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Functionality of plant proteins for comminuted meat systems Paulson, Allan Thomas 1985

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FUNCTIONALITY OF PLANT PROTEINS FOR COMMINUTED MEAT SYSTEMS  by A l l a n Thomas P a u l s o n B.Sc. ( A g r . ) The U n i v e r s i t y of B r i t i s h Columbia, 1973 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1978 f  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF GRADUATE STUDIES (Department  of Food S c i e n c e )  We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d  THE U N I V E R S I T Y OF BRITISH COLUMBIA October, 1985  ©  A l l a n Thomas P a u l s o n , 1985  In  presenting  degree  at  this  the  freely available copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for reference  this or  thesis  thesis by  this  for  his thesis  and study. scholarly  or for  her  Department of  Food Science  The University of British 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  Columbia  November 27, 1985  I further  purposes  gain  the  requirements  I agree  that  agree  may  representatives.  financial  permission.  of  It  shall not  be is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ii ABSTRACT  A three part study i s presented w h i c h examines the f u n c t i o n a l p r o p e r t i e s of p l a n t p r o t e i n s as t h e y r e l a t e to t e x t u r a l and s t a b i l i t y c h a r a c t e r i s t i c s of p r o t e i n - r e p l a c e d meat emulsions. In the f i r s t chapter, the e f f e c t s of cooking  time (25 or 50 min) and  temperature (70 or 95°C) on t e x t u r e , m i c r o s t r u c t u r e and cook s t a b i l i t y of a model meat emulsion system c o n t a i n i n g soy or canola p r o t e i n i s o l a t e were investigated.  The p l a n t p r o t e i n s were added e i t h e r d r y or r e h y d r a t e d at  replacement l e v e l s of 33.3% and 66.7% of the meat p r o t e i n .  Instrumental  t e x t u r e p r o f i l e a n a l y s i s and s t a b i l i t y data r e v e a l e d s e v e r a l complex i n t e r a c t i o n s between experimental v a r i a b l e s ; however, l e v e l of p r o t e i n replacement was predominant, w i t h decreased firmness and i n c r e a s e d y i e l d r e s u l t i n g from i n c r e a s e d replacement of meat p r o t e i n . T h e r m o r h e o l o g i c a l  p r o f i l e s of emul-  sions and p r o t e i n d i s p e r s i o n s demonstrated that the development of e l a s t i c i t y of all-meat emulsions d u r i n g h e a t i n g was e s s e n t i a l l y complete at 75-80°C, while the e l a s t i c i t y of canola or soy p r o t e i n d i s p e r s i o n s c o n t i n u e d w i t h h e a t i n g to 95°C. characteristics  to r i s e  A meat emulsion c o n t a i n i n g canola p r o t e i n d i s p l a y e d  of the all-meat  emulsion and canola  protein dispersion  t h e r m o p r o f i l e s , but the i n c r e a s e d s t r u c t u r e formation from the canola p r o t e i n at h i g h e r  heating  temperatures d i d not f u l l y compensate f o r an i n i t i a l  decrease i n e l a s t i c i t y that r e s u l t e d from the loss of meat p r o t e i n . A l t h o u g h there were s l i g h t d i f f e r e n c e s i n the f a t p a r t i c l e d i s t r i b u t i o n s of the emulsions c o n t a i n i n g p l a n t p r o t e i n , the d i s t r i b u t i o n s had s i m i l a r shapes, where p a r t i c l e s l a r g e r t h a n 50 micrometers approximated a normal d i s t r i b u t i o n , and were thought to be r e l a t i v e l y i n t a c t f a t c e l l s while the number of p a r t i c l e s w i t h diameters of 10-50 micrometers i n c r e a s e d  i n an  e s s e n t i a l l y l o g a r i t h m i c manner as s i z e d e c r e a s e d .  The m i c r o s t r u c t u r e of the  p r o t e i n a c e o u s m a t r i x was a f f e c t e d p r i m a r i l y b y p r o t e i n source, replacement l e v e l and c o o k i n g c o n d i t i o n s . In c h a p t e r 2, t h e r m a l l y i n d u c e d g e l a t i o n (72°C, 30 m i n h e a t i n g ) and emulsification  p r o p e r t i e s of u n m o d i f i e d  and succinylated  canola  protein  i s o l a t e (54% and 84% m o d i f i c a t i o n of free amino groups) were examined over a wide range of pH values (0.0-0.7M).  ( p H 3.5-11.0) a n d sodium c h l o r i d e  concentrations  S u c c i n y l a t i o n improved the g e l a t i o n a b i l i t y of canola  isolate.  For the unmodified i s o l a t e , gels formed at o n l y 4 of 18 combinations of pH and N a C l c o n c e n t r a t i o n , while 12 gels formed from each l e v e l of s u c c i n y l a t i o n under the same c o n d i t i o n s . o n l y i n the presence of N a C l .  Above pH 6.5, s u c c i n y l a t e d p r o t e i n formed gels In g e n e r a l , the f i r m e s t gels were o b t a i n e d  the moderate l e v e l of s u c c i n y l a t i o n . T r a n s l u c e n t  with  and opaque gels r e s p o n d e d  d i f f e r e n t l y to Theological t e s t s and were r e l a t e d i n d i f f e r e n t ways t o the physicochemical a n d Theological p r o p e r t i e s of p r o t e i n d i s p e r s i o n s . viscoelastic  properties  of the t r a n s l u c e n t  p r o t e i n s o l u b i l i t y and h y d r o p h o b i c i t y , r e l a t e d to s o l u b i l i t y , h y d r o p h o b i c i t y , of p r o t e i n d i s p e r s i o n s .  The  gels were a f f e c t e d m a i n l y b y  while those of the opaque gels were zeta p o t e n t i a l and apparent v i s c o s i t y  The types of bonds i n v o l v e d i n g e l formation and  s t a b i l i t y were t e n t a t i v e l y i d e n t i f i e d as h y d r o p h o b i c i n t e r a c t i o n s and h y d r o g e n bonds.  With the s u c c i n y l a t e d i s o l a t e s , gels were formed i n the presence of  calcium ions at a c o n c e n t r a t i o n an order of magnitude less t h a n was r e q u i r e d f o r s i m i l a r g e l s t r e n g t h s w i t h N a C l , w h i c h has i m p l i c a t i o n s f o r e x p l o i t i n g the g e l a t i o n a b i l i t y of s u c c i n y l a t e d p r o t e i n s i n p r o d u c t s where h i g h t r a t i o n s of N a C l are u n d e s i r a b l e .  concen-  iv B o t h e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y were i n c r e a s e d s u c c i n y l a t i o n , but e x h a u s t i v e s u c c i n y l a t i o n was not r e q u i r e d significant related  improvement i n these p r o p e r t i e s .  to protein  behavior of p r o t e i n  to produce a  E m u l s i f i c a t i o n a c t i v i t y was  s o l u b i l i t y , h y d r o p h o b i c i t y , zeta dispersions,  by  p o t e n t i a l and flow  while emulsion s t a b i l i t y appeared to be  m a i n l y a measure of r e s i s t a n c e to creaming and was r e l a t e d to p r o t e i n s o l u b i l i t y , zeta p o t e n t i a l , apparent v i s c o s i t y of p r o t e i n  dispersions,  and the  d i f f e r e n c e i n d e n s i t y between the aqueous and o i l phases. The t h i r d c h a p t e r examined the r e l a t i o n s h i p between t e x t u r a l measurements of c a n o l a i s o l a t e gels o b t a i n e d by means of a p u n c t u r e test w i t h an I n s t r o n t e s t e r , and fundamental r h e o l o g i c a l parameters o b t a i n e d from n o n d e s t r u c t i v e dynamic shear measurements w i t h a Weissenberg Rheogoniometer. A l t h o u g h the force required  to r u p t u r e the gels, as measured by the p u n c t u r e t e s t , was  p o o r l y c o r r e l a t e d w i t h the v i s c o e l a s t i c parameters, the slope of the f o r c e d e f o r m a t i o n c u r v e s to the point  of r u p t u r e was w e l l c o r r e l a t e d  storage and loss m o d u l i of the gels.  I n a d d i t i o n , the area under the f o r c e -  d e f o r m a t i o n c u r v e s to r u p t u r e f o l l o w e d loss tangent of the gels. two  w i t h the  a c u r v i l i n e a r r e l a t i o n s h i p with the  The response of t r a n s l u c e n t and opaque gels t o the  types of r h e o l o g i c a l t e s t s was not i d e n t i c a l , w h i c h i n d i c a t e d that g e l  microstructure  i s an i n f l u e n t i a l  factor  when e v a l u a t i n g  p r o p e r t i e s by d e s t r u c t i v e or n o n d e s t r u c t i v e methods.  gel rheological  TABLE OF CONTENTS Page ABSTRACT  i i  LIST OF T A B L E S  vii  LIST OF FIGURES  ix  ACKNOWLEDGEMENTS  xi  GENERAL INTRODUCTION Chapter 1.  1  MICROSTRUCTURE AND TEXTURE OF MEAT EMULSIONS SUPPLEMENTED WITH PLANT PROTEINS  INTRODUCTION  3  LITERATURE REVIEW  4  MATERIALS AND METHODS A. E x p e r i m e n t a l D e s i g n B. E m u l s i o n P r e p a r a t i o n C. Cook S t a b i l i t y D. T e x t u r e E. T h e r m o r h e o l o g i c a l S c a n n i n g F. L i g h t M i c r o s c o p y G. S c a n n i n g E l e c t r o n M i c r o s c o p y  7 7 7 8 8 9 11 13  RESULTS A. B. C. D.  14 14 16 20 21  AND DISCUSSION Texture Profile Analysis Thermorheological Scanning Cook S t a b i l i t y Microstructure  SUMMARY AND CONCLUSIONS  36  REFERENCES  38  Chapter 2.  GELATION AND EMULSIFICATION PROPERTIES OF UNMODIFIED AND SUCCINYLATED CANOLA PROTEIN ISOLATE  INTRODUCTION  42  LITERATURE REVIEW A. P r o t e i n S u c c i n y l a t i o n B. T h e r m a l l y Induced G e l a t i o n of G l o b u l a r P r o t e i n s C. P r o t e i n s as E m u l s i f i e r s  44 44 47 51  \  vi Page MATERIALS AND METHODS A. S u c c i n y l a t i o n P r o c e d u r e B. D e t e r m i n a t i o n of E x t e n t of S u c c i n y l a t i o n G. P r o t e i n S o l u b i l i t y D. P r o t e i n S u r f a c e H y d r o p h o b i c i t y E. Zeta P o t e n t i a l (Net Charge D e n s i t y ) F. Steady Shear Rheology G. T h e r m a l l y Induced G e l a t i o n 1. Dynamic Shear P r o p e r t i e s of T h e r m a l l y Induced Gels 2. P r o t e i n Content of G e l Exudate H. E m u l s i f i c a t i o n A c t i v i t y and E m u l s i o n S t a b i l i t y I. L i g h t M i c r o s c o p y J. S t a t i s t i c a l A n a l y s e s RESULTS A. B. C. D. E.  AND DISCUSSION Protein Solubility H y d r o p h o b i c i t y and Zeta P o t e n t i a l Steady Shear Rheology and M i c r o s t r u c t u r e T h e r m a l l y Induced G e l a t i o n E m u l s i f i c a t i o n A c t i v i t y and E m u l s i o n S t a b i l i t y  55 55 56 57 57 58 59 60 60 61 61 62 62 65 65 70 78 88 112  SUMMARY AND CONCLUSIONS  124  REFERENCES  127  C h a p t e r 3.  DYNAMIC SHEAR VERSUS PUNCTURE PROBE MEASUREMENTS OF GEL TEXTURE  INTRODUCTION  138  LITERATURE REVIEW  140  MATERIALS AND METHODS A. P u n c t u r e Test  143 143  B. Dynamic V i s c o e l a s t i c P r o p e r t i e s  145  RESULTS AND DISCUSSION  146  SUMMARY AND CONCLUSIONS  154  REFERENCES  155  L I S T OF  TABLES  Chapter 1. T a b l e 1.1.  I n s t r u m e n t a l t e x t u r e p r o f i l e a n a l y s i s of p r o t e i n - r e p l a c e d meat emulsions  Chapter 2. T a b l e 2.1.  O r t h o g o n a l m u l t i p l i e r s f o r t r e n d comparison analyses  T a b l e 2.2.  T r e n d comparison a n a l y s i s of the e f f e c t s of s u c c i n y l a t i o n , pH and N a C l on p r o t e i n s o l u b i l i t y of c a n o l a i s o l a t e  T a b l e 2.3.  T r e n d comparison analyses of the e f f e c t s of s u c c i n y l a t i o n , pH and N a C l on s u r f a c e h y d r o p h o b i c i t y ( S ) and zeta p o t e n t i a l of c a n o l a i s o l a t e Q  Table  2.4.  E f f e c t s of s u c c i n y l a t i o n , pH, N a C l and h e a t i n g on s u r f a c e h y d r o p h o b i c i t y of c a n o l a i s o l a t e  T a b l e 2.5.  Power-law and power-law p l a s t i c flow parameters of 11.4% c a n o l a i s o l a t e d i s p e r s i o n s  T a b l e 2.6.  T r e n d comparison analyses of the e f f e c t s of s u c c i n y l a t i o n , pH and N a C l on apparent v i s c o s i t y of 11.4% c a n o l a i s o l a t e d i s p e r s i o n s  Table  2.7A.  Dynamic shear flow behavior parameters of c a n o l a i s o l a t e gels  Table  2.7B.  Dynamic shear storage behavior parameters of c a n o l a i s o l a t e gels  T a b l e 2.8.  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n of v i s c o e l a s t i c parameters of t h e r m a l l y i n d u c e d c a n o l a i s o l a t e gels  T a b l e 2.9.  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n of v i s c o e l a s t i c parameters of t r a n s l u c e n t gels  Table  2.10.  P r o t e i n content of exudate from t h e r m a l l y i n d u c e d gels of c a n o l a p r o t e i n i s o l a t e  Table  2.11.  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n of v i s c o e l a s t i c parameters of opaque gels  Table  2.12.  T r e n d comparison analyses of the e f f e c t s of s u c c i n y l a t i o n , pH and N a C l on e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y  viii Page T a b l e 2.13.  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n of e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y  119  Rheogoniometer v s . I n s t r o n measurements of gel t e x t u r e  148  Chapter 3. T a b l e 3.1.  ix L I S T OF FIGURES Chapter 1. F i g u r e 1.1. F i g u r e 1.2.  F i g u r e 1.3. F i g u r e 1.4.  F i g u r e 1.5. F i g u r e 1.6. F i g u r e 1.7. F i g u r e 1.8. F i g u r e 1.9.  Page  T y p i c a l force-deformation curves for texture profile a n a l y s i s of meat emulsion samples  10  Storage m o d u l i as a f u n c t i o n of temperature of an a l l meat emulsion, a 66.7% c a n o l a p r o t e i n meat emulsion and a 15% c a n o l a i s o l a t e d i s p e r s i o n  17  Storage moduli of 15% soy i s o l a t e d i s p e r s i o n s as a f u n c t i o n of temperature  19  F a t p a r t i c l e d i s t r i b u t i o n s of meat emulsions c o n t a i n i n g soy or c a n o l a p r o t e i n ; A-C D-E  22 23  LM and SEM m i c r o g r a p h s of a 66.7% soy p r o t e i n meat emulsion cooked at 95°C f o r 50 min  24  LM and SEM m i c r o g r a p h s of a 66.7% c a n o l a p r o t e i n meat emulsion cooked at 95°C f o r 50 min  25  SEM m i c r o g r a p h s of 66.7% c a n o l a p r o t e i n meat emulsions  27  LM and SEM m i c r o g r a p h s of a 33.3% soy p r o t e i n meat emulsion cooked at 95°C f o r 25 min  29  LM and SEM m i c r o g r a p h s of a 33.3% c a n o l a p r o t e i n meat emulsion cooked at 95°C f o r 25 min  30  F i g u r e 1.10. SEM m i c r o g r a p h s of 33.3% c a n o l a p r o t e i n meat emulsions  31  F i g u r e 1.11. LM and SEM m i c r o g r a p h s of an a l l - m e a t emulsion cooked at 70°C f o r 25 min  33  F i g u r e 1.12. LM and SEM m i c r o g r a p h s of an a l l - m e a t emulsion cooked at 95°C f o r 50 min  35  Chapter 2. F i g u r e 2.1.  P r o t e i n s o l u b i l i t y of 11.4% c a n o l a i s o l a t e dispersions  67  F i g u r e 2.2.  S u r f a c e h y d r o p h o b i c i t y ( S ) of c a n o l a i s o l a t e  72  F i g u r e 2.3.  Zeta p o t e n t i a l of c a n o l a i s o l a t e  73  0  X  Page F i g u r e 2.4. F i g u r e 2.5. F i g u r e 2.6.  F i g u r e 2.7.  F i g u r e 2.8. F i g u r e 2.9. F i g u r e 2.10.  Apparent v i s c o s i t y at 10 s " l f o r 11.4% canola isolate dispersions  80  Apparent v i s c o s i t y at 1000 s isolate dispersions  81  - 1  f o r 11.4% canola  L i g h t m i c r o g r a p h s of 11.4% canola i s o l a t e d i s p e r s i o n s ; A-D E-H  85 86  Storage m o d u l i and dynamic v i s c o s i t i e s of 5.2% SA. pH 6.5, 0.7M N a C l c a n o l a i s o l a t e g e l as a f u n c t i o n of o s c i l l a t o r y frequency  90  Storage modulus at 10 s " i s o l a t e gels  93  Loss modulus at 10 s i s o l a t e gels  - 1  1  f o r 11.4% canola  f o r 11.4% canola 94  Loss tangent at 10 s"* f o r 11.4% canola isolate gels  F i g u r e 2.11. Storage and loss m o d u l i at 10 s " c a n o l a i s o l a t e gels F i g u r e 2.12. E m u l s i f i c a t i o n a c t i v i t y of c a n o l a dispersions  1  f o r 11.4% 98 isolate  F i g u r e 2.13. E m u l s i o n s t a b i l i t y of c a n o l a i s o l a t e d i s p e r s i o n s Chapter 3. F i g u r e 3.1. F i g u r e 3.2. F i g u r e 3.3. F i g u r e 3.4. F i g u r e 3.5.  95  114 117  F o r c e - d e f o r m a t i o n curve from I n s t r o n p u n c t u r e test of p r o t e i n gels  144  R u p t u r e f o r c e v s . storage modulus f o r canola i s o l a t e gels  147  R u p t u r e slope v s . storage modulus f o r canola i s o l a t e gels  147  R u p t u r e slope v s . loss modulus f o r canola i s o l a t e gels  150  R u p t u r e area v s . loss tangent f o r c a n o l a i s o l a t e gels  150  xi ACKNOWLEDGEMENTS I  would like to express ray sincere appreciation to ray supervisor,  Dr. Marvin A. Tung, for his support  and advice over the course of this  research project. The comments and suggestions of Dr. William D. Powrie and Dr. Shuryo Nakai of the Dept. of Food Science, and Dr. Jim Shelf ord of the Dept. of Animal Science are also appreciated. I would like to dedicate this thesis to my wife, Judy, for her understanding and patience during the time required to complete my graduate study. Financial support for these studies was provided by a grant from the Natural Sciences and Engineering Research Council of Canada.  1 GENERAL INTRODUCTION  The  f u n c t i o n a l behavior of p r o t e i n s i n food systems r e s u l t s from complex  i n t e r a c t i o n s among the composition, s t r u c t u r e and p h y s i c o c h e m i c a l of the p r o t e i n s per  properties  se, t h e i r i n t e r a c t i o n s w i t h other components such as  l i p i d s and c a r b o h y d r a t e s , and the nature of the environment i n w h i c h they are associated  ( K i n s e l l a , 1976,  1979;  references  l i s t e d at end  of Chapter 1).  T y p i c a l f u n c t i o n a l p r o p e r t i e s of food p r o t e i n s i n c l u d e such d i v e r s e phenomena as foaming, e m u l s i f i c a t i o n and their  measurement  gelation. Protein functional properties  have been e x t e n s i v e l y r e v i e w e d  (e.g., B r i s k e y ,  and 1970;  K i n s e l l a , 1976, 1979; Schoen, 1977). T h e r e i s c u r r e n t l y a t r e n d toward the use of non-meat p r o t e i n sources as r e p l a c e m e n t s and  e x t e n d e r s i n p r o d u c t s c o n t a i n i n g animal p r o t e i n .  In  a d d i t i o n to n u t r i t i o n a l v a l u e , these p r o t e i n s must possess f u n c t i o n a l p r o perties which maintain used.  or improve the q u a l i t y of foods i n w h i c h t h e y are  A p r o m i s i n g area of r e s e a r c h has been the i n c o r p o r a t i o n of non-meat  p r o t e i n s i n t o f r a n k f u r t e r - t y p e comminuted meat p r o d u c t s .  At  low  levels,  replacement p r o t e i n s have been suggested to act as e m u l s i f i e r s or serve  as  b i n d i n g agents p r e v e n t i n g water and f a t r e l e a s e d u r i n g p r o c e s s i n g (Smith et a l . , 1973). At h i g h l e v e l s , however, the r e d u c e d s t r u c t u r e - f o r m i n g p r o p e r t i e s of the replacement p r o t e i n s can l e a d to d e t r i m e n t a l e f f e c t s on p r o d u c t t e x t u r e (Comer, 1979; R a n d a l l et a l . , 1976). The  present  i n v e s t i g a t i o n examines the  f u n c t i o n a l behavior  of  plant  p r o t e i n s as t h e y r e l a t e to t e x t u r a l and s t a b i l i t y c h a r a c t e r i s t i c s of p r o t e i n r e p l a c e d meat emulsions. The s t u d y was d i v i d e d i n t o three p a r t s d e a l i n g w i t h the t e x t u r e and m i c r o s t r u c t u r e of a model meat emulsion system c o n t a i n i n g soy  2 or canola p r o t e i n i s o l a t e , the e f f e c t of s u c c i n y l a t i o n on the e m u l s i f i c a t i o n and t h e r m a l l y induced g e l a t i o n p r o p e r t i e s of canola p r o t e i n i s o l a t e , and the r e l a t i o n s h i p s between fundamental v i s c o e l a s t i c parameters of t h e r m a l l y induced canola p r o t e i n gels and t e x t u r e measurements o b t a i n e d w i t h a p u n c t u r e t e s t .  3  CHAPTER 1 TEXTURE AND MICROSTRUCTURE OF MEAT EMULSIONS SUPPLEMENTED WITH PLANT PROTEINS  INTRODUCTION The use of p l a n t p r o t e i n s as e x t e n d e r s or replacements f o r meat p r o t e i n i n f r a n k f u r t e r - t y p e comminuted meat p r o d u c t s has been the subject of much investigation.  A t h i g h l e v e l s of replacement, the t e x t u r e of these p r o d u c t s  u s u a l l y becomes soft and mushy.  Normal f r a n k f u r t e r p r o c e s s i n g  schedules  do not employ temperatures greater t h a n a p p r o x i m a t e l y 75°C a n d i t has been found that h i g h e r temperatures are o f t e n r e q u i r e d f o r the d e n a t u r a t i o n and g e l a t i o n of many g l o b u l a r p r o t e i n s (Catsimpoolas and Meyer, 1970; Hermansson, 1979).  The o b j e c t i v e s of t h i s r e s e a r c h were to i n v e s t i g a t e the e f f e c t s of  processing  c o n d i t i o n s on t e x t u r e , cook s t a b i l i t y and m i c r o s t r u c t u r e  of a  model meat emulsion system i n which l a r g e amounts of meat p r o t e i n have been r e p l a c e d w i t h soy or c a n o l a p r o t e i n i s o l a t e .  Soy p r o t e i n i s used i n a wide  v a r i e t y of foods i n c l u d i n g b a k e r y p r o d u c t s , c e r e a l s , d a i r y foods and comminuted meats ( K i n s e l l a , 1979).  Canola i s the major o i l s e e d c r o p grown i n  Canada, a n d c a n o l a p r o t e i n s have been found to possess good e m u l s i f i c a t i o n , water and f a t a b s o r p t i o n ,  and w h i p p i n g p r o p e r t i e s ( S o s u l s k i et a l . , 1976;  Thompson et a l . , 1982). A l t h o u g h these r e p o r t s have i n d i c a t e d poor g e l a t i o n p r o p e r t i e s of c a n o l a p r o t e i n , G i l l and T u n g (1978) found that the 12S f r a c t i o n of r a p e s e e d p r o t e i n underwent t h e r m a l l y i n d u c e d g e l a t i o n under a wide range of pH and i o n i c c o n d i t i o n s , but temperatures other t h a n 100°C were not t e s t e d .  4 LITERATURE REVIEW  F i n e l y comminuted meat p r o d u c t s such as f r a n k f u r t e r s or bologna are commonly p r e p a r e d b y c h o p p i n g l e a n meat i n a b r i n e s o l u t i o n to form a p r o t e i n a c e o u s s l u r r y i n w h i c h animal f a t i s t h e n f i n e l y d i v i d e d and d i s p e r s e d . The r e s u l t i n g b a t t e r , r e s e m b l i n g an o i l i n water emulsion (Hansen, 1960), i s s u b s e q u e n t l y cooked to form a p r o d u c t i n which the f a t g l o b u l e s are e n t r a p p e d w i t h i n a r i g i d p r o t e i n m a t r i x . P r e v i o u s work has documented the r o l e of the s a l t - s o l u b l e meat p r o t e i n s myosin and actomyosin i n emulsion f o r m a t i o n and s t a b i l i z a t i o n t h r o u g h c o a t i n g the f a t d r o p l e t s w i t h a stable membrane (Hansen, 1960; H e g a r t y et a l . , 1963; S w i f t et a l . , 1961).  Reviews by S a f f l e (1968) and  Webb (1974) c o n s i d e r e d e m u l s i f i c a t i o n to be the p r i m a r y f a c t o r r e s p o n s i b l e f o r the  s t a b i l i t y of such p r o d u c t s . Other workers have suggested that too much  emphasis has been p l a c e d on the importance of e m u l s i f i c a t i o n (van den Oord and V i s s e r , 1973). Theno and Schmidt (1978) examined the m i c r o s t r u c t u r e of t h r e e c o m m e r c i a l l y a c c e p t a b l e f r a n k f u r t e r s and found that o n l y one c o u l d be c a l l e d a t r u e meat emulsion. While these p r o d u c t s may not be emulsions i n the s t r i c t est sense, the term "meat e m u l s i o n " has been i n common use f o r many years and is r e t a i n e d i n the present study. A t t e n t i o n has s h i f t e d from the e m u l s i f i c a t i o n p r o p e r t i e s of meat p r o t e i n s to t h e i r involvement i n m a t r i x f o r m a t i o n t h r o u g h t h e r m a l l y i n d u c e d g e l a t i o n , w i t h the entrapment of f a t and the development of the c h a r a c t e r i s t i c t e x t u r e of the p r o d u c t . S e v e r a l r e c e n t r e v i e w s have examined t h i s aspect. Schmidt et a l . (1981) gave an overview of the p r o t e i n m a t r i x i n comminuted meat p r o d u c t s w i t h r e s p e c t to g e l a t i o n and v i s c o e l a s t i c p r o p e r t i e s of meat p r o t e i n s , b i n d i n g between chunks of meat, and e m u l s i f i c a t i o n .  A c t o n et a l . (1983) d i s c u s s e d  meat emulsions i n terms of p r o t e i n - w a t e r i n t e r a c t i o n , p r o t e i n - l i p i d a s s o c i a -  5 t i o n , p r o t e i n - p r o t e i n a g g r e g a t i o n , and t h e i r i n t e r r e l a t i o n s h i p s w i t h r e s p e c t to the environmental  c o n d i t i o n s d u r i n g comminution and thermal p r o c e s s i n g .  Schmidt (1984) p o i n t e d out the d i v e r s i t y and c o m p l e x i t y of comminuted meat p r o c e s s i n g , and emphasized the need f o r more r e s e a r c h on the e f f e c t s of the s p e c i e s of meat u t i l i z e d , d e s i r e d composition, degree of comminution, mechani c a l a c t i o n such as t u m b l i n g finished products.  or massaging, and  thermal  treatment  on  the  Z i e g l e r and A c t o n (1984) and Asghar et a l . (1985) d e t a i l e d  the d e n a t u r a t i o n , a g g r e g a t i o n and g e l a t i o n r e a c t i o n s of muscle p r o t e i n s .  Lee  (1985) surveyed the m i c r o s t r u c t u r a l aspects of meat emulsion formation  and  stabilization.  He  i n d i c a t e d that the m i c r o s t r u c t u r e of meat emulsions i s  i n f l u e n c e d by numerous f a c t o r s i n c l u d i n g type of meat, f a t and other i n g r e d i e n t s , l e v e l s of s a l t , moisture  and f a t , and the comminution process.  The  major changes to the m i c r o s t r u c t u r e of meat emulsions were i n the p a t t e r n s of fat d i s t r i b u t i o n , w h i c h r e f l e c t e d f a t s t a b i l i z a t i o n .  He r e v i e w e d the evidence  for the emulsion and nonemulsion t h e o r i e s of fat s t a b i l i z a t i o n and  concluded  that a l t h o u g h b o t h t h e o r i e s should be c o n s i d e r e d , from p h o t o m i c r o g r a p h i c  data  and p h y s i c a l a n a l y s i s , the nonemulsion t h e o r y should r e c e i v e more c o n s i d e r a tion. Several  authors  have attempted  to r e l a t e  f u n c t i o n a l p r o p e r t i e s of  non-meat p r o t e i n s to t h e i r performance as i n g r e d i e n t s i n comminuted meat products.  Thomas et a l . (1973) and  L a u c k (1975) suggested a r e l a t i o n s h i p  between apparent v i s c o s i t y of the f o r m u l a t i o n and c o o k i n g loss. (1975) and  Hermansson and  Akesson  (1975a,b) r e l a t e d moisture  Hermansson loss i n a  heated l e a n meat system c o n t a i n i n g non-meat p r o t e i n s to salt c o n c e n t r a t i o n , protein  type  proteins.  and  amount, s w e l l i n g , v i s c o s i t y and  T o r g e r s e n and  g e l a t i o n of the  added  Toledo (1977), w o r k i n g w i t h novel p r o t e i n s i n a  6 comminuted meat system, found a s i g n i f i c a n t negative protein  solubility  and f a t b i n d i n g ,  textural mechanical properties.  c o r r e l a t i o n between  as w e l l as s o l u b i l i t y at 100°C and  The more s o l u b l e p r o t e i n s allowed  increased  fat r e l e a s e on c o o k i n g and the cooked p r o d u c t s had lower m e c h a n i c a l s t r e n g t h . Comer  (1979) s t a t e d that the performance of f i l l e r s i n comminuted meat  p r o d u c t s was b e t t e r i n d i c a t e d by g e l a t i o n and water b i n d i n g t h a n e m u l s i f y i n g ability.  Cassens et a l . (1975) observed that t e x t u r e d soy f l o u r i n a f r a n k -  f u r t e r emulsion r e t a i n e d i t s morphology a f t e r p r o c e s s i n g and suggested that i t m e r e l y took up space w i t h i n the matrix.  P a u l s o n et a l . (1984) r e p o r t e d that  the s t a b i l i t y and firmness of model f r a n k f u r t e r s c o n t a i n i n g m o d i f i e d  plant  p r o t e i n s were i n f l u e n c e d m a i n l y by v a r i a b l e s r e l a t i n g p r o t e i n - l i p i d  inter-  actions  such  as f a t a b s o r p t i o n ,  p r o p e r t i e s of the added p r o t e i n s .  hydrophobicity,  and o i l e m u l s i f i c a t i o n  7 MATERIALS AND A.  METHODS  Experimental Design A model meat emulsion f o r m u l a t i o n c o n t a i n i n g 10.5% beef p r o t e i n , 29%  p o r k f a t , 57.5% water, and 2.5% sodium c h l o r i d e was used as a c o n t r o l .  Beef  p r o t e i n was r e p l a c e d w i t h e i t h e r soy or c a n o l a p r o t e i n i s o l a t e at e i t h e r 33.3 or 66.7% (w/w). L a u c k (1975) found that the h y d r a t i o n state of a whey p r o t e i n p r o d u c t i n f l u e n c e d the s t a b i l i t y of f r a n k f u r t e r s .  To test t h i s e f f e c t , the  i s o l a t e s were added e i t h e r d r y or r e h y d r a t e d o v e r n i g h t i n d i s t i l l e d water (3:1 (w/w), H 2 0 : i s o l a t e ) . 50 min.  The emulsions were t h e n cooked at 70 or 90°C f o r 25 or  The e f f e c t s of ( i ) replacement, ( i i ) p r o t e i n source, ( i i i ) p r e t r e a t -  ment, ( i v ) cook temperature and (v) cook time on t e x t u r e and cook s t a b i l i t y of the f i n i s h e d p r o d u c t s were t e s t e d u s i n g a 2 f r a c t i o n a l f a c t o r i a l e x p e r i m e n t a l 5  d e s i g n and a n a l y s i s of v a r i a n c e .  P r o d u c t m i c r o s t r u c t u r e was examined u s i n g  l i g h t m i c r o s c o p y (LM) and s c a n n i n g e l e c t r o n microscopy  B.  (SEM).  Emulsion Preparation B o n e l e s s beef c h u c k and pork b a c k f a t were p u r c h a s e d from a l o c a l ab-  b a t o i r , trimmed vacuum packaged  of v i s i b l e  f a t and  meat t r a c e s r e s p e c t i v e l y , minced  s e p a r a t e l y i n 450 g l o t s , t h e n f r o z e n at -35°C.  and  P r i o r to  use, beef and b a c k f a t were a l l o w e d to thaw at 4°C, t h e n kept on i c e when t a k e n from the coldroom. Soy p r o t e i n i s o l a t e (%N(d.b.)=14.56) and c a n o l a p r o t e i n i s o l a t e (%N(d.b.)=14.42) were p u r c h a s e d from the POS  Pilot Plant  Corp. (Saskatoon, S K ) . L a b o r a t o r y scale emulsion batches were p r e p a r e d w i t h a S o r v a l l Omnimixer ( I v a n S o r v a l l , Inc., Norwalk, C T ) .  The Omnimixer was m o d i f i e d to allow  the j a r to be moved up and down r e l a t i v e to the b l a d e s to give improved  8 c h o p p i n g of the e n t i r e sample  ( M o r r i s o n et a l . , 1971).  Ground beef, s a l t ,  d i s t i l l e d water and p l a n t p r o t e i n were b l e n d e d f o r 25 s at h a l f speed, pork b a c k f a t was added, and the emulsion formed by c h o p p i n g at f u l l speed f o r 2 x 30 s w i t h i n t e r m e d i a t e s c r a p i n g and hand m i x i n g . F i n a l emulsion temperatures a f t e r c h o p p i n g r a n g e d from 16-18°C. The emulsions were s t u f f e d into s t a i n l e s s s t e e l tubes (2.54 cm diam. x 10 cm l o n g ) , capped, t h e n cooked i n a water b a t h at 70 or 95°C f o r 25 or 50 min. W h i t i n g and M i l l e r (1984) e v a l u a t e d a l a b o r a t o r y b l e n d e r and a food processor f o r making model f r a n k f u r t e r emulsions and c o n c l u d e d t h a t , a l t h o u g h the f r a n k f u r t e r s were not i d e n t i c a l to those p r o d u c e d by commercial machines, the  d i f f e r e n c e s were not of a magnitude  that were c o n s i s t e n t l y d e t e c t e d .  Smaller scale machines have the advantages of b e i n g i n e x p e n s i v e , economical w i t h i n g r e d i e n t s , and t i m e - s a v i n g .  C.  Cook S t a b i l i t y The  cooked emulsions were c o o l e d i n i c e water f o r 10 min, removed  from the tubes and weighed.  Cook s t a b i l i t y was determined as the weight  r a t i o of the cooked to uncooked emulsions, e x p r e s s e d as a p e r c e n t a g e .  D.  Texture T e x t u r e of the cooked emulsions was e v a l u a t e d at room temperature by  an i n s t r u m e n t a l t e x t u r e p r o f i l e a n a l y s i s (TPA) u s i n g a Model 1122 I n s t r o n U n i v e r s a l T e s t i n g Machine ( I n s t r o n Corp., Canton, MA) (Bourne, 1968).  Bite  s i z e d c y l i n d e r s of cooked emulsions (12 mm l o n g , 23 mm diam.) were s u b j e c t e d to two c o n s e c u t i v e compressive strokes between f l a t p l a t e f i x t u r e s to 75% of t h e i r o r i g i n a l h e i g h t at a c r o s s h e a d speed of 100 mm/min and a c h a r t speed of  9 1000 mm/min. The r e s u l t i n g f o r c e - d e f o r m a t i o n c u r v e s were a n a l y z e d for rupture f o r c e , f i r s t b i t e hardness, second b i t e hardness, s p r i n g i n e s s and cohesiveness ( F i g u r e 1.1).  B.  Thermorheological Scanning The dynamic shear response to small amplitude s i n u s o i d a l o s c i l l a t i o n of  all-meat and 66.7% c a n o l a ( d r y ) s u b s t i t u t e d emulsions, and 15% (w/w)  disper-  sions of soy and c a n o l a i s o l a t e i n 3.5% s a l i n e was e v a l u a t e d as a f u n c t i o n of temperature w i t h a Model R.19 Weissenberg Rheogoniometer (Sangamo-Schlumberger L t d . , Bognor R e g i s , U.K.) (1984).  by a method s i m i l a r to that of B e v e r i d g e et a l .  The Rheogoniometer was equipped w i t h a m o d i f i e d F e r r a n t i - S h i r l e y  lower p l a t e n and a 7.5 cm f l a t upper p l a t e n supported by a no. 8 t o r s i o n bar (87.5 Pa cm3  fim'l).  The  lower p l a t e n was connected to a Haake Model FP  c i r c u l a t i n g h e a t e d - r e f r i g e r a t e d b a t h (Haake Inc., Saddle Brook, NJ) w i t h a Haake PG 11 temperature programmer and a Haake DK 51 r e f r i g e r a t e d b a t h .  The  sample was p l a c e d i n the c e n t e r of the lower p l a t e n and the upper p l a t e n was lowered to a gap t h i c k n e s s of 1.5 mm.  To prevent d r y i n g of the sample at the  edges, a masking tape dike was formed around the c i r c u m f e r e n c e of the lower p l a t e n and p a r a f f i n o i l was sample.  i n t r o d u c e d to a depth s u f f i c i e n t to cover the  O s c i l l a t i o n was at a constant f r e q u e n c y of 5.96 s ~ l w i t h a maximum  s t r a i n amplitude of 0.875%, which elasticity  was  w i t h i n the range  f o r samples r e p r e s e n t a t i v e of those t e s t e d .  of l i n e a r The  visco-  sample  was  o s c i l l a t e d f o r 10 min to e q u i l i b r a t e the sample temperature p r i o r to h e a t i n g . The temperature p r o f i l e w i t h i n the gap was measured w i t h a c o p p e r - c o n s t a n t a n thermocouple embedded i n the approximate c e n t e r of the bottom p l a t e n so that the thermocouple  t i p was  f l u s h w i t h the p l a t e n s u r f a c e .  M i l l i v o l t signals  10  _  First  r,  bite  _ •^m.  Seconds U  « . . .  force  y  Distance  _  Cohesiveness =  HI = H a r d n e s s Rupture  bite  A1  Springiness  /  77  =  D2  /  H2  AI 0  r*-D2—H  F i g u r e 1.1. T y p i c a l f o r c e - d e f o r m a t i o n c u r v e s f o r t e x t u r e p r o f i l e a n a l y s i s of meat emulsion samples.  11 were r e c o r d e d w i t h a D i g i t e c Model 1268 d a t a l o g g e r ( U n i t e d Systems Corp., Dayton, OH) and c o n v e r t e d to temperature. The samples were heated from 20°C to e i t h e r 70°C or 95°C at a r a t e of 2 C°/min. When the d e s i r e d temperature was r e a c h e d , the c o o l i n g b a t h was a c t i v a t e d and the sample was c o o l e d at 2 C°/min back to 20°C. The d e l a y between h e a t i n g and c o o l i n g caused a temperature overshoot of 2-3 C°. Thermal e x p a n s i o n of the metal p l a t e n s n e c e s s i t a t e d adjustment of the gap every 5 or 10 C°; the extent of adjustment was determined by p r e l i m i n a r y e x p e r i m e n t a t i o n . O s c i l l a t o r y i n p u t was c o n t i n u o u s throughout the e x p e r i m e n t a l p e r i o d and measurements were t a k e n e v e r y 5 or 10 C°. The amplitudes of the i n p u t ( s t r a i n ) and output ( s t r e s s ) v o l t a g e s i g n a l s , and the phase d i f f e r e n c e between them, were monitored w i t h a T r o n o t e c Model 703A d i g i t a l phasemeter ( T r o n o t e c Inc., F r a n k l i n , N J ) , and dynamic r h e o l o g i c a l parameters were c a l c u l a t e d from the equations of Walters (1968). The storage modulus (G ) i s a measure of the 1  energy s t o r e d e l a s t i c a l l y i n the sample, the loss modulus (G") i s a measure of the energy d i s s i p a t e d as heat, and the loss tangent ( n u m e r i c a l l y equal to G"/G') r e f l e c t s the r e l a t i v e p r o p o r t i o n of v i s c o u s to e l a s t i c c h a r a c t e r i n the sample. and  As a m a t e r i a l undergoes g e l a t i o n , i t becomes more e l a s t i c i n nature  the storage modulus w i l l  decrease.  i n c r e a s e while the v a l u e s of loss  tangent  F o r these experiments, the e f f e c t of temperature on the storage  modulus of the samples was monitored.  F.  Light Microscopy U n f i x e d p i e c e s of sample were f r o z e n i n l i q u i d n i t r o g e n , a l l o w e d to  e q u i l i b r a t e to -25°C i n a c r y o s t a t microtome, ness of 14 micrometers.  t h e n s e c t i o n e d to a t h i c k -  The s e c t i o n s were a f f i x e d to glass s l i d e s w i t h Mayers  12 glycerol  albumen adhesive p r i o r  to s t a i n i n g .  For l i p i d s , sections  were  immersed i n 100% p r o p y l e n e g l y c o l f o r 6 min, s t a i n e d i n 0.5% Sudan B l a c k B i n p r o p y l e n e g l y c o l f o r 10 min t h e n d i f f e r e n t i a t e d i n 85% p r o p l y e n e g l y c o l f o r 6 min and 50% propylene g l y c o l f o r 2 min. These s e c t i o n s were not c o u n t e r s t a i n e d f o r p r o t e i n as f i n e d e t a i l tended to be o b s c u r e d .  To examine the  p r o t e i n m a t r i x i n d e p e n d e n t l y , separate s e c t i o n s were s t a i n e d w i t h 0.17% light  green  dehydrated Attempts  i n 0.33% a c e t i c w i t h 90%, then  acid  f o r 1 min, r i n s e d  in distilled  100% e t h a n o l , t h e n d e l i p i d a t e d  water,  with xylene.  to d i s t i n g u i s h between p l a n t and meat p r o t e i n s by d i f f e r e n t i a l  s t a i n i n g were u n s u c c e s s f u l . To observe b o t h l i p i d and p r o t e i n s i m u l t a n e o u s l y , the l i p i d was s t a i n e d w i t h 0.5% Sudan IV i n p r o p y l e n e g l y c o l f o r 15 min and c o u n t e r s t a i n e d w i t h l i g h t green f o r 30 sec. The samples were examined and photographed under b r i g h t f i e l d i l l u m i n a t i o n w i t h 5X and 10X o b j e c t i v e s , u s i n g a W i l d M20 microscope and a P e n t a x 35 mm Fat  camera.  p a r t i c l e d i s t r i b u t i o n s of the emulsions c o n t a i n i n g p l a n t p r o t e i n s  heated at 95°C were o b t a i n e d from 18 cm x 24 cm l i g h t m i c r o g r a p h s of s e c t i o n s s t a i n e d f o r l i p i d ( a c t u a l specimen area = 1.89 x 10 square m i c r o m e t e r s ) . 6  One  m i c r o g r a p h was used f o r each treatment. E a c h m i c r o g r a p h was p a r t i t i o n e d into 48 squares (3 cm x 3 cm) to f a c i l i t a t e c o u n t i n g . As not a l l f a t p a r t i c l e s were s p h e r i c a l , the e q u i v a l e n t area diameter of the p a r t i c l e s (the diameter of a circle  h a v i n g the same area as the p a r t i c l e ; F i s c h m e i s t e r , 1968) were  o b t a i n e d u s i n g a c i r c l e template, and c l a s s e d into 32 s i z e c a t e g o r i e s between 10 and 126 micrometers i n diameter.  Those f a t p a r t i c l e s w i t h diameters of  34-126 micrometers (23 s i z e c a t e g o r i e s ) were counted i n a l l 48 squares of each micrograph.  P a r t i c l e s w i t h diameters of 24-34 micrometers (4 s i z e c a t e g o r i e s )  were counted i n 36 squares s e l e c t e d at random, and the counts were p r o j e c t e d  13 to  a n estimated count f o r t h e e n t i r e f i e l d .  Similarly, fat particles with  diameters of 18-24 micrometers (2 s i z e c a t e g o r i e s ) were c o u n t e d i n 14 randomly s e l e c t e d squares, while p a r t i c l e s of 10-18 micrometers i n diameter (3 s i z e c a t e g o r i e s ) were counted i n 5 squares. The i n d i v i d u a l f a t p a r t i c l e d i s t r i b u t i o n s were t h e n combined and averaged to give t h e f o l l o w i n g c o n t r a s t s : (1) 66.7%  soy vs. 66.7% c a n o l a p r o t e i n  substitution,  (2) 33.3% soy vs. 33.3%  c a n o l a , (3) 33.3% soy vs. 66.7% soy, (4) 33.3% c a n o l a vs. 66.7% c a n o l a , (5) soy vs. c a n o l a , and (6) 33.3% vs. 66.7% s u b s t i t u t i o n . distribution samples.  T h u s each f a t p a r t i c l e  i n t h e c o n t r a s t s was the mean of e i t h e r 2 or 4 i n d i v i d u a l  No s t a t i s t i c a l comparative p r o c e d u r e s were performed due to the  c o m p l e x i t y of the d i s t r i b u t i o n s .  G.  Scanning Electron Microscopy Small cubes of cooked emulsions a p p r o x i m a t e l y (4 mm)3 were c r y o f r a c t u r e d  in liquid nitrogen.  Small fragments a p p r o x i m a t e l y (1-1.5 mm)3 were f i x e d i n  4% g l u t e r a l d e h y d e i n 0.07 M phosphate b u f f e r (pH 7) f o r 12-24 h at 4°C. A f t e r r i n s i n g t h r e e times i n phosphate b u f f e r , secondary f i x a t i o n was accomplished w i t h 1% osmium t e t r o x i d e i n phosphate b u f f e r f o r 4 h. A f t e r a second set of phosphate b u f f e r r i n s e s , the samples were d e h y d r a t e d t h r o u g h a graded e t h a n o l s e r i e s f o l l o w e d by exchange of e t h a n o l w i t h a graded s e r i e s of amyl acetate i n 100% e t h a n o l , t h e n 100% amyl acetate f o r 1 h. The samples were d r i e d i n a P a r r c r i t i c a l p o i n t d r y i n g bomb ( P a r r Instrument Co., M o l i n e , I L ) u s i n g l i q u i d C02, mounted on aluminum s t u b s , g o l d coated i n a T e c h n i c s s p u t t e r c o a t i n g u n i t ( T e c h n i c s Inc., A l e x a n d r i a , V A ) , and observed w i t h a Cambridge Stereoscan 250 SEM (Cambridge Instruments (Canada) Inc., M o n t r e a l , PQ) at an a c c e l e r a t i n g v o l t a g e of 20 kV.  14 RESULTS AND A.  DISCUSSION  Texture Profile Analysis A n a l y s i s of v a r i a n c e of the TPA  data r e v e a l e d a complex i n t e r a c t i o n  between e x p e r i m e n t a l f a c t o r s and t e x t u r e p r o f i l e components, although replacement l e v e l was  found to be predominant  o v e r a l l ( T a b l e 1.1).  For rupture  f o r c e , replacement l e v e l was the o n l y s i g n i f i c a n t main e f f e c t (p<0.01) w i t h d e c r e a s e d r u p t u r e f o r c e v a l u e s w i t h i n c r e a s e d replacement l e v e l .  An i n t e r -  a c t i o n was also found between replacement l e v e l and cook temperature (p<0.05); at 66.7% replacement of meat p r o t e i n , the 95°C cook p r o d u c e d h i g h e r r u p t u r e f o r c e v a l u e s t h a n at 70°C, w h i l e at 33.3% replacement the o p p o s i t e was t r u e . F o r f i r s t b i t e hardness and  second b i t e hardness, replacement  level  was a g a i n s i g n i f i c a n t (p<0.01) w i t h d e c r e a s e d hardness v a l u e s at i n c r e a s e d substitution.  Canola p r o t e i n i s o l a t e p r o d u c e d s i g n i f i c a n t l y g r e a t e r hardness  v a l u e s t h a n soy p r o t e i n i s o l a t e (p<0.01).  An i n t e r a c t i o n was found between  cook temperature and pretreatment (p<0.05) w i t h g r e a t e r hardness v a l u e s at 70°C i f the i s o l a t e s were r e h y d r a t e d p r i o r to a d d i t i o n , whereas the 95°C cook p r o d u c e d the o p p o s i t e e f f e c t . S p r i n g i n e s s , e x p r e s s e d as p e r c e n t r e c o v e r y from the o r i g i n a l deformat i o n , was  influenced  by  two  f a c t o r s ; replacement  level  (p<0.01),  where  g r e a t e r s p r i n g i n e s s was found at the 33.3% replacement l e v e l t h a n the 66.7% l e v e l , and cook temperature (p<0.01), where a 95°C cook p r o d u c e d s p r i n g i e r p r o d u c t s t h a n at 70°C. For the c o n t r o l emulsions, cook time appeared to be more important f o r s p r i n g i n e s s t h a n temperature, w i t h a 50 min cook p r o d u c i n g g r e a t e r s p r i n g i n e s s t h a n 25 min, a l t h o u g h s l i g h t l y g r e a t e r s p r i n g i n e s s produced  at 70°C t h a n 95°C.  by any of the f a c t o r s under  Cohesiveness was  investigation.  not a f f e c t e d  was  significantly  15  T a b l e 1 . 1 . I n s t r u m e n t a l t e x t u r e p r o f i l e a n a l y s i s o f p r o t e i n - r e p l a c e d meat eaulslons.  Significant Experimental Factors  Texture Profile Component  33.3% 37.0 + 2.3  66.7% 16.7 + 1.9  33.32 70 C:38.6 + 1.9 95°C:35.3 + 1.2  66.7% 15.4 + 1.6 18.0 + 1.1  33.3% 48.3 + 2.7  66.7% 31.4 +4.1  Soy 37.8 + 10.1  Canola 42.0 + 8.7  Dry 70°C:37.3 + 11.6 95°C:42.4 + 10.4  Rehydrated 40.5 + 10.9 39.4 + 7.9  33.3% 36.6 + 8.1  66.7% 22.9 + 3.1  Soy 28.1 + 8.1  Canola 31.4 + 7.1  Dry 70 C:27.7 + 8.6 95°C:31.8 + 8.6  Rehydrated 30.0 + 9.0 29.5 + 6.6  (i) Replacement Level**  Rupture Force (N)  Control: 62.0 + 3.5 ( i i ) Level x Cook Temp.*  U  First Bite Hardness (N)  (i) Replacement Level** Control: 63.8 + 2.2 ( i i ) Protein Source** ( i i i ) Pretreatment x Cook Temperature*  Second Bite Hardness (N)  (i) Replacement Level** Control: 50.A + 2.5 ( i i ) Protein Source** ( i i i ) Pretreatment x Cook Temperature*  Springiness (%)  (i) Replacement Level** Control: 57.1 + 5.6 ( i i ) Cook temperature**  * p<0.05 ** p<0.01  U  33.3%  51.5 + 5.3 70°C  66.7% 45.8 + 7.2  95°C  16 Patana-Anake  and F o e g e d i n g (1985) also found s i g n i f i c a n t i n t e r a c t i o n s  between h e a t i n g temperature and time of c o o k i n g f o r s t a b i l i t y and t e x t u r a l c h a r a c t e r i s t i c s of meat b a t t e r s c o n t a i n i n g soy p r o t e i n or v i t a l wheat g l u t e n .  B.  Thermorheological Scanning The e f f e c t s of h e a t i n g from 20°C to 95°C on the storage modulus (G ) of 1  an a l l - m e a t emulsion, a 66.7% replacement canola/meat emulsion, and a 15% canola i s o l a t e d i s p e r s i o n are shown i n F i g u r e 1.2. H e a t i n g the a l l - m e a t emulsion to 60°C caused a decrease i n G', but from 60 to 75°C, G' i n c r e a s e d r a p i d l y w i t h little  change between 75 and 95°C.  Upon c o o l i n g to 25°C, G  l o g a r i t h m i c a l l y f o l l o w e d by a sharp r i s e i n G  1  1  increased  w i t h f u r t h e r c o o l i n g to 20°C,  p r o b a b l y as a r e s u l t of s o l i d i f i c a t i o n of the pork fat g l o b u l e s i n the d i s p e r s e d phase of the meat emulsion. D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y (DSC) of the pork b a c k f a t used i n the meat emulsions r e v e a l e d s e v e r a l endothermic  peaks  between 15°C and 50°C, but over 50% of the f a t was melted at 30°C and 75% was melted at 35°C.  Townsend et a l . (1968) and Quinn et a l . (1980) r e p o r t e d  s i m i l a r thermograms f o r p o r k f a t .  Quinn et a l . (1980) also found that the  c h a r a c t e r i s t i c DSC t h e r m o p r o f i l e f o r muscle p r o t e i n s was altered by comminuting w i t h s a l t , r e s u l t i n g i n a s i n g l e endothermic peak w i t h an onset of a p p r o x i m a t e l y 60°C and a maximum at 72°C. These c o r r e s p o n d v e r y c l o s e l y to the temperatures at w h i c h G  1  began to i n c r e a s e (60°C) and t h e n l e v e l e d o f f (75°C). T h u s the  p r o t e i n d e n a t u r a t i o n r e a c t i o n s observed b y DSC  a n a l y s i s appeared  to be  f o l l o w e d v e r y c l o s e l y by a g g r e g a t i o n and g e l a t i o n r e a c t i o n s i n v o l v e d i n the f o r m a t i o n of a s t r o n g c o h e s i v e meat emulsion m a t r i x . The normal p r o c e s s i n g temperature f o r comminuted meat p r o d u c t s ( a p p r o x i m a t e l y 70-75°C) corresponds c l o s e l y to the temperature at w h i c h G  1  no l o n g e r i n c r e a s e d .  Figure 1.2.  Storage moduli as a f u n c t i o n of temperature of an a l l - m e a t emulsion, a 66.7% canola p r o t e i n meat emulsion and a 15% canola isolate d i s p e r s i o n : open symbols i n d i c a t e h e a t i n g , solid symbols i n d i c a t e c o o l i n g . H  18 The meat emulsion i n w h i c h 66.7% of the meat p r o t e i n was r e p l a c e d by c a n ola  protein isolate followed a similar pattern for G  was r e a c h e d at 75°C and G structure formation.  f  1  except that no p l a t e a u  c o n t i n u e d to r i s e to 95°C i n d i c a t i n g  Upon c o o l i n g , t h e r e was l i t t l e change i n G  1  continued  between 95°C  and 60°C, f o l l o w e d b y an i n c r e a s e i n G as c o o l i n g c o n t i n u e d to 20°C. 1  The  storage modulus of a 15% c a n o l a i s o l a t e d i s p e r s i o n d e c r e a s e d w i t h  h e a t i n g to 60°C f o l l o w e d by a steady i n c r e a s e i n G' w i t h f u r t h e r h e a t i n g to 95°C. Upon c o o l i n g , G i n c r e a s e d s t e a d i l y t h r o u g h o u t the e n t i r e tempera1  t u r e range.  As t h i s sample was not a f a t - c o n t a i n i n g emulsion, however, the  t h e r m o r h e o l o g i c a l p r o f i l e s h o u l d be used f o r q u a l i t a t i v e comparisons only. Comparing the three t h e r m o p r o f i l e s , i t i s apparent that replacement of meat p r o t e i n w i t h c a n o l a p r o t e i n p r o d u c e d a meat emulsion that was i n i t i a l l y l e s s e l a s t i c t h a n an all-meat emulsion, and upon h e a t i n g d i s p l a y e d c h a r a c t e r i s t i c s of both the all-meat and a l l - c a n o l a samples.  Thus, u n l i k e the  all-meat emulsion, G c o n t i n u e d to r i s e w i t h h e a t i n g from 75°C to 95°C but at 1  a slower r a t e t h a n between 60°C and 75°C. The i n c r e a s e d s t r u c t u r e formation at the h i g h e r h e a t i n g temperatures was not enough, however, to compensate f o r the e f f e c t s of the l o s s of meat p r o t e i n . The  dynamic shear a n a l y s i s of h e a t i n g 15% soy i s o l a t e d i s p e r s i o n s to  95°C i s shown i n F i g u r e 1.3. Storage modulus d e c r e a s e d s t e a d i l y w i t h i n i t i a l h e a t i n g , b u t at a slower r a t e t h a n f o r the 15% canola i s o l a t e d i s p e r s i o n . U n l i k e c a n o l a i s o l a t e , G c o n t i n u e d to decrease from 60°C to 70°C f o l l o w e d by 1  an  i n c r e a s e i n G' w i t h f u r t h e r h e a t i n g to 95°C.  DSC thermograms of soy  i s o l a t e d i s p e r s i o n s r e v e a l e d two major endotherms w i t h t r a n s i t i o n temperatures of a p p r o x i m a t e l y  67°C and 90°C and maxima at 80°C and 98°C, r e s p e c t i v e l y .  These c o r r e s p o n d  to d e n a t u r a t i o n of the 7S and 11S f r a c t i o n s i n that order  F i g u r e 1.3.  Storage moduli of 15% soy isolate dispersions as a f u n c t i o n of t e m p e r a t u r e : symbols i n d i c a t e h e a t i n g , s o l i d symbols i n d i c a t e c o o l i n g .  open — (• VD  20 (Hermansson, 1978).  L i k e the a l l - m e a t emulsion, the development  of G  1  with  h e a t i n g of the soy i s o l a t e d i s p e r s i o n appeared to be r e l a t e d to the t h e r m a l t r a n s i t i o n s found w i t h DSC.  Montejano et a l . (1984) also r e p o r t e d that major  t r a n s i t i o n s i n r i g i d i t y w i t h h e a t i n g of s o l pastes p r e p a r e d from comminuted f i s h , beef, pork and t u r k e y c l o s e l y p a r a l l e l e d DSC t h e r m o p r o f i l e s of the same samples.  F o r the 15% soy i s o l a t e d i s p e r s i o n , the i n c r e a s e i n G' from 70°C to  97°C was less t h a n that f o r the c a n o l a i s o l a t e d i s p e r s i o n between 60°C and 95°C, but the i n c r e a s e i n G upon c o o l i n g was g r e a t e r f o r the soy i s o l a t e , so 1  that b o t h d i s p e r s i o n s s t a r t e d  and ended up w i t h v e r y s i m i l a r G v a l u e s . 1  H e a t i n g a 15% soy i s o l a t e d i s p e r s i o n to 73°C and t h e n c o o l i n g ( F i g u r e 1.3) p r o d u c e d no i n c r e a s e i n G w h i c h i n d i c a t e d that p r o t e i n d e n a t u r a t i o n i n d u c e d 1  by h i g h e r temperatures was r e q u i r e d f o r g e l f o r m a t i o n .  C.  Cook S t a b i l i t y Cook s t a b i l i t y was a f f e c t e d by replacement l e v e l (98.5% y i e l d at 66.7%  replacement  v s . 97.2% at 33.3% replacement; p<0.01), c o o k i n g temperature  (98.6% at 70°C v s . 97.1% at 95°C; p<0.01), c o o k i n g time (98.1% at 25 min vs. 97.7% at 50 min; p<0.05) and p r o t e i n source ( c a n o l a , 98.1% v s . soy, 97.8%, p<0.05), as w e l l as i n t e r a c t i o n s between p r o t e i n source and pretreatment (p<0.05), p r o t e i n source and c o o k i n g time (p<0.05), and cook temperature and replacement l e v e l (p<0.01).  These v a r i a t i o n s were small when compared to the  a l l - m e a t c o n t r o l emulsions, i n which the y i e l d v a r i e d from 82.0% w i t h a 95°C, 50 min cook to 98.4% w i t h a 70°C, 25 min cook. work of R a n d a l l et a l . (1976)  T h i s i s c o n s i s t e n t w i t h the  and S o s u l s k i et a l . (1977)  who  attributed  improved cook s t a b i l i t y of f r a n k f u r t e r s c o n t a i n i n g p l a n t p r o t e i n s to i n c r e a s e d water h o l d i n g c a p a c i t y , and Schut (1976) who d e s c r i b e d d e c r e a s e d water holding  21 c a p a c i t y of meat p r o t e i n s w i t h i n c r e a s e d s e v e r i t y of t h e r m a l treatment as b e i n g due i n p a r t to p r o t e i n d e n a t u r a t i o n , c o a g u l a t i o n and s h r i n k a g e .  D.  Microstructure M i c r o s t r u c t u r e of the cooked  emulsions was  examined by l i g h t m i c r o -  scopy (LM) and s c a n n i n g e l e c t r o n m i c r o s c o p y (SEM). The f a t p a r t i c l e s r a n g e d i n s i z e from l e s s t h a n 1 micrometer  to 130 micrometers  i n diameter.  d i s t r i b u t i o n s of f a t p a r t i c l e s w i t h diameters of 10-126 micrometers  The were  o b t a i n e d f o r the emulsions c o n t a i n i n g p l a n t p r o t e i n s ( F i g u r e 1.4). F i g u r e 1.5A shows the f a t p a r t i c l e s i n a 66.7% soy s u b s t i t u t e d emulsion cooked at 95°C f o r 50 min.  The p r o t e i n a c e o u s m a t r i x ( F i g u r e 1.5B)  had  an  open, l a c y appearance  w i t h r e g u l a r l y spaced areas of more d e n s e l y s t a i n i n g  protein material.  SEM  An  micrograph  ( F i g u r e 1.5C)  showed f a t p a r t i c l e s  embedded i n the p r o t e i n a c e o u s m a t r i x , s e v e r a l of w h i c h appeared to be deformed perhaps d u r i n g comminution and s t u f f i n g or by c o a l e s c e n c e . The 66.7% c a n o l a s u b s t i t u t e d emulsions had a g r e a t e r number of f a t p a r t i c l e s w i t h diameters of 10-50 micrometers and fewer p a r t i c l e s w i t h diameters g r e a t e r t h a n 50 m i c r o meters ( F i g u r e s 1.4A and 1.6A) and the p r o t e i n m a t r i x had a more compact and l e s s l a c y appearance t h a n the 66.7% soy emulsion ( F i g u r e 1.6B and C ) . A l s o seen were a number of pores and openings i n the f a t p a r t i c l e s ( F i g u r e  1.6C,  arrows) w h i c h may c o r r e s p o n d to pores i n f a t d r o p l e t membranes as r e p o r t e d by B o r c h e r t et a l . (1967) u s i n g t r a n s m i s s i o n e l e c t r o n microscopy.  These were  seen i n other samples as w e l l . The m a t r i x also appeared more g r a n u l a r t h a n i n the s o y - c o n t a i n i n g emulsions. At t h i s replacement l e v e l the c a n o l a emulsions had f i r m e r t e x t u r e but were l e s s s p r i n g y t h a n those c o n t a i n i n g soy p r o t e i n . It was noted that as w e l l as h a v i n g poorer t e x t u r a l a t t r i b u t e s , the emulsions  22 2.5  I  0 2.5  F i g u r e 1.4.  I  20  40  60  80  100  120  I  F a t p a r t i c l e d i s t r i b u t i o n s of meat emulsions c o n t a i n i n g soy or canola protein: (A) 66.7% soy vs. 66.7% canola; (B) 33.3% vs. 66.7% substitution; (C) 33.3% soy vs. 33.3% canola.  23  Figure 1.4.  (cont.) (D) 33.3% vs. 66.7% soy; (E) 33.3% vs. 66.7% canola; (F) soy vs. canola substitution.  F i g u r e 1.5.  66.7% soy p r o t e i n meat emulsion cooked at 95°C f o r 50 min: (A) and ( B ) , l i g h t m i c r o g r a p h s of l i p i d and p r o t e i n s t a i n i n g r e s p e c t i v e l y (bar=200 pim); (C) SEM m i c r o g r a p h (bar=100 ^ra)  F i g u r e 1.6.  66.7% c a n o l a p r o t e i n meat e m u l s i o n cooked at 95°C f o r 50 min: (A) and ( B ) , l i g h t m i c r o g r a p h s of l i p i d and p r o t e i n s t a i n i n g r e s p e c t i v e l y (bar=200 /un); ( C ) SEM m i c r o g r a p h (bar=100 /im).  to  26 c o n t a i n i n g p l a n t p r o t e i n that were cooked at 70°C were v e r y d i f f i c u l t to s e c t i o n f o r LM and tore e a s i l y d u r i n g s t a i n i n g so were not used f o r d e t e r m i n a t i o n of f a t p a r t i c l e d i s t r i b u t i o n s .  The e f f e c t of c o o k i n g c o n d i t i o n s on  m i c r o s t r u c t u r e of the 66.7% canola emulsions i s seen i n F i g u r e 1.7A w h i c h i s an SEM m i c r o g r a p h of a sample cooked at 70°C f o r 25 min and F i g u r e 1.7B w h i c h is a sample cooked at 95°C f o r 50 min. The l e s s severe c o o k i n g  conditions  p r o d u c e d a m a t r i x w i t h a l e s s s t r u c t u r e d and more pasty appearance as w e l l as poorer s p r i n g i n e s s and s o f t e r t e x t u r e t h a n the sample r e c e i v i n g the more severe heat treatment. also f o u n d  with  A s i m i l a r e f f e c t on m i c r o s t r u c t u r e and t e x t u r e was  the 66.7% soy samples, and i s p r o b a b l y  a t t r i b u t a b l e to  enhanced g e l a t i o n of the p l a n t p r o t e i n s under the more severe cooking c o n d i t i o n s , as suggested by the t h e r m o r h e o l o g i c a l p r o f i l e s .  A l l the f a t d r o p l e t  d i s t r i b u t i o n s ( F i g u r e 1 . 4 ) had s i m i l a r shapes; the l a r g e d r o p l e t s ( g r e a t e r t h a n a p p r o x i m a t e l y 50 micrometers i n diameter) approximated a normal d i s t r i b u t i o n , while  below 50 micrometers the p a r t i c l e number  increased  i n an  e s s e n t i a l l y l o g a r i t h m i c manner. The s i z e range of the l a r g e d r o p l e t s was v e r y s i m i l a r to that r e p o r t e d by v a n den Oord and V i s s e r (1973) and Lee (1985) f o r the  cell  diameter of adipose t i s s u e .  T h u s i t appears as t h o u g h the f a t  p a r t i c l e d i s t r i b u t i o n s were the r e s u l t of r e l a t i v e l y i n t a c t f a t c e l l s as w e l l as f i n e l y d i s p e r s e d f a t p a r t i c l e s that were r e d u c e d i n s i z e b y the comminution process.  The m i c r o s t r u c t u r e of meat emulsions i s i n f l u e n c e d by such f a c t o r s  as the types of meat and f a t , the l e v e l s of f a t , moisture and s a l t , the comm i n u t i o n p r o c e s s (e.g. c h o p p i n g s p e e d ) , the v i s c o s i t y of the emulsion, and the c o o k i n g c o n d i t i o n s ( L e e , 1985). At canola  the 33.3% replacement l e v e l , the p r o t e i n m a t r i x of both soy and s u b s t i t u t e d emulsions had a t i g h t e r , l e s s l a c y appearance t h a n at  F i g u r e 1.7. SEM m i c r o g r a p h s of 66.7% c a n o l a p r o t e i n meat emulsions cooked at: (A) 70°C f o r 25 rain, and ( B ) 95°C f o r 50 min (bar=20 Jim).  to  —]  28 the 66.7% replacement l e v e l , w h i l e the m a t r i x of the c a n o l a emulsion a g a i n appeared somewhat more compact than the soy emulsion ( F i g u r e s 1.8B.C and 1.9B.C). The  f a t p a r t i c l e s of the 33.3% soy and c a n o l a emulsions are shown  i n F i g u r e s 1.8A and 1.9A, r e s p e c t i v e l y .  The f a t p a r t i c l e d i s t r i b u t i o n of the  33.3% s u b s t i t u t e d emulsions showed fewer p a r t i c l e s w i t h diameters g r e a t e r t h a n 15 micrometers greater  t h a n the 66.7%  number of p a r t i c l e s  s u b s t i t u t e d emulsions ( F i g u r e 1.4B), but a w i t h smaller diameters.  As  w i t h the  66.7%  replacement l e v e l , the 33.3% c a n o l a emulsions had a g r e a t e r number of f a t p a r t i c l e s w i t h diameters less t h a n 50 micrometers as compared to the 33.3% soy emulsions, w h i l e the 33.3% soy emulsions had more p a r t i c l e s l a r g e r t h a n 50 micrometers  i n diameter ( F i g u r e 1.4C).  The  66.7% soy emulsions had more  fat p a r t i c l e s between 10 and 100 micrometers i n diameter t h a n were found i n the 33% soy emulsions ( F i g u r e 1.4D)  w h i l e 66.7% c a n o l a emulsions had more  p a r t i c l e s between 20 and 55 micrometers and g r e a t e r than 90 micrometers t h a n 33.3% c a n o l a emulsions ( F i g u r e 1.4E). O v e r a l l , both soy and c a n o l a emulsions had s i m i l a r numbers of l a r g e p a r t i c l e s ( F i g u r e 1.4F) but c a n o l a emulsions had more p a r t i c l e s smaller t h a n 50 micrometers i n diameter.  As the f a t p a r t i c l e  d i s t r i b u t i o n s were estimated by a manual c o u n t i n g t e c h n i q u e , o n l y a s m a l l number of f i e l d s c o u l d be examined, w h i c h data.  l i m i t e d the r e l i a b i l i t y of the  Recent developments i n image a n a l y s i s , w h i c h a l l o w f o r r a p i d  l e c t i o n and  col-  p r o c e s s i n g of t h i s type of data, s h o u l d g r e a t l y i n c r e a s e the  e f f i c a c y of a n a l y z i n g not o n l y the f a t p a r t i c l e d i s t r i b u t i o n but also the m a t r i x s t r u c t u r e of meat emulsions (Kempton et a l . , 1982; Kempton and T r u p p , 1983).  The  effect  of c o o k i n g c o n d i t i o n s on m i c r o s t r u c t u r e of the  33.3%  s u b s t i t u t e d emulsions was somewhat s i m i l a r to that seen at the 66.7% l e v e l . F i g u r e 1.10A shows the somewhat amorphous, p a s t y a p p e a r i n g m a t r i x found with a  Figure 1.8. 33.3% soy protein meat emulsion cooked at 95°C for 25 min: (A) and (B), light micrographs of lipid and protein staining respectively (bar=200 (im); (C) SEM micrograph (bar=100 /xm).  F i g u r e 1.9.  33.3% c a n o l a p r o t e i n meat emulsion cooked at 95°C f o r 25 min: (A) and ( B ) , l i g h t m i c r o g r a p h s of l i p i d and p r o t e i n s t a i n i n g r e s p e c t i v e l y (bar=200 (ita); ( C ) SEM m i c r o g r a p h (bar-100 fim).  to o  F i g u r e 1.10. SEM m i c r o g r a p h s of 33.3% canola p r o t e i n meat emulsions cooked at: (A) 70°C for 25 m i n , and (B) 95°C f o r 50 min ( b a r = 2 0 / x m ) .  OJ  32 33.3%  canola emulsion cooked  tured appearance It  at 70°C f o r 25 rain c o m p a r e d to t h e more s t r u c -  o b t a i n e d w i t h a 95°C, 50 m i n h e a t t r e a t m e n t ( F i g u r e 1.10B).  is interesting  to n o t e  that  greater product  f i r m n e s s was  obtained  w i t h a 9 5 ° C c o o k at t h e 66.7% r e p l a c e m e n t l e v e l , w h e r e a s t h e o p p o s i t e t e n d e n c y was  found  at t h e 33.3%  would appear the  replacement  level, e s p e c i a l l y with soy protein.  t h a t at t h e 66.7% r e p l a c e m e n t  It  l e v e l the f u n c t i o n a l b e h a v i o r of  n o n - m e a t p r o t e i n s p r e d o m i n a t e d , w h i l e at t h e 33.3% r e p l a c e m e n t l e v e l , t h e  meat p r o t e i n p r e d o m i n a t e d .  S i e g e l et a l . (1979) s u g g e s t e d t h a t i s o l a t e d  protein  i n t e r f e r e s w i t h the  g e l - f o r m i n g i n t e r a c t i o n s between myosin  cules.  King  (1977)  found  an  interaction  between  the  7S  fraction  soy  moleof  soy  p r o t e i n a n d m y o s i n w h e n e x p o s e d to t e m p e r a t u r e s i n t h e 7 5 - 1 0 0 ° C r a n g e , w h i l e P e n g et a l . (1982a,b) r e p o r t e d a n i n t e r a c t i o n b e t w e e n t h e 11S f r a c t i o n o f s o y protein  and  myosin  at t e m p e r a t u r e s  g r e a t e r t h a n 85°C.  Since these  inter-  a c t i o n s t a k e p l a c e at t e m p e r a t u r e s w h i c h a r e g r e a t e r t h a n t h o s e g e n e r a l l y u s e d i n c o m m i n u t e d meat p r o d u c t s , h i g h l e v e l s o f s o y p r o t e i n s p r o b a b l y a c t o n l y a s a d i l u e n t , d e c r e a s i n g meat p r o t e i n i n t e r a c t i o n s a n d g e l a t i o n , a n d r e s u l t i n g i n softer  product  texture  when  cooked  at  normal  processing  temperatures.  A l t h o u g h no r e p o r t s h a v e b e e n f o u n d s u g g e s t i n g a n i n t e r a c t i o n b e t w e e n c a n o l a proteins  and  meat  indicate  that  proteins,  gelation  of  the  meat  thermorheological profiles protein  i n d e p e n d e n t l y when t h e y are combined The  and  protein  i n a meat e m u l s i o n  isolate  distribution  W i t h a 70°C, 25 m i n  appeared  c o o k ( F i g u r e 1.11)  f a i r l y s i m i l a r to t h a t o f t h e 33.3%  s i o n s , b u t w i t h a s l i g h t l y more o p e n m a t r i x . more  oblong  than  i n the  samples  The  containing  1.2  occur  system.  m i c r o s t r u c t u r e of the a l l - m e a t c o n t r o l e m u l s i o n s v a r i e d  cessing conditions.  also  canola  of F i g u r e  with pro-  the f a t p a r t i c l e  replacement  emul-  l a r g e l i p i d d r o p l e t s were plant  protein,  perhaps  F i g u r e 1.11. A l l - m e a t emulsion cooked at 70°C f o r 25 min: (A) and ( B ) , l i g h t m i c r o g r a p h s of l i p i d and p r o t e i n s t a i n i n g r e s p e c t i v e l y (bar=200 /Am); (C) SEM m i c r o g r a p h (bar=100 / t i n ) .  CO CO  34 reflecting  the  increased  viscosity  w h e n t h e e m u l s i o n was s t u f f e d  of  the  system  and  an orientation  into the stainless steel tubes.  In the  c o o k e d a t 9 5 ° C f o r 50 m i n ( F i g u r e 1 . 1 2 ) t h e r e w e r e m o r e l a r g e a n d sized droplets, the matrix,  perhaps  with thick  as a r e s u l t strands  of droplet  surrounding  coalescence  the droplets  effect sample  intermediate  and shrinkage  in the matrix.  of The  shrunken appearance of the m a t r i x and possible droplet coalescence (arrow) s e e n i n F i g u r e 1.12C. the  sample  probably  shown  T h i s sample had greater firmness and s p r i n g i n e s s  in Figure  a contributing  Differences treatment,  and  replacement  factor  in texture cooking  level.  required for  and  but  to  and  cook  viscosity Randall,  of  salt-soluble  g e l a t i o n of meat  yield, which  the  emulsions  and  in  the  would  their  resulted  small  to  protein  compared not  proteins  at  to  only to  the  fat  as  of  gelation  a l s o as a r e s u l t lower than  of  those  Therefore, by the time  the  to s t r u c t u r e formation, the m a t r i x  had  replacement  affect  resistance  the to  levels  mobility  the  increased  of  cutting  the  fat  action  (Voisey  droplets  of  the  and  during  Omnimixer  along w i t h the thermal processing c o n d i t i o n s , may have  observed  and  matrix  As n o t e d b y F r o n i n g and N e e l a k a n t a n (1971), f o r c h i c k e n  frank-  showing  globules.  differences  pre-  effects  superior  temperatures  proteins.  source,  greater  differences  tensile  in fat  strength  droplet  there  distribution  was  greater  uniformity  the appearance of the fat globules and a heavier m a t r i x of p r o t e i n the  was  Another c o n t r i b u t i n g f a c t o r may have been the decreased apparent  This effect,  furters  due  meat p r o t e i n s but  d e n a t u r a t i o n of the plant  blades.  appearance.  stability  These r e s u l t s may be due  1977), w h i c h  comminution  had much lower  than  effects.  were  plant proteins were able to c o n t r i b u t e already set.  also  these  conditions  p r o p e r t i e s of the r o d - l i k e coagulation  1.11  is  This  may  also  be  a contributing  found between protein replacement  levels  factor in the  to  in  surrounding the  present  textural study.  Figure 1.12. All-meat emulsion cooked at 95°C for 50 min: (A) and (B), light micrographs of lipid and protein staining respectively (bar=200 fim); (C) SEM micrograph (bar=100 fim).  36 SUMMARY AND  CONCLUSIONS  A model meat emulsion system was used to evaluate the effects of cooking time and temperature on texture, microstructure and cook stability of meat emulsions containing soy or canola protein isolate. The plant proteins were incorporated either dry or rehydrated at replacement levels of 33.3% and  66.7%  of the meat protein, and the emulsions were cooked at 70 or 95°C for 25 or 50 min.  Texture of the cooked emulsions was assessed by an instrumental texture  profile analysis (TPA) using an Instron tester, while the storage modulus of emulsions and plant protein dispersions during heating was monitored with a Weissenberg Rheogoniometer. Analysis of TPA  and  stability data revealed several complex interac-  tions between experimental ment was increased  variables; however, level of protein replace-  found to be the predominant factor, with decreased firmness yield  Thermorheological  resulting from  increased  and  replacement of meat protein.  profiles demonstrated that the development of elasti-  city, as indicated by the storage modulus, during heating of all-meat emulsions was essentially complete at 75-80°C, while the elasticity of dispersions of canola or soy protein continued to increase with heating to 95°C. storage modulus of a l l samples increased upon cooling. containing canola  The  A meat emulsion  protein displayed characteristics of both the  all-meat  emulsion and the canola dispersion. Thus, the storage modulus continued to increase with heating to 95°C, but the increased structure formation at higher heating temperatures was not enough to compensate for an initial lower storage modulus that resulted from the loss of meat protein.  37 The microstructure of the cooked emulsions was examined by light microscopy and scanning electron microscopy. ferences  in the  fat  particle  Although there were slight dif-  distributions of the emulsions containing  plant proteins, the distributions had similar shapes, where particles larger than 50 micrometers approximated a normal distribution and were thought to be relatively intact fat cells, while the number of particles with diameters of 10-50 micrometers increased in an essentially logarithmic manner as size decreased.  The microstructure of the proteinaceous matrix was  affected  primarily by protein source, replacement level and cooking conditions.  38 REFERENCES  A c t o n , J . 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DeHoff, R.T. and R h i n e s , F.N. (Eds.). M c G r a w - H i l l , T o r o n t o , ON. F r o n i n g , G.W. a n d N e e l a k a n t a n , S. 1971. E m u l s i f y i n g c h a r a c t e r i s t i c s of p r e r i g o r and p o s t r i g o r p o u l t r y muscle. P o u l t r y S c i . 50:839. Gill,  T.A. and T u n g , M.A. 1978. T h e r m a l l y i n d u c e d r a p e s e e d g l y c o p r o t e i n . J . Food S c i . 43:1481.  Hansen, L . J . 1960. E m u l s i o n formation T e c h n o l . 14:565.  g e l a t i o n of the 12S  i n f i n e l y comminuted sausage. Food  H e g a r t y , G.R., B r a t z l e r , L.J. and P e a r s o n , A.M. 1963. Studies on the emuls i f y i n g p r o p e r t i e s of some i n t r a c e l l u l a r beef muscle p r o t e i n s . J . Food S c i . 28:663.  39 Herraansson, A.-M. 1975. F u n c t i o n a l p r o p e r t i e s of added p r o t e i n s c o r r e l a t e d w i t h p r o p e r t i e s of meat systems. E f f e c t on t e x t u r e of a meat product. J. Food S c i . 40:611. Herraansson, A.-M. 1978. P h y s i c o - c h e m i c a l formation. J . T e x t u r e Stud. 9:33.  aspects of soy p r o t e i n s s t r u c t u r e  Hermansson, A.-M. 1979. A g g r e g a t i o n and d e n a t u r a t i o n i n v o l v e d i n g e l f o r mation. In: F u n c t i o n a l i t y and P r o t e i n S t r u c t u r e . P o u r - E l , A. (Ed.). ACS Symposium S e r i e s 92:81. Hermansson, A.-M. and Akesson, C. 1975a. F u n c t i o n a l p r o p e r t i e s of added p r o t e i n s c o r r e l a t e d w i t h p r o p e r t i e s of meat systems. E f f e c t of c o n c e n t r a t i o n and temperature on w a t e r - b i n d i n g p r o p e r t i e s of model meat systems. J . Food S c i . 40:595. Hermansson, A.-M. and Akesson, C. 1975b. F u n c t i o n a l p r o p e r t i e s of added p r o t e i n s c o r r e l a t e d w i t h p r o p e r t i e s of meat systems. E f f e c t of salt on w a t e r - b i n d i n g p r o p e r t i e s of model meat systems. J . Food S c i . 40:603. Kempton, A.G., Polonenko, D.R., B i s s o n n e t t e , J . and Quinn, J . 1982. A s t a t i s t i c a l e v a l u a t i o n of h i s t o l o g i c a l changes caused by nonmeat p r o t e i n s i n wiener b a t t e r s . Can. Inst. Food S c i . T e c h n o l . J . 15:85. Kempton, A.G. and T r u p p , S. 1983. Image a n a l y s i s of m o r p h o l o g i c a l changes i n wiener b a t t e r s d u r i n g c h o p p i n g and c o o k i n g . Food M i c r o s t r u c t u r e 2:27. K i n g , N.L. 1977. H e a t - i n d u c e d complex formation between myosin and soybean 7S g l o b u l i n s . J . A g r i c . Food Chem. 25:166. K i n s e l l a , J.E. 1976. F u n c t i o n a l p r o p e r t i e s of p r o t e i n s i n foods: a survey. CRC C r i t . Rev. Food S c i . N u t r . 7:219. K i n s e l l a , J.E. 1979. F u n c t i o n a l p r o p e r t i e s of soy p r o t e i n s . Chem. Soc. 56:242.  J . Am. O i l  Lauck, R.M. 1975. The f u n c t i o n a l i t y of b i n d e r s i n meat emulsions. S c i . 40:736. Lee,  J . Food  C M . 1985. M i c r o s t r u c t u r e of meat emulsions i n r e l a t i o n to f a t s t a b i l i z a t i o n . Food M i c r o s t r u c t u r e 4:63.  Montejano, J.G., Hamann, D.D. and L a n i e r , T.C. 1984. Thermorheological c h a n g e s i n s e l e c t e d comminuted muscle systems d u r i n g p r o c e s s i n g . P r o c . IX I n t . C o n g r e s s on Rheology, Mexico, p.273. M o r r i s o n , G.S., Webb, N.B., Blumer, T.N., Ivey, F.J. and Haq, A. 1971. R e l a t i o n s h i p between composition and s t a b i l i t y of sausage-type emulsions. J . Food S c i . 36:426.  40 Patana-Anake, C. and F o e g e d i n g , E.A. 1985. R h e o l o g i c a l and s t a b i l i t y t r a n s i t i o n s i n meat b a t t e r s c o n t a i n i n g soy p r o t e i n c o n c e n t r a t e and v i t a l wheat g l u t e n . J . Food S c i . 50:160. P a u l s o n , A.T., T u n g , M.A., Garland, M.R. and Nakai, S. 1984. F u n c t i o n a l i t y of m o d i f i e d p l a n t p r o t e i n s i n model food systems. Can. I n s t . Food S c i . T e c h n o l . J . 17:202. Peng, I.C, Dayton, W.R., Quass, D.W. and A l l e n , C E . 1982a. I n v e s t i g a t i o n s of soybean 11S p r o t e i n and myosin i n t e r a c t i o n by s o l u b i l i t y , t u r b i d i t y and t i t r a t i o n s t u d i e s . J . Food S c i . 47:1976. Peng, I.C, Dayton, W.R., Quass, D.W. and A l l e n , C E . 1982b. S t u d i e s on the s u b u n i t s i n v o l v e d i n the i n t e r a c t i o n of soybean 11S p r o t e i n and myosin. J . Food S c i . 47:1984. Quinn, J.R., Raymond, D.P. and H a r w a l k a r , V.R. 1980. D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y of meat p r o t e i n s as a f f e c t e d by p r o c e s s i n g treatment. J. Food S c i . 45:1146. R a n d a l l , C.J., Raymond, D.P. and V o i s e y , P.W. 1976. E f f e c t of v a r i o u s animal and vegetable p r o t e i n m a t e r i a l s on r e p l a c i n g the beef component i n a meat emulsion system. Can. I n s t . Food S c i . T e c h n o l . J . 9:216. S a f f l e , R.L.  1968.  Meat emulsions.  S c h m i d t , G.R. 1984. Processing Food M i c r o s t r u c t u r e 3:33.  Adv. Food Res. 16:105.  e f f e c t s on  meat p r o d u c t  microstructure.  S c h m i d t , G.R., Mawson, R.F. and S i e g e l , D.G. 1981. F u n c t i o n a l i t y of p r o t e i n m a t r i x i n comminuted meat p r o d u c t s . Food T e c h n o l . 35:235.  a  Schoen, H.M. 1977. F u n c t i o n a l p r o p e r t i e s of p r o t e i n s and t h e i r measurement. In: Food P r o t e i n s . Whitaker, J.R. and Tannenbaum, S.R. (Eds.), p.387. AVI P u b l i s h i n g Co. Westport, CT. S c h u t , J . 1976. Meat emulsions. In: Food Emulsions. F r i b e r g , S. (Ed.). M a r c e l Dekker, New York, NY. S i e g e l , D.G., C h u r c h , K.E. and Schmidt, G.R. 1979. Gel s t r u c t u r e of nonmeat p r o t e i n s as r e l a t e d to t h e i r a b i l i t y to b i n d meat p i e c e s . J . Food S c i . 44:1276. Smith, G.C, Juhn, H., C a r p e n t e r , Z.L., M a t t i l , K.F. and C a t e r , C M . 1973. E f f i c a c y of p r o t e i n a d d i t i v e s as emulsion s t a b i l i z e r s i n f r a n k f u r t e r s . J . Food S c i . 38:849. S o s u l s k i , F., H u m b e r t , E.S., B u i , K. and Jones, J.D. 1976. Functional p r o p e r t i e s of r a p e s e e d f l o u r s , c o n c e n t r a t e s and i s o l a t e . J . Food. S c i . 41:1349. S o s u l s k i , F., Humbert, E.S., L i n , M.J.Y. and C a r d , J.W. 1977. Rapeseedsupplemented wieners. Can. I n s t . Food S c i . T e c h n o l . J . 10:9.  41 S w i f t , C.E., L o c k e t t , C. and F r y a r . A.J. 1961. Comminuted meat emulsions the c a p a c i t y of meats f o r e m u l s i f y i n g f a t . Food T e c h n o l . 15:468. Theno, D.M. and Schmidt, G.R. 1978. M i c r o s t r u c t u r a l comparisons commercial f r a n k f u r t e r s . J . Food S c i . 43:845.  of three  Thomas, M.A., Baumgartner, P.A., B o a r d , P.W. and Gipps, P.G. 1973. E v a l u a t i o n of some non-meat p r o t e i n s f o r use i n sausage. J . Food T e c h n o l . 8:175. Thompson, L.U., L i u , R.F.K. and Jones J.D. 1982. Functional properties and food a p p l i c a t i o n s of rapeseed p r o t e i n c o n c e n t r a t e . J . Food S c i . 47:1175. T o r g e r s e n , H. and Toledo, R.T. 1977. P h y s i c a l p r o p e r t i e s of p r o t e i n p r e p a r a t i o n s r e l a t e d to t h e i r f u n c t i o n a l c h a r a c t e r i s t i c s i n comminuted meat systems. J . Food S c i . 42:1615. Townsend, W.E., Witnauer, L.P., R i l o f f , J.A. and S w i f t , C.E. 1968. Comminuted meat emulsions: d i f f e r e n t i a l t h e r m a l a n a l y s i s of f a t t r a n s i t i o n s . Food T e c h n o l . 22:319. v a n den Oord, A.H.A. and V i s s e r , P.R. 1973. P r o p e r t i e s and d i s t r i b u t i o n of fat i n minced meat p r o d u c t s . F l e i s c h w i r t s c h a f t 53:1427. Voisey, P.W. and R a n d a l l , C.J. 1977. Stud. 8:339.  A v e r s a t i l e food rheometer.  J . Texture  Walters, K. 1968. B a s i c Concepts and Formulae f o r the Rheogoniometer. Sangamo C o n t r o l s L t d . , Bognor R e g i s , E n g l a n d . Webb, N.B. 1974. E m u l s i o n technology. P r o c . Meat Ind. Res. Conf., American Meat I n s t i t u t e F o u n d a t i o n , A r l i n g t o n , VA. p . l . W h i t i n g , R.C. and M i l l e r , A.J. 1984. E v a l u a t i o n of a food p r o c e s s o r for making model meat emulsions. J . Food S c i . 49:1222. Z i e g l e r , G.R. and A c t o n , J.C. 1984. Mechanisms of g e l f o r m a t i o n by p r o t e i n s of muscle t i s s u e . Food T e c h n o l . 38:77.  42 CHAPTER 2 GELATION AND EMULSIFICATION PROPERTIES OF UNMODIFIED AND SUCCINYLATBD CANOLA PROTEIN ISOLATE  INTRODUCTION  Canola i s the number one o i l s e e d c r o p i n Canada (Downey et a l . , 1974), and number f i v e worldwide ( O h l s o n and Anjou, 1979).  A l t h o u g h food uses of  p r o t e i n from Canola meal have been l i m i t e d u n t i l r e c e n t l y , i t i s p o t e n t i a l l y an important p r o t e i n source. Problems i n the past w i t h a n t i t h y r o i d a c t i v i t y have been l a r g e l y overcome by the b r e e d i n g of v a r i e t i e s low i n g l u c o s i n o lates.  C a n o l a p r o t e i n has a balance of e s s e n t i a l amino a c i d s that has been  shown to be s u p e r i o r to any other known v e g e t a b l e p r o t e i n (Jones, 1979; Ohlson and Anjou, 1979). perties  A number of r e p o r t s have documented the f u n c t i o n a l p r o -  of c a n o l a p r o t e i n  p r o d u c t s (Jones, 1980; N a k a i et a l . , 1980a,b;  P a u l s o n et a l . , 1984; S o s u l s k i et a l . , 1976; Thompson et a l . , 1982); t h e y showed good e m u l s i f i c a t i o n , w h i p p i n g and f a t - h o l d i n g p r o p e r t i e s , but poor gelation properties.  When used i n meat emulsion systems, the q u a l i t y of the  f i n a l p r o d u c t s were degraded  due to a s o f t , mushy t e x t u r e that p r o b a b l y  r e s u l t e d from poor g e l a t i o n p r o p e r t i e s of the p r o t e i n ( S o s u l s k i et a l . , 1977; Thompson et a l . , 1982; Chapter 1, t h i s t h e s i s ) .  H i g h p r o t e i n s o l u b i l i t y has  been suggested to be an important requirement f o r optimum p r o t e i n g e l a t i o n (Balmaceda et a l . , 1976).  At l o w - a c i d pH v a l u e s such as are e n c o u n t e r e d i n  meat emulsions, the s o l u b i l i t y of c a n o l a p r o t e i n i s r e l a t i v e l y low (Hermansson et a l . , 1974; Ohlson and A n j o u , 1979; POS C o r p o r a t i o n , p e r s o n a l communication).  43 Chemical  modification  of p r o t e i n s  with  succinic  anhydride  has  been  d e m o n s t r a t e d to improve p r o t e i n s o l u b i l i t y , and to enhance t h e r m a l s t a b i l i t y . Little  work has been r e p o r t e d ,  however, on the f u n c t i o n a l b e h a v i o r of u n -  m o d i f i e d a n d s u c c i n y l a t e d p r o t e i n s u n d e r c o n d i t i o n s e m p l o y e d i n meat e m u l s i o n s y s t e m s , a n d t h e r e h a v e b e e n few i n v e s t i g a t i o n s i n t o t h e f u n c t i o n a l of s u c c i n y l a t e d c a n o l a p r o t e i n . i n meat  emulsion  induced gelation. and  T h e f u n c t i o n a l p r o p e r t i e s o f most  systems are r e g a r d e d  to be  emulsification  and  properties importance thermally  T h e p r e s e n t s t u d y was u n d e r t a k e n t o e x a m i n e t h e g e l a t i o n  emulsification properties  o v e r a v a r i e t y of p H  of unmodified and s u c c i n y l a t e d  isolate  and sodium c h l o r i d e c o n c e n t r a t i o n s ,  a n d to  r e l a t e the g e l a t i o n and e m u l s i f i c a t i o n r e s u l t s to p h y s i c o c h e m i c a l and  rheolo-  gical properties  conditions  canola  o f t h e i s o l a t e u n d e r t h e same  conditions.  44  L I T E R A T U R E REVIEW  A.  Protein Succinylation C h e m i c a l m o d i f i c a t i o n of p r o t e i n s has been r o u t i n e l y p r a c t i c e d as a  technique to s t u d y s t r u c t u r e , c o n f o r m a t i o n , a c t i v e s i t e r e s i d u e s and enzymatic mechanisms i n the f i e l d of p r o t e i n c h e m i s t r y , but the c h e m i c a l m o d i f i c a t i o n of food p r o t e i n s to improve f u n c t i o n a l i t y has been less w e l l s t u d i e d .  Reviews by  F e e n e y (1977), K i n s e l l a and Shetty  (1977) and  (1979), Meyer and Williams  S h u k l a (1982) have documented the e f f e c t s of v a r i o u s m o d i f y i n g agents and methods on f u n c t i o n a l and p h y s i c o c h e m i c a l p r o p e r t i e s of food p r o t e i n s . S u c c i n i c a n h y d r i d e i s the most f r e q u e n t l y used c h e m i c a l agent f o r p r o t e i n derivatization  ( K i n s e l l a , 1976).  Succinic  anhydride  along  with  acetic  a n y h y d r i d e are u s u a l l y the a c y l a t i n g agents of choice because of t h e i r ease of use, r e l a t i v e s a f e t y , low cost, and a b i l i t y to produce modified p r o t e i n s w i t h enhanced f u n c t i o n a l i t y ( S p i n e l l i et a l . , 1975). and  I n a d d i t i o n , since s u c c i n i c  a c e t i c a c i d s are present i n t h e t r i c a r b o x y l i c a c i d c y c l e , t h e i r p r o t e i n  d e r i v a t i v e s are l e s s l i k e l y to be t o x i c compared to many other c h e m i c a l m o d i f y i n g agents ( F r a n z e n , 1977). N - a c y l a t i o n of p r o t e i n s w i t h s u c c i n i c a n h y d r i d e at a l k a l i n e pH i n t r o d u c e s an a n i o n i c s u c c i n a t e chains.  group i n the n u c l e o p h i l i c groups of amino a c i d side  S u c c i n i c a n h y d r i d e r e a c t s p r i m a r i l y w i t h the e p s i l o n - a m i n o group of  l y s i n e b u t other f u n c t i o n a l groups such as t y r o s y l (Gounaris and Perlmann, 1967), and s u l f h y d r y l (Habeeb, 1967) have also been r e p o r t e d to have undergone succinylation.  Rates of s u c c i n y l a t i o n are i n v e r s e l y r e l a t e d to the pK of the  n u c l e o p h i l i c groups b e i n g s u c c i n y l a t e d and t h e r e f o r e are i n f l u e n c e d by the pH of the r e a c t i o n environment i n a d d i t i o n to b e i n g a f f e c t e d b y s t e r i c f a c t o r s  45 ( K i n s e l l a and S h e t t y , 1979; S h u k l a , 1982). The h y d r o x y l groups of s e r i n e and t h r e o n i n e are weak n u c l e o p h i l e s and solution,  while  are not e a s i l y a c y l a t e d i n aqueous  a c y l a t i o n of h i s t i d i n e  and  cysteine  i s seldom  observed  since the r e a c t i o n p r o d u c t s h y d r o l y z e i n aqueous s o l u t i o n ( K i n s e l l a  and  Shetty, 1979). Food p r o t e i n s that have been s u c c i n y l a t e d i n c l u d e soy ( F r a n z e n and K i n s e l l a , 1976a), a l f a l f a  leaf  ( F r a n z e n and  K i n s e l l a , 1976b), beef heart  ( E i s e l e et a l . , 1981), s u n f l o w e r ( C a n e l l a et a l . , 1979; K a b i r u l l a h and W i l l s , 1982), f i s h ( G r o n i n g e r , 1973; G r o n i n g e r and M i l l e r , 1979), r a p e s e e d (Thompson and Cho, 1984a,b), egg albumen (Ma and Holme, 1982), wheat (Grant.,1973), oat (Ma, 1984) and peanut (Beuchat, 1977), to mention a few.  The i n t r o d u c t i o n of  s u c c i n a t e anions i n c r e a s e s the net n e g a t i v e charge of the p r o t e i n molecules w h i c h r e s u l t s i n a l t e r e d p r o t e i n c o n f o r m a t i o n (Gounaris and Perlmann, 1967; Habeeb, 1967; Oppenheimer et a l . , 1967; R i o r d a n and V a l l e e , 1964) and i n c r e a s e d p r o p e n s i t y of the p r o t e i n s to d i s s o c i a t e into s u b u n i t s ( B e u c h a t , 1977; Grant, 1973).  In a d d i t i o n , s u c c i n y l a t i o n has been r e p o r t e d to i n c r e a s e  p r o t e i n s o l u b i l i t y (Beuchat, 1977; F r a n z e n and K i n s e l l a , 1976a,b; Habeeb et a l . , 1958; Oppenheimer et a l . , 1967), lower the i s o e l e c t r i c point ( F r a n z e n and K i n s e l l a , 1976a; G r o n i n g e r , 1973), improve e m u l s i f i c a t i o n p r o p e r t i e s ( C h i l d s and Park, 1976; F r a n z e n and K i n s e l l a , 1976a; Johnson and B r e k k e , 1983) and foaming p r o p e r t i e s ( C h i l d s and P a r k , 1976; Sato and Nakamura, 1977), i n c r e a s e w a t e r - h o l d i n g and o i l - h o l d i n g c a p a c i t i e s ( C h i l d s and Park, 1976), improve f l a v o r ( F r a n z e n and K i n s e l l a , 1976b) and i n c r e a s e t h e r m a l s t a b i l i t y (Ma and Holme, 1982; Sato and Nakamura, 1977).  S u c c i n y l a t e d egg y o l k p r o t e i n s have  been f o u n d to be u s e f u l i n mayonnaise and s a l a d d r e s s i n g s (Evans and Irons,  46 1971) while  s u c c i n y l a t e d soy p r o t e i n has been p a t e n t e d f o r use i n coffee  whitener f o r m u l a t i o n s  ( M e l n y c h y n and Stapley, 1973).  The e f f e c t s of s u c c i n y l a t i o n of p r o t e i n s on t h e i r n u t r i t i v e value and t o x i c i t y have not been r e s o l v e d .  Decreased i n v i t r o d i g e s t i b i l i t y has been  r e p o r t e d f o r a number of s u c c i n y l a t e d p r o t e i n s , p a r t i c u l a r l y i n the release of l y s i n e ( C h e n et- a l . , 1975; G r o n i n g e r and M i l l e r , 1979; Matoba and Doi, 1979) as w e l l as p r o t e i n e f f i c i e n c y r a t i o ( B j a r n a s o n and C a r p e n t e r , 1969; Creamer et a l . , 1971; G r o n i n g e r , 1973).  Others, however, have f o u n d o n l y s l i g h t e f f e c t s  of s u c c i n y l a t i o n on i n v i t r o d i g e s t i b i l i t y ( E i s e l e et a l . , 1981; Johnson and B r e k k e , 1983) and amino a c i d c o m p o s i t i o n ( E i s e l e et a l . , 1981), while (1984) r e p o r t e d  Ma  improved d i g e s t i b i l i t y of s u c c i n y l a t e d oat p r o t e i n s perhaps  as a r e s u l t of i n c r e a s e d s o l u b i l i t y , d i s s o c i a t i o n and u n f o l d i n g of the p r o t e i n molecules making them more a c c e s s i b l e to p r o t e o l y t i c enzymes.  K i n s e l l a and  Shetty (1979) i n d i c a t e d that s u c c i n y l a t i o n of yeast p r o t e i n s p r i o r to p r o t e i n e x t r a c t i o n p r o t e c t e d some amino a c i d s and d i d not reduce l y s i n e c o n c e n t r a tions.  F r a n z e n and K i n s e l l a (1976a), however, found l i t t l e change i n the  amino a c i d p r o f i l e of s u c c i n y l a t e d soy p r o t e i n w i t h the e x c e p t i o n of decreased l y s i n e content.  T h e y suggested that t h i s e f f e c t may be m i n i m i z e d by d e c r e a -  s i n g the extent of m o d i f i c a t i o n and s u p p l e m e n t i n g the d i e t w i t h l y s i n e , but c a u t i o n e d that d e r i v a t i z e d p r o t e i n s i n t e n d e d as f u n c t i o n a l i n g r e d i e n t s s h o u l d not be the major source of d i e t a r y p r o t e i n i n a f a b r i c a t e d food. al.  (1981) f o u n d  that  u n m o d i f i e d and a c e t y l a t e d  E i s e l e et  beef heart m y o f i b r i l l a r  p r o t e i n had r a t - P E R v a l u e s s l i g h t l y greater t h a n f o r c a s e i n while those of succinylated  p r o t e i n were s l i g h t l y less t h a n f o r c a s e i n , but o v e r a l l were  comparable to c a s e i n as w e l l as to lean beef (Brekke and E i s e l e , 1981).  They  f o u n d no d i f f e r e n c e s i n body/organ weight r a t i o s , o v e r a l l depot body fat or  47 acute t o x i c problems i n r a t s f e d u n m o d i f i e d , a c e t y l a t e d or s u c c i n y l a t e d protein.  T h e y also suggested the e x i s t e n c e of an enzyme that was capable of  d e a c y l a t i n g the a n h y d r i d e moiety from the p r o t e i n .  G r o n i n g e r and M i l l e r  (1979) also noted that a c e t y l a t e d p r o t e i n gave a b e t t e r growth response t h a n s u c c i n y l a t e d p r o t e i n and c o n c l u d e d that the a c y l a t i n g agent, type of p r o t e i n and  extent of m o d i f i c a t i o n a l l i n f l u e n c e p r o t e i n u t i l i z a t i o n and n u t r i t i o n a l  quality.  B.  T h e r m a l l y I n d u c e d G e l a t i o n of G l o b u l a r  Proteins  Food gels c o n s i s t of a c o n t i n u o u s phase of i n t e r c o n n e c t e d  particles  and/or macromolecules i n t e r m i n g l e d w i t h a c o n t i n u o u s l i q u i d phase such as water (Powrie and T u n g , 1976). G e l l i n g agents are g e n e r a l l y present at l e v e l s of 10% or less and form a t h r e e - d i m e n s i o n a l  m a t r i x such that the system  behaves as a soft s o l i d yet r e t a i n s many p r o p e r t i e s c h a r a c t e r i s t i c of the f l u i d component.  As a r u l e , to o b t a i n gels from g l o b u l a r p r o t e i n s r e q u i r e s  protein concentrations  an order  g e l a t i o n of p o l y s a c c h a r i d e  of magnitude h i g h e r  or g e l a t i n d i s p e r s i o n s .  than i s required f o r Noncovalent  bonding  i n v o l v e d i n p r o t e i n g e l c r o s s - l i n k i n g i n c l u d e s i o n i c b o n d i n g between c h a r g e d amino a c i d side c h a i n s or as s a l t b r i d g e s , hydrogen b o n d i n g at s p e c i f i c s i t e s , or n o n - s p e c i f i c h y d r o p h o b i c i n t e r a c t i o n s . The i r r e v e r s i b l e nature of some p r o t e i n gels suggests the formation of covalent bonds such as d i s u l f i d e , or h i g h l y i r r e v e r s i b l e d e s t r u c t i o n of t e r t i a r y and/or q u a t e r n a r y s t r u c t u r e . The types of bonds i n a p r o t e i n g e l v a r y q u a n t i t a t i v e l y and q u a l i t a t i v e l y w i t h d i f f e r e n t types of p r o t e i n and the g e l a t i o n environment.  Catsimpoolas and  Meyer (1970) suggested that h y d r o g e n and i o n i c bonds s t a b l i z e the gel network of soy p r o t e i n , a l t h o u g h d i s u l f i d e bonds have been i m p l i c a t e d as w e l l ( C i r c l e  48 et a l . , 1964). V o u t s i n a s et a l . (1983b) i m p l i c a t e d h y d r o p h o b i c i t y of u n f o l d e d p r o t e i n s along w i t h s u l f h y d r y l groups i n the t h e r m a l f u n c t i o n a l p r o p e r t i e s (thickening, coagulation Shimada and M a t s u s h i t a  and  gelation)  of  globular  proteins.  Similarly,  (1980a) suggested the involvement of d i s u l f i d e bonds  and h y d r o p h o b i c i n t e r a c t i o n s , but not i o n i c a t t r a c t i o n s , i n t h e r m a l l y i n d u c e d g e l a t i o n of egg albumin. p h o b i c i t y was  Hayakawa and N a k a i (1985b) c o n c l u d e d that h y d r o -  i n v o l v e d i n b o t h s t r e n g t h and c o a g u l a b i l i t y of ovalbumin and  that s u l f h y d r y l groups were i n v o l v e d i n g e l a t i o n while net charge played important r o l e i n c o a g u l a t i o n .  As there i s a minimum p r o t e i n  an  concentration  below w h i c h g e l a t i o n does not o c c u r , an e f f e c t i v e o v e r l a p p i n g of f u n c t i o n a l groups between adjacent p r o t e i n molecules or d i s s o c i a t e d s u b u n i t s i s n e c e s s a r y for  g e l formation.  L i p a t o v and  P r o s h l y a k o v a (1961) stated that f o r d i l u t e  s o l u t i o n s the p r o b a b i l i t y of formation of i n t r a m o l e c u l a r bonds i s independent of c o n c e n t r a t i o n ,  whereas i n t e r m o l e c u l a r  increases i n concentration.  bonding increases  sharply  with  T h e r e f o r e the c r i t i c a l c o n c e n t r a t i o n for g e l a t i o n  c o r r e s p o n d s to the point at w h i c h i n t e r m o l e c u l a r bonds b e g i n to be formed i n preference The  to i n t r a m o l e c u l a r bonds.  mechanisms of g e l a t i o n of g l o b u l a r p r o t e i n s are not yet  understood. (1948) who  The  most g e n e r a l l y a c c e p t e d hypothesis  suggested a two  was  completely  proposed by F e r r y  step mechanism w h i c h b e g i n s w i t h an i n i t i a t i o n  step i n v o l v i n g u n f o l d i n g or d i s s o c i a t i o n of the p r o t e i n molecules, f o l l o w e d by an a g g r e g a t i o n  step i n w h i c h a g g r e g a t i o n and a s s o c i a t i o n r e a c t i o n s o c c u r and  under a p p r o p r i a t e thermodynamic c o n d i t i o n s may gel network s t r u c t u r e .  It was  r e s u l t i n the formation of a  suggested that the r a t e of each step r e l a t i v e  to the other i n f l u e n c e s the c h a r a c t e r i s t i c s of the g e l . The slower the second step  (aggregation)  r e l a t i v e to the  first  (denaturation),  the  better  the  49 p a r t i a l l y u n f o l d e d c h a i n s can o r i e n t themselves p r i o r to a g g r e g a t i o n , and the finer  w i l l be the g e l network.  Such g e l s would show lower o p a c i t y and  g r e a t e r e l a s t i c i t y and w a t e r - h o l d i n g c a p a c i t y t h a n i f random a g g r e g a t i o n and denaturation occurred  s i m u l t a n e o u s l y or  i f aggregation  occurred  before  denaturation. Hermansson (1978, 1979a,b) d e s c r i b e d a g l o b u l a r p r o t e i n g e l as a state i n t e r m e d i a t e between a p r o t e i n s o l and a p r e c i p i t a t e .  I f the p r o t e i n c o n c e n -  t r a t i o n exceeds some c r i t i c a l l e v e l , a balance between p r o t e i n - p r o t e i n and p r o t e i n - s o l v e n t i n t e r a c t i o n s may F a c t o r s t h a t a f f e c t t h i s balance  be a c h i e v e d and a g e l may such  as pH,  type and  t h e n be formed. q u a n t i t y of s a l t ,  p r o t e i n c o n c e n t r a t i o n and h e a t i n g c o n d i t i o n s w i l l a l t e r the c h a r a c t e r i s t i c s of the g e l . Hegg et a l . (1979) s t u d i e d the e f f e c t s of n e u t r a l s a l t s and pH  on  the t h e r m a l a g g r e g a t i o n of ovalbumin and r e p o r t e d that the appearance of the heated d i s p e r s i o n s was r e l a t e d to the a g g r e g a t i o n and d e n a t u r a t i o n temperat u r e s of ovalbumin  under the d i f f e r e n t c o n d i t i o n s of pH and i o n i c s t r e n g t h .  T r a n s p a r e n t gels were formed when the a g g r e g a t i o n temperature was greater than or equal to the d e n a t u r a t i o n temperature  while opaque gels, g e l - l i k e p r e c i -  p i t a t e s or p r e c i p i t a t e s were p r o g r e s s i v e l y formed as the a g g r e g a t i o n temperat u r e d r o p p e d below the d e n a t u r a t i o n temperature. i n i t i a l l y more d e n a t u r e d  T h u s i t appeared as i f an  p r o t e i n s t r u c t u r e accompanied g e l f o r m a t i o n , i n  c o n t r a s t to the s i t u a t i o n that gave r i s e to p r e c i p i t a t i o n . Tombs (1970, 1974) suggested t h a t gels are formed from g l o b u l a r p r o t e i n s as a r e s u l t of a g g r e g a t i o n of p r o t e i n molecules to form s t r a n d s f o l l o w e d by i n t e r a c t i o n of the s t r a n d s to form the g e l network. Since random a g g r e g a t i o n of  s p h e r i c a l p a r t i c l e s might be  expected  to l e a d to l a r g e r  approximately  s p h e r i c a l p a r t i c l e s , the author proposed that g e l a t i o n must r e s u l t from f a i r l y  50 s p e c i f i c h i g h l y o r i e n t e d i n t e r a c t i o n s w h i c h he l i k e n e d to a " s t r i n g of beads" model. P r o t e i n molecules do not show completely random a g g r e g a t i o n because the s u r f a c e s of p r o t e i n molecules are not uniform w i t h r e s p e c t to the p r o b a b i l i t y that  contact  will  lead to adhesion.  Heating protein  dispersions  i n d u c e s c o n f o r m a t i o n a l changes w h i c h tend to increase p r o t e i n - p r o t e i n interactions.  I w a b u c h i and S h i b a s a k i (1981) noted that t h e r m a l l y d e n a t u r e d p r o t e i n s  are g e n e r a l l y less u n f o l d e d  t h a n c h e m i c a l l y d e n a t u r e d p r o t e i n s and r e t a i n  r e g i o n s of o r d e r e d s t r u c t u r e . T h e r e f o r e the " s t r i n g of beads" model f o r the aggregation  process may  involve only moderately unfolded  and s t i l l g l o b u l a r  p r o t e i n molecules, an hypothesis that i s i n agreement w i t h m i c r o s c o p i c ( B e v e r i d g e et a l . , 1984; C l a r k et a l . , 1981; Tombs, 1970, A l t h o u g h a great  d e a l of work has  canola  protein.  1974).  been done on examination of  g e l a t i o n behavior of soy p r o t e i n , l i t t l e has  been r e p o r t e d  S o s u l s k i et a l . (1976) r e p o r t e d  data  on  g e l a t i o n of  that r a p e s e e d  c o n c e n t r a t e s , and i s o l a t e had poor g e l a t i o n p r o p e r t i e s , a l t h o u g h  the  flours,  concentrates  and i s o l a t e showed e x c e l l e n t water and f a t - h o l d i n g c a p a c i t y and the i s o l a t e was  high  i n o i l e m u l s i f i c a t i o n and  w h i p p i n g c h a r a c t e r i s t i c s . Rapeseed  p r o d u c t s were s u p e r i o r to soybean p r o d u c t s i n most f u n c t i o n a l t e s t s . Thompson et a l . (1982) also r e p o r t e d concentrate. the  12S  poor g e l a t i o n p r o p e r t i e s f o r r a p e s e e d p r o t e i n  G i l l and T u n g (1976, 1978a) examined the g e l a t i o n behavior of  g l y c o p r o t e i n f r a c t i o n of rapeseed by both r h e o l o g i c a l and  micro-  s c o p i c a l t e c h n i q u e s and r e p o r t e d g e l a t i o n at p r o t e i n c o n c e n t r a t i o n s as low as 4.5 p e r c e n t w i t h measurable t h i c k e n i n g at 1 percent p r o t e i n . Gel s t r e n g t h and m i c r o s t r u c t u r e were a f f e c t e d by pH and N a C l c o n c e n t r a t i o n where the strongest gels were formed under c o n d i t i o n s at w h i c h both pH and i o n i c s t r e n g t h were high.  A l t h o u g h the mechanism of g e l a t i o n and  the bonds i n v o l v e d i n gel  51 f o r m a t i o n and s t a b i l i t y were not f u l l y e l u c i d a t e d , the authors c o n c l u d e d that some d i s u l f i d e b o n d i n g was i n v o l v e d but that i o n i c and hydrogen bonds were not l i k e l y to be major f a c t o r s i n the g e l c r o s s - l i n k s .  Jones (1980) examined the  e f f e c t s of t r y p s i n and potassium l i n o l e a t e on m i c r o s t r u c t u r e and v i s c o e l a s t i c p r o p e r t i e s of heated c a n o l a i s o l a t e d i s p e r s i o n s and r e p o r t e d that as h e a t i n g temperature  i n c r e a s e d to 95°C, the storage modulus of 10 p e r c e n t i s o l a t e  d i s p e r s i o n s g e n e r a l l y i n c r e a s e d due to the f o r m a t i o n of a g e l - l i k e m a t r i x capable of s t o r i n g more of the d e f o r m a t i o n energy.  Scanning electron micro-  s c o p i c examination of the heated d i s p e r s i o n s r e v e a l e d the presence of small p r o t e i n aggregates of less t h a n one micrometer i n diameter which i n t e r a c t e d to form l a r g e r aggregates that c o u l d be observed under the l i g h t microscope i n unheated and heated d i s p e r s i o n s . T r y p s i n h y d r o l y z e d samples tended to have lower e l a s t i c i t y perhaps because of decreased aggregate s i z e and i n c r e a s e d solubility.  C.  P r o t e i n s as E m u l s i f i e r s P r o t e i n s have the a b i l i t y to promote and s t a b i l i z e o i l - i n - w a t e r (o/w)  emulsions.  The r o l e of p r o t e i n s i n e m u l s i f i c a t i o n has been r e v i e w e d b y  C h e r r y et a l . (1979), H a i l i n g (1981), K i n s e l l a (1976) and McWatters and C h e r r y (1981), w h i l e emulsion f o r m a t i o n has been r e v i e w e d by Gopal (1968) and emulsion s t a b i l i t y by K i t c h e n e r and M u s s e l l w h i t e (1968). P r o t e i n s are able to a c t as e m u l s i f y i n g  agents  due to t h e i r  large  m o l e c u l a r weights and by v i r t u e of p o s s e s s i n g both h y d r o p h i l i c and hydrophobic p r o p e r t i e s s i m u l t a n e o u s l y , w h i c h enables the p r o t e i n s to be adsorbed at the o i l / w a t e r i n t e r f a c e and decrease the i n t e r f a c i a l t e n s i o n between the two phases.  T h i s l e s s e n s the m e c h a n i c a l energy r e q u i r e d to produce  a given  52 emulsion droplet  s i z e (Cante  compactness, e l a s t i c i t y , and  et a l . , 1979).  In a d d i t i o n , the  e l e c t r i c a l p r o p e r t i e s of the  strength,  interfacial  film  around the o i l d r o p l e t s i n f l u e n c e emulsion s t a b i l i t y (Powrie and Tung, 1976). P r o t e i n s , however, are l e s s e f f e c t i v e t h a n other commonly used s u r f a c t a n t s i n reducing  interfacial  tension  (Bennett  et a l . , 1968), and  p r e d i c t i o n of  p r o t e i n e m u l s i f y i n g b e h a v i o r i s much more d i f f i c u l t t h a n f o r e m u l s i f i e r s as  the  f u n c t i o n a l p r o p e r t i e s of a g i v e n  i n f l u e n c e d by the i o n i c environment, i n p a r t i c u l a r pH, nature and  valency  non-protein  p r o t e i n are  greatly  i o n i c s t r e n g t h , the  of ions as w e l l as the presence of other  non-protein  components (Cante et a l . , 1979). The amino a c i d composition and sequence as w e l l as secondary, t e r t i a r y and q u a t e r n a r y s t r u c t u r e h e l p govern the e f f e c t i v e n e s s of p r o t e i n e m u l s i f i e r s (Powrie and Tung, 1976). Many r e p o r t s have suggested a d i r e c t r e l a t i o n s h i p between the e m u l s i f y i n g p r o p e r t i e s of p r o t e i n s and t h e i r aqueous s o l u b i l i t y (Crenwelge et a l . , K i n s e l l a , 1976;  P e a r s o n et a l . , 1965;  Yasumatsu et a l . , 1972), while  have f o u n d a poor c o r r e l a t i o n between p r o t e i n s o l u b i l i t y and properties  (Aoki  et a l . , 1981;  McWatters and  1974; others  emulsifying  Holmes, 1979a,b; Smith  a l . 1973; Wang and K i n s e l l a , 1976). R e c e n t l y , p r o t e i n surface  et  hydrophobicity  has r e c e i v e d a t t e n t i o n for i t s r o l e i n e m u l s i f i c a t i o n . K e s h a v a r z and  Nakai  (1979) determined the surface h y d r o p h o b i c i t y of s e v e r a l p r o t e i n s by chromatographic  and  p a r t i t i o n t e c h n i q u e s and  found a s i g n i f i c a n t c o r r e l a t i o n w i t h  i n t e r f a c i a l t e n s i o n of the p r o t e i n s s t u d i e d .  Kato and N a k a i (1980) and Nakai  et a l . (1980b) determined s u r f a c e h y d r o p h o b i c i t y by a f l u o r o m e t r i c method and reported  s i g n i f i c a n t c o r r e l a t i o n s w i t h i n t e r f a c i a l t e n s i o n and  emulsifying  a c t i v i t y of the p r o t e i n s , while Kato et a l . (1981) f o u n d that p a r t i a l  de-  n a t u r a t i o n of ovalbumin and lysozyme improved t h e i r e m u l s i f i c a t i o n p r o p e r t i e s ,  53 which  were  l i n e a r l y correlated  V o u t s i n a s et a l . (1983a) and  with  surface  hydrophobicity.  However,  L i - C h a n et a l . (1984) found that a balance  between p r o t e i n s o l u b i l i t y and s u r f a c e h y d r o p h o b i c i t y was r e q u i r e d f o r optimum e m u l s i f i c a t i o n by b o t h non-meat and meat p r o t e i n s . Methods f o r i n v e s t i g a t i n g the e m u l s i f y i n g p r o p e r t i e s of p r o t e i n s i n c l u d e emulsification  capacity,  e m u l s i f i c a t i o n a c t i v i t y and  emulsion  stability.  E m u l s i f i c a t i o n c a p a c i t y (EC) i s u s u a l l y d e f i n e d as the maximum amount of o i l that can be e m u l s i f i e d by a p r o t e i n s o l u t i o n to phase i n v e r s i o n under s t a n d a r d conditions.  T h i s method was  o r i g i n a l l y devised  by Swift et a l . (1961) to  s t u d y the f a c t o r s that i n f l u e n c e meat emulsions, but has since been used as a g e n e r a l method to compare p r o t e i n e m u l s i f y i n g p r o p e r t i e s .  EC measurements,  however, are not s o l e l y a p r o p e r t y of the p r o t e i n under test but also r e f l e c t f a c t o r s such as speed of b l e n d i n g , r a t e of o i l a d d i t i o n , p r o t e i n c o n c e n t r a - . t i o n , and  type of equipment used (Pearce and K i n s e l l a , 1978; T o r n b e r g  and  Hermansson, 1977). E m u l s i f i c a t i o n a c t i v i t y (EA)  r e f l e c t s the a b i l i t y of a p r o t e i n to form  and s t a b i l i z e an emulsion ( K i t c h e n e r and M u s s e l l w h i t e , 1968) and i s measured by d e t e r m i n i n g the p a r t i c l e s i z e d i s t r i b u t i o n of the d i s p e r s e d phase e i t h e r w i t h a C o u l t e r Counter, by m i c r o s c o p y or by s p e c t r o t u r b i d i t y (Walstra et a l . , 1969).  A simple r e l a t i o n s h i p e x i s t s between t u r b i d i t y and  the  interfacial  area of an emulsion ( K e r k e r , 1969). P e a r c e and K i n s e l l a (1978) have developed a s p e c t r o t u r b i d i m e t r i c test f o r e m u l s i f i c a t i o n a c t i v i t y that has found wide acceptance.  A l t h o u g h i t i s not a p r o p e r t y of the test m a t e r i a l alone but i s  a p r o p e r t y of the system as a whole, the measurement i s simple, r a p i d , and t h e o r e t i c a l l y sound, and i s more l i k e l y to be r e l a t e d to p r a c t i c a l performance t h a n e m u l s i f i c a t i o n t e s t s more commonly used.  54  Emulsions may  d e s t a b i l i z e by creaming, f l o c c u l a t i o n , and c o a l e s c e n c e ,  w h i c h may o c c u r s i n g l y or i n c o m b i n a t i o n ( H a i l i n g , 1981).  Methods used f o r  d e t e r m i n i n g emulsion s t a b i l i t y i n c l u d e s e p a r a t i o n of the phases under the i n f l u e n c e of a g r a v i t a t i o n a l f i e l d (e.g., Yasumatsu et a l . , 1972), measuring the number and s i z e of d r o p l e t s as a f u n c t i o n of time w i t h a C o u l t e r Counter ( M i t a et a l . , 1973), or by  spectrophotometrically following  the decay of  t u r b i d i t y of an emulsion w i t h time (Pearce and K i n s e l l a , 1978).  55 M A T E R I A L S AND  Canola  isolate  ( v a r . Tower) was  C o r p o r a t i o n (Saskatoon, S K ) . tion  The  of c a n o l a meal f o l l o w e d by  METHODS  o b t a i n e d from  the POS  Pilot Plant  i s o l a t e was p r e p a r e d by a l k a l i n e e x t r a c acid  precipitation, neutralization,  and  r e c o v e r y of the p r o t e i n by s p r a y d r y i n g (POS C o r p o r a t i o n , p e r s o n a l communication).  P r o t e i n content (Nx5.5; Tkachuk, 1969)  was  74.9%  (w.b.) as d e t e r -  mined by the micro K j e l d a h l method of Concon and S o l t e s s (1973).  A.  Succinylation Procedure  S u c c i n y l a t i o n was c a r r i e d out by a procedure s i m i l a r to t h a t of Groninger (1973) and Hoagland (1966). One h u n d r e d and t e n grams of c a n o l a i s o l a t e was d i s p e r s e d by s t i r r i n g i n 2 L of d i s t i l l e d water at room temperature, and the pH was  a d j u s t e d to 8.0 w i t h 4N NaOH.  4.296 g of s u c c i n i c a n h y d r i d e  (MCB  Reagents, Norwood, OH) was added to the d i s p e r s i o n i n s i x a p p r o x i m a t e l y equal increments w i t h constant s t i r r i n g over a 75 min time p e r i o d . T h i s amounted to 5.2%  of the p r o t e i n i n the d i s p e r s i o n .  The  m i c r o s t r u c t u r e of the d i s -  p e r s i o n was monitored by l i g h t m i c r o s c o p y and t h i s amount of s u c c i n i c anhyd r i d e was  j u s t s u f f i c i e n t to s o l u b i l i z e p r o t e i n aggregates i n the d i s p e r -  s i o n . The pH was m a i n t a i n e d between 8.0 and 8.5 w i t h 4N NaOH d u r i n g s u c c i n y lation.  A f t e r the pH had s t a b i l i z e d , the d i s p e r s i o n was d i a l y z e d f o r 44 h  against f o u r changes of d i s t i l l e d water at 4°C ( S p e c t r a p o r membrane t u b i n g no. 1, Spectrum M e d i c a l I n d u s t r i e s , Inc., Los Angeles, CA), a f t e r which the s u c c i n y l a t e d i s o l a t e was r e c o v e r e d by l y o p h i l i z a t i o n . l a t e d i s o l a t e was r e f e r r e d to as "5.2% SA".  T h i s b a t c h of s u c c i n y -  A second b a t c h was s u c c i n y l a t e d  56 i n a s i m i l a r manner w i t h  11.715 g of s u c c i n i c a n h y d r i d e (14.2% SA),  and  r e c o v e r e d as above.  B.  D e t e r m i n a t i o n of E x t e n t of S u c c i n y l a t i o n The  n i n h y d r i n assay of F r i e d m a n et a l . (1984) was  extent of s u c c i n y l a t i o n . acetate-dimethyl  Two  mL  used to q u a n t i f y the  of d i s t i l l e d water and  2 mL  of l i t h i u m  s u l f o x i d e n i n h y d r i n reagent were added to d u p l i c a t e 1-3  mg  p r o t e i n samples i n l a r g e test tubes. The tubes were p l a c e d i n a b o i l i n g water b a t h f o r 15 min, cooled i n an ice bath, and 6.0 mL added w i t h v o r t e x i n g . p a r t i c l e s and C a r e y 210  The  s o l u t i o n s were c e n t r i f u g e d to remove i n s o l u b l e  the absorbance of each sample was  Spectrophotometer  against a reagent blank.  of 50% e t h a n o l - w a t e r were  The  (Varian  measured at 570 nm  with a  Instrument D i v i s i o n , Palo A l t o ,  CA)  absorbance i n d i c a t e d the number of free amino  groups a v a i l a b l e f o r r e a c t i o n w i t h the n i n h y d r i n r e a g e n t , and the d i f f e r e n c e i n absorbance between s u c c i n y l a t e d and extent of s u c c i n y l a t i o n . The  extent of m o d i f i c a t i o n of free amino groups as  determined by t h i s method was 14.2%  SA  isolate.  unmodified p r o t e i n s r e f l e c t e d the  54%  f o r the 5.2%  SA i s o l a t e and  84%  for the  A l t h o u g h i t i s p o s s i b l e to s u c c i n y l a t e a l l n u c l e o p h i l i c  groups on amino a c i d r e s i d u e s (Gounaris and Perlmann, 1967), the epsilon-amino group of l y s i n e i s most r e a d i l y s u c c i n y l a t e d because of i t s r e l a t i v e l y low  pK  and i t s s t r o n g n u c l e o p h i l i c nature ( F r a n z e n and K i n s e l l a , 1976a; Thompson and Cho,  1984a).  The  u n m o d i f i e d and  protein s o l u b i l i t y , surface  succinylated  hydrophobicity,  p r o p e r t i e s as a f u n c t i o n of N a C l c o n c e n t r a t i o n (3.5, 5.0, 6.5, 8.0, 9.5, and  11.0).  i s o l a t e s were examined for  zeta p o t e n t i a l and  rheological  (0.0M, 0.35M and 0.7M)  and  pH  57 C.  Protein Solubility The s o l u b i l i t y of unheated 11.4% (w/w) i s o l a t e d i s p e r s i o n s was determined  by c e n t r i f u g a t i o n of the d i s p e r s i o n s at 27,000 x g f o r 30 min and a n a l y z i n g the s u p e r n a t a n t f o r n i t r o g e n by micro K j e l d a h l (Concon and S o l t e s s , 1973). P r o t e i n s o l u b i l i t y was e x p r e s s e d as the percentage of the p r o t e i n i n t h e d i s p e r s i o n that was r e c o v e r e d i n the supernatant.  No s i g n i f i c a n t d i f f e r e n c e s  were found i n p r o t e i n content of the i s o l a t e s as a r e s u l t of s u c c i n y l a t i o n . The d i s p e r s i o n s were p r e p a r e d b y m i x i n g  2.8 g of i s o l a t e i n d i s t i l l e d  water, a d d i n g e i t h e r 0, 2.0 or 4.0 g of a 25.6% N a C l s o l u t i o n to make the f i n a l c o n c e n t r a t i o n 0.0M, 0.35M, or 0.7M N a C l , a d j u s t i n g the pH w i t h 2N NaOH or 2N HC1 and a d d i n g d i s t i l l e d water to 25 g. The pH was r e c h e c k e d and i f n e c e s s a r y adjusted w i t h a drop or two of 2N NaOH or 2N HC1. The d i s p e r s i o n s were used to evaluate p r o t e i n s o l u b i l i t y , steady shear r h e o l o g i c a l behavior, m i c r o s t r u c t u r e by l i g h t microscopy, and t h e r m a l l y i n d u c e d g e l a t i o n p r o p e r t i e s of the u n m o d i f i e d and s u c c i n y l a t e d i s o l a t e s .  D.  P r o t e i n Surface  Hydrophobicity  P r o t e i n surface h y d r o p h o b i c i t y  ( S ) was determined u s i n g l - a n i l i n o - 8 0  naphthalene s u l f o n a t e (ANS) as a h y d r o p h o b i c probe. B r i t t o n and Robinson-type u n i v e r s a l b u f f e r s ( B r i t t o n , 1956) were made o n e - q u a r t e r s t r e n g t h to pH 3.5, 5.0, 6.5, 8.0, 9.5 and 11.0 and c o n t a i n e d otherwise  s p e c i f i e d , these b u f f e r s were  0.0M, 0.35M or 0.7M N a C l . used  throughout  Unless  t h i s study f o r  examining the e f f e c t s of pH and N a C l c o n c e n t r a t i o n on f u n c t i o n a l and p h y s i c o chemical  p r o p e r t i e s of the canola  isolates.  The i s o l a t e s were  serially  d i l u t e d w i t h b u f f e r of the d e s i r e d pH and N a C l c o n c e n t r a t i o n to o b t a i n p r o t e i n c o n c e n t r a t i o n s r a n g i n g from 0.0005 to 0.015% (w/v).  T e n m i c r o l i t e r s of ANS  58 (8.0mM i n 0.01M  phosphate b u f f e r , pH 7.0) were added to 2 mL  p r o t e i n s o l u t i o n s . The  of the d i l u t e d  f l u o r e s c e n c e i n t e n s i t y ( F I ) of A N S - p r o t e i n  gates was measured w i t h an Aminco-Bowman S p e c t r o f l u o r o m e t e r A m e r i c a n Instrument  Co. Inc., S i l v e r S p r i n g s , MD).  conju-  (No. 4-8202,  E x c i t a t i o n and  emission  wavelengths were 390 nm and 470 nm, r e s p e c t i v e l y . The FI r e a d i n g was standard i z e d by a d j u s t i n g the s p e c t r o f l u o r o m e t e r r e a d i n g f o r ANS  i n methanol to 30%  f u l l s c a l e . Net FI for each d i l u t i o n was o b t a i n e d by s u b t r a c t i n g the FI of a p r o t e i n b l a n k without ANS.  The  slopes of the p l o t s of net FI v e r s u s percent  p r o t e i n were c a l c u l a t e d by least squares l i n e a r r e g r e s s i o n , and s p e c i f i e d as S.  H y d r o p h o b i c i t y was  0  also determined a f t e r h e a t i n g 0.1%  d i s p e r s i o n s i n the a p p r o p r i a t e b u f f e r at 72°C f o r 30 min.  The  (w/v)  protein  hydrophobicity  measured was that w h i c h was "exposed" by the heat treatment and was designated S. e  T h i s method d i f f e r e d from a s i m i l a r d e t e r m i n a t i o n by Townsend and N a k a i  (1983) and  Voutsinas  et a l . (1983b) i n that those  investigators included  sodium d o d e c y l s u l f a t e i n the test s o l u t i o n and heated i t to a h i g h e r temperature.  E.  Zeta P o t e n t i a l (Net C h a r g e D e n s i t y ) Net charge d e n s i t y of the i s o l a t e s was measured as zeta p o t e n t i a l  w i t h a Laser Zee Model 501 p a r t i c l e m i c r o e l e c t r o p h o r e s i s apparatus (Pen Inc., B e d f o r d H i l l s , NY) at an a p p l i e d p o t e n t i a l d i f f e r e n c e of 150 V.  (mV) Kem  Protein  d i s p e r s i o n s f o r zeta p o t e n t i a l measurement were p r e p a r e d by homogenizing 5 mL of a 0.1% mL  (w/v)  p r o t e i n d i s p e r s i o n i n the  of 3,3'-dimethylbiphenyl  appropriate buffer with  ( A l d r i c h C h e m i c a l Co., Milwaukee, Wl)  0.15  with a  B r i n k m a n n P o l y t r o n at 2200 r.p.m. f o r 20 s, and then d i l u t i n g the r e s u l t i n g emulsions 5 0 - f o l d w i t h the a p p r o p r i a t e b u f f e r .  59 F.  S t e a d y Shear  Rheology  The flow p r o p e r t i e s of unheated 11.4% i s o l a t e d i s p e r s i o n s under steady shear were e v a l u a t e d at 21°C over a shear r a t e range of 4.3 to 1000 s ~ l u s i n g 5 cm  diameter 2 degree cone/plate f i x t u r e s w i t h a Model R.19  Rheogoniometer.  Weissenberg  The instrument was c a l i b r a t e d by measuring the r e l a t i o n s h i p  between shear s t r e s s and  shear r a t e of c e r t i f i e d v i s c o s i t y s t a n d a r d o i l s  (Cannon Instrument Co., State C o l l e g e , P A ) . The goodness of f i t , as i n d i c a t e d by the c o e f f i c i e n t of d e t e r m i n a t i o n ( r ) from least squares l i n e a r r e g r e s s i o n , 2  of shear s t r e s s [a, where rj= oV?)  P a ) , shear r a t e  (7.  s )  and  - 1  v i s c o s i t y (7), Pa  s,  data to the power-law or power-law p l a s t i c flow models f o r  each d i s p e r s i o n were determined u s i n g a program w r i t t e n f o r an A p p l e 11+ microcomputer. f i t t e d model.  The c o e f f i c i e n t of d e t e r m i n a t i o n was at least 0.99 f o r each The models may  be s t a t e d as: a = m7  Power-law: Power-law p l a s t i c : where m (or m ) 1  O-Oy  (1)  n  = m "y ' ,  (2)  n  i s the c o n s i s t e n c y c o e f f i c i e n t (Pa s ) , n (or n ) i s the flow n  behavior i n d e x ( d i m e n s i o n l e s s ) , and  1  0"y i s the y i e l d s t r e s s ( P a ) . The y i e l d  s t r e s s was estimated from the Casson Model ( e q u a t i o n 3) by e x t r a p o l a t i n g the rheogram to zero shear r a t e ; hence Casson:  a  1 / 2  Oy =  k . 2  0  = ko + k i ?  1  /  (3)  2  F o r equations 1 and 2, p l o t s of l o g 0" or l o g ( a - Oy) v e r s u s log"? are l i n e a r , where the i n t e r c e p t at 1 s " l i s l o g m or m  1  and the slope i s n or n . 1  of apparent v i s c o s i t y at shear r a t e s of 10 s*" and 1000 s " 1  from the power-law or power-law p l a s t i c flow parameters.  1  Values  were c a l c u l a t e d  60 G.  Thermally Induced Gelation  1.  Dynamic Shear P r o p e r t i e s o f T h e r m a l l y I n d u c e d Gels Seven to e i g h t grams of 11.4% i s o l a t e d i s p e r s i o n s were heated i n capped  plastic  s c i n t i l l a t i o n vials  (i.d.=22 mm,  f i c ) f o r 30 min i n a 72°C water bath. running  20 mL  capacity, Fisher  Scienti-  The samples were c o o l e d under c o l d  t a p water and t h e n allowed to e q u i l i b r a t e to room temperature f o r  a p p r o x i m a t e l y two hours. The samples that formed gels were i n i t i a l l y examined for " g e l s t r e n g t h " u s i n g a p u n c t u r e t e s t ( d e s c r i b e d i n C h a p t e r 3 ) . The dynamic v i s c o e l a s t i c p r o p e r t i e s of the gels were then o b t a i n e d  u s i n g the  Weissenberg Rheogoniometer equipped w i t h 5 cm diameter p a r a l l e l p l a t e fixtures at a gap t h i c k n e s s of 1 mm.  The gels were c a r e f u l l y removed from the v i a l s ,  u n d i s t u r b e d p o r t i o n s were s l i c e d to a t h i c k n e s s of s l i g h t l y greater t h a n 1 mm, and p l a c e d on the bottom p l a t e n . The top p l a t e n , s u p p o r t e d b y a no. 7 t o r s i o n bar (9.4 Pa cm3 fim~l),  was then c a r e f u l l y lowered to a gap t h i c k n e s s of 1 mm  to avoid a i r pockets between the p l a t e n s . sample was avoided  Evaporation  of water from the  by a p p l y i n g a t h i n l a y e r of s i l i c o n e o i l to the exposed  edge of the g e l . A small s i n u s o i d a l l y v a r y i n g o s c i l l a t o r y s t r a i n of maximum amplitude of 1.88% was imposed over a f r e q u e n c y (CO ) range of 0.19 to 19 s~*. The amplitudes of the i n p u t ( s t r a i n ) and output ( s t r e s s ) voltage s i g n a l s , and the phase d i f f e r e n c e between them, were monitored w i t h a T r o n o t e c Model 703A d i g i t a l phasemeter. From these data, v a l u e s of the storage modulus (G , Pa; a 1  measure of the e n e r g y s t o r e d e l a s t i c a l l y per c y c l e of s i n u s o i d a l d e f o r m a t i o n ) , loss modulus (G", Pa; the e n e r g y d i s s i p a t e d as heat per c y c l e ) , l o s s tangent (the tangent of the phase angle between the s t r e s s and s t r a i n waves and numerically  equal  to G"/G', t h u s r e f l e c t i n g  the r e l a t i v e p r o p o r t i o n s of  v i s c o u s to e l a s t i c components of the samples) and the dynamic v i s c o s i t y ( f]\  61 Pa s where r/'=G"/OJ) were c a l c u l a t e d from the equations of Walters (1968) u s i n g a program w r i t t e n f o r an Apple 11+ microcomputer.  As p l o t s of G' or 77'  v e r s u s co were l i n e a r on l o g a r i t h m i c c o o r d i n a t e s , the slope and i n t e r c e p t of each l i n e were determined by least squares l i n e a r r e g r e s s i o n .  V a l u e s of G*,  G" and loss tangent at a f r e q u e n c y of 10 s""* were c a l c u l a t e d from the f o l l o w i n g equations: G' = a c o  (4)  b  where a ( s t o r a g e c o e f f i c i e n t ) i s the i n t e r c e p t (at co=l s " l ) and b ( s t o r a g e index) i s the slope of a l o g - l o g plot; and, rj l  = c co  (5)  d _ 1  where c i s the dynamic shear c o n s i s t e n c y c o e f f i c i e n t (Pa sd) and d i s the dynamic shear flow b e h a v i o r i n d e x .  2.  P r o t e i n Content o f G e l Exudate The  g e l s were c e n t r i f u g e d at 27,000 x g f o r 30 min; the p r o t e i n i n the  exudate was determined as f o r the unheated d i s p e r s i o n s and e x p r e s s e d as a percentage of the t o t a l g e l p r o t e i n .  H.  E m u l s i f i c a t i o n A c t i v i t y and E m u l s i o n S t a b i l i t y E m u l s i f i c a t i o n a c t i v i t y (EA) was determined i n a manner s i m i l a r to the  method of Pearce and  Kinsella  (1978).  Four m i l l i l i t e r s of a 0.5%  (w/v)  p r o t e i n d i s p e r s i o n i n b u f f e r of the d e s i r e d pH and N a C l c o n c e n t r a t i o n and 4 mL of c o r n o i l ( F i s h e r S c i e n t i f i c ) were homogenized i n a S o r v a l l Omnimixer w i t h a micro-attachment assembly at 1880 r.p.m. f o r 1 min.  A 50 m i c r o l i t e r sample  was immediately taken from the bottom of the c o n t a i n e r and d i l u t e d i n 10 mL of b u f f e r c o n t a i n i n g 0.1% (w/v) sodium d o d e c y l s u l f a t e . Subsequent a l i q u o t s were  62 removed i n a s i m i l a r manner at a p p r o p r i a t e time i n t e r v a l s as determined  by  p r e l i m i n a r y e x p e r i m e n t a t i o n . The absorbance of the d i l u t e d emulsions at 500 nm  was measured w i t h a S p e c t r o n i c 20 spectrophotometer  Rochester, NY).  The  ( B a u s c h and Lomb,  i n i t i a l A500 measurement was t a k e n to be the e m u l s i -  f i c a t i o n a c t i v i t y , w h i l e emulsion s t a b i l i t y (ES) was  d e f i n e d as the time i n  minutes r e q u i r e d f o r A50Q to decrease to o n e - h a l f that of the emulsion at zero time.  I.  L i g h t Microscopy The unheated 11.4 percent i s o l a t e d i s p e r s i o n s were observed under a W i l d  M-20 microscope equipped w i t h a P e n t a x ME 35mm camera. U n s t a i n e d a l i q u o t s of the d i s p e r s i o n s were examined under phase c o n t r a s t and b r i g h t f i e l d K o h l e r illumination.  P h o t o g r a p h i c images were r e c o r d e d on Kodak P l u s - X b l a c k and  white f i l m and developed w i t h M i c r o d o l - X developer.  J.  S t a t i s t i c a l Analyses The  e f f e c t s of s u c c i n y l a t i o n , pH  and  N a C l c o n c e n t r a t i o n on  protein  s o l u b i l i t y , h y d r o p h o b i c i t y , zeta p o t e n t i a l , apparent v i s c o s i t y , e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y were a n a l y z e d by a t r e n d comparison  procedure  based on s i n g l e degree of freedom o r t h o g o n a l p o l y n o m i a l c o n t r a s t s f o r the treatments.  With q u a n t i t a t i v e treatments as used i n the present study, the  types of mean comparison  t e c h n i q u e s t h a t f o c u s on the s p e c i f i c treatments  t e s t e d are not adequate; i n s t e a d , a more a p p r o p r i a t e approach i s to examine the f u n c t i o n a l r e l a t i o n s h i p between treatment and response that covers the e n t i r e range of the treatment l e v e l s t e s t e d (Gomez and Gomez, 1984).  For each  treatment, a set of m u t u a l l y o r t h o g o n a l s i n g l e degree of freedom c o n t r a s t s was  63 c o n s t r u c t e d where t h e f i r s t c o n t r a s t r e p r e s e n t e d the f i r s t degree polynomial ( l i n e a r ) while the second c o n t r a s t r e p r e s e n t e d the second degree ( q u a d r a t i c ) polynomial.  A l t h o u g h the degree of the polynomial t h a t c a n be t e s t e d may be  up to one l e s s t h a n t h e number of treatment  l e v e l s ( i . e . up to q u a d r a t i c for  s u c c i n y l a t i o n or N a C l c o n c e n t r a t i o n and up to q u i n t i c f o r pH) the q u a d r a t i c polynomial was the maximum degree that was examined. The o r t h o g o n a l m u l t i p l i e r s f o r the treatment c o n t r a s t s were determined as o u t l i n e d by Gomez and Gomez (1984) and are presented c o n t r a s t s f o r each treatment  i n T a b l e 2.1. The l i n e a r and q u a d r a t i c  and t h e i r i n t e r a c t i o n s were a n a l y z e d f o r s i g n i -  f i c a n c e by f o r w a r d stepwise m u l t i p l e r e g r e s s i o n (Kleinbaum and K u p p e r , 1978) u s i n g the MIDAS s t a t i s t i c a l computer program (Fox and G u i r e , 1976) a v a i l a b l e on the UBC Amdahl 470/V8 mainframe computer. In a d d i t i o n , backward stepwise m u l t i p l e r e g r e s s i o n analyses  were used to examine t h e e f f e c t s of p r o t e i n  solubility,  zeta p o t e n t i a l and steady  hydrophobicity,  shear r h e o l o g i c a l  p r o p e r t i e s on e m u l s i f i c a t i o n a c t i v i t y , emulsion s t a b i l i t y , and dynamic shear parameters of t h e r m a l l y i n d u c e d  gels.  Table 2.1.  O r t h o g o n a l m u l t i p l i e r s f o r t r e n d comparison analyses Orthogonal Polynomial C o e f f i c i e n t  Treatment A.  Linear  Succinylation (% M o d i f i c a t i o n ) 0 54 84  B.  NaCl  -23 4 19  5 -14 9  -1 0 1  1 -2 1  -5 -3 -1 1 3 5  5 -1 -4 -4 -1 5  (M)  0.0 0.35 0.7  2:  Quadratic  PJL 3.5 5.0 6.5 8.0 9.5 11.0  65 RESULTS AND  A.  DISCUSSION  Protein Solubility P r o t e i n s o l u b i l i t y was s i g n i f i c a n t l y a f f e c t e d by the l i n e a r and  quadratic  e f f e c t s of s u c c i n y l a t i o n and pH as w e l l as i n t e r a c t i o n e f f e c t s between these v a r i a b l e s and an i n t e r a c t i o n e f f e c t of N a C l and pH (Table 2.2).  Succinylation  markedly enhanced p r o t e i n s o l u b i l i t y at a l k a l i n e and low a c i d pH ( F i g u r e The  s o l u b i l i t y p r o f i l e s of the s u c c i n y l a t e d i s o l a t e s were s i m i l a r to those of  succinylated  f i s h , l e a f , and  i s o l a t e without below pH (POS  2.1).  5.  soy  p r o t e i n ( S h u k l a , 1982).  NaCl exhibited a gradual  T h i s was  The  unmodified  i n c r e a s e i n s o l u b i l i t y above  and  s i m i l a r to the s o l u b i l i t y p r o f i l e of the s t a r t i n g meal  C o r p o r a t i o n , p e r s o n a l communication). Canola i s o l a t e i s a complex mixture  molecular  weights and  isoelectric points.  of p r o t e i n s w i t h a wide range of L o n n e r d a l et a l . (1977) found at  least n i n e t e e n d i s t i n c t p r o t e i n f r a c t i o n s w i t h i s o e l e c t r i c p o i n t s from pH to pH  4.5  10 i n a r a p e s e e d i s o l a t e where the p r o t e i n was e x t r a c t e d i n the presence  of sodium hexametaphosphate to increase y i e l d .  T h e y also found that  an  i s o l a t e d e r i v e d from a l k a l i n e e x t r a c t i o n of d e f a t t e d r a p e s e e d meal f o l l o w e d by a c i d n e u t r a l i z a t i o n was r i c h i n a c i d i c p r o t e i n s and c o n t a i n e d a l l of the h i g h molecular  weight p r o t e i n s from the meal (pI=4-7). A p p r o x i m a t e l y twenty to  f o r t y percent 13,000 D) 1972).  The  molecular  and  of the r a p e s e e d p r o t e i n s have a low molecular an i s o e l e c t r i c point close to pH  weight (about  11 ( L o n n e r d a l  and  Janson,  other rapeseed p r o t e i n s can be d i v i d e d into three groups w i t h weights of 50,000 to 75,000, 150,000 and  320,000 w i t h  isoelectric  p o i n t s s p r e a d out i n the i n t e r v a l from pH 4 to 8 ( L o n n e r d a l , 1975). a u t h o r s have r e p o r t e d on the f r a c t i o n a t i o n , i s o l a t i o n , and  Other  physicochemical  66  Table 2 . 2 .  T r e n d comparison a n a l y s i s o f t h e e f f e c t s o f s u c c i n y l a t i o n , pH and N a C l on p r o t e i n s o l u b i l i t y o f c a n o l a I s o l a t e .  Dependent V a r i a b l e  Protein R  2  Independent V a r i a b l e  Coefficient  F-prob.  10.652  0.0000  -3.093  0.0034  15.275  0.0000  -5.406  0.0000  4.319  0.0001  -5.559  0.0000  NaCl x pH  -2.293  0.0265  Constant  35.363  Solubility  Succinylation  = 0.905  Succinylation  2  S.E.a = 11.724  PH  F-prob. = 0.0000  pH  n - 54  S u c c i n y l a t i o n x pH  2  Succinylation x pH  a  Standard error of estimate.  2  100 90 80 70 60 50 40 30 20 10  0  10090SO  7060 50 40 30 20 10  0  100 90 80 70 60  e i n s o l u b i l i t y of 11.4% c a n o l a i s o l a t e d i s p e r s i o n s : unmodified; (B) 5.2% SA; (C) 14.2% SA.  68 and  f u n c t i o n a l p r o p e r t i e s of v a r i o u s p r o t e i n f r a c t i o n s (e.g., B h a t t y et a l . ,  1968;  Finlayson  1970;  et a l . , 1969;  M a c K e n z i e , 1975).  G i l l and  By  T u n g , 1976,  1978a,b; Goding et a l . ,  c o n t r a s t , p r o t e i n i s o l a t e s from most  other  o i l s e e d s c o n t a i n o n l y a few p r o t e i n s and u s u a l l y d i s p l a y a s o l u b i l i t y p r o f i l e where the s o l u b i l i t y i s low  at the i s o e l e c t r i c point but  w i t h pH  on e i t h e r side of the p i .  also may  c o n t a i n RNA,  increases r a p i d l y  In a d d i t i o n to p r o t e i n , canola i s o l a t e s  a c i d i c p o l y s a c c h a r i d e s , p h y t i c a c i d and  acidic poly-  phenols e x t r a c t e d from the meal (Aman and G i l l b e r g , 1977). S u c c i n y l a t i o n has t y p i c a l l y been found to increase p r o t e i n s o l u b i l i t y , a l t e r p r o t e i n c o n f o r m a t i o n by promoting u n f o l d i n g and i n c r e a s i n g d i s s o c i a t i o n of s u b u n i t s as w e l l as s h i f t i n g the i s o e l e c t r i c point to lower v a l u e s .  The  a l t e r e d c o n f o r m a t i o n of s u c c i n y l a t e d p r o t e i n s r e s u l t s from the replacement of s h o r t - r a n g e a t t r a c t i v e f o r c e s (ammonium, c a r b o x y l ) w i t h s h o r t - r a n g e repulsive forces  (succinate  c a r b o x y l , native c a r b o x y l )  c o m b i n a t i o n of i n t r a and  (Habeeb et a l . , 1958).  The  i n t e r m o l e c u l a r charge r e p u l s i o n promotes p r o t e i n  u n f o l d i n g and p r o d u c e s fewer p r o t e i n - p r o t e i n and more p r o t e i n - w a t e r a c t i o n s , w i t h the r e s u l t that aqueous s o l u b i l i t y i s enhanced. As net  internegative  charge i s p r o p o r t i o n a l to the extent of s u c c i n y l a t i o n , s o l u b i l i t y of  canola  i s o l a t e i n c r e a s e d as the number of s u c c i n y l a t e d groups i n c r e a s e d , a l t h o u g h the s l i g h t increase i n o v e r a l l s o l u b i l i t y w i t h 84% m o d i f i c a t i o n of amino groups as compared to 54% m o d i f i c a t i o n demonstrated that exhaustive s u c c i n y l a t i o n was not r e q u i r e d to s u b s t a n t i a l l y improve s o l u b i l i t y .  A s i m i l a r e f f e c t was  noted  by F r a n z e n and K i n s e l l a (1976a) w i t h soy p r o t e i n . T h e r e also appeared to be a s l i g h t s h i f t of the i s o e l e c t r i c r e g i o n to a lower pH w i t h i n c r e a s e d s u c c i n y l a t i o n , w h i c h may  have i m p l i c a t i o n s f o r the s u c c e s s f u l employment of s u c c i n y -  l a t e d p r o t e i n s i n l o w - a c i d foods such as comminuted meat systems.  69 The  e f f e c t of N a C l on the u n m o d i f i e d i s o l a t e was  to increase p r o t e i n  s o l u b i l i t y ( s a l t i n g - i n ) i n the a c i d i c r e g i o n and decrease s o l u b i l i t y ( s a l t i n g out) i n the a l k a l i n e r e g i o n ( F i g u r e 2.1A). These r e s u l t s are s i m i l a r to those of Rhee et a l . (1972) and McWatters and Holmes (1979a) for peanut p r o t e i n , and McWatters and Holmes (1979b) f o r soy p r o t e i n .  Schut (1976) suggested that  N a C l causes a s h i f t i n the i s o e l e c t r i c point to a more a c i d i c pH as a r e s u l t of s p e c i f i c i o n b i n d i n g e f f e c t s . Since i n o r g a n i c anions are bound to p r o t e i n s more s t r o n g l y t h a n i n o r g a n i c c a t i o n s due  to t h e i r smaller h y d r a t e d r a d i i ,  anions are able to a t t a i n a c l o s e r p r o x i m i t y to the p r o t e i n molecule and able to " s c r e e n "  are  the c h a r g e d groups of the p r o t e i n s more e f f e c t i v e l y t h a n  c a t i o n s , the e f f e c t i v e n e s s b e i n g  i n accordance w i t h the Hofmeister s e r i e s .  Thus, w i t h the a d d i t i o n of N a C l and  the s e l e c t i v e b i n d i n g of the c h l o r i d e  anions, the p r o t e i n would have an excess of negative c h a r g e s at the pH of the o r i g i n a l i s o e l e c t r i c point and more a c i d i s t h e r e f o r e needed to r e a c h the isoelectric point.  new  A l t h o u g h not s p e c i f i c a l l y a l l u d e d to, the e f f e c t s of N a C l  on the p r o t e i n s o l u b i l i t y p r o f i l e s of peanut p r o t e i n (McWatters and Holmes, 1979a; Rhee et a l . , 1972)  and  soy p r o t e i n (McWatters and  appeared to support such an hypothesis.  A l t h o u g h t h i s also appeared to be the  case i n the present study, the hypothesis s o l u b i l i t y p r o f i l e s alone as t h e y d i d not region.  Holmes, 1979b)  c o u l d not be f u l l y v e r i f i e d by the go f a r enough i n t o the a c i d  pH  The m i c r o e l e c t r o p h o r e t i c m o b i l i t y data ( S e c t i o n B) however, t e n d s to  support t h i s p r o p o s a l . For s u c c i n y l a t e d canola d e c r e a s e d s o l u b i l i t y i n the  i s o l a t e , N a C l i n c r e a s e d s o l u b i l i t y at pH a l k a l i n e pH  solubility.  r e g i o n , but  at pH  3.5  5 and  there  was  A l t h o u g h p r o t e i n s o l u b i l i t y at pH  less  v i r t u a l l y no  e f f e c t on  t h a n 3.5 was  not determined, s e v e r a l r e p o r t s have demonstrated a f a i l u r e of  70 succinylated  p r o t e i n to r e s o l u b i l i z e  at pH  values  more a c i d i c  than  the  i s o e l e c t r i c p o i n t (e.g., Chen et a l . , 1975; Hoagland, 1966; Oppenheimer et al.,  1967).  Franzen  and  Kinsella  (1976a) a t t r i b u t e d t h i s e f f e c t  to  an  i n s u f f i c i e n t number of h y d r o p h i l i c c a t i o n i c groups r e m a i n i n g a f t e r s u c c i n y l a t i o n to overcome a g g r e g a t i n g  B.  f o r c e s at the p l .  H y d r o p h o b i c i t y and Zeta P o t e n t i a l The treatment e f f e c t s on s u r f a c e h y d r o p h o b i c i t y ( S ) and zeta p o t e n t i a l Q  (z.p.) of c a n o l a i s o l a t e are shown i n T a b l e 2.3 respectively.  S  0  and  F i g u r e s 2.2  and  2.3,  decreased i n a l i n e a r manner w i t h extent of s u c c i n y l a t i o n  while the e f f e c t of N a C l v a r i e d w i t h pH.  At pH 3.5 and 5 for the  unmodified  i s o l a t e and pH 3.5 for the s u c c i n y l a t e d i s o l a t e , N a C l d e c r e a s e d h y d r o p h o b i c i t y while the opposite e f f e c t was e f f e c t was  to decrease S  Q  found at h i g h e r pH v a l u e s .  i n a c u r v i l i n e a r manner as pH  The  increased.  decreased r a p i d l y from pH 3.5 to 6.5 and more s l o w l y t h e r e a f t e r . (1974) found a s i m i l a r pH d e n s i t y lipoprotein:ANS  overall  The  h y d r o p h o b i c i t i e s of heated  p e r s i o n s ( S ) were o v e r a l l o n l y s l i g h t l y h i g h e r t h a n S e  D  S  Q  Ghosh et a l .  e f f e c t on the f l u o r e s c e n c e of human serum  complexes.  pH  (Table 2.4).  lowdis-  This did  not n e c e s s a r i l y mean that h e a t i n g p r o d u c e d l i t t l e change i n p r o t e i n c o n f o r mation, however, as F r a n k s and  (1975) noted that r e l a t i v e l y  few  peptide r e s i d u e s need to be exposed to the solvent i n order to render  the  n a t i v e conformation Zeta  Eagland  of the p r o t e i n u n s t a b l e .  p o t e n t i a l became more e l e c t r o n e g a t i v e i n a l i n e a r manner w i t h  s u c c i n y l a t i o n as a r e s u l t of the i n c r e a s i n g number of s u c c i n a t e groups on the p r o t e i n molecules.  carboxyl  A l t h o u g h the e f f e c t s of pH and N a C l were  i n t e r d e p e n d e n t , the o v e r a l l pH e f f e c t was to i n c r e a s e e l e c t r o n e g a t i v i t y i n a  71  T a b l e 2.3.  T r e n d comparison a n a l y s e s o f t h e e f f e c t s o f s u c c i n y l a t i o n , pH and NaCl on s u r f a c e h y d r o p h o b i c i t y ( S ) and s e t a p o t e n t i a l o f c a n o l a i s o l a t e (n=54). 0  Dependent Variable  Coefficient  Succinylation  -1.282  0.0025  -32.767  0.0000  19.085  0.0000  7.954  0.0026  -7.002  0.0036  Hydrophobicity (S ) D  R  2  = 0.891  S.E.  a  - 51.336  F-prob. = 0.0000  PH pH  2  NaCl x pH NaCl x pH  2  157.070  Constant Succinylation  -0.226  0.0000  = 0.943  NaCl  10.156  0.0000  S.E. = 4.790  NaCl  -3.402  0.0000  F-prob. = 0.0000  pH  -3.418  0.0000  1.419  0.0000  1.905  0.0000  -1.042  0.0000  -0.601  0.0001  Zeta Potential R  2  pH  2  2  NaCl x pH NaCl x pH  2  NaCl x pH 2  Constant a  F-prob.  Independent Variable  Standard error of estimate.  2  -23.135  600  i g u r e 2.2.  A  Surface h y d r o p h o b i c i t y ( S ) of canola i s o l a t e : (A) u n m o d i f i e d ; (B) 5.2% SA; (C) 14.2% SA. 0  igure 2.3  Zeta potential of canola isolate: (B) 5.2% SA; (C) 14.2% SA.  (A) unmodified;  Table 2.4.  E f f e c t s o f s u c c i n y l a t i o n , pH, NaCl and heating on surface hydrophobicity o f canola I s o l a t e . NaCl C o n c e n t r a t i o n ( M o l a r )  Anhydride  0  5.2  14.2  0.35  0. 0  Succinic (%)  S b  pH  S  e  So  Se  3. 5  533.6  586.0  457.5  505.4  440.3  482.0  5. 0  263.3  249.5  222.9  230.1  236.4  214.8  6. 5  81.6  112.6  146.9  151.7  160.5  146.5  8. 0  68.0  78.3  133.3  139.8  126.2  145.6  9. 5  57.3  78.2  129.3  111.8  122.2  129.8  11. 0  33.4  32.6  62.8  69.5  91.7  85.4  3. 5  578.5  605.7  429.0  461.3  382.2  477.3  5. 0  105.8  119.4  144.6  168.1  202.2  149.3  6. 5  52.8  49.5  109.3  103.9  125.4  136.1  8. 0  34.6  43.1  80.5  96.1  97.1  116.4  9. 5  37.9  37.3  82.2  80.1  78.3  106.9  11. 0  21.9  16.8  66.7  59.7  83.7  77.8  3. 5  520.9  609.5  433.4  480.4  331.2  340.9  5. 0  77.5  117.3  143.4  145.5  144.8  138.3  6. 5  36.6  37.7  73.9  83.0  106.8  108.6  8. 0  23.2  20.2  65.7  78.0  103.4  103.6  9. 5  25.2  22.5  61.7  69.0  89.9  92.2  11. 0  21.1  15.4  71.3  58.5  73.0  63.9  H y d r o p h o b i c i t y o f unheated d i s p e r s i o n s . ^ H y d r o p h o b i c i t y o f heated d i s p e r s i o n s .  a  So  0. 7  75 c u r v i l i n e a r manner, not u n l i k e the e f f e c t of pH on S . The o v e r a l l e f f e c t of Q  i n c r e a s i n g N a C l c o n c e n t r a t i o n to 0.35M was to decrease zeta p o t e n t i a l r a p i d l y , w i t h l i t t l e change i n zeta p o t e n t i a l between 0.35M and 0.7M N a C l .  T h i s was  not unexpected as at u n i v a l e n t e l e c t r o l y t e c o n c e n t r a t i o n s of a p p r o x i m a t e l y 0.1 m o l a l , the t h i c k n e s s of the e l e c t r i c  double  layer  surrounding colloidal  p a r t i c l e s , ( t o w h i c h the zeta p o t e n t i a l i s d i r e c t l y r e l a t e d ) , i s suppressed and becomes of n e g l i g i b l e p r o p o r t i o n s . T h i s r e s u l t s i n marked changes i n the p r o p e r t i e s of f l e x i b l e macromolecules such as p r o t e i n s ( F r a n k s and E a g l a n d , 1975).  B o t h s u c c i n y l a t i o n and N a C l appeared to lower the i s o e l e c t r i c point;  i.e., t h e pH at w h i c h the zeta p o t e n t i a l was zero s h i f t e d to a more a c i d i c pH as s u c c i n y l a t i o n and N a C l c o n c e n t r a t i o n i n c r e a s e d . In aqueous s o l u t i o n , the c h a r g e d groups of p r o t e i n molecules are a t t r a c t e d to the aqueous phase while nonpolar ( h y d r o p h o b i c ) groups t e n d to a v o i d the aqueous phase and become d i s p l a c e d toward the c e n t e r of the molecule.  However, due to the p r o p o r t i o n of h y d r o p h o b i c to h y d r o p h i l i c r e s i d u e s  as w e l l as c o n s t r a i n t s imposed by the amino a c i d sequence, i t i s not p o s s i b l e for a l l h y d r o p h o b i c groups to be b u r i e d i n the i n t e r i o r of the molecule and those h y d r o p h o b i c r e s i d u e s f o r c e d to remain on the s u r f a c e c o n t r i b u t e to the s u r f a c e h y d r o p h o b i c i t y of the molecule. H y d r o p h o b i c i n t e r a c t i o n s i n p r o t e i n s also serve to b r i n g t o g e t h e r groups that c a n p a r t i c i p a t e i n hydrogen or i o n i c b o n d i n g i n t h e absence of water, t h u s each of t h e types of b o n d i n g aids i n f o r m a t i o n of the o t h e r s i n d e t e r m i n i n g p r o t e i n c o n f o r m a t i o n (White et a l . , 1973). Charged  colloidal  particles  such as p r o t e i n molecules i n an i o n i c  environment are s u r r o u n d e d by an e l e c t r i c a l double l a y e r of ions; an inner r e g i o n of s t r o n g l y a d s o r b i n g c o u n t e r i o n s and an o u t e r r e g i o n where the ions  76 are  d i f f u s e l y d i s t r i b u t e d a c c o r d i n g to a balance between e l e c t r i c a l f o r c e s and  random t h e r m a l motion ( D i c k i n s o n and S t a i n s b y , 1982).  The  zeta p o t e n t i a l  r e f e r s to the e l e c t r i c a l p o t e n t i a l at the s u r f a c e of shear between the i n n e r " S t e r n " l a y e r and the d i f f u s e o u t e r l a y e r (Powrie and T u n g , 1976). The DLVO (Derjaguin-Landau-Verwey-Overbeek) s t a b i l i t y of a c o l l o i d a l  t h e o r y of c o l l o i d a l s t a b i l i t y r e l a t e s the  s u s p e n s i o n to a balance between van der Waals  a t t r a c t i v e f o r c e s and e l e c t r o s t a t i c r e p u l s i v e f o r c e s (from e l e c t r i c a l f o r c e s of  identical sign).  When the double l a y e r i s t h i c k and r e p u l s i v e f o r c e s  exceed a t t r a c t i v e f o r c e s , f l o c c u l a t i o n w i l l be r e s i s t e d and the s u s p e n s i o n will  be  stable.  The  a d d i t i o n of n e u t r a l s a l t s , however, decreases the  e f f e c t i v e r a d i u s of the i o n i c potential.  double  l a y e r w i t h a l o w e r i n g of the  zeta  The e f f e c t of ions on the double l a y e r depends on t h e i r v a l e n c y  and c o n c e n t r a t i o n .  With monovalent c o u n t e r i o n s such as sodium or potassium,  the  double l a y e r w i l l be t h i c k because of the low charge to volume r a t i o of  the  ions, and the shear plane w i l l l i e i n a r e g i o n of h i g h p o t e n t i a l .  With  m u l t i v a l e n t ions, the charge to volume r a t i o i s much l a r g e r and most of the c o u n t e r i o n charge w i l l l i e w i t h i n the shear p l a n e , w h i c h w i l l t h e r e f o r e be i n a r e g i o n of much lower p o t e n t i a l t h a n f o r monovalent ions.  Since the added  salt does not a f f e c t the van der Waals a t t r a c t i v e f o r c e s , the r e p u l s i v e energy b a r r i e r s between c o l l o i d a l p a r t i c l e s i s lowered or lost and f l o c c u l a t i o n result.  may  The S c h u l z e - H a r d y r u l e d e s c r i b e s the minimum c o n c e n t r a t i o n of ions  n e c e s s a r y to cause f l o c c u l a t i o n as b e i n g a p p r o x i m a t e l y p r o p o r t i o n a l to the s i x t h power of the charge and i s almost independent of the i o n i c species ( D i c k i n s o n and S t a i n s b y , 1982). monovalent  T h e r e f o r e , f a r fewer m u l t i v a l e n t ions t h a n  ions are r e q u i r e d to cause f l o c c u l a t i o n .  77 Melander and H o r v a t h (1977) and S a l a h u d d i n et a l . (1983) d i s c u s s e d the i n t e r p l a y of e l e c t r o s t a t i c  and  hydrophobic  interactions which result i n  s a l t i n g - i n and s a l t i n g - o u t of p r o t e i n s i n s o l u t i o n . T h e y suggested an inverse r e l a t i o n s h i p between the r e l a t i v e s u r f a c e h y d r o p h o b i c i t y and the frequency of c h a r g e d groups. As the c h a r g e d groups are assumed to be e x c l u s i v e l y on the p r o t e i n s u r f a c e , a h i g h e r f r e q u e n c y of c h a r g e d groups i s l i k e l y to r e s u l t i n a lower f r e q u e n c y of non-polar groups on the s u r f a c e , w i t h a r e s u l t a n t decrease in  surface  hydrophobicity  w i t h i n c r e a s i n g charge frequency.  Thus  with  i n c r e a s e d s u c c i n y l a t i o n , the charge f r e q u e n c y and e l e c t r o n e g a t i v i t y i n c r e a s e d and the s u r f a c e h y d r o p h o b i c i t y decreased. the  e f f e c t i v e charge f r e q u e n c y  on  In the presence of N a C l , however,  the p r o t e i n molecules d r o p p e d w i t h a  r e s u l t a n t increase i n the number of h y d r o p h o b i c groups exposed at the s u r face.  I n t e r m o l e c u l a r h y d r o p h o b i c i n t e r a c t i o n s between exposed h y d r o p h o b i c  amino a c i d r e s i d u e s of p r o t e i n molecules are t h e r e f o r e l i k e l y to be  important  in salt-induced protein-protein interactions. For c a n o l a i s o l a t e under the c o n d i t i o n s of s u c c i n y l a t i o n , pH and s t r e n g t h of the present  study, S  Q  and  zeta p o t e n t i a l were r e l a t e d by  ionic the  quadratic relationship: S  D  where S  = 14.552 z.p. + 0.158 Q  z.p.  2  + 355.54 (R = 2  0.901, p<0.001, n=54)  (6)  i n c r e a s e d s l o w l y i n i t i a l l y and t h e n more r a p i d l y as e l e c t r o n e g a t i v i t y  decreased. Hydrophobicity  was  measured w i t h the f l u o r e s c e n t probe ANS  which is  composed of aromatic r i n g s . Hayakawa and N a k a i (1985a) suggested that protein h y d r o p h o b i c i t y may  be  c l a s s i f i e d into two  a l i p h a t i c , as  i n f l u e n c e d by  respectively.  These may  aromatic  and  h y d r o p h o b i c i t i e s , aromatic  and  a l i p h a t i c amino a c i d r e s i d u e s ,  be r e l a t e d i n d i f f e r e n t ways to p r o t e i n f u n c t i o n -  78 ality.  L i - C h a n et a l . (1985) found that both aromatic and a l i p h a t i c hydropho-  b i c i t i e s were s i g n i f i c a n t p r e d i c t o r s of e m u l s i f y i n g and f a t - b i n d i n g p r o p e r t i e s of s a l t s o l u b l e muscle p r o t e i n s , but Hayakawa and N a k a i (1985b) found  no  d i f f e r e n c e between the two types of h y d r o p h o b i c i t y measurements i n r e l a t i o n to the thermal p r o p e r t i e s of ovalbumin.  In the present study, o n l y ANS  hydro-  p h o b i c i t y was measured, as d i f f i c u l t y was e x p e r i e n c e d i n o b t a i n i n g h y d r o p h o b i c i t y measurements w i t h the a l i p h a t i c h y d r o p h o b i c i t y probe c i s - p a r i n a r i c a c i d (CPA)  at pH 5 and lower.  (1985) who  S i m i l a r d i f f i c u l t y was e x p e r i e n c e d by L i - C h a n et a l .  suggested that h y d r o p h o b i c i t y measurements w i t h t h i s probe at low  pH must be i n t e r p r e t e d w i t h c a u t i o n since CPA was found to become l e s s s o l u b l e at pH  v a l u e s below 5.  The  quantum y i e l d of the ANS  probe i s i n s e n s i t i v e  to pH i n t h i s range ( G i b r a t and Grignon, 1982), however, so ANS was  used t h r o u g h o u t t h i s study as an index of  C.  S t e a d y Shear R h e o l o g y and  fluorescence  hydrophobicity.  Microstructure  A l l d i s p e r s i o n s f o l l o w e d power-law or power-law p l a s t i c flow (Table  2.5).  The  d i s p e r s i o n s were p s e u d o p l a s t i c  behavior  (shear r a t e t h i n n i n g )  as  i n d i c a t e d by a flow behavior i n d e x (n) l e s s t h a n 1.0 f o r each d i s p e r s i o n .  As  the shear r a t e i n c r e a s e d , the p a r t i c l e s may have a l i g n e d w i t h the shear p l a n e s and o f f e r e d l e s s r e s i s t a n c e to flow, t h e r e b y e x h i b i t i n g decreased v i s c o s i t y . The  apparent v i s c o s i t i e s of each d i s p e r s i o n at 10 s  i n F i g u r e s 2.4 and  2.5, r e s p e c t i v e l y . The  and 1000 s~* are shown  - 1  s u c c i n y l a t e d i s o l a t e s had  higher  apparent v i s c o s i t i e s t h a n the u n m o d i f i e d i s o l a t e under n e a r l y a l l c o n d i t i o n s of pH and i o n i c s t r e n g t h . s"  1  and 1000 s "  1  The  treatment e f f e c t s on apparent v i s c o s i t y at 10  are p r e s e n t e d i n T a b l e 2.6.  At 10 s  the apparent v i s c o s i t y  - 1  i n c r e a s e d w i t h s u c c i n y l a t i o n and pH, while at 1000 s " , i n t e r a c t i o n s between 1  T a b l e 2.5.  Power-law and power-law p l a s t i c f l o w parameters o f 11.4Z c a n o l a I s o l a t e d i s p e r s i o n s .  NaCl Concentration  0.0 Succinic Anhydride (%)  (Molar)  0.35  0.7  m o r m' (mPa s )  n o r n'  cy (mPa)  m o r m' (mPa s )  n o r n'  (jy (mPa)  3.5 5.0 6.5 8.0 9.5 11.0  6.2 47.3 23.2 33.1 160.2 1280  0.943 0.693 0.865 0.917 0.777 0.506  0 168 51 0 0 0  7.4 22.4 57.6 59.6 26.6 553.6  0.921 0.813 0.733 0.747 0.909 0.590  0 0 206 196 24 0  18.3 34.2 89.5 85.3 40.1 245.8  3.5 5.0 6.5 8.0 9.5 11.0  43.2 53.1 228.3 358.2 410.8 1269  0.718 0.853 0.721 0.688 0.670 0.563  153 0 0 0 0 0  43.6 861.0 567.8 320.4 329.6 1268  0.710 0.570 0.604 0.667 0.667 0.512  159 0 0 0 0 0  28.9 301.1 608.1 353.4 405.5 1215  0.740 0.677 0.589 0.655 0.648 0.510  73 0 0 0 0 0  3.5 5.0 6.5 8.0 9.5 11.0  41.2 905.2 197.2 265.3 279.0 1191  0.683 0.601 0.755 0.758 0.754 0.597  322 0 0 0 0 0  45.8 1423 379.6 279.2 202.9 1083  0.690 0.539 0.643 0.698 0.732 0.543  186 0 0 0 0 0  59.9 1475 706.7 275.9 320.0 873.8  0.685 0.532 0.566 0.698 0.665 0.561  267 0 0 0 0 0  pH  n  n  m o r m' (mPa s ) n  n o r n'  Oy (raPa)  0.790 0 0.763 0 0.699 476 407 0.722 76 0.819 0 . 6 9 4 1486  F i g u r e 2.4.  A p p a r e n t v i s c o s i t y at 10 s"* f o r 11.4% c a n o l a i s o l a t e d i s p e r s i o n s : (A) unmodified; (B) 5.2% SA; (C) 14.2% SA. 1  80  70  Legend A OM NoCI  60  O 0.35M NaCl 0.7M NoCI  50  40  30  20  10  0  1  4  1  5  1 6  1  I  7  1  1  10  I  8  9  11  1 8  1  1  r  9  10  11  80  Legend  70 &  OM NoCI  60  50  40  SO  20  10  0  -I  4  1  I  I  5  6  7  80 70 60 50 4030 20 100  -I  4  1  5  I  I  6  7  1  I  8 P  9  1  10  I  It  H  v i s c o s i t y at 1000 s " f o r 11.4% canola isolate : (A) u n m o d i f i e d ; (B) 5.2% SA; (C) 14.2% SA. 1  82  T a b l e 2.6.  T r e n d comparison a n a l y s e s o f t h e e f f e c t s o f s u c c i n y l a t i o n , pH and N a C l on apparent v i s c o s i t y o f 11.AZ c a n o l a i s o l a t e d i s p e r s i o n s (n=54).  Dependent Variable  Independent Variable  Coefficient  Succinylation  3.475  0.0003  19.986  0.0001  "I) Apparent Viscosity (10 s" R  2  = 0.404  S.E.a - 113.91  pH Constant  F-prob.  166.230  F-prob. = 0.0000 Apparent Viscosity (1000 s~l) R  a  2  = 0.682  Succinylation  0.566  0.0000  pH  2.934  0.0000  -0.0504  0.0283 0.0164  S.E. = 10.665  Succinylation x pH  F-prob. = 0.0000  NaCl x pH  -1.294  Constant  28.530  Standard error of estimate.  2  83 s u c c i n y l a t i o n and the square of pH and between N a C l and pH were s i g n i f i c a n t a l o n g w i t h s u c c i n y l a t i o n and pH as main e f f e c t s . K i n s e l l a and S h e t t y (1979) r e p o r t e d i n c r e a s e d v i s c o s i t y w i t h s u c c i n y l a t i o n of a r a c h i n and c o n c l u d e d that a decrease i n v i s c o s i t y e x p e c t e d w i t h p r o t e i n d i s s o c i a t i o n by s u c c i n y l a t i o n was more t h a n c o u n t e r b a l a n c e d by u n f o l d i n g of the d i s s o c i a t e d components and the r e s u l t a n t increase i n hydrodynamic volume. In e x t r e m e l y d i l u t e d i s p e r s i o n s the t o t a l v i s c o s i t y e f f e c t i s simply the sum  of the e f f e c t s caused by each of the i n d i v i d u a l suspended p a r t i c l e s , but  as the c o n c e n t r a t i o n  of the d i s p e r s e d  phase i n c r e a s e s , the e f f e c t s of the  suspended p a r t i c l e s are no longer independent. The flow b e h a v i o r of c o n c e n t r a t e d p r o t e i n d i s p e r s i o n s r e f l e c t s complex i n t e r a c t i o n s between p r o t e i n and solvent as w e l l as p r o t e i n - p r o t e i n i n t e r a c t i o n s as i n f l u e n c e d by  particle  s i z e , shape and hydrodynamic i n t e r a c t i o n s . The apparent v i s c o s i t y of p r o t e i n d i s p e r s i o n s may  r i s e e x p o n e n t i a l l y w i t h c o n c e n t r a t i o n ( T u n g , 1978). F r i s c h  and Simha (1956) suggested that the hydrodynamic volume and shape of the p r o t e i n are the most important f a c t o r s g o v e r n i n g the flow p r o p e r t y of the p r o t e i n . The hydrodynamic volume i s dependent upon the m o l e c u l a r s i z e and the degree of h y d r a t i o n of the molecule i n s o l u t i o n .  P r o t e i n i n s o l u t i o n can  e x i s t i n a wide range of s i z e s from s i n g l e molecules to l a r g e aggregates. Since the degree of a g g r e g a t i o n of  the  a f f e c t s the hydrodynamic or e f f e c t i v e volume  p r o t e i n , i t g r e a t l y a f f e c t s the  dispersions.  Lee and Rha  r h e o l o g i c a l p r o p e r t i e s of  protein  (1979) r e p o r t e d that the p a r t i c l e s i z e d i s t r i b u t i o n  of soy p r o t e i n d i s p e r s i o n s a f f e c t e d the hydrodynamic volume and v i s c o s i t y of the d i s p e r s i o n ; d i s p e r s i o n s w i t h l a r g e r p a r t i c l e s were higher  in viscosity  t h a n d i s p e r s i o n s w i t h small p a r t i c l e s . T h e y suggested that the s i z e and shape of the p r o t e i n aggregates were l a r g e l y determined by h y d r o p h o b i c e f f e c t s .  84 Thus such f a c t o r s as p r o t e i n s o l u b i l i t y , the s i z e , shape and number of a g g r e gates, hydrodynamic volume, p r o t e i n - s o l v e n t , and p r o t e i n - p r o t e i n i n t e r a c t i o n s a l l c o n t r i b u t e to the flow p r o p e r t i e s of p r o t e i n d i s p e r s i o n s under steady shear. For the u n m o d i f i e d i s o l a t e , l i g h t m i c r o g r a p h s r e v e a l e d that the d i s p e r sions were l a r g e l y composed of r e l a t i v e l y s p h e r i c a l aggregates w h i c h r a n g e d i n s i z e from the l i m i t of r e s o l u t i o n of the l i g h t microscope to more t h a n  100  micrometers i n diameter ( F i g u r e 2.6A-D). In view of the h i g h p r o t e i n c o n c e n t r a t i o n of the i s o l a t e , the aggregates were assumed to be mostly p r o t e i n . Jones (1980) r e p o r t e d  a similar microstructure  f o r canola i s o l a t e p r e p a r e d  e i t h e r w i t h or without potassium l i n o l e a t e or t r y p s i n . Wolf and Baker (1980) f o u n d that i s o l a t e d soy p r o t e i n was collapsed roughly  l a r g e l y composed of whole, b r o k e n  s p h e r i c a l p r o t e i n bodies.  and  B e v e r i d g e et a l . (1984) noted  that the p r o t e i n bodies of the soy i s o l a t e Promine-D d i d not d i s s o l v e comp l e t e l y i n water and Promine-D gels had v e r y l a r g e p a r t i c l e s (up to 100 m i c r o meters i n diameter) embedded i n a g e l matrix. At 10 s ~ l the apparent v i s c o s i t y of the d i s p e r s i o n s w i t h N a C l was low  up  to pH 9.5, while the d i s p e r s i o n s without N a C l i n c r e a s e d i n v i s c o s i t y from pH 8 to 11.  At pH  6.5  and  8, the h i g h e r apparent v i s c o s i t i e s of the  dispersions  w i t h N a C l r e f l e c t e d the y i e l d s t r e s s of these d i s p e r s i o n s w h i c h a f f e c t e d the apparent v i s c o s i t i e s at low r a t e s of shear. of y i e l d s t r e s s was n e g l i g i b l e . s " l and 1000 s "  1  and  - 1  i n c r e a s e i n apparent v i s c o s i t y at both 10  as the pH i n c r e a s e d to 11 was p r o b a b l y a r e s u l t of i n c r e a s e d  p r o t e i n s o l u b i l i t y and Ishino  The  At 1000 s , however, the e f f e c t  s w e l l i n g of the aggregates at t h i s pH.  Okamoto (1975) suggested that the  increased  In a d d i t i o n ,  v i s c o s i t y of  soy  p r o t e i n d i s p e r s i o n s at h i g h pH may have been caused by u n f o l d i n g , d i s s o c i a t i o n and  i n t e r a c t i o n of the p r o t e i n molecules.  85  Figure 2.6. A-D, Light micrographs of 11.4% dispersions of unmodified canola isolate: (A) pH 3.5, 0.0M NaCl (BF); (B) pH 9.5, 0.0M NaCl (PC); (C) pH 11.0, 0.0M NaCl (PC); (D) pH 11.0, 0.7M NaCl (PC); BF=bright field, PC=phase contrast, bar=200 (im.  86  Figure 2.6. (cont.) E-H, Light micrographs of 11.4% dispersions of succinylated canola isolate (5.2% SA): (E) pH 3.5, 0.0M NaCl (BF); (F) pH 5.0, 0.35M NaCl (PC); (G) pH 8.0, 0.0M NaCl (PC); (H) pH 8.0, 0.35M NaCl (PC), bar=200 /-cm.  87 The  m i c r o s t r u c t u r e of the i s o l a t e s seemed to p l a y an important r o l e i n  the steady shear r h e o l o g i c a l p r o p e r t i e s of the i s o l a t e s .  F o r the unmodified  i s o l a t e the p r o t e i n appeared to be present as s p h e r i c a l l y shaped aggregates w h i c h seemed to remain e s s e n t i a l l y i n t a c t except f o r the sample at pH  11  without  pH  N a C l ( F i g u r e 2.6C).  The  aggregates appeared to s w e l l as the  i n c r e a s e d , and phase c o n t r a s t i l l u m i n a t i o n was r e q u i r e d to best v i s u a l i z e the aggregates, i n d i c a t i n g a s o f t e n i n g of aggregate m i c r o s t r u c t u r e . a g g r e g a t e s were i n t a c t and s o l u b i l i t y was s l i p past  one  another  apparent v i s c o s i t y was  with low.  little  When the  low t h e y would have been able to  interaction during  steady shear,  thus  At pH 11 where the d i s p e r s i o n s w i t h N a C l s t i l l  had m o s t l y i n t a c t aggregates ( F i g u r e 2.6D), the i n c r e a s e d apparent v i s c o s i t y was p r o b a b l y the r e s u l t of a more v i s c o u s c o n t i n u o u s phase due to i n c r e a s e d p r o t e i n s o l u b i l i t y , u n f o l d i n g and  d i s s o c i a t i o n , i n a d d i t i o n to s w e l l i n g and  s o f t e n i n g of the aggregates w h i c h would increase t h e i r hydrodynamic volume and aggregate-aggregate interactions. The  flow behavior of the s u c c i n y l a t e d i s o l a t e s also appeared to r e f l e c t  an i n t e r a c t i o n between the s o l u b l e and d i s p e r s e d phases. For both 5.2% SA and 14.2%  SA d i s p e r s i o n s , p r o t e i n s o l u b i l i t y was  t h i s pH  most of the  p r o t e i n was  present  at a minimum near pH 3.5.  At  as aggregates of v a r i o u s s i z e s  d i s p e r s e d i n a t h i n c o n t i n u o u s phase e s s e n t i a l l y devoid of s o l u b l e p r o t e i n ( F i g u r e 2.6E); thus under steady shear the apparent v i s c o s i t y of these d i s p e r sions was  low.  Between pH  d r a m a t i c a l l y so that at pH  3.5  and  6.5  the p r o t e i n s o l u b i l i t y  increased  5 the d i s p e r s i o n s were i n an intermediate state  where the p r o t e i n aggregates were d i s p e r s e d i n c o n t i n u o u s phases of v a r y i n g p r o t e i n c o n c e n t r a t i o n and  apparent v i s c o s i t y ( F i g u r e 2.6F).  The  combined  e f f e c t of i n t e r a c t i o n s of the aggregates w i t h each other as i n f l u e n c e d  by  88 t h e i r s i z e and shape and the v i s c o s i t y of the c o n t i n u o u s phase l e d to v e r y high  apparent v i s c o s i t i e s f o r some of the d i s p e r s i o n s , e s p e c i a l l y those  s u c c i n y l a t e d w i t h 14.2% SA. A s i m i l a r e f f e c t on the v i s c o s i t y of soy p r o t e i n d i s p e r s i o n s i n t h i s pH range was r e p o r t e d by Lee and Rha (1979) who suggested that the increase  i n apparent v i s c o s i t y was r e l a t e d to the growth of the  particle size. Above pH 6.5, the apparent v i s c o s i t y was e s s e n t i a l l y that of the s o l u b l e p r o t e i n w i t h some c o n t r i b u t i o n from i r r e g u l a r l y shaped i n s o l u b l e m a t e r i a l that may have been c e l l w a l l fragments ( F i g u r e 2.6G), while at pH 11 the i n c r e a s e d v i s c o s i t y may have r e s u l t e d from a l k a l i i n d u c e d p r o t e i n u n f o l d i n g and perhaps d i s s o c i a t i o n i n t o s u b u n i t s of molecules not a l r e a d y d i s s o c i a t e d by s u c c i n y l a tion  (Ishino  and Okamoto, 1975; Whitaker, 1980).  The c o n t i n u o u s phase  of s u c c i n y l a t e d i s o l a t e d i s p e r s i o n s took on a s l i g h t l y coarser appearance as NaCl concentration increased aggregation.  ( F i g u r e 2.6H), p r o b a b l y as a r e s u l t of p r o t e i n  A decrease i n p r o t e i n s o l u b i l i t y also r e s u l t e d from  increased  N a C l c o n c e n t r a t i o n i n t h i s pH range. A l t h o u g h the 5.2% SA i s o l a t e i s shown i n Figure  2.6, b o t h  succinylated  i s o l a t e s had s i m i l a r m i c r o s t r u c t u r e s  with  r e s p e c t to pH and N a C l .  D.  Thermally Induced Gelation T h e r m a l l y i n d u c e d gels or g e l - l i k e m a t e r i a l s were formed at t w e n t y - e i g h t  of a p o s s i b l e f i f t y - f o u r combinations of s u c c i n y l a t i o n , N a C l c o n c e n t r a t i o n and pH.  V i s u a l l y , t h e y appeared as t r a n s l u c e n t g e l s , opaque g e l s , or g e l - l i k e  precipitates.  T h i s was s i m i l a r to the e f f e c t s of n e u t r a l s a l t s and pH on the  appearance of ovalbumin gels (Hegg et a l . , 1979). F o r u n s u c c i n y l a t e d  canola  89 i s o l a t e o n l y four of a p o s s i b l e e i g h t e e n gels formed, while twelve gels formed for each of the two l e v e l s of s u c c i n y l a t i o n . The storage modulus (G ) f o r each g e l i n c r e a s e d s l i g h t l y , w i t h o s c i l l a t o r y 1  frequency, while the dynamic v i s c o s i t y (ry ) decreased i n a l i n e a r manner over 1  the same f r e q u e n c y range when p l o t t e d on l o g a r i t h m i c c o o r d i n a t e s .  A repre-  s e n t a t i v e rheogram i s shown i n F i g u r e 2.7; a l l samples had s i m i l a r s t r a i g h t l i n e dynamic shear behavior.  The equation of each l i n e was determined by  l e a s t - s q u a r e s l i n e a r r e g r e s s i o n (Table 2.7A.B), and v a l u e s of storage modulus, loss modulus (G") and loss tangent were c a l c u l a t e d f o r a f r e q u e n c y of 10 s"*. The  e f f e c t of f r e q u e n c y  the flow behavior  on the dynamic v i s c o s i t y of the gels was s i m i l a r to  of p s e u d o p l a s t i c f l u i d s under steady  shear.  F o r many  polymer s o l u t i o n s , shear r a t e dependent apparent v i s c o s i t y c l o s e l y p a r a l l e l s f r e q u e n c y dependent dynamic v i s c o s i t y over the same range of shear r a t e and frequency  ( C o x and Merz, 1958), but t h i s was not examined i n the present  study. For the u n s u c c i n y l a t e d i s o l a t e , gels were formed at pH 9.5 i n the absence of N a C l and at pH 11 at a l l N a C l c o n c e n t r a t i o n s . although  the g e l at pH 11 without  character.  As seen i n F i g u r e  A l l gels were opaque  N a C l had a great d e a l of t r a n s l u c e n t  2.8A, as pH i n c r e a s e d  i n c r e a s e d s l i g h t l y for the gels without NaCl.  from 9.5 to 11, G  1  At pH 11, G i n c r e a s e d drama1  t i c a l l y as N a C l c o n c e n t r a t i o n i n c r e a s e d to 0.35M, f o l l o w e d by a decrease i n G' from 0.35M to 0.7M N a C l .  The treatment e f f e c t s on the loss modulus of each  g e l p a r a l l e l e d the e f f e c t s on storage modulus except f o r the gels without N a C l where a s l i g h t decrease i n G" was seen as the pH was r a i s e d from 9.5 to 11 ( F i g u r e 2.9A). The loss tangent of the gels without N a C l decreased as the pH i n c r e a s e d from 9.5 to 11 ( F i g u r e 2.10A), while at pH 11, the l o s s tangent  ^  3.5  JD O O CO  rj)  Frequency (Log Scale) Figure 2.7. Storage moduli and dynamic viscosities of 5.2% SA, pH 6.5, 0.7M NaCl canola isolate gel as a function of oscillatory frequency. VO o  T a b l e 2.7A.  Dynamic shear f l o w b e h a v i o r parameters o f c a n o l a i s o l a t e  gels.  NaCl Concentration (Molar)  Succinic anhydride (%)  pH  c (Pa s )  0  9.5  21.09  0.210 0.996  11.0  13.65  0.163  0.986  5.0  2.45 0.270  0.990  6.5  5.80 0.245 -1  0.982  5.2  8.0 9.5 11.0 14.2  5.0  138.5  -  r2  -  0.139 0.999  6.5  3.56  8.0  -  -  -  —  —  —  9.5 11.0 Did not gel.  d  d  0.7  0.35  0.0  0.230 0.974  c (Pa s ) d  107.2  d  -  r2  c (Pa s )  -  -  d  d  -  r2  -  0.142  0.999  98.25  0.142  0.999  80.19  0.123  0.992  37.90  0.157  0.990  88.39  0.115  0.992  0.084  0.999  40.74 0.116  0.999  80.88  0.111  0.999  36.83 0.126  0.999  84.42 0.119  0.999  25.50 0.089  0.988  68.39  0.033  0.999  0.111  0.999  202.9  167.9  138.7  0.092  0.999  33.48 0.098  0.999  66.97 0.096  0.999  10.30  0.105  0.999  31.46  0.112  0.996  12.27  0.118  0.996  30.90 0.112  0.996  10.39  0.072  0.999  28.82 0.105  0.992  T a b l e 2.7B.  Dynamic shear s t o r a g e b e h a v i o r parameters o f c a n o l a i s o l a t e  gels.  NaCl Concentration (Molar) 0.35  0.0 Succinic Anhydride (%) 0  PH 9.5  b  -  b  r  -  -  2  a (Pa s ) b  -  b  r2  -  -  670.4  0.103  0.988  517.7  0.105  0.968  5.0  10.44 0.144  0.891  328.9  0.095  0.990  150.7  0.096  0.972  6.5  28.36 0.155 0.950 _1  510.0  0.120  0.992  565.6  0.133  0.992  224.2  0.113  0.988  395.9  0.118  0.988  230.9  0.100  0.992  435.8  0.108  0.982  178.0  0.071  0.980  489.3  0.077  0.986  9.5  -  11.0  -  5.0  531.1  6.5 8.0 9.5 11.0 ^ i d not gel.  0.978  a (Pa s )  0.952  8.0  14.2  70.77 0.130 108.2  r2  b  b  0.109  11.0 5.2  a (Pa s )  0.7  -  -  -  -  -  0.130  0.984  556.7  0.131  0.986  484.0  0.121  0.986  10.30 0.127  0.822  212.9  0.096  0.996  362.4  0.104  0.986  59.24 0.056  0.974  188.7  0.101  0.996  -  65.60 0.109  0.984  205.0  0.101  0.988  —  54.21  187.9  0.086  0.992  -  -  -  0.109 0.976  F i g u r e 2.8.  Storage modulus at 10 s " f o r 11.4% canola isolate gels: (A) u n m o d i f i e d ; (B) 5.2% SA; (C) 14.2% SA. 1  94 250-  200-  n •3  150-  Legend EZ3 OM NoCI Ea 0.35M NoCI K ] 0.7M NoCI  o  m  100-  50-  6.5  9.5  250  200-  _3  •3  250  200-  o .0-.  Figure 2.9. Loss modulus at 10 s" for 11.4% canola isolate gels: (A) unmodified; (B)5.2% SA; (C) 14.2% SA. 1  95  Figure 2.10. Loss tangent at 10 s" 1 for 11.4% canola isolate gels: (A) unmodified; (B) 5.2% SA; (C) 14.2% SA.  96 increased  with NaCl concentration.  Since  the l o s s tangent r e f l e c t s the  p r o p o r t i o n of v i s c o u s to e l a s t i c c h a r a c t e r i n a v i s c o e l a s t i c m a t e r i a l ( F e r r y , 1980), the gels became p r o p o r t i o n a t e l y more e l a s t i c as the pH i n c r e a s e d to 11 and as N a C l c o n c e n t r a t i o n decreased at pH 11. For t h e i s o l a t e m o d i f i e d w i t h 5.2% s u c c i n i c anhydride  (54% m o d i f i c a t i o n  of amino g r o u p s ) , gels formed at pH 5 and 6.5 both w i t h and without w h i l e from pH 8.5 to 11, gels formed o n l y i n the presence of N a C l .  NaCl,  The gels  at pH 5 were v e r y opaque and p a s t y while a l l others were t r a n s l u c e n t and springy.  F o r the pH 5 g e l s , i n c r e a s i n g the N a C l c o n c e n t r a t i o n to 0.35M  i n c r e a s e d G' and G" f o l l o w e d b y a decrease i n these parameters at 0.7M N a C l ( F i g u r e s 2.8B and 2.9B), F o r the t r a n s l u c e n t gels (pH 6.5 and above), G' and G" i n c r e a s e d as N a C l c o n c e n t r a t i o n i n c r e a s e d . B o t h G and G" were greatest at 1  pH 6.5 f o r each l e v e l of N a C l .  With the e x c e p t i o n of the sample at pH 6.5,  0.35M NaCl, t h e l o s s tangent decreased, i n d i c a t i n g that the e l a s t i c component of each g e l i n c r e a s e d as pH i n c r e a s e d . The l o s s tangent was dependent on N a C l c o n c e n t r a t i o n as w e l l ( F i g u r e 2.10B). Gels without N a C l h a d a h i g h e r loss tangent t h a n gels w i t h 0.35M NaCl, w h i l e at each pH except 11, gels w i t h 0.7M N a C l had a h i g h e r l o s s tangent t h a n gels w i t h 0.35M N a C l . The i s o l a t e s u c c i n y l a t e d w i t h 14.2% s u c c i n i c anhydride of amino groups) had s i m i l a r  g e l a t i o n behavior  (84% m o d i f i c a t i o n  to the 5.2% SA i s o l a t e .  Opaque, p a s t y gels were formed at pH 5 at a l l N a C l c o n c e n t r a t i o n s  while  t r a n s l u c e n t gels formed at pH 6.5 and above. From pH 8.5 to 11, gels formed o n l y i n the presence of N a C l , w h i l e at pH 6.5, the sample without N a C l d i d not quite form a s e l f - s u p p o r t i n g g e l but t h i c k e n e d c o n s i d e r a b l y upon h e a t i n g and appeared t r a n s l u c e n t and e l a s t i c .  F o r the t r a n s l u c e n t g e l s , both G' and G"  i n c r e a s e d w i t h N a C l c o n c e n t r a t i o n , while f o r the opaque g e l s , G' and G" were  97 h i g h e s t at 0.35M N a C l ( F i g u r e s 2.8C and 2.9C). Thus f o r a l l opaque gels, G and  1  G" f i r s t i n c r e a s e d and t h e n decreased as N a C l i n c r e a s e d while f o r a l l  t r a n s l u c e n t gels these parameters i n c r e a s e d as N a C l i n c r e a s e d .  F o r a l l gels  there was a close a s s o c i a t i o n between G' and G" where b o t h the e l a s t i c and v i s c o u s components i n c r e a s e d s i m u l t a n e o u s l y  ( F i g u r e 2.11).  The e f f e c t s of pH and N a C l on the l o s s tangent of the 14.2% SA gels are shown i n F i g u r e 2.IOC. As expected, the l o s s tangent f o r the g e l at pH 6.5 without N a C l , w h i c h appeared to be on the g e l t h r e s h o l d , was s u b s t a n t i a l l y higher  t h a n f o r the other  g e l s w h i c h were a l l s e l f - s u p p o r t i n g .  A l l loss  t a n g e n t s were s u b s t a n t i a l l y l e s s t h a n 1.0, however, i n d i c a t i n g that a l l gels were p r o p o r t i o n a t e l y more e l a s t i c t h a n v i s c o u s .  F o r the s e l f - s u p p o r t i n g  t r a n s l u c e n t g e l s , n e i t h e r pH nor i o n i c s t r e n g t h g r e a t l y a f f e c t e d the l o s s tangent.  The opaque gels had a higher loss tangent t h a n the s e l f - s u p p o r t i n g  t r a n s l u c e n t gels; thus even t h o u g h b o t h G' and G" were h i g h e r f o r the opaque gels, there was p r o p o r t i o n a t e l y l e s s e l a s t i c t h a n v i s c o u s c h a r a c t e r . The h i g h v i s c o e l a s t i c m o d u l i of the opaque gels appeared to c o n t r a d i c t the r e s u l t s of a p u n c t u r e t e s t as w e l l as v i s u a l o b s e r v a t i o n .  With the  p u n c t u r e test ( C h a p t e r 3) the f o r c e r e q u i r e d to r u p t u r e the opaque g e l s was of the  same order  or l e s s t h a n f o r the t r a n s l u c e n t g e l s .  appeared pasty, l a c k e d s p r i n g i n e s s and syneresed r e a d i l y .  The opaque  gels  A similar effect  was noted by G i l l and T u n g (1978a) w i t h t h e r m a l l y i n d u c e d gels from the 12S f r a c t i o n of r a p e s e e d . T h e y f o u n d that gels at pH 6.0 had lower apparent v i s c o s i t y i n steady shear but h i g h e r v i s c o e l a s t i c parameters i n dynamic shear t h a n gels at h i g h e r  pH.  Microscopic  examination showed the presence of  a g g r e g a t e s i n the pH 6.0 g e l and the a u t h o r s h y p o t h e s i z e d that the aggregates were r e s p o n s i b l e f o r h i g h l y e l a s t i c r e c o v e r i e s under the n o n - d e s t r u c t i v e small  900-r 800700O  ^  600  V)  D 3 TJ o  500  * 400 rj) O O  300 200  Legend  CO  tn  A  H  Unmodified  O 5.2% Succ. An. • 14.2% Succ. An.  100SL  50  100  150  200  Loss Modulus (Pa)  F i g u r e 2.11. Storage and loss moduli at 10 s " for 11.4% canola i s o l a t e gels ( s o l i d symbols i n d i c a t e opaque g e l s ) . 1  250  99 deformations a p p l i e d i n dynamic t e s t i n g .  Under steady shear c o n d i t i o n s ,  however, the f o r c e s between a g g r e g a t e s would be b r o k e n and the aggregates would t h e n be able to move r e a d i l y w i t h r e s p e c t to one another. As p r e v i o u s l y noted, the d i s p e r s i o n s that formed opaque gels i n the present s t u d y a l l c o n t a i n e d r e l a t i v e l y l a r g e p r o t e i n aggregates to some extent, and these gels would be expected to behave d i f f e r e n t l y under n o n - d e s t r u c t i v e and d e s t r u c t i v e t e s t i n g t h a n the t r a n s l u c e n t g e l s , w h i c h appeared to have a r e l a t i v e l y homogeneous g e l matrix. To  T h i s w i l l be d e s c r i b e d i n d e t a i l i n Chapter 3.  date, n e a r l y a l l r e p o r t s p u b l i s h e d on the thermal response of s u c -  c i n y l a t e d food p r o t e i n s have i n d i c a t e d i n c r e a s e d heat s t a b i l i t y and a decrease or  loss of g e l a t i o n a b i l i t y  (Kinsella  and Shetty,  1979), p r e s u m a b l y as a  r e s u l t of i n c r e a s e d charge r e p u l s i o n between molecules (Ma and Holme, 1982; Sato and Nakamura, 1977). I n c o n t r a s t , M i l l e r and G r o n i n g e r (1976) found that the g e l a t i o n a b i l i t y of f i s h p r o t e i n concentrate the amino groups were s u c c i n y l a t e d .  was improved when 43-59% of  C h o i et a l . (1981) r e p o r t e d that l i m i t e d  s u c c i n y l a t i o n of cottonseed p r o t e i n i n c r e a s e d the g e l s t r e n g t h of 20% p r o t e i n d i s p e r s i o n s , and Montejano et a l . (1984) found that s u c c i n y l a t e d egg white r e q u i r e d h i g h e r h e a t i n g temperatures f o r g e l a t i o n , but the gels had s i g n i f i c a n t l y g r e a t e r s t r e n g t h and d e f o r m a b i l i t y at f a i l u r e t h a n those from native egg white.  I n the present study, the e l e c t r o n e g a t i v i t y i n d u c e d b y s u c c i n y -  l a t i o n appeared to r e d u c e or prevent g e l a t i o n i n the absence of N a C l while the a d d i t i o n of N a C l overcame the charge r e p u l s i o n and allowed close a p p r o a c h and aggregation  of the p r o t e i n molecules i n t o a g e l upon h e a t i n g .  This i s  s u p p o r t e d b y the o b s e r v e d e f f e c t s of s u c c i n y l a t i o n l e v e l and i o n i c s t r e n g t h where G and G" d e c r e a s e d as s u c c i n y l a t i o n i n c r e a s e d from 54% to 84% of amino 1  groups.  S u c c i n y l a t i o n p r o g r e s s i v e l y i n c r e a s e d the e l e c t r o n e g a t i v i t y of the  100 protein molecules, while increasing the NaCl concentration overcame this effect. unsuccinylated  progressively  The effect of pH also supports this hypothesis, as the  isolate formed gels only at high pH (i.e. at high electro-  negativity) while succinylated isolates formed the firmest gels in the lower end of the pH range in which gels formed. There appeared to be an optimum NaCl concentration for maximization of the viscoelastic moduli as indicated by the effect of NaCl on the opaque gels, where the moduli first increased as NaCl concentration increased from 0.0M to 0.35M and then decreased to 0.7M NaCl, but the optimum did not appear to be reached with the translucent gels. It was apparent that degree of succinylation, pH and NaCl concentration were all important variables in the gel-forming ability of canola isolate and the viscoelastic properties of the gels. As these variables also influenced protein solubility, hydrophobicity,  zeta potential and flow behavior, which  in turn were expected to influence the viscoelastic properties of the gels, the relationships among these factors were examined by multiple regression analysis. Since the gels dissolved in 8M urea or 6M guanidine hydrochloride, covalent bonds such as disulfide were believed not to be involved in gel formation or stabilization. For multiple regression analysis, the viscoelastic parameters of the gels were used as dependent variables while potential independent variables included  power-law flow parameters from 11.4% isolate dispersions, zeta  potential, surface hydrophobicity  before  ( S ) or after ( S ) heating, and Q  e  protein solubility. When all gels were considered in the analysis, solubility, S , and their Q  interactions with each other and zeta potential (z.p.) accounted for 72% of the  variation in G  1  (Table  2.8). For G", solubility  (curvilinear), z.p.  101  T a b l e 2.8.  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n o f v i s c o e l a s t i c p a r a meters o f t h e r m a l l y induced c a n o l a i s o l a t e g e l s (n=28).  Dependent Variable  Independent Variable  Storage Modulus R  2  = 0.720  S.E.  a  - 156.18  F-prob. = 0.0000  Solubility So Solubility x S  Q  Solubility x Z.P. S  0  Coefficient -35.523  0.0000  -9.189  0.0004  0.291  0.0000  -0.200  0.0203  0.449  x Z.P.  0.0002  Constant  2.587 x 10  Loss Modulus  Solubility  8.695  R  Solubility  2  = 0.653  Zeta Potential  F-prob. = 0.0002  Zeta Potential  Loss Tangent R  a  2  S.E. = 44.24  S  2  = 0.510  e  2  x Z.P.  0.0137  0.202  0.0314  -0.482  -0.254  F-prob. = 0.0006  Constant  Standard error of estimate.  16.596  Solubility  S.E. = 0.0558  0.0063 0.0050  42.122  S Solubility x Z.P.  3  -0.711 x io-i  Constant  e  F-prob.  0.110 -0.637 0.174  X  io-i  X  IO"  X  IO"  X  IO"  0.0158  2  2  4  0.0002 0.0464 0.0015  102 (curvilinear)  and  the  i n t e r a c t i o n between S  e  and  z.p. were  significant  independent v a r i a b l e s (R =0.653) while for the l o s s t a n g e n t , s o l u b i l i t y , 2  and the i n t e r a c t i o n between s o l u b i l i t y and  S  e  z.p. were s i g n i f i c a n t (R =0.510). 2  Schmidt (1981), however, emphasized that the importance of s u b j e c t i v e or qualitative  e v a l u a t i o n of p r o t e i n g e l s should  not  be  underestimated,  as  m e a s u r a b l y s t r o n g p r o t e i n gels may range i n appearance from t r a n s l u c e n t and e l a s t i c to c u r d - l i k e and  opaque.  As p r e v i o u s l y noted, the gels c o u l d  d i v i d e d into two major c l a s s e s based on v i s u a l o b s e r v a t i o n , t r a n s l u c e n t opaque, and  i t has been h y p o t h e s i z e d  be and  t h a t , due to t h e i r m i c r o s t r u c t u r e , the  v i s c o e l a s t i c p r o p e r t i e s of these c l a s s e s of gels may  differ.  The  translucent  gels v a r i e d i n c h a r a c t e r from f i r m and s p r i n g y to a t h i c k m a t e r i a l that  was  g e l - l i k e and s p r i n g y but was not s e l f - s u p p o r t i n g . The opaque gels r a n g e d from g e l - l i k e pasty  precipitates with very  little  e l a s t i c i t y to a g e l that  was  l a r g e l y t r a n s l u c e n t w i t h some opaque c h a r a c t e r . The t r a n s l u c e n t g e l s i n c l u d e d a l l gels formed from each s u c c i n y l a t e d i s o l a t e at pH 6.5 or h i g h e r (18 t o t a l ) while the opaque gels i n c l u d e d those formed at pH 5 from each s u c c i n y l a t e d i s o l a t e p l u s the gels from u n m o d i f i e d i s o l a t e (10 t o t a l ) . For the t r a n s l u c e n t g e l s , G' was where G' was  w e l l d e s c r i b e d by s o l u b i l i t y and  i n v e r s e l y r e l a t e d to s o l u b i l i t y and  (R =0.775; T a b l e 2.9). 2  When the apparent v i s c o s i t y at 10 s  unheated d i s p e r s i o n s was  allowed  1  e  - 1  G  1  e  S  e  (r/io) of the  as an independent v a r i a b l e , 7)IQ  together a c c o u n t e d f o r 77.0% of the v a r i a t i o n i n G . S  p o s i t i v e l y r e l a t e d to  S,  and  i n c r e a s e d as r j  1 0  S  e  and  increased. A l t h o u g h the w a t e r - h o l d i n g  sively, little  has  been r e p o r t e d  c a p a c i t y of g e l s has been examined e x t e n on  the r e l a t i o n s h i p between the  protein  content of the gel exudate and t e x t u r a l p r o p e r t i e s . The percentage of p r o t e i n  103 T a b l e 2.9.  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n o f v i s c o e l a s t i c meters o f t r a n s l u c e n t g e l s (n-18).  Dependent Variable  Independent Variable  Storage Modulus R  2  = 0.775  S.E.  a  = 116.74  Solubility s  Coefficient -25.755  F-prob. 0.0162  4.089  e  para-  0.0243  Constant  2.329 X 1 0  S  7.662  0.0000  0.750  0.0190  3  F-prob. = 0 . 0 0 0 0  Storage Modulus R  2  - 0.770  S.E.  = 117.89  e  VlO  Constant  -491.380  F-prob. = 0 . 0 0 0 0  Storage Modulus R  2  = 0.963  S.E.  = 47.324  Gel Solubility Gel Solubility  2  -44.062  0.0000  0.234  0.0000  Constant  2 . 0 9 9 X 103  S  0.222 X i o - i  0.0000  -0.266 X i o - i  0.0053  F-prob. = 0 . 0 0 0 0  Loss Modulus R  2  = 0.882  S.E. = 1 8 . 4 8  2  Solubility x S  e  Constant  89.538  S S  -2.360  F-prob. = 0 . 0 0 0 0  Loss Modulus R  2  - 0.888  S.E.  Loss Tangent R  = 0.900  S.E.  = 0.024  F-prob. = 0 . 0 0 0 0  a  2  - 18.624  F-prob. = 0 . 0 0 0 0  2  e  Standard error of estimate.  0.0417  0.239 X i o - i  0.0015  0.155  0.0229  Constant  47.714  s  -0.387 X i o - i  0.0000  0.382 X 10-3  0.0001  - 0 . 1 1 7 X 10-5  0.0004  e  Se  2  Se Constant 3  1.390  104  f o u n d i n the gel exudate a f t e r c e n t r i f u g a t i o n i s presented i n T a b l e 2.10.  The  storage modulus of the t r a n s l u c e n t gels f o l l o w e d a c u r v i l i n e a r r e l a t i o n s h i p w i t h exudate p r o t e i n content that a c c o u n t e d f o r more t h a n 96% of the v a r i a t i o n i n G' (Table 2.9).  As exudate p r o t e i n decreased, G' i n c r e a s e d i n a c u r v i -  l i n e a r manner. T h e r e f o r e , f o r these gels i t appeared as i f G  1  was r e l a t e d to  the number of p r o t e i n molecules t a k i n g p a r t i n j u n c t i o n zones and c r o s s - l i n k s and was p r o b a b l y the r e s u l t of a g r e a t e r number of s i m i l a r bonds r a t h e r t h a n a few covalent l i n k a g e s .  F o r the opaque gels there were no s i g n i f i c a n t r e l a -  t i o n s h i p s between g e l exudate p r o t e i n and the v i s c o e l a s t i c parameters. The l o s s modulus (G") of t r a n s l u c e n t gels was w e l l d e s c r i b e d by S the i n t e r a c t i o n of s o l u b i l i t y of unheated d i s p e r s i o n s and S  2 e  (R =0.882). 2  e  plus G"  i n c r e a s e d i n an upward c u r v i l i n e a r manner w i t h S , and G" g e n e r a l l y i n c r e a s e d e  A l t e r n a t i v e l y , when 7)IQ was allowed as an i n d e p e n -  as s o l u b i l i t y d e c r e a s e d . dent v a r i a b l e , TJIQ, S with  G,  G"  1  e  and S  followed  2 e  a c c o u n t e d for 88.8% of the v a r i a t i o n i n G".  a curvilinear  (R =0.836), where G"  increased  decreased.  be  2  T h i s may  relationship with  gel  solubility  at a f a s t e r r a t e as g e l p r o t e i n s o l u b i l i t y  the r e s u l t of l i m i t e d p o l y m e r i z a t i o n  of p r o t e i n  molecules p r o d u c i n g i n e f f e c t l a r g e r a g g r e g a t e s or longer strands but complete c r o s s - l i n k i n g or i n t e g r a t i o n into the t h r e e - d i m e n s i o n a l The  As  without  g e l matrix.  loss tangent of the t r a n s l u c e n t gels was d e s c r i b e d by a c u b i c f i t of  S  e  (R =0.900) where minimum v a l u e s of loss tangent were f o u n d at intermediate  S  e  v a l u e s , while loss tangent i n c r e a s e d at low and h i g h v a l u e s of S .  2  e  This  i m p l i e d that even t h o u g h both G' and G" i n c r e a s e d w i t h S , optimum development e  of the e l a s t i c p o r t i o n of the g e l as compared to the v i s c o u s p o r t i o n o c c u r r e d when S  e  was n e i t h e r too l a r g e nor too s m a l l . At low l e v e l s of h y d r o p h o b i c i t y ,  T a b l e 2.10.  P r o t e i n c o n t e n t o f exudate from t h e r m a l l y induced g e l s o f c a n o l a protein Isolate (percent).  NaCl C o n c e n t r a t i o n Succinic Anhydride  (%)  0  5.2  pH  0.0  9.5  0.35  0.7  50.0  -  -  11.0  68.7  31.7  25.5  5.0  27.6  25.7  26.0  6.5  94.3  44.4  40.3  8.0  _1  57.6  43.8  61.6  48.4  67.1  44.0  9.5 11.0  14.2  -  5.0  21.6  27.9  29.8  6.5  97.0  62.4  48.1  79.1  63.6  80.3  61.7  78.5  68.5  8.0 9.5 11.0  *Did not g e l .  (Molar)  -  —  106 t h e r e would be fewer areas on the s u r f a c e s of the p r o t e i n molecules f o r three d i m e n s i o n a l network f o r m a t i o n , but l i m i t e d h y d r o p h o b i c i n t e r a c t i o n s would allow f o r a g g r e g a t i o n of p r o t e i n molecules to form s t r a n d s as proposed Tombs (1970, 1974), thus i n c r e a s i n g the v i s c o u s Alternatively, i f protein hydrophobicity p r o p o r t i o n of the surface was  component of the  by  system.  was v e r y h i g h and a r e l a t i v e l y large  able to take part i n h y d r o p h o b i c i n t e r a c t i o n s ,  a g g r e g a t i o n would be e x p e c t e d to be l e s s o r d e r e d , g i v i n g l a r g e r more a p p r o x i m a t e l y s p h e r i c a l p a r t i c l e s and structure.  The  a coarser,  less w e l l o r i e n t e d  gel network  l a r g e r the aggregates, the smaller would be the c o n t r i b u t i o n  from each p a r t i c l e i n the g e l network (Tombs, 1974), hence the p r o p o r t i o n  of  e l a s t i c to v i s c o u s components of the system would be e x p e c t e d to decrease even t h o u g h both G  1  and  G"  would  increase.  For the opaque gels, G' was  a f f e c t e d by S ,  v i s c o s i t y of the unheated d i s p e r s i o n s  a c c o u n t e d for less t h a n 45%  apparent  (Table 2.11), while s i g n i f i c a n t v a r i -  ables i n f l u e n c i n g G" were p r o t e i n s o l u b i l i t y , S loss tangent, o n l y s o l u b i l i t y was  zeta p o t e n t i a l and  e  and T)IQ (R =0.886). For  the  2  e  a s i g n i f i c a n t independent v a r i a b l e , but i t  of the v a r i a t i o n i n loss tangent.  The  visco-'  e l a s t i c p r o p e r t i e s of the opaque gels appeared to be i n f l u e n c e d not o n l y the  g e l matrix, but  also by the a g g r e g a t e s that l e n d o p a c i t y to the  by  gels.  Thus, the a n a l y s i s of f a c t o r s i n f l u e n c i n g v i s c o e l a s t i c i t y of the opaque gels was by  c o m p l i c a t e d , not o n l y by the p r o p e r t i e s of the c o n t i n u o u s phase but also s u c h f a c t o r s as the s i z e , shape, number and  i n t e r n a l s t r u c t u r e of  the  a g g r e g a t e s w h i c h i n t u r n may depend on extent of s u c c i n y l a t i o n , pH, and i o n i c strength.  S i n c e the f a c t o r s i n f l u e n c i n g the f o r m a t i o n and p h y s i c a l p r o p e r t i e s  of the a g g r e g a t e s would not n e c e s s a r i l y be the same as f o r the g e l matrix, may  i n f a c t oppose one  and  another, i t i s d i f f i c u l t to make a m e a n i n g f u l i n t e r -  107  T a b l e 2.11.  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n o f v i s c o e l a s t i c parameters o f opaque g e l s (n=»10).  Dependent Variable  Independent Variable  Storage Modulus R  2  = 0.919  S.E.  8  -  120.17  F-prob. = 0 . 0 0 6 2  s  e  Zeta Potential ^10  S  e  x Z.P.  Loss Modulus R  = 0.886  S.E. = 3 4 . 2 5  *7l0  F-prob. = 0 . 0 0 3 1  Constant  Loss Tangent R  2  = 0.449  S.E. = 0 . 0 5 4  F-prob. = 0 . 0 3 4 0  a  Solubility  Standard error of estimate.  Solubility Constant  2  F-prob.  -12.650  0.0038  44.716  0.0020  1.588  0.0024  -0.338  0.0076  1.724 x 1 0  Constant  2  Coefficient  3  -1.381  0.0427  -0.494 x i o - i  0.0063  0.576  0.0007  197.01  2  - 0 . 2 3 8 x lO" 4 0.308  0.0339  108 pretation of the mode of action of the predictive factors on the system as a whole. Gelation is a protein aggregation phenomenon where attractive and repulsive forces between protein molecules and the solvent are so balanced that a well ordered three dimensional network or matrix is formed which is capable of trapping or immobilizing large amounts of solvent. Ionizable amino acids play an important role in determining electrostatic interactions between protein molecules, and therefore factors such as salts, pH and temperature influence the balance between attractive and repulsive forces in the system (Wall, 1979). During denaturation, hydrophobic and hydrogen bonds buried in the interior of the molecule become exposed and reform in a manner different from the native structure (Buttkus,  1974).  Thus regions of the molecule  originally involved in the stability of the native form become available for intermolecular interactions and a three dimensional network can form, provided that there  are at least two  attractive sites per molecule (Clark et al.,  1981). Therefore it appears as if optimum gelation conditions occur when the attractive forces set free by denaturation are just strong enough to counteract electrostatic repulsion, and an ordered limited aggregation can take place resulting in a gel network. The nature of the gel would be determined by the number of bonding sites available on each protein molecule, their spacial distribution and their relative bonding strengths under the prevailing conditions of pH and ionic strength.  Hegg et al. (1979) noted that ovalbumin  gels were formed at an intermediate  state between a high level of charge  repulsion which gave solubility and a low level that gave rise to precipitation. In a medium where ovalbumin underwent gelation, however, the gels became increasingly transparent as the net charge repulsion increased such as by  109  i n c r e a s i n g the pH  or d e c r e a s i n g  the i o n i c s t r e n g t h .  found by E g e l a n d s d a l (1980) where c l e a r and  A similar effect  was  uniform ovalbumin gels were  p r o d u c e d by d i r e c t e d l i n e a r a g g r e g a t i o n whereas more i r r e g u l a r networks were formed by e s s e n t i a l l y random a g g r e g a t i o n repulsion.  w h i c h o c c u r r e d at low e l e c t r o s t a t i c  Hermansson (1982) found that blood plasma gels were coarser  and  more r a n d o m l y a g g r e g a t e d at pH 7 t h a n 9.5 due to the lower net negative charge at pH  7.  W a t e r - b i n d i n g p r o p e r t i e s also decreased as the degree of random  aggregation  of the g e l network i n c r e a s e d .  The  author suggested that gel  t e x t u r e was i n f l u e n c e d at the m o l e c u l a r l e v e l by the o r g a n i z a t i o n of molecules into strands or aggregates and on the c o l l o i d a l l e v e l by such f a c t o r s as the strength  and  d e f o r m a b i l i t y of s t r a n d s , the  s t r e n g t h , type and  number of  j u n c t i o n zones as w e l l as the d e n s i t y and s i z e d i s t r i b u t i o n of aggregates and conglomerates (Hermansson, 1982).  Moreover, i t was  v i s c o s i t y of the c o n t i n u o u s phase should not be  pointed  out that the  overlooked.  P r e v i o u s s t u d i e s w i t h s u c c i n y l a t e d p r o t e i n have found i n c r e a s e d t h e r m a l s t a b i l i t y and decreased or r e t a r d e d h e a t - i n d u c e d g e l a t i o n or c o a g u l a t i o n . and Holme (1982) found that the t h e r m o c o a g u l a t i o n of egg albumen was g r e s s i v e l y d e c r e a s e d and  e l i m i n a t e d by i n c r e a s i n g the extent  of  Ma pro-  succiny-  l a t i o n and  t h e r e f o r e the amount of e l e c t r o s t a t i c r e p u l s i o n between p r o t e i n  molecules.  Balmaceda et a l . (1976) suggested t h a t , w i t h i n f i n i t e l i m i t s , a  h i g h degree of p r o t e i n s o l u b i l i t y i s n e c e s s a r y for o p t i m a l p r o t e i n g e l a t i o n . In the present study, the s o l u b i l i t y of canola p r o t e i n under low a c i d a l k a l i n e c o n d i t i o n s was  g r e a t l y improved by  and  s u c c i n y l a t i o n as a r e s u l t of  i n c r e a s e d e l e c t r o n e g a t i v i t y of the p r o t e i n molecules, but t h i s also appeared to  increase  strength.  t h e r m o s t a b i l i t y and  With the  reduce  g e l a t i o n a b i l i t y at low  a d d i t i o n of N a C l , however, the  charge r e p u l s i o n  ionic was  110 p r o g r e s s i v e l y r e d u c e d a l l o w i n g f o r p r o t e i n - p r o t e i n i n t e r a c t i o n s and g e l a t i o n when a p r o p e r balance between p r o t e i n - p r o t e i n and p r o t e i n - s o l v e n t i n t e r a c t i o n was a c h i e v e d .  N a C l promotes a g g r e g a t i o n  due to r e d u c t i o n of the  d i f f u s e p a r t of the e l e c t r i c double l a y e r , and may also induce c o n f o r m a t i o n a l changes i n the p r o t e i n molecules (Fennema, 1977) w h i c h may a l t e r the number of bonding sites for gelation. Heating  p r o t e i n d i s p e r s i o n s also i n d u c e s c o n -  f o r m a t i o n a l changes w h i c h tend to i n c r e a s e p r o t e i n - p r o t e i n i n t e r a c t i o n (Tombs, 1974). The a d d i t i o n of N a C l has also been found to improve the g e l s t r e n g t h of many other t h e r m a l l y p r o c e s s e d p r o t e i n systems (e.g. Hermansson, 1982; Schmidt and I l l i n g w o r t h , 1978; Schmidt et a l . , 1978; Shiraada and M a t s u s h i t a , 1981). A l t h o u g h not examined i n depth, low l e v e l s of d i v a l e n t c a t i o n s were able to induce g e l a t i o n i n heated d i s p e r s i o n s of s u c c i n y l a t e d canola i s o l a t e .  This  was s u s p e c t e d to be a r e s u l t not o n l y of enhanced d e p r e s s i o n of the e l e c t r i c double-layer  but also the c a p a c i t y of d i v a l e n t c a t i o n s to l i n k two p r o t e i n  molecules together b y t h e i r c a r b o x y l groups. P r e l i m i n a r y experiments demons t r a t e d that 0.035M CaCl2 i n 11.4% d i s p e r s i o n s of both 5.2% SA and 14.2% SA canola i s o l a t e s at pH 6.8 gave s i m i l a r g e l s t r e n g t h s to those o b t a i n e d 0.7M N a C l as determined by a p u n c t u r e t e s t .  with  P r o t e i n p r e c i p i t a t i o n and  t h e r e f o r e no g e l a t i o n o c c u r r e d w i t h 0.35M CaCl2. A s i m i l a r r e s u l t was found by Schmidt et a l . (1978) who r e p o r t e d that the maximum g e l s t r e n g t h of whey p r o t e i n c o n c e n t r a t e gels was o b t a i n e d w i t h 5 to 20mM CaCl2 compared to 0.1 to 0.3M N a C l . the  T h i s f i n d i n g may be of p r a c t i c a l i n t e r e s t as i t demonstrates that  g e l a t i o n a b i l i t y of s u c c i n y l a t e d p r o t e i n s may be u t i l i z e d i n p r o d u c t s  where h i g h l e v e l s of s a l t s such as N a C l may be d e t r i m e n t a l to p r o d u c t q u a l i t y or u n d e s i r a b l e f o r h e a l t h reasons.  Ill The  major types of bonds involved in gel formation and stability were  tentatively identified as hydrophobic interactions and hydrogen bonds. It seems likely that hydrophobic groups largely contributed to gel formation since the gels formed during heating.  In this temperature range, hydrophobic  interactions increase with temperature (Ben-Nairn, 1980).  Schmidt (1981)  suggested that hydrophobic interactions are important to dissociative-associative reactions which initiate the gelation process and contribute to layering or thickening of the gel matrix strands upon cooling. Shimada and  Matsushita  (1980b) found a relationship between the proportion of hydrophobic groups in the amino acid profile of proteins and the type of gel formed upon heating, while Ma and Holme (1982) showed hydrophobicity to be involved in gelation of egg albumen. Hydrophobic and acidic residues dominate the amino acid profile of rapeseed proteins, while basic amino acids are in relatively low concentration (Sosulski and Sarwar, 1973). In the present study, the gels increased in firmness upon cooling, thus implicating the involvement of hydrogen bonds which are weakened by increasing temperature (Joesten and Schaad, 1974). Oakenfull and Scott (1984) reported that a combination of hydrogen bonds and hydrophobic interactions stabilized gels formed from high methoxy pectins. Schmidt (1981) suggested that hydrogen bonds stabilize gel structure and allow for a more open orientation necessary for water immobilization. Although the effects of different heating conditions were not examined in detail, it was  noted that temperatures up to 160°C did not destroy the  gelation ability of the isolates.  This is in contrast to soy protein, for  which temperatures in excess of 125°C have been reported to result in a metasol which did not form a gel upon cooling (Catsimpoolas and Meyer, 1970). This is of potential importance for the use of succinylated canola protein in  112 a retorted product.  Other authors have reported on the effects of heating  conditions on gel formation.  Hermansson (1982) found that heating  plasma protein above an optimal gelation temperature caused an  blood  increased  tendency toward protein-protein interactions with a partial disruption of the gel network due to local aggregation phenomena. A similar effect was noted by Tombs (1970) with bovine serum albumin and Schmidt and Illingworth (1978) who found that heating whey protein at temperatures above 110°C produced gels with more visual syneresis than gels formed at lower temperatures. al. (1978) found that whey protein dispersions with 0.2M while a temperature of 90°C was  Schmidt et  NaCl gelled at 75°C  required for gelation in distilled water.  Egelandsdal (1984), however, found that hardness of ovalbumin gels  was  independent of the temperature to which it was heated and suggested that the rate of gel formation was more important for gel hardness than the degree of protein unfolding.  In view of these reports, the effects of heating con-  ditions on the gelation properties of succinylated canola protein could form the basis of further studies.  E.  E m u l s i f i c a t i o n A c t i v i t y and E m u l s i o n S t a b i l i t y The effects of succinylation, pH and NaCl on emulsification activity (EA)  are presented in Table 2.12 and Figure 2.12.  EA was significantly affected by  succinylation (linear and quadratic), NaCl (linear and quadratic), pH (linear) as well as an interaction between all three factors. In general, EA increased with extent of succinylation, but the rate of increase was greater between 0% and 54% modification of amino groups than between 54% and 84% modification. NaCl at both 0.35M and 0.70M increased EA compared to the samples without salt, but the highest EA overall was  at 0.35M indicating first an increase  Table 2.12. Trend comparison analyses o f the e f f e c t s o f s u c c i n y l a t i o n , pH and NaCl on e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y (n=54). Dependent V a r i a b l e  Emulsification R  2  =  S.E.  R  2  Activity  0.945 a  =  F-prob.  Emulsion  Independent V a r i a b l e  -  S.E.  0.0312  NaCl  = 0.0000  NaCl  of  estimate.  0.0432  0.146 X i o - i  0.0072  2  0.0113 0.0000  S u c c . x N a C l x pH  0.330 X 10-3  0.0005  Constant  0.399  0.370  0.0000  -0.144  0.0265  -3.950  0.0000  2.756  0.0000  0.416 X i o - i  0.0003  -0.206 X i o - i  0.0395  N a C l x pH  -1.049  0.0000  Constant  17.208  2  PH  Succinylation x pH  Standard error  0.0000  0.328 X i o - i  S u c c i n y l a t i o n x pH  a  2  PH  NaCl  0.0000  F-prob.  - 0 . 8 8 0 X 10-3  -0.793 X lO"  2  Succinylation  = 4.634 =  2  Succinylation  0.897  F-prob.  0.227 X l O "  Succinylation Succinylation  Stability  Coefficient  2  F i g u r e 2.12. E m u l s i f i c a t i o n a c t i v i t y of c a n o l a i s o l a t e d i s p e r s i o n s : (A) unmodified; (B) 5.2% SA; (C) 14.2% SA.  115 and then a decrease in EA as ionic strength increased. However, this effect depended on pH and extent of succinylation. In addition, EA increased in an overall linear manner with  pH.  Succinylation has often been reported to improve the emulsifying properties of proteins (e.g., Childs and  Park, 1976;  Franzen and  Kinsella,  1976a,b; Thompson and Cho, 1984b). Waniska et al. (1981) reported that above pH 5, the emulsifying activity of succinylated bovine serum albumin (BSA) markedly improved compared to native BSA,  which may  was  be a reflection of  increased solubility and somewhat looser structure of the modified protein, thus facilitating diffusion of the protein to the oil-water interface and rearrangement within the interfacial film. Pearce and Kinsella (1978) found that succinylated yeast proteins gave the smallest droplets in o/w compared to proteins from other sources.  emulsions  Since EA is related to interfacial  area, EA increases as droplet size decreases.  Succinylation also results in  an increase in the molecular surface area of a protein, apparently as a result of electrical repulsion forces between identical charges; the  increased  surface area gives improved surfactant properties (Watanabe and Arai, 1982). In addition, emulsification proceeds more rapidly if the surface tension is decreased by a combination of surfactants (Dickinson and Stainsby, 1982). Succinylation increases the heterogeneity of protein mixtures as a result of different extents of modification of the proteins as well as different degrees of subunit dissociation (e.g., Grant, 1973; Klotz and Keresztes-Nagy, 1962), and this may  aid in emulsion formation.  The effect of NaCl on EA may be related to its effect on protein adsorption at the o/w  interface. Bennett et al. (1968) stated that the addition of  electrolytes may act favorably or unfavorably on emulsion stability depending  116 on whether they diminish charge repulsions or increase the electrical potential of the  ionized layer of the  interfacial film.  Waniska et al. (1981)  reported an effect of NaCl on emulsifying activity of BSA  similar to that of  the present study, where EA first increased and then decreased with addition of NaCl. They suggested that at low ionic strength, high charge repulsion at the o/w  interface decreased the amount of protein adsorbed at the interface.  For the present data, at intermediate ionic strength, EA increased probably as a result of neutralization of surface charges which reduced electrostatic repulsion and facilitated an increased rate of protein adsorption and greater protein-protein interactions. However, at high ionic strength, EA dropped, possibly because of a reduced rate of protein transfer to the o/w interface. A similar effect of NaCl on the emulsifying properties of peanut and  soy  protein was reported by McWatters and Holmes (1979a,b). Mita et al. (1973) found that for five different proteins studied, the largest droplets tended to be formed near the isoelectric point.  In the present study EA increased with  pH, which suggests that droplet size decreased as the pH went farther away from the isoelectric region. The treatment effects on emulsion stability are presented in Table 2.12 and Figure 2.13.  As with EA, ES increased with succinylation but at a slower  rate between 54% and 84% modification than between 0% and 54% modification. There were significant interaction effects between pH and NaCl concentration and  between succinylation and  pH  (linear and  quadratic), but overall, ES  decreased in a linear manner with NaCl and increased linearly with pH. As already described, succinylation, pH solubility, hydrophobicity, isolate dispersions.  and  NaCl influenced protein  zeta potential, and apparent viscosity of canola  Since these factors were expected to affect EA and  ES,  Figure 2.13. Emulsion stability of canola isolate dispersions: (A) unmodified; (B) 5.2% SA; (C) 14.2% SA.  118 multiple regression analyses were used to examine the relative influence of each factor so as to obtain a better understanding of the manner in which the treatments affected emulsification. For the regression involving emulsification activity, solubility and the square of solubility as well as interaction effects between solubility and S , Q  zeta potential and flow behavior index (n) accounted for 89.3% of the variability of EA (Table 2.13). The quadratic effect of solubility indicated that EA increased rapidly at low levels of solubility but more slowly as solubility increased. bility  with  The presence of solubility along with the interaction of soluS  Q  and zeta potential as significant  independent  variables  underscored the importance of a balance between hydrophile and lipophile in emulsion formation (Aoki et al., 1981) as well as the involvement of electrical repulsion forces between droplets in retarding coalescence during emulsification.  The  importance  of a hydrophilic-lipophilic  balance  was  emphasized by Voutsinas et al. (1983b) who reported that regardless of protein solubility, as surface hydrophobicity increased, emulsification activity and emulsion stability initially increased and then decreased, and Li-Chan et al. (1984) who showed the importance of solubility, hydrophobicity, and their interaction in emulsifying properties of salt-soluble meat protein.  The  interaction between protein solubility and flow behavior index in the present study was also probably related to resistance to coalescence during emulsification.  The overall effect of the flow behavior index was to increase EA  with a decrease in n, indicating that as the continuous phase of the dispersions became more pseudoplastic, a greater interfacial area in the emulsions became stabilized.  The physical meaning of a flow behavior index less than  1.0 is that the apparent  viscosity of the dispersion decreases  with an  119  T a b l e 2.13.  Dependent  M u l t i p l e r e g r e s s i o n models f o r p r e d i c t i o n o f e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y (n=54).  Variable  Emulsification R  2  Independent  Solubility  Activity  Solubility  = 0.893  S.E.a  = 0.0433  S  n  R  2  Stability = 0.900  S.E.  0  2  x Solubility  Z.P. x S o l u b i l i t y  F-prob. = 0.0000  Emulsion  Variable  b  x Solubility  Standard error of estimate.  b  Power-law f l o w b e h a v i o r  Index.  -0.264 X 10"-4  0.0000  0.409 X 10"-4  0.0004  -0.572 X 10"-2  0.0000  0.690 X 10"-2  Constant  a  0.0000  S o l u b i l i t y x ^JOOO  ADensity  F-prob. = 0.0000  0.0000  -0.490 X 10"-4  0.239  Zeta P o t e n t i a l  2  F-prob.  0.141 X 10"-1  Constant  Zeta P o t e n t i a l  = 4.430  Coefficient  0.0000  -0.268  0.0032  -0.590 X 10"-2  0.0026  -212.790 21.674  0.0077  120 increase in shear rate. At the high shear rates encountered at the blades of the Oranimixer used to create the emulsions, the dispersions would have had a low  apparent viscosity, thereby facilitating breakup of the o i l phase and  emulsion formation.  As the o i l droplets flowed away from the blades into the  bulk dispersion, o i l droplets in dispersions with greater pseudoplasticity would have encountered a higher apparent viscosity at the low rates of shear in  the bulk dispersion than if the dispersion was less  pseudoplastic.  Droplets in a highly viscous dispersion would tend to have fewer collisions and therefore less coalescence and demulsification than in a dispersion of lower viscosity, and this would be reflected in the EA. The  multiple regression model for emulsion stability is presented in  Table 2.13. In addition to protein solubility, hydrophobicity, zeta potential and rheological parameters, the difference in density between the o i l phase and the aqueous phase at each NaCl concentration was included as an independent variable in the regression. potential, along  Zeta potential and the square of zeta  with the density difference between the dispersed and  continuous phases, and the interaction between protein solubility and apparent viscosity at 1000 s " l of aqueous dispersions accounted for 90% of the variation in emulsion stability. In general, emulsions became more stable as both protein solubility and apparent viscosity of the continuous phase increased, as the difference in density between the two phases decreased, and as the charge repulsion between protein molecules at the interface increased. The nonlinear effect of charge repulsion indicated that changes in zeta potential had a greater effect on stability when charge repulsion was high compared to when it was low.  121 ' E m u l s i o n s may d e s t a b i l i z e b y c r e a m i n g ( t h e movement o f d r o p l e t s u n d e r the influence  of a g r a v i t a t i o n a l f i e l d ) , f l o c c u l a t i o n (the c l u s t e r i n g together of  droplets), ones).  and/or  coalescence  (the merging  of small  droplets  into  larger  Examination of the s i g n i f i c a n t independent v a r i a b l e s i n d i c a t e d  emulsion  stability  as determined  r e s i s t a n c e to c r e a m i n g .  in this  study  was  mainly  that  a measure of  C r e a m i n g o c c u r s as a r e s u l t of t h e d e n s i t y d i f f e r e n c e  between t h e d i s p e r s e d and c o n t i n u o u s p h a s e s as a f f e c t e d b y s u c h f a c t o r s as t h e viscosity  of the c o n t i n u o u s  surfactant  molecules (Dickinson  Creaming rate  droplet  and Stainsby,  size, and the charge  on the  1982; P o w r i e a n d T u n g , 1976).  i s r e l a t e d to S t o k e s ' e q u a t i o n : 2r2g(Ad)  =  where V  phase,  m  977  i s the v e l o c i t y of droplet  1  movement, r i s d r o p l e t  '  radius, g i s the  g r a v i t a t i o n a l f o r c e , A d i s t h e d e n s i t y d i f f e r e n c e b e t w e e n t h e two p h a s e s , a n d 77 i s t h e a p p a r e n t v i s c o s i t y o f t h e c o n t i n u o u s p h a s e ( P o w r i e a n d T u n g , 1976). Thus,  emulsion  droplet  radius,  tinuous  phases,  flocculation  stability a small and  with  respect  density  high  to creaming  difference  is favored  between the dispersed  v i s c o s i t y of the continuous  and coalescence  example, t h e h e t e r o g e n e i t y  by a  phase.  small  and conCreaming,  are not i n d e p e n d e n t p r o c e s s e s , however.  of the droplet  d i s t r i b u t i o n may  For  a f f e c t not o n l y  c r e a m i n g r a t e b u t also f l o c c u l a t i o n a n d c o a l e s c e n c e as a r e s u l t of c o l l i s i o n s between  fast  moving  large  droplets  and  slow  moving  small  f l o c c u l a t i o n a n d / o r c o a l e s c e n c e do o c c u r , c r e a m i n g r a t e since  flocculation increases  increases greatest  actual droplet  the effective droplet  size.  With respect  size  droplets.  If  w o u l d be e n h a n c e d while  coalescence  t o pH, e m u l s i o n s t e n d t o h a v e  s t a b i l i t y to c o a l e s c e n c e at t h e i s o e l e c t r i c p o i n t .  Cohesiveness and  122 rigidity of protein surface films are usually greatest at the isoelectric point where electrostatic repulsions between different parts of a single protein molecule as well as between protein molecules are minimized. The cohesiveness and rigidity imparted by the compact protein molecules opposes deformation and rupture of the interfacial lamella, thus providing stability against coalescence (Hailing, 1981). There is a tendency for droplet diameter to be highest at the isoelectric point (Mita et al., 1973) and the rate of flocculation increases as the pH is adjusted closer to the isoelectric point (Hailing, 1981). Ionized  surfactants help to stabilize o/w emulsions by means of an  electric double layer in the aqueous phase.  For protein emulsifiers this  becomes more important as the pH goes away from the isoelectric point. Surface charges can originate from ionization of groups on ionic emulsifiers, adsorption  of ions on nonionic  emulsifier layers, or frictional  contact  between droplet surfaces and the continuous phase (Powrie and Tung, 1976). The  electrical charge is proportional to the surface area; the smaller the  droplet, the larger is the relative surface relative charge.  area  and the higher  The repulsive energy is a function of kH where H Q  is the 0  is the  distance between the droplets and k"" is the effective radius of the double 1  layer (Powrie and Tung, 1976). Charged oil droplets can interact with one another via their electrical double layers which leads to a reduction in the mean creaming speed due in part to drag from the "ionic atmosphere" and a microelectrophoresis effect (Dickinson and Stainsby, 1982). The surface activity of a protein is a function of the ease with which a protein can migrate to, adsorb at, unfold and rearrange at an interface, and therefore the aqueous solubility of a protein is closely related to its  123 surface  activity  (Kinsella, 1976).  As  protein molecules are  relatively  hydrophilic, the bulk of the adsorbed molecule is probably located on the aqueous side of the oil-water interface with the hydrophilic groups directed toward the aqueous phase and hydrophobic groups toward the o i l phase.  The  importance of solubility is related to several aspects of emulsion formation and  stability.  The  ease of emulsion formation  is affected by the rate at  which protein is transferred from the bulk phase and penetrates into the  o/w  interface, irrespective of whether or not the protein stays at the interface to help stabilize the emulsion (Canella et al., 1979).  It is possible then  for a protein to have a high emulsification activity but poor stability, and this was present created process. creased  indeed the case for several of the treatment combinations of the study.  However, since proteins adsorb relatively slowly at newly  oil-water  interfaces, high  protein  solubility facilitates this  Pearce and Kinsella (1978) noted that emulsion droplet size deas protein concentration  increased, probably as a result of  increased rate of adsorption at the. freshly formed o/w  an  interfaces. Smaller  oil droplets should enhance stability with respect to creaming. Proteins  may  cause flocculation of emulsions, however, by acting as "polymer bridges" between molecules (Hailing, 1981), and excessive emulsification may depletion of surfactant  and  emulsion instability.  The  lead to  role of insoluble  protein is not clear. Franzen and Kinsella (1976b) suggested that granular, insoluble proteins  separate from the o i l phase or just float on  the o i l  surface where they remain inert and contribute little toward emulsification. Others, however, have suggested that insoluble proteins may provide a steric hindrance to coalescence by adsorbing at oil-water interfaces and  reducing  inter-droplet contact (Smith et al., 1973; Powrie and Tung, 1976; Paulson et al., 1984; Kitchener and Mussellwhite,  1968).  124 SUMMARY AND  CONCLUSIONS  Thermally induced gelation and oil emulsification properties of unmodified and  succinylated canola  protein isolate were examined over a wide  range of pH values (pH 3.5-11.0) and sodium chloride concentrations  (0.0-  0.7M). Reaction of the isolate with succinic anhydride at 5.2% and 14.2% of the protein weight resulted in 54% and 84% modification of free amino groups, respectively. Succinylation markedly enhanced protein solubility at alkaline low  acid pH,  but exhaustive succinylation was  and  not required to produce a  large increase in solubility. Surface hydrophobicity decreased, and electronegativity (zeta potential) increased as succinylation increased. lation  also appeared to cause a slight  shift  in the  Succiny-  isoelectric  region  to lower pH values, which may have implications for the successful employment of succinylated protein in low-acid foods such as comminuted meat products. Protein dispersions followed power-law or power-law plastic flow behavior.  The rheological properties appeared to reflect the microstructure of  the dispersions; the succinylated isolates had than the  higher apparent viscosities  unmodified isolate under nearly all conditions of pH  and  ionic  strength. Thermally induced gels were formed by heating 11.4% dispersions of the isolates at 72°C for 30 min.  Succinylation improved the gelation ability of canola  protein. For the unmodified isolate, gels formed at only 4 out of 18 combinations of pH and NaCl concentration, while 12 gels formed for each of the succinylated isolates under the same conditions. The unmodified isolate formed gels at pH 9.5 and 11.0 while the succinylated isolates formed gels from pH 5.0 to 11.0, but above pH 6.5 only in the presence of NaCl. In general, the firmest gels were obtained with the moderate level of succinylation.  125 Overall, the gels were divided into two types based on visual observation, opaque and translucent, and each type appeared to respond in a different manner to rheological tests, and were related in different ways to the physicochemical and rheological properties  of protein dispersions.  The  viscoelastic properties of the translucent gels reflected the properties of the gel matrix, and multiple regression analyses indicated that the viscoelastic parameters were governed mainly by protein solubility and hydrophobicity.  The viscoelastic properties of the opaque gels appeared to be  influenced by both the gel matrix and insoluble aggregates, and were related to  s o l u b i l i t y , hydrophobicity,  protein dispersions.  zeta potential and apparent viscosity of  The types of bonds involved  in gel formation and  stability were tentatively identified as hydrophobic interactions and hydrogen bonds. With the succinylated isolates, gels were formed in the presence of calcium ions at a concentration an order of magnitude less than was required for similar gel strengths  with NaCl, which has implications for exploiting  the gelation ability of succinylated protein in products where high concentrations of NaCl are undesirable. Both emulsification activity and emulsion stability were improved by succinylation.  Emulsification activity was related to protein solubility,  hydrophobicity,  zeta potential and flow behavior of protein dispersions,  while emulsion stability appeared to be mainly a measure of resistance to creaming and was related to protein solubility, zeta potential, apparent viscosity of protein dispersions, and the difference in density between the aqueous and oil phases. Succinylation of 54% and 84% of the free amino groups improved the gelation  and emulsification properties of canola  protein isolate under  126  e n v i r o n m e n t a l and processing c o n d i t i o n s similar to those employed i n comminuted meat p r o d u c t s .  Exhaustive s u c c i n y l a t i o n was not r e q u i r e d to b r i n g  about improvements i n e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y , while the v i s c o e l a s t i c p r o p e r t i e s of t h e r m a l l y induced gels appeared to be best w i t h moderate s u c c i n y l a t i o n .  I t remains to be determined whether the  quality  c h a r a c t e r i s t i c s of comminuted meat p r o d u c t s w i l l be m a i n t a i n e d when meat p r o t e i n is r e p l a c e d by s u c c i n y l a t e d canola p r o t e i n .  I n a d d i t i o n , the e f f e c t s  of s u c c i n y l a t i o n on the n u t r i t i v e value of canola p r o t e i n , and on the g e l a t i o n p r o p e r t i e s of other p r o t e i n s under the c o n d i t i o n s of pH and ionic s t r e n g t h employed i n comminuted meat p r o d u c t s are subjects for f u r t h e r i n v e s t i g a t i o n .  127 REFERENCES Aman, P. and Gillberg, L. 1977. Preparation of rapeseed protein isolates: a study of the distribution of carbohydrates in the preparation of rapeseed protein isolates. J. Food Sci. 42:1114. Aoki, H., Taneyama, O., Orirao, N. and Kitagawa, I. 1981. Effect of lipop h i l i z a t i o n of soy protein on its emulsion stabilizing properties. J. Food Sci. 46:1192. 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Chem. 36:719.  \  138  CHAPTER 3 DYNAMIC SHEAR VERSUS PUNCTURE PROBE MEASUREMENTS OF GEL TEXTURE  INTRODUCTION R h e o l o g i c a l test methods f o r gels (1)  generally  fundamental tests that measure well d e f i n e d  f a l l into three  categories:  r h e o l o g i c a l p a r a m e t e r s , (2)  e m p i r i c a l t e s t s t h a t d e p e n d o n t h e g e o m e t r y o f t h e i n s t r u m e n t u s e d a n d do n o t provide  fundamental  simulate  rheological  conditions  (Mitchell,  that  a  quantities,  material  will  a n d (3) i m i t a t i v e be  that  to i n p r a c t i c e  1976).  Fundamental tests, which a i d i n elucidating performed  subjected  tests  at small  strains  o n t h e sample  instrumentation with complex c a l c u l a t i o n s .  gel structure,  using  relatively  are usually sophisticated  T h e test p r o c e d u r e s c a n be v e r y  time c o n s u m i n g a n d t h e r e s u l t s m a y n o t be w e l l c o r r e l a t e d w i t h s e n s o r y p a n e l tests  o n t h e same  imitative  tests  material  are usually  (Mohsenin  and Mittal,  of the large  deformation  1977).  Empirical  or failure  and  type, are  p e r f o r m e d r a p i d l y with r e l a t i v e l y simple c o m p u t a t i o n of r e s u l t s , a n d u s u a l l y are and  better  with panel tests than are fundamental tests  M i t t a l , 1977; Wood, 1979).  strength" the  correlated  based  same o r d e r  (Mitchell, control  on rupture as tests  1976).  because  tests  which  Empirical  However, single point will  small  methods  deformations  are commonly  peak force  without used  rupture  for quality  of t h e i r simplicity, r a p i d i t y and r e p r o d u c i b i l i t y , although  r e s u l t s may be d e p e n d e n t o n t h e type o f test employed. the  measurements of " g e l  not always r a n k a s e r i e s of gels i n  involve  test  (Mohsenin  a n d i n i t i a l slope from  Wood (1979) m e a s u r e d  a compression test performed with an  139 Instron Universal Testing Machine, and the Bloom number for a series of seven gels prepared from agar, xanthan gum plus locust bean gum and carrageenan plus locust bean gum and found that the ranking order for the seven gels using the three different parameters was not the same. Penetration or puncture testing is among the simplest to perform and most widely used methods for the measurement of textural characteristics of food products.  Although few studies have looked  at the relationship between  measurements obtained from empirical test methods and fundamental rheological studies, empirical tests are often used in situations where fundamental tests would be more appropriate but limitations on time, expertise and availability of instrumentation  dictate that an empirical test be used.  It would  be  desirable then, for fundamental rheological properties to be approximated or predicted from an empirical test method. The objective of this study was to examine the relationships between textural measurements of canola isolate gels obtained  by means of a puncture test with an Instron Universal Testing  Machine, and fundamental rheological parameters obtained from dynamic shear measurements with a Weissenberg  Rheogoniometer.  140 LITERATURE REVIEW -  All polymer dispersions, including gels, possess both fluid-like viscous behavior and solid-like elastic behavior, and are thus termed "viscoelastic". Information about the nature and rates of configurational rearrangements and the disposition and interaction of macromolecules in their short-range and long-range interrelations can be obtained from the measurement of viscoelastic properties  (Ferry, 1980).  Methods used to study viscoelasticity usually  employ very low stress or strain so as to be nondestructive and hence minimize alteration of internal structure.  Such methods include creep compliance  testing where a small constant stress is applied to a sample and the resulting strain is followed with time, stress relaxation tests where a predetermined strain is suddenly imposed on a sample and kept constant while the relaxation of stress with time is measured, and dynamic testing where small sinusoidal strains are imposed on a sample and the resulting stress and difference in phase between the strain and stress waves are measured (Tung, 1978). Dynamic testing has been performed on a number of food materials including ovalbumen gels (Van  Kleef et al., 1978), carrageenan gels (Elliott and  Ganz, 1975),  alginate gels (Segeren et al., 1974), rapeseed protein gels (Gill and Tung, 1978; Jones, 1980)  and gelatin gels (Nijenhuis, 1974). Mitchell (1976, 1980)  has extensively reviewed the testing and rheological properties of gels, while Tung (1978) reviewed the rheology of protein dispersions including protein gels. The puncture test is one of the most widely used techniques for measuring food texture and consists of measuring the force (and often the resulting deformation) required to push a probe into a food material.  Some puncture  tests merely deform the material without rupture while others are performed to  141 a depth that causes irreversible crushing  and flow of the food material.  Although puncture tests are mechanically  simple,  complex.  they are theoretically  Bourne (1979) reviewed the theory and application of puncture  testing of food materials.  The punch is usually a cylindrical metal probe but  numerous other shapes have been used. Bourne (1966) showed that the yieldpoint force from a puncture test was directly proportional to both the area and perimeter of the punch. Kamel and deMan (1977) reported that the force readings for penetration of gelatin gels were related to the probe size and shape, but there was no relationship between the force readings and penetration speed in the range of speeds tested.  Mohsenin and Gohlich (1962)  reported no significant difference in the shape of force-deformation  curves  with penetration speeds of 0.05 to 2.5 cm/min. Rasmussen (1974) studied the texture of gels made from gelatin, agar, carrageenans and combinations of these with locust bean gum and guar gum, with a puncture test using an Instron. The author defined several characteristics of the gels from the force-deformation curves. Hermansson (1982) examined the texture of blood plasma gels by puncture and compression tests using an Instron and reported that although the information obtained generally  by each test  agreed, the compression test better indicated gel structural  differences as a function of pH, while the puncture test was better for assessing the effects of temperature and salt induced changes at a constant pH.  The initial slope of the stress-strain curve of compression tests is a  common index of gel texture. Hermansson (1982) however, found that in spite of large differences in gel structure due to variation of pH, there were no differences in the initial slopes of the stress-strain curves.  Mitchell and  Blanshard (1976a) found a poor relationship between initial slopes and rupture  142 forces from compression t e s t s of a l g i n a t e gels and concluded t h a t the p r i m a r y molecular textural  weight response.  of  the The  g e l l i n g m a t e r i a l was a g o v e r n i n g f a c t o r interpretation  of  the  stress-strain  compression or p u n c t u r e t e s t s is not s t r a i g h t f o r w a r d , however.  in  curves  the from  Calzada and  Peleg (1978) noted t h a t an a p p a r e n t l y l i n e a r r e g i o n of a s t r e s s - s t r a i n curve may be the r e s u l t of two a n t a g o n i s t i c e f f e c t s such as f r a c t u r e and compression r a t h e r t h a n b e i n g due to an e l a s t i c r e g i o n .  143 MATERIALS AND METHODS Thermally-induced  gels from unmodified or succinylated canola isolate  were prepared as described in Chapter 2. Canola isolate was succinylated with 5.2 percent or 14.2 percent succinic anhydride  (on a protein basis) which  resulted in 54 percent and 84 percent modification of amino groups, respectively.  Dispersions of the unmodified and succinylated isolates (11.4% w/w)  at pH 3.5, 5.0, 6.5, 8.0, 9.5, or 11.0 containing 0.0, 0.35, or 0.7M NaCl were heated at 72°C for 30 min.  After cooling in an ice bath and equilibration to  room temperature, the samples that formed self-supporting gels were evaluated for texture with a puncture  test and for viscoelastic properties with a  Weissenberg Rheogoniometer.  A.  Puncture Test The Puncture  test was conducted  with a Model 1122 Instron Universal  Testing Machine using an 8ramdiameter cylindrical steel probe with a rounded end (Magness-Taylor puncture probe; Bourne, 1968). The probe was driven into each gel to a depth of 12 mm at a crosshead speed of 50 mm/min and a recording chart speed of 200 mm/min. Tests were conducted in duplicate for each sample and the results averaged. The force-deformation curves were analyzed for rupture force (the force at which gel rupture occurred) and the distance the probe had travelled to the point of rupture (Figure 3.1). From these measurements were calculated the rupture slope (rupture force/rupture distance), and rupture area (1/2 rupture force x rupture distance) which approximated the area under the force-deformation curve to the point of rupture.  144  Rupture Force (RF)  0)  P  Rupture Slope = Rl/RD  Rupture Area = (RFxRD)/2  Rupture Distance (RD)Distance (mm)  Figure 3.1. Force-deformation curve from Instron puncture test for protein gels.  145 B.  Dynamic V i s c o e l a s t i c P r o p e r t i e s Dynamic shear behavior of the gels was assessed as described in Chapter  2. After the puncture tests were completed, the gels were carefully removed from the containers in which they were formed and the bottom portion of each gel cylinder, which was undisturbed by the puncture test, was used for dynamic testing by a Weissenberg Rheogoniometer equipped with 5 cm diameter parallel plate fixtures.  The storage modulus, loss modulus and loss tangent of each  gel were calculated for a frequency of 10 s * from dynamic shear rheograms -  obtained over a frequency range of 0.19 to 19 s * . -  Simple  linear, multiple linear and polynomial regressions using the  dynamic shear parameters as dependent variables and the puncture test measurements as potential independent variables were performed with the MIDAS statistical computer program (Fox and Guire, 1976) on the U.B.C. Amdahl 470/V8 mainframe computer.  146 RESULTS AND DISCUSSION Twenty-seven self-supporting gels that were formed under various conditions of pH, sodium chloride concentration and extent of protein succinylation were tested. The physical appearance of the gels ranged from opaque and pasty to translucent and springy as described in Chapter 2. The relationship between rupture force of the puncture test and storage modulus at 10 s * is shown in Figure 3.2. Although significant (p<0.01, Table -  3.1), rupture force alone accounted for less than thirty percent of the variation in storage modulus. Of the twenty-seven gels tested, seventeen were classified as translucent and ten were opaque, although one of the opaque gels had  a great deal of translucent character and seemed to be  between the two types.  intermediate  Rupture force appeared to be related in a curvilinear  manner to the nine gels with the most opaque character, but there was a poor relationship between rupture force and storage modulus for the translucent gels (Figure 3.2). The relationship between rupture slope and storage modulus is shown in Figure  3.3.  For a l l gels, a highly significant correlation was observed  between the two variables ( r = 0.857, Table 3.1). 2  For the translucent gels,  rupture slope accounted for nearly ninety-five percent of the variation in storage modulus (Table 3.1). For the opaque gels, storage modulus was also well described by rupture slope (R = 0.857, p<0.001), but the slope of the 2  linear relationship between the two variables was greater  than for the  translucent gels. Thus for a given storage modulus, the rupture slope of the translucent gels was higher  than for the opaque gels, which indicated a  difference in response of opaque and translucent gels to the two types of gel texture measurement. The storage modulus of the opaque gels also followed a  147 900 800700.p-, 600 V) D "5 T5 O  500H  ©  400 H  g) O  300-  00  (7) 200  • o  Legend  •  A Unmodified O 5.2% Succ. An.  A  •  14.2% Succ. An.  •  100 i  0  100  50  150  300  250  200  Rupture Force (mN) F i g u r e 3.2.  Rupture force vs. storage modulus for canola isolate gels ( s o l i d symbols indicate opaque g e l s ) .  900 800700-  &  600-  in  3 TJ O 0)  O) O i_  o  500400300-  Legend  CO  tn  A Unmodified  9  200-  O 5.2% Succ. An.  •  100-  •  14.2% Succ. An.  o 10  20  30  40  50  60  70  80  90  Rupture Slope (mN/mm) F i g u r e 3.3.  Rupture slope vs. storage modulus for canola isolate gels ( s o l i d symbols indicate opaque g e l s ) .  148 T a b l e 3.1.  Rheogoniometer v s . I n s t r o n measurements o f g e l t e x t u r e .  Dependent Variable A.  Independent Variable  Coefficient  F-prob.  2.275 x IO 104.03  3  0.0033  A l l g e l s (n=27)  Storage Modulus r = 0.296** S.E. - 224.2  Rupture Force Constant  Storage Modulus r = 0.857*** S.E. = 101.0  Rupture Slope Constant  12.12 x 103 -4.260  0.0000  Loss Modulus R - 0.840*** S.E. • 28.19  Rupture Slope Rupture Area Constant  2.912 x 10 •133.34 27.964  0.0000 0.0001  Loss Tangent R = 0.800*** S.E. = 0.029  Rupture Area Rupture Area Constant  2  a  2  2  2  B.  2  3  -0.536 0.389 0.321  0.0000 0.0085  T r a n s l u c e n t g e l s (n=17)  Storage Modulus r = 0.946*** S.E. = 54.11  Rupture Slope Constant  10.94 x 10 -4.992  3  0.0000  Loss Modulus R = 0.940*** S.E. = 13.19  Rupture Slope Rupture Area Constant  2.433 x 103 -80.123 16.229  0.0000 0.0038  Storage Modulus r = 0.857*** S.E. = 126.0  Rupture Slope Constant  13.83 x 10 4.156  3  0.0001  Storage Modulus R = 0.958*** S.E. = 78.44  Rupture Slope Rupture Area Rupture Area Constant  7.961 x 10 40.321 0.192 -52.134  3  0.0058 0.0091 0.0087  Loss Modulus r = 0.779*** S.E. - 41.30  Rupture Slope Constant  3.469 x 103 1.552  0.0007  2  m  2  C.  Opaque G e l s (n=10)  2  2  2  **, p<0.01; ***, p<0.001 Standard error of estimate. a  2  149 curvilinear relationship with rupture  area, where  storage modulus first  increased and then decreased as rupture area increased. Rupture slope and rupture area (quadratic) together accounted for nearly ninety-six percent of the variation in storage modulus for these gels (Table 3.1). The relationship between loss modulus and rupture slope is shown in Figure 3.4. For all gels, loss modulus was well predicted by rupture slope and rupture area which together accounted  for eighty-four percent of the  variation in loss modulus (Table 3.1). Loss modulus increased as rupture slope increased and rupture area decreased. For the translucent gels, rupture slope and rupture area together accounted  for ninety-four percent of the  variation in loss modulus, while for the opaque gels rupture slope alone accounted for nearly seventy-eight percent of the variation in loss modulus. The loss tangent of the gels was well described by the rupture area of the puncture test (Figure 3.5, Table 3.1). As rupture area increased, loss tangent  decreased  in a curvilinear manner.  The quadratic polynomial of  rupture area accounted for eighty percent of the variation in loss tangent. No improvement in predictive ability was obtained by subdividing the gels into translucent and opaque for the analysis of loss tangent. In dynamic testing, the storage modulus is a measure of the energy stored per cycle of sinusoidal deformation, the loss modulus is a measure of the energy dissipated as heat, while the loss tangent is the proportion of energy dissipated to energy recovered per cycle (Ferry, 1980). The force-deformation (stress-strain) relationship obtained from a puncture  or compression  test  indicates how a material deforms under load. The force-deformation curve represents the force required to obtain a given deformation at any point in the test while the area under the curve to the point of rupture is a measure of the energy required to rupture the sample.  150 250  200-  CD  150  Z3  o CO  to o  100-  Legend A Unmodified  50-  B  O  5.2%  S u c c .  •  14.2%  An.  S u c c .  An.  cO 1 - T —  10  - T —  30  20  40  50  60  - i —  70  Rupture Slope (mN/mm)  80  90  F i g u r e 3.4. R u p t u r e slope v s . loss modulus f o r c a n o l a i s o l a t e g e l s ( s o l i d symbols i n d i c a t e opaque g e l s ) .  0.4-1  Legend 0.3c  A  Unmodified  O  5.2%  •  14.2%  Succ. S u c c .  An. An.  V  o> c tn tn o  0.2 °  0.1  100  O  D  D  A  °  O  i 200  i 300  0 ^  i 400  i 500  Rupture Area (mN mm)  i  600  i 700  r 800  F i g u r e 3.5. R u p t u r e area v s . loss tangent f o r c a n o l a i s o l a t e gels ( s o l i d symbols i n d i c a t e opaque g e l s ) .  151 The  initial slope of the force-deformation curve has been variously  described as the elasticity modulus, apparent elasticity, rigidity, stiffness, deformability, and initial tangent (Hermansson, 1982; Mohsenin and Mittal, 1977). Mitchell (1976, 1980) and Mitchell and Blanshard (1976a,b) stated that the rigidity modulus of a gel is not necessarily related to its rupture strength, as the rigidity modulus is influenced primarily by short, stiff chains present in the gel whereas the rupture strength is governed by the number of long, flexible chains remaining after the cross-links joined by the short, stiff chains have ruptured. They suggested that this may explain why the rigidity modulus was independent of molecular weight above a certain critical value, while the rupture strength appeared to be strongly dependent on molecular weight (Mitchell and Blanshard, 1976a,b). Mohsenin (1970) and Mohsenin and Mittal (1977), however, stated that elasticity as defined by Hooke's Law  does not really exist in food materials, as even some very hard  materials show some unrecoverable deformation upon unloading. Hermansson (1982) and Mohsenin and Mittal (1977) suggested that the use of the term "elasticity modulus" be  avoided  in the case of non-linear stress-strain  behavior or where the upper limit of the elastic region is unknown. Calzada and Peleg (1978) also cautioned that an apparently linear region of a stressstrain curve may not be due to an elastic region but may be a result of two antagonistic effects such as fracture and  compression.  Mohsenin (1970)  suggested that a "modulus of deformability" be used instead, where the total elastic and plastic deformation at some selected point on the force-deformation curve is taken for computing the strain.  Using this concept, Mohsenin  and Mittal (1977) computed a modulus of deformability for apple tissue at onehalf the total deformation to the point of inflection, and as expected found no correlation between it and the force of deformation at the bioyield point.  152 They explained  this by pointing out that the bioyield point is a failure  phenomenon whereas the modulus of deformability is an indication of the stiffness of the tissue when small forces are applied, so the two measurements should not be expected to be correlated. In the present study, the rupture slope was highly correlated with the storage and loss moduli of the gels, and was faster and simpler to obtain than either the initial slope of the force-deformation deformability as defined by Mohsenin (1970).  curve or the modulus of  In addition, rupture area as  calculated from the force and deformation at rupture, although an approximation of the true area, was simpler to calculate and was well correlated with the loss tangent of the gels, and along with rupture slope was a significant predictor of storage modulus for the opaque gels and loss modulus for all gels. The different relationship between the dynamic viscoelastic parameters and the puncture test measurements for the translucent and opaque gels was probably the result of differences in microstructure between the two types of gels. Although the microstructure of the gels per se was not examined, light micrographs of the unheated dispersions revealed the presence of numerous insoluble aggregates in the dispersions that formed opaque gels, while the dispersions that formed translucent gels were relatively homogeneous (see Chapter 2). Under the small deformation, nondestructive conditions employed in dynamic testing, the moduli obtained would be the result of contributions from both the insoluble aggregates and the interaggregate gel matrix in the case of the opaque gels, but from the homogeneous gel matrix alone in the case of translucent  gels.  With the large deformations employed by the  puncture test, however, the force-deformation curves for the opaque gels would reflect mainly the strength of the interaggregate  matrix and the friction  153 between aggregates as they move with respect to one another. Thus for a given storage modulus, opaque gels would be expected to have a smaller rupture slope than translucent  gels where the rupture slope and dynamic viscoelastic  parameters reflect the microstructure of the homogeneous matrix, and this was indeed the case. Gill and Tung (1978) found a similar response to dynamic and steady shearing conditions of gels from the 12S fraction of rapeseed protein that contained aggregates. They hypothesized that the aggregates gave highly elastic recoveries under dynamic shear, but under steady shear the forces between the aggregates would be broken and the aggregates would be able to move with respect to one another resulting in lower apparent viscosities than from gels with an homogeneous matrix.  154 SUMMARY AND CONCLUSIONS The relationships between a large deformation puncture test and a nondestructive dynamic test method for assessing the rheological properties of canola protein gels were examined. Although the force required to rupture the gels, as measured by the puncture test, was poorly correlated with the viscoelastic parameters, the slope of the force-deformation  curves to the  point of rupture was well correlated with the storage and loss moduli of the gels.  In addition, the area under the force-deformation  curves followed a  curvilinear relationship with the loss tangent of the gels.  Of the 27 gels  tested, 17 were translucent and 10 were opaque. The responses of the translucent and opaque gels to the two types of rheological tests were not identical, which indicated that gel microstructure may be an influential factor when evaluating gel texture by destructive or nondestructive  methods.  Although excellent correlations between puncture test measurements and viscoelastic parameters for canola protein gels were demonstrated, it is not certain that similar results would be obtained with gels from other gelation conditions, protein sources or concentrations, or with non-protein gels where the types of bonds involved in gel formation and stabilization may influence the results. Although more work is required to clarify these relationships, the present data provide a framework on which to base future research in this area.  155 REFERENCES Bourne, M.C. 1966. Measurement of shear and compression components of puncture tests. J. Food Sci. 31:282. Bourne, M.C. 1968. Texture profile of ripening pears. J. Food Sci. 33:223. Bourne, M.C. 1979. Theory and application of the puncture test in food texture measurement. In: Food Texture and Rheology. Sherman, P. (Ed.). Academic Press, New York, NY. Calzada, J.F. and Peleg, M. 1978. Mechanical interpretation of compressive stress-strain relationships of solid foods. J. Food Sci. 43:1087. Elliott, J.H. and Ganz, A.J. 1975. Gel characterization with the Weissenberg rheogoniometer: application to carrageenan gels. J. Food Sci. 40:394. Ferry, J.D. 1980. York, NY.  Viscoelastic Properties of Polymers, 3rd ed. Wiley, New  Fox, D.J. and Guire, K.E. 1976. Documentation for MIDAS, 3rd ed. The Statistical Research Laboratory, The University of Michigan, Ann Arbor, MI. Gill,  T.A. and Tung, M.A. 1978. Thermally induced gelation of the 12S rapeseed glycoprotein. J. Food Sci. 43:1481.  Hermansson, A.-M. 1982. Gel characteristics - compression and penetration of blood plasma gels. J. Food Sci. 47:1960. Jones, L.J. 1980. Functional properties of modified oilseed protein concentrates and isolates. M.Sc. Thesis, University of British Columbia, Vancouver, B.C. Kamel, B.S. and deMan, J.M. 1977. Some factors affecting gelatin gel texture evaluation by penetration testing. J. Texture Stud. 8:327. Mitchell, J.R. 1976.  Rheology of gels.  J. Texture Stud. 7:313.  Mitchell, J.R. 1980. The rheology of gels.  J. Texture Stud. 11:315.  M i t c h e l l , J.R. and Blanshard, J.M.V. 1976a. alginate gels. J. Texture Stud. 7:219.  Rheological properties of  Mitchell, J.R. and Blanshard, J.M.V. 1976b. Rheological properties of pectate gels. J. Texture Stud. 7:341. Mohsenin, N.N. 1970. Physical Properties of Plant and Animal Materials Structure, Physical Characteristics and Mechanical Properties. Gordon and Breach Science Publishers, New York, NY.  156 Mohsenin, N.N. and Goehlich, H. 1962. Techniques for determination of mechanical properties of fruits and vegetables as related to design and development of harvesting and processing machinery. J. Agric. Eng. Res. 7:300. Mohsenin, N.N. and Mittal, J.P. 1977. Use of rheological terms and correlation of compatible measurements in food texture research. J. Texture Stud. 8:395. Nijenhuis, K. te 1974. Investigation into the ageing process of gel systems by the measurement of their dynamic moduli. Dechema-Monogr. 77:177. Rasraussen, J. 1974. Gel texture in foods - its relationship to choice of gelling agent, formulae and processing conditions. Dechema-Monogr. 77:187. Segeren, A.J.M., Boskamp, J.V. and van den Tempel, M. 1974. Rheological and swelling properties of alginate gels. Faraday. Diss. Chem. Soc. 57:255. Tung, M.A. 1978.  Rheology of protein dispersions.  J. Texture Stud. 9:3.  Van Kleef, F., Boskamp, J. and van den Tempel, M. 1978. Determination of the number of cross-links in a protein gel from its mechanical and swelling properties. Biopolymers 17:225. Wood, F.W. 1979. Psychophysical studies on liquid foods and gels. In: Food Texture and Rheology. Sherman, P. (Ed.), p.21. Academic Press, New York, NY.  Publications (cont.) G i l l , T.A. and Paulson, A.T. 1982. Localization, characterization and partial purification of TMAD-ase. Comp. Biochem. Physiol. 71B:49. Paulson, A.T., Vanderstoep, J . and Porritt, S.W. 1980. Enzymatic brcwning of peaches: effects of gibberellic acid and ethephon on phenolic compounds and polyphenoloxidase activity. J . Food Sci. 45:341. Paulson, A.T., Vanderstoep, J. and Eaton, G.W. 1979. Effects of gibberellic acid and ethephon on enzymatic browning of "Redhaven" peaches. HortScience 14:711. Douglas, M., Vanderstoep, J . and Paulson, A.T. 1977. Effect of gibbe r e l l i c acid and ethephon on ascorbic acid content and ascorbic acid oxidase activity of Redhaven peaches. Can. Inst. Food S c i . Technol. J . 10:233.  

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