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Studies on the hydrophobicity of proteins and enzymes Voutsinas, Leandros Panagis 1982

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STUDIES ON THE HYDROPHOBICITY OF PROTEINS AND ENZYMES by LEANDROS PANAGIS |VOUTSINAS B.Sc, Uni v e r s i t y of Thessaloniki, Greece, 1969 M.Sc, The Un i v e r s i t y of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1982 (c) Leandros Panagis Voutsinas, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Leandros P. Voutsinas Department Of Food Science The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 21, 1982 ABSTRACT This t h e s i s deals w i t h s t u d i e s on the hydrophobicity of p r o t e i n s and enzymes and i s d i v i d e d i n four chapters summarized se p a r a t e l y below. (1) T r a d i t i o n a l methods of coagulating m i l k f o r the manufacture of cheese s u f f e r from two major drawbacks, namely, the high cost of the enzyme and the f a c t that they are batch systems. An obvious s o l u t i o n to both problems would be the use of immobilized enzymes to coagulate m i l k . Hydrophobic adsorption o f f e r s c e r t a i n p o t e n t i a l advantages over other techniques of enzyme i m m o b i l i z a t i o n . The o b j e c t i v e of t h i s part of the t h e s i s was to immobilize the m i l k c l o t t i n g enzymes chymosin and pepsin on v a r i o u s hydrophobic c a r r i e r s and to assess t h e i r s u i t a b i l i t y f o r continuous coagulation of skimmilk. A l l enzyme-carrier preparations e x h i b i t e d high i n i t i a l a c t i v i t y on exposure to m i l k . However, the d e a c t i v a t i o n r a t e s were very high. The main reason f o r t h i s r a p i d d e a c t i v a t i o n appeared to be the l o s s of enzyme from the c a r r i e r s , s i n c e s o l u b l e a c t i v i t y was detected i n a l l enzyme pre p a r a t i o n s . The enzyme l o s s was due to the p h y s i c a l desorption of enzyme from the c a r r i e r s as w e l l as to the r e l a t i v e l y r a p i d leakage of the l i g a n d from the c a r r i e r (phenoxyacetyl c e l l u l o s e ) . The best enzyme p r e p a r a t i o n was obtained w i t h phenoxyacetyl c e l l u l o s e . However, a study i n d i c a t e d that the continuous coagulation of skimmilk w i t h proteases immobilized on the hydrophobic supports used i n t h i s study was not economically f e a s i b l e . (2) The f a t b i n d i n g c a p a c i t y (FBC) of food p r o t e i n s i s an e s s e n t i a l f u n c t i o n a l property. However, f a t b i n d i n g as determined by e x i s t i n g methods has been mainly a t t r i b u t e d to p h y s i c a l entrapment of the o i l r a t h e r than to the b i n d i n g w i t h p r o t e i n s . A simple t u r b i d i m e t r i c i i i method, thus, was developed for determining the FBC of various proteins. The t u r b i d i t y was dependent on wavelength, blending time and volume of o i l . The regression equation for p r e d i c t i n g FBC was: FBC (%) = 30.271 + 1.381 S - 0.014 S x s o o where S q and s are surface hydrophobicity and s o l u b i l i t y index, respectively. 2 A highly s i g n i f i c a n t c o r r e l a t i o n (R = 0.802, P < 0.01) was found between S , S x s, and FBC of 11 food proteins tested. Advantages of o o the method developed include the small amount of sample required and the f a c t that the measured values would r e f l e c t the true f a t binding capacity of proteins by minimizing the fat-entrapping e f f e c t s . (3) The objectives of t h i s part of the thesis were to determine the e f f e c t s of heating on the emulsifying properties of selected food proteins, and, to assess the value of S q as a predictor of these properties. The r e s u l t s obtained indicated that the emulsifying properties of the proteins studied were d i f f e r e n t l y affected by heating, and that heat-denaturation was not always accompanied by l o s s of f u n c t i o n a l i t y , but, on the contrary, resulted i n great improvement. The emulsifying properties could well be predicted s o l e l y on the basis of S l e v e l but not on the basis of s o l u b i l i t y l e v e l , which indicated that S o o i s a very important property determining protein f u n c t i o n a l i t y . However, the emulsion a c t i v i t y , emulsion s t a b i l i t y and fat binding of the proteins studied could be well explained and more accurately predicted by S q and s o l u b i l i t y together. (4) The objectives of t h i s part were to evaluate the thermal properties (thickening, coagulation and gelation) of selected food proteins and to assess the value of hydrophobicity as t h e i r predictor. The r e s u l t s obtained indicated that the average (S ) and not the surface e i v hydrophobicity was important for these properties. The thermal properties studied could not be explained by either the average hydrophobicity or su l f h y d r y l s alone. Instead, they could well be predicted using average hydrophobicity and sulfhydryls together. TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS V LIST OF TABLES v i i i . LIST OF FIGURES i x ACKNOWLEDGEMENT x Chapter I. Coagulation of Skimmilk with Proteases Immobilized on Hydrophobic C a r r i e r s 1 INTRODUCTION 2 LITERATURE REVIEW 4 A. Introduction to immobilized enzymes 4 (a) Methods for enzyme immobilization . . . . 4 1. Adsorption 4 2. Entrapment 8 3. Cross-linking 9 4. Covalent binding 10 (b) Properties of immobilized enzymes . . . . 12 (c) Advantages of immobilized enzymes . . . . 15 (d) P o t e n t i a l applications of immobilized enzymes i n the food industry 16 B. Continuous coagulation of skimmilk with immobilized proteases 18 (a) Mechanism of milk coagulation and t h e o r e t i c a l basis f o r the system . . . . 18 (b) Advantages of using immobilized proteases i n cheese manufacture 19 (c) Enzymes, c a r r i e r s , methods of immobilization and reactor designs used i n preparing immobilized m i l k - c l o t t i n g proteases 21 (d) A c t i v i t y and s t a b i l i t y of immobilized m i l k - c l o t t i n g proteases 23 MATERIALS AND METHODS 27 A. Materials . . 27 B. Preparation of hexyl-substituted epichloro-hydrin cross-linked desulfated Sepharose 6B . 28 C. Immobilization of pepsin on hexyl-ECD-Sepharose 6B 29 D. Immobilization of pepsin and chymosin on phenoxyacetyl c e l l u l o s e 29 E. Immobilization of pepsin on activated carbon . 30 F. Immobilized enzyme assays 31 v. PAGE i i v i . PAGE RESULTS AND DISCUSSION 33 A. Immobilization of pepsin on hexyl-ECD-Sepharose 6B • 33 B. Immobilization of pepsin and chymosin on phenoxyacetyl c e l l u l o s e 39 C. Immobilization of pepsin on activated carbon 44 D. Economics of using immobilized proteases f o r the continuous coagulation of skimmilk . . . . 48 E. General Discussion and Conclusions 53 REFERENCES 54a Chapter I I . A Simple Turbidimetric Method for Determining the Fat Binding Capacity of Proteins 61 INTRODUCTION 62 MATERIALS AND METHODS 63 A. Materials 63 B. Methods 64 (a) Fat binding capacity determination . . . 64 (b) Protein (surface) hydrophobicity , determination 66 (c) S o l u b i l i t y index determination 67 (d) S t a t i s t i c a l analysis 67 RESULTS AND DISCUSSION 68 A. Fat binding capacity 68 B. Comparison of the FBC of various proteins . . 75 C. S t a t i s t i c a l analysis 77 D. Mechanism of f a t absorption 83 CONCLUSIONS 85 REFERENCES 86 Chapter I I I . Relationships of Hydrophobicity to Emulsifying Properties of Heat Denatured Proteins 88 INTRODUCTION 89 LITERATURE REVIEW 92 A. S o l u b i l i t y 92 B. Hydrophobicity 93 C. Formation and s t a b i l i z a t i o n of emulsions . . . 95 v i i . PAGE MATERIALS AND METHODS 100 Materials 100 Methods 101 A. Preparation ( i n s o l u b i l i z a t i o n ) of pr o t e i n samples 101 B. S o l u b i l i t y index, hydrophobicity and f a t binding capacity determinations 103 C. Emulsifying a c t i v i t y index (EAI) determination 104 D. Emulsion s t a b i l i t y index (ESI) determination. 104 E. V i s c o s i t y determination . 104 F. S t a t i s t i c a l analysis 105 RESULTS AND DISCUSSION 105 A. E f f e c t of heat treatment on emulsifying properties 105 B. S t a t i s t i c a l analysis . 113 CONCLUSIONS . . . . . 123 REFERENCES 125 Chapter IV. Relationships between Protein Hydrophobicity and Thermal Properties of Food Proteins 130 INTRODUCTION 131 LITERATURE REVIEW 132 MATERIALS AND METHODS 135 Materials 135 Methods 135 A. Thickening determination 135 B. Heat coagulation determination 136 C. Gelation measurement 137 D. Hydrophobicity determination 137 E. Determination of s u l f h y d r y l and d i s u l f i d e groups of proteins 138 F. S t a t i s t i c a l analysis 139 . RESULTS AND DISCUSSION 139 CONCLUSION 150 REFERENCES 151 GENERAL CONCLUSIONS 154 v i i i . LIST OF TABLES PAGE Chapter I. Table 1 - P o t e n t i a l a p p l i c a t i o n s of immobilized enzymes i n the food industry . . . . 17 Table 2 - Pepsin and chymosin immobilized on hydrophobic supports for the continuous coagulation of skimmilk 35 Table 3 - Ca l c u l a t i o n of the economics of using immobilized proteases for the continuous coagulation of skimmilk 36 Chapter I I . Table 1 - Relationship between protein hydrophobicity, s o l u b i l i t y index, and f a t binding capacity of various proteins 76 Table 2 - M u l t i p l e regression model for p r e d i c t i o n of f a t binding capacity of various food proteins 80 Chapter I I I . Table 1 - Relationships between protein hydrophobicity, s o l u b i l i t y index, emulsifying a c t i v i t y , emulsion s t a b i l i t y and f a t binding capacity of various proteins 106 Table 2 - E f f e c t of heating on the apparent v i s c o s i t y of some proteins at 20 C I l l Table 3 - Regression models f o r p r e d i c t i o n of emulsifying and f a t binding properties of heat denatured proteins 114 Chapter IV. Table 1 - Relationships between protein hydrophobicities, s u l f h y d r y l s , s u l f h y d r y l s + reduced d i s u l f i d e s and thermal properties of various proteins 140 Table 2 - Mul t i p l e regression models for pr e d i c t i o n of thermal properties of various proteins 144 i x . LIST OF FIGURES PAGE Chapter I. F i g . 1. C l o t t i n g a c t i v i t y of pepsin and chymosin immobilized on phenoxyacetyl c e l l u l o s e for the continuous coagulation of skimmilk . . . 41 F i g . 2. C l o t t i n g a c t i v i t y of pepsin-untreated activated carbon preparations used i n the continuous coagulation of skimmilk 49 F i g . 3. C l o t t i n g a c t i v i t y of pepsin-HCl-treated activated carbon preparations used i n the continuous coagulation of skimmilk 50 Chapter I I . F i g . 1. E f f e c t of wavelength on absorbance . . 69 F i g . 2. E f f e c t of blending time on absorbance at 600 nm 71 Fi g . 3. Standard curve for fat-binding determination 72 F i g . 4. Flow diagram f o r the determination of FBC of proteins 73 F i g . 5. Relationship between hydrophobicity and f a t binding capacity of food proteins . . . 79 F i g . 6. Fat binding capacity response surface contour as a function of hydrophobicity (S ) and s o l u b i l i t y index (s) 81 Chapter I I I . F i g . 1. Emulsifying a c t i v i t y index response surface contour as a function of hydrophobicity ( S q ) and s o l u b i l i t y index (s) 118 F i g . 2. Emulsion s t a b i l i t y index response surface contour as a function of hydrophobicity ( S q ) and s o l u b i l i t y index (s) 120 Chapter IV. F i g . 1. Thickening response surface contour as a function of S and SH content 145 e F i g . 2. Heat coagulation response surface contour as a function of S and SH content . . . 147 e X . ACKNOWLEDGEMENT I wish to express my sincere gratitude to my research supervisor, Dr. S. Nakai, for h i s constant advice, assistance, encouragement and constructive c r i t i c i s m throughout the course of t h i s study, and i n the preparation of the th e s i s . I am also thankful to the members of my supervisory committee, Drs. W. D. Powrie and P. M. Townsley of the Department of Food Science and to Dr. B. Roufogalis of the Faculty of Pharmaceutical Sciences, for the i r i n t e r e s t i n and review of t h i s t h e s i s . F i n a l l y , I would l i k e to thank my wife, Maria, f o r her encourage-ment and patience during my years of graduate studies. 1. Chapter I Coagulation of Skimmilk with Proteases Immobilized on Hydrophobic C a r r i e r s 2. INTRODUCTION T r a d i t i o n a l methods of curd formation i n the manufacture of cheese s u f f e r from two major drawbacks; that i s , the high cost of rennet and the labour intensiveness of batch systems (Chefyan et a l . , 1975b). A s o l u t i o n to both problems would be the use of immobilized p r o t e o l y t i c enzymes to c l o t milk, since the enzyme can be reused and a continuous throughput of milk can be employed. The major problem encountered by Cheryan et a l . (1975a, 1975b, 1976) and Taylor et a l . (1977) was a rapid loss i n a c t i v i t y of the immobilized enzymes. The longest active l i f e t i m e of proteases was about two days of continuous treatment of milk. Other disadvantages encountered i n these studies were the high cost of c a r r i e r s , the lengthy and complicated immobilization methods and the excessive desorption of pepsin from alumina into the e f f l u e n t milk. Hydrophobic adsorption o f f e r s c e r t a i n p o t e n t i a l advantages over other techniques f o r enzyme immobilization (Brash and Lyman, 1969'; Butler, 1975). These are: (1) e s s e n t i a l l y i r r e v e r s i b l e enzyme binding; (2) the enzymes t y p i c a l l y r e t a i n a very high percentage of t h e i r native a c t i v i t y ; (3) since hydrophobic i n t e r a c t i o n s tend.to s t a b i l i z e p r otein conformation, immobilized enzymes show increased resistance to denaturation; (4) r e u s a b i l i t y of the c a r r i e r ; (5) s i m p l i c i t y and convenience; and (6) r e p r o d u c i b i l i t y i n preparing enzyme c a t a l y s t s . The objectives of t h i s part of the thesis were to 3. immobilize the m i l k - c l o t t i n g enzymes, chymosin and pepsin, on several hydrophobic c a r r i e r s and to assess t h e i r s u i t a b i l i t y f o r continuous coagulation of skimmilk. 4. LITERATURE REVIEW A. Introduction to immobilized enzymes (a) Methods for enzyme immobilization There have been a large number of methods reported i n the l i t e r a t u r e for immobilizing enzymes. Most of these methods can be c l a s s i f i e d under one of the following four general categories: 1. Adsorption onto a water-insoluble c a r r i e r . 2. Entrapment, by occlusion within cross-linked gels or by encapsulation within microcapsules, hollow f i b e r s , and f i b e r s . 3. Cross-linking of the enzyme by b i - or m u l t i - f u n c t i o n a l reagents. 4. Covalent binding to a reactive insoluble c a r r i e r , through functional groups which are not involved i n the a c t i v i t y of the enzyme. 1. Adsorption H i s t o r i c a l l y , the e a r l i e s t method of enzyme immobilization, namely adsorption, i s also the easiest way of preparing enzyme-carrier conjugates. Adsorption of an enzyme can be achieved by simply mixing an enzyme solu t i o n with the finely-powdered adsorbent for some period of time a f t e r which the excess enzyme Is washed o f f the i n s o l u b l e adsorbent. A wide v a r i e t y of s o l i d s have been used to adsorb enzymes. Some of the most popular adsorbents are activated carbon, organic polymers, glass, mineral s a l t s , and s i l i c a g e l . Adsorption for immobilizing enzymes has the following advantages: (1) i t i s cheap and e a s i l y c a r r i e d out, since no reagents are required and the process involves only a minimum of a c t i v a t i o n steps (Barker and Kay, 1975); (2) the enzyme i s le s s l i k e l y to be denatured during 5. the process of immobilization when compared with chemical methods of immobilizing enzymes ( K i l a r a and Shahani, 1979); (3) the nature of the i n t e r a c t i o n s are such that a high l e v e l of a c t i v i t y i s retained by the adsorbed enzyme; (4) a wide v a r i e t y of adsorbents can be used; (5) r e u s a b i l i t y of the adsorbent (Carr and Bowers, 1980); and (6) adsorption can y i e l d reproducible preparations of the immobilized enzyme. The binding forces between an enzyme and a c a r r i e r may be i o n i c , hydrophobic, hydrogen bonds, and/or Van der Waal's i n t e r a c t i o n s , depending on the nature of the surface of the c a r r i e r . Since the bond energies are weak desorption of the bound enzyme can be brought about by changes i n temperature, pH, i o n i c strength or the presence of substrate (Barker and Kay, 1975). Another disadvantage of adsorption immobilization i s that the adsorption surface i s non-specific, i . e . , the surface does not s e l e c t i v e l y bind the enzyme, but w i l l adsorb proteins or other substances to which i t i s exposed i n the course of i t s use (Barker and Kay, 1975). A su i t a b l e adsorbent should possess high a f f i n i t y for the enzyme and cause minimal denaturation (Brodelius, 1978; Goldstein and Manecke, 1976). The r e v e r s i b i l i t y of the enzyme attachment by adsorption requires a c o n t r o l of process conditions such as i o n i c strength, pH of the reactant so l u t i o n , and temperature, i n order to avoid desorption of the enzyme. If desorption of the enzyme i s detrimental to the o v e r a l l process, such as contaminating the product or allowing the c a t a l y t i c process to proceed i n s o l u t i o n , t h i s r e v e r s i b l e binding may be a serious 6. drawback (Stanley and Olson, 1974). I r r e v e r s i b l e behaviour of enzymes has been reported, however, for a number of adsorption systems (Sundaram and Crook, 1971). Moreover, i t i s possible to s t a b i l i z e the enzyme which has been temporarily adsorbed onto the matrix by c r o s s - l i n k i n g the enzyme molecules i n a chemical reaction subsequent to i t s adsorption (Barker and Kay, 1975). Thus, some of the objectionable desorption c h a r a c t e r i s t i c s can be removed, but c a r r i e r r e g e n e r a b i l i t y i s s a c r i f i c e d (Carr and Bowers, 1980). By using the p r i n c i p l e s of a f f i n i t y chromatography, more s p e c i f i c techniques f o r adsorption of enzymes have ..been developed. For example, "hydrophobic chromatography" has been used to achieve the v i r t u a l l y i r r e v e r s i b l e adsorption of several enzymes onto N-alkyl d e r i v a t i v e s of Sepharose (Hofstee, 1973c; Visser and S t r a t i n g , 1975). The bound enzymes ( a l k a l i n e phosphatase, l a c t a t e dehydrogenase, urease, and xanthine oxidase) were not e a s i l y desorbed by aqueous solvents such as 1 M NaCl. The strong binding of these a c i d i c enzymes to N-alkyl-Sepharose, known to carry r e s i d u a l p o s i t i v e charges could be a t t r i b u t e d to a combination of hydrophobic and e l e c t r o s t a t i c i n t e r a c t i o n s (Hofstee, 1973a, 1973b, 1973c). Therefore, i n s o l u b l e polymers for use as b i o s p e c i f i c adsorbents i n a f f i n i t y chromatography should not contain groups which would cause a decrease i n s p e c i f i c i t y . Porath j2t a l . (1973) have described the adsorption of proteins on a completely nonionic substituent of agarose as a new approach to hydrophobic adsorption. In aqueous solutions, the adsorbent showed an a f f i n i t y f o r solutes with hydrophobic groups. Nonionic agarose exhibited hydrophobic i n t e r a c t i o n with hydrophobic regions on the molecular surface of proteins and other solutes. Porath 7. and h i s coworkers (Caldwell et a l . , 1976a, 1976b, 1976c, 1976d), subsequently, studied the immobilization of enzymes through hydrophobic i n t e r a c t i o n s . They immobilized fS-amylase and amyloglucosidase through adsorption onto hexyl-substituted epichlorohydrin-crossj-linkedagarose gels (Sepharose 6B), and subsequently used these adsorbates f o r continuous hydrolysis of starch over extended periods of time. The adsorption was rapid and the retention of the c a t a l y t i c a c t i v i t y upon immobilization was high for both enzymes. The operational s t a b i l i t y of the adsorbates was good during month-long continuous substrate percolation. A c t i v i t y leakage (due to release of enzyme) was demonstrated but was low. A second type of hydrophobic c a r r i e r has been produced by the reaction of phenoxyacetyl chloride with c e l l u l o s e (Butler, 1975). Butler (1975) reported that phenoxyacetyl c e l l u l o s e very strongly bound a l l 10 of the enzymes tested. The bound enzymes, which were not desorbed by 1 M (NH^^SO^ or moderate concentrations (25 - 50%) of nonaqueous solvents (e.g. ethylene g l y c o l ) , but which e f f e c t i v e l y desorbed i n solutions containing nonionic detergents (0.1% T r i t o n X-100 i n b u f f e r ) , exhibited nearly complete retention of c a t a l y t i c a c t i v i t y . Butler (1975), moreover, suggested that the strong noncovalent adsorption of the enzymes was due s o l e l y to hydrophobic i n t e r a c t i o n s . The fac t that a l l of the 10 enzymes tested were strongly bound to phenoxyacetyl c e l l u l o s e suggested that a f f i n i t y f o r ce r t a i n hydrophobic groups may be a general c h a r a c t e r i s t i c of enzymes and other proteins. Phenoxyacetyl c e l l u l o s e i s commercially a v a i l a b l e from Regis Chemical Co., under the trade name ENZORB-A. 8. 2. Entrapment In p r i n c i p l e , a l l entrapment methods are based on confining an enzyme i n the l a t t i c e of a polymer matrix or enclosing an enzyme i n a semipermeable membrane. Immobilization by entrapment, therefore, d i f f e r s from other methods of immobilization (e.g. covalent binding or cross-li n k i n g ) i n the respect that the enzyme molecules are free i n sol u t i o n , but r e s t r i c t e d i n the room by a gel l a t t i c e or a membrane. The structure of the entrapping polymer should thus be t i g h t enough to prevent the enzyme from d i f f u s i n g out and at the same time be loose enough to allow d i f f u s i o n of substrate and product. The most popular matrices for entrapment include polyacrylamide, s i l i c o n e rubber, starch and s i l i c a g e l . Occlusion within cross-linked polyacrylamide gels has been the most widely used entrapment technique. In general, entrapment techniques have^the following advantages: (1) as there i s no bond formation between the enzyme and the polymer matrix, entrapment provides a method which i s generally applicable to any enzyme (Barker and Kay, 1975; Goldstein and Manecke, 1976); (2) enzymes can be contained without any chemical modification of bonding that may lead to a c t i v i t y loss (Pitcher, 1980a); (3) they allow high l o c a l concentration of enzymes; and (.4) multiple enzyme systems can be handled r e a d i l y since entrapment i s e s s e n t i a l l y the method l i v i n g c e l l s use to r e t a i n t h e i r enzyme systems (Pitcher, 1980a). Entrapment techniques, however, have several l i m i t a t i o n s : (1) the g e n e r ality of t h i s method (due to advantage No. 1) i s l i m i t e d by the fa c t that entrapment methods are suitable mainly for enzymes that u t i l i z e substrates of molecular weights low enough to d i f f u s e through the matrix (Goldstein and Manecke, 1976). This disadvantage may serve as a l i m i t a t i o n for t h e i r use i n food 9. technology, since food systems often contain macromolecules ( K i l a r a and Shahani, 1979); (2) entrapment r e s u l t s i n enzyme loss by leakage of the enzyme from the matrix. This i s due to the broad d i s t r i b u t i o n i n the pore s i z e of the polymer. I t should be noted, however, that u l t r a -f i l t r a t i o n methods may be used to overcome the problem of leakage, because of the uniform pore si z e of the semipermeable membranes (Barker and Kay, 1975); (3) none of the s t a b i l i z i n g e f f e c t s of bonding to a r i g i d support are a v a i l a b l e to the entrapped enzyme (Pitcher, 1980a); (4) reproducible preparations are d i f f i c u l t to make (Stanley and Olson, 1974); and (5) mass transfer l i m i t a t i o n s i n conjunction with the phys i c a l properties of the entrapped enzyme devices places some constraints on the reactor design (Pitcher, 1980a). 3. Cross-linking This immobilization method i s based on the formation of intermolecular cross-linkages between the enzyme molecules or between protein and enzyme molecules by means of b i - or m u l t i - f u n c t i o n a l reagents. The most commonly used b i f u n c t i o n a l reagent i s glutaraldehyde, a dialdehyde that reacts with primary amines to form stable linkages. Although the matrix may contain j u s t enzyme molecules, i t i s usually i n the i n t e r e s t of economy to copoly-merize the enzyme with an i n e r t protein such as albumin i n order to increase the bulk of the f i n a l product (Trevan, 1980). The advantages of the cross-l i n k i n g are i t s r e l a t i v e s i m p l i c i t y and the fact that a sin g l e reagent can be used to prepare numerous enzyme de r i v a t i v e s . This method has the following disadvantages: (1) c r o s s - l i n k i n g an enzyme to i t s e l f i s both expensive and i n e f f i c i e n t , as part of the enzyme w i l l be i n e v i t a b l y functioning as a support, r e s u l t i n g i n r e l a t i v e l y low enzymic a c t i v i t y 10. (Barker and Kay, 1975; K i l a r a and Shahani, 1979); (2) the polymerization of the enzyme d i r e c t l y with b i f u n c t i o n a l reagents s u f f e r s from a lack of s e l e c t i v i t y (Carr and Bowers, 1980), that i s , i t i s extremely d i f f i c u l t to c o n t r o l intramolecular c r o s s - l i n k i n g while obtaining a high degree of intermolecular c r o s s - l i n k i n g ; (3) i t i s d i f f i c u l t to c o n t r o l the si z e and mechanical properties of the polymer; and (4) many enzymes are s e n s i t i v e to the coupling reagents and lose a c t i v i t y i n the process (Guilbault, 1975). Cross-linking of the enzyme, however, has proven valuable i n combination with other immobilization techniques. For example, enzymes adsorbed on insoluble supports have been cross-linked to minimize leakage of the enzyme from the support and thus to increase > s t a b i l i t y of the immobilized enzyme preparation (Goldman et a l . , 1968; L i u et a l . , 1975; Taylor et a l . , 1977). 4. Covalent binding The covalent binding method involves the l i n k i n g of enzymes and water-insoluble c a r r i e r s by covalent bonds, and i s the most intensely studied of the enzyme immobilization techniques. The f u n c t i o n a l groups of enzymes a v a i l a b l e for covalent binding include N-terminal amino groups, e-amino groups of l y s i n e and arginine, C-terminal carboxyl groups, 3- and y-carboxyl groups of aspartic and glutamic acids, r e s p e c t i v e l y , phenol group of tyrosine, the s u l f h y d r y l group of cysteine, the hydroxyl groups of serine and threonine, the imidazole group of h i s t i d i n e , and the indole group of tryptophan (Barker and Kay,. 1975; K i l a r a and Shahani, 1979). Of these, the most widely used are.the f i r s t three. In coupling reactions, these f u n c t i o n a l groups react with c a r r i e r s containing reactive groups such as diazonium, acid azide, isocyanate and halides. Undoubtedly, the greatest 11. advantage of t h i s method i s the d u r a b i l i t y of the d e r i v a t i v e s . Thus, v a r i a t i o n s i n pH, i o n i c strength, substrate, solvents, and temperature w i l l not normally cause the enzyme desorption problem which i s usually exhibited by c e r t a i n adsorbed enzyme systems (Guilbault, 1975; Pitcher, 1980a). Binding i s not n e c e s s a r i l y permanent, however, as diazo and s u l f u r -s u l f u r linkages have been reported to be unstable under c e r t a i n conditions (Stanley and Olson, 1974). Another advantage i s that enzymes attached by covalent binding may be s t a b i l i z e d by v i r t u e of being t i e d down i n several places (Falb, 1972). The major disadvantage of t h i s method i s that the. reaction conditions required for covalent binding are r e l a t i v e l y complicated and not p a r t i c u l a r l y mild. Covalent binding, therefore, frequently a l t e r s the conformational structure of the enzymes and may change thei r r e a c t i v i t y . Moreover, in, the extreme case, the a c t i v e s i t e of the enzyme may be blocked through the chemical reaction used i n the attachment reaction and the enzyme i s rendered i n a c t i v e (Falb, 1972). Covalent binding has not been found to be used as extensively i n the food processing industry as one might expect from the numerous a r t i c l e s describing d i f f e r e n t chemical coupling schemes. One obvious reason, i s that many of the highly toxic reagents used i n the coupling processes would not be s u i t a b l e for applications where they would come i n contact with foods (Pitcher, 1980a). From the above de s c r i p t i o n of the methods for enzyme immobilization, i t i s evident that there i s no s i n g l e method that i s u n i v e r s a l l y applicable, and a number of factors (such as enzyme s t a b i l i t y , mechanical properties and cost of the c a r r i e r , as well as c a r r i e r r e g e n e r a b i l i t y and capacity for enzyme binding) have to be considered p r i o r to choosing a method. In p r a c t i c e , therefore, i t i s necessary to f i n d a s u i t a b l e method and conditions 12. for the immobilization of a p a r t i c u l a r enzyme according to the intended a p p l i c a t i o n . (b) Properties of immobilized enzymes The properties of an. immobilized enzyme.system can be quite d i f f e r e n t from those of the corresponding soluble enzyme system. These changes can be a t t r i b u t e d to a l t e r a t i o n s of the enzyme conformation or to the physico-chemical nature of the c a r r i e r . The a c t i v i t y of enzymes almost always decreases as a r e s u l t of the immobilization. This decrease may be ascribed to the following factors (Chibata, 1978): (1) f u n c t i o n a l groups of e s s e n t i a l amino acids i n the a c t i v e center of the enzyme p a r t i c i p a t e i n binding the enzyme to water-insoluble c a r r i e r s ; (2) conformational change of the enzyme occurs when the enzyme i s bound to an i n s o l u b l e c a r r i e r ; and (3) the enzyme may be bound without loss of a c t i v i t y , but the i n t e r a c t i o n of substrate with the enzyme i s affected by s t e r i c hindrance. The extent of the decrease i n a c t i v i t y depends on the immobilization technique, conditions required for enzymic treatment, c h a r a c t e r i s t i c s of supporting materials, d i f f u s i o n rates of substrate to and product from the enzyme, and molecular weight of the substrate (Olson and Richardson, 1974). One of the most important features of an immobilized enzyme used i n a large scale operation i s i t s s t a b i l i t y . Most enzymes when immobilized show a greater s t a b i l i t y than t h e i r soluble counterparts. It has been suggested that t h i s increased s t a b i l i t y could be the r e s u l t of reduced conformational i n a c t i v a t i o n , reduced a u t o l y s i s (autodigestion), and reduced attack by r e a c t i v e solutes due to s t e r i c s h i e l d i n g (Brodelius, 1978; Trevan, 1980). S t a b i l i t y of immobilized enzymes r e f e r s to several factors including operational s t a b i l i t y , storage s t a b i l i t y , and temperature 13. and pH s t a b i l i t y . The operational s t a b i l i t y of the a c t i v i t y of immobilized enzymes i s one of the most important factors a f f e c t i n g the success of i n d u s t r i a l i z a t i o n of an immobilized system. Operational s t a b i l i t y i s usually expressed as h a l f - l i f e . The h a l f - l i f e of various immobilized enzymes varies widely. A few immobilized enzymes (e.g. glucose isomerase, amino acylase) have long h a l f - l i v e s permitting i n d u s t r i a l a p p l i c a t i o n . Most of them, however, exhibit short h a l f - l i v e s . The storage s t a b i l i t y of an immobilized enzyme becomes important when the enzyme preparation i s used only i n t e r m i t t e n t l y . Immobilized enzymes may exhibit better storage s t a b i l i t y than soluble enzymes. The storage s t a b i l i t y of 50 immobilized enzyme systems have been reviewed by Melrose (1971). Of these, 12 systems showed no differ e n c e i n s t a b i l i t y r e l a t i v e to t h e i r soluble counterparts, 8 showed decreased s t a b i l i t y and 30 exhibited greater storage s t a b i l i t y . This indicates that one can generally expect enhanced storage s t a b i l i t y . The c a t a l y t i c a c t i v i t y of enzymes increases with elevation of the temperature. However, as enzymes are proteins and thus are susceptible to heat denaturation ( i n a c t i v a t i o n ) , the enzyme reaction cannot be p r a c t i c a l l y c a r r i e d out at high temperature. I f the heat s t a b i l i t y of an enzyme i s , therefore, increased by immobilization, the p o t e n t i a l u t i l i z a t i o n of such enzymes w i l l be extensive. The heat s t a b i l i t y of many immobilized enzymes has been studied, and there are examples showing increases, no change, and decreases of heat s t a b i l i t y of enzymes upon immobilization. S t a b i l i t y of immobilized enzymes toward v a r i a t i o n i n pH values depends upon physical and chemical c h a r a c t e r i s t i c s of the enzyme c a r r i e r and chemical modification of the enzyme brought about by binding of enzyme to c a r r i e r . I t has been reported, that polyanionic d e r i v a t i v e s of proteases (tr y p s i n , chymotrypsin and papain) exhibited improved s t a b i l i t i e s toward 14. a l k a l i n e pH values (Goldstein, 1970; Levin e t ' a l . , 1964). On the other hand, po l y c a t i o n i c d e r i v a t i v e s possessed greater s t a b i l i t y i n the a c i d i c pH range (Goldman et _al., 1971). S h i f t s i n pH optima upon immobilization have been found for many enzymes. The changes i n optimum pH (and pH-a c t i v i t y curve) depend on the charge of the water-insoluble c a r r i e r and/ or of the enzyme protein. The s h i f t i n g of the pH optimum for an enzyme may be r a t i o n a l i z e d as a microenvironmental e f f e c t . The environment i n the immediate v i c i n i t y of an immobilized enzyme often d i f f e r s from that i n the bulk phase. Thus, i f the enzyme i s bound to a p o l y e l e c t r o l y t i c support, e l e c t r o s t a t i c i n t e r a c t i o n s w i l l lead to an unequal d i s t r i b u t i o n of the ions between the c a r r i e r phase and the bulk phase. Within a polyanionic c a r r i e r (e.g. carboxymethyl c e l l u l o s e ) the concentration of the p o s i t i v e l y charged ions w i l l be higher than i n the external sol u t i o n . Consequently, the pH in s i d e such a c a r r i e r w i l l be lower than i n the surrounding media and the pH optimum of the immobilized enzyme w i l l be s h i f t e d toward a higher value ( i . e . more a l k a l i n e pH)to compensate for the increased l o c a l hydrogen-ion concentration. On the other hand, i n a po l y c a t i o n i c c a r r i e r (e.g. DEAE-cellulose), the opposite e f f e c t w i l l be expected, i . e . the pH optimum for the immobilized enzyme w i l l be lower than for the native enzyme (Brodelius, 1978; Carr and Bowers, 1980). By proper s e l e c t i o n of support material, therefore, such s h i f t s i n pH optima could permit the use of enzymes i n environments which would normally i n a c t i v a t e them. Thus, i t may be advantageous to r a i s e the pH optimum of acid proteases i n t r e a t i n g foods at pH values closer to n e u t r a l i t y . On the other hand, s h i f t i n g the pH toward a c i d i c values could aid i n c o n t r o l l i n g microbial growth during continuous processing or i t could increase the a c t i v i t y of enzymes i n a c i d i c foods (Olson and Richardson, 1974). 15. (c) Advantages of Immobilized enzymes The r a t i o n a l e for the replacement of e x i s t i n g a p p l i c a t i o n s of soluble enzymes by immobilized enzymes i s based on the following advantages which immobilized enzymes can.offer over soluble enzymes i n many areas such as study of enzymes, a n a l y t i c a l biochemistry, pharmacology, and i n d u s t r i a l processing including food processing (Barker and Kay, 1975; Olson and Korus, 1977; Taylor et a l . , 1976): (1) an immobilized enzyme acts as a stable b i o c a t a l y s t to bring about chemical reactions on a continuous basis. In t h i s respect, the enzyme cost would be lower; (2) the reaction i s e a s i l y terminated by separating the substrate from the enzyme; (3) the enzyme i s not l e f t i n the product. Thus, enzymes can be used which are presently unusable f or various reasons (e.g. m i c r o b i a l rennets). Moreover, not only the cost of heat i n a c t i v a t i o n of the enzyme i s saved but also the detrimental e f f e c t of Cheating to some foods i s avoided; (4) increased pH and temperature s t a b i l i t y ; (5) choice of continuous or batch operation; (.6) better q u a l i t y c o n t r o l of the produce (because of the continuous process); (7) les s product i n h i b i t i o n due to the easier separation of the product and substrate; (8) advantageous use of multiple enzyme systems; (9) greater reactor design f l e x i b i l i t y due to the large number of d i f f e r e n t p h y s i c a l forms of s o l i d matrices; and (10) p o t e n t i a l operation over greater pH range by modifying the charge c h a r a c t e r i s t i c s of the c a r r i e r . Immobilized enzymes have p o t e n t i a l disadvantages which include: (1) the cost of support and immobilization procedure; (.2) the lower a c t i v i t y or loss of enzyme a c t i v i t y upon immobilization; (3) the i n a c t i v a t i o n with continued operation; (4) greater i n i t i a l plant investment; (.5) more t e c h n i c a l l y complex process r e q u i r i n g more s k i l l e d supervision; and (6) applicable mainly to soluble substrates. 16. Although the above generalizations can provide some guidelines, the f i n a l considerations which determine whether i t i s advantageous to immobilize an enzyme depend on the p a r t i c u l a r process one i s considering (Olson and Korus, 1977). (d) P o t e n t i a l applications of immobilized enzymes i n the food industry Although every f i e l d that u t i l i z e s enzymes has found p o t e n t i a l uses of immobilized enzymes, t h e i r greatest p o t e n t i a l i s i n the food industry. Table 1 summarizes some of the enzymes used i n the food industry and t h e i r a p p l i c a t i o n s . Despite the large volume of research work c a r r i e d out on the p o t e n t i a l a p p l i c a t i o n s of immobilized enzymes, r e l a t i v e l y few processes using immobi-l i z e d enzymes have been commercialized. Currently there are only three immobilized systems i n commercial use due to t h e i r economy and p r a c t i c a l i t y . These are the glucose isomerase system f o r producing high fructose corn syrup (HFCS), the amino acylase system used for the r e s o l u t i o n of D- and L-amino acids, and the p e n i c i l l i n acylase system used i n the pharmaceutical industry to obtain 6-aminopenicillanic acid ( K i l a r a and Shahani, 1979). The large-scale use of immobilized enzymes i n food processing has been prevented by two major problems (Cheryan, 1974). One major problem has been the frequent plugging of the reactors possibly due to m i c r obial growth i n the columns, but more probably due to the presence of suspended matter i n the substrate which clogs the i n t e r s t i c e s of the c a t a l y s t bed. This problem, however, can be overcome by the proper s e l e c t i o n and design of the reactor and proper choice of operating parameters. Another major problem has been the r e l a t i v e l y poor s t a b i l i t y c h a r a c t e r i s t i c s shown by some immobilized enzymes i n contact with 17. Table 1 - P o t e n t i a l applications of immobilized enzymes i n the food industry Enzyme Appli c a t i o n Amino acylase a-Amylase 3-Amylase Catalase C e l l u l a s e 6-Galactosidase Glucose oxidase Glycoamylase Glucose isomerase Invertase Lipase Naringinase Papain, F i c i n , Bromelain Resolution of D- and L-amino acids Starch degradation ( l i q u e f a c t i o n ) High maltose syrups Cold S t e r i l i z a t i o n of milk Convert c e l l u l o s e to glucose Hydrolyze lactose i n milk and whey Deoxygenate foods; desugar eggs Production of glucose from starch Production of fructose from glucose Hydrolyze sucrose from production of invert sugar and confections Enzymic modification of f l a v o r of foods Debitter c l a r i f i e d c i t r u s j u i c e s C h i l l p r o o f i n g of beer Pectinase Pepsin, Rennin, Chymotrypsin or other proteases F r u i t j u i c e c l a r i f i c a t i o n and y i e l d improvement Continuous coagulation of skimmilk for cheese-making; production of protein hydrolysates Peroxidases Cold s t e r i l i z a t i o n of foods Sulfhydryl oxidase Elimination of the cooked f l a v o r i n UHT s t e r i l i z e d milk Trypsin I n h i b i t i o n of oxidative r a n c i d i t y i n milk 18. t h e i r natural substrates ( e s p e c i a l l y protein-containing substrates). Thus, the immobilized enzyme preparations tend to lose a c t i v i t y too r a p i d l y f o r commercial a c c e p t a b i l i t y . B. Continuous coagulation of skimmilk with immobilized proteases (a) Mechanism of milk coagulation and t h e o r e t i c a l basis for the system Milk i s a complex f l u i d c onsisting of numerous proteins, f a t , lactose and minerals. The major proteins are the caseins, comprising 80% of the t o t a l protein. The remaining proteins, whey proteins, are soluble i n the serum. Casein i s a quite complicated mixture of proteins,which can be generally f r a c t i o n a t e d i n t o K--;~ and y-caseins although, many other subfractions have been described. The casein proteins are present i n milk as stable m icelles (agglomerated macromolecular s p h e r i c a l p a r t i c l e s ) with the various components together as "calcium caseinate". A d d i t i o n a l .: complexity occurs with calcium phosphate as well as with magnesium and c i t r a t e ions (Gordon and Kalan, 1974). When milk i s coagulated, i t i s the s t a b i l i t y of t h i s casein m i c e l l e that i s disrupted. Coagulation of milk by m i l k - c l o t t i n g enzymes i s a remarkably complex process. In t h i s process, two rather d i s t i n c t phases can be distinguished: a primary or enzymatic phase and a secondary or non-enzymatic phase (Ernstorm and Wong, 1974). In the primary phase the p r o t e o l y t i c enzyme cleaves a s p e c i f i c phenylalanyl-methionine bond of K-casein, releasing a negatively charged hydrophilic glycomacropeptide and para - K-casein, thereby d e s t a b i l i z i n g the m i c e l l e s . In the secondary phase, which requires calcium, there i s a ph y s i c a l aggregation of the d e s t a b i l i z e d m icelles which r e s u l t s i n the formation of a coagulum. The mechanism of m i c e l l e aggregation i s not completely elucidated, but the negative charge on micelles i s reduced by the loss of the glycopeptides allowing further 19. i n t e r a c t i o n of micelles (Green and C r u t c h f i e l d , 1971; Green and Marshall, 1977; Payens, 1966; Pearce, 1976). It i s f e a s i b l e to develop a system for continuous coagulation of milk employing immobilized p r o t e o l y t i c enzymes because of a p e c u l i a r i t y of the coagulation process. The large temperature c o e f f i c i e n t (Q-^Q) O R the secondary phase (Q^Q=11-12) but the low Q^Q of the primary phase (Q^Q=2) permits separation of the two phases by lowering the temperature i n the immobilized enzyme reactor (Cheryal et a l . , 1975a). Thus, the immobilized enzyme retains s u f f i c i e n t a c t i v i t y at lower temperatures (e.g. 5 - 15°C) to complete the primary enzymatic phase but c l o t t i n g does not occur u n t i l a f t e r the milk i s removed from the immobilized enzyme reactor and warmed. The two phases can also be separated by maintaining the milk at high pH (6.7), since both phases of enzymatic milk coagulation are pH dependent - and the secondary phase i s highly pH dependent. A 30-fold decrease i n the c l o t t i n g time, when the pH of the secondary phase was lowered from 6.7 to 5.6, has been reported by Cheryan et a l . (1975a). (b) Advantages of using immobilized proteases i n cheese manufacture C l o t t i n g milk with immobilized proteases for the manufacture of cheese o f f e r s the following s p e c i f i c (and potential) advantages over the t r a d i t i o n a l method: (1) there has been an ever increasing worldwide shortage (and thus high cost) of c a l f rennet for the past 20 years, created by the increasing demand for both beef and cheese, prompting use of other s u i t a b l e proteases i n t r a d i t i o n a l cheesemaking systems (Taylor et _al., 1976). However, these substitutes have some l i m i t a t i o n s (due to excessive p r o t e o l y s i s , defective f l a v o r , body and texture, color development and the production of toxins and pathogens). Shortage of m i l k - c l o t t i n g enzymes may 20. be eased by employing immobilized enzymes which can be reused; (2) since the immobilized enzyme does not contaminate the curd, other proteases, which are not s u i t a b l e f or use i n the soluble form because of excessive uncontrolled p r o t e o l y s i s , could be used i n an immobilized state. Thus, i t may be possible to substitute a less expensive, less desirable, but more r e a d i l y a v a i l a b l e enzyme which normally cannot be used, such as crude m i c r o b i a l proteases, instead of commercially a v a i l a b l e m i l k - c l o t t i n g enzymes (Richardson and Olson, 1974); (3) the lack of contamination of cheese with m i l k - c l o t t i n g enzymes would allow, moreover, separation of the m i l k - c l o t t i n g and cheese-ripening functions of immobilized proteases. Consequently, the immobilized proteases could be chosen for t h e i r optimal m i l k - c l o t t i n g a c t i v i t y and for the e f f e c t s on cheese y i e l d and c h a r a c t e r i s -t i c s of cheese curd, while soluble proteases added to the curd could be selected f or t h e i r optimum cheese-ripening action (Taylor et^ al., 1979). The enzyme used i n the t r a d i t i o n a l process i s chosen for i t s m i l k - c l o t t i n g capacity as well as for i t s influence on cheese-ripening process, that i s , the choice of enzyme i s a compromise between i t s m i l k - c l o t t i n g and cheese-ripening a c t i v i t i e s (Brodelius, 1978; Richardson and Olson, 1974). As a r e s u l t , cheese-ripening i s much l e s s c o n t r o l l a b l e i n the t r a d i t i o n a l method; (4) greater f l e x i b i l i t y and control over cheese ripening and merchandising. Thus, i f le s s enzyme i s added, the cheese could be stored longer (because of the longer ripening time), allowing better coordination between seasonal f l u c t u a t i o n s i n milk supply and cheese demand (Taylor et a l . , 1979); and (5) immobilized proteases could f i t into a continuous, semi-continuous or batch cheese-making process, depending upon requirements (Taylor'et a l . , 1979). The above s p e c i f i c advantages, of a continuous cheese making process 21. based on an Immobilized protease system help i n explaining the continual e f f o r t s made to develop such a system. (c) Enzymes, c a r r i e r s , methods of immobilization arid reactor designs used  i n preparing immobilized m i l k - c l o t t i n g proteases Several p r o t e o l y t i c enzymes such as chymotrypsin, Mucor miehei proteases, papain, pepsin, chymosin, rennet and t r y p s i n have been immobilized for use i n milk coagulation. Although a d i r e c t comparison of the performance of these c a t a l y s t s i s d i f f i c u l t (since the c a r r i e r and immobilization technique have also a great e f f e c t on the c a t a l y s t a c t i v i t y ) , Taylor et a l . (1976) suggested that, based on the l i t e r a t u r e , pepsin appears to be the best immobilized protease for coagulating milk. Pepsin, an enzyme with a low pH optimum and not p a r t i c u l a r l y a c t i v e nor stable at the normal pH of milk, i s quite a c t i v e and stable upon immobilization (Cheryan et a l . , 1975b; Cheryan et a l . , 1976). Presumably immobilization of pepsin places i t i n a microenvironment of lower pH than the bulk s o l u t i o n which s t a b i l i z e s i t s t e r t i a r y structure (Taylor et a l . , 1979). Rennet was found to be more act i v e but l e s s stable than pepsin (Cheryan at a l . , 1975b). I t i s i n t e r e s t i n g to note that, contrary to the behaviour of commercial c a l f rennet, chymosin exhibited very low (Arima et _al., 1974; Green and C r u t c h f i e l d , 1969; Thonart'_et'al. , 1978) or no a c t i v i t y at a l l (Cheryan et a l . , 1975b) when immobilized. Since most commercial rennet preparations contain bovine pepsin, i t has been suggested by Cheryan et a l . (1975b) that the a c t i v i t y observed with immobilized rennet was probably due to the immobilized bovine pepsin. In order for the coagulation of milk with immobilized proteases to be commercially f e a s i b l e , the c a r r i e r material should be reasonably 22. inexpensive, p h y s i c a l l y s u i t a b l e and stable for the reactor design, non-toxi c , and allow good enzyme-substrate contact and proper flow of substrate. A great v a r i e t y of c a r r i e r materials have been used to immobilize m i l k - c l o t t i n g proteases. Agarose (Angelo and Shahani, 1979; Arima et a l . , 1974; Green and C r u t c h f i e l d , 1969), aminoethyl c e l l u l o s e (Arima et a l . , 1974; Green and C r u t c h f i e l d , 1969), and p a r a f f i n wax (Savangikar and Joshi, 1978; Shindo et a l . , 1980a, 1980b) are some of the organic c a r r i e r s used for immobilizing m i l k - c l o t t i n g enzymes. However, the most successful immobilized enzyme-carrier preparations have been obtained with inorganic c a r r i e r s such as alumina, porous glass, and t i t a n i a (Cheryan et a l . , 1975b; Cheryan el: a l . , 1976; Taylor et a l . , 1977). Covalent attachment and adsorption are the two major methods reported i n the l i t e r a t u r e f o r immobilizing m i l k - c l o t t i n g proteases. Entrapment of m i l k - c l o t t i n g enzyme within a gel or membrane has not been attempted because such a method w i l l not be workable since the substrate (casein) i s too large to d i f f u s e i n and out of the enzyme-trapping matrix. Covalent binding has been the most widely used method for immobilizing milk-c l o t t i n g enzymes. This i s due to the fa c t that covalent binding allows l i t t l e or no desorption of the m i l k - c l o t t i n g enzymes in t o the curd (Cheryan et a l . , 1975a, 1975b; F e r r i e r et a l . , 1972; Hicks et a l . , 1975). Although t h i s method i s preferable for laboratory studies, for p r a c t i c a l a p p l i c a t i o n to commercial cheese-making, i t may be too complicated and c o s t l y . On the other hand, adsorption i s a simple, mild and inexpensive method, but desorption of protease may be a problem, since the i o n i c strength of milk may promote enzyme desorption. Prevention of the desorption of the immobilized m i l k - c l o t t i n g enzyme i s very important i n 23. order to minimize c a t a l y s t i n a c t i v a t i o n and to c o n t r o l p r o t e o l y s i s i n cheese curd during aging. One of the great advantages of using immobilized enzymes to coagulate m i l k i s the f l e x i b i l i t y i n r e a c t o r design. Three common r e a c t o r designs -f l u i d i z e d bed, packed bed and s t i r r e d tank - have been reported i n the l i t e r a t u r e . The packed bed r e a c t o r i s u s u a l l y l i m i t e d by m i l k plugging of the column. The plugging problem can be avoided by u s i n g a continuously s t i r r e d tank r e a c t o r , but the shear f o r c e s developed l i m i t the types of c a r r i e r s to those having s u f f i c i e n t s t r u c t u r a l i n t e g r i t y (Taylor j i t a l . , 1976). Plugging can a l s o be e l i m i n a t e d by using a f l u i d i z e d bed r e a c t o r . Cheryan ejt _ a l . (1975b) reported that f l u i d i z e d bed r e a c t o r performed equal or s u p e r i o r to the f i x e d bed r e a c t o r under almost a l l c o n d i t i o n s . Another advantage of f l u i d i z e d bed i s that i t can be used w i t h c a r r i e r s (e.g. porous g l a s s ) that would d i s i n t e g r a t e i n a s t i r r e d tank r e a c t o r . (d) A c t i v i t y and s t a b i l i t y of immobilized m i l k - C l o t t i n g proteases Performance of an immobilized enzyme-carrier p r e p a r a t i o n depends upon both a c t i v i t y and s t a b i l i t y . A c a t a l y s t p r e p a r a t i o n w i t h high i n i t i a l a c t i v i t y and a slow i n a c t i v a t i o n r a t e i s p r e f e r r e d . A r e l a t i v e l y great number of s t u d i e s have been conducted on the i m m o b i l i z a t i o n of m i l k - c l o t t i n g enzymes and t h e i r use i n cheese production. Green and C r u t c h f i e l d (.1969) prepared enzymically a c t i v e i n s o l u b l e d e r i v a t i v e s of chymotrypsin and chymosin, using cyanogen bromide a c t i v a t e d agarose as the c a r r i e r ( m a t r i x ) . They a l s o immobilized chymosin on aminoethyl c e l l u l o s e . Each of the i n s o l u b l e enzyme d e r i v a t i v e s apparently c a t a l y s e d the c l o t t i n g of m i l k , but i n a l l cases t h i s a c t i v i t y was shown to be almost e n t i r e l y due to enzyme rel e a s e d i n t o s o l u t i o n (milk) from 24. the c a r r i e r . Thonart ej: a l . (1978) immobilized chymosin on glass bead by d i a z o t i z a t i o n . The i n i t i a l a c t i v i t y of t h i s preparation was very low (8 - 10% of the a c t i v i t y of free enzyme), and a f t e r one hour of continuous coagulation of milk, immobilized chymosin retained only 40% of i t s i n i t i a l a c t i v i t y . It was, furthermore, suggested by these authors that t h i s loss of a c t i v i t y was due to adsorption of milk protein onto the c a r r i e r and desorption or s o l u b i l i z a t i o n of the active enzyme molecules from the c a r r i e r . Ohmiya et_ al. (1978, 1979) immobilized an a l k a l i n e protease from B a c i l l u s s u b t i l i s on an anion exchange r e s i n (Dowex MWA-1) by g l u t a -raldehyde and studied the f e a s i b i l i t y of using t h i s preparation for cheese-making. They concluded that the a l k a l i n e protease could be used for cheese making as a m i l k - c l o t t i n g enzyme instead of c a l f rennet, i f the p r o t e o l y t i c a c t i v i t y to release s i a l i c a c i d was properly c o n t r o l l e d . These workers, however, f a i l e d to report any economic study substantiating t h e i r claim. Shindo and coworkers (Shindo and Arima, 1979a, 1979b; Shindo et a l . , 1980a, 1980b) have extensively studied the preparation, properties and u t i l i z a t i o n of immobilized chymosin i n cheese-making. Immobilized chymosin prepared by p a r a f f i n wax showed r e l a t i v e l y high s t a b i l i t y to repeated enzymatic reaction (Shindo et a l . , 1980a). Furthermore, Gouda type cheese was produced by Shindo e_t _al. (1980b) with t h i s immobilized chymosin preparation. These authors concluded that immobilized chymosin using p a r a f f i n wax could be useful f or cheese-making i f a s u i t a b l e manufacturing method was applied. I t should be noted, however, that no economic comparison between free and immobilizing chymosin preparation was reported, and therefore, these authors f a i l e d to reveal any economic advantage of t h e i r batch method over the t r a d i t i o n a l cheese-making process. Angelo and Shahani (1979) immobilized rennet on 25. Sepharose 4B through covalent bonding with an objective to assess i t s s u i t a b i l i t y for milk coagultion i n continuous cheese-making. Immobilized rennet coagulated skimmilk within an average of 18 min (range 7 to 30 min). A decline i n the milk c l o t t i n g a c t i v i t y of a packed bed reactor of immobilized rennet occurred i n 3 hr run. No "soluble a c t i v i t y " of immobilized rennet was detected. The most successful system among the immobilized m i l k - c l o t t i n g proteases reported i n the l i t e r a t u r e appear to be that described by Taylor et a l . (1977). The three best pepsin-carrier preparations were obtained with t i t a n i a (controlled pore ceramic) and glass (zirconium oxide-coated c o n t r o l l e d pore), using a covalent attachment procedure (glutaraldehyde) and alumina (controlled pore ceramic), using adsorption at pH 1.2. The reactor was operated as a f l u i d i z e d bed at low temperature, and coagulation of the milk was achieved by a c i d i f y i n g (pH 6.1) and then warming (30°C) a f t e r the milk emerged from the reactor. Although the i n i t i a l c l o t t i n g a c t i v i t y was high, the a c t i v e l i f e t i m e of the c a t a l y s t was not more than 2 days. Loss of enzymatic a c t i v i t y was very rapid during the i n i t i a l stages of continuous coagulation of skimmilk. The rate of lo s s of a c t i v i t y was slower but exponential during subsequent operation of the reactor. Inactivation during use of the reactor appeared to be caused in part by deposition of proteins and peptides on the immobir-. .: l i z e d enzyme (Cheryan et a l . , 1975"; F e r r i e r et a l . , 1972). Accumulation of nitrogenous material and s i a l i c acid p a r a l l e l e d the i n i t i a l rapid loss i n enzymatic a c t i v i t y . Both whey proteins and caseins appeared to be responsible for loss i n a c t i v i t y . In addition, desorption of pepsin from the alumina c a r r i e r during the continuous reactor operation was observed (Taylor et a l . , 1977). I n a c t i v a t i o n during continuous usage was minimized by precoating the c a r r i e r with proteins (BSA) and then coupling pepsin to 26. the coated supports (Cheryan et a l . , 1976). Coating the supports with proteins probably increased c a t a l y s t s t a b i l i t y by decreasing the p o s i t i v e charge of the c a r r i e r and thus decreasing the adsorption of negatively charged milk proteins. Attempts to restore the a c t i v i t y of ina c t i v a t e d c a t a l y s t s were only p a r t i a l l y successful. Thus, washing immobilized pepsin with water restored some a c t i v i t y but maximum regeneration was obtained by washing with d i l u t e hydrochloric acid (0.1 N) or urea at pH 3.5 (Cheryan et al., 1975b; Taylor et a l . , 1977). On subsequent reuse of the regenerated immobilized pepsin, a rapid loss i n a c t i v i t y was observed and the c a t a l y s t exhibited a memory i n returning to the a c t i v i t y l e v e l p r i o r to regeneration. Reasons for t h i s memory e f f e c t , which has been observed with porous glass as well as alumina have not been defined. Furthermore, two other attempts to regenerate immobilized pepsin a c t i v i t y using polyethylene g l y c o l and T r i t o n X-100 resulted i n complete loss of a c t i v i t y (Taylor et a l . , 1977). Taylor ej: a l . (.1977) suggested that pepsin adsorbed on alumina may have commercial p o t e n t i a l for cheese making.and promised an economic study which has never been been reported. It should be pointed out that the encouraging r e s u l t s presented by Taylor e_t a l . (.1977) were obtained by using controlled-pore alumina as enzyme c a r r i e r which i s an expensive chromatographic material. However, adsorption of pepsin to a number of inexpensive industrial-grade aluminas did not reproduce the c a t a l y t i c a c t i v i t y obtained with the expensive controlled-pore alumina (Skogberg, 1976; Taylor et a l . , 1977). Differences i n surface area and/or surface charge may be responsible for the poor r e s u l t s obtained with the industrial-grade aluminas (Taylor et_ a l . , 1979). Olson and Richardson (1979) of the same group i n t h e i r l a s t communication suggested that immobilized enzymes may have a p p l i c a t i o n i n cheese manufacturing i f the economics of the process are improved, and that t h i s would, require cheaper enzyme supports, more complete immobilization of enzyme, better enzyme s t a b i l i t y on exposure to milk or means of regenerating l o s t enzymatic a c t i v i t y . According to Sardinas (1976) the undeniable promise of the continuous coagulation of milk with immobilized proteases i s s u f f i c i e n t challenge to assure i t s continued development. MATERIALS AND METHODS A. Materials D i s t i l l e d , deionized water was used throughout except for washing of c a t a l y s t s , which was done with d i s t i l l e d water. A l l chemicals used i n t h i s study were reagent grade or better. Pasteurized skimmilk, pH 6.7, was purchased from the l o c a l market. Enzymes used included pepsin (from porcine stomach mucosa, c r y s t a l l i z e d and l y o p h i l i z e d ) , from Sigma Chemical Company, Saint Louis, Mo., and rennin (N.F.), from N u t r i t i o n a l Biochemical Corporation, Cleveland, Ohio. Other reagents included epichlorohydrin, from Fisher S c i e n t i f i c Company, F a i r Lawn, N.J.; Sodium borohydride (NaBH^), from Sigma Chemical Company, Saint Louis, Mo.; 1-bromohexane, from Eastman Kodak Co., Rochester, N.Y. Support materials ( c a r r i e r s ) used include Sepharose 6B, from Pharmacia Fine Chemicals, Uppsala, Sweden; Phenoxyacetyl c e l l u l o s e , containing 0.3 to 0.5 phenoxyacetyl groups per glucose moiety, from Regis Chemical Co., Morton Grove, 111.;- and activated carbon 12 x 20 mesh, from Darco ICI America Inc., Wilmington, Del. 28. B. Preparation of hexyl-substituted epichlorohydrin-crosslinked  flesulfat'ed Sepharose 6B Agarose g e l of type Sepharose 6B was- s t a b i l i z e d through c r o s s l i n k i n g with epichlorohydrin p r i o r to the introduction of the a l i p h a t i c ether substituents. Epichlorohydrin crosslinked desulfated Sepharose 6B (ECD-Sepharose 6B) was prepared according to the method of Porath et a l . (1971) as follows: 500 ml of swollen agarose beads were mixed at room temperature with 500 ml of IN NaOH containing 50 ml epichlorohydrin and 2.5 g sodium borohydride. The mixture was heated to 60°C for 2 hr with adequate s t i r r i n g . The crosslinked gel so obtained was washed with hot d i s t i l l e d water to neutral pH. 500 ml of 2N NaOH and 2.5 g NaBH^ were added to 500 ml of the suspension and the mixture was heated i n an autoclave at 120°C for 1 hr. The gel was washed with 1.5 L of hot solu t i o n of IN NaOH containing 0.5% NaBH^ and then with 1.5 L of cold s o l u t i o n of the same composition. The gel was quickly transferred to a beaker containing f i n e l y crushed i c e and ac e t i c ac i d was added to bring the pH down to 4.0. The gel was again transferred to a Blichner funnel and washed with hot d i s t i l l e d water (to remove the remaining traces of boric acid) and f i n a l l y with i c e water. The gel was stored as a suspension i n 0.02% sodium azide s o l u t i o n . Hexyl-substituted ECD-Sepharose 6B (hexyl-ECD-Sepharose 6B) was prepared according to the method of Caldwell et a l . (1975) as follows: 100 mg of sedimented ECD-Sepharose 6B, suspended i n 100 ml of 5 N NaOH containing 0.5% NaBH^, were mixed with 50 ml of 1-bromohexane. The reaction mixture was kept at 100°C f o r 35 hr under gentle s t i r r i n g . A f t e r c a r e f u l washing with water, and subsequently with ethanol, u n t i l a neutral pH was reached, the product was again washed with d i s t i l l e d water and stored as an aqueous suspension. C. Immobilization of pepsin on hexyl-ECD-Sepharose 6B Slurry of the gel was packed i n a column of 1.0 cm i n t e r n a l diameter and the amount adjusted so as to give a bed volume of 0.6 ml. The bed was washed with d i s t i l l e d water for s e t t l i n g and then with 10 ml of 0.1 M acetate buffer pH 4.6 containing 3 M NaCl for e q u i l i b r a t i o n . A so l u t i o n of pepsin (10 mg porcine pepsin i n 3 ml of 0.1 M acetate buffer containing 3 M NaCl) was passed through the gel which was subsequently washed with the e q u i l i b r a t i o n buffer u n t i l the eluate showed n e g l i g i b l e UV absorption at 280 nm. The amount of enzyme adsorbed at t h i s point was estimated from the d i f f e r e n c e i n absorbance at 280 nm of the pepsin s o l u t i o n before and a f t e r adsorption to the g e l . The column was then washed with d i s t i l l e d water pH 6.7 u n t i l the pH of the eluate was 6.7. Subsequently, cold pasteurized milk pH 6.7 was applied to the column which was operated as a fixed bed reactor at a flow rate of 8 ml/hr i n a cold room (4°C). D. Immobilization of pepsin and chymosin on phenoxyacetyl c e l l u l o s e To 2.5 g of phenoxyacetyl c e l l u l o s e (PAC) s u f f i c i e n t mixture of ethanol-water (1:1) was added to cover the phenoxyacetyl c e l l u l o s e powder i n a beaker. The r e s u l t i n g s l u r r y was s t i r r e d and the trapped a i r bubbles were allowed to r i s e . The s l u r r y was then poured into a column (1 x 30 cm). The bed was washed with 30 ml of ethanol-water to s e t t l e packing, then with 30 ml of deionized water to expose the phenoxyacetyl c e l l u l o s e to aqueous environment, and f i n a l l y with 30 ml of 0.1 M acetate buffer pH 4.6. A s o l u t i o n of pepsin (15 mg pepsin i n 4 ml 0.1 M acetate buffer pH 4.6 containing 3 M NaCl) was pumped through the PAC column. The unbound 30. enzyme was washed from the bed by passing fresh buffer through the column. The column was, subsequently, washed with d i s t i l l e d water pH 6.7, u n t i l the pH of the ef f l u e n t was 6.7. Then, cold pasteurized skimmilk was applied to the column. A constant flow rate of 36 ml/hr was maintained with a p e r i s t a l t i c pump. For chymosin immobilization, a dispersion of 300 mg rennin i n 5 ml of 0.1M acetate buffer, pH 6.2 containing 3M NaCl was centrifuged at 6,950 x g (7,500 rpm) for 10 min and only the supernatant was applied to the column. E. Immobilization of pepsin on activated carbon The activated carbon granules (12 x 20 mesh) were ground i n a mortar with a pestle and they were sieved with standard sieves (30/40 mesh) to obtain 425-589 um diameter p a r t i c l e s f o r enzyme immobilization, washed thoroughly with 1 L of warm deionized water and dried at 105°C overnight. The carbon, thus obtained, was designated as untreated activated carbon (UAC). Hydrochloric acid-treated activated carbon (TAC) was prepared according to the method of Cho and Bailey (1979) as follows: 20 g of UAC were treated i n a Soxhlet extraction apparatus for 48 hr with 1.7N HC1. The carbon p a r t i c l e s were then rinsed with warm d i s t i l l e d water and then warm deionized water u n t i l no chl o r i d e ion was detected and dried at 110°C overnight. Chloride ion detection was done according to Hogness et^ a l . (1966). Two methods of enzyme immobilization were investigated. Adsorption and covalent binding. The adsorption immobilization was c a r r i e d out as follows: 100 mg of pepsin were dissolved i n 12.5 ml of d i s t i l l e d water. To t h i s s o l u t i o n 12.5 ml of 0.4N HC1 were added (dropwise with gentle s t i r r i n g ) to bring the pH down to 1.2. This enzyme so l u t i o n was poured 31. into a 125 ml f l a s k and a 5 g of UAC or TAC were added. The mixture was gently shaken at room temperature for 30 min on a shaking bath. The immobilized preparation was thoroughly washed with 4 L of water at pH 3.5 and 0.5 L water at pH 6.7, and then transferred into a column (1.6 x 20 cm). Covalent immobilization of pepsin was c a r r i e d out according to the method of Cho and Bailey (1977). Thus, 5 g of UAC or TAC were mixed with 10 ml of 0.1M acetate buffer pH 4.6. To t h i s s l u r r y 100 mg of water soluble diimide [l^cyclohexyl-3-(2-morpholino-ethyl)-carbodiimide metho-p-toluene sulfonate] was added with gentle shaking. A f t e r 10 min, 100 mg of pepsin i n 5 ml buffer was added, and the immobilization was c a r r i e d out at room temperature for 2 hr with mild shaking. F i n a l l y , the immobilized enzyme-carbon preparation was washed with 100 ml of 0.1M acetate buffer and then with 3 L of d i s t i l l e d water pH 6.7 p r i o r to trans f e r into the reactor. The column was operated as a f l u i d i z e d bed (upward flow), since preliminary studies with a f i x e d bed column proved t h i s design to be unsuitable due to plugging of the column. F. Immobilized enzyme assays A c t i v i t y of m i l k - c l o t t i n g enzymes i s normally expressed as the time (min) for milk to c l o t under standardized conditions a f t e r adding the enzyme to the milk (Ernstrom and Wong, 1974). C l o t t i n g time i s re l a t e d inversely to enzymic a c t i v i t y (Hicks et a l . , 1975). Immobilized enzyme preparations i n t h i s study were assayed for a c t i v i t y and s t a b i l i t y as described by Cheryan et a l . (1975b). A c t i v i t y was expressed as c l o t t i n g time, which was the-time i n minutes for e f f l u e n t skimmilk from the reactor to c l o t under standardized conditions a f t e r treatment with immobilized 32. enzyme. E f f l u e n t skimmilk was c o l l e c t e d i n a 5 ml graduated cylinder cooled i n an ice-bath and 2 ml sample was quickly added to a prewarmed (30°C) 125 ml f l a s k with a pipette c h i l l e d i n i c e . The required amount of 2N phosphoric acid was added (generally about 28 yl/2 ml skimmilk) to bring the pH of the milk to 6.1. The f l a s k was then quickly attached to the arm of a rotor (forming a constant angle of about 30° with the water surface) and placed i n a water bath of 30°C and rotated continuously at a speed of about 10 rpm. The timer was started j u s t a f t e r the f l a s k was immersed into the water. C l o t t i n g time was taken when the f i r s t v i s i b l e signs of coagulum formed on the sides of the f l a s k . "Soluble a c t i v i t y " of an immobilized enzyme preparation i s the a c t i v i t y due to desorption or s o l u b i l i z a t i o n of the enzyme from the c a r r i e r . This a c t i v i t y was determined by adding the whey from a coagulated milk sample to a fresh untreated milk sample (2 ml) at the same pH and measuring the c l o t t i n g time as described above. The c l o t t i n g time of the mixture was used as an index of soluble a c t i v i t y . Catalyst s t a b i l i t y was described by the rate of c a t a l y s t deactivation at any point of operation (time on stream) of the reactor. Since the rates of deactivation of m i l k - c l o t t i n g enzymes were shown to be logarithmic with the time (Cheryan et a l . , 1975b), s t a b i l i t i e s on exposure to milk were most conveniently expressed i n terms of "D values", i . e . . the time i n hours for the c l o t t i n g time to increase by one logarithmic cycle. The "D value" was determined from the p l o t of log c l o t t i n g time versus time on stream by drawing a tangent to the c l o t t i n g time curve. The time for the tangent to transcend one log cycle was a "D value". Higher D values would i n d i c a t e greater ca t a l y s t s t a b i l i t y . 33. RESULTS AND DISCUSSION A. Immobilization of pepsin on hexyl-ECD-Sepharose 6B Sepharose has been widely used as a support material f or enzyme immobilization (Angelo and Shahani, 1979; Green and C r u t c h f i e l d , 1969; Caldwell et a l . 1975, 1976a, 1976b, 1976c, 1976d). I t s usefulness i s mainly due to features such as a large porosity i n combination with mechanical s t a b i l i t y . If the immobilized enzyme i s to serve as a ca t a l y s t for a reaction involving a high molecular weight substrate, i t i s evident that the porosity of the c a r r i e r w i l l be of importance for substrate and product transports. Agarose can be prepared i n a beaded form well suited for column chromatographic procedures i n which r e s o l u t i o n of large molecules i s desired. Caldwell et a l . (1975) have reported on the immobilization of 3-amylase based on hydrophobic i n t e r a c t i o n s between the enzyme and hydrocarbon chains attached to a c a r r i e r g e l . A hexyl-substituted agarose (hexyl-ECD-Sepharose 6B) produces an absorbate of high s t a b i l i t y s u i t a b l e f o r month-long continuous starch hydrolysis at room temperature. Furthermore, Caldwell et a l . (1976c) immobilized amyloglucosidase through adsorption onto the same c a r r i e r (hexyl-ECD-Sepharose 6B). This preparation exhibited high retention of i t s a c t i v i t y and was used for continuous production of glucose from starch for three months with only 40% reduction i n i t s a c t i v i t y . Hexyl-Sepharose i s an adsorbent quite permeable and highly r e s i s t a n t to chemical and b i o l o g i c a l attack (Caldwell et a l . , 1976b). Crosslinking with epichlorohydrin renders the agarose polymer stable without changing i t s molecular sieving properties or i t s a b i l i t y to swell i n water (Porath et a l . , 1971). One advantage of hexyl-ECD-Sepharose 6B as a c a r r i e r i s the fact that i t i s a non-charged c a r r i e r 34. (as opposed to cyanogen bromide activated agarose d e r i v a t i v e s ) . Therefore, i t should not exhibit mixed ionic-hydrophobic adsorption but, instead, "pure" hydrophobic in t e r a c t i o n s with the hydrophobic regions on the molecular surface of proteins (Porath ej: a^l. , 1973). Therefore, neither desorption of the enzyme from the c a r r i e r (due to increased i o n i c strength of the substrate) nor adsorption of protein from the substrate on the c a r r i e r (due to e l e c t r o s t a t i c i n t e r a c t i o n s ) should be expected. Another advantage of hexyl-ECD Sepharose 6B i s the great s t a b i l i t y of the ether bond (linkage) between ligand and the matrix ( c a r r i e r ) as opposed to the unstable bond obtained with cyanogen bromide-activated Sepharose r e s u l t i n g i n the leakage of the ligand from the matrix i n a l k a l i n e medium (Matsumoto et a l . , 1979). As i t can be seen from Table 2, pepsin immobilized on hexyl-ECD Sepharose 6B exhibited small i n i t i a l c l o t t i n g a c t i v i t y and extremely rapid i n a c t i v a t i o n . Thus, a l i m i t of 10 min coagulation time at 30°C (considered as the maximum desirable c l o t t i n g time for evaluation purposes) was reached a f t e r 5 hr of continuous operation at a flow rate of 8 ml/hr (Table 3). Binding capacity of t h i s c a r r i e r was 3.7 mg pepsin per 0.6 ml g e l , corresponding to a binding e f f i c i e n c y of 40%. No "soluble a c t i v i t y " of immobilized pepsin preparation was detected i n the treated milk. It should also be reported that rennin exhibited no a c t i v i t y at a l l when immobilized on hexyl-ECD-Sepharose 6B. Although the reasons for the small i n i t i a l a c t i v i t y and extremely rapid i n a c t i v a t i o n of pepsin-hexyl-ECD-Sepharose 6B preparation were not investigated, the following (reasons) are very p l a u s i b l e . One reason for the low i n i t i a l a c t i v i t y exhibited by t h i s preparation may be the " s t e r i c hindrance" by the carrier, when'-'the enzyme approaches the substrate. This e f f e c t has been also reported by Table 2 - Pepsin and chymosin immobilized on hydrophobic supports f o r the continuous coagulation of skimmilk C l o t t i n g time (min) D value (hr) Support Enzyme Immobil. Method 4 Time 14 on 20 - stream (hr) 30 4 14 20 PAC Pepsin Adsorption 0.75(7.4) b 5.2(59.6) b 7.6 8.9 8.5 23.0 55.0 PAC Chymosin Adsorption 0.69(5.7) 4.8(48.7) 7.4 10.0 7.5 24.0 41.7 UAC Pepsin Adsorption 0.50(3.0) 4.5(36.1) 6.8 9.9 6.0 24.5 45.5 TAC Pepsin Adsorption 0.80(4.5) 5.5(41.3) 7.1 9.9 8.5 23.5 36.0 UAC Pepsin Covalent B. 0.85(7.2) 6.1(54.8) 8.0 9.5 8.4 35.0 61.0 TAC Pepsin Covalent B. 0.35(3.2) 2.1(24.2) 4.1 6.8 10.0 22.0 38.0 Hexyl-ECD-Sepharose 6B Pepsin Adsorption 8.05(0.0) PAC, Phenoxyacetyl c e l l u l o s e UAC, Untreated activated carbon TAC, HCl-treated activated carbon 'Number i n parenthesis shows corresponding soluble a c t i v i t y Average of two t r i a l s 4) value i s a measure of s t a b i l i t y of the immobilized enzyme, as defined i n the text Table 3 - Ca l c u l a t i o n of the economics of using immobilized proteases f o r the continuous coagulation of skimmilk (Reactor: pH 6.7, Temper. 4°C; Coagulation: pH 6.1, Temper. 30°C) Enzyme-Carrier Preparation Flow Rate (ml/hr) Time on c Stream (hr) Amount of Enzyme Applied (mg) Amount of Milk Treated (L) Enzyme Required Per L Milk (mg) Pepsin-PAC(Ad.) b 36 33 15 6.5 2.3 Rennin-PAC(Ad.) 36 30 300 5.5 54.5 Pepsin-UAC(Ad.) 100 30 100 22.7 4.4 Pepsin-TAC(Ad.) 100 30 100 14.8 6.8 Pepsin-UAC(C.B.) 100 32 100 13.2 7.6 Pepsin-TAC(C.B.) 100 '45 100 32.7 3.1 Pepsin-Hexyl-ECD-Sepharose 6B(Ad.) 8 5 10 0.08 125.00 Pepsin (soluble) — 0.84 Rennin NF (soluble) — 17.60 aPAC, Phenoxyacetyl c e l l u l o s e UAC, Untreated activated carbon TAC, HCl-treated activated carbon ^Letters i n parenthesis i n d i c a t e immobilization method: Ad., adsorption; C.B., covalent binding C l o t t i n g time l i m i t of 10 min reached 37. Green and C r u t c h f i e l d (1969). Their chymosin-agarose (Sepharose 2B) d e r i v a t i v e d i s p l a y e d very low or zero a c t i v i t y on c a s e i n m i c e l l e s i n m i l k , although i t d i d hydrolyse K - c a s e i n . However, K - c a s e i n i s only about one-f i f t h of the s i z e of a casein m i c e l l e , so s t e r i c hindrance was l e s s . A second m a n i f e s t a t i o n of s t e r i c hindrance was deduced from the data obtained f o r K - c a s e i n as substrate i n d i c a t i n g that the agarose-chymosin d e r i v a t i v e e x h i b i t e d constant enzymic a c t i v i t y r e g a r d l e s s of the amount of enzyme bound. Green (1980) has r e c e n t l y drawn an i d e a l i z e d v e r s i o n of chymosin-agarose d e r i v a t i v e of Green and C r u t c h f i e l d (1969). This model suggested that the s t e r i c hindrance may have prevented even the surface of the c a s e i n m i c e l l e from making contact w i t h most of the enzyme molecules. The author concluded that s t e r i c hindrance i s l i k e l y to be a serious problem w i t h immobilized enzymes, p a r t i c u l a r l y i f the substrate i s l a r g e . The s t e r i c hindrance e f f e c t i s expected to be more pronounced i n the hexyl-ECD-Sepharose used i n t h i s study than i n the simple (noncross-linked) Sepharose of Green and C r u t c h f i e l d (1969). This i s due to the f a c t t h a t , the e p i c h l o r o h y d r i n c r o s s - l i n k i n g increases the s t a b i l i t y and the r i g i d i t y « of the g e l but, at the same time, decreases i t s p e r m e a b i l i t y , and t h i s w i l l be a s e r i o u s problem i n enzymatic treatment of high molecular weight substances (e.g. c a s e i n m i c e l l e s ) due to d i f f u s i o n a l l i m i t a t i o n In the s u b s t r a t e supply. Another reason f o r the low i n i t i a l a c t i v i t y e x h i b i t e d by the pepsin-hexyl-ECD-Sepharose 6B p r e p a r a t i o n may be masking of the a c t i v e center of a c e r t a i n number of enzyme molecules upon i m m o b i l i z a t i o n . These molecules become immediately i n a c t i v e and, t h e r e f o r e , do not c o n t r i b u t e to the a c t i v i t y of the immobilized p r e p a r a t i o n . The extremely f a s t i n a c t i v a t i o n of the pepsin-hexyl-ECD-Sepharose -6B d e r i v a t i v e could not be due to m i c r o b i a l growth, s i n c e p a s t e u r i z e d f r e s h 38. milk and low temperature (4 C) could not f a c i l i t a t e such a growth. Moreover, adsorption of milk proteins on the gel could also be eliminated as possible cause of the observed i n a c t i v a t i o n , since hexyl-ECD-Sepharose 6B i s a non-charge c a r r i e r and, thus, does not promote ..electrostatic i n t e r a c t i o n s . It i s suggested that one possible reason for the rapid i n a c t i v a t i o n observed may be denaturation of the enzyme d e r i v a t i v e . This denaturation may be due to c e r t a i n conformtional changes of the enzyme. Such changes could be due to a steady increase i n the number of attachment points, with a r e s u l t i n g increase i n binding s t a b i l i t y , as the molecule experiences free motion. A multipoint attachment w i l l , however, impose a s t r a i n on the conformation, with subsequent loss of a c t i v i t y (Caldwell ^ t a l . , 1976b). Caldwell et a l . (1976b) reported that the enzymatic decay suffered by the B-amylase adsorbate i n continuous operation could not only be ascribed to enzyme leakage, but also to a denaturation of the enzyme attached to hexyl-ECD-Sepharose 6B. Subsequently, Caldwell et a l . (1976d) investigated the e f f e c t of substituent density (on the gel) on the a c t i v i t y and s t a b i l i t y of the adsorbed enzyme. They found that hydrophobic immobilization of g-amylase on hexyl-ECD-Sepharose gels was optimally performed, as far as enzyme a c t i v i t y and s t a b i l i t y were concerned, with gels of a hexyl to galactose r a t i o around 0.5. ...A lower degree of s u b s t i t u t i o n (0.3) resulted i n lower a c t i v i t y and f a s t e r decay of enzymatic a c t i v i t y due to s u b s t a n t i a l enzyme leakage from the g e l . On the other hand, an adsorbate with higher degree of s u b s t i t u t i o n (0.7) also exhibited lower a c t i v i t y and less favorable operational s t a b i l i t y . It i s possible that the degree of s u b s t i t u t i o n (0.5) of the gel used i n t h i s study may have not been s a t i s f a c t o r y f or optimum a c t i v i t y and e s p e c i a l l y s t a b i l i t y of the pepsin 39. d e r i v a t i v e , r e s u l t i n g i n rapid loss i n a c t i v i t y by a gel induced denaturation. Observations i n d i c a t i n g that too many linkages to the supporting medium may be harmful to the retention of enzyme a c t i v i t y have been reported previously for covalently bound proteins by Datta et_ al (1973) and Zabriskie et al. (1973). They concluded that higher degrees of su b s t i t u t i o n , while advantageous for the creation of a strong i n t e r a c t i o n between the gel and the enzyme, might enhance denaturation and thus lead to lower a c t i v i t y y i e l d s . B. Immobilization of pepsin and chymosin on Phenoxyacetyl c e l l u l o s e Phenoxyacetyl c e l l u l o s e was chosen as support material i n t h i s study, because i t has been shown to be a unique medium for immobilizing enzymes and other proteins (Butler, 1975). Advantages of phenoxyacetyl c e l l u l o s e as a support material include (Butler, 1975): (1) hydrophobic binding of proteins to phenoxyacetyl c e l l u l o s e i s strong and e s s e n t i a l l y i r r e v e r s i b l e under most working conditions (Butler, 1975; Regis Chemical Co.); (2) enzymes immobilized on phenoxyacetyl c e l l u l o s e e x h i b i t nearly complete retention.of t h e i r c a t a l y t i c a c t i v i t y ; (3) preparation of the adsorbent ( c a r r i e r ) i s extremely f a c i l e , v e r s a t i l e , and inexpensive; (4) preparation of the enzyme-carrier conjugate i s convenient, simple and mild; (5) phenoxyacetyl c e l l u l o s e contains no charged groups, thus permitting true hydrophobic enzyme immobilization without i n t e r f e r i n g i o n i c e f f e c t s ; and. (6) immobilization of an enzyme on phenoxyacetyl c e l l u l o s e i s r e v e r s i b l e , desorption being accomplished through the use of nonionic detergents, and t h i s permits r e u s a b i l i t y of the c a r r i e r . From the differ e n c e i n absorbance at 280 nm of the enzyme so l u t i o n before and a f t e r immobilization (adsorption), i t was estimated that 4.8 mg 40. pepsin were adsorbed per g of phenoxyacetyl c e l l u l o s e ; t h i s represents binding e f f i c i e n c y of 80%. Results of pepsin and chymosin immobilized on phenoxyacetyl c e l l u l o s e are presented i n Table 2. As i t can be seen, both preparations showed high i n i t i a l a c t i v i t y on exposure to milk. However, the deactivation rates were very high. The nature of the los s i n c l o t t i n g a c t i v i t y i s shown i n Figure 1, which presents m i l k - c l o t t i n g a c t i v i t i e s of enzyme-phenoxyacetyl preparations during continuous exposure to skimmilk. It can be seen, that the enzyme i n a c t i v a t i o n occurred i n two stages: the f i r s t decay was rapid whereas the second decay was more gradual. Inactivation rates at d i f f e r e n t points of operation i n the reactor are expressed i n t h i s study as "D values" and are reported i n Table 2. Very low D values (8.5 hr for pepsin and 7.5 hr for chymosin) at 4 hr on the stream i n d i c a t e that these preparations were very unstable during the f i r s t phase of i n a c t i v a t i o n . Higher D values for both preparations at 14 and 20 hr on the stream i n d i c a t e that both became progressively more stable. Decay patterns s i m i l a r to those observed i n t h i s study with pepsin and chymosin ( i . e . two phase patterns) have been reported repeatedly for pepsin and c a l f rennet immobilized on d i f f e r e n t c a r r i e r s and used for continuous coagulation of milk (Cheryan et a l , , 1975b; Cheryan et a l . , 1976; Taylor et a l . , 1977). In these studies, the f i r s t rapid decay was at t r i b u t e d to deposition of protein and peptides on the immobilized protease. Accumulation of nitrogenous materials and s i a l i c acid p a r a l l e l e d the i n i t i a l rapid loss i n enzymic a c t i v i t y . Both whey proteins and casein appeared to be responsible for loss i n enzymic a c t i v i t y . The support materials used i n those studies (e.g. porous glass, alumina) were charged having a net p o s i t i v e charge at the pH of the milk which presumably 100 r ^ 6 0 h H 0 10 20 30 40 50 Time on st rea m ( hr F i g . 1. C l o t t i n g a c t i v i t y of pepsin and chymosin immobilized on phenoxyacetyl c e l l u l o s e f or the continuous coagulation of skimmilk: (•) pepsin; (A) chymosin. 42. f a c i l i t a t e d the adsorption of milk protein and peptides, which are negatively charged at the pH of milk (6.7). Causes of the second gradual enzymic decay of immobilized proteases remained elusive, although c e r t a i n whey components could be implicated. The semilogarithmic pl o t s of a c t i v i t y versus time obtained with pepsin - and chymosin - phenoxyacetyl c e l l u l o s e preparations suggest that two a c t i v i t y l o s s mechanisms were involved i n , the more rapid one a f f e c t i n g only a c e r t a i n f r a c t i o n of the enzyme. A number of possible i n a c t i v a t i o n causes can be eliminated. Thus, under the conditions of experiment ( i . e . operation at 4°C and use of fresh pasteurized milk) microbial growth could not account for the r e l a t i v e l y rapid i n a c t i v a t i o n rates observed. Moreover, i n a c t i v a t i o n due to adsorption of proteinaceous material on the phenoxyacetyl c e l l u l o s e can be ruled out, since phenoxyacetyl c e l l u l o s e i s a non-charged c a r r i e r and, therefore, does not f a c i l i t a t e e l e c t r o s t a t i c i n t e r a c t i o n s with the negatively charged milk proteins. One obvious reason responsible for the f i r s t rapid decay of enzymic a c t i v i t y seems to be the leakage (desorption) of enzyme from the c a r r i e r , since soluble a c t i v i t y , which i s an i n d i c a t i o n of enzyme desorption, was detected i n both enzyme preparations (see numbers i n parenthesis i n Table 2). As i t can be seen from Table 2, about 10% of the a c t i v i t y exhibited by the immobilized preparations at 4 and 14 hr on the stream was due to desorbed enzyme. Soluble a c t i v i t i e s were also observed for milk samples taken at subsequent time i n t e r v a l s (20 and 30 hr on the stream), but these a c t i v i t i e s were small leading to long c l o t t i n g times, and thus re q u i r i n g time-consuming and tedious measurements. That i s why these soluble a c t i v i t i e s were not exactly timed and, therefore, are not included i n Table 2. Although enzyme binding to phenoxyacetyl c e l l u l o s e has been reported to be 43. i r r e v e r s i b l e under most working conditions (Butler, 1975; Regis Chemical Co.), i t i s evident from the r e s u l t s of Table 2 that enzyme was l o s t from the immobilized systems. According to Pitcher (1980b) the enzyme can be l o s t from a system because of desorption, severing of chemical bonds or erosion of the support material. A portion of the enzyme leakage observed here may have been due to changes i n pH, i o n i c strength and temperature, since hydrophobic binding i s influenced by these f a c t o r s . For example, as the pH increases and temperature decreases the hydrophobic i n t e r a c t i o n diminishes. The e f f e c t of these changes, i f any, i s expected to be more profound during the i n i t i a l stage of the operation. Although phenoxyacetyl c e l l u l o s e i s claimed as a medium being r e l a t i v e l y i n s e n s i t i v e to such changes (Regis Chemical Co.), i t i s possible that changes i n pH, i o n i c strength or temperature may lead to the desorption of the weakly (loosely) bound enzyme molecules, since not a l l of the enzyme molecules are equally well bound to the c a r r i e r due to i t s usually nonhomogeneous surface. Another mechanism by means of which the enzyme has been l o s t from the phenoxyacetyl c e l l u l o s e d e r i v a t i v e s seems to be the leakage of the ligand from the c a r r i e r , since Carr and Bowers (1980) commented that the use of phenoxyacetyl c e l l u l o s e as a matrix i s l i m i t e d mainly by the r e l a t i v e l y rapid degradation of the ester bond (between phenoxyacetyl groups and c e l l u l o s e ) r e s u l t i n g i n a slow leakage of the enzyme from the r e s i n . Such leakage of the ligand from the c a r r i e r i s a very common problem and has been con s i s t e n t l y reported for derivatives of cyanogen bromide-activated Sepharose used as c a r r i e r s f o r enzyme immobilization (Matsumoto et a l . , 1979). A t h i r d mechanism which may be p a r t l y responsible for the observed leakage of enzyme from the c a r r i e r i s the competitive displacement of the enzyme from the c a r r i e r (held through hydrophobic in t e r a c t i o n s ) by c e r t a i n 44. milk proteins. It seems, therefore, that the f i r s t rapid decay i n a c t i v i t y observed here was probably due to both physical desorption of loo s e l y bound enzyme and slow leakage of the ligand-enzyme complex from the c a r r i e r . The second decay of enzymic a c t i v i t y was probably due only to the continued slow leakage of the ligand-enzyme complex from the c a r r i e r . C. Immobilization of pepsin on activated carbon Activated carbon has a long h i s t o r y as support material i n enzyme immobilization and a r e l a t i v e l y great number of papers have appeared on i t s use (Cho and Bailey, 1977, 1978, 1979; Gol'dfel'd et a l . , 1966; Nelson and G r i f f i n , 3916; Nelson and Hitchcock, 1921; Tosa et a l . , 1966). Its usefulness i s due to the f a c t that activated carbon has many a t t r a c t i v e properties f or use as immobilized enzyme support. Activated carbon i s cheap, possesses mechanical strength, and can be obtained i n several forms including porous structures with a v a r i e t y of pore s i z e 3 d i s t r i b u t i o n s (Cho and Bailey, 1977). I t s skeleton density (0.75 g/cm ) i s such that activated carbon immobilized enzymes can be f l u i d i z e d much easier than enzymes immobilized on other inorganic supports such as 3 porous glass of which skeleton density i s about 2.4 g/cm . Furthermore, activated carbon i s already employed i n numerous food, medical, and f i n e chemical processing operations, so that the material i s r e l a t i v e l y f a m i l i a r i n these i n d u s t r i e s . Questions of possible secondary influences on process performance owing to the enzyme support are therefore minimized, enhancing a c c e p t a b i l i t y of the immobilized enzyme system (Cho and Bailey, 1978). Activated carbon i s a highly porous carbonaceous material, prepared by carbonizing and a c t i v a t i n g organic substances of mainly 45. vegetable o r i g i n s . T y p i c a l l y , a c t i v a t i o n s are conducted by chemicals, CO^, 0^ or steam at high temperatures. Boehm et a l . (1964) detected carboxylic groups, phenolic hydroxyl group, and other oxides on the carbon surfaces. Two types of activated carbon (untreated and HCl-treated) and two immobilization procedures were investigated i n t h i s study. Treatment of activated carbon with HC1 was intended to clean the carbon p e l l e t s and thus improve access to external and i n t e r n a l surface. This washing step was to be followed by treatment with n i t r i c a cid, an o x i d i z i n g agent. The motivation for such oxidative pretreatment was derived from the postulated chemistry for diimide-mediated enzyme immobilization (Weetall, 1975). This process involves peptide bond formation between enzyme amino groups and carboxyl groups on the carbon surface. Therefore, by increasing the number of carboxyl groups on the carbon surface, oxidative pretreatment may increase the enzyme loading. It should be noted at t h i s point, however, that treatment of HCl-washed activated carbon with n i t r i c acid had an adverse e f f e c t , since i t resulted i n immediate c l o t t i n g as the milk came i n contact with the n i t r i c acid-treated carbon i n the column. This c l o t t i n g was probably due to r e s i d u a l n i t r a t e ions s t i l l e x i s t i n g within the carbon pores, despite the excessive washing with water. Moreover, n e u t r a l i z a t i o n of n i t r i c acid-treated carbon before use as a c a r r i e r did not have any b e n e f i c i a l e f f e c t i n avoiding the immediate c l o t t i n g within the column. As a r e s u l t , the n i t r i c acid treatment was discontinued and thus i s not included i n Table 2. Adsorption of pepsin was c a r r i e d out at pH 1.2, which i s close to i t s i s o e l e c t r i c pH, since i t has been repeatedly reported that the maximum adsorption of proteins occurs at t h e i r i s o e l e c t r i c point (Armstrong and Chesters, 1964; Brash and Lyman, 46. 1971; McLaren, 1954). Moreover, Taylor et^ a l . (1977) have reported that pepsin was best adsorbed on alumina (a charged c a r r i e r ) at pH 1.2 and that i o n i c a t t r a c t i o n was not the strong force contributing to the adsorption. Adsorption of pepsin on activated carbon at pH 1.2 i s due to hydrophobic i n t e r a c t i o n s between the hydrophobic surface of activated carbon and hydrophobic groups present on the surface of pepsin. The binding e f f i c i e n c y of activated carbon could not be determined due to the black color of the washings a f t e r immobilization. Since i t has been reported that the enzyme loadings obtained with activated carbon are very s i m i l a r to those obtained with porous glass (Cho and Bailey, 1977), 100 mg of pepsin were used for immobilization. Results of pepsin immobilized on activated carbon are presented i n Table 2. It i s evident that, covalent binding of pepsin on HCl-treated carbon (TAC) produced the c a t a l y s t with the highest i n i t i a l a c t i v i t y , presumably because of the higher enzyme loading achieved by the carbodiimide immobilization process. On the other hand, covalent binding of pepsin on untreated activated carbon (UAC) gave a c a t a l y s t which inact i v a t e d l e s s r a p i d l y . Adsorption of pepsin on UAC produced i n i t i a l a c t i v i t y higher than the covalent binding of pepsin on the same type of carbon (probably because of increased enzyme desorption), but i n a c t i v a t e d more r a p i d l y . A l l enzyme-carriers preparations were p e r i o d i c a l l y s t e r i l i z e d by r i n s i n g with 0.05 M hydrogen peroxide with no loss i n enzymatic a c t i v i t y . This, i n conjunction with the low temperature (4°C) of operation of the reactor and the use of fresh pasteurized milk, excluded the possible i n a c t i v a t i o n of the preparations due to microbial growth. Moreover, i n a c t i v a t i o n due to adsorption of proteinaceous material on the carbon was rejected, because the carbon was negatively charged at the pH of the milk 47. and, therefore, repelled rather than adsorbed the negatively charged milk proteins and peptides. The i n a c t i v a t i o n of a l l pepsin-activated carbon c a t a l y s t s was a t t r i b u t e d to the desorption of enzyme since soluble a c t i v i t i e s were detected i n a l l cases. The contribution of soluble enzyme to the c l o t t i n g a c t i v i t y of each preparation was r e l a t i v e l y greater for adsorption than for covalent immobilization (Table 2). In other words, the portion of enzymic a c t i v i t y (exhibited by each preparation) due to desorbed enzyme was greater for the adsorbed than for the covalently bound enzymes probably due to the l e s s stable binding obtained with adsorption. However, desorption of even covalently bound enzymes has been repeatedly reported i n the l i t e r a t u r e (Stanley and Olson, 1974; Taylor et a l . , 1977). On the other hand, the desorption of adsorbed pepsin was probably due to the increase i n the pH (from 1.2 to 6.7), although the preparations were equ i l i b r a t e d at pH 6.7 before being exposed to milk. The adsorption-desorption mechanism of pepsin on activated carbon may be as follows. At pH 1.2 hydrophobic a f f i n i t y was very strong between pepsin and activated carbon due to the f a c t that a l l carboxyl groups were not d i s s o c i a t e d . Thus, the enzyme molecules were adsorbed by hydrophobic binding on l o c i of the carbon each of which included at l e a s t one hydrophobic group. As the pH was increased from 1.2 to 6.7, the carboxyl groups of the adsorbing l o c i i n the activated carbon were gradually converted to the d i s s o c i a t e d form, simultaneously reducing the hydrophobicity. At pH 6.7, the enzyme was retained on the carbon by the remaining hydrophobic a f f i n i t y (due to phenolic groups) minus the e l e c t r o s t a t i c repulsion (produced by the dissociated carboxyl groups). The above adsorption-e l u t i o n mechanism of enzymes constitutes the p r i n c i p l e of "hydrophobic-i o n i c chromatography" described by Sasaki et a l . (1979). 48. Looking at the Figures 2 and 3 as well as Table 2, i t can be seen that the i n a c t i v a t i o n rates were i n i t i a l l y high and subsequently l e v e l l e d o f f . These observed differences i n the i n a c t i v a t i o n rates were probably due to the sequence of enzyme desorption. Thus, enzyme molecules adsorbed on the surface may have been desorbed f a s t e r than those adsorbed within the pores of the carbon. It also i s possible that the strength of adsorption of d i f f e r e n t enzyme molecules was varying due to nonexistence of homogeneous d i s t r i b u t i o n of hydrophobic binding s i t e s on the carbon surface. This e f f e c t could give r i s e to loosely bound enzyme molecules, which would desorb quickly, and strongly bound enzyme molecules which would desorb l e s s r a p i d l y . Taylor jit al_. (1977) have also reported that most desorption of pepsin (about 45%), immobilized by p h y s i c a l adsorption on alumina at pH 1.2 and used f o r continuous coagulation of milk, took place during the f i r s t 12 hr on the stream and that a f t e r 25 hr s l i g h t l y more than ha l f (50%) the pepsin had desorbed. The above assumption of the desorption of adsorbed pepsin because of the increase i n the pH of the medium i s supported by the findings of Kikawa (1926) who reported that pepsin was best adsorbed on animal charcoal at pH 1 or 2, and that the adsorbed enzyme was leached from the charcoal by a phosphate s o l u t i o n of pH 6.8. D. Economics of using immobilized proteases f o r the continuous coagulation  of skimmilk Besides the obvious saving i n labor costs and improved e f f i c i e n c y expected when an enzyme i s immobilized (due to the conversion of the process from batch i n t o continuous), the major consideration w i l l be the savings i n enzyme costs (Cheryan, 1974). The operational s t a b i l i t y and h a l f - l i f e 49. 4 0 r c E 0) E c o o 0.2 0 10 20 3 0 40 5 0 Time on st ream ( h r ) F i g . 2. C l o t t i n g a c t i v i t y of pepsin-untreated activated carbon preparations used i n the continuous coagulation of skimmilk: ( • ) adsorption; (•) covalent binding. 50. F i g . 3. C l o t t i n g a c t i v i t y of pepsin-HCl-treated activated carbon preparations used i n the continuous coagulation of skimmilk: ( • ) adsorption; (•) covalent binding. 51. of the immobilized enzyme have a profound importance i n t h i s respect, since the p o t e n t i a l for reuse of the c a t a l y s t cannot be r e a l i z e d unless the immobilized enzyme i s s u f f i c i e n t l y stable under operational conditions. If the immobilized enzyme must be con t i n u a l l y replaced, then l i t t l e w i l l be gained by the use of an immobilized enzyme process, except an increase i n cost (Trevan, 1980). The net outcome of an immobilized enzyme process should be to produce a product which i s les s expensive than a comparable product produced by a soluble enzyme process. Table 3 shows the c a l c u l a t i o n of the economics of using proteases immobilized on hydrophobic supports f o r the continuous coagulation of skimmilk i n cheese manufacture. This economic study was based on determining the amount of enzyme required to coagulate 1 L of skimmilk i n 10 min at 30°C, using both the soluble and insolu b l e (immobilized) form of the same enzyme. Since the secondary phase of coagulation i s r e l a t i v e l y i n a c t i v e at the normal pH of milk (6.7), the assays for c l o t t i n g a c t i v i t y were performed with the pH of the treated milk (effluent) decreased to 6.1, where the coagulation times were much f a s t e r . As i t can be seen from Table 3, the best enzyme preparation among those tested was attained with phenoxyacetyl c e l l u l o s e as c a r r i e r . The l i m i t of 10 min c l o t t i n g time at 30°C (considered as the maximum desirable c l o t t i n g time for evaluation purposes) was reached a f t e r 33 hr of continuous operation of the pepsin c a t a l y s t at a flow rate of 36 ml/hr. It was calculated that 6.5 and 5.5 L of skimmilk was coagulated with 15 mg pepsin and 300 mg rennin (N.F.), r e s p e c t i v e l y . The c l o t t i n g e f f i c i e n c i e s of these c a t a l y s t s were 2.7 and 3.1 times lower than those of the soluble forms of pepsin and rennin, r e s p e c t i v e l y . As f a r as the activated carbon i s concerned, the best preparation was obtained with pepsin covalently bound to HC1-52. treated activated carbon (TAC). It should be noted here, however, that the d i r e c t comparison of phenoxyacetyl c e l l u l o s e and activated carbon preparations i n terms of t h e i r e f f i c i e n c i e s as c a t a l y s t s for continuous coagulation may be inappropriate, since t h e i r c l o t t i n g e f f i c i e n c i e s were calculated on the basis of the amount of enzyme added i n the immobilization step and not on the amount of enzyme bound by the c a r r i e r . This i s due to the i n a b i l i t y of spectrophotometrical determination of the binding e f f i c i e n c y of activated carbon as already mentioned because of the black color of the washings a f t e r immobilization. It should be pointed out that d i f f e r e n t types of activated carbon e x i s t e x h i b i t i n g d i f f e r e n t surface properties r e s u l t i n g from d i f f e r e n t preparative methods (Garten and Weiss, 1957). I t seems l o g i c a l , therefore, that i f the pepsin binding e f f i c i e n c y of Darco activated carbon used i n t h i s study was lower .than the expected average of 20 mg pepsin/g carbon, then the use of 100 mg pepsin must have been a waste of enzyme. There are two major factors that could improve the economics of using immobilized milk c l o t t i n g enzymes (Cheryan, 1974). These are: (1) optimization of the y i e l d or complete immobilization of the enzyme on each c a r r i e r ; and (.2) d i l u t i o n of the treated milk as i t comes out of the reactor with fresh untreated milk, which would g r e a t l y increase output ( i . e . amount of milk that could be coagulated). Even a f t e r taking both of the above factors into consideration, the data i n Table 3 suggest that the continuous coagulation of skimmilk with proteases immobilized on the hydrophobic supports studied i s not economically a t t r a c t i v e . 53. E. General Discussion and Conclusions In p r a c t i c e , the use of an immobilized enzyme for continuous coagulation of skimmilk depends on several c r i t i c a l points including separation of the enzymic and c l o t t i n g phases so that c l o t t i n g does not occur i n the enzyme reactor, high enzymatic a c t i v i t y , s u f f i c i e n t l y long retention of enzymatic a c t i v i t y under operating conditions, freedom from microbial growth, and production of a normal product ( F e r r i e r et a l . , 1972; Taylor et a l . , 1979). Furthermore, use of a cheap enzyme c a r r i e r that would not adsorb deactivating proteinaceous materials, and employment of a mild, simple (one step) and inexpensive immobilization procedure are also important considerations contributing to the economics of an immobilized system. In t h i s study, both cheap and non-charged (phenoxyacetyl c e l l u l o s e ) or negatively charged (activated carbon) c a r r i e r s e s s e n t i a l l y not adsorbing proteinaceous material were used. In addition, the simplest, mildest and cheapest immobilization method a v a i l a b l e ( i . e . adsorption) was mainly employed. Separation of the enzymic and c l o t t i n g stages was accomplished by using low temperature and milk at i t s normal pH. A l l enzyme-carrier preparations exhibited high i n i t i a l a c t i v i t y . The major problem encountered was the rapid reduction i n enzymatic a c t i v i t y . ' The main reason of t h i s rapid c a t a l y s t i n a c t i v a t i o n appeared to be the loss of enzyme from the c a r r i e r s , since soluble a c t i v i t y was detected i n a l l enzyme-carrier preparations. The enzyme loss was due to the ph y s i c a l desorption of enzyme from the c a r r i e r (activated carbon), mainly caused by the increase i n pH, as well as to the r e l a t i v e l y rapid leakage of the ligand from the c a r r i e r (phenoxyacetyl c e l l u l o s e ) . This desorption of the enzyme into the eff l u e n t milk, however, should not pose a problem since the soluble form 54. of the enzyme i s inactivated during cheese manufacture ( c i t e d i n Taylor et a l . , 1977). The best enzyme preparation (based on the amount of enzyme used i n the immobilization step) was obtained with phenoxyacetyl c e l l u l o s e as the c a r r i e r . According to the data presented i n t h i s study i t i s concluded that continuous coagulation of milk with proteases immobilized on the hydrophobic c a r r i e r s studied appears to be economically unfeasible. Further studies on other c a r r i e r s and better (more e f f i c i e n t ) immobilization procedures that w i l l give c a t a l y s t s of greater a c t i v i t y and e s p e c i a l l y s t a b i l i t y are necessary to allow more favorable economic projections. The need f or solving the problem created by the shortage of rennet i s so acute and the advantages of a continuous coagulation process are so great that any further attempt to assess the economic f e a s i b i l i t y of any immobilized enzyme system for the continuous coagulation of milk i s believed to be absolutely j u s t i f i a b l e . 54a. REFERENCES 1. Angelo, I. A. and Shahani, K. M. 1979. Coagulation of milk with rennet immobilized on Sepharose-4B. J . Dairy S c i . 62 (suppl. 1): 64. 2. Arima, S., Shimazaki, K., Yamazumi, T. and Kanamaru, Y. 1974. Preparation on insoluble d e r i v a t i v e s of rennin. Preliminary report. Coupling of rennin with Sepharose and aminoethylcellulose. 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P a r i s , France. 80. Trevan, M. D. 2980. Immobilized enzymes - An introduction and applications i n biotechnology. John Wiley and Sons, N.Y. 81. Tosa, T., Mori, T., Fuse, N. and Chibata, I. 1966. Studies on continuous enzyme reactions. 1. Screening of c a r r i e r s f o r preparation of water-insoluble aminoacylase. Enzymologia 31:214. 60 . 82. Vis s e r , J . and Stra t i n g , M. 1975. Enzymatic behaviour of lipoamide dehydrogenase isoenzymes immobilized on N-alkyl Sepharose matrices. FEBS L e t t . 57:183. 83. Weetall, H. H. 1975. Immobilization by covalent attachment and by entrapment. In "Immobilized Enzymes f o r I n d u s t r i a l Reactors". Ed. Messing, R. A. Academic Press, N.Y. 84. Zabriskie, D., O l l i s , D. F. and Burger, M. M. 1973. A c t i v i t y and s p e c i f i c i t y of covalently immobilized wheat germ ag g l u t i n i n toward c e l l surfaces. Biotechnol. Bioeng. 15:981. Chapter II A Simple Turbidimetric Method for Determining  the Fat Binding Capacity of Proteins 62. INTRODUCTION The a b i l i t y of proteins to bind fa t i s a very important f u n c t i o n a l property for such applications as meat replacers and extenders, p r i n c i p a l l y because i t enhances f l a v o r retention and reputedly improves mouthfeel ( K i n s e l l a , 1976). The key rol e ( s ) of fat i n food f l a v o r i n g has been i l l u s t r a t e d by K i n s e l l a (1975) and i t s capacity to improve f l a v o r carry-over i n simulated foods during processing i s apparent. Soy proteins have been added to ground meats to promote fat absorption or f a t binding, and thus decrease cooking losses and maintain dimensional s t a b i l i t y i n the cooked product (Wolf and Cowan, 1975). Fat separation i s a well known major problem i n processed meat-in-sauce-or-gravy type products. This problem can be prevented by incorporating into these products (canned or frozen meat/sauce products) a combination of soy protein ingredients ( i . e . an extruded soy protein concentrate, a soy protein i s o l a t e and l e c i t h i n ) designed to emulsify, bind, and s t a b i l i z e f a t s (Morris, 1980). On the other hand, i n some foods such as pancakes and doughnuts, the addition of soy f l o u r helps prevent excessive absorption during f r y i n g (Johnson, 1970). Fat absorption of proteins i s usually measured by adding excess l i q u i d f a t ( o i l ) to a protein powder, thoroughly mixing and holding, centrifuging and determining the amount of bound or absorbed o i l - t o t a l minus free (Lin et a l . , 1974; Wang, and K i n s e l l a , 1976). The amount of o i l and protein sample, kind of o i l , holding and centrifu g i n g conditions, and units of expression have varied s l i g h t l y from one inv e s t i g a t o r to another (Hutton and Campbell, 1981). The mechanism of fat absorption i s not cl e a r . However, Wang and 63. K i n s e l l a (1976) have a t t r i b u t e d f a t absorption, as assessed by the above method, mostly to physi c a l entrapment of the o i l ; i n support of t h i s a c o r r e l a t i o n c o e f f i c i e n t of 0.95 was found between bulk density and fat absorption by a l f a l f a leaf proteins. Chemical modification of protein, which increases bulk density concomitantly enhances fat absorption (Franzen, 1975). The objective of t h i s part of the th e s i s , was to develop a simple method for determining the a b i l i t y of proteins to bind f a t . The development of the method and a comparison of the fat binding capacities of several food proteins w i l l be presented. MATERIALS AND METHODS A. Materials Bovine serum albumin (#A-4503), 3-lactoglobulin (#L-6879 from milk) and ovalbumin (//A-5503) were a l l purchased from Sigma Chemical Co., St. Louis, Mo. Soy protein i s o l a t e was obtained from General M i l l s , Inc., Minneapolis, Min. Promine-D was purchased from Central Soya Co., Chicago, I I . Pea protein i s o l a t e (M 412-046), Century c u l t i v a r f i e l d pea, was received from POS P i l o t Plant Corp., Uni v e r s i t y of Saskatchewan, Saskatoon, Sask. Rapeseed protein i s o l a t e and sunflower protein i s o l a t e were prepared by the method of Nakai et a l . (1980). G e l a t i n , Bloom 300, was purchased from United States Biochemical Corp., Cleveland, Oh. Whey protein concentrate (75%) was obtained from Sodispro Technol., St. Hyacinthe, Que. Whole casein was prepared by the following method: approximately 4.5 L of raw milk was heated u n t i l i t reached a temperature 64. of 45°C, centrifuged at 4,100 x g for 10 min and cooled 20 min at 20°C. The upper f a t layer was discarded and the lower skimmilk portion was d i l u t e d with water on a 1:1(V/V) basis. The d i l u t e d sample was heated to 40°C and the pH was adjusted to 4.6 with 1 N HC1. The mixture was f i l t e r e d through cheese c l o t h , the casein p r e c i p i t a t e was washed with water and the excess water was squeezed from the cheese c l o t h . The p r e c i p i t a t e d casein was dissolved i n water and the pH was adjusted to 7.75 so that the casein was t o t a l l y s o l u b i l i z e d . The s o l u t i o n was heated to 40°C and the p r e c i p i t a t i o n with HC1 was repeated. The casein p r e c i p i t a t e was dissolved i n water and the pH was adjusted to 7.0 at which the casein was t o t a l l y dissolved. The so l u t i o n was centrifuged at 4,100 x G for 10 min and the supernatant was c o l l e c t e d and freeze-dried. — Corn o i l was from Fisher S c i e n t i f i c Company, F a i r Lawn, N.J. Urea, ACS reagent, 99+%, was obtained from A l d r i c h Chemical Company, Inc., Milwaukee, Wis. Metaphosphoric acid was from J . T. Baker Chemical Co., P h i l l i p s b u r g , N.J. B. Methods (a) Fat binding capacity determination To 40 mg of freeze-dried protein sample i n a 15 ml glass centrifuge tube 1.5 ml of corn o i l was added. The contents were s t i r r e d and sonicated, with a Braun-Sonic 1510 sonicator (Braun Instruments, San Francisco, Ca) f i t t e d with a needle probe, at 100 watts for 1 min to disperse the protein sample. Af t e r holding at room temperature for 30 min the tube was centrifuged at 3,020 x G for 20 min. The free o i l was pipetted o f f and 2 ml of d i s t i l l e d water was added. O i l adhered to the sides of the tube was removed by scraping the sides with a glass rod. 65. Then, i n order to remove any o i l that might have been entrapped i n the form of f i l m beneath the protein p r e c i p i t a t e , the p r e c i p i t a t e was gently scraped from the bottom of the tube and any o i l found was taken to the top ( i . e . surface of water) with the glass rod. Subsequently, 1 ml of 0.1 N metaphosphoric acid (pH 2.1) was added and the tube was centrifuged at . 4,340 x g for 15 min. The supernatant was pipetted o f f . The p r e c i p i t a t e was, then, c a r e f u l l y washed with d i s t i l l e d water (3 - 4 ml) without dispersing i t . The supernatant was pipetted o f f . F i n a l l y the tube walls were cleaned with a disposable (paper) wiper (to remove any trace of o i l , i f any existed). 0.3 ml of d i s t i l l e d water was added and mixed well with the glass rod. A digestion medium of 20 ml of 7 M urea i n 50% H^SO^ was measured into a graduated c y l i n d e r . An aliq u o t of about 2 ml of t h i s d i g e s t i o n medium was added into the tube, the contents were mixed well with a glass rod, and then transferred into an Omni-mixer homogenizing chamber. The centrifuge tube was washed twice with about 2 ml of digestion medium. These washings and the remainder of the digestion medium i n the graduated cy l i n d e r were poured into the homogenizer chamber. The mixture was homogenized for 30 sec at speed s e t t i n g 1, and then poured into a 50 ml beaker. The sample was held for 30 min at room temperature and then the absorbance was taken at 600 nm i n a Spectronic 20 (Bausch and Lomb, Rochester, N.Y.), spectrophotometer with a round cuvette against the diges-t i o n medium. The absorbance was stable for at least 1 hr. The volume (ml) of o i l bound was determined from the standard curve. The protein content of the combined supernatants (#2 and #3) was, subsequently, determined by the Phenol-Biuret method (Brewer et a l . , 1974) to c a l c u l a t e the amount of protein l o s t i n these supernatants during the handling of the p r e c i p i t a t e . The amount of l o s t p r o t e i n was converted to the amount of the o r i g i n a l sample, 66. since the p r o t e i n content of the sample was known, and t h i s value was subtracted from the 40 mg of the s t a r t i n g sample. This c a l c u l a t i o n gave the amount of o i l i n ml bound by the corrected amount of protein sample. The f a t binding capacity of the sample (expressed as %) was then calculated as the volume of o i l i n ml bound by 100 g of protein sample. The standard curve was constructed as follows: To 40 mg of soy protein i s o l a t e i n a 30 ml beaker, increasing amounts of corn o i l were added (0 to 100 y l ) . While mixing with a glass rod, 0.3 ml of d i s t i l l e d water was added (to f a c i l i t a t e mixing) followed by 20 ml of digestion medium (7 M urea i n 50% R^SO^) and further mixing. The mixture was transferred i n t o an Omni-mixer chamber and homogenized for 30 sec at speed se t t i n g 1, and then poured into a 50 ml beaker. The sample was held for 30 min at room temperature and the absorbance was then taken at 600 nm i n a Spectronic 20 with a round cuvette against the digestion medium. (b) Protein (surface) hydrophobicity determination Protein surface hydrophobicity was f l u o r o m e t r i c a l l y determined according to the method of Kato and Nakai (1980) a f t e r s l i g h t modification. Each protein sample (2 ml) was s e r i a l l y d i l u t e d with 0.01 M phosphate buffer, pH 7.4, to obtain protein concentrations ranging from 0.00156% to 0.05%. Two sets of protein samples were prepared ( i . e . two tubes for each protein concentration). Ten y l of c i s - p a r i n a r i c a c i d s o l u t i o n were added only to one set of tubes. The p a r i n a r i c acid-protein conjugate was then excited at 325 nm and the r e l a t i v e fluorescence i n t e n s i t y was measured at 420 nm i n an Aminco-Bowman spectrofluorometer, using s l i t width of 0.5 mm. The method was standardized by adjusting the r e l a t i v e fluorescence i n t e n s i t y reading of the fluorometer to 7.4/10 f u l l scale (by turning the s e n s i t i v i t y knob) when 10 y l of c i s - p a r i n a r i c acid s o l u t i o n was added to 2 ml of decane. Then, the fluorescence readings of the protein samples were taken. The net fluorescence i n t e n s i t y at each protein concentration was determined by subtracting the fluorescence i n t e n s i t y of each sample without c i s - p a r i n a r i c acid from the fluorescence i n t e n s i t y of the corresponding sample containing c i s - p a r i n a r i c acid. The i n i t i a l slope ( S q ) of the fluorescence i n t e n s i t y vs. protein concentration p l o t was used as an index of the protein surface hydrophobicity. The i n i t i a l slope was determined by l i n e a r regression analysis using a Monroe (Orange, N.J.) 1880 programmable c a l c u l a t o r . (c) S o l u b i l i t y index determination Protein samples (1%, w/v, i n 0.01 M phosphate buffer pH 7.4) were dispersed by s t i r r i n g with a magnetic s t i r r e r f o r 5 min and then blended i n a Sorval Omnimixer at speed s e t t i n g 5 for 1 min. The pH of each dispersion was adjusted to 7.4 by adding 1 N NaOH. For 100% soluble proteins the blending step was eliminated. A portion of each protein suspension was then centrifuged at 27,000 x G for 30 min. Aliquots of the suspension and the supernatant a f t e r c e n t r i f u g a t i o n were d i l u t e d and the protein contents were determined by the Phenol-Biuret method (Brewer et a l . , 1974). The percent s o l u b i l i t y index (s) was taken as the r a t i o of the protein content of the supernatant to that of the suspension. (d) S t a t i s t i c a l analysis Simple and multiple l i n e a r regression analyses were done by using a Monroe 1880 programmable c a l c u l a t o r . Backwards stepwise multiple regression analysis and surface v i s u a l i z a t i o n p l o t t i n g were done at the 68. Un i v e r s i t y of B r i t i s h Columbia using an Amdahl 470 V/8 computer. Independent v a r i a b l e s used i n the backwards stepwise regression analysis included surface hydrophobicity ( S q ) , s o l u b i l i t y index (s) , i n t e r a c t i o n ofSQ and s, and quadratic powers of S q and s. RESULTS AND DISCUSSION A. Fat binding capacity Wavelength dependence of t u r b i d i t y Figure 1 shows the e f f e c t of wavelength on absorbance of o i l - p r o t e i n systems. Plot 1 was obtained by mixing 40 mg soy protein i s o l a t e (used as a standard protein) with 50 y l corn o i l and 20 ml 7 M urea i n 50% H^SO^ (used as a digestion medium) with a glass rod, homogenizing the mixture for 30 sec, holding f or 30 min at room temperature and then scanning with a Beckman DB spectrophotometer. Plot 2 was obtained by scanning the same sample with a Spectronic 20 spectrophotometer. Plots 3 and 4 were obtained using the Spectronic 20 instrument when protein or o i l was omitted from the mixture, r e s p e c t i v e l y . As i t can be seen, the absorbance values read from the Beckman DB spectrophotometer were higher than those measured using the Spectronic 20 spectrophotometer. This i s probably due to the d i f f e r e n t cuvette-to-photodetector distance and thus to the d i f f e r e n t angle of acceptance of the l i g h t by the photodetector as suggested i n a study of turbidimetry by Pearse and K i n s e l l a (1978). It i s also evident from Figure 1 that as the wavelength was increased the absorbance by a l l samples decreased. In the present study a wavelength of 600 nm was used because of the n e g l i g i b l e absorbance by the protein. Moreover, the 69. F i g . 1. E f f e c t of wavelength on absorbance. 70. Spectronic 20 spectrophotometer with a round cuvette was chosen over the Beckman DB spectrophotometer since the l a t t e r gave nonreproducible absorbance values. A s i m i l a r phenomenon was observed by Nakai and Le (1970) who a t t r i b u t e d the a b i l i t y of round c e l l s to give reproducible readings to t h e i r focusing e f f e c t . T u r b i d i t y dependence on blending time The e f f e c t of blending time during f a t binding capacity determination on the absorbance at 600 nm i s shown i n Figure 2. A gradual r i s e i n absorbance and then attainment of a steady state with increasing blending time was observed. In t h i s study a blending time of 30 sec was chosen. Standard curve for FBC determination The standard curve obtained i s shown i n Figure 3. The regression equation was Y = 0.008 X + 0.012, where Y i s the absorbance at 600 nm and X i s the amount of bound o i l i n ul.' The c o r r e l a t i o n c o e f f i c i e n t (r) was 0.9996 and the standard error of estimate (S ) 0.008. y .x Comments on the method for FBC determination A flow diagram of the developed method i s shown i n Figure 4. The use of 0.1 N metaphosphoric acid was necessary f o r preventing s o l u b i l i z a t i o n , thereby l o s i n g soluble proteins (e.g. 3-lactoglobulin, BSA, whey, casein) during t h e i r resuspension i n water (after the f i r s t c e n t r i f u g a t i o n ) . I n i t i a l l y , other protein p r e c i p i t a n t s (e.g. ethanolic solutions, mercuric chl o r i d e , acetate buffer pH 4.6, 8% s i l i c o t u n g s t i c acid i n 1.2 M p e r c h l o r i c acid) were added a f t e r protein resuspension to p r e c i p i t a t e the s o l u b i l i z e d protein. These p r e c i p i t a n t s were found, however, to be unsuitable since 71. Fig. 2. Effect of blending time on absorbance at 600 nm. 0 2 0 4 0 6 0 8 0 100 A m o u n t o f o i l ( u l ) F i g . 3. Standard curve for fat-binding determination. 73. 40 mg protein sample +1.5 ml corn o i l Sonicate f o r 1 min I Hold f o r 30 min @ R.T. I Centrifuge at 3,020 x g for 20 min Supernatant #1 Protein ppt. (free o i l ) | Add 2 ml H o0 I 2 Gently l i f t ppt. i d d 1 ml 0.1 N HP0 o I 3 Centrifuge at 4,340 x g for 15 min Supernatant #2 Protein ppt (free o i l + H 20) Wash with H 20 Supernatant #3 Protein ppt (free o i l + H 20) | Add 0.3 M H.O and mix I 2 Add 2 ml dig. medium and mix well I Transfer tube content into homog. chamber Wash tube twice with 2 ml dig. med. i Add 16 ml dig. med. I Homogenize f or 30 sec I Transfer i n t o beaker and hold for 30 min J a k e A600nm Calculate F.B.C. Figure 4 - Flow diagram for the determination of FBC of proteins 74. the p r o t e i n p r e c i p i t a t e obtained was very f i r m and not d i s p e r s i b l e by the subsequent homogenization step or because of the nonreproducible absorbance reading obtained ( s i l i c o t u n g s t i c a c i d ) . T r i c h l o r o a c e t i c a c i d (TCA) i n high concentrations (10 - 12%) was found e f f e c t i v e i n p r e c i p i t a t i n g the soluble proteins tested and, t h e i r p r e c i p i t a t e s were e a s i l y dispersed i n the digestion medium. However, when 10% TCA was used for FBC determination of r e l a t i v e l y i n s o l u b l e proteins (e.g. soy p r o t e i n ) , the protein p r e c i p i t a t e obtained was very firm and d i f f i c u l t to disperse. Another problem associated with the use of TCA as a protein p r e c i p i t a n t i s that TCA possesses a hydrophobic group, and therefore, i t may react with hydrophobic s i t e s of the protein thus releasing some of the bound o i l . The use of protein p r e c i p i t a n t s may probably be c r i t i c i z e d because many of them (such as TCA, p i c r i c a c i d , s a l i c y l s u l p h o n i c acid) cause protein denaturation (Perlmann and Herrmann, 1938), which influence the f a t binding by proteins. Thus, metaphosphoric aci d , a known strong protein p r e c i p i t a n t , was used. It was found very e f f i c i e n t i n p r e c i p i t a t i n g soluble proteins (causing instantaneous formation of a p r e c i p i t a t e ) , and moreover, a l l protein p r e c i p i t a t e s obtained were e a s i l y dissolved by the digestion medium. However, the main advantage of metaphosphoric acid as a protein p r e c i p i t a n t i s the f a c t that metaphosphoric acid has been repeatedly shown not to cause pro t e i n denaturation (Briggs, 1940; Perlman, 1938; Perlmann and Herrmann, 1938). Briggs (1940) concluded that the metaphosphate-protein reaction can be regarded as a. complex .in which the.negative multivalent (polymerized) metaphosphate ion i s linked to the p o s i t i v e (amino) groups of the protein by a s a l t - l i k e bond of very low d i s s o c i a t i o n tendency ( i . e . i o n i z i n g capacity). Thus, when the s o l u t i o n containing protein and metaphosphate i s dialyzed at pH>7, the metaphosphate 75. i s r e a d i l y removed and the protein i s obtained with a l l of i t s o r i g i n a l properties unchanged (Briggs, 1940). The protein-metaphosphoric acid complex i s also e a s i l y s o l u b i l i z e d by s a l t addition (Perlmann and Herrmann, 1938). After homogenizing and holding t h e p r o t e i n sample for 30 min (to allow the a i r bubbles to r i s e to the surface), some proteins (e.g. soy protein) formed a t h i n usually broken foam layer f l o a t i n g on the sample surface. In t h i s case, an aliquot was taken for absorbance measurement by dipping a pasteur pipette through a hole of the foam layer into the sample dispersion. If the foam layer was not well broken, the beaker containing the sample dispersion was s l i g h t l y i n c l i n e d . Thus, the layer was moved to a d i r e c t i o n opposite to that of the i n c l i n a t i o n , allowing the taking of an aliquot without disturbing the foam layer. The formation of the foam layer was due to the big volume of the homogenizer's container (100 ml) r e l a t i v e to that of the sample (20 ml) allowing the incorporation of a i r into the sample. It s formation, therefore, can be avoided by using a small container (e.g. 25 ml). B. Comparison of the FBC of various proteins Fat absorption of proteins i s affected by the p r o t e i n source, extent of processing and/or composition of protein, p a r t i c l e s i z e , and temperature (Hutton and Campbell, 1981). The FBC values for some food proteins are given i n Table 1. As i s seen, soy protein i s o l a t e and sunflower protein i s o l a t e bound more o i l than the other proteins tested On the other hand, 3-lactoglobulin bound the least amount of o i l among a l l the proteins tested. The FBC of the proteins i n Table 1 was determined by using the equation derived from the standard curve without any Table 1 - Relationship between protein hydrophobicity, s o l u b i l i t y index, and fat binding capacity of various proteins. Hydrophobicity S o l u b i l i t y index (%) b c FBC (%) Protein Sample <V (s) I II Ovalbumin 6.0 100.0 37.7 560.0 Casein 28.0 100.0 10.1 95.0 Soy protein i s o l a t e 95.0 26.4 105.9 161.0 Promine D 39.0 29.1 85.3 175.0 Pea protein i s o l a t e 66.0 42.6 92.3 145.0 Gelatin 5.0 15.3 19.1 100.0 Sunflower protein i s o l a t e 47.0 31.0 105.8 230.0 Rapeseed protein i s o l a t e 55.0 44.0 66.2 140.0 Whey protein 182.0 88.7 52.2 220.0 Bovine serum albumin 325.0 100.0 25.0 340.0 3-Lactoglobulin 426.0 100.0 4.2 210.0 Average of duplicate determinations ' l : Determined according to the new method described here I I : Determined according to the method of Wang and K i n s e l l a (1976) %: ml oil/100 g sample (dry weight) c o r r e c t i o n , since the reagent blank (protein plus digestion medium) of d i f f e r e n t proteins had an absorbance value very close to 0.025 (absorbance of reagent blank of soy protein i s o l a t e used as a standard protein f or construction of the standard curve). In the case of rapeseed protein i s o l a t e and Promine D (another commercial soy protein i s o l a t e ) , however, since t h e i r reagent blanks had absorbance values of 0.9 and 0.05, respectively, a c o r r e c t i o n was made to compensate for these excessive blank (>0.025) absorbances. Thus, 0.065 (=0.09 - 0.025) and 0.025 (=0.05 - 0.025) were subtracted from the absorbances observed for rapeseed and Promine D, res p e c t i v e l y , and then, these net absorbance values were entered into the equation of the standard curve for FBC determination. Table 1 also includes the FBC values of the same food proteins determined by the method of L i n et a l . (1974) a f t e r i t s s l i g h t modification by Wang and K i n s e l l a (1976). It i s noteworthy that ovalbumin and (3-lactoglobulin had very high FBC values while the present method yielded considerably lower values. C. S t a t i s t i c a l analysis Regression analysis was used to quantify the r e l a t i o n s h i p between FBC of 11 food proteins and various independent v a r i a b l e s . Simple l i n e a r regression analysis showed no s i g n i f i c a n t c o r r e l a t i o n between protein surface hydrophobicity ( S Q ) and FBC. M u l t i p l e l i n e a r regression analysis of S q , s o l u b i l i t y (s) and FBC also did not show any s i g n i f i c a n t c o r r e l a t i o n . However, when simple l i n e a r regression analysis was applied to c o r r e l a t e S q and FBC of only 8 proteins of Table 1 ( i . e . g-lactoglobulin, B S A and whey protein were excluded), the c o e f f i c i e n t of determination was 2 s i g n i f i c a n t (r = 0.6191, p < 0.05). The regression equation was 78. FBC(%) = 22.78+0.9976S , and the standard error of estimate (S ) was o xy 22.21. This c o r r e l a t i o n can be seen i n Figure 5. Moreover, backwards stepwise regression analysis between FBC and various independent variables showed a highly s i g n i f i c a n t c o r r e l a t i o n 2 between S , i n t e r a c t i o n of S and s (S x s ) , and FBC (R = 0.8017, o o o P < 0.01). A multiple regression model for p r e d i c t i o n of S q and x s e f f e c t s on FBC i s presented i n Table 2. FBC was p o s i t i v e l y affected by S whereas the i n t e r a c t i o n of S with s o l u b i l i t y had a negative e f f e c t o o on i t . The 3 (normalized c o e f f i c i e n t ) values i n t h i s model suggest that both S and S x s were equally important i n determining the FBC o o 2 2 of these proteins. The r ( c o e f f i c i e n t of determination) and R ( c o e f f i c i e n t of multiple determination) values ind i c a t e the percentage of v a r i a t i o n i n a dependent va r i a b l e accounted for by i t s regression on the independent v a r i a b l e or v a r i a b l e s , r e s p e c t i v e l y ; the higher the value, the greater the a c c o u n t a b i l i t y . Comparing simple and multiple regression models i n terms of t h e i r a b i l i t y to more accurately predict the FBC of proteins, i t i s obvious that the l a t t e r i s the model of choice, since 80.17% of the v a r i a t i o n i n FBC could be accounted for by the multiple regression model of Table 2 (as opposed to 61.91% of the v a r i a t i o n i n FBC of only 8 proteins accounted for by the simple regression model). Figure 6 shows the response surface contour of the FBC (of the 11 food proteins of Table 1) as a function of S q and s o l u b i l i t y index. As i t can be seen, the iso-response l i n e with the highest FBC value corresponds to r e l a t i v e l y medium S Q (75 - 125) and low s o l u b i l i t y (20 - 48%) values. As S decreased below or increased above these values the FBC o decreased. A l l the above r e s u l t s , therefore, suggest that S q plays a very important r o l e i n the f a t binding process. F i g . 5. Relationship between hydrophobicity (S ) and f a t binding capacity of food proteins. 1, ovalbumin; 2, casein; 3, soy protein i s o l a t e ; 4, Promine D; 5, pea protein i s o l a t e ; 6, g e l a t i n ; 7, sunflower p r o t e i n i s o l a t e ; 8, rapeseed p r o t e i n i s o l a t e . Table 2 - Multiple regression model for pr e d i c t i o n of fat binding capacity of various food proteins. Dependent Variable Regression F - r a t i o F - p r o b a b i l i t y Beta v a r i a b l e description c o e f f i c i e n t value Fat binding capacity (R2=0.8017; Standard error of estimate = 19.01; F-pro-b a b i l i t y = 0.0015) S x s o Constant 1.381 -0.014 30.271 21.77 25.91 8.41 0.002 0.001 0.020 5.034 -5.492 0.793 n = 11 Fig. 6. Fat binding capacity response surface contour as a function of hydrophobicity ( S Q ) and solubility index (s). 82. It should be noted that no c o r r e l a t i o n (simple or multiple of any form) was found for FBC of the same food proteins determined by the method of Wang and K i n s e l l a (1976) with S or S and s. o o The f i n d i n g by t h i s study that high protein s o l u b i l i t y negatively affected the FBC of proteins has some resemblance with the r e s u l t s of the work of Torgersen and Toledo (1977) who correlated p h y s i c a l properties of proteins with t h e i r f unctional c h a r a c t e r i s t i c s i n comminuted meat systems. They found a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t between s o l u b i l i t y and f a t binding (fat release on cooking), which meant that the more soluble the protein additives the lesser the f a t binding properties of the system to which these protein preparations were used. Moreover, Dippold (1961) reported that a doughnut mix containing 4% soy f l o u r of high s o l u b i l i t y (NSI of 80%) absorbed about 50% l e s s f a t than; the same mix containing 4% soy f l o u r of low s o l u b i l i t y (NSI of 60%). Although the aforementioned studies were conducted on complex food systems and the f a t binding capacity determined d i f f e r e n t l y (as f a t release or absorption on cooking), and therefore a d i r e c t comparison with the f i n d i n g i n t h i s study may be inappropriate, i t i s l i k e l y that high s o l u b i l i t y of proteins has an adverse e f f e c t on t h e i r f a t binding c a p a b i l i t y . One possible reason for the adverse e f f e c t of high s o l u b i l i t y on the FBC of proteins observed i n t h i s study i s the conformation of the soluble proteins (BSA, 8-lactoglobulin, whey protein) which does not permit t h e i r binding s i t e s (hydrophobic side chains) to be s t e r i c a l l y a v a i l a b l e for i n t e r a c t i o n with the o i l (hydrocarbon chains). The explanation i s supported by the f a c t that BSA, B-lactoglobulin (Pham and Nakai, 1981) and whey proteins (Morr, 1979) have mainly a - h e l i c a l conformation as opposed to the random or B-pleated sheet conformation of soy protein 83. (Wolf, 1972). Another reason may be a l i m i t e d access of o i l to hydrophobic s i t e s of soluble (100%) proteins due to the presence of an excessive number of polar groups forming a b a r r i e r around the surface hydrophobic groups (of p r o t e i n ) . The f i n d i n g of t h i s study that with increasing S q the FBC i s increased and subsequently decreased (at high S q values) may be explained by taking i n t o considerat ion the suggestion of Wolf and Cowan (1975) that fat absorption may be another aspect of e m u l s i f i c a t i o n , since i n ground meat products f a t absorption or binding appears to involve formation and s t a b i l i z a t i o n of an emulsion. According to Aoki jit al. (1981), however, the emulsifying properties of proteins ultimately depend on the s u i t a b l e balance between hydrophile and l i p o p h i l e , and do not n e c e s s a r i l y increase as the proteins become more l i p o p h i l i c . These s i t u a t i o n s are s i m i l a r to the concept of the required HLB (hydrophile-lipophile balance) values of f a t s and the HLB values of surfactants for e m u l s i f i c a t i o n . D. Mechanism of fat absorption Factors a f f e c t i n g the p r o t e i n - l i p i d i n t e r a c t i o n include protein conformation, protein-protein i n t e r a c t i o n s , and the s p a t i a l arrangement of the l i p i d phase r e s u l t i n g from the l i p i d - l i p i d i n t e r a c t i o n (Hutton and Campbell, 1981). Non-covalent bonds, such as hydrophobic, e l e c t r o s t a t i c , and hydrogen, are the forces involved i n the p r o t e i n - l i p i d i n t e r a c t i o n s . Hydrogen bonding i s of secondary importance i n l i p i d - p r o t e i n complexes, although i t i s i n d i r e c t l y important i n hydrophobic bonding (Karel, 1973), since i n aqueous media the water-water i n t e r a c t i o n s by hydrogen bonding i s much stronger than the i n t e r a c t i o n between water and nonpolar groups, thus giving r i s e to hydrophobic bonding between nonpolar groups. E l e c t r o s t a t i c 84. a t t r a c t i o n can occur between the negatively charged phosphate groups of phospholipids and p o s i t i v e l y charged protein groups (such as l y s y l or guanidyl) or between a p o s i t i v e l y charged group i n the phospholipid (e.g. choline) and a negatively charged amino acid side chain (e.g. a s p a r t y l ) . A r e l a t e d mode of binding i s the formation of s a l t bridges between a negatively charged amino acid side chain and a negatively charged phosphate group of a phospholipid v i a divalent calcium or other metal ions (Karel, 1973; Pomeranz, 1973; Ryan, 1977). Hydrophobic bonding i s l i k e l y to play a major r o l e i n s t a b i l i z i n g the i n t e r a c t i o n s of both polar and nonpolar l i p i d s with proteins (Ryan, 1977). Moreover, nonpolar dispersion or Van der Waals forces become important when i n t e r -acting groups are near (Karel, '1973) and'may play a r o l e i n a t t r a c t i o n between nonpolar groups i n systems i n which hydrophobic bonds are impossible because of l i m i t e d water (Pomeranz1973). As with the protein-protein i n t e r a c t i o n s , i t i s not possible to a t t r i b u t e p r o t e i n - l i p i d i n t e r a c t i o n s to any single s p e c i f i c kind of molecular force (Ryan, 1977). However, according to Wall (1979) l i p i d s bind to proteins mainly through association with hydrophobic groups. In the method described i n t h i s study e l e c t r o s t a t i c a t t r a c t i o n does not seem to play any r o l e i n l i p i d - p r o t e i n i n t e r a c t i o n , since the o i l used was refined and so should have a n e g l i g i b l e amount of phospholipids. The 2 fa c t that a highly s i g n i f i c a n t c o e f f i c i e n t of determination (R = 0.8017) was observed between S , S x s, and FBC of the food proteins tested o o suggest that S i s a major determinant of FBC of proteins. 85. CONCLUSIONS A simple turbidimetric method was developed for determining the f a t binding capacity (FBC) of various proteins. The t u r b i d i t y was dependent on wavelength, blending time and volume of o i l . A highly s i g n i f i c a n t c o r r e l a t i o n (R = 0.8017, P < 0.01) was found between S , S x s, and FBC o o of 11 food proteins tested. Advantages of t h i s method include: (1) the small amount (40 mg) of sample required as compared to 4 g (Sosulski ^ t aJL., 1976), 1 g (Sathe and Salunkhe, 1981), or 0.5 g (Lin et a l . , 1974; Wang and K i n s e l l a , 1976) required by the e x i s t i n g methods; and (2) the f a t absorption, as determined by the present method, can be at t r i b u t e d to binding of o i l by the protein and not to the entrapment. 86. REFERENCES 1. Aoki, H., Taneyama, D. , Orimo, N. and Kitagawa, I. 1981. E f f e c t of l i p o p h i l i z a t i o n of soy protein on i t s emulsion s t a b i l i z i n g properties. J . Food S c i . 46:1192. 2. Brewer, J . M., Pesce, A. J. and Ashworth, R. B. 1974. Experimental Techniques i n Biochemistry. P r e n t i c e - H a l l , Inc., Englewood C l i f f s , N.J. 3. Briggs, D. R. 1940. The metaphosphoric acid-protein reaction. J . B i o l . Chem. 134:261. 4. Dippold, M. W. 1961. Cited by Johnson, D. W. 1970. Functional properties of o i l s e e d proteins. J . Amer. O i l Chem. Soc. 47:402. 5. Franzen, K. 1975. Cited by K i n s e l l a , J . E. 1976. Functional properties of proteins i n foods: a survey. C r i t . Rev. Food S c i . Nutr. 7:219. 6. Hutton, C. W. and Campbell, A. M. 1981. Water and f a t absorption. In "Protein F u n c t i o n a l i t y i n Foods". Ed. Cherry, J . P. ACS Symposium Series 147, Amer. Chem. S o c , Wash., D.C. 7. Johnson, D. W. 1970. Functional properties of o i l s e e d proteins. J . Amer. O i l Chem. Soc. 47:402. 8. Karel, M. 1973. P r o t e i n - l i p i d i n t e r a c t i o n s . J . Food S c i . 38:756. 9. Kato, A. and Nakai, S. 1980. Hydrophobicity determined by a fluorescence probe method and i t s c o r r e l a t i o n with surface properties of proteins. Biochim. Biophys. Acta 624:13. 10. K i n s e l l a , J . E. 1975. Butter f l a v o r . Food Technol. 29(5):82. 11. K i n s e l l a , J . E. 1976. Functional properties of proteins i n foods: a survey. C r i t . Rev. Food S c i . Nutr. 7:219. 12. L i n , M. J . Y., Humbert, E. S. and Sosulski, F. W. 1974. Certain functional properties of sunflower meal products. J . Food S c i . 39:368. 13. Morr, C. V. 1979. Conformation and f u n c t i o n a l i t y of milk proteins. In " F u n c t i o n a l i t y and protein structure". Ed. Pour-El, A. Amer. Chem. Soc. Symposium Series 92, Washington, D.C. 14. Morris, C. E. 1980. Soy protein system binds f a t . Food Eng. 52(9):28. 15. Nakai, S., Ho, L., Tung, M. A. and Quinn, J . R. 1980. S o l u b i l i z a t i o n of rapeseed, soy and sunflower protein i s o l a t e s by surfactant and proteinase treatments. Can. Inst. Food S c i . Technol. J . 13:14. 16. Nakai, S. and Le, A. C. 1970. Spectrophotometry determination of protein and f a t i n milk simultaneously. J . Dairy S c i . 53:276. 87. 17. Pearse, K. N. and K i n s e l l a , J . E. 1978. Emulsifying properties of proteins: evaluation of a turbidimetric technique. J . Agr i c . Food Chem. 26:716. 18. Perlmann, G. 1938. On the preparation of c r y s t a l l i z e d egg albumin metaphosphate. Biochem. J. 32:931. 19. Perlmann, G. and Herrmann, H. 1938. On the reaction between metaphosphoric acid and egg albumin. Biochem. J . 32:926. 20. Pham, A.-M. and Nakai, S. 1981. Contributions of s t e r i c parameters to f u n c t i o n a l i t y of food proteins. 1. P r e d i c t i o n program of secondary structure from protein sequence according to the method of Chou and Fasman. 24th Annual Conference, Can. Inst. Food S c i . Technol., paper no. 30. 21. Pomeranz, Y. 1973. Interaction between g l y c o l i p i d s and wheat f l o u r macromolecules i n breadmaking. Adv. Food Res. 20:153. 22. Ryan, D. S. 1977. Determinants of the fun c t i o n a l properties of proteins and protein derivatives i n foods. In "Food Proteins. Improvement through chemical and Enzymatic Modifications". Eds. Feeney, R. E. and Whitaker, J . R. Adv. Chem. Ser. 160, Amer. Chem. Soc., Wash., D.C. 23. Sathe, S. K. and Salunkhe, D. K. 1981. Functional properties of the great northern bean (Phaseolus v u l g a r i s L.) proteins: emulsion, foaming, v i s c o s i t y , and gelation properties. J . Food S c i . 46:71. 24. Sosulski, F., Humbert, E.S., Bui, K. and Jones, J . D. 1976. Functional properties of rapeseed f l o u r s , concentrates and i s o l a t e . J . Food S c i . 41:1349. 25. Torgersen, H. and Toledo, R. T. 1977. Physical properties of protein preparations r e l a t e d to t h e i r f u n c t i o n a l c h a r a c t e r i s t i c s i n comminuted meat systems. J . Food S c i . 42:1615. 26. Wall, J . S. 1979. Properties of proteins contributing to func-t i o n a l i t y of cereal foods. Cereal Foods World 24(7):288. 27. Wang, J . C. and K i n s e l l a , J . E. 1976.Functional properties of novel proteins: a l f a l f a l e a f protein. J . Food S c i . 41:286. 28. Wolf, W. J . 1972. P u r i f i c a t i o n and properties of the proteins. In "Soybeans: Chemistry and Technology". Eds. Smith, A. K. and C i r c l e , S. J . AVI Publishing Co., Westport, Conn. 29. Wolf, W. J . and Cowan, J . C. 1975. Soybeans as a food source. CRC Press, Inc., Cleveland, Ohio. Chapter III Relationships of Hydrophobicity to Emulsifying Properties of Heat Denatured Proteins 89. INTRODUCTION To be useful and successful i n food a p p l i c a t i o n s , proteins i n addition to providing e s s e n t i a l amino acids, should i d e a l l y possess several desirable c h a r a c t e r i s t i c s referred to as fun c t i o n a l properties (Wang and K i n s e l l a , 1976). Moreover, according to Johnson (1970) the fu n c t i o n a l and physical properties, rather than the n u t r i t i o n a l value, of protein i n protein-containing products w i l l l a r g e l y determine t h e i r a c c e p t i b i l i t y as ingredients i n prepared foods. Functional properties of proteins connote those physicochemical properties which a f f e c t the behaviour of proteins i n food systems during preparation, processing, storage and consumption ( K i n s e l l a , 1979). These properties r e f l e c t the composition and conformation of the proteins, t h e i r i n t e r a c t i o n s with other food components, and they are affected by processing treatments and the environment ( K i n s e l l a , 1979; K i n s e l l a and Shetty, 1979). The a b i l i t y of protein to aid the formation and s t a b i l i z a t i o n of emulsions i s c r i t i c a l for many applications i n chopped, comminuted meats, cake batters, coffee whiteners, milks, mayonnaise, salad dressings, and frozen desserts. In these products varying emulsifying and s t a b i l i z i n g c a p a cities are required because of the d i f f e r i n g composition and stresses to which these products are subjected ( K i n s e l l a , 1979). Moreover, the a b i l i t y of proteins to bind f a t s i s a very important functional property for such applications as meat replacers and extenders, p r i n c i p a l l y because i t enhances f l a v o r retention and reputedly improves mouthfeel ( K i n s e l l a , 1976). Soy proteins have been added to ground meats to promote f a t absorption or f a t binding, and thus decrease cooking losses and maintain dimensional s t a b i l i t y i n the cooked product (Wolf and Cowan, 1975). 90. To evaluate the emulsifying properties of a protein i t s s o l u b i l i t y p r o f i l e i s usually determined, because of i t s usefulness as an excellent index of protein f u n c t i o n a l i t y ( K i n s e l l a , 1976). Good s o l u b i l i t y can markedly expand p o t e n t i a l applications of a protein ( K i n s e l l a , 1976). Denaturation, on the other hand, implicates loss of protein f u n c t i o n a l properties and i s usually.measured as a loss of s o l u b i l i t y (Nakai and Powrie, 1981). Generally, surfactant properties are related to the aqueous s o l u b i l i t y of proteins ( K i n s e l l a , 1976). A p o s i t i v e c o r r e l a t i o n between s o l u b i l i t y and the a b i l i t y of a protein to emulsify and s t a b i l i z e an emulsion has been reported'.in many studies (Crenwelge et: a l . , 1974; Pearson et a l . , 1965; Swift and Sulzbacher, 1963; Volkert and K l e i n , 1979; Yasumatsu et a l . , 1972). Many authors point to evidence, however, that emulsifying properties and s o l u b i l i t y are not well correlated (Aoki et a l . , 1980; McWatters arid Cherry, 1975; McWatters and Holmes, 1979a, 1979b; Smith et a l . , 1973; Wang and K i n s e l l a , 1976). Thus, Smith and coworkers (1973) found that no s i g n i f i c a n t c o r r e l a t i o n existed between s o l u b i l i t y and emulsifying capacity or emulsion s t a b i l i t y of the proteins studied. Wang and K i n s e l l a (1976) reported that the emulsifying capacity of a l f a l f a l e a f proteins showed no s i g n i f i c a n t c o r r e l a t i o n with the s o l u b i l i t y , since these proteins showed maximum emulsifying capacity at pH 5 where the protein s o l u b i l i t y was at the lowest l e v e l . The same authors, moreover, suggested that proteins remaining soluble i n t h i s pH region had a higher emulsifying capacity than those s o l u b i l i z e d above and below t h i s pH region. McWatters and Cherry (1975) and McWatters and Holmes (1979a, 1979b) also found that high l e v e l s of nitrogen s o l u b i l i t y were not nec e s s a r i l y associated with maximum emulsifying capacity. Moreover, Aoki et a l . , (1980) reported that the pH-emulsion s t a b i l i t y 91. p r o f i l e of soy protein (11S protein r i c h f r a c t i o n ) did not correspond to i t s p H - s o l u b i l i t y p r o f i l e . The protein hydrophobicity has been l a t e l y r e c e i v i n g much attention since the hydrophobic i n t e r a c t i o n s are considered to play important r o l e s i n the fun c t i o n a l properties of food proteins (Kato et a l . , 1981; K i n s e l l a , 1979). Keshavarz and Nakai (1979) reported a s i g n i f i c a n t c o r r e l a t i o n between surface hydrophobicity (determined by hydrophobic chromatography and hydrophobic p a r t i t i o n techniques) and i n t e r f a c i a l tension of the proteins studied. Kato and Nakai (1980) subsequently reported that the surface hydrophobicity (determined f l u o r o m e t r i c a l l y ) showed s i g n i f i c a n t c o r r e l a t i o n s with i n t e r f a c i a l tension aid emulsifying a c t i v i t y of the proteins studied. Their r e s u l t s suggest that the em u l s i f i c a t i o n of o i l with protein can be explained using the concept of protein hydrophobicity. Nakai et a l . (1980b) also reported that the e f f e c t i v e (surface)hydrophobicity showed good c o r r e l a t i o n s with i n t e r -f a c i a l tension and emulsifying a c t i v i t y of the plant proteins studied. It i s noteworthy that these authors observed a closer c o r r e l a t i o n of em u l s i f i c a t i o n capacity with hydrophobicity than with s o l u b i l i t y . Kato et a l . (1981) reported that the emulsifying properties of ovalbumin and lysozyme were markedly improved by p a r t i a l denaturation r e s u l t i n g from heat - and SDS - treatments, and were l i n e a r l y correlated with surface hydrophobicity. Moreover, Voutsinas and Nakai (.1981), using a new turjbidimetric method they developed, found that surface hydrophobicity was correlated with f a t binding capacity of the food proteins studied. While many factors influence the performance of proteins i n food systems, heat treatment i s one of the most important and i s very often used during the processing of protein products. This study was i n i t i a t e d 92. to elucidate the observation that the emulsifying capacity of soy protein was not adversely affected even by t e x t u r i z a t i b n which caused a loss i n s o l u b i l i t y . The objectives of t h i s part of the thesi s , therefore, were to determine the e f f e c t of heating on the emulsifying properties of selected food proteins, and, to assess the value of surface hydrophobicity as a predictor of the emulsifying properties of these proteins (because of the aforementioned contradictions among s c i e n t i s t s with respect to value of s o l u b i l i t y as an index of the emulsifying properties of a pr o t e i n ) . LITERATURE REVIEW A. S o l u b i l i t y S o l u b i l i t y i s an experimentally measurable property that can y i e l d information about the fun c t i o n a l behaviour as well as the physico-chemical nature of the proteins (Shen, 1981). Bigelow (1967) proposed that two s t r u c t u r a l features, namely charge frequency and hydrophobicity, are the factors which influence protein s o l u b i l i t y . The higher the charge frequency and the lower the hydrophobicity, the higher would be the s o l u b i l i t y . S o l u b i l i t y i s , also, affected by a magnitude, of fac t o r s , namely, protein source (Sosulski and Bakal, 1969), p a r t i c l e s i z e of the product (Johnson, 1970), processing h i s t o r y , minor and major treatments i n i t s preparation and processing, heating ( K i n s e l l a , 1976), protein concentration (Betschart, 1974; K i n s e l l a , 1976), presence of other ingredients such as s a l t s (Hermansson, 1973; M a t t i l , 1971) and carbohydrates (Tybor'et'al., 1975), and the experimental conditions of s o l u b i l i t y determination, e.g., pH (Hermansson, 1973; Kodagoda et a l , 93. 1973), temperature (Hermansson, 1973; Shen, 1976), mixing procedures and c e n t r i f u g a l force (Hermansson, 1973). B. Hydrophobicity Much e f f o r t has been made to quantify the hydrophobic character of proteins due to the importance of hydrophobic i n t e r a c t i o n s for t h e i r s t a b i l i t y , conformation and function (Bigelow, 1967; Tanford, 1962). Ea r l y studies were mainly concerned with the t o t a l hydrophobicity of protein (Tanford, 1962), calculated as the sum of the side chain hydrophobicities of a l l residues (constituent amino a c i d s ) , or the average hydrophobicity (Bigelow, 1967), calculated from the t o t a l hydrophobicity divided by the number of residues. However, as Melander and Horvath (1977) pointed out, protein functions such as s o l u b i l i t y and chromatographic behaviour depend on the hydrophobic surface properties, because the nonpolar residues buried i n the i n t e r i o r of the native protein are not believed to a f f e c t d i r e c t l y these phenomena. Therefore, the above mentioned parameters are not expected to y i e l d information about the surface hydrophobicity, which i s l i k e l y to be of great b i o l o g i c a l and technological s i g n i f i c a n c e (Melander and Horvath, 1977). In most native proteins, some hydrophobic groups remain exposed at the molecular surface or i n crevices (Tanford, 1972), and the hydrophobic side chains occur more frequently on the surface of the protein that had been assumed (Klotz, 1970). Thus, many attempts have l a t e l y been made to determine the e f f e c t i v e or surface hydrophobicity of proteins that correlates well with the propensity of protein molecule to p a r t i c i p a t e i n hydrophobic i n t e r a c t i o n s . Keshavarz and Nakai (1979) applied hydrophobic a f f i n i t y 94. chromatography and hydrophobic p a r t i t i o n to determine the e f f e c t i v e hydrophobicity of various proteins. However, the former method i s time consuming and the l a t t e r s u f f e r s from the low s o l u b i l i t y of some proteins i n the phase systems used. Therefore, development of a simple quantitative method f o r protein e f f e c t i v e hydrophobicity determination was highly desirable. Thus, Kato and Nakai (1980) used a fluorescent probe, c i s -p a r i n a r i c a c i d , to determine the e f f e c t i v e hydrophobicity of proteins. c i s - P a r i n a r i c a c i d i s a natural polyene f a t t y a c i d , thus, i t can r e a d i l y simulate natural l i p i d - p r o t e i n i n t e r a c t i n g systems (Nakai and Powrie, 1981). According to Sklar et al_. (1976), c i s - p a r i n a r i c acid possesses the advantages of the fluorescent probe techniques ( i . e . great detection s e n s i t i v i t y , s e n s i t i v i t y of probes to t h e i r environment, and the large number of parameters that can be monitored continuously and on a rapid time s c a l e ) , while minimizing the disadvantages ( i . e . perturbations and i n a b i l i t y to predict the l o c a t i o n of the probe). Since the structure and the conformation of c i s - p a r i n a r i c acid c l o s e l y resemble those of normally occurring, c i s - p a r i n a r i c a c i d i s expected to cause minimal perturbations i n the systems, and moreover, i t s l o c a t i o n and o r i e n t a t i o n r e l a t i v e to the surroundings i s predictable (Sklar et a l . , 1976). c i s - P a r i n a r i c acid i s p r a c t i c a l l y non-fluorescent i n water (quantum y i e l d , Q < 0.001) but fluoresces with d i f f e r e n t quantum y i e l d s i n organic solvents or aqueous solutions of d i f f e r e n t proteins. Thus, several l i n e s of evidence support the notion that hydrophobic i n t e r a c t i o n s are responsible for the enhancement of c i s - p a r i n a r i c acid fluorescence. They include: (1) the increases i n quantum y i e l d with decreases i n the d i e l e c t r i c constant of the solvent (Sklar e t / a l . , 1977); (2) the high r e l a t i v e fluorescence values observed i n solutions of proteins (e.g. bovine serum albumin, 95. 3-lactoglobulin, K-casein) known to possess hydrophobic binding s i t e s as opposed to low values obtained with solutions of other proteins such as ovalbumin, conalbumin, etc. (Kato and Nakai, 1980); and (3) the increase i n the fluorescent i n t e n s i t y observed a f t e r denaturation of proteins, e.g. ovalbumin and lysozyme (Kato and Nakai, 1980; Kato et^ al., 1981). The good c o r r e l a t i o n (r = 0.97) observed by Kato and Nakai (1980) between e f f e c t i v e hydrophobicity determined f l u o r o m e t r i c a l l y and e f f e c t i v e hydrophobicity of the same proteins determined by the hydrophobic p a r t i t i o n method, suggests that t h e i r fluorescence probe method can be r e l i a b l y used for quantitative estimation of protein e f f e c t i v e hydro-phobicity. The fluorescence probe method has the following advantages: (1) i t i s much simpler and quicker than the hydrophobic chromatography and hydrophobic p a r t i t i o n techniques reported by Keshavarz and Nakai (1979). Thus, f or one analysis of protein e f f e c t i v e hydrophobicity, the fluorescence probe method requires only 10 min compared to approximately 2 hr for the hydrophobic p a r t i t i o n and 5 hr for the hydrophobic chromato-graphy (due to slow e f f l u e n t flow r a t e ) ; and (2) i t i s one of few methods that can be used f o r determining the e f f e c t i v e hydrophobicity of insoluble proteins. C. Formation and s t a b i l i z a t i o n of emulsions An emulsion can be most simply defined as a dispersion of one l i q u i d i n another with which i t i s immiscible. In an emulsion, the dispersed droplets are commonly referred to as the dispersed, d i s -continuous or i n t e r n a l phase, and the medium i n which they are dispersed i s the continuous or external phase. The most common method for 96. preparing an emulsion i s by mechanically dispersing one bulk l i q u i d phase i n another. Emulsions of f a t s and water are thermodynamically unstable because of the p o s i t i v e free energy caused by i n t e r f a c i a l tension ( K i n s e l l a , 1979). Thus, an emulsion w i l l r a p i d l y separate into two d i s t i n c t phases upon standing unless a t h i r d phase, an adsorbed surfactant, i s present i n the i n t e r f a c e to s t a b i l i z e i t (Morr, 1981). Proteins are examples of hydrocolloids that exhibit unique surfactant properties due to t h e i r large molecular weights and t h e i r m u l t i p l i c i t y of hydrophobic and hydro-p h i l i c residues, each of which exhibit a spectrum of a f f i n i t i e s for the polar and nonpolar phases i n the emulsion system (Morr, 1981). The amino acid composition and sequence, as well as the secondary, t e r t i a r y and quaternary structure, are major factors which govern t h e i r effectiveness as surfactants (Powrie and Tung, 1976). The major way to s t a b i l i z e an emulsion i s to develop an energy b a r r i e r which prevents coalescence (Karel, 1973). According to Karel (1973), the mechanisms of the a c t i v a t i o n b a r r i e r common i n foods are: (1) Film formation at the oil-water i n t e r f a c e with strong s t e r i c hindrance to coalescence (association of i n d i v i d u a l d r o p l e t s ) . The strength, compactness, and e l a s t i c i t y of i n t e r f a c i a l films around droplets greatly influence the s t a b i l i t y of an emulsion (Powrie and Tung, 1976). Cumper and Alexander (1950) proposed that f i l m formation by proteins at an i n t e r f a c e occurs i n three stages: (a) d i f f u s i o n of protein molecules to the i n t e r f a c e where they are adsorbed i n the globular form. Yamauchi et a l . (1980) considered that the adsorption of protein at f i r s t occurs by means of hydrophobic i n t e r a c t i o n s between the protein and a f a t surface; (b) surface denaturation by u n c o i l i n g of the adsorbed globular 97. protein; and (c) aggregation of the unrolled polypeptide chain into a coagulum l a r g e l y devoid of surface a c t i v i t y and consequently forced out of the i n t e r f a c e by the spreading pressure of the native protein under-going surface denaturation. The k i n e t i c s of f i l m formation i s very much influenced by the composition-conformation of the protein, v i s c o s i t y of the protein dispersion, pH, ions, temperature and energy input (Tornberg, 1978). (2) E l e c t r o s t a t i c repulsion between charged groups located i n the o i l -water i n t e r f a c e . With regard to o i l i n water (0/W) emulsions, surface charges can o r i g i n a t e from i o n i z a t i o n of groups on i o n i c e m u l s i f i e r s , adsorption of ions from the aqueous phase on nonionic e m u l s i f i e r layer, or f r i c t i o n a l contact between droplet surfaces and the aqueous medium (Powrie and Tung, 1976). (3) Formation of hydration layers outside the o i l droplets, because water-orienting hydrophilic groups are present at the surface. (A) Low i n t e r f a c i a l tension can s t a b i l i z e emulsions by allowing large droplet deformations, thus increasing the amount of l i q u i d between c o l l i s i o n surfaces. V i s c o s i t y i s an important phy s i c a l property of emulsions i n terms of emulsion formation and s t a b i l i t y . When the v i s c o s i t y of the continuous phase i s too low, the increased movement of p a r t i c l e s (droplets) w i l l cause f l o c c u l a t i o n and f i n a l l y coalescence. Emulsions can be s t a b i l i z e d by increasing the v i s c o s i t y of the continuous phase. Three d i f f e r e n t methods have been used i n i n v e s t i g a t i n g the emulsi-fying properties of proteins: emulsifying capacity (EC), emulsion s t a b i l i t y (ES), and emulsifying a c t i v i t y (EA). EC i s usually defined as the volume of o i l (ml) that can be emulsified by a standard amount of 98. protein (g), before phase inversion or collapse of emulsion occurs. There are many fa c t o r s ( i . e . equipment design, shape of container, speed of blending, rate of o i l addition, kind of o i l , pH of the medium, protein source, protein concentration, s a l t , etc.) i n the determination that can a f f e c t the EC of proteins. Thus, EC i s not s o l e l y a property of the protein under test but rather i s a property of the emulsion system, the equipment and method used to produce the emulsion (Tornberg and Hermansson, 1977). Furthermore, i n cases where very viscous emulsions are formed, mixing of o i l into the emulsion may be i n e f f i c i e n t or incomplete and the observed EC value erroneous (Pearse and K i n s e l l a , 1978). Emulsion s t a b i l i t y (ES) r e f e r s to the a b i l i t y of a protein to form an emulsion that remains unchanged for a p a r t i c u l a r duration, under s p e c i f i c conditions ( K i n s e l l a , 1976). ES i s commonly measured i n terms of the amount of o i l and/or cream separating from an emulsion during a c e r t a i n period of time at a stated temperature and g r a v i t a t i o n a l f i e l d (Acton and S a f f l e , 1970). The time required f or a s p e c i f i e d degree of breakdown to occur i s also used as a measure of s t a b i l i t y (Pearson et_ a l , 1965). Several workers centrifuged heated emulsions and expressed the ES i n terms of the height of the cream layer as a percentage of the i n i t i a l height of the emulsion (Wang and K i n s e l l a , 1976; Yasumatsu et a l . , 1972). However, according to Pearse and K i n s e l l a (1978), the process occurring during the ce n t r i f u g a t i o n of the emulsion may hot be c h a r a c t e r i s t i c of those occurring i n a stored or heated emulsion. Thus, ES measured by the method of Yasumatsu et^ aJ_. (1972) may not be a v a l i d i n d i c a t i o n of emulsion s t a b i l i t y . Emulsifying a c t i v i t y (EA) r e f l e c t s the a b i l i t y of the protein to aid i n emulsion formation and s t a b i l i z a t i o n of the newly created emulsion (Kitchener and Musselwhite, 1968). EA i s measured by determining 99. the p a r t i c l e s i z e d i s t r i b u t i o n of the dispersed phase by microscopy, Coulter counting, or s p e c t r o t u r b i d i t y (Walstra et a l , 1969). Results from microscopic techniques take much time and show poor r e p r o d u c i b i l i t y , while the Coulter counter method i s more reproducible (Waniska et^ al_, 1981) . The s p e c t r o t u r b i d i t y method i s simple, rapid, and t h e o r e t i c a l l y sound (Kerker, 1969) and provides information about the average diameter and p a r t i c l e s i z e d i s t r i b u t i o n . Because of the problems and shortcomings of many of the t r a d i t i o n a l approaches and the need for more r e l i a b l e methods for the q u a n t i f i c a t i o n of emulsifying properties of proteins by techniques re q u i r i n g small quantities of protein, Pearse and K i n s e l l a (1978) evaluated the p o t e n t i a l of turbidimetry f or measuring e m u l s i f i c a t i o n . These authors proposed two indexes, emulsifying a c t i v i t y index (EAI) and emulsion s t a b i l i t y index (ESI), for the cha r a c t e r i z a t i o n of emulsifying agents, e s p e c i a l l y proteins. The EAI i s related to the i n t e r f a c i a l area of the emulsion and has units 2 of area of i n t e r f a c e (m ) s t a b i l i z e d by unit weight of protein (g). The EAI i s a function of o i l volume f r a c t i o n , p rotein concentration, and of the type of equipment used to prepare the emulsion. The emulsion breakdown can be monitored by determining the ESI from the decrease i n absorbance with time r e s u l t i n g from the i r r e v e r s i b l e reduction i n the i n t e r f a c i a l area (brought about by the processes of coalescence and o i l i n g - o f f ) . These two indexes (EAI"and ESI) are e a s i l y measured and, from t h e o r e t i c a l and p r a c t i c a l considerations, seem more l i k e l y to be rel a t e d to p r a c t i c a l performance of products than the EC, ES, and EA tes t s which are commonly used (Pearse and K i n s e l l a , 1978). 100. MATERIALS AND METHODS Materials Pea protein i s o l a t e (a), Century c u l t i v a r f i e l d pea, was obtained from POS P i l o t Plant Corp., University of Saskatchewan, Saskatoon, Sask. Pea protein i s o l a t e (b), Pro-Pulse W100, was obtained from G r i f f i t h Laboratories Ltd., Scarborough, Ont. V i t a l wheat gluten, Whetpro 75%, was supplied by I n d u s t r i a l Grain Products Ltd., Thunder Bay, Ont. Canola protein i s o l a t e was prepared by the method of Nakai et^ a l . (1980a). The protein content of i s o l a t e batches (a) and (b) was 84% and 88%, res p e c t i v e l y . Myosin was prepared according to the method of Perry (1955) with s l i g h t modification as follows: the p e c t o r a l i s s u p e r f i c i a l i s and profundus muscles of a f r e s h l y slaughtered male chicken ( b r o i l e r 11 - 12 weeks old)were ground (after being c h i l l e d i n crushed ice) with a meat grinder and immediately extracted with 3 volumes of cold KCl-potassium phosphate buffer, pH 6.5. The suspension was s t i r r e d slowly for 15 min and then allowed to stand at 4°C for 45 min. The extract was subsequently centrifuged f or 1 hr at 1,000 x g. C l a r i f i c a t i o n of the crude myosin extract was achieved by gentle f i l t r a t i o n of the supernatant through a Whatman No. 4 f i l t e r paper. The pH of the f i l t r a t e was adjusted to 6.5, i f necessary, with NaOH. The crude myosin was then p r e c i p i t a t e d by the slow addition, with s t i r r i n g , of 400 ml of crude extract to 3.6 L of d i s t i l l e d deionized water. Af t e r overnight s e t t l i n g , the myosin was c o l l e c t e d by ce n t r i f u g a t i o n for 1 hr at 12,000 x g. The resultant gel was dissolved i n one volume of 0.01 M phosphate buffer pH 7.4 containing 0.6 M NaCl, and the pH was adjusted to 7.4 with NaOH. A l l the other materials used i n t h i s study were exactly s i m i l a r to those described i n the materials section of the second chapter of t h i s t h e s i s . 101. Methods A. Preparation ( i n s o l u b i l i z a t i o n ) of protein samples For hydrophobicity, s o l u b i l i t y , emulsifying a c t i v i t y index, emulsion s t a b i l i t y index and f a t binding capacity determinations, the following protein samples were prepared: Bovine Serum Albumin (BSA) 1% s o l u t i o n i n d i s t i l l e d water. Heated: the pH was adjusted to 4.0 and then the s o l u t i o n was heated at 100°C for 5 min and homogenized i n an Omni-Mixer (Ivan S o r v a l l , Norwalk, Con.) for 1 min at speed se t t i n g 1 (lowest speed). Control: not heated. g-Lactoglobulin 1% s o l u t i o n i n d i s t i l l e d water. Heated: the pH was adjusted to 1.0, heated at 100°C for 15 min and then homogenized for 1 min at speed se t t i n g 1. Soy Protein Isolate 1% aqueous dispersion, s t i r r e d on magnetic s t i r r e r for 5 min, and then, homogenized for 1 min at speed s e t t i n g 5. Samples 1 - 4 : pH 5.5, heated at 100°C for 0.25, 0.5, 1.0, and 2.0 min, r e s p e c t i v e l y . Sample 5: pH 5.5, autoclaved at 121°C for 15 min. Sample 6: pH 7.2, autoclaved at 121°C for 15 min. Promine-D 1% dispersion i n 0.01 M phosphate buffer, pH 7.4, s t i r r e d magnetically for 5 min and homogenized for 1 min at speed s e t t i n g 5. Ovalbumin 1% aqueous s o l u t i o n . Samples 1 - 4: pH 5.6, heated at 80°C for 1.5, 2.0, 2.5, and 3.0 min, r e s p e c t i v e l y . Sample 5: pH 5.6, heated at 100°C for 5 min. Sample 6: pH 1.0, heated at 100°C for 15 min. 102. Pea Protein Isolate (a) 1% aqueous dispersion, s t i r r e d magnetically f or 5 min, and then, homogenized for 1 min at s e t t i n g 5. Samples 1 - 4 : pH 5.8, heated at 80°C f o r 1, 2, 4 and 7 min, res p e c t i v e l y , and then, homogenized for 10 sec. Pea Protein Isolate (b) 1% dispersion i n 0.01 M phosphate buffer, pH 7.4, was magnetically s t i r r e d 5 min, and then, homogenized for 1 min at speed s e t t i n g 5. V i t a l Gluten Samples 1 - 5 : 1% dispersions i n 0.5, 1.0, 1.5, 1.75 and 2.0 N ac e t i c a c i d , r e s p e c t i v e l y . Then, the samples were magnetically s t i r r e d for 5 min, homogenized 15 sec at speed s e t t i n g 1, and heated at 100°C for 30 min. Canola Protein Isolate (a) 1% aqueous dispersion, was s t i r r e d f o r 5 min, and then, homogenized for 1 min at speed s e t t i n g 5. Samples 1 - 4 : pH 5.5, heated at 100°C for 0.5, 1.0, 1.5 and 2.0 min, r e s p e c t i v e l y , and then homogenized for 5 sec at speed s e t t i n g 1. Sample 5: pH 7.2, autoclaved at 121°C f o r 10 min. Canola Protein Isolate (b) 1% dispersion i n 0.01 M phosphate buffer, pH 7.4, was s t i r r e d f o r 5 min, and then, homogenized for 1 min at speed s e t t i n g 5. Sunflower Protein Isolate 1% dispersion i n 0.01 M phosphate buffer, pH 7.4, was s t i r r e d for 5 min, and then, homogenized for 1 min at speed s e t t i n g 5. Whey Protein 1% s o l u t i o n i n 0.03 M C a C l 0 . Samples 1 - 3 : pH 6.0, heated at 80°C 103. for 4, 5 and 15 min, respectively. Sample 4: pH 5.8, heated at 80°C for 15 min. Whole Casein Control: 1% s o l u t i o n i n 0.01 M CaCl 2,pH 7.4. Samples 1 and 2: 1% s o l u t i o n i n 0.01 M CaCl^, pH 7.4, autoclaved at 121°C f o r 5 and 20 min, r e s p e c t i v e l y . Sample 3: 1% s o l u t i o n i n 0.02 M CaC^, pH 7.4, autoclaved at 121°C for 20 min. G e l a t i n Control: 1% dispersion i n 0.01 M phosphate buffer, pH 7.4, s t i r r e d 5 min, and then, homogenized for 1 min at speed s e t t i n g 5. Heated: 0.5% dispersion i n 0.01 M phosphate buffer, pH 7.4, heated at 75°C f o r 2.5 min with s t i r r i n g . Myosin To form a stock s o l u t i o n for determination of hydrophobicity, s o l u b i l i t y , and other functional properties, the o r i g i n a l gel was d i l u t e d 2X with 0.3 M NaCl i n 0.01 M phosphate buffer, pH 7.4. The c o n t r o l sample was unheated and samples 1 and 2 were heated at 75°C for 1 and 5 min, r e s p e c t i v e l y . P r i o r to a n a l y s i s , a l l heated and unheated (control) protein samples -except Promine-D, pea protein (b), canola protein (b), sunflower protein, g e l a t i n , and myosin - were dialysed against 0.01 M phosphate buffer, pH 7.4, containing 0.02% sodium azide. A f t e r hydrophobicity, s o l u b i l i t y , EAI and ESI were determined, the protein samples were freeze-dried and subsequently used for fa t binding capacity (FBC) determinations. B. S o l u b i l i t y index, hydrophobicity and f a t binding capacity determinations These protein properties were determined as described i n the. second 104. chapter of t h i s t h e s i s . C. Emulsifying a c t i v i t y index (EAI) determination The EAI was determined by the turbidimetric technique of Pearse and K i n s e l l a (1978). To prepare emulsion, each protein sample was d i l u t e d to a concentration of 0.5% with 0.01 M phosphate buffer, pH 7.4. Two ml of corn o i l and 6 ml of the d i l u t e d protein dispersion were homogenized together i n an Omni-mixer with a micro-attachment (Ivan S o r v a l l , Inc., Norwalk, CN) at speed s e t t i n g 1 for 1 min at 20°C. D. Emulsion s t a b i l i t y index (ESI) determination ESI was determined by a modification of the method of Pearse and K i n s e l l a (1978) as follows: the emulsion, prepared as described above (EAI), was held at room temperature and aliquots (0.1 ml) were taken d i r e c t l y from the bottom of the container, containing the emulsion, at d i f f e r e n t time i n t e r v a l s and d i l u t e d to 50 ml (500 x d i l u t i o n ) with 0.01 M phosphate buffer, pH 7.4, containing 0.1% sodium dodecyl s u l f a t e (SDS). The absorbance of d i l u t e d emulsions at 500 nm was then recorded with a Beckman DB spectrophotometer. The h a l f - l i f e (min) of the absorbance decay with time, determined gra p h i c a l l y , was used as ESI. E. V i s c o s i t y determination V i s c o s i t y measurements of 0.5% protein dispersions at 20°C were made using a Brookfield Synchro-Lectric viscometer, Model LVT f i t t e d with a UL adapter, at 60 rpm. 105. F. S t a t i s t i c a l analysis Simple and multiple l i n e a r regression analyses were used to determine the r e l a t i o n s h i p s between hydrophobicity, s o l u b i l i t y and emulsifying properties of the protein samples. These analyses were ca r r i e d out by using a Monroe 1880 programmable c a l c u l a t o r . In addition, backwards stepwise multiple regression analyses and surface v i s u a l i z a t i o n p l o t t i n g were c a r r i e d out using an Amdahl 470 V/8 computer at the University of B r i t i s h Columbia. Five independent v a r i a b l e s were used i n the i n i t i a l equation i n the backwards stepwise procedure including surface hydrophobicity (S Q)> s o l u b i l i t y index ( s ) , i n t e r a c t i o n of S q and s, and quadratic powers of S q and s. Dependent v a r i a b l e s included EAI, ESI, and FBC. RESULTS AND DISCUSSION A. E f f e c t of heat treatment on emulsifying pro.per.t-ies Table 1 shows the r e l a t i o n s h i p s of hydrophobicity and s o l u b i l i t y index of selected proteins with t h e i r emulsifying properties. It i s evident that, for most proteins under a given set of conditions, protein s o l u b i l i t y decreased as heating time increased due to the progressive denaturation of the protein. G e l a t i n , as expected, was completely s o l u b i l i z e d on heating due to the rupture of hydrogen bonds which are responsible for i t s i n s o l u b i l i t y . As protein denaturation progressed, as seen by the decrease i n protein s o l u b i l i t y , the hydrophobicity usually increased. This i s due to the gradual exposure of hydrophobic amino acid residues of native proteins (which are usually buried " i n the i n t e r i o r of the molecules) as a r e s u l t of the protein unfolding. In the case of Table 1 - Relationships between protein hydrophobicity, s o l u b i l i t y index, emulsifying a c t i v i t y , emulsion s t a b i l i t y and f a t binding capacity of various p r o t e i n s a Protein Hydrophobicity S o l u b i l i t y index (%) EAI ES I b (min) FBC(%) < S o > (s) (m2/g) I II Bovine serum albumin - control 325.0 100.0 148 108.50 91.50 25.0 Bovine serum albumin - heated 304.0 26.8 140 90.00 109.00 54.4 3-Lactoglobulin - control 426.0 100.0 96 27.20 37.00 4.2 B-Lactoglobulin - heated 192.0 6.4 51 25.30 27.50 16.5 Soy i s o l a t e - control 95.0 26.4 42 6.65 25.20 90.0 - sample 1 97.0 26.4 51 7.30 26.00 86.3 - sample 2 131.0 24.0 56 5.55 17.00 81.7 - sample 3 150.0 14.2 48 1.00 5.13 76.0 - sample 4 144.0 4.2 58 0.98 14.03 73.5 - sample 5 160.0 8.2 50 0.76 11.20 68.4 - sample 6 128.0 77.2 76 112.50 100.00 54.8 Promine-D 39.0 29.1 65 1.10 5.80 85.3 Ovalbumin - cont r o l 6.0 100.0 24 0.56 0.70 42.7 - sample 1 38.0 90.6 34 0.63 1.10 45.9 - sample 2 87.0 75.8 35 1.10 1.80 57.5 - sample 3 95.0 70.6 45 1.08 1.70 66.9 - sample 4 142.00 48.9 49 5.85 8.40 82.5 - sample 5 269.0 10.6 78 24.30 138.00 102.6 (continued) Table 1 - continued Protein Hydrophobicity S o l u b i l i t y index (%) EAI ESI^_(mln) FBC(%) (S Q) (s) (m2/g) I II Ovalbumin - sample 6 296.0 46.0 136 12.60 108.00 101.3 Pea i s o l a t e (a) - control 66.0 42.6 61 3.67 15.50 66.4 - sample 1 77.0 34.7 66 0.60 0.42 53.9 - sample 2 104.0 29.5 35 0.56 0.31 51.6 - sample 3 100.0 25.2 37 0.85 0.16 50.4 - sample 4 121.0 20.2 25 1.45 0.16 57.5 Pea i s o l a t e (b) 59.0 58.0 54 1.60 17.60 78.9 Gluten - sample 1 65.0 71.6 70 0.36 17.60 38.0 - sample 2 59.0 79.0 69 0.55 25.00 41.8 - sample 3 65.0 85.6 82 0.28 23.80 34.8 - sample 4 60.0 89.6 74 0.80 26.60 39.9 - sample 5 57.0 93.3 69 0.78 23.30 32.2 Canola i s o l a t e (a) - control 65.0 28.3 60 0.54 3.76 33.9 - sample 1 75.0 15.9 63 0.49 2.80 29.1 - sample 2 77.0 10.5 49 0.17 1.27 29.2 - sample 3 86.0 3.6 41 0.12 0.45 35.1 - sample 4 78.0 2.9 40 0.14 0.47 42.5 - sample 5 101.0 21.2 50 2.20 8.40 35.7 Canola i s o l a t e (b) 55.0 44.0 66 0.25 3.35 56.0 Sunflower i s o l a t e 47.0 31.0 75 1.40 5.30 105.8 (continued) Table 1 - continued Protein Hydrophobicity <So> S o l u b i l i t y index (%) (s) EAI (m2/g) E S I b I (min) II FBC(%; Whey protein - control 182.0 88.7 87 50.30 87.50 74.5 - sample 1 211.0 75.2 98 61.30 102.00 75.2 - sample 2 164.0 65.5 89 63.00 104.00 72.5 - sample 3 128.0 50.4 82 48.30 104.50 100.8 - sample 4 132.0 61.6 85 46.30 108.00 99.4 Casein - control 28.0 100.0 58 1.70 39.30 11.3 - sample 1 30.0 76.2 56 9.50 35.00 18.1 - sample 2 21.0 71.5 49 14.75 24.30 16.7 - sample 3 23.0 70.2 50 12.50 16.80 14.0 Gelatin - control 5.0 15.3 46 4.65 3.10 19.1 G e l a t i n - heated 6.0 100.0 59 10.20 9.40 Myosin - control 14.0 C 100.0C 43 c 36.00 c - sample 1 44.0 C 50.9 C 50° 22.40° - sample 2 54.0 C 16.5 C 48° i o . o o c average of duplicate determinations b l : 0.1 M NaCl added; I I : NaCl not added ° 0 . 3 M NaCl added 109. whey protein, i t can be seen, that excessive heating resulted i n a decrease of hydrophobicity, due probably to p a r t i c i p a t i o n of some of the exposed hydrophobic groups i n hydrophobic i n t e r a c t i o n s . For casein, heating did not r e s u l t i n any s u b s t a n t i a l change of i t s hydrophobicity and t h i s was expected, since casein e x i s t s i n a random c o i l conformation. The r e s u l t s of Table 1 also i n d i c a t e that for samples having the same s o l u b i l i t y , the more hydrophobic the protein, the greater are i t s emulsifying properties. Looking, s p e c i f i c a l l y , at the r e s u l t s of soy p r o t e i n i n Table 1, i t can be seen that, the EAI of a l l heated samples was s l i g h t l y greater than than of the c o n t r o l . The ESI, however, was i n i t i a l l y s l i g h t l y increased by heating, but subsequently started to decrease as the s o l u b i l i t y progressively decreased;; that i s , s o l u b i l i t y became an i n c r e a s i n g l y important factor c o n t r o l l i n g t h i s property. The higher EAI values of heated soy protein samples were probably due to t h e i r increased hydrophobicity values. On the other hand, the observed decrease i n ESI upon heating must be due to the decrease i n s o l u b i l i t y mainly as a r e s u l t of protein-protein i n t e r a c t i o n , and secondarily because of the increase i n hydrophobicity. The proteins studied here can be broadly divided i n t o 4 categories according to the e f f e c t of heating on t h e i r emulsifying properties. The f i r s t category includes BSA, gluten, and whey protein whose one emulsifying property (EAI) was not s u b s t a n t i a l l y affected by heating, whereas the other (ESI) was improved by heating.' The second category includes soy protein and myosin whose one emulsifying property (EAI) was s l i g h t l y improved by heating, whereas the other (ESI) was decreased. The t h i r d category includes 8-lactoglobulin, pea, canola, and casein, whose both 110. emulsifying properties were adversely affected by heating. F i n a l l y the fourth category includes ovalbumin and g e l a t i n whose both emulsifying properties were markedly, improved upon heating. I t i s evident, therefore, that heating did not have the same e f f e c t on the emulsifying properties of d i f f e r e n t proteins. The improvement of emulsifying properties of g e l a t i n upon heating was mainly due to the increase i n s o l u b i l i t y since i t s hydrophobicity was not changed by heating. Kato and Nakai (1980), and Kato et a l . (1981) reported that the emulsifying properties (EAI and ESI) of ovalbumin and lysozyme were markedly improved upon heating. They also found that t h i s improvement was correlated with the higher hydrophobicities of heat denatured protein samples as compared to those of the native proteins. Their r e s u l t s , moreover, indicated that the more hydrophobic the proteins, the greater the decrease i n i n t e r f a c i a l tensions and the increase i n emulsifying properties. During the handling of the ovalbumin samples we noticed that the apparent v i s c o s i t i e s of>heat-denatured samples were greater than that of the c o n t r o l . This i s evident i n Table 2, which shows the apparent v i s c o s i t y changes of some proteins upon heating. I t i s suggested, therefore, that higher v i s c o s i t y appears to be another factor contributing to the great improvement of emulsifying properties of heat denatured ovalbumin samples. As shown i n Table 1, the FBC of d i f f e r e n t proteins was d i f f e r e n t l y affected by heating. While the FBC of canola (rapeseed) protein was generally not affected by heating, the FBC's of BSA, 8-lactoglobulin, ovalbumin, whey protein, and casein were p o s i t i v e l y a f f e c t e d , and the FBC's of soy and pea proteins were adversely affected by heating. It i s generally accepted that protein denaturation i s undesirable Table 2 - E f f e c t of heating on the apparent v i s c o s i t y of some proteins at 20°C Apparent v i s c o s i t y Protein (Pa.s x 10" 3) Whey - con t r o l 1.14 - sample 1 1.14 - sample 2 1.14 - sample 3 1.16 - sample 4 1.20 BSA - c o n t r o l 1.13 BSA - heated 1.24 Ovalbumin - control 1.10 - sample 1 1.13 - sample 2 1.13 - sample 3 1.16 - sample 4 1.17 - sample 5 1.18 - sample 6 1.90 0.5% pr o t e i n i n 0.01 M phosphate buffer, pH 7.4 112. since i t adversely a f f e c t s protein f u n c t i o n a l i t y . However, as shown i n t h i s study, emulsifying and f a t binding properties of some proteins (ovalbumin, whey protein) can be improved by denaturation. According to Morr (1979) denaturation of the whey protein molecule, i f produced at the proper stage of the protein concentrate i s o l a t i o n / u t i l i z a t i o n process, can improve the f u n c t i o n a l i t y . The improvement i n f u n c t i o n a l i t y i s probably due to an unfolding of the molecule to expose hydrophobic amino acid residues, thus making the protein more amphiphilic and capable of o r i e n t i n g at the oil-water i n t e r f a c e (Morr, 1979). A great improvement of emulsifying properties (EAI and ESI) of ovalbumin and lysozyme by heat denaturation was also reported by Kato and Nakai (1980) and Kato et a l . (1981). Moreover, another example of protein denaturation r e s u l t i n g i n improvement of f u n c t i o n a l i t y reported by Aoki et: a l . (1981), who determined the e f f e c t of alcohol modification of soy p r o t e i n on i t s emulsion s t a b i l i z i n g properties. The soy protein was denatured with 50% alcohol (ethanol or n-propanol) by treatment at 35°C for 2 hr. The emulsion s t a b i l i z i n g properties of soy protein modified with ethanol or n-propanol decreased with increasing the s o l u b i l i t y , whereas the emulsion s t a b i l i z i n g properties of the unmodified (control) soy protein increased. Aoki _et'al. (1981) a t t r i b u t e d the improved emulsion s t a b i l i t y brought about by the a l c o h o l modification of soy protein to the perturbation and unfolding of the hydrophobic i n t e r i o r structure of the native soy protein by alcohol, and to the r e s u l t i n g increase i n the exposed hydrophobic amino acid residues. It should be noted, that the increased emulsion s t a b i l i z i n g properties of soy protein between pH 2 and 7, achieved through alcohol modification by Aoki et a l . (1981), i s very important because soy protein can be expected to play a s i g n i f i c a n t r o l e i n the 113. s t a b i l i z a t i o n of a wide range of food emulsions, a l l meat emulsions f a l l i n g well within these pH l i m i t s . B. S t a t i s t i c a l Analysis Regression equations (models) for p r e d i c t i o n of the emulsifying and fat binding properties from hydrophobicity and s o l u b i l i t y data of heat denatured proteins of Table 1 are presented i n Table 3. Simple l i n e a r 2 regression analyses showed that the c o e f f i c i e n t s of determination (r ) between S and EAI, and between S and ESI (determined without NaCl) were o ' o highly s i g n i f i c a n t (P < 0.001). No s i g n i f i c a n t c o r r e l a t i o n was found, however, between s o l u b i l i t y and EAI or ESI. Moreover, no c o r r e l a t i o n was found between S q or s o l u b i l i t y and FBC. Although s i g n i f i c a n t c o r r e l a t i o n s were found between S and ESI as well as s o l u b i l i t y and ESI o determined i n the presence of 0.1 M NaCl, however, such c o r r e l a t i o n s are not considered r e l i a b l e since the e f f e c t of 0.1 M NaCl on s o l u b i l i t y was not determined. It i s well known that even 0.1 M NaCl may p o s i t i v e l y or negatively a f f e c t the s o l u b i l i t y of a protein. Sodium chloride was used i n the ESI t e s t , to obtain a general idea on the s a l t s e n s i t i v i t y of the emulsions formed by d i f f e r e n t proteins. As seen i n Table 1, NaCl even at the low concentration used had generally a detrimental e f f e c t upon the s t a b i l i t y of the emulsions. NaCl exerted i t s negative e f f e c t on emulsion s t a b i l i t y probably by reducing the charge on the surface of the p a r t i c l e s ( o i l globules) and by withdrawing water from t h e i r hydrated surfaces. M u l t i p l e l i n e a r regression analyses (Table 3) between S , s o l u b i l i t y 2 and EAI or ESI, showed highly s i g n i f i c a n t c o r r e l a t i o n s (R values were 0.5419 and 0.4336, P < 0.001, r e s p e c t i v e l y ) . In order to determine Table 3 - Regression models for prediction of emulsifying and f a t binding properties of heat denatured proteins. Dependent Regression Variable Regression v a r i a b l e analysis description c o e f f i c i e n t F - r a t i o F - p r o b a b i l i t y t-value 3-value EAI (n=52; r = 0.464, P<0.001; S.E.=18.95) Simple l i n e a r Constant S 41.162 0.202 EAI (n=52; R = 0.542, P<0.001; S.E. =17.78) Multiple l i n e a r Constant S 29.283 0.207 0.226 7.202 2.887 A A A A A 0.698 0.280 R2= EAI (n=52; 0.583, PO.001; S.E.b=17.40) Backwards multiple nonlinear Constant S 16.877 0.214 0.931 -0.007 4.531 58.77 7.83 4.73 0.039 0.000 0.008 0.035 0.720 1.151 -0.894 ESI „ (n=49; r = 0.377, P<0.001; S.E.b=30.98) Simple l i n e a r Constant S 0.880 0.274 ESI „ (n=49; R = 0.434, P<0.001; S.E.b=29.53) Multiple l i n e a r Constant S -14.308 0.278 0.295 5.613,, 2.153 A A A 0.623 0.239 ESI 7 (n=49; R = 0.584, P<0.001; S.E.b=26.70) Backwards multiple nonlinear Constant S o s Sr>X S -69.463 0.565 2.034 -0.004 -0.012 18.02 34.31 13.62 10.08 5.92 0.000 0.000 0.001 0.003 0.019 1.268 1.651 -0.779 -1.044 (continued) Table 3 - continued Dependent Regression Variable Regression v a r i a b l e analysis description c o e f f i c i e n t F - r a t i o F - p r o b a b i l i t y t - v a l u e a B-value FBC „ Backwards Constant 4.895 0.16 0.691 (n=48; R = multiple S 0.451 13.84 0.001 1.445 0.473, P<0.001; nonlinear s° 1.398 10.49 0.002 1.602 S.E.b=20.97) si -0.001 10.15 0.003 -1.270 s 2 -0.014 11.79 0.001 -1.745 a*: P<0.05; **: P<0.01; ***: P<0.001 ^Standard error of estimate 116. whether both hydrophobicity and s o l u b i l i t y had a s i g n i f i c a n t e f f e c t on the emulsifying properties of proteins studied, t h e i r p a r t i a l regression c o e f f i c i e n t s were tested for s i g n i f i c a n c e by the Student's t - t e s t . This test showed (Table 3) that both p a r t i a l regression c o e f f i c i e n t s were highly s i g n i f i c a n t , and thus, i t was concluded that both hydrophobicity and s o l u b i l i t y s i g n i f i c a n t l y affected the emulsifying properties. Moreover, i n order to determine the r e l a t i v e importance of surface hydrophobicity and s o l u b i l i t y i n estimating the values of emulsifying properties, the B-values (standard p a r t i a l regression c o e f f i c i e n t s or normalized c o e f f i c i e n t s ) for hydrophobicity and s o l u b i l i t y were calculated (Table 3). I t was found that hydrophobicity was almost twice as u s e f u l as s o l u b i l i t y i n estimating or p r e d i c t i n g the emulsifying properties of the proteins studied. I t should, also, be reported here that multiple l i n e a r regression analysis showed no s i g n i f i c a n t c o r r e l a t i o n between S Q, s o l u b i l i t y and FBC of the proteins studied. In an attempt to improve the c o e f f i c i e n t s of m u l t i p l e determination 2 (R ) of Table 3, a backwards stepwise multiple regression analysis was applied to the data of Table 1. The m u l t i p l e regression models, thus obtained, are shown i n Table 3. As shown i n Table 3, the backwards stepwise multiple regression analysis gave a highly s i g n i f i c a n t c o r r e l a t i o n between S q, s o l u b i l i t y and FBC of the 48 p r o t e i n samples of Table 1. The r e l a t i o n s h i p , however, was not l i n e a r but quadratic. Moreover, comparing the models of Table 3, i t i s obvious, that the models obtained 2 by stepwise regression analysis have higher R values and lower S.E. (standard error of the estimate) values than the multiple and simple regression models. It was, therefore, concluded that these models should be used i n p r e d i c t i n g the values of emulsifying properties of the 117. proteins studied. Our discussion, therefore, w i l l be confined within these models. The EAI of the proteins studied was s i g n i f i c a n t l y affected by S Q , ' 2 s o l u b i l i t y and the square of s o l u b i l i t y (Table 3). As i t i s known, R values i n d i c a t e the percentage of v a r i a t i o n i n a dependent v a r i a b l e accounted f o r by i t s regression on the independent v a r i a b l e s . The 2 higher the value, the greater the a c c o u n t a b i l i t y . The R value for EAI was 0.583, i n d i c a t i n g that 58.3% of the v a r i a b i l i t y i n EAI of the protein studied could be accounted for by the 3 independent v a r i a b l e s stated above. Comparison of the g-values of these variables indicated that the most important independent v a r i a b l e i n the model was s o l u b i l i t y . In t h i s model, the s t a t i s t i c a l s i g n i f i c a n c e of the square of s o l u b i l i t y index indicates that as the value of s o l u b i l i t y index increases, the e f f e c t of that v a r i a b l e declines, i . e . , the response of EAI to increasing l e v e l s of s o l u b i l i t y may be depicted as a c u r v i l i n e a r graph rather than a s t r a i g h t l i n e . Response surface plots (contours) were generated by computer to aid i n v i s u a l i z i n g the e f f e c t s of S q and s o l u b i l i t y on the f u n c t i o n a l properties studied. As i t can be seen i n Figure 1, regardless of the s o l u b i l i t y , as hydrophobicity was increased the EAI was i n i t i a l l y increased and then decreased. Moreover, at low and medium hydrophobicity values, increasing s o l u b i l i t y l e v e l s increased the EAI. However, at very high S q values, s o l u b i l i t y did not appear to play a s i g n i f i c a n t r o l e i n the a b i l i t y of a protein to form an oil-in-water emulsion. The ESI of the proteins studied was s i g n i f i c a n t l y affected by S^, s o l u b i l i t y index, the i n t e r a c t i o n of S q and s o l u b i l i t y index, and the 2 square of s o l u b i l i t y index (Table 3). The R value of the model was 0.5842, i n d i c a t i n g that 58.42% of the v a r i a b i l i t y i n ESI could be 00 119. accounted f or by the aforementioned 4 independent v a r i a b l e s . A comparison of the g-values of these variables indicated that the most important independent v a r i a b l e i n the model was s o l u b i l i t y . Figure 2 shows the E S I contour pl o t as a function of S q and s o l u b i l i t y index. As shown, regardless of the s o l u b i l i t y , as S q was increased the E S I was i n i t i a l l y increased and then decreased. At low and medium S values, increasing o s o l u b i l i t y index l e v e l s increased E S I . However, at very high S q values, s o l u b i l i t y index did not appear to be important for the s t a b i l i t y of emulsion. The FBC of the proteins studied was s i g n i f i c a n t l y affected by S Q , s o l u b i l i t y index, and the square powers of these two v a r i a b l e s (Table 3). The R 2 of the model was 0.473 (P < 0.001), i n d i c a t i n g that 47.3% of the v a r i a b i l i t y i n FBC could be accounted f or by the 4 independent v a r i a b l e s . Moreover, a comparison of the 3-values of these v a r i a b l e s indicated that the most important independent.variable i n the model was the square of s o l u b i l i t y index. The s t a t i s t i c a l s i g n i f i c a n c e of quadratic powers of S q and s o l u b i l i t y i n the FBC model of Table 3, ind i c a t e s that as the value of S or s o l u b i l i t y index i s increased, t h e i r e f f e c t s on FBC o J ' decline ( i . e . the response of FBC to increasing l e v e l s of S^ or s o l u b i l i t y can be depicted as a c u r v i l i n e a r graph). The f i n d i n g of t h i s study that with increasing S^ the emulsifying properties were i n i t i a l l y increased and then decreased can be explained by taking into account the fac t that the emulsifying properties of proteins ultimately depend on the sui t a b l e balance between the hydrophile and l i p o p h i l e , and do not ne c e s s a r i l y increase as the proteins become more l i p o p h i l i c (Aoki et; a l . , 1981). Thus, as Aoki et^ a l . (1981) reported, the excessive denaturation of the soy protein by n-propanol resulted i n Fig. 2. Emulsion stability index response surface contour as a function of hydrophobicity (So> and solubility index (s). 121. a lower emulsion s t a b i l i z i n g properties (as compared to the moderate denaturation by ethanol). Needless to say, there e x i s t many complicated f a c t o r s , e.g., molecular s i z e , molecular f l e x i b i l i t y , charge, etc., besides the balance of hydrophile and l i p o p h i l e which p a r t i c i p a t e s i n determining the emulsifying properties of proteins (Aoki jit al_., 1981) . Wolf and Cowan (1975) reported that i n ground meat products fat absorption or binding appeared to involve formation and s t a b i l i z a t i o n of an emulsion. Thus, they suggested that f a t absorption may simply be another aspect of e m u l s i f i c a t i o n . This suggestion helps to explain the observation i n t h i s study that hydrophobicity had a c u r v i l i n e a r e f f e c t on FBC, b y . u t i l i z i n g the concept of h y d r o p h i l e - l i p o p h i l e balance discussed above. Voutsinas and Nakai (1981) also observed that with increasing S the FBC was i n i t i a l l y increased and then decreased, o The findings of t h i s study concerning the r e l a t i o n s h i p between s o l u b i l i t y and emulsifying, properties are contrary to the general b e l i e f that s o l u b i l i t y i s the best index of protein emulsifying properties, that i s , the higher the s o l u b i l i t y of a protein the better i t s emulsifying properties. These findings, however, are i n agreement with the r e s u l t s of Smith et a l , (1973), McWatters and Cherry (1975), Wang and K i n s e l l a (.1976), McWatters and Holmes (1979a, 1979b), and Aoki et a l . (1980), who reported that emulsifying properties of proteins cannot be predicted s o l e l y on the basis of protein s o l u b i l i t y l e v e l . Pearson et a l . (1965) reported that only that f r a c t i o n of protein which i s soluble can function as an e f f e c t i v e emulsifying agent. Moreover, Franzen and K i n s e l l a (1976) suggested that, as a p r o t e i n becomes more soluble, i t forms layers around the f a t droplet to f a c i l i t a t e a s s o c i a t i o n with the aqueous phase. Granular, inso l u b l e proteins, however, separate 122. from the o i l phase or ju s t f l o a t on the o i l surface where they remain i n e r t and contribute l i t t l e toward e m u l s i f i c a t i o n . S i m i l a r l y , soluble proteins enclose the f a t globule and render the emulsion more stable to heat treatment. Also, according to B u l l (1972), the surface a c t i v i t y of a protein i s a function of the ease with which the protein can migrate to, adsorb at, unfold, and rearrange at an i n t e r f a c e . Therefore, s o l u b i l i t y i n the aqueous phase, i s c l o s e l y r e l a t e d to surface a c t i v i t y of the proteins ( K i n s e l l a , 1979). Many authors, on the other hand, have reported that high l e v e l s of s o l u b i l i t y were not ne c e s s a r i l y associated with maximum emulsifying properties (Aoki et aj_., 1980; McWatters and Cherry, 1975; McWatters and Holmes, 1979a, 1979b; Smith et a l . , 1973; Wang and K i n s e l l a , 1976). F l i n t and Johnson (1981) i n an i n t e r e s t i n g study evaluated the f i l m formation by soy protein ( i s o l a t e ) at an oil-water i n t e r f a c e for the pH range 1 - 1 0 . D e f i n i t e f i l m s were seen at a l l pH values below that of the i s o e l e c t r i c point (~4.6) of the protein and up to pH 6.5. At pH 5.4, despite the low s o l u b i l i t y strong f i l m formation was found to occur. However, beyond t h i s point the strength of f i l m formation gradually decreased u n t i l at an upper l i m i t of pH 7.5 the presence of an i n t e r f a c i a l layer could barely be seen. The marked pH dependence of f i l m formation on the a l k a l i n e side of the i s o e l e c t r i c point was at t r i b u t e d by F l i n t and Johnson (1981) to the fac t that with increasing pH the prot e i n becomes more soluble i n the aqueous phase and consequently less l i k e l y to be brought out of s o l u t i o n (coagulated) at the phase boundary. The a b i l i t y of soy protein to form a f i l m even at very low pHs, where the s o l u b i l i t y (of the soy,protein) i s high, suggested that t h i s phenomenon may not be linked s o l e l y to s o l u b i l i t y but also to the a v a i l a b i l i t y of 1 2 3 . l i p o p h i l i c groups for binding at the oil-water i n t e r f a c e . Changes i n the conformation of the proteins present may occur at a c i d pHs which enhance the combination of the protein and o i l leading to the formation of i n t e r f a c i a l f i l m described ( F l i n t and Johnson, 1981). The f i n d i n g of t h i s study that high s o l u b i l i t y had a negative e f f e c t on FBC i s i n agreement with the r e s u l t s of Voutsinas and Nakai (1981) who a t t r i b u t e d the low FBC of soluble proteins to t h e i r conforma-t i o n (mainly h e l i c a l ) which may not permit t h e i r binding s i t e s to be s t e r i c a l l y a v a i l a b l e for i n t e r a c t i o n with o i l or to the l i m i t e d access of o i l (hydrocarbon chains) to the protein binding s i t e s due to a possible b a r r i e r around them formed by the excessive number of protein polar groups. CONCLUSIONS The emulsifying properties of the food proteins studied here were d i f f e r e n t l y affected by heating. Thus, for some proteins heating did not have a s u b s t a n t i a l e f f e c t on one emulsifying property but i t affected p o s i t i v e l y or negatively the other properties, whereas for other proteins heating had a p o s i t i v e or negative e f f e c t on a l l emulsifying properties. It was, therefore, demonstrated that heat-denaturation i s not always accompanied by l o s s of emulsifying properties. This i s p a r t i c u l a r l y true for protein whose S^ was greatly increased upon heating. On the.other hand, f o r proteins of low S q whose S q was s l i g h t l y increased by heating, t h e i r emulsifying properties generally were i n i t i a l l y increased but subsequently decreased, since the decreasing 124'. s o l u b i l i t y became an increasingly important factor c o n t r o l l i n g t h e i r emulsifying properties. Results.of t h i s study ind i c a t e that the emulsifying properties of the proteins studied (native and heat denatured) could well be pre-d i c t e d solely., on the basis of protein l e v e l but not on the basis of protein s o l u b i l i t y l e v e l . It was, therefore, demonstrated that surface hydrophobicity i s a very important property governing - protein f u n c t i o n a l i t y and can be used as a r e l i a b l e predictor of emulsifying properties. I t was also shown that both hydrophobicity and s o l u b i l i t y data should be taken i n t o consideration i n order to explain and more accurately predict the emulsifying and f a t binding properties of the heat denatured proteins. 125. REFERENCES 1. Acton, J . C. and S a f f l e , R. L. 1970. S t a b i l i t y of oil-in-water emulsions. 1. E f f e c t s of surface tension, l e v e l of o i l , v i s c o s i t y and type of meat protein. J . Food S c i . 35:852. 2. Aoki, H., Taneyama, 0. and Inami, M. 1980. 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Soc. 84:4240. 46. Tanford, C. 1972. Hydrophobic free energy, m i c e l l e formation and the a s s o c i a t i o n of proteins with amphiphiles. J . Mol. B i o l . 67:59. 47. Tornberg, E. 1978. Ph.D. Thesis, Lund Inst, of Technol., Lund, Sweden. Cited by K i n s e l l a , J . E. 1979. J . Amer. O i l Chem. Soc. 56:242. 48. Tornberg, E. and Hermansson, A. M. 1977. Functional c h a r a c t e r i z a t i o n of protein s t a b i l i z e d emulsions: e f f e c t of processing. J . Food S c i . 42:468. 49. Tybor, P. T., D i l l , C. W. and Landmann, W. A. 1975. Functional properties of protein i s o l a t e d from bovine blood by a continuous p i l o t process. J . Food S c i . 40:155. 50. Volkert, M. A. and K l e i n , B. P. 1979. Protein d i s p e r s i b i l i t y and emulsion c h a r a c t e r i s t i c s of f l o u r soy products. J . Food S c i . 44:93. 51. Voutsinas, L. P. and Nakai, S. 1981. A simple tu r b i d i m e t r i c method for determining the fa t binding capacity of proteins. Abstract 26. 24th Annual Conference, Can. Inst. Food S c i . Technol. Winnipeg. 52. Walstra, P., Ourtwijn, H. and deGraaf, J . J . 1969. Neth. Milk Dairy J . 23:12. Cited by Waniska e t a l . 1981. J . Agric. Food Chem. 29:826. 53. Wang, J . C. and K i n s e l l a , J . E. 1976. Functional properties of novel proteins: a l f a l f a l e a f proteins. J. Food.Sci. 41:286. 54. Waniska, R. D., Shetty, J . K. and K i n s e l l a , J . E. 1981. P r o t e i n -s t a b i l i z e d emulsions: e f f e c t s of modification on the emulsifying a c t i v i t y of bovine serum albumin i n a model system. J . Agric. Food Chem. 29:826. 55. Wolf, W. J. and Cowan, J. C. 1975. Soybeans as a Food Source. CRC Press, Inc., Cleveland, Ohio. 129. Yamauchi, K., Shimizu, M. and Kamiya, T. 1980. E m u l s i f y i n g p r o p e r t i e s of whey p r o t e i n . J . Food S c i . 45:1237. Yasumatsu, K., Sawada, K., Moritaka, S., M i s a k i , M., Toda, J . , Wada, T. and I s h i i , K. 1972. Whipping and e m u l s i f y i n g p r o p e r t i e s of soybean products. A g r i c . B i o l . Chem. 36:719. Chapter IV Relationships Between Protein Hydrophobicity and Thermal Properties of Food Proteins 131. INTRODUCTION The u t i l i z a t i o n of proteins as food ingredients i s l a r g e l y determined by t h e i r f unctional properties such as emulsifying a c t i v i t y , emulsion s t a b i l i t y , foaming capacity, water-holding capacity, f a t absorption or fat binding, and thermal properties ( i . e . thickening, coagulation and g e l a t i o n ) . V i s c o s i t y changes can be used to evaluate the thickening power of proteins, a property of p r a c t i c a l i n t e r e s t i n f l u i d foods, e.g., soups, beverages, batters, etc. ( K i n s e l l a , 1976). The a b i l i t y of protein to form a g e l and provide a s t r u c t u r a l matrix f o r holding water, sugars, and food ingredients i s useful i n food applications and i n new product development, because i t provides an added dimension to protein function-a l i t y ( K i n s e l l a , 1976, 1979). Moreover, the g e l l i n g properties of protein contribute to texture i n ground meat - and simulated ground meat - products such as frankfurters and luncheon meats, and give chewiness to the products. Because the g e l l i n g a b i l i t y i s very important i n the manufacture of processed meats, the use of many nonmeat proteins i n processed meat products depends upon t h e i r c o m p a t i b i l i t y with meat proteins, but mostly upon t h e i r capacity to form gels, under normal processing conditions ( K i n s e l l a , 1976). Wheat gluten forms a gel when heated and may be used i n f i s h - and meat - based products as a g e l l i n g / s t r u c t u r a l agent ( K i n s e l l a , 1976). Both whey and soy proteins form gels, when dispersions of 8% or more are heated and cooled (Hermansson, 1972, 1975; Hermansson and Akesson, 1975). Thus, these are s u i t a b l e f u n c t i o n a l proteins for processed meats. Coagulation and curd formation i s an important f u n c t i o n a l property 132. of proteins, e.g., soy protein and casein, which form t o f u (soy-curd) and cheese, res p e c t i v e l y ( K i n s e l l a , 1976). Egg albumin i s a key ingredient i n many food products because of i t s a b i l i t y to coagulate upon heating (Shimada and Matsushita, 1980a). Coagulated proteins may provide body to a good product. The c e l l s of bread possess coagulated proteins (Powrie and Nakai, 1981). With the recent increase of i n t e r e s t i n the food uses of edible proteins, the d e s i r a b i l i t y of quantitative information on t h e i r f u n c t i o n a l properties has become more apparent. The objectives of the present study, therefore, were to evaluate the thermal properties of selected food proteins and to assess the value of hydrophobicity as a predictor of these f u n c t i o n a l properties. LITERATURE REVIEW The terms gelation and coagulation are not very c l e a r l y defined (Schmidt, 1981). Gelation may be t h e o r e t i c a l l y defined as a protein aggregation phenomenon i n which polymer-polymer and polymer-solvent in t e r a c t i o n s are so balanced that a well ordered t e r t i a r y network or matrix i s formed (Schmidt, 1981). This semi-elastic matrix i s capable of immobilizing or entrapping large amounts of water i n addition to other food components. On the other hand, coagulation i s a random protein aggregation i n which polymer-polymer i n t e r a c t i o n s are favored over polymer-solvents reactions, r e s u l t i n g i n a less e l a s t i c , l e s s hydrated structure than that of a protein g e l . E m p i r i c a l l y , there i s unavoidable overlap i n t h i s terminology. At the macroscopic l e v e l , i t may be d i f f i c u l t to 133. d i f f e r e n t i a t e between a highly solvated coagulum (or coagel) from a true protein gel (Schmidt, 1981). It has long been recognised (Ferry, 1948; C i r c l e et a l , 1964; Catsimpo6las and Meyer, 1970) that a gel can be made from many native proteins by heating them i n concentrated aqueous s o l u t i o n at s u i t a b l e pH and i o n i c strength. The mechanism suggested by Ferry i n 1948 i s s t i l l the most generally accepted heat-induced protein gelation mechanism. This mechanism i s a two-stage process and involves an i n i t i a l denaturation of native protein into unfolded polypeptides ( f i r s t step), which then gradually associate to form the gel network under appropriate conditions (second step). For a given rate of denaturation, the smaller the a t t r a c t i v e forces between chains of denatured protein, the slower the second step of the gelation process w i l l be; accordingly, the higher w i l l be the concentration of free denatured protein that accumulates as an i n t e r -mediary i n the course of gelation. The higher the concentration of these long-chain molecules, the f i n e r the gel network should be. Since the temperature c o e f f i c i e n t s of denaturation are enormous, i t i s to be expected that a temperature increase would accelerate the f i r s t step far more than the second, leading to a f i n e r structure (Ferry, 1948). However, conditions of the cooling step, which i s usually required to permit gel a t i o n , also a f f e c t physical c h a r a c t e r i s t i c s of gels ( K i n s e l l a , 1976). Upon cooling, the uncoiled polypeptides associate to form the network. In complex globular protein systems aggregation may occur more randomly and simultaneously with the i n i t i a l step (Schmidt, 1981). Thus, Tombs (1970, 1974) concluded from aggregation studies that the higher the randomness of aggregation the more l i k e l y i t i s that a coagel i s obtained instead of a g e l . 134. In general sense there are two basic types of heat-induced gel structures, namely r e v e r s i b l e and i r r e v e r s i b l e , depending on the conditions involved (Schmidt, 1981). In the r e v e r s i b l e gelation, a s o l or progel condition can be obtained upon heating which i s usually accompanied by increased v i s c o s i t y . This progel "sets" to form a gel upon cooling. This type of gel can usually be melted to reform the progel upon subsequent heating suggesting that the aggregation step i s r e v e r s i b l e . Gelatin gels may also be characterized as r e v e r s i b l e gels (Stainsby, 1977). I r r e v e r s i b l e gels w i l l soften or shrink with subsequent heating, but melting or reversion to the progel does not occur under p r a c t i c a l conditions (Schmidt, 1981). In contrast to g e l a t i n gels, whose s t a b i l i t y i s unaffected and most of whose properties are only s l i g h t l y changed by wide v a r i a t i o n s of concentration, pH, and s a l t content, denatured protein gels can be formed only under highly s p e c i f i c conditions with j u s t the r i g h t balance of a t t r a c t i v e and repulsive forces between polypeptide chains (Ferry, 1948). Thus, the method of preparation of protein, i t s concentration, pH, temperature and duration of heating, cooling conditions, the presence of s a l t s , t h i o l s , s u l f i t e , and/or l i p i d s a l l influence the properties of the gels formed ( C i r c l e et a l . 1964; Catsimpoolas and Meyer, 1970). In addition to e f f e c t s of the s i z e , shape and arrangement of the primary protein strands comprising•the gel network, the c h a r a c t e r i s t i c s of protein gels are affected by i n t r a - and i n t e r - s t r a n d c r o s s - l i n k i n g . Protein gels may be cross-linked by s p e c i f i c bonding at s p e c i f i c s i t e s on the protein strands or by nonspecific bonding occurring along the protein strand. Hydrogen bonding, d i s u l f i d e bridging and hydrophobic a t t r a c t i o n s play major r o l e s i n c r o s s - l i n k i n g and s t a b i l i z i n g the structure of a 135. protein gel or coagulum (Schmidt, 1981). The nature and degree of cross-l i n k i n g would vary with the type of protein and gel a t i o n environment (Schmidt, 1981). MATERIALS AND METHODS Materials H C l - s o l u b i l i z e d gluten was prepared according to the method of Wu et a l . (1976) as follows; a 5% dispersion of v i t a l gluten (Whetpro 75%) i n 0.05 N HC1 was autoclaved at 121°C for 15 min. The pH was adjusted to 7.0 and the sample was dialyzed 2 days against running water, one day against d i s t i l l e d water, and then, freeze-dried. The protein content of the dry product as determined by the micro-Kjeldahl method was 74.5%. A l l other proteins and reagents were the same as those used i n chapters II and III of t h i s t h e s i s . Methods A. Thickening determination Aqueous dispersions of 8% (W/W) protein were prepared for selected food proteins by s t i r r i n g f o r 5 min on a magnetic s t i r r e r and then homogenizing i n a Sorval Omni-mixer at speed s e t t i n g 1 for 1 min. The a i r was removed from the s l u r r i e s under vacuum. The experiment was conducted at pH 7.0, the adjustment being made with 1 N NaOH. Two d i f f e r e n t models of Brookfield (Cooksville, Ont.) Synchro-Lectric viscometers were used to measure the v i s c o s i t y of protein dispersions. Thus, a model LVT, f i t t e d with a UL adapter and operated at 6 rpm, was used 136. for measuring the v i s c o s i t y of the unheated protein dispersions at 24 C. A model RVT, f i t t e d with a jacketed, cylinder attachment (small sample adapter) and a spindle No. SC4-21, was used for measuring the v i s c o s i t y of the heated samples at 90°C as follows: the protein dispersion was transferred to the small sample adapter, heated to 90°C by a c i r c u l a t i n g bath, and when the temperature of the dispersion reached the 90°C mark, the viscometer was turned on and continuously operated at 100 rpm. V i s c o s i t y readings were taken at one min i n t e r v a l s f o r 5 min. The di f f e r e n c e between f i n a l (heated for 5 min at 90°C) and i n i t i a l (unheated, 24°C) v i s c o s i t y indicated the v i s c o s i t y gained or l o s t during the heating process and was used as a measure of the thickening power of the protein. B. Heat coagulation determination Heat c o a g u l a b i l i t y was determined according to the method of Balmaceda et a l . (.1976) as follows: a 5% (W/W) protein dispersion i n water was prepared by s t i r r i n g for 5 min on a magnetic s t i r r e r . The pH was adjusted to 7.0 with 1 N NaOH and the dispersion was then centrifuged at 27,000 x g for 30 min. The protein content of the supernatant was determined by the Phenol-Biuret method (Brewer et; j i l . 1974). A portion (10 ml) of the supernatant was heated i n a centrifuge . b o t t l e (hermetically closed) at 98 - 100°C for 30 min and then cooled to room temperature. The heated sample was subsequently centrifuged at 27,000 x g for 15 min, f i l t e r e d through Whatman No. 2 f i l t e r paper, and the protein content of the f i l t r a t e was determined. The heat c o a g u l a b i l i t y (HC) of the sample was calculated from the following equation: HC = Protein cont. supernatant (%) - protein cont. f i l t r a t e (%) x 100 Protein cont. supernatant (%) 137. G. Gelation measurement Aqueous dispersions of 10% (W/W) protein were prepared f o r selected food proteins by s t i r r i n g on a magnetic s t i r r e r for' 5 min. The pH was adjusted to 7.0, and an aliquot (15 ml) of each dispersion was transferred into a closed container, heated at 100°C for 40 min and then cooled i n an ice-bath to room temperature. The g e l l i n g a b i l i t y of each protein was q u a l i t a t i v e l y determined by v i s u a l l y judging the c h a r a c t e r i s t i c s of the f i n a l product and assigning to i t a value corresponding to a r a t i n g scale of -4 to +3. The r a t i n g scale was as follows: -4, smooth l i q u i d ; -3, s l i g h t feathering ( s l i g h t l y granular l i q u i d ) ; -2, moderate feathering; -1, syneresis (after inversion of the container the structure collapsed); .0, viscous g e l - l i k e semi-liquid; +1, s o f t g e l , +2, medium ge l ; and +3, firm g e l . D. Hydrophobicity determination Surface hydrophobicity ( S q ) was determined as described i n Chapter I I of t h i s t h e s i s . The hydrophobicity of the same proteins was also determined f l u o r o m e t r i c a l l y with c i s - p a r i n a r i c acid as a probe, a f t e r the protein dispersions were heated at 100°C for 10 min i n the presence of 1.5% sodium dodecyl s u l f a t e (Townsend, 1982). This hydrophobicity was designated as S g and i t was shown by Townsend (1982) to be l i n e a r l y correlated with the average hydrophobicity values of Bigelow (1967) for various proteins. Thus,'.in t h i s study S^ w i l l be used as an index of the average hydrophobicity of proteins. 138. E. Determination of s u l f h y d r y l and d i s u l f i d e groups of proteins The s u l f h y d r y l (free and buried SH) and t o t a l s u l f h y d r y l (SH + reduced SS) groups of the proteins were determined according to the method of Beveridge et _al. (1974) a f t e r s l i g h t modification as follows: (1) soy protein i s o l a t e , Promine-D, pea protein i s o l a t e (a), canola i s o l a t e (b), sunflower i s o l a t e , H C l - s o l u b i l i z e d gluten. A 75 mg sample was suspended i n 1 ml of T r i s - g l y c i n e buffer, pH 8.0, containing 0.12% EDTA, denoted as T r i s - G l y ; 4.7 g of guanidine hydrochloride was added, and the volume made to 10 ml. For SH determination, to 1 ml of t h i s s o l u t i o n was added 4 ml of 8 M urea i n T r i s - G l y and then 0.05 ml of Ellman's reagent (4 mg of 5, 5' -d i t h i o b i s - 2 - nitrobenzoic acid i n 1 ml Tris- G l y ) was added. For t o t a l SH (SH + reduced SS) determination, to 1 ml of the protein s o l u t i o n was added 0.05 ml of 2-mercaptoethanol and 4 ml of 8 M urea i n T r i s - G l y , and the mixture was incubated for 1 hr at 25°C. Aft e r an a d d i t i o n a l 1 hr incubation with 10 ml of 12% TCA, the tubes were centrifuged at 5,000 x g, for 10 min. The p r e c i p i t a t e was twice resuspended i n 5 ml of 12% TCA and centrifuged to remove • . 2-mercaptoethanol. The p r e c i p i t a t e was dissolved i n 10 ml of 8 M urea i n T r i s - G l y and the color was developed with 0.04 ml of Ellman's reagent. (2) for casein and whey proteins. For SH deter-mination, 10 mg of sample was dissolved i n 5 ml of 8 M urea i n T r i s - G l y , and then, 0.04 ml of Ellman's reagent was added. For t o t a l SH determination, 10 mg of sample was dissolved i n 5 ml of 10 M urea i n T r i s - G l y . Then, 0.1 ml of 2-mercaptoethanol was added and mixed with a Vortex. The mixture was held at 25°C for 1 hr. A f t e r p r e c i p i t a t i o n and washing of protein as f o r the other proteins above (1), the p r e c i p i t a t e was dissolved i n 10 ml of 8 M urea i n T r i s - G l y . An aliquot of 3 ml was taken and 0.05 ml of Ellman's reagent was added to i t for color development. 139. Absorbance was measured at 412 nm on a Beckman DB spectrophotometer. The amount of SH and t o t a l SH was calculated from the following equation: uM SH/g = 73.53 A .D , where A,,„ = the absorbance at 412 nm; 412 412 C C = the sample concentration i n mg solids/ml; and D = the d i l u t i o n f a c t o r . F. S t a t i s t i c a l a n alysis S t a t i s t i c a l analysis of the data obtained i n t h i s study was done as described i n chapter I II of t h i s t h e s i s . Five independent v a r i a b l e s were used i n the i n i t i a l equation i n the backwards stepwise multiple regression analysis including hydrophobicity ( S q or S £ ) , su l f h y d r y l s (SH or SH + reduced SS), i n t e r a c t i o n of hydrophobicity and s u l f h y d r y l s , and the quadratic powers of hydrophobicity and s u l f h y d r y l s . It should be mentioned that the models for the p r e d i c t i o n of the thermal properties as well as the fu n c t i o n a l properties studied i n Chapters II and III of t h i s thesis were selected on the basis of the s t a t i s t i c a l s i g n i f i c a n c e of F - p r o b a b i l i t i e s of the p a r t i a l regression c o e f f i c i e n t s . RESULTS AND DISCUSSION Table 1 shows the r e l a t i o n s h i p s of hydrophobicities and sulfhydryls of various proteins with t h e i r thermal properties. As can be seen, the proteins studied exhibited d i f f e r e n t behaviour upon heating. Thickening of proteins was measured as the increase i n apparent v i s c o s i t y of protein dispersions upon heating. Among the proteins studied whole casein and Promine-D showed some thinning (decrease i n v i s c o s i t y ) upon heating, Table 1 - Relationships between protein hydrophobicities, s u l f h y d r y l s , s u l f h y d r y l s + reduced d i s u l f i d e s and thermal properties of various p r o t e i n s . 3 Protein Surface hydropho-b i c i t y ( So> Average hydropho-b i c i t y ( s e ) SH (uM/g prot.) SH+ r.SS (yM/g prot.) Thickening app. v i s -c o s i t y (Pa.s x 10 ) Heat co-agulation (%) Gelation r a t i n g ^ Soy i s o l a t e 95 822 3.2 56.1 12.5 0.0 0 Promine-D 39 927 3.2 57.7 -26.0 0.0 0 Pea i s o l a t e 66 277 12.1 54.8 2.2 0.0 -4 Canola i s o l a t e 65 950 7.4 90.2 32.2 47.7 -1 Sunflower i s o l a t e 47 597 6.5 126.9 84.8 22.6 -1 Whole casein 28 725 1.4 3.5 -0.8 4.8 -3 Whey protein 182 387 20.2 248.4 105.1 9.9 +1 HC l - s o l u b i l i z e d gluten 17 349 2.8 101.9 6.2 0.0 -4 Average of duplicate determinations 'For gelation r a t i n g scale see Materials and Methods 141. whereas a l l others showed d i f f e r e n t degrees of thickening, with whey protein e x h i b i t i n g the greatest thickening power. Heat coagulation was determined as % l o s s i n s o l u b i l i t y of a protein a f t e r heating (98 - 100°C for 30 min). As seen from Table 1, soy i s o l a t e , Promine-D, pea i s o l a t e and H C l - s o l u b i l i z e d gluten did not show any coagulation on heating, whereas canola i s o l a t e , sunflower i s o l a t e , whole casein, and whey protein showed d i f f e r e n t degrees of -. coagulation, with canola i s o l a t e e x h i b i t i n g the greatest c o a g u l a b i l i t y . The observation that whole casein exhibited a small coagulation was unexpected, since i t i s well known that casein i s very r e s i s t a n t towards heat coagulation. However, protein s o l u b i l i t y i s affected by a magnitude of f a c t o r s , such as conditions of s o l u b i l i t y determination, processing h i s t o r y , the presence of other ingredients i n the sample, etc. ( K i n s e l l a , 1976), and t h i s can account for the observed coagulation of casein. The g e l l i n g a b i l i t y of proteins i s usually determined q u a n t i t a t i v e l y by measuring the v i s c o s i t y of the gel with Helipath viscometers ( C i r c l e et a l . , 1964; Catsimpoolas and Meyer, 1970, 1971; Hermansson, 1972; Hermansson and Akesson, 1975). However, Fleming et a l . (1975) reported that v i s c o s i t y was not d i r e c t l y associated with the gelation property; a soybean concentrate (Isopro) showed a high v i s c o s i t y a f t e r heating but remained granular and pourable. S i m i l a r l y , the high v i s c o s i t y of the sunflower concentrates studied did not i n d i c a t e gel formation since the products formed an i r r e g u l a r structure with a foamy appearance. On the other hand, the)importance of q u a l i t a t i v e evaluation of protein gel systems 142. has been l a t e l y discussed by Schmidt (1981). For the aforementioned reasons the g e l l i n g a b i l i t y of the proteins i n t h i s study was q u a l i t a t i v e l y evaluated by v i s u a l l y observing the c h a r a c t e r i s t i c s of the f i n a l product. As seen from Table 1, whey was the only protein that showed a true gel structure upon heating and cooling. Soy i s o l a t e and Promine-D formed a viscous g e l - l i k e semi-liquid (not a self-supported g e l ) . A l l other proteins showed eit h e r some degree of coagulation (feathering or syneresis) or remained as smooth l i q u i d s (pea i s o l a t e and H C l - s o l u b i l i z e d gluten). The r e s u l t s of t h i s study can not e a s i l y be compared with r e s u l t s of other studies due to lack of consistent methodology. For example, protein concentration, temperature and duration of heating, cooling conditions (mainly duration of cooling), pH, etc., vary considerably among d i f f e r e n t studies. For instance, some studies have been conducted at the natural pH of each product (Sosulski jet al., 1976). It should be noted here, however, that when during preliminary studies heat coagulation was conducted at the natural pH of each product, a l l products exhibited d i f f e r e n t degrees of coagulation as opposed to the r e s u l t s of Table 1. This i s , of course, due to the smaller e l e c t r o s t a t i c repulsions between protein molecules at pHs lower than 7.0 (but above the i s o e l e c t r i c p o i n t ) . Thus, the a t t r a c t i v e forces generated between protein molecules by thermal unfolding predominated over the repulsive forces (due to the protein charge) r e s u l t i n g i n coagulum formation. It can be mentioned, however, that Hermansson (1972) reported that gelation occurred for Promine-D and whey protein concentrate (WPC) but not for caseinate (10% protein d i s p e r s i o n s ) . She also observed that the gel strength of Promine-D increased to a maximum (corresponding to 80°C) and then decreased as the temperature was raised above t h i s point ( i n contrast to the gel strength 143. of WPC which increased w i t h i n c r e a s i n g heating temperature). M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n of hyd r o p h o b i c i t y , s u l f h y d r y l s , and s u l f h y d r y l s + reduced d i s u l f i d e s e f f e c t s on thermal f u n c t i o n a l p r o p e r t i e s studied are presented i n Table 2. As shown, two models were obtained f o r p r e d i c t i o n of the t h i c k e n i n g power of p r o t e i n s . In the f i r s t model, t h i c k e n i n g was s i g n i f i c a n t l y a f f e c t e d by S^ (average h y d r o p h o b i c i t y ) , the i n t e r a c t i o n of S and s u l f h y d r y l s , and the square 2 of S . The R value of t h i s model was 0.961, i n d i c a t i n g that 96.1% of the v a r i a b i l i t y i n t h i c k e n i n g could be accounted f o r by the aforementioned 3 independent v a r i a b l e s . A comparison of the g-values of these v a r i a b l e s i n d i c a t e d t h a t S^ was the primary determinant of t h i c k e n i n g . This model a l s o i n d i c a t e s that had a c u r v i l i n e a r e f f e c t on t h i c k e n i n g , that i s , as the value of increased the t h i c k e n i n g was i n i t i a l l y increased and then decreased. This trend can a l s o be seen i n Figure 1, which shows the t h i c k e n i n g response surface contour as a f u n c t i o n of S^ and SH content. As shown, re g a r d l e s s of the SH content, the response of t h i c k e n i n g to i n c r e a s i n g l e v e l s of S increased and then decreased. Moreover, i t i s e evident that at low S^ va l u e s , i n c r e a s i n g SH content increased the t h i c k e n i n g power, but at medium and high S g v a l u e s , SH content d i d not seem to s i g n i f i c a n t l y a f f e c t t h i c k e n i n g . In the second model (Table 2) th i c k e n i n g was s i g n i f i c a n t l y a f f e c t e d by S e, the sum of s u l f h y d r y l s and reduced d i s u l f i d e s , the i n t e r a c t i o n as w e l l as the square powers of these 2 two independent v a r i a b l e s . The R value of t h i s model was 0.995. Comparing^ both r e g r e s s i o n models of Table 2, i t seems that the l a t t e r i s most r e l i a b l e i n p r e d i c t i n g the t h i c k e n i n g power of p r o t e i n s , s i n c e i t s 2 R and S.E. values are higher and s m a l l e r , r e s p e c t i v e l y . The heat c o a g u l a b i l i t y of p r o t e i n s was s i g n i f i c a n t l y a f f e c t e d by the Table 2 - M u l t i p l e regression models for prediction of thermal properties of various proteins Dependent v a r i a b l e Variable d e s c r i p t i o n Regression c o e f f i c i e n t F - r a t i o F - p r o b a b i l i t y 5-value Thickening (R = 0.961, P < 0.01: S.E. 11.79) Constant S e S e x SH S 2 e -202.675 0.754 0.016 -0.001 33.04 71.96 38.98 0.005 0.001 0.003 4.461 0.867 -4.857 Thickening (R 2 = 0.995, P < 0.01; S.E. 5.79) Constant S e SH+ r.SS S p.x SH+ r.SS S 2 3 e 2 (SH+ r.SS) 2.469 0.349 -2.293 0.004 -0.001 0.004 21.16 33.00 58.99 82.36 33.49 0.035 0.029 0.017 0.012 0.029 2.065 -3.710 2.665 -3.616 1.906 Heat coagulation (R 2 = 0.740, P <c 0.05; S.E. a = 10.21) Constant SH S x SH e -5.618 -2.495 0.009 6.443 13.870 0.050 0.014 -0.930 1.364 Gelation (R 2 = 0.740, P < 0.05; S.E. a = 1.16) Constant S e 2 (SH) Z -6.543 0.006 0.012 11.48 9.913 0.020 0.025 0.895 0.831 Gelation (R 2 = 0.810, P < 0.05; S.E. a = 1.00) Constant S e SH+ r.SS -7.202 0.006 0.022 14.42 15.33 0.013 0.011 0.809 0.834 Standard error of estimate 146. SH content and the i n t e r a c t i o n of S and SH content (Table 2) . The R*" e value of the model was 0.740, i n d i c a t i n g that 74.0% of the v a r i a b i l i t y i n heat coagulation could be accounted for by these two v a r i a b l e s . A comparison of the 8-values of these variables indicated that the i n t e r a c -t i o n of S g and SH content was the most important v a r i a b l e i n the model. Figure 2 shows the heat coagulation response surface contour as a function of S g and SH content. As shown i n t h i s Figure, regardless of the SH content, as S^ increased the heat c o a g u l a b i l i t y was i n i t i a l l y increased and then decreased. The region of the maximum response (heat coagulation) was l o c a l i z e d i n the region defined by r e l a t i v e l y high l e v e l of S and r e l a t i v e l y low l e v e l s of SH content. I t should be noted that e J no s i g n i f i c a n t c o r r e l a t i o n was found between S^, SH + reduced SS and heat coagulation. Two models were obtained for p r e d i c t i o n of the g e l l i n g a b i l i t y of proteins (Table 2). In the f i r s t model, gelation was" s i g n i f i c a n t l y affected by S g and the square of s u l f h y d r y l s . The 8-values of t h i s model suggested that both independent variables were equally important i n determining the g e l l i n g a b i l i t y of the proteins studied. In t h i s model, the s t a t i s t i c a l s i g n i f i c a n c e of the square of s u l f h y d r y l content in d i c a t e s that the response of g e l a t i o n to increasing l e v e l s of SH can be depicted as a c u r v i l i n e a r graph rather than a s t r a i g h t l i n e . In the second model, gel a t i o n was s i g n i f i c a n t l y affected by S £ and the sum of sulfhydryls and reduced d i s u l f i d e s . I t i s obvious from Table 2, that the r e l a t i o n s h i p between S^, SH + reduced SS and gelation was l i n e a r . Both independent va r i a b l e s of t h i s model were equally important for determining the g e l l i n g a b i l i t y of proteins. Comparing both regression models, i t seems that 2 the l a t t e r i s most r e l i a b l e i n p r e d i c t i n g g e l a b i l i t y since i t s R and 148. S.E. values were higher and smaller, r e s p e c t i v e l y . It should be noted, that as expected, no c o r r e l a t i o n (simple or multiple l i n e a r ) was found between surface hydrophobicity (S Q) and any of the thermal properties studied. The r e s u l t s of t h i s study concerning the importance of S e, SH and SS contents for the thermal properties of proteins are i n agreement with the r e s u l t s of most previous studies attempting to i d e n t i f y the types of bonds responsible f o r protein gelation or coagulation ( C i r c l e ej: a l . , 1964; Furukawa et a l . , 1979; Fukushima, 1980; H i l l i e r et a l . , 1980; Itoh et a l , 1980a, 1980b; Shimada and Matsushita, 1980a, 1980b, 1981). C i r c l e e_t al. (1964) reported that the s p e c i f i c d i s u l f i d e - r e d u c i n g agents, sodium s u l f i t e and cysteine, profoundly decreased the v i s c o s i t y of both unheated and heated 10% dispersions of soy i s o l a t e , and prevented g e l a t i o n . This f i n d i n g was interpreted as being i n d i c a t i v e of p a r t i c i p a t i o n of d i s u l f i d e bonds (through s u l f h y d r y l - d i s u l f i d e chain reaction) i n the gel a t i o n process. Furukawa e_t a l . (1979) demonstrated that the gel network of i s o l a t e d soy protein was formed through c r o s s - l i n k i n g by d i s u l f i d e , hydrogen and hydrophobic bonds. The findings of C i r c l e et^ a l . (1964) and Furukawa et a l . (1979) are i n contrast to the f i n d i n g of Catsimpoolas and Meyer (1970) who reported that the bonds involved i n the sol-progel and g e l t r a n s i t i o n s appeared to be p r i m a r i l y of noncovalent nature ( i . e . hydrogen, hydrophobic and i o n i c bonds). H i l l i e r ejt a l . (1980) at t r i b u t e d the heat-induced gelation of whey powders to d i s u l f i d e cross-l i n k i n g . They showed conclusively that the rate of g e l l i n g (taken as an index of the g e l l i n g a b i l i t y of a protein preparation) depended on the su l f h y d r y l content of the whey powders, and that i t was the amount of t o t a l SH groups that was important, not the free SH; t h i s was expected, 149. since at 80°C, the standard temperature used i n measuring g e l l i n g time, there could be l i t t l e d i f f e r e n c e i n the r e a c t i v i t y of free and masked SH groups ( H i l l i e r et a l . , 1980). The same authors, however, found a poor c o r r e l a t i o n between the rate of g e l l i n g and t o t a l SH content and suggested there were important d i f f e r e n c e s , i n addition to SH content, between the whey powders which affected t h e i r rate of g e l l i n g and t h e i r appearance a f t e r g e l a t i o n . Itoh et a l . (1980a, 1980b) reported that i n the formation of polymeric protein molecules during the heat gelation of carp actomyosin SH groups were involved (through formation of SS bonds). According to Schmidt (1981) hydrophobic i n t e r a c t i o n s are important to d i s s o c i a t i v e - a s s o c i a t i v e reactions which i n i t i a t e the g e l a t i o n process. These a t t r a c t i o n s could also be involved i n layering or thickening of the gel network strands upon cooling which r e s u l t s i n improved strength and s t a b i l i t y . According to Fukushima (1980) the tofu-gel (a g e l , although i s also r e f e r r e d to as curd, prepared by coagulation of soybean milk with calcium s a l t ) made from 7S gl o b u l i n i s mostly s t a b i l i z e d by hydrophobic bonds, while the tofu-gel made from 11S g l o b u l i n i s s t a b i l i z e d by both d i s u l f i d e bonds through s u l f h y d r y l - d i s u l f i d e interchange reaction and hydrophobic bonds. This i s the reason why 7S tofu-gel i s s o f t , while 11S tofu-gel i s remarkably harder (Saio et a l . 1971). Shimada and Matsushita (1980a) based on t u r b i d i t y studies suggested that the thermocoagulable matrix formation of egg albumin was due to d i s u l f i d e c r o s s - l i n k i n g (by s u l f h y d r y l -d i s u l f ide exchange) and hydrophobic i n t e r a c t i o n s . The same authors (Shimada and Matsushita, 1980b, 1981), subsequently concluded that the mechanism of protein thermocoagulation i s l a r g e l y dependent on hydrophobic i n t e r a c t i o n s among proteins. CONCLUSIONS Results of t h i s study i n d i c a t e that the thermal properties of the proteins studied could not be explained s o l e l y on the basis of average hydrophobicity or sulf h y d r y l s (SH or the sum of SH and reduced SS). However, i t was demonstrated that these f u n c t i o n a l properties could be r e l i a b l y predicted on the basis of both average hydrophobicity and sul f h y d r y l s . I t was, thus, concluded that these v a r i a b l e s are the main determinants of the thermal properties studied. 151. REFERENCES 1. Balmaceda, E. A., Kim, M. K., Franzen, R. , Mardones, B., and Lugay, J . C. 1976. Protein f u n c t i o n a l i t y : standard t e s t s . Presented at the 36th annual meeting of the Inst, of Food Technol., Anaheim, CA. 2. Beverldge, T., Toma, S. J . and Nakai, S. 1974. Determination of the SH-- and SS - groups i n some food proteins using Ellman's reagent. J . Food S c i . 39:49. 3. Bigelow, C. C. 1967. On the average hydrophobicity of proteins and the r e l a t i o n between i t and protein structure. J . Theoret. B i o l . 16:187. 4. Brewer, J. M., Pesce, A. J . and Ashworth, R. B. 1974. Experimental Techniques i n Biochemistry. Prentice-Hall.Inc., Englewood C l i f f s , N.J. 5. Catsimpoolas, N. and Meyer, E. W. 1970. Gelation phenomena of soybean globulins. 1. Protein-protein i n t e r a c t i o n s . Cereal Chem. 47:559. 6. Catsimpoolas, N. and Meyer, E. W. 1971. Gelation phenomena of soybean globulins. 3. P r o t e i n - l i p i d i n t e r a c t i o n s . Cereal Chem. 48:159. 7. C i r c l e , S. J . , Meyer, E. W. and Whitney, R. N. 1964. Rheology of soy protein dispersions. E f f e c t of heat and other factors on g e l a t i o n . Cereal Chem. 41:157. 8. Ferry, J . D. 1948. Protein gels. Adv. Protein Chem. 4:1. 9. Fleming, S. E., Sosulski, F. W. and Hamon, N. W. 1975. Gelation and thickening phenomena of vegetable protein products. J . Food S c i . 40:805. 10. Fukushima, D. 1980. Deteriorative changes of proteins during soybean food processing and t h e i r use i n foods. In "Chemical d e t e r i o r a t i o n of proteins". Eds. Whitaker, J . R. and Fujimaki, M. Amer. Chem. Soc. Symposium Series 123, Washington, D.C. 11. Furukawa, T., Ohta, S. and Yamamoto, A. 1979. Texture-structure relationship.in heat-induced soy protein gels. J . Texture Studies 10:333. 12. Hermansson, A.-M. 1972. Functional properties of protein for foods: swelling. Lebensmitt.-Wiss. u. Technol. 5:24. 13. Hermansson, A.-M. 1975. Functional properties of added proteins correlated with properties of meat systems. E f f e c t on texture of a meat product. J . Food S c i . 40:611. 152. 14. Hermansson, A.-M. and Akesson, C. 1975. Functional properties of added proteins correlated with properties of meat systems. E f f e c t of concentration and temperature on water-binding properties of model meat systems. J . Food S c i . 40:595. 15. H i l l i e r , R. M., Lyster, R. L. J . and Cheeseman, G. C. 1980. Gelation of reconstituted whey powders by heat. J . S c i . Food Agric. 31:1152. 16. Itoh, Y., Yoshinaka, R. and Ikeda, S. 1980a. Changes of higher molecular weight of protein molecules during the g e l formation of carp actomyosin by heating and p a r t i c i p a t i o n of SH groups i n these changes. B u l l . Jap. Soc. S c i . F i s h . 46:617. 17. Itoh, Y., Yoshinaka, R. and Ikeda, S. 1980b. Formation of polymeric molecules of protein r e s u l t i n g from intermolecular SS bonds formed during the g e l formation of carp actomyosin by heating. B u l l . Jap. Soc. S c i . F i s h . 46:621. 18. K i n s e l l a , J . E. L976. Functional properties of proteins i n foods: a survey. C r i t . Rev. Food S c i . Nutr. 7:219. 19. K i n s e l l a , J . E. 1979. Functional properties of soy protein. J . Amer. O i l Chem. Soc. 56:242. 20. Powrie, W. D. and Nakai, S. 1981. Processing e f f e c t s on protein systems. In " U t i l i z a t i o n of protein resources". Eds. Stanley, D. W., Murray, E. D. and Lees, D. H. Food and N u t r i t i o n Press, Inc., Westport, CT. 21. Saio, K., Kajikawa, M. and Watanabe, T. 1971. Food processing c h a r a c t e r i s t i c s of soybean proteins. Part 2. E f f e c t of s u l f h y d r y l groups on ph y s i c a l properties of tof u - g e l . Agric. B i o l . Chem. 35:890. 22. Schmidt, R. H. 1981. Gelation and coagulation. In "Protein, f u n c t i o n a l i t y i n foods". Ed. Cherry, J . P. Amer. Chem. Soc. Symposium Series 147, Washington, D.C. 23. Shimada, K. and Matsushita, S. 1980a. Thermal coagulation of egg albumin. J . Agric. Food Chem. 28:409. 24. Shimada, K. and Matsushita, S. 1980b. Relationship between thermocoagulation of proteins and amino acid compositions. J . Agric. Food Chem. 28:413. 25. Shimada, K. and Matsushita, S. 1981. E f f e c t s of s a l t s and denaturants on thermocoagulation of proteins. J . Agric. Food Chem. 29:15. 26. Sosulski, F., Humbert, E. S., Bui, . K. and Jones, J . D. 1976. Functional properties of rapeseed f l o u r s , concentrates and i s o l a t e . J . Food S c i . 41:1349. 153. 27. Stainsby, G. 1977. The g e l a t i n gel and the s o l - g e l transformation. In "The science and technology of g e l a t i n " . Eds. Ward, A. G. and Courts, A. Academic Press, N.Y. 28. Tombs, M. P. 1970. A l t e r a t i o n s of proteins during processing and the formation of structures. In "Proteins as human food", Ed. Lawrie, R. A. Butterworths, London. 29. Tombs, M. P. 1974. Gelation of globular proteins. Faraday Discuss. Chem. Soc. 57:158. Cited by Hermansson, A.-M. 1979. Aggregation and denaturation involved i n gel formation. In " F u n c t i o n a l i t y and protein structure". Ed. Pour-EL, A. Amer. Chem. Soc. Symposium Series 92, Washington, D.C. 30. Townsend, A.-A. E. 1982. Relationship between physicochemical properties of proteins and t h e i r foaming c h a r a c t e r i s t i c s . Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia. 31. Wu, C. H., Nakai, S. and Powrie, W. D. 1976. Preparation and properties of a c i d - s o l u b i l i z e d gluten. J . Agric. Food Chem. 24:504. 154. GENERAL CONCLUSIONS The r e s u l t s of the study on hydrophobic immobilization of proteases included i n the f i r s t chapter of t h i s t hesis indicated that a l l immobilized enzyme preparations exhibited high i n i t i a l a c t i v i t y . The major problem encountered was the rapid reduction i n enzymatic a c t i v i t y due to the l o s s of enzyme from the hydrophobic c a r r i e r s . An economic study showed that continuous coagulation of skimmilk with p r o t e o l y t i c enzymes immobilized on the hydrophobic c a r r i e r s employed was u n l i k e l y to be f e a s i b l e . Therefore, t h i s study was discontinued and our i n t e r e s t was then s h i f t e d to the r e l a t i o n s h i p of protein hydrophobicity to f u n c t i o n a l i t y . In the second chapter a new, simple turbidimetric method was developed for determining the a b i l i t y of protein to bind f a t , since f a t absorption or f a t binding, as assessed by the e x i s t i n g methods, was a c t u a l l y a measure of o i l which was p h y s i c a l l y entrapped into protein tested. The r e s u l t s i n t h i s study showed that the t u r b i d i t y due to o i l was dependent on the wavelength used for t u r b i d i t y measurement, blending time and volume of o i l . The f a t binding capacity of 11 food proteins tested was p o s i t i v e l y affected by surface hydrophobicity and negatively by an i n t e r a c t i o n of surface hydrophobicity and s o l u b i l i t y . According to the r e s u l t s obtained i n the t h i r d chapter, heating did not always deteriorate the emulsifying properties of the proteins. Instead, for proteins, of which surface hydrophobicity was markedly increased upon heating, t h e i r emulsifying properties were s u b s t a n t i a l l y improved. This improvement was a t t r i b u t e d to the greater surface hydrophobicity as well as to increased v i s c o s i t y of these heat denatured 155. protein samples. However, for proteins with low surface hydrophobicity, of which hydrophobicity was only s l i g h t l y increased by heating, t h e i r emulsifying properties were generally adversely affected by heating probably due to decrease i n s o l u b i l i t y . Simple l i n e a r regression a n a l y s i s of the obtained data revealed that surface hydrophobicity, but not s o l u b i l i t y , could be used as a r e l i a b l e predictor of the emulsifying properties of heat denatured proteins. I t i s , therefore, p o s s i b l e that surface hydrophobicity i s a very important property governing protein f u n c t i o n a l i t y . However, backwards stepwise multiple regression analysis showed that both surface hydrophobicity and s o l u b i l i t y should be taken into account i n p r e d i c t i n g the emulsifying and f a t binding properties of heat denatured proteins. Regression analysis of the data obtained i n the fourth chapter showed that average and not the surface hydrophobicity of proteins was important for heat gelation, coagulation and thickening. Simple l i n e a r regression analysis demonstrated that neither average hydrophobicity nor s u f l h y d r y l content could by i t s e l f explain these thermal properties. However, when the average hydrophobicity and s u l f h y d r y l content were both used i n multiple regression a n a l y s i s , the thermal properties of proteins could be accurately predicted. 

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