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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 (Brassica  c a m p e s t r i s L. v a r . E c h o ) By  LAKSHMAN PUNNIYADASA KODAGODA B.Sc.  University  o f C e y l o n , 1961  M.Sc.  University  of. T o k y o ,  1965  A T H E S I S SUBMITTED I N P A R T I A L FULFILMENT OF THE REQUIREMENTS  FOR THE DEGREE  OF  DOCTOR OF PHILOSOPHY  i n t h e Department of Food We a c c e p t t h i s required  Science  thesis  as c o n f o r m i n g  t o the  standard  THE UNIVERSITY OF B R I T I S H M a r c h , 197 2.  COLUMBIA  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y  of B r i t i s h Columbia, I agree t h a t  the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be  granted by  permission.  Department of  Food S c i e n c e  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  March 2U,  Department or  I t i s understood t h a t copying or  of t h i s t h e s i s f o r f i n a n c i a l g a i n written  the Head of my  1972.  s h a l l not be  publication  allowed without  my  ABSTRACT A three  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  rapeseed f l o u r s e q u e n t i a l l y with a c i d , and  0.02  N s o d i u m h y d r o x i d e has  f i r s t water extract contained t e i n s whereas the basic  p r o t e i n s , which migrate to the  arginine  This  can  partially  hydrochloric  been d e v e l o p e d . neutral  acid extract c a t h o d e by be  The  contained  pH  explained  pro-  8.8 by  gel higher  content. Ash  approximately 0.05  content i n the 36%,  was  second h y d r o c h l o r i c  r e d u c e d t o l e s s t h a n 10%  acid by  extract,  treating  M oxalic acid. The  w a t e r e x t r a c t may  be  a good s u p p l e m e n t t o  v e g e t a b l e p r o d u c t s because of high The  water, hydrochloric  7,  a c i d and  41  and  61%  i n the  i n the  whey d u r i n g  prepara-  respectively for  the  sodium h y d r o x i d e e x t r a c t i o n s  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 o n .  were l o s t  other  l y s i n e content.  approximate y i e l d s of proteins  t i o n o f i s o l a t e s were 11,  f o r the  N  m a i n l y a c i d i c and  second h y d r o c h l o r i c  electrophoresis.  with  w a t e r , 0.1  from  and  Proteins  i s o e l e c t r i c p r e c i p i t a t i o n , with  a concomitent l o s s of a considerable  amount o f c y s t i n e  and  methionine. The  emulsifying  b e t t e r than that corn o i l per  of the  10 0 mg  capacity  of the water e x t r a c t  other extracts:  45  and  35 ml  of protein i s o l a t e respectively.  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 , b u t  the  was  of The  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 electric  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  p r e c i p i t a t i o n caused  d e c r e a s e i n l o a f volume. centrates  The  an a p p r o x i m a t e  bread prepared with  d e c r e a s e d l o a f v o l u m e by a b o u t The  a d d i n g 0.5%  l o a f v o l u m e was  Atmul  manufactured  124  10 t o  restored  the  any  by  t o t h e dough. hydro-  i n l o a f v o l u m e o f 10  to  With the i s o l a t e s from  s i n g l e stage  t h e l o a f v o l u m e was  sodium  restored  without  increase. The  with  an i n c r e a s e  s o d i u m h y d r o x i d e and  hydroxide extractions  con-  ( a mono- and d i g l y c e r i d e m i x t u r e  observed over the c o n t r o l .  third  the  i n most c a s e s  W i t h t h e i s o l a t e s f r o m t h e f i r s t w a t e r and s e c o n d  15% was  15%  20%.  by A t l a s C h e m i c a l I n d u s t r i e s )  chloric acid extracts  iso-  bread prepared from the concentrates t o g e t h e r  e m u l s i f i e r showed a d e c r e a s e o f 12%  except the t h i r d  i n l o a f volume  sodium h y d r o x i d e e x t r a c t w h i c h had a  6%  increase. 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 m e r i n g u e when t h e p r o t e i n t i o n was  i s o l a t e from the h y d r o c h l o r i c  acid  u s e d a t a l e v e l o f 3% r e p l a c i n g e g g w h i t e  extracprotein,  an e v e n b e t t e r r e s u l t t h a n w h i p p i n g t h e e g g w h i t e a l o n e . All  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 .  centrates  except the water e x t r a c t  showed a s l i g h t  The  con-  decrease.  TABLE OF CONTENTS  ABSTRACT TABLE OF CONTENTS LIST  OF  FIGURES  LIST OF TABLES LIST OF PLATES ACKNOWLEDGEMENTS PART I ISOLATION OF PROTEINS FROM RAPESEED C B r a s s i c a c a m p e s t r i s L. v a r . Echo) FLOUR CHAPTER I II III  INTRODUCTION LITERATURE REVIEW EXPERIMENTAL MATERIALS AND PROCEDURES Materials Procedures D e t e r m i n a t i o n of n i t r o g e n D e t e r m i n a t i o n of m o i s t u r e and ash D e t e r m i n a t i o n of l i p i d s D e t e r m i n a t i o n of phosphorus D e t e r m i n a t i o n of c a l c i u m D e t e r m i n a t i o n of t o t a l  sulfur  Determination of carbohydrates D e t e r m i n a t i o n of crude f i b e r D e t e r m i n a t i o n o f i s o t h i o c y a n a t e . and thiooxazolidones  - ivCHAPTER III  PAGE 2. P r o c e d u r e s  (continued)  C a l i b r a t i o n standard  curves f o r  protein determination  24  Slab  g e l electrophoresis  24  Disc  g e l electrophoresis  25  Isoelectric  focusing  26  pH m e a s u r e m e n t  _  26  Amino a c i d a n a l y s i s  27  Cystine  27  determination  Tryptophan determination  28  Ultracentrifugation  28  3. E x t r a c t i o n a n d p r e p a r a t i o n o f i s o l a t e s Three stage e x t r a c t i o n procedure Single  30 30  stage 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 treatment f o r decreasing ash content i n the hydrochloric acid extract (SA)  34  Preparation  of  concentrates  34  Preparation  of  isolates  34  Preparation  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  pH-solubility profiles  36  - Preparation  of the extracts  o f crude myrosinase  36  S o l u b i l i t y measurements o f i s o l a t e s and IV  concentrates  RESULTS C a l i b r a t i o n curves f o r proteins  37 38 38  -  V  -  CHAPTER IV  P  RESULTS  G  E  (Continued)  pH-solubility  profile of the extracts  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 hydrochloric acid extract  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 concentrates  45  Electrophoresis  53  Isoelectric  56  Amino A c i d  focusing composition  64  Ultracentrifugation  71  M a t e r i a l balance of 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  75  Yield  of protein  washing procedure  isolated  ,  78  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 V  A  DISCUSSION  78 81  PART I I FUNCTIONAL PROPERTIES OF THE  RAPESEED  PROTEIN FRACTIONS VI VII VIII  INTRODUCTION  89  LITERATURE REVIEW  90  EXPERIMENTAL MATERIALS AND PROCEDURES  97  1. - M a t e r i a l s  97  2.  Procedures  98  M i c r o l o a v e s b a k i n g method (10 grams)  98  Crust  99  o  colour  determination  - vi -  CHAPTER VIII  PAGE 2. P r o c e d u r e s  (continued)  Determination of the emulsifying capacity  99  Stability  t e s t s f o remulsions  100  Determination of whippability  100  Manufacture o f spreads  102  Enriched ice IX  milk  Microloaves  o f bread  106  Emulsifying  capacity  116  Emulsion s t a b i l i t y  118  Whippability  118  Drip  119  The s p r e a d s f r o m i s o l a t e s  119  ice  XI  104  RESULTS  Enriched  X  i m i t a t i o n i c e cream and  i m i t a t i o n i c e c r e a m and  milk  121  DISCUSSION  122  GENERAL CONCLUSION APPENDIX  125 126  LITERATURE CITED  129  L I S T OF FIGURES FIGURE 1.  PAGE FLOW DIAGRAM OF THE THREE STAGE EXTRACTION PROCEDURE  2.  32  FLOW DIAGRAM OF THE SINGLE STAGE EXTRACTION PROCEDURE  3.  33  E F F E C T OF PROTEIN CONCENTRATION AND HYDROCHLORIC ACID EXTRACT  4.  OF THE WATER  ON ABSORBANCE  E F F E C T OF PROTEIN CONCENTRATION  OF THE SODIUM  HYDROXIDE EXTRACTS ON ABSORBANCE 5.  40  pH-SOLUBILITY PROFILES OF THE WATER AND HYDROCHLORIC ACID EXTRACTS  6.  41  pH-SOLUBILITY PROFILES OF THE SODIUM HYDROXIDE EXTRACTS  7.  42  EFFECT OF OXALIC ACID ON CALCIUM  REMOVAL  FROM HYDROCHLORIC ACID EXTRACT 8.  43  EFFECT OF OXALIC ACID TREATMENT OF THE HYDROCHLORIC ACID EXTRACT  ON PROTEIN  CONCENTRATIONS  9.  39  SLAB GEL ELECTROPHORESIS PROTEIN ISOLATES PREPARED PRECIPITATION  44  OF THE RAPESEED BY I S O E L E C T R I C 54  - viii -  FIGURE 10  PAGE SLAB GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN WHEY AFTER ISOELECTRIC PRECIPITATION  11.  55  DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION  12.  57  DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLOROACETIC ACID PRECIPITATION  13.  DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN CONCENTRATES  . 14.  59  DISC GEL ELECTROPHORESIS OF THE RAPESEED PROTEIN WHEY AFTER ISOELECTRIC PRECIPITATION  15.  58  60  ISOELECTRIC FOCUSING OF THE RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION  16.  61  ISOELECTRIC FOCUSING OF THE RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLOROACETIC ACID PRECIPITATION  17.  62  ISOELECTRIC FOCUSING OF THE RAPESEED PROTEIN CONCENTRATES  63  -  i x  -  FIGURE 18.  PAGE SCHLIEREN ISOLATE  P A T T E R N OF THE WATER  EXTRACT  .  72  19.  SCHLIEREN  P A T T E R N OF THE H C 1 E X T R A C T  20.  SCHLIEREN  P A T T E R N OF THE NaOH E X T R A C T  ISOLATE  ISOLATE 21.  22.  7H  SCHLIEREN  P A T T E R N OF THE S I N G L E  HYDROXIDE  EXTRACT  STAGE  SODIUM  7H  ISOLATE  MICROLOAVES PREPARED  FROM THE  ISOLATES  -  WHOLE B R E A D 23.  108  MICROLOAVES PREPARED  FROM THE I S O L A T E S  -  CUT L O A V E S 24.  MICROLOAVES  109 PREPARED  FROM THE  CONCENTRATES  -  WHOLE B R E A D 25.  110  MICROLOAVES PREPARED  FROM THE C O N C E N T R A T E S  -  CUT L O A V E S 26.  MICROLOAVES  111 PREPARED  FROM P R O T E I N  ISOLATE  TO STUDY THE E F F E C T ON VOLUME AND COLOUR  -  WHOLE B R E A D 27.  112  MICROLOAVES PREPARED STUDY THE E F F E C T CUT L O A V E S  73  FROM P R O T E I N  ISOLATE  OF VOLUME AND COLOUR  TO  113  MICROLOAVES PREPARED FROM SOYBEAN PROTEIN ISOLATE TO STUDY THE E F F E C T OF E M U L S I F I E R S ON VOLUME - WHOLE BREAD MICROLOAVES PREPARED FROM SOYBEAN PROTEIN ISOLATE TO STUDY THE EFFECT OF E M U L S I F I E R S ON VOLUME - CUT LOAVES  L I S T OF TABLES TABLE I  PAGE PROXIMATE A N A L Y S I S OF RAPESEED PROTEIN ISOLATES PREPARED BY ISOELECTRIC PRECIPITATION  II  46  PROXIMATE A N A L Y S I S OF RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLOROACETIC A C I D PRECIPITATION  III  47  PROXIMATE ANALYSIS OF RAPESEED PROTEIN CONCENTRATES PREPARED BY THE THREE  STAGE  AND SINGLE STAGE EXTRACTION IV  48  AMINO A C I D COMPOSITION OF RAPESEED PROTEIN CONCENTRATES PREPARED BY THE THREE  STAGE  AND SINGLE STAGE EXTRACTION V  65  AMINO ACID COMPOSITION OF RAPESEED PROTEIN ISOLATES PREPARED BY I S O E L E C T R I C PRECIPITATION  VI  66  AMINO A C I D COMPOSITION OF RAPESEED PROTEIN ISOLATES PREPARED BY TRICHLOROACETIC A C I D PRECIPITATION  VII  67  AMINO A C I D COMPOSITION OF RAPESEED PROTEIN WHEY AFTER I S O E L E C T R I C P R E C I P I T A T I O N  68  - xii  -  TABLE VIII IX X XI  PAGE PROTEIN MATERIAL BALANCE  76  PROTEIN LOSSES DURING WASHING PROCEDURE  77  Y I E L D S OF PROTEINS ISOLATED  '  S O L U B I L I T Y MEASUREMENTS  80  XII  COMPOSITION OF THE IMITATION MILK PRODUCTS  XHIa  LOAF VOLUMES AND CRUST COLOUR MEASUREMENTS OF ISOLATES  XHIb  XV  105  107  LOAF VOLUMES AND CRUST COLOUR MEASUREMENTS OF CONCENTRATES  . XIV  79  - .  EMULSIFYING CAPACITY AND EMULSION S T A B I L I T Y  107 117  MERINGUE S P E C I F I C VOLUME AND DRIP MEASUREMENTS  •  12 0  LIST OF PLATES PLATE I  PAGE AMINCO DISTILLATION APPARATUS FOR NITROGEN  II III IV  '•  EXTRACTION APPARATUS - LOURDES BLENDER EMULSIFICATION EQUIPMENT - OMNI MIXER  17 31 101  EQUIPMENT FOR WHIP MEASUREMENTS SUNBEAM MIXMASTER  10 3  ACKNOWLEDGEMENTS The and  author wishes t o express h i s sincere  a p p r e c i a t i o n t o Dr. Shuryo N a k a i ,  D e p a r t m e n t o f Food S c i e n c e ,  Professor,  University of British  for his supervision, indispensable suggestions during  Associate  gratitude  Columbia,  g u i d a n c e and h e l p f u l  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  of this  dissertation. The  author would a l s o l i k e t o express h i s  sincere  t h a n k s t o D r . W i l l i a m D. P o w r i e , C h a i r m a n , D e p a r t m e n t o f Food S c i e n c e f o r e n a b l i n g leave  him t o obtain the extension of  from t h e Government o f C e y l o n . The  author wishes t o express h i s thanks 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 official Dr.  duties  during  t h e course o f t h i s  s t u d y , and t o  J.W.L. P e i r i s , D e p u t y D i r e c t o r A g r i c u l t u r e  of t h e C e n t r a l A g r i c u l t u r a l Research I n s t i t u t e , of A g r i c u l t u r e , Ceylon, f o r i n i t i a t i n g The  (Research) Department  t h e program.  author 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 Office) for providing  (formally the External A i d  f i n a n c i a l aid enabling  h i m t o come t o  C a n a d a f o r a d v a n c e d s t u d y b y way o f t h e g e n e r o u s Colombo Plan  scholarship.  - xv The and  d e f i n i t i o n f o r rapeseed  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  protein  concentrates  once from f l o u r and t h o s e  w h i c h w e r e 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. It that  i s generally  soyprotein  products  for soyprotein  a minimum o f 70 a n d 9 0 % p r o t e i n  free basis, respectively.  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 treated  t o decrease  glucosinolates  However,  protein  With  1.  an i n t e n t i o n extraction f o r  c o n t e n t o f t h e c o n c e n t r a t e s and  extraction  Second h y d r o c h l o r i c  Single  which  (N x 6.25) a r e  below:  F i r s t water  Third  rapeseed  attempted.  i s o l a t e s on a m o i s t u r e a n d f a t f r e e b a s i s tabulated  (N x 6.25)  before e x t r a c 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 r e m o v i n g t h e s e r e s i d u a l l i p i d s were  as  i n t h i s s t u d y was h e a t -  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 .  The  products  i s o l a t e s and c o n c e n t r a t e s a r e d e f i n e d  containing  on a m o i s t u r e  accepted  acid  sodium hydroxide s t a g e NaOH e x t r a c t i o n  These r e s u l t s a r e v e r y c l o s e soybean p r o d u c t s .  Concentrates  Isolates  64. 6  86. 8  45. 8  86. 6  75. 6  87. 5  76. 0  • 9 1 .3  t o the value obtained f o r  PART I  ISOLATION OF PROTEIN FRACTIONS FROM RAPESEED FLOUR ( B r a s s i c a campestris L. v a r . Echo)  \  CHAPTER I INTRODUCTION Rapeseed ties  ( B r a s s i c a sp) i s produced  i n Canada e s p e c i a l l y  i n large  quanti-  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 oils.  F i f t y p e r c e n t o f t h e o i l seed c r o p i n Canada i s  rapeseed  (29).  With t h e l a r g e tonnage  produced  annually f o rthe o i l ,  crop  t h e r e s u l t i n g m e a l h a s become  an i m p o r t a n t s o u r c e o f m e a l p r o t e i n . produced  o f t h e o i l seed  Compared t o t h e m e a l  p r e v i o u s l y , t h e p r e s e n t day m e a l s a r e o f a b e t t e r  q u a l i t y due t o i m p r o v e d  t e c h n i q u e s used  i n the processing of  t h e m e a l and f l o u r . The  g l u c o s i n o l a t e s which are the p r e c u r s o r s o f  toxic principles  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  and m e a l a r e t h e two m a j o r utilization The  limiting  Smith  i n human f o o d s .  and c o n c e n t r a t e s may a l s o be  another obstacle f o r the u t i l i z a t i o n foods.  factors against the  o f them as a p r o t e i n supplement  colour of the isolates  flour  o f them i n s u p p l e m e n t a r y  (84) i n d i c a t e d t h a t t h e y i e l d  of isolated  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 l o w f o r c o m m e r c i a l production. Chichester  The y i e l d  o b t a i n e d f r o m r a p e s e e d by Owen a n d  ( 6 5 ) 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 1 8 % .  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%. it  i s noted 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  i s o l a t e d by i s o e l e c t r i c  precipitation.  Thus  rapeseed  2. Most of the recent been c a r r i e d out  work on rapeseed p r o t e i n  at the N a t i o n a l  Regional L a b o r a t o r y , Saskatoon. been conducting e x t e n s i v e properties  Research C o u n c i l , Finlayson  Prairie  et_ aJL. (37)  work on p h y s i c a l and  have  chemical  of rapeseed p r o t e i n f r a c t i o n s p u r i f i e d by  chromatographic t e c h n i q u e s .  has  various  Bhatty e_t aJL. (14) have i s o l a t e d  d i f f e r e n t p r o t e i n f r a c t i o n s from rapeseed p r o t e i n s o l u b l e i n s a l t s o l u t i o n s by u s i n g  column chromatography.  Rapeseed ( B r a s s i c a napus L. var. Nugget) at l e a s t 9 c h r o m a t o g r a p h i c a l l y d i s t i n g u i s h a b l e components.  Of t h e s e , there  S ) , which t o g e t h e r contained  and  the  12  chains and  protein  are 2 major p r o t e i n s  1.7  35%  of the  a l a r g e m o l e c u l a r weight (14).  have shown t h a t the g r a p h i c a l l y and  12  S p r o t e i n f r a c t i o n was  at pH  8.0  and  9.5,  but  values below 3.5  In t h i s study r e s e a r c h  and  protein These workers  chromato-  dissociated into  and  was  i n urea s o l u t i o n s .  i n i t i a t e d to f i n d  economical source of vegetable p r o t e i n i s o l a t e s f o r supplementation of p r o t e i n d e f i c i e n t d i e t s . o b j e c t i v e was  S  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  s o l u t i o n s between pH subfractions  (12  seed p r o t e i n  S p r o t e i n i s an aggregate of s e v e r a l has  contained  The  the  primary  to develop an e x t r a c t i o n procedure to  from rapeseed f l o u r n e a r l y  an  obtain  c o l o r l e s s p r o t e i n 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 f u n c t i o n a l pro-  p e r t i e s s u i t a b l e f o r food p r o c e s s i n g .  The  proteins  3.  resulting and  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  soybean p r o t e i n i s o l a t e s  evaluate t h e i r  importance.  and  concentrates i n order  to  CHAPTER I I LITERATURE REVIEW O r i g i n o f B r a s s l e a n a p u s L. and L. i s n o t w e l l d o c u m e n t e d .  The  Brassica campestris  earliest direct reference to  the o i l s e e d rapes i s found i n the a n c i e n t S a n s k r i t o f 200  t o 1500  B.C.  (82).  form found w i t h i n t h i s  Sinskaia  writings  (83) a f t e r a s t u d y o f  s p e c i e s i n Europe  and A s i a ,  suggested  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 would  u l t i m a t e l y be  Sinskaia  located i n Asia.  rape  On t h e o t h e r h a n d  (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 f o 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 h a v e o r i g i n a t e d u n d e r m a r i t i m e c l i m a t e s u c h as Mediterranean.  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  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  I n w e s t e r n C a n a d a r a p e s e e d was i n 1942,  of marine  first  t i o n i s the 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 i s not s u b j e c t t o h i g h  d u r i n g p r o c e s s i n g , a meal comparable  t o soybean  Rapeseed i s a crop w e l l adapted t o p r o d u c t i o n has  s u p p l y o f t h e s e e d has As a c o n s e q u e n c e  g r o w n commer-  A major by-product of t h i s o i l e x t r a c -  solvent e x t r a c t i o n which  The  lines.  a war m e a s u r e 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  engines.  conditions.  European  stage i n the develop-  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  cially  the  temperatures i s produced.  Canadian  increased rapidly  g r a d u a l l y exceeded  until  d o m e s t i c demand.  i n r e c e n t y e a r s C a n a d a h a s e x p o r t e d more  rapeseed than a l l other countries  combined.  Accurate s t a t i s t i c s not r e a d i l y a v a i l a b l e .  of trade i n rapeseed  H o w e v e r , i t has  t h a t t h e m a j o r p r o d u c i n g c o u n t r i e s had  been  meal  are  established  an a n n u a l e x p o r t  of  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 m e a l i n t h e p e r i o d to  1962,  (22).  I n t h e y e a r 1968/69 t h e p r o d u c t i o n h a d  i n c r e a s e d t o 5,323,000 t o n s and was  a s l i g h t decrease  Of t h i s  f o r t h e y e a r 1969/70 t h e r e  w i t h 5,250,000 t o n s o f r a p e s e e d  a p p r o x i m a t e l y 75% i s p r o d u c e d In  1958  and  Canada 2 t y p e s o f r a p e s e e d  B r a s s i c a ri apu s L. (Polish rape).  (Argentine rape)  and  (23).  consumed i n A s i a .  are  produced:  Brassica campestris  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  a g r e a t e r p o t e n t i a l f o r s e e d and  o i l than the P o l i s h  L  has  rape.  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 r e p r e f e r r e d , i n Canada, b e c a u s e t h e y m a t u r e 10 t o 14 d a y s e a r l i e r f o r m s a r e g r o w n , as e v e n t h e most h a r d y varieties will of  western  not c o n s i s t e n t l y  Canada.  The  oils,  s u r v i v e on t h e o p e n  acreage  soybean,  peanut,  oil  and  Arlo  Canada.  c o t t o n s e e d and  l a r g e amount o f m e a l e n a b l e s  crushed  sunflower  has  50%  (29).  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 rapeseed  plains  of western  i s the l a r g e s t e x p o r t e r of rapeseed  the o i l seed crop The  rape  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  e x c e e d e d by  Canada w h i c h  turnip  t u r n i p r a p e v a r i e t i e s E c h o and  o c c u p y 70 t o 80% o f t h e r a p e s e e d Rapeseed r a n k s  ( 3 0 ) . Thus summer  a  substantial  f o r consumption.  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  50% o i l m e a l and  the remainder  i s moisture.  The 40% The  of  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 is presently u t i l i z e d The  a b o u t 4 0%  f o r l i v e s t o c k and  poultry  meal c u r r e n t l y produced d i f f e r  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  l e s s heat during processing  and  the  crude p r o t e i n , feeding.  from those  that  subjected  to  amount o f o i l l e f t  in  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 h i c h damage t o t h e Because of the  p r o t e i n had  reduction  occurred  i n the  during  processing.  amount o f h e a t u s e d i n  c e s s i n g t h e m e a l s a r e o f much b e t t e r q u a l i t y t h a n produced years  l b pressure  p r e p a r e d by  and  Hill  solvent  a f f e c t methionine.  C l a n d i n i n and the  a t 121°C  sunflower  at  o i l meal  e x t r a c t i o n caused a marked d e s t r u c t i o n These t r e a t m e n t s however d i d  Morrison  that the n u t r i t i o n a l value processing  (1) f o u n d t h a t a u t o c l a v i n g  dry h e a t i n g  of l y s i n e i n the meal.  as t h e  those  ago.  A l e x a n d e r and 15  pro-  e t a l . (59  of sunflower  t e m p e r a t u r e was  Robblee  (19)  p r o t e i n from sunflower  and  60)  observed  o i l meal  increased.  a t t r i b u t e d the  not  increased  However,  poor q u a l i t y of  o i l meal t o e x c e s s i v e  processing  temperature. I t has  been r e p o r t e d  by  Craig  (24-) t h a t  growing c o n d i t i o n s of rapeseed a f f e c t the seed c o n s t i t u e n t s . of f a t t y during  a c i d s was  growth.  The  For  instance  r e l a t e d t o the  the  composition  degree of  environmental  of  unsaturation conditions  g l u c o s i n o l a t e ( f o r m e r l y known  t h i o g l u c o s i d e ) content  the  as  of the rapeseed i s p r o p o r t i o n a l to  7. the by  available sulfur i n the s o i l . W e t t e r (99) and J o s e f s s o n  T h i s has been d e m o n s t r a t e d  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  that plants of Brassica  n a p u s L. v a r . n u g g e t grown i n s o i l s produced seeds w i t h  deficient i n sulfur  a lower content 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 o f t h e 12 S g l o b u l i n t h a n s e e d s p r o d u c e d f r o m fertilized  plants  availability protein  of s o i l  variety.  s u l f u r h a s a s i g n i f i c a n t e f f e c t on  a r e many c o m p o s i t i o n a l  to species, This  from  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 self-fertile  rape and under f i e l d c o n d i t i o n s  they  largely self-pollinated (62). Bell  (13) i n d i c a t e d i n a review t h a t  m e a l when i n c o r p o r a t e d or higher  In a r e p o r t  published  detrimental  and d e t r i m e n t a l  t o growth.  by C l a n d i n i n e t a l . ( 2 0 ) i t was rape  ( B r a s s i c a n a p u s L.) was more  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 percent Argentine  rapeseed  into poultry rations at levels of  was g o i t r o g e n i c  proved that Argentine  Ten  inconsis-  differences  i s m a i n l y due t o t h e h i g h l y  nature o f Argentine  10%  These f i n d i n g s suggest t h a t t h e  composition o f rapeseed i s h i g h l y  Thus t h e r e  species  are  adequate  synthesis. The  tent.  (37).  behaviour  (Brassica campestris L . ) .  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 , w h e r e a s much a s 1 5 % P o l i s h r a p e s e e d m e a l i n the  r a t i o n d i d not r e s u l t i n appreciable  growth  depressions.  Five percent Argentine  r a p e s e e d m e a l was shown t o d o u b l e 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 was r e q u i r e d  15% P o l i s h rapeseed  t o p r o d u c e a s i m i l a r e f f e c t , on c h i c k s .  r e s u l t s were o b t a i n e d  by R e n n e r e t a l .  Limitations of using  meal  Similar  (75).  r a p e s e e d i s due t o t h e  p r e s e n c e o f l o w 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 s e e d s , some o f w h i c h may be r e l e a s e d causing  metabolic  t h r o u g h enzyme a c t i o n  disturbance.  C l a n d i n i n e t a l . (20) r e p o r t e d  that  isothiocyanate  c o n t e n t was m a r k e d l y a f f e c t e d by v a r i e t y and no effect tent  o f l o c a t i o n was a p p a r e n t .  on t h e o t h e r  consistent  The t h i o o x a z o l i d i n e c o n -  h a n d was n o t o n l y  a f f e c t e d by v a r i e t y , b u t  a l s o a p p e a r e d 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 The gluconapin,  three  major g l u c o s i n o l a t e s o f rapeseed meal a r e  glucobrassicanapin  and p r o g o i t r 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-CH -CH 0  CH =CH-CH(OH)CH 2  2  conditions.  respectively (5).  In  gluconapin  and  On enzyme h y d r o l y s i s  these g l u c o s i n o l a t e s produce 3-butanyl i s o t h i o c y a n a t e , pentyl isothiocyanate respectively.  and 5 - v i n y l o x a z o l i d i n e  Sandburg and H o l l y  4-  thione,  (77) i n d i c a t e d t h a t  this  s y s t e m i s made up o f 2 e n t i t i e s , n a m e l y t h i o g l u c o s i d a s e a n d sulfatase.  I t was s u g g e s t e d by P i t t - R i v e r ( 6 8 ) 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 be  converted t o 5-vinyl thiooxazolidone G m e l i n and V i r t a n e n  might  by o x i d a t i o n .  (43) suggested that t h e  goitrogenic effect of thiooxazolidones  i s due t o t h e f a c t  9.  that they i n h i b i t  the synthesis  o f t h y r o i d hormones.  e f f e c t c a n n o t be o v e r c o m e by h i g h  This  doses o f i o d i n e .  Another type of g o i t r o g e n i c  substance  belonging  t o t h e B r a s s i c a f a m i l y h a s a p r i m a r y i n f l u e n c e on t h e u p t a k e o f i o d i n e by t h e t h y r o i d g l a n d .  This  p r e v e n t e d by i n c r e a s i n g t h e amount Literature  indicates that salts  represents  this  i n f l u e n c e c a n be  o f i o d i n e dose ( 4 3 ) .  of thiocyanate  (SCN~)  type of c r u c i f e r s .  I t h a s been p r o v e d by E t t l i n g e r a n d L u n d e e n ( 3 6 ) analytically rearranges  that s i n i g r i n  during  i s the potassium s a l t  enzyme c l e a v a g e t o a l l y l  which  isothiocyanate.  F e e d i n g e x p e r i m e n t s h a v e shown t h a t i n t a c t thioglucosides  are not p a r t i c u l a r l y harmful 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 s u c h as oxazolidinethiones  and n i t r i l e s  isothiocyanates,  w h i c h a r e l i b e r a t e d by  myrosinase i n the seed i n t h e presence o f moisture. oxazolidinethiones  are mainly responsible  ment o f t h e t h y r o i d ( 1 8 ) . t o be t h e compound  thiocyanates may  f o r the  Oxazolidinethiones  enlarge-  also  p r i m a r i l y , i f not e n t i r e l y ,  for goitrogenicity.  The  appear  responsible  However i t h a s b e e n shown t h a t  iso-  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 ,  which  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 types  of rapeseed  compounds.  A survey o f the Canadian rapeseed  cultivers  ( B r a s s i c a n a p u s 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 within species o l a t e content (100). were r e l a t i v e l y  cultivar difference i n glucosin-  However d i f f e r e n c e s b e t w e e n  cultivars  s m a l l and none o f t h e c u l t i v a r s was  found t o  10. be  free or p r a c t i c a l l y  free of g l u c o s i n o l a t e s .  f r o m J a p a n and  I n d i a were f o u n d t o be  isothiocyanate  and  or  5-vinyl  I n p r e s e n t day is  destroyed i n the  the  goitrogenic  glucoside  f a c t o r s are  complex  production from the  a l . (32)  et  substrate  left  by  and  the  i n the  They were a l s o a b l e  f l o u r and  the  (33)  by  to  this  to obtain  a gray meal.  a  Ballester  f o r meal  detoxi-  glucosinolates.  most e c o n o m i c a l s o l u t i o n h o w e v e r little  o r no  sulfur  containing  upon g l u c o s i d i c h y d r o l y s i s . has  c o n t e n t o f t h e s e compounds i n t h e of s u l f u r a p p l i e d ,  m a r k e d e f f e c t on  s e e d s , but  some v a r i e t i e s a r e  only  It is the  regardless  total of  consistently  the low  isothiocyanates. Until  containing chain  for  oxazolidinethiones  air clarification  shown t h a t s u l f u r f e r t i l i z a t i o n  in total  for  seeds, or t o s e l e c t l i n e s which produce  harmless isothiocyanates  level  and  effective in eliminating  safest  method  enzyme r e s p o n s i b l e  aqueous e x t r a c t i o n  e i t h e r to breed s t r a i n s with  glucosides  and  original  developed a water washing technique  The  enzyme  steam heat  bound i n t h e  glucosinolate.  f r e e , creamy c o l o u r e d  a l . (9)  by  h a v e d e v e l o p e d a wet  f i b r o u s h u l l s by  f i c a t i o n t h a t was  is  processing  m y r o s i n a s e , the  remove g l u c o s i n o l a t e s  hull  t e c h n i q u e s , the  of t o x i c isothiocyanates  process the  4-pentyl  (101).  Eapan e t i n a c t i v a t i n g the  free of  oxazolidine-2-thione.  processing  initial  Cultivars  fatty  20  1960  a l l v a r i e t i e s of rapeseed produced o i l  t o 2 5%  acid.  e r u c i c a c i d , a m o n o u n s a t u r a t e d 22  S t e f a n s s o n and  Hougen (87)  carbon  i s o l a t e d rapeseed  11. s t r a i n s which produced o i l p r a c t i c a l l y  free of e r u c i c acid.  O l e i c a c i d r e p l a c e d e r u c i c a c i d as the major c o n s t i t u e n t o f these  oils.  Species  c o n t a i n i n g o l e i c a c i d at l e v e l s  than 70% have been obtained.  Beare et a l . (11)  higher  showed the  d e f i c i e n c y o f s a t u r a t e d f a t t y a c i d s would r e s u l t i n u n d e s i r a b l e 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 r e s i d u a l o i l i n the meal when used as f e e d , or f o r the p r e p a r a t i o n o f or  isolates  concentrates. I t has  been r e p o r t e d by S o s u l s k i and  Bakal  (86)  that a c i d p r e c i p i t a t i o n o f a l k a l i e x t r a c t s o f p r o t e i n from rapeseed gave low y i e l d s with brownish c o l o r . i s o l a t e s obtained t o the  formation  from sunflower  dark green i n c o l o r  There i s no evidence  i n rapeseed.  b e l i e v e d t o be due  due  The  that  chlorogenic  development o f a green c o l o r i s  to o x i d a t i o n o f c h l o r o g e n i c a c i d , a t a n n i n  l i k e compound present was  protein  of an i n s o l u b l e complex between c h l o r o g e n i c  a c i d and p r o t e i n (84). a c i d occurs  was  The  i n a p p r e c i a b l e amounts i n sunflower  seeds  e x p l a i n e d by M a l i c et a l . (56). Zdenek et a l . (104)  have used  countercurrent  e x t r a c t i o n f o r p r o t e i n from rapeseed meal. batch  In comparison  e x t r a c t i o n under s i m i l a r c o n d i t i o n s , e x t r a c t s  by c o u n t e r c u r r e n t bicarbonate  e x t r a c t i o n with sodium hydroxide  contained  f e a s i b l e due  obtained or sodium  more dry matter, t o t a l n i t r o g e n  precipitatable protein.  T h i s procedure i s not  t o the complexity  o f the  Gheyasuddin et a l . (41)  with  and  economically  operation.  obtained  almost c o l o r l e s s  sunflower p r o t e i n i s o l a t e s by m a i n t a i n i n g the pH a t d u r i n g e x t r a c t i o n w i t h 0.2 5% sodium s u l f i t e .  The  10.5  precipi-  t a t e d p r o t e i n s were washed w i t h 50% aqueous i s o p r o p y l probably able t o break H-bonds, t o produce isolates.  alcohol,  the n e a r l y white  I t i s known t h a t polyphenols at a c i d pH  remain  combined with p r o t e i n s by u n u s u a l l y s t r o n g H-bonds as shown by Lommis and B a t t a i l e  (54).  Stronger H-bond b r e a k i n g  compounds, such as 50% aqueous e t h a n o l , w i l l be even more e f f e c t i v e i n p r e v e n t i n g the i n t e r a c t i o n between polyphenols and p r o t e i n s .  However such compounds denature  p r o t e i n s by  destroying t h e i r native structure. Goding of  et a l . (44) has a l s o r e p o r t e d p u r i f i c a t i o n  12 S g l o b u l i n i s o l a t e d  from each of two  s p e c i e s o f rapeseed  ( B r a s s i c a napus L. and .Brassica campestris L . ) .  They have  been shown t o be s i m i l a r in. terms o f amino a c i d  composition,  amino t e r m i n a l amino a c i d , number o f s u b f r a c t i o n s and carbohydrate  the  content.  While  the present work was  i n p r o g r e s s , Owen and  C h i c h e s t e r (65) r e p o r t e d a process f o r the p r o d u c t i o n of n o n t o x i c rapeseed p r o t e i n i s o l a t e s and an a c c e p t a b l e feed by-product.  They e x t r a c t e d the rapeseed presscake w i t h  sodium c h l o r i d e s o l u t i o n , p r e c i p i t a t e d with a c i d and o b t a i n e d an i s o l a t e with l i g h t  tan c o l o r a f t e r repeated washing w i t h  water. Tape e t a l . (90) developed procedure  f o r the removal  an aqueous e x t r a c t i o n  o f t h i o g l u c o s i d e s from  crushed  rapeseed  t o produce a f l o u r s u i t a b l e  The  d e f a t t e d rapeseed  oil  extraction  50%  and  of  and  f l o u r and  meal produced  air clarification  30% p r o t e i n r e s p e c t i v e l y .  e a c h f r a c t i o n was Lo  almost  and H i l l  from rapeseed  The  filtration  dialysis  a g a i n s t running water  rapeseed  milk.  and  protein sodium  product  chloride coats  and  original  c o n t a i n i n g 61 t o  76%  showed a c o n s i d e r a b l y g r e a t e r  lower content o f crude  s i m i l a r and  concentrates  up t o 75% o f t h e  s i n o l a t e than d i d the s t a r t i n g m a t e r i a l . c o m p o s i t i o n was  over  freeze dry the d e s a l t e d  meal n i t r o g e n i n the form o f products  c o n t e n t o f a s h and  contain  t o remove t h e s e e d  method y i e l d s  The  standard  t h i o g l u c o s i d e content  e x t r a c t i n g w i t h 10%  f o l l o w e d by  (N X 6 . 2 5 ) .  by  free.  solution  protein  consumption.  techniques  (53) have p r e p a r e d  m e a l by  The  f o r human  the phosphorus  f i b e r and  gluco-  The  acid  amino  l e v e l d i d not  change d u r i n g p r o c e s s i n g . Janson gel  filtration  rapeseed a broad  ( 4 9 ) has  r e p o r t e d columns f o r l a r g e s c a l e  on p o r o u s g e l s f o r t h e f r a c t i o n a t i o n  p r o t e i n s and  insulin.  From t h i s m e t h o d he  peak c o n t a i n i n g a t l e a s t  Korolezuk of and  nitrogenous  approximately Ruttowski  I  10  and  15 t o 2 0,000. extraction  meal i n r e l a t i o n  to  pH  80% o f t o t a l n i t r o g e n a t  pH  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  compounds more t h a n  A l l of  g r e a t e r than  (52) s t u d i e d t h e  compounds o f r a p e s e e d  temperature.  nitrogenous  and  obtained  4 main components.  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 with molecular weights  of  of  14.  9.5 t o 10 a n d 30 t o 45°C. a r o u n d 60°C g a v e a b o u t 5 5 %  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 extractability.  CHAPTER I I I EXPERIMENTAL MATERIALS AND 1.  The E c h o ) was  rapeseed  a generous  Materials  flour gift  PROCEDURES  ( B r a s s i c a c a m p e s t r i s L.  f r o m t h e Food R e s e a r c h  o f t h e Canada Department o f A g r i c u l t u r e , Ottawa. 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  developed  al.  ( 9 0 ) s o as t o f r e e t h e p r o d u c t o f t o x i c  and  hulls. Residual  t i n g with proteins  o i l i n t h e f l o u r was  petroleum ether (57). for electrophoresis  chloroform:methanol  var.  Institute The  by Tape e_t thioglucosides  r e m o v e d by  extrac-  For the p r e p a r a t i o n  t h e f l o u r was  extracted  (2:1 v/v) m i x t u r e t o improve  of with  the  resolution. All  c h e m i c a l s were a n a l y t i c a l  from F i s h e r S c i e n t i f i c pH  3 t o 10 was  Co.  grade  L t d . , Vancouver,  o b t a i n e d from Pharmacia  obtained B.C.  Ampholine  U p p s a l a , Sweden.  ,2. A.  Determination  Semi m i c r o tillation  apparatus  tion of total presence mixture  Procedures of nitrogen  K'j.eldahl p r o c e d u r e  u s i n g an A m i n c o  ( s e e P l a t e 1) was u s e d f o r t h e d e t e r m i n a -  nitrogen.  of potassium  The m a t e r i a l s w e r e d i g e s t e d i n t h e  s u l f a t e : s e l e n i u m powder (100:1  t o g e t h e r w i t h a piece o f copper wire w i t h  ted s u l f u r i c  acid.  dis-  concentra-  F i f t y m l o f t h e d i s t i l l a t e was  in  3% b o r i c a c i d  c o n t a i n i n g a few d r o p s o f m i x e d  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 .  w/w)  collected  indicator, 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 t h e 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.  Determination  Moisture s t a n d a r d A.O.A.C. C.  of moisture  and ash  and a s h w e r e d e t e r m i n e d  according t o the  1970 p r o c e d u r e ( 6 ) .  Determination  of lipids  L i p i d s were e x t r a c t e d f r o m t h e i s o l a t e s t r a t e s by w e t t i n g w i t h 1.0 m l o f a l c o h o l and t h e n hydrochloric acid  a l c o h o l was a d d e d and t h e m i x t u r e  then  10.0 m l  ( 2 4 : 1 1 by v o l u m e ) was added and h y d r o l y z e d  f o r 40 m i n . a t 80°C i n a w a t e r b a t h . ml  and c o n c e n -  t r a n s f e r r e d t o Mojonnier  A f t e r h y d r o l y s i s 10.0 was c o o l e d .  These were  G 3 F a t 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 m l o f e t h e r added i n 3 e q u a l portions.  The f l a s k s were s t o p p e r e d  f o r 1.0 m i n . , t h e n  20.0 m l p e t r o l e u m  and shaken v i g o r o u s l y ether  (b.p.  a d d e d a n d s h a k e n a g a i n v i g o r o u s l y f o r 1.0 m i n .  30-60°C) The e t h e r  PLATE I AMINCO DISTILLATION APPARATUS DETERMINATION  FOR NITROGEN  18. l a y e r was a l l o w e d t o s e p a r a t e o t h e r l a y e r was t h e n filtered  through  e t h e r pass f r e e l y  from t h e aqueous phase.  The  d r a w n o f f a s much a s p o s s i b l e a n d  a c o t t o n wool packed stem o f f u n n e l t o l e t i n t o weighed beakers  c o n t a i n i n g broken  glass. Reextracted the remaining  aqueous phase i n t h e  t u b e t w i c e w i t h 15.0 m l e a c h o f e t h e r a s d e s c r i b e d  before.  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  beaker.  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 f e w m l o f equal  volumes o f e t h e r and p e t r o l e u m  slowly evaporated beakers for  on a s t e a m b a t h  The  i n an o v e n a t 100°C  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 constant weight. fat  The e t h e r was  i n s i d e a fume h o o d .  c o n t a i n i n g t h e f a t were d r i e d  90 m i n .  ether.  by a c i d  The r e s u l t s a r e e x p r e s s e d  as %  hydrolysis.  D.  Determination  Phosphorus  o f phosphorus  c ontent  of the isolates  and c o n c e n -  t r a t e s was e s t i m a t e d by t h e m e t h o d o f F i s k a n d Subbarow (38).  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 w e t o x i d a -  t i o n and t h e f o r m a t i o n o f p h o s p h o m o l y b d i c a c i d which i s q u a n t i t a t i v e l y reduced was m e a s u r e d by  complex  to heteropholyblue  spectrophotometry.  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 d i g e s t e d w i t h 6.0 m l o f n i t r i c loric  acid  evolved  color,  acid:  (5:1:2'by volume) m i x t u r e  f o r 2 to 3 hrs.  sulfuric until  acid:  white  were perch-  acid  fumes  On c o o l i n g t h e d i g e s t was t r a n s -  f e r r e d t o a v o l u m e t r i c f l a s k with 0.1 N h y d r o c h l o r i c a c i d . To a l i q u o t s o f the d i g e s t was added 5.0 ml o f 2.5% ammonium molybdate i n 5 N s u l f u r i c a c i d and d i l u t e d t o 45 ml i n a 50 ml v o l u m e t r i c f l a s k . 15.4%  To t h i s was added 2.0 ml o f  l-amino 2-naphthol 4 - s u l f o n i c a c i d  dry mixture  i n warm water.  : sulfite-bisulfite  Made up t o volume and t h e contents  w e l l mixed, the absorbance measured at 6 80 nm a f t e r 25 mins. i n a Bausch and Lomb S p e c t r o n i c 20 C o l o r i m e t e r / S p e c t r o photometer.  C o n c e n t r a t i o n o f phosphorus was read from a  standard curve prepared with sodium phosphate. E.  Determination  o f Calcium  Calcium was determined N t a i l i a n a s and Whitney (61). and  by the method d e s c r i b e d by  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 were ashed at 400°C f o r 2 h r s . i n a m u f f l e  furnace.  The ash was d i s s o l v e d i n 0.1 N h y d r o c h l o r i c a c i d  and made t o volume.  The a l i q u o t s o f the e x t r a c t were added  5.0 ml o f 0.024 M disodium  dihydrogen  a c e t a t e d i h y d r i d e and 8 N potassium pH t o 13 f o l l o w e d  ethylenediamine  hydroxide  b y 3 drops o f 0.2% c a l c i n e  to increase (aminomethyl-  f l u r o s c e i n ) i n d i c a t o r i n d i l u t e sodium h y d r o x i d e . of the sample becomes pink.  Back t i t r a t e with  0.0 24 M c a l c i u m c h l o r i d e s o l u t i o n , prepared c a l c i u m carbonate  tetra-  The c o l o r  standard  by d i s s o l v i n g  i n minimum amount o f h y d r o c h l o r i c a c i d  and d i l u t i n g t o volume.  The e n d p o i n t i s marked by a c o l o r  change i n s o l u t i o n t o a permanent green c o l o r . t i o n was c a r r i e d out a g a i n s t a b l a c k background.  The t i t r a -  20. The  amount o f c a l c i u m was c a l c u l a t e d a f t e r making  c o r r e c t i o n s f o r the b l a n k s .  R e s u l t s are expressed as  percentages. F.  Determination o f t o t a l  T o t a l s u l f u r was determined  sulfur i n the i s o l a t e s and  c o n c e n t r a t e s o f rapeseed p r o t e i n s by the method o f B a r d s l e y and L a n c a s t e r (10).  The samples were mixed with 0.5 g  sodium b i c a r b o n a t e thoroughly and an a d d i t i o n a l 0.5 g b i carbonate was l a i d over and was fused at 600°C f o r 4 h r s . The  fused m a t e r i a l was then e x t r a c t e d with 2 5 ml o f 0.46%  sodium dihydrogen phosphate i n 2 N a c e t i c a c i d i n 3 p o r t i o n s . A f t e r the r e a c t i o n s u b s i d e s , allow t o stand f o r 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 a l i q u o t s add 1.0 ml o f 0.5% f r e s h l y prepared gum a c a s s i a i n 50% a c e t i c a c i d , and 1.0 ml 6 N h y d r o c h l o r i c a c i d , and 0.5 g f i n e l y ground barium  chloride.  A f t e r one min. c o n t e n t s were shaken t o d i s s o l v e the barium chloride. in  The absorbance  was read a f t e r 5 mins. a t 420 nm  a S p e c t r o n i c 20 spectrophotometer.  The amount o f s u l f u r  was read o f f from a standard curve prepared with magnesium sulfate. G'.  Determination o f Carbohydrates  Carbohydrates  were determined  i n the rapeseed  pro-  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 by a method developed by Dubois et  a l . (31) w i t h a few m o d i f i c a t i o n s .  w i t h 1.0 ml o f 5% aqueous phenol  Samples were mixed  (w/w) f o l l o w e d by 5.0 ml  concentrated pipet.  sulfuric  a c i d added d i r e c t l y w i t h a wide  opening  A f t e r s t a n d i n g f o r 10 m i n s . t h e c o n t e n t s w e r e m i x e d  and , t h e  a b s o r b a n c e was  spectrophotometer.  The  read o f f from a standard H.  r e a d a t 480  nm  on a S p e c t r o n i c  concentration of carbohydrate  20 was  curve.  Determination  of crude  fiber  C r u d e f i b e r d e t e r m i n a t i o n i s b a s e d 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 , which i s probably  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 o f crude  pentosans.  due  largely  The  amount  f i b e r i s roughly t h a t p o r t i o n of food which 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 f i b e r i s estimated  from the  animals.  l o s s on i g n i t i o n o f  Crude  dried  residue remaining  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  sulfuric  0.313  a c i d and  N osdium h y d r o x i d e  under  specific  conditions. Samples o f t h e rapeseed w e r e m i x e d w i t h 0.5  g predigested asbestos  0.255 N s u l f u r i c a c i d a d d e d and digesting apparatus, w i t h a few and  chips.  10 0 m l  of  i n 15 m i n s .  together  diluted with  water  F i l t e r the d i g e s t buchner f u n n e l  mesh s c r e e n , w i t h s u c t i o n .  35 m l b o i l i n g w a t e r and  and  concentrates  f o r 30 m i n s . i n  drops of a n t i f o r m A emulsion  a California polyethylene  w i t h a 200 with  boiled  adjusted to b o i l  c o n t a i n i n g some b o i l i n g  through  i s o l a t e s and  The  fitted  beaker i s r i n s e d  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 m l b o i l i n g w a t e r .  The  residue i s then t r a n s -  f e r r e d back t o the beaker and b o i l e d f o r 30 mins. with 100 boiling  0.313  N sodium hydroxide as d e s c r i b e d b e f o r e .  ml  The  beaker i s r i n s e d with 35 ml b o i l i n g water and the r e s i d u e i s washed w i t h 15 ml of b o i l i n g  0.255 N s u l f u r i c a c i d and  then  with 3 p o r t i o n s of 25 ml b o i l i n g water f o l l o w e d by 15 ml of alcohol. The r e s i d u e i s removed from the f u n n e l by t a p p i n g i n t o an ashing d i s h and heated f o r 2 h r s . at 130°C. then c o o l e d i n a d e s i c c a t o r and weighed.  It i s  Then the dry  r e s i d u e i s i g n i t e d at 600°C f o r 30 mins., c o o l e d i n a d e s i c c a t o r and reweighed.  A blank d e t e r m i n a t i o n was  t e d under the same c o n d i t i o n s .  Percentage  crude f i b e r  c a l c u l a t e d by d e t e r m i n i n g the l o s s of weight minus the l o s s i n weight  conduc-  on  was  ignition  o f the asbestos blank m u l t i p l i e d  by 10 0 and d i v i d e d by the weight  of sample.  I.  D e t e r m i n a t i o n of i s o t h i o c y a n a t e and t hiooxazolidones  The  i s o t h i o c y a n a t e content was  determined by the  a r g e n t r i m e t r i c method of Viehoever e t a l . (96), and Andre and M a i l l e  ( 3 ) , i n which the mustard  forming s u b s t i t u t e d t h i o u r e a .  The  o i l r e a c t s with ammonia  l a t t e r decomposes i n  ammonical s i l v e r n i t r a t e forming i n s o l u b l e s i l v e r and monosubstituted then determined  carbodiamide.  sulfide  The unreacted s i l v e r i s  v o l u m e t r i c a l l y by the Volhard method u s i n g  potassium t h i o c y a n a t e s o l u t i o n .  The t h i o o x a z o l i d o n e s were  determined by the method d e s c r i b e d by Astwood et 'al.  (7)  and m o d i f i e d by Wetter  (98).  Samples o f the i s o l a t e s and c o n c e n t r a t e s approximately myrosinase" buffer  30 mg were mixed with 1.5 ml o f 0.5% "crude  i n 0.9% sodium c h l o r i d e  pH 4.0 added and macerated.  and 10.0 ml c i t r a t e The contents were then  shaken f o r 2 h r s . at 2 5°C i n a mechanical mixture was then d i s t i l l e d tus.  weighing  The d i s t i l l a t e ,  5 ml 0.1 N s i l v e r  shaker.  The  i n an Aminco d i s t i l l a t i o n  approximately  appara-  25 ml was c o l l e c t e d i n  n i t r a t e c o n t a i n i n g 1.25 ml 10% ammonium  h y d r o x i d e , surrounded  by an i c e and water bath.  c o n t a i n e d o n l y the crude myrosinase  The c o n t r o l  solution.  For i s o t h i o c y a n a t e The d i s t i l l a t e , with an a i r condenser water bath.  was h e a t e d  fitted  f o r 30 mins. i n a b o i l i n g  The contents o f the f l a s k was made up o f 30 ml  after f i l t r a t i o n . nitric  i n an a l l g l a s s apparatus,  To t h e f i l t r a t e was added 1.0 ml o f 6 N  a c i d and 0.5 ml o f 8% f e r r i c  ammonium s u l f a t e , and  t h i s mixture was t i t r a t e d t o a f a i n t salmon c o l o r end p o i n t w i t h 0.01 N potassium t h i o c y a n a t e f r e s h l y d i l u t e d 0.1 N stock  from an  solution.  For thiboxazo1idone s The  suspension a f t e r d i s t i l l a t i o n  was  filtered  through Whatmann No. 42 f i l t e r paper and the pH o f t h e f i l t r a t e was a d j u s t e d t o 10.5 with 1.0 N sodium h y d r o x i d e . One  m i l l i l i t e r o f t h i s s o l u t i o n was e x t r a c t e d twice with  5.0 ml o f anhydrous e t h y l e t h e r .  The absorban.ce  o f the  e t h e r l a y e r was measured a t 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 meter. the  spectrophoto-  The a b s o r b a n c e a t 248 nm was c o r r e c t e d b y s u b t r a c t i n g  average value J.  o f a b s o r b a n c e a t 2 30 nm a n d 266 nm.  C a l i b r a t i o n standard curves f o r protein determination  A calibration of the p r o t e i n content diluting  standard  curve  for the determination  o f t h e e x t r a c t s were p r e p a r e d  t h e e x t r a c t s from water, a c i d  a l i q u o t s o f these teins  i n a Beckman DB  contained  f r o m 0.5 t o 8.0 mg  and base so t h a t  d i f f e r e n t concentrations o f pro-  approximately.  For the absorbance determination the d i l u t e sulfuric  by  1.0 t o 10.1 m l o f  e x t r a c t s were a c i d i f i e d w i t h 2 d r o p s o f 2 N  a c i d and t h e volume made t o 20.0 m l w i t h  a f t e r t h e e x t r a c t s w e r e made e q u a l  i n volume.  3 M  The  urea  contents  were m i x e d a n d t h e a b s o r b a n c e m e a s u r e d a t 2 80 nm i n a Beckman DB  spectrophotometer. N i t r o g e n was d e t e r m i n e d b y 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 bance d e t e r m i n a t i o n . and  and c o n c e n t r a t e s  The n i t r o g e n was c o n v e r t e d  a p l o t was made a g a i n s t K.  as f o r t h e a b s o r to protein  absorbance.  Slab 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 o u t 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 M Tris  g e l s made i n 0.175  - 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  a n d 0.1  M 2 - m e r c a p t o e t h a n o l as d e s c r i b e d b y Raymond ( 7 4 ) a n d Tombs (91).  The g e l p l a t e s w e r e p r e c o n d i t i o n e d b e f o r e  application  of samples.  The  samples of the  whey w e r e d i s s o l v e d i n 0.175 taining  8 M u r e a and  0.1  M Tris glycine buffer  M 2 - m e r c a p t o e t h a n o l and  f i l t e r paper s t r i p s  and  Electrophoresis  c a r r i e d out  The  was  applied to slots  p l a t e s w e r e s t a i n e d i n 1%  m i n s . and  destained  g r o u n d was  i s o l a t e s , concentrates  i n 7%  amido b l a c k  Disc  gel  cm  0.1  i n t e r n a l diameter glass (26)  with  s a m p l e s w e r e t a k e n up 8 M u r e a and  d r o p o f 0.01% electrolytes  0.1  a few  modifications  M 2 - m e r c a p t o e t h a n o l and  contained  3 milliamperes  1.0%  20 w i t h  an  the  gels  t u b e f o r 75 m i n s .  f o r 30 m i n s . i n 12.5% i n 10%  The  glass  blue  and  proThe  8.6  contain-  mixed w i t h  8.6  v o l t s at a  t r i c h l o r o a c e t i c a c i d before  g e l s were d e s t a i n e d  t o the  i n t e r n a l marker.  a t 125  6 M  long  (63).  T r i s - g l y c i n e b u f f e r pH  c a r r i e d out  per  as  aqueous c o o m a s s i e b r i l l i a n t 10.0%  gels containing  tubes according  e l e c t r o p h o r e s i s were r e m o v e d f r o m t h e in  a c l e a r back-  i n T r i s g l y c i n e b u f f e r pH  bromophenol blue  e l e c t r o p h o r e s i s was of  U°C.  acid for 5  M 2 - m e r c a p t o e t h a n o l w e r e made i n 6 cm  cedure of Davis  ing  at  electrophoresis  Seven p e r c e n t p o l y a c r y l a m i d e  0.6  a t 2 5 mA  obtained. L.  u r e a and  plates.  i n 7%  acetic acid until  con-  soaked i n  i n the  f o r 18 h r s .  and  a The  and current  g e l tubes a f t e r t u b e s and  R 250 use,  stained  diluted 1 after  trichloroaceticacid.  trichloroacetic acid.  to  fixing The  M.  Isoelectric  Isoelectric  focusing  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 S v e n s s o n (95), Wrigley poolas  ( 8 8 ) , Vesterberg  ( 1 0 3 ) , K e n r i c k and M a r g o l i s  and S v e n s s o n  ( 5 1 ) and  Catsim-  ( 1 6 ) 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  a m p h o l i n e pH 3 t o 1 0 , i n t h e p r e s e n c e o f u r e a .  The  electro-  p h o r e s i s was done a t 25 0 v o l t s w i t h t h e c u r r e n t f a l l i n g 50 mA t o 12 mA d u r i n g t h e f i r s t was m a i n t a i n e d  30 m i n s . and e l e c t r o p h o r e s i s  f o r a f u r t h e r 60 m i n s . w i t h 0.4%  a c i d i n the anodic  hydroxide  adjusted  i n the cathodic  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 w e r e r e m o v e d and  s t a i n e d i n .2% b r o m o p h e n o l b l u e acid  phosphoric  c h a m b e r and 0.5% e t h a n o l d i a m i n e  t o pH 10.5 w i t h 1.0 N p o t a s s i u m chamber.  from  solution i n ethanol  : acetic  : w a t e r ( 5 0 : 5 : 4 5 by v o l u m e ) f o r 1 h r . and d e s t a i n e d  washing i n ethanol  : acetic acid  : ether  (30:5:65 by volume)  a s 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 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 N.  pH  by  f o r 12 t o  f o r photography.  measurement  The g e l s a f t e r i s o e l e c t r i c were c u t c o r r e s p o n d i n g  focusing i n duplicate  t o t h e s t a i n e d b a n d s and w e r e  m a c e r a t e d i n 0.5 m l w a t e r a f t e r 4 h r s . t h e pH was  determined  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.  1 3 - 6 3 9 - 9 2 ) i n a Beckman d i g i t a l pH m e t e r . In another  experiment the gels a f t e r  f o c u s i n g were c u t i n t o 0.5 m l w a t e r .  5 mm  isoelectric  l o n g s e c t i o n s and s u s p e n d e d i n  The pH o f t h e s e  were r e a d  after  4 hrs.  0.  Amino a c i d a n a l y s i s  Hydrolyses 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 c a r r i e d out i n s e a l e d h y d r o l y s i s tubes f o r 24 h r s . at 110°C  with 6 N (constant  boiling)hydrochloric acid.  The  samples were d i s s o l v e d i n 0.05 N sodium hydroxide and representative  a l i q u o t s were f r e e z e d r i e d i n the presence o f  1.0 ml 0.2 5 mM n o r l e u c i n e 5.0 ml h y d r o c h l o r i c  as i n t e r n a l standard.  To these  a c i d were added and f r o z e n and vacuum  was a p p l i e d a f t e r f l u s h i n g with n i t r o g e n a f o r c e d r a f t oven.  The h y d r o l y s a t e  and h y d r o l y z e d i n  was f i l t e r e d  through a  s i n t e r e d f u n n e l and evaporated i n r o t a r y evaporator  repeat-  e d l y by d i s s o l v i n g i n water t o remove r e s i d u a l h y d r o c h l o r i c acid. The  F i n a l l y , the r e s i d u e was taken i n c i t r a t e b u f f e r pH 2.2.  a n a l y s i s was performed with a Phoenix Micro Amino  Analyzer  Model M 6800, Moore-Stein system. P.  Cystine  determination  C y s t i n e was determined as c y s t e i c a c i d f o l l o w i n g performic and  a c i d o x i d a t i o n as d e s c r i b e d  Moore ( 5 8 ) .  by Schram e t a l . (78)  Samples o f the i s o l a t e s , concentrates  whey weighing 2 t o 3 mg were mixed w i t h 0.5 ml  and  performic  a c i d a t 0°C and h e l d  f o r 16 h r s . at the same temperature ( 4 6 ) .  The  a c i d was removed by f r e e z e d r y i n g  excess p e r f o r m i c  sodium hydroxide t r a p s .  using  The h y d r o l y s i s was performed w i t h  2.5 ml o f 6 N h y d r o c h l o r i c a c i d f o r 20 h r s . at 110°C i n d r a f t f o r c e d oven. a s i n t e r e d funnel  The h y d r o l y s a t e  was f i l t e r e d  through  ( f i n e ) t o remove any p r e c i p i t a t e s and  28. t h e n h y d r o c h l o r i c a c i d was r e m o v e d on a r o t a r y under reduced in  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  2.0 m l c i t r a t e b u f f e r pH 2.2. Q.  Tryptophan  The  t r y p t o p h a n was d e t e r m i n e d  method o f I n g l i s To  evaporator  determination  and Leavers  ( 4 7 ) , w i t h a few m o d i f i c a t i o n s .  1 ml o f c o n c e n t r a t e d s u l f u r i c  m l o f 20% s o d i u m h y d r o x i d e  by t h e c o l o r i m e t r i c  a c i d , s l o w l y p i p e t t e d 0.5  i n 40% m e t h a n o l ,  t h a n 2 mg s a m p l e s  o f t h e p r e p a r a t i o n s i n 0.4 m l w a t e r w e r e a d d e d , a n d warmed f o r 20 s e e s .  To t h i s m i x t u r e  1.0 m l c o n c e n t r a t e d s u l f u r i c o f 8% p o t a s s i u m  add 4 m l g l a c i a l acid with mixing  p e r s u l f a t e reagent  acetic  acid,  and 2 drops  and t h e a b s o r b a n c e  read  a t 550 nm a f t e r 20 m i n s . 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.  Tryptophan  standard tryptophan The of Edelhock  c o n c e n t r a t i o n was r e a d o f f f r o m solution.  amount o f t r y p t o p h a n  c a l c u l a t e d b y t h e method  ( 3 4 ) by t a k i n g a b o u t one t h i r d  t y r o s i n e determined  a  t h e amount o f  i n amino a c i d a n a l y s i s a g r e e w i t h t h e  above d e t e r m i n a t i o n s . R.  Ultracentrifugation  U l t r a c e n t r i f u g a t i o n o f t h e rapeseed isolates  was made u s i n g a Beckman L2 6 5B a n a l y t i c a l  centrifuge. and  protein ultra-  A l l s e d i m e n t a t i o n a n a l y s e s w e r e made a t 2 5°C  59,000 r.p.m. The  protein isolates  f o r sedimentation  analyses  were  prepared  a c c o r d i n g to. method of L i u (55) by b l o c k i n g  the s u l f h y d r y l groups, f o l l o w e d by r e v e r s i b l e b l o c k i n g of amino groups as d e s c r i b e d by Dixon e t a l . (28). To 100  mg  p r o t e i n i s o l a t e s , 5 g urea and  phosphate b u f f e r f o l l o w e d by 4.6 pH a d j u s t e d t o 6.7 of was  5% e t h y l e n e d i a m i n e - t e t r a a c e t i c a c i d was f o r 15 mins.  added 15 u l o f 2-mercaptoethanol and at  37°C.  C  was  was  added 150  c o n i c anhydride  a f t e r 5 mins. at  was  a d j u s t e d to finally  and t r e a t e d with 0.83  with  The  d e s a l t e d i n i t i a l l y by d i a l y s i s i n running  citra-  while  8 and  The  protein solution tap water at  4°C f o l l o w e d by d e m i n e r a l i z e d water f o r 2 days. s o l u t i o n s were then a d j u s t e d t o pH  to  5 N sodium hydroxide.  a f t e r 10 t o 15 mins.  the c l e a r supernatants  ml  at room temperature  m a i n t a i n i n g the pH at 8 by adding  The d i a l y s e d  after centrifugation  were f r e e z e d r i e d .  For the sedimentation samples were d i s s o l v e d i n 0.1 The  6.6.  hours.  with s t i r r i n g  base uptake ceased  was  mg  pH of the d i a l y s e d s o l u t i o n s were a d j u s t e d  8 w i t h 1 N sodium hydroxide  was  nitrogen  f o r 15 mins.  d i a l y s e d i n running water and  d e m i n e r a l i z e d water f o r 24 The  added and  2  room temperature the pH of the mixture T h i s mixture  the  Then 0.0 9 ml  incubated  2H 0) and  M  To the mixture  To the l a t t e r r e d u c t i o n mixture  sodium t e t r a t h i o n a t e (Na S 0 . 2 4 6  0.1  ml water were added and  with 1 N sodium hydroxide.  bubbled i n t o the mixture  2 ml  analyses, the f r e e z e d r i e d M T r i s - g l y c i n e b u f f e r pH  c o n c e n t r a t i o n o f the p r o t e i n s i n s o l u t i o n were  1%.  8.5.  30. 3.  E x t r a c t i o n and p r e p a r a t i o n  of isolates  A.  Three stage  The  f l o u r was m i x e d w i t h w a t e r a t 4°C i n t h e r a t i o  e x t r a c t i o n procedure  o f 1 : 15 a n d b l e n d e d f o r 3 m i n . u s i n g a L o u r d e s M o d e l blender  s e t a t 80, ( s e e P l a t e 2 ) .  The m i x t u r e  was  a t 10,000 x g f o r 15 m i n . a t U°C i n a S o r v a l l RC2-3 fuge. The  The  centri-  residue  were  filtered.  f r o m t h e above was e x t r a c t e d  twice  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 the water e x t r a c t .  extracted with pH 10.  A f t e r 3 successive  given  i n Figure  hydroxide  The r e s i d u e was f u r t h e r  0.02 t o 0.01 N s o d i u m h y d r o x i d e  c o m b i n e d and f i l t e r e d . is  clarified  The r e s i d u a l m e a l was r e - e x t r a c t e d t w i c e a s b e f o r e .  combined s u p e r n a t a n t s  using  NM-1A  a t 40°C a t  e x t r a c t i o n s supernatants  The f l o w d i a g r a m f o r t h e e x t r a c t i o n  1.  B.  S i n g l e stage  The  f l o u r was e x t r a c t e d  sodium hydroxide  a t pH 10 a n d 40°C.  3 times  The m i x t u r e  extraction  with  0.01 N s o d i u m  was b l e n d e d f o r  3 m i n s . and c e n t r i f u g e d a t 10,000 x g f o r 15 m i n s . combined s u p e r n a t a n t s  were f i l t e r e d .  e x t r a c t i o n i s given i n Figure The procedures,  The  Flow diagram f o r t h e  2.  e x t r a c t s from t h e t h r e e and s i n g l e  stage  except 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 ,  w e r e made 0.15% w i t h r e s p e c t cipitation  were  t o sodium s u l f i t e  of isolates or preparations  of  before  pre-  concentrates.  PLATE I I EXTRACTION APPARATUS  - LOURDES  BLENDER  32  Rapeseed f l o u r (15g) Blend 3 min with 22 5 ml water at 4°C and c e n t r i f u g e  $  Residue  Supernatant (SW) F i l t e r , add 0.15% Na S0 and f r e e z e dry  Blend 3 min with HC1 at pH 2 and 6 0°C and c e n t r i f u g e  o  Concentrate Isolates  (CW)  (P.W i  1  g P W) y 2  r  Supernatant (SA)  Residue  A d d 0.0 5 M o x a l i c a c i d and centrifuge  Blend 3 min with NaOH at pH 10, and 40°C and c e n t r i f u g e  Supernatant (SA°) F i l t e r and f r e e z e dry  Residue (discard)  Concentrate (CA)  Supernatant (SB)  Residue (Discard)  F i l t e r , add 0.15% N a S 0 and f r e e z e dry Concentrate (CB o  Isolates Supernatants  o  (P B & P B ) 1  i  2  t  (SW, SA° 8 SB) A d j u s t t o the I s o e l e c t r i c p o i n t s w arm t o 35°C and c e n t r i f u g e  Sediments  Wash with water and 50% e t h a n o l ^ Isolates d i s s o l v e at pH ,5 and f r e e z e dry^^ P.W,P.A° £ P.B) l i i Supernatants (SW, SA S SB) Add 12.5% t r i c h l o r o a c e t i c a c i d warm to. 3 5°C and c e n t r i f u g e r  S ediments  Wash with water and 50% e t h a n o l d i s s o l v e at pH 8.5 and f r e e z e dry (P W,P A & P B ) J  F  b U l t i L e b  J  t  F i g u r e 1.  t  Flow diagram o f t h e t h r e e stage . e x t r a c t i o n procedure  t  R a p e s e e d F l o u r (10 g ) Blend  3 min w i t h  NaOH a t pH 10 a n d 40°C a n d c e n t r i f u g e  Supernatant  (SS)  Residue (discard)  F i l t e r , add 0.15% Na S0 2  A d j u s t pH 5.2, and warm t o 3 5°C centrifuge  Freeze dry  Concentrate (CS)  3  Sediment  P^  Whey P SU  1  S e d i m e n t P.  t  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  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  Freeze d r y  I s o l a t e _P S i  Figure  Whey P. SU  Add 1 2 . 5 % TCA, warm t o 35°C a n d c e n trifuge  2.  Isolate  P.S t  Flow diagram o f t h e s i n g l e stage sodium hydroxide e x t r a c t i o n p r o c e s s .  In another p a r a l l e l before  r e m o v a l o f o i l was  e x t r a c t e d by t h e t h r e 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 extracted the  a t pH v a l u e s  s e r i e s of experiments the  above.  f r o m 9.5  degree o f d e n a t u r a t i o n  and s i n g l e  Also the f l o u r  t o 11.5 i n o r d e r  of the i s o l a t e  flour  was  t o determine  at the high  pH  values. C.  O x a l i c a c i d treatment f o r decreasing 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)  To 2 5.0 m l a l i q u o t s 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 w e r e a d d e d 0.01 t o 0.0 8 M o x a l i c a c i d . were a l l o w e d  t o stand  for 1 hr.  The s o l u t i o n s  The p r e c i p i t a t e w h i c h  formed  was r e m o v e d b y c e n t r i f u g a t i o n and t h e r e s u l t i n g s u p e r n a t a n t was u s e d t o d e t e r m i n e t h e n i t r o g e n b y t h e K j e l d a h l m e t h o d . The r e s i d u e s  left  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  a s h e d 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 determination and  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  Whitney ( 6 1 ) . D.  Preparations  The c o n c e n t r a t e s freeze  f o r concentrates were o b t a i n e d  f r o m t h e e x t r a c t s by  drying. E.  Preparation  of isolates  The e x t r a c t s w e r e a d j u s t e d isoelectric  to their  respective  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 s o d i u m h y d r o x i d e and t h e n warmed t o 35°C f o r 10 m i n s . precipitates  The  f o r m e d w e r e c e n t r i f u g e d f o r 15 m i n s . a t 15,000  x g and w a s h e d 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 with dissolved  50% e t h a n o l .  i n w a t e r a t pH 8.5 a n d f r e e z e  water extract  extract  dried  (P^).  The  (SW) was a d j u s t e d t o pH 2.6, a n d t h e h y d r o -  chloric acid extract xide  The w a s h e d i s o l a t e s w e r e  (SA) t o pH 3.6.  and t h e s i n g l e  stage sodium h y d r o x i d e  w e r e a d j u s t e d t o pH 4.2 and 5.2 Aliquots  The t h i r d s o d i u m h y d r o extract  respectively.  of the extracts  were a l s o p r e c i p i t a t e d by  a d d i n g 1 2 . 5 % t r i c h l o r o a c e t i c a c i d a n d 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  described and  were washed as  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  then freeze . F.  dissolved  dried. Preparation  o f whey  proteins  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 G.  dried  t o obtain  calcium.  t o obtain  extract isolates  with  f o r 1 h r . t h e m i x t u r e was  a supernatant r e l a t i v e l y free of  0.5 N s o d i u m h y d r o x i d e , and i n a n o t h e r , t h e  was a d j u s t e d t o 1 2 . 5 % w i t h  trichloroacetic acid.  The  f o r m e d w e r e warmed t o 35°C f o r 10 m i n s . and s e p a r a -  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 m i n s .  l a t e s were w a s h e d t w i c e w i t h with  was made 0.0 5 M  I n 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  ted  acid extract  a c i d and a f t e r s t a n d i n g  centrifuged  P^U.  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 oxalic  t h e whey p r o t e i n  50% a l c o h o l .  The i s o -  w a t e r a t 50°C a n d t h e n  twice  The p r e c i p i t a t e s w e r e t h e n d i s s o l v e d i n  water at pH and  8.5  and  f r e e z e dried,, t o o b t a i n i s o l a t e s P_^A°  PA . 0  H.  p H - s o l u b i l i t y p r o f i l e s o f the  The  determination  f u n c t i o n of pH  of the c o n s e c u t i v e  h y d r o c h l o r i c a c i d and c a r r i e d out 4  of n i t r o g e n  as o u t l i n e d below.  adjustment were  m ade  the pH o f  Preparation  equal and  the  The nitrogen  t o the method  from rapeseed meal.  allowed t o stand  f o r 1 h r . before  F i v e hundred  ml o f water at 4°C blending.  The  and  slurry  at 4,000 x g f o r 15 mins. to separate the  natant from the r e s i d u a l meal.  The  mixed w i t h an equal volume of 90% p r e c i p i t a t e formed was p r e c i p i t a t e was residue  at  of "crude myrosinase"  mixed w i t h 1800  The  volumes  supernatant whey. of  N  respective aliquots.  grams o f meal was  The  The  centrifuged  prepared a c c o r d i n g  by Wrede (101)  centrifuged  the  P l o t s were made o f the whey  Myrosinase was described  from 2 to 11.  used f o r the d e t e r m i n a t i o n  by the K j e l d a h l method.  I.  2 5 ml a l i q u o t s of  N h y d r o c h l o r i c a c i d or 0.5  15,00 0 x g f o r 15 mins. t o o b t a i n  nitrogen against  a  e x t r a c t i o n with water,  To  sodium hydroxide to a d j u s t the pH  supernatant whey was  s o l u b i l i t y as  sodium hydroxide e x t r a c t s , were  e x t r a c t s were added 0.5  a f t e r pH  extracts  was  was  super-  supernatant obtained  ethanol  at 4°C  and  was  the  c e n t r i f u g e d at 14,000 x g f o r 15 mins.  washed w i t h  d i s s o l v e d i n 400  7 0% e t h a n o l  and  centrifuged.  ml o f d i s t i l l e d water  and  37. then c e n t r i f u g e d the  soluble  f r a c t i o n w h i c h was  The y i e l d was until  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 filtered  0.52% and t h e p r e p a r a t i o n  lyophilized.  was s t o r e d  a t -20°C  required. J.  S o l u b i l i t y measurements o f and concentrates  Solubility  of the preparations  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 . centrifuged  isolates were d e t e r m i n e d i n These were  a t 27,000 x g f o r 30 m i n s . and t h e c l e a r  n a t a n t was u s e d f o r t h e n i t r o g e n K j e l d a h l method. ted  and  determination  fraction  and i n r e l a t i o n  o f sample o r i g i n a l l y taken f o r t h e  super-  by t h e  The p e r c e n t a g e o f s o l u b i l i t y was  f o r the soluble  then  calcula-  t o t h e amount  determination.  \  CHAPTER IV RESULTS A.  C a l i b r a t i o n curves f o r p r o t e i n s  As seen i n F i g u r e s 3 and 4, the p l o t o f absorbance a g a i n s t mg n i t r o g e n i n the e x t r a c t s shows a l i n e a r ship.  relation-  Each curve has a d i f f e r e n t g r a d i e n t due t o the  d i f f e r e n t absorbance  at 280 nm- The n i t r o g e n content o f the  e x t r a c t s was c a l c u l a t e d from these curves as a r o u t i n e procedure. B.  p H - s o l u b i l i t y p r o f i l e o f the e x t r a c t s  The pH-nitrogen s o l u b i l i t y F i g u r e s 5 and 6.  curves are shown i n  From a study o f the pH-nitrogen  solubility  p r o f i l e s o f the rapeseed p r o t e i n e x t r a c t s i t i s seen 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 o i n t s at pH 3.6 and 7.  that  (SA) showed 2 minimum  From these the lower pH showed a  l a r g e r amount o f p r o t e i n p r e c i p i t a t e d than the h i g h e r pH point.  With the water e x t r a c t the shape o f the curve was  not h y p e r b o l i c  as w i t h the sodium hydroxide e x t r a c t s , but  a minimum p o i n t was observed i n the r e g i o n o f pH 2.6, w i t h a broad r e g i o n on e i t h e r s i d e .  The t h i r d sodium hydroxide  e x t r a c t showed a d i s t i n c t minimum whey n i t r o g e n content a t pH 4.2.  The s i n g l e stage sodium hydroxide e x t r a c t showed a  minimum around the pH 5.2. C.  I n f l u e n c e of o x a l i c a c i d treatment on hydrochloric acid extract  A gradual  i n c r e a s e i n the c a l c i u m content i n the  g u r e 3.  Effect of protein  concentration of the  f i r s t water extract...o...o., chloric acid extract Protein  ©  and s e c o n d ®  on  hydro  absorbance  d e t e r m i n e d by K j e l d a h l method  and t h e  a b s o r b a n c e m e a s u r e d a t 2 80 nm i n 3 M u r e a .  1.4  1.2  1.0 LU  o <  .8  CQ OC  O CO CQ <  .6  O 2  3 mg  4  P R O T E I N  g u r e 4. third single  Effect of protein  concentration  sodium h y d r o x i d e e x t r a c t step  P r o t e i n was  of the  - . - o - . - o - . - and  sodium hydroxide e x t r a c t  ©  d e t e r m i n e d by K j e l d a h l method  a b s o r b a n c e m e a s u r e d a t 280 nm  i n 3 M urea.  •-. and  ABSORBANCE O  '— 1  co  • cn  \  41.  Figure 5. p H - S o l u b i l i t y p r o f i l e s o f the f i r s t water extract  ...©...©..., and second h y d r o c h l o r i c  acid extract  --o—o.  20  0 1  2  3  4  5  pH  6  7  8  9  42.  Figure  6.  p H - S o l u b i l i t y p r o f i l e s of the t h i r d  hydroxide extract  sodium  - . - o . - . - , and t h e s i n g l e  sodium hydroxide e x t r a c t  a  9 — .  stage  g u r e 7. the  Effect  of the o x a l i c acid treatment  second h y d r o c h l o r i c  calcium removal.  acid extract  on  Q LU >  15 —o —  o <  oc  LU hOC X LU 3  o _J <  o  10  cc  /  LL  /  o  / o 2 X10  3 -2  4 M  5  OXALIC  8 ACID  4H.  F i g u r e 8.  E f f e c t of the o x a l i c a c i d treatment o f the  second h y d r o c h l o r i c content.  acid extract  on  protein  6.6  6.0 1  2  3 X 10~ M 2  4  5  OXALIC  6 ACI D  7  8  45,  Also with the increasing oxalic  acid  concentra-  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  decrease  as i l l u s t r a t e d  r e a c h i n g t h e maximum o f 0.05 M o x a l i c p r o t e i n content of o x a l i c  acid  decreased  again.  i n F i g u r e 8.  acid  After  concentration the  The r e s i d u e a f t e r a d d i t i o n  and c e n t r i f u g a t i o n  contained approximately  9%  protein.  a.  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 concentrates  The  results  Moisture The  are t a b u l a t e d i n Tables  I , I I and I I I .  determinations  moisture  content  isoelectric precipitation  o f the i s o l a t e s prepared  P^. r a n g e d f r o m 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  higher,  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 s e c o n d  chloric  P.A a n d P^A h a d t h e h i g h e s t  acid  isolates  i  by  t  hydro-  moisture  &  content. The those  concentrates  of the isolates.  chloric acid 6.7%.  showed m o i s t u r e  The f i r s t  e x t r a c t s had moisture  The t w o s o d i u m h y d r o x i d e  moisture b.  contents  levels  w a t e r and second levels  similar to hydro-  of approximately  e x t r a c t s c o n c e n t r a t e s had  o f 3.6%.  Protein determination The  p r o t e i n content  trichloroacetic acid  of the isolates  precipitation  (P^_) was  o b t a i n e d by slightly  higher than that obtained  f o rthe i s o e l e c t r i c  (P.).  f o r t h e second h y d r o c h l o r i c a c i d  The v a l u e o b t a i n e d  precipitation  TABLE  1.  PROXIMATE ANALYSIS OF THE RAPESEED PROTEIN ISOLATES FROM ISOELECTRIC P R E C I P I T A T I O N  P.W l P^  P S i  (a)  Cal  Moisture  Protein  CarboFat hydrate — 2.9 4.2  Ash  Ca  4.7  72.5  5.3  71.6  1.3  2.3  73.9  2.4  77.3  % 5.0  0.16  3.31  0.22  1.12  0.89  4.8  9.0  1.37  1.82  0.64  1.80  0.55  2.8  5.8  3.3  1.25  2.50  0.27  1.31  0.30  3.4  5.2  5.3  0.15  2.52  0.47  1.22  0.65  For d e t a i l s o f a b b r e v i a t i o n s  s e e p a g e s 32 and 3 3 .  P  S  Crude I s o t h i o Fiber cyanate  TABLE I I PROXIMATE ANALYSIS OF THE RAPESEED PROTEIN ISOLATES FROM TRICHLOROACETIC ACID PRECIPITATION  Mois-  ture  Protein  Carbo- Fat hydrate  Ash  Ca  P  S  Crude I s o t h i o F i b e r cyanate  _%  83. 3  0. 7  5.3  1.2  0.73  0.69  0 .17  0.85  0.70  PA  5. 6  7 8.7  0 .9  5.2  1.2  1.12  1.15  0 . 38  1.67  0 . 56  P B  3.2  81.9  1.1  5.4  1.1  0 . 84  2 . 30  0 . 26  1.24  0 . 22  ps  5.1  82 . 7  0.6  4.3  1.2  0 .49  1. 34  0 . 39  1.33  0.42  t  t  t  (a)  F o r 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 I PROXIMATE ANALYSIS OF RAPESEED CONCENTRATES  Moisture  Protein  CarboFat hydrate  Ash  PROTEIN  Ca  P  S  Crude I s o t h i o Fiber cyanate  ._%  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 cfetails o f a b b r e v i a t i o n s  s e e p a g e s 32 a n d 33.  extract isolate isolate P . treatment  was  P_^. was  72% w h i c h  i s low compared t o t h e  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 22% w h i c h  i s f a r t o o l o w f o r an i s o l a t e .  o t h e r i s o l a t e s P^ and P^ h a d v a l u e s r a n g i n g f r o m P r o t e i n content second and  single  stage  approximately c.  water  e x t r a c t s were  determination  t i o n i s o l a t e s P^_W  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^  and P^A h a d a s l i g h t l y  except  f o r the t h i r d  i s o l a t e s P^B a n d P^S w h i c h  acid  The l i p i d  g r e a t e r amount o f  sodium h y d r o x i d e  extract  w e r e 5.4 and 4.3% l i p i d  respecP^S.  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 higher than the values obtained f o r t h e i r f o r the h y d r o c h l o r i c acid  d.  Carbohydrate  isolates  extract.  content  The c a r b o h y d r a t e  content of the i s o l a t e s  P^ were  s l i g h t l y higher than those prepared  by t r i c h l o r o a c e t i c  precipitation P .  content ranged  to  3.4%.  The c a r b o h y d r a t e  acid  from  1.3  The i s o l a t e s P-S h a d 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 second  ranged  precipita-  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  except  and  63% p r o t e i n .  "Lipid  lipid,  37  e x t r a c t s (CB a n d CS) h a d  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  total  72 t o 84%.  The c o n c e n t r a t e s f r o m t h e t h i r d  sodium h y d r o x i d e  The l i p i d from  A l l  i n t h e c o n c e n t r a t e s from t h e  h y d r o c h l o r i c a c i d and f i r s t  52% r e s p e c t i v e l y .  acid  and P.B  showed a v a l u e o f 2.8%.  The  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%. isolates  P  had  The  trichloroacetic acid  a carbohydrate  precipitated  content ranging from  0.64  to  1.08%. Carbohydrate h i g h e r as e x p e c t e d c e n t r a t e CW  had  than t h a t of the i s o l a t e s .  a v e r y h i g h v a l u e o f 11.3%.  c o n c e n t r a t e s ranged isolates  P^  e.  to  9%.  i n t h e i r carbohydrate  36.5  ash  t o 9%,  ash c o n t e n t  and  the o x a l i c  The The  water  rest  resemble  con-  of  the  the  content.  P^  v a r i e d from  3  content of the i s o l a t e from the h y d r o c h l o r i c  (P^A)  The  was  which  decreased  by o x a l i c  was  higher than other  still  of the i s o l a t e s acid  Therefore  treatment  acid  acid  treatment isolates.  P^ w e r e a p p r o x i m a t e l y  increased i t for isolate  the t r i c h l o r o a c e t i c a c i d  done w i t h o u t o x a l i c  treatment  1.1% P^A  0  precipitation  f o r the h y d r o c h l o r i c  extract. 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 ,  b e t w e e n 14 t o 19% extract  (CA).  hydroxide f.  The  except  ' Calcium  t o 1.4%.  34%  f o r the h y d r o c h l o r i c acid  ash c o n t e n t  e x t r a c t was  The 0.14  which  ash c o n t e n t o f t h e i s o l a t e s  from  acid  t o 4.5%  The  extract  was  3.1  content  The  2.0%.  from  Ash  acid  to  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  f o r the s i n g l e  hydrochloric acid  sodium  16.7%.  content  calcium content of the i s o l a t e s The  stage  P^ r a n g e d  c a l c i u m content of the o x a l i c e x t r a c t P.A°  had  from  acid treated  the highest value of  1.4%.  The  trichloroacetic acid  contained  0.5  t o 1.1%  l o w v a l u e o f 0.5%. acid  precipitated  calcium.  The  The  p r o t e i n i s o l a t e s Pi-  isolate P S  showed  t  calcium content of the  extract before oxalic  acid  treatment  the  hydrochloric  was  approximately  2.6%. The 0.6  t o 0.8%  w h i c h was  c o n c e n t r a t e s had  except  2.6%  a calcium content ranging  f o r the h y d r o c h l o r i c a c i d  s i m i l a r t o the i s o l a t e  extract  from  (CA)  P.A. l  g.  Total The  isolates  sulfur  s u l f u r content of the i s o e l e c t r i c  P^ was  precipitated  precipitated  higher than that of the t r i c h l o r o a c e t i c  isolates  P^.  The  i s o l a t e Pj_A  had  0  a  concentra-  t i o n o f 0.64%  which i s n e a r l y t w i c e that present i n the  i s o l a t e P^A.  The  i s o l a t e s P^B,  of  and  0.17%  0.24,  0.22  sodium h y d r o x i d e 0.39%  and  respectively.  extract isolates  P^W The  P^S  and  had  lower  single P^S  had  values  stage 0.47  and  respectively. The  was  P^W  acid  quite  trates 0.64%  CS  amount o f s u l f u r p r e s e n t i n t h e  s i m i l a r t o t h a t i n the i s o l a t e s , except and  CA h a v i n g  0.6 0 and  i n the r e s p e c t i v e i s o l a t e s  sulfur  content  h.  0.11% P^S  i n t h e c o n c e n t r a t e CA  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°. CB h a d  concentrates  a s u l f u r content of  concen-  c o m p a r e d t o 0.47  and  P-^A  0  .  Thus t h e  i s v e r y much l e s s  The  and  c o n c e n t r a t e s CW  than and  0.3%.  P h o s p h o r u s 'content The  phosphorus content of the i s o l a t e s  P-j_ and  P^  v a r y f r o m 0.7 t o 3.3%.  The amount o f p h o s p h o r u s i n t h e i s o -  lates P  t h e i s o l a t e s P^.  and  t  was l o w e r t h a n  The i s o l a t e s  P^A w e r e 1.8 a n d 1 1 . 6 % p h o s p h o r u s w i t h a n d w i t h o u t  acid treatment i s o l a t e P^W.  respectively.  0  oxalic  .The h i g h e s t v a l u e was i n t h e  The t r i c h l o r o a c e t i c  acid precipitated  P-tB a l s o h a d a h i g h v a l u e o f 2.3% p h o s p h o r u s . the phosphorus i n t h e t h i r d  Pj_A  sodium h y d r o x i d e  isolate  The amount o f extract isolates  P-j_B a n d P^B d i d n o t show much o f a d i f f e r e n c e a s n o t e d f o r the other  isolates. The c o n c e n t r a t e s h a d p h o s p h o r u s c o n t e n t s  similar  to 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.  t r a t e s r a n g e d f r o m 0.9 t o 2.0%. hydroxide  extract concentrate  The o t h e r  The s i n g l e  stage  CS h a d t h e l o w e s t  was c l o s e t o t h a t o b t a i n e d by Lo a n d H i l l  concen-  sodium  v a l u e and  (1971) w i t h a  con-  o f t h e i s o l a t e s P^ r a n g e d  from  c e n t r a t i o n o f 1.1%. i.  Crude  fiber  Crude f i b e r  content  0.9 t o 1 . 1 % , w h i l e t h e i s o l a t e s P^ h a d a s l i g h t l y r a n g e f r o m 1.1 t o 1.8%. extract isolate  The s e c o n d s t a g e h y d r o c h l o r i c a c i d  P^A° h a d t h e h i g h e s t v a l u e 1.8%.  d a t a f o r soybean p r o t e i n i s o l a t e s  trate  f r o m 2.3 t o 3.7%.  Published  i s 0.7%.  The c o n c e n t r a t e s h a d a c r u d e higher, ranging  higher  f i b e r content  slightly  The w a t e r e x t r a c t c o n c e n -  CW h a d 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 h a d 3.1%.  The s o y b e a n p r o t e i n c o n c e n -  t r a t e s w e r e 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 by i s o e l e c t r i c  precipitation  trichloroacetic  of the isolates  h a d h i g h e r amounts t h a n t h e  acid precipitated  isolates  hydrochloric acid extract isolates. isolate  P^W  prepared  P , except the  The f i r s t  water e x t r a c t  h a d 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  basic  e x t r a c t i s o l a t e s P.B and P.B h a d v a l u e s o f 0.32 a n d 0.22% i t r e s p e c t i v e l y f o r both which are the lowest values obtained. The  isolates  P.S l  isothiocyanate  and P.S h a d v a l u e s o f 0.65 a n d 0.42% t  respectively.  The c o n c e n t r a t e s h a d a l o w e r r a n g e f o r t h e i s o t h i o cyanate  c o n t e n t was f r o m 0.2 t o 0.37%.  The w a t e r e x t r a c t  c o n c e n t r a t e h a d t h e h i g h e s t v a l u e o f 0.37%. T h e r e 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 the  isolates E.  and c o n c e n t r a t e s  present  by t h e m e t h o d u s e d .  Electrophoresis  (a) S l a b g e l As i n d i c a t e d in  slab g e l electrophoresis of isolates  P^  F i g u r e 9, m o s t o f t h e a c i d i c and n e u t r a l p r o t e i n s w e r e  extracted w i t h i n the f i r s t  stage whereas t h e second  stage  hydrochloric acid extracted only the basic protein fractions. The r e s i d u a l f r a c t i o n s a p p e a r 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 of the s i n g l e  e x t r a c t , o f w h i c h p a t t e r n was s i m i l a r t o t h a t  stage  sodium h y d r o x i d e  extract.  The  isolate  ure  9.  Slab  rapeseed  gel electrophoretic pattern  protein isolates  precipitation. 7%  prepared  Stained  i n 0.2%  extract  isolate  by  of  the  isoelectric  Amido b l a c k  in  acetic acid.  W - first  water  P.W l  A - second B - third  hydrochloric  acid extract  sodium h y d r o x i d e e x t r a c t  S - s i n g l e s t a g e NaOH e x t r a c t  isolate  isolate  isolate  P^S.  P^A P^B  55.  Figure  10.  Slab  g e l electrophoretic pattern  r a p e s e e d p r o t e i n whey (U) a f t e r p r e c i p i t a t i o n , P-j_U. black acetic  Stained  of the  isoelectric  with  0.2%  i n 7% a c e t i c a c i d a n d d e s t a i n e d  Amido i n 5%  acid.  W - f i r s t water e x t r a c t  i s o l a t e whey J  A - second h y d r o c h l o r i c B - third  P.WU x  a c i d e x t r a c t i s o l a t e whey  sodium h y d r o x i d e e x t r a c t  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  i s o l a t e whey P^SU.  P^AU  P^BU  P^. a n d c o n c e n t r a t e s  showed s i m i l a r  The whey f r o m t h e i s o l a t e s precipitation isolates.  patterns. obtained  after  isoelectric  showed a number o f bands c o r r e s p o n d i n g  This  i s shown i n F i g u r e  10.  w a t e r e x t r a c t t e n d s t o show a t r a i l i n g  to their  The whey f r o m t h e effect,  (b) D i s c g e l Disc g e l electrophoresis of the isolates and  the concentrates  a r e shown i n F i g u r e s  electrophoresis patterns distinct  number o f b a n d s .  isolates  and c o n c e n t r a t e  components.  This  i s true  f o r the different  P^ and P  1 1 , 12 a n d 13. preparations  The show a  Thus f o r e x a m p l e t h e w a t e r e x t r a c t show a s i m i l a r  separation  of protein  f o r a l l t h e e x t r a c t s t h a t w e r e 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  show a s i m i l a r o a t t e r n t o t h a t o f t h e i s o l a t e  14 a l s o  P.. l  F„  Isoelectric  focusing  The pH r a n g e 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 to obtain  a c o m p l e t e i s o e l e c t r i c s p e c t r u m o f b o t h a c i d i c and  basic proteins. I t was s e e n f r o m t h e s e e x p e r i m e n t s a s i n d i c a t e d i n Figures  15 , 16 a n d 17 t h a t t h e m a j o r i t y  isolates  P.A°, P.B and P.S h a v e i s o e l e c t r i c p o i n t s  pH 7 a n d 1 0 , e x c e p t f o r t h e i s o l a t e P^W 3 and 7.  This  concentrates. precipitated acidic  of the protein  p i c t u r e was t r u e  between  which l i e s  between  f o r the i s o l a t e P  and t h e  As s e e n i n t h e s e f i g u r e s some o f t h e p r o t e i n s during  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 t h e  s i d e a t t h e t o p o f t h e column.  A fair  amount o f  g u r e 11.  Disc gel electrophoretic pattern  rapeseed p r o t e i n i s o l a t e s precipitation. Strained 10%  with  Buffer:  Tris  the  isoelectric  g l y c i n e , pH  coomassie b r i l l i a n t  trichloroacetic  W - first  p r e p a r e d by  of  8.6.  b l u e R 250  in  acid.  water e x t r a c t  isolate  P.W I  A - second h y d r o c h l o r i c  acid extract isolate  B - t h i r d sodium h y d r o x i d e e x t r a c t S - s i n g l e s t a g e NaOH e x t r a c t  isolate  isolate  P^S.  P^A  P-j_B  w  A  +  B  S  gure  12.  Disc  rapeseed  gel electrophoretic  protein  i s o l a t e s prepared  acetic acid precipitation. pH  8.6.  R 250  Stained  pattern  with  by  Buffer:  coomassie  of the trichloro-  Tris  glycine,  brilliant  blue  i n 10% t r i c h l o r o a c e t i c a c i d .  W - first A - second B - third  water  extract  hydrochloric  isolate  P^W  acid extract  sodium h y d r o x i d e e x t r a c t  S - s i n g l e s t a g e NaOH e x t r a c t  isolate  isolate  i s o l a t e P-j-S.  P^A  P^_B  F i g u r e 13.  Disc g e l e l e c t r o p h o r e t i c pattern of the  rapeseed p r o t e i n concentrates. g l y c i n e , pH  8.6.  Buffer:  S t a i n e d w i t h coomassie  b l u e R 2 5 0 , i n 10% t r i c h l o r o a c e t i c W - first  water e x t r a c t concentrate  Tris brilliant  acid. 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 B - third  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 - 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.  CB  CA  ure  14.  Disc g e l electrophoretic pattern  of the  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 tation. with  Buffer:  T r i s g l y c i n e , pH 8.6.  coomassie b r i l l i a n t  trichloroacetic W - first  b l u e R 2 50 i n 1 0 %  acid.  w a t e r e x t r a c t whey  A - second h y d r o c h l o r i c B - third  Staine  P^WU  a c i d e x t r a c t whey  s o d i u m h y d r o x i d e e x t r a c t whey  S - s i n g l e s t a g e NaOH e x t r a c t whey P^SU.  P^AU  P.BU l  g u r e 15.  Isoelectric  focusing  of the rapeseed  protein  isolates  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 ,  at  pH r a n g e  3 and 10.  blue  W - first  S t a i n e d i n 0.2%  water e x t r a c t  A - second h y d r o c h l o r i c  isolate  bromophenol  P^W  acid extract  B - t h i r d sodium h y d r o x i d e e x t r a c t S - s i n g l e s t a g e NaOH e x t r a c t  isolate  P^A  i s o l a t e Pj_B  i s o l a t e Pj_S.  g u r e 16.  Isoelectric  isolates  of the rapeseed  p r e p a r e d by t r i c h l o r o a c e t i c  t i o n , a t pH r a n g e phenol  focusing  3 and  10.  acid  protein  precipita-  S t a i n e d i n 0.2%  bromo-  blue.  W - first  water extract  A - second h y d r o c h l o r i c B - t h i r d sodium  isolate  P^W  acid extract  hydroxide extract  S - s i n g l e s t a g e NaOH e x t r a c t  isolate  isolate  isolate  P S. t  P^A P^B  g u r e 17.  Isoelectric  focusing of the rapeseed p r o t e i n  c o n c e n t r a t e s , a t pH r a n g e 3 and 10.  Stained i n  0.2%  bromophenol b l u e . W - f i r s t water e x t r a c t concentrate  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 B - third S - single  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 t a g e NaOH e x t r a c t c o n c e n t r a t e  CS.  CB  CA  p r o t e i n , p a r t i c u l a r l y from the. a c i d i c and b a s i c migrate t o the b a s i c The  extracts,  s i d e a t t h e bottom o f the icol'umn.  r e s u l t s f o r the whey p r o t e i n s  showed s i m i l a r  p a t t e r n s as f o r the i s o l a t e s . When i s o e l e c t r i c f o c u s i n g  was c a r r i e d i n g e l con-  t a i n i n g no urea e s p e c i a l l y the h y d r o c h l o r i c  acid  extract  i s o l a t e s tend t o remain a t the top o f the column. G.  Amino a c i d  composition  The  amino a c i d composition o f the i s o l a t e s and con  c e n t r a t e s are given i n Tables 4, 5, 6 and 7.  In the concen  t r a t e s CW and CA t h e r e were h i g h e r l e v e l s o f g l u t a m i c 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 addition  and P.A° r e s p e c t i v e l y .  In  CA had a 70% more p r o l i n e than the i s o l a t e P^A°,  w h i l e P.B had 15% lower p r o l i n e than i n the c o n c e n t r a t e CB. The  i s o l a t e s P-B and P.S r e v e a l e d  a lower l e v e l o f glutamic  a c i d , methionine and p h e n y l a l a n i n e , however, 3 5 and 25% 39 and 25% and 20 and 2 5% l e s s than those i n the concent r a t e s CB and CS r e s p e c t i v e l y . a The  The i s o l a t e P^S a l s o had  3 8% h i g h e r p r o l i n e content than the c o n c e n t r a t e s CS. remaning amino a c i d s  were h i g h e r i n the i s o l a t e .  The  had  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^ t a s i m i l a r p a t t e r n 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° i n d i c a t e d h i g h e r l y s i n e ,  a r g i n i n e , methionine and p r o l i n e threonine, aspartic acid, leucine  f o r the water e x t r a c t , and and t y r o s i n e  f o r the  .6 5. TABLE I V AMINO ACID COMPOSITION OF RAPESEED PROTEIN CONCENTRATES FROM THE DIFFERENT FRACTIONS COMPARED TO THAT OF RAPESEED AND SOYBEAN PROTEIN CONCENTRATES (G AMINO ACID PER 16 G NITROGEN)  Soybean^  Rapeseed  6.7 5.1 5.0  Single NaOH extract 6.3 5.3 4.9  6.0 3.6 3.7  11. 3 3.9 6.0  Three stage e x t r a c t i o n HC1 NaOH Water  a  Asp. Thr. Ser.  6.9 6.8 4.6  4.1 4.5 4.9  Glu.  17.0 3.4 6.8  19. 5 4.9 5.7  19 . 3 5.3 5.1  15.9 4.8 5.4  16 .4 5.6 4.6  18.5 4.0 4.6  Pro. Gly.  Ala. Cys.# Val.  6.2 1.3 4.1  5.2 2.7 4.5  5.0 2.4 5. 3  6.0 2.4 5.0  4.0 1.9 4.6  5.3 1.3 5.6  Met.#  2.1  2.5  3.1  2. 5  1.6  1.3  He. Leu. Tyr.  3.0 5.5 3.8  3.8 7.0 3.0  4.4 8.0 3.5  4.0 7.2 3. 3  3.8 6.8 2. 3  5.3" 8.1 4.4  Phe. Lys. His.  2.2 6.9 5.1  3.7 6.6 3.5  4.2 4.6 2.1  3.9 6.6 3.4 .  3.7 6.2 2.7  5.6 6.7 2.6  Arg. Try. * Amm.  4.8 1.4 2.4  5.5 1.1 2.2  3.7 1.3 1.6  5.0 1.2 2.1  5.8 0.8  6.5 1.4  95.9  95.5  Total  94.3  95 . 3  -  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 S c h r a m e t " a 1. ( 1 9 5 4 ) and Moore ( 1 9 6 3 ) .  *  Tryptophan  d e t e r m i n e d by t h e method o f I n g l i s a n d L e a v e r s  ( 1964). a.  V a l u e s f r o m Lo a n d H i l l  (19 71) .  b.  V a l u e s f r o m Van E t t e n e t a l . (.1959).  TABLE V AMINO ACID COMPOSITION OF RAPESEED PROTEIN ISOLATES 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 COMPARED TO SOYBEAN PROTEIN ISOLATES AND RAPESEED PREPARATION B l CG. AMINO ACID PER 16 G NITROGEN) N.  Three stage e x t r a c t i o n NaOH HC1 Water Asp. Thr. Ser.  7.1 5.3 4.5  Glu. Pro. Gly.  11.9 2. 3 5.4  10 .9 2.9 5.9  12.5 4.5 5. 5  Ala. Cys. # Val.  6.3 0.9 5.0  5.9 1.5 5.3  Met.#  1.7  He. Leu. Tyr. Phe. Lys. His. Arg. Try. * Amm. Total  6.5 7.2 5.2  Soybean protein  Bl. a  10. 5 3. 5 4.7  10.4 4.2 4.0  12 .0 3.5 5.1  17 .6 5.1 3.9  22.2 5.0 5.8  5.1 2.4 5. 3  5.4 2.0 4.9  4.1 1.5 4.8  4.5 1.8 5.3  2.0  1.9  1.9 "  1.5  1.3  3.5 6.3 3.9  4.5 8.5 4.8  4.0 7.1 3. 2  3.5 6.5 3.4  4.6 7. 8 3. 5  4.3 8.0 2. 8  2.6 9.1 5.5  3.4 7.2 5.6  3.4 5.0 4.2  2.9 8.6 5.8  5.1 6.2 2.4  5.1 3.2 1,9  7.8 1.5 2.0 91.1  8.7 1.8  6.7 1.2 2 .4 87 .4  9.7 1.3 1.9 9 2.6  6.0 1.2  7.6 0. 7 I .1 95.9  •  2  .14  9 7.1  6.2 5.4 4.4  Single NaOH extract 6.2 5.7 4.6  —  93.9  #  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 m e t h o d s o f S c h r a m e t a l . ( 1 9 5 4 ) a n d Moore ( 1 9 6 3 ) .  *  Tryptophan (1964).-  a.  V a l u e s f r o m Lo a n d H i l l Skidmore E n t e r p r i s e s .  b.  Values from F i n l a y s o r i e t a l . (1968).  d e t e r m i n e d by t h e m e t h o d o f I n g l i s a n d L e a v e r s (.1971) f o r A s s a y p r o t e i n C - l  TABLE. V I AMINO ACID COMPOSITION OF RAPESEED PROTEIN ISOLATES PREPARED BY- TCA P R E C I P I T A T I O N COMPARED TO C A S E I N AND RAPESEED FLOUR (G AMINO. ACID PER 16 G NITROGEN)  Asp. Thr. Ser. Glu. Pro. Gly.  Three stage e x t r a c t i o n Water HC1 NaOH [ 7.2 5.3 6.1 7.2 5.8 5.9 4.3 4.7 4.5  Single NaOH extract 6.1 5.6 4.0  Casein  a  Rapeseed flour b  7.2 4.9 6.4  6.4 3.0 4.1  14.9 4.6 6.9  13.6 4.4 5.4  14.3 5.9 5.6  14.0 5.1 5.6  22.7 11.5 2.7  17.6 5.1 4.4  Ala. Cys.# Val.  6.7 0.7 4.4  5.5 3.2 4.7  5.0 0.9 5.3  5.3 2.4 5.2  3.0 0.3 7.3  3.8 0.9 2.5  Met.#  1.2  2.2  0.9  2.3.  2.8  1.7  He. Leu. Tyr.  3.3 6.5 4.0  3. 6 7.3 3.5  4.0 7.5 3.4  3.8 7.2 4.6  6.1 9.3 6.4  1.9 5.3 2.2  Phe. Lys. His.  2.3 8.3 4.5  3.5 7.5 5.8  3.4 6.7 4.8  3.1 7.6 5.6  5.0 8.3 3.1  3.3 4.4 2.2  7.5 1.3 2.6 94.7  7.8 1.7 2.3 95.3  4.1 2.1-  3.8  Arg. Try.* Amm. Total  6.0 1.5 1.6 94.0  8.5 1.3, X. 5 93.4  =  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 m e t h o d s o f S c h r a m e t aJL. (19 54) and Moore ( 1 9 6 3 ) .  *  Tryptophan  by t h e m e t h o d o f I n g l i s and L e a v e r s  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 Lo and H i l l (19 7 1 ) .  Chem. N o r w i c k , N.Y.  b. V a l u e s f r o m Tape e t a l . (19 7 0 ) .  (1964). from  TABLE V I I AMINO ACID COMPOSITION. OF RAPESEED PROTEIN WHEY AFTER 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 THOSE OF SOYBEAN PROTEIN WHEY (G AMINO ACID PER 16 G NITROGEN)  T h r e e stage' e x t r a c t i o n HC1 NaOH Water 7.1 7. 3 5.1  Asp. Thr. Ser.  16 .6 4.6 8.4  Glu. Pro. Gly.  2.2 2. 8 5.2  2.2 3. 8 3.4 28.9 8.9 4.1  20.7 5.6 6.1  Single NaOH extract 4.2 5.1 4.7  Soy whey  23.5 8.0 6. 3  14.6 5.4 4.7  2.7'  2. 3  2.2  3.0 6.7 1.3  2.9 5.8 2.1  4.6 7. 2 4.2  4.2 4.8 2. 8  2. 2 7. 5 5.5  2.6 9. 5 5. 6  4.2 8.4 3.5  2.8 4.2  5.5 5.3 3. 9  3.5 2.7 5.1  5.0 11.1 4.2.  - Met.#  2.1  1.8  0.9  He. Leu. Tyr.  2.8 5.4 3.4  3.1 6.4 1.1  Phe. Lys. His.  1.9 10 . 3 4.9  #  7.2 1.1 2.8 10 2.7  2. 8  4.2  6.6 1.7 3.6  Arg. Try. * Amm. . Total  13.9 5.4 6.7  5.2 2.3 5.3  Ala. Cys. # Val.  4.0 11. 5 8.1  FAO  7.7 8.8 0.4 0.4 3.1 4.2 109.9 . . 10 3. 7  7.4 0.7 2.8 113. 7  6.8 1.5 1.4 107 . 6  1.4  P e r f o r m i c a c i d o x i d a t i o n separate d e t e r m i n a t i o n s , by the. m e t h o d s o f S c h r a m e t a l . ( 1 9 5 4 ) a n d Moore ( 1 9 6 3 ) .  *  Method o f I n g l i s  and Leaver  C1964).  a.  Values  b.  F o o d A g r i c u l t u r e O r g a n i z a t i o n recommended  from Rackis e t a l . (1971). pattern.  hydrochloric acid P^A. t  The i s o l a t e s  higher levels and  extract  P^S.  P.B t  than  than  those  i n the isolates  and P.S h a d most o f t h e a m i n o a c i d s a t t  the i s o e l e c t r i c p r e c i p i t a t e d  The i s o l a t e d  P^W  The amino a c i d  distribution.  islates  h a d 2 0 t o 44% more l y s i n e  other i s o l a t e i n the three stage e x t r a c t i o n  of the i s o l a t e s  P W and  composition  P^B  than  procedure.  o f t h e two p r e p a r a t i o n 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 The i s o l a t e P W h a d 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. l  The i s o l a t e P.A h a d a h i g h e r s u l f u r c o n t a i n i n g t  amino a c i d  together with glutamic acid  P.A . 0  &  &  than  i n the i s o l a t e  The i s o l a t e P.B h a d a h i g h e r c y s t i n e  content  and l o w e r  isolate  P.B. t  lysine  and g l u t a m i c a c i d  I n t h e i s o l a t e P^S t  tion of tryptophan, and  tyrosine  lower l e v e l s o f arginine  and than  methionine i n the  there i s a higher °  methionine than  concentra-  and g l u t a m i c  i n the i s o l a t e  Among t h e 8 i s o l a t e s p r e p a r e d , t h e  acid  P.S.  hydrochloric acid  extract  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  acids.  The i s o l a t e P^B h a d 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  cystine.  I t was o b s e r v e d  amino a c i d  any  that the i s o l a t e s  P^S  and P S  c o n c e n t r a t i o n s averaging those present  i s o l a t e s by t h r e e s t a g e  extraction,  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 The v a l u e s o b s e r v e d  except  and  had  i n the  f o r lower  i n isolate  amino  proline  P.S.  by F i n l a y s o n e t a l . ( 3 7 ) f o r  t h e f r a c t i o n A I V S 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 isolate  and p r o l i n e .  B I had a s i m i l a r amino a c i d  But t h e i r  composition  except  for a  70. higher proline,  aspartic  concentrations of lysine the  acid  and g l u t a m i c a c i d ;  and t y r o s i n e .  These d e v i a t i o n s i n  amino a c i d s i s demished i n t h e i s o l a t e s It i s interesting  and l o w e r  P^.  t o note t h a t these 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 % ) ,  glycine  alanine  (14 t o 3 2 % ) ,  histidine  58%), acid (33  (18 t o 3 4 % ) ,  and a r g i n i n e (32 t o 4 1 % ) ,  t o 50%),  lysine  (10 t o 3 8 % ) , glutamic acid  and p r o l i n e  i n t h e soybean p r o t e i n had  a 20% l e s s  lysine  (25 t o 3 5 % ) ,  and l o w e r l e v e l s (30 t o 3 8 % ) ,  (42 t o  of  aspartic  phenylalanine  (12 t o 43%) c o m p a r e d t o t h o s e  isolates  (53).  present  Only t h e i s o l a t e s  c o n t e n t than t h e soybean  P^B  protein  isolate. Compared t o c a s e i n h i g h e r amounts o f t h r e o n i n e cystine  (61 t o 86%),  the  isolates  the  other fractions  The  isolates  the  amino a c i d The  P.W  P  (53) the i s o l a t e s (8 t o 3 7 % ) ,  and a r g i n i n e  alanine  (39 t o 5 8 % ) .  (41 t o 5 3 % ) , In  addition  a n d P.S h a d h i g h e r amounts o f l y s i n e  while  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 .  were q u i t e  s i m i l a r t o casein with regard t o  composition. amino a c i d  after isoelectric  soybean p r o t e i n soybean p r o t e i n ,  c o n t e n t i n r a p e s e e d whey  protein  p r e c i p i t a t i o n was h i g h e r t h a n t h a t o f t h e  respective isolates  P..  isolates  The same phenomenon was o b s e r v e d f o r (72).  Compared t o t h e whey  t h e whey f r o m i s o l a t e s  t h r e o n i n e , a l a n i n e and l y s i n e leucine  P^ showed  and p h e n y l a l a n i n e .  P^W  from  contained  and l o w e r a s p a r a t i c  The whey f r o m i s o l a t e s  higher  acid P.A  iso0  was  higher i n histidine  and g l u t a m i c a c i d .  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  The whey f r o m and h i s t i d i n e  exceptionally high concentration of cystine.  those  rapeseed  higher  p r o t e i n concentrates prepared  (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  trate  from  i n t h e o t h e r wheys. The  Hill  and an  The whey  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 than  isolate  CS e x c e p t  b y Lo a n d  t o t h e concen-  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 , 2 1 % c y s t i n e , 35% m e t h i o n e , 3 1 % t y r o s i n e , a n d 39% tryptophan single  a n d 1 2 % more p r o l i n e .  stage  containing  extraction  Thus i t a p p e a r s t h a t  this  e n d o r s e s t h e good r e c o v e r y o f s u l f u r  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 s o y b e a n p r o t e i n c o n c e n t r a t e s , t h e  rapeseed  protein concentrate  phenylalanine 19  CS c o n t a i n s h i g h e r amounts o f  a n d i s o l e u c i n e by 30 a n d 25% r e s p e c t i v e l y and  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  acid  contents.  Thus t h e 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 rapeseed  amount p r e s e n t  protein concentrate  CS c o m p a r e d t o t h e  i n t h e s o y b e a n p r o t e i n c o n c e n t r a t e may be an  advantage t o t h e wide use o f t h i s p r e p a r a t i o n . H.  Ultracentrifugation  Ultracentrifugation pattern of the f i r s t t r a c t i s o l a t e prepared in  F i g u r e 18.  experimental  water ex-  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  T h e r e were t h r e e p e a k s o b s e r v e d c o n d i t i o n s used.  The r e s p e c t i v e  c o e f f i c i e n t o f t h e t h r e e p e a k s were 5.4S, 3.OS  under t h e sedimentation a n d 0.9S.  g u r e 18.  S c h l i e r e n patterns of the f i r s t water  i s o l a t e P^W. pH 8.5  C o n c e n t r a t i o n 1.0%;  extract  0.1 M T r i s - g l y c i n e  c o n t a i n i n g 6 M u r e a and 0.1 M  2-mercaptoethanol  The p i c t u r e s were t a k e n 2 5 m i n s ( u p p e r ) and 60 m i n s ( l o w e r ) a f t e r r e a c h i n g 59,000  r.p.m.  g u r e 19.  Schlieren pattern  acid extract isolate  A°.  0.1  M T r i s - g l y c i n e pH  0.1  M 2-mercaptoethanol.  20 m i n s a f t e r r e a c h i n g  of the  8.5  second  hydrochloric  Concentration containing The  6 M urea  p i c t u r e was  59,000 r.p.m.  1.0%; and  taken  F i g u r e 20.  Schlieren pattern of the t h i r d  extract isolate  P^B.  sodium hydroxide  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 a n d 0.1 M 2-mercaptoethanol.  The p i c t u r e was t a k e n 65 m i n s  a f t e r r e a c h i n g 59,000  Figure  21.  r.p.m.  Schlieren pattern of the single  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.  stage  sodium  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 a n d 0.1 M 2 - m e r c a p t o e t h a n o l . m i n s a f t e r r e a c h i n g 59,000  The p i c t u r e was t a k e n 60 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 hydrochloric  acid extract  precipitation  o f the second  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  i s p r e s e n t e d i n F i g u r e 19.  o b s e r v e d by t h i s p r e p a r a t i o n . under the c o n d i t i o n s  A s i n g l e p e a k was  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  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  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 hydroxide extract  and s i n g l e  1.9S.  o f the t h i r d sodium  sodium h y d r o x i d e e x t r a c t  isolate  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 h a d s e d i m e n t a t i o n coefficients are  o f 0.2S a n d O.HS  respectively.  shown i n F i g u r e s 2 0 a n d 2 1 .  extract  i s o l a t e had a l a r g e  disappeared within I.  The t h i r d s o d i u m h y d r o x i d e  3 minutes.  balance of 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 was l o w . the that  patterns  number o f f a s t m o v i n g p e a k s w h i c h  the f i r s t  Material  These  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  This i s evident i n the high l e v e l s o f nitrogen  r a p e s e e d whey o b t a i n e d f r o m d i f f e r e n t f r a c t i o n s much o f t h e e x t r a c t e d  nitrogen  loss  protein  in  indicating  was n o t p r e c i p i t a t e d .  The  i n t h e whey f r o m 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 w h a t was o b s e r v e d f o r t h e i s o e l e c t r i c precipitation. j.  This i s given i n Table  Loss o f p r o t e i n  during washing  A-considerable loss of protein the  washing process.  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  procedure  was o b s e r v e d  The l o s s o f p r o t e i n  trichloroacetic acid precipitated  protein  VIII.  by w a s h i n g t h e  isolateswas  isolatesP^.  i n w a s h i n g i s shown i n T a b l e  IX.  during  greater  This loss of  than  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 d r y m a t t e r i n o r i g i n a l flour).  Extract ~ F i r s t water  extract  Second a c i d i c Third basic  extract  extract Total  First basic  extract  Whey P  i  Whey P  5.5  1.6  0.4  5.8  3.2  0.5  5.4  0.9  20.4 33.7 26.8  10.2 4.6  1.8 1.5  \  TABLE IX PROTEIN LOSSES FROM WASHING THE ISOLATES WITH WATER AND 50% ALCOHOL Cas p e r c e n t a g e of i s o l a t e s } .  Water washing p  i  p  Alcohol  washing  t .  T o t a l washing •  P.  x  P.  t  1st  water e x t r a c t  0.02  0.41  0.01  0.01  0.03  0.42  2nd  acidic  0.02  1.09  0.02  0.38  0.04  1.47  3rd  basic  0.11  ].17  0.04  0.77  0.15  2.94  0.15  3.67  0.07  1.16  0.22  4.83  0.09  2.51  0.11  2.49  0.20  5.00  extract extract  Total 1st b a s i c  losses  extract  K.  Yield of protein  The y i e l d s o f p r o t e i n Table  X.  isolated i s o l a t e d are tabulated i n  W i t h 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 t 35°C t h e  y i e l d s o b t a i n e d were 1 0 . 8 , 7.2 the three stage extraction.  extraction  and 4 1 . 5 % r e s p e c t i v e l y f o r  and 51.2% f o r t h e s i n g l e  For the t r i c h l o r o a c e t i c acid  precipitation  t h e y i e l d s o b t a i n e d were 1 4 . 2 , 14.7 and 53.8% f o r the three stage e x t r a c t i o n stage  and 70.3% f o r t h e s i n g l e  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 i s o l a t e s P^W  and P^A°  that the s o l u b i l i t y  were a p p r o x i m a t e l y  r e s p e c t i v e l y , w h i l e the sodium hydroxide I  respectively  extraction. L.  P.B  stage  of the  59 and 4 9 % extract  isolates  and P.S h a d a l o w e r v a l u e o f 4 2 . 5 % . I  The c o n c e n t r a t e s three stage e x t r a c t i o n CA and CB r e s p e c t i v e l y . extract  concentrate  had a h i g h e r s o l u b i l i t y  f o r the  7 7 , 80 and 7 1 % f o r t h e s a m p l e s The s i n g l e  stage  CW,  sodium h y d r o x i d e  CS h a d 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 r e t a b u l a t e d i n Table X I .  TABLE X THE Y I E L D S OF PROTEINS  Water e x t r a c t  ISOLATED  Acid extract  Basic  extract  Single basic extract  Isoelectric precipitation  10.8  7.2  41.5  61.2  Trichloroacetic acid precipitation  14.2  14.7  53.8  70.3  TABLE XI SOLUBILITY MEASUREMENTS OF THE ISOLATES AND .CONCENTRATES EXPRESSED AS PERCENTAGES OF PREPARATIONS I s o l a t e P,  Concentrate  F i r s t water e x t r a c t  58.7  76.9  Second a c i d  extract  48.5  80.3  Third basic  extract  42.1  70.8  F i r s t basic  extract  43 . 2  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 regions  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 g r e e n  alkaline product,  w h i l e a r o u n d pH 10 t h e c o l o u r was c r e a m y y e l l o w . ing  of colour at higher  tractability trolling  pH i s due m a i n l y  of pigments present  The d a r k e n -  t o t h e g r e a t e r ex-  i n the flour.  Thus by c o n -  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 c a n be k e p t  lighter.  The i s o l a t e o b t a i n e d  showed some t r a i l i n g electrophoresis. achieved  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  A greater  at higher  by e x t r a c t i n g a t pH o v e r 10  recovery  o f p r o t e i n c a n be  pH-regions, but the colour o f the p r o t e i n  was n o t s a t i s f a c t o r y . As s e e n f r o m 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 o b s e r v e d  that  a t t h e o p t i m u m 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. to  combine and p r e c i p i t a t e s a l t s  Thus o x a l c i a c i d was  able  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 r e m o v e d . The n a t u r e  o f t h e curve  that the p r o t e i n content reaching the  shown i n F i g u r e  i n the supernatant  initial  increased  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  second, h y d r o c h l o r i c a c i d e x t r a c t .  6 indicates after  ( 0 . 0 5 M) o f  The i n c r e a s e a f t e r t h e  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  proteinate.  A f t e r reaching  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 t h e r e was a s u d d e n d e c r e a s e i n t h e p r o t e i n observed.  This  content  s u d d e n d e c r e a s e may be due t o s a l t i n g o u t o f  82.  some p r o t e i n . As shown i n the T a b l e s I and I I , t h e i s o l a t e s p r e 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  o f the  ex-  t r a c t s showed h i g h e r v a l u e s f o r m o i s t u r e and p r o t e i n w h i l e 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.  had  h i g h e r v a l u e s f o r c a r b o h y d r a t e s , a s h , phosphorus, s u l f u r thiocyanates.  The  f a t and crude f i b e r c o n t e n t was g e n e r a l l y  about t h e same f o r both p r e p a r a t i o n s . be  and  These r e s u l t s would  e x p e c t e d because o f the h i g h e r p u r i t y o f 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 c o n t e n t and l o w e r c o n c e n t r a t i o n s o f o t h e r compounds. The  c o n c e n t r a t e s , as e x p e c t e d , show a l o w e r  percen-  tage o f p r o t e i n and h i g h e r l e v e l s o f m o i s t u r e , a s h , 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 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^.  The most i n t e r e s t i n g  f e a t u r e o f t h e c o n c e n t r a t e s was  l o w e r i s o t h i o c y a n a t e c o n t e n t compared t o the two  isolates.  The r e s u l t s f o r t h e second h y d r o c h l o r i c a c i d t r a c t i s o l a t e a f t e r t h e t r e a t m e n t w i t h 0.05 showed t h a t not o n l y t h e ash c o n t e n t was 9%,  but a l s o the phosphorus c o n t e n t was  M oxalic  reduced decreased  the  ex-  acid  from 36.5 from  t o 1.8%.  T h i s r e d u c t i o n o f phosphorus on o x a l i c a c i d  ment may  be due t o complex c a l c i u m s a l t s formed d u r i n g  to  11.6 treat-  precipitation. The  v a l u e s f o r the p r o t e i n i s o l a t e s o b t a i n e d were  low compared t o t h e p u b l i s h e d d a t a 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 such as soybean, c o t t o n seed and s u n f l o w e r .  This  83.  may be due t o t h e h i g h a s h a n d l i p i d • The h i g h c a r b o h y d r a t e  content i n the i s o l a t e s .  content of the f i r s t  e x t r a c t c o n c e n t r a t e may be due t o t h e e a s y sugars the  during the extraction.  isolates  prepared  solubility  The c a r b o h y d r a t e  from t h e water  water of the  content of  e x t r a c t was l o w due t o  the p r e c i p i t a t i o n o f the p r o t e i n s during 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 h a v e c o n t a i n e d m o s t o f t h e c o n taminating  carbohydrates. S u l f u r c o n t e n t was l o w e r 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 c o n c e n t r a t e CA, t h a n 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 content of  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 .  sulfur during precipitation  incomplete fractions  precipitation  into  This  t h e whey may be due t o  of the s u l f u r  containing protein  or peptides. The h i g h p h o s p h o r u s c o n t e n t i n t h e f i r s t  extract  loss  i s o l a t e P^W  water  may be due t o t h e h i g h s o l u b i l i t y  p h y t i c compounds p r e s e n t i n t h e r a p e s e e d c o n c e n t r a t e s the second  hydrochloric acid  even h i g h e r phosphorus l e v e l caused t h e compounds i n t h e a c i d  flour.  of the  Among t h e  extract  CA h a d an  by t h e s o l u b i l i z a t i o n o f  media.  As m e n t i o n e d 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 t h e second acid of  hydrochloric acid  treatment  extract  was r e d u c e d  i s o l a t e P^A a f t e r  by a p p r o x i m a t e l y  calcium i n the h y d r o c h l o r i c acid  acid  treatment  i s due t o t h e a b i l i t y  5 0%.  oxalic The r e d u c t i o n  i s o l a t e P^A on of oxalic  acid  oxalic t o combine  84.  and  precipitate  content  salts  i n the extract.  i n the i s o l a t e s  and c o n c e n t r a t e s  G e n e r a l l y t h e ash c o n t e n t 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 protein isolates. may  were h i g h .  of the i s o l a t e s  and  reported f o r other  con-  vegetable  This 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  be a t t r i b u t e d t o t h e amounts o f c a l c i u m p r e s e n t  The a s h c o n t e n t by  Generally the calcium  i n them.  of the hydrochloric acid extract p r e c i p i t a t e d  trichloroacetic  a c i d was 1.0%, w h i l e t h e i s o l a t e  a f t e r o x a l i c acid treatment  was  2.0%.  prepared  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 which causes the coprecipitation  of calcium salts with the protein during  loroacetic acid  precipitation.  C r u d e f i b e r c o n t e n t was especially P^A.  fairly  high i n the i s o l a t e s ,  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  The h i g h v a l u e may  supernatant  t h a t tends  pH a d j u s t m e n t s . and  trich-  be due t o t h e f i n e p a r t i c l e s  i n the  to p r e c i p i t a t e with the p r o t e i n s during  The p u b l i s h e d d a t a f o r s o y b e a n p r o t e i n i s o l a t e  c o n c e n t r a t e w e r e 0.7  and 3.0%  respectively.  From t h e r e s u l t i t was a p p a r e n t  that 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 observed cyanate  f o r any o f t h e o t h e r p r e p a r a t i o n s .  This high  that  isothio-  content i n d i c a t e d i n the water e x t r a c t i s probably  to the higher e x t r a c t a b i l i t y It  due  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 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  three stage procedure  the i s o t h i o c y a n a t e content  The i s o t h i o c y a n a t e p r e s e n t  decreases.  i n water e x t r a c t i s o l a t e s  and  85.  concentrates than  that  exept  procedure  f o r the single  the single  approximately Slab  second  extraction fractions  acid  a t pH  improve  the n u t r i t i o n a l  fractions  Considerable  find  o u t methods  isoelectric  being  contains  the highest Amino  that  could  amount  acid  amount  isolate  whey.  this  be  i s higher  used  water  and  indicated  simple  acids  from  i n food  of protein and  basic  protein  three  stage  the  protein  rapeseed  processing to  l a b o r a t o r y by which  from  r e v e a l e d an  loss  to  fraction  the  e t al_. (72) f o r soybean  acids. single  extremely  of cystine  phenomenon  Work  using  c o n t a i n i n g amino  isolate  The  i n the  i n the future..  o f t h e whey  This  lost  i t i s necessary  to determine  of sulfur  extract  i s being  loss  out i n this  as p o s s i b l e . Rackis  P^  to separate  amino  composition  hydroxide  by  extract,  extract  p r o t e i n s from  precipitation  chromatography  a s much  observed  higher  value.  concentration of cystine.  prevented  25%  precipitation  of the f i r s t  isolates  Thus w i t h  carried  column  sodium  hydroxide  of preventing this  sephadex  high  extracts  containing important  from  hydroxide  isoelectric  i t i s possible  These  stage  sodium  and n e u t r a l  8.8.  procedure  currently  by  gel electrophoresis  whey  approximately  sodium  prepared  flour.  is  stage  stage  of acidic  confirmed  were  8%.  hydrchloric  separation as  this  f o r the isolates  where by  by  must  be  i s similar  to  protein  86.  distinct  Each p r o t e i n f r a c t i o n from the rapeseed  f l o u r shows  c h a r a c t e r i s t i c s r e g a r d i n g the amino a c i d  composition  when compared t o c a s e i n and soybean p r o t e i n i s o l a t e s . c o u l d be i n f e r r e d t h a t rapeseed utilized  It  protein isolates i f properly  a f t e r p r e p a r a t i o n , much o f the present demands f o r  high q u a l i t y p r o t e i n s c o u l d be overcome without  encountering  difficulties. As observed  from the amino a c i d composition o f the  s i n g l e stage sodium hydroxide  c o n c e n t r a t e CS c o n t a i n s a  h i g h e r s u l f u r content amino a c i d compared t o the soybean prot e i n concentrate.  T h i s s i t u a t i o n may be o f some b e n e f i t t o  the consumer as i s o l a t e s tend t o l o s e 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 t r a c e n t r i f u g a t i o n p a t t e r n s f o r the rapeseed t e i n i s o l a e s from the f i r s t water, P^W and second  pro-  hydro-  c h l o r i c a c i d e x t r a c t , P.A i n d i c a t e d t h a t l a r g e molecules broken down i n t o u n i t s o f s m a l l m o l e c u l a r weights.  have  This  d i s s o c i a t i o n i s due t o the b l o c k i n g o f the s u l f h y d r y l and amino group p r e s e n t i n the i s o l a t e , t o f a c i l i t a t e z a t i o n o f the i n s o l u b l e p r o t e i n s . obtained f o r the t h i r d  very low sedimentation  S i m i l a r l y the r e s u l t s  sodium hydroxide  s i n g l e stage sodium hydroxide  solubili-  e x t r a c t P^B and the  e x t r a c t i s o l a t e P^S i n d i c a t e d  coefficients.  The r e s u l t s p u b l i s h e d by Bhatty e t 'al. (14) showed sedimentation c o e f f i c i e n t s o f 12S and 1.7S f o r t h e i r ation B l .  prepar-  The r e s u l t s o b t a i n e d i n t h i s study, 1.9S value f o r  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, c l o s e l y agree with  their  87. sedimentation  coefficient.  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 isolates  from the three stage  e x t r a c t i o n procedure  precipi-  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 from the s i n g l e  stage e x t r a c t i o n procedure,  while the y i e l d s  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 are approximately for  t h e two p r o c e d u r e s .  than  t h e same  The l o w 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 of the three stage e x t r a c t i o n procedure  should  h a v e 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 proteins. There i s a great l o s s o f p r o t e i n d u r i n g washing the i s o l a t e s prepared  by t r i c h l o r o a c e t i c  acid  precipitation,  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 trichloroacetic  ability  a c i d f o r s m a l l p e p t i d e s w h i c h may  be  of  lost  during the washing operations. The s o l u b i l i t y  o f the i s o l a t e s i n water i s not very  high f o r preparations.  T h i s i n s o l u b i l i t y may  result  heat  from the i n i t i a l  w h i c h i t was  treatment  o f rapeseed  have been denatured  decreased  points.  of the i s o l a t e s .  c e d u r e was r e a s o n a b l y  was  only  58%.  The p r o t e i n c o u l d iso-  A l l t h e s e may h a v e p l a y e d a r o l e i n t h e  prepared  while the single  and  d u r i n g t h e a d j u s t m e n t o f pH t o t h e i r  solubility  concentrates  meal t o  subjected during the preparation of f l o u r  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.  electric  have been a  stage  The s o l u b i l i t y  from the t h r e e stage  extraction  of the pro-  g o o d w i t h v a l u e s r a n g i n g f r o m 70 t o 8 0 % , sodium hydroxide  extract concentrate  PART I I FUNCTIONAL PROPERTIES OF THE PROTEIN  FRACTIONS  RAPESEED  CHAPTER VI INTRODUCTION The  modern use  c o n c e n t r a t e s f o r food  is  of i s o l a t e d p r o t e i n s  and  protein  i n an e a r l y stage of development.  Progress w i l l depend l a r g e l y on t e c h n o l o g i c a l and search a p p l i e d to t h e i r p r o d u c t i o n .  basic  At present the  re-  chief  f u n c t i o n i s to improve the n u t r i t i o n a l q u a l i t y o f the bread  by  supplementation o f the p r o t e i n i n wheat f l o u r . Rapeseed p r o t e i n i s o l a t e s and u s e f u l n u t r i t i o n a l l y because of the h i g h containing  and  b a s i c amino a c i d s .  c o n c e n t r a t e s may  be  content of s u l f u r  T h e r e f o r e these p r e p a r a -  t i o n s could become r i c h sources of supplementary m a t e r i a l foods d e f i c i e n t i n them.  Another important f e a t u r e  seed p r o t e i n i s o l a t e s i s the  absence of any  and  o f rape-  beany f l a v o u r  c h a r a c t e r i s t i c to soybean p r o t e i n i s o l a t e s . processing  for  With methods of  techniques f o r 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 , i t  is  v ery l i k e l y t h a t rapeseed meal w i l l become a good source  of  p r o t e i n t h a t can be used l i k e the  p r o t e i n i s o l a t e s and  conventional  vegetable  c o n c e n t r a t e s t h a t are a v a i l a b l e e a s i l y  today. At t h i s stage of our r e s e a r c h  we  are h o p e f u l  f i n d i n g 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 areas of processing,  f o r i n s t a n c e , water h o l d i n g  capacity  bakery p r o d u c t s ;  c o n t r a o l of f a t a b s o r p t i o n ;  characteristics;  gel formation;  properties  in coffee;  and  desserts.  frozen  aerating  food  i n meat or  viscosity  texturability; capacity  of  feathering  i n whipped toppings  CHAPTER V I I LITERATURE REVIEW R a p e s e e d p r o t e i n i s o l a t e s and not been so f a r f u l l y supplementation  i n any  or f o r t i f i c a t i o n  commodities such ducts.  utilized  Reference  as b r e a d ,  c o n c e n t r a t e s have  significant extent, for  or enrichment  i c e cream, spreads  to recent l i t e r a t u r e  o f common f o o d and meat p r o -  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 the above p r o d u c t s  commercially  (85).  soybean p r o t e i n i s o l a t e s were u s e d ,  I t was  n o t e d t h a t when  t h e r e were  certain  i m p r o v e m e n t s 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 These p r o p e r t i e s i n c l u d e water s t a b l i z i n g , t h i c k e n i n g and ( 4 , 4 2 , and  foods.  binding, emulsifying,  f i l m and  dough f o r m i n g  ability  48).  It  i s known t h a t t h e a d d i t i o n o f 3% s o y b e a n  t o albumen improved a shorter time.  protein  t h e w h i p , g i v i n g a h i g h e r whip volume i n  R e c e n t l y T s e n 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 bread.  Bread  being  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 h u n g r y malnourished  people  i n developing countries.  This  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 w h i c h significantly  enrichwill  improve the n u t r i t i o n a l v a l u e of bread  s a c r i f i c i n g other q u a l i t i e s  of the f i n i s h e d  without  product.  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 c h a n g e d some o f t h e s t r u c t u r a l and  or  has  f u n c t i o n a l p r o p e r t i e s of  the product  such as f l a v o u r , c o l o u r t e x t u r e , appearance,  aroma and volume.  I t has been i m p o s s i b l e t o add any s i g -  n i f i c a n t amounts o f nonwheat n u t r i e n t a d d i t i v e s t o bread without  losing desirable qualities. With the use o f i s o l a t e d p r o t e i n f r a c t i o n s as a  b a s i s f o r the f o r m u l a t i o n o f duly a c c e p t a b l e , h i g h p r o t e i n food products  at reasonably  low c o s t s have been  developed  (21). There i s a c o n s i d e r a b l e need t o improve the prot e i n supply i n order t o overcome famine and d i s a s t e r i n underdeveloped areas o f the world. o f improving production  The c o n v e n t i o n a l means  p r o t e i n supply i s by i n c r e a s i n g a g r i c u l t u r a l  and c o n v e r s i o n  o f p l a n t t o animal p r o t e i n can be  p r o p e r l y and e f f i c i e n t l y e x p l o i t e d . much i n t e r e s t i n new sources  of protein:  y e a s t grown on petroleum, chemical photosynthesis  At present  and by p l a n t c e l l c u l t u r e ( 2 ) .  been made d u r i n g the past 15 years p r o t e i n sources  the most important  another  advantages have  in utilizing  vegetable  i n the f i g h t a g a i n s t p r o t e i n shortage ( 7 9 ) .  p r o t e i n s p r o p e r l y processed one  from the sea  s y n t h e s i s , improved  There i s no doubt t h a t important  Probably  fish  there i s  advantage made i s t h a t  vegetabl  and e f f i c i e n t l y combined with  can r e p l a c e animal p r o t e i n food i n n u t r i t i o n o f  man and, expand the a v a i l a b i l i t y o f d i e t a r y p r o t e i n , now so s c a r c e (15).  This has been p o s s i b l e because o f accumulated  knowledge o f p r o t e i n n u t r i t i o n i n d i c a t i n g t h a t amino a c i d  p a t t e r n , n o t p r o t e i n a s s u c h , i s t h e most i m p o r t a n t i n determining  the n u t r i t i v e  Considering t i n g vegetable  value  o f the food p r o t e i n ( 7 3 ) .  the s i g n i f i c a n t  p r o t e i n i n t o animal  quite l o g i c a l that research  inefficiency  w o r k e r s i n Food S c i e n c e  f r o m I n d i a by C h a n d r a s e k h a r a  t h a t toned, a n d d o u b l e t o n e d m i l k c a n be made b y  r e c o n s t i t u t e d skim m i l k  vegetable  s o l i d s w i t h cow m i l k .  t o n e d m i l k , i s now a i m e d a t r e p l a c i n g  Miltone  imported  s k i m m i l k p o w d e r 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 mainly  costs  respectively (97).  I t has been r e p o r t e d  mixing  protein  $3.20, w h i l e t h a t f r o m soybean and wheat  31 and 1 1 c e n t s  e t a l . (17)  have  proteins i n  I t i s known t h a t 1 p o u n d o f u t i l i z a b l e  from beef costs only  o f conver-  p r o t e i n , i t is' perhaps  g i v e n due t h o u g h t t o t h e d i r e c t u s e o f v e g e t a b l e human f o o d .  factor  from peanuts.  mixed w i t h g l u c o s e , The  miltone  and  then cooled  The d e o d o u r i z e d  protein solutioni s  v i t a m i n p r e m i x and s t a n d a r d i z e d  thus obtained  protein,  milk.  i s h o m o g e n i z e d and p a s t e u r i z e d  t o 4°C a n d b o t t l e d .  I m i t a t i o n m i l k h a s 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 h a d i n 196 8.  In a d d i t i o n t o being  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 cost advantageous product  creates  difficulties  d u p l i c a t i o n o f m o u t h f e e l and b o d y .  i n achieving  Because o f u s i n g p r o t e i n  derivates i n imitation milk products,  the preparation solids,  lacks  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  milk  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- a n d  diglyerides.  I m i t a t i o n c h o c o l a t e milk d r i n k r e q u i r e s an  a d d i t i o n a l q u a n t i t y o f kappa carrageenan to s t a b i l i z e  the  cocoa f i b e r s , because of the .low p r o t e i n l e v e l s of the d r i n k . Phosphates or c i t r a t e s a l t s are o c c a s i o n a l l y added to h e l p 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 p r o t e i n . These s a l t s not necessary with  0.1  except when h i g h l y denatured  to 0.2% o f the s a l t s  and t r y p t o p h a n ,  a v a i l a b l e at reasonable difficulty. a c i d thus food  p r o t e i n i s used  (76).  F o r t i f i c a t i o n of foods with l y s i n e , threonine  s y n t h e t i c methionine,  which are  commercially  c o s t s , can be done without  much  However, t h e r e i s a p o s s i b i l i t y t h a t the amino  supplemented i s r e a d i l y l o s t d u r i n g cooking  processing.  a f f e c t f l a v o u r and  Furthermore, the f r e e amino a c i d s c o l o u r through d e g r a d a t i o n  with other food components, f o r i n s t a n c e by degradation  or amino-carbonyl r e a c t i o n .  l a t i o n of n u t r i t i o n a l l y w e l l balanced the primary  are  or  and may  interaction  Strecker  Therefore  the  iso-  p r o t e i n must s t i l l  be  importance i n food p r o c e s s i n g .  F i j i m a k i e t 'al. (39) have developed  a method of  r e c t i f y i n g amino a c i d imbalance i n most vegetable  proteins  by d i g e s t i n g p r o t e i n with a p r o t e i n h y d r o l y z i n g enzyme and r e s y n t h e s i z e d a p r o t e i n l i k e substance  ( p l a s t i n ) with  the  h e l p o f a p r o t e i n a s e a f t e r condensing the h y d r o l y s a t e . They c o u l d even couple  an amino a c i d e s t e r such as methionine  e t h y l e s t e r d i r e c t l y to p l a s t i n molecule i n s t e a d of u s i n g 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 content  by  s i x t o seven  the  s u l f u r c o n t a i n i n g amino a c i d  times.  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  m eans o f i n c r e a s i n g the  b a l a n c e o f amino a c i d s literatures  on  mentation.  P o l l o c h and  1%  u n h e a t e d soy  decrease the It a  soy  nutritive  (35).  f l o u r and  value  Diser soy  (27)  grits  Geddes (69)  with a  has  as p r o t e i n  reported  f l o u r improves the b r e a d , but  supple-  work i n w h i c h higher  levels  l o a f volume. i s w e l l known t h a t g l u t e n p r o t e i n c o m p l e x i s  with other  f l o u r c o n s t i t u e n t s t o f o r m dough t h a t  retain  and  produce bread of acceptable  P e n c e e t a l . (67) lated  better  reviewed  f u n c t i o n a l p r o t e i n system which i n t e r a c t s w i t h  gas  affords  itself will  l o a f volume.  showed t h a t c r u d e a l b u m e n f r a c t i o n  from water s o l u b l e s i s r e s p o n s i b l e  and  f o r the  iso-  larger loaf  volume. H a r r i s e t a l . (45) of  wheat f l o u r with  mentation with lipids  5% n o n f a t  f r o m wheat f l o u r  freshness  soy  Daftary  ment was  mainly  showed t h a t  dry milk.  e t a l . (25) due  supplementation  equivalent  Small  (70).  to  amounts o f  have i n d i c a t e d t h a t t h e Pomeranz e t a l .  a v a i l a b l e sucroesters' t o 16%  G l y c o l i p i d s i n which the  supplepolar and  Preliminary investiga-  to glycolipids.  commercially  that  s u b s t a n t i a l l y improve volume  d e l e t e r i o u s e f f e c t s up  volume.  f l o u r was  r e t e n t i o n of bread  t i o n s by  the  5%  illustrated  soy  (71)  counteract  b e a n f l o u r on  fatty  improve-  a c i d s are  loaf  directly  attached  t o the sugar moiety as i n s u c r o e s t e r s  were as  e f f e c t i v e improvers as g l y c o s y l g l y c e r i d e s i n r e s t o r i n g l o a f volume o f bread baked with s h o r t e n i n g defatted  flour.  from petroleum  ether  Comparison o f the e f f e c t o f s y n t h e t i c  g l y c o l i p i d s i n d i c a t e d t h a t both hydrophobic and hydrogen bonds are p l a y i n g important r o l e s i n bread'making. there  i s an o p t i m a l h y d r o p h i l e  - l i p o p h i l e balance  which s i n g l e l i p i d o r l i p i d mixtures i n c r e a s e  Possibly (6M-) at  l o a f volume  o f bread. Dairy products can e n t i r e l y o r p a r t l y p l a n t products i n d i f f e r e n t ways.  be r e p l a c e d by  For example i t i s the use  o f modern technology t o make from vegetable o i l s , that is'^very much l i k e b u t t e r .  margarine  I t i s f u r t h e r p o s s i b l e t o make  m i l k , i c e cream and cheese from milk o r p l a n t p r o t e i n and vegetable o i l s .  I t could  a l s o be p o s s i b l e t o r e p l a c e , i n due  c o u r s e , milk o r animal p r o t e i n with p l a n t p r o t e i n , and milk o r animal f a t with vegetable f a t ( 4 ) . We cannot overlook t h e o r i e n t a l technology f o r making food based on o i l seed p r o t e i n s . examples, soy m i l k , bean curd  To give a few  and fermented products have  been i n demand f o r many c e n t u r i e s . Pearson e t a l . (66) found that sodium soy p r o t e i n a t e t o be a poor e m u l s i f i e r i n the u s u a l pH range o f meat and suggested that i t probably does n o t serve any major function i n emulsifying  f a t when added t o sausage p r o d u c t s .  S w i f t e t a l . (89) s t a t e d t h a t the p a r t i c i p a t i o n o f water  soluble protein  i n emulsion- f o r m a t i o n  a d d i t i o n o f sodium c h l o r i d e . of  o il  e m u l s i f i e d per  increased  as  the  total  i s d e p e n d e n t on  They r e p o r t e d  m i l l i g r a m of  salt  the  t h a t the  soluble  amount  protein  amount o f p r o t e i n i n s o l u t i o n  was  decreased. S h e i k h e t a l . (80) centrates  f o r the  have  preparation  -  used rapeseed p r o t e i n  of enriched  bread containing  bread.  dietary  studies  on  skim milk  vitamin  mineral  mixtrue together with rapeseed or  p r o t e i n c o n c n e t r a t e s h a v e shown t h a t high  p r o t e i n , calcium  and  They showed t h a t t h e r e d i g e s t i o n of various With the  The  powder  was  only  little  and  other  bread  i r o n than unsupplemented  had bread.  differences  in  breads.  d e v e l o p m e n t o f new  c e r t a i n p r i n c i p l e s must be acceptability.  enriched  con-  There are  met  protein rich  to ensure  food,  commercial  s e v e r a l c r i t e r i a enumerated  B e h e r e t a l . (12)  and  concerning the  develop-  m e n t , 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  products  as  basic material  follows:  locally be  and  The not  fully  e c o n o m i c a l and  portation. influence w e l l as  Van  u t i l i z e d must be  exploited already.  s h o u l d have l o n g  T h e y must be and  Veen (94)  by  f r e e o f any  must h a v e an  The  p r o d u c t must  storage l i f e t o x i c or  a g r e e a b l e t a s t e and  available  for  trans-  deleterious odour,  other desirable physical c h a r a c t e r i s t i c s .  as  CHAPTER  VIII  EXPERIMENTAL MATERIALS AND  PROCEDURES  1. M a t e r i a l s A l l m a t e r i a l s f o r 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,  e n r i c h e d f l o u r produced by Robin Hood M u l t i f o o d L t d . , Montreal.  : Eggs were a g i f t  S c i e n c e , U.B.C., f r e s h l y  from the Department o f P o u l t r y  laid.  Corn o i l and coconut o i l . Margarine, Dalewood brand, manufactured Westminster  Foods L t d . , North Burnaby,  by  B.C.  Y e a s t , Fleischman a c t i v e dry y e a s t , Standard Brands L t d . , Vancouver. I n s t a n t skim m i l k powder, Seven Farms, W e s t f a i r Food L t d . , Vancouver. E m u l s i f i e r s , Atmul 124- and Atmos 300 by A t l a s Chemical I n d u s t r i e s .  manufactured  98.  2. .  Procedure  (  A.  Microloaves  Bread Shogren  e t a l .  preparations by v  15  g  2.5 *  min,  dough The  35°C  and  by  was  then  shape.  dough  by  were  a  wire  dough pans  baked were  mesh One  and  made  the loaf  was  5.2  at  x  210°C  removed  a  Atmul  and  with  plexiglas  cm  f o r 10  a  0.3  plate  rapeseed  min.  The  period.  dough  was  and molded  a t t h e bottom.  from  potassium  spatula f o r  f o r 60  min. i n an  g  and  this The  t o proof  immediately  salt f o r  5%  f o r 105  emulsifiers.  contain-  0 . 0 5 mg  35 ° C t h r o u g h o u t  2.3  g  added  protein  prepared  38°C  0.15  with  by m i x i n g  allowed  was  containing  o f  5%  124- ( a m o n o -  together  without on  brew  was  t o ferment  a t about  with  water,at  this  flour  formed  r o l l i n g  The mold  microloaves on  was  and allowed  were  38°C i n greased loaves  To  g  The  ml  flour)  t o t h e method  extract,  bath.  a n d 10  maintained  punched  malt  or concentrate  hand  controls  i n 6.7  emulsifier  mixture)  The  v  g  (10 gram,  modifications  f o r flour.  g  water  method  according  some  yeast  0.05  0.05  isolate  bromate.  g  sucrose,  diglyceride protein  (81) with  0.2 5  min. i n a  margarine  prepared  substituted  suspending  i n g 0.6  was  baking  t o  min. a t The  oven.  t h e pans  proofed  The and  baked cooled  tray. hour  after  baking  volume  was  measured  the microloaves by  dwarf  were  rapeseed  weighed  displace-  ment. In isolates  from  another 1  series  t o 10% was  o f experiments used  rapeseed  f o r the manufacture  protein o f  microloaves. 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 o n b r e a d m a k i n g was c o m p a r e d w i t h isolate.  followed loaves  In this B.  Crust  colour  determination  The  method d e s c r i b e d  f o rthe determination  The o u t e r  of crust colour.  size.  by  mechanically  in  Ten  milliliters  shaking  (dry weight)  2 5 m l o f 50% e t h a n o l stoppered Erlenmeyer  f o r4 hr. flasks.  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  f o r 15 m i n . a n d t h e a b s o r b a n c e o f t h e c l e a r  supernatant  s o l u t i o n d e t e r m i n e d a t 46 0 nm. from d u p l i c a t e  The m e a s u r e m e n t s a r e a v e r a g e s  extractions.  C.  Determination of the emulsifying capacity  The  method o f S w i f t et_ a l . ( 8 9 ) was f o l l o w e d f o r  determination  isolates the  The m i c r o -  a n d d r i e d f o r 24 h r .  Five hundred m i l l i g r a m o f t h i s with  the  ( 3 5 ) was  and ground i n a m o r t a r t o a f i n e  c r u s t was t h e n e x t r a c t e d  g  by E h l e and J a n s e n  one s i x t e e n t h o f a n i n c h o f c r u s t was  scraped o f f from t h e s l i c e s particle  protein  3 t o 5% s o y b e a n p r o t e i n i s o l a t e was u s e d .  1 h r . a f t e r b a k i n g were s l i c e d  a t 75°C.  rapeseed  of the emulsifying  and c o n c e n t r a t e s  isolates  capacity  from rapeseed f l o u r .  and c o n c e n t r a t e s  containing  of the protein Samples o f  100 mg i n 30 m l 1  N s o d i u m c h l o r i d e w e r e h e a t e d t o 80°C f o r 5 m i n . a n d b l e n d e d in  a n o m n i m i x e r f o r 2 m i n . a t c a . 100 0 r.p.m. ( S e e P l a t e  III)  100' m  To t h i s was and m i x i n g was  a d d e d 20 m l c o r n o i l and  f o r 30 s e c . a t c a . 13,000 r.p.m.  c o n t i n u e d a t t h e r a t e o f 0.8  p o i n t was  b e g i n h i g h speed  reached.  The  ml per  emulsion  cutting  A d d i t i o n of o i l  sec  till  the  break  f o r m e d p e r s i s t e d , and  finally  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 m a r k e d 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 s u d d e n d e c r e a s e oil  was,immediately  transition.  The  i n 'Viscosity.-  t e r m i n a t e d on o b s e r v a t i o n o f  t o t a l o i l a d d e d was  noted.  c a p a c i t y i s r e p o r t e d as t h e m l o i l p e r 100 or  Stability  Stability "» m e t h o d o f P e a r s o n c o r n o i l was  tests  e t 'al.  (66).  added a t t h e  of  abrupt  The e m u l s i f y i n g mg  protein  isolate  emulsions  The was  was  determination  the  optimum l e v e l o f o i l o b t a i n e d added t o t h e p r o t e i n  The  The  tubes  solution  emulsions  Determination of  the time  c e n t r i f u g e tubes  to the  recorded.  whippability  Whip t e s t s were p e r f o r m e d  as. d e s c r i b e d by  S i x t y gram o f f r e s h egg  w h i p p e d a t s p e e d 10  blend  formed were  were allowed to stand t i l l  p h a s e s w e r e s e p a r a t e d and  '. e t a l . -(AO) .  the  r a t e as f o r t h e e m u l s i f y i n g  t r a n s f e r r e d t o 15 m l c o n i c a l g r a d u a t e d  E~.  by  i n 3 0 m l o f 1 N s o d i u m c h l o r i d e and  1 3 , 0 0 0 r.p.m. f o r 2 m i n .  t h e 12 ml mark.  determined  For t h i s  s ame  from t h e above e x p e r i m e n t c o n t a i n i n g 10 0 mg  for  of the emulsions  capacity determination.  two  addition  concentrate. D.  at  The  Garibaldi  a l b u m e n a t 25°C  i n a Sunbeam m i x m a s t e r f o r 4 0  sec.  was (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 0.25 r a p e s e e d  speed t o 6 w i t h  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 adding 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  addition.  C o n t r o l s were c a r r i e d  i n t h e same way  without either protein isolates or concentrates incorporated into the sucrose. 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 weight on  a  from t h e  o f t h e m e r i n g u e i n a f u n n e l o f known v o l u m e 10 m l m e a s u r i n g  cylinder.  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 which expansion o f t h e whip.  sitting  i sthe  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 m i n . i n t e r v a l s . F.  Manufacture  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 a n d o r coconut  o i l were used  i n these experiments.  w e r e warmed t o room t e m p e r a t u r e c o n c e n t r a t e s from t h e s i n g l e t i o n were b l e n d e d  The s o l i d  fats  25°C a n d 0.5 g i s o l a t e s o r  stage sodium  hydroxide  extrac-  f o r 30 s e c . a n d h e a t e d t o 80°C f o r 5 m i n .  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  at ca.  13,000 r.p.m. f o r 45 s e c . a n d 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 w e r e p l a c e d i n p a p e r  cups  and w e r e 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 a n i c e a n d s a l t bath.  T h e s e w e r e 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 a n d t h e  consistency o f the product  noted.  PLATE IV EQUIPMENT FOR WHIP MEASUREMENT  - SUNBEAM  MIXMASTER  104.  Composition of blends: No. 1  Margarine  No. 2  Margarine  : corn o i l : coconut  No. 3  Margarine  : c o r n o i l (1:3 by wt)  No. 4  Margarine  : coconut  No. 5  Margarine  : c o r n o i l ( 1 : 1 by w t ) .  G.  o i l (2:0.5:0.5 by wt)  o i l ( 1 : 3 by w t )  E n r i c h e d i m i t a t i o n i c e c r e a m and i c e m i l k  T h e s e m i l k p r o d u c t s w e r e 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 s k i m m i l k p o w d e r a n d d i s s o l v i n g i n 9 0 ml water for to  a n d h e a t i n g t o 80°C f o r 5 m i n .  T h i s was  blended  30 s e c . a t c a . 1,000 r.p.m. i n an omni m i x e r a n d c o o l e d room temperature.  M a r g a r i n e , s u c r o s e , s p a n 20 a s  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  or corn  syrup  w e r e a d d e d a s 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. a n d 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 . b y h e a t i n g t o 80°C.  After pasteurization the  p r o d u c t s w e r e c o o l e d a n d 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 ice  and s a l t , w i t h c o n s t a n t  agitation.  TABLE X I I  COMPOSITION OF IMITATION I C E CREAM, CHOCOLATE ICE CREAM, AND I C E MILK. THE BASIC INGREDIENTS CONTAIN SKIM MILK POWDER AND PROTEIN ISOLATES FROM RAPESEED.  Composition  I c e Cream  Chocolate drink  Ice milk  Margarine (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  Vanilla  1.0  -  -  (drops)  Cocoa s y r u p (g)  -  15.0  -  CHAPTER I X RESULTS A.  Microloaves o f bread  W i t h t h e p r e l i m i n a r y e x p e r i m e n t s on t h e p r e p a r a - 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 v o l u m e was d e c r e a s e d by a p p r o x i m a t e l y 10 t o 1 5 % when 5% r a p e s e e d i s o l a t e s were s u b s t i t u t e d  f o rflour.  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% d e c r e a s e i n l o a f v o l u m e . e m u l s i f i e r s t h e l o a f volumes were r e s t o r e d The  protein  By u s i n g  for isolates.  c o n c e n t r a t e s d i d n o t show much i m p r o v e m e n t i n t h e l o a f  volume w i t h which  emulsifiers.  The b e s t e m u l s i f i e r was A t m u l  i n c r e a s e d t h e l o a f volume o f t h e b r e a d p r e p a r e d . As s e e n i n T a b l e X I I I , t h e b r e a d p r e p a r e d  5% r a p e s e e d p r o t e i n i s o l a t e s Atmul  124  containing  from  0.5% e m u l s i f i e r  1 2 4 , showed some i m p r o v e m e n t i n l o a f v o l u m e 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 h a d l o a f v o l u m e s h i g h e r by a p p r o x i m a t e l y 10 a n d 14% r e s p e c t i v e l y from sodium  over the c o n t r o l .  hydroxide extracts  i n t h e l o a f volume. cate experiments.  The  isolates  showed no s i g n i f i c a n t  A l l r e s u l t s were o b t a i n e d from When t h e r e s u l t s d i f f e r e d  experiment The  centrates with approximately  i s illustrated  dupli-  greatly  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 b r e a d in this  change  prepared  i n F i g u r e s 22 a n d 23.  b r e a d p r e p a r e d f r o m f l o u r c o n t a i n i n g 5% c o n 0.5% A t m u l  124 showed a d e c r e a s e o f  8% e x c e p t f o r t h e 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  P.W  56  b  0.04  P.A l P.B l P.S i Control  58  a  0.04  50  c  0.07  50  c  0.04  1  460 nm  0.025  51°  ' a ' j 'b' a n d ' c ' a r e t h r e e g r o u p s s i g n i f i c a n t l y d i f f e r e n t a s shown b y D u n c a n New M u l t i p l e Range T e s t . ( A p p e n d i x Table 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 CW  48  CA  4 7  CBQ  57  CS  47  Control  51*>  c  c a  c  (ml)  A b s o r b a n c e 460 nm 0.03 0.035 0.06 0.04 0.025  ' a ' , 'b' a n d ' c ' a r e t h r e e g r o u p s s i g n i f i c a n t l y d i f f e r e n t a s shown b y D u n c a n New M u l t i p l e Range T e s t . ( A p p e n d i x Table I I ) .  F i g u r e 22. protein  M i c r o - l o a v e s (10 g r a m s ) b a k e d isolates.  containing W - first  From l e f t  from  to right,  rapeseed  bread  the following: water e x t r a c t  isolate  P.W 1  A - second h y d r o c h l o r i c B - third  acid extract  sodium h y d r o x i d e e x t r a c t  S - s i n g l e s t a g e NaOH e x t r a c t C - the  control.  isolate  isolate  isolate  P^S  P^A P^B  GO  <  >  g u r e 23.  M i c r o - l o a v e s (.10 g r a m s ) b a k e d  protein isolates. of bread containing  From l e f t the  from rapeseed  to r i g h t , cut loaves  following:  W - f i r s t water extract  isolate  A - second h y d r o c h l o r i c  acid extract  B - third  sodium h y d r o x i d e e x t r a c t  S - s i n g l e s t a g e NaOH e x t r a c t C - the  P^W  control.  isolate  isolate  isolate  P^S  P^A P^B  CO  g u r e 24.  Microloaves  (10 g r a m s ) b a k e d f r o m  protein concentrates. containing  From l e f t  rapeseed  to right,  bread  the following:  W - f i r s t water extract  concentrate  A - second h y d r o c h l o r i c  acid extract concentrate  B - third  CW  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 - s i n g l e s t a g e NaOH e x t r a c t C - the c o n t r o l .  concentrate  CS  CB  CA  111.  Figure  25.  Microloaves  (10 g r a m s ) b a k e d f r o m  protein concentrates.  From l e f t  rapeseed  t o r i g h t , the cut  loaves bread containing the f o l l o w i n g : W - f i r s t water e x t r a c t concentrate  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 B - third S - single  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 t a g e NaOH e x t r a c t c o n c e n t r a t e  C - the control.  CS  CB  CA  DQ  112.  Figure  26.  M i c r o l o a v e s (10 g r a m s ) b a k e d f r o m t h e  stage sodium h y d r o x i d e e x t r a c t rapeseed  flour.  From l e f t  isolate  to right,  P^S  bread  from containing  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 , C - the  control.  single  and  gure  27.  M i c r o l o a v e s (10 g r a m s ) b a k e d f r o m t h e s i n  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 .rapeseed bread  flour.  P_^S  from  From l e f t . t o r i g h t , c u t l o a v e s  c o n t a i n i n g 1, 2, 4, 6, 8 and 10  p r o t e i n and C - t h e  control.  percent  CD  CO  O  114.  Figure  28.  protein  Microloaves isolate  (10 g r a m s ) b a k e d f r o m s o y b e a n  containing  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 b e l o w :  Emulsifier Bread N o . l 2 3 4 5 6  Atmul Atmos Atmul Atmos Atmul  12 4 300 12 4 300 124  Protein  3% 3% 5% 5% 5%  isolate  soybean p r o t e i n soybean p r o t e i n soybean p r o t e i n soybean p r o t e i n rapeseed p r o t e i n  115.  Figure  29.  protein  Microloaves  (10 gram) b a k e d f r o m  isolates containing  0.5% e m u l s i f i e r s .  composition of the bread i s given  Emulsifier Bread N o . l 2 3 4 5 6  Atmul Atmos Atmul Atmos Atmul  124 30 0 124 300 124  soybean The  below:  Protein isolate concentration 3% 3% 5% 5% 5%  soybean p r o t e i n soybean p r o t e i n soybean p r o t e i n soybean p r o t e i n rapeseed p r o t e i n  116. the.third 12%  s o d i u m h y d r o x i d e e x t r a c t w h i c h had  over t h a t of the  i n Figures  24  The t i o n on and  and  The  from the  28  of  the  and  29  5%  isolate  The  f o r the  crust colour  control.  was The  value  than t h a t of the  tabulated  and  l o a f volume.  was  slightly  The  better  isolate.  l o a f volume  i s o l a t e s and  higher  obtained  concentrates  sodium h y d r o x i d e than that  f o r the  concentrate  control.  bigger  isolate.  third  a little  hydroxide extract i s o l a t e  the  soybean p r o t e i n  protein  bread, except f o r the  a  e f f e c t of  a s l i g h t l y higher 3%  extract preparation f o r the  at  26  prepared.  the  a t 0.5%  bread containing  containing  bread  illustrate  e m u l s i f i e r Atmos 300  than the  was  observed  third  sodium  three  times  These r e s u l t s  are  i n Table X I I I . B.  Emulsifying  From t h e isolate  emulsifying 2 8%.  other  p r o t e i n f r o m s o y b e a n on  p r o t e i n i s o l a t e had  t h a t the  i s shown i n F i g u r e s  1% r a p e s e e d p r o t e i n  i n d i c a t e d t h a t A t m u l 124  greater  concentra-  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  than the 5%  crumb c o l o u r  bread containing  e m u l s i f i e r s on  The  illustrated  e f f e c t of rapeseed p r o t e i n i s o l a t e  Figures  of  25.  l o a f v o l u m e t h a n any  results  increase  c o n t r o l . . These r e s u l t s are  l o a f v o l u m e and  27.  an  This  capacity  r e s u l t s shown i n T a b l e XIV from the  capacity  first  than the  i t is  w a t e r e x t r a c t has  other  i s o a l t e s by  experiment suggests that the  water  evident a  higher  approximately  extract  117 .  TABLE X I V THE EMULSIFYING CAPACITY OF THE RAPESEED PROTEIN ISOLATES AND CONCENTRATES, EXPRESSED AS M I L L I L I T E R S CORN O I L PER 100 MILLIGRAM PROTEIN ISOLATES OR CONCENTRATE AND THE EMULSION S T A B I L I T Y I N MINUTES.  Volume o f o i l  (ml)  Stability  P.W  45.0  10  P. A° l  36.1  200  P.B l  32 . 3  8  P.S l  37.2  8  -  3  1  Control CW  33.1  7  CA  27.4  200  CB  25.2  7  CS  29 . 3  6  (min)  118. isolate  i s f a r s u p e r i o r to the  c a p a c i t y o f 45 m l lowest with  value  was  32 m l p e r The  corn  o i l per  w i t h the  100  mg  r e s t w i t h an 100  third  mg  emulsifying  protein isolate.  sodium h y d r o x i d e  isolate  protein.  concentrates  showed a s i m i l a r t r e n d b u t  a lower e m u l s i f y i n g capacity of approximately  25%  the  third  isolates.  Here a g a i n  hydroxide extract  t h e w e a k e s t was  Emulsion  stability  The  stability  of the  expected, except f o r i s o l a t e  e m u l s i o n s t o b r e a k was  and  concentrate The  of  sodium  u n d e r 10 m i n .  3 min.  The  not  from  f o r most w h i l e  i s o l a t e and  concentrate min.  as  the  time taken 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 t o o k o v e r 200 signs  than f o r  e m u l s i o n s f o r m e d was  second h y d r o c h l o r i c a c i d e x t r a c t .  only  the  with  concentrate.  C.  c o n t r o l was  The  the the from  t o show  any  separation. D.  Whippability  (a)  Expansion  The  ability  of the  isolates  t o whip the  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 i n c r e a s i n g the  s p e c i f i c v o l u m e by  l a t e s decreased the These r e s u l t s  are  When t h e determination,  the  10%  expansion a b i l i t y  tabulated  i n Table  concentrates specific  while by  egg  capable the  white of  other  approximately  iso16%.  XV.  were used f o r t h e  whip  volume d e c r e a s e d i n a l l s a m p l e s ,  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 h y d r o c h l o r i c a c i d and  single  the  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 was  s e e n f r o m T a b l e XV t h e d r i p f r o m t h e m e r i n g u e  negligible  for a l lisolates  and c o n c e n t r a t e s p r e p a r e d  from the t h r e e stage e x t r a c t i o n procedure d u r i n g the 30  min.  The  single  stage sodium  tions during this period up t o 9 0 m i n .  first  hydroxide extract prepara-  showed a d r i p o f 1.0  a l l p r e p a r a t i o n s showed 1.0  ml.  t o 3.5  With ml  drip,  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 , c o m p a r e d t o 4.5 d r i p i n the c o n t r o l . t r a t e s was  The  drip  o b t a i n e d from the  sodium h y d r o x i d e E.  The  be  and  in this  third  extract. spreads from  isolates  From p r e l i m i n a r y e x p e r i m e n t s c o n d u c t e d isolates  ml  concen-  h i g h e r than t h a t noted from the i s o l a t e s  time p e r i o d except f o r the c o n c e n t r a t e s from the  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  conveniently incorporated into butter like  the found  products  t h a t t h e s e c o u l d be u s e d as s p r e a d s w i t h h i g h p r o t e i n tents.  Among t h e  refrigerated  time  p r e p a r a t i o n s t h a t were s t o r e d  c o n d i t i o n s a t 4°C,  t h e s a m p l e No.  a f t e r 5 m o n t h s , w h i l e t h e s a m p l e No.  3 was  so con-  under  5 was  f i r m , and  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 .  to  soft the  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  or  TABLE XV. WHIP TEST OF RAPESEED PROTEIN ISOLATES AND CONCENTRATES, THE S P E C I F I C VOLUME AND DRIP OF MERINGUE WITH TIME.  Specific  volume  D r i p o f meringue 1.0 h r 0.5 h r  (ml) 1.5 h r  2.96  -  -  -  P.A°  ,3.74  -  0.6  1.0  P.B  2 . 81  0. 3  1.0  1.8  P.S  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  Control  3.40  1.0  2.1  4.5  P.W 1 1  1 l  121.  c o c o n u t o i l w i t h i s o l a t e s and spread ditions  emulsions  t h a t c a n be  f o r long periods of F.  Enriched  Enriched t u r e d a t 1 t o 2%  easily  s t o r e d under r e f r i g e r a t e d  con-  time.  imitation  i s o l a t e s without  l e v e l t h e r e was  i s o l a t e s , i n t o the  i c e c r e a m and  i m i t a t i o n milk products  f l a v o u r s i n t o the product. 5%  concentrates, to prepare  c o u l d be  i m p a r t i n g any  manufac-  undesirable  When t h e i s o l a t e s w e r e u s e d  a detectable off-flavour product.  ice milk  from  the  at  CHAPTER  X  DISCUSSION As  seen from t h e r e s u l t s o b t a i n e d  f r o m t h e manu-  f a c t u r e o f bread c o n t a i n i n g the rapeseed p r o t e i n i s o l a t e s from t h e f i r s t the  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 ,  l o a f v o l u m e was i n c r e a s e d by 10 t o 14%.  evident  Thus i t i s  that the water s o l u b l e fractions^ s p e c i a l l y the  a l b u m i n s a r e 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 . is  P^ ,  This  s i m i l a r t o what was o b s e r v e d by P e n c e 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 rthe hydrochloric acid extract  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  of the protein  fraction. 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  bread prepared hydroxide  with the concentrate  from t h e t h i r d  e x t r a c t , w h i c h was t h e o n l y  sodium  s a m p l e t h a t was  able  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 .  This  the  v o l u m e 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 content  may  h ave a l a r g e percentage o f g l y c o l i p i d s I t was o b s e r v e d  the  from Figures  26 a n d 2 7 , t h a t  a gradual  others.  isolate  I t i s also  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  decrease i n l o a f volume.  protein i s o l a t e the bigger The d a r k e n i n g  with  o v e r 6% c o l o r o f t h e b r e a d  t e n d s t o d a r k e n t o p r o d u c e a b r o w n i s h crumb. observed that with  which  and p h o s p h o l i p i d s .  increase i n concentration o f rapeseed p r o t e i n  from t h e s i n g l e sodium h y d r o x i d e ,  is  increase of  there  W i t h 1% r a p e s e e d  l o a f v o l u m e was o b t a i n e d ,  than  o f t he crumb may be due t o t h e  123.  b r o w n i n g r e a c t i o n s t a k i n g p l a c e b e t w e e n t h e amino a c i d s a n d sugars i n the bread. concentration  a darker  in the n u t r i t i o n a l Crust  Thus w i t h an i n c r e a s e  crumb i s p r o d u c e d c a u s i n g  colour results  showed t h a t t h e t h i r d  and c o n c e n t r a t e  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 in this preparation.  high  extract isolate lipids  t o produce  emulsifying capacity of the f i r s t  could  water  be due t o t h e p r e s e n c e o f p h o s p h o -  o r g l y c o l i p i d s w h i c h enhance t h e e m u l s i f y i n g  The h i g h  t he w a t e r e x t r a c t i s o l a t e  solubility  of this  capacity.  stability  except 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  w h i c h was e x t r e m e l y s t a b l e . of  acids  crust.  w a t e r e x t r a c t i s o l a t e P_^W h a d a g r e a t e r  others  contents  I t i s p o s s i b l e that browning r e a c t i o n  colour i n the bread The  absor-  glycolipids  or caramelization reaction taking place  a darker  sodium  had higher  e n h a n c e d by t h e t y p e o f c a r b o h y d r a t e a n d amino  present  The  a decrease  value.  hydroxide extract i s o l a t e  is  i n the i s o l a t e  emulsifying  than  isolate capacity  c o u l d be due t o t h e g r e a t e r  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 to give emulsion 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  isolate  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 effect. The  whip t e s t i n d i c a t e s t h e r e  d i f f e r e n c e among t h e i s o l a t e s .  i s a kind of  The h y d r o c h l o r i c  i s o l a t e P-A c a u s e d a 1 0 % i n c r e a s e  i n specific  acid  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  partly, to the  b a s i c nature of the p r o t e i n f r a c t i o n s present i n the The fic  c o n c e n t r a t e s were u n a b l e  volume o f t h e m e r i n g u e .  chloric acid  and  single  decreased  the s p e c i f i c  the water  e x t r a c t had The  sodium h y d r o x i d e e x t r a c t  a 19%  e x t r a c t c o n c e n t r a t e was  preparation.  a p p r o x i m a t e l y 2%,  hydro-  while  decrease.  e x t r a c t i s o l a t e and The  third  sodium  a b l e to decrease  This behaviour of the t h i r d  speci-  concentrates  d r i p i n m e r i n g u e c o u l d be d e c r e a s e d  hydrochloric acid isolate.  i s due,  the  Among t h e m t h e s e c o n d  v o l u m e by  a b l y by u s i n g t h e w a t e r  trate  t o improve  isolate.  by  78%  considerwith  hydroxide  t h e d r i p by  sodium h y d r o x i d e e x t r a c t  65%. concen-  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  the  CHAPTER GENERAL  XI  CONCLUSION  From t h e r e s u l t s o b s e r v e d it  w as c o n c l u d e d  from t h i s  investigation  t h a t by u s i n g t h e t h r e e s t a g e  procedure,  protein fractions rich  a c i d s such  as l y s i n e  and  i n certain essential  o f elegant chromatographic i s mainly  this  f r a c t i o n s , e s p e c i a l l y the water  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 , b e h a v e d i f f e r e n t l y a p p l i e d t o food p r o c e s s i n g such  as b r e a d  emulsion  ability  trate this  of  important.  These i n d i v i d u a l  f o r m a t i o n and w h i p p i n g  intriguing results  methods.  due t o t h e p r e s e n c e  l a r g e amounts o f c a l c i u m i s a l s o a n a d v a n t a g e , a s element i s n u t r i t i o n a l l y  amino  s u l f u r - c o n t a i n i n g amino a c i d s w e r e  s e p a r a t e d w i t h o u t t h e use A h i g h ash content which  extraction  from t h e t h i r d  when  manufacture, o f egg a l b u m e n . The  sodium h y d r o x i d 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 f r a c t i o n i n t h e most e c o n o m i c a l  concen-  i n order to u t i l i z e way i n t h e f o o d  pro-  cessing industry. From t h e r e s u l t s o b t a i n e d s o f a r , d i f f e r e n t protein isolates for  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  special-purposes i n different  stage of t h i s tions bakery  food products.  At 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  i n other areas  others  applica-  o f f o o d p r o c e s s i n g , s u c h a s meat o r  p r o d u c t s , c a n be a c c o m p l i s h e d  i n the future.  APPENDIX  127 . TABLE I ANALYSIS OF VARIANCE WITH RAPESEED  Source  **  of variation  OF LOAF VOLUME  PROTEIN  ISOLATES  Degree o f freedom  Mean s q u a r e  Replicates  5  24.32  Isolates  4  80.60  Error  20  0.46  Total  29  Significant  at probability  Duncan New M u l t i p l e  -  Range T e s t :  significantly different  from t h e i s o l a t e  f r o m t h a t o f P^W,  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 the  175.23**  0.01  Loaf volumes o f t h e bread p r e p a r e d P^A is  F-ratio  and t h e s e i n  o f t h e i s o l a t e s and  control.  Isolate  treatment  Mean v a l u e  Control 51. 33  P.W  P .A  l  X  56 . 12  58.00  P.B  P.S  i  l  50.33  50.17  128 . TABLE I I ANALYSIS OF VARIANCE OF LOAF VOLUME WITH RAPESEED PROTEIN  Source  **  of variation  CONCENTRATES  Degree o f freedom  Mean s q u a r e  Replicates  5  0.38  Concentrates  4  115.9 8  Error  20  1.35  Total  29  Significant  at p r o b a b i l i t y  Duncan New M u l t i p l e  Range T e s t :  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  the c o n t r o l the r e s t  from t h e rapeseed different  and t h e s e i n t u r n a r e d i f f e r e n t  o f the c o n c e n t r a t e s , which  Concentrate treatment Mean v a l u e  85.91**  0.01  Loaf volumes o f bread p r e p a r e d protein  F-Ratio  Control  CW  51.33  47.67  are  from t h a t o f  significantly  from  s i m i l a r t o one a n o t h e r  CA 46 .67  CB 57.00  CS 46 .83  129 .  LITERATURE CITED 1.  A l e x a n d e r , J.C. and H i l l , D.C, 1952 . The e f f e c t o f h e a t on l y s i n e and m e t h i o n i n e i n s u n f l o w e r o i l m e a l . J . N u t r i t i o n , 48: 149.  2.  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