Utility of Enzymes in Solubilization of Seed and Leaf Proteins

18 Utility of Enzymes in Solubilization of Seed and Leaf Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 29, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch018

Proteins E . A. CHILDS, J. L. FORTE, and Y. KU a

b

Department of Food Technology and Science, University of Tennessee, Knoxville, Tenn. 37901

The use o f enzymes f o r solubilization o f seed and leaf p r o t e i n s has been s t u d i e d as a means o f overcoming difficulties p r e s e n t e d by the v a r y i n g c o n d i t i o n o f seed and l e a f m a t e r i a l a v a i l a b l e f o r p r o c e s s i n g . The variations a r e the result o f d i f f e r e n c e s i n the original c o n d i t i o n o f the m a t e r i a l and its p o s t - h a r v e s t treatment. E x t r a c t i o n o f P r o t e i n from O i l s e e d s O i l s e e d aleurin p r o t e i n s a r e found in c y t o p l a s m i c vacuoles. Examples i n c l u d e the 7S and 11S g l o b u l i n s of soybeans ( 1 ) , a r a c h i n from peanuts (2,3), edistin from hemp seed ( 4 ) , vicilin and legumin from pea seeds ( 5 ) , and vicilin and legumin from Vicia f a v a ( 6 ) . Based on the similarity o f amino a c i d sequences as meas u r e d by the D i f f e r e n c e Index s u g g e s t e d by Metzger ( 7 ) , D i e c k e r t and D i e c k e r t (8) have s u g g e s t e d the r e s e r v e p r o t e i n s o f leguminous seeds a r e q u i t e homologous and a r e u s u a l l y hexamers o r t e t r a m e r s o f a disulfide bridged s u b u n i t w i t h an A - S - S - B s t r u c t u r e . These similarities i n (a) c y t o p l a s m i c l o c a t i o n and c o m p a r t m e n t a l i z a t i o n and (b) amino a c i d sequence o f a variety o f o i l s e e d p r o t e i n s o f f e r s a u n i f y i n g view o f the c o n d i t i o n s f a c e d in e x t r a c t i n g p r o t e i n s from o i l s e e d s f o r p r o d u c t i o n o f p r o t e i n concentrates or isolates. Several techniques a r e a v a i l a b l e f o r production o f seed p r o t e i n c o n c e n t r a t e s and i s o l a t e s . Protein i s o l a t es c a n be p r e p a r e d from d e f a t t e d seeds by e x t r a c t i o n i n d i l u t e a l k a l i , f o l l o w e d by i s o e l e c t r i c p r e c i p i t a t i o n o f Current Address: D e d e r i c h C o r p o r a t i o n , P.O. Box 7, Hubertus, WI 53033 Current Address: Biology Division, Oak Ridge N a t i o n a l L a b o r a t o r i e s , Oak Ridge, TN 37830 a

b

304 Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 29, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch018

18.

CHILDS

ET

AL.

Solubilization of Seed and Leaf Proteins

305

the p r o t e i n a t an a c i d i c pH, and d r y i n g t h e p r e c i p i t a t ed m a t e r i a l a f t e r washing. P r o t e i n c o n c e n t r a t e s may be produced from seeds by e x t r a c t i o n o f low m o l e c u l a r w e i ght m o l e c u l e s a t pH 4.0 w i t h the p r o t e i n r e m a i n i n g i n the i n s o l u b l e m a t e r i a l f o r d r y i n g . Sugarman (9) d e v e l oped methodology f o r p r o d u c i n g p r o t e i n c o n c e n t r a t e s from f u l l f a t seeds. An aqueous e x t r a c t i o n i s p e r f o r m ed and the r e s u l t i n g e m u l s i o n broken by s h e a r f o r c e s a t an a p p r o p r i a t e temperature and pH. These methods a r e e f f i c i e n t i f the seed has n o t been heated t o an e x t e n t t h a t w i l l cause d e n a t u r a t i o n of the p r o t e i n . I f d e n a t u r a t i o n has o c c u r r e d , e x t r a c t a b l e p r o t e i n y i e l d s w i l l drop t o l e s s than 20% i n soybeans o r c o t t o n s e e d (10,11). Because most o i l s e e d s a r e a l s o an o i l s o u r c e , they may undergo a hexane e x t r a c t i o n and then be s u b j e c t e d t o h e a t and m o i s t u r e t o a i d i n d e s o l v e n t i z a t i o n (13,14). In s p e c i f i c commodities such as soybeans, h e a t may a l s o be u t i l i z e d t o maximize the n u t r i t i o n a l q u a l i t y o f t h e p r o t e i n (15). Thus the p r o b a b i l i t y o f u t i l i z i n g seeds c o n t a i n i n g denatured p r o t e i n s f o r p r o d u c t i o n of p r o t e i n i s o l a t e s and c o n c e n t r a t e s seems l i k e l y . In c o t t o n s e e d , M a r t i n e z e t a l (12) have suggested the lowered s o l u b i l i t y o f p r o t e i n from h e a t e d seeds i s the r e s u l t o f c y t o p l a s m i c p r o t e i n s " g l u i n g " the c y t o p l asmic v a c u o l e s c o n t a i n i n g the s t o r a g e p r o t e i n s t o g e t h e r so t h e y cannot d i s p e r s e when the c e l l i s r u p t u r e d d u r i ng g r i n d i n g . In o r d e r t o i n c r e a s e the e x t r a c t i o n o f p r o t e i n from h e a t d e n a t u r e d s u b s t r a t e s , t e c h n i q u e s which c h e m i c a l l y o r m e c h a n i c a l l y break the c y t o p l a s m i c p r o t e i n s h o l d i n g the a l e u r i n v a c u o l e s t o g e t h e r must be developed. Should t h e i n s o l u b i l i z a t i o n be the r e s u l t o f the b i n d i n g o f i n d i v i d u a l p r o t e i n s o r s t o r a g e vacuol e s t o o t h e r c e l l u l a r c o n s t i t u e n t s (e.g. f i b e r p o l y m e r s ) , t h e s e bonds would a l s o have t o be b r o k e n . E x t r a c t i o n o f P r o t e i n from

Leaves

L e a f p r o t e i n s o c c u r t h r o u g h o u t the c y t o p l a s m o f i n d i v i d u a l c e l l s w i t h 30-40% o f the p r o t e i n l o c a l i z e d i n the c h l o r o p l a s t (16). E x t r a c t i o n o f t h e s e p r o t e i n s has been a c c o m p l i s h e d by p u l p i n g t h e l e a v e s f o l l o w e d by p r e s s i n g t o s e p a r a t e the p r o t e i n a c e o u s j u i c e from the l e a f f i b e r . The j u i c e i s then h e a t e d t o c o a g u l a t e the p r o t e i n and the c o a g u l a t e d p r o t e i n and j u i c e a r e s e p a r a t e d by a p p r o p r i a t e means (17). When t h e s e c h l o r o p l a s t p r o t e i n s a r e e x t r a c t e d , l a r g e amounts o f pigments a r e a l s o f r e e d from the c e l l u l a r m a t r i x t o y i e l d a green p r o d u c t . The p r e s e n c e o f t h e pigments has lowered l e a f p r o -

Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


306

E N Z Y M E S IN

FOOD AND

B E V E R A G E PROCESSING

t e i n c o n c e n t r a t e a c c e p t a b i l i t y as a human f o o d . This has l e d t o development o f p r o c e s s e s w h i c h produce a f i b e r f r e e p r o t e i n - c a r o t e n o i d complex f o r use as an ani m a l f e e d (18,19,20,21,22). R e c e n t l y , a number o f s o l v e n t e x t r a c t i o n and d i f f e r e n t i a l h e a t i n g t e c h n i q u e s have been d e v e l o p e d t o produce c o l o r l e s s o r l o w - c o l o r e d p r o t e i n c o n c e n t r a t e s (23,24,25,26,27,28,29,30,31,32,33). P r o d u c t i o n o f any o f the above p r o d u c t s r e q u i r e s the a v a i l a b i l i t y o f non-heated l e a f p r o d u c t s . Approxi m a t e l y 45% o f the l e a f p r o t e i n can be e x t r a c t e d from f r e s h a l f a l f a l e a v e s (33) w h i l e o n l y 5-15% o f a v a i l a b l e p r o t e i n can be e x t r a c t e d from a l f a l f a d r i e d a t 75-150°C (34) . Approaches t o E x t r a c t i o n o f Heat Denatured P r o t e i n From Seeds and L e a v e s Two e x p e r i m e n t a l approaches have been e v a l u a t e d i n experiments t o i n c r e a s e the y i e l d o f e x t r a c t e d p r o t e i n from h e a t - d e n a t u r e d seeds and l e a f s : enzymatic d i g e s t i o n and u l t r a s o n i c d i s p e r s i o n . C e l l u l a s e s and p r o t e a s e s have been used t o h y d r o l y z e p o r t i o n s o f the p l a n t m a t r i x t o a l l o w more e f f i c i ent d i s p e r s i o n and e x t r a c t i o n o f p r o t e i n s . Hang e t a l (35) showed pea bean p r o t e i n e x t r a c t a b i l i t y was i n c r e a sed by t r e a t m e n t w i t h A s p e r g i l l u s n i g e r c e l l u l a s e and Abdo and K i n g (36) and S r e e k a n t i a h e t a l , (37) reported i n c r e a s e d n i t r o g e n e x t r a c t i o n from soybeans, c h i c k p e a s and sesame f o l l o w i n g c e l l u l a s e t r e a t m e n t . Conversely Lu and K i n s e l l a (32) found a n o n - s i g n i f i c a n t i n c r e a s e i n a l f a l f a meal e x t r a c t a b i l i t y f o l l o w i n g c e l l u l a s e treatment. In our l a b o r a t o r i e s , c e l l u l a s e has n o t i n c r e a s e d p r o t e i n e x t r a c t i o n y i e l d s from a l f a l f a meal, screw e x p r e s s e d c o t t o n s e e d meal, o r hexane d e f a t t e d soybean meal. H y d r o l y s i s o f seed p r o t e i n s has been i n t e n s i v e l y investigated. A r z u e t a l (38) d e s c r i b e d the h y d r o l y s i s o f c o t t o n s e e d cake p r o t e i n s by 10 d i f f e r e n t p r o t e o l y t i c enzyme p r e p a r a t i o n s o f b a c t e r i a l , f u n g a l , p l a n t , and a n i m a l o r i g i n . A l l enzymes h y d r o l y z e d c o t t o n s e e d p r o tein. P r o t e o l y t i c enzymes have a l s o been shown t o hydr o l y z e soybean (39) and sesame meal (37) p r o t e i n s . In none o f t h e s e s t u d i e s was the e f f i c i e n c y o f p r o t e i n e x t r a c t i o n from a food s u b s t r a t e measured. The use o f u l t r a s o n i c energy t o i n c r e a s e s o l u b i l i z a t i o n o f p r o t e i n from h e a t d e n a t u r e d s o u r c e s i s a new development. Wang (10) i n c r e a s e d the e f f i c i e n c y o f p r o t e i n e x t r a c t i o n from a u t o c l a v e d soybean f l a k e s from 16% t o 58% by a p p l i c a t i o n o f u l t r a s o n i c waves. In a d d i t i o n , the u l t r a c e n t r i f u g e p a t t e r n s o f the e x t r a c t e d p r o t e i n s



18.

CHiLDS E T

AL.

307


were t h e same o f t h o s e o f p r o t e i n s e x t r a c t e d from nona u t o c l a v e d samples by c o n v e n t i o n a l t e c h n i q u e s . M o l i n a and La Chance (40) i n c r e a s e d the e x t r a c t a b i l i t y o f p r o t e i n from h e a t t r e a t e d c o c o n u t meal by t r e a t m e n t w i t h a v a r i e t y o f p r o t e o l y t i c enzymes. In those e x p e r i m e n t s , the meal was i n c u b a t e d w i t h the p r o t e a s e and the r e s i d u e e x t r a c t e d w i t h d i l u t e a l k a l i . Y i e l d s o f up t o 80% were o b t a i n e d . In experiments w i t h c o t t o n s e e d meal and a l f a l f a meal (11,41), M o l i n a and L a Chanes's t e c h n i q u e s were modified (Figure 1). I n s t e a d o f s h a k i n g , the samples were s t i r r e d and s e p a r a t i o n o f the s o l i d s u b s t r a t e from the l i q u i d was a c c o m p l i s h e d by g r a v i t y f i l t r a t i o n through c o t t o n organdy r a t h e r than by c e n t r i f u g a t i o n .

25g Meal + Enzyme + 100 ml

liquid

I Stir

30 min.

a t 50°C

I Filter Filtrate (Enzyme F r a c t i o n )

Residue Suspend i n 100 0.75% NaOH Stir

30 min.

ml

of

a t 60oc

Filtrate (NaOH F r a c t i o n ) Figure 1. Flow sheet for proteolytic enzyme-chemical extraction of protein from heat-denatured substrates

P r o t e o l y t i c enzyme p r e t r e a t m e n t i n c r e a s e d e x t r a c t a b i l i t y o f p r o t e i n from h e a t d e n a t u r e d c o t t o n s e e d meal (Table I ) . W i t h o u t enzyme t r e a t m e n t , o n l y 15% o f the c o t t o n s e e d p r o t e i n was e x t r a c t e d by a two-step c h e m i c a l e x t r a c t i o n (11) . F i c i n t r e a t m e n t i n c r e a s e d the amount o f p r o t e i n e x t r a c t i o n a p p r o x i m a t e l y 2.5 t i m e s . Trypsin t r e a t m e n t was most e f f e c t i v e , c a u s i n g an approximate f i v e f o l d i n c r e a s e i n e x t r a c t i o n w i t h g r e a t e r than 65% of p r o t e i n b e i n g e x t r a c t e d .


308

E N Z Y M E S I N FOOD A N D B E V E R A G E PROCESSING

T a b l e I . E f f i c i e n c y o f a two-step p r o t e o l y t i c enzyme-chemical t e c h n i q u e f o r e x t r a c t i o n o f p r o t e i n from c o t t o n s e e d and a l f a l f a meal p r o t e i n s . A l l d a t a a r e t h e mean o f t h r e e r e p l i c a t e s + s t a n d a r d d e v i a t i o n (From 1 1 ) .


Enzyme None Papain Ficin Trypsin

Total % Kjeldahl Protein Extracted C o t t o n s e e d Meal A l f a l f a Meal 15.1 16.0 42.7 69.4

+ + + +

2.3 2.4 1.8 5.7

21.9 23.6 34.1 60.2

+ + + +

4.6 2.8 11.3 5.7

Without enzyme t r e a t m e n t , o n l y 21.9% o f t h e a l f a l f a p r o t e i n was e x t r a c t e d (Table I ) . As w i t h c o t t o n s e e d meal, t r e a t m e n t w i t h p a p a i n d i d n o t markedly improve the e f f i c i e n c y o f e x t r a c t i o n (23.6%). F i c i n treatment i n c r e a s e d t h e amount o f p r o t e i n e x t r a c t e d a l m o s t twof o l d (34.1). T r y p s i n t r e a t m e n t was t h e most e f f e c t i v e , c a u s i n g an approximate t h r e e f o l d i n c r e a s e i n e x t r a c t i o n w i t h g r e a t e r than 60% o f a v a i l a b l e p r o t e i n b e i n g e x t r a cted. Parameters A f f e c t i n g t h e E n z y m a t i c - C h e m i c a l E x t r a c t i o n Process pH. E x p e r i m e n t s have been performed i n which t h e amount o f p r o t e i n e x t r a c t e d d u r i n g t h e enzymatic h y d r o l y s i s s t e p and the d i l u t e a l k a l i e x t r a c t i o n s t e p s were q u a n t i f i e d s e p a r a t e l y as a f u n c t i o n o f t h e pH o f t h e enzyme i n c u b a t i o n . T r y p s i n t r e a t m e n t was more e f f e c t i v e than f i c i n o r b r o m e l a i n a t pH's from 4.0 - 9.5 (Table I I ) . There was f i n i t e b u t l i t t l e v a r i a t i o n amongst the enzyme f r a c t i o n s . T r y p s i n t r e a t m e n t , howe v e r , r e s u l t e d i n a two t o t h r e e f o l d i n c r e a s e i n t h e p r o t e i n c o n t e n t o f t h e NaOH f r a c t i o n r e l a t i v e t o f i c i n or bromelain treatment. T h i s would s u g g e s t t h a t t r y p s i n a c t s on t h e s u b s t r a t e i n a manner which a l l o w s the p r o t e i n t o become more d i s p e r s a b l e i n d i l u t e a l k a l i . A t l e s s than pH 6.5, b r o m e l a i n was t h e most e f f i c i e n t i n t h e enzyme e x t r a c t i o n s t e p i n a l f a l f a meal (Table I I I ) . A t pH's g r e a t e r than 6.5, t r y p s i n and b r o m e l a i n were more e f f e c t i v e than f i c i n . As w i t h c o t t o n s e e d , t h e m a j o r i t y o f t h e p r o t e i n was e x t r a c t e d i n t h e d i l u t e a l k a l i s t e p s u g g e s t i n g the enzymes a r e a c t i n g i n a manner which a l l o w s t h e p r o t e i n t o become more d i s p e r s a b l e i n t h e a l k a l i .



2. 10+0.7

0.70+0.2

1.40+0.6

3.40+0.2

2.51+0.8

9.0

1. 80+0.9

0.60+0.2

1.30+0.7

3.40+0.4

2.48+0.9

8.0

1. 80+0.9

0.60+0.1

1.30+0.6

2.55+0.4

2.50+0.6

7.0

1.30+0.2

1. 60+1.2

0.40+0.1

1.15+0.7

2.60+0.6

2.40+0.4

6.0

1.10+0.3

1. 30+0.2

0.80+0.2

1.10+0.6

2.30+0.4

2.45+0.5

5.0

1.30+0.3

1.10+0.1

1.20+0.2

0.70+0.2

1. 30+0.6

0.70+0.2

1.20+0.8

2.05+0.1

1.80+0.4

4.0

1.40+0.3

0. 80+0.2

Enz .

0.50+0.2

NaOH F r a c .

Bromelain Frac. NaOH F r a c .

0.75+0.3

Enz.

Ficin Frac.

0.50+0.2

1.20+0.2

Enz.

3.0

pH

Trypsin Frac. NaOH F r a c .

Grams o f p r o t e i n e x t r a c t e d from 25 g o f c o t t o n s e e d meal i n a two s t e p p r o t e o l y t i c enzyme-chemical e x t r a c t i o n t e c h n i q u e u t i l i z i n g t r y p s i n , b r o m e l a i n , o r ficin. E x t r a c t a b i l i t y i s p r e s e n t e d as a f u n c t i o n o f t h e pH o f the enzyme r e a c t i o n media.

Table II


I—»

1

3

•Α

•ή.

CO

δ*

«Η.

r

ο

CO

η

S

3

00


0.53+0.1

0.62+0.1

0.95+0.1

0.97+0.1

1.10+0.1

0.72+0.1

0.78+0.1

0.92+0.2

1.00+0.2

1.05+0.2

1.05+0.2

4.0

5.0

6.0

7.0

8.0

9.0

1.22+0.2

0.52+0.2

0.75+0.1

Trypsin Frac. NaOH F r a c .

3.0

Enz.

0.98+0.1

0.49+0.2 0.80+0.1

0.48+0.1

0.98+0.1

0.55+0.2 0.83+0.1

0.92+0.1

0.90+0.2

0.55+0.2

0.53+0.1

0.52+0.1

1.10+0.1

0.82+0.1

0.81+0.1

0.41+0.0

0.68+0.1

1.00+0.1

0.43+0.2

0.80+0.0

0.68+0.1

0.74+0.1

0.39+0.1

0.38+0.0

1.20+0.3

Bromelain NaOH F r a c . Frac.

0.72+0.2

Enz.

0.72+0.1

Ficin Frac. NaOH F r a c .

0.34+0.1

Enz.

Grams o f p r o t e i n e x t r a c t e d from 25 g o f a l f a l f a meal i n a two s t e p p r o t e o l y t i c enzyme-chemical e x t r a c t i o n t e c h n i q u e u t i l i z i n g t r y p s i n , b r o m e l a i n o r f i c i n . E x t r a c t a b i l i t y i s p r e s e n t e d as a f u n c t i o n o f t h e pH o f t h e enzyme r e a c t i o n media.

Table I I I



18.

CHiLDS E T

AL.

311


One p o s s i b l e h y p o t h e s i s t o e x p l a i n the above d a t a i s t h a t the p r o t e i n s a r e bound t o a polymer l i g a n d a f t e r h e a t treatment and the p r o t e o l y t i c enzyme c l e a v e s the two m o l e c u l e s . A c a n d i d a t e polymer might be d i e t a r y fiber. To t e s t t h i s h y p o t h e s i s , a N e u t r a l D e t e r g e n t F i b e r (NDF) e x t r a c t o f c o t t o n s e e d meal was prepared (Van S o e s t , 1973). C o t t o n s e e d meal samples were a l s o produced from the e x t r a c t i o n r e s i d u e s . I t was n o t e d t h a t the Ν c o n t e n t o f the NDF samples from t r y p s i n t r e a t e d c o t t o n s e e d meal was much lower than t h a t from o t h e r samples (41) . T h e r e f o r e , t r y p s i n was a b l e t o c l e a v e p r o t e i n - f i b e r bonds more e f f i c i e n t l y than o t h e r enzymes. T h i s s t r o n g l y s u g g e s t s , b u t n o t c l e a r l y prove, the s u p e r i o r performance o f t r y p s i n may be based on i t s a b i l i t y to c l e a v e f i b e r - p r o t e i n bonds. T h i s a c t i o n c o u l d r e l e a s e n o n - h y d r o l y z e d p r o t e i n , r a t h e r than w a t e r s o l u b l e fragments; and the whole p r o t e i n might then e x h i b i t i t s normal non-denatured s o l u b i l i t y c h a r a c t e r istics in dilute alkali. T h i s h y p o t h e s i s i s g i v e n add i t i o n a l c r e d e n c e by r e c e n t o b s e r v a t i o n s t h a t 60% o f N i t r o g e n s o l u b i l i z e d from c o t t o n s e e d by t r y p s i n t r e a t ment has a m o l e c u l a r w e i g h t g r e a t e r than 1000 d a l t o n s (42) . R e l a t i o n s h i p o f enzyme c o n c e n t r a t i o n t o p r o t e i n extraction. The r e l a t i o n s h i p o f enzyme c o n c e n t r a t i o n to p r o t e i n e x t r a c t i o n i n c o t t o n s e e d was c u r v i l i n e a r (Table I V ) . Treatment w i t h a t r y p s i n c o n c e n t r a t i o n o f 0.03g/25g meal caused g r e a t e r than 75% o f the p r o t e i n to be e x t r a c t e d . A t g r e a t e r than 0.03g enzyme/25g meal, t h e r e was a lowered r a t e o f i n c r e a s e i n p r o t e i n e x t r a c t ability.

Table

IV

E f f e c t o f T r y p s i n C o n c e n t r a t i o n on the E f f i c i e n c y o f a Two-Step T r y p s i n - C h e m i c a l E x t r a c t i o n o r P r o t e i n From C o t t o n s e e d and A l f a l f a Meal Amount o f Trypsin/25g Substrate 0 0.01 0.03 0.06 0.12 0.24

T o t a l % Kjedahl P r o t e i n Extracted Cottonseed 16.4 18.2 78.4 82.6 86.5

+ + + + +

2.3 1.7 3.1 4.3 6.1

Meal

Alfalfa 21.8 27.3 41.7 57.5 64.3 73.4

+ + + + + +


Meal 2.1 1.4 4.8 7.3 2.8 4.8

312

ENZYMES

IN FOOD A N D B E V E R A G E

PROCESSING

The r e l a t i o n s h i p o f t r y p s i n c o n c e n t r a t i o n t o e x t r a c t a b l e p r o t e i n was q u i t e d i f f e r e n t f o r a l f a l f a meal ( T a b l e I V ) . The e f f i c i e n c y o f the p r o c e s s i n c r e a s e d w i t h i n c r e a s i n g amounts o f t r y p s i n up t o 0.24g t r y p s i n / 25g a l f a l f a meal. T h i s i s 8 times the c o n c e n t r a t i o n used i n c o t t o n s e e d meal.


E f f e c t o f U l t r a s o n i c Energy on the Enzyme-Chemical Technique Because o f the p o t e n t i a l advantages a s s o c i a t e d w i t h use o f the u l t r a s o n i c t e c h n i q u e (e.g., c a v i t a t i o n o f sample t o c r e a t e new s i t e s f o r enzyme a c t i v i t y , i n creased mixing e f f i c i e n c y , decreased r e a c t i o n times, and r e d u c e d energy e x p e n d i t u r e s ) , e x p e r i m e n t a l s t u d i e s have been performed t o determine the e f f e c t s o f u l t r a s o n i c energy on the p r o t e o l y t i c enzyme-chemical t e c h n i q u e i n c o t t o n s e e d and soybean meal. The combined e n z y m a t i c - c h e m i c a l - u l t r a s o n i c t e c h n i q u e i n c r e a s e d the e f f i c i e n c y o f t o t a l p r o t e i n e x t r a c t i o n from c o t t o n s e e d meal o v e r enzyme use a l o n e ( T a b l e V ) . In soybean meal, the i n c r e a s e i n e x t r a c t i o n e f f i c i e n c y was a p p r o x i m a t e l y 10%.

Table V S o l u b i l i z a t i o n o f c o t t o n s e e d and soybean meal p r o t e i n by e n z y m a t i c - c h e m i c a l t e c h n i q u e s w i t h and w i t h o u t u l t r a s o n i c energy

Technique T r y p s i n , no ultrasonic energy T r y p s i n , 200 accoustical watts

Total % Kjeldahl Protein Extracted C o t t o n s e e d Meal Soybean Meal

65.66+4.98

80.72+3.85

72.99 + 3.18

91.32

+

4.70

The p r e s e n c e o f u l t r a s o n i c energy a l s o i n c r e a s e s the r a t e o f t h e e n z y m a t i c - c h e m i c a l p r o c e d u r e w i t h o u t u l t r a s o n i c energy, a p p r o x i m a t e l y 120 minutes was r e q u i r e d f o r the e x t r a c t i o n (11,42,4 3 ) . With the a d d i t i o n o f u l t r a s o n i c energy, t h e r e were no s i g n i f i c a n t d i f f e r ences i n the i n the p e r c e n t a g e p r o t e i n e x t r a c t e d f o r s o n i c a t i o n times v a r y i n g from 1 - 1 6 min o f t r y p s i n c o t t o n s e e d meal m i x t u r e s ( T a b l e V I ) . These d a t a s u g g e s t


18.

CHILDS ET AL.

313


t h a t s o n i c a t i o n times o f 1 min a r e adequate. The same was t r u e f o r soybean meal where 90% o f a v a i l a b l e p r o t e i n was e x t r a c t e d i n one minute. Therefore, u l t r a s o n i c energy i n c r e a s e s t h e e f f i c i e n c y o f enzyme s o l u b i l i c a t i o n procedures.


T a b l e VI E f f e c t o f S o n i c a t i o n Time on E x t r a c t i o n o f P r o t e i n From C o t t o n s e e d and Soybean Meal Sonication Time (Min) 1 2 4 8 12 16

Total % Kjeldahl Protein Extracted C o t t o n s e e d Meal Soybean Meal 70.0 67.5 67.5 73.2 61.3 71.6

Functionality of Extracted

+ + + + + +

1.2 1.8 3.4 6.1 9.0 7.8

93.7 93.9 95.3 94.8

+ + + +

4.2 2.4 3.8 2.7

Protein

P r o t e i n e x t r a c t e d from c o t t o n s e e d meal by t h e enzy m a t i c c h e m i c a l p r o c e s s i s q u i t e f u n c t i o n a l . The s o l u b i l i t y o f p r o t e i n c o n c e n t r a t e s produced by i s o e l e c t r i c p r e c i p i t a t i o n was t y p i c a l o f s o l u b i l i t y o f c o t t o n s e e d p r o t e i n as a f u n c t i o n o f pH (42). S o l u b i l i t y was minimal a t a c i d i c pH's and i n c r e a s e d i n b a s i c pH's (Crenwelge e t a l . , 1974). There were no marked d i f f e r ences i n s o l b u i l i t y i n 0.1 M NaCl v s w a t e r . T h i s sugge s t s l i t t l e o r no i n t e r a c t i o n between pH and i o n i c strength. There were no s i g n i f i c a n t d i f f e r e n c e s i n t h e w a t e r o r o i l - h o l d i n g c a p a c i t i e s o f t h e samples (Table V I I ) . However, t h e e m u l s i f y i n g c a p a c i t y o f t h e NaOH f r a c t i o n produced by t h e combined t e c h n i q u e was s i g n i f i c a n t l y g r e a t e r than t h a t o f o t h e r f r a c t i o n c o n c e n t r a t e s . It has o f t e n been s u g g e s t e d t h a t e m u l s i f y i n g c a p a c i t y i s a f u n c t i o n o f m o l e c u l a r r a d i u s (45) and t h e s e d a t a tend to c o n f i r m t h a t o b s e r v a t i o n s i n c e t h e h i g h e s t e m u l s i f i c a t i o n c a p a c i t y was n o t e d w i t h t h e c o n c e n t r a t e having the l o w e s t amount o f low m o l e c u l a r w e i g h t n i t r o g e n ( 4 2 ) .


ENZYMES IN FOOD AND BEVERAGE PROCESSING

314

Table V I I


F u n c t i o n a l i t y o f c o t t o n s e e d meal p r o t e i n i s o l a t e s p r e p a r e d by the p r o t e l y t i c enzyme-chemical t e c h n i q u e (PC) and the u l t r a s o n i c - e n z y m a t i c (UE) t e c h nique. ( A l l d a t a a r e t h e mean o f t h r e e r e p l i c a t e s + standard deviation) Oilholding capacity (ml/g)

Sample Enzyme fraction (PC) NaOH fraction (PC) NaOH fraction (UE)

4.03

+ 1.10

Waterholding capacity (ml/g)

2.43

+ 0.86

Emulsifying capacity (ml/g)

333

+

4.0

3.53 + 0.32

2.80 + 0.26

443 + 4.0

3.66 + 0.15

2.73 + 0.15

586 + 2.5

In summary, i n v e s t i g a t i o n s w i t h e x t r e m e l y h e a t de n a t u r e d s u b s t r a t e s (NSI 0.15) such as c o t t o n s e e d meal, a l f a l f a meal and soybean meal have shown t h a t t r y p s i n t r e a t m e n t w i l l a l l o w e f f i c i e n t p r o t e i n e x t r a c t i o n . The e f f i c i e n c y o f t h i s e x t r a c t i o n can be markedly i n c r e a s e d by i n t e r f a c i n g u l t r a s o n i c energy w i t h t h e system. Iso l a t e s produced from the e x t r a c t e d p r o t e i n s a r e f u n c t i o n al. REFERENCES 1. Koshiyama, I. 1972. Agri. Biol. Chem. 36:62. 2. Altschul, A.M., N . J . N e u c e r e , A.A. Woodham, and J.M. Dechary. 1964. N a t . 203:501. 3. Daussant, J., N . J . N e u c e r e , and L . Y . Y a t s u . 1969. P l a n t P h y s i o l . 44:471. 4. S t . A n g e l o , A.J., L . Y . Y a t s u , and A.M. Altschul. 1968. A r c h . Biochem. B i o p h y s . 124:199. 5. V a r n e r , J.E. and G. S c h i d l o v s k y . 1963. P l a n t P h y s i o l . 38:139. 6. Graham, Τ . A . and B . E . S . Gunning. 1970. N a t . 228:81. 7. M e t z g e r , Η . , M.B. S h a p i r o , J.E. Mosiman, and J.E. V i n t o n . N a t . 219:1166. 8. D i e c k e r t , J.W. and M.C. D i e c k e r t . 1976. J Fd Sci. 41:475. 9. Sugarman, N. 1956. U.S. P a t e n t 2, 762, 820.



18.

CHILDSETAL.


315

10. Wang, L.C. 1975. J F d Sci. 40:549. 11. C h i l d s , E.A. 1975. J Fd Sci. 40:78. 12. M a r t i n e z , W . H . , Berardi, L.C. and L.A. G o l d b l a t t . 1970. J A g r F d Chem. 1 8 : 9 6 1 . 13. V i x , H.L.E. 1968. Oil Mill G a z . 72:53. 14. M a r t i n e z , W . H . 1969. P r o c . XVII C o t t o n P r o c Clin. ARS Report 72-69:18. 15. Circle, S.J. and A.K. S m i t h . 1972. "Soybeans:Chemistry and T e c h n o l o g y . " 638. AVI P u b l i s h i n g C o . 16. N e u r a t h , H. and K. Bailey. 1973. "The P r o t e i n s . " 542. Academic P r e s s . 17. Pirie, N.W. 1971. "Leaf P r o t e i n : I t s Agronomy, P r e p a r a t i o n , Q u a l i t y and U s e . " IBP Handbook #20. Blackwell Scientific. 18. K n u c k l e s , B.E., R.R. S p e n c e r , M.E. L a z a r , E.M. Bickoff. and G.O. K o h l e r . 1970. J A g r F d Chem. 18:1086. 19. K o h l e r , G.O. and E.M. Bickoff. 1970. T h i r d Int'l Congress o f Food S c i e n c e and T e c h n o l o g y . Wash, D.C. 20. Lazar,M.E., R.R. S p e n c e r , B.E. K n u c k l e s , and E.M.J. B i c k o f f . 1971. J A g r Fd Chem. 19:944. 21. Miller, R.E., R.H. Edwards, M.E. L a z a r , E.M. Bickoff, and G.O. K o h l e r . 1972. J Agr Fd Chem. 20:1151. 22. S p e n c e r , R.R., A.C. M o t t o l a , E.M. B i c k o f f , J.P. C l a r k , and G.O. K o h l e r . 1971. J A g r Fd Chem. 19:504. 23. Chayen, I.H., R.H. S m i t h , G.R. T r i s t r a m , D. Thirkell, and T . Webb. 1961. J S c i Fd A g r . 12:502. 24. Hartman, G.H., W.R. A k e s o n , and M.A. Stohmann. 1967. J A g r F d Chem. 1 5 : 7 4 . 25. Huang, K.T., M.C. T a o , M. B o u l e t , R.R. Riel, J.P. Julien, and G.J. G r i s s o m . 1971. Can I n s t Fd T e c h . 4:85. 26. W i l s o n , R.F., and J.M.A. Lilley. 1965. J Sci F d A g r . 16:173. 27. B y e r s , M. 1967. J S c i Fd A g r . 18:34. 28. Cowlishaw, S.J., D.E. E y l e s , W . F . Raymond, and J.M.A. Tilley. 1956. J S c i F d A g r . 7:775. 29. deFremery, D., E.M. B i c k o f f , and G.O. K o h l e r . 1973. J A g r F d Chem. 20:1155. 30. Henry, K.M. and J.E. F o r d . 1965. J Sci Fd Agric. 16: 425. 31. L e x a n d e r , K., R. C a r l s o n , V . S c h a l e n , A. Simonsson, and T . L u n d b o r g . 1970. A n n a l . A p p l . Biol. 66:193. 32. Subba-Rau, B.H., S. Mahadeviah, and N . S i n g h . 1969. J S c i F d A g r . 20:355. 33. Edwards, R.H., R.E. Miller, D. deFremery, B.E. K n u c k l e s , E.M. B i c k o f f , and G.O. K o h l e r . 1975. J Agr F d Chem. 23:620. 34. L u , P.S. and J.E. Kinsella. 1972. J Fd Sci. 37:94. 35. Hang, Y.D., W . F . W i l k e n s , A.S. Hill, K.H. S t e i n k r a u s , and L.R. H a c k l e r . 1970. J A g r Fd C h . 1 8 : 9 .



316

E N Z Y M E S I N FOOD A N D B E V E R A G E PROCESSING

36. Abdo, K.M. and K.W. K i n g . 1967. J A g r Fd Chem. 15:83. 37. S r e e k a n t i a h , K.R., H. E b i n e , J. O h t a , and M . Nakans. 1969. F d T e c h . 23:1055. 38. A r z u , Α., H. Mayorga, J. G o n z a l e s , and C . R o l z . 1972. J A g r F d Chem. 20:805. 39. F u j i m a k i , M . H. K a t o , S. Arai, and E. Tamaki. 1968. Fd T e c h . 22:889. 40. M o l i n a , M . R . and P . A . L a C h a n c e . 1973. J Fd Sci. 38: 607. 41. C h i l d s , Ε . A . 1976. U n p u b l i s h e d d a t a . 42. C h i l d s , E.A. and J.L. Forte'. 1976. J F d Sci. 41: 652. 43. K u , Y . and E.A. Childs. 1976. Tenn Fm Hm Sci. 97:26. 44. C r e n w e l g e , D.D., C . W . Dill, P . T . T y b o r , and W . A . Landmann. 1974. J Fd Sci. 39:175. 45. C a r p a n t e r , J.L. and R.L. Saffle. 1965. Fd T e c h . 1567.


Utility of Enzymes in Solubilization of Seed and Leaf Proteins

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