Chemical Deterioration of Proteins - American Chemical Society

10 Deteriorative Changes of Proteins During Soybean

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 12, 2015 | http://pubs.acs.org Publication Date: May 28, 1980 | doi: 10.1021/bk-1980-0123.ch010

Food Processing and Their Use in Foods DANJI FUKUSHIMA Kikkoman Foods, Inc., Walworth, WI 53184

Various deteriorative changes occur in proteins during food processing and food storage, even under mild conditions. However, a deteriorative change for one purpose can be a favorable one for another purpose. For instance, some meat proteins change physical properties during frozen storage, resulting in loss of chewing qualities and/or functional properties such as binding or emulsifying properties (1). Therefore, this change during frozen storage is a deteriorative one for meats. However, this change is an advantage for the manufacture of a soybean protein product known as "kori-tofu" described later. Another example where a deteriorative change from one aspect can be a favorable one from another aspect is the insolubilization of soybean protein during evaporation (2,3). This insolubilization of proteins is a deteriorative one for the manufacture of "yuba", another soybean protein product described later. The deterioration of physical properties of proteins during food processing and/or storage described above are due to irreversible insolubilization of the proteins. Irreversible insolubilization occurs when unfolded molecules come c l o s e enough t o combine i n t e r m o l e c u l a r l y . Such molecular condensations u s u a l l y occur during d r y i n g , f r e e z i n g , heating and n e u t r a l i z a t i o n o f mol e c u l a r charges of p r o t e i n s o l u t i o n s . T h e r e f o r e , the processes a s s o c i a t e d w i t h i r r e v e r s i b l e i n s o l u b i l i z a t i o n can be c l a s s i f i e d by the patterns o f these molecular condensations. Soybean p r o t e i n s i r r e v e r s i b l y i n s o l u b i l i z e d through n e u t r a l i z a t i o n o f charges are widely used i n the p r o d u c t i o n of t o f u i n the O r i e n t . The present paper deals with these changes and t h e i r use f o r food p r o d u c t i o n . D e t e r i o r a t i v e Changes of Soybean P r o t e i n During Drying and T h e i r Use i n Foods I r r e v e r s i b l e I n s o l u b i l i z a t i o n o f Soybean P r o t e i n During D r y ing. Soymilk i s an economical h i g h - p r o t e i n food o f h i g h n u t r i t i v e v a l u e produced by g r i n d i n g soaked whole soybeans w i t h water, h e a t 0-8412-0543-4/80/47-123-211$07.25/0 © 1980 American C h e m i c a l Society In Chemical Deterioration of Proteins; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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ing the r e s u l t a n t mixture, and then removing the r e s i d u e t o g i v e a s t a b l e emulsion. About 65%, 83%, and 13%, r e s p e c t i v e l y , o f t o t a l s o l i d s , p r o t e i n , and f a t contained i n whole soybeans are found i n the soy m i l k . A n t i - n u t r i t i v e f a c t o r s i n soybeans, such as t r y p s i n i n h i b i t o r s , hemagglutinins, e t c . , are a l s o e x t r a c t e d . Therefore, soy m i l k must be heated f o r i n a c t i v a t i o n o f these a n t i n u t r i t i v e f a c t o r s as w e l l as avoidance of o f f - f l a v o r s . However, soy m i l k powder produced from a heated soy m i l k i s not e a s i l y d i s persed i n t o water when r e c o n s t i t u t e d before d r i n k i n g . This i s a r e s u l t o f i n s o l u b i l i z a t i o n of the heated p r o t e i n which occurred during d r y i n g . T h i s i n s o l u b i l i z a t i o n occurs even when d r y i n g i s c a r r i e d out at room temperature or by l y o p h i l i z a t i o n . This i n d i cates t h a t the process o f evaporation o f water during d r y i n g i s r e s p o n s i b l e f o r the i n s o l u b i l i z a t i o n . E f f e c t of the heating c o n d i t i o n s o f soy m i l k before d r y i n g on i t s r e d i s p e r s i b i l i t y a f t e r d r y i n g i s not simple. The upper two curves (designated as (a)) i n F i g u r e 1 show the e f f e c t o f tempera t u r e and time of heating before d r y i n g o f soy m i l k on the amounts of the p r o t e i n i n s o l u b i l i z e d during d r y i n g . In t h i s f i g u r e , 10 ml of heated soy m i l k i n a 250 ml beaker was d r i e d i n a 50°C constant temperature room f o r l 6 hours. According t o F i g u r e 1, i n s o l u b i l i z a t i o n during d r y i n g of raw soy m i l k without heating was s m a l l ; i n s o l u b i l i z a t i o n was at a maximum a f t e r 10 minutes o f heating and then decreased g r a d u a l l y with longer heating times at 100 and 120O C. In order t o determine the mechanism o f t h i s i n s o l u b i l i z a t i o n , -SH b l o c k i n g reagents were added t o soy m i l k heated under the con d i t i o n which caused maximum i n s o l u b i l i z a t i o n during d r y i n g . The r e s u l t a n t soy m i l k was d r i e d and the amount o f i n s o l u b i l i z e d p r o t e i n was measured, as shown i n F i g u r e 2. As shown i n t h i s f i g u r e , the amount o f i n s o l u b i l i z e d p r o t e i n decreased s h a r p l y w i t h the a d d i t i o n of N-ethylmaleimide (NEMl) or sodium-£-chloromercuribenzoate (PCMB) and reached a constant v a l u e at around 2 X 10"^ M of e i t h e r reagent. As the concentrations of f r e e -SH groups o f t h i s soy m i l k were around k and 2 Χ 10~^ M i n unheated and heated m i l k , r e s p e c t i v e l y , t h i s c o n c e n t r a t i o n of NEMI or PCMB c o i n c i d e s w i t h the c o n c e n t r a t i o n o f f r e e -SH groups i n the heated soy m i l k . This may i n d i c a t e t h a t the f r e e -SH groups present i n the heated soy m i l k p r o t e i n take p a r t i n the i n s o l u b i l i z a t i o n o f the p r o t e i n during d r y i n g . There are two mechanisms f o r the molecular p o l y m e r i z a t i o n by d i s u l f i d e bonds. One i s the p o l y m e r i z a t i o n through an i n t e r molecular d i s u l f i d e bond formed by o x i d a t i o n between the two f r e e -SH groups l o c a t e d on d i f f e r e n t p r o t e i n molecules. The other mechanism i s p o l y m e r i z a t i o n through an i n t e r m o l e c u l a r d i s u l f i d e bond formed by an interchange r e a c t i o n between f r e e -SH groups and d i s u l f i d e bonds which are l o c a t e d i n t e r m o l e c u l a r l y . In order t o determine whether the d i s u l f i d e p o l y m e r i z a t i o n o f heated soy m i l k p r o t e i n occurs through the f i r s t or second mecha nism, i t i s necessary t o measure the d i s u l f i d e and -SH content o f 0

In Chemical Deterioration of Proteins; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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Figure 1. Ε feet of heating of soy milk before drying and effect of addition of N-ethylmaleimide (NEMI) to heated soy milk on the insolubilization of protein after drying. The curves are: (a), dried without adding NEMI; (b), dried after adding NEMI; and (C), the values of (a) minus the values of (b). Curve (a) indicates total amount of insolubilized protein; curve (b) indicates the amount of protein insolubilized by mechanisms other than by intermolecular disulfide bond formation; and curve (c) indicates the amount of protein insolubilized through disulfide bond polymerization (3).



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Figure 2. Effect of the concentration of N-ethylmaleimide (NEMI) or sodium p-chloromercuribenzoate (PCMB) added to heated soy milk (100°C, 20 min) before drying on the insolubilization of the soy milk protein after drying (β).



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the p r o t e i n . The heated soy milk p r o t e i n was found t o c o n t a i n only one or two -SH groups per mole (average molecular weight) whereas there were t e n times as many d i s u l f i d e groups. The presence o f o n l y one or two f r e e -SH groups per mole o f soy m i l k p r o t e i n r u l e s out d i s u l f i d e p o l y m e r i z a t i o n v i a o x i d a t i o n o f -SH groups because: ( l ) the p r o b a b i l i t y t h a t the one or two -SH groups w i l l r e a c t i n t e r m o l e c u l a r l y i s s m a l l , (2) even though two -SH groups might r e a c t i n t e r m o l e c u l a r l y the molecules w i l l polymerize only t o dimers when each molecule has one -SH group, and t o onedimensional polymers o n l y i f a l l the molecules c o n t a i n two -SH groups. Therefore, l a r g e amounts of i n s o l u b i l i z e d p r o t e i n through t h i s mechanism cannot be expected. On the other hand, e x i s t e n c e of l a r g e numbers o f d i s u l f i d e bonds i n each molecule suggests t h a t i n t e r c h a i n d i s u l f i d e p o l y m e r i z a t i o n o f the heated soy m i l k p r o t e i n occurs through an interchange r e a c t i o n between -SH and d i s u l f i d e groups. One or two -SH groups i n each molecule c o u l d r e a c t r e a d i l y w i t h any o f the s e v e r a l a c c e s s i b l e d i s u l f i d e bonds of another molecule and consequently the interchange r e a c t i o n between the -SH and d i s u l f i d e groups occurs t o form a new i n t e r m o l e c u l a r d i s u l f i d e bond. By t h i s r e a c t i o n a new f r e e -SH group appears, which can take p a r t i n another i n t e r m o l e c u l a r r e a c t i o n with d i s u l f i d e bonds t o form a new i n t e r m o l e c u l a r d i s u l f i d e bond. Thus, the i n t e r change r e a c t i o n can proceed s u c c e s s i v e l y , producing new i n t e r molecular d i s u l f i d e bonds and new -SH groups, as shown i n F i g u r e 3. Through t h i s interchange mechanism, the i n t e r m o l e c u l a r d i s u l f i d e bonds can l i n k at m u l t i p l e s i t e s on each molecule, r e s u l t ing i n a three-dimensional p o l y m e r i z a t i o n and i n s o l u b i l i z a t i o n o f the molecules. There i s another o b s e r v a t i o n which i n d i c a t e s t h a t polymerizat i o n during d r y i n g i n heated woy m i l k occurs through the d i s u l f i d e bond interchange r e a c t i o n . I n s o l u b i l i z a t i o n o f the p r o t e i n was increased r a t h e r than decreased by the a d d i t i o n o f a small amount of d i s u l f i d e bond s p l i t t i n g reagents such as mereaptoethanol, sodium s u l f i t e , e t c . , as shown i n F i g u r e k. Further a d d i t i o n o f these reagents decreased i n s o l u b i l i z a t i o n . The explanation appears t o be as f o l l o w s . In the presence of l a r g e amounts o f d i s u l f i d e b o n d - s p l i t t i n g reagents, a l l the d i s u l f i d e bonds o f the p r o t e i n are s p l i t and formation of i n t e r m o l e c u l a r d i s u l f i d e bonds does not occur. However, i n the presence of a s m a l l amount of these reagents, f o r example 10~3 M, some o f the d i s u l f i d e bonds are s p l i t t o produce new -SH groups. In t h i s case, some o f the d i s u l f i d e bonds are not s p l i t . T h e r e f o r e , the i n c r e a s e i n -SH groups act as i n i t i a t o r s of the interchange r e a c t i o n . Thus, i n s o l u b i l i z a t i o n through p o l y m e r i z a t i o n i s i n c r e a s e d by the d i s u l f i d e bond interchange r e a c t i o n with the i n c r e a s e d number o f -SH groups. These r e s u l t s agree with those f o r the -SH and d i s u l f i d e bond interchange r e a c t i o n s i n plasma albumin (5.,6). Since part o f the i n s o l u b i l i z a t i o n of heated soy m i l k p r o t e i n during drying occurs through the s u l f h y d r y l / d i s u l f i d e bond i n t e r change r e a c t i o n , experiments were c a r r i e d out so as t o d i s t i n g u i s h



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Schematic diagram of unfolding of native protein molecules and their intermolecular polymerization (7)



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Effect of added mercaptoethanol and Na SO on the of soy milk proteins after drying (2). g

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q u a n t i t a t i v e l y between p r o t e i n i n s o l u b i l i z e d through the s u l f h y d r y l / d i s u l f i d e bond interchange r e a c t i o n and p r o t e i n i n s o l u b i l i zed by other methods. The f r e e -SH groups o f soy m i l k heated f o r the times i n d i c a t e d i n F i g u r e 1 were then blocked by N-ethylmaleimide (NEMI), the p r o t e i n d r i e d and the amount o f i n s o l u b i l i z e d p r o t e i n determined. The r e s u l t s are shown i n the middle two curves designated as (b) i n F i g u r e 1 . I t i s q u i t e c l e a r from the d i f f e r e n c e between the curves with and without NEMI t h a t some o f the i n s o l u b i l i z a t i o n o f p r o t e i n occurs through the s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n , which i s shown i n the lower two curves designated (c) i n F i g u r e 1. The decrease of extent o f i n s o l u b i l i z a t i o n through d i s u l f i d e bond p o l y m e r i z a t i o n r e s u l t i n g from prolonged h e a t i n g , ( F i g . 1 ( c ) ) , i n d i c a t e s t h a t l o s s o f some o f the f r e e -SH groups o f the p r o t e i n o c c u r r e d during the h e a t i n g . This -SH l o s s was more r a p i d at 120°C than at 100°C and i s a t t r i b u t e d t o o x i d a t i o n by 0 present i n soy m i l k . Free -SH groups r e a c t r a p i d l y i n the presence o f oxygen at h i g h temperature, formi n g (probably) s u l f e n i c a c i d (-SOH), s u l f i n i c a c i d ( - S 0 H ) , and s u l f o n i c a c i d (SO3H) groups. O x i d a t i v e reagents, such as hydrogen p e r o x i d e , a l s o remove the f r e e -SH groups o f soy m i l k p r o t e i n . Next, i n order t o determine whether the i n s o l u b i l i z a t i o n brought about by means other than p o l y m e r i z a t i o n through d i s u l f i d e bonds i s due t o hydrophobic bonds, the s o l u b i l i t y behavior o f i n s o l u b i l i z e d p r o t e i n i n which the -SH groups o f soy m i l k were blocked was t e s t e d a f t e r d r y i n g w i t h sodium d o d e c y l s u l f a t e (SDS), a hydrophobic bond d i s r u p t i n g agent. Almost a l l the p r o t e i n i n s o l u b i l i z e d during d r y i n g of the -SH blocked soy m i l k were s o l u b i l i z e d by 0.5% SDS at n e u t r a l pH, i n d i c a t i n g some i n s o l u b i l i z a t i o n occurs by means other than p o l y m e r i z a t i o n by s u l f h y d r y l / d i s u l f i d e bond interchange. T h i s i n s o l u b i l i z a t i o n may be due t o i n t e r m o l e c u l a r p o l y m e r i z a t i o n through hydrophobic i n t e r a c t i o n s . Thus, i t i s concluded t h a t i n s o l u b i l i z a t i o n of soy m i l k p r o t e i n during d r y i n g occurs both through i n t e r m o l e c u l a r d i s u l f i d e bonds formed by interchange between the -SH and d i s u l f i d e groups of the molecules and through i n t e r m o l e c u l a r hydrophobic i n t e r a c t i o n . When soy m i l k was not heated before d r y i n g and, t h e r e f o r e the p r o t e i n s i n soy m i l k were i n a n a t i v e s t a t e , the i n s o l u b i l i z e d p r o t e i n a f t e r d r y i n g was around l6% r e g a r d l e s s of the presence o r absence o f NEMI. T h i s may be a r e s u l t o f few or no d i s u l f i d e bonds on the s u r f a c e o f the n a t i v e p r o t e i n t o interchange w i t h the -SH groups. When soy m i l k i s heated, however, the n a t i v e t h r e e dimensional s t r u c t u r e o f the molecules are d i s r u p t e d and as a r e s u l t the f r e e -SH groups, many d i s u l f i d e bonds, and most o f the hydrophobic groups, formerly b u r i e d i n s i d e the molecules, are exposed ( F i g . 3 ) . When the exposed r e s i d u e s are brought i n t o c l o s e p r o x i m i t y as a r e s u l t o f d r y i n g both d i s u l f i d e bonds and hydrophobic bonds, formed i n t e r m o l e c u l a r l y by mechanisms d e s c r i b e d above, c o n t r i b u t e t o the i n s o l u b i l i z a t i o n o f soy m i l k . Longer times and higher temperatures d u r i n g h e a t i n g of soy m i l k b e f o r e d r y i n g i n c r e a s e d the number o f exposed hydrophobic groups, i n 2

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c r e a s i n g the amount of p r o t e i n i n s o l u b i l i z e d by i n t e r m o l e c u l a r hydrophobic i n t e r a c t i o n (middle two curves o f (b) i n F i g . l ) . Thus, the complicated phenomena observed f o r i n s o l u b i l i z a t i o n o f the p r o t e i n o f heated soy m i l k may be e x p l a i n e d by these mechanisms. There i s another phenomenon, regarded as a d e t e r i o r a t i v e change i n the p r o t e i n o f soy m i l k , caused a l s o by the evaporation o f water. T h i s i s a f i l m formation on the s u r f a c e of soy m i l k , which occurs when heated soy m i l k i s kept open t o the a i r . T h i s phenomenon i s observed not only i n heated soy m i l k but a l s o i n heated cow*s m i l k . F i l m formation o f soy m i l k occurs only when the soy m i l k i s heated above 60°C and t h e r e i s evaporation o f water from the s u r f a c e o f the soy m i l k . The mechanism of p r o t e i n i n s o l u b i l i z a t i o n i s b a s i c a l l y the same as t h a t of soy m i l k powder produced from heated soy m i l k (k). When water i s removed from the s u r f a c e o f heated soy m i l k by e v a p o r a t i o n , the molecular concent r a t i o n o f p r o t e i n near the s u r f a c e i n c r e a s e s l o c a l l y and the exposed r e a c t i v e groups of the denatured molecules come c l o s e enough t o i n t e r a c t i n t e r m o l e c u l a r l y both by hydrophobic i n t e r a c t i o n s and through the s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n t o form a p o l y m e r i z a t i o n ( f i l m ) on the s u r f a c e . The upper s i d e o f the f i l m contains more hydrophobic amino a c i d s because o f o r i e n t a t i o n o f the hydrophobic p o r t i o n s o f the unfolded molecules t o the atmosphere r a t h e r than i n t o the aqueous s o l u t i o n . Use o f D e t e r i o r a t i v e Changes of P r o t e i n During Evaporation f o r Food P r o d u c t i o n . In Japan, t h e r e i s a t r a d i t i o n a l product c a l l e d "yuba" manufactured by i r r e v e r s i b l e i n s o l u b i l i z a t i o n o f soy m i l k p r o t e i n during evaporation. The f i l m formation o f heated soy m i l k d e s c r i b e d above i s u t i l i z e d f o r p r o d u c t i o n of yuba. Yuba p r o d u c t i o n was s t u d i e d i n d e t a i l by Okamoto et a l . (8^,9.). In the making of yuba, soy m i l k i s put i n t o an open, shallow pan and heated above 80°C. The f i l m , formed on the s u r f a c e by h e a t i n g and evaporation o f water, i s skimmed from the s u r f a c e r e p e t i t i v e l y with a f i n e s t i c k and d r i e d by warm a i r . More than 80% o f the soy m i l k s o l i d s can be recovered from soy m i l k as yuba. Samples o f yuba are shown i n F i g u r e 5 · Yuba i s a v e r y n u t r i t i o u s p r o t e i n food composed of 8.7% water, 5 2 . 3 $ p r o t e i n , 2k.1% fat, 11.9$ carbohydrate, and 3 . 0 $ ash. Yuba, w i t h a m e a t - l i k e t e x t u r e , i s used as an i n g r e d i e n t i n v a r i o u s d i s h e s a f t e r seasoning. D e t e r i o r a t i v e Changes of Soybean P r o t e i n A f t e r F r e e z i n g and T h e i r Use f o r Foods I r r e v e r s i b l e I n s o l u b i l i z a t i o n o f Soybean P r o t e i n A f t e r F r e e z ing. I t i s w e l l known t h a t d e t e r i o r a t i v e changes occur i n p r o t e i n s during f r o z e n storage. Hashizume e t a l . (10^,11^12.) have i n v e s t i g a t e d the i n s o l u b i l i z a t i o n of soybean p r o t e i n a f t e r f r e e z i n g . Comparison o f these r e s u l t s w i t h those o f i n s o l u b i l i z a t i o n of heated soy m i l k p r o t e i n during d r y i n g i n d i c a t e t h a t p r o t e i n i n s o l -



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Figure 5.

Samples of "yuba'



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u b i l i z a t i o n a f t e r f r e e z i n g and during d r y i n g occur e s s e n t i a l l y by the same mechanisms. Both are i n s o l u b i l i z e d through s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n s and hydrophobic i n t e r r a c t i o n s , which occur when p r o t e i n molecules are brought c l o s e together through c o n c e n t r a t i o n . In the evaporation the molecules are brought enough t o r e a c t i n t e r m o l e c u l a r l y by removal of water, whereas i n f r e e z i n g they come c l o s e enough t o r e a c t by removal o f water by formation o f i c e c r y s t a l s . When a p r o t e i n s o l u t i o n i s f r o z e n , the p r o t e i n molecules are concentrated i n t o the unfrozen water s o l u t i o n which e x i s t s among the i c e c r y s t a l s . The amount of t h i s unfrozen water depends upon the r a t e of f r e e z i n g and temperat u r e used, the temperature o f f r o z e n storage and the k i n d of sol u t i o n i n which the p r o t e i n s were d i s s o l v e d . The lower the temperature, the lower the amount o f unfrozen water. At a v e r y low temperature, such as - 3 0 ° C , the amount of unfrozen water i s v e r y s m a l l and t h e r e f o r e , i n s o l u b i l i z a t i o n w i l l not occur by the above mechanisms i f the p r o t e i n s o l u t i o n i s f r o z e n r a p i d l y . At - 3 ° t o - 5 ° C , however, most f r o z e n foods c o n t a i n about 1 0 - 2 0 $ o f unf r o z e n water among the i c e c r y s t a l s i n which the p r o t e i n molecules are concentrated. In such concentrated s o l u t i o n s , the s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n and hydrophobic i n t e r a c t i o n des c r i b e d above can occur r e a d i l y , r e s u l t i n g i n p r o t e i n i n s o l u b i l i zation. I n s o l u b i l i z a t i o n o f heated soybean p r o t e i n a f t e r f r e e z i n g occurs f o r the same reasons as i n s o l u b i l i z a t i o n o f heated soybean p r o t e i n during d r y i n g . F i g u r e 6 shows the e f f e c t of time o f f r o z e n storage on the i n s o l u b i l i z a t i o n o f heated soybean p r o t e i n s o l u t i o n f o l l o w i n g f r e e z i n g and the s o l u b i l i t y of the i n s o l u b i l i z ed p r o t e i n i n urea and/or mercaptoethanol. As shown by curve (a) i n F i g u r e 6 , the heated soybean p r o t e i n was i n s o l u b i l i z e d r a p i d l y during f r o z e n storage. Most o f the i n s o l u b i l i z e d p r o t e i n c o u l d be s o l u b i l i z e d by urea alone, as long as the time o f f r o z e n storage i s s h o r t , but the p r o t e i n became more i n s o l u b l e i n urea as the time o f f r o z e n storage i n c r e a s e d (curve ( b ) ) . A l l the i n s o l u b l e p r o t e i n could not be s o l u b i l i z e d , u n t i l mercaptoethanol i s added t o u r e a (curve ( c ) ) . T h i s i n d i c a t e s t h a t i n s o l u b i l i z a t i o n o f heated p r o t e i n s d u r i n g f r o z e n storage occurs mainly by hydrophobic bonds i n the i n i t i a l stage o f the storage but d i s u l f i d e bonds are g r a d u a l l y formed on longer storage. As a r e s u l t , the p r o t e i n was not s o l u b i l i z e d by urea o n l y . The d i s u l f i d e bonds formed d u r i n g f r o z e n storage are probably formed through s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n s , j u s t as i n heated soy m i l k powder d u r i n g d r y i n g , s i n c e i n s o l u b i l i z a t i o n during f r o z e n storage was a l s o a c c e l e r a t e d by a d d i t i o n of s m a l l amounts o f mercaptoethanol ( F i g . 7). In order t o determine whether formation of these hydrophobic and d i s u l f i d e bonds was caused by c o n c e n t r a t i n g the p r o t e i n molec u l e s i n t o the l i q u i d phase among the i c e c r y s t a l s , heated soybean p r o t e i n s o l u t i o n was concentrated t o about 60% water content at room temperature u s i n g carbowax (polyethylene g l y c o l 6 0 0 0 ) and



222


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(DAYS)

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Figure 6. The insolubilization of soybean protein during frozen storage at — 5°C and their solubility behavior in urea and mercaptoethanol (ME) (10).



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Processing

Agricultural and Biological Chemistry

Figure 7. Increase in the insolubilization of soybean protein during frozen storage at —5°C by the addition of small amounts of mercaptoethanol (ME), indicating the promotion of a sulfhydryl/disulfide interchange reaction by a disulfide bond splitting agent (10)



224

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DETERIORATION OF PROTEINS

then stored at +5°C without being f r o z e n . Measurements of amount of i n s o l u b i l i z e d p r o t e i n and i t s s o l u b i l i t y i n urea and mercaptoethanol are shown i n F i g u r e 8. The r e s u l t s show t h a t p r o t e i n i n s o l u b i l i z a t i o n occurred i n the sample stored i n a concentrated s t a t e without being f r o z e n j u s t as i t d i d i n the sample stored i n a f r o z e n s t a t e without p r i o r concentration ( F i g . 6 ) . Similar s o l u b i l i t y curves i n urea and mercaptoethanol were obtained f o r these two samples ( F i g s . 6 and 8 ) , i n d i c a t i n g t h a t i n s o l u b i l i z a t i o n r e s u l t e d from concentrating the s o l u t i o n s (by evaporation or by f r e e z i n g ) p e r m i t t i n g s u l f h y d r y l / d i s u l f i d e interchange and hydrophobic r e a c t i o n s t o occur. As described above, i n s o l u b i l i z a t i o n does not occur g e n e r a l l y i n n a t i v e p r o t e i n s . However, i f a n a t i v e p r o t e i n has -SH and d i s u l f i d e groups on i t s s u r f a c e , i n s o l u b i l i z a t i o n may occur during f r o z e n storage even i n n a t i v e p r o t e i n molecules. In t h i s i n s o l u b i l i z a t i o n , c o n t r i b u t i o n of the hydrophobic bonds i s l e s s than i n the denatured p r o t e i n because most of the hydrophobic residues are b u r i e d i n s i d e the n a t i v e p r o t e i n molecule. Therefore, the c o n t r i b u t i o n of d i s u l f i d e bonds t o p r o t e i n i n s o l u b i l i z a t i o n during f r o z e n storage can be assessed more c l e a r l y i n n a t i v e p r o t e i n s than i n heated ones. For example, 11S g l o b u l i n ( g l y c i n i n e ) , one of the major components of soybean g l o b u l i n s (Table l ) , contains -SH and d i s u l f i d e groups on the surface of the n a t i v e molecule. When a s o l u t i o n of n a t i v e 11S g l o b u l i n was s t o r e d i n the f r o z e n s t a t e at -5°C, p r e c i p i t a t i o n occurred and s e v e r a l polymerized molecules were present even i n the supernatant as shown i n F i g u r e 9(b) of the d i s c - g e l e l e c t r o p h o r e t i c p a t t e r n s . A d d i t i o n of d i s u l f i d e - s p l i t t i n g agents, such as mercaptoethanol, t o t h i s sol u t i o n stored i n the f r o z e n s t a t e , however, s o l u b i l i z e d the p r e c i p i t a t e s and the s o l u b l e polymers were depolymerized as shown i n F i g u r e 9 ( c ) . Moreover, when -SH b l o c k i n g agents, such as NEMI, were added t o n a t i v e 11S g l o b u l i n s o l u t i o n before f r e e z i n g i n s o l u b i l i z a t i o n d i d not occur on f r o z e n storage. These observations i n d i c a t e t h a t i n s o l u b i l i z a t i o n o f n a t i v e 11S g l o b u l i n during f r o z e n storage occurred p r i m a r i l y through d i s u l f i d e bond formation and hydrophobic bonds were not p r i m a r i l y r e s p o n s i b l e f o r t h i s i n solubilization. When the 11S g l o b u l i n s o l u t i o n was stored i n a concentrated s t a t e without being f r o z e n , polymerization of the molecules occurr e d ; they had the same s o l u b i l i t y behavior as s o l u t i o n s stored i n a f r o z e n s t a t e without being concentrated as shown i n Figure 9(d) and ( e ) . Therefore, the d i s u l f i d e bonds formed during f r o z e n storage of n a t i v e 11S g l o b u l i n , as w e l l as i n heated soybean prot e i n , were caused by the concentrating of the p r o t e i n molecules i n t o the l i q u i d phase among the i c e c r y s t a l s . A s p e c i a l sponge-like t e x t u r e produced as a r e s u l t o f i n s o l u b i l i z a t i o n of unfolded p r o t e i n molecules through both hydrophobic i n t e r a c t i o n s and s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n s , caused by concentrating the p r o t e i n s i n t o the l i q u i d phase present among the i c e c r y s t a l s during f r o z e n storage, i s shown i n F i g u r e 10.



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Table 1,

Major P r o t e i n Components o f Soybean*

P r o t e i n Components

P r o t e i n Contents

By u l t r a centrifuge

By immu nology

By immu nology

By u l t r a centrifuge

2S Glob ulin

o(-Conglycinine

13.8$

15.0$

0-Conglycinine

27.9$ 3.0$

m

ME ADDED TO (b)

ι m-

AFTER CONCENTRATED

m*

ME ADDED TO (d)

Agricultural and Biological Chemistry Figure 9. Disc-gel electrophoretic patterns of US soubean globulin stored in a frozen or concentrated state, (a), original solution; (b), after 2 days storage in a frozen state at —5°C; (c), after the addition of 0.01 M mercaptoethanol (ME) to solution (b); (d), after 2 days of storage in a concentrated state (unfrozen); and (e), after the addition of 0.01 M mercaptoethanol to solution (d) (10).



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Use of D e t e r i o r a t i v e Changes i n P r o t e i n During Frozen Storage i n Food Production. There i s a very unique product made by use of the changes d e s c r i b e d above during f r o z e n storage. T h i s i s a soybean p r o t e i n product c a l l e d " k o r i - t o f u " which was o r i g i n a l l y developed i n ancient Japan i n the regions with s e v e r e l y c o l d winters. The f i r s t step of k o r i - t o f u making i s the production of soy m i l k curd from soy m i l k , u s i n g calcium s a l t s as a coagulant, j u s t as the f i r s t step o f cheese making i s t h a t o f m i l k curd from cow's m i l k using rennet as a coagulant. The i n i t i a l soy m i l k curd, c a l l e d " t o f u " , possesses a f r a g i l e and g e l a t i n o u s t e x t u r e as described l a t e r . The second step of k o r i - t o f u making i s f r o z e n storage of the t o f u curd. The t o f u curd i s f r o z e n at -10°C r a p i d l y and then kept at -1° t o -3°C f o r 2 t o 3 weeks. During t h i s f r o z e n storage, i n t e r m o l e c u l a r i n t e r a c t i o n o f the p r o t e i n occurs i n the l i q u i d phase which surrounds each c r y s t a l through the mechanisms d e s c r i b e d above. As a r e s u l t , the t e x t u r e of the soy m i l k curd a f t e r thawing has changed d r a m a t i c a l l y from a f r a g i l e and g e l a t i n o u s t e x t u r e t o a strong and sponge-like t e x t u r e with a great many holes where the i c e c r y s t a l s e x i s t e d . The f i n a l step of k o r i - t o f u making i s a d r y i n g process. A f t e r thawing, the drying can be c a r r i e d out very e a s i l y by f i r s t squeezing out most of the water i n s i d e the curd and then blowing a warm a i r current on the m a t e r i a l . The f i n a l product i s u s u a l l y 20 gram square pieces as shown i n F i g u r e 11. I t i s a very n u t r i t i o u s product which cont a i n s ( t y p i c a l l y ) 53-5$ p r o t e i n , 26.5% o i l , 1.0% carbohydrate, 2.5% ash, and 10.5% water. Before p r e p a r a t i o n f o r e a t i n g , k o r i t o f u i s r e c o n s t i t u t e d by soaking i n hot water. The rehydrated k o r i - t o f u can imbibe a l a r g e amount of seasoning s o l u t i o n and i s u s u a l l y used as an i n g r e d i e n t i n v a r i o u s dishes a f t e r cooking with seasonings. A meat-like chewiness and f l a v o r can be given t o the r e c o n s t i t u t e d k o r i - t o f u , depending upon the method o f cooking. K o r i - t o f u i s mass produced i n modern f a c t o r i e s where about 30,000 metric tons o f soybeans are used f o r i t s production annually i n Japan.

R e v e r s i b l e and I r r e v e r s i b l e I n s o l u b i l i z a t i o n o f Soybean P r o t e i n and T h e i r Use f o r Foods I t i s v e r y important i n food p r o c e s s i n g whether soybean p r o t e i n i s r e v e r s i b l y or i r r e v e r s i b l y i n s o l u b i l i z e d , s i n c e i r r e v e r s i b l e i n s o l u b i l i z a t i o n g e n e r a l l y r e s u l t s i n d e t e r i o r a t i o n of the p h y s i c a l p r o p e r t i e s of the p r o t e i n . I r r e v e r s i b l e i n s o l u b i l i z a t i o n occurs when unfolded molecules are brought c l o s e enough, through water e v a p o r a t i o n , f r e e z i n g o f water or the n e u t r a l i z a t i o n o f mol e c u l a r charges, t o form i n t e r m o l e c u l a r bonds. In F i g u r e 12, r e v e r s i b l e and i r r e v e r s i b l e i n s o l u b i l i z a t i o n s are c l a s s i f i e d schem a t i c a l l y according t o the p a t t e r n s of the condensation o f the molecules.



228


Japanese Society of Food Science and Technology

Figure 10.

Schematic diagram of the insolubilization frozen storage (11)

Figure 11.

of soybean protein during

Samples of "kori-tofu*


FUKusHiMA

(1)

Soybean Food

REVERSIBLE


(a)

(2)

229

Processing

INSOLUBILIZATION

SOLUBLE STATE ( F A R FROM I S O E L E C T R I C POINT)

(FOLDED (b)

IRREVERSIBLE

INSOLUBILIZATION

(a)

STATE

SOLUBLE

MOLECULES)

PRECIPITATED (ISOELECTRIC

(UNFOLDED

(c)

FROZEN

STATE POINT)

MOLECULES) STATE

Journal of Japan Soy Sauce Research Institute Figure 12. Schematic diagram for the mechanisms of reversible and irreversible insolubilization of soybean protein (7)



230


U s u a l l y , r e v e r s i b l e i n s o l u b i l i z a t i o n occurs when t h e p r o t e i n molecules a r e i n a n a t i v e s t a t e as shown i n F i g u r e 1 2 - ( l ) . The s u r f a c e o f n a t i v e p r o t e i n molecules contains p r i m a r i l y t h e hydrop h i l i c amino a c i d r e s i d u e s and, even though molecules may contact each other during i s o e l e c t r i c p r e c i p i t a t i o n , through c o n c e n t r a t i o n by evaporation o f water and by f r e e z i n g , i r r e v e r s i b l e intermolecul a r bonds are not g e n e r a l l y formed among the molecules. Even i n the n a t i v e s t a t e however, i r r e v e r s i b l e i n s o l u b i l i z a t i o n through a s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n may occur when f r e e -SH and d i s u l f i d e bonds are l o c a t e d a t t h e s u r f a c e o f the molecules. This has a l r e a d y been d e s c r i b e d above f o r the i n s o l u b i l i z a t i o n o f n a t i v e 11S g l o b u l i n during f r o z e n storage. Free -SH groups are a l s o v e r y s e n s i t i v e t o o x i d a t i o n even by air. Native soybean p r o t e i n i n s o l u t i o n became l e s s s o l u b l e when f r o z e n immediately a f t e r p r e p a r a t i o n than i t d i d when f r o z e n a f t e r storage f o r 2 days ( F i g . 13). The same behavior was found f o r heated soybean p r o t e i n f r o z e n immediately or a f t e r two days. These r e s u l t s tend t o i n d i c a t e t h a t t h e one or two -SH groups i n soybean p r o t e i n become o x i d i z e d a f t e r storage f o r two days i n t h e unfrozen s t a t e . Thus, i n t e r m o l e c u l a r d i s u l f i d e bond formation could not occur as d e s c r i b e d i n F i g u r e 3. R e v e r s i b l y i n s o l u b i l i z e d soybean p r o t e i n products possess v a r i o u s f u n c t i o n a l p r o p e r t i e s , such as b i n d i n g , e m u l s i f i c a t i o n e f f e c t , e t c . These f u n c t i o n a l i t i e s may appear when t h e n a t i v e p r o t e i n molecules are unfolded during h e a t i n g i n food p r o c e s s i n g . Therefore these products, such as soybean p r o t e i n i s o l a t e , a r e u s e f u l as b i n d e r s o r e m u l s i f i e r s f o r sausage, hams, e t c . On the other hand, i r r e v e r s i b l e i n s o l u b i l i z a t i o n occurs among unfolded p r o t e i n molecules. In unfolded soybean p r o t e i n molecules, the -SH, d i s u l f i d e and hydrophobic amino a c i d s i d e chains o f t h e molecules a r e exposed, but t h e molecules remain s o l u b l e when t h e c o n c e n t r a t i o n i s not t o o h i g h , as shown i n F i g u r e 12(2a). A t y p i c a l example o f t h i s type o f product i s soy m i l k . When thé u n f o l d ed soybean p r o t e i n molecules a r e concentrated so t h a t contact a mong them i s enhanced, however, i r r e v e r s i b l e i n s o l u b i l i z a t i o n o c curs through both s u l f h y d r y l / d i s u l f i d e interchange and hydrophobic i n t e r a c t i o n s . As d e s c r i b e d above, molecules may be brought t o gether by c o n c e n t r a t i o n o f t h e molecules through removal o f water by evaporation and through removal o f water by f r e e z i n g . Other methods o f b r i n g i n g t h e molecules together are through n e u t r a l i z a t i o n o f charges by adding s a l t or a c i d i f y i n g agents, and by ext e n s i o n and o r i e n t a t i o n o f p r o t e i n s . A t y p i c a l example o f charge n e u t r a l i z a t i o n i n food p r o d u c t i o n i s t h e manufacture o f t o f u , a soybean p r o t e i n food consumed i n l a r g e amounts i n Japan. When calcium s u l f a t e i s added t o heated soy m i l k , t h e soy m i l k i s coagulated. This i s due t o decrease o f the negative charge on the p r o t e i n as a r e s u l t o f b i n d i n g o f C a t o t h e n e g a t i v e l y charged a c i d i c amino a c i d r e s i d u e s o f t h e p r o t e i n molecules. Therefore the unfolded molecules can aggregate, owing t o the decrease o f e l e c t r o s t a t i c r e p u l s i o n , and then form 2 +



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0

2 PERIOD

4 OF FROZEN

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6 STORAGE

8 (DAYS)

Japanese Society of Food Science and Technology

Figure 13. Comparison of rates of insolubilization during frozen storage between soybean protein solutions frozen immediately after preparation (heated and unheated) and frozen after 2 days of storage (heated and unheated). The heated samples were held at 100°C for 5 min prior to freezing (11).



232


an i r r e v e r s i b l e coagulate. Instead o f c a l c i u m s a l t s , glucono-£l a c t o n e i s o f t e n used. The g l u c o n o - i - l a c t o n e i s hydrolyzed t o g l u c o n i c a c i d d u r i n g h e a t i n g and acts as an a c i d i f y i n g agent. In t h i s case, the negative charge on the p r o t e i n i s decreased by p r o t o n a t i o n o f the -COO" o f t h e a c i d i c amino a c i d r e s i d u e s . Tofu i s a white g e l a t i n o u s curd with a unique t e x t u r e i n which l a r g e amounts o f water are h e l d ( F i g . l U ) . The t e x t u r e i s s o f t , smooth, and e l a s t i c . T y p i c a l percentages o f water, p r o t e i n , o i l , carbohydrate and ash i n t o f u are 88.0$, 6.0$, 3 · 5 $ 1 . 9 $ and 0.6$, respectively. In Japan, 270,000 m e t r i c tons o f whole soybeans and 65,000 m e t r i c tons o f d e f a t t e d soybean meal are used i n making t o f u and i t s d e r i v a t i v e s . An example where extension and o r i e n t a t i o n o f p r o t e i n molec u l e s i s used t o b r i n g them t o g e t h e r f o r i n t e r a c t i o n i s i n a r t i f i c i a l meat products, i n c l u d i n g t e x t u r e d p r o t e i n products. In such products, d i s u l f i d e bonds, hydrophobic bonds and hydrogen bonds are formed among the p r o t e i n s extended as f i b e r s as shown i n F i g u r e 12(2e). I r r e v e r s i b l e i n s o l u b i l i z a t i o n of p r o t e i n s may occur mainly through formation o f both i n t e r m o l e c u l a r d i s u l f i d e and hydrophobic bonds. The product can be q u i t e d i f f e r e n t depending on the r e l a t i v e c o n t r i b u t i o n o f these two types o f bonds. The hydrophobic bonds are formed among the hydrophobic amino a c i d s i d e chains cont r i b u t e d by v a l i n e , l e u c i n e , i s o l e u c i n e , p h e n y l a l a n i n e , e t c . These s i d e chains share a common l a c k o f a f f i n i t y f o r water and are pushed together out o f the network o f water molecules i n order that water may preserve i t s s t r u c t u r e . Each hydrophobic bond i s a weak bond (1-2 k c a l / m o l e ) , but they may make a s i g n i f i c a n t c o n t r i b u t i o n t o s t a b i l i z a t i o n o f the polymerized s t a t e i f t h e r e are enough exposed hydrophobic residues among the molecules. In c o n t r a s t , d i s u l f i d e bonds are covalent and strong (80-100 k c a l / mole). T h e r e f o r e , the amount o f i n t e r m o l e c u l a r d i s u l f i d e bond formation w i l l have a major i n f l u e n c e on the p h y s i c a l p r o p e r t i e s o f the i n s o l u b i l i z e d p r o t e i n s . For i n s t a n c e , t h e r e i s a marked d i f f e r e n c e between the p h y s i c a l p r o p e r t i e s o f t o f u g e l made from 7S and 11S g l o b u l i n s . 7S g l o b u l i n (/0-conglycinine) does not cont a i n f r e e -SH groups and only two d i s u l f i d e bonds per molecule, whereas 11S g l o b u l i n has a number o f f r e e -SH groups and a l a r g e number o f d i s u l f i d e bonds (Table l ) . T h e r e f o r e , the t o f u g e l made from 7S g l o b u l i n i s mostly s t a b i l i z e d by hydrophobic bonds, w h i l e the t o f u g e l made from 11S g l o b u l i n i s s t a b i l i z e d by both d i s u l f i d e bonds formed through the s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n and hydrophobic bonds. T h i s i s the reason why 7S t o f u g e l i s s o f t and l e s s e l a s t i c , while 11S t o f u g e l i s much more e l a s t i c (10,13). The same d i f f e r e n c e s can be seen between the p h y s i c a l p r o p e r t i e s of yuba produced from 7S and 11S g l o b u l i n s . Yuba f i l m made from 11S p r o t e i n i s much stronger than when made from 7S p r o t e i n ( l U ) . 9



10.

FUKUSHiMA


Figure 14.

Samples of "tofu*


233

234



E f f e c t o f D e t e r i o r a t i v e Changes of Soybean P r o t e i n During Heating on Enzyme D i g e s t i b i l i t y Enzyme D i g e s t i b i l i t y and Y i e l d o f Soy Sauce. There are v a r i o u s kinds of t r a d i t i o n a l soybean p r o t e i n foods i n the O r i e n t . In a d d i t i o n t o soy m i l k , t o f u , k o r i - t o f u and yuba described so f a r , there are fermented soy sauce, miso, n a t t o , sufu and temphe. Soy sauce was introduced i n t o Japan during the 7th century by Buddhist p r i e s t s and has been developed i n t o the present-day Japanese type o f soy sauce, c h a r a c t e r i z e d by an e x c e l l e n t aroma and f l a v o r , through c e n t u r i e s of a r t i s t r y . Recently, fermented soy sauce has become popular with Western people. Manufacture o f fermented soy sauce i s composed o f three processes, the k o j i making process, the b r i n e fermentation process, and the r e f i n i n g process. For k o j i production, A s p e r g i l l u s species are i n o c u l a t e d onto the cooked s o l i d mixture o f soybeans and wheat and c u l t u r e d f o r ho t o k5 hours under c i r c u l a t i n g a i r of constant temperature and humidity. The c u l t u r e d s o l i d mash, c a l l e d k o j i , i s then mixed with a b r i n e (NaCl) s o l u t i o n o f Ik t o 15 percent by weight. During t h i s b r i n e fermentation, the p r o t e i n i n the soybeans and wheat i s hydrolyzed by proteases from the A s p e r g i l l u s s p e c i e s . T h i s i s i n c o n t r a s t t o a chemical soy sauce made by h y d r o l y s i s o f p r o t e i n s with HCI. Therefore, d i g e s t i b i l i t y of the p r o t e i n s by enzymes i s one o f the most important f a c t o r s i n the making o f fermented soy sauce because i t i s c l o s e l y r e l a t e d to the y i e l d of soy sauce. The d i g e s t i b i l i t y of soybean and wheat p r o t e i n s by the enzymes i s markedly i n f l u e n c e d by the c o n d i t i o n s o f heat treatment of the soybeans. Native soybean p r o t e i n i s q u i t e r e s i s t a n t t o p r o t e o l y s i s because of i t s compact conformation. The r a t e o f p r o t e o l y s i s i s dependent on the degree of u n f o l d i n g o f the subs t r a t e p r o t e i n molecules as shown i n F i g u r e 15. A c c o r d i n g l y , when soybean p r o t e i n i s used as substrate f o r proteases, the p r o t e i n molecules must be unfolded by some treatment, such as heating. However, heat treatment o f the p r o t e i n may decrease the r a t e o f p r o t e o l y s i s . Extended heating of soybean p r o t e i n decreases the r a t e of p r o t e o l y s i s as shown i n Figure l 6 . Therefore, denaturat i o n of the p r o t e i n leads t o b e t t e r p r o t e o l y s i s but too much heat treatment decreases the r a t e o f p r o t e o l y s i s by causing other changes i n the p r o t e i n . Factors which a f f e c t the r a t e and extent o f enzymatic hydrol y s i s of proteins include: ( l ) the substrate s p e c i f i c i t y of the enzymes, (2) m o d i f i c a t i o n o f the amino a c i d s i d e chains o f the substrate p r o t e i n s , and (3) the three-dimensional s t r u c t u r e o f the substrate p r o t e i n s . I t i s e s s e n t i a l that the a c t i v e center o f the enzymes be able t o b i n d with s p e c i f i c amino a c i d residues o f the substrate p r o t e i n . The n a t i v e soybean p r o t e i n molecules are comp l e t e l y f o l d e d and t h e r e f o r e the s p e c i f i c amino a c i d residues r e quired by the enzymes may not be a v a i l a b l e . This i s why n a t i v e


10.


FUKUSHIMA

235

2


M

0

30

60

90

120

180

0

DIGESTION TIME (MIN)

10

20

30

40

DEGREE OF UNFOLDING OF PROTEIN MOLECULES (da* )

Cereal Chemistry Figure 15. Relationship between the degree of unfolding of US globulin mole cules and their susceptibility to proteolysis. In Figure 15B, Δα is calculated as (a — a )/(ao ' — a ) in the Moffitt-Young equation for optical rotatory dispersion. The samples were treated at 20° C for 90 min at the indicated pH and then neutralized (15). 0

Sample

0

Native

Urea denatured

Native

0

0

7

60

180

600

TIME OF HEAT TREATMENT AT 120'C (MIN)

Cereal Chemistry Figure 16. The effect of heat treatment of soybean protein on the maximum extent of enzymatic hydrolysis by proteases of Aspergillus species (A)



236


soybean p r o t e i n cannot be hydrolyzed by enzymes r e a d i l y . In cont r a s t , when the p r o t e i n i s unfolded by heat treatment enzymatic h y d r o l y s i s w i l l proceed r a p i d l y because the enzyme-specific amino a c i d residues of the substrate are a v a i l a b l e as shown i n Figure IT. However, i t i s known that some amino a c i d residues of p r o t e i n s are modified during heating through r e a c t i o n with other compounds or through c r o s s - l i n k i n g . For i n s t a n c e , ^ - and €-amino groups may be modified by r e a c t i o n with aldehyde compounds such as glucose, while l y s i n e , s e r i n e , c y s t i n e , t h r e o n i n e , a r g i n i n e , h i s t i d i n e , tryptophan, a s p a r t i c a c i d and glutamic a c i d may be m o d i f i ed t o l y s i n o a l a n i n e or other compounds through f - e l i m i n a t i o n and c r o s s - l i n k i n g during heat treatment o f p r o t e i n s ( l U - 1 9 ) . The a l k a l i n e proteases o f A s p e r g i l l u s species used i n soy sauce manufacture are s p e c i f i c f o r t y r o s i n e , phenylalanine, l e u c i n e , l y s i n e , and a r g i n i n e residues i n p r o t e i n s . I t has been shown that l y s i n e , a r g i n i n e and c y s t i n e o f the soybean p r o t e i n s are p a r t l y destroyed or modified during heat treatment of d e f a t t e d soybean f l o u r i n the presence of water (Table 2). Since some of these amino acids are e s s e n t i a l f o r maximum h y d r o l y s i s by the enzymes of A s p e r g i l l u s s p e c i e s , t h e i r d e s t r u c t i o n or m o d i f i c a t i o n w i l l r e s u l t i n a decrease i n the degree of maximum h y d r o l y s i s by enzyme. This i s one of the reasons why maximum h y d r o l y s i s of the p r o t e i n was decreased by prolonged heating ( F i g . l 6 ) . A l s o , duri n g prolonged heating new i n t e r m o l e c u l a r or i n t r a m o l e c u l a r i n t e r a c t i o n s among the hydrophobic residues o f the unfolded p r o t e i n w i l l a l s o r e s u l t i n a decrease of enzymatic h y d r o l y s i s . With due c o n s i d e r a t i o n of the e f f e c t o f heating on d i g e s t i b i l i t y of soybean p r o t e i n , v a r i o u s i n v e s t i g a t i o n s were c a r r i e d out using high-temperature - short-time treatment f o r dénaturâtion of the soybean p r o t e i n f o r use i n making soy sauce. A high temperat u r e treatment achieved maximum u n f o l d i n g of the soybean p r o t e i n . A v e r y short time treatment minimized the other d e t e r i o r a t i v e changes. Therefore the y i e l d of soy sauce, based on weight of p r o t e i n of the s t a r t i n g soybean, has increased from 65$ of 20 years ago t o almost 90$ at the present. Enzyme D i g e s t i b i l i t y and N u t r i t i v e Value o f P r o t e i n . Decreased d i g e s t i b i l i t y of soybean p r o t e i n w i t h an increase o f time of heat treatment i s a l s o observed f o r t r y p s i n and pepsin as w e l l as f o r the enzymes from A s p e r g i l l u s s p e c i e s . T h i s decrease i n t r y p s i n and pepsin d i g e s t i b i l i t y gives decreased n u t r i t i v e v a l u e s , j u s t as the decrease i n h y d r o l y s i s by the enzymes from A s p e r g i l l u s species gave a decrease i n y i e l d of soy sauce. The i n f l u e n c e on d i g e s t i b i l i t y o f the d e s t r u c t i o n of amino a c i d s i d e chain residues during heating w i l l be l a r g e r f o r t r y p s i n than f o r other proteases because the amino a c i d s , l y s i n e and arginene, s p e c i f i c f o r t r y p t i c h y d r o l y s i s are more s e n s i t i v e t o d e s t r u c t i o n during heating(Table 2). The a c t i o n of t r y p s i n on unheated soybean p r o t e i n preparat i o n s i s p a r t i c u l a r l y low i n comparison w i t h other enzymes. This



FUKUSHiMA

Soybean

Food

237

Processing

(1) ENZYMATIC HYDROLYSIS OF FOLDED PROTEIN MOLECULE

(NATIVE PROTEIN)

(2) ENZYMATIC HYDROLYSIS OF UNFOLDED PROTEIN MOLECULE

(DENATURED PROTEIN)

Journal of Japan Soy Sauce Research Institute Figure 17.

Schematic explanation for enzymatic hydrolysis of denatured proteins


238

C H E M I C A L DETERIORATION

Table 2.

The D e s t r u c t i o n o f Some Amino Acids During the Heating o f Defatted Soybean F l o u r P r o t e i n * Hours a t 126°C

No heatM

™ ° acids

treatk

OF PROTEINS

0.5

1

2

Hours a t 115°C h

0.5

1

2

1.

m e i r


(Amino a c i d r e s i d u e #/l00 g r . p r o t e i n ) Gly k.l Ala k.k Val 5.U Ile 5-2 8.k Leu Asp 12.2 Glu 19.7 Lys 6.3 Arg 7.6 His 2.1* Phe 5.1 Tyr 3.3 Pro 5.5 Trp 1.1 Met 0.98 Half Cys 1.3 Ser 6.1* Thr k.6

k.k

k.l k.5

k.2 k.6

U.O k.5

k.2 k.k

5.5 5.1 8.5 12.0 19.2 6.0 7.5 2.6 k.9 3.2 5Λ 1.1 1.0 1.3 6.3

5.U 5.2 8.1* 12.2 19.5 5.9 6.8 2.3 5.3 3.1 5.5 1.1 1.0 1.2 6.2

5.5 5.0 8.5 12.0 19.1* 5.8 6.3 2.3 5.1 3.2 5.U 1.1 1.0 1.1 6.1

5-5 5-0 8.5 12.2 19.8 5.1* 6.1 2.3 5.1 3.3 5.6 1.1 1.1 0.9 6.2

5.7 5.1 8Λ 12.1 19-5 5.6 6.2 2.5 5.2 3.2 5.3 1.0 1.0 1.2 6.0

k.6

k.5

k.k

U.l

k.5

k.5

k.O k.3

k.l k.2

1.0 1.0 6.2

5.5 5.1 8.1» 12.2 19.6 5.1 5.9 2.5 5-0 3.2 5.5 1.0 1.0 1.0 6.0

5.1 1*.9 8.3 12.2 19.5 U.O 1*.5 2.1* 5.0 3.1 5.5 0.9 1.0 0.8 5.7

k.6

k.6

k.5

k.l k.5 5.5 5.2 8.5 12.1 19.5 5.6 6.3 2.3 5.2 3.3

5Λ 1.1

*Taken from Table I o f T a i r a et a l . (20). Agricultural and Biological Chemistry

Japanese Society of Miso Science and Technology

Figure 18. Schematic representation of effect of heat treatment on soybean protein and its hydrolysis patterns by various enzymes. Pattern A, pepsin and other acid proteinases; pattern B, the proteinases having an optimum near neu trality, such as papain, bacteria neutral proteinase, Aspergillus alkaline proteinase, Aspergillus neutral proteinase; and pattern C, trypsin and in vivo nutritional values (21). In Chemical Deterioration of Proteins; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


10.

FUKusHiMA


239

i s due t o t r y p s i n i n h i b i t o r s which are present i n soybeans. T h e r e f o r e , increase i n t r y p t i c d i g e s t i b i l i t y by heating i s a t t r i b uted t o i n a c t i v a t i o n of t r y p s i n i n h i b i t o r s as w e l l as u n f o l d i n g of the n a t i v e p r o t e i n molecules. The d i g e s t i o n of heated or unheated soybean p r o t e i n s by v a r i ous enzymes i s s c h e m a t i c a l l y compared w i t h the n u t r i t i v e values i n F i g u r e 1 8 . P a t t e r n A i s t y p i c a l o f pepsin where, because o f low pH o f the r e a c t i o n , the p r o t e i n does not have t o be denatured p r i or t o a d d i t i o n t o the r e a c t i o n . P a t t e r n Β i s t y p i c a l of enzymes such as p a p a i n , b a c t e r i a l n e u t r a l protease e t c . where p r i o r d é n a t u r â t ion of the substrate p r o t e i n i s r e q u i r e d but there are no i n h i b i t o r s of the enzyme p r e s e n t . P a t t e r n C i s t y p i c a l of t r y p s i n where p r i o r heat treatment of the substrate p r o t e i n i s r e q u i r e d t o destroy i n h i b i t o r s o f t r y p s i n as w e l l as t o denature the p r o t e i n for d i g e s t i o n . The decrease i n d i g e s t i b i l i t y with prolonged h e a t ing i n a l l three cases i s due t o m o d i f i c a t i o n o f the substrate p r o t e i n as described above. Conclusion D e t e r i o r a t i o n of the p h y s i c a l p r o p e r t i e s of p r o t e i n s d u r i n g food processing or food storage can be a s c r i b e d p r i m a r i l y t o an i r r e v e r s i b l e i n s o l u b i l i z a t i o n of p r o t e i n s . However, a d e t e r i o r a t i v e change f o r one purpose can be a f a v o r a b l e one f o r another purpose. In Japan, f o r i n s t a n c e , the i r r e v e r s i b l e i n s o l u b i l i z a t i o n of soybean p r o t e i n s has been u t i l i z e d e f f e c t i v e l y f o r product i o n of soybean p r o t e i n f o o d s , such as t o f u , k o r i - t o f u , and yuba. G e n e r a l l y , i r r e v e r s i b l e i n s o l u b i l i z a t i o n occurs when unfolded p r o t e i n molecules are brought c l o s e enough together t o combine intermolecularly. This molecular condensation u s u a l l y occurs as a r e s u l t o f evaporation of water, f r e e z i n g of water, and n e u t r a l i z a t i o n of molecular charges which r e s u l t s i n i n t e r m o l e c u l a r polymeri z a t i o n among the unfolded molecules. The bonds r e s p o n s i b l e f o r the i n t e r m o l e c u l a r p o l y m e r i z a t i o n are both the d i s u l f i d e bonds formed by s u l f h y d r y l / d i s u l f i d e interchange r e a c t i o n and i n t e r a c t i o n among the hydrophobic amino a c i d residues l o c a t e d i n the u n f o l d e d polypeptide chains of the molecules. During heating of soybean p r o t e i n , d e t e r i o r a t i v e changes may occur which decrease enzymatic d i g e s t i b i l i t y . These changes are the r e s u l t of both the m o d i f i c a t i o n o f the enzyme-specific amino a c i d residues o f soybean p r o t e i n s and hydrophobic bonds formed a mong the exposed hydrophobic amino a c i d residues d u r i n g prolonged heating. Literature Cited

1. 2.

Fukushima, D. Proceedings of Western Hemisphere Nutrition Congress II 1968, American Medical Association, 1969, p. 1. Fukushima, D.; Van Buren, J. P. Cereal Chem., 1970, 47, 571.


240

CHEMICAL DETERIORATION OF PROTEINS

3. Fukushima, D.; Van Buren, J. P. Cereal Chem., 1970, 47, 687. 4. Fukushima, D. Cereal Chem., 1969, 46, 405. 5. Huggins, C.; Tapley, D. F.; Jensen, Ε. V. Nature, 1951, 167, 592.

6. Hospelhorn, V. D.; Cross, B.; Jensen, Ε. V. J. Am. Chem.


Soc.,

1954, 76, 2827.

7. Fukushima, D. J. Japan Soy Sauce Res. Inst., 1977, 3, 22. 8. Okamoto, S. J. Japanese Soc. Food Sci. Technol., 1977, 24, 40. 9. Okamoto, S. Cereal Foods World, 1978, 23, 256. 10. Hashizume, K.; Kakiuchi, K.; Koyama, E.; Watanabe, T. Agr. Biol. Chem., 1971, 35, 449. 11. Hashizume, K.; Kosaka, K.; Koyama, E.; Watanabe, T. J. Japanese Soc. Food Sci. Technol., 1974, 21, 136. 12. Hashizume, K. Chemistry and Biology (Japan), 1977, 15, 301. 13. Saio, K.; Kamiya, M.; Watanabe, T. Agr. Biol. Chem., 1969, 33, 1301.

14. Shirai, M.; Watanabe, K.; Okamoto, S. J. Japanese Soc. Food Sci. Technol., 1974, 21, 324. 15. Fukushima, D. Cereal Chem., 1968, 45, 203. 16. Hurrell, R. F.; Carpenter, K. J. Abst., ACS, 1976, 172, 42. 17. Finley, J. W.; Friedman, M. Abst., ACS, 1976, 172, 46. 18. Sternberg, M.; Kim, C. Y. Abst., ACS, 1976, 172, 39. 19. Ziegler, K.; Schmitz, I.; Zahn, H. Abst., ACS, 1976, 172, 101.

20.

Taira, Ha; Taira, Hi; Sugimura, K.; Sakurai, Y. Agr. Biol. Chem., 1965, 29, 1074. 21. Fukushima, D. Miso Sci. Technol. Japan, 1973, No. 232, p. 2. RECEIVED

October

18, 1979.


Chemical Deterioration of Proteins - American Chemical Society

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