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Rheological and ultrastructural properties of wheat gluten Cumming, Daniel Brian 1974

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RHEOLOGICAL AND ULTRASTRUCTURAL PROPERTIES OF WHEAT GLUTEN BY DANIEL BRIAN CUMMING B.S.A. University of B r i t i s h Columbia M.Sc. University of Guelph A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Food Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1974. I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Food Science T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e December 5, 1974. i . ABSTRACT A three part study i s presented i n which the u l t r a s t r u c t u r a l and dynamic shear properties of modified and unmodified rehydrated commercial, v i t a l wheat gluten are investigated. Both scanning and transmission electron microscopy were employed to observe the u l t r a s t r u c t u r a l interactions of starch, protein and l i p i d f r a c t i o n s of rehydrated gluten and f l o u r doughs. Dynamic shear pro-perties of rehydrated gluten were studied with a cone and plate rheometer i n o s c i l l a t o r y shear mode, and the e f f e c t of adjusting the levels and nature of the protein, starch and l i p i d phases was also observed using both electron microscopy and dynamic shear t e s t i n g . Gross stressing of gluten and f l o u r specimens resulted i n noticeable a l t e r a t i o n to t h e i r u l t r a s t r u c t u r a l appearance, manifested as a d i r e c t i o n a l o r i e n t a t i o n of the l i p o i d a l inclusions i n gluten and the formation of a d e f i n i t e f i b e r or sheeted structure i n f l o u r dough. Removal of free l i p i d seemed to a f f e c t protein-protein as well as starch-protein i n t e r a c t i o n as evidenced by the microscopic appearance. Gluten proved to be a i d e o l o g i c a l l y complicated material requiring multiple regression analysis to adequately describe the relationships of dynamic shear storage and loss moduli, dynamic v i s c o s i t y and dynamic shear loss tangent with factors such as o s c i l l a t o r y frequency and cycle, speci-men moisture content and age, and amplitude of s t r a i n input. Moisture content of the specimens and o s c i l l a t o r y c y c l i n g had s i g n i f i c a n t influence over the storage and loss moduli and apparently were responsible for the f a i l u r e of simple relationships to account for v a r i a t i o n i n the rheological parameters for pooled or even rep l i c a t e d samples. It was found that over the range of o s c i l l a t o r y frequencies employed (0.075 - 0.949 sec "S the storage modulus or e l a s t i c component increased with increasing frequency. However, the loss modulus or viscous component was found to have a greater rate of increase and consequently the loss/storage r a t i o or loss tangent showed a s h i f t of the v i s c o e l a s t i c response toward the viscous end of the behavorial continuum. Adding of l i p i d (wheat germ o i l and unemulsified, hydrogenated rapeseed shortening), and pearled wheat starch and petroleum ether extraction of free l i p i d had no s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on the nature of the v i s c o e l a s t i c response. The l e v e l of response was affected markedly, ranging from an intercept value for dynamic v i s c o s i t y (vs. o s c i l l a t o r y frequency) of 8330 poise for 5% shortening to 31,050 poise for 30% starch. Increasing l e v e l of l i p i d from 0% free l i p i d to 5% added wheat germ o i l caused a general decrease i n response l e v e l . However, increasing l i p i d l e v e l beyond about 3% did not result i n any s i g n i f i c a n t change i n response. L i p i d seemed attracted to starch granule surfaces and began to coalesce as evidenced by u l t r a s t r u c t u r a l appearance as the l e v e l of added l i p i d increased. The rheological behavior of gluten i n both modified and unmodified form agreed well with a recent molecular model for wheat gluten v i s c o e l a s t i c i t y based on hydrogen bonding. iv'. GENERAL TABLE OF CONTENTS PAGE ABSTRACT i TABLE OF CONTENTS (GENERAL) i v TABLE OF CONTENTS (CHAPTER 1) v TABLE OF CONTENTS (CHAPTER 2) v i TABLE OF CONTENTS (CHAPTER 3) v i i LIST OF TABLES ix LIST OF FIGURES x i ACKNOWLEDGEMENTS xv GENERAL INFORMATION 1 CHAPTER 1 3 CHAPTER 2 22 CHAPTER 3 53 LITERATURE CITED 100 V . CHAPTER 1. THE ULTRASTRUCTURAL RELATIONSHIPS OF THE COMPONENT PARTS OF COMMERCIAL VITAL WHEAT GLUTEN : PAGE 1.1 Introduction 3 1.2 Literature Review 4 1.3 Methods and Materials 5 1.3.1 General sample preparation 5 1.3.2 Scanning electron microscopy 5 1.3.3 Transmission electron microscopy 6 1.3.4 Light microscopy 6 1.4 Results and Discussion . 7 1.4.1 Microstructure of wheat fl o u r doughs and rehydrated gluten 7 1.4.2 Ultrastructure of wheat f l o u r dough 7 1.4.3 Gluten composition and character 11 1.4.4 Ultrastructure of rehydrated wheat gluten 13 1.4.5 Ultrastructure of freshly prepared gluten 19 1.5 Summary and Conclusions 20 C H A P T E R 2 . A N I N V E S T I G A T I O N OF S E L E C T E D R H E O L O G I C A L P R O P E R T I E S OF C O M M E R C I A L WHEAT G L U T E N V I 2 . 1 2 . 2 2 . 3 2 . 4 2 . 5 P A G E I n t r o d u c t i o n 22 L i t e r a t u r e R e v i e w 24 M e t h o d s a n d M a t e r i a l s 2 8 2 . 3 . 1 S a m p l e p r e p a r a t i o n 2 8 2 . 3 . 2 R h e o l o g i c a l m e a s u r e m e n t 29 2 . 3 . 4 E x p e r i m e n t a l c a l c u l a t i o n s 33 2 . 3 . 5 S t a t i s t i c a l p r o c e d u r e s 33 R e s u l t s a n d D i s c u s s i o n 34 2 . 4 . 1 P r e l i m i n a r y f i n d i n g s - a n o m a l o u s b e h a v i o r 34 2 . 4 . 2 D e f i n i t i o n o f p r i m a r y e x p e r i m e n t 39 2 . 4 . 3 S i m p l e l i n e a r r e g r e s s i o n 39 2 . 4 . 4 M u l t i p l e r e g r e s s i o n 39 2 . 4 . 5 S t a n d a r d p a r t i a l r e g r e s s i o n c o e f f i c i e n t s 40 2 . 4 . 6 E v a l u a t i o n o f s t a t i s t i c a l r e l a t i o n s h i p s f o r n 1 , G ' , G " , a n d t a n jz$ 4 3 2 . 4 . 7 E f f e c t o f s p e c i m e n m o i s t u r e c o n t e n t 44 2 . 4 . 8 T h e e f f e c t o f o s c i l l a t o r y f r e q u e n c y 4 5 2 . 4 . 9 E x p l a n a t i o n o f v i s c o e l a s t i c i t y b y m o l e c u l a r t h e o r y 46 S u m m a r y a n d C o n c l u s i o n s 4 9 v i i . C H A P T E R 3 . T H E E F F E C T OF S P E C I F I C M O D I F I C A T I O N S ON T H E U L T R A S T R U C T U R E AND R H E O L O G Y O F C O M M E R C I A L V I T A L WHEAT G L U T E N ... P A G E 3 . 1 I n t r o d u c t i o n 52 3 . 2 L i t e r a t u r e R e v i e w 53 3 . 3 M e t h o d s a n d M a t e r i a l s 57 3 . 3 . 1 S a m p l e p r e p a r a t i o n 57 3 . 3 . 2 R h e o l o g i c a l M e a s u r e m e n t 5g 3 . 3 . 3 S t a t i s t i c a l p r o c e d u r e s 59 3 . 3 . 4 E l e c t r o n m i c r o s c o p y 59 3 . 4 R e s u l t s a n d ' D i s c u s s i o n s 60 3 . 4 . 1 T h e e f f e c t o f a d d e d w h e a t g e r m o i l o n r e h y d r a t e d v i t a l w h e a t g l u t e n 6 0 3 . 4 . 1 . 1 T h e e f f e c t o f w h e a t g e r m o i l o n g l u t e n r h e o l o g y -c o m p l e x r e l a t i o n s h i p s 6 0 3 . 4 . 1 . 2 T h e e f f e c t o f w h e a t g e r m o i l l e v e l o n g l u t e n r h e o l o g y - s i m p l e r e l a t i o n -s h i p s 64 3 . 4 . 1 . 3 G e n e r a l r h e o l o g i c a l t r e n d s gg 3 . 4 . 1 . 4 T h e e f f e c t o f w h e a t g e r m o i l o n t h e u l t r a s t r u c t u r a l c h a r a c t e r o f w h e a t g l u t e n gg 3 . 4 . 2 T h e e f f e c t o f a d d e d s h o r t e n i n g o n r e h y d r a t e d v i t a l w h e a t g l u t e n gg 3 . 4 . 2 . 1 T h e e f f e c t o f r a p e s e e d s h o r t e n i n g o n g l u t e n r h e o l o g y - c o m p l e x r e l a t i o n -s h i p s gg • 3 . 4 . 2 . 2 T h e e f f e c t o f s h o r t e n i n g l e v e l o n g l u t e n r h e o l o g y - s i m p l e r e l a t i o n s h i p s 71 v i i i . C H A P T E R 3 ( C o n t i n u e d ) P A G E 3 . 4 . 2 . 3 G e n e r a l r h e o l o g i c a l t r e n d s 74 3 . 4 . 2 . 4 T h e e f f e c t o f s h o r t e n i n g o n t h e u l t r a s t r u c t u r a l c h a r a c t e r o f w h e a t g l u t e n 74 3 . 4 . 3 T h e e f f e c t o f f r e e l i p i d r e m o v a l o n r e h y d r a t e d v i t a l w h e a t g l u t e n 7 6 . 3 . 4 . 3 . 1 T h e e f f e c t o f f r e e l i p i d e x t r a c t i o n o n g l u t e n r h e o l o g y r- c o m p l e x r e l a t i o n s h i p s 78 3 . 4 . 3 . 2 T h e e f f e c t o f f r e e l i p i d e x t r a c t i o n o n g l u t e n r h e o l o g y <- s i m p l e r e l a t i o n s h i p s 78 3 . 4 . 3 . 3 T h e e f f e c t o f f r e e l i p i d e x t r a c t i o n o n t h e u l t r a s t r u c -t u r a l c h a r a c t e r o f g l u t e n 8 2 3 . 4 . 4 T h e e f f e c t o f a d d e d s t a r c h o n r e h y d r a t e d v i t a l w h e a t g l u t e n 82 3 . 4 . 4 . 1 T h e e f f e c t o f a d d e d s t a r c h o n g l u t e n r h e o l o g y ~ c o m p l e x r e l a t i o n s h i p s 83 3 . 4 . 4 . 2 T h e e f f e c t o f s t a r c h l e v e l o n g l u t e n r h e o l o g y - s i m p l e r e l a t i o n s h i p s 86 3 . 4 . 4 . 3 G e n e r a l r h e o l o g i c a l t r e n d s 88 3 . 4 . 4 . 4 T h e e f f e c t o f s t a r c h o n t h e u l t r a s t r u c t u r a l c h a r a c t e r o f g l u t e n 88 3 . 4 . 5 T h e i n f l u e n c e o f l i p i d o n g l u t e n b e h a v i o r 91 3 . 4 . 6 T h e i n f l u e n c e o f s t a r c h o n g l u t e n b e h a v i o r 95 3 . 5 S u m m a r y a n d C o n c l u s i o n s 97 I X , TABLE LIST OF TABLES PAGE 1 Experimental operating conditions for Weissenberg Rheogoniometer 31 2 L i s t of symbols employed for dynamic shear studies 32 3 Stepwise multiple l i n e a r regression equations related to the rheological response of wheat gluten subjected to dynamic shear testing 41 4 Standard p a r t i a l regression c o e f f i c i e n t s for the multiple regression equations related to the rheological response of commercial wheat gluten subjected to dynamic shear testing 4 2 5 Stepwise multiple l i n e a r regression response of equations related to the rheological response of WGO modified commercial wheat gluten subjected to dynamic shear testing 61 6 Standard p a r t i a l regression c o e f f i c i e n t s for the multiple regression equations related to the rheological response of WGO modified commercial wheat gluten subjected to dynamic shear testing 62 7 Stepwise multiple l i n e a r regression equations related to the rheological response of RS modified commercial wheat gluten subjected to dynamic shear t e s t i n g 70 8 Standard p a r t i a l regression equations related to the rheological response of RS modified commercial wheat gluten subjected to dynamic shear t e s t i n g , 7 2 9 Stepwise multiple l i n e a r regression equations related to the rheological response of ether extracted commercial wheat gluten subjected to dynamic shear measurement 79 10 Standard p a r t i a l regression c o e f f i c i e n t s for the multiple regression equations related to the rheological response of ether extracted commercial wheat gluten subjected to dynamic shear testing LIST OF TABLES (Continued) TABLE 11 Stepwise multiple l i n e a r regression equations related to the rheological response of starch modified commercial wheat gluten subjected to dynamic shear tes t i n g 12 Standard p a r t i a l regression c o e f f i c i e n t s for the multiple regression equations related to the rheological response of starch modified commercial wheat gluten subjected to dynamic shear testing x i LIST OF FIGURES - CHAPTER 1 FIGURE . PAGE 1 Light micrograph of relaxed wheat f l o u r dough 8 2 Light micrograph of stressed defatted wheat flou r dough 8 3 Light micrograph of relaxed fresh gluten 8 4 Light micrograph of stressed fresh gluten 8 5 Electron micrographs of relaxed f l o u r doughs; a (TEM), b (SEM) 10 6 Electron micrographs of stressed f l o u r doughs; a (TEM), b (SEM) 10 7 Electron micrographs of relaxed defatted f l o u r doughs; a (TEM), b (SEM) 12 8 Electron micrographs of stressed defatted f l o u r doughs; a (TEM), b (SEM) 12 9 Electron micrographs of relaxed rehydrated V i t a l gluten; a (TEM), b (SEM) 14 10 Electron micrographs of stressed rehydrated v i t a l gluten; a (TEM) - FL = free l i p i d , BL = bound l i p i d , b (SEM) 14 11 Electron micrographs of relaxed defatted v i t a l gluten (rehydrated); a (TEM),b (SEM). 17 12 Electron micrographs of stressed defatted v i t a l gluten (rehydrated); a (TEM) -in s e r t magnification X 35,000, b (SEM) 17 13 Electron micrographs of relaxed freshly prepared gluten; a (TEM), b (SEM) 18 14 Electron micrographs of stressed freshly prepared gluten; a (TEM), b (SEM) 18 L I S T OF FIGURES - CHAPTER 2 T y p i c a l o s c i l l a t o r y s h e a r r e s u l t s f o r p u r e l y e l a s t i c ( a ) , p u r e l y v i s c o u s ( b ) , and v i s c o e l a s t i c r e s p o n s e ( c ) . S o l i d l i n e r e p r e s e n t s s t r a i n and t h e b r o k e n l i n e r e p r e s e n t s s t r e s s S c h e m a t i c r e p r e s e n t a t i o n o f t h e W e i s s e n b e r g R h e o g o n i o m e t e r The e f f e c t o f o s c i l l a t o r y f r e q u e n c y on G" and G" f o r r e h y d r a t e d wheat g l u t e n The e f f e c t o f s t r a i n a m p l i t u d e on G' and G" r e s p o n s e f o r r e h y d r a t e d wheat g l u t e n . The e f f e c t o f o s c i l l a t o r y c y c l i n g on G' and G" r e s p o n s e f o r r e h y d r a t e d wheat g l u t e n . x i i i . LIST OF FIGURES - CHAPTER 3 FIGURES . PAGE 20 The e f f e c t of varied oscillatory-frequency (log to) on dynamic v i s c o s i t y (log n..') of wheat gluten for f i v e l e v e l s of added wheat germ o i l . 6 ^ 21 The e f f e c t of added wheat germ o i l on the rheological response of wheat gluten 67 22 Electron micrographs of rehydrated wheat gluten with various l e v e l s of wheat germ o i l . Scanning electron micrographs a (1%) , b (.3%) and c (5% WGO) . Trans-mission electron micrograph; d (5% WGO) 8^ 23 The e f f e c t of varied o s c i l l a t o r y frequency (log co) on dynamic v i s c o s i t y (log n' ) of wheat gluten for f i v e l e v e l s of added rapeseed shortening ^ 24 The e f f e c t of added rapeseed shortening on the rheological response of wheat gluten 7 5 25 Electron micrographs of rehydrated wheat gluten with added rapeseed shortening. Scanning electron micrograph; a (4% RS) -edge view. Transmission electron micrograph b (3% RS) 77 26 The e f f e c t of varied o s c i l l a t o r y frequency (log to) on dynamic v i s c o s i t y (log n 1 ) of wheat gluten for various le v e l s and types of l i p i d . 81 27 Scanning electron micrograph of rehydrated wheat gluten with free l i p i d extracted 77 28 The e f f e c t of o s c i l l a t o r y frequency (log to) on dynamic v i s c o s i t y (log n ' ) of wheat gluten with four l e v e l s of added starch 87 29 The e f f e c t of added wheat starch on the rheological response of wheat gluten 89 Electron micrographs of rehydrated wheat gluten with various l e v e l s of added starch. Scanning electron micrographs; a (5%), b (30% starch) and c (30% starch-fracture surface). Transmission electron micrograph; d (30% starch). ACKNOWLEDGEMENTS The author, wishes to express his thanks to Vr. M . A . Tung, Vr. W.V. Powrie, Vr. S. Nakai, Vr. J . F . Richards and Vr. J . Vanderstoep, Vepartment o{ food Science and Vr. E.O. Myborg, Vepartment o{ A g r i c u l t u r a l Engineering {or guidance throughout the course o{ t h i s i n v e s t i g a t i o n . The assistance o{ many other members, sta{{ and students, o{ the Vepartment o{ Food Science is also g r a t e f u l l y acknowledged. The use o{ electron microscopes in both the Vepartment o{ Biology and the Vepartment o{ Metallurgy is greatly appreciated. A special recognition must be made to my wi{e, Judi, without whose understanding and encouragement i t would not have been possible to begin or complete t h i s undertaking. GENERAL INTRODUCTION Wheat gluten has been known, studied and u t i l i z e d for many years. The unique behavioral character of gluten has made i t of utmost importance to the q u a l i t y of bread doughs and other bakery products. Gluten "develops" from glutenin and g l i a d i n as a r e s u l t of hydration and mixing of wheat f l o u r . A crude protein f r a c t i o n known as v i t a l wheat gluten can be i s o l a t e d from wheat endosperm material. A number of commercial processes e x i s t but the most common method for accomplishing t h i s i s o l a t i o n i s to "wash" the starch and soluble constituents from the gluten phase with water. The gluten protein i s subsequently dried and marketed i n a powdered form for use i n various applications including the strengthening of doughs. Gluten i s an agglomeration of proteins which forms only after wheat fl o u r i s hydrated and worked. The term i s often used as i f gluten were a definable protein e n t i t y . In fact, i t consists of an agglomeration of water insoluble proteins of d i f f e r i n g character and molecular weight held together by a number of forces including covalent, i o n i c , hydrogen and hydrophobic bonds as well as Van der Waals forces. The r e l a t i v e amounts of the component parts vary widely with wheat variety (Pomeranz, 1971b). The v i s c o e l a s t i c properties of wheat fl o u r doughs are largely attributable to the glutenous network that forms during dough development. The investigation reported herein was designed to further elucidate the rheological and u l t r a s t r u c t u r a l nature of a commercially prepared v i t a l wheat gluten. The study was divided into three parts dealing with the ultrastructure of gluten and f l o u r doughs, the dynamic shear properties of rehydrated v i t a l gluten^and the e f f e c t of s p e c i f i c additives and modifications on the u l t r a s t r u c t u r a l and dynamic shear character of v i t a l wheat gluten. I t was intended that the findings of th i s work would be important both to the improvement and understanding of t r a d i t i o n a l dough processes and to the i d e n t i f i c a t i o n and development of new and novel processes related to protein t e x t u r i z a t i o n . CHAPTER 1: THE ULTRASTRUCTURAL RELATIONSHIPS OF THE : COMPONENT PARTS OF COMMERCIAL VITAL WHEAT GLUTEN 1.1. INTRODUCTION Since the interactions of the component parts of any system are largely responsible for the c h a r a c t e r i s t i c chemical and physical properties of that system, i t seemed worthwhile to consider the in t e r r e l a t i o n s h i p s of the various components of v i t a l wheat gluten. Although considerable, work has been done i n the area of microscopic study of wheat "dough" systems, i t has been directed at the ultimate f u n c t i o n a l i t y of wheat fl o u r i n bread doughs. The unique properties of hydrated gluten when subjected to stress have s t i r r e d some intere s t in using these properties i n the ever expanding area of texturized vegetable protein technology. A better understanding of the contribution of each part of the gluten mass would appear to be required to r e a l i z e the optimum u t i l i z a t i o n of the o v e r a l l f u n c t i o n a l i t y of the system. 1.2 LITERATURE REVIEW Over the past few years the electron microscope has proven to be a useful tool for studying the u l t r a -structure of various fractions and forms of wheat f l o u r . Aranyi and Hawrylewicz (1969) employed the scanning electron microscope (SEM) to observe portions of the wheat kernel, endosperm material, f l o u r and dough. These authors reported the extreme s u i t a b i l i t y of t h i s instrument f o r the examina-t i o n of such materials. Simmonds (1972a, b) made use of the transmission electron microscope (TEM) to investigate the ultrastructure of endosperm and wheat dough. Seckinger and Wolf (1970) studied the properties of endosperm proteins from, hard and soft wheats. Endosperm material was allowed to disperse on water droplets, transferred to grids and then observed with the TEM. The structure of various p u r i f i e d protein fractions from wheat endosperm was examined by Orth et a l . (1973a, b) using SEM technique. Bernardin and Kasarda (1973a, b) reported on the formation mechanism of f i b r i l s from hydrated endosperm protein. These authors have put forward a theory for f i b r i l formation i n doughs that involved the rupturing and r o l l i n g back of a sheeted protein structure. The present report i s concerned with u l t r a -s t r u c t u r a l i n t e r r e l a t i o n s h i p s of the component parts of commercial v i t a l wheat gluten. .5. 1.3 METHODS AND1'- MATERIALS 1.3.1 General Sample Preparation Samples of commercially prepared v i t a l wheat gluten and flo u r (50% straight run/50% f i r s t clears) were obtained from Ogilvie Flour M i l l s Ltd. Samples of gluten and flo u r were defatted with petroleum ether and a fresh gluten sample was prepared by handwashing gluten from the f l o u r . Each of these f i v e separate specimens was rehydrated to a "dough". Half of each sample was allowed to rest 45 min p r i o r to f i x a t i o n . The other portion was stressed by kneading and working and immediately fixed. At the time of f i x a t i o n the entire sample was placed d i r e c t l y in excess 2% glutaraldehyde i n phosphate buffer (pH 7.2, 0.15M) where the small specimens suitable for EM embedding and sectioning were cut from the mass. Fixation was accomplished by immersing the specimens i n glutaraldehyde for 45 minutes followed by 1.5 hours i n 1% osmium tetroxide (Os04) i n the phosphate buffer. After f i x a t i o n the specimens were washed with buffer and de-hydrated by a stepwise exchange with ethanol. 1.3.2 Scanning Electron Microscopy After dehydration some samples from each treat-ment were segregated and allowed to a i r dry on f i l t e r paper. These specimens were mounted with s i l v e r paint on aluminum stubs and gold coated i n a Balzers Vacuum Evaporator p r i o r to observation with a Cambridge Stereoscan Mk.II scanning electron microscope operated at 2 0Kv. Images were recorded on Agfa 100 professional f i l m (ASA 100). 1.3.3 Transmission Electron Microscopy The remaining specimens were i n f i l t r a t e d with Spurr's medium by a stepwise replacement of the alcohol within the sample. The resi n was cured for 8.5 hours at 70°C. Sections for both l i g h t and transmission electron microscopy were cut from the hardened r e s i n blocks using a Carl Reichert 0mU3 Ultramicrotome. Thin sections (approximately 60o£) were mounted on uncoated 4 00 mesh copper grids and stained with uranyl acetate p r i o r to observation with an AEI Corinth 27 5 trans-mission electron microscope at 60 KV, Photographic record of the images was made using Agfa Gevatex T51P 70mm f i l m . 1.3.4 Light' Microscopy Thick sections cut for l i g h t microscopy were stained with toluidine blue i n 1% borax. Photographic record was made of these specimens using Kodak high speed Ektachrome f i l m (Tungsten 3200, ASA 125). ' 7.. 1.4 RESULTS AND DISCUSSION 1.4.1 Microstructure of Wheat Flour Dough and Rehydrated  GlutenT ' The present communication w i l l consider three broad component categories: protein, l i p i d , and starch. Light microscope examination was employed to observe the general orientation and s t r u c t u r a l arrangement of the various specimens. Since only the most basic information such as presence or absence of stress and starch content was evident, micrographs of only some of the treatments are presented here. It could be seen that the relaxed protein i n the flo u r (Figure 1) formed a network structure i n r e l a t i o n to the starch phase. In the case of gluten (Figure 3), the protein was observed to be present i n far greater proportion and formed an amorphous mass, which could be oriented under stress (Figure 4) though to a lesser extent than the protein phase of f l o u r (Figure 2). A general observation that could be made on examining the various specimens i s that the larger starch granules are removed from the gluten during i t s preparation. The presence or absence of l i p i d i s not r e a d i l y obvious under the conditions of observation for l i g h t microscopy (Figure 2). 1.4.2 Ultrastructure of Wheat Flour Dough Figure 5a and 5b are TEM and SEM micrographs of relaxed f l o u r dough. In Figure 5b many starch granules can 1 0 L i m F i g . l . L i g h t m i c r o g r a p h o f r e l a x e d wheat f l o u r dough. F i g . 2 . L i g h t m i c r o g r a p h o f s t r e s s e d d e f a t t e d wheat f l o u r dough. 10 L i m F i g . 3 . L i g h t m i c r o g r a p h o f r e l a x e d f r e s h g l u t e n . F i g . 4 . L i g h t m i c r o g r a p h o f s t r e s s e d f r e s h g l u t e n . be seen both on the surface and embedded p a r t i a l l y or completely i n the protein matrix. I t i s quite evident that there i s no p a r t i c u l a r orientation of the protein phase but that i n cross section (Figure 5a) the gluten forms a network around the starch granules. Figure 6a and 6b show the same material in a stressed condition. The protein phase can now be seen to be quite oriented and fibrous i n nature. This i s e s p e c i a l l y evident from the SEM micrograph (Figure 6b). The gluten also seems to form thin sheets over the starch i n certain areas, while forming d e f i n i t e fib.er-like structures i n others. The f i b e r formation theory of Bernardin and Kasarda (1973a) may explain t h i s . The evidence that these workers present suggested that the f i b r i l s r e s u l t from the rupturing and r o l l i n g back of a sheeted structure. TEM studies of t h i s stressed fl o u r dough (Figure 6a) demonstrate the u l t r a s t r u c t u r a l r e l a t i o n s h i p of the component parts i n cross section. If one imagines that Figure 6a represents a section cut perpendicular to the plane of the surface shown in Figure 6b, i t becomes quite clear just how the two micrographs are related. I t would appear from Figures 5 and 6 that the starch and protein phases are p h y s i c a l l y i n t e r r e l a t e d i n the o v e r a l l microstructure of the dough but l i t t l e evidence was observed either i n these micrographs or other similar specimens that there existed any d i r e c t physical or chemical in t e r a c t i o n of these two phases. However, upon extraction of the free l i p i d phase, F i g . 6 . E l e c t r o n m i c r o g r a p h s o f s t r e s s e d f l o u r d oughs; a (TEM), b (SEM). numerous instances of f i b r i l s adhering to starch granules could be observed with the SEM. Figure 7b shows a relaxed defatted fl o u r dough and an excellent example of protein adhering to the surface.of a starch.granule. The protein phase i t s e l f appears to become far less regular and more l i k e a folded sheet, quite d i s s i m i l a r to Figure lb and published SEM micrographs of dough structure (Aranyi and Hawrylewicz, 1969). Figure 7a shows a closer association of the Starch and protein phases than was observed i n the untreated specimens. Stressing of t h i s defatted material produces the results demonstrated by Figure 8a and 8b. The most notable feature of these micrographs i s the evidence of d i r e c t i o n a l orientation of the minute osmiophilic inclusions as seen i n Figure 8a. This w i l l be discussed further i n r e l a t i o n to the various gluten samples examined. 1.4.3. Gluten Composition and Character Preparation of v i t a l gluten involves, among other things, the removal of a large portion of the starch phase of wheat f l o u r . On a dry weight basis wheat flou r generally contains about 75% starch and 16% protein (Bushuk, 1971) while v i t a l wheat gluten on the average w i l l contain 15-20% starch and 80% protein (Pomeranz, 1971a). I t has been widely reported i n the l i t e r a t u r e that development of gluten involves i n t e r a c t i o n of free l i p i d with the protein phase F i g . 8 . E l e c t r o n m i c r o g r a p h s o f s t r e s s e d d e f a t t e d f l o u r douqhs; a (TEM), b (SEM). 13. to produce a bound lip o p r o t e i n component. These interactions have been reviewed by Pomeranz (1971a) i n some d e t a i l . Free , l i p i d per s_e apparently plays l i t t l e d i r e c t role i n the rheology or function of doughs. I t was shown by Simmonds (1972b) and confirmed by c e r t a i n segments of the present investigation that an apparent free l i p i d phase characterized by regularly shaped osmiophilic inclusions (FL i n Figure 10a) could be extracted with petroleum ether. A second l i p i d containing phase apparently was bound and not removed with extraction by petroleum ether (BL i n Figure 10a). Simmonds (1972b) considers the o r i g i n of t h i s phase to be rough endoplasmic reticulum. This seems to be confirmed by an investigation conducted previously by the same author on mature wheat endosperm (Simmonds, 1972a). 1.4.4 Ultrastructure of Rehydrated Wheat Gluten One of the most obvious differences between the various gluten preparations and the f l o u r doughs i s the lack of network structure of any consequence i n the gluten samples. The network system in both relaxed and stressed flour specimens arises as r e s u l t of the presence of starch granules dispersed throughout the enveloping protein matrix. In rehydrated gluten specimens starch i s present i n such r e l a t i v e l y small amounts that i t takes the r o l e of l i t t l e more than an inc l u s i o n , having l i t t l e apparent e f f e c t on the microstructure of the system. When considering the p o s s i b i l i t i e s of t e x t u r i z i n g wheat gluten this f a c t may be important. Without a r e l a t i v e l y i n e r t "spacer" F i g . 9 . E l e c t r o n m i c r o g r a p h s o f r e l a x e d r e h y d r a t e d v i t a l g l u t e n ; a (TEM), b (SEM). F i g . 1 0 . E l e c t r o n m i c r o g r a p h s o f s t r e s s e d r e h y d r a t e d v i t a l g l u t e n ; a (TEM), F L - f r e e l i p i d , BL = bound l i p i d , b (SEM). .15. phase in the system a true f i b e r i z a t i o n .of unmodified wheat gluten may be d i f f i c u l t . Figure 9a i s a t y p i c a l TEM f i e l d for relaxed v i t a l gluten. The o v e r a l l appearance i s that of an amorphous mass with numerous osmiophilic inclusions of the types discussed previously. The corresponding SEM micrograph (Figure 9b) reveals a rather featureless plane surface when compared to f l o u r doughs. As can be seen from Figure 10a, when v i t a l gluten i s stressed there i s consider-able orientation of the various components within the mass. This phenomenon was reported previously by Crozet et a l . (1966). However, r e l a t i v e l y speaking, t h i s mass remains quite continuous when compared to the flo u r specimens. The surface structure of t h i s specimen i s represented by Figure 10b and i n certain places suggests a layered or overlapping Structure. This may explain the dark p a r a l l e l l i n e s i n Figure 10a. The layers seen i n Figure 10b are about 3-5 ym across and the average spacing between the dark l i n e s i n the TEM micrographs was found to be 3 ym. The exact nature of the dark lines i s not c l e a r . They may be the e f f e c t of a certain degree of overlap of two layers or they may be a l i p o i d a l deposit demonstrating a c e r t a i n degree of osmiophilia. Certainly no such structures were observed i n the defatted v i t a l gluten specimens. Relaxed defatted v i t a l gluten (Figure 11a) exhibits a reduction i n the number of inclusions regarded as free l i p i d and an altered uniformity to the background density of the protein matrix. I t i s 16. quite unlikely that removal of the l i p i d phase resulted d i r e c t l y i n empty spaces i n the matrix since.defatting was performed p r i o r to rehydration and development of the gluten dough. Figure l i b shows the surface appearance of t h i s specimen. Typical f i e l d s of such samples demonstrated rather coarse or discontinuous surfaces r e l a t i v e to untreated speci-mens (Figures 9b, 10b, 13b, and 14b). The TEM micrograph of stressed defatted y i t a l gluten (Figure 12a) shows considerable orientation and separation of the protein matrix. This can be observed even at magnifications, of about X35, 000 (Insert Figure 12a). I t may be that a p a r t i a l l y bound l i p i d complex i s responsible for the interaction of the gluten proteins. In addition, t h i s l i p i d phase would appear to be at least p a r t i a l l y extractable with petroleum ether. It should be noted that i f the theory of incorporation of free l i p i d into the protein complex during gluten development i s accepted, we are not r e f e r r i n g to the free l i p i d inclusions which are also removed by ether extraction. That i s , the extraction was done on the v i t a l gluten and any l i p i d - p r o t e i n complex formed during gluten development should have been present at the time of extraction. I t has been shown by Olcott and Mecham (1947) that as dough development continues, free l i p i d content i s reduced. Removal of some portion of th i s complexed l i p i d seems to have occurred as indicated by the more p a r t i -culate appearance of relaxed gluten and the oriented, separated Fig.12. Electron micrographs of stressed defatted v i t a l gluten (rehydrated); a (TEM) - i n s e r t magnifica-t i o n X 35,000, b (SEM). Fig.13. Electron micrographs of relaxed freshly prepared gluten; a (TEM), b (SEM). f i b r i l structure of the stressed sample. Very l i t t l e actual material has been removed but the r e s u l t seems to be a reduced a b i l i t y to form an interconnected continuous mass. 1.4.5 Ultrastructure of Freshly Prepared Gluten The f i n a l two sets of micrographs (Figure 13 and 14) are of gluten freshly prepared from the fl o u r used i n t h i s study. This part of the experiment was conducted as a control procedure to provide a comparison of v i t a l gluten with fresh gluten. As can be seen by comparing Figures 13 with 9, and 14 with 10, there are no great differences between s i m i l a r l y treated samples. When a number of speci-mens were compared the differences exhibited i n the various micrographs presented were no more marked than the difference between f i e l d s i n any given specimen. ' ... 20. 1.5 SUMMARY AND CONCLUSIONS Protein, starch and l i p i d s play an important part i n the ultrastructure of commercial v i t a l wheat gluten. In the preparation of v i t a l gluten a large part of the starch phase of wheat fl o u r i s removed and that which remains tends to be small i n regard to p a r t i c l e s i z e . The tendency of the protein phase to form a fibrous network i n either stressed or relaxed samples i s destroyed or at lea s t reduced i n the r e l a t i v e absence of starch granules. Starchy-protein interactions seem to be affected by the removal of free l i p i d as was evidenced by the presence of protein f i b r i l s adhering to the surface of starch grains in defatted but not untreated samples. This was apparently true for both gluten and fl o u r samples though i t was not as c l e a r l y evident with gluten as with f l o u r doughs. Protein-protein i n t e r a c t i o n also seemed to be altered by the removal of some part of the l i p i d material from v i t a l gluten. Protein f i b r i l s were quite.evident i n stressed defatted gluten and i n the unstressed specimen the protein matrix, though demonstrating no p a r t i c u l a r orientation, was seen to be quite granular or discontinuous i n appearance. It i s l i k e l y that the empty spaces between the protein masses were occupied by free water pr i o r to EM preparation procedures. This indicates that rehydration was affected by the action of the l i p i d extraction procedure. The presence of these spaces may also indicate that disruption of the p r o t e i n - l i p i d complex formed daring gluten development hinders complete i n t e r a c t i o n of the protein phase within i t s e l f . Such basic changes i n the u l t r a s t r u c t u r e of wheat gluten could well be expected to a l t e r i t s ultimate functional properties. A better understanding of exactly how these properties are and can be altered w i l l be of considerable importance i n the preparation of modified vegetable protein products based wholly or i n part on v i t a l wheat gluten. 22. CHAPTER 2; AN INVESTIGATION OF SELECTED RHEOLOGICAL PROPERTIES OF COMMERCIAL WHEAT GLUTEN 2.1 INTRODUCTION . A considerable l i t e r a t u r e exists with regard to the rheological properties of wheat doughs. In p a r t i c u l a r t h i s research relates most often to t e n s i l e properties and to the f u n c t i o n a l i t y of wheat fl o u r i n baking applications, e s p e c i a l l y bread. Some work has been done on gluten per se but most often the te s t material has been a flou r dough. The unique functional properties of gluten seem to be related to i t s v i s c o e l a s t i c nature. Few other vegetable proteins from commonly used grains or seeds behave i n exactly the same way or to the same extent as wheat gluten (Ewart, 1972) . Most of the t r a d i t i o n a l methods of rheological testing applied to dough involve large deformation testing and though t h i s i s of i n t e r e s t , the same general methods are not suitable for small deformation and short time measurements. For t h i s purpose dynamic testing with a small deformation sinusoidal input, i s more appropriate. U n t i l r e l a t i v e l y recently the necessary test equipment had not been read i l y available. The present investigation was carried out with a Weissenberg Rheogoniometer, an instrument capable of dynamic shear measurement. In Chapter 1 the f i b r i l forming a b i l i t y of gluten and the influence of various factors on t h i s was reported. In t h i s chapter, dynamic shear properties of gluten are investigated. The influence of s p e c i f i c test conditions and procedures on these properties are reported. 2.2 LITERATURE REVIEW Rheological testing of glutenous masses has cen-tered largely i n the area of dough technology. An evolution has occurred from early, unsophisticated consistency measure-ment through to very basic rheological studies performed more recently. Many of the e a r l i e r methods have found very p r a c t i c a l uses i n the baking industry. Bloksma (1971) reviews t h i s l i t e r a t u r e i n considerable d e t a i l . Although useful as processing aids, many of the e a r l i e r tests and instruments did not provide p a r t i c u l a r l y meaningful information oh the rheological behavior of these dough systems. Rasper et a l . (1974) employed an Instron Universal Testing Machine to examine the e f f e c t of chemical improvers on doughs. The techniques of t h i s work and experiments l i k e i t are of a more d e f i n i t i v e nature than much preceding investigation, but require considerable testing and data manipulation to produce useful information regarding behavior of the doughs examined. A more e a s i l y handled and more fundamental type of testing appears to be dynamic shear measurement. Ferry (1961) provides very clear explanations of procedures and terms including the ,relationships between dynamic and steady shear measurement. In dynamic testing very small deformations and short time spans can be employed to study rheological 25. c h a r a c t e r i s t i c s of the test material. If a sinusoidal s t r a i n i s imposed on a sample which responds i n a l i n e a r fashion, a sinusoidal stress w i l l r e s u l t . For a purely e l a s t i c material, the stress w i l l be i n phase with the s t r a i n (Figure 15a). For a purely viscous material the stress w i l l be i n phase with the s t r a i n rate but 90° out of phase with the s t r a i n (Figure 15b). Most food and b i o l o g i c a l materials show an intermediate response that i s between purely viscous (Newtonian) and purely e l a s t i c (Hookean) and possesses both an e l a s t i c and a viscous component. Such materials are y i s c o e l a s t i c i n nature and can be represented generally by Figure 15c. From the curves developed for any given specimen e l a s t i c and viscous components, known as the dynamic shear storage modulus (G1) and dynamic shear loss modulus (G"), can be calculated. In the l i n e a r v i s c o e l a s t i c range dough behavior would be expected, according to theory, to be independent of s t r a i n amplitude but dependent on frequency of o s c i l l a t i o n (Smith, et a l . 1970). I t has been shown however by these authors, as well as others (Hibberd and Wallace, 1966) that dough does i n fact display an amplitude dependence. Most of the work reported to date has dealt with f l o u r doughs tested i n either simple t e n s i l e or shear modes. L i t t l e has been reported on i s o l a t e d gluten subjected to dynamic te s t i n g . Smith et a l . (1970) seem to be the only authors to work on gluten using dynamic shear measurement. c TIME Fig,15, T y p i c a l o s c i l l a t o r y shear r e s u l t s f o r purely e l a s t i c (a), purely viscous (b), and v i s c o e l a s t i c response ( c ) . S o l i d l i n e repre-sents s t r a i n and the broken l i n e represents s t r e s s . Even then the experiments with gluten were only a small part of a larger report on doughs. A l l reports seem to equate the physical properties of doughs and gluten with bread making. 28 2.3 METHODS AND MATERIALS 2.3.1 Sample Preparation A sample of commercial v i t a l wheat gluten was obtained from O g i l v i e . Flour M i l l s Ltd., Montreal. Gluten was rehydrated to approximately 55% moisture, worked by hand to d i s t r i b u t e moisture as evenly as possible and pressed at room temperautre, for 24 hours between aluminum plates to a thickness of approximately 2-3 mm. Normally, batches of 200 g t o t a l weight were prepared. After 24 hours the gluten sheet was removed from the plates and c i r c u l a r sample were cut from the mass for mounting i n the Weissenberg Rheogoniometer. These were wrapped i n d i v i d u a l l y i n aluminum f o i l with edges folded and crimped t i g h t l y , main-tained a further six hours at room temperature to allow d i s s i p a t i o n of stored energy r e s u l t i n g from handling, and f i n a l l y stored at le a s t 4 8 hours at 4°C p r i o r to t e s t i n g . Because i t was expected that moisture content and stored energy r e s u l t i n g from preparation would play s i g n i f i -cant roles i n the rheological behavior of gluten, moisture determinations were done on each specimen a f t e r rheological measurement and a record was kept of time post preparation when the actual testing was done. Moisture determinations were performed • on specimens by weighing before and aft e r drying at 100°C for 24 hours. 23. 2.3.2 Rheological Measurement Dynamic shear measurements were performed with the Weissenberg Rheogoniometer using cone/plate f i x t u r e s . The instrument was set up and used according to the sp e c i f i c a t i o n s of Table 1. The cone/plate geometry employs a truncated cone set. with a s p e c i f i c gap between platens corresponding to the truncation length. During loading, the specimen was gently and slowly extruded from the gap as the spec i f i e d spacing was reached. Since t h i s caused a stressing of the specimen, a one hour period was allowed for sample relaxation p r i o r to test i n g . To avoid drying of the edge of the specimen, rough, trimming was performed when the specimen was compressed to within about 200 ym of the desired thickness. The gap was then brought slowly to within a few ym of the f i n a l setting and the extruded gluten coated with a s i l i c o n e o i l to minimize drying. Just p r i o r to test i n g , f i n a l adjustment of the gap was made and excess sample trimmed o f f with a razor blade or s c a l p e l . For dynamic shear measurements, the instrument was operated i n the o s c i l l a t o r y mode. (See Figure 16 for i d e n t i f i c a t i o n of components.) A sinusoidal s t r a i n input was imposed on the specimen at the lower platen by a mechanical sine wave generator activated by the drive motor. Energy transmitted through the specimen was detected with a trans-ducer system connected to the top platen suspended by a 30. A = Drive motor B = Gearbox C = Sine.wave generator D = Strain wave transducer E = Truncated cone F = Gap at truncation G = F l a t Platen H = A i r bearing J = Torsion bar K = Radius arm L = Torsion head transducer H B Fig.16. Schematic representation of the Weissenberg Rheogoniometer. TABLE 1. EXPERIMENTAL OPERATING CONDITIONS FOR WEISSENBERG RHEOGONIOMETER Platen Diameter 5 cm Platen Geometry = cone and plate Cone Angle 2° 1' 39" Gap Setting = 103 ym Torsion Bar Constant ». 10.9.0 X 10 3 dyne cm/ym Temperature of Measurement = 23-25°C O s c i l l a t o r y Frequency = 0.075 - 0.9^  Strain Amplitude = 75 - 150 ym torsion bar through an a i r bearing. The a i r bearing provides for v i r t u a l l y f r i c t i o n l e s s rotation of the sensing head and the torsion bar r e s i s t s upper platen rotation which i s detected by movement of an armature within the stator of the LVDT transducer. For small deformations the shear stress signal provides a sinusoidal output wave which i s recorded on a s t r i p chart along with the input s t r a i n s i g n a l . Although, i n order to sense stress some movement must occur at the torsion head transducer, i d e a l l y the stress to s t r a i n displacement r a t i o should be kept small i f stress i s to be measured accurately. For t h i s reason experimental conditions were chosen so that the stress to st r a i n displacement r a t i o was less than 1%. Since glutenous materials f a l l between the viscous and e l a s t i c models described previously the stress wave i s found within 0 to 90° i n advance of the s t r a i n wave.. By measuring t h i s phase angle, input and output amplitudes and applying cer t a i n other known and controlled parameters, a number of basic rheological c h a r a c t e r i s t i c s of the specimen can be evaluated. Table 2 i s a l i s t of symbols used i n calculations and the i r meanings. TABLE 2. LIST OF SYMBOLS EMPLOYED FOR DYNAMIC SHEAR STUDIES C = Number of o s c i l l a t o r y cycles G. = Dynamic shear storage modulus (dyne/cm ) G" - Dynamic shear loss modulus (dyne/cm2) I m - Maximum value for movement at o s c i l l a t o r y input transducer turn) M - Moisture content of specimen (%, wet basis) S m ^ Maximum value for movement at torsion head transducer (ym) d = Platen diameter (cm) k = Torsion bar constant (dyne cm/ym) t = Time post preparation when tested (hr) P a = Cone angle (degrees) n.' - Dynamic v i s c o s i t y (poise) 0 = Phase difference between platens (degrees) tan 0 = Dynamic shear loss tangent - O s c i l l a t o r y frequency (sec ^) 2.3.4 E xp e r line n t a l Calculations Four s p e c i f i c rheological values were determined for the various samples. These were the dynamic shear storage ( G 1 ) and loss (G") moduli, dynamic v i s c o s i t y ( n 1) and loss tangent (tan czJ) calculated by the following equations: r , _ 2160 k a Sm , ... G - ^—^ COS 0 [1] d I m G " = 2 1 6 ° k a S m s i n 0 [2] 3 d I m n' =91 [3] tan 0 = [4] A l l other calculations were simple manipulations of these values. 2.3.5 S t a t i s t i c a l Procedures The data derived from the various measurements were analyzed s t a t i s t i c a l l y using simple l i n e a r as well as stepwise multiple l i n e a r regressions. These procedures were performed using the IBM 370/168 computer and the UBC TRIP (Triangular Regression Package) program. 2.4 RESULTS AND DISCUSSION 2.4.1 Preliminary Findings - Anomalous Behavior As described i n the Literature Review c e r t a i n general r e s u l t s were expected, based on theory. As w i l l be clear upon examining portions of the following data, practice a,nd theory did not coincide for commercial wheat gluten. I t was found that many factors played a r o l e i n determining the ultimate rheological behavior of t h i s material. One of the anomalies encountered was the incidence of hysteresis, i n r e s u l t s for G1 and G" over a range of o s c i l l a t o r y frequencies. Figure 17 i s an example of t h i s phenomenon. A second non-classical r e s u l t was encountered although i t was not e n t i r e l y unexpected (Smith et a l . , 1970). The curves of Figure 18 demonstrate an apparent s t r a i n amplitude ( y m ) dependence for G' and G". Hysteresis i n the curves of Figure 17 did not form a closed loop. If testing was continued over the range of frequencies successive curves were formed of s i m i l a r shape but greater magnitude. A large portion of the increase i n G1 and G" r e l a t i v e to s t r a i n amplitude, as well as the con-tinuing increase of these values when subjected to repeated back and forth testing through the range of frequencies seemed to be related to the extent of o s c i l l a t o r y c y c l i n g . This i s demonstrated by the curves of Figure 19. These values 35 . . 25 i n 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CO ( s e c - 1 ) Fig.17. The effect of oscillatory frequency on G' and G" for rehydrated wheat gluten. 36; 2 5 0.00 0.10 0.20 0.30 r m Fig.18. The e f f e c t of s t r a i n amplitude on G' and G" response f o r r e h y d r a t e d wheat g l u t e n . 37 . for G1 and G" are the same res u l t s used to generate Figure 18. In both figures a break i n the curves i s evidenced at a s t r a i n amplitude of about 0.088. Testing was done such that the s t r a i n amplitudes were varied from high to low and therefore the high amplitudes of Figure 18 correspond to the low cycle values of Figure 19. Up to the indicated break point, i t would appear that the increase i n G1 and G" could be related very well with the number of o s c i l l a t o r y cycles to which the specimen had been subjected. In a s i m i l a r manner the hysteresis i n Figure 17 could be related to the e f f e c t of c y c l i n g . In fact, when a specimen was o s c i l l a t e d at one frequency (0.299 s e c - 1 ) and amplitude (=^ 0.088 ym) over 40 cycles, G' and G" increased 3 0 0 Q and 1300 dyne/cm2 respec-t i v e l y . Within the experimental error inherent i n the system such an increase could account for the discrepancies between G' and G" values for successive tests at the same frequency. The preceding r e s u l t s have been presented to demonstrate a trend or pattern that was observed repeatedly for s i m i l a r l y treated specimens. The complexing e f f e c t of M and C made i t d i f f i c u l t to duplicate experiments exactly or to demonstrate simple relationships for combined data. As a r e s u l t , simple l i n e a r regression analyses did not provide much meaningful information. Fig.19. The e f f e c t of o s c i l l a t o r y cycling on G and G" response for rehydrated wheat gluten. 39. 2.4.2 D e f i n i t i o n of Primary Experiment An experiment was conducted i n which specimens were t e s t e d at one frequency and amplitude over a number of c y c l e s . The t e s t i n g was repeated over a range of frequencies (0.188 to 0.949 sec--1-) and maximum shear s t r a i n (0.066 to 0.132). Moisture content and time a f t e r p r e p a r a t i o n were noted. The r e s u l t s of t h i s experiment were analyzed using both simple and stepwise m u l t i p l e l i n e a r r e g r e s s i o n procedures. G', G", • n.', tan ft and t h e i r logs were considered as dependent v a r i a b l e s w h i l e to, ym., C, t , M, t h e i r squares and logs were P used as independent v a r i a b l e s . 2.4.3 Simple L i n e a r Regression Very few of the simple regressions demonstrated s i g n i f i c a n t r e l a t i o n s h i p s at P < 0.05 and those that, d i d had r e l a t i v e l y low c o e f f i c i e n t s of determination. The 2 regressions of n' on l o g to, tan ft on co , l o g n' oh l o g to o and l o g tan ft on eg were notable exceptions. The e f f e c t of M on G1 and l o g G' were a l s o of some i n t e r e s t . I t had been noted p r e v i o u s l y t h a t the specimen, moisture content seemed to have a considerable e f f e c t on the magnitude of G' when a l l other v a r i a b l e s were held constant. 2.4.4 M u l t i p l e Regression Stepwise, m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s i s revealed the complexity of the behavior, of g l u t e n . The equations developed to des cribe t h i s behavior i n terms of the v a r i a b l e s s t u d i e d a r e p r e s e n t e d i n T a b l e 3 . T h e s e e q u a t i o n s t a k e t h e f o r m : Y = b Q + b ^ + b 2 x 2 + . + b n x n [5 ] . T h e r e l a t i v e a b i l i t y o f t h e s e e q u a t i o n s t o d e s c r i b e t h e v a r i o u s r e l a t i o n s h i p s i s r e f l e c t e d b y t h e R 2 v a l u e s ( c o e f f i c i e n t o f m u l t i p l e d e t e r m i n a t i o n ) a l s o p r e s e n t e d i n T a b l e 3 . T h e s e v a l u e s a r e c o m m o n l y u s e d t o i n d i c a t e t h e c o m b i n e d a b i l i t y o f t h e i n d e p e n d e n t v a r i a b l e s i n t h e e q u a t i o n t o e x p l a i n t h e v a r i a t i o n i n t h e d e p e n d e n t v a r i a b l e . I n t h i s p a r t i c u l a r c a s e i t c a n b e s e e n t h a t R- v a l u e s r a n g e f r o m . 5 2 f o r e x p l a n a t i o n o f t h e v a r i a t i o n i n l o g G " t o . 9 6 o f t h e V a r i a t i o n i n l o g n ' . 2 E v e n t h o u g h t h e R v a l u e s a r e s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t , i n s e v e r a l i n s t a n c e s t h e e q u a t i o n s a r e f a r f r o m a b l e t o g i v e a c o m p l e t e e x p l a n a t i o n o f v a r i a t i o n i n t h e d e p e n d e n t v a r i a b l e , p a r t i c u l a r l y i n t h e c a s e s o f G ' , G " a n d t h e i r l o g a r i t h m s . T h i s m a y b e d u e i n p a r t t o t h e a b s e n c e o f s o m e u n i d e n t i f i e d f a c t o r f r o m t h e e q u a t i o n . I t m a y a l s o b e . r e l a t e d t o s o m e e x t e n t t o t h e f a c t t h a t m e a s u r e m e n t s w e r e m a d e u n d e r c o n d i t i o n s t h a t d i d n o t f a l l s t r i c t l y w i t h i n a l i n e a r v i s c o e l a s t i c r a n g e . 2 . 4 . 5 S t a n d a r d P a r t i a l R e g r e s s i o n C o e f f i c i e n t s T h e r e l a t i v e i m p o r t a n c e o f t h e c o n t r i b u t i n g v a r i a b l e s c a n b e o b t a i n e d b y c a l c u l a t i n g s t a n d a r d p a r t i a l r e g r e s s i o n c o e f f i c i e n t s (b^') ( S t e e l a n d T o r r i e , 1 9 6 0 ) . T h e s e v a l u e s c a n TABLE 3. STEPWISE MULTIPLE LINEAR REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING REGRESSION COEFFICIENTS* Dependent Variables G' G" V tan jzS log G' log G" log n ' log tan f6 b o 78350 29030 71590 3.277 5.733 4.888 4.793 2.360 t o - 1 .782 0.04 6 6 0.1451 0.3553 - 1 . 685 2 t o 2475 39960 1.040 0.9714 log t o -90840 0.8039 -1 .231 0.7669 loa M 2' '•V -24020 - 15 .53 - 7 .026 - 29 . 63 -0 .5060 -0.0004 -0 .0003 . -0 .0004 log M. -1 .118 - 1 .074 C 82.10 30. 74 0.0018 0.0016 c 2 3.271 ; -0 .00003 t 2 P - 0 .2807 R 2 0.537 0. 534 0.929 0.728 0.557 0.519 0.961 0 . 706 * Regression c o e f f i c i e n t s are provided only for those independent variables s i g n i f i c a n t at P < 0.05. TABLE 4. STANDARD PARTIAL REGRESSION COEFFICIENTS FOR THE MULTIPLE REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING. STANDARD PARTIAL REGRESSION COEFFICIENTS Dependent V a r i a b l e s G' G" tan l o g G1 l o g G" l o g n 1 l o g tan 0 03 -11. 60 0.2032 0.5754 0.4155 -12.22 2 CO 0. 5986 0.9691 7. 860 8.186 l o g co -1.736 4. 790 -1.318 5.093 l o g T m M 2 -0. 2505 -0.2201 -0. 7279 -0. 6564 -0.2774 -0.7824 -0.5336 -0.2098 l o g M - o . 2300 -0.2462 C 0. 3274 0. 2443 0.2996 0.2421 c 2 0.1215 0.0625 t 2 P -0.1254 to be compared w i t h i n an e q u a t i o n t o a s c e r t a i n t h e r e l a t i v e i m p o r t a n c e o f t h e i n d e p e n d e n t v a r i a b l e s c o n t r i b u t i n g s i g n i -f i c a n t l y t o t h e e x p l a n a t i o n o f v a r i a t i o n i n t h e d e p e n d e n t v a r i a b l e . T h e s e b^V v a l u e s were c a l c u l a t e d f o r t h e e q u a t i o n s d e v e l o p e d and a r e r e p o r t e d i n T a b l e 4. 2.4.6 E v a l u a t i o n o f S t a t i s t i c a l R e l a t i o n s h i p s f o r  n', G', G" and t a n 0 Dynamic v i s c o s i t y i s q u i t e s t r o n g l y c o r r e l a t e d w i t h 0 3 . T h i s i s p a r t l y due t o t h e f a c t t h a t G" i s r e l a t e d t o 03 and t h a t n.1 i s c a l c u l a t e d f r o m E q u a t i o n [3] w h i c h c o m b i n e s G" and OJ. M, C and t c o n t r i b u t e t o t h e o v e r a l l r e l a t i o n s h i p i n a s t a t i s t i c a l l y s i g n i f i c a n t way as w e l l . I n t h e l o g n ' e q u a t i o n 96% o f t h e v a r i a t i o n i n l o g n' i s e x p l a i n e d by v a r i a t i o n o f t h e p e r t i n e n t i n d e p e n d e n t v a r i a b l e s , t h u s making i t t h e most c o m p l e t e d e s c r i p t i o n o f g l u t e n b e h a v i o r d e v e l o p e d i n t h i s e x p e r i m e n t . T h i s i s o f c o n s i d e r a b l e i m p o r t a n c e f o r some p u r p o s e s , b u t doe s n o t a l l o w t h e o p t i o n o f d i r e c t l y i d e n t i f y i n g t h e r e l a t i v e c o n t r i b u t i o n o f t h e v i s c o u s and e l a s t i c components t o t h e o v e r a l l b e h a v i o r o f t h e s p e c i m e n . G 1 and G" a r e t h e u s u a l i n d i c a t o r s o f t h e e l a s t i c and v i s c o u s components r e s p e c t i v e l y ; however, t h e c o m p l e t e n e s s o f d e s c r i p t i o n o f t h e b e h a v i o r o f g l u t e n by e i t h e r G' o r G" as r e f l e c t e d by t h e d e v e l o p e d e q u a t i o n s l e a v e s some room f o r improvement.. The o t h e r p a r a m e t e r i n v e s t i g a t e d , t a n 0, i s c a l c u l a t e d f r o m G' and G" a c c o r d i n g t o E q u a t i o n [4] and t h e R" values for tan ft and log tan ft are much improved over those for G', G" and the i r logarithms. The equation for tan ft i s able to explain 73% of i t s v a r i a t i o n and according to Table 4 i s strongly related to frequency of o s c i l l a t i o n . Though i t i s s t i l l not possible to separate the viscous and e l a s t i c components completely, t h e i r r e l a t i v e contributions to specimen behavior can be estimated by the magnitude of tan ft. When tan ft i s zero, the response i s e n t i r e l y e l a s t i c . As ft approaches 90°, tan ft indicates the approach of a purely viscous response. A s h i f t of tan ft i n either d i r e c t i o n from any given response indicates the e f f e c t of the experimental input (e.g. to) on the v i s c o e l a s t i c behavior of the specimen i n terms of the r e l a t i v e contributions of the viscous and e l a s t i c components. 2,4,7 .Effect of Specimen Moisture Content Using the developed equations to evaluate the behavioral c h a r a c t e r i s t i c s one finds several inte r e s t i n g r e s u l t s . In the case of moisture content there tends to be a negative r e l a t i o n s h i p with the dependent variables. The greater the moisture content the less the magnitude of the various dependent variables. This was the greatest consequence for G','G" and th e i r logs. I t was not possible to control closely the moisture content, thus i t varied from about 53 to 57%. Prelimianry experiments indicated an extreme sensi-t i v i t y of G' to thi s small f l u c t u a t i o n . The implication of t h i s f i n d i n g i s t h a t m o i s t u r e c o n t e n t h a s i t s g r e a t e s t e f f e c t o n t h e m a g n i t u d e o f r e s p o n s e , e x e r t i n g a l e s s e r i n f l u e n c e o v e r t h e r a t i o o f t h e v i s c o u s a n d e l a s t i c c o m p o n e n t s o f o v e r a l l v i s c o e l a s t i c b e h a v i o r . A s i m i l a r s e n s i t i v i t y o f g l u t e n b e h a v i o r t o m o i s t u r e l e v e l h a s b e e n r e p o r t e d b y B a r n e y e t a l . (19 65 ) f o r l o n g t i m e , l a r g e d e f o r m a t i o n t e s t i n g . T h e s e a u t h o r s s u g g e s t e d t h a t d y n a m i c t e s t i n g m a y s h o w a c o n t i n u u m t o t h i s r e s p o n s e f o r s h o r t t i m e , s m a l l d e f o r m a t i o n t e s t i n g . T h e r e s u l t s o f . t h i s e x p e r i m e n t s e e m t o c o n f i r m t h e i r t h e o r y . 2 . 4 . 8 T h e E f f e c t o f O s c i l l a t o r y F r e q u e n c y T h e r e s p o n s e o f G 1 t o v a r i a t i o n i n co i n t h i s e x p e r i m e n t i s v e r y s m a l l a c c o r d i n g t o t h e r e s u l t s o f m u l t i p l e r e g r e s s i o n a n a l y s i s . T h i s i s n o t a n e x p e c t e d r e s u l t . a n d m a y i n f a c : t b e r e l a t e d t o t h e c o m p l e x i t y o f t h e i n t e r a c t i o n s b e t w e e n v a r i a b l e s . C e r t a i n l y , p r e l i m i n a r y e x p e r i m e n t s s u g g e s t e d s e n s i t i v i t y o f G ' t o co. F i g u r e 17 d e m o n s t r a t e s s u c h a r e l a t i o n s h i p e v e n w h e n c y c l i n g e f f e c t i s t a k e n i n t o a c c o u n t . T h e l i t e r a t u r e a l s o i n d i c a t e s t h a t G ' w i l l i n c r e a s e 2 W i t h i n c r e a s i n g co ( S m i t h e t a l . 1 9 7 0 ) . S i n c e t h e R v a l u e s f o r G ' a n d l o g G 1 a r e n o t h i g h ( T a b l e 3 ) , t h e v a r i a t i o n i n g l u t e n b e h a v i o r , a s r e f l e c t e d b y t h e s e p a r a m e t e r s , i s n o t c o m p l e t e l y e x p l a i n e d . T h i s m a y b e d u e i n p a r t t o t h e n a r r o w r a n g e o f o s c i l l a t o r y f r e q u e n c i e s e m p l o y e d a n d t o t h e i n t e r -a c t i o n o f v a r i a b l e s . A n o t h e r p o s s i b l e f a c t o r c o u l d b e t h e influence of an unidentified independent variable contributing to v a r i a t i o n i n G 1 . The s t a t i s t i c a l description of tan cz> i s far more complete and demonstrates an inte r e s t i n g trend. Over the range of co employed tan $z5 increases with oo. The implication of t h i s finding i s that the true response of gluten to increasing co i s a more viscous behavior. This does not imply that gluten behaves i n a viscous manner at the highest to used. Actually, the G' or e l a s t i c component i s s t i l l about twice as large as the G" or viscous component. I t i s t h i s fact that determines the apparent character of gluten, that i s , a r e l a t i v e l y e l a s t i c material. 2.4.9 Explanation of V i s c o e l a s t i c i t y by Molecular Theory With th i s apparent e l a s t i c i t y i n mind i t i s , at f i r s t , somewhat d i f f i c u l t to envision the increased viscous response re s u l t i n g from increased co. The explanation may l i e i n the . nature of the chemical interactions responsible for the physical behavior of gluten. Bernardin and Kasarda (1973b) suggested that v i s c o e l e a s t i c behavior i n gluten and glutenous doughs i s related to hydrogen bonding. The e l a s t i c and viscous responses can be explained by the same general mechanism according to these authors. In t h e i r theory i t i s suggested that a m u l t i p l i c i t y of hydrogen bonds e x i s t between protein molecules and protein f i b r i l s within the gluten network. E l a s t i c response occurs when bonds are deformed and then return to the minimum energy state. I f bonds are ruptured others can reform along the f i b r i l s s t i l l allowing for e l a s t i c behavior. At the same time, viscous flow i s i n no way in h i b i t e d since after s u f f i c i e n t energy has been supplied to rupture the bonds, the f i b r i l s are free to s l i p along one another u n t i l a new position i s attained, whereupon bonds reform and an e l a s t i c response i s once again possible. The findings of the present experiment f i t very well into t h i s model when one considers the t o t a l v i s c o e l a s t i c response. As energy input i s increased the response tends toward a viscous response. In other words, s u f f i c i e n t energy i s being put into the system to break bonds and cause flow. By the nature of dynamic testing these deformations are small and the specimen i s not noticeably deformed. The changes that are occurring w i l l be at an u l t r a s t r u c t u r a l l e v e l . The results for n 1 seem to indicate compliance with t h i s model as well. At low to (0.075 s e c - 1 ) the value for dynamic 5 v i s c o s i t y i s r e l a t i v e l y high {- 1.0 X 1.0 poise) while at higher to (0.949 sec "*") the value of n 1. i s much lower (- 1.5 X 10^ poise). At low frequency the rate of shear or energy input i s such that moire bonds are able to deform storing energy, restructuring the system and giving a r e l a t i v e l y e l a s t i c response as well as demonstrating greater resistance to gross deformation as r e f l e c t e d by n 1. When the rate of shearing i s increased and the system i s unable to store the energy added, the tendency to flow i s evident, the ac t i v a t i o n energy for 48. bond rupture i s exceeded and much less energy i s required to bring about a given deformation. Since the actual rate of shear i s continually changing during dynamic measurement and gross deformation i s prevented within the system, a response state or condition i s set up for the specimen based on the test conditions p r e v a i l i n g . Certainly a lower n' suggests an increased tendency of the specimen to flow under the applied deformation and t h i s correlates well with the i n d i c a t i o n of the tan jzS behavior that increased to results i n a s h i f t toward the viscous end of the v i s c o e l a s t i c response continuum. It must be understood that the present experiment was not designed to test the theory of Bernardin and Kasarda. As a r e s u l t , other models, e.g. that of Ewart (1972). involving nodes of entanglement, may f i n d support at le a s t i n part from the findings reported here. However, on c r i t i c a l l y reviewing theory published to date only the model proposed by Bernardin and Kasarda i s completely compatible with the findings of this study. 2.5 SUMMARY AND CONCLUSIONS Hydrated wheat gluten has a very complex rheological nature in dynamic shear. Although some s t a t i s t i c a l l y s i g n i f i -cant simple l i n e a r relationships existed between a number of the variables, few of these were s u f f i c i e n t l y precise to indicate that a simple rel a t i o n s h i p was capable of describing the behavior of the various dependent variables under the conditions of te s t i n g . The one exception to t h i s may have been the rel a t i o n s h i p of dynamic v i s c o s i t y . (n 1) and o s c i l l a t o r y frequency (to) i n the form of log n\ vs. log t o . The simple 2 l i n e a r regression showed an r = 0.925. A l l other parameters investigated required a multiple regression analysis to account more f u l l y for the v a r i a t i o n i n the dependent variables. The storage modulus or e l a s t i c component was found to be very sensitive to moisture^being the most important single factor in the multiple l i n e a r regression equations for the storage modulus. Though moisture content did not c o n t r i -bute as heavily to the response of the loss modulus, i t was found to be important. This dependency on moisture content i s demonstrated by the magnitude of the experimental values for these parameters as moisture varies. The p r a c t i c a l importance of t h i s l i e s i n the fact that moisture content i n gluten and glutenous materials i s d i f f i c u l t to control and, moreover, the response to a r e l a t i v e l y small v a r i a t i o n i n 5 0 . moisture content (+ 3%) i s disproportionately large. Moisture content did not have a large e f f e c t on the r a t i o of viscous to e l a s t i c behavior of gluten. The equations for loss tangent contain moisture as a s i g n i f i c a n t independent variable (Table 3) but i t s importance i s far overshadowed by the influence of o s c i l l a t o r y frequency (Table 4). Working of the specimen was also found to e f f e c t the storage and loss moduli. Cycling at any given amplitude and frequency caused an increase i n magnitude of both para-meters. This can be observed i n the p r a c t i c a l c h a r a c t e r i s t i c known as work hardening which can be observed during dough mixing operations. The multiple regression for loss tangent does not contain a c y c l i n g component indicating that working, under the experimental conditions employed, does not have an influence on the proportional contribution of storage and loss moduli for rehydrated gluten. One of the most important findings of t h i s study i s that within the l i m i t s of the test procedures and condi-tions, gluten exhibits an increased viscous response with increased o s c i l l a t o r y frequency. This finding i s quite com-patible with the recently proposed mechanism for visco-e l a s t i c response of gluten proposed by Bernardin and Kasarda (1973b), Indications are that increased energy input causes disruption of sequential hydrogen bonds on surfaces of protein f i b r i l s and molecules allowing for e l a s t i c response at low energies with viscous flow developing as energy input increases but with an a b i l i t y to reform hydrogen bonds at the new p o s i t i o n attained thus restoring e l a s t i c response. This corresponds to the finding that storage modulus tends to increase s l i g h t l y or remain stable with energy input and remain the more dominant component of v i s c o e l a s t i c behavior but that the proportionate contribution of loss modulus i s increased with increased energy input. Developing new foods based on wheat gluten and improving processes and products already incorporating gluten w i l l depend on an understanding and awareness of the responses of the gluten mass to i t s environment. Cl e a r l y , gluten i s a complex material with complex responses to any manipulative procedure. Proper control of the various c h a r a c t e r i s t i c s of wheat gluten should lead to useful modifications, and improved processes for gluten based products CHAPTER 3. THE EFFECT OF SPECIFIC MODIFICATIONS ON THE ULTRASTRUCTURE AND RHEOLOGY OF COMMERCIAL VITAL WHEAT GLUTEN 3.1 INTRODUCTION The preceding two chapters have dealt with the basic ultrastructure and rheology of commercial v i t a l wheat gluten. Reported i n t h i s section are the r e s u l t s of various modifying treatments designed to expand on the findings of the preceding experiments. The role of starch, l i p i d and protein were d i s -cussed in some d e t a i l , e s p e c i a l l y i n Chapter 1. A number of theories were discussed regarding the role of these components in the ultrastructure of rehydrated gluten. Fundamental rheological properties were examined;, and discussed i n the second chapter. The intention of the experiments reported here was to combine the techniques of u l t r a s t r u c t u r a l and rheological examination to investigate the e f f e c t of various lev e l s of starch, l i p i d and protein and of d i f f e r e n t kinds of l i p i d s on gluten properties. It was expected that such information would prove useful i n possible p r a c t i c a l applications related to both tex t u r i z a t i o n and more t r a d i t i o n a l dough technology. 3.2 LITERATURE REVIEW Much has been written on the f u n c t i o n a l i t y of gluten and on the e f f e c t s of various additives and fl o u r components. However, t h i s information i s almost e n t i r e l y concerned with bread dough and various baking applications. In general, t h i s l i t e r a t u r e i s reviewed very well by . Pomeranz (1971a,1973). In p a r t i c u l a r , Bloksma (1971) provides considerable information on the rheology and chemistry of doughs. This information i s augmented by a review of composi-tio n and functional properties of fl o u r components (Pomeranz, 1971b) and by Mecham's (1971) treatment of dough response to l i p i d s . Much of the l i t e r a t u r e pertinent to the present report has been reviewed i n Chapter 1 and 2 with reference to ultrastructure and rheology. I t i s possible to rather a r b i t r a r i l y divide the l i t e r a t u r e s p e c i f i c a l l y related to the following experiments into basic or th e o r e t i c a l and applied or p r a c t i c a l treatments. It must be repeated that most of the reported work relates to dough and baking technology. This i s p a r t i c u l a r l y true of the applied reports. There are some exceptions to t h i s , including a growing patent l i t e r a t u r e related to texturized gluten products. Hartman (1966) reported a method for rehydrating gluten to form a high protein textured food and Kjelson (1965) claimed a method for preparing a fibrous protein product based on glute 54 Sternberg (1973) discussed the control of dough structure r e l a t i v e to gluten and includes SEM micrographs to demon-strate the e f f e c t of gluten development during dough mixing. Similar observations were reported previously by Aranyi and Hawrylewicz (1969). P r a c t i c a l studies of dough f u n c t i o n a l i t y generally involve the use of t r a d i t i o n a l dough mixing and testing equipment. This f i e l d has been extensively investigated and the l i t e r a t u r e i s r i c h with such reports. These experi-ments are related to the e f f e c t of various factors ranging from use of modifying additives to f l o u r h i s t o r y . A l l these are of extreme importance to s p e c i f i c p r a c t i c a l processes. This l i t e r a t u r e was reviewed extensively by Pomeranz (1971a) The more basic or t h e o r e t i c a l l i t e r a t u r e relates to the interactions of various components of flo u r or gluten with regard to chemistry, structure and rheological response Although the majority of these papers have dough technology as the underlying area of i n t e r e s t , they tend to deal more generally with the chemistry of the system as i t relates to s t r u c t u r a l and functional properties. Grosskreutz (1961) reported transmission electron microscope and X-ray d i f f r a c t i o n r e s u l t s and postulated an early model for gluten structure and functional properties. The model was based on protein p l a t e l e t s and involved s l i p planes containing l i p o p r o t e i n situated between the p l a t e l e t s The theory has now largely been discounted but the work marks t h e b e g i n n i n g o f a n u m b e r o f s t u d i e s t h a t h a v e f u r t h e r e l u c i d a t e d g l u t e n u l t r a s t r u c t u r e a n d . p r o p o s e d n e w m o d e l s f o r t h e r h e o l o g i c a l b e h a v i o r . B e r n a r d i n a n d K a s a r d a ( 1 9 7 3 a , b ) h a v e c o n t r i b u t e d c o n s i d e r a b l e i n f o r m a t i o n o n g l u t e n u l t r a -s t r u c t u r e a n d h a v e p r o p o s e d a t h e o r y o f g l u t e n v i s c o -e l a s t i c i t y i n v o l v i n g h y d r o g e n b o n d i n g a s t h e m a j o r f a c t o r ( 1 9 7 3 b ) . S e c k i n g e r a n d W o l f ( 1 9 7 0 ) r e p o r t e d f i n d i n g s o n t h e u l t r a s t r u c t u r a l c h a r a c t e r o f a n u m b e r o f f r a c t i o n s o f w h e a t e n d o s p e r m . O r t h e t a l . ( 1 9 7 3 ) p r e s e n t e d a n S E M s t u d y o f g l u t e n i n f r o m s e v e r a l s o u r c e s a n d w e r e a b l e t o d e m o n s t r a t e d i f f e r e n c e s a c c o r d i n g t o o r i g i n . T h e w o r k o f S i m m o n d s ( 1 9 7 2 a , b ) h a s c o n t r i b u t e d s i g n i f i c a n t l y t o t h e u n d e r s t a n d i n g O f t h e n a t u r e a n d o r i g i n o f m a n y o f t h e m i c r o s c o p i c o b j e c t s o b s e r v e d w i t h t h e T E M . C r o z e t e t a l . ( 1 9 6 6 ) m a d e a r e l a t i v e l y e a r l y r e p o r t o n u l t r a s t r u c t u r a l r e l a t i o n s h i p s o f w h e a t f l o u r c o m p o n e n t s w h i c h t h e y l a t e r e x p a n d e d ( 1 9 7 4 ) . A s m e n t i o n e d i n C h a p t e r 2 , m o s t r h e o l o g i c a l s t u d i e s r e l a t e d t o w h e a t f l o u r a n d g l u t e n h a v e b e e n d o n e e i t h e r w i t h i n s t r u m e n t s i n v o l v i n g s t e a d y s h e a r ( W a l l a n d B e c k w i t h , 1 9 6 9 ) o r t e n s i l e p r o p e r t i e s m e a s u r e m e n t ( R i n d e e t a l . 1 9 7 0 , T s c h o e g e l e t a l . 1 9 7 0 , a n d R a s p e r e t a l . 1 9 7 4 ) . M o s t o f t h e . m o r e b a s i c p a p e r s t e n d t o d i s c u s s f i n d i n g s i n t e r m s o f t h e s u s p e c t e d o r p r o p o s e d c h e m i s t r y o f t h e s y s t e m . I t w o u l d a p p e a r t h a t f e w e r m o d i f i c a t i o n s t u d i e s h a v e b e e n a t t e m p t e d i n t h e t h e o r e t i c a l i n v e s t i g a t i o n s t h a n i n t h e a p p l i e d w o r k . 5 6 . This statement should be q u a l i f i e d i n that many modification techniques have been u t i l i z e d but these are s p e c i f i c chemical treatments employed to i s o l a t e various protein fractions (Or.th et a l . 1973a,b) and to a l t e r the chemistry of the system i n order to predict or describe the mechanism of gluten behavior (e.g. Barney et a l . 1 9 6 5 ) . Generally they involve chemicals and procedures which would not y i e l d an edible product. The experiment reported herein was designed to investigate the interaction of.component parts of wheat gluten r e l a t i v e to basic ultrastructure and rheological behavior without resorting either to specialized i s o l a t i o n procedures or to dr a s t i c chemical treatments. 57. 3.3 METHODS AND MATERIALS Pr o c e d u r e s employed i n the f o l l o w i n g e x p e r i m e n t s were g e n e r a l l y t h e same as those d e s c r i b e d i n Ch a p t e r 1 and 2. Both u l t r a s t r u c t u r a l and r h e o l o g i c a l methods were used t o i n v e s t i g a t e the e f f e c t s of a l t e r i n g s t a r c h , p r o t e i n and l i p i d c o n t e n t o f g l u t e n specimens. 3.3.1 Sample P r e p a r a t i o n Samples were p r e p a r e d f o r dynamic shear t e s t i n g as d e t a i l e d i n Chapter 2 except t h a t v a r i o u s a d j u n c t s were i n c o r p o r a t e d o r m o d i f i c a t i o n s made b e f o r e c o m b ining t h e g l u t e n and w a t e r . Wheat germ o i l , m anufactured by Propharm L a b o r a t o r i e s , , Vancouver, and hydrogenated r a p e s e e d s h o r t e n i n g c o n t a i n i n g no added e m u l s i f i e r were added t o g l u t e n a t 1, 2, 3, 4, and 5% o f t h e v i t a l g l u t e n w e i g h t . The wheat germ o i l and s h o r t e n i n g were added t o the dry g l u t e n and mixed f o r 5 minutes a t low speed i n a Hobart K i t c h e n a i d M i x e r p r i o r t o r e h y d r a t i o n o f the m o d i f i e d g l u t e n specimens. Batches f o r t e s t i n g were p r e p a r e d by combining g l u t e n and water i n such p r o p o r t i o n t h a t t h e g l u t e n , i n c l u d i n g a d j u n c t , made up 45% by weight o f the f i n a l m i x t u r e . Samples o f d e f a t t e d g l u t e n p r e p a r e d as d e s c r i b e d i n Chapter 1 were r e h y d r a t e d and r e a d i e d f o r measurement i n a s i m i l a r manner. P e a r l e d wheat s t a r c h , o b t a i n e d from O g i l v i e F l o u r M i l l s L t d . , M o n t r e a l , was added t o t h e v i t a l g l u t e n a t 5, 10, 20 and 30% o f the g l u t e n w e i g h t . M o i s t u r e c o n t e n t was a d j u s -t e d t o m a i n t a i n a c o n s t a n t p r o t e i n m o i s t u r e r a t i o among s t a r c h 58. l e v e l s . To f a c i l i t a t e starch d i s t r i b u t i o n , i t was found necessary to combine the starch with the appropriate weight of water followed by the gluten to make up the f i n a l sample. Since 30% added starch constituted a considerable d i l u t i o n of the protein content, moisture l e v e l was adjusted accordingly to maintain the protein/moisture (Pr/M) x'atio at a constant Value (- 0.86). This corresponds generally to the Pr/M r a t i o used for the gluten specimens employed i n Chapter 2. To calculate protein/moisture r a t i o s , gluten protein content was determined on duplicate samples of v i t a l wheat gluten using the Technicon Autoanalyser. The Autoanalyser employs a c l a s s i c a l Kjeldahl nitrogen determination with t o t a l nitrogen detected c o l o r i m e t r i c a l l y at 630 nm for a blue indophenol complex (Autoanalyser Method No. 146-71A). Protein content was calculated from Kjeldahl nitrogen by using a con-version factor of 5.7. Moisture contents of gluten, as well as rehydrated specimens, were determined as described i n Chapter 2. 3.3.2 Rheological Measurement Rheological procedures were b a s i c a l l y unchanged from Chapter 2. Duplicate batches were employed with at least duplicate r e p l i c a t i o n from each batch. A constant (maximum) s t r a i n amplitude was employed (- 0.110) and frequency of o s c i l l a t i o n was varied from 0.075 s e c - 1 to 0.949 s e c - 1 . 3.3.3 S t a t i s t i c a l Procedures Simple and multiple l i n e a r regression analyses and l i n e comparisons were performed on the rheological data obtained using an IBM 370/168 computer and the UBC TRIP program. Specimen protein content i n grams (Pr) i t s square and logarithm, treatment l e v e l and i t s square were considered in the analyses as independent variables i n addition to those variables detailed i n Chapter 2, Section 2.4.2. 3.3.4 Electron Microscopy Scanning electron microscopy procedures were modified i n that samples were gold/paladium coated and observed with an ETEC Scanning Electron Microscope operated at 20 Kv. Images were recorded on 4 by 5 inch Polaroid positive/negative f i l m . A l l EM specimens were prepared by mounting i n the Weissenberg Rheogoniometer and under steady shear taken 3 2 to a constant stress (16.6 X 10 dynes/cm ). The specimen was then immediately removed from the gap, placed i n glutaraldehyde and cut into small pieces suitable for even-tual EM observation. The purpose of t h i s procedure was to produce specimens with known rheological history. 3.4 RESULTS AND DISCUSSION 3.4.1 The E f f e c t of Added Wheat Germ O i l on -Rehydrated, V i t a l Wheat Gluten '. : Fatty material, usually i n the form of la r d or shortening, i s commonly added to dough products to improve baking properties. In order to assess the contribution of the f a t phase to gluten behavior, l i p i d l e v e l and type were modified, p r i o r to examination u l t r a s t r u c t u r a l l y and i d e o l o g i c a l l y . Wheat germ o i l was chosen as one of the adjuncts because i t provided a means of evaluating the e f f e c t of the type of l i p i d normally found i n gluten. The most apparent finding of t h i s study was that the s t a t i s t i c a l analysis of the re s u l t s showed an increased complexity with regard to interacting variables. 3.4.1.1 The e f f e c t of wheat germ o i l on gluten rheology -complex relat i o n s h i p s . Stepwise multiple l i n e a r regression analyses were performed on the rheological data for both a pooled sample of wheat germ o i l (WGO) modified gluten and the i n d i v i d u a l additive l e v e l s . The multiple regression equations and t h e i r c o e f f i c i e n t s of multiple determination fo.r the pooled data are presented i n Table 5. The corresponding standard p a r t i a l regression c o e f f i c i e n t s can be found in.Table 6. Compared to unmodified gluten the equations for TABLE 5. STEPWISE MULTIPLE LINEAR REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF WGO MODIFIED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING REGRESSION COEFFICIENTS* Dep e n d e n t V a r i a b l e s G 1 G" t a n ft l o g G' l o g G" l o g n ' l o g t a n ft b -183800 139000 361500 0.2434 • 17.79 11. 20 10.52 -0.6775 CO 3013 135900 -0.1363 0.1380 C O 2 -54050 0.1563 l o g co 7825 2956 -112900 0.3416 0.3109 -0.6900 % WGO 7 6 8 .3 0.0075 0.0367 0.0453 0.0064 % WGO2 -169.7 -189.0 -238.2 -0.0054 -0.0095 -0.017 Y 2 m -4.504000 -49300 1 . 3 4 3 -1.034 C 2 -3.703 P r 2 301400 30760 120000 0.9883 1.666 1.800 l o g P r -210100 t 2 rS 0.8423 0.00002 -0.000004 P l o g t -70770 -4959 -21160 -2.105 -0.2834 Cr l o g M -162500 -72400 -233600 0.9602 -5.460 -3.948 -3.883 0.8249 R 2 0.87 0 0.840 0.927 0.681 0.880 0.879 0.964 0.706 * R e g r e s s i o n c o e f f i c i e n t s a r e p r o v i d e d o n l y f o r t h o s e i n d e p e n d e n t v a r i a b l e s s i g n i f i c a n t a t P < 0.05. TABLE 6 . STANDARD PARTIAL REGRESSION COEFFICIENTS FOR THE MULTIPLE REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF WGO MODIFIED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING STANDARD PARTIAL REGRESSION COEFFICIENTS Dependent Variables G' G" n' tan 0 log G1 log G" log n ' log tan 0 CO 0 . 3 4 8 7 2 . 0 3 5 - 0 . 3 3 7 8 0 . 7 2 4 4 2 CO - 0 . 8 5 8 9 0 . 7 0 3 4 log co o, 6 6 9 8 0 . 4 1 7 7 - 2 . 0 6 4 0 . 9 1 2 6 0 . 7 8 3 2 - 0 . 9 3 6 2 % WGO 0 . 4 4 1 6 0 . 1 5 8 0 0 . 3 7 61 0 . 2 5 0 0 0 . 1 6 6 9 % WGO2 - 0 . 3 3 3 4 - 0 . 6 1 3 0 - 0 . 1 0 0 0 - 0 . 3 7 5 0 - 0 . 2 9 5 8 - 0 . 5 2 9 4 Y 2 m 2 log Y m - 0 . 0 . 8 8 6 8 9 7 7 2 - 0 . 1 6 1 0 - 0 . 1 5 3 6 C 2 - 0 . 2 6 4 2 P r 2 1. 6 2 8 0 . 2 7 4 2 0 . 1 3 8 4 0 . 1 8 8 7 0 . 2 6 4 8 0 . 1 5 4 1 log Pr - 1 . 3 2 1 t p 2 1. 1 8 8 0 . 9 9 7 5 - 0 . 0 8 9 5 l o g t - 1 . 4 1 1 - 0 . 1 6 3 2 - 0 . 0 9 0 1 - 1 . 4 8 3 0 . 1 6 6 3 P log M - 0 . 3 1 4 2 - 0 . 2 3 1 1 - 0 . 0 9 6 5 0 . 1 1 2 3 - 0 . 3 7 3 2 0 . 2 2 4 6 - 0 . 1 1 9 0 0 . 1 1 9 4 the.WGO t r e a t e d s p e c i m e n s were c o n s i d e r a b l y more co m p l e x . The 2 R v a l u e s ( T a b l e 5) were g e n e r a l l y - i m p r o v e d i n d i c a t i n g t h a t a g r e a t e r amount o f t h e v a r i a t i o n i n t h e d e p e n d e n t v a r i a b l e s was e x p l a i n e d by v a r i a t i o n i n t h e i n d e p e n d e n t v a r i a b l e s . T h i s i n c r e a s e d c o m p l e x i t y c a n be a c c o u n t e d f o r i n p a r t by t h e f a c t t h a t f i v e new p o t e n t i a l v a r i a b l e s b a s e d on t h e l e v e l o f a d d i t i v e .(% WGO) and s p e c i m e n p r o t e i n c o n t e n t (Pr) were i n t r o d u c e d t o t h e m u l t i p l e r e g r e s s i o n . The l e v e l Of WGO w^s s t a t i s t i c a l l y s i g n i f i c a n t i n e a c h o f t h e e q u a t i o n s f o r t h e v a r i o u s r h e o l o g i c a l p a r a m e t e r s i n v e s t i g a t e d . P r o t e i n c o n t e n t was i n c l u d e d s i n c e a d d i t i o n o f t h e WGO was made t o t h e d r y g l u t e n p r i o r t o r e h y d r a t i o n t o a p p r o x i m a t e l y 55% M and t h e r e f o r e c o n s t i t u t e d a d i l u t i o n o f t h e p r o t e i n i n t h e f i n a l s p e c i m e n . P r o t e i n c o n t e n t was f o u n d t o be a s t a t i s -t i c a l l y s i g n i f i c a n t c o n t r i b u t o r t o t h e r e g r e s s i o n f o r a l l r h e o l o g i c a l p a r a m e t e r s e x c e p t t a n 0 . The same s t e p w i s e m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s e s were p e r f o r m e d f o r e a c h r h e o l o g i c a l p a r a m e t e r a t e a c h l e v e l o f o i l . O s c i l l a t o r y f r e q u e n c y and e i t h e r M o r P r were f o u n d t o be s i g n i f i c a n t c o n t r i b u t o r s t o t h e r e g r e s s i o n e q u a t i o n s f o r G" and n.' a t a l l l e v e l s o f added o i l . T h e s e f a c t o r s were a l s o o f i m p o r t a n c e t o G' and t a n 0 b u t n o t q u i t e as c o n s i s t e n t l y as f o r G" and n.''., O t h e r v a r i a b l e s c o n t r i b u t e d s i g n i f i c a n t l y t o t h e r e g r e s s i o n e q u a t i o n s b u t d i d n o t a p p e a r t o have any p a r t i c u l a r p a t t e r n i n t h e r e l a t i o n s h i p s . F o r a l l dependent variables and a l l l e v e l s of o i l the R values were very high, generally being greater than 0.90. 3.4.1.2 The e f f e c t of wheat germ o i l l e v e l on gluten  rheology - simple relationships Simple l i n e a r regression analyses were performed for each dependent variable vs. each independent variable and l e v e l of added o i l . It was found that the regression of log n ' on log u) provided equations with very high c o e f f i c i e n t s of determination ( r 2 > 0.90). These relationships are presented graphically i n Figure 20. These l i n e s were s t a t i s t i c a l l y analyzed as to t h e i r r e l a t i v e difference o v e r a l l , by slope and by magnitude of intercept (Ostle, 1963) . The same procedure was carried out for the other dependent variables and quite similar patterns of si g n i f i c a n c e arose. None of the slopes proved to be s i g n i f i c a n t l y d i f f e r e n t , but in certain cases l e v e l or magnitude of response, as well as the equation i n general, did prove to be s i g n i f i c a n t at P < 0.05. The 1% l i n e was s i g n i f i c a n t l y d i f f e r e n t from a l l the other l i n e s . The 2% l i n e was also found to be d i f f e r e n t from the 3% and 4% but not the 5% l i n e . Observing the various l i n e s of Figure 20, i t can be seen that o i l addition of 1, 2, and 3% had.an e f f e c t on the magnitude of log n ' . Once about 3% WGO was added further increase to 5% WGO had no s i g n i f i c a n t e f f e c t as indicated by the overlap and trans-position of l i n e s from the apparently l o g i c a l sequence. 65 4 .9 4.7 h 4.5 h O) o 4.3 h 4.1 h 3 9 - 1 . 2 - 0 . 8 - 0 . 4 L o g CO F i g . 20. T h e e f f e c t o f v a r i e d o s c i l l a t o r y f r e q u e n c y ( l o g to) o n d y n a m i c v i s c o s i t y ( l o g n ! ) ° f w h e a t g l u t e n f o r f i v e l e v e l s o f a d d e d w h e a t g e r m o i l . 3 . 4 . 1 . 3 G e n e r a l r h e o l o g i c a l t r e n d s F i g u r e 21 d e m o n s t r a t e s t h e r e s p o n s e o f G 1 , G " a n d n ' t o l e v e l o f o i l a d d e d . I t c a n b e s e e n t h a t a g e n e r a l d e c r e a s e i s s h o w n f o r e a c h o f t h e p a r a m e t e r s a s o i l l e v e l i n c r e a s e s . A t t h e h i g h e r l e v e l s o f WGO, t h e e f f e c t c a n b e s e e n t o d i m i n i s h . T h e s e f i n d i n g s a g r e e w i t h t h e i n t e r p r e t a t i o n o f t h e r e s u l t s p r e s e n t e d i n F i g u r e 2 0 . 3 . 4". 1. 4 T h e e f f e c t o f w h e a t g e r m o i l o n t h e u l t r a s t r u c t u r a l  c h a r a c t e r o f w h e a t g l u t e n — ~ T h e r h e o l o g i c a l b e h a v i o r o f WGO m o d i f i e d w h e a t g l u t e n c a n p e r h a p s b e e x p l a i n e d b y m i c r o s c o p i c e x a m i n a t i o n o f t h e c o m p o n e n t p a r t s o f t h e s y s t e m . F i g u r e s 2 2 a - c d e m o n s t r a t e t h a t a s t h e l e v e l o f o i l i n c r e a s e s t h e r e i s a t e n d e n c y f o r t h e o i l t o c o a l e s c e r a t h e r t h a n m i x t h r o u g h o u t t h e p r o t e i n m a t r i x . S t a r c h g r a n u l e s a p p e a r a s m o r e o r l e s s r e g u l a r l y s h a p e d s p h e r e s o f v a r y i n g s i z e w h i l e l i p i d , o f t e n s e e n t o b e a s s o c i a t e d w i t h t h e s t a r c h g r a n u l e s , i s o b s e r v a b l e i n t h e s c a n n i n g e l e c t r o n m i c r o g r a p h s a s i r r e g u l a r g l o b u l e s . F i g u r e 2 2 d i s a t r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f a 5% a d d e d WGO s p e c i m e n . T h e l a r g e , d a r k s t a i n i n g m a s s i s a l i p i d d r o p l e t , a s a r e t h e s m a l l e r o s m i o p h i l i c s t r u c t u r e s a d j a c e n t t o i t . T h e m i c r o g r a p h s p r e s e n t e d a r e , i n g e n e r a l , s i m i l a r t o t h o s e o f C h a p t e r 1 , s u g g e s t i n g t h a t t h e d i f f e r e n c e s o b s e r v e d c a n b e r e l a t e d t o t r e a t m e n t . O n l y t h e e d g e o f t h e s p e c i m e n d i s c s h o w e d t h e r e s u l t s o f t h e r h e o l o g i c a l t r e a t m e n t . S i n c e t h e e f f e c t s o f m o u n t i n g , t r i m m i n g , e t c . a r e m o s t e v i d e n t o n t h i s 67. F i g . 2 1 . The e f f e c t of added wheat germ o i l on the rheological response of wheat gluten. Electron micrographs of rehydrated wheat gluten with various lev e l s of added wheat germ o i l . Scanning electron micrographs; a (1%), b (3%), and c (5% WGO). Transmission electron micrograph d (5% WGO). surface, i t was f e l t that t h i s was not a r e l i a b l e u l t r a -s t r u c t u r a l specimen upon which to base a discussion. As a r e s u l t of the r e l a t i v e l y fast ( 1 8 - 2 0 sec) half relaxation times of the gluten specimens and slow i n f i l t r a t i o n i t was not possible to preserve evidence of the rheological t r e a t -ment i n the mass of the specimen. 3 . 4 . 2 The E f f e c t of Added Shortening on Rehydrated  V i t a l Wheat Gluten Unemulsified rapeseed shortening was used as an adjunct i n v i t a l wheat gluten i n order to evaluate the e f f e c t of a "hard" f a t on gluten rheology. The procedures involved were the same as those discussed i n r e l a t i o n to added wheat germ o i l . Levels of 1 - 5 % added hydrogenated rapeseed shortening (% RS) were employed. 3 . 4 . 2 . 1 The e f f e c t of rapeseed shortening on gluten  rheology - complex rela t i o n s h i p Stepwise multiple l i n e a r regression analysis of the pooled data for shortening resulted i n a series of equations describing the i n t e r a c t i o n of the variables tested (Table 7 ) . As with the WGO series, the equations proved to involve more factors than the corresponding equations for untreated gluten. The completeness of explanations of v a r i a -t i o n i n the dependent variables as r e f l e c t e d by the several c o e f f i c i e n t s of determination (Table 7) was generally improved r e l a t i v e to s i m i l a r r e s u l t s f o r gluten alone. TABLE 7. STEPWISE MULTIPLE LINEAR REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF RS MODIFIED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING REGRESSION COEFFICIENTS* D e p e n d e n t V a r i a b l e s G' G" n' t a n 0 l o g G' l o g G" l o g n ' l o g t a n 0 180900 209900 1987000 9.858 8 .196 8 .876 19.28 0.3953 0) -0.0898 0)2 -0.1167 -0.1211 l o g 0) 6131 3478 -66730 0.2659 0.2736 -0.7229 0.0390 % RS -3961 -2602 -0.039 2 -0.1034 -0.1391 -0.1390 -0.0401 % R S 2 589.8 408 .2 0.0069 0.0154 0.0223 0.0222 0.0070 Y 2 m l o g Y m -93.20 -53.20 900.0 1690000 8.610 9.285 C 0.0072 C2 -0.0003 P r 2 -158400 -82290 -174300 -5.161 -5.2109 -5.152 l o g P r 156700 83010 223100 5.255 5.430 5.351 t p 2 -0.00001 -0.00001 P l o g t _ -22650 -13380 -0.6631 M 2 P -8.17.0 -2.991 0.0001 -0.0002 -0.0002 -0.0002 R 2 0.863 0.879 0.914 0.774 0.916 0.930 0.982 0.758 * R e g r e s s i o n c o e f f i c i e n t s a r e p r o v i d e d o n l y f o r t h o s e i n d e p e n d e n t v a r i a b l e s s i g n i f i c a n t a t P < 0.05* Standard p a r t i a l regression c o e f f i c i e n t s are provided i n Table 8 to allow comparison of the r e l a t i v e importance of variables i n the i n d i v i d u a l equations. I t can be seen that for G', G", n1 and the i r logarithms, the single most important factor was protein content (in the 2 form Pr ). In the case of tan 0 and i t s logarithm, the %RS was found to be the most important single factor. When multiple regression equations were developed at each of the various shortening levels the R2 values, i n general, approached unity and certa i n trends were noted. For G', G", n' and tan 0 at most le v e l s of added shortening OJ i n some form was involved i n the equations as an indepen-dent variable. In the case of G", protein content was a s i g n i f i c a n t variable at every o i l l e v e l and, i n regard to G', n' and tan 0, was involved i n most of the equations. For G1 and n' the number of o s c i l l a t o r y cycles appeared as a s i g n i f i c a n t factor i n many of the regressions. 3.4.2.2 The e f f e c t of shortening l e v e l on gluten  rheology - simple relationships Simple l i n e a r regressions were performed on a l l combinations of dependent and independent variables for.each l e v e l of shortening. As with the WGO data log n 1 vs. log OJ 2 ' . demonstrated the highest r values and these relationships were used to i l l u s t r a t e specimen response to shortening content (Figure 23). TABLE 8 . STANDARD PARTIAL REGRESSION COEFFICIENTS FOR THE MULTIPLE REGRESSION • EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF RS MODIFIED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING. STANDARD PARTIAL REGRESSION COEFFICIENTS Dependent V a r i a b l e s GV G" n 1 t a n ft l o g G' l o g G" l o g n ' l o g t a n ft to - 0 . 2 6 0 9 to 2 - 0 . 2 7 2 0 - 0 . 1 4 5 0 l o g to 0.6704 0 . 5 6 8 1 - 1 . 3 2 5 0 . 9 4 3 1 0 . 7 3 2 8 - 0 . 9 9 5 0 0 . 2 8 1 3 . % RS - 1 . 5 8 9 - 1 . 5 5 9 - 0 . 9 8 3 2 - 1 . 3 4 6 - 1 . 3 6 7 - 0 . 7 0 1 9 - 1 . 0 6 1 % R S 2 1 . 4 4 7 1 . 4 9 6 0 . 9 6 4 2 1 . 2 2 6 1 . 3 4 0 0 . 6 8 5 7 1 . 1 3 3 Y 2 - 0 . 3 9 0 8 - 0 . 6 0 1 3 l o g y m C - 0 . 2 5 4 8 - 0 . 5 0 3 4 - 0 . 8 0 2 0 - 0 . 1 9 1 5 0 . 7 3 9 2 c 2 - 0 . 7 8 6 2 P r 2 - 1 7 . 2 6 1 3 . 3 9 - 3 . 4 4 8 - 1 8 . 2 4 • 1 3 . 9 1 - 7 . 0 6 5 l o g P r 1 . 8 4 9 1 . 4 6 3 0 . 4 7 8 0 2 . 0 1 2 1 . 5 7 0 0 . 7 9 4 9 t 2 • P l o g t - 0 . 1 3 5 0 - 0 . 1 0 1 9 - 0 . 1 8 2 9 - 0 . 1 6 1 4 - 0 . 0 6 7 4 2 ^ M^ - 0 . 3 0 1 5 - 0 . 1 6 4 9 0 . 1 2 6 1 - 0 . 2 3 9 4 - 0 . 1 8 0 8 - 0 . 0 9 2 9 4 . 9 73. 4 . 7 L \ \ \ 4.5 co o 4 . 3 4.1 3 .9 S h o r t e n i n g , % 2 - A 3 - • 4 - o 5 - A \ V \ \ \ \ V - 1 . 2 - 0 . 8 - 0 . 4 0 . 0 Log O) Fig.23. The e f f e c t of varied o s c i l l a t o r y frequency (log w) on dynamic v i s c o s i t y (log n') of wheat gluten for f i v e l e v e l s of added rapeseed shortening. When the r e s u l t i n g l i n e s were compared s t a t i s -t i c a l l y i t was found that none of the slopes d i f f e r e d s i g n i f i c a n t l y (P < 0.05). Magnitudes of n l as well as the ov e r a l l equations were found to be s i g n i f i c a n t l y d i f f e r e n t between 1%RS and a l l other levels of shortening. As with the WGO experiment i t was found that l i n e s corresponding to the high levels of shortening became displaced from a l o g i c a l numerical sequence on the ordinate. The conclusion arrived at must be that addition of a small amount of shortening s i g n i f i c a n t l y reduces the. magnitude of n 1 but beyond a certa i n point the e f f e c t i s l o s t . 3.4.2.3. General rheological trends Figure 24 p a r a l l e l s Figure 21 i n that i t i s a plot of the mean values of G 1, G" and n' for the various levels of shortening. As with WGO i t can be seen that addition of shortening results i n a decrease i n magnitude of a l l three rheological parameters, followed by a l e v e l l i n g off i n the response. This would seem to corroborate the findings detailed i n the preceding section (3.4.2.2). 3.4.2.4 The ef f e c t of shortening on the u l t r a s t r u c t u r a l  character of wheat gluten Figure 25a i s a scanning electron micrograph of the edge of a specimen disc (4%RS). S t r i a t i o n s are quite obvious, running diagonally across the specimen face. This c h a r a c t e r i s t i c was common for v i r t u a l l y a l l the 75. 4.5 h V4 .3 h o O CO A . O CO o 3.9 h 3.7 0 Fig.24. 1 2 3 4 5 R a p e s e e d S h o r t e n i n g (%) The e f f e c t of added rapeseed shortening on the rheological response of wheat gluten. specimens examined, regardless of treatment. However, two features of this surface indicate that drying has occurred and may make interpretation hazardous. The face has become concave, a r e s u l t of the f l a t surfaces of the disc spreading upon release from the Rheogoniometer gap. The interface of the surface and the edge of the specimen can be seen to have curled i n toward the edge probably causing the second d e t r i -mental e f f e c t , rupturing of the specimen surface. Such a rupture i s evident i n the upper r i g h t of Figure 25a. Figure 25b demonstrates i n t e r a c t i o n of starch with the protein matrix. I t can also be seen quite c l e a r l y that the starch granule present at the lower l e f t i s i n intimate contact with an o i l droplet. This phenomenon was observed for both WGO and shortening. The a t t r a c t i o n of l i p i d to the surface of starch granules has been reported previously (Crozet and Guilbot, 1974). 3.4.3 The E f f e c t of Free L i p i d Removal on Rehydrated  V i t a l Wheat Gluten Petroleum ether extracted wheat gluten was examined r h e o l o g i c a l l y and u l t r a s t r u c t u r a l l y i n the same manner as the specimens containing wheat germ o i l and shortening. Free l i p i d per se i s not expected to influence rheological behavior. However, the a v a i l a b i l i t y of free l i p i d to complex with the protein phase may be important. Fig,25. Electron micrographs of rehydrated wheat gluten with added rapeseed shortening. Scanning electron micrograph; a (4% RS) - edge view. Transmission electron micrograph b (3% RS) . Fig.27. Scanning electron micrograph of rehydrated wheat gluten with free l i p i d extracted. 7 8 . The f i n d i n g s r e p o r t e d h e r e i n r e l a t e t o t h e e f f e c t o f r e m o v i n g f r e e l i p i d p r i o r t o m i x i n g and s u b s e q u e n t p r o t e i n -l i p i d i n t e r a c t i o n . 3 . 4 . 3 . 1 The e f f e c t o f f r e e l i p i d e x t r a c t i o n on g l u t e n  r h e o l o g y - complex r e l a t i o n s h i p s T a b l e 9 c o n s i s t s o f t h e e q u a t i o n s d e v e l o p e d f o r t h e d e p e n d e n t v a r i a b l e s a l o n g w i t h t h e c o r r e s p o n d i n g c o e f f i c i e n t s o f m u l t i p l e d e t e r m i n a t i o n . The e q u a t i o n s r e s e m b l e t h o s e o f u n t r e a t e d g l u t e n ( T a b l e 5) i n t h a t f e w e r i n d e p e n d e n t v a r i a b l e s a r e i n v o l v e d t h a n i n t h e c a s e s w i t h added wheat germ o i l and r a p e s e e d s h o r t e n i n g . One s t r i k i n g d i f f e r e n c e f r o m t h e u n t r e a t e d g l u t e n c a n be o b s e r v e d i n t h e s t a n d a r d p a r t i a l r e g r e s s i o n c o e f f i c i e n t s o f T a b l e 1 0 . M o i s t u r e c o n t e n t o n l y e n t e r s i n t o two o f t h e e i g h t e q u a t i o n s and was n o t f o u n d t o be t h e m a j o r . c o n t r i b u t o r t o v a r i a t i o n i n any o f t h e r e l a t i o n s h i p s f o r e x t r a c t e d g l u t e n . 3 .4.3.2 The e f f e c t o f f r e e l i p i d e x t r a c t i o n on g l u t e n  r h e o l o g y - s i m p l e r e l a t i o n s h i p s As w i t h t h e p r e c e d i n g t r e a t m e n t s a s e r i e s o f s i m p l e l i n e a r r e g r e s s i o n a n a l y s e s was p e r f o r m e d . L o g n ' 2 and l o g co p r o v e d t o be h i g h l y c o r r e l a t e d ( r = 0 . 9 3 2 8 , P < 0 . 0 1 ) . T h i s r e l a t i o n s h i p i s . p r e s e n t e d g r a p h i c a l l y i n F i g u r e 26 a l o n g w i t h t h e c o r r e s p o n d i n g r e g r e s s i o n l i n e s f o r 1% WGO and 1% RS. The e x i s t e n c e o f d i f f e r e n c e s between t h e s e l i n e s TABLE 9. STEPWISE MULTIPLE LINEAR REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF ETHER EXTRACTED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR MEASUREMENT l o g C V ^ a b l e s G ' G " n ' t a n l o S G ' 1°9" G" l o g n 1 l o g t a n ^ b 74350 285-400 345700 4.131 -5.057 3.706 4.697 1.575. o l o g to 10430 -93430 0.3530 -0.9796 -0.6175 v 2 25400000 -115.2 -256.0 'm l o g Y 314500 16.67 -8.575 C 509.4 0.0314 0.7484 0.3634 2 0.00002 fcP l o q t 76590 31590 296100 y P M -1237 l o g M 8.529 R 2 0.964 0.994 0.983 0.907 0.898 0.932 0.995 0.923 TABLE 10. STANDARD PARTIAL REGRESSION COEFFICIENTS FOR THE MULTIPLE REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF ETHER EXTRACTED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING Dependent V a r i a b l e s G' G" TT t a n ft l o g G' l o g G" l o g n1 l o g t a n ft l o g co 2 m l o g y Y m l o g C t 2 P l o g t £ M l o g M 0.8883 -1.401 1.267 -0.5379 -0.3805 -0.7224 0.4138 0.62 28 0.3834 0.1672 0.2610 •0.2867 0.5709 0.9897 -1.575 1.066 -0.2616 0.6241 -17.54 0.399-9 0.2187 Log. 26. The e f f e c t of varied o s c i l l a t o r y frequency (log co) on dynamic v i s c o s i t y (log n.') of wheat gluten for various l e v e l s and types of l i p i d . was tested for s t a t i s t i c a l l y . I t was found that the n' - co rela t i o n s h i p for ether extracted gluten was s i g n i f i c a n t l y d i f f e r e n t from the l i p i d added specimens both on an o v e r a l l basis and on magnitude of response. The WGO and shortening regressions were not s i g n i f i c a n t l y d i f f e r e n t o v e r a l l but did d i f f e r i n magnitude of response (P < 0.05) with the WGO specimens producing the higher n 1 values. None of the slopes was found to.be s i g n i f i c a n t l y d i f f e r e n t (P < 0.05). 3.4.3.3 The e f f e c t of free l i p i d extraction on the  u l t r a s t r u c t u r a l character of gluten Figure 27 demonstrates the roughened surface features noted i n Chapter 1 for ether extracted gluten specimens. Again i t was not possible to correlate rheolo-g i c a l history with u l t r a s t r u c t u r a l appearance. As can be seen by comparing Figure 2 7 with the SEM photographs i n Figure 25 and 22 and those i n Chapter 1 r e l a t i n g to f u l l f a t gluten specimens, there i s an apparent, a l t e r a t i o n to the surface microstructure upon extraction of free l i p i d with petroleum ether. 3.4.4 The E f f e c t of Added Starch on Rehydrated V i t a l  Wheat Gluten Because some of the r e s u l t s of Chapter 1 indicated that starch might influence the sheeting and f i b e r forming properties of gluten, pearled wheat starch was added back to v i t a l gluten at lev e l s up to 30% of gluten weight (wet 8 3 . basis) p r i o r to specimen preparation. The e f f e c t s of t h i s modification on the rheological and u l t r a s t r u c t u r a l properties of rehydrated v i t a l gluten are reported i n the following sections. 3.4.4.1 The e f f e c t of added starch on gluten rheology -complex relationships The r e s u l t s of stepwise multiple l i n e a r regression analyses and the corresponding standard p a r t i a l regression c o e f f i c i e n t s are presented i n Tables 11 and 12 respectively. Only three independent variables entered into these equations, co, C and M. In contrast to unmodified gluten the equations for pooled starch had M (log M) as the most important factor contributing to the v a r i a t i o n of log n' (Table 12). Two dependent variables, n1 and log tan ft had log C as the major contributor while G 1 , G", tan ft, log G 1 and log G" were most strongly influenced by v a r i a t i o n i n to. Moisture content was a factor i n every equation. When multiple regression analyses were performed on the data of each l e v e l of starch i t was found that co was of importance to the regressions at a l l l e v e l s of starch for n' and log n1. Protein was involved i n the equations for n ' , G" and t h e i r logarithms at most l e v e l s . For several variables, G ' , G", tan ft and t h e i r logarithms when co loses significance C becomes an important contributor. TABLE 11. STEPWISE MULTIPLE LINEAR REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF STARCH MODIFIED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHAR TESTING REGRESSION COEFFICIENTS* Dependent Variables G' G" V tan 0 log G' log G" log n 1 log tan 0 b o 831900 441900 2027000 1.3078 15.52 16.41 16.4 6 0.74 07 03 0.1879 log o) 35120 25680 0.4121 0.6771 -0.3242 0.1797 C 7535 -0.0066 -0.0134 0.0065 log C -26710 -22780 -316500 -0.5794 -0.5803 -0.3226 log M -440600^ -227700 -1015000 -0.4810 -6.234 -6.619 -6.644 -0 .4212 R 2 0.758 0.660 0.77 5 0.281 0.823 0.766 0.903 0.303 * Regression coefficients, are provided only for those independent variables s i g n i f i c a n t at P < 0.05. TABLE 1 2 . STANDARD PARTIAL REGRESSION COEFFICIENTS FOR THE MULTIPLE REGRESSION EQUATIONS RELATED TO THE RHEOLOGICAL RESPONSE OF STARCH MODIFIED COMMERCIAL WHEAT GLUTEN SUBJECTED TO DYNAMIC SHEAR TESTING STANDARD PARTIAL REGRESSION COEFFICIENTS D e p e n d e n t V a r i a b l e s G' G" n1 t a n ft l o g G' l o g G" l o g n 1 l o g t a n ft CO 1 . 1 1 2 l o g co 0 . 9 4 0 6 1 . 1 7 7 0 . 8 2 0 8 1 . 1 7 0 - 0 . 3 6 0 7 1 . 4 9 2 C . 0 . 7 1 1 1 - 0 . 7 2 1 7 - 0 . 4 0 4 1 - 0 . 8 1 7 3 l o g C . - 0 . 5 2 1 5 - 0 . 6 5 3 4 - 1 . 2 3 5 - 0 . 6 2 6 7 - 0 . 4 0 4 0 - 1 . 6 7 7 l o g M - 0 . 7 3 9 4 - 0 . 6 5 4 0 - 0 . 3 9 6 4 - 0 . 2 1 7 7 - 0 . 7 7 8 0 - 0 . 7 1 6 8 ' - 0 . 4 6 3 2 - 0 . 2 1 9 2 3 6 . O s c i l l a t o r y frequency and C were found to be correlated (r = 0.948). This was also true of some of the other variables i n the reported multiple regressions. The method of c a l c u l a -t i o n employed excludes l i n e a r combinations of variables already i n the regression equation. This requires that r e s u l t s be interpreted c a r e f u l l y . I t was found that starch l e v e l did not enter the multiple regression equations for any of the rheological parameters tested. At the same time, starch content shows s t a t i s t i c a l l y s i g n i f i c a n t relationships with a l l the dependent variables. In each case M i s s l i g h t l y more highly correlated with these variables than % starch. Moisture i s also highly correlated with starch l e v e l (r = 0.940, P < 0.05). As a r e s u l t , inclusion of starch l e v e l i n the equation does not ca,use a s i g n i f i c a n t reduction i n the residual sums of squares, thus % starch i s excluded as a variable i n the multiple regression. As long as t h i s i s understood and care taken, the r e s u l t s can s t i l l be interpreted. 3.4.4.2 The e f f e c t of starch l e v e l on gluten rheology -simple relationships ~~ Simple l i n e a r regression analyses of a l l the variables investigated proved once again that the highest c o r r e l a t i o n was between log n 1 and log to. These r e s u l t s are presented graphically i n Figure 28. The significance of the relationships of these equations one to the other were X X • X X X X X X N • X x x X X XX X X X X X \ \ \ X x . \ \ X X w x X . \ S t a r e h , 1 5 - o 10 - • 2 0 - ° 3 0 - • X X X X X X N D 88. tested s t a t i s t i c a l l y and each l i n e was found to be s i g n i f i -cantly d i f f e r e n t from a l l other l i n e s for magnitude of i n t e r -cept. A l l were s i g n i f i c a n t l y d i f f e r e n t on an o v e r a l l basis, except the 10% and 2 0% li n e s r e l a t i v e to each other. None of the slopes were found to d i f f e r s i g n i f i c a n t l y (P > 0.05). •3.4.4.3 General rheological trends When the mean values for the logarithms of G', G" and n' were plotted against added starch, a steady increase in a l l three dependent variables was observed with increasing starch content (Figure 29). This r e s u l t was seen.to be exactly opposite to the e f f e c t of adding l i p i d . 3 . 4 . 4 . 4 The e f f e c t of starch on the u l t r a s t r u c t u r a l character of gluten ' Increasing le v e l s of starch seemed to enhance the sheeting character of gluten. Figure 30a i s a scanning electron micrograph of the f l a t surface of a 5% added starch specimen di s c . The glutenous material can be seen to be quite continuous i n appearance. Figure 30b demonstrates the influence of added starch (30%) as the proteinaceous material forms i t s e l f around the starch granules. In many areas the protein layer i s quite thin suggesting what might be c a l l e d a sheeted structure. The manner i n which the protein organizes i t s e l f around the starch granules i s c l e a r l y demonstrated by Figure 30c. This i s a part of the specimen i n Figure 30b 89. 0 10 2 0 3 0 Wheat Starch (%) F i g . 2 9 . T h e e f f e c t o f a d d e d w h e a t s t a r c h o n t h e r h e o l o g i c a l r e s p o n s e o f w h e a t g l u t e n . 90 . Fig,30. E l e c t r o n micrographs of rehydrated wheat glut e n w i t h various l e v e l s of added s t a r c h . Scanning e l e c t r o n micrographs; a ( 5 % ) , b (30% starch) and c (30% s t a r c h - f r a c t u r e s u r f a c e ) . Transmission e l e c t r o n micrograph; d (30% s t a r c h ) . broken at r i g h t angles to the f l a t face of the specimen di s c , i . e . an edge surface. The holey appearance i s a r e s u l t of starch granules being dislodged during the fracturing procedure The fourth micrograph (Figure 30d) produced with the TEM shows a starch granule embedded i n the protein matrix. The dark features i n the starch granule are probably the r e s u l t of swelling of the starchy material during preparation procedures (Cumming and Lee, 1974). They found that for both wheat and potato starch, sectioning onto water (the general EM ultramicrotoming procedure) these features appeared even in thick sections. With some d i f f i c u l t y (related to surface tension) specimens of potato could be thick sectioned into and retrieved from 100% ethanol. None of the sections obtained i n t h i s manner demonstrated the folded i n t e r i o r . I t i s i n t e r e s t i n g to note that the granule i s surrounded by a large number of minute osmiophilic bodies. No other staining procedure was applied to t h i s specimen, thus i t may be that these structures constitue a layer of l i p i d or the remnants of a membrane structure surrounding the starch granule. Simmonds (1972a) has reported finding membrane residues around starch granules i n wheat endosperm material. 3.4.5 The Influence of L i p i d on Gluten Behavior The actual role of l i p i d material i n the physical behavior of doughs appears to be i n some confusion. Bloksma (1971) reviewed recent findings of research related to f l o u r and added l i p i d s . The contribution of l i p i d material to the baking character of bread doughs seems to be dependent on many factors. The present experiment was not related d i r e c t l y to a p a r t i c u l a r application and was designed to investigate fundamental rheological and u l t r a s t r u c t u r a l properties of wheat gluten. The f i r s t three treatments covered by t h i s chapter r e l a t e to presence, type and l e v e l of l i p i d . I t was found that the various treatments had s i g n i -f i c a n t e f f ects on gluten rheology. Within the addition experiments, l e v e l of added l i p i d from 1 - 5 % did not show a s i g n i f i c a n t influence on slope of the simple regression l i n e s (Figures 21 and 23) for log n 1 vs. log to for either WGO or RS. When the 1% le v e l s for WGO and RS were compared with the extracted gluten the slopes were found to be not s i g n i f i c a n t l y d i f f e r e n t . The int e r p r e t a t i o n of these r e s u l t s seems to be that within the bounds of the experiment, l i p i d l e v e l and type had no influence on the rate of change of log n 1 with log to. This was generally true for a l l the dependent variables tested against to. I t was found for simple l i n e a r regression equations of log n 1 vs. log to that s i g n i f i c a n t differences existed between some level s of l i p i d content. In most cases where l e v e l was of si g n i f i c a n c e , the equations for the l i n e s were found to be s i g n i f i c a n t l y d i f f e r e n t on an o v e r a l l basis. A l l s t a t i s t i c s quoted are at P < 0.05 but i n most cases where l e v e l of l i p i d content was s i g n i f i c a n t the p r o b a b i l i t y l e v e l was P < 0.01. As previously stated i n sections 3.4.1.2 and 3.4.2.2,. l i p i d content could be correlated to a s i g n i f i c a n t change i n response only up to a l e v e l in the range of 3% added l i p i d . Beyond t h i s l e v e l the decrease i n log n 1 related to increase i n l i p i d became non-significant, l i n e s became transposed from the predicted orientation and SEM and TEM micrographs indicated possible coalescence of the l i p i d material. If the hydrogen bonding theory of Bernardin and Kasarda (1973b) quoted i n Chapter 2 as a l i k e l y mechanism, for gluten v i s c o e l a s t i c i t y holds true, there must be an explanation of the role of l i p i d i n a l t e r i n g that behavior. Where l i p i d l e v e l was of s i g n i f i c a n c e , i t would appear to be related to the magnitude rather than the fundamental nature of the response. A number of potential bonding s i t e s e x i s t i n the protein mass and probably account for the uptake of free l i p i d during dough development (Olcott and Mecham, 194 7). I f hydrogen bonding i s l a r g e l y responsible for the v i s c o e l a s t i c response of gluten i t may be that binding of l i p i d reduces the number of available hydrogen bonding s i t e s by causing a. state of hindrance to be set up at various points along the molecule. With fewer available in t e r a c t i o n s i t e s the expected reaction would be a diminished response. Since the nature of the v i s c o e l a s t i c mechanism would not l i k e l y be changed the r e l a t i v e proportion of viscous to e l a s t i c response (tan 0) would not be expected to change i n r e l a t i o n to l e v e l of l i p i d . For the pooled data of % WGO and of % RS, the simple l i n e a r regression of tan 0 on l e v e l was not s i g n i f i c a n t (P > 0.05). This would indicate that the r a t i o G"/G' did not change appreciably with l e v e l of l i p i d . If the proposed model holds, t h i s would be the expected r e s u l t . Kasarda et a l . ( 1 9 7 1 ) reviewed the r o l e of water i n the structure of gluten. I t was suggested that water occupies pot e n t i a l hydrogen bonding s i t e s and may, i f present to an excessive degree, cause "weakening" of a dough. As discussed i n the previous chapter, moisture content seems to have a marked e f f e c t on the rheological behavior of gluten. The e f f e c t of l i p i d addition tended to diminish to non-significance upon continued increase of l e v e l up to 5% of v i t a l gluten weight. Electron microscopy revealed aggregations of f a t t y material with increased l e v e l s of l i p i d . The explanation of these r e s u l t s l i k e l y involves two factors. The i n a b i l i t y of the mixing procedure to disperse the l i p i d phase intimately and completely throughout the gluten mass and the inherent p o t e n t i a l of the protein to bind available l i p i d are probably the major contributors to the res u l t s observed. With improved mixing procedures more l i p i d may have been induced to disperse throughout and bind with the protein mass but ultimately i t would seem that lack of bonding s i t e s would become the determining f a c t o r . i n the 95. influence of l i p i d on gluten behavior. The petroleum ether extracted specimen would s t i l l be expected to have bound l i p i d present i n the protein matrix since very gentle extraction only seems to a f f e c t free l i p i d . The magnitude of rheological response shows an increase as a re s u l t of l i p i d extraction since l i t t l e further l i p i d binding occurs on rehydration and mixing. According to the theory proposed here more pote n t i a l bonding s i t e s are ph y s i c a l l y available for in t e r a c t i o n . 3.4.6 The Influence of Starch oh Gluten Behavior Increasing starch content results i n a considerable increase i n magnitude of rheological response, e.g. at 5% added starch the intercept value of n ' when plotted against to was 58,100 poise, while at 30% starch n 1 was found to be 193,500 poise. The e f f e c t may be related, i n part, to M. Starch w i l l absorb a small amount of free water thus rendering i t unavailable to the protein. This would i n turn free more hydrogen bonding s i t e s for protein-protein i n t e r a c t i o n . In l i g h t of the s e n s i t i v i t y of gluten to M and the fact that the Pr/M r a t i o was maintained constant regardless of starch l e v e l , thus lowering available moisture s l i g h t l y with increasing starch, moisture a v a i l a b i l i t y may account for the observed behavior. A further contributing factor may be the increased 96. orientation of protein into sheets and f i b r i l s surrounding the starch granules. Orientation tends to increase the magnitude of v i s c o e l a s t i c response (Ewart, 1972). There i s just such an increase with increased starch ' l e v e l . A further p o s s i b i l i t y may l i e i n the in t e r a c t i o n of starch and protein providing what might be c a l l e d anchoring points for the protein. In Chapter 1, SEM studies of defatted fl o u r speci-mens showed proteinaceous f i b e r s adhering to the surface of starch granules (Figure 7b). Other specimens which were not defatted did not seem to show t h i s . However, i n many instances the protein was seen to form a continuous sheet over the starch surface and i n TEM studies the starch granule seems generally to contact the protein mass at some point on i t s periphery. It i s noted that when l i p i d material began to coalesce at the higher addition le v e l s i t was often associated with starch granules. If in t e r a c t i o n took place at the starch surface with an excess amount of l i p i d present, the ultimate r e s u l t might be reduced protein interaction with the starch. In view of the rheological r e s u l t s related.to l i p i d l e v e l , t h i s would seem to be a rather minor factor. 3 . 5 SUMMARY AND C O N C L U S I O N S I t w a s f o u n d t h a t t h e r h e o l o g i c a l a n d u l t r a s t r u c -t u r a l p r o p e r t i e s o f g l u t e n c a n b e s i g n i f i c a n t l y a l t e r e d b y m a n i p u l a t i o n o f s p e c i m e n p r o t e i n , s t a r c h , l i p i d a n d m o i s t u r e l e v e l s . A d d i n g f a t i n t h e f o r m o f w h e a t g e r m o i l a n d r a p e s e e d s h o r t e n i n g c a u s e d a d e c r e a s e i n t h e m a g n i t u d e o f t h e d y n a m i c s h e a r s t o r a g e ( G ' ) a n d l o s s ( G " ) m o d u l i a n d d y n a m i c v i s c o s i t y (n') f o r i n c r e a s i n g l e v e l s o f a d d e d l i p i d u p t o a b o u t 3% o f t h e w e i g h t o f v i t a l g l u t e n u s e d i n t h e m i x . I t m a y b e t h a t l i p i d i s a t t r a c t e d t o h y d r o p h o b i c b o n d i n g >' s i t e s i n t h e p r o t e i n c r e a t i n g a s t e r i c h i n d r a n c e w h i c h r e d u c e s t h e n u m b e r o f p o t e n t i a l h y d r o g e n b o n d i n g s i t e s o r a t l e a s t w e a k e n s t h e i n t e r a c t i o n s . S i n c e i t i s e x p e c t e d t h a t o n l y a f i n i t e n u m b e r o f s u i t a b l e l i p i d b i n d i n g s i t e s w o u l d b e p r e s e n t i n t h e p r o t e i n p h a s e a l i m i t f o r l i p i d u p t a k e m u s t b e a t t a i n e d b a s e d b o t h o n a v a i l a b i l i t y o f p o t e n -t i a l s i t e s a n d e f f i c i e n c y o f m i x i n g o f t h e s a m p l e . T h e r h e o l o g i c a l r e s u l t s o f t h i s e x p e r i m e n t i n d i c a t e t h a t t h e r e i s a p p a r e n t l y n o s i g n i f i c a n t e f f e c t o f l i p i d a d d i t i o n o r t y p e ( w h e a t g e r m o i l o r r a p e s e e d s h o r t e n i n g ) t o t h e f u n d a m e n t a l r e s p o n s e o f g l u t e n . T h e l o s s t a n g e n t ( t a n ft) w a s n o t c o r r e l a t e d s i g n i f i c a n t l y w i t h l e v e l o f a d d e d l i p i d f o r e i t h e r t y p e o f f a t u s e d . T h i s s u g g e s t s t h a t l i p i d c o n t e n t w i t h i n t h e l i m i t s o f t h e e x p e r i m e n t h a d n o e f f e c t o n t h e 98 . on'the fundamental nature of the v i s c o e l a s t i c response but only on the magnitude. Loss tangent was found to be related to o s c i l l a t o r y frequency (co) i n the same way as i t was for untreated gluten: increased o s c i l l a t o r y frequency produced a more viscous response though the o v e r a l l behavior was s t i l l more notably e l a s t i c . These findings lend credence to the theory that protein-protein i n t e r a c t i o n i s being hindered or reduced but not altered i n nature. The influence of starch in the system i s s i m i l a r to that of added l i p i d i n that the nature of the rheological behavior does not seem to change. However, i n general, starch promotes an e f f e c t opposite to that of added l i p i d i n that i t causes a considerable increase i n the magnitude of response. The two most important effects of starch on the system are probably the reduction of available water r e s u l t i n g i n increased ihterprotein hydrogen bonding, and firming of the structure through promotion of orientation of the protein mass. Starch-protein interactions at granule surfaces l i k e l y provide anchor points which allow the maintainance of the protein web i n bread dough. Combining an understanding of the p r a c t i c a l con-siderations of dough technology and protein modification techniques with the information presented, i t may be possible to improve old processes and develop new ones. It was suggested in Chapter 1 that starch or some other spacer phase would aid in orientation of the protein mass. The findings presented here seem to verify this. Magnitude of response can be altered by starch or l i p i d addition without altering the nature of gluten behavior. It has become evident that moisture content of the gluten mass is of c r i t i c a l importance and must be carefully controlled during processing i f the desired end product is to be achieved. Manipulation of a l l four components: protein, starch,lipid and water have a measurable effect on the behavior of glutenous masses. Careful control of a l l of these factors combined with various energy input systems can be expected to yield improved, as well as new and novel, processes and products. Many other modification treatments are possible, but control of the proportions of components and the amount and nature of energy input w i l l give the processor the a b i l i t y to adjust magnitude and nature of rheological behavio of gluten based products. It is hoped that the information reported herein w i l l aid in the better understanding of how this endpoint may be achieved. 100. LITERATURE CITED Aranyi, C. and Hawrylewicz, E.J. 1969. Application of scanning .electron microscopy to cereal specimens. Cereal Science Today 14: 230. Barney, J.E., Pollock, H.B. and Bolze, C C . 1965. A study of the relat i o n s h i p between v i s c o e l a s t i c proper-t i e s and the chemical nature of wheat gluten and glutenin. . Cereal Chem. 42: 215. Bernardin, J.E.. and Kasarda, D.D. 1973a. Hydrated protein f i b r i l s from wheat endosperm. Cereal Chem. 50: 529. Bernardin, J.E. and Kasarda, D.D. 1973b. The microstructure of wheat protein f i b r i l s . Cereal Chem. 50: 735. Bloksma, A.H. 1971. Chap..11: Rheology and chemistry of dough. Wheat Chemistry and Technology (2nd Ed.) American Association of Cereal Chemists Inc., St. Paul, Minn. Bushuk, W. 1971. Ultrastructure related to wheat processing and products. Symposium: Seed proteins. G.E. Inglett, Ed. Avi Pub. Co., Westport, Conn. Crozet, N., Gallant, D., Godon, B., Guilbot, A. and P e t i t , L. 1966. Structure submicroscopique des fragments proteiques de farine de ble et du gluten separe par l i x i v i a t i o n . Ind. Aliment. Agr. (Paris) 5: 559. Crozet, N., Godon, B., P e t i t , L. and Guilbot, A. 1974. Submicroscopic structure of wheat flou r and gluten proteins. Cereal Chem. 51: 288. Crozet, N. and Guilbot, A. 1974., A note on the influence of Osmium f i x a t i o n on wheat fl o u r l i p i d s observa-t i o n by scanning electron microscopy. Cereal Chem. 51: 300. Cumming, D.B. and Lee, L.P. 1974. The influence of section preparation on the u l t r a s t r u c t u r a l appearance of starch granules. Unpublished data. 101 Ewart, J.A.D. 1972. A modified hypothesis for the structure and rheology of gl u t e l i n s . J. S c i . Fd. Agric. 23: 687. Ferry, J.D. 1961. Viscoelastic Properties of Polymers. Chap. 1, 2 John Wiley and Sons Inc., New York. Grosskreutz, J.-'C. 1961. A lip o p r o t e i n model of wheat gluten structure. Cereal Chem. 38: 336. Hartman, W.E. 1966. Method of making rehydrated gluten products. U.S. Pat. 3,290,152. Hibberd, G.E. and Wallace, W.J. 1966. Dynamic v i s c o e l a s t i c behavior of wheat flou r doughs. I. Linear aspects Rheologica Acta 5: 193. Kasarda, D.D.,Nimmo, C C . and Kohler, G.O. 1971. Proteins and the amino acid composition of wheat fractions Wheat chemistry and Technology (Y. Pomeranz, ed.) Amer. Assoc. Cereal Chem. Inc., St. Paul, Minn. Kjelson, N.A. 1965. Fibrous gluten product and process for preparing said product. U.S. Pat. 3,197,310. Mecham, D.K. 1971. Chap. 8: Li p i d s . Wheat Chemistry and Technology (2nd ed.). Assoc. of Cereal Chem., Inc., St. Paul, Minn. Olcott, H.S. and Mecham, D.K. 1947. Characterization of wheat gluten. I. P r o t e i n - l i p i d complex formation during doughing of f l o u r s . Lipoprotein nature of the glutenin f r a c t i o n . Cereal Chem.24: 407. Grth, R.A., Dronzek, B.L. and Bushuk, W. 1973a. Studies of glutenin. IV. Microscopic structure and i t s rela t i o n s to bread making q u a l i t y . Cereal Chem. 50: 688. Orth, R.A., Dronzek, B.L. and Bushuk, W. 1973b. Scanning electron microscopy of bread wheat proteins fractionated by gel f i l t r a t i o n . Cereal Chem. 50: 696. Ostle, B. 1963. S t a t i s t i c s i n Research - Chap. 8. (Revised second e d i t i o n ) . Iowa State University Press, Ames, Iowa. Pomeranz, Y. (ed.)- 1971a. Wheat Chemistry and Technology (2nd ed.). Am. Assoc. of Cereal Chem., Inc., St. Paul, Minn. Pomeranz, Y. 1971b, Chap. 12: Composition and f u n c t i o n a l i t y of wheat-flour components. Wheat Chemistry and Technology (2nd ed.). Am. Assoc. of Cereal Chem., Inc., St. Paul, Minn. Pomeranz, Y. (ed.). 1973. Indu s t r i a l Uses of Cereals, Am. Assoc. of Cereal Chem., Inc., St. Paul, Minn. Rasper, V., Rasper, J. and deMan, J.M. 1974. Stres s - s t r a i n relationships of chemically improved unfermented doughs. I: The evaluation of data obtained at large deformations i n simple t e n s i l e mode. J. Text. Studies 4: 438. Rinde, J.A., Tschoegl, N.W. and Smith, T.L. 1970. Large-deformation and rupture properties of wheat f l o u r gluten. Cereal Chem. 47: 225. Seckinger, H.L. and Wolf, M.J. 1970. Electron microscopy of endosperm protein from hard and soft wheats. Cereal Chem. 47: 236. Simmonds, D.H. 1972a. The ultrastructure of the mature wheat endosperm. Cereal Chem. 49: 212. Simmonds, D.H. 1972b. Wheat grain morphology and i t s relationship to dough structure. Cereal Chem. 49: 324. Smith, J.R., Smith, T.L. and Tschoegl, N.W. 1970. Rheological properties of wheat flou r doughs. I I I . Dynamic shear modulus and i t s dependence on amplitude frequency and dough composition. Rheologica Acta 9: 239. Steel, R.G.D. and Torrie, J.H. 1960. Chap. 14. Pr i n c i p l e s and Procedures of S t a t i s t i c s . McGraw-Hill Book, Inc., New York. Sternberg, G. 197 3. P r a c t i c a l gluten structure control. Bakers Digest 47: 34. Technicon Autoanalyser - Industrial Method No. 146-71A, Technicon I n d u s t r i a l Corporation, Tarrytown, N.Y. .103. Tschoegal, N.W., Rinde, J.A. and Smith, T.L. 1970. Rheological properties of wheat f l o u r doughs. I. Method for determining the large deformation and rupture properties i n simple tension. J . S c i . Fd. Agric. 21: 65. Wall, J.S. and Beckwith, A.C. 1969. Relationships between structure and rheological properties of gluten proteins. Cereal Science Today 14: 16. 

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