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

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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. 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).

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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

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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).

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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,

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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

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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 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.