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Isolation and functional properties of protein fractions from rapeseed flour (Brassica campestris L.… Kodagoda, Lakshman Punniyadasa 1972

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ISOLATION AND FUNCTIONAL PROPERTIES OF PROTEIN FRACTIONS FROM RAPESEED FLOUR ( B r a s s i c a c a m p e s t r i s L. v a r . Echo) By LAKSHMAN PUNNIYADASA KODAGODA B.Sc. U n i v e r s i t y o f C e y l o n , 1961 M.Sc. U n i v e r s i t y of. Tokyo, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f Food S c i e n c e We a c c e p t t h i s t h e s i s as 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 UNIVERSITY OF BRITISH COLUMBIA March, 197 2. 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 for 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 for 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 Food Science The University of B r i t i s h Columbia Vancouver 8, Canada Date March 2U, 1972. ABSTRACT A t h r e e s t a g e method o f e x t r a c t i o n o f p r o t e i n s from r a p e s e e d f l o u r s e q u e n t i a l l y w i t h w a t e r , 0.1 N h y d r o c h l o r i c a c i d , and 0.02 N sodium h y d r o x i d e has been d e v e l o p e d . The f i r s t w a t e r e x t r a c t c o n t a i n e d m a i n l y a c i d i c and n e u t r a l p r o -t e i n s whereas the second h y d r o c h l o r i c a c i d e x t r a c t c o n t a i n e d b a s i c p r o t e i n s , w h i c h m i g r a t e t o t h e cathode by pH 8.8 g e l e l e c t r o p h o r e s i s . T h i s can p a r t i a l l y be e x p l a i n e d by h i g h e r a r g i n i n e c o n t e n t . Ash c o n t e n t i n t h e second h y d r o c h l o r i c a c i d e x t r a c t , a p p r o x i m a t e l y 36%, was r e d u c e d t o l e s s t h a n 10% by t r e a t i n g w i t h 0.05 M o x a l i c a c i d . The w a t e r e x t r a c t may be a good supplement t o o t h e r v e g e t a b l e p r o d u c t s because o f h i g h l y s i n e c o n t e n t . The a p p r o x i m a t e y i e l d s o f p r o t e i n s i n the p r e p a r a -t i o n o f i s o l a t e s were 11, 7, 41 and 61% r e s p e c t i v e l y f o r t h e w a t e r , h y d r o c h l o r i c a c i d and sodium h y d r o x i d e e x t r a c t i o n s and f o r t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i o n . P r o t e i n s were l o s t i n t h e whey d u r i n g i s o e l e c t r i c p r e c i p i t a t i o n , w i t h a c o n c o m i t e n t l o s s o f a c o n s i d e r a b l e amount o f c y s t i n e and m e t h i o n i n e . The e m u l s i f y i n g c a p a c i t y o f t h e w a t e r e x t r a c t was b e t t e r t h a n t h a t o f t h e o t h e r e x t r a c t s : 45 and 35 ml o f c o r n o i l p e r 10 0 mg o f p r o t e i n i s o l a t e r e s p e c t i v e l y . The c o n c e n t r a t e s showed a s i m i l a r t r e n d , but t h e volume o f o i l - i i -was a p p r o x i m a t e l y 2 5% l e s s f o r a l l p r e p a r a t i o n s . I n b a k i n g s t u d i e s , t h e r e p l a c e m e n t o f 5% o f t h e wheat f l o u r w i t h r a p e s e e d p r o t e i n i s o l a t e s p r e p a 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 caused an approximate 10 t o 15% d e c r e a s e i n l o a f volume. The b r e a d p r e p a r e d w i t h t h e con-c e n t r a t e s d e c r e a s e d l o a f volume by about 20%. The l o a f volume was r e s t o r e d i n most c a s e s by a d d i n g 0.5% Atmul 124 ( a mono- and d i g l y c e r i d e m i x t u r e m a n u f a c t u r e d by A t l a s C h e m i c a l I n d u s t r i e s ) t o t h e dough. With t h e i s o l a t e s from t h e f i r s t w a t e r and second h y d r o -c h l o r i c a c i d e x t r a c t s an i n c r e a s e i n l o a f volume o f 10 t o 15% was o b s e r v e d o v e r t h e c o n t r o l . W i t h t h e i s o l a t e s from t h e t h i r d sodium h y d r o x i d e and s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i o n s t h e l o a f volume was r e s t o r e d w i t h o u t any i n c r e a s e . The b r e a d p r e p a r e d from t h e c o n c e n t r a t e s t o g e t h e r w i t h e m u l s i f i e r showed a d e c r e a s e o f 12% i n l o a f volume e x c e p t t h e t h i r d sodium h y d r o x i d e e x t r a c t which had a 6% i n c r e a s e . Whip t e s t s showed t h e b e s t e x p a n s i o n o f meringue when t h e p r o t e i n i s o l a t e from t h e h y d r o c h l o r i c a c i d e x t r a c -t i o n was used a t a l e v e l o f 3% r e p l a c i n g egg w h i t e p r o t e i n , an even b e t t e r r e s u l t t h a n w h i p p i n g the egg w h i t e a l o n e . A l l o t h e r i s o l a t e s showed a d e c r e a s e i n e x p a n s i o n . The con-c e n t r a t e s e x c e p t t h e w a t e r e x t r a c t showed a s l i g h t d e c r e a s e . TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF PLATES ACKNOWLEDGEMENTS PART I ISOLATION OF PROTEINS FROM RAPESEED CBrassica campestris L. var. Echo) FLOUR CHAPTER I INTRODUCTION I I LITERATURE REVIEW I I I EXPERIMENTAL MATERIALS AND PROCEDURES M a t e r i a l s Procedures Determination of n i t r o g e n Determination of moisture and ash Determination of l i p i d s Determination of phosphorus Determination of calcium Determination of t o t a l s u l f u r Determination of carbohydrates Determination of crude f i b e r Determination of th i o o x a z o l i d o n e s isothiocyanate. and - i v -CHAPTER PAGE I I I 2. P r o c e d u r e s ( c o n t i n u e d ) C a l i b r a t i o n s t a n d a r d c u r v e s f o r p r o t e i n d e t e r m i n a t i o n 24 S l a b g e l e l e c t r o p h o r e s i s 24 D i s c g e l e l e c t r o p h o r e s i s 25 I s o e l e c t r i c f o c u s i n g 26 pH measurement _ 26 Amino a c i d a n a l y s i s 27 C y s t i n e d e t e r m i n a t i o n 27 Try p t o p h a n d e t e r m i n a t i o n 28 U l t r a c e n t r i f u g a t i o n 28 3. E x t r a c t i o n and p r e p a r a t i o n o f i s o l a t e s 30 Three s t a g e e x t r a c t i o n p r o c e d u r e 30 S i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i o n 30 O x a l i c a c i d t r e a t m e n t f o r d e c r e a s i n g ash c o n t e n t i n t h e h y d r o c h l o r i c a c i d e x t r a c t (SA) 34 P r e p a r a t i o n o f c o n c e n t r a t e s 34 P r e p a r a t i o n o f i s o l a t e s 34 P r e p a r a t i o n o f whey p r o t e i n s , 35 P r e p a r a t i o n o f i s o l a t e s from o x a l i c a c i d t r e a t e d h y d r o c h l o r i c a c i d e x t r a c t (SA°) 35 p H - s o l u b i l i t y p r o f i l e s o f t h e e x t r a c t s 36 - P r e p a r a t i o n o f crude m y r o s i n a s e 36 S o l u b i l i t y measurements o f i s o l a t e s and c o n c e n t r a t e s 37 IV RESULTS 38 C a l i b r a t i o n c u r v e s f o r p r o t e i n s 3 8 - V -CHAPTER P A G E IV RESULTS ( C o n t i n u e d ) p H - s o l u b i l i t y p r o f i l e o f t h e e x t r a c t s 38 I n f l u e n c e o f o x a l i c a c i d t r e a t m e n t on h y d r o c h l o r i c a c i d e x t r a c t 38 C o m p o s i t i o n o f t h e 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 45 E l e c t r o p h o r e s i s 53 I s o e l e c t r i c f o c u s i n g 56 Amino A c i d c o m p o s i t i o n 64 U l t r a c e n t r i f u g a t i o n 71 M a t e r i a l b a l a n c e o f i s o l a t e s 75 Loss o f P r o t e i n d u r i n g w ashing p r o c e d u r e 7 5 Y i e l d o f p r o t e i n i s o l a t e d , 78 S o l u b i l i t y measurements o f the i s o l a t e s • and c o n c e n t r a t e s 7 8 V DISCUSSION 81 PART I I FUNCTIONAL PROPERTIES OF THE RAPESEED PROTEIN FRACTIONS VI INTRODUCTION 89 V I I LITERATURE REVIEW 9 0 V I I I EXPERIMENTAL MATERIALS AND PROCEDURES 97 1. - M a t e r i a l s 97 2. P r o c e d u r e s 9 8 M i c r o l o a v e s b a k i n g method (10 grams) 98 C r u s t c o l o u r d e t e r m i n a t i o n 99 o - v i -CHAPTER PAGE V I I I 2. P r o c e d u r e s ( c o n t i n u e d ) D e t e r m i n a t i o n o f the e m u l s i f y i n g c a p a c i t y 99 S t a b i l i t y t e s t s f o r e m u l s i o n s 100 D e t e r m i n a t i o n o f w h i p p a b i l i t y 100 Manuf a c t u r e o f spreads 102 E n r i c h e d i m i t a t i o n i c e cream and i c e m i l k 104 IX RESULTS M i c r o l o a v e s o f b r e a d 106 E m u l s i f y i n g c a p a c i t y 116 E m u l s i o n s t a b i l i t y 118 W h i p p a b i l i t y 118 D r i p 119 The spreads from i s o l a t e s 119 E n r i c h e d i m i t a t i o n i c e cream and i c e m i l k 121 X DISCUSSION 122 XI GENERAL CONCLUSION 125 APPENDIX 126 LITERATURE CITED 129 LIST OF FIGURES FIGURE PAGE 1. FLOW DIAGRAM OF THE THREE STAGE EXTRACTION PROCEDURE 32 2. FLOW DIAGRAM OF THE SINGLE STAGE EXTRACTION PROCEDURE 3 3 3. EFFECT OF PROTEIN CONCENTRATION OF THE WATER AND HYDROCHLORIC ACID EXTRACT ON ABSORBANCE 39 4. EFFECT OF PROTEIN CONCENTRATION OF THE SODIUM HYDROXIDE EXTRACTS ON ABSORBANCE 40 5. pH-SOLUBILITY PROFILES OF THE WATER AND HYDROCHLORIC ACID EXTRACTS 41 6. pH-SOLUBILITY PROFILES OF THE SODIUM HYDRO-XIDE EXTRACTS 4 2 7. EFFECT OF OXALIC ACID ON CALCIUM REMOVAL FROM HYDROCHLORIC ACID EXTRACT 4 3 8. EFFECT OF OXALIC ACID TREATMENT OF THE HYDROCHLORIC ACID EXTRACT ON PROTEIN CONCENTRATIONS 44 9. SLAB GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION 54 - v i i i -FIGURE PAGE 10 SLAB GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN WHEY AFTER ISOELECTRIC PRECIPITATION 5 5 11. DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION 5 7 12. DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLORO-ACETIC ACID PRECIPITATION 58 13. DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN CONCENTRATES 59 . 14. DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN WHEY AFTER ISOELECTRIC PRECIPITATION 6 0 15. ISOELECTRIC FOCUSING OF THE RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPI-TATION 61 16. ISOELECTRIC FOCUSING OF THE RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLOROACETIC ACID PRECIPITATION 62 17. ISOELECTRIC FOCUSING OF THE RAPESEED PROTEIN CONCENTRATES 6 3 - i x -F I G U R E P A G E 1 8 . S C H L I E R E N P A T T E R N OF THE WATER E X T R A C T I S O L A T E . 72 1 9 . S C H L I E R E N P A T T E R N OF THE H C 1 E X T R A C T I S O L A T E 7 3 2 0 . S C H L I E R E N P A T T E R N OF THE NaOH E X T R A C T I S O L A T E 7H 2 1 . S C H L I E R E N P A T T E R N OF THE S I N G L E STAGE SODIUM H Y D R O X I D E E X T R A C T I S O L A T E 7H 2 2 . M I C R O L O A V E S P R E P A R E D FROM THE I S O L A T E S -WHOLE BREAD 108 2 3 . M I C R O L O A V E S P R E P A R E D FROM THE I S O L A T E S -CUT LOAVES 109 2 4 . M I C R O L O A V E S P R E P A R E D FROM THE CONCENTRATES -WHOLE BREAD 110 2 5 . M I C R O L O A V E S P R E P A R E D FROM THE CONCENTRATES -CUT L O A V E S 1 1 1 2 6 . M I C R O L O A V E S P R E P A R E D FROM P R O T E I N I S O L A T E TO STUDY THE E F F E C T ON VOLUME AND COLOUR -WHOLE BREAD 112 2 7 . M I C R O L O A V E S P R E P A R E D FROM P R O T E I N I S O L A T E TO STUDY THE E F F E C T OF VOLUME AND COLOUR -CUT L O A V E S 113 MICROLOAVES PREPARED FROM SOYBEAN PROTEIN ISOLATE TO STUDY THE EFFECT OF EMULSIFIERS ON VOLUME - WHOLE BREAD MICROLOAVES PREPARED FROM SOYBEAN PROTEIN ISOLATE TO STUDY THE EFFECT OF EMULSIFIERS ON VOLUME - CUT LOAVES LIST OF TABLES TABLE PAGE I PROXIMATE ANALYSIS OF RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION 46 I I PROXIMATE ANALYSIS OF RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLOROACETIC ACID PRECIPITATION 47 I I I PROXIMATE ANALYSIS OF RAPESEED PROTEIN CONCENTRATES PREPARED BY THE THREE STAGE AND SINGLE STAGE EXTRACTION 48 IV AMINO ACID COMPOSITION OF RAPESEED PROTEIN CONCENTRATES PREPARED BY THE THREE STAGE AND SINGLE STAGE EXTRACTION 6 5 V AMINO ACID COMPOSITION OF RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION 66 VI AMINO ACID COMPOSITION OF RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLOROACETIC ACID PRECIPITATION 67 V I I AMINO ACID COMPOSITION OF RAPESEED PROTEIN WHEY AFTER ISOELECTRIC PRECIPITATION 68 - x i i -TABLE PAGE V I I I PROTEIN MATERIAL BALANCE 76 IX PROTEIN LOSSES DURING WASHING PROCEDURE 77 X YIELDS OF PROTEINS ISOLATED ' 79 XI SOLUBILITY MEASUREMENTS 80 X I I COMPOSITION OF THE IMITATION MILK PRODUCTS 105 X H I a LOAF VOLUMES AND CRUST COLOUR MEASUREMENTS OF ISOLATES 107 X H I b LOAF VOLUMES AND CRUST COLOUR MEASUREMENTS OF CONCENTRATES - . 107 . XIV EMULSIFYING CAPACITY AND EMULSION STABILITY 117 XV MERINGUE SPECIFIC VOLUME AND DRIP MEASUREMENTS • 12 0 LIST OF PLATES PLATE PAGE I AMINCO DISTILLATION APPARATUS FOR NITROGEN '• 17 II EXTRACTION APPARATUS - LOURDES BLENDER 31 III EMULSIFICATION EQUIPMENT - OMNI MIXER 101 IV EQUIPMENT FOR WHIP MEASUREMENTS -SUNBEAM MIXMASTER 10 3 ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s h i s s i n c e r e g r a t i t u d e and a p p r e c i a t i o n t o Dr. Shuryo N a k a i , A s s o c i a t e P r o f e s s o r , Department o f Food S c i e n c e , 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 , f o r h i s s u p e r v i s i o n , i n d i s p e n s a b l e g uidance and h e l p f u l s u g g e s t i o n s d u r i n g t h e i n v e s t i g a t i o n and p r e p a r a t i o n o f t h i s d i s s e r t a t i o n . The a u t h o r would a l s o l i k e t o e x p r e s s h i s s i n c e r e t h a n k s t o Dr. W i l l i a m D. P o w r i e , Chairman, Department o f Food S c i e n c e f o r e n a b l i n g him t o o b t a i n t h e e x t e n s i o n o f l e a v e from t h e Government o f C e y l o n . The a u t h o r w i s h e s t o e x p r e s s h i s t h a n k s t o t h e Department o f A g r i c u l t u r e , C e y l o n , f o r r e l e a s i n g him from o f f i c i a l d u t i e s d u r i n g t h e c o u r s e o f t h i s s t u d y , and t o Dr. J.W.L. P e i r i s , Deputy D i r e c t o r A g r i c u l t u r e ( R e s e a r c h ) o f t h e C e n t r a l A g r i c u l t u r a l R e search I n s t i t u t e , Department o f A g r i c u l t u r e , C e y l o n , f o r i n i t i a t i n g t h e program. The a u t h o r acknowledges w i t h t h a n k s , t h e Canadian I n t e r n a t i o n a l Development Agency ( f o r m a l l y t h e E x t e r n a l A i d O f f i c e ) f o r p r o v i d i n g f i n a n c i a l a i d e n a b l i n g him t o come t o Canada f o r advanced s t u d y by way o f t h e generous Colombo P l a n s c h o l a r s h i p . - xv -The d e f i n i t i o n f o r r a p e s e e d 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 as p r o d u c t s e x t r a c t e d once from f l o u r and t h o s e w h i c h were p r o c e s s e d one s t e p f u r t h e r , r e s p e c t i v e l y , was used. I t i s g e n e r a l l y a c c e p t e d f o r s o y p r o t e i n p r o d u c t s t h a t s o y 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 a re d e f i n e d as p r o d u c t s c o n t a i n i n g a minimum o f 70 and 90% p r o t e i n (N x 6.25) on a m o i s t u r e f r e e b a s i s , r e s p e c t i v e l y . However, r a p e s e e d f l o u r used f o r e x t r a c t i o n o f p r o t e i n i n t h i s s t u d y was h e a t -t r e a t e d t o de c r e a s e g l u c o s i n o l a t e s b e f o r e e x t r a c t i o n , w h i c h made r e s i d u a l f a t e x t r a c t i o n d i f f i c u l t . W i th an i n t e n t i o n o f p r a c t i c a l a p p l i c a t i o n , no d r a s t i c s o l v e n t e x t r a c t i o n f o r removing t h e s e r e s i d u a l l i p i d s were a t t e m p t e d . The p r o t e i n c o n t e n t o f the c o n c e n t r a t e s and i s o l a t e s on a m o i s t u r e and f a t f r e e b a s i s (N x 6.25) are t a b u l a t e d below: C o n c e n t r a t e s I s o l a t e s F i r s t w a t e r e x t r a c t i o n 64. 6 86. 8 Second h y d r o c h l o r i c a c i d 45. 8 86. 6 T h i r d sodium h y d r o x i d e 75. 6 87. 5 S i n g l e s t a g e NaOH e x t r a c t i o n 76. 0 • 91. 3 1. These r e s u l t s are v e r y c l o s e t o the v a l u e o b t a i n e d f o r soybean p r o d u c t s . PART I ISOLATION OF PROTEIN FRACTIONS FROM RAPESEED FLOUR (Brassica campestris L. var. Echo) \ CHAPTER I INTRODUCTION Rapeseed ( B r a s s i c a sp) i s produced i n l a r g e q u a n t i -t i e s i n Canada e s p e c i a l l y f o r i t s o i l used i n t h e manufacture o f e d i b l e p r o d u c t s such as m a r g a r i n e , s h o r t e n i n g s and e d i b l e o i l s . F i f t y p e r c e n t o f t h e o i l seed crop i n Canada i s ra p e s e e d ( 2 9 ) . With t h e l a r g e tonnage o f t h e o i l seed c r o p produced a n n u a l l y f o r t h e o i l , t h e r e s u l t i n g meal has become an i m p o r t a n t s o u r c e o f meal p r o t e i n . Compared t o t h e meal produced p r e v i o u s l y , t h e p r e s e n t day meals a r e o f a b e t t e r q u a l i t y due t o improved t e c h n i q u e s used i n t h e p r o c e s s i n g o f t h e meal and f l o u r . The g l u c o s i n o l a t e s which a r e t h e p r e c u r s o r s o f t o x i c p r i n c i p l e s and t h e h i g h f i b e r c o n t e n t o f r a p e s e e d f l o u r and meal a r e t h e two major l i m i t i n g f a c t o r s a g a i n s t t h e u t i l i z a t i o n o f them as a p r o t e i n supplement i n human f o o d s . The c o l o u r o f t h e i s o l a t e s and c o n c e n t r a t e s may a l s o be a n o t h e r o b s t a c l e f o r t h e u t i l i z a t i o n o f them i n supplementary f o o d s . Smith (84) i n d i c a t e d t h a t t h e y i e l d o f i s o l a t e d p r o t e i n s from s u n f l o w e r and f l u x meal were low f o r comm e r c i a l p r o d u c t i o n . The y i e l d o b t a i n e d from r a p e s e e d by Owen and C h i c h e s t e r (65) u s i n g t h e i r e x t r a c t i o n p r o c e d u r e was 18%. S o s u l s k i and B a k a l (86) had a p r o t e i n r e c o v e r y o f 25%. Thus i t i s n o t e d t h a t t h e r e i s a l o s s o f p r o t e i n from r a p e s e e d i s o l a t 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 . 2. Most of the recent work on rapeseed protein has been carried out at the National Research Council, P r a i r i e Regional Laboratory, Saskatoon. Finlayson et_ aJL. (37) have been conducting extensive work on physical and chemical properties of rapeseed protein fractions p u r i f i e d by various chromatographic techniques. Bhatty e_t aJL. (14) have i s o l a t e d d i f f e r e n t protein fractions from rapeseed protein soluble i n s a l t solutions by using column chromatography. Rapeseed (Brassica napus L. var. Nugget) contained at least 9 chromatographically distinguishable protein components. Of these, there are 2 major proteins (12 S and 1.7 S), which together contained 35% of the seed protein and the 12 S protein i s an aggregate of several protein chains and has a large molecular weight (14). These workers have shown that the 12 S protein f r a c t i o n was chromato-graphically and e l e c t r o p h o r e t i c a l l y homogeneous i n s a l t solutions between pH 8.0 and 9.5, but dissociated into subfractions at pH values below 3.5 and i n urea solutions. In t h i s study research was i n i t i a t e d to f i n d an economical source of vegetable protein i s o l a t e s for the supplementation of protein d e f i c i e n t d i e t s . The primary objective was to develop an extraction procedure to obtain from rapeseed f l o u r nearly c o l o r l e s s protein i s o l a t e s with d i f f e r e n t c h a r a c t e r i s t i c s which had some functional pro-perties suitable for food processing. The proteins 3. r e s u l t i n g from t h i s s t u d y were compared w i t h t h a t o f c a s e i n and soybean 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 i n o r d e r t o e v a l u a t e t h e i r i m p o r t a n c e . CHAPTER I I LITERATURE REVIEW O r i g i n o f B r a s s l e a napus L. and B r a s s i c a c a m p e s t r i s L. i s not w e l l documented. The e a r l i e s t d i r e c t r e f e r e n c e t o t h e o i l s e e d r a p e s i s found i n t h e a n c i e n t S a n s k r i t w r i t i n g s o f 200 t o 1500 B.C. ( 8 2 ) . S i n s k a i a (83) a f t e r a s t u d y o f form found w i t h i n t h i s s p e c i e s i n Europe and A s i a , s u g g e s t e d t h a t t h e c e n t e r o f o r i g i n o f b o t h t u r n i p and t u r n i p r a p e would u l t i m a t e l y be l o c a t e d i n A s i a . On t h e o t h e r hand S i n s k a i a (83) n o t e d t h a t a l l c u l t i v a t e d A s i a n t u r n i p rape i s o f t h e summer fo r m , and t h u s c o n c l u d e d t h a t w i n t e r t u r n i p r a p e must have o r i g i n a t e d under m a r i t i m e c l i m a t e such as t h e M e d i t e r r a n e a n . I t i s p o s s i b l e t h a t t h e I n d i a n and European v a r i e t i e s were s e p a r a t e d a t an e a r l y s t a g e i n t h e d e v e l o p -ment o f t h e s p e c i e s and e v o l v e d a l o n g d i f f e r e n t l i n e s . I n w e s t e r n Canada r a p e s e e d was f i r s t grown commer-c i a l l y i n 1942, a war measure t o s u p p l y o i l f o r l u b r i c a t i o n o f marine e n g i n e s . A major b y - p r o d u c t o f t h i s o i l e x t r a c -t i o n i s t h e meal. W i t h t h e p r e s e n t use o f p r e p r e s s -s o l v e n t e x t r a c t i o n w h i c h i s not s u b j e c t t o h i g h t e m p e r a t u r e s d u r i n g p r o c e s s i n g , a meal comparable t o soybean i s produced. Rapeseed i s a c r o p w e l l adapted t o Canadian c o n d i t i o n s . The p r o d u c t i o n has i n c r e a s e d r a p i d l y u n t i l s u p p l y o f t h e seed has g r a d u a l l y exceeded d o m e s t i c demand. As a consequence i n r e c e n t y e a r s Canada has e x p o r t e d more r a p e s e e d t h a n a l l o t h e r c o u n t r i e s combined. A c c u r a t e s t a t i s t i c s o f t r a d e i n r a p e s e e d meal a r e n o t r e a d i l y a v a i l a b l e . However, i t has been e s t a b l i s h e d t h a t the major p r o d u c i n g c o u n t r i e s had an a n n u a l e x p o r t o f a p p r o x i m a t e l y 30,000 m e t r i c t o n s o f meal i n t h e p e r i o d 1958 t o 1962, ( 2 2 ) . I n t h e y e a r 1968/69 th e p r o d u c t i o n had i n c r e a s e d t o 5,323,000 t o n s and f o r t h e y e a r 1969/70 t h e r e was a s l i g h t d e c r e a s e w i t h 5,250,000 t o n s o f r a p e s e e d ( 2 3 ) . Of t h i s a p p r o x i m a t e l y 75% i s produced and consumed i n A s i a . I n Canada 2 t y p e s o f r a p e s e e d a r e produced: B r a s s i c a ri apu s L. ( A r g e n t i n e rape) and B r a s s i c a c a m p e s t r i s L ( P o l i s h r a p e ) . Of t h e 2 s p e c i e s t h e A r g e n t i n e v a r i e t y has a g r e a t e r p o t e n t i a l f o r seed and o i l t h a n t h e P o l i s h r a p e . But t h e v a r i e t i e s o f t h e l a t t e r a re p r e f e r r e d , i n Canada, because t h e y mature 10 t o 14 days e a r l i e r ( 3 0 ) . Thus summer forms a r e grown, as even t h e most hardy t u r n i p rape v a r i e t i e s w i l l n o t c o n s i s t e n t l y s u r v i v e on t h e open p l a i n s o f w e s t e r n Canada. The t u r n i p rape v a r i e t i e s Echo and A r l o occupy 70 t o 80% o f t h e r a p e s e e d acreage o f w e s t e r n Canada. Rapeseed r a n k s f i f t h i n w o r l d p r o d u c t i o n among v e g e t a b l e o i l s , exceeded by soybean, peanut, c o t t o n seed and s u n f l o w e r Canada which i s t h e l a r g e s t e x p o r t e r o f r a p e s e e d has 50% o f t h e o i l seed c r o p ( 2 9 ) . The l a r g e amount o f meal e n a b l e s a s u b s t a n t i a l q u a n t i t y o f p r o t e i n t o be a v a i l a b l e f o r consumption. The r a p e s e e d c r u s h e d i n modern m i l l s y i e l d s a p p r o x i m a t e l y 40% o i l and 50% o i l meal and t h e r e m a i n d e r i s m o i s t u r e . The 6 . p r o t e i n r i c h meal o r f l o u r , h a v i n g about 4 0% crude p r o t e i n , i s p r e s e n t l y u t i l i z e d f o r l i v e s t o c k and p o u l t r y f e e d i n g . The meal c u r r e n t l y produced d i f f e r from t h o s e t h a t were a v a i l a b l e p r e v i o u s l y . Modern meals a r e s u b j e c t e d t o l e s s h e a t d u r i n g p r o c e s s i n g and t h e amount o f o i l l e f t i n t h e meal i s g r e a t l y r e d u c e d , compared t o e x p e l l e r m e a l , i n w hich damage t o t h e p r o t e i n had o c c u r r e d d u r i n g p r o c e s s i n g . Because o f the r e d u c t i o n i n t h e amount o f h e a t used i n p r o -c e s s i n g t h e meals a r e o f much b e t t e r q u a l i t y t h a n t h o s e produced y e a r s ago. A l e x a n d e r and H i l l (1) found t h a t a u t o c l a v i n g a t 15 l b p r e s s u r e and d r y h e a t i n g a t 121°C s u n f l o w e r o i l meal p r e p a r e d by s o l v e n t e x t r a c t i o n caused a marked d e s t r u c t i o n o f l y s i n e i n t h e meal. These t r e a t m e n t s however d i d not a f f e c t m e t h i o n i n e . M o r r i s o n e t a l . (59 and 60) o b s e r v e d t h a t t h e n u t r i t i o n a l v a l u e o f s u n f l o w e r o i l meal i n c r e a s e d as t h e p r o c e s s i n g t e m p e r a t u r e was i n c r e a s e d . However, C l a n d i n i n and Robblee (19) a t t r i b u t e d t h e poor q u a l i t y o f t h e p r o t e i n from s u n f l o w e r o i l meal t o e x c e s s i v e p r o c e s s i n g t e m p e r a t u r e . I t has been r e p o r t e d by C r a i g (24-) t h a t t h e g r owing c o n d i t i o n s o f r a p e s e e d a f f e c t t h e c o m p o s i t i o n o f seed c o n s t i t u e n t s . For i n s t a n c e t h e degree o f u n s a t u r a t i o n o f f a t t y a c i d s was r e l a t e d t o t h e e n v i r o n m e n t a l c o n d i t i o n s d u r i n g growth. The g l u c o s i n o l a t e ( f o r m e r l y known as t h i o g l u c o s i d e ) c o n t e n t o f t h e r a p e s e e d i s p r o p o r t i o n a l t o 7. th e a v a i l a b l e s u l f u r i n t h e s o i l . T h i s has been demonstrated by Wetter (99) and J o s e f s s o n and A p p e l q v i s t ( 5 0 ) . E a r l i e r i t was r e p o r t e d t h a t p l a n t s o f B r a s s i c a napus L. v a r . nugget grown i n s o i l s d e f i c i e n t i n s u l f u r produced seeds w i t h a l o w e r c o n t e n t o f c e r t a i n p r o t e i n f r a c t i o n s and r e s u l t e d i n d i f f e r e n t c h r o m a t o g r a p h i c b e h a v i o u r o f t h e 12 S g l o b u l i n t h a n seeds produced from adequate f e r t i l i z e d p l a n t s ( 3 7 ) . These f i n d i n g s suggest t h a t t h e a v a i l a b i l i t y o f s o i l s u l f u r has a s i g n i f i c a n t e f f e c t on p r o t e i n s y n t h e s i s . The c o m p o s i t i o n o f r a p e s e e d i s h i g h l y i n c o n s i s -t e n t . Thus t h e r e a r e many c o m p o s i t i o n a l d i f f e r e n c e s from s p e c i e s t o s p e c i e s , v a r i e t y t o v a r i e t y and even w i t h i n a v a r i e t y . T h i s i s m a i n l y due t o t h e h i g h l y s e l f - f e r t i l e n a t u r e o f A r g e n t i n e rape and under f i e l d c o n d i t i o n s t h e y a r e l a r g e l y s e l f - p o l l i n a t e d ( 6 2 ) . B e l l (13) i n d i c a t e d i n a r e v i e w t h a t r a p e s e e d meal when i n c o r p o r a t e d i n t o p o u l t r y r a t i o n s a t l e v e l s o f 10% o r h i g h e r was g o i t r o g e n i c and d e t r i m e n t a l t o growth. I n a r e p o r t p u b l i s h e d by C l a n d i n i n e t a l . (20) i t was proved t h a t A r g e n t i n e rape ( B r a s s i c a napus L.) was more d e t r i m e n t a l t o growth o f c h i c k s and g o i t r o g e n i c t h a n t h e meal o r i g i n a t i n g from P o l i s h rape ( B r a s s i c a c a m p e s t r i s L . ) . Ten p e r c e n t A r g e n t i n e rape meal i n t h e r a t i o n d e p r e s s e d c h i c k g r o w t h , whereas much as 15% P o l i s h r a p e s e e d meal i n t h e r a t i o n d i d not r e s u l t i n a p p r e c i a b l e growth d e p r e s s i o n s . F i v e p e r c e n t A r g e n t i n e r a p e s e e d meal was shown t o double t h e t h y r o i d t o body w e i g h t r a t i o s w h i l e 15% P o l i s h r a p e s e e d meal was r e q u i r e d t o produce a s i m i l a r e f f e c t , on c h i c k s . S i m i l a r r e s u l t s were o b t a i n e d by Renner e t a l . ( 7 5 ) . L i m i t a t i o n s o f u s i n g r a p e s e e d i s due t o t h e pr e s e n c e o f low m o l e c u l a r w e i g h t s u l f u r compounds i n t h e se e d s , some o f whi c h may be r e l e a s e d t h r o u g h enzyme a c t i o n c a u s i n g m e t a b o l i c d i s t u r b a n c e . C l a n d i n i n e t a l . (20) r e p o r t e d t h a t i s o t h i o c y a n a t e c o n t e n t was markedly a f f e c t e d by v a r i e t y and no c o n s i s t e n t e f f e c t o f l o c a t i o n was a p p a r e n t . The t h i o o x a z o l i d i n e con-t e n t on t h e o t h e r hand was not o n l y a f f e c t e d by v a r i e t y , but a l s o appeared t o be i n f l u e n c e d by t h e e n v i r o n m e n t a l c o n d i t i o n s . The t h r e e major g l u c o s i n o l a t e s o f r a p e s e e d meal a r e g l u c o n a p i n , g l u c o b r a s s i c a n a p i n and p r o g o i t r i n . I n g l u c o n a p i n and p r o g o i t r i n t h e r a d i c a l R i s CH =CH-CH0-CH and CH 2=CH-CH(OH)CH 2 r e s p e c t i v e l y ( 5 ) . On enzyme h y d r o l y s i s t h e s e g l u c o s i n o l a t e s produce 3 - b u t a n y l i s o t h i o c y a n a t e , 4-p e n t y l i s o t h i o c y a n a t e and 5 - v i n y l o x a z o l i d i n e t h i o n e , r e s p e c t i v e l y . Sandburg and H o l l y (77) i n d i c a t e d t h a t t h i s system i s made up o f 2 e n t i t i e s , namely t h i o g l u c o s i d a s e and s u l f a t a s e . I t was su g g e s t e d by P i t t - R i v e r (68) t h a t t h e main mustard o i l o f r a p e s e e d , 3 - b u t e n y l i s o t h i o c y a n a t e might be c o n v e r t e d t o 5 - v i n y l t h i o o x a z o l i d o n e by o x i d a t i o n . Gmelin and V i r t a n e n (43) su g g e s t e d t h a t t h e g o i t r o g e n i c e f f e c t o f t h i o o x a z o l i d o n e s i s due t o t h e f a c t 9. t h a t t h e y i n h i b i t t h e s y n t h e s i s o f t h y r o i d hormones. T h i s e f f e c t cannot be overcome by h i g h doses o f i o d i n e . A n o t h e r type o f g o i t r o g e n i c s u b s t a n c e b e l o n g i n g t o the B r a s s i c a f a m i l y has a p r i m a r y i n f l u e n c e on t h e uptake o f i o d i n e by t h e t h y r o i d g l a n d . T h i s i n f l u e n c e can be p r e v e n t e d by i n c r e a s i n g the amount o f i o d i n e dose ( 4 3 ) . L i t e r a t u r e i n d i c a t e s t h a t s a l t s o f t h i o c y a n a t e (SCN~) r e p r e s e n t s t h i s t y p e o f c r u c i f e r s . I t has been p r o v e d by E t t l i n g e r and Lundeen (36) a n a l y t i c a l l y t h a t s i n i g r i n i s the p o t a s s i u m s a l t w h i c h r e a r r a n g e s d u r i n g enzyme c l e a v a g e t o a l l y l i s o t h i o c y a n a t e . F e e d i n g e x p e r i m e n t s have shown t h a t i n t a c t t h i o g l u c o s i d e s are not p a r t i c u l a r l y h a r m f u l but r a t h e r t o x i c due t o t h e i r h y d r o l y t i c p r o d u c t s such as i s o t h i o c y a n a t e s , o x a z o l i d i n e t h i o n e s and n i t r i l e s which are l i b e r a t e d by m y r o s i n a s e i n the seed i n t h e p r e s e n c e o f m o i s t u r e . The o x a z o l i d i n e t h i o n e s a re m a i n l y r e s p o n s i b l e f o r t h e e n l a r g e -ment o f the t h y r o i d ( 1 8 ) . O x a z o l i d i n e t h i o n e s a l s o appear t o be the compound p r i m a r i l y , i f not e n t i r e l y , r e s p o n s i b l e f o r g o i t r o g e n i c i t y . However i t has been shown t h a t i s o -t h i o c y a n a t e s can c y c l i z e t o form o x a z o l i d i n e t h i o n e s , w h i c h may account f o r the r a t h e r s i m i l a r e f f e c t s o f the 2 t y p e s o f r a p e s e e d compounds. A s u r v e y o f t h e Canadian r a p e s e e d c u l t i v e r s ( B r a s s i c a napus L. and B r a s s i c a c a m p e s t r i s L.) i n d i c a t e s a s i g n i f i c a n c e w i t h i n s p e c i e s c u l t i v a r d i f f e r e n c e i n g l u c o s i n -o l a t e c o n t e n t ( 1 0 0 ) . However d i f f e r e n c e s between c u l t i v a r s were r e l a t i v e l y s m a l l and none o f the c u l t i v a r s was found t o 10. be f r e e o r p r a c t i c a l l y f r e e o f g l u c o s i n o l a t e s . C u l t i v a r s from Japan and I n d i a were found t o be f r e e o f 4 - p e n t y l i s o t h i o c y a n a t e and o r 5 - v i n y l o x a z o l i d i n e - 2 - t h i o n e . I n p r e s e n t day p r o c e s s i n g t e c h n i q u e s , t h e enzyme i s d e s t r o y e d i n t h e i n i t i a l p r o c e s s i n g by steam h e a t and t h e g o i t r o g e n i c f a c t o r s are l e f t bound i n t h e o r i g i n a l g l u c o s i d e complex ( 1 0 1 ) . Eapan e t a l . (32) have d e v e l o p e d a wet method f o r i n a c t i v a t i n g the m y r o s i n a s e , the enzyme r e s p o n s i b l e f o r the p r o d u c t i o n o f t o x i c i s o t h i o c y a n a t e s and o x a z o l i d i n e t h i o n e s from t h e s u b s t r a t e g l u c o s i n o l a t e . They were a l s o a b l e t o remove g l u c o s i n o l a t e s by aqueous e x t r a c t i o n (33) by t h i s p r o c e s s t h e f i b r o u s h u l l s by a i r c l a r i f i c a t i o n t o o b t a i n a h u l l f r e e , creamy c o l o u r e d f l o u r and a gray meal. B a l l e s t e r e t a l . (9) d e v e l o p e d a w a t e r washing t e c h n i q u e f o r meal d e t o x i -f i c a t i o n t h a t was e f f e c t i v e i n e l i m i n a t i n g g l u c o s i n o l a t e s . The s a f e s t and the most e c o n o m i c a l s o l u t i o n however i s e i t h e r t o b r e e d s t r a i n s w i t h l i t t l e o r no s u l f u r c o n t a i n i n g g l u c o s i d e s i n the s e e d s , o r t o s e l e c t l i n e s which produce o n l y h a r m l e s s i s o t h i o c y a n a t e s upon g l u c o s i d i c h y d r o l y s i s . I t i s shown t h a t s u l f u r f e r t i l i z a t i o n has marked e f f e c t on t h e t o t a l c o n t e n t o f t h e s e compounds i n the s e e d s , but r e g a r d l e s s o f t h e l e v e l o f s u l f u r a p p l i e d , some v a r i e t i e s are c o n s i s t e n t l y low i n t o t a l i s o t h i o c y a n a t e s . U n t i l 1960 a l l v a r i e t i e s o f r a p e s e e d produced o i l c o n t a i n i n g 20 t o 2 5% e r u c i c a c i d , a monounsaturated 22 carbon c h a i n f a t t y a c i d . S t e f a n s s o n and Hougen (87) i s o l a t e d r a p e s e e d 11. strains which produced o i l p r a c t i c a l l y free of erucic acid. Oleic acid replaced erucic acid as the major constituent of these o i l s . Species containing o l e i c acid at lev e l s higher than 70% have been obtained. Beare et a l . (11) showed the deficiency of saturated f a t t y acids would r e s u l t in undesirable e f f e c t s of the o i l and p a r t i c u l a r l y the residual o i l i n the meal when used as feed, or for the preparation of i s o l a t e s or concentrates. It has been reported by Sosulski and Bakal (86) that acid p r e c i p i t a t i o n of a l k a l i extracts of protein from rapeseed gave low yi e l d s with brownish color. The protein i s o l a t e s obtained from sunflower was dark green i n color due to the formation of an insoluble complex between chlorogenic acid and protein (84). There i s no evidence that chlorogenic acid occurs i n rapeseed. The development of a green color i s believed to be due to oxidation of chlorogenic acid, a tannin l i k e compound present i n appreciable amounts in sunflower seeds was explained by Malic et a l . (56). Zdenek et a l . (104) have used countercurrent extraction for protein from rapeseed meal. In comparison with batch extraction under s i m i l a r conditions , extracts obtained by countercurrent extraction with sodium hydroxide or sodium bicarbonate contained more dry matter, t o t a l nitrogen and p r e c i p i t a t a b l e protein. This procedure i s not economically f e a s i b l e due to the complexity of the operation. Gheyasuddin et a l . (41) obtained almost c o l o r l e s s sunflower protein i s o l a t e s by maintaining the pH at 10.5 during extraction with 0.2 5% sodium s u l f i t e . The p r e c i p i -tated proteins were washed with 50% aqueous isopropyl alcohol, probably able to break H-bonds, to produce the nearly white i s o l a t e s . It i s known that polyphenols at acid pH remain combined with proteins by unusually strong H-bonds as shown by Lommis and Ba t t a i l e (54). Stronger H-bond breaking compounds, such as 50% aqueous ethanol, w i l l be even more e f f e c t i v e i n preventing the i n t e r a c t i o n between polyphenols and proteins. However such compounds denature proteins by destroying t h e i r native structure. Goding et a l . (44) has also reported p u r i f i c a t i o n of 12 S globulin i s o l a t e d from each of two species of rapeseed (Brassica napus L. and .Brassica campestris L.). They have been shown to be s i m i l a r in. terms of amino acid composition, amino terminal amino acid, number of subfractions and the carbohydrate content. While the present work was i n progress, Owen and Chichester (65) reported a process for the production of nontoxic rapeseed protein i s o l a t e s and an acceptable feed by-product. They extracted the rapeseed presscake with sodium chloride s o l u t i o n , p r e c i p i t a t e d with acid and obtained an i s o l a t e with l i g h t tan color a f t e r repeated washing with water. Tape et a l . (90) developed an aqueous extraction procedure for the removal of thioglucosides from crushed r a p e s e e d t o produce a f l o u r s u i t a b l e f o r human consumption. The d e f a t t e d r a p e s e e d f l o u r and meal produced by s t a n d a r d o i l e x t r a c t i o n and a i r c l a r i f i c a t i o n t e c h n i q u e s c o n t a i n o v e r 50% and 30% p r o t e i n r e s p e c t i v e l y . The t h i o g l u c o s i d e c o n t e n t o f each f r a c t i o n was a l m o s t f r e e . Lo and H i l l (53) have p r e p a r e d p r o t e i n c o n c e n t r a t e s from r a p e s e e d meal by e x t r a c t i n g w i t h 10% sodium c h l o r i d e s o l u t i o n f o l l o w e d by f i l t r a t i o n t o remove the seed c o a t s and d i a l y s i s a g a i n s t r u n n i n g w a t e r and f r e e z e d r y t h e d e s a l t e d r a p e s e e d m i l k . The method y i e l d s up t o 75% o f t h e o r i g i n a l meal n i t r o g e n i n the form o f p r o d u c t s c o n t a i n i n g 61 t o 76% p r o t e i n (N X 6.25). The p r o d u c t showed a c o n s i d e r a b l y g r e a t e r c o n t e n t o f ash and l o w e r c o n t e n t o f crude f i b e r and g l u c o -s i n o l a t e t h a n d i d t h e s t a r t i n g m a t e r i a l . The amino a c i d c o m p o s i t i o n was s i m i l a r and t h e phosphorus l e v e l d i d n o t change d u r i n g p r o c e s s i n g . J a n s o n (49) has r e p o r t e d columns f o r l a r g e s c a l e g e l f i l t r a t i o n on porous g e l s f o r t h e f r a c t i o n a t i o n o f r a p e s e e d p r o t e i n s and i n s u l i n . From t h i s method he o b t a i n e d a b r o a d peak c o n t a i n i n g a t l e a s t 4 main components. A l l o f t h e s e have been b a s i c p r o t e i n s h a v i n g I g r e a t e r t h a n 10 and w i t h m o l e c u l a r w e i g h t s a p p r o x i m a t e l y 15 t o 2 0,000. K o r o l e z u k and R u t t o w s k i (52) s t u d i e d t h e e x t r a c t i o n o f n i t r o g e n o u s compounds o f r a p e s e e d meal i n r e l a t i o n t o pH and t e m p e r a t u r e . They o b t a i n e d h i g h e x t r a c t a b i l i t y o f n i t r o g e n o u s compounds more t h a n 80% o f t o t a l n i t r o g e n a t pH 14. 9.5 t o 10 and 30 t o 45°C. F o r t h e r e g i o n o f pH 2 t e m p e r a t u r e s around 60°C gave about 55% e x t r a c t a b i l i t y . CHAPTER I I I EXPERIMENTAL MATERIALS AND PROCEDURES 1. M a t e r i a l s The r a p e s e e d f l o u r ( B r a s s i c a c a m p e s t r i s L. v a r . Echo) was a generous g i f t from t h e Food Research I n s t i t u t e o f t h e Canada Department o f A g r i c u l t u r e , Ottawa. The f l o u r was p r o c e s s e d by a p r o c e d u r e d e v e l o p e d by Tape e_t a l . (90) so as t o f r e e the p r o d u c t o f t o x i c t h i o g l u c o s i d e s and h u l l s . R e s i d u a l o i l i n the f l o u r was removed by e x t r a c -t i n g w i t h p e t r o l e u m e t h e r ( 5 7 ) . For t h e p r e p a r a t i o n o f p r o t e i n s f o r e l e c t r o p h o r e s i s the f l o u r was e x t r a c t e d w i t h c h l o r o f o r m : m e t h a n o l (2:1 v/v) m i x t u r e t o improve t h e r e s o l u t i o n . A l l c h e m i c a l s were a n a l y t i c a l grade o b t a i n e d from F i s h e r S c i e n t i f i c Co. L t d . , Vancouver, B.C. Ampholine pH 3 t o 10 was o b t a i n e d from Pharmacia U p p s a l a , Sweden. ,2. P r o c e d u r e s A. D e t e r m i n a t i o n o f n i t r o g e n Semi m i c r o K'j.eldahl p r o c e d u r e u s i n g an Aminco d i s -t i l l a t i o n a p p a r a t u s (see P l a t e 1) was used f o r t h e d e t e r m i n a -t i o n o f t o t a l n i t r o g e n . The m a t e r i a l s were d i g e s t e d i n t h e presence o f p o t a s s i u m s u l f a t e : s e l e n i u m powder (100:1 w/w) m i x t u r e t o g e t h e r w i t h a p i e c e o f copper w i r e w i t h c o n c e n t r a -t e d s u l f u r i c a c i d . F i f t y ml o f the d i s t i l l a t e was c o l l e c t e d i n 3% b o r i c a c i d c o n t a i n i n g a few drops o f mixed i n d i c a t o r , by steam d i s t i l l a t i o n w i t h 40% sodium h y d r o x i d e . P r o t e i n was c a l c u l a t e d by m u l t i p l y i n g the n i t r o g e n c o n t e n t by t h e c o n s t a n t 5.7. B. D e t e r m i n a t i o n o f m o i s t u r e and ash M o i s t u r e and ash were d e t e r m i n e d a c c o r d i n g t o the s t a n d a r d A.O.A.C. 1970 proced u r e ( 6 ) . C. D e t e r m i n a t i o n o f l i p i d s L i p i d s were e x t r a c t e d from t h e i s o l a t e s and concen-t r a t e s by w e t t i n g w i t h 1.0 ml o f a l c o h o l and t h e n 10.0 ml h y d r o c h l o r i c a c i d (24:11 by volume) was added and h y d r o l y z e d f o r 40 min. a t 80°C i n a w a t e r b a t h . A f t e r h y d r o l y s i s 10.0 ml a l c o h o l was added and t h e m i x t u r e was c o o l e d . These were then t r a n s f e r r e d t o M o j o n n i e r G 3 Fat E x t r a c t i o n F l a s k s . The c o n t a i n e r was r i n s e d w i t h 20.0 ml o f e t h e r added i n 3 e q u a l p o r t i o n s . The f l a s k s were s t o p p e r e d and shaken v i g o r o u s l y f o r 1.0 min., then 20.0 ml p e t r o l e u m e t h e r (b.p. 30-60°C) added and shaken a g a i n v i g o r o u s l y f o r 1.0 min. The e t h e r PLATE I AMINCO DISTILLATION APPARATUS FOR NITROGEN DETERMINATION 18. l a y e r was a l l o w e d t o s e p a r a t e from t h e aqueous phase. The o t h e r l a y e r was t h e n drawn o f f as much as p o s s i b l e and f i l t e r e d t h r o u g h a c o t t o n wool packed stem o f f u n n e l t o l e t e t h e r pass f r e e l y i n t o weighed be a k e r s c o n t a i n i n g b r o k e n g l a s s . R e e x t r a c t e d t h e r e m a i n i n g aqueous phase i n t h e tube t w i c e w i t h 15.0 ml each o f e t h e r as d e s c r i b e d b e f o r e . Draw t h e e t h e r l a y e r c o n t a i n i n g t h e f a t i n t o t h e same be a k e r . Wash t h e f u n n e l and t h e t i p o f t h e f u n n e l w i t h a few ml o f e q u a l volumes o f e t h e r and p e t r o l e u m e t h e r . The e t h e r was s l o w l y e v a p o r a t e d on a steam b a t h i n s i d e a fume hood. The bea k e r s c o n t a i n i n g t h e f a t were d r i e d i n an oven a t 100°C f o r 90 min. These were t h e n a l l o w e d t o s t a n d i n a i r and weighed t o c o n s t a n t w e i g h t . The r e s u l t s a r e e x p r e s s e d as % f a t by a c i d h y d r o l y s i s . D. D e t e r m i n a t i o n o f phosphorus Phosphorus c o n t e n t o f t h e i s o l a t e s and concen-t r a t e s was e s t i m a t e d by t h e method o f F i s k and Subbarow ( 3 8 ) . The o r g a n i c m a t t e r was d e s t r o y e d by t h e wet o x i d a -t i o n and t h e f o r m a t i o n o f phosphomolybdic a c i d complex w h i c h i s q u a n t i t a t i v e l y r e d u c e d t o h e t e r o p h o l y b l u e c o l o r , was measured by s p e c t r o p h o t o m e t r y . Samples o f t h e i s o l a t e s and c o n c e n t r a t e s were d i g e s t e d w i t h 6.0 ml o f n i t r i c a c i d : s u l f u r i c a c i d : p e r c h -l o r i c a c i d (5:1:2'by volume) m i x t u r e u n t i l w h i t e a c i d fumes e v o l v e d f o r 2 t o 3 h r s . On c o o l i n g t h e d i g e s t was t r a n s -ferred to a volumetric flask with 0.1 N hydrochloric acid. To aliquots of the digest was added 5.0 ml of 2.5% ammonium molybdate i n 5 N s u l f u r i c acid and d i l u t e d to 45 ml i n a 50 ml volumetric f l a s k . To t h i s was added 2.0 ml of 15.4% l-amino 2-naphthol 4-sulfonic acid : s u l f i t e - b i s u l f i t e dry mixture i n warm water. Made up to volume and the contents well mixed, the absorbance measured at 6 80 nm af t e r 25 mins. i n a Bausch and Lomb Spectronic 20 Colorimeter/Spectro-photometer. Concentration of phosphorus was read from a standard curve prepared with sodium phosphate. E. Determination of Calcium Calcium was determined by the method described by Ntailianas and Whitney (61). The samples of the i s o l a t e s and concentrates were ashed at 400°C for 2 hrs. i n a muffle furnace. The ash was dissolved i n 0.1 N hydrochloric acid and made to volume. The aliquots of the extract were added 5.0 ml of 0.024 M disodium dihydrogen ethylenediamine t e t r a -acetate dihydride and 8 N potassium hydroxide to increase pH to 13 followed b y 3 drops of 0.2% calcine (aminomethyl-fluroscein) i n d i c a t o r i n d i l u t e sodium hydroxide. The color of the sample becomes pink. Back t i t r a t e with standard 0.0 24 M calcium chloride s o l u t i o n , prepared by di s s o l v i n g calcium carbonate i n minimum amount of hydrochloric acid and d i l u t i n g to volume. The end point i s marked by a color change i n solution to a permanent green color. The t i t r a -t i o n was ca r r i e d out against a black background. 20. The amount of calcium was calculated a f t e r making corrections for the blanks. Results are expressed as percentages. F. Determination of t o t a l s u l f u r Total s u l f u r was determined i n the i s o l a t e s and concentrates of rapeseed proteins by the method of Bardsley and Lancaster (10). The samples were mixed with 0.5 g sodium bicarbonate thoroughly and an additional 0.5 g b i -carbonate was l a i d over and was fused at 600°C for 4 hrs. The fused material was then extracted with 2 5 ml of 0.46% sodium dihydrogen phosphate i n 2 N acetic acid i n 3 portions. After the reaction subsides, allow to stand for some time and f i l t e r through a Whatman No. 1 f i l t e r paper. To 10.0 ml aliquots add 1.0 ml of 0.5% fre s h l y prepared gum acassia i n 50% acetic acid, and 1.0 ml 6 N hydrochloric acid, and 0.5 g f i n e l y ground barium chloride. A f t e r one min. contents were shaken to dissolve the barium chloride. The absorbance was read a f t e r 5 mins. at 420 nm in a Spectronic 20 spectrophotometer. The amount of su l f u r was read o f f from a standard curve prepared with magnesium su l f a t e . G'. Determination of Carbohydrates Carbohydrates were determined i n the rapeseed pro-t e i n i s o l a t e s and concentrates by a method developed by Dubois et a l . (31) with a few modifications. Samples were mixed with 1.0 ml of 5% aqueous phenol (w/w) followed by 5.0 ml c o n c e n t r a t e d s u l f u r i c a c i d added d i r e c t l y w i t h a wide o p e n i n g p i p e t . A f t e r s t a n d i n g f o r 10 mins. t h e c o n t e n t s were mixed and , t h e absorbance was r e a d a t 480 nm on a S p e c t r o n i c 20 s p e c t r o p h o t o m e t e r . The c o n c e n t r a t i o n o f c a r b o h y d r a t e was r e a d o f f from a s t a n d a r d c u r v e . H. D e t e r m i n a t i o n o f crude f i b e r Crude f i b e r d e t e r m i n a t i o n i s based on t h e m a t e r i a l not s o l u b l e i n a c i d and a l k a l i , w h i c h i s p r o b a b l y due l a r g e l y t o c e l l u l o s e t o g e t h e r w i t h l i g n i n and p e n t o s a n s . The amount o f crude f i b e r i s r o u g h l y t h a t p o r t i o n o f f o o d w h i c h i s not a p p r e c i a b l y d i g e s t i b l e by most m o n o g a s t r i c a n i m a l s . Crude f i b e r i s e s t i m a t e d from t h e l o s s on i g n i t i o n o f d r i e d r e s i d u e r e m a i n i n g a f t e r d i g e s t i o n o f m a t e r i a l w i t h 0.255 N s u l f u r i c a c i d and 0.313 N osdium h y d r o x i d e under s p e c i f i c c o n d i t i o n s . Samples o f t h e r a p e s e e d i s o l a t e s and c o n c e n t r a t e s were mixed w i t h 0.5 g p r e d i g e s t e d a s b e s t o s and 10 0 ml o f 0.255 N s u l f u r i c a c i d added and b o i l e d f o r 30 mins. i n d i g e s t i n g a p p a r a t u s , a d j u s t e d t o b o i l i n 15 mins. t o g e t h e r w i t h a few drops o f a n t i f o r m A e m u l s i o n d i l u t e d w i t h w a t e r and c o n t a i n i n g some b o i l i n g c h i p s . F i l t e r t h e d i g e s t t h r o u g h a C a l i f o r n i a p o l y e t h y l e n e buchner f u n n e l f i t t e d w i t h a 200 mesh s c r e e n , w i t h s u c t i o n . The b e a k e r i s r i n s e d w i t h 35 ml b o i l i n g w a t e r and washed, the r e s i d u e w i t h 3 p o r t i o n s o f 25 ml b o i l i n g w a t e r . The r e s i d u e i s t h e n t r a n s -ferred back to the beaker and boiled for 30 mins. with 100 ml b o i l i n g 0.313 N sodium hydroxide as described before. The beaker i s rinsed with 35 ml b o i l i n g water and the residue i s washed with 15 ml of b o i l i n g 0.255 N s u l f u r i c acid and then with 3 portions of 25 ml b o i l i n g water followed by 15 ml of alcohol. The residue i s removed from the funnel by tapping into an ashing dish and heated for 2 hrs. at 130°C. It i s then cooled i n a desiccator and weighed. Then the dry residue i s ig n i t e d at 600°C for 30 mins., cooled i n a desiccator and reweighed. A blank determination was conduc-ted under the same conditions. Percentage crude f i b e r was calculated by determining the loss of weight on i g n i t i o n minus the loss i n weight of the asbestos blank mul t i p l i e d by 10 0 and divided by the weight of sample. I. Determination of isothiocyanate and t hiooxazolidones The isothiocyanate content was determined by the argentrimetric method of Viehoever et a l . (96), and Andre and Maille (3), i n which the mustard o i l reacts with ammonia forming substituted thiourea. The l a t t e r decomposes i n ammonical s i l v e r n i t r a t e forming insoluble s i l v e r s u l f i d e and monosubstituted carbodiamide. The unreacted s i l v e r i s then determined volumetrically by the Volhard method using potassium thiocyanate solution. The thiooxazolidones were determined by the method described by Astwood et 'al. (7) and modified by Wetter (98). Samples of the i s o l a t e s and concentrates weighing approximately 30 mg were mixed with 1.5 ml of 0.5% "crude myrosinase" i n 0.9% sodium chloride and 10.0 ml c i t r a t e buffer pH 4.0 added and macerated. The contents were then shaken f o r 2 hrs. at 2 5°C i n a mechanical shaker. The mixture was then d i s t i l l e d i n an Aminco d i s t i l l a t i o n appara-tus. The d i s t i l l a t e , approximately 25 ml was col l e c t e d i n 5 ml 0.1 N s i l v e r n i t r a t e containing 1.25 ml 10% ammonium hydroxide, surrounded by an ice and water bath. The control contained only the crude myrosinase solution. For isothiocyanate The d i s t i l l a t e , i n an a l l glass apparatus, f i t t e d with an a i r condenser was heated for 30 mins. i n a b o i l i n g water bath. The contents of the fl a s k was made up of 30 ml af t e r f i l t r a t i o n . To the f i l t r a t e was added 1.0 ml of 6 N n i t r i c acid and 0.5 ml of 8% f e r r i c ammonium s u l f a t e , and th i s mixture was t i t r a t e d to a f a i n t salmon color end point with 0.01 N potassium thiocyanate fr e s h l y d i l u t e d from an 0.1 N stock solution. For thiboxazo1idone s The suspension a f t e r d i s t i l l a t i o n was f i l t e r e d through Whatmann No. 42 f i l t e r paper and the pH of the f i l t r a t e was adjusted to 10.5 with 1.0 N sodium hydroxide. One m i l l i l i t e r of t h i s solution was extracted twice with 5.0 ml of anhydrous ethyl ether. The absorban.ce of the ether layer was measured at 230 nm, 248 nm and 266 nm a g a i n s t an e t h y l e t h e r b l a n k i n a Beckman DB s p e c t r o p h o t o -meter. The absorbance at 248 nm was c o r r e c t e d by s u b t r a c t i n g the average v a l u e o f absorbance a t 2 30 nm and 266 nm. J . C a l i b r a t i o n s t a n d a r d c u r v e s f o r p r o t e i n d e t e r m i n a t i o n A c a l i b r a t i o n s t a n d a r d curve f o r t h e d e t e r m i n a t i o n o f t h e p r o t e i n c o n t e n t o f the e x t r a c t s were p r e p a r e d by d i l u t i n g t h e e x t r a c t s from w a t e r , a c i d and base so t h a t a l i q u o t s o f t h e s e c o n t a i n e d 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 p r o -t e i n s from 0.5 t o 8.0 mg a p p r o x i m a t e l y . F o r the absorbance d e t e r m i n a t i o n 1.0 t o 10.1 ml o f the d i l u t e e x t r a c t s were a c i d i f i e d w i t h 2 drops o f 2 N s u l f u r i c a c i d and t h e volume made t o 20.0 ml w i t h 3 M u r e a a f t e r t h e e x t r a c t s were made e q u a l i n volume. The c o n t e n t s were mixed and t h e absorbance measured at 2 80 nm i n a Beckman DB s p e c t r o p h o t o m e t e r . N i t r o g e n was de t e r m i n e d by t h e K j e l d a h l method u s i n g t h e same d i l u t i o n s and c o n c e n t r a t e s as f o r the a b s o r -bance d e t e r m i n a t i o n . The n i t r o g e n was c o n v e r t e d t o p r o t e i n and a p l o t was made a g a i n s t absorbance. K. S l a b g e l e l e c t r o p h o r e s i s S l a b g e l e l e c t r o p h o r e s i s was c a r r i e d out i n h o r i z o n t a l p l a t e s u s i n g 10% p o l y a c r y l a m i d e g e l s made i n 0.175 M T r i s - g l y c i n e b u f f e r pH 8.8 c o n t a i n i n g 4.5 M u r e a and 0.1 M 2-me r c a p t o e t h a n o l as d e s c r i b e d by Raymond (74) and Tombs ( 9 1 ) . The g e l p l a t e s were p r e c o n d i t i o n e d b e f o r e a p p l i c a t i o n o f samples. The samples o f the i s o l a t e s , c o n c e n t r a t e s and whey were d i s s o l v e d i n 0.175 M T r i s g l y c i n e b u f f e r c o n -t a i n i n g 8 M u r e a and 0.1 M 2 - mercaptoethanol and soaked i n f i l t e r paper s t r i p s and a p p l i e d t o s l o t s i n the p l a t e s . E l e c t r o p h o r e s i s was c a r r i e d out f o r 18 h r s . a t 2 5 mA a t U°C. The p l a t e s were s t a i n e d i n 1% amido b l a c k i n 7% a c i d f o r 5 mins. and d e s t a i n e d i n 7% a c e t i c a c i d u n t i l a c l e a r back-ground was o b t a i n e d . L. D i s c g e l e l e c t r o p h o r e s i s Seven p e r c e n t p o l y a c r y l a m i d e g e l s c o n t a i n i n g 6 M u r e a and 0.1 M 2 - mercaptoethanol were made i n 6 cm l o n g and 0.6 cm i n t e r n a l d i a m e t e r g l a s s tubes a c c o r d i n g t o the p r o -cedure o f D a v i s (26) w i t h a few m o d i f i c a t i o n s ( 6 3 ) . The samples were t a k e n up i n T r i s g l y c i n e b u f f e r pH 8.6 c o n t a i n -i n g 8 M u r e a and 0.1 M 2 - mercaptoethanol and mixed w i t h a drop o f 0.01% bromophenol b l u e as an i n t e r n a l marker. The e l e c t r o l y t e s c o n t a i n e d T r i s - g l y c i n e b u f f e r pH 8.6 and e l e c t r o p h o r e s i s was c a r r i e d out a t 125 v o l t s a t a c u r r e n t o f 3 m i l l i a m p e r e s p e r tube f o r 75 mins. The g e l tubes a f t e r e l e c t r o p h o r e s i s were removed from t h e g l a s s t u b e s and s t a i n e d i n 1.0% aqueous coomassie b r i l l i a n t b l u e R 250 d i l u t e d 1 t o 20 w i t h 10.0% t r i c h l o r o a c e t i c a c i d b e f o r e u s e , a f t e r f i x i n g t h e g e l s f o r 30 mins. i n 12.5% t r i c h l o r o a c e t i c a c i d . The g e l s were d e s t a i n e d i n 10% t r i c h l o r o a c e t i c a c i d . M. I s o e l e c t r i c f o c u s i n g I s o e l e c t r i c f o c u s i n g was done a c c o r d i n g t o t h e methods d e s c r i b e d by Svensson ( 8 8 ) , V e s t e r b e r g and Svensson ( 9 5 ) , W r i g l e y ( 1 0 3 ) , K e n r i c k and M a r g o l i s (51) and C a t s i m -p o o l a s (16) by d i s c e l e c t r o p h o r e s i s u s i n g g e l s c o n t a i n i n g ampholine pH 3 t o 10, i n t h e presence o f u r e a . The e l e c t r o -p h o r e s i s was done at 25 0 v o l t s w i t h the c u r r e n t f a l l i n g from 50 mA t o 12 mA d u r i n g t h e f i r s t 30 mins. and e l e c t r o p h o r e s i s was m a i n t a i n e d f o r a f u r t h e r 60 mins. w i t h 0.4% p h o s p h o r i c a c i d i n t h e a n o d i c chamber and 0.5% e t h a n o l d i a m i n e a d j u s t e d t o pH 10.5 w i t h 1.0 N p o t a s s i u m h y d r o x i d e i n t h e c a t h o d i c chamber. The g e l s a f t e r e l e c t r o p h o r e s i s were removed and s t a i n e d i n .2% bromophenol b l u e s o l u t i o n i n e t h a n o l : a c e t i c a c i d : w a t e r (50:5:45 by volume) f o r 1 h r . and d e s t a i n e d by washing i n e t h a n o l : a c e t i c a c i d : e t h e r (30:5:65 by volume) as r e p o r t e d by Awdeh ( 8 ) . The g e l s were d e s t a i n e d f o r 12 t o 24 h r s . and were s t o r e d i n 7% a c e t i c a c i d f o r photography. N. pH measurement The g e l s a f t e r i s o e l e c t r i c f o c u s i n g i n d u p l i c a t e were c u t c o r r e s p o n d i n g t o t h e s t a i n e d bands and were macerated i n 0.5 ml w a t e r a f t e r 4 h r s . the pH was d e t e r m i n e d u s i n g F i s h e r e l e c t r o d e s -- m i c r o p r o b e c o m b i n a t i o n ( C a t . No. 13-639-92) i n a Beckman d i g i t a l pH meter. I n a n o t h e r e x p e r i m e n t t h e g e l s a f t e r i s o e l e c t r i c f o c u s i n g were c u t i n t o 5 mm l o n g s e c t i o n s and suspended i n 0.5 ml water . The pH o f t h e s e were r e a d a f t e r 4 h r s . 0. Amino acid analysis Hydrolyses of the i s o l a t e s , concentrates, and whey were car r i e d out i n sealed hydrolysis tubes for 24 hrs. at 110°C with 6 N (constant boiling)hydrochloric acid. The samples were dissolved i n 0.05 N sodium hydroxide and representative aliquots were freeze dried i n the presence of 1.0 ml 0.2 5 mM norleucine as i n t e r n a l standard. To these 5.0 ml hydrochloric acid were added and frozen and vacuum was applied a f t e r flushing with nitrogen and hydrolyzed i n a force draft oven. The hydrolysate was f i l t e r e d through a sintered funnel and evaporated in rotary evaporator repeat-edly by dis s o l v i n g i n water to remove residual hydrochloric acid. F i n a l l y , the residue was taken i n c i t r a t e buffer pH 2.2. The analysis was performed with a Phoenix Micro Amino Analyzer Model M 6800, Moore-Stein system. P. Cystine determination Cystine was determined as cysteic acid following performic acid oxidation as described by Schram et a l . (78) and Moore (58). Samples of the i s o l a t e s , concentrates and whey weighing 2 to 3 mg were mixed with 0.5 ml performic acid at 0°C and held for 16 hrs. at the same temperature (46). The excess performic acid was removed by freeze drying using sodium hydroxide traps. The hydrolysis was performed with 2.5 ml of 6 N hydrochloric acid for 20 hrs. at 110°C i n draft forced oven. The hydrolysate was f i l t e r e d through a sintered funnel (fine) to remove any pr e c i p i t a t e s and 28. then h y d r o c h l o r i c a c i d was removed on a r o t a r y e v a p o r a t o r under reduced p r e s s u r e a t U0°C. The r e s i d u e was t a k e n up i n 2.0 ml c i t r a t e b u f f e r pH 2.2. Q. Tryptophan d e t e r m i n a t i o n The t r y p t o p h a n was d e t e r m i n e d by the c o l o r i m e t r i c method o f I n g l i s and Lea v e r s ( 4 7 ) , w i t h a few m o d i f i c a t i o n s . To 1 ml o f c o n c e n t r a t e d s u l f u r i c a c i d , s l o w l y p i p e t t e d 0.5 ml o f 20% sodium h y d r o x i d e i n 40% m e t h a n o l , t h a n 2 mg samples o f t h e p r e p a r a t i o n s i n 0.4 ml w a t e r were added, and warmed f o r 20 se e s . To t h i s m i x t u r e add 4 ml g l a c i a l a c e t i c a c i d , 1.0 ml c o n c e n t r a t e d s u l f u r i c a c i d w i t h m i x i n g and 2 drops of 8% p o t a s s i u m p e r s u l f a t e r e a g e n t and t h e absorbance r e a d a t 550 nm a f t e r 20 mins. i n a S p e c t r o n i c 20 s p e c t r o p h o t o -meter. Try p t o p h a n c o n c e n t r a t i o n was r e a d o f f from a s t a n d a r d t r y p t o p h a n s o l u t i o n . The amount o f t r y p t o p h a n c a l c u l a t e d by t h e method o f E d e l h o c k (34) by t a k i n g about one t h i r d t he amount o f t y r o s i n e d e t e r m i n e d i n amino a c i d a n a l y s i s agree w i t h t h e above d e t e r m i n a t i o n s . R. U l t r a c e n t r i f u g a t i o n U l t r a c e n t r i f u g a t i o n o f t h e rap e s e e d p r o t e i n i s o l a t e s was made u s i n g a Beckman L2 6 5B a n a l y t i c a l u l t r a -c e n t r i f u g e . A l l s e d i m e n t a t i o n a n a l y s e s were made a t 2 5°C and 59,000 r.p.m. The p r o t e i n i s o l a t e s f o r s e d i m e n t a t i o n a n a l y s e s were prepared according to. method of Liu (55) by blocking the su l f h y d r y l groups, followed by r e v e r s i b l e blocking of amino groups as described by Dixon et a l . (28). To 100 mg protein i s o l a t e s , 5 g urea and 2 ml 0.1 M phosphate buffer followed by 4.6 ml water were added and the pH adjusted to 6.7 with 1 N sodium hydroxide. Then 0.0 9 ml of 5% ethylenediamine-tetraacetic acid was added and nitrogen was bubbled into the mixture for 15 mins. To the mixture was added 15 u l of 2-mercaptoethanol and incubated for 15 mins. at 37°C. To the l a t t e r reduction mixture was added 150 mg sodium tetrathionate (Na S 0 C. 2H 0) and a f t e r 5 mins. at 2 4 6 2 room temperature the pH of the mixture was adjusted to 6.6. This mixture was dialysed i n running water and f i n a l l y with demineralized water for 24 hours. The pH of the dialysed solutions were adjusted to 8 with 1 N sodium hydroxide and treated with 0.83 ml c i t r a -conic anhydride with s t i r r i n g at room temperature while maintaining the pH at 8 by adding 5 N sodium hydroxide. The base uptake ceased a f t e r 10 to 15 mins. The protein solution was desalted i n i t i a l l y by d i a l y s i s i n running tap water at 4°C followed by demineralized water for 2 days. The dialysed solutions were then adjusted to pH 8 and a f t e r centrifugation the c l e a r supernatants were freeze dried. For the sedimentation analyses, the freeze dried samples were dissolved i n 0.1 M T r i s - g l y c i n e buffer pH 8.5. The concentration of the proteins i n solution were 1%. 30. 3. E x t r a c t i o n and p r e p a r a t i o n o f i s o l a t e s A. Three s t a g e e x t r a c t i o n p r o c e d u r e The f l o u r was mixed w i t h water a t 4°C i n t h e r a t i o o f 1 : 15 and b l e n d e d f o r 3 min. u s i n g a Lourdes Model NM-1A b l e n d e r s e t a t 80, (see P l a t e 2 ) . The m i x t u r e was c l a r i f i e d a t 10,000 x g f o r 15 min. a t U°C i n a S o r v a l l RC2-3 c e n t r i -f u g e . The r e s i d u a l meal was r e - e x t r a c t e d t w i c e as b e f o r e . The combined s u p e r n a t a n t s were f i l t e r e d . The r e s i d u e from t h e above was e x t r a c t e d t w i c e u s i n g 0.1 N and 0.05 N h y d r o c h l o r i c a c i d a t pH o f 2 a t 60°C as d e s c r i b e d f o r t h e w a t e r e x t r a c t . The r e s i d u e was f u r t h e r e x t r a c t e d w i t h 0.02 t o 0.01 N sodium h y d r o x i d e a t 40°C a t pH 10. A f t e r 3 s u c c e s s i v e e x t r a c t i o n s s u p e r n a t a n t s were combined and f i l t e r e d . The f l o w diagram f o r t h e e x t r a c t i o n i s g i v e n i n F i g u r e 1. B. S i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i o n The f l o u r was e x t r a c t e d 3 ti m e s w i t h 0.01 N sodium h y d r o x i d e a t pH 10 and 40°C. The m i x t u r e was b l e n d e d f o r 3 mins. and c e n t r i f u g e d a t 10,000 x g f o r 15 mins. The combined s u p e r n a t a n t s were f i l t e r e d . Flow diagram f o r t h e e x t r a c t i o n i s g i v e n i n F i g u r e 2. The e x t r a c t s from t h e t h r e e and s i n g l e s t a g e p r o c e d u r e s , e x c e p t the second h y d r o c h l o r i c a c i d e x t r a c t , were made 0.15% w i t h r e s p e c t t o sodium s u l f i t e b e f o r e p r e -c i p i t a t i o n o f i s o l a t e s o r p r e p a r a t i o n s o f c o n c e n t r a t e s . PLATE I I EXTRACTION APPARATUS - LOURDES BLENDER 32 Rapeseed f l o u r (15g) $ Supernatant (SW) F i l t e r , add 0.15% NaoS0 Blend 3 min with 22 5 ml water at 4°C and centrifuge Residue and freeze dry Concentrate (CW) Isolates (P.W1 g P W2) i y r Blend 3 min with HC1 at pH 2 and 6 0°C and centrifuge Supernatant (SA) Residue Supernatant (SA°) F i l t e r and freeze dry Concentrate (CA) Add 0.0 5 M oxal i c acid and centrifuge Residue (discard) Blend 3 min with NaOH at pH 10, and 40°C and centrifuge Residue (Discard) Supernatant (SB) F i l t e r , add 0.15% Na oS0 o and freeze dry Concentrate (CB Isolates ( P iB 1 & P tB 2) Supernatants (SW, SA° 8 SB) Adjust to the I s o e l e c t r i c points w arm to 35°C and centrifuge Sediments Wash with water and 50% ethanol ^ ,5 and freeze dry^^ Isolates P.W,P.A° £ P.B) l i i dissolve at pH Supernatants (SW, SA S SB) Add 12.5% t r i c h l o r o a c e t i c acid warm to. 3 5°C and centrifuge r S ediments Wash with water and 50% ethanol dissolve at pH 8.5 and freeze dry J - b U l t i L e b F J (PtW,PtA & P tB) Figure 1. Flow diagram of the three stage . extraction procedure Rapeseed F l o u r (10 g) Bl e n d 3 min w i t h NaOH a t pH 10 and 40°C and c e n t r i f u g e S u p e r n a t a n t (SS) Fr e e z e d r y C o n c e n t r a t e (CS) F i l t e r , add 0.15% N a 2 S 0 3 A d j u s t pH 5.2, and warm t o 3 5°C c e n t r i f u g e Sediment P ^ Whey P. SU Wash w i t h w a t e r and 50% a l c o h o l A d j u s t pH 8.6 Freeze d r y Whey P tSU I s o l a t e _PiS Residue ( d i s c a r d ) Add 12.5% TCA, warm t o 35°C and cen-t r i f u g e 1 Sediment P. Wash w i t h w a t e r and 50% a l c o h o l A d j u s t pH 8. Freeze d r y I s o l a t e P.S t F i g u r e 2. Flow diagram o f t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i o n p r o c e s s . I n a n o t h e r p a r a l l e l s e r i e s o f e x p e r i m e n t s the f l o u r b e f o r e removal o f o i l was e x t r a c t e d by the t h r e e and s i n g l e s t a g e p r o c e d u r e , as d e s c r i b e d above. A l s o t h e f l o u r was e x t r a c t e d a t pH v a l u e s from 9.5 t o 11.5 i n o r d e r t o d e t e r m i n e the degree o f d e n a t u r a t i o n o f the i s o l a t e at the h i g h pH v a l u e s . C. O x a l i c a c i d t r e a t m e n t f o r d e c r e a s i n g ash c o n t e n t i n the h y d r o c h l o r i c a c i d e x t r a c t (SA) To 2 5.0 ml a l i q u o t s o f the h y d r o c h l o r i c a c i d ex-t r a c t were added 0.01 t o 0.0 8 M o x a l i c a c i d . The s o l u t i o n s were a l l o w e d t o s t a n d f o r 1 h r . The p r e c i p i t a t e which formed was removed by c e n t r i f u g a t i o n and the r e s u l t i n g s u p e r n a t a n t was used t o d e t e r m i n e the n i t r o g e n by t h e K j e l d a h l method. The r e s i d u e s l e f t a f t e r o x a l i c a c i d t r e a t m e n t were ashed and t a k e n up i n 0.1 N h y d r o c h l o r i c a c i d f o r t h e c a l c i u m d e t e r m i n a t i o n u s i n g t h e EDTA t i t r a t i o n method o f N t a i l i a n a s and Whitney ( 6 1 ) . D. P r e p a r a t i o n s f o r c o n c e n t r a t e s The c o n c e n t r a t e s were o b t a i n e d from t h e e x t r a c t s by f r e e z e d r y i n g . E. P r e p a r a t i o n o f i s o l a t e s The e x t r a c t s were a d j u s t e d t o t h e i r r e s p e c t i v e i s o e l e c t r i c p o i n t s by a d d i n g 0.5 N h y d r o c h l o r i c a c i d o r 0.5 N sodium h y d r o x i d e and t h e n warmed t o 35°C f o r 10 mins. The p r e c i p i t a t e s formed were c e n t r i f u g e d f o r 15 mins. a t 15,000 x g and washed t w i c e w i t h w a t e r a t 50°C, f o l l o w e d by two washings w i t h 50% e t h a n o l . The washed i s o l a t e s were d i s s o l v e d i n wa t e r a t pH 8.5 and f r e e z e d r i e d ( P ^ ) . The w a t e r e x t r a c t (SW) was a d j u s t e d t o pH 2.6, and t h e h y d r o -c h l o r i c a c i d e x t r a c t (SA) t o pH 3.6. The t h i r d sodium h y d r o -x i d e e x t r a c t and t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t were a d j u s t e d t o pH 4.2 and 5.2 r e s p e c t i v e l y . A l i q u o t s o f the e x t r a c t s were a l s o p r e c i p i t a t e d by a d d i n g 12.5% t r i c h l o r o a c e t i c a c i d and h e a t i n g t o 35°C f o r 10 mins. The i s o l a t e s a f t e r c e n t r i f u g a t i o n were washed as d e s c r i b e d f o r t h e i s o e l e c t r i c p r e c i p i t a t i o n , and d i s s o l v e d and t h e n f r e e z e d r i e d . . F. P r e p a r a t i o n o f whey p r o t e i n s The whey o b t a i n e d a f t e r t h e 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 f r e e z e d r i e d t o o b t a i n t h e whey p r o t e i n P^U. G. P r e p a r a t i o n o f i s o l a t e s from o x a l i c a c i d t r e a t e d h y d r o c h l o r i c a c i d e x t r a c t (SA°) The h y d r o c h l o r i c a c i d e x t r a c t was made 0.0 5 M w i t h o x a l i c a c i d and a f t e r s t a n d i n g f o r 1 h r . t h e m i x t u r e was c e n t r i f u g e d t o o b t a i n a s u p e r n a t a n t r e l a t i v e l y f r e e o f c a l c i u m . In one e x p e r i m e n t t h e s u p e r n a t a n t was a d j u s t e d t o pH 3.6 w i t h 0.5 N sodium h y d r o x i d e , and i n a n o t h e r , t h e e x t r a c t was a d j u s t e d t o 12.5% w i t h t r i c h l o r o a c e t i c a c i d . The i s o l a t e s formed were warmed t o 35°C f o r 10 mins. and s e p a r a -t e d by c e n t r i f u g a t i o n a t 15,000 x g f o r 15 mins. The i s o -l a t e s were washed t w i c e w i t h w a t e r a t 50°C and th e n t w i c e w i t h 50% a l c o h o l . The p r e c i p i t a t e s were t h e n d i s s o l v e d i n water at pH 8.5 and freeze dried,, to obtain i s o l a t e s P_^ A° and P A 0 . H. p H - s o l u b i l i t y p r o f i l e s of the extracts The determination of nitrogen s o l u b i l i t y as a function of pH of the consecutive extraction with water, hydrochloric acid and sodium hydroxide extracts, were car r i e d out as outlined below. To 2 5 ml aliquots of the 4 e x t r a c t s were added 0.5 N hydrochloric acid or 0.5 N sodium hydroxide to adjust the pH from 2 to 11. The volumes a f t e r pH adjustment were m ade equal and centrifuged at 15,00 0 x g for 15 mins. to obtain supernatant whey. The supernatant whey was used for the determination of nitrogen by the Kjeldahl method. Plots were made of the whey nitrogen against the pH of t h e respective aliquots. I. Preparation of "crude myrosinase" Myrosinase was prepared according to the method described by Wrede (101) from rapeseed meal. Five hundred grams of meal was mixed with 1800 ml of water at 4°C and allowed to stand for 1 hr. before blending. The s l u r r y was centrifuged at 4,000 x g for 15 mins. to separate the super-natant from the r e s i d u a l meal. The supernatant obtained was mixed with an equal volume of 90% ethanol at 4°C and the p r e c i p i t a t e formed was centrifuged at 14,000 x g for 15 mins. The p r e c i p i t a t e was washed with 7 0% ethanol and centrifuged. The residue was dissolved i n 400 ml of d i s t i l l e d water and 37. t h e n c e n t r i f u g e d t o remove any i n s o l u b l e m a t e r i a l t o o b t a i n t h e s o l u b l e f r a c t i o n w h i c h was f i l t e r e d and l y o p h i l i z e d . The y i e l d was 0.52% and t h e p r e p a r a t i o n was s t o r e d a t -20°C u n t i l r e q u i r e d . J . S o l u b i l i t y measurements o f i s o l a t e s and c o n c e n t r a t e s S o l u b i l i t y o f t h e p r e p a r a t i o n s were d e t e r m i n e d i n w a t e r a t 2 5°C by m i x i n g them t h o r o u g h l y . These were th e n c e n t r i f u g e d a t 27,000 x g f o r 30 mins. and t h e c l e a r s u p e r -n a t a n t was used f o r t h e n i t r o g e n d e t e r m i n a t i o n by t h e K j e l d a h l method. The p e r c e n t a g e o f s o l u b i l i t y was c a l c u l a -t e d f o r t h e s o l u b l e f r a c t i o n and i n r e l a t i o n t o t h e amount o f sample o r i g i n a l l y t a k e n f o r t h e d e t e r m i n a t i o n . \ CHAPTER IV RESULTS A. Cal i b r a t i o n curves for proteins As seen i n Figures 3 and 4, the plot of absorbance against mg nitrogen in the extracts shows a l i n e a r r e l a t i o n -ship. Each curve has a d i f f e r e n t gradient due to the di f f e r e n t absorbance at 280 nm- The nitrogen content of the extracts was calculated from these curves as a routine procedure. B. pH - s o l u b i l i t y p r o f i l e of the extracts The pH-nitrogen s o l u b i l i t y curves are shown i n Figures 5 and 6. From a study of the pH-nitrogen s o l u b i l i t y p r o f i l e s of the rapeseed protein extracts i t i s seen that the second hydrochloric acid extract (SA) showed 2 minimum points at pH 3.6 and 7. From these the lower pH showed a larger amount of protein p r e c i p i t a t e d than the higher pH point. With the water extract the shape of the curve was not hyperbolic as with the sodium hydroxide extracts, but a minimum point was observed i n the region of pH 2.6, with a broad region on eithe r side. The t h i r d sodium hydroxide extract showed a d i s t i n c t minimum whey nitrogen content at pH 4.2. The single stage sodium hydroxide extract showed a minimum around the pH 5.2. C. Influence of oxalic acid treatment on hydrochloric acid extract A gradual increase in the calcium content i n the gure 3. E f f e c t o f p r o t e i n c o n c e n t r a t i o n o f t h e f i r s t w a t e r e x t r a c t . . . o . . . o . , and second h y d r o c h l o r i c a c i d e x t r a c t © ® on absorbance P r o t e i n d e t e r m i n e d by K j e l d a h l method and t h e absorbance measured a t 2 80 nm i n 3 M u r e a . 1.4 L U o < CQ OC O CO CQ < 1.2 1.0 .8 .6 O 2 3 4 m g P R O T E I N gure 4. E f f e c t o f p r o t e i n c o n c e n t r a t i o n o f t h e t h i r d sodium h y d r o x i d e e x t r a c t -.-o-.-o-.- and s i n g l e s t e p sodium h y d r o x i d e e x t r a c t © •-. P r o t e i n was d e t e r m i n e d by K j e l d a h l method and absorbance measured a t 280 nm i n 3 M u r e a . A B S O R B A N C E O '—1 co • cn \ 41. Figure 5. pH-Solubility p r o f i l e s of the f i r s t water extract ...©...©..., and second hydrochloric acid extract - - o — o . 2 0 0 1 2 3 4 5 6 7 8 9 pH 42. F i g u r e 6. p H - S o l u b i l i t y p r o f i l e s o f the t h i r d sodium h y d r o x i d e e x t r a c t -.-o.-.-, and the s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t a 9 — . gure 7. E f f e c t o f t h e o x a l i c a c i d t r e a t m e n t the second h y d r o c h l o r i c a c i d e x t r a c t on c a l c i u m r e m o v a l . Q LU > o LU OC 3 o _J < o < oc h-X LU o cc LL 15 10 / / / o 2 3 -2 —o — 4 5 X10 M O X A L I C A C I D 8 4H . Figure 8. E f f e c t of the o x a l i c acid treatment of the second hydrochloric acid extract on protein content. 6.6 6.0 1 2 3 4 5 6 7 8 X 10~2M O X A L I C ACI D 45, A l s o w i t h t h e i n c r e a s i n g o x a l i c a c i d c o n c e n t r a -t i o n t h e r e was an i n c r e a s e o f 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 an i n i t i a l d e c r e a s e as i l l u s t r a t e d i n F i g u r e 8. A f t e r r e a c h i n g t h e maximum o f 0.05 M o x a l i c a c i d c o n c e n t r a t i o n t h e p r o t e i n c o n t e n t d e c r e a s e d a g a i n . The r e s i d u e a f t e r a d d i t i o n o f o x a l i c a c i d and c e n t r i f u g a t i o n c o n t a i n e d a p p r o x i m a t e l y 9% p r o t e i n . D. C o m p o s i t i o n o f t h e 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 The r e s u l t s a r e t a b u l a t e d i n T a b l e s I , I I and I I I . a. M o i s t u r e d e t e r m i n a t i o n s The m o i s t u r e c o n t e n t o f t h e i s o l a t e s p r e p a 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 P^ . ranged from 2.4 t o 5.3%, w h i l e t h e range f o r t h e i s o l a t e s P^_ was s l i g h t l y h i g h e r , t h a t i s 3.2 t o 5.6%. Among t h e i s o l a t e s t h e second h y d r o -c h l o r i c a c i d i s o l a t e s P.A and P^A had t h e h i g h e s t m o i s t u r e i t & c o n t e n t . The c o n c e n t r a t e s showed m o i s t u r e l e v e l s s i m i l a r t o t h o s e o f t h e i s o l a t e s . The f i r s t w a t e r and second h y d r o -c h l o r i c a c i d e x t r a c t s had m o i s t u r e l e v e l s o f a p p r o x i m a t e l y 6.7%. The two sodium h y d r o x i d e e x t r a c t s c o n c e n t r a t e s had m o i s t u r e c o n t e n t s o f 3.6%. b. P r o t e i n d e t e r m i n a t i o n The p r o t e i n c o n t e n t o f t h e i s o l a t e s o b t a i n e d by t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n (P^_) was s l i g h t l y h i g h e r t h a n t h a t o b t a i n e d f o r t h e i s o e l e c t r i c p r e c i p i t a t i o n ( P . ) . The v a l u e o b t a i n e d f o r t h e second h y d r o c h l o r i c a c i d TABLE 1. PROXIMATE ANALYSIS OF THE RAPESEED PROTEIN ISOLATES FROM ISOELECTRIC PRECIPITATION M o i s - P r o t e i n Carbo- F a t Ash Ca P S Crude I s o t h i o -t u r e h y d r a t e F i b e r c y a n a t e Cal — % P.W 4.7 72.5 2.9 4.2 5.0 0.16 3.31 0.22 1.12 0.89 l P ^ 5.3 71.6 1.3 4.8 9.0 1.37 1.82 0.64 1.80 0.55 2.3 73.9 2.8 5.8 3.3 1.25 2.50 0.27 1.31 0.30 P iS 2.4 77.3 3.4 5.2 5.3 0.15 2.52 0.47 1.22 0.65 (a) For d e t a i l s o f a b b r e v i a t i o n s see pages 32 and 33. TABLE I I PROXIMATE ANALYSIS OF THE RAPESEED PROTEIN ISOLATES FROM TRICHLOROACETIC ACID PRECIPITATION Mois-t u r e P r o t e i n Carbo-hydrate Fat Ash Ca P S Crude F i b e r I s o t h i o -cyanate 83. 3 0 . 7 5.3 1.2 _ % 0.73 0.69 0 .17 0.85 0.70 P t A 5 . 6 7 8.7 0 .9 5.2 1.2 1.12 1.15 0 . 38 1.67 0 . 56 P tB 3.2 81.9 1.1 5.4 1.1 0 . 84 2 . 30 0 . 26 1.24 0 . 22 p ts 5.1 82 . 7 0.6 4.3 1.2 0 .49 1. 34 0 . 39 1.33 0.42 (a) For d e t a i l s of abbreviations see pages 32 and 33. TABLE I I I PROXIMATE ANALYSIS OF RAPESEED PROTEIN CONCENTRATES M o i s -t u r e P r o t e i n Carbo-h y d r a t e F a t Ash Ca P S Crude F i b e r I s o t h i o -c y a n a t e CW ( a ) 6.6 5 2.4 11. 3 5.4 14.0 ._% 0.61 1.70 0. 32 3 . 35 0. 37 CA 6 . 8 37 . 3 4.5 4.7 33.6 2 . 57 6 . 31 0.11 3 .05 0 . 34 CB 3.6 62 . 8 3.0 5.9 19 . 3 0.57 1.9 0 0.31 2 .09 0 . 20 CS 3.6 63.5 4.1 5.3 16.7 0.74 0.80 0.60 2 . 21 0 . 24 (a) F o r c f e t a i l s o f a b b r e v i a t i o n s see pages 32 and 33. e x t r a c t i s o l a t e P_^. was 72% which i s low compared t o t h e i s o l a t e P . The i s o l a t e o b t a i n e d b e f o r e t h e o x a l i c a c i d t r e a t m e n t was 22% wh i c h i s f a r t o o low f o r an i s o l a t e . A l l o t h e r i s o l a t e s P^ and P^ had v a l u e s r a n g i n g from 72 t o 84%. P r o t e i n c o n t e n t i n t h e c o n c e n t r a t e s from t h e second h y d r o c h l o r i c a c i d and f i r s t w a ter e x t r a c t s were 37 and 52% r e s p e c t i v e l y . The c o n c e n t r a t e s from t h e t h i r d and s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t s (CB and CS) had a p p r o x i m a t e l y 63% p r o t e i n . c. " L i p i d d e t e r m i n a t i o n The l i p i d c o n c e n t r a t i o n o f t h e i s o l a t e s P^ ranged from 4.0 t o 5.8%, w h i l e t h e t r i c h l o r o a c e t i c a c i d p r e c i p i t a -t i o n i s o l a t e s P^_W and P^A had a s l i g h t l y g r e a t e r amount o f t o t a l l i p i d , e x c e p t f o r t h e t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e s P^B and P^S which were 5.4 and 4.3% l i p i d r e s p e c -t i v e l y , b e i n g l o w e r t h a n f o r t h e i s o l a t e s P^B and P^S. The l i p i d c o n t e n t o f t h e c o n c e n t r a t e s were s l i g h t l y h i g h e r t h a n t h e v a l u e s o b t a i n e d f o r t h e i r i s o l a t e s e x c e p t f o r the h y d r o c h l o r i c a c i d e x t r a c t . d. C a r b o h y d r a t e c o n t e n t The c a r b o h y d r a t e c o n t e n t o f t h e i s o l a t e s P^ were s l i g h t l y h i g h e r t h a n t h o s e p r e p a r e d by t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n P . The c a r b o h y d r a t e c o n t e n t ranged from 1.3 t o 3.4%. The i s o l a t e s P-S had t h e l a r g e s t v a l u e o f 3.4% w h i l e t h e i s o l a t e s P.W and P.B showed a v a l u e o f 2.8%. The second s t a g e h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P.A was t h e 50. l o w e s t w i t h 1.3%. The t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e d i s o l a t e s P had a c a r b o h y d r a t e c o n t e n t r a n g i n g from 0.64 t o 1.08%. Ca r b o h y d r a t e c o n t e n t o f t h e c o n c e n t r a t e s were h i g h e r as e x p e c t e d t h a n t h a t o f t h e i s o l a t e s . The w a t e r con-c e n t r a t e CW had a v e r y h i g h v a l u e o f 11.3%. The r e s t o f t h e c o n c e n t r a t e s ranged from 3.1 t o 4.5% which resemble t h e i s o l a t e s P^ i n t h e i r c a r b o h y d r a t e c o n t e n t . e. Ash c o n t e n t The ash c o n t e n t o f t h e i s o l a t e s P^ v a r i e d from 3 t o 9%. The ash c o n t e n t o f t h e i s o l a t e from t h e h y d r o c h l o r i c a c i d e x t r a c t (P^A) was d e c r e a s e d by o x a l i c a c i d t r e a t m e n t from 36.5 t o 9%, which was s t i l l h i g h e r t h a n o t h e r i s o l a t e s . The ash c o n t e n t o f t h e i s o l a t e s P^ were a p p r o x i m a t e l y 1.1% and t h e o x a l i c a c i d t r e a t m e n t i n c r e a s e d i t f o r i s o l a t e P^A 0 t o 2.0%. T h e r e f o r e t h e t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n was done w i t h o u t o x a l i c a c i d t r e a t m e n t f o r t h e h y d r o c h l o r i c a c i d e x t r a c t . The ash c o n t e n t o f t h e c o n c e n t r a t e s were h i g h e r , between 14 t o 19% e x c e p t 34% f o r t h e h y d r o c h l o r i c a c i d e x t r a c t (CA). The ash c o n t e n t f o r t h e s i n g l e s tage sodium h y d r o x i d e e x t r a c t was 16.7%. f . ' C a l c i u m c o n t e n t The c a l c i u m c o n t e n t o f t h e i s o l a t e s P^ ranged from 0.14 t o 1.4%. The c a l c i u m c o n t e n t o f t h e o x a l i c a c i d t r e a t e d h y d r o c h l o r i c a c i d e x t r a c t P.A° had t h e h i g h e s t v a l u e o f 1.4%. The t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e d p r o t e i n i s o l a t e s Pi-c o n t a i n e d 0.5 t o 1.1% c a l c i u m . The i s o l a t e P tS showed t h e low v a l u e o f 0.5%. The c a l c i u m c o n t e n t o f t h e h y d r o c h l o r i c a c i d e x t r a c t b e f o r e o x a l i c a c i d t r e a t m e n t was a p p r o x i m a t e l y 2.6%. The c o n c e n t r a t e s had a c a l c i u m c o n t e n t r a n g i n g from 0.6 t o 0.8% e x c e p t f o r t h e h y d r o c h l o r i c a c i d e x t r a c t (CA) which was 2.6% s i m i l a r t o t h e i s o l a t e P.A. l g. T o t a l s u l f u r The s u l f u r c o n t e n t o f t h e i s o e l e c t r i c p r e c i p i t a t e d i s o l a t e s P^ was h i g h e r t h a n t h a t o f t h e t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e d i s o l a t e s P^. The i s o l a t e Pj_A 0 had a c o n c e n t r a -t i o n o f 0.64% which i s n e a r l y t w i c e t h a t p r e s e n t i n t h e i s o l a t e P^A. The i s o l a t e s P^B, P^ W and P^ W had l o w e r v a l u e s o f 0.24, 0.22 and 0.17% r e s p e c t i v e l y . The s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i s o l a t e s P^S and P^S had 0.47 and 0.39% r e s p e c t i v e l y . The amount o f s u l f u r p r e s e n t i n t h e c o n c e n t r a t e s was q u i t e s i m i l a r t o t h a t i n t h e i s o l a t e s , e x c e p t concen-t r a t e s CS and CA h a v i n g 0.6 0 and 0.11% compared t o 0.47 and 0.64% i n t h e r e s p e c t i v e i s o l a t e s P^S and P-^A0 . Thus t h e s u l f u r c o n t e n t i n t h e c o n c e n t r a t e CA i s v e r y much l e s s t h a n t h a t p r e s e n t i n t h e i s o l a t e P^A°. The c o n c e n t r a t e s CW and CB had a s u l f u r c o n t e n t o f 0.3%. h. Phosphorus 'content The phosphorus c o n t e n t o f t h e i s o l a t e s P-j_ and P^ v a r y from 0.7 t o 3.3%. The amount o f phosphorus i n t h e i s o -l a t e s P t was l o w e r t h a n t h e i s o l a t e s P^. The i s o l a t e s Pj_A 0 and P^A were 1.8 and 11.6% phosphorus w i t h and w i t h o u t o x a l i c a c i d t r e a t m e n t r e s p e c t i v e l y . .The h i g h e s t v a l u e was i n t h e i s o l a t e P^W. The t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e d i s o l a t e P-tB a l s o had a h i g h v a l u e o f 2.3% phosphorus. The amount o f t h e phosphorus i n t h e t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e s P-j_B and P^B d i d not show much o f a d i f f e r e n c e as n o t e d f o r t h e o t h e r i s o l a t e s . The c o n c e n t r a t e s had phosphorus c o n t e n t s s i m i l a r t o the i s o l a t e s . The h i g h e s t amount among t h e c o n c e n t r a t e s , 6.3% was o b s e r v e d i n t h e p r e p a r a t i o n CA. The o t h e r concen-t r a t e s ranged from 0.9 t o 2.0%. The s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e CS had t h e l o w e s t v a l u e and was c l o s e t o t h a t o b t a i n e d by Lo and H i l l (1971) w i t h a con-c e n t r a t i o n o f 1.1%. i . Crude f i b e r Crude f i b e r c o n t e n t o f t h e i s o l a t e s P^ ranged from 0.9 t o 1.1%, w h i l e t h e i s o l a t e s P^ had a s l i g h t l y h i g h e r range from 1.1 t o 1.8%. The second s t a g e h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A° had t h e h i g h e s t v a l u e 1.8%. P u b l i s h e d d a t a f o r soybean p r o t e i n i s o l a t e s i s 0.7%. The c o n c e n t r a t e s had a crude f i b e r c o n t e n t s l i g h t l y h i g h e r , r a n g i n g from 2.3 t o 3.7%. The wa t e r e x t r a c t concen-t r a t e CW had t h e v a l u e o f 3.7% w h i l e t h e h y d r o c h l o r i c a c i d e x t r a c t c o n c e n t r a t e had 3.1%. The soybean p r o t e i n concen-t r a t e s were r e p o r t e d t o c o n t a i n 3%. j . I s o t h i o c y a r i a t e and' t h i o o x a z o l i ' d o n e .content The i s o t h i o c y a n a t e c o n t e n t o f t h e i s o l a t e s p r e p a 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 had h i g h e r amounts t h a n t h e t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e d i s o l a t e s P , e x c e p t t h e h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e s . The f i r s t w a t e r e x t r a c t i s o l a t e P^ W had a h i g h v a l u e o f 0.9%, w h i l e t h e t h i r d b a s i c e x t r a c t i s o l a t e s P.B and P.B had v a l u e s o f 0.32 and 0.22% i t r e s p e c t i v e l y f o r b o t h which are t h e l o w e s t v a l u e s o b t a i n e d . The i s o l a t e s P.S and P.S had v a l u e s o f 0.65 and 0.42% l t i s o t h i o c y a n a t e r e s p e c t i v e l y . The c o n c e n t r a t e s had a l o w e r range f o r t h e i s o t h i o -c y a n a t e c o n t e n t was from 0.2 t o 0.37%. The water e x t r a c t c o n c e n t r a t e had t h e h i g h e s t v a l u e o f 0.37%. There was no d e t e c t a b l e t h i o o x a z o l i d o n e p r e s e n t i n t h e i s o l a t e s and c o n c e n t r a t e s p r e s e n t by t h e method used. E. E l e c t r o p h o r e s i s (a) S l a b g e l As i n d i c a t e d s l a b g e l e l e c t r o p h o r e s i s o f i s o l a t e s P^ i n F i g u r e 9, most o f t he a c i d i c and n e u t r a l p r o t e i n s were e x t r a c t e d w i t h i n t h e f i r s t s t a g e whereas t h e second s t a g e h y d r o c h l o r i c a c i d e x t r a c t e d o n l y t h e b a s i c p r o t e i n f r a c t i o n s . The r e s i d u a l f r a c t i o n s appear to. be e x t r a c t e d i n t h e t h i r d sodium h y d r o x i d e e x t r a c t , o f which p a t t e r n was s i m i l a r t o t h a t o f t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t . The i s o l a t e ure 9. S l a b g e l e l e c t r o p h o r e t i c p a t t e r n o f t h e r a p e s e e d p r o t e i n i s o l a t e s p r e p a 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 . S t a i n e d i n 0.2% Amido b l a c k i n 7% a c e t i c a c i d . W - f i r s t w a t e r e x t r a c t i s o l a t e P.W l A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e P^B S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e P^S. 55 . F i g u r e 1 0 . S l a b g e l e l e c t r o p h o r e t i c p a t t e r n o f t h e ra p e s e e d p r o t e i n whey (U) a f t e r i s o e l e c t r i c p r e c i p i t a t i o n , P-j_U. S t a i n e d w i t h 0.2% Amido b l a c k i n 7% a c e t i c a c i d and d e s t a i n e d i n 5% a c e t i c a c i d . W - f i r s t w a t e r e x t r a c t i s o l a t e whey P.WU J x A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e whey P^AU B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e whey P^BU S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e whey P^SU. P^ . and c o n c e n t r a t e s showed s i m i l a r p a t t e r n s . The whey from the i s o l a t e s o b t a i n e d a f t e r i s o e l e c t r i c p r e c i p i t a t i o n showed a number o f bands c o r r e s p o n d i n g t o t h e i r i s o l a t e s . T h i s i s shown i n F i g u r e 10. The whey from t h e w a t e r e x t r a c t tends t o show a t r a i l i n g e f f e c t , (b) D i s c g e l D i s c g e l e l e c t r o p h o r e s i s o f t h e i s o l a t e s P^ and P and the c o n c e n t r a t e s are shown i n F i g u r e s 11, 12 and 13. The e l e c t r o p h o r e s i s p a t t e r n s f o r t h e d i f f e r e n t p r e p a r a t i o n s show a d i s t i n c t number o f bands. Thus f o r example the w a t e r e x t r a c t i s o l a t e s and c o n c e n t r a t e show a s i m i l a r s e p a r a t i o n o f p r o t e i n components. T h i s i s t r u e f o r a l l the e x t r a c t s t h a t were made. The whey p r o t e i n i s o l a t e s shown i n F i g u r e 14 a l s o show a s i m i l a r o a t t e r n t o t h a t o f the i s o l a t e P.. l F„ I s o e l e c t r i c f o c u s i n g The pH range 3 t o 10 f o r e l e c t r o f o c u s i n g e n a b l e s one t o o b t a i n a complete i s o e l e c t r i c spectrum o f b o t h a c i d i c and b a s i c p r o t e i n s . I t was seen from t h e s e e x p e r i m e n t s as i n d i c a t e d i n F i g u r e s 15 , 16 and 17 t h a t the m a j o r i t y o f t h e p r o t e i n i s o l a t e s P.A°, P.B and P.S have i s o e l e c t r i c p o i n t s between pH 7 and 10, e x c e p t f o r the i s o l a t e P^ W which l i e s between 3 and 7. T h i s p i c t u r e was t r u e f o r t h e i s o l a t e P and the c o n c e n t r a t e s . As seen i n t h e s e f i g u r e s some o f the p r o t e i n s p r e c i p i t a t e d d u r i n g e l e c t r o p h o r e s i s and m i g r a t e d t o the a c i d i c s i d e a t t h e top o f t h e column. A f a i r amount o f gure 11. D i s c g e l e l e c t r o p h o r e t i c p a t t e r n o f t h e r a p e s e e d p r o t e i n i s o l a t e s p r e p a 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 . B u f f e r : T r i s g l y c i n e , pH 8.6. S t r a i n e d w i t h coomassie b r i l l i a n t b l u e R 250 i n 10% t r i c h l o r o a c e t i c a c i d . W - f i r s t w a t e r e x t r a c t i s o l a t e P.W I A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e P-j_B S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e P^S. w A + B S gure 12. D i s c g e l e l e c t r o p h o r e t i c p a t t e r n o f t h e r a p e s e e d p r o t e i n i s o l a t e s p r e p a r e d by t r i c h l o r o -a c e t i c a c i d p r e c i p i t a t i o n . B u f f e r : T r i s g l y c i n e , pH 8.6. S t a i n e d w i t h coomassie b r i l l i a n t b l u e R 250 i n 10% t r i c h l o r o a c e t i c a c i d . W - f i r s t w a t e r e x t r a c t i s o l a t e P^ W A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e P^ _B S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e P-j-S. F i g u r e 13. D i s c g e l e l e c t r o p h o r e t i c p a t t e r n o f t h e r a p e s e e d p r o t e i n c o n c e n t r a t e s . B u f f e r : T r i s g l y c i n e , pH 8.6. S t a i n e d w i t h coomassie b r i l l i a n t b l u e R 250, i n 10% t r i c h l o r o a c e t i c a c i d . W - f i r s t w a t e r e x t r a c t c o n c e n t r a t e CW A - second h y d r o c h l o r i c a c i d e x t r a c t c o n c e n t r a t e CA B - t h i r d sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e CB S - s i n g l e s t a g e NaOH e x t r a c t c o n c e n t r a t e CS. ure 14. D i s c g e l e l e c t r o p h o r e t i c p a t t e r n o f t h e r a p e s e e d p r o t e i n whey a f t e r i s o e l e c t r i c p r e c i p i t a t i o n . B u f f e r : T r i s g l y c i n e , pH 8.6. S t a i n e w i t h coomassie b r i l l i a n t b l u e R 2 50 i n 10% t r i c h l o r o a c e t i c a c i d . W - f i r s t w a ter e x t r a c t whey P^WU A - second h y d r o c h l o r i c a c i d e x t r a c t whey P^AU B - t h i r d sodium h y d r o x i d e e x t r a c t whey P.BU l S - s i n g l e s t a g e NaOH e x t r a c t whey P^SU. gure 15. I s o e l e c t r i c f o c u s i n g o f t h e r a p e s e e d p r o t e i n i s o l a t e s p r e p a 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 , a t pH range 3 and 10. S t a i n e d i n 0.2% bromophenol b l u e W - f i r s t w a t e r e x t r a c t i s o l a t e P^ W A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e Pj_B S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e Pj_S. gure 16. I s o e l e c t r i c f o c u s i n g o f t h e r a p e s e e d p r o t e i n i s o l a t e s p r e p a r e d by t r i c h l o r o a c e t i c a c i d p r e c i p i t a -t i o n , a t pH range 3 and 10. S t a i n e d i n 0.2% bromo-p h e n o l b l u e . W - f i r s t w a t e r e x t r a c t i s o l a t e P^ W A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e P^B S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e P t S . gure 17. I s o e l e c t r i c f o c u s i n g o f t h e r a p e s e e d p r o t e i n c o n c e n t r a t e s , a t pH range 3 and 10. S t a i n e d i n 0.2% bromophenol b l u e . W - f i r s t w a t e r e x t r a c t c o n c e n t r a t e CW A - second h y d r o c h l o r i c a c i d e x t r a c t c o n c e n t r a t e CA B - t h i r d sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e CB S - s i n g l e s t a g e NaOH e x t r a c t c o n c e n t r a t e CS. protein, p a r t i c u l a r l y from the. a c i d i c and basic extracts, migrate to the basic side at the bottom of the icol'umn. The r e s u l t s for the whey proteins showed s i m i l a r patterns as for the i s o l a t e s . When i s o e l e c t r i c focusing was carr i e d i n gel con-tai n i n g no urea e s p e c i a l l y the hydrochloric acid extract i s o l a t e s tend to remain at the top of the column. G. Amino acid composition The amino acid composition of the i s o l a t e s and con centrates are given i n Tables 4, 5, 6 and 7. In the concen trates CW and CA there were higher lev e l s of glutamic a c i d , cystine and methionine, 30 and 45%, 33 and 45% and 22 and 2 more than i n the i s o l a t e s P.W and P.A° respectively. In addition CA had a 70% more proline than the i s o l a t e P^A°, while P.B had 15% lower proline than i n the concentrate CB. The i s o l a t e s P-B and P.S revealed a lower l e v e l of glutamic acid, methionine and phenylalanine, however, 3 5 and 25% 39 and 25% and 20 and 2 5% less than those i n the concen-trates CB and CS respectively. The i s o l a t e P^S also had a 3 8% higher proline content than the concentrates CS. The remaning amino acids were higher i n the i s o l a t e . The t r i c h l o r o a c e t i c acid p r e c i p i t a t e d i s o l a t e s P^ t had a s i m i l a r pattern as the i s o e l e c t r i c p r e c i p i t a t e d i s o l a t e s P.. l The i s o l a t e s P^ W and P^A° indicated higher l y s i n e , arginine, methionine and proline for the water extract, and threonine, aspartic acid, leucine and tyrosine for the .6 5. TABLE IV AMINO ACID COMPOSITION OF RAPESEED PROTEIN CONCEN-TRATES FROM THE DIFFERENT FRACTIONS COMPARED TO THAT OF RAPESEED AND SOYBEAN PROTEIN CONCENTRATES (G AMINO ACID PER 16 G NITROGEN) Three Water s t a g e e x t r a c t i o n HC1 NaOH S i n g l e NaOH e x t r a c t R a p e s e e d a Soybean^ Asp. 6.9 4.1 6.7 6 . 3 6.0 11. 3 Thr. 6 . 8 4.5 5.1 5.3 3.6 3.9 Ser. 4.6 4.9 5.0 4.9 3.7 6.0 Glu. 17.0 19. 5 19 . 3 15.9 16 .4 18.5 P r o . 3.4 4.9 5.3 4.8 5.6 4.0 G l y . 6 . 8 5.7 5.1 5.4 4.6 4.6 A l a . 6.2 5.2 5.0 6.0 4.0 5.3 Cys.# 1.3 2.7 2.4 2.4 1.9 1.3 V a l . 4.1 4.5 5 . 3 5.0 4.6 5.6 Met.# 2.1 2.5 3.1 2 . 5 1.6 1.3 H e . 3.0 3.8 4.4 4.0 3.8 5.3" Leu. 5.5 7.0 8.0 7.2 6.8 8.1 T y r . 3.8 3.0 3.5 3. 3 2 . 3 4.4 Phe. 2.2 3.7 4.2 3.9 3.7 5.6 Ly s . 6.9 6.6 4.6 6.6 6.2 6.7 H i s . 5.1 3.5 2.1 3.4 . 2.7 2.6 A r g . 4.8 5.5 3.7 5.0 5 . 8 6 . 5 Tr y . * 1.4 1.1 1.3 1.2 0 . 8 1.4 Amm. 2.4 2.2 1.6 2.1 - -T o t a l 94.3 95 . 3 95.9 95.5 84.6 82.7 # P e r f o r m i c a c i d o x i d a t i o n s e p a r a t e d e t e r m i n a t i o n s , by t h e method o f Schram e t " a 1. (1954) and Moore (1963). * Try p t o p h a n d e t e r m i n e d by t h e method o f I n g l i s and L e a v e r s ( 1964). a. V a l u e s from Lo and H i l l (19 71) . b. V a l u e s from Van E t t e n e t a l . (.1959). TABLE V AMINO ACID COMPOSITION OF RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION COMPARED TO SOYBEAN PROTEIN ISOLATES AND RAPESEED PREPARATION B l CG. AMINO ACID PER 16 G NITROGEN) N. Three s t a g e e x t r a c t i o n S i n g l e Soybean B l . Water HC1 NaOH NaOH e x t r a c t p r o t e i n a Asp. 7.1 6 . 5 6.2 6.2 10. 5 10.4 Thr. 5.3 7.2 5.4 5.7 3 . 5 4.2 Ser. 4.5 5.2 4.4 4.6 4.7 4.0 G l u . 11.9 10 .9 12.5 12 .0 17 .6 22.2 P r o . 2 . 3 2.9 4.5 3.5 5.1 5.0 G l y . 5.4 5.9 5 . 5 5.1 3.9 5.8 A l a . 6 . 3 5.9 5.1 5.4 4.1 4.5 Cys. # 0.9 1.5 2.4 2.0 1.5 1.8 V a l . 5.0 5.3 5 . 3 4.9 4.8 5.3 Met.# 1.7 2.0 1.9 1.9 " 1.5 1.3 H e . 3.5 4.5 4.0 3.5 4.6 4.3 Leu. 6 . 3 8.5 7.1 6 . 5 7 . 8 8.0 T y r . 3.9 4.8 3 . 2 3.4 3 . 5 2 . 8 Phe. 2.6 3.4 3.4 2.9 5.1 5.1 L y s . 9.1 7.2 5.0 8.6 6.2 3.2 H i s . 5.5 5.6 4.2 5.8 2.4 1,9 A r g . 7.8 8.7 6.7 9.7 6.0 7.6 Tr y . * 1.5 1.8 1.2 1.3 1.2 0 . 7 Amm. 2.0 • 2 .14 2 .4 1.9 — I . 1 T o t a l 91.1 9 7.1 87 .4 9 2.6 93.9 95.9 # P e r f o r m i c a c i d o x i d a t i o n s e p a r a t e d e t e r m i n a t i o n s , by t h e methods o f Schram e t a l . (1954) and Moore (1963). * T r y p t o p h a n d e t e r m i n e d by t h e method o f I n g l i s and L e a v e r s (1964).-a. V a l u e s from Lo and H i l l (.1971) f o r Assay p r o t e i n C - l Skidmore E n t e r p r i s e s . b. V a l u e s from F i n l a y s o r i e t a l . (1968). TABLE. VI AMINO ACID COMPOSITION OF RAPESEED PROTEIN ISOLATES PREPARED BY- TCA PRECIPITATION COMPARED TO CASEIN AND RAPESEED FLOUR (G AMINO. ACID PER 16 G NITROGEN) Three s t a g e e x t r a c t i o n S i n g l e C a s e i n a Rapeseed Water HC1 NaOH NaOH f l o u r b [ e x t r a c t  Asp. 7.2 5.3 6.1 6.1 7.2 6.4 Thr. 7.2 5.8 5.9 5.6 4.9 3.0 Ser. 4.3 4.7 4.5 4.0 6.4 4.1 G l u . 14.9 13.6 14.3 14.0 22.7 17.6 P r o . 4.6 4.4 5.9 5.1 11.5 5.1 G l y . 6.9 5.4 5.6 5.6 2.7 4.4 A l a . 6.7 5.5 5.0 5.3 3.0 3.8 Cys.# 0.7 3.2 0.9 2.4 0.3 0.9 V a l . 4.4 4.7 5.3 5.2 7.3 2.5 Met.# 1.2 2.2 0.9 2 . 3 . 2.8 1.7 H e . 3.3 3. 6 4.0 3.8 6.1 1.9 Leu. 6.5 7.3 7.5 7.2 9.3 5.3 T y r . 4.0 3.5 3.4 4.6 6.4 2.2 Phe. 2.3 3.5 3.4 3.1 5.0 3.3 L y s . 8.3 7.5 6.7 7.6 8.3 4.4 H i s . 4.5 5.8 4.8 5.6 3.1 2.2 A r g . 6.0 8.5 7.5 7.8 4.1 3.8 T r y . * 1.5 1 . 3 , 1.3 1.7 2 . 1 -Amm. 1.6 X. 5 2.6 2.3 = -T o t a l 94.0 93.4 94.7 95.3 113.1 72.6 # P e r f o r m i c a c i d o x i d a t i o n s e p a r a t e d e t e r m i n a t i o n s , by t h e methods o f Schram e t aJL. (19 54) and Moore (1963 ). * T r y p t o p h a n by t h e method o f I n g l i s and L e a v e r s (1964). a. V a l u e s f o r c a s e i n , S h e f f i e l d Chem. N o r w i c k , N.Y. from Lo and H i l l (19 7 1 ) . b. V a l u e s from Tape e t a l . (19 7 0 ) . TABLE V I I AMINO ACID COMPOSITION. OF RAPESEED PROTEIN WHEY AFTER ISOELECTRIC PRECIPITATION, AND THOSE OF SOYBEAN PROTEIN WHEY (G AMINO ACID PER 16 G NITROGEN) Three stage' e x t r a c t i o n S i n g l e Soy whey FAO Water HC1 NaOH NaOH e x t r a c t Asp. 7.1 2.2 2.2 4.2 13.9 Thr. 7 . 3 2 . 8 3 . 8 5.1 5.4 2 . 8 Ser . 5.1 5.2 3.4 4.7 6.7 G l u . 16 .6 20.7 28.9 23.5 14.6 P r o . 4.6 5.6 8.9 8.0 5.4 G l y . 8.4 6.1 4.1 6 . 3 4.7 A l a . 6.6 5.5 3.5 5.0 5.2 Cys. # 1.7 5.3 2.7 11.1 2.3 V a l . 3.6 3 . 9 5.1 4.2. 5.3 4.2 - Met.# 2.1 1.8 0.9 2.7' 2 . 3 2.2 H e . 2.8 3.1 3.0 2.9 4.6 4.2 Leu. 5.4 6.4 6.7 5.8 7 . 2 4.8 T y r . 3.4 1.1 1.3 2.1 4.2 2 . 8 Phe. 1.9 4.0 2 . 2 2.6 4.2 2.8 Ly s . 10 . 3 11. 5 7 . 5 9 . 5 8.4 4.2 H i s . 4.9 8.1 5.5 5 . 6 3.5 A r g . 7.2 8.8 7.7 7.4 6.8 T r y . * 1.1 0.4 0.4 0.7 1.5 1.4 Amm. 2.8 3.1 4.2 2.8 1.4 . T o t a l 10 2.7 109.9 . . 10 3. 7 113. 7 107 . 6 # P e r f o r m i c a c i d o x i d a t i o n s e p a r a t e d e t e r m i n a t i o n s , by the. methods o f Schram e t a l . (1954) and Moore (1963). * Method o f I n g l i s and Le a v e r C1964). a. V a l u e s from R a c k i s e t a l . (1971). b. Food A g r i c u l t u r e O r g a n i z a t i o n recommended p a t t e r n . h y d r o c h l o r i c a c i d e x t r a c t than those i n t h e i s o l a t e s P W and P^A. The i s o l a t e s P.B and P.S had most o f the amino a c i d s a t t t t h i g h e r l e v e l s than the i s o e l e c t r i c p r e c i p i t a t e d i s l a t e s P^B and P^S. The i s o l a t e d P^ W had 2 0 t o 44% more l y s i n e t h a n any o t h e r i s o l a t e i n the t h r e e s t a g e e x t r a c t i o n p r o c e d u r e . The amino a c i d c o m p o s i t i o n o f the two p r e p a r a t i o n s o f t h e i s o l a t e s P. and P. showed some d i f f e r e n c e s i n t h e i r 1 t d i s t r i b u t i o n . The i s o l a t e P W had a h i g h e r t h r e o n i n e c o n t e n t t h a n P.W. The i s o l a t e P.A had a h i g h e r s u l f u r c o n t a i n i n g l t & & amino a c i d t o g e t h e r w i t h g l u t a m i c a c i d than i n t h e i s o l a t e P.A 0. The i s o l a t e P.B had a h i g h e r c y s t i n e and m e t h i o n i n e c o n t e n t and l o w e r l y s i n e and g l u t a m i c a c i d than i n the i s o l a t e P.B. In the i s o l a t e P^S t h e r e i s a h i g h e r c o n c e n t r a -t t ° t i o n o f t r y p t o p h a n , t y r o s i n e m e t h i o n i n e and g l u t a m i c a c i d and l o w e r l e v e l s o f a r g i n i n e than i n t h e i s o l a t e P.S. Among t h e 8 i s o l a t e s p r e p a r e d , t h e h y d r o c h l o r i c a c i d e x t r a c t c o n t a i n e d h i g h e r l e v e l s o f most o f t h e e s s e n t i a l amino a c i d s . The i s o l a t e P^B had h i g h e r g l u t a m i c a c i d , p r o l i n e and c y s t i n e . I t was o b s e r v e d t h a t t h e i s o l a t e s P^S and P S had amino a c i d c o n c e n t r a t i o n s a v e r a g i n g t h o s e p r e s e n t i n t h e i s o l a t e s by t h r e e s t a g e e x t r a c t i o n , e x c e p t f o r l o w e r p r o l i n e and h i g h e r h i s t i d i n e and a r g i n i n e i n i s o l a t e P.S. The v a l u e s o b s e r v e d by F i n l a y s o n e t a l . (37) f o r the f r a c t i o n AIVS f o r amino a c i d s were l o w e r e x c e p t f o r h i g h e r amounts o f g l u t a m i c a c i d and p r o l i n e . But t h e i r i s o l a t e BI had a s i m i l a r amino a c i d c o m p o s i t i o n e x c e p t f o r a 70. h i g h e r p r o l i n e , a s p a r t i c a c i d and g l u t a m i c a c i d ; and l o w e r c o n c e n t r a t i o n s o f l y s i n e and t y r o s i n e . These d e v i a t i o n s i n th e amino a c i d s i s demished i n t h e i s o l a t e s P^. I t i s i n t e r e s t i n g t o n o t e t h a t t h e s e i s o l a t e s had a h i g h e r range o f t h r e o n i n e (35 t o 5 1 % ) , g l y c i n e (25 t o 3 5 % ) , a l a n i n e (18 t o 3 4 % ) , l y s i n e (14 t o 3 2 % ) , h i s t i d i n e (42 t o 5 8 % ) , and a r g i n i n e (10 t o 3 8 % ) , and l o w e r l e v e l s o f a s p a r t i c a c i d (32 t o 4 1 % ) , g l u t a m i c a c i d (30 t o 3 8 % ) , p h e n y l a l a n i n e (33 t o 5 0 % ) , and p r o l i n e (12 t o 43%) compared t o t h o s e p r e s e n t i n the soybean p r o t e i n i s o l a t e s ( 5 3 ) . Only the i s o l a t e s P^B had a 20% l e s s l y s i n e c o n t e n t than t h e soybean p r o t e i n i s o l a t e . Compared t o c a s e i n (53) the i s o l a t e s P^ showed h i g h e r amounts o f t h r e o n i n e (8 t o 3 7 % ) , a l a n i n e (41 t o 5 3 % ) , c y s t i n e (61 t o 8 6 % ) , and a r g i n i n e (39 t o 58 % ) . I n a d d i t i o n t h e i s o l a t e s P.W and P.S had h i g h e r amounts o f l y s i n e w h i l e the o t h e r f r a c t i o n s were l o w e r t h a n t h o s e p r e s e n t i n c a s e i n . The i s o l a t e s P were q u i t e s i m i l a r t o c a s e i n w i t h r e g a r d t o the amino a c i d c o m p o s i t i o n . The amino a c i d c o n t e n t i n rapeseed whey p r o t e i n a f t e r 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 h i g h e r than t h a t o f t h e r e s p e c t i v e i s o l a t e s P.. The same phenomenon was o b s e r v e d f o r soybean p r o t e i n i s o l a t e s ( 7 2 ) . Compared t o the whey from soybean p r o t e i n , t h e whey from i s o l a t e s P^ W c o n t a i n e d h i g h e r t h r e o n i n e , a l a n i n e and l y s i n e and l o w e r a s p a r a t i c a c i d i s o -l e u c i n e and p h e n y l a l a n i n e . The whey from i s o l a t e s P.A 0 was h i g h e r i n h i s t i d i n e and g l u t a m i c a c i d . The whey from i s o l a t e P^S was h i g h i n g l y c i n e , g l u t a m i c a c i d and h i s t i d i n e and an e x c e p t i o n a l l y h i g h c o n c e n t r a t i o n o f c y s t i n e . The whey from P.A° i n d i c a t e d c o n c e n t r a t i o n s o f b a s i c amino a c i d s h i g h e r t h a n t h o s e i n the o t h e r wheys. The r a p e s e e d p r o t e i n c o n c e n t r a t e s p r e p a r e d by Lo and H i l l (53) had s i m i l a r aminco a c i d c o m p o s i t i o n t o t h e concen-t r a t e CS e x c e p t l o w e r v a l u e s by 30% t h r e o n i n e , 2 2% s e r i n e , 3 3% a l a n i n e , 21% c y s t i n e , 35% me t h i o n e , 31% t y r o s i n e , a n d 39% t r y p t o p h a n and 12% more p r o l i n e . Thus i t appears t h a t t h i s s i n g l e s t a g e e x t r a c t i o n endorses the good r e c o v e r y o f s u l f u r c o n t a i n i n g and o t h e r e s s e n t i a l amino a c i d s . Compared t o t h e soybean p r o t e i n c o n c e n t r a t e s , t h e r a p e s e e d p r o t e i n c o n c e n t r a t e CS c o n t a i n s h i g h e r amounts o f p h e n y l a l a n i n e and i s o l e u c i n e by 30 and 25% r e s p e c t i v e l y and 19 and 89% h i g h e r t h r e o n i n e and s u l f u r c o n t a i n i n g amino a c i d c o n t e n t s . Thus the h i g h c o n c e n t r a t i o n o f s u l f u r c o n t a i n i n g amino a c i d i n r a p e s e e d p r o t e i n c o n c e n t r a t e CS compared t o t h e amount p r e s e n t i n t h e soybean p r o t e i n c o n c e n t r a t e may be an advantage t o the wide use o f t h i s p r e p a r a t i o n . H. U l t r a c e n t r i f u g a t i o n U l t r a c e n t r i f u g a t i o n p a t t e r n o f t h e f i r s t w a t e r ex-t r a c t i s o l a t e p r e p a 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 i s g i v e n i n F i g u r e 18. There were t h r e e peaks obse r v e d under t h e e x p e r i m e n t a l c o n d i t i o n s used. The r e s p e c t i v e s e d i m e n t a t i o n c o e f f i c i e n t o f the t h r e e peaks were 5.4S, 3.OS and 0.9S. gure 18. S c h l i e r e n p a t t e r n s o f t h e f i r s t w a t e r e x t r a c t i s o l a t e P^ W. C o n c e n t r a t i o n 1.0%; 0.1 M T r i s - g l y c i n e pH 8.5 c o n t a i n i n g 6 M u r e a and 0.1 M 2 - m e r c a p t o e t h a n o l The p i c t u r e s were t a k e n 2 5 mins (upper) and 60 mins ( l o w e r ) a f t e r r e a c h i n g 59,000 r.p.m. gure 19. S c h l i e r e n p a t t e r n o f the second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e A°. C o n c e n t r a t i o n 1.0%; 0.1 M T r i s - g l y c i n e pH 8.5 c o n t a i n i n g 6 M u r e a and 0.1 M 2 - m e r c a p t o e t h a n o l . The p i c t u r e was t a k e n 20 mins a f t e r r e a c h i n g 59,000 r.p.m. F i g u r e 20. S c h l i e r e n p a t t e r n o f t h e t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e P^B. C o n c e n t r a t i o n 1.0%; 0.1 M T r i s - g l y c i n e pH 8.5 c o n t a i n i n g 6 M u r e a and 0.1 M 2-m e r c a p t o e t h a n o l . The p i c t u r e was t a k e n 65 mins a f t e r r e a c h i n g 59,000 r.p.m. F i g u r e 21. S c h l i e r e n p a t t e r n o f t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i s o l a t e P^S. C o n c e n t r a t i o n 1.0% 0.1 M T r i s - g l y c i n e pH 8.5 c o n t a i n i n g 6 M u r e a and 0.1 M 2- m e r c a p t o e t h a n o l . The p i c t u r e was t a k e n 60 mins a f t e r r e a c h i n g 59,000 r.p.m. The u l t r a c e n t r i f u g a t i o n p a t t e r n o f the second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e p r e p a 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 i s p r e s e n t e d i n F i g u r e 19. A s i n g l e peak was o b s e r v e d by t h i s p r e p a r a t i o n . The s e d i m e n t a t i o n c o e f f i c i e n t under the c o n d i t i o n s d e s c r i b e d i n t h i s e x p e r i m e n t was 1.9S. The u l t r a c e n t r i f u g a t i o n p a t t e r n o f the t h i r d sodium h y d r o x i d e e x t r a c t and s i n g l e sodium h y d r o x i d e e x t r a c t i s o l a t e p r e p a 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 had s e d i m e n t a t i o n c o e f f i c i e n t s o f 0.2S and O.HS r e s p e c t i v e l y . These p a t t e r n s a r e shown i n F i g u r e s 2 0 and 21. The t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e had a l a r g e number o f f a s t moving peaks which d i s a p p e a r e d w i t h i n t h e f i r s t 3 m i n u t e s . I . M a t e r i a l b a l a n c e o f i s o l a t e s The r e c o v e r y o f p r o t e i n s 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 low. T h i s i s e v i d e n t i n the h i g h l e v e l s o f n i t r o g e n i n the r a p e s e e d whey o b t a i n e d from d i f f e r e n t f r a c t i o n s i n d i c a t i n g t h a t much o f the e x t r a c t e d p r o t e i n was n o t p r e c i p i t a t e d . The n i t r o g e n l o s s i n t h e whey from t r i c h l o r o a c e t i c a c i d p r e c i p i -t a t i o n was l e s s t h a n what was o b s e r v e d f o r the i s o e l e c t r i c p r e c i p i t a t i o n . T h i s i s g i v e n i n T a b l e V I I I . j . Loss o f p r o t e i n d u r i n g washing p r o c e d u r e A - c o n s i d e r a b l e l o s s o f p r o t e i n was o b s e r v e d d u r i n g t h e washing p r o c e s s . The l o s s o f p r o t e i n by washing the t r i c h l o r o a c e t i c a c i d p r e c i p i t a t e d i s o l a t e s w a s g r e a t e r than i n the i s o e l e c t r i c p r e c i p i t a t e d i s o l a t e s P ^ . T h i s l o s s o f p r o t e i n i n washing i s shown i n T a b l e IX. 76. TABLE V I I I PROTEIN MATERIAL BALANCE FROM THE PREPARATIONS OF RAPESEED PROTEIN EXTRACTS AND ISOLATES (as p e r c e n t a g e o f dry m a t t e r i n o r i g i n a l f l o u r ) . E x t r a c t ~ Whey P i Whey P F i r s t w a t e r e x t r a c t 5.5 1.6 0.4 Second a c i d i c e x t r a c t 5.8 3.2 0.5 T h i r d b a s i c e x t r a c t 20.4 5.4 0.9 T o t a l 33.7 10.2 1.8 F i r s t b a s i c e x t r a c t 26.8 4.6 1.5 \ TABLE IX PROTEIN LOSSES FROM WASHING THE ISOLATES WITH WATER AND 50% ALCOHOL Cas percentage of i s o l a t e s } . 1st water e x t r a c t 2nd a c i d i c e x t r a c t 3rd b a s i c e x t r a c t Total losses 1st b a s i c extract Water washing p i p t . A l c o h o l washing 0.02 0.41 0.02 1.09 0.11 ].17 0.15 3.67 0.09 2.51 0.01 0.01 0.02 0.38 0.04 0.77 0.07 1.16 0.11 2.49 T o t a l washing P. P. • x t 0.03 0.42 0.04 1.47 0.15 2.94 0.22 4.83 0.20 5.00 K. Y i e l d o f p r o t e i n i s o l a t e d The y i e l d s o f p r o t e i n i s o l a t e d a re t a b u l a t e d i n T a b l e X. W i t h the 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 t 35°C th e y i e l d s o b t a i n e d were 10.8, 7.2 and 41.5% r e s p e c t i v e l y f o r the t h r e e s t a g e e x t r a c t i o n and 51.2% f o r t h e s i n g l e s t a g e e x t r a c t i o n . For the t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n t h e y i e l d s o b t a i n e d were 14.2, 14.7 and 53.8% r e s p e c t i v e l y f o r t h e t h r e e s t a g e e x t r a c t i o n and 70.3% f o r the s i n g l e s t a g e e x t r a c t i o n . L. S o l u b i l i t y measurements o f t h e i s o l a t e s and c o n c e n t r a t e s . The r e s u l t s i n d i c a t e t h a t t h e s o l u b i l i t y o f the i s o l a t e s P^ W and P^A° were a p p r o x i m a t e l y 59 and 49% r e s p e c t i v e l y , w h i l e the sodium h y d r o x i d e e x t r a c t i s o l a t e s P.B and P.S had a lo w e r v a l u e o f 42.5%. I I The c o n c e n t r a t e s had a h i g h e r s o l u b i l i t y f o r the t h r e e s t a g e e x t r a c t i o n 77, 80 and 71% f o r t h e samples CW, CA and CB r e s p e c t i v e l y . The s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e CS had a l o w e r s o l u b i l i t y o f 5 8%. These r e s u l t s a re t a b u l a t e d i n Table X I . TABLE X THE YIELDS OF PROTEINS ISOLATED Water e x t r a c t A c i d e x t r a c t B a s i c e x t r a c t S i n g l e b a s i c e x t r a c t I s o e l e c t r i c p r e c i p i t a t i o n 10.8 7.2 41.5 61.2 T r i c h l o r o a c e t i c a c i d p r e c i p i t a -t i o n 14.2 14.7 53.8 70.3 TABLE XI SOLUBILITY MEASUREMENTS OF THE ISOLATES AND .CONCENTRATES EXPRESSED AS PERCENTAGES OF PREPARATIONS Isolate P, Concentrate F i r s t water extract Second acid extract Third basic extract F i r s t basic extract 58.7 48.5 42.1 43 . 2 76.9 80.3 70.8 58.4 CHAPTER V DISCUSSION The e x t r a c t i o n o f r a p e s e e d f l o u r a t h i g h a l k a l i n e r e g i o n s o v e r pH 10 r e s u l t e d i n a y e l l o w i s h green p r o d u c t , w h i l e around pH 10 t h e c o l o u r was creamy y e l l o w . The d a r k e n -i n g o f c o l o u r a t h i g h e r pH i s due m a i n l y t o t h e g r e a t e r ex-t r a c t a b i l i t y o f pigments p r e s e n t i n t h e f l o u r . Thus by con-t r o l l i n g t h e pH o f t h e s o l u t i o n t h e c o l o u r can be k e p t l i g h t e r . The i s o l a t e o b t a i n e d by e x t r a c t i n g a t pH o v e r 10 showed some t r a i l i n g as o b s e r v e d i n t h e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . A g r e a t e r r e c o v e r y o f p r o t e i n can be a c h i e v e d a t h i g h e r p H - r e g i o n s , but t h e c o l o u r o f t h e p r o t e i n was not s a t i s f a c t o r y . As seen from F i g u r e 5, a f t e r r e a c t i n g t h e h y d r o -c h l o r i c a c i d e x t r a c t w i t h o x a l i c a c i d , i t was ob s e r v e d t h a t a t t h e optimum c o n c e n t r a t i o n o f 0.0 5 M t h e r e was no f u r t h e r i n c r e a s e i n t h e c a l c i u m removed. Thus o x a l c i a c i d was a b l e t o combine and p r e c i p i t a t e s a l t s i n t h e e x t r a c t up t o t h e maximum amount o f c a l c i u m was removed. The n a t u r e o f t h e cur v e shown i n F i g u r e 6 i n d i c a t e s t h a t t h e p r o t e i n c o n t e n t i n t h e s u p e r n a t a n t i n c r e a s e d a f t e r r e a c h i n g t h e optimum o x a l i c a c i d c o n c e n t r a t i o n (0.05 M) o f th e second, h y d r o c h l o r i c a c i d e x t r a c t . The i n c r e a s e a f t e r t h e i n i t i a l d e c r e a s e may be a t t r i b u t e d t o s o l u b i l i z a t i o n o f c a l c i u m p r o t e i n a t e . A f t e r r e a c h i n g t h e optimum o x a l i c a c i d concen-t r a t i o n t h e r e was a sudden d e c r e a s e i n the p r o t e i n c o n t e n t o b s e r v e d . T h i s sudden d e c r e a s e may be due t o s a l t i n g out o f 82. some p r o t e i n . As shown i n the Tables I and I I , the i s o l a t e s pre-pared by t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n P of the ex-t r a c t s showed higher values f o r moisture and p r o t e i n while the i s o l a t e s prepared 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 P. had higher values f o r carbohydrates, ash, phosphorus, s u l f u r and t h i o c y a n a t e s . The f a t and crude f i b e r content was g e n e r a l l y about the same f o r both p r e p a r a t i o n s . These r e s u l t s would be e xpected because of the higher p u r i t y of the t r i c h l o r o -a c e t i c a c i d p r e c i p i t a t i o n P^ w i t h g r e a t e r p r o t e i n content and lower concentrations of other compounds. The concentrates, as expected, show a lower percen-tage of p r o t e i n and higher l e v e l s of moisture, ash, c a l c i u m , phosphorus and crude f i b e r . The s u l f u r content ranges s i m i l a r l y t o i s o l a t e s prepared 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 P^. The most i n t e r e s t i n g f eature of the concentrates was the lower i s o t h i o c y a n a t e content compared to the two i s o l a t e s . The r e s u l t s f o r the second h y d r o c h l o r i c a c i d ex-t r a c t i s o l a t e a f t e r the treatment w i t h 0.05 M o x a l i c a c i d showed th a t not only the ash content was reduced from 36.5 t o 9%, but a l s o the phosphorus content was decreased from 11.6 t o 1.8%. This r e d u c t i o n of phosphorus on o x a l i c a c i d t r e a t -ment may be due t o complex calcium s a l t s formed during p r e c i p i t a t i o n . The values f o r the p r o t e i n i s o l a t e s obtained were low compared to the published data f o r other vegetable p r o t e i n i s o l a t e s such as soybean, cotton seed and sunflower. This 83. may be due t o the h i g h ash and l i p i d c o n t e n t i n the i s o l a t e s . • The h i g h c a r b o h y d r a t e c o n t e n t o f t h e f i r s t w a t e r e x t r a c t c o n c e n t r a t e may be due t o the easy s o l u b i l i t y o f t h e sugars d u r i n g the e x t r a c t i o n . The c a r b o h y d r a t e c o n t e n t o f the i s o l a t e s p r e p a r e d from t h e w a t e r e x t r a c t was low due t o t h e p r e c i p i t a t i o n o f t h e p r o t e i n s d u r i n g t h e i r p r e p a r a t i o n . The whey t h e r e f o r e s h o u l d have c o n t a i n e d most o f the con-t a m i n a t i n g c a r b o h y d r a t e s . S u l f u r c o n t e n t was l o w e r i n the h y d r o c h l o r i c a c i d e x t r a c t c o n c e n t r a t e CA, than i n t h e i s o l a t e P.A. Whereas ' 1 f o r t h e o t h e r e x t r a c t i o n s , t h e c o n c e n t r a t e s had a h i g h e r s u l f u r c o n t e n t than i n t h e i r r e s p e c t i v e i s o l a t e s . T h i s l o s s o f s u l f u r d u r i n g p r e c i p i t a t i o n i n t o the whey may be due t o i n c o m p l e t e p r e c i p i t a t i o n o f t h e s u l f u r c o n t a i n i n g p r o t e i n f r a c t i o n s o r p e p t i d e s . The h i g h phosphorus c o n t e n t i n the f i r s t w a t e r e x t r a c t i s o l a t e P^ W may be due t o the h i g h s o l u b i l i t y o f t h e p h y t i c compounds p r e s e n t i n t h e rap e s e e d f l o u r . Among the c o n c e n t r a t e s the second h y d r o c h l o r i c a c i d e x t r a c t CA had an even h i g h e r phosphorus l e v e l caused by t h e s o l u b i l i z a t i o n o f the compounds i n the a c i d media. As mentioned e a r l i e r t h e c a l c i u m c o n t e n t o f the second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A a f t e r o x a l i c a c i d t r e a t m e n t was reduced by a p p r o x i m a t e l y 5 0%. The r e d u c t i o n o f c a l c i u m i n the h y d r o c h l o r i c a c i d i s o l a t e P^A on o x a l i c a c i d t r e a t m e n t i s due t o the a b i l i t y o f o x a l i c a c i d t o combine 84. and p r e c i p i t a t e s a l t s i n t h e e x t r a c t . G e n e r a l l y the c a l c i u m c o n t e n t i n the i s o l a t e s and c o n c e n t r a t e s were h i g h . G e n e r a l l y t h e ash c o n t e n t o f the i s o l a t e s and con-c e n t r a t e s were h i g h e r t h a n t h o s e r e p o r t e d f o r o t h e r v e g e t a b l e p r o t e i n i s o l a t e s . T h i s h i g h ash l e v e l i n the p r e p a r a t i o n s may be a t t r i b u t e d t o the amounts o f c a l c i u m p r e s e n t i n them. The ash c o n t e n t o f t h e h y d r o c h l o r i c a c i d e x t r a c t p r e c i p i t a t e d by t r i c h l o r o a c e t i c a c i d was 1.0%, w h i l e t h e i s o l a t e p r e p a r e d a f t e r o x a l i c a c i d t r e a t m e n t was 2.0%. T h i s c o u l d be due t o the b u f f e r i n g a c t i o n o f o x a l i c a c i d w hich causes t h e co-p r e c i p i t a t i o n o f c a l c i u m s a l t s w i t h t h e p r o t e i n d u r i n g t r i c h -l o r o a c e t i c a c i d p r e c i p i t a t i o n . Crude f i b e r c o n t e n t was f a i r l y h i g h i n the i s o l a t e s , e s p e c i a l l y i n the second h y d r o c h l o r i c a c i d e x t r a c t p r e p a r a t i o n P^A. The h i g h v a l u e may be due t o t h e f i n e p a r t i c l e s i n t h e s u p e r n a t a n t t h a t tends t o p r e c i p i t a t e w i t h the p r o t e i n s d u r i n g pH a d j u s t m e n t s . The p u b l i s h e d d a t a f o r soybean p r o t e i n i s o l a t e and c o n c e n t r a t e were 0.7 and 3.0% r e s p e c t i v e l y . From the r e s u l t i t was a p p a r e n t t h a t the i s o t h i o c y a n a t e l e v e l o f t h e w a t e r e x t r a c t p r e p a r a t i o n s were h i g h e r t h a n t h a t o b s e r v e d f o r any o f the o t h e r p r e p a r a t i o n s . T h i s h i g h i s o t h i o -c y a n a t e c o n t e n t i n d i c a t e d i n t h e w a t e r e x t r a c t i s p r o b a b l y due t o t h e h i g h e r e x t r a c t a b i l i t y o f t h i s m a t e r i a l i n w a t e r a t 4°C. I t i s seen t h a t w i t h s u c c e s s i v e e x t r a c t i o n s i n t h e t h r e e s t a g e p r o c e d u r e t h e i s o t h i o c y a n a t e c o n t e n t d e c r e a s e s . The i s o t h i o c y a n a t e p r e s e n t i n w a t e r e x t r a c t i s o l a t e s and 85. c o n c e n t r a t e s by t h i s p r o c e d u r e were a p p r o x i m a t e l y 25% h i g h e r t h a n t h a t f o r t h e s i n g l e s t a g e s o d ium h y d r o x i d e e x t r a c t , e x e p t f o r t h e i s o l a t e s p r e p a 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 where t h e s i n g l e s t a g e s o d i u m h y d r o x i d e e x t r a c t i s h i g h e r by a p p r o x i m a t e l y 8%. S l a b g e l e l e c t r o p h o r e s i s o f t h e f i r s t w a t e r and s e c o n d h y d r c h l o r i c a c i d e x t r a c t s i s o l a t e s P^ i n d i c a t e d s e p a r a t i o n o f a c i d i c and n e u t r a l p r o t e i n s f r o m b a s i c p r o t e i n as c o n f i r m e d a t pH 8.8. Thus w i t h t h i s s i m p l e t h r e e s t a g e e x t r a c t i o n p r o c e d u r e i t i s p o s s i b l e t o s e p a r a t e t h e p r o t e i n f r a c t i o n s c o n t a i n i n g i m p o r t a n t amino a c i d s f r o m r a p e s e e d f l o u r . T h e s e f r a c t i o n s c o u l d be u s e d i n f o o d p r o c e s s i n g t o i m p r o v e t h e n u t r i t i o n a l v a l u e . C o n s i d e r a b l e amount o f p r o t e i n i s b e i n g l o s t i n t h e whey f r o m i s o e l e c t r i c p r e c i p i t a t i o n and i t i s n e c e s s a r y t o f i n d o u t methods o f p r e v e n t i n g t h i s l o s s i n t h e f u t u r e . . Work i s c u r r e n t l y b e i n g c a r r i e d o u t i n t h i s l a b o r a t o r y by u s i n g s ephadex column c h r o m a t o g r a p h y t o d e t e r m i n e w h i c h f r a c t i o n c o n t a i n s t h e h i g h e s t amount o f s u l f u r c o n t a i n i n g amino a c i d s . Amino a c i d c o m p o s i t i o n o f t h e whey f r o m t h e s i n g l e s t a g e s o d i u m h y d r o x i d e e x t r a c t i s o l a t e r e v e a l e d an e x t r e m e l y h i g h c o n c e n t r a t i o n o f c y s t i n e . T h i s l o s s o f c y s t i n e must be p r e v e n t e d as much as p o s s i b l e . The phenomenon i s s i m i l a r t o t h a t o b s e r v e d by R a c k i s e t a l _ . (72) f o r s o y b e a n p r o t e i n i s o l a t e whey. 8 6 . Each protein f r a c t i o n from the rapeseed f l o u r shows d i s t i n c t c h a r a c t e r i s t i c s regarding the amino acid composition when compared to casein and soybean protein i s o l a t e s . It could be infer r e d that rapeseed protein i s o l a t e s i f properly u t i l i z e d a f t e r preparation, much of the present demands for high quality proteins could be overcome without encountering d i f f i c u l t i e s . As observed from the amino acid composition of the single stage sodium hydroxide concentrate CS contains a higher sulf u r content amino acid compared to the soybean pro-t e i n concentrate. This s i t u a t i o n may be of some benefit to the consumer as i s o l a t e s tend to lose t h i s e s s e n t i a l amino acid during p r e c i p i t a t i o n . U l tracentrifugation patterns for the rapeseed pro-t e i n isolaes from the f i r s t water, P^ W and second hydro-c h l o r i c acid extract, P.A indicated that large molecules have broken down into units of small molecular weights. This d i s s o c i a t i o n i s due to the blocking of the sulf h y d r y l and amino group present i n the i s o l a t e , to f a c i l i t a t e s o l u b i l i -zation of the insoluble proteins. Si m i l a r l y the r e s u l t s obtained for the t h i r d sodium hydroxide extract P^B and the single stage sodium hydroxide extract i s o l a t e P^S indicated very low sedimentation c o e f f i c i e n t s . The r e s u l t s published by Bhatty et 'al. (14) showed sedimentation c o e f f i c i e n t s of 12S and 1.7S f o r t h e i r prepar-ation B l . The re s u l t s obtained i n t h i s study, 1.9S value for hydrochloric acid i s o l a t e P>A, clos e l y agree with t h e i r 87. s e d i m e n t a t i o n c o e f f i c i e n t . From t h e r e s u l t s o b t a i n e d i t i s e v i d e n t t h a t t h e i s o l a t e s from t h e t h r e e s t a g e e x t r a c t i o n p r o c e d u r e p r e c i p i -t a t e d by t r i c h l o r o a c e t i c a c i d g i v e y i e l d s o f 14% more t h a n from t h e s i n g l e s t a g e e x t r a c t i o n p r o c e d u r e , w h i l e t h e y i e l d s f o r t h e 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 r e a p p r o x i m a t e l y t h e same f o r t h e two p r o c e d u r e s . The low v a l u e f o r t h e 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 t h e t h r e e s t a g e e x t r a c t i o n p r o c e d u r e s h o u l d have r e s u l t e d due t o o n l y p a r t i a l p r e c i p i t a t i o n o f t h e p r o t e i n s . There i s a g r e a t l o s s o f p r o t e i n d u r i n g washing t h e i s o l a t e s p r e p a r e d by t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n , w h i c h c o u l d be due t o t h e s t r o n g p r e c i p i t a t i n g a b i l i t y o f t r i c h l o r o a c e t i c a c i d f o r s m a l l p e p t i d e s which may be l o s t d u r i n g t h e washing o p e r a t i o n s . The s o l u b i l i t y o f t h e i s o l a t e s i n w a t e r i s not v e r y h i g h f o r p r e p a r a t i o n s . T h i s i n s o l u b i l i t y may have been a r e s u l t from the i n i t i a l h eat t r e a t m e n t o f ra p e s e e d meal t o wh i c h i t was s u b j e c t e d d u r i n g t h e p r e p a r a t i o n o f f l o u r and d u r i n g t h e e l i m i n a t i o n o f t o x i c compounds. The p r o t e i n c o u l d have been d e n a t u r e d d u r i n g t h e adjustment o f pH t o t h e i r i s o -e l e c t r i c p o i n t s . A l l t h e s e may have p l a y e d a r o l e i n t h e d e c r e a s e d s o l u b i l i t y o f t h e i s o l a t e s . The s o l u b i l i t y o f t h e c o n c e n t r a t e s p r e p a r e d from t h e t h r e e s t a g e e x t r a c t i o n p r o -cedure was r e a s o n a b l y good w i t h v a l u e s r a n g i n g from 70 t o 80%, w h i l e t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e was o n l y 58%. PART I I FUNCTIONAL PROPERTIES OF THE RAPESEED PROTEIN FRACTIONS CHAPTER VI INTRODUCTION The modern use of i s o l a t e d proteins and protein concentrates for food i s i n an early stage of development. Progress w i l l depend larg e l y on technological and basic re-search applied to t h e i r production. At present the chief function i s to improve the n u t r i t i o n a l q u a l i t y of the bread by supplementation of the protein i n wheat f l o u r . Rapeseed protein i s o l a t e s and concentrates may be useful n u t r i t i o n a l l y because of the high content of s u l f u r containing and basic amino acids. Therefore these prepara-tions could become r i c h sources of supplementary material for foods d e f i c i e n t i n them. Another important feature of rape-seed protein i s o l a t e s i s the absence of any beany flavour c h a r a c t e r i s t i c to soybean protein i s o l a t e s . With methods of processing and techniques for eliminating glucosinolates, i t i s v ery l i k e l y that rapeseed meal w i l l become a good source of p r o t e i n that can be used l i k e the conventional vegetable protein i s o l a t e s and concentrates that are available e a s i l y today. At t h i s stage of our research we are hopeful of finding more useful applications i n other areas of food processing, for instance, water holding capacity i n meat or bakery products; contraol of fat absorption; v i s c o s i t y c h a r a c t e r i s t i c s ; gel formation; t e x t u r a b i l i t y ; feathering properties i n coffee; aerating capacity i n whipped toppings and frozen desserts. CHAPTER V I I LITERATURE REVIEW Rapeseed 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 have not been so f a r f u l l y u t i l i z e d i n any s i g n i f i c a n t e x t e n t , f o r s u p p l e m e n t a t i o n o r f o r t i f i c a t i o n o r e n r i c h m e n t o f common f o o d commodities such as b r e a d , i c e cream, spreads and meat p r o -d u c t s . R e f e r e n c e t o r e c e n t l i t e r a t u r e i n d i c a t e s t h a t soybean p r o t e i n i s o l a t e s have been s u c c e s s f u l l y used t o e n r i c h t h e above p r o d u c t s c o m m e r c i a l l y ( 8 5 ) . I t was n o t e d t h a t when soybean p r o t e i n i s o l a t e s were us e d , t h e r e were c e r t a i n improvements i n some p h y s i c a l p r o p e r t i e s o f c e r t a i n f o o d s . These p r o p e r t i e s i n c l u d e w a t e r b i n d i n g , e m u l s i f y i n g , s t a b l i z i n g , t h i c k e n i n g and f i l m and dough f o r m i n g a b i l i t y ( 4 , 42, and 4 8 ) . I t i s known t h a t t h e a d d i t i o n o f 3% soybean p r o t e i n t o albumen improved t h e whip, g i v i n g a h i g h e r whip volume i n a s h o r t e r t i m e . R e c e n t l y Tsen and Hoover (92) r e p o r t e d improvements o f n u t r i t i o n a l v a l u e o f b r e a d . Bread b e i n g a b a s i c f o o d s t u f f t h e w o r l d o v e r , p r e s e n t s a l o g i c a l means o f i m p r o v i n g t h e p r o t e i n i n t a k e i n t h e d i e t o f many hungry o r m a l n o u r i s h e d p e o p l e i n d e v e l o p i n g c o u n t r i e s . T h i s e n r i c h -ment o r f o r t i f i c a t i o n must be done a t l e v e l s which w i l l s i g n i f i c a n t l y improve t h e n u t r i t i o n a l v a l u e o f bread w i t h o u t s a c r i f i c i n g o t h e r q u a l i t i e s o f t h e f i n i s h e d p r o d u c t . U n f o r t u n a t e l y , t h e a d d i t i o n o f p r o t e i n t o wheat f l o u r has changed some o f t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f the product such as flavour, colour texture, appearance, aroma and volume. It has been impossible to add any s i g -n i f i c a n t amounts of nonwheat nutrient additives to bread without l o s i n g desirable q u a l i t i e s . With the use of i s o l a t e d protein fractions as a basis f o r the formulation of duly acceptable, high protein food products at reasonably low costs have been developed (21). There i s a considerable need to improve the pro-t e i n supply i n order to overcome famine and disast e r i n underdeveloped areas of the world. The conventional means of improving protein supply i s by increasing a g r i c u l t u r a l production and conversion of plant to animal protein can be properly and e f f i c i e n t l y exploited. At present there i s much i n t e r e s t i n new sources of protein: f i s h from the sea yeast grown on petroleum, chemical synthesis, improved photosynthesis and by plant c e l l culture (2). There i s no doubt that important advantages have been made during the past 15 years i n u t i l i z i n g vegetable protein sources i n the f i g h t against protein shortage (79). Probably the most important advantage made i s that vegetabl proteins properly processed and e f f i c i e n t l y combined with one another can replace animal protein food i n n u t r i t i o n of man and, expand the a v a i l a b i l i t y of dietary protein, now so scarce (15). This has been possible because of accumulated knowledge of protein n u t r i t i o n i n d i c a t i n g that amino acid p a t t e r n , not p r o t e i n as s u c h , i s t h e most i m p o r t a n t f a c t o r i n d e t e r m i n i n g t h e n u t r i t i v e v a l u e o f t h e f o o d p r o t e i n ( 7 3 ) . C o n s i d e r i n g t h e s i g n i f i c a n t i n e f f i c i e n c y o f c o n v e r -t i n g v e g e t a b l e p r o t e i n i n t o a n i m a l p r o t e i n , i t i s ' perhaps q u i t e l o g i c a l t h a t r e s e a r c h workers i n Food S c i e n c e have g i v e n due tho u g h t t o t h e d i r e c t use o f v e g e t a b l e p r o t e i n s i n human f o o d . I t i s known t h a t 1 pound o f u t i l i z a b l e p r o t e i n from b e e f c o s t s $3.20, w h i l e t h a t from soybean and wheat c o s t s o n l y 31 and 11 c e n t s r e s p e c t i v e l y ( 9 7 ) . I t has been r e p o r t e d from I n d i a by Chandrasekhara e t a l . (17) t h a t toned, and double t o n e d m i l k can be made by m i x i n g r e c o n s t i t u t e d skim m i l k s o l i d s w i t h cow m i l k . M i l t o n e v e g e t a b l e t o n e d m i l k , i s now aimed a t r e p l a c i n g i m p o r t e d s k i m m i l k powder by i n d i g e n o u s l y a v a i l a b l e v e g e t a b l e p r o t e i n , m a i n l y from p e a n u t s . The d e o d o u r i z e d p r o t e i n s o l u t i o n i s mixed w i t h g l u c o s e , v i t a m i n premix and s t a n d a r d i z e d m i l k . The m i l t o n e t h u s o b t a i n e d i s homogenized and p a s t e u r i z e d and t h e n c o o l e d t o 4°C and b o t t l e d . I m i t a t i o n m i l k has l o s t much o f t h e p o p u l a r i t y w h i c h i t had i n 196 8. I n a d d i t i o n t o b e i n g i n f e r i o r t o cow's m i l k , t h e low p r o t e i n l e v e l r e q u i r e d t o produce a c o s t advantageous p r o d u c t c r e a t e s d i f f i c u l t i e s i n a c h i e v i n g d u p l i c a t i o n o f m o u t h f e e l and body. Because o f u s i n g p r o t e i n d e r i v a t e s i n i m i t a t i o n m i l k p r o d u c t s , t h e p r e p a r a t i o n l a c k s t h e r e s i d u a l e m u l s i f y i n g power o f n o n f a t m i l k s o l i d s , so t h e d e f i c i e n c y i s made up by a d d i n g a h i g h e r l e v e l o f mono- and d i g l y e r i d e s . Imitation chocolate milk drink requires an a d d i t i o n a l quantity of kappa carrageenan to s t a b i l i z e the cocoa f i b e r s , because of the .low protein lev e l s of the drink. Phosphates or c i t r a t e s a l t s are occasionally added to help i n the c o l l o i d a l s o l u b i l i z a t i o n of the protein. These s a l t s are not necessary except when highly denatured protein i s used with 0.1 to 0.2% of the s a l t s (76). F o r t i f i c a t i o n of foods with synthetic methionine, l y s i n e , threonine and tryptophan, which are commercially available at reasonable costs, can be done without much d i f f i c u l t y . However, there i s a p o s s i b i l i t y that the amino acid thus supplemented i s r e a d i l y l o s t during cooking and food processing. Furthermore, the free amino acids may a f f e c t flavour and colour through degradation or i n t e r a c t i o n with other food components, for instance by Strecker degradation or amino-carbonyl reaction. Therefore the i s o -l a t i o n of n u t r i t i o n a l l y well balanced protein must s t i l l be the primary importance i n food processing. Fijimaki et 'al. (39) have developed a method of r e c t i f y i n g amino acid imbalance i n most vegetable proteins by digesting protein with a protein hydrolyzing enzyme and resynthesized a protein l i k e substance (plastin) with the help of a proteinase a f t e r condensing the hydrolysate. They could even couple an amino acid ester such as methionine ethyl ester d i r e c t l y to p l a s t i n molecule instead of using methionine-containing protein hydrolysate. As a r e s u l t t h e y were a b l e t o i n c r e a s e t h e s u l f u r c o n t a i n i n g amino a c i d c o n t e n t by s i x t o seven t i m e s . Soybean p r o t e i n s u p p l e m e n t a t i o n o f b r e a d a f f o r d s a m eans o f i n c r e a s i n g t h e n u t r i t i v e v a l u e w i t h a b e t t e r b a l a n c e o f amino a c i d s ( 3 5 ) . D i s e r (27) has r e v i e w e d l i t e r a t u r e s on soy f l o u r and soy g r i t s as p r o t e i n s u p p l e -m e n t a t i o n . P o l l o c h and Geddes (69) r e p o r t e d work i n which 1% unheated soy f l o u r improves t h e b r e a d , but h i g h e r l e v e l s d e c r e a s e t h e l o a f volume. I t i s w e l l known t h a t g l u t e n p r o t e i n complex i s a f u n c t i o n a l p r o t e i n system w h i c h i n t e r a c t s w i t h i t s e l f and w i t h o t h e r f l o u r c o n s t i t u e n t s t o form dough t h a t w i l l r e t a i n gas and produce b r e a d o f a c c e p t a b l e l o a f volume. Pence e t a l . (67) showed t h a t crude albumen f r a c t i o n i s o -l a t e d from w a t e r s o l u b l e s i s r e s p o n s i b l e f o r t h e l a r g e r l o a f volume. H a r r i s e t a l . (45) i l l u s t r a t e d t h a t s u p p l e m e n t a t i o n o f w h e a t f l o u r w i t h 5% soy f l o u r was e q u i v a l e n t t o s u p p l e -m e n t a t i o n w i t h 5% n o n f a t d r y m i l k . S m a l l amounts o f p o l a r l i p i d s from wheat f l o u r s u b s t a n t i a l l y improve volume and f r e s h n e s s r e t e n t i o n o f b r e a d ( 7 0 ) . P r e l i m i n a r y i n v e s t i g a -t i o n s by D a f t a r y e t a l . (25) have i n d i c a t e d t h a t t h e improve-ment was m a i n l y due t o g l y c o l i p i d s . Pomeranz e t a l . (71) showed t h a t c o m m e r c i a l l y a v a i l a b l e s u c r o e s t e r s ' c o u n t e r a c t t h e d e l e t e r i o u s e f f e c t s up t o 16% soy bean f l o u r on l o a f volume. G l y c o l i p i d s i n w h i c h the f a t t y a c i d s are d i r e c t l y attached to the sugar moiety as in sucroesters were as e f f e c t i v e improvers as glycosylglycerides i n restoring l o a f volume of bread baked with shortening from petroleum ether defatted f l o u r . Comparison of the ef f e c t of synthetic g l y c o l i p i d s indicated that both hydrophobic and hydrogen bonds are playing important roles in bread'making. Possibly there i s an optimal hydrophile - l i p o p h i l e balance (6M-) at which single l i p i d or l i p i d mixtures increase l o a f volume of bread. Dairy products can e n t i r e l y or pa r t l y be replaced by plant products in d i f f e r e n t ways. For example i t i s the use of modern technology to make from vegetable o i l s , margarine that is'^very much l i k e butter. It i s further possible to make milk, i c e cream and cheese from milk or plant protein and vegetable o i l s . It could also be possible to replace, i n due course, milk or animal protein with plant protein, and milk or animal fat with vegetable fat (4). We cannot overlook the o r i e n t a l technology for making food based on o i l seed proteins. To give a few examples, soy milk, bean curd and fermented products have been i n demand for many centuries. Pearson et a l . (66) found that sodium soy pro-teinate to be a poor em u l s i f i e r i n the usual pH range of meat and suggested that i t probably does not serve any major function i n emulsifying fat when added to sausage products. Swift et a l . (89) stated that the p a r t i c i p a t i o n of water s o l u b l e p r o t e i n i n emulsion- f o r m a t i o n i s dependent on t h e a d d i t i o n o f sodium c h l o r i d e . They r e p o r t e d t h a t t h e amount o f o i l e m u l s i f i e d p e r m i l l i g r a m o f s a l t s o l u b l e p r o t e i n i n c r e a s e d as t h e t o t a l amount o f p r o t e i n i n s o l u t i o n was d e c r e a s e d . S h e i k h e t a l . (80) have - used rapeseed p r o t e i n con-c e n t r a t e s f o r t h e p r e p a r a t i o n o f e n r i c h e d b r e a d . The d i e t a r y s t u d i e s on b r e a d c o n t a i n i n g skim m i l k powder and v i t a m i n m i n e r a l m i x t r u e t o g e t h e r w i t h r a p e s e e d o r o t h e r p r o t e i n c o n c n e t r a t e s have shown t h a t e n r i c h e d b r e a d had h i g h p r o t e i n , c a l c i u m and i r o n t h a n unsupplemented b r e a d . They showed t h a t t h e r e was o n l y l i t t l e d i f f e r e n c e s i n d i g e s t i o n o f v a r i o u s b r e a d s . W i t h t h e development o f new p r o t e i n r i c h f o o d , c e r t a i n p r i n c i p l e s must be met t o ensure c o m m e r c i a l a c c e p t a b i l i t y . There are s e v e r a l c r i t e r i a enumerated by Beher e t a l . (12) and Van Veen (94) c o n c e r n i n g th e d e v e l o p -ment, t e s t i n g and i n t r o d u c t i o n o f p r o t e i n r i c h new p r o d u c t s as f o l l o w s : The b a s i c m a t e r i a l u t i l i z e d must be a v a i l a b l e l o c a l l y and not f u l l y e x p l o i t e d a l r e a d y . The p r o d u c t must be e c o n o m i c a l and s h o u l d have l o n g s t o r a g e l i f e f o r t r a n s -p o r t a t i o n . They must be f r e e o f any t o x i c o r d e l e t e r i o u s i n f l u e n c e and must have an a g r e e a b l e t a s t e and odour, as w e l l as o t h e r d e s i r a b l e p h y s i c a l c h a r a c t e r i s t i c s . CHAPTER VIII EXPERIMENTAL MATERIALS AND PROCEDURES 1. Materials A l l materials for t h i s work were purchased from the l o c a l market. Wheat f l o u r , Robin Hood a l l purpose, enriched f l o u r produced by Robin Hood Multifood Ltd., Montreal. : Eggs were a g i f t from the Department of Poultry Science, U.B.C., freshly l a i d . Corn o i l and coconut o i l . Margarine, Dalewood brand, manufactured by Westminster Foods Ltd., North Burnaby, B.C. Yeast, Fleischman active dry yeast, Standard Brands Ltd., Vancouver. Instant skim milk powder, Seven Farms, Westfair Food Ltd., Vancouver. Emul s i f i e r s , Atmul 124- and Atmos 300 manufactured by Atlas Chemical Industries. 98. 2. (. P r o c e d u r e A. M i c r o l o a v e s b a k i n g m e t h o d ( 1 0 g r a m , f l o u r ) B r e a d was p r e p a r e d a c c o r d i n g t o t h e m e t h o d o f S h o g r e n e t a l . ( 8 1 ) w i t h some m o d i f i c a t i o n s w i t h 5% p r o t e i n p r e p a r a t i o n s s u b s t i t u t e d f o r f l o u r . T h e b r e w was p r e p a r e d b y s u s p e n d i n g 0.2 5 g y e a s t i n 6.7 m l w a t e r , a t 3 8 ° C c o n t a i n -v i n g 0.6 g s u c r o s e , 0.05 g m a l t e x t r a c t , a n d 0.15 g s a l t f o r 15 m i n . i n a 3 5 ° C w a t e r b a t h . T o t h i s was a d d e d 0.3 g m a r g a r i n e a n d 0.05 g e m u l s i f i e r A t m u l 124- ( a mono- a n d d i g l y c e r i d e m i x t u r e ) a n d 10 g f l o u r c o n t a i n i n g 5% r a p e s e e d p r o t e i n i s o l a t e o r c o n c e n t r a t e t o g e t h e r w i t h 0.0 5 mg p o t a s s i u m b r o m a t e . T h e d o u g h was f o r m e d b y m i x i n g w i t h a s p a t u l a f o r 2.5 min, v b y h a n d a n d a l l o w e d t o f e r m e n t f o r 105 m i n . T h e * d o u g h was m a i n t a i n e d a t a b o u t 3 5 ° C t h r o u g h o u t t h i s p e r i o d . T h e c o n t r o l s w e r e made w i t h o u t e m u l s i f i e r s . T h e d o u g h was t h e n p u n c h e d b y r o l l i n g o n a p l e x i g l a s p l a t e a n d m o l d e d t o s h a p e . T h e m o l d d o u g h was a l l o w e d t o p r o o f f o r 60 m i n . a t 3 8 ° C i n g r e a s e d p a n s 5.2 x 2.3 cm a t t h e b o t t o m . T h e p r o o f e d l o a v e s w e r e b a k e d a t 2 1 0 ° C f o r 10 m i n . i n a n o v e n . T h e b a k e d m i c r o l o a v e s w e r e r e m o v e d i m m e d i a t e l y f r o m t h e p a n s a n d c o o l e d o n a w i r e m e s h t r a y . One h o u r a f t e r b a k i n g t h e m i c r o l o a v e s w e r e w e i g h e d a n d t h e l o a f v o l u m e was m e a s u r e d b y d w a r f r a p e s e e d d i s p l a c e -m e n t . I n a n o t h e r s e r i e s o f e x p e r i m e n t s r a p e s e e d p r o t e i n i s o l a t e s f r o m 1 t o 1 0 % was u s e d f o r t h e m a n u f a c t u r e o f m i c r o l o a v e s . A l s o t h e e f f e c t o f e m u l s i f i e r s on soybean p r o t e i n i s o l a t e on b r e a d making was compared w i t h r a p e s e e d p r o t e i n i s o l a t e . I n t h i s 3 t o 5% soybean p r o t e i n i s o l a t e was used. B. C r u s t c o l o u r d e t e r m i n a t i o n The method d e s c r i b e d by E h l e and Ja n s e n (35) was f o l l o w e d f o r t h e d e t e r m i n a t i o n o f c r u s t c o l o u r . The m i c r o -l o a v e s 1 h r . a f t e r b a k i n g were s l i c e d and d r i e d f o r 24 h r . a t 75°C. The o u t e r one s i x t e e n t h o f an i n c h o f c r u s t was s c r a p e d o f f from t h e s l i c e s and ground i n a m o r t a r t o a f i n e p a r t i c l e s i z e . F i v e hundred m i l l i g r a m o f t h i s ( d r y w e i g h t ) c r u s t was t h e n e x t r a c t e d w i t h 2 5 ml o f 50% e t h a n o l f o r 4 h r . by m e c h a n i c a l l y s h a k i n g i n s t o p p e r e d E r l e n m e y e r f l a s k s . Ten m i l l i l i t e r s o f t h e e x t r a c t was c e n t r i f u g e d a t 15,000 x g f o r 15 min. and t h e absorbance o f t h e c l e a r s u p e r n a t a n t s o l u t i o n d e t e r m i n e d a t 46 0 nm. The measurements a r e avera g e s from d u p l i c a t e e x t r a c t i o n s . C. D e t e r m i n a t i o n o f t h e e m u l s i f y i n g c a p a c i t y The method o f S w i f t et_ a l . (89) was f o l l o w e d f o r t h e d e t e r m i n a t i o n o f 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 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 from r a p e s e e d f l o u r . Samples o f th e i s o l a t e s and c o n c e n t r a t e s c o n t a i n i n g 100 mg i n 30 ml 1 N sodium c h l o r i d e were h e a t e d t o 80°C f o r 5 min. and b l e n d e d i n a n omni m i x e r f o r 2 min. a t c a . 100 0 r.p.m. (See P l a t e I I I ) 100' m To t h i s was added 20 ml c o r n o i l and b e g i n h i g h speed c u t t i n g and m i x i n g f o r 30 s e c . a t c a . 13,000 r.p.m. A d d i t i o n o f o i l was c o n t i n u e d a t t h e r a t e o f 0.8 ml per s e c t i l l t h e break p o i n t was r e a c h e d . The e m u l s i o n formed p e r s i s t e d , and f i n a l l y c o l l a p s e d , t h e t r a n s i t i o n b e i n g marked by g r a d u a l i n c r e a s e , f o l l o w e d by a sudden d e c r e a s e i n 'Viscosity.- The a d d i t i o n o f o i l w a s , i m m e d i a t e l y t e r m i n a t e d on o b s e r v a t i o n o f a b r u p t t r a n s i t i o n . The t o t a l o i l added was n o t e d . The e m u l s i f y i n g c a p a c i t y i s r e p o r t e d as t h e ml o i l per 100 mg p r o t e i n i s o l a t e o r c o n c e n t r a t e . D. S t a b i l i t y t e s t s f o r e m u l s i o n s S t a b i l i t y o f t h e e m u l s i o n s was d e t e r m i n e d by t h e "» method o f Pear s o n e t ' a l . ( 6 6 ) . F o r t h i s d e t e r m i n a t i o n t h e c o r n o i l was added a t t h e s ame r a t e as f o r t h e e m u l s i f y i n g c a p a c i t y d e t e r m i n a t i o n . T h e optimum l e v e l o f o i l o b t a i n e d from t h e above e x p e r i m e n t was added t o t h e p r o t e i n s o l u t i o n c o n t a i n i n g 10 0 mg i n 3 0 ml o f 1 N sodium c h l o r i d e and b l e n d at 13,000 r.p.m. f o r 2 min. The e m u l s i o n s formed were t r a n s f e r r e d t o 15 ml c o n i c a l g r a d u a t e d c e n t r i f u g e t u b e s t o t h e 12 ml mark. The t u b e s w e r e a l l o w e d t o s t a n d t i l l t h e two phases were s e p a r a t e d and t h e time r e c o r d e d . E~. D e t e r m i n a t i o n o f w h i p p a b i l i t y Whip t e s t s were performed as. d e s c r i b e d by G a r i b a l d i '. e t a l . -(AO) . S i x t y gram o f f r e s h egg albumen a t 25°C was whipped at speed 10 i n a Sunbeam mixmaster f o r 4 0 s e c . (See PLATE I I I EMULSIFICAT.ION EQUIPMENT - OMNI MIXER 102 . or P l a t e I V ) . T h i s was c o n t i n u e d a f t e r c h a n g i n g speed t o 6 w i t h 0.25 r a p e s e e d p r o t e i n i s o l a t e s o r c o n c e n t r a t e s c o n t a i n i n g 46 g s u c r o s e by a d d i n g i n 3 e q u a l p o r t i o n s w i t h 4 s e c . w h i p p i n g a f t e r each a d d i t i o n . C o n t r o l s were c a r r i e d i n t h e same way w i t h o u t e i t h e r p r o t e i n i s o l a t e s o r c o n c e n t r a t e s i n c o r p o r a t e d i n t o t h e s u c r o s e . The s p e c i f i c g r a v i t y was c a l c u l a t e d from t h e w e i g h t o f t h e meringue i n a f u n n e l o f known volume s i t t i n g on a 10 ml measuring c y l i n d e r . The r e c i p r o c a l o f t h e s p e c i f i c g r a v i t y g i v e s t h e s p e c i f i c volume wh i c h i s t h e e x p a n s i o n o f t h e whip. The d r i p was a l s o r e c o r d e d f o r 1.5 h r . a t 30 min. i n t e r v a l s . F. M a n u f a c t u r e o f spreads B l e n d s o f 50 g o f m a r g a r i n e , c o r n o i l and o r coconut o i l were used i n t h e s e e x p e r i m e n t s . The s o l i d f a t s were warmed t o room t e m p e r a t u r e 25°C and 0.5 g i s o l a t e s o r c o n c e n t r a t e s from t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c -t i o n were b l e n d e d f o r 30 s e c . and h e a t e d t o 80°C f o r 5 min. T h i s was t h e n c o o l e d t o room t e m p e r a t u r e and mixed a t c a . 13,000 r.p.m. f o r 45 s e c . and t h e n c o n t i n u e d a t c a . 1,000 r.p.m. f o r 60 s e c . The b l e n d s were p l a c e d i n paper cups and were f r o z e n w i t h c o n s t a n t a g i t a t i o n i n an i c e and s a l t b a t h . These were s t o r e d at- 4°C f o r p e r i o d s o f t i m e and t h e c o n s i s t e n c y o f t h e p r o d u c t n o t e d . PLATE IV EQUIPMENT FOR WHIP MEASUREMENT - SUNBEAM MIXMASTER 104. C o m p o s i t i o n o f b l e n d s : No. 1 M a r g a r i n e No. 2 M a r g a r i n e : c o r n o i l : coconut o i l (2:0.5:0.5 by wt) No. 3 M a r g a r i n e : c o r n o i l (1:3 by wt) No. 4 M a r g a r i n e : coconut o i l (1:3 by wt) No. 5 M a r g a r i n e : c o r n o i l (1:1 by w t ) . G. E n r i c h e d i m i t a t i o n i c e cream and i c e m i l k These m i l k p r o d u c t s were made by b l e n d i n g 0.5 g i s o l a t e s w i t h 12 g skim m i l k powder and d i s s o l v i n g i n 9 0 ml w a t e r and h e a t i n g t o 80°C f o r 5 min. T h i s was blended f o r 30 s e c . a t c a . 1,000 r.p.m. i n an omni m i x e r and c o o l e d t o r o o m t e m p e r a t u r e . M a r g a r i n e , s u c r o s e , span 20 as e m u l s i f i e r , g e l a t i n as s t a b i l i z e r , and v a n i l l a o r c o r n s y r u p were added as i n d i c a t e d i n T a b l e X I I . The m i x t u r e was b l e n d e d a g a i n f o r 30 s e c . a t 13,000 r.p.m. and a t 1,000 r.p.m. f o r 60 s e c . The e m u l s i o n s formed were t h a n p a s t e u r i z e d f o r 30 s e c . by h e a t i n g t o 80°C. A f t e r p a s t e u r i z a t i o n t h e p r o d u c t s were c o o l e d and f r o z e n by s u r r o u n d i n g i n a b a t h o f i c e and s a l t , w i t h c o n s t a n t a g i t a t i o n . T A B L E X I I COMPOSITION OF IMITATION ICE CREAM, CHOCOLATE ICE CREAM, AND ICE MILK. THE BASIC INGREDIENTS CONTAIN SKIM MILK POWDER AND PROTEIN ISOLATES FROM RAPESEED. C o m p o s i t i o n I c e Cream C h o c o l a t e I c e m i l k d r i n k M a r g a r i n e (g) 10.0 10.0 4.0 Sucrose (g) 15.0 12.0 15.0 Span 20 (g) 0.1 0.1 0.1 G e l a t i n (g) 0.3 0.3 0.3 V a n i l l a ( d r o p s ) 1.0 - -Cocoa s y r u p (g) - 15.0 -CHAPTER IX RESULTS A. M i c r o l o a v e s o f bread With the p r e l i m i n a r y e x p e r i m e n t s on the prepara--t i o n o f b r e a d c o n t a i n i n g no e m u l s i f i e r s t h e l o a f volume was de c r e a s e d by a p p r o x i m a t e l y 10 t o 15% when 5% r a p e s e e d p r o t e i n i s o l a t e s were s u b s t i t u t e d f o r f l o u r . With the c o n c e n t r a t e s a t 5% t h e r e was a 2 0% de c r e a s e i n l o a f volume. By u s i n g e m u l s i f i e r s the l o a f volumes were r e s t o r e d f o r i s o l a t e s . The c o n c e n t r a t e s d i d not show much improvement i n t h e l o a f volume w i t h e m u l s i f i e r s . The b e s t e m u l s i f i e r was Atmul 124 whic h i n c r e a s e d t h e l o a f volume o f the bread p r e p a r e d . As seen i n T a b l e X I I I , the b r e a d p r e p a r e d from 5% r a p e s e e d p r o t e i n i s o l a t e s c o n t a i n i n g 0.5% e m u l s i f i e r Atmul 124, showed some improvement i n l o a f volume f o r t h e f i r s t w a t e r and second h y d r o c h l o r i c a c i d e x t r a c t i o n s . These two p r e p a r a t i o n s had l o a f volumes h i g h e r by a p p r o x i m a t e l y 10 and 14% r e s p e c t i v e l y o v e r t h e c o n t r o l . The i s o l a t e s from sodium h y d r o x i d e e x t r a c t s showed no s i g n i f i c a n t change i n the l o a f volume. A l l r e s u l t s were o b t a i n e d from d u p l i -c a t e e x p e r i m e n t s . When t h e r e s u l t s d i f f e r e d g r e a t l y a d d i t i o n a l e x p e r i m e n t s were c a r r i e d o u t . The br e a d p r e p a r e d i n t h i s e x p e r i m e n t i s i l l u s t r a t e d i n F i g u r e s 22 and 23. The b r e a d p r e p a r e d from f l o u r c o n t a i n i n g 5% con-c e n t r a t e s w i t h 0.5% Atmul 124 showed a de c r e a s e o f a p p r o x i m a t e l y 8% e x c e p t f o r the c o n c e n t r a t e p r e p a r e d from 107 . . TABLE X I I I - a THE LOAF VOLUMES OF BREAD AND THE COLOUR OF BREAD CRUST CONTAINING 5% RAPESEED PROTEIN ISOLATES Volume l o a f (ml) Absorbance 460 nm P.W 1 5 6b 0.04 P.A l 5 8 a 0.04 P.B l 5 0 c 0.07 P.S i 5 0 c 0.04 C o n t r o l 51° 0.025 'a' j 'b' and 'c' a r e t h r e e groups s i g n i f i c a n t l y d i f f e r e n t as shown by Duncan New M u l t i p l e Range T e s t . (Appendix T a b l e I ) . TABLE X I I I - b THE LOAF VOLUMES OF BREAD AND THE COLOUR OF BREAD CRUST CONTAINING 5% RAPESEED PROTEIN CONCENTRATES / Volume l o a d (ml) Absorbance 460 nm CW 4 8 c 0.03 CA 4 7 c 0.035 CBQ 5 7 a 0.06 CS 47 c 0.04 C o n t r o l 51*> 0.025 ' a ' , 'b' and 'c' a r e t h r e e groups s i g n i f i c a n t l y d i f f e r e n t as shown by Duncan New M u l t i p l e Range T e s t . (Appendix T a b l e I I ) . F i g u r e 22. Micro-loaves (10 grams) baked from r a p e s e e d p r o t e i n i s o l a t e s . From l e f t t o r i g h t , b r e a d c o n t a i n i n g t h e f o l l o w i n g : W - f i r s t w a t e r e x t r a c t i s o l a t e P.W 1 A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e P^B S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e P^S C - t h e c o n t r o l . GO < > gure 23. Micro-loaves (.10 grams) baked from r a p e s e e d p r o t e i n i s o l a t e s . From l e f t t o r i g h t , c u t l o a v e s o f b r e a d c o n t a i n i n g t h e f o l l o w i n g : W - f i r s t w a t e r e x t r a c t i s o l a t e P^ W A - second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e P^A B - t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e P^B S - s i n g l e s t a g e NaOH e x t r a c t i s o l a t e P^S C - t h e c o n t r o l . CO gure 24. M i c r o l o a v e s (10 grams) baked from r a p e s e e d p r o t e i n c o n c e n t r a t e s . From l e f t t o r i g h t , b r e a d c o n t a i n i n g t h e f o l l o w i n g : W - f i r s t w a t e r e x t r a c t c o n c e n t r a t e CW A - second h y d r o c h l o r i c a c i d e x t r a c t c o n c e n t r a t e CA B - t h i r d sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e CB S - s i n g l e s t a g e NaOH e x t r a c t c o n c e n t r a t e CS C - t h e c o n t r o l . 111. F i g u r e 25. M i c r o l o a v e s (10 grams) baked from r a p e s e e d p r o t e i n c o n c e n t r a t e s . From l e f t t o r i g h t , t h e c u t l o a v e s b r e a d c o n t a i n i n g t h e f o l l o w i n g : W - f i r s t w a t e r e x t r a c t c o n c e n t r a t e CW A - second h y d r o c h l o r i c a c i d e x t r a c t c o n c e n t r a t e CA B - t h i r d sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e CB S - s i n g l e s t a g e NaOH e x t r a c t c o n c e n t r a t e CS C - t h e c o n t r o l . DQ 112. F i g u r e 26. M i c r o l o a v e s (10 grams) baked from t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t i s o l a t e P^S from r a p e s e e d f l o u r . From l e f t t o r i g h t , b r e a d c o n t a i n i n g 1, 2, 4, 6, 8 and 10 p e r c e n t p r o t e i n i s o l a t e , and C - t h e c o n t r o l . gure 27. M i c r o l o a v e s (10 grams) baked from t h e s i n s t a g e sodium h y d r o x i d e e x t r a c t i s o l a t e P_^ S from .rapeseed f l o u r . From l e f t . t o r i g h t , c u t l o a v e s b r e a d c o n t a i n i n g 1, 2, 4, 6, 8 and 10 p e r c e n t p r o t e i n and C - t h e c o n t r o l . CD CO O 114. F i g u r e 28. M i c r o l o a v e s (10 grams) baked from soybean p r o t e i n i s o l a t e c o n t a i n i n g 0.5% e m u l s i f i e r s . The c o m p o s i t i o n o f the b r e a d i s g i v e n below: E m u l s i f i e r P r o t e i n i s o l a t e Bread N o . l 2 Atmul 12 4 3 Atmos 300 4 Atmul 12 4 5 Atmos 300 6 Atmul 124 3% soybean p r o t e i n 3% soybean p r o t e i n 5% soybean p r o t e i n 5% soybean p r o t e i n 5% r a p e s e e d p r o t e i n 115. F i g u r e 29. M i c r o l o a v e s (10 gram) baked from soybean p r o t e i n i s o l a t e s c o n t a i n i n g 0.5% e m u l s i f i e r s . The c o m p o s i t i o n o f t h e b r e a d i s g i v e n below: E m u l s i f i e r P r o t e i n i s o l a t e c o n c e n t r a t i o n Bread N o . l 2 Atmul 124 3% soybean p r o t e i n 3 Atmos 30 0 3% soybean p r o t e i n 4 Atmul 124 5% soybean p r o t e i n 5 Atmos 300 5% soybean p r o t e i n 6 Atmul 124 5% r a p e s e e d p r o t e i n 116. t h e . t h i r d sodium h y d r o x i d e e x t r a c t which had an i n c r e a s e o f 12% o v e r t h a t o f t h e c o n t r o l . . These r e s u l t s are i l l u s t r a t e d i n F i g u r e s 24 and 25. The e f f e c t o f r a p e s e e d p r o t e i n i s o l a t e c o n c e n t r a -t i o n on l o a f volume and crumb c o l o u r i s shown i n F i g u r e s 26 and 27. The b r e a d c o n t a i n i n g 1% r a p e s e e d p r o t e i n i s o l a t e from t h e s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t had a b i g g e r l o a f volume t h a n any o f t h e o t h e r b r e a d p r e p a r e d . F i g u r e s 28 and 29 i l l u s t r a t e the e f f e c t o f e m u l s i f i e r s on p r o t e i n from soybean on the l o a f volume. The r e s u l t s i n d i c a t e d t h a t Atmul 124 a t 0.5% was s l i g h t l y b e t t e r t h a n the e m u l s i f i e r Atmos 300 a t 5% soybean p r o t e i n i s o l a t e . The 5% p r o t e i n i s o l a t e had a s l i g h t l y h i g h e r l o a f volume t h a n th e b r e a d c o n t a i n i n g 3% p r o t e i n i s o l a t e . The c r u s t c o l o u r f o r t h e i s o l a t e s and c o n c e n t r a t e s c o n t a i n i n g b r e a d , e x c e p t f o r t h e t h i r d sodium h y d r o x i d e e x t r a c t p r e p a r a t i o n was a l i t t l e h i g h e r t h a n t h a t o b s e r v e d f o r t h e c o n t r o l . The v a l u e o b t a i n e d f o r t h e t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e and c o n c e n t r a t e was t h r e e 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 c o n t r o l . These r e s u l t s a r e t a b u l a t e d i n T a b l e X I I I . B. E m u l s i f y i n g c a p a c i t y From t h e r e s u l t s shown i n T a b l e XIV i t i s e v i d e n t t h a t t h e i s o l a t e from the f i r s t w a t e r e x t r a c t has a h i g h e r e m u l s i f y i n g c a p a c i t y t h a n th e o t h e r i s o a l t e s by a p p r o x i m a t e l y 2 8%. T h i s e x p e r i m e n t s u g g e s t s t h a t t h e w ater e x t r a c t 117 . TABLE XIV THE EMULSIFYING CAPACITY OF THE RAPESEED PROTEIN ISOLATES AND CONCENTRATES, EXPRESSED AS MILLILITERS CORN OIL PER 100 MILLIGRAM PROTEIN ISOLATES OR CONCENTRATE AND THE EMULSION STABILITY IN MINUTES. Volume o f o i l (ml) S t a b i l i t y (min) P.W 1 45.0 10 P. A° l 36.1 200 P.B l 32 . 3 8 P.S l 37.2 8 C o n t r o l - 3 CW 33.1 7 CA 27.4 200 CB 25.2 7 CS 29 . 3 6 118. i s o l a t e i s f a r s u p e r i o r t o t h e r e s t w i t h an e m u l s i f y i n g c a p a c i t y o f 45 ml c o r n o i l p e r 100 mg p r o t e i n i s o l a t e . The l o w e s t v a l u e was w i t h t h e t h i r d sodium h y d r o x i d e i s o l a t e w i t h 32 ml p e r 100 mg p r o t e i n . The c o n c e n t r a t e s showed a s i m i l a r t r e n d but w i t h a l o w e r e m u l s i f y i n g c a p a c i t y o f a p p r o x i m a t e l y 25% t h a n f o r t h e i s o l a t e s . Here a g a i n the weakest was t h e t h i r d sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e . C. E m u l s i o n s t a b i l i t y The s t a b i l i t y o f t h e e m u l s i o n s formed was not as e x p e c t e d , e x c e p t f o r i s o l a t e and c o n c e n t r a t e from t h e second h y d r o c h l o r i c a c i d e x t r a c t . The t i m e t a k e n f o r t h e e m u l s i o n s t o break was u n d e r 10 min. f o r most w h i l e t h e c o n t r o l was o n l y 3 min. The i s o l a t e and c o n c e n t r a t e from t h e h y d r o c h l o r i c a c i d e x t r a c t t o o k o v e r 200 min. t o show any s i g n s o f s e p a r a t i o n . D. W h i p p a b i l i t y (a) E x p a n s i o n The a b i l i t y o f t h e i s o l a t e s t o whip the egg w h i t e shows t h a t t h e h y d r o c h l o r i c a c i d e x t r a c t was c a p a b l e o f i n c r e a s i n g t h e s p e c i f i c volume by 10% w h i l e t h e o t h e r i s o -l a t e s d e c r e a s e d th e e x p a n s i o n a b i l i t y by a p p r o x i m a t e l y 16%. These r e s u l t s a r e t a b u l a t e d i n T a b l e XV. When t h e c o n c e n t r a t e s were used f o r t h e whip d e t e r m i n a t i o n , t h e s p e c i f i c volume d e c r e a s e d i n a l l samples, 119. w i t h t h e w a t e r e x t r a c t h a v i n g a g r e a t e r l o s s o f 19% and t h e h y d r o c h l o r i c a c i d and s i n g l e sodium h y d o x i d e e x t r a c t s o n l y 2%. (b) D r i p As seen from T a b l e XV t h e d r i p from t h e meringue was n e g l i g i b l e f o r a l l i s o l a t e s and c o n c e n t r a t e s p r e p a r e d from t h e t h r e e s t a g e e x t r a c t i o n p r o c e d u r e d u r i n g t h e f i r s t 30 m i n . The s i n g l e s t a g e sodium h y d r o x i d e e x t r a c t p r e p a r a -t i o n s d u r i n g t h i s p e r i o d showed a d r i p o f 1.0 ml. With t i m e up t o 9 0 min. a l l p r e p a r a t i o n s showed 1.0 t o 3.5 ml d r i p , e x c e p t f o r t h e w a t e r e x t r a c t i s o l a t e , compared t o 4.5 ml d r i p i n t h e c o n t r o l . The d r i p o b t a i n e d from t h e concen-t r a t e s was h i g h e r t h a n t h a t n o t e d from t h e i s o l a t e s i n t h i s t i m e p e r i o d e x c e p t f o r t h e c o n c e n t r a t e s from t h e t h i r d sodium h y d r o x i d e e x t r a c t . E. The spreads from i s o l a t e s From p r e l i m i n a r y e x p e r i m e n t s conducted t h e i s o l a t e s and c o n c e n t r a t e s o f r a p e s e e d p r o t e i n was found t o be c o n v e n i e n t l y i n c o r p o r a t e d i n t o b u t t e r l i k e p r o d u c t s so t h a t t h e s e c o u l d be used as spreads w i t h h i g h p r o t e i n con-t e n t s . Among t h e p r e p a r a t i o n s t h a t were s t o r e d under r e f r i g e r a t e d c o n d i t i o n s a t 4°C, t h e sample No. 5 was s o f t a f t e r 5 months, w h i l e t h e sample No. 3 was f i r m , and t h e r e s t o f t h e samples were h a r d l i k e m a r g a r i n e . Thus i t i s p o s s i b l e by p r o p e r b l e n d i n g o f m a r g a r i n e , c o r n o i l and o r TABLE XV. WHIP TEST OF RAPESEED PROTEIN ISOLATES AND CONCENTRATES, THE SPECIFIC VOLUME AND DRIP OF MERINGUE WITH TIME. S p e c i f i c volume D r i p o f 0.5 h r meringue 1.0 h r (ml) 1.5 P.W 1 2.96 - - -P.A° 1 ,3.74 - 0.6 1.0 P.B 1 2 . 81 0. 3 1.0 1.8 P.S l 2. 88 1.0 1.8 2 . 7 CW 2.76 - 1.5 2.6 CA 3.33 0.2 2.1 3.5 CB 3.18 - 1.0 1.6 CS 3. 31 1.0 2.0 3.2 C o n t r o l 3.40 1.0 2.1 4.5 h r 121. coconut o i l w i t h i s o l a t e s and c o n c e n t r a t e s , t o p r e p a r e e a s i l y s p r e a d e m u l s i o n s t h a t can be s t o r e d under r e f r i g e r a t e d con-d i t i o n s f o r l o n g p e r i o d s o f t i m e . F. E n r i c h e d i m i t a t i o n i c e cream and i c e m i l k E n r i c h e d i m i t a t i o n m i l k p r o d u c t s c o u l d be manufac-t u r e d a t 1 t o 2% i s o l a t e s w i t h o u t i m p a r t i n g any u n d e s i r a b l e f l a v o u r s i n t o t h e p r o d u c t . When the i s o l a t e s were used a t 5% l e v e l t h e r e was a d e t e c t a b l e o f f - f l a v o u r from t h e i s o l a t e s , i n t o t h e p r o d u c t . CHAPTER X DISCUSSION As seen from t h e r e s u l t s o b t a i n e d from t h e manu-f a c t u r e o f b r e a d c o n t a i n i n g t h e r a p e s e e d p r o t e i n i s o l a t e s P^ , from t h e f i r s t w a t e r and second h y d r o c h l o r i c a c i d e x t r a c t i o n s , t h e l o a f volume was i n c r e a s e d by 10 t o 14%. Thus i t i s e v i d e n t t h a t t h e wa t e r s o l u b l e f r a c t i o n s ^ s p e c i a l l y t h e albu m i n s a re r e s p o n s i b l e f o r t h e l o a f volume i n c r e a s e . T h i s i s s i m i l a r t o what was ob s e r v e d by Pence e t a l . ( 6 7 ) . The l a r g e l o a f volume o b t a i n e d f o r t h e h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e may be due t o t h e b a s i c n a t u r e o f t h e p r o t e i n f r a c t i o n . The most i n t r i g u i n g o b s e r v a t i o n was made w i t h t h e b r e a d p r e p a r e d w i t h t h e c o n c e n t r a t e from t h e t h i r d sodium h y d r o x i d e e x t r a c t , which was t h e o n l y sample t h a t was a b l e t o i n c r e a s e t h e l o a f volume o f t h e b r e a d . T h i s i n c r e a s e o f t h e volume may be a t t r i b u t e d t o t h e h i g h e r f a t c o n t e n t w h i c h may h ave a l a r g e p e r c e n t a g e o f g l y c o l i p i d s and p h o s p h o l i p i d s . I t was o b s e r v e d f r o m F i g u r e s 26 and 27, t h a t 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 o f r a p e s e e d p r o t e i n i s o l a t e from t h e s i n g l e sodium h y d r o x i d e , o v e r 6% c o l o r o f t h e b r e a d t ends t o dark e n t o produce a b r o w n i s h crumb. I t i s a l s o o b s e r v e d t h a t w i t h an i n c r e a s e i n p r o t e i n c o n c e n t r a t i o n t h e r e i s a g r a d u a l d e c r e a s e i n l o a f volume. With 1% r a p e s e e d p r o t e i n i s o l a t e t h e b i g g e r l o a f volume was o b t a i n e d , t h a n o t h e r s . The d a r k e n i n g o f t he crumb may be due t o t h e 123. b r owning r e a c t i o n s t a k i n g p l a c e between the amino a c i d s and sug a r s i n t h e b r e a d . Thus w i t h an i n c r e a s e i n t h e i s o l a t e c o n c e n t r a t i o n a d a r k e r crumb i s produced c a u s i n g a d e c r e a s e i n t h e n u t r i t i o n a l v a l u e . C r u s t c o l o u r r e s u l t s showed t h a t t h e t h i r d sodium h y d r o x i d e e x t r a c t i s o l a t e and c o n c e n t r a t e had h i g h e r a b s o r -bance i n d i c a t i n g t h e p r e s e n c e o f h i g h e r g l y c o l i p i d s c o n t e n t s i n t h i s p r e p a r a t i o n . I t i s p o s s i b l e t h a t browning r e a c t i o n i s enhanced by t h e t y p e o f c a r b o h y d r a t e and amino a c i d s p r e s e n t o r c a r a m e l i z a t i o n r e a c t i o n t a k i n g p l a c e t o produce a d a r k e r c o l o u r i n t h e br e a d c r u s t . The h i g h e m u l s i f y i n g c a p a c i t y o f t h e f i r s t w a ter e x t r a c t i s o l a t e c o u l d be due t o t h e presence o f phospho-l i p i d s o r g l y c o l i p i d s w hich enhance t h e e m u l s i f y i n g c a p a c i t y . The w a t e r e x t r a c t i s o l a t e P_^W had a g r e a t e r s t a b i l i t y t h a n o t h e r s e x c e p t t h e second h y d r o c h l o r i c a c i d e x t r a c t i s o l a t e w h i c h was e x t r e m e l y s t a b l e . The h i g h e m u l s i f y i n g c a p a c i t y o f t he w a t e r e x t r a c t i s o l a t e c o u l d be due t o t h e g r e a t e r s o l u b i l i t y o f t h i s compared t o t h e o t h e r s . The p r o t e i n s o l u t i o n forms a p r o t e c t i v e c o a t i n g around t h e o i l d r o p l e t s t o g i v e e m u l s i o n s t a b i l i t y . The h y d r o c h l o r i c a c i d i s o l a t e w h i c h i s e x t r e m e l y s t a b l e may be a t t r i b u t e d t o t h e same e f f e c t . The whip t e s t i n d i c a t e s t h e r e i s a k i n d o f d i f f e r e n c e among t h e i s o l a t e s . The h y d r o c h l o r i c a c i d i s o l a t e P-A caused a 10% i n c r e a s e i n s p e c i f i c volume 124. compared t o t h e o t h e r s . T h i s c o u l d be due p a r t l y , t o t h e b a s i c n a t u r e o f t h e p r o t e i n f r a c t i o n s p r e s e n t i n t h e i s o l a t e . The c o n c e n t r a t e s were u n a b l e t o improve t h e s p e c i -f i c volume o f t h e meringue. Among them th e second h y d r o -c h l o r i c a c i d and s i n g l e sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e s d e c r e a s e d t h e s p e c i f i c volume by a p p r o x i m a t e l y 2%, w h i l e t h e w a t e r e x t r a c t had a 19% d e c r e a s e . The d r i p i n meringue c o u l d be d e c r e a s e d c o n s i d e r -a b l y by u s i n g t h e w a t e r e x t r a c t i s o l a t e and by 78% w i t h h y d r o c h l o r i c a c i d i s o l a t e . The t h i r d sodium h y d r o x i d e e x t r a c t c o n c e n t r a t e was a b l e t o d e c r e a s e t h e d r i p by 65%. T h i s b e h a v i o u r o f t h e t h i r d sodium h y d r o x i d e e x t r a c t concen-t r a t e i s due, as e x p a l i n e d e a r l i e r , t o t h e c o m p o s i t i o n o f t h e p r e p a r a t i o n . CHAPTER XI GENERAL CONCLUSION From t h e r e s u l t s o b s e r v e d from t h i s i n v e s t i g a t i o n i t w as c o n c l u d e d t h a t by u s i n g t h e t h r e e s t a g e e x t r a c t i o n p r o c e d u r e , p r o t e i n f r a c t i o n s r i c h i n c e r t a i n e s s e n t i a l amino a c i d s such as l y s i n e and s u l f u r - c o n t a i n i n g amino a c i d s were s e p a r a t e d w i t h o u t t h e use o f e l e g a n t c h r o m a t o g r a p h i c methods. A h i g h ash c o n t e n t w h i c h i s m a i n l y due t o t h e p r e s e n c e o f l a r g e amounts o f c a l c i u m i s a l s o an advan t a g e , as t h i s element i s n u t r i t i o n a l l y i m p o r t a n t . These i n d i v i d u a l f r a c t i o n s , e s p e c i a l l y t h e w a t e r and h y d r o c h l o r i c a c i d i s o l a t e s , behave d i f f e r e n t l y when a p p l i e d t o f o o d p r o c e s s i n g such as b r e a d m a n u f a c t u r e , e m u l s i o n f o r m a t i o n and w h i p p i n g a b i l i t y o f egg albumen. The i n t r i g u i n g r e s u l t s from t h e t h i r d sodium h y d r o x i d e concen-t r a t e s h o u l d be f u r t h e r i n v e s t i g a t e d i n o r d e r t o u t i l i z e t h i s f r a c t i o n i n the most e c o n o m i c a l way i n t h e f o o d p r o -c e s s i n g i n d u s t r y . From t h e r e s u l t s o b t a i n e d so f a r , d i f f e r e n t p r o t e i n i s o l a t e s seem t o be f u n c t i o n i n g b e t t e r t h a n o t h e r s f o r s p e c i a l - p u r p o s e s i n d i f f e r e n t f o o d p r o d u c t s . A t t h i s s t a g e o f t h i s s t u d y i t i s h o p e f u l t h a t more u s e f u l a p p l i c a -t i o n s i n o t h e r a r e a s o f f o o d p r o c e s s i n g , such as meat o r b a k e r y p r o d u c t s , can be a c c o m p l i s h e d i n t h e f u t u r e . APPENDIX 127 . TABLE I ANALYSIS OF VARIANCE OF LOAF VOLUME WITH RAPESEED PROTEIN ISOLATES Source o f v a r i a t i o n Degree o f freedom Mean square F - r a t i o R e p l i c a t e s 5 24.32 I s o l a t e s 4 80.60 175.23** E r r o r 2 0 0.46 T o t a l 29 ** S i g n i f i c a n t a t p r o b a b i l i t y 0.01 Duncan New M u l t i p l e Range T e s t : Loaf volumes o f t h e br e a d p r e p a r e d from the i s o l a t e P ^ A - i s s i g n i f i c a n t l y d i f f e r e n t from t h a t o f P^W, and t h e s e i n t u r n d i f f e r s i g n i f i c a n t l y from t h e r e s t o f t h e i s o l a t e s and the c o n t r o l . I s o l a t e t r e a t m e n t C o n t r o l P.W l P .A X P.B i P.S l Mean v a l u e 51. 33 56 . 12 58.00 50.33 50.17 128 . TABLE I I ANALYSIS OF VARIANCE OF LOAF VOLUME WITH RAPESEED PROTEIN CONCENTRATES Source o f v a r i a t i o n Degree o f freedom Mean square F - R a t i o R e p l i c a t e s 5 0.38 C o n c e n t r a t e s 4 115.9 8 85.91** E r r o r 2 0 1.35 T o t a l 29 ** S i g n i f i c a n t at p r o b a b i l i t y 0.01 Duncan New M u l t i p l e Range T e s t : L o a f volumes o f bread p r e p a r e d from the r a p e s e e d p r o t e i n c o n c e n t r a t e CB i s s i g n i f i c a n t l y d i f f e r e n t from t h a t o f the c o n t r o l and t h e s e i n t u r n are d i f f e r e n t s i g n i f i c a n t l y from t h e r e s t o f t h e c o n c e n t r a t e s , which are s i m i l a r t o one a n o t h e r C o n c e n t r a t e C o n t r o l CW CA CB CS t r e a t m e n t Mean v a l u e 51.33 47.67 46 .67 57.00 46 .83 129 . LITERATURE CITED 1. 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