Chemical Deterioration of Proteins - American Chemical Society

9 Deterioration of Food Proteins by Binding Unwanted Compounds Such as Flavors, Lipids and Pigments SOICHI ARAI

Downloaded by CORNELL UNIV on September 27, 2016 | http://pubs.acs.org Publication Date: May 28, 1980 | doi: 10.1021/bk-1980-0123.ch009

Department of Agricultural Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Proteins often interact with other compounds found in bio logical materials. The retention and stability of a flavor generally is markedly improved in the presence of protein. This is the result of some type of protein-flavor interaction which decreases the volatility of the flavor. When a flavor is an un desirable one, such interaction may cause the protein to be organoleptically unacceptable as human food. It is often a very formidable task to remove a protein-bound flavor completely in order to prepare a bland protein. Many lipids and natural pig ments, physiologically significant to living systems, are important in foods. While they often provide desirable color and texture to food, both lipids and pigments may cause undesirable color changes and deteriorative changes in proteins. In a few cases toxic constituents may be formed. Therefore, interaction of constituents in foods may be both boon and bane (1). The present paper reviews some of the undesirable effects resulting from the interaction of flavor constituents, lipids and pigments with proteins. Our l a b o r a t o r y as w e l l as others have c o n t r i b u t e d to t h i s

knowledge.

I n t e r a c t i o n of P r o t e i n s with F l a v o r s Examples of i n t e r a c t i o n of p r o t e i n w i t h both v o l a t i l e and n o n - v o l a t i l e f l a v o r c o n s t i t u e n t s are a v a i l a b l e . One example i s the i n t e r a c t i o n between g e l a t i n and s e v e r a l n o n - v o l a t i l e f l a v o r n u c l e o t i d e s : 5'-GMP, 5'-IMP, 5 -AMP and 5 -CMP. S a i n t - H i l a i r e and Solms (2) e q u i l i b r a t e d s o l u t i o n s of 5 - 90 mM n u c l e o t i d e i n 0.004 % g e l a t i n at pH 6.5 and determined bound n u c l e o t i d e by u l t r a v i o l e t spectroscopy. They analyzed the r e s u l t s by use of the Scatchard equation: f

r /

f

(n - r ) = KC

(1)

where r i s the average number of moles of bound l i g a n d per mole of p r o t e i n , η i s the maximum number of moles of l i g a n d bound per mole 0-8412-0543-4/80/47-123-195$05.00/0 © 1980 American Chemical Society Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


196

C H E M I C A L DETERIORATION O F PROTEINS

of p r o t e i n , Κ i s a b i n d i n g constant and C i s the molar concentra t i o n of non-bound l i g a n d a t e q u i l i b r i u m (3). The potent f l a v o r p o t e n t i a t o r s , 5'-GMP and 5'-IMP, gave Κ values of 235 and 112 mM-1, r e s p e c t i v e l y , w h i l e 5'-AMP and 5'-CMP, known t o have poor f l a v o r - p o t e n t i a t i n g a c t i v i t i e s , had much smaller Κ v a l u e s . No other r e p o r t s of p r o t e i n i n t e r a c t i o n w i t h such n o n - v o l a t i l e f l a v o r s are a v a i l a b l e . More a t t e n t i o n has been given t o i n t e r a c t i o n of p r o t e i n s w i t h v o l a t i l e f l a v o r s , e s p e c i a l l y w i t h v o l a t i l e carbonyIs. Nawar (4) found that a d d i t i o n of g e l a t i n t o s o l u t i o n s of a homologous s e r i e s of 2-alkanones caused decreases i n t h e i r v o l a t i l i t i e s . Hawrysh and S t i n e (5) reported on the r e t e n t i o n of 2-alkanones i n a model system that simulated b l u e - v e i n cheese. A s i m i l a r but more sys tematic experiment was c a r r i e d out by Franzen and K i n s e l l a (6). By headspace a n a l y s i s v i a gas chromatography, they q u a n t i f i e d the r e t e n t i o n of a v a r i e t y of v o l a t i l e aldehydes and ketones by food p r o t e i n s such as a-lactalbumin, bovine serum albumin, l e a f p r o t e i n concentrate, s i n g l e - c e l l p r o t e i n and soy p r o t e i n preparations. Although the quantity of r e t a i n e d f l a v o r depended on the type, amount and composition of p r o t e i n as w e l l as on the presence of l i p i d s , i t was c l e a r that p r o t e i n decreased t o some extent the v o l a t i l i t i e s o f f l a v o r s by adsorbing or occluding them. For exam p l e , the a d d i t i o n of p r o t e i n t o an aqueous system c o n t a i n i n g 1hexanal caused a 9 - 23 % decrease i n i t s v o l a t i l i t y . Gremli (7) a l s o determined the headspace composition of a model system of a 10:1 mixture o f soy p r o t e i n and aldehyde i n water. The percent r e t e n t i o n of aldehydes were as f o l l o w s : 1-hexanal, 3 7 - 44 %; 1heptanal, 62- 72 %; 1-octanal, 8 3 - 85 %; 1-nonanal, 9 0 - 9 3 %; 1decanal, 94- 97 %; 1-undecanal, 9 6 - 100 %; 1-dodecanal, 94- 100%; 2-hexenal, 68- 75 %; 2-heptenal, 82- 88 %; 2,6-nonadienal, 9 0 - 9 8 %; 2,4-nonadienal, 92 - 97 %; 2-decenal, 100 %; and 2-dodecenal, 100 %. I n these experiments, the l a s t two v o l a t i l e compounds behaved as i f they were completely n o n - v o l a t i l e . Beyeler and Solms (8) c a l c u l a t e d the b i n d i n g constant (K) f o r s e v e r a l v o l a t i l e compounds i n the presence of soy p r o t e i n and bovine serum albumin by the f o l l o w i n g equation: r = KC

(2)

where C i s the molar c o n c e n t r a t i o n of f r e e l i g a n d a t e q u i l i b r i u m and r i s the average number of moles bound l i g a n d per mole of p r o t e i n . The data showed that the Κ v a l u e s f o r aldehydes a r e g e n e r a l l y l a r g e r than those f o r other c l a s s e s of compounds. Strong i n t e r a c t i o n of v o l a t i l e aldehydes occur n a t u r a l l y i n soy p r o t e i n products (9). A r a i et a l . (10) found that 1-hexanal i s one of the major odorants of soybean and that t h i s aldehyde i n t e r a c t s r e a d i l y with soy p r o t e i n . In order t o determine whether the i n t e r a c t i o n i s enhanced by d e n a t u r a t i o n of p r o t e i n , A r a i e t a l . (9) d i d three experiments under d i f f e r e n t c o n d i t i o n s . In the f i r s t experiment ( I ) , an a c i d - p r e c i p i t a t e d f r a c t i o n of

Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


9.

ARAI

Binding

Unwanted

Compounds

197

n a t i v e soy p r o t e i n (10 g) was d i s s o l v e d i n water (100 ml) and v a r i o u s amounts of 1-hexanal were added. The r e s u l t i n g mixtures were v i g o r o u s l y a g i t a t e d at 20°C f o r 5 h i n a sealed f l a s k f i l l e d with n i t r o g e n . Each sample was then f r e e z e - d r i e d . In the second experiment ( I I ) , v a r i o u s amounts of 1-hexanal were added t o the a c i d - p r e c i p i t a t e d p r o t e i n (10 g) d i s s o l v e d i n water (100 ml). The mixtures, i n f l a s k s equipped with condensers, were heated at 90°C f o r 1 h with vigorous s t i r r i n g and then f r e e z e - d r i e d . In the t h i r d experiment ( I I I ) , i d e n t i c a l hexanal/protein mixtures to those above were heated at 90°C f o r 24 h under r e f l u x c o n d i t i o n s and then l y o p h i l i z e d . A l i q u o t s of the f r e e z e - d r i e d samples were d i s s o l v e d i n a NaOH s o l u t i o n ( f i n a l pH ca. 13) to l i b e r a t e the 1hexanal bound by p r o t e i n . Gas chromatography was used to d e t e r mine the l i b e r a t e d 1-hexanal. F i g u r e 1 shows the q u a n t i t y of 1hexanal bound depended upon the heat treatment of p r o t e i n as w e l l as upon the amount of aldehyde i n i t i a l l y added. A r a i et a l . (9) obtained a b i n d i n g constant (K) f o r the i n t e r a c t i o n of 1-hexanal with the p a r t i a l l y denatured soy p r o t e i n (Experiment I I above) from g e l f i l t r a t i o n data analyzed by u s i n g B e i d l e r ' s equation: η / (S - η) = KC

(3)

where C i s the c o n c e n t r a t i o n of t o t a l l i g a n d , η i s the amount of bound l i g a n d when the i n i t i a l t o t a l l i g a n d c o n c e n t r a t i o n equals C, and S i s the amount when the l i g a n d c o n c e n t r a t i o n has reached a maximum (11). The a n a l y s i s gave Κ =173 M~l and S = 0.847 mg/g pro t e i n . The S v a l u e i n d i c a t e s that the p a r t i a l l y denatured soy pro t e i n bound 1-hexanal to almost 0.1 % of i t s weight. An a d d i t i o n a l study (9) demonstrated that h y d r o l y s i s of the p r o t e i n decreased the 1-hexanal b i n d i n g a b i l i t y (Table I ) . The amount of 1-hexanal l i b e r a t e d by the enzyme treatment c o r r e l a t e d w e l l with the degree of h y d r o l y s i s of p r o t e i n (Table I ) . A r a i et a l . (12) have a l s o reported that f r e e tryptophan i n an enzymatic h y d r o l y s a t e of soy p r o t e i n r e a c t s with 1-hexanal to form the condensation products, l-pentyl-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-3-carboxylie a c i d ( I ) , l-pentyl-4,9-dihydro-3H-pyrido[3,4-b]indole-3-carboxylic a c i d (II) and l-pentyl-9H-pyrido[3,4-b]indole ( I I I ) . Related com pounds are discussed l a t e r with respect to potent mutagenicity. V o l a t i l e aldehydes, i n c l u d i n g 1-hexanal, may be p r i m a r i l y r e s p o n s i b l e f o r the beany odor of soybean (10, 13, 14). They a r e present even i n d e f a t t e d soybean f l o u r . Recently, Chiba et a l . (15) have deodorized soybean f l o u r by treatment with aldehyde dehydrogenase from bovine l i v e r . Deodorization was a r e s u l t of c o n v e r t i n g aldehydes to a c i d s , e.g., 1-hexanal to c a p r o i c a c i d . They postulated that both f r e e and bound aldehydes can a c t as sub s t r a t e s f o r t h i s dehydrogenase. Consequently, enzymatic treatment r e s u l t e d i n a product without beany odor.



198


0

50

100

200

1000

Amount of 1-hexanal added (mg/kg p r o t e i n ) Agricultural and Biological Chemistry

Figure 1. Binding of 1-hexanal by soy protein. I, H and III refer to the first, second and third experiments, respectively. For details see text (9).

Table I . R e l a t i o n s h i p between the degree of h y d r o l y s i s of soy p r o t e i n * and the amount of 1-hexanal r e t a i n e d Degree of h y d r o l y s i s * * (%) 0 22.1 41.6 72.7 99.8

Amount of 1-hexanal r e t a i n e d (mg / kg hydrolysate) 6.67 5.20 2.41 1.03 0.05

* P a r t i a l l y denatured soy p r o t e i n (see t e x t ) . ** H y d r o l y s i s was performed a t pH 2.8 with a m i c r o b i a l a c i d protease (Molsin). Each degree of h y d r o l y s i s was measured w i t h 10 % t r i c h l o r o a c e t i c a c i d (TCA) and r e presented as (TCA-soluble Ν / t o t a l N ) x 100. From A r a i et a l . (9) Agricultural and Biological Chemistry


ARAI

Binding Unwanted Compounds


9.


199

200

C H E M I C A L DETERIORATION OF PROTEINS


I n t e r a c t i o n of P r o t e i n w i t h L i p i d s L i p i d s a r e o f t e n a nuisance i n e x t r a c t i o n of p r o t e i n s . For example, i n p r e p a r i n g l e a f p r o t e i n concentrate a p r o t e i n - l i p i d complex i s formed f r e q u e n t l y a f f e c t i n g the p r o t e i n e x t r a c t i o n e f f i c i e n c y (16). N u t r i t i o n a l l y , the complex i s disadvantageous because i t r e s i s t s d i g e s t i o n by proteases (17). Shenouda and P i g o t t (18) found that the formation of protein-bound l i p i d s can cause a low e f f i c i e n c y of e x t r a c t i o n of p r o t e i n from f i s h . Hydrophobic bonding probably p l a y s an important r o l e i n p r o t e i n - l i p i d i n t e r a c t i o n s . Mohammadzadeh-k et a l . (19) reported p r o t e i n i n t e r a c t i o n with completely a p o l a r compounds such as a l i p h a t i c hydrocarbons. Shenouda and P i g o t t (20) have studied the i n t e r a c t i o n of p o l a r l i p i d s w i t h p r o t e i n . Using a s p i n - l a b e l technique, they demonstrated that heat-denatured myosin from f i s h muscle bound p o l a r l i p i d s more t i g h t l y than n e u t r a l t r i g l y c e r i d e s . They showed that a solvent with higher p o l a r i t y permits b e t t e r s e p a r a t i o n of l i p i d s from p r o t e i n (21). S i m i l a r examples of the i n t e r a c t i o n of p o l a r l i p i d s w i t h p r o t e i n s have been found i n peas (22) and i n soy p r o t e i n a f t e r heat-denaturation (23). According t o Noguchi e t a l . (24), soy p r o t e i n curd, prepared by heat-denaturation of p r o t e i n followed by s a l t i n g - o u t , contains a v a r i e t y of p o l a r l i p i d s i n c l u d i n g p h o s p h a t i d y l c h o l i n e , phosphatidylethanolamine and p h o s p h a t i d y l i n o s i t o l . They found that a s i g n i f i c a n t amount of these l i p i d s i s probably i n a protein-bound s t a t e r e s i s t a n t t o e x t r a c t i o n w i t h chloroform/methanol. When the curd was incubated with an a c i d protease ( M o l s i n ) , most of the bound p h o s p h o l i p i d s were l i b e r a t e d t o an extent dependent on the i n c u b a t i o n time. Ohtsuru et a l . (25) have r e c e n t l y i n v e s t i g a t e d the behavior of p h o s p h a t i d y l c h o l i n e i n a model system that simulated soy milk. They used s p i n - l a b e l l e d p h o s p h a t i d y l c h o l i n e (PC*) synthesized from egg l y s o l e c i t h i n and 1 2 - n i t r o x i d e s t e a r i c a c i d anhydride. The ESR spectrum of a mixture of PC* (250 yg) and n a t i v e soy p r o t e i n (20 mg) homogenized i n water by s o n i c a t i o n resembled that observed f o r PC* alone before s o n i c a t i o n . However, when PC* (250 yg) was sonicated i n the presence of heat-denatured soy p r o t e i n (20 mg), s p l i t t i n g of the ESR s i g n a l occurred. On t h i s b a s i s , they postul a t e d the e x i s t e n c e of two phases: PC* making up a f l u i d l a m e l l a phase and PC* immobilized probably due t o the hydrophobic i n t e r a c t i o n w i t h t h e denatured p r o t e i n . In a study of a soy-milk model, Ohtsuru e t a l . (25) reported that a t e r n a r y p r o t e i n - o i l - P C * comp l e x occurred when the three m a t e r i a l s were subjected t o s o n i c a t i o n under t h e proper c o n d i t i o n . Based on data from the ESR study, a schematic model has been proposed f o r the r e v e r s i b l e formationdeformation of the t e r n a r y complex i n soy m i l k ( F i g u r e 2 ) . Serious problems a r i s e when protein-bound l i p i d s undergo a u t o x i d a t i o n f o l l o w e d by decomposition. C a s t e l l (26) showed t h a t formaldehyde formed by the decomposition of a u t o x i d i z e d f i s h o i l can cause toughened t e x t u r e of f i s h p r o t e i n . St. Angelo and Ory



9.

ARAI

201


Agricultural and Biological Chemistry

Figure 2. A model for the formation-deformation of a ternary protein-oil-phosphatidylcholine complex. The protein molecule is represented as a large open circle and the phosphatidylcholine molecule as a small filled circle having two tails. The large filled circle represents an oil particle (25).



202

C H E M I C A L DETERIORATION OF PROTEINS

(27) reported on the u n d e s i r a b l e e f f e c t s of unsaturated l i p i d s on p r o t e i n ; t h e i r a u t o x i d a t i o n produced aldehydes and ketones which reacted w i t h some amino a c i d r e s i d u e s . Roubal and Tappel (28) and Roubal (29) showed that p r o t e i n d e t e r i o r a t i o n could be i n i t i ated by f r e e r a d i c a l s o r i g i n a t i n g from l i p i d p e r o x i d a t i o n . Per o x i d a t i o n r e s u l t e d i n o x i d a t i o n of some amino a c i d r e s i d u e s as w e l l as c r o s s - l i n k i n g of p r o t e i n s v i a malonaldehyde formed. K a r e l ' s group at M.I.T. has a l s o studied the e f f e c t of pero x i d i z i n g methyl l i n o l e a t e i n the presence of lysozyme and other s e l e c t e d food p r o t e i n s i n model systems ( 3 0 - 3 3 ) . ESR s t u d i e s i n d i c a t e d that p r o t e i n f r e e r a d i c a l s were formed as a r e s u l t of the i n t e r a c t i o n , e s p e c i a l l y when the r e a c t i n g system was main tained at a water a c t i v i t y of 0.75. The f r e e r a d i c a l s p r i m a r i l y showed c e n t r a l s i n g l e t l i n e s , a t t r i b u t a b l e to carbon-centered r a d i c a l s w i t h g=2.004 and guanidyl nitrogen-centered r a d i c a l s with g=2.0027. S u b s t a n t i a l evidence i n d i c a t e d formation of f r e e r a d i c a l s w i t h the s i d e - c h a i n s of tryptophan, h i s t i d i n e , l y s i n e and a r g i n i n e r e s i d u e s of p r o t e i n . P r o t e i n s c o n t a i n i n g c y s t e i n e a l s o e x h i b i t e d downfield shoulders at g=2.015 and 2.023 that were e s s e n t i a l l y i d e n t i c a l to peaks obtained w i t h f r e e c y s t e i n e . Based on these data, K a r e l and coworkers postulated that f r e e r a d i c a l t r a n s f e r to p r o t e i n occurs v i a complex formation of the f o l l o w i n g type: PH

+

LOOH +

[ΡΗ···ΙΌ0Η] +

Ρ· +

LOO* +

H0

(5)

2

where PH and LOOH r e f e r t o p r o t e i n and l i p i d hydroperoxide, r e s p e c t i v e l y . An i n t e r m o l e c u l a r r e a c t i o n of Ρ· may then take p l a c e l e a d i n g t o p r o t e i n p o l y m e r i z a t i o n of the type Ρ - ( Ρ ) - Ρ · . I t was shown that lysozyme polymerized ( c o v a l e n t l y ) during i n c u b a t i o n with p e r o x i d i z i n g methyl l i n o l e a t e , w i t h a decrease i n s o l u b i l i t y as w e l l as i n enzyme a c t i v i t y . Such a p e r o x i d e - i n i t i a t e d r a d i c a l r e a c t i o n can cause chemical d e t e r i o r a t i o n of wheat g l i a d i n , bovine serum albumin and ovalbumin as w e l l . Free r a d i c a l s trapped i n matrices of p r o t e i n s , p a r t i c u l a r l y of denatured p r o t e i n s , can be kept s t a b i l i z e d over a long p e r i o d of time (29). undoubtedly, food p r o t e i n s undergo r a d i c a l - i n d u c e d damage during t h e i r prolonged storage i n the presence of unsaturated l i p i d s . η

I n t e r a c t i o n of P r o t e i n s w i t h Pigments and Related

Compounds

Synthetic dyes can i n t e r a c t w i t h p r o t e i n s . Experiments have been c a r r i e d out to assess b i n d i n g c a p a c i t i e s of p r o t e i n s toward a v a r i e t y of food dyes ( 3 4 " 26). These i n c l u d e azo dyes (new coccine, amaranth, orange G, e t c . ) , triphenylmethane dyes (guinea green, b r i l l i a n t blue FCF, a c i d v i o l e t 6B, etc.) and isoxanthene dyes (rose bengal, e r y t h r o s i n e , eosine, e t c . ) . An example of p h y s i o l o g i c a l importance i s the work of Tokuma and Terayama (37). They i d e n t i f i e d a l c o h o l dehydrogenase as a target p r o t e i n f o r b i n d i n g of a c a r c i n o g e n i c aminoazo dye. I t i s , however, beyond



9.

ARAI

Binding

Unwanted

Compounds

203

the scope of t h i s a r t i c l e to d e a l w i t h these t o p i c s ; t h e r e f o r e the d i s c u s s i o n w i l l concentrate on the i n t e r a c t i o n of p r o t e i n s w i t h n a t u r a l l y o c c u r r i n g pigments and r e l a t e d compounds. S p e c i a l problems a r i s e during e x t r a c t i o n of p r o t e i n s from green leaves and algae. Much e f f o r t has been devoted to separat i o n of c h l o r o p h y l l s , carotenoids and other photosynthetic p i g ments from l e a f p r o t e i n s (38 - 44). A tight chlorophyll-protein complex formed, f o r example, during greening of e t i o l a t e d bean leaves i s a nuisance (45). A s i m i l a r example i s c h l o r o p h y l l l i p o p r o t e i n complex formation i n c h l o r o p l a s t s of C h l o r e l l a (46). Feeding t e s t s using r a t s showed that the complex was p r i m a r i l y r e s p o n s i b l e f o r the low n u t r i t i v e v a l u e of the C h l o r e l l a p r o t e i n . A r a i et a l . (47) attempted t o e x t r a c t an a l k a l i - s o l u b l e p r o t e i n from the blue-green a l g a , S p i r u l i n a maxima, and another one from the non-sulfur purple bacterium, Rhodopseudomonas capsulatus. In both cases, pretreatment of dry c e l l s w i t h ethanol removed most of the photosynthetic pigments. However, the extracted p r o t e i n s were s t i l l brown. Gel chromatography on Sephadex G-15 showed that the remaining pigment was t i g h t l y bound t o p r o t e i n . The pigment could be separated from p e p s i n - t r e a t e d p r o t e i n on a Sephadex column. A l g a l pigments i n c l u d e the s o - c a l l e d b i l i p r o t e i n s such as phycoe r y t h r i n s and phycocyanins which are c o v a l e n t l y bound to p r o t e i n (48). Higher p l a n t s c o n t a i n a chromoprotein c a l l e d phytochrome, which occurs i n two i n t e r c o n v e r t i b l e forms, P R and PpR, w i t h abs o r p t i o n maxima a t 665 nm and 725 nm, r e s p e c t i v e l y . Walker and B a i l e y (49) b e l i e v e that the i n t e r c o n v e r t i b l e photoreaction of phytochrome may be involved i n the photoregulation of growth, development, adaptation and other f u n c t i o n s of higher p l a n t s . RUdiger (50) proposed a model of the two forms of phytochrome i n v o l v i n g c o v a l e n t l y bound l i n e a r t e t r a p y r r o l e . The chromophore i s a b i l e pigment. Fry and Mumford (51) have i s o l a t e d a chromophore-containing peptide by d i g e s t i n g phytochrome w i t h protease; t h e r e f o r e i t may be p o s s i b l e to use p r o t e o l y s i s to depigment the chromoprotein. Besides the above-mentioned pigments, a v a r i e t y of c l o s e l y r e l a t e d c o l o r l e s s compounds occur widely i n p l a n t s . Among these are polyphenols, some of which have a potent tanning a c t i v i t y . Because of t h e i r s p e c i f i c a f f i n i t y f o r p r o t e i n s , tannins have been used i n p r o t e i n a n a l y s i s . The method can be Improved f u r t h e r by c h a r a c t e r i z i n g the mode of p r o t e i n - t a n n i n i n t e r a c t i o n (52) and by o p t i m i z i n g the c o n d i t i o n s f o r q u a n t i t a t i v e a n a l y s i s (53, 54). Tannins o f t e n cause n u t r i t i o n a l d e f i c i e n c i e s and t o x i c i t i e s as shown f o r the p r o t e i n extracted from high-tannin species of sorghum (55). Another example i s the i n h i b i t o r y e f f e c t of oak l e a f t a n n i n on the t r y p t i c d i g e s t i o n of p r o t e i n s (56). Chlorogenic a c i d , a ubiquitous depside-type tannin, a l s o can a s s o c i a t e t i g h t l y w i t h l e a f p r o t e i n s , a f f e c t i n g t h e i r t r y p t i c d i g e s t i b i l i t y (57,58). Dryden and S a t t e r l e e (59) showed that chlorogenic a c i d added t o c a s e i n r e a c t s c o v a l e n t l y to prevent the p r o t e i n from undergoing


204

CHEMICAL

DETERIORATION O F PROTEINS

i n v i v o d i g e s t i o n . The growth of Tetrahymena on t h e chlorogenic a c i d - t r e a t e d c a s e i n was decreased. Other polyphenols a l s o r e a c t with p r o t e i n s , f o r example, w i t h a r a c h i n (60). Phenolic compounds i n c l u d i n g f l a v o n o l s , although c o l o r l e s s , can be enzymatically and/or non-enzymatically o x i d i z e d t o p i g ments. When such o x i d a t i o n takes p l a c e i n the presence of p r o t e i n , the p r o t e i n may become pigmented. I g a r a s h i et a l . (61) measured the e f f e c t of a number of phenolic compounds on browning of c a s e i n s o l u t i o n a t pH 6.8 (Table I I ) . The r e s u l t s i n d i c a t e that t h e p o s i t i o n o f the OH groups of the phenolic compounds determines the c o l o r i n t e n s i t y . I n p a r t i c u l a r , 3- and 7-OH groups on the flavone r i n g bearing 3 - and 4'-0H groups appear t o be r e q u i r e d f o r maximum browning of the c a s e i n s o l u t i o n . From a n u t r i t i o n a l point of view i t should be noted that a v a i l a b l e l y s i n e o f c a s e i n decreased i n accordance with the brown c o l o r i n t e n s i t y . Igarashi et a l . (62) a l s o showed that i n v i t r o d i g e s t i b i l i t y of c a s e i n decreased when the p r o t e i n was incubated with q u e r c e t i n . In t h i s case a s i g n i f i c a n t degree of damage occurred t o methionine as w e l l as t o l y s i n e . Horigome and Kandatsu (63) prepared a F u k i ( P e t a s i t e s japoni c u s miq.; a t r a d i t i o n a l food p l a n t i n Japan) acetone-powder w i t h a high o-diphenol oxidase a c t i v i t y . When the powder was added to a mixture of c a f f e i c a c i d and c a s e i n , c a s e i n was g r a d u a l l y p i g mented. A feeding t e s t w i t h r a t s demonstrated that there was a c l o s e r e l a t i o n s h i p between the decrease i n b i o l o g i c a l v a l u e of the pigmented c a s e i n and i t s c o l o r i n t e n s i t y . F i n a l l y , examples of t h e e f f e c t of p r o t e i n i n t e r a c t i o n with f l u o r e s c e n t compounds a r e a v a i l a b l e . Lohrey et a l . (64) demons t r a t e d that a p h o t o s e n s i t i z i n g e f f e c t (photodynamic e f f e c t ) appeared when r a t s were f e d on a d i e t c o n t a i n i n g l e a f p r o t e i n concentrate prepared from lucerne (Medicago s a t i v a ) . Skin l e s i o n s of v a r y i n g s e v e r i t y up t o t h e sloughing o f ears and t a i l s occurred when the f e d r a t s were i l l u m i n a t e d w i t h n a t u r a l d a y l i g h t through window g l a s s . E x t r a c t s from blood plasma and l i v e r s of r a t s given the l e a f p r o t e i n concentrate contained phaeophorbide-a which i s a f l u o r e s c e n t compound d e r i v e d from c h l o r o p h y l l - a by removal of magnesium and p h y t o l . T h i s f i n d i n g i s supported by the work of Isobe e t a l . (65) who have c l e a r l y shown that phaeophorbide-a does cause h y p e r s e n s i t i z a t i o n i n r a t s . Much more a t t e n t i o n i s c u r r e n t l y being paid t o carcinogeni c i t y o f f l u o r e s c e n t compounds. A t y p i c a l example may be a f l a t o x i n (66). Beckwith et a l . (67) have studied the i n t e r a c t i o n between corn p r o t e i n and a f l a t o x i n B]_. Tryptophan d e r i v a t i v e s a l s o a r e of i n t e r e s t because of t h e i r p o s s i b l e c a r c i n o g e n i c i t y . Sugimura e t a l . (68) i d e n t i f i e d t h e f o l l o w i n g two mutagenic p r i n c i p l e s : 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (I) and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (II) i n a p y r o l y z a t e of tryptophan. Subsequently, Yoshida e t a l . (69), i n i n v e s t i g a t i o n s on f a c t o r s inducing mutagenicity i n Salmonella typhimurium TA 98, i d e n t i f i e d two r e l a t e d compounds: 2-amino-9H-pyrido[2,3-b]indole


1


ARAI

Binding

Unwanted

Compounds

Table I I . E f f e c t of phenolic compounds added t o a c a s e i n s o l u t i o n on i t s browning during i n c u b a t i o n *


Optical

density

Phenolic compounds

at 470 nm

at 520 nm

Chlorogenic a c i d Caffeic acid Catechol Quercetin Kaempherol Myricetin D ihydr οquer cet i n Protocatechuic a c i d Phloroglucinol Azaleatin Rhamnetin Quercitrin Rutin Luteolin

0.521 0.751 0.850 0.941 0.206 1.505 0.590 0.314 0.780 0.778 0.206 0.078 0.047 0.196

0.396 0.413 0.510 0.724 0.174 1.007 —

0.260 0.331 0.510 0.174 0.055 0.033 0.174

* Casein (1 g) was d i s s o l v e d i n 100 ml of 0.1 M phosphate (pH 6.8) c o n t a i n i n g 0.1 mM phenolic com pound. The s o l u t i o n was r e f l u x e d a t 80°C f o r 10 h under a e r a t i o n p r i o r t o measurement of o p t i c a l densities. From I g a r a s h i et a l . (61) Journal of the Agricultural Chemical Society of Japan


206


(III) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (IV) i n a pyrol y z a t e of soy p r o t e i n . These f l u o r e s c e n t compounds could be protein-bound i n roasted foods. D e t a i l e d experiments on the i n t e r a c t i o n between food p r o t e i n s and such compounds of t o x i c o l o g i c a l importance a r e being c a r r i e d out i n Japan.


Discussion P r o t e i n s can bind f l a v o r c o n s t i t u e n t s , e s p e c i a l l y v o l a t i l e carbonyls. When bound t o p r o t e i n , some v o l a t i l e aldehydes behave as i f they were n o n - v o l a t i l e compounds and a r e r e t a i n e d over a long p e r i o d of time during storage. Such i n t e r a c t i o n may cause chemical as w e l l as o r g a n o l e p t i c a l d e t e r i o r a t i o n of food p r o t e i n s . As exemplified by soy p r o t e i n , i n t e r a c t i o n of p r o t e i n w i t h 1-hexanal i s promoted by heat denaturation. The i n t e r a c t i o n i s decreased by t r e a t i n g a heat-denatured soy p r o t e i n w i t h protease; the aldehyde i s l i b e r a t e d to an a p p r e c i a b l e extent. L i p i d s can a c t as precursors of a v a r i e t y of a l i p h a t i c c a r bonyls w i t h o b j e c t i o n a b l e f l a v o r s . P r o t e i n s i n t e r a c t with l i p i d s as w e l l . The i n t e r a c t i o n i s p r i m a r i l y through hydrophobic bonding. A d d i t i o n a l l y , a type of p o l a r i n t e r a c t i o n may be i n v o l v e d , p a r t i c u l a r l y when phospholipids take p a r t . In the case of soy milk, a t e r n a r y p r o t e i n - o i l - p h o s p h a t i d y l c h o l i n e complex probably occurs. A s e r i o u s problem a r i s e s when l i p i d s undergo p e r o x i d a t i o n i n the presence of p r o t e i n . Free r a d i c a l s o r i g i n a t i n g from hydroperoxides t r a n s f e r t o s e v e r a l s p e c i f i c amino a c i d r e s i d u e s of p r o t e i n . As a r e s u l t , the p r o t e i n undergoes r a d i c a l induced polymerization, with l o s s of s o l u b i l i t y and other o r i g i n a l p r o p e r t i e s i n c l u d i n g n u t r i t i v e value. N a t u r a l l y o c c u r r i n g pigments a l s o a r e bound by p r o t e i n s t o a greater or l e s s e r extent depending on chemical s t r u c t u r e . Photos y n t h e t i c pigments o f t e n obstruct the process f o r preparing pure l e a f p r o t e i n concentrate. A c h l o r o p h y l l - l i p o p r o t e i n complex o c c u r r i n g i n the a l g a l c h l o r o p l a s t f r a c t i o n i s a nuisance i n that i t r e s i s t s i n v i v o as w e l l as i n v i t r o d i g e s t i o n . Of p h y s i o l o g i c a l and t o x i c o l o g i c a l importance i s the p o s s i b i l i t y that some f l u o r e s cent compounds with p h o t o s e n s i t i z i n g and mutagenic a c t i v i t i e s could remain i n foods i n a protein-bound s t a t e . Besides pigments, c l o s e l y r e l a t e d compounds occur n a t u r a l l y . Among these a r e phenolic compounds. Chlorogenic a c i d , a u b i q u i tous compound, i n t e r a c t s r e a d i l y with p r o t e i n s , a f f e c t i n g t h e i r d i g e s t i b i l i t y . Many other common phenolic compounds, although c o l o r l e s s , undergo enzymatic and/or non-enzymatic o x i d a t i o n to pigments. When such pigmentation takes p l a c e with p r o t e i n s , d e t e r i o r a t i v e changes r e s u l t . Examples given i n t h i s chapter suggest c a u t i o n i n the use of some p r o t e i n s f o r food. A great d e a l of time and e f f o r t has been spent i n attempting t o remove f l a v o r s , l i p i d s , pigments, e t c . from p r o t e i n s . T h e i r treatment w i t h proteases may be g e n e r a l l y u s e f u l


9.

ARAI

Binding

Unwanted

Compounds

207

t o o l f o r t h i s purpose. Use of non-protease enzymes a l s o seems p r o m i s i n g , although f u r t h e r s t u d i e s are needed to provide information a p p l i c a b l e i n the i n d u s t r i a l production of wholesome p r o t e i n s for human consumption.


Literature Cited 1. Feeney, R. E., in "Evaluation of Proteins for Humans", Bodwell, C. E., Ed.; Avi Publ. Co., 1977, p. 233. 2. Saint-Hilaire, P.; Solms, J . , J. Agric. Food Chem., 1973, 21, 1126. 3. Scatchard, G., Ann. N.Y. Acad. Sci., 1949, 51, 660. 4. Nawar, W. W., J. Agric. Food Chem., 1971, 19, 1057. 5. Hawrysh, Z. J . ; Stine, C. M., J. Food Sci., 1973, 38, 7. 6. Franzen, K. L.; Kinsella, J. E., J. Agric. Food Chem., 1974, 22, 675. 7. Gremli, Η. Α., J. Amer. Oil Chem. Soc., 1974, 51, 95A. 8. Beyeler, M.; Solms, J., Lebensm.-Wiss. u. Technol., 1974, 7, 217. 9. Arai, S.; Noguchi, M.; Yamashita, M.; Kato, H.; Fujimaki, Μ., Agric. Biol. Chem. Japan, 1970, 34, 1569. 10. Arai, S.; Noguchi, M.; Kaji, M.; Kato, H.; Fujimaki, Μ., Agric. Biol. Chem. Japan, 1970, 34, 1420. 11. Beidler, L. M., J. Gen. Physiol., 1954, 38, 133. 12. Arai, S.; Abe, M.; Yamashita, M.; Kato, H.; Fujimaki, Μ., Agric. Biol. Chem. Japan, 1971, 35, 552. 13. Qvist, I. H.; Von Sydow, E. C. F., J. Agric. Food Chem., 1974, 22, 1077. 14. Wolf, W. J., J. Agric. Food Chem., 1975, 23, 136. 15. Chiba, H.; Takahashi, N.; Yoshikawa, M.; Sasaki, R., Abstracts of Papers, p. 509, Annual Meeting of the Agricultural Chemical Society of Japan, Nagoya, 1978. 16. Buchanan, R. Α., J. Sci. Food Agr., 1969, 20, 359. 17. Buchanan, R. Α., Br. J. Nutr., 1969, 23, 533. 18. Shenouda, S. Y. K.; Pigott, G. M., J. Food Sci., 1975, 40, 523.

19. Mohammadzadeh-k, Α.; Feeney, R. E.; Samuels, R. B.; Smith, L. M., Biochim. Biophys. Acta, 1967, 147, 583. 20. Shenouda, S. Y. K.; Pigott, G. M., J . Food Sci., 1974, 39, 726. 21. Shenouda, S. Y. K.; Pigott, G. M., J. Food Sci., 1975, 40, 520. 22. Hayder, M.; Hadziyev, D., J. Food Sci., 1973, 38, 772. 23. Kamat, V. B.; Graham, G. E.; Davis, M. A. F., Cereal Chem., 24. 25. 26.

1978, 55, 295. Noguchi, M.; Arai, S.; Kato, H.; Fujimaki, M., J . Food Sci., 1970, 35, 211. Ohtsuru, M.; Kito, M.; Takeuchi, Υ., Agric. Biol. Chem. Japan, 1976, 40, 2261. Castell, C. H., J . Amer. Oil Chem. Soc., 1971, 48, 645.



208


27. St. Angelo, A. J.; Ory, R. L . , J. Agric. Food Chem., 1975, 23, 141. 28. Roubal, W. T.; Tappel, A. L., Arch. Biochem. Biophys., 1966, 113, 150. 29. Roubal, W. T., J. Amer. Oil Chem. Soc., 1970, 47, 141. 30. Karel, M.; Schaich, K.; Roy, R. B., J. Agric. Food Chem., 1975, 23, 159. 31. Schaich, Κ. M.; Karel, M., J. Food Sci., 1975, 40, 456. 32. Kanner, J.; Karel, M., J. Agric. Food Chem., 1976, 24, 468. 33. Schaich, Κ. M.; Karel, Μ., Lipids, 1976, 11, 392. 34. Aizawa, H., J. Japanese Soc. Food Nutr., 1969, 22, 231. 35. Aizawa, H., J. Japanese Soc. Food Nutr., 1969, 22, 240. 36. Aizawa, H.; Takeyama, I., J. Japanese Soc. Food Nutr., 1969, 22, 235. 37. Tokuma, Y.; Terayama, Η., Biochem. Biophys. Res. Commun., 1973, 54, 341. 38. Arkcoll, D. B., J. Sci. Food Agr., 1969, 20, 600. 39. Arkcoll, D. B., J. Sci. Food Agr., 1973, 24, 437. 40. Arkcoll, D. B.; Holden, M., J. Sci. Food Agr., 1973, 24, 1217. 41. Knuckles, B. E.; de Fremery, D.; Bickoff, Ε. M.; Kohler, G. O., J. Agric. Food Chem., 1975, 23, 209. 42. Igarashi, K.; Sakamoto, Y.; Yasui, T., J. Agric. Chem. Soc. Japan, 1976, 50, 67. 43. Little, A. C., J. Food Sci., 1977, 42, 1570. 44. Bray, W. J.; Humphries, C.; Ineritei, M. S., J. Sci. Food Agr., 1978, 29, 165. 45. Argyroudi-Akoyunoglou, J. H.; Feleki, Z.; Akoyunoglou, G., Biochem. Biophys. Res. Commun., 1971, 45, 606. 46. Ishii, T.; Kandatsu, M.; Kametaka, M., J. Japanese Soc. Food Nutr., 1974, 27, 103. 47. Arai, S.; Yamashita, M.; Fujimaki, M., J. Nutr. Sci. Vitaminol., 1976, 22, 447. 48. O hEocha, C., in "Biochemistry of Chloroplasts", Vol. I, Goodwin, T. W., Ed.; Academic Press, 1966, p.411. 49. Walker, T. S.; Bailey, J. L . , Biochem. J., 1970, 120, 613. 50. Rűdiger, W., Liebig's Ann. Chem., 1969, 723, 208. 51. Fry, K. T.; Mumford, F. Ε., Biochem. Biophys. Res. Commun., 1971, 45, 1466. 52. Van Buren, J. P.; Robinson, W. B., J. Agric. Food Chem., 1969, 17, 772. 53. Hoff, J. E.; Singleton, Κ. I., J. Food Sci., 1977, 42, 1566. 54. Hagerman, A. E.; Butler, L. G., J. Agric. Food Chem., 1978, 26, 809. 55. Guiragossian, V.; Chibber, Β. A. K.; Van Scoyoc, S.; Jambunathan, R.; Mertz, E. T.; Axtell, J. D., J. Agric. Food Chem., 1978, 26, 219. 56. Feeney, P. P., Phytochem., 1969, 8, 2119. 57. Lahiry,N.L.; Satterlee, L. D., J. Food Sci., 1975, 40, 1326. 58. Lahiry, N. L.; Satterlee, L. D.; Hsu, H. W.; Wallace, G. W., J. Food Sci., 1977, 42, 83.



9.

ARAI


209

59. Dryden, M. J.; Satterlee, L. D., J. Food Sci., 1978, 43, 650. 60. Neucere, N. J.; Jacks, T. J.; Sumrell, G., J. Agric. Food Chem., 1978, 26, 214. 61. Igarashi, K.; Shishido, N.; Yasui, T., J. Agric. Chem. Soc. Japan, 1978, 52, 499. 62. Igarashi, K.; Suzuki, M.; Majima, T.; Yasui, T., J. Agric. Chem. Soc. Japan, 1978, 52, 219. 63. Horigome, T.; Kandatsu, M., J. Japanese Soc. Food Nutr., 1971, 24, 253. 64. Lohrey, E.; Tapper, B.; Hove, E. L., Br. J. Nutr., 1974, 31, 159. 65. Isobe, Α.; Sasaki, R.; Kimura, S., J. Japanese Soc. Food Nutr., 1977, 30, 99. 66. Butler, W. Η., in "Aflatoxin", Goldblatt, L. Α., Ed.; Academic Press, 1969, p. 223. 67. Beckwith, A. C.; Vesonder, R. F.; Ciegler, Α., J. Agric. Food Chem., 1975, 23, 582. 68. Sugimura, T.; Kawachi, T.; Nagao, M.; Yahagi, T.; Seino, Y.; Okamoto, T.; Shudo, K.; Kosuge, T.; Tsuji, K.; Wakabayashi, K.; Iitaka, Y.; Itai, A., Proc. Japan Academy, 1977, 53, 58. 69. Yoshida, D.; Matsumoto, T.; Yoshimura, R.; Matsuzaki, T., Biochem. Biophys. Res. Commun., 1978, 83, 915. RECEIVED

October

18, 1979.


Chemical Deterioration of Proteins - American Chemical Society

Recommend Documents