Functionality and Protein Structure

8 Functional Properties of Microwave-Heated Soybean Proteins

Downloaded by TUFTS UNIV on November 30, 2016 | http://pubs.acs.org Publication Date: March 13, 1979 | doi: 10.1021/bk-1979-0092.ch008

D. L. ARMSTRONG and D. W. STANLEY Department of Food Science, University of Guelph, Guelph, N1G 2W1, Ontario, Canada T. J. MAURICE General Foods Limited, Cobourg, Ontario, Canada

Although the functional properties of proteins are of the greatest concern to the food scientist, our knowledge of them remains empirical. Observations are but rarely related back to fundamental physicochemical properties such as conformation. In many cases it is impossible even to connect one study with another since the researcher is faced with a myriad of techniques used for measuring protein functionality. It is of great importance to gain an understanding of the relationship between structure and functionality so that the latter can be correctly predicted, manipulated and controlled, if only to insure better utilization of protein in a world where population and malnutrition are growing daily. This study stems from an attempt to find alternatives to the present method of obtaining protein from heated, solvent extracted soy flakes. As the primary process can cause protein denaturation, insolubilization, impairment of flavour, functionality and color and may possibly affect its nutritive value, i t would seem that a better approach might be the direct isolation of soy protein using less harsh conditions. One method to achieve this is the preparation of an aqueous extract of soaked beans (soymilk) from which the protein is subsequently precipitated at the isoelectric point, a method favoured for recovering soy proteins with unimpaired functionality (1). However, i f the protein is not exposed to elevated temperatures the lipoxidase systems in the soybeans promote off-flavour in the soymilk, and trypsin inhibitors, which have a retarding effect on growth laboratory animals, will not be destroyed. A technique has been developed for processing soymilk in which the beans are soaked in water and then ground in water at a temperature above 80°C to inactivate the lipoxidase enzymes before they can have a significant effect on flavour (2). Although this eliminates the beany flavour previously associated with soymilk the protein is still exposed to high temperatures American Chemical a l

S o c i e t y

® ^ïsoWmljiIw Washington, 0. C.and 20036 Pour-El; Functionality Protein Structure ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

148

FUNCTIONALITY

A N D PROTEIN

STRUCTURE

•which c a n l e a d t o d e n a t u r a t i o n a n d i n s o l u b i l i z a t i o n . A l s o , t h e a b i l i t y of t h i s procedure to i n a c t i v a t e t r y p s i n i n h i b i t o r s i s d o u b t f u l s i n c e H a c k l e r et_ a l . (3) e s t i m a t e d t h a t s o y m i l k p r e p a r e d a t a m b i e n t t e m p e r a t u r e s r e q u i r e d 120 m i n a t 9 0 ° C t o r e d u c e t r y p s i n i n h i b i t o r a c t i v i t y t o 5$ o f t h e o r i g i n a l l e v e l . We h a v e a t t e m p t e d t o e m p l o y m i c r o w a v e h e a t i n g t o i n a c t i v a t e t h e o b j e c t i o n a b l e t r y p s i n i n h i b i t o r s and l i p o x i d a s e s y s t e m s . T h i s p r o c e s s has the advantages o f b e i n g e a s i l y c o n t r o l l e d and h a v i n g g r e a t p e n e t r a t i o n so t h a t a s h o r t e r t r e a t m e n t may b e u s e d w i t h a concomitant decrease i n p e r i p h e r a l o v e r - h e a t i n g .


MATERIALS AND METHODS Soybeans A l l s o y b e a n s u s e d w e r e f r o m t h e same l o t a n d v a r i e t y (Harrowsoy 6 5 ) . Proximate analyses i n d i c a t e d 37.6$ p r o t e i n (Ν x 5 . 5 0 ) , 2 0 . 2 $ f a t , 6 . 9 $ m o i s t u r e a n d 1 1 . 0 $ crude f i b e r . Preparation of

Soymilk

A 100 g sample o f s o y b e a n s was s o a k e d f o r 12 h i n ^50 m l o f t a p w a t e r at ambient t e m p e r a t u r e . This increased the moisture l e v e l o f the beans t o 6 2 . 1 $ . I f t h e b e a n s r e c e i v e d no h e a t t r e a t m e n t t h e y were p r o c e s s e d i m m e d i a t e l y . D r a i n e d beans were ground i n 1 £ o f tap water (20°C) i n a 1 g a l s t a i n l e s s s t e e l Waring Bien dor at h i g h speed f o r 3 min. The r e s u l t a n t s l u r r y was p a s s e d t h r o u g h a s m a l l h o u s e h o l d c e n t r i f u g a l s e p a r a t o r ( j u i c e e x t r a c t o r ) which trapped the s o l i d p a r t i c l e s i n a t r i p l e layer of cheesecloth. T h e s o y m i l k was s t o r e d a t 0 - ^ ° C u n t i l needed. Heating

Treatments

M i c r o w a v e i r r a d i a t e d s o y b e a n s were p r o d u c e d a f t e r t h e i n i t i a l s o a k i n g p r o c e d u r e u s i n g a L i t t o n microwave oven (Model 5 5 0 , f r e q u e n c y 21+50 M H , o u t p u t 1250 w a t t s ) . T h e b e a n s were p l a c e d i n t h e c e n t r e o f t h e o v e n on a p a p e r p l a t e i n a l a y e r about t h r e e beans t h i c k . Samples were t r e a t e d f o r 3 0 , 6 0 , 90 o r 120 s e c . S o y m i l k was t h e n p r e p a r e d i m m e d i a t e l y as p r e v i o u s l y described. The m e t h o d o f M a t t i c k and Hand (2_) a l s o was u s e d t o prepare soymilk. The b l e n d e r was p r e h e a t e d w i t h a b o i l i n g w a t e r r i n s e a n d b o i l i n g w a t e r was a d d e d t o t h e d r a i n e d b e a n s ; t h e minimum t e m p e r a t u r e o f t h e s l u r r y d u r i n g g r i n d i n g was 8 o ° C . Z

I s o l a t i o n o f Soymilk P r o t e i n s F i g u r e 1, a m o d i f i c a t i o n o f t h e m e t h o d u s e d b y P u s k i a n d M e l n y c h y n ( Λ ) , shows t h e p r e p a r a t i v e p r o c e d u r e u s e d t o i s o l a t e 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 o f s o y m i l k p r o t e i n ( R D P ) .

Pour-El; Functionality and Protein Structure ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


(discard)

Sediment

Figure 1.

Supernatant - RDP

(discard)

Supernatant - Whey

Centrifuge (500 χ g, 30 min)

0.1 Ν citric acid

Adjust to pH 4.6 w/

Supernatant

Scheme for isolation of acid precipitate protein from soymilk, adapted from Ref. 4


buffer (pH 7.0)

Dissolve in 0.3 M citrate-phosphate

buffer (pH 4.5, 2x)

Wash w/ 0.1 M citrate-phosphate

Sediment

Sediment (discard)


Soymilk


Ο

a*

a

S3

ι

ο

ι m

150

FUNCTIONALITY


Determination of Residual Trypsin

A N DPROTEIN

STRUCTURE

Inhibitor

The m e t h o d o f L e a r m o n t h (5_) as a d a p t e d b y V a n B u r e n et_ a l . (6) -was f o l l o w e d t o d e t e r m i n e r e s i d u a l amounts o f t r y p s i n i n h i bitor. T h i s method i s b a s e d upon t h e a b i l i t y o f t r y p s i n t o r e t a r d g e l f o r m a t i o n and t r y p s i n i n h i b i t o r t o suppress t h e r e t a r dation. The s o u r c e o f t r y p s i n was a n e x t r a c t p r e p a r e d u s i n g 30 g o f c o m m e r c i a l b a r l e y m a l t f l o u r a n d 100 m l o f d i s t i l l e d w a t e r . T h i s was s t i r r e d f o r 1 h , t h e m i x t u r e a l l o w e d t o s e t t l e o v e r night and the supernatant f i l t e r e d . A b u f f e r e d g e l a t i n s o l was made b y d i s s o l v i n g 8 g g e l a t i n a n d 1 g d i s o d i u m h y d r o g e n c i t r a t e i n 100 m l d i s t i l l e d w a t e r . A mixture o f 5 ml d i s t i l l e d w a t e r , 5 m l g e l a t i n s o l a n d 5 m l m a l t e x t r a c t was i n c u b a t e d a t 3 5 ° C f o r 2 h a n d t h e n p l a c e d i m m e d i a t e l y i n an i c e w a t e r b a t h . This c o n t r o l h a d an a v e r a g e s e t t i n g t i m e o f 2 0 . 3 m i n . T r y p s i n i n h i b i t o r was a s s a y e d b y u s i n g s o y m i l k i n p l a c e o f t h e d i s t i l l e d w a t e r ; the presence o f t r y p s i n i n h i b i t o r l e s s e n s the time f o r gel formation. R e l a t i v e % t r y p s i n i n h i b i t o r was e x p r e s s e d a s : Relative

% t r y p s i n i n h i b i t o r = 100 -

Six determinations Sensory

were

done

s e t t i n g time

(soymilk)

setting

(water)

i n duplicate

time

f o r each

χ

1

Q

Q

sample.

Analysis

S o y m i l k s a m p l e s were e v a l u a t e d f o r o d o u r b y a p a n e l composed o f 1 7 - 2 0 u n t r a i n e d members. P a n e l i s t s were a s k e d t o e v a l u a t e t h e s a m p l e s f o r b e a n y o d o u r o n an u n s t r u c t u r e d 10 cm s c a l e r a n g i n g from " n o p e r c e p t i b l e odour ( θ ) t o "extreme odour" (lO) by p l a c i n g a v e r t i c a l mark on t h e l i n e a t a p o i n t c h o s e n t o r e f l e c t t h e i r o p i n i o n o f the sample. D a t a were r e c o r d e d as t h e d i s t a n c e from t h e l e f t h a n d , 0 e n d , o f t h e s c a l e t o t h e v e r t i c a l mark. The j u d g e s a l s o i n d i c a t e d w h e t h e r t h e o d o u r was n o t o b j e c t i o n able, s l i g h t l y objectionable or very objectionable. Three r e p l i c a t e s were p e r f o r m e d . 11

Functional

Analyses

Samples u s e d f o r a n a l y s e s o f f u n c t i o n a l p r o p e r t i e s were t h e r e d i s s o l v e d p r o t e i n f r a c t i o n s (RDP) f r o m t h e u n h e a t e d , t h e 6 θ sec microwave t r e a t m e n t and from t h e h o t water t r e a t m e n t s t h a t had s u b s e q u e n t l y been f r e e z e - d r i e d , a p r o c e d u r e r e p o r t e d t o r e s u l t i n minimum d e n a t u r a t i o n (7_). At l e a s t s i x determinations were done f o r e a c h t e s t . Solubility, A 1 g s a m p l e o f p r o t e i n was a d d e d t o e i t h e r 100 m l o f 0 . 3 M c i t r a t e - p h o s p h a t e b u f f e r r a n g i n g f r o m pH 2 . 0 t o 8 . 5 o r t o 100 m l o f pH 7 . 0 c i t r a t e - p h o s p h a t e b u f f e r e x t e n d i n g i n i o n i c s t r e n g t h from 0.005 t o 1.00. The m i x t u r e s were b l e n d e d


8.

ARMSTRONG E T AL.

Microwave-Heated

Soybean

Proteins

151


f o r 5 m i n i n a S o r v a l l O m n i m i x e r a t 3000 r p m , c e n t r i f u g e a , f o r 30 m i n a t 1000 χ g a n d 20 m l o f t h e s u p e r n a t a n t a n a l y z e d f o r p r o t e i n . Emulsion Capacity and Stability. A 0 . 5 g sample o f t h e f r e e z e - d r i e d p r o t e i n f r a c t i o n was r e d i s s o l v e d i n a minimum o f 0 . 3 M c i t r a t e - p h o s p h a t e b u f f e r a t pH 7 . 0 a n d m i x e d t h o r o u g h l y •with 50 m l o f 1 M N a C l f o r 1 min i n a S o r v a l l O m n i m i x e r a t 1000 rpm i n a one p i n t Mason j a r s e t i n a w a t e r b a t h ( 2 0 ° C ) . Crisco o i l (50 ml) was a d d e d t o t h e j a r a n d an e m u l s i o n f o r m e d b y m i x i n g a t 500 rpm w i t h s i m u l t a n e o u s a d d i t i o n o f o i l a t t h e r a t e o f 1 ml/min u n t i l the emulsion broke. The e n d p o i n t was d e t e r m i n e d b y m o n i t o r i n g e l e c t r i c a l r e s i s t a n c e w i t h an o h m e t e r . As t h e e m u l s i o n b r o k e a s h a r p i n c r e a s e (.1 ΚΩ t o 35-^0 ΚΩ) was n o t e d . Emul s i o n c a p a c i t y was e x p r e s s e d as t h e t o t a l volume o f o i l r e q u i r e d t o r e a c h t h e i n v e r s i o n p o i n t p e r mg p r o t e i n . T h i s method i s s i m i l a r t o t h a t u s e d by C a r p e n t e r and S a f f l e (8) f o r sausage emulsions. To e s t a b l i s h e m u l s i o n s t a b i l i t y t h e same p r o c e d u r e was u s e d e x c e p t t h a t 100 m l o f o i l was a d d e d a n d a s t a b l e e m u l s i o n f o r m e d b y b l e n d i n g a t 1000 rpm f o r 1 m i n . A 100 m l a l i q u o t was t r a n s f e r r e d t o a g r a d u a t e c y l i n d e r a n d a l l o w e d t o s t a n d a t room t e m p e r a t u r e . O b s e r v a t i o n s were made o f t h e volume o f t h e o i l , e m u l s i o n and w a t e r p h a s e s a t 3 0 , 6 0 , 90 a n d 180 m i n . Bulk Density. B u l k d e n s i t y d e t e r m i n a t i o n s were made u s i n g a Scott paint volumeter ( F i s h e r S c i e n t i f i c Co.) with a 1 i n brass r e c e i v e r to c o l l e c t the sample. 3

Foam Volume. The d e t e r m i n a t i o n o f f o a m a b i l i t y was c a r r i e d o u t u s i n g t h e p r o c e d u r e o f Hermansson et_ a l . ( 9 ) w i t h m o d i f i c a tions. A 1 g sample o f t h e f r e e z e - d r i e d p r o t e i n f r a c t i o n was h o m o g e n i z e d w i t h 90 m l o f c i t r a t e - p h o s p h a t e b u f f e r i n a S o r v a l l O m n i m i x e r a t 3000 r p m . The r e s u l t a n t foam a n d l i q u i d were t r a n s f e r r e d t o a 250 m l g r a d u a t e d c y l i n d e r a n d t h e m i x e r cup w a s h e d w i t h 10 m l o f b u f f e r . The cup was d r a i n e d f o r 2 m i n a n d t h e c y l i n d e r a l l o w e d t o s t a n d f o r 30 m i n a t w h i c h t i m e t h e foam volume was m e a s u r e d . The i n f l u e n c e o f pH a n d i o n i c s t r e n g t h on foam volume was e s t a b l i s h e d u s i n g t h e b u f f e r s y s t e m s p r e v i o u s l y described. Wettability. W e t t i n g t i m e was d e t e r m i n e d b y d r o p p i n g 1 g o f sample f r o m a h e i g h t o f 10 cm o n t o t h e s u r f a c e o f a 0 . 3 M c i t r a t e - p h o s p h a t e b u f f e r i n a b e a k e r 7 cm i n d i a m e t e r a n d m e a s u r i n g t h e t i m e r e q u i r e d f o r a l l t h e sample t o w e t , as e v i d e n c e d by a complete c o l o r change. The i n f l u e n c e o f pH was e s t a b l i s h e d u s i n g the b u f f e r system p r e v i o u s l y d e s c r i b e d . Water Swelling Water Binding and Dispersion Indices. The methods u s e d were m o d i f i c a t i o n s o f t h o s e e m p l o y e d b y R a s e k h ( l O ) . A 1 g s a m p l e was a d d e d t o 20 m l d i s t i l l e d w a t e r i n a p r e w e i g h e d , g l a s s s t o p p e r e d g r a d u a t e d c y l i n d e r , m i x e d w i t h a g l a s s r o d and 3


152

FUNCTIONALITY

A N D PROTEIN

STRUCTURE

s h a k e n v i g o r o u s l y f o r 3 m i n . The m i x t u r e was a l l o w e d t o s t a n d f o r 1 h a t room t e m p e r a t u r e a n d t h e foam l a y e r w a s h e d down w i t h 5 m l d i s t i l l e d w a t e r f o l l o w e d "by a f u r t h e r h o u r o f s t a n d i n g . At t h i s p o i n t t h e d i s p e r s i o n i n d e x c o u l d be d e t e r m i n e d by removing a 5 ml a l i q u o t from the midpoint o f the suspension and d r y i n g i t at 1 0 5 ° C f o r 18 h , i t s v a l u e b e i n g t h e p e r c e n t a g e d r y w e i g h t o f t h e t o t a l sample w e i g h t . A l t e r n a t i v e l y , t h e s u p e r n a t a n t was s e p a r a t e d f r o m t h e s e d i m e n t b y d é c a n t a t i o n , t h e volume o f t h e s e d i m e n t r e c o r d e d and t h e weight o f t h e sediment d e t e r m i n e d b e f o r e a n d a f t e r d r y i n g a t 1 0 5 ° C f o r 15 h . T h e p a r a m e t e r s were c a l c u l a t e d as :


Water

swelling index =

, _ . ,. . . Water b i n d i n g i n d e x T T

S w e l l e d volume P r e c i p i t a t e weight

Weight o f water i n = - — ~ — 7 — Ζ :—π P r e c i p i t a t e weight

sediment

Scanning E l e c t r o n Microscopy R e p r e s e n t a t i v e f r e e z e - d r i e d samples num s t u b s u s i n g c o n d u c t i v e p a i n t , c o a t e d (60:ko) i n a T e c h n i c s s p u t t e r c o a t e r and Autoscan scanning e l e c t r o n microscope at

were mounted o n a l u m i with gold/palladium e x a m i n e d i n an E T E C 10 k V .

Electrophoresis S a m p l e s were e l e c t r o p h o r e s e d u s i n g a 1% p o l y a c r y l a m i d e s y s t e m ( s t a c k s a t pH 8 . 9 , r u n s a t pH 9 · 5 ) . Sample l o a d i n g was b e t w e e n 5 a n d 15 μ ΐ . A c u r r e n t o f k m a / g e l was a p p l i e d w i t h t h e c a t h o d e i n t h e u p p e r b a t h o f a M o d e l 1200 C a n a l c o s y s t e m u n t i l t h e b r o m p h e n o l b l u e t r a c k i n g dye h a d moved 3 . 8 cm t h r o u g h t h e r u n n i n g g e l . The r e s u l t i n g g e l s were s t a i n e d f o r 1 h w i t h a n i l i n e b l u e - b l a c k ( 0 . 5 $ i n 1% a c e t i c a c i d ) , d e s t a i n e d w i t h 1% a c e t i c a c i d i n a Model l 8 0 1 Canalco quick g e l d e s t a i n e r and s u b s e q u e n t l y s c a n n e d a t 620 nm w i t h a J o y c e - L o e b l Chromos c a n densitometer. Differential

Scanning Calorimetry

A DuPont M o d e l 990 t h e r m a l a n a l y z e r e q u i p p e d w i t h a M o d e l 910 DSC c e l l b a s e was u s e d f o r d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y . Samples w e r e a n a l y z e d as 15$ (w/w) s o l u t i o n s o f f r e e z e - d r i e d RDP w h i c h h a d b e e n d i a l y z e d t o remove e x c e s s b u f f e r s a l t s . A heating r a t e o f 5 ° C / m i n was u s e d ; r u n s were p e r f o r m e d i n a n i t r o g e n a t m o s p h e r e ( 5 ^ p s i ) . A known w e i g h t o f w a t e r was u s e d i n t h e r e f e r e n c e p a n t o b a l a n c e t h e h e a t c a p a c i t y o f t h e sample p a n . The i n s t r u m e n t a l s e n s i t i v i t y was 0 . 0 0 5 ( m e a l / s e c ) / i n . Heats o f t r a n s i t i o n (ΔΗ) were c a l c u l a t e d as c a l o r i e s / g p r o t e i n :


8.

ARMSTRONG ET AL.

Microwave-Heated

ΔΗ = ^

Soybean

Proteins

153

(60 BEAqs)

where A = peak area, M = sample mass, C = sample c o n c e n t r a t i o n , Β = time base s e t t i n g , Ε = c e l l c a l i b r a t i o n c o e f f i c i e n t , Δqs = Y a x i s range. The t r a n s i t i o n temperature was taken as the temperature at the peak maximum. RESULTS AND

DISCUSSION


Yield P r o t e i n r e c o v e r i e s were e s t a b l i s h e d f o r soymilk, RDP and "whey" f r a c t i o n s as a percentage of the p r o t e i n i n the i n t a c t beans. Table I gives the comparative r e c o v e r i e s f o r the s i x treatments. I t may be seen t h a t i n c r e a s i n g the microwave t r e a t ment s i g n i f i c a n t l y reduced the p r o t e i n y i e l d i n soymilk and RDP. These r e s u l t s p a r a l l e l e d previous f i n d i n g s which i n d i c a t e d t h a t the degree t o which soy p r o t e i n s can be d i s s o l v e d at n e u t r a l pH depends upon the extent o f h e a t i n g during p r o c e s s i n g ( l l ) . Although more p r o t e i n was l o s t i n the g r i n d i n g step a f t e r micro wave p r o c e s s i n g than the hot water treatment, the corresponding RDP s gave a higher p r o t e i n y i e l d ; twice as much p r o t e i n was recovered from the 60 sec microwave i r r a d i a t e d sample as from t h a t t r e a t e d w i t h hot water. Thus, more p r o t e i n was a c i d pre c i p i t a t e d u s i n g moderate microwave h e a t i n g while hot water g r i n d i n g produced p r o t e i n s t h a t were i n i t i a l l y water s o l u b l e but could not be p r e c i p i t a t e d at pH k.6. The l a t t e r have probably been heat denaturated to the p o i n t t h a t they e a s i l y form i n s o l u b l e aggregates and were removed during the c e n t r i f u g a t i o n steps. 1

R e s i d u a l T r y p s i n I n h i b i t o r Levels Table I I gives the r e s u l t s o f r e s i d u a l t r y p s i n i n h i b i t o r l e v e l s f o r the various soymilk p r e p a r a t i o n s . The 90 and 120 sec microwave treatments were the most e f f e c t i v e i n i n a c t i v a t i n g the t r y p s i n i n h i b i t o r complex w h i l e hot water t r e a t e d and unheated samples showed the highest l e v e l s . I t i s not s u r p r i s i n g t o f i n d t h a t microwave p r o c e s s i n g i s more e f f i c i e n t than hot water i n suppressing t r y p s i n i n h i b i t o r c o n s i d e r i n g the r a p i d p e n e t r a t i o n o f food m a t e r i a l by microwaves and the s u s c e p t i b i l i t y o f p r o t e i n a c t i o n t o s m a l l heat induced changes i n t e r t i a r y s t r u c t u r e . Hence, C o l l i n s and McCarty (.12) found microwaves produced a more r a p i d d e s t r u c t i o n o f endogenous potato enzymes (polyphenol o x i dase and peroxidase) than hot water heating. I t i s d i f f i c u l t t o judge a safe l e v e l o f t r y p s i n i n h i b i t o r but Van Buren et_ a l . (6) have shown t h a t maximum p r o t e i n e f f i ciency r a t i o s are obtained when at l e a s t 90% of the t r y p s i n


154

FUNCTIONALITY A N D PROTEIN STRUCTURE

Table I .

I n f l u e n c e o f h e a t i n g methods on p r o t e i n recovery.

P r o t e i n recovery (% o f p r o t e i n i n whole bean)


Treatment

Milk

RDP

Whey

Unheated

77.7°

59.7

d

30 sec microwave

72.6°

U6.3

C

60 sec microwave

1*1. 3*

21.8

90 sec microwave

3U.8

a

11.3

a

10.3

C

120 sec microwave

30.7

a

8.6

a

11.2

C

Hot water

7l.U

5.1

a

c

10.7

9.1°>

c

7.6 '

b

7.6 '

b

a

b

a

a

a,b c,d ç l e a p i n g s i m i l a r s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (P < 0.05) 9

o l u n ] n s

Table I I .

I n f l u e n c e of h e a t i n g methods on r e s i d u a l t r y p s i n inhibitor levels.

Treatment

Relative trypsin i n h i b i t o r l e v e l (%)

Unheated

9k.6

30 sec microwave

5^.9

6θ sec microwave

13.5

d

90 sec microwave

0

120 sec microwave

0

Hot water

b

a

73.5°

Numbers b e a r i n g s i m i l a r s u p e r s c r i p t s do not d i f f e r s i g n i f i c a n t l y (p

ο

a

Ω

8.

ARMSTRONG

E T A L .

Microwave-Heated

Soybean

Proteins

171


e x p o s e more h y d r o p h o b i c g r o u p s a n d l e a d t o e v e n l a r g e r a n d i n s o l u b l e aggregates. The l o s s o f e l e c t r o s t a t i c r e p u l s i o n between e l e c t r i c a l double l a y e r s o f l i k e s i g n c o u l d o c c u r a t t h i s p o i n t , c o n c o m i t a n t "with a l o s s o f b o u n d w a t e r , a n d make L o n d o n Van d e r W a a l * s f o r c e s i m p o r t a n t . The i n t e r f a c i a l a r e a o f t h e p r o t e i n w o u l d i n c r e a s e as u n f o l d i n g p r o c e e d s . U n f o l d i n g o f n a t i v e p r o t e i n subunits would be expected t o b e a c c o m p a n i e d b y a s h a r p d r o p i n ΔΗ b u t t h e d i s s o c i a t i o n o f s u b u n i t s may n o t demand u n f o l d i n g . Thus, w h i l e P r i v a l o v and K h e c h i n a s h v i l i (2j0) showed f o r f i v e s i n g l e c h a i n p r o t e i n s t h a t ΔΗ v a r i e d d i r e c t l y w i t h d e n a t u r a t i o n t e m p e r a t u r e , o u r d a t a ( F i g u r e 8) e x h i b i t s enough l a c k o f l i n e a r i t y t o q u e s t i o n t h e c o n c l u s i o n t h a t w i t h a l a r g e , s u b u n i t e d p r o t e i n o n l y an u n f o l d i n g phenomenon i s b e i n g o b s e r v e d . I t may b e t h a t t h e i n i t i a l p a r t o f the curve represents subunits d i s s o c i a t i n g . Obviously, f u r t h e r w o r k on t h e f u n d a m e n t a l a s p e c t s o f t h e r m a l d e n a t u r a t i o n i n t h i s soybean system i s needed. ACKNOWLEDGEMENTS T h i s w o r k was s p o n s o r e d i n p a r t b y t h e N a t i o n a l R e s e a r c h C o u n c i l , t h e D e p a r t m e n t o f I n d u s t r y , T r a d e a n d Commerce a n d t h e O n t a r i o M i n i s t r y o f A g r i c u l t u r e and Food. Technical assistance was p r o v i d e d b y M r s . C. B u r g e s s , M r s . B . Holmes a n d M i s s P . Pierson.

ABSTRACT Aqueous extracts of soybeans were prepared from beans that had been either microwave processed or ground in hot water and protein fractions were acid precipitated to establish the effects of the processing methods on functional properties. Soymilk proteins which had received no heat treatment retained optimal functional properties (with the exception of foaming and those properties relating to the interaction of proteins with water) and gave the highest yield of protein. Residual trypsin inhi bitor levels and objectionable odours were, however, highest in this preparation. Proteins from soymilk prepared by the hot water method exhibited poor isoelectric precipitation and functional properties, a low level of objectionable odour but high levels of trypsin inhibitor. An initial microwave exposure gave a superior product compared to the conventional process and higher protein solubility at certain pH's than even the unheated sample. Electrophoresis, scanning electron microscopy and differential scanning calorimetry were used to suggest that these observations resulted from the action of heat in first disso ciating protein subunits followed by their aggregation and unfolding.


172

FUNCTIONALITY AND PROTEIN STRUCTURE

LITERATURE CITED 1. 2. 3.


4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Smith, A.K. and Circle, S.J. "Soybeans: Chemistry and Technology", Smith, A.K. and Circle, S.J., Eds., AVI Publishing Co., Westport, Conn., 1972. Mattick, L.R. and Hand, D.B., J. Agric. Food Chem. (1969) 17, 15. Hackler, L.R., Van Buren, J.P., Steinkraus, K.H., El Rawi, I. and Hand, D.B., J. Food Sci. (1965) 30, 723. Puski, G. and Melnychyn, P., Cereal Chem. (1968) 45, 192. Learmonth, E.M., J. Sci. Food Agric. (1952) 3, 54. Van Buren, J.P., Steinkraus, K.H., Hackler, L.R., El Rawi, I. and Hand, D.B., J. Agric. Food Chem. (1964) 12, 524. Kinsella, J . E . , Crit. Rev. Food Sci. Nutr. (1976) 7, 219. Carpenter, J.A. and Saffle, R.L., J. Food Sci. (1964) 29, 774. Hermansson, A.M., Sivik, B. and Skjöldebrand, C., Lebensm. Wiss. Technol. (1971) 4, 201. Rasekh, J., J. Milk Food Technol. (1974) 37, 78. Wolf, W.J. and Tamura, T., Cereal Chem. (1969) 46, 331. Collins, J.L. and McCarthy, I.E., Food Technol. (1969) 23, 337. Hermansson, A.M., "Proteins in Human Nutrition", Porter, J.W.G., and Rolls, B.A., Eds., Academic Press, London, 1973. Hermansson, A.M., Olsson, D. and Holmberg, Β., Lebensm. Wiss. Technol. (1974) 7, 176. Nash, A.M., Kwolek, W.F. and Wolf, W.J., Cereal Chem. (1971) 48, 360. Lee, C.H. and Rha, C., J. Food Sci. (1978) 43, 79. Cumming, D.B., Stanley, D.W. and deMan, J.M., J. Food Sci. (1973) 38, 320. Catsimpoolas, Ν., Cereal Chem. (1969) 46, 369. Bau, H.M., Poullain, B., Beaufrand, M.J. and Debry, G., J. Food Sci. (1978) 43, 106. Privalov, P.L. and Kechinashvili, N.N., J. Mol. Biol. (1974) 86, 665.

RECEIVED

October 17, 1978.


Functionality and Protein Structure

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