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Isolation and functional properties of protein from the whey of acid-modified gluten Dosanjh, Bakhshish S. 1975

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ISOLATION AND FUNCTIONAL PROPERTIES OF PROTEIN FROM THE WHEY OF ACID-MODIFIED GLUTEN by BAKHSHISH S. DOSANJH _ M . S c , Pb. A g r i . U n i v . I n d i a , 1969 A t h e s i s s u b m i t t e d i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r t h e d e g r e e o f M a s t e r o f S c i e n c e i n t h e ' D e p a r t m e n t o f FOOD SCIENCE F a c u l t y o f A g r i c u l t u r a l S c i e n c e s We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h C o l u m b i a S e p t e m b e r , 1975 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission f o r extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication ,of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of p &o Jb <T//tT>//T/4r The University of B r i t i s h Columbia 2075 Wesbrook Place, Vancouver, Canada V6T 1W5 ABSTRACT I n s o l u b i l i t y of gluten i n water and s a l t solutions l i m i t s i t s u t i l i z a t i o n i n the food industry. To overcome t h i s d i f f i c u l t y , gluten can be s o l u b i l i z e d by mild acid treatment (47). Gluten i n 0.02 N HCl was heated at 120°C for 30 minutes and water soluble protein f r a c t i o n was pre-c i p i t a t e d at pH 4.7. The remaining whey possessed about 9% of the o r i g i n a l protein i n the gluten. Experiments were carried out to i s o l a t e the pro-t e i n from whey and to assess i t s functional properties. Upon adjusting the whey to pH 1.0, about 44% of t o t a l N was precipitated, but the y i e l d was increased to 74% by heating the a c i d i f i e d whey to 90°C for 10 minutes. The addition of f e r r i c chloride (45 mg FeCl2"6H20 per l i t r e of whey with 0.4% protein) to the a c i d i f i e d whey before heat-ing increased the y i e l d to about 84%. Hexametaphosphate (HMP) (128 mg sodium hexametaphosphate per l i t r e of whey) re-duced the brown colour of the Fe-containing p r e c i p i t a t e . E l ution of HMP-Fe-protein through Sephadex G-200 column using 0.1 M phosphate buffer revealed four d i s t i n c t peaks. Molecular weights of peaks were calculated to be (I) > 100,000, (II) 62,000, (III) 29,000 and (IV) < 10,000. Ash content of f i n a l product was high (8.8%) due to use of s o d i u m h y d r o x i d e f o r n e u t r a l i z a t i o n . /Amino a c i d a n a l y s i s showed s i g n i f i c a n t l y l o w e r amount o f l y s i n e i n HMP-Fe-pro-t e i n c o m p a r e d t o u n h y d r o l y z e d g l u t e n . N i'inetyrjxkne p o i n t two. p e r c e n t N i n H M P - F e - p r o t e i n wa w a t e r s o l u b l e and h a d e x c e l l e n t e m u l s i f y i n g c a p a c i t y . Foama-b i l i t y was p o o r b u t i m p r o v e d g r e a t l y when l o w e r e d t o pH 5.0 b e f o r e w h i p p i n g . I s o e l e c t r i c p r e c i p i t a t e s a t pH 6.2 a n d pH 8.2 w e r e a l s o c o l l e c t e d f r o m t h e whey. A n o t h e r s a m p l e was p r e p a r e d by u l t r a f i l t r a t i o n o f t h e s u p e r n a t a n t a f t e r p r e c i p i t a t i o n a t pH 8.2. T h e s e t h r e e s a m p l e s w e r e c o m p a r e d w i t h H M P - F e - p r o t e i n c o m p l e x . The y i e l d o f a l l t h e t h r e e s a m p l e s was l o w e r b u t f u n c t i o n a l p r o p e r t i e s w e r e e q u a l t o o r b e t t e r a s c o m p a r e d t o H M P - F e - p r o t e i n . The a mino a c i d b a l a n c e was s i g n i f i c a n t l y b e t t e r i n t h e s e s a m p l e s . - i v -TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 4 M o d i f i c a t i o n o f G l u t e n 6 P r e p a r a t i o n o f F e r r i c Whey 8 EXPERIMENTAL M a t e r i a l s : 10 M e t h o d s o f P r e p a r a t i o n : P r e p a r a t i o n o f A c i d - M o d i f i e d G l u t e n Whey - 10 I s o l a t i o n o f P r o t e i n F r a c t i o n s f r o m A c i d - M o d i f i e d G l u t e n Whey A. T h r e e S t e p P r o c e d u r e 10 B. P r e p a r a t i o n o f H e x a m e t a p h o s p h a t e C o n t a i n i n g F e r r i c P r o t e i n (HMP-F e - p r o t e i n ) 14 M e t h o d s o f C h e m i c a l A n a l y s i s : N i t r o g e n D e t e r m i n a t i o n 14 M o i s t u r e D e t e r m i n a t i o n 16 A s h D e t e r m i n a t i o n 16 F a t D e t e r m i n a t i o n 16 I r o n D e t e r m i n a t i o n 17 P h o s p h o r u s D e t e r m i n a t i o n 17 C a r b o h y d r a t e D e t e r m i n a t i o n 17 N i n h y d r i n T e s t 18 C h l o r a n i l T e s t 18 - v -EXPERIMENTAL: Cont'd. P a g Amino Acid Analysis 19 Agarose Gel Electrophoresis 19 Gel F i l t r a t i o n of HMP-Fe-protein 20 S o l u b i l i t y C h a r a c t e r i s t i c s : pH-Solubility P r o f i l e of Acid-Modified Gluten Whey 20 S o l u b i l i t y of HMP-Fe-protein 21 Functional Properties: Emulsifying Capacity 21 Emulsion S t a b i l i t y 22 Foam Preparation 2 3 Foam S t a b i l i t y 23 RESULTS AND DISCUSSION: Ef f e c t of pH on Protein S o l u b i l i t y : A. Increase i n pH 24 B. Decrease i n pH 26 Ef f e c t of Heating on the A c i d i f i e d Gluten Whey 26 Ef f e c t of F e r r i c Chloride Addition to A c i d i f i e d Whey Before Heating 2 8 E f f e c t of Washing 29 Molecular Weight Determination of HMP-Fe-protein 32 Amino Acid Composition 34 Functional Properties of Gluten Whey Proteins Emulsifying Capacity 38 Emulsion S t a b i l i t y 40 - V I -P a g e F u n c t i o n a l P r o p e r t i e s o f G l u t e n Whey P r o t e i n s : C o n t ' d . F o a m i n g P r o p e r t i e s o f D i f f e r e n t P r o t e i n F r a c t i o n s 42 SUMMARY AND CONCLUSIONS REFERENCES 49 51 - v i i -L I S T OF TABLES TABLE Page I . E f f e c t o f h e a t on t h e p r o t e i n p r e c i p i t a t i o n o f a c i d i f i e d g l u t e n whey 27 I I . % N i t r o g e n r e c o v e r e d f r o m g l u t e n whey by d i f f e r e n t m e t h o d s 31 I I I . C h a r a c t e r i s t i c s o f H M P - F e - p r o t e i n 33 I V . Amino a c i d c o n t e n t o f d i f f e r e n t p r o t e i n f r a c t i o n s f r o m whey o f a c i d - m o d i f i e d g l u t e n 36 ) V. E m u l s i f y i n g c a p a c i t y and s t a b i l i t y o f d i f f e r e n t p r o t e i n f r a c t i o n s 39 V I . F o a m a b i l i t y a n d foam s t a b i l i t y o f d i f f e r e n t p r o t e i n f r a c t i o n s 43 V I I . E f f e c t o f c o n c e n t r a t i o n on f o a m a b i l i t y a n d foam s t a b i l i t y o f H M P - F e - p r o t e i n 45 V I I I . E f f e c t o f t e m p e r a t u r e on f o a m a b i l i t y a n d foam s t a b i l i t y on H M P - F e - p r o t e i n 4 6 I X . E f f e c t o f pH on f o a m a b i l i t y a n d foam s t a b i -l i t y o f H M P - F e - p r o t e i n 48 - V l l l -L I S T OF F I G U R E S F I G U R E P r e p a r a t i o n o f s o l u b i l i z e d g l u t e n S e p a r a t i o n o f d i f f e r e n t p r o t e i n f r a c -t i o n s f r o m a c i d - m o d i f i e d g l u t e n w h e y P r e p a r a t i o n o f H M P - F e - p r o t e i n E f f e c t o f p H o n t h e n i t r o g e n s o l u b i l i t y o f w h e y f r o m a c i d - m o d i f e d g l u t e n E f f e c t o f w a s h i n g s (A ) o n t h e r e m o v a l o f s o l u b l e s o l i d s (B ) o n t h e l o s s o f p r o t e i n G e l f i l t r a t i o n p a t t e r n o f H M P - F e - P r o t e i n o f S e p h a d e x G-200 u s i n g 0.1 M p h o s p h a t e b u f f e r ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude and appreciation to Dr. S. Nakai, Professor, Department of Food Science, University of B r i t i s h Columbia and Dr. W.D. Powrie, Chairman, Department of Food Science, University of B r i t i s h Columbia, who supervised t h i s study and provided encouragement and advice during the investigation and preparation of t h i s t h e s i s . Appreciation i s also extended to Dr. M.A. Tung, Department of Food Science for useful c r i t i c i s m and invaluable suggestions during the preparation of t h i s t h e s i s . Suggestions from fellow graduate students, es p e c i a l l y C.Y. Ma and C P . Fung, were greatly appreciated. F i n a l l y , my deep gratitude goes to my wife, Kashmir, without whose constant encouragement and u n f a i l i n g support, t h i s study would have been impossible. / 1. INTRODUCTION In spite of the r e l a t i v e l y high cost (per pound) of animal protein, the consumption of meat, eggs, and dairy products i s far greater than that of vegetable proteins. The d i s t i n c t preference for animal protein foods may be due to long-standing food preferences and to th e i r balance, of ess e n t i a l amino acids. It i s known that the vegetable pro-teins are d e f i c i e n t i n one or more e s s e n t i a l amino acids. During the l a s t two decades, oilseeds have att r a c -ted great i n t e r e s t as a pot e n t i a l source of protein for human consumption.- This i n t e r e s t i s due to serious protein deficiency i n parts of the world and to increasing cost of many food ingredients from animal sources (40; 22) . Oilseeds were p a r t i c u l a r l y well-adapted as a vegetable pro-t e i n source because of being a by-product of the edible o i l i n d ustries. The primary c r i t e r i o n for foods designed to a l l e v -i ate hunger i n developing countries i s maximum n u t r i t i o n at the minimum cost. However, foods designed to compete on a more sophisticated market must possess functional character-i s t i c s , aesthetic q u a l i t y and moderate cost comparable to conventional products (41). Oilseed protein concentrates and i s o l a t e s have been used i n meat emulsion 'formulations 2. as well as for o v e r a l l cost reduction i n the meat indus-t r y for many years (38). Oilseed proteins have also been used as aerating agents i n whipped toppings and frozen desserts (46) . They are also used i n confections such as nougats and marshmallow, and as p a r t i a l replacement of egg whites i n cake formulations (22). Wheat proteins i n the form of f l o u r have been used i n the baking industry since ancient times. The r e l a t i v e abundance and low cost of wheat gluten focuses attention on the possible use of t h i s protein i n d i f f e r e n t food products other than bread. The main drawback i n i t s wider food applications i s i t s i n s o l u b i l i t y i n aqueous solution. To overcome t h i s d i f f i c u l t y , attempts have been made to s o l u b i l i z e gluten with mild acid hydrolysis. This method has been quite successful without a f f e c t i n g the protein structure extensively (47). Protein i n acid-treated gluten was recovered 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 at pH 4.7. The supernatant, aft e r centrifugation, s t i l l included 8 to 9% of the o r i g i n a l gluten. From the aspect of the economics and p r a c t i c a l use of t h i s method i n i n d u s t r i a l manufacturing, i t i s neces-sary to recover most of the protein. The objectives of th i s study were: 1. To recover the proteins l e f t i n the whey aft e r preparation of acid-treated gluten. 2. To investigate the functional properties of the recovered whey protein f r a c t i o n . 3. Foamability and emulsification properties of the recovered protein f r a c t i o n s were studied. 4. LITERATURE REVIEW I n 1745 (26) i t was r e c o r d e d t h a t B e c c a r i , P r o -f e s s o r o f M e d i c i n e i n t h e Anatomy and C h e m i s t r y I n s t i t u t e o f B o l o g n a , I t a l y , f o r m e d a d o u g h f r o m w h e a t f l o u r a n d i s o l a t e d g l u t e n b y w a s h i n g t h i s w i t h w a t e r . T h i s was t h e e a r l i e s t r e c o r d i n t h e l i t e r a t u r e on g l u t e n a n d t h e f l o u r p r o t e i n s . Wheat f l o u r c o n t a i n s 5 - 15% p r o t e i n b y w e i g h t o f t o t a l s o l i d s . The t o t a l p r o t e i n s i n f l o u r h a v e b e e n r o u g h l y d i v i d e d i n t o two f r a c t i o n s , t h e w a t e r - s o l u b l e f r a c t i o n a n d t h e w a t e r - i n s o l u b l e f r a c t i o n . The w a t e r -i n s o l u b l e f r a c t i o n i s known a s g l u t e n . G l u t e n c a n be s e p a r a t e d f r o m w h e a t f l o u r b y m i x i n g t h e f l o u r w i t h a b o u t a n e q u a l amount o f w a t e r t o f o r m an e l a s t i c d o u g h a n d s u b s e q u e n t l y w a s h i n g o u t t h e s t a r c h g r a n u l e s t o f o r m an 8 0 % - f l o u r f r a c t i o n . I n 1 8 2 0 , T a d d e i (5) s e p a r a t e d g l u t e n i n t o two - f r a c -t i o n s b y e x t r a c t i o n w i t h a l c o h o l , n a m e l y a n a l c o h o l - s o l u b l e f r a c t i o n a n d a n a l c o h o l - i n s o l u b l e f r a c t i o n . He g a v e t h e name g l i a d i n e t o t h e a l c o h o l - s o l u b l e f r a c t i o n a n d zimome t o t h e . f r a c t i o n n o t s o l u b l e i n a l c o h o l . I n h i s m o n o g r a p h i n 1907, O s b o r n e (24) u s e d t h e t e r m g l i a d i n f o r t h e a l c o h o l - s o l u b l e f r a c t i o n and g l u t e n i n 5. for the alcohol-soluble f r a c t i o n , which i s soluble i n an alkaline solution. The fractions separated by the alcohol extraction technique are i n roughly equal amounts. Amino acid compositions of glutenin and g l i a -din d i f f e r s l i g h t l y e s p e c i a l l y in glutamic acid (present largely as glutamine) and proline. The amount of gluta-mic acid and proline i s r e l a t i v e l y high i n both fractions, glutamine being about one i n every three and proline about one i n every seven amino acid residues (48, 49). Low contents of ionizable amino acid residues, e.g. lys i n e , h i s t i d i n e and arginine i n addition to r e s t r i c t e d i o n i z a t i o n of glutamic acid and arginine are responsible for the low water s o l u b i l i t y of gluten proteins (7, 37). Gliadin and glutenin d i f f e r i n t h e i r physical properites, most notably in t h e i r v i s c o e l a s t i c i t y . Hydrated g l i a d i n i s cohesive, but with low e l a s t i c i t y , whereas hydrated glutenin i s both cohesive and e l a s t i c (12). Gliadin has a molecular weight of 25,000 to 100,000 whereas the glutenin f r a c t i o n has a molecular*, weight of more than 10 (11). Gliadins have larger amounts of pro-l i n e , glutamic acid, cystine, isoleucine, phenylalanine and amide nitrogen than glutenin, whereas glutenin has larger amounts of glycine, lysine and tryptophan (15). 6. M o d i f i c a t i o n o f G l u t e n A s g l u t e n i s i n s o l u b l e i n w a t e r , m e t h o d s h a v e b e e n u s e d t o m o d i f y i t t o i n c r e a s e i t s s o l u b i l i t y i n a q u e o u s s o l u t i o n . Once i t i s s o l u b l e i n w a t e r / i t c a n be u s e d i n p r e p a r a t i o n o f d r i n k s , e m u l s i o n s a n d f o a m s . Use o f m o d i f i e d w h e a t g l u t e n i n f o o d foams was s t a r t e d due t o r e l a t i v e a b u n d a n c e a n d l o w c o s t o f g l u t e n ( 3 7 ) . K r u l l a n d W a l l (29) i n 1966 p r o v e d t h a t a m i d e g r o u p s o f g l u t e n p r o t e i n s p l a y an i m p o r t a n t r o l e i n t h e a g g r e g a t i o n o f g l u t e n p r o t e i n s t h r o u g h h y d r o g e n b o n d i n g . Holme a n d B r i g g s (20) i n 1959 h y d r o l y z e d t h e a m i d e g r o u p s o f g l u t a m i n e r e s i d u e s i n g l i a d i n w i t h 0.008 t o 0.04 N H C l . S u c h t r e a t m e n t i n c r e a s e d t h e s o l u b i l i t y o f t h e p r o t e i n i n w a t e r . I n 1 9 6 3 , B e c k w i t h e t a l . (6) w e r e a b l e , t o r e d u c e c o h e s i v e n e s s o f g l u t e n by m e t h o x y l a t i o n o f a m i d e g r o u p s . The p r o d u c t was s o l u b l e i n a q u e o u s m e t h a n o l . L a s z t i t y (30) i n 1969 m o d i f i e d g l u t e n b y d e a m i -d a t i o n . , m e t h y l a t i o n , e s t e r i f i c a t i o n a n d a c y l a t i o n a n d b y b l o c k i n g o f t h i o l g r o u p s w i t h N - e t h y l m a l e i m i d e a n d s t u d i e d i t s i m p o r t a n t r h e o l o g i c a l p r o p e r t i e s . I n t e r f e r e n c e o f a m i d e g r o u p s a n d a c y l a t i o n o f p r i m a r y a m i n o g r o u p s w e r e r e p o r t e d t o c a u s e r a d i c a l c h a n g e s i n t h e r h e o l o g i c a l b e h a v i o u r o f g l u t e n . G r a n t (18) i n 1973 m o d i f i e d w h e a t f l o u r w i t h s u c -c i n i c a n h y d r i d e s u s p e n d e d i n a q u e o u s d i o x a n e . T h i s t r e a t -7. ment was a b l e t o s o l u b . i l i . z e .95 % o f t h e p r o t e i n f r a c t i o n o f t h e w h e a t f l o u r . M c D o n a l d a n d P e n c e (33) r e p o r t e d t h a t d e a m i -d i z e d g l i a d i n i s c o m p a t i b l e w i t h egg w h i t e . T hey p r e -p a r e d d e a m i d i z e d g l i a d i n b y d i s p e r s i n g g l i a d i n i n 0.07 N HC1 a t a c o n c e n t r a t i o n o f 5.5% and h e a t i n g f o r 3 h o u r s a t 96-98°C. D e a m i d i z e d g l i a d i n a l o n e o r a s a m i x t u r e w i t h egg w h i t e , made e x c e l l e n t m e r i n g u e s h e l l s , d i v i n i t y c a n d y , f l u f f y c a k e i c i n g s a n d s t a b i l i z e d b u t t e r c r e a m c a k e i c i n g s . Gagen a n d Holme (17) d e a m i d i z e d g l u t e n by h y d r o -l y s i s o f g l u t e n u n d e r r e f l u x u s i n g 0.15 t o 1.0 N h y d r o -c h l o r i c a c i d . P r o t e i n s o l u b i l i t y i n c r e a s e d w i t h h y d r o -l y s i s o f g l u t e n . P o o r f u n c t i o n a l p r o p e r t i e s ( i . e . f o a m a b i l i t y , w h i p p a b i l i t y a n d b a k i n g ) o f h y d r o l y z e d g l u t e n w e r e r e p o r t e d a s c o m p a r e d t o u n h y d r o l y z e d g l u t e n . Wu (47) p r e p a r e d s o l u b l e g l u t e n u s i n g 0.02 N t o 0.5 N h y d r o c h l o r i c a c i d a n d 1.75 t o 8.75 N a c e t i c a c i d a t d i f f e r e n t - t e m p e r a t u r e s (100-121°C) f o r . d i f f e r e n t l e n g t h s o f t i m e . She o b s e r v e d t h e f o l l o w i n g r e s u l t s : 1. 8 5 - 9 2 % o f o r i g i n a l g l u t e n was o b t a i n e d i n s o l u b i l i z e d f o r m a f t e r h y d r o l y s i s a n d d i a l y s i s and 7 0 - 78% 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 a f t e r h y d r o l y s i s . 2. G l u t e n was s o l u b i l i z e d t o a l e v e l o f d e a m i d a -t i o n a s l o w a s 1 0 % . 8. 3. The amount o f u n h y d r o l y z e d a m i d e n i t r o g e n i n t h e t r e a t e d s a m p l e s was d e p e n d e n t m a i n l y o n t h e c o n -c e n t r a t i o n o f a c i d u s e d . 4. M o l e c u l a r w e i g h t o f h y d r o l y s a t e d e c r e a s e d w i t h i n c r e a s e i n a c i d c o n c e n t r a t i o n . 5. A c i d m o d i f i c a t i o n o f g l u t e n r e m a r k a b l y i m p r o v e d t h e e m u l s i f y i n g c a p a c i t y a n d e m u l s i f y i n g s t a b i l i t y . P r e p a r a t i o n o f F e r r i c Whey B l o c k e t a l . (8) i n 1953 m a n u f a c t u r e d f e r r i l a c t i n f r o m c h e e s e whey and pH a d j u s t m e n t f o l l o w e d b y f r e e z i n g i t t o c r y s t a l l i z e t h e p r o t e i n and t h e n f i l t e r e d i t . I t was m o d i f i e d b y Imado e t a l . (21), w h e r e t h e y u t i l i z e d w h o l e whey w i t h o u t s e p a r a t i o n o f f e r r i l a c t i n a f t e r t h e a d d i t i o n o f f e r r i c c h l o r i d e . P o l y p h o s p h a t e was a d d e d t o p r e v e n t t h e b r o w n c o l o r o f t h e p r o d u c t due t o f e r r i c i o n s . S t o r -age s t u d i e s o f m i l k f o r t i f i e d w i t h t h i s p r o d u c t showed no v i t a m i n d e s t r u c t i o n o r f l a v o r d e t e r i o r a t i o n . J o n e s e t a _ l . (23) i n 19 72 u s e d " F e r r i - p o l y p h o s p h a t e " t o p r e c i p i t a t e whey p r o t e i n s a t pH 3.2 - 4.0. F e r r i -p h o s p h a t e i s a s o l u b l e c o m p l e x o f f e r r i c i o n s w i t h a l o n g c h a i n p o l y p h o s p h a t e . The l y o p h i l i z e d whey p r o d u c t was w h i t e , f l u f f y and h a d a m i l d a c i d i c f l a v o r . The p r o d u c t h a d l o w p r o t e i n c o n t e n t (22%) w i t h c o n s i d e r a b l y h i g h c o n -t e n t o f p h o s p h a t e (39% ~P00K) . H i g h p h o s p h a t e c o n t e n t i s 9. u n d e s i r a b l e as i t may d i s t u r b t h e k i d n e y f u n c t i o n e s p e c -i a l l y i n b a b i e s . A m a n t e a e t a _ l . (2) i n 1974 p r e p a r e d a s o l u b l e f e r r i c whey p r o t e i n c o n t a i n i n g more t h a n 80% p r o t e i n b y an i m p r o v e d h e a t i n g m e thod u s i n g an a c i d pH. Whey was c o n c e n t r a t e d 3 t o 4 t i m e s a n d h e a t e d t o 92°C f o r 15 m i n u t e s a f t e r : a d d i n g F e C l ^ a n d a d j u s t i n g t o pH 2.5. A f t e r c e n t r i f u g a t i o n , t h e s e d i m e n t was washed w i t h d i l u t e HC1 a t pH 2.5, d i s s o l v e d a n d d r i e d . H i g h s o l u b i l i t y o f t h e d r i e d p r o t e i n was o b t a i n e d b y a c i d w a s h i n g o f t h e p r e c i p i t a t e b y t h e method o f H a r w a l k e r and Emmons (19). I r o n i n f e r r i c whey p r o t e i n was a v a i l a b l e t o g r o w i n g c h i c k s . 10. EXPERIMENTAL M a t e r i a l s 1. V i t a l g l u t e n u s e d f o r t h e e x p e r i m e n t s was p r o v i d e d b y R e s e a r c h L a b o r a t o r y o f J o h n L a b a t t , L t d . , M o n t r e a l . 2. A l l t h e r e a g e n t s w e r e a n a l y t i c a l g r a d e . 3. C o r n o i l u s e d f o r e m u l s i f y i n g c a p a c i t y a n d s t a b i l i t y was l a b o r a t o r y g r a d e p u r c h a s e d f r o m F i s h e r S c i e n t i f i c Co. L t d . M e t h o d s o f P r e p a r a t i o n P r e p a r a t i o n o f A c i d - M o d i f i e d G l u t e n Whey: Whey u s e d i n t h e e x p e r i m e n t s was p r e p a r e d b y t h e me t h o d o f Wu (47) a s f o l l o w s : 5% s o l u t i o n o f v i t a l g l u t e n i n 0 . 0 2 N H C 1 was a u t o -c l a v e d a t 121°C (15 p s i g ) f o r 30 m i n u t e s . The pH o f t h e h y d r o l y s a t e was a d j u s t e d t o 4.7 t o p r e c i p i t a t e t h e a c i d -m o d i f i e d g l u t e n . The s u p e r n a t a n t (whey) o b t a i n e d a f t e r c e n t r i f u g a t i o n o f t h e m i x t u r e a t 10,000 x g f o r 10 m i n u t e s was u s e d ' f o r t h i s s t u d y ( F i g . 1 ) . I s o l a t i o n o f P r o t e i n F r a c t i o n s f r o m A c i d - M o d i f i e d G l u t e n Whey: A. T h r e e S t e p P r o c e d u r e : U s i n g minimum s o l u b i l i t y p o i n t s , t h e f o l l o w i n g p r o c e d u r e was a d o p t e d ( F i g . 2 ) : 11, 5% g l u t e n s u s p e n s i o n i n 0.02 N H C l A u t o c l a v e a t 121 C f o r 30 m i n u t e s H y d r o l y s a t e A d j u s t t o pH C e n t r i f u g e p p t S u s p e n d i n w a t e r a n d a d j u s t t o pH 7.5 C e n t r i f u g e S u p e r n a t a n t (whey) p p t S u p e r n a t a n t f r e e z e d r y S o l u b i l i z e d g l u t e n F i g u r e 1: P r e p a r a t i o n o f s o l u b i l i z e d g l u t e n 12. ;Whey + 1 N NaOH t o pH 6.2 pH 6.2 p p t S u p e r n a t a n t + I N NaOH t o pH 8.2 pH 8.2 p p t S u p e r n a t a n t U l t r a f i l t r a t i o n o n PM10 a t 40 l b s p r e s s u r e C o n c e n t r a t e P e r m e a t e (U.F.) F i g u r e 2: S e p a r a t i o n o f d i f f e r e n t p r o t e i n f r a c t i o n s f r o m a c i d - m o d i f i e d g l u t e n whey A. T h r e e S t e p P r o c e d u r e : ( c o n t ' d ) S t e p I : Whey was a d j u s t e d t o pH 6.2 and t h e p r e c i p i t a t e was r e m o v e d b y c e n t r i f u g a t i o n a t 4,000 x g . The p r e -c i p i t a t e was w a s h e d w i t h d i s t i l l e d w a t e r a nd f r e e z e -d r i e d . S t e p I I : The s u p e r n a t a n t f r o m S t e p I was a d j u s t e d t o pH 8.2 a n d c e n t r i f u g e d a t 4,000 x g t o remove t h e p r e -c i p i t a t e . T h e * p r e c i p i t a t e was washed w i t h d i s t i l l e d w a t e r and f r e e z e - d r i e d . S t e p I I I : U l t r a - f i l t r a t i o n was u s e d t o r e c o v e r t h e p r o t e i n i n t h e s u p e r n a t a n t a f t e r S t e p I I . U l t r a - f i l t r a t i o n was c a r r i e d o u t o n a s m a l l l a b o r a t o r y - s c a l e a p p a r a t u s ( M o d e l 5 2 , A m i c o n C o r p o r a t i o n , L e x i n g t o n , Ky.,U.S.A.) h a v i n g t h e maximum i n t e r n a l v o l u m e o f 65 m l . I t , h a s a c y l i n d r i c a l c o n t a i n e r w i t h h o r i z o n t a l p o r o u s s u p p o r t i n g d i s c f o r c e l l u l o s e a c e t a t e membrane u s e d f o r f i l t r a t i o n o f t h e s o l u t i o n . S o l u t i o n was c o n t i n u o u s l y s t i r r e d i m m e d i a t e l y a b o v e t h e s u p p o r t i n g d i s c . The c y l i n - -d e r was c o n n e c t e d t o a f i b r e g l a s s l i q u i d r e s e r v o i r ( M o d e l RG-3, A m i c o n C o r p o r a t i o n , L e x i n g t o n , K y . , U.S.A.) w h i c h s u p p l i e d l i q u i d t o t h e u l t r a - f i l t r a t i o n c e l l . The c e l l u l o s e a c e t a t e membrane u s e d i n t h i s e x -p e r i m e n t was t y p e PM 10 (a c u t o f f m o l e c u l a r w e i g h t o f 1 0 , 0 0 0 ) . The membrane was c o n d i t i o n e d b e f o r e u s e t o remove a d d e d g l y c e r i n e . I t was d o n e by r i n s i n g t h e mem-b r a n e i n a b e a k e r o f d i s t i l l e d w a t e r f o r one h o u r w i t h one c h a n g e . A p r e s s u r e o f 4 0 p s i g w a s a p p l i e d f r o m an a i r c y l i n d e r . T e m p e r a t u r e s u s e d f o r t h e e x p e r i m e n t w e r e 25°C and 4°C. B. P r e p a r a t i o n o f H e x a m e t a p h o s p h a t e C o n t a i n i n g F e r r i c P r o t e i n ( H M P - F e - p r o t e i n ) : A m a n t e a m e t h o d (2) was u s e d w i t h some m o d i f i -c a t i o n s . The p r o c e d u r e was a s f o l l o w s : One l i t r e o f g l u t e n whey s t d r r e d ^ - w i t h 0.15 ml o f 30% f e r r i c c h l o r i d e s o l u t i o n , was a d j u s t e d t o pH 1.0 w i t h H C l a n d was h e a t e d a t 9 0°C f o r 10 m i n u t e s . A f t e r c o o l i n g to„25°C, t h e p r e c i p i t a t e was r e m o v e d b y c e n t r i f u -g a t i o n a t 4,000 x g . F e - P r o t e i n p r e c i p i t a t e was w a s h e d w i t h 20 v o l u m e s o f w a t e r a t pH 1.0, r e d i s s o l v e d a t pH 7.0 and f r e e z e - d r i e d . S o d i u m h e x a m e t a p h o s p h a t e was a d d e d a t t h e r a t e o f 128 m g / l i t r e o f g l u t e n whey j u s t b e f o r e d r y i n g ( F i g . 3 ) . M e t h o d s o f C h e m i c a l A n a l y s i s N i t r o g e n D e t e r m i n a t i o n : F o r r o u t i n e w o r k , n i t r o g e n was d e t e r m i n e d b y an i n d o p h e n o l m e t h o d ( 3 5 ) . 0.5 m l o f s a m p l e s o l u t i o n was d i g e s t e d w i t h 0.3 m l o f t h e d i g e s t i o n m i x t u r e c o n t a i n i n g Whey + 9.3 mg Fe/1 Adjust to pH 1.0 Heat at 9 0°C for 10 min. Cool Centrifuge Fe-protein ppt Wash at pH 1.0 and dissolve at pH 7.0 Centrifuge Supernatant Discard ppt I scard Supernatant + 128 mg of Sod. Hexametaphosphate/1 Freeze dry HMP-Fe-protein Figure 3: Preparation of HMP-Fe-protein 100 m l o f 6.7 N H^SO^, 4 g p o t a s s i u m s u l p h a t e , 1 d r o p o f S e 0 3 C l a n d 9 0mg C u S O 4 ' 5 H 2 0 . A f t e r c o o l i n g , t h e i n s i d e o f a t e s t t u b e was w a s h e d w i t h 2 m l w a t e r . 1.6 m l o f 5% p h e n o l f o l l o w e d b y 1 m l o f 2 N NaOH w e r e a d d e d t o t h e d i g e s t e d s a m p l e w i t h t h o r o u g h m i x i n g . The v o l u m e was made up t o 10 m l w i t h c a r b o n a t e b u f f e r , (pH 9.8) and 1.6 m l o f s o d i u m h y p o c h l o r i t e was m i x e d a nd a l l o w e d t o s t a n d f o r 30 m i n u t e s . The c o l o u r d e v e l o p e d i n s o l u t i o n was m e a s u r e d a t 610 nm. N i t r o g e n c o n c e n t r a t i o n i n t h e s a m p l e was r e a d f r o m t h e s t a n d a r d c u r v e d r a w n w i t h d i f f e r e n t c o n c e n t r a t i o n s o f ammonium s u l p h a t e a n d a b s o r b a n c e . A m i c r o - K j e l d a h l m e thod (4) was u s e d a s t h e s t a n d a r d m e t h o d . A f a c t o r o f 5.7 was u s e d t o c a l c u l a t e p r o t e i n f r o m t o t a l n i t r o g e n . M o i s t u r e D e t e r m i n a t i o n : M o i s t u r e was d e t e r m i n e d a f t e r h e a t i n g t h e s a m p l a t 98°C u n t i l i t r e a c h e d t h e c o n s t a n t w e i g h t . A s h D e t e r m i n a t i o n : A s h c o n t e n t was d e t e r m i n e d b y a s h i n g t h e s a m p l e a t 600°C i n a m u f f l e f u r n a c e f o r s i x h o u r s ( 3 ) . F a t D e t e r m i n a t i o n : F a t . c o n t e n t was d e t e r m i n e d b y m e t h o d o f T s e n e t ( 4 5 ) . The s o l v e n t s y s t e m was a m i x t u r e o f m e t h y l a l c o h o l c h l o r o f o r m a n d w a t e r ( 2 : 1: 0.8 v / v / v ) . W a t e r r e q u i r e d was c a l c u l a t e d i n c l u d i n g t h e w a t e r p r e s e n t i n t h e s a m p l e . A f t e r b l e n d i n g t h e s a m p l e w i t h t h e s o l v e n t m i x -t u r e , t h e m i x t u r e was c e n t r i f u g e d a n d c h l o r o f o r m l a y e r was r e c o v e r e d , f i l t e r e d , e v a p o r a t e d u n d e r n i t r o g e n a n d d r i e d i n a vacuum o v e n a t 50°C t o c o n s t a n t w e i g h t . I r o n D e t e r m i n a t i o n : I r o n was d e t e r m i n e d b y u s i n g t h e method o f C a r t e r ( 9 ) . A f t e r d e p r o t e i n i z a t i o n , c o l o u r was d e v e l o p e d w i t h f e r r o z i n e and m e a s u r e d a t 562 nm a g a i n s t w a t e r i n a Beckman DB s p e c t r o p h o t o m e t e r . P h o s p h o r u s D e t e r m i n a t i o n : P h o s p h o r u s was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a f t e r w e t a s h i n g u s i n g s u l p h u r i c a c i d ( 3 4 ) . A 0.2 m l o f 0.5% s a m p l e s o l u t i o n was d i g e s t e d w i t h s u l p h u r i c a c i d and h y d r o g e n p e r o x i d e , c o o l e d a n d d i l u t e d w i t h w a t e r t o 3 m l . A f t e r r e d u c t i o n w i t h s o d i u m s u l p h a t e , p h o s p h o m o l y b -d i c a c i d . .. c o m p l e x was f o r m e d b y a d d i n g ammonium p a r a -m o l y b d a t e s o l u t i o n . B l u e c o l o u r was p r o d u c e d a f t e r r e d u c -t i o n w i t h a s c o r b i c a c i d a n d was m e a s u r e d a t 822 nm i n a B a u s c h a n d Lomb S p e c t r o n i c 20 c o l o r i m e t e r . C o n c e n t r a t i o n o f p h o s p h o r u s i n t h e s a m p l e was r e a d f r o m t h e s t a n d a r d c u r v e p r e p a r e d w i t h s t a n d a r d monosodium p h o s p h a t e . C a r b o h y d r a t e Determination: C a r b o h y d r a t e s i n t h e H M P - F e - p r o t e i n w e r e d e t e r m i n e d 18. by t h e method o f D u b o i s e t a l . ( 1 3 ) . One m l o f 1% p r o -t e i n s o l u t i o n i n w a t e r was m i x e d w i t h 1.0 m l o f 5% a q u e o u s p h e n o l s o l u t i o n . F i v e m l o f c o n e . I^SO^ w e r e a d d e d d i r e c t l y i n t h e t e s t t u b e c o n t a i n i n g s a m p l e and p h e n o l . A f t e r 10 m i n u t e s , a b s o r b a n c e was m e a s u r e d a t 480 nm o n a Beckman DB s p e c t r o p h o t o m e t e r . C a r b o h y d r a t e c o n t e n t was r e a d f r o m t h e s t a n d a r d c u r v e p r e p a r e d w i t h s t a n d a r d g l u c o s e s o l u t i o n ' s . N i n h y d r i n T e s t : The N i n h y d r i n t e s t was done t o c h e c k t h e amino a c i d c o n t e n t o f f i n a l p e ak e l u t e d f r o m t h e S e p h a d e x G-200 c o l u m n w i t h 0.1 M p h o s p h a t e b u f f e r . N i n h y d r i n r e a g e n t was p r e p a r e d b y d i s s o l v i n g 1 g o f n i n h y d r i n and 0.15 g o f h y d r i n d a t i n i n 37.5 m l m e t h y l c e l l o s o l v e . T w e l v e a n d o n e -h a l f m l o f a c e t a t e b u f f e r w e r e a d d e d t o a d j u s t t h e pH t o 5.5; One m l o f n i n h y d r i n r e a g e n t was m i x e d w i t h 1.0 m l o f s a m p l e , s o l u t i o n , s h a k e n and h e a t e d i n a b o i l i n g w a t e r b a t h f o r 15 m i n u t e s . Then i t was d i l u t e d w i t h 3 m l o f e t h a n o l ( 5 0 : 5 0 ) , s h a k e n a n d t h e a b s o r b a n c e r e a d a t 570 nm on a Beckman DB S p e c t r o p h o t o m e t e r a g a i n s t b l a n k p r e p a r e d i n t h e same way w i t h o u t s a m p l e . C h l o r a n i l T e s t : T h i s t e s t was a l s o u s e d t o d e t e r m i n e t h e a m i n o a c i d s a n d s m a l l p e p t i d e s i n t h e l a s t p e ak e l u t e d f r o m t h e S e p h a d e x G-200 c o l u m n w i t h 0.1 M p h o s p h a t e b u f f e r . To 1 m l o f e a c h f r a c t i o n , 2 m l c h l o r a n i l s o l u t i o n , 2 m l o f b o r a t e b u f f e r (pH 9.0) a n d 5 m l w a t e r w e r e a d d e d . C o l o u r was d e v e l o p e d b y h e a t i n g i n a w a t e r b a t h a t 65il°C f o r one h o u r . A b s o r b a n c e was m e a s u r e d a t 350 nm i n 1 cm c e l l i n a Beckman DB S p e c t r o p h o t o m e t e r a g a i n s t b l a n k p r e -p a r e d i n t h e same way w i t h o u t ' s a m p l e . Amino A c i d A n a l y s i s A f r e e z e - d r i e d s a m p l e was h y d r o l y z e d w i t h p - t o l u e n e s u l f o n i c a c i d a n d 3 - ( 2 - a m i n o e t h y l ) i n d o l e a t 110°C f o r 24 h o u r s (31) a f t e r c y s t e i n b l o c k i n g b y p y r i d y l e t h y l a t i o n ( 1 0 ) . Amino a c i d s w e r e d e t e r m i n e d on a s i n g l e c o l u m n s y s -tem (Durrum Chem. C o r p . , P a l o A l t o , C a l i f . ) a t t a c h e d t o P h o e n i x Amino A c i d A n a l y z e r M o d e l M 6800. A g a r o s e G e l E l e c t r o p h o r e s i s F i f t y g o f u r e a was d i s s o l v e d i n 200 m l o f b a r b i t a l b u f f e r , pH 8.6, 0.05 M w i t h 0.035% EDTA a n d 0.6 m l o f 2-m e r c a p t o e t h a n o l was a d d e d t o i t . T h i s s o l u t i o n was u s e d f o r e q u i l i b r a t i o n o f a g a r o s e g e l and s a m p l e p r e p a r a t i o n . E q u i l i b r a t i o n o f g e l was done by s o a k i n g t h e p l a t e i n t h i s s o l u t i o n f o r 30 m i n u t e s . T h r e e p e r c e n t s a m p l e s o l u t i o n was p r e p a r e d i n t h e s o l u t i o n - . ^ d e s c r i b e d a b o v e . One p i o f s a m p l e s o l u t i o n was a p p l i e d t o t h e a g a r o s e f i l m s l o t w i t h a m i c r o l i t r e d i s p e n s e r w i t h d i s p o s a b l e t i p . E l e c t r o p h o r e s i s was c a r r i e d o u t f o r one h o u r i n t h e A C I c a s s e t t e e l e c t r o p h o r e s i s c e l l and t h e 20. power s u p p l y u n i t a t 4°C u s i n g t h e same b u f f e r s o l u t i o n . P r o t e i n b a n d s w e r e s t a i n e d b y i m m e r s i n g t h e f i l m i n 1% amid o b l a c k 10 B s o l u t i o n i n 5% a c e t i c a c i d f o r 5 m i n u t e s . E x c e s s d y e was washed w i t h 5% a c e t i c a c i d u n t i l a c l e a r b a c k g r o u n d was o b t a i n e d w h i c h u s u a l l y t o o k one m i n u t e o r l e s s . Then f i l m was d r i e d i n a c o n v e c t i o n o v e n a t 75-80°C. G e l F i l t r a t i o n o f H M P - F e - p r o t e i n H M P - F e - p r o t e i n was d i s s o l v e d i n 0.1 M p h o s p h a t e b u f f e r . T h r e e m l o f s a m p l e s o l u t i o n c o n t a i n i n g 200 mg o f p r o t e i n was a p p l i e d t o a 2.5 x 80 cm c o l u m n p a c k e d w i t h S e p h a d e x G-200. I t was e l u t e d w i t h 0.1 M p h o s p h a t e b u f f e r a t pH 7.4 i n t h e u p w a r d d i r e c t i o n . The t o t a l v o l u m e v/as 450 m l a n d t h e f l o w r a t e was 25 m l p e r h o u r . The e l u t i o n p r o f i l e was o b t a i n e d b y r e a d i n g t h e a b s o r b a n c e o f e a c h f r a c t i o n a t 280 nm. The f r a c t i o n v o l u m e o f 3 m l o r 4 m l was u s e d . The c o l u m n was p r e - s t a n d a r d i z e d w i t h t h e s t a n d a r d p r o t e i n s o l u t i o n s t o r e a d t h e m o l e c u l a r w e i g h t o f e l u t i o n p e a k s ( 3 2 ) . S o l u b i l i t y C h a r a c t e r i s t i c s p H - S o l u b i l i t y , P r o f i l e o f A c i d - M o d i f i e d G l u t e n Whey: N i t r o g e n s o l u b i l i t y as a f u n c t i o n o f pH i n whey was d e t e r m i n e d b y t h e method o f Kodagoda e t a l . (28) w i t h some m o d i f i c a t i o n . T w e n t y - t w o m l o f whey was a d j u s t e d f r o m pH 1.0 t o pH 10.7 u s i n g 1.0 N HC1 o r 1.0 N NaOH a t a pH i n t e r v a l o f 0.5, a n d t h e t o t a l v o l u m e was made up t o 25 m l w i t h w a t e r . A f t e r c e n t r i f u g a t i o n a t 16,000 x g f o r 20 m i n u t e s , n i t r o g e n i n t h e s u p e r n a t a n t was d e t e r m i n e d a n d p l o t t e d a g a i n s t pH. S o l u b i l i t y o f H M P - F e - p r o t e i n : A 500 mg s a m p l e was b l e n d e d i n 50 m l w a t e r a t 25°C i n a L o u r d e s M o d e l MM-LA M u l t i m i x e r ( L o u r d e s I n s t r u -ment C o r p o r a t i o n , New Y o r k ) a t a s p e e d s e t t i n g o f 10. A f t e r c e n t r i f u g a t i o n a t 2000 x g f o r 20 m i n u t e s , n i t r o g e n i n t h e s u p e r n a t e n t was d e t e r m i n e d . S o l u b i l i t y was e x p r e s s e d as a p e r c e n t a g e o f t o t a l n i t r o g e n i n t h e s a m p l e . F u n c t i o n a l P r o p e r t i e s ; E m u l s i f y i n g C a p a c i t y : E m u l s i f i c a t i o n c a p a c i t y o f t h e p r o t e i n - s a m p l e -was d e t e r m i n e d by t h e m e t h o d o f S w i f t e t a l . (42) w i t h some m o d i f i c a t i o n s . I n a one p i n t w i d e - m o u t h e d mason j a r , 100 mg o f s a m p l e was d i s s o l v e d i n 30 m l o f d i s t i l l e d w a t e r . Then 10 ml o f c o r n o i l was a d d e d . I t was b l e n d e d i n an omni m i x e r a t 8,600 rpm. More o i l was ad d e d c o n t i n u o u s l y a t a r a t e o f 10 m l p e r m i n u t e f r o m a b u r e t . A v i s c o u s o i l - i n - w a t e r t y p e e m u l s i o n was f o r m e d . A f t e r some t i m e , t h e v i s c o s i t y d e -c r e a s e d a b r u p t l y , i n d i c a t i n g t h e e m u l s i o n b r e a k a g e p o i n t . A t t h i s p o i n t , a d d i t i o n o f o i l was t e r m i n a t e d i m m e d i a t e l y . T o t a l amount o f o i l a d d e d was r e c o r d e d . E m u l s i f i c a t i o n was r e p o r t e d as t h e m l o i l p e r 100 mg o f p r o t e i n s a m p l e . A l l e x p e r i m e n t s w e re done a t pH 7.0. S a m p l e s w e r e k e p t i n i c e d u r i n g b l e n d i n g t o p r e v e n t t e m p e r a t u r e i n c r e a s e , b y h i g h s p e e d m i x i n g o p e r a t i o n s . E m u l s i o n S t a b i l i t y : The p r o c e d u r e u s e d f o r d e t e r m i n a t i o n o f e m u l s i o n s t a b i l i t y was s i m i l a r t o t h a t o f P e a r s o n e t a l . ( 3 6 ) . C o r n o i l , 5% l e s s t h a n t h e e m u l s i f i c a t i o n c a p a c i t y , was b l e n d e d w i t h 100 mg o f a p r o t e i n s a m p l e i n 30 m l w a t e r f o r 2 m i n u t e s a t 8600 rpm i n an Omni M i x e r . E a c h e m u l s i o n was t h e n t r a n s f e r r e d t o a g r a d u a t e d c y l i n d e r and a l l o w e d t o s t a n d a t 25°C. E m u l s i o n s t a b i l i t y was r e p o r t e d as t h e t i m e r e q u i r e d f o r p h a s e s e p a r a t i o n o f t h e e m u l s i o n a f t e r t h e b l e n d i n g t i m e . E m u l s i o n : : s t a b i l i t y was a l s o m e a s u r e d by a d d i n g 5 m l o f o i l t o 25 m l o f 1% p r o t e i n s o l u t i o n i n w a t e r , b l e n d -i n g 2 m i n u t e s a t 8600 rpm i n t h e Omni M i x e r and a l l o w i n g i t t o s t a n d a t . 25°C ( 4 4 ) . Time r e q u i r e d f o r a p p e a r a n c e o f o i l d r o p l e t s a t t h e s u r f a c e o f t h e e m u l s i o n was r e c o r d e d a s e m u l s i o n s t a b i l i t y . Foam P r e p a r a t i o n : The p r o c e d u r e o f K o d a g o d a e t aJL. (27) was u s e d w i t h some m o d i f i c a t i o n . A 0.5% p r o t e i n s o l u t i o n a t room t e m p e r a t u r e (25°C) was w h i p p e d i n a Sunbeam M i x m a s t e r a t a s p e e d s e e t i n g o f 10 f o r 5 m i n u t e s . Foam e x p a n s i o n r a t i o was c a l c u l a t e d f r o m t h e v o l u m e s b e f o r e a nd a f t e r w h i p p i n g u s i n g : Foam e x p a n s i o n r a t i o . = V o l u m e a f t e r w h i p p i n g - Volume b e f o r e w h i p p i n g V o l u m e b e f o r e w h i p p i n g Foam S t a b i l i t y : I m m e d i a t e l y a f t e r w h i p p i n g , f oam was t r a n s f e r r e d t o a g r a d u a t e d c y l i n d e r . The amount o f l i q u i d d r a i n e d f r o m t h e foam was r e c o r d e d a f t e r 5, 1 5 , 30, 60, 90 and 120 m i n u t e s . Foam s t a b i l i t y was c a l c u l a t e d a s t h e v o l u m e o f l i q u i d d r a i n e d a f t e r 30 m i n u t e s . RESULTS AND DISCUSSION G l u t e n was d e a m i d i z e d u s i n g t h e m e t h o d o f Wu (47) o u t l i n e d i n F i g . 1. A f t e r a d j u s t i n g t h e pH t o 4.7, whey was remo v e d b y c e n t r i f u g i n g t h e h y d r o l y z a t e a t 10,000 x g . E f f e c t o f pH o n P r o t e i n S o l u b i l i t y pH o f t h e whey a f t e r s e p a r a t i o n f r o m a c i d - m o d i f i e d g l u t e n was b e t w e e n 4.5 and 4.7. E f f e c t o f pH on t h e s o l u -b i l i t y o f whey p r o t e i n was e x a m i n e d b y i n c r e a s i n g a n d d e c r e a s i n g i t s pH. A c o m p l e t e p H - s o l u b i l i t y p r o f i l e o f a c i d - m o d i f i e d g l u t e n whey i s shown i n F i g . 4. A. I n c r e a s e i n pH: S e v e n t e e n p e r c e n t p r o t e i n was s e p a r a t e d o u t f r o m t h e whey a t pH 6.2. A n o t h e r s a m p l e o f whey was a d j u s t e d t o pH 8.2 w h i c h r e s u l t e d i n 27.5% p r o t e i n p r e c i p i t a t i o n . A t h r e e - s t e p p r o c e d u r e was a d o p t e d a s s t a t e d i n M e t h o d s o f P r e p a r a t i o n . The c o m b i n e d y i e l d f r o m S t e p I a n d S t e p I I was o n l y 3 2.5% a s c o m p a r e d t o t h e e x p e c t e d 4 4 . 5 % N y i e l d , s u g g e s t i n g an o v e r l a p p i n g p r e c i p i t a t i o n a l t h o u g h t h e p r e c i p i -t a t e s w e r e n o t e x a c t l y s i m i l a r . As shown b y c o l u m n c h r o m a -t o g r a p h y a n d a g a r o s e g e l e l e c t r o p h o r e s i s , t h e pH 8.2 p p t had more l o w m o l e c u l a r w e i g h t p r o t e i n s t h a n t h e pH 6.2 p p t . U l t r a f i l t r a t i o n (U.F.) was u s e d t o r e c o v e r p r o -t e i n s r e m a i n i n g a f t e r S t e p I I . A t room t e m p e r a t u r e , o n l y 30 m l o f t h e p e r m e a t e was c o l l e c t e d a f t e r 48 h o u r s o f f i l t r a t i o n p r o c e s s . The r u n a t c o l d room t e m p e r a t u r e (4°C) d i d n o t i m p r o v e t h e s p e e d s i g n i f i c a n t l y ; a f t e r 48 h o u r s , t h e t o t a l p e r m e a t e c o l l e c t e d was 49 m l . The g e l f i l t r a -t i o n p a t t e r n o f t h e U.F. c o n d e n s a t e showed p r o t e i n o f h i g h m o l e c u l a r w e i g h t s i m i l a r t o t h e pH 8.2 p p t a l o n g w i t h l o w m o l e c u l a r w e i g h t p e p t i d e s . B. D e c r e a s e i n pH: pH o f whey was l o w e r e d b y a d d i n g 0.1 N H C l . No c h a n g e was n o t i c e d u n t i l pH 3.7, b e l o w w h i c h t u r b i d i t y i n c r e a s e d c o n t i n u o u s l y . As shown i n F i g . 4, n i t r o g e n p r e c i p i t a t e d a t pH 1.0 was 4 4 . 0 % o f t o t a l N i n whey. E f f e c t o f H e a t i n g on t h e A c i d i f i e d G l u t e n Whey E f f e c t o f h e a t o n a c i d i f i e d whey was c a r r i e d o u t by i n c r e a s i n g t h e t e m p e r a t u r e a t 10° i n t e r v a l s up t o 95°C a f t e r a d j u s t i n g t h e whey t o pH 1.0. H o l d i n g t i m e o f 10 m i n u t e s was u s e d i n a l l e x p e r i m e n t s . As shown i n T a b l e I , t h e y i e l d o f p r e c i p i t a t e d p r o t e i n i n c r e a s e d w i t h a n i n c r e a s e i n t e m p e r a t u r e u n t i l i t l e v e l l e d o f f a t 90°C. By h e a t i n g t h e whey t o 90°C a t pH 1.0, 74% o f t h e t o t a l N i n whey p r e c i p i t a t e d . T a b l e I : E f f e c t o f h e a t o n t h e p r o t e i n p r e c i p i t a t i o n o f a c i d i f i e d g l u t e n whey T e m p e r a t u r e % N (°C) P r e c i p i t a t e d 3 20 44.0 30 48.3 40 51.7 50 58.4 60 65 . 3 70 69.9 80 73.6 90 74.0 95 74.2 S a m p l e s w e r e a d j u s t e d t o pH 1.0 b e f o r e h e a t i n g . a V a l u e s a r e t h e mean o f two r e a d i n g s . 28 . E f f e c t o f F e r r i c C h l o r i d e A d d i t i o n t o A c i d i f i e d Whey  B e f o r e H e a t i n g When 0.15 m l o f 30% f e r r i c c h l o r i d e ( F e C l 3 ' 6 H 2 0 ) was a d d e d t o one l i t r e o f whey a n d t h e s o l u t i o n was a c i d i -f i e d t o pH 1.0 a n d h e a t e d t o 90°C, a b o u t 8 4.8% o f t o t a l N i n t h e whey, was p r e c i p i t a t e d . The method i s o u t l i n e d i n F i g u r e 3. Hexameta-p h o s p h a t e was a d d e d t o t h e f e r r i c c h l o r i d e - t r e a t e d whey b e f o r e f r e e z e d r y i n g t o d e c r e a s e t h e b r o w n c o l o u r o f t h e d r i e d p r o d u c t . Amount o f f e r r i c c h l o r i d e , h e a t i n g t e m p e r a t u r e , h e a t i n g t i m e a n d pH w e r e o p t i m i z e d u s i n g T a g u c h i ' s m e t h o d ( 4 3 ) . A l t h o u g h e a c h e f f e c t was n o t s t a t i s t i c a l l y s i g n i -f i c a n t , t h e e f f e c t c u r v e s showed t h e f o l l o w i n g t e n d e n c y : 1. The amount o f p r e c i p i t a t e d p r o t e i n d e c r e a s e d w i t h t h e i n c r e a s e i n pH. pH 1.0 g a v e t h e b e s t r e s u l t s , h e n c e was u s e d i n f u r t h e r e x p e r i m e n t s . 2. The a d d i t i o n o f 0.15 m l o f 30% f e r r i c c h l o r i d e t o a l i t r e o f whey., c a u s e d maximum p r e c i p i t a t i o n o f whey p r o t e i n . P r e c i p i t a t e d N d e c r e a s e d w i t h e i t h e r i n c r e a s e o r d e c r e a s e i n t h e v o l u m e o f t h e f e r r i c c h l o r i d e s o l u t i o n . 3. By i n c r e a s i n g t h e t e m p e r a t u r e up t o 70°C t h e y i e l d o f p r e c i p i t a t e d p r o t e i n i n c r e a s e d , t h e n 3. ( c o n t ' d ) d e c r e a s e d . T h o u g h 4% l e s s N w a s p r e c i p i t a t e d a t 90°C a s c o m p a r e d t o 70°C, 90°C w a s u s e d i n t h e f i n a l p r o c e d u r e b e c a u s e o f e a s y s e p a r a t i o n o f t h e p r e c i p i t a t e . T h e p r e c i p i t a t e f o r m e d a t 70°C w a s s o g e l a t i n o u s t h a t i t w a s d i f f i c u l t t o s e p a r a t e i f r o m t h e s u p e r n a t a n t u n l e s s v e r y h i g h c e n t r i f u g i n g s p e e d w a s a p p l i e d . T h i s g e l - l i k e s t r u c t u r e d e c r e a s e d w i t h t h e i n c r e a s e i n h e a t -i n g t e m p e r a t u r e . A t 90°C, t h e p r e c i p i t a t e f o r m e d b e c a m e r e a d i l y s e p a r a b l e . T h e N y i e l d s o f p r e c i p i t a t e s o b t a i n e d a r e s h o w n i n T a b l e I I . E f f e c t o f W a s h i n g T h r e e w a s h i n g s w e r e a p p l i e d t o t h e f e r r i c - p r o t e i n p r e c i p i t a t e . T h e s e w a s h i n g s w e r e q u i t e c r i t i c a l a s i s s e e n i n F i g u r e 5. U p t o t w o w a s h i n g s , t h e s o l i d s r e m o v e d w e r e o n l y a b o u t h a l f t h e s o l i d s r e m o v e d b y t h e t h i r d w a s h -i n g . H o w e v e r , w h e n t h e f o u r t h w a s h i n g w a s a p p l i e d , t h e p r o t e i n l o s s d r a s t i c a l l y i n c r e a s e d . T h e p H o f w a s h w a t e r w a s i m p o r t a n t ; p H 1.0 w a t e r w a s m o s t e f f i c i e n t f o r r e m o v a l o f s a l t s . W h e n r e d i s s o l v e d i n w a t e r a t p H 7.0, F e - p r o t e i n s h o w e d e x c e l l e n t s o l u b i l i t y (99.2%). T h i s v a l u e w a s d e t e r m i n e d a f t e r a h o l d i n g p e r i o d o f 24 h o u r s a t 4°C. T h e s o l u t i o n 30. Figure 5: E f f e c t of washings (A) on the removal of S.S. (B) on the loss of protein. *S.S.= % Soluble Solids i n the wash water. T a b l e I I : % N i t r o g e n r e c o v e r e d f r o m a c i d - m o d i f l e d g l u t e n whey by d i f f e r e n t m ethods M e t h o d pH 6.2 pH 8.2 pH 1.0 H e a t e d 90- C F e r r i c o f P r o t e i n P r e c i p i - P r e c i p i - > P r e c i p i - f o r 10 m i n . C h l o r i d e r e c o v e r y t a t i o n t a t i o n t a t i o n a t pH 1.0 M e t h o d Y i e l d (% N) 17.0 27.5 44.0 74.0 84 .8 V a l u e s a r e t h e mean o f t h r e e e x p e r i m e n t s . was s l i g h t l y v i s c o u s b u t c l e a r . An a n a l y s i s o f t h e f i n a l p r o d u c t i s g i v e n i n T a b l e I I I . The f i n a l p r o d u c t , a f t e r f r e e z e d r y i n g c o n s i s t e d o f 80.48% p r o t e i n , w i t h a t r a c e o f l i p i d (0.32%) a n d c a r -b o h y d r a t e ( 0 . 1 2 % ) . A s h c o n t e n t was r e l a t i v e l y h i g h (8.8%) e v e n a f t e r t h r e e w a s h i n g s . I t c a n be a t t r i b u t e d t o a l a r g e amount o f N a C l f o r m e d when p r e c i p i t a t e was d i s s o l v e d a n d a d j u s t e d t o pH 7.0 b e f o r e f r e e z e d r y i n g . O b v i o u s l y , a p a r t o f h i g h a s h c o n t e n t i s d e r i v e d f r o m s o d i u m h e x a m e t a -p h o s p h a t e a d d e d p r i o r t o f r e e z e d r y i n g t o i m p r o v e t h e c o l o u r o f t h e f i n a l p r o d u c t . A n a l y s i s showed 0.80% p h o s p h o r u s w h i c h i s a l m o s t e q u a l t o t h e amount o f p h o s p h o r u s a d d e d as s o d i u m h e x a m e t a p h o s p h a t e . H M P - F e - p r o t e i n h a d a g r e y i s h t i n g e , a l l o t h e r F e - f r e e p r o t e i n f r a c t i o n s w e r e w h i t e . B e f o r e t h e a d d i t i o n o f s o d i u m h e x a m e t a p h o s p h a t e , F e - p r o t e i n was b r o w n i n c o l o u r due t o t h e p r e s e n c e o f i r o n o x i d e . When s o d i u m h e x a m e t a -p h o s p h a t e was a d d e d , HMP-Fe c o m p l e x was f o r m e d w h i c h was o f r e l a t i v e l y w h i t e c o l o u r t h u s i m p r o v i n g t h e c o l o u r o f -the f i n a l p r o d u c t . M o l e c u l a r W e i g h t D e t e r m i n a t i o n o f H M P - F e - P r o t e i n A 2.5 cm x 80 cm S e p h a d e x G-200 c o l u m n was u s e d f o r m o l e c u l a r w e i g h t d e t e r m i n a t i o n o f H M P - F e - p r o t e i n by e l u t i n g w i t h 0.1 M p h o s p h a t e b u f f e r a t pH 7.4. F o u r T a b l e I I I : C h a r a c t e r i s t i c s o f H M P - F e - p r o t e i n C o m p o s i t i o n 0 Y i e l d a ' C  g p r o t e i n . b c j. P r o t e i n A s h Fe P h o s p h o r u s S o l u b i l i t y C o l o u r o f J 0.446 80.48 8.8 0.078 0.80 99.2 g r e y i s h w h i t e a F r e e z e d r i e d p r o d u c t f r o m 100 m l whey, k On d r y b a s i s . ° V a l u e s a r e mean o f t h r e e e x p e r i m e n t s . p e a k s were, o b t a i n e d ( F i g . .6), a nd t h e i r m o l e c u l a r w e i g h t s a r e r e c o r d e d i n t h e f o l l o w i n g t a b l e a s r e a d f r o m t h e c a l i b r a t e d g r a p h : P e a k ^ S i ^ ^ y l ^ ^ - ^ i S ^ t 1 >100,000 2 62,000 3 29,000 4 <10,000 The m a j o r p e a k h a d m o l e c u l a r w e i g h t o f a b o u t 62,000 w h i c h i s v e r y s i m i l a r t o t h e r e s u l t s f r o m SDS d i s c g e l e l e c t r o -p h o r e s i s (16) . P e a k 4 h a d a m o l e c u l a r w e i g h t o f l e s s t h a n 10,000 w h i c h s u g g e s t s t h a t some l o w m o l e c u l a r w e i g h t p e p t i d e s , a r e p r e c i p i t a t e d u n d e r t h e s e c o n d i t i o n s w h i c h was c o n f i r m e d by t h e c h l o r a n i l a n d n i n h y d r i n t e s t s . The c h l o r a n i l t e s t h a s b e e n p r e v i o u s l y u s e d f o r s m a l l p e p t i d e s a n d a m i n o a c i d s ( 1 ) . The n i n h y d r i n t e s t h a s b e e n u s e d f o r d e t e c t i o n o f f r e e amino g r o u p s . B o t h t e s t s showed p o s i t i v e r e s u l t s c o n f i r m i n g t h e p r e s e n c e o f s m a l l p e p t i d e s o r amino a c i d s i n t h i s p e a k . Amino A c i d C o m p o s i t i o n Amino a c i d c o m p o s i t i o n o f d i f f e r e n t whey p r o t e i n f r a c t i o n s i s g i v e n i n T a b l e I V . The m a j o r a mino a c i d i n a l l t h e f r a c t i o n s i s g l u t a m i c a c i d f o l l o w e d b y p r o l i n e . Figure 6: Gel f i l t r a t i o n pattern of HMP-Fe-protein on Sephadex G-200 using 0.1 M phosphate buffer Table IV: Amino acid content of d i f f e r e n t protein fractions recovered from whey of acid treated gluten (g/16 g N) Amino Acid Untreated gluten HMP-Fe-protein pH 6.2 ppt. pH 8.2 ppt. U.F Aspartic acid 3.64 2.62 3.60 5.15 3.90 Threonine 2.31 2.50 2.85 3.41 3.15 Serine 5.40 5.88 4.84 5.42 5.10 Glutamic acid 27.44 26.26 28.82 24.03 30.78 Proline 17.41 19.85 11.35 8.87 12.33 Glycine 3.69 1.90 3.27 3.61 4.29 Alanine 2.87 2.28 3.17 4.40 3.44 Valine 3.20 3.66 4.10 4.20 3.51 Methionine 1.41 1.53 1.44 1.80 1.62 Isoleucine 3.20 3.81 4.43 4.04 2.81 Leucine 5.42 7.80 7.01 8.46 6.56 Tyrosine 3.64 2.73 2.85 3.21 3.30 Phenylalanine 6.05 6.39 4.21 4.28 3.69 Lysine 1.55 0.83 2.19 4.62 2.22 Hist i d i n e 1.88 1.97 2.02 2.91 1.85 Tryptophan Trace Trace 0.98 1.07 -Arginine 3.24 2.59 3.77 5.91 3.71 Cysteine 1.94 1.99 2.23 2.82 2.97 T h i s t r e n d i s v e r y s i m i l a r t o t h e one o b t a i n e d i n t h e v i t a l g l u t e n . Among t h e f r a c t i o n s , h i g h e s t g l u t a m i c a c i d v a l u e was o b t a i n e d w i t h U.F. p r o t e i n w h i l e H M P - F e - p r o t e i n c o n -t a i n e d t h e g r e a t e s t amount o f p r o l i n e . The h i g h e s t c o n t e n t o f t h r e o n i n e , v a l i n e , m e t h i o n i n e , l e u c i n e , l y s i n e a n d t r y p t o p h a n was f o u n d i n t h e pH 8.2 p r e c i p i t a t e w h e r e a s H M P - F e - p r o t e i n h a d h i g h e s t p h e n y -l a l a n i n e . The pH 6.2 p r o t e i n p r e c i p i t a t e was h i g h i n i s o l e u c i n e . L y s i n e i s t h e m o s t l i m i t i n g amino a c i d i n H M P - F e - p r o t e i n w h i c h may h a v e b e e n d e c r e a s e d due t o s e v e r e t e m p e r a t u r e c o n d i t i o n s u s e d f o r p r o t e i n r e c o v e r y . A b e t t e r e s s e n t i a l a m i n o a c i d s b a l a n c e was f o u n d i n t h e pH 6.2 p r e c i p i t a t e , t h e pH 8.2 p r e c i p i t a t e a n d U.F. due t o l o w e r c o n t e n t s o f one o r b o t h o f a s p a r a t i c a c i d a n d p r o l i n e t h a n H M P - F e - p r o t e i n . B u t H M P - F e - p r o t e i n h a d an amino a c i d p a t t e r n v e r y s i m i l a r t o v i t a l g l u t e n e x c e p t f o r l y s i n e w h i c h i s l e s s t h a n t h e amount i n u n t r e a t e d g l u t e n . F u n c t i o n a l P r o p e r t i e s o f G l u t e n Whey P r o t e i n s E v e n t h o u g h n u t r i t i o n i s t h e m a i n a i m i n new p r o d u c t d e v e l o p m e n t , i t i s e q u a l l y i m p o r t a n t t o s e l e c t a p r o t e i n w h i c h a l s o m a i n t a i n s g o o d f u n c t i o n a l p r o p e r t i e s . F o a m a b i l i t y , e m u l s i f y i n g c a p a c i t y a n d e m u l s i o n s t a -b i l i t y a r e i m p o r t a n t f u n c t i o n a l p r o p e r t i e s o f a p r o t e i n . 38. E m u l s i o n s a r e i m p o r t a n t i n t h e meat i n d u s t r y . I n a meat e m u l s i o n , s u c h a s w i e n e r s , p r o t e i n s w o r k as e m u l s i f y i n g a g e n t s i n o i l - i n - w a t e r t y p e e m u l s i o n s . I n c a k e s , m e r i n g u e and d e s s e r t t o p p i n g s , p r o t e i n s a r e u s e d as a e r a t i n g a g e n t s t o make g o o d s t a b l e f o a m s . I n d u s t r y i s a l w a y s l o o k i n g f o r new e m u l s i f y i n g and f o a m i n g a g e n t s o f g o o d q u a l i t y t o r e p l a c e more c o s t l y o n e s , e.g. s o d i u m c a s e i n a t e o r e g g w h i t e . Wheat g l i a d i n s h a v e b e e n r e p o r t e d t o h a v e good f o a m i n g p r o p e r t i e s a n d h a v e b e e n s u g g e s t e d as e g g w h i t e s u b s t i t u t e s (33). The p o s s i b l e u s e o f g l u t e n whey p r o -t e i n s f o r e m u l s i f i c a t i o n a n d f o a m i n g w e r e e x a m i n e d as a p a r t o f t h e i r f u n c t i o n a l p r o p e r t i e s . E m u l s i f y i n g C a p a c i t y 1 D i f f e r e n t p r o t e i n f r a c t i o n s f r o m g l u t e n whey showed good e m u l s i f y i n g c a p a c i t y ( T a b l e V ) . V i t a l g l u t e n was i n -f e r i o r (20.8 m l o i l / 1 0 0 mg s a m p l e ) a s c o m p a r e d t o t h e s o y i s o l a t e u s e d a s s t a n d a r d (32.0 m l o i l / 1 0 0 mg s a m p l e ) . The pH 6.2 p r e c i p i t a t e h a d e x c e l l e n t e m u l s i f y i n g c a p a c i t y , a l m o s t -one a n d h a l f t i m e s g r e a t e r t h a n t h a t o f t h e s o y i s o l a t e . The pH 8.2 p r e c i p i t a t e showed a s l i g h t l y i n f e r i o r e m u l s i f y i n g c a p a c i t y a s c o m p a r e d t o t h e s o y i s o l a t e b u t s t i l l b e t t e r t h a n t h a t o f t h e u n t r e a t e d g l u t e n . Table V: Emulsifying capacity and s t a b i l i t y of d i f f e r e n t protein fractions Sample Emu l s i f i -cation Soy V i t a l pH 6.2 pH 8.2 u F a H M P - F e ~ Isolate Gluten ppt ppt * * protein Capacity 3 2 > 0 2 Q Q 4 5 _ Q 2 9 3 3 Q e 3 2 3 (ml o i l / 100 mg sample) (0.57)* (0.17) (0.57) (0.34) (0.67) (0.67) S t a b i l i t y 1 3 37.25 20.75 48.25 38.0 42.75 114.0 (min.) (0.48) (0.25) (0.77) (0.41) (0.48) (0.70) a Sample prepared by u l t r a f i l t r a t i o n . k Values are average of four observations. * Standard error of the mean. 40. H M P - F e - p r o t e i n h a d t h e e m u l s i f y i n g c a p a c i t y c o m p a r -a b l e t o t h a t o f t h e s o y i s o l a t e . A s p r o t e i n c o n c e n t r a t i o n , r a t e o f a d d i t i o n o f o i l , pH o f p r o t e i n s o l u t i o n , c o m p o s i t i o n o f o i l , s p e e d o f b l e n d -i n g a n d t e m p e r a t u r e o f b l e n d i n g a r e i m p o r t a n t f a c t o r s t o a f f e c t t h e e m u l s i f i c a t i o n p r o p e r t i e s , c a r e was t a k e n t o k e e p a l l t h e f a c t o r s c o n s t a n t . E m u l s i o n S t a b i l i t y : S t a b i l i t y o f f o r m e d e m u l s i o n i s a n o t h e r i m p o r t a n t c r i t e r i o n t o compare t h e e m u l s i f y i n g p r o p e r t y o f a p r o t e i n . L o n g e r s t a b i l i t y i s u s u a l l y d e s i r a b l e . S t a b i l i t i e s o f d i f f e r e n t p r o t e i n f r a c t i o n s a r e g i v e n i n T a b l e V. U n t r e a t e d g l u t e n h ad p o o r e m u l s i o n s t a -b i l i t y . A l l t h e p r o t e i n s s e p a r a t e d f r o m whey had b e t t e r e m u l s i o n s t a b i l i t y a s c o m p a r e d w i t h t h e s o y i s o l a t e . HMP-F e - p r o t e i n h a d e x c e l l e n t e m u l s i o n s t a b i l i t y w h i c h i s more t h a n t h r e e t i m e s t h a t o f t h e s o y i s o l a t e . H M P - F e - p r o t e i n s o l u t i o n i n w a t e r was more v i s c o u s a s c o m p a r e d t o o t h e r p r o t e i n f r a c t i o n s . T h i s c o u l d be a r e a s o n f o r h i g h s t a b i -l i t y o f t h e e m u l s i o n p r e p a r e d f r o m H M P - F e - p r o t e i n . M e t h o d s o f P e a r s o n e t a l . (36) a n d T r a u t m a n (44) w e r e u s e d t o mea-s u r e t h e e m u l s i o n s t a b i l i t y . B o t h m e t h o d s showed s i m i l a r r e s u l t s e x c e p t f o r o c c a s i o n a l l o w e r v a l u e s by T r a u t m a n ' s m e t h o d . Some i m p o r t a n t c o n c l u s i o n s w e r e d r a w n f r o m t h e . e m u l s i f i c a t i o n e x p e r i m e n t s : 4 1 . 1. Once t h e j a r was h e a t e d , e m u l s i o n s t a b i l i t y -d e c r e a s e d p r o b a b l y due t o a d e c r e a s e i n v i s c o s i t y o f t h e p r o t e i n s o l u t i o n o r t o d e n a t u r a t i o n o f p r o t e i n p r i o r t o e m u l s i o n f o r m a t i o n ( 3 8 ) . An i c e b a t h was u s e d d u r i n g b l e n d i n g t o o v e r c o m e t h i s d i f f i c u l t y . 2. V a l u e s f o r e m u l s i f i c a t i o n w e r e e a s i l y a f f e c t e d i f w a s h i n g d e t e r g e n t was n o t t h o r o u g h l y r e m o v e d f r o m t h e b l e n d e r j a r s . F a i r l y g o od r e p e a t a b i l i t y was p r o d u c e d w i t h s e v e n w a t e r w a s h i n g s g i v e n t o e n s u r e t h e c o m p l e t e r e m o v a l o f r e s i d u a l d e t e r g e n t f r o m t h e j a r . C h r o m i c a c i d w a s h i n g s w e r e t r i e d b u t no i m p r o v e m e n t o v e r t h e w a t e r w a s h i n g was n o t i c e d . 3. V e r y l o w b l e n d i n g s p e e d d i d n o t a f f o r d p r o p e r m i x i n g w h i l e v e r y h i g h s p e e d e x c e s s i v e l y b r o k e t h e o i l d r o p l e t s t o v e r y s m a l l s i z e . T h e r e f o r e , p r o p e r c o n s t a n t s p e e d was e x t r e m e l y i m p o r t a n t t o o b t a i n r e p r o d u c i b l e r e s u l t s t h r o u g h o u t a l l e x p e r i m e n t s . V e r y s m a l l o i l d r o p l e t s a r e u n d e s i r a b l e as t h e y i n c r e a s e t h e s u r f a c e a r e a w h i c h w i l l n o t be p r o p e r l y c o v e r e d by t h e same amount o f p r o t e i n . A f t e r e c o n o m i c s and a c c e p t a b i l i t y s t u d y , HMP-Fe-p r o t e i n , due t o t h e e x c e l l e n t e m u l s i o n s t a b i l i t y , mayu.be u s e d i n meat p r o c e s s i n g . An i m p o r t a n t p r o p e r t y o f s a u s a g e e m u l s i o n i s t h a t t h e y s h o u l d be s t a b l e d u r i n g t h e t i m e b e t w e e n p r e p a r a t i o n and c o o k i n g w h i c h g e n e r a l l y e x t e n d s o v e r a few h o u r s ( 3 8 ) . F o a m i n q P r o p e r t i e s o f D i f f e r e n t P r o t e i n F r a c t i o n s ; F o a m a b i l i t y was m e a s u r e d a s a r a t i o o f t h e i n c r e a s e d v o l u m e t o t h e o r i g i n a l v o l u m e o f 50 m l o f 0.5% s o l u t i o n a n d foam s t a b i l i t y was t h e d r a i n v o l u m e o f t h i s foam a f t e r 30 m i n u t e s o f w h i p p i n g . • F o a m i n g p r o p e r t i e s o f d i f f e r e n t p r o t e i n f r a c t i o n s a r e g i v e n i n T a b l e V I . The pH 6.2 p r e c i p i t a t e a n d t h e pH 8.2 p r e c i p i t a t e h a d b e t t e r foam e x p a n s i o n a n d s t a b i l i t y t h a n t h o s e o f v i t a l g l u t e n . U.F. showed a n i n t e r e s t i n g t r e n d w i t h one a n d a h a l f t i m e s g r e a t e r . e x p a n s i o n r a t i o t h a n , t h a t o f v i t a l g l u -t e n b u t was l e s s s t a b l e . H M P - F e - p r o t e i n h a d p o o r f oam i n t e r m s o f e x p a n s i o n a n d s t a b i l i t y . F r o m t h e r e s u l t s , i t seems t h a t f o r h i g h f o a m a b i l i t y , t h e p r o d u c t s h o u l d b e ^ p a r t i a l l y s o l u b l e i n w a t e r . I n c a s e o f H M P - F e - p r o t e i n t h e p r o d u c t was c o m p l e t e l y s o l u b l e , b u t t h e foam e x p a n s i o n r a t i o i s v e r y c l o s e t o s o y i s o l a t e w h i c h i s h a r d l y s o l u b l e i n w a t e r . I n a l l o t h e r c a s e s , foam e x p a n s i o n seems t o be r e l a t e d t o t h e w a t e r s o l u b i l i t y . Foams made f r o m s a m p l e s w i t h h i g h e x p a n s i o n r a t i o showed h i g h s t a b i l i t y e x c e p t U.F. T h i s was p r o b a b l y due t o l o w m o l e c u l a r w e i g h t c o m p o n e n t s w h i c h w e r e u n a b l e t o h o l d t h e b u b b l e s f o r m e d d u r i n g w h i p p i n g . T a b l e V I : F o a m a b i l i t y and foam s t a b i l i t y o f d i f f e r e n t p r o t e i n f r a c t i o n s , Foam E x p a n s i o n S a mple ^ .. = ^ R a t i o 3 D r a i n V o l u m e 3 (ml) 5 15 30 60 90 120 M i n u t e s U n t r e a t e d g l u t e n 4.0 14 30 41 47 47 47 47 H M P - F e - p r o t e i n 1.8 10 25 37 42 46.5 47 47 pH 6.2 p p t 5.9 2 15 28 36 44 46 46 pH 8.2 p p t 6.4 2 13 19 25 40 44 45 U.F . 5.9 12 26 37.5 47 47 47 47 Soy I s o l a t e 1.7 3 13 20 : 29 37 42 43 S t a r t v o l . was 50 ml o f 0.5% s a m p l e c o n c e n t r a t i o n . a V a l u e s a r e mean o f t h r e e e x p e r i m e n t s . A l l t h e e x p e r i m e n t s w e r e done a t pH 7.0, 25°C t e m p e r a t u r e a nd w i t h b e a t i n g t i m e o f 6 m i n u t e s . I t i s known (14) t h a t f o a m a b i l i t y ( e x p a n s i o n a nd s t a b i l i t y ) i s a f f e c t e d b y p r o t e i n c o n c e n t r a t i o n , pH, b e a t i n g t i m e a n d s p e e d , t e m p e r a t u r e a nd s o l u b i l i t y . E x p e r i m e n t s w e r e done t o i m p r o v e t h e f o a m a b i l i t y o f H M P - F e - p r o t e i n a n d t o e s t a b l i s h c o n d i t i o n s u n d e r w h i c h we c a n u t i l i z e t h i s p r o d u c t m o s t e f f i c i e n t l y . The f o l l o w i n g f a c t o r s w e r e f o u n d e f f e c t i v e i n i m p r o v i n g i t s f o a m i n g p r o p e r t i e s : 1. P r o t e i n C o n c e n t r a t i o n : E f f e c t o f c o n c e n t r a t i o n o n f o a m i n g p r o p e r t i e s g i v e n i n T a b l e V I I . When t h e c o n c e n t r a t i o n o f H M P - F e - p r o t e i n was i n c r e a s e d f r o m 0.5% t o 1.5%, b e t t e r foam e x p a n s i o n was o b t a i n e d t h a n t h a t o f v i t a l g l u t e n . I t was f u r t h e r i m p r o v e d a s t h e c o n -c e n t r a t i o n was i n c r e a s e d t o 4% b u t s l i g h t l y d e c l i n e d a t t h e c o n c e n t r a t i o n s o v e r 4%. S t a b i l i t y i n c r e a s e d c o n t i n u o u s l y w i t h t h e i n c r e a s e i n c o n c e n t r a t i o n . 2. T e m p e r a t u r e : E f f e c t o f t e m p e r a t u r e o n f o a m i n g p r o p e r t i e s i s g i v e n i n T a b l e V I I I . E f f e c t o f t e m p e r a t u r e o n f o a m i n g p r o p e r t i e s was e x a m i n e d w i t h 1.5% H M P - F e - p r o t e i n s o l u t i o n . S a m p l e s a t d i f f e r e n t t e m p e r a t u r e s r a n g i n g f r o m 25°C t o 85°C w e r e b l e n d e d u n d e r 1 T a b l e \ V I I : E f f e c t o f c o n c e n t r a t i o n o n f o a m a b i l i t y and foam s t a b i l i t y o f H M P - F e - p r o t e i n C o n c e n t r a t i o n s Foam E x p a n s i o n R a t i o Foam S t a b i l i t y 0 .5% 1.74 ( 0 . 1 8 ) * 45.0 ( 0 . 2 1 ) * 1 .0% 3. 65 (0.04) 39.5 (0.14) 1 .5% 4 . 36 (0.03) 33.66 (0:34) 2 . 0% 5. 21 (0.04) 29.3 (0.34) 3 . 0% 5.58 (0.03) 26.3 (0.11) 4 .0% 5.65 (0.03) 19.33 (0.19) 5 .0% 5.56 (0.01) 15. 7 (0.33) a D r a i n v o l u m e 50 m l o f e a c h (ml) i n 30 m i n u t e s c o n c e n t r a t i o n . S t a r t v o l u m e was b V a l u e s a r e t h e mean o f f o u r e x p e r i m e n t s . * S t a n d a r d , e r r o r o f t h e mean. T a b l e V I I I : E f f e c t o f t e m p e r a t u r e on f o a m a b i l i t y a n d foam s t a b i l i t y o f H M P - F e - p r o t e i n T e m p e r a t u r e F o a I n E x p a n s i o n R a t i o b Foam S t a b i l i t y 3 ' 25 4.36 (0.03)* 33.66 (0.34)* 40 4.45 (0.03) 32.75 (0.10) 55 5.08 (0.05) 31.25 (0.11) 70 5.18 (0.03) 29.25 (0.11) 85 5.13 (0.05) 26.25 (0.11) Drain v o l u m e (ml) i n 30 m i n u t e s . S t a r t v o l u m e was 50 m l . V a l u e s a r e t h e mean " o f f o u r e x p e r i m e n t s . * S t a n d a r d e r r o r o f t h e mean. t h e same b l e n d i n g c o n d i t i o n s . Up t o 70 C, f oam e x p a n s i o n r a t i o i n c r e a s e d b u t d e c r e a s e d a t h i g h e r h e a t i n g t e m p e r a -t u r e s . Foam s t a b i l i t y was i m p r o v e d w i t h h e a t i n g . Temp-e r a t u r e d e c r e a s e d s o l u b i l i t y o f p r o t e i n due t o a g g r e g a t i o n . I t was q u i t e n o t i c e a b l e a t 85°C w h i c h p r o b a b l y c a u s e d a d e c r e a s e i n t h e foam e x p a n s i o n r a t i o . 3. pH E f f e c t o f pH o n t h e f o a m i n g p r o p e r t i e s i s g i v e n i n T a b l e I X . U s i n g 1.5% s a m p l e s o l u t i o n , t h e pH was a d j u s t e d b e t w e e n 3 a n d 8. E x p a n s i o n r a t i o i n c r e a s e d f r o m pH 3 t o pH 5 t h e n d e c r e a s e d w i t h i n c r e a s i n g pH. K e s h a v a r z (25) e x p l a i n e d t h a t i n s o l u b i l i t y i n c r e a s e s f o a m a b i l i t y due t o t h e f a c t t h a t i n s o l u b l e p r o t e i n s t e n d t o o r i e n t o n t h e s u r f a c e o f g a s b u b b l e s , i n c r e a s e t h e l a m e l l a r d i a m e t e r s e p a r a t i n g t h e b u b b l e s a n d p r e v e n t t h e i r c o a l e s -c a n c e . I t seems t r u e i n t h i s c a s e b e c a u s e s a m p l e was s o l -u b l e a t pH 7.0, b u t t h e s o l u b i l i t y d e c r e a s e d a s t h e pH was l o w e r e d . A s i m i l a r t r e n d was n o t i c e d i n f oam s t a b i l i t y . Foam made w i t h s o l u t i o n a t pH 5.0 was m o s t s t a b l e w i t h a d e c r e a s e i n s t a b i l i t y a s t h e pH d e v i a t e d f r o m 5.0. B e a t i n g t i m e was s t a n d a r d i z e d a t 6 m i n u t e s f o r a l l t h e e x p e r i m e n t s . Foams made w i t h l e s s b e a t i n g t i m e h a d h i g h e x p a n s i o n r a t i o b u t l e s s s t a b i l i t y . B e a t i n g l o n g e r t h a n 6 m i n u t e s d e c r e a s e d t h e foam v o l u m e s u b s t a n t i a l l y . T a b l e I X : E f f e c t o f pH o n f o a m a b i l i t y a n d foam s t a b i l i t y o f H M P - F e - p r o t e i n pH Foam E x p a n s i o n R a t i o Foam S t a b i l i t y 3.0 4.0 ( 0 . 0 4 ) * 46.0 ( 0 . 0 0 ) * 4.0 4. 42 (0.03) 34.5 (0.34) 5.0 4. 86 (0.01) 15. 7 (0.33) 6.0 4.62 (0.03) 30.75 (0.11) 7.0 4.36 (0.34) 33. 66 (0.34) 8.0 3.98 (0.03) 41. 3 (0.34) a D r a i n v o l u m e (ml) i n 30 m i n u t e s . S t a r t v o l u m e was. 50 m l . V a l u e s a r e t h e mean o f f o u r e x p e r i m e n t s . * S t a n d a r d e r r o r o f t h e mean. SUMMARY AND CONCLUSIONS A. I s o l a t i o n o f P r o t e i n f r o m t h e Whey o f A c i d - m o d i f i e d G l u t e n 1. A b o u t 84% o f t h e p r o t e i n i n t h e g l u t e n whey was r e c o v e r e d b y h e a t i n g a f t e r a d d i t i o n o f f e r r i c c h l o r i d e a t pH. 1.0. 2. T h r e e w a s h i n g s w i t h pH 1.0 w a t e r y i e l d e d 8 0 . 48% p r o t e i n i n t h e d r i e d p r o d u c t . 3. A d d i t i o n o f s o d i u m h e x a m e t a p h o s p h a t e b e f o r e d r y i n g i m p r o v e d t h e c o l o r o f t h e end p r o d u c t . 4. The m a j o r p e a k o f H M P - F e - p r o t e i n e l u t e d b y g e l f i l t r a t i o n was e s t i m a t e d t o h a v e a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 6 2,000. 5. Amino a c i d a n a l y s i s o f H M P - F e - p r o t e i n showed l o w e r l y s i n e c o n t e n t t h a n o t h e r f r a c t i o n s . 6. P r o t e i n f r a c t i o n s r e c o v e r 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 h a d b e t t e r e s s e n t i a l amino a c i d b a l a n c e a s c o m p a r e d t o H M P - F e - p r o t e i n a l t h o u g h t h e y i e l d was c o n s i d e r a b l y l o w e r . B. F u n c t i o n a l P r o p e r t i e s o f D i f f e r e n t P r o t e i n F r a c t i o n s 1. E m u l s i f y i n g c a p a c i t y o f a l l t h e f r a c t i o n s r e c o v e r e d was b e t t e r t h a n t h a t o f v i t a l g l u t e n . The pH 6.2 p r e c i p i t a t e g a v e t h e b e s t e m u l s i f y i n g c a p a c i t y . H M P - F e - p r o t e i n showed r e s u l t s c o m p a r a b l e t o s o y i s o l a t e . 50. B. Cont'd 2. Emulsion s t a b i l i t y of a l l f r a c t i o n s was b e t t e r than v i t a l g l u t e n but HMP-Fe-protein showed t h r e e times b e t t e r s t a b i l i t y as compared to soy i s o l a t e . 3. Foam expansion and s t a b i l i t y of HMP-Fe-protein was i n f e r i o r as compared to other p r o t e i n f r a c -t i o n s and v i t a l g l u t e n . The pH 8.2 p r e c i p i t a t e showed the best r e s u l t s . 4. F o a m a b i l i t y of HMP-Fe-protein was g r e a t l y improved by changing pH of the sample s o l u t i o n to 5.0. 5. 99.2% of the n i t r o g e n i n HMP-Fe-protein was s o l u b l e i n water. From the r e s u l t s i t i s c l e a r t h a t the F e C l ^ method was q u i t e s u c c e s s f u l f o r r e c o v e r i n g p r o t e i n from whey of a c i d - m o d i f i e d g l u t e n . A study of f u n c t i o n a l p r o p e r t i e s shows t h a t recovered p r o t e i n may be t r i e d i n emulsions and as egg white s u b s t i t u t e . 5 1 . REFERENCES 1. A l - S u l i m a n y , F. a n d A. Townshend. 1 9 7 3 . S p e c t r e — p h o t o m e t r i c d e t e r m i n a t i o n o f m i c r o g r a m amounts o f a m i n o a c i d s w i t h c h l o r a n i l . A n a l . C h i m i c a A c t a 6 6:195. 2. A m a n t e a , G.F. , C M . K a s o n , S. N a k a i , D.B. B r a g g a n d D.B. Emmons. 1974. P r e p a r a t i o n o f f e r r i c whey p r o t e i n b y h e a t i n g . Can. I n s t . F o o d S c i . Tech:. J . 7:199. 3. A s s o c i a t i o n o f O f f i c i a l A g r i c u l t u r a l C h e m i s t s . 1970. 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