Enzyme Modification of Proteins - ACS Symposium Series (ACS

13 Enzyme Modification of Proteins R. DIXON PHILLIPS and L A R R Y R. BEUCHAT

Downloaded by GEORGETOWN UNIV on October 29, 2014 | http://pubs.acs.org Publication Date: March 6, 1981 | doi: 10.1021/bk-1981-0147.ch013

Department of Food Science, University of Georgia College of Agriculture Experiment Station, Experiment, GA 30212

Functionality can be defined as the physio-chemical behavior which proteins exhibit while interacting with other constituents of multi-component food systems. The basis of this behavior is, of course, the chemical nature of proteins: long chains of covalently bonded amino acid residues, the side groups of which may be charged, polar, or hydrophobic. These chains are coiled and arranged into a variety of configurations and may be associated with other chains through covalent or non-covalent bonds. Such macro-systems assume shapes which vary from globular to fiberous and exhibit a wide range of functional behavior, as shown in Figure 1 and described elsewhere in this volume. See also the extensive review by Kinsella (1). The topic of this chapter deals with the effect of proteolytic enzyme action on functionality. As noted by Whitaker (2), protein functionality can be altered by many different types of enzymes. However, since the great majority of enzymes which modify protein behavior in food systems are proteases, we will confine our discussion to them. The use of proteases f o r m o d i f i c a t i o n of p r o t e i n f u n c t i o n a l i t y is an ancient a r t . O r i g i n a l l y , the enzymes were e i t h e r endogenous to the food ( e . g . , aging of meat) or part of an added bio-system ( e . g . m i c r o b i a l c u l t u r e s in cheeses) (3). With increased knowledge of what enzymes are and how they work, t h e i r d e l i b r a t e i s o l a t i o n and a d d i t i o n to food systems became widely p r a c t i c e d . The motivation f o r using proteases to a l t e r p r o t e i n f u n c t i o n a l i t y in food i s t w o - f o l d : (a) The need to convert food source p r o t e i n s to more p a l a t a b l e or useful forms ( i n c r e a s i n g l y important with the advent of "novel" p r o t e i n s ) ; and (b) the rather s p e c i f i c way in which proteases accomplish t h i s g o a l . Proteases exert t h e i r i n f l u e n c e s by c a t a l y z i n g the cleavage o r , more r a r e l y , the synthesis of peptide bonds (Figure 2 ) . The breaking of peptide bonds r e s u l t s in three major m o d i f i c a t i o n s : (a) An increase in the number of polar groups ( - N H 4 , - C O 2 " ) , and an increase in the h y d r o p h i l i c i t y of the product; (b) a

0097-6156/81/0147-0275$06.00/0 © 1981 American Chemical Society

In Protein Functionality in Foods; Cherry, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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Figure 2. Formation of an acyl-enzyme intermediate by peptide bond scission and nucleophilic attack by an amine group to form a new peptide bond (transpeptidation), or by water to release the shortened chain (hydrolysis) β)



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decrease in molecular weight of the peptide c h a i n s , although not n e c e s s a r i l y of the t o t a l aggregates; (c) a p o s s i b l e a l t e r a t i o n in molecular c o n f i g u r a t i o n . Thus peptide bond breakage may be the f i r s t step in a complex s e r i e s of changes which a l t e r s f u n c t i o n a l i t y , e . g . , by d i s s o c i a t i o n of subunits or by opening a compact globular s t r u c t u r e to expose the hydrophobic i n t e r i o r to an aqueous phase. The net synthesis of peptide bonds (as i s claimed in the p l a s t e i n r e a c t i o n ) would be expected to decrease the number of polar groups, and the h y d r o p h i l i c i t y , to increase molecular weight, and to a f f e c t c o n f i g u r a t i o n . The foregoing d e s c r i p t i o n of the e f f e c t s of proteases i s admittedly a s i m p l i f i e d one when we consider the complexity of food systems containing not only many constituents besides p r o t e i n , but a l s o a great v a r i e t y of d i f f e r e n t p r o t e i n s . Likewise the enzymes which modify f u n c t i o n a l i t y are u s u a l l y m u l t i f u n t i o n a l mixtures, whether endogenous or exogenous. Nevertheless, t h i s d e s c r i p t i o n i s h e l p f u l in understanding the e f f e c t s of proteases on f u n c t i o n a l i t y . We have chosen to discuss enzyme m o d i f i c a t i o n of proteins in terms of changes in various f u n c t i o n a l p r o p e r t i e s . Another approach might have been to consider s p e c i f i c substrates f o r protease a c t i o n such as meat and m i l k , legumes and c e r e a l s , and the novel sources of food p r o t e i n such as leaves and microorganisms (4). A l t e r n a t i v e l y , the proteases themselves provide categories f o r d i s c u s s i o n , among which are t h e i r source (animals, p l a n t s , microorganisms), t h e i r type ( s e r i n e - , s u l f h y d r y l - , and metalloenzymes), and t h e i r s p e c i f i c i t y (endo- and exopeptidases, aromatic, a l i p h a t i c , or basic residue bond specificity). See Yamamoto (3) f o r a review of p r o t e o l y t i c enzymes important to f u n c t i o n a l i t y . P l a s t e i n Reaction The protease-catalyzed synthesis of peptide bonds i s known as the p l a s t e i n r e a c t i o n ( 5 J . P l a s t e i n i t s e l f i s defined as the product formed by t h i s r e a c t i o n which is i n s o l u b l e in t r i c h l o r o a c e t i c acid solutions (6). The p l a s t e i n r e a c t i o n has been most e x t e n s i v e l y i n v e s t i g a t e d by researchers in Japan (5^, 6, 7_ 8, 9). These s c i e n t i s t s have reviewed various aspects of p l a s t e i n and the p l a s t e i n r e a c t i o n and i t s importance to p r o t e i n f u n c t i o n a l i t y and n u t r i t u r e (6, 8, 10). The c o n d i t i o n s necessary Tor the p l a s t e i n r e a c t i o n have been reviewed by Fujimaki et a l . (8), and compared to those necessary f o r p r o t e o l y s i s by Arai et a l . (6). The substrate f o r the s y n t h e t i c r e a c t i o n must c o n s i s t of low molecular weight p e p t i d e s , p r e f e r a b l y in the tetramer to hexamer range. These are u s u a l l y produced from proteins by protease a c t i o n . A number of p r o t e o l y t i c enzymes and p r o t e i n substrates have been i n v e s t i g a t e d f o r producing p l a s t e i n r e a c t i o n s u b s t r a t e s . The most often used proteases are pepsin (9, Y\), and papain (]2 U) but others 9

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i n c l u d i n g a c i d , s e r i n e , and s u l f h y d r y l proteases have been described ( 8 ) . Substrate proteins have been derived from soy (14), casein and z e i n (6), gluten ( 7 ) , milk whey (5^, 15), microorganisms (5, _7, 9 j , l e a v e s (16J, and egg albumin~T5, 11)· Arai et a l . [5) TnvestTgated the e f f e c t s of substrate h y d r o p h i l i c i t y and concluded that a combination of h y d r o p h i l i c and hydrophobic peptides was o p t i m a l . They proposed a parameter, β / α , r e l a t e d to h y d r o p h i l i c i t y and found that maximum y i e l d s of p l a s t e i n are obtained when 3/a - 0 . 5 . Amino acids may a l s o be incorporated along with peptides into p l a s t e i n s provided they are present in the r e a c t i o n mixture as e s t e r s . Aso et a l . (12) s t u d i e d the s p e c i f i c i t y of amino a c i d e s t e r i n c o r p o r a t i o n and found that the r e a c t i o n v e l o c i t y increased with the hydrophobicity of both the amino a c i d s i d e chain and the alcohol moiety. In contrast to peptide bond cleavage but in keeping with the law of mass a c t i o n , synthesis i s promoted by high substrate concentration (20 - 40%, w/v). At , J[2, 16). A number of other enzymes are discussed by Fujimaki et al.~~[8). Yamamoto (3) described the c h a r a c t e r i s t i c s of p r o t e a s e - a c t i v e s i t e s . Amino a c i d residues are arranged in such a way as to accommodate the s u b s t r a t e and to d e s t a b i l i z e the target bond (Figures 3 and 4 ) . Serine proteases such as chymotrypsin and s u l f h y d r y l proteases such as papain are s i m i l a r in the way they accomplish t h i s bond a c t i v a t i o n . The acyl-enzyme intermediates i l l u s t r a t e d in Figures 3 and 4 are c e n t r a l to both l y t i c and s y n t h e t i c r e a c t i o n s . The most s t r a i g h t f o r w a r d mechanism f o r peptide bond formation would be r e v e r s a l of p r o t e o l y s i s promoted by the very high substrate concentrations. The f i r s t step in such a mechanism would be formation of a peptidyl-enzyme intermediate with l o s s of water (the reverse of r e a c t i o n b, Figure 3 ) . A l t e r n a t i v e l y , the acyl-enzyme intermediate could be formed with s c i s s i o n of a peptide bond (Figure 2 ) . The second step in both mechanisms would be n u c l e o p h i l i c attack by the amino group of a peptide or amino a c i d e s t e r on the a c t i v e acyl intermediate to form the new bond (Figures 2 and 4 ) . The f i r s t of these mechanisms i s condensation; the second, t r a n s p e p t i d a t i o n . There i s evidence that both mechanisms are a c t i v e in the p l a s t e i n r e a c t i o n (8, 2 5 ) , although the l a t e r i s thermodynamically favored (10). The properties of p l a s t e i n s are q u i t e d i f f e r e n t from those of the s t a r t i n g peptide mixture. In a d d i t i o n to i n s o l u b i l i t y in



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Figure 3.

Possible mechanism for (a) formation and (b) breakdown of acyl-enzyme (chymotrypsin) intermediate (3)

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Schematic of the active site of papain with peptide and amino acid ester in place β)



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t r i c h l o r o a c e t i c acid s o l u t i o n s , p l a s t e i n s are u s u a l l y i n s o l u b l e in ethanol and acetone and often i n s o l u b l e in water and buffers ( 6 ) . The p r e v i o u s l y mentioned advantage of hydrophobic reactants i s p a r t i a l l y due to the i n s o l u b i l i t y of the products in water. The r e a c t i o n i s thus s h i f t e d toward products. A d d i t i o n a l l y , an organic solvent such as acetone may be added to the r e a c t i o n mixture to f u r t h e r decrease the s o l u b i l i t y of products (b). G e l l i n g i s often observed during the p l a s t e i n r e a c t i o n . Tsai et a l . (17) studied the e f f e c t of substrate concentration on gel rheology. They observed that stronger gels were formed as substrate concentration i n c r e a s e d , e s p e c i a l l y above 20%. Edwards and Shipe (11) i n v e s t i g a t e d the r e l a t i o n s h i p of gel strength to the degree of" h y d r o l y s i s of p e p s i n - d i g e s t e d egg albumin and to the enzyme used in the p l a s t e i n r e a c t i o n . They reported that gel strength decreased as h y d r o l y s i s time increased from 4 to 24 hours and that the use of pepsin r e s u l t e d in the strongest gels followed by ot-chymotrypsin then papain. Perhaps the most c o n t r o v e r s i a l aspect of the p l a s t e i n r e a c t i o n has been the molecular weight of r e s u l t i n g products. E a r l y work (18) i n d i c a t e d very high molecular weights (250,000 - 500,000) as determined by u l t r a - c e n t r i f u g a l a n a l y s i s . Arai et a l . (6) found the upper l i m i t of p l a s t e i n formed from a soy p r o t e i n h y ï ï r o l y z a t e to be ^30,000 as measured by gel e x c l u s i o n chromatography. Hofsten and L a l a s i d i s (15), however, reported that p l a s t e i n s subjected to gel exclusion chromatography in 50% a c e t i c a c i d showed no increase in molecular s i z e over that of the r e a c t a n t s . Monti and Jost (]9_) reached the same conclusion based on gel chromatography in DMSO and on a n a l y s i s of α - a m i n o nitrogen in plasteins. Hofsten and L a l a s i d i s (15) noted that hydrophobic peptides showed unusual e l u t i o n behavior on sephadex gels in water or d i l u t e b u f f e r s , providing a p o s s i b l e explanation f o r d i f f e r e n c e s in t h e i r r e s u l t s compared to those of A r a i et a l . (6). Based on the s o l u b i l i t y and chromatographic behavior of p l a s t e i n s , Hofsten and L a l a s i d i s (15) concluded that gel formation was due to nonpolar i n t e r a c t i o n s of small hydrophobic p e p t i d e s . The o r i g i n a l a p p l i c a t i o n of the p l a s t e i n r e a c t i o n to foods was the removal of b i t t e r peptides from enzyme hydrolyzates of soy p r o t e i n (6). Peptides containing aromatic or a l i p h a t i c s i d e chain amino a c i ï ï s , e s p e c i a l l y in the terminal p o s i t i o n s , have been i d e n t i f i e d with b i t t e r n e s s of enzyme hydrolyzates (8). Subjecting hydrolyzates to the p l a s t e i n r e a c t i o n e l i m i n a t e s thTs b i t t e r n e s s e i t h e r by moving the hydrophobic residue to the i n t e r i o r of the peptide or by c l e a v i n g i t during t r a n s p e p t i d a t i o n . Other f u n c t i o n a l improvements of proteins that may be accomplished by use of the p l a s t e i n r e a c t i o n have been reviewed by Fujimaki et a l . (8). In a d d i t i o n to b i t t e r p e p t i d e s , unwanted i m p u r i t i e s which c o n t r i b u t e to o f f f l a v o r s , odors or c o l o r s may be e l i m i n a t e d from p r o t e i n s by enzyme h y d r o l y s i s , p u r i f i c a t i o n of the hydrolyzate, and r e s y n t h e s i s by the p l a s t e i n r e a c t i o n (Figure 5 ) . An improvement in the s o l u b i l i t y p r o f i l e of soy p r o t e i n was


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Figure 5.

Scheme for removing impurities from protein substrate by hydrolysis, purification, and resynthesis via the plastein reaction (S)



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demonstrated by p r o t e i n - c a t a l y z e d i n c o r p o r a t i o n of glutamic a c i d into soy p r o t e i n hydrolyzates (7). The production of food gels could a l s o be an important r o l e f o r the p l a s t e i n r e a c t i o n (10). Another major a p p l i c a t i o n of the p l a s t e i n r e a c t i o n i s n u t r i t i o n a l improvement of proteins by the incorporation of l i m i t i n g amino acids (8). A p l a s t e i n containing approximately 7% methionine was produce? from soy p r o t e i n hydrolyzate and L-methionine ethyl e s t e r in the presence of papain. This material was shown to be u t i l i z e d as a source of methionine in the r a t , producing a PER of 3.38 when incorporated into soy p r o t e i n d i e t s to give a methionine l e v e l of 2.74% of p r o t e i n . S i m i l a r l y , l y s i n e has been incorporated into gluten hydrolyzate and l y s i n e , threonine and tryptophan have been i n d i v i d u a l l y incorporated into zein h y d r o l y z a t e s . Lysine, methionine, and tryptophan were incorporated simultaneously into hydrolyzates of p r o t e i n from photosynthetic o r i g i n . A very i n t e r e s t i n g a p p l i c a t i o n of t h i s procedure involved the preparation of low-phenylalanine p l a s t e i n s from a combination of f i s h protein concentrate and soy p r o t e i n i s o l a t e by a p a r t i a l h y d r o l y s i s with pepsin then pronase to l i b e r a t e mainly phenylalanine, t y r o s i n e , and tryptophan, which were then removed on sephadex G-15. Desired amounts of t y r o s i n e and tryptophan were added back in the form of ethyl esters and a p l a s t e i n s u i t a b l e f o r feeding to i n f a n t s a f f l i c t e d with phenylketonuria was produced. E r i k s e n and Fagerson (_10), while c i t i n g the p o t e n t i a l of the p l a s t e i n r e a c t i o n to the food i n d u s t r y , l i s t e d several constraints: (a) The p o s s i b l e problems of scale-up to i n d u s t r i a l s i z e d o p e r a t i o n s ; (b) the presence of r e s i d u a l enzymes; (c) the p o s s i b i l i t y that t o x i c peptides could be produced; and (d) the stigma attached to s y n t h e t i c f o o d s . Yamashita et a l . (_7) mentioned economic c o n s t r a i n t s due to cost of enzymes and e s t a b l i s h i n g s a f e t y , which must be determined through animal feeding s t u d i e s . Recent work has addressed some of these constraints. Yamashita et a l . (13) i d e n t i f i e d r e a c t i o n c o n d i t i o n s that allowed the incorporation oT"methionine into soy p r o t e i n in a single step. This involved a r e a c t i o n mixture that was 20% by weight soy i s o l a t e , 1 nM in c y s t e i n e (enzyme a c t i v a t o r ) , an i n i t i a l pH of 10, a papain to substrate r a t i o to 1:100, a temperature of 3 7 ° C , and a r e a c t i o n time 8 hours. When a 20:1 mixture of soy protein and methionine ethyl ester was incubated under these c o n d i t i o n s , 81.5 mole percent of the L-methionine was incorporated. P a l l a v i c i n i et a l . (16) u t i l i z e d α - c h y m o t r y p s i n immobilized on c h i t i n to c a t a l y z e p l a s t e i n formation from l e a f p r o t e i n hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by s o l u b l e enzymes. M o d i f i c a t i o n of S p e c i f i c Functional Solubility.

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p r o p e r t i e s of soy p r o t e i n i s o l a t e as r e l a t e d to the extent of treatment with a neutral protease from A s p e r g i l l u s oryzae. Heat treatment of i s o l a t e s decreased the s o l u b i l i t y of n i t r o g e n , but with i n c r e a s i n g enzyme treatment l e s s p r o t e i n was rendered insoluble. Untreated c o n t r o l s did not show s i g n i f i c a n t s o l u b i l i t y at pH 4.5 in the presence of 0.03M CaCl2, but p r o t e i n s were i n c r e a s i n g l y s o l u b l e as treatment progressed. The author points out that increased acid s o l u b i l i t y would be advantageous in the u t i l i z a t i o n of soy proteins in a c i d i c foods, whereas calcium t o l e r a n c e is important when calcium a d d i t i o n i s needed f o r improved n u t r i t i o n such as in i m i t a t i o n d a i r y products. Enzyme treatment to increase a c i d s o l u b i l i t y (2Λ) has a l s o been described. Exposure of proteins to heat has been shown to adversely a f f e c t p e p t i z a t i o n properties (22). The c r i t i c a l denaturation temperature f o r peanut p r o t e i n in meal i s above 118°C (dry heat) and above 8 0 ° C at 100% r e l a t i v e humidity. Although heat treatments r o u t i n e l y employed in a majority of peanut o i l m i l l s are i n s u f f i c i e n t l y c o n t r o l l e d to prevent denaturation of meal p r o t e i n , the authors suggest that temperature and moisture c o n t r o l during processing can be maintained at l e v e l s s u f f i c i e n t to achieve o i l e x t r a c t i o n without d r a s t i c denaturation of p r o t e i n . Better and Davidsohn (23) reported that the s u s c e p t i b i l i t y of proteins in o i l s e e d presscllce and s o l v e n t - e x t r a c t e d meal was s t r o n g l y a f f e c t e d by heat treatment. Working with peanut and coconut presscake, they demonstrated that the stronger the heat treatment, the higher are the concentrations of pepsin r e q u i r e d to make p e p t i z a t i o n more complete. This i s an important f a c t to bear in mind when o i l s e e d press or e x t r a c t i o n residues are being used as raw m a t e r i a l s f o r production of i n d u s t r i a l p r o t e i n . Nitrogen s o l u b i l i t y of defatted peanut f l o u r as a f f e c t e d by h y d r o l y s i s with pepsin, bromelain and t r y p s i n was i n v e s t i g a t e d by Beuchat et a l . (24). P r o f i l e s of e n z y m a t i c a l l y hydrolyzed f l o u r in water were markedly d i f f e r e n t from t h e i r r e s p e c t i v e c o n t r o l s . S o l u b i l i t i e s f o r pepsin, b r o m e l a i n - , and t r y p s i n - t r e a t e d samples were 64, 46, and 30% of t o t a l n i t r o g e n , r e s p e c t i v e l y , at pH 4 . 0 , compared to l e s s than 15% f o r nontreated f l o u r s . Although hydrolyzed samples had higher nitrogen s o l u b i l i t i e s than t h e i r r e s p e c t i v e pH-adjusted c o n t r o l s in the a l k a l i n e pH range, they were l e s s s o l u b l e than samples that had received no heat or pH adjustment. The i n f l u e n c e of change in i o n i c strength of peanut f l o u r s l u r r y r e s u l t i n g from the a d d i t i o n of HC1 and NaOH during pH adjustment was c i t e d as a p o s s i b l e reason f o r a l t e r a t i o n s in nitrogen s o l u b i l i t i e s at a l k a l i n e pH v a l u e s . Beuchat et a l . (24) a l s o examined the nitrogen s o l u b i l i t y p r o f i l e s f o r enzymatically hydrolyzed and c o n t r o l peanut f l o u r samples in 0.03M C a (as C a C l 2 ) . S o l u b i l i t i e s of c o n t r o l s between pH 2.0 and 5.0 in 0.031 C a s o l u t i o n s were s i m i l a r to those noted f o r water; however, very l i t t l e increase in nitrogen s o l u b i l i t y of c o n t r o l s was noted in the pH 5.0 t c 11.0 range. 2 +

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S i m i l a r data were reported by Rhee et a l . (25). They noted that at pH 2.0 to 3 . 0 , peanut p r o t e i n e x t r a c t a b i l i t y was enhanced in 0.01 to 0.10M CaCl2 but suppressed in s o l u t i o n s c o n t a i n i n g 0.25M or higher CaCl2Treatment of peanut f l o u r with pepsin, bromelain and t r y p s i n g r e a t l y increased the nitrogen s o l u b i l i t y at pH 2.0 to 11.0 in 0.03M C a (24). Pepsin treatment r e s u l t e d in the greatest i n c r e a s e . Lowest s o l u b i l i t i e s in the p r o f i l e s were at pH 4.0 to 5 . 0 , where values of 81, 57, and 38% were measured f o r p e p s i n - , b r o m e l a i n - , and t r v p s i n - t r e a t e d f l o u r s , r e s p e c t i v e l y . A concentration of 0.03M C a ^ has been p r e s c r i b e d as a minimum in the formulation of i m i t a t i o n m i l k . Evidence from the study reported by Beuchat et a l . (24) suggests that enzymatic h y d r o l y s i s of peanut f l o u r modifies protein to the extent that i t is h i g h l y s o l u b l e in 0.03M C a at a pH range normally a s s o c i a t e d with f l u i d m i l k . Further s t u d i e s are r e q u i r e d to assess the e f f e c t of enzyme-induced p r o t e o l y s i s on o r g a n o l e p t i c p r o p e r t i e s of hydrolyzed peanut protein s o l u t i o n s . Changes in s o l u b l e proteins of peanut f l o u r caused by p r o t e o l y t i c enzyme d i g e s t i o n as detected by gel e l e c t r o p h o r e s i s (24) resemble those r e s u l t i n g from fermentation of peanut meal wrth fungi (26) and growth of fungi on v i a b l e peanut kernels (27). "Standard" peanut p r o t e i n e l e c t r o p h o r e t i c patterns are d i s t i n c t l y modified as a r e s u l t of treatment with commercial proteases or fungal growth. Biochemical transformations in proteins include decomposition of large molecular weight g l o b u l i n s such as arachin to smaller components, followed by r a p i d q u a n t i t a t i v e and q u a l i t a t i v e decreases in these l a t t e r c o n s t i t u e n t s as fungal enzymatic a c t i v i t y progresses. Thus, nitrogen s o l u b i l i t y changes d r a m a t i c a l l y . The use of l i v e fungal c u l t u r e s rather than enzymes extracted therefrom to modify the f u n c t i o n a l i t y of defatted peanut f l o u r was i n v e s t i g a t e d by Quinn and Beuchat (28). Fungi not known to produce mycotoxins were s t u d i e d : A s p e r g i l l u s elegans, A. oryzae, Mucor h i e m a l i s , Neurospora s i t o p h i l a , and Rhizopus οngosporus. Each of the fungi increased the nitrogen s o l u b i l i t y of peanut f l o u r in the pH range of 3.0 to 6.0 compared to the control. In p a r t i c u l a r , M. hiemalis increased the s o l u b i l i t y at pH 4.0 to 5.0 from less than 5% in a nontreated c o n t r o l to about 34% in the f r e e z e - d r i e d ferment. Although ferments were demonstrated to be nontoxic, there may be s p e c i a l r e g u l a t o r y problems a s s o c i a t e d with i n c o r p o r a t i o n of new m i c r o b i a l l y - d e r i v e d or modified ingredients into food products f o r the purpose of functionality. Sekul et a l . (29) studied the nitrogen s o l u b i l i t y p r o p e r t i e s of enzyme-hydrolyzed peanut p r o t e i n s . A deionized water d i s p e r s i o n of peanut f l o u r (1:10, w/v) was t r e a t e d with papain (0.5% t o t a l volume) at 45°C f o r 15 min. S o l u b i l i t y was t e s t e d over a range of pH 1 to 9. In g e n e r a l , papain treatment improved s o l u b i l i t y at a l l l e v e l s examined except pH 2 and 8 (Figure 6 ) . The authors suggest that h y d r o l y s i s of peanut proteins in aqueous 2 +

+


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s l u r r i e s of defatted f l o u r by papain should be of commercial interest. For beverage a p p l i c a t i o n where high p r o t e i n s o l u b i l i t y is desired (milk-type d r i n k s , a c i d pH f r u i t - f l a v o r e d beverages, dry soup, sauce, or gravy mixes), p a r t i a l l y hydrolyzed peanut proteins seem to have an advantage over unhydrolyzed p r o t e i n s . A l s o , papain i s one of the l e a s t expensive FDA-approved vegetable enzymes; there are economic or FDA h e a l t h - r e l a t e d r e s t r i c t i o n s on the a d d i t i o n of animal or m i c r o b i a l enzymes to food f o r m u l a t i o n s ; and studies have shown that papain i s f r e e of peptidase a c t i v i t y


(30).

Fontaine et a l . ( 3 1 ) presented data comparing the s o l u b i l i t y behavior of proteins of peanut and cottonseed meals, proteins of corresponding d i a l y z e d meals, and i s o l a t e d p r o t e i n s . While the shapes of the p H / s o l u b i l i t y curves f o r cottonseed and peanut meals d i f f e r e d , the response of proteins to the removal of d i a l y z a b l e meal c o n s t i t u e n t s was s i m i l a r . Data i n d i c a t e d the presence of natural m a t e r i a l s in both meals which decreased the s o l u b i l i t y of meal nitrogen at c e r t a i n a c i d pH values but exerted no e f f e c t at a l k a l i n e pH values. Thus procedures f o r s o l u b i l i z i n g proteins by treatment with p r o t e o l y t i c enzymes should a l s o be designed with c o n s i d e r a t i o n of the i n f l u e n c e of non-protein c o n s t i t u e n t s . The r e l a t i v e a c t i v i t i e s of commercially a v a i l a b l e p r o t e o l y t i c enzymes and h e m i c e l l u l a s e were tested on cottonseed cake by Arzu et a l . ( 3 2 J . These included f i c i n concentrate, three b a c t e r i a l p r o t e i n a s e s , papain concentrate, bromelain concentrate, p e p s i n , t r y p s i n , a c i d fungal protease, and fungal h e m i c e l l u l a s e . The most a c t i v e enzymes were two b a c t e r i a l proteinases and the ones derived from higher plants (bromelain, papain, and f i c i n ) , which s o l u b i l i z e d about 4 0 % of the i n i t i a l i n s o l u b l e p r o t e i n . Activity shown by the h e m i c e l l u l a s e was the lowest, b a r e l y f a c i l i t a t i n g the s o l u b i l i z a t i o n of nitrogenous compounds, contrary to what has been reported by Hang et a l . (32U 3 4 ) on mung and pea beans employing c e l l u l a s e , where a large s o l u ï ï T l i z a t i o n was n o t i c e d . Gossypol markedly i n h i b i t e d the a c t i v i t y of b a c t e r i a l proteases. Sreekantiah et a l . ( 3 5 ) evaluated defatted sesame and peanut meals, and four kinds o f T e a n s - c h i c k p e a , green gram, black gram, and f i e l d bean - f o r p r o t e i n e x t r a c t i o n c h a r a c t e r i s t i c s a f t e r treatment with enzymes. Three commercial p r o t e o l y t i c enzymes derived from A s p e r g i l l u s species and one from Trametes sanguina were t e s t e d . The protease from T. sanguina was best f o r hydrolyzing sesame meal and chiclcpea. In the case of sesame meal, the s o l u b l e p r o t e i n increased fom 9 . 4 8 to 6 4 . 1 2 % while the increase in amino nitrogen was from 0 . 6 to 9 . 2 % . Appreciable i n c r e a s e s , both in s o l u b l e and amino n i t r o g e n , were observed in t r e a t e d bengal gram. Results showed that uncooked substrates were not attacked by enzymes. Adjustment of pH to 3 . 0 by d i l u t e a c i d a f t e r heat treatment was congenial f o r the h y d r o l y s i s of p r o t e i n in sesame meal and chickpea. The amino a c i d composition of e x t r a c t s of enzyme-hydrolyzed proteins may d i f f e r somewhat from that of untreated m a t e r i a l s . In the case of sesame meal, there



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was an increase in the percentage of a l l e s s e n t i a l amino acids except methionine, when compared to the defatted meal. In chickpea, even though the percentage of protein in the f r e e z e - d r i e d extract was higher, there was a reduction in l y s i n e , h i s t i d i n e , arginine and l e u c i n e . Hermansson et a l . (36) used pepsin and papain to s o l u b i l i z e rapeseed p r o t e i n concentrate. Papain had a lower s o l u b i l i z i n g e f f e c t than d i d pepsin. However, the f a c t that pepsin has an optimum pH f o r a c t i v i t y at about 1.6, f a r below the pH range of most f o o d s , made i t p o s s i b l e to study the e f f e c t s of c o n t r o l l e d hydrolysis. At pH 7 . 0 , a l l hydrolysates were more s o l u b l e than the o r i g i n a l rapeseed protein concentrate. The use of enzymes f o r s o l u b i l i z a t i o n of seed and l e a f proteins has been studied as a means of overcoming d i f f i c u l t i e s presented by the varying c o n d i t i o n of seed and l e a f material a v a i l a b l e f o r processing (37). U l t r a s o n i c energy was reported to increase the e f f i c i e n c y of enzyme s o l u b i l i z a t i o n procedures. The e f f e c t s of various conditions of h y d r o l y s i s of cottonseed and a l f a l f a meal p r o t e i n with t r y p s i n were d e f i n e d . Several p r o t e o l y t i c enzymes have been shown to enhance the s o l u b i l i t y of f i s h p r o t e i n concentrate (38). Product i n h i b i t i o n and s e l f d e s t r u c t i o n of enzymes occurred, so that rates of h y d r o l y s i s decreased with time. The e l i m i n a t i o n or i n a c t i v a t i o n of enzymes used to t r e a t proteins i s a c r i t i c a l prdblem once the desired m o d i f i c a t i o n in f u n c t i o n a l i t y i s achieved. In many i n s t a n c e s , product i n h i b i t i o n or s e l f d e s t r u c t i o n does not occur as noted above f o r f i s h p r o t e i n concentrate. As stated by Puski (20), i f heat i n a c t i v a t i o n i s used, the proteins may be denaturecTand r e v e r t to i n s o l u b l e forms. Washing out the enzyme at i t s i s o e l e c t r i c point would a l s o remove a portion of the protein which i s s o l u b i l i z e d by the enzyme. I n a c t i v a t i o n of enzymes by chemical means may a l s o cause s i g n i f i c a n t changes in the p r o t e i n . Thus, while d e s i r e d f u n c t i o n a l m o d i f i c a t i o n s of food ingredients may be obtained through enzyme treatment, the problem of l a t e n t enzyme a c t i v i t y in food formulations must be addressed. Emulsifying Capacity and S t a b i l i t y . Zakaria and McFetters (39) s t u d i e d the e f f e c t s of pepsin h y d r o l y s i s of heated soy protein i s o l a t e . Treatment was c a r r i e d out between pH 1.0 and 4.0. At short h y d r o l y s i s times, there was a r a p i d increase in the free amino groups in the p r o t e i n and a corresponding increase in e m u l s i f i c a t i o n a c t i v i t y (expressed as the volume percentage of the emulsified layer). The e m u l s i f i c a t i o n a c t i v i t y d e c l i n e d as the incubation time was increased (Figure 7 ) . However, only small increases in f r e e amino groups occurred during the period of decreased a c t i v i t y . Good emulsion s t a b i l i t y p r o p e r t i e s were observed in hydrolysates with high emulsion a c t i v i t y . The authors suggest that e i t h e r h y d r o l y s i s of a few key soy p r o t e i n peptide bonds r e s u l t s in r e l a t i v e l y large changes in e m u l s i f i c a t i o n


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I.Or

—1

1

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2

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10

Nitrogen solubility (Kjeldahl) of peanut proteins: P.H.P. = partial hy drolysis by 5% papain; CON. = control (no papain) (2%).

Figure 6.

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Libbensmittel-Wissenschaft & Technologie

Figure 7. Emulsifying activity (expressed as volume percentage of the emulsified layer) of pepsin hydrolysates of soy protein as a function of pH and hydrolysis time. No hydrolysis (A); 2 h (A); 8 h (X); 17 h Ο; 24 h (Ο) (39).



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p r o p e r t i e s or that changes other than p r o t e o l y s i s occur which affect functionality. A d l e r - N i s s e n and Olsen (40) studied the i n f l u e n c e of peptide chain length on the t a s t e ancPfunctional properties of enzymatically modified soy p r o t e i n . The emulsifying c a p a c i t y of modified proteins could be improved s i g n i f i c a n t l y compared to unmodified c o n t r o l samples by c o n t r o l l i n g the extent of h y d r o l y s i s . The emulsifying c a p a c i t y of soy p r o t e i n i s o l a t e can be increased by treatment with neutral fungal protease (20); however, enzyme treatment decreased emulsifying s t a b i l i t y . It was t h e o r i z e d that enzyme d i g e s t i o n of proteins increases the number of peptide molecules a v a i l a b l e at the o i l - w a t e r i n t e r f a c e , and thus, a l a r g e r area may be "covered", r e s u l t i n g in the e m u l s i f i c a t i o n of more o i l . However, s i n c e these peptides are smaller and l e s s globular compared to those in untreated soy i s o l a t e , they may form a thinner layer around the o i l d r o p l e t s , thus, r e s u l t i n g in a less s t a b l e emulsion. Enzyme treatment may have a l s o exposed buried hydrophobic groups which r e s u l t e d i n improved h y d r o p h i l i c - l i p o p h i l i c balance f o r better emulsification. Burnett and Gunther (41) and Gunther (42) have patented procedures f o r the commercial production of a w i ï i p p i n g protein by p a r t i a l h y d r o l y s i s of soy p r o t e i n . The e m u l s i f y i n g c a p a c i t y and the v i s c o s i t y of emulsions of peanut proteins p a r t i a l l y hydrolyzed with papain are reported to be g e n e r a l l y lower than f o r unmodified f l o u r (29). Products from papain h y d r o l y s i s of peanut protein d i f f e r s u b s t a n t i a l l y from those described by Beuchat et a l . (24), who used bromelain, pepsin, and t r y p s i n . These researchers reported that emulsion c a p a c i t i e s of peanut f l o u r were higher in water than in 0.5M NaCl and that heating d i d not r e s u l t in s u b s t a n t i a l changes in the c a p a c i t y of f l o u r to emulsify o i l . However, enzymatic d i g e s t i o n of p r o t e i n s completely destroyed the emulsifying c a p a c i t y of the flour. Apparently h y d r o l y s i s a l t e r e d p r o t e i n surface a c t i v i t y strengths and the a b i l i t y of peanut p r o t e i n to s t a b i l i z e o i l - i n - w a t e r emulsions. Hermansson et a l . (36) examined the s t a b i l i t y r a t i n g s f o r p r o t e a s e - t r e a t e d emulsions of rapeseed protein concentrate at pH 5.5 and 7 . 0 . The use of carboxymethylcellulose in conjunction with pepsin h y d r o l y s i s gave approximate t e n - f o l d increases in s t a b i l i t y r a t i n g s f o r emulsions. Kuehler and S t i n e (43) studied the f u n c t i o n a l p r o p e r t i e s of whey p r o t e i n with respect to emulsifying c a p a c i t y as a f f e c t e d by treatment with three p r o t e o l y t i c enzymes. Two m i c r o b i a l proteases and pepsin were examined. The emulsion c a p a c i t y decreased as prot e o l y s i s continued, suggesting that there i s an optimum mean molec u l a r s i z e of the whey p r o t e i n s c o n t r i b u t i n g to e m u l s i f i c a t i o n . Considerable e f f o r t has been devoted to the improvement of f u n c t i o n a l properties of f i s h p r o t e i n concentrate. S p i n e l l i et a l . (44) conducted a study to determine the f e a s i b i l i t y of modifying m y o f i b r i l l a r f i s h proteins by p a r t i a l l y hydrolyzing them


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with p r o t e o l y t i c enzymes. Increases in both emulsion s t a b i l i t y and c a p a c i t y (g o i l / g protein) were achieved by p a r t i a l hydrolysis. The emulsion c a p a c i t y , as well as s o l u b i l i t y , of s p r a y - d r i e d enzyme-modified m y o f i b r i l l a r protein were about 5% lower than those of f r e e z e - d r i e d samples. The authors concluded that f i s h p r o t e i n i s o l a t e s possessing valuable f u n c t i o n a l p r o p e r t i e s can be prepared from m y o f i b r i l l a r p r o t e i n s that are p a r t i a l l y hydrolyzed with a p r o t e o l y t i c enzyme. Caution i s advised, however, regarding the long-term storage s t a b i l i t y of such products due to chemical a l t e r a t i o n s of r e s i d u a l l i p i d s . Foaming Capacity and S t a b i l i t y . Pepsin d i g e s t i o n of soy p r o t e i n has been proposed as a method f o r making a whipping p r o t e i n f o r egg albumen replacement (42_, 45) and f o r extenders f o r albumen in bakery and c o n f e c t i o n e r y formulations (46). Puski (20), on the other hand, reported that although treatment of soy p r o t e i n i s o l a t e with fungal protease increased foam volume, s t a b i l i t y was reduced to z e r o . The lack of s t a b i l i t y was a t t r i b u t e d in part to heat treatment which may have denatured the large p r o t e i n components s u f f i c i e n t l y so that they could not act as a s t a b i l i z i n g component. Limited d i g e s t i o n of globular soy proteins with rennin affords a modified p r o t e i n preparation which r e t a i n s a high molecular weight (47). Whipping q u a l i t y , measured by foam volume and s t a b i l i t y , was superior in comparison with native p r o t e i n s . The l i m i t e d rennin p r o t e o l y s i s of soy was i d e n t i f i e d as a key f a c t o r in f u n c t i o n a l i t y , s i n c e t h i s m o d i f i c a t i o n conferred improved solubility. P a r t i a l h y d r o l y s i s of peanut p r o t e i n s with papain s i g n i f i c a n t l y increases both foaming c a p a c i t y and foam volume (29). When the pH was f i r s t adjusted to 4, then back to 8 . 2 , foaming c a p a c i t y increased t h r e e - f o l d . While the s t a b i l i t y of foam at acid pH (6.3/6.7) was low ( l e s s than 30 min), s t a b i l i t y at pH 8.2 was long l a s t i n g , suggesting greater p o t e n t i a l f o r p a r t i a l l y hydrolyzed peanut proteins in nonacidic pH foods. The authors suggested that papain-modified peanut p r o t e i n f l o u r should f i n d use in products such as frozen d e s s e r t s , s o f t mix i c e creams, dessert and p i e toppings. Pepsin and papain hydrolysates of rapeseed p r o t e i n concentrate increased foam volumes and decreased drainage compared to the untreated c o n t r o l (3(6). Foaming p r o p e r t i e s could be f u r t h e r enhanced by adding a s t a b i l i z e r such as carboxymethylcellulose. Foaming or w h i p p a b i l i t y c h a r a c t e r i s t i c s of whey p r o t e i n as a f f e c t e d by treatment with three p r o t e o l y t i c enzymes were evaluated by Kuehler and Stine (43). The s p e c i f i c volume of foams increased i n i t a l l y as a r e s u l t of treatment, then decreased with time at a more r a p i d rate compared to nontreated whey (Figure 8 ) . A l i m i t e d amount of h y d r o l y s i s appears to be d e s i r a b l e to increase foam volume but foam s t a b i l i t y is g r e a t l y decreased as a r e s u l t of such h y d r o l y s i s . The authors suggest that t h i s i s probably due to



290

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i n c r e a s i n g the polypeptide content i n i t i a l l y which allows more a i r to be i n c o r p o r a t e d . However, the polypeptides do not have the strength required to maintain a s t a b l e foam and f u r t h e r h y d r o l y s i s l i k e l y r e s u l t s in peptides which lack any c a p a c i t y to s t a b i l i z e the a i r c e l l s of the foam. Limited p r o t e o l y s i s may be advantageous f o r u t i l i z i n g whey p r o t e i n s in foams s i n c e s p e c i f i c volume was increased by as much as 25%. The decrease in s t a b i l i t y which r e s u l t s from l i m i t e d h y d r o l y s i s can be retarded by adding s t a b i l i z e r s such as carboxymethylcellulose. P r o t e o l y t i c enzymes derived from A s p e r g i l l u s oryzae and Streptomyces qriseus enhance the foaming capacity of frozen whole egg products (48). More r e c e n t l y Grunden et a l . (49) examined the e f f e c t s of several crude p r o t e o l y t i c enzymes on f u n c t i o n a l p r o p e r t i e s of egg albumen, as measured by angel food cake volume and foam volume and s t a b i l i t y . Papain, bromelain, t r y p s i n , f i c i n , and a fungal protease were e v a l u a t e d . Volumes of a l l cakes c o n t a i n i n g enzyme-treated albumen were comparable to or better than the c o n t r o l . Differences were noted in the texture of angel food cakes made from control albumen and those made from enzyme-treated albumen. Unlike the c o n t r o l cakes, those c o n t a i n i n g hydrolyzed albumen had a coarse and gummy t e x t u r e . Off odors and f l a v o r s were detected in cakes made from albumen that had been t r e a t e d with bromelin or fungal protease. Treatment of albumen with t r y p s i n , bromelain and fungal protease produced s i g n i f i c a n t l y greater volumes of foam compared to stored c o n t r o l albumen (49). However, a l l enzyme treatments had i n f e r i o r foam s t a b i l i t y when compared to c o n t r o l s . Both the r a t e and amount of foam c o l l a p s e was greater in enzyme-treated samples. The f r e s h control produced the most s t a b l e foam. Changes in aeration p r o p e r t i e s of bromelain-modified s u c c i n y l a t e d f i s h proteins were studied by Groninger and M i l l e r (50). The e f f e c t s of whipping time, p r o t e i n c o n c e n t r a t i o n , pH and a d d i t i v e s ( s a l t , sugar, v a n i a l l a f l a v o r i n g and f a t ) on foam volume and s t a b i l i t y were determined. Volume and s t a b i l i t y were increased as a r e s u l t of m o d i f i c a t i o n . In comparison to egg white and soy p r o t e i n , enzyme-hydrolyzed, s u c c i n y l a t e d f i s h p r o t e i n had a lower foam volume but was more s t a b l e . Hydrolyzed, s u c c i n y l a t e d p r o t e i n formed foams over the pH range of 3.0 to 9 . 0 ; foams produced at a pH below 7.0 showed lower s t a b i l i t y . A l s o , there was not a s i g n i f i c a n t decrease in the whipping p r o p e r t i e s of the hydrolyzed, s u c c i n y l a t e d f i s h p r o t e i n in the i s o e l e c t r i c range of approximately pH 4 . 5 . This i s in contrast to unhydrolyzed soy p r o t e i n , f o r example, which has a pronounced decrease in foam volume and s t a b i l i t y in the i s o e l e c t r i c range (51). A d d i t i v e s had s u b s t a n t i a l e f f e c t s on the aeration p r o p e r t i e s of bromelain-modified s u c c i n y l a t e d f i s h protein (50). Foam volume was increased with up to 2% sodium c h l o r i d e in the system; however there was a decrease in foam s t a b i l i t y when greater than 0.3% s a l t was used. Sucrose, at concentrations up to 50%, increased foam stability. When f a t was added to t r e a t e d f i s h p r o t e i n d i s p e r s i o n s


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previous to whipping, foam formation was i n h i b i t e d . When f a t was added to a p r o t e i n foam a f t e r i t was whipped to a maximum volume, the foam f l a t t e n e d . Water Uptake and R e t e n t i o n . The water binding c a p a c i t y of soy p r o t e i n i s o l a t e can be increased by treatment with neutral fungal protease (20). Since the number of f r e e amino and carboxyl groups increases as a r e s u l t of d i g e s t i o n and because moisture uptake by p r o t e i n s i s proportional to the number of i o n i c groups present (52), i t i s not s u r p r i s i n g that moisture uptake i s increased by enzyme treatment. The e f f e c t s of fungal fermentation on the moisture adsorption and r e t e n t i o n properties of defatted peanut f l o u r have been reported (28). At 8 and 2 1 ° C , l i t t l e d i f f e r e n c e was noted between the moisture contents of f r e e z e - d r i e d ferments and untreated samples e q u i l i b r a t e d at r e l a t i v e humidities ranging from 14 to 75%. However, marked changes were noted above 75% e q u i l i b r i u m r e l a t i v e humidity (ERH), where the c o n t r o l samples did not adsorb as much moisture as d i d the ferments. These d i f f e r e n c e s were a t t r i b u t e d to an increased r a t i o of exposed h y d r o p h i l i c to hydrophobic groups r e s u l t i n g from fermentation. Changes in the c a p a c i t y of defatted peanut f l o u r to adsorb moisture as a r e s u l t of treatment with protease were i n v e s t i g a t e d by Beuchat et a l . (24). Hydrolysis with pepsin, bromelain, and t r y p s i n caused the peanut f l o u r to adsorb more water than nontreated c o n t r o l s at s p e c i f i c ERH v a l u e s . Increased water adsorbing c a p a c i t i e s of enzyme-treated peanut p r o t e i n s are probably r e l a t e d to increased numbers of p o l a r s i t e s , such as carboxyl and amino groups, which appear on p r o t e i n s as a r e s u l t of hydrolysis. P e p t i z a t i o n and permanent c o n f i g u r a t i o n a l changes may occur during heat treatment or exposure to a c i d i c and a l k a l i n e pH c o n d i t i o n s , i n f l u e n c i n g the c a p a c i t y of seed f l o u r s to adsorb moisture. Better and Davidsohn (23) noted that heat and pH may a l t e r the e f f e c t i v e n e s s of using pepsin to s o l u b i l i z e p r o t e i n s in peanut meal. From a p r a c t i c a l viewpoint, the increased water-adsorbing c a p a c i t y of enzyme-treated f l o u r at s p e c i f i c ERH values may have important i m p l i c a t i o n s in the formulation of intermediate-moisture foods. At an ERH of 60%, a l e v e l regarded as minimal f o r the growth of microorganisms in f o o d s t u f f s , Beuchat et a l . (24) reported that the e q u i l i b r i u m moisture content of nontreated peanut f l o u r was 10% compared to 14% f o r f l o u r that had been t r e a t e d with pepsin (Figure 9 ) . Conceivably, a food product could be developed using p e p s i n - d i g e s t e d f l o u r which would be s a f e from the standpoint of not supporting m i c r o b i a l growth and yet contain s i g n i f i c a n t l y more water than a product formulated using nontreated f l o u r . Such a product might not e x h i b i t the c h a r a c t e r i s t i c mouth-drying sensation often a s s o c i a t e d with vegetable p r o t e i n s .



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1

'

1

2


ι

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L.

3

4

TIME (hr)

Journal of Food Science

Figure 8. Effect of enzymatic hydrolysis on specific volume of foam obtained by whipping a heated whey protein sol (4% w/w, 85°C, 6 min whipping) (43)

οI

•

20

1

40

1

60

1

1

80 100

EQUILIBRIUM RELATIVE HUMIDITY (%)

Journal of Agricultural and Food Chemistry

Figure 9. Moisture adsorption isotherms of enzyme-treated and nontreated peanut flour: no pH-heat treatment (% %); 50-min treatment at 50°C followed by 10 min at 90°C ( ); pepsin, pH 2.0 (A); bromelain, pH 4.5 O / trypsin, pH 7.6 during heat treatment. All samples were adjusted to pH 6.9 prior to freezedrying and analytical examination (24).


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A p p l i c a t i o n to Food Systems Fungal enzymes have been used f o r hundreds of year to prepare and modify f o o d s t u f f s . However, modern i n d u s t r i a l enzyme technology probably s t a r t e d with Takamine ( 5 3 J and h i s work with A. oryzae. Today many i n d u s t r i a l enzymes used to modify f u n c t i o n a l p r o p e r t i e s of foods and food ingredients are of fungal origin ( 5 4 ) . M i c r ô ï ï i a l p r o t e o l y t i c enzymes have been used f o r decades to improve f u n c t i o n a l c h a r a c t e r i s t i c s of aged wheat f l o u r s . The r o l e of proteases in r e l a t i o n to production of bread was reviewed in 1 9 4 6 ( 5 5 ) . Improvement in q u a l i t y has been evidenced by changes in handling q u a l i t y of doughs, improved e l a s t i c i t y and texture of the gluten washed from such doughs, and s u b s t a n t i a l increases in l o a f volume of bread ( 5 6 ) . Swanson and Andres ( 5 7 J reported that the amount of gluten t ï ï â t could be recovered from doughs t r e a t e d with papain decreased with increased enzyme concentration and the r e s t p e r i o d of the doughs was lengthed. Oka et a l . ( 5 8 ) s t u d i e d the a c t i o n of pepsin on g l u t e n i n and reported that the cleavage of only a few peptide bonds r a p i d l y produced large molecular polypeptides. Verma and McCalla ( 5 6 ) studied the action of p e p s i n , papain and a commercial fungal protease on wheat g l u t e n . A l l enzymes acted e f f e c t i v e l y on dispersed g l u t e n ; however, the a c t i o n of d i f f e r e n t enzymes produced d i f f e r e n t types of d i g e s t i o n products. Depending upon desired handling c h a r a c t e r i s t i c s of bread doughs prepared from t r e a t e d wheat f l o u r , various types of protease treatments can be s e l e c t e d . Proteases are used in the baking industry to improve handling p r o p e r t i e s of doughs, e l a s t i c i t y and texture of g l u t e n , and l o a f volume ( 5 9 ) . Fungal proteinases are widely used and have assumed a more important r o l e than the amylases due to the general low l e v e l of n a t u r a l l y o c c u r r i n g proteinases in f l o u r , to s t r i c t c o n t r o l of mixing times, and the d e s i r a b i l i t y of doughs with optimum handling p r o p e r t i e s . B a c t e r i a l proteinases are used in general food production and in the production of baked goods such as c r a c k e r s , c o o k i e s , and many snack f o o d s . Bromelain and papain are approved a d d i t i v e s but have not been widely used f o r the production of white bread. Beuchat ( 6 0 ) i n v e s t i g a t e d the performance of enzyme-hydrolyzed defatted peanut f l o u r in a cookie formula. Flour s l u r r i e s were t r e a t e d with pepsin at pH 2 . 0 , bromelain at pH 4 . 5 , and t r y p s i n at pH 7 . 6 . A f t e r readjustment to pH 6 . 9 , m a t e r i a l s were f r e e z e d r i e d , p u l v e r i z e d ( 6 0 - m e s h ) , and then s u b s t i t u t e d f o r wheat f l o u r at 5 , 1 5 , and 2 5 % . Adjustment of peanut f l o u r to pH 2 . 0 , as well as treatment with pepsin at t h i s pH, g r e a t l y improved the handling c h a r a c t e r i s t i c s of dough in which these f l o u r s were i n c o r p o r a t e d . Use of peanut f l o u r s t r e a t e d at pH 4 . 5 , with or without bromelain, and at pH 7 . 6 , with or without t r y p s i n , improved handling p r o p e r t i e s of cookie dough. These doughs d i d not tend to crumble



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and compared f a v o r a b l y in texture to that of 100% wheat f l o u r control. Enzyme h y d r o l y s i s of peanut f l o u r a l s o a l t e r e d the p h y s i c a l c h a r a c t e r i s t i c s of baked cookies (60). With the exception of the bromelain h y d r o l y s a t e , the use of peanut f l o u r in cookies r e s u l t e d in increased s p e c i f i c volume when compared to the 100% wheat f l o u r control. Untreated peanut f l o u r s u b s t i t u t i o n reduced the diameter and increased the height of c o o k i e s ; however, treatment with p r o t e o l y t i c enzymes reversed the behavior. As evidenced by s u b s t a n t i a l increases in spread r a t i o s , the diameter of cookies containing t r e a t e d f l o u r s increased p r o p o r t i o n a t e l y more than did the h e i g h t . These data promote the f e a s i b i l i t y of decreasing or i n c r e a s i n g the spread of cookies through the a d d i t i o n of various amounts of untreated or enzyme-treated peanut f l o u r . The f u n c t i o n a l i t y of bromelain-hydrolyzed s u c c i n y l a t e d f i s h p r o t e i n has been t e s t e d in a dessert t o p p i n g , a s o u f f l e , and both c h i l l e d and frozen desserts (50). Taste panel evaluations revealed that no f i s h l i k e odors or f l a v o r s were d e t e c t e d . The c o m p a t i b i l i t y of enzyme-treated f i s h p r o t e i n in these diverse food systems points up the p o t e n t i a l value of such a product to the food processing industry. K i n s e l l a {]) reviewed the p r i n c i p a l categories of f u n c t i o n a l p r o p e r t i e s of proteins in foods and o u t l i n e d various f a c t o r s a f f e c t i n g them from the point of view of the food chemist and technologist. Enzymatic m o d i f i c a t i o n of proteins a p p l i c a b l e to foods i s reviewed by Whitaker (2). Described b r i e f l y are present uses of p r o t e o l y t i c enzymes f o r modifying proteins through p a r t i a l hydrolysis. Major emphasis i s placed on those enzymes which b r i n g about aggregation of p r o t e i n s , c r o s s - l i n k formation, and s i d e chain m o d i f i c a t i o n through p o s t - t r a n s l a t i o n a l changes in the polypeptide c h a i n . Richardson (4>) reviewed the changes in proteins f o l l o w i n g a c t i o n of enzymes. Various exogenous and endogenous enzymes which a l t e r f u n c t i o n a l properties are d i s c u s s e d . Selected food systems, e . g . , meat, m i l k , and dough p r o t e i n s , are emphasized to r e l a t e the extent to which p r o l e o l y t i c enzymes play a r o l e in modifying and i n f l u e n c i n g f u n c t i o n a l properties of foods. The need f o r b a s i c research d i r e c t e d toward determining the importance of s t r u c t u r a l features involved in various types of p r o t e i n f u n c t i o n a l i t y i s stressed. It i s also pointed out that empirical f u n c t i o n a l t e s t s on p r o t e i n should be c o r r e l a t e d with performance of the p r o t e i n in given food products. Conclusions The expanded use of enzymes to modify properties has great promise f o r the food advantages of using proteases compared to t h e i r s p e c i f i c i t y , t h e i r e f f e c t i v e n e s s at

protein functional industry. Major other agents include low concentrations and



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under mild c o n d i t i o n s , and t h e i r general s a f e t y , thus e l i m i n a t i n g the n e c e s s i t y f o r removing them from f i n i s h e d products in most instances. E x i s t i n g uses of proteases in foods have been discussed in the foregoing s e c t i o n . Expanding such a p p l i c a t i o n s in the f u t u r e depends upon our a b i l i t y to c o n t r o l both the processes themselves and t h e i r c o s t s . The development of continuous reactors u t i l i z i n g f r e e or immobilized enzymes w i l l address each of these constraints. Furthermore, our understanding of the chemical basis f o r the various f u n c t i o n a l properties of proteins must be expanded (4). That i s , we must learn how amino a c i d content and molecular c o n f i g u r a t i o n of food proteins are r e l a t e d to t h e i r f u n c t i o n a l properties. This goal i s made more d i f f i c u l t by the f a c t that secondary, t e r t i a r y , and quarternary s t r u c t u r e s of proteins are l i k e l y to be q u i t e d i f f e r e n t when e x e r t i n g f u n c t i o n a l e f f e c t s in food systems as compared to s t r u c t u r e s of the same proteins in d i l u t e s o l u t i o n s and in t h e i r native s t a t e s . The way in which s p e c i f i c actions of proteases a f f e c t p r o t e i n s t r u c t u r e must a l s o be s t u d i e d so that c o r r e l a t i o n s with changes in f u n c t i o n a l p r o p e r t i e s can be made (61).

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Enzyme Modification of Proteins - ACS Symposium Series (ACS

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