Food Protein Deterioration - American Chemical Society

12 Protein Structure and Functional Properties: Emulsification and Flavor Binding Effects

Downloaded by UCSF LIB CKM RSCS MGMT on December 2, 2014 | http://pubs.acs.org Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch012

JOHN E. KINSELLA Cornell University, Institute of Food Science, Ithaca, NY 14853

Proteins are the p r i n c i p a l structural and functional compo nents of many food systems; e.g., meat, cheese, gelatin, egg white and many cereal products. In addition, proteins are being used increasingly to fabricate and facilitate the engineering of new foods such as protein beverages and extruded foods. These and other applications depend upon the physicochemical properties of protein ingredients, c o l l e c t i v e l y referred to as the functional properties. Proteins per se as dry powders, have very limited appeal to potential users or consumers. To facilitate their use in foods and their conversion to desirable ingredients they must possess appropriate functional properties following interactions with other food components; e.g., water, carbohydrates or l i p i d s , during processing. Functional properties of proteins are those physicochemical properties of proteins which affect their behavior i n food systems during preparation, processing, storage, and con sumption, and contribute to the quality and organoleptic a t t r i b u tes of food systems (1). Generally, n u t r i t i o n a l properties are not included in this category. Several typical classes of func tional properties are shown i n Table I . There are numerous examples of functional properties i n t r a d i t i o n a l foods; e . g . , v i s c o e l a s t i c i t y of wheat gluten, texture, succulence and color of myofibrillar proteins i n meats, curd for mation of caseins, and whippability of egg white proteins. Different food applications require different characteristics; e . g . , i n beverages, proteins should be soluble; i n comminuted meats, they should have emulsion s t a b i l i z i n g and g e l l i n g proper ties ; and i n whipped toppings they must s t a b i l i z e the foam. The type of functional properties required i n a protein or a protein mix varies with the particular food system i n question. Thus, i n meat systems, water binding, s o l u b i l i t y , emulsifying a c t i v i t y , adhesiveness, swelling, viscosity and gelation are typical proper ties of proteins that determine their usefulness and impact on the f i n a l quality. These c r i t e r i a are c r i t i c a l , and as the number of processed and fabricated foods increase, greater reliance w i l l be placed on the consistent performance of ingredients i n specific food formulations. 0097-6156/82/0206-0301 $07.50/0 © 1982 American Chemical Society In Food Protein Deterioration; Cherry, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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Table I.

T y p i c a l f u n c t i o n a l p r o p e r t i e s performed by p r o t e i n s i n food systems.


Functional Property Solubility Water a b s o r p t i o n and b i n d i n g Viscosity Gelation Cohesion-adhesion

Elasticity

Emulsification Fat

absorption

Flavor-binding Foaming

Mode o f A c t i o n Protein solvation Hydrogen-bonding o f w a t e r , Entrapment o f water T h i c k e n i n g , Water b i n d i n g Protein matrix formation and s e t t i n g P r o t e i n a c t s as adhesive material Hydrophobic bonding i n gluten, Disulfide links i n gels F o r m a t i o n and s t a b i l i z a t i o n o f f a t emulsions Binding of free f a t A d s o r p t i o n , entrapment, release Form s t a b l e f i l m s t o e n t r a p gas

Food S y s t e m Example Beverages Meats, Sausages, B r e a d s , Cakes Soups, G r a v i e s Meats, Curds, Cheese Meats, Sausages, Baked g o o d s , Pasta products Meats, Bakery

Sausages, Bologna, Soup, Cakes Meats, Sausages, Donuts Simulated meats, Bakery, e t c . Whipped t o p p i n g s , Chiffon desserts, Angel cakes

Though t h e i m p o r t a n c e o f f u n c t i o n a l p r o p e r t i e s have b e e n r e c o g n i z e d b y commodity s p e c i a l i s t s f o r many y e a r s , t h e w i d e s p r e a d awareness o f t h e i r i m p o r t a n c e t o food s c i e n c e and t e c h n o l o g y i s r e l a t i v e l y recent. T h i s h a s been a c c e l e r a t e d by t h e i n c r e a s e d e m p h a s i s o n f o o d p r o c e s s i n g , m a n u f a c t u r i n g , a n d f o r m u l a t i o n . The u n i v e r s a l attempts t o u t i l i z e l e s s expensive sources o f p r o t e i n s t o f a b r i c a t e new f o o d a n a l o g s , t o s i m u l a t e t r a d i t i o n a l f o o d s , t o e x t e n d t r a d i t i o n a l f o o d s , a n d t o d e v e l o p new f u n c t i o n a l i n g r e d i e n t s have a c c e n t u a t e d t h e need f o r i n f o r m a t i o n on f u n c t i o n a l properties of proteins. Factors A f f e c t i n g Functional Properties S e v e r a l f a c t o r s and p r o c e s s i n g s t e p s a f f e c t t h e f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s . Thus, t h e p r o t e i n source c a n a f f e c t functionality. I n t h e c a s e o f meat t h e age o f t h e a n i m a l , w h i c h i s r e l a t e d to the q u a n t i t y o f connective t i s s u e , can markedly a f f e c t t h e f u n c t i o n a l p r o p e r t i e s . The p r o g r e s s i v e m o d i f i c a t i o n of t h e c o l l a g e n molecules v i a c r o s s l i n k i n g i n c r e a s e s t h e toughness

In Food Protein Deterioration; Cherry, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


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of meat. In the case of egg white there i s a p r o g r e s s i v e t h i n ning of the egg white with storage time. The g l u t e n i n content v a r i e s immensely with the d i f f e r e n t v a r i e t i e s of wheat, and t h i s a f f e c t s the f u n c t i o n a l p r o p e r t i e s o f f l o u r s prepared from these cereals. Both i n t r i n s i c and a p p l i e d f a c t o r s i n f l u e n c e the observed f u n c t i o n a l p r o p e r t i e s of p r o t e i n s . The inherent molecular p r o p e r t i e s of the p r o t e i n per se ( s i z e , shape, conformation, whether n a t i v e o r denatured), the methods and c o n d i t i o n s o f i s o l a t i o n ( r e f i n i n g , d r y i n g , s t o r a g e ) , the degree of p u r i f i c a t i o n , (processing a l t e r a t i o n s ) and m o d i f i c a t i o n by p h y s i c a l (heat), chemical ( d e r i v a t i z a t i o n , h y d r o l y s i s ) or enzymatic processes, a l l i n f l u e n c e performance i n food systems. The methods employed i n determining a p a r t i c u l a r f u n c t i o n a l property, the composition of the assay system (model o r food), the p r o t e i n concentration, the pH, temperature, i o n c o n c e n t r a t i o n and composition, o x i d a t i o n / r e duction p o t e n t i a l , the type of equipment/machinery used (geometry, c o n f i g u r a t i o n ) , energy i n p u t , temperature c o n t r o l s o f the system and the method of measurement can a l l a f f e c t the observed prope r t y (1-6). Thus, m u l t i p l e i n t e r a c t i o n s are i n v o l v e d , and f o r the d e r i v a t i o n of u s e f u l data a l l experimental v a r i a b l e s should be defined and c o n t r o l l e d . F a i l u r e to do so renders the data redundant f o r most researchers and v i t i a t e s v a l i d comparisons of data from d i f f e r e n t l a b o r a t o r i e s . The l i t e r a t u r e i s r e p l e t e with papers on the f u n c t i o n a l prope r t i e s of p r o t e i n s prepared by d i f f e r e n t methods as measured by a v a r i e t y of methods under d i f f e r i n g c o n d i t i o n s ( 6 ) . The tendency has been f o r each i n v e s t i g a t o r to devise methods and/or c o n d i t i o n s to s u i t a p a r t i c u l a r s i t u a t i o n with l i m i t e d concern f o r the v a l i d i t y , comparative v a l u e , o r experimental design to f a c i l i t a t e e l u c i d a t i o n of the b a s i s of the p h y s i c a l property being s t u d i e d . Thus, much o f the data i n the l i t e r a t u r e are of very l i m i t e d value and d e f i n i t e l y cannot be used i n the systematic t a b u l a t i o n of p h y s i c a l p r o p e r t i e s i n a standardized format. This s i t u a t i o n r e f l e c t s and i s f u r t h e r complicated by the heterogeneous mixture of p r o t e i n s being s t u d i e d ; the very complex s e r i e s of i n t e r a c t i o n s which govern expression o f a p a r t i c u l a r f u n c t i o n a l property (and which are not amenable to easy measurement); the m u l t i p l e i n t e r a c t i n g f a c t o r s which impact on the aggregate e f f e c t and the l a c k of appropriate m e a s u r i n g devices which can a c c u r a t e l y quant i f y the r e s u l t a n t of these i n t e r a c t i o n s as a f u n c t i o n o f i n herent p r o p e r t i e s , i n t e r a c t i o n s per se and environmental f a c t o r s . Because of the complexity of food systems, the heterogeneity of the p r o t e i n s and the v a r i e t y o f f u n c t i o n s r e q u i r e d i n the d i f f e r e n t food systems f o r which they are used, i t has been d i f f i c u l t to standardize t e s t s f o r measuring f u n c t i o n a l p r o p e r t i e s (1,6). I t i s impossible to g e n e r a l i z e concerning the a v a i l a b l e information and d i f f i c u l t to e x t r a p o l a t e data from one system to p r e d i c t the behavior o f that p a r t i c u l a r p r o t e i n i n other systems. Measurements, i n order to have more general a p p l i c a b i l i t y ,



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must be based upon the fundamental p r o p e r t i e s of the system. In order to p r e d i c t the behavior of p r o t e i n s i t i s necessary to determine t h e i r b a s i c composition and physicochemical p r o p e r t i e s under s p e c i f i e d c o n d i t i o n s . However, to date, there are few methods that can be v a l i d l y or u n i v e r s a l l y used to p r e d i c t the behavior of p r o t e i n s i n food systems. In measuring the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s , i d e a l l y , e q u i l i b r i u m c o n d i t i o n s should be a t t a i n e d so that thermodynamic c r i t e r i a are met and so that the p a r t i c u l a r measurement r e f l e c t s the p h y s i c a l p r o p e r t i e s of the p r o t e i n i n question. For many f u n c t i o n a l p r o p e r t i e s a number of o p e r a t i o n a l d e f i n i t i o n s have been used to d e f i n e those part i c u l a r p r o p e r t i e s . Each has i t s advantages and disadvantages, and, of course because thermodynamic c r i t e r i a are r a r e l y a t t a i n e d , thermodynamic a n a l y s i s cannot e a s i l y be a p p l i e d (7). E v a l u a t i o n of a p a r t i c u l a r property or a p p l i c a t i o n i n food systems u s u a l l y i n c l u d e s t e s t s i n model systems; i . e . , systems that mimic the food system i n a l i m i t e d way and that are s e l e c t e d to h i g h l i g h t the performance of that p a r t i c u l a r f u n c t i o n . U l t i m a t e l y , of course, a l l f u n c t i o n a l a t t r i b u t e s should be evaluated i n food systems ( 6 ) . The method of e x t r a c t i o n and r e f i n i n g can have a marked e f f e c t on the f u n c t i o n a l p r o p e r t i e s o f i s o l a t e d p r o t e i n s . This depends upon the solvent used, the degree o f heating, the presence or absence of a l k a l i , pH, i o n s , mode of p r e c i p i t a t i o n and d r y i n g , and c o n d i t i o n s of storage (1-8). Temperature markedly a f f e c t s f u n c t i o n a l p r o p e r t i e s ; e.g., high temperatures which i n duce denaturation may impair f u n c t i o n a l p r o p e r t i e s . Time-temperature r e l a t i o n s h i p s used i n d r y i n g markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s ; e.g., spray versus drum d r y i n g a f f e c t s the whipping p r o p e r t i e s of egg white (9) . The exposure o f p r o t e i n s to d i f f e r ent ions during p r e p a r a t i o n can a f f e c t f u n c t i o n a l p r o p e r t i e s (10). Moisture l e v e l s and storage temperature can a f f e c t i n t e r a c t i o n s during storage and thereby impact on f u n c t i o n a l p r o p e r t i e s . The presence of r e a c t i v e components such as unsaturated f a t t y a c i d s and reducing sugars can r e s u l t i n chemical i n t e r a c t i o n s and thereby markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s . To f a c i l i t a t e the development of p r o t e i n s with p a r t i c u l a r f u n c t i o n a l p r o p e r t i e s i t i s f i r s t necessary to define which physicochemical p r o p e r t i e s are most important. To achieve t h i s i t i s very necessary to develop good techniques f o r measuring funct i o n a l p r o p e r t i e s and d e v i s i n g methods which e l u c i d a t e r e l a t i o n ships between s t r u c t u r e and f u n c t i o n . L i m i t e d advances have been made i n t h i s area though i n t e r e s t has i n c r e a s e d . Obviously, i n approaching t h i s challenge one should s e l e c t a w e l l c h a r a c t e r i z e d p r o t e i n and use i t i n a model system wherein f u n c t i o n a l p r o t e i n s are the major a c t i v e reagents ; e.g., protein-based foams, meat systems, cheese curds, e t c . When such r e l a t i o n s h i p s are understood i t may be f e a s i b l e to modify inexpensive p r o t e i n s and impart the necessary f u n c t i o n a l p r o p e r t i e s . For t h i s the s t r u c t u r a l features of the p r o t e i n , molecular p r o p e r t i e s , and


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i n t e r a c t i o n s r e q u i r e d f o r p a r t i c u l a r f u n c t i o n s need to be understood.


P r o t e i n Structure and F u n c t i o n a l

Relationships

The s t r u c t u r e and chemical p r o p e r t i e s of food components determine t h e i r behavior and u l t i m a t e l y the a c c e p t a b i l i t y o f many foods. One challenge to the modern food s c i e n t i s t i s to define the chemical and/or p h y s i c a l a t t r i b u t e s that are r e s p o n s i b l e f o r these d e s i r a b l e p r o p e r t i e s and apply t h i s knowledge toward the development of f u n c t i o n a l i n g r e d i e n t s and the f a b r i c a t i o n of new foods. The amino a c i d composition, the sequence of amino a c i d s , the degree of m o d i f i c a t i o n of the nascent polypeptides during synthes i s , the manner i n which secondary and t e r t i a r y f o l d i n g occurs and p o s s i b l e a s s o c i a t i o n s between polypeptides a l l a f f e c t the f i n a l s t r u c t u r e and p h y s i c a l p r o p e r t i e s of d i f f e r e n t food prot e i n s . These s t r u c t u r e s i n turn vary i n t h e i r response to environmental f a c t o r s (temperature, pH, i o n i c s t r e n g t h ) , which a l s o a f f e c t f u n c t i o n a l p r o p e r t i e s (Table I I ) . Table I I .

S t r u c t u r e of polypeptides and i n t e r a c t i o n s that i n f l u e n c e f u n c t i o n a l p r o p e r t i e s of food p r o t e i n s .

Amino a c i d composition (major groups) Amino a c i d sequence (segments/polypeptides) Secondary/tertiary conformation (compact/coil) Surface charge, hydrophobicity, p o l a r i t y S i z e , shape (topography) Quaternary s t r u c t u r e s Secondary i n t e r a c t i o n s ( i n t r a - and i n t e r - p e p t i d e ) Hydrogen bonding, i o n i c , Van der Waals, 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 . D i s u l f i d e / s u l f h y d r y l content Environmental c o n d i t i o n s (pH, 0/R s t a t u s , s a l t s , temperature) Amino A c i d s . The r e l a t i v e amounts o f d i f f e r e n t amino a c i d s and t h e i r d i s p o s i t i o n i n the polypeptide chain a f f e c t f u n c t i o n a l p r o p e r t i e s . Because hydrophobic e f f e c t s are important f o r c e s determining the p h y s i c a l behavior of p r o t e i n s the content and d i s p o s i t i o n of apolar amino a c i d s ( l e u c i n e , v a l i n e , i s o l e u c i n e , a l a nine, p r o l i n e , phenylalanine, tryptophan, t y r o s i n e and methionine) s i g n i f i c a n t l y a f f e c t the s t r u c t u r e and f u n c t i o n of p r o t e i n s (11-13). The shape of p r o t e i n molecules r e f l e c t the i n t e r n a l i z a t i o n of the maximum number o f apolar amino a c i d s , and the subun i t s of many o l i g o m e r i c p r o t e i n s a s s o c i a t e v i a hydrophobic i n t e r a c t i o n s (14). The content of apolar amino a c i d s (which range from 25-35% f o r most p r o t e i n s ) a f f e c t conformation, h y d r a t i o n , s o l u b i l i t y , denaturation and g e l a t i o n p r o p e r t i e s . Shimada and Matushita (15) showed that p r o t e i n s ; e.g., soybean, bovine serum



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FOOD

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a l b u m i n , c o n a l b u m i n , c o n t a i n i n g f r o m 2 6 - 3 1 % a p o l a r amino a c i d s formed g e l s upon h e a t i n g , w h e r e a s p r o t e i n s w i t h >31%, c o a g u l a t e d . K a t o and N a k a i (16) showed a c l o s e r e l a t i o n s h i p b e t w e e n t h e h y d r o p h o b i c i t y o f p r o t e i n s and t h e i r e m u l s i f y i n g p r o p e r t i e s . The p r e s e n c e o f c h a r g e d amino a c i d s e n h a n c e s 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 which are s i g n i f i c a n t i n s t a b i l i z i n g globular pro t e i n s and i n w a t e r b i n d i n g ; e . g . , a s p a r t i c and g l u t a m i c a c i d b i n d 6-7 m o l e c u l e s w a t e r p e r c h a r g e d r e s i d u e ( 1 7 ) . This i s important f o r many f u n c t i o n a l p r o p e r t i e s ( h y d r a t i o n , s o l u b i l i t y , g e l a t i o n , surfactancy). P o l a r amino g r o u p s a r e c h e m i c a l l y r e a c t i v e and e n gage i n h y d r o g e n - b o n d i n g , w h i c h i n f l u e n c e s c o n f o r m a t i o n o f a h e l i x and 3-sheet s t r u c t u r e s . T h e s e may be m o d i f i e d ; e . g . , a c y l a t e d , t o manipulate the p h y s i c a l p r o p e r t i e s o f food p r o t e i n s (18)· The h y d r o p h i l i c amino a c i d s on t h e s u r f a c e o f g l o b u l a r proteins impart molecular f l e x i b i l i t y (19). Hence, p r o t e i n s w i t h a l a r g e p r o p o r t i o n o f h y d r o p h i l i c r e s i d u e s may be good f u n c t i o n a l p r o t e i n s ; e.g., egg w h i t e p r o t e i n s . C y s t e i n e and c y s t i n e s i g n i f i c a n t l y a f f e c t s t r u c t u r e and f u n c tion of proteins. S u l f h y d r y l g r o u p s may be o x i d i z e d t o f o r m d i s u l f i d e bonds ( i n t r a - and i n t e r m o l e c u l a r ) , t h i o l s a n d d i s u l f i d e s can u n d e r g o i n t e r c h a n g e r e a c t i o n s w h i c h m a r k e d l y a f f e c t s t r u c t u r e and f u n c t i o n ; e . g . , g l u t e n f o r m a t i o n ( 2 0 ) . Reduction of d i s u l f i d e bonds can s i g n i f i c a n t l y a f f e c t m o l e c u l a r p r o p e r t i e s such as t h e u n f o l d i n g o f g l u t e n i n artel enhance t h e d i g e s t i b i l i t y o f s o y 11S. D i s u l f i d e c r o s s l i n k s are important i n s t a b i l i z i n g the t e r t i a r y structure of proteins. The f o r m a t i o n o f t h e B - l a c t o globulin/κ-casein d i s u l f i d e l i n k e d c o m p l e x e s and i m p r o v e s b a k i n g p r o p e r t i e s o f m i l k powders ( 2 1 ) . G e n e r a l l y , e x c e p t i n c a s e s where a p a r t i c u l a r t y p e o f amino a c i d p r e d o m i n a t e s , k n o w l e d g e o f amino a c i d c o m p o s i t i o n i s o f l i m i t e d value i n p r e d i c t i n g t e r t i a r y s t r u c t u r e or p h y s i c a l proper t i e s o f p r o t e i n s ; e . g . , α-lactalbumin and l y s o z y m e h a v e v e r y s i m i l a r amino a c i d c o m p o s i t i o n b u t d i f f e r m a r k e d l y i n p r o p e r t i e s ; t h e mere r e p l a c e m e n t o f g l u t a m i n e w i t h v a l i n e c h a n g e s t h e c o n f o r m a t i o n and p r o p e r t i e s o f h e m o g l o b i n , and c a s e i n v a r i a n t s w i t h m i n o r d i f f e r e n c e s i n amino a c i d s show s m a l l d i f f e r e n c e s i n h e a t s t a bility. Protein Structure. The p a r t i c u l a r s e q u e n c e o f amino a c i d s i n a p r o t e i n d e t e r m i n e s i t s s t r u c t u r e , c o n f o r m a t i o n and p r o p e r ties. Knowledge o f p r o t e i n s t r u c t u r e and t h e s t a b i l i t y o f d i f f e r ent c o n f o r m a t i o n s i s p e r t i n e n t t o u n d e r s t a n d i n g f u n c t i o n a l prop e r t i e s o f f o o d p r o t e i n s ( T a b l e I I ) . The s t r u c t u r e o f p r o t e i n i s c a t e g o r i z e d as s e c o n d a r y , t e r t i a r y , and q u a t e r n a r y , a c c o r d i n g t o the s p a t i a l arrangement o f t h e p o l y p e p t i d e c h a i n s . I n a n aqueous environment primary polypeptides tend t o c o i l i n a c h a r a c t e r i s t i c f a s h i o n t o form l o c a l i z e d secondary s t r u c t u r e s ; i . e . , α - h e l i x o r B - p l e a t e d s h e e t . The d r i v i n g f o r c e f o r t h i s f o l d i n g i s h y d r o p h o b i c i n o r i g i n due t o t h e f a v o r a b l e change i n f r e e e n e r g y o f s o l v e n t upon f o l d i n g o f t h e p o l y p e p t i d e ( 1 1 - 1 3 ) . The s e c o n d a r y s t r u c t u r e s a r e s t a b i l i z e d by hydrogen b o n d i n g .



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The t e r t i a r y s t r u c t u r e r e f e r s to the p r e f e r r e d three-dimen s i o n a l arrangement of the f o l d e d polypeptide chains. In a t y p i c a l t e r t i a r y s t r u c t u r e the polypeptides are t i g h t l y f o l d e d to give a compact molecule i n which most of the p o l a r groups of the amino a c i d s are l o c a t e d on the outer s u r f a c e and are hydrated. Most o f the apolar groups are i n t e r n a l i n the hydrophobic region from which water i s e s s e n t i a l l y excluded (a few Η-bonded water mole cules may remain). The p r o l i n e residues are l o c a t e d at the bends or folds i n the polypeptide chain where i s o l e u c i n e , s e r i n e and charged amino a c i d residues (α-helix d i s r u p t i n g molecules) a l s o tend to be l o c a t e d . These f o l d regions l a c k h e l i c a l s t r u c ture tending to be random (11, 12). The gross conformation; i . e . , t e r t i a r y s t r u c t u r e of most g l o b u l a r p r o t e i n s tend to be s i m i l a r i n general o r g a n i z a t i o n . The degree of f o l d i n g and r e l a t i v e pro p o r t i o n of α-helix, 3-sheet o r random c o i l v a r i e s immensely among p r o t e i n s . Some p r o t e i n s are l o o s e l y f o l d e d and the t e r t i a r y s t r u c t u r e i s s t a b i l i z e d by d i s u l f i d e bonds, others are compactly f o l d e d with many S-S bonds (lysozyme, g l y c i n i n ) . Cytochrome C has no α-helix and many residues are i n the extended 3-conformat i o n . The polypeptides i n f i b r o u s p r o t e i n s ( c o l l a g e n , myosin, f i b r i n o g e n ) are mostly α - h e l i x , myoglobin has mostly 70% α-helix; however other g l o b u l a r p r o t e i n s serum albumin, lysozyme, r i b o n u c l e a s e , are made up o f f o l d e d polypeptides with extensive regions of unordered s t r u c t u r e and p r o t e i n s l i k e c a s e i n s , e l a s t i n which possess very l i t t l e order i n manner of polypeptide f o l d i n g . The compactness and extent of i n t e r p e p t i d e i n t e r a c t i o n s or bonding markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s (22). Both e n t r o p i e and e n t h a l p i c f a c t o r s c o n t r i b u t e to the f o l d ing of p r o t e i n s , but i t i s g e n e r a l l y f e l t that the dominant d r i v i n g f o r c e i s the negative entropy e f f e c t caused by the ex posure of polypeptides to the aqueous solvent phase (10, 11). This r e s u l t s i n the unfavorable s t r u c t u r i n g of the water i n the immediate v i c i n i t y of the hydrophobic peptide m o i e t i e s . Conse quently, f o l d i n g of hydrophobic regions out of the water i n t o the p r o t e i n i n t e r i o r i n c r e a s e s the entropy of the p r e v i o u s l y s t r u c tured water. Though the enthalpy of t h i s process i s u s u a l l y s l i g h t l y p o s i t i v e and t h e r e f o r e unfavorable, the l a r g e p o s i t i v e entropy g r e a t l y favors f o l d i n g . Hydrophobic i n t e r a c t i o n s are n o n s p e c i f i c because they r e s u l t from solvent p r o p e r t i e s r a t h e r than a t t r a c t i v e bonding between s p e c i f i c groups. Once apolar groups are forced w i t h i n a c r i t i c a l d i s t a n c e , Van der Waals forces, 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 , and other i n t e r a c t i o n s may c o n t r i b u t e to s t a b i l i z a t i o n of s t r u c t u r e (11, 13, 23). Peturbat i o n of e n t r o p i e r e l a t i o n s h i p s can a l t e r f u n c t i o n a l p r o p e r t i e s of p r o t e i n s (10). Many polypeptides with molecular weights exceeding 50,000 daltons tend to a s s o c i a t e and form quaternary s t r u c t u r e s . From the amino a c i d composition of known p r o t e i n s , i t has been ob served that p r o t e i n s c o n t a i n i n g more than 28 mole percent o f



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p a r t i c u l a r amino a c i d s ( v a l i n e , p r o l i n e , l e u c i n e , i s o l e u c i n e and p h e n y l a l a n i n e ) t e n d t o s e l f - a s s o c i a t e (14). T h u s , t h e α a n d β c h a i n s o f hemoglobin a s s o c i a t e , whereas m y o g l o b i n , w h i c h has l e s s t h a n 28% a p o l a r amino a c i d s , does n o t s e l f - a s s o c i a t e . An e x p l a n a t i o n f o r t h i s phenomenon i s t h a t b e l o w a c r i t i c a l f r a c t i o n o f h y d r o p h o b i c r e s i d u e s i t i s p o s s i b l e t o b u r y a l l o f t h e s e com p l e t e l y w i t h i n t h e m o l e c u l e . When t h i s f r a c t i o n i s e x c e e d e d t h e f o l d i n g o f m o l e c u l e c a n n o t accomodate t h e s e i n t e r n a l l y and some a r e e x p o s e d on t h e s u r f a c e . When enough s u c h r e s i d u e s a p p e a r on the s u r f a c e h y d r o p h o b i c p a t c h e s w i l l be c r e a t e d , a n d t h e s e c a n o n l y be p r o t e c t e d o r removed f r o m t h e p o l a r medium by s e l f - a s s o ciation. I n many i n s t a n c e s f o r c e s o t h e r t h a n h y d r o p h o b i c i n t e r actions are involved i n maintaining multi-subunit structures. S e v e r a l m a j o r f o o d p r o t e i n s show q u a t e r n a r y s t r u c t u r e . C a s e i n s a s s o c i a t e s t r o n g l y v i a h y d r o p h o b i c f o r c e s ; t h e a c i d i c and b a s i c s u b u n i t s o f s o y U S and t h e component p o l y p e p t i d e s o f g l u t e n i n a r e d i s u l f i d e l i n k e d , and t h e s u b u n i t s o f m y o s i n a s s o c i a t e v i a h y d r o p h o b i c 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 . These s t r u c t u r e s markedly a f f e c t the f u n c t i o n a l p r o p e r t i e s o f these p r o t e i n s . The f o o d s c i e n t i s t i s f a c e d w i t h t h e s i t u a t i o n w h e r e i n t h e p r o t e i n s w i t h w h i c h he i s w o r k i n g p o s s e s s t h e t e r t i a r y a n d q u a t e r n a r y s t r u c t u r e s and c o n f o r m a t i o n t h a t p r e s u m a b l y have e v o l v e d t o p e r f o r m s p e c i f i c b i o l o g i c a l f u n c t i o n s and were n o t n e c e s s a r i l y d e s i g n e d f o r p a r t i c u l a r f o o d a p p l i c a t i o n s . I n most i n s t a n c e s t h e p h y s i c a l p r o p e r t i e s o f p a r t i c u l a r food p r o t e i n s have d i c t a t e d t h e manner i n w h i c h t h e y a r e u s e d o r consumed. T h u s , t h e p e c u l i a r nature o f m i l k p r o t e i n s w h i c h under a p p r o p r i a t e c o n d i t i o n s form curds l e d t o the development o f cheese. I n the case o f soy pro t e i n s , t h e i r a b i l i t y t o c o a g u l a t e i n t h e p r e s e n c e o f c a l c i u m and h e a t i n g t o form t o f u f a c i l i t a t e d t h e i r p r e s e r v a t i o n , improved t h e i r d i g e s t i b i l i t y and q u a l i t y a t t r i b u t e s . The p a r t i c u l a r com p o s i t i o n , s t r u c t u r e and c o n f o r m a t i o n o f e g g w h i t e p r o t e i n made i t the p r e m i e r w h i p p i n g p r o t e i n and l e d t o t h e e v o l u t i o n o f foamb a s e d f o o d s and b a k e r y p r o d u c t s . The f i b r o u s s t r u c t u r e o f t h e a c t i n and m y o s i n i n meats i s r e s p o n s i b l e f o r t h e t e x t u r e o f m u s c l e f o o d s , and t h e p r o d u c t i o n o f l e a v e n e d b r e a d s r e f l e c t t h e u n i q u e v i s c o e l a s t i c p r o p e r t i e s o f wheat g l u t e n . The consumer h a s been c o n d i t i o n e d t o t h e s e f o o d s , h e n c e much e f f o r t w i l l be r e q u i r e d i n t r y i n g to s i m u l a t e these p r o p e r t i e s u s i n g other pro t e i n i n g r e d i e n t s . I n order t o m a n i p u l a t e these p r o t e i n s and/or s i m u l a t e them w i t h o t h e r f u n c t i o n a l p r o t e i n s , i t i s i m p o r t a n t t o u n d e r s t a n d t h e i r s t r u c t u r e and p h y s i c a l p r o p e r t i e s and t o e l u c i d a t e t h e r e l a t i o n s h i p b e t w e e n s t r u c t u r e and s p e c i f i c f u n c t i o n a l properties. Non-Covalent Forces S t a b i l i z i n g P r o t e i n S t r u c t u r e . Because f u n c t i o n a l p r o p e r t i e s a r e r e l a t e d t o s t r u c t u r e and s i n c e changes i n s t r u c t u r e o f food p r o t e i n s f r e q u e n t l y a r e important i n a spe c i f i c f u n c t i o n ( s u r f a c e d e n a t u r a t i o n o f egg w h i t e d u r i n g w h i p p i n g o r t h e u n r a v e l i n g o f g l u t e n p r o t e i n s d u r i n g dough d e v e l o p m e n t ) ,



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i t i s important to consider the forces r e s p o n s i b l e f o r the n a t i v e s t r u c t u r e of p r o t e i n s and f a c t o r s which e f f e c t these i n r e l a t i o n to t h e i r f u n c t i o n i n the n a t i v e o r denatured s t a t e . The forces i n v o l v e d i n s t a b i l i z i n g the t e r t i a r y s t r u c t u r e of p r o t e i n s may i n c l u d e hydrogen bonding, Van der Walls f o r c e s , d i pole 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 , and hydrophobic a s s o c i a t i o n s . Covalent d i s u l f i d e bonds are important i n s e v e r a l p r o t e i n s . The nature and importance o f these i n t e r a c t i o n s are described i n Chapter 13_ of t h i s volume (10). The s t a b i l i t y of the quaternary s t r u c t u r e o f p r o t e i n s i s governed by the same general c l a s s e s o f s t a b i l i z i n g i n t e r a c t i o n s , p a r t i c u l a r l y hydrophobic i n t e r a c t i o n s , i o n c r o s s b r i d g i n g (Ca-casein) and d i s u l f i d e c r o s s l i n k i n g ; e.g., a c i d i c and b a s i c subunits of 11S p r o t e i n s and g l u t e n i n subunits. Manipulation of these f o r c e s to a l t e r the p h y s i c a l p r o p e r t i e s o f these p r o t e i n s provides an approach f o r improving f u n c t i o n a l p r o p e r t i e s of some food p r o t e i n s . Surface P r o p e r t i e s / E m u l s i f y i n g A c t i v i t y . Surface a c t i v i t y c l o s e l y r e f l e c t s the s t r u c t u r a l features of p r o t e i n s and these a f f e c t the usefulness of p r o t e i n s i n emulsions. In protein-based emulsions the p r o t e i n f u n c t i o n s by forming an i n t e r f a c i a l f i l m . The p h y s i c a l p r o p e r t i e s of t h i s f i l m matrix and i t s surface c h a r a c t e r i s t i c s determine i t s c a p a c i t y to form and s t a b i l i z e emul sions . Several researchers have studied the f i l m forming proper t i e s of p r o t e i n s , the p r o p e r t i e s of p r o t e i n f i l m s and t r i e d to c o r r e l a t e these with e m u l s i f y i n g p r o p e r t i e s (22, 24-28)· During the formation of an emulsion under i d e a l c o n d i t i o n s , the s o l u b l e p r o t e i n d i f f u s e s to and concentrates a t the o i l - w a t e r i n t e r f a c e once the i n t e r f a c i a l e l e c t r o s t a t i c b a r r i e r i s overcome. The pro t e i n , depending upon i t s s t r u c t u r a l s t a b i l i t y (compactness, f l e x i b i l i t y , charge, d i s u l f i d e bonds, hydrophobicity) tends to unfold to e s t a b l i s h a new thermodynamic e q u i l i b r i u m . The hydrophobic segments or loops o r i e n t i n the apolar o i l phase while the p o l a r and charged segments tend to occupy the aqueous phase. The forma t i o n of an i n t e r f a c i a l f i l m occurs i n s e q u e n t i a l stages, a l l o f which are i n f l u e n c e d by the molecular p r o p e r t i e s of the p r o t e i n . The i n i t i a l d i f f u s i o n of n a t i v e molecules to the i n t e r f a c e i s concentration dependent and v a r i e s with the p r o t e i n (24). The newly a r r i v e d molecule must penetrate the i n t e r f a c e and u n f o l d . The r a t e and extent of these events i s p r o t e i n dependent and i s a f f e c t e d by the surface pressure and c o m p r e s s i b i l i t y of the p r o t e i n f i l m already formed (25). F i n a l l y , rearrangement o f ad sorbed surface denatured p r o t e i n molecules occurs to a t t a i n the lowest free energy s t a t e . S o l u b i l i t y of p r o t e i n i s an important p r e r e q u i s i t e f o r f i l m formation because r a p i d migration to and adsorption a t the i n t e r face i s c r i t i c a l . This i s p a r t i c u l a r l y r e l e v a n t i n f l u i d emul s i o n s . D i f f e r e n t p r o t e i n s r e q u i r e d i f f e r e n t times to e q u i l i b r a t e and form a f i l m . These events are r a p i d f o r l o o s e , f l e x i b l e pro t e i n s l i k e β-casein, intermediate f o r g l o b u l i n s (BSA) and slow



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f o r p r o t e i n s w i t h a r i g i d t e r t i a r y s t r u c t u r e l i k e lysozyme o r soy I I S (24) · Graham and P h i l l i p s (24) r e l a t e d t h e p h y s i c a l p r o p e r t i e s o f p r o t e i n f i l m s made w i t h d i f f e r e n t p r o t e i n s t o e m u l s i o n f o r m a t i o n and s t a b i l i t y . They o b s e r v e d t h a t d i f f e r e n t p r o t e i n s behave d i f f e r e n t l y a t i n t e r f a c e s ; e.g., a t low c o n c e n t r a t i o n s , 3 - c a s e i n s p r e a d s o u t a t t h e i n t e r f a c e . As t h e s u r f a c e c o n c e n t r a t i o n p r o g r e s s i v e l y i n c r e a s e s l o o p i n g of the a p o l a r r e g i o n s i n t o the o i l phase o c c u r s . Subsequent compression of these loops occurs to y i e l d a condensed f i l m . Further increases i n protein c o n c e n t r a t i o n does n o t i n c r e a s e f i l m p r e s s u r e o r r e d u c e s u r f a c e t e n s i o n t h o u g h t h e t h i c k n e s s i n c r e a s e s due t o a d s o r p t i o n o f m u l t i layers. Lysozyme m o l e c u l e s do n o t u n f o l d as r e a d i l y as 3 - c a s e i n and take l o n g e r to form a f i l m . The f i l m i s composed o f l a y e r s o f minimally unfolded molecules. B o v i n e serum a l b u m i n b e h a v e s i n an i n t e r m e d i a t e f a s h i o n ; i . e . , i t u n f o l d s t o a l i m i t e d e x t e n t presumably because the d i s u l f i d e bonds p r e v e n t complete l o s s o f i t s t e r t i a r y s t r u c t u r e . B e c a u s e i t p o s s e s s e s a s i g n i f i c a n t numb e r o f h y d r o p h o b i c d o m a i n s b o v i n e serum a l b u m i n i s q u i t e s u r f a c e active. I t r e a d i l y l o c a t e s a t an i n t e r f a c e and u n f o l d s t o p e r m i t p o l a r and a p o l a r segments t o p r o t r u d e i n t o t h e aqueous and l i p i d phases r e s p e c t i v e l y w h i l e the b u l k of the m o l e c u l e occupies the interface. A d j a c e n t m o l e c u l e s may a s s o c i a t e v i a h y d r o p h o b i c 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 to form a cohesive film.. I n model s y s t e m s t h e i n t e r f a c i a l c o n c e n t r a t i o n o f p r o t e i n and t h e t h i c k n e s s o f t h e i n t e r f a c i a l f i l m a r e a f f e c t e d by t h e t y p e and c o n c e n t r a t i o n o f p r o t e i n i n s o l u t i o n (22, 2 4 ) . B o v i n e serum a l b u m i n i s more e f f e c t i v e i n s t a b i l i z i n g e m u l s i o n s t h a n 3 - c a s e i n o r l y s o z y m e . I t forms a s t r o n g e r f i l m a t lower p r o t e i n l e v e l s , r e f l e c t i n g a g r e a t e r number o f i n t e r m o l e c u l a r i n t e r a c t i o n s . In the case of food emulsions the r a t e of i n t e r f a c i a l f i l m f o r m a t i o n and t h e t y p e and p r o p e r t i e s o f t h e f i l m f o r m e d a r e g r e a t l y a f f e c t e d by t h e m e c h a n i c s o f e m u l s i o n f o r m a t i o n ; i . e . , s h e a r i n g , m i x i n g , d e n a t u r a t i o n , o x i d a t i o n , and t h i s , p e r h a p s , has more i m p a c t on e m u l s i o n f o r m a t i o n t h a n t h e p r o p e r t i e s o f t h e p r o t e i n p e r se (j4, 29). However, i t i s i m p o r t a n t t o r e c o g n i z e t h a t t h e p h y s i c a l and r h e o l o g i c a l p r o p e r t i e s o f t h e f i l m , o n c e f o r m e d , i s c r i t i c a l i n governing emulsion s t a b i l i t y . Globular proteins w i t h s u f f i c i e n t f l e x i b l e d o m a i n s can r a p i d l y f o r m f i l m s and d e press i n t e r f a c i a l t e n s i o n . Furthermore, they r e t a i n r e s i d u a l t e r t i a r y s t r u c t u r e which f a c i l i t a t e s extensive molecular i n t e r a c t i o n s ( e l e c t r o s t a t i c , h y d r o p h o b i c , d i s u l f i d e , Van d e r W a a l s forces). These c o n t r i b u t e t o t h e r h e o l o g i c a l p r o p e r t i e s ( s h e a r r e s i s t a n c e , v i s c o e l a s t i c i t y , d i l a t a t i o n a l modulus, i n c o m p r e s s i b i l i t y ) o f the f i l m w h i c h enhance f i l m s t a b i l i t y . However, t h e i r i n d i v i d u a l c o n t r i b u t i o n s to emulsion s t a b i l i t y remains u n c l e a r . G e n e r a l l y the s t a b i l i t y of p r o t e i n based emulsions are i n f l u e n c e d by t h e p r o p e r t i e s o f t h e i n t e r f a c i a l f i l m m a t e r i a l s and t h e v i s c o s i t y o f t h e c o n t i n u o u s p h a s e ( T a b l e I I I ) .


12.

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Table I I I .

Emulsification

and

Flavor

Binding

311

Factors a f f e c t i n g formation and s t a b i l i t y of p r o t e i n based emulsions.

PROTEIN I n t r i n s i c p h y s i c a l p r o p e r t i e s of the p r o t e i n : s i z e , shape, compactness, surface charge, hydrophobicity, solubility, f l e x i b i l i t y , d i s u l f i d e / t h i o l groups PROPERTIES OF ADSORBED PROTEIN FILM Thickness; r h e o l o g i c a l p r o p e r t i e s ( v i s c o s i t y , cohesiveness, e l a s t i c i t y ) ; net charge and d i s t r i b u t i o n ; degree of hydrat i o n ( h y d r o p h i l i c , p o l a r , charged groups) ENVIRONMENTAL FACTORS pH, i o n s , temperature PROCESSING PARAMETERS Shear f o r c e s , temperature, o i l composition, d r o p l e t s i z e , viscosity CONTINUOUS PHASE Viscosity In a packed emulsion the p o l y h e d r a l o i l d r o p l e t s are separated by an aqueous l a m e l l a r l a y e r . The v i s c o s i t y of t h i s l a y e r determines the r a t e a t which adjacent globules can approach each other. E l e c t r o s t a t i c and s t e r i c f a c t o r s r e t a r d the approach and coalescence of f a t d r o p l e t s . The i n t e g r i t y of the p r o t e i n f i l m and i t s s t a b i l i t y to shock (thermal, a g i t a t i o n , aging) i s governed by i t s p h y s i c a l and r h e o l o g i c a l p r o p e r t i e s (thickness, v i s c o s i t y , f l e x i b i l i t y and cohesiveness). Thinning of the f i l m r e s u l t s i n weak segments that may upon shock, rupture and coal e s c e . Hence, the r e s t o r a t i v e nature of the p r o t e i n f i l m i t s e l a s t i c i t y , d i l a t a t i o n a l modulus, shear v i s c o s i t y determines i t s i n t r i n s i c s t a b i l i t y (24-30). Coalescence of o i l d r o p l e t s r e s u l t i n g from the breakdown of the aqueous l a m e l l a i s a major f a c t o r i n emulsion i n s t a b i l i t y . Graham and P h i l i p s (24) concluded that the d i s j o i n i n g pressure s t a b i l i z i n g the l a m e l l a i s mostly a f f e c t e d by e l e c t r o s t a t i c and s t e r i c f a c t o r s c o n t r i b u t e d by the p r o t e i n s i n the f i l m . Thus, while c a p i l l a r y s u c t i o n , Van der Waals f o r c e s , and g r a v i t y tend to cause t h i n n i n g o f the aqueous l a m e l l a these are counteracted by e l e c t r o s t a t i c and s t e r i c f a c t o r s c o n t r i b u t e d by the surface l a y e r s of the adjacent p r o t e i n f i l m s . Thus, the surface charge, the s i z e , nature of the degree of h y d r a t i o n o f exposed groups a l l impair the approach and coalescence o f adjacent f i l m s . For the formation of a s t a b l e emulsion the i n t e r f a c i a l p r o t e i n f i l m should be as t h i c k as p o s s i b l e with good cohesive and r h e o l o g i c a l p r o p e r t i e s , be w e l l hydrated, contain exposed p o l a r and charged groups, and possess a net negative charge (24, 26). Current Research;

Experimental Procedures

Much current research

i s concerned with the determination o f



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the e m u l s i f y i n g p r o p e r t i e s o f food p r o t e i n s . A wide d i v e r s i t y o f m o d e l s y s t e m s and d i f f e r e n t c o n d i t i o n s have b e e n u s e d t o d e t e r mine e m u l s i f i c a t i o n p r o p e r t i e s o f numerous p r o t e i n s , b u t u s e f u l c o m p a r i s o n s o f methods and r e s u l t s a r e d i f f i c u l t (1, 4_, 6^, 3 1 ) . E m u l s i f y i n g a c t i v i t y r e f l e c t s the a b i l i t y of the p r o t e i n to a i d e m u l s i o n f o r m a t i o n and t o s t a b i l i z e t h e n e w l y c r e a t e d e m u l s i o n . E m u l s i f y i n g a c t i v i t y c a n be e a s i l y m e a s u r e d by m a k i n g an e m u l s i o n and d e t e r m i n i n g t h e p a r t i c l e s i z e d i s t r i b u t i o n o f t h e d i s p e r s e d phase by m i c r o s c o p y o r s p e c t r o t u r b i d i t y . I n each procedure the a v e r a g e d i a m e t e r o f t h e d i s p e r s e d p h a s e i s d e t e r m i n e d , and f r o m t h e s e d a t a t h e i n t e r f a c i a l a r e a c a n be c a l c u l a t e d . The s p e c t r o t u r b i d i t y method i s s i m p l e , r a p i d and t h e o r e t i c a l l y sound and p r o v i d e s i n f o r m a t i o n a b o u t t h e a v e r a g e d i a m e t e r and p a r t i c l e s i z e distribution. The method i s a p p l i c a b l e t o e m u l s i o n s w i t h a v e r a g e p a r t i c l e s i z e ; i . e . d i a m e t e r s b e t w e e n 0.2-8 μ ( 2 6 , 32, 33). The o p t i c a l density of d i l u t e d emulsions i s d i r e c t l y r e l a t e d to the i n t e r f a c i a l area; i . e . , the s u r f a c e area of a l l the d r o p l e t s , f o r c o a r s e emulsions (See T a b l e I ? ) ( 3 4 ) . S e v e r a l t y p e s o f b l e n d e r s and h o m o g e n i z e r s and many s i z e s and s h a p e s o f c o n t a i n e r s have b e e n u s e d i n e m u l s i o n p r e p a r a t i o n ( 4 , 2 9 ) . These i n s t r u m e n t s v a r y i n t h e i r a b i l i t y t o f o r m an emulsion; i . e . , 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 o i l drop l e t s v a r y and f r e q u e n t l y i n g r e d i e n t f a c t o r s a f f e c t i n g e m u l s i f y i n g a c t i v i t y a r e o v e r r i d d e n by t h e c h a r a c t e r i s t i c s o f t h e equipment used. The J a n k e - K u n k e l h o m o g e n i z e r h a s c o n s i s t e n t l y p r o v e d t o be the b e s t f o r p r o d u c i n g model e m u l s i o n s i n o u r l a b o r a t o r y (29). U s i n g model s y s t e m s and t h e p r o c e d u r e s d e s c r i b e d by P e a r c e and K i n s e l l a ( 3 4 ) and W a n i s k a , e t a l . ( 2 9 ) some o f t h e s t r u c t u r a l f a c t o r s and i n t e r a c t i o n s a f f e c t i n g e m u l s i o n f o r m a t i o n and s t a b i l i t y are being studied. Electrostatic Effects. The i m p o r t a n c e o f 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 on t h e f o r m a t i o n and s t a b i l i z a t i o n o f p r o t e i n - b a s e d e m u l s i o n s i s r e f l e c t e d by t h e e f f e c t s o f c h a r g e a l t e r a t i o n v i a p r o t e i n m o d i f i c a t i o n , pH v a r i a t i o n , and i o n c o n c e n t r a t i o n o n e m u l s i o n f o r m a t i o n and s t a b i l i t y ( 2 4 , 2 7 , 28, 29, 3 1 , 5 2 ) .

T a b l e IV.

E m u l s i f y i n g a c t i v i t y o f s u c c i n y l a t e d y e a s t proteins.. 2 -1 Emulsifying A c t i v i t y (M .g ) pH 6.5 pH 8

Modification Yeast Yeast Yeast Yeast

protein protein protein protein

(unmodified) (24% s u c c i n y l a t e d ) (62% succinylated) (88% s u c c i n y l a t e d )

From P e a r c e and K i n s e l l a

8 no 262 322

59

204 332 341

(34).



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Emulsification

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Flavor

313

Binding

S u c c i n y l a t i o n of r e l a t i v e l y i n s o l u b l e p r o t e i n s markedly improves the e m u l s i f y i n g c a p a c i t y of p l a n t p r o t e i n s (35). Yeast p r o t e i n i n which an i n c r e a s i n g number of l y s i n e residues were s u c c i n y l a t e d showed a progressive increase i n emulsion a c t i v i t y (Table IV). This i s a t t r i b u t e d to enhanced s o l u b i l i t y and expanded molecular s t r u c t u r e which enhanced emulsion formation. However, e l e c t r o s t a t i c r e p u l s i o n between the n e g a t i v e l y charged, e m u l s i f i e d d r o p l e t s enhanced s t a b i l i t y . Waniska, e_t a l . (29) studied the e f f e c t s of pH and s t r u c t u r a l m o d i f i c a t i o n on the e m u l s i f y i n g p r o p e r t i e s of p r o t e i n s i n c l u d i n g bovine serum albumin which possesses e x c e l l e n t e m u l s i f y ing a c t i v i t y (EA). The EÂ of bovine serum albumin (BSA) was two to t h r e e f o l d b e t t e r than other food p r o t e i n s (Table V) r e f l e c t i n g the favorable molecular c h a r a c t e r i s t i c s of BSA; i . e . , molecular s i z e , a b i l i t y to unfold to a l i m i t e d extent and allow polypept i d e s to r e o r i e n t at the i n t e r f a c e , surface hydrophobicity, d i s u l f i d e l i n k a g e s which maintained s u f f i c i e n t t e r t i a r y s t r u c t u r e and a balance of charged groups (24, 25)· Table V.

The r e l a t i o n s h i p between the pH and r e l a t i v e emsusifyi n g c a p a c i t i e s of v a r i o u s p r o t e i n s .

pH of solution

BSA

3 4 5 6 7 8 9 10

0.20 0.60 0.70 0.73 0.77 0.80 0.82 0.68

Absorbance (550 nm) of d i l u t e d emulsion soy Oval3S-Lg i s o l a t e bumin c a s e i n R-BSA S-BSA 0.15 0.53 0.56 0.51 0.48 0.47 0.42 0.30

0.01 0.03 0.60 1.20 1.40 1.23 1.24 1.20

0.18 0.25 0.34 0.31 0.32 0.33

-

-

0.01 0.10 0.30 0.30 0.35 _

-

0.26 0.25 0.21 0.21 0.22 0.26

-

0.02 0.01

-

0.07 0.08 0.08 0.10

soy lis 0.20 0.38 0.42 0.47 0.45 0.43 0.24 0.22

The e m u l s i f y i n g c a p a c i t y was determined by t u r b i d i m e t r y (29, 34); BSA - bovine serum albumin; R-BSA - a l l d i s u l f i d e s reduced; S-BSA - s u c c i n y l a t e d BSA; 3 - L g - B - l a c t o g l o b u l i n and U S = soy glycinin. Between pH 3 and 4 there was a s i g n i f i c a n t i n c r e a s e i n emuls i f y i n g a c t i v i t y of BSA. At pH 4 BSA undergoes an a c i d induced molecular expansion, presumably caused by e l e c t r o s t a t i c r e p u l s i o n , which r e s u l t s i n d i s r u p t i o n of some hydrophobic i n t e r a c t i o n s (36). This f a c i l i t a t e d emulsion formation. The EA of BSA p r o g r e s s i v e l y increased between pH 4 and 9 i n d i c a t i n g that as net charge i n creased the a b i l i t y to form a f i l m was enhanced. At pH 9.0 BSA undergoes an a l k a l i n e expansion (36) which may i n v o l v e some d i s u l f i d e cleavage and d i s r u p t i o n of hydrophobic a t t r a c t i o n s (37). The EA of BSA a b r u p t l y decreased a t ρH 9.0 r e f l e c t i n g the a l t e r e d t e r t i a r y s t r u c t u r e as d i s u l f i d e bonds were broken. These data



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FOOD P R O T E I N

DETERIORATION

r e f l e c t the importance o f a c e r t a i n degree o f s t r u c t u r a l s t a b i l i t y ( t e r t i a r y s t r u c t u r e ) f o r optimum e m u l s i o n f o r m a t i o n . Above pH 9, EA d e c r e a s e d ; t h i s may r e f l e c t t h e a l k a l i i n d u c e d h y d r o l y s i s o f some d i s u l f i d e bonds and p o s s i b l y some c r o s s l i n k i n g following ^-elimination reactions. The i m p o r t a n c e o f t e r t i a r y and s e c o n d a r y s t r u c t u r e i s r e v e a l e d by t h e a l m o s t c o m p l e t e e l i m i n a t i o n o f EA f o l l o w i n g t r e a t ment o f BSA w i t h u r e a ( 8 M ) . T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e s u g g e s t i o n t h a t w h i l e d e n a t u r e d BSA may o c c u p y t h e i n t e r f a c e the l a c k o f o r d e r e d s t r u c t u r e p r e v e n t s t h e f o r m a t i o n o f a c o h e s i v e continuous f i l m . T h i s was c o r r o b o r a t e d by t h e EA o f BSA i n w h i c h a l l t h e 17 d i s u l f i d e bonds w e r e r e d u c e d b y d i t h i o t h r e i t o l (0.01M). T h e r e was a s i g n i f i c a n t r e d u c t i o n i n EA, e s p e c i a l l y above pH 5.0. T h u s , t h e t e r t i a r y c o n f o r m a t i o n s t a b i l i z e d by d i s u l f i d e bonds a r e a p p a r e n t l y i m p o r t a n t i n t h e f o r m a t i o n o f a s t a b l e c o h e s i v e mem brane around the o i l d r o p l e t s . The r e d u c e d BSA was more s e n s i t i v e t o pH i n d i c a t i n g g r e a t e r s e n s i t i v i t y t o e l e c t r o s t a t i c r e p u l s i o n s . S u c c i n y l a t i o n , which s i g n i f i c a n t l y increased the net negative c h a r g e and c a u s e s m o l e c u l a r e x p a n s i o n m a r k e d l y i m p r o v e d t h e EA o f BSA b e t w e e n pH 5 and 1 0 . The maximum was o b s e r v e d a t pH 6 and subsequently decreased, p o s s i b l y because the n e t n e g a t i v i t y be came e x c e s s i v e and i m p a i r e d f o r m a t i o n o f a c o h e s i v e f i l m . Pre sumably s u c c i n y l a t i o n f a c i l i t a t e d m o l e c u l a r u n f o l d i n g a t t h e i n t e r f a c e , enhanced e l e c t r o s t a t i c r e p u l s i o n between e m u l s i f i e d d r o p l e t s and t h e c h a r g e d g r o u p s , b e i n g h i g h l y h y d r a t e d , may h a v e i n c r e a s e d the v i s c o s i t y o f t h e l a m e l l a r phase thereby r e t a r d i n g coalescence. M e t h y l a t i o n o f t h e ε-amino g r o u p s o f l y s i n e w h i c h u n l i k e s u c c i n y l a t i o n d i d n o t i n t r o d u c e a n e g a t i v e charge, b u t reduced t h e n e t p o s i t i v e c h a r g e b e l o w ρH 8.5, d i d n o t s i g n i f i c a n t l y a l t e r EA o f BSA. These o b s e r v a t i o n s i n d i c a t e t h a t i n d i v i d u a l e f f e c t s ; i . e . , n e t c h a r g e , a r e m o d i f i e d by m o l e c u l a r s t r u c t u r e and f l e x i b i l i t y , and optimum f u n c t i o n a l i t y i s o b t a i n e d o n l y when t h e s e a r e a t an optimum b a l a n c e . I o n s a t r e l a t i v e l y l o w c o n c e n t r a t i o n s ( 0 . 1 - 0.3 M) i n f l u ence e l e c t r o s t a t i c e f f e c t s i n e m u l s i o n s . Thus, t h e o i l / w a t e r i n t e r f a c e possesses a c o n c e n t r a t i o n o f charges which tend t o r e p e l approaching charged p r o t e i n s . T h i s r e s u l t s i n a reduced r a t e o f p r o t e i n a d s o r p t i o n , and u n f o l d i n g , t h u s i n h i b i t i n g t h e f o r m a t i o n o f a n i n t e r f a c i a l f i l m ( 2 5 ) . A t a p a r t i c u l a r pH, t h e n e t c h a r g e o n t h e p r o t e i n may r e s u l t i n e x c e s s i v e i n t e r m o l e c u l a r r e p u l s i o n and i m p a i r t h e i n t e r m o l e c u l a r a s s o c i a t i o n s n e c e s s a r y f o r the f o r m a t i o n o f a c o h e s i v e f i l m . S a l t s a t low c o n c e n t r a t i o n s f u n c t i o n by c o u n t e r a c t i n g s u c h e l e c t r o s t a t i c e f f e c t s v i a c o u n t e r i o n b i n d i n g , t h e r e b y p e r m i t t i n g a d s o r p t i o n and f i l m f o r m a t i o n . F u r t h e r m o r e , t h i s weakens c o u l o m b i c i n t e r a c t i o n s , e s p e c i a l l y i n aqueous s o l u t i o n s w h i c h i n t u r n p e r m i t s more e x t e n s i v e h y d r o p h o b i c i n t e r a c t i o n s between p r o t e i n s a t t h e i n t e r f a c e . Waniska e t a l . ( 2 9 ) o b s e r v e d t h a t b o t h c h l o r i d e and s u l f a t e a n i o n s a t c o n c e n t r a t i o n s o f z e r o t o 0.1 M m a r k e d l y e n h a n c e d t h e e m u l s i f y i n g


12.

KINSELLA

Emulsification

and Flavor

315

Binding

a c t i v i t y of BSA. C h l o r i d e enhanced EA s l i g h t l y up to 0.6 M, but the ' s a l t i n g i n ' e f f e c t o f sodium c h l o r i d e reduced the r a t e o f ab s o r p t i o n at the i n t e r f a c e a t s a l t concentrations above 0.6 M. S u l f a t e , a water s t r u c t u r e promoting s a l t ( ' s a l t i n g o u t ) favored the t r a n s f e r of BSA to the i n t e r f a c e a t maximum r a t e s a t concen t r a t i o n s from 0.1 to 1M. Thus, ions i n a d d i t i o n to i n f l u e n c i n g coulombic i n t e r a c t i o n s , may a l s o a f f e c t conformational s t a b i l i t y of p r o t e i n s , e s p e c i a l l y at higher concentrations v i a the chaotrop i c e f f e c t s (10). P r o t e i n s with d i f f e r e n t molecular s i z e s and s t r u c t u r e s showed markedly d i f f e r e n t EA under i d e n t i c a l c o n d i t i o n s . Soy 11S p r o t e i n and a r a c h i n are o l i g o m e r i c p r o t e i n s , around 330,000 d a l tons, with over 40 d i s u l f i d e c r o s s l i n k s and very l i t t l e (~12%) h e l i c a l s t r u c t u r e . These are s t r u c t u r a l l y very s t a b l e p r o t e i n s yet U S had a much greater EA compared to a r a c h i n . This obser v a t i o n i s being f u r t h e r explored. 3-Casein i s a h i g h l y amphip a t h i c molecule that i s surface a c t i v e and r a p i d l y forms f i l m s :nd f a c i l i t a t e s e m u l s i f i c a t i o n , but because f i l m s o f β-casein are weak, emulsion s t a b i l i t y i s poor. Ovalbumin i s reasonably hydro phobic, but tends to denature e a s i l y and coagulate r e s u l t i n g i n weak i n t e r f a c i a l f i l m s .


1

H y d r o p h i l i c E f f e c t s . The h y d r o p h i l i c character o f glycopro t e i n s may improve t h e i r surface a c t i v i t y . Conceivably the carbo hydrate moieties because o f t h e i r h y d r a t i o n provide s t e r i c s t a b i l i z a t i o n i n emulsions. To study the e f f e c t of h y d r o p h i l i c groups we c o v a l e n t l y a c y l a t e d d i f f e r e n t carbohydrate groups to 3 - l a c t o g l o b u l i n which contains 16 f r e e amino groups and 25 c a r boxyl groups a v a i l a b l e f o r m o d i f i c a t i o n . 3-Lactoglobulin has a low i n t r i n s i c v i s c o s i t y (0.034 dl/g) r e f l e c t i n g i t s g l o b u l a r na ture (38). I t has an average hydrophobicity of 1060 compared to 1000 and 980 f o r BSA and ovalbumin, r e s p e c t i v e l y (39). 3-Lacto g l o b u l i n i s reasonably surface a c t i v e ; i . e . surface tension = 60, i n t e r f a c i a l tension 11.0 and e m u l s i f y i n g a c t i v i t y index 151; bovine serum albumin has corresponding values of 58, 10.3 and 166, r e s p e c t i v e l y (16). A c t i v a t e d c y c l i c carbonate d e r i v a t i v e s of maltose and 3c y c l o d e x t r i n were l i n k e d to 3 - l a c t o g l o b u l i n v i a the f r e e amino groups on the p r o t e i n (R-NH-CO-maltose). Glucosamine was a t tached to 3 - l a c t o g l o b u l i n f o l l o w i n g carbodiimide a c t i v a t i o n o f the free carboxyl groups (R-CO-NH-glucose)· The number o f groups attached to the 3 - l a c t o g l o b u l i n was v a r i e d by manipulating r e a c t i o n c o n d i t i o n s (27). From s t u d i e s of the molecular p r o p e r t i e s ( v i s c o s i t y , c i r c u l a r dichroism, fluorescence and UV d i f f e r e n c e spectroscopy) g l y c o s y l a t i o n of 3 - l a c t o g l o b u l i n with maltose caused greater hy d r a t i o n , molecular expansion and e l o n g a t i o n as the number of c a r bohydrate residues added was i n c r e a s e d . Fluorescence and UV s p e c t r a together with r a t e s of p r o t e o l y s i s i n d i c a t e d a more open t e r t i a r y s t r u c t u r e and some d i s o r d e r i n g of the molecule ; i . e . ,



316

FOOD PROTEIN

DETERIORATION

r e d u c e d amount o f α h e l i x . As more m a l t o s e g r o u p s were added t h e s u r f a c e h y d r o p h o b i c i t y was d e c r e a s e d and n e t n e g a t i v e c h a r g e was increased. M o d i f i c a t i o n w i t h glucosamine caused l i t t l e c o n f o r m a t i o n a l changes e x c e p t a t h i g h l e v e l s of m o d i f i c a t i o n ; i . e . , when 22 r e s i d u e s were g l y c o s y l a t e d some l o s s o f s e c o n d a r y s t r u c t u r e became e v i d e n t (27). Some s u r f a c e a c t i v e p r o p e r t i e s o f t h e g l y c o s y l a t e d 3 - l a c t o g l o b u l i n were s t u d i e d t o d e t e r m i n e i f g l y c o s y l a t i o n a f f e c t e d these. Because g l y c o s y l a t i o n , e s p e c i a l l y w i t h m a l t o s e , caused a more expanded s t r u c t u r e t h e g a i n i n f r e e e n e r g y d u r i n g p r o t e i n a d s o r p t i o n s h o u l d be l o w e r and t h e r a t e o f a d s o r p t i o n a t an o i l w a t e r i n t e r f a c e s h o u l d be l e s s . However, g l y c o s y l a t i o n may a l s o f a c i l i t a t e u n f o l d i n g and r e o r i e n t a t i o n a t t h e i n t e r f a c e and a l t e r s u r f a c e a r e a o c c u p i e d by e a c h m o l e c u l e . I n i t i a l l y the p r o p e r t i e s o f f i l m s o f 3 - l a c t o g l o b u l i n and m o d i f i e d 3 - l a c t o g l o b u l i n were studied. The s o l u b l e g l o b u l a r 3 - l a c t o g l o b u l i n (Lg) a d s o r b e d r a p i d l y and p a r t l y u n f o l d e d t o f o r m a f a i r l y t i g h t l y p a c k e d c o n d e n s e d film. Maximum s u r f a c e p r e s s u r e o f 3-Lg was o b s e r v e d a t pH 5.0 s l i g h t l y b e l o w t h e i s o e l e c t r i c p o i n t ( 5 . 2 5 ) o f 3-Lg. T h u s , when n e t c h a r g e was c l o s e t o z e r o more p r o t e i n was a d s o r b e d b e c a u s e 3 - l a c t o g l o b u l i n had a more compact s t r u c t u r e . As pH was i n c r e a s e d t h e e l e c t r o n e g a t i v e c h a r g e c a u s e d r e p u l s i o n and p r o b a b l y f e w e r p r o t e i n segments o c c u p i e d t h e i n t e r f a c e . S y s t e m a t i c s t u d i e s of f i l m s r e v e a l e d t h a t g l y c o s y l a t i o n of 3 - l a c t o l o b u l i n w i t h m a l t o s e , g l u c o s a m i n e o r c y c l o d e x t r i n r e d u c e d the r a t e s o f a d s o r p t i o n and r e a r r a n g e m e n t a t t h e i n t e r f a c e . T h i s was a t t r i b u t e d to l o w e r f r e e energy g a i n , the i n c r e a s e d v i s c o s i t y , the s t e r i c h i n d r a n c e by c a r b o h y d r a t e s , t h e s t a b i l i z i n g e f f e c t o f t h e c a r bohydrate moiety a g a i n s t u n f o l d i n g , a l t e r a t i o n of net charge, and r e d u c e d s u r f a c e h y d r o p h o b i c i t y o f t h e m o d i f i e d 3-lactoglobu l i n (27)· Though g l y c o s y l a t i o n r e s u l t e d i n a r e d u c t i o n o f e l e c t r o s t a t i c r e p u l s i o n the c a r b o h y d r a t e s a p p a r e n t l y r e s t r i c t e d c l o s e m o l e c u l a r p a c k i n g i n t h e i n t e r f a c i a l f i l m , i n h i b i t e d un f o l d i n g o f t h e h y d r o p h o b i c r e g i o n s t o t h e a p o l a r p h a s e and s i g n i f i c a n t l y i n c r e a s e d t h e amount o f w o r k r e q u i r e d f o r e a c h m o l e c u l e t o p e n e t r a t e the i n t e r f a c e . The g e n e r a l c o n c l u s i o n o f t h e s e s t u d i e s were t h a t w h i l e g l y c o s y l a t i o n e n h a n c e d t h e h y d r o p h i l i c c h a r a c t e r o f 3-Lg i t c o n c u r r e n t l y d e c r e a s e d t h e number o f i o n i z a b l e g r o u p s and p e r h a p s more i m p o r t a n t l y , i t r e s t r i c t e d t h e e x p r e s s i o n o f t h e h y d r o p h o b i c p r o p e r t i e s a t the i n t e r f a c e t h e r e b y r e d u c i n g the s u r f a c e a c t i v i t y o f 3 - l a c t o g l o b u l i n as i n d i c a t e d by p r o p e r t i e s o f i n t e r facial films. B e c a u s e t h e h y d r o p h i l i c g l y c o s y 1 - s u b s t i t u e n t s w o u l d be e x p e c t e d t o e n h a n c e e m u l s i o n f o r m a t i o n and s t a b i l i t y v i a h y d r a t i o n and s t e r i c e f f e c t s , the e m u l s i f y i n g p r o p e r t i e s o f g l y c o s y l a t e d 3 - l a c t o g l o b u l i n were e x a m i n e d ( T a b l e V I ) .


12.

KINSELLA

Table VI.

Protein Preparation

Emulsification

and

Flavor

317

Binding

E f f e c t s of g l y c o s y l a t i o n of 3 - l a c t o g l o b u l i n on e m u l s i f y i n g p r o p e r t i e s a t d i f f e r e n t pH v a l u e s . Emulsion A c t i v i t y (OD

3

1:10 D i l u t i o n s )

Stability O i l Separation


pH

3L Β C D Ε F G

5 0.20 0.24 0.23 0.21 0.25 0.26 0.12

6.5 0.23 0.30 0.29 0.26 0.21 0.32 0.10

(%)

pH 7.5 0.26 0.40 0.31 0.26 0.34 0.29 0.23

5 9.0 7.5 6.8 8.0 9.5 12.3 9.9

6.5 13.0 8.2 11.5 11.4 12.8 7.9 10.0

7.5 11.6 6.1 13.0 10.8 8.5 9.8 9.8

Emulsions were prepared with 0.5% p r o t e i n and measured as r e ported by Waniska et_ a l . (29). Legend: 3-Lactoglobulin (3Lg) with amino or carboxyl groups modified with JB 7 m a l t o s y l ; £ U m a l t o s y l ; I) 3 3 - c y c l o d e x t r i n ; _E 6 glucosamine ; JF 16 glucosamine and G_ 12 glucosamineoctaosyl residues, respectively. I s o e l e c t r i c p o i n t s 3Lg 5.2; Β = 4.8; C = 4.3; D = 4.3; G = 5.5.

G l y c o s y l a t i o n o f 3 - l a c t o g l o b u l i n improved emulsion formation and s t a b i l i t y . This was i n c o n t r a s t to i t s general e f f e c t s on the p r o p e r t i e s of i n t e r f a c i a l f i l m s . However, the energy input i n emulsion p r e p a r a t i o n may have overcome some of the energy b a r r i e r s encountered i n the f i l m s t u d i e s where e q u i l i b r i u m c o n d i t i o n s p r e v a i l e d . O v e r a l l the data are c o n s i s t e n t with the sug g e s t i o n that the increased h y d r o p h i l i c nature of the p r o t e i n en hances emulsion p r o p e r t i e s by s t a b i l i z i n g the aqueous i n t e r f a c e through enhanced h y d r a t i o n and perhaps s t e r i c hindrance prevent i n g coalescence of the o i l droplets· However, d e r i v a t i z a t i o n a l t e r e d e l e c t r o s t a t i c and hydrophobic i n t e r a c t i o n s w i t h i n and between the polypeptides and these may have impeded attainment of maximum e m u l s i f y i n g p r o p e r t i e s . E m u l s i f y i n g a c t i v i t y increased with increased pH i n d i c a t i n g that as the net e l e c t r o n e g a t i v i t y i n c r e a s e d e l e c t r o s t a t i c r e p u l s i o n f a c i l i t a t e d formation and s t a b i l i z a t i o n of a greater number of o i l d r o p l e t s . The number and s i z e o f h y d r o p h i l i c groups i n troduced a f f e c t e d both EA and emulsion s t a b i l i t y . The attachment of, seven maltose groups i n t o 3 - l a c t o g l o b u l i n was s u p e r i o r to 11 groups i n improving e m u l s i f y i n g p r o p e r t i e s . Conceivably the lower number of maltose groups provided l e s s s t e r i c hindrance to i o n i c and hydrophobic i n t e r a c t i o n s between the polypeptides i n the i n t e r f a c e . The 3 - c y c l o d e x t r i n had l i t t l e impact on emulsi f y i n g p r o p e r t i e s . The e m u l s i f y i n g p r o p e r t i e s of 3 - l a c t o g l o b u l i n , modified with glucosamine r e s i d u e s , were g e n e r a l l y improved.



318

FOOD P R O T E I N DETERIORATION

Near the i s o e l e c t r i c range of the d e r i v a t i z e d p r o t e i n , emulsion formation was good. P r o t e i n s have a low net charge i n t h i s pH range and maximum hydrophobic i n t e r a c t i o n s are p o s s i b l e (40). Therefore, i t i s p o s s i b l e that small glucose d e r i v a t i v e s (even when 16 carboxyl groups were d e r i v a t i z e d ) d i d not completely impede hydrophobic i n t e r a c t i o n s at t h i s pH. D e r i v a t i z a t i o n with a l a r g e amino sugar, i . e . , glycosamineoctaose, impaired emulsion formation, but s t a b i l i t y of the emulsion was good. These data i n d i c a t e the importance of net charge, h y d r o p h i l i c i n t e r a c t i o n s and s t e r i c e f f e c t s i n emulsion formation and emulsion s t a b i l i z a t i o n and confirm the p o s i t i v e e f f e c t s of hydrop h i l i c groups. However, l a r g e , bulky h y d r o p h i l i c residues may i n t e r f e r e with hydrophobic i n t e r a c t i o n s causing excessive s t e r i c hindrance and a c t u a l l y reduce e m u l s i f y i n g p r o p e r t i e s . Hydrophobic E f f e c t s . The i o n i c , h y d r o p h i l i c and hydrophobic c h a r a c t e r i s t i c s of p r o t e i n a f f e c t t h e i r surface a c t i v i t y and functional properties. In an emulsion one s i d e ' of the p r o t e i n f i l m , i . e . , that exposed to the o i l phase, should be prepondera n t l y hydrophobic. The hydrophobicity i n f l u e n c e s adsorption and o r i e n t a t i o n of p r o t e i n at the i n t e r f a c e (22). The s i d e of the f i l m that i s exposed to the continuous aqueous phase has most of the p o l a r and charged groups exposed. Hydrophobic i n t e r a c t i o n s s t a b i l i z e the conformation of p r o t e i n s i n the n a t i v e s t a t e i n s o l u t i o n . At an i n t e r f a c e the normal thermodynamic e q u i l i b r i u m i s disturbed. The apolar segments of the polypeptides tend to u n f o l d , r e o r i e n t and occupy the l e s s p o l a r o i l phase. T h i s phenomenon depends upon the amphipathic a t t r i b u t e s of the p r o t e i n , a property required f o r surface a c t i v i t y . The hydrophobicity of p r o t e i n s i s r e l a t e d to t h e i r contents of apolar amino a c i d s . The r e l a t i v e h y d r o p h o b i c i t i e s of these; i . e . , t r p , i l e u , t y r , phe, pro, l e u , v a l , l y s , meth, cys/2, a l a , arg are 3.00, 2.95, 2.85, 2.65, 2.60, 2.40, 1.7, 1.5, 1.3, 1.0, 0.75, 0.75 K c a l / r e s i d u e r e s p e c t i v e l y (13). The average hydrophob i c i t y (HQ) r e p r e s e n t i n g the t o t a l hydrophobicity d i v i d e d by the t o t a l number of residues was c a l c u l a t e d f o r s e v e r a l p r o t e i n s by Bigelow (39). P r o t e i n s have HQ values ranging from 1000-12000 c a l / r e s i d u e (Table V I I ) . While general r e l a t i o n s h i p s are apparent, i t should be remembered that the d i s p o s i t i o n of the apolar residues (sequence and whether l o c a t e d i n t e r n a l l y , on the s u r f a c e , or on f l e x i b l e segments) a f f e c t p h y s i c a l p r o p e r t i e s more than the average hydrophobic amino a c i d content. L e s l i e (41) r e l a t e d the strong NMR s i g n a l s of the apolar residues i n soy p r o t e i n s to the e m u l s i f y i n g p r o p e r t i e s and Kato and Nakai (16) reported good c o r r e l a t i o n s between molecular hydrophobicity and s u r f a c t a n t p r o p e r t i e s . They s u c c e s s f u l l y q u a n t i f i e d e f f e c t i v e hydrophobicity; i . e . , true surface hydrop h o b i c i t y , using cii>-parinaric a c i d , an e x c e l l e n t f l u o r e s c e n t probe f o r measuring hydrophobicity of food p r o t e i n s . Proteins were reacted with c i s - p a r i n a r i c a c i d . The conjugates were 1


12.

KINSELLA

Emulsification

and

Flavor

319

Binding

e x c i t e d a t 325 nm and fluorescence i n t e n s i t y measured at 420 nm. The i n i t i a l slope ( S ) obtained by p l o t t i n g fluorescence against p r o t e i n c o n c e n t r a t i o n i n d i c a t e s s u r f a c e hydrophobicity. The energy t r a n s f e r e f f i c i e n c y of conjugates a t zero p r o t e i n concen t r a t i o n (T ) also r e f l e c t hydrophobicity. These two values were h i g h l y c o r r e l a t e d and corresponded to data obtained using hydro phobic chromatography (42). 0


Table V I I .

The average hydrophobicity of some food p r o t e i n s . MW 1 0

Tropomyosin Ovomucoid Collagen ( s o l u b l e c a l f ) Lysozyme Myosin Actin Gliadin Conalbumin Myoglobin Ovalbumin BSA α-Lactalbumin α-Casein 3-Lactoglobulin Zein

-

27 80 15 500 57 120 87 17 46 65 16

-17 50

3

HQ

NPS

870 920 960 970 1020 1050 1080 1080 1090 1110 1120 1150 1200 1230 1310

0.20 0.25 0.19 0.26 0.28 0.31

-

0.31 0.32 0.34 0.32 0.34 0.38 0.37

-

Ρ 1.94 1.50 0.89 1.18 1.43 1.15

-

1.30 1.12 0.92 1.22 1.11 1.27 0.96

-

Charge 0.47 0.30 0.16 0.16 0.35 0.27

-

0.32 0.34 0.24 0.33 0.28 0.24 0.28

-

MW = mol. weight; HQ = t o t a l hydrophobicity d i v i d e d by t o t a l number of r e s i d u e s ; NPS « sum o f Trp, l i e , T y r , Phe, Pro, Leu, and V a l residues as a f r a c t i o n of t o t a l residues ; Ρ = p o l a r i t y r a t i o roughly a l l p o l a r s i d e c h a i n s / a l l nonpolar s i d e chains and charge i s sum of Asp, G l u , H i s , L y s , Arg as f r a c t i o n of t o t a l amino a c i d s . From Bigelow (39).

Bovine serum albumin, κ-casein and 3 - l a c t o g l o b u l i n showed high S values (Table VIII) r e f l e c t i n g the b i n d i n g o f c i s - p a r i n a r i c a c i d to t h e i r hydrophobic r e g i o n s . The surface hydropho b i c i t y showed strong c o r r e l a t i o n s w i t h surface tension, i n t e r f a c i a l t e n s i o n and e m u l s i f y i n g a c t i v i t y o f these p r o t e i n s . This was f u r t h e r demonstrated by s t u d i e s i n d i c a t i n g that the p r o g r e s s i v e thermal denaturation o f lysozyme increased surface hydrophobicity and the EA was c o n c u r r e n t l y enhanced (3)· These data i n d i c a t e that hydrophobic i n t e r a c t i o n s are important, par t i c u l a r l y f o r the i n t e r m o l e c u l a r i n t e r a c t i o n s and a s s o c i a t i o n s which enhance the cohesiveness w i t h i n the p r o t e i n f i l m s surround i n g f a t d r o p l e t s i n emulsions. The above s e c t i o n s demonstrate some r e l a t i o n s h i p s between the molecular p r o p e r t i e s of food p r o t e i n s and t h e i r emulsion p r o p e r t i e s . However, much research i s needed to determine the optimum q u a n t i t a t i v e r e l a t i o n s h i p s between i n d i v i d u a l molecular Q


320

FOOD PROTEIN

DETERIORATION

p r o p e r t i e s ( c h a r g e , h y d r o p h o b i c i t y and h y d r o p h i l i c i t y ) , e n v i r o n m e n t a l p a r a m e t e r s , and p a r t i c u l a r t y p e s o f f o o d e m u l s i o n s .


Table V I I I .

Relative Hydrophobicity (fluorescence i n t e n s i t y , S ) S u r f a c e T e n s i o n , I n t e r f a c i a l T e n s i o n and E m u l s i f y i n g A c t i v i t y Index Values of Various P r o t e i n s . 0

S

0

Bovine serum albumin 1400 k-Casein 1300 3-lactoglobulin 750 Trypsin 90 Ovalbumin 60 Conalbumin 70 Lysozyme 100

Surface Tension (Dynes/cm)

57.9 54.1 59.8 64.1 61.1 63.7 64.0 From K a t o and

P r o t e i n Ligand

Interactions - Flavor

Interfacial Tension (Dynes/cm)

Emulsifying Activity Index (m g 1)

10.3 9.5 11.0 12.0 11.6 12.1 11.2

166 185 151 93 57 105 55

Nakai

2

(16)

Binding

P r o t e i n s b e i n g s u r f a c e a c t i v e can b i n d l i p i d s , f a t t y a c i d s , a l d e h y d e s , k e t o n e s , t a n n i n s , c h l o r o g e n i c a c i d , e t c . , and a d v e r s e l y a f f e c t f u n c t i o n a l and n u t r i t i o n a l p r o p e r t i e s ( 4 3 , 4 4 ) . The b i n d i n g o f low amounts o f s u r f a c e a c t i v e compounds and l i p i d s t o f o o d p r o t e i n s may a l t e r t h e i r s t a b i l i t y and t h e r m a l p r o p e r t i e s . Thus, t h e b i n d i n g o f l a u r i c a c i d t o egg w h i t e p r o t e i n s e n h a n c e s t h e i r t h e r m a l s t a b i l i t y (45) and low m o l e c u l a r w e i g h t a l c o h o l s o r c a r b o n y l s a t l o w c o n c e n t r a t i o n s s t a b i l i z e b o v i n e serum a l b u m i n (46). The b i n d i n g o f f l a v o r s t o f o o d components, e s p e c i a l l y p r o t e i n s i s o f p a r t i c u l a r i m p o r t a n c e , and i t i s a p r o b l e m t h a t has b e e n d r a m a t i z e d as f o o d t e c h n o l o g i s t s h a v e t r i e d t o f a b r i c a t e a n a l o g s and new f o o d s f r o m n o v e l p r o t e i n s ( 4 7 ) . More f u n d a m e n t a l i n f o r m a t i o n concerning the nature of f l a v o r - p r o t e i n i n t e r a c t i o n i s needed to h e l p s o l v e problems o f o f f - f l a v o r b i n d i n g , to a i d the d e v e l o p m e n t o f more s u c c e s s f u l f l a v o r i n g p r o c e d u r e s and u n d e r s t a n d t h e mechanism and t h e r m o d y n a m i c s o f f l a v o r r e l e a s e when f o o d i s chewed. Because the p e r c e i v e d f l a v o r i s important i n d e t e r m i n i n g f o o d a c c e p t a b i l i t y , t h e phenomenon o f f l a v o r b i n d i n g and r e l e a s e i s extremely s i g n i f i c a n t . The o f f - f l a v o r s t h a t become a s s o c i a t e d w i t h p r o t e i n s l i m i t s t h e i r a p p l i c a t i o n . T h i s i s e x e m p l i f i e d by many soy p r o t e i n p r e p a r a t i o n s w h i c h , t h o u g h p o s s e s s i n g good



12.

KINSELLA

Emulsification

and

Flavor

321

Binding

f u n c t i o n a l p r o p e r t i e s , have l i m i t e d use because of the impact o f bound o f f - f l a v o r s (48). To develop e f f e c t i v e methods f o r the r e moval o f o f f - f l a v o r s we have been studying f l a v o r - p r o t e i n i n t e r a c t i o n s i n simple model systems composed of soy p r o t e i n s and carbonyl compounds and determining b i n d i n g using e q u i l i b r i u m d i a l y s i s (49). Binding isotherms i n d i c a t e d a p r o g r e s s i v e i n c r e a s e i n b i n d i n g o f carbonyls with t h e i r c o n c e n t r a t i o n . There were four binding s i t e s per 100,000 molecular weight soy p r o t e i n f o r 2heptanone, 2-octanone and 2-nonanone, r e s p e c t i v e l y . The b i n d i n g a f f i n i t i e s i n c r e a s e d with chain length of the carbonyls i n d i c a t i n g that hydrophobic i n t e r a c t i o n s are i n v o l v e d (Table I X ) . There was a t h r e e f o l d i n c r e a s e i n Keq f o r each increment of methylene group (-CH^) i n the chain l e n g t h o f the carbonyl which c o r r e sponded to a change i n f r e e energy of -600 c a l o r i e s / C H 2 group. S i m i l a r r e s u l t s were obtained with bovine serum albumin except that i n the case of soy p r o t e i n and BSA the Keq f o r nonanone was 930 and 1800 M~l r e s p e c t i v e l y . This r e f l e c t s the d i f f e r e n c e i n s t r u c t u r e of these p r o t e i n s and the greater surface hydropho b i c i t y i n BSA which possesses s i x b i n d i n g s i t e s f o r these c a r bonyls (50). Table

IX.

Thermodynamic constants f o r i n t e r a c t i o n s of carbonyls with soy p r o t e i n s i n model systems. K

Ligand

2-Nonanone 2-Nonanone 2-Nonanone 2-Nonanone

AG

η

native native native native native

25 25 25 25 25

4 4 4 4 4

310 930 541 1094

-2.781 -3.395 -4.045 -3.725 -4.141

(succinylated) native native

25 25 5

2 4 2

850 930 2000

-3.992 -4.045 -4.221

25

4

1240

-4.215

7S 25 (0.03M S a l t ) 25 (0.5M S a l t ) 25

4

930

4.040

-8

290

-

-

2-Heptanone 2-0ctanone 2-Nonanone 5-Nonanone 1-Nonanal 2-Nonanone 2-Nonanone 2-Nonanone

eq

Temp °C

Protein

(heated

IIS IIS

90°C)

1

(M" )

no

(Kcal/mole)

-

These data s t r o n g l y support hydrophobic b i n d i n g and the AG value f o r the b i n d i n g of each C H 2 group, i . e . , -550 and -600 cal/-CH« group f o r BSA and soy p r o t e i n c l o s e l y approximate the -540 c a l / m o l e - C H 2 observed f o r the t r a n s f e r of a C H 2 group from water to an apolar s o l v e n t (51). Further evidence of hydrophobic i n t e r a c t i o n i s obtained by comparing the b i n d i n g of nonanol, 2-nonanone and 5-nonanone to soy p r o t e i n (Table I X ) . The b i n d i n g


322

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c o n s t a n t Κ d e c r e a s e d as t h e k e t o g r o u p was moved t o w a r d t h e m i d d l e o f t h e c h a i n ; i . e . , Keq was 1094, 930 and 541 M" for t h e s e t h r e e c a r b o n y l s r e f l e c t i n g t h e s t e r i c h i n d r a n c e c a u s e d by t h e k e t o g r o u p , e s p e c i a l l y when l o c a t e d n e a r t h e c e n t e r o f t h e hydrocarbon chain. The b i n d i n g a f f i n i t y o f s u c c i n y l a t e d soy p r o t e i n f o r 2-nonanone was s l i g h t l y r e d u c e d compared t o t h e n a t i v e p r o t e i n , and t h e number o f b i n d i n g s i t e s was r e d u c e d t o two r e f l e c t i n g m a j o r c o n f o r m a t i o n a l changes i n t h e s o y p r o t e i n i n t h e n e i g h b o r h o o d of the b i n d i n g s i t e s . Though t h e two b i n d i n g s i t e s have a l m o s t t h e same i n t r i n s i c b i n d i n g c o n s t a n t as t h e n a t i v e soy p r o t e i n , t h e b i n d i n g c a p a c i t y ( m o l e s l i g a n d bound p e r mole p r o t e i n ) o f t h e s u c c i n y l a t e d s o y was d e c r e a s e d . T h e r e f o r e , suc c i n y l a t i o n d e c r e a s e d f l a v o r b i n d i n g and i m p r o v e d t h e f l a v o r o f s u c c i n y l a t e d soy p r o t e i n as n o t e d p r e v i o u s l y ( 5 2 ) . A t l o w t e m p e r a t u r e (5°C) t h e number o f b i n d i n g s i t e s was r e d u c e d t o two, b u t t h e s e had s i g n i f i c a n t l y g r e a t e r b i n d i n g c o n s t a n t s r e f l e c t i n g changes i n the c o n f o r m a t i o n o f soy p r o t e i n a t low t e m p e r a t u r e s . The b i n d i n g c o n s t a n t was s i g n i f i c a n t l y i n c r e a s e d (30%) f o l l o w i n g h e a t i n g o f soy (90°C, 1 h r ) , b u t t h e r e were s t i l l f o u r b i n d i n g s i t e s . The e n h a n c e d b i n d i n g upon h e a t i n g corroborates the d a t a o f A r a i ejt a l . (53) and i n d i c a t e s t h a t t h e t h e r m a l p r o c e s s i n g o f soy p r o t e i n may e x a c e r b a t e t h e p r o b l e m o f o f f - f l a v o r b i n d i n g by i n c r e a s i n g t h e b i n d i n g a f f i n i t y o f t h e soy p r o t e i n f o r c a r b o n y l s . The e f f e c t s o f h e a t i n g o f s o y p r o t e i n on l i g a n d b i n d i n g seemed i n c o n s i s t e n t w i t h t h e k n o w l e d g e t h a t soy U S d i s s o c i a t e s , and t h e r e f o r e h e a t i n g s h o u l d i n c r e a s e the number o f b i n d i n g s i t e s . T h e r e f o r e , we s t u d i e d t h e b i n d i n g o f c a r b o n y l s t o U S and 7S, t h e m a j o r p r o t e i n components o f s o y b e a n . S i g n i f i c a n t l y , t h e r e was n e g l i g i b l e b i n d i n g o f nonanone t o t h e 11S f r a c t i o n , w h e r e a s t h e b i n d i n g t o 7S was c o m p a r a b l e t o t h a t f o r w h o l e soy p r o t e i n . This s e l e c t i v e i n t e r a c t i o n o b v i o u s l y r e f l e c t s the d i f f e r e n c e s i n the m o l e c u l a r s t r u c t u r e , c o n f o r m a t i o n and s u r f a c e p r o p e r t i e s o f t h e s e two p r o t e i n s . The 7S p r o t e i n (17,500 d a l t o n s ) i s c o m p r i s e d o f t h r e e s u b u n i t s o f d i f f e r e n t s i z e s and amino a c i d c o m p o s i t i o n ( 5 4 ) . The U S p r o t e i n (320,000 d a l t o n s ) c o n t a i n s s i x a c i d i c and s i x basic subunits (55). The s p a t i a l a r r a n g e m e n t o f t h e s u b u n i t s i n t h e s e two p r o t e i n s may be s u c h t h a t t h e 7S p r o t e i n has h y d r o phobic r e g i o n s which are a c c e s s i b l e f o r l i g a n d b i n d i n g , whereas i n t h e c a s e o f U S p r o t e i n s u c h h y d r o p h o b i c r e g i o n s may be b u r i e d i n s i d e t h e p r o t e i n o r be a t t h e p o i n t s o f c o n t a c t o r a s s o c i a t i o n b e t w e e n s u b u n i t s , and h e n c e may n o t be a c c e s s i b l e t o t h e l i g a n d . B e c a u s e the weak i n t e r a c t i o n b e t w e e n U S and 2-nonanone may r e f l e c t i t s unique q u a t e r n a r y s t r u c t u r e , then changes i n t h i s s t r u c t u r e may a f f e c t t h e b i n d i n g a f f i n i t y f o r 2-nonanone. The o l i g o m e r i c s t r u c t u r e o f soy 11S i s i n f l u e n c e d by i o n i c s t r e n g t h ; i . e . , a t 0.5 M i o n i c s t r e n g t h t h e p r o t e i n has a s e d i m e n t a t i o n c o e f f i c i e n t o f U S , b e l o w 0.1 M i o n i c s t r e n g t h i t d i s s o c i a t e s i n t o ' h a l f U S ' u n i t s ( 5 6 ) . A t the i o n i c s t r e n g t h u s e d i n o u r i n i t i a l s t u d y t h e U S was i n h a l f 11S f o r m and p e r h a p s had a


1



12.

KINSELLA

Emulsification

and

Flavor

Binding

323

r e l a t i v e l y high surface charge which impaired b i n d i n g . A t higher i o n i c (0.5 M) s t r e n g t h when the e l e c t r o s t a t i c r e p u l s i o n i s weakened, the i n t a c t U S i s formed, and t h i s i s accompanied by a marked increase i n nonanone b i n d i n g . This o b s e r v a t i o n i s s i g n i f i c a n t because i f the U S e x i s t s i n an environment of low i o n i c strength i n the soybean seed or i f low i o n i c s t r e n g t h prev a i l s during m i l l i n g of the seed, the 11S f r a c t i o n which i s ^35% of soy p r o t e i n , should possess l e s s o f f - f l a v o r s . Because hydrophobic i n t e r a c t i o n s are the predominant f o r c e s i n v o l v e d i n b i n d i n g of apolar organic l i g a n d s to p r o t e i n i t may be f e a s i b l e to remove o f f - f l a v o r s by c h a o t r o p i c agents which weaken hydrophobic i n t e r a c t i o n s (57). Thus, we determined the e f f e c t s o f i n c r e a s i n g urea concentrations on f l a v o r b i n d i n g c h a r a c t e r i s t i c s of soy p r o t e i n . There was a p r o g r e s s i v e decrease i n b i n d i n g a f f i n i t y f o r nonanone as urea was increased from 0 to 4.5 M. I t i s known that the denaturing a c t i o n of urea i n v o l v e s a hydrophobic mechanism that favors exposure of nonpolar residues i n the p r o t e i n to s o l v e n t environment (58). As these conformat i o n a l changes occurred i n the soy p r o t e i n s the b i n d i n g a f f i n i t y concomitantly decreased. At low concentrations of urea the b i n d i n g a f f i n i t y of soy f o r nonanone was decreased s i g n i f i c a n t l y ; i . e . , by ^50 and 70% a t 1.5 and 3.0 M urea, r e s p e c t i v e l y . This observation may have p r a c t i c a l i m p l i c a t i o n s . Thus, r e v e r s i b l e d i s s o c i a t i o n o f soy p r o t e i n subunits; e.g., using urea or other d i s s o c i a t i n g agent may f a c i l i t a t e the removal o f o f f - f l a v o r s such as carbonyls, because b i n d i n g i s an e q u i l i b r i u m process. Thus, u l t r a f i l t r a t i o n of d i l u t e urea s o l u t i o n s of undenatured soy p r o t e i n s may be u s e f u l f o r the p r e p a r a t i o n of bland soy protein. Conclusion The f u n c t i o n a l p r o p e r t i e s of p r o t e i n s r e f l e c t the inherent molecular p r o p e r t i e s of the p r o t e i n s ( s i z e , shape, conformation, molecular f l e x i b i l i t y , s u s c e p t a b i l i t y to d e n a t u r a t i o n ) , the manner i n which they i n t e r a c t with other food c o n s t i t u e n t s and how they i n t e r a c t with the p r e v a i l i n g environmental c o n d i t i o n s . Thus numerous i n t r i n s i c and e x t r i n s i c f a c t o r s a c t i n g i n concert d e t e r mine the f i n a l e f f e c t . T h i s s i t u a t i o n makes i t extremely c h a l l e n g i n g to the food s c i e n t i s t to develop standard methods based on the physicochemical behavior of p r o t e i n i n a simple system. Because of the complexity o f the systems i n v o l v e d , i t i s obvious that the conventional e m p i r i c a l methods f o r comparing the funct i o n a l p r o p e r t i e s of p r o t e i n s w i l l continue to be used. Recogn i z i n g t h i s , food s c i e n t i s t s should s t r i v e to standardize the methods as much as p o s s i b l e so that published data can be v a l i d l y used to compare the r e l a t i v e f u n c t i o n a l i t y of d i f f e r e n t p r o t e i n preparations. There i s a c o n t i n u i n g need f o r dedicated b a s i c research (Table X) d i r e c t e d toward the e l u c i d a t i o n of the physicochemical


324



bases o f s p e c i f i c f u n c t i o n a l p r o p e r t i e s ; e.g., g e l a t i o n , s u r f a c e a c t i v i t y , f i l m formation. W h i l e i t may n o t be p o s s i b l e t o d e f i n e each f u n c t i o n a l property i n mathematical terms, the knowl edge g a i n e d f r o m f u n d a m e n t a l r e s e a r c h w i l l p r o v i d e a c l e a r e r u n d e r s t a n d i n g o f t h e p h y s i c a l and c h e m i c a l r e l a t i o n s h i p s i n v o l v e d . This i n f o r m a t i o n w i l l enable t h e food s c i e n t i s t t o s e l e c t t h e most a p p r o p r i a t e p r o t e i n f o r a s p e c i f i c f u n c t i o n a l p r o p e r t y o r a p p l i c a t i o n and a l s o e n a b l e t h e s c i e n t i s t t o m o d i f y t h e p r o t e i n s to o p t i m i z e d e s i r a b l e p h y s i c a l p r o p e r t i e s . T a b l e X.

Some r e s e a r c h properties.

needs i n t h e a r e a o f f u n c t i o n a l

S t a n d a r d i z i n g the d e f i n i t i o n o f f u n c t i o n a l p r o p e r t i e s D e v e l o p m e n t o f s t a n d a r d i z e d methods b a s e d o n p h y s i c a l p r o p e r t i e s R e l a t i n g f u n c t i o n a l p r o p e r t i e s t o s t r u c t u r a l f e a t u r e s and secondary i n t e r a c t i o n s M a n i p u l a t i o n o f p r o t e i n s t r u c t u r e by c h e m i c a l o r p h y s i c a l alterations T e s t i n g i n food systems Acknowledgments The a u t h o r g r a t e f u l l y a c k n o w l e d g e s t h e s u p p o r t and d i s c u s s i o n s o f D r s . J . S h e t t y , S. Damodaran, a n d R. W a n i s k a , some o f whose d a t a a r e p r e s e n t e d i n t h i s p a p e r . F i n a n c i a l s u p p o r t f r o m t h e N a t i o n a l S c i e n c e F o u n d a t i o n G r a n t #CPE 80-18394 i s g r a t e f u l l y acknowledged.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.

Kinsella, J . E. CRC Crit. Rev. Food Sci. Nutr., 1976, 7, 219. Cherry, J . P., Ed. "Protein Functionality in Foods", American Chemical Society Symp. Series #147: Washington, DC, 1981. Hermansson, A. M. In "Problems in Human Nutrition"; Porter, J. & Rolls, Β., Eds., Acad. Press, New York, 1973; p. 407. Tornberg, E . ; Hermansson, A. M. J . Food Sci., 1977, 42, 468; 1978, 43, 1553. Pour-El, Α., Ed. "Functionality and Protein Structure"; American Chemical Society Symp. Series #92: Washington, DC, 1980. Pour-El, A. In "Protein Functionality in Foods"; Cherry, J. P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981; p. 1. Shen, J . In "Protein Functionality in Foods"; Cherry, J . P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981. Smith, Α.; Circle, A. J . "Soybeans: Chemistry and Technology"; Avi Publ. Co.: Westport, CT, 1978.


KINSELLA

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Galyean, R.; Cotterill, O. J . Food Sci., 1979, 44, 1365. Damodaran, S.; Kinsella, J . E. This volume Chap. 13. Kauzman, P. Advances Protein Chem., 1959, 14, 1. Anfinsen, C.; Scheraga, H. Adv. Protein Chem., 1975, 29, 205. Tanford, C. "The Hydrophobic Effect"; Wiley, J., Ed., New York, 1973. Van Holde, N. In "Food Proteins"; Whitaker, J . & Tannenbaum, S., Eds.; Avi Publ. Co.; Westport, CT, 1971; p. 1. Shimada, K.; Matushita, S. J . Agr. Food Chem., 1980, 28, 413. Kato, A.; Nakai, S. Can. Inst. Food Sci. & Technol. J., press, 1981. Bull, H.; Breese, K. Arch. Biochem. Biophys., 1968, 128, 488. Feeney, R.; Whitaker, J., Eds.; "Food Proteins: Improvement Through Chemical and Enzymatic Modification"; Advances in Chem. Series 160, American Chemical Society: Washington, DC, 1977. Williams, R. J . Biol. Rev., 1979, 54, 389. Ewart, J . J . Sci. Food Agr., 1972, 23, 687. Morr, C. J . Dairy Sci., 1975, 58, 977. Phillips, M. C. Food Technol., 1981, 35, 50. Brandts, J . In "Structure and Stability of Macromolecules"; Timasheff, S. & Fasman, G., Eds., Marcel-Dekker: New York, 1969, Chap. 3. Graham, D. E . ; Phillips, M. C. In "Theory and Practice of Emulsion Technology"; Smith, A. L., Ed., Acad. Press: New York, 1976; p. 75. MacRitchie, F. Adv. In Protein Chem., 1978, 32, 283. Halling, P. J . Crit. Rev. Food Sci. and Nutr., 1981, 12, 155. Waniska, R.; Kinsella, J . E. Unpubl. research, 1982. Tornberg, E. In "Functionality and Protein Structure"; Pour-El, Α., Ed.; American Chemical Society Symp. Series #92: Washington, DC, 1976, p. 105. Waniska, R.; Shetty, J.; Kinsella, J . E. J . Agr. Food Chem., 1981, 29, 826. Kinsella, J . E. Food Chem. (Lond.) 1982 (press). McWatters, K; Cherry, J . P. In "Protein Functionality in Foods"; Cherry, J . P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981, p. 217. Mulder, H.; Walstra, P. "The Milk Fat Globule: Emulsion Science as Applied to Milk Products"; Pub. Commonwealth Agr. Bureau: Bucks, England, 1974. Walstra, P.; Ourtwijn, H.; deGroaf, J . Neth. Milk Dairy J., 1969, 23, 12. Pearce, K.; Kinsella, J . E. J . Agr. Food Chem., 1976, 26, 716.

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15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

Emulsification

and

Flavor

Binding

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12.



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FOOD P R O T E I N

DETERIORATION

35. Kinsella, J. E.; Shetty, J. In "Functionality and Protein Structure"; Pour-El., Ed.; American Chemical Society Symp. Series #92: Washington, DC, 1979, p. 37. 36. Leonard, W.; Foster, J. J. Biol. Chem., 1961, 236, 2262. 37. Aoki, K.; Murata, M.; Hiramatus, K. Anal. Biochem., 1974, 59, 16. 38. McKenzie, H. "Milk Proteins: Chemistry and Technology"; Acad. Press: New York, 1971; p. 257. 39. Bigelow, C. J. Theor. Biol., 1967, 16, 187. 40. Kuntz, L . ; Kauzmann, W. Adv. Protein Chem., 1974, 28, 239. 41. Leslie, R. B. J. Am. Oil Chem. Soc., 1976, 56, 290. 42. Kato, A.; Makai, S. Biochem. Biophys. Acta., 1980, 624, 13. 43. Kinsella, J. E. In "Criteria of Food Acceptance"; Solms, J. & Hall, R., Eds.; Forster Verlog A. G.: Zurich, Switzerland, 1981, p. 140. 44. Blouin, F.; Zarins, Z.; Cherry, J. In "Protein Functionality in Foods"; Cherry, J. P., Ed.; American Chemical Society Symp. Series #147: Washington, DC, 1981, p. 21. 45. Hegg, P. O.; Lofquist, B. J. Food Sci., 1974, 39, 1231. 46. Damodaran, S.; Kinsella, J. E. J. Biol. Chem., 1980, 255, 8503. 47. Kinsella, J. E. Crit. Rev. Food Sci. Nutr., 1978, 10, 147. 48. Kinsella, J. E. J. Am. Oil Chem. Soc., 1979, 56, 242. 49. Damodaran, S.; Kinsella, J. E. J. Agr. Food Chem., 1981, 29, 1253. 50. Damodaran, S.; Kinsella, J. E. J. Agr. Food Chem., 1980, 28, 567. 51. Abraham, M. J. Am. Chem. Soc., 1980, 102, 5910. 52. Franzen, K.; Kinsella, J. E. J. Agr. Food Chem., 1976, 24 914. 53. Arai, S.; Noguchi, M.; Kato, H.; Fujimaki, M. Agr. Biol. Chem., 1970, 34, 1420 54. Thanh, V.; Shibasaki, K. J. Agr. Food Chem., 1978, 26, 692. 55. Badley, R.; Atkinson, D.; Oldani, D.; Green, J.; Stubbs, J. Biochem. Biophys. Acta., 1975, 412, 216. 56. Koshiyama, L. Int. J. Peptide & Protein Res., 1972, 4, 167. 57. Damodaran, S.; Kinsella, J. E. J. Biol. Chem., 1981, 256, 3394. 58. Wetlaufer, D.; Malik, S.; Stoller, L . ; Coffin, R. J. Am. Chem. Soc., 1964, 86, 508. RECEIVED June

1, 1982.


Food Protein Deterioration - American Chemical Society

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