was consistently higher than that of oils from the arid inland areas. Bernhard ( 1958a) applied gas-partition chromatography to the examination of lemon oil and found that this technique may be used to show gross compositional differences between lemon oils and also to detect adulterants. Low-temperature chromatography, a procedure recently devised by Clements (1958), may lead to a more complete knowledge of the constituents of the hydrocarbon fraction of citrus oils.

B. IN CITRUSJUICES 1. Source The characteristic flavors of citrus juices are due largely to essential oils derived from the peels. In 1932, Davis made a histological examina-

w

s

TABLE XVII VOLATILE CONSTITUENTS OF CITRUS OILS'

Oil analysis Extraction Hydrocarbons

Carbonyl compounds

Sweet orange (Calif., Florida, Italy, French Guinea)

Bitter orange (Italy)

Expressed Expressed ca. 95% ca. 92% d-Limoneneb d-Lmonene Myrcene Terpinolene( ?) a-Terpinene (?) Ocimene ( ?) Cadinene(1)

1-2 % n-Octanal n-Decand n-Dodecanal n-2Decen-1-a1 Citral Acetaldehyde

ca. 0.8% n-Nonanal n-Decanal n-Dodecanal

Mandarin (Italy, Florida)

Natsudaidai (Japan)

Grapefruit (Florida)

Expressed Expressed ca. 96% ca. 95% d-Limonene d-Limonene Cadinene(?) a-Pinene 8-Pinene Camphene y-Phellandrene p-Cymene

Expressed 90 %Y d-Lmonene a-Pinene Bisabolene Cadinene

ca. 1% n-Octanal n-Decanal Citral

n-Decanal

ca. 1.5% n-Octanal n-Decanal Citral Citronella1 Acetaldehyde

Lemon (Calif., Italy) Expressed 8590% d-Limonene a-Pinene 8-Pinene 7-Terpinene Bisabolene Cadinene Camphene 8-Phellandrene Octylene(?) ca. 2.5% Citral n-Octanal n-Nonanal n-Decanal n-Dodecanal Citronellal Methylheptenone Acetaldehyde

Lime (Mexico) Distilled 75-80 % d-Limonene a-Pinene 8-Pinene Dipentene Bisabolene

4

.r IY! r

0

z ca. 2.5% Citral n-Octanal n-Nonanal n-Decanal n-Dodecanal Furfural

TABLE XVII (Continued)

Oil analysis Alcohols and esters

Sweet orange (Calif., Florida, Italy, French Guinea) 0.05-1.5%

n-Nonanol d-Linalo61 n-Decanol Nerol Geraniol(?) d--a-Terpineol Farnesol Nerolidol Methyl anthranilate

Acids

0

b

Formic acid Acetic acid Caprylic acid Capric acid

4

Bitter orange (Italy) ca. 2.5% Linalyl acetate Linalool Terpineol Neryl acetate Geranyl acetate Citronellyl acetate Decyl pelargonate

Formic acid Acetic acid Pelargonic acid Cinnamic acid

Mandarin (Italy, Florida) ca. 1% Linalool Citronellol Methyl-Nmethylanthralinate Terpineol Nerol Linalyl and Terpenyl acetates

Natsudaidai (Japan) ca. 1% Linalool Terpineol Nonanol

Grapefruit (Florida)

Lemon (Calif., Italy)

Lime (Mexico)

2-4 % c2. 2% 2-4 % n-Octanol and Lbalool and Geraniol acetate acetate Linalool n-Nonanol a-Terpineol a-Terpineol Linalool and Nerol (prob. Borneo1 acetate as acetate) n-Decanol Geraniol Citronellol (as ester?) (prob. as Geranyl acetate) acetate Geraniol and Terpineol acetate Methyl anthranilate Methyl anthranilate Acetic acid Acetic acid Acetic acid Caprylic acid Caprylic acid Caprylic acid Capric acid Capric acid Capric acid Lauric acid

From Guenther (1949). Kesterson and Hendrickson (1953b). Burdick (1954b), Agricultural Research Service (1956), and Calvarano (1958b). The compounds in bold type are thought to be the major components of the reapective fractions.

E n

F n

o

2

0

r

336

J. F. KEFFORD

tion of citrus juice sacs and reported the presence of minute oily deposits which he considered to contain essential oils. This report has persisted in the literature in spite of the subsequent work of King (1947a), who showed that these deposits are lipoidal in nature (see Section X,B). Nevertheless a number of workers have claimed that some essential oil is present in the endocarp of oranges. As a result of determinations on carefully hand-peeled oranges, Rice et a2. (1952) stated that orange juices contain about 0.005% of oil derived from the endocarp. Hand-peeled oranges, treated with potassium permanganate to destroy any peel oil left on the surface, gave a juice containing 0.0005% oil (Blair et al., 1952). Using the same technique, Morgan et d. (1953) obtained juices with oil contents ranging from 0.0007 to 0.004%, depending on the variety and maturity of the fruit. On the other hand, Guyer and Boyd (1954) could detect no volatile oil in the juice from oranges which were steamed, hand-peeled, and extracted on a mechanical extractor. In any case, the presence or absence of essential oil in the juice vesicles has little practical significance since commercially-extracted orange juices may contain 0.01 to 0.1% of volatile oil, most of which is derived from the peel. In handextracted orange juices, Rakieten et &. (1951) reported oil contents ranging from 0.001 to 0.103%. 2. Composition

A painstaking study of the volatile flavoring substances present in orange and grapefruit juices was made by Kirchner and his co-workers at the Fruit and Vegetable Chemistry Laboratory, Pasadena, California (Kirchner et al., 1953; Kirchner and Miller, 1953, 1957). Freshlyreamed juices, freshly-canned juices, canned grapefruit juice stored 4 years at room temperature, and canned orange juice stored 3 years were examined (1500 to 3000 gallons of each juice being processed by low-temperature vacuum distillation to recover the volatiles) . The compounds identified and their approximate quantitative distribution are tabulated in Tables XVIII and XIX. Most of the constituents previously identified in orange and grapefruit peel oils were present in the volatile oils from the juices. I n the processing and storage of canned orange and grapefruit juices, the major change in the composition of the volatile constituents was a loss of hydrocarbons and an increase in oxygen-containing compounds, probably as a result of acid-catalyzed hydration reactions. In grapefruit juice, the d-limonene lost during storage could be accounted for almost quantitatively as linalool monoxide and a-terpineol. Since linalool monoxide resembles in aroma the volatile flavoring constituents

337

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

TABLE XVIII VOLATILE FLAVORING CONSTITUENTS OF GRAPEFRUIT JUKCE"

Constituent

Fresh juice (m@;./kg.)

Freshly canned juice (mg./kg.)

Volatile water-soluble constituents 1.45 0.33 Acetaldehyde Furf ural 0 Trace 0 0 Acetone 400 400 Ethanol 0.2 0.2 Methanol 0 1.9 Acetic acid 0 4.8 Unsaturated acid A (CeHs02) 0 1.9 Unsaturated acid B (CeH802) 0 Trace Acid G Trace 0 Hydrogen sulfide Volatile oil constituents 20.9 Total oil 17.3 Hydrocarbons 15.7 d-Limonene 1.4 D-Caryophyllene 0.10 Akryophyllene Trace a-Pinene 0.11 C16H24 Trace C16H28 3.6 Nonhydrocarbons Alcohols 0.03 a-Terpineol 0.16 Linaloijl 0.30 Carveol 0.05 Geraniol 0.19 C16H240(Caryophyllene alcohol?) 0.12 3-Hexen-1-01 Carbonyl compounds 0.1 Citral 0.1 Carvone 0.45 G15H220(ketone) ' 0.20 C 1 2 H 2 ~ 0(ester) 2 Oxides 0.37 Linalotil monoxide 0.80 Caryophyllene oxide 0.32 Other oxides 0.40 Polyoxygenated compounds Nitrogen compounds Trace N-methyl methyl anthranilate Trace CI3HI6N(substituted indole?)

26.0 19.2 17.7 1.4 0.10 Trace 0.11 Trace 6.8 0.88 0.23 0.30 0.05 0.23

Canned juice stored 4 yr. (mdkg.1 0.6 8.2 0.1 460 23 23.3 2.9 1.6 Trace Trace

27.6 12.4 11.2 0.87 0.12 Trace 0.14 0 15.2 2.02

0.08

0

0.27 0 0.54 0

0.1 0.1 0.42 0.20

0.1 0.1 0.62 0.20

2.03 0.80 0.32 0.86

8.95 0.27 0.32 0.97

Trace Trace

0 0

0

b

From Kirchner et al. (1953); Kirchner and Miller (1953). The compounds in bold type are those which showed major changes in processing or storage.

338

J. F. KEFFORD

TABLE XIX VOLATILE FLAVORING CONSTITUENTSOF ORANGEJUICE' Fresh juice (mg./kg.)

Constituent

Freshly canned juice (mg./lrg.)

Volatile water-soluble constituents Acetaldehyde 3.0 3.0 Furfuralb Trace Trace Acetone Trace Trace Diacetyl Ethanol 380 550 Methanol Present 0.8 Acetic acid 5.8 2.8 Propionic acid Butyric acid Isovaleric acid Unsaturated acid A (C~H802) 0.1 0.1 Hydrogen sulfide Trace Trace

Total oil Hydrocarbons d-Limonene p-Myrcene a-Thujene CIKH24 (1) C I K H Z(11) ~ Nonhydrocarbons Alcohols a-Terpineol Linalool Carveol n-Hexan-1-01 n-Octan-1-01 n-Decan-1-01 3-Hexen-1-01 C16H280 (I, farnesol?) CIKHZEO (11) CTHIEOZ Carbonyl compounds n-Hexanal n-Octanal n-Decanal n-a-Dodecanal( ?) Citronella1 C I ~ H Z(I, , ~aldehyde?) C16H240 (11, aldehyde?) C1,H220 (ketone?) Carvone

Volatile oil constituents 91.6 88.4 80.1 1.98 0.30 5.8 0.20 3.2

b

Canned juice stored 3 yr. (mg./kg.) 0.8 5.I Trace Trace 484 62 18.6 0.1 Trace 0.4 0.7 None

76.4 71.0 63.6 1.14 0.30 5.78 0.18 5 4

54.4 46.0 43.0 0.69 0.03 2.04 0.21 8.4

0.32 0.93 0.06 0.10 0.21 0.10 0.10 0.07 0.14 0.07

1.72 1.10 0.17 0.14 0.23 0.09 0.18 0.24 0.16 0.08

4.08 0.12 0.86 0.08 0.19 0.07 0.06 0.23 0 0.09

0.04 0.06 0.05 0.OG 0.04 0.14 0.07 0.09 0

0.03 0.06 0.04 0.06 0.04 0.10 0.12 0.09 0

0 0 0.02 0 0.02 0.15 0 0.09 0.08

I

T H E CHEMICAL CONSTITUENTS OF CITRUS FRUITS

339

TABLE XIX (Continued)

Constituent Esters Ethyl isovalerate Ethyl ester of acid A Methyl a-ethyl-n-caproate Citronellyl acetate Terpinyl acetate Polyoxygenated compounds Other compounds a

b

Fresh juice (mg.bg.1

0.01 0.03 0.06 0.10 0.08

0.12 -

Freshly canned juice (mg./kg.) 0.01 0.03 0.10 0.04 0.01 0.15 -

Canned juice stored 3 yr. (mg./kg. 1

0 0 0.02 0.02

0 0.75 0.59

From Kirchner and Miller (1967). The compounds in bold type are those which showed major changes in processing or storage.

originally present, canned grapefruit juice may not show marked flavor deterioration in storage. In stored, canned orange juice, no linalool monoxide was found, but a-terpineol increased greatly in concentration. There was a general loss of the carbonyl compounds to which the characteristic flavor of orange oil is attributed. Most significant, however, was a 30% loss in total oil content, presumably to the aqueous phase. Among the water-soluble constituents, marked increases in concentration were observed for furfural (presumably derived from sugars and ascorbic acid), for methanol (presumably from hydrolysis of pectins), and for lowmolecular weight fatty acids, particularly acetic acid. The flavor deterioration in canned orange juice in storage, leading to the appearance of “stale” off-flavors, appears to be due mainly to reactions among the nonvolatile water-soluble constituents. This view is confirmed by the fact that similar deterioration occurs in juices canned after removal of the volatile oils. From infrared spectrograms of the volatile oils from stored canned orange juices, Blair et aZ. (1952) obtained evidence for the acidcatalyzed hydration of d-limonene to form a-terpineol. I n model systems consisting of limonene dispersed in citrate buffer solutions, held for 7 days at 175OF., further hydration and dehydration reactions occurred, leading to the formation of terpinenes, cineoles, and terpinolene. Such compounds were held by Blair et al. (1952) to be responsible for the L G terpeney” off-flavors that predominate in high-oil orange juices after storage; but no terpinenes, cineoles, or terpinolene were found by Kirchner’s group. Alvey and Cahn (1956) have claimed that a steam-volatile con-

340

J. F. KEFFORD

stituent of orange juice, which was not identified but was possibly terpenoid, was responsible for producing characteristic nausea symptoms in susceptible human subjects.

3 . Diacetyl and Acetoin

An off-flavor in concentrated citrus juices, described as similar to the flavor of buttermilk, has been attributed to diacetyl (CH,CO. COCH,) produced, together with the related compound acetoin (CH,CHOH. COCH,) by microbial fermentation. Kirchner and Miller (1957) found only a trace of diacetyl in the volatile flavoring substances of canned orange juice (Table XIX). In fresh orange juice, Hill et al. (1954), using an analytical method involving distillation, found about 0.2 p.p.m. of diacetyl. Beisel et al. (1954) used a gas-stripping technique which avoided heating the sample, and they found much lower diacetyl contents (0.03 to 0.05 p.p.m.) in fresh juices. However, when they used a distillation method, they found diacetyl contents in the range 0.5 to 1 p.p.m., and they attributed the discrepancy to the presence of a volatile degradation product of sugars, which gave a positive VogesProskauer reaction. Serini (1956) reported that acetoin was usually absent from citrus fruits, but Swift and Veldhuis (1957) appear to believe that diacetyl and acetoin may be naturally present in oranges in significant amounts. They found, using a distillation procedure, that peel juices showed consistently higher diacetyl contents than pulp juices throughout the season. The weight of evidence on this matter suggests that diacetyl and acetoin are present in very low amounts, if at all, in fresh juices; the larger quantities detected are evidently microbial in origin or else represent artifacts produced during analysis. XII. NONVOLATILE CONSTITUENTS OF CITRUS OILS

In addition to the volatile constituents, citrus peel oils contain a nonvolatile waxy residue in varying amounts, depending on the method of oil extraction. The residue consists of waxes, hydrocarbons, steroids, triterpenes, and substituted coumarins; it is probably derived from the cuticle wax of the fruit (Markley et at., 1937).

A. COUMARIN DERIVATIVES By chromatographic separation on silicic acid columns, cold-pressed lemon oil was separated into about 25 fractions, several of which deposited crystalline solids (Stanley, 1958; Stanley and Vannier, 1957a). From these deposits, a number of pure compounds were isolated, and

THE CHEMICAL CONSTITUENTS O F CITRIJS FIlUITS

341

most of them were identified as coumarins or furocoumarins with hydroxyl, methoxyl, and isoprenoid substituents. Compounds of this type that have been identified in citrus oils are listed in Table XX. Lemon, lime, and bergamot oils appear to cont,sin a greater variety of substituted coumarins than the other citrus oils. Column chromaTABLE XX COUMARINCOMPOUNDS IN CITRUSOILS

Oil Lemon"

Limeb

BergamotC

Sweet Orangec Grapefruitd Natsudaidaia

Approximate concentration in oil (mg./100 9.)

Compound

Limettin (citropten,5,7-dimethoxycoumarin) 5-Geranoxy-7-methoxycoumarin Bergamotin (5-geranoxypsoralen) 8-Geranoxypsoralen Byakangelicin [5-methoxy-S-(2,3-dihydroxyisopent any1oxy)-psoralen] Unidentified compounds Limettin 5-Geranox y -7-metlioxy coumarin Bergaptol (5-hydroxypsoralen) Bergamotin SGeranoxypsoralen Isopimpinellin (5,s-dimethoxypsoralen) Limettin 5-Geranoxy-7-methoxycoumarin Bergaptol Bergapten (5-methoxypsoralen) Bergamotin Aurapten (7-methoxy-8-epoxyisopentanylcoumarin) 7-Geranoxycoumarin Umbelliferone (7-hydroxycoumarin) Aurapten Umbelliferone Bergaptol Isoimperatorin (auraptinI5-isopentenyloxypsoralen)

mco Coumarin

From From c From d From a From b

Psoralen

Stanley (1958) and Stanley and Vannier (1957a). Caldwell and Jones (1945) and Stanley (1958). Geissman and Hinreiner (1952). Rodighiero and Caporale (1954), and Stanley (1958). Croaby (1954) and Vannier and Stanley (1958). Nomura (1950) and Matsuno (1956).

342

J. F. KEFFORD

tography of oil of bitter orange yielded only small amounts of crystalline coumarin derivatives (Stanley and Vannier, 1957b). Bernhard (ly58b) examined the occurrence of substituted coumarins in lemon juice which was specially prepared to avoid contamination with peel oil. Chromatographic separation of petroleum ether extracts revealed the presence of eight substances, five of which were tentatively identified as limettin, bergamotin, byakangelicin, 8-geranoxypsoralen, and oxypeucedanin ( 5-epoxyisopentanyloxypsoralen). The substituted coumarins have characteristic ultraviolet absorption spectra which have been applied in procedures for the identification of pure cold-pressed lemon oils and for the detection of adulteration with other citrus oils (Sale, 1953; Stanley and Vannier, 1957b). A more sensitive fluorometric method for the detection of grapefruit oil in lemon oil, developed by Vannier and Stanley (1958), depends on the fact that grapefruit oil contains 7-geranoxycoumarin which is absent from lemon oil. This compound is hydrolyzed to the strongly fluorescent 7hydroxycoumarin (umbelliferone) which is detectable in very low concentrations by visual or photoelectric fluorheby.

B. STEROIDS AND TRITERPENOIDS From the unsaponifiable fraction of the nonvolatile residue of grapefruit peel oil, Weizman et al. (1955) isolated p-sitosterol (22-dihydrostigmasterol) , and friedelin, a saturated triterpenoid ketone previously known from cork and lichens. Accompanying the p-sitosterol in grapefruit and orange peel oils, Weizman and Mazur (1958) found a new sterol, citrostadienol, which was subsequently shown to be 4 ~ r n e t h y l A7’24(28)-~tigma~taden-3p-ol (Mazur et al., 1958). Swift ( 195213) had previously recovered p-sitosteryl-D-ghcoside from Florida Valencia orange juice in a yield of 0.004%, and Ma and Schaffer (1953) isolated p-sitosterol and its D-glucoside from dried grapefruit residues consisting mainly of peels and seeds. XIII. FLAVONOIDS

Citrus fruits contain yet another group of distinctive chemical substances which belongs to the broad category of flavonoid compounds. Bate-Smith (1954) has reviewed the occurrence and significance of flavonoid compounds in foods in general. The flavonoids present in citrus fruits comprise flavanone and flavone glycosides and also some highly methoxylated flavanones and flavones which are known to occur in no other natural source. The methoxylated compounds are thought to be present in the fruit in solu-

THE C H E M I C A L C O N S T I T U E N T S OF C I T R U S F R U I T S

343

tion in the oil sacs, while the glycosides are distributed generally throughout the tissues. A. IDENTIFICATION OF CITRUSFLAVONOIDS Present knowledge of the identity and distribution of flavonoids in the various species of citrus fruits is summarized in Table XXI; only the most recent relevant references are given. Attempts have been made to trace patterns of flavonoid distribution associated with taxonomic relationships (Swingle, 1948). Recent work (Horowitz, 1956, 1957) has revealed, however, that citrus fruits contain much more complex mixtures of flavonoids than were formerly suspected to be present. Nevertheless, some broad patterns may be distinguished. Thus, sweet oranges, mandarins, lemons, and citrons contain hesperidin as the principal flavonoid, while grapefruit and pumelos contain naringin. When the two groups come together as they are thought to do in natsudaidai (Swingle, 1948), both hesperidin and naringin are present. Satswnelo, however, a hybrid of similar parentage, is unique among citrus fruits in containing rutin in amounts up to 3.2% in the dry peel of green fruits. Rutin was not detectable in Valencia oranges, grapefruit, or lemons (Krewson and Couch, 1948). The sour oranges (Citrus aurantium) show some anomalies which merit further investigation. In the immature fruit of European varieties, Karrer (1949) found only hesperidin and neohesperidin; hesperidin predominated in samples from Italy, and neohesperidin predominated in samples from Spain. These two flavanone glycosides differ only in the sugar residue. Hesperidin is the 7-p-~-rhamnosido-6-~-glucoside (i.e., the rutinoside) of I-hesperetin (Arthur et al., 1956), while neohesperidin is probably the corresponding rhamnosido-4-glucoside (Zemplkn et aZ., 1938). Other workers, however, have found naringin to be the principal fIavonoid in C . aurantiun varieties, e.g., Australian Seville oranges (Kefford and Chandler, 1950), bitter oranges in Greece (Alivertis, 1958), and the Japanese varieties Kabusu and cyathifera. The latter varieties have been classified as C . aurantium on morphological grounds, but Hattori et al. (1952) consider that Citrus grandis parentage is more likely. In 1886, Tanret claimed to have found i n C.aurantium hesperidin together with “isohesperidin” and “aurantamarin.” The latter compounds have never been confirmed as pure chemical entities, and the names should be allowed to disappear from the literature. Hesperidin chalcone has been reported to occur in lemon peel in the form of a complex with protein (Wawra and Webb, 1942), but this

TABLE XXI FUVONOIDS IN CITRUS FRUITS Fruit Sweet orange

Sour orange

Variety 4 Var.

Batavia 2 Var. Amara Kamala Seville

Mandarin

Region Florida

Flavonoids identified

Aglycone

Hesperidin

(I) 3‘,5-OH,4’-OCH3,7-R~ Hesperetin

Eriodictin

(I) 3’,4’,5-OH,7-R*

Eriodictyol

India Hesperidin Greece Hesperidin Italy, Spain Hesperidin Neohesperidin (I) 3’,50H,4’-OCH3,7-Rz Hesperetin India Hesperidin (11) 3,4’,6,7,8-OCHa Auranetin (I) 4’, 5-OH, 7-R 1 Naringenin Australia Naringin

Kabusu eyathifera

Japan Japan Japan

Naringin Rhoifolin Hesperidin

Tangerine Tangerine

Sicily Russia Florida

Hesperidin Hesperidin Tangeritin

Tankan

Formosa

Hesperidin

-

Formula and substituents”

Nobiletin Ponkan

Japan

Ponkanetin

Grapefruit

5 Var.

Florida

Naringin

Pumelo

Shaddock

India

Naringin

Matheepala India Buntan Japan Thong Dee Florida

Naringin Naringin Naringin

(11) 3’,4’,5,6,7,%0CH, (I) 4‘,5,6,7,8-OCH3

References Hendrickson and Kesterson (1954a) Bruckner and SzentGyorgyi (1936) Patnayak et al. (1942) Exarchos (1958) Karrer (1949) Karrer (1949) Patnayak et al. (1942) Murti et al. (1948) Kefford and Chandler ( 1950) Hattori et al. (1952) Hattori et al. (1952) Iwasaki (1936): Naito et al. (1942) Sara and Micale (1957) Kotidi (1950) Nelson (1934); Goldsworthy and Robinson (1957) Tsukamoto and Ohtaki

-

(1941) Kesterson and Hendricksoa (1953s) Seshadri and Veeraraghaviah (1940) Patnavak et al. (1942) Hattoii et al. (1949) . Kesterson and Hendrickson (1953a)

Meyer

Florida

Hesperidin Eriodictin Diosmin Limocitrin Hesperidin ( ?)

Ponderosa

Japan

Citronin

Lime

Tahiti

Florida

Hesperidin ( ?)

Citron

Naranja Dabba

India India

Hesperidin Hesperidin

Citrus

-

China China Japan

Hesperidin Neohesperidin Naringin

Lemon

Mixed

kotokad Citrus fuscab Trifoliate orange

Temple orange Tangelo Satsumelo Natsudaidai a

-

California

(11) 3’,50H,4’-OCH8,7-R3 Diosmetin

-

(11) 3,4’,5,7-OH,3‘,8-0CHa

(I) ~ - O H , ~ ’ - O C H ~ , ~ - RCitronetin I

Horowits (1956, 1957) Horowits (1956, 1957) Horowitz (1956, 1957) Horowits (1956, 1957) Hendrickson and Kesterson (1954a) Yamamoto and Oshima (1931); Simpson and Whalley (1955) Hendrickson and Kesterson (19544 Patnayak et al. (1942) Patnayak et al. (1942) Hsu and Tominaga (1949) Hsu and Tominaga (1949) Hattori and Shimokoriyama (1956) Hattori and Shimokoriyama (1956)

Poncirin

(I) 5-OH,4’-OCH,,7-Rs

Isosakuranetin

Citrof olioside

(I) 5-OH,4‘-OCH3,7-Ra

Citrofoliol (I-isosakuranetin) Sannie and Sosa (1949) Hendrickson and Kesterson (1954s) Hendrickson and Kesterson (1954a) Quercetin Krewson and Couch (1948) Nomura (1953a, 1954) Nomura (1953a, 1954)

-

Florida

Hesperidin(?)

Orlando

Florida

Hesperidin( 1)

-

Florida Japan

Rutin Hesperidin Naringin

(11) 3’,4‘,5,7-OH,3-R1

n 0

I

Structural formulas and aubetituenta. i

RI = ~-rhamnosido-6-glueoayl. RI = 0-rhamnosido-Pplucosyl. RI

(I) Flavanone 6

Not mentioned in Swingle’a (1948) classification.

=

Unidentified augar residue, presumed to be Ri.

(11) Flavone w

h

346

J. F. KEFFORD

report must be regarded with scepticism since Shimokoriyama (1957) has questioned the identity of the hesperidin chalcone which Wawra and Webb prepared. The occurrence in citrus fruits of a great variety of interrelated flavonoid compounds invites conjecture about the biogenetic processes by which they are synthesized (cf., Seshadri, 1951; Geissman and Hinreiner, 1952). It may be postulated that from common precursors having the C,-C,C, skeleton, biosynthesis may proceed in several different directions, according to the presence or absence of specific enzyme systems. For instance, a number of corresponding pairs of flavanone and flavone, representing different levels of oxidation of the heterocyclic ring, occur in citrus fruits, e.g., hesperidin and diosmin, naringin and rhoifolin, and rutin and eriodictin. Again, hesperidin and eriodictin represent a pair differing only in the methylation of one hydroxyl group. When a pair of these compounds occurs in the same fruit, enzyme-controlled interconversion may reasonably be assumed. Shimokoriyama (1957) has postulated that chalcones may be intermediates in the biosynthesis of flavonoids and has produced evidence for the presence in citrus peels of an enzyme, designated “flavanone synthease,” which converts phloroglucinol-type chalcone glycosides to flavanone glycosides.

B. DISTRIBUTION OF CITRUSFLAVONOIDS The role of the flavonoids in the metabolism of citrus fruits is obscure, but the fact that they may make up as much as 75% of the total solids of the fruit in the first stages of development suggests a significant physiological function. Karrer (1949) found up to 28.5% hesperidin and up to 10% neohesperidin on a dry basis in the smallest fruits examined of Italian and Spanish sour oranges. Similarly, Florida workers (Kesterson and Hendrickson, 1952, 1953a; Hendrickson and Kesterson, 1954a) found up to 75% naringin on a dry basis in grapefruit %-in. in diameter, and up to 35% hesperidin in oranges of that size; in both fruits the flavonoid content decreased to 2 to 3% at maturity. Kesterson and Hendrickson (1958) found hesperidin to be the predominating flavonoid in Florida lemons. It has been generally observed that the absolute flavonoid content per fruit increases during the growth of citrus fruits up to a certain diameter and then becomes approximately constant; it follows that the concentration decreases as the fruits mature. This trend with maturity has been observed in sweet oranges and mandarins (Hendrickson and Kesterson, 1954a; Iwasaki, 1936; Naito et al., 1942), in sour oranges (Karrer, 1949), in grapefruit and shaddocks (Kesterson and Hendrick-

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

347

son, 1952, 1953a), and in natsudaidai (Shioiri et al., 1953; Nomura, 1954). Hendrickson and Kesterson ( 1954b) obtained maximum yields of hesperidin and naringin early in the season. From the basis of 10 to 15 lb. of crude product per ton of wet peel, the yields declined at the rate of about 1 lb. per ton per month with advancing maturity. Davis (1947) determined the flavonoid content of various parts of mature grapefruit, lemons, oranges, and tangerines and found the descending order of concentration to be: core, albedo, segment membranes, flavedo, juice vesicles, and juice (cf., Maurer et al., 1950; Burdick, 1954). In grapefruit 90%, and in oranges 70 to 80% of the flavonoids present in the whole fruit are located in the albedo, rag, and pulp (Kesterson and Hendrickson, 1953a; Hendrickson and Kesterson, 1954a). Davis (1947) also found that juice withdrawn from the juice vesicles of grapefruit by means of a glass needle contained almost as much naringin as juice expressed from the separated juice vesicles. Evidently naringin is present in solution in the juice, but additional amounts enter the juice fraction during the operations of mechanical juice extraction. Atkins and Rouse (1935) found that the flavonoid content of Valencia orange juice depended on the method of juice extraction and ranged from 0.035 to 0.065%. The higher the pulp content of the juice, the higher was the flavonoid content. Commercial Florida orange juices contained 0.03 to 0.05% flavonoids throughout the season (Swift and Veldhuis, 1957) while “peel juices” contained 0.16 to 0.3%, the content declining towards the end of the season. Similarly, Florida grapefruit juices contained 0.02 to 0.04% naringin (Kesterson and Hendrickson, 1953a).

C. PROPERTIES OF CITRUSFLAVONOIDS

I . General Hesperidin is almost tasteless, and its presence in orange juice is generally not apparent to the consumer. Naringin, however, is intensely bitter and is responsible for the characteristic bitterness of grapefruit, Seville oranges, and marmalades made from these fruits. The constitution of naringin offers no clue as to why it alone among the flavanone glycosides should exhibit the property of intense bitterness; the aglycone, naringin, is not bitter. The flavanone glycosides are sparingly soluble in water, e.g., naringin has a solubility of 0.50 g./liter at 2OOC. (Pulley, 1936), while hesperidin is almost completely insoluble in water, cold or hot; under some conditions the flavanone glycosides may crystallize out in citrus products. Hesperidin crystals are commonly found in frosted oranges

348

J. F. KEFFORD

(Samisch and Cohen, 1952; Alderman and Godfrey, 1953) and occasionally in canned orange juice (Von Loesecke, 1954). Naringin also crystallizes in frosted grapefruit and in processed grapefruit products, in the form of round white aggregates (Boyes et al., 1945; Keenan, 1946).

2. Analytical Hendrickson and Kesterson (1957) critically examined methods for the analysis of citrus flavonoids and concluded that the colorimetric method of Davis (1947), depending on the production of a yellow color on the addition of alkali in the presence of diethylene glycol, was most suitable for routine assaying. However, Ting (1958) found that grapefruit juice contains substances other than naringin which form a yellow color in alkaline diethylene glycol. This interference may be circumvented by applying the Davis procedure before and after enzymic hydrolysis of the naringin present. Ting has shown that enzymes, capable of hydrolyzing naringin (to naringenin, glucose, and rhamnose) are present in some commercial pectolyzing enzymes of fungal origin. Hendrickson et al. (1958) have recently devised an ultraviolet absorption method for the determination of naringin which gives results in agreement with the Davis procedure. A paper test also based on the reaction of flavonoids with alkali has been suggested for distinguishing citrus juices from substitutes (Dickinson and Harris, 1950). For the detection of orange peel in orange beverages, Born (1957) proposed a test which appears to depend on the chromatographic separation of a highly methylated flavonoid, probably tangeritin (Stanley, 1958).

3. Pharmacologicail Citrus flavonoids have no known function in human nutrition although they have recently come to be designated as “bioflavonoids” because of some evidence of pharmacological properties, e.g., a synergistic association with ascorbic acid and a favorable effect on capillary fragility (“vitamin P” effect). These properties of flavonoid compounds have been reviewed by Scarborough and Bacharach (1949), and Martin and Szent-Gyorgyi (1955). It would appear however that most of the therapeutic claims lack support from well-controlled clinical studies. XIV. LIMONOID BITTER PRINCIPLES

The occurrence of the bitter-tasting flavonoid, naringin, in citrus fruits has been discussed in the preceding section. Citrus fruits may also

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349

contain bitter principles that have been designated limonoids (Kefford, 1955) since the compound occurring most commonly is limonin.

A. SOMEGENERALOBSERVATIONS

1. Distribution of Bitter Principles Limonin was first isolated from the seeds of lemons and bitter oranges by Bernays in 1841. Since then it has been found to occur widely in the seeds, bark, and roots of other members of the Rutaceae family. Present knowledge of the occurrence of limonin and related bitter principles in citrus fruits is summarized in Table XXII. Limonoid bitter principles have been shown, at least qualitatively, to be present in the seeds and in all the structural tissues of citrus fruits, especially in the segment walls, the albedo, and the core, and even in the walls of the juice vesicles (Higby, 1938; Samisch and Ganz, 1950).

2. Effect of Maturity The amounts of bitter principles in all parts of citrus fruits decrease with advancing maturity (Higby, 1938; Emerson, 1949; Samisch and Ganz, 1950). The crude bitter principle content of the dried peel of Navel oranges on rough lemon rootstock decreased from 0.10 to 0.06% while that of Valencias on rough lemon stock decreased from 0.07 to 0.00% during three months (Chandler, 1958b). Rockland et al. (1957) have claimed that disappearance of the bitter principles from oranges continues by accelerated metabolic processes during storage at temperatures of 80 to 90°F. after removal from the tree. Limonoid bitter principles may cause bitterness in extracted citrus juices. Fortunately, however, the bitter principles disappear, at least from the endocarp of most citrus fruits, by the time they reach optimal maturity. But some varieties, notably Navel oranges and to some extent Shamouti oranges (Samisch and Ganz, 1950), retain significant amounts of bitter principles at normal maturity, and bitterness in the juice then becomes an important practical problem. 3 . Effect of Rootstock The rate of disappearance of the limonoid bitter principles with maturity is greatly influenced by the rootstock on which the scion variety is grafted. Marsh (1953), working in California, observed that bitterness disappeared early in the season from Navel oranges grown on grapefruit and trifoliate orange stocks, late in the season from fruit on stocks of sweet and sour oranges and Navel cutting, and not at all from fruit on rough lemon stock. Similar observations were made in

TABLE XXII LIMONOID BITTERPRINCIPLES m CITRUSFRUITS ~

Fruit

Variety

Origin

Part of fruit

Sweet orange

-

-

Orange and grapefruit Grapefruit Tangerine Pumelo Lemon Sweet lime Natsudaidai

0

b

Navel Valencia Navel Valencia

California California California Catlifornia

Seeds Peel, pulp Peel, pulp, seeds Pulp, juice Seeds

Shamouti Navel Valencia Mixed

Israel Australia Australia Florida

Peel, pulp Peel, juice Peel, seeds Seed oil

-

Shaddock I

-

-

Florida Russia India India

Seed oil Peel Seeds Seeds Seeds California Seeds Sicily Seeds India Japan Peel, pulp Japan Seeds

Identified only by solubility behavior. Designated “lemonine” in the abstract.

Bitter principles isolated Limonin, isolimonin Limonin, isolimonin Limonin, isolimonin Lim0Xlil-l Limonin, nomilin, substance X Limonino Limonin, limonexic acid Limonin, limonexic a,cid Nomilin, limonin, obacunone Limonin Limonid Limonin, neolimonin Limonin, isolimonin Citrolimonin Limonin, nomilin Limonin,. isolimonina Limonin Compound C2.H32Ol8 Limonin, isolimonin, ‘‘hydrolimoninic acid ’’

~~~

~~~~~~~~~~~~

References

-

Koller and Czerny (1936, 1937) Etigby (1938) EIigby (1938) Emerson (1948) Emerson (1948) Samisch and Ganz (1950) Chandler and Kefford (1951, 1953) Chandler and Kefford (1951, 1953) Emerson (1951)

4

Nolte and Von Loesecke (1940) Xotidi (1950) Mookerjee (1940) Seshadri and Veeraraghaviah (1940) Feist and Schulte (1936); Brachvogel (1952) Emerson (1948) Averna and Petronici (1955) Seshsdri (1943) Inagaki (1951) Nomura (1952)

0

T ?! M

2 0

U

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

351

Australia by Kefford et al. (1952). Navel oranges from trifoliate orange, tangelo, and Cleopatra mandarin stocks gave the least bitter juices; fruit from sweet orange, East Indian lime, and sweet lime stocks gave juices intermediate in bitterness; and juices from fruit on Kusaie lime and rough lemon stocks were most bitter. Even juices from Valencia oranges on rough lemon stocks were bitter up to a n advanced stage of maturity. Determinations of the amounts of crude bitter principle in the peels of these fruits showed parallel trends (Chandler, 1958b). Thus, no bitter principle could be recovered from the peel of Navel and Valencia oranges on trifoliate orange stocks, while Navels on rough lemon stock yielded 0.03 to 0.10% bitter principle on a dry basis, and Valencias on rough lemon stock yielded 0 to 0.07%, the highest contents being found in the least mature fruits. 4 . Delayed Bitterness in Orange Juice

A most intriguing aspect of the problem of bitterness in the juice of varieties such as Navel oranges is the fact that the juice is not bitter immediately after extraction but becomes so after a few hours at room temperature, or within a few minutes if the juice is heated. To explain this phenomenon, a number of workers (Higby, 1938; Emerson, 1948, 1949; Samisch and Ganz, 1950) have postulated that the structural tissues of the fruit contain a nonbitter precursor substance which diffuses into the juice where it is slowly converted into the bitter principle. Emerson (1948, 1949) considered that the precursor might be a diacid, or a monoacid-lactone, or a glycoside which is stable at the pH of the tissues but is converted to limonin at the pH of the juice. He was unable, however, to isolate the precursor substance, and Chandler (1958b) also sought such a substance without success. It appears to the reviewer and his fellow workers that the hypothesis of a precursor substance is unnecessary and that the phenomenon of delayed bitterness can be explained on physical grounds. Initially, limonin is present in the juice as a constituent of the tissue fragments that make up the suspended solids. Because of its low solubility in orange juice, it will take an appreciable time for the limonin to diffuse from these particles and to reach a concentration of about 2 p.p.m., which is necessary for the juice to taste distinctly bitter. This process will be accelerated by heating. When finely-divided limonin or limonin adsorbed on alumina is suspended in water acidified to pH 3.5, the liquor is not bitter after 8 hr. at room temperature, but becomes bitter on further standing or on heating (Chandler, 1958b). Samisch and Ganz (1950) observed delayed bitterness both in reamed juices and in albedo suspensions dispersed in water. In the latter case, there was no bitterness if the solid particles

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were removed immediately after dispersion. Although this observation is interpreted as supporting the hypothesis of a precursor substance, it may equally well be cited in support of the physical mechanism.

B. CHEMISTRY OF LIMONIN Limonin is a high-melting compound (m.p. 302-304°C. in vacuumsealed tube) with a very low solubility in water. It appears to have no important physiological properties (Emerson, 1949) except its intense bitterness which is detectable down to a limonin concentration of 0.75 p.p.m. The empirical formula of limonin is C,,H,,O, but its molecular constitution is not yet known. The fact that the structure of limonin is not yet established, although the compound has been known for over 100 years and is readily isolated from common fruits, demonstrates forcibly the intractable nature of limonin as a subject for chemical investigation. Early studies, reviewed by Geissman and Tulagin (1946), present a story of frustration and disappointment. Workers in several European, Japanese, and American schools of organic chemistry took up the investigation of limonin only to drop it again with little progress achieved. About 1945, the technological importance of bitterness in Navel orange juice in California and Australia, where approximately half of the oranges grown are Navels, prompted further attacks on the problem of the structure of limonin by chemists who were primarily interested in food science. These investigators advanced the subject substantially (Emerson, 1948, 1951,1952; Chandler and Kefford, 1951,1953; Kefford et at., 1951 ;Chandler, 1958b) . More recently when a successful solution to the limonin problem seemed imminent, organic chemists in universities have taken up the subject again (Melera et at., 1957; Corey et al., 1958). It is now generally agreed that limonin consists of a perhydronaphthalene nucleus, to which are attached two cyclic ether groups and a furan ring; and also that it is related to two other bitter principles whose constitutions also defied elucidation until very recently, namely, marmbiin, the bitter principle of horehound (Marrubium uutgare) and columbin, a bitter principle from the columba root (Jatrorrhizap a t m t a ) . Partly by analogy with these bitter principles and as an attempt to represent the known reactions of limonin in structural terms, Chandler (1958b) has put forward a tentative structure (Formula 111). Experience with other natural products suggests that this proposed structure is unlikely to stand without modification, but that eventually a molecular configuration for limonin generally acceptable to all the workers in the field will be established.

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A

0(111) Limonin

C. OTHER BITTERPRINCIPLES The other limonoid bitter principles listed in Table XXII are assumed to be related to limonin although no direct relationships have been established. They are, in general, bitter lactones containing 25 to 28 carbon atoms and 7 to 10 oxygen atoms, and they frequently occur together with limonin. Specific comments may be made on some of these compounds: (1) Obacunone, C,,H,,,O,, was isolated by Emerson (1951) from citrus seeds and is known to occur in other Rutaceae. It has been shown by Kubota and Tokoroyama (1957) to have a 3-substituted furan ring, in common with limonin and columbin. (2) Nomilin, C,,H,,O,, was first found in citrus seeds by Emerson (1948, 1951), who presented evidence indicating that nomilin is probably acetoxydihydroobacunone with the acetoxy group p to one of the lactone carbonyl groups. Dean and Geissman (1958) have recently proposed partial structures for nomilin (Formula N) and obacunone (Formula V). The placing of the two lactone rings immediately adjacent in Formulas (IV) and (V) represents a fundamental difference from the structure of Formula (111) proposed for limonin. It is unlikely, however, on general grounds that limonin and nomilin are radically different in structure, and therefore a reconciliation of the proposed structures must be sought.

(IV) Nomilin

(V) Obacunone

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J. F. KEFFORD

(3) Citrolimonin, C,,H,,O,, isolated from lemon seeds (Table XXII), is very similar to limonin in physical and chemical properties, and the two compounds are almost certainly identical. (4) Isolimonin, C,,H,,O,, and neolimonin, C,,H,,O,, are two compounds reported by earlier workers (Table XXII) , which have not been recovered from citrus peels and seeds in recent studies, and their separate identity from limonin is doubtful. It appears likely that the preparations originally isolated were solvates of limonin, which are known to be formed very readily and to show considerable stability. ( 5 ) Limonexic acid, C,,H,,O,,, is a bitter principle occurring together with limonin, particularly in citrus seeds. It was shown (Chandler and Kefford, 1953) to be an oxidation product of limonin, and it may be a natural degradation product, but it is not thought to be an artifact produced during the processes of extraction and purification. (6) Substance X, (C9Hlz04)n,a bitter principle isolated in small amounts from Valencia orange seeds (Emerson, 1948), closely resembles limonexic acid in physical properties, but Emerson considers that the two compounds are not identical (see Chandler and Kefford, 1953). XV. RESEARCH NEEDS

When present knowledge of the chemical constitution of citrus fruits is compared with the information available to Braverman in 1949, it is evident that substantial progress has been made in the ten years surveyed in this review. I n fact it might reasonably be claimed that the catalog of chemical compounds in citrus fruits is approaching completion. Nevertheless, the record in several fields is still untidy, and there is room f o r wider application of the new techniques of partition and gas chromatography to bring order to our knowledge of, for instance, the volatile flavoring constituents of citrus oils and the flavonoids of citrus fruits. The major need, however, of the food scientists and horticulturists who are interested in citrus fruits is for knowledge that will permit them to control fruit composition in desired directions by treatment of the tree. Attention has been drawn to the gross effects on composition of many agricultural practices. It is necessary now to explore more deeply the physiology of the tree and the fruit in order to understand the mechanisms of biosynthesis, in the hope that it may be possible to control the natural processes so that the plant produces more solids, or more acid, or more ascorbic acid according to the appropriate need. In particular, a greater understanding is needed of the mechanism of genetic control of fruit composition which leads to the gross differ-

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ences between the kinds and varieties of citrus fruits and also to the more subtle differences between budwood strains (Cohen, 1956) and nucellar embryonic lines (Cameron et al., 1957). The experiments of Erickson (1958) on the grafting of developing fruits from one variety to another are of particular interest in this connection. Another major influence on fruit composition is that of the rootstock, but the mechanism of this effect also awaits elucidation. How does the rootstock control the sweetness, the acidity, and even the bitterness of the fruit on the scion variety? Is it by control of the supply of organic and inorganic nutrients from the roots (see Section VI), or is it by secretion of chemical agents into the sapstream of the tree? It is obvious from the record that much of our present knowledge of the composition of citrus fruits has been contributed by plant physiologists. From them also must come the answers to the forementioned problems. A second major need for research lies in a direction that has been only briefly touched upon in this review: that is the need for greater knowledge of the reactions of the constituents of citrus fruits when they are processed into citrus products. Many of the studies on citrus juice composition that have been reviewed were prompted by the problems of flavor and color deterioration in processed c i m s juices. The solutions to those problems are still incomplete, but it is now generally accepted that anaerobic reactions of citrus juice constituents are mainly responsible (Guyer and Boyd, 1954; Kefford et al., 1959). In such reactions, phosphatides, amino acids, sulfur compounds, terpenes, and volatile flavoring substances probably all take part, but their respective roles have not been resolved (Swift, 1951). In the attack on the outstanding problems of quality deterioration in processed citrus products, comprehensive information on the nature and concentrations of the constituents of the raw fruits is an essential first step.

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63, 53.

ERRATA: VOL. VIII Page 145, line 1, 2.72 g. not 2.82 Page 147, lines 7-8, formula to read:

100

[{loo - ( A + E ) k ) A x K ]

Page 147, lines 9-10, lc is the coefficient of solubility in water and is 1.0020 at 15°C. K is the coefficient of solubility in alcohol and is 3.1993.

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AUTHOR INDEX Numbers in boldface indicate the page on which the reference is listed at the end of the article.

A

Baier, W. E., 289, 291, 306, 312, 313,

Aamlid, K., 203, 217 Adam, W. B., 69, 70, 71, 76, 77, 88, 89, 102, 114, 142 Adams, E., 36, 47 Adams, M. C., 185, 214 Adova, A,, 38, 4, 57 Akerberg, E., 72, 148 Alderman, D. C., 348, 355 Aldrich, B. B., 6, 55 Alexander, 0. R., 86, 142 Alivertis, N., 343, 355 Allen, H. E., 37, 39, 47 Almendinger, V. V., 309, 355 Alvey, C., 339, 355 Anderson, A. J., 74, 142 Anderson, E. E., 30,44, 53, 300, 370 Andreassen, E. G., 95, 144 Andrew, M. M., 35, 47 Antoniani, C., 36, 47 Arigoni, D., 352, 365 Armbruster, G., 71, 143 Arnold, N., 33, 47 Arthur, H. R., 343, 356 Asboe-Hansen, G., 9, 47 Asenjo, C. F., 312, 356 Ashmarin, I. P., 11, 47 Astbury, W. T., 12, 47 Atkins, C. D., 303, 304, 306, 307, 311, 320, 321,347,356, 368 Auerbach, E., 39, 48, 50 Averna, V., 319, 323, 331, 350, 356, 366,

Bailey, C. H., 184, 214 Bailey, G. F., 325, 327, 358, 359 Bailey, K., 5, 6, 13,22, 48 Bain, J. M., 295, 306, 316, 356 Baker, B. E., 41, 49 Baker, G. L., 182, 214 Balls, A. K., 35, 48,323, 362 Barker, J., 93, 148 Baron, M., 183, 214 Barretto, A., 271, 276, 340, 361 Barron, E. S. G., 6,22, 57 Bartholomew, E. T., 289, 291, 292, 293, 294, 295, 296, 298, 300, 301, 302, 308, 309, 314, 319, 330, 332, 356,

356, 362

367 Avidor, Y., 323, 356 Ayres, T. B., 75, 143 Axelrod, B., 322, 323, 356

B Bacharach, A. L., 348, 368 Backinger, G., 205, 217

369 Batchelor, L. D., 294, 357 Bate-Smith, E. C., 1, 10, 11, 14, 15, 17, 20, 21, 48, 329, 342, 357 Batjer, L. P., 190, 214 Bauer, E., 266,267, 277 Baumann, A., 33, 34, 51 Baumann, J., 234, 273 Beadle, B. W., 79, 80, 151 Bear, R. S., 9, 48 Beard, F. J., 42, 58 Beavens, E. A., 185, 218, 245, 281, 336, 349, 367 Becerra de la Flor, J., 293, 357 Beck, K., 38, 48 Bedford, C. L., 79, 143 Bednarczyk, W., 91, 143 Beech, F. W., 223, 229, 230, 240, 241, 243, 245, 246, 247, 256, 270, 273,

274 Behrens, J., 233, 274 Beidler, L. M., 210, 214 Beisel, C. G., 309, 340, 355, 357 Bendall, J. R., 11, 14, 15, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 35, 48 Bendix, G. H., 75, 81, 143 375

376

AUTHOR INDE X

Benk, E., 291, 320, 357 Bennett, B. B., 79, 80, 146 Bergrnann, M., 36, 50 Bernays, F., 349, 357 Bernhard, R. A,, 333, 342, 357 Berry, J. A., 92, 144, 224, 228, 253, 263,

274 Berry, J. M., 252, 263, 278, 284 Berth, L., 135, 137, 143 Bhatia, B. S., 302, 369 Biale, J. B., 309, 372 Bicknell, F., 91, 143 Billmeyer, F. W., 214 Binkley, A. M., 208, 217 Bioletti, F. T., 256, 274 Bionda, G., 313, 357 Birdsall, E. L., 185, 214 Birkner, M. L., 30, 39, 40, 41, 42, 43,

58, 59 Bitters, W. P., 294, 357, 360 Bitting, A. W., 99, 133, 143 Blair, J. R., 75, 143 Blair, J. S., 324, 336, 339, 357 Blanck, F. C., 154, 212, 214 Block, R. J., 94, 148 Blondel, L., 291, 293, 357 Blum, J. J., 12, 26, 27, 49, 55 Bock, G., 39, 49 Boehi, A,, 250, 274 Boggs, M. M., 101, 111, 143, 144 Bohart, C. S., 97, 149 Bohart, G. S., 76, 86,92, 144 Bohrer, C. W., 129, 143 Bonney, V. B., 97,99, 101, 143 Borasky, R., 9, 49 Borbiro, M., 6, 58 Borgstrom, G., 254, 274 Borkowski, G., 205, 220 Born, R., 348, 357 Bossen, D., 186, 214 Botts, J., 12, 26, 27, 55 Boucek, R. J., 46, 56 Bouma, D., 299, 357 Bourne, E. J., 66, 149 Bouton, P. E., 24, 31, 33, 49 Bowen, J. F., 271, 274 Bowen, W. J., 8, 58 Bowes, J. H., 9, 49 Boyd, J. M., 336,355, 360

Boyes, W. W., 348, 357 Boyle, F. P., 73, 151 Bozler, E., 26, 27, 33, 49 Brachvogel, L., 350, 357 Brady, A. L., 101, 150 Braverman, J. B. S., 254, 256, 274, 277, 286, 308. 354, 357 Brenner, S., 89, 143, 145 Brenner, W. D., 80, 146 Brewer, D. B., 10, 49 Briant, A. M., 95, 143 Brice, B. A,, 170, 172, 214 Briza, K., 202, 214 Brown, J. G., 211, 215 Bruckner, V., 346, 357 Brunner, H., 247,248, 274 Brush, M. K., 84,89,95, 143, 146 Buchanan, B. F., 39, 60 Buchi, W., 226,236, 258,271, 274 Buffa, A., 320, 357 Burdick, E. M., 242, 274, 291, 295, 312, 317, 335, 347, 357, 365 Burger, J., 293, 357 Burger, M., 73, 77, 86,94, 143 Burgoin, A. M., 95, 144 Burkhardt, G. J., 104, 147,204, 205, 217 Burns, R. M., 300, 371 Burroughs, L. F., 267, 274 Burton, L. V., 132, 134, 137, 144

C Caabeiro, J. C., 317, 357 Cahill, V. R., 30, 31, 32, 33, 34, 36, 59 Cahn, A., 339, 355 Cain, R. F., 295, 364 Caldwell, A. G., 341, 358 Caldwell, E., 299, 363 Caldwell, J. S., 79, 80, 86, 140, 144, 202, 206, 215 Callow, E. H., 21, 30, 49 Calvarano, M., 309, 335, 358 Cameron, E. J., 83, 85, 89, 144, 224, 236,

274, 284 Cameron, J. W., 355, 358 Camp, A. F., 298,299, 300, 369 Campbell, A. A., 26, 31, 56

377

AUTHOR INDEX

Campbell, H., 97, 108, 127, 140, 144, 146, 149 Canals, A. M., 312, 356 Candee, F. W., 101, 144 Cantino, B. C., 263, 283 Caporale, G., 341, 367 Carey, E. J., 29, 49 Carman, G. E., 300, 356, 371 Carr, J. G., 223, 224, 225, 226, 265, 266, 267, 268, 270, 274, 275, 284 Carr, J. W., 41, 49 Carson, H. L., 244, 275 Casas Carraminana, A,, 293, 360 Casimir, D. J., 68, 74, 124, 125, 126, 130, 133, 134, 135, 136, 137, 147, 148 Castelli, T., 223, 232,233, 243, 244, 275 Catalano, M., 293, 296, 364 Caul, Jean F., 210, 215 Centonze, M., 293, 359 Challinor, S. W., 267, 275 Chambers, R., 25, 49 Chandler, B. V., 328, 343, 344, 349, 350, 351, 352,354, 358, 363 Chapman, H. D., 299, 358 Charpentik, Y., 261, 264, 269, 270, 271, 272, 275, 282 Chase, J. T., 84, 151 Cheldelin, V. H., 85, 144 Chichester, C. O., 156, 175, 217, 218 Chitre, R. G., 71, 94, 144 Chou, Y.-C., 293, 364 Christel, W. F., 102, 144 Christian, W. A., 289, 291, 307, 365 Chrzaszcz, T., 237, 275 Ciferri, R., 232, 237, 275 Clark, D. S., 229, 230, 233, 238, 275, 284 Clark, G. L., 211, 216 Clark, W. L., 175, 219 Clements, R. L., 333, 358 Clifcorn, L. E., 73, 81, 82, 83, 84, 85, 88, 89, 128, 133, 134, 137, 143, 144, 145, 146 Clifford, P. A., 101, 143 Coggins, C. W., 301, 358, 361 Cohen, A., 292, 293, 294, 295, 296, 297, 298, 302, 303, 307, 311, 348, 355, 358, 368 Coke, E. J., 38, 49 Collet, R. A., 36, 37, 60 Colombo, S., 36, 49

Cook, G. A., 25,49 Cook, J. A,, 294, 358 Coonen, N. H., 89, 145 Cordova, R. M. A., 293, 358 Corey, E. J., 352, 358 Corse, J., 211, 215 Corsi, A,, 4, 7, 50, 56 Couch, J. F., 343, 364 Coulson, D. M., 318, 358 Cowgill, G. R., 312, 364 Cover, R., 189, 215 Cover, S., 44,50 Crandall, P. R., 305,306,314, 369 Crocker, E. C., 210, 211, 215 Cromwell, W. R., 318, 358 Crosby, D. G., 341, 358 Crosby, M. W., 95, 144 Cruess, W. V., 91, 146, 249, 254, 255, 256, 274, 275 Cruickshank, B., 10, 50 Culpepper, C. W., 79, 80, 86, 90, 140, 144 Cunningham, E., 85, 150 Curl, A. L., 302, 324, 325, 327, 328, 358, 359 Cuzzocrea, G., 293, 359 Czarnecki, H. J., 36, 54 Czerny, H., 351, 364

D Damm, A,, 236, 275 Danielson, C. E., 68, 70, 72, 144 Dasa Rao, C. J., 331, 359 Davey, C. L., 19, 20, 48 Davidson, S., 201, 204, 219 Davis, E. G., 78, 144 Davis, G. K., 319, 359 Davis, J. J., 229, 230, 233, 275 Davis, R. B., 182, 183, 185, 186, 215 Davis, R. N., 311, 359 Davis, W. B., 323, 333, 347, 348, 359 Davison, S., 101, 150 Day, T. D., 35, 50 Deaker, E. M., 292, 293, 311, 359 Dean, F. M., 353, 359 Dean, R. W., 340, 357 Deans, R., 30, 53 Deatherage, F. E., 29, 30, 31, 32, 33, 34, 36, 47, 50, 53, 59, 211, 218

378

AUTHOR I NDEX

De Borinquen Segundo, O., 312, 356 Decker, R. W., 104, 144, 201, 205, 215 de Fremery, D., 18, 21, 23, 26, 31, 50, 56 de la Puente Pouch, M. T., 39, 50 Dernaree, K. D., 105, 144 Dempsey, W. H., 183, 217 Derse, P. H., 73, 77, 86, 94, 143 Desrosier, D. W., 207, 218 Dessens, H. B., 293, 370 Deszyck, E. J., 299, 300, 301, 309, 359, 370, 371 Devescovi, M., 311, 367 de Villiers, A. J. R., 348, 357 DeWeese, D., 182, 183, 185, 186, 215 Deysher, E. F., 123, 1843, 219 Dickinson, D., 102, 142, 348, 359 Diehl, H. C., 92, 144,253, 274 Dietrich, W. C., 76, 86, 92, 144, 331, 371 Dietz, J. H., 304, 359 Di Giacomo, A., 293, 359 Dillman, C. A., 309, 355 Dimick, K. P., 211, 215, 249, 275 Dinsa, H. S., 297, 367 Doesburg, J. J., 106, 144, 203, 215 Dole, K. K., 182, 217 Domercq, S., 223, 231, 233, 244, 275, 281 Donald, J. R., 251, 275 Dorfrnan, A., 9, 50 Doty, D. M., 36, 39, 42, 48, 50, 58 Douglas, M., 228, 275 Draczynski, M., 227, 233, 277, 282 Drake, M. P., 36, 50 Draudt, H. N., 30, 36, 53 Driggers, J. C., 319, 359 Dubuisson, M., 5, 50 Dunlop, S. G., 89, 143 Dunn, H. C., 331, 332, 359 Dunn, L. C., 193, 219 Dupaigne, P., 291, 359

E Eagon, R. G., 251, 276 Eddy, C. W., 317, 366 Edgar, A. D., 208, 217 Edin, M. D., 228, 275 Edwards, G. J., 348, 361

Elehwany, N., 101, 111, 144, 183, 209, 215 El-Gharbawi, M., 44.45,46, 50 Ellenberger, H. A., 80, 82, 83, 84, 85, 89, 145 Elvehjem, C. A,, 71, 81, 82, 84, 85, 88, 89, 135, 144, 146, 150, 151 Embden, G., 19, 50 Embleton, T. W., 299, 300, 359 Emch, F., 246, 276 Emerson, 0. H., 349, 350, 351, 352, 353, 354, 359, 360 Engel, R. W., 91, 144 Engelhardt, V. A,, 22, 50 Eny, D. M., 307,308,309,310,314,369 Eolkin, D., 187, 215 Ephraim, A., 293, 314, 360 Erdos, T., 8, 58 Erickson, L. C., 301, 310, 355, 360 Eriksson, E., 72, 148 Erlandsen, R. F., 306, 364 Eskew, R. K., 336, 365 Esty, J. R., 224, 274 Evans, G., 108,109, 145 Evers, C. F., 127, 140, 151 Exarchos, K., 346, 360 Ezell, B. D., 79, 80, 86, 90, 140, 144

F Fabian, F. W., 224, 245, 249, 250, 276 Fabre, P., 257, 276 Fagerson, I., 211, 215 Falk, G., 27, 50 Falk, K. B., 312, 313, 316, 317, 319, 336, 367 Fanska, J. R., 84, 151 Farag6, S., 343, 372 Farber, L., 212, 215 Fardig, 0. B., 80, 82, 83, 85, 89, 145 Farrel, L., 231, 278 Farrer, K. T. H., 80, 144 Feaster, J. F., 81, 86, 142, 145 Feigen, G. A,, 5, 50 Feist, K,, 351, 360 Fellers, C. R., 30, 44, 53, 80, 90, 144, 145, 185, 215 Fenn, L. S., 99, 145 Fenton, F., 83, 84, 88, 90, 95, 143, 144, 145, 146, 147, 151

AUTHOR INDEX

Ferro, M., 212, 215 Fessler, 5. H., 47, 50 Fickle, B. E., 95, 144 Finch, A. H., 299, 363 Fisher, D. F., 293, 294, 295, 296, 301, 361 Fitzgerald, G. A,, 80, 88, 90, 145, 146 Fleischmann, O., 35, 51 Flesch, P., 266, 267, 276, 277 Flynn, L. M., 87, 145 Folinazzo, J. E., 228, 252, 274, 284 Forgacs, J., 252, 276 Fornachon, J. C. M., 265, 276 Fowler, H. D., 72, 145 Fox, Margaret M., 336, 337, 363 Frakas, D. F., 93, 145 Francia, F. R., 293, 360 Francis, F. S., 169, 215 Fratoni, A., 293, 360 Freed, M., 89, 145 Friedrnan, M. E., 174, 215 Friess, S. L., 318, 358 Frith, H. J., 299, 360 Frost, H. B., 355, 358 Fruton, 3. S., 36, 50, 51 Fuertes Polo, C., 293,307, 360

G Gantner, G., 33, 54 Ganz, D., 349, 350,351, 368 Garber, M. J., 301, 361 Garcia Alverez, R., 293, 360 Garcia de la Noceda, H., 312, 355 Gardner, F. E., 294,299, 358, 367 Gardner, J., 79, 149 Gardner, M. E., 211, 215 Geddes, 3. A., 179, 220 Geise, C. E., 206, 216 Geissman, T. A., 341, 346, 352, 353, 359, 360 Gelfan, S., 5, 12, 50 Gerard, R. W., 5, 27, 50, 51 Gergely, J., 6, 37, 51 Gerlaugh, P., 30, 53 Getty, M. R., 293,295,301, 368 Gewasini, C., 36, 49 Gibbons, N. E., 21, 51 Giffee, J. W., Jr., 36, 50

3 79

Gifford, P. S., 208, 217 Gillespy, T. G., 129, 145, 257, 276 Ginger, B., 30, 39,40,41, 42,43, 58, 59 Ginsburg, L., 348, 357 Glascoff, W. G., 130, 145 Glazier, E. R., 352, 358 Gleason, P., 66, 67, 97, 149 Glenn, J. J., 167, 216 Godar, E. M., 324,336,339,357 Godfrey, G. H., 348, 355 Goldberg, A. O., 35, 38, 58 Goldblith, S. A., 36, 56, 88, 93, 145, 149 Goldhammer, H., 39,49 Goldsworthy, L. J., 344, 360 Golovkin, N. A., 22, 51 Goodall, G. E., 294, 360 Goodall, M. C., 26, 51 Goresline, H. E., 245, 281 Gortner, W. A., 138, 147 GottschaII, G. Y., 38, 39, 41, 51 Gould, W. A., 181, 182, 183, 185, 186, 215, 216 Gouvea, M. A., 6, 37, 51 Gowen, P. L., 126, 127, 145 Graham, R. W., 108,109, 145 Grant, E. L., 188, 216 Grant, N. H., 35, 51 Grau, R., 33, 34, 35, 44, 51, 52 Green, C. R., 251, 276 Green, N. M., 39, 56 Greenwood, D. A,, 81, 145 Grevers, G., 106, 144,203, 215 Grey, T. C., 26, 56 Griffiths, F. P., 185, 215, 295, 328, 364 Griffiths, J. T., 300, 371 Griswold, R. M., 44, 51 Grogg, B., 185, 216 Grohmann, H., 261,262,276,280 Gross, J., 8, 9, 51, 53 Guenther, E., 333,335, 360 Guerrant, N. B., 80, 82, 83, 84, 88, 89, 145 Guggolz, J., 66, 147 GuguSeviC-RistiC, M., 293, 360 Guild, L. P., 293, 312, 313, 316, 365, 366 Gunison, J. B., 224, 228, 279 Guthneck, B. T., 35,47 Gutmann, H. R., 36, 51 Gutschmidt, J., 102, 108, 145

380

AUTHOR INDEX

Guyer, R. B., 63, 74, 87, 93, 104, 111, 145, 147,336,355, 360

H Haas, A. R. C., 291, 294, 301, 315, 332, 360 Haas, W., 127, 145 Hale, H. P., 25, 49 Hall, E. G., 291, 360 Hall, J. L., 35, 52, 55 Hall, L. A,, 211, 216 Haller, M. H., 201, 216 Halliday, E. G., 84, 89, 95, 143, 146 Halter, P., 235, 236, 278 Hamm, R., 14, 18, 32, 33, 34, 35, 44, 51, 52 Hand, D. B., 65, 71, 106, 139, 140, 148, 149, 175, 183, 216, 219 Hankins, 0. G., 25, 37, 42, 53, 58 Hansen, E. C., 223,237, 238,241,276 Hanson, H. L., 26, 29, 31, 52, 54, 68, 74, 111, 133, 143, 147 Hanson, J., 4, 52, 54 Hard, M. M., 79, 88, 143, 145 Harding, P. L., 289, 293, 294, 295, 298, 300, 301, 360, 361, 364 Hardy, F., 328, 361 Harriman, H., 36, 50 Harris, F. J. T., 348, 359 Harris, K. W., 95, 144 Harris, P. L., 91, 145 Harris, R. S., 293, 312, 313, 365, 366 Harrison, A. P., Jr., 254, 276 Harrison, D. L., 29, 40, 41, 52, 53 Harsham, A., 29, 50 Hart, W. J., 172, 217 Hartman, J. D., 210, 211, 212, 216 Hasegawa, M., 344, 361 Hasselbach, W., 27, 52 Hatschek, D., 181, 216 Hattori, S., 343, 344, 345, 361 Haut, I. C., 204, 209,212, 217 Hay, P. P., 40,41, 53 Hays, G. L., 252, 282 Heald, F. D., 195, 216 Heberlein, D. G., 73, 81, 82, 84, 85, 88, 89, 134, 137, 143, 144, 145, 146 Heid, J. L., 156, 216 Hein, L. W., 73, 77, 86, 94, 143

Heinrich, F., 234, 255, 277 Heinzelman, D. C., 328, 364 Hellstrom, V., 293, 361 Hendrickson, R., 290, 333, 335, 344, 345, 346, 347, 348, 361, 363 Hening, J. C., 97, 100, 147, 183, 216 Henry, R. E., 75, 143 Hepburn, J. S., 37, 53 Herbst, E. J., 317, 361 Herschel, W. H., 184, 216 Hershberger, T. V., 30, 53 Hield, H. Z., 301, 358, 361 Higby, R. H., 349, 350,351, 361 Highberger, J. H., 9, 53 Hilbert, G. E., 66, 67, 146 Hilditch, T. P., 331, 332, 359 Hilgeman, R. E., 301, 361 Hill, A. G. S., 10, 50 HiI1, A. V., 11, 53 Hill, E. C., 271, 276, 340, 361 Hill, T. L., 12, 26, 27, 55 Hillig, F., 212, 216 Hiner, R. L., 25, 30, 37, 53 Hinman, W. F., 84, 89, 95, 143, 146 Hinreiner, E., 341, 346, 360 Hiwatari, Y., 317, 361 Hochstrasser, R., 224, 225, 236, 250, 259, 271, 272, 276, 278 Hodgkiss, W., 36, 53 Hofmann, E., 251, 260, 276, 278 Hogan, A. G., 87, 145 Holm, L., 191, 218 Holmquist, J. W., 73, 93, 128, 133, 137, 145, 146 Holz, G., 272, 276, 284 Homeyer, P. G., 206, 216 Horanic, G. E., 294, 358 Horner, G., 69, 70, 71, 72, 76, 77, 78, 88,94, 142, 146 Horowitz, R. M., 343, 345, 361, 362 Horspool, R. P., 295, 301, 317, 318, 356, 372 Horvath, B., 7, 54 Howard, A., 20, 21, 24, 25, 28, 31, 33, 49, 53,230, 278 Howard, L. B., 140, 146 Hrnciar, G., 298, 367 Hsu, H.-Y., 345, 362 Hucker, G. J., 260, 276 Huelin, F. E., 311, 323, 362

381

AUTHOR I N D E X

Huffington, J. M., 114, 117, 118, 119, 150 Huggart, R. L., 304, 368 Hui, W. H., 343, 355 Hull, W. O., 306, 362 Hulnie, A. C., 262, 276 Hunt, S. M. V., 8, 53 Hunter, H. A,, 80, 83, 89, 90, 137, 148, 151 Hunter, R. S., 168, 216 Husaini, S. A., 30, 36, 53 Huskins, C. W., 329, 330, 362 Hussein, A. A., 323, 362 Hutchins, M. C., 79, 80, 86, 90, 140, 144 Huxley, H. E., 4, 52, 53, 54

I Ichikawa, N., 344, 362 Ide, L. E., 63, 74, 87, 104, 111, 147, 204, 209, 217 Inagaki, C., 293, 311, 312, 351, 362 Ingalls, R., 80, 146 Ingram, M., 229, 230, 231, 245, 256, 267,273, 276, 277, 283 Irish, J. H., 249, 255, 275 Irving, E. A., 10, 54 Irving, G. W., Jr., 36, 50 Iseda, S., 317, 362 Ishirnaru, K., 310,344, 346, 366 Ito, S., 302, 308, 317, 362 Ives, M., 85, 146 Iwasaki, Y., 310, 344, 346, 362

J Jablonski, J. R., 311, 365 Jackson, J. M., 81, 145 Jackson, R. K., 211, 215 Jacobs, M. B., 184, 216 Jacquot, R., 261, 271 Jang, R., 318, 320, 321, 322, 323, 356, 362, 364 Jansen, E. F., 318, 320, 321, 322, 323, 362, 364 Jeger, O., 352, 365 Jenkins, R. R., 88, 90, 146

Jenkins, W. F., 189, 208, 216 Jenny, J., 251, 260, 277, 281 Jeppson, L. R., 300, 367 Jerchel, D., 266, 267, 277 Jodidi, S. L., 99, 146 Jones, C. L., 251, 275 Jones, E. R. H., 341, 358 Jones, N. R., 36, 53, 57 Jones, W. W., 299, 300, 315, 316, 359, 363 Joslyn, M. A., 75, 91, 92, 146, 175, 181, 216, 249, 254, 256, 277, 283, 316, 317,319, 363, 371 Judd, D. B., 158,162,216

K Kanao, M., 343, 344, 361 Kaniuga, Z., 98, 146 Karibian, D., 6, 37, 51 Karrer, W., 343, 344,346, 363 Kastelic, J., 30, 35, 44, 54, 55, 60 Katz, Y. H., 114, 119, 121, 146 Kazakov, S., 190, 216 Keenan, G. L., 348, 363 Kefford, J. F., 287, 297, 343, 344, 34.9, 350, 351,352, 354, 355, 358, 363 Keith, S. C., Jr., 252, 253, 277 Keller, G. J., 185, 220, 319, 336, 337, 363, 367 Kelley, G. G., 39, 59 Kelley, L., 79, 146 Kelley, L. T., 293, 312, 313, 316, 366 Kemmerer, A. R., 311, 359 Kemp, W. B., 102, 104, 151, 203, 220 Kenten, R. H., 9, 49 Kenyon, E. M., 333, 367 Kertesz, Z. I., 65, 68, 69, 91, 97, 98, 146, 148, 183, 190, 207, 216, 219 Kessler, H., 259, 281 Kesterson, J. W., 290, 333, 335, 344, 345, 346, 347, 348, 361, 363 Keyahian, T., 39, 48, 50 Khan, M-U-D, 328, 363 Kies, M. W., 38, 41,51 Killian, J. T., 167, 216 Kimball, L. B., 190, 216 Kimura, S., 328, 369 King, C. G., 31, 55, 90, 145

382

AUTHOR INDE X

King, Gladys, S., 330, 336, 363 Kirchner, J. G., 319, 336, 337, 339, 340, 363 Kirkpatrick, J. D., 299, 300, 359 Kirn, J. E., 36, 54 Kitchel, R. L., 340, 357 Klatzo, I., 7, 54 Kline, E. A., 30, 60 Kloecker, A., 237, 277 Klose, A. A., 26, 31, 54, 56 Klosterman, E. W., 30, 59 Klotz, I. M., 33, 54 Klotz, L. J., 291, 332, 360 Knapp, E. P., 244, 275 Kobayashi, T., 220 Koch, J., 227, 277 KGrmendy, L., 33, 35, 54 Kolkwitz, R., 251, 277 Koller, G., 351, 364 Komatsu, T., 310, 344, 362 Koo, R. J., 297, 315, 364 Koonz, C. H., 25, 57 Korey, S., 22, 54 Korkes, S., 265, 277 Kostytschew, S., 261, 277 Kotidi, E. P., 344, 350, 364 Koutler-Andersson, E., 72, 148 Kramer, A., 63, 65, 71, 72, 73, 74, 76, 77, 78, 87, 94, 101, 104, 106, 109, i l l , 144, 146, 147, 154, 155, 156, 158, 162, 167, 169, 170, 171, 172, 174, 183, 187, 188, 201, 202, 203, 204, 205, 206, 207, 209, 212, 215, 216, 217, 219 Kramer, H., 10, 54 Kraybill, H. R., 79, 80, 81, 145, 151 Kreger-van Rij, N. J. W., 230, 278 Krehl, W. A,, 312, 364 Krewson, C. F., 343, 364 Krezdorn, A. H., 295, 364 Krieger, C. H., 73, 77, 86, 94, 143 Kringstad, H., 82, 147 Kroemer, K., 231,234, 255, 277 Krumbholz, G., 231, 233, 277 Krumperman, P. H., 263, 278 Kubota, T., 353, 364 Kiihne, W., 6, 54 Kulkavni, P. S., 182, 217 Kunkel, R., 208, 217

Kunkle, L. E., 30, 31, 32, 33, 34, 36, 53, 59 Kuschinsky, G., 7, 22, 54, 58

1 Lafar, F., 235, 278 Lafourcade, S., 272, 282 Laki, K., 7, 8, 54 Lambion, R., 265, 278 Lana, E. P., 186, 217 Larson, J. A., 40, 54 Larson, R. E., 79, 149 Laskovskaya, I. N., 38, 57 Lataste-Dorolle, C.,36, 37, 60 Lawrence, J. M., 323, 356 Lawrie, R. A., 3, 11, 15, 16, 20, 21, 24, 25, 28, 31, 33, 49, 53, 54 Lee, F. A., 73, 79, 92, 97, 100, 112, 138, 147, 151, 208, 217 Lee, J. B., 63, 67, 68, 71, 86, 87, 89, 148, 151 Leffler, F., 44, 51 Legault, A. R., 68, 74, 127, 133, 140, 141, 147, 150 Leinati, A. L., 36, 54 Leinbach, L. R., 68, 133, 147 Leonard, S., 183, 217 Lepper, H. A., 101, 143 Lewis, W. E., 298, 361 Lieske, R., 251, 260, 278 Lime, B. J., 295, 328, 364 Lindquist, F. E., 76, 86, 92, 144 Lindsay, C. W., 306, 362 Lindwall, R. C., 333, 370 Lineweaver, H., 26, 54, 320, 321, 364 Link, H. L., 135, 147 Lipman, F., 15, 55 Lipscomb, A. G., 182, 217 Lisanti, L. E., 293,296, 364 Lissoni, A., 36, 55 Little, A. C., 156, 217 Little, K., 10, 54 Lochhead, A. G., 231, 278 Loconti, J. D., 183, 216 Lodder, J., 230, 278 Long, W. G., 298, 364 Lo Presto, V., 293, 359 Lorand, L., 26, 55

AUTHOR INDEX

Lougheed, T. C., 41,49 Love, E. F. J., 25, 49 Lowe, B., 29,30, 52, 55, 56, 60 Loy, H. W., 35, 55 Lu, Y.-C., 293, 364 Lueck, R. H., 104, 148 Liithi, A., 224, 225, 226, 227, 230, 235, 236, 246, 250, 259, 267, 269, 271, 272, 278, 279 Luh, B. S., 183, 217 Lund, A., 238,249,278 Lunde, G., 82, 147 Lynch, L. J., 63, 64, 65, 67, 68, 74, 96, 98, 100, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 130, 131, 132, 133, 134, 135, 136, 137, 147, 148, 202, 203, 217, 351, 363 Lyubimova, M. N., 22, 50

M Ma, C. N., 343, 356 Ma, R. M., 342, 364 Mabrouk, A. F., 211,218 McArdle, F. J., 207, 218 McBride, B. H., 35, 47 McCall, E. R., 328, 364 McCance, R. A., 77, 147 McCarthy, J. F., 31, 55 Maclay, W. D., 302, 306, 364 McClung, L. S., 231,232,233,243, 279 McClurg, B. R., 29, 52, 56 McColloch, R. J., 185, 218 McCready, R. M., 66, 147, 149, 302, 306, 364 MacDonnell, L. R., 320, 321, 322, 362,

364 McFarlane, V. H., 253, 279 McIntosh, J. A., 90, 147 Mack, G. L., 86, 148 McKee, H. S., 63, 67, 68, 70, 71, 86, 87, 89,94, 148 McKenzie, H. A., 355,363 MacKenzie, V. E., 95, 143 Mackinney, G., 75, 146, 156, 174, 175, 215, 217, 218, 324, 328, 363, 366 Mackintosh, D. L., 35, 52

383

McLean, A. R. M., 251, 275 MacLean, M., 212, 216 MacMasters, M. M., 66, 67, 146 McNelly, A. M., 293, 312, 313, 316,

366 McWethy, J. A., 38, 55 Madsen, L. L., 25, 37, 53 Magnani, N., 311, 367 Magness, J. R., 201, 218 Maharg, L., 39, 59 Mahoney, C. H., 80, 83, 89, 90, 148, 210, 212, 217, 219 Maier, V. P., 40, 41, 45, 58 Makino, M., 220 Makower, B., 249, 275 Makower, R. U., 100, 137, 148 Malecki, G. J., 75, 101, 148, 150, 201, 204, 219 Manchester, T. C., 312, 323, 356, 364 Mangel, M., 39, 59 Marcus, O., 241, 242, 279 Markley, K. S., 340, 364 Marloth, R. H., 294, 295, 365 Marsh, B. B., 13, 20, 22, 23, 24, 25, 26, 3i,33, 48, 55 Marsh, G. L., 174, 215, 263, 282, 283, 294, 302, 316, 349, 365 Marshall, C. R., 223, 224, 229, 234, 235, 239, 240, 241, 242, 243, 245, 247, 248, 279 Marshall, J. R., 324, 357 Marshall, R. E., 249, 276 Martin, A. J. P., 211, 218 Martin, C. J., 35, 55 Martin, G. J., 348, 365 Martin, R., 35, 58 Martin, W. M., 103, 104, 148,202, 218 Martinant, V., 223, 232, 239, 279 Mason, J. M., 186, 218 Masters, J. E., 336, 339, 357 Masure, H. P., 97, 149 Matheson, N. A., 8, 53 Mathews, M. B., 9, 50 Matlack, M. B., 328, 329, 365 Matsubara, K., 293, 302, 366 Matsunaga, M., 293, 366 Matsuno, T., 341, 365 Matsushita, A., 317, 362 Matthews, W. A., 210, 219

384

AUTHOR INDE X

Mattson, S., 72, 148 Matzik, B., 227, 279, 308, 365 Maurer, R. H., 347, 365 Maynard, L. A., 79, 80, 150 Maynard, N., 30, $2, 58 Mazia, D., 4, 58 Mazur, Y.,342, 365, 372 Medoff, S., 81, 84, 85, 88, 89, 134, 145 Mehlenbacker, V. C., 169, 218 Mehlitz, A., 227, 279, 308, 365 Mehrhof, N. R., 319, 359 Meisels, A., 31.2, 372 Melera, A., 352, 365 Mellon, M. G., 170, 218 Melms, D., 185, 216 Melvick, D., 84, 95, 149 Mercer, W. A,, 128, 148 Meskhi, A., 265, 278 Meyer, K., 10, 55 Meyer, K. F., 224, 228, 279 Micale, A,, 319, 344, 365, 368 Miers, J. C., 76, 86, 144 Mihilyi, E., 6, 8, 37, 55 Mikhailov, A. N., 9, 58 Miller, E. E., 191, 218 Miller, E. V., 297, 311, 324, 365, 372 Miller, I., 312, 313, 316, 317, 319, 336, 367 Miller, J. M., 317, 318, 319, 336, 337, 339, 340, 363, 365 Miller, M., 30, 44, 55 Miller, M. W., 244, 281 Millis, N. F., 224, 226, 258, 265, 271, 279 Minard, R. A., 176, 177, 180, 218 Mirimanyan, V. A., 302, 365 Mirna, A,, 51 Miro, J. C., 39, 55 Mitchell, H. H., 94, 148 Mitchell, R. S., 63, 64, 65, 67, 68, 74, 96, 98, 100, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 130, 131, 132, 133, 134, 135, 136, 137, 147, 148, 202, 203, 217 Miura, H., 307, 332, 347, 369 Miyada, D. S., 40, 41, 44, 45, 55, 58 Mommaerts, W. F. H. M., 5, 6, 55 Money, R. W., 289,291, 307,365

Moncrieff, R. W., 211, 218 Monselise, J. J., 293, 314, 360 Monzini, A,, 36, 47, 55 Mookerjee, A,, 350, 365 Moore, E. L., 311, 356 Moore, P. W., 300, 371 Morales, M. F., 12, 26, 27, 49, 55 Moran, T., 23, 25, 56 Morgan, A. F., 94, 148 Morgan, D. A,, 336, 365 Morgan, R. H., 313,314, 365 Morris, E. O., 267, 279 Morris, H. J., 76, 92, 144 Morris, 0. M., 201, 218 Morris, T. N., 93, 148 Mottern, H. H., 317, 366 Mouton, R. F., 36, 37, 60 Moyer, J. C., 65, 68, 71, 80, 106, 108, 123, 139, 140, 148, 149, 183, 216 Moyls, A. W., 271, 274 Mozolowski, W., 18, 56 Mrak, E. M., 231, 232, 233, 243, 244, 279, 281 Miihlberger, F. H., 261, 262, 276, 280 Miiller-Thurgau, H., 223, 237, 239, 260, 262,263,264, 265, 280 Miiller, V., 80, 150 Muenchow, A. F., 99, 149 Munechika, T., 31 7, 366 Muniz, A. I., 312, 356 Munsell, H. E., 293, 312, 313, 316, 365, 366 Murray, H. C., 71, 83, 143, 149 Murti, V. V. S., 344, 366

N Nagel, C. W., 340, 357 Naito, H., 310, 344, 346, 366 Nakayama, O., 307,332,347,369 Nardone, R. M., 35, 56 Natarajan, C. P., 324, 366 Nauer, E. M., 300, 371 Neergaard, P., 236,258, 280 Nehring, K., 70, 94, 149 Nelson, E. K., 317, 340, 344, 364, 366 Nestel, L., 70, 94, 148 Neubert, A. M., 128, 151,259, 282 Neuman, R. E., 46, 56 Neumann, H. J., 76, 86, 144

385

AUTHOR INDEX

Neurath, H., 35, 39, 56, 58 Newman, B., 312, 313, 316, 317, 319, 336, 367 Nicholls, M. J., 66, 149 Nickerson, D., 165, 167, 218 Nickerson, J. T. R., 36, 56 Niehaus, Ch. J. G., 232, 242, 280 Nielsen, J. P., 66, 67, 97, 127, 149 Nielson, B. W., 185, 218 Niethammer, A,, 241, 242, 280 Niewiarowicz, A., 36, 56 Nightingale, G., 293, 312, 313, 316, 365,

Owens, G., 79, 149 Owens, H. S., 66, 147, 149

P Paddock, L. S., 39, 57 Pagenstedt, B., 185, 218 Pallesen, H. R., 65, 71, 139, 140, 148,

149 Palmore, J. I., 97, 143 Park, S. E., 228, 283 Parker, E. R., 299, 300, 301, 315, 316,

363, 370

366 Niinivaara, F. P., 33, 56 Nikolaeva, N. V., 35, 57 Nolte, A. J., 319, 351, 366 Nomura, D., 293, 302, 308, 317, 341, 345,347,351, 366 Nomura, M., 263, 280, 282 Nonnecke, I. L., 67, 69, 86, 103, 104,

151 Nossal, P. M., 265, 280 Nottingham, P. M., 30, 44, 56 Nutting, G. C., 182, 183, 218, 220 Nutting, M. D., 76, 92, 144 Nygren, V. E., 65, 149

Parker, M. E., 128, 149 Parnas, J. K., 18, 56 Parsons, J., 41, 42, 59 Partmann, W., 42, 56 Pasteur, L., 223, 281 Patnayak, K. C., 344, 345, 366 Patron, A,, 293, 366 Patten, C. G., 211, 215 Patterson, W. I., 212, 216 Paul, P., 29, 56, 79, 80, 146 Pearce, W. E., 252, 282 Peat, S., 66, 149 Pederson, C. S., 245, 246, 249, 260, 276,

281, 283

0 Ochoa, S., 265, 277, 280 OConnor, R. T., 328, 364 OHara, M. B., 84, 85, 88, 145 Ohtalti, T., 344, 371 Oken, D. E., 46, 56 Olliver, M., 235, 236, 239, 250, 258, 280 Olsen, A., 82, 147 Olson, H. R., 36, 56 Ooeda, M., 310,344,346, 366 Orlovskaya, G. V., 9, 58 Orr, M. L., 85, 151 Oser, B. L., 84, 94, 95, 149 Oser, M., 84, 95, 149 Oshima, Y.,345, 372 Osterwalder, A,, 223, 233, 234, 235, 237, 251, 255, 259, 262, 263, 265, 280,

281 Otake, S., 31, 56 Ottosson, L., 63, 67, 149 Owen, R. F., 84, 90, 151

Pennisi, L., 293, 312, 366 Pepkowitz, L. P., 79, 149 Perry, J. H., 175, 178, 181, 218 Perry, S. V., 4, 6, 7, 22, 23, 24, 25, 26,

48, 50, 56 Pershina, L. I., 22, 51 Peterson, H. B., 63, 67, 86, 149 Peterson, R., 21, 57 Petronici, C., 319, 323, 331, 350, 356,

366, 367 Peynaud, E., 223, 231, 233, 244, 266, 267, 269, 271, 272, 275, 281, 282 Phaff, H. J., 244, 275, 281 Phillips, G. W. M., 336, 365 Phillips, J. D., 223, 262, 270, 274, 275,

281 Pierce, J. C., 42, 58 Pilcher, R. W., 83, 85, 89, 144 Pilgrim, F. J., 211, 218 Plagge, A. R., 30, 60 Plummer, J., 80, 146 Pohle, W. D., 169, 218

386

AUTHOR INDEX

Pollard, A., 223, 229, 262, 270, 274, 275, 281 Pollard, A. E., 85, 146 Pollard, L. H., 63, 67, 86, 149 Ponting, J. D., 249, 275 Pool, M. F., 18, 21, 23, 26, 31, 50, 54, 56 Porchet, B., 230, 281 Portzehl, H., 6, 22, 27, 56, 57, 59 Potter, A. L., 66, 149 Potter, F. E., 183, 184, 219 Powers, J. J., 321, 367 Pratt, D. E., 321, 367 Prescott, F., 91, 143 Prijol, A. C., 39, 55 Primo Y6fera, E., 293, 360 Proctor, B. E., 36, 56, 88, 93, 101, 145, 149, 150,201,204, 219,333,367 Prudent, I., 30, 57 Ptak, L. R., 81, 84, 85, 88, 89, 134, 143, 145 Pulley, G. N., 37, 39, 57, 347, 367

Q Quackenbush, F. W., 324, 367 Quaife, M. L., 91, 145

R Raacke, I. D., 70, 150 Rahourn, W. J., 324, 367 Rafferty, J. P., 79, 146 Raistrick, H., 236, 281 Rakieten, M. L.,e312, 313, 316, 317, 319, 336, 367 Ramakrishnan, C. V., 309, 310, 371 Ramshottom, J. M., 25, 28, 29, 30, 37, 39, 57 Ramsey, R. C., 289,308,309, 369 Randall, J. T., 4, 5, 9, 57 Randhawa, G. S., 297, 367 Rangaswami, S., 344,345, 366 Ransford, J. R., 175, 183, 216, 219 Rao, C. R., 319, 369 Rauen, H. M., 311, 367 Raveaux, R., 261, 277 Rayner, D. S., 299, 358 Rebour, H., 295, 367 Recca, J. A., 244, 281

Rees, M., 232, 281 Reinke, H. G., 324, 357 Reitz, H. J., 296, 297, 301, 359, 369, 370 Rendle, T., 235, 236, 239, 250, 258, 280 Rentschler, H., 269, 269, 270, 272, 281, 283 Reuther, W., 298,299,300, 367 Riaz-Ur-Rahman, 31 1, 367 RiEreau-Gayon, J., 269, 271, 272, 275, 282 Rice, R. G., 319,336,337, 363, 367 Richardson, L. R., 85, 151 Richardson, W. D., 185, 219 Richert, P. H., 255, 275 Riehl, L. A., 300, 367 Riester, D. W., 336, 339, 357 Rietsch, M., 223, 232, 279 Riley, J. P., 33'1, 332, 359 Rippel-Baldes, A., 269, 282 Ritchell, E. C., 73, 128, 133, 137, 146 Robbins, R. C., 87, 90, 150 Robertson, R. N., 63, 67, 68, 70, 71, 86, 87, 89, 94, 148 Robinson, R., 3M, 360 Robinson, W. B., 65, 68, 71, 139, 140, 148, 149, 175, 183, 219 Rockland, L. B., 317, 318, 349, 365, 367, 371 Rodgers, B. L., 190, 214 Rodighiero, G., 341, 367 Rodriguez, J. R., 300, 367 Rogers, H. P., 104, 147, 204, 205, 217 Romwalter, A., 240, 241, 282 Roper, E., 111, 150 Rose, A. H., 267, 275 Rose, D., 21, 51, 57 Ross, E., 88, 145 Ross, H. H., 194, 219 Roth, W., 186, 219 Rouse, A. H., 303, 304, 306, 307, 320, 321,347,356, 359, 367, 368 Rowan, K. F., 70, TI, 150 Rowe, S. C., 99, 143 Rowell, K. M., 340, 357 Rowinski, P., 36,37, 60 Roy, W. R., 299, 367 Royo Iranzo, J., 293, 307, 360 Rutgus, R., 185, 219 Ruth, W. A., 252, 276

AUTHOR INDEX

Ruyle, E. H., 252, 282 Ryer, R., 111, 36, 50 Rygg, G. L., 293, 295, 301, 368 Ryynaenen, T., 33, 56

5 Sacco, T., 293, 368 Sadiq Ali, 311, 367 Safina, G., 317,318,319,320, 368 Sakaguchi, K., 263, 280, 282 Sakasegawa, H., 302, 308, 317, 362 Sale, J. W., 342, 368 Sallans, H. R., 261, 282 Sallee, E. D., 104, 148 Samisch, Z., 293, 294, 295, 296, 302, 303, 307, 311, 348, 349, 350, 351, 368 Samuels, C. E., 212, 219 Sannie, C., 345, 368 Sarl, R., 318, 319, 320, 3444, 365, 368 Sato, H., 220 Savitsky, A., 211, 219 Sayre, C. B., 113, 114, 150 Scarborough, H., 348, 368 Schaffer, P. S., 342, 364 Schaffner, K., 352, 365 Schanderl, H., 227, 233, 241, 242, 255, 266, 277, 282 Schenck, A. M., 155, 219 Schiffner, G., 227, 277 Schmidt, C. F., 73, 128, 133, 137, 146 Schmitt, F. O., 9, 53 Schmitt, H. P., 212, 219 Schmitthenner, F., 227, 251, 259, 282 Schneider, A., 66, 103, 150, 203, 219 Scholz, U., 228, 282 Schomer, H. A., 324, 365 Schopfer, W. H., 80, 150 Schormuller, J., 38, 44, 48 Schramm, G., 6, 57 Schulte, L., 351, 360 Schultz, H. G., 211, 218 Schweigert, B. S., 35, 47 Schwerdtfeger, E., 70, 94, 149 Scott-Blair, G. W., 187, 219 Scott, L. E., 63, 74, 80, 83, 87, 89, 90, 104, 111, 147, 148, 204, 209, 210, 217, 219 Scudder, G. C., 298, 367

387

Seaton, H. L., 114, 117, 118, 150, 212, 219 Serini, G., 340, 368 Serrano, P., 312, 356 Seshadri, T. R., 331, 344, 345, 346, 350 359, 366, 368 Sesseler, M., 293, 370 Shadboh, C. A., 191, 218 Shaw, A. M., 176, 177, 178, 219 Shearer, P. S., 29, 30, 52, 60 Shepherd, A. D., 306, 364 Sherman, M. S., 340, 364 Shewan, J. M., 36, 57 Shifrine, M., 244, 281, 282 Shimokoriyama, M., 324, 343, 344, 345, 346, 361, 369 Shioiri, H., 307, 328, 332, 347, 369 Siddappa, G. S., 302,319, 369 Sidewell, Arthur P., 104, 144, 201, 205, 215 Silviera, V., 66, 147, 149 Simpson, T. H., 345, 369 Sinclair, W. B., 289, 291, 292, 293, 294, 295, 296, 298, 301, 302, 305, 306, 307, 308, 309, 310, 314, 317, 319, 330,332, 356, 369, 372 Singer, T. P., 6, 22, 57 Singh, I., 46, 57 Singh, S. I., 46, 57 Sinnott, E. W., 193, 219 Sites, J. W., 296, 297, 298, 299, 300, 301, 315, 359, 364, 369, 370, 371 Sizer, I. W., 45, 57 Sloan, H. J., 37, 58 Slomp, G., 352, 358 Smart, H. F., 224, 282 Smit, C. J. B., 316, 317, 319, 371 Smith, E. L., 36, 39, 47, 57 Smith, G., 236, 281 Smith, H. R., 106, 147, 167, 169, 171, 202, 209, 217 Smith, M. C., 299, 363 Smith, M. H., 65, 71, 72, 73, 76, 78, 147 Smith, P. F., 298,299, 300, 367, 370 Smith, W. H., Jr., 44,50 Smock, R. M., 259, 282 Smorodintzev, I. A., 35, 38, 44, 57, 58 Snell, E. E., 85, 150, 317, 361 Snellman, O., 8, 58 Snoke, J. E., 35, 58

388

AUTHOR INDEX

Sondheimer, F., 342, 365 Soost, R. K., 355, 358 Sosa, A., 345, 368 Soule, M. J., 298,299, 361, 364 Spadoni, M. A., 293, 360 Sparling, B. L., 87, 89, 90, 151 Spencer, F. T., 261, 282 Spicer, S. S., 8, 58 Spiegelberg, C. H., 282 Spoon, W., 293, 370 Spragg, S. P., 71, 150 Srivastava, H. C., 302, 370 Stadtman, T . C., 263, 282, 283 Stalder, L., 238, 240, 264, 282 Stanley, J., 184, 219 Stanley, W. L., 211, 219, 333, 340, 341, 342, 348, 370, 371 Stanworth, J., 69, 70, 71, 76, 77, 88, 142 Stapp, C., 242, 283 Stark, F. C., 119, 150, 210, 219 Stenstrom, E. C., 293, 295, 370, 372 Stepat, W., 90, 144 Stephens, I. M., 311,323, 362 Stephens, T. S., 295, 328, 364 Sterling, C., 40, 41, 45, 58 Stern, I., 313, 314, 370 Stern, R. M., 89, 145 Stevens, J. W., 313, 370 Stevens, T. W., 291,312, 313, 356 Stewart, G. F., 29, 52 Stewart, W. S., 300, 301, 356, 370 Stier, H. C., 102, 104, 151 Stimson, C. R., 79, 80, 150 Stofberg, F. J., 300, 370 Stotz, E. H,, 65, 68, 148, 183, 219 Stotz, E. H., 65, 68, 148 Stover, R., 6, 59 Strachan, C. C., 271, 274 Strachan, G., 67, 69, 86, 103, 104, 151 Strandine, E. J., 29, 30, 57 Strasburger, L. V., 65, 97, 150 Strodtz, N. H., 75, 143 Strong, F. M., 81, 82, 83, 84, 85, 88, 89, 135, 146, 150, 151 Sugawara, N., 212, 219 Sukh, Dev., 352, 358 Sunday, M. B., 293, 294, 295, 296, 299,

361 Suri. B. R., 35, 58 Suzuki, M., 220

Swanson, H. A,, 306, 364 Swanson, M. H., 37, 58 Swanson, W. J., 91, 145 Swift, L. J., 293, 295, 314, 329, 330, 331, 340,342,347, 355,362, 370,371 Swingle, W. T., 287, 288, 292, 343, 345,

371 Swinzow, H., 293, 366 Synge, R. L. M., 211, 218 Szent-Gyorgyi, A., 2, 4, 5, 7, 11, 12, 13, 18,23, 34, 58, 348, 365 Szent-Gyorgyi, A. G., 4, 5, 6, 8, 26, 37, 51, 55, 58, 346, 357

T Tada, S., 263, 282 Takahashi, H., 263, 282 Takahashi, S., 308, 366 Talburt, W. F., 68, 74, 76, 86, 127, 133, '140, 141, 144, 147, 150 Tanner, F. W., 224, 227,252, 276, 283 Tanner, H., 262, 269, 270, 272, 281, 283 Tanret, C., 343, 371 Tappel, A. L., 35, 40, 41, 44, 45, 55, 58 Tarver, M. G., 155, 219 Tate, D. R., 205, 220 Tauschanoff, W., 23.3, 277 Taylor, G. F., 201, 218 Taylor, 0. C., 300, 371 Taylor, R. E., 5, 51 Tchelistcheff, A., 265, 283 Teply, L. J., 73, 77, 86, 94, 143 Tettamanti, A. K., 343, 372 Thompson, J. F., 23, 24, 25, 55 Thompson, M. L., 85, 150 Thompson, P. C. O., 355, 363 Thompson, W. L., 300, 371 Ting, S. V., 302, 309, 348, 371 Tischer, R. A., 186, 217 Tischer, R. G., 206, 216 Tobey, H. L., 80, 146 Tobinaga, S., 352, 358 Todhunter, E. N., 87, 89, 90, 150 Toepfer, E. W., 85, 151 Tokoroyama, T., 353, 364 Toldby, V., 190, 219 Tolle, W. E., 210,211,212, 216 Tominaga, T., 345. 362 Tomiyama, T:,212, 219

389

AUTHOR INDE X

Tomlin, S. G., 10, 54 Torfason, W. E., 67, 69, 86, 103, 104, 151 Townsley, P. M., 316, 317, 319, 371 Trefethen, I., 83, 84, 151 Tressler, D. K., 79, 80, 86, 88, 90, 127, 140, 145, 146, 147, 148, 150, 151, 229, 249, 281, 283 Troescher, C. B., 293, 312, 313, 316, 365, 366 Tsao, T.-C., 12, 58 Tsukamoto, T., 344, 371 Tulagin, V., 352, 360 Turba, F., 7, 22, 54, 58 Turner, A., Jr., 170, 172, 214 Turner, D. H., 68, 69, 70, 71, 72, 150, 151 Turner, J. F., 63, 64, 65, 67, 68, 69, 72, 147, 151 Turrell, F. M., 318, 371 Tustanvosky, A. A,, 9, 58 Tuzson, P., 325, 372 Twigg, B. A., 157, 188, 217, 220 Tytell, A. A., 46, 56

U Ulrich, R., 259, 283 Underwood, J. C., 185, 220, 317, 349, 367, 371 Urbain, M. W., 36, 54

V Vahtras, K., 72, 148 Vail, G. E., 35,40,41, 52, 53 Van Atta, G. R., 331, 371 Van der Laats, J. E., 293, 371 Van Duyne, F. O., 84, 90, 151 Van Horn, C. W., 299, 363 Vannier, S. H., 333, 340, 341, 342, 370, 371 Varma, T. N. S., 309, 310, 371 Vas, K., 256, 283 Vaughn, R. H., 224, 263, 265, 274, 278, 282, 283, 340, 357 Vavich, M. G., 80, 82, 83, 84, 85, 89, 145 Veeraraghaviah, J., 331, 344, 350, 359, 368

Veldhuis, M. K., 128, 151, 293, 295, 302, 314, 329, 330, 336, 340, 347, 359, 362, 365, 371 Vetsch, U., 227, 236, 250, 259, 267, 271, 278, 279 Vickery, J. R., 25, 49 von Kiraly, A., 240, 241, 282 Von Loesecke, H. W., 37, 39, 57, 291, 319,348, 351, 366, 371 von Schelhorn, M., 230, 249, 254, 255, 273, 283

W Wachter, J. P., 36, 50 Wadley, F. M., 294, 361 Wagenknecht, A. C., 73, 92, 147, 151 Wagner, J. R., 81, 82, 83, 84, 85, 88, 89, 135, 151 Waibel, C. W., 347, 365 Walker, E. D., 302, 364 Walkley, V. T., 223, 224, 229, 234, 235, 239, 240, 241, 242, 243, 245, 247, 248, 279 Wallace, A,, 315, 316, 372 Wallace, G. I., 228, 283, 284 Wallace, R. H., 229, 230, 233, 238, 275, 284 Walls, E. P., 80, 83, 89, 90, 98, 102, 104, 137, 148, 151,203, 220 Wander, I. W., 299, 370 Wang, H., 30, 39, 40, 41, 42, 58, 59 Warneford, F. H. S., 328, 361 Watanabe, S., 26, 59 Watts, B. M., 2, 59 Wawra, C. W., 343, 371 Wearmouth, W. G., 202, 220 Webb, B. H., 183, 184,219 Webb, J. L., 343, 371 Webber, H. J., 287,288,292, 372 Weber, H. H., 6, 27, 52, 57, 59 Webster, H. L., 19, 59 Wedding, R. T., 295, 300, 317, 318, 319, 367, 372 Weiner, S., 39, 59 Weir, C. E., 30, 39, 40, 41, 42, 43, 58, 59 Weizman, A., 342, 365, 372 Welch, M., 101, 150, 201, 204, 219 Wenzel, F. W., 271, 276, 340, 361 West, E. S., 180, 220 Westbrook, G. F., 293,295, 370, 372

390

AUTHOR INDEX

Whalley, W. B., 345, 369 Whitaker, J. R., 39, 44, 45, 46, 50, 59 Whitcombe, J., 79, 97, 100, 138, 147 White, J. W., Jr., 170, 214 Whitehead, E. I., 36, 56 Whiting, G. C., 223, 262, 270, 275, 281, 284 Whittenberger, R. T., 182, 183, 216, 220 Wicker, C. R., 179, 220 Widdowson, E. M., 77, 147 Wiederhold, E., 311, 356 Wiele, M. B., 26, 31, 54 Wierbicki, E., 30, 31, 32, 33, 34, 36, 47, 59 Wiercinski, F. J., 27, 59 Wilcox, E. B., 63, 67, 86, 149 Wilcox, M. S., 79, 80, 86, 90, 140, 144 Wilder, C. J., 212, 219 Wilder, H. K., 203, 220 Wiley, R. C., 186, 210, 218, 220 Wilharm, G., 272, 276, 284 Wilkie, D. R., 13, 60 Williams, A. H., 223, 270, 275 Williams, A. J., 238, 284 Williams, B. E., 39, 60 Williams, C. C., 236, 284 Williams, J. N., Jr., 71, 94, 144 Williams, L. O., 293, 312, 313, 316, 365, 366 Williams, 0. B., 236, 284 Williams, R. J., 85, 144 Williams, V. B., 87, 145 Willis, J. B., 352, 363 Wilson, B. L., 205, 220 Wilson, D. E., 106, 149 Winegarden, M. W., 30, 60

Winston, J. R., 293, 294, 295, 296, 297, 324, 361, 365, 372 Wishnetsky, T., 175, 219 Witter, L. D., 228, 252, 274, 284 Wodicka, V. O., 89, 143, 145 Wolf, J., 307, 308, 372 Wolfe, J. C., 84, 90, 151 Wolford, E. R., 127, 149 Woll, E., 242, 284 Worthington, 0. S., 210, 220 Wortmann, J., 242, 284

Y Yamakawa, M., 220 Yamamoto, J., 31, 56 Yamamoto, R., 345, 372 Yamashita, T., 344, 362 Yeatman, J. N., 104, 144, 201, 205, 215 Pone, Y., 212, 219 York, G. K., 128, 148 Young, R. E., 309, 372 Young, W. J., 25, 49

Z Zachariadd, P. A., 9, 60 Zaides, A. L., 9, 58 Zechmeister, L., 325, 372 ZemplBn, G., 343, 372 Zender, R., 36, 37, 60 Zhigalov, V. P., 35,38, 58 Zidan, Z. I., 315, 316, 372 Zielinski, Q., 193, 220 Zook, E. G., 85, 151 Zscheile, F. P., 79, 80, 151

SUBJECT INDEX A ATP in muscle contraction, 6, 11-13, 1 4 2 6 in muscle relaxation, 26-28 Acids, in citrus fruits, 291-302, 307-309 in citrus peels, 309-310 organic, in fruit juices, 262-271 Actin, 4, 6, 11-12 F-actin, 7 globular (or G-actin), 7 molecular weight, 7 polymerization of, 7 Actomyosin, 8 Adenosine triphosphate, see ATP Aging of meat, artificial, 37-46 Alcohols, in fruit juices, 259-262 Amino acids, in peas loss in processing, 71 relation to maturity, 70-71 Amylopectin, molecular weight, 66 Amylose, molecular weight, 66 Ascorbic acid, as additive to fruit juices, 256-257 Ash, i n peas, 76-78

B Bacteria, in noncitrous fruit juices, 223228 acetic acid bacteria, 225 effects on malic acid, 264-267 lactic acid bacteria, 225-227 other bacteria, 227-228 Beef, freezing of, 2 4 2 5 tenderness of, 28-29 effect of salt on, 32-35 Beef muscle, rigor in, 20 Brix/acid ratio, 289, 291, 295, 297, 307

C Calcium in muscle chemistry, 26-28 Carbohydrates in citrus juices pectic substances, 303-304 sugars, 302-303

Carbohydrates in citrus pulps and peels, 304-307 Carbon dioxide, use in precessing fruit juices, 250-251 Cathepsins, see Enzymes, proteolytic Centrifugation, of fruit juices, 247 Chicken muscle, rigor in, 21 Chlorophyll, in peas changes in processing, 74-76 conversion to pheophytins, 75-76 Chlorosis, i n peas, 74 Chromatographic separation, in flavor chemistry, 211 Chromatography, paper, use in anlysis of fruit juices, 262 Citric acid, in fruit juices, 269-270 Citrus fruits components, 287, 289 composition of factors affecting, 292-302 variability, 291-292 distribution, 293 varieties, 287, 288 Cold storage, in treatment of fruit juices, 252-254 Collagen amino acid content, 9 definition, 8 physiological function of, 8 polysaccharides in, 9 properties, 8-9 Collastromin, see Collagen Colorimetry, 162-165 Color perception, 159-161 standards of illumination, 159 Connective tissues, 3 collagen in, 8-9 ground substance of, 10 mucopolysaccharides in, 10

D Defects in food quality, 192-197 classification of, 193-195 instrumental measurement, 195-1 97 Dehydrofreezing, 65, 140-141 391

392

SUBJECT I N D E X

E Elastin chemical composition, 9 polysaccharides in, 10 preparation, 9 properties, 9-10 Endomysium, see Connective tissues Enzymes in citrus fruits acetylesterase, 322 depolymerizing, 321 glutamic acid decarboxylase, 323 others, 323-324 pectinesterase, 320-321 peroxidase, 323 phosphatase, 322 Enzymes, in peas, 91-93 Enzymes, proteolytic as evaluating agents, 44-46 in muscle, 35-37 as tenderizers, 40-46

F Fibrils, 3-4 Filaments, 4 Filtration, of fruit juices, 248 Fining, of fruit juices, 247-248 Fish muscle, rigor in, 22 Flavenoids in citrus fruits, 342 distribution, 346-347 identification, 343-346 properties, 347-348 Fluidity, 181 Frost, effects on fruit composition, 301302 ci

Gas chromatography, use in flavor chemistry, 2, 21 1 Genetic factors in fruit composition, 292 Glycerin, in fruit juices, 261-262

H Hardness meter, 106 Heat treatment, of fruit juices, 249-250 Horse muscle, rigor in, 20 Hunter color meter, 168-169 Hydration of proteins in muscle, 32-35

I Inorganic constituents in citrus fruits, 313-3 15 Iron content, in peas, 78

L Limonoids in citrus fruits, 348-349 chemistry, 352-353 delayed bitterness, 351-352 distribution, 349 effect of maturity on, 349 effect of rootstock on, 349 Lipids, in citrus fruits, 329-332 Lipids, in peas, 73-74

M Magnesium in muscle chemistry, 26-28 Malic acid, in fruit juices, 264-269 Maturity, effects o n fruit composition, 294-296 Maturometer, 63, 74, 105, 115, 119-121, 122 Meat color of, 2, 3 criteria for evaluating, 2 flavor of, 2 flavor of frozen, 23-26 juiciness of, 2 measurement of tenderness, 40-41 water-binding capacity, 2 Microorganisms i n noncitrous fruit juices chemical changes induced by, 259272 control of, 247-257 effects on appearance, 257-259 relation to climate, 243-244 relation to insects, 244 relation to season, 249 sources, 237-246 varieties, 222-237 washing, effects on, 245 Moisture, effects on fruit composition, 301 Mold, in noncitrus fruit juices, 234-236 effects on malic acid, 269 Munsell color system, 165-168 Muscle

393

SUBJECT INDEX

amino acid content i n aging, 36-37 contractile elements in, 2 extracellular components, 3 intracellular material, 3 literature on, 1-2 nonprotein components, 5 proteins of, 5-10 shortening in thawing, 23-26 Muscle contraction active phase, 12 actomyosin in, 11-12 experimental models, 13-14 relaxed phase, 12 schematic of, 12 theories of, 10-13 Muscle, red, 3 Muscle relaxation, 12, 26-28 chemical factors in, 26-28 Muscle, white, 3 Myofibrils, see Fibrils Myogen, 5 enzymes in, 5-6 Myoglobin, and meat color, 3 Myosin, 4, 6-7, 11-12 function in contraction, 6 “heavy,” 6-7 “light,” 6-7 molecular weight, 6 molecule schematic, 7 peptide chains in, 6 splitting of ATP, 22-23 Myosin B, see Actomyosin

N Newtonian liquids, 176-177 Nitrogen compounds in citrus fruits amino acids, 316-318 analytical applications, 319-320 bases, 316-318 containing sulfur, 318-319 factors affecting, 315-316 proteins, 319 Nitrogen supply, effects on fruit composition, 298-299 Nomographs, color, 172-174 Non-Newtonian materials, 177-179 dilatants, 187 plastics, 178 psendoplastics, 177-1 78

Nonvolatile constituents of citrus oils coumarin derivatives, 340-342 steroids, 342 triterpenoids, 342

0 Optimal harvest time (O.H.T.), 63, 64, 69, 70, 114-116

P Pasteurization, see Heat treatment Peas, dry storage, 62 Peas, green blanching, 65, 70, 71, 73, 75, 76, 81, 83, 88, 91, 93, 132-134 chemical composition, 6’2-92 relation to maturity, 62-63 cleaning, 128-129 color, 75-76 drying, 139-141 field transport, 12C128 flavor, 73-74, 75, a7 freezing, 90, 92, 93, 138-139 grade standards, 111-113 harvesting methods, 122-126 maturity, 95-122 measurement of, 96-122 prediction of, 113-122 nutritive value, 93-95 research needs, 141-142 scheduled planting, 121-122 size grading, 129-130 specific gravity, 99-101, 134-138 texture, T2-73, 75, 78, 87 water content, 63-67 yield prediction, 122 Perimysium, 3 pH, effects on microorganisms in fruit juices, 226-228, 252-257 Phosphorus compounds, in peas loss in processing, 72-73 relation to maturity, 71-72 relation to texture, 72-73 Phosphorus supply, effects on fruit composition, 299 Pigments in citrus fruits blood oranges, 328 grapefruit, 328

394

SUBJECT INDEX

limes, 328-329 oranges and tangerines, 324-328 Position on tree, effects on fruit composition, 296-298 Potassium supply, effects on fruit composition, 299-300 Procollagen, see Collagen Proteins, in peas relation to maturity, 70-71 loss in processing, 71 Protomyosin, 6

Q Quality in food appearance, 157-197 control methods, 156 flavor, 209-212 correlated measurements, 2 t 2 odor, 21 1-212 taste, 210-211 human evaluation, 154155 instrumentation, 155-157 sampling, 156 kinesthetics, 197-209 classification, 198-199 instrumental measurement, 201-209 principles of measurement, 199-201 methodology, 213-214 principles of measurement and control, 154157 significant correlations, 155 terminology, 213

R Rabbit muscle, rigor in, 19-20, 21 Reticulin, 3, 10 Rigor mortis ammonia liberation in, 18-19 ATP decrease in, 14-26 differences from muscle contraction, 11 effects of glucose on, 21 effects of preslaughter conditions on, 20-21 glycogen content of muscle, 15-23 glycolosis in, 14-22 insulin injections and, 21 Marsh-Bendall factor, 18, 28 onset of, 14-23

p H in, 11 resolution of, 26-37 chemical changes in, 26-28 connective tissues, 30-31 dissolution of Actomyosin, 31-32 histology of, 29-30 temperature in, 1% work done in, 11 Rootstock, effects on fruit composition, 292-294

5 Salt, effect on meat tenderness, 32-35 Sarcolemma, 3 Sarcomere, 3-4 Shear press, 104-105 Size and shape, measurement of, 187192 Size, effects on fruit composition, 298 Spectrophotometry in color measurement, 161-162 in flavor chemistry, 211-ZI2 Sprays, effects on fruit composition, 300-301 copper, 301 hormonal, 301 lead arsenate, 300-301 oil, 300 Starch, see nlso Amylose and Amylopectin Starch, in peas, 65-68 measurement of, 67-68 relation to maturity, 67-68 relation to texture, 67 Stroma, 3, 4 Succulometer, 106 Sugars, in peas loss in processing, 69-70 relation to maturity, 68-69 Sulfur dioxide, use in fruit juices, 254957

T Tartaric acid, in fruit juices, 262-264 Tenderizers commercial, 38-40 distribution of in meat, 41 enzymes used, 38-39 histological effects. 4 1 4 4 producers of, 38 ’

395

SUBJECT INDEX

Tenderometer, 63, 67, 74, 87, 101, 103, 105, 116 Texturemeter, 102-1 03 Thaw rigor, 11, 23-26, see also Rigor mortis Thixotropy, 178-1 79 Tin uptake, in peas, 78 Trace elements, effects on fruit composition, 300 Tropomyosin, 7-8 molecular weight, 8 properties, 8

carotene, 78-80 folic acid, 85-86 inositol, 85-86 miscellaneous, 91 niacin, 84-85 pantothenic acid, 85-86 pyridoxine, 85-86 riboflavin, 83-84 thiamine, 80-83 vitamin A, 78-80 Volatile constituents in citrus fruit i n juices, 333-340 in peels, 332-333

V Viners, pea, 123-126 Viscosity in food, 175-187 absolute, 180 apparent, 180-181 formula for, 180 measurement of, 181-187 relative, 181 Vitamins in citrus fruits ascorbic acid, 310-31 I others, 311-312 Vitamins, in peas, 78-91 ascorbic acid, 86-90 biotin, 85-86

W Whale muscle, rigor in, 22

X X-protein, 4 Y

Yeasts, in noncitms fruit juices, 228-234 in apple juice, 229-230 in concentrates, 230-231 effect on malic acid, 267-268 i n grape juice, 231-234

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