Advances in Food Research, Volume 28

ADVANCES IN FOOD RESEARCH VOLUME 28

Contributors to This Volume A. Asghar J. J. A. Heffron R. L. Henrickson Lea Hyvonen Pekka Koivistoinen G. Mitchell N. R. Reddy D. K. Salunkhe S. K. Sathe Valdemiro C. Sgarbieri Felix Voirol John R. Whitaker

ADVANCES IN FOOD RESEARCH VOLUME 28

Edited by

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

E. M. MRAK

G. F. STEWART

University of California Davis, California

University of California Davis, California Editorial Board

JOHN AYRES E. M. FOSTER S. GOLDBLITH J. HAWTHORNE J. F. KEFFORD

S. LEPKOVSKY D. REYMOND EDWARD SELTZER W. M. URBAIN

1982

ACADEMIC PRESS A Subsidiary of Harcolut Brace lovanovich, Publishers

New York Paris

London

San Diego San Francisco S b Paulo Sydney

Tokyo Toronto

COPYRIGHT @ 1982, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published b y ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W I

LIBRARY OF

7DX

CONGRESS CATALOG CARD

NUMBER:48-7808

ISBN 0-12-016428-0 PRINTED IN THE UNITED STATES O F AMERICA 82 83 84 85

9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORS TO VOLUME28 ................................................. GEORGEF. STEWART..........................................................

VII IX

Phytates in Legumes and Cereals N. R. Reddy, S. K. Sathe, and D. K. Salunkhe Introduction . . . . . . . . . . . . . . . Chemistry . . . . . . . . . . . . . . . . . 111. Nutritional Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Summary and Conclusions . . . . . . . . . . . V. Future Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

11.

23 74 75 75

Physical, Chemical, and Nutritional Properties of Common Bean (Phaseolus) Proteins Valdemiro C. Sgarbieri and John R. Whitaker

I.

Introduction .

..........

Chemical Properties of Isolated Storage Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . Amino Acid Composition and Nutritional Properties of Proteins from Several Phaseolus Species and Varieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Toxicity Associated with Phaseolus Proteins: Lectins; Inh Enzymes and Other Factors ........................ V. Influence of Storage and Processing on Chemical and Nut ...................... Bean Roteins . . . . . . . . . . . . . . . . VI. Additional Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

94 94

111.

102 112 144 148 151

Porcine Stress Syndromes G. Mitchell and J. J. A. Heffron I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Predictive Tests ...................... .......... 111. Etiology of Porcine Stress Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Future Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ...................................................

167 178 187 21 1 216 217 V

vi

CONTENTS

Chemical. Biochemical. Functional. and Nutritional Characteristics of Collagen in Food Systems A . Asghar and R . L . Henrickson I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Morphology of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ 111. Chemistry of Collagen ............ IV . Metabolism of Collagen V . Factors Affecting Collagen Composition and Structure ...................... VI . Functional Properties of Collagen in Food Systems . . . . . . . VII . Nutritional Aspects of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Food Uses of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

232 233 240 261 274 287 312 322 331 333

Food Technological Evaluation of Xylitol Lea Hyviinen. Pekka Koivistoinen. and Felix Voirol I . Introduction .... ... I1 . The Occurre .............................. 111. Physicochemical and Food Technological Properties of Xylitol . . . . . . . . . . . . . . . IV . Food Applications V . Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

373 374 382 392 399 399 400 405

CONTRIBUTORS TO VOLUME 28 Numbers in parentheses indicate the pages on which the authors' contributions begin.

A. Asghar,' Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078 (231) J. J. A. Heffron, Department of Biochemistry, University College, Cork, Ireland (167)

R. L. Henrickson, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078 (231) Lea Hyvijnen, Department of Food Chemistry and Technology, University of Helsinki, 00710 Helsinki 71, Finland (373) Pekka Koivistoinen, Department of Food Chemistry and Technology, University of Helsinki, 00710 Helsinki 71, Finland (373) G . Mitchell, Department of Physiology, University of the Witwatersrand, Medical School, Johannesburg, South Africa (167)

N . R. Reddy,' Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322 (1)

D . K . S a l ~ n k h e Department ,~ of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322 (1) S. K . Sathe,4Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322 (1)

Valdemiro C. Sgarbieri, Department of Food and Nutrition Planning, Faculty of Food and Agricultural Engineering, University of Campinas, Campinas, Brazil (93) Felix Voirol, Xyrojin Ltd., 6340 B a r , Switzerland (373) John R. Whitaker, Department of Food Science and Technology, University of California, Davis, California 95616 (93)

'Present address: Department of Food Technology, University of Agriculture, Fasialaband, Pakistan. 'Present address: Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061. 3Present address: Mahatma Phule Agricultural University, Rahuri, Maharashtra, India 413722. 4Present address: Department of Nutrition and Food Science, Muscle Biology Group, University of Arizona, Tucson, Arizona 85721. vii

This Page Intentionally Left Blank

George F. Stewart 1908-1982

This Page Intentionally Left Blank

ADVANCES I N ~ O O ORESEARCH, VOL.

28

PHYTATES IN LEGUMES AND CEREALS N. R. REDDY,' S . K. SATHE,* AND D. K. SALUNKHE3 Department of Nutrition and Food Sciences. Utah State University, Logan, Utah

1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Historical Background. . . . . . . . . . . . . . . . . . . . . . . . B. Structure of Phytic Acid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Biological Function ............................ D. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..........

111.

IV. V.

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

C. Phytase Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nutritional Implications .................... B . Effects of Phytate on Mineral Bioavailability . . . . . . . . . . . . . . . . . . . . . C. Effects of Processings on Phytates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Methods for Removal of Phytates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. Summary and Conclusions . . . . . . . . . . . Future Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ..........................................

I. A.

1 1

4 7 7 12 15 15 18 21 23 23 29 42 67 74 75 75

INTRODUCTION

HISTORICAL BACKGROUND

Knowledge of phytic acid had its beginning in the discovery by Hartig (1855, 1856), who isolated small particles or grains (which were not starch grains) from 'Present address: Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061. *Present address: Department of Nutrition and Food Science, Muscle Biology Group, University of Arizona, Tucson, Arizona 85721. 3Present address: Mahatma Phule Agricultural University, Rahuri, Maharashtra, India 413722. 1 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-016428-0

2

N. R. REDDY ET AL HC(OH)OP,

I I HC(OH)OP/ FIG. 1 .

,(OH) 0 \(OH)

Structural formula proposed by Posternak.

the seeds of various plants. He considered them an essential reserve product designed to play an important role in the germination of the seed and the growth of the plant. In 1872, Pfeffer differentiated the grains described by Hartig into three groups: (1) crystals of calcium oxalate, (2) a protein substance, and (3) a compound giving no reactions for protein, fat, or inorganic salts and was found in all of the 100 different seeds which he examined. Pfeffer described the third type as having rounded surfaces, assuming spheroidal shapes, and frequently twinning so as to present a convoluted appearance. Pfeffer named this third group of grains “globoids,” and these globoid particles were found to be free of nitrogen, but to contain calcium, magnesium, and phosphorus. Organic matter was noted in the globoids and the suggestion was then made that the substance was a phosphate combined with a carbohydrate. In 1894, Palladin, while studying the proteins of Indian mustard (Sinupis niger), obtained a substance from the fat-free, finely ground seeds which was soluble in 10% sodium chloride, but precipitated on heating. He found that this substance was soluble in cold and insoluble in hot water. By filtering off the permanent coagulum, reheating the filtrate, and filtering while hot, he obtained a fairly pure product that was rich in phosphorus and contained calcium and magnesium, but no nitrogen. Palladin’s work was later confirmed by Schulze and Winterstein (1896), who also expressed the opinion that the compound discovered by this chemical procedure was identical with Pfeffer’s “globoid” particles. The following year (1897), Winterstein suggested “inosite-phosphoric acid” as the proper name for the compound, since it yielded inosite and phosphoric acid on hydrolysis. The most extended study of this substance was made by Posternak (1900, 1903a, 1904, 1905) and his findings were presented in eight papers. He successfully prepared it in pure form and studied its physical and chemical properties, speculating on its constitution and biological function. In the early stages of his work, he (Posternak) rejected the name suggested by Winterstein and proposed a structural formula which did not include the inositol (inosite) ring. He gave the name “phytin” (derived from Greek) to the substance and under this trade name it has long been marketed by a chemical firm in Basel, Switzerland. Posternak

FIG. 2.

Structural formula of anhydro-oxymethylene diphosphoric acid proposed by Posternak.

3

PHYTATES IN LEGUMES AND CEREALS H

HO P‘ HOo>

H

- 0 - C -C - 0 - P

I I

HO H

H

HO FIG. 3. Structural formula proposed by Suzuki etal. (1907). C6H18024P6,MW 660; C P = 28.18%.

=

10.91%;

first constructed the formula from his chemical analyses (see Fig. 1). After further detailed chemical tests, Posternak proposed a second formula (see Fig. 2). Posternak opined that inosite is synthesized from the products of hydrolysis, when the “phytin” is heated under pressure with mineral acids. A number of chemists have expressed doubt concerning the probability of such a formation of inosite (inositol). In 1907, Suzuki and his co-workers obtained inosite from “phytin” by the action of an enzyme in rice bran, from which they concluded that inosite was an integral part of the “phytin” molecule and constructed a formula (Fig. 3) to describe their views on the structure of the phytin molecule. Neuberg (1908) came to a conclusion similar to that of Suzuki et al. (1907) regarding the presence of inosite. He obtained inosite and furfurol on mixing ‘‘phytin” with phosphoric acid and distilling under reduced pressure, and he also showed that furfurol can be obtained from inosite. He proposed the structural formula shown in Fig. 4. Levene (1909), working with a preparation from hempseed, believed that the “phytin” of hempseed grain contained phosphate, inosite, and a carbohydrate of the pentose group in its molecule. His work was later criticized by Neuberg, who claimed that there were impurities in the preparation. Later, Starkenstein (1908,

HO HC --CH

I

:! ‘\O/

t

0 OH bfOH OH

FIG. 4. Structure proposed by Neuberg (1908). C6H18024P6.MW 714; C 26.05%.

=

10.085%; P

=

4

N. R. REDDY ET AL.

I I

HO - C - c - OH

OH

HO

I

I P=O

O=P

OH/‘OJ\OH FIG. 5.

Structure proposed by Starkenstein (1911). C6H18027P6, MW 714; C = 10.985%; P

=

26.05%.

1910, 1911) offered the formula shown in Fig. 5. He argued that the phosphoric acid was in the pyro form. In 1910, Contardi demonstrated that phytin is a salt of inosite-phosphoric acid, which he prepared from rice bran and which gave analyses identical with that of a synthetic preparation. Within a few years, it became generally acceptable that phytic acid was the hexaphosphate of myoinositol (Fig. 6) (Anderson, 1912a-d; Starkenstein, 1914; Posternak, 1921). B.

STRUCTURE OF PHYTIC ACID

The structure of phytic acid, a naturally occurring antinutrient in seeds, had been the subject of controversy. For years the controversy was centered around the structure proposed by Anderson (1914) (Fig. 7A) and the structure suggested by Neuberg (1908) (Fig. 7B). Several additional structures for phytic acid have also been proposed (Courtois, 1954). The controversial issue has been the iso0 HO-!-OH

OH H0’

.

0.

P’

O’ ‘OH

9

HO

HO-P-OH II

0 FIG. 6 . Most accepted structure proposed by Anderson (1912a-d). C6H18024P6.

PHYTATES IN LEGUMES AND CEREALS

5

FIG. 7.

Proposed structures for phytic acid. Structure A was suggested by Anderson (1914) and structure B by Neuberg (1908).

meric conformation of the phosphate groups within the compound and whether three strongly bound water molecules were incorporated into the structure. Several studies have been published to support each of the structures proposed by Neuberg and Anderson (Beck, 1948; Otolski, 1935; Posternak and Posternak, 1929; Wrenshall and Dyer, 1941; Bourdillon, 1953; Earley, 1944; Otolski, 1937; Courtois and Mason, 1950). From a chemical hydrolysis study, Desjobert and Fleurent (1954) concluded that phytic acid behaved in a manner consistent with the Anderson model. Barre ef al. (1954) also supported Anderson's structure after studying the titration and conducting curves. The evidence reviewed by Posternak (1965) suggests that only 12 dissociable hydrogens per molecule can be detected by potentiometric titration in aqueous solution; this has been recently confirmed by Maddaiah et al. (1964). This evidence supports the Anderson structure for phytic acid. Some results appeared to favor the Neuberg structure (Beck, 1948; Fischler and Kurten, 1932; Otolski, 1935; Wrenshall and Dyer, 1941). Brown et al. (1961) found that only 12 acid hydrogens were titratable in aqueous solution and 18 hydrogens could be detected in glacial acetic acid solution. Six of the 18 hydrogens were too weakly acidic to be ordinarily titratable in water. Elemental analysis, titrations of sodium phytate with metal ions, and titration of phytic acid solutions containing an excess metal ion by Brown ef al. (1961) all supported the Neuberg structure.

6

N. R. REDDY ET AL

Although several studies have been published to support the structures of Anderson and Neuberg, most recent evidence employing nuclear magnetic resonance (Johnson and Tate, 1969) and X-ray crystallography (Fennessey and Nowacki, 1968; Truter and Tate, 1970; Blank et d . , 1971; Costello et d . , 1976) left little doubt that the structure proposed by Anderson was in fact the predominant form found in plant materials. According to Smith and Clark (195 1) and Cosgrove (1966), soil phytate is a mixture of polyphosphates which includes several isomers of inositol, namely, neo-, myo-, chiro-, and scillo-inositol hexaphosphate. The phytic acid which was claimed by Rapoport (1940) and Rapoport and Guest (1941) to be in the erythrocytes of birds and turtles was recently identified by Johnson and Tate (1969) to be the myo-inositol 1,3,4,5,6pentaphosphate. The nomenclature for inositol phosphates has been revised (IUPAC-IUB, 1968). The new nomenclature for phytic acid is myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate). Recently, Weingartner and Erdman ( 1978) proposed a partially dissociated Anderson-based structure (Fig. 8) for phytic acid that might occur at a neutral pH (Fig. 8A). At neutral pH, phosphate groups have either one or two negatively charged oxygen atoms. It is apparent that various cations could strongly chelate between two phosphate groups or weakly within a phosphate group (Fig. 8B). The relative binding strengths of various metal ions to phytic acid differs appreciably.

A

FIG. 8. Structure of phytic acid (A) and phytic acid chelate (B) at neutral pH. From Erdman ( 1979).

PHYTATES IN LEGUMES AND CEREALS

7

C. BIOLOGICAL FUNCTION Phytic acid has been generally regarded as the primary storage form of both phosphate and inositol in almost all seeds (Cosgrove, 1966). Three physiological roles have been suggested for phytic acid in seeds: (1) storage of phosphorus (Hall and Hodges, 1966; Asada et al., 1969), ( 2 ) storage of energy (Biswas and Biswas, 1965), and (3) initiation of dormancy (Sobolev and Rodionova, 1966). However, in 1970, Williams presented evidence that phytic acid serves only as a source of phosphorus and cations for the germinating seed. Phytic acid may also act as a phosphagen during germination (Asada et al., 1969). It has been suggested that phytic acid acts as a carrier or storage site for trace minerals during plant growth on the basis of its strong chelating powers (Weildlein, 1951). However, no evidence was presented to support this hypothesis (Cosgrove, 1966). Recently, Gupta and Venkatasubramanian (1975) have suggested that phytic acid plays a mycological role in the field, preventing aflatoxin production in soybean seeds by making zinc unavailable to the mold. D.

OCCURRENCE

Phytic acid (phytate), myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) is one of the widespread occurrences in plant seeds and/or grains (Posternak, 1903b; Rose, 1912; Averill and King, 1926; Belavady and Banerjee, 1953; O’Dell, 1979), roots and tubers (Rose, 1912; McCance and Widdowson, 1935), nucleated erythrocytes of birds and turtles (Rapoport, 1940; Rapoport and Guest, 1941; Oshima et al., 1964; Johnson and Tate, 1969), and organic soils (Dyer et al., 1940; Caldwell and Black, 1958). It is primarily present as a salt of monoand divalent cations (Ca2+, Mg2 , and K + ) , it is a chelating agent for cations, and it is a form of storage of cations as well as phosphorus in many seeds (Sobolev, 1966; Cosgrove, 1966). Phytate rapidly accumulates in seeds during the ripening period (Asada and Kasai, 1959, 1962; Asada et al., 1969; Sobolev, 1966; Makower, 1969; Abernethy et al., 1973; Nahapetian and Bassiri, 1975) accompanied by other storage substances such as starch and lipids. The accumulation site of phytic acid in monocotyledonous seeds (rice, wheat, barley, etc.) and dicotyledonous seeds (castor, peanuts, cottonseeds, beans, etc.) was aleurone particles or grains in the aleurone layer and globoids (which are one of the inclusions of the protein body), respectively (Sobolev, 1966; Tanaka et al., 1974; Lott and Buttrose, 1978). A typical structure of the protein body of dicotyledons with two types of inclusions is illustrated in Fig. 9. The proportion of phytic acid is up to 6&80% of the dry weight of globoids of dicotyledons (Sobolev, 1966; Lui and Altschul, 1967). The presence of phytic acid within the globoid of dicotyledonous seeds has been shown for a wide range of plants: Curcurbita maxima (Lott, 1975; Lott et al., 1978; Lott and Vollmer, +

8

N. R. REDDY ET AL External Single Membrane

Crystalloid (proteins) *

2m,

FIG. 9. Schematic view of a typical protein body as exemplified by a complete protein body with two kinds of inclusions in castor bean endosperm. From Pemollet (1978).

1979); Gossypium hirsutum (Lui and Altschul, 1967); Linum usitatissimum and Cucumis sativus (Poux, 1965); Arachis hypogea (Dieckert et al., 1962; Sharma and Dieckert, 1975); Ricinus communis (Sobolev and Rodionava, 1966; Suvorov et al., 1970; Surorov and Sobolev, 1972; Tully and Beevers, 1976; Pernollet, 1978); Glycine m a , Clianthus formosus (sturt desert pea), and Acacia sp. (Lott and Buttrose, 1978); and Vicia faba (Lott and Buttrose, 1978; Morris et al., 1970). In case of monocotyledonous seeds, phytin is located mainly in the globoids of aleurone grains (protein bodies). Aleurone grain is mainly present in aleurone layer cells of cereals and it has two inclusions (Jacobsen et al., 1971; Ogawa et al., 1975) (see Fig. 10): (1) globoids-enclosed by the globoid cavity and contain high amounts of phytate and (2) protein<arbohydrate bbdies. Presence of phytic acid within the globoids of monocotyledonous seeds has been demonstrated in Avena sativa (Buttrose, 1978), Hordeum vulgare (Jacobsen et al., 1971; Liu and Pomeranz, 1975), Tritium aestivum (Tanaka et al., 1974), High Phytin Containing

Coat (Protein and Carbohydrates)

FIG. 10. Schematic diagram showing an aleurone particle with inclusions, as in rice grains. From Ogawa et al. (1975).

9

PHYTATES IN LEGUMES AND CEREALS

Oryza sativu L. (Tanaka et al., 1973, 1977; Ogawa et al., 1975), and Sorghum bicolor L. (Adams and Novellie, 1975). A typical composition of globoids from mono- and dicotyledonous seeds is presented in Table I. Lui and Altschul (1967) reported that the isolated globoids from cottonseeds had low amounts of protein, carbohydrates, and lipids, and 60 and 10% of phytic acid and metals, respectively (Table I). Major components of globoids from peanut (Sharma and Dieckert, 1975) were as follows: protein (35.1%), phytic acid (28.0%), and metals (calcium, magnesium, and potassium) (5.0%). The globoidal fraction accounted for about 50% of the total magnesium and phytic acid, 13% of the potassium, and 80% of the calcium in the aleurone grains (protein bodies) of peanuts. Ogawa et al. (1975) reported that over 90% of the compounds of the isolated particles of rice were phytic acid, potassium, and magnesium. Isolated particles had low amounts of carbohydrates and protein. They concluded that phytic acid exists in rice as the salts of metals, mainly potassium and magnesium (Ogawa et a l . , 1977, 1979a; Tanaka et al., 1977). Current literature suggests that phytate primarily occurs as potassium-magnesium salt in rice (Ogawa et al., 1975, 1979b), wheat (Tanaka et ul., 1974), broad beans (Lott and Buttrose, 1978), castor (Suvorov and Sobolev, 1972),

TABLE I COMPOSITION OF GLOBOIDS FROM COTTONSEEDS, PEANUTS, AND RICE

Composition Nitrogen Protein Carbohydrate Phytic acid Organic phosphorus Inositol Metals Potassium Magnesium Calcium Moisture

Globoids of cottonseeda

Globoids of peanutsb

Isolated particles of ricer

(%)

(%I

(%)

0.70

-

13.85 13.21

35.1 N.S.d 28.0 7.3 7.0

6.40 1.70 1.30 9.71

2.0 2.5 0.5 8.6

-

1.35 -

1.26 0.47 67.23 -

18.89 10.83 1.39

aPercentage is calculated as weight of the composition in grams per 100 grams of the globoids (for details, see Lui and Altschul, 1967). %loboids obtained after centrifugation at 20,000 g (for details, see Sharma and Dieckert, 1975). ‘‘Isolated particles (globoids) isolated from aleurone layer of rice (for details, see Ogawa el al., 1975). dN.S., Not significant.

10

N. R. REDDY ET AL

TABLE I1 PHYTIC ACID PHOSPHORUS AND PHYTIC ACID CONTENT OF CEREALS, LEGUMES, OTHER OIL SEEDS, AND THEIR PROTEIN PRODUCTS Phytic acid phosphorus

Phytic acida

(%)

(%)

0.17-0.38 0.24 0.32 0.25 0.10

0.62-1.35 0.84 1.14 0.89 0.34

Lolas et al. (1976) Chang et al. (1977) O’Dell et al. (1972b) de Boland et al. (1975) Reddy and Salunkhe (1980)

0.04-0.05 0.04 0.62 0.25 0.28 0.14-0.53

O.l&O. 19

Rye Barley Oats Milo Sorghum (low tannin)

0.27 0.27-0.33 0.22-0.29 0.28 0.16

0.97 0.97-1.16 0.79-1.01 0.99 0.57

Sorghum (high tannin)

0.27

0.96

Toma and Tabekhia (1979) Toma and Tabekhia (1979) Harland and Harland (1980) de Boland et al. (1975) de Boland er al. (1975) Singh and Sedeh (1979); Singh and Reddy (1977) Singh and Reddy (1977) Lolas et al. (1976) Lolas et a/. (1976) Wheeler and Ferrel (1971) Radhakrishnan and Sivaprasad (1980) Radhakrishnan and Sivaprasad (1980)

0.41 0.48 0.19 0.08 0.12 0.28-0.4

1.46 1.70 0.66 0.28 0.44 1.OGI.47

Reddy et al. (1978) Reddy et al. (1978) Kumar et al. (1978) Kumar et al. (1978) Kumar et al. (1978) Lolas et al. (1976); de Boland et al. (1975)

0.29 0.25 0.33 0.11 0.19 0.5 I 0.2 1-0.45 0.15-0.21 0.15-0.3 1 0.16-0.46

I .03 0.89 1.20 0.40 0.67 1.80 0.741.58 0.55-0.75 0.54-1.10 0.56-1.63

Chang et a/. (1977) Chang et al. (1977) Welch et al. (1974) Chen and Pan (1977) Chen and Pan (1977) Bramsnaes and Olsen (1979) Lolas and Markakis (1975) Lolas and Markakis (1975) Lolas and Markakis (1975) Lolas and Markakis (1975)

Name Cereals Wheat Wheat (hard red winter) Wheat (soft) Rice (long-grain brown) Rice (polished long grain) Rice (polished medium grain) Rice (polished short grain) Wild rice Corn (commercial) Corn (high lysine) Triticale

Legumes Black gram (whole seed) Black gram (cotyledons) Green gram (whole) Chick-pea Cowpea Soybeans California small white beans Lima beans Peas Dwarf gray pea Early Alaskan pea Faba beans (dehulled) Navy beans Small white beans Red Mexican beans Great Northern beans

0.14 2.20 0.89 0.99 0.5G1.89

Reference

11

PHYTATES IN LEGUMES AND CEREALS

TABLE I1 (Confinued) Phytic acid phosphorus

Phytic acido

(%)

(%)

Name

Reference

Pinto beans

0.17-0.55

0.61-1.95

Red kidney beans

0.34-0.58

1.20-2.06

Peanut meal (dehulled, defatted)

0.48

1.70

Dieckert et a / . (1962)

1.33 1.45 1.33

4.71 5.16 4.72

Toma et a/. (1979) Toma et a / . (1979) Toma e f al. (1979)

I .46 1.12

5.18 2.00-3.98

0.83

2.94

de Boland et af. (1975) Anderson et al. (1976); Ohlson and Anjou (1979) Wozenski and Woodburn

Other Oil Seeds Sesame seed (whole) Sesame seed (dehulled) Sesame seed (roasted) Sesame seed meal (defatted) Rapeseed

Cottonseed flour (glanded)

Lolas and Markakis (1975); Iyer et al. (1980) Lolas and Markakis (1975); Iyer et al. (1980)

( 1975)

Cottonseed flour (glandless)

1.35

4.80

Wozenski and Woodburn ( 1975)

Cottonseed toasted kernels (glandless)

0.73

2.60

Wozenski and Woodburn ( 1975)

Protein Products Soyflakes (defatted) Soy protein isolate

0.43 0.12-0.33

1.52 0.43-1.17

Soy protein concentrate Soybean meal (full-fat or defatted and hulled) Rapeseed protein concentrate

0.35-0.6 1

1.24-2.17

de Boland et al. (1975) Wallace and Satterlee (1977); Ranhotra et a / . (1974a) Ranhotra et al. (1974a)

0.39-0.45

I .4GI.60

Erdman ( 1979)

1.49-2.I 1

5.30-7.50

Wheat protein concentrate

0.53-0.76

1.88-2.70

Wheat gluten

0.60

2.13

Erdman (1979);Ohlson and Anjou (1979) Ranhotra (1972); Ranhotra et a / . (1974a) Nelson and Potter (1979); Wallace and Satterlee ( 1977)

“Calculated phytic acid content, assuming 28.20% phosphorus in the molecule.

12

N. R. REDDY ET AL

sesame (O’Dell and de Boland, 1976), and Cucurbita sp. (Lott, 1975; Lott et al., 1978), and as calcium-magnesium-potassium salt in soybeans (Ford et al., 1978). E.

PHYTATE CONTENT

Phytic acid phosphorus and phytic acid contents of cereals, legumes, other oil seeds, and their protein products are presented in Table 11. The amount of phytic acid varies from 0.50 to 1.89% in cereals (except polished rice), from 0.40 to 2.06% in legumes, from 2.00 to 5.20% in oil seeds except soybeans and peanuts (grouped under legumes), and from 0.40 to 7.50% in protein products. In polished rice (long-, medium-, and short-grain) phytic acid content ranged from 0.14 to 0.34%. A range of 1.21-1.62 mmol of phytic acid (on the basis of myoinositol values) for proso- and foxtail-type millets has been recently reported (Becker and Lorenz, 1978). Among cereals, legumes, and other oil seeds, sesame had higher amounts of phytic acid. In many cases, the phytic acid content is not considered to be absolute and may vary depending upon the variety andlor cultivar, climatic conditions, locations, irrigation conditions, type of soil, and year during which they are grown. Bassiri and Nahapetian (1 977) observed that wheat varieties grown under dry land conditions had lower concentrations of phytate than the ones grown under irrigated conditions. Nahapetian and Bassiri (1976), Singh and Reddy (1977), Singh and Sedeh (1979), and Miller et al. (1980a,b) reported variations in phytic acid content of triticales, wheat, rye, and oats grown at different locations and in different years. Phytic acid phosphorus in several seeds and grains constitutes the major portion of total phosphorus (Nelson et al., 1968). For example, phytic acid phosphorus represents the following percentages of the total phosphorus: 81.0% in brown rice, 51.0% in polished long-grain rice, 60.&80.0% in wheat, 66.0-70.0% in barley, 59.0-66.0% in oats, 18.0-53.1% in triticale, 83.0-88.0% in corn, 71.7% in low-tannin and 88.9% in high-tannin sorghum, 70.0% in California small white beans, 79.0% in black gram, 50.@58.% in soybeans, 69.0% in green gram, 40.0% in chick-pea, 38.0% in cowpeas, 57.0-81 .O% in navy beans, 68.&72.0% in red kidney beans, and 55.0-77.0% in Great Northern beans (O’Dell et al., 1972b; Lolas and Markakis, 1975; Lolas et al., 1976; Chang et al., 1977; Kumar et al., 1978; Reddy et al., 1978; Singh and Reddy, 1977; Radhakrishnan and Sivaprasad, 1980; Reddy and Salunkhe, 1981). In cereals, phytic acid is associated with specific components within the grain and can be preferentially extracted with those components. Phytic acid concentration in morphological components or parts of cereal grains is presented in Table 111. The endosperm of wheat and rice kernels is almost devoid of phytic acid, because it is concentrated in the germ and aleurone layers (pericarp) of the kernel cells. Corn differs from most other studied cereals in that 88.0% of the phytic acid is concentrated in the germ

13

PHYTATES IN LEGUMES AND CEREALS

TABLE I11 PHYTIC ACID CONCENTRATION IN MORPHOLOGICAL COMPONENTS OF CEREALSO Phytic acid P

Phytic acidb

Cereal

Sample

(%I

(%)

Corn

Commercial hybrid Endosperm Germ Hull High lysine Endosperm Germ Hull Soft Endosperm Germ Hull Aleurone Brown Endosperm Germ Pericarp

0.25 0.01 1.80 0.02 0.27 0.01 1.61 0.07 0.32

0.89 0.04 6.39 0.07 0.96 0.04 5.72 0.25 1.14 0.004 3.91 0.00 4.12 0.89 0.01 3.48 3.37

Corn

Wheat

Rice

0.001 1.10 0.00 1.16 0.25 0.004 0.98 0.95

Distribution‘ (%o) -

3.20 88.00 0.40 -

3.00 88.90 1.50 -

2.20 12.90 0.00 87.10 1.20 7.60 80.0

“Calculated from the data of O’Dell er al. (1972b) and all the values are based on air dry weight. bPhytic acid content calculated by assuming that it contains 28.20% phosphorus. CPercentageof element in the component part.

portion of the kernel (O’Dell et al., 1972b). Corn endosperm had small amounts (3.2%) of phytic acid. Rice and wheat germ contained appreciable amounts of phytic acid, but the major portion of phytic acid was in the aleurone layers. In rice, of the total phytic acid, 84.0-88.0% was reported to be in bran (Resurreccion et al., 1979). In dicotyledonous seeds, including legumes and oil seeds, phytates are distributed throughout the cotyledon and located within the subcellular inclusions of aleurone grains or protein bodies. Data on phytic acid content of whole grain, flour, and bran for triticales, wheat, and rye are presented in Table IV. Because most of the phytic acid in cereals is located in the aleurone layers (bran), milling of cereals and subsequent separation of bran results in significant reduction of phytic acid in flours. Data in Table IV support this (bran has higher amounts of phytic acid than does the flour) (Reddy, 1976). Harland and Oberleas (1977) evaluated several soy protein-based food products for their phytate content by employing an ion-exchange chromatographic method. The concentration of phytate in 10 soy protein-based meat substitutes ranged from 0.12 to 1.63% (Table V). Harland and Harland (1980) reported that

TABLE IV PHYTIC ACID CONTENT IN TRITICALE, WHEAT, AND RYE AND THEIR MILLED FRACTIONSp BRAN, AND FLOUR Phytic acidb (%) Cultivar

Whole grain

Triticales 6TA 131 6TA 203 6TA 204 6TA 205 6TA 385 6TA 418 6TA 419 6TA 514 72-S NB-69150 K.S. Bulk Mark IV 8X Triticale

Bran

Flour

0.81C 1.08 0.52 0.49 1.47 0.59 I .25 0.83 0.52 1.27 1.89 0.88 1.35

0.47~ 0.61 0.47 0.52 0.67 0.61 0.78 0.32 0.47 0.61 0.47 0.31 0.98

0.25c 0.19 0.33 0.19 0.41 0.33 0.48 0.20 0.36 0.45 0.37 0.33 0.18

0.94

0.85

0.20

0.97

0.61

0.33

Wheat

Arthur-7 I RY e Abruzzi

‘‘Milled fractions obtained after milling with Brabender Quadrumat Junior Mill. hCalculated from data of Reddy (1976) assuming 28.20% phosphorus in the phytic acid. cData expressed on dry weight basis.

TABLE V PHYTATE CONTENT OF SOYBEAN-BASED FOODSTUFFSa Phytate contenth Foodstuff

(%)

TVPc pork TVP bacon TVP ham TVP beef TVP beef chunks TVP bacon + vitamins TVP unflavored + vitamins Chicken analog Ham analog Textured soy concentrate

1.42 0.95 1.26 1.36 1.36 1.15 1.63 0.27 0.12 I .50

aSource: Harland and Oberleas (1977). hValues are based on the ion-exchange phosphorus assay method. (‘TVP, Textured vegetable protein.

15

PHYTATES IN LEGUMES AND CEREALS TABLE VI PHYTATE IN BREADSO Phytate Breads

(% dry wt.)

Corn bread Whole wheat RYe Pumpernickel Raisin French White

1.36 0.56 0.41 0.16 0.09 0.03 0.03

“Source: Harland and Harland (1980).

the phytate content in different breads varied from 0.03% in French and white breads to 1.36% in corn bread (Table VI).

II. CHEMISTRY A.

ANALYTICAL METHODS FOR PHYTIC ACID DETERMINATION

Many foods and seeds contain myo-inositol hexaphosphate as an important source of phosphorus, and accurate methods for its determination are needed. The measurement of phytate in any material requires an initial extraction. Dilute hydrochloric acid (HCI) and trichloroacetic acid (TCA) are the most common extractants used for extraction. Methods for the analysis of phytate have been extensively reviewed (Oberleas, 1971; Cosgrove, 1966). There are no specific reagents that identify phytate, nor does it have a characteristic absorption spectrum. The determination of phytate is dependent on estimating the components of inositol or phosphate or upon establishing a stoichiometric relationship between phytate and certain cations that can be measured easily. The most common method used for phytic acid determination utilizes the precipitation method based on the method of Heubner and Stadler (1914). This method is based on the principle that phytate forms an insoluble stable complex with ferric ion in dilute acid solution and presumably is the only phosphate compound with that property. Inorganic phosphate is not precipitated under these conditions and it was believed that organic phosphates other than phytic acid also remain in the solution. In the original procedure, the phytate-containing extracts were titrated with standardized ferric chloride solution (using ammonium thiocyanate as an internal indicator). Formation of white ferric phytate precipitate during titration makes it difficult to judge the end point in this method. Harris and Mosher (1934) modi-

16

N. R. REDDY ET AL.

fied this method by titrating beyond the end point, removing the femc phytate precipitate by filtration, and determining the excess iron colorimetrically. Precipitation methods can be divided into two categories: (1) the direct method and (2) the indirect method. In the direct method, insoluble stable ferric inositol hexaphosphate precipitate is removed, and the phosphorus content of the precipitate is determined after wet ashing or hydrolysis; or alternatively, the inositol content of the precipitate is determined. In the indirect method, a known standard quantity of ferric chloride is added to the extract and the concentration of unprecipitated ferric iron is measured by a standard colorimetric method. The accuracy of phytate determination depends on the satisfactory precipitation of ferric phytate from the acid extract and the known iron-to-phosphorus conversion factor. Although there are several factors that may affect the accuracy of the results, the direct methods are among the most popular ones for determining phytate in food- and feedstuffs (McCance and Widdowson, 1935) due to ease of handling and fair reproducibility. McCance and Widdowson (1935) have modified the indirect method. The modification involves direct measurement of phosphorus in the precipitate by converting ferric phytate to soluble sodium phytate using sodium hydroxide. The phosphorus content of the soluble sodium phytate is then determined colorimetrically after wet acid digestion. The long time requirement for the acid digestion in this method, however, is undesirable. To overcome this problem, several researchers (Pons et al., 1953; Hegge and Rein, 1948; Oberleas, 1964) modified this method. Hegge and Rein (1948) suggested hydrolysis of soluble sodium salt and the direct estimation of resulting free inositol by the periodate oxidation method. Oberleas (1964) claimed improved reproducibility and reduced analysis time. The indirect method for phytate determination was first introduced by Young (1936). In this method, the loss of added ferric iron, due to precipitation as ferric phytate, is determined by measuring the remaining iron in the solution in the form of ferric thiocyanate by the standard colorimetric method. The accuracy of this method depends on the efficiency of precipitation of ferric phytate having a known constant iron-to-phosphorus ratio. Young (1936) used an experimentally determined factor of 1.06 to convert iron to phosphorus, whereas the theoretical factor was 1.20. In a later investigation, however, Earley (1944) found that under carefully controlled conditions, the theoretical value of the conversion factor was applicable. Most workers (Earley, 1944; Young, 1936; Samotus and Schwimmer, 1962; Wheeler and Ferrel, 1971; Magrill, 1972; Eklund, 1975) experimentally obtained Fe:P ratios of 3.54.6:6 while determining phytate in several food products and seeds and grains. A ratio of 4:6 (Fe:P) is now generally used in determination of phytate in foods. This ratio supports the Anderson structure for phytic acid, which favors the formation of ferric phytate from the trivalent cation. The indirect methods are generally more convenient and re-

PHYTATES IN LEGUMES AND CEREALS

17

producible. However, when the phytic acid level in the food under examination is low, the indirect method is subject to large errors because the results are based on a small difference of two relatively large numbers (Makower, 1970). Samotus and Schwimmer (1962) have pointed out that high results by the indirect method can be due to the presence of reducing substances such as the ascorbic and chlorogenic acids. Reduction of Fe3 to Fez by these reducing agents can yield high values. Addition of 30% hydrogen peroxide prior to precipitation with ferric chloride was suggested to overcome this difficulty. Marrese et al. (1961) compared three methods for phytate determination in plant materials and found that the volumetric and gravimetric methods gave much higher values compared to the method based on ion-exchange chromatography which separates the hexaphosphate from other inorganic and lower inositol phosphates. The lower inositol phosphates may react chemically like inositol hexaphosphate (phytic acid), leading to high results for phytic acid determination. By definition, however, these lower phosphates can not be regarded as phytic acid (Anderson, 1956). In the case of mature seeds and grains such as wheat, corn, rice, soybean, sesame, and others, the concentration of lower phosphates appears to be insignificant (de Boland et al., 1975). Presence of excess iron in the extract (either by too much addition of ferric chloride or due to naturally occurring high iron content in the food itself) shifts the equilibrium resulting in the formation of soluble ferric phytate, thus leading to the underestimation of phytate content (Anderson, 1963). According to the findings of Anderson (1963), recovery of phytate in barley was only 80% when the soluble iron-to-phytate phosphorus ratio was about 0.33:l.O. Thus when extracting material contains great excess of iron, it poses problems in phytate determination. With proper modifications the indirect method has been recently used in the microdetermination of phytate in the solution or bound to hydroxyapatite in concentrations up to M (Magrill, 1972), however. Makower (1970) reported that the indirect method is not suitable for determination of small amounts of phytic acid in immature beans. In another study, Wheeler and Ferrel (1971) reported a modified McCance and Widdowson (1935) method to determine phytic acid in cereals and their mill feed fractions by using a known ratio of Fe:P. In this method, phytate is extracted with trichloroacetic acid and precipitated as femc phytate. The ferric phytate is then converted to ferric hydroxide (precipitate) and soluble sodium phytate by adding sodium hydroxide and boiling. The precipitate is then dissolved in dilute acid and the iron content is determined colorimetrically. The phytate content is calculated from the iron concentration by assuming a constant Fe:P molecular ratio of 4:6 in the precipitate. Recently, Harland and Oberleas (1977) developed yet another method for determination of phytic acid in textured vegetable proteins using an ion-exchange procedure. The method involved concentrating phytic acid on an anion-exchange resin, stripping the resin of contaminating inorganic phosphate with 0.05 M sodium chloride, and eluting phytate with 0.7 M +

+

18

N. R. REDDY ET AL

sodium chloride. The eluate was then digested and inorganic phosphate determined by the standard colorimetric method. The method, however, gave results slightly lower than those of the iron precipitation method. The method has been claimed to be reproducible. Ellis et al. (1977) reported a method for phytate determination in the presence of high inorganic phosphate. This method is also based on precipitation and determination of phosphate colorimetrically . Oberleas (197 1) has reviewed methods for qualitative and quantitative determination of phytic acid, including paper, thin-layer, and ion-exchange chromatographic methods. Methods for determination of phytate in specific foods have also been described. One such example is the method described by Makower (1970) for determination of phytic acid in beans. In this method, the beans are extracted with an acid, the phytate is converted to ferric phytate by adding ferric chloride solution, ferric phytate in turn is converted to ferric hydroxide (by using sodium hydroxide), and the iron content of ferric hydroxide is determined by the colorimetric method. He concluded that the colorimetric determination of iron in ferric phytate (after initial conversion to ferric hydroxide) has the advantage of greater speed and convenience, and improved separation from interfering ironcontaining materials in comparison with the iron or phosphorus determination in ashed ferric phytate. In summary, most of the methods for phytic acid determination are based on precipitation of phytic acid wherein the phytate is converted to ferric phytate by adding ferric chloride solution. Addition of small amounts of sodium sulfate solution aids the formation of ferric phytate precipitate. The precipitate is then converted to ferric hydroxide. The iron content of the ferric hydroxide is then determined colorimetrically , after acid hydrolysis of the precipitate. Alternatively, the phosphorus content of the ferric phytate precipitate can be determined colorimetrically after the wet-acid hydrolysis of the ferric phytate precipitate. The iron or the phosphorus content is then related to phytic acid concentration. Care must be taken to use conditions so that the ratio of Fe:P is maintained as close to 4:6 as possible, when using the indirect method for phytic acid determination. Reddy et al. (1978) have found that using o-phenanthroline reagent or a,a-dipyridyl reagent, instead of the recommended potassium thiocyanate, is more convenient for colorimetric determination of iron in acid solution.

B . PHYTATE-PROTEIN-MINERAL

INTERACTIONS

The titration curve for phytic acid has been reported to have two inflections (Maddaiah et al., 1964; Crean and Haisman, 1963; Vohra et al., 1965). Crean and Haisman (1963) reported that the two inflections were at pH 4.9 and 8.1, respectively. They also stated that of the 12 replaceable hydrogen atoms of phytic acid, six are strongly dissociated with a pK value of about 1.8, two are

PHYTATES IN LEGUMES AND CEREALS

19

weakly dissociated (pK value of about 6.3), and the remaining four are so feebly acidic (pK value about 9.7) that they cannot be determined. Blank et al. (1971) reported the structural characterization of phytate employing X-ray crystallography. They reported that phytate has the hexaorthophosphate ester structure and that the phosphates at positions C-1, 3 , 4 , 5 , and 6, are axially disposed with that of the C-2 equatorial. Phytic acid in free form is quite unstable (Vohra et al., 1965) and decomposes to yield orthophosphoric acid. The ability of phytic acid to complex with proteins and particularly with minerals has been a subject of investigation for several reasons but predominantly from chemical and nutritional viewpoints. Crystalline preparation of phytate-protein complex at pH 4.0 has been reported by Bourdillon (195 1) from the Great Northern bean (Phaseolus vulgaris L.) and several physiochemical properties were reported. The interaction between phytic acid and proteins is thought to be of ionic type (de Rham and Jost, 1979). The interaction between phytate and proteins leads to decreased solubility of proteins (Bourdillon, 1951; Courtois and B a d , 1953; Smith and Rackis, 1957; Cheryan, 1980). It has also been shown that calcium ions interact with protein and phytate to further decrease the solubility of proteins (Wolf and Briggs, 1959; Saio et al., 1967). Barr6 (1956) reported that the phytate-protein complexes are less subject to proteolytic attack than the same protein alone. Complexation between phytate and proteins has been reported for several proteins, including those from bran, wheat, and oats (Hill and Tyler, 1954); the Great Northern beans (Bourdillon, 1951); corn germ, soybean flakes, and sesame meal (O’Dell and de Boland, 1976); soybeans (Saio et al., 1967, 1968; Wang, 1971; de Rham and Jost, 1979; Okubo et al., 1976; Rackis and Anderson, 1977; Omosaiye and Cheryan, 1979); peanuts and cottonseed (Fontaine et al., 1946); and black gram (Reddy, 1981). Phytic acid is known to form complexes with proteins at both acidic and alkaline pH. Omosaiye and Cheryan (1979) and Cheryan (1980) offered the following explanation:

Low pH: This is attributed to strong charge effects. Six of 12 dissociable protons of phytic acid are strongly dissociated with a pK of about 1.8 (Crean and Haisman, 1963). Thus phytate is strongly negatively charged below the acidic pH range (pH of about 2.0), while proteins are strongly positively charged at pH 2.0. As a result, phytate-protein complex could be formed under such circumstances (Hill and Tyler, 1954; Saio et al., 1967; Okubo et al., 1975, 1976). This complexation, it is suggested (Smith and Rackis, 1957), is rapid and followed by nonionic irreversible reactions. It infers, therefore, that this nonionic irreversible reaction confers the stability to the phytate-protein complex. High pH: Strong protein-phytate interaction is suggested at alkaline pH (O’Dell and de Boland, 1976), which is apparently different than at low pH in that the electrostatic effects are minor at alkaline pH. Multivalent cations such as

20

N. R. REDDY ET AL.

Ca2+ (at certain necessary concentrations) are thought to mediate such phytate-protein complexes (Saio et al., 1967; Okubo et al., 1976). It is therefore suggested that such ternary complexes of phytic acid be formed by the following mechanisms (Omosaiye and Cheryan, 1979): Cation Protein

+ phytic acid

+ cation + phytic acid

(cation-phytic acid)

(1)

(protein-cation-phytic acid)

(2)

Saio et al. (1967) suggested that these ternary complexes are fairly labile in alkaline solution. Conceivably, if proteins are to form complexes with phytic acid, they must have some electrical charge on them, which is possible at below or above the isoelectric pH of the protein. This is evident from several studies indicating involvement of lysyl, histidyl, arginyl, and amino terminal groups in the formation of phytate-protein complexation (Hill and Tyler, 1954; Okubo et al., 1975; Omosaiye and Cheryan, 1979; de Rham and Jost, 1979; Saio et al., 1967; Cosgrove, 1966). Reddy and Salunkhe (198 1) suggested that phytate-protein complexes did not form in black gram albumins at pH 6.40 (which is near to the isoelectric pH of black gram albumins), supporting the view that protein must to be in a charged state to form the phytate-protein complex. Nutritional implications of phytate-protein complexes are still under scrutiny (O’Dell, 1979). The technological importance is, however, appreciated (Hartman, 1979). The reduced solubility of proteins as a result of protein-phytate complexes can adversely affect certain functional properties of proteins which are dependent upon their hydration and solubility, such as hydrodynamic properties (viscosity, gelation, etc.), emulsifying capacity, foaming and foam performance, and dispersibility in aqueous media. In addition, reduced bioavailability of phosphorus is a distinct possibility. Phytate-mineral complexes are well-documented (Oberleas, 1973; Erdman, 1979). Phytate, being a strong acid, forms a variety of salts with heavy metals such as zirconium, thorium, titanium, and uranium in 6 N acid (Alimarin and Tozel, 1957; Ryabchikov et al., 1956). The solubility of such metal complexes of phytate is pH dependent, as would be expected. The nutritionally important minerals, such as calcium, magnesium, copper, iron (Fe2+ and Fe3+), and others form complexes with phytic acid resulting in reduced solubility of the metals. Synergistic effects of divalent metal ions in the formation of metal-phytate complexes have also been reported (Oberleas, 1964, 1973; Oberleas et al., 1966a,b). The decreasing order of metal complexation has been reported to be Cu2+ > Zn2+ > Co2+ > Mn2+ > Fe3+ > Ca2+ (Oberleas, 1973) at pH 7.4. Nutritionally, more important is the fact that at pH 6 (approximate pH of the duodenum, where maximum absorption of divalent metal ions takes place) max-

PHYTATES IN LEGUMES AND CEREALS

21

imum precipitation of zinc phytate or zinc-calcium-phytate occurs. Similar observations are reported for copper, calcium, and phytate complexes (Oberleas, 1973). Decreased iron availability due to iron-phytate complexes is also of concern (Anonymous, 1967; Koepke and Stewart, 1964; Murray and Stein, 1970a,b; Multani et al., 1970; Erdman, 1979). Magnesium availability also decreases through complexation with phytate (Seelig, 1964). In conclusion, most of the available information on phytate-mineral interactions stems from studies on soy proteins. The contribution of fiber and other food constituents capable of binding with minerals and the proteins will undoubtedly have effects on such interactions. Also the data on soy proteins may not be completely applicable to other types of beans and food systems. C.

PHYTASE ENZYME

Phytase (meso-inositol hexaphosphate phosphohydrolase, EC 3.1.3.8) is widely distributed in plants, animals, and fungi (Cosgrove, 1966). Phytase dephosphorylates free inositol phosphates. Several investigators have isolated and characterized phytases from different sources, including cereals such as triticale (Singh and Sedeh, 1979), wheat (Peers, 1953; Nagai and Funahashi, 1962; Lim and Tate, 1973), corn (Chang, 1967), barley (Preece and Gray, 1962), and rice (Suzuki et al., 1907; Yoshida et al., 1975); beans such as navy beans (Lolas and Markakis, 1977), mung beans (Mandal and Biswas, 1970; Mandal et al., 1972; Maiti et al., 1974; Maiti and Biswas, 1979), dwarf beans (Gibbins and Norris, 1963), and California small white beans (Chang, 1975); and animals such as rats, chickens, calves, and humans (Bitar and Reinhold, 1972). Phytase has been found in fungi, bacteria, and plant leaves as well (Patwardhan, 1937). The first preparation of phytase was reported by Suzuki et al. (1907) from rice bran, and that the intestine has the ability to split phosphate from phytic acid was first shown by Patwardhan (1937), who attributed this action to phytase. Although the presence of phytase activity in human intestine is demonstrated (Bitar and Reinhold, 1972), the importance of this activity insofar as phytate destruction is concerned is not known. The phytase acts on inositol hexaphosphate to yield inositol and orthophosphate, via inositol penta- to monophosphates as intermediary products. Plant seeds are rich in phytate and both phytate and phytase are present in most plant seeds. Phytase activity usually increases on germination (Peers, 1953; Mayer, 1958; Ashton and Williams, 1958; Gibbins and Norris, 1963; Mandal and Biswas, 1970; Mandal et al., 1972; Fordham et al., 1975; Walker, 1974; Chen and Pan, 1977; Kuvaeva and Kretovich, 1978). The mode of action of phytase has remained controversial. Maiti et al. (1974) reported that degradation of phytate by phytase occurs in a stepwise manner starting with dephosphorylation from position 6 followed by removal of phosphorus from positions 5 and 4, 1 and

TABLE VII OPTIMUM pH, TEMPERATURE, AND MICHAELIS-MENTEN CONSTANT K , OF PHYTASES FROM CEREALS AND LEGUMES Optimum Phytase source

P"

Triticale Corn Wheat flour Wheat bran Rice aleurone particles Navy bean California small white bean Dwarf french bean Mung beans (germinating)

5.4 5.6 5.15 5.0 4.0-5.0 5.3 5.2 5.2 7.5

Temperature ("C)

45 50 55 -

45 50 60 40 57

Michaelis-Menten constant K , ( M )

0.22 0.99 0.33 0.57

x 10-3

x 10-3 x 10-3 x 10-3 -

0.018 x 10-3 2.22 x 10-4 0.15 x 10-3 0.65 x 10-3

Reference Singh and Sedeh (1979) Chang (1967) Peers (1953) Nagai and Funahashi (1962) Yoshida et al. (1975) Lolas and Markakis (1977) Chang (1 975) Gibbins and Norris (1963) Mandal and Biswas (1970)

23

PHYTATES IN LEGUMES AND CEREALS

3, or 1 and 4, the phosphate at position 2 being stable, and stated that this mode of action was different than that reported by others (Lim and Tate, 1973). Lim and Tate (1973) showed that the F-2 fraction from wheat bran phytase attacked the phytate molecule at the 2, D-4, and 5 positions. Maiti et al. (1974) concluded that this difference in mechanism was probably due to the differences in phytases of mung bean and wheat bran. The pH and temperature optima for phytases from certain cereals and legumes are summarized in Table VII. In general, the optimum pH for the phytase activity appears to be in the range 4-7.5. The optimum temperature for phytase activity varies somewhat from source to source but appears to be in the high temperature range (4540°C). This high optimal temperature for the activity of phytase may be advantageous in food processing operations involving high temperatures (such as drying or baking) (Ranhotra, 1972, 1973; Ranhotra et al., 1974b). Phytases from different cereals have been shown to be resistant to dry heat (McCance and Widdowson, 1944). The ability of man to hydrolyze phytates remains controversial (Ranhotra and Loewe, 1975). The hydrolysis of phytate which occurs in the digestive tract is probably due to microbial phytases or nonenzymatic cleavage (Nicolaysen and Njaa, 1951; Hegsted et al., 1954; Subrahmanyan et al., 1955). Phytase inhibitors include phytate precipitants such as Cu2 , Zn2 , Fe3 , Ca2+, F- , inorganic phosphorus, and phytate; whereas activators include Ca2 , Mg2 , CN - , SCN- , oxalate (although they can act as inhibitors as well under certain conditions), and vitamin D (Long, 1961). +

+

+

+

+

Ill. NUTRITIONAL IMPLICATIONS A.

DIGESTION OF PHYTATE AND ITS BIOAVAILABILITY

In mature cereal grains, legumes, and oil seeds, the major portion of the total phosphorus is present in the form of phytic acid (phytate). Phytase hydrolyzes phytate into inositol and phosphates or phosphoric acid. The availability of phosphorus when present in the form of phytate depends on the species, the age of the experimental animal, and the level of phytase activity in the intestinal tracts of the specific species. Phytate is regarded generally as being less biologically available than the most inorganic phosphorus. Reid et al. (1947) reported that sheep could utilize natural phytate and that most of the hydrolysis occurred in the rumen in less than 8 hr. Raun et al. (1956), using an artificial rumen technique, showed that rumen microorganisms from a steer hydrolyzed calcium phytate, suggesting the presence of phytase. Since then, several studies (Mathur, 1951; Plumlee et al., 1955; Tillman and Brethour, 1958; Lofgreen, 1960; Ellis and Tillman, 1961; Wilson, 1975) reported that ruminants (sheep and cattle)

24

N. R. REDDY ET AL

were able to utilize most of the dietary phytate. Biological values for dietary phytate in sheep and dairy cattle were stated to be 66 and 50%, respectively (Mathur, 1951; Lofgreen, 1960). Recently, Nelson et al. (1976) studied the hydrolysis of natural phytate phosphorus from soybean meal, sorghum grains, and corn meal in the intestinal tract of calves and steers. No phytate phosphorus was found in the feces of steers, but traces of phytate phosphorus were recovered in the feces of younger calves (Table VIII). No phytate was recovered from the contents of the rumen, abomasum, small and large intestines of calves fed a diet composed primarily of soybean meal and sorghum grains. They concluded that the initial phytate hydrolysis occurred in the rumen and was complete before the feed reached the other parts of the digestive system. Swine can utilize variable amounts of phytate phosphorus. From several studies (Bayley and Thompson, 1969; Woodman and Evans, 1948; Besecker et al., 1967; Noland et al., 1968), summarized in Table IX, involving several techniques, the availability of phosphorus from phytate phosphorus source has been found to range from 20 to 60%, with an average value of 33% for pigs weighing 50 to 90 Ib. There is some indication that the ability of the swine to utilize phytate phosphorus improves with age. A recent study of Calvert et al. (1978) also demonstrated that natural phytate from barley and corn is poorly available to growing swine. Pierce et al. (1977) studied the availability of phytate phosphorus to growing pigs receiving wheat- and/or corn-based diets. They concluded that the growing pigs (1 1-14 kg) were able to grow and develop normally when fed wheat- or corn-based diets containing 0.30% phytate with supplemental phosphorus as dicalcium phosphate. Overall performance and development was impaired when pigs were fed similar diets but containing 0.38% phytate. Phytate in cereal grains and other plant foodstuffs has long been considered to be virtually unavailable to chicks or rats when supplied either in its natural form or extracted as the calcium or sodium salts (Nelson, 1967). Most of the research has focused on the availability of phosphorus from phytate phosphorus to poulTABLE VIII HYDROLYSIS OF PHYTATE PHOSPHORUS BY CALVES AND MATURE STEERSa Phytate phosphorus

Steersb Calvesc

Intake (8)

Excreted

Hydrolyzed

(s)

(%)

71 20

0 0.06

100

aSource: Nelson ef a / . (1976). bSteers, 9 months of age, with average weight of 200 kg. “Calves, 56 days old.

99

25

PHYTATES IN LEGUMES AND CEREALS TABLE IX BIOLOGICAL VALUE OF PHYTATE PHOSPHORUS FROM SWINE

Researcher Bayley and Thompson Woodman and Evans Besecker et al. Noland et al. Average

Year 1969

1948 1967 1968

Pig weight (lb)

Biological availability

60 5C-90 50

20-30

Growing

3C-60 2540

(%I 3 M O

18-24

try. Extensive studies have been done in this area. Heuser et al. (1945), McGinnis et al. (1944), Singsen et al. (1947), Gillis et al. (1949), and Sunde and Bird (1956) noticed that natural phytate was a poor source of phosphorus for various species of poultry. In contrast, Sieburth et al. (1952) reported that the phosphorus in finely ground whole wheat flour was almost completely available to chicks for growth but was less available than inorganic phosphate for bone deposition. Temperton et al. (1965a-c) concluded that pullet chicks less than 4 weeks old, growing pullets reared to 18 weeks of age, and laying hens were able to utilize effectively the organic sources of phosphorus for growth and bone formation. Several investigators had fed poultry with various isolated impure phytates as a source of phosphorus. Lowe et al. (1939) reported that chicks did not efficiently utilize phytate phosphorus isolated from wheat bran. Singsen and Mitchell (1945) and Matterson et al. (1946) found calcium-magnesium phytate to be a poor source of phosphorus for the turkey poult. Gillis et al. (1948) showed that chicks were unable to utilize relatively pure calcium phytate. Conversely, Harms et al. (1962) and Waldroup et al. (1964) concluded that the phosphorus in phytic acid was highly available to the chick. Gillis et al. (1953) studied the quantitative utilization of phytate phosphorus by white leghorn hens. They found that phosphorus from isolated calcium phytate was biologically less available (less than 50%) than that from dicalcium phosphate as indicated by mortality, egg production, and bone mineral changes. In other studies (Waldroup et al., 1967; Singsen et al., 1969) phytate phosphorus was found to be 30 and 80% available to laying hens. A summary of biological values from several studies for the laying hen is shown in Table X. As with other animals, few investigators have studied the quantitative biological utilization of phytate phosphorus by chicks and turkeys. Gillis et al. (1957) fed chicks and turkeys 32P-labeledcalcium phytate and 32P-labeledmonosodium orthophosphate and then measured the amount of radioactivity retained in the tibia. They concluded that chicks used only 10% of the phosphorus from calcium

26

N. R. REDDY ET AL TABLE X BIOLOGICAL VALUE OF PHYTATE PHOSPHORUS (CALCIUM PHYTATE) FOR LAYING HENS Biological availability Researcher

Year

(%I

Gillis et al. Waldroup et al. Singsen er al. Average

1953 1967 1969

50 30 80 54

~

phytate as effectively as that from monosodium orthophosphate and the corresponding utilization of calcium phytate phosphorus by the turkey was less than 2%. Ashton et al. (1960) fed 32P-labeled calcium phytate and observed that 4week-old chicks retained approximately 20% of the phytate phosphorus compared to 6-week-old chicks that retained 3 6 4 9 % of the phytate phosphorus consumed. They concluded that chicks utilized only 20% of the supplied phytate phosphorus. Temperton and Cassidy (1964a) reported that chicks retained approximately 60% of the phytate phosphorus in their body. They also indicated that the chicks utilized phytate phosphorus from foodstuffs of plant origin for deposition in the growing bones. This was confirmed by their later study (Temperton and Cassidy, 1964b). Nelson et al. (1968, 1971) found that the addition of a mold phytase preparation to a diet containing natural phytate phosphorus increased the availability of phytate phosphorus to chicks. A review by Peeler (1972) indicates phytate phosphorus is intermediate in biological availability for adult poultry, but is very low in biological availability for young poultry. Nelson ( 1976) employed the chromic oxide balance method to measure the amount of natural phytate hydrolyzed by chicks and laying hens. He found that 4- and 9-week-old chicks and laying hens (single comb white leghorn), respectively, hydrolyzed 0, 3, and 8% of the natural phytate when the diet contained corn as the only grain source (Table XI). He observed that 4- and 9-week-old chicks and laying hens hydrolyzed 8, 13, and 13%, respectively, of natural phytate when 50% of the corn was replaced by wheat in the diet (Table XII). Adverse effects on phosphorus retention in growing chicks by phytic acid from soybean, palm kernel, cottonseed, and rapeseed meals are also reported (Nwokolo and Bragg, 1977). As discussed earlier, a wide disagreement has existed between investigators on the ability of poultry to utilize phytate. The disagreements could be due to variations in their experimental methods and materials. These variables include species differences, age of the test animals, the source of phytate, criteria of response, and the levels of calcium and vitamin D used in the experimental diets. Some of these factors have been well discussed by Nelson (1967).

27

PHYTATES IN LEGUMES AND CEREALS TABLE XI AVERAGE PHYTATE HYDROLYSIS BY CHICKS AND LAYING HENS” Phytate hydrolyzed (%) Poultry

Diet l b

Diet 2‘

Chicks, 4 weeks old Chicks, 9 weeks old Laying hens

0 ? 0.9 3 2 1.0 8 ? 1.7

8 ? 0.8 13 2 0.5 13 ? 1.2

“Source: Nelson (1976). bDiet 1 consisted of 54.05% ground yellow corn, 37.00% soybean meal (49% protein), 5.0% soybean oil, and other salt and vitamin mixtures. cDiet 2 consisted of 27.025% wheat, 27.025% ground yellow corn, 37.0% soybean meal, 5% soybean oil, and other salt and vitamin mixtures.

Ranhotra et al. (1974b) fed rats bread-based diets containing increasing levels of phytate for 6 weeks and then measured the availability of phytate phosphorus to rats in terms of their growth rate, serum inorganic phosphorus, phytate hydrolysis, and phosphorus retention. They found the amount of phytate hydrolyzed increased with increased dietary phytate levels (Table XII) and concluded that the availability of phytate phosphorus to rats was not affected. The retention of inorganic phosphorus decreased with the increase of dietary phytic acid levels. TABLE XI1 EFFECT OF PHYTIC ACID ON THE RETENTION OF INGESTED PHOSPHORUS” Phytic acid phosphorus hydrolyzed

mg

Total phosphorus retained %

mgc

%

78.6

21.8

79.4

B (179.2)

1220.0

75.3

C (358.4)

2391.7

83.8

D (537.6)

3351.1

81.1

4152.5 (4104.9) 4414.0 (3 181.7) 3845.3 (1395.6) 3545.5

Dietb A (44.8)

75.1 74.3 71.0

(-1 Ranhotra et al. (1974b). bValues within parentheses refer to the amount (milligrams/lOO grams) of dietary phytic acid phosphorus.
28

N. R. REDDY ET AL TABLE XI11 EFFECT OF AGE ON THE HYDROLYSIS OF PHYTATE BY RATSU Phytate hydrolyzed Age (weeks) 4 8 12 16 20 Mature

Experiment 1

(%)b

Experiment 2

71'

74' 54* 38; 36?$ 32$

-

-

39*

"Source: Nelson and Kirby (1979). bMeans with the same superscript (',

*, t , or $) are not significantly different.

Nelson and Kirby (1 979) conducted balance trials to determine the effects of age and diet composition on the ability of rats to hydrolyze natural phytate. They observed significant decreases in phytate hydrolysis during each of the first two 4-week periods (Table XIII). Weanling and mature rats, respectively, hydrolyzed 71 and 39% of phytate in a corn-soybean meal-based diet. They also noted that dietary variables in the basal diet influenced the amount of phytate hydrolyzed by both weanling and mature rats. The presence of wheat in a basal diet increased phytate hydrolysis in both weanling and mature rats (Table XIV). Although experiments to determine the bioavailability of phosphorus from legume phytates in humans have not been performed, it is estimated t h a 4040% of the phytate phosphorus in cereals is available to man (McCance and Widdowson, 1935). Subrahmanyan et al. (1955) have reported that 85% of the phytate phosphorus in ragi was hydrolyzed during digestion, although phytate splitting activity in humans is not yet clearly established. TABLE XIV EFFECT OF DIET COMPOSITION ON THE HYDROLYSIS OF PHYTATE BY WEANLING AND MATURE RATSU Phytate hydrolyzed (%) Diet Basal dietb Wheatc

Weanling rats

Mature rats

71 78

27 41

[ISource: Nelson and Kirby (1979). bBasal diet contained 71.72% ground yellow corn and 23.0% soybean meal (49% protein). =Wheat-based diet contained 35.86% ground yellow corn, 35.86% wheat, and 23.0% soybean meal.

PHYTATES IN LEGUMES AND CEREALS

B.

29

EFFECTS OF PHYTATE ON MINERAL BIOAVAILABILITY

Experiments with animals have suggested that phytic acid in plant foods complexes with dietary essential minerals such as calcium, zinc, iron, and magnesium and makes them biologically unavailable for absorption (Oberleas, 1973; O’Dell, 1979). The mechanism by which phytate affects mineral nutrition is not clearly understood. Most of the investigations (Maddaiah et al., 1964; Vohra et al., 1965; O’Dell, 1969; Oberleas, 1973) suggest that the formation of insoluble phytate-metal complexes in the intestinal tract prevents the metal absorption. The formation of these complexes is pH dependent. Data from several laboratories (Bruce and Callow, 1934; Reinhold et al., 1973; Bronner et al., 1954; Krebs and Mellanby, 1943; O’Dell et al., 1964; Mellanby, 1950) indicate that cereal diets or isolated phytate from plant sources interfere with mineral utilization. In most studies, the availability of minerals has been examined in relation to the administration of ionic salts. Results obtained under such conditions may not represent the level of absorption of minerals present in other natural sources. Rackis and Anderson (1977) reported that reduced availability of essential minerals by either phytate or phytate-protein complexes in legumes and other protein foods depends on several factors such as the following: 1. The ability of endogenous carriers in the intestinal mucosa to absorb essential minerals bound to phytate and other dietary substances 2. The concentration of phytic acid in foodstuffs 3. The concentration of minerals in the foodstuffs 4. The digestion or hydrolysis of phytate by phytase enzyme in the intestine 5 . Phytase inhibition 6. Processing of products or methods of processing Other food constituents, such as the dietary fiber, polysaccharides, oxalates, and polyphenolic compounds, may also play a major role in mineral bioavailability. Dietary fiber in whole wheat bread accounts for most of the poor availability of minerals (Reinhold et al., 1974, 1975, 1976; Ismail-Beigi et al., 1977). Conversely, Davies et al. (1977) suggested that phytate rather than fiber largely determines the availability of zinc for absorption. Davies and co-workers (Davies and Nightingale, 1975; Davies and Olpin, 1979; Davies and Reid, 1979; Davies et al., 1977) have published several reports- dealing with the bioavailability of different minerals from high-phytate and high-fiber cereals and legumes. Recently, two reviews (O’Dell, 1979; Erdman, 1979) appeared on this subject with primary emphasis on soy proteins and other oilseed products. With the possibility of increased utilization in the future of other legume proteins in developed countries and the existent high consumption of beans in underdeveloped countries, much remains unknown about the detrimental effects of phytate on mineral bioavailability in relation to human nutrition.

30

N. R. REDDY ET AL.

1. Calcium

Interest in the relation of phytate to nutrition began with the demonstration by Mellanby (1921, 1925) that certain cereals were anticalcifying and rachitogenic for puppies. He found that more severe rickets in puppies developed when the diet consisted of mainly oatmeal, maize, or whole wheat flour. Later Mellanby (1929) found that the rachitogenic agent could be destroyed by boiling the cereals with 1% HCl and also by germination, but he was unable to identify the compound at that time. The compound responsible for such effects was later shown to be phytic acid (inositol hexaphosphoric acid) (Bruce and Callow, 1934; Harrison and Mellanby, 1939). Later, Mellanby (1949) induced rickets in puppies raised on a low-calcium diet containing phytate, whereas controls raised on the same diet without phytate did not become rachitic. McCance and Widdowson (1942a,b) first reported the dietary influence of phytate on calcium in humans and concluded that the absorption of calcium from the diets could be improved by adding calcium salts or by removing the phytic acid from the diet. Later on it was widely accepted that phytic acid was an anticalcifying agent to man (Walker, 1951). Since then it has been considered that a high concentration of phytic acid in cereals makes calcium unavailable for absorption by forming an insoluble calcium-phytate complex, resulting in growth reduction and rickets. The effect of phytic acid on calcium metabolism has been thought to be temporary and the rachitogenic effect of phytic acid has been questioned (Cruickshank et al., 1945; Walker et al., 1948; Cullumbine et al., 1950; Walker, 1951). It was later shown that human subjects adapted to a high-phytate diet after a short period of time and suffered no ill effects. Whether such adaptation is due to enhanced production of intestinal phytase is not known. There is no evidence that human intestinal secretions ever have phytase activity (Walker et al., 1948; Walker, 195 1). The normal bone and teeth calcification throughout the world in several populations who depend almost exclusively on cereal diets (thus high phytate consumption) (Davidson and Passmore, 1970) suggest the human adaptability toward high phytate consumption, however. Several investigators (Ford et af., 1972a,b, 1977; Wills etal., 1972; Berlyne et al., 1973; Reinhold et al., 1973, 1976) have reported that the nutritional rickets and osteomalacia in the populations of Northem India, Pakistan, Iran, and among the Bedouins are related to the high phytate intake in the form of chapatis (unleavened bread), the major mode of cereal consumption. Other investigators (Hill, 1972; Toetia and Toetia, 1972), however, suggest that vitamin D deficiency or the interference in vitamin D metabolism by phytate is responsible for such effects and are independent of calcium absorption in the intestine. The relevance of Mellanby’s classical puppy experiments to man or even to other animal species is uncertain. Taylor (1965) suggested that the appearance of rachitic symptoms could be explained by the fact that metal ions, such as calcium

PHYTATES IN LEGUMES AND CEREALS

31

or magnesium, present in the diet are bound strongly by the soluble phytate (such as phytic acid or sodium phytate), making them unavailable. Further, metalbound phytic acid is not readily hydrolyzed in the pH range of the digestive tract to supply inorganic phosphate. The resulting phosphate deficiency provides an added factor contributing to the appearance of rickets. Van Den Berg et al. (1972) showed a new relationship between phytic acid, mineralization, and vitamin D. They have shown that polyphosphate esters of inositol are highly potent inhibitors of in virro calcification of rachitic rat cartilage and rat arota, and that such esters or phytate itself, injected particularly in small doses (1 mg inorganic phosphorus per kilogram of body weight daily), can prevent arotic calcification in the vitamin D intoxicated rat. However, so far there is no convincing evidence that phytic acid hydrolyzates can inhibit the mineralization of ricket rat cartilage in vitro. This subject requires further study before firm conclusions can be reached. Legumes contain large amounts of calcium; that is why few studies have been reported in the literature related to the bioavailability of calcium from legumes, including oil seeds. Erdman (1979) reported that rats derive only 10% of their calcium requirement from the soy, when fed diets containing 40% soy flodr. Nwokolo and Bragg (1977) demonstrated that the availability of calcium from protein supplements (soy bean meal, cottonseed meal, rapeseed meal, and palm kernel meal) was adversely affected by phytic acid in growing chicks. Erdman et al. (1978) conducted experiments to investigate the effects of the presence of various types of soy products (full-fat soy flour, freeze-dried soy beverage, and a commercial soy concentrate) in rat diets on the bioavailability of calcium added in incremental levels to diets as calcium carbonate. A slope-ratio assay was used to compare the regression of femur calcium upon the addition of calcium to soy and casein diets. They concluded that calcium added as carbonate to soy products was as available as calcium carbonate added to casein diets. Weingartner et al. (1979) investigated the effect of soybean hulls on the bioavailability of calcium from soy-based diets and found that inclusion of soybean hulls in soy flour-based diets had no significant effect upon the bioavailability of added calcium. 2. Magnesium During physiological studies on rats, Roberts and Yudkin (1960) observed magnesium deficiency symptoms, which appeared to be related to dietary factors. The symptoms were aggravated by addition of sodium phytate to caseinbased diets and they suggested that cases of magnesium deficiency of unknown origin in other animals could be due to excess dietary phytate. McWard (1969) reported that addition of 4% phytic acid-soy protein complex to a purified diet containing 75 ppm of supplemental magnesium depressed chick growth and increased mortality as a result of decreased bioavailability of magnesium. They

32

N. R. REDDY ET AL

also demonstrated that addition of 500 ppm EDTA (ethylenediaminetetraacetic acid) to a purified diet containing 4% phytic acid did not change the availability of magnesium to chicks. Guenter and Sell (1974) evaluated the “true” availability of magnesium from different foodstuffs (cereals, cereal products, and legumes) as compared to MgS04.7H,0 by chick bioassay. “True” availability of magnesium from MgS04.7H,0 was 57.4% and was assigned a relative availability index of 100 (Table XV). Magnesium availability in oats and soybean meal reportedly is higher than for MgS04.7H,0. The relative availabilities for the foodstuffs tested by Guenter and Sell were as follows in descending order: oats, 144; soybean meal (48.5% oil), 105; wheat, 99; corn, 97; barley, 95; Great Northern beans, 89; peas, 84; cream of wheat, 76; polished rice, 74; oatmeal cereal (baby food), 67; rice cereal (baby food), 64; and oats (minute breakfast), 58. Availability of magnesium from processed cereal foods is lower than that of the whole grain from which they were obtained (Table XV). Reinhold et al. (1976) and Nwokolo and Bragg (1977) showed that the presence of phytic acid decreased the availability of magnesium. Recently, Forbes et al. (1979) evaluated the bioavailability of magnesium from three soybean products-full-fat soy flour, freeze-dried soy beverage, and a commercial soy protein concentrate-as

TABLE XV TRUE AND RELATIVE AVAILABILITIES OF MAGNESIUM FROM FOODSTUFFSa Availability Relative index

Test ingredient MgS04 7H,0C Oats Soybean meal (48.5% oil) Wheat Corn Barley Great Northern beans Peas Cream of wheat Polished rice Oatmeal cereal (baby food) Rice cereal (baby food) Minute breakfast oats

57.4 82.9 60.4 56.6 55.9 54.2 51.1 48.3 43.8 42.2 38.6 36.9 33.1

100 144 105 99 97 95 89 84 76 14 61 64 58

aSource: Compiled from Guenter and Sell (1974). hTrue availability is defined as the proportion (%) of dietary magnesium absorbed during the passage through the digestive tract. cThe mean for MgS04 . 7H20 is the average of seven experiments with four birds each.

PHYTATES IN LEGUMES AND CEREALS

33

compared to MgCO, added to purified casein diets by a slope-ratio bioassay procedure. They concluded that magnesium is highly available from full-fat soy flour and freeze-dried soy beverage, whereas magnesium from commercial soy protein concentrate was about 80% as available as that from the inorganic source, MgCO,. Lo et al. (1978) supported the view that magnesium is highly available from soy protein products. 3 . Iron

Phytate, one of the most widely recognized inhibitors, was reported more than three decades ago to impair iron absorption (Widdowson and McCance, 1942; Moore et al., 1943; McCance et al., 1943). The effect of phytate on iron absorption has been studied both in humans (Sharpe et al., 1950; Foy et al., 1959; Hussain and Patwardhan, 1959; Turnball et al., 1962; Apte and Venkatachalam, 1962, 1964) and in animals (Sathe and Krishnamurthy, 1953; Davies and Nightingale, 1975). Results obtained by several investigators are conflicting regarding the effect of phytate on iron bioavailability. McCance et al. (1943) observed that addition of sodium phytate to white bread decreased iron absorption and they attributed the lower iron balances in persons consuming brown bread to its higher phytate content. In a study with adolescent boys, Sharpe et al. (1950) found that addition of sodium phytate to milk (0.2 g/200 ml) decreased iron absorption 15-fold. However, the same quantity of phytate in the form of oatmeal had much less effect. Using iron utilization for red cell production in normal human adults, Turnbull et al. (1962) demonstrated that the addition of sodium phytate to 5 mg of ferrous ascorbate resulted in the reduction of iron absorption by 50%. Several other researchers (Sathe and Krishnamurthy, 1953; Hussain and Patwardhan, 1959; Apte and Venkatachalam, 1964; Elwood et al., 1968; Berlyne et al., 1973; Davies and Nightingale, 1975) have also found that addition of phytate to foods or the high phytate content of foods substantially reduced the bioavailability of dietary iron. Others have found that feeding relatively high levels of phytate had little or no adverse effect on iron availability and absorption in animals and humans (Fuhr and Steenback, 1943; Foy et al., 1959; Cowan et al., 1966a,b; Callender and Warner, 1970; Ranhotra et al., 1974b; Liebman and Driskell, 1979). Much of this controversy may be due to the differing types of experimental animals, designs, and techniques used by these investigators, and to the presence of an intestinal phytase that might liberate iron as the phytate is degraded. Patwardhan (1937), Pileggi (1959), Roberts and Yudkin (1961), and Ranhotra et al. (1974b) demonstrated that rats do possess an intestinal phytase. Its quantitative activity has not been thoroughly investigated. The presence of phytase has been suggested as the probable reason why rats can utilize the iron of phytate-rich cereals and other foodstuffs. Phytase activity in the intestinal mucosa of man, chicken, and calf has also been reported (Bitar and

34

N. R. REDDY ET AL

Reinhold, 1972). Whether the in vitro measurement of enzyme activity represents the in vivo activity is not known. Recently, Welch and Campen (1975) found that intrinsic labeled iron (59Fe) in mature soybeans was more available than iron in immature soybeans, even though mature beans had approximately three times more phytic acid. They further concluded that apparently the mature seeds contain a factor other than phytate which impaired iron availability. It has also been reported that iron availability from soybeans and its protein products ranged from 28.5 to 80.0% (or inorganic iron) in rat studies (Monsen, 1974; Welch and Campen, 1975; Steinke and Hopkins, 1978). Steinke and Hopkins (1978) compared hemoglobin repletion in rats fed one of the three soy protein isolates with that in rats fed ferrous sulfate. They obtained a mean relative iron bioavailability of 61% for soy Hard wheat bran butanol extraction water extraction

I

I

Residue

Extracts

extraction with 1-.1.2 M NaCl o r NH, acetate

Residue

(discarded)

Extract concentrated by ultrafiltration saturated with (NH,),SO, filtered

I

I

Precipitate

Filtrate

(discarded)

diafiltered against 5-6 volumes of H,O

2

Diafiltered folution

1

freeze-dried

Diafiitrate

(discarded)

Buff-colored solid (H,O soluble)

chromatographed on BioGel P-4 or Sephadex G-25 iron-containing fractions made to 60-70’%by volume ethanol White precipitate Monoferric phytate FIG, 11. ( 1976b).

Isolation and purification of monoferric phytate from wheat bran. From Moms and Ellis

35

PHYTATES IN LEGUMES AND CEREALS TABLE XVI RELATIVE BIOLOGICAL VALUE TO THE RAT OF THE IRON IN WHEAT AND ITS MILLING FRACTIONS0 Hard wheat

Soft wheat

Iron source

(%)

(%)

Whole grain Bran Germ Shorts Ferrous ammonium sulfate Fe(NH& . 6H20

90 86 92 92 100

88 98 91 100

u s o w e : Moms and Ellis (1976b).

protein isolates and further stated that inorganic iron added to diets containing isolated soybean protein had bioavailabilities similar to that of the iron present in the soybean. These (Steinke and Hopkins, 1978) data suggest that the isolated soybean proteins are a good source of dietary iron and can be of significant value to the human diet. Morris and Ellis (1976a,b) isolated and characterized (see Fig. 11) a major iron component from hard wheat bran as monoferric phytate and showed that over 60% of iron in wheat bran is present as monoferric phytate. Several studies (Morris and Ellis, 1976a,b; Lipschitz et al., 1979; Ellis and Morris, 1979) demonstrated the monoferric phytate is a good source of iron and in rats and dogs has a high availability like that of dietary inorganic iron or ferrous ammonium sulfate. Morris and Ellis (1976b) evaluated wheat and its milling fractions for bioavailability of iron to the rat. They found that relative biological values for iron in wheat and its milling fractions-bran, germ, and shorts-varied from 86 to 92% with an average of about 90%, based on response to ferrous ammonium sulfate (Table XVI). They further tested several whole wheat breads, ready to eat cereal, and instant cereal for iron bioavailability by rat bioassay and the results are shown in Table XVII. Relative biological value of the iron in whole wheat TABLE XVII RELATIVE BIOLOGICAL VALUE OF THE IRON IN WHEAT PRODUCTSa Product

Relative biological valueb (%)

Whole wheat breads Ready-to-eat whole wheat Ready-to-eat bran Instant whole wheat, to be served hot

53-1 15 100 89 12

aSource: Moms and Ellis (1976b). bRelative biological value is based on response to Fe(NH& . 6H20 = 100.

36

N. R . REDDY ET AL.

breads (purchased from the grocery store) varied from 53 to 115%. The instant cereal that is served hot had a lower relative biological value than ready-to-eat whole wheat and bran.

4. Zinc The first direct evidence that zinc deficiency may develop in animals fed a diet composed of plant products (corn and soybean meal) was discovered by Tucker and Salmon (1955) in pigs. The zinc deficiency in animals was characterized by depression in growth and severe skin lesions. They showed that it could be cured or prevented by zinc supplementation. Later it was shown in chicks (O’Dell and Savage, 1957; Morrison and Sarett, 1958), swine (Smith et al., 1962), rats (Forbes and Yohe, 1960; Forbes, 1961, 1964), and humans (Prasad e t a l . , 1963). Animals fed on animal protein-based diets containing the same level of zinc grew normally without any deficiency symptoms. It was then found that the outstanding difference between animal and plant sources of protein is their phytic acid content. O’Dell (1969) hypothesized that phytate associated with plant proteins is responsible for decreased availability of zinc in foods prepared from seeds. To test this hypothesis, phytic acid was added to a casein-based diet (animal protein) and the growth responses of chicks fed this diet and those fed soybean proteins as the only source of protein were compared (O’Dell and Savage, 1960). Phytic acid decreased the bioavailability of zinc in the casein-based diet and produced symptoms similar to that observed among animals fed soybean protein diets containing a comparable level of phytate. O’Dell and Savage (1960) attributed the decreased bioavailability of zinc to formation of a complex between zinc and phytate. Maddiah et al. (1964) studied the influence of sodium phytate on the availability of zinc in chicks and reported that zinc formed the most stable (insoluble) complex with phytic acid in the physiological pH ranges and reduced the zinc availability to chicks. Likuski and Forbes (1964) also observed zinc deficiency symptoms in chicks after feeding them casein-based diets containing added free amino acids and phytic acid. Several studies support the view that phytic acid does decrease the bioavailability of zinc in chicks (Maddaiah et al., 1964; O’Dell et al., 1964; Nwokolo and Bragg, 1977), swine (Oberleas et al., 1962), and rats (Oberleas et al., 1966b; Likuski and Forbes, 1965; Makdani et al., 1975; Davies and Nightingale, 1975). The effect of phytate on the possibility of zinc deficiency in humans was strengthened by reports from Egypt (Prasad et al., 1963; Sandstead et al., 1965) and Iran (Reinhold, 1971, 1973; Halsted et al., 1972; Reinhold et al., 1973, 1976) that zinc deficiency occurred under conditions where unleavened flat bread was consumed in greater amounts than leavened bread. These flat breads are prepared from high-extraction wheat meal and contain higher amounts of phytic acid than do the leavened bread (Table XVIII). Franz (1978) employed slope-ratio methods (without an initial depletion period)

37

PHYTATES IN LEGUMES AND CEREALS TABLE XVIII PHYTATE CONCENTRATIONS OF IRANIAN FLAT BREADSO

Bread

Extraction (%)

Phytate ( W 1 0 0 g)

Treatment

Bazari Sangak Tanok

75 85-90 9 G 100

326 388 684

Leavened Leavened Unleavened

aSource: Compiled from data of Reinhold (1975a,b).

and measured the relative zinc bioavailability from several cereals. Whole rice and brown rice had low relative zinc bioavailability (0.51 or less, in contrast to 1.OO in zinc sulfate), and whole wheat flour and unleavened bread had medium values (0.73-0.75). Refined cereal products, namely, white flour, leavened and unleavened white breads, and polished white rice had relatively high zinc bioavailability (0.87-1.09) as did the leavened whole wheat bread (1.05). The high bioavailability of zinc observed by Franz (1978) in white polished rice and white flour was probably due to the reduction in phytic acid content caused by milling these cereals. Kratzer et al. (1959) reported that autoclaving soybean protein increased the availability of zinc to turkey poults. They suggested that the increased zinc availability from autoclaved soybean protein is due to destruction of phytic acid. This was later confirmed by O’Dell (1962), who found that most (88%) of the phytate was destroyed in isolated soybean protein during autoclaving at 115°C for 4 hr. In contrast, Lease (1966) autoclaved sesame meal for 4 hr at 15 psi and noticed only a small (22%) reduction in phytate content despite the fact that there was a marked increase in zinc availability to chicks. Obviously, there is a difference between soybean protein and sesame meal in responding to heat treatment. The mechanism of how zinc is held by phytic acid is still unknown. Zinc may be held by one or several bonds to the phytic acid and breaking of one of these bonds by autoclaving would allow it to become available to the chick. It is also likely that zinc may be held by something other than the phytic acid which was destroyed by autoclaving and thus released zinc to be used in the presence of phytic acid (Lease, 1966). Lease (1967) also evaluated the bioavailability of zinc from zinc-phytate complexes isolated from oil seed meals in chicks by an in vitro digestion method. He showed that the zinc of sesame meals and safflower was present in an insoluble, nondialyzable Ca-Mg-Zn-phytate complex at the intestinal pH and was poorly available to experimental chicks. Calcium has also been known to accentuate the effect of phytate on zinc availability. The interrelationship among calcium, phytate, and zinc has been studied in detail by Oberleas et al. (1962, 1966a), O’Dell et al. (1964), and

38

N. R . REDDY ET AL.

Likuski and Forbes (1965) in different species of animals. Calcium aggravated zinc deficiency when it was added to diets based on plant products that were high in phytate or diets with added phytic acid. In the absence of phytate, excess calcium had no effect on zinc availability. It thus appeared that calcium and zinc have a synergistic effect in the precipitation of phytate. To determine the mechanism whereby these two minerals interact with phytic acid, Byrd and Matrone (1965) studied the influence of calcium on incorporation of zinc into the phytate complex. They found that when the molar ratio of calcium to zinc was 1: 1 or 2: 1, the calcium decreased the complexing of zinc to the phytate; when the ratio of calcium to zinc was 100:1, the presence of excess calcium in the solution increased incorporation of zinc to the point where 99% of the zinc was present as the phytate. A similar effect was also found by Oberleas et al. (1966b). It is understandable that when zinc and calcium are present at high levels, calcium competes for positions on the phytate molecule, thereby reducing the amount of zinc precipitate. However, under practical feeding conditions those rations associated with the development of parakeratosis usually contained 30 to 100 ppm zinc and 1 to 2% calcium. The presence of excess calcium may provide an explanation for zinc deficiency symptoms observed in the above case. It was postulated that calcium increases the total cationic environment sufficiently to initiate a co-precipitation with zinc to form Zn-Ca-phytate. This resultant complex has been shown to be less soluble than Zn-phytate at pH 6.0, the approximate pH of the upper intestine, where most of the absorption of these divalent cations occurs. Therefore, in the presence of excess calcium zinc would not be as available for intestinal absorption and zinc deficiency would be aggravated. The calcium phytate (insoluble) has been reported (O’Dell et al., 1964) to have little effect on zinc availability and only the soluble phytate in the diet has shown to have the binding effect. Addition of EDTA chelating agent in the diet has been shown to neutralize the deleterious effect of soluble phytate on the availability of zinc and to increase zinc absorption both in chicks (O’Dell et al., 1964) and in rats (Oberleas et al., 1966a). The mechanism by which EDTA makes the zinc more available to animals is not clearly established. It has been suggested (O’Dell, 1969) that EDTA competes with phytate in chelating zinc to form a Zn,EDTA complex and thus interferes with the formation of Ca-Zn-phytate. The soluble Zn-EDTA complex can either be absorbed as such or can release zinc ion to the intestinal absorption and thus counteract the detrimental effect of the phytate. Various bioassays (growth rate, slope ratios of both weight gain and total femur zinc) have been employed to measure quantitatively the bioavailability of zinc in cereal- and legume-based foodstuffs. Values relative to casein, nonfat milk, and zinc salts (ZnCO, and ZnSO,) are shown in Table XIX. In general, zinc in plant seed products except peas (mature and immature) is less available either to chicks or rats than in animal products such as nonfat milk and casein,

PHYTATES IN LEGUMES AND CEREALS

41

protein isolates (or concentrates) by identical procedures using acid precipitation method. One acid-precipitated product was freeze-dried without neutralization and the other was neutralized prior to freeze dehydration. These two soy products were then fed to experimental rats to determine the zinc bioavailability. They found that zinc from acid-precipitated protein concentrate (without neutralization) produced excellent growth in rats, whereas rats fed acid-precipitated protein concentrate (neutralized) had significantly reduced growth response. They attributed these differences in growth response by rats to the processing conditions employed during the preparation of protein concentrates. Similar concerns have been raised by several investigators (Rackis et al., 1975; Rackis and Anderson, 1977; Erdman and Forbes, 1977). Several studies have been reported about the possible role of fiber in the mineral bioavailability in cereals and legumes (Reinhold et al., 1975, 1976; Ismail-Beigi et al., 1977; Oberleas and Harland, 1977). These investigators suggest that dietary fiber may also contribute, along with phytate, to reduced mineral bioavailability from several cereals and legumes. Such synergistic effects of fiber were investigated by Reinhold et al. (1973), who found that the unleavened whole wheat bread containing high fiber had a more detrimental effect on zinc balance than did sodium phytate supplied daily at the same level as that in the bread. In contrast to these observations, several other studies suggest that fiber does not contribute to reduced mineral bioavailability . Investigations by Davies et al. (1977) suggest that phytate, rather than fiber in the bran is the major determinant in the reduction of zinc availability to rats. Sandstead et al. (1978) tested five sources of dietary fiber, including wheat bran, and concluded that fiber did not have any effect on zinc balance in adult men. Guthrie and Robinson (1978) conducted zinc balances in four young women, with and without daily supplements of wheat bran (14 g/day), and found no overall difference in zinc metabolism. A recent study by Weingartner et al. (1979) showed that addition of soybean hulls (approximately 50% of the dietary fiber and 5% of the phytic acid of the whole soybean) to soy flour-based diets had no significant effect on the bioavailability of native zinc or of added calcium to rats. They also suggest that soybean hull fiber plays no role in reducing mineral absorption when fed at levels normally found in whole soybean products.

5 . Other Minerals The effects of phytate on the absorption and availability of other minerals (other than Ca, Mg, Zn, and Fe) have also been studied. For example, Davis et al. (1961) reported that diets containing an isolated soybean protein with phytate reduced the availability of manganese and copper for the chick. In a study with rats, Davies and Nightingale (1975) found that the addition of 1% phytate to an

42

N. R. REDDY ET AL.

egg albumin diet significantly reduced the absorption and availability of copper and manganese. C.

EFFECTS OF PROCESSINGS ON PHYTATE

I. Cooking The correlation of phytate with the cooking quality of peas was first suggested by Mattson (1946). He showed that the cooking quality of peas deteriorated when the level of phytate phosphorus in peas was low. He also suggested that phytic acid acted as a precipitant for calcium and magnesium, which were then unable to cross-link the uronic acid groups of pectin. Later workers were unable to obtain a good correlation between phytate content and cooking quality. A thorough examination of the problem by Crean and Haisman (1963) also fails to support Mattson’s hypothesis. Crean and Haisman (1963) studied the interaction between phytic acid and the divalent cations4alcium and magnesium-during the cooking of dried peas. They found that phytic acid in dried peas exists wholly as a water-soluble salt (probably potassium phytate), but on cooking, some of it combines with the calcium and magnesium in the pea to form insoluble calcium and magnesium phytate (Table XXI). Crean and Haisman (1963) further studied the rate of conversion of soluble phytate to its insoluble form in peas during cooking in distilled water with the addition of calcium chloride (Fig. 12). Equilibrium was approached within 5-10 min, but even at high concentrations of calcium chloride (2 x 10W2Ca eq./liter) only 60% of the available phytate in the peas was converted to the insoluble form. They obtained similar results with magnesium chloride, a maximum of 43% of the phytate being complexed when the peas were cooked in 2 X lo-* Mg eq./liter. Finally, Crean and Haisman (1963) concluded that the influence of phytate on the texture of cooked peas was small. Rosenbaum and Baker (1969) studied the distribution of phytic acid in pea cotyledons and the movement of calcium ions during cooking. Their results indicated that the higher cookability of the interior of the pea is not associated TABLE XXI EFFECTS OF COOKING ON PHYTIC ACID PHOSPHORUS CONTENT IN DRIED PEASa

Legume

Water extractionh

HC1 extractionb

Dried pea flour Peas after cooking

1.94 1.44

1.90 1.76

aSource: Crean and Haisman (1963). bEquivalents of phosphorus x 103/10 g dry weight of peas

39

PHYTATES IN LEGUMES AND CEREALS TABLE XIX BIOAVAILABILITY OF ZINC IN VARIOUS FOODSTUFFS

Product ZnC03 Nonfat milk Nonfat milk Casein Peas, immature Peas, mature Whole fat soy flour Whole fat soy flour Soybean meal, defatted Soy protein isolate Soybased infant formula Sesame meal Wheat Wheat Corn Corn High lysine corn High lysine corn Rice Rice

Assay animal Rat or chick chick Rat Rat Rat Rat Rat Rat Chick Rat Rat Chick Chick Rat Chick Rat Chick Rat Chick Rat

Value Method Growth rate Growth rate Growth rate Growth rate Absorption Absorption Slope ratio-femur Slope ratio-gain Growth rate Growth rate Slope ratio-femur Growth rate Growth rate Growth rate Growth rate Growth rate Growth rate Growth rate Growth rate Growth rate

(%)

100 82 79 84 95 75 34 54 67 44 67 59 59 38 63 57 65 55 62 39

Reference O’Dell et al. (1972a) O’Dell er al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a) Welch et al. (1974) Welch er al. (1974) Forbes and Parker (1977) Forbes and Parker (1977) O’Dell et al. (1972a) Forbes and Yohe (1960) Momcilovic et al. (1976) O’Dell et al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a) O’Dell et al. (1972a)

because of the presence of high phytate in plant seed products. The availability of zinc depends on the species to be used for study. For example, rats utilized less zinc from plant seed products than did chicks (Table XIX). According to the chick assays, the zinc availability in legume- and cereal-based products ranged from 59 to 67%. In the case of mature peas, the availability of zinc is comparable to that of nonfat milk, even though mature peas contained about 1.23%phytic acid (Welch et al., 1974). Welch et al. (1974) reported that most of the zinc in mature peas is present in the form of a soluble anionic complex (MW < 1000) which does not contain phytic acid. They further reported that cooking (autoclaving) the mature peas did not affect the availability of zinc to rats (Table XX). Franz (1978) noted high (9498%) and medium availability of zinc, respectively, from lima and California small white beans compared to an inorganic zinc source. Recently, Forbes et al. (1979) evaluated several soybean products (fullfat soy flour, freeze-dried soy beverage, and a commercial soy concentrate) for bioavailability of zinc to rats by employing a slope-ratio assay method. They found that zinc was poorly available from soy concentrate compared to from other soybean products. Forbes et al. (1979) further suggested that the poor

40

N. R. REDDY ET AL. TABLE XX EFFECT OF AUTOCLAVING ON BIOAVAILABILITY OF ZINC IN PEASa Assay animal

Value

Product Peas, mature, raw Peas, mature, autoclaved ZnS04

Rat Rat Rat

67.0 67.4 83.6

(%)

USource: Compiled from data of Welch et al. (1974).

availability of zinc from soy concentrate was due to the presence of high phytate in it. Soy concentrate had the highest molar ratio of phytate to zinc. Forbes and Parker (1977) have shown that zinc added to rat diets in the form of full-fat whole soy flour was significantly less biologically available than zinc added as zinc carbonate to an egg white diet. Bioavailability of zinc also varies from product to product. For example, zinc was better utilized from a full-fat soy flour than from a soy concentrate or soy isolate (Rackis et al., 1975; Rackis and Anderson, 1977; Erdman et al., 1978; Forbes et al., 1979). Rackis et al. (1975), Rackis and Anderson (1977), and Erdman (1981) suggest that the difference in bioavailability of zinc from soy protein products is most likely due to the use of various processing conditions employed during their preparation. These investigators believe that the type of phytate-protein-mineral complexes formed during processing, rather than the specific phytate content, are responsible for the reduced mineral absorption in soy products. More research is needed to identify the processing steps that affect formation of phytate-protein-mineral complexes. Reinhold et al. (1974) observed an increase in availability of zinc in whole wheat leavened bread as a result of phytate hydrolysis during yeast fermentation of bread dough. A study by Ranhotra et al. (1978) also showed that bioavailability of zinc in soy-fortified bread-based diets remained unaffected, probably because most of the phytate was hydrolyzed during breadmaking. Use of the phytate-zinc molar ratio to predict the zinc bioavailability from high-phytate foods was suggested by Oberleas (1975). Davies and Olpin (1979), in studying zinc availability from different commercial textured vegetable proteins, concluded that the phytate-zinc molar ratio was a valid indicator of zinc bioavailability . This was further supported by the investigations by Harland and Harland (1980) on phytate hydrolysis in several breads. Recently, it has been suggested to the nutrition committee (Anonymous, 1979) that poor mineral status may arise when one protein source is substituted for another without careful evaluation of the phytate-zinc molar ratio. Erdman et al. (1980) demonstrated that significant differences in zinc bioavailability were present in soy products with similar composition and phytate-zinc molar ratios. They prepared two soy

43

PHYTATES IN LEGUMES AND CEREALS

FIG. 12. Rate of formation of insoluble phytate in peas cooked in calcium chloride solutions. Calcium ions added to the cooking liquid equivalent X lo4 per 10 g dry peas. ( I ) 0; (2) 2.44; (3) 4.88; and (4) 9.76. From Crean and Haisman (1963).

with a higher phytic acid content. Recently, Kumar et al. (1978) investigated the effects of cooking on characteristics of legumes-green gram, cowpea, and chickpea. Their results indicate that the cooking processes decreased both waterand acid-extractable phytate phosphorus in all three legumes (Table XXII). They also observed a little change in the ratio of phytate phosphorus/total phosphorus in the uncooked and cooked legumes (in only HC1-extracted legumes). The poor extractability of phytate phosphorus with water and HC1 noticed by Kumar et al. (1978) in all three cooked legumes could be due to the formation of insoluble complexes between phytate phosphorus and other components in legumes during cooking, which subsequently could not be extracted with water or HCl. Reddy et al. (1978) did not find any breakdown of phytate phosphorus during cooking of TABLE XXII EFFECTS OF COOKING ON WATER AND ACID-EXTRACTABLE PHYTIC ACID PHOSPHORUS IN GREEN GRAM, COWPEA, AND CHICKPEASa Phytic acid phosphorusb (%)

Legume Green gram Uncooked Cooked Cowpea Uncooked Cooked Chickpea Uncooked Cooked

Water extracted

Retained

Acid extracted

0.142 0.080

100.00 56.30

0.150

100.00 81.20

0.090 0.032

100.00 35.56

0.123 0.090

100.00 73.20

0.056 0.036

100.00 64.29

0.078 0.075

100.00 96.15

aSource: Kumar et al. (1978). bEach value is expressed on a dry weight basis.

0.185

Retained

44

N. R. REDDY ET AL

black gram seeds and cotyledons (Fig. 13). Whatever losses in total phosphorus and phytate phosphorus they observed during short-time cooking were due to leaching of those components into the cooked water. Cooking for 45 min at 1 15°C caused small losses of phosphorus and phytate into cooked water, which may have been due to reabsorption of phytate by beans from cooked water. Phytic acid in mung beans is not completely destroyed by cooking, and 58-85% of phytic acid remained after autoclaving for 30 min at 120°C (AVRDC-Mung Bean Report, 1976). Since phytate can be detrimental to the absorption of essential trace elements, de Boland et al. (1975) investigated the rate of destruction of phytate during heat processing (autoclaving) of cereal and oil seed products and inositol hexaphosphate. They stated that the amount of water added during autoclaving had little or no effect on the rate of phytate destruction. Results show that 30-min autoclaving reduced the phytate content of cereals and oil seed products by less than 10% (Fig. 14). Autoclaving inositol hexaphosphate in aqueous solution (pH 6.0) resulted in nearly an 80% loss of iron-precipitable phosphorus in 2 hr, and approximately 50% was lost within 1 hr. The next most labile source of phytate is that in isolated soy protein-nearly 70% loss in 2 hr

5.50

5.25 5.00

4.15

-

:- I

amm....mm...a Total P in whole bean --o Phytate P in whole bean c--m Total P in cotyledons L - 8 - 1 6 Phytatc P incotyledons

.r;

?c+

2 4.50 - !\i$<*, ., E 4.25

,,"

7' p ............,,,,,,,,,,,,,,,,~,,,,,.,,,,,,,,,~,,,~,~,~~~.~,.~~~.~~.~~~~.~~~~~~~ - -- p ,i-.,';' i p /' *".'.** , -*----aH--* ,.*' = ' \*%.

a3

4.00

0

L

< - \'..p 315-

c

3.50

c"

3.25 300

-

2.15

-

2.50

-

.

,.,,,1111111111.

010//

*'#'

*/*Mo

0 *.P"O

\',\'*

/o0-

.**g;/;

0

e<m :.,

-HA#-

5

10

I

1

15

I

1

20

25

30

1

I

35

40

45

FIG. 13. Effects of cooking on total phosphorus and phytate phosphorus contents in black gram seeds and cotyledons. Cooking was carried out at 10 psi ( I 16°C) for different time lengths (bean-towater ratio of 1 :4).At the end of cooking, cooking water was discarded and samples were immediately lyophilized for phytate phosphorus and total phosphorus analyses. Total phosphorus in whole bean (O.....) and in cotyledons (W- - - -W); phytate phosphorus in whole bean (0- -0) and in cotyledons (O.-.-O).From Reddy et al. (1978).

45

PHYTATES IN LEGUMES AND CEREALS

100

-

80 -x - 70 60 90

0

2

z 50 Y

I-

2

’ >

40

-

30-

10 -

20

..*..

Rice Wheat

~ ~ 1 ControlCornGrrm 0 ” ~

\

High Lynne Corn Germ ,.hL.. -**#

0

Soyban Flakes Serama Meal

Soybean Protein IRP 100) Phytate pH6 I l l 1

0 1 5 3 0 60 120 AUTOCLAVING TIME (min)

240

FIG. 14. Rate of phytate loss during autoclaving phytate and moist slurries of various cereal and oil seed products at 115°C: rice (W- - -U),wheat (0-- -0),control corn germ (O....O),high-lysine corn germ (O-U), soybean flakes (A- - -A),sesame meal (A-- -A),soybean protein (RP-100) ( O - - . O ) , and phytate, pH 6 (@I-*). From de Boland and O’Dell (1975).

(Fig. 14). Phytate in other products is relatively stable. Losses of phytate varied from 25% in sesame meal to 5% in rice and wheat in 2 hr at 115°C. Lease (1966) observed no decrease in the phytate content of sesame meal after autoclaving for 2 hr and only a 22% decrease after 4 hr (Table XXIII). The reports by de Boland er al. (1975) and Lease (1966) suggest that the rate of phytate destruction, probably by hydrolysis, is influenced by its protein and/or cation environment. Tabekhia and Luh (1980) studied the effects of cooking and canning on phytic acid retention in dry beans, black-eyed beans, red kidney beans, mung beans, TABLE XXIII EFFECTS OF AUTOCLAVING ON THE PHYTIC ACID CONTENT OF SESAME MEAL0 Time at 15 psi (hr)

Total phosphorus

Phytic acid phosphorus

(%)

(%)

Content

Remaining

0 2

1.50

4

-

1.oo 1.03 0.78

3.57 3.68 2.78

100 103 78

aSource: Lease (1966).

-

Phytic acid (%)

46

N. R. REDDY ET AL

and pink beans. Cooking the beans in water containing 3% NaCl (beanswater 1:4 w/v) at 100°C for 3 hr resulted in a slight decrease in phytic acid in blackeyed and red kidney beans, but more reduction in mung and pink beans (Table XXIV). However, canning the beans with brine containing 3% NaCl (the beansto-brine ratio was 200:750 w/v) at 115.5"C for 3 hr showed a significant breakdown in phytic acid in all four bean varieties (Table XXIV). The retention of phytic acid in the canned black-eyed beans was only 8.50%, whereas that in the other three varieties ranged from 25.10 to 32.40%. Tabekhia and Luh (1980) further found that canning resulted in a decrease in phytic acid with an accompanying increase in inorganic phosphorus. Iyer el al. (1980) observed a reduction in the phytate content of pinto, Great Northern, and red kidney beans during a combined process of soaking and cooking (Table XXV). They soaked the beans for 18 hr in distilled water or a combined salt solution (see Table XXV for details). Hydrolysis was higher in beans soaked and cooked in distilled water than those soaked and cooked in salt solution. The investigations of Toma and Tabekhia (1979) indicated that cooking wellmilled rice in domestic tap water reduced phytic acid content by = 70% (Table XXVI), whereas cooking well-milled rice in distilled deionized water did not reduce phytic acid content. TABLE XXIV EFFECTS OF COOKING AND CANNING ON PHYTIC ACID RETENTION IN BEANSa,b

Bean variety Black eyed beans Raw dry beans Cooked beans Canned beans Red kidney beans Raw dry beans Cooked beans Canned beans Mung beans Raw dry beans Cooked beans Canned beans Pink beans Raw dry beans Cooked beans Canned beans

Phytic acid

Phytic acid retained

(%)

(%I

1.148 0.995 0.098

100.00 86.70 8.50

1.170 1.080 0.260

100.00 92.30 30.30

0.204 0.130 0.066

100.00 63.70 32.40

0.503 0.370 0.126

100.00 73.60 25.10

~

OEach val'ue is the mean of five determinations and is expressed on a dry weight basis. "Source: Tabekhia and Luh (1980).

TABLE XXV EFFECTS OF SOAKING AND COOKING ON PHYTIC ACID PHOSPHORUS CONCENTRATIONS IN THE GREAT NORTHERN, PINTO, AND RED KIDNEY BEANSO ~

Great Northern

Pinto

Phytic acid P

Hydrolysis

Treatment

(mgk)

(%)

Phytic acid P (mg/g)

Control (dry beans) Soakingb + cooking SoakingC + cooking

4.60

0.00

5.50

1.10

76.10

4.00

13.04

Red kidney Hydrolysis

Phytic acid P (mg/g)

Hydrolysis

0.00

5.80

0.00

2.10

61.80

2.50

56.80

4.10

25.50

4.00

31.30

(%I

aSource: Iyer et a / . (1980). bSoaked for 18 hr in distilled water at room temperature (22°C) before quick cooking. CSoaked for 18 hr in a combination of salt solutions (2.5% sodium chloride + 1.5% sodium bicarbonate tripolyphosphate), pH 7.0, at room temperature (22°C) before quick cooking.

+

0.5% sodium carbonate

(%)

+

1.0% sodium

48

N. R. REDDY ET AL TABLE XXVI EFFECTS OF COOKING ON PHYTIC ACID RETENTION IN WELL-MILLED RICE VARIETIES"

Variety Terso Raw rice Rice cooked Rice cooked M-5 Raw rice Rice cooked Rice cooked S-6 Raw rice Rice cooked Rice cooked

Phytic acidb

Phytic acid retainedb

(%I

(%)

with distilled deionized water with domestic tap water

0.192 0.188 0.0.54

100.00 98.00 28.00*

with distilled deionized water with domestic tap water

0.140 0.135 0.045

100.00 96.30 32.10*

with distilled deionized water with domestic tap water

0.137 0.135 0.042

100.00 98.20 30..50*

USource: Toma and Tabekhia (1979). bEach value is the average of six determinations and is expressed on a dry weight basis *Significant at the 5% level.

In summary of these studies, it appears that destruction and/or reduction of phytate during cooking is dependent on several factors. For instance, the rate of destruction of phytate by autoclaving is low when it is associated with proteins and/or cations in natural products such as cereal and oil seed products. In many cases, the cations and proteins associated with phytates in natural products are not known.

2.

Germination

The phytate is utilized as a source of inorganic phosphate during seed germination and the inorganic form becomes available for purposes of plant growth and development. The liberation of phosphate from phytate occurs by enzyme hydrolysis. Phytase is the currently accepted enzyme, which is responsible for the complete hydrolysis of phytate (inositol hexaphosphate) into inositol and phosphate. Several seeds or grains are known to contain phytase enzyme and its activity varies widely. Oats, maize, and some millets have negligible phytase activity; barley has moderate activity; wheat and rye have high activity (Long, 1961). Belavady and Banerjee (1953) reported the absence of phytase activity in some of the ungerminated legumes. However, they noticed phytase activity in germinated legumes. Phytase activity reportedly increased during germination of

49

PHYTATES IN LEGUMES AND CEREALS

several seeds (Peers, 1953; Cosgrove, 1966; Chang, 1967; Mandal and Biswas, 1970; Walker, 1974; Lolas and Markakis, 1977; Kuvaeva and Kretovich, 1978). The principal function of phytase in seeds or grains is to liberate phosphate from phytate during germination and other processes (fermentation, soaking, and autolysis). Germination reduces and/or eliminates considerable amounts of phytate from the seeds or grains. The effects of germination on the extent of phytate hydrolysis in various cereal grains are presented in Table XXVII. All the grains were steeped for 2 days at room temperature and then germinated at 25°C. Except in the case of rye, complete hydrolysis of phytate did not occur during 7day germination (Mellanby, 1950). On the seventh day of germination, wheat, yellow corn, white corn, barley, and oats still contained 47.4, 33.0, 50.0, 34.8, and 67.2% of the original phytate, respectively (Table XXVII). Ashton and TABLE XXVII EFFECT OF GERMINATION ON PHYTIC ACID PHOSPHORUS AND PHYTIC ACID OF CEREALSa,b

Cereal Wheat

Time (days) 0 1

2 3 4 5 6 7

Yellow corn

0 1

2 3 4 5 6 7

White corn

0 1 2 3 4

5 6 7

Phytic acid phosphorus (mgig)

Phytic acidC (mg/g)

Phytic acid hydrolyzed

2.32 2.00 2.00 1.61 1.53 1.39 1.25 1.10

8.24 7.10 7.10 5.72 5.43 4.93 4.44 3.91

0.00 13.80 13.80 30.60 34.05 40.08 46.10 52.60

2.12 1.64 1.31 1.17 1.15 0.76

7.53 5.82 4.65 4.15 4.08 2.70

0.00 22.64 38.21 44.81 45.75 64.15

-

-

0.70

2.49

67.00

1.80 1.75 1.53 1.33 1.15 1.15

6.39 6.21 5.43 4.72 4.08 4.08

0.00 2.86 15.00 26.11 36.1 1 36.11

-

-

0.90

3.20

50.00

(a)

(continued)

50

N . R. REDDY ET AL TABLE XXVII (Continued)

Cereal Barley

Time (days) 0 1

2 3 4 5 6 7 Oats

0 1

2 3 4 5 6 7 0 1 2 3 4 5 6

Phytic acid phosphorus (mgk)

Phytic acidc (mgig)

Phytic acid hydrolyzed

v@)

1.98 1.56 1.39 1.17 1.14 0.82

7.03 5.54 4.93 4.15 4.05 2.91

0.00 21.21 29.80 40.90 42.42 58.60

0.69

2.45

65.20

1.95 1.60 1.58 I .49 1.37 1.30 1.34 1.31

6.92 5.68 5.61 5.29 4.86 4.62 4.76 4.65

0.00 17.95 19.00 23.60 29.74 33.33 31.28 32.82

1.68 1.21 0.59

5.96 4.30 2.09

0.00 27.98 64.88

-

-

-

-

0.12 0.00

0.43 0.00

92.86 100.00

“Germinated at a temperature of 22-26°C. bSource: Mellanby (1950). cPhytic acid calculated by assuming that it contains 28.20% phosphorus.

Williams (1958) found that germination of oats was accompanied by a gradual breakdown of phytate and simultaneous release of inorganic phosphorus. The investigations of Hall and Hodges (1966) indicate that phytic acid disappeared completely from the oat endosperm during 8-day germination. In the case of wheat, Mihailovic et al. (1965) observed that phytate had completely disappeared by the seventh day of germination. Using circular paper chromatography, they examined extracts of wheat made at various stages of germination and concluded that the enzymic hydrolysis occurred in a stepwise manner with the formation of intermediates-penta-, tetra-, tri-, di-, and monophosphates of myo-inositol. This conclusion is in agreement with the results of in vitro and in vivo studies of Sobolev (1963).

51

PHYTATES IN LEGUMES AND CEREALS

TABLE XXVIII EFFECT OF GERMINATION ON TOTAL PHOSPHORUS, PHYTIC ACID PHOSPHORUS, AND PHYTIC ACID CONTENTS OF LEGUMES

Time (days)

Total phosphorus (mg/g)

Phytic acid phosphorus (Wg)

Phytic acida (mg/g)

Pigeon peab

0 1 3 5

3.68 3.68 3.68 3.68

0.35 0.34 0.34 0.28

1.24 1.21 1.21 0.99

0.00 2.42 2.42 20.16

Chick-peab

0

3.51 3.51 3.51 3.51

1.24 1.21 1.11 0.73

4.40 4.30 3.94 2.59

0.00 2.27 10.45 41.14

0.87 0.88 0.67 0.41

3.09 3.12 2.38 1.46

0.00

0.00 22.98 52.75

Legume

1

3 5

Lentils*

0 I 3

Phytic acid hydrolyzed (%)

5

3.16 3.16 3.16 3.16

Cowpeab

0 1 3 5

4.16 4.16 4.16 4.16

0.62 0.59 0.41 0.31

2.20 2.09 1.46 1.10

0.00 5.00 33.64 50.00

Garden peab

0 1 3 5

3.32 3.32 3.32 3.32

0.91 0.62 0.42 0.28

3.23 2.20 1.49 0.99

0.00 31.89 53.87 69.35

Dwarf grey peaC

0

-

5

2.48 1.94

8.80 6.89

0.00 21.70

0

-

5

-

1.13 0.59

4.01 2.09

0.00 47.88

0 5

-

-

1.86 1.20

6.60 4.26

0.00 35.45

0

3.56 3.56 3.56 3.56

0.81 0.74 0.45 0.32

2.88 2.63 1.60 1.14

0.00 8.68 44.44 60.42

5.20 4.80 5.00 5.00 5.00 5.00 5.10

4.10 3.70 3.30 3.40 3.20 3.10 3.00

14.56 13.14 11.72 12.07 11.36 11.01 10.65

0.00 9.75 19.51 17.10 21.98 24.38 26.85

Early Alaska peac SoybeanC Black gramC

1

3 5

Black gramd

0 1

2 3 4 5 6

(continued)

52

N. R. REDDY ET AL. TABLE XXVIII (Conrinued)

Legume

Green gramb

Time (days)

(%)

9.94 9.23 7.81 7.10

31.73 36.61 46.36 51.24

0

3.74 3.74 3.74 3.74

0.88 0.81 0.38 0.36

3.12 2.88 1.35 1.28

0.00 7.69 56.73 58.97

4.40 4.51 4.45 4.45 4.40 4.40

0.58 0.48 0.42 0.40 0.40 0.40

2.05 1.70 IS O 1.42 1.42 1.43

0.00 17.07 26.83 30.73 30.73 30.24

5.40 5.40 5.35 5.35 5.35 5.40

3.23 2.99 2.41 2.03 0.92 0.73

11.48 10.92 8.57 7.20 3.28 2.59

0.00 4.88 25.35 37.28 71.43 77.44

5.45 5.40 5.40 5.45 5.40 5.40

3.30 2.99 2.95 2.18 2.18 2.11

11.70 10.63 10.48 7.73 7.75 7.50

0.00 9.15 m.43 33.93 33.76 35.90

5.00 5.05 5.00 5.00 5.00 5.00

1.42 1.23 1.02 0.96 0.87 0.76

5.03 4.38 3.62 3.40 3.09 2.69

0.00 12.92 28.03 32.41 38.57 46.52

0 2 3 4 5 0 1

2 3 4 5

0 1

2 3 4 5 Pink beanse

Phytic acid hydrolyzed

2.80 2.60 2.20 2.00

1

Red kidney beanse

Phytic acid" (mgig)

5.30 5.40 5.20 5.50

3 5

Black-eyed beanse

bgk)

Phytic acid phosphorus (mg/g)

7 8 9 10 1

Green grame

Total phosphorus

0 1

2 3 4 5

aPhytate calculated by assuming that it contains 28.20% phosphorus. bBelavady and Banerjee (1953). CChen and Pan (1977). dReddy er al. (1978). =Tabekhia and Luh (1980).

53

PHYTATES IN LEGUMES AND CEREALS

The percentages of phytate hydrolyzed at various stages of germination of different beans are presented in Table XXVIII. During germination, hydrolysis of phytate in legumes varied greatly. The percentage of phytate hydrolyzed in legumes (pigeon pea, chick-pea, lentils, cowpea, garden peas, dwarf peas, early peas, soybeans, black gram, green gram, red kidney beans, pink beans, and black-eyed beans) ranged from 20.16 to 77.44% after germination for 5 days (Table XXVIII). The maximum amount of phytate was hydrolyzed in black-eyed beans, i.e., 77.44% during 5-day germination. In legumes, complete hydrolysis of phytate did not take place during 5 days of germination. Disappearance of phytate during germination depends on the phytase activity. A rapid rise in phytase activity was observed after 48-hr germination of bush beans (Walker, 1974). Chen and Pan (1977) found that phytase activity increased 227, 807, and 3756% in soybeans, dwarf grey peas, and early Alaska peas, respectively, after 5-day germination. There exists a good correlation between phytate breakdown and phytase activity of the seeds during germination. Such correlations have been demonstrated for bush beans (Walker, 1974), mung beans (Mandal et al., 1972), navy beans (Lolas and Markakis, 1977), cotton (Ergle and Guin, 1959), dwarf grey pea (Fig. 15), early Alaska pea (Fig. 16), and soybean seeds (Fig. 17) during germination (Chen and Pan, 1977). Ergle and Guin (1959) reported that most of the phytate disappeared in cottonseeds after 6 days of germination. Recently, Reddy et al. (1978) found a negative significant correlation between total phosphorus and phytate phosphorus during black gram germination.

- 0.7 I - 0.6 p 8

.-2 E

. - -e 0.5

m

.m

0.4

n

- 0.3 k .-.5 .- 0.2 ; m

c >

0

1

2

3

4

0.1

s a. c

5

Days

- -0) and phytase activity (0-0) during FIG. 15. Changes in phytate phosphorus content (0dwarf gray sugar pea germination. From Chen and Pan (1977).

54

N. R. REDDY ET AL

0.3

0 Days

FIG. 16. Changes in phytate phosphoms content (0-- -0)and phytase activity (0-0) during early Alaska pea germination. From Chen and Pan (1977).

FIG. 17. Changes in phytate phosphorus content soybean germination. From Chen and Pan (1977).

(0- -0)and phytase activity (0-0) during

55

PHYTATES IN LEGUMES AND CEREALS

3 . Fermentation and Breadmaking

Fermentation of cereals and legumes appreciably reduces the phytate content due to endogenous phytase of cereals and legumes and that of added yeast and other useful microorganisms. Hydrolysis of phytate in the bread depends on the amount of yeast added to the dough and the time of dough fermentation. The phytate destruction in flour by phytase during different modes of fermentation and preparation has been studied by Widdowson (1941). Some of the results obtained by Widdowson (1941) are presented in Table XXIX. The percentage of phytate (85%) hydrolyzed in the bread prepared from 70% extraction white flour is much higher than that in the bread prepared from 92% extraction wheat meal flour (31%). Addition of yeast to the preparations considerably increased the phytate hydrolysis. He found the least hydrolysis of phytate in bread and other products prepared without addition of yeast. Pringle and Moran (1942) determined the effect of fermentation time on the destruction of phytate in doughs made with 85% extraction flour and found 59, 64, and 76% phytate hydrolysis in TABLE XXIX DESTRUCTION OF PHYTIC ACID PHOSPHORUS DURING FERMENTATION AND COOKING OF FOODSa

Nature of flour used White flour (70% extraction) National wheat meal (85% extraction) Wheat meal (92% extraction)

White flour with added sodium phytate

Nature of product Bread made with yeast Bread made with yeast Bread made with yeast Baking powder bread Steamed pudding Pastry Baking powder bread Steamed pudding Pastry

aCalculated from the data of Widdowson (1941)

Phytic acid P present originally in flour (mg/100 g)

Amount of phytic acid P hydrolyzed (mg/100 9)

Phytic acid P hydrolyzed

51

43.4

85.0

127

87.6

69.0

214

66.3

31.0

214

10.7

5.0

214

34.2

16.0

214 214

0.0 32.1

0.0 15.0

214

128.4

60.0

214

32.1

15.0

(%)

56

N. R . REDDY ET AL

bread baked after 3, 5, and 8 hr, respectively, of fermentation. The relatively high optimum temperature of phytase may also permit some hydrolysis during the initial stages of baking. Ranhotra (1972) investigated phytic acid hydrolysis during breadmaking. The results of hydrolysis of phytic acid during different steps of baking with white flour, blend A (containing 30% wheat protein concentrate), and all wheat protein concentrate (WPC) blend are shown in Fig. 18. The wheat protein concentrate was richer in phytic acid and contained about 92% phosphorus in the form of phytic acid. Most of the phytic acid is hydrolyzed during fermentation (storage time, 210 min) in the preparation of white bread with a simultaneous increase in inorganic phosphorus. In blend A, hydrolysis of phytic acid and the increase in inorganic phosphorus were substantial during fermentation. With all wheat protein concentrate blend, hydrolysis of phytic acid was rather slow during sponge time but greatly increased during the subsequent 30-min period (floor time), accompanied by a sharp increase in inorganic phosphorus level. Ranhotra (1972) also observed an inverse relationship between the phytic acid initially present in the unbaked blend and the amount of phytic acid hydrolyzed during baking when white flour was increasingly replaced by WPC (Fig. 19). Hydrolysis of phytic acid was greater in the blends with 30% or less substitution of WPC. However, the rate of phytic acid hydrolysis progressively decreased during breadmaking as the WPC incorporation was increased to greater than 30% in the blends. The level of inorganic phosphorus continued to rise during baking with each increase of WPC apparently due to an accelerated hydrolysis, notably at high WPC levels of residual phosphorus. In later studies, Ranhotra (1973) reported that the decreased rate of phytic acid hydrolysis observed in blends with increased (more than 30% incorporation) WPC during breadmaking was due to increased inhibition of phytase and/or rephosphorylation of partially hydrolyzed phytic acid. Ranhotra et al. (1974) demonstrated that all of the phytate in wheat bread and more than 3/4 of the phytate in soy-fortified wheat flour (soy lo%, wheat 90%) was hydrolyzed during the process of breadmaking apparently due to phytases in the wheat and/or yeast. Ranhotra et al. (1974a) also investigated the effect of yeast addition on hydrolysis of phytic acid during breadmaking. When soy-fortified (10% soy, 90% wheat) bread was made without yeast, less than half of the phytate was hydrolyzed (Table XXX). Hydrolysis increased substantially when yeast was added, and at levels normally used in the bread formulation (9 g), the phytate hydrolysis is maximum. Increased hydrolysis was accompanied by increase in the level of available phosphorus. Harland and Harland (1980) investigated the effect of yeast addition and fermentation time on phytate hydrolysis in breads prepared from rye flour, white flour (all-purpose wheat flour), and whole wheat flour. The major reduction of phytate occurred in three breads during the first 2 hr of rising (Table XXXI) and only a small decrease was observed in next 6 hr of rising. Phytate reduction was greater in rye bread than in whole wheat bread after doubling yeast. The percentage of

PHYTATES IN LEGUMES AND CEREALS

57

A

f 150 130

[

FIG. 18. Hydrolysis of phytic acid during various steps in baking: (A) phytic acid phosphorus; (B) inorganic phosphorus. WPC, Wheat protein concentrate; blend A, 30% WPC plus 70% white flour; flour, 100% white flour. From Ranhotra (1972).

520

3

400

300 200 100

g

0 150 120

90 60

30

':;[mTh 0

RESIDUAL P (in bread)

.

0 WPC %

0 10 20 30 40 50 70 90 100 F L O b R . % 100 90 80 70 60 50 30 10 0

FIG. 19. Effect of the amount of WPC (wheat protein concentrate) on hydrolysis of phytic acid during baking. Values represent the total (mg) in unbaked ingredients or the resultant pup loaf. Values for percent phytic acid hydrolyzed are shown on top of bars. From Ranhotra (1972).

58

N. R. REDDY ET AL

TABLE XXX EFFECT OF YEAST ON THE HYDROLYSIS OF PHYTIC ACID DURING BREADMAKING IN SOY-FORTIFIED WHEAT FLOURu,b Yeast (gllb loaf) 0

3

9

15

307.2 167.0 45.6

307.2 77.7 74.7

307.2 37.2 87.9

307.2 42.4 86.2

Phytic acid phosphorus In bread ingredients representing 1-1b loaf (mg/loaf) In bread (mg/loaO Hydrolyzed (0.0) uPhytase activity in yeast was 135 unitsllO0 g. bSource: Ranhotra et al. (1974a).

phytate was decreased from 0.78 to 0.21% in rye bread, from 0.03 to 0.02% in white bread, and from 0.64 to 0.43% in whole wheat bread during 8 hr of rising after yeast doubling (i.e., addition of two packets of yeast to the respective doughs). Reinhold (1975a) studied destruction of phytate by yeast fermentation in sponges or doughs prepared from Iranian whole wheat meals of different extraction rates (Bazari, Sangak, and Tanok wheat meals with respective extraction TABLE XXXI PERCENT PHYTATE IN BREADS PREPARED WITH INCREASED YEAST AND FERMENTATION TIMEu Rising time (hr)

Yeast added No yeast

1 package

2 packages

Rye bread

0.78 0.77 0.76 0.76

0.80 0.41 0.34 0.37

0.43 0.28 0.23 0.21

0.04 0.03 0.02 0.01 Whole wheat bread 0.64 0.56 0.48 0.42

0.03 0.02 0.02 0.02

White bread

0.03 0.03 0.03 0.03 0.64 0.59 0.59 0.59

uSource: Harland and Harland (1980)

0.60 0.57 0.47 0.43

PHYTATES IN LEGUMES AND CEREALS SANGAK

59

TANOK A

B

0

t UlL 0 2 4 1 6

hl ! 4 16

0 2 416 HOURS

FIG. 20. (A) Production of acid-soluble phosphorus which accounts for nearly all of the phytate phosphorus that was destroyed in the two whole meals of lower extraction rates, but not in that of the Tanok meal. (B) Rapid disappearance of phytate by action of yeast in sponges of whole wheat meals of Bazari (75-85% extraction rate), Sangak (85-90% extraction rate), and Tanok (95-100% extraction rate). From Reinhold (1975a).

rates of 75-85, 85-90, and 95-100%). Phytate was destroyed or hydrolyzed rapidly in whole meals of Bazari and Sangak with a simultaneous increase in acid-soluble phosphorus content (Fig. 20). About 1/3 of the phytate disappeared within 2 hr of fermentation in Bazari and Sangak sponges. The decrease of phytate in Tanok whole meal was much less. Retardation of phytate hydrolysis persisted after 4 hr of fermentation and was still evident after 16 hr. Reinhold (1975a) suggested the following factors for slow phytate hydrolysis in Tanok whole meal dough: The presence of inhibitors such as calcium and other metal ions, which form stable salts resistant to phytase attack 2. The existence of phytate in combination with certain proteins in the form of complexes in seeds, which form may not be vulnerable to phytase attack 3. The high concentration of phytate in the whole meal, which inhibits phytase action 1.

Sudarmadji and Markakis (1977) studied the changes in phytic acid during tempeh preparation by fermenting boiled soybeans with Rhizopus oligosporus.

60

N. R. REDDY ET AL TABLE XXXII PHYTIC ACID CONTENT OF SOYBEANS AND TEMPEHo,b

Phytic acid Sample

(%a)

Soybeans, raw Soybeans, soaked Soybeans, boiled Tempeh

1.41 1.43 1.23 0.96

Phytic acid hydrolyzed (%)

0.00 0.00 13.99 32.88

aData expressed on a dry weight basis. bSource: Sudarmadji and Markakis (1977).

Boiling the soybeans resulted in a reduction (14.0%) of phytic acid (Table XXXII). The phytic acid was reduced by about 1/3 in soybeans as a result of fermentation with mold (Rhizopus oligosporus). The decrease of phytic acid was accompanied by an increase in inorganic phosphorus. They concluded that the reduction in phytic acid obtained was due to the action of the enzyme, phytase, which was produced by mold during fermentation. The effect of natural fermentation on phytic acid in black gram, rice, and black gram and rice blends was investigated by Reddy and Salunkhe (1980). The black gram and rice blend contained black gram and rice in a 1:l ratio (w/v). After 45 hr of fermentation, about 13.3 and 48.8% of phytic acid was hydrolyzed in black gram, and black gram and rice blends, respectively (Table XXXIII) with subsequent increases in inorganic phosphorus. In the case of rice blend, fermentation for 8 hr resulted in complete hydrolysis of the phytic acid with a simultaneous increase in inorganic phosphorus. Reddy and Salunkhe (1980) prepared a fermented steamed product, Idli, by steaming 20-hr-fermented black gram and rice blend that contained low amounts (0.15%) of phytic acid. 4.

Soaking, Autolysis, and Other Processes

There have been many reports of the presence of a phytase in cereals and legumes. McCance and Widdowson (1944) have demonstrated that a pH of 4.50 and a temperature of 43-58°C are the best for phytase action. Mellanby (1950) reported that the phytate hydrolysis in whole wheat was slow compared to that in ground wheat when both were incubated at 45°C and pH 4.50 (maintained by using a sodium acetate-acetic acid buffer). The rates of hydrolysis of phytate by phytase in ground wheat and whole wheat under the above-described conditions are shown in Fig. 21. Phytate hydrolysis in whole intact grain was slow and still contained about 17% (or 38 mg) of phytate at the end of 12 hr. In the ground grain, on the other hand, all of the phytate was hydrolyzed within 1 hr. Dif-

61

PHYTATES IN LEGUMES AND CEREALS TABLE XXXIII EFFECTS OF FERMENTATION ON TOTAL PHOSPHORUS AND PHYTIC ACID CONTENTS IN BLACK GRAM, RICE, AND BLACK GRAM AND RICE BLENDSa

Fermentation time (hr) Black gram Raw 0= 4 8 12 16 20 24 45 Rice Raw OC

4 8 12 16 20 24 45

Phytic acid hydrolyzed

Total phosphorus (mgk)

Phytic acid phosphorus (mgk)

Phytic acidb (mgig)

5.60 5.61 5.59 5.43 5.62 5.57 5.61 5.69 5.77

4.80 4.80 4.76 4.66 4.66 4.62 4.62 4.22 4.16

17.04 17.04 16.90 16.54 16.54 16.40 16.40 14.98 14.77

0.00 0.00 0.82 2.93 2.93 3.76 3.76 12.09 13.32

1.89 1.96 1.75 1.79 1.83 1.79 1.96 1.96 1.96

0.97 0.65 0.07 0.00 0.00 0.00 0.00 0.00 0.00

3.44 2.31 0.25 0.00 -

-

0.00 32.85 92.73 100.00 -

3.71 3.84 3.75 3.10 3.71 3.74 3.72 3.89

2.52 2.15 1.93 1.61 1.55 1.51 1.38 1.29

8.95 7.63 6.85 5.72 5.50 5.36 4.90 4.58

0.00 14.75 23.46 36.09 38.55 40.11 45.25 48.83

-

(%)

Black gram and rice OC

4 8 12 16 20 24

45

a s o w e : Reddy and Salunkhe (1980). bcalculated phytic acid (phytate) assuming 28.20% phosphorus in the molecule.
ferences similar to those between whole and ground wheat, as regards the rate of destruction of phytate, were found in other cereals by Mellanby (1950). The variations in the rate of hydrolysis of phytate observed in ground wheat compared to whole wheat might be due to the mechanical effect of the grinding in that it broke up the grain, thereby allowing the phytate and the phytase to come

62

N. R. REDDY ET AL

0

1

2

3

4

5 6 7 TIME (hr)

8

9 1 0 1 1 1 2

FIG. 21. Rate of phytate hydrolysis by phytase in wheat (wheat was stirred in water at pH 4.50 and temperature 45°C). From Mellanby (1950).

into contact. He also studied the rate of hydrolysis of phytate by phytase in other cereals at 45°C and pH 4.50 and the results are presented in Table XXXIV. All the grains, with the exception of corn, were ground to approximately the same degree of fineness. The corn sample was somewhat coarser. Rye and wheat had the greatest phytase activity; in rye, all of the phytate (168 mg/100 g) was hydrolyzed in 45 min, and in wheat, the corresponding time for the hydrolysis of complete phytate (232 mg/100g) was 60 min. The phytase of oats, yellow corn, and white corn acted much more slowly, so that even after 12 hr a large part of the phytate remained intact in these grains. In barley, all the phytate was completely hydrolyzed in 2 hr. Glass and Geddes (1959) showed increased inorganic phosphorus and decreased phytate in wheat stored at increased moisture content and temperature. In another study, Ferrel (1978) showed that over 80% of the phytate was hydrolyzed in hard red winter wheat during 4-hr autolysis at temperatures of 35 and 55°C and pH 5.20 (maintained by 0.1 M acetate buffer). The behavior of barley phytin during malting and brewing processes has been studied (Preece et al., 1960). They demonstrated that most of the phytate had disappeared during the malting and brewing processes. Kon et al. (1973) found an increase in inositol and inorganic phosphorus following addition of phytase to a California small white bean slurry. They found that on average, 65% degradation of phytic acid occurred upon incubating the beans for 20 hr at 55°C. Becker et al. (1974) also noted a similar decrease in phytic acid during incubation of California small white bean slurry. They observed increases of 88 and 77%, respectively, for inorganic phosphorus and inositol after 48-hr incubation. They reported a pH optimum of 5.2 for the appearance of inositol. The effects of time, temperature, pH, soaking, and heating on the autolysis of phytate of California small white beans were evaluated by Chang et al. (1977). At 50"C, the hydrolysis of phytate from the beans was 31.0% and reached a maximum of 49% at 60°C (Table XXXV). However, little effect was found at

TABLE XXXIV RATE OF HYDROLYSIS OF PHYTIC ACID BY PHYTASES IN VARIOUS GROUND CEREALS AT 45°C AND pH 4.50a.b Wheat

Rye

Barley

Oats

Yellow corn

White corn

Phytic acid Phytic acid Phytic acid Phytic acid Phytic acid Phytic acid Time of Phytic acid hydrolyzed Phytic acid hydrolyzed Phytic acid hydrolyzed Phytic acid hydrolyzed Phytic acid hydrolyzed Phytic acid hydrolyzed incubation (mg/100 g) (%) (mgi100 g) (%) (mgi100 g) (%) (mgi100 g) (%) (mgi100 g) (%) (mgi100 g) (%) min min min min min 1 hr 2hr 4 hr 6 hr 12 hr

0 7 15 30 45

232 190 137 98 50 0

0.0 18.1 40.9 57.8 78.4 100.0

168 156 125 23 0 0

0.0 7.1 25.6 86.3 100.0 -

75 36 0

0.0 3.0 17.2 44.4 62.2 81.8 100.0

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

198 I92 164 110

194

0.0

212

0.0

-

-

-

-

-

-

-

-

-

-

__

-

-

-

-

-

181 156 154 152 144

6.7 19.6 20.6 21.6 25.8

194 189 180 176 157

8.5 10.8 15.1 17.0 25.9

180 -

-

175 153 141

2.8 15.0 21.7

130

27.8

0.0

aExperimental conditions: 5 g of ground cereal were suspended in 25 ml of acetate-acetic buffer and shaken for the whole incubation period (temperature 45°C. buffer pH 4.50). bSource: Mellanby (1950).

64

N. R. REDDY ET AL

TABLE XXXV EFFECT OF TEMPERATURE ON THE AUTOLYSIS AND DIFFUSION OF PHYTATE IN CALIFORNIA SMALL WHITE BEANS",b Distribution of phytate after heat treatment Temperature ("C)

Bean (mg/g)

23 40 50 60 70 80 90

2.88 2.55 1.98 1.04 1.13 1.14 1.11

Phytate hydrolyzed

Water

Total phytate after treatment (mg/g)

0.00 0.00 Trace 0.42 1.21 1.23 1.44

2.88 2.55 1.98 1.46 2.34 2.47 2.55

0 11 31 49 19 14

(%I

11

"Experimental conditions: 5 g of dry beans were incubated with 50 ml of distilled water at the indicated temperature for 3 hr. bSource: Chang et al. (1977).

temperatures below 50°C or above 70°C. These results suggest an initiation of enzyme activity at about 60°C and inactivation of the enzyme at 70°C. They also reported that after 10 hr of incubation of beans at 60"C, only a negligible amount of phytic acid was found in the beans, approximately 75% of the total phytic acid being hydrolyzed, and 25% being diffused into the water in which the beans were incubated. They further found an apparent decrease in phytate throughout the pH range of 4.0-5.8 with greatest decrease between pH range of 5.0-5.5 (about 5661% of phytate hydrolysis during 36 hr of incubation) (Table XXXVI). A TABLE XXXVI EFFECT OF pH ON AUTOLYSIS AND DIFFUSION OF PHYTATE AT 50°C IN CALIFORNIA SMALL WHITE BEANS" Phytate (mg/g dry bean)

Phytate hydrolyzedb

P"

Bean

Water

Total

("/.I

4.0 4.5 5.0 5.5 5.8

1.20 0.77 0.27 0.27 1.39

0.41 0.88 0.99 0.84 0.55

1.61 1.65 1.26 1.11 1.94

44 43 56 61 33

~~~~~~~~~~~

"Source: Chang et al (1977) bBased on 2 88 mg phytate per gram of dry bean Expenmental conditions. 5 g of dry beans were incubated in 40 ml of 0 2 M acetate buffer at vanous pH for 24 hr

65

PHYTATES IN LEGUMES AND CEREALS TABLE XXXVII EFFECT OF 60°C PREHEATING ON HYDROLYSIS OF PHYTATE IN CALIFORNIA SMALL WHITE BEANS UNDER VARIOUS CONDITIONSa

Conditions Dry beans soaked at room temperature Overnight Dry beans incubated at 60°C with 10 volumes of distilled water for 4 hr Rehydrated beansb exposed to 60°C water-saturated air for 6 hr Dry beans incubated in distilled water at 60°C for 1 hr, then transferred to 50°C water or to pH 5.0 buffer solution for 3 hr Rehydrated beansb frozen overnight, thawed at room temperature, and then exposed to 60°C water-saturated air for 6 hr

Phytate Phytate hydrolyzed ( W g bean) 2.88

0.00

1.16 1.97

60.00 31.00

1.63

43.00

1.45

50.00

"Source: Compiled from the data of Chang (1975) and Chang et af. (1977). bBeans were soaked in distilled water at room temperature for about 16 hr.

considerable amount of phytate from the beans was also diffused into the medium under these time and temperature conditions. Hydrolysis of phytate was higher in beans when they were incubated in distilled water at 60°C for 4 hr than when they were incubated at 50°C either in distilled water alone or in acetate buffer for 3 hr (Table XXXVII). The low hydrolysis of phytate in rehydrated beans exposed to water-saturated air at 60°C for 6 hr was also reported by these investigators. The effect of 60°C preheating treatment has been studied in several beans (California small white, mung, soy, and lima beans) and wheat by Chang et al. (1977). A 36.9% decrease in phytate content was observed in soybeans when incubation was carried out at room temperature instead of at 60°C (Table XXXVIII) by them. Greater hydrolysis of phytate was noted in lima beans, California small white beans, and wheat slurries when incubated at 60"C, however. Ferrel (1978) found that the autolysis of phytate in beans was slow at both 35 and 55°C. Tabekhia and Luh (1980) demonstrated a decrease of 7.7, 8.1, 13.2, and 19.1%, respectively, for black-eyed, red kidney, mung, and pink beans, on soaking these beans for 12 hr at 24°C in tap water. Iyer et al. (1980) found that when pinto, Great Northern, and red kidney beans were soaked in distilled water for 18 hr at room temperature, the phytate content of beans was appreciably reduced (52.7, 69.6, and 51.7%, respectively) (Table XXXIX). However, they noticed a somewhat lessened phytate hydrolysis when the beans were soaked in a mixed salt solution (2.5% sodium chloride + 1.5% sodium bicarbonate + 9.5% sodium carbonate + 1.0% sodium tripolyphosphate) at pH 7.0 and room temperature of 21°C. The reasons for such differential phytate hydrolysis remain obscure at this time, however (Iyer, 1979).

TABLE XXXVIII EFFECTS OF SOAKING AND HEATING ON HYDROLYSIS OF PHYTATE IN SEEDS OF VARIOUS SPECIESa.b CSW beanc

Conditions Untreated powder Whole beans soaked in water at room temperature overnight Slurry incubated in water at 60°C overnight Whole beans incubated in water at 60°C for 5 hr Rehydrated whole beans incubated at 60°C in water-saturated air overnight

Phytate (mg/g)

Phytate hydrolyzed

Mung bean

(%)

Phytate (wig)

2.58

-

2.88

Phytate hydrolyzed

Soybean

(%)

Phytate (mg/g)

2.21

0.0

0.0

2.16

0.75

74.0

1.08

1.76

Lima bean

Phytate hydrolyzed (%)

Phytate (mg/g)

3.06

0.0

2.3

1.93

1.26

43.0

3.03

1

62.5

-

-

-

38.6

1.38

37.6

2.59

Phytate hydrolyzed

Wheat Phytate hydrolyzed

(%)

Phytate (mg/g)

2.30

-

2.36

0.0

36.9

2.46

0.0

2.26

4.2

.o

0.48

80.5

0.85

64.00

-

1.36

44.7

1.81

23.3

18.0

0.84

65.9

1.72

27.1

(%)

uExperimental conditions: 5 g of sample were used in each experiment, except lima bean in which 10 g of sample were used. For wet incubation 10 volumes of distilled water were used as the medium. bSource: Chang et al. (1977). “CWS bean, California small white bean

67

PHYTATES IN LEGUMES AND CEREALS TABLE XXXIX EFFECTS OF SOAKING ON PHYTATE OF THE GREAT NORTHERN, KIDNEY, AND PINTO BEANSo Great Northern

Treatment

Phytate (mg/g)

Control Soakingb Soakingc

4.6 1.4 4.2

Phytate hydrolyzed

0.0 69.6 8.7

Kidney

Phytate (mg/g) 5.8 2.8 4.5

Pinto

Phytate hydrolyzed (%)

Phytate (mglg)

0.0 51.7 22.4

5.5 2.6 4.4

Phytate hydrolyzed

0.0 52.7 18.2

oSource: Iyer et al. (1980). bSoaked for 18 hr in distilled water at room temperature (22°C). CSoakedin a combination of salt solutions (2.5%sodium chloride + 1.5% sodium bicarbonate + 0.5% sodium carbonate + 1% sodium tripolyphosphate) for 18 hr at room temperature (22"C),pH 7.0.

D.

METHODS FOR REMOVAL OF PHYTATES

Several processing methods (germination, soaking, autoclaving, autolysis, fermentation, special treatments, etc.) discussed in the previous section are shown to reduce or remove considerable amounts of phytate in cereals and legumes. In certain cases, phytic acid can be selectively removed by mechanical processes such as milling. For example, about 89% of the phytic acid in corn is concentrated in germ and can be removed by milling followed by germ separation (O'Dell et al., 1972b). In certain cereals such as wheat, rice, triticale, and rye, phytate is concentrated in the outer layers (bran) and hence normal milling (which involves bran removal) should remove appreciable amounts of phytate from these cereals (Reddy, 1976; Donnelly and Tabekhia, 1977). Polishing the rice has been shown to remove significant amounts of phytate (Resurreccion et al., 1979; Tabekhia and Luh, 1979). Differential solubility methods to precipitate selectively and remove phytate from soybeans have been investigated. Such methods usually involve extraction with water or alkali followed by careful pH adjustment so that the phytate (insoluble and precipitated) can be removed by centrifugation or filtration. McKinney et al. (1949) reported about 80% removal of phytate from the alkaline soybean extract by precipitation with calcium and barium ions and subsequent centrifugation (Table XL). In their studies they could recover about 80% of the soybean meal nitrogen in the process of protein concentration (while removing the phytate simultaneously) described above. They further showed that phosphorus-free soy protein suitable for fundamental studies could be prepared from

68

N. R. REDDY ET AL TABLE XL PRECIPITATION OF PHYTATE FROM SOYBEAN MEAL EXTRACT WITH CALCIUM AND BARIUM IONSa

Extracting agent

Phosphorus in protein

Extract PH

0.2% NaOH

9.7

0.2% NaOH

9.7

NaOH

1&11

0.1% NaOH

9.5

0.1% NaOH

9.5

0.1% Ca(OH)2

9.2

0.1% Ca(OH)2 1.0% Ba(OH)2

9.2 -

Special treatment

(%)

0.2% Ba(OH)* added, pH 10.9; centrifuged to remove precipitate 0.2% Ba(0H) added; adjusted to pH 9.5 and heated to 80°C; centrifuged Used meal after leaching with 0.5 N C1,CCOOH; 0.2% Ba(OH)2 added to alkaline dispersion; centrifuged 5% BaCI2 added; held at 5°C for 17 hr; centrifuged; Na2S03 added to remove Ba; clarified and precipitated 5% BaCI2 added; held at 5°C for 17 hr; centrifuged; dialyzed 0.7% NaOH added; held at 5"C, pH 11.5 for 17 hr; centrifuged; dialyzed Heated to 85°C; centrifuged (precipitate discarded) Extract set 2 hr; centrifuged

0.46 0.27 0.22

0.18 0.32 0.20 0.65 0.20

aSource: McKinney et al. (1949).

wet-acid soy curd after dialyzing the curd against 1 N sodium chloride solution. Phytate could also be removed from the wet soy protein curd by mixing it with a saturated solution of sodium or ammonium sulfate. Goodnight et al. (1976, 1978) prepared a low-phytate soy protein isolate using high pH (Fig. 22). This process used the phytate precipitation at high pH followed by centrifugation or filtration. Details of the process are as follows: Step 1: Soy flakes with high protein dispersibility index were extracted with water at high pH (9-10) with a water-to-flakes ratio of 16:l and the slurry was centrifuged for 15-20 min at 5000 g. CENTRIFUGATION p H 9 1 SOY FLAKE

soy pH 1

EXTRACT

SPENT FLAKE step 1 Extraction

FIG. 22.

CENTRIFUGATION 1 1t H 7

Tre%<

LOW-PHYTATE SOY EXTRACT

PHYTATE step 2 Phytate Removal

1

ULTRAFILTRATION

LOW-PHYTATE SOY PROTEIN

SOLUBLE CHO, ASH Step 3 Purification

Process outline for phytic acid removal from soy protein. From Hartman (1979).

69

PHYTATES IN LEGUMES AND CEREALS

Step 2: The phytate in the supernatant obtained in step 1 was precipitated by adjusting the pH of the supernatant to 11-12. The precipitated phytate was then removed by either centrifugation or by vacuum filtration and the pH of soy extract (after removing the phytate) was rapidly adjusted to 7.0 with dilute HCI. Temperature during the above processing was controlled between 20 and 30°C. Step 3: The soy extract was then subjected to ultrafiltration to obtain a lowphytate soy protein isolate.

These investigators (Goodnight et al., 1976) found that the pH and temperature were both critical for efficient removal of phytate without protein degradation. The protein isolate thus prepared had 93% of the original soy protein and the phytate content was reduced from the original 2.6% to 0.1% (i.e., 96.15% of the phytate was removed). Later, de Rham and Jost (1979) developed three pilot plant-scale processes for the low-phytate soy protein isolate production. The main features of these processes as compared to the classical process for soy isolate production are summarized in Table XLI. In a process at pH 11.5 they obtained a low-phytate soy protein isolate with 0.18% phytate and 72% protein. Lysinoalanine formation, which generally occurs at such high pH values and which is nutritionally toxic, was not detected in soy protein isolates prepared in this manner (Goodnight et al., 1976; Hartman, 1979). De Rham and Jost (1979) evaluated the above-described protein isolates for composition and nutritional value (Table XLII) and found that the isolate prepared at pH 5.5 had a protein TABLE XLI ISOLATE PRODUCTION PROCESSES PERFORMED AT THE PILOT PLANTO 4.5 classical

5.5 11.515.5

NaCllUF

Extraction of the defatted soy flour with water at pH 8.2, centrifugation, washing of the residue, acidification to pH 4.5, washing of the precipitate, lyophilization Identical to 4.5 except the precipitation at pH 5.5, and the omission of the washing steps Identical to 4.5 except an extraction at pH 11.5 (NaOH), precipitation at pH 5 . 5 , and the omission of washing steps Extraction of the defatted soy flour with 10% NaCl solution, centrifugation (first juice), washing of the residue with water, centrifugation, addition of NaCl to 10% (second juice). Separate filtration and ultrafiltration-diafiltration of the first and second juices, lyophilization of the UF concentrates NaCl/UF Process

Nitrogen yield (%) Phytate level (%)

4.5

5.5

1 1.515.5

First juice

Second juice

78

60 0.60

72 0.18

60 0.14

19 i .5

1.84

“Source: de Rham and Jost (1979).

70

N. R. REDDY ET AL. TABLE XLII COMPOSITION AND NUTRITIONAL EVALUATION OF THE LOW-PHYTATE SOY ISOLATES" Process

Protein (% N x 6.25) Phytate (%) Ashes (as is) (%) Na (%) K (%) Ca (%) Mg (%) Ashes (neutralized) (%) Amino acids (g116 g N) Ile Leu LYs Metb Cysb Phe TY Thr Val PER (casein 3.2) Trypsin inhibitoF PER (+ 1% phytate) PER (IOO"C, 10 min) Trypsin inhibitoF

4.5

5.5

1135.5

NaCI/UF first juice

86 1.84 1.3 0.01 0.06 0.06 0.04 4.2

93 0.60 2.2 0.07 0.4 0.06 0.08 2.7

90 0.18 2.1 0.4 0.4 0.06 0.04 2.7

84 0.14 10.4 3.8 0.2 0.04 0.05 10.4

3.8 7.1 4.5 1.6 1.3 4.3 2.8 3.3 3.5 1.7 375

4.1

4.3 7.6 4.6 1.5 1.2 5.0 2.7 3.6 4.2 1.9 270 1.7 -

4.3 8.6 5.9 1.3 1.3 4.7 3.8 3.9 4.4

-

2.1 110

7.6 3.9 1.6 1.4 4.7 3.2 3.6 4.0 2.2 120 2.0 2.7 40

1.5

860 1.6 -

"Source: de Rham and Jost (1979). bPerformic acid oxidation. =Arbitrary units per milligram of nitrogen. Standard error of the mean for PER values = 0.1,

efficiency ratio (PER) (a PER of 2.2 compared to 3.2of casein) better than those of other protein isolates. A significant change in soy protein at pH around 5.5 is known to occur (Fontaine et al., 1946; de Rham and Jost, 1979). Using this principle, Ford et al. (1978)developed a process to prepare soy protein isolate. They employed either a combination of low pH (3.54.0) and high calcium concentration (0.04 M ) or a high pH (5.CL5.5) and low calcium concentration (0.0025 M ) to remove about 90% (or more) of the phytate. The latter combination (high pH and low calcium concentration) afforded high (90%) zinc recovery in the curd together with high recoveries of protein (90-93%), fat (98-loo%), and iron (9696%). De Rham and Jost (1979)also prepared a soy protein isolate by precipitating proteins at pH 5.5 with no salt addition (Table XLI). At pH 5.5

PHYTATES IN LEGUMES AND CEREALS

71

soy proteins are insoluble, whereas phytic acid remains in solution. The insoluble proteins could then be removed to yield low-phytate (about 60% phytate removed) protein isolate (de Rham and Jost, 1979). Several studies have shown that phytic acid complexes with proteins as well as metal cations at both alkaline and acid pH levels (O’Dell and de Boland, 1976; Omosaiye and Cheryan, 1979; de Rham and Jost, 1979; Hill and Tyler, 1954). At acid pH (4.0-4.5) the phytate-protein complexation is thought to be of ionic type, resulting in an insoluble phytate-protein complex precipitate. The presence of excess calcium ions is shown to reduce the extent of such complexation (Hill and Tyler, 1954). Phytate removal by dialysis and diafiltration in the presence of 0.5 M calcium chloride has also been suggested (Okubo et al., 1975) (Fig. 24). At acidic pH both cationic protein groups and calcium compete for phytic acid binding. The higher affinity of calcium for phytic acid may prevent the phytic acid-protein complexation, thus allowing the production of the low-phytate protein isolates described in the above processes. McKinney and Solars (1949) employed sulfurous acid extraction at pH 2.30 to extract the soy proteins, followed by isoelectric precipitation (using NaOH) and dialysis to prepare a lowphytate soy protein isolate. The protein isolate thus prepared had 1.7-3.0% phytic acid which could not be removed by dialysis in acid conditions probably due to the phytate-protein complexation at low pH of protein extraction. A combined application of dialysis and anionic exchange resin has been investigated by Smith and Rackis (1957) in an attempt to remove the free phytic acid and its salts at different pH ranges (Table XLIII). They reported that about 78% of the phytic acid could be removed without appreciable loss of proteins by such a method. Dialysis and such methods seem impractical on a commercial scale, however.

72

N. R. REDDY ET AL

TABLE XLIII REMOVAL OF PHOSPHORUS AND NITROGEN COMPOUNDS FROM WATER EXTRACT OF THE SOYBEAN MEAL BY DIALYSIS AND ANION EXCHANGEa Phosphorus removed (%)

Nitrogen removed (%)

40.5 72.0 52.0 56.5 65.0 82.3 78.2 78.5

Dialysis 24 hr, pH 7.20 Dialysis 48 hr, pH 6.50 Dialysis 48 hr, pH 8.70 Anion exchange,b pH 7.50 Anion exchange, pH 7.00 Anion exchange, pH 6.50 Dialysis + anion exchange,c pH 7.00 Dialysis + anion exchange,c pH 7.00

7.5 7.0 28.0 ppt 4.0 7.5 24.0 10.1 9.2

“Source: Smith and Rackis (1957). bDowex- 1-X 10 ‘Dialysis at pH 7.20 (24 hr) followed by Dowex-]-XI0 at pH 7.00

100 80 Y

2 60 I-

2 Y

t

- 40 0

5 5 a

5 a

a 20

x

10

1

0

1

1

1

1

1

i

l

2 4 6 8 1 0 1 2 1 4 ULTRAFILTRATE VOLUMES, Vg

FIG. 24. Removal of phytate (as measured by phosphorus remaining in retentate) during diafiltration through PM-30 membrane: (1) pH 8.50, 0.015 M sodium borate, 65°C; (2) pH 8.50, 0.01 M sodium carbonate/bicarbonate, 6 5 T , 0.05 M EDTA initially present in the extract; (3) pH 7.10, sodium carbonateibicarbonate, 65°C; (4) pH 5.0, water, 65°C; ( 5 ) pH 3.0, water, 25°C; (6) pH 3.0, water, 0.5 M calcium chloride, 25°C. From Okubo et al. (1975).

73

PHYTATES IN LEGUMES AND CEREALS

Ultrafiltration seems promising for selective removal of low-molecular-weight components such as the phytic acid. Ultrafiltration offers the advantages of mild processing conditions and selectivity (ability to discriminate different molecules of differing size and shape). Okubo er al. (1975) employed ultrafiltration for specific removal of phytic acid from soybeans by first dissociating protein-bound phytate. Phytate dissociation was effected by phytase (at pH 5.2), addition of 0.5 M CaCl, at pH 3.0, or by adding 0.05 M EDTA (Na+ salt) at pH 8.5. The results of their study are presented in Fig. 24. These investigators also established conditions for phytate-protein dissociation (Table XLIV) at, above, and below the isoelectric point of soy globulins for the preparation of low-phytate soy protein isolates. Later, Omosaiye and Cheryan (1979) reported a 90% reduction in phytate by employing a two-stage discontinuous diafiltration (ultrafiltration + re-ultrafiltration) operation at pH 6.7 (Table XLV). They recommend use of alkaline pH over acid pH (2.0) for such operations. These same processes may be applicable to other legumes and need further investigations for optimization. Phytate autolysis has been also investigated in California small white beans (Chang et al., 1977). The incubation conditions included (1) incubation of beans in buffer (pH 5.5) at 50°C for 24 hr, (2) incubation of presoaked beans in water at 60°C for 10 hr, and (3) exposure of whole beans to water-saturated air at 60°C. They reported that about 33-90% of the phytate was removed by such processes TABLE XLIV DISSOCIATION O F PHYTIN-PROTEIN COMPLEX MEASURED BY DIALYSIS FOR 6 DAYS AT 2 5 T a

PH 8.5

8.5

1.2

5.5

2.0

Solvent conditions 0.026 M borate buffer (Na+) + EDTA - EDTA 0.05 M Tris-HCI buffer (Cl-) + EDTA - EDTA 0.05 M Tris-HCI buffer (CI-) + EDTA - EDTA Water + EDTA - EDTA 0.01 N HCI

Phosphorus removed

("/.I

Phosphorus content (g/IOO g protein)

80 65

0.24 0.42

12 50

0.33 0.60

85 82

0.18

95 95 31

0.06 0.06 0.82

0.21

"Source: Okubo er al. (1975). bOriginal extract used in the experiments contained 1.20 g phosphorus per 100 g protein

74

N. R. REDDY ET AL

TABLE XLV COMPOSITION OF SOY PROTEIN CONCENTRATES PRODUCED BY ULTRAFTLTRATION AT pH 6.7 (% DRY WEIGHT BASIS)" Concentrate Original soybean Water extract (VCR 1) Ultrafiltered (VCR 5 ) Re-ultrafiltered (R 5 )

Protein

Fat

Phytate

Ash

Otherh

43.3 48.3 56.7 59.1

24.0 26.4 32.5 34.2

1.27 1.68 0.823 0.064

4.71 5.34 3.43 2.85

26.12 18.28 6.55 3.19

aSource: Ornosaiye and Cheryan (1979). bBy difference. Includes fiber, carbohydrate, etc.

and that these processes caused similar removal of phytate from mung beans, lima beans, and wheat. The genetic approach for the selection of low-phytate wheat varieties has been studied (Bassiri and Nahapetian, 1977) and may be extended to other cereals and legumes. It seems at the present time, however, that such an approach may not be successful at least in the near future due to the lengthy procedures involved in it.

IV.

SUMMARY AND CONCLUSIONS

Phytic acid, myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate serves as the main phosphorus store in mature seeds and grains. In cereals and legumes, phytic acid content ranges from 0.14 to 2.05%, which accounts for 18 to 88% of the total phosphorus. Globoids are the primary storage bodies for phytic acid in legume seeds and cereal grains. Phytic acid has a strong binding capacity to form complexes with multivalent cations and proteins. Most of the phytate-metal complexes are insoluble at physiological pH and hence render several complexed minerals biologically unavailable to animals and humans. Published reports indicate that phytic acid interacts with proteins to form complexes at acidic pH and low cation concentration by ionic interactions, whereas at alkaline pH, divalent cations mediate such formation of complexes between phytic acid and proteins. Phytate-protein complexes are more resistant to proteolytic digestion compared to protein alone at low pH and are thought to be responsible for the reduced bioavailability of the minerals. Very little is known about the interaction chemistry and in many cases, the mechanism(s) by which phytic acid interferes in mineral bioavailability and forms complexes with proteins and/or minerals is (are) not clearly understood. Animal feeding trails indicate that the foods containing high levels of phytate interfere with absorption of several minerals such as calcium, magnesium, zinc, and iron. Processing methods such as soaking,

PHYTATES IN LEGUMES AND CEREALS

75

cooking, germination, fermentation, autoclaving, canning, milling, and membrane filtration can reduce or eliminate appreciable amounts of phytic acid from cereals and legumes.

V.

FUTURE RESEARCH NEEDS

Although the existence of phytic acid has been recognized for more than 125 years, our current knowledge about it is far from adequate. Both cereals and legumes are globally important dietary components which contain appreciable quantities of phytic acid. It is therefore important to develop a thorough understanding of the nutritional and technological implications of phytic acid. To accomplish this, several aspects need to be investigated and evaluated. One such area is the form(s) of naturally occurring phytic acid. The precise chemical form(s) in which phytic acid occurs in cereals and legumes needs to be investigated. Such knowledge will help to follow clearly the subsequent form(s) during processing. This demands the understanding of phytic acid synthesis in vivo, which can also help in the development of low-phytic acid lines through breeding. One of the reasons for the lack of identification of naturally occurring form(s) of phytic acid, to date, is that there is no direct method for its determination. Therefore, there is clearly a need for development of such a method. Phytic acid interacts with several other food components, such as proteins and minerals, leading to undesirable effects such as reduced mineral bioavailability and altered protein functionality. Improved understanding of the underlying mechanisms of such interactions should enable better control and subsequent minimization of undesirable effects accompanying such interactions. In view of the recent emphasis on reducing phytic acid in foods through processing, determination of threshold level(s) at which phytic acid has deleterious effects will help in the development of adequate processing methods.

REFERENCES Abernathy, R . H., Paulsen, G. M . , and Ellis, R., Jr. 1973. Relationship among phytic acid, phosphorus, and zinc during maturation of winter wheat. J . Agric. Food Chem. 21, 282. Adams, C. A,, and Novellie, L. 1975. Composition and structure of protein bodies and spherosomes isolated from ungerminated seeds of Sorghum bicolor (Linn)Moench. Plant Physiol. 55, 1. Alimarin, I. P., and Tozel, L. Z. 1957. Determination of zirconium with phytin. Ins?. Absch. Neorg. Khim. 3, 114; Chem. Abstr. 52, 26591, (1958). Anderson, G . 1956. The identification and estimation of soil inositol phosphates. J , Sci. Food Agric. 7, 437. Anderson, G . 1963. Effect of irodphosphorus ratio and acid concentration on the precipitation of ferric inositol hexaphosphate. J . Sci. Food Agric. 14, 352.

76

N. R. REDDY ET AL.

Anderson, G. H . , Hams, L., Rao, A. V., and Jones, J. D. 1976. Trace mineral deficiencies in rats caused by feeding rapeseed flours during growth, gestation, and lactation. J . Nutr. 106, 1166. Anderson, R. J. 1912a. N . Y . Agric. Exp. Stn., Geneva, Tech. Bull. 19, 21. Anderson, R. J. 1912b. Phytin and phosphoric acid esters of inosite. J . Biol. Chem. 11, 471. Anderson, R. J. 1 9 1 2 ~ .Phytin and phosphoric acid esters of inosite. J . Biol. Chem. 12, 97. Anderson, R. J. 1912d. The organic phosphoric acid compound of wheat bran. 111. Phytin. J . Biol. Chem. 12, 447. Anderson, R. J. 1914. A contribution to the chemistry of phytin. J . Biol. Chem. 17, 171. Anonymous. 1967. Effect of phytate on iron absorption. Nutr. Rev. 25, 218. Anonymous. 1979. Phytate and zinc bioavailability. Nutr. Rev. 37, 365. Apte, S . V., and Venkatachalam, P. S . 1962. Iron absorption in human volunteers using high phytate cereal diets. Indian J . Med. Res. 50, 516. Apte, S . V., and Venkatachalam, P. S . 1964. The influence of dietary calcium on absorption of iron. Indian J . Med. Res. 52, 213. Asada, K., and Kasai, Z. 1959. Formation of phytin and its role in the ripening process of rice plant. Mem. Res. Inst. Food Sci., Kyoto Univ. 18, 32. Asada, K., and Kasai, Z. 1962. Formation of myo-inositol and phytin in ripening rice grains. Plant Cell Physiol. 3, 397. Asada, K., Tanaka, K., and Kasai, Z. 1969. Formation of phytic acid in cereal grains. Ann. N . Y . Acud. Sci. 165, 801. Ashton, W. M., and Williams, P. C. 1958. The phosphorus compounds of oats. I. The content of phytate phosphorus. J . Sci. Food Agric. 9, 505. Ashton, W. M., Evans, C., and Williams, P. C. 1960. Phosphorus compounds of oats. 11. The utilization of phytate phosphorus by growing chicks. J . Sci. Food Agric. 11, 722. Averill, H . P., and King, C. G . 1926. The phytin content of food stuffs. J . Am. Chem. SOC. 48, 724. AVRDC-Mung Bean Report. 1976. Nutritional chemistry. In “Asian Vegetable Research and Development Center-Mung Bean Report,” p. 28. Shanhua, Taiwan. Baker, E. C., Mustakas, G . C., Erdman, J. W., Jr., and Black, L. T. 1981. The preparation of soy products with different levels of native phytate of zinc bioavailability studies. J . Am. Oil Chem. SOC. 58, 54. Barrk, R., Curtois, J. E . , and Wormser, G. 1954. Etude de la structure de l’acide phytique au moyCn de ses courbes de titration et de la conductivitk de ses solutions. Bull. SOC.Chim. Biol. 36,455. B a d , M. R. 1956. Influence de I’acide phytique sur la digestion pepsique de diffkrentes proteinas. Ann. Pharm. Fr. 14, 182. Bassiri, A., and Nahapetian, A. 1977. Differences in concentrations and interrelationships of phytate, phosphorus, magnesium, calcium, zinc, and iron in wheat varieties grown under dryland and imgated conditions. J . Agric. Food Chem. 25, 1118. Bayley, H. S . , and Thompson, R. G. 1969. Phosphorus requirements of growing pigs and effect of steam pelleting on phosphorus availability. J . Anim. Sci. 28, 484. Beck, G . 1948. Mikrochem. Ver. Mikrochim. Acta 34, 62 (cited from Brown et a / . , 1961). Becker, R., and Lorenz, K. 1978. Saccharides in proso and foxtail millets. J . Food Sci. 43, 1412. Becker, R., Olson, A. C., Frederick, D. P., Kon, S . , Gumbmann, M. R., and Wagner, J. R. 1974. Conditions for autolysis of a-galactosides and phytic acid in California small white beans. J . Food Sci. 39, 766. Belavady, B., and Banerjee, S . 1953. Studies on the effect of germination on the phosphorus values of some common Indian pulses. Food Res. 18, 223. Berlyne, G. M., BenAri, J., Nord, E., and Shainkin, R. 1973. Bedouin osteomalacia due to calcium deprivation caused by high phytic acid content of unleavened bread. Am. J . Clin. Nutr. 26, 910. Besecker, R. J., Jr., Plumlee, M. P., Pickett, R. A., and Conrad, J. H. 1967. Phosphorus from barley grain for growing swine. J . Anim. Sci. 26, 1477.

PHYTATES IN LEGUMES AND CEREALS

77

Biswas, S., and Biswas, B. B. 1965. Enzymatic synthesis of guanosine triphosphate. Biochim. Biophys. Acta 108, 710. Bitar, K., and Reinhold, J. G. 1972. Phytase and alkaline phosphatase activities in intestinal mucosae of rat, chicken, calf, and man. Biochim. Biophys. Acta 268, 442. Blank, G. E., Pletcher, J., and Sax, M. 1971. The structure of myo-inositol hexaphosphate, dodecasodium salt octama-contahydrate: A single crystal X-ray analysis. Biochem. Biophys. Res. Commun. 44, 319. Bourdillon, J. 1951. A crystalline bean seed protein in combination with phytic acid. J.Bio1. Chem. 189, 65. Bourdillon, J. 1953. N . Y . State Dep. Health, Annu. Rep. Div. Lab. Res. 23 (cited from Brown et a / . , 1961). Bramsnaes, F., and Olsen, H. S. 1979. Development of field pea and faba bean proteins. J . Am. Oil Chem. SOC. 56, 450. Bronner, F., Harris, R. S., Maletskos, C. J., and Brenda, C. E. 1954. Studies in calcium metabolism: Effect of food phytates on calcium45 uptake in children on low-calcium breakfasts. J . Nutr. 54, 523. Brown, E. C., Heit, M. L., and Ryan, D. E. 1961. Phytic acid: An analytical investigation. Can. J . Chem. 39, 1290. Bruce, H. M . , and Callow, H. K. 1934. Cereals and rickets. The role of inositol hexaphosphoric acid. Biochem. J . 28, 57. Buttrose, M. S. 1978. Manganese and iron in globoid crystals of protein bodies from Avena and Casuarina. Aust. J . Plant Physiol. 5, 631. Byrd, C. A., and Matrone, G. 1965. Investigations of chemical basis of zinc-calcium-phytate interaction in biological systems. Proc. SOC. Exp. Biol. Med. 119, 347. Caldwell, A. G., and Black, C. A. 1958. Inositol hexaphosphate. 111. Content in soils. Soil Sci. SOC. Am. Proc. 22, 296. Callender, S . T., and Warner, G. T. 1970. Iron absorption from brown bread. Lancet 1, 546. Calvert, C. C., Besecker, R. J., Plumlee, M. P., Cline, T. R., and Forsyth, D. M. 1978. Apparent digestibility of phosphorus in barley and corn for growing swine. J . Anim. Sci. 47, 420. Chang, C. W. 1967. Study of phytase and fluoride effects in germinating corn seeds. Cereal Chem. 44, 129. Chang, R., Schwimmer, S . , and Burr, H. K. 1977. Phytate: Removal from whole dry beans by enzymatic hydrolysis and diffusion. J . Food Sci. 42, 1098. Chang, R. H. 1975. Removal of phytic acid from beans by potentiation of in situ phytase. Ph.D. Dissertation, University of California, Berkeley. Chen, L. H., and Pan, S. H. 1977. Decrease of phytates during germination of pea seeds (Pigsum saliva). Nutr. Rep. Int. 46, 125. Cheryan, M. 1980. Phytic acid interactions in food systems. CRC Crit. Rev. Food Sci. Nutr. 13, 297. Contardi, A. 1910. Phosphoric esters of some polyvalent alcohols and of some carbohydrates. Atti Accad. Naz. Lincei, Cl. Sci. Fis., Mat. Nut., Rend. 19, 823. Cosgrove, D. J. 1966. The chemistry and biochemistry of inositol polyphosphates. Rev. Pure Appl. Chem. 16, 209. Costello, A. J. R., Glonek, T., and Myers, T. C. 1976. Phosphorus-31 nuclear magnetic resonance-pH titrations of myoinositol hexaphosphate. Carbohydr. Res. 46, 159. Courtois, J. E. 1954. J . Jpn. Biochem. SOC. 26, 85 (cited from Oberleas, 1971, p. 100). Courtois, J. E., and B a d , R. 1953. Bull. SOC. Chim. Biol. 35, 913 (cited from Brown eral., 1961). Courtois, J. E., and Masson, M. 1950. Phytase. XVI. Compounds obtained after partial hydrolysis of phytin. Bull. SOC. Chim. Biol. 32, 326. Cowan, J. W., Esfahani, M., Salji, J. P., and Azzam, S . A. 1966a. Effect of phytate on iron absorption in the rat. J . Nutr. 90, 423.

78

N. R. REDDY ET AL

Cowan, J. W., Esfahani, M., Salji, J. P., and Nahapetian, C. 1966b. Nutritive value of middleeastem food-stuffs. 111. Physiological availability of iron in selected foods common to middleeast. J . Sci. Food Agric. 18, 227. Crean, D. E. C., and Haisman, D. R. 1963. The interaction between phytic acid and divalent cations during the cooking of dried peas. J . Sci. Food Agric. 14, 824. Cruickshank, E. W. H., Duckworth, J . , Kosterlitz, H. W . , and Wamock, G. W. 1945. The digestibility of the phytate P of oatmeal in adult man. J . Physiol. (London) 104, 41. Cullumbine, H., Basnayake, V., Lemottee, J., and Wickramanayake, T. W. 19.50. Mineral metabolism on rice diets. Br. J . Nutr. 4, 101. Davidson, S. S . , and Passmore, R. 1970. “Human Nutrition and Dietetics,” p. 105. Williams & Wilkins, Baltimore, Maryland. Davies, N. T., and Nightingale, R. 1975. The effects of phytate on intestinal absorption and secretion of zinc, and whole body retention of zinc, copper, iron, and manganese in rats. Br. J . Nutr. 34, 243. Davies, N. T., and Olpin, S . E. 1979. Studies on the phytate: Zinc molar contents in diets as a determinant of zinc availability to young rats. Br. J . Nutr. 41, 590. Davies, N. T., and Reid, H. 1979. An evaluation of the phytate, zinc, copper, iron, and manganese contents of and zinc availability from soy-based textured vegetable protein meat substitutes or meat extenders. Br. J . Nutr. 41, 579. Davies, N. T., Hristic, V., and Flett, A. A. 1977. Phytate rather than fibre in bran as the major determinant of zinc availability to rats. Nutr. Rep. Int. 15, 207. Davis, K. R. 1981. Proximate composition, phytic acid, and total phosphorus of selected breakfast cereals. Cereal Chem. 58, 347. Davis, P. N., Norris, L. C., and Kratzer, F. H. 1961. Interference of soybean proteins with the utilization of trace minerals. J . Nutr. 77, 217. de Boland, A,, Gamer, G. B., and O’Dell, B. L. 1975. Identification and properties of phytate in cereal grains and oilseed products. J . Agric. Food Chem. 23, 1186. de Lange, D. I . , Joubert, C. P., and du Preez, S . F. M. 1961. The determination of phytic acid and factors which influence its hydrolysis in bread. Proc. Nutr. Soc. South Afr. 2, 69. de Rham, O., and Jost, T. 1979. Phytate-protein interactions in soybean extracts and low-phytate soy protein products. J . Food Sci. 44, 596. Desjobert, A,, and Fleurent, P. 19.54. Influence de la rCaction du mileen sur l’hydrolyse chimique de I’inositol-hexaphosphate.Considerations sur la constitution de ce derive. Bull. Soc. Chim. Biol. 36, 475. Dieckert, J . W., Snowden, J . E., Jr., Moore, A. T., Heinzelman, D. C., and Altschul, A. M. 1962. Composition of some subcellular fractions from seeds of Arachis hypogaea. J . Food Sci. 27, 321. Donnelly, B. J., and Tabekhia, M. M. 1977. Durum wheat and its milled products: Effect of variety and environment on the phytic acid content. Cereal Foods World 22, 483 (Abstr. No. 180). Dyer, W. J., Wrenshall, C. L . , and Smith, G. R. 1940. The isolation of phytin from soil. Science 91, 319. Earley, E. B. 1944. Stoichiometric relations of iron and phosphorus in ferric phytate and a method for quantitative determination of phytin phosphorus. Ind. Eng. Chem., Anal. Ed. 16, 389. Eklund, A. 1975. The contents of phytic acid in protein concentrates prepared from Niger seed, sunflower, rapeseed, and poppy seed. Upsala J . Med. Sci. 80, 5 . Ellis, L. C., and Tillman, A. D. 1961. Utilization of phytin phosphorus in wheat bran by sheep. J . Anim. Sci. 20, 606. Ellis, R., and Morris, E. R. 1979. Effect of sodium phytate on stability of monoferric phytdte complex and the bioavailability of the iron to rat. Nutr. Rep. In?. 20, 739. Ellis, R., and Morris, E. R. 1981. Relation between phytic acid and trace metals in wheat bran and soybean. Cereal Chem. 58, 850.

PHYTATES IN LEGUMES AND CEREALS

79

Ellis, R., Moms, E. R., and Philpot, C. 1977. Quantitative determination of phytate in the presence of high inorganic phosphate. Anal. Biochem. 77, 536. Elwood, P. C., Newton, D., Eakins, J. D., and Brown, D. A. 1968. Absorption of iron from bread. Am. J . Clin. Nutr. 21, 1162. Erdman, J. W., Jr. 1979. Oilseed phytates: Nutritional implications. J . Am. Oil Chem. SOC.56,736. Erdman, J. W., Jr. 1981. Bioavailability of trace minerals from cereals and legumes. Cereal Chem. 58, 21. Erdman, J. W., Jr., and Forbes, R. M. 1977. Mineral bioavailability from phytate containing foods. Food Prod. Dev. 11, 46. Erdman, J. W., Jr., Weingartner, K. E., Parker, H. M., and Forbes, R. M. 1978. Bioavailability of calcium, zinc, and magnesium in soy protein diets. Fed. Proc., Fed. Am. Soc. Exp. B i d . 37, 891 (Abstr. No. 3563). Erdman, J. W., Jr., Weingartner, K. E . , Mustakas, G. C., Schmutz, R. D., Parker, H. M., and Forbes, R. M. 1980. Zinc and magnesium bioavailability from acid precipitated and neutralized soybean protein products. J . Food Sci. 45, 1193. Ergle, D. R., and Guin, G. 1959. Phosphorus compounds of the cotton embryos and their changes during germination. Plan? Physiol. 34, 476. Evans, W. J . , and Pierce, A. G. 1981. Calcium-phytate complex formation studies. J . Am. Oil Chem. Soc. 58, 850. Fardiaz, D., and Markakis, P. 1981. Degradation of phytic acid in oncom (Fermented peanut press cake). J . Food Sci. 46, 523. Fennessey, J. P., and Nowacki, W. 1968. Rontgen-Daten des 38-hydrates des Natriumsalzes der phytinsaure. Helv. Chim. Acra 51, 1475. Ferrel, R. E. 1978. Distribution of bean and wheat inositol phosphate esters during autolysis and germination. J . Food Sci. 43, 563. Fischler, F., and Kurten, F. 1932. Biochem. 254, 138 (cited from Brown et al., 1961). Fontaine, T. D., Pons, W. A,, Jr., and Irving, G . .W. 1946. Protein phytic acid relationship in peanuts and cottonseed. J . Biol. Chem. 164, 487. Forbes, R. M. 1961. Excretory patterns and bone deposition of zinc, calcium, and magnesium in the rat as influenced by zinc deficiency, EDTA, and lactose. J . Nutr. 74, 194. Forbes, R. M. 1964. Mineral utilization in the rat. 111. Effects of calcium, phosphorus, lactose, and source of protein in zinc deficient and in zinc-adequate diets. J . Nutr. 83, 225. Forbes, R. M . , and Parker, H. M. 1977. Biological availability of zinc in and as influenced by whole fat soy flour in rat diets. Nutr. Rep. Int. 15, 681. Forbes, R. M., and Yohe, M. 1960. Zinc requirements and nitrogen balance studies with rats. J . Nutr. 70, 53. Forbes, R. M . , Weingartner, K. E., Parker, H. M., Bell, R. R., and Erdman, J. W., Jr. 1979. Bioavailability to rats of zinc, magnesium, and calcium in casein-, egg-, and soy proteincontaining diets. J . Nutr. 109, 1652. Ford, J . A,, Colhoun, E. M., McIntosh, W. B., and Dunnigan, M. G. 1972a. Nutritional rickets in immigrants. Br. Med. J . 2, 677. Ford, J. A,, Colhoun, E. M., McIntosh, W. B., and Dunnigan, M. G . 1972b. Biochemical response of late rickets and osteomalacia to a chupaty-free diet. Br. Med. J . 3, 446. Ford, J. A,, McIntosh, W. B., and Dunnigan, M. G. 1977. A possible relationship between high extraction cereal and rickets and osteomalacia. Adv. Exp. Med. Biol. 81, 353. Ford, J. R., Mustakas, G. C., and Schmutz, R. D. 1978. Phytic acid removal from soybeans by a lipid protein concentrate process. J . Am. Oil Chem. SOC.55, 371. Fordham, J. R., Wells, C. E., and Chen, L. R. 1975. Sprouting of seeds and nutrient composition of seeds and sprouts. J . Food Sci. 40, 552. Foy, H., Kondi, A , , and Austin, W. H. 1959. Effect of dietary phytate on fecal absorption of radioactive femc chloride. Nature (London) 183, 691.

80

N. R. REDDY ET AL

Franz, K. B. 1978. Bioavailability of zinc from selected cereals and legumes. Ph.D. Dissertation, p. 264. University of California, Berkeley. Fuhr, I., and Steenback, H. 1943. The effect of dietary calcium, phosphorus, and vitamin D on the utilization of iron. J . Biol. Chem. 147, 59. Gibbins, L. N., and Noms, F. W. 1963. Phytase and acid phosphatase in the dwarf bean (Phaseolus vulgaris). Biochem. J . 86, 67. Gillis, M. B., Noms, L. C., and Heuser, G. F. 1948. The utilization by the chick of phosphorus from different sources. J . Nurr. 35, 195. Gillis, M. B., Noms, L. C . , and Heuser, G. F. 1949. The effect of phytin on the phosphorus requirement of the chick. Poult. Sci. 28, 283. Gillis, M. B., Noms, L. C., and Heuser, G. F. 1953. Phosphorus metabolism and requirements of hens. Poult. Sci. 32, 977. Gillis, M. B., Keane, K. W., and Collins, R. A. 1957. Comparative metabolism of phytate and inorganic P32 by chicks and poults. J . Nutr. 78, 155. Glass, R. L., and Geddes, W. F. 1959. Grain storage studies. XXVII. Inorganic phosphorus content of deteriorating wheat. Cereal Chem. 36, 86. Goodnight, K. C., Hartman, G. H., and Marquardt, R. F. 1976. Aqueous purified soy protein and beverage. U. S . Patent 3,995,071. Goodnight, K. C., Hartman, G. H., and Marquardt, R. F. 1978. Low-phytate, isoelectric precipitated soybean protein isolate. U. S. Patent 4,072,670. Guenter, W., and Sell, J. L. 1974. A method for determining “true” availability of magnesium from foodstuffs using chickens. J . Nurr. 104, 1446. Gupta, S. K . , and Venkatasubramanian, T. A. 1975. Production of aflatoxin on soybeans. Appl. Microbiol. 29, 834. Guthrie, B . E., and Robinson, M. F. 1978. Zinc balance studies during wheat bran supplementation. Fed. Proc., Fed. Am. SOC. Exp. Biol. 37, 254 (Abstr. No. 219). Hall, J. R., and Hodges, T. K. 1966. Phosphorus metabolism of germinating oat seeds. Plant Physiol. 41, 1459. Halsted, J. H., Ronaghy, H. A,, Abadi, P., Haghshenass, M., Amirhakimi, R. M., Barakat, R. M., and Reinhold, J. G. 1972. Zinc deficiency in man: The Shiraz experiment. Am. J . Med. 53, 277. Harland, B. F., and Harland, J. 1980. Fermentative reduction of phytate in rye, white, and whole wheat breads. Cereal Chem. 57, 226. Harland, B. F., and Oberleas, D. 1977. A modified method for phytate analysis using an ionexchange procedure: Application to textured vegetable proteins. Cereal Chem. 54, 827. Harms, R. H., Waldroup, P. W., Shirley, R. L., and Ammerman, C. B. 1962. Availability of phytic acid phosphorus for chicks. Poult. Sci. 41, 1189. Hams, R. S . , and Mosher, L. M. 1934. Estimation of phytin phosphorus. Ind. Eng. Chem., Anal. Ed. 6, 320. Harrison, D. C., and Mellanby, E. 1939. Phytic acid and the rickets-producing action of cereals. Biochem. J . 33, 1660. Hartig, T. 1855. Uber das klebermehl. Bot. Ztg. 13, 881. Hartig, T. 1856. Weitere Mittheilungen uber klebermehl. Bot. Zrg. 14, 257. Hartman, G. H., Jr. 1979. Removal of phytate from soy proteins. J . Am. Oil Chem. SOC.56, 731. Hegge, M. H. M., and Rein, J. F. 1948. Pharm. Weekbl. 87, 801 (cited from Brown et a l . , 1961). Hegsted, D. M . , Trulson, M. F., and Stare, F. J. 1954. Role of wheat and wheat products in human nutrition. Physiol. Rev. 34, 221. Heubner, W . , and Stadler, H. 1914. Uber eine titrations methode zur bestimmung des phytins. Biochem. 2. 64, 422. Heuser, G. F., Norris, L. C., McGinnis, J., and Scott, M. L. 1943. Further evidence of the need for supplementing soybean meal chick rations with phosphorus. Poulr. Sci. 22, 269.

PHYTATES IN LEGUMES AND CEREALS

81

Hill, L. F. 1972. Phytic acid and serum-calcium. Lancet 2 , 769. Hill, R., and Tyler, C. 1954. The reaction between phytate and protein. J . Agric. Sci. 44, 324. Hoff-Jorgensen, E., Anderson, O., and Nielsen, G . 1946. The effect of phytic acid on the absorption of calcium and phosphorus. Biochem. J . 40, 555. Hunter, J. E. 1981. Iron availability and absorption in rats fed sodium phytate. J . Nutr. 111, 841. Hussain, R., and Patwardhan, V. N. 1959. Influence of phytate on absorption of iron. IndianJ. Med. Res. 47, 676. Ismail-Beigi, F., Faraji, B., and Reinhold, J. G . 1977. Binding of zinc and iron to wheat bread, wheat bran, and other components. Am. J . Clin. Nutr. 30, 1721. IUPAC-IUB. 1968. The nomenclature of cyclitols. Eur. J . Biochem. 5, 1. Iyer, V. G . 1979. Production of quick-cooking beans (Phaseolus vulgaris L.): Investigations on antinutrients and quality. M.S. Thesis, Utah State University, Logan. Iyer, V. G . , Salunkhe, D. K., Sathe, S. K., and Rockland, L. B. 1980. Quick-cooking beans (Phaseolus vulgaris L.). 11. Phytates, oligosaccharides, and antienzymes. Qual. Plant.-Plant Foods Hum. Nutr. 30, 45. Jacobsen, J. V., Knox, R. B., and Pyliotis, N. A. 1971. The structure and composition of aleurone grains in the barley aleurone layer. Planta 101, 189. Johnson, L. F., and Tate, M. E. 1969. Structure of “phytic acids.” Can. J . Chem. 47, 63. Kelsay, J. L. 1981, Effect of diet fiber level on bowel function and trace mineral balances of human subjects. Cereal Chem. 58, 2. Knorr, D., Watkins, T. R., and Carlson, B. L. 1981. Enzymatic reduction of phytate in whole wheat breads. J . Food Sci. 46, 1866. Koepke, J. A,, and Stewart, W. B. 1964. Role of gastric secretion in iron absorption. Proc. SOC. Exp. Biol. Med. 115, 927. Kon, S . , Olson, A. C., Frederick, D. P., Eggling, S . B., and Wagner, J. R. 1973. Effect of different treatments on phytate and soluble sugars in California small white beans. J . Food Sci. 39, 215. Kratzer, F. H., Allred, J. B., Davis, P. N., Marshall, B. J., and Vohra, P. 1959. The effect of autoclaving soybean protein and the addition of ethylenediaminetetraacetic acid on biological availability of dietary zinc for turkey poults. J . Nutr. 68, 313. Krebs, H. A , , and Mellanby, E. 1943. The effect of national wheat-meal on the absorption of calcium. Biochem. J . 37, 366. Kumar, K. G . , Venkataraman, L. V., Jaya, T. V., and Krishnamurthy, K. S . 1978. Cooking characteristics of some germinated legumes: Changes in phytins, Ca+ +,Mg+ +,and pectins. J . Food Sci. 43, 85. Kuvaeva, E. B., and Kretovich, V. L. 1978. Phytase of germinating pea seeds. Sov. Plant Physiol. (Engl. Transl.) 25, 290. Latta, M., and Eskin, M. 1980. A simple and rapid colorimetric method for phytate determination. J . Agric. Food Chem. 28, 1313. Lease, J. G . 1966. The effect of autoclaving sesame meal on its phytic acid content and on the availability of its zinc to the chick. Poult. Sci. 45, 237. Lease, J. G . 1967. Availability to the chick of zinc-phytate complexes isolated from soy oilseed meals by an in vitro digestion method. J . Nutr. 93, 523. Levene, U. 1909. Biochem. Z . 16, 399 (cited from Rose, 1912). Liebman, M., and Driskell, J. 1979. Dietary phytate and liver iron repletion in iron-depleted rats. Nutr. Rep. Int. 19, 281. Likuski, H. J. A., and Forbes, R. M. 1964. Effect of phytic acid on the availability of zinc in amino acid and casein diets fed to chicks. J . Nutr. 84, 145. Likuski, H. J. A., and Forbes, R. M. 1965. Mineral utilization in the rat. IV. Effects of calcium and phytic acid on the utilization of dietary zinc. J . Nutr. 85, 230.

82

N. R. REDDY ET AL.

Lim, P. E., and Tate, M. E. 1973. The phytase. 11. Properties of phytase fractions FI and F2 from wheat bran and myoinositol phosphates produced by fraction F2. Biochim. Biophys. Acta 302, 316. Lipschitz, D. A., Simpson, K. M., Cook, J. D., and Morris, E. R. 1979. Absorption of monoferric phytate by dogs. J . Nutr. 109, 1154. Liu, D. J . , and Pomeranz, Y . 1975. Distribution of minerals in barley at the cellular level by X-ray analysis. Cereal Chem. 52, 620. Lo, G. S . , Collins, D. W . , Steinke, F. J . , and Hopkins, D. T. 1978. Effect of isolated soy protein on bioavailability of magnesium. Fed. Proc., Fed. Am. Soc. Exp. Biol. 37, 667 (Abstr. No. 2386). Lo, G. S., Settle, S . L., Steinke, F. H., and Hopkins, D. T. 1981. Effect of phytate: Zinc molar ratio and isolated soy protein on zinc bioavailability. J . Nutr. 111, 2223. Lofgreen, G. P. 1960. The availability of the phosphorus in dicalcium phosphate, bone meal, soft phosphate, and calcium phytate for mature wethers. J . Nutr. 70, 58. Lolas, G. M., and Markakis, P. 1975. Phytic acid and other phosphorus compounds of beans (Phaseolus vulgaris L.). J . Agric. Food Chem. 23, 13. Lolas, G . M., and Markakis, P. 1977. The phytase of navy bean (Phaseolus vulgaris). J . Food Sci. 42, 1094. Lolas, G. M . , Palamidis, N., and Markakis, P. 1976. The phytic acid-total phosphorus relationship in barley, oats, soybeans, and wheat. Cereal Chem. 53, 867. Long, C. 1961. Phytase. In Biochemists’ Handbook,” p. 259. Van Nostrand-Reinhold, Princeton, New Jersey. Lott, J. N. A. 1975. Protein body composition in Cucurbita maxima cotyledons as determined by energy dispersive X-ray analysis. Plant Physiol. 55, 913. Lott, J. N. A , , and Buttrose, M. K. 1978. Globoids in protein bodies of legume seed cotyledons. Aust. J . Plant Physiol. 5, 89. Lott, J. N. A., and Vollmer, C. M. 1979. Composition of globoid crystals from embryo protein bodies in five species of Cucurbita. Plant Physiol. 63, 307. Lott, J. N. A., Greenwood, J. S . , Vollmer, C. M., and Buttrose, M. S . 1978. Energy dispersive Xray analysis of P, K, Mg, and Ca in globoid crystals in protein bodies from different regions of Cucurbita maxima embryos. Plant Physiol. 61, 984. Lowe, J. T., Steenbock, H., and Krieger, C. H. 1939. Cereals and rickets. IX. The availability of phytin-P to the chick. Poult. Sci. 18, 40. Lui, N. S . T . , and Altschul, A. M. 1967. Isolation of globoids from cottonseed aleurone grain. Arch. Biochem. Biophys. 121, 678. McCance, R. A., and Widdowson, E. M. 1935. Phytin in human nutrition. Biochem. J . 29B, 2694. McCance, R. A,, and Widdowson, E. M. 1942a. Mineral metabolism of healthy adults on white and brown bread varieties. J . Physiol. (London) 101, 44. McCance, R. A,, and Widdowson, E. M. 1942b. Mineral metabolism on dephytinized bread. J . Physiol. (London) 101, 304. McCance, R. A., and Widdowson, E. M. 1944. Activity of the phytase in different cereals and its resistance to dry heat. Nature (London) 153, 650. McCance, R. A., Edgecombe, C. N., and Widdowson, E. M. 1943. Phytic acid and iron absorption. Lancet 2, 126. McGinnis, J., Nonis, L. C., and Heuser, G. F. 1944. Poor utilization of phosphorus in cereals and legumes by chicks for bone development. Poult. Sci. 23, 157. McKinney, L. L., and Sollars, W. F. 1949. Extraction of soybean protein with sulfurous acid. Ind. Eng. Chem., Anal. Ed. 41, 1058. McKinney, L. L., Sollars, W. F . , and Setzkorn, E. A. 1949. Studies on the preparation of soybean protein free from phosphorus. J . Biol. Chem. 178, 117.

PHYTATES IN LEGUMES AND CEREALS

83

McWard, G. W. 1969. Effects of phytic acid and ethylenediaminetetraacetic acid (EDTA) on the chick’s requirement for magnesium. Poult. Sci. 48, 791. Maddaiah, V . T., Kurnick, A. A,, and Reid, B. L. 1964. Phytic acid studies. Proc. Soc. Exp. Biol. Med. 115, 391. Magrill, D. S. 1972. Microdetermination of phytate in solution or bound to hydroxyapatite. Anal. Biochem. 49, 522. Maiti, I. B., and Biswas, B. B. 1979. Further characterization of phytase from Phaseolus aureus. Phytochemistv 18, 316. Maiti, I. B., Majumdar, A. L., and Biswas, B. B. 1974. Purification and mode of action of phytase from Phuseolus vulgaris. Phytochemistry 13, 1047. Makdani, D., Mickelsen, O., Ullrey, D., and Ku, P. K. 1975. Effect of phytic acid on rat growth and availability of zinc, copper, iron, and calcium. Fed. Proc., Fed. Am. SOC. Exp. Biol. 34, 926 (Abstr. No. 4004). Makower, R. U. 1969. Changes in phytic acid and acid-soluble phosphorus in maturing pinto benas. J . Sci. Food Agric. 20, 82. Makower, R. U. 1970. Extraction and determination of phytic acid in beans (Phaseolus vulgaris). Cereal Chem. 47, 288. Mandal, N. C., and Biswas, B. B. 1970. Metabolism of inositol phosphates. I. Phytase synthesis during germination in cotyledons of mung beans, Phaseolus aureus. Plan? Physiol. 45, 4. Mandal, N. C., Burman, S . , and Biswas, B. B. 1972. Isolation, purification, and characterization of phytase from germinating mung bean. Phytochemistry 11, 495, Marrese, J . , Duell, R. W . , and Sprague, M. A. 1961. A comparison of three current methods for the analysis of phytin phosphorus. Crop Sci. 1, 80. Mathur, M. L. 1951. Assimilation of phytin phosphorus by dairy cows. Indian J . Vet. Sci. Anim. Husb. 23, 243. Matterson, L. D., Scott, H. H., and Singsen, E. P. 1946. The influence of sources of phosphorus on relative efficiency of vitamin D3 and cod liver oil in promoting calcification in poults. J . Nutr. 31, 599. Mattson, S . 1946. The cookability of yellow peas: A colloid-chemical and biochemical study. Acta Agric. Sci. 2, 185. May, L., Morris, E. R., and Ellis, R. 1980. Examination by Mossbauer spectroscopy of the chemical identity of iron phytate in wheat. J . Agric. Food Chem. 28, 1004. Mayer, A. M. 1958. The breakdown of phytin and phytase activity in germinating lettuce seeds. Enzymologia 19, 1. Mellanby, E. 1921. Med. Res. Counc. Spec. Rep. Ser. (G.B.), SRS-61. Mellanby, E. 1925. Med. Res. Counc. Spec. Rep. Ser. (G.B.), SRS-93. Mellanby, E. 1929. Med. Res. Counc. Spec. Rep. Ser. (G.B.), SRS-140. Mellanby, E. 1949. The rickets-producing and anticalcifying action of phytate. J . Physiol. (London) 109, 488. MeHanby, E. 1950. “A Story of Nutritional Research,” p. 263. Williams & Wilkins, Baltimore, Maryland. Mihailovic, M. L., Antic, M., and Hadzijev, D. 1965. Chemical investigation of wheat. VIII. Dynamics of various forms of phosphorus in wheat during its ontogenesis. The extent and mechanism of phytic acid decomposition in germinating wheat grain. Plant Soil 23, 117. Miller, G. A., Youngs, V. L., and Oplinger, E. S . 1980a. Environmental and cultivar effects on oat phytic acid concentration. Cereal Chem. 57, 189. Miller, G. A , , Youngs, V. L., and Oplinger, E. S. 1980b. Effect of available soil phosphorus and environment on the phytic acid concentration in oats. Cereal Chem. 57, 192. Momcilovic, B., Belonji, B., Giroux, A , , and Shah, B. G. 1976. Bioavailability of zinc in milk and soy protein based infant formulas. J . Nutr. 106, 913.

84

N. R. REDDY ET AL

Monsen, E. R. 1974. Validation of an extrinsic iron label in monitoring absorption of nonheme food iron in normal and iron-deficient rats. J . Nutr. 104, 1490. Moore, C. V., Minnich, V., and Dubach, R. 1943. Absorption and therapeutic efficacy of iron phytate. J . Am. Diet. Assoc. 19, 841. Moms, E. R., and Ellis, R. 1976a. Isolation of monoferric phytate from wheat bran and its biological value as an iron source to the rat. J . Nutr. 106, 753. Moms, E. R., and Ellis, R. 1976b. Phytate as a camer for iron: A breakthrough in iron fortification of foods? Baker’s Dig. 50, 28. Moms, E. R., and Ellis, R. 1980. Effect of dietary phytatelzinc molar ratio on growth and bone zinc response of rats fed semipurified diets. J . Nutr. 110, 1037. Moms, E. R., and Ellis, R. 1980. Bioavailability to rats of iron and zinc in wheat bran: Response to low-phytate bran and effect of the phytateizinc molar ratio. J . Nutr. 110, 2000. Morris, E. R., and Ellis, R. 1981, Phytate-zinc molar ratio of breakfast cereals and bioavailability of zinc to rats. Cereal Chem. 58, 363. Moms, G. F. I . , Thurman, D. A,, and Boulter, D. 1970. The extraction and chemical composition of aleurone grains (protein bodies) isolated from seeds of Viciafaba. Phytochemistry 9, 1707. Momson, A. B., and Sarett, H. P. 1958. Zinc deficiency in the chick. J . Nutr. 65, 267. Multani, J. S., Cepumeck, C. P., Davis, P. S . , and Saltman, P. 1970. Biochemical characterization of gastroferrin. Biochemistry 9, 3970. Murray, J., and Stein, N. 1970a. The effect of antigastric mucosal antibodies on iron absorption in experimental achylia gastrica. Proc. SOC. Exp. Biol. Med. 133, 86. Murray, J., and Stein, N. 1970b. Gastric secretions and iron absorption in rats. In “Trace Element Metabolism in Animals” (C. F. Mills, ed.), p. 37. Livingstone, Edinburgh. Nagai, Y., and Funahashi, S. 1962. Phytase (myo-inositol hexaphosphate phosphohydrolase) from wheat bran. Part I. Purification and substrate specificity. Agric. Biol. Chem. 26, 794. Nahapetian, A,, and Bassiri, A. 1975. Changes in concentrations and interrelationships of phytate, phosphorus, magnesium, calcium, and zinc in wheat during maturation. J . Agric. Food Chem. 23, 1179. Nahapetian, A , , and Bassiri, A. 1976. Variations in concentrations and interrelationships of phytate , phosphorus, magnesium, calcium, zinc, and iron in wheat varieties during two years. J . Agric. Food Chem. 24, 947. Nelson, K. J., and Potter, N. N. 1979. Iron binding by wheat gluten, soy isolate, zein, albumin, and casein. J . Food Sci. 44, 104. Nelson, T. S. 1967. The utilization of phytate phosphorus by poultry-A review. Poult. Sci. 46, 862. Nelson, T. S. 1976. The hydrolysis of phytate phosphorus by chicks and laying hens. Poult. Sci. 55, 2262. Nelson, T. S., and Kirby, L. K. 1979. Effect of age and diet composition on the hydrolysis of phytate phosphorus by rats. Nutr. Rep. Int. 20, 729. Nelson, T. S., Ferrara, L. W., and Storer, N. L. 1968. Phytate phosphorus content of feed ingredients derived from plants. Poult. Sci. 47, 1372. Nelson, T. S . , Shieh, T. R., Wodzinski, R. J., and Ware, J. H. 1968. The availability of phytate phosphorus in soybean meal before and after treatment with a mold phytase. Poult. Sci. 47, 1842. Nelson, T. S . , Shieh, T. R., Wodzinski, R. J . , and Ware, J. H. 1971. Effect of supplemental phytase on the utilization of phytate phosphorus by chicks. J . Nutr. 101, 1289. Nelson, T. S., Daniels, J. B., Hall, J. R., and Shields, L. G. 1976. Hydrolysis of natural phytate phosphorus in the digestive tract of calves. J . Anim. Sci. 42, 1509. Neuberg, C. 1908. Beziehung des cyclischen inosits zu den aliphatischen zuckem. Biochem. Z . 9, 551.

PHYTATES IN LEGUMES AND CEREALS

85

Nicolaysen, R., and Njaa, L. R. 1951. Investigations on the effect of phytic acid on the absorption of calcium in rats, pigs, and man. Acta Physiol. Scund. 22, 246. Noland, P. R., Funderburg, M., and Johnson, Z. 1968. Phosphorus availability in a practical diet for swine. J . Anim. Sci. 27, 1155. Nwokolo, E. N., and Bragg, D. B. 1977. Influence of phytic acid and crude fibre on the availability of minerals from four protein supplements in growing chicks. Can. J . Anim. Sci. 57, 475. Oberleas, D. 1964. Dietary factors affecting zinc availability. Ph.D. Dissertation, University of Missouri, Columbia. Oberleas, D. 1971. The determination of phytate and inositol phosphates. In “Methods of Biochemical Analysis” (D. Glick, ed.), p. 87. Wiley (Interscience), New York. Oberleas, D. 1973. Phytates. In “Toxicants Occumng Naturally in Foods,” 2nd ed., p. 363. Natl. Acad. Sci., Washington, D.C. Oberleas, D. 1975. Factors influencing availability of minerals. Proc. West. Hemisphere Nutr. Congr., 4th, 1974 p. 156. Oberleas, D., and Harland, G . R. 1977. Nutritional agents which affect metabolic zinc status. In “Zinc Metabolism: Current Aspects in Health and Disease” (G. J. Brewer and A. S. Prasad, eds.), p. 11. Alan R. Liss, New York. Oberleas, D., Muhrer, M. E., O’Dell, B. L., and Kintner, L. D. 1961. Effects of phytic acid on zinc availability in rats and swine. J . Anim. Sci. 20, 945. Oberleas, D., Muhrer, M. E., and O’Dell, B. L. 1962. Effects of phytic acid on zinc availability and parakeratosis in swine. J . Anim. Sci. 21, 57. Oberleas, D., Muhrer, M. E., and O’Dell, B. L. 1966a. The availability of zinc from foodstuffs. In “Zinc Metabolism” (A. S. Prasad, ed.), p. 225. Thomas, Springfield, Illinois. Oberleas, D., Muhrer, M. E., and O’Dell, B. L. 1966b. Dietary metal-complexing agents and zinc availability in the rat. J . Nutr. 90, 56. O’Dell, B. L. 1962. Mineral availability and metal binding constituents of the diet. Proc.--Cornell Nutr. Conf. p. 77. O’Dell, B. L. 1969. Effect of dietary components upon zinc availability: A review with original data. Am. J . Clin. Nutr. 22, 1315. O’Dell, B. L. 1979. Effect of soy protein on trace mineral bioavailability. In “Soy Protein and Human Nutrition’’ (H. L. Wilcke, D. T. Hopkins, and D. H. Waggle, eds.), p. 187. Academic Press, New York. O’Dell, B. L., and de Boland, A. 1976. Complexation of phytate with proteins and cations in corn germ and oilseed meals. J . Agric. Food Chem. 24, 804. O’Dell, B. L., and Savage, J. E. 1957. Symptoms of zinc deficiency in the chick. Fed. Proc., Fed. Am. SOC. Exp. Biol. 16, 394 (abstr.). O’Dell, B. L., and Savage, J. E. 1960. Effect of phytic acid on zinc availability. Proc. SOC. Exp. Biol. Med. 103, 304. O’Dell, B. L., Yohe, J. M., and Savage, J. E. 1964. Zinc availability in the chick as affected by phytate, calcium, and ethylenediamine-tetra-acetate.Poult. Sci. 43, 415. O’Dell, B. L., Burpo, C. E., and Savage, J. E. 1972a. Evaluation of zinc availability in foodstuffs of plant and animal origin. J . Nutr. 102, 653. O’Dell, B. L., de Boland, A,, and Koirtyohann, R. 1972b. Distribution of phytate and nutritionally important elements among the morphological components of cereal grains. J . Agric. Food Chem. 20, 718. Ogawa, M., Tanaka, K., and Kasai, Z. 1975. Isolation of high phytin containing particles from rice grains using an aqueous polymer two phase system. Agric. Biol. Chem. 39, 695. Ogawa, M., Tanaka, K., and Kasai, Z. 1977. Note on the phytin-containing particles isolated from rice scutellum. Cereal Chem. 54, 1029.

86

N. R. REDDY ET AL

Ogawa, M., Tanaka, K., and Kasai, 2. 1979a. Energy-dispersive X-ray analysis of phytin globoids in aleurone particles of developing rice grains. Soil Sci. Planr Nurr. 25, 437. Ohlson, R., and Anjou, K. 1979. Rapeseed protein products. J . Am. Oil Chem. Soc. 56, 431. Okubo, K., Waldrop, A. B., Iacobucci, G. A,, and Myers, D. V. 1975. Preparation of low-phytate soybean protein isolate and concentrate by ultrafiltration. Cereal Chem. 52, 263. Okubo, K., Myers, D. V., and Iacobucci, G. A. 1976. Binding of phytic acid to glycinin. Cereal Chem. 53, 513. Omosaiye, O., and Cheryan, M. 1979. Low-phytate, full fat soy protein product by ultrafiltration of aqueous extracts of whole soybeans. Cereal Chem. 56, 58. Oshima, M., Taylor, T. G., and Williams, A. 1964. Variations in the concentration of phytic acid in the blood of the domestic fowl. Biochem. J . 92, 42. Otolski, S. 1935. J . Pharm. Chim. 21, 170 (cited from Brown et al., 1961). Otolski, S . 1937. Arch. Chem. Farm. 3, 231 (cited from Brown et al., 1961). Palladin, W. 1894. Z. Biol. 31, 191 (cited from Rose, 1912). Patwardhan, V. N . 1937. The occurrence of phytin-splitting enzyme in the intestines of albino rats. Biochem. J . 31, 560. Peeler, H. T. 1972. Biological availability of nutrients in seeds: Availability of major mineral ions. J . Anim. Sci. 35, 695. Peers, G. F. 1953. The phytase of wheat. Biochem. J . 53, 102. Pemolett, J . C. 1978. Protein bodies of seeds: ultrastructure, biochemistry, biosynthesis, and degradation. Phytochemistry 17, 1473. Pfeffer, W. 1872. Untersuchungen uber die proteinkomer und die Bedeu des Asparagin beim Keimen der Samen. Jahrb. Wiss. Bot. 8, 429. Pierce, A. B., Doige, C. E., Bell, J. M., and Owen, B. D. 1977. Availability of phytate phosphorus to the growing pig receiving isonitrogenous diets based on wheat or corn. Can. J. Anim. Sci. 57, 573. Pileggi, V. J. 1959. Distribution of phytase in the rat. Arch. Biochem. Biophys. 80, 1 . Plumlee, M. P., Kennington, M. H., and Beeson, W. M. 1955. Utilization of phosphorus from various sources by growing-fattening swine. J . Anim. Sci. 14, 1220. Pons, W. A,, Stansbury, M. F., and Hoffpauir, C. L. 1953. An analytical system for determining phosphorus compounds in plant materials. J. Assoc. Of. Anal. Chem. 36, 492. Posternak, S. 1900. Rev. Gen. Bot. 12, 5-24, 65-73 (cited from Rose, 1912). Postemak, S. 1903a. C. R . Hebd. Seances Acad. Sci. 137, 202-203, 337-339 (cited from Rose, 1912). Postemak, S. 1903b. C. R . Seances Soc. Biol. Ses Fil. 55, 119&1192 (cited from Rose, 1912). Postemak, S . 1904. Bull. Soc. Chim. 33, 116 (cited from Rose, 1912). Postemak, S. 1905. Sur la composition chimique et la signification des grains d’aleurone. C. R . Hebd. Seances Acad. Sci. 140, 322. Postemak, S. 1921. The synthesis of inositohexaphosphoric acid. Helv. Chim Acta 4, 150; Chem. Abstr. 15, 1891 (1921). Postemak, S., and Postemak, T. 1929. Sur la configuration de I’inosite inactive. Helv. Chim. Acta 12, 1165. Postemak, T. 1965. “The Cyclitols,” p. 227. Holden-Day, San Francisco, California. Poux, N. 1965. Localisation de l’activit6 phosphatasique acide et des phosphates dans les grains d’aleurone. I . Grains d’aleurone refermont i la fois globoides et crystdlloides. J . Microsc. (Paris) 4, 771. Prasad, A. S., Miale, A., Jr., Farid, Z . , Sandstead, H. H., Schulert, A. R., and Darby, W. J . 1963. Biochemical studies on dwarfism, hypogonadism, and anemia. Arch. Intern. Med. 111, 407. Preece, I. A., and Gray, H. J . 1962. Studies on phytin. 11. Preliminary study of some barley phosphatase. J . Inst. Brew. 68, 66.

PHYTATES IN LEGUMES AND CEREALS

87

Preece, I. A., Gray, H. J., and Wadham, A. T. 1960. Studies on phytin. I. Inositol phosphates. J . Inst. Brew. 66, 487. Pringle, W. J. S., and Moran, T. 1942. Phytic acid and its destruction in baking. J . Soc. Chem. Ind., London 61, 108. Rackis, J. J., and Anderson, R. L. 1977. Mineral availability in soy protein products. Food Prod. Dev. 11, 38. Rackis, J. J., McGhee, J. E., Honig, D. H., and Booth, A. N. 1975. Processing soybeans into foods: Selected aspects of nutrition and flavor. J . Am. Oil Chem. Soc. 52, 249A. Radhakrishnan, M. R., and Sivaprasad, J. 1980. Tannin content of sorghum varieties and their role in iron bioavailability. J . Agric. Food Chem. 28, 55. Ranhotra, G . S. 1972. Hydrolysis during breadmaking of phytic acid in wheat protein concentrate. J . Food Sci. 37, 12. Ranhotra, G . S. 1973. Factors affecting hydrolysis during breadmaking of phytic acid in wheat protein concentrate. Cereal Chem. 50, 355. Ranhotra, G . S., and Loewe, R. J. 1975. Effect of wheat phytase on dietary phytic acid. J . FoodSci. 40, 940. Ranhotra, G . S., Loewe, R. J., and Puyat, L. V. 1974a. Phytic acid in soy and its hydrolysis during breadmaking. J . Food Sci. 39, 1023. Ranhotra, G . S., Loewe, R. J., and Puyat, L. V. 1974b. Effect of dietary phytic acid on the availability of iron and phosphorus. Cereal Chem. 51, 323. Ranhotra, G . S., Lee, C., and Gelroth, J. A. 1978. Bioavailability of zinc in soy-fortified wheat bread. Nutr. Rep. Int. 18, 487. Rapoport, S. 1940. Phytic acid in avian erythrocytes. J . Biol. Chem. 135, 403. Rapoport, S., and Guest, G. M. 1941. Distribution of acid-soluble phosphorus in the blood cells of various vertebrates. J . Biol. Chern. 138, 269. Raun, A., Cheng, E., and Burroughs, W. 1956. Phytate phosphorus hydrolysis and availability to rumen microorganisms. J . Agric. Food Chem. 4, 869. Reddy, N. R. 1976. Milling and biochemical characteristics of triticale. M.S. Thesis, Alabama A & M University, Normal. Reddy, N. R. 1981. Effects of cooking and germination of black gram and fermentation of black gram and rice blend on phytates, a-amylase inhibitor, phytohemagglutinins, and flatulence producing factors. Ph.D. Dissertation, Utah State University, Logan. Reddy, N. R., and Salunkhe, D. K. 1980. Effects of fermentation on phytate phosphorus and minerals of black gram, rice, and black gram rice blends. J . Food Sci. 45, 1708. Reddy, N. R., and Salunkhe, D. K. 1981. Interactions between phytate, protein, and minerals in whey fractions of black gram. J . Food Sci. 46, 564. Reddy, N. R . , Balakrishnan, C. V., and Salunkhe, D. K. 1978. Phytate phosphorus and mineral changes during germination and cooking of black gram (Phaseolus mungo L.) seeds. J . Food Sci. 43, 540. Reid, R. L., Franklin, M. C., and Hallsworth, E. G. 1947. The utilization of phytate phosphorus by sheep. Aust. Vet. J . 23, 136. Reinhold, J. G . 1971. High phytate content of Iranian bread: A possible cause of zinc deficiency. Am. J . Clin. Nutr. 24, 1204. Reinhold, J. G. 1973. Phytate concentrations of leavened and unleavened bread. Ecol. FoodNutr. 1, 187. Reinhold, J. G. 1975a. Phytate destruction by yeast fermentation in whole wheat meals. J . Am. Diet. Assoc. 66, 38. Reinhold, J . G . 1975b. Zinc and mineral deficiencies in man: The phytate hypothesis. In “Review of Basic Knowledge” (A. Chavez, H. Bourges, and S. Basta, eds.), p. 115. Karger, Basel.

88

N. R. REDDY ET AL.

Reinhold, J. G . , Nasr, K., Lahimgarzadeh, A,, and Hedayati, H. 1973. Effects of purified phytate and phytate rich bread upon metabolism of zinc, calcium, phosphorus, and nitrogen in man. Lancet 1, 283. Reinhold, J. G., Parsa, A,, Karimian, N., Hammick, J. W., and Ismail-Beigi, F. 1974. Availability of zinc in leavened and unleavened whole meal breads as measured by solubility and uptake by rat intestine in vitro. J . Nutr. 104, 976. Reinhold, J. G., Ismail-Beigi, F., and Faradji, B. 1975. Fibre vs phytate as determinant of the availability of calcium, zinc, and iron of bread stuffs. Nutr. Rep. Int. 12, 75. Reinhold, J. G . , Faradji, B., Abadi, P., and Ismail-Beigi, F. 1976. Decreased absorption of calcium, magnesium, zinc, and phosphorus by humans due to increased fiber, and phosphorus consumption as wheat bread. J . Nutr. 106, 493. Resurreccion, A. P., Juliano, B. O., and Tanaka, Y. 1979. Nutrient content and distribution in milling fractions of rice grain. J . Sci. Food Agric. 30, 475. Roberts, A. H., and Yudkin, J. 1960. Dietary phytate as a possible cause of magnesium deficiency. Nature (London) 185, 823. Roberts, A. H., and Yudkin, J. 1961. Effect of phytate and other dietary factors on intestinal phytase and bone calcification in the rat. Br. J . Nutr. 15, 457. Rose, A. R. 1912. A resume of the literature on inosite phosphoric acid, with special reference to the relation of that substance to plants. Biochem. Bull. 2, 21. Rosenbaum, T. M., and Baker, B. E. 1969. Constitution of leguminous seeds. VII. Ease of cooking field peas (Pisum sativum L.) in relation to phytic acid content and calcium diffusion. J . Sci. Food Agric. 20, 709. Ryabchikov, D. I., Belyaeva, V. K., and Ermakov, A. M. 1956. Use of phytic acid in the analytical chemistry of thorium. Zh. Anal. Khim. 11, 658; Chem. Abstr. 51, 7940e (1957). Saio, K., Koyama, E., and Watanabe, T. 1967. Protein-calcium-phytic acid relationships in soybean. I. Effects of calcium and phosphorus on solubility characteristics of soybean meal protein. Agric. Biol. Chem. 31, 110. Saio, K., Koyama, E . , and Watanabe, T. 1968. Protein-calcium-phytic acid relationships in soybean. 11. Effects of phytic acid on combination of calcium with soybean meal protein. Agric. Biol. Chem. 32, 448. Samotus, B., and Schwimmer, S. 1962. Indirect method for determination of phytic acid in plant extracts containing reducing substances. Biochim. Biophys. Acta 57, 389. Sandstead, H. H., Shukry, A. S . , Prasad, A. S . , Gabr, M. K., El Hifney, A., Mokhtar, N., and Darby, W. J. 1965. Kwashiorkar in Egypt. I. Clinical and biochemical studies, with special reference to plasma, zinc, and serum lactic dehydrogenase. Am. J . Clin. Nutr. 17, 15. Sandstead, H. H., Klevay, L. M., Munoz, J. M . , Jacob, R. A , , Logan, G. M . , Jr., Dintzis, F. R., Inglett, G. E., and Shuey, W. C. 1978. Human zinc requirements: Effects of dietary fiber on zinc metabolism. Fed. Proc., Fed. Am. SOC. Exp. Biol. 37, 254 (Abstr. No. 218). Sathe, V., and Krishnamurthy, K. 1953. Phytic acid and absorption of iron. Indian J . Med. Res. 41, 453. Schulze, E., and Winterstein, E. 1896. Hoppe-Seyler’s Z . Physiol. Chem. 40, 120 (cited from Rose, 1912). Seelig, M. S . 1964. The requirement of magnesium by the normal adult. Am. J . Clin. Nutr. 14, 342. Sharma, C. B., and Dieckert, J. W. 1975. Isolation and partial characterization of globoids from aleurone grains of Aruchis hypogaea seeds. Physiol. Plant. 33, 1 . Sharma, C. B., Goel, M . , and Irshad, M. 1978. Myoinositol hexaphosphate as a potential inhibitor of a-amylases. Phytochemistry 17, 201. Sharpe, L. M., Peacock, W. C . , Cooke, R., and Hams, R. S . 1950. The effect of phytate and other food factors on iron absorption. J . Nutr. 41, 433. Sieburth, J. F., McGinnis, J., Wahl, T., and McLaren, B. A. 1952. The availability of the phosphorus in unifine flour for the chick. Poult. Sci. 31, 813.

PHYTATES IN LEGUMES AND CEREALS

89

Singh, B., and Reddy, N. R. 1977. Phytic acid and mineral compositions of triticales. J. Food Sci. 42, 1077. Singh, B., and Sedeh, H. G. 1979. Characteristics of phytase and its relationship to acid phosphatases and certain minerals in triticale. Cereal Chem. 56, 267. Singsen, E. P., and Mitchell, H. H. 1945. Phosphorus in poultry nutrition. I. The relation between phytin and different sources of vitamin D. PoultSci. 24, 479. Singsen, E. P., Matterson, L. D., and Scott, H. M. 1947. Phosphorus in poultry nutrition. 111. The relationship between the source of vitamin D and the utilization of cereal phosphorus by the poult. J . Nurr. 33, 13. Singsen, E. P.,Matterson, L. D., Tlustohowicz, J. J., and Pudelkiewicz, W. J. 1969. Phosphorus in the nutrition of the adult hen. 11. The relative availability of phosphorus from several sources for caged layers. Poult. Sci. 48, 387. Smith, A. K.,and Rackis, J. J. 1957. Phytin elimination in soybean protein isolation. J. Am. Chem. SOC. 79, 633. Smith, D. H., and Clark, F. E. 1951. Anion-exchange chromatography of inositol phosphates from soil. Soil Sci. 72, 353. Smith, K. T., and Rotruck, J. T. 1981. Chemical and biological studies of phytate-mineral interactions. J. Am. Oil Chem. SOC. 58, 573A. Smith, W. H., Plumlee, M. P., and Beeson, W. M. 1962. Effect of source of protein on zinc requirement of the growing pig. J. Anim. Sci. 21, 399. Sobolev, A. M. 1963. Enzymatic hydrolysis of phytin in viva and in germinating seeds. Sov. Plant Physiol. (Engl. Transl.) 9, 263. Sobolev, A. M. 1966. On the state of phytin in the aleurone grains of mature and germinating seeds. Sov. Plant Physiol. (Engl. Transl.) 13, 177. Sobolev, A. M., and Rodionova, M. A. 1966. Phytin synthesis by aleurone grain in ripening sunflower seeds. Sov. Plant Physiol. (Engl. Transl.) 13, 958. Starkenstein, E. 1908. inosituria and the physiological significance of inosite. Z . Exp. Pathol. Ther. 5, 378. Starkenstein, E. 1910. The biological importance of inositephosphoric acid. Biochem. 2. 30, 56. Starkenstein, E. 1911. Ion action of phosphoric acids. Biochem. Z . 32, 243. Starkenstein, E. 1914. Pharmacological action of acids which precipitate calcium and of magnesium salts. Arch. Exp. Pathol. Pharmakol. 77, 45. Steinke, F. H., and Hopkins, D. T. 1978. Biological availability to the rat of intrinsic and extrinsic iron with soybean protein isolates. J . Nutr. 108, 481. Subrahmanyan, V . , Narayana Rao, M., Rama Rao, G., and Swaminathan, M. 1955. The metabolism of nitrogen, calcium, and phosphorus in human adults on a poor vegetarian diet containing Ragi (Eleusine coracana). Br. J . Nutr. 9, 350. Sudarmadji, S., and Markakis, P. 1977. The phytate and phytase of soybean tempeh. J. Sci. Food Agric. 28, 381. Sunde, M. L., and Bird, H. R. 1956. A critical need of phosphorus for the young pheasant. Poult. Sci. 35, 424. Suvorov, V. I., and Sobolev, A. M. 1972. Cationic composition of aleurone grains of seeds. Sov. Plant Physiol. (Engl. Transl.) 19, 486. Suvorov, V. I., Buzulukova, N. P., Sobolev, A. M., and Sveshnikova, I. N. 1970. Structure and chemical composition of globoids from aleurone grains of castor seeds. Sov. Plant Physiol. (Engl. Transl.) 17, 1020. Suzuki, U., Yoshimura, K . , andTakaishi, M. 1907. Bull. Coll. Agric. TokyoImp. Univ. 7:495-512 (cited from Mellanby, 1950). Tabekhia, M. M., and Luh, B. S. 1979. Effect of milling on macro and micro-minerals and phytate of rice. Dtsch. Lebensm.-Rundsch. 75, 57.

90

N. R. REDDY ET AL

Tabekhia, M. M., and Luhn, B. S. 1980. Effect of germination, cooking, and canning on phosphorus and phytate retention in dry beans. J . Food Sci. 45, 406. Tanaka, K., Yoshida, T., Asada, K., and Kasai, Z. 1973. Subcellular particles isolated from aleurone layer of rice seeds. Arch. Biochem. Biophys. 155, 136. Tanaka, K., Yoshida, T., and Kasai, Z. 1974. Radioautographic demonstration of the accumulation site of phytic acid in rice and wheat grains. Plant Cell Physiol. 15, 147. Tanaka, K., Ogawa, M., and Kasai, Z. 1977. The rice scutellum. 11. A comparison of scutellar and aleurone electron-dense particles by transmission electron microscopy including energy-dispersive X-ray analysis. Cereal Chem. 54, 684. Tangendjaja, B., Buckle, K. A , , and Wootton, M. 1980. Analysis of phytic acid by high-performance liquid chromatography. J . Chromatogr. 197, 274. Tangendjaja, B., Buckle, K. A,, and Wootton, M. 1981. Dephosphorylation of phytic acid in rice bran. J . Food Sci. 46, 1021. Tangkongchitr, U., Seib, P. A,, and Hoseney, R. C. 1981. Phytic acid. I. Determination of three forms of phosphorus in flour, dough, and bread. Cereal Chem. 58, 226. Tangkongchitr, U., Seib, P. A , , and Hoseney, R. C. 1981. Phytic acid. 11. Its fate during breadmaking. Cereal Chem. 58, 229. Taylor, T. G . 1965. The availability of the calcium and phosphorus of plant materials for animals. Proc. Nutr. SOC. 24, 105. Temperton, H. F., and Cassidy, J. 1964a. Phosphorus requirements of poultry. I. The utilization of phytin phosphorus by the chick as indicated by balance experiments. Br. Poult. Sci. 5, 75. Temperton, H. F., and Cassidy, J. 1964b. Phosphorus requirements of poultry. 11. The utilization of phytin phosphorus by the chick for growth and bone formation. Br. Poult. Sci. 5, 81. Temperton, H. F., Dudley, J . , and Pickering, G . J. 1965a. Phosphorus requirements of poultry. IV. The effects on growing pullets of feeding diets containing no animal protein or supplementary phosphorus. Br. Poult. Sci. 6, 125. Temperton, H. F., Dudley, J., and Pickering, G . J. 1965b. Phosphorus requirements of poultry. V. The effects during the subsequent laying year of feeding growing diets containing no animal protein or supplementary phosphorus. Br. Poult. Sci. 6 , 135. . requiremen'ts of poultry. VI. Temperton, H. F., Dudley, J., and Pickering, G . J. 1 9 6 5 ~Phosphorus The phosphorus requirements of growing pullets between 8 and 18 weeks of age. Br. Poult. Sci. 6 , 143. Tillman, A. D., and Brethour, J. R. 1958. Utilization of phytin phosphorus by sheep. J . Anim. Sci. 17, 104. Toetia, S . P. S . , and Toetia, M. 1972. Nutritional rickets in immigrants. Br. Med. J . 2, 669. Toma, R. B., and Tabekhia, M. M. 1979. Changes in mineral elements and phytic acid contents during cooking of three California rice varieties. J . Food Sci. 44, 619. Toma, R. B., Tabekhia, M. M., and Williams, J. D. 1979. Phytate and oxalate contents in sesame seeds (Sesamum indicum L.). Nutr. Rep. Int. 20, 25. Truter, M. R., and Tate, M. E. 1970. J . Chem. SOC.B . 70:40 (cited by Oberleas, 1971, p. 90). Tucker, H. F., and Salmon, W. D. 1955. Parakeratosis or zinc deficiency disease in the pig. Proc. SOC. Exp. Biol. Med. 88, 613. Tully, R. E., and Beevers, H. 1976. Protein bodies of castor bean endosperm: Isolation, fractionation, and the characterization of protein components. Plant Physiol. 58, 710. Turnbull, A., Cleton, F., Finch, C. A., Thompson, L., and Martin, J. 1962. Iron absorption. IV. The absorption of hemoglobin iron. J . Clin. Invest. 41, 1897. Van Den Berg, C. J . , Hill, C. F., and Stanbury, S. W. 1972. Inositol phosphates and phytic acid as inhibitors of biological calcification in the rat. Clin. Sci. 43, 377. Verma, S. C., Lal, B. M., and Prakash, V. 1964. Changes in the chemical composition of the seed parts during ripening of Bengal gram (Cicer arietinum) seed. J . Sci. Food Agric. 15, 25.

PHYTATES IN LEGUMES AND CEREALS

91

Vohra, P., Gray, G. A,, and Kratzer, F. H. 1965. Phytic acid-metal complexes. Proc. Soc. Exp. Biol. Med. 120, 447. Waldroup, P. W., Ammerman, C. B., and Harms, R. H. 1964. The availability of phytic acid phosphorus for chicks. 11. Comparison of phytin phosphorus sources. Poult. Sci. 43, 426. Waldroup, P. W . , Simpson, C. F., Damron, B. L., and Harms, R. H. 1967. The effectiveness of plant and inorganic phosphorus in supporting egg production in hens and hatchability and bone development in chick embryos. Poult. Sci. 46, 659. Walker, A. R. P. 1951. Cereals, phytic acid, and calcification. Lancer 2, 244. Walker, A. R. P., Fox, F. W., and Irving, J. T. 1948. Studies in human mineral metabolism: The effect of bread rich in phytate phosphorus on the metabolism of certain mineral salts with special reference to calcium. Biochem. J. 42, 452. Walker, K . A. 1974. Changes in phytic acid and phytase during early development of Phaseolus vulgaris. Planta 116, 91. Wallace, G. W., and Satterlee, L. D. 1977. Calcium binding and its effects on the properties of several food protein sources. J . Food Sci. 42, 473. Wang, L. C. 1971. Effect of phytate on isoelectric focusing of soybean whey proteins. Cereal Chem. 48, 229. Weildlein, E. R. 1951. Bibliogr. Ser. Mellon Insr. Bull. 6 (cited by Cosgrove, 1966). Weingartner, K. E., and Erdman, J. W . , Jr. 1978. Bioavailability of minerals in human soybean foods. Ill. Res. 20, 4. Weingartner, K. E., Erdman, J. W., Jr., Parker, H. M . , and Forbes, R. M. 1979. Effect of soybean hull upon the bioavailability of zinc and calcium from soy flour-based diets. Nutr. Rep. In?. 19, 223. Welch, R. M., and Campen, R. V. 1975. Iron availability to rats from soybeans. J . Nutr. 105, 253. Welch, R. M . , House, W. A., and Allaway, W. H. 1974. Availability of zinc from pea seeds to rats. J . Nutr. 104, 733. Wheeler, E. L., and Ferrel, R. E. 1971. A method for phytic acid determination in wheat and wheat fractions. Cereal Chem. 48, 312. Widdowson, E. M. 1941. Phytic acid and the preparation of food. Nature (London) 148, 219. Widdowson, E. M., and McCance, R. A. 1942. Iron exchange of adults on white and brown bread diets. Lancet 1, 588. Williams, S. G. 1970. The role of phytic acid in the wheat grain. Plant Physiol. 45, 376. Wills, M. R., Day, R. C., Phillips, J. B., and Bateman, F. C. 1972. Phytic acid and nutritional rickets in immigrants. Lancet 1, 771. Wilson, W. M. D. 1975. Calcium phytate as a source of phosphorus for ruminants. Ph.D. Dissertation, University of Illinois, Urbana-Champaign. Winterstein, E. 1897. Ber. Dtsch. Chem. Ges. 30, 2299 (cited from Rose, 1912). Wolf, W. J., and Briggs, D. R. 1959. Purification and characterization of 1 IS component of soybean proteins. Arch. Biochem. Biophys. 85, 186. Woodman, H. E., and Evans, R. E. 1948. Nutrition of the bacon pig. XIII. The minimum level of protein intake consistent with the maximum rate of growth. J . Agric. Sci. 38, 354. Wozenski, J., and Woodburn, M. 1975. Phytic acid (myoinositol hexaphosphate) and phytase activity in four cottonseed products. Cereal Chem. 52, 665. Wrenshall, C. L., and Dyer, W. J. 1941. Organic phosphorus in soil. 11. The nature of the organic phosphorus compounds, nucleic acid derivatives, and phytin. Soil Sci. 51, 235. Yoshida, T., Tanaka, K., and Kasai, Z. 1975. Phytase activity associated with isolated aleurone particles of rice grains. Agric. B i d . Chem. 39, 289. Young, L. 1936. The determination of phytic acid. Biochem. J. 30, 252. Young, V. R., and Janghorbani, M. 1981. Soy protein in human diets in relation to bioavailability of iron and zinc: A brief overview. Cereal Chem. 58, 12.

92

N. R. REDDY ET AL

Zuidenveg, E. R. P., Hamers, L. F., de Bruin, S. H . , and Hilbers, C. W. 1981. Equilibrium aspects of the binding of myo-inositol hexakis phosphate to human hemoglobin as studied by 3'P NMR and pH-stat techniques. Eur. J . Biochem. 118, 85. Zuidenveg, E. R. P., Hamers, L. F., Rollema, H . S . , de Bruin, S . H . , and Hilbers, C. W. 1981. 3 1 P NMR study of the kinetics of binding of myo-inositol hexakis phosphate to human hemoglobin: Observation of fast-exchange kinetics in high affinity systems. Eur. J . Biochem. 118, 95.

ADVANCES I N FOOD RESEARCH, VOL.

28

PHYSICAL, CHEMICAL, AND NUTRITIONAL PROPERTIES OF COMMON BEAN (PHASEOLUS) PROTEINS VALDEMIRO C. SGARBIERI Department of Food and Nutrition Planning, Faculty of Food and Agricultural Engineering, University of Campinas, Campinas, Brazil

JOHN R . WHITAKER Department of Food Science and Technology, University of California, Davis. CaliJornia

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biosynthesis and Storage of Proteins in the Protein Bodies. Physical and Chemical Properties of Isolated Storage Proteins . . . . . . . . . . . . . . . . . . . . . . A. Biosynthesis of Storage Proteins ............... B. Identification and Quantitation of Storage Proteins. . . . . . . . . . . . . . . . . C. Protein Body Membrane Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Composition of the Protein Body Storage Proteins. . . . . . . 111. Amino Acid Composition and Nutritional Properties of Proteins .................... Several Phaseolus Species and Varieties. . . . A. Amino Acid Composition of Bean Protei .................... B. Amino Acid Composition of Protein Isolates and Fractions. . . . . . . . . . C. Biological Value of Bean Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . D. Digestibility of Bean Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Biological Availability of Amino Acids. . . . . . . . . . . . . . . . . IV. Toxicity Associated with Phaseolus Proteins: Lectins; Inhibitors of Digestive Enzymes and Other Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Lectins (Phytohemagglutinins) . . . . . . . . . . . . . . . . . . . . B. Protein Inhibitors of Digestive Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . V. Influence of Storage and Processing on Chemical and Nutritional Properties ..... of Bean Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Additional Research Needs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

94 94 95 96 98 99 102 102 105 106 109 111 112 112 128 144 148 151

93 Copyright 0 1982 by Academic Press. Inc. All nghts of reproduction in any form reserved. ISBN 0- 12-016428-0

94

VALDEMIRO C. SGARBIERI AND JOHN R . WHITAKER

I.

INTRODUCTION

According to Aykroyd and Doughty (1964), the family Leguminosae includes approximately 600 genera, with about 13,000 species. Out of this great number, only about 10 to 12 species are of economic importance today. Dietary surveys indicate that bean consumption is high in some countries, and that the amount and type of legume seed consumed vary in different parts of the world (Aykroyd and Doughty, 1964; Stanton et al., 1966). For example, the genera Pisum and Viciu and the species Lens esculenta are, in general, more popular in the Middle East. In the Far East and Africa, species of Dolichos, Vigna, and Cujanus are of greater importance, whereas in North, Central, and South America the species Phaseolus vulgaris is the most consumed. The genus Phaseolus includes all species of legume seeds normally known as common beans. Based on archeological investigations, Kaplan (1965) suggested that common beans originated on the American Continent, specifically in the southern United States, Mexico, Central America, and the northern part of South America, particularly in the regions of the Incaic culture. Radioactive carbon measurements indicate that the species Phaseolus vulgaris was adapted to the ecological and cultivation conditions of Central America about 7000 years ago, being one of the oldest cultivated plants in that region of the world (Kaplan, 1965). It was introduced into Europe in the sixteenth century and since then it has become a very important crop in several regions of the world. The content of protein in Phaseolus species and cultivars varies from about 18 to above 35%. It is obvious that where daily bean intake is high it provides significant amounts of protein, calories, and other nutrients. The biological and nutritional values of Phaseolus proteins are limited by a number of factors: amino acid composition of the proteins; digestibility of the proteins and biological availability of the amino acids; presence of toxic proteins and other antinutritional factors. In contrast to some legume seeds, for example, soybean (Glycine m u ) , common bean proteins have not been studied extensively with respect to their composition, and physicochemical and biological properties. This article is an attempt to draw together the present knowledge on composition, nutritive value, toxicity, physical, chemical, and biological properties of the best-known common bean (Phaseolus)proteins and to encourage additional research to close the gaps in our knowledge of this high-protein food source.

II. BIOSYNTHESIS AND STORAGE OF PROTEINS IN THE PROTEIN BODIES. PHYSICAL AND CHEMICAL PROPERTIES OF ISOLATED STORAGE PROTEINS In legume seeds, the cotyledons form the bulk of the seed and synthesize most of the proteins. Proteins laid down during seed development and utilized as a

PROPERTIES OF COMMON BEAN PROTEINS

95

nitrogen and carbon source during germination are termed storage proteins; in legumes 80% of the seed proteins may be storage proteins. In the developing cotyledon there are two phases of growth, an initial one with intensive cell division followed by a longer period of growth by cell expansion. During the expansion growth, about 95% of the storage proteins are synthesized (Briarty et al., 1969; Millerd et al., 1971; Smith, 1973; Millerd and Spencer, 1974). In describing the accumulation of storage proteins during seed development, various aspects should be considered: (1) When does the synthesis of storage proteins begin and what is the period or phase of maximum accumulation? (2) What proportion of total seed protein is storage protein? (3) What is the number of proteins and the quantitative contribution of each storage protein? A.

BIOSYNTHESIS OF STORAGE PROTEINS

Hall et al. (1972), using polyacrylamide gel electrophoresis (PAGE) to study the change in protein profile of the French bean (Phaseolus vulgaris) as a function of maturity, found a continuously increasing proportion of storage proteins. These authors noted that up to a length of about 9-1 1 mm the embryo grew until it filled the space available within the embryo sac. Therefore, the electrophoretic profile of proteins separated from 5- to 10-mm seeds represents proteins contributed by the seed coat, the immature cotyledons, and the embryonic axis. A short lag followed this stage of embryo growth, and it seemed that messenger ribonucleic acid (mRNA) for globulins was synthesized in the newly forming cotyledon cells at this time (Hall et al., 1972). A rapid increase of storage proteins is visible in gels run with extracts from 10- to 13-mm seeds. Protein profiles for seeds larger than 13 mm did not change markedly, although the total protein content per seed continued to increase during this phase of maturation. The relatively large proportion of storage protein (Rf 0.384.42) tended to dilute out the other protein species so that it appeared as a greatly overloaded band in the extracts from 15-mm seeds. During the period of active synthesis and deposition of protein in the beans (seed maturation) some of these proteins are passed to the Golgi vesicles, where they may become glycosylated. The products are then either secreted into vacuoles or, while in the endoplasmic vesicles, formed into small particles; a combination of these two processes may also occur. In all cases, the end products are electron-dense organelles of 1- to 10-p.m diameter called protein bodies (Varner and Schidlovsky, 1963; Dieckert and Dieckert, 1976). When the seed is mature these organelles, together with starch particles, completely fill the cotyledonous cells (Opik, 1966, 1968). Protein bodies from Phaseolus have been isolated by differential centrifugation in various media based on density gradients (Barker et al., 1976) or by combination of filtration in a high-sucrose medium followed by discontinuous density centrifugation (Pusztai et al., 1978).

96

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

B . IDENTIFICATION AND QUANTITATION OF STORAGE PROTEINS Research on identification and quantification of the storage proteins in beans (Phaseolus) goes back several decades (Osborne, 1894; Waterman and Johns, 1921). These investigators referred to three proteins in Phaseolus (phaseolin, phaselin, and conphaseolin). Osborne (1894) reported that phaseolin accounted for about 20% of the dry weight of the seed; that is, it accounted for 85% of the total crude protein. The two other fractions, phaselin and conphaseolin, made up 2% (Osborne, 1894) and 0.35-0.40% (Waterman et al., 1923), respectively. More recent investigations gave slightly different numbers and quantities of bean storage proteins, probably due to the use of different varieties and different techniques by various investigators. The main storage protein, corresponding to phaseolin in the nomenclature of Osborne (1894) and Waterman et al. (1923), has been given different names: fraction E (Jaffe and Hannig, 1965); glycoprotein I1 (Pusztai and Watt, 1970); G1 fraction (Sun et al., 1974; Sun and Hall, 1975); a-component (Ishino and Ortega, 1975); 7 S component (Barker et al., 1976; Vaintraub et al., 1976), and vicilin (Bollini and Chrispeels, 1978). Sun and Hall (1975) characterized two globulin fractions from dry bean seeds (Phaseolus vulgaris, cv. Tendergreen) which they named G1 and G2. The main fraction GI was essentially insoluble in deionized water but began to dissolve in 0.1 N NaCl. At 0.2 N NaCl, G1 was completely solubilized. The isoelectric point of G1 was in the range of pH 4.4-4.6. The G2 fraction was about 20% soluble in water and the solubility increased with increasing sodium chloride concentration. Fraction G2 was completely soluble in 0.05 N NaCl solution. The isoelectric point for G2 was reported to be pH 3.7. Sodium dodecyl sulfate (SDS)-PAGE indicated that G2 (lower mobility in the gel) and GI contained three polypeptide subunits, respectively. In a more recent study Sun et al. (1978) investigated the timing of synthesis and the quantitative relationship of G1 and G2 storage proteins in the seeds for the cultivar Tendergreen. They confirmed their previous result (Hall et al., 1972) that low levels of protein were extractable from cotyledons of young seeds smaller than 9 mm, but for those greater than 13 mm a dramatic increase in extractability occurred. In cotyledons of 20-mm seeds, reached 37 days after flowering (37 DAF), the protein content was 75 times that for 9-mm (14 DAF) seeds. The seeds increased in length by about 0.5 m d d a y and in protein by about 0.3 mg/day for a cotyledon pair over the active phase of protein accumulation. On a dry weight basis, cotyledons contained 16% protein at the 20-mm (37 DAF) seed stage and 20% at maturity. On drying, the seeds (more than 45 DAF) decreased in length from 21 to 13 mm. Sun et al. (1978) were not able to demonstrate any accumulation of G1 protein in cotyledons which were less than 2 mm or up to the time the seed attained 6 mm (10 DAF) by use of PAGE, rocket immunoelectrophoresis, and polysome-di-

PROPERTIES OF COMMON BEAN PROTEINS

97

rected protein synthesis. Over the 48-hr period between 13 and 15 DAF, the cotyledons more than doubled in length and small amounts of G1 protein subunits (MW 47,000 and 43,000) could be detected. The major burst of G1 synthesis started abruptly at 16 DAF when the cotyledons attained 10 mm in length. Presumably, the genetic information for the GI polypeptide subunits is derepressed at this stage; however, the biochemistry of the regulatory events for the biosynthesis of storage proteins in Phaseolus has not been elucidated. Mature dry seeds of the cultivar Tendergreen contained 20% protein, of which about 50% was globulin (Romero et al., 1975). In the cultivar Tendergreen, G1 is composed of three subunits of MW 43,000,47,000, and 53,000 (McLeester et al., 1973). These subunits were clearly identified in the 9-mm stage of the seeds but were not detectable in extracts from 8-mm seeds. Polypeptides of the G2 fractions were also detectable in the 9-mm seeds. Three polypeptides (MW 34,000, 32,000, and 30,000) could be detected in the G2 fraction, but the smallest one only became visible in the electrophoretogram of 19-mm seeds. At this stage, the 32,000-MW band stained more intensely, whereas the amount of the largest polypeptide remained constant throughout growth. It has been suggested (McLeester et al., 1973) that the G2 fraction contains several different proteins. In a recent publication, Bollini and Chrispeels (1978) described the isolation and characterization of two major proteins from bean extracts (Phaseolus vulgaris L., cv Greensleeves) which they identified as vicilin and phytohemagglutinin (lectin). Vicilin, a 6.9 S protein fraction at neutral pH, associated to an 18.0 S form at pH 4.5 and had three nonidentical subunits with MW 52,000, 49,000, and 46,000. Phytohemagglutinin, a 6.4 S protein fraction, had two nonidentical subunits with MW 34,000 and 36,000. According to these investigators, vicilin is apparently identical with the glycoprotein I1 described by Pusztai and Watt (1970) and with globulin G1 described by McLeester et al. (1973), whereas phytohemagglutinin is identical with globulin G2 of McLeester et al. (1973). Both proteins are located in the protein bodies and are catabolized during the course of seedling growth, indicating that they are reserve proteins. Vicilin, isolated in its 18.0 S form from the cotyledons of young seedlings, contained substantial quantities of smaller polypeptides, in addition to the three previously reported (McLeester et al., 1973). Legumin, the 11 S protein found in large amounts in many species of the Leguminosae family, appears to be only a minor protein component of Phaseolus vulgaris, according to Derbyshire and Boulter (1976). According to Pusztai and Watt (1970), glycoprotein I1 isolated by their method contained a single subunit of MW 40,000, which makes it difficult to determine its relation to vicilin and the GI globulin. However, based on the common properties of these proteins, several investigators (McLeester et al., 1973; Derbyshire e t a / . , 1976; Bollini and Chrispeels, 1978) share the opinion that they are identical.

98

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Phytohemagglutinin (lectin) has been characterized from a number of cultivars of Phaseolus vulgaris and accounts for about 10% of the total protein content of the cotyledons (Liener, 1976). By using SDS-PAGE, Bollini and Chrispeels ( 1978) found that phytohemagglutinin had two nonidentical subunits of MW 36,000 and 34,000, confirming the work of other investigators (Allan and Crumpton, 1971; Oh and Conard, 1972; Pusztai and Watt, 1974). According to Bollini and Chrispeels (1978), the reserve nature of phytohemagglutinin and of vicilin was demonstrated by their parallel catabolism during seedling growth and their localization in the protein bodies. They interpreted the presence of smaller polypeptides in the 18.0 S or 6.9 S proteins to be the result of partial proteolytic degradation of the subunits as the seedling grows. Evidence that the main storage proteins are localized in protein bodies of legume seeds and are catabolized during seedling growth has been given also by Barker et al. (1976) for Phaseolus vulgaris cv “Seafarer” and by Ericson and Chrispeels (1973) for Phaseolus aureus. C.

PROTEIN BODY MEMBRANE PROTEINS

Pusztai et al. (1979~)isolated the protein body membranes of Phaseolus vulgaris cv. “Processor” by the technique of Croy (1977). When analyzed by SDS-PAGE, the protein body membrane gave five polypeptide components of MW 16,000, 20,000, 25,000, 30,000, and 78,000. Of these, the 30,000-MW component gave only one band by regular electrophoresis and had a mobility similar to that of pure samples of lectins. Indeed, the recovered 30,000-MW polypeptide was a powerful lectin. The 25,000- and 78,000-MW polypeptides were invariably present as the major and, possibly, integral membrane components of protein bodies of the cotyledonous cells of Phaseolus vulgaris. The amounts of the other polypeptide (MW 20,000 and 30,000) components were somewhat more variable and depended on the method of preparation (Croy, 1977). The 20,000-MW polypeptide exhibited a strong tendency to aggregate. When it was dissolved in 5 M guanidine.HC1 and passed through a BioGel A 5m column, it gave two main components. Some aggregated material emerged at the void volume, whereas the main component eluted at 1.9 times the void volume, which correlated well with the expected molecular weight of 20,000. According to Pusztai and co-workers (1979c), the 25,000-MW polypeptide purified from protein body membrane of Phaseolus vulgaris was similar in amino acid composition to the so-called structural protein found in chloroplast (Criddle, 1969; Thornber, 1975), and in endoplasmic reticulum and glyoxysomal membranes (Bowden and Lord, 1976), suggesting that most internal membranes of plant cells may require similar polypeptides for as yet unknown structural roles.

PROPERTIES OF COMMON BEAN PROTEINS

99

The 20,000-MW polypeptide isolated by Pusztai et al. (1979~)has not yet been identified as any known Phaseolus vulgaris protein. D.

COMPOSITION OF THE PROTEIN BODY STORAGE PROTEINS

As described in Section I11 of this review, the main amino acid deficiency of bean protein as a whole is the low contents of methionine, cysteine, and cystine, making the sulfur-containing amino acids the most limiting ones for nutritional purposes. These amino acids are particularly low in the main storage (7.0 S or 18.0 S) globulins. According to various workers (Pusztai and Watt, 1970; Hall et al., 1972; Ishino and Ortega, 1975; Barker et al., 1976), the value for methionine ranged from 0.57 to 0.94% (g/lOO g protein; average 0.75%), for half cystine from 0.12 to 0.40% (average 0.27%), for tryptophan from 0.8 to 1.0%, and for lysine from 4.7 to 6.4% (average 5.5%). The low content of these essential amino acids, particularly the sulfur-containing amino acids, the poor digestibility of these globulin fractions (Seidl et al., 1969; Romero and Ryan, 1978; Liener and Thompson, 1980), and the fact that these two globulin fractions may represent 50-75% of the total bean protein, seem to be very important factors in determining the low nutritive value of the bean proteins. Another important characteristic of the composition of several bean proteins, including the two major storage proteins, is that they contain various amounts of covalently linked carbohydrate (glycoproteins). The monosaccharides associated with different protein fractions include mannose, galactose, glucose, glucosamine, xylose, fucose, arabinose, and rhamnose (Jaffk and Hannig, 1965). Of 10 different protein fractions isolated, mannose .was present in the largest amounts in all of them; glucosamine, galactose, and xylose were present in nine of them, fucose in eight of the fractions, and arabinose and rhamnose in only two fractions. Pusztai (1965) reported a glycoprotein in kidney bean seeds. In 1966, Pusztai confirmed the occurrence of mannose and 2-glucosamine as sugar components of a minor, purified protein which he named glycoprotein I. Glucosamine seems to be commonly present in fairly high concentrations in seeds of kidney bean (Phaseolus vulgaris; Pusztai, 1965). Later, Pusztai and Watt (1970) isolated a major glycoprotein from kidney bean (glycoprotein 11) which contained 4.5% neutral sugars and 1% glucosamine. This sugar content has been confirmed by other investigators (Racusen and Foote, 1971) for the main storage protein of Phaseolus vulgaris, var. pencil pod wax. Some glycoproteins from bean contain higher contents of carbohydrate, such as the lectin isolated by Junqueira and Sgarbieri (1981) which contained 8.30% neutral sugar (as mannose) and 2.21% glucosamine. The a-amylase inhibitor of red kidney bean contains about 13% carbohydrate (Wilcox and Whitaker, 1981).

TABLE I SOME CHEMICAL AND PHYSICAL PROPERTIES OF THE TWO MAJOR STORAGE PROTEIN FRACTIONS FROM PHASEOLUS SEEDS Protein fractions Properties Sedimentation coefficient (S) pH 7.0 pH 4.5

GlU

6.9=

G2b

6.4~

18.OC

(X

P . vulgaris cv “Greensleeves”

P . aureus coccineus lunatus nanus vulgaris P . vulgaris cv “negro mecentral” P . vulgaris cv “Haricot”

8.0d 7.4d 6.3d 6.6d 7.3d 7.4e 7.w Molecular weight pH 7.0

Species and var. or cv

P. P. P. P.

10-3)

pH 3.4-6.6 Subunit molecular weight

(X

170e 15Id 140~ 56W 52.0, 49.0, 46.0= 56.0, 50.0, 47.0, 43.0d 49.0, 4 3 . 0 ~ 50.0, 47.0, 32.0, 23.0h 43 .&35.W

36.0, 34.OC

P . vulgaris cv “negro mecentral” P . vulgaris P . vulgaris P . vulgaris cv “Haricot” P . vulgaris cv “Greensleeves” P. vulgaris P. vulgaris P . vulgaris cv “Seafarer” P . vulgaris “Haricot”

Solubility in NaCl solution ( M ) Isoelectric point (pn Sulfur-containing amino acid (@I00g protein) Methionine

4-Cystine

Neutral sugar content (%) Glucosarnine content (%) Hemagglutinin activity

O.I-O.2’J 4.44.6, 5.4f

P . vulgaris cv. “Tendergreen” P. vulgaris cv “Tendergreen” P . vulgaris cv “Haricot”

0.7W 0.80h 0.94k 0.69f 0.3W 0.40h 0.38k 0.28f 4.50k 4.46f 1.08k 0.99f Very low

P. P. P. P. P. P. P. P. P. P. P. P. P.

aG1 = glycoprotein I1 = a-globulin = vicilin = fraction E. bG2 = phytohernagglutinin globulin.

cFrom Bollini and Chrispeels (1978). dData given by Derbyshire et a/. (1976) from various investigators eFrom Ishino and Ortega (1975). fFrom Pusztai and Watt (1970). gFrom Liener and Thompson (1980). *From Barker ef al. (1976). ‘The intervals indicate beginning and complete solubilization. jFrorn Sun and Hall (1975). kFrorn Racusen and Foote (1971).

High

vulgaris vulgaris cv “Seafarer” vulgaris var. pencil pod vulgaris cv “Haricot” vulgaris vulgaris cv “Seafarer” vulgaris var. pencil pod vulgaris cv “Haricot” vulgaris var. pencil pod vulgaris cv “Haricot” vulgaris var. pencil pod vulgaris cv “Haricot” vulgaris spp.

wax

wax wax wax

102

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

A compilation of known physical and chemical properties for the main storage proteins of Phaseolus seeds is given in Table I.

Ill. AMINO ACID COMPOSITION AND NUTRITIONAL PROPERTIES OF PROTEINS FROM SEVERAL PHASEOLUS SPECIES AND VARIETIES Bean proteins were first studied by Osborne (1894). He found that the extractable proteins were mostly globulins, a fact which was later confirmed and emphasized by other investigators (Waterman et al., 1923; Saint-Paul et al., 1956), who found three globulins in Phaseolus seeds. A.

AMINO ACID COMPOSITION OF BEAN PROTEIN

There are a large number of investigations showing that total bean protein and isolated bean protein fractions are deficient in sulfur-containing amino acids (methionine, cysteine, and cystine), but that they contain high concentrations of lysine (Tandon et al., 1957; Bressani et al., 1961; King, 1964; Baldi and Salamini, 1973; Jaffk and Hannig, 1965; Evans and Bandemer, 1967; Kakade and Evans, 1965a,b; Kelly, 1971, 1975; Moraes and Angelucci, 1971; Palmer e f al., 1973; Jaff6 and Briicher, 1974; Pusztai et al., 1979b; Sgarbieri et al., 1979; Sgarbieri, 1980; V. C. Sgarbieri and A. S . Pompeu, unpublished; V. C. Sgarbieri, P. L. Antunes, and R. G . Junqueira, unpublished). Tandon et al. (1957) showed that methionine and tryptophan were low in 25 varieties of beans (Phaseolus vulgaris) of Central America, whereas lysine was high in all varieties. Methionine ranged from 0.80 to 1.39% (g amino acid/100 g protein) (average 1.17%), tryptophan from 0.56 to 0.94% (average 0.68%), and lysine from 7.22 to 9.22% (average 8.46%). The total protein concentration found in these 25 bean varieties ranged from 20.1 to 27.9% (average 24.1%). Baldi and Salamini (1973) studied the amino acid composition of 22 species of Phaseolus. They found a range for methionine of 0.70-1.55%, for 4-cystine of 0.55-2.28% and for lysine of 5.6&8.23%. King (1964) did extensive amino acid determinations of six Haitian commercial cultivars of Phaseolus vulgaris (pois blanc, pois rouge, pois beurre, pois noir, pois jeune, and pois valet) and on Phaseolus lunatus (pois souche). He found a range of 7.31-8.20% of the protein (average 7.90%) for lysine, 0.44-0.98% (average 0.71%) for i-cystine, and 0.43-0.58% (average 0.50%) for methionine for the P. vulgaris varieties; for P. lunatus, the values were as follows: lysine, 7.52%; i-cystine, 0.89%; and methionine, 0.47%. Jaffk and Briicher (1974) studied the protein and sulfur-amino acid content of 100 lines of beans. They found an average value of 1.12% for methionine,

PROPERTIES OF COMMON BEAN PROTEINS

103

0.98% for cystine, and 22.7% for the total protein. Pusztai et al. (1979b) studied the composition and nutritional value of 13 cultivars of kidney bean (Phaseolus vulgaris). They found the following values for the sulfur-containing amino acids, reported in g amino acid/100 g protein: methionine, 1.1-1.8%; i-cystine, 0.7-0.9%. For lysine the values were 5.1-7.5%. A study of 60 varieties of Phaseolus vulgaris (V. C. Sgarbieri, M. A. M. Galeazzi, R. G. Junqueira, and L. D. Almeida, unpublished) gave the following range for the total protein (% N X 6.25) and for total methionine (g/lOO g protein) on a dry basis: the total protein content varied from 21.1 to 36.0%, whereas the methionine varied from 0.4 to 1.48% of the protein; the nonprotein nitrogen ranged from 0.4 to 1.27% (also on a dry basis). In this work, the ratio of globulins to albumins was determined to range from 1.OO to 3.3 1 for the 60 varieties. More extensive protein and amino acid determinations were done by V. C. Sgarbieri and A. S . Pompeu (unpublished). A total of 120 varieties were analyzed and the results were as follows (g/100 g crude protein): lysine, 5.56-9.60% (average 7.31%); methionine, 0.84-1 SO% (average 1.10%); i-cystine, traces-l.29%. Acid hydrolysis could have been responsible for a significant destruction of i-cystine. The protein content in the 120 varieties ranged from 18.9 to 34.0% (average 25.2%) on a dry basis. These data are summarized in Table 11. Although the cited references by no means represent a complete list of published work on protein and amino acid contents of Phaseolus, the data given are sufficient to show the range and mean values for protein and for the nutritionally most important amino acids of beans. Methionine is important because it is the first limiting essential amino acid in beans. Although cysteine and cystine are not essential amino acids, they are nevertheless important because methionine is an intermediate in the biosynthesis of cysteine and cystine; therefore, cysteine and cystine have a sparing effect on the utilization of methionine by animals. Lysine is important because legume seed proteins are used as important dietary complements for cereal seed proteins, which have a higher proportion of sulfur-containing amino acids than the legumes but are deficient in lysine. Kelly (1971), using a microbiological assay technique, determined the percentage of available methionine in 3600 cultivars of Phaseolus vulgaris from the crop years 1968 and 1969. From these, 82 cultivars were selected in 1968 as having greater than 33% more microbiologically available methionine in the mature seed than the navy bean (Sanilac) used as the standard. From the 82 cultivars selected in 1968, 63 still contained 33% more available methionine than the standard in 1969. The “Sanilac” navy bean was selected as the standard because of its genetic purity, widespread commercial use, and relatively constant methionine level (- 1.O% of protein) when cultivated under different conditions. The standard lot of navy bean contained 3.9% nitrogen and 0.98% total methionine (g/100 g crude protein), of which 42% was biologically available. From these studies, it was concluded that the level of methionine in mature

TABLE 11 AMINO ACID AND PROTEIN COMPOSITION OF SEEDS FROM PHASEOLUS gil00 g proteino Protein Sample

Met

25 varieties of Phaseolus vulgaris of Central America

0.8CL1.39 ( I . 17)b 0.7CL1.55 0.43-0.58 (0.50) (1.12) 1.1-1.8 0.41.48

22 species of Phaseolus 6 cultivars of Phaseolus vulgaris 100 lines of beans 13 cultivars of kidney bean ( P . vulgaris) 60 varieties of Phaseolus vulgaris 120 varieties of Phaseolus vulgaris

0.84-1.50

(1.10)

CYS

0.55-2.28 0.44-0.98 (0.71) (0.98) 0.7-0.9 Trace 1.29

Trp

LYS

(%)

0.56-0.94 (0.68)

7.22-9.22 (8.46) 5.6C8.23 7.3 1-8.20 (7.90)

20.1-27.9 (24.1)

(22.7)

ODetermined by Kjeldahl analysis and using the factor 6.25 to convert N content to protein content. bAverage values given in parentheses.

Tandon et al. (1957) Baldi and Salamini (1973) King (1 964)

5.1-7.5 5.5&9.90 (7.31)

Reference

21.1-36.0 18.9-34.0 (25.2)

Jaff6 and Briicher (1974) Pusztai et al. (1979b) Sgarbieri et al. (1980b) Sgarbieri and Pompeu (1980)

PROPERTIES OF COMMON BEAN PROTEINS

105

seeds of the common bean is determined genetically and sufficient variation exists within the species to permit improvement through hybridization and selection. Hackler and Dickson (1 973) found very high correlations in the inheritance for different amino acids. This indicates the possibility of selecting for more than one amino acid at the same time. The positive correlation of 0.78 found between methionine and lysine is of great significance because in any breeding program to increase methionine it is also of the greatest importance to keep lysine content high. B.

AMINO ACID COMPOSITION OF PROTEIN ISOLATES AND FRACTIONS

Amino acid composition has also been determined for protein isolates and isolated protein fractions from beans (Jaff6 and Hannig, 1965; Palmer et al., 1973; Ishino and Ortega, 1975; V. C. Sgarbieri, P. L. Antunes, and R. G. Junqueira, unpublished). Jaff6 and Hannig (1965) separated 10 protein fractions (A, B-a, B-P, CI-a, CI-p, CII, D, E, F, and GII) from seeds of black kidney beans by ammonium sulfate fractionation and free-boundary electrophoresis. Amino acids and sugars were determined in all fractions. The most abundant amino acids in the different fractions were aspartic acid, glutamic acid, and serine. Important differences were found in the relative amounts of cystine, histidine, methionine, and tryptophan. In fraction A, in contrast to all other fractions, the amount of leucine present was smaller than that of isoleucine. No proline was found in fraction GII. Fractions B-P and F had very similar amino acid compositions and small amounts of hydroxylysine were detected in both fractions. The contents of some amino acids (mol %) were as follows: methionine, ranged from 0 (B-a) to 1.34 (fraction D); i-cystine, from 0 (B-a) to 4.23 (CI-a); tryptophan, from 0.21 (GII) to 2.07 (A); and lysine, from 4.22 (CII) to 6.88 (B-P). Five different sugars were present in most of the fractions, namely, fucose, xylose, mannose, galactose, and an amino sugar. Rhamnose and arabinose were found only in fractions CI and GII. Total sugar content in the different fractions ranged from 1.7 (fraction D) to 22.1% (fraction GII). Palmer et al. (1973) studied the total nitrogen content and amino acid composition of whole kidney bean “haricot” as well as of the albumin and globulin fractions of beans before and after 4 and 8 days of germination. The albumin and globulin fractions had no tryptophan. Half cystine content was higher in the albumin fraction than in the globulin or whole bean (2.6% (g/100 g protein) versus 1.0% in whole bean or globulin] and it increased to 4.2% on the eighth day of germination. Methionine was around 1.0% and did not change with germination; the other amino acids also did not change significantly. The total

106

VALDEMIRO C. SGARBlERl AND JOHN R. WHITAKER

nitrogen was found to increase with germination in proportion to total dry matter. Germination also caused an improvement in nutritive value in spite of the nearly doubling of the trypsin inhibitor activity by the eighth day of germination. This observation led the authors to rule out trypsin inhibitors as the main toxic components of raw beans. The amino acid composition of Phaseolus vulgaris, var. ‘‘negro mecentral,” was studied by Ishino and Ortega (1975). They reported the amino acid composition of the whole seed, the globulin fraction, the protein fraction precipitated with acid at pH 4.1, a globulin protein (a-component) isolated from the globulin fraction, and a fraction comparable to the glycoprotein I1 isolated by Pusztai and Watt (1970). They showed that all five materials had very similar amino acid compositions except for methionine, which was 1.11,0.98, 1.94,0.71, and 0.57 g % of protein, for the total seed, the total globulin fraction, the acid precipitate, the a-globulin component, and the glycoprotein 11, respectively. The a-cystine was highest (0.87 mol % of protein) for the acid precipitate and lowest (0.12 mol %) for the a-globulin component. The authors found the isolated a-component to be identical with the glycoprotein I1 isolated by Pusztai and Watt (1970). From a nutritional point of view, it is significant that the a-globulin component, which accounted for 50% of the total globulin fraction and about 30% of the total bean protein, was very poor in sulfur-containing amino acids. V. C. Sgarbieri, P. L. Antunes, and R. G. Junqueira (unpublished) determined the amino acid composition of the albumin and globulin fractions of a pink Brazilian bean variety (Phaseolus vulgaris L. var. “Rosinha G2”). The amino acid compositions of the two fractions were similar except for the contents of tryptophan and sulfur-containing amino acids, which were higher in the albumin fraction than in the globulin fraction. C.

BIOLOGICAL VALUE OF BEAN PROTEINS

The biological value of bean proteins in general is low as compared to most other food proteins. The beneficial effect of supplementing the bean protein, in rat diets, with the sulfur-containing and other limiting essential amino acids has been demonstrated by several investigators (Jaffe, 1950a; Kakade and Evans, 1965a,b; Evans and Bandemer, 1967; Moraes e Santos and Dutra de Oliveira, 1972; Antunes and Markakis, 1977; Antunes et al., 1979; V. C. Sgarbieri, P. L. Antunas, and R. G. Junqueira, unpublished). Jaff6 (1950a) reported that addition of 0.3% methionine to a rat diet based on various kinds of legume seeds as sources of protein increased the protein efficiency ratio (PER); however, the increase was not the same in all cases. In the case of Phaseolus vulgaris (black) the PER increased from values of 0.0-0.9 to 3.5-3.8; for Phaseolus vulgaris (red) PER increased from 0.0 to 1.7; for

PROPERTIES OF COMMON BEAN PROTEINS

107

Phaseolus vulgaris (white) the improvement was from 1.2 to 2.7. This differential effect of methionine addition could be due to one or both of the following reasons: (1) in some legume seeds, including Phaseolus, methionine may not be the only limiting essential amino acid; and (2) it may be that the presence of other types of chemical compounds (antinutrients) in the seeds partially inhibit the full utilization of the added methionine. Kakade and Evans (1965a) separated navy bean protein into five fractions, based on differential solubility in various solvents, adsorption on bentonite-celite mixture (1: l), and precipitation with ammonium sulfate (0.75 saturation). All five fractions (unheated) depressed rat growth when included in the autoclaved bean diet. One of the fractions (fraction 4, which precipitated with 0.75-saturated ammonium sulfate) was the most toxic. One percent of this fraction added to the autoclaved bean (PER = 1.5) decreased the PER to negative values. Most of the lectin activity was in fraction 5, soluble in 0.75-saturated ammonium sulfate; however, this fraction was not nearly as toxic as fraction 4. The authors suggested that lectins might not be the most toxic compounds in navy bean and assumed that the lectin of fraction 5 could be identical with the nontoxic lectin isolated by Rigas and Osgood (1955). Evans and Bandemer (1967) studied the nutritive value of several species of legume seeds, including some varieties of Phaseolus vulgaris (Sanilac beans, red kidney beans, black beans, and red beans). Red kidney bean was the most limiting in sulfur-amino acid content when compared to the FA0 reference pattern (Food and Agriculture Organization/World Health Organization, 1973). The relative nutritive value of the Sanilac bean increased from 0 to 38 by autoclaving (121"C, 15-30 min) and that of red kidney bean, from 0 to 5 1. Addition of 2.2% methionine to the Sanilac bean protein increased rat growth (relative to unsupplemented) by 171%, and 2.4% methionine addition to red kidney bean protein increased relative growth by 400%. Moraes e Santos and Dutra de Oliveira (1972) extracted the proteins of Brazilian bean (Phaseolus vulgaris, var. Goiano Precoce) with 1% sodium chloride. The salt extract was adjusted to pH 4.0 and the precipitate was collected and dried in hot air at 60°C (acid-insoluble fraction); the supernatant was autoclaved (127"C, 15 min) and the coagulated protein was separated (acid-soluble fraction). A third fraction (total protein isolate) was obtained by acidifying the salt extract to pH 4.0 and immediately autoclaving (127"C, 15 min); the precipitate was separated and dried in a hot air current (60°C). The total protein isolate had 64.2% protein and represented 65% of the total bean protein. The acid-insoluble fraction contained 66.5% protein and represented 32.5% of the total bean protein. The acid-soluble fraction contained 59.1% protein and accounted for 32.5% of the total bean protein. The raw bean caused the death of all six rats within the period of the experiment (28 days) when fed at a 10% protein level in the diet; the cooked bean gave a PER of 1.46. When the diet containing the cooked bean was

108

VALDEMIRO C. SGARBIERI AND JOHN R . WHITAKER

supplemented with 0.2% m-methionine, the PER increased to 2.48. The total protein isolate (above) produced the death of 33% of the rats, but when supplemented with methionine (same level as above) produced no death and gave a PER of 2.2. The acid-insoluble fraction produced the death of 83% of the animals in the absence of methionine and 50% death with 0.2% DL-methionine added in the diet. The acid-soluble fraction caused 50% death in the absence of added methionine, whereas it gave a PER of 2.3 when 0.2% DL-methionine was added to the diet. Antunes and Markakis (1977) showed that the biological value of the navy bean protein is markedly improved by mixing it with ground Brazil nuts (Bertholletia excelsa), which are exceptionally rich in methionine (6.18 g/16 g N). Diets prepared with 10%protein from navy bean and autoclaved for 10 min at 121°C gave a PER of 1.53 as compared with 2.5 for casein. When mixtures of bean and Brazil nuts in the proportion 80:20, 90:10, and 9 5 5 were made and used to prepare the 10% protein diet, the PER values were 2.42, 2.16, and 1.95, respectively. Antunes et al. (1979) showed that methionine can be infused into beans by soaking in a 5% DL-methionine solution for 1 hr at 50°C. By this procedure the methionine content was raised from 1.2 to 24 g/lOO g bean protein and the increase in weight due to absorbed water was only 40% of the weight of the original bean. The infused beans, after drying, were mixed 1:7 (w/w) with original beans. The mixture, after cooking, still contained 3% methionine on a protein basis. Infusion with methionine raised the bean PER from 0.9 to 2.6 and the biological efficiency of a 10% bean protein diet from 9.4 to 26.6%. The effectiveness of supplementing Brazilian bean (Phaseolus vulgaris, var. Rosinha G2) protein with methionine was also demonstrated by V. C. Sgarbieri, P. L. Antunes, and R. G. Junqueira (unpublished). Supplementation with 3% DLmethionine and 2% L-cysteine (protein basis) improved the PER of a 10% protein diet for rats as follows: whole flour, from 1.17 to 2.47; total protein isolate, from 0.70 to 4.00; albumin fraction, from 0.50 to 3.98; globulin fraction, from 0.98 to 4.47; and protein of insoluble residue (from protein extraction), from 0.87 to 3.27. All samples were autoclaved 15 min at 121°C prior to use in the preparation of the diets. Most interesting was the observation that supplementation with the same amount of methionine improved nutritional performance (PER) to a different extent for the different protein fractions. All isolated protein fractions gave better response to sulfur-amino acid supplementation than the whole flour, indicating the presence of substances in the whole flour (whole bean) capable of interfering with bean protein utilization by the rat. Such compounds evidently are heat stable, but their chemical nature is still not known. Differences in response to methionine supplementation were also reported by Evans and Bandemer (1967). They found that the same level of methionine in soybean- and navy bean-containing diets resulted in better growth for the soy-

PROPERTIES OF COMMON BEAN PROTEINS

109

bean diet than for the navy bean diet. They observed (Evans et al., 1974) that soybean containing the same original level of methionine as navy bean required less methionine supplementation to produce the maximum growth of rats. Evans and Bauer (1978), studying the nutritive value of whole navy bean and protein fractions isolated from navy beans with and without addition of methionine, concluded that the autoclaved navy bean contains one or more dialyzable substance(s) which partially inhibit rat growth. This substance(s) was not identified. According to these authors, the presence in navy beans of heat-resistant dialyzable growth inhibitors would explain the difference in response to methionine supplementation as compared with soybeans. D.

DIGESTIBILITY OF BEAN PROTEINS

According to various investigators (Jaff6, 1950b; Jaff6 and Vega Lette, 1968; Seidl et al., 1969; Romero and Ryan, 1978; Sgarbieri et al., 1979; V. C. Sgarbieri, P. L. Antunes, and R. G. Junqueira, unpublished; Liener and Thompson, 1980), the low digestibility observed for bean proteins is one of the main causes of their low nutritive value. Jaff6 (1950b) reported an in vivo true digestibility of 76.8, 79.5, and 84.1%, respectively, for the protein of black, pink, and white beans (Phaseolus vulgaris). Digestibility appears to decrease as the content of pigment in the seed coat increases. The pigments are, in general, phenolic compounds and it is likely that they interact with the bean proteins, decreasing their digestibility and utilization. Jaff6 and Vega Lette (1968) reported an inhibitory effect of raw beans on intestinal proteolysis which was not caused by trypsin inhibitor. In assays with young rats kept on a diet containing 40% of raw bean meal from a cultivar of very low trypsin inhibitor and lectin activities, growth and nitrogen retention were low as compared to the control animals fed the corresponding rations prepared with heated beans. A casein supplement did not increase weight gain but supplementation with digested casein did. According to the authors, the low trypsin inhibitor activity of the bean seed and the fact that trypsin inhibitor seemed not to interfere significantly with in vivo digestion in rats (Pusztai, 1967) suggested that the trypsin inhibitor could not have been the cause for the poor growth and nitrogen retention. Even after proper heating, the apparent protein digestibility of the black kidney bean was low, although considerably improved over the uncooked beans (Jaffe and Vega Lette, 1968). Seidl et al. (1969) isolated a major globulin fraction (fraction E) from black kidney bean which represented 30% of the total bean protein. This globulin fraction was resistant to hydrolysis by pepsin, trypsin, chymotrypsin, papain, ficin, hurain, and subtilisin. After denaturation of this protein by heat or urea, only slight enzymatic hydrolysis could be detected. The activity of all seven proteinases on appropriate synthetic substrates was

110

VALDEMIRO C. SGARBIERI A N D JOHN R. WHITAKER

inhibited by the bean globulin, although the specific activity of the inhibitor was considerably lower than that of the trypsinxhymotrypsin inhibitor. The name “globulin proteinase inhibitor” was proposed for this protein. Because the fraction E globulin represented a large percentage of the total bean protein and was partially inhibitory and resistant to proteolysis, even after heat or urea denaturation, Seidl et al. (1969) concluded that this protein fraction was responsible, at least in part, for the low digestibility and low nutritive value of black kidney bean protein. Further evidence for the resistance of globulin components of beans to proteolytic digestion comes from the work of Romero and Ryan (1978) and Liener and Thompson (1980). Romero and Ryan (1978) studied the in vitro digestibility of the GI fraction from three varieties of Phaseolus vulgaris (Improved Tendergreen, PI 207222, and BBL 240) by pepsin, trypsin, and chymotrypsin. By following the extent of enzyme hydrolysis by SDS-gel electrophoresis they concluded that there are a certain number of peptide bonds which are quite labile to trypsin in the main Phaseolus globulin (Gl). On the other hand, a number of relatively large peptides were resistant to further trypsin hydrolysis. A substantial increase in digestibility by all three proteolytic enzymes was measured after heat treatment of the GI globulin. Romero and Ryan (1978) concluded that there are conformational constraints on enzymatic hydrolysis of the native GI molecule. They also verified that the concentration of tannins in different seed lines did not correlate with the in vitro susceptibility to hydrolysis of the protein, but added tannins decreased the in vitro susceptibility to enzymatic hydrolysis. Tannins have been shown to interfere with protein digestibility by a number of workers; these include in vitro studies (Barbara and Starkey, 1966; Feeny, 1969; Ramachandra et al., 1977) and in vivo studies (Nelson et al., 1975; Marquardt et al., 1977; Ward et al., 1977). Recently, Liener and Thompson (1980) found that rats fed diets containing native GI globulin lost weight and the weight loss was not significantly different from those animals which had received a protein-free diet. Rats fed heated GI grew to a limited extent, but their weight gain was still markedly lower than that of the rats on a casein diet. The improvement in growth response to GI produced by heat treatment was accompanied by a marked increase in the true digestibility of the protein, from 57 to 92.5% compared with 95% for casein. Despite the very low level of trypsin inhibitor activity of purifed unheated GI, the sizes of the pancreas of rats fed this protein were significantly larger than those of the animals receiving heated GI, casein, or the protein-free diets. In a similar study, El-Hag et al. (1978) reported that the digestibility of fraction E, isolated from red kidney bean according to Seidl et al. (1969), increased from 62.5 to 71.5% on heating. Sgarbieri et al. (1979) reported on the digestibility and nutritive value of four Brazilian varieties of Phaseolus vulgaris (“Rico 23,” black; “Rosinha G2,”

PROPERTIES OF COMMON BEAN PROTEINS

111

pink; “Carioca,” brownish; and “Pirat2 1,” brown). The PER values for total protein in the autoclaved (15 min, 121°C) whole flour from these varieties were 0.75, 0.85, 1.12, and 1.32 for Carioca, Rosinha G2, Piratii 1, and Rico 23, respectively. Digestibility of the proteins was 44, 45, 45, and 39% for the unheated beans and 64.5, 58,69.5, and 52% for the autoclaved beans. With the variety “Rosinha G2,” Sgarbieri et al. (1980a) found marked differences in digestibility between the total protein in the whole flour (59%), the total protein isolate (73%), the albumin fraction (72%), and the globulin fraction (83%). These preparations were all autoclaved for 15 min at 121°C prior to preparation of the rat diets. The effect of heat treatment (97”C, 30 min) on in virro digestibility (pepsin plus pancreatin) of the preparations (above) from Rosinha G2 was different for the different samples. The change in digestibility as a result of heat treatment was as follows: whole flour, from 27.5 to 39.0%; total protein isolate, from 52.0 to 59.5%; albumin fraction, from 46.0 to 42.5%; and globulin, from 67.0 to 87.0%. It is evident that the globulin fraction not only had the higher digestibility when unheated but also showed the greatest improvement due to heat treatment. On the other hand, the whole flour protein and the albumin fraction had very low digestibility prior to heating, which changed little after heating. In fact, the in vitro digestibility of the albumin fraction decreased from 46.0% (unheated) to 42.5% after heating (97”C, 30 min). The very low in vitro digestibility of the proteins in the whole flour and in the albumin fraction was attributed primarily to the trypsin-chymotrypsin inhibitor activity, which was very high in these Samples. The failure of heat treatment at 97°C to improve appreciably the digestibility in these samples was because the trypsin-chymotrypsin inhibitor from this variety of bean is unusually heat-resistant and very difficult to inactivate in acidic or neutral solutions. On the other hand, inactivation of the trypsin-chymotrypsin inhibitor in whole (intact) and water-soaked beans was accomplished with much less heat treatment. E.

BIOLOGICAL AVAILABILITY OF AMINO ACIDS

Another important factor which contributes to the low nutritive value of bean proteins is the low biological availability of some of the amino acids. Sgarbieri et al. (1979) reported percentage availability of methionine for the rat ranging from 29.3% in the variety “Carioca” to 40.6% for “Rico 23.” With the variety “Rosinha G2,” methionine availability for the rat was found to be as follows: in the whole flour, 46.0%; in total protein isolate, 34.0%; in the globulin fraction, 51.0%; in the albumin fraction, 5.8%; and in the insoluble residue from protein extraction, 4.6% (V. C. Sgarbieri, P. L. Antunes, and R. G . Junqueira, unpublished). The very low biological availability found in the albumin fraction and the insoluble residue of this variety are certainly due to various factors, some of which are probably not known. Some of the factors are as follows: (1)

112

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Maillard-type reactions and other types of reactions may take place due to the carbohydrate present in both the albumin fraction and in the insoluble residue. (2) The albumin fraction contains proteins with substantial numbers of disulfide bonds. It was shown (V. C. Sgarbieri, P. L. Antunes, and R. G. Junqueira, unpublished) that breaking the disulfide bonds by sulfitolysis or by reduction with dithiothreitol greatly increased the in vitro digestibility of the albumin fraction. (3) It was also shown, by comparing the lysine content of the acid hydrolysate with that determined by the trinitrobenzenesulfonic acid (TNBS) method, that the available lysine in the whole flour, total protein isolate, albumin fraction, globulin fraction, and insoluble residue were 68.4, 68.4, 83.8, 80.5, and 43.1 %, respectively. Evans et al. (1974) studied the availability of methionine and cystine in soybean and navy bean (Phaseolus vulgaris, var. Sanilac) employing microbiological assays with Leuconostoc mesenteroides P-60. They found a methionine availability of 51% and a cystine availability of 75% in navy bean compared with 74 and 89%, respectively, in the soybean. Evans and Bauer (1978), using a metabolic balance technique, found 50% of the methionine and 41% of the cystine available to the rat in the navy bean protein. Kakade et al. ( 1 969) studied the availability of cystine to the rat from a trypsin inhibitor isolated from navy bean. A diet containing a mixture of synthetic amino acids lacking cystine was compared with the same basic diet containing 0.15% added L-cystine. When the native trypsin inhibitor was added as a source of cystine instead of free L-cystine, the rats lost weight, indicating the nonavailability of cystine from the trypsin inhibitor. However, when the trypsin inhibitor was denatured (2 hr at 120°C) before adding to the diet, the weight gain and the efficiency of the diet were comparable to those of diets containing free Lcystine. A more detailed discussion of the toxicity associated with bean (Phaseolus) proteins will be the subject of Section IV of this article. IV. TOXICITY ASSOCIATED WITH PHASEOLUS PROTEINS: LECTINS; INHIBITORS OF DIGESTIVE ENZYMES AND OTHER FACTORS

A.

THE LECTINS (PHYTOHEMAGGLUTININS) 1.

Occurrence and Nomenclature

According to Boyd ( 1963), hemagglutinating activity was first described by Stillmark in 1888 in extracts of castor bean (Ricinus communis) and the first plant lectin found to be blood group specific was from lima bean (Phaseolus lunatus).

PROPERTIES OF COMMON BEAN PROTEINS

113

Lectins are glycoproteins with the unique property of being able to bind saccharides and saccharide-containing proteins in a highly specific fashion. In addition to erythrocyte agglutination, the lectins can interact with other types of cells. Nowell (1960b) showed that lectin from red kidney bean (Phaseolus vulgaris) transforms lymphocytes from peripheral blood into large and morphologically primitive cells, which then undergo mitosis. This phenomenon is accompanied by an increase in protein and nucleic acid biosynthesis. Nordman et al. (1964) verified that Phaseolus lectin can also cause agglutination of leucocytes. At very low concentrations of lectin they observed agglutination of erythrocytes only. As the concentration of lectin was increased, separate agglutinates of erythrocytes and leucocytes were observed, whereas at high lectin concentration mixed agglutinate was formed. Aub et al. (1963) also demonstrated that certain lectins combine with rat tumor cells or trypsin-treated cells of the same tissue but not with intact unmodified cells. Burger and Goldberg (1967) suggested that the common feature of the sensitivity of tumor cells and trypsintreated cells to the lectin was the presence of exposed glycopeptides (binding sites) which were probably not exposed in the intact cell surface. A survey of 2663 plant species showed that over 800 of them contained hemagglutinating activity (Allen and Brilliantine, 1969). In the Leguminosae family, over 600 species and varieties have been shown to contain lectins (Toms and Western, 1971). In addition to higher plant cells, proteins capable of specific interaction with carbohydrate-containing substances are also found in living cells from animals and microorganisms; for example, invertebrates (Khalap et al., 1970; Scott, 1972), fish (Kothbauer and Schenkel-Brunner, 1975), fungi (Coulet et al., 1970; Seeger and Weidmann, 1972; Fujita et al., 1974), bacteria (Gilboa-Garber, 1972), lichen (Howe and Barrett, 1970), and mammalian tissues (Roberts and Boursnell, 1974; Stockert et al., 1974). The ubiquitous presence of these proteins in nature poses difficult problems of terminology and nomenclature. Boyd and Shapleigh (1954d) proposed the term “lectin” (Latin, legere = elect, to choose) to denote those substances which agglutinate red blood cells and which show a high degree of specificity toward different blood groups of human and animals. It was proposed (Liener, 1976) that the term “lectin” be used as a generic term to denote all sugar-specific proteins; further specification as phytolectins, zoolectins, or mycolectins indicates that the lectin is of plant, animal, or microbial origin. Several excellent review articles have been published on the lectins (Boyd, 1963; Jafft5, 1969, 1980; Sharon and Lis, 1972; Lis and Sharon, 1973; Cohen, 1974; Liener, 1976; Goldstein and Hayes, 1978). The present article will be restricted to lectins (phytolectins) which have been extracted and purified from plants of the genus Phaseolus.

114

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

2 . Distribution and Physiological Importance in the Plant In the Phaseolus species, lectins have been found mainly in the seeds. Susplugas and Coulet (1954) suggested that lectins found in the primordial leaves come from the cotyledons. They further suggested that lectins are synthesized in the leaves of the plant, but do not accumulate in the leaves because they are transported and stored in the seed cotyledons. The lectins are localized in the cytoplasm of the cotyledons and embryonic cells (Mialonier et al., 1973), where they appear during the time of early development and differentiation of the embryo (Howard et al., 1972). During germination of the seed the content of lectin decreased at a rate parallel to the loss of the reserve proteins (RougC, 1974a,b). The lectins make up 2-10% of the protein of numerous leguminous seeds, suggesting that they may play an important role in the physiology of the plant. Some of the suggested functions for the lectins have been reviewed (Toms and Western, 1971; Sharon and Lis, 1972) and summarized by Liener (1976) as follows: (1) they act as antibodies to counteract soil bacteria; (2) they serve to protect plants against fungal attack by inhibiting fungal polysaccharases; (3) they serve in transportation or storage of sugars; (4) they attach glycoprotein enzymes in organized multienzyme systems; and ( 5 ) they play a key role in the development and differentiation of embryonic cells. More recent investigations indicate that lectins may play a role in the symbiotic relationship between leguminous plants and bacteria (Hamblin and Kent, 1973; Bohlool and Schmidt, 1974). Hamblin and Kent (1973) found that lectins are present in the roots of Phaseolus vulgaris and that a strain of Rhizobium phaseoli, when treated with these lectins, was capable of agglutinating erythrocytes. On the other hand, Bohlool and Schmidt (1974) found that soybean lectin binds only to those bacteria which are known to nodulate the soybean. The lectins might therefore account for the specific interaction of legumes with certain bacteria in the nitrogen-fixing process. Howard et al. (1972) detected the appearance of lectin during early development and differentiation of the seed embryo, indicating a possibility of involvement of the lectins in the embryogenesis and germination of the seeds. Concanavalin A, the lectin from jack bean (Canavalia ensiformis), and the lectin from Phaseolus vulgaris were shown to stimulate the germination of pollen (Southworth, 1975). Addition of black bean (Phaseolus vulgaris) lectin to the normal diet of the bruchid beetle larvae kills them (Janzen et al., 1976). These larvae can eat lectinfree cow pea (Vigna unguiculata) seeds but not Phaseolus vulgaris seeds. It was concluded (Janzen et al., 1976) that “a major part of the adaptive significance of lectins in black bean and other legume seeds is to protect them from attack by insect seed predators. ”

PROPERTIES OF COMMON BEAN PROTEINS

115

In contrast to the interesting and important functions attributed to the lectins in the physiology and relationship of plants with bacteria and fungi are the observations of Briicher et al. (1969) and Palozzo and Jaff6 ( 1 969) that many strains of Phaseolus vulgaris apparently do not contain lectin. 3.

Composition and Physicochemical Properties

More than 200 species of Phaseolus have been shown to exhibit hemagglutinating activity (NowakovA and Kocoureck, 1974). The Phaseolus species can be divided into three groups according to their hemagglutinating capacity and specificity: 1. A group represented by lima bean (P. lunatus) contains lectins which agglutinate specifically blood type A cells (Boyd, 1962). Lima bean lectin also precipitates blood group A and B secretor saliva but does not precipitate secretor saliva of type 0, and the saliva of any nonsecretors (Boyd and Shapleigh, 1954b; Boyd et al., 1955). 2. Most Phaseolus vulgaris varieties and cultivars contain lectins which react nonspecifically with human erythrocytes of all blood groups. The mitogenic property exhibited by many lectins was first discovered in red kidney bean (Phaseolus vulgaris) extract (Hungerford et al., 1959; Nowell, 1960a,b). 3. A third group of Phaseolus species was described in which the proteins show no apparent hemagglutinating activity (Briicher et al., 1969; Palozzo and Jaffe, 1969).

In this section our discussion will be restricted to Phaseolus lectins which have been characterized as to composition and physicochemical properties. a. Lima Bean (P. lunatus) Lectins. The physicochemical properties and composition of lima bean lectins were reviewed recently by Goldstein and Hayes (1978). Lima bean lectin was the first lectin shown (by Boyd in 1945) to exhibit bloodgroup specificity, although the observation was only reported much later (Boyd, 1962). Kriipe (1956) described inhibition of the lima bean lectin by 2acetamido-2-deoxy-~-galactose and reported that the lectin did not react with chicken, guinea pig, mouse, rabbit, or sheep erythrocytes. Miikela (1957) showed that lima bean lectin reacted equally well in saline or serum with normal or papain-treated human erythrocytes of type A but not with bovine erythrocytes. The most inhibitory sugar for lima bean lectin is methyl-2-deoxy-2-(p-nitrobenzamid0)-a-D-galactopyranoside, followed by methyl-2-acetamido-2-deoxy-a-D-galactopyranoside (Goldstein and Hayes, 1978). Lima bean lectins have been purified by several methods. Gould and Scheinberg (1970), employing salt fractionation in conjunction with pH adjustment

116

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

followed by gel filtration on BioGel, isolated two active lectin components from the lima bean (Phaseolus lunatus, var. thorogreen). The components, designated I1 and 111 by order of elution from the gel filtration column, were essentially pure by electrophoresis in polyacrylamide gel and by ultracentrifugation. These components had MW 269,000 and 138,000, respectively. Galbraith and Goldstein (1970, 1972a,b) used specific adsorption of lima bean lectin, followed by elution with 2-acetamido-2-deoxy-~-galactoseto isolate components I1 and 111, which were electrophoretically pure and had MW 247,000 and 124,000, respectively. A more recent procedure for isolating and purifying lima bean lectins employed affinity chromatography with concanavalin A-Sepharose (Con A-Sepharose; Bessler and Goldstein, 1973). Some properties of the two lectins isolated from lima bean were studied by Galbraith and Goldstein (1972a) and by Gould and Scheinberg (1970). On treatment with disulfide bond-reducing agents (1,4-dithiothreitol or 2-mercaptoethanol) in SDS, components I1 and 111 gave only 3 1,000-MW subunits. On the other hand, PAGE in the presence of 1% SDS alone gave subunits of MW 60,000 (Gould and Scheinberg, 1970). Both groups of workers (Gould and Scheinberg, 1970; Galbraith and Goldstein, 1972a) described several other properties of lima bean lectins. The amino acid compositions were similar for components I1 and 111. Neither contained methionine, but each contained two i-cystine residues per subunit of 30,000 MW. They had a high content of aspartic acid, serine, and leucine. Titration of components I1 and 111 with 5,5’-dithiobis(2-nitrobenzoicacid), in the absence or presence of 8 N urea or SDS, gave one sulfhydryl group per 31,000 MW. However, after reduction with 1,Cdithiothreitol, two sulfhydryl gToups could be titrated. Lectins I1 and I11 are glycoproteins containing 3-5% carbohydrate as mannose, fucose, and 2-amino-2-deoxyglucose, with traces of arabinose and xylose. Carbohydrate analysis of the glycosyl moiety (Goldstein et al., 1974) gave 4 residues of mannose, 2 of 2-amino-2-deoxyglucose and 0.5 of fucose. The presence of and the essentiality of some inorganic cations for the activity of lima bean lectins I1 and 111 were also demonstrated (Galbraith and Goldstein, 1970, 1972a). Cations, such as Mn2+ and Ca2+, were bound to the purified lectins. Removal of Mn2+ lowered the hemagglutination titer by 75%. Chelating agents such as ethylenediaminetetraacetate (EDTA) completely inhibited the precipitin reaction between lima bean lectin component 111 and type A blood group substance. Several divalent-metal cations restored activity. Addition of Ca2 , Fez , Mg2 , Mn2 , Ni2 , Sr2 , or Zn2 gave equivalent activation. The relative specific titers for the activity of lima bean lectin components I1 and 111 on type A human erythrocytes were 5100 and 1300, respectively. b. Phaseolus vulgaris Lectins. Phaseolus vulgaris lectins are a more complex system of isoglycoproteins than the lima bean lectins. They are not inhibited by simple monosaccharides or derived sugars but only by more complex carbo+

+

+

+

+

+

+

PROPERTIES OF COMMON BEAN PROTEINS

117

hydrate moieties, normally attached to glycoproteins or peptides. The complete structural requirements for the binding are still not defined. They agglutinate erythrocytes and leukocytes to a different extent and also exhibit stimulating action on lymphocytes, resulting in morphological changes and division (mitosis). Due to the large number of varieties of Phaseolus vulgaris studied, the differences in techniques of extraction and purification, and the different properties studied by the various investigators, the published results are quite difficult to correlate. Red Kidney Bean Lectins. The most extensively investigated lectins in this group are those from red kidney bean (Allen et a l . , 1969; Yachnin and Svenson, 1972; Yachnin et al., 1972; Miller et al., 1973). Allen et al. (1969), in investigating a commercial lectin preparation (P-PHA) from red kidney bean, reported 17 different protein bands by PAGE. Chromatography of the same sample on CM-Sephadex and Sephadex G-150 gave several proteins with distinct mitogenic and hemagglutinating capacity. The most mitogenic protein isolated (L-PHA) was homogeneous by several criteria and had considerable leukoagglutinating activity. A fraction containing at least two closely related proteins (H-PHA) had 250 times the hemagglutinating activity of L-PHA. This fraction had some leukoagglutinating activity and was slightly less mitogenic than the L-PHA. The amino acid and carbohydrate composition of the two fractions were similar, but H-PHA contained about twice as much carbohydrate as L-PHA, and had a slightly higher molecular weight by gel filtration. Both fractions contained 2-amino-2-deoxyglucose and mannose, and somewhat less xylose and arabinose or fucose. It has been proposed, primarily by Yachnin and his colleagues (Allen et al., 1969; Yachnin and Svenson, 1972; Yachnin et al., 1972; Miller et al., 1973, 1975), that red kidney bean lectins are tetrameric molecules, and thaf they are formed through different combinations of two slightly different subunits (L and R) which give rise to a family of five isolectins. The existence of two different subunits in P . vulgaris lectins was confirmed by several other investigators (Weber and Grasbeck, 1968; Allan and Crumpton, 1971; Oh and Conard, 1972 Weber et al., 1972). The subunits can be distinguished by N-terminal amino acid sequence, isoelectric points, and biological properties. The leukoagglutinin (L-PHA) contains four identical L subunits, with an isoelectric point of 5.25 and serine as the N-terminal amino acid. This protein shows a strong affinity for lymphocyte receptors, but little affinity for erythrocyte receptors. The erythroagglutinating isolectin (H-PHA) is formed of four identical R subunits with an isoelectric point of 5.95 and contains alanine as the N-terminal residue. This protein exhibits strong affinity for erythrocyte membrane receptors. The three mitogenic isolectins contain various proportions of L and R subunits (LR,, L,R,, L,R). The mixed leukocyte-erythrocyte agglutinat-

118

VALDEMIRO C. SGARBIERI AND JOHN R . WHITAKER

ing activity (mixed agglutination) exhibited by these isolectins must be a reflection of their hybrid structures. They also show lymphocyte mitogenic activity proportional to their content of L subunit. The L and R subunits have been isolated in homogeneous form by isoelectric focusing in 8 M urea (Miller et a l . , 1973, 1975). They have identical molecular weights, around 34,000, and both lack methionine, cysteine, and cystine. The subunits differ in amino acid sequence from residues 1 to 7 (amino terminal), but are identical in positions 8 to 24 and in the C-terminal residue. The 12th residue in each unit is a glycosylated asparagine residue; the carbohydrate composition of both subunits is identical. The great similarity between the subunits is apparent and, according to the above-cited authors, the difference in biological properties between the two subunits is the result of relatively small differences in primary structure. A lectin high in leukoagglutinating activity has been purified (Weber, 1969; Rasanen et a l . , 1973) by fractional precipitation with ethanol, followed by ionexchange chromatography on DEAE-cellulose and SP-Sephadex and by exclusion chromatography on Sephadex G- 150. The crystalline lectin was homogeneous by various criteria and was formed of four L supnits of MW 3 1,000 to give an aggregate molecular weight of 126,000 (S 20,w = 6.87 S). This glycoprotein lacked sulfur-containing amino acids, but contained high proportions of aspartic acid, leucine, serine, threonine, and valine. It contained 2amino-2-deoxyglucose and mannose as the only carbohydrates and Mn2 and Ca2 , which are essential for both leukoagglutinating and lymphocyte-stimulating activities. Harms-Ringdahl and co-workers (1 973; Harms-Ringdahl and Jornvall, 1974) isolated a mitogenic factor from red kidney beans which, unlike the mitogens previously described from this material, had no effect on RNA biosynthesis by chicken spleen lymphocytes. However, it did stimulate the biosynthesis of RNA by a bacterial system of plasmolyzed bacterial cells. The most unusual feature of this mitogen was its low molecular weight, about 10,000, and its unusually high content of cystine. The authors compared this protein with a pokeweed mitogen which also stimulated RNA biosynthesis in their bacterial system. Black Kidney Bean Lectins. Jaffi and Hannig (1965) and Jaffk et al. (1972) isolated and characterized the proteins from a black kidney bean variety. Their analysis showed proteins that had both hemagglutinating and mitogenic properties. These lectins were also glycoproteins. Rabbit anti-serum prepared against proteins from black kidney beans cross-reacted with the water-soluble proteins from white and red kidney beans. However, each variety of kidney bean gave quite different immunoelectrophoretic patterns. White Kidney Bean Lectins cv “Haricot” and “Processor. Isolectins from “Haricot” kidney bean (Phaseolus vulgaris) were purified and studied by Pusztai and Watt (1974). They found two types of glycoprotein subunits of MW +

+

”

PROPERTIES OF COMMON BEAN PROTEINS

119

30,000 and 35,000. Although these glycoproteins were shown to be agglutinins of red and white blood cells, they had negligible effects on lymphocyte transformation. Pusztai and Stewart (1978) separated two groups of lectins from Phaseolus vulgaris cv. “Processor” based on their different solubilities at pH 5.0. The albumin isolectins were comprised of five major lectin components with isoelectric points between 4.6 and 5.2. The globulin isolectins, partly overlapping with the albumins on isoelectric focusing, contained several more lectin components with higher isoelectric points. The two groups had similar amino acid and sugar composition but were only partly identical by immunochemical criteria. They also had a common protomer molecular weight of 119,000. Both groups contained, in a slow equilibrium with the protomer, just over 10% of dimer and oligomers. Pusztai and Stewart (1978) also reported the presence in the albumin fraction of a smaller 2 S unit in equilibrium with the albumin protomer. The isolectins found in “Haricot” kidney bean (Pusztai and Watt, 1974) were quite different in number, size, and physicochemical behavior from those found in the “Processor” cultivar (Pusztai and Stewart, 1978). Due to variations among cultivars and preparations and to the existence of two distinct groups (albumin and globulin) of isolectins with distinct physicochemical and immunological properties, Pusztai and Stewart (1978) suggested that it is likely that more than five isolectins occur naturally in the different varieties and cultivars of Phaseolus vulgaris. Pusztai et al. (1979~)isolated a polypeptide (MW 30,000) with very strong hemagglutinating activity from the protein body membrane of the “Processor” cultivar. It agglutinated rabbit erythrocytes at concentrations as low as 1 pg/ml (similar to Difco P hemagglutinin). When dissolved in 5 M guanidine-HC1 and chromatographed on BioGel A 5m, the 30,000-MW polypeptide gave two main fractions. Both fractions recovered from the BioGel column were active as lectins and both gave one band of MW 30,000 on SDS-gel electrophoresis. Isoelectric focusing gave one major component (PI = 5.8) and three relatively minor lectin components with more acidic isoelectric points. The membrane lectin from Phaseolus vulgaris cv. “Processor” gave reactions of identity with the globulin lectin in double diffusion tests but gave no appreciable reaction with the anti-albumin lectin serum which had been produced against albumin isolectins (Pusztai and Stewart, 1978). According to the authors, their results suggested that the lectin prepared from Phaseolus vulgaris protein body membranes is similar to the globulin isolectin glycoproteins described previously (Pusztai and Watt, 1974; Pusztai and Stewart, 1978). Pusztai and his co-workers (1979b) have found that over 10% of some Phaseolus vulgaris seed proteins can be accounted for as lectins. Two-thirds of the total lectin content of the seeds seems to be albumin isolectins and appears to be matrix or storage proteins which are synthesized late during seed maturation.

120

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

It has been suggested that the globulin isolectins, which are synthesized early during seed development (A. Pusztai, personal communication), are more intimately involved in the formation and stabilization of the evolving protein body membranes. Navy Bean Lectins. Andrews (1974) isolated a lectin from the navy bean which had both strong erythroagglutinating and leukoagglutinating activities. It was characterized as a tetramer with four identical subunits of MW 32,000. Although it had a strong resemblance in size, amino acid composition, and carbohydrate content to the lectins of the red kidney bean, the fact that it displayed both kinds of activities makes the proposed structure inconsistent with the tetrameric structure of the red kidney bean lectin composed of identical subunits as proposed by Yachnin and his co-workers (1972). An alternative explanation would be that the same subunit, in this instance, is able to agglutinate either erythrocytes or leukocytes. Wax Bean Lectins. A lectin from the wax bean (Phaseolus vulgaris, var. Sure Crop Stringless Wax) was purified by Takahashi and co-workers (1967). Its amino acid composition was similar to that of the red kidney bean lectins (no sulfur-containing amino acids, and high content of aspartic acid, serine, and threonine). The oligosaccharide moiety contained, in addition to mannose and 2amino-2-deoxyglucose, glucose, arabinose, galactose, fucose, and xylose. Takahashi and Liener (1968) later reported the isolation from the same lectin preparation of a glycopeptide with a carbohydrate composition different from that of the original preparation. Sela and co-workers (1973) isolated two lectins from the wax bean (Phaseolus vulgaris, var. brittle-wax) that also contained arabinose, glucose, galactose, and fucose, in addition to mannose. The lectins were tetramers (125,000 -+ 5000 MW) comprising identical subunits (MW 30,000). Other Lectins from Phaseolus Varieties. Dahlgren and his colleagues (1970) isolated two lectins from Phaseolus vulgaris, var. blue lake. One of them (PHAa”) was purified to homogeneity. It had MW 83,000, considerably lower than most Phaseolus vulgaris lectins. These data have been confirmed by Hoglund and Dahlgren ( 1970). Their lectin (PHA-a”) had erythroagglutinating, leukoagglutinating, and mitogenic properties. A lectin from Phaseolus vulgaris, var. red, was isolated by conventional protein purification procedures (Mialonier et al., 1973). The lectin, localized in the cytoplasm of the cotyledon and embryo cells of the seed, was a glycoprotein containing 6.6% carbohydrate, high amounts of aspartic acid, serine, and tryptophan, but no sulfur-containing amino acids. It had MW 128,000. Moreira and Perrone (1977), using extraction at pH 4.2 followed by ammonium sulfate precipitation and DEAE-cellulose chromatography, isolated two protein fractions with lectin activity, i.e., LcPA and LcPB, from Phaseolus vulgaris, var. “Rico 23.” The fraction of LcPA was homogeneous by disc gel electrophoresis and isoelectric focusing. This lectin (LcPA) had MW 100,000,

PROPERTIES OF COMMON BEAN PROTEINS

121

an isoelectric point at pH 5.1, and an extinction coefficient at 280 nm of 7.85. It contained 9.11% neutral sugars, 1.44% amino sugars, and no methionine, and had a very low content of $-cystine, but a high content of acidic and hydroxylated amino acids. Junqueira and Sgarbieri (198 1) isolated a lectin from Phaseolus vulgaris, var. “Rosinha G2,” by affinity chromatography on Con A-Sepharose and exclusion chromatography on Sephadex G-200 with the following characteristics: It was a glycoprotein with 8.3% neutral carbohydrate and 2.1% glucosamine; it had a very low content of sulfur-containing amino acids and high proportions of aspartic acid, serine, threonine, and tryptophan. It had pZ in the range pH 5.5-5.7 and MW 136,000. Calculations of data from Sephadex gel chromatography and amino acid analysis gave a molecular radius of 43 A, a partial specific volume ( 9 ) of 0.75 ml-g-I, and a frictional ratioflfo of 1.3 for this glycoprotein. Specificity of Phaseolus vulgaris Lectins. The specificity of binding of Phaseolus lectins to cell membranes was studied by Kornfeld and his colleagues (S. Kornfeld and Kornfeld, 1969; R. Kornfeld and Kornfeld, 1970, 1974; Kornfeld et al., 1972; Leseney et al., 1972). They found that treatment of human erythrocytes with trypsin released a soluble glycopeptide (MW 10,000) that bound to purified Phaseolus vulgaris lectin, abolishing its erythroagglutinating and lymphocyte-stimulating properties. The glycopeptide was digested with pronase and the products were chromatographed on DEAE-cellulose. Carbohydrate and amino acid composition, plus the sequential cleavage of sugars from the nonreducing end with specific glycosidases, led to the following structure for the carbohydrate moiety. a-Ac-Neu

2

.1 6 P-D-Galp

P-D-Galp

1

1

.1

.1

3 24 P-D-G~cNAc~

3,4 P-D-G~cNAc~

1

1

.1

.1

? 2 a-D-Manp( 1+2)-ci-~-Manp(I+?)-D-GlcNAcp+Asn

The glycopeptide was an excellent inhibitor of P . vulgaris lectin-induced cell agglutination. Removal of the sialic acid residue by neuraminidase treatment did not affect the activity, whereas treatment of the desialized product with p-Dgalactosidase essentially destroyed the ability of the glycopeptide to inhibit hemagglutination.

TABLE I11 PHYSICOCHEMICAL PROPERTIES OF LECTINS ISOLATED FROM SEEDS OF SEVERAL SPECIES AND CULTIVARS OF PHASEOLUS Species and cultivars P . lunatus Lima bean

sZOw

MW Designation ( X lo-))

pl

( X 1 0 - 1 3 S)

Neutral“ Aminob sugar sugar

D2o.w u

(A)

(XIO-7

V

cmZ.sec-’) (ml.g-’)

$!fa

(%)

(%)

Lectin I1

247

3-5

+

Lectin 111

124

3-5

+

P-PHA

E-PHA L-PHA L-PHA PHA-a’ Black kidney bean PHA-A

128 128.4 150 140 126 83-91 126-130

White kidney bean D3-alb 11-glb Wax bean WBH F-111

118 4.8; 4.9 119.7 5.15; 5.65 121-132 5.5 125

P . vulgaris Red kidney bean

P . vulgaris “Rico 23” “Rosinha G2”

LcPA

CII-p

100.3 136

6.5 6.5 6.5 5.0 5.1

6.5 7.05 7. I 6.8 6.87

4.9

5.9

5.1 5.5-5.7

ONeutral sugars expressed as % mannose. bAmino sugars expressed as % glucosamine.

4.8 5.08

48

1.255 1.3 1.3 1.45

0.683

6.84 6.76 5.37

6.9

4.91

0.738 0.75 0.75 0.728

1.56

3.7

0.712 0.715 0.728

5.0

0.75

1.3

4.9

43

8.02

2.39

6.8 7.34 10.0 5.03

3.1 2.84 3.2 0.68

6.66 6.03 7.3 8.0

1.64 1.62 0.9

9.11 8.3

1.44 2.12

Reference

Galbraith and Goldstein (1970, 1972a.b) Galbraith and Goldstein (1970, 1972a,b) Rigas et a l . (1972) Rigas and Osgood (1955) Weber et al. ( 1972) Weber et al. (1972) Rasanen et al. (1973) Dahlgren er a / . (1970) Jaffe and Gaede (1959), Jaff6 and Hannig ( 1 965) Pusztai and Stewart (1978) Pusztai and Stewart (1978) Takahashi et al. (1967) Sela et al. (1973) Moreira and Perrone (1977) Junqueira and Sgarbieri (1981)

PROPERTIES OF COMMON BEAN PROTEINS

123

The purification of a second glycopeptide from erythrocytes has also been described by Kornfeld and Kornfeld (1969) in which both P-D-galactosyl residues were penultimate to sialic acid. Neuraminidase treatment did not affect its potency as an inhibitor. Two glycopeptides isolated from immunoglobulin G (Kornfeld et al., 1971) and glycopeptides derived from fetuin (Spiro, 1964) and from transferrin (Jamieson, 1966), each containing mannose residues in the core chain as well as Gal + GlcNAc sequences in the outer chains, proved to be inhibitors. From these studies it was suggested that a branched structure containing at least two terminal P-D-galactopyranoyl groups or residues that are penultimate to sialic acid are required for efficient binding to Phaseolus vulgaris lectins (see above structure). Uhlenbruck et al. (1970) reported that a trisaccharide, P-D-Galp( 1+3,4)-P-DGlcNAcp( 1+2)-~-Man, is an essential component of glycoproteins reactive with the red kidney bean lectins. This trisaccharide has been synthesized by Kaifu and Osawa (1976) and found to inhibit 0-erythrocyte hemagglutination by P. vulgaris lectins. The physicochemical properties of some isolated Phaseolus lectins are shown in Table 111. 4 . Nutritional and Medical Importance

A number of reviews have been published on the toxicity and nutritional importance of the lectins (Liener, 1962, 1974, 1979; Jaff6, 1975, 1980). Suggestions that lectins are responsible, at least in part, for the toxicity and low nutritive value of uncooked or improperly cooked beans has been made frequently since the beginning of the century. Differences of opinion are found in the older literature (Weinhaus, 1909; Schneider, 1911; Liining and Bartels, 1926; Goddard and Mendel, 1929) as to the toxicity of lectins of Phaseolus seeds. However, most of the controversies have originated from differences in the material studied. De Muelenaere (1965) studied the hemagglutinating activity in several varieties of Phaseolus vulgaris seeds; he reported a range from 155,000 hemagglutinating units (HU)/g protein for the Natal Round Yellow bean to 3200 HU/g protein for the butter bean. Autoclaving the bean rapidly reduced the hemagglutinating titer. In all cases, the extract was toxic at hemagglutination activity above 1000 HU/g protein when injected intraperitoneally. He concluded that lectin itself is not the factor responsible for bean toxicity. Jack Sword bean (Canavalia ensiformis) is extremely toxic although the hemagglutinating activity was only 1200 HU/g protein, whereas Haricot bean (Phaseolus vulgaris) with a hemagglutinating activity of 40,000 HU/g protein was not toxic. Jaff6 (1962) made a detailed study of the toxicity of various lectin fractions derived from Phaseolus vulgaris. He found large differences in toxicity when the different fractions were injected into mice, with a definite positive correlation

124

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

between toxicity and hemagglutinating activity. One of the most toxic fractions (LD,, of 50 mg/kg body weight) was 0.0014 and 0.025 times as toxic as ricin and diphtheria toxin, respectively. This fraction, named phaseolotoxin A, was reported to inhibit strongly the growth of rats. When active lectin was present in the diet there was a pronounced decrease in nutrient absorption as well as in the digestibility of proteins and fats (Jaff6 et al., 1955; Jaff6 and Camejo, 1961; Jaff6, 1962). Absorption of glucose was decreased by 50% in rat intestinal loops perfused with solutions containing lectins. The authors suggested that lectins, in a similar fashion as they combine with red blood cells, also combine with cells of the intestinal mucosa and microvilli, thus causing a nonspecific interference with intestinal absorption and nutrient utilization. Active hemagglutinating fractions isolated from red kidney bean and from black beans were fed to rats at various levels in a basal diet containing 10% casein (Honovar et al., 1962). A definite inhibition of growth was observed at levels of lectin as low as 0.5% of the diet, although in the case of black beans this level did not cause the death of any of the animals. Growth inhibition was more pronounced with the red kidney bean lectin as compared with the black bean lectin. Red kidney bean lectin, at a level of 1.5% of the diet, killed all the rats in a week, whereas 4.6% black bean lectin was necessary to cause the same lethal effect. Lectins from different varieties of beans were shown to exhibit different levels of toxicity (Jaff6, 1968; Jaff6 and Vega Lette, 1968). From a study of the effects of a number of protein fractions separated by chromatography from the seeds of Phaseolus vulgaris and Glycine man, Stead et al. (1966) also found that there was no direct correlation between hemagglutination and toxicity. This study suggested that the two properties were apparently associated with different factors. On the basis of agglutination tests with erythrocytes from different animals, Jaff6 et al. (1972) showed the existence of four different types of lectins and suggested that, on this basis, beans could be classified into four distinct groups: (1) type A beans contained lectins which agglutinated rabbit, trypsin-treated cow, and pronase-treated hamster erythrocytes; (2) type B beans contained lectins which agglutinated rabbit and pronase-treated hamster erythrocytes but did not agglutinate trypsin-treated cow erythrocytes; (3) type C beans contained lectins which agglutinated pronase-treated hamster and trypsin-treated cow erythrocytes; (4) type D beans contained lectins which agglutinated only pronase-treated hamster erythrocytes. Lectins from types A and C, which agglutinated trypsin-treated cow blood cells, were toxic when injected intraperitoneally into rats and mice, whereas types B and D, which did not agglutinate trypsin-treated cow erythrocytes, were nontoxic. Jaff6 and Briicher (1972) and Jaffe et al. (1972) also observed that heating extracts of types A and C beans eliminated hemagglutination before toxicity

PROPERTIES OF COMMON BEAN PROTEINS

125

when rabbit blood cells were used; only by using trypsin-treated cow erythrocytes did the elimination of hemagglutination and toxicity coincide. Based on these observations, the authors recommended the test with trypsintreated cow erythrocytes for detecting toxic beans and for monitoring the adequacy of heat treatment for elimination of Phaseolus vulgaris lectin toxicity. In this same series of investigations, Jaff6 and Briicher (1972) presented evidence for the presence in one commercial variety of black bean “Cubagua” of seeds containing different types of lectins. Twenty-four percent of the seeds was type A containing toxic lectins and 76% was type B (nontoxic). These different types of lectins could be separated by ion-exchange chromatography (DEAE-cellulose column). The proportions of seeds with toxic and nontoxic lectins suggested that the inheritance for this factor was of simple Mendelian type. Jaff6 et al. (1972) described the inheritance for type A lectin as a single dominant genetic trait which facilitates the detection of the factor in a population of seeds and its elimination by breeding programs. Another interesting observation (Jafft5, 1975) was that the toxic lectins appeared mainly in the pigmented varieties of beans rather than in white or nonpigmented ones. Lima et al. (1980) have studied the hemagglutinating, mitogenic, and toxic properties of 16 Brazilian varieties of Phaseolus vulgaris. All varieties agglutinated trypsin-treated bovine or rabbit blood red cells and induced mitosis in human lymphocyte culture with different intensities but none showed activity on non-trypsin-treated bovine red blood cells. The extracts from several beans were highly toxic when injected intraperitoneally into mice. The toxic samples had a high agglutinating capacity against both trypsin-treated bovine and rabbit blood cells and were also highly mitogenic; however, several samples with high mitogenic activity were not toxic when injected intraperitoneally into mice. A toxic factor which inhibited rat growth has been isolated from navy beans (Phaseolus vulgaris) by Evans et al. (1973). On the basis of its hemagglutinating and mitogenic activities, as well as its physical properties, this protein fraction was judged to be similar, if not identical, to the lectins previously described in other varieties of Phaseolus vulgaris. The toxicity of lectins in kidney bean has been extensively studied by Pusztai and co-workers (1975; Pusztai and Palmer, 1977). Pusztai et al. (1975) isolated and partially characterized the toxic constituents of a kidney bean protein isolate. They showed that both the albumin and the globulin fractions were toxic when added to a 5% casein diet, with the albumin fraction being more toxic than the globulin fraction. They identified lectins in each of the two fractions and concluded that the toxicity was directly related to the hemagglutinating titer of these two fractions. Pusztai and Palmer (1977) purified the kidney bean (Phaseolus vulgaris) lectins of both protein fractions by affinity chromatography on a fetuinSepharose 4B column. The net protein utilization (NPU) of rats fed on a 5%casein diet was strongly depressed by these pure lectin preparations. The extent

126

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

of toxicity was directly related to the lectin content of the diet. On the other hand, they demonstrated that lectin-free kidney bean protein preparations were nontoxic for rats. Based on these findings they suggested that the toxic principle of kidney bean is identical with the lectins. Recently, Antunes and Sgarbieri (1980) showed that the toxicity of raw bean (Phaseolus vulgaris, var. Rosinha G2") was associated with various protein fractions. The observed order of toxicity (based on the time to kill all rats in a group of 10) was as follows: whole flour > albumin > globulin > insoluble residue (after extraction). In the same paper they reported on the amount of heat treatment required to eliminate the toxicity from the various fractions and from whole beans. It was observed that limited heat treatment (2-3 min at 97°C) of the water-soaked whole bean improved the nutritional value close to the maximum, even though considerable lectin and protease inhibitor activities still remained. V. C. Sgarbieri, P. L. Antunes, and R. G . Junqueira (unpublished) found that the addition of lectin isolated from the Rosinha G2 bean to an adequate 10%casein rat diet impaired protein digestibility and depressed the growth of the rats appreciably, although this diet did not kill the rats nearly as fast as the unheated whole flour. They also reported that digestibility was higher in the isolated protein fractions than in the whole flour and that supplementation with sulfurcontaining amino acids improved the biological value of isolated bean protein fractions much more than that of the bean flour. Based on these results, it was suggested that certain varieties of bean (Phaseolus vulgaris) contain at least three classes of toxic compounds: (1) extremely heat-labile and highly toxic compounds of unknown nature which might have been responsible for the rapid killing of the animals; (2) lectins and perhaps other compounds of medium toxicity which require greater heat treatment for complete destruction; and (3) heat-resistant compounds of low toxicity which contribute to the low digestibility and low nutritive value of certain bean varieties. These might be phenolic compounds, phytic acid, saponins, and possibly other compounds of unidentified nature. The mechanism of the antinutritional effect of the lectins is not completely understood, although it has been the focus of attention for many years. Jaff6 (1960) proposed that the toxic effect of lectins, when ingested orally, may be caused by their ability to bind to specific receptor sites on the surface of the intestinal epithelial cells. Support for this hypothesis came from investigations of in vitro and in vivo intestinal absorption of several nutrients (Jaff6 and Camejo, 1961). It was shown that lectin can cause a decrease in the intestinal absorption of sugars, lipids, and proteins in the rat. Further support for this hypothesis came from Etzler and Branstrator (1974), who found that a number of different lectins reacted in vitro with the crypts and/or villi of the intestine at different regions depending on the specificity of the lectin. Since the surface-bound lectins are known to produce profound physiological effects on the cells with which they "

PROPERTIES OF COMMON BEAN PROTEINS

127

interact, one of these effects could be a serious impairment in the ability of these cells to absorb nutrients from the intestinal tract, thus causing inhibition of growth and, in extreme cases, even death. Serious damage to intestinal epithelial cells and microvilli due to lectins was shown by light and electron microscopy (Pusztai et al., 1979a,b). The lectins were shown to react with intestinal cells in vivo and to cause a disruption of many of the brush borders of duodenal and jejunal enterocytes. Although depressed to a certain extent, absorption still occurred probably through the nondisrupted cells of the small intestine. In addition, abnormal absorption of potentially harmful substances, lectinrelated or of bacterial origin, could also occur possibly as a direct effect of the disruption caused by the lectins on the enterocytes. Pusztai et al. (1979a,b) also found a very high urinary nitrogen excretion in the animals receiving diets containing active lectins. They suggested that the overall effect of lectin toxicity is due to a systemic derangement of the metabolism starting with perturbation of absorption and selectivity of the biological membrane and increased tissue catabolism. In line with the theory that abnormal absorption of harmful substances occurs due to disruption of the enterocytes by lectins, Jayne-Williams (1973) and JayneWilliams and Burgess (1974) observed that germ-free Japanese quail were able to tolerate the toxic effects of raw navy beans much better than conventional birds. Similar observation was made for germ-free rats (Rattray et al., 1974) and for chicks (Hewitt et al., 1973). It was suggested (Jayne-Williams and Burgess, 1974) that the binding of lectins to the cells of the intestinal mucosa might interfere with the normal defense mechanism of these cells. In the presence of lectins, normally innocuous intestinal bacteria could not be prevented from passing from the lumen of the intestine into the lymph, blood, and other tissues of the body, thereby increasing the observed toxicity and decreasing the animal’s resistance to the toxic effects. It has been generally assumed that the lectins are heat-labile substances which are destroyed under normal conditions of domestic or industrial preparation of foods. However, during this review we came across some reports (Griebel, 1950; Korte, 1972; Anonymous, 1976) in which lectin activity was detected in processed food and could have been responsible for reported toxicity. Korte ( I 972) reported that lectin activity was found in 22% of Phaseolus vulgaris and maize mixtures prepared and cooked under African village conditions. In these cases signs of toxicity, such as vomiting and diarrhea, and of malabsorption were frequently observed among children consuming the mixture. The same author mentioned that in 1948 bean flakes were supplied to the population of Berlin. A cooking time of 15 min had been recommended, after which the flakes were apparently soft enough to be consumed. When a massive poisoning among the population was reported (Griebel, 1950), it was concluded that the heating time

128

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

had not been adequate to destroy all the toxins contained in the beans. More recently a case of toxicity by uncooked red kidney bean in humans was described in Great Britain (Anonymous, 1976). Due to their diversified properties, such as their ability to combine with different types of physiologically important substances and with cells and also to stimulate cell modifications and cell division, lectins have been the object of great interest to scientists in medical research. According to Boyd (1963) the following uses have been made of lectins in applied and basic research: (1) determination of subgroups of blood groups A and AB (Boyd and Shapleigh, 1954a); (2) diagnosis of saliva secretors (Boyd and Shapleigh, 1954b); ( 3 ) grouping of blood into A, B, and 0 (Saint-Paul, 1961; Boyd and Shapleigh, 1954~); (4) MN blood grouping; (5) separation of A and 0 cells; (6) population studies of blood character G4 (Schwarzfischer and Liebrich, 1962); (7) stimulation of mitosis (Hungerford et al., 1959; Nowell, 1960a,b); (8) investigations in cancer research (Nungester and Van Halsema, 1953); (9) discovery of new animal donors for immune sera (Levine et al., 1957); (10) study of blood group substances (Morgan and Watkins, 1956). B.

PROTEIN INHIBITORS OF DIGESTIVE ENZYMES

There are two types of protein inhibitors of digestive enzymes in common beans. These are the inhibitors of the serine group of proteolytic enzymes and of the a-amylases from animal and insect sources. Much more work has been reported on the inhibitors of proteolytic enzymes, particularly the inhibition of trypsin. Protein inhibitors of the other three groups of proteolytic enzymes have not been reported in common beans; however, this may be due to lack of research in this area.

I.

Protein Inhibitors of the Proteolytic Enzymes (Protease Inhibitors)

The most investigated of the protease inhibitors in legumes are the two major inhibitors found in soybeans (Glycine m m ) . The Kunitz inhibitor was crystallized from soybeans in 1946 (Kunitz, 1946) and the Bowman-Birk inhibitor was discovered by Bowman (1946) that same year. Other trypsin inhibitors are also present in the soybean (Kassell, 1970) but have not been studied as extensively. The two major protease inhibitors of soybeans differ markedly in properties. The Kunitz inhibitor has MW 21,500 (Yamamoto and Ikenaka, 1967), contains two disulfide bonds per molecule, and contains many more glycine, valine, leucine, isoleucine, and arginine residues than does the Bowman-Birk inhibitor (Table IV). The Kunitz inhibitor stoichiometrically inhibits 1 mole of trypsin per mole but inhibits chymotrypsin nonstoichiometrically. The Bowman-Birk inhibitor has MW 7975 (Yamamoto and Ikenaka, 1967), contains seven disulfide

PROPERTIES OF COMMON BEAN PROTEINS

129

bonds per molecule (Odani and Ikenaka, 1973), has no glycine, and is relatively poor in valine, leucine, isoleucine, and arginine (Table IV). The Bowman-Birk inhibitor stoichiometrically , and simultaneously, inhibits 1 mole of trypsin and 1 mole of chymotrypsin per mole of inhibitor. A Bowman-Birk type protease inhibitor has also been isolated from soybeans which inhibits 2 moles of trypsin per mole of inhibitor and does not inhibit chymotrypsin (Odani and Ikenaka, 1976). It contains arginine at both of the binding sites (Odani and Ikenaka, 1978). The primary structures of the Kunitz and Bowman-Birk inhibitors are shown in Figs. 1 and 2, respectively. It is clear that the two inhibitors have evolved through separate pathways. The Bowman-Birk soybean protease inhibitor appears to be a good prototype of the Phaseolus protease inhibitors to be discussed in the remainder of this section. Because of its smaller size and the more extensive disulfide bonds, the Bowman-Birk inhibitor is much more stable to heat and high and low pH than is the Kunitz inhibitor. a. Occurrence and Nomenclature. Proteins which inhibit trypsin are generally assumed to be ubiquitous. Therefore, it is not surprising that trypsininhibitory activity has been reported in essentially all Leguminosae examined. V. C. Sgarbieri, M. A. M. Galeazzi, R. G. Junqueira, and L. D. Almeida (unpublished) have measured the trypsin- and chymotrypsin-inhibitory activity of 60 cultivars of Phaseolus vulgaris. All contained inhibitors of both trypsin and chymotrypsin, although the ratio of the two inhibitory activities was not constant. The most extensive work on the trypsin inhibitors of beans has been with species other than Phaseolus vulgaris. Trypsin inhibitors have been purified and partly characterized from the soybean (Glycine max L.; Kunitz, 1946; Wu and Scheraga, 1962; Birk, 1961, 1976b; Birk et al., 1963; see above), the Jack bean (Canavalia ensiformis; Ubatuba, 1955), the field bean (Dolichos lablab; Sohonie and Ambe, 1955), the broad bean (Viciafaba; Warsy et al., 1974), red gram (Cajanus cajan L.; Tawde, 1961), black-eyed pea (Vigna sinensis; Ventura and Xavier Filho, 1967), and sweet pea (Lathyrus odoratus; Weder and Belitz, 1969). Inhibitors investigated from the genus Phaseolus include the runner bean (Phaseolus coccineus L.; Weder et al., 1975; Weder and Hory, 1976), the lima bean (Phaseolus lunatus; Jones et al., 1963), and the mung bean (Phaseolus aureus Roxb.; Baumgartner and Chrispeels, 1976), as well as several cultivars of Phaseolus vulgaris (see below). Among the Phaseolus vulgaris, inhibitors have been purified and partly characterized from the following cultivars: the white kidney bean (Pusztai, 1966), the navy bean (Wagner and Riehm, 1967; Whitley and Bowman, 1975), the French bean (Belitz and Fuchs, 1972, 1973), the Great Northern bean (Wilson and

130

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER TABLE COMPOSITION, MOLECULAR WEIGHT, AND BINDING

Amino acid Asp Glu GlY Ala Val Leu

Ile Sei”

Thi” !-Cyci Met Pro Phe TY+ Trp

His LYS Sugar 0 MW Tdmole ChTrlmole

Clvcine m u

Phoseolus lunarus

Phoseolus coccmeus

Soybean

Lima bean(

French hean“

Kunitr” 13.5

8.9 7.8 4. I 7.2 6.9 6.9 5.5

3.6 2.0 I .2 5.I 45 1.9 0.9 1.1

5.8 46 0 21.500 1
BuwmanBirk”

I

11

111

IV

11.0 6.9 0 4.0 I.o I.9 19 87 I .9 12 7 1 1 5.9 2.0 1.9 0.3 09 4.8 I .9

143 7.0

165 6.4 0.04 38

13.2 72 0.Y5 3.8

14.0 74 I I 2.8

12

1.2

1 1

1.1

3.4 4.6 12 4 14.5 0 6.9 1.2 I 0 6.1 49 2.3

3.7 4.8 I2 3 16.9 0 72 1.2 I 0 3.6 4.9 2.4

3.1 4.8 15

3.3 4.4 I3

7975 1 I

8408 I

8291 I I

1.1

3.6

2

3

4

2 0 6.0 4.2 2.1

15.2 0 7.2 2.1 I 0 63 4.0 2.2

1 2 6 12.7 10.3 9.4 4.4 4.7 42 42 2.1 2.2 3.8 4.1 3.4 3.3 12.2 12.5 5.5 53 11.6 12.3 0 0 6.3 57 1.8 1.6 14 1.3 4.7 4.8 5 2 5.1 3.0 3.4

11.9 11.1 5.6 53 3.1 3.7 35 11.0 51 9.2 0 5.6 2.0 1.3

9892 I I

9423 I I

9230 I
9100 I

5

16 1 0 7 I 1.X

5

42 5.8 3.6

Phasmlus uurePu.7 Roxb.

Mung bean? 12.3 8.6 25 3.7 I2 2.5 2.5 12.3 3.7 9.9 25 7.4 I.2 1.2 0 49 7.4 4.9

0

im

9330 I
8113


aThe amino acid and sugar compositions are expressed as moles per 10,000MW protein. ”From Yamamoto and Ikenaka (1967). cFrom Jones et a/. (1963). dFrom Belitz et a/. (1972). eFrom Chu e t a / . (1964). fFrom Whitaker and Sgarbieri (1981). ZFrom Pusztai (1966).

Laskowski, 1973; Birk, 1976c), the pinto bean (Wang, 1975), and the Brazilian pink bean (Rosinha G2; Whitaker and Sgarbieri, 1981; Sgarbieri and Whitaker, 1981). The inhibitors are generally named as an inhibitor of the first protease against which they have been tested, usually trypsin. As shown by the data of Table IV, all the inhibitors listed inhibit trypsin usually at a ratio of 1:1, although the three inhibitors from Brazilian pink beans and the navy bean inhibit 2 moles of trypsin per mole of inhibitor. These four inhibitors have MW 20,000-23,000, whereas the other inhibitors, showing a 1:l ratio in inhibition of trypsin, have MW 8000-10,000. Could the trypsin binding site of the inhibitors of the Brazilian pink bean and navy bean have become duplicated via a mutation? The inhibitors

131

PROPERTIES OF COMMON BEAN PROTEINS IV PROPERTIES OF SEVERAL TRYPSIN INHIBITORS FROM BEANSO Phaseoh vulpris

Brazilian pink bead

A

10.0 6.5 2.1 2.8 0 1.8 3.5 10.7 4.2 9.2 0.5 5.2 16 05 0 4.7 4.6 2.7 0.5 20,000 2 1

B

C

10.0 6.6 2.0 3.0 0 2.0 3.7 10.7 4.6 10.5

10.6 6.9 1.2 3.0 0 2.0 3.7 11.0 4.8 10.5

0.5

0.5

5.4 1.7 0.5 0 4.8 4.0 2.8 0.5 20,000 2 I

5.5

1.6 0.5 0 4.0 4.0 2.0 0.5 20.000 2 1

Great Northernh

Kidney bean8 14.4 7.3 3.2 4.7 1.8 3.0 4.2 14.8 7. I 14.2 1.1 6.8 1.4 1.4 0.8 3.7 4.5 35 0

I

I1

13.6 7.4 1.3 3.8

12.3 6.0 2.2 2.4 1.9 2.3 3.2 14.9 5.7 14.7 2.3 6.9 0 2.1 0 3.3 4.6 3.2 0 8371 1 0.2

1.1

3.5 3.6 13.0 3.8 16.1 0 7.5 1.4 1.2 0 3.5 4.8 4.0 0 10,ooo 8086 1 1" 0.2 1"

IIIb 12.5 7.9 2.1 3.3 0 2.3 4.1 13.1 5.4 14.4 0 7.1 23 1.1 0 5.6 4.5 3.2 0 8884 1

1

Navy bean' I3 1 7.6 2.0 3.4 1.0 2.7 3.9 15.2 5.9 13.1 0.6 7.1 1.6 1.6 0 4.5 4.8 3.2 0 23,000 2 N.D.0

Pinto k a n i

Phaseolus

I

Avg. Range

II

10.5 6.2 2.0 2.8 0.9 I .8 3.1 12.1 4.3 16.0 0.8 5.4 0.9 I .2

11.0 7.3 2.0 3.2 0.0 1.9 3.6 12.6 4.2 15.9 00 5.9 1.6 0.7 -

4.0 4.3 3.2

5.0

~

19,000

5.2 3.3

12.5 7.6 2.2 3.5 1.1 2.8 3.8 12.7 4.8 13.4 0.5 6.5 1.5 1.2 0 4.6 4.8 31

1G-16 &I0

M 3-5 Ck3 2-4 3-5

11-15 3-7 9-17 Ck2 5-8 Ck2 1-2 %I 3 4 &7 2-5 &2

19,000

hFrom Wilson and Laskowski (1973). 'From Wagner and Riehm (1967). jFrom Wang (1975). kCorrected for loss on hydrolysis. 'Determined as cysteic acid. mKrahn and Stevens (1971) "Pusztai (1968). ON.D., Not determined.

listed in Table IV also inhibit chymotrypsin, although in the cases of the Kunitz soybean inhibitor, the three isoinhibitors of the French bean, and isoinhibitors I and I1 of the Great Northern bean, the inhibition is nonstoichiometric (meaning that even at excess inhibitor there is less than 1 mole of inhibitor bound per mole of chymotrypsin). Some other serine proteases, such as elastase and plasmin, are inhibited by some bean inhibitors. Because these inhibitors decrease the activity of both trypsin and chymotrypsin, they are frequently referred to as trypsin-chymotrypsin inhibitors. As we shall see later, this is appropriate because these two serine proteases probably bind at independent sites on the inhibitor. An inhibitor, specific for the chymotrypsins but which does not inhibit trypsin or subtilisin, isolated from winged bean (Psophocarpus tetragonolobus L.) has

132

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

63

FIG. 1.

Primary structure of Kunitz soybean trypsin inhibitor. From Koide and Ikenaka (1973)

30

70 FIG. 2. Primary structure of Bowman-Birk inhibitor from soybeans. Residues at the two reactive sites are shown as solid black circles. From Odani and Ikenaka (1973).

PROPERTIES OF COMMON BEAN PROTEINS

133

been described recently (Kortt, 1981). This is the only reported case of an inhibitor which binds with chymotrypsin but not trypsin. Multiple molecular forms of the protease inhibitors are often found (Table IV). For example, four isoinhibitors from lima beans, three from French beans, three from Brazilian pink beans, and three from Great Northern beans have been purified. These are generally named, using Roman numerals or the alphabet, in the order in which they are eluted from a DEAE-cellulose anion-exchange column. b. Distribution and Physiological Significance in the Plant. In beans, the highest concentrations of protease inhibitors are in the seeds, although Birk (1976a) showed that the inhibitors may be found in other parts of the plant. The tomato and potato protease inhibitors have been shown conclusively to be present in the leaves of these plants (Green and Ryan, 1972). In double bean (Faba vulgaris) and field bean (Dolichos lablab) plants, the inhibitor was reported to be present in seeds, leaves, stems, and roots of the plant, with the highest concentration in the seeds (Ambe and Sohonie, 1956). However, Kapoor and Gupta (1978) reported that protease inhibitors of the soybean are not found in other parts of the plant, regardless of the stage of growth. They suggested that the inhibitor is synthesized in the seed with the initiation of seed formation and that it is not translocated from other parts of the plant to the seed. The cotyledons contained about 83% of the total trypsin inhibitor activity of the seed with no inhibitor in the embryo. There appears to be a paucity of data on this for the common beans. The inhibitors account for about 0.2-2% of the total soluble protein of the bean seed. For example, Whitaker and Sgarbieri (1981) reported the protease inhibitors of the Brazilian pink bean accounted for about 0.20% of the total protein; the trypsin inhibitors accounted for 0.27% of the protein in the kidney bean according to Abramova and Chernikov (1964) and up to 2% iu soybeans according to Kozlowska et al. (1976). The values of 5-10% cited by Richardson (1977) appear to be high. The physiological significance of the protease inhibitors in bean seeds is not known. Only in the case of the mung bean has it been shown that the trypsin inhibitor is active against a protease found in the cotyledon (Baumgartner and Chrispeels, 1976). It is well known that the level of inhibitors varies with different stages of growth, suggesting that the inhibitors may be physiologically important to the organism (Richardson, 1977). Ryan (1973) and Richardson (1977) have suggested that the inhibitors may have evolved as defense mechanisms against attack by insects and microorganisms. Certainly this appears to be the case in potatoes, where infestation with Colorado potato beetle larvae leads to elevated levels of protease Inhibitor-I in the leaves (Green and Ryan, 1972). Much more work on this point is needed for the beans.

134

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

c . Composition and Physicochemical Properties. The amino acid composition of isolated protease inhibitors from Phaseolus are given in Table IV. Comparison of the amino acid composition shows them to be quite similar to the Bowman-Birk inhibitor from soybean and quite similar to each other. In comparison to other proteins, they all are low in glycine, valine, methionine, phenylalanine, tyrosine, and tryptophan, relatively low in leucine, isoleucine, and threonine, and quite high in 4-cystine, serine, and proline. There is no cysteine in the inhibitors which have been analyzed for this amino acid. Most of the inhibitors have MW 8000-10,000 with the three isoinhibitors from Brazilian pink beans and the inhibitor of navy beans being the apparent exceptions. These four inhibitors are the only ones found to contain a small amount of carbohydrate. Whether the similar amino acid composition of the inhibitors is indicative of a similar primary structure can be only partly answered at the moment. Lima bean isoinhibitors I and IV are quite identical in sequence from residues I 1 to 81 (Fig. 3; using the numbering for isoinhibitor IV). Isoinhibitor IV has 8 amino acid residues at the N-terminal end and 2 at the C-terminal end, not found in isoinhibitor I (Stevens et a l . , 1974). There is considerable homology among the Bowman-Birk soybean trypsin inhibitor, lima bean trypsin inhibitor IV, and Great Northern trypsin isoinhibitor 11, as shown in Fig. 4. However, the trypsinbinding sites are near the N-terminal end of the Bowman-Birk inhibitor (Lys,,) and lima bean isoinhibitor IV (Lys,,) and near the carboxyl terminal end of the Great Northern isoinhibitor I1 (Ar&. This is the chymotrypsin-binding site for

IV I

10 Ser-Gly-His-His-Glu-His-Ser-Thr-Asp-Glx-Pro-Ser-Glx-Ser-Ser-LysAsp-G1 x-Pro-Ser-Glx-Ser-Ser-Lys-

IV

20 30 Pro-Cys (Cys ,Asx)Hi s (Cys ,Cys )Thr- Lys-Ser- Ile- Pro-Pro-G1 n-CysPro-Cys (Cys ,Asx)Hi s (Cys ,A1 a ,Cys)Thr-Lys-Ser- I1 e-Pro-Pro-G1 n-Cys-

I

IV I IV I

IV I IV I

{k:

40 Arg-Cys-:~:-Asp-~~~-Arg-Leu-Asp-Ser-Cys-Hi

s-Ser-Ala-Cys-Lys-SerArg-Cys-Thr-Asp-Leu-Arg-Leu-Asp-Ser-Cys-His-Ser-Ala-Cys-Lys-Ser50 6o (Cys ,Asx Thr ,Asx)I 1 eCys- I1 e-Cys-Thr-Leu-Ser- I 1 e-Pro-A1 a-G1 n-Cys-Val Cys-I1 e-Cys-Thr-Leu-Ser-I1 e-Pro-A1 a-G1 n-Cys-Val (Cys ,Asx,Asx) Ile-

-A;T

70

80

Asp-Phe-Cys-Tyr-G1 u-Pro-Cys-Lys-Ser-Ser-Hi s- Ser-Asp-Asp-AspAsx-Asp-Phe-Cys-Tyr-G1 u-Pro-Cys-Lys (Ser,Ser ,Hi s ,Ser ,Asx .Asx, Asx, Asn-Asn-Asn Asx)

FIG. 3. The complete amino acid sequences of lima bean trypsin inhibitor variants IV and I. From Stevens et al. (1974).

PROPERTIES OF COMMON BEAN PROTEINS

BB LB

135

Asp- Asp-G1 u-Ser-Ser-Lys-Pro-Cys10 Ser-Gly-Hi s-Hi s-G1 u-Hi s-Ser-Thr-Asp-G1 x-Pro-Ser-G1 x-Ser-Ser-Lys-Pro-Cys10

BE

Cys-Arg-Cys-Ser-Asp-

GB

Cys,Ile,Cys,Thr,Asx,

20 LB

Cys-Arg-Cys-Thr-AspSer

BB GB LB Phe

BB GB LB BE

?Pro-Ser-Glu-----Asp-Asp-Lys-Glu-Asn 70

GB

80 Lys Ser-Asx-Ser-Gly-Glx-Asx-Asx

LB

&Ser-Ser-Hi

t

80 s-Ser-Asp-Asp-Asp-Asn-Asn-Asn

FIG. 4. Amino acid sequences of Bowman-Birk soybean trypsin inhibitor (BB; Odani and Ikenaka, 1972), lima bean trypsin inhibitor IV (LB; Stevens et al., 1974), and Great Northern trypsin inhibitor I1 (GB; Wilson and Laskowski, 1974).

the Bowman-Birk inhibitor (Leu,,) and lima bean isoinhibitor IV (Leu,,). This difference may account for the poor binding of chymotrypsin to the Great Northem isoinhibitor I1 and the exceptionally good binding with elastase (Ala,, as possible binding site), which is noncompetitive with trypsin binding (Wilson and Laskowski, 1973). Great Northern isoinhibitors I and IIIb must be closely homologous since the peptide maps of these two proteins are very similar. On the other hand, isoinhibitors I and I1 have no peptides in common (Wilson and Laskowski, 1973). It is quite important to obtain peptide maps for all isolated inhibitors of Phaseolus in the future, as one method of assessing similarities and differences among these inhibitors.

136

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

A characteristic of the Bowman-Birk type inhibitors of beans is their great stability, in contrast to the Kunitz soybean inhibitor. Brazilian pink bean inhibitors retained 100% of their activity after heating for 15 rnin at 97°C (Whitaker and Sgarbieri, 1981), were stable at pH 2 and 12 at 25"C, and were not digested by pepsin at pH 2. Lima bean inhibitors were stable on heating for 15 min at 90°C at pH 5 or 7, or when kept for 24 hr at 23°C in 0.0 1 M NaOH or for 24 hr at 40°C in 0.01 M HC1 (Fraenkel-Conrat et al., 1952). Field bean inhibitor lost 59% of its activity in 30 min and 86% in 60 min at 100°C and pH 7.2. Autoclaving at 15 lb pressure destroyed 93% of the activity in 30 min. It was stable for a week at 15°C between pH 3 and 10 (Banerji and Sohonie, 1969). White kidney bean inhibitor did not lose activity in 2 hr at pH 2 and 37°C (Pusztai, 1968) even in the presence of pepsin. Lima bean inhibitors were resistant to pepsin for 24 hr at pH 2 (Fraenkel-Conrat ef al., 1952) and pH 1.5 (Jones et al., 1963). Because of the low tryptophan and tyrosine content of the inhibitors, the absorbance at 280 nm is much lower than that for an average protein. For example, A!&;% for the Brazilian pink bean inhibitors was found to be 0.274 (Sgarbieri and Whitaker, 1981). A:&% reported for the Bowman-Birk soybean inhibitor was 0.44 (Yarnamoto and Ikenaka, 1967), whereas A:gA% for the three Great Northern isoinhibitors was as follows: I, 0.354; 11, 0.231; and IIIb, 0.340 (Wilson and Laskowski, 1973). The isoelectric points of the inhibitors are generally between pH 4 and 5. Some examples are the following: Bowman-Birk soybean inhibitor, 4.0 (Yamamoto and Ikenaka, 1967); white kidney bean inhibitor, 5 (Pusztai, 1968); and field bean inhibitor, 4.7 (Banerji and Sohonie, 1969). Some typical partial specific volumes for these inhibitors are as follows: Brazilian pink bean inhibitors, 0.69 (Sgarbieri and Whitaker, 1981); Bowman-Birk soybean inhibitor, 0.69 (Yamamoto and Ikenaka, 1967); lima bean inhibitor fraction 6, 0.725 (Haynes and Feeney, 1967); navy bean inhibitor, 0.693 (Wagner and Riehm, 1967); and black-eyed pea inhibitor, 0.69 (Ventura and Xavier Filho, 1967). The Brazilian pink bean inhibitors have been reported to have a frictional ratioJf0 of 1.14 (Sgarbieri and Whitaker, 1981), whereasJfo for the black-eyed pea inhibitor was reported to be 1.47 (Ventura and Xavier Filho, 1967). The bean protease inhibitors form quite strong complexes with trypsin and often less tight complexes with chymotrypsin. For example, the three isoinhibitors isolated from Brazilian pink beans (Sgarbieri and Whitaker, 1981) gave the following Ki values, determined at 35°C and pH 7.8: with trypsin-Inhibitor A, 8.5 X 10-lOM;InhibitorB, 1.8 X 10WIOM;InhibitorC, 6.8 X 10W'OM; with chymotrypsin-Inhibitor A, 4.4 X l o W 7M ;Inhibitor B, 2.8 X l o W 8M ; Inhibitor C, 3.0 X 10W8 M. The ratios of Ki values for chymotrypsin compared to trypsin are as follows: Inhibitor A, 520; Inhibitor B, 160; Inhibitor C, 44. Other reported Ki values for trypsin are as follows: Bowman-Birk soybean inhibitor, M (with chymotrypsin, 5.0 X l o p 7 M ; Birk, 1968); lima bean, 4 X 5.6 X

PROPERTIES OF COMMON BEAN PROTEINS

137

M (Grob, 1950); mung bean, 10-9-10- lo M (Chu and Chi, 1963); field bean, 3.3 X M (Banerji and Sohonie, 1969); green gram, 6.1 X M (Honovar and Sohonie, 1959). Therefore, the Kivalues for trypsin inhibitors generally are in the range of 10- l o M ,with those for chymotrypsin inhibitors in the range of 10W8 M , although the three isoinhibitors of French beans and isoinhibitors I and I1 of Great Northern beans are reported to form nonstoichiometric complexes with chymotrypsin (Table IV), indicating that Kimay be larger for these complexes. The binding sites for trypsin and chymotrypsin on the Bowman-Birk soybean inhibitor are on different parts of the inhibitor molecule (Fig. 2). Treatment of the inhibitor with cyanogen bromide cleaves the molecule at Met,, and on treatment with pepsin the peptide bond between Asp,, and Phe,, is hydrolyzed, separating the molecule into two fragments. One fragment contains the trypsinbinding site, the other contains the chymotrypsin-binding site (Odani and Ikenaka, 1973). Separation of the trypsin- and chymotrypsin-binding sites by cyanogen bromide treatment has also been reported for the lima bean inhibitor (Feinstein and Feeney, 1967). Sgarbieri and Whitaker (1981) showed that cyanogen bromide treatment of the three isoinhibitors purifed from Brazilian pink beans gave two fragments (by gel electrophoresis) with little loss in trypsinand chymotrypsin-inhibitory activity. In fact, the trypsin-inhibitory activity of isoinhibitor B increased 80% over the control as a result of cyanogen bromide treatment, with no change in chymotrypsin-inhibitory activity. There are two types of trypsin inhibitors, based on the amino acid at the specific binding site on the inhibitor. One type contains a lysine residue, such as the Bowman-Birk soybean inhibitor and the lima bean inhibitor (see Fig. 4), and the other type contains an arginine residue, such as isoinhibitor I1 of the Great Northern bean (Fig. 4). Isoinhibitor I from Great Northern beans has lysine at the binding site (Wilson and Laskowski, 1973). The three isoinhibitors of Brazilian pink beans are lysine-type inhibitors (Sgarbieri and Whitaker, 1981) as is the trypsin-chymotrypsin inhibitor from chick-peas (Cicer arietinum) (Smirnoff et al., 1979). The two types of trypsin inhibitors can be differentiated on the basis of loss of activity with chemical modifying reagents. Loss of inhibitor activity following reductive alkylation is indicative of lysine-type inhibitors, whereas loss of activity on modification with p-hydroxyphenylglyoxal is indicative of arginine-type inhibitors (Yamasaki et al., 1980). Subsequent work has shown that the specific binding site for chymotrypsin contains a tyrosine, phenylalanine, leucine, or methionine residue, whereas that for elastase contains an alanine or serine residue (Laskowski and Kato, 1980). Location of the specific binding site for trypsin and chymotrypsin on the inhibitor can be determined by taking advantage of the discovery that incubation of inhibitor with protease at acidic pH (3.2-3.5) leads to hydrolysis of the peptide bond of the inhibitor at the binding site (Ozawa and Laskowski, 1966).

TABLE V HOMOLOGY AMONG SOME OF THE TRYPSIN AND CHYMOTRYPSIN INHIBITORS FROM LEGUMINOSAEO Amino acid sequence Inhibitor

Chymotrypsin site

Trypsin site I b

Bowman-Birk (soybean) Lima bean Runner bean Garden bean (Great Northern, isoinhibitor 11) Kunitz (soybean)

..Cys-Thr-Lys

...Cys-Thr-Lys26...Ik-

Tyr-Lys-

I 6-Ser-Asn-Pro..

...Ile-

. Pro.. .

Ser-IleSerAln-Pro(?).

..Cys-Thr-Args3-Ser-Met-Pro.. . ..‘Ser- Tyr-Arge3-I1eArg-Phe.. .

OAdapted from Hory and Weder (1976). hThe arrow indicates the specific recognition point involving P I and Pi residues

..

Ib

Cys-Thr-Leu4s-Ser-11ePro... ...Ile- Cys-Thr-Leu,,-Ser-IIePro.. . ... A s p V a l - Ala-LeuSer-Pro(?)

PROPERTIES OF COMMON BEAN PROTEINS

139

Determination of the newly formed carboxyl and amino terminal residues indicates the location of the active site. These types of studies have been extended to the lima bean inhibitor (Krahn and Stevens, 1970) and the French bean (Belitz and Fuchs, 1973). The amino acid sequences around the trypsin- and chymotrypsin-binding sites for several inhibitors are shown in Table V. There appears to be more homology among the lysine-type inhibitors than the arginine-type inhibitors. In most of the inhibitors serine is in the Pi position of the binding site. Much research has been done with an objective of determining the mechanism of inhibition by these specific protease inhibitors. While specific hydrolysis of the peptide bond at the binding site on the inhibitor occurs, especially at low pH (see above), the evidence that this hydrolysis is a key step in the inhibitory process is not convincing. Complexation between inhibitor and inactive derivatives of proteases is often just as tight as with the native protease (Feinstein and Feeney, 1966; Fossum and Whitaker, 1968; Ako et al., 1974). X-Ray crystallographic data appeared to indicate that the complex is probably an adduct with a tetrahedral intermediate state approaching a covalent bond (Sweet et al., 1974; Tschesche, 1974). However, more recent refinements of the X-ray crystallographic maps do not support the earlier interpretation. 13C-NMR studies appear to conclusively rule out formation of a tetrahedral or covalent intermediate in formation of the complex (Hunkapiller et a l . , 1979; Baillargeon et a l . , 1980; Richarz et a l . , 1980). Jibson et al. (1981) have recently shown that a conformational change in trypsin probably does not occur on binding with the Bowman-Birk soybean inhibitor or with chick-pea trypsin inhibitor. A complete explanation for the mutually high specificity of the protease and inhibitor is still not available. d . Nutritional Sign$cance and Medical Importance. The first interest in the proteinase inhibitors was shown by nutritionists concerned with the effect of these inhibitors in feeds and foods on animal and human nutrition. Weanling rats fed raw bean flour, prepared from Phaseolus vulgaris, var. Rosinha G2, all died before completion of a 21 -day feeding experiment (Antunes and Sgarbieri, 1980). It is well known that heat treatment of beans decreases their toxicity and improves their nutritional quality. As shown by removing the protease inhibitors of soybean flour by affinity chromatography, perhaps 40% of the heat improvement (in soybeans) may be the result of destruction of the protease inhibitors (Kakade et al., 1973). However, improvement on heating is also due to destruction of the lectins (see Section IV,A), goitrogenic substances, possibly amylase inhibitors, and enhanced digestibility due to denaturation of the proteins (Kakade et a l . , 1973; Rackis, 1974; Boonvisut and Whitaker, 1976; Savaiano et al., 1977). Cooking of beans in order to destroy the protease inhibitors and lectins is not without its problems because prolonged cooking decreases the digestibility and amino acid availability of beans. There are likely to be still other unaccounted-for compounds in beans which are also responsible for the decrease in nutritional quality.

140

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Cooking beans definitively improves their nutritional quality. When whole soaked beans are heated, the protease inhibitors are destroyed very rapidly (Antunes and Sgarbieri, 1980), but in the isolated form they are quite resistant to heat denaturation as described above. Under some conditions, the protease inhibitors are destroyed much more slowly and inhibitor activity may still be detected after 30-60 min of heating at 100°C (V. C. Sgarbieri, P. L. Antunes, and R. G. Junqueira, unpublished). The reason for this great variability in observed heat stability of the protease inhibitors is not known. Considerable information is available on the physiological response of feeding active protease inhibitors, especially the soybean trypsin inhibitor. Presence of active soybean trypsin inhibitor in the small intestine increases the formation of a humoral pancreazymic-like substance that markedly stimulates external secretion by the pancreas (Khayambashi and Lyman, 1969). Lyman and his colleagues (Green and Lyman, 1972; Lyman et al., 1974; Schneeman and Lyman, 1975) found that the presence of active protease inhibitors in the small intestine increased the secretion of proteases, apparently in an effort to compensate for loss of proteases due to complexation with the inhibitors. It has been proposed (Liener, 1979) that there is a feedback mechanism (Fig. 5) which controls the level of proteases in the small intestine. On continuous feeding of raw soybeans, the pancreas also becomes larger as a result of hyperplasia and hypertrophy of the pancreatic cells (Madar et al., 1976), apparently because it is required to work increasingly harder in its attempt to supply the required level of proteases. There is also the possibility that some of the growth retardation observed on feeding raw bean flour is due to unavailability of sulfur-containing amino acids, especially cystine from trypsin inhibitors (Kakade et al., 1969; Madar et al., 1979). As shown by the data of Table IV, the protease inhibitors have a high cystine content. Most of the other proteins in beans are quite deficient in cystine. The lectins probably also contribute to growth inhibition as described in Section IV,A. CCK

TRYPSINOGEN 4 (pancreas)

\

,b (mucosa) /

.TrI

*/

PROTEOLYSIS

Tr

- TrI

FIG. 5 . Effect of trypsin inhibitor on the biosynthesis and secretion of trypsinogen. CCK, Cholecystokinin; TrI, trypsin inhibitor; Tr, trypsin. From Liener (1979).

PROPERTIES OF COMMON BEAN PROTEINS

141

Most of the nutritional work has been done with soybeans. There is a great deal of additional work needed on beans of the genus Phaseolus. With the availability of methods of purification of the protease inhibitors and of lectins, it will be quite useful to study the effect of incorporation of these proteins into casein-containing diets at approximately the level in which they occur in beans (-0.2-0.3% of the protein for the protease inhibitors and 8-12% for the lectins). V. C. Sgarbieri, P. L. Antunes, and R. G . Junqueira (unpublished) found no effect of addition of isolated Brazilian pink bean trypsin-chymotrypsin inhibitors on rats fed a casein diet (1 % of the casein) as judged by a 5-day nitrogen balance experiment. To our knowledge the effect of feeding the purified protease inhibitor for longer periods has not been properly evaluated. While there is currently great interest in the medical implications and applications of the protease inhibitors found in the various organs and fluid of humans (Whitaker, 1981), essentially nothing is available on the medical importance of the protease inhibitors from beans. 2 . Protein Inhibitors of a-Amylase Proteins that inhibit a-amylases from animals and insects have been reported in a variety of plants. These plants include wheat (Chrzaszcz and Janicki, 1933, 1934; Kneen and Sandstedt, 1943, 1946), rye (Kneen and Sandstedt, 1946), mangos (Mattoo and Modi, 1970), taro root (Narayano Rao et al., 1967, 1970), acorns (Stankovic and Markovic, 196&1961), and beans (Bowman, 1945; Hernandez and Jaffi, 1968). Polypeptide inhibitors of a-amylases are found in Streptomyces (Murao et al., 1979; Namiki et al., 1979; Ueda et al., 1979). In this article, we shall deal only with the amylase inhibitors of legumes, and in particular the common bean. a. Occurrence in Beans. Jaffi et al. (1973) reported the presence of aamylase inhibitory activity in 79 of 95 legume cultivars tested. The most active extracts were from a kidney bean (Phaseolus vulgaris) cultivar. Powers and Whitaker (1977a) measured the a-amylase inhibitor concentration in six varieties of beans with the following results (activity expressed in units per milligram protein): red kidney bean (Phaseolus vulgaris), 41 .O; California white bean (Phaseolus vulgaris), 19.9; cowpea (Vigna sinensis), 16.4; garbanzo bean (Cicer arietinum), 0; Western lima bean (Phaseolus lunatus), 0; and Westley lima bean (Phaseolus lunatus), 0. Marshall and Lauda (1975) also found that the white kidney bean had high a-amylase inhibitor activity. Sgarbieri (1980) measured the a-amylase inhibitory activity of extracts from seeds of 11 species of legume including 26 varieties and cultivars. The inhibitory activity ranged from 7.3 to 4056 unitslg raw flour.

142

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

b. Distribution and Physiological Importance of a-Amylase Inhibitors. The a-amylase inhibitors are concentrated in the cotyledon of the seed. In some preliminary studies, J. R. Powers and J. R. Whitaker (unpublished) were not able to find the inhibitor in the seedlings when measured from the earliest stage of the shoot up to 8 days. In the more mature shoot chlorophyll presented some problems in the assay. Therefore, more definitive studies are needed on the question of whether the amylase inhibitor appears in the vegetative and root parts of the plant. It would be useful to use immunoelectrophoretic techniques in the studies. The physiological role of the a-amylase inhibitors in the bean seeds has not been elucidated. The inhibitors are not active against the a- or P-amylases of the beans (Jaffi et al., 1973; Powers and Whitaker, 1977a) or other higher plant amylases (Jaffi et al., 1973; Powers and Whitaker, 1977a). Powers and Whitaker (1977a) reported that a-amylases from the red kidney bean, malt from two sources, and barley were not inhibited. This is true of a-amylase inhibitors from other plant sources as well (Kneen and Sandstedt, 1946) with the possible exception of the a-amylase inhibitors in corn (Blanco-Labra and Iturbe-Chitias, 1981). The inhibitors are thought not to be inhibitory of microbial a-amylases, although some of the reports are conflicting in this regard (Jaff6 et al., 1973; Marshall and Lauda, 1975; Powers and Whitaker, 1977a). P. E. Granum (personal communication) has found that the rye a-amylase inhibitor actually increases the activity of Bacillus marcesens a-amylase. Powers and Whitaker (1977a) found no inhibition of Bacillus subtilis and Aspergillus oryzae aamylases by red kidney bean amylase inhibitor. The bean amylase inhibitors do inhibit insect larva a-amylases. J. R. Powers (personal communication) has found that all but one insect larva a-amylase tested is inhibited by red kidney bean amylase inhibitor. On this basis, it has been proposed that the physiological role of the a-amylase inhibitor in beans is to protect the seed against insect attack. More work is needed on this point. The a-amylase inhibitors from beans are generally inhibitory of human salivary a-amylase, porcine pancreatic a-amylase, and insect a-amylases but not of higher plant and microbial a-amylases. c. Composition and Physicochemical Properties. Bean a-amylase inhibitors have been purified to homogeneity from the white (Marshall and Lauda, 1975) and red (Powers and Whitaker, 1977a) kidney beans. The two inhibitors are very similar in properties. Hernandez and Jaff6 (1968) have also partially purified an a-amylase inhibitor from kidney beans. The molecular weights of the inhibitors prepared by the three groups were in the range of 45,000-49,OOO. They are glycoproteins. Marshall and Lauda (1975) reported the white kidney bean inhibitor contained 9-10% carbohydrate whereas the red kidney bean had 8.6% carbohydrate (Powers and Whitaker, 1977a). Based on SDS-PAGE, the red kidney bean a-amylase inhibitor appears to be

PROPERTIES OF COMMON BEAN PROTEINS

143

composed of four subunits of three different types. The three polypeptide chains had apparent molecular weights of 15,000-17,000, 12,00&15,000, and 11,000-12,000. By polyacrylamide disc gel isoelectric focusing electrophoresis, the purified red kidney bean a-amylase inhibitor gave a major band at pH 4.65 and a minor band (-5% of the total) at pH 4.5 (Powers and Whitaker, 1977a). The amino acid composition of the red kidney bean a-amylase inhibitor (Powers and Whitaker, 1977a) is quite different from that of the two inhibitors isolated from wheat (Shainkin and Birk, 1970) or the protease inhibitors from beans. In contrast to the protease inhibitors, the red kidney bean a-amylase inhibitor has no proline, has two cystine residues, and is relatively high in tryptophan, tyrosine, valine, and glycine residues. The a-amylase inhibitor of red kidney beans represented 5.6% of the total water-soluble protein of the bean (Powers and Whitaker, 1977a), whereas the protease inhibitors of the Brazilian pink bean were only 0.20% of the total protein (Whitaker and Sgarbieri, 1981). Only a single a-amylase inhibitor was found in white (Marshall and Lauda, 1975) and red (Powers and Whitaker, 1977a) kidney beans, whereas two to six isoinhibitors have been reported in wheat (Shainkin and Birk, 1970; Saunders and Lang, 1973; Silano et al., 1973; O'Donnell and McGeeney, 1976). The red kidney bean a-amylase inhibitor was stable at 60°C for 20 min (Powers and Whitaker, 1977a). It was stable at pH 6.3 to 7.8 for 3 hr at 45°C; however, up to 15 and 42% of the activity was lost at pH 5.1 and 3.5, respectively, after 3 hr at 45°C. The kidney bean a-amylase inhibitors form a 1:l complex with porcine pancreatic a-amylase (Marshall and Lauda, 1975; Powers and Whitaker, 1977a) with a dissociation constant of 3.5 x lo-" M (Powers and Whitaker, 1977b). The rate of inactivation of porcine pancreatic a-amylase by kidney bean aamylase inhibitors is quite slow (Marshall and Lauda, 1975; Powers and Whitaker, 1977b). For 4.46 X 10V8 M enzyme and 2.16 X 1OW8M inhibitor at pH 6.9, it took more than 210 min to reach maximum inhibition, whereas at pH 5 (the pH optimum) under the same conditions the rate was about 10 times faster (Powers and Whitaker, 1977b). The rate of inactivation was markedly affected by ionic strength (p)of the solution, being five times faster at 0.92 than at 0.14 p. The rate of inactivation of a-amylase by the inhibitor was markedly dependent upon temperature of the reaction. At 20°C little inactivation occurred, whereas at 37°C the rate was quite rapid (Marshall and Lauda, 1975; Powers and Whitaker, 1977b). The activation energy for this inactivation was 39.5 kcal/mole (Powers and Whitaker, 1977b). The specific groups involved in recognition between porcine pancreatic aamylase and red kidney bean a-amylase inhibitor and the mechanism of inhibition are not known. Of great interest is the role of the carbohydrate portion of the

144

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

inhibitor given that it inhibits a carbohydrate-splitting enzyme, a-amylase. Periodate oxidation of the red kidney bean a-amylase inhibitor caused complete loss of inhibitor activity (Powers and Whitaker, 1977b). However, E. Wilcox and J. R. Whitaker (unpublished) have enzymatically removed up to 70% of the carbohydrate with no loss of activity. The carbohydrate chains obtained by digesting away the protein portion with a protease do not inhibit a-amylase even at 1000 times the concentration of inhibitor needed for complete inhibition of a-amylase. The a-amylase-red kidney bean a-amylase inhibitor complex has no activity. Yet it can still bind to maltose (Powers and Whitaker, 1977b), a competitive inhibitor of a-amylase, and to Sephadex and to starch (E. Wilcox and J. R. Whitaker, unpublished). These data indicate that a binding site of a-amylase is still available in the complex even though the complex is not catalytically active. At the moment, all the data available indicate that the binding of the inhibitor does not occur at the active site of a-amylase. Rather, it appears that binding occurs somewhere away from the active site and then a slow conformational change occurs (39.5 kcal/mol activation energy) which inactivates the catalytic ability of the enzyme. This hypothesis is supported by the observation that rye aamylase inhibitor actually activates the a-amylase from Bacillus marcesens (P. E. Granum, personal communication). d. Nutritional Significance and Medical Importance. Rather limited work has been done in this area. While the inhibitor is relatively stable, there is no evidence on whether it survives the cooking of beans. When pure red kidney bean a-amylase inhibitor was fed to rats in a casein diet at a level equivalent to that which would be obtained by eating 70 g of raw beans per day per rat, there was no decrease in rate of growth of the rats in relation to the control (Savaiano et al., 1977). On the other hand, J. J. Marshall (personal communication) has reported that the white kidney bean a-amylase inhibitor had a marked effect on the growth of mice. Jaffi and Vega Lette (1968) reported fecal starch from rats fed raw white kidney beans. Lang et al. (1974) reported a reduction of growth rate and increased fecal starch levels when rats were fed on a caseinktarch diet containing purified wheat a-amylase inhibitor.

V.

INFLUENCE OF STORAGE AND PROCESSING ON CHEMICAL AND NUTRITIONAL PROPERTIES OF BEAN PROTEINS

Common beans (Phaseolus vulgaris) are normally harvested with about 20% moisture in the seeds and dried to about 10% water content before storage or consumption. This is done to prevent molding and to decrease quality deterioration during storage. Prior to consumption the beans may be subjected to the following treatments: (1) storage under different environmental conditions; ( 2 )

PROPERTIES OF COMMON BEAN PROTEINS

145

soaking in water or different salt solutions; (3) cooking at normal or increased pressure; and (4) frying after cooking. It is well known that dry beans undergo deterioration during storage, including a decrease in water absorption capacity (hardshell), increase in cooking time (loss of cookability), and alteration in texture, color, and flavor, which lead to a loss of commercial value. These changes have been well documented by several investigators (Dexter et al., 1955; Morris and Wood, 1956; Muneta, 1964; Takayama et al., 1965; Bourne, 1967; Burr et al., 1968; Kon, 1968; Burr, 1973; Antunes and Sgarbieri, 1979). Common observations by the above workers were the following: (1) Under certain conditions of storage certain cultivars of beans develop hardshell which results in failure to rehydrate. This phenomenon is favored by low relative humidity in the storage atmosphere and low water content in the seeds but it also seems to be a characteristic of certain varieties or cultivars. The hardshell can be eliminated or reversed by heat treatment. (2) A hardening or loss of cookability of the cotyledons occurs. This is an irreversible phenomenon and the rate of its development depends primarily on the temperature of storage and the water content of the seeds or the relative humidity of storage atmosphere. High temperature and high humidity both accelerate the hardening. Along with studies on deterioration of commercial value, organoleptic and cooking properties of beans as a function of time, and conditions of storage, a few studies have also considered the loss of nutritive properties of beans stored for various times under different conditions (Molina et al., 1974, 1975, 1976; Antunes and Sgarbieri, 1979). Molina et al. (1974) studied the interrelationship between soaking time, cooking time, and nutritive value for the black bean (Phaseolus vulgaris, var. S-19N) cultivated in Guatemala as a function of time and conditions of storage. They stored the beans for 3 months at 22-25°C and 60-70% relative humidity and also for 6 months at 21°C and 77% relative humidity. Their main findings were as follows: (1) there was an increase in the cooking time (121°C) from 10 to 30 min for both sets of conditions; (2) there was a significant decrease in the protein efficiency ratio (PER) as a result of storage under both sets of conditions, which was proportional to storage time; (3) the solubility of the proteins in water and salt solutions decreased with storage time; (4) they reported an increase of total methionine and of available lysine, with no correlation with the decrease of PER. Antunes and Sgarbieri (1979) studied the influence of storage conditions on dry bean (Phaseolus vulgaris, var. Rosinha G2) for 6 months. The storage conditions were as follows: A, 12°C and 52% relative humidity (RH); B, 25°C and 65% RH; and C, 37°C and 76% RH. The hydration capacity of the beans remained constant under condition A, increased slightly under condition C, and decreased to less than 50% of the original under condition B. Changes in the

146

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

percentages of hardshell beans were inversely proportional to hydration capacity. The texture (hardness) of the cooked beans increased from 200 to 250 kg force for extrusion in the Instron apparatus for conditions A and B and to values greater than 500 kg after 4 months of storage under condition C. Cooking time increased from 60 min to 95, 1 16, and 300 min for the conditions A , B, and C, respectively. The PER dropped from 1.01 to 0.66, 0.43, and 0.10 for storage conditions A, B, and C, respectively. The main cause for the drop in PER was the decrease in availability of sulfur-containing amino acids. Methionine availability dropped from 46.3% to 43.1, 38.2, and 27.6%, whereas cystine availability decreased from 51.6% to 45.8, 43.0, and 30.0% under conditions A, B, and C, respectively. Protein digestibility changed from 62.4% to 58.9, 57.1, and 54.4% for conditions A, B, and C, respectively. Addition of 3% methionine (protein basis) to the beans stored for 6 months under conditions A, B, and C raised the PER from 0.66, 0.43, and 0.10 to the values 2.45, 2.46, and 2.40, respectively, without significantly affecting the protein digestibility. It thus became evident that along with deterioration of commercial, organoleptic, and cooking attributes, prolonged or improper storage of beans can also cause a marked decrease in protein biological value mainly through a decrease in the biological availability of the most limiting essential amino acid, methionine. The influence of storage on chemical and biological alterations of bean protein was studied by Durigan (1979) for Phaseolus vulgaris, var. “mulatinho,” which was stored at 21°C and 71 and 80% RH during a period of 8 months. Protein solubility and digestibility did not change appreciably but available lysine dropped from 8.17 to 4.87 and 4.18%, respectively, for the 71 and 80% RH storage. Methionine availability changed from 42.9 to 22.5 and 22.4% after 4 months and to 19.1 and 18.9% after 8 months of storage at 71 and 80% RH, respectively. The PER changed from 1.27 to 0.66 and 0.18 at 71% RH after 4 and 8 months, respectively. Decrease in biological value of bean protein on storage is associated with the heat treatment necessary to cook the bean after storage and prior to consumption. Cooking of dry beans is necessary not only to tenderize the seed coat and cotyledon and develop acceptable flavor and texture, but also to eliminate toxic factors (Section IV) and to make bean protein more digestible and nutritionally more available. Dry beans are usually prepared for food by soaking in water at ambient temperature for 12-16 hr (overnight) and cooking at atmospheric or higher pressure (pressure cooker) in fresh boiling water, with or without addition of condiments. Cooking time to reach the proper texture depends upon variety, time after harvest, storage history, and other quality-dependent factors. Cooking is a function of temperature and time of heating. In practice two temperatures are used, that of boiling water (- 100°C) or 121°C in the autoclave. Bressani et al. (1963), based on determination of the PER, found 10 to 30 min at 121°C to be the optimum limit for heat treatment of black beans (Phaseolus vulgaris). They

PROPERTIES OF COMMON BEAN PROTEINS

147

also found that cooking 4 hr at 100°C gave the same PER as autoclaving the bean for 30 min. Gomez Brenes ef al. (1973) and La Belle and Hackler (1973) reported 10 min at 121°C to be the best heat treatment for the bean. Antunes and Sgarbieri (1980) studied the effect of heat treatment on elimination of toxicity and changes in the nutritive value of whole beans and of different protein fractions from a Brazilian pink bean (Phaseolus vulgaris, var. Rosinha G 2 ) . When fed to weanling rats the unheated whole flour and six different protein fractions were toxic; all rats died before completing a 21-day feeding experiment. Most of the toxicity was eliminated by heating the water-soaked beans 2.5 min at 97"C, but maximum PER was attained at 10 min at the same temperature. Not all trypsin inhibitor and hemagglutinating activities had been eliminated by the heat treatment for 10 min at 97°C. Autoclaving (15 min, 121°C) decreased the availability of lysine in the whole flour and in the insoluble solids (residue left from extraction) by 36.7 and 29.3%, respectively, whereas it did not affect significantly lysine availability in the isolated protein fractions. However, heating the isolated protein fractions at 121°C for 7.5 min significantly decreased the PER for the rat. The actual mechanisms by which the beans become hard-to-cook and the biological value of the bean proteins is decreased are not yet understood. Bigelow and Fitzgerald (1918) observed that the contact of bean with hard water caused them to become hard to cook and that this hardening effect could be minimized by adding sodium carbonate to the water. Mattson (1946), on studying some biochemical and colloidal properties of peas during cooking, suggested that the cookability properties depended on the valence of cations combined with the pectic material forming the middle lamella of the cotyledon cells. He observed that when the medium contained only monovalent ions, including H , the peas were easily cooked but they became hard to cook when calcium or magnesium was present. Based on the assumption that inorganic salts play an important role in the major constituents of the seed cells (seed coats and cotyledons), Rockland (1964) tested the effect of a variety of compounds on the cooking characteristics of legume seeds. Rockland and Metzler (1967) reported on a process for obtaining quick-cooking beans based on the following: (1) loosening the seed coat by either a brief blanching in steam or boiling water or vacuum infiltration; (2) soaking the whole seed for 1-24 hr, depending upon variety, cultivar, and storage history, at 20°C in dilute solutions of food-grade salts (NaCl, Na,PO,, NaHCO,, and Na,CO,; the composition was optimized for different types of legumes); and (3) draining to remove excess solution. On the basis of chemical, light microscope, scanning electron microscope, and electrophoresis studies of the bean proteins (Rockland and Jones, 1974; Rockland, 1978), it was concluded that the cooking process involves at least four distinct chemical and/or physical changes: (1) partial release of calcium and +

148

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

magnesium into the cooking water; (2) rapid intracellular starch gelatinization; (3) gradual plasticization or partial solubilization of components of the middle lamella and separation of bean cells along the planes of the cell wall without cell rupture; and (4) progressive slow denaturation of proteins. Based on these experimental observations, Rockland and Jones (1974) suggested that the separation of bean cells during cooking may be related to the transposition or removal of divalent cations, particularly calcium and magnesium from bridge positions within the pectinaceous matrix of the middle lamella. According to Rockland and Hahn (1977), the new process did not affect the PER of any of the legume seeds. The storage stability was greatly increased and the processed whole legume required only 10 to 15% of the cooking time required by analogous standard water-soaked seeds.

VI. ADDITIONAL RESEARCH NEEDS Common beans contain from 18 to 35% protein; however, their biological value is much lower than expected on a protein and amino acid composition basis. This low biological value is due to a combination of factors. Common beans are deficient in the sulfur-containing amino acids and methionine is the first limiting essential amino acid. The level and biological availability of methionine is low and decreases even further on storage, with the result that the biological value of bean flour may be essentially zero after 6 months storage at 37°C and 76% relative humidity. Addition of free methionine is effective in markedly improving the biological value of the stored dry bean protein. Extensive research is needed on the genetics of bean proteins specifically with respect to improvement of the methionine content. More data are needed on the methionine biosynthetic pathway in beans. Research is also needed to elucidate the reaction(s) by which methionine becomes biologically inaccessible on storage in order that the commercial storage conditions might be modified as required. Is there an association between the peroxidation of the polyunsaturated fatty acids of beans and the loss of methionine (as methionine sulfone?)? Research on the biological availability of all essential amino acids of bean protein is needed as well as the conditions of storage, processing, and cooking which influence availability. Much basic research will be necessary in order to elucidate the actual chemical and/or biochemical mechanisms (reactions) leading to hardening of the seed cotyledons with time and under certain conditions of storage. Common beans contain a number of antinutritional compounds, including the trypsin-chymotrypsin inhibitors, amylase inhibitors, lectins, cyanogenic glycosides, phytic acid, phenolic compounds, saponins, flatuents, and esterogens. The nutritional significance of each of these compounds is not clear, in part, because

PROPERTIES OF COMMON BEAN PROTEINS

149

most of the rat feeding studies have been done with bean flour. More nutritional studies are needed in which the isolated compounds are fed individually in welldefined casein diets. Even though feeding experiments have been done with the native isolated protease inhibitors, the amylase inhibitors and the lectins, there is still no general agreement about the effect of each of these compounds on the nutritional value of beans. There appears to be little correlation between the protease inhibitor content of various varieties of beans and their nutritional quality. Pure red kidney bean a-amylase inhibitor, fed in massive doses to rats on a starch-containing diet, did not affect significantly the growth rate, whereas feeding pure white kidney bean a-amylase inhibitor resulted in a marked growth retardation in mice. The question remains whether these results were due to differences in the species or in the feeding. Quantitatively, the lectins represent 8-10% of the total protein of beans. Some native lectins are toxic, others are nontoxic when fed to rats. Why? Lectins have been shown to bind to the glycoproteins of the microvilli of the intestinal wall. Does this binding of lectins decrease the absorptive capacity and the ability of the cells of the microvilli to transfer food constituents across the membrane? Do the lectins disrupt the integrity of the cell membranes, permitting intestinal bacteria to leak into the blood and lymph systems? Do the lectins cause a more rapid turnover of the components of the small intestinal membrane, thereby causing additional (energy) stress on the animal? Are the denatured bean lectins still capable of binding to the mucosa cell membrane, causing rupture of membrane microvilli and cell degradation? Feeding of bean-containing diets to laboratory animals often results in increased loss of nitrogen and nucleic acids in the feces and of nitrogen in the urine. Do the increased losses found in the feces come directly from the food consumed or are they of endogenous origin? It is imperative that research be carried out to distinguish between excreta of food origin and of body origin in the feces of animals receiving bean- or bean component-containing diets. Is the increased urinary nitrogen loss a result of a systemic immunological reaction leading to an increased catabolism of body proteins? What are the possible factors (beans constituents) responsible for these observed losses? Is the low digestibility of bean protein related to some inherent property of some of the proteins or to stimuli caused by one or more of the proteins or nonprotein components which then lead to increased loss of endogenous nitrogen? Research results indicate that all of the above events probably occur. However, little is known about the relative importance of each of these factors and how much effect they have on the nutritional and biological values of beans. Much of this work has been done on isolated gut segments or on rats. No work is available on the effect of these lectins on humans. The biological activity of the protease inhibitors, amylase inhibitors, and lectins can be destroyed by proper cooking of beans. However, there is substan-

150

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

tial evidence that residual activity remains in some heat-processed beans and bean products. How much is retained under various cooking regimes in different cultures is not known. The protease inhibitors have markedly different heat stabilities in different varieties of beans for unknown reasons. In some varieties, trypsin inhibitory activity is destroyed in less than 10 min of cooking; in other varieties trypsin inhibitory activity can be detected after 60 min at 100°C. The trypsin inhibitors of whole Brazilian pink beans are completely inactivated by heating at 100°C for 10 min; on the other hand, the inhibitors are more stable in bean flour and the isolated inhibitors retain 100% activity after 15 min at 100°C or when held at pH 2 or 11 for 15 min at 37°C. What is the reason for this difference in stability? Several researchers have shown that some varieties of beans contain substantial amounts of a globulin which is quite resistant to proteolysis, even after heat treatment. What is unique about this globulin? What effect does this globulin, with low levels of protease inhibitory activity, have on the nutritional quality of beans? Should it, or can it, be bred out of beans? Can conditions such as use of reducing reagents increase its heat lability? Much of the definitive work on protease inhibitors of legumes has been on soybeans. Comparatively little is known about the amounts and molecular properties of the protease inhibitors of common beans. In those few cases where the inhibitors have been purified and studied in common beans, the protease inhibitors are of the Bowman-Birk type with substantial amounts of cystine. Although the amino acid composition of the purified inhibitors are similar, in only a few cases have homology studies been made. As a start, it would be useful to compare the tryptic peptide maps of these inhibitors. Why are there great differences in the heat stabilities of the various protease inhibitors? More information is needed on the genetics of the protease inhibitors biosynthesis and whether these inhibitors can be bred out of beans without substantially affecting the sulfur-amino acid composition of the bean. Primary attention has been given to the trypsin inhibitors of beans. More recently, some attention has been given to chymotrypsin inhibitory activity. Do these two activities always reside in the same protein but at different loci? Are there other proteins inhibitory of other types of proteases such as the metallo- and carboxyproteases? Much data indicate that all the toxic compounds in beans have not yet been discovered. The presence of these unidentified substances may be responsible for the lack of correlation often found between biological value and component composition. Bean proteins are not as readily hydrolyzed in vitro by proteases as might be anticipated from the amino acid composition. The reasons for this are not clear and certainly this area is deserving of much more research.

PROPERTIES OF COMMON BEAN PROTEINS

15 1

We do not believe that definitive answers to most of the questions raised above will come through the continued extensive feeding of animals on bean flour from different varieties of beans, certainly not unless there is an associated detailed analysis of the biologically active constituents as well as other constituents of beans. Much effort is expended on improving the yield, pest resistance, drought tolerance, and harvestability through genetic breeding programs. Relatively little research is being done on retention of quality of beans through proper storage conditions, and on improving the methionine content and biological quality of beans, and even less research is being done on elucidating the molecular composition of beans and the ability to modify this composition through genetic and cultural practices. Yet common beans are a major high-protein food for a large portion of the world’s population. We hope this article will stimulate more research in the areas identified above.

REFERENCES Abramova, E. P., and Chemikov, M. P. 1964. Proteinase inhibitor content in the seeds of some leguminous seeds. Vopr. Pitan. 23, 13-16. Ako, H., Foster, R. J., and Ryan, C. A. 1974. Mechanism of action of naturally occurring proteinase inhibitors. Studies with anhydrotrypsin and anhydrochymotrypsin purified by affinity chromatography. Biochemistry 13, 132-139. Allan, D., and Crumpton, M. J. 1971. Fractionation of the phytohemagglutinin of Phaseolus vulgaris by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Biochem. Biophys. Res. Commun. 44, 1143-1 148. Allen, L. W., Svenson, R. H., and Yachnin, S . 1969. Purification of mitogenic proteins derived from Phaseolus vulgaris: Isolation of potent and weak phytohemagglutinin possessing mitogenic activity. Proc. Natl. Acad. Sci. U.S.A. 63, 334-341. Allen, N. K., and Brilliantine, L. 1969. A survey of hemagglutinins in various seeds. J . Immunol. 102, 1295-1299. Ambe, K. S . , and Sohonie, K. 1956. Trypsin inhibitor in plant metabolism. Experientia 12, 302-303. Andrews, A. T. 1974. Navy (haricot) bean (Phaseolus vulgaris) lectin. Isolation and characterization of two components from a toxic agglutinating extract. Biochem. J . 139, 421-429. Anonymous. 1976. Unusual outbreak of food poisoning. Br. Med. J . 2, 1268. Antunes, A. J., and Markakis, P. 1977. Protein supplementation of navy beans with Brazil nuts. J . Agric. Food Chem. 25, 109&1098. Antunes, P. L., and Sgarbieri, V. C. 1979. Influence of time and conditions of storage on technological and nutritional properties of a dry bean (Phaseolus vulgaris, L.) variety Rosinha G2. J . Food Sci. 44, 1703-1706. Antunes, P. L., and Sgarbieri, V. C. 1980. Effect of heat treatment on the toxicity and nutritive value of dry bean (Phaseolus vulgaris var. Rosinha G2) proteins. J . Agric. Food Chem. 28,935-938. Antunes, P. L., Sgarbieri, V. C., and Garmti, R. S. 1979. Nutrification of dry bean (Phaseolus vulgaris L.) by methionine infusion. J . Food Sci. 44, 1302-1305.

152

VALDEMIRO C. SGQRBIERI AND JOHN R. WHITAKER

Aub, J. C . , Tieslau, C . , and Lankester, A. 1963. Reactions of normal and tumor cell surfaces to enzymes, I. Wheat-germ lipase and associated mucopolysaccharides. Proc. Natl. Acad. Sci. U.S.A. 50, 613-619. Aykroyd, W. R., and Doughty, J. 1964. Legumes in human nutrition. FA0 Nutr. Stud. 19, 1-138. Baillargeon, M. W., Laskowski, M., Jr., Neves, D. E., Porubcan, M. A., Santini, R. E., and Markley, J. L. 1980. Soybean trypsin inhibitor (Kunitz) and its complex with trypsin. Carbon-I3 nuclear magnetic resonance studies of the reactive site arginine. Biochemistry 19, 5703-57 10. Baldi, G., and Salamini, F. 1973. Variability of essential amino acid content in seeds of 22 Phaseolus species. Theor. Appl. Genet. 43, 75-78. Banerji, A. P., and Sohonie, K. 1969. Trypsin inhibitor from field beans (Dolichos lablab). Isolation, purification and properties of a trypsin inhibitor from field beans. Enzymologia 36, 137-152. Barbara, J., and Starkey, R. L. 1966. Effect of plant tannins on decomposition of organic substances. Soil Sci. 101, 17-23. Barker, R. D.,Derbyshire, E., Yanvood, A , , and Boulter, D. 1976. Purification and characterization of the major storage proteins of Phaseolus vulgaris seeds, and their intracellular and cotyledonary distribution. Phytochemistry 15, 751-757. Baumgartner, B., and Chrispeels, M. J. 1976. Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans. Plant Physiol. 58, 1-6. Belitz, H.-D., and Fuchs, A. 1972. Complexes of a Phaseolus vulgaris inhibitor with a- and ptrypsin. Chem. Mikrobiol., Technol. Lebensm. 1, 132-134. Belitz, H.-D., and Fuchs, A. 1973. Die reaktiven Zentren des Trypsin-Chymotrypsin-Inhibitors PVI 3 aus Phaseolus vulgarus var. nanus. Z. Lebensm.-(inters. -Forsch. 152, 129-133. Belitz, H.-D., Fuchs, A , , Nitsche, G., and Al-Sultan, T. 1972. Proteinase inhibitors of Phaseolus vulgaris var. nanus: Isolation and comparison with inhibitors of other Phaseolus species. Z. Lebensm.-Linters. -Forsch. 150, 215-220. Bessler, W., and Goldstein, I. J. 1973. Phytohemagglutinin purification: A general method involving affinity and gel chromatography. FEBS Lett. 34, 58-62. Bigelow, W. D., and Fitzgerald, F. F. 1918. Suggestions for canning pork and beans. Natl. Canners’ Assoc., Res. Lab., Bull. 15, 1-31. Birk, Y. 1961. Purification and some properties of a highly active inhibitor of trypsin and achymotrypsin from soybeans. Biochim. Biophys. Acra 54, 378-381. Birk, Y. 1968. Chemistry and nutritional significance of proteinase inhibitors from plant sources. Ann. N . Y . Acad. Sci. 146, 388-399. Birk, Y. 1976a. Proteinase inhibitors from legume seeds. In “Methods in Enzymology” (L. Laszlo, ed.), Vol. 45, Part B, pp. 697-700. Academic Press, New York. Birk, Y. 1976b. Trypsin and chymotrypsin inhibitors from soybeans. In “Methods in Enzymology” (L. Laszlo, ed.), Vol. 45, Part B, pp. 700-707. Academic Press, New York. Birk, Y. 1976c. Trypsin isoinhibitors from garden beans (Phaseolus vulgaris). In “Methods in Enzymology” (L. Laszlo, ed.), Vol. 45, Part B, pp. 710-716. Academic Press, New York. Birk, Y . , Gertler, A., and Khalef, S. 1963. A pure trypsin inhibitor from soya beans. Biochem. J . 87, 281-284. Blanco-Labra, A., and Iturbe-ChiAas, F. A. 1981. Purification and characterization of an a-amylase inhibitor from maize (Zea maize). Food Biochem. 5, 1-17. Bohlool, B. B., and Schmidt, E. L. 1974. Lectins: A possible basis for specificity in the Rhizobiumlegume root nodule symbiosis. Science 185, 269-271. Bollini, R., and Chrispeels, M. J . 1978. Characterization and subcellular localization of vicilin and phytohemagglutinin, the two major reserve proteins of Phaseolus vulgaris L. Planta 142, 29 1-298.

PROPERTIES OF COMMON BEAN PROTEINS

153

Boonvisut, S . , and Whitaker, J. R. 1976. Effect of heat, amylase, and disulfide bond cleavage on the in virro digestibility of soybean proteins. J . Agric. Food Chem. 24, 113Cb1135. Bourne, M. C. 1967. Size, density and hardshell in dry beans. Food Technol. 21, 335-338. Bowden, L., and Lord, J. M. 1976. Similarities in the polypeptide composition of glyoxysomal and endoplasmic reticulum membranes from castor-bean endosperm. Biochem. J . 154, 491499. Bowman, D. E. 1945. Amylase inhibitor of navy beans. Science 102, 358-359. Bowman, D. E. 1946. Differentiation of soy bean antitryptic factors. Proc. SOC.Exp. Biol. Med. 63, 547-550. Boyd, W. C. 1962. “Introduction to Immunochemical Specificity.” Wiley, New York. Boyd, W. C. 1963. The lectins: Their present status. Vox Sang. 8, 1-32. Boyd, W. C., and Shapleigh, E. 1954a. Diagnosis of subgroups of blood groups A and AB by use of plant agglutinins (lectins). J . Lab. Clin. Med. 44, 235-237. Boyd, W. C., and Shapleigh, E. 1954b. Separation of individuals of any blood group into secretors and non-secretors by use of plant agglutinin (lectin). Blood 9, 1195-1198. . relations of blood group antigens as suggested by Boyd, W. C., and Shapleigh, E. 1 9 5 4 ~Antigenic tests with lectins. J . Immunol. 73, 22&231. Boyd, W. C., and Shapleigh, E. 1954d. Specific precipitating activity of plant agglutinins (lectins). Science 119, 419. Boyd, W. C., Shapleigh, E., and McMaster, M. 1955. Immunochemical behavior of a plant agglutinin (lectin). Arch. Biochem. Biophys. 55, 226-234. Bressani, R., Elias, L. G., and Navarette, D. A. 1961. The essential amino acid content of samples of black beans, red beans, rice beans, and cowpeas of Guatemala. J . FoodSci. 26, 525528. Bressani, R., Elias, L. G . , and Valiente, A. T. 1963. Effect of cooking and of amino acid supplementation on the nutritive value of black beans (Phaseolus vulgaris L.). Br. J . Nufr. 17,69-78. Briarty, L. G., Coult, D. A , , and Boulter, D. 1969. Protein bodies of developing seeds of Viciafaba. J . EXP. Bot. 20, 358-372. Briicher, O., Wecksler, M., Levy, A,, Polozzo, A,, and Jaffk, W. G. 1969. Comparison of phytohemagglutinins in wild beans (Phaseolus aborigineus) and in common beans (Phaseolus vulgaris) and their inheritance. Phytochemistry 8, 1739-1743. Burger, M. M., and Goldberg, A. R. 1967. Identification of a tumor-specific determinant on neoplastic cell surfaces. Proc. Natl. Acad. Sci. U.S.A. 57, 359-366. Burr, H. K. 1973. Effect of storage on cooking qualities, processing, and nutritive values of beans. Arch. Latinoam. Nutr . 23, 81-92. Burr, H. K . , Kon, S., and Morris, H. J. 1968. Cooking rates of dry beans as influenced by moisture content and temperature and time of storage. Food Technol. 22, 336-338. Chrzaszcz, T . , and Janicki, J. 1933. “Sisto-amylase,” ein natiirlicher paralysator der amylase. Biochem. 2. 260, 354-368. Chrzaszcz, T., and Janicki, J. 1934. XLI. The inactivation of animal amylase by plant paralysers and the presence of inactivating substances in solutions of animal amylase. Biochem. J . 28, 29&304. Chu, H.-M., and Chi, C.-W. 1963. The isolation and crystallization of two trypsin inhibitors of low molecular weight from mung bean Phaseolus aureus. Acta Biochim. Biophys. Sin. 3, 229-241. Chu, H.-M., Lo, S.-S., Jen, M.-H., Chi, C.-W., and Tsao, T.-C. 1964. Trypsin inhibitor from mung bean. 11. The relation between components A and B and some chemical characteristics of the inhibitor. Acta Biochim. Biophys. Sin. 4, 588-597; Chem. Abstr. 62, 165378 (1965). Cohen, E., ed. 1974. Biomedical perspectives of agglutinins of invertebrate and plant origins. Ann. N . Y . Acad. Sci. 234, 1 4 1 2 . Coulet, M . , Mustier, J., and Guillot, J . 1970. Hemagglutinins of fungi. Rev. Mycol. 35, 71-89. Criddle, R. S. 1969. Structural proteins of chloroplasts and mitochondria. Annu. Rev. Plant Physiol. 20, 23P-252.

154

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Croy, R. R. D. 1977. The localization of the major proteins and proteolytic enzymes in Phaseolus vulgaris, L. at germination. Ph.D. Thesis, University of Aberdeen, Scotland. Dahlgren, K., Porath, J . , and Lindahl-Kiessling, K. 1970. On the purification of phytohemagglutinins from Phaseolus vulgaris seeds. Arch. Biochem. Biophys. 137, 3 0 6 3 14. De Muelenaere, H. J. H. 1965. Toxicity and haemagglutinating activity of legumes. Nature (London) 206, 827-828. Derbyshire, E., and Boulter, D. 1976. Isolation of legumin-like protein from Phaseolus aureus and Phaseolus vulgaris. Phytochemistry 15, 41 1-414. Derbyshire, E., Wright, D. J . , and Boulter, D. 1976. Legumin and vicilin, storage proteins of legume seeds. Phyrochemistry 15, 3-24. Dexter, S. T., Anderson, A. L., Pfahler, P. L., and Benne, E. J. 1955. Responses of white pea beans to various humidities and temperatures of storage. Agron. J . 47, 246-250. Dieckert, J. W., and Dieckert, M. C. 1976. The chemistry and cell biology of vacuolar proteins of seeds. J . Food Sci. 41, 475-482. Durigan, J. F. 1979. Influincia do tempo e das condicBes de estocagem solre as propriedades qenimicas, fisico-mecbnicas e nutritionais do feijio mulatinho (Phaseolus vulgaris, L.). M.S. Thesis, University of Campinas, Sio Paulo, Brazil. El-Hag, N., Haard, N. F., and Morse, R. E. 1978. Influence of sprouting on the digestibility coefficient, trypsin inhibitor and globulin proteins of red kidney beans. J . Food Sci. 43, 1874-1 875. Ericson, M. C., and Chrispeels, M. J. 1973. Isolation and characterization of glucosamine-containing storage glycoproteins from the cotyledons of Phaseolus aureus. Plant Physiol. 52, 98- 104. Etzler, M. E . , and Branstrator, M. L. 1974. Differential localization of cell surface and secretory components in rat intestinal epithelium by use of lectins. J . Cell Biol. 62, 329-343. Evans, R. J., and Bandemer, S. L. 1967. Nutritive value of legume seed proteins. J . Agric. Food Chem. 15,439-443. Evans, R. J., and Bauer, D. H. 1978. Studies of the poor utilization of the rat of methionine and cystine in heated dry bean seed (Phaseolus vulgaris). J . Agric. Food Chem. 26, 779-784. Evans, R. J., Pusztai, A , , Watt, W. B., and Bauer, D. H. 1973. Isolation and properties of protein fractions from navy beans (Phaseolus vulgaris) which inhibit growth of rats. Biochim. Biophys. Acta 303, 175-184. Evans, R. J., Bauer, D. H., Sisak, K. A., and Ryan, P. A. 1974. The availability for the rat of methionine and cystine contained in dry bean seed. J . Agric. Food Chem. 22, 13S133. Feeny, P. P. 1969. Inhibitory effect of oak leaf tannins on the hydrolysis of proteins by trypsin. Phytochemistry 8, 21 19-2126. Feinstein, G., and Feeney, R. E. 1966. Interaction of inactive derivatives of chymotrypsin and trypsin with protein inhibitors. J . Biol. Chem. 241, 5183-5189. Feinstein, G., and Feeney, R. E. 1967. Chemical modification of the methionine residues in turkey ovomucoid. Biochim. Biophys. Acta 140, 55-61, Food and Agriculture Organization/World Health Organization (FAOIWHO). 1973. Energy and protein requirements. W . H . O . Tech. Rep. Ser. 522, 1-118. Fossum, K., and Whitaker, J. R. 1968. Ficin and papain inhibitor from chicken egg white. Arch. Biochem. Biophys. 125, 367-375. Fraenkel-Conrat, H., Bean, R. C., Ducay, E. D., and Olcott, H. S. 1952. Isolation and characterization of a trypsin inhibitor from lima beans. Arch. Biochem. Biophys. 37, 393-407. Fujita, Y . , Oishi, K., and Aida, C. 1974. A novel hemagglutinin produced by Aspergillus niger. J . Biochem. (Tokyo) 76, 1347-1349. Galbraith, W., and Goldstein, I. J. 1970. Phytohemagglutinins: A new class of metalloproteins. Isolation, purification and some properties of the lectin from Phaseolus lunatus. FEBS Lett. 9, 197-20 1.

PROPERTIES OF COMMON BEAN PROTEINS

155

Galbraith, W., and Goldstein, I. J . 1972a. Phytohemagglutinin of the lima bean (Phaseolus lunatus). Isolation, characterization and interaction with type-A blood group substance. Biochemistry 11, 39763984. Galbraith, W., and Goldstein, I. J . 1972b. Lima bean (Phaseolus lunatus) lectin. In “Methods in Enzymology” (V. Ginsburg, ed.), Vol. 28, Part B, pp. 318-323. Academic Press, New York. Gilboa-Garber, N. 1972. Purification and properties of hemagglutinin from Pseudomonas aeruginosa and its reaction with human blood cells. Biochim. Biophys. Actu 273, 165-173. Goddard, V. R., and Mendel, L. B. 1929. Plant hemagglutinins with special reference to a preparation from navy bean (Phaseolus vulgaris). J . Biol. Chem. 82, 447463. Goldstein, I . J . , and Hayes, C. E. 1978. The lectins: Carbohydrate-binding proteins of plants and animals. Adv. Carbohydr. Chem. Biochem. 35, 127-340. Goldstein, I. J., Reichert, C. M., and Misaki, A. 1974. Interaction of concanavalin A with model substrates. Ann. N. Y . Acad. Sci. 234, 283-296. G6mez Brenes, R. A , , Elias, L. G., Molina, M. R., de la Fuente, G., and Bressani, R. 1973. Changes in chemical composition and nutritive value of common beans and other legumes during house cooking. Arch. Latinoam. Nutr. 23, 93-108. Gould, N. R., and Scheinberg, S. L. 1970. Isolation and partial characterization of two anti-A hemagglutinins from P. lunatus. Arch. Biochem. Biophys. 137, 1-1 1. Green, G. M . , and Lyman, R. L. 1972. Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin inhibitor-induced hypersection in rats. Proc. SOC.Exp. Biol. Med. 140, 612. Green, T. R . , and Ryan, C. A. 1972. Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175, 776777. Griebel, C. 1950. Erkrankungen durch Bohnenflocken (Phaseolus vulgaris L.) und Platterbsen (Lathyrus tingitanus L.). 2. Lebensm.-Unters. -Forsch. 90, 191-197. Grob, D. 1950. Proteolytic enzymes. 111. Further studies on protein, polypeptide, and other inhibitors of serum proteinase, leucoproteinase, trypsin and papain. J . Gen. Physiol. 33, 103-124. Hackler, L. R., and Dickson, M. H. 1973. A comparison of the amino acid and nitrogen content of pods and seeds of beans (Phaseolus vulgaris L.). Search 3(5). Hall, T. C., McLeester, R. C., and Bliss, F. A. 1972. Electrophoretic analysis of protein changes during the development of the French bean fruit. Phytochemistry 11, 647-649. Hamblin, J . , and Kent, S. P. 1973. Possible role of phytohaemagglutinin in Phaseolus vulgaris L. Nature (London), New Biol. 245, 28-30. Harms-Ringdahl, M., and Jornvall, H. 1974. Structural studies of a polypeptide that stimulates RNA synthesis: A component obtained from red kidney beans, the source of phytohemagglutinins. Eur. J . Biochem. 48, 541-547. Harms-Ringdahl, M . , Fedorcsak, I., and Ehrenberg, L. 1973. Isolation of substances that stimulate RNA synthesis from red kidney bean: Their activities in lymphocytes and Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 70, 569-573. Haynes, R., and Feeney, R. E. 1967. Fractionation and properties of trypsin and chymotrypsin inhibitors from lima beans. J . Biol. Chem. 242, 5378-5385. Hernandez, A,, and Jaffk, W. G . 1968. Inhibitor of pancreatic amylase from beans (Phaseolus vulgaris). Acta Cient. Venez. 19, 183-185. Hewitt, D., Coates, M. E., Kakade, M. L., and Liener, I. E. 1973. A comparison of fractions prepared from navy (haricot) beans (Phaseolus vulgaris L.) in diets for germ-free and conventional chicks. Br. J . Nutr. 29, 423435. Hoglund, S . , and Dahlgren, K. 1970. On the morphology of phytohemagglutinin from Phaseolus vulgaris seeds. Eur. J . Biochem. 17, 23-26. Honovar, P. M., and Sohonie, K. 1959. Trypsin inhibitor from green gram (Phuseolus aureus). J . Sci. Ind. Res., Sect. C 18, 202-206.

156

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Honovar, P. M., Shih, C. V., and Liener, I. E. 1962. Inhibition of the growth of rats by purified hemagglutinin fractions isolated from Phaseolus vulgaris. J . Nutr. 77, 109-1 14. Hory, H.-D., and Weder, J. K . P. 1976. Trypsin and chymotrypsin inhibitors in leguminosae. VII. Partial amino acid sequence of the trypsin and chymotrypsin inhibitor PCI 3 from Phaseolus coccineus. Z . Lebensm.-Unters. -Forsch. 162, 349-356. Howard, I. K., Sage, H. J., and Horton, C. B. 1972. Studies on the appearance and location of hemagglutinins from a common lentil during the life cycle of the plant. Arch. Biochem. Biophys. 149, 323-326. Howe, M. L., and Barrett, J. T. 1970. Studies on a hemagglutinin from the lichen Parmelia michauxiana. Biochim. Biophys. Acta 215, 97-104. Hungerford, D. A,, Donnelly, A. I . , Nowell, P. C., and Beck, S . 1959. The chromosome constitution of a human phenotypic intersex. Am. J . Hum. Genet. 11, 215-236. Hunkapiller, M. W . , Forgac, M. D., Yu, E. H., and Richards, J. H. 1979. Carbon-13 NMR studies of the binding of soybean trypsin inhibitor to trypsin. Biochem. Biophys. Res. Commun. 87, 25-3 I . Ishino, K., and Ortega, M. L. 1975. Fractionation and characterization of major reserve proteins from seeds of Phaseolus vulgaris. J . Agric. Food Chem. 23, 529-533. Jaffk, W. G. 1950a. The comparative biological value of some legumes of importance in the Venezuelan diet. Arch. Venez. Nutr. 1, 107-126. Jaff6, W. G . 1950b. Protein digestibility and trypsin inhibitor activity of legume seeds. Proc. Soc. Exp. Biol. Med. 75, 219-220. Jaffi, W. G. 1960. Uber phytotoxine aus Bohnen (Phaseolus vulgaris). Arzneim. Forsch. 10, 1012- 1016. Jaff6, W. G. 1962. Blutagglutinierende und toxische Eiweissfraktionen aus Bohnen. Experientia 18, 7677. Jaff6, W. G . 1968. Toxic factors in legumes. Arch. Latinoam. Nutr. 18, 205-218. Jaff6, W. G. 1969. Hemagglutinins. In “Toxic Constituents of Plant Foodstuffs” (I. E. Liener, ed.), pp. 69-101. Academic Press, New York. Jaff6, W. G. 1975. Factors affecting the nutritional value of beans. In “Nutritional Improvement of Food Legumes by Breeding” (M. Milner, ed.), pp, 4 3 4 8 . Protein Advisory Group of the United Nations System, New York. Jaffe, W. G. 1980. Hemagglutinins (lectins). In “Toxic Constituents of Plant Foodstuf$” (1. Liener, ed.), 2nd ed., pp. 73-102. Academic Press, New York. Jaffk, W. G., and Briicher, 0. 1972. Toxicidad y especificidad de diferentes fitohemaglutininas de frijoles (Phaseolus vulgaris). Arch. Latinoam. Nutr. 22, 267-28 1. Jaff6, W. G., and Briicher, 0. 1974. El contenido de nitrbgeno total y aminoicidos azufrados en diferentes lineas de frijoles (Phaseolus vulgaris). Arch. Latinoam. Nutr. 24, 107-1 13. Jaff6, W. G . , and Camejo, G. 1961. The action of a toxic protein, isolated from black beans (Phaseolus vulgaris), on intestinal absorption in rats. Acta Cienr. Venez. 12, 59-61. Jaffe, W. G . , and Gaede, K . 1959. Purification of a toxic haemagglutinin from black beans (P$aseolus vulgaris). Nature (London) 183, 1329-1330. Jaffe, W. G., and Hannig, K. 1965. Fractionation of proteins from kidney beans (Phaseolus vulgaris). Arch. Biochem. Biophys. 109, 8Ck91. Jaff6, W. G., and Vega Lette, C. L. 1968. Heat-labile growth-inhibiting factors in beans (Phaseolus vulgaris). J . Nutr. 94, 203-210. Jaff6, W. G., Planchart, A , , Paez Pumar, J . I., Torrealba, R., and Franceschi, D. N. 1955. New studies on a toxic factor in black beans (Phaseolus vulgaris). Arch. Venez. Nutr. 6 , 195-205. Jaff6, W. G., Briicher, O., and Pallozzo, A. 1972. Detection of four types of specific phytohemagglutinins in different lines of beans (Phaseolus vulgaris). Z . Immunitaetsforch., Exp. Klin. Immunol. 142, 439447.

PROPERTIES OF COMMON BEAN PROTEINS

157

Jaffk, W. G . , Moreno, R., and Wallis, V. 1973. Amylase inhibitors in legume seeds. Nutr. Rep. Int. 7, 169-174. Jamieson, G. A. 1966. Glycoprotein structure of human transfernin and ceruloplasmin. Protides Eiol. Fluids 14, 71-74. Janzen, D. H., Juster, H. B., and Liener, I. E. 1976. Insecticidal action of the phytohemagglutinin in black beans on a bruchid beetle. Science 192, 795-796. Jayne-Williams, D. J . 1973. Influence of dietary jack beans (Canavalia ensiformis) and of concanavalin A on the growth of conventional and qnotobiotic Japanese quail (Coturnixjaponica). Nature (London), New Biol. 243, 15G151. Jayne-Williams, D. J., and Burgess, C.D. 1974. Further observations on the toxicity of navy beans (Phaseolus vulgaris) for Japanese quail (Coturnix japonica). J . Appl. Bacteriol. 37, 149-169. Jibson, M. D., Birk, Y., and Bewley, T. A. 1981. Circular dichroism spectra of trypsin and chymotrypsin complexes with Bowman-Birk or chick pea trypsin inhibitor. Int. J . Pept. Protein Res. 18, 26-32. Jones, G., Moore, S., and Stein, W. H. 1963. Properties of chromatographically purified trypsin inhibitors from lima beans. Biochemistry 2, 66-71, Junqueira, R. G., and Sgarbieri, V . C. 1981. Isolation and general properties of lectins from the bean (Phaseolus vulgaris L., var. Rosinha G2). J . Food Biochem. 5, 165-179. Kaifu, R., and Osawa, T. 1976. Synthesis of O-P-~-galactopyranosyl-(1~4)-0-(2-acetamid0-2deoxy-P-o-glucopyran0syl)-( I+2)-~-mannose and its interaction with various lectins. Carbohydr. Res. 52, 179-185. Kakade, M. L., and Evans, R. J. 1965a. Growth inhibition of rats fed navy bean fractions. J . Agric. Food Chem. 13, 4 5 W 5 4 . Kakade, M. L., and Evans, R. J. 1965b. Nutritive value of navy beans (Phaseolus vulgaris). Br. J . Nutr. 19, 269-276. Kakade, M. L., Arnold, R. L., Liener, I. E., and Waibel, P. E. 1969. Unavailability of cystine from trypsin inhibitors as a factor contributing to the poor nutritive value of navy beans. J . Nutr. 99, 34-42. Kakade, M. L., Hoffa, D. E., and Liener, I. E. 1973. Contribution of trypsin inhibitors to the deleterious effects of unheated soybeans fed to rats. J . Nutr. 103, 1772-1778. Kaplan, L. 1965. Archeology and domestication in American Phaseolus (beans). Econ. Bot. 19, 358-368. Kapoor, A. C., and Gupta, Y. P. 1978. Trypsin inhibitor activity in soybean seed as influenced by stage of its development and different treatments, and the distribution in its anatomical parts. Indian J . Nutr. Diet. 15, 429-433. Kassell, B. 1970. Trypsin and chymotrypsin inhibitors from soybeans. In “Methods in Enzymology” (G. E. Perlmann and L. Lorand, eds.), Vol. 19, pp. 853-862. Kelly, J. F. 1971. Genetic variation in the methionine levels of mature seeds of common beans (Phaseolus vulgaris L.). J . Am. SOC. Hortic. Sci. 96, 561-563. Kelly, J. F. 1975. Increasing protein quantity and quality. In “Nutritional Improvement of Food Legumes by Breeding” (M. Milner, ed.), pp. 179-184. Protein Advisory Group of the United Nations System, New York. Khalap, S . , Thompson, T. E., and Gold, E. R. 1970. Haemagglutination and haemagglutinationinhibition reactions of extracts from snails and sponges. Vox Sung. 18, 501-526. Khayambashi, H., and Lyman, R. L. 1969. Secretion of rat pancreas perfused with plasma from rats fed soybean trypsin inhibitor. Am. J . Physiol. 217, 6 4 6 6 5 1. King, K. W. 1964. Development of all plant food mixture using crops indigenous to Haiti: Amino acid composition and protein quality. Econ. Bot. 18, 31 1-322. Kneen, E., and Sandstedt, R. M. 1943. An amylase inhibitor from certain cereals. J . Am. Chem. SOC.65, 1247.

158

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Kneen, E., and Sandstedt, R. M. 1946. Distribution and general properties of an amylase inhibitor in cereals. Arch. Biochem. Biophys. 9, 235-249. Koide, T., and Ikenaka, T. 1973. Studies on soybean trypsin inhibitors. 3. Amino-acid sequence of the carboxyl-terminal region and the complete amino-acid sequence of soybean trypsin inhibitor (Kunitz). Eur. J . Biochem. 32, 417431. Kon, S. 1968. Pectic substances of dry beans and their possible correlation with cooking time. J . Food Sci. 33, 437-438. Kornfeld, R., and Kornfeld, S . 1970. The structure of a phytohemagglutinin receptor site from human erythrocytes. J . Biol. Chem. 245, 2536-2545. Kornfeld, R., and Kornfeld, S. 1974. Structure of membrane receptors for plant lectins. Ann. N. Y . Acad. Sci. 234, 276282. Kornfeld, R., Keller, J., Baenziger, J., and Kornfeld, S . 1971. The structure of the glycopeptide of human yG myeloma proteins. J . Biol. Chem. 246, 3259-3268. Kornfeld, R., Gregory, W. T., and Komfeld, S. A. 1972. Red kidney bean (Phaseolus vulgaris) phytohemagglutinin. In “Methods in Enzymology” (V. Ginsburg, ed.), Vol. 28, Part B, pp. 344-349. Kornfeld, S . , and Kornfeld, R. 1969. Solubilization and partial characterization of a phytohemagglutinin receptor site from human erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 63, 1439-1446. Korte, R. 1972. Heat resistance of phytohemagglutinins in weaning food mixtures containing beans (Phaseolus vulgaris). Ecol. Food Nutr. 1, 303-307. Kortt, A. A. 1981. Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus L.). Biochim. Biophys. Acta 657, 212-221. Kothbauer, H., and Schenkel-Brunner, H. 1975. Hemagglutinins in fish eggs: Comparative studies on different Salmonidae species. Comp. Biochem. Physiol. A . 50A, 27-29. Kozlowska, H., Borowski, J., and Elkowicz, K. 1976. Correlation between the activity of trypsin inhibitors and the protein content in soybean products. Zesz. Nauk. Akad. Ro1n.-Tech. Olsztynie, Tecnol. Zywn. 10, 47-52; Chem. Abstr. 88, 5001C (1978). Krahn, J., and Stevens, F. C. 1970. Lima bean trypsin inhibitor. Limited proteolysis by trypsin and chymotrypsin. Biochemistry 9, 26462652. Krahn, J., and Stevens, F. C. 1971. Stoichiometry of the interaction of lima bean protease inhibitor with trypsin and (or) chymotrypsin. FEES Left. 13, 339-341. Kriipe, M. 1956. “Blutgruppenspezifische Pflanzliche Eiweisskorper (Phytagglutinine).” Enke, Stuttgart. Kunitz, M. 1946. Crystalline soybean trypsin inhibitor. J . Gen. Physiol. 29, 149-154. La Belle, R. L., and Hackler, L. R. 1973. Preparation and utilization of dry, canned and pre-cooked beans. Arch. Latinoam. Nutr. 23, 109-120. Lang, J. A,, Chang-hum, L. E., Reyes, P. S . , and Briggs, G. M. 1974. Interference of starch metabolism by a-amylase inhibitors. Fed. Proc., Fed. Am. SOC.Exp. B i d . 33, 718 (Abstr.). Laskowski, M., Jr., and Kato, I. 1980. Protein inhibitors of proteinases. Annu. Rev. Biochem. 49, 593-626. Leseney, A. M., Bourrillon, R . , and Kornfeld, S. 1972. The nature of the erythrocyte receptor site for Robinia pseudoaccacia phytohemagglutinin. Arch. Biochem. Biophys. 153, 83 1-836. Levine, P., Celano, M. J., Lange, S . , and Berliner, V. 1957. On anti-M in horse sera. VoxSang. 2, 433439. Liener, I. E. 1962. Toxic factors in edible legumes and their elimination. Am. J . Clin. Nurr. 11, 28 1-298. Liener, I. E. 1974. Phytohemagglutinins: Their nutritional significance. J . Agric. Food Chem. 22, 17-22. Liener, I. E. 1976. Phytohemagglutinins (phytolectins). Annu. Rev. Plant Physiol. 27, 291-319. Liener, I. E. 1979. Significance for humans of biologically active factors in soybeans and other food legumes. J . Am. Oil Chem. SOC. 56, 121-129.

PROPERTIES O F COMMON BEAN PROTEINS

159

Liener, I. E., and Thompson, R. M. 1980. In vitro and in vivo studies on the digestibility of the major storage protein of the navy bean (Phaseolus vulgaris). Qual. Plant.-Plant Foods Hum. Nutr. 30, 13-25. Lima, A. L., Mancini Filho, J., Domingues, J. B., and Lajolo, F. M. 1980. Propriedades hemaglutinantes, mitogCnica e t6xica de variedades Brasileiras de feijdes (Phaseolus vulgaris, L.). Rev. Farm. Bioquim. Univ. Sao Paulo 16, 145-154. Lis, H., and Sharon, N. 1973. The biochemistry of plant lectins (phytohemagglutinins). Annu. Rev. Biochem. 42, 541-574. Luning, O., and Bartels, W. 1926. Uber die Giftigkeit der Weissen Bohnen. Z . (Inters. Lebensm. 51, 220-228. Lyman, R. L., Olds, B. A,, and Green, G. M. 1974. Chymotrypsinogen in the intestine of rats fed soybean trypsin inhibitor and its inability to suppress pancreatic enzyme secretions. J . Nutr. 104, 105-110. McLeester, R. C., Hall, T. C., Sun, S. M., and Bliss, F. A. 1973. Comparison of globulin proteins from Phaseolus vulgaris with those from Viciafaba. Phytochemistry 12, 85-93. Madar, Z., Tencer, Y., Gertler, A,, and Birk, Y. 1976. The comparative effect of prolonged feeding with raw and heated soybean meal on the growth response, pancreatic enlargement and pancreatic enzymes of chicks. Nutr. Metab. 20, 234-242. Madar, Z., Gertler, A , , and Birk, Y. 1979. The fate of the Bowman-Birk trypsin inhibitor from soybeans in the digestive tract of chicks. Comp. Biochem. Physiol. A 62A, 1057-1061. Makela, 0. 1957. Studies in hemagglutinins of leguminosae seeds. Ann. Med. Exp. Biol. Fenn. 35, Suppl. 1 1 , 1-156. Marquardt, R. R., Ward, A. T., Campbell, L. D., and Cansfield, P. E. 1977. Purification, identification and characterization of growth inhibitor in faba beans (Vicia faba, L. var. minor). J . Nutr. 107, 1313-1324. Marshall, J. J., and Lauda, C. M. 1975. Purification and properties of phaseolamin, an inhibitor of a-amylase from the kidney bean Phaseolus vulgaris. J . Biol. Chem. 250, 803&8037. Mattoo, A. K., and Modi, V. V. 1970. Partial purification and properties of enzyme inhibitors from unripe mangoes. Enzymologia 39, 237-247. Mattson, S. 1946. The cookability of yellow peas. Acta Agron. Suecana 2, 185-231. Mialonier, G., Privat, J. P., Monsigny, M., Kahlen, G . , and Durand, R. 1973. Isolement, proprittks physico-chimiques et localisation in vivo d’une phytohimagglutine (lectine) de Phaseolus vulgaris L. (var. rouge). Physiol. Veg. 11, 519-537. Miller, J . B., Noyes, C., Heinrikson, R., Kingdon, H. S . , and Yachnin, S. 1973. Phytohemagglutinin mitogenic proteins. J . Exp. Med. 138, 939-951. Miller, J. B., Hsu, R., Heinrikson, R., and Yachnin, S. 1975. Extensive homology between the subunits of the phytohemagglutinin mitogenic proteins derived from Phaseolus vulgaris. Proc. Natl. Acad. Sci. U.S.A. 72, 1388-1391. Millerd, A,, and Spencer, D. 1974. Changes in RNA-synthesizing activity and template activity in nuclei from cotyledons of developing pea seeds. Aust. J . Plant Physiol. 1, 331-341. Millerd, A,, Simon, M., and Stern, H. 1971. Legumin synthesis in developing cotyledons of Vicia faba L. Plant Physiol. 48, 419425. Molina, M. R., de la Fuente, G., and Bressani, R. 1974. Interrelaciones entre tiempo de remojo, tiempo de cocci6n, valor nutritivo y otras caracteristicas del frijol (Phaseolus vulgaris). Arch. Latinoam. Nutr. 24, 469483. Molina, M. R., de la Fuente, G., and Bressani, R. 1975. Interrelationships between storage, soaking time, cooking time, nutritive value and other characteristics of the black bean (Phaseolus vulgaris). J . Food Sci. 40, 587-591. Molina, M. R., Baten, M. A., Gdmez Brenes, R. A., King, K. W., and Bressani, R. 1976. Heat treatment: A process to control the development of the hard-to-cook phenomenon in black beans (Phaseolus vulgaris). J . Food Sci. 41, 661-666.

160

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Moraes, R. M., and Angelucci, E. 1971. Chemical composition and amino acid contents of Brazilian beans (Phaseolus vulgaris). J. Food Sci. 36, 493494. Moraes e Santos, T., and Dutra de Oliveira, J . E. 1972. Valor nutrivo de fracdes proteicas, isoladas de freijio (Phaseolus vulgaris). Arch. Latinoam. Nutr. 22, 547-560. Moreira, R. A,, and Perrone, 1. C. 1977. Purification and partial characterization of a lectin from Phaseolus vulgaris. Plant Physiol. 59, 783-787. Morgan, W. T. J., and Watkins, W. M. 1956. The product of the human blood group A and B genes in individuals belonging to group AB. Nature (London) 177, 521-522. Moms, H. J., and Wood, E. R. 1956. Influence of moisture content on keeping quality of dry beans. Food Technol. 10, 225-229. Muneta, P. 1964. The cooking time of dry beans after extended storage. Food Technol. 18, 12461241, Murao, S . , Ohyama, K . , Murai, H., Gotoo, A., Matsui, Y., Fukuhara, K., Miyata, S . , Sumida, M., and Arai, M. 1979. Amylase inhibitors from Streptomyces species. J . Jpn. SOC. Srarch Sci. 26, 157-1 64. Namiki, S . , Kangouri, K., Nagate, T., Sugita, K., Hara, H., Mori, E., Ohmura, S., and Ohzehi, M. 1979. Studies on the amylase inhibitors from Streptomyces calvus TM-521 and their physiological activities. J . Jpn. SOC. Starch Sci. 26, 134-144. Narayano Rao, M . , Shurpalekar, K. S., and Sundaravalli, 0. E. 1967. An amylase inhibitor in Colocasia esculenta. Indian J . Biochem. 4, 185. Narayano Rao, M . , Shurpalekar, K. S . , and Sundaravalli, 0. E. 1970. Purification and properties of an amylase inhibitor from colocasia (Colocasia esculenta) tubers. Indian J . Biochem. 7, 24 1-243. Nelson, T. S . , Stephenson, E. L., Burgos, A,, Floyd, J . , and York, J. 0. 1975. Effect of tannin content and dry matter digestion on energy utilization and average amino acid availability of hybrid sorghum grains. Poult. Sci. 54, 1620-1623. Nordman, C. T., de la Chapelle, A,, and Grasbeck, R. 1964. The interrelations of erythroagglutinating, leucoagglutinating and leucocyte-mitogenic activities in Phaseolus vulgaris phytohaemagglutinin. Acta Med. Scand., Suppl. 412, 49-58. Nowakovl, N., and Kocourek, J. 1974. Studies on phytohemagglutinins. XX. Isolation and characterization of hemagglutinins from scarlet runner seeds (Phaseolus coccineus). Biochim. Biophys. Acta 359, 326333. Nowell, P. C. 1960a. Differentiation of human leukemic leucocytes in tissue culture. Exp. Cell Res. 19, 267-277. Nowell, P. C. 1960b. Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res. 20, 4 6 2 4 6 6 . Nungester, W. J., and Van Halsema, G. 1953. Reaction of certain phytoagglutinins with FlexnerJobling carcinoma cells of the rat. Proc. SOC. Exp. Biol. Med. 83, 863-866. Odani, S . , and Ikenaka, T. 1972. Studies on soybean trypsin inhibitors. IV. Complete amino acid sequence and the antiproteinase sites of Bowman-Birk soybean proteinase inhibitor. J . Biochem. (Tokyo) 71, 839-848. Odani, S., and Ikenaka, T. 1973. Scission of soybean Bowman-Birk proteinase inhibitor into two small fragments having either trypsin or chymotrypsin inhibitory activity. J. Biochem. (Tokyo) 74, 857-860. Odani, S., and Ikenaka, T. 1976. The amino acid sequences of two soybean double headed proteinase inhibitors and evolutionary consideration on the legume proteinase inhibitors. J . Biochem. (Tokyo) 80, 641-643. Odani, S . , and Ikenaka, T. 1978. Studies on soybean inhibitors. XII. Linear sequences of two soybean double-headed trypsin inhibitors, D-I1 and E-I. J . Biochem. (Tokyo) 83, 737-745.

PROPERTIES OF COMMON BEAN PROTEINS

161

O’Donnell, M. D., and McGeeney, K. F. 1976. Purification and properties of an a-amylase inhibitor from wheat. Biochim. Biophys. Acta 422, 159-169. Oh, Y.H . , and Conard, R. A. 1972. Further studies on mitogenic components of Phuseolus vulgaris phytohemagglutinin: Subunit structure. Arch. Biochem. Biophys. 152, 631-637. Opik, H. 1966. Changes in cell fine structure in the cotyledons of Phaseolus vulgaris, L. during germination. J . Exp. Bot. 17, 427439. Opik, H. 1968. Development of cotyledon cell structure in ripening Phaseolus vulgaris seeds. J . Exp. Bot. 19, 64-76. Osborne, T. B. 1894. The proteids of the kidney bean (Phaseolus vulgaris). J . Am. Chem. SOC. 16, 633-643, 703-712, 757-764. Ozawa, K., and Laskowski, M., Jr. 1966. The reactive site of trypsin inhibitors. J . Biol. Chem. 241, 3955-3961. Palmer, R., McIntosh, A,, and Pusztai, A. 1973. The nutritional evaluation of kidney beans (Phaseolus vulgaris). The effect on nutritional value of seed germination and changes in trypsin inhibitor content. J . Sci. Food Agric. 24, 937-944. Palozzo, A,, and Jaff6, W. G. 1969. Immunoelectrophoretic studies with bean proteins. Phytochemistry 8, 1255-1258. Powers, J. R., and Whitaker, J. R. 1977a. Purification and some physical and chemical properties of red kidney bean (Phaseolus vulgaris) a-amylase inhibitor. J . Food Biochem. 1, 217-238. Powers, J . R., and Whitaker, J. R. 1977b. Effect of several experimental parameters on combination of red kidney bean ( Phaseolus vulgaris) a-amylase inhibitor with porcine pancreatic a-amylase. J . Food Biochem. 1, 239-260. Pusztai, A. 1965. A study on the glycosamine containing constituents of the seeds of kidney beans (Phaseolus vulgaris). Biochem. 1.94, 604-610. Pusztai, A. 1966. The isolation of two proteins, glycoprotein I and a trypsin inhibitor, from the seeds of kidney bean (Phaseolus vulgaris). Biochem. J . 101, 379-384. Pusztai, A. 1967. Studies on the extraction of nitrogenous and phosphorus-containing materials from the seeds of kidney beans (Phaseolus vulgaris). Biochem. J . 94, 61 1-616. Pusztai, A. 1968. General properties of a protease inhibitor from the seeds of kidney beans. Eur. J . Biochem. 5, 252-259. Pusztai, A , , and Palmer, R. 1977. Nutritional evaluation of kidney beans (Phaseolus vulgaris):The toxic principle. J . Sci. Food Agric. 28, 62G623. Pusztai, A,, and Stewart, J.C. 1978. Isolectins of Phaseolus vulgaris: Physicochemical studies. Biochim. Biophys. Acta 536, 3 8 4 9 . Pusztai, A,, and Watt, W. B. 1970. Glycoprotein 11. The isolation and characterization of a major antigenic and non-hemagglutinating glycoprotein from Phaseolus vulgaris. Biochim. Biophys. Acta 207, 413431. Pusztai, A,, and Watt, W. B. 1974. Isolectins of Phaseolus vulgaris, a comprehensive study of fractionation. Biochim. Biophys. Acta 365, 57-71. Pusztai, A., Grant, G., and Palmer, R. 1975. Nutritional evaluation of kidney beans (Phaseolus vulgaris):The isolation and partial characterization of toxic constituents. J . Sci. Food Agric. 26, 149-1 56. Pusztai, A,, Stewart, J . C., and Watt, W. B. 1978. A novel method for the preparation of protein bodies by filtration in high (over 70%, w/v) sucrose containing media. Plant Sci. Lett. 12,9-15. Pusztai, A,, Clarke, E. M. W . , and King, T. P. 1979a. The nutritional toxicity of Phaseolus vulgaris lectins. Proc. Nutr. SOC. 38, 115-120. Pusztai, A,, Clarke, E. M. W., King, T. P., and Stewart, J. C. 1979b. Nutritional evaluation of kidney beans (Phaseolus vulgaris): Chemical composition, lectin content and nutritional value of selected cultivars. J . Sci. Food Agric. 30, 843-848.

162

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Pusztai, A., Croy, R. R. D., Stewart, J. S . , and Watt, W. B. 1979c. Protein body membranes of Phaseolus vulgaris cultivar processor cotyledons: Isolation and preliminary characterization of constituent proteins. New Phyrol. 83, 371-378. Rackis, J. J. 1974. Biological and physiological factors in soybeans. J . Am. Oil Chem. Soc. 51, 161A-1 74A. Racusen, D., and Foote, M. 1971. The major glycoprotein in germinating bean seeds. Can. J . Bor. 49, 2107-2111. Ramachandra, G . , Virupaksha, T. K., and Shadaksharaswamy, M. 1977. Relationship between tannin levels and in virro protein digestibility in finger millet (Eleusine coracana Gaertn.). J . Agric. Food Chem. 25, 1101-1 104. Rasanen, V., Weber, T. H., and Grasbeck, P. 1973. Crystalline kidney bean leucoagglutinin. Eur. J . Biochem. 38, 193-200. Rattray, E. A. S., Palmer, R., and Pusztai, A. 1974. Toxicity of kidney beans (Phaseolus vulgaris L.) to conventional and gnotobiotic rats. J . Sci. Food Agr. 25, 1035-1040. Richardson, M. 1977. The proteinase inhibitors of plants and micro-organisms. Phytochemistry 16, 159- 169. Richarz, R., Tschesche, H., and Wiithrich, K . 1980. Carbon-I3 nuclear magnetic resonance studies of the selectively isotope-labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin. Biochemistry 19, 571 1-5715. Rigas, D. A , , and Osgood, E. E. 1955. Purification and properties of the phytohemagglutinin of Phaseolus vulgaris. J . Biol. Chem. 212, 607-615. Rigas, D. A,, Head, C., and Eginitis-Rigas, C. 1972. Biophysical characterization of a mitogenic erythro- and lympho-agglutinating phytohemagglutinin (PPHA) of Phaseolus vulgaris. Physiol. Chem. Phys. 4, 153-165. Roberts, T. K., and Boursnell, J. C. 1974. The inhibitory action of phosphatidyl compounds on boar seminal haemagglutinin. J . Reprod. Ferril. 41, 489492. Rockland, L. B. 1964. Effects of hydration in salt solutions on cooking rates of lima beans. U . S., Agric. Res. Sent., ARS ARS 74-32. Rockland, L. B. 1978. Relationship between fine structure and composition and development of new food products from legumes. In “Postharvest Biology and Biotechnology” (H. 0. Hultin and M. Milner, eds.), Chapter 11. Food and Nutrition Press, Westport, Connecticut. Rockland, L. B., and Hahn, D. M. 1977. Quick-cooking grain legumes. An alternative food processing technology. Grain Legume Process., Pap. FAOIUNEP Expert Consult., 1977. Rockland, L. B., and Jones, F. T. 1974. Scanning electron microscope studies on dry beans. Effects of cooking on the cellular structure of cotyledons in rehydrated large lima beans. J . Food Sci. 39, 342-346. Rockland, L. B., and Metzler, E. A. 1967. Quick-cooking lima and other dry beans. Food Technol. 21, 344-348. Romero, J., and Ryan, D. S. 1978. Susceptibility of the major storage protein of the bean, Phaseolus vulgaris L., to in virro enzymatic hydrolysis. J . Agric. Food Chem. 26, 784788. Romero, J., Sun, S. M., McLeester, R. C., Bliss, F. A,, and Hall, T. C. 1975. Heritable variation in a polypeptide subunit of the major storage protein of the bean, Phaseolus vulgaris L. Plant Physiol. 56, 7 7 6 7 7 9 . Rougk, P. 1974a. Etude de la phytohkmagglutinine de graines de lentille au cours de la germination et des premiers stades du dCveloppement de la plante. Evolution dans le cotylCdons. C. R. Hebd. Seances Acad. Sci., Ser. D 278, 449452. RougC, P. 1974b. Etude de la phytohkmagglutinine des graines de lentille au cours de la germination et des premiers stades du dkvelopment de la plante. Evolution dans les racines, les tiges et les feuilles. C. R. Hebd. Seances Acad. Sci., Ser. D 278, 3083-3086.

PROPERTIES OF COMMON BEAN PROTEINS

163

Ryan, C. A. 1973. Proteolytic enzymes and their inhibitors in plants. Annu. Rev. Plant Physiol. 24, 173-196. Saint-Paul, M. 1961. Les hhagglutinines vCgCtales. Transfusion 4, 3-37. Saint-Paul, M., Douglas-Le Bourdelles, F., and Fine, J. M. 1956. Contribution 1’Ctude biochimique de I’hemagglutinine de Phaseolus vulgaris. C. R. Hebd. Seances Soc. Biol. Ses Fil. 150, 1742- 1747. Saunders, R . M., and Lang, J. A. 1973. a-Amylase inhibitors in Triticum aestivum: Purification and physical chemical properties. Phytochemistry 12, 1237-1241. Savaiano, D. A., Powers, J. R., Costello, M. J., Whitaker, J. R., and Clifford, A. J. 1977. The effect of an a-amylase inhibitor on the growth rate of weanling rats. Nun. Rep. Int. 15, 443449. Schneeman, B. O., and Lyman, R. L. 1975. Factors involved in the intestinal feedback regulation of pancreatic enzyme secretion in the rat. Proc. Soc. Exp. Biol. Med. 148, 897-903. Schneider, E. C. 1911. The haemagglutinating and precipitating properties of the bean (Phaseolus). J . Biol. Chem. 11, 47-59. Schwarzfischer, V. F., and Liebrich, K. 1962. Populationsgenetische untersuchungen iiber das Blutkorperchenmerkmal Gy. Blut 8, 161-166. Scott, M. T. 1972. Partial characterization of the hemagglutinating activity in hemolymph of the American cockroach (Periplaneta americana). J . Invertebr. Pathol. 19, 6&7 1. Seeger, R., and Weidmann, R. 1972. Zum Vorkommen von Hamolysinen und Agglutininen in hoheren Pilzen (Basidiomyceten). Arch. Toxikol. 29, 189-217. Seidl, D., Jaff6, M., and Jaff6, W. G. 1969. Digestibility and proteinase inhibitory action of a kidney bean globulin. J . Agric. Food Chem. 17, 1318-1321. Sela, B. A,, Lis, H., Sharon, N., and Sachs, L. 1973. Isolectins from wax bean with differential agglutination of normal and transformed mammalian cells. Biochim. Biophys. Acta 310, 273-277. Sgarbieri, V. C. 1980. Estudo do conteudo e de algumas caracteristicas das proteinas de sementes de plantas leguminosas. Cienc. Cult. (Sao Paulo) 32, 78-84. Sgarbieri, V. C., and Whitaker, J. R. 1981. Partial characterization of trypsin-chymotrypsin inhibitors from bean (Phaseolus vulgaris L. var. Rosinha (32): Chemical and physical properties. J . Food Biochem. 5, 215-232. Sgarbieri, V. C., Antunes, P. L., and Almeida, L. D. 1979. Nutritional evaluation of four varieties of dry beans (Phaseolus vulgaris, L.). 1. Food Sci. 44, 130&1308. Shainkin, R., and Birk, Y. 1970. a-Amylase inhibitors from wheat: Isolation and characterization. Biochim. Biophys. Acta 221, 502-5 13. Sharon, N., and Lis, H. 1972. Lectins: Cell-agglutinating and sugar-specific proteins. Science 177, 949-959. Silano, V., Pocchiari, F., and Kasarda, D. D. 1973. Physical characterization of a-amylase inhibitors from wheat. Biochim. Biophys. Acta 317, 139-148. Smirnoff, P., Khalef, S . , Birk, Y., and Applebaum, S. W. 1979. Trypsin and chymotrypsin inhibitor from chick peas. Int. J . Pept. Protein Res. 14, 18&192. Smith, D. L. 1973. Nucleic acid, protein and starch synthesis in developing cotyledons of Pisum arvensel. Ann. Bot. (London) [N.S.] 37, 795-804. Sohonie, K., and Ambe, K. S . 1955. Crystalline trypsin inhibitors from the Indian field bean and the double bean. Nature (London) 175, 508-509. Southworth, D. 1975. Lectins stimulate pollen germination. Nature (London) 258, 600-602. Spiro, R. G. 1964. Periodate oxidation of the glycoprotein fetuin. J . Biol. Chem. 239, 567-573. Stankovic, S . C., and Markovic, N. D. 1960-1961. A study of amylase inhibitors in the acorn. Glas. Hem. Drus. Beograd 25-26, 51%525; Chem. Abstr. 59, 3084d (1963).

164

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Stanton, W. R., Doughty, J., Orraca-Tetteh, R., and Steele, W. 1966. “Grain Legumes in Africa.” Food and Agriculture Organization of the United Nations (FAO), Rome. Stead, R. H., de Muelenaere, H. J. H., and Quicke, C. V. 1966. Trypsin inhibition, hemagglutination, and intraperitoneal toxicity in extracts of Phaseolus vulgaris and Glycine m a . Arch. Biochem. Biophys. 113, 703-708. Stevens, F. C., Wuerz, S . , and Krahn, J. 1974. Structure-function relationships in lima bean protease inhibitor. Buyer-Symp. 5, 344-354. Stockert, R. J., Morrell, A. G . , and Scheinberg, I. H. 1974. Mammalian hysatic lectin. Science 186, 365-366. Sun, S. M., and Hall, T. C. 1975. Solubility characteristics of globulins from Phaseolus seeds in regard to their isolation and characterization. J . Agric. Food Chem. 23, 184189. Sun, S. M., McLeester, R. C., Bliss, F. A., and Hall, T. C. 1974. Reversible and irreversible dissociation of globulins from Phaseolus vulgaris seed. J . Biol. Chem. 249, 21 1&2121. Sun, S. M., Mutschler, M. A., Bliss, F. A., and Hall, T. C. 1978. Protein synthesis and accumulation in bean cotyledons during growth. Plant Physiol. 61, 918-923. Susplugas, J., and Coulet, M. 1954. “Repartition de I’hCmagglutinine du Phaseolus vulgaris, L., spCcialement au moment de la germination.” SOC.Pharm., Montpellier. Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H., and Blow, D. M. 1974. Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6-A resolution. Biochemistry 13, 42124228. Takahashi, T., and Liener, I. E. 1968. Isolation and composition of a glycopeptide from a phytohemagglutinin of Phaseolus vulgaris. Biochim. Biophys. Acta 154, 56CL564. Takahashi, T., Ramachandramurthy, P., and Liener, I. E. 1967. Some physical and chemical properties of a phytohemagglutinin isolated from Phaseolus vulgaris. Biochim. Biophys. Actu 133, 123-133. Takayama, K. K., Muneta, P., and Wiese, A. C. 1965. Lipid composition of dry beans and its correlation with cooking time. J . Agric. Food Chem. 13, 269-272. Tandon, 0. B., Bressani, R., Schrimshaw, N. S . , and Le Beau, F. 1957. Nutritive value of beans. Nutrients in Central American beans. J . Agric. Food Chem. 5, 137-142. Tawde, S. 1961. Isolation and partial characterization of Cajanus cajon trypsin inhibitor. Ann. Biochem. Exp. Med. 21, 359-366. Thomber, J. P. 1975. Chlorophyll-proteins: Light harvesting and reaction center components of plants. Annu. Rev. Plant Physiol. 26, 127-158. Toms, G. C., and Westem, A. 1971. Phytohaemagglutinins. In “Chemotaxonomy of Leguminosae” (J. B. Harbome, D. Boulter and B. L. Tumer, eds.), pp. 367462. Academic Press, New York. Tschesche, H. 1974. Biochemistry of natural proteinase inhibitors. Angew. Chem., Int. Ed. Engl. 13, 1C28. Ubatuba, F. B. 1955. Occurrence of a trypsin inhibiting factor in the seeds of Canavalia ensiformis. Rev. Bras. Biol. 15, 1-8. Ueda, S., Koba, Y., and Chaen, H. 1979. Amylase inhibitor produced by Streptomyces sp. no. 280. J . Jpn. SOC. Starch Sci. 26, 145-156. Uhlenbruck, G., Wintzer, G., Salfner, B., Schumacher, K., Oerkermann, H., Hirschmann, W. D., and Alzer, G. 1970. Studies on the nature of the PHA receptor. Klin. Wochenschr. 48, 1369- 1370. Vaintraub, I. A., Bassiiner, R., and Shutov, A. D. 1976. The action of trypsin and chymotrypsin on the reserve proteins of some leguminous seeds. Nahrung 20, 763-771. Varner, J. E., and Schidlovsky, G. 1963. Intracellular distribution of proteins in pea cotyledons. Plant Physiol. 38, 139-144.

PROPERTIES OF COMMON BEAN PROTEINS

165

Ventura, M. M., and Xavier Filho, J. 1967. A trypsin and chymotrypsin inhibitor from black-eyed pea (Vigna sinensis). I. Purification and partial characterization. An. Acad. Bras. Cienc. 38, 553-566. Wagner, L. P., and Riehm, J. P. 1967. Purification and partial characterization of a trypsin inhibitor isolated from the navy bean. Arch. Biochem. Biophys. 121, 672-677. Wang, D. 1975. A crystalline protein-proteinase inhibitor from pinto bean seeds. Biochim. Biophys. Acta 393, 583-596. Ward, A. T., Marquardt, R. R., and Campbell, L. D. 1977. Further studies on the isolation of thermolabile growth inhibitor from faba bean (Vicia faba L. var. minor). J . Nutr. 107, 1325-1 334. Warsy, A. S., Norton, G., and Stein, M. 1974. Protease inhibitors from broad bean. Isolation and purification. Phytochemistry 13, 248 1-2486. Waterman, H. C., and Johns, C. 0. 1921. Studies on the digestibility of proteins in vitro. I. The effect of cooking on the digestibility of phaseolin. J . Biol. Chem. 46, 9-17. Waterman, H. C., Johns, C. O., and Jones, D. B. 1923. Conphaseolin. A new globulin from the navy bean (Phaseolus vulgaris). J . Biol. Chem. 55, 93-104. Weber, T., and Grasbeck, R. 1968. Physio-chemical characterization of a lymphocyte-stimulating leucoagglutinin from red kidney bean (Phaseolus vulgaris). Scand. J . Clin. Lab. Invest. 21, Suppl. 101, 14. Weber, T. H. 1969. Isolation and characterization of a lymphocyte-stimulating leucoagglutinin from red kidney bean (Phaseolus vulgaris). Scand. J . Clin. Lab. Invest. 24, Suppl. 111, 1-80. Weber, T. H., Aro, H., and Nordman, C. T. 1972. Characterization of lymphocyte stimulating blood cell-agglutinating glycoproteins from red kidney beans (Phaseolus vulgaris). Biochim. Biophys. Acta 263, 94-105. Weder, J., and Belitz, H.-D. 1969. Trypsin and chymotrypsin inhibitors in foods. Dtsch. Lebensm.Rundsch. 65, 78-83. Weder, J. K. P., and Hory, H.-D. 1976. Trypsin and chymotrypsin inhibitors in leguminosae. VI. Further properties of the inhibitors from Phaseolus coccineus. Chem. Mikrobiol., Technol. Lebensm. 4, 161-169. Weder, J. K. P., Kassubek, A,, and Hory, H.-D. 1975. Trypsin and chymotrypsin inhibitors in leguminosae. V. Isolation and characterization of some inhibitors from Phaseolus coccineus. Chem. Mikrobiol., Technol. Lebensm. 4, 79-84. Weinhaus, 0. 1909. Zur biochemie des phasins. Biochem. Z . 18, 22&260. Whitaker, J . R. 1981. Naturally occurring peptide and protein inhibitors of enzymes. In “Impact of Toxicology on Food Processing” (J. C. Ayres and J. E. Kirschman, eds.), pp. 57-104. Avi Press, Westport, Connecticut. Whitaker, I. R., and Sgarbieri, V. C. 1981. Purification and composition of the trypsin-chymotrypsin inhibitors of Phaseolus vulgaris L. var. Rosinha G2. J . Food Biochem. 5, 197-213. Whitley, E. J., Jr., and Bowman, D. E. 1975. Isolation and properties of navy bean proteinase inhibitor component I. Arch. Biochem. Biophys. 169, 42-50. Wilson, K. A,, and Laskowski, M., Sr. 1973. Isolation of three isoinhibitors of trypsin from garden bean, Phaseolus vulgarus, having either lysine or arginine at the reactive site. J . B i d . Chem. 248, 75G762. Wilson, K. A , , and Laskowski, M., Sr. 1974. The partial linear sequence of garden bean inhibitor I1 and location of the protease reactive sites. Buyer-Symp. 5 , 286-290. Wu, Y. V., and Scheraga, H. A. 1962. Studies of trypsin inhibitor. I. Physicochemical properties. Biochemistry 1, 698-705. Yachnin, S., and Svenson, R. H. 1972. The immunological and physicochemical properties of mitogenic proteins derived from Phaseolus vulgaris. Immunology 22, 87 1-883.

166

VALDEMIRO C. SGARBIERI AND JOHN R. WHITAKER

Yachnin, S . , Allen, L. W., Baron, J. M., and Svenson, R. H. 1972. The potentiation of phytohemagglutinin-induced lymphocyte transformation by cell-cell interaction; a matrix hypothesis. Cell. Immunol. 3, 569-589. Yamamoto, M., and Ikenaka, T. 1967. Studies on soybean trypsin inhibitors. I. Purification and characterization of two soybean trypsin inhibitors. J . Biochem. (Tokyo) 62, 141-149. Yamasaki, R. B., Vega, A,, and Feeney, R. E. 1980. Modification of available arginine residues in proteins by p-hydroxyphenylglyoxal. Anal. Biochem. 109, 32-40.

ADVANCES I N FOOD RESEARCH, VOL.

28

PORCINE STRESS SYNDROMES G . MITCHELL Department of Physiology, University of the Witwatersrand, Medical School, Johannesburg, South Africa

J. J. A. HEFFRON Department of Biochemistry, University College, Cork, Ireland

Introduction .............................. .............................. Porcine 11. Predictive Te ........................... A. Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Serological Tests.. . . . . . . . . . . . . . . . . . . . . . . . . . C. Hematological Tests . . . . . ................... D. Muscle Biopsies and Halothane Exposure . . . . . . . . . . . . . . . . . . . . . . . . ....................................... E. Conclusions 111. Etiology of Porcine Stress Syndromes . . . . . . . . . . ....... A . Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... .................. B. Muscle Metabolism. . . . . . . . IV. General Conclusions . . . . . . . . V. Future Research. . . . . . . . . . . . ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

167 169

181

182 186 187 187 192 216 217

I. INTRODUCTION In recent years there has been a steadily increasing body of literature written on the subject of porcine stress syndromes. There are several reasons for this. First, as Paterson and Allen (1972) point out, the pork and bacon industries depend ultimately for their profitability on the healthy growth of pig muscle. Any study, therefore, that attempts to improve diagnostic methods and to devise 167 Copyright 0 1982 by Academic Press, Inc All rights of reproduction in any fonn resewed. ISBN 0-12-016428-0

168

G. MITCHELL AND J. J. A . HEFFRON

means of eradication of muscle diseases in pigs, is important. In this context, the porcine stress syndromes were, in 1972, estimated to be costing the United States swine industry $230-320 milliodyr and since some breeds of pigs are more severely affected than American breeds, the cost was estimated to be greater in other countries (Williams et al., 1978a). A 1977 report estimated that acute stress deaths alone cost the United Kingdom pig industry &780,000/yr and confirmed that elsewhere, losses were likely to be more severe (Winstanley, 1979). For example, about 30% of South African Landrace and German Landrace pigs have been found to be susceptible to stress (Harrison et al., 1968; Heffron and Mitchell, 1975a,b; Mitchell and Heffron, 1975a). In these circumstances, research which seeks to understand the cause of and eliminate the disease has vast economic implications. Another stimulus for research into porcine stress syndromes has been that the pig is a useful model for the human malignant hyperthermia syndrome. Indeed, much of the research on porcine stress syndromes is directed towards elucidating the human rather than the porcine syndromes, especially now that there is evidence that humans susceptible to malignant hyperthermia (MH) are also susceptible to other forms of stress (Wingaard, 1974; Lister et al., 1975; Wingaard and Gatz, 1978). The use of the pig as a model for the human syndrome began after Hall et al. (1966) reported the deaths of three pigs undergoing halothane and suxamethonium anesthesia. Two years later Harrison et al. (1968) reported hyperthermia in pigs given halothane alone. The descriptions provided by these two reports tallied well with descriptions of the human syndrome (Stephen, 1967; Wilson et al., 1967) and it has been generally assumed, therefore, that the human and porcine syndromes are identical (e.g., Britt and Kalow, 1970b; Nelson, 1973; Okumura et al., 1979), although this view has been questioned (Berman and Kench, 1973; Heffron and Mitchell, 1975~).Nevertheless, the pig has been, and is, extensively studied physiologically, biochemically, and pathologically to provide information on the cause of MH, its course, and its treatment. A further development of the study of the pig as a model for human MH has been the discovery that pigs dying of porcine MH develop the pale, soft, exudative pork syndrome (Allen et al., 1970a; Nelson, 1973; Nelson et al., 1974; Cheah and Cheah, 1979). Consequently, identification of pigs likely to develop pale, soft, exudative pork is widely made on the susceptibility of pigs to MH (Eikelenboom and Minkema, 1974; Dimarco et al., 1976) and the drug-induced syndrome is considered to be another feature of the general stress syndrome (Hende et al., 1976). Another consequence of the study of porcine stress syndromes has been the realization that similar syndromes occur in many species. Uncontrollable hyperthermia during anesthesia has been recorded in dogs (Bagshaw et al., 1978; Short and Paddleford, 1973), cats (De Jong et al., 1974), horses (Klein, 1975; Waldronmease, 1978), fallow deer (Pertz and Sundberg, 1978), chicks (Korczyn

PORCINE STRESS SYNDROMES

169

et al., 1980), and rabbits (Lefever and Rosenberg, 1980). Further, cattle suffering from muscular hypertrophy show the same postmortem changes in muscle that occur in the muscle of susceptible pigs (Holmes et al., 1973). In addition, wild animals (Hofmeyr et al., 1973) and birds (Henschel and Louw, 1978) after severe and prolonged exercise develop the signs characteristic of the exerciseassociated stress of pigs. Thus it can be argued that the study of the porcine stress syndromes can provide insight into stress and its treatment in many species of animal. For all these reasons studies of the porcine stress syndromes have been carried out. In general, research has concentrated on the establishment of an unequivocal predictive test for pigs carrying the trait, on the nature of the underlying lesion, and on treatment and prevention of the syndromes. Despite intensive study, however, little consensus has been reached in any area. PORCINE STRESS SYNDROMES Before any research on the porcine stress syndromes can be discussed, it is important to establish the characteristics of each type. Three porcine stressrelated syndromes have been described: porcine stress syndrome (PSS), malignant hyperthermia (MH), and the pale, soft, exudative pork syndrome (PSE). 1. Porcine Stress Syndrome

PSS is characterized by acute death induced by natural stressors such as transport, high ambient temperature, exercise, fighting, service, and parturition (Patterson and Allen, 1972). These stresses result in progressive dyspnea, hyperthermia, disseminate vasoconstriction (blotchiness), and the rapid onset of rigor mortis after death (Nelson, 1973), and were first observed and described in 1967 (Topel et al., 1967). Since many animals that have no genetic or other predisposition to stress die after severe stress, showing signs similar to those described for pigs, it is important to distinguish between animals which succumb to stress and those which have an inherited predisposition and succumb to stress. The purpose of such a classification (Table I) is to circumscribe and describe the disease process which many animals are subject to when exposed to stress, so that the residue of changes unique to stress-susceptible pigs becomes clear and criteria for the inherited defect in stress-susceptible pigs can be delineated. In this context it has long been established that excessive exercise can be harmful. For example, exertional myopathy (Azoturia, Monday Morning Disease) of horses is characterized by muscle stiffness during exercise, hyperthermia, sweating, myoglobinuria, a lactacidosis, and increases in serum creatine phosphokinase (SCPK) and lactate dehydrogenase activity (Axt et al., 1968; Hammel and Raker, 1972; Diethmuller and Wels, 1972). This disease seems to be precipitated by rapid glycogenolysis and the production of lactate in poorly perfused muscle. At postmortem examination the muscles are pale. In zebras an identical syn-

TABLE I CLASSIFICATION OF STRESS SYNDROMESo ~

~

Stress syndromes Inherited Predisposition:

Nil

Cause of onset:

Exercise

Antemortem C[ussifcation

Signs

Resistant after training Nil

“Homozygote”

“PSS” t SCPK t “C f Lactate t Catecholamines 1 PH Muscle stiffness

.‘Heterozygote”

Not known

Exercise

Drugs

Slaughter

Drugs

True PSS As “PSS”

MH As “PSS”

-

Not known

MH As “PSS”

+

t

T,, T4

J. Cortisol

Postmortem Classification Signs

Nil Nil

Species

Horses Antelope

“PSE” t “C J Muscle pH A Muscle ATP J Muscle PC Muscle edema Rapid rigor Horses Zebra Antelope Birds Man Pigs

PSE

?

PSE As “PSE”

PSE As “PSE”

As “PSE”

Not known

Pigs Man

Pigs Man

Pigs Cattle

Dogs Cats Horses Chicks Rabbits

OThis table indicates that many animals with no genetic predisposition to stress syndromes can develop an increase ( t ) in serum creatine phosphokinase (SCPK) activity, a rise in body temperature (“C), as well as ante- and postmortem changes in muscle metabolism as shown by raised plasma lactate and decreased ( 4 ) plasma and muscle pH, and decreased muscle adenosine triphosphate (ATP) and phosphocreatine (PC). All of these changes also occur in pigs. The only apparent difference between pigs and other animals is that in susceptible pigs changes in plasma thyroid hormones and cortisol also occur.

172

G. MITCHELL AND J. J. A. HEFFRON

drome has been described and called “capture myopathy” or “overstraining disease” (Hofmeyr et al., 1973; Young and Bronkhorst, 1971). It occurs most frequently when the animals are manhandled, restrained, or chased to exhaustion. The zebras show raised SCPK activity, raised plasma glucose and lactate, and raised hematocrit and rectal temperature. Muscle degeneration similar to that occurring in pigs also occurs. These changes have also been reported in several different species of antelope (Harthoorn et al., 1974; Gericke and Hopmeyr, 1976) and birds (Young, 1967; Henschel and Louw, 1978). It also seems likely that the disease reported in fallow deer by Pertz and Sundberg (1978) as MH was “PSS.” In addition, all of the characteristic signs of PSS have been noted in pigs that have no predisposition to stress (Marple et al., 1974) and in pigs which are susceptible to stress (Nelson et al., 1974). In all of these cases it has been assumed that the cause of the changes is an increase in serum catecholamines in response to stress, although catecholamine levels have not been measured in all cases. Harthoorn (1976), however, has shown that in wild animals accustomed to being chased, plasma catecholamine levels do not increase and the animals do not develop typical signs of stress. Further, Hende et al. (1976) have recorded increases in serum catecholamines in pigs during exercise, and Hall et al. (1977a) have, by infusing catecholamines, produced in pigs changes similar to those that occur in wild animals undergoing stress. Thus catecholamines raise plasma glucose levels, stimulate muscle glycogenolysis, which, together with increased work load in muscle, will result in a lactacidosis, and lower pH. The low pH leads to changes in cell membrane integrity and intracellular enzymes such as CPK, and electrolytes leak into plasma. The increase in cell lactate, which is osmotically active, contributes to the rise in hematocrit (Hende et al., 1976). The characteristic skin blotchiness, changes in heart and respiratory rate, cardiac fibrillation, and reduced heat loss are all consequences of the actions of catecholamines (Ludvigsen, 1957; Nelson et al., 1974; Williams et al., 1975). Another feature of the syndrome is that rigor mortis occurs rapidly after death. The two well-established causes of rigor are a lack of ATP (Szent-Gyorgi, 1944) and low muscle pH (Bate-Smith and Bendall, 1947). Since plasma inorganic phosphate levels increase in wild animals (Hofmeyr et al., 1973) and in pigs during stress (Berman et al., 1970) and ATP depletion occurs in pigs (Nelson et al., 1972), a lack of ATP seems likely to contribute to rigor. A low muscle pH is an intrinsic feature of the syndrome and thus rigor results. These observations must make it clear that severe stress can cause death, with all the signs typical of PSS, in animals which have no inherited susceptibility to stress. The signs of PSS do not occur exclusively, therefore, in stress-susceptible pigs. However, there is little doubt that PSS will occur more easily in such pigs. Thus, although muscle activity and an increase in serum catecholamines can

PORCINE STRESS SYNDROMES

173

precipitate PSS, these changes alone do not explain the unusual sensitivity some pigs have to stress. This point is emphasized by the syndrome of MH. 2 . Malignant Hyperthermia MH is a pharmacogenetic disease. There is, therefore, a genetic defect which, when it is exploited by a drug, precipitates MH. Thus MH does not occur in animals that do not have the genetic defect and so wild animals, stress-resistant pigs, and other animals do not develop MH when exposed to halothane, succinylcholine, or other triggering drugs. The few MH-like reactions which have been reported in some animals (see Table I) undergoing anesthesia should be regarded as disturbances of temperature regulation until a genetic component is established. In the context of this article they should, therefore, be disregarded. The most common triggers of porcine MH are the gaseous anesthetic halothane (2-bromo-2-chloro- 1,1,1-trifluoroethane) and the muscle relaxant succinylcholine (Gronert and Theye, 1976b). However, other volatile anesthetics such as methoxyfluorane, diethyl ether, fluoroxene, and chloroform have produced MH in pigs (Harrison et al., 1969; Hall et al., 1972). It can be assumed also that all breeds of pig are susceptible although morbidity varies considerably. Thus Pietrain are most susceptible and Largewhite pigs and their crosses are least susceptible (McLoughlin, 1971). In South Africa the incidence among Largewhite pigs has been put at 0.5% in a sample of 763 and in Landrace pigs, 14.4% in a sample of 1317 (R. T. Naude, personal communication). The first description of MH in pigs was that of Hall et al. (1966), in which both halothane and succinylcholine were used. In 1968 Harrison et al. described halothane-induced MH. Whatever the trigger, typically the first clinical sign is a tachycardia of 200-300 beats/min. Blood pressure stays normal until a decrease in cardiac output occurs terminally. Muscle rigidity appears within 1-4 min of the onset of anesthesia. An increase in respiratory rate to 125 breathdmin, a blotchy cyanosis indicating vasoconstriction (since PaO, is normal), and an increase in rectal temperature at a rate of about 1°C per 10 min follow. A combined respiratory and metabolic acidosis develops 15-20 min after the tachycardia, venous blood becomes extremely desaturated, and a hyperkalemia of 11-12 mmol/liter develops. Changes in the composition of plasma have also been described (Berman et al., 1970) which start immediately after halothane is given. The first sign is an increase in oxygen consumption. A shift of water from plasma to the inter- and intracellular spaces (both serum sodium and total protein increase) and a shift of magnesium, calcium, potassium, and inorganic phosphate follow. A rise in plasma glucose and lactate occurs as well as increases in SCPK activity (Woolf et al., 1970), catecholamines (Hende et a l . , 1976; Gronert et a l . , 1976a, 1977a;

174

G . MITCHELL AND J. J. A. HEFFRON

Hall et al., 1975; Lucke et al., 1976), and cortisol (Mitchell and Heffron, 1981a). Plasma pH decreases to 6.6 within 20 min. These physiological and biochemical changes are invariably fatal, are clearly similar to those occumng in PSS, and are summarized in Figs. 1 and 2 . On the basis of these changes it is widely assumed that pigs with an inherited predisposition to PSS have the same defect as pigs susceptible to MH. Therefore, PSS and MH are essentially similar diseases: they differ in that PSS results from muscle activity of either natural or artificial origin, and MH results from the action of volatile anesthetic agents. Both also result in PSE (Table I). A -20

IOOO]

, p-----."

450 /

Plasma

-15 L

-10 K+(mmol liter-')

-5 0 100

0

80

75

Arterial pH 70 40

20

'

'

20

C

16 Oxygen

consumption (ml rnin-l kg-')

20 16

12 xic lactate

8 4

l2

Lactate (mmol liter-')

4 I

4

Control

.,

65

n

4

4

%me

Rigor

1-370 HAL/S Ch

4

Death

4

Rigidity FIG. 1. Changes in blood gases, hormones, and electrolytes during an episode of MH. The time scale is C.50 min. HAL/S.Ch., Halothaneisuccinylcholine.(A) V, Cortisol; K + ; A , catecholamines. (B) pH; 0, PaC02. (C) 0 , VO,; 0, lactate. Data from Hall et al. (1973, Gronert and Theye (1976a), Gronert et al. (1976a, 1977b), and Berman e r a / . (1970).

A,

175

PORCINE STRESS SYNDROMES A

Rectal temperature

("0

421 J 41

B

51 60

C 1 l2 61

O L i

\\\

I

1

HAL

t Rigidity

1

I

I

,

I

Time

t

Death

FIG. 2. Changes in rectal temperature (A) and metabolism (B, C) during an episode of MH. Note that rigor, indicated by the arrow, occurs at pH 6.2 (Bate-Smith and Bendall, 1947). The time scale is G 2 3 min. (C) 0 ,G-6-P; 0,ATP; PC. HAL, Halothane; Barb., barbiturate. Data from Mitchell et al. (1980).

A,

3. Pule, SOB,Exudative Pork Syndrome Historically, PSE was the first of the porcine stress syndromes to be described. Ludvigsen (1957) provided the first substantial report of PSE, however, WismerPedersen ( 1 959) has pointed out that over 60 years ago pale and watery pork, which developed in some pigs after slaughter by conventional methods, caused difficulties in the sausage industry in Germany. In a series of papers, Ludvigsen (1957) described a disease he termed muscle degeneration, in pigs dying acutely after stress. The disease was characterized by muscle discoloration, malodor, and edema. The affected meat was useless since the disease, to a great extent, ruins the quality of the fresh meat and meat

176

G. MITCHELL AND J. I. A. HEFFRON

products. Ludvigsen concluded from his experiments that the disease resulted from poor circulation and hypoxia. In 1959 Wismer-Pedersen found a significant positive correlation between water-holding capacity of muscle, its pH, and the rate of pH change postmortem. He also noted that pigs likely to have muscles which exude water, have a 45-min postmortem pH of less than 6.0 and high muscle ATPase activity. WismerPedersen also suggested that since the cause of the low pH was lactate, glycolysis had been stimulated. Further, he inferred that stress was involved in that pigs producing PSE had usually undergone severe stress during transport to the abattoir. Thus catecholamines were the likely, but not proven, cause of glycolysis. An additional conclusion of Wismer-Pedersen (1959) and Lawrie (1960) was that because glycolysis is also controlled by the relative concentrations of the adenine nucleotides, either ATPase activity must be increased or muscle activity must be increased. These observations were confirmed by Kastenschmidt et al. (1966), who found that fast glycolyzing muscle, in addition to having a low lactate-induced pH, had low ATP (2.54 kmol/g) and creatine phosphate levels immediately postmortem. These findings imply breakdown of creatine phosphate and failure of its restitution. Another factor in the development of PSE found to be important was high muscle temperature in combination with low muscle pH (Wismer-Pedersen and Briskey, 1961; McLoughlin, 1963). PSE only developed in muscle with a high rate of glycolysis and a temperature of 36-40°C, produced apparently by the high rate of glycolysis. This combination of changes results in alteration of sarcoplasmic and myofibrillar proteins, a fall in ATP levels, and the muscle becoming pale, soft, and exudative as it goes into rigor. As Szent-Gyorgi (1944) has noted “rigor and insolubility of actomyosin are the different consequences of one and the same condition-a lack of ATP.” In summarizing these observations, Briskey et al. (1966) have stated that rapid glycolysis should result in high glucose-6-phosphate levels and low fructose- 1,6diphosphate, ATP, and creatine phosphate levels. These conclusions have more recently received support. Schmidt et al. (1972) have correlated stress susceptibility with high glucose-6-phosphate levels, and Sair et al. (1970) and Lister et al. (1970) have shown that PSE develops in pigs with low muscle levels of highenergy phosphates. These studies also concluded that the muscle of PSE-susceptible pigs either responded to anoxia or that it is made anoxic more easily than PSE-resistant muscle. More importantly, these studies also suggested that since the muscle relaxant curare did not prevent PSE, the rapid glycolysis was not a result of muscle activity. On the other hand, magnesium anesthesia did prevent PSE. Since magnesium depresses glycolysis, (Regen et al., 1964), inhibits myosin ATPase (Needham, 1971), and prevents ATP breakdown by competing for Ca2+ binding sites (Dubois et al., 1943), whereas catecholamines facilitate or

PORCINE STRESS SYNDROMES

177

promote these changes, the cause of PSE in PSE-susceptible pigs was an unusual sensitivity to the effects of catecholamines on glycolysis. A clear link was, therefore, established between PSS and PSE in that catecholamines, rapid uncontrolled glycolysis, and low levels of ATP and phosphocreatine are crucial to both. All of these studies satisfactorily explained Ludvigsen’s ( 1957) observation that pigs dying of PSS developed PSE, and also pinpointed the crucial factors necessary for the development of PSE. In summary, PSE develops as a result of catecholamine-stimulated glycolysis. Glycolysis proceeds at a rate exceeding the capacity of aerobic metabolism and independent of the inhibitory effects of low pH. Consequently large amounts of lactate are produced while muscle temperature is high. Protein precipitation occurs and water leaves the cytoplasm to enter the intercellular space, thus giving the muscle a watery appearance. The paleness of the muscle results from protein denaturation and catecholamine-mediated vasoconstriction. Blood vessels also collapse as a result of muscle stiffness and associated high tissue pressure. All that remained was for a similar link to be shown between MH and PSE. This link was provided by Harrison et al. (1969) and Nelson et al. (1974), who noticed that although initial ATP levels in both normal and susceptible pigs were similar, PSE was characterized by a rapid depletion of ATP, and halothane, which causes MH, could precipitate ATP depletion. In addition, the same breeds of pig develop PSE and MH (Nelson, 1973) and exercise before exposure to halothane results in quicker onset of MH and PSE in susceptible pigs (Hende et al., 1976). It has thus become well established that pigs dying of MH will develop PSE. However, recent evidence suggests that not all pigs susceptible to PSE are also susceptible to MH (Mitchell and Heffron, 1980a). Further, the metabolic changes induced by slaughter and which result in PSE differ from those induced by halothane and which result in PSE (Mitchell and Heffron, 1981b). These findings are discussed below. 4.

Conclusions

This article provides substantial evidence that the three stress syndromes PSS, MH, and PSE have much in common, and establishes criteria against which the defect in stress-susceptible pigs can be assessed. All three syndromes are associated with a high rate of glycolysis and the production of lactate-the crucial metabolic changes. Further, PSS and MH are linked by the common denominators of a genetic defect and PSE (Fig. 1). PSE can occur independently of the genetic defect. The unique characteristic, therefore, which separates those animals with unusual sensitivity to stress from those

178

G. MITCHELL AND J. J . A. HEFFRON

developing PSE after the stress of severe exercise or the stress associated with slaughter, is a genetic defect. Thus animals without the defect do not develop drug-induced MH. The genetic defect associated with stress susceptibility is itself not lethal unless the animals are stressed. Indeed, stress-susceptible pigs grow almost as well as resistant animals. In addition, susceptible pigs are not more sensitive to changes in metabolism: they are killed by changes that would kill most animals. Thus the first criterion for the defect associated with stress susceptibility is that it must be minor in that it does not interfere with growth, but severe enough to allow rapid and fatal development of metabolic changes in the presence of an appropriate stimulus. Additional criteria are that the defect must be present in many tissues and that the changes in metabolism in these tissues must be similar to, or enhanced by, hormones, especially catecholamines. Similarly the syndromes can develop in the absence of drugs, but volatile anesthetics and muscle relaxants will precipitate them. The defect, therefore, is latent and is not induced by drugs. Identification of pigs carrying the defect and analysis of the nature of the defect is discussed below.

II. PREDICTIVE TESTS A.

GENETICS

Identification of pigs susceptible to any of the stress syndromes is of great importance. The finding of a quick, cheap, nonlethal, and unequivocal means of identifying stress-susceptible pigs should lead to the rapid elimination of pigs carrying the trait. Moreover, such a test would provide researchers selecting pigs for experiments, a means of identifying susceptible pigs before exposing them to the often fatal triggering agents. This is of special significance in those experiments which test putative therapeutic or prophylactic drugs. It is clear that if pigs in these experiments are not identified unequivocally, proper interpretation of the efficacy of drugs cannot be made. Indeed, much of the confusion in the literature concerning drugs in particular and the stress syndromes in general can be attributed to imperfect identification of pigs. In general, tests have been based on the assumption that MH, PSS, and PSE are identical, differing only in the cause of onset (Nelson, 1973). The attractiveness of a single syndrome theory is that finding a pathognomonic sign for PSS, MH, or PSE will, if it is selected against, lead to eradication of all three. On this basis numerous tests for predisposition to stress have been used. Further, this theory depends on the assumption that a single genetic defect is the cause of stress susceptibility, yet the genetic basis of the diseases is unclear.

PORCINE STRESS SYNDROMES

179

Breeding and other experiments have suggested that one major recessive gene with complete penetrance (Eikelenboom et al., 1978; Smith and Bampton, 1977), high penetrance (Andresen, 1979a), incomplete penetrance (Ollivier et al., 1975), or variable penetrance (Cheah and Cheah, 1979) forms the basis of inheritance. On the other hand, a single autosomal dominant gene with complete penetrance (Hall et al., 1966; Allen et al., 1970b; Jones et al., 1972) or two different dominant genes (Britt et al., 1978) have been implicated. Despite this lack of consensus, an important result of these experiments has been to reveal that a range of phenotypes exist. Five phenotypes have been distinguished on the basis of response to halothane anesthesia (Britt et al., 1978), four on the basis of rate of postmortem glycolysis (Wismer-Pedersen and Briskey, 1961), three on the basis of hormone studies (Judge et al., 1966), three on the basis of heat production and muscle pH (Williams et al., 1978a), three on the basis of SCPK activity (Allen et al., 1970b; Jones et al., 1972; Patterson and Allen, 1972), three on the basis of SCPK, muscle pH, and halothane sensitivity (Mitchell and Heffron, 1980a), and three on the basis of the concentration of glutathione peroxidase in erythrocytes (Schanus et al., 1979). Therefore, the genetic defect is not absolute in all animals carrying the trait. However, the gene for susceptibility to halothane-induced MH is closely linked to H blood types and the locus for the enzyme phosphohexose isomerase, and is associated with meat quality. Thus H blood types (Rasmussen and Christian, 1976) and plasma phosphohexose isomerase activity could indicate susceptibility to halothane and poor meat quality traits (Andresen, 1979b, 1980; Eikelenboom, 1981) but not necessarily with complete accuracy. The difficulties of finding a suitable and accurate predictive test is thus obvious. Nevertheless, much research has been done and the tests that have been used can be classified into three types: those that examine variables in the blood, those that analyze muscle biopsies, and those in which intact individuals are exposed to triggering agents. B.

SEROLOGICAL TESTS

Serological tests have focused on the measurement of serum enzymes. Serum pyrophosphate levels have been suggested for use (Addis et al., 1978), but in pigs they have only been measured during an episode of MH (Berman et al., 1970). Several enzymes have been measured in attempts to find a suitable test. Lactate dehydrogenase levels has been found to be high in susceptible pigs and increase in the serum of pigs after exercise and during MH (Pollock et al., 1973; Sybe,sma and Eikelenboom, 1969), although enzyme activity correlates poorly with susceptibility (Watanabe et al., 1979; Hende et al., 1976). Serum glutamate oxalate transferase has also been found to be high in pigs before and during MH

180

G . MITCHELL AND J . J . A . HEFFRON

(Sybesma and Eikelenboom, 1969). Aldolase is also raised in the plasma of susceptible pigs (Eikelenboom and Minkema, 1974). However, all of these enzymes are found in most tissues of the body, and an increase in activity in plasma need not be specific for a muscle defect. Since muscle is the major site of metabolic changes in MH, PSS, and PSE a more specific enzyme marker is desirable. For this reason, by far the most widely used enzyme is SCPK since nearly all SCPK is found in the cytoplasm of cardiac or skeletal muscle cells (Baskin and Deaner, 1970). The use of SCPK as a test in pigs was considered only after it had been shown to indicate muscle disease (Hess et al., 1964) and muscle injury (Tammisto and Airaksinen, 1966) in man. In 1970 its use as a test of predisposition to MH was reported in both man (Isaacs and Barlow, 1970) and in pigs (Allen et al., 1970b; Woolf et al., 1970). Halothane exposure increased SCPK activity in susceptible pigs, a finding which lent support to its use as a predictive test (Woolf et al., 1970). Following these initial reports a large number of studies have suggested that in man SCPK activity is nonspecific, correlates poorly with susceptibility to MH, and is highly variable (Britt et al., 1976; Ellis et al., 1975). With this background and despite some reports that MH can be well predicted by SCPK activity, especially where a familial predisposition to MH exists (Isaacs, 1973), the measurement of SCPK as a predictive test has fallen into disrepute. This is unfortunate because in pigs virtually all reports show that in those pigs susceptible to stress or undergoing MH, SCPK levels are raised (Allen et al., 1970b; Woolf et al., 1970; Jones et al., 1972; Nelson et al., 1974; Eikelenboom and Minkema, 1974; Mitchell and Heffron, 1975b). Significant differences between normal and susceptible pigs have, however, been difficult to show because of variability (Nelson et al., 1974; Woolf ef al., 1970; Hall et al., 1972), overlap between normal and susceptible pig values (Watanabe et al., 1979), and a lack of appreciation of the causes of variation and the limitations of the test. Thus Hende et al. (1976), while finding significant differences between susceptible and resistant pigs, have also stated that SCPK activity is only useful when other diagnostic features are present. However, in a fairly extensive analysis of the causes of variability and the use of SCPK in pigs (Mitchell and Heffron, 1975a; Heffron and Mitchell, 1975a,b), three factors could be identified which contribute greatly to the variation in SCPK activity referred to in the literature and which give rise to uncertain interpretation of increases in SCPK. These factors are a diurnal variation which can increase SCPK activity by 50%, muscle activity which can increase activity 4-fold in 60 min, and age. Of these, age is most important. Maximum SCPK activity is well correlated with peak protein anabolism, which occurs between 11 and 28 weeks of age (Fig. 3). During this phase no significant differences between SCPK activity of susceptible and resistant pigs can be shown and SCPK has no diagnostic value. On the other hand, clear differences in SCPK values

181

PORCINE STRESS SYNDROMES 3000 2 50C 2 000 Serum CPK I U /Iirer

1500 I000

. ’,

500 0 0

4

8

I2

16

.

20

.

24

28

Age (weeks)

FIG. 3 . Variation of SCPK activity with age. Note that peak protein anabolism (weeks 12-16) coincides with peak SCPK activity. 0 , CPK; 0, wt (weight). Data from Mitchell and Heffron (1975a) and Heffron and Mitchell (1975a,b).

between susceptible and resistant pigs can be shown if SCPK activity is measured before 11 or after 28 weeks of age. The data from these studies also emphasized that a genetic or familial predisposition to stress in pigs was essential before SCPK activity could be properly interpreted. In view of this, the value of SCPK does not lie in the absolute values of the enzyme activity, and it is not possible to establish “basal” values against which all pigs can be assessed. However, in a breed of pig in which there is a known predisposition, high SCPK values relative to littermates can indicate predisposition, especially where extraneous factors, as discussed above, are minimized. C.

HEMATOLOGICAL TESTS

Hematological tests have analyzed platelet function, blood groups, and erythrocyte fragility in attempts to find a predictive test. In general, the rationale has promised much, but the results have been disappointing. Platelets have many characteristics similar to muscle and, therefore, platelet metabolism has been examined and found to be abnormal in human patients (Solomons et al., 1978). Similarly, abnormal platelet aggregation (Zsiginond et al., 1978) and increased platelet factor I11 (Addis et al., 1978) have been found to correlate with susceptibility to MH. However, the difficult techniques associated with these tests preclude their general use in pigs. With regard to blood groups, some preliminary work has shown that two H blood types ( d a and -/-) in pigs correlate well with susceptibility to PSS (Rasmussen and Christian, 1976), and Andresen (1979b, 1980) has correlated H blood types and susceptibility to halotliane. This work has yet to be extended.

182

G. MITCHELL AND J. J. A. HEFFRON

Tests of erythrocyte fragility as a means of identifying stress susceptibility in pigs were suggested by the observation that hemolysis occurs during MH (Britt and Kalow, 1968) and MH-susceptible patients have increased erythrocyte fragility (Reske-Nielsen, 1978). Further, as there is considerable leakage of myoglobin, potassium, and SCPK into plasma during MH (Berman et al., 1970), it has been suggested that MH may be characterized by a general membrane defect which could be identified by increased fragility of erythrocytes (Britt and Kalow, 1970b) because of the similarity between red cell membranes and other cell and subcellular membranes (Bjerrum and Bog-Hansen, 1976). On this premise Harrison and Verburg (1973) tested osmotic fragility in pigs and found significant differences between resistant and susceptible pigs in sodium chloride concentrations of 0.077-0.094 M . King et ul. (1976) have also found significant differences in fragility but in the range of 0.120-0.137 M sodium chloride. Cheah and Cheah (1978) have shown that at 0.103 M sodium chloride susceptible pig erythrocytes release more than 4 times the hemoglobin than do erythrocytes from resistant pigs. Susceptible pigs also have a deficiency of glutathione peroxidase (Schanus et al., 1979) which should facilitate hemolysis. On the other hand, some detailed studies have not recorded hemolysis during MH in pigs (Berman et al., 1970; Nelson et al., 1974), which suggests that the particular circumstances which cause hemolysis occur rarely. On the grounds that changes in plasma osmolarity are likely to be small in pigs during episodes of stress, another study (Heffron and Mitchell, 1981) tested the possibility that changes in temperature and pH may initiate hemolysis. Further, it was argued that since halothane is metabolized to bromide, chloride, and trifluoroacetate, these metabolites may cause hemolysis. This study confirmed an increased fragility of erythrocytes at 0.077-0.103 M sodium chloride and showed that low pH exacerbated differences. However, high temperatures reduced fragility and halothane metabolites had no detectable effect. Thus although osmotic fragility may be a useful additional test, especially in combination with low pH, it is not, at this stage, wholly reliable. D.

MUSCLE BIOPSIES AND HALOTHANE EXPOSURE

In man, susceptibility to MH is well correlated with clinical myopathies (Denborough et al., 1970). However, no single myopathy predisposes to MH and, furthermore, since the incidence of myopathy is far higher than the incidence of MH, it can be argued that myopathy is an incidental rather than a specific finding. In pigs, muscle biopsies have been examined by light microscopy, electron microscopy, and biochemistry for defects and also have been exposed to triggering agents. Microscopic examination has revealed motor end-plate abnormalities

PORCINE STRESS SYNDROMES

183

(Swatland and Cassens, 1972), low mitochondria1 content, and regeneration of fibers (Muir, 1970). However, since the incidence of clinical myopathy is low (Venable, 1973) compared to the incidence of stress susceptibility and, as Hall et al. (1980b) point out, susceptible pigs grow well unless exposed to undue stress, microscopic analysis of pig muscle has not been considered to be of value. The usual test performed on muscle biopsies to predict susceptibility to MH is an in vitro contracture test, in which a muscle strip is exposed to drugs which precipitate MH (e.g., halothane, succinylcholine, and caffeine). In man, this test is the most specific available (Kalow et al., 1977; Nelson et al., 1977). In pigs, the variability of contraction produced by drugs is so great that the test is unsuccessful (Okumura et al., 1979), although Gronert (1979) has reported that the caffeine contracture test is a reliable indication of MH susceptibility in Poland China swine. A recurring theme in all of these reports is that since the accuracy of muscle biopsy tests is unproven, their cost is high, they require precisely controlled environments and techniques, and the surgical risk is unacceptable, muscle biopsies are of no value for use as a screening technique. It is for these reasons that serological and hematological tests have been more widely investigated. Nevertheless, muscle tissue is considered to be the key tissue in MH and PSS, and should provide a basis for distinguishing susceptible and resistant pigs. Indeed, muscle biopsies have been studied exhaustively biochemically and several biochemical differences have been found between “susceptible” and “normal” muscle. However, few of these studies have been directed toward establishing a predictive test, mainly for the reasons discussed above. But an attempt to establish calcium content of muscle as a test has proved inconclusive (Britt et al., 1975a). On the other hand, Schmidt et al. (1972) have shown that susceptible pigs have raised glucose-6-phosphate levels. Also, Harrison et al. (1969) have found that halothane-induced depletion of ATP in muscle is a good indicator of susceptibility, although there are technical limitations to this test (Teeter et al., 1969). An extrapolation of the data of Harrison et al. (1969) is that since halothane triggers changes in ATP, brief challenge with halothane will identify stresssusceptible pigs, and this test is now used throughout the world (Dimarco et al., 1976; King et al., 1976; Eikelenboom and Minkema, 1974; Sybesma and Eikelenboom, 1978). A genetic basis for the halothane test has also been reported (Eikelenboom et al., 1978). Some difficulties with regard to the halothane test have been noted. First, ever since Harrison et al. (1968) produced MH in pigs using halothane it has been accepted that once initiated and in the absence of therapy, MH is invariably fatal in pigs. This assumption suggests that in those experiments in which brief challenge is followed by recovery, the pigs are not truly susceptible and could be undergoing the initial reaction responses to

184

G . MITCHELL AND J: J. A. HEFFRON

halothane (Mitchell et al., 1980) or simply variable responses to halothane (Ahern et al., 1980). On the other hand, if the pigs do not survive, then they cannot be used for experiments and are also a commercial loss. A possible explanation for the variable responses noted is that the concentration of halothane used is not standardized. The concentration is crucial despite the observation of Berman et al. (1970) that the process of MH once initiated continues independent of halothane concentration in the tissue. For example, at a concentration of 10% halothane in oxygen, the time course of MH is 8 min (Mitchell and Heffron, 1975b), whereas at 1%, the time course is 5&70 min (Gronert et al., 1976a). Thus it is surprising that different concentrations have been used in the halothane exposure test. Concentrations of 5% halothane in oxygen (Eikelenboom et al., 1978) and 4% halothane in an approximately 3: 1 oxygen-nitrous oxide mixture have been used (Sybesma and Eikelenboom, 1969). Christian and co-workers have used 6% halothane as a test at 7 weeks of age (Dimarco et al., 1976) but have recommended 2-6% for general use, while using 10% to precipitate MH. Since 1-3% halothane (Hall et al., 1980b; Berman el al., 1970) will cause MH, it is difficult to reconcile that 6% is nonfatal in one circumstance and 1% is fatal in another, although responses to halothane may be age dependent. Perhaps more important than these difficulties is that the basis of the halothane 8

6

nkg4 week

2

0

0

,

2

I

4

6

8

I

I

I

I

10

12

14

16

18

Time (weeks)

FIG. 4. Weekly mass of three groups of pigs, No significant differences between the three groups could be shown. 0 , Normal; 0, MH/PSE; PSE. Data from Mitchell and Heffron (1981b).

A,

185

PORCINE STRESS SYNDROMES

0

1

2

3

24

Time (hours,postmortem)

FIG. 5 . Rate of change of postmortem glycolysis in three groups of pigs. There are no significant differences between MH/PSE (0) and PSE (A) pigs, but both of these groups have a significantly higher rate of postmortem glycolysis than normal pigs. 0 ,Normal. Data from Mitchell and Heffron (1980a).

test is suspect. The basis is that identifying pigs susceptible to MH also identifies those susceptible to PSS and PSE. However, in a study using 10% halothane (Mitchell and Heffron, 1980a) to cause MH, and in which SCPK activity was measured and muscle biopsies were analyzed, it was shown that three groups of pigs can be identified. The first group (MH/PSE) is sensitive to halothane, and has high SCPK activity, a high rate of postmortem glycolysis (Fig. 4), and a muscle pH < 6.0, which indicates that the development of PSE is likely (McLoughlin, 1971). These pigs also grow almost as well as normal pigs (Fig. 5 ) and show marked stimulation of glycolysis and diversion of glucose to lactate when exposed to halothane. Moreover, they are easily identified using exposure to halothane, SCPK activity, or muscle biochemistry, and thus potential losses can be prevented or contained. The second group of pigs (PSE) is halothane resistant, has SCPK activity significantly less than that of the MH/PSE group, but also has a high rate of postmortem glycolysis and a muscle pH < 6.0 (Fig. 4). These pigs cannot be identified using halothane, SCPK, or muscle biochemistry. Further, because they grow at the slowest rate of the three groups (Fig. 5 ) , they represent a potentially greater source of economic loss: Pigs likely to develop PSE muscle not only result in postmortem losses but also result in premortem losses since feed efficiency is poor. The third group of pigs (normal) is halothane resistant, and has relatively low SCPK activity, a low rate of postmortem glycolysis (Fig. 4), and a high muscle pH. They also grow best. An implication of these data is that the use of halothane challenge and the

186

G. MITCHELL AND I. J. A. HEFFRON

measurement of SCPK activity detect only animals susceptible to MH. These findings add another limitation to SCPK measurements in addition to those already discussed, and show that not all pigs susceptible to PSE develop MH. The use of these tests will not, therefore, eliminate pigs susceptible to PSE. Another implication of these data is that MHiPSE syndrome is different in some respects from the PSE syndrome. For example, it seems likely that pigs susceptible to PSE lose control of skeletal muscle metabolism in response to stress, an increase in plasma catecholamine concentration, and anoxia (Allen et af., 1970a; Lister et al., 1970), whereas MH/PSE pigs undergo nonhypoxic lactate production (Gronert er af., 1977b). Moreover, these data support the view of Williams et al. (1978a) that pigs “homozygous” for the MH trait are more severely affected than those that are “heterozygous.” Heterozygote carriers also have low meat quality scores, occupying an intermediate position between reactors and noncarriers (Eikelenboom, 1981). Further, if the reaction to halothane is inherited as a single recessive gene with complete penetrance (McGloughlin et al., 1980; Eikelenboom et al., 1978; Smith and Bampton, 1977), then the suggestion that heterozygotes are less severely affected may have a genetic basis (Eikelenboom, 198 1). These data also confirm a long-standing assumption that stress susceptibility is a disease of “modern” agricultural practice in which genetic selection for heavily muscled pigs, having a high growth rate and a high feed conversion ratio, has resulted in a myopathy (Nelson, 1973; Hall et al., 1980b). Thus pigs reacting to halothane have a slow growth rate, an inferior meat quality, lower back fat thickness, and a high total meat percentage (Eikelenboom, 1981; McGloughlin et al., 1980).

E. CONCLUSIONS There is no doubt that since the incidence of stress susceptibility varies between breeds, an unequivocal test is needed that will distinguish susceptible pigs in a herd or breed in which a known predisposition to stress exists. The attempts described above to find such a test suggest that since the MH (or PSS) and PSE syndromes can occur separately, using a test which identifies only one of them will not eliminate the trait. Also, none of the tests discussed when used individually is perfect. Halothane challenge will identify unequivocally MH/PSE pigs but at the risk of a potentially fatal outcome. Muscle biopsies are equivocal and have the additional disadvantage of being difficult and time-consuming. Similarly, fragility tests are not wholly reliable. Of all the tests, SCPK activity is most useful since it has reasonable accuracy and minimum risk, and is easy and cheap. However, SCPK will identify only pigs susceptible to MH/PSE. It is plain, therefore, that a more comprehensive search for adequate tests or combination of tests is essential.

PORCINE STRESS SYNDROMES

Ill.

187

ETIOLOGY OF PORCINE STRESS SYNDROMES A.

HORMONES

From the discussion in Section 11, it is clear that a common denominator in the development of PSS, MH, and PSE is rapid glycolysis and the production of lactate. Since hormonal changes are rapid and associated with stress, hormones have been implicated. Indeed, since most of the signs of the syndromes can be explained by the effects of “stress” hormones, it seems unnecessary to invoke the presence of any defect in stress-susceptible pigs other than undue sensitivity to hormones. Thus catecholamines, thyroid hormones, and cortisol, the prime hormonal regulators of glycolysis, have been studied. Sex steroids, although possibly of importance in human stress syndromes (Crawford, 1972; Stovner et al., 1976; Kawane et al., 1979), are not important in the porcine syndromes (Patterson and Allen, 1972; Lucke, 1977; Lister et al., 1972; Eikelenboom and Weiss, 1972). 1. Catecholamines

There is no doubt that sympathetic nerve activity associated with stress causes the release of adrenaline and noradrenaline (Westfall, 1977). It is not surprising, therefore, that in pigs undergoing MH and in pigs undergoing natural stress, catecholamine levels increase in the plasma (Gronert et al., 1976a, 1977a; Hall et al., 1975; Lucke et al., 1976) to values as high as 45 p,g/liter (Lucke et al., 1976) and to values usually greater than 25 kg/liter (Hall et al., 1975). There is also little doubt that the underlying changes associated with capture stress in wild animals (Harthoorn, 1976) and those occurring in pigs exposed to exercise (Hende et al., 1976) are partly the result of the actions of catecholamines. It has also been argued that in both man and pigs a common link between preoperative excitement and anxiety and MH is catecholamines (Berman, 1973; Hende et al., 1976; Lister et al., 1975; Mogenson et al., 1974; Wingaard, 1974; Wingaard and Gatz, 1978). In addition, in man pheochromocytoma, monoamine oxidase inhibitors, and tricyclic antidepressant drugs have caused rigor and deaths with MHlike signs (Denborough et al., 1962; Ellis, 1973). However, it is necessary to establish a pivotal role for catecholamines in MH or PSS before concluding that unusual sensitivity to catecholamines is the primary defect in the syndromes. Whether catecholamines are involved primarily or secondarily has often been argued (Williams et al., 1978b; Gronert, 1978). The evidence which supports a primary role for catecholamines is that infusions of catecholamines into susceptible pigs causes PSS, whereas specific blockade of a-adrenergic receptors, epidural anesthesia, adrenalectomy , prevention of secretion of catecholamines from

188

G. MITCHELL AND J. J. A . HEFFRON

the adrenal gland by using bretylium, and depletion of catecholamines using reserpine all prevent or attenuate suxamethonium-induced PSS (Kerr et al., 1975; Lister et al., 1974, 1976; Hall et al., 1977b; Lucke et al., 1978; Williams et al., 1978b). An explanation of these data is that catecholamines, via an a receptor, potentiate release of acetylcholine at the neuromuscular junction (Bowman and Nott, 1969; Lister et al., 1974) and thus mimic and complement injections of SUXamethonium. Susceptible pigs have more motor terminals than resistant pigs (Swatland and Cassens, 1972) and so there is an anatomical substrate for this idea. Moreover, blockade of a-adrenergic receptors prevents muscle fasciculation and rigor (Lister et al., 1974), prevents an increase in serum lactate (Lister et al., 1970), and reduces the rate of increase of plasma potassium and rectal temperature (Lister et al., 1974). Further evidence which is consistent with this idea is that pancuronium, an acetylcholine antagonist, is as effective as phentolamine (an a-receptor antagonist) in preventing suxamethonium-induced PSS (Lister et al., 1976). A second explanation for the effect of infusions of catecholamines is that they cause vasoconstriction and hence muscle hypoxia. Hypoxia is important in the development of PSE (Lister et al., 1970). &-Receptor blocking agents protect the muscle by preventing vasoconstriction. Vasodilatation stimulated by P-receptor agonists also prevents both hypoxia and increases in serum lactate in susceptible animals (Lister et al., 1970; Weiss et al., 1974), thus emphasizing the role of hypoxia. On the other hand, blockade of p receptors combined with an infusion of a dose of noradrenaline, which when given alone produces only small increases in temperature, will precipitate fatal hyperthermia (Hall et al., 1977a). This finding suggests that the a-receptor-mediated constriction uncovered by this maneuver results in a lethal tissue hypoxia in stress-susceptible animals. A third explanation for the effect of catecholamines is that they stimulate padrenergic receptors, although this seems unlikely based on the evidence discussed above. However, glycogenolysis in muscle is stimulated by activation of p receptors via adenyl cyclase and CAMP (Harris et al., 1965; Sutherland and Rall, 1960), though specific stimulation of P-receptors using isoprenaline does not cause hyperthermia (Hall et al., 1977a). Nevertheless, stimulation of muscle metabolism combined with vasoconstriction, both results of a rise in plasma catecholamines, are synergistic. Further, since halothane stimulates P-adrenergic receptors (Price et al., 1970) and susceptible pigs have increased adenyl cyclase activity (Willner et al., 1979), a common mechanism exists for stimulation of metabolism by either stress or halothane. Although these explanations appear separate, it is obvious that increased release of catecholamines from the adrenal medulla or sympathetic nerves may facilitate release of acetylcholine, will cause vasoconstriction, and will stimulate muscle glycogenolysis, all of which will result in the rapid onset of anaerobic

PORCINE STRESS SYNDROMES

189

glycolysis. Therefore, there seems to be overwhelming evidence for a primary role for catecholamines in the development of PSS or MH and PSE. Indeed the changes in cardiac function (Gronert et al., 1978), hyperglycemia (Lucke et al.: 1976), skin blotchiness, cyanosis, and lactacidosis (Harrison et al., 1968) can be explained by the effects of catecholamines. In addition, once the process has been initiated, release of catecholamines is self-perpetuating, as the most potent causes of catecholamine secretion are acidosis (Nahas et al., 1974) and increased plasma-ionized calcium (Douglas et al., 1963). Both of these conditions exist in PSS and MH. However, if catecholamines did play a primary role, then blockade of adrenergic receptors should reverse established PSS or MH, but neither can be blocked or reversed by a-or P-receptor antagonists (Lister et al., 1976) or by neuromuscular blockade (Hall et al., 1976b; Harrison, 1973). Furthermore, doses of catecholamines which produce fatal responses in susceptible pigs are without significant effect in resistant pigs (Hall et al., 1977a). Thus it seems likely that catecholamines can exacerbate metabolic changes which are dependent on a defect beyond the adrenergic receptors. If this were not so, then blockade of the receptors, and thus the stimulus, should stop the reaction. This is not the case. Further, Altrogge et al. (1980) have shown that urinary levels of catecholamines are similar in both resistant and susceptible pigs, which implies that a primary abnormality in catecholamine secretion or metabolism is not present. Perhaps the most convincing evidence that catecholamines are not involved primarily, at least in MH, is that complete inhibition of catecholamine release and neuromuscular blockade do not protect susceptible animals from halothane (Gronert et al., 1977a; Harrison, 1973). In these circumstances, halothane must be acting beyond the motor end plate and the adrenergic receptors, to produce the changes which can be enhanced by catecholamines. Thus, catecholamines, although integral to the syndromes, cannot be considered to be part of the primary lesion. 2.

Thyroid Hormones

It is well recognized that thyroid hormones regulate tissue metabolic rates. Hypermetabolism of tissues and hyperthermia are characteristic of PSS and MH, and an a priori link between thyroid hormone activity and stress seems likely (Williams et al., 1978a). Furthermore, thyroxine decreases calcium uptake and binding by sarcoplasmic reticulum (Ash et al., 1972), can alter the structural integrity of mitochondria (Tapley, 1956; Tapley and Cooper, 1956), and stimulates the release of calcium ions from cardiac (Harris et al., 1979) and skeletal muscle mitochondria (Heffron and Harris, 1981). Thus two organelles involved in muscle contraction and metabolism are affected by the action of thyroid hormones. Consequently, hyperthyroidism results in the rapid development of rigor after

190

G. MITCHELL AND J. J. A. HEFFRON

death (Hoet and Marks, 1926), a fast rate of fall of muscle pH, low muscle ATP, and the development of PSE (Marple et al., 1975). Since stress-susceptible pigs have high resting thyroid hormone levels (Judge et al., 1966, 1968; Eighmy et al., 1978), a link between porcine stress syndromes and high thyroid hormone activity is established. Further, injections of large doses of triiodothyronine exacerbate PSS (Lister, 1973), whereas thyroidectomy retards lactate accumulation in muscle (Marple et al., 1975). Hyperthyroidism as a cause of PSE is, however, incompatible in some respects with results of Ludvigsen (1957) and Lister (1973). Ludvigsen inhibited thyroid gland activity and produced PSEPS. Lister reported that triiodothyronine levels decrease during PSS as catecholamines increase, a finding confirmed by Christenson (1973). The probable reason for the fall in thyroid hormones is increased peripheral utilization. An explanation for the development of PSEPS when thyroid hormone levels are low is that low levels of the hormone could produce a fall in oxidative phosphorylation and, consequently, facilitation of onset of anaerobic metabolism (Judge et al., 1968). This seems likely since Lister was able to treat PSS by injecting low doses of thyroid hormones. Thus both high and low thyroid activity could lead to PSE. On balance though, the evidence indicates that thyroid levels are likely to be higher in stress-susceptible pigs and to remain high during an episode of stress (Williams et al., 1978a). In these circumstances, since sympathetic nerves can directly influence thyroid secretion, and thyroid hormones have a permissive action on adrenergic receptors (Melander et al., 1974), high thyroid levels could facilitate development of both MH and PSS. Nevertheless, since PSS can develop in animals unlikely to have aberrant thyroid activity, thyroid hormones cannot be considered to be essential for the onset of either MH or PSS, as claimed by Williams et al. (1978a). 3. Corticosteroids

Another hormone which has a permissive action on catecholamines is cortisol. Moreover, cortisol is a major regulator of glycogen metabolism, has vasodilator properties, and can affect muscle water content (Conway and Hingerty, 1953). However, whereas a role for catecholamines is fairly clear-cut, a role for cortisol in the porcine stress syndromes is not. Ludvigsen (1957) first drew attention to the possibility that adrenal insufficiency could contribute to the development of PSE. He was able to prevent PSE by injecting 50 mg of hydrocortisate before exercise. The explanation offered was that the interaction between catecholamines and cortisol maintained muscle microcirculation and thus prevented hypoxia. The idea of a role for cortisol has been supported by other data (Judge et al., 1966, 1968; Tope1 et al., 1967; Briskey et al., 1966). Judge et al. (1966, 1968) also found a positive correlation

PORCINE STRESS SYNDROMES

191

between low urinary 17-ketosteroids and PSE and between low urinary 17hydroxycorticosteroids and the length of the delay phase (as defined by Briskey et al., 1966) to onset of rigor and total time of completion of rigor. Stresssusceptible pigs have also been shown to have lower resting cortisol levels (Sebranek et al., 1973; Mitchell and Heffron, 1981a) and a lower net increase in plasma 17-hydroxycorticosteroids after heat stress and exercise (Forrest et al., 1968) in comparison to stress-resistant pigs. Further, injections of adrenocorticotropic hormone (ACTH) do not produce the same increase in plasma cortisol in susceptible pigs as they do in resistant pigs (Sebranek et al., 1973; Mitchell and Heffron, 1981a). All of these data indicate an adrenocortical insufficiency. However, virtually all of the studies have also found increased plasma ACTH levels (Marple 1972; Marple and Cassens, 1973; Sebranek et al., 1973), whereas Ludvigsen (1957) noted low pituitary ACTH concentration. Thus, Marple and Cassens (1973) have argued that since resting cortisol levels are low and ACTH levels high (60 and 200%, respectively, of normal), there is no adrenal insufficiency but rather increased metabolism of cortisol and reduced negative feedback on ACTH releasing factor. They supported this contention by finding a 5-fold increase in the rate of turnover of infused cortisol in susceptible pigs (Marple and Cassens, 1973). These experiments, however, only show that susceptible pigs can metabolize cortisol which is in plasma. They do not answer the crucial question of whether susceptible pigs can secrete cortisol, although clearly rapid metabolism of any secreted cortisol will compound the effects of a deficiency. There is some evidence for adrenal insufficiency and for the fact that the insufficiency is a result of reduced stimulation of the adrenal cortex by ACTH. This evidence is that halothane which causes the release of cortisol (Oyama et al., 1968; Oyama and Takiguchi, 1970; Mitchell and Heffron, 1981a) has a greater effect than ACTH in stress-susceptible pigs on both rate of increase and total increase of plasma cortisol (Mitchell and Heffron, 1981a). Thus the gland cannot be exhausted and there must be biosynthesis of cotisol, but it seems to have reduced sensitivity to ACTH. This defect would be compounded by the finding of Sebranek et al. (1973) that there is a disturbance in feedback control of ACTH release: dexamethasone which normally inhibits ACTH release does not do so in stress-susceptible pigs. Thus decreased secretion of cortisol resulting from an attenuated effect of ACTH and reduced inhibition of ACTH release, would adequately explain the high plasma ACTH, low plasma cortisol, decreased response to injected ACTH and stress, and low urinary excretion of steroid metabolites, all of which are characteristic of stress-susceptible pigs. The consequences of reduced secretion (and increased metabolism) would be glycogenolysis (Dimarco et al., 1976), vasoconstriction, and the facilitation of PSS and MH. However, a lack of cortisol is not essential to the development of the syndromes. Lister et al. (1972) have not shown reduced responses in susceptible pigs to injected ACTH and Marple et al. (1974) have shown that despite a 6-

192

G. MITCHELL AND J. J. A. HEFFRON

to 10-fold increase in ACTH and a 2- to 3-fold increase in cortisol, stressresistant pigs can develop a lactacidosis and may die and develop rigor within a few minutes of death, after exposure to severe stress. Moreover, hydrocortisol will not reverse established MH either in in vitro models (Isaacs et al., 1975) or in vivo in pigs (Hall et al., 1977b) and one study (Jedlika et al., 1976) has suggested that heat stress increases plasma 17-hydroxysteroid levels in pigs, with the highest increase occurring in pigs with poorest meat quality. Despite this discrepancy, it seems likely that in stress-susceptible pigs inhibition of ACTH secretion is deficient and secretion of cortisol is deficient. It seems unlikely that metabolism of cortisol is greater since urinary excretion of cortisol metabolites is low. It is also unlikely that adrenocortical insufficiency is essential to the development of PSS, MH, or PSEPS, although reduced secretion of cortisol in response to stress will facilitate their onset. 4.

Conclusions

The data reviewed above suggest that catecholamines, thyroid hormones, and reduced secretion of cortisol all play contributory roles in the development of the syndromes, both pre- and postmortem. The changes in circulating hormone levels are summarized in Fig. 1. Hypermetabolism, and the associated production of lactate and heat, causes changes in the permeability of cell membranes, loss of electrolytes and enzymes to plasma, muscle stiffness and necrosis, and death. All can be attributed to the effects of hormones. However, halothane causes metabolic changes before catecholamine levels rise (Gronert et al., 1976a) and without affecting thyroid hormone levels (Eighmy et al., 1978) while causing substantial increases in plasma cortisol (Mitchell and Heffron, 198 la). Thus halothane must be exploiting a defect which is not concerned with the primary effects of hormones, but which produces similar signs to those produced by hormones. Further, the defect must be such that should hormone levels change, all the characteristic changes of PSS, MH, and PSE will develop, even in the absence of halothane or other drugs. Thus sensitivity to catecholamines, hyperthyroidism, and adrenal insufficiency, although integral to the syndromes, must be relegated to a secondary role in them. The primary lesion must lie elsewhere. B.

MUSCLE METABOLISM

The previous discussions have suggested that the lesion caused by the genetic defect underlying PSS and MH is latent and can be exacerbated by the action of hormones or drugs. It is generally assumed that a site of the genetic defect in stress-susceptible pigs is in skeletal muscle. No other tissue can produce the large amounts of lactate and SCPK that are characteristic of PSS and MH. Moreover, muscle rigidity is almost pathognomonic for the syndromes and hyperthermia

193

PORCINE STRESS SYNDROMES

r

Sarcolemmoa

l

Z-line

FIG. 6 . Diagram of the principal functional structures of a mammalian skeletal muscle fiber: neuromuscular junction, sarcolemma, transverse tubule, mitochondrion, and contractile proteins.

seems to depend on muscle contraction. However, although muscle tissue undoubtedly contains the defect, liver (Hall et al., 1980a) and cerebral tissue (Artru and Gronert, 1980) may carry the defect but react less conspicuously. Indeed, Britt and Kalow (1970b) proposed that the defect was a general membrane defect and there is some evidence to support this: erythrocytes are more fragile (Harrison and Verburg, 1973; Heffron and Mitchell, 1981; Ollivier et al., 1975), ACTH interaction with adrenal cortex cell membrane receptors is attenuated (Mitchell and Heffron, 1981a), and muscle cell membranes are more permeable in susceptible than in resistant pigs. Despite the possibility that the defect is widespread, muscle tissue has been used in experiments to uncover the lesion to the exclusion of virtually all other tissues. Further, in skeletal muscle there are, broadly, only four organelles which could be affected to produce the signs of PSS and MH. These are the neuromuscular junction and its associated structures, the sarcoplasmic reticulum, the contractile proteins, and the mitochondria (Fig. 6). All have been studied in some detail. 1. Neuromuscular Junction

The neuromuscular junction, the sarcolemma, and the T tubules function as the trigger mechanism for muscle contraction and it is possible, therefore, that if an abnormality exists here, it would facilitate the onset of PSS or MH. Evidence

194

G. MITCHELL AND J . J . A. HEFFRON

that the sarcolemma is abnormal is that stress-susceptible pigs have higher resting levels of SCPK activity than resistant animals (Heffron and Mitchell, 1975b). Since the major source of SCPK is muscle CPK, increased SCPK implies leakage of the enzyme into plasma. There is also evidence that halothane can increase sarcolemma permeability: halothane causes release of CPK from normal muscle cells (Innes and Stromme, 1973) but causes a greater release from muscle cells of susceptible pigs (Woolf er al., 1970), as well as causing release of myoglobin and other constituents of muscle cells which are not released from normal cells (Berman et al., 1970). Additional evidence that the sarcolemma of muscle of susceptible pigs is abnormal has been provided by Gallant et al. (1979). They showed that halothane, at concentrations which initiate MH, depolarizes the membranes of susceptible but not those of normal Poland China pigs. Some anatomical evidence that there is a neuromuscular junction abnormality also exists. Swatland and Cassens (1972) have shown that in stress-susceptible pigs there is a greater mean maximum end plate diameter, greater terminal sprouting, and more double end plates in both red and white muscle than in normal pigs. They concluded that there is a relationship between neural growth, muscle hypertrophy, and stress susceptibility. Muir ( 1970) has provided evidence that there is an ongoing myopathy in stress-susceptible pigs which affects superficial as well as intracellular membranes. In man, neural defects have been postulated on the basis of SCPK studies (Starkweather et al., 1973), other biochemical studies (Heffron and Isaacs, 1976), and electromyographic studies (McComas et al., 1974). Further, both denervated muscle and muscle from MHsusceptible patients are hypersensitive to halothane (Moulds and Danborough, 1974). However, in a more detailed study Moulds (1978) concluded that MH muscle was not similar to denervated muscle. Although they have not been studied in any detail, some evidence exists that the T-tubule system may be involved. This evidence is that glycerine, which disrupts the T system, prevents suxamethonium-induced contractures and diminishes halothane-induced contractures. Nevertheless, halothane and caffeine in combination produce contractures in the presence of glycerine (Okumura et al., 1980). These experiments suggest, therefore, that part of the action of suxamethonium is on the T system, but they also suggest that halothane, although acting on the T system and the sarcolemma, must also act elsewhere in muscle cells. In pigs, there is also other convincing evidence that the site of a more definitive lesion is beyond the motor end plate. This evidence is that acetylcholine does not induce in virro contracture in muscle from susceptible pigs (Okumura er al., 1980) and that premedication of susceptible pigs using tubocurarine (Hall et al., 1976b) or curare (Harrison, 1971, 1973), while preventing onset of suxamethonium-induced PSS (or MH), will not prevent halothane-induced MH or

PORCINE STRESS SYNDROMES

195

prevent in vitro contractures induced by halothane (Okumura et al., 1980). In addition, dantrolene, which probably acts beyond the end plate (Ellis et al., 1977), will reverse MH (Harrison, 1975; Gronert et al., 1976b), whereas neuromuscular blockade will not (Hall et al., 1976b). Further data to support the contention that a triggering mechanism defect is not primary are that 4-aminopyridine does not cause PSS or MH (Hall et al., 1980~).4-Aminopyridine increases acetylcholine secretion by nerves and increases muscle contractility by prolonging the action potential. Thus since PSS or MH is not caused by 4aminopyridine, a compound which acts on the sarcolemma, the sarcolemma is unlikely to be the site of the primary lesion. But even though the data suggest that the neuromuscular junction and its associated structures are not the key site of the genetic defect, the facts that the sarcolemma stores 10% of the muscle cell calcium (Sulakhe et al., 1973), that the T-tubule system is involved in depolarization, and that calcium initiates muscle contraction mean that all these components of the triggering mechanism could contribute to the onset of PSS and MH. It can be predicted from this observation that if the nerve-muscle interface is not stimulated, then the onset of halothane-induced MH should be slower than the onset of suxamethonium-induced PSS. This prediction is generally true in that the mean onset time for halothane-induced MH is 18-20 min and that for suxamethonium is 1-2 min (Hende et al., 1976; Gronert et al., 1976a). In this context it has been suggested that there is less inhibition of release of acetylcholine at neuromuscular junctions of susceptible pigs (Altrogge et al., 1980). In these experiments urinary concentration of dopamine was found to be significantly less in susceptible pigs than in resistant pigs. Since dopamine inhibits the excitatory action of motor nerves to skeletal muscle, Altrogge et al. (1980) have suggested that overstimulation of neuromuscular junctions occurs in susceptible pigs. However, as noted above, acetylcholine does not induce contraction of muscle from susceptible pigs in vitro (Okumura et al., 1980) and 4-aminopyridine, which increases acetylcholine release from nerves, does not cause MH (Hall et al., 1980~).Nevertheless, it can be argued that if the concentration of halothane is increased so that both the triggering mechanisms and subcellular effects of halothane occur simultaneously, the onset of halothane-induced MH will be rapid. However, the fact that the syndromes can occur in the absence of muscle activity suggests that the contribution of a defect in the triggering mechanisms to the onset of signs is small, and the key lesion is more likely to be elsewhere in the muscle cell. 2. Sarcoplasmic Reticulum The onset of muscle rigidity or stiffness in pigs is one of the first macroscopic signs of an impending episode of MH or PSS and is not clinically distinguishable from rigor. The onset of rigidity occurs within 1-4 min of initiation of halothane

I96

G. MITCHELL AND J. J . A. HEFFRON

anesthesia (Harrison et al., 1968; Berman and Kench, 1973; Mitchell et al., 1980) and long before there is depletion of ATP and a fall in muscle pH to 6.2the two well-established causes of rigor (Szent-Gyorgi, 1944; Bate-Smith and Bendell, 1947). The rigidity cannot, therefore, be rigor and must be a result of some other cause. For these reasons the theory often presented to account for the changes seen is that the rigidity is caused by sustained high levels of cytoplasmic calcium (Britt and Kalow, 1970b). Since 80% of all calcium in skeletal muscle is sequestered in the sarcoplasmic reticulum (SR) (Sulakhe er al., 1973), by implication the SR must be the site of the basic lesion. This view is widely held (Britt, 1979). There is no doubt that calcium plays an important role in the regulation of muscle contraction and associated events, and thus in the porcine stress syndromes. For example, after release from storage depots myoplasmic calcium M to produce contraction (Bianchi, must reach a concentration of 5 x 1973). The calcium binds to troponin (Ebashi et al., 1967), producing a conformational change in troponin (Han and Benson, 1970) which exposes actin and myosin binding sites and allows the formation of actomyosin (Weber and Herz, 1961). This process requires energy from ATP, and calcium indirectly simultaneously stimulates actomyosin ATPase, which hydrolyzes the ATP to produce energy (Weber and Herz, 1961) (Fig. 6). Calcium also activates phosphorylase kinase b (Ozawa and Ebashi, 1967) by action on its 6 subunit, calmodulin (Cohen, 1979), thus stimulating glycogenolysis. It is clear, therefore, that a rapid and sustained increase in myoplasmic calcium would not only cause muscle contraction, but also contribute to the generation of lactate. The sources of calcium are the extracellular fluid space, the sarcolemma, the mitochondria, and the SR. The facts that plasma calcium increases initially in MH (Berman et al., 1970) and that the sarcolemma is normally impermeable to extracellular calcium suggest an outward movement of calcium into the extracellular space rather than the reverse (Britt, 1979). Further, since the sarcolemma and mitochondria each contain 10% of the cell calcium, their contribution to raising myoplasmic calcium from 10V8 M to l o p 6M must be small. By far the most likely source of calcium is, therefore, the SR. These arguments have led to much study of the SR, using procaine and isolated SR. The rationale for the use of procaine stems from Bianchi’s (1968) finding that procaine, because of its high pK,, can enter cells easily in a charged lipophilic form at pH 7.4. Once in the cell, it antagonizes calcium influx into the myoplasm from the SR, thus explaining Feinstein’s (1963) observation that procaine will inhibit caffeine-induced contracture of skeletal muscle. The work of Feinstein (1963), together with a chance and apparently successful administration of procaine during an episode of MH (Beldavs et al., 1971) and the finding that procaine will reverse halothane-induced skeletal muscle contracture in vitm (Moulds and Demborough, 1972; Harrison and Verburg,

PORCINE STRESS SYNDROMES

197

19731, provided circumstantial evidence that excessive leakage of calcium from the SR was the cause of MH. These preliminary data also led to many attempts to use procaine as a means of treating established MH. Some of these attempts were reported to be successful in man (Beldavs et al., 1971; Brebner and Jozefowicz, 1974; Giosa, 1975; Hoivik and Stovner, 1975; Keaney and Ellis, 1971; Noble et al., 1973; Relton et al., 1972; Strobel, 1971) and in pigs (Harrison, 1971, 1973). On the other hand, several reports have indicated that procaine is ineffective in both man and pigs in reversing MH (Clarke and Ellis, 1975; Flewellyn and Nelson, 1979; Gronert et al., 1976b; Hall et al., 1972; Hall and Lister, 1974; MacLachlan and Forrest, 1974; Mitchell and Heffron, 1975b,c). The only possible way that procaine can reverse the established syndrome is to stimulate calcium ATPase-the enzyme responsible for pumping cytoplasmic calcium into SR. But in experiments designed to assess this (Green et al., 1976), significant inhibition of the enzyme occurred at concentrations of procaine ( 1-5 mM) which had reversed in vitro contracture (Feinstein, 1963; Moulds and Denborough, 1972), which suggests that procaine will exacerbate MH rather than reverse it. Indeed procaine’s main action as reported by Feinstein (1963) and Bianchi (1968) is in preventing calcium release from the SR. Thus if procaine is to be effective, than pretreatment only would be effective. In this context Feinstein (1963) showed that procaine could prevent caffeine-induced contractures of skeletal muscle only if procaine was added to the medium before caffeine. Further, since Feinstein used concentrations of procaine in the 1-5 mM range, it is possible that contractures occurring when procaine was added after caffeine were partly a result of inhibition of ATPase-coupled calcium uptake by the SR. Although pretreatment with procaine will theoretically prevent PSS or MH, Hall et al. (1972; Hall and Lister, 1974) have shown that pretreatment of pigs with procaine does not prevent onset of the syndromes or reduce rigidity. However, this finding and the failure of procaine to reverse MH are also predictable because the procaine concentrations required to reverse contracture in vitro are not attainable clinically. The concentrations required to reduce caffeine-induced contracture range from 1.8 to 3.7 mM (Feinstein, 1963; Weber and Herz, 1968), whereas reversal of halothane-induced contracture requires 5 mM (Moulds and Denborough, 1972). A concentration of 5 mM corresponds to a dose of 6.8 g for a circulating volume of 5 liters, yet Wikinski et al. (1970) have found that after accidental, and near fatal, intravenous injection of 4 g of procaine, the highest blood level was 0.41 mM. Assuming a linear response, 6.8 g would produce a maximal blood concentration of 0.7 &-a value 3-7 times less than the concentration of procaine required in vitro, and a dose 4-7 times greater than the lethal dose for pigs (Mitchell and Heffron, 197%). The recommended dose of procaine for reversal of the syndrome in pigs-30-40 mg/kg followed by an infusion of 0.2 mg/kg/min (Harrison, 197i)-also exceeds the toxic dose for

198

G . MITCHELL AND J. J. A. HEFFRON

pigs by %fold. In addition, Hall and Lister (1974) have reported that 2.1 mM procaine inhibits myocardial contraction. Thus it has been established that procaine should be disregarded as a therapeutic agent. The findings that procaine is useful and those which suggest it is not, are difficult to reconcile. However, as Hall et al. (1975) point out, it is impossible to ascribe the reversal of MH to procaine when its use has commenced after the previous administration of numerous other drugs. This observation raises a second point. Most treatment regimes include bicarbonate to counteract the acidosis. Harrison (197 1) originally used procaine after bicarbonate and Hoivik and Stovner (1975) have observed that procaine is more useful after correction of the acidosis. Thus it is possible that the pH is crucial to whether or not procaine is effective at the low concentrations attainable clinically. Bianchi ( 1968) has pointed out that pH < 7.2 results in decreased entry into cells of tetracaine, which has a similar structure and pK, to those of procaine. Another possible explanation for procaine’s reported success is that it may reduce the secretion of catecholamines from the adrenal medulla. Rubin et al. ( 1967) have reported that 0.3 mM tetracaine inhibits catecholamine secretion. However, this explanation is unlikely since specific adrenergic blockade does not reverse MH. It can also be argued that survival of humans, but not pigs, after procaine may be coincidental since MH in humans is only fatal in 60-70% of the cases (Britt and Kalow, 1970a). In pigs though, it is possible that those pigs surviving halothane anesthesia after pretreatment with procaine were not properly identified as being susceptible to MH. On balance then, these observations suggest that procaine is unlikely to be of use therapeutically in MH or PSS. More importantly, these data suggest that the SR is not the site of the basic biochemical lesion, a suggestion supported by the fact that 4-aminopyridine, which indirectly prolongs depolarization of the SR, does not cause MH or PSS (Hall et al., 1980~).However, the SR cannot be excluded on these data alone since there is evidence that the SR function is altered both directly and indirectly by halothane. The evidence for a direct effect is that the rate of and total calcium uptake by the SR obtained from normal pigs and undergoing halothane anesthesia are, respectively, 11 and 13% less than the rate of and total calcium uptake obtained during barbiturate anesthesia (Heffron and Mitchell, 1976), a finding supported by other data (Brucker et al., 1973; Denborough et al., 1973). In this context it has been established that halothane can produce conformational changes in the protein component of the SR (Augustin and Hasselbach, 1973), and although this is reversible in normal animals, it may not be so in affected animals. It is also possible that the 1% of cell CPK which is bound to the SR and which may play a significant role in calcium transport into the SR during muscle relaxation by providing ATP (Baskin and Deaner, 1970), may not function in the presence of the halothane-induced distortion of the SR membrane.

PORCINE STRESS SYNDROMES

199

Over and above its direct effects on SR, halothane, by causing metabolic changes in susceptible animals, can also have indirect effects on the SR. Thus the SR isolated from susceptible pigs undergoing halothane anesthesia accumulates about 60% less calcium than it accumulates during barbiturate anesthesia (Heffron and Mitchell, 1976). Further, Britt et al. (1975a) have argued that the capacity of the SR, isolated from susceptible pigs, to bind calcium, already 50% of expected, is reduced further by halothane anesthesia in vivo. Since normal calcium uptake by the SR of susceptible pigs has been reported in the absence of halothane (Berman and Kench, 1973; Nelson et al., 1972) and in v i m in the presence of halothane (Britt et al., 1975b), the case for indirect effects is strengthened. The two most likely indirect causes of alteration of SR function are hyperthermia and acidosis. Raising the temperature of the SR undoubtedly reduces its calcium uptake (Nelson and Bee, 1979). Moreover, the onset of decreased binding in susceptible pig SR occurs at lower temperatures than in normal SR (Nelson and Bee, 1979). The explanation for decreased binding is that at temperatures between 37 and 40°C the processes governing calcium accumulation and ATPase activity are uncoupled, and calcium efflux from the SR proceeds rapidly (Inesi et al., 1973). Inesi et al. (1973) have concluded that this is due to a thermally induced change in the protein conformation of the SR membranes. However, during MH significant temperature increases occur a relatively long time after the effects of putative SR malfunction (muscle rigidity) have become evident (Fig. 2). Thus temperature-induced changes are not likely to cause SR malfunction initially but may eventually contribute to it. Acidosis will also affect SR function. McIntosh et a?. (1977; McIntosh and Berman, 1974) have shown that the SR of stress-susceptible pigs has decreased calcium binding, despite high ATPase activity, and that a decreasing pH enhanced this uncoupling. Further, although originally suggesting that the phospholipids in the SR membranes contained a higher proportion of unsaturated fatty acids, they concluded, like Inesi et al. (1973), that the decreased calcium binding was not related to abnormal phospholipids, but that low pH caused protein denaturation. Low pH, therefore, increased calcium transport and calcium efflux, and since this ultimately reduces pH further by stimulating glycolysis, results in a cyclic and uncontrolled calcium release. However, the elegant experiments which suggested this sequence of events were done at pH 5 . 4 5 . 7 , far below that which occurs in muscle during MH or PSS. These observations suggest that the SR from stress-susceptible pigs is altered not only by halothane directly, but also by high temperatures, and perhaps more importantly, by low pH. The consequences of these changes are high myoplasmic calcium, muscle rigidity, and metabolic changes. Nevertheless, in the most detailed and careful study yet of the calcium-binding properties of the SR, Gronert et al. (1979), although confirming that changes in SR activity do occur,

200

G . MITCHELL AND J. J. A. HEFFRON

have concluded that the differences between the SR of susceptible and normal pigs are insufficient to explain the onset of MH and PSS in susceptible pigs. Nevertheless, they do conclude that if secondarily involved, SR malfunction will contribute to the changes occurring in MH and PSS. 3.

Contractile Proteins

The possibility that neither the sarcolemma nor the SR are primarily involved in PSS and MH led to speculation that the contractile proteins themselves may be abnormal. Thus Fuchs (1975) suggested that there may be a loss of calcium sensitivity in the contractile proteins of muscle during episodes of MH, rather than an increase in myoplasmic calcium concentration. Fuchs (1975) and Fuchs et al. (1975) have shown that at temperatures slightly greater than those occurring physiologically, there is a loss of calcium dependence for the occurrence of superprecipitation of natural actomyosin (NAM) prepared from frog, rabbit, and human muscle. It has also been shown (Levy and Fleisher, 1965) that increased concentrations of ATP can “protect” against a loss of calcium sensitivity and that low concentrations of magnesium will activate superprecipitation of NAM (de Villafranca and Waksmonski, 1970). On the other hand, high concentrations of magnesium inhibit superprecipitation (Weber and Winicut, 1961) and cause clearing of established superprecipitation of NAM (de Villafranca and Waksmonski, 1970). Since cell ATP levels ultimately fall during MH (Mitchell et al., 1980; Berman et a l . , 1970; Harrison et al., 1968; Nelson et al., 1974) and magnesium enters the plasma (Berman et al., 1970; Hall et al., 1975), it seems entirely possible that muscle contraction during MH or PSS can become independent of calcium as temperature increases, and if ATP and Mg2+ leave the cells. The experiments on NAM reported above and those reported in the sole study on superprecipitation in susceptible and normal pigs (Green et al., 1980) have assumed that NAM is a legitimate model for the complex intermolecular rearrangements which occur during muscle contraction. Although there is some questioning of this assumption, in that intramolecular changes may also occur, it is generally accepted that superprecipitation of NAM as a model of the sliding filament theory of contraction is at least reliable, if not perfect (de Villafranca and Waksmonski, 1970). Thus Green et al. (1980) found that NAM from pig muscle behaves in the same way as that of frog, rabbit, and human muscle in that calcium sensitivity is lost at temperatures 2 4 ° C higher than normal physiological temperatures. More importantly, however, the results suggested that the contractile proteins are not involved primarily in the onset of MH and PSS. The evidence for this is that there are no significant differences between NAM from halothane-sensitive pigs and that from halothane-resistant pigs, when ATP and Mg2+ levels are similar. Nonetheless, the data did suggest that the onset of superprecipitation

20 1

PORCINE STRESS SYNDROMES

occurs at lower temperatures (43°C) in susceptible animals than those (45°C) in resistant animals. Further, disturbances in the concentrations of ATP and Mg2+ can lead to rapid onset of superprecipitation, an effect potentiated by increasing temperature (Fig. 7). These experiments suggested, however, that it is more likely that rigor is potentiated than that rigidity is produced by a loss of calcium sensitivity since hyperthermia, a fall of muscle ATP, and leakage of Mg2 into plasma occur late in the development of PSS and MH (Figs. 1 and 2). Nevertheless, the muscle stiffness during exercise noted in pigs susceptible to MH and PSS (Ludvigsen, 1957; Hende et al., 1976) and horses susceptible to exertional myopathy (Hammel and Raker, 1972) may be partly explained by a hyperthermia-induced loss of calcium sensitivity of contractile proteins. It must also be noted that the effect of pH on calcium sensitivity of pig muscle, as yet unknown, could also be important. An important implication of these results is that attempts to reverse established MH or PSS by decreasing myoplasmic calcium must be futile once calcium sensitivity is lost. The drug most commonly used to decrease calcium during MH, and thus reverse MH, is dantrolene, an amino-hydantoin derivative (Snyder et al., 1967). Dantrolene has been thought to act by pumping calcium into the SR (Snyder et al., 1967). However, it has been shown that dantrolene does not stimulate calcium-dependent ATPase of the SR (Green et al., 1976) and its solubility in physiological media suggests that it acts superficially, an idea supported by the finding that dantrolene antagonizes the effects of 4-aminopyridine, which acts on the sarcolemma (Bowman et al., 1977). Thus it could reverse halothane-induced contractures (Anderson and Jones, 1976; Nelson and Bee, 1979; Gallant et al., 1979) by preventing muscle depolarization (Ellis et al., 1977; Gallant et al., 1979; Bowman et al., 1977) or by inhibiting calcium influx from extracellular fluid (Putney and Biamchi, 1973), thus raising the mechanical threshold and resting membrane potential (Gallant et al., 1979). But as halothane +

I

I

(.-.-.. (.-.-..

I I

0 2-

t-5555 -50 50

I

2

-$

E!

0

u)

-45

a

-40

P

't35 -35

0' 0

5

10 15 Time (minutes)

20

30 25

FIG. 7. The effect of magnesium and ATP concentrations on superprecipitation of NAM (see text). - -) 1.0 mM Mgz+ + 1 mM ATP; (-.-.) 4.0 mM Mg*+ + 1 mM ATP; + 4 mM ATP. Data from Green et a / . (1980).

(-) Temperature; (- (- - -) I .O mM Mg*+

202

G . MITCHELL AND J . J. A . HEFFRON

acts both at and beyond the end plate, these maneuvers should not reverse halothane-induced MH. The fact that dantrolene does reverse the syndrome suggests, therefore, that its effect is more likely to be due to its action in preventing calcium efflux from the SR (Ellis et al., 1977; Anderson and Jones, 1976; Hainaut and Desmedt, 1974), which implies that it has a procaine-like action, yet unlike procaine, it has been used successfully and consistently in both pigs and man to prevent onset of and to reverse MH (Harrison, 1975, 1977; Pandit et al., 1979; Gronert et al., 1976b; Friesen et a l . , 1979). Moreover, it not only reduces rigidity but also reverses metabolic changes. Its effect may, therefore, be the result of a combination of more than one of the above actions and it may act at other sites, for example, at the level of calmodulin. It is also significant that in none of the reports mentioned above does the temperature of successfully treated patients or pigs exceed a temperature of 43°C-the temperature at which superprecipitation occurs (Green et al., 1980). Further, the in vitro studies which have illustrated the effectiveness of dantrolene have been done at temperatures less than 43°C and usually at 37°C. Moreover, in another report there is a clear indication that the higher the temperature, the less effective dantrolene is in reversing MH (Liebenschutz et al., 1979). In a patient whose body temperature was 43"C, resolution of MH after injection of dantrolene took 9 hr, whereas at 40°C resolution took 2 hr 40 min. Thus if dantrolene is to be effective, then it must be used before the rectal (and muscle) temperature is so high that contraction of muscle occurs independently of calcium. In summary, the data reviewed here suggest that the contractile proteins of susceptible pigs are not primarily involved in the onset of MH. However, they can become involved secondarily, lose their calcium sensitivity, and potentiate the reaction. 4 . Mitochondria

The data reviewed so far suggest that the motor end plate, the sarcolemma, the SR, and the contractile proteins are not primarily involved in the development of PSS and MH, although their functioning may be abnormal and may also be disturbed by the metabolic changes occurring during MH and PSS. Thus, only mitochondria of the major organelles in muscle cells remain as a possible site of a major defect. The earliest suggestion that mitochondria may be involved in MH and PSS was made by Wilson et al. (1966) when they showed that uncoupling of oxidative phosphorylation by 2,4-dinitrophenol produced similar changes to those occurring in Mh. Also in 1966, Snodgrass and Piras assessed the effect of halothane on rat liver mitochondria and found, using very high, nonclinical concentrations of halothane, that oxidative phosphorylation was uncoupled at all

PORCINE STRESS SYNDROMES

203

three oxidation sites. Further, a mitochondrial structural change was induced by high concentrations of halothane in that after exposure to halothane, motochondria were able to oxidize exogenous NADH. Fink and Kenny (1968), using cultures of normal cells and clinical concentrations of halothane, showed that halothane increased glucose uptake and lactate production, while inhibiting oxygen consumption. To explain these observations, they concluded that the dihydroxyacetone shuttle in which extramitochondrial NADH is transported into mitochondria as a-glycerophosphate, was inhibited by halothane. The lack of cytoplasmic NAD+ consequent on this inhibition, and which is necessary for the conversion of lactate to pyruvate, resulted in an increase in lactate production. Fink and Kenny (1968) also observed that inhibition of pyruvate entry into mitochondria would produce the same result. To support these views, they argued that since mitochondria contain 40% lipids and anesthetics are highly lipid soluble, the effect of halothane was to disrupt the mitochondrial membrane. Further, the higher the lipid solubility, the more potent the agent in producing changes. Thus halothane, which is the most common cause of MH, is 100-fold more lipid soluble than nitrous oxide, which usually does not cause MH. Fink and Kenny (1968) also showed that inhibition of mitochondrial respiration must lead to a deficit of adenosine triphosphate (ATP). Oxidative ATP was reduced from 133 to 38 pmo1/106 cells. Glycolytic ATP cannot, therefore, compensate for loss of oxidative ATP, a finding which could explain the marked fall in cell ATP observed during MH (Nelson et al., 1974; Harrison et al., 1968; Mitchell et al., 1980). Since the report of Fink and Kenny (1968), several workers have shown that halothane affects mitochondrial respiration. Inhibition of NADH oxidation has been reported by Cohen et al. (1969), Harris et al. (1971), Williams et al. (1978a), and Rosenberg and Haugaard (1973). Inhibition of mitochondrial respiration and a subsequent decrease in ability to produce ATP has been reported by Cohen et al. (1969), Gatz and Jones (1969), Brucker et al. (1973), and Eikelenboom and van der Borgh (1973). In support of these findings, Miller and Hunter (1970) showed a dose-dependent effect of halothane. At low concentrations a reversible inhibition of the electron transfer chain in the region of NADH dehydrogenase and limited uncoupling occurred. At higher concentrations inhibition of respiration resulted. Harris et al. (1971) also found a dose-related effectlow doses prevented oxidation of NADH-linked substrates and high nonclinical doses damaged the membranes since exogenous NADH could be oxidized after exposure. Denborough et al. (1973) have also shown that halothane inhibits respiration and uncouples oxidative phosphorylation, and that mitochondria exposed to halothane have a decreased ability to produce ATP. However, Brucker et al. (1973) have argued that a decrease in ATP is not due to uncoupling since DNP-induced uncoupling did not produce the same effects as halothane. Williams el al. (1978a) have also found no uncoupling, whereas Eikelenboom

204

G . MITCHELL AND J . J. A . HEFFRON

and van der Bergh (1973) have shown that the decreased ability to synthesize ATP is a result of inhibition, not uncoupling, of oxidative phosphorylation. On the other hand, Brooks and Cassens (1973), although finding that high temperature reduces the phosphorylation efficiency of mitochondria, have found together with Campion et al. (1974) no differences in the respiratory rate of mitochondria from susceptible and resistant pigs, and Campion and Tope1 (1975) have concluded that although functional uncoupling may occur, mitochondrial abnormalities are not important-a view supported by Britt et al. (1975~).However, Brooks and Cassens (1973) and Campion et al. (1974) did not use halothane or stress to precipitate latent differences. On balance, therefore, it seems that in the absence of halothane mitochondrial activity is similar in both susceptible and resistant pigs, although susceptible pigs may have reduced ATP synthesis. Low concentrations of halothane will depress mitochondrial respiration and higher concentrations will lead to uncoupling of oxidative phosphorylation. There are compelling arguments, however, that neither of these constitutes a major lesion. For example, inhibition of respiration should decrease oxygen consumption, heat production, and carbon dioxide production, yet virtually all reports record increases in these (Berman et al., 1970; Gronert et al., 1976a). Further, the amount of heat generated by uncoupling does not account for the heat produced during MH (Berman et al., 1970; Harris et al., 1971). More importantly, uncoupling of oxidative phosphorylation and inhibition of the electron transport chain is not unique to halothane. Methoxyflurane, trichloroethylene, and chloroform will all inhibit respiration or uncouple oxidation (Hall et al., 1973; Nahnvold and Cohen, 1973; Snodgrass and Piras, 1966), yet none of these cause MH as consistently as halothane. Also, thiobarbiturates act as uncouplers (Aldridge and Parker, 1960; Hall et al., 1980b) but do not produce MH and even suppress its development (Gatz and Jones, 1969; Hall et al., 1972; Harrison et al., 1969; Mitchell and Heffron, 1981b). Barbiturates also inhibit the NADH-CoQ reductase complex (Harris et al., 1971) without causing MH. These findings make it impossible for inhibition of respiration or uncoupling of phosphorylation to be responsible for all of the signs of MH, and consequently halothane must have additional effects. Moreover, in the absence of halothane, PSS occurs with identical signs. Thus it cannot be argued that halothane or halothane-induced effects are essential to the syndromes: the latent lesion must be able to produce the signs in the absence of triggering drugs. For these reasons attention has turned to the role of mitochondria in the regulation of myoplasmic calcium. As noted above, in an early article Snodgrass and Piras (1966) reported that very high concentrations of halothane prevented calcium accumulation by mitochondria. Their finding has been confirmed in a study using clinical concentrations of halothane (Heffron and Gronert, 1977). Further, volatile anesthetics can displace calcium from membranes, such as

PORCINE STRESS SYNDROMES

205

mitochondrial membranes, which are rich in lipids (Blaustein and Goldmin, 1966). The idea of a defect in calcium regulation associated with a defect in mitochondrial function is also supported by the finding that low ATP levels lead to release of calcium from mitochondria (Lehninger, 1970). In addition, mitochondria can accumulate calcium at the expense of ATP and when ATP levels decline, by the use of high-energy intermediates. The use of high-energy intermediates precludes formation of ATP, and calcium may thus cause uncoupling of oxidative phosphorylation (Bianchi, 1973). Several workers have shown that mitochondria from stress-susceptible pigs accumulate less calcium than normal pig mitochondria (Britt et al., 197%; Heffron and Gronert, 1977; Gronert and Heffron, 1979; Cheah and Cheah, 1976, 1978; Rosenberg and Haugaard, 1973) and that increasing temperature (Cheah and Cheah, 1978), anaerobiosis (Cheah and Cheah, 1976), and halothane (Rosenberg and Haugaard, 1973) will further decrease calcium uptake into mitochondria. An additional conclusion that has been reached (Britt et al., 197%; Cheah and Cheah, 1978) is that decreased calcium uptake is consistent with a mitochondrial membrane defect, since calcium is usually associated with a phospholipid lattice. More recently Cheah and Cheah (1979), while confirming the membrane defect, have indicated that reduced calcium binding is a result of rapid calcium efflux, although the method used in these studies is subject to error (Scott and Jeacocke, 1980). In general, the data suggest that a defect in calcium accumulation by mitochondria is probable, and thus such a defect would contribute to raising myoplasmic calcium levels. However, this defect is unlikely to be a major cause of MH or PSS (Gronert and Heffron, 1979), mainly because mitochondria sequester only 10% of cell calcium (Sulakhe et al., 1973). All of these studies on mitochondrial function have been discussed with a view to establishing a role for depletion of ATP and/or increase in myoplasmic calcium as initiating events in MH or PSS (Fig. 8). Both of these ideas have been proposed (Britt, 1979; Ahern et al., 1980). However, as an increase in myoplasmic calcium will only explain muscle rigidity and not the diversion of glucose metabolism to lactate or the release of catecholamines, a secondary role for calcium seems likely. Similarly, cell ATP levels fall only when MH is well established (Mitchell et al., 1980; Berman et al., 1970; Fig. 2 ) , thus a fall in ATP does not initiate the syndromes. Nonetheless, the observations reported on mitochondrial function, when allied to the observations that halothane at clinical concentrations can cause structural changes in the membranes of mitochondria (Harris et al., 1971; Fink and Kenny, 1968; Chang et al., 1975) and that any putative lesion must also account for changes in the absence of drugs, lead one to conclude that the membrane defect may cause other changes. In this context, it has been shown that one of the earliest changes occurring during the syndromes is an increase in blood lactate (Gronert et al., 1976a), a

206

G . MITCHELL AND J. J. A. HEFFRON

neural input

1 acetylcholine I

catecholamines

~

FIG. 8. The principal events and sources of energy for muscle contraction. Note that under aerobic conditions NADH and pyruvate are transported into mitochondria with little lactate production.

change preceded by a decrease in muscle cell creatine phosphate (CP) (Ahern et al., 1980). Several other reports have also noted that a decrease in CP is characteristic of PSS and MH (Mitchell et al., 1980; Ahern et al., 1980; Somers et al., 1977) and even essential for the development of PSE (Sair et al., 1970). Associated with the increase in lactate is a decrease in muscle pH and an increase in muscle glucose-6-phosphate (G-6-P) levels (Mitchell et al., 1980; Schmidt et al., 1972; Ahern et al., 1980; Fig. 2). The inference to be drawn from these data is that the lesion produces a marked stimularion of glycogenolysis, diversion of glycolysis to lactate, and compromised CP production. A crucial feature of these studies is that muscle samples obtained during barbiturate anesthesia show that stress-susceptible pigs already have significantly higher levels of G-6-P and significantly lower muscle pH and CP (Table 11). Thus the lesion producing these features is present before exposure to drugs and in the absence of stress. Moreover, after exposure to halothane, normal pigs also show an increase in G-6-P and decreases in pH and CP, which, since the pigs survive, are reversible. On the other hand, after exposure to halothane, susceptible pigs show a 5-fold increase in G-6-P, a rapid decrease in pH, and a reduction of CP to 60% of resting levels

TABLE I1 MUSCLE METABOLISM BEFORE AND AFTER HALOTHANE" Prehalothane (barbiturate) Oxidative phosphorylation

Glycolysis

Normal pigs MH pigs Nb P C

Halothane

G-6-P (wok)

PH

0.76 -+ 0.18 1.47 k 0.28 21 C0.05

7.23 t 0.03 7.08 t 0.03 21 <0.005

ATP (vol/g) 5.66 5.62 21 NS

C

IT

0.37 0.36

OData from Mitchell et al. (1980). b N , Number of pigs. = P , Probability calculated using Student's t-test; NS, not significant.

Oxidative phosphorylation

Glycolysis

PC OLmoW

G-6-P (wok)

22.93 2 1.53 15.80 IT 1.60 21 <0.005

2.75 2 0.50 7.33 t 0.64 20 <0.001

PH 6.93 2 0.03 6.42 ? 0.06 20 <0.001

ATP (wollg) 6.10 5.27 20 NS

C

?

0.59 0.45

PC (w-Wd 16.74 C 1.63 6.51 t 1.08 20 <0.001

208

G. MITCHELL AND J . J . A. HEFFRON

within 5 min (Mitchell et al., 1980), depletion of CP within 30 min (Ahern et al., 1980), and death. Halothane is, therefore, exaggerating the changes detailed above, which are similar to those produced by anaerobic glycolysis (Table 111). These changes could be produced in four ways. First, halothane could be inhibiting electron transport or uncoupling oxidative phosphorylation, and thus inhibiting ATP production and causing breakdown of CP to supplement ATP levels. Further, hydrolysis of CP (and ATP) ultimately results in an increase in adenosine diphosphate (ADP) and adenosine monophosphate (AMP), which stimulate glycolysis allosterically at the level of phosphofructokinase. However, since halothane is not unique as an inhibitor of mitochondria1 respiration or as an uncoupling agent, this explanation is unlikely. Second, a more attractive explanation for the production of lactate is that adenylate kinase, which catalyzes ATP + AMP 2ADP, is inhibited by halothane in susceptible pigs. If this were so, then the ratio of ATP:AMP:ADP could not be regulated and the rapid onset of glycolysis should ensue (Sachsenheimer et al., 1977). However, this argument relies on a highly specific interaction between halothane and adenylate kinase, so this explanation, like the first, does not account for exercise- or stress-induced PSS. Moreover, no differences between susceptible and resistant pigs have as yet been shown either for concentrations of adenylate kinase or for interaction between halothane and adenylate kinase. In addition, this and the first explanations do not explain diversion of glucose metabolism to lactate: they only explain stimulation of glycolysis. Third, an explanation for the production of lactate that would also account for the decrease in CP is that NADH oxidation is inhibited. If NADH oxidation is inhibited, then oxygen consumption should decrease during MH or PSS. However, while CP is decreasing and lactate increasing, oxygen consumption is increasing (Gronert et al., 1976a). Fourth, these observations suggest that a less specific explanation and one that accounts for onset of glycolysis, production of lactate, and a decrease in CP must be sought. In this context reports have suggested that halothane inhibits the NADH-a-glycerophosphate shuttle (Fink and Kenny , 1968) and inhibits pyruvate dehydrogenation (Husain and Paradise, 1973; Mitchell et al., 1980). Both stress-susceptible and normal pigs are affected this way, but susceptible pigs are affected more severely (Mitchell et al., 1980; Ahern et al., 1980). The consequences of such a defect should be a reduction of ATP production and a decrease in CP levels as a result of decreased reconstitution or increased breakdown. The subsequent rise in ADP and AMP would stimulate glycolysis and result in the production of lactate. The oxidation of pyruvate to acetyl-coenzyme A (CoA), which allows the use of pyruvate in the tricarboxylic acid cycle in mitochondria, depends on the group of enzymes (pyruvate dehydrogenase, dihydrolipoyl transacetylase, di-

TABLE I11 EFFECT OF HALOTHANE ON NORMAL AND MH-SUSCEPTIBLE PIGSa

Barbiturate Halothane Nb P'

0.76 f 0.18 2.75 f 0.50 25 (0.001

7.23 f 0.03 6.93 f 0.03 25 <0.001

5.66 6.10 25 NS

f

f

0.37 0.59

22.93 f 1.53 16.74 f 1.63 25 <0.02

aData from Mitchell et al. (1980). b N , Number of pigs. c P , Probability calculated using Student's t-test; NS, not significant

1.47 f 0.28 7.33 2 0.64 16 <0.001

7.08 2 0.03 6.42 5 0.06 16 <0.001

5.62 f 0.36 5.27 2 0.45 16 NS

15.80 f 1.60 6.51 f 1.08 16 <0.001

210

G. MITCHELL AND J. J . A . HEFFRON

hydrolipoyl dehydrogenase, pyruvate dehydrogenase kinase, pyruvate dehydrogenase phosphatase) which lie in the mitochondrial membrane. Disruption of this function of the membrane, together with inhibition of the NADH shuttle, can only result in the generation of lactate. Further, since halothane may stimulate diphosphofructophosphatase (Clark et al., 1973) and the phosphofructokinase of susceptible pigs is not initially inhibited by low pH (Kastenschmidt et al., 1966), there is enhancement of glycolysis in the presence of halothane and lactate production would be rapid. Further support for the idea of a halothaneenhanced mitochondrial membrane defect is that susceptible pigs and patients show abnormal mitochondrial inclusions and swollen, disrupted, and degenerating mitochondria (Isaacs et al., 1973; Muir, 1970; Reske-Nielsen, 1978; Hull er al., 1978; Heffron and Isaacs, 1976). In addition, halothane exerts its anesthetic effects on membranes rich in lipids by causing their expansion (Glauber, 1976; Johnson and Bangham, 1969; Johnson and Miller, 1970). Pathological effects of halothane have also been shown to include disruption of mitochondrial membranes (Chang et al., 1975). Thus as Patterson and Allen (1972) suggested, halothane becomes dissolved in the lipoprotein membranes of mitochondria, disorganizing their structure, destroying the spatial relationships of the enzyme complexes, and causing both diversion of glucose metabolism to lactate and depletion of CP. In pigs carrying the genetic defect, gross changes in already defective membranes are precipitated. In resistant animals, less severe, but similar, changes result. Although these data mainly refer to studies using halothane to precipitate the syndrome of MH, it is clear that if the mitochondria of stress-susceptible pigs are not able to metabolize pyruvate or to transport NADH, then an increase in glycolysis of whatever cause will aggravate the preexisting dependence on glycolysis for energy. Thus any substance which either initiates muscle depolarization or further disturbs the integrity of organelle membranes will cause a severe lactacidosis in susceptible animals. This observation could explain why a very wide range of drugs, and exercise, may cause PSS or MH. Moreover, since catecholamines stimulate glycolysis and produce hypoxia, low cortisol will facilitate glycogenolysis and the development of tissue hypoxia, and thyroid hormones can alter the integrity of mitochondrial membranes, cause calcium release from mitochondria, and are permissive on the actions of catecholamines, a clearer secondary role for hormonal abnormalities in the onset of PSS and MH is established.

5.

Conclusions

The results reviewed in this section indicate that the sarcolemma, T tubules, SR, and mitochondria of susceptible pigs have functional defects. There is also a likelihood that the contractile proteins of such pigs are more sensitive to tempera-

PORCINE STRESS SYNDROMES

21 1

ture than their normal counterparts. Of these defects, the mitochondrial defect seems to be most important in the onset of PSS or MH and, therefore, PSE. Since only mitochondria of the organelles discussed are ubiquitous, a mitochondrial defect will explain why many tissues show abnormal function during episodes of stress. The metabolic change measured during MH or PSS are the sum of metabolic changes in all tissues.

IV. GENERAL CONCLUSIONS Although it is possible for clinically normal pigs to develop stress syndromes, the data presented here argue that pigs most likely to develop MH and PSS have a “homozygote” genetic defect involving a single gene. These pigs also develop PSE musculature as a result of the rapid development of a low muscle pH while muscle temperature is still high. PSE will also develop in these pigs after slaughter by conventional means. However, PSE may develop in animals which have a “heterozygote” genetic defect and which do not develop MH or PSS. A whole range of responses to stress or drugs can, therefore, be envisaged and breed vviations, variation in the rapidity of onset of signs, and the difficulty of finding unequivocal predictive tests all can be accounted for by variation in the severity of the inherited defect. All the currently available data also suggest that the inherited defect is in membranes, an idea first proposed by Britt and Kalow (1970b). Both the phospholipid (Britt et al., 1975c) and protein components (McIntosh et al., 1977) of membranes have been implicated. Further, all the membranes in which a defect exists have calcium-related functions or actions. In this context, since the bulk of the evidence also suggests that a single gene is involved in inheritance of the trait, it seems likely that a single amino acid substitution has taken place in all calcium-binding membranes, or possibly in calmodulin (Mitchell and Heffron, 1980b). It is also possible that the severity with which a membrane is affected depends on the number of the specific amino acid substitutions. Such an explanation would clarify the data reporting different degrees of membrane malfunction in a wide range of membranes. Thus although mitochondria may be involved metabolically in a major way and so determine the onset of metabolic changes, the SR or sarcolemma may have more marked defects at a molecular level. A general, but nevertheless specific, defect of this kind would explain the most striking feature of the syndromes-muscle stiffness or rigidity. This rigidity is not rigor since it occurs before a decrease in ATP and before muscle pH has reached 6.2, the two established causes of rigor. The cause of rigidity must, therefore, be associated with an increase in calcium. Since halothane will induce muscle contraction in vitro (Okumura et al., 1979; Nelson et al., 1977), depolarize skeletal muscle membranes (Gallant et al., 1979; Kendig and Bunker, 1972),

212

G . MITCHELL AND J . J . A . HEFFRON

disrupt the sarcolemma (Innes and Stromme, 1973), affect T-tubules (Okumura et al., 1980), cause conformational changes in the SR (Augustin and Hasselbach, 1973), and reduce mitochondrial uptake of calcium (Heffron and Gronert, 1977), it is likely that halothane can cause release of calcium from storage sites and prevent or attenuate reuptake. Similarly, succinylcholine, although not apparently altering the SR or mitochondrial function, will raise myoplasmic calcium by prolonging membrane depolarization, especially if the SR and mitochondrial functions are intrinsically defective. These observations support the idea of a rapid and sustained increase in cell calcium, at least during MH. In PSS, however, muscle stiffness develops after metabolic changes have occurred, and furthermore, develops in the absence of drugs. Moreover, changes in SR function do not occur unless halothane concentrations exceed tissue concentrations of 1.2%, thus indicating that SR malfunction is not likely at anesthetic concentrations of halothane. Also, although high myoplasmic calcium will explain rigidity, it will not explain lactate production or the characteristic release of catecholamines. For these reasons a central role for calcium is unlikely. On the other hand, diversion of glucose metabolism resulting from a mitochondrial membrane lesion will explain the finding that a rise in lactate is the first measurable change occurring in MH or PSS. Lactate will also cause the release of catecholamines (Nahas et al., 1960) and by inhibiting SR function, will depress calcium uptake by the SR (McIntosh et al., 1977). This sequence of events will also explain the onset of the syndromes after exercise and their more rapid development if drugs are superimposed on exercise (Hende et al., 1976). However, this sort of mitochondrial malfunction should lead to a fall in ATP levels and a decrease in oxygen consumption. Yet neither occurs initially during the syndromes. The most likely explanation for the maintenance of ATP is that it is replenished from CP and glycolysis. CP levels are 3-fold higher (approximately 25 pmol/g tissue) than ATP (approximately 8 p.mol/g tissue), and ATP levels do not fall until CP levels fall below ATP levels (Fig. 2). In addition, onethird of the ATP produced from oxidative phosphorylation can be replaced by glycolytic ATP (Fink and Kenny, 1968). In these circumstances, ATP levels will only decrease when glycogen levels fall. Thus ATP levels fall when glycogen levels have decreased by 50% (Berman et al., 1970) and when G-6-P levels decrease (Fig. 2). There must also be adequate ATP production for growth since stress-susceptible pigs grow almost as well as normal pigs (Mitchell and Heffron, 1981b). Further, it would be difficult to explain the sustained muscle contraction which precedes rigor if ATP levels were inadequate. Thus it cannot be claimed that a lack of ATP is responsible for decreased calcium uptake by the SR, or that a fall in ATP level is the key event of MH or PSS (Britt, 1979; Ahern et al., 1980). Nevertheless, low ATP and CP levels are integral to PSE and so PSE will only develop after ATP has been depleted (Sair et al., 1970).

PORCINE STRESS SYNDROMES

213

The increase in oxygen consumption (Gronert et al., 1976a, 1977b; Berman et al., 1970) is less easy to explain. Hydrolysis of ATP does not account for increased oxygen consumption as suggested by Gronert and Theye (1976a). Increased oxygen consumption can only be explained either by increased production of ATP or by uncoupling of oxidative phosphorylation. Further, since normal pigs (Gronert and Theye, 1976a) and normal cells (Fink and Kenny, 1968) show decreased oxygen consumption after exposure to halothane, the increased oxygen consumption in susceptible pigs must be related to the genetic defect. The most reasonable conclusion is that increased oxygen consumption is a result of uncoupling of oxidative phosphorylation. Low concentrations of halothane will cause uncoupling (Cohen et al., 1969; Miller and Hunter, 1970) and high myoplasmic Ca2+ levels can induce uncoupling (Bianchi, 1973). Also the abrupt decrease in CP levels which occurs when halothane anesthesia starts (Fig. 2; Somers et al., 1977) and which is occurring while oxygen consumption is increasing, also suggests uncoupling. However, oxygen consumption falls precipitously as metabolism becomes increasingly disordered. Another interesting observation associated with the increase in oxygen consumption is that lactate is produced while oxygen consumption is increasing, thus the lactate cannot be anaerobic lactate typical of exercise. Nevertheless, the production of lactate is of key significance in causing the signs of both MH and exercise-induced stress, but it is important to emphasize that the lactate of stresssusceptible animals is of different etiology from that produced in normal animals during exercise. During strenuous exercise oxygen consumption increases maximally, but the supply of oxygen is not adequate to oxidize NADH produced during glycolysis and so maintain the supply of NAD . Instead lactate is formed to generate NAD+ . In MH, tissue perfusion is maintained normally for 15-20 min, yet oxygen consumption increases only 3-fold during that time, although it can increase by 10- to 20-fold (50% inhibition of state 3 respiration; Gronert and Heffron, 1979) and lactate is produced (Gronert et al., 1977b). The lactate generated in these circumstances must reflect decreased dehydrogenation of pyruvate and/or transport of NADH in the face of stimulated glycogenolysis and glycolysis. The lactate is significant because it alters membrane integrity, (Parpart et al., 1947) and cell electrolytes and enzymes leak into the plasma. Lactate, by lowering cell pH, will alter SR function and also produce a metabolic acidosis. The acidosis in turn stimulates the secretion of catecholamines, which mediate vasoconstriction and further stimulate glycolysis. The vasoconstriction, which occurs despite the vasodilatory effects of low tissue pH, is enhanced by the increase in tissue pressure associated with muscle contraction and will not only produce tissue hypoxia and so cause anaerobic glycolysis (Lister et al., 1970), but will also reduce heat loss (Williams et al., 1975). These effects of catecholamines will be compounded by low cortisol levels and high thyroid hormone levels, +

214

G . MITCHELL AND J. J. A. HEFFRON

which seem to be features of stress-susceptible pigs and which themselves may produce changes independently of catecholamines. The production of lactate, the increase in oxygen consumption, the decrease in muscle CP, and the muscle rigidity which characterize the onset of the syndromes, are associated with a rise in rectal temperature of about 1°C per 6-10 min. To produce this heat requires an estimated 250 kJ/sec (Mitchell et al., 1980) or 150 kcal (Berman and Kench, 1973), depending on the mass of the pigs. Glycogenolysis cannot produce this alone, even if accelerated substrate cycling occurs (Clark et al., 1973), because glycogen stores are inadequate (Mitchell et al., 1980). Further, as oxidative phosphorylation is reduced during MH or PSS and carcass temperatures remain high postmortem, nonoxidative mechanisms must play a role, in conjunction with reduced heat loss. Although reduced heat loss has been shown (Williams et al., 1975), there is controversy as to whether nonoxidative mechanisms can account for the additional heat produced. Three sources of nonoxidative heat are uncoupling of oxidative phosphorylation, hydrolysis of high-energy compounds, and metabolism of lactate. In the best analysis of the origin of heat during MH (Hall et al., 1976a), it has been suggested that initially heat production relies on aerobic metabolism. From figures given by Wang et al. (1969), a minute volume of 5 literdmin will supply the 1 liter of oxygen required to raise the temperature of a 30-kg pig 1" in 6 min. However, once muscle temperature reaches 40°C (within 1 min; Fig. 2), aerobic metabolism is not sufficient to sustain the temperature rise. Thereafter, metabolism of lactate becomes the main source of heat. Despite these observations, the contribution of nonoxidative mechanisms has been put at only 25% (Berman and Kench, 1973) and 50% (Hall et al., 1976a), and the source of heat must presently remain an enigma. Nevertheless, rectal temperature clearly rises, and rises significantly only in susceptible animals. Thus although the effects of high body temperature are deleterious to tissues of all animals, it is only in susceptible animals that the consequences of the rise in temperature are seen-SR function is decreased (Nelson and Bee, 1979), myosin ATPase activity is enhanced (Hartshorne et al., 1972), and calcium sensitivity of actomyosin may be lost (Green et al., 1980). In summary, therefore, the onset of MH or PSS is associated with an increase in glycolysis and a rise in myoplasmic calcium levels. The diversion of pyruvate to lactate causes a metabolic acidosis. The rise in myoplasmic calcium levels causes muscle contraction. The acidosis and a rise of serum calcium levels enhances catecholamine release. The hypermetabolism and peripheral vasoconstriction which follow catecholamine release cause a rise in body temperature. Thus the classical signs of muscle rigidity, hyperthermia, and lactacidosis of porcine stress syndromes develop (Fig. 9).

neural input

catecholamines

acetylcholine succinylcholine

I thyroid Qormones

1

halothane

FIG. 9. A possible etiology of the signs of MH and PSS in stress-susceptible pigs. Note that lactate is produced because pyruvate and NADH transport are diminished (see Fig. 8).

216

G. MITCHELL AND J . J . A. HEFFRON

V.

FUTURE RESEARCH

From the foregoing, it should be clear that a great volume of research has been devoted to the etiology, mode of inheritance, detection, and treatment of animals susceptible to stress. The poor-quality pork derived from stress-susceptible pigs has been studied most extensively and the data have been reviewed by Cassens et al. (1978). It is now generally accepted that no major problems exist in this area, from a research point of view, although the disease itself is still of great economic importance. We believe that the following are the principal areas for future research. First, since the stress-susceptible syndrome is most probably a true, if subclinical, myopathy, it is essential to identify unequivocally the locus of the defect in the muscle fiber. However, although muscle, because of its bulk, produces the most conspicuous changes in metabolism, the inescapable conclusion is that all tissues will be affected by stress or drugs and that the severe metabolic changes occurring during the porcine stress syndromes are the sum of metabolic changes in all tissues. Thus any defect identifiable in muscle should also be identifiable in other tissues. Although considerable research indicates that stress susceptibility and malignant hyperthermia are a disease involving membranes with calciumtransporting functions, the data reviewed above suggest that the mitochondria1 membranes, especially those of muscle, deserve the most attention since an abnormality in them would most easily explain how the stress syndromes are precipitated by drugs, on the one hand, and exercise, on the other. In this context also, investigation of the initial nonhypoxic lactate accumulation, the origin of heat, and the effects of a decrease in pH on the contractile proteins of stresssusceptible animals are urgently needed. These proposed studies should take into account the changing role of the mitochondrion in cell metabolism recently proposed (e.g., McCormack and Denton, 1980). Thus, the mitochondria may also regulate the intramitochondrial calcium concentration, in turn modulating the activities of at least two regulatory enzymes of the Krebs cycle (isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase) in addition to pyruvate dehydrogenase phosphatase. The role of the relatively recently discovered calcium regulatory protein, calmodulin (Means and Dedman, 1980), in sensitizing mitochondrial oxidative metabolism to calcium must also be established. In the context of the porcine stress syndromes, a defect at this site would explain the phenomenon of nonhypoxic lactate formation as well as provide a link with the elevated myoplasmic calcium concentration believed to be an early, if not primary, event in the etiology of the stress syndromes. Indeed, research directed towards measuring myoplasmic calcium concentrations during episodes of stress would be of value. It should also be pointed out that no research on the possibility that calmodulin itself could be defective, has been undertaken. Abnormal calmodulin would explain the ubiquitous nature of the defect in the porcine stress

PORCINE STRESS SYNDROMES

217

syndromes and provide a basis for the single autosomal recessive gene mode of their inheritance. If this is the case, then proper elucidation of the defect resides in amino acid sequencing of calmodulin or other calcium-binding proteins. The idea that the stress syndromes are the result of a “cascade” effect consequent on small changes in the calcium-regulating functions of the various membrane systems could then be considered. Second, another area of research is the role of the adrenal medullary and cortical hormones and that of thyroid hormones in the stress syndromes. In particular, the mechanism of decreased cortical response to ACTH in susceptible pigs and the role of the specific calcium-releasing effect of thyroxine on mitochondria should be reconsidered in the light of the comments made above. Although the effects of these hormones are unlikely to be primary in the etiology of stress, their abnormal secretion undoubtedly potentiates or exacerbates already unstable metabolic states. Third, the area of identification of pigs must be developed. Although the whole animal halothane exposure test is now used by several groups of researchers to identify stress-susceptible pigs, this method will not detect all pigs which produce PSE meat (Mitchell et al., 1980a). Thus, development of a noninvasive test, which is both simple to carry out and accurate, is essential for the elimination of such animals from breeding herds. It is also important to establish the mode of inheritance of the syndromes so that tests can be assessed with benefit of a genetic profile. In this context, animals resistant to halothane but which nevertheless produce PSE meat are most probably heterozygotes, and they are likely to show intermediate sensitivities to halothane and to have intermediate SCPK activity and red cell fragility. Further, the muscle biopsy contracture test should be evaluated to see if the heterozygotes show intermediate sensitivities to halothane and to halothane and caffeine. Finally, we wish to make a plea for standardization of methodologies, both at a fundamental research level and in the methods of detection. Many of the results reviewed above cannot be related or compared because of widely varying enzyme assay conditions and methods of membrane preparation. This is particularly true in the studies of the action of anesthetics and drugs on mitochondria and the SR; however, there seems little doubt that the comment also applies to in vitro contracture tests as well as other assay techniques. Without standardization the task of unequivocally resolving the problems of porcine stress syndromes will be prolonged.

REFERENCES Addis, P. B., Britt, B. A , , Henderson, A. R., and Nealon, D. A . 1978. Muscle creatine kinase isoenzymes in human and porcine subjects susceptible to malignant hyperthermia. In “Malig-

218

G . MITCHELL AND J. J . A. HEFFRON

nant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 227-232. Grune & Stratton, New York. Ahern, C. P., Somers, C. J . , Wilson, P . , and McLoughlin, J. V. 1980. Halothane-induced malignant hyperthermia: Creatine phosphate concentration in skeletal muscle as an early indicator of the onset of the syndrome. J. Comp. Pathol. 90, 177-186. Aldridge, W. N . , and Parker, V. H. 1960. Barbiturates and oxidative phosphorylation. Biochem. J. 76, 47-5 6. Allen, W. M., Berret, S . , Harding, J. D. J . , and Patterson, D. S . P. 1970a. Experimentally induced acute stress syndrome in Pietrain pigs. Vel. Rec. 87, 64-69. Allen, W. M., Berret, S . , Harding, J. D. J., and Patterson, D. S . P. 1970b. Plasma levels of muscle enzymes in the Pietrain pig in relation to the acute stress syndrome. Vet. Rec. 87, 410-411. Altrogge, D. M . , Topel, D. G., Cooper, M. A , , Hallberg, J.W., and Draper, D. D. 1980. Urinary and caudate nuclei catecholamine levels in stress-susceptible and normal swine. J. Anim. Sci. 51, 74-77. Anderson, I. L., and Jones, E. W. 1976. Effect of dantrolene sodium on in vitro halothane induced contraction of susceptible muscle. Anesthesiology 44,57-61. Andresen, E. 1979a. Associative additive effects of alleles of the H and PHI loci on the quality of meat from Danish Landrace pigs. Acta Agric. Scand. 29, 321-325. Andresen, E. 1979b. Linkage disequilibrium responsible for association between susceptibility to the malignant hyperthermia (MHS) and PHI types in Danish Landrace pigs. Acta Agric. Scand. 29, 369-373. Andresen, E. 1980. Association between susceptibility to the malignant hyperthermia syndrome (MHS) and H blood types in Danish Landrace pigs explained by linkage disequilibrium. Livest. Prod. Sci. 7, 155-162. Artru, A. A , , and Gronert, G . A. 1980. Cerebral metabolism during porcine malignant hyperthermia. Anesthesiology 53, 121-126. Ash, A. S. F., Besch, H. R., Harigaya, S . , and Zaimis, E. 1972. Changes in activity of sarcoplasmic reticulum fragments and actomyosin isolated from skeletal muscle of thyroxine treated cats. J. Physiol. (London) 224, 1-19. Augustin, J., and Hasselbach, W. 1973. Changes of the fluorescence of I-anilo-8-naphthalenesulphonate associated with the membranes of the sarcoplasmic reticulum induced by general anaesthetics. Eur. J. Biochem. 39, 75-84. Axt, J., Richter, W., and Ott, A. 1968. Vitamin C content in the blood serum of various animal species under stress. 111. Effect of work stress on serum ascorbic acid and blood sugar in the horse. Arch. Exp. Veterinaermed. 22, 1165-1173. Bagshaw, R. J., Cox, R. H . , Knight, D. H . , and Detwiler, D. R. 1978. Malignant hyperthermia in a greyhound. J. Am. Vet. Med. Assoc. 172, 61-62. Baskin, R. J., and Deaner, D. W. 1970. A membrane bound creatine phosphokinase in fragmented sarcoplasmic reticulum. 1.Biol. Chem. 245, 1345-1347. Bate-Smith, E. C., and Bendall, J . R. 1947. Rigor mortis and adenosine triphosphate. J. Physiol. (London) 106, 177-1 8 5 . Beldavs, J., Small, V., Cooper, D. V., and Britt, B. A. 1971. Postoperative malignant hyperthermia: A case report. Can. Anaesth. Soc. J . 18, 202-212. Berman, M. C. 1973. Role of preoperative management in the development of malignant hyperthermia. Lancet 2, 743. Berman, M. C . , and Kench, J . E. 1973. Biochemical features of malignant hyperthermia in Landrace pigs. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 287-297. Thomas, Springfield, Illinois. Berman, M. C., Harrison, G. G., Bull, A. B., and Kench, J. E. 1970. Changes underlying halothane-induced malignant hyperthermia in Landrace pigs. Nature (London) 225, 653-655.

PORCINE STRESS SYNDROMES

219

Bianchi, C. P. 1968. Pharmacological actions on excitation-contraction coupling in striated muscle. Fed. Proc., Fed. Am. Soc. Exp. Biol. 27, 126131. Bianchi, C. P. 1973. Cell calcium and malignant hyperthermia. In “Malignant Hyperthermia” (R. A. Gordon, D. A. Britt, and W. Kalow, eds.), pp. 147-151. Thomas, Springfield, Illinois. Bjenum, 0. J., and Bog-Hansen, T. C. 1976. Human erythrocyte membrane proteins analysed as a model system. Biochim. Biophys. Acta 455, 6 6 8 9 . Blaustein, M. P., and Goldman, D. E. 1966. Action of anionic and cationic nerve blocking agents: Experiment and interpretation. Science 153, 429432. Bowman, W. C., and Nott, M. W. 1969. Action of sympathomimetic amines and their antagonists on skeletal muscle. Pharmacol. Rev. 21, 27-72. Bowman, W. C., Khan, H. H., and Savage, A. 0. 1977. Some antagonists of dantrolene sodium on the isolated diaphragm muscle of the rat. J . Pharm. Pharmacol. 29, 616625. Brebner, J., and Jozefowicz, J. A. 1974. Procainamide therapy of malignant hyperthermia: A case report. Can. Anaesth. SOC. J . 21, 9 6 1 0 5 . Briskey, E. J . , Kastenschmidt, L. L., Forrest, L. C., Beecher, G. R., Judge, M. D., Cassens, R. G., and Hoestra, W. G. 1966. Biochemical aspects of post-mortem changes in porcine muscle. J . Agric. Food Chem. 14, 201-207. Britt, B. A. 1979. Etiology and pathophysiology of malignant hyperthermia. Fed. Proc., Fed. Am. SOC.Exp. Biol. 38, 4 4 4 8 . Britt, B. A,, and Kalow, W. 1968. Hypenigidity and hyperthermia associated with anaesthesia. Ann. N . Y . Acad. Sci. 151, 947-958. Britt, B. A., and Kalow, W. 1970a. Malignant hyperthermia: A statistical review. Can. Anaesth. Soc. J . 17, 293-3 15. Britt, B. A., and Kalow, W. 1970b. Malignant hyperthermia: Aetiology unknown. Can. Anaesth. SOC. J . 17, 316330. Britt, B. A,, Endrenyi, L., Barclay, R. L., and Cadman, D. L. 1975a. Total calcium content of skeletal muscle isolated from humans and pigs susceptible to malignant hyperthermia. Br. J . Anaesth. 47, 647-649. Britt, B. A,, Endrenyi, L., and Cadman, D. L. 1975b. Calcium uptake into muscle of pigs susceptible to malignant hyperthermia. Br. J . Anaesth. 47, 65G653. Britt, B. A,, Endrenyi, L., Cadman, D. L., Fan, H. M., and Fung, H. Y. 1 9 7 5 ~ .Effects of halothane on mitochondria1 respiration and calcium accumulation. Anesthesiology 42, 292-300. Britt, B. A., Endrenyi, L., Peters, P. L., Kwong, F. H. F., and Kadijevic, L. 1976. Screening of malignant hyperthermia susceptible families by creatine phosphokinase measurement and other clinical observations. Can. Anaesth. Soc. J . 23, 263-284. Britt, B. A,, Kalow, W., and Endrenyi, L. 1978. Malignant hyperthermia-pattern of inheritance in swine. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 195-21 1. Grune & Stratton, New York. Brooks, G . A., and Cassens, R. G. 1973. Respiratory functions of mitochondria isolated from stresssusceptible and stress-resistant pigs. J . Anim. Sci. 37, 688-691. Brucker, R. F., Williams, C. H., Popinigis, J., Galvez, T. L . , Vail, W. J., and Taylor, C. A. 1973. In vitro studies on liver mitochondria and skeletal muscle sarcoplasmic reticulum fragments isolated from hyperpyrexic swine. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 23&270. Thomas, Springfield, Illinois. Campion, D. R., and Topel, D. G. 1975. A review of the role of swine skeletal muscle in malignant hyperthermia. J . Anim. Sci. 41, 779-786. Campion, D. R . , Topel, D. G., Christian, L. L., and Stromer, M. H. 1974. Mitochondria1 traits of muscles from stress susceptible pigs. J . Anim. Sci. 39, 201. Cassens, R. G., Marple, D. N., and Eikelenboom, G. 1978. Animal physiology and meat quality. Adv. Food Res. 21, 71-155.

220

G. MITCHELL AND J. J. A. HEFFRON

Chang, L. W., Dudley, A. W., and Lee, Y. K. 1975. Ultrastructural changes in the kidney following chronic exposure to low levels of halothane. Am. J . Pathol. 78, 225-242. Cheah, K. S . , and Cheah, A. M. 1976. The trigger for PSE condition in stress-susceptible pigs. J . Sci. Food Agric. 27, 1137-1 144. Cheah, K. S., and Cheah, A. M. 1978. Calcium movements in skeletal muscle mitochondria of malignant hyperthermic pigs. FEBS Lett. 95, 307-310. Cheah, K. S . , and Cheah, A. M. 1979. Mitochondria1 calcium erythrocyte fragility and porcine malignant hyperthermia. FEBS Lett. 107, 265-268. Christenson, N. J . 1973. Plasma noradrenaline and adrenaline in patients with thyrotoxicosis and myxoedema. Clin. Sci. Mol. Med. 45, 163-171. Clark, M. G., Williams, C. H . , and Pfeifer, W. F. 1973. Accelerated substrate cycling of fructose-6phosphate in the muscle of malignant hyperthermia pigs. Nature (London) 245, 99-101. Clarke, I. M. C., and Ellis, F. R. 1975. An evaluation of procaine in the treatment of malignant hyperpyrexia. Br. J . Anaesth. 47, 17-21. Cohen, D. 1979. The hormonal control of glycogen metabolism in mammalian muscle by multivalent phosphorylation. Biochem. SOC. Trans. 7, 459480. Cohen, P. J., Marshall, B. E., and Lecky, J. H. 1969. Effects of halothane on mitochondria1oxygen uptake: Site of action. Anesthesiology 30, 337. Conway, E. J., and Hingerty, D. 1953. A comparison of the effects of cortisone and deoxycorticosterone on various aspects of organic and inorganic metabolism. Biochem. J . 55,447454. Crawford, J. S. 1972. Hyperpyrexia during pregnancy. Lancet 1, 1244. De Jong, R. H., Heavner, J. E., and Amory, D. W. 1974. Malignant hyperpyrexia in the cat. Anesthesiology 41, 608-609. Denborough, M. A , , Forster, J. F. A,, Lovell, R. R. H., Maplestone, P. A,, and Villiers, J . D. 1962. Anaesthetic deaths in a family. Br. J . Anaesth. 34, 395-396. Denborough, M. A., Ebeling, P., King, J. O., and Zapf, P. 1970. Myopathy and malignant hyperpyrexia. Lancet 1, 1138-1 140. Denborough, M. A., Hird, F. J . R., King, J. O., Marginson, M. A,, Mitchelson, K. R., Nayler, W. G., Rex, M. A., Zapf, P., and Condron, R. J. 1973. Mitochondria1 and other studies in Australian Landrace pigs affected with malignant hyperthermia. In ‘‘Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 229-237. Thomas, Springfield, Illinois. de Villafranca, G. W., and Waksmonski, C. A. 1970. Superprecipitation of horseshoe crab and rabbit myosin B. Int. J . Biochem. 1, 29-38. Diethmuller, H., and Wels, A. 1972. Effect of training on thoroughbreds. I. Muscle specific enzymes. Zentralbl. Veterinaermed. 7, 537-545. Dimarco, N. M., Beitz, D. C., Young, J. W., Topel, D. G., and Christian, L. L. 1976. Gluconeogenesis from lactate in liver of stress-susceptible and stress-resistant pigs. J . Nutr. 106, 71&716. Douglas, W. W., and Rubin, R. P. 1963. The mechanism of catecholamine release from the adrenal medulla and the role of calcium in stimulus-secretion coupling. J . Physiol. (London) 167, 288-310. DuBois, K. P., Albaum, H. G., and Potter, V. R. 1943. Adenosinetriphosphate in magnesium anaesthesia. J . Biol. Chem. 147, 699-704. Ebashi, S . , Ebashi, F., and Kodama, A. 1967. Troponin as the calcium receptive protein in the contractile system. J . Biochem. (Tokho) 62, 137-138. Eighmy, J. J., Williams, C. H., and Anderson, R. R. 1978. The fulminant hyperthermia-stress syndrome: Plasma thyroxine and tri-iodothyronine levels in susceptible and normal pigs and man. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 161-173. Grune & Stratton, New York.

PORCINE STRESS SYNDROMES

22 1

Eikelenboom, G. 1981. Hereditary aspects of susceptibility to stress in meat animals. In “The Problem of Dark-cutting in Beef” (D. E. Hood and P. V. Tarrant, eds.). Commission of European Communities, Brussels (in press). Eikelenboom, G., and Minkema, E. 1974. Prediction of PSE with a non-lethal test for the halothaneinduced porcine MH syndrome. Neth. J. Vet. Sci. 99, 421426. Eikelenboom, G., and van der Bergh, S. G. 1973. Mitochondria1 metabolism in stress-susceptible pigs. J. Anim. Sci. 37, 692-696. Eikelenboom, G., and Weiss, G. M. 1972. Breed and exercise influence on T4 and PSE. J. Anim. Sci. 35, 1096. Eikelenboom, G., Minkema, D., van Eldik, P., and Sybesma, W. 1978. Inheritance of the malignant hyperthermia syndrome in Dutch Landrace swine. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 141-146. Grune & Stratton, New York. Ellis, F. R., Clarke, I. M., Modgill, M . , Cume, S., and Hamman, D. G. F. 1975. Evaluation of creatine phosphokinase in screening patients for malignant hyperpyrexia. Br. Med. J. 3, 511-513. Ellis, F. R. 1973. Malignant hyperthermia. Anaesthesia 28, 245-252. Ellis, K. O., Morgan, K G., and Bryant, S. H. 1977. The mechanism of action of dantrolene sodium. J. Pharmacol. Exp. Ther. 201, 138-147. Feinstein, M. B. 1963. Inhibition of caffeine rigor and radiocalcium movements by local anaesthetics in frog sartorius muscle. J . Gen. Physiol. 47, 151-172. Fink, B. R., and Kenny, G. E. 1968. Metabolic effects of volatile anaesthetics in cell culture. Anesthesiology 29, 505-516. Flewellyn, E. H., and Nelson, T. E. 1979. A comparison of dantrolene and procainamide on in vivo muscle twitch of malignant hyperthermia susceptible pigs. Fed. Proc., Fed. Am. SOC. Exp. Biol. 37, 331. Forrest, J.C., Will, J. A,, Schmidt, G. R., Judge, M. D., and Briskey, E. J. 1968. Homeostasis in animals (Sus domesticus) during exposure to a warm environment. J. Appl. Physiol. 24, 33-39. Friesen, C. M., Brodsky, J. B., and Dillingham, M. F. 1979. Successful use of dantrolene sodium in human malignant hyperthermia syndrome: A case report. Can. Anaesth. SOC.J. 26, 319-321. Fuchs, F. 1975. Thermal inactivation of the calcium regulatory mechanism of human skeletal muscle actomyosin. Anesthesiology 42, 584-589. Fuchs, F., Hartshorne, D. J., and Barnes, E. M. 1975. ATPase activity and superprecipitation of skeletal muscle actomyosin of frog and rabbit: Effect of temperature on calcium sensitivity. Comp. Biochem. Physiol. B B51, 165-170. Gallant, E. M., Godt, R. E., and Gronert, G. A. 1979. Role of plasma membrane defect of skeletal muscle in malignant hyperthermia. Muscle Nerve 2, 491494. Gatz, E. E., and Jones, J. R. 1969. Effects of six general anaesthetics upon mitochondria1 oxidative phosphorylation: Possible site and mechanism of malignant hyperpyrexia. Fed. Proc., Fed. Am. SOC. Exp. Biol. 28, 356. Gericke, M. D., and Hofmeyr, J. M. 1976. Aetiology and treatment of capture stress and myopathy in springbok. S. Afr. J. Sci. 72, 28. Giosa, N. 1975. Procaine for malignant hyperthermia. N. Engl. J. Med. 292, 869-870. Glauber, H. S. 1976. Mechanisms of action of inhalation anaesthetics. S. Afr. J. Med. Sci. 41, 305-326. Green, R. A., Heffron, J. J. A,, and Mitchell, G. 1976. Effects of potassium, procaine and dantrolene on the calcium-dependent and ‘basal’ ATPase activity of sarcoplasmic reticulum of skeletal muscle. Gen. Pharmacol. 7, 361-363. Green, R. A,, Mitchell, G., and Heffron, J. J. A. 1980. Effects of temperature, adenosine triphosphate and magnesium concentrations on the contraction of actomyosin isolated from halothanesensitive and insensitive German Landrace p g s . Er. J . Anaesth. 52, 319-323.

222

G. MITCHELL AND J. J. A. HEFFRON

Gronert, G. A. 1978. Experimental malignant hyperthermia: A reply. Anesthesiology 49, 59-60. Gronert, G. A. 1979. Muscle contractures and adenosine triphosphate depletion in porcine malignant hyperthermia. Anesth. Analg. (Cleveland) 58, 367-371. Gronert, G. A , , and Heffron, J. J. A. 1979. Skeletal muscle mitochondria in porcine malignant hyperthermia: Respiratory activity, calcium functions, and depression by halothane. Anesth. Analg. (Cleveland) 58, 76-81. Gronert, G. A,, and Theye, R. A. 1976a. Halothane-induced porcine malignant hyperthermia: Metabolic and hemodynamic changes. Anesthesiology 44, 36-43. Gronert, G. A,, and Theye, R. A. 1976b. Suxamethonium induced malignant hyperthermia. Er. J. Anaesth. 48, 513-517. Gronert, G. A,, Milde, J. H., and Theye, R. A. 1976a. Porcine malignant hyperthermia induced by halothane and succinylcholine. Anesthesiology 44, 124132. Gronert, G. A,, Milde, J. H., and Theye, R. A. 1976b. Dantrolene in porcine malignant hyperthermia. Anesthesiology 44,488-495. Gronert, G. A., Milde, J. H., and Theye, R. A. 1977a. Role of sympathetic activity in porcine malignant hyperthermia. Anesthesiology 47, 41 1-415. Gronert, G. A,, Heffron, J. J. A,, Milde, J. H., and Theye, R. A. 1977b. Porcine malignant hyperthermia: Role of skeletal muscle in increased oxygen consumption. Can. Anaesth. Soc. J. 24, 103-109. Gronert, G. A , , Theye, R. A , , Milde, J. H., and Tinker, J. H. 1978. Catecholamine stimulation of myocardial oxygen consumption in porcine malignant hyperthermia. Anesthesiology 49, 33c337. Gronert, G. A., Heffron, J. J. A,, and Taylor, S. R. 1979. Skeletal muscle sarcoplasmic reticulum in porcine malignant hyperthermia. Eur. J. Pharmacol. 58, 179-1 87. Hainaut, K., and Desmedt, J.E. 1974. Effect of dantrolene sodium on calcium movements in single muscle fibres. Nature (London) 252, 728-730. Hall, G. M., and Lister, D. 1974. Procaine and malignant hyperthermia. Lancet 1, 208. Hall, G. M., Kirtland, S . J., and Baum, H. 1973. The inhibition of mitochondria1 respiration by inhalational anaesthetic agents. Er. J . Anaesth. 45, 1005-1009. Hall, G. M., Lucke, J. N., and Lister, D. 1975. Treatment of porcine malignant hyperthermia. Anaesthesia 30, 308-317. Hall, G. M . , Bendall, J. R., Lucke, J. N., and Lister, D. 1976a. Porcine malignant hyperthermia. 11. Heat production. Er. J. Anaesth. 48, 305-308. Hall, G. M., Lucke, J. N., and Lister, D. 1976b. Porcine malignant hyperthermia. IV. Neuromuscular blockade. Er. J . Anaesth. 48, 1135-1 141. Hall, G. M., Lucke, J. N., and Lister, D. 1977a. Porcine malignant hyperthermia. V. Fatal hyperthermia in Pietrain pigs, associated with infusion of alpha adrenergic agonists. Er. J . Anaesth. 49, 855-863. Hall, G. M., Lucke, J. N., and Lister, D. 1977b. Failure of methylprednisolone in porcine malignant hyperthermia. Lancet 2, 1359. Hall, G. M . , Lucke, J. N., Lovell, R., and Lister, D. 1980a. Porcine malignant hyperthermia. VII. Hepatic metabolism. Er. J . Anaesth. 52, 11-17. Hall, G . M . , Lucke, J. N., and Lister, D. 1980b. Malignant hyperthermia. Pearls out of swine. E r . J . Anaesth. 52, 165-171. Hall, G. M., Cooper, G. M . , Lucke, J. N., and Lister, D. 1980c. 4-Aminopyridine fails to induce porcine malignant hyperthermia. Er. J . Anaesth. 52, 707. Hall, L. W . , Woolf, N., Bradley, J. W. P., and Jolly, D. W. 1966. Unusual reaction to suxamethonium chloride. Er. Med. J. 2, 1305. Hall, L. W . , Trim, C. M., and Woolf, N. 1972. Further studies of porcine malignant hyperthermia. Er. Med. J . 2. 145-148.

PORCINE STRESS SYNDROMES

223

Hammel, E. P., and Raker, C. W. 1972. Exertional myopathy. In “Equine Medicine and Surgery,” pp. 587-591. Am. Vet. Publ., Illinois. Han, M. H., and Benson, E. S. 1970. Conformational changes in troponin induced by calcium. Biochem. Biophys. Res. Commun. 3, 378-384. Harris, E. J., Al-Shaikhaly, M., and Baum, H. 1979. Stimulation of mitochondria1 calcium ion efflux by thio-specific reagents and by thyroxine. Biochem. J . 182, 455-464. Harris, R. A., Munroe, J., Fanner, B., Kim, K. C., and Jenkins, P. 1971. Action of halothane upon mitochondria1 respiration. Arch. Biochem. Biophys. 142, 435444. Harris, W. S . , Schoenfield, C. D., Brooks, R. H., and Weisster, A. M. 1965. Receptor mechanism for the metabolic and circulatory action of epinephrine in man. J . Clin. Invest. 44, 1058. Hamson, G. G. 1971. Anaesthetic induced malignant hyperpyrexia: A suggested method of treatment. Br. Med. J . 3, 454-456. Harrison, G . G. 1973. The effect of procaine and curare on the initiation of anaesthetic-induced malignant hyperpyrexia. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 271-286. Thomas, Springfield, Illinois. Harrison, G. G. 1975. Control of the malignant hyperpyrexic syndrome in MHS swine by dantrolene sodium. Br. J . Anaesth. 47, 62-65. Harrison, G. G. 1977. The prophylaxis of malignant hyperthermia by oral dantrolene sodium in swine. Br. J . Anaesth. 49, 315-317. Harrison, G. G., and Verburg, C. 1973. Erythrocyte osmotic fragility in hyperthermia-susceptible swine. Br. J . Anaesth. 45, 131-133. Harrison, G. G., Biebuyck, J. F., Terblanche, J., Dent, D. M., Hickman, R., and Saunders, J. 1968. Hyperpyrexia during anaesthesia. Br. Med. J . 3, 594-595. Harrison, G. G., Saunders, S. J., Biebuyck, J. F., Hickman, R., Dent, D. M., Weaver, V . , and Terblanche, J. 1969. Anaesthetic-induced malignant hyperpyrexia and a method for its prediction. Br. J . Anaesth. 41, 844-855. Harthoorn, A. M. 1976. Physiology of capture myopathy. Ph.D. Thesis, University of Pretoria. Harthoorn, A. M., van der Walt, K., and Young, E. 1974. Possible therapy for capture myopathy in captured wild animals. Nature (London) 247, 577. Hartshome, D. J., Barnes, E. M., Parker, L., and Fuchs, F. 1972. The effect of temperature on actomyosin. Biochim. Biophys. Acta 267, 19g202. Heffron, J. J. A,, and Gronert, G. A. 1977. Calcium binding and respiratory activity of skeletal muscle mitochondria of pigs susceptible to the malignant hyperthermia syndrome. Ir. J . Med. Sci. 146, 87. Heffron, J. J. A , , and Harris, E. J. 1981. The effect of temperature on sodium stimulated and basal calcium efflux from cardiac and skeletal muscle mitochondria. Biochem. SOC. Trans. 9, 82-83. Heffron, J. J. A , , and Isaacs, H. 1976. Malignant hyperthermia syndrome-evidence for denervation changes in human skeletal muscle. Klin. Wochenschr. 54, 865-867. Heffron, J. J. A,, and Mitchell, G. 1975a. Age dependent variation of serum creatine phosphokinase levels in pigs. Experientia 31, 657-658. Heffron, J. J. A., and Mitchell, G. 1975b. Diagnostic value of serum creatine phosphokinase activity for the porcine malignant hyperthermia syndrome. Anesth. Analg. (Cleveland) 54, 5 3 6 539. Heffron, J. J. A , , and Mitchell, G. 1975c. Procaine for malignant hyperthermia. N . Engl. J . Med. 292, 266267. Heffron, J . J. A., and Mitchell, G. 1976. Calcium uptake by sarcoplasmic reticulum of muscle of pigs susceptible to malignant hyperthermia. Proc. Int. Pig Vet. SOC. Congr., 1976 p T6. Heffron, J. J. A,, and Mitchell, G. 1981. Influence of temperature, pH, halothane and its metabolites on the osmotic fragility of erythrocytes of malignant hyperthermia susceptible and resistant pigs. Br. J . Anaesth. 53, 499-504.

224

G. MITCHELL AND J. J. A. HEFFRON

Hende, C. v. d., Lister, D., Muylle, E., Ooms, L., and Oyaert, W. 1976. Malignant hyperthermia in Belgian Landrace pigs rested or exercised before exposure to halothane. Br. J . Anaesth. 48, 821-829. Henschel, J. R., and Louw, G. N. 1978. Capture stress, metabolic acidosis and hyperthermia in birds. S. Afr. J . Sci. 74, 305-306. Hess, J. W., Macdonald, R. P . , Frederick, R. J., Jones, R. N., Neely, J., and Gross, D. 1964. Serum creatine phosphokinase (CPK) activity in disorders of heat and skeletal muscle. Ann. Intern. Med. 61, 1015-1028. Hoet, J. P., and Marks, H. P. 1926. Observation on the onset of rigor mortis. Proc. R . SOC.London, Ser. B 100, 75-86. Hofmeyr, J. M., Louw, G . N., and du Preez, J. S . 1973. Incipient capture myopathy as revealed by blood chemistry of chased zebras. Madoqua Ser. I 7, 45-50. Hoivik, B., and Stovner, J. 1975. Procaine and malignant hyperthermia. Lancet 2, 185. Holmes, J. H. G., Ashmore, C. R., and Robinson, D. W. 1973. Effects of stress on cattle with hereditary muscular hypertrophy. J . Anim. Sci. 36, 684694. Hull, M. T., Muller, J., and Albrecht, W. 1978. Morphologic abnormalities in a case of malignant hyperthermia. Anesthesiology 48, 223-228. Husain, S., and Paradise, R. R. 1973. Effects of halothane on substrate metabolism in rat cerebral cortex slices. Fed. Proc., Fed. Am. SOC. Exp. Biol. 32, 2825. Inesi, G . , Millman, M., and Eletr, S. 1973. Temperature induced transitions of function and structure in sarcoplasmic reticulum membranes. J . Mol. Biol. 81, 483-504. Innes, R. K. R., and Stromme, J. H. 1973. Rise in serum creatine phosphokinase associated with agents used in anaesthesia. Br. J . Anaesth. 45, 185-190. Isaacs, H. 1973. High serum creatine phosphokinase levels in asymptomatic members of the families of patients developing malignant hyperpyrexia-a genetic study. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 331-338. Thomas, Springfield, Illinois. Isaacs, H., and Barlow, M. B. 1970. The genetic background to malignant hyperpyrexia revealed by serum creatine phosphokinase estimations in asymptomatic relatives. Br. J . Anaesth. 42, 1077-1084. Isaacs, H., Frere, G., and Mitchell, J. A. 1973. Histological, histochemical and ultramicroscopic findings in muscle biopsies from carriers of the trait for malignant hyperthermia. Br. J . Anaesth. 45, 860-868. Isaacs, H., Heffron, J. J. A,, and Badenhorst, M. 1975. Predictive tests for malignant hyperpyrexia. Br. J . Anaesth. 47, 1075-1080. Jedlika, J., Mojto, J., and Sidor, V. 1976. Correlation between 17-OHCS levels influenced by heat stress and some basic carcass value indices in pigs. Anim. Prod. 23, 243-248. Johnson, S . M., and Bangham, A. D. 1969. The action of anaesthetics on phospholipid membranes. Biochim. Biophys. Acta 193, 92-104. Johnson, S. M., and Miller, K. W. 1970. Antagonism of pressure and anaesthesia. Nature (London) 228, 75-76. Jones, E. W., Nelson, T. E., Anderson, I. L., Kerr, D. D., and Bumap, T. K. 1972. Malignant hyperthermia of swine. Anesthesiology 36, 42-5 1. Judge, M. D., Briskey, E. J., and Meyer, R. K. 1966. Endocrine related postmortem changes in porcine muscle. Nature (London) 212, 287-288. Judge, M. D., Briskey, E. J., Cassens, R. G., Forrest, J. C., and Meyer, R. K. 1968. Adrenal and thyroid function in stress-susceptible pigs (Sus domesticus). Am. J . Physiol. 214, 146151. Kalow, W., Britt, B. A,, and Richter, A. 1977. The caffeine test of isolated human muscle in relation to malignant hyperthermia. Can. Anaesth. Soc. J . 24, 67&694. Kastenschmidt, L. L., Hoekstra, W. G . , and Briskey, E. J. 1966. Metabolic intermediates in skeletal muscles with fast and slow rates of postmortem glycolysis. Nature (London) 212, 288-289.

PORCINE STRESS SYNDROMES

225

Kawane, M . , Mono, M., Ohtani, M., Kikuchi, H., Yuge, O . , Senami, M., and Chikasue, F. 1979. Statistical review of malignant hyperthermia in Japan. Hiroshima J . Anesth. 15, Suppl. 54. Keaney, N. P., and Ellis, F. R. 1971. Malignant hyperpyrexia. Br. Med. J . 4, 49. Kendig, J. J., and Bunker, J. P. 1972. Alterations in muscle resting potentials and electrolytes during halothane and cyclopropane anesthesia. Anesthesiology 36, 128-131. Kerr, D., Wingaard, D. W., and Gatz, E. E. 1975. Prevention of porcine malignant hyperthermia by epidural block. Anesthesiology 42, 307-3 11. King, W. A., Ollivier, L., and Basrur, P. K. 1976. Erythrocyte osmotic response test on malignant hyperthermia susceptible pigs. Ann. Genet. Sel. Anim. 8, 537-540. Klein, L. V. 1975. Case report: A hot horse. Vet. Anesth. 2, 4 1 4 2 . Korczyn, A. D., Shavit, S . , and Shlosberg, I. 1980. Chick as a model for malignant hyperpyrexia. Eur. J . Pharmacol. 61, 187-189. Lawrie, R. A. 1960. Postmortem glycolysis in normal and exudative longissimus dorsi muscles of the pig in relation to so-called white muscle disease. J . Comp. Pathol. 70, 273-295. Lefever, G. S . , and Rosenberg, H. 1980. Aminophylline, halothane and malignant hyperpyrexia in the rabbit. Fed. Proc., Fed. Am. SOC.Exp. Biol. 39, 295. Lehninger, A. L. 1970. Mitochondria and calcium ion transport. Biochem. J . 119, 129-138. Levy, H. M., and Fleisher, M. 1965. Studies of the superprecipitation of actomyosin suspension as measured by change in turbidity. 1. Effects of adenosine triphosphate concentration and temperature. Biochim. Biophys. Acta 100, 479-490. Liebenschutz, F., Mai, C., and Pickerodt, W. W. A. 1979. Increased carbon dioxide production in two patients with malignant hyperpyrexia and its control by dantrolene. Br. J . Anaesth. 51, 899-903. Lister, D. 1973. Correction of adverse response to suxamethonium of susceptible pigs. Br. Med. J . 1, 208-210. Lister, D., Sair, R. A,, Will, J. A,, Schmidt, G. R., Cassens, R. G., Hoekstra, W. G., and Briskey, E. J. 1970. Metabolism of striated muscle of stress susceptible pigs breathing oxygen or nitrogen. Am. J . Physiol. 218, 102-107. Lister, D., Lucke, J. N., and Perry, D. N. 1972. Investigation of the hypothalamic-pituitary-adrenal axis in mesomorphic types of pigs. J . Endocrinol. 53, 505-506. Lister, D., Hall, G. M., and Lucke, J. N. 1974. Catecholamines in suxamethonium-induced hyperthermia in Pietrain pigs. Br. J . Anaesth. 46, 803-804. Lister, D., Hall, G. M., and Lucke, J. N. 1975. Malignant hyperthermia: A human and porcine stress syndrome. Lancet 1, 519. Lister, D., Hall, G. M., and Lucke, J. N. 1976. Porcine malignant hyperthermia. 111. Adrenergic blockade. Br. J . Anaesth. 48, 831-838. Lucke, J. N. 1977. Malignant hyperthermia in a parturient Poland China sow. Br. J . Anaesth. 49, 1070. Lucke, J. N., Hall, G. M., and Lister, D. 1976. Porcine malignant hyperthermia. I. Metabolic and physiological changes. Br. J . Anaesth. 48, 297-304. Lucke, J. N., Denny, H., Hall, G. M., Lovell, R., and Lister, D. 1978. Porcine malignant hyperthermia. VII. Effects of bilateral adrenalectomy and pretreatment with bretylium on halothaneinduced response. Br. J . Anaesth. 50, 241-246. Ludvigsen, J. 1957. On the hormonal regulation of vasomotor reactions during exercise with special reference to the action of adrenal cortical steroids. Acta Endocrinol. (Copenhagen) 26, 406416. McComas, A. J . , Sica, R. E. P., Upton, A. R. M., and Petito, F. 1974. Sick motoneurones and muscle disease. Ann. N . Y . Acad. Sci. 228, 261-279. McCormack, J. G., and Denton, R. M. 1980. Role of calcium ions in the regulation of intramitochondria1 metabolism. Biochem. J . 190, 95-105.

226

G. MITCHELL AND J. J. A . HEFFRON

McGloughlin, P., Ahern, C. P., Butler, M., and McLoughlin, J. V. 1980. Halothane-induced malignant hyperthermia in Irish pig breeds. Livest. Prod. Sci. 7, 147-154. McIntosh, D. B., and Berman, M. C. 1974. Neutral lipid and phospholipid composition of normal and myopathic skeletal muscle of pigs. S. Afr. Med. J . 48, 1221. McIntosh, D. B., Berman, M. C., and Kench, J. E. 1977. Characteristics of sarcoplasmic reticulum from slowly glycolysing and from rapidly glycolysing pig skeletal muscle post-mortem. Biochem. J . 166, 387-398. MacLachlan, D., and Forrest, A. L. 1974. Procaine and malignant hyperthermia. Lancet 1, 355. McLoughlin, J . V. 1963. Studies on pig muscle. 11. The effect of rapid postmortem pH fall on the extraction of sarcoplasmic and myofibrillar proteins of post-rigor muscle. Ir. J . Agric. Res. 2 , 115-124. McLoughlin, J. V. 1971. The death reaction and metabolism post-mortem of porcine skeletal muscle. In “Condition and Meat Quality of Pigs” (J. C. M. Hessel-de-Heer, G. R . Schmidt, and W. Sybesma, eds.), pp. 123-132. Pudoc, Wageningen. Marple, D. N., and Cassens, R. G. 1973. Increased metabolic clearance of cortisol by stress susceptible swine. J . Anim. Sci. 36, 1139-1 142. Marple, D. N., Judge, M. D., and Aberle, E. D. 1972. Pituitary and adrenocortical function of stress-susceptible swine. J . Anim. Sci. 35, 995-1000. Marple, D. N., Jones, D. J., Alliston, C. W., and Forrest, J. C. 1974. Physiological and endocrinological changes in response to terminal heat stress in swine. J . Anim. Sci. 39, 79-82. Marple, D. N., Nachreiner, R. F., McGuirre, J. A,, and Squires, C. D. 1975. Thyroid function and muscle glycolysis in swine. J . Anim. Sci. 41, 799-803. Means, A. R., and Dedman, J. R. 1980. Calmodulin-an intracellular calcium receptor. Nature (London) 285, 73-77. Melander, A . , Ericson, L. E., and Sindler, F. 1974. Sympathetic regulation of thyroid hormone secretion. Life Sci. 14, 237-246. Miller, R. N., and Hunter, F. E. 1970. The effect of halothane on electron transport, oxidative phosphorylation and swelling of rat liver mitochondria. Mol. Pharmacol. 6, 67-77. Mitchell, G . , and Heffron, J . J. A. 1975a. Factors affecting serum creatine phosphokinase activity in pigs. J . S. Afr. Vet. Assoc. 46, 145-148. Mitchell, G., and Heffron, J. J. A. 1975b. Procaine in porcine malignant hyperthermia. Br. J . Anaesth. 47, 667-668. . the toxicity of procaine for pigs. J . South Afr. Vet. Mitchell, G., and Heffron, J. J. A. 1 9 7 5 ~On Assoc. 46, 259-260. Mitchell, G . , and Heffron, J. J. A. 1980a. The occurrence of pale, soft, exudative musculature in Landrace pigs susceptible and resistant to the malignant hyperthermia syndrome. Br. Vet. J . 136, 500-506. Mitchell, G., and Heffron, J. J. A. 1980b. Porcine stress syndromes: A mitochondria1 defect? S. Afr. J . Sci. 76, 546-55 1. Mitchell, G . , and Heffron, J. J. A . 1981a. Plasma cortisol levels in pigs susceptible and resistant to malignant hyperthermia. J . Sourh Afr. Vet. Assoc. 52, 109-1 12. Mitchell, G., and Heffron, J. J. A. 1981b. Some muscle and growth characteristics of pigs susceptible to stress. Br. Vet. J . 137, 374380. Mitchell, G., Heffron, J. J. A,, and van Rensburg, A. J. J. 1980. A halothane induced biochemical defect in muscle of normal and malignant hyperthermia susceptible Landrace pigs. Anesth. Analg. (Cleveland) 59, 250-256. Mogenson, J. V . , Misfeldt, B., and Hanel, H. 1974. Preoperative excitement and malignant hyperthermia. Lancet 1, 461. Moulds, R. F. W. 1978. The site of the abnormality in MH muscle: A comparison of MH muscle and denervated muscle. In “Maiignant Hyperthermia” (J. A . Aldrete and B. A. Britt, eds.), pp. 49-65. Grune & Stratton, New York.

PORCINE STRESS SYNDROMES

227

Moulds, R. F. W . , and Denborough, M. A. 1972. Procaine in malignant hyperpyrexia. Br. Med. J . 4, 526-528. Moulds, R. F. W., and Denborough, M. A. 1974. Identification of susceptibility to malignant hyperpyrexia. Br. Med. J . 2, 245-247. Muir, A. R. 1970. Normal and regenerating skeletal muscle fibres in Pietrain pigs. J . Comp. Pathol. 80, 137-143. Nahas, G. G . , Ligou, J. C., and Mehlman, B. 1960. Effects of pH changes on oxygen uptake and plasma catecholamine levels in the dog. Am. J . Physiol. 198, 6C66. Nahnvold, M. L., and Cohen, P. J. 1973. The effects of forane and fluoroxene on mitochondria1 respiration: Correlation with lipid solubility and in vivo potency. Anesthesiology 38, 437444. Needham, D. M. 1971. “Machina Camis,” pp. 152-153. Cambridge Univ. Press, London and New York. Nelson, T. E. 1973. Porcine stress syndromes. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 191-197. Thomas, Springfield, Illinois. Nelson, T. E., and Bee, D. E. 1979. Temperature perturbation studies of sarcoplasmic reticulum from malignant hyperthermia pig muscle. J . Clin. Invest. 64, 895-901. Nelson, T. E., Jones, E. W., Venable, J. H., and Ken, D. D. 1972. Malignant hyperthermia of Poland China swine. Anesthesiology 36, 52-56. Nelson, T. E., Jones, E. W., Henrickson, R. L., Falk, S. N., and Kerr, D. D. 1974. Porcine malignant hyperthermia: Observations on the occurrence of pale, soft, exudative musculature among susceptible pigs. Am. J . Vet. Res. 35, 347-350. Nelson, T. E., Austin, K. L., and Denborough, M. A. 1977. Screening for malignant hyperpyrexia. Br. J . Anaesth. 49, 169-172. Noble, W. H., McKee, D., and Gates, B. 1973. Malignant hyperthermia with rigidity successfully treated with procainamide. Anesthesiology 39, 4 5 M 5 1. Okumura, F., Crocker, B. D., and Denborough, M. A. 1979. Identification of susceptibility to malignant hyperpyrexia in swine. Br. J . Anaesth. 51, 171-176. Okumura, F., Crocker, B. D., and Denborough, M. A. 1980. Site of the muscle cell abnormality in swine susceptible to malignant hyperthermia. Br. J . Anaesth. 52, 377-383. Ollivier, L., Sellier, P., and Monin, G . 1975. Determinisme gCnCtique du syndrome d’hyperthermie maligne chez le porc de Pietrain. Ann. Genet. Sel. Anim. 7 , 159-166. Oyama, T. S . , and Takiguchi, K. T. 1970. Plasma levels of ACTH and cortisol in man during halothane anaesthesia and surgery. Anesth. Analg. (Cleveland) 49, 363-366. Oyama, T. S . , Shibata, F., Matsumoto, M., Takiguchi, M., and Kudo, T. 1968. Effects of halothane anaesthesia and surgery on adrenocortical function in man. Can. Anaesth. Soc. J . 15, 258266. Ozawa, E., and Ebashi, S. 1967. Requirement of calcium ion for the stimulating effect of cyclic AMP on muscle phosphorylase b kinase. J . Biochem. (Tokyo) 62, 285-286. Pandit, S. K., Kothary, S. P., and Cohen, P. J. 1979. Orally administered dantrolene for prophylaxis of malignant hyperthermia. Anesthesiology 50, 156-158. Parpart, A. K., Lorenz, P. B., Parpart, E. R., Gregg, J. R . , and Chase, A. M. 1947. The osmotic resistance (fragility) of human red cells. J . Clin. Invest. 26, 636-654. Patterson, S. P., and Allen, W. M. 1972. Biochemical aspects of some pig muscle disorders. Br. Vet. J . 128, 101-111. Pertz, C., and Sundberg, J. P. 1978. Malignant hyperthermia induced by etorphine and xylazine in a fallow deer. J . Am. Vet. Med. Assoc. 173, 1243. Pollock, R . A,, Standefer, J. C., Hildebrandt, P. K., Goodwin, B., and Li, T-K. 1973. Malignant hyperthermia in the American Landrace pig. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 224228. Thomas, Springfield, Illinois. Price, H. L., Skovestead, P., Pauca, A. L., and Cooperman, L. M. 1970. Evidence for beta-receptor activation produced by halothane in normal man. Anesthesiology 32, 389-395.

228

G. MITCHELL AND J. J . A. HEFFRON

Putney, J. W., and Bianchi, C. P. 1973. Effect of dantrolene on E-C coupling in skeletal muscle. Fed. Proc., Fed. Am. SOC.Exp. Biol. 32, 772. Rasmussen, B. A., and Christian, L. L. 1976. H blood types in pigs as predictors of stress susceptibility. Science 191, 947-948. Regen, D. M., Young, D. A. B., and Davis, W. W. 1964. Adjustment of glycolysis to energy utilization in perfused rat heart. J . Biol. Chem. 239, 381-384. Relton, J.E. S., Steward, D. J., Creighton, R. E., and Britt, B. A. 1972. Malignant hyperpyrexia: A therapeutic and investigative regimen. Can. Anaesth. SOC. J . 19, 200-205. Reske-Nielsen, E. 1978. Malignant hyperthermia in Denmark: Survey of a family study and investigation into muscula morphology in ten additional cases. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 287-327. Grune & Stratton, New York. Rosenberg, H., and Haugaard, N. 1973. The effects of halothane on metabolism and calcium uptake in mitochondria of the rat liver and brain. Anesthesiology 39, 4&53. Rubin, R. P., Feinstein, M. B., Jaanus, S. D., and Paimre, M. 1967. Inhibition of catecholamine secretion and calcium exchange in perfused cat adrenal glands by tetracaine and magnesium. J . Pharmacol. Exp. Ther. 155, 463471. Sachsenheimer, W . , Pai, E. F., Schulz, G. E., and Schirmer, R. H. 1977. Halothane binds in the adenine-specific niche of crystalline adenylate kinase. FEBS Lett. 79, 3 10-3 12. Sair, R. A,, Lister, D., Moody, W. G., Cassens, R. G., Hoekstra, W. G., and Briskey, E. J. 1970. Action of curare and magnesium on striated muscle of stress-susceptible pigs. Am. J . Physiol. 218, 108-114. Schanus, E., Glass, R., and Lovrien, R. 1979. Molecular basis for malignant hyperthermia involving erythorcyte glutathione peroxidase. Fed. Proc., Fed. Am. SOC. Exp. Biol. 38, 571. Schmidt, G. R., Zuidam, L., and Sybesma, W. 1972. Biopsy technique and analysis for predicting pork quality. J . Anim. Sci. 34, 25-29. Scott, I. G., and Jeacocke, R. E. 1980. Interference by cytochromes in the measurement of calcium with murexide. FEBS Lett. 109, 93-98. Sebranek, J. G., Marple, D. N., Cassens, R. G., Briskey, E. J., and Kastenschmidt, L. L. 1973. Adrenal response to ACTH in the pig. J . Anim. Sci. 36, 4 1 4 4 . Short, C. E., and Paddleford, R. R. 1973. Malignant hyperthemia in the dog. Anesthesiology 39, 462463. Smith, C., and Bampton, P. R. 1977. Inheritance of reaction to halothane anaesthesia in pigs. Genef. Res. 29, 287-292. Snodgrass, P. J., and Piras, M. M. 1966. The effects of halothane on rat liver mitochondria. Biochemistry 5, 1140-1149. Snyder, H. R., Davis, C. S . , Bickerton, R. K., and Halliday, R. P. 1967. l(5-arylfurfurylidene) amino hydantoins. A new class of muscle relaxants. J . Med. Chem. 10, 807-810. Solomons, C. C., Tan, S . , and Aldrete, J. A. 1978. Platelet metabolism and malignant hyperthermia. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 221-225. Grune & Stratton, New York. Somers, C. J., Wilson, P., Ahern, C. P., and McLoughlin, J. V. 1977. Energy phosphate turnover and glycolysis in skeletal muscle of the Pietrain pig: The effects of premedication with azaperone and pentobarbitone anaesthesia. J . Comp. Pathol. 87, 177-183. Starkweather, W. H., Zsigmond, E. K., Duboff, G. S., and Flynn, K. 1973. Creatine phosphokinase isoenzyme patterns in a malignant hyperthermic family. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 339-349. Thomas, Springfield, Illinois. Stephen, C. R. 1967. Fulminant hypeithermia during anesthesia and surgery. JAMA, J . Am. Med. ASSOC.202, 178-182. Stovner, J., Innes, K. R., and Holen, A. 1976. Ten cases of malignant hyperthermia in Norway. Can. Anaesth. SOC.J . 23, 518-526.

PORCINE STRESS SYNDROMES

229

Strobel, G. E. 1971. Treatment of anaesthetic induced malignant hyperpyrexia. Lancet 1, 40. Sulakhe, P. V., Drummond, G. I., and Nig, D. C. 1973. Calcium binding by skeletal muscle sarcolemma. J . Biol. Chem. 248, 415M157. Sutherland, E. W . , and Rall, T. W. 1960. The relation of adenosine 4’, 5’ phosphorylase to the actions of catecholamines and other hormones. Pharmacol. Rev. 12, 265-299. Swatland, H. J., and Cassens, R . G. 1972. Peripheral innervation of muscle from stress-susceptible pigs. J . Comp. Pathol. 82, 229-236. Sybesma, W . , and Eikelenboom, G . 1969. Malignant hyperthermia syndrome in pigs. Neth. J . Vet. Sci. 2, 155-160. Sybesma, W., and Eikelenboom, G. 1978. Methods of predicting pale, soft, exudative pork and their application in breeding programmes-A review. Meat Sci. 2, 79-90. Szent-Gyorgi, A. 1944. Studies on muscle. Acta Physiol. Scand. 9, Suppl. 25. Tammisto, T., and Airaksinen, M. 1966. Increase of creatine kinase activity in serum as a sign of muscular injury caused by intermittently administered suxamethonium during halothane anaesthesia. Br. J . Anaesth. 38, 51e515. Tapley, D. F. 1956. The effect of thyroxine and other substances on the swelling of isolated rat liver mitochondria. J . Biol. Chem. 222, 325-339. Tapley, D. F., and Cooper, C. 1956. The effect of thyroxine and related compounds on oxidative phosphorylation. J . Biol. Chem. 222, 341-349. Teeter, C . , Cam, S. C., Tsai, R., and Briskey, E. J. 1969. A cryobiopsy technique for assessing metabolite levels in skeletal muscle. Proc. SOC.Exp. Biol. Med. 131, 5-7. Topel, D. G., Merkel, R. A., and Wismer-Pedersen, J. 1967. Relationship of plasma 17-hydroxycorticosteroid levels to some physical and biochemical properties of porcine muscle. J . Anim. Sci. 26, 311-315. Venable, J. H. 1973. Skeletal muscle structure in Poland China pigs, suffering from malignant hyperthermia. In “Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), pp. 208-223. Thomas, Springfield, Illinois. Waldronmease, E. 1978. Correlation of postoperative and exercise-induced equine myopathy with the defect malignant hyperthermia. Proc. Am. Assoc. Equine Practitioners 24, 95-99. Wang, J . E., Moffitt, E. A,, and Rosevear, J. W. 1969. Oxidative phosphorylation in acute hyperthermia. Anesthesiology 30, 439-442. Watanabe, S . , Akita, T., Mikami, H., Mizuho, A , , and Jinbu, M. 1979. Evaluation of CPK and LDH, in screening pigs for malignant hyperthermia. Hiroshima J . Anesth. 15, 124. Weber, A,, and Herz, R. 1961. Requirement for calcium in the synaeresis of myofibrils. Biochem. Biophys. Res. Commun. 6, 364-368. Weber, A., and Herz, R. 1968. The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J . Gen. Physiol. 52, 75e759. Weber, A,, and Winicur, S. 1961. Dependence of superprecipitation of actomyosin on the concentration of ionised calcium. Fed. Proc., Fed. Am. SOC. Exp. Biol. 20, 300. Weiss, G. M., Topel, D. G., Siers, D. G., and Ewan, R. C. 1974. Influence of adrenergic blockade upon some endocrine and metabolic parameters in a stress-susceptible and fat strain of swine. J . Anim. Sci. 38, 591-597. Westfall, T. C. 1977. Local regulation of adrenergic neurotransmission. Physiol. Rev. 57,659-712. Wikinski, J. A,, Usubiaga, J.E., and Wikinski, P. W. 1970. Cardiovascular and neurological effects of 4000 mg of procaine. JAMA, J . Am. Med. Assoc. 213, 621-623. Williams, C. H., Houchins, C., and Shanklin, M. D. 1975. Pigs susceptible to energy metabolism in the fulminant stress syndrome. Br. Med. J . 3, 411-413. Williams, C. H., Shanklin, M. D., Hedrick, H. B., Muhrer, M. E., Stubbs, D. H., Krause, G. F., Payne, C. G., Benedict, J. D., Hutcheson, D. P., and Lasley, J. F. 1978a. The fulminant hyperthermia-stress syndrome: Genetic aspects, hemodynamic, and metabolic measurements in

230

G. MITCHELL AND J. J. A . HEFFRON

susceptible and normal pigs. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 113-140. Grune & Stratton, New York. Williams, C. H., Hoech, G. P., and Roberts, J. T. 1978b. Experimental malignant hyperthermia. Anesthesiology 49, 5&59. Willner, J. H., Cem, C. J., and Wood, D. S. 1979. Malignant hyperthermia: Abnormal cyclic AMP metabolism in skeletal muscle. Neurology 29, 557. Wilson, R. D., Nichols, R. J., Dent, T. E., and Allen, C. R. 1966. Disturbances of the oxidative phosphorylation mechanisms as a possible etiological factor in sudden unexplained hyperthermia occurring during anesthesia. Anesthesiology 27, 23 1-232. Wilson, R. D., Dent, T. E., Traber, D. L., McCoy, N. R., and Allen, C. R. 1967. Malignant hyperpyrexia with anaesthesia. JAMA, J. Am. Med. Assoc. 202, 183-186. Wingaard, D. W. 1974. Malignant hyperthermia. A human stress syndrome. Lance? 2, 1450. Wingaard, D. W., and Gatz, E. E. 1978. Some observations on stress-susceptible patients. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 363-372. Grune & Stratton, New York. Winstanley, M. 1979. A cure for stress. New Sci. 84, 594596. Wismer-Pedersen, J. 1959. Quality of pork in relation to rate of pH change postmortem. Food Res. 24, 71 1-727. Wismer-Pedersen. J., and Briskey, E. J. 1961. Rate of anaerobic glycolysis versus structure in pork muscle. Nature (London) 189, 318-320. Woolf, N., Hall, L. W., Thorne, C., Down, M., and Walker, R. 1970. Serum creatine phosphokinase levels in pigs reacting abnormally to halogenated anaesthetics. Br. Med. J. 3, 386-387. Young, E. 1967. Leg paralysis in the greater flamingo and the lesser flamingo following capture and transportation. Int. Zoo1 Yearb. 7 , 226227. Young, E., and Bronkhorst, P. J. L. 1971. Overstraining disease in game. Afr. Wildl. 25, 51-52. Zsigmond, E. K., Penner, J., and Kothary, S. P. 1978. Normal erythrocyte fragility and abnormal platelet aggregation in MH families: A pilot study. In “Malignant Hyperthermia” (J. A. Aldrete and B. A. Britt, eds.), pp. 213-219. Grune & Stratton, New York.

ADVANCES IN FOOD RESEARCH, VOL.

28

CHEMICAL, BIOCHEMICAL, FUNCTIONAL, AND NUTRITIONAL CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS A . ASGHAR' AND R.L. HENRICKSON Oklahoma Agricultural Experiment Station, Oklahoma State Universiry, Stillwater, Oklahoma

I. Introduction . . ......................................... 11. Morphology of ................................... A. Different Genetic Types of Collagen. . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Isolation and Identification of Collagen Types . . . . . . . . . . . . . . . . . . . . ...... 111. Chemistry of Collagen . . . . . . . . . . . . . , . , . . . . . . . . . . . . . . A. Amino Acid Composition.. . . . . . . . . . . . . . , . . . . . . . , . . . . . . . . . . . . . B. Molecular Organization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. C. Amino Acid Sequences.. . . . . . . . . . . ............. D. Functional Role of Amino Acids . . . E. Type and Nature of Interchain Cross.................... F. Interaction with Carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Polysaccharides of Connective Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Immunochemistry of Collagen ........................... ..... I. Functions of Collagen in Tissues.. . . . . . . . . . . . . . . . . IV. Metabolism of Collagen . . . . . . . . , . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Biosynthesis on Polyribosomes. . . . . . ................... B. Catabolism of Collagen.. . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Factors Affecting Collagen Composition and Structure . . . . . . . . . . . . . . . . . A. Antemortem Factors . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Postmortem Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. VI. Functional Properties of Collagen in Food Systems . . . . . . . . . . A. Water Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............................. .... B. Swelling. .. . . . . , . , . . . . C. Emulsifyi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Foaming. .......................................... E. Viscoelasticity . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . , . . . . . . . . . . . . .

232 233 233 238 240 24 1 245 241 250 25 1 260 26 1 266 266 261 261 212 214 215 284 287 288 306 309 311 312

'Present address: Department of Food Technology, University of Agriculture, Fasialaband, Pakistan.

23 I Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved ISBN 0-12-016428-0

232

A. ASGHAR AND R. L. HENRICKSON VII.

VIII.

Nutritional Aspects of Collagen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. A. Protein Quality Assays .................................. B. Digestibility of Collage C. Biological Value and PER of Collagen .......................... D. Possible Fortification Methods of Collagen.. ..................... Food Uses of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Production of Edible Fibrous Collagen . . . . . . . . . . . . . . . . C.

IX.

312 313 318 319 320 322

Various Uses of Collagen as Gelatin

Research Needs ......................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

331 333

INTRODUCTION

The unique features of connective and skeletal tissues are their diversity and morphological character. In addition, these tissues are composed basically of varying proportions of similar extracellular constituents. Advances in connective tissue research have revealed the presence of several kinds of such tissue whose organization is designed by nature to perform specific biological functions in the animal body. The functional properties of connective tissue in vivo are mainly determined by the macromolecular organization of the collagen structure, which is the principal component of connective tissue. For example, collagen constitutes over 70% of the dry weight of skin, tendon, and cartilage (Grant and Jackson, 1976), yet the physical properties of each tissue are different. The general agreement among scientists is that various macromolecular structures result from distinct genetically determined types of collagen (Harwood, 1979). Evolutionary processes probably gave rise to various types of collagen by altering the amino acid sequence but preserving the general structural features to perform different biochemical functions in the body. Apart from the physiological significance of collagen in various tissues, its bearing on the texture of meat is also well recognized. For that matter, both the quantity of collagen and its quality characteristics (extent of cross-linkages) are important (Bailey, 1972; Asghar and Yeates, 1978). From a nutritional point of view, collagen is an incomplete protein since it is limiting in some essential amino acids such as methionine, lysine, and threonine, and is practically devoid of tryptophan (McClain ef al., 1971). However, collagen, on account of its unique structural characteristics, possesses many potential functional properties (moisturizing, binding, texturizing , lubricating, viscoelastic, emulsifying, synergistic), which it can impart under appropriate conditions in various food systems. Collagen also has many other industrial uses. These facts probably enticed Battista (1975) to state, “Nature has produced in collagen a polymer architecture

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

233

of remarkable sophistication, an ‘engineering’ achievement that qualifies collagen for a role of far greater versatility and complexity than any other known man-made or natural high-molecular-weight polymer. This article is designed to review the current information on the biology, chemistry, biochemistry, and nutritional aspects of collagen with special reference to its potential uses in various food systems. ”

II. MORPHOLOGY OF COLLAGEN Morphologically, connective tissue consists of three distinct components: fibrous proteins, ground substance, and cells. The major fibrous proteins include mainly collagen with some elastin and reticulin. The ground substance occupies the extracellular space of the connective tissue as a viscous fluid derived from plasma (Fitton-Jackson, 1964), which is composed of globular mucoprotein. The proteins are associated with mucopolysaccharides, such as hyaluronic acid, chondroitin sulfates A, B, and C, keratosulfate, heparitin sulfate, and heparin in the form of galactosamine or glucosamine. The proportion of the various mucopolysaccharides in ground substance varies in different tissues. Generally two types of cell populations have been recognized in the extracellular space: fixed and wandering cells. The fixed cells comprise fibroblasts, mesenchyme cells, and adipose fat storage cells. The wandering cells comprise mast cells, macrophages or histiocytes, lymph cells, eosinophiles, and plasma cells, and are concerned mainly with controlling infection (Fitton-Jackson, 1965; Schubert and Hamerman, 1968; Forrest et al., 1975). A.

DIFFERENT GENETIC TYPES OF COLLAGEN

Before 1970, all vertebrate collagens were regarded as a simple class of molecules composed of two a1 chains and one a2 chain, with only minor heterogeneity in composition between species. During the last decade, this view has been changed by the discoveries of several genetically distinct forms of collagen having different chemical composition of a 1 chains. About 10 distinct collagen types have been reported so far with different degrees of precision, and these are believed to be the products of at least 10 nonallelic structural genes (Harwood, 1979). However, the presence of five types of a chains, namely, al(I), al(II), aI(III), al(IV), and a2 chains, is well established in collagen molecules from various sources (Miller, 1973; Epstein, 1974; Johnson et al., 1974; Epstein and Munderloh, 1975; Slutskii and Simkhovich, 1980). These a chains constitute various types of collagen which are genetically distinct and differ in primary structure. Many researchers succeeded recently in isolating the mRNA (Diaz de Le6n et al., 1977; Monson and Goodman, 1978), and in cloning

234

A. ASGHAR AND R . L. HENRICKSON

the DNA fragments corresponding to parts of the message (Lehrach et al., 1978; Sobel et al., 1978; Graves et al., 1979). I.

Collagen Types

The salient features of the four forms of collagen are shown in Table I. Type I collagen is composed of two identical al(1) chains and one a 2 chain and denoted as [a1(1)]2[a2(1)].It is found in mature skin, tendon, bone, and cornea. Type I1 collagen from cartilage is composed of three identical al(I1) chains and is designated [cxl(H)13. Type I11 collagen, found in human fetal dermis and the cardiovascular system, is composed of three identical al(II1) chains and is named (al(III)l3. Type IV collagen is found in the basement membrane. This collagen is composed of three identical al(1V) chains and is designated [al(IV)],. Some recent reports have indicated the existence of molecular heterogeneity within collagen types (Crouch et al., 1980). Tissue culture studies by Benya et al. (1977, 1978) on rabbit chondrocytes have shown that dedifferentiated chondrocytes in four subcultures also produced the type I trimer, [a1(I)I3, in addition to type I and 111 collagens, and two new pepsin-resistant collagen chains X and Y having the chain composition X,Y. They considered the new chains as the product of expression of two different collagen genes. The possible existence of additional a chains in the basement membrane of human placenta has also been indicated by Burgeson et al. (1976) and Burgeson and Hollister (1977). The new chains were designated as a A and a B , which constituted collagen type [aA(aB),]. Chung et al. (1976) have indicated the presence of additional chains, designated as A and B, in several human tissues. These chains are believed to constitute collagen [A], and [BI3 types, which closely resembled type IV collagen but lack cysteine. Two other polypeptide chains of collagen, called CP55 (Chung et al., 1976) and CP45 (Mayne et al., 1977a,b), have been identified. A study by Butler et al. (1977) has indicated that al(I1) chain in nasal cartilage is the product of more than one structural gene, which produces two types of al(I1) chains, called al(I1)-Major and cYl(I1)-Minor.They could be classified as a2(II) if they are the product of a genetic locus different from that for al(1I) chain (Bornstein and Sage, 1980). Recently, Davison et al. (1979) have also identified AB collagen from bovine cornea. They designated it as type VI on the assumption that Little and Church (1978) had already reported the presence of type V collagen in mouse embryo. Stenn et al. (1979) have also found AB, collagen. A critical assessment of the data on the above-mentioned new chains from various sources by Bornstein and Sage (1980) suggests that A and B chains, and the recently discovered C chain (Sage and Bornstein, 1979), have many common features. They believe that there is now enough evidence to categorize XY, (Benya et al., 1977), aAaB2 (Burgeson and Hollister, 1977), and type VI (Davison et al., 1979) as collagen type V, whereas A, B, and C chains may be

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

235

designated as cil(V), a2(V) and a3(V) chain, respectively, in accordance with the accepted nomenclature. The collagen type V reported by Little and Church (1978) may be regarded as type I trimer procollagen since it is closely related to the latter. Somewhat divergent views have emerged from the studies which attempted to assess the chromosomal linkage of genes coding for different collagen types by following the culture hybridization technique. For instance, gene coding in the case of collagen type I has been found on chromosomes 17 (Sunder-Raj et al., 1977) and 7 (Sykes and Solomon, 1978). Recently, Kefalides (1979) has also assigned the gene coding for collagen type IV to chromosome 17. Bomstein and Sage (1980) have emphasized that collagen types, by definition, are the products of different genetic loci and are nonallelic. Hence, the term “genetic polymorphism,” which has frequently been used in the literature to describe the molecular heterogeneity of collagen (Grant and Jackson, 1976), should be avoided because genetic polymorphism generally indicates structural differences that arise in proteins coded for by different alleles at a single genetic locus. The hemoglobin variants and immunoglobulin allotypes are the examples of genetic polymorphism. 2.

Collagen Types in Muscle

It appears that very few, if any, of the collagen types have a unique distribution in different tissues. More than one type seem to be present in a particular tissue. The inter- and intramuscular connective tissue (mainly collagen) has long been classified on a histological basis as the epimysium, perimysium, and endomysium (Cassens, 1971; Lawrie, 1974). Little was known about the isomorphic composition of collagen present in these layers of connective tissue until the late 1970s (Bailey and Sims, 1977; Duance et al., 1977; Wu, 1978; Bailey et al., 1979). All of the collagen types, except type 11, have been found to exist in skeletal muscle. According to Bailey and Sims (1977), type I collagen, [(~1(1)]~(~2(1), is the major component of epimysium and perimysium, whereas type IV, [ ( ~ l ( I v ) ] ~ is , confined to the endomysium. Type I11 collagen, [(~1(111)]~, was mainly identified in the perimysium and to a lesser extent in the endomysium. However, Wu’s studies ( 1978) on intramuscular bovine connective tissue indicated the presence of two different types of (Y chains in type IV collagen, namely aA(IV) and aB(IV). Both of these chains contain less alanine and glycine, and more threonine, glutamic acid, leucine, and hydroxylysine than do collagen types I and 111. The aB(1V) chain has slower electrophoretic mobility than aA(IV) chain, but both are resistant to peptic digestion. Wu (1978) has further shown that there were 5-10 mg of type 111, 10-20 mg of type IV, and 50-150 mg of type I collagen in 100 g of fresh muscle from good-grade steers. Some studies have suggested that type I11 collagen is essential for normal tensile

N

m

W

TABLE 1 DISTINCT FEATURES OF FOUR TYPES OF COLLAGEN AND OTHER NEWLY DISCOVERED a CHAINS IN DIFFERENT TISSUESO

Characteristics a-Chain composition Molecular formula Carbohydrate content Hydroxylation of lysineb

Type I collagen

Type 11 collagen

Type Ill collagen

Type IV collagen

2al (I) and la2(I) [a~(I)Iz[a2(~)1 10% 0.5% in al chain 0.8% in a2 chain

3aI(lI)

3a I(II1) [al(III)Is

3a I (IV) [al(IV)l3

Hydroxylation of prolineh 1. 4-Isomer -11% in -10% in 2. 3-Isomer -0.1% in GIycosylationb 1. Hydroxylysine0.06% in galactose 0.15% in 2. Hydroxylysine0.30% in galactose-glucosyl galactose

a1 chain

a2 chain both chains al chain a2 chain both chains

[al(II)I3 1w c

2.3%

-10.0%

-0.2%

10%

0.5%

-12.5% ~

1w o 4.5-5.7%

13%

Newly discovered a-chains A

“41,

B [Bls

-

-

2.2%

3.9%

-11%

1%

0.7%

1w o

-I%

CP55

-

4.8%

6.5% Absent

0.4%

0.01%

0.2%

0.3%

0.5%

0.7%

0.5%

0.08%

3.2%

0.5%

2.9%

4.1%

Amino acidsb I . Glycine 2. Alanine 3. Cysteine Fibril size

Occurrence

33% I I% Absent Relatively large and bulky

Skin, bone, tendon, lung, aorta, muscle ligament, dentin, blood vessels

33%

>33%

<33%

<33%

>33%

wa

< 10%

< 10%

< 10%

< 10%

Present

Absent

Absent

-

-

-

1

Absent Relatively delicate and narrow Cartilage, cornea, retinal tissue

Present Smaller than

-

<33%

< 10% Absent

-

type I

Skin, artery, uterus, muscle lung, blood vessels, intestine

Basement membrane, muscle, lens capsule

aThese data have been derived from the sources mentioned in the footnotes of Table 11. bResiduesll00 amino acid residues.

N w -4

33%
Amniotic and Skin, liver, chorionic epithelial membrane basement of placenta membrane

Aorta, skin, liver, placenta, smooth muscle cell membrane

Endothelial basement membrane

238

A. ASGHAR AND R. L. HENRICKSON

strength of skin and intestinal tissues (Eyre and Glimcher, 1972; Pope 1975). B.

et

al.,

ISOLATION AND IDENTIFICATION OF COLLAGEN TYPES

Traditionally collagen has been regarded as an insoluble protein. However, Go11 (1965) has quoted Orekhovitch et al. (1948) as saying that a French researcher, Zachariades, showed in 1900 that collagen could be solubilized in dilute acetic acid. Later on, many different conditions were explored to solubilize collagen by varying pH and the salt concentrations of the extracting medium. Most of the methods fall into two categories: (1) those in which extraction was carried out at pH 3-4 with low salt concentration, the extracted product being designated as acid-soluble collagen or procollagen (Orekhovitch, 1958); and (2) those in which the extraction was performed at pH close to 7 with high salt concentration, yielding a product called neutral salt-soluble collagen or tropocollagen (Gross et al., 1955). Further characterization of these collagen extracts revealed monodispersed tropocollagen, composed of different molecular weight compounds (Orekhovitch and Shpikitar, 1955), the lighter one designated a-and the heavier ones as P-components (PI PI2, P.-J (Grassman etal., 1961; Piez et al., 1961, 1963). Collagen has also been partitioned into four classes on the basis of solubility in different buffer systems (Goll, 1965): neutral salt-soluble, acid-soluble, alkalisoluble, and alkali-insoluble. The collagen, in cold neutral salt solution, represents newly synthesized molecules in a loose state, that is, a chain monomers (Jackson and Bentley, 1960). However, Gross (1964a) believes that it is the low temperature of the solution which induces solubility rather than the presence of salt, as little collagen may be extracted at body temperature. The hydrogenbonding capacity of water increases at a low temperature and may facilitate hydration and disruption of the molecules (Kauzmann, 1964). The acid-soluble fraction generally comprises two different aggregates besides a subunits. One, designated as P2 or P, is composed of two a1 chains, and the second, P, or P12, consists of one a1 and a2 chain linked by ester-type covalent bonds (Piez et al., 1961; Gallop, 1964). A small amount of a trimer, y-component, has also been reported in acid extract (Altgelt et al., 1961). Salt-soluble collagen was found to consist exclusively of 90% a chains and some P forms (lo%), whereas acid-soluble collagen is about 10% a chains and 90% in the P form (Piez et al., 1965; Delaunay and Bazin, 1974). The schematic diagrams in Fig. 1 show the native and some reconstituted forms of collagen. Only 50% as many ionizable groups are present in acid-soluble as in salt-soluble collagen. The remaining groups are possibly involved in cross-linkages (Hartman and Bakerman, 1966). On the other hand, alkali-soluble and insoluble fractions represent mature collagen aggregates of higher degree.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

239

FIG. 1 . The fibroblasts (a) produce tropocollagen molecules (b), which overlap to form native collagen (c). The newly synthesized tropocollagen is soluble in cold salt solution (d) and forms reconstituted fibrils like native collagen on warming. The native collagen can also be dispersed in acetic acid (e), which on reacting with adenosine triphosphate (ATP) produces nonoverlapping segment long-spacing (SLS) collagen (f), whereas on reacting with glycoprotein it yields fibrous long-spacing (FLS) collagen (8). From Seifter and Gallop (1966).

Different collagen types are generally isolated in the native form from various tissue by differential salt precipitation at neutral pH with NaCl (Kefalides, 1971, 1972; Trelstad et al., 1970, 1976; Chung and Miller, 1974; Burgeson et al., 1976) or (NH,),SO,, or by EtOH (Trelstad et al., 1976). For example, collagen types I11 and I precipitate in the ranges of 1.5 to 1.7 M and 2.2 to 2.5 M NaCl, respectively, whereas types I1 and IV precipitate in the range of 4.0 to 4.4 M NaCl. Further purification is performed by ion-exchange chromatography using mostly carboxymethyl cellulose and occasionally molecular sieve chromatography (Piez, 1967; Chung and Miller, 1974; Epstein, 1974). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has also been used to separate different species of collagen (Eyre and Muir, 1975a,b; Burgeson et al., 1976; Bailey and Sims, 1976; Scott and Veis, 1976; Scott et al., 1976; Fessler and Fessler, 1978; Reiser and Last, 1980). The latest methods involved differential denaturation and renaturation (Chandra-Rajan, 1978) and covalent chro-

240

A. ASGHAR AND R. L. HENRICKSON

matography on activated thiol-Sepharose (Angermann and Barrach, 1979) to separate different types of collagen.

111.

CHEMISTRY OF COLLAGEN

Numerous comprehensive reviews have appeared on the chemistry and biochemistry of collagen and procollagen (Ramachandran, 1968; Kuhn, 1969; Traub and Piez, 1971; Gallop et al., 1972; Fietzek and Kuhn, 1976; Bornstein, 1974; Martin et al., 1975; Piez, 1976; Prockop et al., 1976; Bornstein and Traub, 1979). Collagen, a glycoprotein, is the longest of all protein molecules and is composed of tropocollagen monomers which are 300 nm long and 1.5 nm in diameter (Piez, 1967; Woodhead-Galloway et al., 1975; Hanvood, 1979). Each tropocollagen monomer comprises three polypeptide (Y chains, each having a molecular weight of 95,000. In view of the discovery of different types of collagen, the three a chains in a tropocollagen monomer may be identical (as in the case of collagen types 11, 111, and IV) or different (Fig. 2). An example of the latter case is type I collagen, whose monomers are composed of two a 1 chains and one 012 chain (Piez, 1966, 1967; Kuhn, 1969; Miller, 1973; Fietzek and Kuhn, 1976). Each a chain is coiled into a left-handed helix with about three amino acids per turn, but the trimers are supercoiled in a right-handed helix (Piez et al., 1963; Ramachandran and Ramakrishnan, 1976). It will be obvious from the following discussion that the sequence and symmetry of the individual collagen molecules are well established. How collagen molecules aggregate to form the functional units (fibrils) of connective tissue has been a matter of speculation. Grassmann (1965) stated that the linear polymerization of tropocollagen monomers produces collagen fibrils which are arranged into parallel bundles (in tendon) or into a three-dimensional irregular network (in skin, cartilage, bone, and teeth) of macroscopic structures. It also appears that the organization of bundles is affected by the type of collagen present in a particular tissue (Nowack et al., 1976b; Lapikre et al., 1977). The three-dimensional molecular arrangements of collagen have been discussed in great depth by E. J . Miller (1976). He has critically evaluated the three principal models, namely heuristic (Smith, 1968), tetragonal (Miller and Parry, 1973), and hexagonal (Katz and Li, 1974) and their modifications, presented by different scientists to account for the array of collagen molecules. According to these models, the collagen molecules are considered to be arranged in long fivestranded microfibrils with 1 0 and 4 0 axial intermolecular staggers (where D = 668 A). The microfibrils are supercoiled with a pitch of about 700 p\, and arranged face to face throughout the fibril on a tetragonal lattice of side 38.5 A. E. J. Miller (1976) concluded that there is general agreement among different

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

24 1

A

Triple helix 1052 residues

Triple helix 1101residues 0

C c

Disulfide linkages

FIG. 2. The helical part of the a chains is supercoiled in a right-handed helix in different types of collagen. Type I collagen consists of two identical a chains and one different a chain (A), whereas types I1 and I11 collagen are composed of three identical a chains (B and C). In the case of type I11 collagen, interchain disulfide bonds are also present within the helical region (C).

views on certain points. For instance, a collagen fibril is regarded as a single crystal in which collagen molecules are not parallel to the fibril axis, but they are tilted about 4". A vector with length 38 A is considered important in the lateral arrangement of the molecules, as is the fact that the collagen molecules, shifted axially by 40, are covalently linked in the fibril. Some aspects of the threedimensional molecular arrangements of collagen have not been established, however, since none of the models satisfy all the necessary requirements (E. J. Miller, 1976). Although studies by Fraser ef al. (1976) support tetragonal patterns, very recently Hulmes and Miller (1979) presented evidence in favor of quasi-hexagonal packings. A.

AMINO ACID COMPOSITION

Twenty or twenty-one different amino acids are known to be present in different collagen types. The overall amino acid composition of mammalian collagen type I is shown in Fig. 3. The amino acid composition of collagen is unique in some respects among other proteins. For example, it is extraordinarily rich in glycine and proline, and contains large amounts of hydroxyproline, whereas tryptophan is absent. Cysteine is present only in collagen types 111, IV, and CP55, and methionine is the only sulfur-containing amino acid in collagen types I and 11. It can be seen that 33% of the total amino acid residues consist of

242

A. ASGHAR AND R. L. HENRICKSON

FIG. 3. Diagrammatic representation of the amino acid composition and relative proportions of acidic, hydroxy, polar, and nonpolar amino acids of type I collagen, [oll(I)]2[2a].

glycine, about 12% of proline, 11% each of alanine and hydroxyproline, total imino acid residues being about 23%. Relatively small amounts of each of the other 14 amino acids account for the remaining proportion of the residues. The content of hydroxylysine, histidine, phenylalanine, isoleucine, tyrosine, and sulfur-containing amino acids is about 1% or less for each. Polar amino acid residues constitute about 40% of the molecule, of which 11% are basic and 9% acidic amino acids and about 17% are hydroxy amino acids. About 5% of the aspartic and glutamic acid residues are present as amides. There are approximately four asparagines for every glutamine in collagen (Cassel and McKenna, 1953). About 60% of the molecule consists of nonpolar (hydrophobic) amino acid residues. Table I1 presents the amino acid composition of different isoformic collagen ci chains. It shows that major variations occur in the content of 3- or 4-hydroxyproline, glutamic acid, proline, valine, isoleucine, leucine, hydroxylysine, lysine, and histidine in different types of a chains, and hence in collagen. Though hydroxyproline and hydroxylysine are considered to be specific to collagen, these residues have been found in the complement component C l q (Reid, 1974) and in elastin (Gosline, 1976; Sandberg, 1976; Franzblau, 1971).

TABLE I1 AMINO ACID COMPOSITION OF DIFFERENT a CHAINS OF COLLAGEN ISOLATED FROM VARIOUS TISSUES Residuesilo0 amino acid residues

3-Hydroxyproline 4-Hydrox yproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Half-cystine Methionine Isoleucine Leucine

1.1

114 46 18 35 77 118 330 1I9 19 ND 5.3 8.2 20

1.2 105 45 18 30 70 114 33 1 105 35 ND 4.7 16 33

2.0 99 42 20 27 89 121 333 100 18 ND 9 9 26

1 .o

97 50 23 28 93 113 336 102 23 ND 5.7 8.8 26

ND 121 48 15 41 71 102 355 92 16 2 7 13 21

ND' 125 42 13 39 71 107 350 96 14 2 8 13 22

11 130 51 23 37 84 61 310 33 29 8 10 30 54

2.5 109 50 26 31 84 97 319 52 27 ND 11

16 35

2.9 109 50 19 26 91 118 322 46 18 ND 8 19 39

7 113 49 29 34 86 98 346 54 28 0 11 12 33

10 105 49 21 25 95 120 334 45 22 0 9 15 38

0 65 78 16 27 104 92 318 41 21 20 8 20 24 (continued)

TABLE I1 (Conrinued) Residues/ I00 amino acid residues

Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine 3- + 4-OH-Proline Proline 3- 4-OH-Proline OH-Lysine Lysine + OH-Lysine

+ +

2.1 13 10 27 4 49 0.49

3.2 11 12 20 10 51 0.48

13 14 22 2 51 0.46

1.1 12 18 17 2.4 43 0.46

2 8 5 30 6 46 0.54

3 8 5 30 6 46 0.54

6 27 44 10 10 33 0.70

1.8 14 24 18 11 68 0.54

2.1 12 35 20 7.5 50 0.49

0 10 22 12 8 48 0.55

3 11 39 13 6 40 0.49

18 15 48 18 3 64 0.41

0.27

0.38

0.39

0.51

0.14

0.14

0.81

0.57

0.64

0.65

0.75

0.73

1

OHuman placental membrane type 1 (Y chain composition from Burgeson et al. (1976) bHuman articular cartilage type I1 composition calculated from Miller and Lunde (1973). cLathyritic chick xiphoid cartilage type I1 collagen composition from Trelstad et al. (1972). dHuman skin type 111 collagen Composition from Epstein (1974). eHuman dermis, aorta, and uterine leiomyoma type 111 composition from Chung and Miller (1974). fHuman glomerular basement membrane type IV composition from Kefalides (1971). gChains isolated from human placental membranes [from Burgeson et a/. (1976)l. hAmino acid composition of chains isolated by Chung e t a / . (1976). 'ND, Not determined.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

B.

245

MOLECULAR ORGANIZATION

The synthesis of a protein in living cells involves placing the amino acids in the proper sequence, determined by the genetic code, and linking them together by a peptide linkages. The polypeptide chains of the native proteins, however, are not freely flexible (random coils) due to certain restrictions imposed by the requirement that bond angles be maintained close to certain fixed degrees. The peptide bond is stabilized by delocalization of n-electrons of the C=O bond into the C-N bond. This imposes restrictions on the rotation about the C-N bond; however, side groups of amino acids may freely rotate around the a-carbon. The resonance hybrid possesses about 40% double-bond character in the C-N bond, whereas 60% double-bond character remains with C = O bond (Cram and Cram, 1978). The planar geometry of the peptide linkage is as follows:

However, the primary structure of collagen has some unusual features. For instance, some degree of nonplanar distortion in collagen structure is now considered possible to account for its increased stability. The peptide linkages in other proteins invariably have a trans configuration, whereas in the collagen structure a cis configuration is also possible at those places where proline and hydroxyproline are involved in the peptide linkages. As the free-energy difference in the two geometrical isomers is insignificant, either one of the stereo forms of peptide bond can occur to suit the requirement of the structure (Badger and Pullin, 1954; Wyckoff et al., 1970; Ramachandran and Ramakrishnan, 1976).

@ and 9, respectively, denote the dihedral angles of rotation between C-N and C--C bonds. Since the a-carbon of proline and hydroxyproline is covalently linked to the N atom to form a five-membered ring, the dihedral angle r$ about the C - C bond can only be close to -60" (Ramachandran and Ramakrishnan, 1976). Another unusual feature of the collagen structure was once thought to be the presence of y-glutamyl peptide linkage (Gallop et al., 1960), however, in some natural peptides, glutamic acid has been found to occur in such linkages (Harding, 1965; Seifter and Gallop, 1966). Franzblau et al. (1963, 1970) have re-

246

A. ASGHAR AND R. L. HENRICKSON

ported that about 40% of the COOH groups of glutamic acid in the primary chain of collagen are involved in peptide linkages through the y-COOH group. The amorphous regions (polar) of the a chains mainly contain this type of intramolecular bond (Rojkind and Gallop, 1963) and presumably serve to disrupt the helical structure imposed by the crystalline regions, hence providing regions of flexible, loose conformation at specific intervals along the collagen fibrils (Seifter et al., 1965; Gallop et al., 1965, 1967). A chemical method was used to determine how much of y-COOH groups of glutamic acid residues are free or bound in y-glutamyl linkage. The groups were converted into hydroxamic acid by reacting with carbodiimide and hydroxylamine followed by dinitrophenylation, Lossen reaction, and acid hydrolysis (Franzblau et al., 1963). However, the reaction was not precisely specific since some amide groups were also affected. Hall (1976) suspected such isopeptide linkages (y-glutamyl) were artifacts brought about during hydrolysis of collagen in the preparatory steps. The use of a sequence of proteases and peptidases, specific for a peptide bond, which would hydrolyze a protein completely to its amino acids, would be a more suitable approach to ascertain the exact nature of the bonds. By following such an approach, Bensusan (1969) successfully released all the glutamic acid residues in collagen by using a mixture of proteolytic enzymes which did not cleave model peptides containing y-glutamyl bonds. Hence this study did not support the presence of y-glutamyl bonds in collagen. At one time the presence of ester bonds was also regarded as one of the special features of the collagen fibril. The first experimental evidence was based on the use of such nucleophilic reagents as hydroxylamine or hydrazine for cleaving the probable ester bonds under defined conditions of pH and temperature (Gallop et al., 1959). Since the cleavage of some special imide bonds was also possible under those conditions, the term “ester-like’’ bonds thereafter has been used to describe them (Gallop, 1964). It was proposed that the four subunits of a chain (Petruska and Hodge, 1964) are linked by three pairs of ester-like bonds, and that the two aspartic acid residues in the C-terminal region of the a chain provide the paired acyl donors for ester-like intrachain bonding (Blumenfeld and Gallop, 1962). One of the esters involves the a-COOH of one aspartyl residue, and the other involves the (3-COOH group of the second aspartyl residues (Seifter et al., 1965). It was also ascertained that glutamyl residues were not acyl donors for ester-like bonds. Although the nature of the alcohol function (if an ester is present) or amide function (if a special imide is present) has never been determined. Seifter.and Gallop (1966) proposed the possibility that the “bound masked aldehyde” that occurs in collagen, represents ester formation between aspartic acid residues and hemiacetal or vinyl alcohol functions. Contrary to this, the present consensus does not support the presence of ester-like linkages in collagen (Hall, 1976; Bornstein and Traub, 1979).

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

C.

247

AMINO ACID SEQUENCES

Presently, the complete sequence of the 1052 amino acid residues in the al(1) chain is known (Hulmes et al., 1973; Fietzek and Kiihn, 1976) and about twothirds of the amino acid sequence of the a2(I) chain has also been established (Fietzek and Kiihn, 1976). Some data on peptides, derived by hydrolyzing with cyanogen bromide from other collagen types, are also available (Dixit et al., 1975; E. J. Miller, 1976; Fietzek et al., 1977). The latest reports, together with the earlier findings, suggest that 50-60% of an a chain consists of nonpolar sequences, which are expressed as tripeptides with the general formula Gly-X-Y (where X is generally proline and Y is mostly hydroxyproline). About two-thirds of the X and Y positions are occupied by a variety of other amino acids which, although they decrease the stability of the triple helical, are essential for the organization of collagen at the fibrils level. These amino acids tend to be clustered in groups of hydrophilic and hydrophobic residues. The precise sequence of amino acids in the X and Y positions in various a chains is different. These sequences represent the crystalline regions. Figure 4 presents the details of such a sequence schematically for a tropocollagen molecule. However, the original proposal of Petruska and Hodge (1964), that a chains of tropocollagen are built from smaller subunits which are joined approximately end-to-end by ester-like bonds (Fig. 4), is no longer considered valid (Traub and Piez, 1971). As mentioned earlier, each tropocollagen unit consists of three polypeptide strands of a chains. Each chain contains alternative amorphous and crystalline regions (Bear, 1952). When viewed under an electron microscope, the regions composed mainly of the nonpolar amino acid residues, and representing crystalline zones, appear light (Fig. 4). The amorphous regions (pyrrolidinepoor), containing ionic polar amino acids which can take up stain, appear as dark bands (Grassman et al., 1956; Kiihn, 1960; Hanning and Nordwig, 1965; Chapman and Hardcastle, 1974). It may be pointed out that the intrachain subunits proposition of Petruska and Hodge (1964) requires that the a chains have different repeating distributions of ionic groups. However, the studies by Tkocz and Kiihn (1969) suggest that a 1 and a 2 chains must contain a similar distribution of ionic groups. There is no difference of opinion that the tropocollagen fibrils are arranged relative to adjacent molecules so as to allow for staggered overlap of one another by about three-quarters of their length (Fig. I). The cross-linkages (hydrogen and covalent bonds) bind the tropocollagen molecules to their laterally placed neighbors (Bruns and Gross, 1973; Bruns et al., 1973). There is a sequence of 24 amino acids with basic and acidic residues in the amorphous regions. The basic parts contain two hydroxylysine, three arginine, and one histidine residues (but no proline or hydroxyproline) in the a1 chain (Chapman, 1974). The acidic part of the amorphous region contains a sequence

...-

-. .>\

I< I

'SEGMENT LONG SPACING' (SLSI FORM

-. -

.-.

- _ _. .

y - h - t w o r ~ ~ ~ ~ w + G I ~ - P ~ ~~y - -Pro < G4 n.1

PEPTIDES

DIALYZABLE STALLINE'

NON DIALYZABLE PEPTIDES '*AMORPHOUS' (BAND)

RAPIDLY DIALYZABLE "CRYSTALLINE'

lNTLRB,AND

/ /

PEPTIDES ,' "AMORPHO~S" (BAND) \

i

\

ly-Pro (Gly, GluZ Asp, Lysi AloZ SwlX f Pro

(Gljs, Alol. Serq.Glu1l IGlyj. Alai. %r,. Valtl l G l y j . A b q . Val,. Thrll

FIG. 4 Schematic presentation of the collagen fibnl packing, showing the sequence of subunit structure From Gallop (1964) Courtesy of Little, Brown and Company, Boston

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

249

Non helical segment Collagen-like segment Globular segment

Nonhelical COOH-terminal domain

Triple helical domain

Nonhelical NHJerminal domain

FIG. 5 . Schematic representation of type I tropocollagen monomer, composed of two identical pro-a1 chains (solid lines) and one pro-a2 chain (dashed line). The central part of the molecule is triple helical. The nonhelical COOH-terminal domain contains the interchain disulfide bonds, whereas the nonhelical NH2-terminal region is composed of a presumably globular segment, a short collagen-like segment, and a nonhelical segment where cleavage by amino-terminal protease occurs.

of 16 amino acids with only one imino acid, two lysine, two aspartic, and one glutamic residues. This helical region seems important for generating interchain cross-linkages. These observations seem to be at variance with earlier ideas regarding collagen structure (Astbury, 1940; Pauling and Corey, 1951). Earlier information on collagen structure also gave the impression that the helicity extends throughout the molecule. However, recent advances have revealed two structurally and functionally distinct regions in each a chain: a central triple helical region composed of 1011 amino acid residues, and the N- and C terminal nonhelical regions (Fig. 5 ) composed of 9-25 residues (Kuhn, 1969). The sequence of amino acids in the helical and nonhelical regions has been determined for collagen [al(I)],[a2(1)] from calf skin (Hulmes et al., 1973; Fietzek and Kuhn, 1976). 1 . Triple-Helical Regions The triple-helical regions are composed of chains of tripeptide units of the general formula (Gly-X-Y), in all types of collagen. However, the distribution of amino acids between the X and Y positions is uneven (Fietzek and Kuhn, 1976). In the case of al(1) chains, glutamic acid and such hydrophobic amino acids as leucine and phenylalanine generally occupy the X position, whereas threonine and arginine are present in the Y position. Other hydrophobic amino acid residues (valine and isoleucine) are distributed randomly, whereas hydrophilic amino acids (aspartic acid and lysine) are almost evenly distributed between X and Y positions. The same seems to be true for the a 2 chain, except that valine is mainly present in the Y position, whereas threonine is evenly distributed. Generally hydroxylation of proline at C-4 and of lysine at C-5 occur when

250

A . ASGHAR AND R . L. HENRICKSON

these amino acid residues are at the Y position, and hydroxylation of 3-hydroxyproline occurs when proline is at the X position in both of the a chains. While 54% of the lysine in the a 2 chain is hydroxylated, only 5-8% is hydroxylated in the a 1 chain, generally at the Y position (Fietzek and Kuhn, 1976). The hydrophobic amino acid residues (valine, leucine, isoleucine, methionine, phenylalanine, and tyrosine) are relatively more abundant in the a 2 chain than in the al(1) chain and they are evenly distributed (Tkocz and Kuhn, 1969).

2. Nonhelical Regions The physicochemical evidence provided by Boedtker and Doty (1956) suggested that collagen molecules have dangling chain peptide appendages at one or both ends which are not in triple-helical conformation. These regions are also referred to as telopeptides by Rubin et al. (1963) and are devoid of hydroxyproline. Sixteen amino acid residues with the same sequence have been found in the N-terminal telopeptide of the a 1 chain of type I collagen from different species. The one variation is leucine, which occupies the N-2 position instead of methionine, in the case of calf skin collagen. Generally the N-terminal region is high in hydrophobic amino acids. Another common feature is the presence of a lysine residue at the N-8 position (Fietzek and Kuhn, 1976), which may be oxidized to an aldehyde derivative by lysine oxidases for generating intra- and intermolecular bonds (Gallop et al., 1972; Robins et al., 1973). In contrast to the al chain, the number of amino acids in the N-terminal end of the a 2 chain varies significantly from 9 to 11 among different species. Lysine may be present at the 5th, 6th, or 7th position. Despite these differences, both the a 1 and a 2 chains begin with glutamic acid as the N-terminal amino acid (Kang and Gross, 1970; Rauterberg et al., 1972a; Fietzek et al., 1974b; Becker et al., 1975b). The investigations on the nonhelical C-terminal end of the al(1) chain of collagen from different species have shown the presence of 25 amino acids, of which hydrophobic amino acids account for the major proportion. This region is longer than the N-telopeptide region. An oxidizable lysine residue was found at the C-16 position in the case of rabbit skin collagen and at the C-17 position in that of calf skin collagen (Rauterberg et al., 1972b; Becker et al., 1975b). D.

FUNCTIONAL ROLE OF AMINO ACIDS

The peptide linkage formed by a-NH, and a-COOH of different amino acids (other than proline and hydroxyproline) contains the NH group, which can participate in hydrogen bonding, and hence contributes to the stability of the helix of proteins. However, proline and hydroxyproline serve different purposes in the collagen structure. Walton (l974), in reviewing the collagen function in tissues,

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

25 1

emphasized the role of imino acid residues in the a 1 chain sequence in determining the pitch of the collagen superhelix. It has been mentioned in the previous section that the N atom of the proline and hydroxyproline residues is linked with a-C to form a rigid five-membered ring structure, hence there is no freedom of rotation about the N-C bond (the dihedral angle c$ = -60"). According to Schimmel and Flory (1968), the pyrrolidine rings impose conformational restraints on residues preceding but not following them in the peptide chain sequence. Besides, as there is no H atom in imino peptide linkage, it cannot participate in hydrogen bond formation. These characteristics of imino acids are important in disruption of the helicity of polypeptide chains. On the other hand, hydroxyproline plays a part in the stability of collagen's minor and superhelix by hydrogen bonding, which involve the oxygen of hydroxyproline's hydroxyl group with the backbone of the collagen triple helix via a water dipole (von Hippel, 1967; Ramachandran and Ramakrishnan, 1976). The ability of the a chains to attain the triple-helical conformation and its thermal stability depends not only on the content of the proline and hydroxyproline residues (Gustavson, 1955; Josse and Harrington, 1964; Sakakibra et al., 1973; Berg and Prockop, 1973; Jimenez et al., 1973; Jimenez and Yankowski, 1975), but also on the distribution of these residues along the chains (Berg and Prockop, 1973). Segal (1969) has shown more specifically that with proline-containing tripeptides, the stability of the helix falls in the following order: Gly-Pro-Pro > Gly-Pro-Y > Gly-X-Pro > Gly-X-Y. Regarding the role of hydroxyproline, Sakakibra et al. (1973) have indicated that it imparts exceptional stability when present in the Y position. Generally, collagen type [al(I)],[a2(1)] and [al(I)I3 are more stable than type [a2],(Tkocz and Kuhn, 1969). The distribution of polar and hydrophobic amino acid residues determines the ordered aggregation of molecules into fibrils (Highberger et al., 1971; Fietzek et al., 1974a). The acidic and basic amino acid residues generally occur in close proximity to each other, concentrated more in some regions than others; hence they are important in interchain cross-linking with adjacent molecules in the formation of collagen fibrils (Tkocz and Kuhn, 1969). Unlike hydroxyproline, which is found only in the helical regions of the molecule, hydroxylysine may occur in both the helical and nonhelical N-terminal region, where it plays an important role in intermolecular cross-linkings (Tanzer, 1973; Bailey et al., 1973, 1974). E.

TYPE AND NATURE OF INTERCHAIN CROSS-LINKAGES

The early studies indicated the presence of different types of interchain crosslinkings in collagen. Despite the fact that tyrosine content is quite low in collagen, some reports have assigned a special role to tyrosine in the aggregation of soluble collagen (Bensusan and Hoyt, 1958; Bensusan and Scanu, 1960; Hodge

252

A. ASGHAR AND R. L. HENRICKSON

et al., 1960). Deasy (1962) reported the presence of peroxide cross-links (-U-O-) in collagen. It was assumed that the phenolic group of two tyrosine residues of adjacent chains oxidized to form a di-p-(2-amino-2-carboxyethy1)phenyl peroxide linkage. LaBella and Paul (1965) supported the existence of such cross-links in collagen, but Sinex (1968) disagreed with their presence. Joseph and Bose (1962) indicated that about 30% of the guanidinyl group of arginine residues in collagen are cross-linked to the a-COOH group of glutamic acid residues. They have further shown that guanidinyl-carboxyl cross-linkages increase with the age of the rat (Joseph and Bose, 1962). Veis and Schlueter (1963) speculated on an important role for serine and threonine residues in hard collagenous tissue (dentin), where they may be involved in PO,-mediated (ester) cross-linkages. Some reports have also indicated that about 30-40% of the ENH, group residues form e-lysyl peptide linkage to provide branching points between chains (Mechanic and Levy, 1959; Joseph and Bose, 1962). It appears that the evidence provided in favor of all these postulated crosslinkages has been suggestive rather than conclusive. A critical evaluation of the experimental evidence regarding different chemical bonds in collagen led Harding (1965) to conclude that y-glutamyl peptide bonds exist in the primary chain, but that none are present in cross-linking between collagen chains. On the other hand, P-aspartyl peptide bonds do not exist in any significant amounts, whereas the existence of interchain elysyl peptide bonds is improbable. During the past decade much new information on this aspect has become available, thereby changing some of the previous concepts dramatically. According to prevailing views, the involvement of aromatic side chains by tyrosine and phenylalanine has not been supported (Hall, 1976) and the ester-like bonds have not been proved in type I collagen (Bornstein and Traub, 1979). Three general groups of cross-linkages in collagen have been defined. First, those linkages which fix the overlap of the ends of a chains are called head-to-tail bonds; second, those reducible cross-linkages which stabilize the aggregation of these chains are denoted as side-to-side bonds; and the third group is end-to-end bonds (Kuhn, 1969; Zimmermann et al., 1970). These cross-linkages originate by different modes of action. The following types of bonds have been precisely defined in collagen by modern methodologies.

I . Hydrogen Bond It is known that hydrogen linked covalently to an electronegative donor (e.g., N, 0) can form a second weak bond with another electronegative atom (acceptor). The latter is called a hydrogen bond. The common types of hydrogen bond in proteins are those between NH.-.N, NH...O, and OH...O atoms of amino acids, and they have an energy content of 3-5 kcal/mole (Ramachandran, 1968).

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

b'

H

0

O

II

I

-C-C-N-C-C-N-C-C-N-C-

8 ..

.

II

(chain A)

I

I H

Y

far away 4'. for H bond

i

I

0

0

'H

!I

-C-C-N-C-C-N-C-C-N-CI II II I 0 H

I

(a) One-bonded structure

z

/

H

-C-

I

C- N-C-C-N-

25 3

II C-C-N-C-

I

H

II

(chain A)

I

H

II

I

-C-C-N-C-C-N-C-C-N-( I II

i

I0

O II

II

(chain B)

(chain B)

I

H

J H Pol\

(b) Water-bridged structure

z -C-

H I

C-NII

II

I

'

C

-\O , -H

*O

H

It

-C-C-N-C-C-N-CI

II C-CI

N- C-

I

c, ,c

-.'0-H

Y0\H.

'.o

O

C- C-N-

I

II

(chain A)

B /

4' 0

II C-N-

I H

C-

(chain B)

(c) Water-bridged structure with hydroxyproline

FIG. 6. The nature of hydrogen bonds between two adjacent chains (A and B) of the collagen fibril. The structure is actually three-dimensional; the one-dimensional representation shown above with parallel chains is a simplification. After Ramachandran and Ramakrishnan (1976).

254

A . ASGHAR AND R. L. HENRICKSON

These bonds are important in stabilizing the secondary structure and packing of collagen molecules (Harrington, 1964); hence they fix the shape of the protein molecule in a specific conformation. In native collagen, the tropocollagen chains are oriented so that the NH group of the third peptide linkage of one chain may form a hydrogen bond with the COOH group of the third peptide linkage of an adjacent chain. It should be emphasized that imino peptide (involving pyrrolidine) bonds, lacking one hydrogen atom, cannot form intramolecularly hydrogen-bond stabilized (Y helical structures (Veis, 1964), although an interchain hydrogen bond can be formed between the OH group of an hydroxyproline residue on one peptide chain and the COOH group on an adjacent chain (Gustavson, 1956). However, opinions differ as to the number of hydrogen bonds in collagen. Rich and Crick (1961) argued in favor of a “one-bonded structure,” that is, only one hydrogen bond for every three residues (Traub, 1969), whereas Ramachandran et al. (1962) believed in a “two-bonded structure,” that is, two hydrogen bonds for every three residues. Figure 6 depicts the “one-bonded’’ and “two-bonded’ ’ structures. Ramachandran and Chandrasekaran (1968) offer an alternative “two-bonded’’ structure which reconciles the two views by indicating that one hydrogen bond is directly between adjacent polypeptide chains and the other hydrogen bond forms via a water molecule. In the revised “two-bonded’’ structure, the OH group of hydroxyproline has been assumed to perform two functions: it forms a hydrogen bond with the bridging water molecule to increase the stability of a triple-chain protofibril, and another hydrogen bond (cross-link) between one triple-helical chain and a neighboring triple-helical chain (Fig. 6C). The “two-bonded’’ structure seems to conform better to the experimental evidence (Harrington, 1964; Berendsen, 1972; Yee et al., 1974; Suzuki et al., 1980). It has been further shown that the 4-OH group of hydroxyproline in the Y position of (Gly-X-Y), is in the trans configuration with respect to the COOH group of hydroxyproline so as to perform these functions (Schubert and Hamerman, 1968; Ramachandran and Ramakrishnan, 1976). Salem and Traub (1975) have also suggested the involvement of glutamine in the formation of hydrogen bridges in the helical region of the cx chains. According to them, glutamine at the Y position in the [Gly-X-Y], tripeptide chain of helical regions may form a hydrogen bridge with the carbonyl group of the peptide bond (the aspargine side chain is too short for this reaction). 2. Hydrophobic Bonds Although the nonpolar amino acids glycine and alanine constitute nearly 44% of the collagen molecule, yet they may contribute little to the nonpolar van der Waals interactions (hydrophobic bonding) due to their small side groups (Veis, 1964). Contrary to this, some studies have indicated possible hydrophobic in-

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

255

teraction between adjacent propyl residues on different chains (Segal, 1969; Yonath and Traub, 1969). However, the side group of other nonpolar amino acids may form inter- and intramolecular hydrophobic bonds (Schnell, 1968; Heidemann and Hill, 1969) in the nonpolar segments (interband regions) of a chains (Schubert and Hamerman, 1968). It is now known that the a 2 chain contains more hydrophobic amino acid residues than the a 1 chain. Thus, the former chain contributes considerably to the hydrophobic character of type I collagen (Fietzek and Kuhn, 1976). Moreover, maximal interaction of the polar and nonpolar regions of adjacent molecules is facilitated by an axial stagger (D= 233 amino acid residues) within the fibrils. Thus, both electrostatic forces and hydrophobic interactions are important in aggregation of collagen molecules into fibrils. 3. Ionic Bonds

Salt (ionic) linkages have been considered relatively unimportant in stabilizing the collagen structure as compared to hydrogen bonding (Weir and Carter, 1950), but Weinstock et al. (1967) believe that most of the ionic sites form interand intramolecular salt linkages. For instance, Salem and Traub (1975) have suggested that arginine at the Y position can be involved in an electrostatic interaction with glutamic acid in the X position of an adjacent chain. 4.

Covalent Bonds

a . Disulfide Linkages. Interchain disulfide (-S-S-) bonds have been found in the C-terminal extraglobular peptide region (called the P,-a chain) of procollagen a chains of all types. But, the P, chains are enzymatically cleaved before the procollagen molecule is secreted into the extracellular space (Martin et al., 1975; Bomstein and Traub, 1979). Consequently, disulfide bonds are not present in collagen types I and I1 due to the absence of cysteine residues in their tropocollagen chains. However, interchain disulfide bonds have been reported in the helical region of type 111 collagen (Harwood, 1979) and in the glycoprotein extensions (terminal regions) of type IV collagen (Kefalides, 1973). Both of these collagens contain appreciable amounts of cysteine residues. b. Cross-LinkagesInvolving Lysine and Hydroxylysine. It is now well documented that covalent interchain (intermolecular) cross-linkages in different collagen types originate from the reaction of aldehydes, derived from oxidative deamination of the E-NH, group of lysine and hydroxylysine residues (Tanzer, 1973, 1976; Bailey, 1969, 1974; Gallop and Paz, 1975). The lysine and hydroxylysine residues in both terminal regions of the aI(1) chain and in the N-terminal region of the a 2 chain may be oxidized to a-aminoadipic acid a-semialdehyde (Traub and Piez, 1971; Stark et a l . , 1971a; Gallop et a l . , 1972). This occurs by

256

A. ASGHAR AND R. L. HENRICKSON

oxidative deamination of the E-NH, group of lysine and hydroxylysine residues by a copper ion-dependent lysine oxidase (Siegel, 1974, 1979). The resultant carbonyl compound then reacts with the E-NH, group of lysine or hydroxy- or glycosylated hydroxylysine present in adjacent molecules. The intermolecular cross-links are formed by a series of aldimine or ketoimine (Schiff base) and aldol condensation reactions, leading to the formation of highly stable pyridinium compounds such as desmosine and isodesmosine (Gallop et al. 1972; Robins et al., 1973; Bailey et al., 1974; Tanzer, 1976); their amount increases with the age of the animal. These cross-linkages have been identified following a mild reductive reaction of collagen fibrils with borohydride, which stabilizes the bonds and makes the isolation of linked amino acids possible. However, Bailey et al. (1974) expressed the feeling that the identification of these compounds from borohydridetreated collagen may not necessarily provide proof that their nonreduced forms function as intermolecular bonds in vivo, because the initial condensation reaction might have been favored by the alkaline-reducing conditions. Based on the known sequence of amino acids in the al(1) chain, it is now proposed that the C-terminal lysine may react with hydroxylysine at position 87 and with lysine at position 327, whereas the N-terminal amino group forms a linkage only with hydroxylysine at position 927. One side-to-side bond can only result from the reactions of the C-terminus with the regions at 327 and 564 (Kang, 1972; Miller, 1971b; Dixit and Bensusan, 1973; Becker et al., 1975a). The following are some of the important reactions which are believed to proceed from the condensation of carbonyl derivative (a-aminoadipic acid 6-semialdehyde) with other functional groups of amino acids through a series of complex mechanisms (Figs. 7 and 8). 1. Hydroxylysinonorleucine: These linkages can be developed by two reactions: first, by condensation of hydroxylysine and a-aminoadipic acid &semialdehyde; second, by condensation of lysine and 6-hydroxy a-aminoadipic acid a-semialdehyde. Such linkages were first identified by Bailey and Peach (1968) in collagen from calf and rat tendons. Later on, many studies substantiated these findings in skin (Franzblau et al., 1970; Tanzer et al., 1970; Bensusan, 1972; Bailey and Lapiere, 1973). In the case of calf skin, hydroxylysine and a-aminoadipic acid &semialdehyde are mainly involved in the cross-linkage (Tanzer and Mechanic, 1970; Nicholls and Bailey, 1980). 2. Lysinonorleucine: Lysinonorleucine links have been found in skin collagen from different species (Bailey, 1970; Kang et al., 1970; Mechanic and Tanzer, 1970; Bensusan, 1972), tendon (Shimokomaki et al., 1972; Cannon and Davison, 1973), and basement membrane (Tanzer and Kefalides, 1973). 3. Dihydroxylysinonorleucine: The occurrence of these cross-linkages in collagen from bone and dentin was suggested by Bailey et al. (1969); Davis and Bailey (1971) identified the actual structure. Later studies reported the pres-

257

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

--N

FH2 N

H

I H C=O

‘C’

J

4,Cu2+

I1

CH I YH(OH) (FHz)*

H

-

y2

I

N II CH I

-N,H,c=o

7

+ hydroxylysine

-N/h=O H I

lysyl oxidase

+ lysine

(FH,)s CHO a-Aminoadipic acid-

Dehydrohydroxylysinonorleucine

( p ,

-N+c=o H I

6-semialdehyde (allysine)

J

-

Dehydrolysinonorleucine

+ allysine

nuc leophilic condensation

H -N\H,C=O C

H -N,E,C

I I

1

(CH,),

I

$H

O=C,H ,C-(CH,),-CH-NH-CH,-C I HNI OH

-t

I

(FHA CH2 CH-CHO

hydroxylysine

H

=O

I I

H

Hydroxymerodesmosine

I O=C\H ,CHN I

I

H I --N,H,C=O C I (CH,), y

I y.

OH

-E/$c=o I

1

Dehydrohydroxymerodesmosine

FIG. 7.

Merodesmosine

1 (+A

I I

(CH,),

1;1

-N+C=O H

J

H I -N,E,C=O

2

C=CH-N

I

-N’$\C=O H I

I

Aldol

CH It (CH,),- CH-N=CH-C

“H

I

I

I

I

H C=O

I

+ histidine

H I -“,H,C=O C

I

CH c-cH,-NH-(cH,),-c’ II (CH,),

-N/$C=O

I

i

+lysine

(CH,),

(FHJ,

-N/ g\ C

H I -N,H,C=O C I (CH,),

=O

I

Aldol histidine

CH2 -N’&.c=o H I

CH I1 C-CH=NI (FHJ,

I

H C=O (CH,),-C< NH

-N’$\C=O

I Dehydromerodesmosine

Cross-linking reactions involving lysine derivative as intermediates

ence of such cross-linkages in skin and tendon collagen (Mechanic et al., 1971; Bensusan, 1972; Eyre and Glimcher, 1972; Forrest et al., 1972; Bailey and Lapikre, 1973). 4. Aldol histidine: The presence of aldol histidine cross-linkages has been suggested in bovine skin and basement membrane but not in other collagens (Fainveather et al., 1972; Tanzer et al., 1973b). This type of unreduced

I

258

A. ASGHAR AND R. L. HENRICKSON

-HN,H,C

I

F

=0

(CH,), CH(OH)

I

7% NH,

H I --N H C=O ‘C’

Hydroxylysine r e s i d u e

H I --N,H,c=o C I (yJ* CH(OH) I CH II

‘ CH(OH)

-E+c=o I Dehydrohydroxylysinonorleucine Amadori

1

I

6 -Hydroxy , a -aminoadipic acid- 6 - semialdehyde (hydroxyallysine) I

rearrangement

+ allysine

--N H H c=o I

‘C’

Lysino-5ketonorleucine

H -N’H

CHO

Hydroxyaldol I + histidine H -N,H

C’

c, c=o I

Dehydrodihydroxylysinonorleucine

4 Amadori H -N,H

I

rearrangement

F’

I c=O

Hydroxylysino- 5ketonorleucine

I C=O

I

(p CH(OH)

I

Hydroxyaldol histidine

FIG. 8. Cross-linking reactions involving hydroxylysine derivative as intermediates.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

259

linkage is quite labile in acid (Kang et al., 1970; Davison et al., 1972; Robins and Bailey, 1973b, 1975). 5 . Hydroxyaldol histidine: Housley et al. (1975) have reported the presence of hydroxyaldol histidine linkages in collagen which are not reducible and are stable. They involve condensation of allysine, hydroxylysine, and histidine (Tanzer et al., 1973b). 6. Hydroxypyridiniurn: Very recently, Eyre and Oguchi (1980) have identified hydroxypyridinium cross-links in skeletal collagen. The formation of these cross-links involves two aldehyde intermediates, formed from hydroxylysine residues. Among all these compounds only hydroxylysinonorleucine and dihydroxylysinonorleucine have been isolated from collagen in significant amounts. Their content in collagen seems to be related with the extent of lysine hydroxylation in the N- and C-terminal regions of the ci chains (Balazs, 1977). In fact, the crosslinkages, originated from lysine and hydroxylysine, are distributed in varying amounts in collagen of different tissue and species (Hall, 1976). Some tissues contain only one type of cross-link, some two, and others may contain more depending upon the ci chain’s composition (Bailey and Robins, 1976). An explanation of these observations could be that the degree of hydroxylation of the lysine residues and the proximity of the reacting groups in the terminal telopeptide of different a chains may determine the nature of the cross-linkages. It seems that aldehydes, formed by lysyl oxidase, are involved in two main types of cross-linkages. First, intramolecular linkage may be formed to join a chains by aldol condensation. Second, intermolecular cross-linkages are formed mainly by Schiff base reaction between an aldehyde derived from lysine, hydroxylysine, or glycosylated hydroxylysine and the E-NH, group of another lysine, hydroxylysine, or glycosylated hydroxylysine (Bailey et al., 1974). Generally these Schiff bases are unstable, but more stable linkages develop (on shifting the double bond) in the formation of keto derivatives. Further dehydration and oxidative reactions, and the formation of complexes with imidazole of histidine results in very stable structures. It has been shown also that cross-linkages originating from hydroxylysine are relatively more stable than those formed by lysine (Bailey et al., 1977; Miller and Robertson, 1973). However, much information on the chemical nature of the cross-linkages has been derived from studies on tissue other than muscle. Little is known about the chemistry of crosslinkages of intramuscular collagen. In summary, the increase in the mechanical stability and the progressive decrease in solubility of collagenous tissues in certain solvents coincide with gradual increase in intermolecular cross-linkages. The mechanisms of these changes is very complex, involving many variables like noncovalent interactions, dehydration, extent of glycosylation, packing of molecules, and location,

260

A . ASGHAR A N D R. L. HENRICKSON

number, and chemical structure of cross-linkages (Tanzer, 1976). The hereditary disorders, such as the Ehler-Danlos syndrome and hydroxylysine deficiency, are reported to cause abnormal cross-linking in the dermis of affected individuals (Eyre and Glimcher, 1972; Mechanic, 1972). Apart from this, homocysteinuria may disturb the process of cross-linking in collagen due to interaction of homocysteine with the aldehyde group of polypeptide chains (Kang and Trelstad, 1973).

F. INTERACTION WITH CARBOHYDRATES It is well documented that certain carbohydrates are covalently bound with collagen as an integral part of its structure. Earlier studies reported the presence of different sugars, such as glucose, galactose, mannose, rhamnose, ribose, and fucose, associated with collagen. Some of these (glucose and galactose) were found linked with acid-soluble (Highberger et al., 1964) and insoluble collagen (Hormann, 1965), whereas other sugars were detected in the neutral salt-soluble collagen (Highberger et al., 1964). However, the prevailing view is that only glucose and galactose are covalently bound with collagen either as monomer or disaccharide in vertebrate tissue (Spiro, 1969). Other sugars may be impurities resulting from inadequate purification of the collagen samples. The presence of mannose and N-acetylglucosamine in the C-terminal region of pro-a1 chain has been reported (Guzman et al., 1978). Divergent views also exist as to how the carbohydrate moiety is linked to collagen. On the basis of earlier evidence, Harding (1965) concluded that an ester linkage may form between the COOH group of aspartic acid residues and the OH function of the hexoses. Bensusan (1965) suggested that themgars may be linked covalently to the E-NH, group of lysine by forming N-D-glycosyl linkage or they may bond to the N-terminal amino acids. However, there is more evidence that the disaccharide of glucose and galactose (M. J . Spiro and Spiro, 1971; R. G. Spiro and Spiro, 1971a-c) is glycosidically linked to the OH group of hydroxylysine (and threonine and serine) in some invertebrate collagen (Lee and Lang, 1968). The presence of glycosyl hydroxylysine and galactosyl hydroxylysine at positions 87 and 681 in the a1 chain, and at positions 87 and 174 in the a 2 chain has been reported by many workers (Butler and Cunningham, 1966; Butler, 1968, 1970; Aguilar et al., 1973). The linkage between hexose monomers with hydroxylysine residue in collagen is shown on the next page. Different opinions have been expressed as to the functional role of carbohydrates in the collagen structure. According to some researchers, the bulky carbohydrate group might direct the regular stagger of tropocollagen molecules by requiring a particular “fit” (Morgan et al., 1970; Piez et al., 1970). Some workers say that carbohydrate could mediate cross-linking without being incorporated into a cross-link (Bailey et al., 1970; Spiro, 1972), whereas others have

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

26 1

H O I I1

NH,- CH,- CH- CH,- CH,- C- C I

I NH

‘ 2 Hydroxylysine residue

Hw -0 H

O -Galactose~

-Glucose

HO

H

OH

assigned an interacting role to carbohydrate between collagen and mucopolysaccharides (Traub and Piez, 1971).

G . POLYSACCHARIDES OF CONNECTIVE TISSUE The polysaccharides of connective tissue are not an integral part of the collagen molecule. They in fact constitute the aqueous phase (ground substance) of the extracellular space in which collagen fibrils are embedded. The ground substance is believed to perform many functions in the tissues (Schubert and Hamerman, 1968). It provides the actual homeostatic environment of the cells and takes up their metabolites. It acts as a barrier against bacterial invasion. Besides, marked changes occur in the extracellular protein during differentiation, regeneration of cells, and hormonal action. The polysaccharide complexes of connective tissue can be divided into two distinct groups: first, the glycoproteins consisting of protein molecules to which monosaccharides or oligosaccharides are covalently bound; and second, proteoglycans consisting of polysaccharide-protein complexes, in which the polysaccharide moiety makes up a major part of the whole molecule. The important polysaccharides found in association with various connective tissues are shown in Table 111, and their structures are depicted in Fig. 9. The molecular weight of these polysaccharides varies from 15 X lo4 to 15 X lo6, and the number of monosaccharide units in a molecule may vary from 50 to 50,000. Detailed information on the chemistry of proteoglycans and mucopolysaccharides is available in many excellent reviews (Muir, 1964; Fitton-Jackson, 1964; Schiller, 1966; Schubert and Hamerman, 1968; Merkel, 1971; Montreuil, 1980). The

I H U L b 111

THE MUCOPOLYSACCHARIDES (GLYCOSAMINOGLYCANS) ASSOCIATED WITH CONNECTIVE TISSUE Sulfates/ period* Polysaccharide (81ycosaminoglycan)

Synonyms

Hexosamine

Hexuronate

__ Hyaluronic acid Glucosamine Glucuronate Chondroitin Chondroitin 4-sulfate Chondroitin sulfate A Galactosamine Glucuronate

Chondroitin 6-sulfate Chondroitin sulfate C Galactosamine Glucuronate Dermatan 4-sulfate Keratan sulfate

Chondroitin sulfate B-heparin Kerato sulfate

Heparin Heparin sulfate

Heparitin sulfate Heparin monosulfate

from Schubert (1964).

Galactosamine Iduronate

Hexose

Ester Amide

-

0.0

-

-

1.0

-

-

1.0

-

-

1.0

-

Glucosamine

-

Galactose

1.0

-

Glucosamine

Glucuronate

-

1.5

I .O

Glucosamine

Glucuronate

-

0.5

0.5

Repeating unit D-Ghcuronic acid + 2-acetamido-2-deoxy-~-glucose D-GhCUrOniC acid + 2-acetamido-2-deoxy-~-galactose D-(;hJcuronic acid + 2-acetamido-2-deoxy-4-O-sulfo-~galactose D-(;hCUrOniC acid + 2-acetamido-2-deoxy-6-0-sulfo-~galactose L-Iduronic acid + 2-acetamido-2-deoxy-4-0-sulfo-~galactose D-(;ahCtOSe + 2-acetamido-2-deoxy-6-O-sulfo-~galactose D-Giucuronic acid f 2-deoxy-2-sulfoamino-o-g~ucose (both residues can also contain 0-sulfate groups) D-Giucuronic acid + 2-deoxy-2-sulfoamino-o-g~ucose (also containing 0-sulfate) and D-glucuronic acid + 2-acetamido-2-deoxy-~-glucose

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

263

polysaccharide components of proteoglycans containing amino sugars coupled to either a neutral sugar residue or a uronic acid are termed glycosaminoglycans. Other names used for defining the compounds are deoxyglycosylaminoglycans and glucuronyldeoxyaminoglycosylglycans (Schubert and Hamerman, 1968). As can be seen in Fig. 9, the basic unit of mucopolysaccharides (glycosaminoglycans) is closely related to glucose, linked by glucosidic bond. In fact, each polysaccharide is composed of two different saccharide units, which repeat alternatively along the chain without branching. In all cases one of the saccharide moieties is hexosamine (mostly D-glucosamine, in some cases Dgalactosamine), whereas the other unit is generally a uronic acid derivative (either D-glucuronic or L-iduronic acid). The NH, group of hexosamine is never free; it is either acetylated or sulfated. Consequently, these polysaccharides have a high negative charge density as carboxylate, ester sulfate, or amide sulfate (Schubert, 1964). At physiological pH they exist as polyanions and are generally associated with an equivalent amount of Na+ counterion. However, in the native tissues the counterions may be of several kinds depending on the salt content of the surrounding tissue. The accumulation of counterions in close proximity to the polyanionic chain reduces the net charge of the mucopolysaccharides. This effect is called “shielding.” Hyaluronic acid is one of the major polysaccharides of the ground substance. It is an unsulfated glycosaminoglycan, composed of D-glucuronic acid and Nacetylglucosamine linked alternately by p-1,4 and 1,3 glucosidic linkages. The chondroitin, heparin, and their sulfated derivatives have identical repeating units, consisting of glucuronic acid linked by 1,3 glucosidic bond to galactosamine, whereas the pairs are joined by 1,4 glucosidic linkage. Keratin is composed of glucosamine and galactose residues, sulfated at the C-6 position. Dermatin is a co-polymer of iduronic acid and galactosamine. The relative proportion of these mucopolysaccharides and their degree of sulfation vary among species, tissues, and with age (Hall, 1976). In vivo the connective tissue polysaccharides do not occur free, but are linked to protein. Earlier studies indicated that polysaccharides are covalently bound to a globular protein through the OH group of the serine residue by way of galactose (Dorfman, 1964; Roden, 1965) to constitute the mucopolysaccharides. The latest structure that emerges for a mucopolysaccharide is that of a “bottle brush” in which the bristles may be regarded as the carbohydrate chains (35-65 in number) linked by a neutral trisaccharide to a protein core (Laurent, 1974; Phelps, 1974; Schubert and Hamerman, 1968). The protein core, consisting of 2000-3000 amino acid residues, has a globular portion and a linear portion, with a total length of 340 nm (Phelps, 1974). On the other hand, Anderson and Jackson (1972) and Heikkinen (1973) have suggested some possible linkages between tropocollagen polymers via pro-

264

A. ASGHAR AND R. L. HENRICKSON

A

NHCOCH,

H

OH

H

NHCOCH, n

D-Glu&onic acid

N - Acetyl-Dglucosamine

n

2500

B

n

D-Glu&Ironic acid

N-Acetyl-b-galactosamine

n = 60

4- sulfate

C

n

L-Iduronic acid

N-Acetyl-D-galactosamine 4- sulfate

D

n

D-Glucuronic

acid

N-Acetyl-D-galactosamine 6 -sulfate

265

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

COO@Na@

E -0 I

H H loH D-Glucuronic acid

D-Glucosamine N-sulfate. 6 sulfate

i!THSOpNa@

n = 10-15

1

An

F NHCOCH,

H

OH

H

Nncocn: n

D-Galactose

N-Acetyl-D-glucosamine 6 -sulfate

n

=

10-20

FIG. 9. Structure of the repeating saccharide units in different mucopolysaccharides of connective tissue. (A) Sodium hyaluronate; linkages p-1,3 and p-1,4.(B) Sodium chondroitin sulfate A; linkages p-1,3 and p- 1,4.(C) Sodium chondroitin sulfate B (dermatan sulfate); linkages p-1,3and p-1,4.(D) Sodium chondroitin sulfate C; linkages p-1,3and p-1,4.(E) Sodium heparitin; linkages C X - D - ~(F) , ~ Sodium . keratan sulfate; linkages p-1,3 and p-1,4.

teoglycans, whereas other groups of protein may be linked through glycoproteins. According to Mathews (1970) proteoglycans, by virtue of a high negative charge, react directly with collagen to form macromolecular complexes having different biophysical properties and functions. Obrink (1974) stated that sulfated polysaccharides and proteoglycans (except keratin sulfate and hyaluronic acid) bind electrostatically to collagen under physiological ionic conditions and the binding increases with increasing chain length and charge density. According to Blackwell and Gelman (1974) the strength of interactions depend, in addition, on the side chain length of basic amino acid residues of collagen, or the degree of sulfation of the polysaccharide chain, and on the position and orientation of the sulfate and carboxyl groups of the polysaccharide chain. The intensity of the interaction between collagen and polysaccharide is also influenced by the pH of the medium, being highest at 3.0 for heparin and chondroitin sulfates A and C (Cundall et al., 1979), since they contain ester-sulfate ( R U S O , -) groups, which are not protonated at pH > 1 (Schubert and Hamerman, 1968). Hence, these groups can interact at pH > 1 with positively charged groups on collagen molecules.

266

A . ASGHAR A N D R. L. HENRICKSON

H.

IMMUNOCHEMISTRY OF COLLAGEN

The antigens (or allergens) in food are identified mostly with protein fractions. The ability of the food to incite immunological reaction is associated sometimes with the nature of protein molecules, their stability against physical agents (heat, cold, oxidation), H + concentration, enzymic action, and their ability to pass through the wall of the gastrointestinal tract with little or no alteration in molecular configuration (Straws, 1964). The allergy is an immunological phenomenon, considered as antigen-antibody reaction. The mediator of this phenomenon is probably histamine or a histamine-like compound, and the chain of reactions can be interrupted by histamine antagonists (Perlman, 1964). Generally, the protein antigens have two operational immunological properties. One is called antigenicity, which denotes the capacity to interact with secreted antibodies, and the other is immunogenicity, which indicates the capacity of a protein to stimulate antibody synthesis. According to the current concept on the cellular basis of the immune response, the interaction of antigen determinants with antigen receptors occurs on the surface of antibody-producing precursor cells, called B cells. Optimal antibody production, however, requires for most antigens the cooperation between B cells and thymus-derived T cells (Katz and Benacerraf, 1972; Gershon, 1974). Probably macrophages also participate in this cooperation (Feldmann, 1974). So far as the antigenic properties of collagen are concerned, a number of studies have shown that the major antigenic sites reside in the C- and N-terminal regions (Schmitt et al., 1964; Timpl et al., 1970, 1972, 1973; Furthmayr and Timpl, 1971; Rauterberg et al., 1972a; Becker et al., 1972, 1975b). It has been further shown that unoxidized or oxidized E-NH, groups of lysine in the terminal regions bind with antibodies (Rauterberg et al., 1972a). More detailed information on the immunochemistry of collagen and procollagen is available in a number of comprehensive reviews (O’Dell, 1968; Kirrane and Glynn, 1968; Timpl, 1976; Furthmayr and Timpl, 1976). 1. FUNCTIONS OF COLLAGEN IN TISSUES Generally speaking, the main functions of fibrous biopolymers are structural, providing physical strength, cementing the cells together, and at the same time serve a sieve-like function for passage of metabolites from cell to cell (Bettleheim, 1974). In this respect, collagen is an unusual biopolymer, having a high tensile strength and being virtually inextensible. Nature, as Bailey stated, has utilized these unique characteristics to perform various mechanical functions in a variety of ways throughout the animal body. For example, in the case of skin and muscle, fascia collagen acts as a flexible network to contain other tissues in register. In the case of tendons and ligaments, collagen not only connects one tissue to another but also transmits tensional stress and resists the compressional

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

267

force (Flint, 1973; Miller, 1980). It also imparts some degree of flexibility to calcified tissue like bone and a certain amount of structural rigidity to cartilage (Viidik, 1973; Gathercole and Keller, 1974; Phelps, 1974). It constitutes the fine membrane that functions as a filtration barrier in the kidney, and constitutes the channels for solid and liquid transport. In all these tissues, collagen has been found in different but highly ordered fashions to suit the registered mechanical function of the respective tissue. However, the optimum functioning of the various tissues depends not only on the amount and types of collagen but also on the relative proportion of other associated constituents (Bailey, 1974). The mucopolysaccharides associated with collagens are largely responsible for the water sorptive and retentive capacity of the tissues. They also create osmotic pressures, at physiological concentration, of a magnitude that is important to the living organism (Bettleheim, 1974; Laurent, 1974). As the polysaccharides also bind counterions, they affect the diffusion of ions through the connective tissues (Preston et al., 1972). Some workers have suggested that mucopolysaccharides protect collagenous tissue from enzymic attack (Eyre and Muir, 1975b; Osebold and Pedrini, 1976). Etherington (1977) espouses the view that the type and quantity of associated mucopolysaccharides are important factors in determining the degree of resistance of the collagen molecules to enzymic vulnerability.

IV. METABOLISM OF COLLAGEN The macromolecular components of collagen are synthesized in large part at a ribosomal-messenger RNA complex within the cytoplasm of a family of mesenchymal cells, which include fibroblasts, chondroblasts, and osteoblasts (Fitton-Jackson, 1964). The fibroblasts are quite familiar cells which synthesize connective tissue as a part of the repair process or normal embryogenesis. However, the existence of several genetically distinct (Y chains in the collagen from different tissues suggests selective gene expression for collagen biosynthesis in certain cell types (Grant and Jackson, 1976). Fibroblasts, osteoblasts, and odontoblasts in cell culture synthesize mainly type I collagen. The same seems to be true for human smooth muscle cells (Layman and Titus, 1975), whereas human skin fibroblasts are reported to produce also a good proportion of type 111 collagen (Lichtenstein e f al., 1975). Chondroblasts produce only type I1 collagen, whereas the basement membrane is synthesized either by epithelial or endothelial cells. A.

BIOSYNTHESIS ON POLYRIBOSOMES

The latest information on the biosynthesis of collagen (procollagen (Y chains) at the subcellular level is available in a number of comprehensive reports (Martin et al., 1975; Brownell and Veis, 1975; Grant and Jackson, 1976; Prockop et al., 1976; Beachey et al., 1979; Harwood, 1979). There is now general agreement

268

A. ASGHAR AND R. L. HENRICKSON

that the a chains of all the procollagens are synthesized about 50% larger than the ultimate a chains of the collagen, and have peptide extensions at both the C- and N-terminal ends (Grant et al., 1972; Dehm et al., 1972; Byers et al., 1974; Tanzer et al., 1974; Fessler et al., 1975a; Harwood et al., 1977; Williams et al., 1976). These extensions are designated as P,a and P,a chain, respectively (Martin et al., 1975). As depicted in Fig. 5, the P,a chain consists of three distinct structural domains: a globular NH,-terminal region, a central collagenlike region, and a short globular region (Becker et al., 1977; J. H. Fessler and Fessler, 1978). The PCa chain has only globular conformation (Olsen et al., 1977). The cysteine residues in the N-terminal region create only intrachain disulfide bonds, whereas they create both inter- and intrachain disulfide bonds in the C-terminal region. The extraglobular peptide portions (Sherr et al., 1973; Murphy et al., 1975) containing sugars, cystine and cysteine amino acids (Furthmayr et al., 1972; Clark and Kefalides, 1976) on the terminal ends are enzymatically cleaved before the molecule is liberated into the extracellular space (Bornstein, 1974; Martin et al., 1975; J. H. Fessler and Fessler, 1978; Prockop et al., 1979). The extra terminal peptides (sometime termed “registration peptides”) presumably facilitate alignment of the three cx chains, and permit the remaining molecule to coil into the in-register triple helix (Grant and Jackson, 1976). The presence of interchain disulfide bonds in the terminal regions also seems to play a role in helix formations (Schofield et al., 1974; Uitto and Prockop, 1974). It has been shown that the individual a chains of tropocollagen are assembled as single polypeptides rather than from short peptides (Bornstein, 1970; Vuust and Piez, 1972). The polycistronic mRNA synchronizes the proper balance of the synthesis of dissimilar tropocollagen subunits, that is, a 1 and a 2 chains, in the case of type I collagen (Vuust and Piez, 1972) at 2:l ratio (Kerwar et al., 1972; Prichard et al., 1974; Harwood et al., 1975a). This suggests that the two mRNAs may be present in a similar ratio. Further, the collagen precursors are synthesized by heavy polyribosomes with a sedimentation value 300 to 400 S (Lazarides and Lukens, 1971; Harwood et al., 1974a,d, 1975b; Pawlowski et al., 1975; Vuust, 1975) on tight membranebound polyribosomes (Rosbash and Penman, 1971; Harrison et al., 1974; Harwood et al., 1975b). It takes 6-7 min for the synthesis of pro-al(I), pro-a2, and pro-al(II1) chains in vivo (Miller et al., 1973), possibly due to the greater demand for glycyl-tRNA and propyl-tRNA in the translation of the long [-Gly-XY-1, triple sequence of the collagen molecule (Grant and Jackson, 1976). On the contrary, translation of mRNA globular proteins may need only about 2 min. At present two hypotheses exist regarding the initial synthesis of a chain (s) precursor. According to one view, the precursor of the three chains may be synthesized initially as a very long single peptide chain (Church et al., 1971, 1974; Tanzer et al., 1974; Park et al., 1975), analogous to the synthesis of the

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

269

insulin polypeptide (Steiner et al., 1974), and then cleaved into three pro-a chains. There is, however, little evidence in favor of this concept. The other view, based on pulse-chase studies, supports simultaneous translation of pro-al(I) and pro-alt(1) chains (Vuust and Piez, 1970, 1972; Harwood et al., 1973, 1975a, 1977; Schofield et al., 1974; Uitto and Prockop, 1974; Byers et al., 1975; Fessler et al., 1975b) by monocistronic mRNA (Diaz de Le6n et al., 1977) or polycistronic mRNA (Harwood, 1979). According to Williamson (1969), generally there are two patterns of genetic control for heteropolymeric proteins. First, the cistrons coding for the different subunits may be adjacent on the genome, and hence give rise to a polycistronic mRNA which ensures a balanced production of the dissimilar polypeptide chains. Second, the subunits may be coded by separate cistrons, linked or unlinked, to give rise to monocistronic mRNA molecules, which are separately translated. However, the most precise control of balanced synthesis of dissimilar subunits is expected from polycistronic mRNA, which ensures balance and synchronization. But, the balanced production of subunits by different monocistronic mRNAs would be liable to disturbance. Hydroxylation of proline and lysine and glycosylation of hydroxylysine are the important changes which occur after the transitional period at the subcellular level in pro-a chains (Cutroneo et al., 1974). 1. Hydroxylation of Proline and Lysine

As the mRNAs carry no codons for the hydroxy amino acids found in collagen, they must be synthesized during the posttransitional period by enzymic hydroxylation of proline and lysine residues, already incorporated in procollagen. Nonhydroxylated collagen molecules are also denoted as protocollagen (Grant and Prockop, 1972). Hydroxylation of proline (generally at the Y position) is brought about by prolyl hydroxylase (Cardinale and Udenfriend, 1974; Prockop et al., 1976) mainly as trans-4-hydroxyproline in different collagens (Fietzek and Kuhn, 1976), except in type IV collagen, where 3-isomers (at the X position) have been found in fair amount (Gryder et al., 1975; Adams and Frank, 1980). Considerably divergent views exist as to the site of hydroxylation at the subcellular level (Bornstein, 1974; Prockop et al., 1976). The general consensus of opinion is that it occurs before the polypeptides are released from the ribosomes. Again, contrary to the initial finding that prolyl hydroxylase is a soluble cytoplasmic enzyme, it is now well documented that this enzyme is membrane bound, associated with cisternae of rough endoplasmic reticulum for hydroxylation (Harwood et al., 1974c, 1975b) before helix formation (Ryhanen and Kivirikko, 1974a,b). The hydroxylation process also requires Fe2 , ascorbate, and decarboxylation +

270

A . ASGHAR AND R. L. HENRICKSON

*-

of 2-ketoglutarate to succinic acid and CO, (Grant and Jackson, 1976) as follows:

[Gly--X-Proln

ascorbate

2-Ketoglutarate

[GI~-X-PJ;-],~

Suciinate

The same sorts of events are involved in the hydroxylation of lysine except that the reaction is catalyzed by lysyl hydroxylase (Guzman et al., 1975). Many research workers have shown that hydroxylation of the a chains is important for the formation of the triple-helical structure of collagen (Fessler and Fessler, 1978; Olsen et al., 1975; Harwood et al., 1974a,b, 1977). Many studies have shown differences in the extent of hydroxylation of proline residues in rat and calf skins, with rat skin being more hydroxylated than the calf skin (Fietzek et al., 1972; Fietzek and Kuhn, 1973, 1974, 1975). The extent of hydration is positively related with the T , value of collagen (Jimenez et a / . , 1973; Berg and Prockop, 1973). 2.

Glycosylation of Hydroxylysine

According to Grant et al. (1975), the disaccharide a-D-glUCOSyl (1,2)-0-p-Dgalactose, associated with collagen, has a significant bearing on its functional and structural properties. This sugar is linked with hydroxylysine (at the Y position in the collagen chain) by the glucosidic bond (Morgan et al., 1970; Aguilar et al., 1973), as mentioned earlier in Section III,D,5. Two enzymes, namely collagen UDP-galactosyl transferase and collagen UDP-glucosyl transferase, catalyze the glycosylation of pro-a chains (Spiro, 1972; Risteli and Kivirikko, 1974; Myllyla etal., 1975) in the presence of Mn2+ as cofactor (Myllyla et al., 1975). These enzymes are bound to the internal surface of rough endoplasmic reticulum cistemae (Harwood et al., 1975b), where glycosylation of peptide chains takes place (Brownell and Veis, 1975). As glycosylation could not be achieved with triple-helical collagen in in vitro studies (Myllyla et al., 1975; Risteli et al., 1976), in vivo glycosylation probably occurs before the procollagen chains attain the triple-helical structure in the cistemae of the rough endoplasmic reticulum. Several pathways have been proposed for onward transportation of procollagen (Weinstock and Leblond, 1974; Prockop et al., 1976). However, various studies suggested that the procollagen molecules are then transferred to smooth endoplasmic reticulum (Harwood et al., 1977), and then directed into the Golgi apparatus (Nist et a/., 1975; Olsen et al., 1975), from where they are possibly released by exocytosis through microtubules. Figure 10 presents the overall events of the biosynthetic process of procollagen a chains and their transport into the extracellular spaces at the subcellular level.

NH2

-

- COOH

N-protease

f

I

C-protease

I

Fibril formation

Aldehyde formation Intermolecular cross1inking

FIG. 10. Schematic representation of the sequential events in the biosynthesis of procollagen at the subcellular level and subsequent polymerization in the extracellular space following stepwise cleavage of terminal extensions at NH, and COOH ends. From Grant and Jackson (1976). Courtesy of The Biochemical Society, London.

272

A. ASGHAR AND R. L. HENRICKSON

The polymerization of procollagen monomers to form collagen fibrils takes place stepwise in the extracellular space. It appears that the NH,-terminal region is cleaved first (Morris et al., 1975; Davidson et al., 1975), followed by stepwise removal of the C-terminal extensions of the three a chains of procollagen, consequently giving rise to an insoluble collagen polymer (Byers et al., 1975; Fessler et al., 1975b; Grant and Jackson, 1976). The cleavage of N- and Cterminal extensions is brought about by either the same enzyme or two different enzymes, presumably bound on the outer cell surface (Davidson et al., 1975). Freshly assembled collagen fibrils have little tensile strength, which increases due to the formation of more covalent bonds and cross-linkages (Grant and Jackson, 1976) with maturity as a function of chronological age. It is obvious from the preceding review that biosynthesis of collagen involves a large number of posttranslational reactions. They can be influenced not only by defects in transcription and translations, but also by defects in any of the enzymes involved in the posttranslational steps. Consequently, cells that use the same mRNA may produce collagen of different types. A number of genetic defects in collagen synthesis have been described by Prockop et al. (1979) and Lenaers et al. (1971). B.

CATABOLISM OF COLLAGEN

Earlier studies have indicated that depolymerization of native tropocollagen macromolecules occurs by cleavage of certain bonds in the nonhelical region (Hodge et al., 1960; Hodge and Schmidt, 1960). For example, pepsin or trypsin attacks the region rich in tyrosine and acidic amino acids, and devoid of hydroxyproline (Steven, 1963; Rubin et al., 1963). The prevailing view is that nonspecific peptide hydrolases such as pepsin and chymotrypsin are not capable of disrupting the helical conformation of collagen (Stark et al., 1971b). However, chymotrypsin (Bornstein et al., 1966) and lysosomal isoenzyme (cathepsin B) can convert p and y aggregates of soluble collagen to a chain (Burleigh et al., 1974; Etherington, 1976, 1977) by hydrolyzing the peptide linkage between glycine-isoleucine, or between serine and valine of the a 1 chain in the NH,terminal region. Figure 11 shows the various cleavage sites on the polypeptide chain of a protein by some important proteolytic enzymes. Harris and Krane (1973, 1974) have shown that collagenases hydrolyze the native collagen molecule at a point about % of the length from the NH,-terminal end. Different explanations have been given to explain the mode of action of collagenases on a1 and a 2 chains (McCroskery et a l ., 1973; Gross et al., 1974). According to Weiss (1976), the cleavage site is at the NH,-terminal end of the nonproline helical portion of the molecule between two regions of tripeptides of opposite symmetry of their proline and hydroxyproline content. Thus, initial breaks produce two fragments with lower T , and higher solubility than the intact molecule, without any loss of helical structure (Gross and Nagai, 1965; Sakai

273

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

-Arg-Glv- Leu-Hyp-

-Ala-Gly-Val- Ala-

b

2

3

0

a

CHYMOTRYPSIN

2

x

'1

'1 -Ser-Tyr-Gly-Tyr-

-Hyp-Gly-Glu-Hyp-

N - T e r m i n a l region of a - c h a i n

-GLy-Tyr- AspGlu-

N-Terminal region of o - c h a i n -Gly-Ala-Hyp-Gly-Thr-

-Thr-Gly-lleu-Ser-VaI-Pro-

Pro-Gly-Pro -1leu-Gly-Gln

h4 c -Lys-Ser-Gly-Asp-

FIG. 1 1. Some typical cleavage sites on native collagen and denatured collagen (gelatin) molecules for the action of certain important peptide hydrolases.

and Gross, 1967). On the other hand, Harris and Krane (1974) are of the view that collagenase cleaves through the collagen triple helix at one point without having definite specificity for denatured collagen products. Bazin and Delaunay (1972) reported on a collagenolytic cathepsin, other than B I , which degraded insoluble collagen without affecting triple-helical conformation, whereas other workers have shown that cathepsin B, (EC 3.4.22.1) degrades insoluble collagen at pH 3.5 (StanEikovBand Trnavskjr, 1974). According to Etherington (1974), lysosomal cathepsin B I removes the telopeptide regions. However, increase in the extent of intermolecular cross-linkages renders collagen resistant to collagenase (Harris and Ferrell, 1972).

274

A. ASGHAR AND R. L. HENRICKSON

The studies of many workers (Harris and Krane, 1974; Woolley et al., 1975a,b) have provided strong evidence which indicates that soluble type I1 collagen is degraded by collagenase, but the rate is much slower than for type I collagen. The action is probably hindered by the presence of the covalently bound bulky carbohydrate moiety to the hydroxylysine residue in the a 1(11) chains. There is also evidence that collagenases from different tissues cleave collagen at different sites at different rates (Jeffrey and Gross, 1970; Tokoro et al., 1972; Davison and Berman, 1973; Wahl et al., 1975). A critical evaluation of various studies by Weiss (1976) suggests that denatured collagen is easily catabolized by such noncollagenous peptide hydrolases as trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), pepsin (EC 3.4.23.1), papain, gelatinases (EC 3.4.24.3), and clostridiopeptidase (EC 3.4.24.3). The native collagen is quite resistant. The unusual primary structure of native collagens, and the high levels of proline, hydroxyproline, and glycine residues make collagen an unsuitable substrate for most of the exopeptidases. However, a variety of noncollagenous endopeptidases can cleave certain regions of the molecule, high in either polar or nonpolar residues and low in imino acid content. This gives rise to larger fragments which possibly are not degraded further (Weiss, 1976). Hence, many of the earlier studies, quoted by Piez (1967) and Eisen (1969), where experimental conditions preclude the possibility of denaturation of collagen, are invalid. For instance, some studies have claimed excessive degradation of insoluble collagen by collagenase, which may be due either to rupture of intermolecular bonds by some pretreatment or to contamination of the enzyme preparation with another protease (Weiss, 1976). This is quite apparent from the study by Fugii and Kobayashi (1970), showing that first dissociation of acid-labile intermolecular Schiff base cross-links in the telopeptide region occurs in an acidic media, which sets the peptides free and makes them available for enzymic catabolism. Swelling of collagen at acidic pH results from or is partly responsible for the cleavage of cross-links (Etherington, 1972). The conformation of collagen molecules also determines the rate of enzymic action (Hayashi et al., 1980). More detailed information on in vivo degradation of collagen may be found in a number of reviews (Lapikre and Gross, 1963; Eisen, 1969; Harris et al., 1970; Davison, 1973; Perez-Tamayo, 1973, 1979; Harris and Krane, 1974; Gross, 1976; Weiss, 1976; Harper, 1980).

V.

FACTORS AFFECTING COLLAGEN COMPOSITION AND STRUCTURE

Some in vitro studies have indicated that environmental factors can influence the mechanism for gene selection to produce a particular type of collagen (Deshmukh and Kline, 1976; Mayne et al., 1976; Deshmukh and Sawyer, 1977).

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

275

Many in vivo studies have shown changes in collagen composition and structure by ante- and postmortem factors. An account of those factors is presented in this section. A.

ANTEMORTEM FACTORS

A number of antemortem factors such as sex and sex condition, caloric and nitrogen intake, hormonal status of the animal, age, and dietary components have a significant bearing on the composition of collagen. The available information on these aspects is discussed below. 1. Sex and Sex Condition

The amount of collagen in tissues seems to be influenced by simultaneous activity of anabolic and catabolic systems between sexes. Prost et al. (1975) reported a relationship between the content of connective tissue and sex of the animal, indicating that bovine males have more intramuscular connective tissue than females. A similar trend was found in skin collagen from male and female rats (Hall et a l . , 1974). However, Summers et al. (1978) observed no real difference in the amount of muscular collagen of ewes and wethers. 2.

Caloric and Nitrogen Intake

Gross’s (1954, 1964a) study has shown that salt-soluble collagen disappeared on prevention of growth in rats by caloric restriction. The findings of McCance and his colleagues (Dickerson and McCance, 1960; Widdowson et al., 1960) indicated that extracellular protein content (collagen) increased in the avian and pig skeletal muscle as a result of undernutrition. Similarly Mendes and Waterlow (1958) reported that muscle connective tissue continued to increase in amount during the depletion period of young rats by underfeeding. Other workers (Harkness et a l . , 1958; Summers and Fisher, 1960) have noted similar effects of inadequate diets on body collagen. According to the work of Hagan and Scow (1957) and Montgomery et al. (1964), short-term starvation caused a greater reduction in the sarcoplasmic proteins than in the myofibrillar proteins. The connective tissue remained relatively unaffected. In a study of the influence of zero- and negative-energy balance feeding on different protein fractions of muscle from growing lambs, Asghar and N. T. M. Yeates (1979) found an increase in total stroma protein, including the alkalisoluble and alkali-insoluble stromal fraction. Besides the changes in amount, the physicochemical nature of the stroma fraction was also altered by nutritional stress, whereby the extent of acid-stable cross-linkages significantly increased. Another study by Asghar et al. (1981) on growing rabbits indicated similar effects of maintenance and submaintenance feeding on L. dorsi stromal protein

276

A. ASGHAR AND R. L. HENRICKSON

(collagen). A number of studies have also been reported on the connective tissue as it is influenced by breeds, feeding regimens, and management practices with hope of relating such information to meat tenderness (Wierbicki et a l . , 1955; Paul, 1962; Sharrah et a l . , 1965). The collagen content varies with type of muscle, being more in “slow” than in “fast” muscles (Kovanen et a l . , 1980). 3.

Effect of Vitamins and Minerals

Among the water-soluble vitamins, the role of ascorbic acid in synthesis and maintenance of collagen has been studied intensively (Gould, 1968; Levene et a l . , 1974). There is general agreement that vitamin C deficiency causes a decrease in collagen synthesis without increased catabolism (Robertson, 1964). Gross (1954) believes that the basic defect occurs before the level of synthesis of the collagen molecule, and not in the formation of intramolecular bonds. It seems that hydroxylation of proline and glycosylation of hydroxylysine are impaired in the absence of vitamin C (Barnes, 1975; Berg et a l . , 1976), resulting in a higher hydroxylysine-to-hydroxyprolineratio in pro-a chain than normal. Staudinger et al. (1961) gave a possible mechanism to account for the role of vitamin C in free radical formation and in hydroxylation of collagen. This involves electron transport in microsomes. According to them, the first product of the reaction with oxygen is an OH radical which may enter into the hydroxylation reaction. In some cases hydroxylation may also involve vitamin C-dependent NAD-oxidase as follows:

’?>

(

Transhydrogenase (flajin)

NAD+

Dehydroascorbic acid

Ascorbic acid

Ascorbic acid )Cytochrome oxidase

b,

ZOH-H,b

+

%02

I (.1*

Other studies have indicated that the oxygen of the OH group of hydroxyproline is derived from molecular 0, and not from water (Fujimoto and Tamiya, 1962; Prockop et a l . , 1962a). According to Gould (1968), certain B-complex vitamins are also believed to influence the collagen synthesis either by their involvement in NADP in the proline hydroxylation chain reaction or in Krebs cycle intermediates. He has quoted some studies indicating that deficiency of vitamins B, and B, decreased the synthesis of collagen in the skin of rats. Among the fat-soluble vitamins, deficiencies of vitamins A and D have been noted to affect the synthesis of collagen. Vitamin A deficiency seems to depress collagen synthesis (Robertson and Gross, 1954). Chung and Houck (1964) reported that hypervitaminosis A decreased salt-soluble collagen, and increased the

277

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

acid-soluble fractions without any effect on the insoluble collagen. However, the hydroxylation of proline is not impaired by hypervitaminosis A (Dingle et al., 1966). A toxic dose of vitamin D increased the amount of both collagen and mucopolysaccharides, but Clark and Smith (1964) noted a decrease in mucopolysaccharides. Vitamin E deficiency increased the content of soluble collagen without affecting the total amount, suggesting prevention or alteration in cross-linkage (Gould, 1968). The effect of fat-soluble vitamins may be indirect by influencing the membrane stability and permeability. The information regarding the effect of dietary minerals on collagen synthesis seems very scanty. Some early studies have reported that dietary copper deficiency causes mechanical weakness in the framework of collagen (Coulson and Carnes, 1962; Kimball et al., 1964). It has been established that lysyl oxidase is a Cu2 -containing enzyme, which oxidatively deaminates the E-NH, group of lysine and hydroxylysine to yield aldehydes, which form cross-linkages in collagen (Section 111,E).This may explain the weakening of collagen structures due to Cu2+ deficiency in the animal body. The deficiencies of Fe2 , Mn2 ,and Ca2 may produce a similar effect on collagen. The reason is that hydroxylation of proline and lysine residues in collagen brought about by prolyl and lysyl hydroxylases, respectively, is dependent on the presence of Fe2+ (Section IV,A). The glycosylation of hydroxylysine residues by galactosyltransferase and glucosyltransferase depends on Mn2+. The activity of amino protease and carboxyprotease, which cleave the P,a and P,a region for converting procollagen to collagen, requires Ca2 (Fugii and Tanzer, 1977; Uitto, 1977; Tuderman et al., 1978; Leung et al., 1979). Hence, the deficiency of these minerals may impede in vivo biosynthesis of collagen. +

+

+

+

+

4. Effect of Hormones There is ample experimental evidence that the anti-inflammatory compounds cortisol, glucocorticoids (Dougherty and Berliner, 1968; Vogel, 1974), and prednisolone (Hall et al., 1974) inhibit the synthesis of new collagen fibrils and promote the removal of already formed collagen. Other studies have shown that these corticosteroids decrease the level of posttranslational enzymes which are involved in collagen synthesis (Oikarinen, 1977; Newman and Cutroneo, 1978). However, it has been difficult to establish whether their effect is specific. The adrenocortical steroids also inhibit the formation of mucopolysaccharides, probably by affecting fibroblasts. Castor and Prince (1964) found that glucocorticoids suppressed the metabolic activity of fibroblasts, including collagen synthesis. Kowalewski (1966) noted a decrease in saline-extractable, insoluble, and total hydroxyproline, and an increase in acid-soluble hydroxyproline on administration of corticosteroid. These effects were minimized by administering anabolic

27 8

A . ASGHAR AND R . L. HENRICKSON

androgen-like methandrestendone, HCTH, and 17-ethyl-10-nortestosterone (Schiller and Dorfman, 1955), which antagonizes the action of corticosteroids. On application of 4- 14C-labeled cortisol, corticosterone, and other adrenal corticosteroids, the hormones tended to localize in or on fibroblasts (Schneebeli and Dougherty, 1963) and to inhibit pinocytosis (Berliner and Nabores, 1967). A number of studies have shown a decrease in the uptake of sulfate by various tissues (Clark and Umbreit, 1954; Schiller and Dorfman, 1955; Kowalewski, 1966) by administering these hormones, which inhibit the synthesis of hyaluronic acid in the skin. Manner and Gould (1965) are of the view that hydrocortisone interferes in part with collagen synthesis on the ribosomes. Hypophysectomy has been shown to decrease the uptake of [35S]sulfate in cartilage (Denko and Bergenstal, 1955; Murphy et al., 1956). However, according to Schiller (1966), both hypophysectomized and hypothyroid (thyroidectomy) conditions cause a decrease in condroitin sulfate but an increase in hyaluronic acid. These effects can be reversed by administering growth hormone and thyroxin, respectively. Thyroxin seems to increase the rate of [35S]sulfate incorporation (Dziewiatkowski et al., 1957), whereas the thyrotropin and thiouracil impair the effect of growth hormones. However, Lorenzen’s (1962) study suggested that administration of thyroxin itself caused a decrease in the content of hydroxyproline and hexosamine; their amount increased when thyroxin was injected in rats along with adrenaline. Baker et af. (1959) proposed that thyroidinduced alterations in connective tissue metabolism may be due to a failure of the pituitary to produce thyroid-stimulating hormone (TSH). Parathyroid hormone, presumably parathormone, affects the components of bone matrix as well as the mineral content. It is believed that parathormone has an inhibitory effect on the resorption of the metaphysis, or it may stimulate the production of chondroitin sulfate in the epiphyseal plate (Bronner, 1961; Sheltar et al., 1961). On the contrary, other studies reported an increase in the excretion of hydroxyproline into urine due to parathormone administration (Avioli et al., 1966), possibly by stimulation of collagenase synthesis (Harris and Sjoerdsma, 1966). It has been observed that under diabetic conditions the incorporation of [ 14C]acetate and [35S]sulfate into hyaluronic acid and chondroitin sulfate decreased in skin (Schiller and Dorfman, 1955). This suggested that insulin apparently acts as a stimulant for the synthesis of mucopolysaccharides. The facts that insulin markedly enhances the pinocytosis in fibroblasts (Schneebeli and Dougherty, 1966) and that glucose enters the cells by a pinocytotic process, may well explain the biochemical mechanism by which insulin stimulates mucopolysaccharide synthesis. According to Davidson and Small (1963), the ratio of keratosulfate to chondroitin sulfate C increased as a function of age. However, the growth hormones, estrogen and endrogen, alter this ratio, as is found at younger ages. Estrogens tend to increase the hyaluronic acid content of connective tissue (Muir,

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

279

1965). Some workers have shown that estrogen inhibits resorption of metaphyseal bone (Budy et al., 1952). Similarly, testosterone also increases the amount of hyaluronic acid (Doyle et al., 1964). The growth hormone ACTH is believed to promote collagen synthesis, whereas STH increases the number of fibroblasts and their synthesis of protein in vitro (Dougherty and Berliner, 1968). Harkness (1961) stated in his review that the growth hormones estrogen and deoxycorticosterone favor collagen deposition, whereas parathyroid hormone, cortisol, and related steroids discourage collagen synthesis. On reviewing the role of hormones in connective tissue metabolism, Sinex (1968) concluded that a decrease in insulin, estrogen, androgen, and thyrotropin is likely to favor the formation of dense connective tissue, deficient in mucopolysaccharides. Increase in glucocorticoids and thyroxin also produces similar effects in connective tissue. The mechanisms by which this occurs can only be the subject of speculation. The hormones may act as cofactor, or stimulate a dormant enzyme, to affect the intermolecular bonds. By using [14C]leucine, Gabourel and Fox (1965) observed that hydrocortisone affected the mRNA-tRNA system, but that tRNA or soluble enzymes were not influenced, and that the total amount of RNA decreased. Manner and Gould (1 965) also found a significant decrease in the amount of material in the polysomal fractions. On reviewing the actions of steroid hormones at cellular and molecular levels, Grant (1969) concluded that these hormones possibly intervene at more than one point in the process of transcription, and probably not in the process of translation of the genetic message. Figure 12 summarizes the possible sites of action of steroid hormones at the subcellular level.

Actinomycin D Amino acid DO01

,&-

Nuclear

rncmbmne

FIG. 12. Diagrammatic presentation of possible sites of action of some steroid hormones and inhibitors at the subcellular level. From Grant (1969). Courtesy of The Biochemical Society, London.

280

A. ASGHAR AND R. L. HENRICKSON

5 . Lathyrism

The phenomenon of lathyrism (osteolathyrism or odoratism) seems to have originated from the work of Ponsetti (1954), who first observed the deformities on feeding sweet peas (Lathyrus odoratus) to rats. Later studies indicated that paminopropionitrile (NH,-CH,-CH,-CN) was the causative lathyritic factor in sweet peas; the P-aminopropionitrile interferes with collagen metabolism and hence results in deformities (Levene and Gross, 1959). Further studies have shown that aminoacetonitrile and semicarbazide also exhibit a lathyrogenic effect. This aspect has been reviewed extensively by Tanzer (1965), Levene (1973), and Barrow et al. (1974). Lathyrogens affect the metabolism either by increasing the solubility of collagen (Smith and Shuster, 1962; Tanzer and Hunt, 1964), or by causing defective synthesis of tropocollagen a chains (Martin et al., 1963), which are incapable of forming inter- and intramolecular cross-linkages (Stelder and Stegemann, 1962; Tanzer, 1965). Gallop (1964) espoused the view that lathyrogens may react with COOH groups of the mucopolysaccharide moiety, and hence prevent them from cross-linking. Later studies have indicated that the a chains of collagen from lathyritic animals are normal and potentially capable of cross-linking, but in the presence of a lathyrogen, the E-NH, groups of lysine residues, which are involved in the formation of interchain bonds, are not oxidatively deaminated. In other words, lathyrogens specifically inhibit lysyl oxidase by irreversibly binding with enzyme (Narayanan et al., 1972; Gallop and Paz, 1975). Consequently, cross-links do not form (Page and Benditt, 1967; Miller and Matukas, 1969, 1974; Trelstad et al., 1970; Levene et al., 1972). It has been anticipated that lathyrogens block the 0-glycosidic linkage between the reducing group of hexoses and the peptide chains, and suppress the synthesis of desmosine and isodesmosine from lysine. However, Edvin (1971) was granted a patent in which he claimed to have achieved increased solubility of muscle collagen from old cattle by administering a lathyrogen and hence produced tender meat. If so, the above proposal on the mechanism of lathyrogen’s action do not explain this effect. Again, whether or not a lathyrogen causes any interference in the synthesis of mucopolysaccharide has been a controversial point. Ross’s (1968) review of the literature on this aspect indicated variable findings. Some studies reported inhibition of [35S]sulfate incorporation into tissue, whereas others have shown increased uptake or little effect by administering lathyrogens. Barrow et al. (1974), in their review, differentiated between two types of lathyrisms, namely, neurolathyrism and osteolathyrism. The former occurs in animals and man, while the latter is specific only for rats and turkeys.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

28 1

6. Thiolism Like lathyric compounds, p ,P-dimethylcysteine (penicillamine) was found to decrease the strength and increase solubility of skin collagen when administered to rats (Nimni and Bavetta, 1965; Nimni, 1968; Nimni et al., 1972; Deshmukh et al., 1971). Other thiol compounds, such as cysteamine, produced similar effects on administration to rats (Dasler and Milliser, 1958; Harris e f al., 1974; Siegel, 1977). Harkness and Harkness (1966) also reported a marked decrease (to 10%) in the tensile strength of skin collagen (at pH 7.0-7.5) by thiol compounds even at lo-, M concentration. All of these compounds possibly act as irreversible lysyl oxidase inhibitors (Siegel, 1979). The thiol compounds have been divided into two groups with regard to their effect on collagen (Nimni and Harkness, 1968). The first group of compounds has an NH, group adjacent to the SH group (e.g., cysteine, cysteamine, penicillamine) and is more effective in decreasing the tensile strength, possibly by shifting the pH toward the more alkaline range (> 9.5). The second group of compounds has either no NH, group or a blocked NH, group (e.g., N-acetylpenicillamine, glutathione). These thiol compounds, contrary to those of the first group, tend to increase the tensile strength of collagen above pH 8.0. Halogen produces similar effects (Harkness and Harkness, 1965; Nimni and Harkness, 1968).

7 . Chronological Age Hall (1976) has discussed in detail the macrostructural, microstructural, chemical, and biochemical changes that occur in connective tissue as a function of chronological and biological age. Age-associated changes in the physicochemical stability of collagen have also been treated at length by a number of authors (Jackson and Bentley, 1960; Gross, 1961; Verzar, 1964; Bakerman, 1964; Sinex, 1968; Gutmann, 1977; Selmanowitz et al., 1977; Wada et al., 1980). There is agreement that aggregation and cross-linkages in collagen continue to increase with age, and eventually reach a point at which they become incompatible with normal physiological functions. Thus old collagen is tougher and less hydrated and has thicker and denser fibers than newly synthesized collagen (Gross, 1961). According to Sopata et al. (1974), stable cross-linkages form with time and insolubility results in about 3 weeks after the formation of tropocollagen units. Many other studies have reported decrease in solubility and increase in crosslinkages in collagen with age (Hill, 1966; Herring et al., 1967; Bailey, 1968; Shimokomaki et al., 1972; McClain, 1971; Bailey and Shimokomaki, 1971; Dutson, 1974).

282

A . ASGHAR A N D R. L. HENRICKSON

The quantity of hydroxyproline released (labile collagen) from tendon into Ringer’s solution on heating to 65°C for 50 min was reported to decline with an increase in biological age (Verzar, 1964). Similarly, Go11 et al. (1963, 1964a,b) found 42% labile intramuscular collagen in a 50-day-old calf, and only 2% in a 10-year-old bovine, and attributed this difference to the number and strength of cross-linkages in collagen with age. These observations were substantiated by many other workers (Vognarovi et al., 1968; Wada et al., 1980). A significant correlation between labile collagen and toughness of meat within ovine (Smith et al., 1968) and bovine maturity groups (Field et al., 1970a) suggested that differences in cross-linkage exist even within animals of the same maturity. The differential thermal analysis study by Field et al. (1970b) on hydrothermal shrinkage of collagen from L. dorsi and bicep femoris from the same animals indicated differences in collagen structure between muscles. These observations possibly can now be explained in terms of recently discovered different types of collagens (Section II,A,2). Divergent views have also been expressed concerning the changes in collagen content (in muscle) as a function of age. Some researchers recorded an increase in collagen content with increasing age (Kim et al., 1967; Hunsley et al., 1971; Nakamura et al., 1975), whereas others observed little relation between age and the amount of connective tissue in muscle (Kauffman et al., 1964; Reagan et al., 1976) or in dermis (Hall et al., 1974). Still other studies showed decreasing trends in the amount of collagen in muscle with age, or the amount tended to become constant after the animal attained maturity (Wilson, et al., 1954; Go11 et al., 1963; Hill, 1966; Kurosu, 1979). Gallop’s (1964) proposition was once regarded as a possible explanation for the origin of the cross-linkages during aging. It has been stated herein (Section 111,D,2) that a subunits were believed to be linked through a pair of P-aspartyl ester bonds. During aging, the formation of P and higher aggregates from a chains, according to Gallop’s hypothesis, involved the rearrangement of the paired P-aspartyl ester bonds to link adjacent a chains so as to form inter- as well as intramolecular linkages. Although the review of literature presented by Harding (1965) strengthened this view, it has not been proven experimentally (Sinex, 1968). Some workers (Deasy, 1962; LaBella and Paul, 1965) have suggested the participation of condensation products of tyrosine-oxidized residues in the formation of new cross-linkages. However, Sinex (1968) has questioned the experimental approach used to derive such conclusions. More recent studies have shown that differences in cross-linkages do exist in insoluble collagen derived from various tissues (Bailey, 1968, 1970; Kang, 1972; Kang et al., 1970; Barnes et al., 1974; Robins and Bailey, 1973a; Bailey et al., 1969, 1970, 1973). Some of these cross-linkages (for example, dehydrodihydroxy-lysinonorleucine)are acetic acid-labile, whereas others (such as hy-

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

283

droxylysine-5-ketonorleucineand glucosylamine) are acid-stable. The prevailing view is that the number of acid-labile cross-linkages decreases, whereas the number of acid-stable bonds increases with age in collagen from all tissues (Davison et al., 1972; Bailey et al., 1974). It is also of interest to note that the extent of hydroxylation of lysine, in newly synthesized type I collagen, decreases and that of proline remains constant with age of the animal (Barnes et al., 1974, 1976). The lysine residues in the N-terminal regions of al(1) and a 2 chains, which have a special role in forming intermolecular cross-linkages and hence stabilizing extracellular fibrils (Tanzer, 1973), are hydroxylated to the extent of 50% in embryonic tissues. However, a failure of hydroxylation at these sites occurs in skin collagen (Barnes et al., 1974). Hydroxylation of lysine in other regions of collagen molecules is not affected. Perhaps the changes in the relative distribution of types I and 111 collagen in dermis with age are significant. Both these types are present in an about equal ratio in fetal dermis, but the proportion of type 111 falls to 15-20% of the total collagen with advance in age (Epstein, 1974). If so, then it may involve turning off the particular gene which is responsible for the development of the mRNA for synthesizing type 111 collagen. The findings seem to be contradictory as to the nature of changes with age in the carbohydrate content associated with collagen. Hormann (1965) reported a decrease in carbohydrate concentration, whereas Joseph and Bose (1962) reported an increase with age. The presence of hexosyl-lysine and hexosyl-hydroxylysine in aged tissue has been taken as indicative of linking of glycoprotein to collagen. This suggests another type of intermolecular cross-link which is age dependent and binds the collagen fibrils with glycoprotein molecules that envelop these fibrils (Balazs, 1977). Among the changes in mucopolysaccharides with maturity of collagen, hyaluronic acid content decreases accompanied by an increase in sulfated glycosaminoglycans, especially chondroitin sulfate B (Loewi and Meyer, 1958; Muir, 1964) with a higher charge density (Mathews, 1964), and to some extent heparitin sulfate and keratosulfates (Kaplan and Meyer, 1960; Meyer et al., 1965). It is thought that the substitution of more viscous hyaluronic acid with less viscous sulfated mucopolysaccharides may favor thermal motion of collagen for interaction. It is also likely that chondroitin sulfate, which is very highly charged (Balazs, 1970a,b), may be more shielded by its own associated protein moiety, preventing interaction with collagen. Age-associated changes in reducible components of bovine collagen have been reported by Robins and Bailey (1973a), whereas Mathews (1973) discussed the changes in glycosaminoglycan, indicating that the nonsulfated fraction increases, the sulfated chondrotin fraction decreases, and the ratio of heparitin sulfate to chondroitin sulfate increases with age. According to Heikkinen (1973), all of those factors which determine the particular metabolic activities of the fibroblasts such as oxygen consumption, the enzymic activity of the citric acid cycle,

284

A. ASGHAR AND R. L. HENRICKSON

glycolysis, and pentose phosphate shunt, together with those involved in synthesis of collagen, are depressed in old age. B . POSTMORTEM FACTORS 1. Aging (Ripening)

A review by Asghar and Yeates (1978) shows that many attempts have been made to identify the nature of the postmortem changes in connective tissue (collagen). Those who followed changes in alkali-insoluble content of meat from different animals during aging, failed to identify any change (Wierbicki et al., 1954; Khan and Van den Berge, 1964; Herring et al., 1967; Davey and Gilbert, 1968). Sharp’s (1963) study on collagen based on hydroxyproline estimations did not find any change in beef muscle connective tissue aged for 172 hr at 37°C under aseptic conditions. McClain et al. (1970) noted a decrease in the solubility of collagen from aged bovine muscle. Other workers (Goll, 1965; Asghar, 1969) indicated that subtle conformational changes in collagen molecules on swelling under the influence of postmortem lactic acid accumulation may not necessarily affect the solubility characteristics of collagen. Sensitive procedures such as thermal shrinkage, electrophoresis, and susceptibility to collagenase digestion may well detect postmortem changes in collagen. By following such approaches many studies have demonstrated alteration in the collagen structure during aging of meat (Field et al., 1970c; Kruggel and Field, 1971; Pfeiffer et al., 1972). Although Asghar and Yeates (1978) indicated that “acid-labile’’ bonds might be cleaved by the action of lactic acid on collagen during the ripening process of meat,‘the exact chemical nature of those cross-links still remains to be elucidated. Some studies also reported an increase in a components of collagen during aging due to cleavage of cross-links (Kruggle and Field, 1971; Wu, 1978). The study by Wu (1978) indicated that collagen type I is affected more during aging than type I11 collagen. Divergent results have also been reported on whether or not the mechanical properties of collagen change with changes in pH. Although some workers found little effect on mechanical strength in the pH ranges 4-11 (Hall, 1951) or 5-8 (Partington and Wood, 1963), others reported a considerable drop in the strength of collagen in the pH ranges 7-4 and 10-12 (Harkness and Harkness, 1965). Harkness (1968) has further stated that at pH 6.0, the collagen strength was only 40% of the value at pH 7.5 and only about 20% at pH 5.0. Winegarden (1950) also found that strips of collagenic tissue, aged for 35 days, exhibited a slightly but consistently smaller shear force value than strips aged for only 10 days.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

285

2 . Action of Lysosomal Enzymes The earlier discussion (Section IV,B) has indicated that native collagen is resistant to proteolytic enzyme changes. However, denatured collagen can be hydrolyzed to low-molecular-weight components by a number of nonspecific proteolytic enzymes. In this regard lysosomal enzymes are believed to hydrolyze collagen which has been denatured first by lactic acid during aging. Go11 (1965) has reported the presence of collagenase in lysosomes which play a part in this process. This enzyme has the ability to attack the helical region of native molecules at physiological pH (Gross, 1970). Later studies have shown that cathepsin B, (a thiol-dependent lysosomal protease) can also degrade nonhelical regions of native collagen in the acidic pH range (Morrison et al., 1973; Burleigh et al., 1974; Etherington, 1976, 1977), whereas cathepsin G (Barrett, 1974) and cathepsin D (Parrish and Bailey, 1967; Dingle et a l . , 1971) accelerate the degradation of collagen and proteoglycan. Other lysosornal enzymes which affect mucopolysaccharides may also play a part (Canonico and Bird, 1970; Ono, 1970; Dutson and Lawrie, 1974). 3. Effect of Heating

Heating causes some protein denaturation whereby the noncovalent bonds stabilizing the quaternary, tertiary, and secondary structures of a protein are broken, and the highly organized macromolecules are distorted by the intense thermal collisions, the so-called Brownian motion. The word “denaturation” has been used in different contexts in the past to denote the changes in protein chemistry without any precise definition (Joly, 1955; Colvin, 1964). Presently, its use has been restricted to indicate only the alterations in the secondary or tertiary structure of polymers caused by any process (Kauzmann, 1956). According to Jirgensons and Straumanis (1962), the coiled peptides become unfolded and the secondary bonds are loosened in the process of protein denaturation. These changes alter the properties of protein such as viscosity, optical rotation, X-ray pattern, chemical reactivity, and biological activity, including the exposure of SH groups and changes in shape of the molecule. Changes on the surface of the protein molecules, such as deamination or salt formation, are not involved in denaturation but electrostatic bonds are effected. The original conformation of the molecule remains intact. The coagulation or peptization may take place as secondary processes following denaturation (Ballou et al., 1944). When heated in an aqueous medium, the collagen fibrils shrink. During this phase a part of the solvated water is lost (Fessler, 1965), possibly due to an increase of the hydrophobic interactions. Since the heat of adsorption of water decreases rapidly with increase in temperature (0-60”C), the adsorption of water

286

A . ASGHAR AND R. L. HENRICKSON

should decrease with increasing temperature (Wollenberg, 1952). As the temperature rises above the melting temperature (T,) of the crystalline regions, cohesive forces maintaining the orderly structure are weakened and the superhelix of collagen molecules collapses. The fibrous state of protein is labile from a thermodynamic viewpoint, and the globular form with random order (nonhelical) of chains is the stable configuration of a protein (Mirsky and Pauling, 1936). Thus, the inherent contractile tendency of the fibrils due to increased entropy results in a less orderly arrangement. Consequently, the transformation of collagen molecules into mixed random coiled components starts with continuing hydrothermal heating. The resulting product is a gelatin (von Hippel, 1967). According to Engel (1962) the denaturation of collagen proceeds in two stages. The helical structure is destroyed rapidly on heating, but separation of the chains takes more time. The chains can also be separated in part by warming the tropocollagen solution at pH 4.0 (Orekhovitch, 1958). During the conversion of collagen to gelatin the amount of hydroxyproline in gelatin is inversely related to the maturity (age) of the collagen (Verzar, 1963, 1964; Gross, 1964b). The composition of gelatin is also influenced by some pretreatment of collagen (Gustavson, 1956). For instance, amide groups are little affected in acid-processed collagen, but alkali treatment destroys a significant amount of amide groups and salt linkages. The gelatin formed comprises fragments that have widely different molecular weights ranging from as high as 150,000 to as low as 10,000 (Pouradier and Venet, 1950). Veis and Drake (1963) had observed that gelatin is composed of such p aggregates as pI3, pI2, and p32.On the other hand, Worrall (1965) reported that salt- and acid-soluble collagen on heating at 37-60°C for 15 min gave rise to a and p components consisting of al,1x2, p, and p2 types. The thermal stability of collagen is considered to be directly proportional to the sum of proline and hydroxyproline (Gustavson, 1956; Harrington, 1964; Josse and Harrington, 1964; von Hippel, 1967; Piez, 1968). Rigby (1967) has indicated some inverse correlation between the thermal stability and serine content of collagen. Bailey and Lister (1968) attempted to identify the thermally labile cross-links in collagen. The difference in the hydrothermal stability of sheep skin and bovine collagen is evident from shrinkage temperatures of 60 and 65"C, respectively. They require extensive acid or alkaline pretreatment and application of heat for gelatinization (Gustavson, 1956). The zone of maximum hydrothermal stability is in the pH range 5-7. At high concentrations of H + or OH-, the T, of collagen is lowered. Low concentrations of neutral monovalent salts (0.1 M ) decrease the enthalpy of activation for denaturation of collagen molecules, and weaken the electrostatic bonds which are labile in aqueous salt solutions. However, divalent cations increase collagen stability (Adzet et al., 1979).

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

287

4 . Effect of Cooking Methods

A detailed account of the changes that occur in intramuscular connective tissue (collagen) during the cooking of meat has already been presented in an earlier review by Asghar and Pearson (1980). Various experimental evidence suggests that interstitial collagen partly dissolves during cooking. The extent of dissolution depends on the methods of cooking (roasting, broiling, deep-fat frying, pressure or microwave cooking), the duration of cooking, the internal temperature reached, and the maturity of the animal (age), which determines the extent of cross-linking in the collagen. Bayne et al. (1971) observed that only the alkaliinsoluble collagen decreased during cooking of meat, whereas the salt-soluble fraction remained unaffected. Several scanning electron microscopic studies have revealed progressive denaturation (coagulation) of collagen fibers with increased internal temperature from 50 to 90°C during cooking of meat (Cheng and Parrish, 1976; Jones et ul., 1977; Leander et ul., 1980). Extensive literature is available on the influence of heat processing on food protein quality in general (Altschul, 1958); however, little information has been reported with reference to collagen. Mauron (1972), on reviewing the effects of industrial and domestic processing on food protein quality, concluded that the nutritive value of protein is often improved by moderate heating. Intensive heating causes impairment and reduces the enzymic release of amino acids, especially in foods low in carbohydrates (e.g., meat). The presence of reducing sugars and other aldehydes and autoxidizing fat greatly contributes to heat deterioration of protein, whereas high water content reduces the incidence of heat damage.

VI.

FUNCTIONAL PROPERTIES OF COLLAGEN IN FOOD SYSTEMS

Proteins have no parallel in their structural and textural versatilities. Although nature has designed proteins to perform specific roles in situ, they can display multifunctional properties by appropriate manipulations and processing treatments in different food systems. The functional properties depend on such intrinsic physicochemical characteristics of proteins as amino acid composition and sequence, molecular weight, conformation, and charge distribution on the molecules. The nature and charge density facilitate interactions with other food components such as water, ions, lipids, carbohydrates, vitamins, color, and flavor constituents depending upon the environment (pH, ionic strength, temperature) during preparation, processing, and storage. The functional properties are important for the organoleptic quality of the ultimate product. Different workers (Her-

288

A. ASGHAR AND R. L. HENRICKSON

mansson, 1975; Kinsella, 1976, 1979; Wolf, 1970; Chou and Morr, 1979; Shen and Morr, 1979) have treated these aspects in detail. Though the physicochemical bases of some functional characteristics of proteins are little understood, proteins are not generally functional in the absence of an aqueous phase. Hence, hydration is the first and most critical step in imparting other desirable functional properties (Circle and Smith, 1972), such as swelling, gelatin, solubility, viscosity, wettability, emulsification, cohesion, adhesion, elasticity, and foaming in a food system. These properties of a protein are directly related to the manner in which the protein interacts with water in the product. Thus, the nature of the protein-water interactions in general, and collagen-water interactions in particular, are considered.

A.

WATER BINDING

It is appropriate to consider first the fundamental principles governing the interaction of water molecules with other compounds and protein hydration in general. Water molecules have a unique three-dimensional geometrical structure due to hybridization of two lone pairs of valence electrons in 2s and 2p atomic orbitals of oxygen. The two electron pairs forming the covalent bond are attracted by the nuclei of oxygen and hydrogen, and the other two lone pairs of electrons are attracted only by the oxygen nucleus (Franks, 1975a,b). Thus, the water molecules possess dipolar characteristics due to asymmetric distribution of electrons. The dipolar nature of water molecules facilitates interaction not only with electrolytes carrying positive and/or negative charges, but also with strong electronegative atoms such as nitrogen and oxygen present in different functional groups in the components of a food system. The dipolar character and orientation of water molecules are also responsible for the very high dielectric constant, which lowers the electrostatic attraction between charged ions by forming stable hydrated shells around them. Natural water is composed of H,160 molecules, with small amounts of H,l80, H,I7O, and HDO. According 'to Pauling (1940), four resonance structures of water molecules are possible, of which the following three dominate: H

..0: :..

H+ :0:H-

..

H:O:

..

H'

H

Biophysically, the protein holds water in two forms. One is called the bound, structural, or protective form and the other the free or biologically active form (Hamm, 1975; Fennema, 1976). The bound fraction (0.15-0.28 g/g protein) is firmly held as water of hydration by functional groups of the protein in the form of mono- and multimolecular layers, having ice-like structure (Wismer-Ped-

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

289

ersen, 1971). There is wide disparity among different workers regarding the amount of bound water associated with various functional groups. For example, according to Pauling (19454,each polar group on a protein molecule binds one H,O molecule with the exception of those C O O H groups which are hydrogenbonded to an amide group of glutamine or asparagine. Bull and Breese (1968) reported the binding of six H,O molecules per polar group. Speakman (1944) found the extent of water binding to be in the descending order of polar side chains, amino groups, carboxyl groups, and hydroxyl groups, followed by peptide linkages at intermediate activity of water and finally by the formation of multilayers at higher activity of water. Kanagy (1950)stated that hydration of a protein occurs in steps of various energy content, as hydration involves functional groups of different degrees of strength and reactivity. The deuteron magnetic relaxation study on biopolymers by Glasel (1970)showed that uncharged carboxyl and amide groups interact strongly and imide groups weakly, whereas hydrophobic groups interact little with water. But, Karmas and DiMarco (1970)proposed the involvement of nonpolar amino acid residues in the binding of water. On the other hand, Bull and Breese (1968)associated water binding with the sum of the polar amino acid residues minus the amide groups as expressed by the following equation:

Y

=

-0.97 x lop3 + 6.77X, - 7.63X2,

where Y is moles of bound water per gram of protein at 25°C (RH, 0.92),X is the sum of moles of acidic, basic, and hydroxyl groups per gram of protein, and X, is the sum of moles of amide group per gram of protein. A more comprehensive assessment of the water-binding capacity of different amino acids has been made by Kuntz (1971,1975), based on nuclear magnetic resonance (NMR) studies. Table IV presents the data along with the pK values, isoelectric points (pl), and structure of different amino acids. It shows that the water-binding capacity varies with the nature of the amino acids and the charge at different pH values. Generally, cationic (lysine, arginine, histidine) and anionic (aspartic and glutamic acid) amino acids bind the highest amount of water followed by neutral ones, whereas hydrophobic amino acids bind little water (Kuntz and Kausman, 1974). Kuntz (1975)derived the following equation to estimate the extent of bound water on the basis of the nature of side residues of amino acid in a protein:

where A is grams of bound water per gram of protein;f,,f,, andf, are fractions of charged, polar, and nonpolar amino acid residues, respectively. So far as the free water fraction is concerned, it exists in an ordered form

290

A. ASGHAR AND R. L. HENRICKSON

TABLE IV HYDRATION CAPACITY OF AMINO ACIDS AS DETERMINED BY MAGNETIC RESONANCE STUDIE OF POLYPEPTIDES AND pK VALUE OF AMINO ACIDS<’

PK Amino acid

Symbol

Structure

a-COOH a-NH2 Side group

PI

Hydratio (moles H20/mo amino aci

Ionic A. Negatively charged form Aspartic acid

H

0

\

Asp

p

~

H

I

C-COOI

,

0

Glu

3.65

2.8

6.0

2.19

9.67

4.25

3.2

7.0

9.11

10.07

5.7

1.5

2.18

9.09

13.20

10.9

3.0

1.78

8.97

5.91

1.6

4.0

2.20

8.90

10.28

9.1

4.5

1.88

9.60

3.65

2.8

2.0

9.67

4.25

3.2

2.0

9.11

10.07

5.7

3.0

H

\

~

9.60

NH,

0

Glutamic acid

1.88

I .c--coo-

//C-cH2-cHA

I

0

F 3

Tyrosine

B. Positively charged form Arginine

Tyr -

IH

Arg

+

I I H,N-C-NH-(CH,),+C-COO1 I ( 1

I NH, I+

F 2

IH

Histidine

Lysine

His

+

Lys +

’ I HC=C-CH,+C-COOI H+NH

I

H

+

f I

jNH, ‘H I I

-(cH,),+c-coo-

H,N-CH,

1 1 N H 3

;

C. Neutral or uncharged form Aspartic acid

ASP

Glutamic acid

Glu

Tyrosine

HO

TYr

! H

‘C-CH,+A-COOI I 0 j NH,

4

HO jH I I \C-CH,-CH,tC-COO-

4 0

2.19

$ 1 INH3

29 1

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

TABLE IV (Continued)

PK iino acid

Symbol

4rginine

Arg

,ysine

LYS

Structure

a-COOH

IH : I H,N-C-NH-(CH,),tC-COI1 Dl NH I NH, I+ / H

# I H,N-cH,-(cH,),+c-cooI

(r-NH2

Hydration (moles HzO/mole Side group pl amino acid)

2.18

9.09

13.20

10.9

3.0

2.20

8.90

10.28

9.7

4.5

1.71

0.78

8.33

5.0

1.0

2'02

8.80

-

5.4

2.0

2.17

9.13

-

5.7

2.0

2.21

9.15

-

5.7

2.0

2.16

9.12

-

5.6

2.0

1.82

9.65

-

5.8

4.0

'

NH,

I

Polar or hydrophilic iH

Zysteine

HS- CH,

CYS

$ 1

Asn

C '-

4

COO-

I l

j H P

Asparagine

+c y

3

IH I1 CH,? C --COO'

# I

0

I NH, It

HZN,

3lutamine

Gln

!H I I -C-CH~-CH,fC-COO-

4

' I : NH,

0

:+

Serine

Ser

IH ' I HO--CH,+C--COOI

jH y3 OH; H

Threonine

Thr

I II

CH,-C+C--COO-

I II

H

;y3

H

H ydroxyproline

Pro-OH

I

HO-C4-,CHZ I H,+H--coo-

I H

(continued)

292

A. ASCHAR AND R. L. HENRICKSON TABLE IV (Continued)

PK Amino acid

Symbol

Structure

a-COOH

a-NH:,

Hydration (moles HzO/mole Side group pl amino acid

Nonpolar or hydrophobic

9.13

-

5.5

0.0

2.34

9.60

-

6.0

1.0

2.32

9.62

-

6.0

1.0

2.36

9.60

-

6.0

1.0

2.26

9.62

-

5.9

1.0

2.28

9.21

-

5.7

1.0

2.34

9.69

-

6.0

1.5

9.39

-

5.9

2.0

Phenylalanine

IH

I I

Clycine

HfC-COO-

I +

i7

H3C\ Valine

Val

/

CH’C-COO-

H3C H3C, Leucine

Isolucine

Leu

; M I I3 !H

CH-CH / H3C

I

1

zTC-COOI I

: NH3 IH I I

CH,-CH,-CH+C I I CH3I

Ile

-COO-

1

MI,

IH Methionine

Met

I i

CH,-S-

CH2-CH2+C‘ I

j

I

Alanine

COO-

y 3

IH ! I CH3+ C -COOI

Ala

i NH, I +

Tryptophan

!H

Trp -ooc3g-H 2c;@ f

: I

2.38

I

H (continue

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

293

TABLE IV (Continued)

PK nino acid

Symbol

Sttucture

a-COOH

a-NH2

Hydration (moles HzO/rnole Side group pl amino acid)

OHydration values are taken from Kuntz (1971). Values were determined in the pH range 6 8 at -35”C,except that ilues for uncharged Asp and Glu were measured or extrapolated at pH 4.0, for uncharged Arg at pH 10.0, for charged Lys at pH I@-11, and for charged Tyr at 12.0. The pK and p1 values are taken from Nivard and Tesser 965).

(because of H,O-H,O molecule interaction) with either restricted motional freedom (Ling and Walton, 1976; Ling, 1972) or freely mobile (Cooke and Wien, 1973; Cooke and Kuntz, 1974). This fraction is greatly affected by the spatial structure of the protein, changes in electrostatic forces of the ionic groups, and hydrogen bonding (Hamm, 1975). Changes in the pH value and ionic strength of the medium affect the conformational state of a protein and hence influence the binding sites, making them sterically available or unavailable for interaction with water. The transition of a protein molecule from compact globular to a random coil conformation exposes buried side groups for water binding. However, those proteins which are closely packed, strongly H-bonded, and devoid of ionic groups (e.g., silk fibroin) or stabilized by disulfide linkage (e.g., keratin) are resistant to hydration (Leeder and Watt, 1965). Some of the important biophysical factors governing the binding of “free” water by proteins will now be presented.

I. Effect of H + and OH- Ions A variety of chemical bonds and other forces determine the spatial configuration and conformation of proteins (Jones, 1964). Table V provides a summary of the structural forces, interacting groups constituting various chemical bonds, their energies, and interacting distance. The changes in protein conformation, that is, unfolding of helical structure, can be considered in terms of free energy of these forces by the following equation (Scheraga, 1963):

TABLE V STRUCTURAL FORCES IN PROTEINSU

Mechanism Covalent bond

Electron sharing

Energy (kcalimole) 3& 100

Distances of interaction

Interacting groups

(A) 1-2

C-C,

C-N,

Example

C=O, C-H

C-N-C

s--s Ionic bond

Coulomb attraction between charged groups of opposite sign

1&20

2-3

-NH,+

-c=m I I

-

P

C<\

+

NHz

HN-. f--NH

'.$!--

Hydrogen bond

Hydrogen shared between two electronegative atoms

2-10

2-3

H N-H.. . o=c

on.. . NH;..; NH...;

Van der Waals attractive force Electrostatic repulsive force Van der Waals repulsive forces

Mutual induction of dipole mo- 1-3 ments in electrically apolar groups Coulomb repulsion between 4142/r2 charged groups of like sign llrl2 Repulsion between apolar groups in close proximity

-coo-

3-5

oFrom Jones (1964), courtesy of the AVI Publishing Company, Inc., Westport, Connecticut.

NH;..;

coo-. ..

Intraresidue bonds Peptide Disulfide a-Amino group Lysine Arginine Histidine Aspartic glutamic a-Carboxyl group Amide-carbonyl group Serine, threonine, tyrosine Polar side chains of residues

Apolar groups

Apolar side chains

Polar groups of like sign

Polar side chains

Steric hindrance between side chain groups

All groups

295

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

is the standard free energy of unfolding of the protein structure, and where WUnf Fob, F", pH, FRO, Ecomb, and FElectrepresent the free energy of unfolding of the helical backbone, of covalent cross-linkages, of H bonding, of hydrophobic bonding, of combination of randomly coiled chain with solvent, and of electrostatic bonding, respectively. According to Scheraga (1963), the quantities FE and F" in the above equation are independent of pH because they do not involve ionizable groups. The remaining parameters in the equation are highly pH dependent. For example, if the donor and acceptor groups in a side chain hydrogen bond are ionizable, then the bond will exist only in the pH range where the donor has a proton and the acceptor lacks one. Beyond this pH range, FR will be zero. These facts point out what type of effect can be expected by changing the H + or OH- ion concentrations in the dispersed phase of a protein. As most proteins behave as lyophilic (hydrophilic) polyelectrolyte systems, the forces of solvation and adsorption play an important part at their surface. This can be visualized with reference to electrokinetic phenomena and the amphoteric nature of the protein as follows: A- t

,*

,NH+ COOH

acid PH

< PI

Cationic form

NH:
pI=pH (2=0)

alkali

+

C+

+

HOH

Anionic form

where A, C, and Z. represent anion, cation, and net charge, respectively. As the net charge on a protein molecule is zero at the isoelectric point (PI) due to the formation of an inner salt (zwitterion), other biophysical properties such as the hydration, solubility, electrophoretic migration, viscosity, swelling, conductivity, optical rotation, and osmotic pressure are also minimum, whereas sedimentation velocity, light scattering (Tyndall effect), surface tension, gel strength, and sensitivity to alcohol are maximum (Hartman, 1947; Jirgensons and Straumanis, 1962). Once the dipolar character of the protein is destroyed, ionic reactions (electrovalent) are created, depending upon the structure of the protein and the charge distribution. As can be visualized from the pK values and isoelectric points of different amino acids (Table IV), the ionization of different functional groups takes place in the following order: a-COOH groups of aspartic and glutamic acid residues at pH 3.6-3.8; the p-COOH group of aspartic acid at pH 4.5; the y-COOH group of glutamic acid at pH 4.6; the imidazol group of histidine at pH 6.1-6.3; a-NH, groups at pH 7.5-7.7; the SH group of cysteine at pH 9.1; the phenolic group of tyrosine at pH 9.6-9.8; the E-NH group of lysine at pH 10.4; the guanidyl group of arginine at pH above 12 (Tanford, 1962). Being a protein, collagen is also influenced by H f and OH- concentration. The acidic and basic functional groups govern many of the physical properties of

296

A. ASGHAR AND R. L. HENRICKSON

the collagen fiber as well as determine the reactivity of collagen to acids, bases, and ionic reagents in general. Collagen contains a fair amount of ionic groups, which in the isoelectric zone (pH 7.2) will be charged and form maximum internal salt linkages. The increase in H+ or OH- concentration breaks the internal compensation of ionic side chains and salt links, increasing the net positive or negative charge, respectively. However, the charged sites of the collagen chains do not have the freedom of action as found in the case of soluble amphoteric electrolytes (Gustavson, 1956). Hence collagen binds very small amounts of acid or alkali as compared to soluble protein in the absence of neutral salts. Nevertheless, by increasing H or OH- concentration, the electrostatic repulsion leads to the development of a Donnan membrane potential inside the fibers (Tolman and Steam, 1918). This favors the inflow of water into the fibers. These water molecules are held either electrostatically by charged polar groups or through hydrogen bonding by polar uncharged groups and electronegative atoms. In addition, keto-imide linkage +

-N-),

(-C

II

I

0

occurring in every fourth residue of the collagen backbone chain and having a free carbonyl group, can function more readily as coordinator of water than regular peptide linkage (-C-NH-).

I1

0

During hydration the structure of the fibers is distorted, their length and diameter increase (Tolman and Steam, 1918). Sonsler et al. (1940) found that at 15% water content, protein bound about 260 molecules of water and the distance between amino acid side chains increased from 10.4 to 11.3 A,whereas at 33% water content, the spacing increased to 13 A. Figure 13 shows the pH-dependent hydration curves of food-grade, freezedried collagen from cattle hide as influenced by mono-, di-, and trivalent anions. It appears that C1- and SO,2- tended to depress the hydration of collagen in the isoelectric pH range 5.0-7.0. Beyond these values, C1- increased the hydration sharply. On the contrary, pyrophosphate ions (P20,, - ) relatively increased hydration in the isoelectric zone of collagen (pH 5-7) as compared to C1- and SO,*-, but beyond pH 4 and 9, pyrophosphate did not increase hydration as C1- did. The changes in the hydration curve around pH 7.0 have been ascribed in part to the ionization of the imidazole group of histidine (Gustavson, 1956). Two types of hydration of collagen are recognized depending on the ionic atmosphere (Gustavson, 1956). The hydration of collagen due to ionic groups and their charges in acid or base is regarded as “osmotic swelling.” However, Schut ( 1976) considered such hydration to be different from the osmotic phenomenon. On the other hand, hydration caused by the interaction of ions of

CHARACTERISTICS O F COLLAGEN IN FOOD SYSTEMS

297

al OI

8

10.0

U

.=

F

3

2

c

9.5

.

9.0

c 0

E,

8.5

-a -E,

8.0

-0 -

7.5

I

L

al

C

8 % +

7.0

x f

6.5

m

.s 0

8 0

6.0

5.5

111111111111 2

3 4 5 6

7 8 9 101112

pH value

FIG. 13. Effect of mono-, di-, and polyvalent anions on the pH-dependent hydration curve of foodgrade, freeze-dried collagen from cattle hide. (A) Control (HCIINaOH); (B) 0.1 M NaCl (HClI NaOH); (C) 0. I M Na2S04 (H2S04/NaOH);(D) 0.1 M Na4P207 (H3P04/NaOH). From Asghar and Henrickson (unpublished data).

neutral salts or nonionic reagents with nonionic bonds (e.g., hydrogen bond) of collagen is described as “lyotropic hydration” or “swelling.” There are characteristic differences in the two types of swelling. Although the osmotic or electrostatic swelling results in great volume increase by dilute acid solutions, the process is reversible in contrast to the lyotropic swelling. The osmotic swelling is considered interprotofibrillar, and the integrity of the triplehelical structure of collagen remains intact. On the other hand, the lyotropic agents may alter the water structure around the collagen fibrils, interrupt the interprotofibrillar bonds and internal hydrogen bonds, or by direct binding at some sites interact with internal hydrophobic bonds. These interactions affect the interprotofibrillar structure, and hence irreversible changes may occur in the native peptide chain (Veis, 1964). However, within the fiber, the tropocollagen monomer units are not disrupted by neutral salts except that intertropocollagen bonds are influenced (Ramachandran, 1968).

298

A. ASGHAR AND R. L. HENRICKSON

2 . Effect of Weak and Strong Acids With regard to the effect of acids on the hydration of proteins, Loeb’s (1922) valency rule implies that the same degree of swelling of protein should be produced by isovalent acids in solutions equilibrated to identical pH values. However, acetic acid produces 50% more swelling and a greater degree of peptization of collagen than HC1 does at pH 2.0. It is assumed that the weak acid (e.g., acetic acid) at pH 2.0 allows the removal of protons from the carboxyl groups of collagen (Gustavson, 1956). However, there appears to be neither marked affinity of the anions of the weak acids for the cationic groups of collagen nor any significant effect of the nonionized acid molecules at pH > 2.0. By decreasing the pH of the system to below 2.0, the fixation of molecular acid increases sharply, presumably by forming H bonds with the =CO group of peptide linkages. This acid also produces permanent swelling, which persists even after bringing the pH back to the isoelectric point (pZ). The shrinkage temperature of acetic acid-treated collagen is 12°C lower than that of untreated collagen. Some deamination of collagen also occurs by treatment with 2-3 M acetic acid (Gustavson, 1956). Acetic acid produces both types of swelling since it involves both the electrostatic or osmotic effect and the lyotropic or Hofmeister effect (Gustavson, 1956). The latter effect in fact dominates; it is more in the nature of a specific molecular effect rather than a specific ionic effect, because it is the nonionized acid which acts as the swelling agent by competing with the peptide group involved in intermolecular linking of the protein chain. Acetic acid molecules are believed to rupture some of the hydrogen bonds and also to associate with free =CO group of the peptide linkage (Gustavson, 1956). At pH 4.5, irreversible changes also take place (Hamm, 1960). On the addition of HCl to collagen, some H + cations combine with -COOanions. Thus, the net positive charge on the molecules first increases with the number of discharged carboxyl groups as a function of H concentration. This continues until all -COO - groups have been discharged and all cationic groups are freed. As anions of monovalent strong acids do not have much attraction for the cationic groups of proteins, the anions are electrostatically compensated by the cationic groups on the protein. Consequently, the aqueous phase inside the collagen structure would contain more C1- than H . The differential distribution of the ions between two phases causes a difference in osmotic pressure and in electrical potential. Thus, to equalize the ion concentration in two phases, water flows into collagen molecules and causes swelling (Lloyd and Shore, 1938). The helical structure, however, can withstand the strong repulsive force of the fixed charge up to a degree of ionization of 50%. At higher ionization the helix breaks down and the polypeptide stretches (Katchalsky, 1964). With further increase in H concentration, the total positive charge on collagen becomes +

+

+

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

299

constant; however, the ion (H+ and C1-) concentration in the external phase increases. Accordingly, the excess of diffusible ions in the solid phase first increases to a maximum, which coincides with the pH of maximum swelling of collagen, then declines (Procter and Wilson, 1916). The same reasoning may also account for the fact that the swelling capacity of a dibasic acid (e.g., H,SO,) is only about half that of HC1 at identical pH value. The decline in hydration at very low pH (< 2.0) is explained by the assumption that excess anions from the added acid screen the positive charges (E-NH, , guanidine, and imidazol groups) of the protein and reduce the interpeptide chains’ electrostatic repulsions. Gustavson (1956) concluded that the lyotropic effects of carboxylic acids on collagen are intimately connected with the hydrogen bonding power of the molecules of the acid, and that breaking or weakening of interchain cross-links of hydrogen bonds is the main reaction leading to labilization of the chains. It should be emphasized that views regarding the fate of the acid anions in protein solutions in the acidic pH range are divergent. The proponents of the Donnan theory assume that there should not occur any combination of anions (e.g., Cl-) with cationic centers of the protein, and all the anions are regarded as free in the internal aqueous solution without any restraint (Bolam, 1932; Wilson, 1928). The contrary view, based on thermodynamic reasonings, does not regard the internal solution as a normal aqueous phase and regards all of the anions as associated with the positively charged centers of the protein (Gilbert and Rideal, 1944). Both of these concepts seem equally applicable to the reversible fixation of the anions with the protein, but Gustavson (1956) argued that none of them apply to the irreversible fixation of the anions by cationic protein groups. For example, polymetaphosphoric acid, which probably does not interact with hydrogen bonding groups, irreversibly binds with cationic groups. However, polymetaphosphoric acid may cause some phosphorylation (probably of peptide bonds) at lower pH on prolonged treatment, and may not be implied as irreversible fixation of the anions by cationic protein groups. +

3. Effect of Bases

In the case of a strong base (e.g., NaOH), the osmotic effect is pronounced and the lyotropic effect is minor, provided the treatment is not prolonged. On the other hand, the hydroxides of bivalent metals produce principally lyotropic swelling along with some osmotic effect at pH > 10, where irreversible alterations in ionic and coordinate bonds also occur. Prolonged action of Ca(OH), increases swelling, possibly due to two reactions. First, liberation of 4 0 0 groups by divalent bases occurs (monovalent bases are less effective) (Marriott, 1933) as a result of deamidation of the amide groups of asparagine and glutamine (Bowes and Kenten, 1948a,b). Some decomposition also occurs of the guanidyl

300

A. ASGHAR AND R . L. HENRICKSON

group of arginine to ornithine and urea (Highberger and Stecker, 1941), destruction of hydroxy amino acids (serine and threonine), and hydrolysis of keto-imide linkage (-C-N II 0

-)

I

between proline and hydroxyproline residues (Bowes et al., 1953) during alkali treatment of collagen at pH 12- 14. These changes account for the displacement of the isoelectric point of alkali-treated collagen toward a lower pH (-5.0) range. With these changes cleavage of interchain cross-links in which amide groups are involved increases the probability of the uptake of cations. Second, Ca2+ has a specific effect by forming complexes with OH- groups of hydroxy amino and residues. However, according to Lloyd and Shore (1938), the main reaction in the alkaline region (pH > 10) is the neutralization of cationic charge of the protein and electrostatic balance of the anionic groups by metal cations. In a collagen molecule the probability of getting two - C O O - groups by a Ca2 into its valency sphere is very limited. This scarcity is likely compensated by a weakly negatively charged enolized form of peptidyl bond present in the vicinity. This possibility is strengthened by the study of Carr (1953), who showed that out of 100 COOH groups of bovine serum albumin, only 8 participate in true complex formation with Ca2 . The other 92 groups form only loose combinations of the ordinary electrostatic type. The involvement of the enolized form of peptide bond with Ca2+ is likely to weaken the hydrogen bond. Since Ca(OH),, being a weak alkali, acts slowly, its lyotropic effect would only be observable after prolonged treatment. These changes are believed not to be a result of a Hofmeister-type molecular effect, but rather of a specific ion effect of an electrostatic nature and of the steric conditions of the collagen molecules with regard to the distance between adjacent COOH groups. Generally the swelling action of bases like NaOH and Ca(OH), follows the valency rule of Loeb (1922), provided the treatment is not prolonged. The maximum proton-accepting H + and OH- binding capacity of gelatin is about 0.9 meq/g protein at pH about 2.0 and 12.5, respectively. However, in collagen no maximum uptake of base is reached, presumably because of the guanidyl group involved in stable linkage, which requires still higher pH for its cleavage. +

+

4.

Effect of Various Salts

Before considering the effect of various salts on the hydration and stability of collagen molecules, it would be appropriate to first present a brief account of certain fundamental principles of biophysics which govern the charge and interactions among protein-ion-water systems. As indicated earlier, a protein in solution is regarded as a hydrophilic polyelectrolyte colloidal system. Other ions, if added to such a solution, will be distributed in certain orders and hence modify the electrical properties of the

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

301

Diffusedouble layer

, \

A

B

I

-

,

E

Fixed (Stern) laye!

Mobile layer

~. Distance from surface Of colloid parli~le

FIG. 14. Distribution of charge in the diffuse double layer (fixed and mobile layer) in the liquid phase around the negatively charged colloidal particle (A); and variation in electrical potential (Z) at the interface with increase in distance from surface to the particle (B). E , Nernst potential; <,zeta potential.

colloid. This was first realized by Helmholz (Kruyt, 1952), who proposed that the charges around the colloids are in two layers (Fig. 14). The inner layer adjacent to the colloid surface was referred to as the “fixed” or Stem layer, and the outer layer as “mobile.” However, several workers, quoted by Kruyt (1952) and Eagland (1975b), later proved that the electrical double layer in fact exists as a “diffuse” double layer, the outer one carrying both positive and negative charges. The density of the charge in the electrical double layers decreases from the surface of colloid to the periphery exponentially rather than linearly. The changes in charge density are expressed in terms of Nernst potential ( E ) and electrokinetic or zeta potential (5). The former refers to the total potential drop in charge from the surface of the colloid to the end of the diffuse layers, whereas the latter is confined to the potential drop across the mobile layer only (Hartman, 1947). Many factors can influence either the Nernst and zeta potential by changing the thickness of the electrical double layer, and hence affect the stability and hydration of the colloid particles. For example, thermal and electrostatic forces of the ions in liquid phase can change the distribution of charge in electrical double layers whose thickness is dependent on ion concentration, but independent of the nature of the ions (Jirgensons and Straumanis, 1962). However total drop in electrical potential ( E ) depends solely on the activity of ions and

302

A. ASGHAR AND R. L. HENRICKSON

H + concentration, whereas the zeta potential varies with the nature of the proteins, adsorption potential of ions at the interface, and dielectric constant of the medium (Kruyt, 1952). An increase in concentration of ions in the medium will decrease the thickness of ionic atmosphere composing the outer mobile layer of charges and hence the zeta potential. The value and the sign of zeta potential depend on the magnitude of specific adsorption of anions and cations. According to Friberg (1976), these concepts were further elaborated by Deryagin and Landau (1941) and Verwey and Overbeck (1948) in an attempt to explain the biophysical mechanism responsible for the stability of colloids. According to them, two forces operate on the colloidal particles. First, the van der Waals-London attractive forces which originate in the unsaturated valency fields at the surface between two particles; they decrease with increase of distance between the particles. These forces are responsible for adsorption. Second, the Coulomb electrostatic repulsion between electrical double layers of identical signs, whose intensity is determined by the structure of the ionic layer surrounding the particles, which, in turn, is determined by the electrolytic composition of the dispersed medium. This is known as the DLVO theory after the names of those workers (Eagland, 1975b; Friberg, 1976). Figure 15 depicts the salient feature of this theory, which suggests that if the repulsion potential ( B ) is greater than the attraction potential (A) at any distance

and the ions between lhem form the electric double layer which gives m e to o

j ~ l l e r e n lf r o m :he .on:~nwi med8um o VOP der A’ms

gz

-

i:

.“p

-

FIG. 15. Diagrammatic presentation of the DLVO theory which proposes that colloidal stability is distance dependent on two independent potentials, that is, van der Waal’s attraction potential and the repulsion potential. From Friberg (1976). Courtesy of Marcel Dekker Inc., New York.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

303

between the particles, the colloids will be stable. But, a low value of B , that is, an interparticle potential equal to or less than the height of energy barrier, would favor the precipitation of the particles as soon as they approach each other by the diffusion process (Friberg, 1976). However, other evidence suggests a possible involvement of hydration and solvent structure in the modification of the DLVO theory of colloid stability (Eagland, 1975b). The ions present in the medium with the colloidal systems are also important in relation to the hydration phenomenon. The ions, if added in a colloidal solution, may be bound with the charged sites or remain free and mobile in the aqueous phase, depending upon a number of factors such as pH, nature of the ions, and nature of the colloid particles. For example, metallic ions in solution are assumed to exist as aqua-complex ions in equilibrium with their respective hydroxo complex like a weak base (Furia, 1975), as follows: M(H2O)” Aqua-complex ion +

MOH(m-I)+ + H + Hydroxo complex (weak base)

If so, then the acid ionization constant (pK,) value of the aqua-complex ion would be a decisive factor in determining if the ion would form complexes or remain a free ion at different pH values of the medium (Basolo and Pearson, 1956). For instance, Ca2+, having a pK, value of >12.6, will bind to a negatively charged protein only under highly alkaline conditions, that is, at pH > 12.6. Another contributing factor to hydration is the ionic radius. The highly hydrated ions with large radii are apt to remain at a distance from the oppositely charged centers on protein, whereas less hydrated small-sized ions are able to approach the charged sites more closely and hence more effectively screen the charge on the protein. The ratio of ion radius to ion charge is inversely related to the degree of ion hydration (Fennema, 1976). The polarizing power (charge/ radius or simply the electrical field) of an ion has the ability to alter the net structure of water (Fennema, 1977). On this basis, ions have been classified into two groups. First, the small ions (e.g., L i + , N a + , OH-, HO,+) and multivalent cations (e.g., Ca2+, Mg2+, A13+), which have strong electrical fields, are called “water structure formers” because they increase the viscosity of water by binding strongly with four to six molecules of water adjacent to them. Second, the monovalent ions of large size (e.g., K + , NH4+, C1-, I-), on account of weak electrical fields, tend to reduce the viscosity of water. They are called “water structure breakers” (Sikorski et al., 1976; Fennema, 1976). The effect of added ions of varying valences on oppositely charged colloidal particles is shown hypothetically in Fig. 16. It shows that the addition of univalent anions to the solution of positively charged colloids increases the value of the zeta potential until a maximum is reached. Accordingly, hydration may also increase due to the screening effect of the corresponding cations, which settle in a compact layer close to charged protein groups. The anions are kept farther apart

304

A. ASGHAR AND R. L. HENRICKSON

+r

0

-r

FIG. 16. Hypothetical curves showing the effect of the addition of monovalent (curve a), divalent (curve b), and polyvalent (curve c) ions on the zeta potential of oppositely charged colloidal particles. After Hartman (1947).

from protein in the diffuse double layer. With further increase in ion concentration, the zeta potential approaches zero as the high concentration of ions compresses the double layer around carboxyl groups, and as the repulsion charge decreases, so does the hydration. The zeta potential decreases more rapidly and approaches zero on adding divalent anions. Contrary to this, the addition of polyvalent anions reverses the sign of the zeta potential to negative, and after reaching a peak, it declines to zero with further increase in the polyvalent anion concentration (Hartman, 1947). Similar behavior may be .expected of various cations on a negatively charged colloidal system. This substantiates Loeb’s (1922) statement that polyvalent ions, of opposite sign from that of the protein, have marked influence in reversing the sign of the zeta potential and changing the isoelectric point of the protein, whereas corresponding ions (of the same charge as the protein) are unimportant. As for other lyophilic colloids, the hydration and the stability of proteins in solution depend on two important factors. First, the electrical charge, which is due partly to the internal phase of the protein molecules and partly to the result of adsorbed ions from the medium; and second, the solvation of the molecules. Removal of these two stability factors, by changing H content or by adsorption of other ions and dehydration agents, respectively, leads to appreciation of protein. The relative difference in the adsorption of ions in relation to colloid stability has been explained in terms of the Hardy-Schulze rule, which says that the higher the valence, the greater the coagulating effect of added ions of op+

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

305

posite charge to that of the suspended colloids. That is, the order of adsorption of ions is trivalent > bivalent > monovalent (Hartman, 1947; Jirgensons and Straumanis, 1962). The DLVO theory presents a logical explanation for the destabilizing action of higher concentrations of the neutral salts (which do not absorb) which drops the potential faster with distance (Fig. 17). Although the total repulsion energy at very short distance is not affected, the drop in repulsion potential is appreciable at medium distance. Thus with the compression of the double layer the energy barrier disappears, leading to flocculation. The charge on the ions significantly affects the distance dependence of the potential. Consequently, electrolytes of higher charges are likely to be more effective in destabilizing emulsions (Friberg, 1976). Although this explanation supports the Hardy-Schulze rule, which states that divalent and trivalent ions, respectively, are 50-100 times more effective than monovalent ions in destabilizing an emulsion (Friberg, 1976), it does not have general applicability, because other factors such as specific characteristics of the ions, the colloidal system, and H + concentration can offset this rule. Bull and Breese (1970) also reported disparity in the behavior of anions and cations of the same size. For example, in contrast to cations, the larger the monovalent anion, the greater is the tendency to bind to the protein and the greater is its dehydrating effect upon the protein. The flocculation power also increases with the atomic weight or ionic radii in the lyotropic series (Jirgensons and Addition of salt changes the

The suface potential and w t h it l h

at particle touch

glvlq

Stoblllty

*

FIG. 17. Hypothetical curves showing the effect of added salts (electrolytes) on the potential charge of colloids. The addition of salts may affect the maxima of the total potential at uncharged surface potential of the colloids. From Friberg (1976). Courtesy of Marcel Dekker Inc., New York.

306

A. ASGHAR AND R. L. HENRICKSON

Straumanis, 1962). Besides these considerations, certain salts of weak acids such as phosphate, carbonate, and citrate give rise to various ionic species as a function of pH of the medium. The proportion of different ionic species can be determined by the Henderson-Hasselbach equation (Jenness and Patton, 1959): pH

=

pK

+ log saluacid.

Probably, that is why the increasing effect of salts of weak acid on hydration tends to be greater than could be expected from the valence of the anion alone according to Loeb’s (1922) suggestion. B.

SWELLING

Both electrolytes and nonelectrolytes can affect the swelling capacity and shrinkage temperature of collagen. For example, sucrose and glucose (which decrease the dielectric constant and enhance electrostatic interactions) at concentrations of about 1 and 2 M , respectively, produced maximum swelling and maximum decrease in shrinkage temperature (Lloyd and Garrod, 1948). These substances possibly influence the dielectric content of the media, which in turn affects the electrovalent linkages. High dielectric constant favors neutralization of the charged groups, whereas the low dielectric constant increases strength of electrovalent linkages and hence T , would increase. Although different factors can offset the expected effect, electrolytes have a decisive influence on the biophysical properties (swelling, solubility, gelatin, viscosity, water-binding capacity) of a protein at different ionic strengths and pH values (Hermansson, 1975; Eagland, 1975a). An increase in swelling by acids seems to be the result of the H + ion concentration less the effect of the acid anions, and that of bases was caused by the OH- concentration less the effect of base cations (Jirgensons and Straumanis, 1962). The neutral salts at moderate concentration (1 M ) repress the swelling, with the higher concentration being more effective. It has been stated that the anions in general are more effective than cations for increasing swelling. Anions and cations of salts can be arranged in a series in which the successive ion allows a lesser degree of swelling than the one before. The sequences usually given for different organic and inorganic ions are as follows: Organic anions: Acetate > citrate > tartrate Inorganic anions: CNS- > I- > NO,- > Br,- > C10,- > C1- > SO,*Inorganic cations: Li+ > Na+ > K + > NH4+ > Cs+ > Mg2+ > Ca2+ > Ba2 +

This broad generalization, describing the influence of salts on the properties of gels consisting of proteins or other hydrophilic colloids in water, is known as the Hofmeister or lyotropic series (Hartman, 1947; Kruyt, 1952; Kragh, 1977). The mechanism related to the existence of these series is only partly clear. The

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

307

valency of the ion, however, is very important. According to Kragh (1977), the specific ion effect is also produced by adsorption, which depends on the polarizability and size of the ions. Besides, the specific ion effect occurs in the electrical double layer at the surface since the distribution of ions in the Stem layer depends on the ionic radius. The ions on the left side of the lyotropic series may act as a hydrogen bond breaker if present in high concentration. Steinberg et al. (1960) stated that ions at the extreme left of the lyotropic series have the highest tendency to favor the disordered form (the transformation of helical to random coiled structures) of a protein. With regard to collagen-ion interaction, two views seem to have emerged. Some workers believe in the possibility of direct ion binding to the peptide backbone of collagen (Bello et al., 1956; Mandelkem and Stewart, 1964). Von Hippel and Wong (1962) disagree with this proposition. They opine that ions affect the collagen fold indirectly by interacting with structurally bound water molecules. This conclusion was derived by treating their experimental data according to the following equation:

where K is the equilibrium constant, [SG] is the molar concentration of filled eP binding sites, [S] is the concentration of free salt, and [GI is the molar concentration of binding sites. By reanalyzing the earlier data according to this equation, Bello (1963) argued that ion-peptide bond interaction cannot be ruled out as an explanation of the effect of neutral salts on the collagen fold formation. Loeb (1922) and Thomas and Foster (1925) also developed ionic series corresponding to the Hofmeister series. It appears from those series that weakly hydrated ions (e.g., CNS-) are strongly adsorbed by protein from aqueous solution, whereas strongly hydrated ions (e.g., SO,2-) exhibit little adsorption owing to their dehydration effect on protein. Thus the former have a strong swelling effect on protein at a concentration of 1.0 M ,suggesting that the effect is due to molecules of the salt rather than ions. Further, Hamm (1958) observed that salts with the same cation but different anions, as well as those with the same anion and different cations, did not function according to the lyotropic order in the case of meat protein. If so, then the Hofmeister ion series would lose some of its significance. It has also been reported that the presence of neutral salts decreases swelling in acid and alkaline media and that the anions are more active than cations. The following series indicate in descending order the swelling in acidic and alkaline media: Citrate > tartrate > phosphate > sulfate > acetate > iodide > thiocyanate > nitrate > bromide > chloride and

308

A . ASGHAR AND R. L. HENRICKSON

However, Hamm’s (1957) studies indicated that neutral salts increased the hydration of meat proteins in the basic pH range and caused dehydration in acidic pH. He found an increased hydration effect of cations in the following lyotropic order: Ca2+ < Mg2+ < K + < Na+ < L i t , while that of anions is F- < CI- < Br- < I- < CNS in the alkaline pH range. The anions, however, were more effective than cations in this pH range. On the acid side, anions produced a greater dehydration effect than cations. Sherman’s (1962) study supported some of these findings. Hamm (1957) explained these observations on the assumption that the positively charged groups would repel each other in the acid pH range, resulting in enlargement of the interspace between peptide chains. By adding neutral salt (e.g., NaCI) the positive centers of protein bind the C1- and diminish repulsion and decrease hydration. On the alkaline side, the proteins have an excess negative charge, a part of which is involved in salt linkages with cations, particularly with the bivalent cation, which screens the charge (Hamm, 1958; Bozler, 1955). Bivalent Ca2+ and Mg2+ can also form coordinate links involving --COO-, -NH,, -SH, and - O H groups. The imidazole of histidine preferably binds to Zn2 . Despite these generalizations, the swelling may increase in the presence of both monovalent and polyvalent ions due to their counteraction. The deamination of collagen (with nitrous acid) to replace the strongly basic E-NH, of lysine by a weakly basic OH group markedly increases the swelling capacity in the alkaline pH range (Thomas and Foster, 1926). +

Interaction of Salts and Hydrogen Ions on Swelling As mentioned before, a certain minimum concentration of H + or OH- is needed for breaking the dipolar compensation between polypeptide chains of collagen. By adding a neutral salt (1 M concentration) having a common anion of the acid, the zone of inert reactivity is eliminated, probably due to the increased concentration of the anion. This increase not only eliminates the Donnan effect, but also removes the potential barrier set up against the anions of the strong acid, which is preponderantly balanced electrostatically by, and not combined with, the cationic groups of protein. Consequently the concomitant swelling of protein is also depressed. This explains the depressing effect of NaCl on collagen hydration in the pH range 5-7 (Fig. 13). Procter and Wilson (1916) also found that a neutral salt (e.g., NaCl) depresses the swelling of collagen in HCl by equalization of C1- distribution between internal and external phases. As the concentration of ions in the external phases

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

309

increases, the excess of diffusible ions in the internal phase decreases. In other words, the difference in anion concentration between the two phases will be reduced. Besides, the osmotic pressure of added salt would be a contributing factor. Neutral salts also screen off the polymeric charges and diminish repulsion. The stretching effect is lowered at high salt concentration since greater numbers of anions tend to combine with the cationic sites of protein due to the law of mass action, lowering the net positive charge. If so, then maximum swelling can be expected only in salt-free systems (Katchalsky, 1964). The hydration curve A in Fig. 13 demonstrates the validity of this proposition. However, Procter-Wilson’s (1916) proposition fails to account for the fact that NaCl added to collagen in NaOH solutions did not reduce swelling, as can be seen from curve B in Fig. 13. On the other hand, CaCl, added to Ca(OH), greatly increases swelling of collagen due to lyotropic effects (Kuntzel, 1944). But, curve C in Fig. 13 shows that salt of a dibasic acid (e.g., Na,SO,) markedly suppresses the swelling of collagen (above pH 9) as compared to NaCl in NaOH solutions. The concept of the Donnan effect (Bolam, 1932) also offers no explanation for these observations. It seems that the degree of affinity of anions of the acid for cationic protein groups is important. The SO,,- have greater binding tendency than C1- with collagen. At low pH, H,SO, partly functions as a monobasic acid; this factor also favors the uptake of sulfuric acid over the HCl. All these instances suggest that the influence of neutral salts of different valency combinations or alkaline solutions containing common cations on swelling of collagen cannot be explained by any concepts which have been advanced so far (Gustavson, 1956). Swelling of protein is an important property in foods such as processed meat, custards, and doughs, where proteins are required to imbibe and hold water without dissolving. Wettability is another functional property closely associated with hydration and swelling of proteins. It mainly depends on the hydrophilic-hydrophobic balance, the molecular surface of the protein, and the surface tension of solvent. All these characteristics determine the body and viscosity of some processed meat products (Hermansson and Akesson, 1975; Kinsella, 1979). In this regard the actomyosin and other myofibrillar proteins are thought to have the sole role (Hamm, 1957; Nakayama and Sato, 1971a,b,c). Little consideration has been given to collagen, although it seems to have a greater potentiality for water binding than other proteins. This aspect needs to be ascertained. C.

EMULSIFYING CAPACITY

As indicated earlier, proteins are important functionally in various food biosystems. They have the potentiality to form and stabilize oil/water emulsions in a number of meat and nonmeat products (Wolf and Cowan, 1971; Friberg, 1976),

3 10

A. ASGHAR AND R. L. HENRICKSON

which otherwise, in the absence of an emulsifier, are unstable on account of the positive free energy resulting from interfacial tension. The surface characteristics of a protein are related to the emulsifying capacity and emulsion stability in the product. The charged protein molecules encapsulate the fat globules and lower the interfacial energy between oil/water phases by mutual repulsion, and hence prevent coalescence of fat droplets in the emulsified products (M. J. Y. Lin et al., 1974; Hermansson and Akesson, 1975). The DLVO theory, already mentioned in Section VI,A, well explains the overall mechanism of emulsion formation and stability. Hence, all those factors which can affect the electrical double layer will influence the stability of the emulsion. The opinions of different workers, however, are divergent as to the state of a protein which would result in maximum emulsion capacity. According to Kamat et al. (1978), denatured proteins provide greater emulsion stability than native ones, and optimum performance can be expected at or near their isoelectric point, where the protein adsorption and viscoelasticity at the oillwater interface is maximum and repulsion is minimum. It is assumed that proteins at their isoelectric point stabilize emulsions by a mechanism of adsorption and interfacial denaturation to produce a physical barrier (steric) against coalescence of dispersed phase (Cante et al., 1979). Trumbetas et al. (1979) also showed maximum nonpolar interactions between protein and fat on emulsification at the isoelectric point of the protein (protein solubility at this point is expected to be minimum). These findings support the view of Boyer et al. (1946), who indicated the existence of nonpolar interactions between protein and oil phases, whereas carboxylate groups (in the case of free fatty acids) play only a minor role (Spector, 1975). The chemical forces which confer stability to protein-lipid complexes depend largely on lipid composition. For example, fatty acids can form ester or amide linkages by involving a COOH group with OH or NH, groups of a protein, respectively (Burley, 1971). Theoretically, ionic linkages are also possible between phosphate groups of phospholipid and appropriate charged groups (-NH,+) of proteins. The groups having dipole character can also be expected to interact with ionic groups (Krimm, 1968). However, neutral triglycerides can react with protein only by various weak van der Waals forces, which are composed mainly of London or dispersion forces (Salem, 1962). Besides, the hydrocarbon side chains of the protein are assumed to be in the nonpolar interior of the molecule in aqueous media, where the interactions between nonpolar groups of protein and lipid are favored by the necessity to avoid the surrounding water. Such interactions created by the presence of water between nonpolar groups are known as hydrophobic bonds (Burley, 1971), which probably are mainly responsible for conferring stability to protein-lipid complexes. The strength of these bonds increases with rise in temperature up to 5OoC, whereas the strength of most other bonds decreases (NCmethy, 1967). The stability of ionic bonds is also

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

311

dependent on the dielectric constant of the medium, and for aqueous media, on the ionic strength and pH as well. It may, however, be emphasized that the surface activity of a protein is a function of the ease with which it can unfold, rearrange, and adsorb at an interface. Since the solubility in aqueous phase may change the surface activity, it is not surprising that a number of studies have found a positive relationship between emulsifying capacity of proteins and their solubility (Pearson et al., 1965; Saffle, 1968; Yatsumatsu et al., 1972; Hagerdal and Lofquist, 1978; Pearce and Kinsella, 1978). Other workers also found unstable emulsions at isoelectric points of the protein (Franzen and Kinsella, 1976; Aoki et al., 1980). Similarly, Crenwelge et al. (1974) noted increased emulsifying capacity as the pH of the emulsion diverged from the isoelectric region of the protein. It must be realized that the solubility of protein is a relative property, since all proteins can be solubilized by using appropriate dispersing media. Hence it would be more appropriate to express the emulsion stability in more appropriate terms, such as pH, ionic strength, and protein concentration, rather than solubility. Very recently, Holm and Eriksen (1980) reported that the emulsifying capacity of protein solution increased significantly on removing the low-molecular-weight components by dialysis. The emulsifying capacity of proteins of animal origin, such as myosin, actin, actomyosin, tropomyosin, and sarcoplasmic proteins, have been studied extensively (Fukazawa et al., 1961; Hegarty et al., 1963; Carpenter and Saffle, 1965; Swift, 1965; Maurer et al., 1969; Neelakantan and Froning, 1971; Dawood, 1980). Saffle (1968) and Schut (1976) have reviewed this issue in detail, but little has been reported about the emulsifying capacity of collagen. Being an insoluble protein, collagen may be expected to be of little significance as an emulsifier. However, properly processed and modified food-grade collagen derived from hide may prove a better emulsifier than nonfat dry milk (Satterlee et al., 1973). On the basis of extensive study on protein emulsions, Smith et al. (1973) proposed that very small particles (finely divided protein) can aid emulsion stabilization. Collagen derived from hide can easily conform to these characteristics. Beside this, better results may be expected if a hydrophilic-hydrophobic balance (HHB) of collagen and fatty constituents may be established as proposed by Griffin (1949). The appropriate values of HHB have been reported for other oil and surfactant and protein systems (Becher, 1965; van Eerd, 1971). Despite certain limitations (Boyd et al., 1972), the HHB value provides a useful approximation for making stable emulsions. D.

FOAMING

The ability of a protein to form a stable foam is one of the great functional characteristics in food science, especially in baking technology. Unlike an emul-

312

A. ASGHAR AND R. L. HENRICKSON

sion, the discontinuous phase in foam is the gas droplets, encapsulated by an aqueous film of protein, which lowers the interfacial tension between the gas and water phases (Kinsella, 1979). The presence of lipid destabilizes the protein film and hence is detrimental to foaming (Yatsumatsu et a / . , 1972). Foaming is also affected by pH, protein concentration, temperature (Eldridge et al., 1963), and partial proteolysis (Horiuchi et al., 1978). The latter authors have associated the foam stability with surface hydrophobicity of a protein molecule. The whipping property of a protein is closely related to the foaming capacity. However, no information seems to be available regarding the foaming capacity ,of food-grade collagen derived from hides. E.

VISCOELASTICITY

The unique physicochemical properties of fibrous collagen have been utilized in the fabrication of useful products such as edible collagen sausage casings (Braun and Braun, 1956; Reissmann and Nichols, 1960; Cohen, 1964; Talty, 1969; Kidney, 1970). The making of sausage casing depends on the viscoelastic characteristics of the dispersed collagen. Due to these characteristics, collagen dispersions can serve as a binder and a lubricant. A number of studies have reported the viscometric characteristics of soluble collagen (Runkel and Lange, 1937; Kahn and Witnauer, 1966; Cerny et al., 1970; Whitmore et al., 1972). It has been shown that satisfactory cold dispersion of collagen cannot be made in the pH range 4.3-8.5 without using an additive like guar gum, which reduces reaggregation of unswollen fibrils (Whitmore et al., 1972). The cold acid dispersion at low ionic strength (<1 .O) consists entirely of swollen fibrils at pH < 4.3 (Borasky, 1963) and temperature below 50°C.

VII.

NUTRITIONAL ASPECTS OF COLLAGEN

While the functional properties of proteins can influence the aesthetic value and organoleptic characteristics of different processed food products, the digestibility, biological availability, and relative proportion of the essential amino acids (Table VI) for optimum utilization of nonessential amino acids, determine the nutritional value of the proteins. These variables affect the efficiency of potential biological utilization of dietary proteins for meeting various requirements of the human body (Morrison and McLaughlan, 1972). Physiological availability of amino acids of a protein is also influenced by various processing treatments. For better appreciation of the subsequent discussion on the nutritional aspects of collagen, first a brief explanation of the relevant terminology and biological assaying of protein quality seems appropriate.

313

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

TABLE VI ESSENTIAL AMINO ACIDS FOR GROWTH AND MAINTENANCE OF HUMAN SUBJECTS AND RATS

Amino acid 1. Isoleucine 2. Leucine 3 . Lysine 4. Methioninea 5 . Phenylalanine" 6 . Threnonine 7. Tryptophan 8. Valine 9. Histidine 10. Arginine

For growth in rats

For growth in children

For maintenance of adults

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

?

Not essential Not essential

"Cystine and tyrosine content can spare the need of methionine and phenylalanine, respectively, up to 30-50%. Arginine is also essential for birds. Human subjects can synthesize arginine, but the rate may be limited, hence dietary supply may be needed for optimum growth. Glycine is essential only for birds.

A.

PROTEIN QUALITY ASSAYS

A number of biological- and chemical-based procedures have been described to evaluate the nutritional quality of food proteins. A detailed discussion of those methods can be found in a number of comprehensive reviews (McLaughlin, 1974a,b; Bender, 1975; Pike and Brown, 1975; Bodwell, 1977a,b; Hackler, 1977a,b; Sammonds and Hegsted, 1977). They include nitrogen balance studies with human subjects, growth studies with laboratory animals (generally rats), chemical assays, bioavailability of individual essential amino acids, and other bioassays (Bodwell, 1977b). These methods can also be classified as primary (direct) and secondary (indirect) assays. Another classification could be onepoint assays and multipoint assays (Lachance et al., 1977). The primary assays of protein quality of different foods are directly performed on human subjects for a specific period (35-45 days) to determine the nitrogen balance index. The biological value or net protein utilization can be derived by direct comparison of different food proteins involving human subjects for 12-15 days. The multipoint nitrogen balance index (NBI) assay can be performed in 10 days using adult human subjects (Lachance et al., 1977). For routine and quality control purposes, indirect assays of protein quality are generally performed by using either laboratory animals, mostly rats (Hackler, 1977) or microorganisms, such as Leuconostoc mesenteroidis, Streptococcus faecalis (Horn and Warren, 1961; Horn et al., 1954; Teeri et al., 1956), Strep-

314

A. ASGHAR AND R. L. HENRICKSON

tococcus zymogenes, protozoa Tetrahymena pyriformis or Tetrahymena thermophila WH,, (Evancho et al., 1977; Satterlee et al., 1977, 1979). Proteolytic enzymes such as papain (Buchanan and Byers, 1969) and the pepsin-pancreatin system (Akeson and Stahmann, 1964) were employed to study the digestibility of the protein in vitro and to correlate them with in vivo digestibility. Further improvement in the enzymic method has been achieved by using multienzyme systems such as pepsin-trypsin-pancreatin and trypsin-chymotrypsin-peptidase (Hsu et al., 1977). The data on nitrogen content of diet, carcass, feces, urine (including endogenous and metabolic nitrogen), growth, protein consumed, amino acid content of protein, etc. are collected depending upon the nature of the experiment. The protein quality is then expressed in any one or more of the following terms depending upon the experimental conditions.

I.

Digestibility

The digestibility of a protein is the primary determinant of the availability of its amino acids. It is defined as the proportion of food which is absorbed, that is: Digestibility

=

N in diet

-

N in feces - N in metabolism x 100 N in diet

If the correction for metabolic loss in feces is not made, the value is called an ‘‘apparent digestibility.”

2. Biological Value Biological value (BV) is a single-point assay, based on nitrogen balance. This indicates the proportion of absorbed nitrogen which is retained in the body for maintenance and/or growth (Mitchell, 1924) that is: BV

=

N in diet

-

(N in feces - N metabolic) N in diet - (N in feces

-

(N in urine - N endogenous) x 100. N metabolic)

However, if the correction for metabolic and endogenous losses is not made, the value is termed ‘‘apparent biological value. ’ ’ 3 . Protein Efficiency Ratio

Protein efficiency ratio (PER) is defined as the ratio of weight gain to protein consumed (Osbome et al., 1919) in a specific period (e.g., 28 days), using a single level of protein (10%) in the diet: PER

=

Weight gain by test animal Weight of protein consumed

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

315

This one-point assay is the AOAC’s approved method for determining the nutritive value of proteins, even though it yields variable results (Bender and Doell, 1957) and has some limitations (Jacquot and Peret, 1972; Satterlee et al., 1979). There is now an overwhelming consensus that serious problems arise with PER as a protein quality indicator. This was reflected in a symposium2 on protein efficiency. 4. Net Protein Ratio

The principal drawback in the assay of PER was removed by Bender and Doell (1957) by measuring the net protein ratio (NPR) value of the diet. This was achieved by including a second group of experimental animals, using a proteinfree diet. It was based on the assumption that protein required for preventing weight loss of the protein-free group would be an estimate of the maintenance requirement of the test animals. This two-point assay needs only 10-14 days (McLaughlan, 1974) and NPR is derived from the following expression: NPR

=

Weight gain

+ weight loss by protein-free group Protein consumed

5 . Net Protein Utilization

Earlier Bender and Miller (1953) also suggested the estimation of net protein utilization (NPU) as a measure of protein quality. This single-point assay is identical to NPR, except that the amount of carcass nitrogen is involved in the estimate instead of the body weight of the test animals, as follows: NPU

=

Body N

-

body N of protein-free group N consumed

McLaughlan (1974b) believes that NPR is essentially equal to PER as NPU is equal to BV X protein digestibility.

+ I . 5 , where-

6. Relative Nutritive Value

The relative nutritive value (RNV) provides an estimate of the protein quality by feeding the protein in question to test animals at three levels for about 3 weeks, including a zero protein level (Hegsted et al., 1968). A comparison is then made using lactalbumin as control with the regression lines of body weight to protein intake. The RNV gives values almost identical to the NPR assay (McLaughlan and Keith, 1975). *Overview of outstanding symposium in Food Science and Technology, Food Technol. (Chicago) 31, 69-93 (1977).

316

A. ASGHAR AND R. L. HENRICKSON B

A Weight change on diet

N bal i c e

N growth ratio Net protein ratio

(standard)

Bioloaical

0

10% protein Protein or N intake

N intake

FIG. 18. Relationship among different bioassay methods for determining the nutritive value of proteins. From Allison (1964).

7 . Relative Protein Value The relative protein value (RPV) is a simplified version of the RNV assay, in which the zero protein level is deleted in the procedure (Hegsted and Chang, 1965). Lactalbumin is still used as a control. Consequently, the regression is derived in the region at which protein intake and body weight exhibits a linear relationship. However, the RPV assay is highly influenced by lysine and threonine content of a protein (Hackler, 1977b). For example, lysine-poor proteins tend to give low slopes, which underestimate protein quality of such proteins. Threonine-deficient proteins give steep slopes and tend to overestimate protein quality of these proteins. McLaughlan (1976) has suggested another method for evaluating protein quality, called “relative nitrogen utilization. Figure 18 depicts the salient differences among some of these bioassays of protein quality including the nitrogen balance index (Allison, 1964). Very recently Heger and Frydrych (1980) have reviewed the merits of each of the abovementioned bioassays of protein quality, whereas Evans and Witty (1980) have elaborated the merits and demerits of using protozoa for determining the protein quality of foodstuffs. ”

8.

Chemical (Amino Acid) Score Assays

These assays of protein quality are based on the comparison of amino acid profiles of a protein with whole egg protein, assuming that all of the amino acids are 100% available in vivo, which in fact is not the case (Bender, 1973). The score can be computed, using the following expression: Amino acid score

=

mg of amino acid in 1 g of test protein mg of amino acid in 1 g of reference protein

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

317

Mitchell and Block (1946) used the percentage of the most limiting essential amino acid, based on Liebig’s law of minima, to evaluate protein quality by comparing it with the essential amino acid profile of the whole egg, and designed this percentage as the “chemical score” to equate it with biological value. Oser (1951) considered the total essential amino acids as a better indication of protein quality. The geometric mean percentage of the percentages of all essential amino acids (by comparing with those of whole egg) was named “essential amino acid index” (EAA index). As Oser’s method of calculation differs from that of Mitchell and Block, it gives different results especially with low-quality proteins. However, Kofranyi (1972) remarked that none of the methods correctly interprets the experimental results, since neither takes into account the role of nonessential amino acids. The procedure of McLaughlan and Keith (1975) which involves three amino acids, namely, lysine, methionine, and cysteine, for computing the chemical score may also suffer from the same limitations. A number of research workers attempted to predict PER of the protein from its essential amino acid content. Satterlee et al. (1977) predicted the PER by expressing the essential amino acid profile of the test protein as a percentage of a reference casein’s essential amino acid profile. On the basis of extensive trials on the PER assay and amino acid analysis of different products, Alsmeyer et al. (1974) derived the following regression equations for computing PER from amino acids data: PER PER PER

= = =

-0.684 + 0.456 (leucine) - 0.047 (proline), -0.468 + 0.454 (leucine) - 0.105 (tryptophan), -1.816 + 0.435 (methionine) + 0.780 (leucine) + 0.21 1 (histidine) - 0.944 (tryptophan).

(1) (2) (3)

Equations (1) and (2) are applicable to meat products, whereas Eq. (3) relates to meat-containing cereals, yeast, etc. (Happich et al., 1975). Apart from these, a number of other assays have also been suggested for measuring the nutritional quality of protein. They include assays based on repletion of exhausted nitrogen stores and measurement of plasma-free amino acids (Hartog and Pol, 1972), clinical methods (Scrimshaw and Young, 1972), various modifications of nitrogen-balance tests, the PER and related methods (Jacquot and Peret, 1972; Hartog and Pol, 1972), protein utilization (Kofranyi, 1972; Payne, 1972), bioavailability of amino acids (Gupta et al., 1958; Carpenter et al., 1972), and chemical estimations of certain essential amino acids (Pongpaeu and Guggengeim, 1968; Momson and McLaughlin, 1972; Carpenter and Booth, 1973; Finley and Friedman, 1973; Lakin, 1973; Holsinger and Posati, 1975; Hurrell et al., 1979). Bodwell (1977b), while reviewing the various approaches of protein quality evaluation, concluded that, although the values obtained from rat assays are related to the nutritional value of proteins in general, their significance in terms

318

A. ASGHAR AND R. L. HENRICKSON

TABLE VII FAOiWHO STANDARD FOR ESSENTIAL AMINO ACID REQUIREMENTS FOR ADULT HUMAN SUBJECTS Amount Essential amino acids

I. 2. 3. 4. 5. 6. 7. 8.

(gi100 g protein)

Leucine Phenylalanine-tyrosine Lysine Valine Isoleucine Threonine Methionine-cystine Tryptophan

7.0 6.0 5.5 5.0 4.0 4.0 3.5 1 .O

of protein for humans is not well defined. Moreover, in those cases where a comparative study has been made involving the same samples of proteins with both human and rat (Bodwell, 1977c), no consistent relationships were found between the bioassays of nutritive value in human or rat. Since 1935, different international committees have made a number of recommendations regarding the protein and amino acid requirements of human subjects (KofrBnyi, 1972). In 1965, the F A 0 expert committee on protein requirements recommended a provisional reference, keeping in view the human need for essential amino acids. As it contained excessive amounts of tryptophan and sulfur-containing amino acids, another FAO/WHO Expert Committee suggested a revised standard reference in 1973 (Table VII). It may be pointed out that all of the nonessential amino acids are metabolically glucogenic in a reversible manner, that is, they are interchangeable with each other and with certain energy-yielding carbohydrates. Among the essential amino acids, only a few (arginine, isoleucine, threonine, valine, cysteine) are glucogenic but nonreversibly, others (leucine, phenalalanine, tyrosine, isoleucine) are irreversibly ketogenic. The metabolic fate of a small group of essential amino acids (lysine, histidine, methionine, tryptophan), which are invariably found to be limiting in most food systems, is uncertain (Payne, 1972). B.

DIGESTIBILITY OF COLLAGEN

The nature of catabolic changes in collagen has been discussed in Section IV,B, indicating that native collagen is almost resistant to proteolytic attack, hence is regarded as indigestible. However, denatured collagen can be acted upon by a number of proteolytic enzymes. Cooking and highly acidic conditions in the stomach (pH < 2.0) cause denaturation and unfolding of the triple helix of collagen, making it susceptible to enzymic digestion. Mandl (1961) has reviewed

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

319

the effects of temperature, acids, swelling, liming, and particle size on the susceptibility of collagen to trypsin attack, even though the extent of crosslinkages and steric structure do influence the digestibility (Banga and Balo, 1965). Nutritional trials with rats by Whitmore et al. (1975) have indicated that the apparent digestibility of fibrous collagen was 90%, and that 1 g of collagen was equivalent to 1.5 g of gelatin as an energy source. Moreover, pathological examination of body organs (stomach, intestine, heart, trachea, larynx, lungs, pancreas, liver, kidneys, urinary bladder, spleen, pituitary, thyroid, parathyroid, thymus, brain, adrenals, testes or ovaries, and uterus), revealed no abnormal lesions on feeding the rats with a diet containing up to 20% collagen for 90 days. However, the kidneys of rats fed collagen weighed more than those of rats fed casein. The functional hypertrophy of the kidney was thought to be due to the high nitrogen content of the collagen (18.6% N of collagen vs 15.9% N of casein). The hypertrophy of the kidneys due to high nitrogen content in the diet has been reported by Osborne et al. (1926-1927). The very low nutritional value of gelatin, a derivative of collagen, has been proven by the studies of Chapman et al. (1959) and Rama-Rao et al. (1964). C.

BIOLOGICAL VALUE AND PER OF COLLAGEN

Mitchell and Carman (1926) stated that different meat cuts may be equal in digestibility, but may vary in biological value due to a difference in connective tissue content. In another study, Mitchell et al. (1927) reported that a cut of veal, evidently fibrous, had a BV of only 62, whereas similar beef cuts had a BV of 69. Similarly, DvofAk and Vognarova (1969) obtained a low chemical score from amino acid analysis of cuts rich in connective tissue. Meat from steers was found to have a lower BV than meat from yearling bulls, and Avshalumova (1974) attributed this finding to the content of collagen. The study of Bender and Zia (1976) also provided supporting evidence for Mitchell and Carman’s contention that low-quality meat (shin) with 23.6% connective tissue had an NPU of 69, whereas high-quality meat (filet beef steak) containing 2.5% collagen had an NPU of 82. The NPU of meat generally varies from 62 to 78, the average being 74 (Food and Agriculture Organization, 1968). The NPU of meat decreases on cooking mainly due to the loss of available methionine in the presence of other foodstuffs (Bender and Husaini, 1976). Lee et al. (1978) found a highly significant negative correlation of meat collagen content with PER ( r = -0.98) and essential amino acids content (r = -0.99, p < 0.001). The amino acids composition of collagen has been discussed in Section II1,A. It can be seen that among the essential amino acids, methionine, lysine, and threonine are limiting in native collagen, whereas tryptophan is practically absent. The total indispensible amino acids constitute 26.3-28.4% [al(I), 27.4%; a2(I), 28.4%; al(II), 26.6%; al(III), 26.3%]. The nonessential amino

320

A . ASGHAR A N D R. L. HENRICKSON

acids constitute 71.6-73.7% of the collagen molecules. The bioassay of gelatin by Rama-Rao et al. (1964) has shown that all of the essential amino acids, except arginine, are limiting as compared to the required pattern. These facts suggest that the addition of collagen to formulated meat, to produce high-priced products by restructuring, is likely to lower the nutritive value of the resulting products. This apprehension probably inspired the USDA to propose interim regulations on the nutritional quality of such meat products. Accordingly, a minimum PER of 2.5 and a minimum essential amino acid content of 32% are specified for most of the fabricated products (U.S. Department of Agriculture, 1981). However, Bodwell (1977b) considered that in the use of such quantity-quality breakpoints where PER <2.5, 65 g of protein are required to supply 100% of the maximum value per serving. A PER 2 2.5, requiring 45 g of protein or less, is scientifically illogical and economically costly. By using varying amounts of collagen in the meat product bioassay, Lee et al. (1978) computed the following linear regression equations to estimate the PER of meat products from their collagen or essential amino acid content: PER PER PER

= = =

-0.0229(collagen %) - 3.1528, -0.0632(ten EAA %) - 0.1539, -0.08084(seven EAA %) - 0,1094.

According to these regression equations, a PER of 2.5 corresponds to 28.5% of collagen in the test products, or 32.2% of the amount of seven essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine) or 42% of the amount of ten essential amino acids. This suggests that there will not be any risk of a low PER or imbalance of essential amino acids in the product if the collagen content in the product is less than 28.5%. The earlier finding by Ashley and Fisher (1966) that chicks fed on a. 10% gelatin + 3% casein diet had weight gains equal to those fed on a 13% soy protein + 0.2% methionine diet provides further support for this contention. Similarly Erbersdobler et al. (1970) have found some improvement over the control group in daily gain and feed conversion in male rats by incorporating collagen or gelatin at levels up to 5% of the diet weight. Hence, collagen, if used with balanced protein and within limits, should not decrease the nutritive value of the diet. However, a collagen content more than 28.5% in the diet is likely to lower the PER to below 2.5. Only in such situations would the problem of fortification arise. D.

POSSIBLE FORTIFICATION METHODS OF COLLAGEN

A most convenient way of improving the nutritional value of proteins in a food system is by fortification directly with the required content of the limiting amino

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

32 1

acids. Despite simplicity, this approach has certain inherent drawbacks. Some amino acids, if added in free form, may change the taste and flavor of the product (Beigler, 1969; Yamashita et al., 1970; Klaui, 1974). For instance, methional and dimethyl disulfide formed from methionine in a processed food give an undesirable flavor (Hippe and Warthesen, 1978). Free amino acids are susceptible to Strecker degradation and Maillard-type reactions with 0

II -C

or -CHO

groups of reducing sugars giving a brown coloration (Reynolds, 1963, 1965; Eskin et al., 1971). The are likely to leach out with the aqueous phase during processing (Bressani, 1975; Fujimaki et al., 1977). The addition of free amino acids may also have nutritional implications. A number of studies have shown a difference in the metabolic efficiency with which free amino acids and those of proteins are transported across the intestinal wall. Small peptides are reported to be more readily assimilated than free amino acids (Craft et al., 1968; Matthews et al., 1968; Adibi, 1971; Cheng et al., 1971; Lis et al., 1972; Burston et al., 1972). These problems could be resolved by covalently linking the limiting amino acids either by chemical or enzymatic reaction with the protein to improve its nutritional value (Feeney and Whitaker, 1977). Sheehan and Hess (1955) first suggested the use of carbodiimides as the coupling reagents for peptide synthesis. Based on a similar approach, Bjarnasson-Baumann et al. (1977) achieved improvement in the nutritional assay of whey protein by covalent incorporation of phenylalanine, tyrosine, methionine, and isoleucine. Li-Chan et al. (1979) reported on the fortification of wheat gluten with lysine and Voutsinas and Nakai (1979) enriched soy protein with methionine and tryptophan using similar methods. The casein was also enriched by a similar approach (Puigserver et al., 1979). Fortification of proteins with limiting amino acids has also been achieved enzymically by the plastein reaction (Yamashita et al., 1970, 1975, 1976; Lalasidis and Sjoberg, 1978). Detailed information on this aspect is available in a review by Fujimaki et al. (1977). Briefly, the process requires two enzymic reactions, viz. protein hydrolysis followed by re-synthesis. However, Yamashita et al. (1979a) modified the original procedure into a one-step process, which has been applied to soy protein (Yamashita et al., 1979b). Although covalent binding of limiting amino acids with proteins seems chemically quite feasible, some biochemical complications can arise with these approaches. For example, prior to fortification, hydrolyzing gluten with mild acid also caused deamidation of asparagine and glutamine (Wu et al., 1976), and subsequent enrichment of the hydrolysate with diamino acids such as lysine is

322

A . ASGHAR A N D R. L. HENRICKSON

likely to result in different isopeptide bonds such as a-E,y-a, y-E, p-a, and p-E isopeptide linkages. Some of these isopeptide bonds may not all be readily digestible (Kornguth et al., 1963) even though Mauron (1972, 1973) has stated that E-(y-glutamy1)-L-lysine and E-(a-glutamyl-L-lysine are available as a lysine source to rats. However, isopeptide bond formation can be minimized by using E-NH, group-protected lysine (NE-benzylidenelysine) in the reaction (Li-Chan et al., 1979; Li-Chan and Nakai, 1980). Moreover, N&-benzylidenelysine is reported to be almost 100% utilized as a source of lysine (Finot et al., 1977a), including Schiff base and other lysine derivatives (Finot et al., 1977b). Puigserver et al. (1979) also did not find any adverse effect of feeding chemically enriched casein to rats on plasma amino acid pattern and PER, although their in vitro observations did show lower digestibility of the enriched casein than that of the control. One important requirement in covalent binding of desired amino acids to a protein is that the amino group taking part in the formation of a peptide bond must be nonionized (Snellman, 1965). Empirically this means that the pH of the medium should be about 8.0 or less, if one is to distinguish between the a-NH, group and the E-NH, group of lysine. The E-NH, is essentially all ionized at pH 7.6 (pK, = 10.0), hence it will not be able to react. However, in peptide chains, containing three or more amino acid residues, the pK value of the a-NH, group is in the range 7.6-7.8 and, therefore, such amino groups can react rapidly. All of these findings suggest that covalent binding of limiting amino acids to collagen may also be possible, however, as yet no experimental attempts have been reported. Even though success may be achieved in the laboratory, the commercial feasibility of such approaches appears to be questionable in view of the economics of the process. It is also not known how such chemical enrichment of the proteins with limiting amino acids would affect the functionality of the resulting proteins.

VIII. A.

FOOD USES OF COLLAGEN

PRODUCTION OF EDIBLE FIBROUS COLLAGEN

Animal skins contain the bulk deposit of collagen (Section 11,A); thus, byproducts of the tannery can be utilized in the commercial production of edible fibrous collagen. Montagna et al. (1970) and Mier and Cotton (1976) have provided extensive information on the structure and composition of skin from a biological viewpoint. However, Fig. 19 presents the structural features of skin and other layers diagrammatically as applied by the leather industry. The outer layer (epidermis) is usually removed along with hairs and hairroots (keratin, rich in sulfur amino acids) during preparatory operations. This layer has been of little

323

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

\ Epidermis Grain Sebaceous gland Erector pill muscle

Grainamurn iunctton

Hair root Artery Vein

Corium

Colbgen fibers lnterfibrillar material

Fat

Flesh 1

FIG. 19. Schematic diagram of the bovine hide cross section, showing various layers and different components. From Johns (1977).

TABLE VIII CHEMICAL COMPOSITION OF SKIN Constituents 1 . Water 2. Protein" a. Globular serum protein b. Glycoproteinb c. Collagen i. Neutral salt-solubleO ii. Acid-soluble0 3. Mucopolysaccharidesc a. Hyaluronic acid b. Dermatan sulfate c . Heparin d. Condroitin 4-sulfate e . Condroitin 6-sulfate f. Heparitin 4. Nucleic acidsd a. Ribonucleic acid b. Deoxyribonucleic acid 5. Inorganic matter

"Bowes et al. (1957). bYates (1968); Veis et al. (1960)
Amount 6@65% 3@35% 0.5-0.7% 0.08% 9@95% 0.03-0.6% 0.05-2.6% 0.345% 525 mg/g dry 205 mgig dry 71 mgig dry 69 mg/g dry 68 mg/g dry 58 mg/g dry 1.0% 0.8% 0.2% 0.8%

skin skin skin skin skin skin

324

A. ASGHAR AND R. L. HENRICKSON

commercial importance. The “grain” layers are relatively rich in blood vessels and muscle but are low in collagen content as compared to the “corium” layers, which are mostly composed of collagen (Bowes and Raistrick, 1968) with small amounts of elastin, reticulin, fibroblasts, and some globular serum proteins (Humphrey et al., 1956, 1957; Cooper and Johnson, 1958; Cooper ef al., 1967; Mellon et al., 1960). The inner subcutaneous layer is rich in fatty components. Recently Tajima and Nagai (1980) have shown that the distribution of collagen in different layers of skin also varies significantly. Table VIII, derived from various sources, summarizes the overall composition of skin. It shows that, besides water, collagen is the principal protein, whereas hyaluronic acid and dermatan sulfate are the main mucopolysaccharides in skin. Other mucopolysaccharides are present only in traces (Meyer et al., 1957; Schiller, 1966; Cifonelli and Roden, 1968; Kofoed and Bozzini, 1959; Barker et al., 1969). Whether or not cattle hide contains all the minor mucopolysaccharides is not certain. The lyophobic fraction in the skin consists of triglycerides, waxes, sterol esters, and free fatty acids, with phospholipids being absent (Nicolaides et al., 1968; Wilkinson, 1969). For processing of leather, the lime-treated hides are split in two layers, that is, TABLE IX SALIENT FEATURES OF FIVE COLLAGEN PRODUCTS Flow rate (1bihr)b

Product Feed (limed splits) Product 1

Processing machine“

Particle descriptiono

Moisture contentb (7%)

-

-

76.0

-

2030

487.2

Relatively large, densely matted fibers Relatively small, less dense with bluish coat Separated fiber bundles Shorter individual fibers Shorter individual fibers

78.2

2.38

2200

479.6

85.5

3.06

3300

478.5

83.2

2.79

2860

480.5

82.9

1.17

2800

478.8

86.7

5.91

3600

478.8

Comitrol, 0.06 in.

Product 2

Comitrol, microcut

Product 3

Disc mill

Product 4

Comitrol, 0.2 in. Disc mill

Product 5

<‘From Komanowsky er al. (1974) bFrom Turkot et al. (1978).

Gelatin0 (7% dry wt. basis)

On wet basis

On solid basis

325

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS LIMED SPLITS

A

STRIP

KNIFE 0.1%E E N Z O I C A C I D SOLUTION

P O I N T (DH 5.3)

PR?OUCT PRCDUCT 2

PRCOUCT PRODUCT -4

B (TWO)

SPLITS

DRAINING SCREEN

ACID MIXING TANK

WATER7

STORAGE HOPPER

\

\

FIG.20. Flowsheet for the production of food-grade fibrous collagen from cattle hides. (A) Pilotplant production; (B) commercial production. From Turkot et al. (1978). Courtesy of Institute of Food Technologists, Illinois.

326

A. ASGHAR AND R . L. HENRICKSON

an outer “grain” and inner “flesh” part, with the help of a machine. The “grain splits” are meant for leather production, whereas the “flesh splits” are used for making suede leather and sausage casings (Talty, 1969; Kidney, 1970) and for the production of food-grade fibrous collagen. About 30% of over 45 million hides annually produced in the United States, not suitable for leather-making for various reasons, may represent a low-cost source of fibrous collagen (Whitmore et al., 1972; Komanowsky et al., 1974). However, special processing technology is needed for the production of foodgrade fibrous collagen with strict control on denaturation and microbial contamination of collagen so that it could be used in various food systems. A breakthrough in this respect was made at the Eastern Regional Research Center, Philadelphia (Elias et al., 1970; Whitmore et al., 1970), and finally Komanowsky et al. (1974) developed a process which yields five types of foodgrade collagen to meet these requirements. Table IX shows the important characteristics of the five types of food-grade collagen products. The flowsheets in Fig. 20 present the salient features of the overall process. Briefly, the flesh splits are fed to strip cutters, and the resulting pieces move onto a rotary cutter which reduces them to about %-in. particle size. By means of a conveyor, the collagen particles are transferred to a hide processor containing water, propionic acid, and benzoic acid (1000:3: 1 w/w/w), tumbled there for 4 hr, and then drained on a screen conveyor. The processed pieces are fed to either a comitrol or disc mill by cavity pumps, and then to the microcut depending on the type of desired product. In all cases, the temperature is reduced to 1.7”C before packing, and the products are stored at - 18°C to keep them microbiologically safe for subsequent food uses. B.

PRODUCTION OF MICROCRYSTALLINE COLLAGEN

Another fibrous form of collagen which has been made from hide for various uses is microcrystalline collagen. The preparation procedure for this product has not been published for proprietary reasons. However, Battista (1975) has provided an informative overview of the process from various patents. The salient feature of the processes are as follows. The corium layer, after being split from the hide, is extensively washed and then mechanically comminuted. The diced collagen is allowed to swell under the controlled conditions of the medium, which comprises an ethyl alcohol-water system and is centrifuged intermittently. The collagen is then treated with HCl at a pH of 1.6-2.6 to allow it to react with the available NH, groups of the collagen. This reaction forms a water-insoluble, ionizable, salt of collagen containing about 0.4-0.7 mmol of acid (HCl/g of collagen). By varying the acid concentration, microcrystalline collagen with different functional properties can be produced. The temperature is maintained below 30°C during this process. The

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

327

cake so obtained is air-dried or freeze-dried before milling into a definitive physical form and packing. Besides HCl, other acids (sulfuric, acetic, and lactic acids) can also be used to produce corresponding partial-acid salts. However, from a practical and commercial viewpoint, HCl is preferred over other acids for most end uses of the resulting product. The physical and microscopic properties of the microcrystalline collagen and its various uses have been described in detail by Battista (1975). A microcrystalline collagen product is commercially produced under the trade name of Avitene. C.

VARIOUS USES OF COLLAGEN AS GELATIN

There are three major areas where hide collagen is used extensively in the form of gelatin: in various food products (Gotthoffer, 1945; Idson and Braswell, 1957; Battista, 1975; Courts, 1977), in photography (Cox, 1972; Rose, 1977), and in pharmaceuticals (Chvapil et al., 1973; Wood, 1977; Bhandari, 1978). According to Idson and Braswell (1957), 65% of the total annual production of gelatin in the United States (- 50 million lb) is consumed in edible products such as desserts, marshmallows, candy, consomme, bakery foods, jellied meat, ice cream, and other dairy products, 20% is used in photography, and 10% in pharmaceutical capsules, ointments, cosmetics, coatings, and other emulsions. Extensive information is available on various food uses of collagen in the form of gelatin (Bennett, 1921; Bogue, 1922; Alexander, 1923; Idson and Braswell, 1957; Stainsby, 1958; Jones, 1977; Howell, 1978; Courts, 1980). The use of collagen as gelatin has certain inherent nutritional implicatons. For instance, cattle hide trimmings are treated wtih lime solution (containing some other salts) for a prolonged period to facilitate solubilization of collagen. The essential basic amino acids (e.g., lysine, arginine, histidine) are deaminated (Theis and Jacoby, 1941) or decomposed (Highberger and Stecker, 1941) and the essential hydroxy amino acid (threonine) is destroyed (Bowes et al., 1953) during this process. Probably that is why all of these amino acids (except arginine) have been found limiting in gelatin as evaluated by nutritional trials (Chapman et al., 1959; Rama-Rao et al., 1964). Besides, alkali treatment has been found to induce some other undesirable changes in proteins. These include racemization of amino acids (Masters and Friedman, 1979, 1980; Smith and Silva de Sol, 1980) and formation of lysinoalanine (Sternberg and Kim, 1977; Friedman, 1978). According to Friedman et al. (1981), hydroxide ion-catalyzed p-elimination reactions of serine, threonine, and cystine produce a dehydroalanine intermediate containing a conjugated carbon-carbon double bond (because of the double bond character of the peptide bond). This intermediate then reacts with the E-NH, group of lysine to form lysinoalanine. The changes induced by alkali in a protein also have nutri-

328

A. ASGHAR AND R. L. HENRICKSON

tional implications. For example, metabolism of the protein and the physiological utilization of amino acids are reduced to a great extent (Woodard et al., 1975; Gould and MacGregor, 1977; Friedman, 1977; Friedman et al., 1981). In view of these facts, it seems more appropriate to find various food uses of collagen in its native fibrous form. Although Elias et al. (1970) pointed out some possible uses of collagen in fiber or granule form in meat products, little is known about the functional behavior of fibrous collagen in different food systems. On account of its unique biophysical properties, fibrous collagen could be utilized efficiently to function as a water binder, extender, moisturizer, texturizer, and emulsifier in different food systems. Studies have recently been initiated to learn how fibrous collagen will function in various food systems. D.

POTENTIAL USES IN FOOD SYSTEMS

Investigations are being carried out at the Oklahoma Agricultural Experiment Station, Stillwater, Oklahoma, on various potential uses of five types of foodgrade fibrous collagen (produced by the Eastern Regional Research Laboratory) in various food systems. The main emphasis so far has been on the production of “all-beef‘’ sausages and bakery products, with the substitution of food-grade collagen at different levels (Henrickson, 1980). The major studies made on these aspects are described below. Schalk (1980) investigated the effect of food-grade collagen substitution on some of the functional properties of coarse-beef bologna by replacing lean meat at 10, 20, and 30% levels. He found no significant difference in the volume change, shrinkage (wrinkling), emulsion stability, or the texture of the final cooked product as compared to control samples (Fig. 21), except that color a-

FIG.21. Coarse bologna sausages, prepared from beef with 10, 20, and 30% food-grade collagen from hide added. The cross-sectional area and external surface appearance show no difference. Courtesy of Schalk (1980).

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

329

FIG. 22. Microscopic view of fine bologna sausage emulsion containing 15% food-grade fibrous collagen from cattle hide (A) and the control (B). There was no apparent difference in emulsion characteristics and the distribution of fat droplets (arrow) in either case (magnification X 320). Courtesy of Gielissen (1981).

values (redness) decreased with increased supplementation of collagen. This study suggested that hide collagen is as good as water binder and fat emulsifier as other proteins of the lean meat such as actomyosin, or that the meat used contained more actomyosin than needed to form the emulsion. In a similar study, Gielissen (198 1) prepared fine-emulsion bologna sausages by incorporating collagen (product No. 1) at 5 , 10, and 15% levels, replacing lean meat, but keeping the fat content constant at 25%. In each case the emulsion was found to be stable, except at higher levels of collagen where the emulsion stability tended to decline slightly. The microscopic examination of various emulsions did not show any adverse effect of collagen on the diameter of fat droplets (Fig. 22). These findings are at variance with those of Saffle et al. (1964) and Maurer and Baker (1966), who considered collagen detrimental to the emulsifying capacity of poultry meat. The high collagen content (>15%) in sausages has been regarded as the causative factor of gel pockets, wrinkling of the outer skin, poor peelability, and unstable batters when the product is cooked at a temperature above 65°C (Saffle et al., 1964; Kramlich, 1971). On the contrary, Hamm (1972) found the fewest gel pockets in frankfurter-type sausages in the temperature range of 65-85°C. A recent study by Wiley et al. (1979) indicated that the incidence of gel-pocket formation may increase only if the soluble collagen

330

A. ASGHAR AND R . L. HENRICKSON

content is high in the sausage, whereas total collagen or insoluble collagen content bears little relationship with gel-pocket formation. Slight devaluation of the surface appearance of the cross-sectional area, caused by gel formation, should not be overemphasized in view of certain intrinsic merits of the gel. For example, several studies have shown that protein hydrolysates (Bishov et al., 1967; Bishov and Henick, 1972, 1975) or certain amino acids (Marcuse, 1962; Chang and Linn, 1964; Karel et al., 1966; Hayes et al., 1977) act as synergists for the naturally occurring antioxidants (vitamin E) in retarding the autoxidative changes in the lipid fractions. Karel et al. (1975) have shown that free radicals formed during autoxidation of unsaturated fat are transferred to protein. An electron-spin resonance spectroscopic study by Uchiyama and Uchiyama (1979, 1981) indicated that free radicals were produced directly and retained in the protein fraction mainly during heating or y irradiation. Free radical production was highest in lysine, followed by tryptophan, phenylalanine, glutamic acid, methionine, and tyrosine. Other amino acids had zero value. Whether or not these trace radicals have any physiological implication seems to be a controversial issue. Although Renner and Reichelt (1973) found no evidence of toxic effect in rats due to feeding diets containing trace radicals, other workers believe that the formation of free radicals in food is the causative factor of mutagenicity (Kosuge et al., 1980; Uyteta et al., 1978; Imoto, 1979). Recently Kawashima et al. (1979) provided evidence that a collagen hydrolysate (gelatin), especially the fraction with MW 1300-2500, exhibited an outstanding synergistic effect in depressing autoxidation of unsaturated fatty acids in lard. The synergistic effect of the hydrolysate has been assigned to the proline content (Bishov and Henick, 1972), which is extraordinarily high in collagen. Possibly, proline nitroxide formed from proline acts as a synergist in the oxidizing system (Van der Veen et al., 1970; J. S. Lin et al., 1974). Another possibility may be the complex brown pigments, formed by Maillard-type reactions between reducing sugars and amino acids on heating in the presence of organic acids. Despite the biological significance of such reactions (Erbersdobler, 1977) the resulting brown pigments have been found to display a greater antioxidant capacity than some of the synthetic antioxidants (Griffith and Johnson, 1957; Kirigaya et al., 1968; Hwang and Kim, 1973; Itoh et al., 1975; Kawashima et al., 1977; Tufail, 1977; Lingnert, 1980; Eichner, 1980). Ebro et al. (1979, 1980) conducted a series of studies using food-grade collagen in various bakery products such as beef loaves, whole wheat muffins, sweet wheat loaf (loaf bread), corn meal muffins, plain cakes, applesauce and carrot cake, oatmeal cookies, and plain and whole wheat spatzle (German noodles). These studies found little adverse effect of five types of collagen products on certain quality criteria (e.g., juiciness, chewability) of beef loaves, whereas the overall scores for texture and flavor were higher for loaves containing col-

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

33 1

lagen product No. 4. The 20% addition level gave better firmness to the beef loaf than 10 or 30% levels. Objective measurement of loaf volume and color did not reveal any significant influence of the collagen level or product type using the volume index, lightness index, or dominant wavelength of beef loaf. Ebro et al. (1980) have shown that substitution of air-dried collagen into a plain muffin formulation was not encouraging because the aroma was not acceptable, although the cellular structure was comparable with the reference samples. On the other hand, whole wheat muffins, containing 5% collagen, were as good as the reference samples in aroma but the texture was grainy. This was also true for sweet whole wheat loaf. The organoleptic characteristics of corn meal muffins, containing 10% collagen, were rated equal to the reference samples at the higher levels, but the quality tended to decline. Studies on cakes suggested that white cake is not a suitable medium for collagen supplementation because the granular nature of air-dried collagen resists proper blending with plain cake, which is supposed to have a velvety smooth texture. However, carrot and applesauce cakes containing raisins and nuts appear to be an appropriate medium for fibrous collagen supplementation.

IX. RESEARCH NEEDS A review by Asghar and Pearson (1980) indicated that the tenderness of meat has been viewed traditionally with reference to the amount or solubility of the connective tissue, yet the opinions of different workers have always been conflicting. Microscopic differences in collagen of muscle from the same lamb breed (Poll Merino X Dorset Horn) has been reported as having some “fibrillar” collagen characteristics, whereas others had “smooth” appearances (Asghar, 1969; Asghar and F. M. Yeates, 1968, 1979b). But their significance in relation to tenderness is not known. Fortunately, the discovery of genetically different collagen types (such as collagen type I, [~xI(I)]~[a2(1)], type 11, [~x1(11)]~, type 111, [~x1(111)]~, type IV, [cxl(IV)l3, and perhaps others (Eyre, 1980)), which are believed to be produced by different nonallelic structural genes (Harwood, 1979) in different tissues of various species, has opened new avenues by which meat scientists can approach the issue of meat tenderness. First, attempts can now be made to discover if a correlation exists between the type of collagen in a muscle and tenderness. Second, attention may be directed to exploring whether or not all types of collagen have the same potentialities of forming cross-linkages, which become thermally stable with advancing biological age of the animal. The present state of knowledge regarding the chemical nature of the crosslinkages in collagen has already been discussed in Section II1,D. It may, however, be realized that most of the information on this aspect has been derived from

332

A. ASGHAR AND R. L. HENRICKSON

the studies of collagen from various tissues other than muscle. It is not clearly known what types of cross-linkages predominate in intramuscular collagen from different species of meat animals. A detailed account of the chemistry and biochemistry of the postmortem aging process presented earlier (Asghar and Yeates, 1978) has indicated inconsistent findings of different researchers who relied on the solubility criterion of connective tissue. Since the chemistry of various cross-linkages in collagen is now well established (Tanzer, 1976), it would be worthwhile to examine the postmortem changes in connective tissue of muscle in terms of the chemical linkages which are labile to the actions of lactic acid and/or lysosomal cathepsins. The studies on type I collagen mRNAs have successfully established the gene coding for pro-al(I) and pro-a2(I) chains. This may help in constructing a structural map of monocistronic procollagen mRNA and its probable translation products (Hanvood, 1979). Similar approaches may be applied to investigate mRNAs for other types of collagen for better understanding of their transcriptional and translational control at subcellular levels. Once the fundamental information on the factors responsible for gene coding on individual types of collagen is elucidated, the appropriate genes may be activated to encourage specific types of collagen (Harwood, 1979). Since the synthesis of different types of collagen is under genetic control, it seems feasible to screen the breeding stock for a specific type of collagen which may be associated with tenderness. Thus, by proper genetic manipulation, meat animal breeds with desired collagen types may be evolved. Another important area which merits further investigation is the functional properties of fibrous collagen in different food systems. It has already been mentioned (Section VI,A) that one of the most important functional properties of fibrous collagen in a food system is the water-holding capacity. However, little is known about the different variables affecting this property of collagen except the pH and a few ions. It is also not clear as to how different chemically modified collagen derivatives (e.g., ether, ester, phosphate, sulfate) would behave as moisturizers, binders, emulsifiers, texturizers, and extenders in different food systems. For this type of investigation, the experimental methodology available on starch (Kerr, 1950; Roberts, 1967; Hamilton and Paschall, 1967; Kruger and Rutenberg, 1967; Knight, 1967; Radley, 1968), casein (Southward and Goldman, 1975, 1978), soy proteins (Meyer and Williams, 1977; Richardson, 1977), and cellulose (Ott et al., 1954; Honeyman, 1959; Yarsley et al., 1964) derivatives may well serve as models for the development and evaluation of fibrous collagen derivatives. The modifications can possibly be achieved with different degrees of substitution at hydroxyl groups of the carbohydrate moiety, associated with collagen molecules to impart desired functional properties needed for different food systems.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

333

ACKNOWLEDGMENTS Journal Services Paper 896 of the Oklahoma Agricultural Experiment Station. Financed in part by the USDA Science and Education Administration, Eastem Regional Research Center, Philadelphia, Coop. Agreement 58-32-U-4-8-2. The authors wish to acknowledge the editorial assistance of Dr. George Gorin, Deborah Doray, Susan Johnson, Dorothy Sipe, and Lyn Sweet.

REFERENCES Adams, E., and Frank, L. 1980. Metabolism of proline and hydroxyproline. Annu. Rev. Biochem. 49, 1005. Adams, S. L., Sobel, M. E., and Howard, B. J. 1977. Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous Sarcoma virustransformed chick embryo fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 74, 3399. Abidi, S. A. 1971. Intestinal transport of dipeptides in man: Relative importance of hydrolysis and intact absorption. J . Clin. Invest. 50, 2266. Adzet, J. M., Soler, J . , Morera, A,, and Antonio, A. 1979. Hydrothermal stability of bated hides treated with various salts. AQEK Biol. Tech. 30(11), 301 (in Spanish). Aguilar, J. H., Jacobs, H. G., Butler, W. T., and Cunningham, L. W. 1973. The distribution of carbohydrate groups in rat skin collagen. J . Biol. Chem. 248, 5106. Akeson, W. R., and Stahmann, M. A. 1964. A pepsin-pancreatin digest index of protein quality evaluation. J . Nutr. 83, 257. Alexander, J. 1923. ‘‘Glue and Gelatin,” p. 236. Chem. Catalog Co., New York. Allison, J. B. 1964. The nutritive value of dietary protein. In “Mammalian Protein Metabolism” (H. N. Munro and J. B. Allison, eds.), Vol. 2, p. 41. Academic Press, New York. Alsmeyer, R. H., Cunningham, A. E., and Happich, M. L. 1974. Equations predict PER from amino acid analysis. Food Technol. 28, 34. Altgelt, K . , Hodge, A. J., and Schmitt, F. 0. 1961. Gamma tropocollagen: A reversibly denaturable collagen macromolecule. Proc. Natl. Acad. Sci. U.S.A. 47, 1914. Altschul, A. M. 1958. “Processed Plant Protein Foodstuffs,” pp. 79, 131. Academic Press, New York. Ames, W. M. 1944. The preparation of gelatin. Parts 1-111. J . SOC. Chem. Ind., London 63, 200, 234, 277. Anderson, J. C., and Jackson, D. S. 1972. Isolation of glycoproteins from bovine Achilles tendon and their interaction with collagen. Biochem. J . 127, 179. Angennann, K., and Barrach, H. J. 1979. Purification of procollagen type I1 by covalent chromatography with activated thiol Sepharose 4B. Anal. Biochem. 94, 253. Aoki, H., Taneyama, O., and Inami, M. 1980. Emulsifying properties of soy protein: Characteristics of 7 S and 11 S proteins. J . Food Sci. 45, 535. Asghar, A. 1969. Biophysical, biochemical, and microstructural aspects of lambs muscle, grown under different nutritional states, with particular reference to meat quality. Ph.D. Thesis, University of New England, Australia. Asghar, A,, and Henrickson, R. L. 1981. Influence of different ions on the hydration capacity of food-grade freeze-dried collagen from cattle-hide. J . Food Sci. 47, No. 3. Asghar, A., and Pearson, A. M. 1980. Influence of ante- and postmortem treatments on muscle composition and meat quality. Adv. Food Res. 26, 53. Asghar, A,, and Yeates, F. M. 1968. Effect of growth rate on chemical and histological aspects of muscle. Proc. Aust. SOC. Anim. Prod. 7, 89.

334

A. ASGHAR AND R. L. HENRICKSON

Asghar, A., and Yeates, F. M. 1979. Muscle characteristics and meat quality of lambs grown on different nutritional planes. 3. Effect on ultrastructure of muscle. Agric. Biol. Chem. 43, 445. Asghar, A,, and Yeates, N. T. M. 1977. Correlations between physicochemical and organoleptic characteristics of lamb L. dorsi muscle. J . Sci. Food Agric. 28, 1. Asghar, A., and Yeates, N. T. M. 1978. The mechanism for the promotion of tenderness in meat during postmortem aging process. Crit. Rev. Food Sci. Nutr. 8(3), 1. Asghar, A , , and Yeates, N. T. M. 1979. Muscle characteristics and meat quality of lambs grown on different nutritional planes. 2. Chemical and biochemical effects. Agric. Biol. Chem. 43, 437. Asghar, A., Pearson, A. M . , Magee, W. T., and Tahir, M. A. 1981. Effects of ad libitum, maintenance, and submaintenance feeding and of compensatory growth on some biochemical properties of muscle from weanling rabbits. J . Nutr. 111, 1343. Ashley, J. H., and Fisher, H. 1966. The amino acid deficiencies of gelatin for the growing chicken. Poult. Sci. 45, 541. Astbury, W. T. 1940. The molecular structure of the fibers of the collagen group. J . Int. Soc. Leather Trades’ Chem. 24, 69. Avioli, L. V . , McDonald, J. E., Henneman, P. H., and Lee, S. W. 1966. Relation of parathyroid activity to pyrophosphate excretion. J . Clin. Invest. 45, 1093. Avshalumova, A. D. 1974. [Comparative characteristics of the quality of meat from normal and castrated bulls from Red Steppe breed.] Tr. Dages. S-kh. Inst. 22, 132, cited in Chem. Abstr. 83, 1 1 3782q. Badger, R. M., and Pullin, A. D. E. 1954. The infrared spectrum and structure of collagen. J . Chem. Phys. 22, 1142. Bailey, A. J. 1968. Intermediate labile intermolecular cross-links in collagen fibres. Biochim. Biophys. Acta 160, 447. Bailey, A. J. 1969. The stabilization of the intermolecular crosslinks of collagen with aging. Gerontologia 15, 65. Bailey, A. J. 1970. Comparative studies on the nature of the cross-links in the collagen of various fish tissues. Biochim. Biophys. Acta 221, 652. Bailey, A. J. 1972. The basis of meat texture. J . Sci. Food Agric. 23, 995. Bailey, A. J. 1974. Collagen: General introduction. In “Structure of Fibrous Biopolymers” (E. D. T. Atkins and A. Keller, eds.), p. 115. Buttenvorth, London. Bailey, A. J., and Lapfre, C. M. 1973. Effect of an additional peptide extension of the N-terminus of collagen from dermatosparactic calves on the cross-linking of the collagen fibres. Eur. J . Biochem. 34, 91. Bailey, A. J., and Lister, D. 1968. Thermally labile cross-links in native collagen. Nature (London) 220, 280. Bailey, A. J., and Peach, C. M. 1968. Isolation and structural identification of a labile intermolecular cross-link in collagen. Biochem. Biophys. Res. Commun. 33, 812. Bailey, A. J., and Robins, S. P. 1976. Current topics in the biosynthesis, structure, and function of collagen. Sci. Prog. (Oxford) 63, 421. Bailey, A. J., and Shimokomaki, M. S. 1971. Age related changes in the reducible cross-links of collagen. FEBS Lett. 16, 86. Bailey, A. J . , and Sims, T. J . 1976. Chemistry of collagen cross-links: Nature of the cross-links in the polymorphic forms of dermal collagen during development. Biochem. J . 153, 21 1. Bailey, A. J . , and Sims, T. J. 1977. Meat tenderness: Distribution of molecular species of collagen in bovine muscles. J . Sci. Food Agric. 28, 565. Bailey, A. J., Fowler, L. J., and Peach, C. M. 1969. Identification of two interchain cross-links of bone and dentine collagen. Biochem. Biophys. Res. Cornmun. 35, 663. Bailey, A. J., Peach, C. M., and Fowler, L. J. 1970. Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen. Biochem. J . 117, 819.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

335

Bailey, A. J., Bazin, S . , and Delauney, A. 1973. Changes in the nature of the collagen during development and resorption of granulation tissue. Biochim. Biophys. Actu 328, 383. Bailey, A. J . , Robins, S. P., and Balian, G. 1974. Biological significance of the intermolecular cross-links of collagen. Nature (London) 251, 105. Bailey, A. J., Ranta, M. H., and Nicholls, A. C. 1977. Isolation of a-amino-6-adipic acid from mature dermal collagen and elastin: Evidence for an oxidative pathway in the maturation of collagen and elastin. Biochem. Biophys. Res. Commun. 78, 1403. Bailey, A. J., Restall, D. J., Sims, T. J., and Duance, V. C. 1979. Meat tenderness: Immunofluorescent localization of the isomorphic forms of collagen in bovine muscle of varying texture. J . Sci. Food Agric. 30, 203. Baker, S. P., Gaffney, G . W., Shock, M. W., and Landowne, W. 1959. Physiological responses of 5 middle-aged elderly men to repeated administration of thyroid stimulating hormones. J . Gerontol. 24, 37. Bakerman, S. 1964. Distribution of the a- and P-components in human skin collagen with age. Biochim. Biophys. Actu 90, 621. Balazs, E. A., ed. 1970a. “Chemistry and Molecular Biology of the Intercellular Matrix,” Vol. 1, pp. 195-250. Academic Press, New York. Balazs, E. A , , ed. 1970b. “Chemistry and Molecular Biology of the Intercellular Matrix,” Vol. 2, pp. 1231-1418. Academic Press, New York. Balazs, E. A. 1977. Intercellular matrix of connective tissue. In “Handbook of the Biology of Aging” (C. E. Finch and L. Hayflick, eds.), p. 222. Van Nostrand-Reinhold, Princeton, New Jersey. Balian, G., and Bowes, J. H. 1977. The structure and properties of collagen. In “The Science and Technology of Gelatin” (A. G. Ward and A. Courts, eds.), p. 11. Academic Press, New York. Ballou, G . A,, Boyer, P. D., Luck, J. M . , and Lum, F. G . 1944. The coagulation of human serum albumin. J . Biol. Chem. 153, 589. Banga, I . , and Balo, J. 1965. Correlation between cross-linkages, steric structure, and digestibility of collagen. Biochem. Z . 342, 330. Barker, S. A,, Kennedy, J. F., and Cruickshank, C. N. D. 1969. Identification of acidic mucopolysaccharides from human skin by radioactive incorporation. Curbohydr. Res. 10, 65. Barnes, M. J. 1975. Function of ascorbic acid in collagen metabolism. Ann. N.Y. Acud. Sci. 258, 264. Barnes, M. J., Constable, B. J., Morton, L. F., and Royce, P. M. 1974. Age-related variations in hydroxylation of lysine and proline in collagen. Biochem. J . 139, 461. Barnes, M. J., Morton, L. F., and Levene, C. I. 1976. Synthesis of collagen types I and 111 by pig medial smooth muscle cells in culture. Biochem. Biophys. Res. Commun. 70, 339. Barrett, A. J . 1974. The enzymic degradation of cartilage matrix. I n “Dynamics of Connective Tissue Macromolecules” (P. M. C. Burleigh and A. R. Poole, eds.), p. 189. North-Holland Pub., Amsterdam. Barrow, M. V., Simpson, C. F., and Miller, E. J. 1974. Lathyrism: A review. Q. Rev. Biol. 49(2), 101.

Basolo, F., and Pearson, R. G. 1956. “Mechanisms of Inorganic Reactions,” p. 5 . Wiley, New York. Battista, 0. A. 1975. “Microcrystal Polymer Science,” p. 58. McGraw-Hill, New York. Bayne, B. H., Strawn, S. S . , Hutton, C. W., Backus, W. R., and Meyer, B. H. 1971. Relationship of alkali and Ringer insoluble collagen to tenderness of beef heated at two rates. J . Anim. Sci. 33, 958. Bazin, S., and Delaunay, A. 1972. Role of cathepsins in the degradation of intercellular matrix in granulation. J . Denr. Res. 51, 244. Beachey, E. H., Chiang, T. M., and Kang, A. H. 1979. Collagen-platelet interaction. Int. Rev. Connect. Tissue Res. 8, 1.

336

A. ASGHAR AND R. L. HENRICKSON

Bear, R. S. 1952. The structure of collagen fibrils. Adv. Protein Chem. 7 , 69. Becher, P. 1965. The chemistry of emulsifying agents. “Emulsions: Theory and Practice,” 2nd ed., p. 209. Van Nostrand-Reinhold, Princeton, New Jersey. Becker, U., Timpl, R., and Kiihn, K. 1972. Carboxyterminal antigenic determinants of collagen from calf skin. Localization within discrete regions of the nonhelical sequence. Eur. J . Biochem. 28, 221. Becker, U., Furthmayr, H., and Timpl, R. 1975a. Tryptic peptides from the cross-linking regions of insoluble calf skin collagen. Hoppe-Seyler’s Z . Physiol. Chem. 356, 21. Becker, U., Fietzek, P. P., Furthmayr, H., and Timpl, R. 1975b. Nonhelical sequences of rabbit collagen. Correlation with antigen determinants detected by rabbit antibodies in homologous regions on rat and calf collagen. Eur. J . Biochem. 54, 359. Becker, U., Helle, D., and Timpl, R. 1977. Characterization of the amino-terminal segment in procollagen pa2 chain from dermatosparactic sheep. FEBS Lett. 73, 197. Beigler, M. A. 1969. Practical problems of amino acid fortification. In “Protein-Enriched Cereal Foods for World Needs” (M. Milner, ed.), p. 200. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Bello, J. 1963. On the mechanism of action of neutral salt on the collagen-fold. Biochemistry, 2, 276. Bello, J., Riese, H. C. A,, and Vinograd, J. R. 1956. Mechanism of gelatin of gelatin. Influence of certain electrolytes on the melting of gels of gelatin and chemistry of modified gelatin. J . Phys. Chem. 60, 1299. Bender, A. E. 1973. Chemical scores and availability of amino acids. In “Proteins in Human Nutrition” (J. W. G. Porter and B. A. Rolls, eds.), p. 167. Academic Press, New York. Bender, A. E. 1975. Rat assays for protein quality-a reappraisal. Proc. 9th Int. Congr. Nurririon, Mexico, 1972, Vol. 3, p. 310. Bender, A. E., and Doell, B. H. 1957. Biological evaluation of proteins: A new aspect. Br. 1.Nutr. 11, 140. Bender, A. E., and Husaini, M. 1976. Nutritive value of proteins in canned meat products. J . Food Technol. 11, 499. Bender, A. E., and Miller, D. S . 1953. A brief method of estimating net protein value. Biochem. J . 53, vii. Bender, A. E., and Zia, M. 1976. Meat quality and protein quality. J . Food Technol. 11, 495. Bennett, H. G . 1921. “Animal Proteins,” p. 287. Van Nostrand-Reinhold, Princeton, New Jersey. Bensusan, H. B. 1965. A novel hypothesis for the mechanism of crosslinking in collagen. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 42. Butterworth, London. Bensusan, H. B. 1969. The glutamyl linkage in collagen. Biochemistry 8, 4723. Bensusan, H. B. 1972. Investigation of cross-links in collagen following an enzymic hydrolysis. Biochim. Biophys. Acta 285, 447. Bensusan, H. B., and Hoyt, B. L. 1958. The effect of various parameters on the rate of formation of fibers from collagen solutions. J . Am. Chem. SOC.80, 719. Bensusan, H. B., and Scanu, A. 1960. Fiber formation from solutions of collagen. 11. Role of tyrosyl residues. J . Am. Chem. SOC.82, 4990. Benya, P. D., Padilla, S. R., and Nimni, M. E. 1977. The progeny of rabbit articular chondrocytes synthesize collagen type I, 111 and type I trimer. Biochemistry 16, 865. Benya, P. D., Padilla, S. R., and Nimni, M. E. 1978. Independent regulation of collagen types by chondrocytes during loss of differentiated function in culture. Cell 15, 1313. Berendsen, H. J. C. 1972. Interaction of water and proteins. Proc. FEES Meet. 29, 19. Berg, R. A., and Prockop, D. J. 1973. The thermal transition of a nonhydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple helix of collagen. Biochem. Biophys. Res. Commun. 52, 115.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

337

Berg, R. A., Kao, W. W. Y., and Prockop, D. J. 1976. Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate. Biochim. Biophys. Acta 444,756. Berliner, D. L., and Nabores, C. J., Jr. 1967. Effect of corticosteroids on fibroblast function. RESJ. Reticuloendothel. SOC. 4, 284. Bettleheim, F. A. 1974. Structure and hydration of acidic polysaccharides. In “Structure of Fibrous Biopolymers” (E. D. T. Atkins and A. Keller, eds.), p. 65. Butterworth, London. Bhandari, V. K. 1978. Pharmaceutical grade: Gelatin manufacture. East. Pharm. 21, 21. Bishov, S. J., and Henick, A. S. 1972. Antioxidant effect of protein hydrolyzates. J . Food Sci. 37, 873. Bishov, S. J., and Henick, A. S. 1975. Antioxidant effect of protein hydrolyzates in freeze-dried model systems. J . Food Sci. 40, 345. Bishov, S. J., Masuoka, Y., and Henick, A. S. 1967. Fat quality and stability in dehydrated proteinaceous food mixes. Food Technol. 21, 148A. Bjamasson-Baumann, B., Pfaender, P., and Siebert, G. 1977. Enhancement of the biological value of whey protein by covalent addition into peptide linkage of limiting essential amino acids. Nutr. Metab. 21, Suppl. 1, 170. Blackwell, J., and Gelman, R. A. 1974. Polypeptide-mucopolysaccharide interactions. In “Structure of Fibrous Biopolymers” (E. D. T. Atkins and A. Keller, eds.), p. 93. Butterworth, London. Blobel, G . , and Dobberstein, B. 1975. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membranebound ribosomes of murine myeloma. J . Cell Biol. 67, 835. Blumenfeld, 0. O., and Gallop, P. M. 1962. The participation of aspartyl residues in the hydroxylamine or hydrazine sensitive bonds of collagen. Biochemistry 1, 947. Blumenfeld, 0. O., and Gallop, P. M. 1966. Amino aldehydes in collagen: The nature of a probable cross-link. Proc. Natl. Acad. Sci. U.S.A. 56, 1260. Bodwell, C. E. 1975. Biochemical parameters as indices of protein nutritional value. In “Protein Nutritional Quality of Foods and Feeds” (M. Friedman, ed.), Part 1 , p. 261. Dekker, New York. Bodwell, C. E. 1977a. Biochemical indices in humans. In “Evaluation of Proteins for Humans” (C. E. Bodwell, ed.), p. 119. AVI Publ. Co., Westport, Connecticut. Bodwell, C. E. 1977b. Problems in the development and application of rapid methods of assessing protein quality. Food Technol. 31, 73. Bodwell, C. E. 1977c. The application of animal data to human protein nutrition: A review. Cereal Chem. 54, 958. Boedtker, H., and Doty, P. 1956. The native and denatured states of soluble collagen. J . Am. Chem. SOC. 78, 4267. Bogue, R. H. 1922. “The Chemistry and Technology of Gelatin and Glue,” p. 644. McGraw-Hill, New York. Bolam, T. R. 1932. “The Donnan Equilibria,” p. 25. Bell, London. Borasky, R . , ed. 1963. “Ultrastructure of Protein Fibers,” p. 19. Academic Press, New York. Bornstein, P. 1970. Structure of al-CB8, a large cyanogen bromide fragment from the a 1 chain of rat collagen. The nature of a hydroxylamine-sensative bond and composition of tryptic peptides. Biochemistry 9, 2408. Bornstein, P. 1974. The biosynthesis of collagen. Annu. Rev. Biochem. 43,567. Bornstein, P., and Sage, H. 1980. Structurally distinct collagen types. Annu. Rev. Biochem. 49,957. Bornstein, P., and Traub, W. 1979. The chemistry and biology of collagen. In “The Proteins” (H. Neurath and R. H. Hill, eds.), 3rd ed., Vol. 4, p. 412. Academic Press, New York. Bornstein, P., Martin, G . R., and Piez, K. A. 1964. Intermolecular cross-linking of collagen and the identification of a new f3-component. Science 144, 1220.

338

A. ASGHAR AND R. L. HENRICKSON

Bose, S . M. 1963. The distribution, nature, and linking of sialic acid in skin proteins. Biochim. Biophys. Acta 74, 265. Bowes, J . H., and Kenten, R. H. 1948a. The amino acid composition and tritation curve of collagen. Biochem. J . 43, 358. Bowes, J. H., and Kenten, R . H. 1948b. The effect of alkalis on collagen. Biochem J . 43, 364. Bowes, J . H., and Raistrick, A. S . 1968. The influence of current trends in livestock production on the composition and leather making properties of hides and skin. J . Am. Leather Chem. Assoc. 63, 192. Bowes, J. H., Elliott, R. G., and Moss, J. A. 1953. In “Nature and Structure of Collagen” (J. T. Randall, ed.), p. 199. Butterworth, London. Bowes, J. H., Elliott, R . D., and Moss, J. A. 1955. The composition of collagen and acid-soluble collagen of bovine skin. Biochem. J . 61, 143. Bowes, J. H., Elliott, R. G., and Moss, J. A. 1957. Collagen and the more soluble constituents of skin. J . Soc. Leather Trades’ Chem. 41, 249. Boyd, J., Parkinson, C., and Sherman, P. 1972. Factors affecting emulsion stability, and the HLB concept. J . Colloid Interface Sci. 41, 359. Boyer, P. D., Lum, F. G . , Ballou, G. A,, Luck, J. M., and Rice, R. G. 1946. The combination of fatty acids and related compounds with serum albumin. 1. Stabilization against heat denaturation. J . B i d . Chem. 162, 181. Bozler, E. 1955. Binding of calcium and magnesium by the contractile elements. J . Gen. Physiol. 38, 735. Branwood, A. W. 1963. The fibroblasts. Int. Rev. Connect. Tissue Res. 1, I . Braun, E., and Braun, B. 1956. Production of collagen strands. U.S. Patent 2,747228. Bressani, R. 1975. Addition of amino acids in foods. I n “Nutritional Evaluation of Food Processing” (R. S. Harris and E. Karmas, eds.), p. 568. AVI Publ. Co., Westport, Connecticut. Bronner, F. 1961. Parathyroid effects on sulfate metabolism. In “Parathyroids” (R. 0. Creep and R . V. Talmage, eds.), p. 123. Thomas, Springfield, Illinois. Brownell, A. G., and Veis, A. 1975. The intracellular location of the glycosylation of hydroxylysine of collagen. Biochem. Biophys. Res. Commun. 63, 371. Bruns, R. R., and Gross, J. 1973. Band pattern of the segment-long-spacing form of collagen. Its use in the analysis of primary structure. Biochemistly 12, 808. Bruns, R. R., Trelstad, R. L., and Gross, J. 1973. Cartilage collagen: A staggered substructure in reconstituted fibrils. Science 181, 269. Buchanan, R . A,, and Byers, M. 1969. Interference by cyanide with the measurements of papain hydrolysis. J . Sci. Food Agric. 20, 364. Budy, A. M., Urist, M. R., and McLean, F. G. 1952. The effect of estrogens on the growth apparatus of the bones of immature rats. Am. J . Pathol. 28, 1143. Bull, H. B., and Breese, K. 1968. Protein hydration. 1. Binding sites. Arch. Biochem. Biophys. 128, 488. Bull, H. B., and Breese, K. 1970. Water and solute binding by proteins. Arch. Biochem. Biophys. 137, 299. Burgeson, R . E., and Hollister, D. W. 1977. P a p . , West Coast Connect. Tissue Soc. Meet. p. 5 (cited by Hanvood, 1979). Burgeson, R. E., El-Adli, F. A,, Kaitila, I. I., and Hollister, D. W. 1976. Fetal membrane collagens: Identification of two new collagen alpha chains. Proc. Natl. Acad. Sci. U . S . A . 73, 2579. Burleigh, M. C., Barrett, A. J., and Lazarus, G. S. 1974. Cathepsin B,: A lysosomal enzyme that degrades native collagen. Biochem. J . 137, 387. Burley, R. W. 1971. Lipoproteins. In “Biochemistry and Methodology of Lipids” (A. R. Johnson and J. B. Davenport, eds.), p. 85. Wiley (Interscience), New York.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

339

Burston, D., Addison, J. M., and Matthews, D. M. 1972. Uptake of dipeptides containing basic and acidic acids by rat small intestine in vitro. Clin. Sci. 43, 823. Butler, W. T. 1968. Partial hydroxylation of certain lysines in collagen. Science 161, 796. Butler, W. T. 1970. Chemical studies on the cyanogen bromide peptides of rat skin collagen. The covalent structure of al-CB5, the major hexose-containing cyanogen bromide peptide of a l . Biochemistry 9, 44. Butler, W. T., and Cunningham, L. W. 1966. Evidence for the linkage of a disaccharide to hydroxylysine in tropocollagen. J . Biol. Chem. 241, 3882. Butler, W. T., Finch, J. E., and Miller, E. J. 1977. The covalent structure of cartilage collagen. J . Biol. Chem. 252, 639. Byers, P. H., McKenney, K. H., Lichtenstein, J. R., and Martin, G . R. 1974. Preparation of type 111 procollagen and collagen from rat skin. Biochemistry 13, 5243. Byers, P. H . , Click, E. M., Harper, E., and Bornstein, P. 1975. Interchain disulfide bonds in procollagen are located in a large non-triple-helical COOH-terminal domain. Proc. Natl. Acad. Sci. U.S.A. 72, 3009. Cannon, D. J., and Davison, P. F. 1973. Cross-linking and aging in rat tendon collagen. Exp. Gerontol. 8, 5 1 . Canonico, P. G., and Bird, J W. C. 1970. Lysosomes in skeletal muscle tissue: Zonal centrifugation evidence for multiple cellular sources. J . Cell Biol. 45, 321. Cante, C. J., Franzen, R. W., and Saleeb, F. Z . 1979. Proteins as emulsifiers. Methods for assessing the role. J . Am. Oil Chem. Soc. 56(1), 71A. Cardinale, G. J., and Udenfriend, S. 1974. Prolyl hydroxylase. Adv. Enzymol. 41, 245. Carpenter, J. A., and Saffle, R. L. 1965. Some physical and chemical factors affecting the emulsifying capacity of meat protein extracts. Food Technol. 19, 1567. Carpenter, K. J., and Booth, V. H. 1973. Damage to lysine in food processing: Its measurement and its significance. Nutr. Rev. 43, 424. Carpenter, K . J., McDonald, I., and Miller, W. S. 1972. Protein quality of feeding-stuffs. 5 . Collaborative studies on the biological value of available methionine using chicks. Br. J . Nutr. 27, I . Cam, C. W. 1953. Studies on the binding of small ions in protein solutions with the use of membrane electrodes. 11. The binding of calcium ions in solutions of bovine serum albumin. Arch. Biochem. Biophys. 43, 147. Cassel, J. M., and McKenna, E. 1953. Amide nitrogen of collagen and hide powder. J . Am. Leather Chem. Assoc. 48, 142. Cassens, R. G. 1971. Microscopic structure of animal tissues. In “The Science of Meat and Meat Products” (J. E. Price and B. S. Schweigert, eds.), p. 11. Freeman, San Francisco, California. Castor, W. C . , and Prince, R. K. 1964. Modulation of the intrinsic viscosity of hyaluronic acid formed by human fibroblasts in vitro: The effect of hydrocortisone and colchicine. Biochim. Biophys. Acta 83, 165. Cerny, L., Groug, J., and James, H. 1970. A viscometric study of dilute collagen solutions. Biorheology 6, 161. Chandra-Rajan, J. 1978. Separation of type 111 from type I collagen and pepsin by differential denaturation and renaturation. Biochem. Biophys. Res. Commun. 83, 180. Chang, R. W. H., and Linn, F. M. 1964. Stabilization of linoleic acid by arginine and lysine against autoxidation. J . Am. Oil Chem. SOC. 41, 780. Chapman, D. G., Castillo, R., and Campbell, J. A. 1959. Evaluation of protein in foods. I . A method for the determination of protein efficiency ratios. Can. J . Biochem. Physiol. 37, 679. Chapman, J. A. 1974. The staining pattern of collagen fibrils. I. An analysis of electron micrographs. Connect. Tissue Res. 2, 137.

340

A. ASGHAR AND R. L. HENRICKSON

Chapman, J. A , , and Hardcastle, R. A. 1974. The staining pattern of collagen fibrils. 11. A comparison with patterns computer-generated from the amino acid sequence. Connect. Tissue Res. 2, 151. Cheng, B., Navab, F . , Lis, M. T., Miller, T. N . , and Matthews, D. M. 1971. Mechanisms of dipeptide uptake by rat small intestine in vitro. Clin. Sci. 40, 247. Cheng, C. S . , and Parrish, F. C., Jr. 1976. Scanning electron microscopy of bovine muscle. Effect of heating temperature on ultrastructure. J . Food Sci. 41, 1449. Chou, D. H., and Morr, C. V. 1979. Protein water interactions and functional properties. J . Am. Oil Chem. Soc. 56(1), 53A. Chung, A. C., and Houck, J. C. 1964. Connective tissue. IX. Effect of hypervitaminosis A upon connective tissue chemistry. Proc. Soc. Exp. B i d . Med. 115, 613. Chung, E., and Miller, E. J. 1974. Collagen polymorphism: Characterization of molecules with a chain composition [(uI(III)]~ in human tissues. Science 183, 1200. Chung, E., Keele, E. M., and Miller, E. J. 1974. Isolation and characterization of the cyanogen bromide peptides from the al(II1) chain of human collagen. Biochemistry 13, 3459. Chung, E., Rhodes, E. K . , and Miller, E. J. 1976. Isolation of three collagenous components of probable basement membrane origin from several tissues. Biochem. Biophys. Res. Commun. 71, 1167. Church, R. L., Pfeiffer, S . E., and Tanzer, M. L. 1971. Collagen biosynthesis: Synthesis and secretion of a high-molecular-weight collagen precursor (procollagen). Proc. Natl. Acad. Sci. U.S.A. 68, 2638. Church, R. L., Yaeger, J. S . , and Tanzer, M. L. 1974. Isolation and partial characterization of procollagen fractions produced by clonal strain of calf dermatosparactic cells. J . Mol. B i d . 86, 785. Chvapil, M., Kronenthal, R. L., and van Winkle, W., Jr. 1973. Medical and surgical applications of collagen. Int. Rev. Connect. Tissue Res. 6, 9. Cifonelli, J . A , , and Roden, L. 1968. The isolation of heparitin sulfate from hog skin. Biochim. Biophys. Acta 165, 553. Cintron, C. 1974. Hydroxylysine glycosides in the collagen of normal and scarred rabbit corneas. Biochem. Biophys. Res. Commun. 60, 288. Circle, S. J., and Smith, A. K. 1972. Functional properties of commercial edible soybean products. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), p. 242. AVI Publ. Co., Westport, Connecticut. Clark, C. C., and Kefalides, N. A. 1976. Carbohydrate moieties of procollagen. Incorporation of isotopically labeled mannose and glucosamine into propeptides of procollagen secreted by matrix-free chick embryo tendon cells. Proc. Nutl. Acad. Sci. U.S.A. 73, 34. Clark, C. C., and Kefalides, N. A. 1978. Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen. J . B i d . Chem. 253, 47. Clark, I., and Smith, M. R. 1964. Effects of hypervitaminosis A and D on skeletal metabolism. J . Biol. Chem. 239, 1266. Clark, I., and Umbreit, W. W. 1954. Effect of cortisone and other steroids on the in vitro synthesis of chondroitin sulfate. Proc. Soc. Exp. Biol. Med. 86, 558. Cohen, J . 1964. Benzoic acid treatment of collagen fibers and chrome tannage thereof. U.S. Patent 3,126,433. Colvin, J. R. 1964. Denaturation: A requiem. In “Symposium on Foods: Proteins and Their Reactions” (H. W. Schultz and A. F. Anglemier, eds.), p. 69. AVI Publ. Co., Westport, Connecticut. Cooke, R., and Kuntz, I. D. 1974. The properties of water in biological systems. Annu. Rev. Biophys. Bioeng. 3, 95.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

34 1

Cooke, R., and Wien, R. 1973. Nuclear magnetic resonance studies of intracellular water protons. Ann. N . Y . Acad. Sci. 204, 197. Cooper, D. R., and Johnson, P. 1958. The soluble proteins of bovine hide 11. Extract ion by aqueous sodium phosphate and half-saturated lime. Biochim. Biophys. Acta 30, 590. Cooper, D. R., Russell, A. E., and Davison, R. T. 1967. The soluble proteins of skin. J . Am. Leather Chem. Assoc. 62, 423. Coulson, W. F., and Cames, W. H. 1962. Cardiovascular studies on Cu deficient swine. 11. Mechanical properties of the aorta. Lab. Invest. 11, 1316. Courts, A. 1977. Uses of collagen in edible products. In “Science and Technology of Gelatin” (A. G. Ward and A. Courts, eds.), p. 396. Academic Press, New York. Courts, A. 1980. Properties and uses of gelatin. In “Applied Protein Chemistry” (R. A . Grant, ed.), Chapter 1. Appl. Sci. F’ubl., Essex, England. Cox, R. J., ed. 1972. “Photographic Gelatin.” Academic Press, New York. Craft, 1. L., Geddes, D., Hyde, C. W., Wise, L. J., and Mathews, D. M. 1968. Absorption and malabsorption of glysine and glysine peptides in man. Gut 9, 425. Cram, J. M . , and Cram, D. J. 1978. “The Essence of Organic Chemistry,” p. 260. AddisonWesley, Reading, Massachusetts. Crenwelge, D. D., Dill, C. W., Tybor, P. T., and Landmann, W. A. 1974. A comparison of the emulsification capacities of some protein concentrates. J . Food Sci. 39, 175. Crouch, E., Sage, H., and Bornstein, P. 1980. Structural basis for apparent heterogeneity of collagens in human basement membranes: Type IV procollagen contains two distinct chains. Proc. Natl. Acad. Sci. U.S.A. 77(2), 745. Cundall, R. B., Lawton, J., Murray, D., and Phillips, G. 0. 1979. Polyelectrolyte complexes. Interaction between collagen and polyanions. Int. J . Biol. Macromol. 1(5), 215. Cutroneo, K. R., Guzman, N. A,, and Sharaway, M. M. 1974. Evidence for a subcellular vesicular site of collagen prolyl hydroxylation. J . Biol. Chem. 249, 5989. Dasler, W . , and Milliser, R. V. 1958. Osteolathyrogenic action of mercaptoethylamine and cystamine. Proc. SOC.Exp. Biol. Med. 98, 759. Davey, C. L., and Gilbert, K. V. 1968. Studies in meat tenderness. J . Food Sci. 33, 2. Davidson, E. A,, and Small, W. 1963. Metabolism in vivo of connective tissue mucopolysaccharides. Biochim. Biophys. Acta 69, 445, 459. Davidson, J. M., McEneany, L. S. G . , and Bornstein, P. 1975. Intermediates in the limited proteolytic conversion of procollagen to collagen. Biochemistry 14, 5 188. Davis, N. R., and Bailey, A. J. 1971. Chemical synthesis of an intermolecular cross-link of collagen: A reevaluation of the structure of syndesine. Biochem. Biophys. Res. Commun. 45, 1416. Davison, P. F. 1973. Homeostasis in extracellular tissues: Insights from studies on collagen. CRC Crit. Rev. Biochem. 1, 201. Davison, P. F., and Berman, M. 1973. Comeal collagenase: Specific cleavage of types (2al)la2 and (a1)3collagens. Connect. Tissue Res. 2, 57. Davison, P. F., Cannon, D. J., and Anderson, L. P. 1972. The effects of acetic acid on collagen cross-links. Connect. Tissue Res. 1, 205. Davison, P. F . , Hong, B. S., and Cannon, D. 1979. Quantitative analysis of the collagens in bovine cornea. Exp. Eye Res. 29, 97. Dawood, A. 1980. Factors affecting the functional properties of fish muscle proteins. Ph.D. Thesis, Michigan State University, East Lansing. Deasy , C. 1962. Occurrence of 3-chlorotyrosine in hydrochloric acid hydrolysates of steerhide collagen preparation. J . Am. Leather Chem. Assoc. 57, 576. Dehm, P., Jimenez, S . A,, Olsen, B. R., and Prockop, D. J. 1972. A transport form of collagen from embryonic tendon: Electron microscopic demonstration of an NH2-terminal extension and evi-

342

A. ASGHAR AND R. L. HENRICKSON

dence suggesting the presence of cysteine in the molecule. Proc. Natl. Acad. Sci. U . S . A . 69, 60. Delaunay, A,, and Bazin, S. 1974. Collagen maturation in granulation tissue. In “Connective Tissues: Biochemistry and Pathophysiology” (R. Fricke and F. Hartmann, eds.). SpringerVerlag, Berlin and New York. Denko, C. W., and Bergenstal, D. M. 1955. The effect of hypophysectomy and growth hormone on 35 S fixation in cartilage. Endocrinology 57, 76. Deryagin (Derjaguin), B., and Landau, L. D. 1941. Theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solutions of electrolytes. Acta Phsicochim. URSS 14, 633; Chem. Abstr. 39, 453 (1945). Deshmukh, A,, Deshmukh, K., and Nimni, M. E. 1971. Synthesis of aldehydes and their interactions during the in vitro aging of collagen. Biochemistry 10, 2337. Deshmukh, K., and Kline, W. G. 1976. Characterization of collagen and its precursors synthesized by rabbit articular-cartilage cells in various culture systems. Eur. J . Biochem. 69, I17 Deshmukh, K . , and Sawyer, B. D. 1977. Synthesis of collagen by chondrocytes in suspension culture: Modulation by calcium, 3’, 5’-cyclic AMP and prostaglandins. Proc. Natl. Acad. Sci. U.S.A. 74, 3864. Diaz de L e h , L., Paglia, L., Breitkreutz, D., and Stern, R. 1977. Evidence that the mRNA for collagen is noncistronic. Biochem. Biophys. Res. Commun. 77, 1 1. Dickerson, J. W. T., and McCance, R. A. 1960. Severe undernutrition in growing and adult animals. Br. J . Nutr. 14, 331. Diegelmann, R. F., Bemstein, L., and Peterkofsky, B. 1973. Cell-free collagen synthesis on membrane-bound polysomes of chick embryo connective tissues and the localization of prolyl hydroxylase on the polysome-membrane complex. J . Biol. Chem. 248, 6514. Dingle, J. T., Fell, H. B., and Lucy, J. A. 1966. Synthesis of connective tissue components. The effect of retinol and hydrocortisone on cultured limb-bone rudiments. Biochem. J . 98, 173. Dingle, J. T., Barrett, A. J., and Weston, P. D. 1971. Cathepsin D: Characterization of immunoinhibition and the confirmation of a role in cartilage breakdown. Biochem. J . 123, 1 . Dixit, S . N., and Bensusan, H. B. 1973. The isolation of cross-linked peptides of collagen involving al-CB6. Biochem. Biophys. Res. Commun. 52, I . Dixit, S . N., Kang, A. H., and Gross. J. 1975. Covalent structure of collagen: Amino acid sequence of al-CB3 of chick skin collagen. Biochemistry 14, 1929. Dorfman, A. 1964. Metabolism of acid mucopolysaccharides. In “Connective Tissue: Intercellular Macromolecules,” p. 155. Little, Brown, Boston, Massachusetts. ougherty, T. F., and Berliner, D. L. 1968. The effect of hormones on connective tissue cells. In “Treatise on Collagen” (B. S. Gould, ed.), Vol. 2, Part A, p. 367. Academic Press, New York. Doyle, J., Szirmai, J. A,, and de Tyssonk, E. R. 1964. Connective tissue changes in the rooster comb during regression. Acta Endocrinol. (Copenhagen) 45, 457. Duance, V. C . , Restall, D. J., Beard, H., Bourne, F. J . , and Bailey, A. J. 1977. The location of three collagen types in skeletal muscle. FEBS Lett. 79, 248. Dutson, T. R. 1974. Connective tissue. Meat Res. Conf. 27, 99. Dutson, T. R., and Lawrie, R. A. 1974. Release of lysosomal enzymes during postmortem conditioning and their relationship to tenderness. J . Food Techno/. 9, 43. DvoMk, Z . , and Vognarovh, 1. 1969. Nutritive value of the proteins of veal, beef, and pork determined on the basis of available essential amino acids or hydroxyproline analysis. J . Sci. Food Agric. 20, 146. Dziewiatkowski, D. D., Ferrante, D., Bronner, F., and Okinaka, G . 1957. Turnover of 35 S sulfate in epiphyses and diaphyses of suckling rats. Nature of the 35 S-labelled compounds. J . Exp. Med. 106. 509.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

343

Eagland, D. 1975a. Protein hydration-its role in stabilizing the helix conformation of the protein. In “Water Relations of Foods” (R. B. Duckworth, ed.), p. 73. Academic Press, New York. Eagland, D. 1975b. The influence of hydration on the stability of hydrophobic colloidal systems. In “Water: A Comprehensive Treatise” (F. Franks, ed.), Vol. 5 , p. 1. Plenum, New York. Ebro, L., Hams, J., Henrickson, R. L., and Sneed, J. 1979. Development and evaluation of food products using food grade collagen. Pap., I F T s 39th Annu. Meet. Ebro, L., Harris, J., Henrickson, R. L., and Sneed, J. 1980. “Development and Evaluation of Food Products using Food-grade Collagen, Prog. Rep. No. 1149-3, 1149-7. Oklahoma State University, Stillwater. Edwin, P. J., Jr. 1971. Antemortem method of tenderization of meat. U.S. Patent 3,577,248; Chem. Abstr. 75, 34249b. Eichner, K. 1980. Antioxidative effect of Maillard reaction intermediates. In “Autoxidation in Food and Biological Systems” (M. G. Simic and M. Karel, eds.), p. 367. Plenum, New York. Eisen, A. Z. 1969. Human skin collagenase: Localization and distribution in normal human skin. J . Invest. Dermatol. 52, 442. Elden, H. R. 1968. Physical properties of collagen fibers. Intl. Rev. Connect. Tissue Res. 4, 283. Eldridge, A . C., Hall, P. K., and Wolf, W. J. 1963. Stable foams from unhydrolysed soybean protein. Food Technol. 17, 1592. Elias, S . , Komanowsky, M., Sinnamon, H., and Aceto, N. C. 1970. Converts collagen to food additives. Food Eng. 42(11), 125. Engel, J. 1962. Investigation of the denaturation and renaturation of soluble collagen by light scattering. Arch. Biochem. Biophys. 97, 150. Epstein, E. H. 1974. [al(III)I3 human skin collagen: Release by pepsin digestion and preponderance in fetal life. J . Biol. Chem. 49, 3225. Epstein, E. H., and Munderloh, N. H. 1975. Isolation and characterization of CNBr peptides of human [al(III)13 collagen and tissue distribution of [al(I)]2a2 and [al(III)]3 collagens. J . Biol. Chem. 250, 9304. Erbersdobler, H. F. 1977. The biological significance of carbohydrate-rich lysine cross-linking during heat-treatment of food proteins. Adv. Exp. Med. Biol. 86A, 387. Erbersdobler, V. H., Pfeiffer, G., Wellhauser, R., and Zucker, H. 1970. Effect of high collagen content in food and the development and deposition of connective tissue in the growing rat. Z . Ernaehrungswiss. 10, 25. Eskin, N. A. M . , Henderson, H. M . , and Townsend, R. J. 1971. “The Biochemistry of Foods,” p. 83. Academic Press, New York. Etherington, D. J. 1972. The nature of the collagenolytic cathepsin of rat liver and its distribution in other rat tissues. Biochem. J . 127, 685. Etherington, D. J. 1974. The purification of bovine cathepsin B, and its mode of action on bovine collagens. Biochem. J . 137, 547. Etherington, D. J. 1976. Bovine spleen cathepsin B I and collagenolytic cathepsin: A comparative study of the properties of the two enzymes in the degradation of native collagen. Biochem. J . 153, 199. Etherington, D. J. 1977. The dissolution of insoluble bovine collagens by cathepsin B,, collagenolytic cathepsin and pepsin: The influence of collagen type, age, and chemical purity on suscep[ability. Conn. Tissue Res. 5 , 135. Evancho, G. M., Hurt, H. D., Devlin, P. A , , Landers, R. E., and Ashton, D. H. 1977. Comparison of Tetruhymena pyriformis W and rat bioassays for the determination of protein quality. J . Food Sci. 42, 444. Evans, E., and Witty, R. 1980. Use of Tetrahymena pyriformis to determine the quality of food protein: A review. Can. Inst. Food Sci. Technol. J . 13(2), 50. Eyre, D. R. 1980. Collagen: Molecular diversity in the body’s protein scaffold. Science 207, 1315.

344

A. ASGHAR AND R. L. HENRICKSON

Eyre, D. R., and Glimcher, M. J. 1972. Reducible cross-links in hydroxylysine-deficient collagens of a heritable disorder of connective tissue. Proc. Nut/. Acad. Sci. U.S.A. 69, 2594. Eyre, D. R., and Glimcher, M. J. 1973a. Analysis of a cross-linked peptide from calf bone collagen: Evidence that hydroxylysyl glycoside participates in the cross-link. Biochem. Biophys. Res. Commun. 52, 663. Eyre, D. R . , and Glimcher, M. J. 1973b. Collagen cross-linking. Isolation of cross-linked peptides from collagen of chicken bone. Biochem. J . 135, 393. Eyre, D. R., and Muir, H. 1975a. Characterization of the major CNBr-derived peptides of porcine type I1 collagen. Connect. Tissue Res. 3, 165. Eyre, D. R., and Muir, H. 1975b. Type 111 collagen: A major constituent of rheumatoid and normal human synovial membrane. Connect. Tissue Res. 4, 1 1. Eyre, D. R., and Oguchi, H. 1980. Hydroxypyridinium cross-links of skeletal collagens: Their measurement, properties, and a proposed pathway of formation. Biochem. Biophys. Res. Commun. 92(2), 403. Fairweather, R. B., Tanzer, M. L., and Gallop, P. M. 1972. Aldol histidine, a new trifunctional collagen cross-link. Biochem. Biophys. Res. Commun. 48, 131 1. Feeney, R. E., and Whitaker, J. R., eds. 1977. “Food Proteins: Improvement through Chemical and Enzymic Modifications.” Am. Chem. SOC.,Washington, D. C. Feldmann, M. 1974. Genes, receptors, signals. In “Immune System” (E. E. Sercarz, A. R. Williamson, and C. F. Fox, eds.), p. 497. Academic Press, New York. Fennema, 0. 1976. Water and ice. In “Principles of Food Science” (0.Fennema, ed.), Part I , p. 13. Dekker, New York. Fennema, 0. 1977. Water and protein hydration. In “Food Proteins” (J. R. Whitaker and S. R. Tannenbaum, eds.), p. 50. AVI Publ., Co., Westport, Connecticut. Fessler, J. H. 1965. Collagen fiber formation observed in buoyant density gradients. In “Structure and Function of Connective and Skeletal Tissue’’ (S. Fitton-Jackson, ed.), p. 80. Butterworth, London. Fessler, J. H., and Fessler, L. I. 1978. Biosynthesis of procollagen. Annu. Rev. Biochem. 47, 129. Fessler, L. I., and Fessler, J. H. 1978. Protein assembly of procollagen and effect of hydroxylation. J . Biol. Chem. 249, 7637. Fessler, L. I., Morris, N. P., and Fessler, J. H. 1975a. Procollagen processing to collagen via disulfide-linked triple-stranded intermediate in chick calvaria. Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, 562. Fessler, L. I., Morris, N. P . , and Fessler, J. H . 1975b. Procollagen: Biological scission of amino and carboxyl extension peptides. Proc. Nutl. Acad. Sci. U . S . A . 72, 4905. Field, R. A., Pearson, A. M., and Schweigert, B. S . 1970a. Labile collagen from epimysial and intramuscular connective tissue as related to Warner-Bratzler shear value. Agric. Food Chem. 18(2), 280. Field, R. A,, Pearson, A. M., and Schweigert, B. S. 1970b. Hydrothermal shrinkage of bovine collagen. J . Anim. Sci. 30, 713. Field, R. A,, Pearson, A. M., Koch, D. E., and Merkel, R. A. 1970c. Thermal behavior of porcine collagen as related to postmortem time. J . Food Sci. 35, 113. Fietzek, P. P., and Kiihn, K. 1973. The covalent structure of collagen: Amino acid sequence of the N-terminal region of a2-CB5 from rat skin collagen. FEBS Lett. 36, 289. Fietzek, P. P., and Kiihn, K. 1974. The covalent structure of collagen: Amino acid sequence of the N-terminal region of a2-CB3 from calf skin collagen. Hoppe-Sey/er’s 2. Physiol. Chem. 335, 647. Fietzek, P. P., and Kuhn, K. 1975. The covalent structure of collagen: Amino acid sequence of alCB2, 4, and 5 from calf skin collagen. Eur. J . Biochem. 52, 77.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

345

Fietzek, P. P., and Kiihn, K. 1976. The primary structure of collagen. In?. Rev. Connect. Tissue Res. 7, 1. Fietzek, P. P., Wendt, P., Kell, I., and Kuhn, K. 1972. The covalent structure of collagen: Amino acid sequence of al-CB3 from calf skin collagen. FEBS Lett. 26, 74. Fietzek, P. P., Breitkreutz, D., and Kiihn, K. 1974a. Amino acid sequence of the amino-terminal region of calf skin collagen. Biochim. Biophys. Acta 365, 30.5. Fietzek, P. P., Furthmayr, H., and Kiihn, K. 1974b. Comparative sequence studies on a2-CB2 from calf, human, rabbit, and pig skin collagen. Eur. J . Biochem. 47, 257. Fietzek, P. P . , Allmann, H., Rauterberg, J., and Wacher, E. 1977. Ordering of cyanogen bromide peptides of type I11 collagen. Proc. Nutl. Acud. Sci. U.S.A. 74, 84. Finley, J. W., and Friedman, M. 1973. Chemical methods for available lysine. Cereal Chem. 50, 101. Finot, P. A,, Bujard, E . , Mottu, F., and Mauron, J. 1977a. Availability of the true Schiff‘s bases of lysine. Adv. Exp. Med. Biol. 86B, 343. Finot, P. A , , Mottu, F., Bujard, E., and Mauron, J. 1977b. N-substituted lysines as sources of lysine in nutrition. 174th Nutl. Meet., Am. Chem. Soc., Div. Agric. Food Chem. Fitton-Jackson, S . 1964. Connective tissue cells. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 6, p. 378. Academic Press, New York. Fitton-Jackson, S. 1965. Antecedent phases in matrix formation. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 277. Butterworth, London. Flint, M. H. 1973. The basis of the histological demonstration of tension in collagen. In “The Ultrastructure of Collagen: Its Relation to the Healing of Wounds and to the Management of Hypertrophic Scar” (J. J. Longacre, ed.), p. 60. Thomas, Springfield, Illinois. Food and Agriculture Organization, 1968. “Amino Acid Content of Foods and Biological Data on Protein.” FAO, Rome. Food and Agriculture OrganizationlWorld Health Organization 1965. Report of a joint FAOiWHO expert group. “Protein Requirements,” Tech. Rep. Ser. No. 301. WHO, Geneva. Food and Agriculture Organization/World Health Organization 1973. Ad Hoc Expert Committee. “Energy and Protein Requirements,” Tech. Rep. Ser. No. 522, p. 63. FAO/WHO, Rome. Forrest, J. C., Aberle, E. D., Hedrick, H. B., Judge, M. D., and Merkel, R. A. 1975. “Principles of Meat Science,” pp, 2.5, 82. Freeman, San Francisco, California. Forrest, L., Shuttleworth, A,, Jackson, D. S., and Mechanic, 0 .L. 1972. A comparison between the reducible intermolecular cross-links of the collagens of mature dermis and young dermal scar tissue of the guinea pig. Biochem. Biophys. Res. Commun. 46, 1776. Franks, F. 197Sa. Water, ice, and solution of simple molecules. In “Water Relations of Foods” (R. B. Duckworth, ed), p. 3. Academic Press, New York. Franks, F. 1975b. The hydrophobic interaction. In “Water: A Comprehensive Treatise” (F. Franks, ed.), Vol. 4, p. 136. Plenum, New York. Franzblau, C. 1971. Elastin. Comp. Biochem. 26, Part C, p. 659. Franzblau, C., Gallop, P. M . , and Seifter, S. 1963. The presence in collagen of y-glutamyl peptide linkages. Biopolymers 1, 79. Franzblau, C., Kang, A. H., and Faris, B. 1970. In vitro formation of intermolecular cross-links in chick skin collagen. Biochem. Biophys. Res. Commun. 40, 437. Franzen, K. L . , and Kinsella, J. G . 1976. Functional properties of succinylated and acetylated soy protein. 1.Agric. Food Chem. 24, 788. Fraser, R. D. B., MacRae, T. P., Miller, A., and Rowlands, R. J. 1976. Digital processing of fiber diffraction patterns. J . Appl. Crystullogr. 9, 81. Friberg, S . 1976. Emulsion stability. In “Food Emulsions” (S. Friberg, ed.), p. 1. Dekker, New York.

346

A. ASGHAR AND R. L. HENRICKSON

Friedman, M. 1975. “Protein Nutritional Quality of Foods and Feeds,” Part I , p. 597. Dekker, New York. Friedman, M. 1977. Cross-linking amino acids-stereochemistry and nomenclature. Adv. Exp. Med. B i d . 86A, 1 . Friedman, M. 1978. Inhibition of lysinoalanine synthesis by protein acylation. In “Nutritional Improvement of Food and Feed Proteins” (M. Friedman, ed.). Plenum, New York. Friedman, M., Zahnley, J. C., and Masters, P. M. 1981. Relationship between in vivo digestibility of casein and its content of lysinoalanine and o-amino acids. J . Food Sci. 46, 127. Fugii, K., and Tanzer, M. L. 1977. Osteogensis imperfecta: Biochemical studies of bone collagen. Clin. Orthop. Relat. Res. 124, 271. Fugii, T., and Kobayashi, K. 1970. The effect of pH and borohydride reduction on the solubilization of steerhide collagen by pronase. J . Biochem. (Tokyo) 74, 307. Fujimaki, M., Arai, S . , and Yamashita, M. 1977. Enzymatic protein degradation and resynthesis for protein improvement. Adv. Chem. Ser. 160, 156. Fujimoto, D., and Tamiya, N. 1962. Incorporation of ‘ 8 0 from air into hydroxyproline by chick embryo. Biochem. J . 84, 333. Fukazawa, T., Hashimoto, Y., and Yasui, T. 1961. Effect of some proteins on the binding quality of an experimental sausage. J . Food Sci. 26, 541. Furia, T. E., ed. 1975. “CRC Handbook of Food Additives,” p. 289. Chem. Rubber Publ. Co., Cleveland, Ohio. Furthmayr, H., and Timpl, R. 1971. Characterization of collagen peptides by sodium dodecylsulfate polyacrylamide electrophoresis. Anal. Biochem. 41, 5 10. Furthmayr, H., and Timpl, R. 1976. Immunochemistry of collagens and procollagens. Int. Rev. Connect. Tissue Res. 7, 61. Furthrnayr, H., Timpl, R., Stark, M., Lapikre, C. M., and Kiihn, K. 1972. Chemical properties of the peptide extension in the pro-a1 chain of dermatosparactic skin procollagen. FEBS Lett. 28, 247. Gabourel, J . D., and Fox, K. E. 1965. Effect of hydrocortisone on the size of rat thymus polysomes. Biochem. Biophys. Res. Commun. 18(1), 81. Gallop, P. M. 1964. Concerning some special structural features of the collagen molecule. In “Connective Tissue: lntracellular Macromolecules,” p. 79. Little, Brown, Boston, Massachusetts. Gallop, P. M., and Paz, M. A. 1975. Posttranslational protein modifications, with special attention to collagen and elastin. Physiol. Rev. 55, 418. Gallop, P. M., Seifter, S . , and Meilman, E. 1959. Occurrence of ester-like linkages in collagen. Nature (London) 183, 1659. Gallop, P. M., Seifter, S., Lukin, M., and Meilman, E. 1960. Application of the Lossen rearrangement of dinitrophenyl-hydroxymates to analysis of carboxyl group in model compounds and gelatin. J . Biol. Chem. 235, 2619. Gallop, P. M., Blumenfeld, 0. O., Rojkind, M., and Pax, M. A. 1965. Subunits and their mode of attachment in tropocollagen. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 26. Butterworth, London. Gallop, P. M., Blumenfeld, 0. O., and Seifter, S . 1967. Subunits and special structural features of tropocollagen. In “Treatise on Collagen” (G. N. Ramachandran, ed.), Vol. I , p. 339. Academic Press, New York. Gallop, P. M., Blumenfeld, 0. O., and Seifter, S. 1972. Structure and metabolism of connective tissue proteins. Annu. Rev. Biochem. 41, 617. Gathercole, L. J., and Keller, A. 1974. Light microscopic wave forms in collagenous tissues and their structural implications. In “Structure of Fibrous Biopolymers” (E. D. T. Atkins and A. Keller, eds.), p. 153. Butterworth, London.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

347

Gersh, L., and Catchpole, H. R . 1949. The organization of ground substance and basement membrane and its significance in tissue injury, disease and growth. Am. J . Anat. 85, 457. Gershon, R. K. 1974. T-cell control of antibody production. Contemp. Top. Immunbiol. 3, 1. Gielissen, R. G. 1981. The effect of replacing lean with collagen in fine emulsion bologna. M.S. Thesis, Oklahoma State University, Stillwater. Gilbert, G. A,, and Rideal, E. K. 1944. The combination of fibrous proteins with acids. Proc. R. SOC.London, Ser. A 182, 335. Glasel, J. A. 1970. Participation of water in conformational changes of biopolymers as studies by deuteron magnetic relaxation. J . Am. Chem. Soc. 92, 375. Goldberg, B. 1974. Electron microscopic studies of procollagen from cultured human fibroblasts. Cell 1, 185. Goll, D. E. 1965. Postmortem changes in connective tissue. Proc. Annu. ReciprocalMeat Conj. 18, 161. Goll, D. E., Hoekstra, W. G . , and Bray, R. W. 1963. Age-associated changes in muscle composition. The isolation and properties of collagen residue from bovine muscle. J . Food Sci. 28,503. Goll, D. E., Hoekstra, W. G., and Bray, R. W. 1964a. Age-associated changes in bovine muscle connective tissues. I. Rate of hydrolysis by collagenase. J . Food Sci. 29, 608. Goll, D. E., Hoekstra, W. G., and Bray, R. W. 1964b. Age-associated changes in bovine muscle connective tissue. 11. Exposure to increasing temperature. J . Food Sci. 29, 615. Gosline, J. M. 1976. The physical properties of elastic tissue. Int. Rev. Connect. Tissue Res. 1,211. Gotthoffer, N. R. 1945. “Gelatin in Nutrition and Medicine,” p. 162. Grayslake Gelatin Co., Grayslake, Illinois. Could, B. S. 1968. The role of certain vitamins in collagen formation. In “Treatise on Collagen” (B. S. Could, ed.), Vol. 2 , Part A, p. 323. Academic Press, New York. Could, D. H., and MacGregor, J. T. 1977. Biologic effects of alkali-treated protein and lysinoalanine: An overview. Adv. Exp. Med. Biol. 86A, 29. Grant, J. K. 1969. Actions of steroid hormones at cellular and molecular levels. Essays Biochem. 5 , I. Grant, M. E., and Jackson, D. S. 1976. The biosynthesis of procollagen. Essays Biochem. 12, 77. Grant, M. E., and Prockop, D. J. 1972. The biosynthesis of collagen-a review in three parts. N . Engl. J . Med. 286, 194, 242, 291. Grant, M. E., Kefalides, N. A , , and Prockop, D. J. 1972. The biosynthesis of basement membrane collagen. 11. Synthesis of a precursor form by matrix-free cells and a time-dependent conversion of chains in intact lens. J . Biol. Chem. 247, 3545. Grant, M. E., Harwood, R., and Schofield, J. D. 1975. Recent studies on the assembly, intracellular processing and secretion of procollagen. In “Dynamics of Connective Tissue Macromolecules” (P. M. C. Burleigh and A. R. Polli, eds.), p. 1. North-Holland Publ., Amsterdam. Grassmann, W. 1965. Structure and chemistry of collagen. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 1. Buttenvorth, London. Grassmann, W., Hanning, K., Endres, H., and Riedel, A. 1956. Zur Aminosaureseguenz dez Kollagens. Hoppe-Seyler’s Z . Physiol. Chem. 322, 7 1. Grassmann, W., Hanning, K., and Engel, J. 1961. Des quantitative Verhaltnis Zwischen Q and p Komponente des denaturierten, Isolichen Kollagens in die Ultrazentrifuge Sowie Beschreibung einer Schneller Sedimentierten Komponente. Hoppe-Seyler’s Z . Physiol. Chem. 324, 284. Graves, P. N., Olsen, B. R., and Fietzek, P. P. 1979. Type I pre-collagen chains synthesized in a messenger RNA dependent reticulocyte lysate. Fed. Proc., Fed. Am. SOC. Exp. Biol. 38, 620. Griffin, W. C. 1949. Classification of surfactive agents by “HLB.” J . SOC. Cosmet. Chem. 337, 161. Griffith, T., and Johnson, J. A. 1957. Relation of the browning reaction to storage stability of sugar cookies. Cereal Chem. 34, 159.

348

A. ASGHAR AND R. L. HENRICKSON

Gross, J. 1954. Studies on the formation of collagen. IV. Effect of vitamin C deficiency on neutral salt-extractable collagen of skin. J . Exp. Med. 109, 557. Gross, J . 1961. Aging of connective tissue: The extracellular components. In “Structural Aspects of Aging” (G. H. Bourne, ed.), p. 177. Pittman, London. Gross, J. 1964a. Organization and disorganization of collagen. In “Connective Tissue: Inter-cellular Molecules,” p. 63. Little, Brown, Boston, Massachusetts. Gross, J. 1964b. Thermal denaturation of collagen in the dispersed and solid state. Science 143,960. Gross, J. 1970. The animal collagenases. In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), Vol. 3 , p. 1623. Academic Press, New York. Gross, J. 1976. Aspects of the animal collagenases. In “Biochemistry of Collagen” (G. N. Ramachandram and A. H. Ruddi, eds.), p. 275. Plenum, New York. Gross, J., and Nagai, Y. 1965. Specific degradation of the collagen molecule by tadpole collagenolytic enzyme. Proc. Natl. Acad. Sci. U.S.A. 54, 1197. Gross, J., Highberger, J. H., and Schmitt, F. 0. 1954. Collagen structure considered as states of aggregation of a kinetic unit. The tropocollagen particle. Proc. Natl. Acad. Sci. U.S.A. 40,679. Gross, J., Highberger, J. H., and Schmitt, F. 0. 1955. Extraction of collagen from connective tissue by neutral salt solutions. Proc. Natl. Acad. Sci. U.S.A. 41, 1 . Gross, J., Harper, E., Hams, E. D., Jr., McCroskery, P., Highberger, J. H., Corbett, C., and Kang, A. H. 1974. Animal collagenases: Specificity of action and structure of the substrate cleavage site. Biochem. Biophys. Res. Commun. 61, 605. Gryder, R. M., Lamon, M., and Adams, E. 1975. Sequence position of 3-hydroxyproline in basement membrane collagen: Isolation of 3-hydroxyprolyl-4-hydroxyproline from swine kidney. J . Biol. Chem. 250, 2470. Gupta, J. D., Dakroury, A . M., Harper, A. E., and Elvehjem, C. A. 1958. Biological availability of lysine. J . Nutr. 64, 259. Gustavson, K. H. 1955. The function of hydroxyproline in collagens. Nature (London) 175, 70. Gustavson, K. H. 1956. “The Chemistry and Reactivity of Collagen,” pp. 102, 155, 171, 202. Academic Press, New York. Gutmann, E. 1977. Muscle. In “Handbook of the Biology of Aging” (C. E. Finch and L. Hayflick, eds.), p. 445. Van Nostrand-Reinhold, Princeton, New Jersey. Guzman, N. A , , Rojas, F. J . , and Cutroneo, K . R. 1975. Collagen lysyl hydroxylation occurs within the cisternae of the rough endoplasmic reticulum. Arch. Biochem. Biophys. 172, 449. Guzman, N. A., Graves, P. N., and Prockop, D. J. 1978. Addition to mannose to both the aminoand carboxy-terminal properties of type I1 procollagen occurs without formation of a triple helix. Biochem. Biophys. Res. Commun. 84, 691. Hackler, L. R. 1977a. In vitro indices: Relationships to estimating protein value for the human. In “Evaluation of Proteins for Humans” (C. E. Bodwell, ed.), p. 119. AVI Publ., Westport, Connecticut. Hackler, L. R. 1977b. Methods of measuring protein quality: A review of bioassay procedures. Cereal Chem. 54, 984. Hagan, S . M., and Scow, R. 0. 1957. Effect of fasting on muscle proteins and fat in young rats of different ages. Am. J . Physiol. 188, 91. Hagerdal, B., and Lofqvist, B. 1978. Wettability and surface pressure of myoglobin treated with acetone. J . Food Sci. 43, 27. Hall, D. A. 1951. Elastin from human tissue and from ox ligament. Nature (London) 168, 513. Hall, D. A. 1976. “The Ageing of Connective Tissue,” pp. 1, 13, 67, 79. Academic Press, New York. Hall, D. A,, Reed, F. B., Nuki, G . , and Vince, J. D. 1974. Relative effect of age and corticosteroid therapy on the collagen profiles of dermis from subjects with rheumatoid arthritis. Age Ageing 3, 15.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

349

Hamilton, R. M . , and Paschall, E. F. 1967. Production and uses of starch phosphates. In “Starch: Chemistry and Technology” (R. L. Whistler and E. F. Paschall, eds.), Vol. 2, p. 351. Academic Press, New York. Hamm, R. 1957. [On the water binding capacity of mammalian muscle.] Z. Lebensm.-Unters. -Forsch. 106, 281. Hamm, R. 1958. [The biochemistry of meat salting.] Z . Lebensm.-Unters. -Forsch. 107, 423. Hamm, R. 1960. Biochemistry of meat hydration. Adv. Food Res. 10, 355. Hamm, R. 1972. “Colloid Chemistry of Meat: The Water-binding Properties of Muscle Proteins in Theory and Practice,” p. 53. Parey, Berlin. Hamm, R. 1975. Water-holding capacity of meat. In “Meat” (D. D. A. Cole and R. A. Lawrie, eds.), p. 321. Butterworth, London. Hanning, K . , and Nordwig, A. 1965. General sequence studies on collagen. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 7. Butterworth, London. Happich, M. L., Swift, C. E., and Naghski, J. 1975. Equations for predicting PER from amino acid analysis-A review and current scope of application. In “Protein Nutritional Quality of Foods and Feeds” (M. Friedman, ed.), Part 1, p. 125. Dekker, New York. Harding, J. 1965. Unusual links and cross-links of collagen. Adv. Protein Chem. 20, 109. Harkness, M. L. R., and Harkness, R. D. 1965. Functional alternations in the mechanical properties of collagenous frameworks. In “The Structure and Function of Connective and Skeletal Tissues” (S. Fitton-Jackson, ed.), p. 376. Butterworth, London. Harkness, M. L. R., and Harkness, R. D. 1966. Effect of sulphydryl reagents on the mechanical property of skin. Nature (London) 211, 496. Harkness, M. L. R., Harkness, R. D., and James, W. 1958. The effect of a protein-free diet on the collagen content of mice. J . Physiol. (London) 144, 307. Harkness, R. D. 1961. Biological functions of collagen. Biol. Rev. Cambridge Philos. SOC.36, 399. Harkness, R. D. 1968. Mechanical properties of collagenous tissues. In “Treatise on Collagen” (B. S. Gould, ed.), Vol. 2, Part A, p. 247. Academic Press, New York. Harper, E. 1980. Collagenase. Annu. Rev. Biochem. 49, 1063. Hamngton, W. F. 1964. On the arrangement of the hydrogen bonds in the structure of collagen. J . Mol. Biol. 9, 613. Harris, E. D., and Farrell, M. E. 1972. Resistance to collagenase: A characteristic of collagen fibrils cross-linked by formaldehyde. Biochim. Biophys. Acta 278, 133. Hams, E. D., and Krane, S. M. 1973. Cartilage collagen: Substrate insoluble and fibril form for mammalian collagenase. Trans. Assoc. Am. Physicians 86, 82. Hams, E. D., and Krane, S. M. 1974. Collagenases-A review in three parts. N . Engl. J . Med. 291, 557-564, 605-610, 652-661. Hams, E. D., and Sjoerdsma, A. 1966. Collagen profile in various clinical conditions. Lancet 2, 707. Hams, E. D., Evanson, J. M . , DiBona, D. R., and Krane, S. M. 1970. Collagenase and rheumatoid arthritis. Arthritis Rheum. 13, 83. Hams, E. D., Gonnerman, W . A,, Savage, J., and O’Dell, B. L. 1974. Connective tissue amine oxidase. 11. Purification and partial characterization of lysyloxidase from chick aorta. Biochim. Biophys. Acta 341, 332. Harrison, T. M., Brownlee, G . G . , and Milstein, C. 1974. Studies on polysome membrane interaction in mouse myeloma cells. Eur. J . Biochem. 47, 613. Hartman, B. K., and Bakerman, S. 1966. Titration of salt-extracted human skin collagen. Biochemistry 5 , 2221. Hartman, R. J. 1947. “Colloid Chemistry,” pp. 221, 399, 421. Houghton, Boston, Massachusetts. Hartog, C. D., and Pol, G . 1972. Protein evaluatiQn by means of nitrogen-balance tests. In “Protein

350

A. ASGHAR AND R. L. HENRICKSON

and Amino Acid Functions” (E. J. Bigwood, ed.), Vol. 2, pp. 311, 347, 353. Pergamon, Oxford. Harwood, R. 1979. Collagen polymorphism and messenger RNA. Int. Rev. Connect. Tissue Res. 8, 159. Harwood, R., Grant, M. E., and Jackson, D. S. 1973. The subcellular location of interchain disulfide bond formation during procollagen biosynthesis by embryonic chick tendon cells. Biochem. Biophys. Res. Commun. 55, 1188. Harwood, R., Grant, M. E., and Jackson, D. S. 1974a. Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs. Biochem. J . 142, 641. Harwood, R . , Grant, M. E., and Jackson, D. S. 1974b. Collagen biosynthesis. Characterization of subcellular fractions from embryonic chick fibroblasts and the intracellular localization of protocollagen prolyl and protocollagen lysyl hydroxylases. Biochern. J . 144, 123. Harwood, R., Grant, M. E., and Jackson, D. S . 1974c. Secretion of procollagen: evidence for the transfer of nascent polypeptides across microsomal membranes of tendon cells. Biochem. Biophys. Res. Commun. 59, 947. Hanvood, R., Connolly, A. D., Grant, M. E., and Jackson, D. S. 1974d. Presumptive mRNA for procollagen: Occurrence in membrane bound ribosomes of embryonic chick tendon fibroblasts. FEES Lett. 41, 8 5 . Harwood, R., Bhalla, A. K., Grant, M. E., and Jackson, D. S. 1975a. The synthesis and secretion of cartilage procollagen. Biochem. J . 148, 129. Harwood, R., Durrant, B., Grant, M. E., and Jackson, D. S. 1975b. Procollagen biosynthesis and polysome-membrane interactions in embryonic chick tendon cells. Biochem. SOC. Trans. 3, 914. Hanvood, R., Merry, A. H., Woolley, D. E., Grant, M. E., and Jackson, D. S. 1977. The disulfidebonded nature of procollagen and the role of the extension peptides in the assembly of the molecule. Biochem. J . 161, 405. Hayashi, T., Nakamura, T . , Hori, H., and Nagai, Y. 1980. The degradation rates of type I, 11, and 111 collagen by tadpole collagenase. 1.Eiochem. (Tokyo) 87(3), 809. Hayes, R. E., Bookwalter, G. M., and Bagley, E. G . 1977. Antioxidant activity of soybean flour and derivatives-a review. J . Food Sci. 42, 1527. Hegarty, G . R., Bratzler, L. J . , and Pearson, A. M. 1963. Studies on the emulsifying properties of some intracellular beef muscle proteins. J . Food Sci. 28, 663. Heger, J., and Frydrych, Z . 1980. Rapid methods of protein quality assessments-A review. Eiol. Chem. Factors Anim. Prod.-Ver. (in Polish) 16(4), 293. Hegsted, D. M. 1974. Assessment of protein quality. In “Improvement of Protein Nutriture” p. 64. Food and Nutrition Board, Natl. Acad. Sci.-Natl. Res. Council, Washington, D.C. Hegsted, D. M., and Chang, Y. 1965. Protein utilization in growing rats. 1. Relative growth index as a bioassay procedure. J . Nutr. 85, 159. Hegsted, D. M., Neff, R., and Worcester, J. 1968. Determination of relative nutritive value of proteins: Factors affecting precision and validity. J . Agric. Food Chem. 16, 190. Heidemann, E., and Hill, H. W. 1969. Behaviour of soluble collagen in glycerol-water mixture. Kolloid-Z. Z . Polym. 232(I ) , 674. Heikkinen, E. 1973. Frontiers of matrix biology. In “Aging of Connective Tissues: Skin” (L. Robert and B. Robert, eds.), p. 107. Karger, Basel. Henrickson, R. L. 1980. Hide collagen as food. Symp. New Uses Untanned Hide Collagen, Am. Leather Chem. Assoc. Convent., J . Am. Leather Chem. Assoc. 75(1 I ) , 464. Hermansson, A. M. 1975. Functional properties of proteins for foods: Flow properties. J . Texture Stud. 5 , 425. Hermansson, A. M., and Akesson, C. 1975. Functional properties of added proteins correlated with properties of meat systems (in three parts). J . Food Sci. 40, 595, 603, 61 1.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

35 1

Hermansson, A. M., Olsson, D., and Holmberg, B. 1974. Functional properties of proteins for foods-modification studies on rapeseed protein concentrate. Lebensm.-Wiss. Technol. 7, 176. Herring, H. K., Cassens, R. G., and Briskey, E. J. 1967. Factors affecting collagen solubility in bovine muscles. J . Food Sci. 32, 534. Highberger, J. H., and Stecker, H. C. 1941. The chemical action of saturated lime solutions on collagen. J . Am. Leather Chem. Assoc. 36, 368. Highberger, J . H., Kroner, T. D., and McGarr, J. J. 1964. Cited by Veis (1964, p. 10). Highberger, J. H., Kang, A. H., and Gross, J. 1971. Comparative studies on the amino acid sequence of the a2-CB2 peptides from chick and rat skin collagens. Biochemistry 10, 610. Hill, F. 1966. The solubility of intramuscular collagen in meat animals of various ages. J . Food Sci. 31, 161. Hippe, R. L . , and Warthesen, J. J. 1978. Effect of methionine and N-acetylmethione fortification on the flavor of soy bread and soy milk. J . Food Sci. 43, 793. Hodge, A. J. 1967. Structure at the electron microscopic level. In “Treatise on Collagen” (G. N. Ramachandran, ed.), Vol. 1, p. 185,Academic Press, New York. Hodge, A. J., and Petruska, J. A. 1963. Recent studies with the electron microscope on ordered aggregates of the tropocollagen molecule. In “Aspects of Protein Structure” (G. N. Ramachandran, ed.), p. 289. Academic Press, New York. Hodge, A. J . , and Schmitt, F. 0. 1960. The charge profile of the tropocollagen macromolecule and packing arrangement in native type collagen fibrils. Proc. Natl. Acad. Sci. U.S.A. 46, 186. Hodge, A. J., Highberger, J. H., Deffner, G. G. J., and Schmitt, F. 0. 1960. The charge profile of the tropocollagen macromolecule and the packing arrangement in native-type collagen fibrils. Proc. Natl. Acad. Sci. U.S.A. 46, 197. Holm, F., and Eriksen, S. 1980. Emulsifying properties of undenatured potato protein concentrate. J . Food Technol. 15, 71. Holsinger, V. H., and Posati, L. P. 1975. Chemical estimation of available lysine in dehydrated dairy products. In “Protein Nutritional Quality of Foods and Feeds” (M. Friedman, ed.), Part 1, p. 479. Dekker, New York. Honeyman, J. 1959. “Recent Advances in the Chemistry of Cellulose and Starch,” p. 188. Wiley (Interscience), New York. Horiuchi, T., Fukuskima, D., Sugimoto, H., and Hattori, T. 1978. Studies on enzyme-modified proteins as foaming agents. Effect of structure on foam stability. Food Chem. 3, 35. Hormann, H. 1965. Chemical methods for the conversion of insoluble collagen into tropocollagen. In “Structure and Function of Connective and Skeletal Tissue’’ (S. Fitton-Jackson, ed.), p. 48. Buttenvorth, London. Horn, M. J., and Warren, H. W. 1961. Availability of amino acids to microorganisms. 111. Development of a method for comparison of hydrolysates of foods with synthetic simulated mixtures. J . Nutr. 74, 226. Horn, M. J., Blum, A. E., and Womack, M. 1954. Availability of amino acids to microorganisms. J . Nurr. 53, 375. Housley, T. J., Tanzer, M. L., Henson, E . , and Gallop, P. M. 1975. Collagen cross-linking: Isolation of hydroxyaldol-histidine, a naturally-occuring cross-link. Biochem. Biophys. Res. Commun. 67, 824. Howell, N. 1978. Use of animal proteins in food. Int. Flavours Food Addir. 9(3), 119. Hsu, H. W., Vavak, D. L., Satterlee, L. D., and Miller, G. A. 1977. A multienzyme-automatic recording technique for in virro protein digestibility. J . Food Sci. 42, 1264. Hulmes, D. J. S . , and Miller, A. 1979. Quasi-hexagonal molecular packing in collagen fibrils. Nature (London) 282, 878. Hulmes, D. J . S . , Miller, A,, Parry, D. A. D., Piez, K. A,, and Woodhead-Galloway, J . 1973. Analysis of the primary structure of collagen for the origins of molecular packing. J . Mol. Biol. 79. 137.

352

A. ASGHAR AND R. L. HENRICKSON

Humphrey, J. H., Neuberger, A., and Perkins, D. J. 1956. The detection and estimation of plasma albumin in rat and rabbit skin. Biochem. J . 64,2p. Humphrey, J. H., Neuberger, A , , and Perkins, D. J. 1957. Observations on the presence of plasma proteins in skin and tendon. Biochem. J . 66, 390. Hunsley, R. E., Vetter, R:L., Kline, E. A , , and Burroughs, W. 1971. Effect of age and sex on quality, tenderness, and collagen content of bovine Longissmus muscle. J . Anim. Sci. 33, 933. Hurrell, R. F., Lerman, P., and Carpenter, K. J. 1979. Reactive lysine in food stuff as measured by a rapid dye-binding procedure. J . Food Sci. 44, 1221. Hwang, C. I., and Kim, D. H. 1973. The antioxidant activity of some extracts from various stages of a Maillard-type browning reaction mixture. Korean J . Food Sci. Technol. 5, 84. Idson, B., and Braswell, E. 1957. Gelatin. Adv. Food Res. 7, 235. Imoto, M. 1979. Cancer and free radical. Kagaku (Kyoto) 34, 26. Itoh, H . , Kawashima, K., and Chimbata, I . 1975. Antioxidant activity of brown products of triose sugar and amino acid. Agric. B i d . Chem. 39, 283. Jackson, D. S., and Bentley, J. P. 1960. On the significance of the extractable collagens. J . Biophys. Biochem. Cytol. 7, 37. Jackson, D. S., and Neuberger, A. 1957. Observations on the isoionic and isoelectric point of acid processed gelatin from insoluble and citrate-extracted collagen. Biochim. Biophys. Acta 26, 638. Jackson, D. S., Ayad, S., and Mechanic, G. 1974. Effect of heat on some collagen cross-links. Biochim. Biophys. Acta 336, 100. Jacquot, R., and Peret, J. 1972. Protein efficiency ratio and related methods. In “Protein and Amino Acid Functions” (E. J. Bigwood, ed.), Vol. 2, p. 317. Pergamon, Oxford. Jeffery, J. J., and Gross, J. 1970. A collagenase from rat uterus: Isolation and partial characterization. Biochemistry 9, 268. Jenness, R., and Patton, S. 1959. “Principles of Dairy Chemistry,” Chapter 5, p. 101. Wiley, New York. Jimenez, S. A,, and Yankowski, R. 1975. Hydroxyproline is not required for secretion of triplehelical collagen. Fed. Proc., Fed. Am. SOC. Exp. Biol. 34, Abstr. No. 1944. Jimenez, S. A , , Harsch, M., and Rosenbloom, J. 1973. Hydroxyproline stabilizes the triple-helix of chick collagen. Biochem. Biophys. Res. Commun. 52, 106. Jirgensons, B., and Straumanis, M. E. 1962. “A Short Textbook of Colloid Chemistry,” pp. 127, 350. Macmillan, New York. Johns, P. 1977. Structure and composition of collagen-containing tissues. In “Science and Technology of Gelatin” (A. G. Ward and A. Courts, eds. j, p. 32. Academic Press, New York. Johnson, A. R., Chung, E., and Miller, E. J. (1974). Cited in Miller and Matukas (1974). Joly, M. 1955. Recent studies on the reversible denaturation of proteins. Prog. Biophys. Biophys. Chem. 5, 168. Jones, N. R. 1977. Uses of gelatin in edible products. In “Science and Technology of Gelatin” (A. G. Ward and A. Courts, eds.), p. 366. Academic Press, New York. Jones, R. T. 1964. The structure of proteins. I n “Symposium on Foods: Proteins and Their Reactions” (H. W. Schultz and A. F. Anglemier, eds.), p. 33. AVI Publ. Co., Westport, Connecticut. Jones, S. B., Carrol, R. J., and Cavanaugh, J. R. 1977. Structural changes in heated bovine muscle. A scanning electron microscopy study. J . Food Sci. 42, 125. Joseph, K. T., and B o x , S. M. 1962. Influence of biological aging on the stability of skin collagen in albino rats. In “Collagen” (G. N. Ramachandran, ed.), p. 371. Wiley (Interscience), New York. Josse, J., and Harrington, W. F. 1964. Role of pyrrolidine residues in the structure and stabilization of collagen. J . Mol. B i d . 9, 269.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

353

Kahn, L., and Witnauer, L. 1966. The viscometric behavior of solubilized calf skin collagen at low rates of shear. J . Biol. Chem. 241, 1784. Kamat, V. B., Graham, G . E., and Davis, M. A. F. 1978. Vegetable protein: lipid interactions. Cereal Chem. 55, 295. Kanagy, J. R. 1950. Influence of temperature on the absorption of water vapor by collagen and leather. J . Am. Leather Chem. Assoc. 45, 12. Kang, A. H. 1972. Studies on the location of intermolecular crosslinks in collagen. Isolation of a CNBr peptide containing 6-hydroxylysineonorleucine.Biochemistry 11, 1828. Kang, A. H., and Gross, J. 1970. Amino acid sequence of cyanogen bromide peptides from the amino-terminal region of chick skin collagen. Biochemistry 9, 796. Kang, A. H., and Trelstad, R. L. 1973. A collagen defect in homocystinuria. J . Clin. Invest. 52, 2571, Kang, A. H., Faris, B., and Franzblau, C. 1970. The in virro formation of intermolecular cross-links in chick skin collagen. Biochem. Biophys. Res. Commun. 39, 175. Kaplan, D., and Meyer, K. 1960. Mucopolysaccharides of aoga at various ages. Fed. Proc., Fed. Am. Soc. Exp. B i d . 105, 78. Karel, M., Tannenbaum, J. R., Wallace, D. H., and Maloney, H. 1966. Autooxidation of methyl linoleate in freeze-dried model system. 111. Effect of added amino acids. J . FoodSci. 31, 892. Karel, M . , Schaick, K., and Roy, B. R. 1975. Interaction of peroxidizing methyl linoleate with some protein and amino acids. J . Agric. Food Chem. 23, 159. Karmas, E., and DiMarco, G. R. 1970. Dehydration thermo-profiles of amino acids and proteins. J . Food Sci. 35, 615. Katchalsky, A. 1964. Polyelectrolytes and their biological interactions. Biophys. J . 4, 9. Katchalsky, A , , Cooper, R. E., Upadhyay, J., and Wassermann, A. 1961. Counterion fixation in aliginates. J . Chem. Soc. Part IV, p. 5198. Katz, D. H., and Benacerraf, B. 1972. The regulatory influence of activated T cells on B-cell responses to antigen. Adv. Immunol. 15, 1. Katz, E. P., and Li, S. T. 1974. Structure and function of bone collagen fibrils. J . Mol. Biol. 80, 1. Kauffman, R. G . , Carpenter, 2. L., Bray, R. W . , and Hoekstra, W. G . 1964. Interrelations of tenderness, chronological age, and connective tissue fractions of porcine musculature. J . Agric. Food Chem. 12, 504. Kauzmann, W. 1956. Structural factors in protein denaturation. J . Cell. Comp. Physiol. 47, Suppl. 1, 113. Kauzmann, W. 1964. The three dimensional structure of proteins. Biophys. J . 4, 43. Kawashima, K., Itoh, H . , and Chibata, I. 1977. Antioxidant activity of browning products prepared from low molecular carbonyl compounds and amino acids. J . Agric. Food Chem. 26, 202. Kawashima, K., Itoh, H., and Chibata, I. 1979. Synergistic ternary antioxidant compositions comprising tocopherol, partially hydrolyzate of gelatin and organic acid. Agric. Biol. Chem. 43, 827. Kefalides, N. A. 1971. Isolation of a collagen from basement membranes containing three identical a chains. Biochem. Biophys. Res. Commun. 45, 226. Kefalides, N. A. 1972. Isolation and characterization of cyanogen bromide peptides from basement membrane collagen. Biochem. Biophys. Res. Commun. 47, 1151. Kefalides, N. A. 1973. Structure and biosynthesis of basement membranes. Int. Rev. Connect. Tissue Res. 6, 63. Kefalides, N. A. 1979. Persistence of basement membrane collagen phenotype in hybrids of human vascular endothelium and rodent fibroblasts. Fed. Proc., Fed. Am. Soc. Exp. Biol. 38, 816. Kerr, R. W. 1950. “Chemistry and Industry of Starch,” p. 259. Academic Press, New York. Kerwar, S . S . , Kohn, L. D., Lapikre, C. M . , and Weissback, H. 1972. In vitro synthesis of procollagen on polysomes. Proc. Natl. Acad. Sci. U.S.A. 69, 2727.

354

A . ASGHAR AND R. L. HENRICKSON

Khan, A. W., and Van den Berg, L. 1964. Some protein changes during postmortem tenderization in poultry meat. J . Food Sci. 29, 597. Kidney, A. J . 1970. Method of preparing a collagen sausage casing. U.S. Patent 3,505,084. Kim, C. W., Ho, G. P., and Ritchey, S . J. 1967. Collagen content and subjective score for tenderness of connective tissue in animals of different ages. J . Food Sci. 32, 586. Kimball, D. A , , Coulson, W. F., and Carnes, W. H. 1964. Cardiovascular studies on copperdeficient swine. Exp. Mol. Pathol. 3, 10. Kinsella, J. E. 1976. Functional properties of proteins in foods. A survey. Crir. Rev. Food Sci. Nurr. 1(3), 219. Kinsella, J. E. 1979. Functional properties of soy proteins. J . Am. Oil Chem. Soc. 56(3), 242. Kirigaya, N., Kato, H., and Fujimaki, M. 1968. Studies on antioxidant of nonenzymatic browning reaction products. Agric. Biol. Chem. 3, 287. Kirrane, J . A , , and Glynn, L. E. 1968. Immunology of collagen. Int. Rev. Connect. Tissue Res. 4, I . Kivirikko, K. I., and Mallyla, R. 1979. Collagen glycosyltransferases. Inr. Rev. Connect. Tissue Res. 8, 23. Klaui, H. 1974. The technological aspects of the addition of nutrients to foods. Proc. Int. Congr. Food Sci. Technol., Vol. I , p. 740. Knight, J . W. 1967. Modification and uses of wheat starch. In “Starch: Chemistry and Technology” (R. L. Whistler and E. F. Paschall, eds.), Vol. 2, p. 279. Academic Press, New York. Kofoed, J . A., and Bozzini, C. E. 1969. Differential effect of hypophysectomy on skin and tracheal cartilage glycosaminoglycans. Experientia 25, 23. Kofranyi, E. 1972. Protein and amino acid requirements. In “Protein and Amino Acid Functions” (E. J. Bigwood, ed.), Vol. 2, p. I . Pergamon, Oxford. Komanowsky, M., Sinnamon, H. I . , Elias, S . , Heiland, W. K., and Aceto, N. C. 1974. Production of comminuted collagen for novel applications. J . Am. Leather Chem. Assoc. 69, 410. Kornguth, M. L., Neidle, A,, and Waelsch, H. 1963. The stability and rearrangement of E-Nglutamyl-lysines. Biochemistry 2, 740. Kosuge, T., Tsuji, K., Wakabayashi, K., Okamoto, T., Sudo, K., Iitaka, Y., Itai, A., Sugimura, T . , Kawachi, T . , Nagao, M., Yahagi, T., and Seino, Y. 1980. Isolation and structural studies of mutagenic principles in amino acid pyrolysates. Chem. Pharm. Bull. 26, 61 I . Kovanen, V., Suominen, H., and Hekkinen, E. 1980. Connective tissue of fast and slow skeletal muscle in rats: Effect of endurance training. Acta Physiol. Scand. 108(2), 173. Kowalewski, K. 1966. Effect of an anabolic steroid upon various functions of tissue hydroxyproline in cortisone treated rats. Acta Endocrinol. (Copenhagen) 53, 73. Kragh, A. M. 1977. Swelling, Adsorption and the photographic uses of gelatin. In “Science and Technology of Gelatin” (A. G . Ward and Courts, eds.), p. 439. Academic Press, New York. Kramlich, W. E. 1971. Sausage products. In “Science of Meat and Meat Products” (J. F. Price and B. S . Schweigert, eds.), p. 484. Freeman, San Francisco, California. Krestinger, R. H., Manner, G . , Could, B. S . , and Rich, A. 1964. Synthesis of collagen on polyribosomes. Nature (London) 202, 438. hydrogen bonding in proteins. In “Symposium on Fibrous ProKrimm, S. 1968. C-H...O=C teins” (W. G. Crewther, ed.), p. 84. Butterworth, Sydney, Australia. Kroner, T. D., Tabroff, W., and McGarr, J. J. 1953. Peptides isolated from a partial hydrolysate of steer hide collagen. J . Am. Chem. Soc. 75, 4084. Kruger, L. H., and Rutenberg, M. W. 1967. Production and uses of starch acetates. In “Starch: Chemistry and Technology” (R. L. Whistler and E. F. Paschall, eds.), Vol. 2, p. 369. Academic Press, New York. Kruggel, W. G., and Field, R. A. 1971. Soluble intramuscular collagen characteristics from stretched and aged muscle. J . Food Sci. 36, 11 14. Kruyt, H. R. 1952. “Colloid Science,” p. 262. Elsevier, Amsterdam.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

355

Kuhn, K. 1960. Uber den Ursprung des Querstreifungsmusters bei Kollagen. Leder 11, 110. Kuhn, K. 1969. The structure of collagen. Essays Biochem. 5, 59. Kuntz, I. D., Jr. 1971. Hydration of macromolecules. 3. Hydration of polypeptides. J . Am. Chem. Soc. 93, 514. Kuntz, I . D., Jr. 1975. The physical properties of water associated with biomacromolecules. In “Water Relations of Foods” (R. B. Duckworth, ed.), p. 93. Academic Press, New York. Kuntz, I. D., and Kauzmann, W. 1974. Hydration of proteins and polypeptides. Adv. Protein Chem. 28, 239. Kuntzel, A. 1944. In “Handbuch der Gerbereichemie” (W. Grassman, ed.), Vol. 1, p. 602. Springer, Vienna (cited by Gustavson, 1956). Kurosu, H. 1979. [Biochemical studies on collagen and connectin from human skeletal muscle. Agerelated changes in the properties of elasticity.] Nippon Seikeigeka Gakkai Zasshi 53(1 I ) , 1641; Chem. Abstr. 92, 1081102. LaBella, F. S . , and Paul, G. 1965. Structure ofcollagen from human tendon as influenced by age and sex. J . Gerontol. 20, 54. Lachance, P. A,, Bressani, R., and Elias, L. G. 1977. Shorter protein bioassays. Food Technol. 31, 82. Lakin, A. L. 1973. Evaluation of protein dye-binding procedures. In “Proteins in Human Nutrition” (J. W. G. Porter and B. A. Rolls, eds.), p. 179. Academic Press, New York. Lalasidis, G., and Sjoberg, L. B. 1978. Two new methods of debittering protein hydrolysates and a fraction of hydrolysis with exceptionally high content of essential amino acids. J . Agric. Food Chem. 26, 742. Lapikre, C. M., and Gross, J. 1963. Animal collagenase and collagen metabolism. In “Mechanisms of Hard Tissue Destruction” (R. F. Sognnaes, ed.), Publ. No. 75, p. 663. Am. Assoc. Adv. Sci., Washington, D.C. Lapikre, C. M . , Nusgens, B., and Pierard, G. E. 1977. Interaction between collagen type I and type 111 in conditioning bundles organization. Connect. Tissue Res. 5 , 21. Laurent, T. C. 1974. The properties and function of connective tissue polysaccharides. In “Structure of Fibrous Biopolymers” (E. D. T. Atkins and A. Keller, eds.), p. 27. Butterworth, London. Lawrie, R. A. 1974. “Meat Sciences,” p. 38. Pergamon, Oxford. Layman, D. L., and Titus, J. L. 1975. Synthesis of type I collagen by human smooth muscle cells in vitro. Lab. Invest. 33, 103. Lazarides, E. L., and Lukens, L. N. 1971. Collagen synthesis on polysomes in vivo and in virro. Nature (London), New Biol. 232, 37. Leander, R. C., Hedrick, H. B., Brown, M. F., and White, J. A. 1980. Comparison of structural changes in bovine longissirnus and semitendinosus muscles during cooking. J . Food Sci. 45, 1. Lee, Y., and Lang, D. 1968. D-Galactose, di- and trisaccharides from the earth worm cuticle collagen. J . B i d . Chem. 243, 677. Lee, Y., Elliott, J. G . , Rickansrud, D. A,, and Hagberg, E. C. 1978. Predicting protein efficiency ratio by the chemical determination of connective tissue content in meat. 1.FoodSci. 43, 1359. Leeder, J . D., and Watt, I. C. 1965. The role of amino groups in water absorption by keratin. J. Phys. Chem. 69, 3280. Lehrach, H . , Frischauf, A. M., and Hanahan, D. 1978. Construction and characterization of a 2.5kilobase procollagen clone. Proc. Natl. Acad. Sci. U.S.A. 75, 5417. Lenaers, A., Ansay, M., Nusgens, B. V., and Lapikre, C. M. 1971. Collagen made of extended a chains, procollagen in genetically defective dermatosparactic calves. Eur. J . Biochem. 23, 533. Leung, M. K. K . , Fessler, L. I., and Greenberg, D. B. 1979. Separate amino carboxyl procollagen peptidases in chick embryo tendon. J . Biol. Chem. 254, 224. Levene, C. I. 1973. Lathyrism. In “Molecular Pathology of Connective Tissues” (R. Perez-Tamayo and M. Rojkind, eds.), p. 177. Dekker, New York.

356

A. ASGHAR AND R. L. HENRICKSON

Levene, C. I . , and Bates, C. J . 1975. Ascorbic acid and collagen synthesis in cultured fibroblasts. Ann. N . Y . Acad. Sci. 258, 285. Levene, C. I . , and Gross, J. 1959. Alterations in state of molecular aggregation of collagen induced in chick embryos by p-amino propionitrile (lathyrus factor). J . Exp. Med. 100, 771. Levene, C. I., Bates, C. J . , and Bailey, A . J. 1972. Biosynthesis of collagen cross-links in cultured 3T6 fibroblasts; effect of lathyrogens and ascorbic acid. Biochim. Biophys. Acfa 263, 574. Levene, C. I . , Aleo, J. J., Prynne, C . J . , and Bates, C. J. 1974. The activation of protocollagen proline hydroxylase by ascorbic acid in cultured 3T6 fibroblasts. Biochim. Biophys. Acta 388, 29. Li-Chan, E., and Nakai, S. 1980. Covalent attachment of NE-acetyl lysine, N-benzylidene lysine, and threonine to wheat gluten for nutritional improvement. J . Food Sci. 45, 514. Li-Chan, E., Helbig, N., Holbek, E., Chau, S . , and Nakai, S . 1979. Covalent attachment of lysine to wheat gluten for nutritional improvement. J . Agric. Food Chem. 27(4), 877. Lichtenstein, J. R., Byers, P. H., Smith, B. D., and Martin, G. R. 1975. Identification of the collagenous proteins synthesized by cultured cells from human skin. Biochemistry 14, 1589. Lin, J. S., Smith, V., and Olcott, H. S. 1974. Loss of free-radical signal during induction period of unsaturated lipids containing nitroxide antioxidants. J . Agric. Food Chem. 22, 682. Lin, M. J. Y . , Humbert, E. S . , and Sosulski, F. W. 1974. Certain functional properties of comflower meal products. J . Food Sci. 39, 368. Ling, G . N. 1972. Hydration of macromolecules. In “Water and Aqueous Solutions’’ (R. A. Home, ed.), p. 663. Wiley, New York. Ling, G. N., and Walton, C. L. 1976. What retains water in living cells? Science 191, 293. Lingnert, H. 1980. Application of antioxidative Millard reaction products in foods. J . Am. Oi[ Chem. SOC.57, 319 (Abstr.). Lis, M. T., Crampton, R. F., and Matthews, D. M. 1972. Effect of dietary changes on intestinal absorption of L-methionine and L-methionyl-L-methionine in the rat. Br. J . Nutr. 27, 159. Little, C. D., and Church, R. L. 1978. A new type of collagen produced by mouse cells derived from early embryonic sources. Arch. Biochem. Biophys. 83, 180. Lloyd, D. J., and Garrod, M. 1948. Contribution to the theory of the structure of protein fibers with special reference to the so-called thermal shrinkage of the collagen fiber. Trans. Faraday Soc. 44,441. Lloyd, D. J., and Shore, A. 1938. “Chemistry of Proteins,” p. 380. Churchill, London. Loeb, J. 1922. “Proteins and Theory of Colloidal Behavior.” McGraw-Hill, New York. Loewi, G . , and Meyer, K. 1958. The acid mucopolysaccharides of embryonic skin. Biochim. Biophys. Acta 27, 453. Lorenzen, 1. 1962. Vascular connective tissue under the influence of thyroxin. Acta Endocrinol. (Copenhagen) 39, 605, 615. Lowther, D. A . , Green, N. M . , and Chapman, J. A. 1961. Morphological and chemical studies of collagen formation. J . Biophys. Biochem. Cytol. 10, 373. McClain, P. E. 1971. In vitro aging-like changes in the cross-linking characteristics of collagen from intramuscular connective tissue. Geronrologia 17, 139. McClain, P. E., Creed, G. J., Wiley, E. R., and Hornstein, I. 1970. Effect of postmortem aging on isolation of intramuscular connective tissue. J . Food Sci. 35, 258. McClain, P. E., Wiley, E. R., Bodwell, C. E., and Hornstein, I. 1971. Amino acidcomposition and cross-linking characteristics of collagen from intramuscular connective tissue of striated muscle (Bos taurus). Int. J . Biochem. 2, 121. McCroskery, P. A,, Wood, S . , and Hams, E. D. 1973. Gelatin: A poor substrate for a mammalian collagenase. Science 182, 70. McLaughlan, J. M. 1974a. Nutritional significance of alterations in plasma amino acids and serum proteins. In “Improvement of Protein Nutriture” p. 89. Food Nutr. Board, Natl. Acad. Sci.Natl. Res. Counc., Washington, D.C.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

357

McLaughlan, J. M. 1974b. Evaluation of standard rat assays. In “Nutrients in Processed Foods Proteins” (P. L. White and D. C. Fletcher, eds.), p. 69. Publishing Sciences Group, Inc., Acton, Massachusetts. McLaughlan, J. M. 1976. The relative nitrogen utilization method for evaluating protein quality. J . Assoc. Off. Anal. Chem. 59, 42. McLaughlan, J. M . , and Keith, M. 0. 1975. Bioassays for protein quality. In “Protein Nutritional Quality of Foods and Feeds” (M. Friedman, ed.), Part Part I, p. 79. Dekker, New York. Mandelkem, L., and Stewart, W. E. 1964. Effect of neutral salts on the melting temperature and reconstitution kinetics of ordered collagen structure. Biochemistry 3(8), 1 132. Mandl, I. 1961. Collagenases and elastases. Adv. Enzymol. 23, 163. Manner, G . , and Could, B. S. 1963. Collagen biosynthesis, the formation of hydroxyproline and soluble ribonucleic acid complexes of proline and hydroxyproline by chick embryo in virro and by a subcellular chick embryo system. Biochim. Biophys. Acta 72, 243. Manner, G . , and Could, B. S . 1965. Action of hydrocortisone on the distribution and collagen forming activity of chick embryo polysomes. Fed. Proc., Fed. Am. Sac. Exp. Biol. 24, Suppl. 14-15, 359. Marcuse, R. 1962. The effect of some amino acids on oxidation of linoleic acid and its methyl esters. J . Am. Oil Chem. Soe. 39, 97. Mamott, R . H. 1933. Swelling in alkaline solutions. J . Int. Soc. Leather Trades’ Chem. 17, 178. Martin, G. R., Piez, K. A., and Lewis, M. S. 1963. The incorporation of [‘4C]glycine into the subunits of collagen from normal and lathyritic animals. Biochim. Biophys. Acfa 69, 472. Martin, G. R . , Byers, P. H., and Piez, K. A. 1975. Procollagen. Adv. Enzymol. 42, 167. Masters, P. M., and Friedman, M. 1979. Racemization of amino acids in alkali-treated food proteins. J . Agric. Food Chem. 27, 507. Masters, P. M., and Friedman, M. 1980. Amino acid racemization in alkali-treated food proteinschemistry, toxicology and nutritional consequences. ACS Symp. Ser. 123, 165. Mathews, M. B. 1964. Structural factors in cation binding to anionic polysaccharides of connective tissue. Arch. Biochem. Biophys. 104, 394. Mathews, M. B. 1970. The interactions of proteoglycans and collagen-model system. In “Chemistry and Molecular Biology of Intracellular Matrix” (A. E. Balazs, ed.), Vol. 2, p. 1155. Academic Press, New York. Mathews, M. B. 1973. Glycosaminoglycans in development and aging of vertebrate cartilage. In “Connective Tissue and Aging,” Int. Congr. Ser. 264 (H. G. Vogel, ed.), p. 151. Excerpta Medica, Amsterdam. Matthews, D. M., Craft, I. L., Geddes, D. M., Wise, I. J., and Hyde, C. W. 1968. Absorption of glycine and glycine peptides from the small intestine of the rat. Clin. Sci. 35, 415. Maurer, A. J., and Baker, R . C. 1966. The relationship between collagen content and emulsifying capacity of poultry meat. Poult. Sci. 45, 1317. Maurer, A. J., Baker, R. C., and Vadehra, D. V. 1969. Kind and concentration of soluble protein extracts and their effect on the emulsifying capacity of poultry meat. Food Technol. 23, 575. Mauron, J . 1972. Influence of industrial and household handling on food protein quality. In “Proteins and Amino Acid Functions” (E. J. Bigwood, ed.), Part I, p. 417. Pergamon, Oxford. Mauron, J. 1973. The analyses of food proteins, amino acid composition and nutritive value. In “Proteins in Human Nutrition” (I. W. G. Porter and B. A. Rolls, eds.), p. 139. Academic Press, New York. Mayne, R., Vail, M. S . , and Mayne, P. M. 1976. Changes in type of collagen synthesized as clones of chick chondrocytes grow and eventually lose division capacity. Proc. Nutl. Acad. Sci. U.S.A. 73, 1674. Mayne, R., Vail, M. S., and Miller, E. J. 1977a. Characterization of the collagens synthesized by cultured smooth muscles derived from rhesus monkey thoracic aorta. J . Cell Biol. 75, 155a (abstr .).

358

A. ASGHAR AND R. L. HENRICKSON

Mayne, R., Vail, M. S . , Miller, E. J., Blose, S. H., and Chacko, S. 1977b. Collagen polymorphism in cell cultures derived from guinea pig aortic smooth muscle: Comparison with three populations of fibroblasts. Arch. Biochem. Biophys. 181, 462. Mechanic, G. L. 1972. Cross-linking of collagen in a heritable disorder of connective tissue: Ehlers-Danlos syndrome. Biochem. Biophys. Res. Commun. 47, 267. Mechanic, G. L., and Levy, M. 1959. An c-lysine tripeptide obtained from collagen. J . Am. Chem. Soc. 81, 1889. Mechanic, G. L., and Tanzer, M. L. 1970. Biochemistry of collagen cross-linking. Isolation of a new cross-link, hydroxylysinohydroxynorleucine, and its reduced precursor, dihydroxynorleucine, from bovine tendon. Biochem. Biophys. Res. Commun. 41, 1597. Mechanic, G. L., Gallop, P. M., and Tanzer, M. L. 1971. The nature of cross-linking in collagens from mineralized tissues. Biochem. Biophys. Res. Commun. 45, 644. Melander, W., and Horvith, C. 1977. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins. An interpretation of the lyotropic series. Arch. Biochem. Biophys. 183, 200. Mellon, E. F., Viola, S . J., and Naghski, J. 1960. Stratigraphic distribution of carbohydrate, hexosamine, and hydroxyproline in cattlehides. J . Am. Leather Chem. Assoc. 55, 622. Mendes, C. B., and Waterlow, J. C. 1958. Effect of a low-protein diet, and of refeeding, on the composition of liver and muscle in the weaning rat. Br. J . Nutr. 12, 74. Merkel, R. A. 1971. Carbohydrates. In “The Science of Meat and Meat Products” (J. F. Price and B. S . Schweigert, eds.), p. 145. Freeman, San Francisco, California. Meyer, E. W., and Williams, L. D. 1977. Chemical modification of soy proteins. Adv. Chem. Ser. 160, 52. Meyer, K., Hoffman, P., and Linker, A. 1957. In “Connective Tissue” (R. E. Tunbridge, ed.), p. 86. Blackwell, Oxford. Meyer, K., Anderson, B., Seno, N., and Hoffman, P. 1965. Peptide complexes of chondroitin sulphates and kerato-sulphates. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 164. Butterworth, London. Mier, P. D., and Cotton, D. W. K. 1976. “The Molecular Biology of Skin,” p. 3. Blackwell, Oxford. Miller, A. 1976. Molecular packing in collagen fibrils. In “Biochemistry of Collagen” (G. N. Ranachandran and A. H. Ruddi, eds.), p. 85. Plenum, New York. Miller, A. 1980. A versatile body builder. New Sci. 85( 1194), 470. Miller, A . , and Parry, D. A . D. 1973. Structure and packing of microfibrils in collagen. J . Mol. Biol. 75, 441. Miller, D. S . , and Bender, A. E. 1955. The determination of the net utilization of proteins by a shortened method. Br. J . Nutr. 9, 382. Miller, E. J . 1971a. Isolation and characterization of a collagen from chick cartilage containing three identical (Y chains. Biochemistry 10, 1652. Miller, E. J . 1971b. Collagen cross-linking: Identification of two cyanogen bromide peptides containing sites of intermolecular cross-link formation in collagen. Biochem. Biophys. Res. Commun. 45, 444. Miller, E. J. 1973. A review of biochemical studies on the genetically distinct collagens of the skeletal system. Clin. Orthop. Relut. Res. 92, 260. Miller, E. J. 1976. Biochemical characteristics and biological significance of the genetically distinct collagens. Mol. Cell Biochem. 13, 165. Miller, E. J., and Lunde, L. G. 1973. Isolation and characterization of the cyanogen bromide peptides from the crl(I1) chain of bovine and human cartilage collagen. Biochemistry 12, 3153. Miller, E. J., and Matukas, V. J . 1969. Chick cartilage collagen: A new type of nl chain not present in bone or skin of the species. Proc. Nut/. Acud. Sci. U.S.A. 64, 1264.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

359

Miller, E. J . , and Matukas, V. J. 1974. Biosynthesis of collagen: The biochemist’s view. Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, 1197. Miller, E. J., and Robertson, P. B. 1973. The stability of collagen cross-link derived from hydroxylysyl residues. Biochem. Biophy. Res. Commun. 54, 432. Miller, E. J., Martin, G. R., Piez, K. A , , and Power, M. J. 1967. Characterization of chick bone collagen and compositional changes associated with maturation. J . B i d . Chem. 242, 5481. Miller, E. J., Epstein, E. H., Jr., and Piez, K. A. 1971. Identification of three genetically distinct collagens by cyanogen bromide cleavage of insoluble human skin and cartilage collagen. Biochem. Biophys. Res. Commun. 42, 1042. Miller, E. J . , Woodall, D. L., and Vail, M. S . 1973. Biosynthesis of cartilage collagen. Use of pulse labeling to order the cyanogen bromide peptides in the al(I1) chain. J . Biol. Chem. 248, 1666. Mirsky, A. E., and Pauling, L. 1936. On the structure of native, denatured, and coagulated proteins. Proc. Natl. Acad. Sci. U.S.A. 22, 439. Mitchell, H. H. 1924. A method for determining the biological value of protein. J . B i d . Chem. 58, 873. Mitchell, H. H., and Block, R. J. 1946. Some relationship between the amino acid contents of protein and their nutritive values. J . Biol. Chem. 163, 599. Mitchell, H. H . , and Carman, G. G. 1926. The biological value of the nitrogen of mixtures of paten white flour and animal foods. J . Biol. Chem. 68, 183. Mitchell, H. H., Beadles, J. R., and Kruger, J. H. 1927. The relation of the connective tissue content of meat to its protein value in nutrition. J . Biol. Chem. 73, 767. Monson, J. M., and Goodman, H. M. 1978. Translation of chick calvarial procollagen messenger RNAs by a messenger RNA dependent reticulocyte lysate. Biochemistry 17, 5122. Montagna, W., Bentley, J. P., and Dobson, R. 1970. Adv. Biol. Skin 10, 1-37. Montgomery, R. D., Dickerson, J. W. T., and McCance, R. A. 1964. Severe undernutrition in growing and adult animals. The morphology and chemistry of development and undernutrition in sartorius muscle of the fowl. Br. J . Nutr. 18, 587. Montreuil, J. 1980. Primary structure of glycoprotein glycans. Adv. Carbohydr. Chem. Biochem. 37, 157. Morgan, P. H., Jacobs, H. G., Segrest, J. P., and Cunningham, L. W. 1970. A comparative study of glycopeptides derived from selected vertebrate collagens. J . Biol. Chem. 245, 5042. Moms, N. L, Fessler, L. I., Weinstock, A,, and Fessler, J. H. 1975. Procollagen assembly and secretion in embryonic chick bone. J . Biol. Chem. 250, 5719. Morrison, A. B., and McLdughlan, J. M. 1972. Availability of amino acids in foods. In “Protein and Amino Acid Functions” (E. J. Bigwood, ed.), Part 1 , p. 389. Pergamon, Oxford. Morrison, R . I. G . , Barrett, A. J., Dingle, J. T., and Prior, D. 1973. Cathepsins B, and D: Action on human cartilage protoeoglycans. Biochim. Biophys. Acta 302, 41 1. Muir, H. 1964. Chemistry and metabolism of connective tissue glycosaminoglycans (rnucopolysaccharides). Int. Rev. Connect. Tissue Res. p. 2. Muir, H. 1965. A mucopolysaccharide from human aorta. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, R. D. Harkness, S . M. Partridge, and G. R. Tristram, eds.), p. 137. Butterworth, London. Murphy, W. H . , von der Mark, K., McEneany, L. S . , and Bornstein, P. 1975. Characterization of procollagen-derived peptides unique to the precursor molecule. Biochemistry 14, 3243. Murphy, W. R., Daughaday, W. H., and Hartnett, C. 1956. The effect of hypopbysectomy and growth hormones on the incorporation of labeled sulfate into fibial epiphyseal and nasal cartilage of rat. J . Lab. Clin. Med. 47, 715. Myllyla, R . , Risteli, L., and Kivirikko, K. I. 1975. Glycosylation of galactosylhydroxylysyl residues in collagen in vitro by collagen glucosyltransferase. Inhibition by triple-helical conformation of the substrate. Eur. J . Biochem. 58, 517.

360

A. ASGHAR AND R. L. HENRICKSON

Nakamura, R., Sekoguchi, S . , and Sato, Y . 1975. The contribution of intramuscular collagen to the tenderness of meat from chicken with different ages. Poult. Sci. 54, 1604. Nakayama, T., and Sato, Y. 1971a. Relationship between binding quality of meat and myofibrillar proteins. 3. Contribution of myosin A and actin to rhelogical properties of heated minced meat gel. J . Texture Stud. 2, 75. Nakayama, T . , and Sato, Y . 1971b. Relationship between binding quality of meat and myofibrillar proteins. 2. The contribution of native tropomyosin and actin to the binding of meat. Agric. Biol. Chem. 32, 208. . between binding quality of meat and myofibrillar Nakayama, T., and Sato, Y. 1 9 7 1 ~Relationship proteins. 4. Contribution of native tropomyosin and actin in myosin B to rhelogical properties of heated minced meat gel. J . Texture Stud. 2, 475. Narayanan, A. S., Siegel, R. C . , and Martin, G. R. 1972. On the inhibition of oxidase by p-amino propionitrile. Biochem. Biophys. Res. Commun. 46, 745. Neelakantan, S., and Froning, G . W. 1971. Studies on the emulsifying characteristics of some intracellular turkey muscle proteins. J . Food Sci. 36, 61 3. Nemethy, G. 1967. Hydrophobic interactions. Angew. Chem., Int. Ed. Engl. 6, 195. Newman, R. A,, and Cutroneo, K . R. 1978. Glucocorticoids selectively decrease the synthesis of hydroxylated collagen peptides. Mol. Pharmacol. 14, 185. Nicholls, A. C., and Bailey, A. J. 1980. Identification of cyanogen bromide peptides involved in intermolecular cross-linking of bovine type I11 collagen. Biochem. J . 185, 195. Nicolaides, N., Fu, H., and Rice, G . R. 1968. The skin surface lipids on man compared with those of eighteen species of animal. J . Invest. Dermafol. 51, 83. Nimni, M. E. 1968. A defect in the intramolecular and intermolecular cross-linking of collagen caused by penicillamine. J . Biol. Chem. 243, 1457. Nimni, M. E., and Bavetta, L. A. 1965. Collagen defect induced by penicillamine. Science 150, 905. Nimni, M. E., and Harkness, R. D. 1968. Chemical and mechanical changes in the collagenous framework of skin induced by thiol compounds. Acta Physiol. Acad. Sci. Hung. 33(3), 325. Nimni, M. E., Deshmukh, K., and Gerth, N. 1972. Collagen defect induced by penicillamine. Nature (London),New Biol. 240, 220. Nist, C., von der Mark, K . , Hay, E. D., Olsen, B. R., Bornstein, P., Ross, R., and Dehm, P. 1975. Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies. J . Cell Biol. 65, 75. Nivard, R. J. F., and Tesser, G. E. 1965. Amino acids and related compounds. Compr. Biochem. 6, 143. Nordwig, A , , and Dick, Y . P. 1965. Cleavage with cyanogen bromide of methionyl bonds in collagen. Biochim. Biophys. Acta 47(1), 179. Nowack, H., Hahn, E., and Timpl, R. 1976a. Requirement for T cells in the antibody response of mice to calf skin collagen. Immunology 30, 29. Nowack, H., Gay, S., Wick, G., Becker, U . , and Timpl, R. 1976h. Preparation and use in immunohistology of antibodies specific for type I and type 111 collagen and procollagen. J . Immunol. Methods 12, 1 17. Obrink, B. 1974. Polysaccharides-collagen interactions. In “Structure of Fibrous Biopolymers” (E. D. T. Atkins and A . Keller, eds.), p. 81. Butterworth, London. O’Dell, D. S. 1968. Immunology of collagen and related materials. In “Treatise on Collagen” (B. S . Could, ed.), Vol. 2, Part A, p. 31 1. Academic Press, New York. Oikarinen, A. 1977. Effect of cortisol acetate on collagen biosynthesis and on the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase, and collagen glycosyltransferase in chick embryo tendon cells. Biochem. J . 164, 533. Olsen, B. R., Alper, R., and Kefalides, N. A. 1973. Structural characterization of a soluble fraction from lens-capsule basement membrane. Eur. J . Biochem. 38, 256

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

36 1

Olsen, B. R., Berg, R. A,, Kishida, Y., and Prockop, D. J . 1975. Further characterization of embryonic tendon fibroblasts and the use of immunofemitin technique to study collagen biosynthesis. J . Cell Biol. 64, 340. Olsen, B. R., Guzman, N. A,, Engel, J., Condit, C., and Aase, S . 1977. Purification and characterization of a peptide from the carboxy-terminal region of chick tendon procollagen. Biochemistry 16, 3030. Ono, K . 1970. Lysosomal-type enzymes in beef Longissimus dorsi muscle. J . Food Sci. 35, 256. Orekhovitch, V. N. 1958. Procollagen. Science 127, 1371. Orekhovitch, V. N., and Shpikiter, V. 0. 1955. Molecular weight and asymmetry of procollagen. Biokhimiya 20, 438. Orekhovitch, V. N., Tustanovsky, A. A,, Orekhovitch, K. D., and Plotnikova, N. E. 1948. The procollagen of hide. Biokhimiya 13, 55. Osborne, T. B., Mendel, L. B., and Ferry, E. L. 1919. A method of expressing numerically the growth-promoting value of proteins. J . Biol. Chem. 37, 223. Osborne, T. R., Mendel, L. B., Park, E. A,, and Winternitz, M. C. 1926-1927. Physiological effects of diets unusually rich in protein or inorganic salts. J . Biol. Chem. 71, 317. Osebold, W. R., and Pedrini, V. 1976. Pepsin-solubilized collagen of human nucleus pulposus and annulus fibrosus. Biochim. Biophys. Acra 434, 390. Oser, B. L. 1951. Method for integrating essential evaluation of protein. J . Am. Dier. Assoc. 27, 396. Ott, E., Spurlin, H. M., and Graffin, M. W. 1954. “Cellulose and Cellulose Derivatives,” Part 11, p. 673. Wiley (Interscience), New York. Page, R. C., and Benditt, E. P. 1967. Molecular diseases of connective and vascular tissues. 11. Amine oxidase inhibition by the lathyrogen, p-amino propionitrile. Biochemistry 6, 1142. Park, E. D., Tanzer, M. L., and Church, R. L. 1975. Procollagen synthesis in cell culture: Nascent chain population consistent with polycistronic mRNA. Biochem. Biophys. Res. Commun. 63, 1. Parrish, F. C., Jr., and Bailey, M. E. 1967. Physicochemical properties of bovine muscle particulate cathepsin. J . Agric. Food Chem. 15, 88. Partington, F. R., and Wood, G. C. 1963. Role of noncollagen components in the mechanical behavior of tendon fibers. Biochim. Biophys. Acta 69, 485. Paul, P. 1962. Tenderness and chemical composition of beef. 11. Variations due to animal treatment and to extent of heating. Food Technol. 16, 117. Pauling, L. 1940. “The Nature of the Chemical Bond,” p. 71. Cornell Univ. Press, Ithaca, New York. Pauling, L. 1945. The absorption of water by proteins. J . Am. Chem. SOC. 67, 555. Pauling, L., and Corey, R. B. 1951. The structure of fibrous proteins of the collagen gelatin group. Proc. Natl. Acad. Sci. U.S.A. 37, 272. Pawlowski, P. J . , Gillette, M. T . , Martinell, J., Lukens, L. N., and Furthmayr, H. 1975. Identification and purification of collagen-synthesising polysomes with anticollagen antibodies. J . Biol. Chem. 250, 2135. Payne, P. R. 1972. Protein quality of diets, chemical scores and amino acid imbalances. In “Protein and Amino Acid Functions” (E. J. Bigwood, ed.), Part 2, pp. 259, 381. Pergamon, Oxford. Pearce, N., and Kinsella, J. E. 1978. Emulsifying properties of proteins: Evaluation of a turbidimetric technique. J . Agric. Food Chem. 26, 716. Pearson, A. M., Spooner, M. E., Hegarty, G. R., and Bratzler, L. J. 1965. The emulsifying capacity and stability of soy sodium proteinate, potassium, caseinate, and non-fat dry milk. Food Technol. 19, 1841. Perez-Tamayo, R. 1973. “Molecular Pathology of Connective Tissue,’’ p. 323. Dekker, New York. Perez-Tamayo, R. 1979. The degradation of collagen. Bull. Rheum. Dis. 30(2), 1012. Perlman, F. 1964. Immunochemistry. In “Symposium on Foods: Proteins and Their Reactions” (H. W. Schultz and A. F. Anglemier, eds.), p. 423. AVI Publ. Co., Westport, Connecticut.

362

A. ASGHAR AND R . L. HENRICKSON

Petruska, J. A , , and Hodge, A. J. 1964. A subunit model for the tropocollagen macromolecule. Proc. Natl. Acad. Sci. U.S.A. 51, 871. Pfeiffer, N. E., Field, R. A., Vamell, T. R., Kruggel, W. G., and Kaiser, I. 1972. Effects of postmortem aging and stretching on the macromolecular properties of collagen. J . Food Sci. 37, 897. Phelps, C. F. 1974. The intercellular matrix. In “Structure of Fibrous Biopolymers” (E. D. T. Atkins and A. Keller, eds.), p. 53. Butterworth, London. Piez, K. A. 1966. Collagen. In “The Physiology and Biochemistry of Muscle as a Food” (E. J. Briskey, R. G. Cassens, and J. C. Trautman, eds.), p. 315. Univ. of Wisconsin Press, Madison. Piez, K. A. 1967. Soluble collagen and the components resulting from its denaturation. In “Treatise on Collagen” (G. N. Ramachandran, ed.), Vol. I , p. 210. Academic Press, New York. Piez, K. A. 1968. Cross-linking of collagen and elastin. Annu. Rev. Biochem. 37, 547. Piez, K. A. 1976. Primary structure. In “Biochemistry of Collagen” (G. N. Ramachandran and A . H. Reddi, eds.), p. 1. Plenum, New York. Piez, K. A,, Lewis, M. S . , Martin, G . R., and Gross, J . 1961. Subunits of the collagen molecules. Biochim. Biophys. Actu 53, 596. Piez, K. A,, Eigner, E. A,, and Lewis, M. S . 1963. The chromatographic separation and amino acid composition of the subunits of several collagens. Biochemistry 2, 58. Piez, K. A,, Bornstein, P . , Lewis, M. S . , and Martin, G. R. 1965. The preparation and properties of single and cross-linked chains from vertebrate collagens. In “Structure and Function of Connective and Skeletal Tissues” (S. Fitton-Jackson, ed.), p. 16. Butterworth, London. Piez, K. A., Miller, E. J., Lane, J. M . , and Butler, W. T. 1970. Localization of chemical and structural features in the collagen molecule. In “Chemistry and Biology of the Intercellular Matrix” (E. A. Balazs, ed.), Vol. I , p. 117. Academic Press, New York. Pike, R. L., and Brown, M. L. 1975. “Nutrition: An Integrated Approach,” p. 863. Wiley, New York. Pongpaeu, P., and Guggengeim, K. 1968. The nutritional availability of tryptophan in foods. Nutr. Diem 10, 222. Ponsetti, 1. V. 1954. Lesions of the skeleton and other mesodermal tissue in rats fed sweet pea seeds. J . Bone Joinr Surg., Am. Vol. 36A, 1031, Pope, E. M., Martin, G. R., and Lichtenstein, J. R. 1975. Patients with Ehlers-Danlos syndrome type IV lack type 111 collagen. Proc. Null. Acud. Sci. U.S.A. 72, 1314. Pouradier, J., and Venet, A. M. 1950. Contribution a I’etude de la structure des gelatines (Fr.). J . Chim. Phys. 47, 11. Preston, B. N., Snowden, J . Mck., and Houghton, K. T . 1972. Model connective tissue system. Biopolymers 11, 1645. Pnchard, P. M., Staton, G. W., and Curtoneo, K. R. 1974. In vitro synthesis of collagen peptides on fetal and neonatal rat skin polysomes by rabbit reticulocyte initiation factors. Arch. Biochem. Biophys. 163, 178. Prockop, D. J., Kaplan, A , , and Udenfriend, S . 1962a. Oxygen-18 studies on the conversion of proline to hydroxyproline. Biochem. Biophys. Res. Commun. 9, 162. Prockop, D. J., Peterkofsky, B., and Udenfriend, S . 1962b. Studies on the intracellular localization of collagen synthesis in the intact chick embryo. J . Biol. Chem. 237, 1581. Prockop, D. J., Berg, R. A , , Kivirikko, K. I . , and Uitto, J. 1976. lntracellular steps in the biosynthesis of collagen. In “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddi, eds.), p. 163. Plenum, New York. Prockop, D. J., Kivirikko, K. I . , Tuderman, L . , and Guzman, N. A. 1979. The biosynthesis of collagen and its disorders. N . Engl. J . Med. 301, 13, 77. Procter, H. R., and Wilson, J. A. 1916. The acid-gelatin equilibrium. J . Chem. SOC. 109, 307.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

363

Prost, E., Pelczynska, E., and Kotula, A. W. 1975. Quality characteristics of meat. I. Connective tissue in relation to individual muscles, age, and sex of animals and carcass quality grades. J . Anim. Sci. 41, 534. Puigserver, A . J., Sen, L. C . , Chifford, A . J., Feeney, R. E., and Whitaker, J. R. 1979. Covalent of attachment of amino acids to casein. J . Agric. Food Chem. 27, 1098, 1286. Radley, J. A . 1968. “Starch and its derivatives,” pp. 306, 354. Chapman & Hall, London. Ramachandran, G. N. 1968. Molecular architecture of collagen. J . Am. Leather Chem. Assoc. 63(3), 161. Ramachandran, G . N., and Chandrasekaran, R. 1968. Interchain hydrogen bonds via bound water molecules in the collagen triple helix. Biopolymers 6, 1649. Ramachandran, G. N., and Ramakrishnan, C. 1976. Molecular structure. In “Biochemistry of Collagen” (G. N. Ramachandran and R. Reddi, eds.), p. 45. Plenum, New York. Ramachandran, G. N., Sasisekharan, V . , and Thathachari, Y. T. 1962. Structure of collagen at the molecular level. In “Collagen” (N. Ramanathan, ed.), p. 81. Wiley (Interscience), New York. Ramachandran, G . N., Bansal, M., and Bhatnager, R. S. 1973. A hypothesis on the role of hydroxyproline in stabilizing collagen structure. Biochim. Biophys. Acta 322, 166. Rama-Rao, P. B., Norton, H. W., and Johnson, B. C. 1964. The amino acid composition and the nutritive value of proteins. 5. Amino acid requirements as a pattern for protein evaluation. J . Nutr. 82, 88. Randall, J. T. 1954. Observations on the collagen system. J . Soc. Leather Trades’ Chem. 38, 362. Rauterberg, J., Timpl, R., and Furthmayr, H. 1972a. Structural characterization of N-terminal antigenic determinants in calf and human collagen. Eur. J . Biochem. 27, 231. Rauterberg, J., Fietzek, P., Rexrodt, F., Becker, U . , Stark, M., and Kiihn, K. 1972b. The amino acid sequence of the carboxy-terminal nonhelical cross-link region of the a1 chain of calf skin collagen. FEES Lett. 21, 75. Reagan, J. O., Carpenter, Z . L . , and Smith, G. C. 1976. Age-related traits affecting the tenderness of the bovine longissirnus muscle. J . Anim. Sci. 43, 1198. Reid, K. 1974. A collagen-like amino acid sequence in a polypeptide chain of human CI, (a subcomponent of the first component of complement). Eiochem. J . 141, 189. Reiser, K. M., and Last, J. A. 1980. Quantitation of specific collagen types from lungs of small mammals. Anal. Biochem. 104(1), 87. Reissmann, T. L., and Nichols, J. 1960. Collagen article and manufacture thereof. U.S. Patent 2,9 19,999. Renner, H. W . , and Reichelt, D. 1973. Wholesomeness of high concentration of free radicals in irradiated foods. Zentralbl. Veterinaermed., Reihe B 20, 648; Chem. Abstr. 80, 116918 (1974). Reynolds, T. M. 1963. Chemistry of nonenzymic browning. I. The reactions between aldoses and amines. Adv. Food Res. 12, 1. Reynolds, T. M. 1965. Chemistry of nonenzymic browning. 11. Adv. Food Res. 14, 167. Rhodes, R. K., and Miller, E. J. 1978. Physicochemical characterization and molecular organization of collagen A and B chains. Biochemistry 17, 3442. Rich, A,, and Crick, F. H. C. 1961. The molecular structure of collagen. J . ,4401. Biol. 3, 483. Richardson, T. 1977. Functionality change in proteins following action of enzymes. Adv. Chem. Ser. 160, 185. Rigby, B. J. 1967. Correlation between serine and thermal stability of collagen. Nature (London) 214, 87. Risteli, J. 1977. Effect of prednisolone on the activities of the intracellular enzymes of collagen biosynthesis in rat liver and skin. Biochem. Pharmacol. 26, 1295. Risteli, J., and Kivirikko, K. I. 1974. Activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in the liver of rats with hepatic injury. Biochem. J . 144, 115.

364

A. ASGHAR AND R. L. HENRICKSON

Risteli, L., Myllyla, R., and Kivirikko, K. I. 1976. Partial purification and characterization of collagen galactosyl transference from chick embryos. Biochem. J . 155, 145. Robb-Smith, A. H. T . 1958. The relationship of reticulin to other collagen. In “Recent Advances in Gelatin and Glue Research” (G. Stainby, ed.), p. 38. Pergamon, Oxford. Roberts, H. J . 1967. Starch derivatives. In “Starch: Chemistry and Technology” (R. L. Whistler and E. F. Paschall, eds.). Vol. 2, p. 293. Academic Press, New York. Robertson, W. V. B. 1964. Collagen in mammalian tissue. In “Connective Tissue: Intracellular Macromolecules,” p. 95. Little, Brown, Boston, Massachusetts. Robertson, W. V. B., and Gross, V. 1954. Collagen formation in vitamin A deficient rats. J . Nutr. 54, 81. Robins, S . P . , and Bailey, A. J. 1973a. Relative stabilities of the intermediate reducible cross-links present in collagen fibres. FEBS Lett. 33, 167. Robins, S. P., and Bailey, A. J. 1973b. The chemistry of the collagen cross-links. The characterization of fraction C, a possible artifact produced during the production of collagen fibres with borohydride. Biochem. J . 135, 657. Robins, S. P . , and Bailey, A. J . 1975. The chemistry of the collagen cross-links. Biochem. J . 149, 381. Robins, S. P., Shimokomaki, M . , and Bailey, A. J. 1973. The chemistry of the collagen cross-links. Age-related changes in the reducible components of intact bovine collagen fibres. Biochem. J . 131, 771. Roden, L. 1965. Studies on the carbohydrate protein linkages in the protein complexes of acid mucopolysaccharides. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 141. Butterworth, London. Rojkind, M., and Gallop, P. M. 1963. y-Glutamyl peptides from collagen. Absrr. Collagen Curr., Meet. Am. Chem. Soc. No. 542. Rosbash, M., and Penman, S. 1971. Two classes of membrane-associated ribosomes. J . Mol. Biol. 59, 227. Rose, P. I. 1977. Gelatin. I. General properties. In “Theory: Photographic Process” (T. H. James, ed.), 4th ed., p. 51. Macmillan, New York. Rosenbloom, J., Harsch, M., and Jimenez, S. A. 1973. Hydroxyproline content determines the denaturation temperature of chick tendon collagen. Arch. Biochem. Biophys. 158, 478. Ross, R. 1968. The connective tissue .fiber forming cell. In “Treatise on Collagen” (B. S . Gould, ed.), Vol. 2, Part A, p. 1. Academic Press, New York. Rubin, A. L., Pfahl, D., Speakman, P. T., and Schmitt, F. 0. 1963. Tropocollagen significance of protease-induced alterations. Science 139, 37. Rudall, K. M. 1968. Comparative biology and biochemistry of collagen. I n “Treatise on Collagen” (B. S. Gould, ed.), Vol. 2, Part A , p. 98. Academic Press, New York. Runkel, R., and Lange, G. 1937. Method of producing fibrous materials from animal skins. U.S. Patent 2,090,902. Ryhanen, L., and Kivirikko, K. I. 1974a. Developmental changes in protocollagen lysyl hydroxylase activity in the chick embryo. Biochim. Biophys. Acta 343, 121. Ryhanen, L., and Kivirikko, K . I. 1974b. Hydroxylation of lysyl residues in native and denatured protocollagen by protocollagen lysyl hydroxylase in vitro. Biochim. Biophys. Acra 343, 129. Saffle, R. L. 1968. Meat emulsions. Adv. Food Res. 16, 105. Saffle, R. L . , Carpenter, J. A,, and Moore, D. G. 1964. Peeling ease of frankfurters. I . Effect of chemical composition, heat, collagen, and type of fat. Food Techno/. 18, 130. Sage, H., and Bornstein, P. 1979. Characterization of a novel collagen chain in human placenta and its relation to AB collagen. Biochemistry 18, 381.5. Sakai, T., and Gross, J. 1967. Some properties of the products of reaction of tadpole collagenase with collagen. Biochemistry 6, 518.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

365

Sakakibra, S., Inouye, K., Shudo, K., Kishida, Y., Kobayashi, Y., and Prockop, D. J. 1973. Synthesis of (Pro-Pro-Gly), of defined molecular weights. Evidence for the stabilization of collagen triple helix by hydroxyproline. Biochim. Biophys. Acta 303, 198. Salem, G., and Traub, W. 1975. Conformational implications of amino acid sequence regularities in collagen. FEBS Lett. 15, 94. Salem, L. 1962. The role of long-range forces in cohesion of lipoprotein. Can. J . Biochem. Physiol. 40, 1287. Sammonds, K . W., and Hegsted, D. M. 1977. Animal bioassays. In “Evaluation of Proteins for Humans” (C. E. Bodwell, ed.), p. 68. AVI Publ., Westport, Connecticut. Sandberg, L. B. 1976. Elastin structure in health and disease. Int. Rev. Connect. Tissue Res. 1, 160. Satterlee, L. D., Zachariah, N. Y., and Levin, E. 1973. Utilization of beef or pork skin hydrolyzates as a binder or extender in sausage emulsions. J . Food Sci. 38, 268. Satterlee, L. D., Kendrick, J. G . , and Miller, G. H. 1977. Rapid in vitro assays for estimating protein quality. Food Technol. 31, 78. Satterlee, L. D . , Marshall, H. F . , and Tennyson, J. M. 1979. Measuring protein quality. J . Am. Oil Chem. Soc. 56(3), 103. Schalk, D. H. 1980. Functional properties of bovine hide collagen. M.S. Thesis, Oklahoma State University, Stillwater. Scheraga, H. A. 1963. Intramolecular bonds in proteins. 11. Noncovalent bonds. In “The Proteins’’ (H. Neurath, ed.), 2nd ed., Vol. 1, p. 478. Academic Press, New York. Schiller, S. 1966. Connective and supporting tissues: Mucopolysaccharides of connective tissues. Annu. Rev. Physiol. 28, 137. Schiller, S., and Dorfman, A. 1955. The biosynthesis of mucopolysaccharides in the skin of alloxan diabetic rats. Biochim. Biophys. Acta 16, 304. Schiller, S., and Dorfman, A. 1957. The metabolism of mucopolysaccharides in animals. The effect of cortisone and hydroxycortisone. Endocrinology 60, 376. Schimmel, P. R., and Flory, P. J. 1968. Conformational energies and conformational statistics of copolypepides containing L-proline. J . Mol. Biol. 34, 105. Schmitt, F. O., Levine, L., Drake, M. P., Rubin, A. L., Pfahl, O., and Davison, P. F. 1964. The antigenicity of tropocollagen. Proc. Natl. Acad. Sci. U.S.A. 51, 493. Schneebeli, G. L., and Dougherty, T. F. 1963. The influences of ACTH and cortisol on pinocytosis and phagocytosis by connective tissue cells. Anat. Rec. 145, 372. Schneebeli, G. L., and Dougherty, T. F. 1966. Differences in cortisol inhibitions of heparin- and insulin-induced pinocytosis. Anat. Rec. 154, 51 1 . Schnell, J. 1968. Evidence for the existance of hydrophobic interactions as a stabilizing factor in collagen structure. Arch. Biochem. Biophys. 127, 497. Schofield, J. D., and Prockop, D. J. 1973. Procollagen-A precursor form of collagen. Clin. Orthop. Relat. Res. 97, 175. Schofield, J. D., Uitto, J., and Prockop, D. J. 1974. Formation of interchain disulfide bonds and helical structure during biosynthesis of procollagen by embryonic tendon cells. Biochemistry 13, 1801. Schubert, M. 1964. Intercellular macromolecules containing polysaccharides. Biophys. J . 4, 119. Schubert, M., and Hamerman, D. 1968. “A Primer on Connective Tissue Biology,” p. 5 . Lea & Febiger, Philadelphia, Pennsylvania. Schut, J. 1976. Meat emulsion. In “Food Emulsions” (S. Friberg, ed.), p. 385. Dekker, New York. Scott, P. G . , and Veis, A. 1976. The cyanogen bromide peptides of bovine soluble and insoluble collagens. 11. Tissue specific cross-linked peptides of insoluble skin and dentin collagen. Connect. Tissue Res. 4, 117. Scott, P. G . , Telser, A. G., and Veis, A. 1976. Semiquantitative determination of cyanogen bromide peptides of collagen in SDS-polyacrylamide gels. Anal. Biochem. 70, 25 1.

366

A. ASGHAR AND R. L. HENRICKSON

Scrimshaw, N. S . , and Young, V. R. 1972. Clinical methods for the evaluation of protein quality. In “Protein and Amino Acid Functions” (E. J. Bigwood, ed.), Vol. 2, p. 363. Pergamon, Oxford. Segal, D. M. 1969. Polymers of tripeptides as collagen models. J . Mol. Biol. 43, 497. Segal, D. M., and Traub, W. 1969. Polymers of tripeptides as collagen models. VIII. X-ray studies of four polyhexapeptides. J . Mol. Biol. 43, 519. Seifter, S., and Gallop, P. M. 1966. The structure of proteins. In “The Proteins” (H. Neurath, ed.), Vol. 4, p. 155. Academic Press, New York. Seifter, S . , Franzblau, C., Harper, E . , and Gallop, P. M. 1965. Special aspects of the primary structure of collagen. In “Structure and Function of Connective and Skeletal Tissue” (S. FittonJackson, ed.), p. 21. Buttenvorth, London. Selmanowitz, V. J . , Rizer, R. L., and Orentreich, N. 1977. Aging of skin and its appendages. In “Handbook of the Biology of Aging” (C. E. Finch and L. Hayflick, eds.), p. 496. Van Nostrand-Reinhold, Princeton, New Jersey. Sharp, J. G . 1963. Aseptic autolysis in rabbit and bovine muscle during storage at 37°C. J . Sci. Food Agric. 14, 468. Sharrah, N., Kunze, M. S., and Pangborn, R. M. 1965. Beef tenderness: Sensory and mechanical evaluation of animals of different breeds. Food Technol. 19, 131. Sheehan, J. C., and Hess, G . R. 1955. A new method of forming peptide bonds. J . Am. Chem. SOC. 77, 1067. Sheltar, M. R., Bradford, R. H., Joel, W., and Howard, R. P. 1961. Effects of parathyroid extract on glycoprotein and mucopolysaccharide components of serum and tissue. In “The Parathyroids” (R. 0. Creep and R. V. Talmage, eds.), p. 144. Thomas, Springfield, Illinois. Shen, J. L., and Morr, C. V. 1979. Physicochemical aspects of texturization: Fiber formation from globular protein. J . Am. Chem. Soc. 56(1), 63A. Sherman, P. 1962. The water-binding capacity of fresh pork. 4. The influence of ion absorption from neutrals and polyphosphate on water retention by lean pork. Food Technol. 16(4), 91. Sherr, C. J., Taubman, M. B., and Goldberg, B. 1973. Isolation of adisulfide-stabilized, three chain polypeptide fragment unique to a precursor of human collagen. J . Biol. Chem. 248, 7033. Shimokomaki, M., Elsden, D. F., and Bailey, A. J. 1972. Meat tenderness: Age related changes in bovine intramuscular collagen. J . Food Sci. 37, 892. Siegel, R. C. 1974. Biosynthesis of collagen cross-links: Increased activity of purified lysyl oxidase with reconstituted collagen fibrils. Proc. Nutl. Acud. Sci. U.S.A. 71, 4826. Siegel, R. C. 1977. Collagen cross-linking. J . Biol. Chem. 252, 254. Siegel, R. C. 1979. Lysyl oxidase. Int. Rev. Connect. Tissue Res. 8, 73. Sikorski, 2 . E., Olley, J . , andKostuch, S. 1976. Protein changes in frozen fish. Crit. Rev. FoodSci. Nutr. 8, 97. Sinex, F. M. 1968. The role of collagen in aging. In “Treatise on Collagen” (B. S. Could, ed.), Vol. 2, Part B, p. 410. Academic Press, New York. Slutskii, L. I., and Simkhovich, B. 2. 1980. [Molecular heterogeneity of collagen.] Usp. Sovrem. Biol. 89(1), 141; Chem. Absrr. 92, 192772~. Smith, D. J., and Shuster, R. C. 1962. Biochemistry of lathyrism. I . Collagen biosynthesis in normal and lathyritic chick embryos. Arch. Biochem. Biophys. 98, 498. Smith, G. G., and Silva de Sol, B. 1980. Racemization of amino acids in dipeptides shows COOH > NH2 for nonsterically hindered residues. Science 207, 765. Smith, G. C., Carpenter, Z. L., and King, G. T. 1968. Bovine muscle collagen content and solubility. J . Anim. Sci. 27, 1148. Smith, G. C., Juhn, H., Carpenter, 2. L . , Mattil, K. F., and Carter, C. M. 1973. Efficiency of protein additives as emulsion stabilizers in frankfurters. J . Food Sci. 38, 849. Smith, J. W. 1968. Molecular pattern in native collagen. Nuture (London) 219, 157.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

367

Snellman, 0. 1965. Biosynthesis of collagen performed in vitro. In “Structure and Function of Connective and Skeletal Tissue Proceedings” (S. Fitton-Jackson, ed.), p. 319. Butterworth, London. Sobel, M. E., Yamamoto, T., and Adams, S. L. 1978. Construction of a recombinant bacterial plasmid containing a chick pro-a2 collagen gene sequence. Proc. Natl. Acad. Sci. U.S.A. 75, 5846. Sopata, I . , Wojtecka-Lukasik, E., and Dancewicz, A. M. 1974. Solubilization of collagen fibrils by human leukocytes: Collagenase activated by rheumatoid synovial fluid. Actu Biochim. Pol. 21, 283. Southward, C. R., and Goldman, A. 1975. Co-precipitates-A review. N. Z . J . Dairy Sci. Technol. 10, 101. Southward, C. R., and Goldman, A. 1978. Co-precipitates and their application in food products. 11. Some properties and applications. N . Z . J . Dairy Sci. Technol. 13, 97. Speakman, J. B. 1944. Analysis of the water absorption isotherm of wool. Trans. Furuday SOC. 40, 6. Speakman, P. T. 1971. Proposed mechanism for the biological assembly of collagen triple helix. Nature (London) 229, 24 1. Spector, A . A. 1975. Fatty acid binding to plasma albumin. J . Lipid Res. 16, 165. Spiro, M. J., and Spiro, R. G. 1971. Studies on the biosynthesis of the hydroxylysine-linked disaccharide unit of basement membranes and collagens. 11. Kidney galactosyltransferase. J . Biol. Chem. 246, 4910. Spiro, R . G . 1969. Characterization and quantitative determination of the hydroxylysine-linked carbohydrate units of several collagens. J . Biol. Chem. 244, 602. Spiro, R. G . 1972. Basement membranes and collagens. In “Glycoproteins-Their Composition, Structure, and Function” (A. Gottschalk, ed.), 2nd ed., Part B, p. 964. Elsevier, Amsterdam. Spiro, R. G., and Spiro, M. J. 1971a. Studies on the biosynthesis of the hydroxylysine-linked disaccharide unit of basement membranes and collagens. I. Kidney glucosyltransferase. J . Biol. Chem. 246, 4899. Spiro, R. G . , and Spiro, M. J. 1971b. Studies on the biosynthesis of the hydroxylysine-linked disaccharide unit of basement membranes and collagens. 111. Tissue and subcellular distribution of glycosyltransferase and the effect of various conditions on the enzyme level. J . Biol. Chem. 246, 4919. Sponsler, 0. L., Bath, J. D., and Ellis, J. W. 1940. Water bound to gelatin as shown by molecular structure studies. 1.Phys. Chem. 44, 996. Stainsby, G . , ed. 1958. “Recent Advances in Gelatin and Glue Research.” Pergamon, Oxford. StanEikovB, M., and Trnavsky, K. 1974. Collagenolytic enzymes and acidproteinase activity in granuloma tissue. Experientia 30, 594. Stark, M., Rauterberg, J . , and Kuhn, K. 1971a. Evidence for a nonhelical region at the carboxyl terminus of the collagen molecule. FEBS Lett. 13, 101. Stark, M., Lenaers, A., Lapi&re,C. M., and Kuhn, K. 1971b. Electron optical studies ofprocollagen from skin of dermatosparaxic calves. FEBS Lett. 18, 225. Staudinger, Hj., Krisch, K., and Leonhauser, S. 1961. Role of ascorbic acid in microsomal electron transport and the possible relationship to hydroxylation reactions. Ann. N.Y. Acud. Sci. 92, 195. Steinberg, I . Z . , Harrington, W. F., Berger, A , , Sela, M., and Katchalaski, E. 1960. The conformational changes of poly-L-proline in solution. J . Am. Chem. SOC. 82, 5263. Steiner, D. F., Kemmler, W . , Tager, H. S . , and Peterson, J. D. 1974. Proteolytic processing in the biosynthesis of insulin and other proteins. Fed. Proc., Fed. Am. SOC. Exp. Biol. 33, 2105. Stelder, G . , and Stegemann, H. S . 1962. Incorporation of P-amino-propionitrile into the collagen molecule in lathyrism in the rat. Narurwissenschqften 49, 398.

368

A. ASGHAR AND R. L. HENRICKSON

Stenn, K. S., Madri, J . A , , and Roll, F. J. 1979. Migrating epidermis produces AB2 collagen and requires continual synthesis for movement. Nature (London) 277, 229. Sternberg, M., and Kim, C. Y. 1977. Lysinoalanine formation in protein food ingredients. Adv. Exp. Med. Biol. 86A, 73. Stevens, F. S . 1963. An enzyme system in the gastric secretion capable of reducing the viscosity of native soluble calf-skin collagen. Biochim. Biophys. Actu 77, 466. Steven, F. S., Coy, G. R., and Jackson, D. S . 1972. Evidence for intrachain masked carboxyl groups in polymeric collagen. Biochim. Biophys. Acta 271, 114. Stoltz, M., Timpl, R., and Kiihn, K. 1972. Nonhelical regions in rat collagen al chain. FEBS Lett. 26, 61. Strauss, M. 1964. Food allergens. I n “Symposium on Foods: Proteins and Their Reactions” (H. W. Schultz and A. F. Anglemier, eds.), p. 409. AVI Publ., Westport, Connecticut. Summers, J. D., and Fisher, H. 1960. The nutritional requirements of the protein depleted chicken. 1. Effect of different protein depletion regimes on body composition during depletion. J . Nutr. 72, 153. Summers, R. L., Kemp, J. D., Ely, D. G., and Fox, J. D. 1978. Effect of weaning, feeding system, and sex of lamb on lamb carcass characteristics and palatability. J . Anim. Sci. 47, 622. Sunder-Raj, C. V., Church, R. L., Klobutcher, L. A. and Ruddle, F. H. 1977. Genetics of the connective tissue proteins. Proc. Nutl. Acud. Sci. U.S.A. 74, 4444. Suzuki, E., Fraser, R. D. B., and MacRae, T. P. 1980. Role of hydroxyproline in stabilization of the collagen molecule via water molecules. Int. J . Biol. Macromol. 2( l), 54. Swift, C. E. 1965. The emulsifying properties of meat protein. Proc. Meat Ind. Res. Conf. p. 78. Sykes, B. C., and Solomon, E. 1978. Assignment of a type collagen structural gene to human chromosome. 7. Nature (London) 272, 548. Tajima, S . , and Nagai, Y. 1980. Distribution of macromolecular components in calf dermal connective tissue. Connect. Tissue Res. 7(2), 65. Talty, R. D. 1969. Method of preparing edible tubular collagen casing. U.S. Patent 3,425,847. Tanzer, M. L. 1965. Experimental lathyrism. Int. Rev. Connect. Tissue Res. 3, 91. Tanzer, M. L. 1973. Cross-linking of collagen. Science 180, 561. Tanzer, M. L. 1976. Cross-linking. In “Biochemistry of Collagen” (G. N. Ramachandran and A. H . Reddi, eds.), p. 137. Plenum, New York. Tanzer, M. L., and Hunt, R. D. 1964. Experimental lathyrism: An autoradiographic study. J . Cell Biol. 22, 623. Tanzer, M. L., and Kefalides, N. A. 1973. Collagen cross-links: Occurrence in basement membrane collagens. Biochem. Biophys. Res. Commun. 51, 775. Tanzer, M. L., and Mechanic G. 1968. Collagen reduction by sodium borohydride: Effects of reconstitution, maturation and lathyrism. Biochem. Biophys. Res. Commun. 32, 885. Tanzer, M. L . , and Mechanic, G. 1970. Isolation of lysinonorleucine from collagen. Biochem. Biophys. Res. Commun. 39, 183. Tanzer, M. L., Mechanic G . , and Gallop, P. M. 1970. Isolation of hydroxylysinonorleucine and its lactone from reconstituted collagen fibrils. Biochim. Biophys. Acfu 207, 548. Tanzer, M. L., Fainveather, R., and Gallop, P. M. 1973a. Isolation of the cross-link, hydroxymerodesmosine, from borohydride-reduced collagen. Biochim. Biophys. Actu 310, 130. Tanzer, M. L., Housley, T., Berube, L., Fainveather, R., Franzblau, C., and Gallop, P. M. 1973b. Structure of two histidine-containing cross-links from collagen. J . Biol. Chem. 248, 393. Tanzer, M. L., Church, R. L., Yaeger, J. A , , Wampler, D. E., and Park, E. D. 1974. Procollagen: Intermediate forms containing several types of peptide chains and noncollagen peptide extensions at the NH2 and COOH ends. Proc. Nutl. Acud. Sci. U.S.A. 71, 3009. Teen, A. E., Virchow, W., and Loughlin, M. E. 1956. A microbial method for the nutritional evaluation of proteins. J . Nufr. 59, 587.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

369

Theis, E. R., and Jacoby, T. F. 1941. Studies in animal skin proteins. 11. The effect of calcium hydroxide solution upon the bone amino acid content of collagen. J . Am. Leather Chem. Assoc. 36, 375. Thomas, A. W., and Foster, S. B. 1925. The destructive and preservative effect of neutral salt upon hide substance. Ind. Eng. Chem. 17, 1162. Thomas, A. W., and Foster, S. B. 1926. The behavior of deaminized collagen. Further evidence in favor of the chemical nature of tanning. J. Am. Chem. SOC.48, 489. Thompson, R. C . , and Ballou, J. E. 1954. Studies of metabolic turnover with tritium as a tracer. IV. Metabolically inert lipid and protein fraction. J . Biol. Chem. 208, 883. Timpl, R. 1976. Immunological studies on collagen. In “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddi, eds.), p. 319. Plenum, New York. Timpl, R., Fietzek, P. P., Furthmayr, H., Meigel, W., and Kuhn, K. 1970. Evidence for two antigenic determinants in the C-terminal region of rat skin collagen. FEBS Lett. 9, 1 I . Timpl, R., Furthmayr, H., and Beil, W. 1972. Maturation of the immune response to soluble rat collagen: Late appearance of antibodies to N-terminal sites of the a2 chain. J . fmmunol. 108, 119. Timpl, R., Wick, G., Furthmayr, H . , Lapiere, C. M., and Kuhn, K . 1973. Immunochemical properties of procollagen from dermatosparactic calves. Eur. J . Biochem. 32, 584. Tkocz, C . , and Kuhn, K. 1969. The formation of triple-helical collagen molecules from al or a 2 polypeptide chains. Eur. J . Biochem. 7, 454. Tokoro, Y . , Eisen, A. Z., and Jeffrey, J. J. 1972. Characterization of a collagenase from rat skin. Biochim. Biophys. Acta 258, 298. Tolman, R. C . , and Steam, A. E. 1918. The molecular mechanism of colloidal behavior. I . Swelling of fibrin in acid. J . Am. Chem. Soc. 40, 264. Traub, W. 1969. Polymers of tripeptides as collagen models. J . Mol. Biol. 43, 479. Traub, W . , and Piez, K. A. 1971. The chemistry and structure of collagen. Adv. Protein Chem. 25, 243. Trelstad, R. L. 1974. Human aorta collagens: Evidence for three distinct species. Biochem. Biophys. Res. Commun. 57, 717. Trelstad, R. L . , Kang, A. H . , Igarashi, S., and Gross, J. 1970. Isolation of two distinct collagens from chick cartilage. Biochemistry 9, 4993. Trelstad, R. L., Kang, A . H . , Took, B. P., and Gross, J. 1972. Collagen heterogeneity. Highresolution separation of native, [al(I)12a2 and [al(II)13 and their component a chains. J . Biol. Chem. 247, 6469. Trelstad, R. L., Catanese, V. M., and Rubin, D. F. 1976. Collagen fractionation: Separation of native types I, 11, and 111 by differential precipitation. Anal. Biochem. 71, 114. Trumbetas, J., Fioriti, J. A , , and Sims, R. J. 1979. Study of lipid-protein interaction using pulsed NMR. J. Am. Oil Chem. Soc. 56(10), 890. Tuderman, L . , Kivirikko, K. I., and Prockop, D. J. 1978. Partial purification and characterization of a neutral protease which cleaves the N-terminal properties from procollagen. Biochemistry 16, 2948. Tufail, M. 1977. Effects of organic acid on amino carbonyl reactions. M.S. Thesis, Kyushu University, Fukuoka, Japan. Turkot, V. A . , Komanowsky, M . , and Sinnamon, H. 1. 1978. Comminuted collagen. Estimated cost of commercial production. Food Techno/. 32(4), 48. Uchiyama, S . , and Uchiyama, M. 1979. Free radical production in protein-rich food. J. Food Sci. 44, 1217. Uchiyama, S . , and Uchiyama, M. 1981. Properties of free radicals in proteins and amino acid pyrolysates. J . Food Sci. 46, 113.

370

A. ASGHAR AND R. L. HENRICKSON

Uitto, J. 1977. Biosynthesis of type I1 collagen: Removal of amino- an- carboxy-terminal extensions from procollagen synthesized by chick embryo cartilage cells. Biochemistry 16, 3421. Uitto, J., and Prockop, D. J. 1973. Rate of helix formation by intracellular procollagen and protocollagen. Evidence for a role of disulfide bonds. Biochem. Biophys. Res. Commun. 55, 904. Uitto, J., and Prockop, D. J. 1974. Biosynthesis of cartilage procollagen. Influence of chain association and hydroxylation of prolyl residues on the folding of the polypeptides into the triple-helical conformation. Biochemistry 13, 4586. Uitto, J., Hoffman, H. P., and Prockop, D. J. 1975. Retention of nonhelical procollagen containing cis-hydroxyproline in rough endoplasmic reticulum. Science 190, 1202. U.S. Department of Agriculture 1981. Fed. Regisr. Title 9-Part 318.0 (3). Uyeta, M., Kaneda, T . , Mazaki, M., and Taue, S . 1978. Studies on mutagenicity of food. I . Mutagenicity of food pyrolysates. J . Food Hyg. SOC. J . 19, 216. Van der Veen, J., Weil, J. T., Kennedy, T. E., and Olcott, H. S . 1970. Aliphatic hydroxylamines as lipid antioxidants. Lipids 5, 509. Van Eerd, J. P. 1971. Meat emulsion stability: Influence of hydrophilic-lipophilic balance, salt concentration, and blending with surfactants. J . Food Sci. 36, 1121. Veis, A . 1964. “The Macromolecular Chemistry of Gelatin,” p. 433. Academic Press, New York. Veis, A., and Drake, M. P. 1963. The introduction of intramolecular covalent cross-linkages into ichthyocol tropocollagen with monofunctional aldehydes. J . Biol. Chem. 238, 2003. Veis, A., and Legowik, J. T. 1965. Interchain interactions in collagen fold formation. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 70. Buttenvorth, London. Veis, A., and Schlueter, R. J. 1963. Presence of phosphate mediated cross-linkages in hard tissue collagen. Nature (London) 197, 1204. Veis, A . , Aresey, J., and Cohen, J. 1960. The depolymerization of collagen fibers. J . Am. Leather Chem.Assoc. 5 5 , 548. Verwey, E. J. W . , and Overbeck, J. T. G. 1948. “Theory of the Stability of Lyophobic Colloid.” Elsevier, Amsterdam. Verzar, F. 1963. The aging of collagen. Sci. Am. 203, 104. Verzar, F. 1964. Aging of the collagen fiber. Int. Rev. Connect. Tissue Res. 2, 243. Viidik, A. 1973. Functional properties of collagenous tissues. Int. Rev. Connect. Tissue Res. 6 , 127. Vogel, H. D. 1974. Organ specifity of the effect of D-penicillamine and of lathyrogen (aminoacetonitrile) on mechanical properties of connective and supporting tissue. Arzneim. -Forsch. 24(2), 157. Vognarova, I., DvoMk, Z., and Bohm, R. 1968. Collagen and elastin in different cuts of veal and beef. J . Food Sci. 33, 339. von der Mark, K., and Bornstein, P. 1973. Characterization of the pro-ul chain of procollagen. Isolation of a sequence unique to the percursor chain. J . Biol. Chem. 248, 2285. von Hippel, P. H. 1967. Structure and stabilization of the collagen molecule in solution. In “Treatise on Collagen” (G. N. Ramachandran, ed.), Vol. 1, p. 253. Academic Press, New York. von Hippel, P. H . , and Wong, K . Y. 1962. The effect of ions on the kinetics of formation and stability of the collagen fold. Biochemistry 1, 664. Voutsinas, L., and Nakai, S. 1979. Covalent binding of methionine and tryptophan to soy protein. J . Food Sci. 44, 1205. Vuust, J. 1975. Procollagen biosynthesis by embryonic-chick bone polysomes. Eur. J . Biochem. 60, 41. Vuust, J., and Piez, K. A. 1970. Biosynthesis of the (Y chains of collagen studied by pulse-labeling in culture. J . Biol. Chem. 245, 9201. Vuust, J., and Piez, K. A. 1972. A kinetic study of collagen biosynthesis. J . Biol. Chem. 247, 856. Wada, K., Shirai, K., and Kawamura, A. 1980. Properties of collagens from pigskins of different ages. J . Am. Leather Chem. Assoc. 75, 90.

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

37 1

Wahl, L. M., Wahl, S. M., Mergenhagen, S., and Martin, G. E. 1975. Collagenase production by lymphokine-activated macrophages. Science 187, 261. Walton, A. G. 1974. Synthesis polypeptide models for collagen structure and function. In ‘‘Structure of Fibrous Biopolymers” (E. D. T. Atkins and A. Keller, eds.), p. 139. Butterworth, London. Weinstock, A,, King, P. C., and Wuthier, R. E. 1967. The ion-binding characteristics of reconstituted collagen. Biochem. J . 102, 923. Weinstock, M., and Leblond, C. P. 1974. Formation of collagen. Fed. Proc., Fed. Am. SOC. Exp. Biol. 33, 1205. Weir, C. E., and Carter, J. 1950. Rate of shrinkage of tendon collagen. Further effects of tannage and liquid environment on the activation constants of shrinkage. J . Res. Nutl. Bur. Stand. (U.S.) 44,599. Weiss, J. B. 1976. Enzymic degradation of collagen. Int. Rev. Connect. Tissue Res. 7, 101. Whitmore, R. A,, Jones, H. W . , Windus, W., and Naghski, J. 1970. Preparation of hide collagen for food. J . Am. Leather Chem. Assoc. 65, 382. Whitmore, R. A , , Jones, H. W., Windus, W., and Naghski, J. 1972. Preparation and viscoelastic properties of fibrous collagen dispersion from limed cattlehide splits. J Food Sci. 37, 302. Whitmore, R., Booth, A., Naghski, J., and Swift, C. 1975. Digestibility and safety of limed hide collagen in rat feeding experiments. J . Food Sci. 40, 101. Widdowson, E. M., Dickerson, J. W. T., and McCance, R. A. 1960. Severe undernutrition in growing and adult animals. Br. J . Nufr. 14, 457. Wiedemann, H., Chung, E., Fujii, T., Miller, E. J., and Kiihn, K. 1975. Comparative electron microscope studies on type 111 and type I collagens. Eur. J . Biochem. 51, 363. Wierbicki, E., Kunkle, L. E., Cahill, V. R., and Deatherage, F. E. 1954. The relation of tenderness to protein alteration during postmortem aging. Food Technol. 8, 506. Wierbicki, E., Cahill, V. R., Kinkle, L. E., Klosterman, E. W., and Deatherage, F. E. 1955. Effect of castration on biochemistry and quality of beef. J . Agric. Food Chem. 3, 244. Wiley, E. L., Reagan, J. O., Carpenter, J. A., and Campion, D. R. 1979. Connective tissue profiles of various raw sausage materials. J . Food Sci. 44,919. Wilkinson, D. I. 1969. Variability in composition of surface lipids. The problem of the epidermal contribution. J . Invest. Dermatol. 52, 339. Williams, I. F., Harwood, R., and Grant, M. E. 1976. Triple helix formation and disulphide bonding during the biosynthesis of glomerular basement membrane collagen. Biochem. Biophys. Res. Commun. 70, 200. Williamson, A. R. 1969. The biosynthesis of multichain proteins. Essays Biochem. 5 , 139. Wilson, G. D., Bray, R. W., and Phillips, P. H. 1954. The effect of age and grade on the collagen and elastin content of beef and veal. J . Anim. Sci. 13, 826. Wilson, J. A. 1928. “The Chemistry of Leather Manufacture,” Vol. 1, p. 129. Chem. Catalog Co., New York. Winegarden, M. W. 1950. Physical and histological changes of three connective tissues of beef during heating. Ph.D. Thesis, Iowa State University, Ames. Wismer-Pedersen, J. 1971. Water. In “The Science of Meat and Meat Products” (J. P. Price and B. S . Schewigert, eds.), p. 177. Freeman, San Francisco, California. Wolf, W. J. 1970. Soybean proteins. The functional chemical and physical properties. J . Agric. Food Chem. 18, 969. Wolf, W. J., andcowan, J. C. 1971. Soybeansas aprotein source. CRCCrit. Rev. FoodTechnol. 2 ,

81. Wollenberg, H. G. 1952. Heat of wetting of hide substance between 0” and 60”. J . SOC. Leather Trades’ Chem. 36, 170. Wood, P. D. 1977. Technical and pharmaceutical uses of gelatin. In “Science and Technology of Gelatin” (A. G. Ward and A. Courts, eds.), p. 414. Academic Press, New York.

372

A. ASGHAR AND R. L. HENRICKSON

Woodard, J. C., Short, D. D., Alvarez, M. R., and Reyniers, J. 1975. Biological effects of NE-(o~-2-amino-caroxyethy~)-~-lysine, lysinoalanine. In “Protein Nutritional Quality of Foods and Feeds” (M. Friedman, ed.), Part 2, p. 595. Dekker, New York. Woodhead-Galloway, J., Hukins, D. W. L., and Wray, J . S. 1975. Closest packing of two-stranded coiled coils as a model for the collagen fibril. Biochem. Biophys. Res. Commun. 64, 1237. Woolley, D. E., Lindberg, K. A,, Glanville, R. W., and Evanson, J. M. 1975a. Action of rheumatoid synovial collagenase on cartilage collagen different susceptibilities of cartilage and tendon collagenase attack. Eur. J . Biochem. 50, 437. Woolley, D. E., Glanville, R. W., Crossley, M. J., and Evanson, J. M. 1975b. Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen. Eur. J . Biochem. 54, 611. Worrall, J . 1965. The eff-ct of temperature on the denaturation of tropocollagen. In “Structure and Function of Connective and Skeletal Tissue” (S. Fitton-Jackson, ed.), p. 50. Butterworth, London. Wu, C. H., Nakai, S . , and Powrie, W. D. 1976. Preparation and properties of acid-solubilized gluten. J . Agric. Food Chem. 24, 504. Wu, J. J. 1978. Characteristics of bovine intramuscular collagen under various postmortem conditions. Ph.D. Thesis, Texas A&M University, College Station. Wyckoff, H. W., Tsemoglou, D., Hanson, A. W., Knox, J. R., Lee, B., and Richards, M. 1970. A three-dimensional structure of ribonucleases. J . B i d . Chem. 245, 305. Yamashita, M., Arai, S . , Tsai, S . J., and Fujimaki, M. 1970. Supplementing S-containing amino acids by plastein reaction. Agric. Biol. Chem. 34, 1593. Yamashita, M., Arai, S . , Kokubo, S . , Aso, K., and Fujimaki, M. 1975. Synthesis and characterization of a glutamic acid plastein with greater solubility. J . Agric. Food Chey. 23, 27. Yamashita, M., Arai, S., and Fujimaki, M. 1976. A low-phenylalanine, high-tyrosine plastein as an acceptable dietetic food. Method of preparation by use of enzymic protein hydrolysis and resynthesis. J . Food Sci. 41, 1029. Yamashita, M . , Arai, S., Imaizumi, Y., Amano, Y., and Fujimaki, M. 1979a. A one-step process for incorporation of L-methionine into soy protein by treatment with papain. J . Agric. Food Chem. 21, 52. Yamashita, M., Arai, S . , Imazumi, Y., Amano, Y., and Fujimaki, M. 1979b. A novel one-step process for enzymatic incorporation of amino acids into proteins. Agric. Biol. Chem. 43, 1065, 1069. Yarsley, V. E., Flavell, W., Adamson, P. S., and Perkins, N. G. 1964. “Cellulosic Plastics,” pp. 8, 55. Iliffe, London. Yates, J. R. 1968. A comparison of the efficiency of various extractants for the solubilization of collagen from sheepskin waste. J . Soc. Leather Trades’ Chem. 52, 425. Yatsumatsu, K., Sawada, K . , Moritaka, S . , Misaki, M., Toda, T., Wada, T . , and Ishii, K. 1972. Whipping and emulsifying properties of soybean products. Agric. Biol. Chem. 36, 719. Yee, R. Y., Englander, S. W., and von Hippel, P. M. 1974. Native collagen has a two-bonded structure. J . Mol. Biol. 83, 1. Yonath, A , , and Traub, W. 1969. Polymers of tripeptides as collagen models. J . Mol. Biol. 43,461. Zachariades, P. A. 1900. Des actions diverses des acides sur la substance conjonctive. C. R. Seances SOC. Biol. Ses. Fil. 52, 1 127. Zimmerman, B. K . , Pikkarainen, J . , Fietzek, P. P., and Kuhn, K. 1970. Cross-linkages in collagen. Demonstration of three different intermolecular bonds. Eur. J . Biochem. 16, 217.

ADVANCES IN I-OOD KEStARCH, VOL..

28

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL LEA HYVONEN, PEKKA KOIVISTOINEN, Department of Food Chemistty and Technology, Uriiversig of Helsinki, Helsinki. Finland

FELIX VOIROL Xyrofn Ltd.. Baar, Switzerland

Introduction . .......................... ...... of Xylitol . . . . . . . . . . . . . . . . ...... The Occurren ...... A. Natural Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... B. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Large-Scale Xylitol Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Physicochemical and Food Technological Properties of Xylitol. . . . . . . . . . . A. Physicochemical Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Food Technological Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... A. Confectionery. . . . . . . . . . . . . . . . . . . . . . . . . B. Ice Cream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... ...... c. Yogurt . . . . . . . . . . . . . . . . . . . . . . . . . . D. Jams, Jellies, and Marmalades.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Bakery Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Drinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . V. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

11.

I.

313 314 374 375 378 382 382 389 392 392 396 396 396 398 398 399 399 400

INTRODUCTION

The sensation of sweetness and the concept of a sweetener have undoubtedly been meaningful and important to man throughout his entire existence. During much of the relatively recent culinary history, i.e., the last 15CL200 years, and indeed continuing to the present day “sweetness” and “sweetener” have for 313 Copyright 0 1982 by Academic Press, Inc All rights of reproduction in any form reserved. ISBN 0-12-016428-0

374

LEA HYVONEN ET AL

most people meant the respective taste and functional use of sucrose, which in turn has simply been referred to as “sugar.” The world of food science, however, is not so simple. On the one hand, there are numerous substances which have the property of sweetness and hence have the potential to be used as sweeteners. On the other hand, the various potential sweeteners have many other properties in addition to sweetness which have important and varying functional characteristics, both positive and negative in nature. As knowledge about the various kinds of sweet-tasting substances has increased, it has become generally recognized that there are valid roles which each of them can play. Sweetness and the enhancement of food palatability are, perhaps, the common denominators in the use of any sweetener in foods. The choice of sweetener for a particular food system, however, is based on other considerations as well. The food technologist may require bulking, preservative, or humectant functions, or other physical and chemical properties such as stability to heat processing and storage. Most of these requirements are adequately fulfilled by the traditional sucrose or hydrolyzed starch sweeteners. From the nutritional and health point of view, however, there may also be objectives such as reducing the amount of energy which the sweetening component brings into the food system, avoidance of too rapidly absorbed carbohydrates, or reducing the exposure to types of food which are known to cause dental decay, to note only a few of the more obvious considerations. In recognition of the validity of these other requirements there has been an intensive search in recent years for suitable alternative sweeteners. The search has not been in vain, because there are a number of sweeteners which hold promise in fulfilling some of the divergent special sweetening needs currently being developed and commercialized. One of the most promising of these from the standpoint of special dietary applications, is xylitol, particularly in the areas of noncariogenic confections and disturbances of carbohydrate metabolism, and from the standpoint of fulfilling many of the food technological requirements traditionally expected of the conventional sweeteners. The metabolic pathways of xylitol and the effects of xylitol on human metabolism as well as the tolerability and toxicity of xylitol have been discussed previously in Advances in Food Research by Ylikahri (1979). The dental aspects of xylitol have also been reviewed in this series (Makinen, 1979). The manufacture, properties, and food applications of xylitol are discussed in this article. II. THE OCCURRENCE AND MANUFACTURE OF XYLITOL A.

NATURAL OCCURRENCE

Xylitol occurs widely in nature. Frerejacque (1943) showed the occurrence of xylitol in lichens, seaweed, and yeast. Kratzl and Silbernagel (1963) found

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

375

xylitol in mushrooms (Psalliota campestris). Xylitol has been found in small quantities in many fruits, berries, and vegetables (Table I) (Washiittl et al., 1973; Makinen and Soderling, 1980), and is also a normal metabolic intermediate in mammalian carbohydrate metabolism, including that of man (Hollmann and Touster, 1956, 1957; Bassler, 1972). The normal xylitol concentration of blood is 0.03-0.06 mg/100 ml blood. Commercially produced xylitol is a nature-identical product similar in structure and properties to the natural substance. B.

HISTORY

Xylitol is by no means a new substance, having been first prepared as a syrup 90 years ago almost simultaneously in the laboratories of Bertrand (1891) and Fischer and Stahel (1891). Wolfrom and Kohn (1942) succeeded in obtaining crystalline xylitol upon hydrogenation of highly purified xylose. Carson et al. (1943) demonstrated the existence of two crystalline forms: the stable rhombic and the unstable monoclinic forms. Chiang et al. (1958) reduced xylose to xylitol by Penicillium chrysogenum and Onishi and Suzuki (1966) by yeasts. Later Onishi and Suzuki (1969) produced xylitol from glucose via D-arabitol and D-xylulose by certain yeasts. Since the time when xylitol was found to be a normal intermediate in carbohydrate metabolism (Touster, 1960) there has been an ever-increasing volume of knowledge about its metabolic behavior in parenteral nutrition (e.g., Horecker et al., 1969; Brian and Miller, 1974; Thomas et al., 1974; Ritzel and Brubacher, 1976; Ylikahri, 1979) as well as its use as a sweetener in diabetic diets, which was first considered by Mellinghoff (1961). By the end of the 1960s xylitol had drawn the attention of dental scientists as being possibly less cariogenic than other known nutritive sweeteners. Miihlemann and his colleagues (1970) confirmed this in the rat model. Scheinin and Makinen and their colleagues (1 974, 1975a) found in the Turku sugar studies that when xylitol was substituted for sucrose in the human diet the result was a 90% reduction in the incidence of new carious lesions, as well as indications of a remineralizing effect on existing caries. Later Scheinin et al. (1975b) made a 1year chewing gum study, the findings of which indicated a therapeutic, cariesinhibiting effect of xylitol even for a partial sucrose replacement in the diet. Before 1975 the production of xylitol was centered in Italy, Germany, the Soviet Union, Japan, and China, with the largest quantity being produced in the Soviet Union, where xylitol is the principal nutritive sweetener used in special dietary foods for diabetics. Total world production was estimated to be under 2000 tonslyr. In 1975 the first truly large-scale production of xylitol was begun in Kotka, Finland, at the sucro-chemical plant of the Finnish Sugar Co. Ltd., Helsinki, with a capacity for producing xylitol of over 3000 tons/yr. In 1976 ownership of the Kotka plant was transferred to Xyrofin Ltd., a joint venture

TABLE I OCCURRENCE OF XYLITOL IN FRUITS" Relative ripenessh Fruit Raspberryd (Rubus idaeus) Strawberryd (Fragaria vesca) Red whortleberryd (lingonberry) (Vaccinium vitis idaeu)

Cranberryd (Vaccinium oxycoccus, Oxycoccus quadripetalus)

B il berryd (Vaccinium myrtillus) Sea buckthornd (Hippophae rhamnoides)

Rowan berryd (Sorbus aucuparia)

1

2 3 1

2 1

2 3 4 5 6 1

2 3 4 1

2 3 1

2 3 4 I 2 3 4

A

B

XylitoP

0.030e 0.300 0.420 0.196f 0.740 0.0308 0.040 0.120 0.600 0.740 1.100 0.0128 0.030 0. I28 0.600 0.124e 0.353 2.0 0.310" 0.380 0.400 0.412 0.030h 0.050 0.242 0.410

Unripe, green, hard Half ripe, reddish, hard Ripe, red Half ripe, reddish, hard Ripe, red Unripe, green, hard Unripe, green, hard Unripe, reddish, hard Half ripe, reddish, hard Half ripe, reddish, hard Ripe, red Unripe, reddish, hard Unripe, reddish, hard Half ripe, reddish, hard Ripe Unripe, green, hard Half ripe, reddish, hard Ripe Unripe, slightly orange. hard Unripe, orange, hard Half ripe, orange Ripe, orange Unripe, green, hard Unripe, reddish, hard Half ripe, reddish Ripe, red

7.5 405 26 150 280 58 I1 36 9 64 17 37 18 21 38 28 21 91 15 26 25 160 I30 1 I9 81

Bog whortlebenyd (bog bilbeny) (Vaccinium uliginosum) Cloudberryd

77

0.050e 0.413 1.460 0.250'

Unripe, green, hard Half ripe, bluish Ripe, blue Ripe, yellow

(Rubus chamaemorus) Black curranv

1.oooe

Ripe, black

(Ribes nigrum) Red curranv

0.450g

Ripe, red

100

Unripe, green, hard Ripe Ripe

Plums (a South African variety)' Pruned Bananai Grapd

128 48 67 0 53 20 93 105

White wine (Bordeaux Blanc-77) Dubonnet (-77)

35 135

(Ribes rubrum) Apple (Malusp Apple, Yellow Cinnamon, Apple, Astrakan' Plums (a Romanian variety)'

1

2 3

100

34 85 70

UReprinted from Makinen and Soderling (1980). Copyright 0 by the Institute of Food Technologists. bRelative ripeness is given as extinctions (A) determined from sample homogenates, and by estimating the ripeness visually and observing the collection time (B). cThe values are in micrograms per 1 g of edible portion (fresh weight). dCrown in the wild state. eAt 540 nm. fAt 520 nm. gAt 500 nm. hAt 410 nm. 'At 370 nm. Kultured.

378

LEA HYVONEN ET AL.

established between the Finnish Sugar Co. and F. Hoffmann-La Roche & Co. Ltd., Basel, Switzerland. The annual world production of sugar alcohols was about 345,000 tons in 1978, and of that amount 330,000 tons were sorbitol. The amount of xylitol and mannitol produced was 6000 tons. The production amounts of maltitol, isomaltitol, galactitol, and lactitol amounted to less than 1000 tons/yr (Albert et al., 1980). C.

LARGE-SCALE XYLITOL PRODUCTION

Production of xylitol by means of extraction from its natural sources is impractical and uneconomical because of the relatively small amounts in which it occurs. Xylose, a pentose which can be hydrogenated to xylitol, is known to be widely distributed in plant material. It does not occur in the free state in plants, but is usually in the form of xylan, a polysaccharide composed of D-xylose units, which occur in association with cellulose. Xylose is also found as part of glycosides (Spalt et al., 1973). Despite its wide occurrence in nature, xylose is difficult to produce commercially because of the problems encountered in separating it, particularly from other carbohydrates such as glucose. However, the fact that xylan is more easily hydrolyzed than cellulose provides the technical possibility for xylose extraction and xylitol production. Accordingly, the recovery of xylose from plant materials and its subsequent hydrogenation is the basic principle of xylitol production (Fig. 1). Plant materials which contain a suitable amount of xylans to be used in this process include hardwoods such as birch and beech, oat and cottonseed hulls, corn (maize) cobs, sugar cane bagasse, straw, and various nut shells. The xylan or xylose content of such materials is 2&30% of the dry substance. The choice of raw materials for the manufacture of pure xylitol is important. Most of the alternatives are bulky and of low density. Optimally, therefore, the raw material for large-scale production should be one which is centrally available in large quantities and of relatively high xylan content. In some of the existing processes agricultural by-products are being utilized, e.g., almond shells in Italy and apparently rice and cotton seed hulls, respectively, in China and the Soviet Union. The large Finnish production is based on birchwood chips, whereas other hardwood chips have been utilized in Germany. Xylan-containing sulfite waste from the paper and pulp industries has been proposed as a more economical alternative to hardwoods. Production in the United States will probably be based on corn cobs. All of these raw materials contain relatively small amounts of polymers of other sugars such as glucose, mannose, arabinose, and galactose in their hemicelluloses. The hydrolyzates require extensive purifications and separations to remove these sugars from xylose and xylitol. Nevertheless, it is possible to recover about 50-60% of the xylans as xylitol.

CH20H

CHO

I

I

H-C-OH

H-C-OH

I

Hydrolysis

Hydrogenation

I

H20 t

I

H2

H-C-OH

acid

I

HO-C-H

HO-C-H

I

H-C-OH

+catalyst

I

CH20H

CH20H

Xylitol

D - Xylose

12’5

C5H1005

FIG. 1. Principle of xylitol production. H y d r o l y s i s o f pentasancontaininq r a w m a t e r i a l s p e n t o s e sugar material

Ion exclusion

r--------I I

I I

I

Final purification and c o l o r removal

I I

purified pentase solution Hydrogenation

I

molasses

Fractionation and crystallization

crystallization

1_ _ _ _ _ _ _

+

XYLOSE

polyol s o l u t i o n

I

J

4

XYLlTOL

FIG. 2. Production of xylitol aild xylose

molasses

380

LEA HYVONEN ET AL.

The main steps in the xylitol production process are illustrated in Fig. 2 and described in detail below.

I.

Hydrolysis

In mass production plant material is treated with a dilute acidic solution under heat and pressure to hydrolyze the hemicelluloses and to precipitate the lignins. The monomeric sugars dissolve in the reaction media together with other soluble products. Fortunately, the cellulose is not attacked, otherwise the xylose would be contaminated with large amounts of glucose which would be troublesome and costly to separate. The simultaneous occurrence of undesired side reactions and the considerable nonspecificity are the restrictions of acid hydrolysis. Von Puls et al. (1978) have described the use of immobilized xylanolytic enzymes in the total hydrolysis of xylans. An enzymatic hydrolysis would be a more subtile method without chemicals, high temperatures, and high pressures, but the specificity of xylanases may disturb hydrolysis and therefore a number of different xylanases are required to complete hydrolysis. However, enzyme hydrolysis has not yet been used in mass production. 2. Xylose Purification In the next phase of the process the hydrolysate is processed via a series of complicated purification steps to remove the undesirable by-products. These substances originally comprised part of the hemicelluloses and were solubilized during the hydrolysis. Two basic routes have been reported for the desired purification. These differ in whether or not xylose is isolated as such. a. Isolation ofXylose. A patented process obtaining xylose from vegetable matter uses oxalic acid treatment (Steinert and Lindlar, 1970). Relatively pure crystalline xylose is produced from the hydrolysate by successive operations of ion exchange, decolorization, and crystallization from methanol (Jaffe et al., 1974). In an alternate process xylose is isolated from impurities with alcohol precipitation and crystallized from an aqueous concentrate diluted with acetic acid (Spalt et al., 1973). The pentose-rich solution obtained by acid hydrolysis is purified by mechanical filtration and ion-exclusion techniques for color removal and desalting. This solution is then subjected to chromatographic fractionation to obtain a highly purified solution of xylose (Melaja and Hamalainen, 1977). b. Nonisolation of Xylose. In this approach the hydrolysate is treated in a series of ion-exchange exclusion and decolorization processes to remove all byproducts except the carbohydrates from the main xylose stream. The mixed xylose and other carbohydrates contained in the solution are in a high state of chemical purity (Melaja and Hamalainen, 1977).

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

38 1

3 . Hydrogenation For the conversion to xylitol the isolated xylose dissolved in water or the mixed xylose-carbohydrate solution is hydrogenated at temperatures ranging from 80 to 140°C and hydrogen pressures up to 50 atm, in the presence of a metal catalyst. With the nonisolated xylose stream, all other sugars present are also hydrogenated to their respective polyols (Wisniak et al., 1974). Some 80% of the world production of sugar alcohols is manufactured in batch suspension processes using Raney nickel catalysts (Albert et al., 1980).

4. Xylitol Purification After removal of the catalyst by filtration and ion exchange the hydrogenated solutions are further processed to obtain xylitol by purification, concentration, and crystallization. In the isolated xylose route, decolorization and crystallization from either alcoholic solvents or aqueous solutions have been used for the isolation of pure crystalline xylitol (Jaffe et al., 1974; Melaja and Hamalainen, 1977). Puritied pentose solution

I

From isolated xylose

I

L-- 1 Xylltol

Xyli tol-rich fraction

Crystallization Recycle of xyli tol-rich fraction

X Y l l to1 solution

fractionation

+

Mixed polyols

FIG. 3.

Chromatographic fractionation and crystallization of xylitol.

382

LEA HYVONEN ET AL

2

6

10

1L

lk

22

26

FRACTIONS 18-30

3L 38 T l M E x 10 min

FIG. 4. Distribution of xylitol and other polyols in ion-exchange chromatography. From Melaja and Hamalainen (1977). (1) Arabinitol, (2) xylitol, (3) rnannitol, (4) galactitol, ( 5 ) sorbitol, ( 6 ) unhydrogenated sugars and unknown impurities. Cationic resin: Ca2+ form; bed: 350 cm, 4 22.5 cm; temperature, 49°C; feed, 17 litersihr.

With the nonisolated xylose, the separation of nonxylitol polyols must be made before xylitol crystallization (Fig. 3 ) . This purification has been effectively carried out by ion-exchange chromatographic fractionation with cationic exchange resins (Fig. 4). Pure xylitol is then crystallized from aqueous solutions separated in the fractionation (Melaja and Hamalainen, 1977).

Ill. PHYSICOCHEMICAL AND FOOD TECHNOLOGICAL PROPERTIES OF XYLITOL A.

PHYSICOCHEMICAL PROPERTIES

I.

Structure of Xylitol

Xylitol is a pentahydric sugar alcohol, or pentitol with the empirical formula C,H,,O, and MW of 152.15. Xylitol is a meso compound completely lacking in optical activity in solution. Its structure is indicated in Fig. 5 . 2.

Crystdlization

a. Bimorphism and Melting Point. Wdfrom and Kohn (1942) reported the first successful attempt at crystallization. They obtained hygroscopic crystals,

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

383

CHZOH

I

H-C-OH

I

HO-C-H

I

H-C-OH

I

CHZOH

FIG. 5. The structure of xylitol.

melting at 61°C. Carson et al. (1943), when repeating the former experiment, produced a new form of crystals melting at 93-94.S"C. By seeding with the lowor high-melting material they were able to grow either form. No melting point depression was shown in a mixture of both, but the low-melting form changed into the high-melting form after a few days. The stable form melting at 94°C was found to be orthorhombic, whereas the metastable form melting at 61°C was monoclinic. Apparently the monoclinic form is very elusive, since Kim and Jeffrey (1969), among others, were unable to crystallize the monoclinic form. Instead Kim and Jeffrey (1969) obtained two different morphologies of the orthorhombic form. This behavior is reminiscent of that observed with D-mannitol, in which polymorphism has been reported, but it is difficult to reproduce the crystals (Berman et al., 1968; Kim et al., 1968). All xylitol produced by industrial processes, microbiologically or chemically, is in the orthorhombic form with a melting point of 94°C. b. Supercooled Melts. One of the outstanding properties of xylitol is its capability to form metastable melts under certain conditions. The phenomenon is known for a number of organic and inorganic substances. When completely melted and subsequently cooled to ambient temperature in a closed container, xylitol will remain in the molten state. The melt is colorless, clear, and of a honey-like viscosity. In addition to seeding with xylitol microcrystals, crystallization can be triggered by ultrasonic cavitation or by scratching the container's inner surface (Voirol, 1979). Contaminants, such as dust, soil, iron powder, or sodium chloride, added to the crystals before melting did not influence the metastability of the melt. However, 10% sorbitol or 5% mannitol will cause crystallization of the mixture after 1 hr or S min, respectively (Voirol, 1979). Xylitol melt at 20°C in an open container will crystallize within a few hours wherever dust particles have fallen. A sample of open melt kept for 6 weeks in a low-dust atmosphere did not crystallize, confirming the role of dust in initiating crystallization at the surface. Supercooled melts can be kept stable in closed aluminum tubes, sealed plastic bags, and rubber-stoppered glass flasks (Voirol, 1979).

384

LEA HYVONEN ET AL

3 . Boiling Point

In contrast to sugars (sucrose, glucose, and fructose), xylitol has a distinct boiling point below decomposition. It will show only slight discoloration when boiled at a constant temperature of 216°C under atmospheric pressure (Kracher, 1975a).

4. Specific Heat The specific heat of liquid xylitol between the melting point and 25°C is 167.9 J/g (40.1 cal/g) as determined by differential thermoanalysis (Schildknecht, personal communication). The heat required to bring crystalline xylitol from room temperature to the melting point (AHs),the heat required for melting (AH,), the heat liberated by supercooling back to room temperature (AH,), and the subsequent heat of crystallization (AH,) represent a cyclic process (Fig. 6) in which the energy balance is zero

AH, + AHm + AH,+ AHc = 0 I

I

I

AH,

.

257.7 J l g

AH, (-189.2 J l g )

100 -

A HS 99 L Jlgl

25

50

Room Temperature

75

93 T('C)

Melting Temperature

FIG. 6 . Heat capacity of xylitol ( A H = 0 at 25°C). H,, heat capacity of the solid phase; H,, heat of melting; H,, specific heat of the liquid phase; H,, heat of crystallization. From J. Schildknecht (personal communication).

385

FOOD TECHNOLOGICAL EVALUATION O F XYLITOL

Calorimetric measurements of crystallization heat have shown 189.2 J/g (45.3 cal/g) to be available in supercooled melts. It is difficult to find a substance capable of forming metastable melts with a higher heat of crystallization (Voirol, 1979). 5.

Solubility

The solubility of xylitol is the same as that of sucrose (68 g/lOO g solution) at 30°C. Below that temperature it is less, above it is more soluble than sucrose (Ape1 and Rossler, 1959; Manz et al., 1973; Virtanen, 1973). The increase of xylitol solubility with increasing temperature is significantly greater than that of sucrose solubility (Fig. 7). Xylitol is only slightly soluble in alcohol: 1.2 g/100 g solution of 96% ethanol, and 6.0 g/lOO g of 96% methanol (Kracher, 1975a).

6. Heat of Solution Another remarkable characteristic of xylitol is its endothermic dissolution. The heat required to dissolve 1 g of this pentitol is the highest of known sugars or sugar alcohols (Mangold, personal communication). The heats of solution of the common alternative sweeteners are as follows: sucrose: dextrose: sorbitol: xylitol:

18.1 Jig 59.4 Jig 97.0 J/g 153.0 Jig

(4.34 calig), (14.2 calig), (23.2 calig), (36.6 calig).

In food use this means that the consumption of xylitol in crystalline form results in an actual cooling of the saliva. This property lends a true cooling effect to

I I,

10

20

30

LO

50

60

70

TEMPERATURE ('C)

FIG. 7. Solubility in water of xylitol and sucrose. Data from Virtanen (1973) and Schneider et al. (1968).

386

LEA HYVONEN ET AL.

foods containing solid xylitol. The cooling effect is desirable in some foods, often proclaimed and even patented (Hammond and Streckfus, 1975). Ten percent xylitol reduces the temperature of an aqueous solution by 3"C, whereas the preparation of a 50% xylitol syrup reduces the temperature by 12°C (Voirol, 1980). 7.

Viscosity

The viscosity of sugars and sugar alcohols depends on many factors: solids concentration in solution, molecular weight, temperature, and composition of solids (von Graefe, 1975). Consequently, the viscosity of a xylitol solution is, for instance, significantly lower than that of the sugar alcohol or sugar of a higher molecular weight (Fig. 8). The viscosity of a saturated xylitol solution is significantly lower than that of sucrose, for instance. The viscosities of sugar solutions as well as that of the xylitol solution decreases with increasing temperature (Fig. 9). The temperature dependence of viscosity for a saturated aqueous xylitol solution shown by H. E. Keller (unpublished) is presented in Table 11.

10

20

30

LO

50

60

% SOLIDS(WIWI

FIG. 8. Viscosity of sweetener solutions at 20°C. ( I ) DE 42 glucose syrup, (2) sucrose, ( 3 ) fructose, (4) xylitol. From Nicol (1980).

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

387

10 000

- I000 -a> 0

k v)

0

0

?

100

0

LO

80

'c

TEMPERATURE r C )

FIG. 9. Viscosity of some carbohydrate sweeteners at different temperatures. Glucose syrup DE 40, 78 wt. %; glucose syrup DE 60, 77 wt. %; isoglucose, 70 wt. %; fructose solution, 70 wt. %; xylitol solution, 70 wt. %. From von Hertzen and Lindqvist (1980).

8. Density

Figure 10 shows the lower density of aqueous xylitol solutions as a function of concentration in comparison with solutions of sorbitol and sucrose. The density of a supercooled melt at 20°C was determined to be 1.42 and that of xylitol crystals 1.49, indicating an approximate contraction of 4.7% at the point of crystallization (Voirol, 1980). 9. Hygroscopicity

Sorption isotherms show that an equilibrium moisture content of xylitol is low at relative air humidities lower than 80%, after which the moisture adsorption TABLE I1 TEMPERATURE DEPENDENCE OF VISCOSITY FOR A SATURATED AQUEOUS XYLITOL SOLUTIONa Temperature ("C)

Viscosity (CP)

20 40 60 70 80

37 15 7 5 4

aFrom H . E. Keller (unpublished).

388

LEA HYVONEN ET AL ""

xylitol

sorbitol

LO

20 -

1,000

1.100

1.200

1.300

DENSITY (g/rnl)

FIG. 10. Densities of xylitol, sorbitol, and sucrose solutions as a function of concentration. Data from Hirschmuller (1953) and G. Pongracz (personal communication).

increases sharply (von Schiweck, 1971; Kammerer, 1972). Fructose, sorbitol, and corn starch are distinctly more hygroscopic than xylitol at relative air humidities between 60 and 80% (Fig. 11). There is hardly any difference between the behavior of crystalline and powdered xylitol during storage. Both show an increasing tendency to pick up moisture above 70% relative air humidity. Below 60% relative air humidity they behave similarly to sucrose and powdered sugar (W. J. Mergens, personal communication). Table I11 shows the relative hygroscopicity of sucrose and three sugar alcohols at a high relative air humidity and room temperature. Sorbitol is the most hygroscopic and sucrose the least hygroscopic in these conditions. The moisture pickup of mannitol increases only slightly, whereas that of xylitol clearly increases with time (W. J. Mergens, personal communication).

!Nl&&zzdY fructose

20 20

LO

60

80

corn s t a r c h 100

RELATIVE HUMIDITY (%)

FIG. 11. Adsorption isotherms for crystalline carbohydrates. From Kammerer (1972). Reproduced with permission from Kakao and Zucker.

389

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

TABLE 111 MOISTURE PICKUP OF GRANULAR MATERIAL DURING STORAGE AT 84% RELATIVE HUMIDITY AND 20"Ca Days 1

2 3 4 9 11 18 65

Sucrose

Mannitol

Sorbitol

Xylitol

0.03 0.05 0.05 0.04 0.07 0.08 0.08 0.04

0.51 0.60 0.59 0.58 0.66 0.65 0.67 0.67

1.89 3.20 4.45 5.53 10.53

0.05 0.08 0.14 0.13 0.33 0.68 1.10 13.89

-

15.90 29.00

aFrom W. J. Mergens (personal communication).

B.

FOOD TECHNOLOGICAL PROPERTIES 1.

Caloric Value

Calorimetric determinations have shown xylitol to be isocaloric with most carbohydrates. Its combustion value is 16.7 kJ/g (4.06 kcal/g). 2. Browning Reactions Due to the absence of aldo or keto groups, xylitol does not take part in browning reactions of the Maillard type. This may be regarded as an advantage or a disadvantage when contemplating its use as a food ingredient. No color formation or sweetness reduction was noted in an aqueous xylitol-aspartame solution (27.17 g xylitol + 0.6467 g aspartame/l000 ml) after sterilization (20 min at 121"C), whereas the sweetness of fructose-aspartame solution (34.3 g fructose 0.5571 g aspartame/l000 ml) was noticeably reduced after sterilization. The color of the solution was yellow and had a honey-like flavor, probably due to the Maillard reaction (Hyvonen, 1981). Xylitol even does not caramelize at elevated temperatures (Kammerer, 1971). A slight yellow color formed when heated over 150°C is thought to be due to small amounts of aldose impurities in xylitol (Kracher, 1975a).

+

3 . Fermentation

Most microorganisms are incapable of utilizing xylitol. It has been shown that xylitol is not fermentable by cariogenic oral microorganisms (Gehring et a l . , 1974; Lamas et a l . , 1974). The common baking yeast Saccharomyces cere-

390

LEA HYVONEN ET AL.

visiae cannot ferment xylitol either. The buns sweetened with xylitol did not rise

and even the fermentation of sucrose in the buns, where xylitol was also used, was retarded (Varo et al., 1979; Hyvonen and Espo, 1981b). Salminen and Branen (1978) noted a prolonged fermentation time in presweetened xylitol yogurt. A lower acid production was also noted in xylitolsweetened yogurt than in sucrose-sweetened yogurt by Hyvonen and Slotte (1981). 4 . Sweetness a. Chemical Basis. Xylitol, a meso-pentitol, has little structural similarity to sucrose, but they have been reported to taste almost equally swket on a weight basis (Gutschmidt and Ordynsky, 1961; Yamaguchi et al., 1970a; Hyvonen et al., 1977). Qualitatively the sweetness of xylitol tended to fall near that of fructose and glucose in a three-dimensional space by a multidimensional scaling procedure (Schiffman et al., 1979). In assessing the sweetness of several pentitols, Lindley et al. (1976) found that xylitol was much sweeter than the stereoisomers, L-( -)-arabitol and ribitol. On the basis of molecular models the oxygen-oxygen distances between all four pairs of oxygen atoms of xylitol in a planar “zigzag” conformation is 2.9-3.0 A, which is ideal for eliciting sweetness according to the AH, B theory (Shallenberger and Acree, 1967). A strong IR absorption peak at 3440 cm- suggests that the nonbonded hydroxyl groups must cause the intense sweetness of xylitol, whereas the intramolecular hydrogen bonding reduces the sweetness of ribitol and arabitol (Lindley et al., 1976). b. Relative Sweetness. Relative sweetness of a sweet-tasting compound is determined as the relation of the concentrations needed to evoke the same sweetness perception. Sucrose has mainly been used as the reference. The relative sweetness is dependent on concentration. The relative sweetness of xylitol was found to increase from 86 to 115 as concentration increased from 1 to 20% (Gutschmidt and Ordynsky, 1961). According to Yamaguchi et al. (1970a), the change was from 96 to 118, when concentration increased from 2.5 to 30%. According to Hyvonen et al. (1977), the relative sweetness values of xylitol solutions tasted at room temperature varied from 103 to 115 as compared to 5-20% sucrose references. The sweetness of xylitol was thought to be largely invariant with temperature, since as a sugar alcohol it does not undergo mutarotation in solution (Fratzke and Reilly, 1977). However, this proved incorrect. The relative sweetness of xylitol decreased significantly, for instance, from 103 to 78, when a 5% sucrose reference was used and when the temperature changed from 5 to 50°C (Hyvonen et al., 1977).

39 1

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

In general the relative sweetness of xylitol was noted to be slightly reduced in acid solutions (Hyvonen et al., 1978a). In 0.0175% o-phosphoric acid solution the relative sweetness of xylitol was exceptional. This acid caused a significant reduction (from 103 to 97) in the sweetness of xylitol at refrigerator temperature, and at hot drink temperature the sweetness was significantly higher in the phosphoric acid solution (87) than in the corresponding water solution (80) (Table IV) . c . Synergistic Effects in Xylitol-Containing Mixtures. Synergism is inferred when the sweetness of a mixture of sweeteners is greater than the sum of the sweetnesses of its components. Synergistic effects have been noted especially in the mixtures of sweeteners with greatly diverging chemical structures and dissimilar relative sweetnesses. Weickmann et al. (1969) suggested that synergism is at its maximum when the components of a mixture contribute about the same amount to the sweetness of a mixture, which applies to xylitol-saccharin mixtures also. Yamaguchi et al. (1970b) also reported synergistic interrelationships in xylitol-saccharin and xylitol-cyclamate mixtures. TABLE IV RELATIVE SWEETNESS OF XYLITOL IN WATER AND ACID SOLUTIONS” ~~

Temperature

Acid ( W )

Relative sweetness

6 t 2°C

No acid Citric acid (0.01) Citric acid (0.05) Malic acid (0.007) Malic acid (0.035) Phosphoric acid (0.0035) Phosphoric acid (0.0175) No acid Citric acid (0.01) Citric acid (0.05) Malic acid (0.007) Malic acid (0.035) Phosphoric acid (0.0035) Phosphoric acid (0.0175) No acid Citric acid (0.01) Citric acid (0.05) Malic acid (0.007) Malic acid (0.035) Phosphoric acid (0.0035) Phosphoric acid (0.0175)

103 t 3 100 2 1 102 t 5 105 t 2 102 t 2

23 t 2°C

50

2

3°C

aFrom Hyvonen e t a / . (1978a). Copyright 0 by Forster Publishing, Inc. bDifference significant by t-test at 10% risk level.
104 t 1 97 t I ” 103 t 2 102 2 1 101 2 3 102 t 1 102 t 1 103 t 1

99 80 79 77 79 78 77 87

2 2 2 2

4 2 2 3 2 2 2 4 ?

2

2

1c

392

LEA HYVONEN ET AL

About 5&60% of extra sweetness was noted in aqueous xylitol-saccharin solutions at the predicted isosweetness with a 5% sucrose solution (Hyvonen et al., 1978b). In the corresponding xylitol-cyclamate mixture the degree of synergism was 6&66% at the maximum. At the higher sweetness level, at the predicted isosweetness with a 10% sucrose solution, the degree of synergism in the xylitol-cyclamate mixture was still greater, as high as 7 6 9 8 % in the most ideal combination at each of the temperatures 8, 25, and 50°C (Sipila, 1977; Hyvonen and Sipila, 1977). In xylitol-aspartame mixtures the synergism noted was 77% at maximum (Hyvonen, 1981). Enhanceh sweetness of the sweetener mixtures could be used advantageously in reducing the energy content of sweetened drinks such as coffee, tea, juice, and soft drinks. Conventional sweetness levels with 50-70% less calories could be achieved without the deterioration of other taste qualities (Hyvonen and Sipila, 1977; Hyvonen, et al., 1978b).

IV. FOOD APPLICATIONS

A.

CONFECTIONERY

Not all confections can be made using xylitol as the only sweetener. There are problems in all those sweet preparations which require crystallization inhibitor. Many standard formulations call for both sucrose and glucose syrup in specified proportions. Due to crystallization properties, it is not possible to make fondant creams, chews, toffees, and transparent hard candies with xylitol alone. Glucose should not be used as a crystallization inhibitor in xylitol confectionery because its cariogenicity would preclude the product’s primary intended use. 1. Chewing Gum

Chewing gum is a cariogenic product, since through constant release of sugar during chewing the time of contact with the teeth is quite long and intensive. In normal chewing gum glucose syrup acts as a softener. It has been shown by Kracher (1975b) that a pure xylitol gum can be made by replacing glucose with gum arabic solution. The use of gum arabic has been found necessary because xylitol solutions have insufficient viscosity, which would otherwise appreciably lengthen the mixing times. The kneading operation also can be controlled with an aqueous xylitol solution or with glycerol (Voirol, 1978). Gums made of pure xylitol and others made of polyol mixtures are on the market in a number of countries. The forms manufactured include laminated “sticks,” extruded bubble gum-type blocks, and coated gums. One of the first xylitol-containing chewing gums was given to students four to

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

393

five times a day during 1 year in a caries study carried out by Scheinin et al. (197%). Under a moderately cariogenic diet the regular chewing of xylitol chewing gum was shown to be an effective means of caries prevention. There are only a few processing steps by which xylitol gum manufacture differs slightly from that of the normal sucrose/glucose type (Kracher, 1975b). The xylitol used must be in the form of a powder. Particle size of the powdered xylitol should not exceed 50 p,m. Kneading of the gum base should take place at a temperature on average 10°C below that normally used in sucrose/glucose gum. The unusually low melting point of xylitol entails the danger of caking. The addition of water should be kept at a minimum to avoid hardening. Furthermore, the “shorter” structure may require an adjustment of extruder or roller parameters (Voirol, 1978).

2. Hard Candy Hard caramels (hard candy, high-boiled sweets) represent another class of high-caries-risk confectionery. Since they are mainly consumed by children, the substitution of sucrose by xylitol, yielding a product both noncariogenic and acceptable in taste, would be meaningful indeed. However, the production of hard candy using xylitol is problematic. Trials carried out with high-boiled hard candy made exclusively with xylitol produced a product in which crystallization had already begun during the cooling phase. The drops became brittle (Manz et al., 1973). The industrial manufacture of normal hard candy takes place in either of two ways: pulling and die-cutting of a formable mass or depositing (casting into molds). The plastic method requires the use of glucose syrup, which must be excluded if the product is to be noncariogenic. Because no plastic phase exists in pure xylitol, this leaves the depositing method, for which a procedure has been developed, resulting in a pure xylitol candy. The product is hard as glass and suckable but not transparent. The process involves melting the xylitol, adding natural coloring agents, heating to 120°C to evaporate all water introduced with the color, cooling to slightly below the melting point (92”C), seeding to 25% of the total weight with powdered xylitol, adding crystalline acid and flavor, and mixing. If the mixture is stirred constantly and thermostatically kept between 88 and 92°C in a hopper, melt and microcrystals will coexist as do ice and water at 0°C. The viscosity of the mass at these temperatures is sufficient to use customary nozzles and molds. Teflon-coated aluminum molds with expeller pins give the best results. In contrast to sucrose/glucose deposited sweets, xylitol candies do not need long conveyors or cooling tunnels. Although heat is generated during crystallization, the setting time is ;bout 1 min for a 1.3-g, lenticular-shaped deposit and 3 min for a 4-g, oval deposit (Voirol and Brugger, 1976).

394

LEA HYVONEN ET AL.

3.

Toffees

Soft caramels containing milk solids can be made with xylitol. The characteristic flavor forms when heating the mass to obtain Maillard-type browning reactions between lactose and milk proteins. Xylitol does not participate in these reactions; thus in caramels it is merely a sweetener, not a flavor precursor. The differing properties of xylitol in relation to those of sucrose only have a slight effect on the properties of toffee-like products. Since the proportions of the individual raw materials (proteins, fat, polysaccharides) are not critical, appealing products are obtained by the use of xylitol. Xylitol toffees tend to have a shorter structure, a structure similar to that which would be obtained using a low glucose content (Kracher, 1975b). 4.

Gum Drops

Chewable confectionery using gum arabic, pectin, or gelatin with xylitol as the sweetener tends to harden during storage. Formulations have been developed using a minimum amount of sorbitol to prevent crystallization. Storage tests have shown that the shelf life of combination products exceeds 12 months. Telemetric tests on humans using gum drops of this type have shown that the plaque pH remains above the critical value during consumption, which justifies the claim “tooth saving” according to Swiss regulations (Imfeld, 1977).

5 . Confectionery Jellies Pectin jellies with conventional soluble solids cannot be made with xylitol. Kracher (1975b) states that crystallization will occur if 75% xylitol is used. If the xylitol proportion is lowered, the pectin will no longer gel. A favorable effect has been obtained by slightly increasing the proportion of gelatin or agar-agar.

6. Compressed Tablets Tablets can be compressed either from crystalline material or granulated xylitol. The Finnish market offers peppermint-flavored tablets consisting of 99.25% xylitol. The direct compression technique uses crystalline xylitol (0.6-0.4 mm), 1.6% stearic acid, and flavor (R. Etter, personal communication). The main problem in the process appears to be the friability of the tablets. On a laboratory scale good results have been obtained by sintering the tablet surface in a hot air stream so that only the surface is melted and the core is protected. This process approaches a “coating” procedure in which no coating pan is

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

395

required. It is another example of new technology made possible by the properties of xylitol (low melting point). If a regular coating layer by melting and subsequently crystallizing the surface is desired, the airflow must be laminar and of homogenous temperature over the surface of the tablets. It will be necessary to reverse the flow or turn the tablets over on the sieves for processing the other side. If the initial friability of the tablets is low enough, better results are obtained in a fluidized bed dehydrator (R. Etter, personal communication). 7. Coatings

Xylitol-only coatings have been successfully applied to centers of compressed tablets, hard candies, and chocolate. Certain deviations from the customary technique using sucrose/glucose must be observed. The best results were obtained with a supersaturated (85%) aqueous xylitol solution at 40°C panned in layers in a hot air stream (60°C). Calcium carbonate can be used as an isolation powder, if the centers must not be visible through the coating. Dusting is preceded and followed by application of the warm syrup (W. Thurkauf, M. Grossmann, and K. Munzel, personal communication). Contrary to sucrose, xylitol will not cover irregular surfaces of the centers. Twenty percent of a 1:2 gum arabic-water solution in the coating syrup helps in obtaining a smooth surface. The final coatings may contain pigments if desirable, and wax can be applied for surface polishing. The surface of the finished dragCes is milky-opaque, and natural colors, applied with the final coating syrup, tend to be less stable on it than on sucrose surfaces. Cross sections examined under the microscope seem to indicate a “rougher” surface. Flavors applied in the form of natural oils improve the surface structure. Peppermint oil, added at 0.5% in the syrup during final coatings, results in a pearl-glossy surface (Voirol, 1979, 1980).

8. Chocolate When sucrose is replaced by xylitol in chocolate on a weight basis, some slight changes must be made in the production process, mainly because of the lower viscosity of the xylitol product. The viscosity may be adjusted by the use of additives (Kracher, 1975b). A coarse sandy texture was noted in the xylitol chocolate after storage, when the relative humidity of the atmosphere in the manufacturing locality had exceeded 85%. This may have been caused by a kind of hydrate film around the xylitol particles (Voirol, 1978). The concentration of xylitol in the chocolate bars sold in Finland, the Federal Republic of Germany, and the Soviet Union ranges from 17 to 42% (Voirol, 1979).

396

LEA HYVONEN ET AL

B.

ICE CREAM

In principle, sucrose can be replaced by xylitol on a weight basis in ice cream, however, the melting properties of the product are appreciably altered. Xylitol ice cream has a considerably softer consistency than sucrose ice cream at the same temperatures. Kracher (1975b) reported that no change occurred in xylitol ice cream during storage for 6 months at -24"C, in particular, there was no recrystallization. The melting of xylitol ice cream as well as its overall acceptance were judged to be better than those of the sucrose reference after 3-month storage at -25°C (Hyvonen and Torma, unpublished). C.

YOGURT

Both Salminen and Branen (1978) and Hyvonen and Slotte (1981) found 8% xylitol to be the most preferred concentration in yogurt. Xylitol had no effect on the pH of yogurt when added after incubation. If xylitol was added to the milk before incubation, the pH of the xylitol-sweetened yogurt was distinctly higher (4.4) than that of the corresponding sucrose-sweetened yogurt (4.0) (Hyvonen and Slotte, 1981). The post-incubation-sweetened xylitol yogurt was judged to be as good as the sucrose reference by sensory evaluation. The flavor of the presweetened xylitol yogurt was regarded as poorer than that of the corresponding sucrose reference, mainly due to the lower acidity of the xylitol yogurt according to Hyvonen and Slotte (1981), whereas Salminen and Branen (1978) reported that the lower acidity of the xylitol yogurt was the reason for its more preferred flavor. This could reflect differences in national taste habits. The viscosity of the presweetened xylitol yogurt was lower than that of the sucrose-sweetened yogurt. However, the texture of the xylitol yogurt was not scored lower in the sensory evaluation (Hyvonen and Slotte, 1981). D.

JAMS, JELLIES, AND MARMALADES

In jams, jellies, and marmalades sugar acts as a preserving agent by its osmotic pressure. Xylitol, in addition to its nonfermentability by most yeasts, molds, and bacteria, is an effective preserving agent due to its higher osmotic pressure even at low concentrations. For instance, a 30% xylitol solution and a 70% sucrose solution have about the same osmotic pressure. Sucrose also has an important role in the gelatinization of gel products. Kawabata et al. (1976) studied the effect of sugars and sugar alcohols on the texture of pectin jellies and found that in HM-pectin jellies the jelly strength of the xylitol jelly had a pattern as a function of concentration similar to that of the sucrose jelly (Fig. 12). In LM-pectin jellies the increase of xylitol or sorbitol

397

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

A

55

60

65

70

75

Concentration of sugars (%I

55

60

65

70

75

Concentration of sugars (%I

FIG. 12. Effect of concentration of sucrose, sorbitol, and xylitol on the hardness and adhesiveness of the HM-pectin jelly: (0)sucrose, (0)sorbitol, (0)xylitol. From Kawabata et al. (1976). Reproduced with permission from the Japanese Journal of Nutrition.

concentration did not change the jelly strength; when using sucrose the jelly strength increased with increasing sucrose concentration (Fig. 13). According to Hyvonen and Torma (1981), as well, the gelling properties of xylitol with LM-pectin differed from those of sucrose. A calcium salt addition was needed for gelatinization and yet the texture of the strawberry jam prepared with xylitol was softer than the sucrose-sweetened one. Xylitol jams and marmalades prepared for the Turku sugar studies had good keeping qualities. The tastes and flavors of xylitol products were judged to be better than those of the corresponding sucrose references. Better color stability was noted in xylitol cranberry and strawberry jams than in the sucrose-sweetened jams (Manz et al. , 1973). Good color stability of the xylitol-sweetened strawberry jam was noted also in the studies of Hyvonen and Torma (1981).

10

20

30

LO

50

Concentration of sugars 1%)

10

20

30

LO

50

Concentration of sugars

1%)

FIG. 13. Effect of concentration of sucrose, sorbitol, and xylitol on the hardness and adhesiveness sucrose, ( 0 )sorbitol, (0) xylitol. From Kawabata er al. (1976). of the LM-pectin jelly: (0) Reproduced with permission from the Japanese Journal of Nutrition.

398

LEA HYVONEN ET AL

E.

BAKERY PRODUCTS

For the sake of complete replacement of sucrose by xylitol, the test subjects in the Turku sugar studies were regularly provided with fresh bakery products. If such products ever become of commercial interest, they would have to be weakly cariogenic or more suitable for diabetics. The characteristic baking flavor is the result of a series of nonenzymatic browning reactions dependent on the presence of keto or aldo groups as in added inverted sucrose, fructose, or glucose. With only xylitol added, this flavor precursor is missing. Nevertheless, some browning can be expected from the reducing sugars present in the flour. In fact, xylitol proved to be a good substitute for sucrose in sugar cake. The color and texture of the xylitol cake closely resembled those of sucrose cake. Xylitol was also a good sweetener in this type of product (Hyvonen and Espo, 198 1a). Xylitol cookies were brown-spotted, probably because of the poor solubility of xylitol in the cookie dough, which contained fat. Xylitol cookies were found to be more friable than the sucrose reference and the mouthfeel of xylitol cookies was more finely divided than that of the sucrose cookies (Hyvonen and Espo, 198 1a). The fact that xylitol is not fermented by Saccharomyces cerevisiae was distinctly seen when buns were sweetened with xylitol. The xylitol buns did not rise, having small volumes, moist interiors, and dense textures (Hyvonen and Espo, 1981b). In addition, the presence of xylitol seemed to retard the inversion of sucrose added for the nourishment of yeast into the bun dough (Varo et al., 1979). Consequently, xylitol is not a suitable sweetener in yeast-leavened doughs, in addition to its unsuitability for the nourishment of yeast. F. DRINKS Due to its laxative effect xylitol alone is not recommended in beverages such as soft drinks, where consumption may easily exceed the recommended single dose intake. Hyvonen and Sipila (1 977) used a mixture of xylitol and cyclamate to sweeten citrus-base and cola-type soft drinks. The drinks contained 3.9% xylitol and 0.133% cyclamate. The energy content of the mixture-sweetened drinks was 60%lower than that of the isosweet sucrose reference. Using mixtures of suitable sweeteners is one means to reduce the carbohydrate and energy content of a drink and to produce a dietetic drink also suitable for diabetics. Four percent xylitol proved to be a suitable amount of the sweetener in an UHT-sterilized milk-base chocolate drink. The physical properties (viscosity, color) of xylitol product did not significantly differ from the sucrose reference.

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

399

The sensory properties of the xylitol drink were judged to be as good as those of the sucrose drink, both when fresh and after 1-month storage at room temperature (L. Hyvonen and A. Espo, unpublished).

V.

OUTLOOK

It is realistic to expect only a relatively small future replacement of sugar by xylitol. Health consciousness in the industrialized countries will, however, increase the demand for suitable sugar substitutes. The new alternative sweeteners meet many requirements. They are expected to be physiological substances (well-tolerated, natural, or nature-identical). Their sweetnesses and tastes should be as similar to those of sucrose as possible. Diabetics should be able to consume them when advised of their energy content, and they should not be cariogenic. In food processing they should not pose unusual technological problems. Xylitol fulfills most of these requirements satisfactorily, which makes it not only a valuable alternative to sucrose and sucrose substitutes but also one of the few new discoveries in the field of foods today. However, the production and technological realities dictate that the price of xylitol will never fall to the level of common sugars, which will limit its use. Xylitol’s special characteristics, particularly its dental and metabolic aspects, justify its higher price. Indeed, the Turku sugar studies have shown that 5-10 g xylitol/day as between-meal sweets is as effective in bringing dental caries under control as the complete dietary substitution of xylitol for sucrose. Consequently, a realistic and useful application of xylitol in foods will be in confectionery and snack products, where the unique properties of xylitol can be best utilized.

VI.

RESEARCH NEEDS

1. The published data on the physicochemical properties of xylitol are insufficient. A filling of this gap of knowledge is needed. 2. The threshold level of xylitol to stop microbial growth in substrates containing available carbohydrates is of interest. 3. The gelling properties of different types of xylitol-containing gels, and the inhibition of xylitol crystallization in jellies should be studied. 4. The color stability-improving effect of xylitol should be studied thoroughly. 5 . Crystallization inhibitors for xylitol melts of supersaturated xylitol solutions are needed. Since the primary xylitol application in foods is in noncariogenic sweets, such additives should also fulfill the prerequisite of being noncariogenic. Until a permissible noncariogenic crystallization inhibitor can be found, xylitol chews will be an unsolved problem.

400

LEA HYVONEN ET AL.

6. The possibility of preparing high-acid fruit-flavored xylitol candies which do not become sticky should be clarified. A promising fact is that when xylitol is used, no inversion takes place and no fructose is formed. 7. The combinations of xylitol and artificial sweeteners seem to have many potential applications. A nonsweet and possibly noncaloric bulking agent for these combinations to make the sweetening mixture isosweet with sucrose on a volume basis is worth studying.

REFERENCES Albert, R., Stratz, A,, and Vollheim, G. 1980. Die katalytische Herstellung von Zuckeralkoholen und deren Verwendung. Chem.-Ing.-Tech. 52, 582-587. Apel, A,, and Rossler, G . 1959. Preparation of stable polyol solutions and the resulting product. U.S. Patent 2,917,390. Bassler, K. H. 1972. Physiologische Wirkungen des Zuckeralkohols Xylit. Suesswaren 16(8), 327-330. Berman, H. M., Jeffrey, G. A,, and Rosenstein, R. D. 1968. The crystal structures of a’-and pforms of mnannitol. Acta Crystallogr., Secr B B24, 442449. Bertrand, M. G . 1891. Recherches sur quelques dCrivCs dy xylose. Bull. SOC. Chim. Fr. [3] 5 , 554-557. Brian, M., and Miller, 0. N. 1974. The safety of oral xylitol. In “Sugars in Nutrition” (H. L. Sipple and K. W. McNutt, eds.), pp. 591-606. Academic Press, New York. Carson, J. F., Waisbrot, S. W., and Jones, F. T. 1943. A new form of crystalline xylitol. J . Am. Chem. Soc. 65, 1777-1778. Chiang, C., Sih, G . J., and Knight, S . G. 1958. The conversion of o-xyloseto xylitol by Penicillium chrysogenum. Biochim. Biophys. Aria 29, 664-665. Fischer, E., and Stahel, R. 1891. Zur Kenntniss der Xylose. Ber. Dtsch. Chem. Ges. 24, 528-539. Fratzke, A. R., and Reilly, P. J. 1977. Uses and metabolic effects of xylitol. Process Biochem. 12, 27-30. Frerejacque, M. 1943. Sur la prksence de d-arabitol dans Boletus bovinus L. C. R . Hebd. Seances Acad. Sci. 217, 251. Gehring, F., Makinen, K. K., Larmas, M., and Scheinin, A. 1974. Turku sugar studies. IV. An intermediate report on the differentiation of polysaccharide-forming streptococci (S. mutans). Acta Odontol. Scand. 32, 435444. Gutschmidt, J., and Ordynsky, G. 1961. Bestimmung des Siissungsgrades von Xylit. Dtsch. Lebensm.-Rundsch. 57, 32 1-324. Harnmond, J. E., and Streckfus, T. K. 1975. Xylitol chewing gum. U.S. Patent 3,899,593. Hirschrniiller, H. 1953. Physical properties of sucrose. In “Principles of Sugar Technology” (P. Honig, ed.), pp. 18-74. Am. Elsevier, New York. Hollmann, S . , and Touster, 0. 1956. An enzymatic pathway from L-xylulose to o-xylulose. J . Am. Chem. SOC. 78, 3544. Hollmann, S . , and Touster, 0. 1957. The L-xylulose-xylitol enzyme and other polyol dehydrogenase of Guinea pig liver mitochondria. J . Biol. Chem. 225, 87-102. Horecker, B. L., Lang, K., and Tabagi, Y., eds. 1969. “Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols.” Springer-Verlag, Berlin and New York. Hyvonen, L. 1981. “Fructose-aspartame, xylitol-aspartame, and fructose-saccharin mixtures,” EKT-Ser. 571. University of Helsinki.

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

401

Hyvonen, L., and Espo, A. 1981a. “Replacement of Sucrose in Bakery Products. I. Cakes and Cookies,” EKT-Ser. 569. University of Helsinki. Hyvonen, L., and Espo, A. 1981b. “Replacement of Sucrose in Bakery Products. 11. Yeast Leavened Products,” EKT-Ser. 570. University of Helsinki. Hyvonen, L., and Sipila, L. 1977. “Effects of Temperature and Mixture Combinations on the Synergism between Fructose and Cyclamate, and Xylitol and Cyclamate, and the Application of the Mixtures of Sweetners in Foods,” EKT-Ser. 421. University of Helsinki (in Finnish). Hyvonen, L., and Slotte, M. 1981. “Alternative Sweetening of Yogurt,’’ EKT-Ser. 575. University of Helsinki. Hyvonen, L., and Torma, R. 1981. “Replacement of Sucrose in Strawberry Jam,” EKT-Ser. 561. University of Helsinki. Hyvonen, L., Kurkela, R., Koivistoinen, P., and Merimaa, P. 1977. Effects of temperature and concentration on the relative sweetness of fructose, glucose and xylitol. Lebensm. -Win. Technol. 10, 316-320. Hyvonen, L., Kurkela, R., Koivistoinen, P., and Ala-Kulju, M-L. 1978a. The relative sweetness of fructose, glucose and xylitol in acid solutions at different temperatures. Lebensm.-Win. Technol. 11, 11-14. Hyvonen, L., Kurkela, R., Koivistoinen, P., and Ratilainen, A. 1978b. Fructose-saccharin and xylitol-saccharin synergism. J . Food Sci. 43, 25 1-254. Imfeld, T. 1977. Evaluation of the cariogenicity of confectionery by intra-oral wire-telemetry. Helv. Odontol. Acta 21, 1-28. Jaffe, G. M., Szkrybalo, W., and Weinert, P. H. 1974. Process for producing xylose. U.S. Patent 3,784,408. Kammerer, F. X. 1971. Xylit-der moderne Zuckeraustauschstoff. Suesswaren 21, 887-890. Kammerer, F. X. 1972. Zuckeraustauschstoffe bei der Susswaren-Herstellung. Kakao & Zucker 24(5), 184-190. Kawabata, A., Sawayama, S . , and Kotobuki, S. 1976. Effects of sugars and sugar alcohols on the texture of pectin jelly. Jpn. J . Nutr. 31, 3-10. Kim, H. S . , and Jeffrey, G. A. 1969. The crystal structure of xylitol. Acta Crystallogr. 25, 2607-261 3. Kim, H. S . , Jeffrey, G . A., and Rosenstein, R. D. 1968. Crystal structure of the K form of Dmannitol. Acta Crystallogr., Sect. B B24, 1449-1455. Kracher, F. 1975a. Xylit. Bedeutung-Wirkung-Anwendung. Kakao & Zucker 27(3), 68-70, 72-73, 75. Kracher, F. 1975b. Xylit. Kakao & Zucker 27(4), 108-1 10, 112. Kracher, F. 1977. Xylitol: PropriCtks dietitiques et techniques d’application. Revue des Fabricants de confiserie. Choc., Confiturerie, Biscuiterie 52(9), 14-24. Kratzl, K., and Silbernagel, H. 1963. Uber das Vorkommen von Xylit im Speisepilz Champignon (Psalliota campestris). Narurwissenschaften 50, 154. Larmas, M . , Makinen, K. K., and Scheinin, A. 1974. Turku sugar studies. 111. An intermediate report on the effect of sucrose, fructose and xylitol diets on the numbers of salivary lactobacilli, candida and streptococci. Acta Odonrol. Scand. 32, 423433. Lindley, M. G . , Birch, G. G., and Khan, R. 1976. Sweetness of sucrose and xylitol. Structural considerations. J . Sci. Food Agric. 27, 140-144. Makinen, K. K . 1979. Xylitol and oral health. Adv. Food Res. 25, 137-158. Makinen, K . K., and Soderling, E. 1980. A quantitative study of mannitol, sorbitol, xylitol, and xylose in wild berries and commercial fruits. J . Food Sci. 45, 367-371, 374. Manz, U . , Vanninen, E., and Voirol, F. 1973. Xylitol-its properties and use as a sugar substitute in foods. Pap., Food R.A. Sympo. Sugar Sugar Substitutes, 1973 pp. 1-26. Melaja, A., and Hamalainen, L. 1977. Process for making xylitol. U.S. Patent 4,008,285.

402

LEA HYVONEN ET AL

Mellinghoff, C. H. 1961. Uber die Verwendbarkeit des Xylit als Ersatzzucker bei Diabetikem. Klin. Wochenschr. 39, 447. Moskowitz, H. R. 1971. The sweetness and pleasantness of sugars. Am. J . Psychol. 84, 387405. Miihlemann, H. R., Regolati, B., and Marthaler, T. M. 1970. The effects on rat fissure caries of xylitol and sorbitol. Helv. Odontol. Acta 14, 48-50. Nicol, W. M. 1980. Sucrose in food systems. In “Carbohydrate Sweeteners in Foods and Nutrition” (P. Koivistoinen, and L. Hyvonen, eds.), pp. 151-162. Academic Press, New York. Onishi, H., and Suzuki, T. 1966. The production of xylitol, L-arabinitol and ribitol by yeasts. Agric. Biol. Chem. 30, 1139-1 144. Onishi, H., and Suzuki, T. 1969. Microbial production of xylitol from glucose. Appl. Microbiol. 18, 1031-1035. Ritzel, G., and Brubacher, G., eds. 1976. “Monosaccharides and Polyalcohols in Nutrition, Therapy and Dietetics.” Huber, Bem. Salminen, S . J . , and Branen, A. L. 1978. Xylitol as yoghurt sweetener. Pap., 38rhAnnu. IFTMeet.. 1978 pp. 1-9. Scheinin, A , , Makinen, K. K., and Ylitalo, K. 1974. Turku sugar studies. I. An intermediate report on the effect of sucrose, fructose and xylitol diets on the caries incidence in man. Acta Odontol. Scand. 32, 383-412. Scheinin, A., Makinen, K. K., and Ylitalo, K. 1975a. Turku sugar studies. V. Final report on the effect of sucrose, fructose and xylitol diets on the caries incidence in man. Acta Odontol. Scand. 33, 67-104. Scheinin, A , , Makinen, K. K., Tammisalo, E., and Rekola, M. 1975b. Turku sugar studies. XVIII. Incidence of dental caries in relation to I-year consumption of xylitol chewing gum. Acta Odontol. Scand. 33, 307-316. Schiffman, S. S . , Reilly, D. A., and Clark, T. B. 1979. Qualitative differences among sweeteners. Physiol. Behav. 23, 1-9. Shallenberger, R. S . , and Acree, T. E. 1967. Molecular theory of sweet taste. Nature (London) 216, 48W82. Sipila, L. 1977. “Replacement of Sucrose in Foods,” EKT-Ser. 414. Pro gradu study. University of Helsinki (in Finnish). Spalt, H. A., Chu, C. Y., and Niketas, P. 1973. Production of crystalline xylose. U.S. Patent 3,780,017. Steinert, K., and Lindlar, H. 1970. Verfahren zur Herstellung von Xylose und Xylit. DP 1,935,934. Thomas, D. W., Edwards, J. B., and Edwards, R. G. 1974. Toxicity of parenteral xylitol. In “Sugars in Nutrition” (H. L. Sipple and K. W. McNutt, eds.), pp. 567-590. Academic Press, New York. Touster, 0. 1960. Essential pentosuria and the glucuronate-xylulose pathway. Fed. Proc., Fed. Am. SOC. Exp. Biol. 19, 977-983. Varo, P., Westermarck-Rosendahl, C., Hyvonen, L., and Koivistoinen, P. 1979. The baking behavior of different sugars and sugar alcohols as determined by high pressure liquid chromatography. Lebensm. - W i n . Technol. 12, 153-1 56. Virtanen, J. 1973. Finnish Sugar Co. Unpublished data. Volrol, F. 1978. The value of xylitol as an ingredient in confectionary. In “Xylitol” (J. W. Counsell, ed.), pp. 11-19. Appl. Sci. Publ. Ltd., London. Voirol, F. 1979. Xylitol, its properties and applications. In ‘‘Sugar: Science and Technology” (G. G. Birch and K. J. Parker, eds.), pp. 325-344. Appl. Sci. Publ. Ltd., London. Voirol, F. 1980. Xylitol properties and applications in foods and pharmaceuticals. In “Carbohydrate Sweeteners in Foods and Nutrition” (P. Koivistoinen and L. Hyvonen, eds.), pp. 269-285. Academic Press, New York. Voirol, F., and Brugger, W. 1976. Procedure for the manufacture of non-cariogenic xylitol hard candies. Swiss Patent Application 8633.

FOOD TECHNOLOGICAL EVALUATION OF XYLITOL

403

von Graefe, G. 1975. Zucker und Zuckeralkohole. Technologie und technologisch wichtige Eigenschaften. Stuerke 27, 16@-169. von Hertzen, G., and Lindqvist, C. 1980. Comparative evaluation of carbohydrate sweeteners. I n “Carbohydrate Sweeteners in Foods and Nutrition” (P. Koivistoinen and L. Hyvonen, eds.), pp. 127-149. Academic Press, New York. von Puls, J., Sinner, M., and Dietrichs, H. H. 1978. Tragergebundene xylanolytische Enzyme. 1. Wirkungsweise xylanolytischer Enzyme. Stiierke 30, 294299. von Schiweck, H. 1971. Chemische und physikalische Eigenschaften von Zuckern, Zuckeralkoholen und Calciumsaccharosephosphat. Dtsch. Zuhnaerztl. Z. 26, 1063-1078. von Schneider, F., Reinefeld, E., and Hoffmann-Walbeck, H. P. 1968. Die Rube und ihre Zusammensetzung. In “Technologie des Zuckers” (F. Schneider, ed.), pp. 1-72. Verlag M . & H. Schaper, Hanover. Washiittl, J., Riederer, P., and Bancher, E. 1973. A qualitative and quantitative study of sugar alcohols in several foods. J . Food Sci. 38, 1262-1263. Weickmann, F., Weickmann, H., Fincke, K., Weickmann, F. A,, and Huber, B. 1969. Kalorienarmes, siisstoffhaltiges Zuckeraustauschmittel. German Patent 1,961,769. Wisniak, J., Hershkowitz, M., and Stein, S . 1974. Hydrogenation of xylose over platinum group catalysts. Ind. Eng. Chem. Prod. Res. Dev. 13, 232-236. Wolfrom, M. L., and Kohn, E. J. 1942. Crystalline xylitol. J . Am. Chem. SOC. 64, 1739. Yamaguchi, S . , Yoshikawa, T., Ikeda, S . , and Ninomiya, T. 1970a. Studies on the taste of some sweet substances. Part I. Measurement of the relative sweetness. Agric. Biol. Chem. 34, 181-186. Yamaguchi, S., Yoshikawa, T., Ikeda, S . , and Ninomiya, T. 1970b. Studies on the taste of some sweet substances. Part 11. Interrelationships among them. Agric. Biol. Chem. 34, 187-197. Ylikahri, R. 1979. Metabolic and nutritional aspects of xylitol.’Adv. Food Res. 25, 159-180.

This Page Intentionally Left Blank

A a-Amylase, protein inhibitors of, 141-144 composition, 142-144 distribution, 142 medical importance, 144 nutritional significance, 144 occurrence in beans, 141 physicochemical properties, 142- 144 physiological importance, 142 Amino acid, hydration capacity, 290-293

B Bean, phytic acid content, effect of cooking and canning, 46-47 soaking, 66-67 Bean (Phaseolus) protein amino acid composition, 102-105 amino acids, biological availability, 11 1-1 12 biological value, 106- 109 body membrane proteins, 98-99 composition, 99-102 Bowman-Birk soybean trypsin inhibitor, 135 chemical properties, influence of storage and processing on, 144-148 chymotrypsin, 138 digestibility, 109- 111 digestive enzyme inhibitors, 128- 144 binding, 130 composition, 130 molecular weight, 130 inhibitors of a-amylase, 141-144 composition, 142-1 44 distribution, 142 medical importance, 144 nutritional significance, 144 physicochemical properties, 142- 144

physiological importance, 142 protease, 128-141 distribution in plants, 133 medical importance, 139- 141 nomenclature, 129-133 occurrence, 129- 133 physiochemical properties, 134- 139 significance in plants, 133 trypsin, 131-133 amino acid sequence, 134 biosynthesis of crypsinogen, effect on, 140 isolates, composition, 105-106 lectins in medical importance, 123- 128 nutritional importance, 123- 128 nutritional properties, influence of storage and processing on, 144-148 physiochemical properties, 1 15- 123 protease inhibitors, composition, 134-139 research needs, 148-151 seeds, lectins from, 122 storage, 96-98 biosynthesis of, 95 identification, 96-98 quantitation, 96-98 trypsin inhibitors from, 131 Bowman-Birk soybean trypsin inhibitor, amino acid sequence, 135 Bran, phytic acid content, 14 Bread, phytic acid content, 15 Breadmaking, phytic acid destruction and, 55-60

C Carbohydrate, collagen, interactions with, 260-261 Catecholamine, in porcine stress syndrome, 187-189

405

406 Cereal phytase activity in, 21-23, 63 phytic acid phosphorus content, 10-13 Chymotrypsin, inhibitors, in Leguminosae, 138 Collagen amino acids, functional role, 25&25 I biological value, 319-320 in bologna sausage emulsion, 328-331 carbohydrates, interactions with, 260-261 catabolism, 272-274 chemistry, 240-250 amino acid composition, 241-244 amino acid sequences, 247-250 nonhelical regions, 250 triple-helical regions, 249-250 molecular organization, 245-246 composition, 274-287 antemortem factors, 275-284 antemortem factors, caloric and nitrogen intake, 275-276 chronological age, 28 1-284 hormones, 277-279 lathyrism, 280 sex, 275 thiolism, 28 I vitamins and minerals, 276-277 postmortem factors, 284-287 aging, 284 heating, 285-286 lysosomal enzymes, 285 cross-linking reactions, 257 digestibility, 318-319 DLVO theory, diagrammatic presentation, 302 edible fibrous, production of, 322-326 effect of bases, 299-300 strong acids, 297-299 swelling, 306-309 various salts, 300-306 weak acids, 297-299 emulsifying capacity, 309-31 I foaming, 31 1-312 food-grade, production of, 325 food systems, use in, 328-331 food uses, 322-331 fortification methods, 320-322 function in tissues, 266-267 functional properties, 287-332

INDEX amino acids, hydration capacity, 290-293 protein structural forces, 294 water binding, 288-306 gelatin, use as, 327-328 genetic types, 233-238 hydroxylysine cross-linking reactions, 258 identification of types, 238-240 immunochemistry of, 266 interchain cross-linkages, 251 -260 covalent bonds, 255-260 hydrogen bond, 252-254 hydrophobic bonds, 254-255 ionic bonds, 255 metabolism, 267-272 biosynthesis on polyribosomes, 267-269 glycosylation of hydroxylysine, 270-272 hydroxylation of proline, 269-270 microcrystalline, production of, 326-327 morphology, 233 nutritional aspects, 3 12-322 peptide hydrolase action, 273 polysaccharides of connective tissue and, 26 1-265 products, salient features, 324 protein quality assays, 313-318 biological value, 314 chemical (amino acid) score assays, 316-31 8 digestibility, 314 net protein ratio, 315 net protein utilization, 315 protein efficiency ratio, 314-315 relative nutritive value, 315 research needs, 33 1-332 salt and hydrogen interaction, 308-309 in skin, 323 viscoelasticity, 312 Connective tissue glycosaminoglycans associated with, 26 1-262 mucopolysaccharides associated with, 26 1-262 Copper, phytic acid and bioavailability, 41-42 Cottonseed, globoid composition, 9

D Digestive enzyme, protein inhibitors of, 128-133

407

INDEX

F Fermentation, phytic acid destruction and, 55-60 Fruit, xylitol, occurrence in, 376-377

G Globoid, composition from cottonseed, 9 Glycolysis, in porcine stress syndrome, 185- 186

Monoferric phytate, purification from wheat bran, 34 Mucopolysaccharide, connective tissue, association with, 261-262 Muscle collagen types, 235-128 contraction, sources of energy, 206 metabolism, halothane and, 207-209 in porcine stress syndrome, 202-210 contractile proteins, 200-202 neuromuscular junction, 193- 195 sarcoplasmic reticulum, 195-200

H P Halothane, effect on muscle metabolism, 207-209 Hormone, in porcine stress syndrome, 190-192

I Iron, phytic acid and bioavailability, 33-36

L Lactate, production in porcine stress syndrome, 214 Lectin from bean seeds, 122 in beans, composition, 115-123 physiochemical properties, 1 15- I23 distribution in plants, 114-1 15 importance, 1 14- 1 15 medical and nutritional importance, 123-128 nomenclature, I 12- 1 13 occurrence, 112- 1 13 Legume phytase enzyme of, 21-23 phytic acid phosphorus content, 10-13 Leguminosae, chymotrypsin and trypsin inhibitors, 138

M Malignant hyperthermia, in porcine stress syndrome, 173-175 Manganese, phytic acid and bioavailability, 4 1-42

Pea, phytic acid content, effect of cooking, 42-43 Peanut, globoid composition, 9 Phytate, see Phytic acid Phytic acid activity cereal, 63 wheat, 62 autolysis of effect of pH, 64 effect of temperature, 64 beans, cooking and canning, effect of, 46-47 bioavailability of, 23-28 calcium, 30-31 copper, 41-42 iron, 33-36 magnesium, 31-33 manganese, 4 1-42 zinc, 36-41 biological function, 7 biological value in laying hens, 26 in swine, 25 breadmaking and, 55-60 characterization of, 21-23 content bran, 14 bread, 15 flour, 14 soybean, 60 tempeh, 60 whole grain, 14 cooking, effect on, 42-48

408

INDEX

Phytic acid (coat.) destruction during breadmaking, 55-60 fermentation, 55-60 determination analytical methods, 15- I8 digestion of, 23-28 effect on mineral bioavailability, 29-42 retention of ingested phosphorus, 27 fermentation and, 55-60 germination and, 48-54 in cereals, 49-50 in legumes, 5 1-52 phytase activity changes, 53-54 phytate phosphorus changes, 53-54 globoid composition, 9 historical background, 1-4 hydrolysis, 24-28, 65-67 and baking, 57 in calves and steers, 24 in chicks and laying hens, 27 effect of preheating, 65 soaking, 66 in rats, 28 in Iranian flat breads, 37 in legumes, 1-91 monoferric, purification from wheat bran, 34 occurrence, 7- 12 pH and protein-mineral interactions, 18-2 1 phytase and, in wheat, 62 phytate-protein-mineral interactions, 18-21 phytin-protein complex, dissociation of, 73 processing effect of, 42-48 removal, 67-74 with calcium and barium ions, 68 from soybean meal, 68 from soy protein, 68-74 from soy protein, by diafiltration, 72-74 by dialysis and anion exchange, 72 at pilot plant, 69 by ultrafiltration, 74 research needs, 75 in rice, effect of cooking, 48 in seeds, effect of soaking, 66 in sesame meal effect of autoclaving, 45 in soy isolates, 70 structure, 4-6 Phytic acid phosphorus, content in cereals and legumes, 10-13

Phytohemagglutinin, see Lectin Polysaccharide, collagen and, 261-265 Porcine stress syndrome characterization, 169- 173 classification, 170- 171 etiology of hormones, 187- 192 catecholamines, 187- 189 corticosteroids, 190- 192 thyroid, 189-190 lactate production, 215 muscle metabolism, 192-21 1 muscle metabolism, contractile proteins, 200-202 mitochondria, 202-2 10 neuromuscular junction, 193- 195 sarcoplasmic reticulum, 195-200 malignant hyperthermia, 173- 175 blood gases, 174 electrolytes during epidodes, 174 rectal temperature changes, 175 pale, soft, exudative syndrome, 175-177 predictive tests genetics, 178-179 halothane exposure, 182-186 hematological test, 181-182 muscle biopsies, 182- 186 postmortem glycolysis, 185-1 86 serological tests, 179- 181 Protease inhibitors from beans, 128-141 composition, 1 3 4 1 3 9 distribution in plants, 133 medical importance, 139-141 nomenclature, 129-133 occurrence, 129-133 physiochemical properties, 134- 139 significance in plants, 133

R Rice globoid composition, 9 phytic acid, effect of cooking on, 48

S Sesame meal, phytic acid content, effect of autoclaving, 45 Skin, chemical composition, 323

409

INDEX Soybean, phytic acid content, 60 Soybean meal, phytate removal, 68 Soy protein, phytic acid removal, 68-74 Steroid hormone, effect on collagen formation, 279

T Tempeh, phytic acid content, 60 Thyroid hormone, in porcine stress syndrome, 189- 190 Trypsin, inhibitor effect on biosynthesis of crypsinogen, 140 from beans, 131- 133 in Leguminosae, 138 lima bean, amino acid sequence, 134 W Wheat iron, biological value of, 35 phytase activity in, 62 Wheat bran, monoferric phytate, purification, 34 Whole grain, phytic acid content, 14 A

Xylitol boiling point, 384 browning reactions, 389 caloric value, 389 in chewing gum, 392-393 in chocolate, 395 in coatings, 395 in compressed tablets, 394-395 in confectionery jellies, 394 confectionery uses, 392-395 crystallization, 382-383

density, 387 in drinks, 398-399 fermentation, 389-390 food applications, 392-399 food technological properties, 389-392 in gum drops, 394 in hard candy, 393-394 heat of solution, 385-386 history, 375-378 hygroscopicity, 387-388 in ice cream, 395-396 in jams, 396-398 in jellies, 396-398 large scale production, 378-382 hydrogenation, 381 hydrolysis, 380 purification, 381-382 xylose purification, 380 in marmalades, 3 9 6 3 9 8 occurrence in fruit, 376-377 in nature, 374-375 physicochemical properties, 382-388 research needs, 399-400 solubility, 385 specific heat, 384-385 structure, 382 sweetness, 390-392 chemical basis, 390 synergistic effects, 391-392 in water and acid solutions, 391 viscosity, 386 in yogurt, 396 2

Zinc, bioavailability effect of autoclaving, 40 in foodstuffs, 39-41

This Page Intentionally Left Blank