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

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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 . .

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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

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

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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

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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

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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

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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.

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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.

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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

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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. (<

B. EXTRACELLULAR PROTEINS 1. Collagen Collagen can be simply defined according to Gross (1954) as “that class of fibrous proteins characterized structurally by a particular wideangle X-ray diffraction pattern, and in most, if not all, native species, a distinctive axial periodicity of about 640 A, and chemically by a high content of hydroxyproline, proline, glycine, and low moieties of aromatic amino acids.” All of the above criteria are met by collagen from marine sponges, coelenterates, echinoderms, fishes, and mammals. Collagen is the structural protein of connective tissues and composes the larger part of tendons, ligaments, fascia, and other similar tissues. The physiological function of collagen is to act as an inert, inextensible material, which as tendons transmits the tensions exerted by the muscle. Collagen is quite resistant to changes in length under physiological conditions. Its elastic limit when wet appears to be around 7%. How-

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

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ever, on heating to about 6OoC., moist collagen suddenly contracts to one-third or one-fourth of its normal length and is very rubbery in behavior. It was first shown by ZachariadeS (1900) that collagen is dissolved by certain dilute acids, e.g., 1 :25,000 acetic acid, to give a clear solution. Extraction with citrate buffers (pH 3.0-4.5) also gives a soluble form of collagen (Tustanovsky, 1947). Prolonged boiling with water irreversibly converts collagen to a soluble form (gelatin) during which certain chemical changes take place in the molecule (Bowes and Kenten, 1948; Bowes, 1951). I n general, however, collagen is only slowly attacked by most dilute acids and bases or by enzymes; therefore, other proteins can be separated from it by treatment with these hydrolytic reagents. Under the prolonged influence of acids, alkalies, or salts, collagen takes up a considerable amount of water, the fibers become shorter and thicker and irreversibly altered. Collagen is peculiar in its amino acid make-up. It contains 15.5% hydroxy amino acids [lo% hydroxyproline, 3% serine, 1.8% threonine, and 0.7% hydroxylysine (found only in collagen)], 34% glycine, 12% proline, no cysteine/cystine, and only a small proportion of aromatic amino acids. Collagen is known to contain some polysaccharide material as evidenced by the presence of hexoses, chondroitin sulfate, and hyaluronic acid (Highberger et al., 1951). On the basis of histological evidence, it has also been suggested that collagen is a polyphase system composed mainly of “collastromin” (consisting of a protein-mucopolysaccharide complex and an argyrophilic, fibrillar protein) and procollagen (Tustanovsky et al., 1954). There are a number of recent excellent reviews on collagen and connective tissue (Bear, 1952; Borasky, 1950; Dorfman and Mathews, 1956; Asboe-Hansen, 1954; Randall, 1953; Josiah Macy Foundation, 1950-54). 2. Elastin

Elastin, the structural protein of elastic fibers, has been prepared from the cervical ligament, the ligamentin nuchae and the aorta. It can be recognized by its yellow color, by certain selective staining reactions, and by its appearance under the microscope. Unlike collagen fibers, the elastin fibers branch freely and anastomose with each other. Elastin has great elasticity and very closely resembles rubber in its properties. On heating, the elastic fibers contract. Chemically, collagen and elastin resemble each other only in that both contain a high content of proline and are poor in histidine, cystine, tyrosine, and tryptophan. While collagen contains 34% of polar groups, elastin contains only 7%. Elastin is more resistant than collagen to acids, bases, and enzymes; it cannot be heat-denatured and is unaffected by boiling

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JOHN R. W H I T A K E R

water. It appears to contain an appreciable amount of polysaccharide material.

3 . Reticulin Reticulin is very similar in nature to collagen and there is a great deal of argument as to whether they are identical or different. It has been claimed in studies with the electron microscope that they are identical (Kramer and Little, 1953; Irving and Tomlin, 1954). However, Cruickshank and Hill (1953) have demonstrated that reticulin contains an antigen not present in collagen, so that although the basic fiber structure may be similar there is an additional component in reticulin. Brewer (1957) also found the presence of this second component in reticulin and postulated it may be polysaccharide in nature. He also showed a difference in the birefringence and staining characteristics of collagen and reticulin. 4 . Ground Substance It is apparent that the amorphous matrix of connective tissues must contain substances in transit between the vascular system and the parenchymal cells as well as those components peculiar to connective tissue. There is increasing evidence of a multiplicity of mucopolysaccharides in connective tissue. The complexity of this system has been emphasized by Meyer (1954) who has reviewed the properties of the following six different mucopolysaccharides which have already been demonstrated in connective tissue: (1) hyaluronic acid; (2) chondroitin sulfuric acid A; (3) chondroitin sulfuric acid B; (4) chondroitin sulfuric acid C; (5) chondroitin; and (6) keratosulfate. The acid mucopolysaccharides are best interpreted as polyelectrolytes, and they probably play a large role in the ionic atmosphere of the muscle. Relatively little progress has yet been made on reactions between these polysaccharides and the proteins of muscle. A recent symposia has emphasized the complexity of the ground substance (Ciba Foundation, 1958). IV. CHEMICAL CHANGES ASSOCIATED WITH CONTRACTION AND WITH ONSET OF RIGOR MORTIS

A. THEORIES OF MUSCLE CONTRACTION There are many similarities between the contracted muscle and the rigor muscle; in fact, the observed differences are probably quantitative rather than qualitative. Bate-Smith (1948) noted that one of the primary differences was that muscle does not contract when it goes into

CHEMICAL CHANGES ASSOCIATED WITH A G I N G O F MEAT

11

the rigor state. Thus, while it becomes hard and relatively inextensible (as is also the case with the contracted muscle) the mechanism whereby the proteins become rigid would appear to differ from the mechanism involved in muscular contraction. However, both Bendall (1951) and Lawrie (1953) observed that in muscles held at 37OC. marked shortening occurred simultaneously with the loss of extensibility. However, if the muscles were held at 17OC., very little shortening was observed, even though the muscles lost their extensibility. Shortening in the rigor muscle, then, appears to be dependent upon temperature. BateSmith and Bendall (1949) have shown that the degree of shortening of the muscle depends upon both temperature and pH, and increases as both temperature and pH are increased. Perhaps the difference in shortening observed at 3 7 O and 17OC. is a matter of overcoming the inertia and viscosity of the muscle at the two temperatures. Rendall (1951) has shown that the amount of work done in rigor is about one-third of the maximum recorded by Hill (1949) for a single twitch in frog muscle. It will be shown later that the shortening in rigor begins only when the ATP level is falling rapidly, and is accompanied by a decrease in the extensibility of the muscle. Thus, it appears that the shortening would be restrained by the loss in extensibility. Szent-Gyorgyi (1949) has found the work done by a muscle in thaw rigor is close to that done by a living muscle. However, it would appear that thaw rigor would not be quite identical with normal rigor, as a great deal of damage must be done to the muscle fibers by the freezing and thawing process. Since the demonstration that “myosin” really consisted of actin and myosin, these two proteins have been regarded as the moving parts of the muscle machine. There are other proteins present in the muscle myofibrils which have been discussed previously, but the function of these proteins in contraction is unknown. Only the actomyosin particles (as in actomyosin threads) have been demonstrated to possess contractile properties. Workers in the field of muscle physiology are not yet sure of how myosin and actin associate to form actomyosin. In fact, there are many investigations which do not support the view that there is association. Szent-Gyorgyi and co-workers have focused most of their attention upon myosin as the component that contracts. The postulated intramolecular changes producing contraction are assumed to occur in myosin. While actin appears to be essential for contraction, its exact function is not clear. Perhaps actin may participate in triggering the contraction by modifying the charge on myosin as suggested by Ashmarin (1953). The association of actin and myosin is a precondition

12

J O H N R. WHITAKER

for contraction, but the elementary act of contraction is still claimed to be carried out by myosin (Szent-Gyorgyi, A.,1956, 1957). Szent-Gyorgyi (1957) has recently advanced a modified scheme for the series of events which lead to muscle contraction. He says that it is no longer a mystery why a muscle should contract; rather, it is difficult to see how it remains stretched out. He now speaks of the “stretched” or relaxed phase as the active phase which requires energy for maintenance. His explanation of the relaxed state of muscle places water in a prominent role. The series of events which bring about muscular contraction as depicted by Szent-Gyorgyi and outlined schematically by Gelfan (1958) are: Excitation + destroys the balance of electrostatic forces which keep actin and myosin dissociated in resting muscle (ATP, linked to myosin, contributes to the maintenance of this balance with its four negative charges) + actomyosin + ATP now “activated” by myosin + ATP split by ATPase to ADP -P + “bond energy” of -P transferred to mobile excitation energy + electronic excitation energy (which upon transmission to the myosin molecule, may assume an unusually long-lived form (triplet excitation) by virtue of “liquid ice” or water structures which form a unique system with the myosin subunits and keep the whole myosin chain stretched out when the muscle is at rest) +collapse of water structure+rearrangement of subunits (protomyosins) into a shortened aggregate, i.e., contraction.

+

It is obvious that in such a scheme energy is required to stretch or relax the muscle after it has contracted. The theory of muscular contraction proposed by Morales et al. (1955) which has aroused so much interest recently is very similar to the scheme presented above. In 1953 workers began to concentrate more on the role of actin in contraction. Astbury (1953) found that the X-ray pattern of frog sartorius muscle consisted of separate diagrams for myosin and actin and did not show any interaction between the two on contraction. Tsao and Blum and Morales (Tsao, 1953; Blum and Morales, 1953) concluded that there is no indication that actin and myosin interact in any way during the contraction process. In the electron microscope investigations of Hanson and Huxley described previously it was found that actin is associated with the I band of the sarcomere and myosin is associated with the A band. If the myofibril is made to contract, the only change observed is a shortening of the I band. This observation reverses the classical roles of actin and myosin as depicted by Szent-Gyorgyi since the properties of contractility now appear to be associated with the actin filament. Myosin is relegated practically to the role of an ATPase. There are many theories of muscle contraction but none of these

CHEMICAL CHANGES ASSOCIATED WITH AGING O F M E A T

13

fully explains all the observed facts of contraction. Wilkie (1954) has critically reviewed the theories of muscle contraction up to 1954.

MODELS FOR CONTRACTION AND RELAXATION B. EXPERIMENTAL The physiologist and biochemist often found it more convenient to work with a model system rather than with the actual system. This has been the case in most muscle investigations. There are five experimental models available to the researcher: the actomyosin thread, the glycerolextracted fiber, the homogenized muscle preparation (in addition to the unextracted fiber or the whole muscle). The unextracted fiber and whole muscle have the advantage over the other systems in that they contain all the materials normally necessary for contraction and relaxation. However, this can be a disadvantage as well when quantitative studies are to be made of the effect of certain additives. They have the further disadvantage of being labile. The size of the muscle does not permit the additives to reach equilibrium rapidly in the muscle. Szent-Gyorgyi (1947) first introduced the actomyosin thread. If a solution of actomyosin in 0.6 M KCl is forced through an orifice into a bath containing a more dilute KCl solution, a thin thread is obtained which possesses the properties of contractility and ATPase activity. It functions in the same manner as the muscle upon the addition of magnesium and calcium ions, ATP, etc., and can be kept for a long period of time without losing these properties. Later, Szent-Gyorgyi ( 1949, 1951) introduced the glycerol-extracted muscle fiber. Exhaustive extraction with glycerol removes all the components from the fiber except the proteins. This extracted fiber will retain its ability to contract upon the addition of ATP for several months when stored in the refrigerator in glycerol. The extensive work of Bozler, some of which will be mentioned later, has been carried out using this type of preparation. Bailey and Marsh (Bailey, 1954; Bailey and Marsh, 1952), however, have argued that the actomyosin thread and glycerol-extracted fibers, while retaining many of the properties of the muscle, should not be accepted unreservedly as true models because they lack many of the components of muscle and much of its fibrillar organization. To overcome these disadvantages, they proposed the use of a muscle homogenate. The homogenate, which consists of short pieces of highly organized fiber, allows physical and chemical changes to be followed simultaneously, permits the introduction and removal of components, and is amenable to qualitative and semiquantitative measurements of fiber volume, fiber length, and syneresis. It will be obvious, however, that the removal of components cannot be carried out as readily as with

14

JOHN R. WHITAKER

the actomyosin thread and glycerol-extracted fibers. The homogenate is also labile.

C. RIGORMORTIS Immediately after the slaughter of an animal, the muscles are relaxed, flexible, and soft. However, soon after death the muscles suddenly become rigid, hard, and contracted (if the experiments are carried out at 37OC.) and are in the so-called “rigor” state. The chief chemical events in the development of rigor have been extensively studied and are as follows: (1) glycolysis starts at death and continues at a rate dependent upon the pH; (2) decrease in pH due to formation of lactic acid; (3) fall of creatine phosphate (CP) to about 30% of the initial W

a m i

o

O

I

2

3

4 S 6 7 8 9 HRS. POST-MORTEM

1

0

1

1

FIG.4. Modulus curves calculated from rigor measurements. Curve I: myanesintreated animal; initial pH 7.00, ultimate pH 6.00. Curve 11: untreated animal (some struggling); initial pH 6.50, ultimate pH 5.90. Curve 111: same as in Curve 11. Curve IV: starved myanesin-treated animal (48-72 hr.); initial pH 7.05, ultimate pH 6.50. Curve V: insulin-treated animal (convulsions); initial pH 7.20, ultimate pH 7.20 (Bate-Smith and Bendall, 194Q).

value by the time the pH reaches approximately 6.8; (4) decrease in ATP until it reaches a low level (20 to 35%) depending upon the animal and muscle; at this point glycolysis is almost at a standstill, and the rate of ATP disappearance falls off markedly. At around 80% of the initial ATP value, the muscle suddenly loses its extensibility, becomes hard, and contracts; and (5) liberation of ammonia. Bate-Smith (1948) and Bate-Smith and Bendall ( 1956) have skillfully reviewed some of the changes which occur during the interval between death and the onset of rigor mortis, and they have adequately emphasized the change in pH which is found to occur due to the buildup of lactic acid caused by glycolysis. H a m (1958b.) has also reviewed these chemical changes which occur in post-mortem meat, and he has developed an excellent biochemical theory of rigor mortis. The time interval between death and the onset of rigor mortis is often found to vary greatly among animals. In an excellent work pub-

CHEMICAL CHANGES ASSOCIATED WITH AGING O F MEAT

15

lished in 1949, Bate-Smith and Bendall showed that this time is determined by two considerations: (1) the pH of the muscle at the moment of death (which is determined by activity immediately preceding death); and (2) the glycogen reserve of the muscle. Figure 4 shows the effect of the pH and glycogen content of the muscle at death on the time between death and the onset of rigor mortis. This work, as well as that discussed below, clearly indicates that the time of onset of rigor mortis is not dependent upon the muscle reaching a certain pH. While this work clearly shows the effect of the initial and ultimate pH and glycogen reserve on the time of onset of rigor, it remains for someone to show the effect of these same variations on the rate of aging of meat.

TIME (MIN. POST-MORTEMI

FIG.5. Relation between the CP-P and ATP-P contents and the shortening of psoas muscle during the course of rigor mortis at 37°C. Average initial pH, 7.10; average initial CP-P, 0.80 mg./g.; average initial ATP-P, 0.50 mg./g.; average ultimate pH, 6.50. KEY:-*-.-, shortening as % final; -X-x-, CP-P as % initial; -O+, ATP-P as % initial; ---, rate-ratio x 100 (Bendall, 1951).

Bate-Smith and Bendall (1947) found that the stiffening in rigor mortis is directly related to the disappearance of ATP. During the glycolysis which occurs in normal muscles after death, ATP is being continually synthesized at the rate of 1.5 moles for every mole of lactic acid produced (Lipmann, 1941) . However, this synthesis is counterbalanced by the breakdown of ATP by myosin. As long as there is sufficient glycogen present for the synthesis of ATP the muscle does not pass into rigor. As soon as the glycogen is exhausted, however, the rate of ATP breakdown will excede the rate of synthesis and rigor mortis sets in. Bate-Smith and Bendall (1947) found that the rapid phase of rigor in rabbit muscle started at 85% of the initial ATP value and was completed at 20% of the initial value. Bendall (1951) and Lawrie (1953) have shown that the interval between death and onset of rigor mortis is dependent upon the quantity of creatine phosphate (CP) present at death as well as the amount of glycogen. See Fig. 5 and Table I. Bendall found that the CP content

TABLE I COMPARATIVE DATAO N

Time before onset rapid phase of rigor (min.)

Duration of rapid phase (mh)

Longissimus dorsi Psoas

214 f 57

72 f 24

173 f 37

63 f 17

Diaphragm

148 k 20

75 f 18

Muscle

OLitwrie (1953).

THE

COURSE OP RIGOR MORTIS IN VARIOUS HORSE MUSCLES"

Initial

Onset

6.95

5.97

CP (mg.P/g.)

ATP (mg.P/g.)

PH Final

Initial

Onset

Final

Initial

5.51

0.39

k0.12 +_0.24 f 0 . 1 3

+0.0.5

0.11 k0.07

0.01 ~0.03

f0.04

7.02 kO.04 6.97 f0.06

0.28 f0.06 0.30 f0.M

0.09 *0.05 0.10 k0.04

0.04 k0.03 0.02 k0.02

0.15 f0.01 0.04 f0.03

6.24 F0.23 6.28 F0.20

5.98 rtO.10 5.76 fO.04

4 0

x

Onset

Final

-

-

7

4

0.36 0.01 ~0.01

-

-

E1-3 Lti!

CHEMICAL CHANGES ASSOCIATED W I T H A G I N G O F MEAT

17

of rabbit muscles is decreased to less than 30% of its initial level (<0.25 mg. CP-P/g.) before there is an appreciable breakdown of ATP. Figure 5 summarizes the main chemical events, with the exception of the breakdown of glycogen and production of ammonia, which are associated with rigor mortis in rested rabbits. The time of onset of rigor mortis cannot be delayed beyond definite limits by the glycogen reserve (Bate-Smith and Bendall, 1949). As the p H decreases, the enzymes of the glycolytic system, most of which have pH optima close to 7, become inactive. It appears that p H 5.3 is the limiting value; beyond this, glycolysis is completely inhibited even though glycogen may still be present.

3u 100

->

m

0

: + a o 6.7

@

pH d onset 6.6

6.4

625

6.0

6.5

6.75

6.0

S75

Ultimate pH

FIG.6. Critical level of ATP at 10% decrease in extensibility as function of ultimate pH, and pH at onset of rigor; 17°C. (Bendall, 1951).

Bendall (1951) has found that the onset of rigor mortis is not only dependent upon the ATP level but also upon the maintenance of the rate of synthesis of ATP by glycolysis as well. If the onset of rigor mortis were dependent upon the ATP concentration alone, the muscle should go into rigor mortis at a constant ATP level regardless of the ultimate pH. Bendall has shown this is not true (Fig. 6) and that this effect is independent of the initial pH or CP level. For example, the critical level of ATP at ultimate pH 6.6 is 78% of the resting muscle level, while it is only 30% of the resting level at ultimate p H 5.8. He concluded, that in order to maintain a muscle in its flexible prerigor state, not only must a high level of ATP be maintained but also a balance between resynthesis and breakdown of ATP must be maintained so that the rate-ratio is 90% or higher. The amount of ATP required at the essential sites to maintain the muscle in its prerigor condition is probably quite small compared to the total amount of ATP

18

J O H N R. WHITAKER

present. However, there is a very rapid turnover of ATP at the muscle sites so that the rate of diffusion from the point of glycolysis to these sites becomes an important factor as the rate of glycolysis is reduced because of limiting substrate and/or low pH. Bendall reported the QW of glycolysis, CP destruction rate, and ATP turnover to be 1.63 0.02. However, Hamm (1958b) believes that the delay interval between death and the onset of rigor mortis is due to the presence of the MarshBendall factor which inhibits myosin ATPase rather than to the rate of resynthesis of ATP. The Marsh-Bendall factor becomes inactivated by the calcium ions released with the beginning of the rapid phase, and as soon as the ATP is reduced to a critical level by myosin ATPase the muscle goes into rigor mortis. De Fremery and Pool (1959) have recently shown that treatments which increase the rate of ATP breakdown in poultry (beating, electrical stimulating, cutting, electron irradiation, or increases in temperature) produce a more rapid onset of rigor and tougher meat. Bendall (195 1) is in agreement with the theory advanced above that muscular contraction and rigor mortis are due to the same mechanism. As postulated in Szent-Gyorgyi’s (1957) theory of muscular contraction, the ATP molecule forms an integral part of the charged system which keeps the muscle extended. If these charges are reduced momentarily by a stimulus, a series of events are triggered which lead to contraction. The same events could conceivably occur in rigor mortis where the ATP level drops below a critical level, not through a stimulus, but because the rate of ATP breakdown exceeds the rate of synthesis. Again the result is a contraction. From here on, however, the series of events differ for the contracted muscle and the rigor muscle. The contracted muscle very rapidly restores the ATP to the initial level through breakdown of the CP present, and the muscle relaxes. However, there is no means whereby the rigor muscle can restore its ATP content to the initial level, and it remains contracted and simultaneously loses its extensibility. A reasonable explanation for this loss in extensibility would be the formation of new cross-bonds in the muscle in the prolonged absence of ATP. It should be noted that the times involved here are fractions of a second. If this postulation be true, then the injection of ATP after the muscle has entered rigor would not cause it to relax, i.e., the shortening in rigor is irreversible. It is true that the muscle may become softer because of the “plasticizing” action of ATP, which will be discussed later. Another chemical change which occurs during the onset of rigor mortis is the liberation of ammonia. This has received very little attention. Parnas and Mozolowski (1927) found that ammonia is lib-

*

CHEMICAL CHANGES ASSOCIATED W ITH AGING OF MEAT

19

erated when skeletal muscle is severely stressed or fatigued. Webster (1953a) also found ammonia to be liberated by the muscle when it passes into rigor. Bendall and Davey (1957) have recently examined the mechanism of this ammonia liberation in greater detail. They found that the ammonia comes solely from the adenine nucleotides by the deamination of AMP to IMP (Embden, 1927) and by the direct deamination of ADP (Webster, 1953b); the former is the primary route. The following reactions show the series of events involved in the liberation of ammonia during rigor mortis: Primary source of NH, ATPase

+ IP (2) 2AI)PATP + A M P deaminaee (3) AMP-----t IMP + NHI ( 1 ) ATP-

ADP

myokinase

Secondary source of NH, (1) ADP+ IDP (2) 2IDP S ITP

+ NH, + ADP

The changes which have been found to occur in the adenine nucleotides after death are shown in Figs. 7 and 8. As has been shown by 37OC

3. Time ofter death (min) FIG. 7. Changes in CP, labile-P ( P w ) , N H S , and I N during rigor development in psoas muscles from myanesin-immobilized rabbits at 37°C. (Bendall and Davey, 1957).

previous data there is a decrease in the CP and ATP content. However, this work also shows the buildup of NH,, IN, IMP, and AMP during the onset of rigor mortis. Most of the work reported above has been carried out on rabbit

20

J O H N R. WHITAKER

muscle, while Lawrie (1953) worked with the muscles of the horse. In 1954, Marsh investigated the onset of rigor m o d s in beef muscles. He found that the onset in beef muscle resembled closely that in rabbit muscle as reported by Bate-Smith and Bendall (1949). As in the rabbit, the decrease in extensibility was associated with dephosphorylation of ATP, and when the final pH was low, the fall in pH paralleled these changes. However, he found a significant difference in the effect of temperature on acid production between beef and rabbit muscles. While D P N ~ P NAMP IMP-

I 1

IDP

ATP

1 1 1 1

I

5-

I

ITP

1 1

(01

pN/TPNT\WTiDP

A T

(b)

1

s

4-

' >I

e

.n_

2

4

FIG. 8. Ion-exchange chromatograms of extracted muscles. ( a ) 10 min. after death; ( b ) at 10 hr. after death (broken lines) and 24 hr. after death (solid lines). Temperature 17°C. (Bendall and Davey, 1957).

the pH-versus-time curves for the two at 37OC. were almost identical, a 10°C. decrease in the case of beef had as pronounced an effect as a 2OOC. decrease for rabbit. In other words, the Qlo (the ratio of the reaction rate at temperature t IOOC. to that at temperature t ) for the reaction in beef muscle was found to be twice as large as for rabbit muscle. The possible significance of this finding was not explained. Howard and Lawrie (1956b71957b) have also studied the effect of induced preslaughter conditions on the development of rigor mortis in the muscle of beef steers. They confirmed the work of Marsh (1954) which showed that the general biochemical changes associated with rigor mortis in beef muscle were similar to those found in the rabbit (Bate-Smith and Bendall, 1949; Bendall, 1951) . However, contrary to the

+

CHEMICAL CHANGES ASSOCIATED W I T H AGING O F MEAT

21

results obtained with pigs (Callow, 1936, 1939; Gibbons and Rose, 1950; Rose and Peterson, 1951), rabbits (Bate-Smith and Bendall, 1949; Bendall, 1951), and rats (Rose and Peterson, 1951), neither fasting nor prolonged exercise alone depleted beef muscle glycogen reserves sufficiently to raise the ultimate pH above the normal values of 5.4 to 5.6. A combination of fasting, exercise, and nervous exhaustion or the injection of insulin, however, did lower the glycogen content. The effect of the convulsions produced by insulin injections were striking: the glycogen reserves in both longissirnus dorsi and psoas muscles were completely exhausted, and the ultimate p H values were found to be 7.08. The onset of rigor mortis was found to be almost coincident with the moment of death, and practically no ATP and CP were found in the muscles one hour post mortem. In subsequent work on the effect of massive doses of insulin on beef quality, Howard and Lawrie ( 1 9 5 6 ~ ) found that, because the ultimate pH was high, the meat gave low drip values. However, the eating quality of the meat was seriously affected as the meat was dark in color and poor in flavor and in texture. Also, contrary to the findings on rabbits, pigs, and rats (as reported above), the addition of glucose to the animal prior to slaughter did not affect the interval between death and the onset of rigor mortis nor the ultimate pH. Since the muscles already contained enough glycogen to reach an ultimate pH of 5.44 (which is low enough to stop all glycolytic action), the additional glucose had no effect on the biochemical events associated with rigor mortis. While the post-mortem changes in the psoas, longissimus dorsi, semitendinosus, and semimembranosus muscles of beef are similar, in that they are characterized by anaerobic glycolysis which causes a fall in pH due to lactic acid production and a decrease in CP and ATP content which causes rigor mortis, they differ in the in uiuo store of glycogen and CP, the rate of post-mortem breakdown processes, the interval between death and onset of rigor mortis, buffering power, and in their response to applied treatment (Howard and Lawrie, 1957b). These differences appear to be due to the different functional specialization of these muscles in the animal. De Fremery and Pool (1959) have investigated the biochemical changes in chicken muscle which are related to rigor mortis. In general, the chemical changes following death are qualitatively the same as in the mammalian species. At room temperature the muscle passes into rigor within 2 to 4 hr. post mortem with an ultimate pH around 5.9. If the holding temperature is above 3OoC., there is a marked increase in the rate of ATP disappearance and in the onset of rigor. The muscle is much tougher than the control held at room temperature.

22

JOHN R. WHITAKER

Marsh (1952b) studied rigor mortis in the whale. While, for the most part the post-mortem behavior of whale muscle was found to be similar to that of other mammalian species, there were certain obvious differences. As the whale muscle goes into rigor, it changes from a dry to a very moist state due to syneresis. It was also noted that under certain circumstances glycolysis could be arrested briefly. Despite the fact that there is no synthesis of ATP, the ATP content was found to remain high, and the muscle remained in the relaxed state. Golovkin and Pershina (1957) reported that the muscles of fish stored at 0 to 6°C. for a period of 10 days contained an ever-increasing amount of ATP, which caused the dissociation of the actomyosin complexes. If these results are found to be correct by further experirnentation then it would suggest a different mechanism of rigor mortis in fish. It should now be clear that rigor mortis is the result of the decomposition of ATP to the point where it can no longer maintain the muscle in the relaxed state. The fact that myosin can split ATP, first shown by Engelhardt and Lyubimova (1939), has been confirmed by many workers. From the data of Bendall (1951) which shows that the curve for the rate of ATP turnover closely resembles the pH-versus-activity curve for myosin (Engelhardt and Lyubimova, 1942), it is reasonable to believe that the myosin-ATPase of muscle is the main enzyme responsible for the turnover of ATP. Data of other workers also support this view. It has also been shown that myosin ATPase is a sulfhydryl enzyme (Singer and Barron, 1944; Bailey and Perry, 1947; Kuschinsky and Turba, 1950). There is also impressive evidence to show that, under conditions where the muscle is relaxed, the ATPase activity is low, and that contractility and ATPase activity develop in a parallel fashion. For example, the different reagents vvhich inhibit ATPase activity such as Salyrgan or benzaldehyde, bring about relaxation (Kuschinsky and Turba, 1950, 1951; Portzehl, 1952; Korey, 1950). It would appear from these findings that the way to prevent muscle from going into rigor mortis is to add a sulfhydryl reagent to the muscle to prevent the breakdown of ATP. Bailey and Marsh (1952) hoped to extend the interval between death and rigor mortis by this method. However, they found that the reagents used were not completely effective in inhibiting the ATPase of muscle and that they arrested the resynthesis of ATP by inhibiting the enzymes of the glycolytic system, many of which are also sulfhydryl in nature. The over-all effect, then, was to shorten rather than to extend the interval between death and rigor mortis. It is unfortunate that more work of this nature has not appeared. It is interesting to think of the results which might be achieved if a more specific inhibitor of myosin ATPase could be found and if this were in-

CHEMICAL CHANGES ASSOCIATED WITH AGING O F MEAT

23

fused with ATP just prior to or immediately after slaughter. More specific, naturally occurring inhibitors of myosin ATPase do exist and will be discussed in the section on relaxing factors.

D. THAW RIGOR It is well known that under certain circumstances frozen meat loses considerable water on thawing and becomes very dry and unpalatable. This phenomenon has also been investigated to a limited extent by muscle physiologists interested in the mechanism of contraction (SzentGyorgyi, 1949; Perry, 1950; Marsh and Thompson, 1957). If rapidly frozen muscle is allowed to thaw at room temperature, it goes

t

..-..J. ,

P

[

20

?:.

*

....

.

8.. I 4.

FIG.9. Relation between “drip” and muscle fiber shortening during the onset of thaw rigor. Values as percentages of initial weight and length, respectively (Marsh and Thompson, 1957). into a very pronounced shortening and loses a great deal of its weight. Marsh and Thompson (1957) found that the shortening in thaw rigor depends on freezing the muscle in the prerigor state and the rate of thawing of the muscle. The loss in weight due to drip is also associated with the rate of thawing (see Figs. 9 and 10). At the biochemical level, it appears that the rapid depletion of ATP associated with the thawing diminishes the fluid-holding capacity of the structural proteins of the fibrils (Marsh, 1952a). Moran ( 1929) demonstrated that muscle shortening in thaw rigor can be prevented by a very prolonged thawing at temperatures just below O0C. The extent of shortening in thaw rigor appears to be independent of the time the muscle is in the frozen state. Thaw rigor of excised, prerigor chicken muscles has been investigated by de Fremery and Pool (1959). After rapid freezing (5 to 6 min. in a dry-ice bath), the ATP and glycogen content of the muscle were

24

JOHN R. WHITAKER

measured as the muscle thawed. This treatment greatly increased the rate of glycogen and ATP disappearance. The meat was found to he tougher than the unfrozen controls. Aging can only partially resolve this toughening. The data of Marsh and Thompson (1957, Fig. 9) show that the elastic limit of the muscle-containing membrane is reached with about 50% shortening. Further shortening is only possible if this membrane is ruptured. Such rupture results in the loss of fluids from the muscle. The increased shortening in thaw rigor is considered to be due to the in

TEMPERATURE. “C

FIG. 10. Effect of ambient temperature on “drip” during thawing of muscle strips frozen before rigor. KEY: 0, mean of 20 results at room temperature; X, value for strip frozen after rigor onset (Marsh and Thompson, 1958).

situ syneresis of the actomyosin of the myofibrils produced by the ATP still present in the muscle (Perry, 1950). In view of the above work on the extensive drip which accompanies the shortening associated with thaw rigor, it would not appear feasible to freeze meat right after slaughter. In commercial practice, beef is usually chilled at -0.5O to 1.5OC. for 1 to 3 days before it is frozen. Marsh (1954) suggested that thaw rigor might occur in beef quarters unless 35 hr. were to elapse before the start of freezing. However, the chilling period is inconvenient and expensive when the beef is to be frozen for shipment. Howard and Lawrie (1956a,c, 1957a; Bouton et al., 1957) have extensively investigated the problem of blast-freezing hot beef quarters. Contrary to expectations, thawing of the beef which had been rapidly frozen while still hot resulted in a slightly lower drip

CHEMICAL CHANGES

ASSOCIATED WITH AGING OF MEAT

25

content than the controls which were chilled 1 to 3 days and then frozen. While freezing in a blast tunnel operating at 250 ft. per minute and -35OC. was found to have an adverse effect on the eating quality of the thawed beef (Howard and Lawrie, 1956a), it was subsequently shown that faster freezing with a more powerful blast (1000 ft. per minute and -4OOC.) resulted in meat with as good eating qualities as beef which was normally chilled and then frozen. It was suggested that the beneficial changes caused by freezing were the result of changes at the microstructural level in the muscular tissues. However, according to Moran (1932) a muscle had to be cooled from $5 to - 5 O C . in under 50 minutes to produce the advantages of the fast frozen state. This certainly was not the case when Howard and Lawrie were working with a quarter of beef. It has been shown by Cook et al. (1926) and Moran (1929), and later confirmed by Ramsbottom and Koonz (1939) and Hiner et al. (1945) that the faster a small sample of meat is frozen the lower will be the drip on thawing. It is believed that the beneficial effect of fast freezing over that of slow freezing is that in the latter case ice formation takes place outside the muscle fiber sarcolemma while in fast freezing, ice formation is intrafibrillar, which facilitates reincorporation of water by the muscle protein colloid and thus largely prevents drip (Chambers and Hale, 1932). It should also be remembered that faster freezing results in the formation of much smaller crystals which would do less damage to the membrane. In order to explain the discrepancy between the results expected on the basis of the work by Perry (1950) and Marsh (1954) and the results obtained on whole beef quarters, Howard and Lawrie (1956a) suggested that when freezing begins there is an increasing concentration of reactants in the remaining liquid phase and there may be increased ATP breakdown at the sites of higher ionic concentrations. If this is true then the conditions necessary for thaw rigor, namely, high ATP level in the frozen state and subsequent rapid breakdown on thawing (Bendall and Marsh, 1951), do not exist in the blast-frozen beef quarters. It should be noted also that the quarters of beef were not frozen until long after the onset of rigor mortis and were thawed at +lO°C. Marsh and Thompson (1957) showed that thaw rigor is dependent upon freezing the muscle in the prerigor state and that the amount of drip is associated with the temperature of thawing. In later work, Marsh and Thompson (1958) found that the extreme shortening and drip characteristic of thaw rigor of isolated muscles did not occur if the muscles remained attached to the skeleton and if thawing were not accelerated.

26

JOHN R. WHITAKER

In contrast to the results obtained with excised chicken muscle (de Fremery and Pool, 1959) holding whole chickens or turkeys (which had been frozen in the prerigor state) at 1.7OC. had essentially as much tenderizing effect as an equal period of chilling before freezing (Klose et al., 1959; Pool et al., 1959). Significant tenderization also took place when the frozen carcasses were held at -2.8O to -3.9OC. for several days. V. CHEMICAL CHANGES ASSOCIATED WITH RELAXATION AND WITH RESOLUTION OF RIGOR MORTIS

A. RELAXATIONOF MUSCLE It has been found that the cooperative action of ATP and magnesium can bring about a contraction or a relaxation of actomyosin, depending upon the ionic milieu. At zero or low concentrations of univalent salts (0.05 M KCl), low concentrations of magnesium ions ( M ) accelerated myosin ATPase; however, at higher concentrations of the univalent salts or magnesium ions, the magnesium ions become a powerful inhibitor of ATPase. Recently, attention has been focused on a group of substances which inhibit or reverse the contraction produced by magnesium ions and ATP alone. These substances have been termed “relaxing factors” although under certain conditions they may actually reinforce contraction. Up to the present time, five relaxing factors have been recognized: (1) myokinase or the Marsh-Bendall factor, “M-B factor” (Marsh, 1951, 1952a; Bendall, 1952, 1953a, 195413) ; (2) ATP-creatine transphosphorylase creatine phosphate (Goodall and Szent-Gyorgyi, 1953; Lorand, 1953); (3) pyrophosphate (Bozler, 1954a, 1955; Bendall, 1953b); (4) EDTA (Watanabe, 1955; Bozler, 1954b; Bendall, 1958) ; and (5) A” (Perry and Grey, 1956), if it is present in excess of the magnesium ions, although it may function somewhat differently than the other four relaxing factors. It appears that these substances are quite different from one another. However, as pointed out by Morales et al. (1955), they have a number of features in common: ( a ) it appears that both ATP and magnesium ions are necessary for relaxation; ( b ) in the presence of magnesium ions and ATP the factor is tightly bound to the myosin system since it is very difficult to extract (Bozler, 1954a,b) ; ( c ) the addition of calcium ions abolishes relaxation (Bendall, 1953b; Watanabe, 1955; Marsh, 1952a; Goodall and SzentGyorgyi, 1953). With EDTA as the factor, Watanabe (1955) found that a stoichiometric amount of calcium ions were required to abolish relaxation. However, with PP only small amounts of calcium ions were

+

C H E M I C A L C H A N G E S ASSOCIATED WITH AGING O F MEAT

27

required (Bendall, 1953b); ( d ) in general, if the ionic strength, magnesium ion concentration, or ATP c,oncentration is lowered in the presence of the factors, ATPase activity and contraction are favored over relaxation (Bendall, 1954c; Hasselbach and Weber, 1953; Bozler, 1954b). It does not appear that the relaxation by myokinase or transphosphorylase is directly associated with their ordinary enzymatic properties as there is no correlation between the conditions which favor relaxation and those which favor enzymatic activity. A fundamental property common to all five factors seems to be a strong affinity for magnesium ions. Four of the above five relaxing factors are present in normal living tissue and any proposed mechanism of contraction and relaxation

Relaxed

Contracted

FIG. 11. Schematic diagram of the role of ATP, magnesium and calcium ions, and relaxing factor in the contraction and relaxation of muscle (drawn from the data of Morales et al., 1955).

should consider these factors. There have been a number of theories proposed to explain the mechanism of the combined action of magnesium and calcium ions, ATP and factor (Morales et al., 1955; Bozler, 1954b; Weber and Portzehl, 1954). According to Morales et al. (1955), it appears that in the living, “resting” muscle, the relaxing factor and ATP are both bound to the magnesium ion-containing active site (see Fig. 11 where P is contractile protein and R is relaxing factor). If excitation can cause the introduction of calcium ions, these could combine with and remove the factor away from the active site. According to this hypothesis the ATP, myosin, magnesium ion combination will then contract. According to the theory of Bozler (1954b), calcium ions are attached to the contractile protein during the contraction. However, both agree that the role of added calcium ions is to remove the relaxing factor and thus permit the system to contract. In support of the hypothesis of Morales, Wiercinski (1952) found that microinjections of magnesium ions had little effect on the system while calcium ions caused contraction. Falk and Gerard (1954) found, also in agreement with the above hypothesis, that added ATP did not affect the system. However, this hypothesis does not account for the splitting of ATP, which apparently is intimately associated with contraction.

28

J O H N R. WHITAKER

Howard and Lawrie (1956b) have examined the effect of preslaughter injections of calcium and magnesium ions on the development of rigor mortis and its associated biochemical changes in the muscles of beef steers. Their results, in general, confirm the in uitro findings reported above. The injection of sufficient magnesium ion to cause a rise from 2.22 to 20.5 mg./100 ml. serum caused a prolonged delay in the onset of rigor mortis. Glycogen was also conserved with a resultant decrease in the rate of pH and ATP fall. The injection of calcium ions to cause the serum level to rise from 9.59 to 42.36 mg./100 ml. serum resulted in a very considerable shortening of time of onset of rigor mortis. In a preliminary experiment in which calcium ions were removed by the injection of EDTA, the interval between death and onset of rigor mortis was prolonged (as expected from the theory of Morales et al.) . It is surprising how well the in uiuo and the in uitro experiments confirm each other. Pyrophosphate was also found to be a relaxing factor in vitro. However, Howard and Lawrie (1957b) found that the physiological effect of preslaughter pyrophosphate injection was to promote aerobic glycolysis and to cause extensive ATP breakdown. This is in marked contrast to its in uitro action where it simulates the Marsh-Bendall factor and slows the rate of ATP breakdown. The injection of pyrophosphate alone causes the collapse and death of the animal (Howard and Lawrie, 1956b). I n order to avoid this effect, which they attributed to a sequestering action on calcium ions, they injected calcium borogluconate along with the pyrophosphate. It should be obvious from the above discussion why they obtained markedly different results from those previously found for the in uitro action of pyrophosphate. Earlier, Bendall (1953b) had reported that only small amounts of calcium ions are required to inhibit the action of pyrophosphate.

B. RESOLUTION OF RIGORMORTIS In general there is agreement that the principal chemical changes which occur in muscle during the interval between death and the onset of rigor mortis involve a decrease in glycogen content, pH, ATP, and CP and an increase in lactic acid, ammonia, and the breakdown products of ATP, and eventually the formation of actomyosin to give a rigid, inflexible, tough muscle. However, there is no general agreement among workers on the chemical changes associated with the resolution of rigor mortis. This directly reflects the scarcity of knowledge concerning this most important phase of meat tenderization. It is well known that beef is more tender after being aged for 15 to 30 days at 0 . 5 O to 1.5"C. than it is 1 to 2 days after slaughter. Rams-

CHEMICAL CHANGES ASSOCIATED WITH AGING OF MEAT

29

bottom and Strandine (1949) have shown that beef is quite tender 2 hr. after slaughter. After this they found that the tenderness of beef gradually decreased to a minimum after 1 to 6 days storage at 1.7OC. and then gradually increased until it became more tender than the beef at 2 hr. Deatherage and Harsham (1947) have shown that tenderness is not a smoothly increasing function of the age of the carcass. See Fig. 12. The curve is typical of one in which two processes are going on simultaneously but at different rates. On the basis of this, Deatherage and Harsham postulated that the rapid and slow phases corresponded

t+.

12 ANIMALS

30 i ;

6

Ib

ir

20

21

30.''

8 ANIMALS

Jb

40

DAYS AGED AT 33-35'F.

FIG.12. Effect of aging on tenderness score (Deatherage and Harsham, 1947).

to changes in the proteins of the contractile system and the connective tissues, respectively. The resolution of rigor mortis has also been followed histologically (Harrison et al., 1949; Ramsbottom and Strandine, 1949; Paul et al., 1944; Lowe, 1948; Hanson et al., 1942; Carey, 1940). I n the prerigor state, the muscle fibers were found to be straight or slightly wavy. During the onset of rigor mortis, hard lumps began to appear which were associated with areas of extreme stretch on either side of the contracted lumps. This gave a washboard appearance to the muscle fiber. During the resolution of rigor mortis, the waves began to disappear and the fibers straightened out. Finally, after 4 to I 2 days of aging, a gradual and progressive breaking and rupturing of the fibers

30

JOHN R. WHITAKER

took place. It was postulated that this disintegration of the muscle fibers was due to the action of proteolytic enzymes and/or to the mechanical stresses placed on the stretched fibers. Histologically, two types of disintegration associated with aging were found; one was the increased fragility of the fiber striations and the other was the loss of fiber striations over a more extensive area. Sometimes the sarcolemma was broken, and the material within the sarcolemma had a granular appearance, and the longitudinal and cross-striations disappeared. The changes here should be compared with those associated with the action of added proteolytic enzymes (Wang et al., 1955, 1958). The histological changes which occurred in beef on aging were found to correlate well with the changes in tenderness measured either by instrumentation or organoleptically. Prima facie, it would appear that changes in tenderness brought about by aging could be associated with either one or a combination of the following factors: (1) changes in the connective tissues; (2) dissolution of actomyosin; (3) increased hydration of the proteins; and (4) proteolysis.

1 . Changes in the Connective Tissues It has been stated by a number of workers (Hiner et aZ., 1955; Miller and Kastelic, 1956; Nottingham, 1956; Callow, 1957) that the connective tissues play a large role in determining the tenderness of beef. These tissues are tough, and while collagen is somewhat modified by the cooking process, elastin is not changed. Callow (1957) makes the following statement: “From the point of view of toughness in meat, connective tissue is all important.” Others have found, however, that there is no correlation between the amount of connective tissue and tenderness (Hershberger et al., 1951). It is interesting in this connection that Wang et al. (1958) were able to distinguish organoleptically between “initial tenderness” which they associated with the muscle plasma proteins and the residue remaining which they associated with the amount of connective tissues present. However, it does not appear that the increase in tenderness associated with aging is due to changes in the connective tissue. In their initial work, Deatherage and co-workers (Husaini et al., 1950a,b) appeared to find a decrease in the alkali-insoluble protein during the aging of beef. However, by proper refinement of the method for determining the connective tissues, later work showed that there was, in fact, no change in the connective tissues on aging (Wierbicki et al., 1954, 1955). These views are in general agreement with other workers (Prudent, 1947; Winegarden et al., 1952; Ramsbottom and Strandine, 1949).

CHEMICAL CHANGES ASSOCIATED WITH AGING OF MEAT

31

Bouton et al. (1958) have also recently reported that the soluble nitrogen, alkali-insoluble proteins, and hydroxyproline do not change during the holding of beef.

2. Dissolution of Actomyosin The formation of actomyosin from actin and myosin appears to be the primary cause of the rigid, contracted, inflexible state of rigor mortis. After the onset of rigor mortis, the beef muscle slowly softens over a period of several days; the exact time required is largely dependent on the temperature. The resolution of rigor is more rapid in the chicken and turkey (Klose et al., 1959; Pool et al., 1959). At chill temperatures, most of the tenderization takes place within 4 and 12 hr. in the chicken and turkey, respectively. Coincident with the resolution of rigor mortis the meat becomes more tender. It would seem logical to believe that the resolution is due to a slow breakdown of the actomyosin into actin and myosin, i.e., a reversal of the event that produced rigor mortis in the first place. However, Marsh (1954) concluded that the resolution of rigor mortis did not involve the reversal of the onset of inextensibility since a strip of muscle held for 7 days at 7OC. in nitrogen did not show a decrease in the modulus of elasticity. Wierbicki et al. (1954, 1956) have investigated the role of the dissolution of actomyosin in the changes in tenderness which take place on aging beef. The results of preliminary work in 1954 indicated that there was a correlation between the buffer-extractable protein (actin and myosin) and tenderness. Subsequently, it was found that the temperature of extraction (room temperature) used in this study denatured some of the proteins. When the extraction was carried out below 10°C., so as not to denature the proteins, no correlation was found between buffer-extractable protein and tenderness. Figure 13 shows the changes which occur in the water- and bufferextractable nitrogen on aging beef. It was found that the bufferextractable proteins decreased sharply soon after slaughter. This supports the view that the onset of rigor mortis is associated with the formation of actomyosin. There appears to be a slight increase at 3 to 5 days post mortem and then a gradual decrease in the buffer-extractable proteins. This slow decrease would indicate that some of the proteins are becoming insoluble in the buffer through denaturation or proteinprotein interaction. McCarthy and King (1942) have found that there is a gradual increase in the sulfhydryl content during meat aging which may be the result of denaturation. Otake and Yamamoto (1954) also found that the amount of extractable and precipitable nitrogen from fish passed through a minimum value in 15 to 40 hours at 2OOC.

32

J O H N R. WHITAKER

In view of the evidence it does not appear that actomyosin is dissociated into actin and myosin and therefore cannot account for the changes in tenderness associated with aging beef. Hamm (1958a) found that salt had no effect on the buffer-extractable proteins but a tremendous effect on tenderness. This has been confirmed by Wierbicki et al. 065;

.--.BUFFER .--WATER

EXTRACT

W

0 12 24 HRS.

I

5

10 DAYS

20

30

FIG. 13. Effect of aging on the nitrogen extracted by potassium citrate buffer and by water (buffer pH 5.60, 0.22 M potassium chloride, 0.48 total ionic strength) (Wierbicki et al.,1956).

(1957a). They concluded that an increase in tenderness is not associated with the dissociation and solution of actomyosin. 3 . Increased Hydration of Proteins

The important role played by ions in the functioning muscle has been shown repeatedly by biochemists and physiologists, and this review has indicated the importance of these ions in muscular contraction and relaxation. From the data accumulated in their integrated study of the relation between biochemical changes and the changes in tenderness on aging, it became increasingly clear to Deatherage and co-workers that such factors as tenderness, texture, drip on freezing and thawing, and shrinkage on cooking were all related to the degree of hydration of the muscle proteins. Preliminary experiments (Wierbicki et al., 1954) had shown that the infusion of salt greatly improved 'the waterholding capacity of beef on cooking and on freezing and thawing. In later work (Wierbicki et al., 1956), it was found that both the pH and the juice expressed on cooking changed with the post-mortem age and appeared to be interrelated. This relation is shown in Fig. 14. As has been shown previously by many workers, the pH drops rapidly after slaughter due to formation of lactic acid. The change in juice expressed on cooking was found to change simultaneously with pH in an inverse relationship. These shrinkage losses were found to increase rapidly to a maximum at 24 hr. and then to decrease slowly

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

33

until after 30-days aging they were about the same as the losses observed at 2 to 3 hr. post mortem. Hamm (1956a) found that relaxation was associated with increased hydration and that contraction was associated with dehydration of the muscle protein. About two-thirds of the post-mortem decrease of muscle hydration was found to be due to ATP breakdown while the rest was due to the pH decrease associated with lactic acid production, Marsh (1952a) also found that the depletion of ATP is associated with a diminished fluid-holding capacity of the structural proteins. Bozler (1958) has recently shown that the muscle fibers of rabbit swell during relaxation and shrink during contraction as the result of uptake and release of water, respectively. It is clear that the p H affects the water-holding capacity of the meat by modifying the charges on the protein. Others have also reported that the

FIG.14. Effect of aging on pH and on the juice expressed on cooking (Wierbicki et a!., 1956).

p H profoundly afiects the water-binding capacity of beef and pork (Niinivaara, and Ryynaenen, 1953; Kormendy and Gantner, 1954; Bouton et al., 1957; Grau and Mirna, 1957; Hamm, 1953; Grau et al., 1953b). The isoelectric points of both myosin and actomyosin are close to the minimum p H observed in meats in rigor mortis. An increase in pH would result in an increase in the charge on the protein and thus would result in better water-holding capacity. Klotz (1958) has recently discussed the role of hydration in a variety of the unique features of protein behavior. It should also be recalled that the recent theory of Szent-Gyorgyi for muscular contraction assigns a central role to hydration and the charges on the protein. However, the fact that the pH minimum does not exactly correspond to the juice maximum led Wierbicki et al. (1956) to postulate that biochemical factors other than pH are controlling protein hydration. Arnold et al. (1956) found that during the aging of beef sodium and calcium ions are continuously released by the muscle proteins,

34

J O H N R. WHITAKER

potassium ions are absorbed after the first 24 hr. (this supports A. SzentGyorgyi's contention (1953) that potassium ions are released from the muscle after death), and magnesium ions are released during the first 24 hr. and also between 6 and 13 days, followed by a decrease in released magnesium ions. Because of the large excess of potassium ions which move onto the muscle, there is an increased net charge on the meat proteins which allows greater hydration and tenderness. In further studies on the role of ions in protein hydration and tenderness, Wierbicki et al. (1957a,b) confirmed the dynamic shifts of potassium, sodium, calcium, and magnesium ions involved in the hydration of BEEF

501

/

ROUND MUSCLES, 7-12 DAYS PM. ADDITIVE/ MEAT. 1/10

O 0

2 4 6 8 10 12N CONCN. OF THE ADDITIVES

FIG.15. Relation of added sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or citric acid on juice expressed by meat heated to 70°C. The concentrations noted in parenthesis on each curve indicate the approximate limits of additives from the standpoint of taste (Wierbicki et al., 1957a).

proteins. They found that the added chlorides of sodium, potassium, magnesium, or calcium increase the water-holding capacity of beef protein on cooking and on freezing and thawing (Fig. 15). They concluded that a combination of sodium and magnesium chlorides show the greatest promise in promoting the water-holding capacity of cooked beef and that beef could be made more tender by treating with a solution of these chlorides. While Wierbicki et al. (1957a) found that calcium ions increase the water-holding capacity of cooked beef, other workers (Grau et al., 1953a; Hamm, 1955a, 1956a,b,c) have reported just the opposite. They found that the removal of calcium ions by complexing agents, such as phosphate, citrates, ATP, ADP, EDTA, etc., or by ion exchange increase the water-binding capacity of sausage. These workers as well as others have also reported that salts of the alkali metals, especially

CHEMICAL CHANGES ASSOCIATED W I T H AGING OF MEAT

35

sodium chloride, as well as phosphates, increase the water-binding properties of pork and beef (Bendall, 1954a; Grau and Fleischmann, 1957; Grau and Hamm, 1956, 1957; Hamm, 1953, 1955c, 1957a,b, 1958a; H a m and Grau, 1955; Kormendy, 1955; Suri, 1957). For optimum results the salts should be added while the meat is still warm. It is interesting that Smorodintzev and Nikolaeva (1937) and other workers (Hall et al., 1944a,b; Loy and Hall, 19%) observed that sodium chloride and phosphate increase the water-binding properties and tenderness of meat and that calcium chloride decreases these properties. Apparently, little significance was attached to these results a t the time. Day (1948, 1949) reported that the swelling and shrinkage which takes place in the tissues of a freshly killed animal, as the result of variations in internal salt concentration and of pH, are the result of the separation of the collagen fibrils without visible alteration of the collagen fibrils themselves. He concluded that the changes in salt and pH affect the ground substances. The ionic nature of this material has already been discussed (Section III,B,4). Thus, it would appear that the next logical step for the meat biochemist would be an examination of the site of action of added salts.

4 . Proteolysis After the death of the animal the enzymes of the muscle are still quite active. The activity involved in the breakdown of glycogen to lactic acid has already been discussed. These enzymes become inactive at the onset of rigor mortis due to absence of substrate and/or inhibition by the pH of the media. Recently, the oxidative enzymes of muscle have been investigated (Andrews et al., 1952; Grant, 1955a,b, 1956; Tappel and Martin, 1958). Grant (1955a) reported that the only appreciably active enzymes are succinic dehydrogenase and cytochrome oxidase. The work of these investigators indicate the absence of substrate for the surviving respiratory enzymes in aged muscle. The muscles also contain a number of proteolytic enzymes, and these enzymes are amply supplied with substrate. To distinguish the proteolytic enzymes of tissues from those of the digestive tract, the former are called cathepsins. Balls (1938) has shown that beef muscle contains a cathepsin which is still active in frozen meat and which has a pH optimum around 4.1. Kastelic (1953), Snoke and Neurath ( 1950), Nardone (1952), and Martin (1954) have reported the purification of cathepsins from beef, rabbit, and frog muscle and rat skin, respectively. The kidney and spleen of beef and swine have been shown to possess appreciable proteolytic activity, and these enzymes have been studied

36

JOHN R. WHITAKER

extensively (Fruton et al., 1941; Fruton, 1941, 1946; Gutmann and Fruton, 1948; Smith, 1948; Adams and Smith, 1951). Fruton et al. (1941) showed that on the basis of specificity for synthetic substrates beef spleen contains at least 4 cathepsins. Zender et al. (1958) have recently shown that the psoas muscle of rabbit contains much less proteolytic activity than the kidney, liver, lung, and heart. Proteolytic enzyme activity has also been reported in tissues sterilized by ionizing radiation (Nickerson et al.,1950; Doty and Wachter, 1955; Kirn et al., 1956; Drake et al., 1957). The proteolytic enzymes present a problem in irradiation-sterilization because of their extensive degradation of proteins at relatively high dose rates. Large amounts of tyrosine crystals are reported to accumulate in the stored, irradiated product. For this reason, it will probably be necessary to heat-treat meat sterilized by ionizing radiation. While the data presented leave little doubt that proteolytic enzymes are present in meat, the exact role played by these enzymes in the increase in tenderness associated with aging meat is rather obscure. Deatherage and co-workers (Husaini et al., 1950a,b; Wierbicki et al., 1954) have found that there is no change in the nonprotein nitrogen (not precipitated by trichloroacetic acid) of beef aged up to 15 days. It has also been reported that with the exception of glutamic acid which increased some 300% and alanine and lysine which decreased slightly, the amino acids which resulted from protein breakdown remained constant in fish stored at O°C. (Hodgkiss and Jones, 1955; Shewan and Jones, 1957). Other workers, however, have reported changes in the nonprotein nitrogen during the aging or freezing of meat. Colombo and Gewasini (1955), Niewiarowicz (1956), and Leinati (1957) have reported that the free amino acid content of beef increased on aging for 6 to 18 days at 0" to 4°C. Both Niewiarowicz and Leinati reported the greatest change in the content of alanine, glutamic acid, cystine, and leucine. The values given by Colombo for these amino acids in the order given are: fresh meat, 0.676, 0.900, 1.964, and 0.261 mg./g. meat; of 6-dayold meat, 0.690, 0.900, 2.060, and 0.312 mg./g. meat; and of 12-day-old meat 0.708, 0.946, 2.100 and 0.335 mg./g. meat. Monzini and coworkers in their earlier work (Antoniani and Monzini, 1951; Monzini, 1953) reported that the amino acid nitrogen content of beef increased to 5 to 6% of the total nitrogen when stored at -25 to -4OOC. for 10 months. However, in more recent work (Monzini and Lissoni, 1955), it was reported that the amino acid content decreased on storage at -25 to -4OOC. for 48 months. Olson and Whitehead (1948) found that the total soluble nitrogen increased 41% in beef muscle and 47%

CHEMICAL CHANGES ASSOCIATED WITH AGING O F MEAT

37

in pork muscle after 29-days aging under the usual conditions. Zender et al. (1958) found a decrease in the glycine-soluble proteins and a corresponding increase in amino acid content of rabbit and lamb muscle stored at 25OC. for more than 31 days. Hepburn (1950b) found marked proteolysis in chicken aged at various temperatures. Proteolytic activity has also been reported in beef and chicken stored in the frozen condition (Hepburn, 1950a; Hiner et al., 1951; Swanson and Sloan, 1953). Swanson and Sloan reported that the overall increase in water-soluble nitrogen and nonprotein nitrogen were, respectively, dark meat 13.5% and 22.4%, light meat 14.3% and 27.9%. While they reported a decrease in the amount of amino acids with increasing period of aging, Hepburn found that the amino acid content increased with the period of aging. Much work remains to be done before the contribution of the proteolytic enzymes to the increase in tenderness of meat can be evaluated. Many of the reported changes in the various nitrogen fractions were obtained with meat held much longer than the normal aging period. In most of the work, proteolysis was determined by measuring the amino acids produced. A little thought should show that a protein can be extensively degraded before many amino acids are liberated. I n order to determine if proteolysis is important in meat tenderization it is necessary to measure the initial changes in the protein. The limited action of trypsin and chymotrypsin on myosin produces two components (Gergely, 1950, 1951, 1953; Perry, 1951; MihAlyi and Szent-Gyorgyi, 1953a,b; Gergely et al., 1955). There is a tremendous change in viscosity and the size of the molecules, yet the amount of amino groups liberated is approximately 3 to 4%; within the range of error of most methods of determining these groups. More work is needed on the correlation of solubility and electrophoretic properties of proteins with the aging period. Deatherage and co-workers have shown the way in the solubility studies while Zender et al. (1958) have reported preliminary studies on the electrophoretic changes which occur during aging. VI. ARTIFICIAL “AGING” OF MEAT

A. HISTORICAL ASPECTS When Cortes entered Mexico in 1519, he found the natives wrapping their meat in the leaves of the papaya to tenderize it. Thus, man’s desire to improve upon the natural aging process of meat goes back many centuries. Even though certain tenderizing mixtures had appeared in the patent literature and on the market (Ramsbottom, 1937; Allen, 1938; Pulley and von Loesecke, 1941) and proteolytic enzymes

38

JOHN R. WHITAKER

had been used for determining the quality of meat (Smorodintzev, 1934; Smorodintzev and Laskovskaya, 1935; Smorodintzev and Adova, 1935; Smorodintzev et al., 1939; Beck and Schormiiller, 1937b), it was not until 1942 (Gottschall and Kies) that the artificial tenderization of meat was approached scientifically. More recently, Tappel and coworkers at the University of California and Wang and co-workers at the American Meat Institute Foundation have investigated the use of proteolytic enzymes for the tenderization of beef. Perhaps the greatest impetus to the use of these tenderizers arose in 1955 when the Government gave packers permission to use them at the wholesale level. The influence of the two largest producers of meat tenderizers, Adolph‘s, Limited and Hercules Powder Company, as well as the producers of the proteolytic enzymes should not be underestimated. According to Glenn H. Freeman, who is in charge of Adolph‘s sales to packing houses, meat packers now are using over 20,000 gallons of enzyme tenderizers a month, which is more than double the amount used a year ago and enough to tenderize about six million steaks (McWethy, 1958). Up to the present time artificially tenderized beef has not appeared in the grocery stores but has been sold to restaurants throughout the country which specialize in the “$1.09 steak.” Many of the meat packers are actively investigating the problem of placing this meat in the grocery stores, and it should appear there before much longer. There are many tenderizing preparations available to the housewife, however, and generally she uses them on chuck and pot roasts as well as on steaks. According to a recent report to the fact-finding committee of the American Cattlemen’s Association (Coke, 1957), the use of enzymes for tenderizing meats may have far-reaching consequences in the beef industry. Conceivably, the use of the enzymes could eliminate the feeder yard and the expensive process of holding beef until it has “aged.” However, perchance the reader think this represents utopia in the beef industry, it should be pointed out that there are some disadvantages which will be mentioned later on.

B. NATURE OF

THE

ENZYMES USED

The enzymes that have been investigated for use in tenderizing meats are proteolytic in nature, i.e., they are able to hydrolyze the peptide bonds of the proteins of meat. They can be divided into three groups depending upon their source: (1) Plant-papain, ficin and bromelain. ( 2 ) Animal-trypsin, pepsin, pancreatin, and chymotrypsin.

CHEMICAL CHANGES ASSOCIATED WITH AGING O F MEAT

39

(3) Bacteria or fungus-protease 15, Rhozyme P-11, Rhozyme P-15, Rhozyme A-4, HT proteolytic, fungal amylase, Hydralase D and Hydralase TP, and elastase. The action of polygalacturonase on beef muscle was also investigated briefly (Doty et al., 1952-53; Auerbach et al., 1953-54). It was found that this enzyme liberates appreciable amounts of reducing compounds from beef tissue slices but does not affect tenderness. It is not the function of this review to discuss the nature of the proteolytic enzymes listed above. Because of the increasing importance of proteolytic enzymes, as well as others, in the food industry, it is anticipated that Advances in Food Research will soon present such a review. For a thorough treatment of proteolytic enzymes the following books and reviews should be consulted (Smith, 1951; Neurath and Green, 1954; Annual Reviews of Biochemistry).

C. COMMERCIAL TENDERIZERS There is an ever-increasing number of companies entering the meat tenderizer field. The preparations of these companies contain the proteolytic enzymes papain, ficin, bromelain, and Rhozyme P-I 1 used either separately or in combination with each other. They may be in powdered form or in solution. Extenders include salts (as high as 14 to 17%), hydrolyzed vegetable proteins, monosodium glutamate, as well as others (Weiner et al., 1958). A perusal of some of the patents granted on meat tenderizers will indicate the nature of these preparations (Ramsbottom, 1937; Allen, 1938; Pulley and Von Loesecke, 1941 ; Ramsbottom and Paddock, 1943, 1948; Bock and Goldhammer, 1951; Miro and Prijol, 1954; de la Puente Pouch, 1954; Drug Houses of Australia Ltd., 1954; Williams, 1957; Williams and Buchanan, 1957). Papain has been the most used of tenderizer. This is not surprising in view of the tremendous amount of work that has been done on papain and the scarcity of work on the other three enzymes mentioned above; it should not be construed as indicating that papain is the best enzyme for tenderization. Some of the properties of ficin have been reported recently by Whitaker (1957a,b, 1959). It should be noted that commercial enzyme preparations are not indefinitely stable on the grocer’s shelves and may be found to possess little or no enzymatic activity (Weiner et al., 1958). This points up the need for a good assay for these enzymes which can be correlated with their meat-tenderizing ability. It does not appear that the activity of the enzyme as determined on hemoglobin, casein, or milk necessarily is a good measure of its activity on the meat proteins (Gottschall, 19M; Wang et al., 1958).

40

JOHN R. WHITAKER

D. EVALUATION OF THE EFFECT OF PROTEOLYTIC ENZYMES ON TENDERNESS The effect of tenderization in meat is difficult to treat quantitatively because adequate criteria of what constitutes tenderness is lacking. However, the tenderness produced by the action of proteolytic enzymes is a result of protein breakdown. Although an exact correlation between protein breakdown and tenderization has not been possible, it is found that the more protein digested as determined chemically, the softer the structure of the meat becomes. There are many mechanical means on the market for measuring meat tenderness; however ultimately evaluation must be made by a trained panel of judges. The observed activity of any proteolytic enzyme depends upon such factors as the concentration of active enzyme, optimum pH of activity, optimum temperature for digestion, the time of action, the stability of the enzyme a t the cooking temperature, ability to come in contact with its substrate, and its ability to digest native proteins as compared with the heat-denatured proteins. 1 . Organoleptic Evaluation

There is general agreement among all the investigations carried out that the tenderness of beef is increased by the use of proteolytic enzymes. In a study of the effect of a meat tenderizer on less tender cuts of beef, Hay et al. (1953) found little difference in cooking losses, change in shape, aroma, and flavor between treated and untreated samples, but they found a significant difference in tenderness between the samples as determined by shear and panel studies, Tappel et al. (1956) found a significant difference between the tenderness of the papain-treated samples and that of the untreated controls as determined by shear measurements. Larson (1956) also found this to be true. Weir et al. (1958) reported that tenderizers had a very great effect on tenderness. The changes in the muscle during the normal aging period were found to be without effect upon the tenderizing action of the preparations. I n a more elegant series of taste-panel analyses, Wang et al. (1958) were able to demonstrate a close relationship between enzyme-induced changes in tissue structure and panel response to tenderness differences. The panel members differentiated between initial tenderness and residue after chewing. The initial tenderness was interpreted as being associated with disintegration of the sarcolemma and muscle fiber envelopes, and with reduced muscle fiber extensibility. The residue after chewing was interpreted as being associated with degradation

CHEMICAL CHANGES ASSOCIATED WITH AGING OF MEAT

41

of collagen and elastin fibers. Enzymes of plant origin (ficin, papain, and bromelin) were found to affect both of these groups of structures and properties, while microbial and fungal enzymes (Rhozyme P-11 and fungal amylase) affected principally the initial tenderness. The addition of 2% sodium chloride greatly increased the tenderizing effect of the enzymes. The mechanical and organoleptic analyses have all pointed up certain problems involved in the use of tenderizers. Perhaps the most important is the inability to distribute the enzyme uniformly throughout the meat. Gottschall and Kies (1942) found that papain does not penetrate solid pieces of meat but grinding the meat results in a much better digestion. The work of Tappel et al. (1956) confirmed this and showed the penetration was of the order of 1 to 2 mm. Thus, the surface becomes mushy due to overtenderization while the interior of the meat is unaffected. There are a number of ways this problem can be partially solved. Hay et al. (1953) “forked” the material into the meat, Wang’s group at the American Meat Institute Foundation have used dehydrated steaks, and a number of meat packers are investigating the feasibility of injecting the animal with the tenderizing solution just before or right after slaughter. This would appear to be the best approach as work on antibiotic infusion has shown that this practice is workable. One difficulty that may be foreseen for the injection of the material into the live animal is hemorrhaging produced by action of the proteolytic enzyme upon the blood vessel walls. Enzyme-treated steaks also have a tendency to become mealy (Hay et al., 1953), especially if they are overtenderized. This is readily apparent in the “$1.09” variety of steak sold by many of the steak restaurants. This mealiness is probably caused by the extensive granulations of the endomysial collagen observed (Weir et al., 1958). It has also been reported (Weir et al., 1958) that enzyme-treated steaks had a slightly bitter taste which is easily recognized by the panel. Carr et al. (1956) also reported that enzymic digests of casein have a bitter taste which is attributable to a single polypeptide. Many consumers indicate that the action of the enzymes on meat produces a bland effect. 2. Histological Eualuation Muscle possesses a visible, rather complex organization when viewed under the microscope (see Section I ) . It has been found that when muscle is treated with a proteolytic enzyme certain specific changes are induced in this organization. Wang and co-workers suspected that increases in tenderness, whether as a result of natural aging

42

J O H N R. WHITAKER

or by the application of proteolytic enzymes could be correlated with the observed microscopic changes in the tissue. Wang and Maynard (1955) found that papain, applied in the form of Adolph’s Meat Tenderizer, and Rhozyme P-11, a fungal enzyme supplied by Rohm and Haas Company, have very similar effects on the muscle tissue components. Both were found to attack the muscle fiber proteins, the nuclei of the muscle fibers and of the cells located in the endomysia. However, these enzymes were found to be inactive toward the collagenous and elastic fibers under the conditions used. The action of Rhozyme P-11on freeze-dried pork and beef longissimus dorsi muscles were described by the following sequence of events: thinning of the sarcolemma, disappearance of the nuclei, disintegration of the cross-striations in the muscle fibers, and the merging of the bare (sarcolemma-free) fibers finally to give a mushy appearance to the meat. They reported that the penetration of the enzymes, when applied to raw beef by either powdering or immersion methods, is inadequate. In a continuation of this work Weir et al. (1958) found that the tenderizers did not produce any structural alteration even on the surface when the raw beef was examined histologically. However, on cooking the treated meat, they found extensive granulation of the endomysial collagen and distintegration of the sarcolemma and muscle fiber envelopes. There was no visible change in the nuclei of either muscle fiber or free connective tissue cells or in the muscle fibers, and the only change in elastin was a slight decrease in its staining capacity. The tenderizers also were found to be without effect upon the extensibility of the muscle fibers. It has been reported (Wang et al., 1956) that muscle fiber extensibility is inversely related to tenderness for beef muscle tissue from some groups of animals. Partmann (1955) found that, unless the action of the enzymes is prolonged, they have no effect on the contraction produced by added ATP. It should be noted that certain of these results of Weir et al. (1958) do not agree with the results reported previously. The authors point out this could be due to (1) the papain concentration used was below the “threshold” level or (2) that its action was modified by the hydrolyzed vegetable protein and salts present in the tenderizer. It is regrettable that the authors did not give the concentration of tenderizer used in these experiments. Wang et al. (1958) studied the action of twelve enzymes on freezedried longissimus dorsi and semitendinosus steaks. The steaks were rehydrated for 15 minutes in the enzyme solutions. The following sequence of events was found to occur: the sarcolemma and the muscle fiber envelopes were attacked first and disappeared; next, the nuclei of the muscle fibers, as well as those of the free cells of the endomysia

43

CHEMICAL CHANGES ASSOCIATED W I T H AGING O F MEAT

slowly disappeared; the cross-striations of the actomyosin disappeared, and finally the actomyosin dissolved. The relative activities of the enzymes in producing this change were, in increasing order of activity: untreated, bromelin, papain, Hydralase TP, Hydralase D, fungal amylase, Viokase, protease 15, ficin, or HT proteolytic enzyme. The effect of these enzymes on muscle fiber extensibility was also found to be in the order given above. The enzymes were found to show great specificity for the muscle components. While the enzymes, ficin, bromelin and papain, from tropical plants and the enzymes, trypsin and Viokase, from the pancreas were found to possess collagenase activity, the microbial enzymes, Hydralase TP, Hydralase D, fungal amylase, protease 15, and HT proteolytic, had little or no effect on collagen. However, this latter group of enzymes had some effect on the ground substance of the connective tissue. Ficin and bromelin appeared to be equally effective on collagen, while papain was somewhat less effective (Table 11). The TABLE I1 RELATIVEPOTENCIES OF TWELVE ENZYME PREPARATIONS ON THE MUSCLE BASEDON STRUCTURAL MANIFESTATION' TISSUECOMPONENTS Muscle fibers Enzyme preparations Protease 15 Rhozyme P-11 Rhozyme A-4 HT proteolyt,ic Fungal amylase Hydralase D Hydralase TP Ficin Papain Bromelin Trypsin Viokase

PH 6.4 6.8 7.3 6.9 7.1 7.4 6.9 5.2 5.1 6.3 5.7 5.8

Connective tissue fibers

Actomyosin

Collagen

+++ ++ ++ ++++ +++ +++ ++ +++ ++ Trace ++

Trace Trace Trace Trace

+++b

Elastin

-

+++ + +++ + +

-

++++ ++ -t

+ +

Wang et al. (1957). 6 Sarcolemma not

affected.

following sequences of changes were found for the action of ficin, bromelin, and papain on collagen: (I) liberation of the ground substance; (2) decrease in the staining capacity; (3) loss of both staining capacity and fibrillar character; ( 4 ) reduction to an amorphous material. Only the plant and pancreatic proteolytic enzymes were found to

44

JOHN R. WHITAKER

possess elastase activity. The order of activity found, in increasing order was: Viokase and trypsin, bromelin, papain, and ficin. Ficin was found to possess at least twice the elastase activity of any other enzyme tested. The importance of this finding cannot be overemphasized as it is well known that the connective tissues which bind the muscle fibers together contribute to the toughness of meat (Hiner et al., 1955; Miller and Kastelic, 1956; Nottingham, 1956). Because of its elastic nature, elastin is difficult to chew or break. Also, in contrast to collagen, which is partially hydrolyzed by the cooking process (Griswold and Leffler, 1952; Cover and Smith, 1956), elastin does not become more tender during cooking; if anything it becomes tougher because of its ability to contract when heated.

3 . Chemical Evaluation The use of proteolytic enzymes for evaluating the quality of meat has been suggested by a number of workers. Smorodintzev and Adova (1934, 1935) found that pepsin can be used to determine the quality of beef. They found that first-grade beef is digested 10 to 20% better by pepsin than is second-grade beef. Beck and Schormiiller (1937a) found that pepsin, trypsin, and papain digested the muscle fiber proteins more rapidly than the connective tissues. More recently, Grau and Hamm (1951, 1954) and Hamm (1955b,d) suggested the use of pepsin for determining the amount of connective tissue in meat and therefore the quality of the meat. A chemical investigation of the relative effect of proteolytic enzymes of plant, animal, and microbiological origin has been reported by Miyada and Tappel ( 1956). The investigation was carried out on rehydrated ground, freeze-dried biceps femoris muscle of beef. For evaluation of the effect of the enzymes upon the meat components, the digestion mixture was divided into the soluble-protein fraction, the nonprotein fraction, and the collagen and the elastin fractions; the nitrogen content of each fraction was determined by the Kjeldahl method. They found that the greatest change among the various protein fractions occurred in the transformation of soluble-protein nitrogen to nonprotein nitrogen, which represents the hydrolysis mainly of actomyosin. This finding has been confirmed for ficin by El-Gharbawi and Whitaker (1958) (Fig. 16). While it was concluded that papain, bromelin, ficin, trypsin, and Rhozyme P-I 1 possess the necessary proteolytic activity for use as meat tenderizers, quantitative differences were found among these enzymes. All of these enzymes gave good hydrolysis of the soluble beef proteins. Papain and ficin were found to digest elastin, while Rhozyme P-I 1,

C H E M I C A L CHANGES ASSOCIATED WITH AGING OF M E A T

45

Protease 15, and Rhozyme A-4 showed slight but definite digestion of elastin. I n the case of collagen, the greatest digestion was found with bromelin, ficin, and trypsin, followed by Rhozyme P-11 and papain. It should be noted that these experiments were carried out at 6OoC., the temperature at which collagen is denatured. Pepsin cannot be used in meat tenderizer preparations because it is not active (pH optimum 1 to 2) at the pH of meat (pH 5.6 to 6.3).

240

* Non-protein Nitrogen

Z

nn

LI

I

Collagen Nilroqen

1

d-

Elastin Nitrogen Soluble Protein Nitrogen

Total Soluble Protein Nitrogen I

0

10

1

I

20

30

I 40

I 50

60 ..

ENZYME CONCENTRATION (rng./g.-ieal)

FIG.16. Effect of ficin concentration on the hydrolysis of the different protein fractions of freeze-dried meat. Incubation time, 1 hr.; temp. 60°C. (El-Gharbawi and Whitaker, 1958).

I n further work on the factors which affect the tenderization of beef by papain (Tappel et al., 1956), it was found that the greatest hydrolysis of beef proteins by papain occurred at 60° to 8OOC. This has also been found to be true of the action of ficin on beef proteins (ElGharbawi and Whitaker, 1958). Collagen was not found to be hydrolyzed by either ficin or papain below 6OOC. However, at this temperature there was a rapid hydrolysis of the collagen. This increase in hydrolysis is not due to conversion of the collagen to gelatin by the heat, as shown by the control, but rather is probably due to denaturation of the collagen at this temperature. Sizer (1949a,b) found that, while pepsin digested native collagen, trypsin and chymotrypsin did not hydrolyze collagen unless it was first denatured. It should be noted that the pepsin action was determined in 0.1 N HCI (the usual procedure)

46

JOHN R. WHITAKER

and that collagen fibers swell at this pH. Consequently, the collagen may be denatured by the pH used in the pepsin experiments. Neuman and Tytell (1950) found that native collagen is resistant to the action of trypsin, chymotrypsin, and papain, while Oken and Boucek (1957) reported these enzymes gave small but consistent digestion of collagen in all samples. El-Gharbawi and Whitaker (1958) found the optimum pH for the digestion of the meat components by ficin was 6.6 to 7.0. The addition of sodium chloride to the reaction mixture was found to also increase the rate of hydrolysis of these meat components. VII. CONCLUSIONS

A knowledge of the structure and physiology of the muscle is as essential to the meat biochemist as it is to the biochemist interested in muscular dystrophy. However, the meat biochemist has not contributed his share of information to the general understanding of the muscle; in fact, he has not always used the information which is available to improve his product. The rather small number of research groups throughout the world who have approached the production and processing of meat from the biochemical angle have made outstanding contributions to this field. Yet there is still much to be done. The reason for the delay period between death and rigor mortis is fairly well understood, and the interval between death and the onset of rigor mortis can be controlled with some success. But many questions still remain. Can rigor mortis be eliminated altogether by certain substances such as a combination of ATP and EDTA, myokinase and EDTA, etc.? What effect would this have on the tenderness and flavor of meat? Can the aging period be eliminated altogether by this procedure? What effect will the infusion of proteolytic enzymes into meat immediately after death for tenderizing purposes have on the normal events associated with rigor mortis? Singh and Singh (1955) have reported the relaxation of contracted unstriated muscle by the action of papain and trypsin. The chemical events associated with the resolution of rigor mortis are incompletely understood. It is well established that meat contains proteolytic enzymes and that the conditions during rigor mortis are optimal for their activity. Yet, the role these enzymes play in the resolution of rigor mortis is controversial, largely because most of the workers have used methods which are relatively insensitive to protein breakdown. Perhaps an application of the techniques of electrophoresis, ultracentrifugation, chromatography, and solubility would supply the missing answers.

C H E M I C A L C H A N G E S ASSOCIATED WITH AGING O F M E A T

47

It appears that tenderness is greatly influenced by the water-binding capacity of the proteins. These proteins can bind water as the result of their ionic nature which can be modified by the presence of salts. More information is needed on the mechanism of binding of these ions to the proteins, the proteins involved, and the nature of the water when bound. What role do the mucopolysaccharides play in the water-binding capacity of meats (Fessler, 1957)? Meat tenderizers are having a tremendous influence on the meat industry. However, relatively little is known about the action of these enzymes on the components of meat, and very little more is known about the proteolytic enzymes used in the tenderizers. A fundamental study of the action of purified enzymes on the purified components of meat should go a long way in demonstrating chemically many of the factors involved in meat tenderization. The action of collagenase and elastase on the tenderization of meat should be investigated. These enzymes should allow one to study the effect of the removal of one component at a time on the process of tenderization. If effective, these enzymes might prevent overtenderization, which is worse than no tenderization at all. What effect do these enzymes have on the waterbinding properties of meats? How can they best be applied to the meat? How can overtenderization be prevented? What influence do they have on the flavor constituents of meat? These are only a few of the many problems that are still awaiting answer. If the author has stimulated the reader sufficiently so that he will consider some of these problems with more than a momentarily curiosity then the author has achieved his goal in writing this review. REFERENCES Adams, E., and Smith, E. L. 1951. Proteolytic activity of pituitary extracts. J . Biol. Chern. 191, 651. Allen, H. E. 1938. Tenderizing meats with enzymes. U S. Patent 2,140,781. Andrews, M. M., Guthneck, B. T., McBride, B. H., and Schweigert, B. S. 1952. Stability of certain respiratory and glycolytic enzyme systems in animal tissues.

J . Biol. Chem. 194,715. Antoniani, C., and Monzini, A. 1951. The increase of amino nitrogen of meat preserved by rapid freezing. Preddo 5, (1/3) 14;Chern. Abstr. 47,4006 (1953). Arnold, N., Wierbicki, E., and Deatherage, F. E. 1956. Post mortem changes in the interactions of cations and proteins of beef and their relation to sex and diethylstilbestrol treatment. Food Technol. 10,245. Asboe-Hansen, G. (ed.) 1954. “Connective Tissue in Health and Disease.” Munksgaard, Copenhagen. Ashmarin, I. P. 1953. Enzymic decomposition of adenosinetriphosphoric acid and contraction of actomyosin. Biokhimiya 18, 71; Chern. Abstr. 47, 8132 (1953). Astbury, W.T. 1953. A discussion on the structure of proteins. Proc. Roy. SOC.B141, 1.

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Auerbach, E., Keyahian, T., and Doty, D. M. 1953-54. Effect of enzymes on beef tissue components. Ann, Rept. Am. Meat Inst, Foundation p. 17. Bailey, K. 19%. Tropomyosin: a new asymmetric protein component of muscle. Nature 157, 368. Bailey, K. 1951. End-group assay in some proteins of the keratin-myosin group. Biochem. J . 49, 23. Bailey, K. 1954. In “The Proteins” (H. Neurath, and K. Bailey, eds.), Vol. 2, Part B. p. 954. Academic Press, New York. Bailey, K., and Marsh, B. B. 1952. The effects of sulphydryl reagents on glycolysis in muscle homogenates. Biochim. et Biophys. Acta 9, 133. Bailey, K., and Perry, S . V. 1947. The role of sulphydryl groups in the interaction of myosin and actin. Biochim. et Biophys. Acta 1, 506. Balls, A. K. 1938. Enzyme action in food products at low temperatures. Ice and Cold Storage 41, 85, 101, 143. Bate-Smith, E. C . 1948. The physiology and chemistry of rigor mortis, with special reference to the aging of beef. Advances in Food Research 1, 1. Bate-Smith, E. C., and Bendall, J. R. 1947. Rigor mortis and adenosine triphosphate. J . Physiol. 106, 177. Bate-Smith, E. C., and Bendall, J. R. 1949. Factors determining the time course of rigor mortis. J . Physiol. 110, 47. Bate-Smith, E. C., and Bendall, J. R. 1956. Changes in muscle after death. Brit. Med. Bull. 12, 230. Bear, R. S . 1952. The structure of collagen fibrils. Advances in Protein Chem. 7, 69. Beck, K., and Schomuller, J. 1937a. The proteins of meat. I. The composition of meat proteins. 2. Untersuch. Lebensm. 74,369. Beck, K., and Schijrmuller, J. 1937b. The proteins of meat. 11. The enzymatic digestion of meat proteins. 2. Untersuch. Lebensm. 74, 461. Bendall, J. R. 1951. The shortening of rabbit muscles during rigor mortis: its relation to the breakdown of adenosine triphosphate and creatine phosphate and to muscular contraction. J . Physiol. 114, 71. Bendall, J. R. 1952. Effect of the ‘Marsh factor’ on the shortening of muscle fiber models in the presence of adenosine triphosphate. Nature 170, 1058. Bendall, J. R. 1953a. Further observations on a factor (the ‘Marsh‘ factor) effecting relaxation of ATP-shortened muscle-fiber models, and the effect of Ca and Mg ions upon it. J . Physiol. 121,232. Bendall, J. R. 1953b. Effect of pyrophosphate on the shortening of muscle-fiber models in presence of adenosine triphosphate. Nature 172,586. Bendall, J. R. 1954a. The swelling effect of polyphosphates on lean meat. J . Sci. Food Agr. 5, 468. Bendall, J. R. 1954b. Myokinase as a relaxing factor in muscle. Nature 173, 548. Bendall, J. R. 1954c. The relaxing effect of myokinase on muscle fibers; its identity with the ‘Marsh‘ factor. Proc. Roy. SOC.B142, 409. Bendall, J. R. 1958. Relaxation of glycerol-treated muscle fibers by ethylenediamine tetraacetate. Arch. Biochem. Biophys. 73, 283. Bendall, J. R., and Davey, C. L. 1957. Ammonia liberation during rigor mortis and its relation to changes in the adenine and inosine nucleotides of rabbit muscle. Biochim. et Biophys. Acta 26, 93. Bendall, J. R., and Marsh, B. B. 1951. The biochemistry of muscular tissue in relation to loss of drip during freezing. Proc. Intern, Congr. Refrig. 8th Congr., London, p. 351.

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Blum, J. J., and Morales, M. F. 1953. The interaction of myosin with adenosine triphosphate. Arch. Biochenl. Biophys. 43, 208. Bock, G., and Goldhammer, H. 1951. Meat tenderizing preparations. Australian Patent 141,733. Borasky, R. 1950. Guide to the literature on collagen. U S . Depi. Agr., Bur. Agr. and Ind. Chem. AIC-178. Bouton, P. E., Howard, A., and Lawrie, R. A. 1957. Studies on beef quality. VI. Effects on weight losses and eating quality of further preslaughter treatments. C.S.I.R.O.Diu. Food Preseru. Transp. Tech. Paper 6. Bouton, P. E., Howard, A., and Lawrie, R. A. 1958. Studies on beef quality. VII. The influence of certain holding conditions on weight losses and eating quality of fresh and frozen beef carcasses. C.S.I.R.O. Diu. Food Preseru. Transp. Tech. Paper 8. Bowes, I. H. 1951. Composition of skin-collagen and the effect of alkalis on collagen. Research 4, 155. Bowes, J. H., and Kenten, R. H. 1948. The effect of alkalis on collagen. Biochem. I . 43, 365. Bozler, E. 1954a. Interactions between magnesium, pyrophosphate, and the contractile elements. J . Gen. Physiol. 38, 53. Bozler, E. 1954b. Relaxation in extracted muscle fibers. J . Gen. Physiol. 38, 149. Bozler, E. 1955. The effect of polyphosphates and magnesium on the mechanical properties of extracted muscle fibers. 1. Gen. Physiol. 39, 789. Bozler, E. 1958. Light scattering and water content of extracted muscle fiber as related to contraction and relaxation. Arch. Biochem. Biophys. 73, 144. Brewer, D. B. 1957. Differences in the fine structure of collagen and reticulin as revealed by the polarizing nliscroscope. J . Pathol. Bacteriol. 74, 371. Callow, E. H. 1936. Transport by rail and its after-effects on pigs. Dept. Sci. Ind. Res. (Brit.),Food Invest. Board 1936, 81. Callow, E. H. 1939. The after effects of fasting. Dept. Sci. Ind. Res. (Brit.), Food Invest. Board 1938, 54. Callow, E. H. 1957. Ten years’ work on meat at the Low Temperature Research Station, Cambridge. Food Sci. Abstr. 29, 101. Carey, E. J. 1940. Wave mechanics in striated muscles. XVI. Effects of experimental variation in temperature and of micro-capillarity on the cross striations in muscle. A.M.A. Arch. Pathol. 30, 1041. Carr, J. W., Loughheed, T. C., and Baker, B. E. 1956. Studies on protein hydrolysis. IV. Further observations on the taste of enzymic protein hydrolysates. I . Sci. Food Agr. 7, 629. Chambers, R., and Hale, H. P. 1932. The formation of ice in protoplasm, Proc. Roy. SOC. B110, 336. Ciba Foundation. 1958. “Chemistry and biology of mucoproteins” (G. E. W. Wolstenholme and M. O’Connor, eds.) . Churchill, London. Coke, E. J. 1957. Revolution in the meat industry. Food Processing 18, (12) 32. Colombo, S., and Gewasini, C. 1955. Ripening of butchering animal meat. IV. Quantitative chromatographic analysis of amino acids in fresh and ripened meat. Atti. SOC. ital. sci. vet. 9, 434; Chem. Abstr. 50, 14137 (1956). Cook, G. A., Love, E. F. J., Vickery, J. R., and Young, W. J. 1926. Studies on the refrigeration of meat. 1. Investigations into the refrigeration of beef, Australian I . Exptl. Biol. Med. Sci. 3, 15.

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Corsi, A,, and Perry, S. V. 1958. Soine observations on the localization of illyosin, actin and tropomyosin in the rabbit myofibril. Biochem. J . 68, 12. Cover, S., and Smith, W. H., Jr. 1956. Effect of two methods of cooking on palatability scores, shear force values and collagen content of two cuts of beef. Food Research 21, 312. Cruickshank, B., and Hill, A. G. S. 1953. The histochemical identification of a connective tissue antigen in the rat. J . Pathol. Bacteriot. 66, 283. Day, T. D. 1948. The spread of fluids in connective tissue. I . Pathol. Bacteriol. 60,

150. Day, T. D. 1949. The mode of reaction of interstitial connective tissue with water. J . Physiol. 109, 380. Deatherage, F. E., and Harsham, A. 1947. Relation of tenderness of beef to aging time at 33-35°F.Food Research 12, 164. de Fremery, D., and Pool, M. F. 1959. Biochemical studies with chicken muscle as related to rigor mortis and tenderization. Submitted to Food Research. de la Puente Pouch, M. T. 1954. Tenderizing of meat. Spanish Patent 214,226. Dorfman, A., and Mathews, M.€3. 1956. The physiology of connective tissues. Ann. Rev. Physiol. 18, 69. Doty, D. M., and Wachter, J. P. 1955. Influence of gamma-radiation on proteolytic enzyme activity i n beef muscle. J . Agr. Food Chem. 3, 61. Doty, D. M., Auerbach, E., and Keyahian, T. 1952-53. Enzymic degradation of beef tissue components, particularly sarcolemma. Ann. Rept. Am. Meat Inst. Poundution p. 16. Drake, M. P., Giffee, J. W., Jr., Ryer, R., 111, and Harriman, H. 1957. Proteolytic enzyme activity in irradiation-sterilized meat. Science 125, 23. Drug Houses of Australia, Ltd. 1954. Composition for tenderizing meat. Australian Patent 157,454. Dubuisson, M. 1954. “Muscular Contraction.” Thomas, Springfield, Illinois. El-Gharbawi, M., and Whitaker, J. R. 1958. Unpublished data. University of California, Davis. Embden, G. 1927. The activity substances of striated muscle and the chemistry of muscle contraction. Klin. Wochschr. 6, 628. Engelhardt, V. A., and Lyubimova, M. N. 1939.Myosin and adenosinetriphosphatase. Nature 144, 668. Engelhardt, V. A.,and Lyubimova, M. N. 1942. The mechanochemistry of muscle. Biokhimiya 7,205;Chem. Abstr. 38, 1555 (1944). Falk, G.,and Gerard, R. W. 1954. Effect of micro-injected salts and ATP on the membrane potential and mechanical response of muscle. J . Cell. Comp. Physiol. 43, 393. Feigen, G. A. 1956.Muscle. Ann. Rev. Physiol. 18, 89. Fessler, J. H. 1957. Water and mucopoIysaccharides as structural components of connective tissue. Nature 179, 426. Fruton, J. S. 1941. Proteolytic enzymes as specific agents in the formation and breakdown of protein. Cold Spring Harbor Symposia Quant. Bio2. 9, 21 1. Fruton, J. S. 1946. On the proteolytic enzymes of animal tissues. V. Peptidases of skin, lung and serum. J . Biol. Chem. 166,721. Fruton, J. S., Irving, G. W., Jr., and Bergmann, M. 1941. On the proteolytic enzymes of animal tissues. 111. The proteolytic enzymes of beef spleen, beef kidney and swine kidney. Classification of the cathepsins. J . Biol. Chem. 141, 763. Gelfan, S. 1958.Muscle. Ann. Rev. Physiol. 20, 67.

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tenderizers on the structure and tentlerness of beef. Proc. Ninth fiesearch Conf., Am. Meat Inst. Foundation p. 72. Wang, H., Weir, C. E., Birkner, M. L., and Ginger, B. 1958. Studies on enzymatic tenderization of meat. 111. Histological and panel analyses of enzyme preparations from three distinct sources. Food Research 23, 423. Watanabe, S. 1955. Relaxing effects of EDTA on glycerol-treated muscle fibers. Arch. Biochem. Biophys. 54, 559. Watts, B. M. 1954. Oxidative rancidity and discoloration of meats. Advances in Food Research 5, 1. Weber, H. H. 1957. The biochemistry of muscle. Ann. Rev. Biochem. 26, 667. Weber, H. H., and Portzehl, H. 1954. The transference of the muscle energy in the contraction cycle. Prog. in Biophys. and Biophys. Chem. 4, 60. Weber, H. H., and Stover, R. 1933. The collodial behavior of muscle proteins. IV. The weight of particles of muscle protein and the van der Waal active volume of myogen particles. Biochem. Z. 259, 269. Webster, H. L. 1953a. The deamination and dephosphorylation of adenine mucleotides in muscles. Ph. D. Thesis. Univ. of Cambridge, Cambridge, England. Webster, H. L. 1953b. Direct deamination of adenosine diphosphate by washed myofibrils. Nature 172, 453. Weiner, S., Mangel, M., Maharg, L., and Kelley, G. G. 1958. The effectiveness of commercial papain in meat tenderization. Food Technol. 12, 248. Weir, C. E., Wang, H., Birkner, M. L., Parsons, J., and Ginger, B. 1958. Studies on enzymatic tenderization of meat. 11. Panel and histological analyses of meat treated with liquid tenderizers containing papain. Food Research 23, 41 1. Whitaker, J. R. 1957a. Assay and properties of commercial ficin. Food Research 22, 468. Whitaker, J. R. 1957b. Properties of the proteolytic enzymes of commercial ficin. Food Research 22, 483. Whitaker, J. R. 1959. Properties of the milk-clotting activity of ficin. Food Technol. 13, 86. Wierbicki, E., Kunkle, L. E., Cahill, V. R., and Deatherage, F. E. 1954. The relation of tenderness to protein alterations during post mortem aging. Food Technol. 8, 506. Wierbicki, E., Cahill, V. R., Kunkle, L. E., KIosterman, E. W., and Deatherage, F. E. 1955. Effect of castration on biochemistry and quality of beef. J . Agr. Food Chem. 3, 244. Wierbicki, E., Kunkle, L. E., Cahill, V. R., and Deatherage, F. E. 1956. Post mortem changes i n meat and their possible relation to tenderness together with some comparisons of meat from heifers, bulls, steers, and diethylstilbestrol treated bulls and steers, Food Technol. 10, 80. Wierbicki, E., Cahill, V. R., and Deatherage, F. E. 1957a. Effects of added sodium chloride, potassium chloride, calcium chloride, magnesium chloride and citric acid on meat shrinkage at 70°C. and added sodium chloride on drip losses after freezing and thawing. Food Technol. 11, 74. Wierbicki, E., Kunkle, L. E., and Deatherage, F. E. 1957b. Changes in the waterholding capacity and cationic shifts during the heating and freezing and thawing of meat as revealed by a simple centrifugal method for measuring shrinkage. Food Technol. 11, 69. Wiercinski, F. J. 1952. Relation of pH to the shortening of muscle protoplasm by cations. Federation Proc. 11, 172.

60

JOHN R. WHITAKER

Wilkie, D. R. 1954. Facts and theories about muscle. Progress in Biophys. and Biophys. Chem. 4 , 288. Williams, B. E. 1957. Methods for aging meat. U.S. Patent 2,816,836. Williams, B. E., and Buchanan, B. F. 1957. Enzymatic tenderization of meats. U.S. Patent 2,805,163. Winegarden, M. W., Lowe, B., Kastelic, J., Kline, E. A., Plagge, A. R., and Shearer, P. S. 1952. Physical changes of connective tissues of beef during heating. Food Research 17, 172. Zachariadd, P. A. 1900. Investigation of connective tissue structure; sensitivity of tendon to acids. Compt. rend. SOC. biol. 52, 182, 251, 1127. Zender, R., Lataste-Dorolle, C., Collet, R. A., Rowinski, P., and Mouton, R. F . 1958. Aseptic antolysis of muscle: biochemical and microscopic modifications occurring in rabbit and lamb muscle during aseptic and anaerobic storage. Food Research 23, 305.

THE CHEMISTRY AND TECHNOLOGY OF THE PRESERVATION OF GREEN PEAS

BY L. J. LYNCH,R. S. MITCHELL, AND D. J. CASIMIR Commonwealth Scientific and Industrial Research Organization, Division of Food Preseruation and Trans~iort, Homebush, N e w South Wales, Australia

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Sugars . . . . . . . ......... ........................ E. Nitrogenous Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Phosphorus Compounds. ................................... G. Lipids . . . . . . . . . . . . . . . ..................................... H. Chlorophyll . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... ................... ............................ ....................................................

K. Enzymes . . . . . . . . . . . . . . ..................... ........... L. Nutritive Value ...........................................

111. Maturity ......... ............................................ A. Defiiition . . . . . . . . . . . ........ ....................... B. Measurement . . . . . . . . ....................................... C. Commercial Application of Maturity Measurement, .............. IV. Unit Processes ........................................... A. Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Factory . . . . . . . . . . . . . . . .. ........................... .................................... V. Future Research Requireme References . . . . . . . . . . . . . . . .................. ...........

Page 61 62 69

65 68 70 71 73 74 76 78 91

93 95 95 96 111 122 122 128 141 142

I. INTRODUCTION

Peas are a widespread and important constituent in the diet of most races of people. They are readily accepted and have a high dietetic value since they are a major source of protein and carbohydrate and make a useful contribution to the requirement for accessory food substances. They may be cooked and eaten as green peas shortly after harvest, stored for short periods in the pod, or, after suitable treatment, stored for long periods before consumption. Except for pods from ediblepodded varieties, the seeds are the only part of the plant used for human nutrition. 61

62

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

The earliest method of preservation consisted of allowing the seed to ripen and dry before harvest. Storage in the dry state for indefinitely long periods without deterioration merely depended upon holding in a dry atmosphere and avoiding the depredations of insects and other pests. Dried peas are also soaked and processed in cans to avoid the inconvenience of long soaking and cooking in the home. Modern methods of preservation by canning, freezing, and dehydration involve the use of green peas, which are botanically immature seeds in a succulent condition. Methods used for preservation are based on scientific considerations but have now been reduced to simple routine factory operations by application of technology. The literature on the subject is considerable, and it is not the intention of the authors to include information from all sources, but to present a coherent account of the chemistry and technology involved. Where authors have reached similar conclusions only one or two are quoted. I n the case of disagreement on major matters, the different viewpoints are presented. Emphasis is placed on certain sections such as maturity because of their importance, whereas aspects of processing which are not specific to peas are treated briefly. Since hand operations are expensive in manpower, commercial pea canning was retarded until the introduction of the viner in the late nineteenth century. This innovation imposed the need for extensive mechanical cleaning methods which led to ever-increasing mechanization within modern pea processing factories. Machinery descriptions in this review are limited to detail necessary for an understanding of the technology of the process. Research and the application of the results of research have played a large part in the progress of the industry. This advance will continue as new and modified ways are found to increase eficiency and to improve the quality of preserved peas. II. CHEMISTRY

A. INTRODUCTION A knowledge of the chemical composition of green peas over the accepted range of processing maturities, and of changes in composition during fresh storage, during processing, and during subsequent storage are important in enabling the technologist to understand the influence of harvest maturity and processing variables on the organoleptic acceptability and nutritional status of the processed product. Betmeen the generally accepted limits of satisfactory processing maturity, maturation takes place at a very rapid rate with correspond-

63

PRESERVATION O P GREEN P E A S

ingly rapid changes in chemical composition. Unfortunately, the majority of workers interested in chemical composition of peas have ignored this fact either because of an unwareness of the importance of maturity, or because of a lack of sufficient material and facilities to enable adequate sampling for maturity measurement and concomitant chemical analyses. Chemical composition, physical properties, and organoleptic properties of peas are interrelated. Some of these relationships apply generally, e.g., alcohol-insoluble solids (A.I.S.) and certain physical measures of maturity, whereas others may vary with variety or with growing conditions. A number of workers (Pollard et al., 1947; Kramer et al., 1950) have studied the relationships between amounts of individual constituents and the maturity of the crop, often with the idea of utilizing composition as a criterion of maturity. Chemical methods, although useful for checking the adequacy of a physical measurement, have proved too time-consuming to be of practical importance for crop prediction or for the determination of maturity of raw peas. Peas at the time of harvest and during processing contain a large proportion of water; all composition data in this section are expressed on a wet weight basis unless otherwise specified.

B. WATER Water is the major component of peas, and at processing maturities it ranges from 74 to 81%. Water content of peas has been related to maturity as measured by time (McKee et al., 1955b), by tenderometer reading (T.R.) (Pollard et al., 1947; Ottosson, 1958), and by maturometer reading (M.R.) (Lynch et al., 1955-1957). All these authors found a n approximate linear decrease of per cent moisture with increase in maturity over the range of processing maturities. Lynch et al. (1955-1957) noted that the rate of moisture loss (i.e., maturation) increased after the optimal harvest time (O.H.T.). Ottosson studied changes in dry matter content for a number of varieties grown in successive seasons and in different locations. These results were presented graphically as dry weight-tenderometer relationships and may be expressed as follows: % moisture (wrinkled-seeded) = -0.089 T.R. + 87.25 % moisture (smooth-seeded) = -0.148 T.R. + 92.68.

The regression equation from the results of Pollard et al. (1947) was calculated as: % moisture (wrinkle-seeded) = -0.083

T.R.

+ 88.3

64

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

and from results of Lynch et al. (1955-1957) as: % moisture (wrinkle-seeded) = -0.025 M.R. + 83.6.

The water relationship of a maturing pea crop may be considered in terms of yield of water from the vined peas in pounds per acre. Changes 400

r

t

350 -

300

-

L-

50 -5

I

-4

-3

-2

DAYS

MATURATION O

0

X

A

0

-1

FROM

1

2

0 H.T.

20 LB/DAY

INDEX

SOLUBLE SUGARS STARCH A.I.S. WATER

YIELD I N L E I A C R E

A.I.s.-STARCH

Y I E L D IN LB/ACRE

I,

I,

I#

(x ( x

II

,I

,I

( x

0.35)

It

I,

I,

(X

0.06)

( x

0.55)

1.00) 1.00)

FIG.1. Changes in yield of constituents and maturity with time (Canner No. 75 variety),

with time are shown in Fig. 1 which is based on results obtained by Lynch et al. (1955-1957) from an experimental pea crop. The levelingoff of the rate of increase in water yield after the O.H.T. suggests that this stage has physiological significance. A similar trend is visible in the vines which commence to yellow and dry out about this time, a phenomenon termed “haying-off‘’ within the industry.

PRESERVATION O F GREEN PEAS

65

Nygren (1954) found that a progressive weight loss and an increase in tenderometer reading occurred in vined peas held in field lugs for 6 hr.The storage of fresh and blanched peas in lug boxes for short periods of time (2 hr.) did not show any significant trends in moisture content (Moyer et al., 1952). When raw peas were held in contact with water (Lynch et al., 1955-1957), there was an increase in water content and a decrease in maturometer reading. From data presented by Moyer et al. (1952) and G a m e r and Smith (1947), there appears to be a slight decrease (up to 2%) in moisture content when peas are water-blanched for 3 min., and a small increase when blanching times are longer. These changes are dependent upon time and temperature of the blanch. In the case of steam-blanching, there appears to be a decrease during blanch times of less than 3 min. The moisture content of peas decreases in the quality grader due to uptake of salt and loss of water. Strasburger (1933) suggested that moisture content of canned peas represents a suitable criterion of quality. He found that Alaska peas of moisture content 62% increased to 66% moisture after blanching for 5 min. at 180OF. and to 74% after retorting and subsequent equilibration, Moisture contents of canned peas were found to vary from 72 to 84% for Alaska and from 81 to 83% for sweet varieties. The range in moisture content for sweet varieties is inadequate for the estimation of maturity because of lack of sensitivity in the determination. Loss of moisture occurs during blast-freezing when peas are frozen unpackaged. This slight desiccation does not influence the quality of the product. A drying procedure which reduces the weight to 50% of the blanched weight, followed by freezing, is now in commercial use and is known as “dehydrofreezing.” Moisture loss during storage is dependent on water vapor permeability of the packaging materials and is greater at higher storage temperatures. During dehydration, moisture is removed from the peas to a moisture content less than 7%. The lower the moisture, the slower are the deteriorative chemical changes which take place during storage, The skin is a major barrier to the loss of moisture from the pea, and Moyer et a2. (1956a) increased the rate of water loss by slitting the skins of peas prior to dehydration. The slitting process was also found to increase rate of rehydration.

C. STARCH Starch grains of up to 40 microns in diameter occur in the parenchymatous tissue of pea cotyledons. The hilum is seldom evident, and the grains may be globular, ellipsoidal, or reniform. Irregular protuberances are often observed.

66

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

I . Chemical Composition Common starches are generally recognized as consisting of at least two different compounds, termed “amylose” and “amylopectin.” Amylose gives a strong blue coloration with iodine, while amylopectin gives a weak reddish-purple color. The color of the starch-iodine complex has formed the basis for rapid starch determination in peas and other vegetables by Nielsen (1943) and Nielsen and Gleason (1945). The chemical composition of pea starch as determined by a number of workers i s summarized in Table I, which shows the amylose content TABLE I A M ~ S CONTENT E

OF STARCH I N SMOOTH-SEEDED AND W R I N K L E - S E E D E D VARIETIES

Per cent amylose

0

Smooth-seeded

Wrinkle-seeded

Reference

35 (Alaska) 35-37a 29-3Oa 35 (Alaska)

66 (Perfection) 65-72O 75“ 60-70” 98 (Steadfast)

Potter et al. (1953) McCready et al. (1950) Nielsen and Gleason (1945) Hilbert and MacMasters (1946) Peat et al. (1948) Schneider (1951)

60a

Variety not quoted.

of wrinkle-seeded varieties to be considerably higher than smoothseeded varieties of the Alaska type. McCready et al. (1950) examined the percentage of amylose in starch from peas of various size grades, i.e., from samples of peas of varying maturities, using total starch as a measure of maturity. Their results showed an initial rapid increase in amylose content of total starch to a maximum of 65 to 700/,, when the total starch content of the peas was 4 to 5%. Thereafter, the proportion of amylose in the starch showed no change with further increase in total starch. Potter et al. (1953) found the average molecular weight of amylose and amylopectin from pea starches to be 125,000 and 2,000,000, respectively, for Alaska peas, and 100,000 and 140,000, respectively, for Perfection peas. The amylose from Perfection peas has a molecular weight similar to amyloses from apple, maize, and sago, while that from Alaska peas resembles most cereal and root starches. Schneider (1951) observed that the starch granules of wrinkleseeded peas have an appearance similar to pure crystalline amylose. He suggested that the high amylose content in the starch from wrinkle-

67

PRESERVATION O F GREEN PEAS

seeded peas gave the starch higher water-retaining capacity than the starches from smooth-seeded peas, and in consequence the onset of dehydration was delayed in wrinkle-seeded varieties, allowing the skin of the pea to attain maximum size. Subsequent dehydration caused the cotyledons to shrink in size, and the skin assumed the typical wrinkled appearance. Hilbert and MacMasters (1946) noted that starch from wrinkle-seeded peas has unusual physical properties, in that it did not gelatinize like ordinary starch but formed a suspension and not a paste. They attributed the unusual properties to the high amylose content. 2. Maturity Starch contributes to A.I.S. content of peas and has been shown to increase steadily over the range of processing maturities. Torfason et al. (1956) related starch content to tasters’ texture rating and per cent A.I.S. ; they obtained highly significant correlation coefficients of r = +0.594 and r = +0.614 for the relationships. McKee et al. (1955b) found an increase in per cent starch with time. Nielsen and Gleason (1945) found a regular increase in starch content with increasing tenderometer reading and obtained a correlation coefficient for the relationship of r = +0.98 for the varieties Alaska, Wisconsin, Perfection, Wisconsin Sweet, and Wilt Resistant Perfection. The regression equation determined from these results is: % starch = 0.0444 T.R. -1.66

Pollard et al. (1947), using Nielsen’s method for the rapid determination of starch in vegetables, stated that starch increased by 0.052 to 0.055% for each unit rise in tenderometer reading for the varieties Early Perfection and Perfection, respectively. Correlation coefficients were r = +0.994 for Early Perfection and r = +0.985 for Perfection. The intercept value of regression equations for the relationship per cent starch with tenderometer reading of different size grades varied. This was probably due to the method of starch determination (Nielsen, 1943), which depends on the type of starch present. Ottosson (1958) estimated starch by determination of sugars after hydrolysis of the starch with diastase, and regression lines for the starch tenderometer rela tionships from his average results have been calculated as: % starch (wrinkle-seeded) = 0.04 T.R. -0.6 % starch (smooth-seeded)= 0.079 T.R. -0.45.

Lynch et al. ( 1955-1 95 7) found the relationship % starch (wrinkle-seeded) = 0.0141 M.R.

+1.204.

68

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

Turner and Turner (1957a), working with Canners Perfection, demonstrated that there is a period during the growth of the pea seed when the starch content rises rapidly with a simultaneous fall in sucrose content. Decrease in the rate of starch synthesis coincided with the cessation of water increase, and up to this point, rate of starch synthesis was found to be directly proportional to starch phosphorylase activity. A similar trend between starch content and moisture content was observed by Danielson (1956), working with Profusion peas. The starch content was found to increase in two stages, an initial phase during which the rate of starch formation is small compared with the rate of increase of the moisture content, followed by a stage in which starch increases more rapidly than water. When the yield of starch in pounds per acre is related to crop maturity (Fig. I ) , the increase is approximately linear, showing that accumulation by the crop is regular and increases throughout the range of processing maturities.

3 . Processing Operations No specific studies have been published on the influence of processing variables on the starch content. of peas. Moyer et at. (1952) found no consistent trends in A.I.S. content, which includes starches, over a wide range of blanching temperatures and times. Starch, on the other hand, may be lost from peas with ruptured skins, and the severity of the blanch has been found by Legault et al. (1950) to increase the degree of skin rupture. Lynch et al. (1956-1957) found that the brine of canned peas became less turbid as the blanch time was increased from 0 to 240 seconds in water at 200OF.

D. SUGARS 1. Chemical Composition Sucrose, fructose, glucose, and galactose have been identified, and their concentrations determined over a wide range of maturities by Turner et al. (1957a). The results showed that sucrose accounted for approximately 95 % of the above four sugars. 2. Maturity

A number of workers including McKee et al. (1955b), Danielson (1956), and Turner et al. (1957a:) have studied the influence of maturity on sugar content. All workers agreed that sucrose content increased in the early stages of maturation, but over the range of commercial maturities remained constant and later declined to a low level.

PRESERVATION OF G R E E N PEAS

69

Table I1 shows the results obtained by Turner et al. (1957b) for change in sucrose content during the maturation of a crop of Canners Perfection peas. Danielson stated that the rate of decrease of sugars obeys the conditions for a first-order reaction. Turner et al. (1957a) found that the TABLE I1 CHANGEIN SUCROSECONTENTWITII PEAMATURITY"

Days from flowering

Sucrose ( % fresh wt.)

A.I.S. (%)

12 15 17 19 21 23 25 27 30 33 30 40

3.66 5.91 6.96 7.30 6.79 6.64 6.65 3.85 2.08 1.18 0.80 0.83

6.5 5.6 6.0 6.4 8.4 10.1 12.8 18.7 21.6 26.5 29.4 48.7

sucrose per pea reached a maximum during the period of most rapid starch synthesis, which was also the time of maximum starch phosphorylase activity. The sucrose content at O.H.T. for a number of pea varieties grown under varying conditions was found to lie between 5.5 and 8.8%. Using samples taken from a commercial canning line and hence covering a restricted maturity range, Torfason et al. (1956) found highly significant correlation coefficients of r = -0.597 and r = -0.622 for the relationship between total sugars and maturity, as measured respectively by tenderometer reading and A.I.S.%. The influence of maturity on the yield of sucrose expressed in pounds per acre is shown in Fig. 1.

3 . Processing When raw peas are stored, sugars may be lost through respiration, conversion to starch, or by microbial action. Respiration losses are dependent upon temperature and the degree of damage, and Kertesz (1930) found that peas held for 6 hr. at 77OF. Iost one-third of their sugar content. Soluble sugars are subject to leaching during blanching. Adam et al.

70

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

(1942), using three varieties of peas, found the average losses of sugars to be 15, 20, and 31% when blanched for 1, 3, and 6 min., respectively, in boiling water. Loss during steam-blanching for 3 min. was the same order as for the l-min. water blanch. Distribution of the soluble sugars between the solid and liquid phases takes place in the canning operation, although final equilibration is varied by the common practice of using a brine containing sugar as well as salt.

E. NITROGENOUS COMPOUNDS Nitrogen occurs chiefly in the form of amino acids and proteins which are often referred to as the soluble and insoluble nitrogen fractions, respectively. McKee et al. (1955a) and Nehring and Schwerdtfeger (1957) have examined the amino acid contents of peas and detected the following amino acids: a-alanine, 0-alanine, y-aminobutyric acid, glycine, homoserine, leucine, isoleucine, lysine, methionine, methionine sulfone, methionine sulfoxide, phenylalanine, pipecolinic acid, serine, threonine, valine, arginine, histidine, and tyrosine. Trigonelline and urea, and the amino acid, proline, have also been detected. Danielson (1949) has shown that pea proteins consist of two well defined globulins, viz., vicilin and legumin, and a less well defined albumin fraction with two main components.

1. Maturity The changes in total nitrogen, protein nitrogen, and soluble nitrogen fractions over a wide range of maturities were studied by Rowan and Turner (1957), who found that soluble and protein nitrogen concentrations were initially of the same order, the rate of protein synthesis increasing with a corresponding decrease in soluble nitrogen in the vicinity of the O.H.T. McKee et al. (1955a) studied chromatographically the occurrence of amino acids and other soluble nitrogenous compounds over the full maturity range. Raacke (1957) determined protein content at various stages of maturity and, within the limits of processing maturities, found a slight increase up to the O.H.T. (moisture content of 77.8%) and then a more rapid increase as maturity advanced. The nature of the pea protein was also found to change with maturity; when too immature for commercial use, albumins make up the protein content, but as processing maturity is attained, legumin and vicilin appear and form a progressively greater proportion of the total protein.

PRESERVATION OF GREEN PEAS

71

Spragg (1958) noted similar trends in the proteins of peas during maturation, and a study of the changes in free amino acids indicated that arginine contributed the major proportion of the nitrogen and that glutamine was the outstanding amide present. The arginine fraction of the globulin nitrogen was reported as 20 to 24%. 2. Processing Loss of protein during blanching has been demonstrated by Adam et al. ( 1942) and Kramer and Smith (1947). Adam et al. ( 1942) found average losses of 6, 12, and 14% of the initial protein content when three varieties of peas were blanched for 1, 3, and 6 min. in boiling water. The loss amounted to 3% during steam-blanching for 3 min. Kramer and Smith (1947) found losses of the same order and showed that loss of protein accounted for approximately one-third of the loss of solids during blanching, Moyer et al. (1952) found no change in the two nitrogenous fractions during the storage of raw and blanched peas. They found an insignificant loss of alcohol-insoluble proteins during blanching for fancy and standard grades, but amino acids and other low molecular weight nitrogenous compounds which are soluble in alcohol showed considerable losses from both grades. The losses of nitrogen compounds during blanching were confined to those compounds soluble in 80% ethanol. Blanching and steam-retorting operations both cause denaturation of proteins. There is disagreement in the literature on the influence of canning procedures. Armbruster and Murray (1951) found that the a-amino nitrogen and methionine content of peas was lowered by cannery procedures. Chitre et al. (1950) concluded that the canning operation did not decrease the biological value of nearly mature peas, but enhanced that of immature peas. These findings are contrary to those of other workers.

F. PHOSPHORUS COMPOUNDS Phosphorus occurs in peas in the following compounds: phosphate esters, e.g., the hexose phosphates; phytic acids and corresponding salts; nucleotides, e.g., adenosine diphosphate and triphosphate, guanosine triphosphate, and uridine triphosphate; and inorganic phosphorus compounds. 1. Maturity McKee et al. ( I 955b) and Rowan and Turner (1957) have investigated the phosphorus status of a maturing pea crop and found that the

72

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

amount of phosphorus per pea increased in proportion to increase in seed weight. The percentage of total phosphorus expressed on a fresh weight basis was relatively constant over the range of processing maturities. Turner and Turner (1957b) found that a decrease in concentration of hexose monophosphate occurred at a maturity equivalent to M.R. 180 and continued to a maturity equivalent to M.R. 560 (M.R. calculated from A.I.S. values). This decline in hexose monophosphate is consistent with the increasing rate of utilization of hexose monophosphate in starch synthesis over the maturity range investigated. The latter workers also found that the concentration of reactive phosphate increased over the period of active protein synthesis, and suggested that the concentration of adenosine triphosphate is a factor governing the rate of protein synthesis in pea seeds. Danielson (1956) considered that the syntheses of starch and protein depend on high energy phosphorus bonds, and that the phosphorus in pea seeds is first used for the synthesis of protein and subsequently f o r starch synthesis. Fowler (1957) followed the changes in orthophosphate and phytin content of Sharpe’s 99 Canner and Onward varieties and found that, during the practical canning stage, as determined by tenderometer and maturometer, the orthophosphate content fell and the phytin content rose. Phytic acid is a calcium and magnesium precipitant, and hence determines the amount of calcium and magnesium remaining available for the formation of insoluble pectates which contribute to pea texture. Fowler considered that high concentrations of orthophosphate and phytin (calcium or magnesium salt of phytic acid) in the skin may be responsible for toughness in skin texture. 2. Processing

Horner (1939) reported a decrease in phosphorus content as a result of blanching, whereas Kramer and Smith (1947) did not find significant changes in phosphorus level over a wide range of blanching treatments. Changes of texture during processing have been related by many authors to calcium and magnesium content. Mattson (1946) suggested that phytic acid, being a calcium and magnesium precipitant, prevents the toughening process by inhibiting the formation of insoluble pectates in the cell walls. Mattson et al. (1950) contended that the “cookability” of peas depends upon the phytin content, the pectin of the middle lamella bind-

PRESERVATION O F GREEN PEAS

73

ing the cells together only when it is in the insoluble-calcium saturated condition. If calcium is removed by the formation of insoluble calcium phytates, the pectin becomes saturated with univalent cations, and the peas tend to soften.

G. LIPIDS Of the work carried out on pea lipids, that of Wagenknecht (1957a,b) is the most comprehensive and demonstrates the presence of plasmalogens (a group of glycerophosphatides) in pea lipids. The composition of the phosphatides was found to be 0.39% lecithin (phosphatidyl choline) , 0.73% cephalin (phosphatidyl serine and phosphatidyl ethanolamine), and 2.94% phosphatidyl inositol, all expressed on the basis of total lipid. Fat content determined as ether-solubles by Soxhlet extraction of dried materials has been used by a number of workers. Values of 0.35 k 0.12% for canned sweet peas, and 0.27 f 0.07% for canned Alaska peas were reported by Kramer (1946), and 0.34 t 0.18% for frozen peas by Burger et al. (1956). Kramer and Smith (1947) determined the fat content of peas after various blanching treatments and found, in general, a slight increase in ether-soluble material, followed by a slight decrease when fat content was related to blanch time. The effect of blanching temperature was not marked, but standard grade peas of sieve size 4 contained a greater percentage of fat than those graded as fancy. Lee and Wagenknecht (1951) claimed that rancidity of lipid substances is responsible for the development of off-flavors during the storage of frozen peas as there was an increase in the peroxide number, a higher acid number, and lower iodine number in unblanched Thomas Laxton peas after storage for 5 years at O O F . Wagenknecht et al. (1952) found that raw peas stored for 5 years at O O F . showed a large increase in the acid number of the lipid fraction, probably caused through the action of the enzymes, lipoxidase and lipase. The increase in acid number was suggested as one of the factors influencing chlorophyll-to-pheophytin conversion. It is generally thought that one of the objects of blanching is removal of lipids present in the pea cuticle. This removal is thought to be more complete in the case of water-blanching. Holmquist et al. (1955) suggested the use of a warm sodium hexametaphosphate wash prior to steam-blanching to eliminate lipids which are a possible cause of off -flavor development. The foam-producing ingredient removed by water-blanching is

74

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

probably of a lipid nature and may, in part, be responsible for the offflavors developing during storage of frozen peas which have not been water-blanched.

H. CHLOROPHYLL 1. Chemistry Chlorophyll, the green coloring matter common to the higher plants, is of considerable importance in preserved peas due to its influence on acceptability. Two forms of chlorophyll occur in green plants, and in pea seed the composition of chlorophyll was found by Lynch et al. (1956-1957) to be 62% chlorophyll A and 38% chlorophyll B. During heat-processing, the chlorophylls are for the greater part converted to the corresponding olive-green pheophytms A and B. The chlorophyll content of a pea crop depends on variety, health of the crop, sunlight received, and maturity. The course of breeding and selection of varieties for freezing has been determined in part by the intensity of green color. Lack of chlorophyll, or chlorosis, is a common symptom of deficient nutrition, and the importance of nodule-forming bacteria in the healthy development of leguminous plants has been discussed by Anderson ( 1949). Kramer et al. (1950) stated that holding peas after shelling reduces the green pigment content. They also found that the chlorophyll content of Alaska peas increased with maturity, whereas in Thomas Laxton peas, chlorophyll content reached a maximum at a tenderometer value of 125, and thereafter decreased. Lynch et al. (1956-1957) found that with a Canners 75 crop the chlorophyll content of the peas decreased over the range of practical processing maturities and could be represented by the equation Total chlorophyll mg./100 g. = -0.0217 M.I. +11.62

where M.I. is the maturometer index of the crop. A correlation coefficient of r = -0.991 was obtained. 2. Processing

Legault et a2. (1950) investigated chlorophyll conversion to pheophytin during blanching in steam at 190OF. and 212OF. for various durations. They found no practical differences when the chlorophyll losses at the two temperatures were expressed on the basis of “adequacy of blanch” (as determined by tests for peroxidase inactivation) ,indicating that the rate of chlorophyll loss parallels the rate of peroxidase destruction. The loss of chlorophyll increased with blanch intensity and

PRESERVATION O F GREEN P E A S

75

reached a maximum of approximately 8.5%. From these results, it may be seen that the rate of loss is much more rapid at the higher blanching temperature. During thermal processing of peas, the chlorophylls have their central coordinated magnesium atom replaced by hydrogen ions. Joslyn and MacKinney (1938) showed this degradation to be a first-order reaction with respect to hydrogen ion concentration. With conventional canning procedures, there is practically complete conversion of chlorophylls to pheophytins during the sterilization process. Using hightemperature/short-time processes, particularly in conjunction with some form of agitation to further increase the rate of heating, a commercially sterile pack can be achieved with only moderate destruction of the chlorophylls. However, as the pH of conventionally canned peas is approximately 6.2, the conversion of chlorophylls to pheophytins proceeds rapidly during storage at room temperatures and is practically complete after a few weeks. Storage at lower temperatures serves to retard the rate of color loss. Many attempts have been made to lessen the degree of degradation of chlorophyll during heat-processing and hence to retain the natural green color of peas. All treatments to this end depend upon control of the hydrogen ion concentration by the addition of a buffer to maintain the p H in the vicinity of 8. The most widely publicized commercial process has been the Blair Process described by Blair and Ayres (1943). The procedure described differs from the conventional canning procedure by using: (1) a pretreatment involving the immersion of the peas for 30 to 60 min. in a 2% sodium carbonate solution at room temperature to adjust the p H to a level where the rate of exchange of magnesium from the chlorophylls for hydrogen ions from the solution is slow; (2) a blanch in 0.005 M calcium hydroxide solution; and (3) a salt-sugar canning brine which is 0.020-0.025 M with respect to magnesium hydroxide. These authors stated that approximately 60% of the chorophyll is protected against conversion to pheophytin by this method but that loss on storage is fairly rapid except at low temperature. Blair and Ayres claimed that peas processed by their method had normal canned pea texture, and that the effect of pH adjustment on flavor was considered by tasters to be favorable. Bendix et a2. (1952) described a method using calcium sucrate and alkali to give the range pH 8.0 to 8.5 immediately after canning, while Malecki (1957) used salts of glutamic acid with the addition of calcium and magnesium ions for texture control. All of these processes require careful supervision and are subject to criticism because the addition of alkalis causes certain flavor and texture changes which alter the char-

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

acter of an already accepted product. Further, even when peas are treated by any of these methods, conversion to pheophytins continues, and the amount of chlorophyll retained depends on duration and conditions of storage, leading to a marked variation in color as received by the consumer. In many countries use of coloring dyes is illegal, but in others, peas are dyed green by coal tar dyes which also cause the brine to become colored. The Allen Chlorophyll Co. Ltd. (1957) has patented the addition of soluble sodium chlorophyllins in pH-adjusted brines to impart a natural green color to canned peas. The influence of blanch time and temperature of frozen storage has been studied by Dietrich et al. (1955), who found that a water blanch of 35 to 40 seconds gave minimum conversion of chlorophyll to pheophytin when chlorophyll-pheophytin ratios were determined after storage at various temperatures. The rate of conversion was found to increase with storage temperature, the greatest degree of conversion being observed in the samples blanched for the shorter times and stored at the higher temperature. Dietrich et al. (1957) studying frozen peas found that the extent of conversion of chlorophyll to pheophytin correlated well with the previous temperature history, but that it was complicated by wide variations in loss of chlorophyll during processing. It appears probable that the chlorophyll-pheophytin ratio may be a suitable quality index for frozen peas. A low ratio would indicate poor processing procedures and improper handling or storage temperatures. Time, temperature, and pH conditions for soaking and cooking dehydrated peas, thawing and cooking frozen peas, and cooking canned peas influence the final color of the peas and, hence, their acceptability.

I. ASH The total nonvolatile inorganic constituents of fresh peas determined as ash lies within the range of 0.5 to 1.0% and appears to increase with increase in total solids or maturity. Kramer and Smith (1947) found ash contents of 0.56 to 0.71% for fresh peas of sieve size 4 graded as fancy and standard grades, respectively. Ash contents of 0.67, 0.73, and 0.75% were obtained by Adam et a2. (1942) for fresh Surprise, Lincoln, and Charles I peas with solids contents of 20.5,20.7, and 22.6%, respectively. Kramer (1945) found the average inorganic content of the solid and liquid fractions of canned peas to be very similar and approximately 1%. This is not unexpected as a large proportion of the total inorganic constituents consists of added salt. Kramer also found that the liquid

77

PRESERVATION O F GREEN PEAS

fraction of canned peas contained on the average 38.1% of the total ash of the can contents. The average content of individual inorganic constituents of fresh, frozen, and canned peas as well as the distribution of minerals between the solid and liquid fractions of canned peas is set out in Table 111. The TABLE I11 AVERAGE CONTENT OF MINERAL CONSTITUENTS OF PEAS

Component

Ash (%) Calcium (mg./lOO g.) Phosphorus (mg./100 6.) Iron (mg./100 9.) Copper (mg./100 9.) Magnesium (mg./100 9.) Potassium (mg./100 g.) Sodium (mg./100 6.) Sulfur (mg./100 6.) 0

b

6

Fresh"

Frozenb

0.72" 15.1 104.0 1.88 0.23 30.2 342 0.5 50.0

0.75 20.0 90.0 2.0 24.0 150 129

Canned. (drained solids)

Per cent of total in liquid fraction of cane

1.28 25.7 169 1.87 0.21 24.4 20 1 (260) 43.9

38.1 17.5 25.8 27.7

McCanee and Widdowaon (1946). Burger el al. (1956). KraIner (1945). Adam el al. (1942). Anonymous (1947).

high sodium content of canned peas is due to the presence of sodium chloride in the canning brine. Kramer (1945) found that commercial cans of peas contained approximately 64% drained solids and 36% brine, and that the moisture in solid and liquid fractions was 77% and 92%, respectively, from which it may be calculated that the distribution of water within the can is about 60% in the drained solids fraction and 40% in the brine fraction. If the inorganic constituents are freely soluble, their distribution within the can should be in these proportions. The results of Kramer give a higher content in the solids fraction, which suggests that a proportion of the inorganic constituents are held within the tissue as organic complexes of low solubility. The complexing of inorganic nutrients limits to a certain extent their availability to satisfy nutritional requirements. Special processes such as the Blair process, in which calcium and magnesium salts are added, increase the content of these minerals in canned peas. The use of hard water for washing, blanching, and canning operations has frequently been reported to influence the calcium

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

and magnesium content of peas. Horner (1939) showed that the calcium content of peas increased during blanching, canning, and home cooking and that magnesium, potassium, and phosphorus contents decreased. Calcium affects the texture of peas, and Kramer and Smith (1947) found that raw standard peas of sieve size 4 contained 33 mg./100 g. of calcium, whereas fancy peas of the same size grade contained only 19 mg. / 100 g. Calcium pectates are constituents of cell walls and contribute to their mechanical rigidity, thus influencing texture. Davis (1957) showed tin uptake during storage at 86OF. of peas canned in plain 301 X 411 cans fabricated from hot dipped plate to be lower than tin uptake in five other canned products (peaches, tomato pulp, grapefruit juice, plum jam, and beans). The tin content of canned peas after 72-weeks storage at 86OF. was 25 p.p.m., expressed on the total can contents. Iron contents on the same cans of peas were approximately 12 p.p.m. initially, and 16 p.p.m. after 72-weeks storage at 86OF. Plain cans are thus satisfactory for canning of peas, but some processors prefer to use enameled cans or enameled ends on plain bodies to reduce sulfur staining, which imparts an objectionable appearance to the container.

J. VITAMINS The vitamin value of any processed food is dependent upon the vitamin content of the raw material, severity of processing, storage conditions, and losses during consumer preparation. Vitamins are lost by leaching, oxidation (both enzymic and nonenzymic) , and thermal decomposition. The initial vitamin content, changes with maturity, and the influence of conditions during processing and prior to consumption have been considered by a number of authors. 1 . Carotene (Provitamin A )

Three isomeric carotenoids, a-,p-, y-carotene, and cryptoxanthin possess vitamin A activity. An International Unit of provitamin A has been defined as the biological equivalent of 0.6 pg. of p-carotene. m-Carotene, y-carotene, and cryptoxanthin each have half the vitamin A potency of p-carotene. Peas may be regarded as a good source of vitamin A as they contain 0.4 to 0.6 mg./100 g. of p-carotene, corresponding to a vitamin A content of 670 to 1000 I.U./lOO g. Vitamin A and carotene are insoluble in water and stable at high temperatures in the absence of oxygen, hence losses during processing are not severe. The carotene content of peas varies with variety, values of 0.43, 0.45, and 0.36 mg./lOO g. having been determined for Pride, Shasta,

PRESERVATION O F G R E E N PEAS

79

and Thomas Laxton peas, respectively, by Kelley et al. (1950). Zscheile et al. (1943) found the total carotene content of Thomas Laxton peas to be 0.4 to 0.5 mg./100 g. of which 7 0 4 0 % was p-carotene. Caldwell et al. (1946) examined the carotene content of one or more sieve sizes of a number of varieties. In Pedigree Extra Early and Alaska, there were significant increases in the larger sizes, whereas with the variety Early Harvest an increase in the intermediate size grades was followed by a decrease to the largest size examined. The varieties Laxton’s Progress, Teton, Thomas Laxton, and World Record showed significant decreases in carotene with advancing maturity, while other varieties showed little or no change. Lee and Whitcombe (1945) found that carotene in Alderman peas remained practically constant when expressed on a fresh weight basis, but showed a considerable decrease in the oldest samples when expressed on a dry weight basis. Pepkowitz et al. [ 1944) found only slight changes in carotene content with maturity, whereas Stimson et at. (1939) suggested that the carotene content of more mature peas is less than in younger peas. Kelley et al. (1950) held peas under various conditions prior to canning and found a variation in carotene losses between varieties, the retention being considerably less with the variety Thomas Laxton. Retention of carotene in stored samples of Pride peas was in general greater than retentions in Shasta and Thomas Laxton varieties. Loss of carotene from the variety Pride was 27% when washed and held for 5 hr. in the shade, 18% when washed and held in cold storage for 48 hr., and 15% when held for 26 hr. in snow ice. The variety Shasta lost 20% when samples were held in the shade for 4 and 8 hr. after washing, and 18 to 19% when held in cold storage for 1 day, or in running water for 8 hr., or washed and held in cold storage for 8 hr. Retentions in Thomas Laxton samples were consistently less, losses of 43% occurring after 47 hr. cold storage, and 42% after washing and 23 hr. cold storage. Investigations by Stimson et al. (1939), Zscheile et at. (1943), and Caldwell et al. (1946) indicated that there was little or no loss of carotene from peas as a result of blanching. Bedford and Hard (I 950) found a loss of less than 10% when carotene contents were determined immediately after blanching. Kelley et al. (1950) found that the carotene content of canned peas expressed on a drained weight basis compared favorably with the value determined prior to canning. Carotene contents varied from 0.44 to 0.54, 0.40 to 0.51, and 0.47 to 0.56 mg./100 g. expressed on drained weight basis for Pride, Thomas Laxton, and Shasta varieties, respectively. Re-

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

tentions of carotene in canned peas generally exceeded 100% when expressed on a dry weight or moist weight basis. Numerous workers have reported low losses of vitamin A during storage of canned peas. Guerrant et al. (1948) found a minimum of 82% retention after 2-years storage at 80°F. Stimson et aZ. (1939) found carotene losses during frozen storage for 11 months to be negligible at -4OOF. and to range from 7 to 26% when storage temperature was OOF. Losses of vitamin A during freezing and subsequent frozen storage have been found to be small, but Zscheile et al. (1943) have found losses during frozen storage of unblanched peas. Fitzgerald and Fellers (1938) recorded insignificant losses during freezing. Losses of carotene during dehydration were, in general, found to be small by Caldwell et aZ. (1946), and in some instances there was an apparent increase in carotene content. Mahoney et al. (1946) examined samples of frozen, canned, and dehydrated peas stored for 6 months prior to the determination of carotenes. The carotene contents of the frozen, canned, and dehydrated samples were 1.98, 1.80, and 1.68 mg./100 g., respectively, on a moisture-free basis. I n general, the loss of provitamin A or carotene during home preparation is slight and of the same order as losses occurring during commercial processing. 2. B Group Vitamins a. Vitamin B, (Thiamine). Thiamine is readily soluble in water and is unstable to heat, especially in alkaline media. Consequently, loss of this vitamin occurs during commercial processing operations. Evidence obtained by Schopfer and Miiller (1938) indicates that in pure thiamine solutions destruction by heat is primarily a cleavage to pyrimidine and thiazole derivatives. Farrer (1955) reviewed the thermal destruction of vitamin B, in foods and discussed the influence of commercial processing and domestic cooking procedures on thiamine retention in vegetables. Moyer and Tressler (1943) found no loss of thiamine in peas held for 16 hr. on the vines in the viner shed and for a further 3 hr. between vining and washing. Ingalls et al. (1950) gave thiamine contents of 0 32, 0.49, and 0.45 mg./lOO g. for the three varieties Thomas Laxton, Pride, and Shasta, respectively, after prompt handling. Samples of peas left on the vines in the field after mowing showed no loss of thiamine; in fact, an apparent increase occurred during the holding of unvined Shasta peas. The lowest thiamine retentions reported were in peas held

PRESERVATION O F GREEN PEAS

81

8 to 10 hr. in running water and in samples washed before the holding period. Clifcorn and Heberlein (1943) found that Alaska and sweet peas graded as fancy had a lower thiamine content than those graded as standard. This result suggests a probable increase in thiamine content with increase in maturity. During a study of the influence of blanching on the nutritive value of peas, Heberlein et d. (1950) concluded that losses of thiamine were less than lo%, and that slightly better retentions were obtained with high-temperature/short-time blanches. Wagner et al. (1 947b) gave tables showing the effect of size grade and maturity on vitamin retention, but, as sieve size and maturity are not independent, these tables should be interpreted with caution. The effect of maturity on vitamin loss during blanching may be ascertained if a particular size grade is divided into two or more maturity fractions by gravity separation and the vitamin contents of the fractions determined, This procedure assumes that maturity does not influence the loss of vitamins during gravity separation. Loss of thiamine, which dissolves in water and is not destroyed during blanching, offers opportunities for accurate study of loss of solubles during commercial blanching treatments. Table IV summarizes the results of a number of workers and suggests that thiamine losses during blanching and associated cooling and transporting procedures are due mainly to leaching, as shown by greater retention in steam blanched material and larger size grades which have a lower surface to volume ratio. Strong and Elvehjem (1947) found that 82 to 93% and 79 to 96% of thiamine was retained after water-blanching Alaska and sweet peas, respectively. Retentions after canning were 44 to 64% and 52 to 70% for these varieties. Greenwood et d.(1944), studying vitamin retentions in meat products, found that for each 18OF. rise in temperature the rate of thiamine destruction is approximately doubled, whereas the rate of bacterial destruction increases approximately 1O-fold. This suggests that the use of high-temperature/short-time processes, especially those which involve agitation, should result in greater retentions of heat-labile nutrients such as thiamine. Of four vegetable packs studied, Bendix et al. (1951) showed that only peas (vacuum, brine, and puree packs) gave a completely linear relationship (in accordance with a first-order reaction) when the logarithm of per cent thiamine retention was plotted against time for processing temperatures of 220, 245, 260, and 270OF. The linearity of the relationship between the logarithm of the reaction rate constant and the reciprocal of the absolute temperatures was interpreted as evidence

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

TABLEIV INF-LUENCE OF CONDITIONS OF BLANCH O N THIAMINE RETENTION IN PEAS Type of blanch Water

Commercial blanching

Water

Water

Water Water

Steam

Conditions of blanch Alaska 1 min. 212°F. Ungraded sweet peas Alaska peas Size grade 1 Size grades 4 and 5 Alaska 3 min. 190-200°F. 535-6 min. 190-200°F. 436-5 min. 190-195°F. Sweet peas 6 min. 200°F. 8 min. 175°F. 6 min. 190-200°F. 6-7 min. 190-200°F. From 235 min. 170-180°F. t o 9 min. 200-205°F. Fancy grade peas 3 min. 180°F. 180°F. 6 min. 9 min. 180°F. 3 min. 200°F. 200°F. 6 min. 200°F. 9 min. 1 min. 210°F. 210°F. 2 min. 3 min. 210°F.

Thiamine retention (%)

75 76 76 05-100 88-92 82-88 88-93

Reference

Lunde et al. (1040) Clifcorn and Heberlein (1943)

Wagner el al. (1947a,b)

96 88 87-88 79-82

66-103 97 95 90 84 84 66 102 105 97

Guerrant et al. (1947)

that thiamine loss varies in rate, but not in its nature, over the range of commercial processing temperatures (220 to 270OF.). These authors suggested the use of peas in preference to other vegetables for the evaluation of new thermal processes. Lunde et al. (1940) found that in canned sweet peas the distribution of thiamine was 73% and 27% in solid and liquid fractions, respectively. Wagner et al. (1947a) studied vitamin retentions in samples of peas from a number of canneries; they determined the percentage of the original thiamine content after canning as being within the range of 52 to 70% for sweet peas and 46 to 64% for Alaska peas. The influence of storage time at temperatures of 50, 65, and 80°F.

PRESERVATION O F G R E E N PEAS

83

on thiamine retention of commercially canned peas was shown by Guerrant et al. (1948) to be important. Minimum retentions after 2-years storage were quoted as 62% for Alaska peas and 72% for sweet peas. When recalculated to a n 83%-moisture basis, the results of Mahoney et al. (1946), who used dark-podded Thomas Laxton peas with an initial thiamine content of 0.317 mg./100 g., gave thiamine contents after processing and 6-months storage of 0.184, 0.168, and 0.149 mg./100 g. for frozen, canned (plus liquor), and carbon dioxide-packed dehydrated peas, respectively. After dehydration and 6-months storage, Mahoney et al. (1946) found thiamine contents of 0.874, 0.764, and 0.697 mg./100 g. on a dry weight basis for carbon dioxide, air, and vacuum packs, respectively. Trefethen and Fenton (1951) examined the influence of domestic pressure cooking procedures on thiamine retention of frozen peas and found no significant differences between the procedures used. Retentions varied from 93 & 2.2 to 98 & 2.0%. b. Riboflavin (vitamin B,) . Riboflavin, like other members of the vitamin B group, is soluble in water and insoluble in fats. However, riboflavin is much more stable a t high temperature than thiamine, but it is destroyed by exposure to light, especially in alkaline medium. In the literature, there is no evidence indicating losses due to enzymic or nonenzymic oxidation during commercial processing operations. The riboflavin content of the germ was found by Murray (1948) to be approximately four times as great as that of the endosperm in Alaska variety field peas. Riboflavin retention during blanching is good, and is stated by Cameron et d. (1949) to range from 67 to 87%, with an average retention of 75%. Wagner et al. (1947a) found retentions of 73 to 89% and 63 to 84% for Alaska and sweet peas, respectively, during blanching in commercial hot-water blanchers, while Guerrant et al. (1947) found losses of 10 to 50% during water-blanching but no significant losses during steam-blanching. Losses from fancy grade peas during waterblanching were generally higher than those from standard grade peas of the same sieve size. The riboflavin contents of peas listed by Wagner et al. (194713) when recalculated on an actual sample-weight basis were found to be 0.139, 0.127, 0.109, and 0.115 mg./100 g. for fresh, blanched, floater, and sinker samples, respectively. The lower values for gravity-graded peas suggests that riboflavin was leached out by the brine and supplementary washings associated with gravity grading. Cameron et al. (1949) stated that the over-all retention of riboflavin in canned peas varied from 70 to 100% and averaged 82%. Wagner

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

et al. (1947a) observed that during commercial processing and retorting procedures Alaska peas retained 71 to 103% and sweet peas 71 to 84% of their original riboflavin content. The distribution of riboflavin between solid and liquid is not in the same ratio as the other watersoluble vitamins, and results of Brush et al. (1944) show the solid-toliquid distribution as 70:30 when the solid-to-liquid ratio in the can was 66:34. The riboflavin content of canned peas was found by Guerrant et al. (1948) to decrease with increases in both time and temperature of storage. They found minimum retentions of 60% and 77% and maximum retentions of 92% and 93%, respectively for Alaska and sweet peas after 2-years storage at temperatures of 50, 65, and SOOF. Van Duyne et al. (1950) showed that samples of two varieties of peas with initial riboflavin contents of 0.19 and 0.17 mg./100 g. retained 82% and 86% of the riboflavin after blanching and cooling, and 78% and 90% respectively after 20 hr. in the freezing unit. This result indicated an average loss of approximately 16% during blanching and cooling, with no additional loss during freezing. Guerrant and O’Hara (1953) reported a loss of approximately 22% during the preparation of peas for freezing and little further loss during freezing and subsequent storage at -1O0F. Oser et al. (1943) observed that the loss of riboflavin from peas during cooking was related to the quantity of water used. Van Duyne et a2. (1948) found that, when peas were boiled for 20 min. with an equal weight of water, 75% of the original content was retained in the peas and 23% was leached into the cooking water. When the weight of cooking water was twice that of the peas, the distribution of riboflavin between peas and cooking water was 61:40. Increasing the cooking time by 100% did not significantly influence the loss of riboflavin. The riboflavin content of frozen peas was not significantly influenced by pressure cooking. Retentions of 86 2 5.1% to 92 0.6% were obtained by Trefethen and Fenton ( 1951) . c. Niacin. Niacin is an important member of the B complex and occurs to a large extent in foods as free and combined nicotinamide. Niacin is water-soluble and stable to light and heat in mildly acid or mildly alkaline media. Recalculation of the results of Wagner et al. (1947b) on niacin contents of raw peas gave values of 2.2, 2.4, and 2.8 mg./100 g. when the solids contents were 20.4, 22.3, and 24.4%, respectively. These figures suggest an increase in niacin content with maturity over the range of processing maturities. Heberlein et d.(1950) found niacin retentions of 55 to 98%, with

*

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

85

an average retention of 75% during blanching of peas. Cameron et al. (1949) reported retentions of 59 to 96% and an average retention of 73%. Wagner et al. (1947b) showed niacin losses to be less than 37% for various conditions of water-blanching. From the results of Wagner et al. (1947b) on blanching and subsequent specific gravity grading, it can be calculated that peas with an initial niacin content of 2.43 mg./100 g. contained 1.92 mg./100 g. after blanching, while niacin contents of floaters and sinkers from the specific gravity grader were 1.66 and 2.37 mg./100 g., respectively. This is further evidence for increase in niacin content with increase in maturity. Heberlein et al. (1950) obtained over-all retentions after processing of 48 to 95%, with an average of 69% for canned peas; similar values of 50 to 80% with a n average of 65% were obtained by Cameron et al. ( 1949). Guerrant et al. (1948) found minimum niacin retentions of 79% and 91% for canned Alaska and sweet peas, respectively, during storage for 2 years a t 50, 65, and 80°F. There was no clear-cut relationship between niacin loss and temperature of storage. Guerrant and O’Hara (1953), using peas with an initial niacin content of 1.91 mg./100 g., showed that the niacin content fell to 1.72 mg./ 100 g. after blanching and to 1.45 mg./100 g. after freezing. No further loss occurred during 12-months storage at -1O0F. d . Other B-group vitamins. Cheldelin and Williams (1942) list the following average values for B vitamins in fresh English green peas: pantothenic acid, 0.38 mg./100 g.; pyridoxine, 0.079 mg./100 g.; biotin, 0.0094 mg./100 g.; inositol, 162 mg./lOO g., and folic acid, 0.13 mg./ 100 g. The folic acid content of raw peas was found by Toepfer et al. (1951) to decrease from 0.097 to 0.088 mg./100 g. during 1 day’s storage at room temperature. These authors also report folic acid contents of 0.001 to 0.012 mg./100 g. expressed on the total can contents. Ives et al. (1946) found that the proportion of folic acid in the drained solids fraction of canned peas was 83% (17% in the brine) when determined as S . lactis factor and 66% when determined as L. casei factor. They also showed that 64% and 88% of the pyridoxine and biotin, respectively, were present in the drained solids of canned peas. This indicates that pyridoxine, but not biotin, is distributed within solid and liquid fractions as the other water-soluble vitamins. The concentrations of pyridoxine and biotin are given as 0.046 and 0.0021 mg./100 g. when expressed on the total can contents. Results of biological determinations of folic acid in canned peas varied from 0.0017 to 0.0044 mg./100 g., depending on the test organism used. Thompson et al. (1944) gave an average value of 0.15 mg./100 g. for calcium pantothenate content of canned peas. The survey of nutri-

86

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

ents in frozen foods by Burger et al. (1956) gives average folic acid, pantothenic acid, and pyridoxine contents of frozen sweet peas as 0.020, 0.277, and 0.150 mg./100 g., respectively.

3. Vitamin C (Ascorbic Acid) Vitamin C is stable to heat in the absence of oxygen, but is readily oxidizable and water-soluble; consequently, it is usually lost in greater proportion than any of the other vitamins. Percentage retention has been used to evaluate handling, processing, and storage history of pea samples. Dietrich et al. (1957) followed changes during frozen storage, and found ascorbic acid content to be a reliable index of previous storage history if the original content of the samples were known. Inclusion of the oxidation products, dehydroascorbic acid and diketogulonic acid, in the analysis gave an even more reliable index than ascorbic acid alone. Peas may be considered a good source of vitamin C, fresh peas of most varieties containing from 19 to 31 mg./lOO g., but contents exceeding 60 mg./100 g. have been found with Alaska and Profusion varieties by Alexander and Feaster (1947). Mack and Tressler (1936) compared 17 varieties and found the ascorbic acid content to be within the range 19 to 40 mg./100 g. on field run material. The small-seeded varieties were, in general, higher in ascorbic acid than large-seeded ones, and early maturing varieties tended to have higher ascorbic acid contents than later varieties. Vitamin C content depends upon variety and growing conditions, but trends with maturity within a crop have been noted. Caldwell et al. (1946) determined vitamin C content of various sieve sizes of 5 smooth and 20 wrinkled varieties, and found that, in general, vitamin C content was highest in the youngest samples and decreased with increase in sieve size. McKee et al. (195513) found the content per seed rose steeply from 0.3 mg. per seed 10 days after flowering to 1.7 mg. at 26 days, then remained constant for about 7 days, and at 40 days had decreased to about 1.0 mg. per seed as shown in Fig. 2. During the edible period, ascorbic acid increased at a slower rate than the fresh weight of the peas. Consequently, the percentage of ascorbic acid decreased over the maturation period. Pollard et al. (1947) similarly observed that the ascorbic acid content of two varieties decreased slowly with increasing maturity, and Torfason et al. (1956) found highly significant correlations of: r = -0.662, -0.625, -0.762, and -0.602, between ascorbic acid content and tenderometer, texturemeter, penetrometer, and pressure tester readings, respectively. A significant relationship with r = -0.528 was also observed between ascorbic acid content and tasters’

a7

PRESERVATION O F GREEN P E A S

texture rating. Kramer et al. (1950) found the ascorbic acid content of peas to vary with variety, but to be related to flavor. He found a decrease in ascorbic acid content of Alaska peas with increase in maturity from 100 to 200 tenderometer reading, whereas the ascorbic acid content of Thomas Laxton peas increased until a tenderometer reading of 150 was reached and then declined as maturity increased. Todhunter and Sparling (1938) observed that the seed coats contained approximately twice as much vitamin C per unit weight as the cotyledons, and concluded that the higher percentage of ascorbic acid in the smaller size grades was due to the higher skin/cotyledon ratio. 1.8

w W

1.2

< 0

5 3 30-

0.9

4

u -

:2 0 -

-

0

0.6

0 0

m

n

0

m 4

0 U Q

U VI

10 -

0.3 4

0 10

20 DAYS

30 FROY

40

RLOSSOM

FIG.2. Change in ascorbic acid content of peas with time (from McKee et al., 1955b).

The vitamin C content of peas was reduced by 25% when cut late in the afternoon and allowed to remain in the field overnight before vining (Anonymous, 1952). Flynn et al. (1949) found that 50% of the vitamin C was retained when peas were stored for 4 days a t ambient temperatures, but the retention was 90% when stored at 32OF. Enzymatic oxidation of vitamin C commences at time of harvest, and the subsequent vining operation damages tissue and liberates enzymes responsible for the oxidation of vitamin C, making it desirable to expedite transport and handling. Todhunter and Robbins ( 1941), using both hand-shelled and washed vined peas, observed that peas soaked for 30 min. in water at 77OF. lost to the water 28.4% of their vitamin C if previously blanched for 60 sec. at 212OF. but that no loss occurred with unblanched peas. This suggests that transport of peas in water prior to blanching results

88

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

in negligible losses of soluble nutrients but contact with large volumes of water after blanching is not desirable from this point of view. Loss of vitamin C during blanching has been extensively studied and the results are summarized in Table V. I n general, retention of TABLE V INFLUENCE OF CONDITIONS OF BLANCH ON VITAMIN C RETENTIONI N P u s ~

Type of blanch Water

Water Water Steam Radar (3000 megacycles) Boiling water Steam Dielectric (200 megacycles) Water Water

Water Steam

~

~~

~

Conditions of blanch 3 min. 3 min. 9 min. 9 min. 1 min. 6 min. 3 min.

172°F. 200-208°F. 172°F. 200-205°F.

25 sec. 1.0 min. 2.0 min. 3).i'-min. blanch water-cooled 3 min. 200°F. Peas of T.R. 105 1 min. 185°F. 1 min. 305°F. 3 min. 205°F. 4 m i n . 185°F. 6 min. 205°F. 8 min. 185°F. 60 sec. 199.4"F. 153 sec. 199.4"F. 1 min. 212°F. 2 min. 212°F. 3 min. 212°F.

~

~~~~~~~

Vitamin C retention

(%I

Reference

61-64 53-71 49-57 48-59 71 60 84

Wagner et ul. (19474,)

100 92-98 100 83.7

Proctor and Goldblith (1948)

68

90 100 83 98 75 80 84 64 75 67 67

Adam et al. (1912)

Hard and Ross (1956) Guerrant and O'Hara (1953) Heberlein el al. (1950)

Jenkins el al. (1938)

vitamin C decreases progressively with increase in time and temperature of the blanch. Loss varies with variety and stage of maturity, but within a given variety the loss of ascorbic acid is greatest with the smallest sieve size. Loss depends upon the leaching and oxidizing properties of the heating and cooling media used for blanching and subsequent cooling. Higher retentions are recorded with steam- and dielectric-blanching as loss due to leaching is low, but loss due to oxidation from oxygen

PRESERVATION O F GREEN PEAS

89

present may be higher than with water-blanching. The leaching properties of the cooling water and the oxidizing properties of the cooling air have also to be considered when studying the over-all losses. Guerrant et d.(1947) found that the practice of blanching successive batches of peas in the same blanch water gave no increase in retention of vitamin C. Thus, continued use of the same blanch water is not of advantage from this point of view. Wagner et al. (1947b) found that vitamin C content, expressed on a dry weight basis, of the less mature fraction of peas from a gravity grader, was higher than that of the sinkers. When their results were recalculated on a wet weight basis, the vitamin C content of floaters and sinkers were of the same order. Mahoney et al. (1946) found the vitamin C content of floaters to be less than that of sinkers when expressed as a percentage of the wet weight. These results appear to conflict with those of Todhunter and Sparling (1938) and McKee et al. (1955b) and may possibly be explained by greater vitamin C loss from small, less mature peas during blanching and gravity grading. Percentage retention of vitamin C in canned peas and the percentage distribution between the solid and liquid fractions within the can have been studied by numerous workers. Cameron et al. (1949) found retentions of 45 to 90%, and Heberlein et al. (1950) found retentions of 49 to 81%. The distribution of solid-to-liquid within the can was shown by Brush et al. (1944) to be in the ratio 66:34; the vitamin C distribution was 63 :37, which is of the same order. Vitamin C concentrations were 9.3 and 7.43 mg./lOO g. in the drained solids and 10.6 and 10.0 mg. / 100 g. in the brine of consumer and No. 10 cans, respectively. Adam (1941) found vitamin C retention to decrease with increase in headspace and to vary from 37 to 70% with change in headspace for the varieties Surprise, Lincoln, and Charles I. Numerous workers, including Adam (1941, 1942), have shown that commercial canning operations retain a greater percentage of vitamin C than normal domestic cooking procedures. Ascorbic acid is stable to heat at the normal pH of canned peas, and when the necessary precautions are taken to eliminate oxygen from the headspace, losses are not marked during storage. Guerrant et al. (1948) found that not more than 26% of the ascorbic acid was lost when canned peas were stored for 2 years at 80°F. A useful nomograph showing the influence of time and temperature of storage on vitamin C retention in canned peas has been given by Freed el al. (1949), who found retentions of 93% and 68% after 12-months storage at 35 and IOOOF., respectively. Rrenner et al. (1948) noted losses of 20, 25, and 34% after stwage for 18 months at 70°, 90°, and 10O0F. They found

90

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

the logarithm of the retention of ascorbic acid during canned storage had a linear relation to time, which indicates a fiist-order reaction. Fenton et at. (1936) observed that freezing in itself was not responsible for any loss of vitamin C. Jenkins et al. (1938) found an overall loss of 30% during freezing preparation, 10% loss occurring during blanching and other losses occurring during cooling and washing subsequent to blanching. Fellers and Stepat (1936) and Todhunter and Robbins (1941) reported losses of 20% and 30%, respectively, during freezing operations. Van D u p e et al. (1950) observed losses of 15% after blanching and 22% after cooling. There was no loss during freezing, and losses of 21, 30, 41, and 47% after storage at O O F . for 1, 2, 6, and 9 months. Jenkins et al. (1938) found that frozen peas showed no loss when stored at -40OF. for 9% months and a slight loss when stored at -18OF. Storage at +lO°F. and +16OF. resulted in relatively rapid loss of vitamin C. Todhunter and Robbins (1941) could not determine any vitamin C loss during frozen storage in sealed cans, which reduced the loss through oxidation. Some workers disagree concerning the loss of vitamin C during the thawing of frozen peas. Fenton and Tressler (1938a) and Jenkins et al. (1938) did not find any loss of vitamin C during thawing, whereas Todhunter and Sparling (1938) claimed that peas blanched for 2 min. at 190°F., and frozen, lost 16% after 30 min., 27% after 1 hr., and 25% after 24 hr. at refrigerator temperature. Todhunter and Robbins (1941), McIntosh et al. (1940), and Fenton and Tressler (1938b) report retentions of 42 to 70% during dornestic cooking procedures. Loss of vitamin C was found by Caldwell et at. (1946) to vary with variety, with sieve size, and with length of blanch. Susceptible varieties showed losses in the range 65 to 75%, but the average loss from all varieties was approximately 50% when expressed on a fresh weight basis. In the drying process, the loss of vitamin C for different sizes was in the reverse order to loss during blanching, the smaller sieve sizes losing less during drying than the larger sizes. The final ascorbic acid content of the dried peas was generally found to be greatest in the smaller sieve sizes. Dehydrated peas retained 40 to 50% of their original ascorbic acid content, but 66 to 75% of this may be lost during domestic rehydration and cooking. Using dark-podded Thomas Laxton peas with an ascorbic acid content of 23.5 mg./100 g. when fresh, Mahoney et al. (1946) found that after storage for 6 months the vitamin C content of cooked peas, together with the cooking liquor, was 11.9, 10.7, and 7.6 mg./1’00 g. for canned, frozen, and dehydrated material, respectively.

PRESERVATION O F GREEN PEAS

91

4 . Oiher Vitamins

The tocopherols are stable to heat in the absence of oxygen. a-Tocopherol has the highest vitamin E activity. Harris et al. (1950) state that peas contain insignificant quantities of a-tocopherol, amounting to approximately 5% of a total tocopherol content of 2.1 mg./100 g. found in fresh green peas. A number of substituted naphthoquinones have been shown to possess vitamin K activity. These derivatives are fat-soluble and stable to heat, and Bicknell and Prescott (1942) have estimated that green peas contain 0.3 mg./100 g. of vitamin K (assuming 1000 Dam units = 0.083 mg. vitamin K). Choline is soluble in water, and Engel (1943) showed the choline concentration in peas to be 260 mg./100 g. Bednarczyk (1950) found peas to have a high choline content when compared with other foods and showed that boiling had no apparent effect on the choline content. He found the choline content of Polish peas to be 168 mg./100 g.

K. ENZYMES A large amount of information is available on the subject of enzymes in peas, probably because peas are a convenient source of material for biochemists in the study of enzymes and enzyme systems. Several of these enzymes are of technological importance since they cause deteriorative changes during processing and subsequent storage, due either to their influence on the rate of destruction of nutrients or their role in the development of objectionable off-flavors and odors. Enzymes reported to cause deterioration either in fresh or processed peas include catalase, peroxidase, ascorbic acid oxidase, chlorophyllase, lipase, lipoxidase, a-hydroxy-acid dehydrogenase, and pyruvic decarboxylase. A primary objective of blanching is the destruction of enzymes which cause deteriorative changes during holding periods prior to processing, during storage of dehydrated peas, and even at the low temperatures used commercially for the storage of frozen peas. Current trends in processing (high-temperature/short-time sterilization, aseptic canning, irradiation sterilization) have introduced problems due to enzyme survival or enzyme regeneration. The need for blanching of vegetables prior to freezing was established by Joslyn and Cruess (1929). Kertesz (1933) considered that enzymes were sufficiently inactivated to permit adequate storage without marked loss of quality when 90% of the respiratory activity had been destroyed. He found that blanching times of 20 sec. a t 212'F.,

92

L. J. L Y N C H , R. S. M I T C H E L L , A N D D. J. CASIMIR

60 sec. at 194OF., and 330 sec. at 176OF. were required to achieve this in size grade 3 peas. Diehl and Berry (1933) showed 30 sec. at 210°F., 50 sec. at 190°F., and 105 sec. at 160OF. to be required for inactivation of catalase in size grade 3 Alderman peas. These authors confined their work to small size grades, and more severe blanches would be necessary to give a similar degree of enzyme destruction in larger size grades. Catalase and peroxidase inactivation tests are used commercially to determine adequacy of blanching. Dietrich et aZ. (1955) confirmed the results of Joslyn (1946) who found that peroxidase activity paralleled the formation of off-flavors in vegetables more closely than catalase activity. Dietrich et al. (1955) found that peas blanched sufficiently to inactivate catalase retained chlorophyll and ascorbic acid during subsequent frozen storage. About 50% of the original peroxidase survived the blanch, and flavor deterioration occurred during OOF. and -10°F. storage. The result indicates that peroxidase activity provides a useful measure of the adequacy of the blanch. A minimum conversion of chlorophyll to pheophytin during frozen storage at O O F . and 1O O F . was observed by Dietrich et aZ. ( 1955), following a blanch time of approximately 35 to 40 sec. in boiling water. The greater change with shorter blanch times is presumably due to the influence of residual enzymes during storage, and the slight increases in percentage conversion with longer blanch times is mainly the result of chlorophyll degradation during the blanching process. Maximum retention of ascorbic acid after storage for 6 to 12 months at -30° to 10°F. was obtained with peas blanched for 70 to 90 sec. Wagenknecht and Lee (1958) used model systems in which the enzymes catalase, peroxidase, lipoxidase, and lipase with known specific activity were added to enzymatically inert macerated blanched peas. They assessed off-flavor and chemical deterioration after specified frozen storage periods and found a reasonably good relationship between catalase and peroxidase contents and the extent of off-flavor development. After frozen storage, examination by a taste test panel showed the production of mild off-flavor with catalase and peroxidase, a moderate degree of off-flavor with lipoxidase, and an intensely disagreeable off-flavor with lipase. Color rating by the panel and chlorophyll determinations demonstrated a deteriorative trend similar to that observed with flavor. In samples containing lipase, the normal bright green color was modified to a green-grey. Deterioration of the pea lipids caused by lipase and lipoxidase resulted in increases in acid number and peroxide values. Lee (1958) investigated the carbonyl contents of crude pea lipid. Unsaturated carbonyl compounds were found to occur only in un-

+

PRESERVATION O F GREEN PEAS

93

blanched samples which had been stored at O O F . for 5% years. Fresh peas and stored blanched peas contained saturated carbonyl compounds. Peroxidase is one of the most heat-resistant enzymes occurring in vegetables, and peroxidase activity has been used for the determination of adequacy of blanch, especially in the fields of dehydration and freezing. Guyer and Holmquist (1954), using peas, showed that any process below 25OCF., with an approved lethal ratio, will destroy peroxidase but that processes at temperatures above 25OCF.,with marginal lethal ratios, will result in residual peroxidase activity. Enzyme regeneration was observed when peas were given still-cooks at 250OF. having F values1 of 3, 5, and 7, and also when given agitating processes at 260OF. having F values of 5 , 9, and 12. Although regenerated peroxidasc activity was only 0.1 to 0.4% of the original, enzyme activity was sufficient to produce viney off-flavors after 8-months storage at room temperature. Consequently, processes using higher temperatures may have to be based on enzyme inactivation rather than spore destruction. Frakas et al. (1956) presented a heat inactivation curve for peroxidase in crushed fresh peas, and they concluded that enzyme activity may be a problem when processing temperatures are greater than 255OF. For dehydration and freezing, the blanching process is relied upon completely to inactivate enzymes. I n the determination of blanching procedures, the possibility of enzyme regeneration, which may proceed during storage even at low temperatures, must not be overlooked. Enzyme action, although retarded, is not completely prevented at temperatures used commercially for frozen storage. Morris and Barker (1932) reported that freezing in liquid air and storage at 14cF. and -4OF. for 4 months did not destroy the autolytic enzymes of peas.

L. NUTRITIVEVALUE Peas contribute significant amounts of protein, carbohydrate, vitamins, and minerals to the diet, and, except for the cereal grains, leguminous vegetables are of greater importance as human foods than the seeds of any other plant family. In many regions of the world, legumes form the principal source of protein. Peas form part of the vegetable diet of practically every country, and their general acceptability is an important adjunct to their high nutritional value. As previously discussed, the effects of maturity, environmental conditions, and processing procedure all influence the chemical composition and, hence, the nutritive value of peas. The F value is the equivalent time in minutes at 250°F.for reactions having the same temperature coefficient as the destruction of Clostridiurn botulinurn spores.

94

L. J. L Y N C H , R. S. M I T C H E L L , AND D. J. CASIMIR

Table VI shows the average nutrient content of fresh, frozen, canned, and dehydrated peas and the average daily requirement. These values must be interpreted with caution due to the influence of thawing and rehydration, where applicable, and domestic cooking procedures. TABLE VI AVERAGE CONTENTOF IMPORTANT NUTRIENTSIN PEAS ~

~~~~~

Component Calories Protein (g.) Calcium (mg.) Iron (mg.) Vitamin A (I.U.) Thiamine (mg.) Riboflavin (mg.) Niacin (mg.) Ascorbic acid (mg.)

~~

~

~

Amount present in 100 g. of edible material

Daily requirement of average adult male aged 25 years4

Freshb

Froaenc

Cannedd

Dehydrated"

3200 65 800 12 5000 1 6 1.0 10 75

101 6.7 22.0 1.o 700 0.25 0.15 2.10 28

74 5.4

52 3.5 19.1 1.5 44 4 0.12 0.05 1.04 9.6

66.6 4.4 14.5 1.25 319 0.125 0.108 1.06 18.3

20.0 2.0 670 0.32

0.10 2.05 18.7

Anonymoirs (1953). Morgan (1943-1944). c Burger et aZ. (1956). d Anonymoiie (1957a). 6 Recalculated on an 80 % moisture basis from results of Morgan (1943-1944). b

Chitre et aZ. (1950) suggested that the nutritive value of pea proteins depends upon the nature and distribution of the constituent amino acids, and they and McKee et aZ. (1955a) presented evidence that distribution changes with maturity. Nehring and Schwerdtfeger (1957), using an essential amino acid index suggested by Oser (1951), found that for 3 varieties of peas the essential amino acid index ratings were 69, 72, and 67 compared with a value of 100 for egg protein. Index ratings for a number of proteins correlated well with the biological values reported by Mitchell and Block (1946). There are conflicting reports in the literature on the influence of processing on the nutritive value of pea proteins. These probably arise from neglect to evaluate the influence of maturity on the amino acid composition of the protein. Protein loss up to 22% during 3 min. blanching at 2 1 . 2 O F . has been reported by Horner (1936-1937), and Kramer (1945) has reported that an average of 14.3% of the total protein was found in the liquid portion of canned peas. The influences of commercial processing operations and subsequent storage are discussed in detail under the respective nutrients. Domestic

PRESERVATION O F GREEN PEAS

95

preparation for consumption causes appreciable losses, due mainly to further leaching of soluble nutrients into the cooking water. Canned peas, which only require heating, show relatively slight nutrient changes when heated in the brine from the can, whereas losses to the cooking water are severe with frozen and dehydrated peas. As already mentioned, the liquid fraction of canned peas contains a large proportion of the soluble nutrients, and use of this brine saves nutrient which is otherwise discarded. Similar considerations apply to the liquid used for cooking fresh, frozen, and dehydrated peas. Briant et aZ. (1946a,b) found that frozen peas when cooked lost 46 to 47% of ascorbic acid, 11 to 30% of thiamine, and 9 to 30% of riboflavin. Crosby et al. (1953) found that vitamin C loss in large-scale cooking of frozen peas was 21 to 32% when pressure-cooked in steam and 36 to 48% when watercooked. Vitamin C loss was also found to increase as the proportion of cooking water was increased. Oser et al. (1943) compared cooking yrocedures and found that peas cooked in a minimum of water lost 10% of vitamins and minerals, whereas when a large volume of water was used the loss was 31%, thus stressing the importance of reducing the volume of cooking water to a minimum. Hinman et al. (1945) studied t w o methods of preparing canned sweet peas, one in which the brine was concentrated and one in which the brine was discarded, and found that the brine concentration procedure gave retentions of 46 to 51% and 93 to 95% for vitamin C and thiamine, respectively. When the brine was discarded, vitamin C and thiamine retentions were only 32 to 39% and 63%. Riboflavin retentions were 103 to 108% and 65 to 67%, respectively, for brine-concentrated and brine-discarded methods. The common practice of sulfite treatment of peas prior to dehydration tends to destroy thiamine, but this loss is balanced by greater retention of ascorbic acid due to the antioxidant properties of the sulfite. Retention of valuable nutrients may be achieved by prompt handling from harvest to warehouse, high-temperature short-time blanching, the use of agitated high-temperature short-time processes, and optimum conditions for the storage of the processed material. 111. MATURITY

A. DEFINITION The most important single factor determining the quality of commercially processed green peas is their harvest maturity. This has been recognized by the United States Standards for grades of canned and of frozen peas by the allocation of 40 points in 100 for maturity and

96

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

tenderness in canned peas and 50 points in the frozen product. Points for liquor, color, and defects make up the remaining scores. In the field, pods in a very early stage of development contain small flat pea seeds, which subsequently enlarge to a more or less spherical shape and become edible when they are about 9/32 inch in diameter. From this point, differential growth rates of peas and pod result in compression of the seeds which assume a partly cubical appearance. The last stages of maturation are characterized by cessation of growth when the peas become shrunken and wrinkled and finally transformed to dry seed. The growth curve of peas is typically sigmoid, and harvest for processing takes place during the period of more rapid enlargement. This phase of growth is accompanied by rapid change in chemical composition and in consumer preference. The major changes include loss of moisture and quantitative variation in sugar, starch, cellulose, hemicellulose, and pectin and give an over-all increase in total solids. Maturity as applied to the harvest condition of green peas is a technological term which is not synonymous with maturity as understood by the botanist. Its precise definition is not possible, but it can be said to relate to a particular stage in the natural process of growth and development. The position is further confused because the term “maturity” as used in the processing industry is applied to samples composed of individual peas of widely differing maturities. It would be preferable to substitute the phrase “commercial maturity” which might be defined loosely as the average maturity of a sample of peas at any given stage of development. Despite the difficulty in definition, maturity may be measured satisfactorily for commercial control within practical limits.

B. MEASUREMENT Grade, or maturity determination, of peas is necessary for the prediction of the harvest date, as a basis for grower payment, and for guidance of the processor. Because various stages in the maturity of green peas may be readily observed, grading was originally based on the factors of size, color, hardness, shape, and prominence of the radicle. Grading by personal judgment in this manner is notoriously subject to error by an individual and to differences in opinion between individuals. Lynch and Mitchell (1950) tested the visual grading efficiency of 7 experienced operatives and demonstrated that peas within fancy grade were readily recognizable, but peas marginal in quality were variously graded as fancy, standard, and substandard. The unsatisfactory nature of personal assessment of grade has led to the development of a number of objective procedures which vary in

PRESERVATION OF GREEN PEAS

97

efficiency according to the error inherent in the method. To be of practical value any such means of measurement must be simple and not require a high degree of skill. It must also be rapid enough to permit results to be applied to the material during processing, and of an accuracy sufficient for practical requirements. The methods which have been used in industry may be classified as chemical, physical, and mechanical.

1. Chemical Methods of Measuring Maturity During the development of pea seeds, change occurs in a number of constituents, and theoretically change in any of these can be used as a measure of maturity. However, only a few chemical methods have been proved useful. a. Moisture (or solids) content. Moisture or total solids are simple to estimate and correlate well with other measures of maturity. Strasburger (1933) investigated total solids content of drained canned peas and set a limiting figure of 28% between standard and substandard peas. Determination of moisture by toluene distillation was sufficiently rapid to be of value. Nielsen et al. (1947) gave total solids limits for frozen peas of 18.80% for first grade, and the ranges 18.81 to 21.20% and 21.21 to 24.40% for second and third grades, respectively. b. Starch. Nielsen (1943) developed a rapid method of starch analysis whereby after preliminary treatment the iodine-induced color was compared photoelectrically with that of standard starch prepared from the same variety of peas. Further work was published by Nielsen and Gleason (1945) on the same subject, and a linear relationship was demonstrated between per cent starch and T.R. Nielsen et al. (1947) considered starch and total solids useful for maturity determination. They defined grade 1 as 3.70% starch or less, grade 2 within the range 3.71 to 4.80%, and grade 3 between 4.81 and 6.40%. Lee et al. (1954) criticized this method of rapid starch determination on the ground that different standard starch preparations were required for different pea varieties and within any variety depending upon maturity. Lee et al. (1954) gave no indication of the magnitude of errors introduced through inadequate knowledge of the maturity or of the variety to be tested. c. Insoluble solids. Bonney and Palmore (1934) found that evaluation of the percentage of material remaining after extraction with water gave promise for assessment of maturity, but this procedure was superseded by a similar type of analysis using alcohol as the solvent. The method of measuring the A.I.S. was introduced by Kertesz (1934) who compared it with Strasburger’s total solids determination and found that A.I.S. gave a range ratio of 1 :4 against the ratio of 1:1.8 for total solids.

98

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

Subsequently, Kertesz (1935) modified his technique, and A.I.S. was accepted as a reliable maturity measure. The A.I.S. has been investigated by many workers and has assumed the status of a standard against which other methods of maturity measurement are compared. It has been adopted in the U.S.A. and other countries for official purposes, and details of procedure are set out in the Association of Official Agricultural Chemists (1950). The National Canners Association (1957) suggested a rapid method for A.I.S. determination in which the use of an infrared moisture balance gives the result of a test in 30 min. All A.I.S. methods are empirical, and departure from a standard technique can produce minor differences in the results. The alcohol treatment removes mainly water and sugar and leaves a residue consisting of starch, hemicellulose, fiber, and protein. It has the advantage of being a relatively simple procedure, and a determination by the standard method can be completed within 3 hr. The generally accepted limit for green peas is 23.5% A.I.S. for Alaska-type and 21% for wrinkle-seeded varieties. Lynch and Mitchell (1950) from taste tests determined the range of first quality for size graded canned peas as 11 to 16% A.I.S. 2. Physical Methods of Maturity Memurernent Physical measurements with standard laboratory equipment which have been studied include refractive index, per cent sieve sizes, weight of 100 peas, specific gravity, and viscosity. a. Refractiue index. Walls (1936) found no useful relationship between the refractive index of the juice of raw peas and their maturity grade. Lynch and Mitchell (1950) confirmed this finding. b. Sieve size. Percentages of peas passing through certain sieve sizes were for a time used to grade crops for processing. Since the size grade distribution within a crop depends not only on maturity but also on variety and conditions during growth, the method is not adequate. c. Weight per pea. Pea weight, which for convenience may be expressed as the weight per 1000 peas or per 100 peas, has been shown to vary directly with maturity. Tables published by Lynch and Mitchell (1953) show for a single crop that within each size grade, as well as for an ungraded sample, there is a steady increase in the weight per 100 peas during maturation. Kaniuga (1952) defined limits for quality in terms of the weight of 1000 peas: for both Alaska and Express varieties, from 170 to 210 g.; Delicates, 350 to 390 g.; Lincoln, 340 to 380 g.; and 540 to 600 g. for Miracle, Wintham, and Senator. The weight of peas is closely related to size, but size even within a single variety can vary depending on the conditions of growth; favorable conditions produce

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larger peas at a given maturity than do poor conditions, and the weight per pea therefore provides an inaccurate means of maturity assessment. A variable standard of the type suggested would be difficult to apply in commercial practice. d . Specific gravity. Bitting (1937) reported that differences in specific gravity (S.G.) were used as early as 1894 to separate peas of different maturities during processing. This method became widely used during the decade commencing 1930 when brine flotation and S.G. determinations were used to define grades. United States Standards for grades of frozen peas recommend using the percentage of peas (with skins removed) which sink in brines containing 13, 15, and 16% salt. The time to sink is standardized at 10 sec., the distance at 2 in., and the temperature of the peas and brine should approximate room temperature. For grade A, the sinkers shall not exceed 10% in 13% brine; for grade B not more than 12%in 15% brine; and for grade C not more than 16% in 16% brine. If the percentage of sinkers exceeds the limit for grade C, then the peas cannot be better than grade D. Flotation tests are also used in the grade classification of canned peas. The procedure differs in that the skins are not removed and additional brine strengths of 11% and 13.5% are included. The limits for percentage sinkers also differ from those for frozen peas, and distinction is made between Alaska and sweet varieties. Bonney and Rowe (1936) tested a flotation method and concluded that canned Alaska peas younger than “mature” did not give a significant number of sinkers in 1.12 S.G. brine. Fenn (1942) suggested the following points of technique as essential for reliable sinker tests: check density of solution, discard solution after two tests, commence counting after eight sec., and regard suspended peas as floaters, use sample size not greater than 10 to 20 peas, exclude all broken peas, use peas for only one determination, and allow at least 2 days after packing for equilibration. Even though the method involving removal of skins is tedious, and the sample size is thus necessarily small, it has been widely adopted in freezing preservation, particularly for checking performance of gravity graders and for identification of grade. The measurement of S.G. as such has been investigated by a number of workers. Jodidi (1937), working with raw peas, determined the volume of 500 weighed peas by measuring the water displaced in a graduated cylinder. He found that S.G. was a linear function of the starch:sugar ratio. When compared with quality grades of peas, he obtained a correlation coefficient of r = -0.45. Muenchow (1941) correlated flotation grading with tenderometer for 6 varieties and obtained

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coefficients I = -0.649 to -0.803. Lynch and Mitchell (1950), using loss of weight in water to calculate the S.G. of canned peas, obtained correlation coefficients with A.I.S. (canned) of I = +0.99 and T = +0.89 for 26 and 61 pairs of observations, respectively, in two consecutive seasons. Their regression equations when combined have the form: S.G.

= 1 + (31.5 A.I.S. + 217)10-4

When this equation was applied to quality limits of 11% and 16% A.I.S. for canned peas, the corresponding S.G. values were found to be 1.056 and 1.072. Limits for frozen peas suggested by Lee (1942) were: 1.072 and less for fancy grade; 1.073 to 1.084 for standard; and 1.085 and higher for substandard. Lee et al. (1954) undertook a critical examination of several methods of determining maturity. Frozen peas were boiled for 5 min. in water and cooled. Specific gravity was determined by loss of weight when immersed in water. T o examine the effect of entrapped air, parallel samples were punctured before the boiling stage. Of 51 determinations, 34 of the punctured lots gave higher S.G. and 6 lower, while 11 were equal; the mean value for punctured was 0.0026 greater than the nonpunctured. They obtained correlations of T = f0.95, $0.95, and -0.94 when S.G. was compared with tenderometer, A.I.S., and taste test values, respectively. Makower (1957) thawed peas at 80°F., removed the skins, and weighed 80 to 100 g. into a tared stainless steel basket. The peas were then immersed in 8% sucrose solution, and a vacuum of 29 in. of mercury was applied for 5 min. to remove air from the tissues. The vacuum was released, and the weight of the peas in the sucrose solution was used to calculate the S.G. Sucrose solutions were found to minimize errors due to absorption of salt and loss of water in salt solutions, or errors due to absorption of water when water was used. Four varieties of peas were tested during three seasons to give six sets of data. Thomas Laxton was included each season; when compared with A.I.S. values, they gave correlation coefficients of between T = +0.97 and T = +0.99 for 9 to 13 pairs of observations. The regression equations were also calculated, and the slopes lay between 0.00298 and 0.00399, while the intercepts on the S.G. axis were between 1.0095 and 1.0287. Makower (1957) concluded that, although the regression lines differ, maturity changes for varieties and seasons were of the same order. A regression using the mean of the slopes and of the intercepts may be expressed as follows: S.G. = 1 + (34.0A.I.S. + 199)104 which is of the same order as that obtained for canned peas by Lynch and Mitchell (1950). Makower also related density measurements to

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panel scores from taste tests and obtained correlation coefficients of r = $0.98, +0.97, and +0.98 for 3 seasons. There is, therefore, ample evidence that S.G. measurements can be used as an index of maturity and that the method is applicable to blanched, canned, and frozen peas. The determination is relatively simple and accurate when due precautions are observed. e. Viscosity. Elehwany and Kramer (1956) blended 100 g. of peas with an equal amount of water in a Waring Blendor for exactly 3 min. and measured the viscosity of the mixture in a Stormer viscometer loaded with a 106 g. weight. When compared with A.I.S., a correlation coefficient of r = +0.94 was obtained. They reported that the error of the viscosity measurement was greater than that for A.I.S. determination.

3 . Mechanical Laboratory-Type Instruments Histological and chemical changes with advancing maturity include an increase in the size and number of starch grains within the cell and the addition of pectin, phytin, and celluloses to the cell wall. These changes contribute to greater mechanical rigidity of the pea. A number of instruments have been devised to measure the force required to crush, puncture, or shear samples of peas. This type of measurement is applicable to raw peas and has outstanding advantages of rapidity and ease of operation. Some mechanical devices have been developed to test single peas and small pea samples; they are thus strictly research instruments. a. Denture tenderometer. The most elaborate of the purely research instruments is the denture tenderometer developed by Proctor et al. (1955, 1956). This instrument made use of artificial dentures mechanically operated to simulate Erequency and motion of chewing. The force required to “chew” foods was detected by strain gages and the pattern displayed on a cathode ray tube screen was recorded by photography. Graphs of the force required to “chew” raw and cooked peas were recorded. They showed that less force was required for cooked peas, and raw peas exhibited a sharper yield point. b. Crushing instruments. Bonney et al. (1931) described an apparatus to measure the force required to penetrate completely a test piece by a standard cylindrical rod, or to crush a test material to a specified fraction of its diameter. Weight was added at the rate of 12 g. per second and the end point, preset by means of a micrometer screw gauge for the crushing test, was indicated by a buzzer activated through an adjustable contact. To obtain a constant rate of change of load, mercury was run into a receiver arranged to apply thrust to the active part of the apparatus. Later Candee and Boggs (1941) replaced the

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mercury with lead shot and used an automatic electrically operated device to cut off the flow when the required distance of penetration had been achieved. In a redesigned model, the lead shot was replaced by a weight sliding on a lever. The lever was suspended at one end, and the test piece was attached to the other. The weight was removed by means of a threaded rod turned in the original model by hand and later by an electric motor. The motor was automatically stopped at the end point. To test pea cotyledons, Candee and Boggs crushed them to a quarter of their original thickness, and to test skin texture, three skins placed together in the apparatus were penetrated by means of a metal ball 4$ in. diameter. A number of workers have used crushing tests for comparisons with other maturity ratings or with other physical tests. Adams and Dickinson (1945) determined the force required to crush cotyledons of canned peas to a quarter of their original thickness in equipment with the load increasing at the rate of 12 g. per sec. They obtained highly significant correlations with organoleptic ratings and A.I.S. c. Single pea puncture instruments. Lynch and Mitchell (19451955) used an apparatus which showed the force required to puncture a single pea with a cylindrical pin of %-in. diameter. They found that considerable variation occurred even within size-graded peas: for instance, in 143 peas of size grade 7 the puncture force ranged from 1.29 to 3.40 lb. for single peas. 4 . Commercial Instruments

While tests of single peas or parts of peas are useful for special research purposes, they are impracticable for assessing average maturity of a crop sample or of size graded peas in a factory. Any commercial instrument must be simple to operate and must readily give from one sample a value applicable to a bulk of peas. a. Texturemeter. The texturemeter patented by Christel (1938) is hand-operated and measures the force necessary to drive 25 steel pins through a sample of peas held in a cylinder. Walls et al. (1940) quoted correlations of r = -0.6652, -0.8236, and -0.7537 between texturemeter and grade scores. The mode of action of the Christel texturemeter was investigated by Gutschmidt (1953), who found that maximum force was encountered when the pins were 5 mm. above the bottom of the sample vessel. This position corresponds with the point of puncture of the bottom layer of peas. He found a residual force was still required to m w e the pins when they

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projected below the sample vessel and attributed this to friction between the peas and the pins. To test the effect of different operating conditions, Gutschmidt varied speed of operation, temperature of the peas, fill of the sample vessel, and cleanliness of the vessel and puncturing pins. He found that the readings were similar when tests were made at a normal rate, three times as fast as the normal, or at two-fifths of the normal speed. Air temperatures as normally encountered in field or factory were found not to affect the readings, but tests of peas cooled for transport and cool-stored gave higher values than normal peas. Extremes of fill were found to affect the readings, but, within the range of normal fill, readings were consistent. When the apparatus was allowed to collect broken pea material and was not properly cleaned, the mean readings were not affected, but the standard deviation between readings was increased. Torfason et al. (1956) obtained highly significant correlation coefficients of r = f0.952, +0.953, 3.0.861, and +0.869

when texturemeter readings were compared with T.R., A.I.S., dry matter % , and organoleptic texture ratings, respectively. A modified texturemeter built in Germany was described by Schneider (1955a,b) as having 25 steel puncturing pins and a mechanism for moving the sample of peas upwards against the pins. T h e test cylinder had a capacity of approximately 84 ml. and was 51 mm. in diameter and 41 mm. high. The pins were 5 mm. in diameter and 45 mm. long. Using this instrument, he obtained a correlation coefficient with per cent solids of r = +0.93. b. Tenderometer. The tenderometer developed by Martin (1937a,b) is described as measuring the force required to shear a sample of peas. An upper grid is rotated by an electric motor through a second grid mounted about the same shaft but free to move independently of it. The force is measured by the angular displacement of a weighted pendulum attached to the second grid, and is indicated by a maximum reading pointer on a scale graduated in pounds per square inch of grid surface. The mechanism is mounted on a steel frame and the whole machine weighs several hundred pounds. Lynch and Mitchell (1945-1955) observed that after initial compression the peas are mashed and forced between the grids. The pointer halts temporarily when pea material is extruded from the upper grid, and resumes its travel until maximum registration is reached when the pea mass is extruded also from the lower grid. These observations show that the tenderometer does not measure the shearing resistance of intact

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peas but measures instead the resistance encountered when mashed pea material is forced between two sets of plates. Martin (1937a,b) and Martin et al. (1938) compared tenderometer readings on raw peas with grade scores and with A.I.S. of canned peas and obtained highly significant correlations. The proportions of small sieve sizes in run-of-pod samples were related to tenderometer readings, but points on the graphs for each of three pea varieties showed considerable spread, and even greater spread when all were combined in one graph. These authors also showed that readings on size grades could be combined in proportions to the relative weights of size grades in an ungraded sample to give a calculated value for the mixture close to that obtained by direct measurement. Walls et al. (1940) compared tenderometer readings with grade scores, and for size graded peas obtained correlations between r = -0.77 and r = -0.87 for different size grades. More recently, Torfason et al. (1956) compared tenderometer readings with a number of factors and obtained highly significant correlation coefficients. The values of these coefficients were +0.878 for tasters' texture ratings, +0.973 for A.I.S., $0.911 f o r dry matter, S0.848 for penetrometer, and t 0 . 8 1 4 for pressure tester. c. Miniature tenderorneter. Kramer et al. (1950) described a readily portable miniature tenderometer which has two grids, the upper one being driven through the lower by a hand-operated crank. They stated that this instrument is not as precise as the full-size tenderometer. d . Shear press. Rramer et al. (1951) designed the shear press as a multi-purpose instrument for measuring the force required to cut, shear, or puncture different foods. Samples of peas were placed in a lower grid at the bottom of a boxlike container. The upper grid, in the form of a series of blades, was moved downwards by means of a hydraulically operated piston, using oil supplied by an electrically driven pump. The resistance to movement of the upper grid was measured by a Bourdon gauge connected to a closed cylinder and piston which formed a link between the driven piston and upper grid. Modifications to the shear press have since led to replacement of the hydraulic system by a U spring and dial indicator with electronically operated indicator gauge. A transducer with electronically operated indicator and recorder has also been fitted. A more recent modification (Kramer, 1957; Decker et al., 1957) incorporates a circular spring (proving ring) between the piston and the grid. The deformation of this spring is measured by a n engineer's dial indicator. The proving ring also serves to suspend the grid, thereby avoiding the use of bearings in the measuring system. This design is stated to eliminate a major source of frictional error.

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The mode of action of the shear press is apparently similar to that of the tenderometer. Data supplied by Demaree (1953) was recalculated to give a correlation of r = +0.998 between shear press and tenderometer and a regression: Shear press reading = 7.57 T.R.

+ 0.52.

The low intercept value 0.52 suggests that the physical property of the pea measured by the instruments is the same. e. Maturometer. This machine developed by Lynch and Mitchell (1950) measures the resistance to puncture of 143 peas by steel pins. The peas are located to register with the pins by means of a plate with countersunk holes. The sample plate is wound vertically upwards by hand so that the peas contact the pins which are attached to a platten. Upward displacement of this platten was opposed by four helical springs in the original machine and by a flat (diaphram) spring in commercial models. The movement of the pin plate which corresponds with the pressure of penetration is indicated on a dial, and the maximum thrust in pounds is shown by a riding pointer. A recent modification uses four circular springs which rigidly support the pin plate and eliminate friction in the measuring mechanism. It was found that, so long as the crank was turned at a rate not less than one revolution per sec. readings were unaffected by speed. This rate corresponds to 0.075 in. per sec. travel of the sampler plate, and it has been demonstrated with a motorized model that speeds between 0.05 and 0.4 in. per sec. do not vary the reading. Lynch and Mitchell (1950) related maturometer readings and A.I.S. to obtain a correlation coefficient of r = 0.981 for 50 pairs of observations. Further work over a number of seasons showed that the maturometer-A.I.S. regression equations for different size grades were similar over the range of maturities examined. A single regression equation for conversion was adopted and was expressed in the form: M.R. = 33 A.I.S. (canned)

- 153.

Maturometer readings were found to correlate highly with tenderometer readings; e.g., the correlation coefficients in one season for size grades 2 to 7 varied from r = $0.91 to I^ = 0.99 with a mean value of r = +0.96 for 1I 2 pairs of observations. The maturometer, like the tenderometer, automatically selects the size of the sample, but whereas the tenderometer samples by volume, maturometer sampling is numerical. The initial action of the maturometer is to compress and force the peas against the sides of the counter-

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sunk holes. The pins are forced into the sample, and the reading increases with depth of penetration until the pins pass almost to the center of the peas. At this point, the reading remains constant for a short time and then rises to a maximum just prior to complete puncture. In very young tender peas, penetration is smooth throughout. The hesitation which occurs with older peas corresponds to the rupture of the uppermost cotyledon, while the maximum reading occurs during penetration of the lower cotyledon. A consequence of these observations is that when a mixture of size grades is under test the pins do not penetrate large and small peas successively, but all contribute simultaneously to the final reading. This conclusion has been substantiated by the fact that the maturometer readings of ungraded field run samples do not differ significantly from weighted means calculated from maturometer readings of constituent size grades and their proportions by number in the original mixture of ungraded peas. Moyer et al. (1956b) described a maturometer of their construction in which the pea plate was raised by a hydraulically operated piston forced upwards by oil pressure supplied by an electrically operated pump. The measuring device consisted of a load cell fitted with strain gauges, and the changes sensed by the strain gauges were recorded on a recording potentiometer. Since the pinplate was suspended from the load cell, the need for guides was eliminated from the measuring section and hence friction was avoided. They obtained a highly significant correlation coefficient of r = +0.924 with A.I.S. f. Hardness meter. Doesburg and Grevers (1952) produced in the Netherlands a portable apparatus for measuring the firmness of raw green peas at the farm. Material for test is placed in a brass receptable, square in cross section and with an effective internal height of 9 cm. The floor of the sample container consists of a horizontal grid of brass bars of rectangular cross section. The machine is hand-operated so that the receptable moves upwards against a piston with a gridlike lower surface which forces the pea sample through the lower grid. The piston is spring-loaded, and the compression of the spring is indicated on a scale graduated in kilograms. g. Succulometer. This instrument, described by Kramer and Smith (1946), presses juice from a sample by application of a thrust developed hydraulically and the volume of expressed liquid is measured in milliliters. Lynch and Mitchell (1950), using this measurement on raw peas, obtained a highly significant correlation coefficient of r = -0.92 with A.I.S. The determination is slow by comparison with other instruments.

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5. Comparison of Maturity Measures It has been shown that readings on the instruments described closely follow changes associated with time and maturation of pea crops. There is no absolute measure of maturity, but A.I.S. has assumed the status of a standard against which other methods are compared. Correlation coefficients between the readings of an instrument and parallel measurements of A.I.S. have frequently been used as indications of the ability of an instrument to measure maturity. These are valuable for the purpose, but they do not permit precise comparisons of the reliability of different instruments. The standard deviation calculated from readings on replicate samples from one well-mixed lot of peas has been used in several ways to compare instruments. There are inevitable variations in the maturities of individual peas within samples and differences between the average maturities of replicate samples. The sample-to-sample variation must always be one component of the measured standard deviation, and it may well be the largest one. In addition, there may be instrumental variations, and different instruments do not necessarily measure the same sort of average of the varying maturities within samples. Thus there are always difficulties in interpreting the standard deviations, and this may limit the accuracy of the comparisons between instruments. Standard deviations of readings on different instruments have sometimes been compared directly. This is unsatisfactory because the standard deviation is expressed as a number of scale divisions, and the number of scale divisions corresponding to a given difference in A.I.S. varies from one type of instrument to another. In order to overcome this difficulty, the coefficient of variation (standard deviation expressed as a percentage of the mean) has been used by some workers. This is a n improvement, but it is still not satisfactory because it cannot be assumed that zero instrument reading corresponds to zero A.I.S. for any instrument or that it has the same significance for different instruments. For instance, Lynch and Mitchell ( 1945-1 955) found the following relation between tenderometer readings (T.R.) and maturometer readings (M.R.) : T.R. = 0.34 M.R. + 43.8.

A more satisfactory procedure is to express the standard deviation as the equivalent variation in A.I.S. calculated from the regression equation of instrument reading on A.I.S. This was done by Lynch and Mitchell (1950), and they found that the change in A.I.S. equivalent

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to the standard deviations of replicate measurements with a tenderometer and a maturometer were very similar and almost identical with the standard deviation calculated from replicate determinations of A.I.S. on the same peas. This suggests that, with both instruments, the main source of variation between replicate readings was the variation in maturity between the samples of peas. A point of great practical importance is consistency of readings which should not be unduly influenced by operating conditions or alter after a period of use. The rate of movement of electrically-operated instruments such as the shear press and tenderometer is, for all practical purposes, constant. Hand-turned machines such as the texturemeter and maturometer may be worked at varying speeds, but no differences have been found between readings made over the range of speeds encountered in practice. Campbell (1942) found that temperature influenced readings given by the tenderometer by amounts of 0.22 to 0.27 lb. for I°F., while Gutschmidt (1935), working with the texturemeter, found differences of 0.45 scale divisions for 1OC. Normal atmospheric temperature variations are not of practical significance, particularly if the peas are first washed. When peas are chilled for transport or temporary storage, they should be washed in water to raise the temperature before a determination is made. The beater speed of the viner was found by Moyer et al. (1954) to affect maturometer reading and probably also influences other instruments which do not mash the sample before the maximum reading is reached. Since conditions for maximum efficiency in vining must be held within narrow limits, this should not be important in practice. Continued use and abuse of mechanical instruments in industry may result in inaccurate readings which, being difficult to detect, often remain uncorrected. For most instruments, it is a simple operation to subject the measuring system to static test. Uniform test material is required to check a mechanical instrument during operation, and efforts have been directed towards the discovery of such a material. Graham and Evans (1957) stated: “The Food Machinery and Chemical Corporation describe a procedure for tenderometer standardization and a method for checking the correct position of the pointer in their service manual. Standardizing involves balancing the moment of the counterweight rod and weights in the horizontal position against the moment of a standard weight, using the grid spindle as the fulcrum. This method is entirely mechanical and is quite unreliable when the grids are either slightly worn or distorted.” The Tenderometer Committee of the National Canners Association

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has for a number of years investigated tenderometer operation to find the reason for lack of agreement between different tenderometers after they have been in use for a period of time. Using four special tenderometers, it was found that after 4 years the wear produced on the shearing blades was not correlated with changes in readings of the machines, and the study is being continued. Blade measurements to detect wear will be made when differences in readings are apparent. Lynch and Mitchell (1945-1955) encountered two conditions in tenderometers likely to affect the readings. The most common of these is corrosion of the ball races supporting the grid shaft, and the other is related to shaft distortion. Corrosion of the bearings is caused by penetration of pea juice. This does not appear to give trouble when the machine is in daily use, but after a period of idleness bearings corrode badly and show a tendency to bind. Bent spindles may result from normal use, and the first indication of the defect is frictional resistance in the return handle. In practice, this symptom may be mitigated by moving the handle along the shaft, but such an adjustment does not necessarily eliminate the reading error involved. Various workers have proposed methods for tenderometer standardization. An early suggestion was the use of cigarettes as test media, but these were not found satisfactory. Kramer (1948) proposed a method using A.I.S. as a standard for checking tenderometer readings and as a basis for mechanical adjustment. He recommended that 12 successive readings on a sample of size graded peas be made and that A.I.S. be determined in duplicate on replicate material after canning. Kramer gave a table of equivalent values for tenderometer and A.I.S., canned and raw, and recommended that if the tenderometer varied by more than 5 Ib. from the expected value it was not sufficiently accurate and should be adjusted. The use of peas preserved in alcohol has been suggested for comparison of tenderometer readings with those from a standard machine kept for the purpose. Such peas are softened by the alcohol and permit standardization only over the lower end of the tenderometer scale. Peas stored in crushed ice have been used as test material. A sample of sizegraded peas was thoroughly mixed and stored in Dewar flasks with ice while awaiting simultaneous test in a standard tenderometer and in the machine under test. Graham and Evans (1957) evolved a method using frozen peas which were removed from store and prepared for test. The peas were thawed in a cool room at 40°F. overnight and mixed and sampled into 8-oz. cans so that each can held sufficient peas for a test. The cans were placed at 37OC. for 5 hr. in an incubator in which the air was maintained in a dry condition by the use of calcium chloride.

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One can at a time was removed from the incubator and 5 oz. weighed from it and tested in the tenderometer. This permitted comparison between a standard tenderometer and the one under test. These workers obtained agreement by moving the counter weight on the pendulum; e.g., the mean of 6 readings from one machine differed by 6 lb. from that of the standard machine. By repeated test and adjustment, agreement was obtained when the counter weight had been raised 3/4 in. on the pendulum shaft. This method is open to criticism because: machines are tested only at the low end of the scale; the procedure is involved and inconvenient; adjustment by shifting the weight, to compensate for some defect in the machine itself, is wrong in principle. The angular displacement from the vertical of a weight fixed at a standard distance from the fulcrum of a lever is an accurate means of measuring force. Adjustment should be effected by removal of the cause of the misreading, which in most cases has been attributed to excessive friction due to incorrect clearances. Working with the maturometer, Lynch and Mitchell (1945-1955) have had success in standardizing commercially used machines. As with other instruments, it is a simple matter to determine if the forcesensing mechanism is registering accurately when balanced against a static force. In the case of the maturometer, this may be done by suspending the instrument and loading with known weights. The gauge itself may be checked in the center of the range by means of a special gauge tester, or a dead weight tester may be used to check the gauge over the whole of the range. The gauge check is a prerequisite to further test. It was found that %e-in. sheet asbestos when punctured under the maturometer behaves in similar fashion to peas, insofar as it gives a sharp but not violent yield point. Rectangular pieces of sheet punctured by 55 pins gave convenient maturometer readings. Uniformity trials with sheets of asbestos showed that if one of each of 4 pairs of adjacent pieces is tested in each of 2 maturometers, the means should agree to within 5 lb. when the two machines are in correct adjustment. Using this method a number of machines was compared and found capable of mechanical correction. A new instrument was used as a standard with which to compare commercial instruments. In all cases of maladjustment, readings lower than the standard were obtained. Low readings were found to be due to friction in the bearings or to misalignment, and when adjustments were made the readings agreed with those of the standard. A further and more absolute check was obtained by using sets of asbestos pieces punctured by different numbers of pins. With a sufficientnumber of sets, the effect of variation of the asbestos sheet was reduced, and a regression between the

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reading and the number of pins involved in puncture was calculated. The intercept of this regression was found to have a small but significant negative value f o r the standard maturometer showing that some friction remained. Similar intercepts were obtained with other machines when they had been properly adjusted. Asbestos sheet was found to be affected by humidity, but comparisons between machines at a distance in place or time were obtained by storing test pieces in a closed can. Tests with asbestos led to minor improvements in design and, finally, to a complete redesign of the measuring section. Sliding friction in the bearings was eliminated as shown from the fact that regression of number of punctures against readings had an intercept value indistinguishable from zero.

6. Taste Tests The final criterion with which all methods of maturity measurement must be compared is the taste test. Such tests are subject to variation inherent in biological material, but when carefully designed (Boggs and Hanson, 1949) they can give reliable results. Lynch and Mitchell (1950) concluded from the results of a number of taste tests on canned peas that youngest and oldest peas were least preferred and that those receiving the highest acceptability scores lay within the limits of 11 to 16% A.I.S. Mitchell and Lynch (1954) reported taste tests which showed no lower limit but a well defined upper limit for first quality. Kramer et d.(1950) found that in canned peas, processed rapidly after harvest, acceptability was greatest for peas with a maturity of 110 tenderometer, whereas younger and older peas were not as acceptable. They stated that this agreed with results of a consumer survey conducted by Roper (1941). Elehwany and Kramer (1956) used 3 expert graders to grade 65 samples of 6 varieties of canned sweet peas and compared the grade with A.I.S. in a graph which showed quality limits of less than 11% A.I.S. for fancy grade, 11 to 15% for extra standard, and above 15% for standard. For frozen and dehydrated peas, a younger optimal maturity than for canned peas is generally accepted, and no definite lower maturity limit for first quality has been suggested. C. COMMERCIAL APPLICATION OF MATURITY MEASUREMENT I . Grade Standards for Canned and Frozen Peas Maturity measurement plays an important part in deciding the quality grade of the processed article. The United States Standards for grades of canned and of frozen peas set limits for A.I.S., for the brine

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flotation test, and for the cotyledon-crushing test. Maturity and tenderness scores for the two highest grades (A and B) are based on brine flotation tests and number of ruptured peas, while scores for grade C are based on brine flotation, A.I.S., and crushing tests. For grade C canned peas, the limits for A.I.S. are not more than 23.5% for early (Alaska-type) peas and not more than 21 % for sweet-type peas. A.I.S. is commonly used for assessing quality of the canned product by the manufacturer, whereas the flotation test is normally preferred for evaluation of frozen material. 2. Raw Pea Grading

Peas when received at the factory are tested for maturity usually by means of one or other of the commercial instruments. The reading SO obtained is used to determine rate of payment to the grower and the quality grade of the final product. Type of pack and details of processing procedure may also be decided by the maturity of the peas. Since such important decisions rely on these measurements, it is essential that samples tested are truly representative of the peas in the load. The sample may be obtained by dipping peas out of a number of lug boxes, or in the case of bulk-transported peas by a sampling-tube pushed in at different points in the load. Samples are taken at the viner station or on arrival at the factory. The use of the sampling-tube method with bulk loads should give an adequately representative sample, but samples drawn from lug boxes by the usual method of dipping are not above suspicion. Peas, like other granular material of different particle size, tend to segregate into layers when subject to vibration, and the accessible peas near the top of the lug box will differ in maturity from the bulk of the peas. Lee (1939) carefully sampled loads of peas at a factory by removing 7 or 8 lug boxes from each load and mixing each box of peas before withdrawing a sample. He compared the tenderometer readings from samples obtained in this way with values from the same load derived from factory sampling wherein a can full was taken from one of the uppermost lug boxes in the load. The data he obtained show that in 14 observations of a total of 33 the difference from factory sampling was 5 units or greater. The mean difference over the 33 test samples was 3.8 units and 22 of Lee’s readings were higher than those obtained by the factory. This demonstrates the need for an adequate sampling system to minimize errors. Lynch and Mitchell (1945-1955) overcame the difficulty of sampling a crop delivered at the factory by use of an automatic device consisting of a small “bucket” attached to the inside of a rod washer lo-

PRESERVATION O F G R E E N PEAS

113

cated before the size grader. With each revolution a small sample was dipped from the flow of peas and delivered to a perforated basket. Periodically and at the end of delivery of each load, the basket was removed, the peas mixed, and 4 maturometer readings made for each 10 lb. of peas collected. Provision for automatic sampling a t the viner station or in the processing line is not difficult, and it is the only means of insuring that the sample tested is truly representative of the delivery.

3 . Prediction of Maturity The maturation rate of peas is so rapid that each crop must be harvested quickly and at the exact time to obtain raw material of high quality without sacrificing a considerable percentage of the total yield. The time of harvest must be forecast in advance in order that the crop may be handled expeditiously in field and cannery. Such prediction requires an initial point of reference, an end point, and some means of measuring the difference in time between these points. It is necessary to define precisely the end point or the maturity most suitable for harvest, and unless this can be done it is impossible to measure the accuracy of prediction and to determine the need for amendment to the method. a. Haruest maturity-prediction end point. Personal judgment following field inspection was originally used for assessment of harvest time, and this method is known to be subject to gross error even when applied by experienced field men. It is virtually impossible to assess visually the average maturity of peas in a field, for maturity varies not only from point to point in the field but from node to node on the plant. Judgment in the field is also subject to bias, because the factory field man avoids any tendency to downgrading due to overmaturity and therefore sets the harvest date before the optimal. The grower, on the other hand, is primarily interested in yield, and thus prefers a delayed harvest. The factory field man concentrates on lower pea pods, while the grower tends to observe those which are immature and uppermost on the plant. Disagreement as to harvest time has been a potent source of unsatisfactory canner-grower relations. Some of the objections raised by growers are removed by payment on a sliding scale, such that loss of yieId by early harvest is offset by a higher price rate. Under such conditions it may be considered that crops can be harvested within wide maturity limits, and the chief concern is to maintain an even flow into the factory. A price scale of this type may not completely compensate for early harvest, but at some selected maturities favored by the processor a greater return may be obtained. Sayrc (1952) described a scale of prices and from the 3-year mean yields of experimental plots calculated the return per acre over a range

114

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

of maturities in a freezing variety (Thomas Laxton) and a canning variety (Perfection). For freezing peas, at a tenderometer reading of 95 and a price of $115 per ton, the return was approximately $151.80 per acre; while for a tenderometer value of 135 the price of $50.00 per ton gave only $84.50 per acre; at 80 tenderometer the return was $120.40 per acre. For this variety, a maximum return was obtained at a tenderometer value of 95, while for the canning variety, maximum return was $171.60 per acre at a tenderometer value of 90. These figures illustrate that even with a sliding price scale there can be encouragement for the grower to harvest within narrow limits of maturity. Considering the industry as a whole, a number of costs are more dependent on acreage than yield, and the greater the yield consistent with quality, the larger the return from the many fixed costs. The importance of harvesting as late as possible is obvious when it is considered that the yield, according to data of Lynch and Mitchell (1953), increases by about 10% per day over the range of processing maturities. The most suitable harvest maturity has frequently been defined in terms of tenderometer value of field rwn peas. Seaton and Huffington (1950) used a 100 tenderometer reading as the correct harvest maturity when predicting canning crops by the use of heat units. Katz (1952) stated that many packers and canners harvest near a tenderometer reading of 100 for fancy quality, and 120 for lower quality. Sayre (1953) quoted 85 tenderometer as fancy for Thomas Laxton (freezing variety) and 95 for Perfection peas for canning. Adam (1956) summarized 5-years comparisons of the practical canning stage determined by taste tests with maturity measures. He stated that at the practical canning stage A.I.S. of raw peas was 13.0 10 14.5%, A.I.S. of canned peas was 10.5 to 12.5%, and the tenderometer reading was 110 to 115 for most varieties. Lynch and Mitchell (1953) and Mitchell and Lynch (1954) compounded limits for best quality with yield from crop analysis trials carried over the critical ripening period. These trials involved examination of detailed yield and maturity data of peas picked daily from 7 to 10 days before estimated harvest time to 7 days beyond this point from randomized blocks established in commercial crops. A graph of daily yield is reproduced as Fig. 3. In addition to total daily yields, the graph shows the yields by consecutive summation of the constituent size grades, and yields of peas with maturity less, and greater, than the quality limits of 11% and 16% A.I.S. which correspond with 210 and 375 maturometer reading. The time of maximum yield of first quality peas is the best time to harvest the crop, and this point was called the O.H.T.

115

PRESERVATION O F GREEN PEAS

Taste tests conducted by Mitchell and Lynch (1954) recorded no lower limit for first quality peas. If the lower limit is eliminated from Fig. 3, the maximum yield of peas less than 16% A.I.S. is very close in time to that for the maximum yield between the two limit lines. The upper limit of first quality for quick-frozen peas was found to be less than that for canned peas, from which it was concluded that the O.H.T. occurs at an earlier stage in pea crops grown for freezing. 0

N u)

-

6ooo

I

5000 , S I Z E S 2.3.4.5.6

4000

-

3000

-

(L

< U

m

2

? S T QUALITY , S I Z E S 2.3.4.5 YIELD

.

\ SIZES 2,3,4 \

\

\ \,“LOWER

I

\

.

SIZES 2,3 SIZE 2

0 -7

LIMIT, F I R S T QUALITY

-6

-5

-4

TIME

TO

-3

-2

-1

0

1

2

M ATURITY (DAY S)

FIG.3. Changes in total yield, size grade distribution, and limits for first quality with time for a crop of Canners Perfection variety (from Lynch and Mitchell, 1953).

With these and other canning crops investigated over a period of years, the O.H.T. was shown to occur when the maturometer reading of a representative ungraded crop sample, i.e., the maturometer index (M.I.) was about 250. The O.H.T. for commercial purposes was therefore defined as the time when the M.I. reached 250. If local or national preference is for maturity other than that selected from this work, the same principle can be used to determine the time of optimal harvest. To insure uniformity, a standard procedure was adopted in preparing peas for determination of the M.I. Vined peas were cleaned, and peas

116

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

smaller than 9/32 in. were eliminated by sieving, as these are not normally used commercially and the proportion in which they occur varies with the spread of maturity range within the crop. Moreover, peas which are dry-cleaned before commercial test have their lower size limited by the cleaner screens, which are not necessarily standard sieves nor of uniform efficiency. Mitchell and Lynch (1954) using this method of standardization obtained tenderometer values for the O.H.T. for canning and freezing of 114 and 94, respectively. Other workers have adopted similar methods to standardize samples. A commercial machine which discards all material passing a 9/32-in. screen was produced to clean and standardize samples for tenderometer determinations. This practice has not always been used, and the method of sample preparation is rarely described in the literature. b. Prediction reference point. The starting point for harvest prediction may be time of planting which is known with certainty. Other stages in the life of the crop from which prediction may commence may be less accurately fixed, but since they are nearer the harvest date, the shorter period involved restricts the error. The first visible and definable stage in crop growth is appearance of blossom. Full blossom is described as the point at which all plants first exhibit an open flower. This stage or some selected fraction of full blossom has been used as a reference point. In climates where the transition from winter to summer temperatures is rapid, first blossom and full blossom may occur during a single daylight period. Under these conditions the reference point may be fixed with certainty, Under other climatic conditions, blossoming is spread over a number of days, and the precise location of fractional or full blossom is difficult. Any error in the recognition of the initial reference point is necessarily reflected in the final result. A desirable procedure is to sample the crop within about 10 days of the anticipated harvest time and to measure the maturity of the sample. This gives, in effect, a reference point for short-term prediction. Such preharvest sampling has been carried out by a number of processors who have tested the peas in the tenderometer. Little has been written of the methods adopted and of their reliability. Lynch and Mitchell (1953) described a method for obtaining samples of peas from the growing crop, The maturometer readings on successive daily samples defined the maturity of the crop and allowed calculation of the time of harvest. c. Time to reach maturity. T o estimate the time from a selected reference point to final maturity, use has been made of average times, in days, to reach maturity, heat units, and rate of change of maturity

PRESERVATION OF G R E E N PEAS

117

measures. For short-term prediction, the time from full flowering to maturity, usually between 18 and 21 days, is a useful guide for field men and is widely used as such. The number of days from planting to maturity for different varieties is commonly quoted in seed catalogs. These figures must be regarded as no more than an approximate expression of relative earliness or lateness of varieties since the absolute values are affected by all environmental factors influencing growth and maturation. The average number of days from planting to harvest may be useful for long-term prediction, but can be applied only after data for a number of seasons have been analyzed. Lynch and Mitchell (1953) using information from Tasmania, Australia, related the number of days from planting to maturity (M.I. 250) with the date of planting. For one district, a crop planted on October 19 required 100 days to mature, while from November 28 only 92 days were needed. From linear regressions, the 95% confidence limits were calculated as a maximum of k5.5 days for one district, while for other areas values of &9.0 and k 6 . 0 days were obtained. Prediction by this means was shown to be useful in indicating the times when crops should be inspected for the purpose of applying short-term prediction procedures. It is significant that these confidence limits did not include variation between seasons but are an expression of the spread of maturity of crops planted on the same day in one particular season. Thus crops planted on October 19th in the district quoted required 100 days average to reach M.I. of 250, but as the 95% confidence limits were k 5 . 5 days, 1 crop in 20 planted on that day would have fallen outside the limits of 94.5 and 105.5 days from planting. This variation is a measure of the influence of factors such as soil, aspect, and slope which are not the same for all crops. The heat unit system is based on the assumption that temperature is the most important factor influencing the rate of growth and maturation, and that little or no growth occurs in pea crops below a temperature of 40°F. The number of heat units is determined by integration of the effective temperature from the time of planting or other fixed time. Usually the mean of daily maximum and minimum temperatures in a standard meteorological screen is used. The amount by which the mean for a day exceeds 40°F. is the number of degree days for that day. This figure may be multiplied by 24 and expressed as degree hours. A value obtained from progressive summation is used to indicate the maturity status of the crop. The use ‘of more frequent temperature readings or continuous records has been suggested. A number of contributions to the literature on heat unit control has been summarized by Seaton and Huffington (1950), who investigated

118

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

its commercial application. They stated that Alaska peas normally require 1200 to 1250 degree days from planting to a tenderometer reading of 100, early Sweets from 1200 to 1300 degree days, and late Sweets from 1625 to 1725 degree days. Three intermediate groups required 1400 to 1500, 1500 to 1575, and 1575 to 1650 degree days, respectively. The importance attached to the system can be gauged by the fact that, in 1950, 37% of pea canneries in the United States and in Canada had adopted this method, and the acreage involved was approximately 158,000. A slightly greater acreage of sweet corn was being controlled also in this way, Heat unit predictions may be made at the time of planting, at any time during the growth of the crop, or at one of the various blossom stages. Prediction from planting time is based on average temperature records for any given locality, and the day of predicted harvest is that which corresponds with the number of heat units stated to mature a particular variety. To permit prediction from any stage in the development of a crop, a progressive total o€ the actual heat units is kept. Reference to average temperature values will indicate the period of time necessary to accumulate the balance of heat units to a predetermined value. Some workers combine heat increments with inspection and maturity measurement of preharvest samples, the heat increments being used as a guide to inspection time for the purpose of crop sampling. Prediction from a selected stage of blossom is similar to long-term prediction except that heat unit estimation is superimposed upon a different starting point. Seaton and Huffington (1950) published graphical data to demonstrate the result of heat unit control of 84 commercial pea crops. Some 80% were found to be within + I day of the desired maturity on the predicted date and 94% within +2 days. In another series, 66% of the crops in the test were within *I day, and all were within &2 days of target tenderometer reading. In the latter case, 1600 degree days corresponded with 100 tenderometer, and the tenderometer was found to increase by 1 unit for each additional 3.2-degree days. This agreement is good for heat summations involving the full growing period and suggests an accuracy of prediction to -t-2 days. Such a degree of coincidence has not been generally obtained. The work of Lynch and Mitchell (1953) in Tasmania demonstrated that a total spread of 11 days in crop maturity may occur because of the operation of factors other than screen temperature. Seaton and Huffington. (1950) list a number of important factors which may influence agreement between summed heat units and maturity. These factors include moisture, fertility, slope, aspect, altitude, latitude, frost, and sunlight.

PRESERVATION OF G R E E N PEAS

119

Katz (1952) compared direct heat unit summation with a similar summation which takes account of the fact that the rate of chemical reaction doubles for every ItlOF.-rise in temperature. Both Alaska and Sweet varieties over 3 seasons, 194S44, showed linear relationships between heat accumulation and tenderometer reading. The difference between exponential and direct summation methods was small, and the simpler direct method was thus preferred. The heat unit requirement was found to vary directly with temperature, crops showing more efficient response at lower temperatures. This result was found to be true irrespective of whether crops were compared within or between seasons. In a “hot” season, therefore, the average heat unit requirement is higher than in a “cool” season. From 23 to 30 heat units were required to mature peas of fancy grade from 100 tenderometer to a n inferior grade at 120, whereas differences in crop maturation in different seasons were as great as 77 units. Katz stated that climatological data as now recorded is often inadequate, and thermographs should be placed in the fields to obtain more closely the temperature in the immediate crop vicinities. In spite of factors other than temperature which affect crops, the heat unit system is a widely applied and useful method of crop control, particularly when used to indicate the appropriate time of inspection and sampling for short-term prediction. In this connection, Stark and Huffington (1956) pointed out that, while heat units assist in predicting harvest dates, the exact time of harvest must be determined by a pretesting program to evaluate the maturity of the product. They considered that the accuracy of the heat unit method probably varies considerably with climate and topography. Prediction by heat units from a blossom stage appears to have less error than prediction over a longer period. Some investigators consider that long-term prediction should be checked and perhaps corrected when the blossom stage has been reached. If the blossom stage can be detected with certainty, errors inherent in heat unit systems are reduced but not eliminated. Many processors adopting one or other of the various methods of prediction take preharvest samples for maturity test, and from these they estimate the time required to reach a definite harvest maturity. Preharvest samples can be taken as late as 1 or 2 days before harvest to reduce the error of the prediction. The tenderometer is commonly used for testing preharvest samples, but details of methods have not been published. Lynch and Mitchell (1953) and Mitchell and Lynch (1954) used the maturometer to determine maturity during the preharvest period;

120

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

they evolved a system of prediction based on an end point defined by maturometer index and the use of a predetermined mean rate of increase of M.I. The method was as follows: Areas of 10 acres and less are sampled daily at 4 points along a line across the field, approximately 1 sq. yd. of vines being taken at each point. The first sample is taken at an estimated 7 days before O.H.T., and samples are taken daily thereafter until 2 days before harvest. Vines from the 4 sample areas are bulked, and the M.I. determined. A preliminary prediction is made on the M.I. of the first sample by the assumption of a daily M.I. increase of 20. On the second day, the mean of both days’ values is used, and thereafter the mean of the last three days’ values only is considered. For M.I. means of 2 and 3 days, it is necessary to adjust time in days on an equivalent basis, since the mean M.I. refers to the midpoint in time of the number of days involved in its calculation, e.g., if the readings from a crop are M.I. 165, 175, and 200 on February 6, 7, and 8, then the mean M.I. 180 corresponds in time to February 7th, and the predicted date of harvest (M.I. 250) will be that date plus 3.5 days. This system is practiced by a number of Australian processors, and it has permitted very close control of crops so that the highest yield consistent with quality is obtained throughout the season. The method can be applied to predict any desired M.I. value for harvest. TABLE VII 95% FIDUCIARY LIMITSOF

MATUROMETER INDICES FOR 4 - P O I N T

AND 6 - P O I N T SAMPLESa

0

Date of harvest

4-Point sample

6-Point sample

Jan. 26 Jan. 27 Jan. 28 3-Day mean

36.5 41 .O

18.5 26.5 30.5 13.0

57.0 18.5

Mitchell and Lynch (1956).

Two sources of error are encountered in the Australian prediction procedure: the first is error due to sampling, and the second is error due to variation in rate of maturation. Because the amount sampled is but a small fraction of the planted area, viz: 4 sq. yds. in 10 acres, sampling error is inevitable. Mitchell and Lynch (1956) analyzed the results of a uniformity trial in which 36 sampling points were chosen in a commercial crop and 1-sq. yd. samples were taken daily over 3 days. The results are given in Table VII and show that the 95% fiducial limits for maturity estimations are excessive when based upon 4-point and 6-point sampling on any one day. When these results are consid-

PRESERVATION O F G R E E N PEAS

121

ered on a 3-day running mean value, the error involved is less than a day for 4-point sampling, and a little more than half a day for the 6-point sample. Marked departure from the M.I. 20 per day increase can occur, particularly when the crops are affected by fluctuation in water supply, by root rot, or by frost. I n practice such conditions usually exert their influence simultaneously over a wide growing area, and a correction factor may be readily applied. Analyses of results (Lynch and Mitchell, 1953) of maturometer harvest control of commercial crops has shown that the M.I. of more than 90% of factory deliveries was within a day of the predicted value.

4 . Scheduled Planting A natural result of long-term prediction has been the development of methods for spacing the planting times of peas to obtain a n even flow of material to the processing plant. Systematic planting is designed to permit efficient use of factory capacity with a minimum of intermittent overload. If the length of time from planting to harvest is known throughout the season, either by analysis of days to maturity or by calculation of heat units, it is possible with the aid of graphs to work backwards from harvest time to corresponding planting date. Lynch and Mitchell (1953) expressed the regression of harvest on planting time in the form: H = 103.81 + 0.805P

for the Sheffield district, Tasmania, Australia, where H is harvest date and P is planting date, both expressed as days from October 10th. This result shows that the acreage required to supply the factory for 8 days at harvest should be planted during a 10-day period. When severe conditions occur during the planting period, the heat unit procedure advocated by a number of authors is more appropriate. This procedure averages for a number of years the number of heat units received per day about the harvest time and uses this value to measure currently the time between plantings on the basis of a I-day requirement at harvest. Katz (1952) suggested that the planting schedule be arranged so that the number of acres planted on each planting day will approximate in yield the daily capacity of the cannery during harvest time, and that planting be started at or soon after the mean daily temperature reaches 40°F. and be delayed during periods of lower temperature. Katz further advocated that successive plantings be spaced at intervals equal to the normal heat unit accumulation per day at harvest time. Maturity prediction is thus variously applied throughout the growth

122

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

of a crop, for time spacing of planting, during the progress of the crop to determine time for prediction sampling, and during the last 7 days to insure that harvest is effected at the desired maturity.

5 . Yield Prediction In addition to maturity prediction, pre-estimation of crop yield is important to the processor. Field men can usually estimate yield with sufficient accuracy to be of use to the factory. Lynch and Mitchell (1953) established a method whereby a measured area was sampled at each of 4 points across a field. After vining, the bulked sample of cleaned peas was weighed, and the yield per acre on the sampling day was estimated. T o predict the yield at O.H.T. they used the formula: Y =

Ym X 250

M.I.

where Y is the predicted yield at M.I. 250 while Ym and M.I. are, respectively, the yield and maturometer index ( M I . ) determined from the samples. Preknowledge of vine yield is a further economic factor of importance in determining transport requirements from field to viner station. Lynch and Mitchell found that vine weights changed little during the immediate preharvest period. This permits estimation of vine yield at harvest from the weight of vines in samples taken for yield predictions. IV. UNIT PROCESSES

A. FIELD

1. Harvesting While the technology of green pea preservation is concerned with all aspects of crop production, from the signing of the grower contract to the final harvest, the preparation of the land and cultural practices adopted are within the realm of the agriculturist rather than the food technologist. The latter is, nevertheless, concerned with scheduling of planting dates, prediction of harvest dates, and harvesting methods since these procedures affect the intake into the factory and the quality of the raw product. Vines are cut with a farm mower or some modification of this machine and, after raking into windrows, they are lifted mechanically onto wagons by a green crop loader. Adaptations of the simple mower include side delivery mechanisms which obviate the need for a separate

PRESERVATION O F GREEN PEAS

123

raking operation and devices to raise prostrate vines ahead of the cutters. When the soil surface is irregular, cutting is satisfactory on the ridges, but pods growing in the hollows are not recovered. Lynch and Mitchell (1945-1955) found in one field of this type that approximately 500 Ib. of peas per acre remained unharvested from a 4000-lb. crop. The development of successful vining equipment was the key to establishment of the pea processing industry, as only by its use could peas be economically removed from the pods. In 1883, Madame Faure in France invented a pea sheller which partly achieved this purpose. I n 1889, Scott and Chisholm in the United States patented a machine which threshed the peas direct from the cut vines. Pea viners perform three distinct operations: threshing, conveying, and separation. Threshing results from the impact of a series of beaters on the vines and the impact of the vines against rubber screens. The beaters are attached to a rotating cylinder which is mounted coaxially with a slowly revolving hexagonal reel. The sides of the reel are constructed of rectangular frames holding rubberized screens. The reel is equipped with internal flights which elevate the vines through an angle of about 60° and allow them to fall into the path of the beaters which throw them to the other side of the reel. Passage along the reel is effected by setting the beaters at a n angle so that each blow deflects the vines in a forward direction. I n many viners the angle of the beaters may be readily altered to give varying numbers of blows to vines during passage through the viner. At the inlet of the viner, a conveyer feeds the vines into the reel, and another conveyer at the discharge end removes the threshed vines. Shelled peas are separated from vines by screens on the side of the reel and by a rod grid at the exit end. Broken vines and peas passing through the reel screens are separated by a canvas or aluminum slat apron in the form of a continuous broad belt. During viner operation, the speed of the reel should be constant at about 18 r.p.m. as this gives the optimum degree of elevation of the vines. At greater speeds centrifugal force carries the vines further than the required 60°-angle, and the point of impact with the beaters is not ideal. The effect of beater speed on peas was studied by Moyer et al. (1954), using small lots of vines in an experimental and a commercial viner. The cleaned peas were tested by the maturometer, and readings obtained were related to beater speed. Mitchell and Lynch (1956) conducted similar experiments with commercial viners. In all of these tests, 6 varieties of peas were used, and the damage, as shown by maturometer value, increased in linear fashion with viner speed giving a n average regression of: M.I. = M - 0.57s

124

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

where M.I. is the maturometer reading of the sample at beater speed S, and M a constant which approximates the maturometer reading of hand-shelled peas. Yield measurements of peas from equal weights of vines showed maximum recovery when the beater speed was in the range 165 to 180 r.p.m. Viner operators tend to use excessive speeds, but injury and fragmentation more than off set the additional peas which are recovered. Further experiments by Lynch et al. (1956-1957) showed that the effect of injury is to give progressively greater loss at each stage of

FIG.4. F.M.C. Pea Harvester

(courtesy of Gordon Edge11 & Sons, Limited).

processing through the factory. One practical disadvantage of low beater speed is decrease in viner capacity, but the limiting factor in this regard is more usually related to feed rate. At least four machinery manufacturers have developed pea harvesters which differ mainly in the principle employed for picking up the vines and in the mechanics of threshing. The mobile viner produced by Scott Viner Company is essentially a conventional-type viner. A feed mechanism in front raises the vines from windrows, and shelled peas are discharged at the rear into lug boxes. The Food Machinery Corporation harvester (Fig. 4) has discarded the conventional threshing method. The vines, after elevation into the machine, travel along an up-and-down path towards the rear, where the waste material is discharged after separation from the peas. The

PRESERVATION

OF GREEN PEAS

125

beating action is performed by a series of narrow beaters running the length of spindles with axes across the direction of travel of the vines. The vines move through the machine and are confined between the beaters and a large mesh grid. Rotation of the beaters causes progression of the vines, and the threshed peas, together with some trash and detached pods, pass through the steel mesh. The pods are returned to the threshing section, and the shelled peas are conveyed to a hopper from which lug boxes are filled. A third type of harvester, the McBain Pea Viner (Fig. 5), threshes by use of narrow beaters on large cylinders which lie across the machine. The beaters have adjustable clearances with steel grid concaves.

FIG.5. McBain Pea Viner (courtesy of Gordon Edge11 & Sons, Limited).

Three sets of cylinders and concaves are mounted in series. A mower blade and pickup device in front of the machine cuts and elevates the vines which pass in turn between each cylinder and concave where the peas are threshed. Short aluminum-slatted moving aprons and an air blast separate trash from peas. Mobile harvesters are reputed to cause excessive damage to peas, but this point does not seem to have been adequately checked by careful measurements, and it may, therefore, be in error. Mitchell and Casimir (1957-1958) compared the performance of the latest modified F.M.C. harvester with that of a standard viner running at a beater speed of 175 r.p.m. Two adjacent windrows of cut vines from one-fifth of an acre were used as test material. Maturometer readings and yields were recorded after each of the stages of threshing, dry cleaning, washing, and size grading. The data summarized in Table

126

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

TABLEVIII COMPARISONOF YIELDAND MATUROMETER READINGSOF PEASRECOVERED FROM A HARVESTER AND A STATIONARY VINER' Yield (Ib./acre) Stage measured Vined Dry cleaned Washed Graded peas: Size 2 and 3 Size 3 and 4 Size 5 and over Total a

Stationary viner

Harvester

1936 1769 -

1839 1660

519 672 513 1704

409 591 573 1573

-

Maturometer reading Stationary viner

Harvester

212 228

256 227

148 216 290

156 225 309

Data from Mitchell and Casimir (1957-1958).

VIII show that the yield from the harvester was lower, but much more elaborate sampling of a crop would be needed to establish the significance of such a difference. The maturometer readings showed no evidence of excessive damage by the harvester. This test suggests that pea harvesters of satisfactory performance have been produced. 2. Transport

Vines are loaded in the field, transported to the viner station, unloaded, and fed into viners. I n some cases, vines are unloaded in the viner station yard and moved to the viners by a tractor fitted with a fork lift. The latter method gives a continuous supply to the viners, but it results in a greater lapse of time between cutting and vining. Maturation continues on cut vines, and: if the bulk of vines is large, selfheating occurs and undesirable changes are accelerated. Gowen (1928) found that peas held on the vines for 30 hr. were tougher than those canned immediately, but that this delay had less effect than holding peas for 2 hr. after shelling. Vined peas should be processed without delay, and delivery to the factory should not occupy more than 1 hr. under most conditions. Mitchell and Casimir (1957-1958) tested vined peas stored under cool cannery conditions and found that maturity as measured by the maturometer changed from 207 to 238 in 24 hr. During the first 4 hr., the change was small but beyond that time the aging process proceeded at a n accelerated rate and was accompanied by the development

PRESERVATION O F G R E E N PEAS

127

of off-flavor. Gowen (1928) found a progressive toughening in peas which had been held 2 hr. and longer at 95 to 98OF., while 4 hr. holding resulted in definite off-flavor. Nielsen et al. (1943) found that vined peas held at 76OF. became completely unfit for consumption after 12 hr. Bacterial numbers increased rapidly at 76OF. but slowly at 50°F. They concluded that peas should be packed as soon as possible, but if delay were unavoidable they should be iced to a temperature not higher than 50°F. Talburt and Legault (1950a) found that peas kept at 80°F. developed a detectable off-flavor after 2 hr. The off-flavor increased with longer holding and became objectionable in peas held 4% hr. These workers used bruised hand-shelled peas, machine-shelled peas, washed and unwashed, and machine-shelled peas dipped in 5% sodium propionate to control bacterial growth. The samples were stored under aerobic and anaerobic conditions at 40 to 45OF. and at 75 to 80°F. Talburt and Legault (1950a) concluded that off-flavors were caused by abnormal metabolism resulting from bruising of the peas. I n further experiments simulating commercial conditions, they observed that peas held at 32OF. in iced water had a moderate off-flavor after 8 hr. and showed little change with additional delay up to 32 hr. There was little evidence that immersion in iced water resulted in greater loss of organoleptic quality than holding in air at 32OF. They concluded that the maximum delay from vining to blanching without loss of quality is 1 hr. Peas are transported in lug boxes holding about 40 lb. The lugs may be handled individually or palletized to reduce time and labor demand. The bulk of 40 lb. of peas is small enough for heat of respiration to dissipate without serious temperature rise. When peas are transported for long distances heat dissipation from the load may be assisted by use of dunnage to separate the boxes. Bulk handling in large bins of about 1500 lb. capacity has been used (Anonymous, 1957b). Haas (1956) measured temperature changes in peas stored in perforated containers of 40-kg. capacity and found increases after 12 hr. of 6OC. in the upper layer and 13OC. in the center from initial values of 16 to 18OC. When delay in transport is unavoidable, peas should be kept cool; the simplest method is to add ice to the top of the lug boxes. I n more elaborate systems, the peas with ice and water are transported in special tanks. Peas may be cooled by washing prior to icing, but this operation requires additional machinery and water, which may present problems of supply and maintenance at the viner station. Water from melting ice is stated by Tressler and Evers (1957) to cause leaching and flavor

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deterioration. Iced peas absorb water, and the percentage of skins and splits increases markedly, particularly when peas are overmature.

B. FACTORY I . Cleaning Vined peas require thorough cleaning to remove adherent juice, damaged vines and pods, and extraneous material including earth and weed seeds. Screens are used to separate material larger and smaller than the working range of pea sizes. Air blast eliminates chaff; water softens and removes dirt, floats off light material, and by differential flow separates stones from peas. Further cleaning takes place during froth flotation, in the blancher, and on inspection belts. The viner juice still adherent to peas after cold-water treatment is removed by the hot water in the blancher. Steam-blanching is not as efficient in this respect, and off -flavor in steam-blanched peas has been eliminated by use of a preblanch wash with warm water containing a suitable detergent (Holmquist et al., 1954). The flotation cleaner developed by Veldhuis and Neubert (1944) to separate nightshade berries from peas uses an air-water-oil emulsion. Differential wetting of the berries and peas results in adherence of air bubbles to the berries which are then separated by flotation. In commercial operation, this equipment was efficient in removing foreign materials other than berries. In practice all peas may be passed through the flotation cleaner, or the smaller size grades only may be treated. Wash water must be essentially free from suspended matter, organic matter, and bacteria, and have a low content of dissolved solids (Parker, 1948). To comply with these conditions it may be necessary to soften or purify the water. The demand for large amounts of water in the processing of peas suggested the reuse of water for more than one cannery operation. Mercer and York (1953) recommended that water from selected later stages in the processing line be used in earlier stages in a counter-flow type of system until it served up to four uses. Bacteria are controlled by water chlorination to a level of 0.1 p.p.m. above the break point after each reuse. Reuse water is cooled, when necessary, by the addition of fresh water. It is important that peas emerging from the blancher be sprayed with fresh water before they are flumed in water intended for reuse to avoid contamination and heating of the flume water. The efficiency of a cleaning process is difficult to measure by physical or chemical means, but it is possible to gauge the effect by bacteriological examination of the product at different stages. The level of

PRESERVATION O F GREEN PEAS

129

contamination is a guide to the amount of soil which remains and the cleanliness of the processing equipment. Bohrer (1955) considered that soil is the main source of bacteria entering a cannery and gave results of examination of peas from different stages in a processing line. A population of 2,300,000 per milliliter of peas from the viner was reduced to 45,000 in the washer, and at the blancher the numbers had risen to 80,000. Blanching and associated washing effected a reduction to 45,000 and a figure of 50,000 was noted at the filler. Bohrer pointed out that bacterial numbers were reduced at each washing, and passage through intervening equipment resulted in increase. Only small numbers of heat-resistant organisms capable of causing flat-sour spoilage were found in vined peas. Nevertheless, these were sufficient to establish points of contamination in the factory. Blancher water was found to contain significant numbers of flat-sour organisms. These were removed from the peas in a rod washer fitted with sprays. If washing were omitted or peas were washed by fluming alone, contamination in the final pack was sufficient to give 100% spoilage when suitably incubated. Gillespy (1948) studied the reduction in bacterial contamination by different washing methods and concluded that spray-washing on a grating was the most efficient. Fluming was shown to be useful for removing heavy dirt but should be followed by a spray wash. Bohrer (1955) found that pronounced reduction in bacterial numbers resulted from mechanical agitation such as that encountered in tortuous flumes equipped with water sprays, or when peas were tumbled in rod washers or by strong sprays or paddles. 2. Size Grading

Within any crop of peas, it is generally true that the older peas are the larger and that size grade and maturity are therefore closely related in the commercial maturity range. Hence, size grading is used to separate peas into different maturity classes. Packaging of material of uniform size is an advantage because it enhances the appearance of the pack. Moreover it is often desirable to vary details of processing with size grade to obtain the highest possible quality from the raw product. Size grading involves the use of a series of screens of different sizes. The diameters of the holes in a standard set of screens increase in steps of 1/32 in. from %/32in. to 1452 in. Peas passing through the smallest screen are known as size 1, and those passing successively larger screens in. screen are as sizes 2 to 6, respectively, while peas retained by termed size 7. The orientation of a pea frequently determines whether or not it

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will pass through a standard aperture in a size grader, and consequently rotation of the peas is desirable during grading. Furthermore peas are not rigid bodies and slight changes in shape due to the weight of a mass of peas in the grader may affect grade distribution. These considerations show that adequate length of screen is required for correct presentation and that the grader loading should be kept constant. The screens of the cylindrical grader constitute a rotating perforated cylinder which has its axis sloping downwards towards the discharge end. It consists of a series of sections of different screen size, the largest peas being discharged from the end of the grader. A variant of the cylindrical grader is the twin-reel grader in which two parallel cylinders of smaller diameter are used. In some makes of grader, the reel cross section has an indented outline to form what is known as a clover leaf. The concentric grader and the hydrogeared grader are similar in principle to the cylindrical type. Glascoff ( 1952) described a commercial shaker grader consisting of a series of flat screens mounted one above the other in a frame which is oscillated to insure movement of the peas. Advantages claimed for this machine as compared with cylindrical graders are that it requires less than one-fifth of the floor area, cleaning time is less, and screens may be replaced by others with larger or smaller holes. Damage to the peas was less than with conventional graders. Lynch et al. (1956-1957) regraded samples of commercial- and laboratory-graded peas and found that on the second grading each size grade was again split into two or three grades, some peas being retained on a larger and some on a smaller screen. Size grades from any single crop constitute maturity groups, and the maturity of the peas may be indicated by the mean value for the size grade. Data by Lynch and Mitchell (1953) are presented graphically in Fig. 6 in which maturometer values at the O.H.T. of each size were plotted against the summed yield up to the mid-point of that size. It can be seen that different quality grades may be obtained from this particular crop harvested at O.H.T. by size grading and recombination of the grades. Sizes 5 and 6 are between 11.2% and 16.4% A.I.S., which range approximates the limits of first quality for canning. Sizes 2, 3, and 4 are mainly less than 11% A.I.S., and size grade 7 more than 16% and are outside the maturity range defined as first quality. Lynch and Mitchell (1945-1955) found that large- and smallseeded varieties, when compared at the same crop maturity, had different maturities for corresponding size grades. Each size grade of the large-seeded variety was similar in maturity to one size grade lower in

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PRESERVATION O F GREEN PEAS

the small-seeded variety. Crops grown under favorable conditions were also found to have peas larger at the same mat,urity than less favored crops. Maturity variation between samples of the same size grade obtained from a number of crops delivered at a factory was demonstrated by Lynch and Mitchell (1950). Their graph for size 5 is reproduced

5 000

;4 0 0 0 K

u

< m

2

n _I

-> 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

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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

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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

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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.

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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

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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

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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.

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Burton, L. V. 1938. Quality separation by difference of density. Food In&. 10, 6. Caldwell, J. S., Culpepper, C. W., Ezell, B. D., Wilcox, M. S., and Hutchins, M. C. 1946. The dehydration of peas. Canner 103(13), 13; (14), 30; (15), 20. Cameron, E. J., Pilcher, R. W., and Clifcorn, L. E. 1949. Nutrient retention during canned food production. Am. J . Public Health 39, 756. Campbell, H. 1942. Temperature and tenderometer. Western Canner and Packer 34, 39. Candee, F. W., and Boggs, M. M. 1941. Measuring texture of frozen peas. Western Canner and Packer 33, 44. Cheldelin, V. H., and Williams, R. J. 1942. The B vitamin content of foods. Studies on the vitamin content of tissues 11. Texas Univ. Publ. No. 4237. Chitre, R. G., Williams, J. N. Jr., and Elvehjem, C. A. 1950. Nutritive value of canned foods. XLII. A study of the protein value of young and mature peas (Pisum scrtiuum). J . Nutrition 42, 207. Christel, W. F. 1938. Texturemeter, a new device for measuring the texture of peas. Canning Trade 60 (10) , 34. Clifcorn, L. E., and Heberlein, D. G. 1943. Thiamine retention in canned vegetables. Canning Age 24, 304. Crosby, M. W., Fickle, B. E., Andreassen, E. G., Fenton, F., Harris, K. W., and Burgoin, A. M. 1953. Vitamin retention and palatability of certain fresh and frozen vegetables in large-scale food service. Cornell Univ., Agr. Expt. Sta. Bull. No. 891. Danielson, C. E. 1949. Seed globulins of the Gramineae and Leguminosae. Biochem. J . 44, 387. Danielson, C. E. 1956. Starch formation in ripening pea seeds. Physiol. Plantarum 9, 212. Davis, E. G. 1957. Internal corrosion of food cans. Food Preseru. Quart. 17, 64. Decker, R. W., Yeatman, J. N., Kramer, A,, and Sidwell, A. P. 1957. Modifications of the shear press for electrical indicating and recording. Food Technol. 11, 343. Demaree, K. D. 1953. Personal communication. Diehl, H. C., and Berry, J. A. 1933. Relation of scalding practice and storage temperature to quality retention in frozen pack peas. Proc. Am. SOC.Hort. Sci. 30, 496. Dietrich, W. C., Lindquist, F. E., Bohart, G. S., Morris, H. J., and Nutting, M. D. 1955. Effect of degree of enzyme inactivation and storage temperature on quality retention in frozen peas. Food Research 20,480. Dietrich, W. C., Lindquist, F. E., Miers, J. C., Bohart, G. S., Neumann, H. J., and Talburt, W. F. 1957. The time-temperature tolerance of frozen foods. IV. Objective tests to measure adverse changes in frozen vegetables. Food Technol. 11, 109. Doesburg, J. J., and Grevers, G. 1952. Metingen van de hardheid van verse en geconserveerde tuinbouwproducten. Conserua (The Hague) 1( 5 ) , 150. Elehwany, N., and Kramer, A. 1956. A quick test for measuring average pea maturity, Canner & Freezer, 123, 17. Engel, R. W. 1943. The choline content of animal and plant products. J . Nutrition 25, 441. Farrer, K. T. H. 1955. The thermal destruction of vitamin B, in foods. Advances in Food Research 6, 257. Fellers, C. R., and Stepat, W. 1936. Effect of shipping, freezing and canning on the ascorbic acid (vitamin C) content of peas. Proc. Am. SOC. Hort. Sci. 33, 627.

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Fenn, L. S. 1942. Specific gravity of canned peas as a n index of maturity. Canner 94(17), 18. Fenton, F., and Tressler, D. K. 1938a. Losses of vitamin C during commercial freezing, defrosting and cooking of frosted peas. Food Research 3, 409. Fenton, F., and Tressler, D. K. 1938b. Losses of vitamin C during the cooking of certain vegetables. J . Home Econ. 30, 717. Fenton, F., Tressler, D. K., and King, C. G. 1936. Losses of vitamin C during cooking of peas. I. Nutrition 12, 285. Fitzgerald, G. A., and Fellers, C. R. 1938. Carotene and ascorbic acid content of fresh market and commercially frozen fruits and vegetables. Food Research 3, 109. Flynn, L. M., Williams, V. B., and Hogan, A. G. 1949. Cracked ice and preservation of stored fruits and vegetables. Food Research 14, 231. Fowler, H. D. 1957. The changes i n orthophosphate in relation to phytin formation in maturing peas. I . Sci. Food Agr. 8,333. Frakas, D. F., Goldblith, S. A., and Proctor, B. E. 1956. Stopping storage off-flavors by curbing peroxidase. Food Eng. 28, 52. Freed, M., Brenner, S., and Wodicka, V. 0. 1949. Prediction of thiamine and ascorbic acid stability in stored canned foods. Food Technot. 3, 184. Gillespy, T. G. 1948. Significance and control of initial infection in canned vegetables. Ann. Rept. Fruit Vegetable Preseru, Research Sta., Campden. Uniu. Bristol. p. 25. Glascoff, W. G. 1952. Shaker improves size grading. Food Eng. 24, 99. Gowen, P. L. 1928. Effect on quality of holding shelled peas. Canner 66 (Conv. No.), 112. Graham, R. W., and Evans, G. 1957. Standardization and operation of tenderometers. Food Manuf. 32, 224. Greenwood, D. A,, Kraybill, H. R., Feaster, J. F., and Jackson, J. M. 1944. Vitamin retention in processed meat. Ind. Eng. Chem. 36, 922. Guerrant, N. B., and OHara, M. B. 1953. Vitamin retention in peas and lima beans after blanching, freezing, processing in tin and glass, after storage, and after cooking. Food Technol. 7 , 473. Guerrant, N. B., Vavich, M. G., Fardig, 0. B., Ellenberger, H. A., Stern, R. M., and Coonen, N. H. 1947. Effect of duration and temperature of blanch on vitamin retention by certain vegetables. Ind. Eng. Chem. 39, 1000. Guerrant, N. B., Fardig, 0. B., Vavich, M. G., and Ellenberger, H. E. 1948. Nutritive value of canned food-influence of temperature and time of storage on vitamin content. Ind. Eng. Chem. 40, 2258. Gutschmidt, J. 1953. Ein Beitrag zur Bestimmung des Reifegrades gruner Erbsen mit Hilfe des Texturemeters. Ind. Obst- u. Gemiiseuerwert. 38, 389. Guyer, R. B., and Holmquist, J. W. 1954. Enzyme regeneration in high temperatureshort time sterilized canned foods. Food Technol. 8, 547. Haas, W. 1956. Qualitatsverschlechterung von griinen Erbsen zwischen Ernte und Verarbeitung und ihre Ursachen. Ind. Obst- u. Gemiiseuerwert. 41, 179. Hard, M. M., and Ross, E. 1956. Dielectric scalding of spinach, peas and snap beans, for freezing preservation. Food Technol. 10, 241. Harris, P. L., Quaife, M. L., and Swanson, W. J. 1950. Vitamin E content of foods. J . Nutrition 40, 367. Heberlein, D. G., Ptak, L. R., Medoff, S., and Clifcorn, L. E. 1950. Quality and nutritive value of peas as affected by blanching. Food Technol. 4, 109.

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Hilbert, G. E., and MacMasters, M. M. 1946. Pea starch, a starch of high amylose content. I. niol. Chem. 162, 229. Hinman, W. F., Brush, M. K., and Halliday, E. G. 1945. The nutritive value of canned foods. VII. Effect of small-scale preparation on the ascorbic acid, thiamine and riboflavine content of commercially-canned vegetables. J . Am. Dietet. Assac. 21, 7. Holmquist, J. W., Clifcorn, L. E., Schmidt, C. F., and Ritchell, E. C. 1954. Steam blanching of peas. Food Technol. 8,437. Holmquist, J. W., Clifcorn, L. E., Heberlein, D. G., Schmidt, C. F., and Ritchell, E. C. 1955. Experiments reveal benefits of steam blanching of peas. Food Eng. 27, 105. Horner, G. 19361937. The losses of soluble solids in the blanching of vegetables. Ann. Rept. Fruit Vegetable Preserv. Research Sta., Campden. Uniu. Bristol. p. 37. Horner, G. 1939. The effect of cooking and canning on the mineral constituents of certain vegetables. J . SOC.Chem. Ind. (London) 58, 86. way of preservHoward, L. B., and Campbell, H. 1946. Dehydrofreezing-new ing foods. Food In&. 18, 674. Ingalls, R., Brenner, W. D., Tobey, H. L., Plummer, J., Bennett, B. B., and Paul, P. 1950. The nutritive value of canned foods. IV. Changes in thiamine content of vegetables during storing prior to canning. Food Technol. 4, 264. Ives, M., Pollard, A. E., Elvehjem, C. A., and Strong, F. M. 1946. The nutritive value of canned foods. XVII. Pyridoxine, biotin and “folic acid.” J . Nutrition 31, 347. Jenkins, R. R., Tressler, D. K., and Fitzgerald, G. A. 1938. Vitamin C content of vegetables. VIII. Frozen peas. Food Research 3, 133. Jodidi, S. L. 1937. Maturity test of peas. J . Franklin Inst. 223, 593. Joslyn, M. A. 1946. Enzyme activity index of quality in frozen vegetables. Food In&. 18, 1204. Joslyn, M. A., and Cruess, W. V. 1929. Freezing storage of fruits and vegetables for retail distribution in paraffined paper containers. Part 2. Fruit Products J . 8, 9. Joslyn, M. A., and Mackinney, G. 1938. Rate of conversion of chlorophyll to pheophytin. J . A m . Chem. SOC. 60, 1132. Kaniuga, Z. 1952. New method of estimating the degree of maturity of green peas for canning (based on the 1,000-seed weight). Pmemysl Rolny i Spoiywczy 6, 307; Food Sci. Abstr. 26, 2512 (1954). Katz, Y. H. 1952. The relationship between heat unit accumulation and the planting and harvesting of canning peas. Agron. J . 44, 74. Kelley, L., Bennett, B. B., Rafferty, J. P., and Paul, P. 1950. The nutritive value of canned foods. V. Changes in carotene contents of vegetables during storage prior t o canning. Food Technol. 4, 269. Kertesz, Z. I. 1930. The chemical changes in peas after picking. Plant Physiol. 5, 399; Chem. Abstr. 24, 5390. Kertesz, Z. I. 1933. Inactivating the respiration of cannery peas by heat. Canner 76(11), 7. Kertesz, Z. I. 1934. New objective method to determine maturity of canned peas. Food In&. 6, 168. Kertesz, Z. I. 1935. The chemical determination of quality of canned green peas. N.Y. State Agr. Expt. Sta., (Geneva, N.Y.) Bull. No. 233. Kramer, A. 1945. The nutritive value of canned foods. VIII. Distribution of the

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Proctor, B. E., Davison, S., Maleclti, G. J., and Welch, M. 1955. A recording straingage denture tenderornetrr for foods. I. Instrunlent evaluation and initial tests. Food Technol. 9, 471. Proctor, B. E., Davison, S., and Brady, A. L. 1956. A recording strain gage denture tenderometer for foods. 11. Studies on the masticatory force and motion and the force-penetration relationship. Pood Technol. 10, 327. Raacke, I. 1). 1957. Protein synthesis in ripening pea seeds. I. Analysis of whole seeds. Biochem. J. 66, 101. 11. Development of embryos and seed coats. Biochem. J . 66, 110. Roper, E. 1941. A study of certain attitudes of women towards canned fruits and vegetables. (Report to Nat. Canners’ Assoc.) Rowan, K. F., and Turner, I).H. 1957. Physiology of pea fruits. V. Phosphate compounds i n the developing seed. Australian J . Biol. Sci. 10, 414. Sayre, C. B. 1952. Tenderometer grades, yields, and gross returns of peas. Farm Research (N.Y.) 18(3). Sayre, C. B. 1953. Heat units-a measure of maturity of peas. Farm Research (N.Y.) 19(4), 12. Schneider, A. 1951. Untersuchungen iiber die Eignung von Erbsensorten fur Zwecke der Nasskonservierung. I and 11. Ziichter 21, 97, 275. Schneider, A. 1955a. Uber das Garkochen von Trockenspeiseerbsen und dessen exakte Bestimmung mit Hilfe eines modifizierten Texturemeters. Ziichter 25, 181. Schneider, A. 1955b. Ober den Reifeablauf von Gemiiseerbsen und die Bestimmung des optimalen Pflucktermins mit Hilfe des Texturemeters. Ziichter 25, 302. Schopfer, W. H. and Muller, V. 1938. Decomposition of aneurin (vitamin B,) by heat. Compt. rend. SOC. biol. 128, 372. Seaton, H. L., and Huffington, J. M. 1950. The commercial application of the heat unit system of crop control in the canning industry. Continental Can Co. Research Dept. Crop Production Memo. (Presented to Meeting Am. SOC.Hort. Sci. 1950.) Spragg, S. P. 1958. Protein synthesis in the maturing pea seed. (Presented at a symposium on “Maturation and Germination of Peas” Cambridge, 1957.) Abstract in Chem. & Ind. (London) 1958,486. Stark, F. C., and Huflington, J. M. 1956. Raw product quality control. Canner 122(8), 21. Stimson, C. R., Tressler, D. K., and Maynard, L. A. 1939. Carotene (Vitamin A) content of fresh and frosted peas. Food Research 4, 475. Strasburger, L. V. 1933. The chemical characteristics of canned peas. Canner 76(19), 10. Strong, F. M., and Elvehjem, C. A. 1947. Effect of commercial canning operations on the ascorbic acid, thiamine, riboflavine, and niacin contents of vegetables. Ind. Eng. Chem. 39, 985. Talburt, W. F., and Legault, R. R. 1950a. Time lapse gets top blame for shelled pea off-flavor. Food In&. 22, 1021. Talburt, W. F., and Legault, R. R. 1950b. Dehydrofrozen peas. Food Technol. 4, 286. Thompson, M. L., Cunningham, E., and Snell, E. E. 1944. The nutritive value of canned foods. IV. Riboflavin and pantothenic acid. J . Nutrition 28, 123. Todhunter, E. N., and Robbins, R. C. 1941. Ascorbic acid (Vitamin C) content of garden type peas preserved by the frozen pack method. Wash., State Coll. Agr. Expt. Stas. Bull. No. 408.

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Todhiinter, E. N., and Sparling, B. L. 1938. Vitamin values of garden type peas preserved by the frozen-pack method. I. Ascorbic acid (Vitamin C). Food Research 3, 489. Toepfer, E. W., Zook, E. G., Orr, M. L., and Richardson, L. R. 1951. Folic acid content of foods: microbiological assay by standardized methods and compilation of data from the literature. US.Dept. Agr. Agr. Handbook No. 29. Torfason, W. E., Nonnecke, I. L., and Strachan, G. 1956. An evaluation of objective methods for determining the maturity of canning peas. Can. J . Agr. Sci. 36, 247. Trefethen, I., and Fenton, F. 1951. Effects of four cooking pressures on commercially frozen peas: cooking time, palatability, ascorbic acid, folic acid, thiamine, and riboflavin. Food Research 16, 342. Tressler, D. K., and Evers, C. F. 1957. “The Freezing Preservation of Foods,” 3rd ed. Avi Publ. Co., Westport, Connecticut. Turner, D. H., and Turner, J. F. 1957a. Physiology of pea fruits. 111. Changes in starch and starch phosphorylase i n the developing seed. Australian J . Biol. Sci. 10, 302. Turner, D. H., and Turner, J. F. 1957b. Personal communication. Turner, J. F., Turner, D. H., and Lee, J. B. 1957a. Physiology of pea fruits. IV. Changes in sugars in the developing seed. Australian J . Biol. Sci. 10, 407. Turner, J. F., Turner, D. H., and Lee, J. B. 1957b. Personal communication. Van Duyne, F. O., Chase, J. T., Owen, R. D., and Fanska, J. R. 1948. Effect of certain home practices on the riboflavin content of cabbage, peas, snap beans and spinach. Food Research 13, 162. Van Duyne, F. O., Wolfe, J. C., and Owen, R. D. 1950. Retention of riboflavin in vegetables preserved by freezing. Food Research 15, 53. Veldhuis, M. K., and Neubert, A. M. 1944. Cleaning vined canning peas by froth flotation-removal of nightshade. Western Canner and Packer 36, ( 6 ) , 18. Wagenknecht, A. C. 1957a. Occurrence of plasmalogens in lipids of green peas. Science 126, 1288. Wagenknecht, A. C. 1957b. The lipids of green peas. J . A m . Oil Chemists’ Soc. 34, 509. Wagenknecht, A. C., and Lee, F. A. 1958. Enzyme action and off-flavor in frozen peas. Food Research 23, 25. Wagenknecht, A. C., Lee, F. A., and Boyle, F. P. 1952. The loss of chlorophyll in green peas during frozen storage and analysis. Food Research 17, 343. Wagner, J. R., Strong, F. M., and Elvehjem, C. A. 1947a. Effect of commercial canning operations on the oscorbic acid, thiamine, riboflavin and niacin contents of vegetables. Znd. Eng. Chem. 39, 985. Wagner, J. R., Strong, F. M., and Elvehjem, C. A. 1947b. Effect of blanching on the retention of ascorbic acid, thiamine and niacin in vegetables. I n d . Eng. Chem. 39, 990. Walls, E. P. 1936. Grading and canning tests for raw peas. Canner 84(1), 7. Walls, E. P., and Hunter, H. A. 1938. Grading raw peas for quality; 1937 Investigations. Canner 86(10), 14; ( l l ) , 20. Walls, E. P., Kemp, W. B., and Stier, H. C. 1940. Determining the grade of peas from the raw product. Canner 90(15), 28. Zscheile, F. P., Beadle, B. W., and Kraybill, H. R. 1943. Carotene content of fresh and frozen vegetables. Food Research 8, 299.

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PRINCIPLES AND INSTRUMENTATION FOR THE PHYSICAL MEASUREMENT OF FOOD QUALITY WITH SPECIAL REFERENCE TO FRUIT AND VEGETABLE PRODUCTS* BY AMIHUDKRAMERAND B. A. TWIGG University of Maryland, College Park, Maryland

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

C. Instrumentation ................................................. D. Physical-Chemical Methods. ...................................... E. Correlated Methods.. .......................................... V. Flavor ......................................................... A, Taste .............................................. ....... B. Odor ........................................................... ... ........................... C. Correlated Measurements. VI. Summary and Conclusions.. .................................... A. Current Status ................................................... B. Terminology Standardization. ......................... C. Direct or Correlated Methods.. ........................ References ......................................................... 11. General Principles. ................................................. A. Specification of the Quality Factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reference to Human Evaluation.. ...................... C. Reproducibility of Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Measurement or Control.. ........... ............... E. Direct or Correlated Measurements. . . ............... 111. Appearance Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Color and Gloss.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Viscosity and Consistency. ...... ..... C. Size and Shape. . . . . . . . . . ...... .... ..... D. Defects ........................................................ IV. Kinesthetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Principles of Measurement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I

.

Page 153 153 154

156 '157 157 158 175 197 198 198

208

209 210

.

213 213 213 214

1. INTRODUCTION

Historically the measurement of quality in food parallels the history of food production. From earliest beginnings every food manufacturer, handler, and consumer evaluated the food he used. Thus in this long history of quality evaluation, the various attributes of quality were

* Miscellaneous Publication No. 332, Contribution No. 2953 of the Maryland Agricultural Experiment Station, Department of Horticulture. 153

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measured and decisions made on the basis of sensory, i.e., human evaluation. Although isolated instances of the development of instrumental methods for measuring food quality have appeared during the last century or even earlier, it is only recently that such methods have become available in sufficient numbers so that an attempt can now be made to organize them as a distinct discipline (Blanck, 1955). It is the presentation of such a discipline, which is to replace the unaided human monitor of qualit.y, that is being attempted here. In some areas, available procedures are still meager and incomplete, so that all that can be reported currently may be partial methods that are no more than aids to the human observer of quality. In other areas, progress is so rapid and current that a definitive presentation at this time would be hopeless. There are areas, however, particularly those that can be classified in the categories of appearance and texture, in which solid achievements have been accomplished, and these stand ready for use by the food industry. Since food quality is ultimately evaluated by the human consumer, it appears logical to organize this material in accordance with the human senses involved in this evaluation. Thus, quality measurements may be classified into categories of: appearance as observed by the eye; kinesthetics as felt by hand or mouth; and flavor as tasted and smelled by the mouth and nose. The ear is not ordinarily involved except in such rare instances as the crunchiness of breakfast cereals and some confections. II. GENERAL PRINCIPLES

In the evaluation of the accuracy and usefulness of an instrumental method, certain principles should be recognized, otherwise the methods themselves may lead to gross errors which could be far greater than any incurred through human evaluation (Gamer, 1951a). A. SPECIFICATION OF

THE

QUALITYFACTOR

Although the ultimate goal of this discipline may be the establishment of an instrument which would provide a measurement of over-all quality, it must be recognized that over-all quality is essentially a composite of several distinct attributes. The more completely and precisely that the specific attribute is defined, the more probable is the attainment of a satisfactory instrumental method for its measurement. B. REFERENCE TO HUMAN EVALUATION Here we are faced with a paradox. In attempting to replace human evaluation, we have no recourse but to use human evaluation as the

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ultimate criterion of the accuracy of our proposed instrumental method. The tentative solution lies in the use of validated sensory panels working under controlled conditions. The degree of correlation obtained between such panel scores and the instrumental method may serve to indicate the accuracy of the proposed instrumental procedure, provided that certain conditions are met; namely, that the samples submitted to panel and instrument are essentially identical; that they cover but do not exceed the commercial range of quality for the particular quality attribute; that the number of samples is adequate; that the samples cover all likely conditions and variations, If these conditions are met, a correlation of 0.90 or higher indicates that the instrumental method is a sufficiently accurate measure of the quality attribute, since variations in the instrumental values would thus account for more than 80% of the quality variations found by the panel. Correlation coefficients of 0.80 to 0.89 may be sufficiently high to justify the use of an instrumental method, although in such instances only two-thirds to four-fifths of the variations found by the panel are accounted for by the instrumental method, thus indicating that some factor noted by the panel is not influencing the instrumental values. Correlation coefficients of less than 0.80 do not show sufficiently good agreement between the panel results and the instrumental procedure, so that the replacement of human evaluation with the particular instrumental method is not justified. A statistically significant correlation per se is not sufficient to validate the use of an instrumental method, since the significance of a correlation coefficient is related to sample numbers. Thus for example, if a correlation coefficient between panel scores and a particular instrumental method is based on 200 samples, a correlation coefkient of 0.138 is sufficient to indicate statistical significance, meaning that there is a definite relationship between the two sets of values. However, in such a case, less than one-tenth of the variation in quality as measured by the panel would be accounted for by the instrumental method, while nine-tenths of the variations would be due to factors not so measured (Kramer, 1952). These same correlation and regression techniques may be used to establish scales of values by which the results obtained with the instruments may be interpreted in terms of quality levels (Tarver and Schenck, 1958).

C. REPRODUCIBILITY OF RESULTS If an instrument is to be used for establishing quality level, it must be precise, i.e., results by the instrumental method should be as nearly

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as possible identical for identical samples. Also replicate instruments should provide similar results on identical samples. In this respect, potentially at least, instrumental methods may be decidedly more satisfactory than sensory methods, since human observers do tend to provide different results from one individual to another, or with time, or conditions of the test or test material. Instrument precision is all too often taken for granted. As with any manufactured or natural product, there are variations among individual instruments. It is, therefore, absolutely essential that any instrument proposed for quality evaluation be subject to calibration by means of generally accepted methods. With proper calibration, different individual units of a certain type of instrument can be made to provide essentially similar results at all times (Little et al., 1958). Another problem limiting precision of instrumental, as well as other measurements, is the sampling procedure by which the test material is obtained. Since 100% inspection, particularly in the case of destructive testing, is impractical, selection of that fraction of the lot upon which the measurement is to be made requires careful consideration. Statistical methods for determining minimal sample size for maximum precision are available (Kramer, 1957a).

D. MEASUREMENT OR CONTROL An instrument can be made merely to measure a particular quality attribute. In such instances, the value obtained may be utilized to ascertain the quality level of the lot, or if obtained on the production line, it may be used as an indicator for adjustment. By means of control valves, relays, and the like, the use of the same instrument may be extended to control the quality of the product continuously. For example, a number of containers may be removed from the production line at regular intervals and weighed. The results may be used as part of the quality grade of the product or to determine whether any adjustment in the filler is required. On the other hand, an automatic check weigher can be inserted into the production line which will automatically sort out underweight or overweight containers (Heid, 1958). Thus in effect, an instrumental procedure, once found satisfactory for the mere measurement of a quality attribute, may serve as the precursor to a more elaborate instrument which not only measures the quality level, but also maintains production within prescribed quality control limits by automatic sorting or adjustment. An instrumental procedure may therefore be considered as a first step in the automation of a process.

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E. DIRECTOR CORRELATED MEASUREMENTS When an attribute of quality is clearly defined, and the physical, chemical, anatomical, or physiological basis clearly understood, it is possible and certainly desirable to utilize a n instrument which is capable of measuring the same attribute directly. If, for example, a measure of chewiness is needed for lima beans, an instrument capable of measuring compression and shear forces, such as those exerted by the teeth, can be expected to be more reliable under a variety of conditions than an instrument measuring the lightness of the lima beans, although it can be shown that lima beans become more chewy as they mature and become lighter in color. Under different growing conditions, however, and particularly when different varieties are used, the color test may lead to gross errors, while the compression-shear test remains reliable (Twigg, 1958). Ill. APPEARANCE FACTORS

Factors of quality included in this group are those evaluated with the eye; hence, they are the first to be noticed by the consumer. It is usually on the basis of the impression gained from the appearance of the product alone that it is accepted or rejected. Since this decision is made before the consumer has the opportunity of preparing the food for consumption, these appearance factors are therefore undoubtedly most important in the case of the first purchase, whereas other factors may be more important for repeat purchases. In the case of packaged foods, the container and especially the label are extremely important in their contribution to the appearance of the product. Other factors that may be included in the appearance category are such items as size, shape, pattern, wholeness, and consistency. Most of these may be measured easily by relatively simple devices such as scales, screens, micrometers, planimeters, sedimentation tests, consistometers, or displacement in liquid media. Defects of various kinds also fall into this category. Although they are also usually rather easily measured, they are occasionally more difficult to determine. For example, there is the problem of whether a particular discolored spot is dark enough or large enough to be called a blemish or whether two small light spots are only as unacceptable as one darker or larger spot, etc. Attempting to establish limits for such minutely detailed factors may result in unwieldy and lengthy procedures and specifications. Another approach is through the employment of photographs, models, and drawings such as those being used and developed by the

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A M I H U D K R A M E R A N D B. A . TWIGG

standardization division of the Agricultural Marketing Service, U.S. Department of Agriculture (US. Dept. Agr.) as visual aids in grading for defects, blemishes, and “character” factors.

A. COLORAND GLOSS Color is an appearance property attributable to the spectral distribution of light. Glossiness, transparency, haziness, and turbidity are properties of materials attributable to the geometric manner in which light is reflected and transmitted. Gloss is an appearance characteristic that has been almost entirely overlooked in the objective evaluation of foods, although it is obvious that this factor which is usually termed “finish” in apples, and “shine,” or “polish” in other products is an evident and important factor of appearance in products such as raw apples, tomatoes, and strawberries. It is entirely possible that even one lot of peas may appear more desirable than another because of a difference in gloss. This factor is being measured in the paint industry by the goniophotometer and a gloss meter. Thus, instruments, in their present or modified form, are available which are capable of measuring the gloss factor. However, the work of applying these instruments to the measurement of the gIoss factor in specific foods remains to be done. Recent advances in the physics and engineering of. color measurement have adequately solved the problem of color measurement of foods, per se. This is especially true when color measurement is not complicated by the presence of other appearance factors such as gloss, mottling, and texture. It is now possible to measure the color of foods as well as other items precisely, in terms of internationally accepted units (Judd, 1950). These methods, however, involve rather expensive and elaborate instruments and/or a considerable amount of calculation before the color specifications of the test item may be established. Furthermore, the specification of the color does not in itself indicate consumer preference for that item. Thus, for the practical application of color measurements for use in quality control, or grades and standards, there remain two fruitful areas of research, work on which is still in the beginning stage. These two areas of research are: ( a ) correlation of color specifications to consumer preference, and ( b ) development of the simplest procedures and instrumentation, covering only the color dimensions of interest to the consumer for any given product (Kramer, 1954). 1 . Definition Physically, color is a characteristic of light, measurable in terms of intensity (radiant energy) and wavelength. It arises from the presence

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of light in greater intensities at some wavelengths than at others. Psychologically, it is further limited to the band of the spectrum from 380 to 770 millimicrons (mp), since the human eye is practically insensible to other wavelengths of radiant energy. Thus the phenomenon of color is said to be psychophysical, and is defined as consisting of the characteristics of light . . . light being that aspect of radiant energy of which a h u m n observer is aware through the visual sensations which arise from the stimulation of the retina of the eye [Optical Society of America (O.S.A.), 19441. Color perception is further limited by the light source. Obviously, no color may be detected by the eye, or by an instrument, in the dark, i.e., in the absence of a light source emitting radiant energy in the visual range of the spectrum. A common experience is to purchase some item in a dimly lighted store, only to find that the color of the item is substantially different when viewed in broad daylight. Visual inspection of food items has occasionally given erroneous results because the light source did not emit sufficient radiant energy at the critical wavelengths. To correct this situation, three standard illuminants have been established by the International Commission on Illumination [Commission International de L'Eclairage (C.I.E.), 19311. These are: Illuminant A-incandescent lamp (2854OK.l) ; Illuminant B-noon sunlight (5000°K.1) ; Illuminant C-cloudy daylight, or north light (6800OK.l). Another illustration of the importance of the light source is the existence of metameric pairs. This refers to a pair of objects which may appear to have the same color when irradiated at certain wavelengths, but which appear of distinctly different color when viewed under a light consisting of different wavelengths. It is therefore important when constructing standard color cards, or models, to have the standards nonmetarneric in relation to the product which is to be tested or to do the color comparison only under carefully standardized light. Light may be reflected, transmitted, absorbed, or refracted by the object being illuminated. Reflected light refers to that part of the radiant energy emitted by the light source which reflects from the surface of the illuminated object. Transmitted light passes through the object, while the light which is refracted is also transmitted; however, the angle of transmission is different from the angle of incidence (Fig. I ) . When practically all radiant energy in the visible spectrum is reflected from an opaque surface, the object appears to be white. If light is partially absorbed, more or less equally across the entire visible spectrum, the object appears to be gray. If the absorption is practically comDegrees Kelvin.

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AMIHUD KRAMER A N D B. A. TWIGG

plete, the result is a black object (Fig. 2). This over-all relation of reflection to absorption without regard to specific wavelengths, is termed psychologically lightness, or value. If, however, radiant energy is absorbed at certain wavelengths to a

FIG.1. Paths light may take on striking a substance. I-incident beam; R-reflected light; R’-refracted light; A-absorption i-angle of incidence; r-angle of reflection; r’-angle of refraction.

area;

FIG.2. Reflectance curves of five colored objects-white, black, blue, green, and red.

greater extent than at others, the human observer sees what he popularly thinks of as color, and what is more accurately referred to physically as dominant wavelength, or psychologically as hue. Thus, if the shorter wavelengths of 400 to 500 m p are reflected to a greater extent

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than the other wavelengths, the color is described as blue. Maximum reflection in the medium wavelength range results in a green or yellow impression, while greater reflection at the longer wavelengths of 600 and 700 mp indicate red objects (Fig. 2). The amount of reflection of light at a given wavelength is a third important attribute of color, which in physical terms is called purity, and in psychological terms has been referred to variously as intensity, strength, or chroma.

FIG.3. Geometric diagram of specular and diffuse reflectance,

When light is reflected from a surface evenly at all angles, the impression on the observer is that the product has a flat, or dull finish. This is termed difluse reflectance, as contrasted with specular or directional reflectance, when light is reflected more strongly at a specific angle, or in a beam, as by a mirror which is frequently, but not always, at 4 5 O angle from the incidence of the light. This directional reflectance gives rise to gloss, or sheen, of some materials (Fig. 3). Some of these physical and sensory terms used to denote different color attributes are summarized below: Physical Measurement

Radiant energy Reflectance Dominant wavelength Purity Directional reflectance

Sensory Term Equivalent Light Lightness, value Hue, color Chroma, intensity, strength Gloss, sheen

2. Spect rophotometry The color of any object may be defined comprehensively by measuring the amount of reflection of light from the surface of the object at

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

each wavelength in the visible range of approximately 380 to 770 mp. This determination may be made by the use of a spectrophotometer. For precise results, wavelength bands as narrow as 5 mp should be used. This can be accomplished in such instruments as the Hardy (General Electric) or the Beckman spectrophotometers, which use a prism, or the Bausch and Lomb Spectronic 20, which uses a grating for isolating the spectral bands. Most filter instruments are capable of selecting a less pure wavelength band (about 30 mp) but are much more economical (Kramer, 1958). The light band is directed at the sample, and the light reflected from the sample activates one or more photosensitive cells which transform radiant energy to electrical energy, which is then indicated on some form of a millivoltmeter. If the instrument is a recording spectrophotometer, as the General Electric instrument, the wavelength is changed automatically, and per cent reflectance is recorded continuously for each incident wavelength. Thus, a complete curve of reflection across the entire range of visible wavelengths is obtained. The spectrophotometric curve of reflection characterizes the color of the product. However, in order to transform these data into more meaningful values, additional calculations are needed. These are discussed below.

3 . Tristimulus Colorimetry and the Standard Observer The discussion above indicated that color is three-dimensional (tristimulus). That this is a psychological as well as a physical fact has been well established, since it was found that normal color vision is three-dimensional (Judd, 1950). It thus follows that any color may be described by three numbers, and that a spectrophotometric curve may be reduced to three numbers, which would adequately and accurately describe the color of any substance. A number of tristimulus systems are in use which provide the opportunity for obtaining color values. The standard system, against which other systems should be compared, is the one recommended by the International Committee on Illumination and which is based on the “standard observer,” which is a simulated standard eye, consisting of three primary color filters having exactly specified spectrophotometric curves X , Y , and 2, with X being essentially amber in color; Y , green, and 2, blue. Any spectrophotometric curve of reflectance obtained from the surface of an object can then be integrated in terms of X , Y , and 2 as follows: (1) Determine per cent reflectance at each wavelength band, beginning with 380 mp at intervals of 10 mp up to 770 mp. (2) At each wavelength, multiply per cent reflectance of the test object by the respective values for X , Y , and 2 given in Table I.

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PHYSICAL MEASUREMENT OF FOOD QUALITY

TABLE I REFLECTANCEVALUESFOR THE X , Y , AND Z OF THE STANDARD OBSERVER AT EACH WAVELENGTH FOR ILLUMINANT C, AVERAGEDAYLIGHT Wavelength bl.1

380 90 400 10 20 30 40 450 60 70 80 90 500 10 20 30 40 550 60 70 80 90 600 10 20 30 40 650 60 70 80 90 700 10 20 30 40 750 60 770

Sums yq

xw,

zu,

X

Y

4 19 85 329 1238 2997 3975 3915 3362 2272 1112 363 52 89 576 1523 2785 4282 5880 7322 8417 8984 8949 8325 7070 5309 3693 2349 1361 708 369 171 82 39 19 8 4 2 1 1

-

98041 0.31012

2 9 37 122 262 443 694 1058 1618 2358 340 1 4833 6462 7934 9149 9832 9841 9147 7992 6627 5316 4176 3153 2190 1443 886 504 259 134 62 29 14 6 3 2 1 1 1000000 0.31631

Z

20 89 404 1570 5949 14628 19938 20638 19299 14972 9461 5274 2864 1520 712 388 195 86 39 20 16 10

7 -

2 2

-

-

-

118103 0.37357

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AMIHUD KRAMER A N D B. A. TWIGG

X

FIG.4. The

(z, y ) chromaticity diagram of the C.I.E. system. (The abscissa z is

+ +

the ratio of the tristimulus value X, to the sum of all three (X Y 2).The ordinate y is the ratio of Y to this sum. The parts of the spectrum locus are identified by wavelength in millimicrons. The region bounded by this locus and the straight line joining its extremes represents all chromaticities producible by actual stimuli.)

(3) Total the products for X , Y , and 2 at all wavelength bands. (4)Determine x from: z = ( X )/ ( X + Y 2 ) . (5) Determine y from: y = ( Y ) /( X Y 2 ) . (6) z is then equal to 1 - ( x y ) .

+

+ + +

When using a Hardy (General Electric) recording spectrophotometer, these x, y, and z values may be obtained automatically by the use of an integrator especially designed for this purpose. These x and y values are also known as the chromaticity coordinates which may be plotted o n a chromaticity diagram (Fig. 4), and the color

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of the test object may thus be identified in color space, in terms of officially and internationally recognized color specifications.

4 . Munsell Color System Color identification by means of a recording spectrophotometer requires the use of elaborate and costly instrumentation and is time consuming. Other tristimulus systems have been developed which, although not as precise, are more economic and practical for routine control purposes. Of these systems, the Munsell system of color notations has gained wide usage in the food industry, primarily as the result of the excellent work of Nickerson (1938, 1946) of the U.S. Department of Agriculture. The Munsell system is based on the use of three or four color discs, each of which has been carefully calibrated in terms of the three notations of hue (red, green, etc.), value (lightness or darkness), and White

Purple

Black

FIG.5. Space diagram for the dimensions of the psychological surface-color solid for the Munsell system.

chroma (strength of the color); see Fig. 5 . Each of these terms is expressed on a scale of 10 units, except for chroma which occasionally exceeds 10 for exceptionally strong colors. The dimension of hue is expressed first by the initial of the hue involved, such as 5G which would be a medium green hue. The dimension of value follows as a number over a line, such as 81, which would be a light gray. The dimension of chroma is expressed by a number below the line, such as /3. Thus the notation of 5G8/3 would denote a color about midway between yellow-

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AMIHUD KRAMER A N D B. A. TWIGG

green and blue-green, which is rather light in value and weak in chroma. On the other hand, a notation such as 263/9 would indicate a green which is on the yellow-green side, which is dark in value, and strong in chroma. In the practical use of the Munsell system, three or four overlapping discs are used, two of which cover the range of hue and chroma for the product to be examined, and the remaining disc or two are neutral, that

X

FIG.6, Munsell hue and chroma notations superimposed on a (I,y ) chromaticity diagram. (Munsell notation value equal to 6/. The curved lines radiating from the point (I= 0.310, y = 0.316) define the chromaticities for Munsell renotation hue, and the ovals surrounding this point define the chromaticities for Munsell renotation chromal.)

is, gray discs which provide the adjustment for the dimension of value. These three or four discs are overlapped so that the proportion of each disc which is exposed may be adjusted until the blend of color, obtained by the rapid spinning of the discs, matches the object whose color is being measured. The percentages of each of the three or four discs which is exposed is then recorded, and the results may be interpreted as such, or as a ratio of the two chromatic discs, or these values may be

167

PHYSICAL MEASUREMENT OF FOOD QUALITY

transferred to x, y, z values of the C.I.E. system by calculation, or by the use of chromaticity diagrams on which Munsell notations are superimposed (Billmeyer, 195 1). See Fig. 6. Since X, Y, and 2 vaIues are available in published form for many Munsell notations, these notations may be recalculated in terms of C.I.E. values or x, y, and z, or as is most frequently shown Y, x, and y. For example, four discs are used for a sample of canned green beans, of the Blue Lake variety, and the X , Y , and 2 values are found in reference tables (Glenn and Killian, 1940) and shown in Table 11. TABLE I1 CALCULATION OF C.I.E. COORDINATES FOR A SAMPLEOF CANNEDBEANS FROM MUNSELLNOTATIONS Tabular data for Munscll disc notation __-

GY 5/6 Y 5/6 N 4/ N I/

X (1)

Y (2)

Z (3)

0.1532 0.2177 0.1145 0.1935 0.2022 0.0603 0.1249 0.1280 0.1525 0.0196 0.0200 0.0236

CaIcdations % of disc exposed (4)

0.21 0.42 0.06 0.31

X? (1

+ +

YZ (2 x 4) (3

z 2

x 4)

0.03217 0.04572 0.02404 0.08127 0.08492 0.02533 0.00755 0.00768 0.00939 0.00608 0.00620 0.00732 _

100

x 4)

_

_

~

0.1271 0.1445 0.0661 = 0.3377

Since X Y 2 = 0.337, x = 0.127113377, or 0.376; similarly y = 0.144513377, or 0.428; and z = 1 - (0.3376 0.428), or 0.196. The color of these Blue Lake canned beans may therefore be reported in C.I.E. terns as 0.1445Y, 0.3762, and 0 . 4 2 8 ~To . illustrate the vastness of the chromaticity diagram and the relatively small color differences which are involved in the comparison of different samples of the same food product, C.I.E. values for a sample of Refugee beans, which differ substantially in color from Blue Lake beans, were found to be 0.1758Y, 0.3772, and 0 . 4 3 6 ~(Kramer and Smith, 1946a). The Munsell system of color notations has been of even greater success when used with a disc colorimeter where the angle intensity and type of the light source are controlled, and the lens rotated instead of the color discs (which were left stationary so that the proportion of the different color discs could be changed while the instrument was in action), thus making it possible to match colors in a relatively short time (Nickerson, 1946). Such instruments had been manufactured in limited numbers by Keuffel and Esser, Inc., and Bausch and Lomb, Inc. More

+

~

168

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

recently, Macbeth, Inc., is offering a disc colorimeter with which discs specified by Munsell notations may be used (U.S. Dept. Agr., 1957.) The disc method is not entirely objective since the matching of the color is done by eye.

5 . Hunter Color and Color Diference Meter Hunter ( 1952) developed a tristimulus photoelectric colorimeter which is gaining wide acceptance in the food industry. Its advantage over the spectrophotometric curve method is the great reduction in cost of equipment, its ruggedness, and the rapidity with which meaningful results may be obtained. The instrument consists of three separate circuits and carefully selected filters and photocells, which provide close 100-White

-80

-50

t50

+I00

OiBlack

FIG.7.

Diagram showing the dimensions of the color difference meter, L, a, and b color solid.

approximations of the X , Y , and Z functions of the C.I.E. system. The Hunter Rd (diffuse reflectance) or L (lightness) values are directly comparable to the Y of the C.I.E. system, or value of the Munsell system. The Hunter a values are measures of redness or greenness, and the Hunter b values are measures of yellowness o r blueness (Fig. 7). The a values are functions of X and Y , and b values are functions of 2 and Y . Together, a and b may provide results equivalent to those obtained with the hue and chroma dimensions of the Munsell system. Thus Hunter values may also be converted to z, y, and z values of the C.I.E. system by the use of the following equations: Rd

=

100Y

a = 175~(1.02X- Y)

b = 70fg(Y - 0.8472) (21 20Y) fil = 0.51 (1 20Y)

+

+

PHYSICAL MEASUREMENT O F FOOD QUALITY

169

or when using the L measuring circuit:

L

=

aL = bL =

100 d7 175(1.02X - Y) 70(Y

1/y - 0.8472/Y)

Hunter values can also be converted to Munsell notation by calculations or by interpolation from charts. Francis (1952) developed an attachment by which an average color evaluation may be obtained with materials having a nonuniform color surface.

6 . Abridged Methods Thus far all the color-measuring systems that were discussed are capable of providing all the data necessary for locating the color of any food sample in color space by determining three dimensions of color. In some instances, however, it may be possible to obtain satisfactory estimates of color of some products by the use of less than three measurements if it can be shown that such abridged methods are highly correlated with human evaluation of color quality. One such procedure is a rapid determination of the pigment present in the food, which may be closely related to human evaluation of color (Kramer and Smith, 1946a; Pohle and Mehlenbacker, 1955). This method involves the extraction of the color from the product by the use of one or more solvents, and the measurement of that extract in a spectrophotometer in terms of per cent transmittance of light at a specific wavelength. A translucent solution transmits its own color and absorbs the complementary color. For example, a purple solution of beet juice transmits the purple color but absorbs the complementary green color. Expressed graphically, when the degree of transmission is plotted against wavelength, the lowest per cent transmittance and highest absorption is at the wavelength of 525 mp (green). When the wavelength is increased to about 600 mp (yellow), there is a sharp increase in transmittance and decrease in absorption, and finally when the wavelength is increased to 700 mp (red), practically all the light is transmitted, since the wavelength now approaches the color of the solution itself. In most cases, it is the transmittance of the complementary color that is measured (Kramer and Smith, 1946a). The greater the intensity of a color, i.e., the greater the pigment concentration in the solution, the more completely will the complementary color be absorbed and thus show a lower per cent transmittance of

170

AMIHUD KRAMER A N D B. A. TWIGG

the complementary color, with the colorless solvent serving as reference at 100% transmittance. Thus, the greater the intensity of the pigment, the lower the per cent transmittance. The following procedure can be used: (1) A suitable solvent or combination of solvents should be selected for extracting the desired pigment. ( 2 ) A small representative sample of the product is blended with the selected solvent in a Waring Blendor until a uniform mix is obtained. (3) A dilution of the above mixture is then clarified in a centrifuge (since turbidity interferes with transmittance measurements). (4) The clear extract is decanted and per cent transmittance recorded at the wavelength giving maximum absorption for the pigment. Many color evaluation problems can be solved colorimetrically by matching the liquid system containing the desired constituent in unknown amount with a similar system containing the desired constituent in known amounts (Mellon, 1945). However, errors such as mechanical, optical, reading, dilution, temperature, time, reagent, interfering ions, operator, turbidity, and colloid particles are specific limitations for this method. Comparators of this type may be classified as follows: 1. Standard series type Q. Comparison with solutions (1) Comparator blocks (2) Slide comparators (3) Devices for vertical comparison b. Comparison with glasses 2. Duplication type 3. Dilution type 4. Blanching type

Another type of color comparator is the comparison of the food product with something visually equivalent to the desired, such as a standard. This procedure is used quite frequently by the Agriculture Marketing Service, U.S. Dept. of Agriculture in grading of raw products. An example of this is the colored plastic blocks used in the colorgrading of lima beans (U.S. Dept. Agr., 1955) or of honey (Brice et al., 1956). In order to determine whether abridged methods may be used, tristimulus color data may be submitted to multiple correlations analysis, where the three attributes of color are correlated with panel evaluation. Examples of this procedure are demonstrated in Table I11 (Kramer, 1954), where the tristimulus values of the Hunter instrument are correlated with panel scores.

171

PHYSICAL MEASUREMENT OF FOOD QUALITY

From the data in Table 111, it may therefore be concluded that for frozen lima beans, practically all of the color differences observed by the consumer are covered by a measurement with the L scale, which is directly comparable to the Y of the C.I.E. system. Thus, the consumer is interested in dark lima beans, rather than whether the lima beans are green or yellow green, or the intensity of the greenness. On the other hand, in the case of tomato juice, the L scale is of least importance. In fact, there is no significant difference in the agreement with the consumer panel when only the a and b values are used than when all three values are used. With applesauce we find still another TABLE I11 CORRELATIONS BETWEEN HUNTER COLOR-DIFFERENCE METER VALUESAND PANELSCORES Frozen lima beans

Canned tomato juice

ra

r1.2

r1,z

Hunter L Hunter a Hunter b

-0.836 +O. 552 +O ,635

-0.542 +O ,635 -0.652

+O ,723 +O .604

-

+O. 903 + O . 904

+O .764 +O. 642 f O . 822 $0.908

Rb L. a L. b a. b I,. a. b a

b

+O .842

Canned applesauce 7.1.2

+o ,477

Simple correlations. Multiple correlations.

situation. Apparently, all three values contribute to consumer evaluation, since the multiple correlation involving all three tristimulus values is significantly higher than any single value or any combination of two values (Kramer, 1954). The measurement of color of canned beets may serve as an example of a procedure which is not strictly a color measurement, but which is a good measure of the color of the product. Beets are red only because of the presence of a single pigment; namely, betanin, which is soluble in water. Hence a very simple method for measuring the color of canned beets is to measure the density of the beet liquor in some suitable colorimeter (Kramer and Smith, 1946a). Thus, once the color attributes of interest to the consumer are determined, the simplest possible procedure and equipment may be selected that will do the color evaluation job. I n the case of the frozen lima beans, a tristimulus instrument is not required, but merely one

172

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

which will measure the density, or the darkness of the product. In such cases of monostimulus, it is possible to develop models, or color chips, that may serve as limits for grades. For example, Brice and Turner (1956) developed such models for maple sirup. However, if two or more stimuli are required to specify consumer reaction to color, such models are not practical since an infinite variety of such models could be prepared, all meaning the same thing in terms of consumer preference. Grade limits in such cases may be established by using regression equations, involving all the color values found to be of interest to the consumer. These regression equations may be converted to nomographs. I n the case of frozen lima beans, the regression of panel score in terms TABLE IV

RELATION OF HUNTER L VALUES TO US.DEPT.AGR. SCORE FOR COLOROF FROZENLIMABEANS Grade B

Grade A

L value ~

29 30 31 32 33 34 35

L value

Score ~~

Grade C Score

L value

Score

53 52 51 50 49 48

42 43 44 45 46 47

47 46 45 44 43 42

~

36 37 38 39 40 41

60 59 58 57 56 55 54

of the official U.S. grade (maximum of 60 points for color) was calculated as p = 89.1 - L, where p is the U.S. grade for color, 89.1 is a constant, and L is the value obtained on the L scale of the Hunter color and color-differencemeter. To avoid the need for calculations, this equation may be expressed in terms of a single line curve, or a table (see Table N, Gamer and Hart, 1954). For tomato juice, where it was found that the a and b values adequately cover the color characteristics of interest to the consumer, a multiple regression equation was calculated using the relationship of both these values to panel scores. Here the regression equation was calculated as p = 32.6 0.682a - 1.678b, where p is the U.S. grade for color, 32.6 is a constant, a is the Hunter a value, and b is Hunter b value. For ease of interpretation, this equation may be expressed in terms of a nomograph, as shown in Fig. 8, where all that is required of the inspector is to locate the LZ and b values, then read the U.S. grade directly from the chart.

+

PHYSICAL MEASUREMENT OF FOOD QUALITY

173

b4

FIG.8.

GRADE SCORE

CONVERSION SCALE

120

I SCALE

48 44 42 40

38

FIG.9. US. grades for color of canned applesauce in terms of Hunter L, b values.

o, and

In the case of applesauce, it was found that all three color dimensions were required; hence, the regression equation includes all three Hunter values, as follows: p = 9.40 0.056L 0.802a 0.2523. For the convenience of the inspector, this equation may also be reduced to nomograph form, as shown in Fig. 9, where by the use of a straight

+

+

+

174

A M I H U D KRAMER A N D B. A. T W I G G

edge the iiispector may obtain the grade score for color in a matter of seconds (Kramer, 1954).

7 . Relation to C.I.E. System Whatever method is adopted for any specific color-measuring purpose, it should be possible to relate the results obtained to the C.I.E. system. This is necessary primarily for purposes of comparing color data obtained by different methods or with different instruments. Friedman et &. (1952) have presented a very interesting chromaticity diagram of the C.I.E. system on which they plot results of color data on tomato juice, which they calculated from values obtained by themselves and by others using the Munsell notations, a three-filter Photovolt reflection meter, and the Hunter (Gardner) color and color-difference meter. Their results are reproduced in Fig. 10 to which are added the .3? 0

.34t .33

,321 .3 I

I

I

I

I

I

1

I

I

I

.45

.46

.47

.48

.49

30

.5l

32

33

.54

X

FIG. 10. C.I.E. chromaticity diagram data plotted for values obtained by the use of Munsell discs, Hunter color-difference meter, and Photovolt reflectance meter.

grade limits as obtained from correlation studies and recalculated in terms of the C.I.E. system. From Fig. 10 (where the results as obtained by different methods are all put on a comparable basis), some very definite conclusions may be drawn, as for example: The dividing line between Fancy and Standard as drawn on the basis of Munsell notations and used by official graders is quite similar to the one calcuIated for using the Hunter color and color-difference meter. On the other hand, there is substantial difference between the two methods as to where the standard-substandard line should be drawn. Whereas the present U.S. grading system is based solely on hue, the additional data indicate that chroma also has a very small, but measurable effect.

PHYSICAL MEASUREMENT OF FOOD QUALITY

175

On the other hand, there is no particular point in calculating all quality or inspection data in C . I . E . terms. If the method is sound and reflects consumer acceptance, entirely adequate results are obtainable by reference to such tables or charts as demonstrated above. For example, when US. color grade equivalents were obtained by reference to Fig. 8 from the Hunter a and b values reported by Robinson et al. (1952), a correlation of 0.95 was obtained with U.S. inspectors’ scores as compared with a correlation of 0.90 when these scores were correlated to an a/ b ratio, and 0.94 when compared to calculated hue values. Color problems in different food commodities are discussed by MacKinney and Chichester (1954). B. VISCOSITY AND CONSISTENCY Viscosity or consistency is an appearance factor of great importance with such food products as catsup, juices, creams, fruit preserves, jams, jellies, mayonnaise, gelatins, oils, sirups, and doughs. Measurement of this quality factor may be made not only to indicate the consistency of the finished products, but also as a quality control tool on the raw product, or on the product at various stages of the process, for predicting final consistency. Such a measurement may also be used as an index to the amount of an ingredient which should be added or to the make-up (per cent solids, etc.) of a product. The duration and amount of heat applied in the process may be regulated to some extent by viscosity measurement since heat penetration and consistency are closely related. Viscosity also is a useful criterion of disaggregation o r depolymerization such as occurs in the initial stages of hydrolysis of protein, starch, or pectin. It can also be used in the determination of constitution of polymers, such as rubber and polysaccharides, or in the determination of molecular weight (Joslyn, 1950).

I. Definition Liquids flow as if they were divided into layers. The friction resulting from the resistance to flow between the liquid layers o r the resistance offered by a substance to deformation when subjected to a shearing force is called consistency or apparent viscosity. This resistance is the result of the movement of molecules in the interior of the liquid owing to Brownian motion and the intermolecular force of cohesion. Fluids which are chemically pure and physically homogeneous (Newtonian fluids) have a constant consistency value if the static pressure and temperature are fixed; for such substances the consistency is usually called viscosity, or absolute viscosity (Perry, 1950).

176

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

Since consistency is a measure of the resistance to flow, and since different terms are used to denote the character of flow, it is of utmost importance to understand the different types of flow. The viscosity of Newtonian liquids does not change with a change in rate of shear. Such “true liquids’’ are chemically pure and physically homogeneous; therefore, food products which are suspensions do not fall into this classification. Materials of Newtonian type include: water, most oils, sirups, sugar and gelatin solutions, etc. (Brookfield Engineering Laboratory, a ) . The physical law governing the viscosity of such fluids was defined by Sir Isaac Newton, who assumed that the viscosity, or coefficient of internal friction, of a given fluid was independent of the rate of shear (relative displacement of particles after application

L SHEARING RATE

SHEARINQ FORCE

FIG. 1 I.Viscosity characteristics of Newtonian fluids.

FIG. 12. Geometry of Newtonian-type flow.

of shearing force) or shearing force applied to the fluid (Minard, 1954). For example, if the viscosity of such a fluid was measured by the drag of the fluid on a rotating spindle, it remains the same regardless of the speed of the spindle. A “ . . . flow curve for a Newtonian fluid (Fig. 1 1 ) plotting shearing force against rate of flow or rate of shear, shows that if it is desired to move the fluid twice as fast, it will take twice the push, the shearing force increasing in the same proportion as the rate of shear or flow” (Minard, 1954). Shaw (1950) illustrated this fact by considering each particle of fluid as spherical in shape, like small balls rolling between flat plates (Fig. 12). If the top plate is moved at a constant velocity, the force is uniformly transmitted through each layer, and the frictional resistance of the balls would be constant. The rate of shear of each layer of all

PHYSICAL MEASUREMENT OF FOOD QUALITY

177

layers will vary in direct proportion to changes in force which vary the velocity. Non-Newtonian liquids are materials whose resistance to flow changes with a change in rate of shear. For example, using a rotating spindle-type viscosimeter, the apparent viscosity value obtained would differ with different rotating speeds of the spindle. In the food industry, the majority of consistency measurements are made on products exhibiting this type of behavior. Materials falling into this classification would

t

L

m 0 0

!! > 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

PHYSICAL MEASUREMENT O F FOOD QUALITY

181

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

182

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

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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Mabrouk, A. F., and Deatherage, F. E. 1956. Organic acids in brewed coffee. Food Technol. 10, 165-208. McArdle, F. J., and Desrosier, D. W. 1954. Here’s a rapid method for determining the pericarp content of sweet corn. Canner 118, 9-11. McColloch, R. J., Nielson, B. W., and Beavens, E. A. 1950. Factors influencing the quality of tomato paste I, Pectin changes during processing. Food Technol. 4, 339343. Mackinney, G., and Chichester, C. 0. 1954. The color problem in food. Aduances in Food Research 7 , 301-351. Magness, J. R., and Taylor, G. F. 1925. An improved type of pressure tester for the determination of fruit maturity. U S . Dept. Agr. Circ. No. 350. Martin, A. J. P., and Synge, R. L. M. 1941. A new form of chromatogram employing two liquid phases. Biochem. J. 35, 1358-1368. Martin, W. M. 1937. The tenderometer. Canner 80, 108-112. Mason, J. M., and Wiley, R. C. 1958. Quick quality test for lima beans. Maryland Processors’ Rept. 4 (7), 1-5. Univ. of Maryland, College Park, Md. Mellon, M. G. 1945. “Colorimetry for chemists.” G. Frederick Smith Chem. CO., Columbus, Ohio. Miller, E. E., Shadboh, C. A., and Holm, L. 1956. Use of an optical planimeter for measuring leaf area. Plant Physiol. 31, 384386. Minard, R. A. 1954. An industrial rotational viscometer and its use with materials of varying complexity. Intern. Instr. Congr. Ezposition Instr. SOC.Am., 1st Congr. Minneapolis-Honeywell Regulator Co. 1954. Continuous viscosity measurement. Instrumentation Data Sheet No. 10, 13-lb., Philadelphia, Pennsylvania. Minneapolis-Honeywell Regulator Co. 1955. Water control in cement manufacture. Instrumentation Data Sheet No. 10, 13-lb., Philadelphia, Pennsylvania. Minneapolis-Honeywell Regulator Co. 1957. Ultrasonic system for continuous viscosity measurement. Instrumentation Data Sheet No. 10, I 3 2a, Philadelphia, Pennsylvania. Moncrieff, R. W. 1951. “The Chemical Senses,” 2nd ed. Leonard Hill Ltd., London. Morris, 0. M. 1925. Studies in apple storage. Wash., State Coll., Agr. Expt. Stas. Bull. No, 193. National Canners Assoc. 1941. Grit cells in Kieffer pears. (Mimeo. Rept.) Washington, D.C. Nickerson, D. 1938. Use of I.C.I. tristimulus values in disk colorimetry. U.S. Dept. Agr. Prelim. Rept. Nickerson, D. 1946. Color measurement and its application to the grading of agricultural products. U S . Dept. of Agr. Misc. Publ. No. 580. Nutting, G. C. 1952. Effect of electrolytes on the viscosity of potato starch pastes. J . Colloid Sci. 7 , 128-139. Optical Society of America. Committee on Colorimetry. 1944. The psychophysics of color. J. Opt. SOC.Am. 34, 246. Pagenstedt, B. 1955. Determination of the baking values of wheat by means of physical testing methods. Australasian Baker and Millers’ I . 2, 13-15. Perry, J. H. 1950. “Chemical Engineer Handbook,” 3rd ed., pp. 369-374, 1197-1202, McGraw-Hill, N.Y. Pilgrim, F. J., and Schutz, H. G. 1957. Measurement of the qualitative and quantitative attributes of flavor. “Quartermaster Food and Container Institute. Symposium-Chemistry of Natural Food Flavors,” pp. 47-55. Available from Institute, Chicago. Pohle, W. D., and Mehlenbacker, V. C. 1955. Evaluation of egg color. Food Technol. 9, 565668.

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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 .

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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.

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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

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

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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233

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

235

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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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.

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C. OCCURRENCEON

THE

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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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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.

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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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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

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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

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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

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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-

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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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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

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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-

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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,

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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

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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

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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

262

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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.

MICROORGANISMS IN NONCITRUS JUICES

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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267

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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269

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

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Beech, F. W. 1958b. Some recent work on the microbiology of apple juices (yeasts). Symposium on Fruit Juice Concentrates, Bristo;. Intern. Federation Fruit Juice Prods. Pans, pp. 373-379, Juris, Zurich. Beech, F. W., and Pollard, A. 1955. Acid changes in cider fermentations. I. The significance of acid changes in cider making. Ann. Rept. Long Ashton Research Sta., Bristol pp. 159-162. Behrens, J. 1910. Die Pilzflora auf Trauben und Obstfriichten. (Lafar, ed.), “Handbuch der Technischen Mykologie,” Vol. V, pp. 343361. Fischer Jena. Berry, J. A. 1932a. Microbiology of the frozen pack. Canning Age 13, 251-254. Berry, J. A. 1932b. Bacteria question in cold packing. Western Canner and Packer 23, 17-18. Berry, J. A. 193%. How freezing affects microbal growth. Food In&. 4 (6), 205. Berry, J. A. 1933a. Destruction of microorganisms at various freezing temperatures. J. Bacteriol. 25, 74-75. Berry, J. A. 1933b. Destruction and survival of microorganisms in frozen pack foods. J. Bacteriol. 26, 459470. Berry, J. A. 1935. Microbiological studies of frozen pack berries, with special reference to effects of carbonation. Proc. Am. SOC.Hort. Sci. 33, 224-226. Berry, J. A., and Diehl, H. C. 1934. Freezing storage in relation to microbial destruction and retention of quality in sweet cider. Proc. Am. SOC.Hort. Sci. 32, 157-159. Berry, J. M., and Vaughn, R. H. 1952. Decomposition of tartrates by lactobacilli (An abstract). Proc. Am. SOC. Enologists 2, 135-138. Berry, J. M., Witter, L. D., and Folinazzo, J. F. 1956. Growth characteristics of spoilages in orange juice and concentrate. Food Technol. 10, 553-556. Bioletti, F. T.,and Cruess, W. V. 1922. Ehological investigations. Calif. Uniu., Agr. Expt. Sta., Bull. Nr. 230, 25-64. Boehi, A. 1912. “Ein neues Verfahren zur Herstellung alkoholfreier Obst- und Traubenweine (Kohlensaureverfahren) .” Huber & Co., Frauenfeld. Borgstrom, G. 1954. Preservation of fruit juices by sterilization fitration, chemical preservatives, etc. In “Chemistry and Technology of Fruit and Vegetable Juices” (D. K. Tressler and M. A. Joslyn, eds.), Chapter 5, Avi, New York. Bowen, J. F., Strachan, C. C., and Moyls, A. W. 1953. Butyric acid fermentation in canned pears and tomatoes. Food Technol. 8, 239-244. Braverman, J. B. S . 1955. The mechanism of the interaction of sulphur dioxide with certain sugars. J. Sci. Food Agr. 4,540-547. Brunner, H.1947. Unpublished data. Biichi, W. 1958. Ueber Zusammenhange zwischen Geschmacksveranderungen von Obstsaften und deren Gehalt an Diacetyl und Acetoin. Schweiz. 2. Obst- u. Weinbau 67, 459-462. Burcik, E. 1948. Eine Kritik der Symbiosetheorie von H. Schanderl auf Grund neuerer eigener Untersuchungen. Arch. Mikrobiol. 14, 309-333. Burroughs, L. F., and Carr, J. G. 1956. The amino acid content of cider in relation to the growth of lactic acid bacteria. Ann. Rept. Long Ashton, Agr. Research Sta., Bristol pp. 162-167. Cameron, E. J., and Esty, J. R. 1940. Comments on the microbiology of spoilage in canned foods. Food Research 5, 549-557. Carr, J. G. 1952. The lactic acid bacteria of cider I. Some organisms responsible for the malolactic fermentation. Ann. Rept. Long Ashton, Agr. Research Sta., Bristol pp. 144-150.

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Carr, J. G. 1956. The occurrence and role of lactic acid bacteria in cider making. Ph.D. Thesis, University of Bristol, Bristol, England. Carr, J. G. 1957a. Occurrence and activity of some lactic bacteria from apple juices, ciders and perries. J. Inst. Brewing 63, 436-444). Carr, J. G. 1958. The fate of acetic acid bacteria in a fermenting cider. Symposium on fruit juice concentrates, Bristol. Int. Fed. Fruit Juice Prod. Paris, Juris, Zurich. Cam, J. G., Phillips, J. D., Pollard, A., Whiting, G. C., and Williams, A. H. 1954. Quinic acid metabolism in cider fermentation. Chemistry & Ind. (London) pp.

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342. Challinor, S. W., and Rose, A. H. 1954. Interrelationships between a yeast and a bacterium when growing together in defined medium, Nature 174, 877-878. Charpentib, Y. 1954. Contribution A l’ktude biochimique des facteurs de l’aciditb des vins. Ph.D. Thesis, Universitk de Bordeaux. Bordeaux. Charpentik, Y., RiWreau-Gayon, J., and Peynaud, E. 1951. Sur la fermentation de l’acide citrique par les bactkries malolactiques. Bull. SOC. chim. biol. 33, 1369-

1378. Chrzaszcz, T. 1902. Physarium leucophaeum ferox, eine hefefressende Amobe. Zentr. Bakteriol., P a r a d e d . Abt. I1 8, 43141. Ciferri, R. 1941. Diffusione e ciclo dei lieviti in natura. “Trattato di Enologia di P. G. Garoglio,” Vol. 11, pp. 310-325. Parenthi di G.,Firenze. Clark, D. S., and Wallace, R. H. 1954.Candida malicola nov. sp. isolated from apples. Can. I . Microbiol. 1, 275-276. Clark, D. S., Wallace, R. H., and Davis, J. J. 1954. Yeasts occurring on apples and in apple cider. Can. J. Microbiol. 1, 145-149. Cruess, W. V. 1948. “Commercial fruit and vegetable products,” 3rd ed. McGrawHill, New York. Cruess, W. V., and Irish, J. H. 1933.Fruit beverage investigations. Calif. Univ., Agr. Expt. Sta., Bull. Nr. 359. Cruess, W. V., Richert, P. H., and Irish, J. H. 1931. The effect of hydrogen-ion concentration on the toxicity of several preservatives to microorganisms. Hilgardia 6, 295-314. Damm, A. 1951. Eine neue, haufiger vorkommende Infektion des Siissmostes. Fliissiges Obst. (2) pp. 23. Dimick, K. P., Ponting, J. D., and Makower, B. 1951. Heat inactivation of polyphenolase in fruit purbes. Food Technol. 5,237-241. Domercq, S. 1956. Etude et classification des levures de vin de la Gironde. PbD. Thesis No. 20, Universitk de Bordeaux, Bordeaux. Donald, J. R., Jones, C. L., and McLean, A. R. M. 1924. The effect of carbonation on bacteria in beverages. Am. J . Publ. Health 14, 122-128. Douglas, M., and Edin, M. D. 1930. Some principles regulating the life and death of pathogenic intestinal bacteria in artificial media and fruit juices. Lancet 129, ii,

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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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Wallace, G. I., and Park, S. E. 1953b. Microbiology of frozen foods. V. The behaviour of Clostridium botulinum in frozen fruits and vegetables. J . Infectious Diseases 52, 150-156. Whiting, G. C., and Cam, J. G. 1957. Chlorogenic acid metabolism in cider fermentation. Nature 180, 1479. Wilharm, G., and Holz, G. 1951. Beitrag zur Kenntnis des Acroleins in Obstbranden, Maischen und Mosten. 11. Isolierung acroleinbildender Mikroorganismen und Erzeugung von Acrolein durch die gewonnenen Reinkulturen. Arch. Mikrobiol. 15, 403-413. Williams, C. C., Cameron, E. J., and Williams, 0. B. 1941. A facultatively anaerobic mold of unusual heat resistance. Food Research 6, 69-73. Williams, A. J., Wallace, R. H., and Clark, D. S. 1956. Changes in the yeast population on Quebec apples during ripening. Can. J . Microbiol. 2, 645-648. Witter, L. D., Berry J. M., and Folinazzo, J. F. 1958. The viability of Escherichia coli and a spoilage yeast in carbonated beverages. Food Research 23, 133-142. Woll, E. 1956. Untersuchungen iiber die behauptete Transmutation von Chondriosomen der Hefe zu Bakterien. Zentr. Bakteriol. Parasitenk. Abt. I I 109, 388-398. Wortmann, J. 1897-98. “Handbuch der Technischen Mykologie” Vol. IV. 1907, p. 150.

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

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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

THE CHEMICAL CONSTITUENTS OF CITRUS FRUITS

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

352

J. F. KEFFORD

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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(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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Harding, P. L. 1954. Evaluating palatability in Florida citrus fruits. Proc. Am. SOC. Hort. Sci. 59, 303. Harding, P. L., and Fisher, D. F. 1945. Seasonal changes in Florida grapefruit. US. Dept. Agr., Tech. Bull. No. 886. Harding, P. L., and Lewis, W. E. 1941. Relation of size of fruit to solids, acid and volume of juice in the principal varieties of Florida oranges. Proc. Florida State Hort, SOC.54, 52. Harding, P. L., and Sunday, M. B. 1949. Seasonal changes in Florida tangerines. US.Dept. Agr., Tech. Bull. No. 988. Harding, P. L., and Sunday, M. B. 1953. Seasonal changes in Florida Temple oranges. U.S. Dept. Agr., Tech. Bull. N o . 1072. Harding, P. L., and Wadley, F. M. 1945. Study of quality in Temple oranges. Food Research 10, 510. Harding, P. L., Winston, J. R., and Fisher, D. F. 1945. Seasonal changes in Florida oranges US.Dept. Agr., Tech. Bull. No. 753. Harding, P. L., Soule, M. J., and Sunday, M. B. 1958. Storage studies on Marsh grapefruit 1955-56 season. Citrus Ind. 39, 8. Hardy, F., and Warneford, F. H. S. 1925. Coloring matter of lime juice. Ind. Eng. Chem. 17, 48. Hattori, S., and Shimokoriyama, M. 1956. Seasonal changes i n flavone and flavanone glycosides in the fruit and flowers of Poncirus trifoliata. Sci. Proc. Roy. Dublin SOC.27, 139. Hattori, S., Hasegawa, M., and Kanao, M. 1949. Flavone glycosides. 111. Naringin from the peels of Citrus grandis. Acta Phytochim. (Japan) 15, 199. Hattori, S., Shimokoriyama, M., and Kanao, M. 1952. Studies on flavanone glycosides. IV. The glycosides of ripe fruit peel and flower petals of Citrus Aurantium L. J . Am. Chem. SOC.74, 3614. Hellstrijm, V. 1955. Ascorbic acid content of citrus fruits. VZr. foda 7, 21-24. Hendrickson, R., and Kesterson, J. W. 1954a. Hesperidin, the principal glucoside of oranges. Florida, Univ. Agr. Expt. Stas. (Gainesuille) Bull. No. 545. Hendrickson, R., and Kesterson, J. W. 1954b. Recovery of citrus glucosides. Proc. Florida State Hort. SOC.67, 199. Hendrickson, R., and Kesterson, J. W. 1957. Chemical analysis of citrus bioflavanoids. Proc. Florida State Hort. SOC.70, 196. Hendrickson, R., Kesterson, J. W., and Edwards, G. J. 1958. Ultraviolet absorption technique to determine the naringin content of grapefruit juice. Proc. Florida State Hort. SOC.71, 194. Herbst, E. J., and Snell, E. E. 1949. Putrescine and related compounds as growth factors for Hemophilus parainfluemae 7901. J . B i d Chem. 181, 47. Hield, H. Z., Coggins, C. W., and Garber, M. J. 1958. Gibberellin tested on citrus. Calif. Agr. 12, 9. Higby, R. H. 1938. Bitter constituents of Navel and Valencia oranges. 1. Am. Chem. SOC. 60, 3013. Hilgeman, R. E. 1951. Irrigation of Valencia oranges. Calif. Citrograph 36, 370. Hill, E. C., Wenzel, F. W., and Barreto, A. 1954. Colorimetric method for detection of microbiological spoilage in citrus juices. Food Technol. 8, 168. Hiwatari, Y, 1927. Nitrogenous components from the fruit of Citrus grandis Osbeck, form Buntan. J . Biochem. (Tokyo) 7, 169. Horowitz, R. M. 1956. Flavonoids of Citrus. I. Isolation of diosmin from lemons (Citrus limon). J . Org. Chern. 21, 1184.

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different locations of the tree as a guide to sampling methods and spot-picking for quality. I. Soluble solids in the juice. Proc. A m . SOC.Hort. Sci. 54, I. Sites, J. W., and Reitz, H. J. 1950a. The variation in individual Valencia oranges from different locations of the tree as a guide to sampling methods and spot-picking for quality. 11. Titratable acid and the soluble solids/titratable acid ratio of the juice. Proc. A m . Soc. Hort. Sci. 55, 73. Sites, J. W., and Reitz, H. J. 1950b. The variation in individual Valencia oranges from different locations on the tree as a guide to sampling methods and spotpicking for quality. 111. Vitamin C and juice content of the fruit. Proc. A m . SOC. Hort. Sci. 56, 103. Sites, J. W., Reitz, H. J., and Deszyck, E. J. 1951. Some results of irrigation research with Florida citrus. Proc. Florida State Hort. SOC.64, 71. Sites, J. W., Wander, I. W., and Deszyck, E. J. 1955. Nitrogen sources as related to yield and quality of Ha& oranges. Proc. Florida State Hort. Sac. 68, 65. Smith, P. F. 1955. Relation of boron level to production and fruit quality of grapefruit and oranges. Proc. Florida State Hort. SOC.68, 54. Spoon, W., Sesseler, M., and Dessens, H. B. 1951. Juices of oranges and grapefruit from Surinam. Voeding 12, 256. Srivastava, H. C. 1953. Paper chromatography of fruit juices. J. Sci. Znd. Research (India) 12B, 363. Stanley, W. L. 1958. Citrus flavors. In “Flavor Research and Food Acceptance” (sponsored by A. D. Little, Inc.), p. 342. Reinhold, New York. Stanley, W. L., and Vannier, S. H. 1957a. Chemical composition of lemon oil. I. Isolation of a series of substituted coumarins. I. A m . Chem. SOC.79, 3488. Stanley, W. L., and Vannier, S. H. 1957b. Analysis of coumarin compounds in citrus oils by liquid solid partition. J . Assoc. Offic. Agr. Chemists 40, 582. Stanley, W. L., and Vannier, S. H. 1958. Effects of environmental and processing factors on the citral content of lemon oil. Food Technol. 13, 96. Stanley, W. L., Lindwall, R. C., and Vannier, S. H. 1958. Specific quantitative colorimetric method of analysis for citral i n lemon oil. J. Agr. Food Chem. 6, 858. Stenstrom, E. C., and Westbrook, G. F. 1956. A study of the degrees Brix and Brix/ acid ratios of grapefruit utilized by Florida citrus processors for the seasons 1952-53 through 1955-56. Proc. Florida State Hort. SOC.69, 113. Stern, I. 1943. Analysis of beverages containing citrus juices. Analyst 68, 44. Stern, I. 1954. Analysis of commercial citrus juices. Food 23, 435. Stevens, J. W. 1954. Preparation of dehydrated agar media containing orange juice serum. Food Technol. 8, 92. Stewart, W. S., and Parker, E. R. 1954. Effect of 2,4-D and related substances on fruit-drop, yield, size, and quality of grapefruit. Hilgardia 22 (18), 623. Stofberg, F. J., and Anderson, E. E. 1949. Effects of oil sprays on the yield and quality of Navel and Valencia oranges. Union S. Africa Dept. Agr. Sci. Bull. No.

296. Swift, L. J. 1946. Determination of crude lipids in citrus juices. J. Assoc. Offic. Agr. Chemists 29, 389. Swift, L. J. 1949. Tangerine seed oil. J. A m . Oil Chemists’ Soc. 26, 438. Swift, L. J. 1951. Flavor changes in stored canned orange juice. Proc. Florida State Hort. Soc. 64, 181. Swift, L. J. 1952a. Fatty acids from the lipids from freshly canned Florida Valencia orange juice. Food Research 17, 8 .

THE CHEMICAL CONSTITUENTS O F CITRUS FRUITS

371

Swift, L. J. 1952b. Isolation of 8-sitosteryl-D-glucoside from the juice of Florida Valencia oranges (Citrus sinensis, L.). J . Am. Chem. SOC.74, 1099. Swift, L. J., and Veldhuis, M. K. 1951. Constitution of the juice lipids of the Florida Valencia orange (Citrus sinensis, L.). Food Research 16, 142. Swift, L. J., and Veldhuis, M. K. 1957. Composition of commercial, segment, and peel juices of Florida oranges. J . Agr. Food Chem. 5, 49. Swingle, W. T. 1948. The botany of Citrus. In “The Cirtus Industry” (H. J. Webber and L. D. Batchelor, eds.), Vol. I, p. 129. Univ. Calif. Press, Berkeley, California. Tanret, C. 1886. Some principles present in the peel of the bitter orange. Compt. rend. 102, 518. Taylor, 0. C., Carman, G. E., Burns, R. M., Moore, P. W., and Nauer, E. M. 1956. Effect of oil and parathion sprays on orange size and quality. Calif. Citrograph 41, 452. Thompson, W. L., and Deszyclr, E. J. 1957. Phosphatic insecticides mixed with oil emulsions for scale control, and their effect on fruit quality. Proc. Florida State Hort. SOC.70, 31. Thompson, W. L., Griffiths, J. T., and Sites, J. W. 1951. A comparison of oil emulsion and parathion for the control of scale insects on citrus. Proc. Florida State Hort. SOC.64, 66. Ting, S. V. 1956. Rapid colorimetric methods for simultaneous determination of total reducing sugars and fructose i n citrus juices. J . Agr. Food Chem. 4, 263. Ting, S. V. 1958. Enzymic hydrolysis of naringin in grapefruit. J . Agr. Food Chem. 6, 546. Ting, S. V., and Deszyck, E. J. 1959. Isolation of 1-quinic acid in citrus fruit. Nature 183, 1404. Townsley, P. M., Joslyn, M. A., and Smit, C. J. B. 1953. The amino acids in various tissues of citrus fruits and in orange protein. Food Research 18, 522. Tsukamoto, T., and Ohtaki, T. 1947. Components of Citrus tankan. J. Pharm. SOC. Japan 67, 45. Turrell, F. M. 1950. A study of the physiological effects of elemental sulfur dust on citrus fruits. Plant Physiol. 25, 13. Underwood, J. C., and Rockland, L. B. 1953. Nitrogenous constituents in citrus fruits. I. Some free amino acids in citrus juices determined by small-scale filter paper chromatography. Food Research 18, 17. US. Department of Agriculture. 1954. U.S. Standards for Grades of Canned Grapefruit Juice. October 19th. Agricultural Marketing Service, Washington, D.C. Van Atta, G. R., and Dietrich, W. C. 1944. Valencia orange-seed oil. Oil h Soap 21, 19. Van der Laats, J. E. 1954. Comparative study of the citric acid and vitamin C content of the juice of some citrus fruits. Reu, biol. trop. Uniu. Costa Rica 2, 45. Vannier, S. H., and Stanley, W. L. 1958. Fluorometric determination of 7-geranoxycoumarin in lemon oil: analysis of mixtures of grapefruit oil in lemon oil. J. Assoc. Ofic. Agr. Chemists 41, 432. Vanna, T. N. S.,and Ramakrishnan, C. V. 1956. Biosynthesis of citric acid in citrus fruits. Nature 178, 358. Von Loesecke, H. W. 1954. Orange juice. In “The Chemistry and Technology of Fruit and Vegetable Juice Production” (D. K. Tressler and M. A. Joslyn, eds.), p. 411. Avi, New York. Wawra, C. W., and Webb, J. L. 1942. Isolation of a new oxidation-reduction enzyme from lemon peel (Vitamin P). Science 96, 302.

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Webber, H.J. 1948. Cultivated varieties of Citrus. In "The Citrus Industry" (H. J. Webber and L. D. Batchelor, eds.), Vol. I, p. 475. Univ. Calif. Press, Berkeley, California. Wedding, R. T., and Horspool, R. P. 1955. Compositional changes in juice of Valencia and Washington Navel oranges during fruit development. Calif. Citrograph 40,

106. Wedding, R. T., and Sinclair, W. B. 1954. A quantitative paper-chromatographic study of the amino acid composition of proteins and juice of the orange. Botan. Gaz. 116, 183. Weizmann, A. and Mazur, Y. 1958. Steroids and triterpenoids of citrus fruit. 11. Isolation of citrostadienol. J . Org. Chem. 23, 832. Weizmann, A., Meisels, A., and Mazur, Y. 1955. Steroids and triterpenoids of grapefruit (Citrus paradisi, Macfadyen). I. J . Org. Chem. 20, 1173. Westbrook, G. F., and Stenstrom, E. C. 1957. A study of the degrees Brix and Brix/ acid ratios of oranges utilized by Florida citrus processors for the seasons 195253 through 1956-57. Proc. Florida State Hort. SOC.70,247. Winston, J. R., and Miller, E. V. 1948. Vitamin C content and juice quality of exposed and shaded citrus fruits. Food Research 13, 456. Wolf, J. 1958. Organic acids of citrus fruits. Pruchtsaft-lnd.3 , 93. Yamamoto, R., and Oshima, Y. 1931. A new glucoside, citronin, from the peel of Ponderosa lemon (Citrus limon. Burm. f. ponderosa Hort.) J . Agr. Chem. SOC. Japan 7, 312. Young, R. E., and Biale, J. B. 1956. Carbon dioxide fixation by lemons in a COXenriched atmosphere. Plant Physiol. 31, Suppl., p. xxiii. Zechmeister, L., and Tuzson, P. 1937. Polyene pigment of the orange. 11. Citraurin. Ber. 70B, 1966. Zemplh, G., Tettamanti, A. K., and Farag6, S. 1938. The biose of hesperidin and neohesperidin. Ber. 71B, 2511. Zidan, Z. I., and Wallace, A. 1954. Concentrations of five nutrient elements in orange fruits collected at different stages of maturity. Proc. Am. SOC.Hort. Sci.

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.

373

This Page Intentionally Left Blank

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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