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

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FUNCTIONALITY OF PLANT PROTEINS FOR COMMINUTED MEAT SYSTEMS by A l l a n Thomas Paulson B.Sc. (Agr.) f The Univ e r s i t y of B r i t i s h Columbia, 1973 M.Sc, The Univ e r s i t y of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1985 © A l l a n Thomas Paulson, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Food Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date November 27, 1985 DE-6(3/81) i i ABSTRACT A three part study is presented which examines the functional properties of plant proteins as they relate to te x t u r a l and s t a b i l i t y c h a r a c t e r i s t i c s of protein-replaced meat emulsions. In the f i r s t chapter, the effects of cooking time (25 or 50 min) and temperature (70 or 95°C) on texture, microstructure and cook s t a b i l i t y of a model meat emulsion system containing soy or canola protein isolate were investigated. The plant proteins were added either dry or rehydrated at replacement levels of 33.3% and 66.7% of the meat protein. Instrumental texture p r o f i l e analysis and s t a b i l i t y data revealed several complex i n t e r -actions between experimental variables; however, level of protein replacement was predominant, with decreased firmness and increased y i e l d r e s u l t i n g from increased replacement of meat protein. Thermorheological p r o f i l e s of emul-sions and protein dispersions demonstrated that the development of e l a s t i c i t y of all-meat emulsions during heating was ess e n t i a l l y complete at 75-80°C, while the e l a s t i c i t y of canola or soy protein dispersions continued to ri s e with heating to 95°C. A meat emulsion containing canola protein displayed c h a r a c t e r i s t i c s of the all-meat emulsion and canola protein dispersion thermoprofiles, but the increased structure formation from the canola protein at higher heating temperatures did not f u l l y compensate for an i n i t i a l decrease i n e l a s t i c i t y that resulted from the loss of meat protein. Although there were sli g h t differences in the fat p a r t i c l e d i s t r i b u t i o n s of the emulsions containing plant protein, the d i s t r i b u t i o n s had similar shapes, where p a r t i c l e s larger than 50 micrometers approximated a normal d i s t r i b u t i o n , and were thought to be r e l a t i v e l y intact fat c e l l s while the number of p a r t i c l e s with diameters of 10-50 micrometers increased i n an e s s e n t i a l l y logarithmic manner as size decreased. The microstructure of the proteinaceous matrix was affected p r i m a r i l y by protein source, replacement lev e l and cooking conditions. In chapter 2, thermally induced gelation (72°C, 30 min heating) and emuls i f i c a t i o n properties of unmodified and succinylated canola protein isolate (54% and 84% modification of free amino groups) were examined over a wide range of pH values (pH 3.5-11.0) and sodium chloride concentrations (0.0-0.7M). Succinylation improved the gelation a b i l i t y of canola isolate. For the unmodified isolate, gels formed at only 4 of 18 combinations of pH and NaCl concentration, while 12 gels formed from each le v e l of succinylation under the same conditions. Above pH 6.5, succinylated protein formed gels only i n the presence of NaCl. In general, the firmest gels were obtained with the moderate level of succi n y l a t i o n . Translucent and opaque gels responded d i f f e r e n t l y to Theological tests and were related i n dif f e r e n t ways to the physicochemical and Theological properties of protein dispersions. The visc o e l a s t i c properties of the translucent gels were affected mainly by protein s o l u b i l i t y and hydrophobicity, while those of the opaque gels were related to s o l u b i l i t y , hydrophobicity, zeta potential and apparent v i s c o s i t y of protein dispersions. The types of bonds involved i n gel formation and s t a b i l i t y were t e n t a t i v e l y i d e n t i f i e d as hydrophobic interactions and hydrogen bonds. With the succinylated isolates, gels were formed i n the presence of calcium ions at a concentration an order of magnitude less than was required for similar gel strengths with NaCl, which has implications for e x p l o i t i n g the gelation a b i l i t y of succinylated proteins i n products where high concen-trations of NaCl are undesirable. iv Both emulsification a c t i v i t y and emulsion s t a b i l i t y were increased by succinylation, but exhaustive succinylation was not required to produce a si g n i f i c a n t improvement i n these properties. E m u l s i f i c a t i o n a c t i v i t y was r e l a t e d to p r o t e i n s o l u b i l i t y , hydrophobicity, zeta potential and flow behavior of protein dispersions, while emulsion s t a b i l i t y appeared to be mainly a measure of resistance to creaming and was related to protein s o l u -b i l i t y , zeta potential, apparent v i s c o s i t y of protein dispersions, and the difference i n density between the aqueous and o i l phases. The t h i r d chapter examined the r e l a t i o n s h i p between textural measurements of canola isolate gels obtained by means of a puncture test with an Instron tester, and fundamental rh e o l o g i c a l parameters obtained from nondestructive dynamic shear measurements with a Weissenberg Rheogoniometer. Although the force required to rupture the gels, as measured by the puncture test, was poorly correlated with the vis c o e l a s t i c parameters, the slope of the force-deformation curves to the point of rupture was well correlated with the storage and loss moduli of the gels. In addition, the area under the force-deformation curves to rupture followed a c u r v i l i n e a r r e l a t i o n s h i p with the loss tangent of the gels. The response of translucent and opaque gels to the two types of rhe o l o g i c a l tests was not i d e n t i c a l , which indicated that gel microstructure i s an i n f l u e n t i a l factor when evaluating gel rh e o l o g i c a l properties by destructive or nondestructive methods. TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x i GENERAL INTRODUCTION 1 Chapter 1. MICROSTRUCTURE AND TEXTURE OF MEAT EMULSIONS SUPPLEMENTED WITH PLANT PROTEINS INTRODUCTION 3 LITERATURE REVIEW 4 MATERIALS AND METHODS 7 A. Experimental Design 7 B. Emulsion Preparation 7 C. Cook S t a b i l i t y 8 D. Texture 8 E. Thermorheological Scanning 9 F. Light Microscopy 11 G. Scanning Electron Microscopy 13 RESULTS AND DISCUSSION 14 A. Texture P r o f i l e Analysis 14 B. Thermorheological Scanning 16 C. Cook S t a b i l i t y 20 D. Microstructure 21 SUMMARY AND CONCLUSIONS 36 REFERENCES 38 Chapter 2. GELATION AND EMULSIFICATION PROPERTIES OF UNMODIFIED AND SUCCINYLATED CANOLA PROTEIN ISOLATE INTRODUCTION 42 LITERATURE REVIEW 44 A. Prot e i n Succinylation 44 B. Thermally Induced Gelation of Globular Proteins 47 C. Proteins as Emulsifiers 51 \ v i Page MATERIALS AND METHODS 55 A. Succinylation Procedure 55 B. Determination of Extent of Succinylation 56 G. Protein S o l u b i l i t y 57 D. Prot e i n Surface Hydrophobicity 57 E. Zeta P o t e n t i a l (Net Charge Density) 58 F. Steady Shear Rheology 59 G. Thermally Induced Gelation 60 1. Dynamic Shear Properties of Thermally Induced Gels 60 2. Pro t e i n Content of Gel Exudate 61 H. Emul s i f i c a t i o n A c t i v i t y and Emulsion S t a b i l i t y 61 I. Light Microscopy 62 J. S t a t i s t i c a l Analyses 62 RESULTS AND DISCUSSION 65 A. Prot e i n S o l u b i l i t y 65 B. Hydrophobicity and Zeta P o t e n t i a l 70 C. Steady Shear Rheology and Microstructure 78 D. Thermally Induced Gelation 88 E. Emuls i f i c a t i o n A c t i v i t y and Emulsion S t a b i l i t y 112 SUMMARY AND CONCLUSIONS 124 REFERENCES 127 Chapter 3. DYNAMIC SHEAR VERSUS PUNCTURE PROBE MEASUREMENTS OF GEL TEXTURE INTRODUCTION 138 LITERATURE REVIEW 140 MATERIALS AND METHODS 143 A. Puncture Test 143 B. Dynamic Vis c o e l a s t i c Properties 145 RESULTS AND DISCUSSION 146 SUMMARY AND CONCLUSIONS 154 REFERENCES 155 LIST OF TABLES Chapter 1. Table 1.1. Chapter 2. Table 2.1. Table 2.2. Table 2.3. Table 2.4. Table 2.5. Table 2.6. Table 2.7A. Table 2.7B. Table 2.8. Table 2.9. Table 2.10. Table 2.11. Table 2.12. Instrumental texture p r o f i l e analysis of protein-replaced meat emulsions Orthogonal m u l t i p l i e r s for trend comparison analyses Trend comparison analysis of the effects of succinylation, pH and NaCl on protein s o l u b i l i t y of canola isolate Trend comparison analyses of the effects of succinylation, pH and NaCl on surface hydrophobicity ( S Q ) and zeta potential of canola isolate E ffects of succinylation, pH, NaCl and heating on surface hydrophobicity of canola isolate Power-law and power-law pl a s t i c flow parameters of 11.4% canola isolate dispersions Trend comparison analyses of the effects of succinylation, pH and NaCl on apparent v i s c o s i t y of 11.4% canola isolate dispersions Dynamic shear flow behavior parameters of canola isolate gels Dynamic shear storage behavior parameters of canola isolate gels Multiple regression models for pred i c t i o n of vi s c o e l a s t i c parameters of thermally induced canola isolate gels Multiple regression models for pred i c t i o n of vis c o e l a s t i c parameters of translucent gels P r o t e i n content of exudate from thermally induced gels of canola protein isolate Multiple regression models for pred i c t i o n of vis c o e l a s t i c parameters of opaque gels Trend comparison analyses of the effects of succinylation, pH and NaCl on emulsification a c t i v i t y and emulsion s t a b i l i t y v i i i Page Table 2.13. Multiple regression models for p r e d i c t i o n of emulsification a c t i v i t y and emulsion s t a b i l i t y 119 Chapter 3. Table 3.1. Rheogoniometer vs. Instron measurements of gel texture 148 i x Chapter 1. LIST OF FIGURES Page Figure 1.1. T y p i c a l force-deformation curves for texture p r o f i l e analysis of meat emulsion samples 10 Figure 1.2. Storage moduli as a function of temperature of an a l l -meat emulsion, a 66.7% canola protein meat emulsion and a 15% canola isolate dispersion 17 Figure 1.3. Storage moduli of 15% soy isolate dispersions as a function of temperature 19 Figure 1.4. Fat p a r t i c l e d i s t r i b u t i o n s of meat emulsions containing soy or canola protein; A-C 22 D-E 23 Figure 1.5. LM and SEM micrographs of a 66.7% soy protein meat emulsion cooked at 95°C for 50 min 24 Figure 1.6. LM and SEM micrographs of a 66.7% canola protein meat emulsion cooked at 95°C for 50 min 25 Figure 1.7. SEM micrographs of 66.7% canola protein meat emulsions 27 Figure 1.8. LM and SEM micrographs of a 33.3% soy protein meat emulsion cooked at 95°C for 25 min 29 Figure 1.9. LM and SEM micrographs of a 33.3% canola protein meat emulsion cooked at 95°C for 25 min 30 Figure 1.10. SEM micrographs of 33.3% canola protein meat emulsions 31 Figure 1.11. LM and SEM micrographs of an all-meat emulsion cooked at 70°C for 25 min 33 Figure 1.12. LM and SEM micrographs of an all-meat emulsion cooked at 95°C for 50 min 35 Chapter 2. Figure 2.1. Protein s o l u b i l i t y of 11.4% canola isolate dispersions 67 Figure 2.2. Surface hydrophobicity ( S 0 ) of canola isolate 72 Figure 2.3. Zeta potential of canola isolate 73 X Page Figure 2.4. Apparent v i s c o s i t y at 10 s " l for 11.4% canola isolate dispersions 80 Figure 2.5. Apparent v i s c o s i t y at 1000 s - 1 for 11.4% canola isolate dispersions 81 Figure 2.6. Light micrographs of 11.4% canola isolate dispersions; A-D 85 E-H 86 Figure 2.7. Storage moduli and dynamic v i s c o s i t i e s of 5.2% SA. pH 6.5, 0.7M NaCl canola isolate gel as a function of o s c i l l a t o r y frequency 90 Figure 2.8. Storage modulus at 10 s" 1 for 11.4% canola isolate gels 93 Figure 2.9. Loss modulus at 10 s - 1 for 11.4% canola isolate gels 94 Figure 2.10. Loss tangent at 10 s"* for 11.4% canola isolate gels 95 Figure 2.11. Storage and loss moduli at 10 s" 1 for 11.4% canola isolate gels 98 Figure 2.12. Emulsification a c t i v i t y of canola isolate dispersions 114 Figure 2.13. Emulsion s t a b i l i t y of canola isolate dispersions 117 Chapter 3. Figure 3.1. Force-deformation curve from Instron puncture test of protein gels 144 Figure 3.2. Rupture force vs. storage modulus for canola isolate gels 147 Figure 3.3. Rupture slope vs. storage modulus for canola isolate gels 147 Figure 3.4. Rupture slope vs. loss modulus for canola isolate gels 150 Figure 3.5. Rupture area vs. loss tangent for canola isolate gels 150 xi ACKNOWLEDGEMENTS I would like to express ray sincere appreciation to ray supervisor, Dr. Marvin A. Tung, for his support and advice over the course of this research project. The comments and suggestions of Dr. William D. Powrie and Dr. Shuryo Nakai of the Dept. of Food Science, and Dr. Jim Shelf ord of the Dept. of Animal Science are also appreciated. I would like to dedicate this thesis to my wife, Judy, for her under-standing and patience during the time required to complete my graduate study. Financial support for these studies was provided by a grant from the Natural Sciences and Engineering Research Council of Canada. GENERAL INTRODUCTION 1 The f u n c t i o n a l behavior of proteins i n food systems r e s u l t s from complex interactions among the composition, structure and physicochemical properties of the proteins per se, th e i r interactions with other components such as l i p i d s and carbohydrates, and the nature of the environment i n which they are associated ( K i n s e l l a , 1976, 1979; references l i s t e d at end of Chapter 1). T y p i c a l f u n c t i o n a l properties of food proteins include such diverse phenomena as foaming, emulsification and gelation. P r o t e i n functional properties and t h e i r measurement have been extensively reviewed (e.g., Briskey, 1970; K i n s e l l a , 1976, 1979; Schoen, 1977). There is c u r r e n t l y a trend toward the use of non-meat protein sources as replacements and extenders i n products containing animal protein. In addition to n u t r i t i o n a l value, these proteins must possess func t i o n a l pro-perties which maintain or improve the quality of foods i n which they are used. A promising area of research has been the incorporation of non-meat proteins into frankfurter-type comminuted meat products. At low levels, replacement proteins have been suggested to act as emulsifiers or serve as binding agents preventing water and fat release during processing (Smith et a l . , 1973). At high levels, however, the reduced structure-forming properties of the replacement proteins can lead to detrimental effects on product texture (Comer, 1979; Randall et a l . , 1976). The present investigation examines the f u n c t i o n a l behavior of plant proteins as they relate to t e x t u r a l and s t a b i l i t y c h a r a c t e r i s t i c s of p r o t e i n -replaced meat emulsions. The study was divided into three parts dealing with the texture and microstructure of a model meat emulsion system containing soy 2 or canola p ro te in iso la te , the e f fec t of succ iny la t ion on the emuls i f i ca t ion and thermal ly induced gelat ion proper t ies of canola p ro te in iso la te , and the re la t ionsh ips between fundamental v iscoelast ic parameters of thermal ly induced canola p ro te in gels and tex ture measurements obtained w i th a puncture tes t . 3 CHAPTER 1 TEXTURE AND MICROSTRUCTURE OF MEAT EMULSIONS SUPPLEMENTED WITH PLANT PROTEINS INTRODUCTION The use of plant proteins as extenders or replacements for meat protein in frankfurter-type comminuted meat products has been the subject of much investigation. At high levels of replacement, the texture of these products usually becomes soft and mushy. Normal frankfurter processing schedules do not employ temperatures greater than approximately 75°C and it has been found that higher temperatures are often required for the denaturation and gelation of many globular proteins (Catsimpoolas and Meyer, 1970; Hermansson, 1979). The objectives of this research were to investigate the effects of processing conditions on texture, cook s t a b i l i t y and microstructure of a model meat emulsion system i n which large amounts of meat protein have been replaced with soy or canola protein isolate. Soy protein is used i n a wide va r i e t y of foods i n c l u d i n g bakery products, cereals, dairy foods and com-minuted meats ( K i n s e l l a , 1979). Canola is the major oilseed crop grown i n Canada, and canola proteins have been found to possess good emulsification, water and fat absorption, and whipping properties (Sosulski et a l . , 1976; Thompson et a l . , 1982). Although these reports have indicated poor gelation properties of canola protein, G i l l and Tung (1978) found that the 12S f r a c t i o n of rapeseed protein underwent thermally induced gelation under a wide range of pH and ionic conditions, but temperatures other than 100°C were not tested. 4 LITERATURE REVIEW F i n e l y comminuted meat products such as frankfurters or bologna are commonly prepared by chopping lean meat i n a brine solution to form a pro-teinaceous s l u r r y i n which animal fat is then f i n e l y divided and dispersed. The r e s u l t i n g batter, resembling an o i l i n water emulsion (Hansen, 1960), is subsequently cooked to form a product i n which the fat globules are entrapped within a r i g i d protein matrix. Previous work has documented the role of the salt- s o l u b l e meat proteins myosin and actomyosin i n emulsion formation and s t a b i l i z a t i o n through coating the fat droplets with a stable membrane (Hansen, 1960; Hegarty et a l . , 1963; Swift et a l . , 1961). Reviews by Saffle (1968) and Webb (1974) considered emulsification to be the primary factor responsible for the s t a b i l i t y of such products. Other workers have suggested that too much emphasis has been placed on the importance of emulsification (van den Oord and Visser, 1973). Theno and Schmidt (1978) examined the microstructure of three commercially acceptable frankfurters and found that only one could be ca l l e d a true meat emulsion. While these products may not be emulsions i n the s t r i c t -est sense, the term "meat emulsion" has been i n common use for many years and is retained i n the present study. Attention has shifted from the emulsification properties of meat proteins to t h e i r involvement i n matrix formation through thermally induced gelation, with the entrapment of fat and the development of the c h a r a c t e r i s t i c texture of the product. Several recent reviews have examined th i s aspect. Schmidt et a l . (1981) gave an overview of the protein matrix i n comminuted meat products with respect to gelation and vi s c o e l a s t i c properties of meat proteins, binding between chunks of meat, and emulsification. Acton et a l . (1983) discussed meat emulsions i n terms of protein-water in t e r a c t i o n , p r o t e i n - l i p i d associa-5 t i o n , p r o t e i n - p r o t e i n aggregation, and the i r i n t e r r e l a t i o n s h i p s with respect to the environmental conditions during comminution and thermal processing. Schmidt (1984) pointed out the d i v e r s i t y and complexity of comminuted meat processing, and emphasized the need for more research on the effects of the species of meat u t i l i z e d , desired composition, degree of comminution, mechan-i c a l action such as tumbling or massaging, and thermal treatment on the fini s h e d products. Ziegler and Acton (1984) and Asghar et a l . (1985) detailed the denaturation, aggregation and gelation reactions of muscle proteins. Lee (1985) surveyed the microstructural aspects of meat emulsion formation and s t a b i l i z a t i o n . He indicated that the microstructure of meat emulsions is influenced by numerous factors i n c l u d i n g type of meat, fat and other i n g r e -dients, levels of salt , moisture and fat, and the comminution process. The major changes to the microstructure of meat emulsions were i n the patterns of fat d i s t r i b u t i o n , which r e f l e c t e d fat s t a b i l i z a t i o n . He reviewed the evidence for the emulsion and nonemulsion theories of fat s t a b i l i z a t i o n and concluded that although both theories should be considered, from photomicrographic data and physical analysis, the nonemulsion theory should receive more considera-t i o n . S e v e r a l authors have attempted to relate f u n c t i o n a l properties of non-meat proteins to the i r performance as ingredients i n comminuted meat products. Thomas et a l . (1973) and Lauck (1975) suggested a r e l a t i o n s h i p between apparent v i s c o s i t y of the formulation and cooking loss. Hermansson (1975) and Hermansson and Akesson (1975a,b) related moisture loss i n a heated lean meat system containing non-meat proteins to salt concentration, protein type and amount, swelling, v i s c o s i t y and gelation of the added proteins. Torgersen and Toledo (1977), working with novel proteins i n a 6 comminuted meat system, found a s i g n i f i c a n t negative c o r r e l a t i o n between p r o t e i n s o l u b i l i t y and fat binding, as well as s o l u b i l i t y at 100°C and te x t u r a l mechanical properties. The more soluble proteins allowed increased fat release on cooking and the cooked products had lower mechanical strength. Comer (1979) stated that the performance of f i l l e r s i n comminuted meat products was better indicated by gelation and water binding than emulsifying a b i l i t y . Cassens et a l . (1975) observed that textured soy flour i n a frank-fu r t e r emulsion retained i t s morphology after processing and suggested that i t merely took up space within the matrix. Paulson et a l . (1984) reported that the s t a b i l i t y and firmness of model frankfurters containing modified plant proteins were influenced mainly by variables r e l a t i n g p r o t e i n - l i p i d i n t e r -a c t i o n s such as f a t absorption, hydrophobicity, and o i l emulsification properties of the added proteins. 7 MATERIALS AND METHODS A. Experimental Design A model meat emulsion formulation containing 10.5% beef protein, 29% pork fat, 57.5% water, and 2.5% sodium chloride was used as a control. Beef protein was replaced with either soy or canola protein isolate at either 33.3 or 66.7% (w/w). Lauck (1975) found that the hydration state of a whey protein product influenced the s t a b i l i t y of frankfurters. To test t h i s effect, the isolates were added either dry or rehydrated overnight i n d i s t i l l e d water (3:1 (w/w), H20:isolate). The emulsions were then cooked at 70 or 90°C for 25 or 50 min. The effects of ( i ) replacement, ( i i ) protein source, ( i i i ) p retreat-ment, (iv) cook temperature and (v) cook time on texture and cook s t a b i l i t y of the f i n i s h e d products were tested using a 2 5 f r a c t i o n a l f a c t o r i a l experimental design and analysis of variance. Product microstructure was examined using l i g h t microscopy (LM) and scanning electron microscopy (SEM). B. Emulsion Preparation Boneless beef chuck and pork backfat were purchased from a l o c a l ab-batoir, trimmed of v i s i b l e fat and meat traces respectively, minced and vacuum packaged separately i n 450 g lots , then frozen at -35°C. P r i o r to use, beef and backfat were allowed to thaw at 4°C, then kept on ice when taken from the coldroom. Soy protein isolate (%N(d.b.)=14.56) and canola protein isolate (%N(d.b.)=14.42) were purchased from the POS P i l o t Plant Corp. (Saskatoon, SK). Laboratory scale emulsion batches were prepared with a Sorvall Omni-mixer (Ivan S o r v a l l , Inc., Norwalk, CT). The Omnimixer was modified to allow the j a r to be moved up and down r e l a t i v e to the blades to give improved 8 chopping of the entire sample (Morrison et a l . , 1971). Ground beef, salt, d i s t i l l e d water and plant protein were blended for 25 s at half speed, pork backfat was added, and the emulsion formed by chopping at f u l l speed for 2 x 30 s with intermediate scraping and hand mixing. F i n a l emulsion temperatures after chopping ranged from 16-18°C. The emulsions were stuffed into stainless steel tubes (2.54 cm diam. x 10 cm long), capped, then cooked i n a water bath at 70 or 95°C for 25 or 50 min. Whiting and M i l l e r (1984) evaluated a laboratory blender and a food processor for making model frankfurter emulsions and concluded that, although the f r ankfurters were not i d e n t i c a l to those produced by commercial machines, the differences were not of a magnitude that were consistently detected. Smaller scale machines have the advantages of being inexpensive, economical with ingredients, and time-saving. C. Cook S t a b i l i t y The cooked emulsions were cooled i n ice water for 10 min, removed from the tubes and weighed. Cook s t a b i l i t y was determined as the weight r a t i o of the cooked to uncooked emulsions, expressed as a percentage. D. Texture Texture of the cooked emulsions was evaluated at room temperature by an instrumental texture p r o f i l e analysis (TPA) using a Model 1122 Instron Universal Testing Machine (Instron Corp., Canton, MA) (Bourne, 1968). Bite sized c y l i n d e r s of cooked emulsions (12 mm long, 23 mm diam.) were subjected to two consecutive compressive strokes between f l a t plate f i x t u r e s to 75% of the i r o r i g i n a l height at a crosshead speed of 100 mm/min and a chart speed of 9 1000 mm/min. The r e s u l t i n g force-deformation curves were analyzed for rupture force, f i r s t bite hardness, second bite hardness, springiness and cohesiveness (Figure 1.1). B. Thermorheological Scanning The dynamic shear response to small amplitude sinusoidal o s c i l l a t i o n of all-meat and 66.7% canola (dry) substituted emulsions, and 15% (w/w) disper-sions of soy and canola isolate i n 3.5% saline was evaluated as a function of temperature with a Model R.19 Weissenberg Rheogoniometer (Sangamo-Schlumberger Ltd., Bognor Regis, U.K.) by a method similar to that of Beveridge et a l . (1984). The Rheogoniometer was equipped with a modified F e r r a n t i - S h i r l e y lower platen and a 7.5 cm f l a t upper platen supported by a no. 8 torsion bar (87.5 Pa cm3 fim'l). The lower platen was connected to a Haake Model FP c i r c u l a t i n g heated-refrigerated bath (Haake Inc., Saddle Brook, NJ) with a Haake PG 11 temperature programmer and a Haake DK 51 r e f r i g e r a t e d bath. The sample was placed i n the center of the lower platen and the upper platen was lowered to a gap thickness of 1.5 mm. To prevent drying of the sample at the edges, a masking tape dike was formed around the circumference of the lower platen and p a r a f f i n o i l was introduced to a depth s u f f i c i e n t to cover the sample. O s c i l l a t i o n was at a constant frequency of 5.96 s ~ l with a maximum st r a i n amplitude of 0.875%, which was within the range of linear v i s c o -e l a s t i c i t y for samples representative of those tested. The sample was o s c i l l a t e d for 10 min to equilibrate the sample temperature prior to heating. The temperature p r o f i l e within the gap was measured with a copper-constantan thermocouple embedded i n the approximate center of the bottom platen so that the thermocouple t i p was f l u s h with the platen surface. M i l l i v o l t signals 10 _ F i r s t r, b i t e _ S e c o n d s b i t e _ H I = H a r d n e s s •^m. U C o h e s i v e n e s s = 7 7 A1 R u p t u r e / f o r c e / « . . . D2 S p r i n g i n e s s = H2 y AI D i s t a n c e 0 r * - D 2 — H Figure 1.1. T y p i c a l force-deformation curves for texture p r o f i l e analysis of meat emulsion samples. 11 were recorded with a Digitec Model 1268 datalogger (United Systems Corp., Dayton, OH) and converted to temperature. The samples were heated from 20°C to either 70°C or 95°C at a rate of 2 C°/min. When the desired temperature was reached, the cooling bath was activated and the sample was cooled at 2 C°/min back to 20°C. The delay between heating and cooling caused a temperature overshoot of 2-3 C°. Thermal expansion of the metal platens necessitated adjustment of the gap every 5 or 10 C°; the extent of adjustment was determined by preliminary experimenta-t i o n . O s c i l l a t o r y input was continuous throughout the experimental period and measurements were taken every 5 or 10 C°. The amplitudes of the input (strain) and output (stress) voltage signals, and the phase difference between them, were monitored with a Tronotec Model 703A d i g i t a l phasemeter (Tronotec Inc., F r a n k l i n , NJ), and dynamic rhe o l o g i c a l parameters were calculated from the equations of Walters (1968). The storage modulus (G 1) is a measure of the energy stored e l a s t i c a l l y i n the sample, the loss modulus (G") is a measure of the energy dissipated as heat, and the loss tangent (numerically equal to G"/G') r e f l e c t s the r e l a t i v e proportion of viscous to ela s t i c character i n the sample. As a material undergoes gelation, i t becomes more elas t i c i n nature and the storage modulus w i l l increase while the values of loss tangent decrease. For these experiments, the effect of temperature on the storage modulus of the samples was monitored. F. L i g h t Microscopy Unfixed pieces of sample were frozen i n l i q u i d nitrogen, allowed to equilibrate to -25°C i n a cryostat microtome, then sectioned to a t h i c k -ness of 14 micrometers. The sections were affixed to glass slides with Mayers 12 glycerol albumen adhesive p r i o r to sta i n i n g . For l i p i d s , sections were immersed i n 100% propylene glycol for 6 min, stained i n 0.5% Sudan Black B i n propylene gl y c o l for 10 min then d i f f e r e n t i a t e d i n 85% proplyene gl y c o l for 6 min and 50% propylene glycol for 2 min. These sections were not counter-stained for protein as fine d e t a i l tended to be obscured. To examine the protein matrix independently, separate sections were stained with 0.17% l i g h t green i n 0.33% acetic acid for 1 min, rin s e d i n d i s t i l l e d water, d e h y d r a t e d with 90%, then 100% ethanol, then delipidated with xylene. Attempts to d i s t i n g u i s h between plant and meat proteins by d i f f e r e n t i a l s t a i n i n g were unsuccessful. To observe both l i p i d and protein simultaneously, the l i p i d was stained with 0.5% Sudan IV i n propylene gl y c o l for 15 min and counterstained with l i g h t green for 30 sec. The samples were examined and photographed under b r i g h t f i e l d i llumination with 5X and 10X objectives, using a Wild M20 microscope and a Pentax 35 mm camera. Fat p a r t i c l e d i s t r i b u t i o n s of the emulsions containing plant proteins heated at 95°C were obtained from 18 cm x 24 cm l i g h t micrographs of sections stained for l i p i d (actual specimen area = 1.89 x 10 6 square micrometers). One micrograph was used for each treatment. Each micrograph was partit i o n e d into 48 squares (3 cm x 3 cm) to f a c i l i t a t e counting. As not a l l fat p a r t i c l e s were sp h e r i c a l , the equivalent area diameter of the p a r t i c l e s (the diameter of a c i r c l e having the same area as the p a r t i c l e ; Fischmeister, 1968) were obtained using a c i r c l e template, and classed into 32 size categories between 10 and 126 micrometers i n diameter. Those fat p a r t i c l e s with diameters of 34-126 micrometers (23 size categories) were counted i n a l l 48 squares of each micrograph. P a r t i c l e s with diameters of 24-34 micrometers (4 size categories) were counted i n 36 squares selected at random, and the counts were projected 13 to an estimated count for the entire f i e l d . S i m i l a r l y , fat p a r t i c l e s with diameters of 18-24 micrometers (2 size categories) were counted i n 14 randomly selected squares, while p a r t i c l e s of 10-18 micrometers i n diameter (3 size categories) were counted i n 5 squares. The i n d i v i d u a l fat p a r t i c l e d i s t r i b u -tions were then combined and averaged to give the following contrasts: (1) 66.7% soy vs. 66.7% canola protein substitution, (2) 33.3% soy vs. 33.3% canola, (3) 33.3% soy vs. 66.7% soy, (4) 33.3% canola vs. 66.7% canola, (5) soy vs. canola, and (6) 33.3% vs. 66.7% substitution. Thus each fat p a r t i c l e d i s t r i b u t i o n i n the contrasts was the mean of either 2 or 4 i n d i v i d u a l samples. No s t a t i s t i c a l comparative procedures were performed due to the complexity of the d i s t r i b u t i o n s . G. Scanning E l e c t r o n Microscopy Small cubes of cooked emulsions approximately (4 mm)3 were cryofractured i n l i q u i d nitrogen. Small fragments approximately (1-1.5 mm)3 were fixed i n 4% gluteraldehyde i n 0.07 M phosphate buffer (pH 7) for 12-24 h at 4°C. After r i n s i n g three times i n phosphate buffer, secondary f i x a t i o n was accomplished with 1% osmium tetroxide i n phosphate buffer for 4 h. After a second set of phosphate buffer rinses, the samples were dehydrated through a graded ethanol series followed by exchange of ethanol with a graded series of amyl acetate i n 100% ethanol, then 100% amyl acetate for 1 h. The samples were dried i n a Parr c r i t i c a l point drying bomb (Parr Instrument Co., Moline, IL) using l i q u i d C02, mounted on aluminum stubs, gold coated i n a Technics sputter coating unit (Technics Inc., Alexandria, VA), and observed with a Cambridge Stereoscan 250 SEM (Cambridge Instruments (Canada) Inc., Montreal, PQ) at an acceler-ating voltage of 20 kV. 14 RESULTS AND DISCUSSION A. Texture P r o f i l e Analysis Analysis of variance of the TPA data revealed a complex interaction between experimental factors and texture p r o f i l e components, although replace-ment lev e l was found to be predominant o v e r a l l (Table 1.1). For rupture force, replacement le v e l was the only s i g n i f i c a n t main effect (p<0.01) with decreased rupture force values with increased replacement l e v e l . An i n t e r -action was also found between replacement le v e l and cook temperature (p<0.05); at 66.7% replacement of meat protein, the 95°C cook produced higher rupture force values than at 70°C, while at 33.3% replacement the opposite was true. For f i r s t bite hardness and second bite hardness, replacement l e v e l was again s i g n i f i c a n t (p<0.01) with decreased hardness values at increased substitution. Canola protein isolate produced s i g n i f i c a n t l y greater hardness values than soy protein isolate (p<0.01). An i n t e r a c t i o n was found between cook temperature and pretreatment (p<0.05) with greater hardness values at 70°C i f the isolates were rehydrated prior to addition, whereas the 95°C cook produced the opposite effect. Springiness, expressed as percent recovery from the o r i g i n a l deforma-t i o n , was influenced by two factors; replacement le v e l (p<0.01), where greater springiness was found at the 33.3% replacement l e v e l than the 66.7% l e v e l , and cook temperature (p<0.01), where a 95°C cook produced springier products than at 70°C. For the control emulsions, cook time appeared to be more important for springiness than temperature, with a 50 min cook producing greater springiness than 25 min, although s l i g h t l y greater springiness was produced at 70°C than 95°C. Cohesiveness was not affected s i g n i f i c a n t l y by any of the factors under investigation. 15 Table 1 .1 . Instrumental texture p r o f i l e a n a l y s i s of protein-replaced meat eaulslons. Texture Profile Component Significant Experimental Factors Rupture Force (i) Replacement Level** 33.3% 66.7% (N) Control: 62.0 + 3.5 37.0 + 2.3 16.7 + 1.9 (ii) Level x Cook Temp.* 33.32 66.7% 70UC:38.6 + 1.9 95°C:35.3 + 1.2 15.4 + 1.6 18.0 + 1.1 First Bite (i) Replacement Level** 33.3% 66.7% Hardness (N) Control: 63.8 + 2.2 48.3 + 2.7 31.4 +4.1 (ii) Protein Source** Soy Canola 37.8 + 10.1 42.0 + 8.7 ( i i i ) Pretreatment x Cook Dry Rehydrated Temperature* 70°C:37.3 + 11.6 95°C:42.4 + 10.4 40.5 + 10.9 39.4 + 7.9 Second Bite (i) Replacement Level** 33.3% 66.7% Hardness (N) Control: 50.A + 2.5 36.6 + 8.1 22.9 + 3.1 (ii) Protein Source** Soy Canola 28.1 + 8.1 31.4 + 7.1 ( i i i ) Pretreatment x Cook Dry Rehydrated Temperature* 70UC:27.7 + 8.6 95°C:31.8 + 8.6 30.0 + 9.0 29.5 + 6.6 Springiness (%) (i) Replacement Level** 33.3% 66.7% Control: 57.1 + 5.6 51.5 + 5.3 45.8 + 7.2 (ii) Cook temperature** 70°C 95°C * p<0.05 ** p<0.01 16 Patana-Anake and Foegeding (1985) also found s i g n i f i c a n t interactions between heating temperature and time of cooking for s t a b i l i t y and tex t u r a l c h a r a c t e r i s t i c s of meat batters containing soy protein or v i t a l wheat gluten. B. Thermorheological Scanning The effects of heating from 20°C to 95°C on the storage modulus (G 1) of an all-meat emulsion, a 66.7% replacement canola/meat emulsion, and a 15% canola isolate dispersion are shown i n Figure 1.2. Heating the all-meat emulsion to 60°C caused a decrease i n G', but from 60 to 75°C, G' increased r a p i d l y with l i t t l e change between 75 and 95°C. Upon cooling to 25°C, G1 increased l o g a r i t h m i c a l l y followed by a sharp r i s e i n G1 with further cooling to 20°C, probably as a resul t of s o l i d i f i c a t i o n of the pork fat globules i n the dispersed phase of the meat emulsion. D i f f e r e n t i a l scanning calorimetry (DSC) of the pork backfat used i n the meat emulsions revealed several endothermic peaks between 15°C and 50°C, but over 50% of the fat was melted at 30°C and 75% was melted at 35°C. Townsend et a l . (1968) and Quinn et a l . (1980) reported similar thermograms for pork fat. Quinn et a l . (1980) also found that the ch a r a c t e r i s t i c DSC thermoprofile for muscle proteins was altered by comminuting with salt, r e s u l t i n g i n a single endothermic peak with an onset of approximately 60°C and a maximum at 72°C. These correspond very closely to the temperatures at which G1 began to increase (60°C) and then leveled off (75°C). Thus the protein denaturation reactions observed by DSC analysis appeared to be followed very clo s e l y by aggregation and gelation reactions involved i n the formation of a strong cohesive meat emulsion matrix. The normal processing temperature for comminuted meat products (approximately 70-75°C) corresponds clos e l y to the temperature at which G1 no longer increased. Figure 1.2. Storage moduli as a func t ion of temperature of an a l l -meat emulsion, a 66.7% canola prote in meat emulsion and a 15% canola isolate d ispers ion: open symbols indicate heat ing, sol id symbols indicate coo l ing. H 18 The meat emulsion i n which 66.7% of the meat protein was replaced by can-ola protein isolate followed a similar pattern for G1 except that no plateau was reached at 75°C and Gf continued to ri s e to 95°C i n d i c a t i n g continued structure formation. Upon cooling, there was l i t t l e change i n G1 between 95°C and 60°C, followed by an increase i n G1 as cooling continued to 20°C. The storage modulus of a 15% canola isolate dispersion decreased with heating to 60°C followed by a steady increase i n G' with further heating to 95°C. Upon cooling, G1 increased steadily throughout the entire tempera-ture range. As th i s sample was not a fat-c o n t a i n i n g emulsion, however, the thermorheological p r o f i l e should be used for qualitative comparisons only. Comparing the three thermoprofiles, i t is apparent that replacement of meat protein with canola protein produced a meat emulsion that was i n i t i a l -l y less el a s t i c than an all-meat emulsion, and upon heating displayed charac-t e r i s t i c s of both the all-meat and a l l - c a n o l a samples. Thus, unlike the all-meat emulsion, G1 continued to ri s e with heating from 75°C to 95°C but at a slower rate than between 60°C and 75°C. The increased structure formation at the higher heating temperatures was not enough, however, to compensate for the effects of the loss of meat protein. The dynamic shear analysis of heating 15% soy isolate dispersions to 95°C is shown i n Figure 1.3. Storage modulus decreased steadily with i n i t i a l heating, but at a slower rate than for the 15% canola isolate dispersion. Unlike canola isolate, G1 continued to decrease from 60°C to 70°C followed by an increase i n G' with further heating to 95°C. DSC thermograms of soy isolate dispersions revealed two major endotherms with t r a n s i t i o n temperatures of approximately 67°C and 90°C and maxima at 80°C and 98°C, respectively. These correspond to denaturation of the 7S and 11S fractions i n that order Figure 1.3. Storage moduli of 15% soy isolate dispersions as a f u n c t i o n of temperature: open symbols indicate heat ing, so l id symbols ind icate coo l ing . (—• VD 20 (Hermansson, 1978). Like the all-meat emulsion, the development of G1 with heating of the soy isolate dispersion appeared to be related to the thermal tr a n s i t i o n s found with DSC. Montejano et a l . (1984) also reported that major tra n s i t i o n s i n r i g i d i t y with heating of sol pastes prepared from comminuted f i s h , beef, pork and turkey clo s e l y p a r a l l e l e d DSC thermoprofiles of the same samples. For the 15% soy isolate dispersion, the increase i n G' from 70°C to 97°C was less than that for the canola isolate dispersion between 60°C and 95°C, but the increase i n G1 upon cooling was greater for the soy isolate, so that both dispersions started and ended up with very similar G1 values. Heating a 15% soy isolate dispersion to 73°C and then cooling (Figure 1.3) produced no increase i n G1 which indicated that protein denaturation induced by higher temperatures was required for gel formation. C. Cook S t a b i l i t y Cook s t a b i l i t y was affected by replacement level (98.5% y i e l d at 66.7% replacement vs. 97.2% at 33.3% replacement; p<0.01), cooking temperature (98.6% at 70°C vs. 97.1% at 95°C; p<0.01), cooking time (98.1% at 25 min vs. 97.7% at 50 min; p<0.05) and protein source (canola, 98.1% vs. soy, 97.8%, p<0.05), as well as interactions between protein source and pretreatment (p<0.05), protein source and cooking time (p<0.05), and cook temperature and replacement le v e l (p<0.01). These variations were small when compared to the all-meat control emulsions, in which the yi e l d varied from 82.0% with a 95°C, 50 min cook to 98.4% with a 70°C, 25 min cook. This i s consistent with the work of Randall et a l . (1976) and Sosulski et a l . (1977) who attri b u t e d improved cook s t a b i l i t y of frankfurters containing plant proteins to increased water holding capacity, and Schut (1976) who described decreased water holding 21 capacity of meat proteins with increased severity of thermal treatment as being due i n part to protein denaturation, coagulation and shrinkage. D. Microstructure Microstructure of the cooked emulsions was examined by l i g h t micro-scopy (LM) and scanning electron microscopy (SEM). The fat p a r t i c l e s ranged i n size from less than 1 micrometer to 130 micrometers i n diameter. The d i s t r i b u t i o n s of fat p a r t i c l e s with diameters of 10-126 micrometers were obtained for the emulsions containing plant proteins (Figure 1.4). Figure 1.5A shows the fat p a r t i c l e s i n a 66.7% soy substituted emulsion cooked at 95°C for 50 min. The proteinaceous matrix (Figure 1.5B) had an open, lacy appearance with r e g u l a r l y spaced areas of more densely staining protein material. An SEM micrograph (Figure 1.5C) showed fat p a r t i c l e s embedded i n the proteinaceous matrix, several of which appeared to be deformed perhaps during comminution and s t u f f i n g or by coalescence. The 66.7% canola substituted emulsions had a greater number of fat p a r t i c l e s with diameters of 10-50 micrometers and fewer p a r t i c l e s with diameters greater than 50 micro-meters (Figures 1.4A and 1.6A) and the protein matrix had a more compact and less lacy appearance than the 66.7% soy emulsion (Figure 1.6B and C). Also seen were a number of pores and openings i n the fat p a r t i c l e s (Figure 1.6C, arrows) which may correspond to pores i n fat droplet membranes as reported by Borchert et a l . (1967) using transmission electron microscopy. These were seen i n other samples as well. The matrix also appeared more granular than i n the soy-containing emulsions. At t h i s replacement le v e l the canola emulsions had firmer texture but were less springy than those containing soy protein. It was noted that as well as having poorer t e x t u r a l attributes, the emulsions 2 2 2.5 I I 0 20 40 60 80 100 120 2.5 I Figure 1.4. Fat particle distributions of meat emulsions containing soy or canola protein: (A) 66.7% soy vs. 66.7% canola; (B) 33.3% vs. 66.7% substitution; (C) 33.3% soy vs. 33.3% canola. 23 Figure 1.4. (cont.) (D) 33.3% vs. 66.7% soy; (E) 33.3% vs. (F) soy vs. canola substitution. 66.7% canola; Figure 1.5. 66.7% soy p r o t e i n meat emulsion cooked at 95°C for 50 min: (A) and ( B ) , l i g h t micrographs of l i p i d and p r o t e i n s ta in ing respec t i ve ly (bar=200 pim); (C) SEM micrograph (bar=100 ^ra) Figure 1.6. 66.7% canola pr o t e i n meat emulsion cooked at 95°C for 50 min: (A) and ( B ) , l i g h t micrographs of l i p i d and protein s t a i n i n g r e s p e c t i v e l y (bar=200 /un); (C) SEM micrograph (bar=100 /im). to 26 containing plant protein that were cooked at 70°C were very d i f f i c u l t to section for LM and tore e a s i l y during s t a i n i n g so were not used for deter-mination of fat p a r t i c l e d i s t r i b u t i o n s . The effect of cooking conditions on microstructure of the 66.7% canola emulsions i s seen i n Figure 1.7A which i s an SEM micrograph of a sample cooked at 70°C for 25 min and Figure 1.7B which is a sample cooked at 95°C for 50 min. The less severe cooking conditions produced a matrix with a less structured and more pasty appearance as well as poorer springiness and softer texture than the sample r e c e i v i n g the more severe heat treatment. A similar effect on microstructure and texture was also found with the 66.7% soy samples, and is probably attributable to enhanced gelation of the plant proteins under the more severe cooking con-ditio n s , as suggested by the thermorheological p r o f i l e s . A l l the fat droplet d i s t r i b u t i o n s (Figure 1 . 4 ) had similar shapes; the large droplets (greater than approximately 50 micrometers i n diameter) approximated a normal d i s t r i -bution, while below 50 micrometers the p a r t i c l e number increased i n an ess e n t i a l l y logarithmic manner. The size range of the large droplets was very similar to that reported by van den Oord and Visser (1973) and Lee (1985) for the c e l l diameter of adipose tissue. Thus i t appears as though the fat p a r t i c l e d i s t r i b u t i o n s were the resu l t of r e l a t i v e l y intact fat c e l l s as well as f i n e l y dispersed fat p a r t i c l e s that were reduced i n size by the comminution process. The microstructure of meat emulsions is influenced by such factors as the types of meat and fat, the levels of fat, moisture and salt, the com-minution process (e.g. chopping speed), the v i s c o s i t y of the emulsion, and the cooking conditions (Lee, 1985). At the 33.3% replacement l e v e l , the protein matrix of both soy and canola substituted emulsions had a t i g h t e r , less lacy appearance than at Figure 1.7. SEM micrographs of 66.7% canola protein meat emulsions cooked at: (A) 70°C for 25 rain, and (B) 95°C for 50 min (bar=20 Jim). to —] 28 the 66.7% replacement l e v e l , while the matrix of the canola emulsion again appeared somewhat more compact than the soy emulsion (Figures 1.8B.C and 1.9B.C). The fat p a r t i c l e s of the 33.3% soy and canola emulsions are shown in Figures 1.8A and 1.9A, respectively. The fat p a r t i c l e d i s t r i b u t i o n of the 33.3% substituted emulsions showed fewer p a r t i c l e s with diameters greater than 15 micrometers than the 66.7% substituted emulsions (Figure 1.4B), but a greater number of p a r t i c l e s with smaller diameters. As with the 66.7% replacement l e v e l , the 33.3% canola emulsions had a greater number of fat p a r t i c l e s with diameters less than 50 micrometers as compared to the 33.3% soy emulsions, while the 33.3% soy emulsions had more p a r t i c l e s larger than 50 micrometers i n diameter (Figure 1.4C). The 66.7% soy emulsions had more fat p a r t i c l e s between 10 and 100 micrometers i n diameter than were found i n the 33% soy emulsions (Figure 1.4D) while 66.7% canola emulsions had more pa r t i c l e s between 20 and 55 micrometers and greater than 90 micrometers than 33.3% canola emulsions (Figure 1.4E). Overall, both soy and canola emulsions had similar numbers of large p a r t i c l e s (Figure 1.4F) but canola emulsions had more p a r t i c l e s smaller than 50 micrometers i n diameter. As the fat p a r t i c l e d i s t r i b u t i o n s were estimated by a manual counting technique, only a small number of f i e l d s could be examined, which limited the r e l i a b i l i t y of the data. Recent developments i n image analysis, which allow for r a p i d c o l -l e c t i o n and processing of t h i s type of data, should greatly increase the e f f i c a c y of analyzing not only the fat p a r t i c l e d i s t r i b u t i o n but also the matrix structure of meat emulsions (Kempton et a l . , 1982; Kempton and Trupp, 1983). The effect of cooking conditions on microstructure of the 33.3% substituted emulsions was somewhat similar to that seen at the 66.7% l e v e l . Figure 1.10A shows the somewhat amorphous, pasty appearing matrix found with a Figure 1.8. 33.3% soy protein meat emulsion cooked at 95°C for 25 min: (A) and (B), light micrographs of lipid and protein staining respectively (bar=200 (im); (C) SEM micrograph (bar=100 /xm). Figure 1.9. 33.3% canola protein meat emulsion cooked at 95°C for 25 min: (A) and ( B ) , l i g h t micrographs of l i p i d and protein s t a i n i n g r e s p e c t i v e l y (bar=200 (ita); (C) SEM micrograph (bar-100 fim). to o Figure 1.10. SEM micrographs of 33.3% canola p r o t e i n meat emulsions cooked at: (A) 70°C for 25 min , and (B) 95°C for 50 min (bar=20/xm). OJ 32 33.3% canola emulsion cooked at 70°C for 25 rain compared to the more s t r u c -t u r e d appearance obtained with a 95°C, 50 min heat treatment ( F i g u r e 1.10B). It is i n t e r e s t i n g to note that greater product firmness was obtained with a 95°C cook at the 66.7% replacement l e v e l , whereas the opposite tendency was found at the 33.3% replacement l e v e l , e s p e c i a l l y with soy p r o t e i n . It would appear that at the 66.7% replacement l e v e l the f u n c t i o n a l behavior of the non-meat p r o t e i n s predominated, while at the 33.3% replacement l e v e l , the meat p r o t e i n predominated. S i e g e l et a l . (1979) suggested that i s o l a t e d soy p r o t e i n i n t e r f e r e s with the g e l - f o r m i n g i n t e r a c t i o n s between myosin mole-cu l e s . K i n g (1977) found an i n t e r a c t i o n between the 7S f r a c t i o n of soy p r o t e i n and myosin when exposed to temperatures i n the 75-100°C range, while Peng et a l . (1982a,b) r e p o r t e d an i n t e r a c t i o n between the 11S f r a c t i o n of soy p r o t e i n and myosin at temperatures greater than 85°C. Since these i n t e r -a c t ions take place at temperatures which are greater than those g e n e r a l l y used i n comminuted meat products, h i g h l e v e l s of soy p r o t e i n s p r o b a b l y act o n l y as a d i l u e n t , d e c r e a s i n g meat p r o t e i n i n t e r a c t i o n s and ge l a t i o n , and r e s u l t i n g i n s o f t e r product texture when cooked at normal p r o c e s s i n g temperatures. Alt h o u g h no r e p o r t s have been found suggesting an i n t e r a c t i o n between canola p r o t e i n s and meat pro t e i n s , the t h e r m o r h e o l o g i c a l p r o f i l e s of F i g u r e 1.2 i n d i c a t e that g e l a t i o n of meat p r o t e i n and canola p r o t e i n isolate occur i n d e p e n d e n t l y when they are combined i n a meat emulsion system. The m i c r o s t r u c t u r e of the all-meat c o n t r o l emulsions v a r i e d with p r o -c e s s i n g c o n d i t i o n s . With a 70°C, 25 min cook ( F i g u r e 1.11) the fat p a r t i c l e d i s t r i b u t i o n appeared f a i r l y s i m i l a r to that of the 33.3% replacement emul-sions, but with a s l i g h t l y more open matrix. The large l i p i d d r o p l e t s were also more oblong than i n the samples c o n t a i n i n g plant p r o t e i n , perhaps Figure 1.11. A l l -meat emulsion cooked at 70°C for 25 min: (A) and ( B ) , l i g h t micrographs of l i p i d and p r o t e i n s ta in ing respec t i ve ly (bar=200 /Am); (C) SEM micrograph (bar=100 / t in) . CO CO 34 r e f l e c t i n g t h e i n c r e a s e d v i s c o s i t y o f t h e s y s t e m a n d a n o r i e n t a t i o n e f f e c t w h e n t h e e m u l s i o n w a s s t u f f e d i n t o t h e s t a i n l e s s s t e e l t u b e s . I n t h e s a m p l e c o o k e d a t 9 5 ° C f o r 50 m i n ( F i g u r e 1 .12) t h e r e w e r e m o r e l a r g e a n d i n t e r m e d i a t e s i z e d d r o p l e t s , p e r h a p s a s a r e s u l t o f d r o p l e t c o a l e s c e n c e a n d s h r i n k a g e o f t h e m a t r i x , w i t h t h i c k s t r a n d s s u r r o u n d i n g t h e d r o p l e t s i n t h e m a t r i x . T h e s h r u n k e n a p p e a r a n c e o f t h e m a t r i x a n d p o s s i b l e d r o p l e t c o a l e s c e n c e ( a r r o w ) i s s e e n i n F i g u r e 1 .12C. T h i s s a m p l e h a d g r e a t e r f i r m n e s s a n d s p r i n g i n e s s t h a n t h e s a m p l e s h o w n i n F i g u r e 1.11 b u t a l s o h a d m u c h l o w e r y i e l d , w h i c h w a s p r o b a b l y a c o n t r i b u t i n g f a c t o r t o t h e s e e f f e c t s . D i f f e r e n c e s i n t e x t u r e a n d c o o k s t a b i l i t y d u e t o p r o t e i n s o u r c e , p r e -t r e a t m e n t , a n d c o o k i n g c o n d i t i o n s w e r e s m a l l c o m p a r e d t o t h e e f f e c t s o f r e p l a c e m e n t l e v e l . T h e s e r e s u l t s m a y b e d u e n o t o n l y t o s u p e r i o r g e l a t i o n p r o p e r t i e s o f t h e r o d - l i k e s a l t - s o l u b l e m e a t p r o t e i n s b u t a l s o as a r e s u l t o f c o a g u l a t i o n a n d g e l a t i o n o f m e a t p r o t e i n s a t t e m p e r a t u r e s l o w e r t h a n t h o s e r e q u i r e d f o r d e n a t u r a t i o n o f t h e p l a n t p r o t e i n s . T h e r e f o r e , b y t h e t i m e t h e p l a n t p r o t e i n s w e r e a b l e t o c o n t r i b u t e t o s t r u c t u r e f o r m a t i o n , t h e m a t r i x h a d a l r e a d y s e t . A n o t h e r c o n t r i b u t i n g f a c t o r m a y h a v e b e e n t h e d e c r e a s e d a p p a r e n t v i s c o s i t y o f t h e e m u l s i o n s as r e p l a c e m e n t l e v e l s i n c r e a s e d ( V o i s e y a n d R a n d a l l , 1 9 7 7 ) , w h i c h w o u l d a f f e c t t h e m o b i l i t y o f t h e f a t d r o p l e t s d u r i n g c o m m i n u t i o n a n d t h e i r r e s i s t a n c e t o t h e c u t t i n g a c t i o n o f t h e O m n i m i x e r b l a d e s . T h i s e f f e c t , a l o n g w i t h t h e t h e r m a l p r o c e s s i n g c o n d i t i o n s , m a y h a v e r e s u l t e d i n t h e o b s e r v e d d i f f e r e n c e s i n f a t d r o p l e t d i s t r i b u t i o n a n d m a t r i x a p p e a r a n c e . A s n o t e d b y F r o n i n g a n d N e e l a k a n t a n ( 1 9 7 1 ) , f o r c h i c k e n f r a n k -f u r t e r s s h o w i n g g r e a t e r t e n s i l e s t r e n g t h t h e r e w a s g r e a t e r u n i f o r m i t y i n t h e a p p e a r a n c e o f t h e f a t g l o b u l e s a n d a h e a v i e r m a t r i x o f p r o t e i n s u r r o u n d i n g t h e f a t g l o b u l e s . T h i s m a y a l s o b e a c o n t r i b u t i n g f a c t o r t o t h e t e x t u r a l d i f f e r e n c e s f o u n d b e t w e e n p r o t e i n r e p l a c e m e n t l e v e l s i n t h e p r e s e n t s t u d y . Figure 1.12. All-meat emulsion cooked at 95°C for 50 min: (A) and (B), light micrographs of lipid and protein staining respectively (bar=200 fim); (C) SEM micrograph (bar=100 fim). 36 SUMMARY AND CONCLUSIONS A model meat emulsion system was used to evaluate the effects of cooking time and temperature on texture, microstructure and cook stability of meat emulsions containing soy or canola protein isolate. The plant proteins were incorporated either dry or rehydrated at replacement levels of 33.3% and 66.7% of the meat protein, and the emulsions were cooked at 70 or 95°C for 25 or 50 min. Texture of the cooked emulsions was assessed by an instrumental texture profile analysis (TPA) using an Instron tester, while the storage modulus of emulsions and plant protein dispersions during heating was monitored with a Weissenberg Rheogoniometer. Analysis of TPA and stability data revealed several complex interac-tions between experimental variables; however, level of protein replace-ment was found to be the predominant factor, with decreased firmness and increased yield resulting from increased replacement of meat protein. Thermorheological profiles demonstrated that the development of elasti-city, as indicated by the storage modulus, during heating of all-meat emul-sions was essentially complete at 75-80°C, while the elasticity of dispersions of canola or soy protein continued to increase with heating to 95°C. The storage modulus of all samples increased upon cooling. A meat emulsion containing canola protein displayed characteristics of both the all-meat emulsion and the canola dispersion. Thus, the storage modulus continued to increase with heating to 95°C, but the increased structure formation at higher heating temperatures was not enough to compensate for an initial lower storage modulus that resulted from the loss of meat protein. 37 The microstructure of the cooked emulsions was examined by light micro-scopy and scanning electron microscopy. Although there were slight dif-ferences in the fat particle distributions of the emulsions containing plant proteins, the distributions had similar shapes, where particles larger than 50 micrometers approximated a normal distribution and were thought to be relatively intact fat cells, while the number of particles with diameters of 10-50 micrometers increased in an essentially logarithmic manner as size decreased. The microstructure of the proteinaceous matrix was affected primarily by protein source, replacement level and cooking conditions. 38 R E F E R E N C E S A c t o n , J . C , Z i e g l e r , G.R. and Burge, D.L. 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Food Technol. 38:77. 42 CHAPTER 2 GELATION AND EMULSIFICATION PROPERTIES OF UNMODIFIED AND SUCCINYLATBD CANOLA PROTEIN ISOLATE INTRODUCTION Canola is the number one oilseed crop i n Canada (Downey et a l . , 1974), and number five worldwide (Ohlson and Anjou, 1979). Although food uses of protein from Canola meal have been limited u n t i l recently, i t is p o t e n t i a l l y an important protein source. Problems i n the past with a n t i t h y r o i d a c t i v i t y have been l a r g e l y overcome by the breeding of v a r i e t i e s low i n glucosino-lates. Canola protein has a balance of essential amino acids that has been shown to be superior to any other known vegetable protein (Jones, 1979; Ohlson and Anjou, 1979). A number of reports have documented the funct i o n a l pro-p e r t i e s of canola protein products (Jones, 1980; Nakai et a l . , 1980a,b; Paulson et a l . , 1984; Sosulski et a l . , 1976; Thompson et a l . , 1982); they showed good emulsification, whipping and f a t - h o l d i n g properties, but poor gelation properties. When used i n meat emulsion systems, the quality of the f i n a l products were degraded due to a soft, mushy texture that probably resulted from poor gelation properties of the protein (Sosulski et a l . , 1977; Thompson et a l . , 1982; Chapter 1, th i s t h e s i s ) . High protein s o l u b i l i t y has been suggested to be an important requirement for optimum protein gelation (Balmaceda et a l . , 1976). At low-acid pH values such as are encountered i n meat emulsions, the s o l u b i l i t y of canola protein is r e l a t i v e l y low (Hermansson et a l . , 1974; Ohlson and Anjou, 1979; POS Corporation, personal communica-t i o n ) . 43 Chemical m o d i f i c a t i o n of p r o t e i n s with s u c c i n i c anhydride has been demonstrated to improve p r o t e i n s o l u b i l i t y , and to enhance thermal s t a b i l i t y . L i t t l e work has been r e p o r t e d , however, on the f u n c t i o n a l behavior of un-modified and s u c c i n y l a t e d p r o t e i n s under c o n d i t i o n s employed i n meat emulsion systems, and there have been few i n v e s t i g a t i o n s into the f u n c t i o n a l p r o p e r t i e s of s u c c i n y l a t e d canola p r o t e i n . The f u n c t i o n a l p r o p e r t i e s of most importance i n meat emulsion systems are r e g a r d e d to be e m u l s i f i c a t i o n and therm a l l y i nduced g e l a t i o n . The present study was undertaken to examine the g e l a t i o n and e m u l s i f i c a t i o n p r o p e r t i e s of unmodified and s u c c i n y l a t e d canola isolate over a v a r i e t y of pH c o n d i t i o n s and sodium c h l o r i d e c o n c e n t r a t i o n s , and to r e l a t e the g e l a t i o n and e m u l s i f i c a t i o n r e s u l t s to p h y s i c o c h e m i c a l and r h e o l o -g i c a l p r o p e r t i e s of the iso l a t e under the same c o n d i t i o n s . LITERATURE REVIEW 44 A. P r o t e i n S u c c i n y l a t i o n Chemical modification of proteins has been r o u t i n e l y practiced as a technique to study structure, conformation, active site residues and enzymatic mechanisms i n the f i e l d of protein chemistry, but the chemical modification of food proteins to improve f u n c t i o n a l i t y has been less well studied. Reviews by Feeney (1977), K i n s e l l a and Shetty (1979), Meyer and Williams (1977) and Shukla (1982) have documented the effects of various modifying agents and methods on functi o n a l and physicochemical properties of food proteins. Succinic anhydride is the most frequently used chemical agent for protein d e r i v a t i z a t i o n ( K i n s e l l a , 1976). Succinic anhydride along with acetic anyhydride are usually the acylating agents of choice because of their ease of use, r e l a t i v e safety, low cost, and a b i l i t y to produce modified proteins with enhanced f u n c t i o n a l i t y ( S p i n e l l i et a l . , 1975). In addition, since succinic and acetic acids are present i n the t r i c a r b o x y l i c acid cycle, their protein derivatives are less l i k e l y to be toxic compared to many other chemical modifying agents (Franzen, 1977). N-acylation of proteins with succinic anhydride at alkaline pH introduces an anionic succinate group i n the nucleophilic groups of amino acid side chains. Succinic anhydride reacts p r i m a r i l y with the epsilon-amino group of lysine but other func t i o n a l groups such as ty r o s y l (Gounaris and Perlmann, 1967), and s u l f h y d r y l (Habeeb, 1967) have also been reported to have undergone succin y l a t i o n . Rates of succinylation are inversely related to the pK of the nucle o p h i l i c groups being succinylated and therefore are influenced by the pH of the reaction environment i n addition to being affected by steric factors 45 ( K i n s e l l a and Shetty, 1979; Shukla, 1982). The hydroxyl groups of serine and threonine are weak nucleophiles and are not ea s i l y acylated i n aqueous s o l u t i o n , while acylation of h i s t i d i n e and cysteine is seldom observed since the reaction products hydrolyze i n aqueous solution ( K i n s e l l a and Shetty, 1979). Food proteins that have been succinylated include soy (Franzen and K i n s e l l a , 1976a), a l f a l f a leaf (Franzen and K i n s e l l a , 1976b), beef heart (Eisele et a l . , 1981), sunflower (Canella et a l . , 1979; K a b i r u l l a h and Wills, 1982), f i s h (Groninger, 1973; Groninger and M i l l e r , 1979), rapeseed (Thompson and Cho, 1984a,b), egg albumen (Ma and Holme, 1982), wheat (Grant.,1973), oat (Ma, 1984) and peanut (Beuchat, 1977), to mention a few. The introduction of succinate anions increases the net negative charge of the protein molecules which r e s u l t s i n altered protein conformation (Gounaris and Perlmann, 1967; Habeeb, 1967; Oppenheimer et a l . , 1967; Riordan and Vallee, 1964) and i n -creased propensity of the proteins to dissociate into subunits (Beuchat, 1977; Grant, 1973). In addition, succinylation has been reported to increase protein s o l u b i l i t y (Beuchat, 1977; Franzen and K i n s e l l a , 1976a,b; Habeeb et a l . , 1958; Oppenheimer et a l . , 1967), lower the i s o e l e c t r i c point (Franzen and K i n s e l l a , 1976a; Groninger, 1973), improve emulsification properties (Childs and Park, 1976; Franzen and K i n s e l l a , 1976a; Johnson and Brekke, 1983) and foaming properties (Childs and Park, 1976; Sato and Nakamura, 1977), increase water-holding and o i l - h o l d i n g capacities (Childs and Park, 1976), improve flavor (Franzen and K i n s e l l a , 1976b) and increase thermal s t a b i l i t y (Ma and Holme, 1982; Sato and Nakamura, 1977). Succinylated egg yolk proteins have been found to be useful i n mayonnaise and salad dressings (Evans and Irons, 46 1971) while succinylated soy protein has been patented for use i n coffee whitener formulations (Melnychyn and Stapley, 1973). The effects of succinylation of proteins on the i r n u t r i t i v e value and t o x i c i t y have not been resolved. Decreased i n v i t r o d i g e s t i b i l i t y has been reported for a number of succinylated proteins, p a r t i c u l a r l y i n the release of lysine (Chen et- a l . , 1975; Groninger and M i l l e r , 1979; Matoba and Doi, 1979) as well as protein e f f i c i e n c y r a t i o (Bjarnason and Carpenter, 1969; Creamer et a l . , 1971; Groninger, 1973). Others, however, have found only s l i g h t effects of s u c c i n y l a t i o n on i n v i t r o d i g e s t i b i l i t y (Eisele et a l . , 1981; Johnson and Brekke, 1983) and amino acid composition (Eisele et a l . , 1981), while Ma (1984) reported improved d i g e s t i b i l i t y of succinylated oat proteins perhaps as a res u l t of increased s o l u b i l i t y , dissociation and unfolding of the protein molecules making them more accessible to proteolytic enzymes. K i n s e l l a and Shetty (1979) indicated that succinylation of yeast proteins p r i o r to protein extraction protected some amino acids and did not reduce lysine concentra-tions. Franzen and K i n s e l l a (1976a), however, found l i t t l e change i n the amino acid p r o f i l e of succinylated soy protein with the exception of decreased lysine content. They suggested that t h i s effect may be minimized by decrea-sing the extent of modification and supplementing the diet with lysine, but cautioned that d e r i v a t i z e d proteins intended as fu n c t i o n a l ingredients should not be the major source of dietary protein i n a fabricated food. Eisele et a l . (1981) found that unmodified and acetylated beef heart m y o f i b r i l l a r protein had rat-PER values s l i g h t l y greater than for casein while those of succinylated protein were s l i g h t l y less than for casein, but ov e r a l l were comparable to casein as well as to lean beef (Brekke and Ei s e l e , 1981). They found no differences i n body/organ weight r a t i o s , o v e r a l l depot body fat or 47 acute toxic problems i n rats fed unmodified, acetylated or succinylated protein. They also suggested the existence of an enzyme that was capable of deacylating the anhydride moiety from the protein. Groninger and M i l l e r (1979) also noted that acetylated protein gave a better growth response than succinylated protein and concluded that the acylating agent, type of protein and extent of modification a l l influence protein u t i l i z a t i o n and n u t r i t i o n a l quality. B. Thermally Induced Gelation of Globular Proteins Food gels consist of a continuous phase of interconnected p a r t i c l e s and/or macromolecules intermingled with a continuous l i q u i d phase such as water (Powrie and Tung, 1976). Gelling agents are generally present at levels of 10% or less and form a three-dimensional matrix such that the system behaves as a soft s o l i d yet retains many properties c h a r a c t e r i s t i c of the f l u i d component. As a r u l e , to obtain gels from globular proteins requires protein concentrations an order of magnitude higher than is required for gelation of polysaccharide or gelat i n dispersions. Noncovalent bonding involved i n protein gel c r o s s - l i n k i n g includes ionic bonding between charged amino acid side chains or as salt bridges, hydrogen bonding at sp e c i f i c s i t e s , or non-specific hydrophobic interactions. The i r r e v e r s i b l e nature of some protein gels suggests the formation of covalent bonds such as d i s u l f i d e , or hi g h l y i r r e v e r s i b l e destruction of t e r t i a r y and/or quaternary structure. The types of bonds i n a protein gel vary quantitatively and q u a l i t a t i v e l y with di f f e r e n t types of protein and the gelation environment. Catsimpoolas and Meyer (1970) suggested that hydrogen and ionic bonds stablize the gel network of soy protein, although d i s u l f i d e bonds have been implicated as well ( C i r c l e 48 et a l . , 1964). Voutsinas et a l . (1983b) implicated hydrophobicity of unfolded proteins along with s u l f h y d r y l groups i n the thermal func t i o n a l properties ( t h i c k e n i n g , coagulation and gelation) of globular proteins. Si m i l a r l y , Shimada and Matsushita (1980a) suggested the involvement of d i s u l f i d e bonds and hydrophobic interactions, but not ionic attractions, i n thermally induced gelation of egg albumin. Hayakawa and Nakai (1985b) concluded that hydro-phobic i t y was involved i n both strength and c o a g u l a b i l i t y of ovalbumin and that s u l f h y d r y l groups were involved i n gelation while net charge played an important role i n coagulation. As there is a minimum protein concentration below which gelation does not occur, an effective overlapping of f u n c t i o n a l groups between adjacent protein molecules or dissociated subunits is necessary for gel formation. Lipatov and Proshlyakova (1961) stated that for dil u t e solutions the p r o b a b i l i t y of formation of intramolecular bonds is independent of concentration, whereas intermolecular bonding increases sharply with increases i n concentration. Therefore the c r i t i c a l concentration for gelation corresponds to the point at which intermolecular bonds begin to be formed i n preference to intramolecular bonds. The mechanisms of gelation of globular proteins are not yet completely understood. The most generally accepted hypothesis was proposed by F e r r y (1948) who suggested a two step mechanism which begins with an i n i t i a t i o n step involving unfolding or dissociation of the protein molecules, followed by an aggregation step i n which aggregation and association reactions occur and under appropriate thermodynamic conditions may r e s u l t i n the formation of a gel network structure. It was suggested that the rate of each step r e l a t i v e to the other influences the c h a r a c t e r i s t i c s of the gel. The slower the second step (aggregation) r e l a t i v e to the f i r s t (denaturation), the better the 49 p a r t i a l l y unfolded chains can orient themselves prior to aggregation, and the fi n e r w i l l be the gel network. Such gels would show lower opacity and greater e l a s t i c i t y and water-holding capacity than i f random aggregation and denaturation occurred simultaneously or i f aggregation occurred before denaturation. Hermansson (1978, 1979a,b) described a globular protein gel as a state intermediate between a protein sol and a p r e c i p i t a t e . If the protein concen-t r a t i o n exceeds some c r i t i c a l l e v e l , a balance between pro t e i n - p r o t e i n and protein-solvent interactions may be achieved and a gel may then be formed. Factors that affect t h i s balance such as pH, type and quantity of salt , protein concentration and heating conditions w i l l alter the c h a r a c t e r i s t i c s of the gel. Hegg et a l . (1979) studied the effects of neutral salts and pH on the thermal aggregation of ovalbumin and reported that the appearance of the heated dispersions was related to the aggregation and denaturation tempera-tures of ovalbumin under the different conditions of pH and ionic strength. Transparent gels were formed when the aggregation temperature was greater than or equal to the denaturation temperature while opaque gels, g e l - l i k e p r e c i -pitates or pr e c i p i t a t e s were progressively formed as the aggregation tempera-ture dropped below the denaturation temperature. Thus i t appeared as i f an i n i t i a l l y more denatured protein structure accompanied gel formation, i n contrast to the s i t u a t i o n that gave ri s e to p r e c i p i t a t i o n . Tombs (1970, 1974) suggested that gels are formed from globular proteins as a r e s u l t of aggregation of protein molecules to form strands followed by i n t e r a c t i o n of the strands to form the gel network. Since random aggregation of s p h e r i c a l p a r t i c l e s might be expected to lead to larger approximately sph e r i c a l p a r t i c l e s , the author proposed that gelation must resu l t from f a i r l y 50 s p e c i f i c h i g h l y oriented interactions which he likened to a " s t r i n g of beads" model. Pro t e i n molecules do not show completely random aggregation because the surfaces of protein molecules are not uniform with respect to the prob-a b i l i t y that contact w i l l lead to adhesion. Heating protein dispersions induces conformational changes which tend to increase protein-protein interac-tions. Iwabuchi and Shibasaki (1981) noted that thermally denatured proteins are generally less unfolded than chemically denatured proteins and r e t a i n regions of ordered structure. Therefore the " s t r i n g of beads" model for the aggregation process may involve only moderately unfolded and s t i l l globular protein molecules, an hypothesis that is i n agreement with microscopic data (Beveridge et a l . , 1984; Clark et a l . , 1981; Tombs, 1970, 1974). Although a great deal of work has been done on examination of the gelation behavior of soy protein, l i t t l e has been reported on gelation of c a n o l a p r o t e i n . S o s u l s k i et a l . (1976) reported that rapeseed f l o u r s , concentrates, and isolate had poor gelation properties, although concentrates and isolate showed excellent water and f a t - h o l d i n g capacity and the isolate was h i g h i n o i l emulsification and whipping c h a r a c t e r i s t i c s . Rapeseed products were superior to soybean products i n most funct i o n a l tests. Thompson et a l . (1982) also reported poor gelation properties for rapeseed protein concentrate. G i l l and Tung (1976, 1978a) examined the gelation behavior of the 12S glycoprotein f r a c t i o n of rapeseed by both r h e o l o g i c a l and micro-scopical techniques and reported gelation at protein concentrations as low as 4.5 percent with measurable thickening at 1 percent protein. Gel strength and microstructure were affected by pH and NaCl concentration where the strongest gels were formed under conditions at which both pH and ionic strength were high. Although the mechanism of gelation and the bonds involved i n gel 51 formation and s t a b i l i t y were not f u l l y elucidated, the authors concluded that some d i s u l f i d e bonding was involved but that ionic and hydrogen bonds were not l i k e l y to be major factors i n the gel c r o s s - l i n k s . Jones (1980) examined the effects of t r y p s i n and potassium linoleate on microstructure and vis c o e l a s t i c properties of heated canola isolate dispersions and reported that as heating temperature increased to 95°C, the storage modulus of 10 percent isolate dispersions generally increased due to the formation of a g e l - l i k e matrix capable of storing more of the deformation energy. Scanning electron micro-scopic examination of the heated dispersions revealed the presence of small protein aggregates of less than one micrometer i n diameter which interacted to form larger aggregates that could be observed under the l i g h t microscope i n unheated and heated dispersions. Trypsin hydrolyzed samples tended to have lower e l a s t i c i t y perhaps because of decreased aggregate size and increased s o l u b i l i t y . C. Proteins as Emulsifiers Proteins have the a b i l i t y to promote and s t a b i l i z e o i l - i n - w a t e r (o/w) emulsions. The role of proteins i n emulsification has been reviewed by Cherry et a l . (1979), H a i l i n g (1981), K i n s e l l a (1976) and McWatters and Cherry (1981), while emulsion formation has been reviewed by Gopal (1968) and emulsion s t a b i l i t y by Kitchener and Mussellwhite (1968). Proteins are able to act as emulsifying agents due to their large molecular weights and by virtue of possessing both hydrophilic and hydrophobic properties simultaneously, which enables the proteins to be adsorbed at the oil/water interface and decrease the i n t e r f a c i a l tension between the two phases. This lessens the mechanical energy required to produce a given 52 emulsion droplet size (Cante et a l . , 1979). In addition, the strength, compactness, e l a s t i c i t y , and e l e c t r i c a l properties of the i n t e r f a c i a l film around the o i l droplets influence emulsion s t a b i l i t y (Powrie and Tung, 1976). Proteins, however, are less effective than other commonly used surfactants i n r e d u c i n g i n t e r f a c i a l tension (Bennett et a l . , 1968), and p r e d i c t i o n of protein emulsifying behavior is much more d i f f i c u l t than for non-protein emulsifiers as the f u n c t i o n a l properties of a given protein are greatly influenced by the ionic environment, i n p a r t i c u l a r pH, ionic strength, the nature and valency of ions as well as the presence of other non-protein components (Cante et a l . , 1979). The amino acid composition and sequence as well as secondary, t e r t i a r y and quaternary structure help govern the e f f e c -tiveness of protein emulsifiers (Powrie and Tung, 1976). Many reports have suggested a direct r e l a t i o n s h i p between the emulsifying properties of proteins and th e i r aqueous s o l u b i l i t y (Crenwelge et a l . , 1974; K i n s e l l a , 1976; Pearson et a l . , 1965; Yasumatsu et a l . , 1972), while others have found a poor c o r r e l a t i o n between protein s o l u b i l i t y and emulsifying p r o p e r t i e s (Aoki et a l . , 1981; McWatters and Holmes, 1979a,b; Smith et a l . 1973; Wang and K i n s e l l a , 1976). Recently, protein surface hydrophobicity has received attention for i t s role i n emulsification. Keshavarz and Nakai (1979) determined the surface hydrophobicity of several proteins by chromato-graphic and p a r t i t i o n techniques and found a s i g n i f i c a n t c o r r e l a t i o n with i n t e r f a c i a l tension of the proteins studied. Kato and Nakai (1980) and Nakai et a l . (1980b) determined surface hydrophobicity by a fluorometric method and reported s i g n i f i c a n t correlations with i n t e r f a c i a l tension and emulsifying a c t i v i t y of the proteins, while Kato et a l . (1981) found that p a r t i a l de-naturation of ovalbumin and lysozyme improved thei r emulsification properties, 53 w h i c h were l i n e a r l y correlated with surface hydrophobicity. However, Voutsinas et a l . (1983a) and Li-Chan et a l . (1984) found that a balance between protein s o l u b i l i t y and surface hydrophobicity was required for optimum emulsification by both non-meat and meat proteins. Methods for i n v e s t i g a t i n g the emulsifying properties of proteins include e m u l s i f i c a t i o n c a p a c i t y , emulsification a c t i v i t y and emulsion s t a b i l i t y . E m u l s i f i c a t i o n capacity (EC) is usually defined as the maximum amount of o i l that can be emulsified by a protein solution to phase inversion under standard conditions. This method was o r i g i n a l l y devised by Swift et a l . (1961) to study the factors that influence meat emulsions, but has since been used as a general method to compare protein emulsifying properties. EC measurements, however, are not solely a property of the protein under test but also r e f l e c t factors such as speed of blending, rate of o i l addition, protein concentra-. t i o n , and type of equipment used (Pearce and K i n s e l l a , 1978; Tornberg and Hermansson, 1977). Emu l s i f i c a t i o n a c t i v i t y (EA) r e f l e c t s the a b i l i t y of a protein to form and s t a b i l i z e an emulsion (Kitchener and Mussellwhite, 1968) and is measured by determining the p a r t i c l e size d i s t r i b u t i o n of the dispersed phase either with a Coulter Counter, by microscopy or by s p e c t r o t u r b i d i t y (Walstra et a l . , 1969). A simple r e l a t i o n s h i p exists between t u r b i d i t y and the i n t e r f a c i a l area of an emulsion (Kerker, 1969). Pearce and K i n s e l l a (1978) have developed a spectroturbidimetric test for emulsification a c t i v i t y that has found wide acceptance. Although i t is not a property of the test material alone but is a property of the system as a whole, the measurement is simple, r a p i d , and t h e o r e t i c a l l y sound, and is more l i k e l y to be related to p r a c t i c a l performance than emulsification tests more commonly used. 54 Emulsions may desta b i l i z e by creaming, f l o c c u l a t i o n , and coalescence, which may occur s i n g l y or i n combination ( H a i l i n g , 1981). Methods used for determining emulsion s t a b i l i t y include separation of the phases under the influence of a gr a v i t a t i o n a l f i e l d (e.g., Yasumatsu et a l . , 1972), measuring the number and size of droplets as a function of time with a Coulter Counter (Mita et a l . , 1973), or by spectrophotometrically following the decay of t u r b i d i t y of an emulsion with time (Pearce and K i n s e l l a , 1978). 55 M A T E R I A L S A N D M E T H O D S Canola isolate (var. Tower) was obtained from the POS P i l o t Plant Corporation (Saskatoon, SK). The isolate was prepared by alkaline e xtrac-t i o n of canola meal followed by acid p r e c i p i t a t i o n , n e u t r a l i z a t i o n , and recovery of the protein by spray drying (POS Corporation, personal communica-t i o n ) . P r o t e i n content (Nx5.5; Tkachuk, 1969) was 74.9% (w.b.) as deter-mined by the micro Kje l d a h l method of Concon and Soltess (1973). A . S u c c i n y l a t i o n P r o c e d u r e Succinylation was c a r r i e d out by a procedure similar to that of Groninger (1973) and Hoagland (1966). One hundred and ten grams of canola isolate was dispersed by s t i r r i n g i n 2 L of d i s t i l l e d water at room temperature, and the pH was adjusted to 8.0 with 4N NaOH. 4.296 g of succinic anhydride (MCB Reagents, Norwood, OH) was added to the dispersion i n six approximately equal increments with constant s t i r r i n g over a 75 min time period. This amounted to 5.2% of the protein i n the dispersion. The microstructure of the d i s -persion was monitored by l i g h t microscopy and t h i s amount of succinic anhy-dride was just s u f f i c i e n t to s o l u b i l i z e protein aggregates i n the disper-sion. The pH was maintained between 8.0 and 8.5 with 4N NaOH during succiny-l a t i o n . After the pH had s t a b i l i z e d , the dispersion was dialyzed for 44 h against four changes of d i s t i l l e d water at 4°C (Spectrapor membrane tubing no. 1, Spectrum Medical Industries, Inc., Los Angeles, CA), after which the succinylated isolate was recovered by l y o p h i l i z a t i o n . This batch of succiny-lated isolate was r e f e r r e d to as "5.2% SA". A second batch was succinylated 56 i n a similar manner with 11.715 g of succinic anhydride (14.2% SA), and recovered as above. B. Determination of Extent of S u c c i n y l a t i o n The ninhydrin assay of Friedman et a l . (1984) was used to quantify the extent of s u c c i n y l a t i o n . Two mL of d i s t i l l e d water and 2 mL of lithium acetate-dimethyl sulfoxide ninhydrin reagent were added to duplicate 1-3 mg protein samples i n large test tubes. The tubes were placed i n a b o i l i n g water bath for 15 min, cooled i n an ice bath, and 6.0 mL of 50% ethanol-water were added with vortexing. The solutions were centrifuged to remove insoluble p a r t i c l e s and the absorbance of each sample was measured at 570 nm with a Carey 210 Spectrophotometer (Varian Instrument D i v i s i o n , Palo Alto, CA) against a reagent blank. The absorbance indicated the number of free amino groups available for reaction with the ninhydrin reagent, and the difference in absorbance between succinylated and unmodified proteins r e f l e c t e d the extent of s u c c i n y l a t i o n . The extent of modification of free amino groups as determined by this method was 54% for the 5.2% SA isolate and 84% for the 14.2% SA isolate. Although i t is possible to succinylate a l l nucleophilic groups on amino acid residues (Gounaris and Perlmann, 1967), the epsilon-amino group of lysine is most r e a d i l y succinylated because of i t s r e l a t i v e l y low pK and i t s strong n u c l e o p h i l i c nature (Franzen and K i n s e l l a , 1976a; Thompson and Cho, 1984a). The unmodified and succinylated isolates were examined for protein s o l u b i l i t y , surface hydrophobicity, zeta potential and rheological properties as a function of NaCl concentration (0.0M, 0.35M and 0.7M) and pH (3.5, 5.0, 6.5, 8.0, 9.5, and 11.0). 57 C. P r o t e i n S o l u b i l i t y The s o l u b i l i t y of unheated 11.4% (w/w) isolate dispersions was determined by c e n t r i f u g a t i o n of the dispersions at 27,000 x g for 30 min and analyzing the supernatant for nitrogen by micro Kjeldahl (Concon and Soltess, 1973). Pro t e i n s o l u b i l i t y was expressed as the percentage of the protein i n the dispersion that was recovered i n the supernatant. No s i g n i f i c a n t differences were found i n protein content of the isolates as a result of succinylation. The dispersions were prepared by mixing 2.8 g of isolate i n d i s t i l l e d water, adding either 0, 2.0 or 4.0 g of a 25.6% NaCl solution to make the f i n a l concentration 0.0M, 0.35M, or 0.7M NaCl, adjusting the pH with 2N NaOH or 2N HC1 and adding d i s t i l l e d water to 25 g. The pH was rechecked and i f necessary adjusted with a drop or two of 2N NaOH or 2N HC1. The dispersions were used to evaluate protein s o l u b i l i t y , steady shear r h e o l o g i c a l behavior, microstructure by l i g h t microscopy, and thermally induced gelation properties of the unmodified and succinylated isolates. D. P r o t e i n Surface Hydrophobicity P r o t e i n surface hydrophobicity ( S 0 ) was determined using l - a n i l i n o - 8 naphthalene sulfonate (ANS) as a hydrophobic probe. B r i t t o n and Robinson-type universal buffers ( B r i t t o n , 1956) were made one-quarter strength to pH 3.5, 5.0, 6.5, 8.0, 9.5 and 11.0 and contained 0.0M, 0.35M or 0.7M NaCl. Unless otherwise s p e c i f i e d , these buffers were used throughout this study for examining the effects of pH and NaCl concentration on funct i o n a l and physico-c h e m i c a l properties of the canola isolates. The isolates were s e r i a l l y d i l u t e d with buffer of the desired pH and NaCl concentration to obtain protein concentrations ranging from 0.0005 to 0.015% (w/v). Ten mi c r o l i t e r s of ANS 58 (8.0mM in 0.01M phosphate buffer, pH 7.0) were added to 2 mL of the d i l u t e d protein solutions. The fluorescence i n t e n s i t y (FI) of ANS-protein conju-gates was measured with an Aminco-Bowman Spectrofluorometer (No. 4-8202, American Instrument Co. Inc., Silver Springs, MD). E x c i t a t i o n and emission wavelengths were 390 nm and 470 nm, respectively. The FI reading was standar-dized by adjusting the spectrofluorometer reading for ANS i n methanol to 30% f u l l scale. Net FI for each d i l u t i o n was obtained by subtracting the FI of a protein blank without ANS. The slopes of the plots of net FI versus percent protein were calculated by least squares linear regression, and s p e c i f i e d as S 0. Hydrophobicity was also determined after heating 0.1% (w/v) protein dispersions i n the appropriate buffer at 72°C for 30 min. The hydrophobicity measured was that which was "exposed" by the heat treatment and was designated S e. This method d i f f e r e d from a similar determination by Townsend and Nakai (1983) and Voutsinas et a l . (1983b) i n that those investigators included sodium dodecyl sulfate i n the test solution and heated i t to a higher tempera-ture. E. Zeta P o t e n t i a l (Net Charge Density) Net charge density of the isolates was measured as zeta potential (mV) with a Laser Zee Model 501 p a r t i c l e microelectrophoresis apparatus (Pen Kem Inc., Bedford H i l l s , NY) at an applied potential difference of 150 V. Protein dispersions for zeta potential measurement were prepared by homogenizing 5 mL of a 0.1% (w/v) protein dispersion i n the appropriate buffer with 0.15 mL of 3,3'-dimethylbiphenyl ( A l d r i c h Chemical Co., Milwaukee, Wl) with a Brinkmann Polytron at 2200 r.p.m. for 20 s, and then d i l u t i n g the r e s u l t i n g emulsions 50-fold with the appropriate buffer. 59 F. Steady Shear Rheology The flow properties of unheated 11.4% isolate dispersions under steady shear were evaluated at 21°C over a shear rate range of 4.3 to 1000 s ~ l using 5 cm diameter 2 degree cone/plate fi x t u r e s with a Model R.19 Weissenberg Rheogoniometer. The instrument was c a l i b r a t e d by measuring the r e l a t i o n s h i p between shear stress and shear rate of c e r t i f i e d v i s c o s i t y standard o i l s (Cannon Instrument Co., State College, PA). The goodness of f i t , as indicated by the co e f f i c i e n t of determination ( r 2 ) from least squares linear regression, of shear stress [a, Pa), shear rate ( 7 . s - 1 ) and v i s c o s i t y (7), Pa s, where rj= oV?) data to the power-law or power-law p l a s t i c flow models for each dispersion were determined using a program written for an Apple 11+ microcomputer. The co e f f i c i e n t of determination was at least 0.99 for each f i t t e d model. The models may be stated as: Power-law: a = m 7 n (1) Power-law p l a s t i c : O-Oy = m,"yn' (2) where m (or m1) is the consistency co e f f i c i e n t (Pa s n ) , n (or n 1) i s the flow behavior index (dimensionless), and 0"y is the y i e l d stress (Pa). The y i e l d stress was estimated from the Casson Model (equation 3) by extrapolating the rheogram to zero shear rate; hence Oy = k 0 2. Casson: a 1 / 2 = ko + k i ? 1 / 2 (3) For equations 1 and 2, plots of log 0" or log ( a - Oy) versus log"? are linear, where the intercept at 1 s " l i s log m or m1 and the slope is n or n 1. Values of apparent v i s c o s i t y at shear rates of 10 s*"1 and 1000 s" 1 were calculated from the power-law or power-law p l a s t i c flow parameters. 60 G. Thermally Induced Gelation 1. Dynamic Shear Properties of Thermally Induced Gels Seven to eight grams of 11.4% isolate dispersions were heated i n capped p l a s t i c s c i n t i l l a t i o n v i a l s (i.d.=22 mm, 20 mL capacity, Fisher S c i e n t i -f i c ) for 30 min i n a 72°C water bath. The samples were cooled under cold running tap water and then allowed to equilibrate to room temperature for approximately two hours. The samples that formed gels were i n i t i a l l y examined for "gel strength" using a puncture test (described i n Chapter 3). The dynamic vi s c o e l a s t i c properties of the gels were then obtained using the Weissenberg Rheogoniometer equipped with 5 cm diameter p a r a l l e l plate fixtures at a gap thickness of 1 mm. The gels were c a r e f u l l y removed from the v i a l s , undisturbed portions were s l i c e d to a thickness of s l i g h t l y greater than 1 mm, and placed on the bottom platen. The top platen, supported by a no. 7 torsion bar (9.4 Pa cm3 fim~l), was then c a r e f u l l y lowered to a gap thickness of 1 mm to avoid air pockets between the platens. Evaporation of water from the sample was avoided by applying a t h i n layer of silicone o i l to the exposed edge of the gel. A small s i n u s o i d a l l y varying o s c i l l a t o r y s t r a i n of maximum amplitude of 1.88% was imposed over a frequency (CO ) range of 0.19 to 19 s~*. The amplitudes of the input (strain) and output (stress) voltage signals, and the phase difference between them, were monitored with a Tronotec Model 703A d i g i t a l phasemeter. From these data, values of the storage modulus (G 1, Pa; a measure of the energy stored e l a s t i c a l l y per cycle of sinusoidal deformation), loss modulus (G", Pa; the energy dissipated as heat per c y c l e ) , loss tangent (the tangent of the phase angle between the stress and s t r a i n waves and n u m e r i c a l l y equal to G"/G', thus r e f l e c t i n g the r e l a t i v e proportions of viscous to el a s t i c components of the samples) and the dynamic v i s c o s i t y ( f]\ 61 Pa s where r/'=G"/OJ) were calculated from the equations of Walters (1968) using a program written for an Apple 11+ microcomputer. As plots of G' or 77' versus co were linear on logarithmic coordinates, the slope and intercept of each line were determined by least squares linear regression. Values of G*, G" and loss tangent at a frequency of 10 s""* were calculated from the f o l -lowing equations: G' = aco b (4) where a (storage c o e f f i c i e n t ) is the intercept (at co=l s " l ) and b (storage index) is the slope of a log-log plot; and, rj l = c co d _ 1 (5) where c i s the dynamic shear consistency coeffi c i e n t (Pa sd) and d is the dynamic shear flow behavior index. 2. P r o t e i n Content of Gel Exudate The gels were centrifuged at 27,000 x g for 30 min; the protein i n the exudate was determined as for the unheated dispersions and expressed as a percentage of the t o t a l gel protein. H. E m u l s i f i c a t i o n A c t i v i t y and Emulsion S t a b i l i t y E m ulsification a c t i v i t y (EA) was determined i n a manner similar to the method of Pearce and K i n s e l l a (1978). Four m i l l i l i t e r s of a 0.5% (w/v) protein dispersion i n buffer of the desired pH and NaCl concentration and 4 mL of corn o i l (Fisher S c i e n t i f i c ) were homogenized i n a Sorvall Omnimixer with a micro-attachment assembly at 1880 r.p.m. for 1 min. A 50 m i c r o l i t e r sample was immediately taken from the bottom of the container and d i l u t e d i n 10 mL of buffer containing 0.1% (w/v) sodium dodecyl sulfate. Subsequent aliquots were 62 removed i n a similar manner at appropriate time int e r v a l s as determined by preliminary experimentation. The absorbance of the d i l u t e d emulsions at 500 nm was measured with a Spectronic 20 spectrophotometer (Bausch and Lomb, Rochester, NY). The i n i t i a l A500 measurement was taken to be the emulsi-f i c a t i o n a c t i v i t y , while emulsion s t a b i l i t y (ES) was defined as the time i n minutes required for A50Q to decrease to one-half that of the emulsion at zero time. I. L i g h t Microscopy The unheated 11.4 percent isolate dispersions were observed under a Wild M-20 microscope equipped with a Pentax ME 35mm camera. Unstained aliquots of the dispersions were examined under phase contrast and bright f i e l d Kohler illu m i n a t i o n . Photographic images were recorded on Kodak Plus-X black and white film and developed with Microdol-X developer. J . S t a t i s t i c a l Analyses The effects of succinylation, pH and NaCl concentration on protein s o l u b i l i t y , hydrophobicity, zeta potential, apparent v i s c o s i t y , emulsification a c t i v i t y and emulsion s t a b i l i t y were analyzed by a trend comparison procedure based on single degree of freedom orthogonal polynomial contrasts for the treatments. With quantitative treatments as used i n the present study, the types of mean comparison techniques that focus on the s p e c i f i c treatments tested are not adequate; instead, a more appropriate approach i s to examine the f u n c t i o n a l r e l a t i o n s h i p between treatment and response that covers the entire range of the treatment levels tested (Gomez and Gomez, 1984). For each treatment, a set of mutually orthogonal single degree of freedom contrasts was 63 constructed where the f i r s t contrast represented the f i r s t degree polynomial (lin e a r ) while the second contrast represented the second degree (quadratic) polynomial. Although the degree of the polynomial that can be tested may be up to one less than the number of treatment levels (i.e. up to quadratic for succinylation or NaCl concentration and up to quintic for pH) the quadratic polynomial was the maximum degree that was examined. The orthogonal m u l t i -p l i e r s for the treatment contrasts were determined as outlined by Gomez and Gomez (1984) and are presented i n Table 2.1. The linear and quadratic contrasts for each treatment and their interactions were analyzed for s i g n i -ficance by forward stepwise multiple regression (Kleinbaum and Kupper, 1978) using the MIDAS s t a t i s t i c a l computer program (Fox and Guire, 1976) available on the UBC Amdahl 470/V8 mainframe computer. In addition, backward stepwise multiple regression analyses were used to examine the effects of protein s o l u b i l i t y , hydrophobicity, zeta potential and steady shear r h e o l o g i c a l properties on emulsification a c t i v i t y , emulsion s t a b i l i t y , and dynamic shear parameters of thermally induced gels. Table 2.1. Orthogonal m u l t i p l i e r s f o r trend com-parison analyses Orthogonal Polynomial C o e f f i c i e n t Treatment Linear Quadratic A. Succinylation (% Modification) 0 -23 5 54 4 -14 84 19 9 B. NaCl (M) 0.0 -1 1 0.35 0 -2 0.7 1 1 2: PJL 3.5 -5 5 5.0 -3 -1 6.5 -1 -4 8.0 1 -4 9.5 3 -1 11.0 5 5 65 RESULTS AND DISCUSSION A. P r o t e i n S o l u b i l i t y P r o t e i n s o l u b i l i t y was s i g n i f i c a n t l y affected by the linear and quadratic effects of succinylation and pH as well as interaction effects between these variables and an i n t e r a c t i o n effect of NaCl and pH (Table 2.2). Succinylation markedly enhanced protein s o l u b i l i t y at alkaline and low acid pH (Figure 2.1). The s o l u b i l i t y p r o f i l e s of the succinylated isolates were similar to those of succinylated f i s h , leaf, and soy protein (Shukla, 1982). The unmodified isolate without NaCl exhibited a gradual increase i n s o l u b i l i t y above and below pH 5. This was similar to the s o l u b i l i t y p r o f i l e of the s t a r t i n g meal (POS Corporation, personal communication). Canola isolate is a complex mixture of proteins with a wide range of molecular weights and i s o e l e c t r i c points. Lonnerdal et a l . (1977) found at least nineteen d i s t i n c t protein fractions with i s o e l e c t r i c points from pH 4.5 to pH 10 i n a rapeseed isolate where the protein was extracted i n the presence of sodium hexametaphosphate to increase y i e l d . They also found that an isolate derived from alkaline extraction of defatted rapeseed meal followed by acid n e u t r a l i z a t i o n was r i c h i n acidic proteins and contained a l l of the high molecular weight proteins from the meal (pI=4-7). Approximately twenty to f o r t y percent of the rapeseed proteins have a low molecular weight (about 13,000 D) and an i s o e l e c t r i c point close to pH 11 (Lonnerdal and Janson, 1972). The other rapeseed proteins can be divided into three groups with molecular weights of 50,000 to 75,000, 150,000 and 320,000 with i s o e l e c t r i c points spread out i n the i n t e r v a l from pH 4 to 8 (Lonnerdal, 1975). Other authors have reported on the f r a c t i o n a t i o n , i s o l a t i o n , and physicochemical 66 Table 2 . 2 . Trend comparison a n a l y s i s of the e f f e c t s of s u c c i n y l a t i o n , pH and NaCl on p r o t e i n s o l u b i l i t y of canola I s o l a t e . Dependent Variable Independent Variable C o e f f i c i e n t F-prob. Protein S o l u b i l i t y S uccinylation 10.652 0.0000 R 2 = 0.905 S u c c i n y l a t i o n 2 -3.093 0.0034 S.E.a = 11.724 PH 15.275 0.0000 F-prob. = 0.0000 pH 2 -5.406 0.0000 n - 54 Succinylation x pH 4.319 0.0001 Succiny l a t i o n x pH 2 -5.559 0.0000 NaCl x pH -2.293 0.0265 Constant 35.363 aStandard er r o r of estimate. 100 90 80 70 60 50 40 30 20 10 0 100-90-SO 70-60 50 40 30 20 10 0 100 90 80 70 60 ein s o l u b i l i t y of 11.4% canola isolate dispersions: unmodified; (B) 5.2% SA; (C) 14.2% SA. 68 and f u n c t i o n a l properties of various protein fractions (e.g., Bhatty et a l . , 1968; Finlayson et a l . , 1969; G i l l and Tung, 1976, 1978a,b; Goding et a l . , 1970; MacKenzie, 1975). By contrast, protein isolates from most other oilseeds contain only a few proteins and usually display a s o l u b i l i t y p r o f i l e where the s o l u b i l i t y is low at the i s o e l e c t r i c point but increases r a p i d l y with pH on either side of the p i . In addition to protein, canola isolates also may contain RNA, acidic polysaccharides, phytic acid and a c i d i c poly-phenols extracted from the meal (Aman and G i l l b e r g , 1977). Succinylation has t y p i c a l l y been found to increase protein s o l u b i l i t y , alter protein conformation by promoting unfolding and increasing dissociation of subunits as well as s h i f t i n g the i s o e l e c t r i c point to lower values. The altered conformation of succinylated proteins results from the replacement of short-range attractive forces (ammonium, carboxyl) with short-range repulsive forces (succinate carboxyl, native carboxyl) (Habeeb et a l . , 1958). The combination of i n t r a and intermolecular charge repulsion promotes protein unfolding and produces fewer protein - p r o t e i n and more protein-water i n t e r -actions, with the result that aqueous s o l u b i l i t y is enhanced. As net negative charge is proportional to the extent of succinylation, s o l u b i l i t y of canola isolate increased as the number of succinylated groups increased, although the sli g h t increase i n o v e r a l l s o l u b i l i t y with 84% modification of amino groups as compared to 54% modification demonstrated that exhaustive succinylation was not required to s u b s t a n t i a l l y improve s o l u b i l i t y . A similar effect was noted by Franzen and K i n s e l l a (1976a) with soy protein. There also appeared to be a s l i g h t sh i f t of the i s o e l e c t r i c region to a lower pH with increased s u c c i n y l a -t i o n , which may have implications for the successful employment of succiny-lated proteins i n low-acid foods such as comminuted meat systems. 69 The effect of NaCl on the unmodified isolate was to increase protein s o l u b i l i t y ( s a l t i n g - i n ) i n the acidic region and decrease s o l u b i l i t y ( s a l t i n g -out) in the alkaline region (Figure 2.1A). These res u l t s are similar to those of Rhee et a l . (1972) and McWatters and Holmes (1979a) for peanut protein, and McWatters and Holmes (1979b) for soy protein. Schut (1976) suggested that NaCl causes a s h i f t i n the i s o e l e c t r i c point to a more acidic pH as a result of s p e c i f i c ion binding effects. Since inorganic anions are bound to proteins more strongly than inorganic cations due to th e i r smaller hydrated r a d i i , anions are able to attain a closer proximity to the protein molecule and are able to "screen" the charged groups of the proteins more e f f e c t i v e l y than cations, the effectiveness being i n accordance with the Hofmeister series. Thus, with the addition of NaCl and the selective binding of the chloride anions, the protein would have an excess of negative charges at the pH of the o r i g i n a l i s o e l e c t r i c point and more acid is therefore needed to reach the new i s o e l e c t r i c point. Although not s p e c i f i c a l l y alluded to, the effects of NaCl on the protein s o l u b i l i t y p r o f i l e s of peanut protein (McWatters and Holmes, 1979a; Rhee et a l . , 1972) and soy protein (McWatters and Holmes, 1979b) appeared to support such an hypothesis. Although t h i s also appeared to be the case i n the present study, the hypothesis could not be f u l l y v e r i f i e d by the s o l u b i l i t y p r o f i l e s alone as they did not go far enough into the acid pH region. The microelectrophoretic mobility data (Section B) however, tends to support t h i s proposal. For succinylated canola isolate, NaCl increased s o l u b i l i t y at pH 5 and decreased s o l u b i l i t y i n the alkaline pH region, but at pH 3.5 there was v i r t u a l l y no effect on s o l u b i l i t y . Although protein s o l u b i l i t y at pH less than 3.5 was not determined, several reports have demonstrated a f a i l u r e of 70 succinylated protein to r e s o l u b i l i z e at pH values more ac i d i c than the i s o e l e c t r i c point (e.g., Chen et a l . , 1975; Hoagland, 1966; Oppenheimer et a l . , 1967). Franzen and K i n s e l l a (1976a) attr i b u t e d t h i s effect to an i n s u f f i c i e n t number of hydrophilic cationic groups remaining after succiny-l a t i o n to overcome aggregating forces at the p l . B. Hydrophobicity and Zeta P o t e n t i a l The treatment effects on surface hydrophobicity ( S Q ) and zeta potential (z.p.) of canola isolate are shown i n Table 2.3 and Figures 2.2 and 2.3, respectively. S 0 decreased i n a lin e a r manner with extent of succinylation while the effect of NaCl varied with pH. At pH 3.5 and 5 for the unmodified isolate and pH 3.5 for the succinylated isolate, NaCl decreased hydrophobicity while the opposite effect was found at higher pH values. The o v e r a l l pH effect was to decrease S Q i n a c u r v i l i n e a r manner as pH increased. S Q decreased r a p i d l y from pH 3.5 to 6.5 and more slowly thereafter. Ghosh et a l . (1974) found a similar pH effect on the fluorescence of human serum low-density lipoprotein:ANS complexes. The hydrophobicities of heated d i s -persions ( S e ) were ove r a l l only s l i g h t l y higher than S D (Table 2.4). This d i d not necessarily mean that heating produced l i t t l e change i n protein confor-mation, however, as Franks and Eagland (1975) noted that r e l a t i v e l y few peptide residues need to be exposed to the solvent i n order to render the native conformation of the protein unstable. Zeta potential became more electronegative i n a linear manner with su c c i n y l a t i o n as a re s u l t of the increasing number of succinate carboxyl groups on the protein molecules. Although the effects of pH and NaCl were interdependent, the o v e r a l l pH effect was to increase electronegativity i n a 71 Table 2.3. Trend comparison analyses of the e f f e c t s of s u c c i n y l a t i o n , pH and NaCl on surface hydrophobicity ( S 0 ) and seta p o t e n t i a l of canola i s o l a t e (n=54). Dependent Variable Independent Variable Coefficient F-prob. Hydrophobicity (S D) Succinylation -1.282 0.0025 R2 = 0.891 PH -32.767 0.0000 S.E.a - 51.336 pH2 19.085 0.0000 F-prob. = 0.0000 NaCl x pH 7.954 0.0026 NaCl x pH2 -7.002 0.0036 Constant 157.070 Zeta Potential Succinylation -0.226 0.0000 R2 = 0.943 NaCl 10.156 0.0000 S.E. = 4.790 NaCl2 -3.402 0.0000 F-prob. = 0.0000 pH -3.418 0.0000 pH2 1.419 0.0000 NaCl x pH 1.905 0.0000 NaCl x pH2 -1.042 0.0000 NaCl2 x pH2 -0.601 0.0001 Constant -23.135 aStandard error of estimate. 600 A igure 2.2. Surface hydrophob ic i t y ( S 0 ) of canola isolate: (A) unmodi f ied; (B) 5.2% SA; (C) 14.2% SA. igure 2.3 Zeta potential of canola isolate: (A) unmodified; (B) 5.2% SA; (C) 14.2% SA. Table 2.4. Effects of succinylation, pH, NaCl and heating on surface hydrophobicity of canola Isolate. NaCl Concentration (Molar) Succinic 0. 0 0.35 0. 7 Anhydride (%) pH S b So S e So Se 0 3. 5 533.6 586.0 457.5 505.4 440.3 482.0 5. 0 263.3 249.5 222.9 230.1 236.4 214.8 6. 5 81.6 112.6 146.9 151.7 160.5 146.5 8. 0 68.0 78.3 133.3 139.8 126.2 145.6 9. 5 57.3 78.2 129.3 111.8 122.2 129.8 11. 0 33.4 32.6 62.8 69.5 91.7 85.4 5.2 3. 5 578.5 605.7 429.0 461.3 382.2 477.3 5. 0 105.8 119.4 144.6 168.1 202.2 149.3 6. 5 52.8 49.5 109.3 103.9 125.4 136.1 8. 0 34.6 43.1 80.5 96.1 97.1 116.4 9. 5 37.9 37.3 82.2 80.1 78.3 106.9 11. 0 21.9 16.8 66.7 59.7 83.7 77.8 14.2 3. 5 520.9 609.5 433.4 480.4 331.2 340.9 5. 0 77.5 117.3 143.4 145.5 144.8 138.3 6. 5 36.6 37.7 73.9 83.0 106.8 108.6 8. 0 23.2 20.2 65.7 78.0 103.4 103.6 9. 5 25.2 22.5 61.7 69.0 89.9 92.2 11. 0 21.1 15.4 71.3 58.5 73.0 63.9 aHydrophobicity of unheated dispersions. ^Hydrophobicity of heated dispersions. 75 c u r v i l i n e a r manner, not unlike the effect of pH on S Q. The ov e r a l l effect of increasing NaCl concentration to 0.35M was to decrease zeta potential r a p i d l y , with l i t t l e change i n zeta potential between 0.35M and 0.7M NaCl. This was not unexpected as at univalent electrolyte concentrations of approximately 0.1 molal, the thickness of the e l e c t r i c double layer surrounding c o l l o i d a l p a r t i c l e s , (to which the zeta potential i s d i r e c t l y r e l a t e d ) , i s suppressed and becomes of negl i g i b l e proportions. This res u l t s i n marked changes i n the properties of f l e x i b l e macromolecules such as proteins (Franks and Eagland, 1975). Both succinylation and NaCl appeared to lower the i s o e l e c t r i c point; i.e., the pH at which the zeta potential was zero shif t e d to a more ac i d i c pH as suc c i n y l a t i o n and NaCl concentration increased. In aqueous solution, the charged groups of protein molecules are at-tracted to the aqueous phase while nonpolar (hydrophobic) groups tend to avoid the aqueous phase and become displaced toward the center of the mole-cule. However, due to the proportion of hydrophobic to hydrophilic residues as well as constraints imposed by the amino acid sequence, i t is not possible for a l l hydrophobic groups to be buried i n the in t e r i o r of the molecule and those hydrophobic residues forced to remain on the surface contribute to the surface hydrophobicity of the molecule. Hydrophobic interactions i n proteins also serve to bring together groups that can participate i n hydrogen or ionic bonding i n the absence of water, thus each of the types of bonding aids in formation of the others i n determining protein conformation (White et a l . , 1973). Charged c o l l o i d a l p a r t i c l e s such as protein molecules i n an ionic environment are surrounded by an e l e c t r i c a l double layer of ions; an inner region of strongly adsorbing counterions and an outer region where the ions 76 are d i f f u s e l y d i s t r i b u t e d according to a balance between e l e c t r i c a l forces and random thermal motion (Dickinson and Stainsby, 1982). The zeta potential r e f e r s to the e l e c t r i c a l p o t ential at the surface of shear between the inner "Stern" layer and the diffuse outer layer (Powrie and Tung, 1976). The DLVO (Derjaguin-Landau-Verwey-Overbeek) theory of c o l l o i d a l s t a b i l i t y relates the s t a b i l i t y of a c o l l o i d a l suspension to a balance between van der Waals attr a c t i v e forces and e l e c t r o s t a t i c repulsive forces (from e l e c t r i c a l forces of i d e n t i c a l s i g n ) . When the double layer is th i c k and repulsive forces exceed attractive forces, f l o c c u l a t i o n w i l l be resisted and the suspension w i l l be stable. The addition of neutral salts, however, decreases the effective radius of the ionic double layer with a lowering of the zeta potent i a l . The effect of ions on the double layer depends on thei r valency and concentration. With monovalent counterions such as sodium or potassium, the double layer w i l l be t h i c k because of the low charge to volume r a t i o of the ions, and the shear plane w i l l l i e i n a region of high potential. With multivalent ions, the charge to volume r a t i o i s much larger and most of the counterion charge w i l l l i e within the shear plane, which w i l l therefore be i n a region of much lower potential than for monovalent ions. Since the added salt does not affect the van der Waals attractive forces, the repulsive energy b a r r i e r s between c o l l o i d a l p a r t i c l e s is lowered or lost and f l o c c u l a t i o n may r e s u l t . The Schulze-Hardy r u l e describes the minimum concentration of ions necessary to cause f l o c c u l a t i o n as being approximately proportional to the s i x t h power of the charge and is almost independent of the ionic species (Dickinson and Stainsby, 1982). Therefore, far fewer multivalent ions than monovalent ions are required to cause f l o c c u l a t i o n . 77 Melander and Horvath (1977) and Salahuddin et a l . (1983) discussed the i n t e r p l a y of e l e c t r o s t a t i c and hydrophobic interactions which result i n s a l t i n g - i n and salting-out of proteins i n solution. They suggested an inverse r e l a t i o n s h i p between the r e l a t i v e surface hydrophobicity and the frequency of charged groups. As the charged groups are assumed to be e x c l u s i v e l y on the protein surface, a higher frequency of charged groups is l i k e l y to result i n a lower frequency of non-polar groups on the surface, with a resultant decrease i n surface hydrophobicity with increasing charge frequency. Thus with increased succinylation, the charge frequency and e l e c t r o n e g a t i v i t y increased and the surface hydrophobicity decreased. In the presence of NaCl, however, the effective charge frequency on the protein molecules dropped with a resultant increase i n the number of hydrophobic groups exposed at the sur-face. Intermolecular hydrophobic interactions between exposed hydrophobic amino acid residues of protein molecules are therefore l i k e l y to be important in salt-induced p r o t e i n - p r o t e i n interactions. For canola isolate under the conditions of succinylation, pH and ionic strength of the present study, S Q and zeta potential were related by the quadratic relationship: S D = 14.552 z.p. + 0.158 z.p.2 + 355.54 (R 2= 0.901, p<0.001, n=54) (6) where S Q increased slowly i n i t i a l l y and then more r a p i d l y as elec t r o n e g a t i v i t y decreased. Hydrophobicity was measured with the fluorescent probe ANS which is composed of aromatic ri n g s . Hayakawa and Nakai (1985a) suggested that protein hydrophobicity may be c l a s s i f i e d into two hydrophobicities, aromatic and a l i p h a t i c , as influenced by aromatic and a l i p h a t i c amino acid residues, respectively. These may be related i n different ways to protein f u n c t i o n -78 a l i t y . Li-Chan et a l . (1985) found that both aromatic and a l i p h a t i c hydropho-b i c i t i e s were s i g n i f i c a n t predictors of emulsifying and f a t - b i n d i n g properties of salt soluble muscle proteins, but Hayakawa and Nakai (1985b) found no difference between the two types of hydrophobicity measurements i n r e l a t i o n to the thermal properties of ovalbumin. In the present study, only ANS hydro-phobic i t y was measured, as d i f f i c u l t y was experienced i n obtaining hydropho-b i c i t y measurements with the a l i p h a t i c hydrophobicity probe c i s - p a r i n a r i c acid (CPA) at pH 5 and lower. Similar d i f f i c u l t y was experienced by Li-Chan et a l . (1985) who suggested that hydrophobicity measurements with t h i s probe at low pH must be interpreted with caution since CPA was found to become less soluble at pH values below 5. The quantum y i e l d of the ANS probe is insensitive to pH i n this range (Gibrat and Grignon, 1982), however, so ANS fluorescence was used throughout this study as an index of hydrophobicity. C. Steady Shear Rheology and Microstructure A l l dispersions followed power-law or power-law pl a s t i c flow behavior (Table 2.5). The dispersions were pseudoplastic (shear rate thinning) as indicated by a flow behavior index (n) less than 1.0 for each dispersion. As the shear rate increased, the p a r t i c l e s may have aligned with the shear planes and offered less resistance to flow, thereby e x h i b i t i n g decreased v i s c o s i t y . The apparent v i s c o s i t i e s of each dispersion at 10 s - 1 and 1000 s~* are shown in Figures 2.4 and 2.5, respectively. The succinylated isolates had higher apparent v i s c o s i t i e s than the unmodified isolate under nearly a l l conditions of pH and ionic strength. The treatment effects on apparent v i s c o s i t y at 10 s" 1 and 1000 s" 1 are presented i n Table 2.6. At 10 s - 1 the apparent v i s c o s i t y increased with succinylation and pH, while at 1000 s" 1, interactions between Table 2.5. Power-law and power-law p l a s t i c flow parameters of 11.4Z canola I s o l a t e dispersions. NaCl C o n c e n t r a t i o n ( M o l a r ) 0 .0 0 .35 0 .7 S u c c i n i c A nhydride m o r m' n cy m or m' n (jy m o r m' n Oy (%) pH (mPa s n ) o r n' (mPa) (mPa s n ) o r n' (mPa) (mPa s n ) o r n' (raPa) 3.5 6 .2 0 .943 0 7.4 0 .921 0 18 .3 0 .790 0 5.0 4 7 . 3 0 .693 168 22 .4 0 .813 0 34 . 2 0 .763 0 6 .5 23 .2 0 .865 51 57 .6 0 .733 206 8 9 . 5 0 .699 476 8 .0 33 . 1 0 .917 0 59 .6 0 .747 196 8 5 . 3 0 .722 407 9 .5 160 .2 0.777 0 26 .6 0 .909 24 4 0 . 1 0 .819 76 11 .0 1280 0 .506 0 553 .6 0 .590 0 245 . 8 0 .694 1486 3.5 4 3 . 2 0 .718 153 43 . 6 0 .710 159 28 . 9 0 .740 73 5.0 53 .1 0 .853 0 861 . 0 0 .570 0 301 . 1 0 .677 0 6 .5 228 .3 0 .721 0 567 .8 0 .604 0 608 . 1 0 .589 0 8 .0 358 .2 0 .688 0 320 .4 0 .667 0 353 .4 0 .655 0 9 . 5 410 . 8 0 .670 0 329 .6 0 .667 0 4 0 5 . 5 0 .648 0 11 .0 1269 0 .563 0 1268 0 .512 0 1215 0 .510 0 3 .5 41 . 2 0 .683 322 45 . 8 0 .690 186 59 . 9 0 . 685 267 5.0 905 .2 0 .601 0 1423 0 .539 0 1475 0 .532 0 6 .5 197 .2 0 .755 0 379 .6 0 .643 0 706 .7 0 .566 0 8 . 0 265 . 3 0 .758 0 279 .2 0 .698 0 275 . 9 0 .698 0 9 .5 279 . 0 0 .754 0 202 .9 0 .732 0 320 . 0 0 .665 0 11 .0 1191 0.597 0 1083 0 .543 0 873 . 8 0 .561 0 F i g u r e 2.4. Apparent v i s c o s i t y at 10 s"*1 for 11.4% canola isolate dispersions: (A) unmodified; (B) 5.2% SA; (C) 14.2% SA. 80 70 60 50 40 30 20 10 0 80 70 60 50 40 SO 20 10 0 80 70 60 50 40-30 20 10-0 Legend A OM NoCI O 0.35M NaCl 0.7M NoCI 1 1 1 I 1 1 1 I 4 5 6 7 8 9 10 11 Legend & OM NoCI -I 1 I I 1 1 1 r 4 5 6 7 8 9 10 11 -I 1 I I 1 I 1 I 4 5 6 7 8 9 10 It P H v iscos i t y at 1000 s " 1 fo r 11.4% canola isolate : (A) unmodi f ied; (B) 5.2% SA; (C) 14.2% SA. 82 Table 2.6. Trend comparison analyses of the e f f e c t s of s u c c i n y l a t i o n , pH and NaCl on apparent v i s c o s i t y of 11.AZ canola i s o l a t e dispersions (n=54). Dependent Variable Independent Variable Coefficient F-prob. Apparent Viscosity (10 s" I) Succinylation 3.475 0.0003 R2 = 0.404 pH 19.986 0.0001 S.E.a - 113.91 Constant 166.230 F-prob. = 0.0000 Apparent Viscosity (1000 s~l) Succinylation 0.566 0.0000 R2 = 0.682 pH 2.934 0.0000 S.E. = 10.665 Succinylation x pH2 -0.0504 0.0283 F-prob. = 0.0000 NaCl x pH -1.294 0.0164 Constant 28.530 aStandard error of estimate. 83 succinylation and the square of pH and between NaCl and pH were sig n i f i c a n t along with succinylation and pH as main effects. K i n s e l l a and Shetty (1979) reported increased v i s c o s i t y with s u c c i n y l a t i o n of arachin and concluded that a decrease i n v i s c o s i t y expected with protein dissociation by succinylation was more than counterbalanced by unfolding of the dissociated components and the resultant increase i n hydrodynamic volume. In extremely d i l u t e dispersions the t o t a l v i s c o s i t y effect i s simply the sum of the effects caused by each of the i n d i v i d u a l suspended p a r t i c l e s , but as the concentration of the dispersed phase increases, the effects of the suspended p a r t i c l e s are no longer independent. The flow behavior of concen-trated protein dispersions r e f l e c t s complex interactions between protein and solvent as well as p r o t e i n - p r o t e i n interactions as influenced by p a r t i c l e size, shape and hydrodynamic interactions. The apparent v i s c o s i t y of protein dispersions may r i s e exponentially with concentration (Tung, 1978). F r i s c h and Simha (1956) suggested that the hydrodynamic volume and shape of the protein are the most important factors governing the flow property of the protein. The hydrodynamic volume is dependent upon the molecular size and the degree of hydration of the molecule i n solution. P r o t e i n i n solution can exist i n a wide range of sizes from single molecules to large aggregates. Since the degree of aggregation affects the hydrodynamic or effective volume of the protein, i t greatly affects the rheological properties of protein dispersions. Lee and Rha (1979) reported that the p a r t i c l e size d i s t r i b u t i o n of soy protein dispersions affected the hydrodynamic volume and v i s c o s i t y of the dispersion; dispersions with larger p a r t i c l e s were higher i n v i s c o s i t y than dispersions with small p a r t i c l e s . They suggested that the size and shape of the protein aggregates were l a r g e l y determined by hydrophobic effects. 84 Thus such factors as protein s o l u b i l i t y , the size, shape and number of aggre-gates, hydrodynamic volume, protein-solvent, and p r o t e i n - p r o t e i n interactions a l l contribute to the flow properties of protein dispersions under steady shear. For the unmodified isolate, l i g h t micrographs revealed that the disper-sions were l a r g e l y composed of r e l a t i v e l y spherical aggregates which ranged i n size from the limit of resolution of the l i g h t microscope to more than 100 micrometers i n diameter (Figure 2.6A-D). In view of the high protein concen-t r a t i o n of the isolate, the aggregates were assumed to be mostly protein. Jones (1980) reported a similar microstructure for canola isolate prepared either with or without potassium linoleate or t r y p s i n . Wolf and Baker (1980) found that isolated soy protein was l a r g e l y composed of whole, broken and collapsed roughly spherical protein bodies. Beveridge et a l . (1984) noted that the protein bodies of the soy isolate Promine-D did not dissolve com-pl e t e l y i n water and Promine-D gels had very large p a r t i c l e s (up to 100 micro-meters i n diameter) embedded i n a gel matrix. At 10 s ~ l the apparent v i s c o s i t y of the dispersions with NaCl was low up to pH 9.5, while the dispersions without NaCl increased i n v i s c o s i t y from pH 8 to 11. At pH 6.5 and 8, the higher apparent v i s c o s i t i e s of the dispersions with NaCl r e f l e c t e d the y i e l d stress of these dispersions which affected the apparent v i s c o s i t i e s at low rates of shear. At 1000 s - 1 , however, the effect of y i e l d stress was n e g l i g i b l e . The increase i n apparent v i s c o s i t y at both 10 s " l and 1000 s " 1 as the pH increased to 11 was probably a r e s u l t of increased protein s o l u b i l i t y and swelling of the aggregates at t h i s pH. In addition, Ishino and Okamoto (1975) suggested that the increased v i s c o s i t y of soy protein dispersions at high pH may have been caused by unfolding, dissociation and i n t e r a c t i o n of the protein molecules. 85 Figure 2.6. A-D, Light micrographs of 11.4% dispersions of unmodified canola isolate: (A) pH 3.5, 0.0M NaCl (BF); (B) pH 9.5, 0.0M NaCl (PC); (C) pH 11.0, 0.0M NaCl (PC); (D) pH 11.0, 0.7M NaCl (PC); BF=bright field, PC=phase contrast, bar=200 (im. 86 Figure 2.6. (cont.) E-H, Light micrographs of 11.4% dispersions of succinylated canola isolate (5.2% SA): (E) pH 3.5, 0.0M NaCl (BF) ; (F) pH 5.0, 0.35M NaCl (PC); (G) pH 8.0, 0.0M NaCl (PC); (H) pH 8.0, 0.35M NaCl (PC), bar=200 /-cm. 87 The microstructure of the isolates seemed to play an important role i n the steady shear r h e o l o g i c a l properties of the isolates. For the unmodified isolate the protein appeared to be present as s p h e r i c a l l y shaped aggregates which seemed to remain e s s e n t i a l l y intact except for the sample at pH 11 without NaCl (Figure 2.6C). The aggregates appeared to swell as the pH increased, and phase contrast illumination was required to best v i s u a l i z e the aggregates, i n d i c a t i n g a softening of aggregate microstructure. When the aggregates were intact and s o l u b i l i t y was low they would have been able to s l i p past one another with l i t t l e i n t e r a c t i o n during steady shear, thus apparent v i s c o s i t y was low. At pH 11 where the dispersions with NaCl s t i l l had mostly intact aggregates (Figure 2.6D), the increased apparent v i s c o s i t y was probably the r e s u l t of a more viscous continuous phase due to increased protein s o l u b i l i t y , unfolding and dissociation, i n addition to swelling and softening of the aggregates which would increase th e i r hydrodynamic volume and aggregate-aggregate interactions. The flow behavior of the succinylated isolates also appeared to r e f l e c t an i n t e r a c t i o n between the soluble and dispersed phases. For both 5.2% SA and 14.2% SA dispersions, protein s o l u b i l i t y was at a minimum near pH 3.5. At t h i s pH most of the protein was present as aggregates of various sizes dispersed i n a t h i n continuous phase ess e n t i a l l y devoid of soluble protein (Figure 2.6E); thus under steady shear the apparent v i s c o s i t y of these disper-sions was low. Between pH 3.5 and 6.5 the protein s o l u b i l i t y increased dramatically so that at pH 5 the dispersions were i n an intermediate state where the protein aggregates were dispersed i n continuous phases of varying protein concentration and apparent v i s c o s i t y (Figure 2.6F). The combined effect of interactions of the aggregates with each other as influenced by 88 t h e i r size and shape and the v i s c o s i t y of the continuous phase led to very h i g h apparent v i s c o s i t i e s for some of the dispersions, e s p e c i a l l y those succinylated with 14.2% SA. A similar effect on the v i s c o s i t y of soy protein dispersions i n this pH range was reported by Lee and Rha (1979) who suggested that the increase i n apparent v i s c o s i t y was related to the growth of the pa r t i c l e s i z e . Above pH 6.5, the apparent v i s c o s i t y was ess e n t i a l l y that of the soluble protein with some contribution from i r r e g u l a r l y shaped insoluble material that may have been c e l l wall fragments (Figure 2.6G), while at pH 11 the increased v i s c o s i t y may have resulted from a l k a l i induced protein unfolding and perhaps diss o c i a t i o n into subunits of molecules not already dissociated by s u c c i n y l a -t i o n (Ishino and Okamoto, 1975; Whitaker, 1980). The continuous phase of succinylated isolate dispersions took on a s l i g h t l y coarser appearance as NaCl concentration increased (Figure 2.6H), probably as a resu l t of protein aggregation. A decrease i n protein s o l u b i l i t y also resulted from increased NaCl concentration i n t h i s pH range. Although the 5.2% SA isolate is shown i n F i g u r e 2.6, both succinylated isolates had similar microstructures with respect to pH and NaCl. D. Thermally Induced Gelation Thermally induced gels or g e l - l i k e materials were formed at twenty-eight of a possible f i f t y - f o u r combinations of succinylation, NaCl concentration and pH. V i s u a l l y , they appeared as translucent gels, opaque gels, or g e l - l i k e p r e c i p i t a t e s . This was similar to the effects of neutral salts and pH on the appearance of ovalbumin gels (Hegg et a l . , 1979). For unsuccinylated canola 89 isolate only four of a possible eighteen gels formed, while twelve gels formed for each of the two levels of succinylation. The storage modulus (G 1) for each gel increased slightly, with o s c i l l a t o r y frequency, while the dynamic v i s c o s i t y (ry1) decreased i n a linea r manner over the same frequency range when plotted on logarithmic coordinates. A repre-sentative rheogram is shown i n Figure 2.7; a l l samples had similar s t r a i g h t -line dynamic shear behavior. The equation of each line was determined by least-squares lin e a r regression (Table 2.7A.B), and values of storage modulus, loss modulus (G") and loss tangent were calculated for a frequency of 10 s"*. The effect of frequency on the dynamic v i s c o s i t y of the gels was similar to the flow behavior of pseudoplastic f l u i d s under steady shear. For many polymer solutions, shear rate dependent apparent v i s c o s i t y c l o s e l y p a r a l l e l s frequency dependent dynamic v i s c o s i t y over the same range of shear rate and frequency (Cox and Merz, 1958), but th i s was not examined i n the present study. For the unsuccinylated isolate, gels were formed at pH 9.5 i n the absence of NaCl and at pH 11 at a l l NaCl concentrations. A l l gels were opaque although the gel at pH 11 without NaCl had a great deal of translucent character. As seen i n Figure 2.8A, as pH increased from 9.5 to 11, G1 increased s l i g h t l y for the gels without NaCl. At pH 11, G1 increased drama-t i c a l l y as NaCl concentration increased to 0.35M, followed by a decrease i n G' from 0.35M to 0.7M NaCl. The treatment effects on the loss modulus of each gel p a r a l l e l e d the effects on storage modulus except for the gels without NaCl where a slig h t decrease i n G" was seen as the pH was raised from 9.5 to 11 (Figure 2.9A). The loss tangent of the gels without NaCl decreased as the pH increased from 9.5 to 11 (Figure 2.10A), while at pH 11, the loss tangent ^ 3.5 JD O O C O rj) Frequency (Log Scale) Figure 2.7. Storage moduli and dynamic viscosities of 5.2% SA, pH 6.5, 0.7M NaCl canola isolate gel as a function of oscillatory frequency. VO o Table 2.7A. Dynamic shear flow behavior parameters of canola i s o l a t e g e l s . NaCl Concentration (Molar) 0.0 0.35 0.7 Succinic anhydride c c c (%) pH (Pa s d) d r2 (Pa s d) d r2 (Pa s d) d r2 0 9.5 21.09 0.210 0.996 - - - - - -11.0 13.65 0.163 0.986 107.2 0.142 0.999 98.25 0.142 0.999 5.2 5.0 2.45 0.270 0.990 80.19 0.123 0.992 37.90 0.157 0.990 6.5 5.80 0.245 0.982 88.39 0.115 0.992 167.9 0.084 0.999 8.0 -1 - - 40.74 0.116 0.999 80.88 0.111 0.999 9.5 - - - 36.83 0.126 0.999 84.42 0.119 0.999 11.0 - - - 25.50 0.089 0.988 68.39 0.033 0.999 14.2 5.0 138.5 0.139 0.999 202.9 0.092 0.999 138.7 0.111 0.999 6.5 3.56 0.230 0.974 33.48 0.098 0.999 66.97 0.096 0.999 8.0 - - - 10.30 0.105 0.999 31.46 0.112 0.996 9.5 - - - 12.27 0.118 0.996 30.90 0.112 0.996 11.0 — — — 10.39 0.072 0.999 28.82 0.105 0.992 Did not gel. Table 2.7B. Dynamic shear storage behavior parameters of canola i s o l a t e g e l s . NaCl Concentration (Molar) 0.0 0.35 0.7 Succinic Anhydride (%) PH a (Pa s b) b r2 a (Pa s b) b r 2 a (Pa s b) b r2 0 9.5 70.77 0.130 0.978 - - - - - -11.0 108.2 0.109 0.952 670.4 0.103 0.988 517.7 0.105 0.968 5.2 5.0 10.44 0.144 0.891 328.9 0.095 0.990 150.7 0.096 0.972 6.5 28.36 0.155 0.950 510.0 0.120 0.992 565.6 0.133 0.992 8.0 _1 - - 224.2 0.113 0.988 395.9 0.118 0.988 9.5 - - - 230.9 0.100 0.992 435.8 0.108 0.982 11.0 - - - 178.0 0.071 0.980 489.3 0.077 0.986 14.2 5.0 531.1 0.130 0.984 556.7 0.131 0.986 484.0 0.121 0.986 6.5 10.30 0.127 0.822 212.9 0.096 0.996 362.4 0.104 0.986 8.0 - - - 59.24 0.056 0.974 188.7 0.101 0.996 9.5 - - - 65.60 0.109 0.984 205.0 0.101 0.988 11.0 — 54.21 0.109 0.976 187.9 0.086 0.992 ^ i d not gel. Figure 2.8. Storage modulus at 10 s " 1 fo r 11.4% canola isolate gels: (A) unmodi f ied; (B) 5.2% SA; (C) 14.2% SA. 250-9 4 200-n 150-•3 o m 100-50-Legend EZ3 OM NoCI Ea 0.35M NoCI K ] 0.7M NoCI 6.5 9.5 250 200-_3 •3 o .0-. 250 200-Figure 2.9. Loss modulus at 10 s" 1 for 11.4% canola isolate gels: (A) unmodified; (B)5.2% SA; (C) 14.2% SA. 95 Figure 2.10. Loss tangent at 10 s" 1 for 11.4% canola isolate gels: (A) unmodified; (B) 5.2% SA; (C) 14.2% SA. 96 increased with NaCl concentration. Since the loss tangent r e f l e c t s the proportion of viscous to el a s t i c character i n a vis c o e l a s t i c material (Ferry, 1980), the gels became proportionately more ela s t i c as the pH increased to 11 and as NaCl concentration decreased at pH 11. For the isolate modified with 5.2% succinic anhydride (54% modification of amino groups), gels formed at pH 5 and 6.5 both with and without NaCl, while from pH 8.5 to 11, gels formed only i n the presence of NaCl. The gels at pH 5 were very opaque and pasty while a l l others were translucent and springy. For the pH 5 gels, increasing the NaCl concentration to 0.35M increased G' and G" followed by a decrease i n these parameters at 0.7M NaCl (Figures 2.8B and 2.9B), For the translucent gels (pH 6.5 and above), G' and G" increased as NaCl concentration increased. Both G1 and G" were greatest at pH 6.5 for each le v e l of NaCl. With the exception of the sample at pH 6.5, 0.35M NaCl, the loss tangent decreased, i n d i c a t i n g that the el a s t i c component of each gel increased as pH increased. The loss tangent was dependent on NaCl concentration as well (Figure 2.10B). Gels without NaCl had a higher loss tangent than gels with 0.35M NaCl, while at each pH except 11, gels with 0.7M NaCl had a higher loss tangent than gels with 0.35M NaCl. The isolate succinylated with 14.2% succinic anhydride (84% modification of amino groups) had similar gelation behavior to the 5.2% SA isolate. Opaque, pasty gels were formed at pH 5 at a l l NaCl concentrations while translucent gels formed at pH 6.5 and above. From pH 8.5 to 11, gels formed only i n the presence of NaCl, while at pH 6.5, the sample without NaCl did not quite form a self-supporting gel but thickened considerably upon heating and appeared translucent and e l a s t i c . For the translucent gels, both G' and G" increased with NaCl concentration, while for the opaque gels, G' and G" were 97 highest at 0.35M NaCl (Figures 2.8C and 2.9C). Thus for a l l opaque gels, G1 and G" f i r s t increased and then decreased as NaCl increased while for a l l translucent gels these parameters increased as NaCl increased. For a l l gels there was a close association between G' and G" where both the el a s t i c and viscous components increased simultaneously (Figure 2.11). The effects of pH and NaCl on the loss tangent of the 14.2% SA gels are shown i n Figure 2.IOC. As expected, the loss tangent for the gel at pH 6.5 without NaCl, which appeared to be on the gel threshold, was su b s t a n t i a l l y higher than for the other gels which were a l l self-supporting. A l l loss tangents were s u b s t a n t i a l l y less than 1.0, however, i n d i c a t i n g that a l l gels were proportionately more el a s t i c than viscous. For the self - s u p p o r t i n g translucent gels, neither pH nor ionic strength greatly affected the loss tangent. The opaque gels had a higher loss tangent than the self - s u p p o r t i n g translucent gels; thus even though both G' and G" were higher for the opaque gels, there was proportionately less el a s t i c than viscous character. The high v i s c o e l a s t i c moduli of the opaque gels appeared to contradict the r e s u l t s of a puncture test as well as v i s u a l observation. With the puncture test (Chapter 3) the force required to rupture the opaque gels was of the same order or less than for the translucent gels. The opaque gels appeared pasty, lacked springiness and syneresed r e a d i l y . A similar effect was noted by G i l l and Tung (1978a) with thermally induced gels from the 12S f r a c t i o n of rapeseed. They found that gels at pH 6.0 had lower apparent v i s c o s i t y i n steady shear but higher v i s c o e l a s t i c parameters i n dynamic shear than gels at higher pH. Microscopic examination showed the presence of aggregates i n the pH 6.0 gel and the authors hypothesized that the aggregates were responsible for h i g h l y e l a s t i c recoveries under the non-destructive small 900-r 800-700-O ^ 600 V) D 3 500 TJ o * 400 rj) O O 300 tn 200 H 100-CO SL 50 Legend A Unmodified O 5.2% Succ. An. • 14.2% Succ. An. 100 150 Loss Modulus (Pa) 200 250 Figure 2.11. Storage and loss moduli at 10 s " 1 for 11.4% canola isolate gels (so l id symbols indicate opaque ge ls ) . 99 deformations applied i n dynamic t e s t i n g . Under steady shear conditions, however, the forces between aggregates would be broken and the aggregates would then be able to move r e a d i l y with respect to one another. As previously noted, the dispersions that formed opaque gels i n the present study a l l contained r e l a t i v e l y large protein aggregates to some extent, and these gels would be expected to behave d i f f e r e n t l y under non-destructive and destructive testing than the translucent gels, which appeared to have a r e l a t i v e l y homo-geneous gel matrix. This w i l l be described i n d e t a i l i n Chapter 3. To date, nearly a l l reports published on the thermal response of suc-cinylated food proteins have indicated increased heat s t a b i l i t y and a decrease or loss of gelation a b i l i t y ( K i n s e l l a and Shetty, 1979), presumably as a result of increased charge repulsion between molecules (Ma and Holme, 1982; Sato and Nakamura, 1977). In contrast, M i l l e r and Groninger (1976) found that the gelation a b i l i t y of f i s h protein concentrate was improved when 43-59% of the amino groups were succinylated. Choi et a l . (1981) reported that limited s u c c i n y l a t i o n of cottonseed protein increased the gel strength of 20% protein dispersions, and Montejano et a l . (1984) found that succinylated egg white required higher heating temperatures for gelation, but the gels had s i g n i -f i c a n t l y greater strength and deformability at f a i l u r e than those from native egg white. In the present study, the electr o n e g a t i v i t y induced by succiny-l a t i o n appeared to reduce or prevent gelation i n the absence of NaCl while the addition of NaCl overcame the charge repulsion and allowed close approach and aggregation of the protein molecules into a gel upon heating. This is supported by the observed effects of succinylation l e v e l and ionic strength where G1 and G" decreased as succinylation increased from 54% to 84% of amino groups. Succinylation progressively increased the electr o n e g a t i v i t y of the 100 protein molecules, while increasing the NaCl concentration progressively overcame this effect. The effect of pH also supports this hypothesis, as the unsuccinylated isolate formed gels only at high pH (i.e. at high electro-negativity) while succinylated isolates formed the firmest gels in the lower end of the pH range in which gels formed. There appeared to be an optimum NaCl concentration for maximization of the viscoelastic moduli as indicated by the effect of NaCl on the opaque gels, where the moduli first increased as NaCl concentration increased from 0.0M to 0.35M and then decreased to 0.7M NaCl, but the optimum did not appear to be reached with the translucent gels. It was apparent that degree of succinylation, pH and NaCl concentration were all important variables in the gel-forming ability of canola isolate and the viscoelastic properties of the gels. As these variables also influenced protein solubility, hydrophobicity, zeta potential and flow behavior, which in turn were expected to influence the viscoelastic properties of the gels, the relationships among these factors were examined by multiple regression analysis. Since the gels dissolved in 8M urea or 6M guanidine hydrochloride, covalent bonds such as disulfide were believed not to be involved in gel formation or stabilization. For multiple regression analysis, the viscoelastic parameters of the gels were used as dependent variables while potential independent variables included power-law flow parameters from 11.4% isolate dispersions, zeta potential, surface hydrophobicity before (S Q) or after (S e) heating, and protein solubility. When all gels were considered in the analysis, solubility, SQ, and their interactions with each other and zeta potential (z.p.) accounted for 72% of the variation in G1 (Table 2.8). For G", solubility (curvilinear), z.p. 101 Table 2.8. M u l t i p l e regression models f o r p r e d i c t i o n of v i s c o e l a s t i c para-meters of thermally induced canola i s o l a t e gels (n=28). Dependent Variable Independent Variable Coefficient F-prob. Storage Modulus Solubility -35.523 0.0000 R2 = 0.720 So -9.189 0.0004 S.E.a - 156.18 Solubility x S Q 0.291 0.0000 F-prob. = 0.0000 Solubility x Z.P. -0.200 0.0203 S 0 x Z.P. 0.449 0.0002 Constant 2.587 x 103 Loss Modulus Solubility 8.695 0.0063 R2 = 0.653 Solubility 2 -0.711 x io-i 0.0050 S.E. = 44.24 Zeta Potential 16.596 0.0137 F-prob. = 0.0002 Zeta Potential 2 0.202 0.0314 S e x Z.P. -0.482 X io- i 0.0158 Constant 42.122 Loss Tangent Solubility -0.254 X IO"2 0.0002 R2 = 0.510 S e 0.110 X IO"2 0.0464 S.E. = 0.0558 Solubility x Z.P. -0.637 X IO"4 0.0015 F-prob. = 0.0006 Constant 0.174 aStandard error of estimate. 102 ( c u r v i l i n e a r ) and the i n t e r a c t i o n between S e and z.p. were s i g n i f i c a n t independent variables (R2=0.653) while for the loss tangent, s o l u b i l i t y , S e and the i n t e r a c t i o n between s o l u b i l i t y and z.p. were s i g n i f i c a n t (R2=0.510). Schmidt (1981), however, emphasized that the importance of subjective or qualitative evaluation of protein gels should not be underestimated, as measurably strong protein gels may range i n appearance from translucent and e l a s t i c to c u r d - l i k e and opaque. As previously noted, the gels could be divided into two major classes based on v i s u a l observation, translucent and opaque, and i t has been hypothesized that, due to their microstructure, the v i s c o e l a s t i c properties of these classes of gels may d i f f e r . The translucent gels v a r i e d i n character from firm and springy to a t h i c k material that was g e l - l i k e and springy but was not self-supporting. The opaque gels ranged from g e l - l i k e pasty precipitates with very l i t t l e e l a s t i c i t y to a gel that was l a r g e l y translucent with some opaque character. The translucent gels included a l l gels formed from each succinylated isolate at pH 6.5 or higher (18 total) while the opaque gels included those formed at pH 5 from each succinylated isolate plus the gels from unmodified isolate (10 t o t a l ) . For the translucent gels, G' was well described by s o l u b i l i t y and S e, where G' was inversely related to s o l u b i l i t y and p o s i t i v e l y r e lated to S e (R2=0.775; Table 2.9). When the apparent v i s c o s i t y at 10 s - 1 (r/io) of the unheated dispersions was allowed as an independent variable, 7)IQ and S e together accounted for 77.0% of the v a r i a t i o n i n G1. G1 increased as r j 1 0 and S e increased. Although the water-holding capacity of gels has been examined exten-sive l y , l i t t l e has been reported on the r e l a t i o n s h i p between the protein content of the gel exudate and t e x t u r a l properties. The percentage of protein 103 Table 2.9. M u l t i p l e regression models f o r p r e d i c t i o n of v i s c o e l a s t i c para-meters of translucent gels (n-18). Dependent Variable Independent Variable Coefficient F-prob. Storage Modulus Solubility - 2 5 . 7 5 5 0 .0162 R2 = 0 . 7 7 5 s e 4 . 0 8 9 0 .0243 S.E.a = 116 .74 Constant 2 . 3 2 9 X 1 0 3 F-prob. = 0 . 0 0 0 0 Storage Modulus S e 7 .662 0 . 0 0 0 0 R2 - 0 . 7 7 0 VlO 0 . 7 5 0 0 . 0 1 9 0 S.E. = 117 .89 Constant - 4 9 1 . 3 8 0 F-prob. = 0 . 0 0 0 0 Storage Modulus Gel Solubility - 4 4 . 0 6 2 0 . 0 0 0 0 R2 = 0 . 9 6 3 Gel Solubility 2 0 . 2 3 4 0 . 0 0 0 0 S.E. = 4 7 . 3 2 4 Constant 2 . 0 9 9 X 103 F-prob. = 0 . 0 0 0 0 Loss Modulus S 2 0 . 2 2 2 X i o - i 0 . 0 0 0 0 R2 = 0 . 8 8 2 Solubility x S e - 0 . 2 6 6 X i o - i 0 . 0 0 5 3 S.E. = 1 8 . 4 8 Constant 8 9 . 5 3 8 F-prob. = 0 . 0 0 0 0 Loss Modulus S e - 2 . 3 6 0 0 . 0 4 1 7 R2 - 0 . 8 8 8 S 2 0 . 2 3 9 X i o - i 0 .0015 S.E. - 1 8 . 6 2 4 0 . 1 5 5 0 . 0 2 2 9 F-prob. = 0 . 0 0 0 0 Constant 4 7 . 7 1 4 Loss Tangent s e - 0 . 3 8 7 X i o - i 0 . 0 0 0 0 R2 = 0 . 9 0 0 Se2 0 . 3 8 2 X 10-3 0 . 0 0 0 1 S.E. = 0 . 0 2 4 Se3 - 0 . 1 1 7 X 10-5 0 .0004 F-prob. = 0 . 0 0 0 0 Constant 1.390 aStandard error of estimate. 104 found i n the gel exudate after c e n t r i f ugation i s presented i n Table 2.10. The storage modulus of the translucent gels followed a c u r v i l i n e a r r e l a t i o n s h i p with exudate protein content that accounted for more than 96% of the v a r i a t i o n in G' (Table 2.9). As exudate protein decreased, G' increased i n a c u r v i -l i n e a r manner. Therefore, for these gels i t appeared as i f G1 was related to the number of protein molecules taking part i n j u n c t i o n zones and c r o s s - l i n k s and was probably the result of a greater number of similar bonds rather than a few covalent linkages. For the opaque gels there were no s i g n i f i c a n t r e l a -tionships between gel exudate protein and the v i s c o e l a s t i c parameters. The loss modulus (G") of translucent gels was well described by S e 2 plus the i n t e r a c t i o n of s o l u b i l i t y of unheated dispersions and S e (R2=0.882). G" increased i n an upward c u r v i l i n e a r manner with S e, and G" generally increased as s o l u b i l i t y decreased. A l t e r n a t i v e l y , when 7)IQ was allowed as an indepen-dent variable, TJIQ, S e and S e 2 accounted for 88.8% of the v a r i a t i o n i n G". As w i t h G1, G" f o l l o w e d a c u r v i l i n e a r r e l a t i o n s h i p w i t h g e l s o l u b i l i t y (R2=0.836), where G" increased at a faster rate as gel protein s o l u b i l i t y decreased. This may be the result of limited polymerization of protein molecules producing i n effect larger aggregates or longer strands but without complete c r o s s - l i n k i n g or integration into the three-dimensional gel matrix. The loss tangent of the translucent gels was described by a cubic f i t of S e (R2=0.900) where minimum values of loss tangent were found at intermediate S e values , while loss tangent increased at low and high values of S e. This implied that even though both G' and G" increased with S e, optimum development of the e l a s t i c portion of the gel as compared to the viscous portion occurred when S e was neither too large nor too small. At low levels of hydrophobicity, Table 2.10. P r o t e i n content of exudate from thermally induced gels of canola p r o t e i n I s o l a t e (percent). NaCl Concentration (Molar) Succinic Anhydride (%) pH 0.0 0.35 0.7 0 9.5 50.0 - -11.0 68.7 31.7 25.5 5.2 5.0 27.6 25.7 26.0 6.5 94.3 44.4 40.3 8.0 _1 57.6 43.8 9.5 - 61.6 48.4 11.0 - 67.1 44.0 14.2 5.0 21.6 27.9 29.8 6.5 97.0 62.4 48.1 8.0 - 79.1 63.6 9.5 - 80.3 61.7 11.0 — 78.5 68.5 *Did not g e l . 106 there would be fewer areas on the surfaces of the protein molecules for three dimensional network formation, but limited hydrophobic interactions would allow for aggregation of protein molecules to form strands as proposed by Tombs (1970, 1974), thus increasing the viscous component of the system. Al t e r n a t i v e l y , i f protein hydrophobicity was very high and a r e l a t i v e l y large proportion of the surface was able to take part i n hydrophobic interactions, aggregation would be expected to be less ordered, giving larger more approxi-mately spherical p a r t i c l e s and a coarser, less well oriented gel network structure. The larger the aggregates, the smaller would be the contribution from each p a r t i c l e i n the gel network (Tombs, 1974), hence the proportion of e l a s t i c to viscous components of the system would be expected to decrease even though both G1 and G" would increase. For the opaque gels, G' was affected by S e, zeta potential and apparent v i s c o s i t y of the unheated dispersions (Table 2.11), while s i g n i f i c a n t v a r i -ables i n f l u e n c i n g G" were protein s o l u b i l i t y , S e and T)IQ (R2=0.886). For the loss tangent, only s o l u b i l i t y was a s i g n i f i c a n t independent variable, but i t accounted for less than 45% of the v a r i a t i o n i n loss tangent. The visco-' el a s t i c properties of the opaque gels appeared to be influenced not only by the gel matrix, but also by the aggregates that lend opacity to the gels. Thus, the analysis of factors i n f l u e n c i n g v i s c o e l a s t i c i t y of the opaque gels was complicated, not only by the properties of the continuous phase but also by such factors as the s i z e , shape, number and i n t e r n a l structure of the aggregates which i n t u r n may depend on extent of succinylation , pH, and ionic strength. Since the factors i n f l u e n c i n g the formation and physical properties of the aggregates would not necessarily be the same as for the gel matrix, and may i n fact oppose one another, i t is d i f f i c u l t to make a meaningful i n t e r -107 Table 2.11. M u l t i p l e regression models f o r p r e d i c t i o n of v i s c o e l a s t i c parameters of opaque gels (n=»10). Dependent Variable Independent Variable Coefficient F-prob. Storage Modulus s e - 1 2 . 6 5 0 0 .0038 R2 = 0 .919 Zeta Potential 44 .716 0 .0020 S.E.8 - 120 .17 ^10 1.588 0 .0024 F-prob. = 0 .0062 S e x Z.P. - 0 . 3 3 8 0 .0076 Constant 1.724 x 1 0 3 Loss Modulus - 1 . 3 8 1 0 .0427 R2 = 0 .886 Solubility 2 - 0 . 4 9 4 x i o - i 0 .0063 S.E. = 34 . 25 *7l0 0.576 0 .0007 F-prob. = 0 .0031 Constant 197 .01 Loss Tangent Solubility 2 - 0 . 2 3 8 x lO" 4 0 .0339 R2 = 0 .449 Constant 0 .308 S.E. = 0 .054 F-prob. = 0 .0340 aStandard error of estimate. 108 pretation of the mode of action of the predictive factors on the system as a whole. Gelation is a protein aggregation phenomenon where attractive and repulsive forces between protein molecules and the solvent are so balanced that a well ordered three dimensional network or matrix is formed which is capable of trapping or immobilizing large amounts of solvent. Ionizable amino acids play an important role in determining electrostatic interactions between protein molecules, and therefore factors such as salts, pH and temperature influence the balance between attractive and repulsive forces in the system (Wall, 1979). During denaturation, hydrophobic and hydrogen bonds buried in the interior of the molecule become exposed and reform in a manner different from the native structure (Buttkus, 1974). Thus regions of the molecule originally involved in the stability of the native form become available for intermolecular interactions and a three dimensional network can form, provided that there are at least two attractive sites per molecule (Clark et al., 1981). Therefore it appears as if optimum gelation conditions occur when the attractive forces set free by denaturation are just strong enough to coun-teract electrostatic repulsion, and an ordered limited aggregation can take place resulting in a gel network. The nature of the gel would be determined by the number of bonding sites available on each protein molecule, their spacial distribution and their relative bonding strengths under the prevailing conditions of pH and ionic strength. Hegg et al. (1979) noted that ovalbumin gels were formed at an intermediate state between a high level of charge repulsion which gave solubility and a low level that gave rise to precipita-tion. In a medium where ovalbumin underwent gelation, however, the gels be-came increasingly transparent as the net charge repulsion increased such as by 109 increasing the pH or decreasing the ionic strength. A similar effect was found by Egelandsdal (1980) where clear and uniform ovalbumin gels were produced by directed linear aggregation whereas more i r r e g u l a r networks were formed by e s s e n t i a l l y random aggregation which occurred at low e l e c t r o s t a t i c repulsion. Hermansson (1982) found that blood plasma gels were coarser and more randomly aggregated at pH 7 than 9.5 due to the lower net negative charge at pH 7. Water-binding properties also decreased as the degree of random aggregation of the gel network increased. The author suggested that gel texture was influenced at the molecular level by the organization of molecules into strands or aggregates and on the c o l l o i d a l level by such factors as the strength and deformability of strands, the strength, type and number of j u n c t i o n zones as well as the density and size d i s t r i b u t i o n of aggregates and conglomerates (Hermansson, 1982). Moreover, i t was pointed out that the v i s c o s i t y of the continuous phase should not be overlooked. Previous studies with succinylated protein have found increased thermal s t a b i l i t y and decreased or retarded heat-induced gelation or coagulation. Ma and Holme (1982) found that the thermocoagulation of egg albumen was pro-gressively decreased and eliminated by increasing the extent of succiny-l a t i o n and therefore the amount of e l e c t r o s t a t i c repulsion between protein molecules. Balmaceda et a l . (1976) suggested that, within f i n i t e l i m i t s , a high degree of protein s o l u b i l i t y is necessary for optimal protein gelation. In the present study, the s o l u b i l i t y of canola protein under low acid and alkaline conditions was greatly improved by succinylation as a r e s u l t of increased e l e c t r o n e g a t i v i t y of the protein molecules, but t h i s also appeared to i n c r e a s e t h e r m o s t a b i l i t y and r e d uce gelation a b i l i t y at low ionic strength. With the addition of NaCl, however, the charge repulsion was 110 progressively reduced allowing for p r o t e i n - p r o t e i n interactions and gelation when a proper balance between protein - p r o t e i n and protein-solvent i n t e r -action was achieved. NaCl promotes aggregation due to reduction of the diffuse part of the e l e c t r i c double layer, and may also induce conformational changes i n the protein molecules (Fennema, 1977) which may alter the number of bonding sites for gelation. Heating protein dispersions also induces con-formational changes which tend to increase p r o t e i n - p r o t e i n interaction (Tombs, 1974). The addition of NaCl has also been found to improve the gel strength of many other thermally processed protein systems (e.g. Hermansson, 1982; Schmidt and Il l i n g w o r t h , 1978; Schmidt et a l . , 1978; Shiraada and Matsushita, 1981). Although not examined i n depth, low levels of divalent cations were able to induce gelation i n heated dispersions of succinylated canola isolate. This was suspected to be a resu l t not only of enhanced depression of the e l e c t r i c double-layer but also the capacity of divalent cations to l i n k two protein molecules together by their carboxyl groups. Preliminary experiments demon-strated that 0.035M CaCl2 i n 11.4% dispersions of both 5.2% SA and 14.2% SA canola isolates at pH 6.8 gave similar gel strengths to those obtained with 0.7M NaCl as determined by a puncture test. P r o t e i n p r e c i p i t a t i o n and therefore no gelation occurred with 0.35M CaCl2. A similar r e s u l t was found by Schmidt et a l . (1978) who reported that the maximum gel strength of whey protein concentrate gels was obtained with 5 to 20mM CaCl2 compared to 0.1 to 0.3M NaCl. This f i n d i n g may be of p r a c t i c a l interest as i t demonstrates that the gelation a b i l i t y of succinylated proteins may be u t i l i z e d i n products where high levels of salts such as NaCl may be detrimental to product quality or undesirable for health reasons. I l l The major types of bonds involved in gel formation and stability were tentatively identified as hydrophobic interactions and hydrogen bonds. It seems likely that hydrophobic groups largely contributed to gel formation since the gels formed during heating. In this temperature range, hydrophobic interactions increase with temperature (Ben-Nairn, 1980). Schmidt (1981) suggested that hydrophobic interactions are important to dissociative-associa-tive reactions which initiate the gelation process and contribute to layering or thickening of the gel matrix strands upon cooling. Shimada and Matsushita (1980b) found a relationship between the proportion of hydrophobic groups in the amino acid profile of proteins and the type of gel formed upon heating, while Ma and Holme (1982) showed hydrophobicity to be involved in gelation of egg albumen. Hydrophobic and acidic residues dominate the amino acid profile of rapeseed proteins, while basic amino acids are in relatively low concen-tration (Sosulski and Sarwar, 1973). In the present study, the gels increased in firmness upon cooling, thus implicating the involvement of hydrogen bonds which are weakened by increasing temperature (Joesten and Schaad, 1974). Oakenfull and Scott (1984) reported that a combination of hydrogen bonds and hydrophobic interactions stabilized gels formed from high methoxy pectins. Schmidt (1981) suggested that hydrogen bonds stabilize gel structure and allow for a more open orientation necessary for water immobilization. Although the effects of different heating conditions were not examined in detail, it was noted that temperatures up to 160°C did not destroy the gelation ability of the isolates. This is in contrast to soy protein, for which temperatures in excess of 125°C have been reported to result in a metasol which did not form a gel upon cooling (Catsimpoolas and Meyer, 1970). This is of potential importance for the use of succinylated canola protein in 112 a retorted product. Other authors have reported on the effects of heating conditions on gel formation. Hermansson (1982) found that heating blood plasma protein above an optimal gelation temperature caused an increased tendency toward protein-protein interactions with a partial disruption of the gel network due to local aggregation phenomena. A similar effect was noted by Tombs (1970) with bovine serum albumin and Schmidt and Illingworth (1978) who found that heating whey protein at temperatures above 110°C produced gels with more visual syneresis than gels formed at lower temperatures. Schmidt et al. (1978) found that whey protein dispersions with 0.2M NaCl gelled at 75°C while a temperature of 90°C was required for gelation in distilled water. Egelandsdal (1984), however, found that hardness of ovalbumin gels was independent of the temperature to which it was heated and suggested that the rate of gel formation was more important for gel hardness than the degree of protein unfolding. In view of these reports, the effects of heating con-ditions on the gelation properties of succinylated canola protein could form the basis of further studies. E. E m u l s i f i c a t i o n A c t i v i t y and Emulsion S t a b i l i t y The effects of succinylation, pH and NaCl on emulsification activity (EA) are presented in Table 2.12 and Figure 2.12. EA was significantly affected by succinylation (linear and quadratic), NaCl (linear and quadratic), pH (linear) as well as an interaction between all three factors. In general, EA increased with extent of succinylation, but the rate of increase was greater between 0% and 54% modification of amino groups than between 54% and 84% modification. NaCl at both 0.35M and 0.70M increased EA compared to the samples without salt, but the highest EA overall was at 0.35M indicating first an increase Table 2.12. Trend comparison analyses of the effects of succinylation, pH and NaCl on emulsification a c t i v i t y and emulsion s t a b i l i t y (n=54). Dependent V a r i a b l e I n d e p e n d e n t V a r i a b l e C o e f f i c i e n t F - p r o b . E m u l s i f i c a t i o n A c t i v i t y S u c c i n y l a t i o n 0 . 2 2 7 X l O " 2 0 . 0 0 0 0 R 2 = 0 . 9 4 5 S u c c i n y l a t i o n 2 - 0 . 8 8 0 X 1 0 - 3 0 . 0 4 3 2 S . E . a = 0 . 0 3 1 2 NaCl 0 . 1 4 6 X i o - i 0 . 0 0 7 2 F - p r o b . = 0 . 0 0 0 0 N a C l 2 - 0 . 7 9 3 X l O " 2 0 . 0 1 1 3 PH 0 . 3 2 8 X i o - i 0 . 0 0 0 0 S u c c . x NaCl x pH 0 . 3 3 0 X 1 0 - 3 0 . 0 0 0 5 C o n s t a n t 0 . 3 9 9 E m u l s i o n S t a b i l i t y S u c c i n y l a t i o n 0 . 3 7 0 0 . 0 0 0 0 R 2 - 0 . 8 9 7 S u c c i n y l a t i o n 2 - 0 . 1 4 4 0 . 0 2 6 5 S . E . = 4 . 6 3 4 NaCl - 3 . 9 5 0 0 . 0 0 0 0 F - p r o b . = 0 . 0 0 0 0 PH 2 . 7 5 6 0 . 0 0 0 0 S u c c i n y l a t i o n x pH 0 . 4 1 6 X i o - i 0 . 0 0 0 3 S u c c i n y l a t i o n x p H 2 - 0 . 2 0 6 X i o - i 0 . 0 3 9 5 NaCl x pH - 1 . 0 4 9 0 . 0 0 0 0 C o n s t a n t 17 .208 a S t a n d a r d e r r o r o f e s t i m a t e . Figure 2.12. Emuls i f i c a t i o n a c t i v i t y of canola isolate dispersions: (A) unmodified; (B) 5.2% SA; (C) 14.2% SA. 115 and then a decrease in EA as ionic strength increased. However, this effect depended on pH and extent of succinylation. In addition, EA increased in an overall linear manner with pH. Succinylation has often been reported to improve the emulsifying pro-perties of proteins (e.g., Childs and Park, 1976; Franzen and Kinsella, 1976a,b; Thompson and Cho, 1984b). Waniska et al. (1981) reported that above pH 5, the emulsifying activity of succinylated bovine serum albumin (BSA) was markedly improved compared to native BSA, which may be a reflection of increased solubility and somewhat looser structure of the modified protein, thus facilitating diffusion of the protein to the oil-water interface and rearrangement within the interfacial film. Pearce and Kinsella (1978) found that succinylated yeast proteins gave the smallest droplets in o/w emulsions compared to proteins from other sources. Since EA is related to interfacial area, EA increases as droplet size decreases. Succinylation also results in an increase in the molecular surface area of a protein, apparently as a result of electrical repulsion forces between identical charges; the increased surface area gives improved surfactant properties (Watanabe and Arai, 1982). In addition, emulsification proceeds more rapidly if the surface tension is decreased by a combination of surfactants (Dickinson and Stainsby, 1982). Succinylation increases the heterogeneity of protein mixtures as a result of different extents of modification of the proteins as well as different degrees of subunit dissociation (e.g., Grant, 1973; Klotz and Keresztes-Nagy, 1962), and this may aid in emulsion formation. The effect of NaCl on EA may be related to its effect on protein adsorp-tion at the o/w interface. Bennett et al. (1968) stated that the addition of electrolytes may act favorably or unfavorably on emulsion stability depending 116 on whether they diminish charge repulsions or increase the electrical poten-tial of the ionized layer of the interfacial film. Waniska et al. (1981) reported an effect of NaCl on emulsifying activity of BSA similar to that of the present study, where EA first increased and then decreased with addition of NaCl. They suggested that at low ionic strength, high charge repulsion at the o/w interface decreased the amount of protein adsorbed at the interface. For the present data, at intermediate ionic strength, EA increased probably as a result of neutralization of surface charges which reduced electrostatic repulsion and facilitated an increased rate of protein adsorption and greater protein-protein interactions. However, at high ionic strength, EA dropped, possibly because of a reduced rate of protein transfer to the o/w interface. A similar effect of NaCl on the emulsifying properties of peanut and soy protein was reported by McWatters and Holmes (1979a,b). Mita et al. (1973) found that for five different proteins studied, the largest droplets tended to be formed near the isoelectric point. In the present study EA increased with pH, which suggests that droplet size decreased as the pH went farther away from the isoelectric region. The treatment effects on emulsion stability are presented in Table 2.12 and Figure 2.13. As with EA, ES increased with succinylation but at a slower rate between 54% and 84% modification than between 0% and 54% modification. There were significant interaction effects between pH and NaCl concentration and between succinylation and pH (linear and quadratic), but overall, ES decreased in a linear manner with NaCl and increased linearly with pH. As already described, succinylation, pH and NaCl influenced protein solubility, hydrophobicity, zeta potential, and apparent viscosity of canola isolate dispersions. Since these factors were expected to affect EA and ES, Figure 2.13. Emulsion stability of canola isolate dispersions: (A) unmodified; (B) 5.2% SA; (C) 14.2% SA. 118 multiple regression analyses were used to examine the relative influence of each factor so as to obtain a better understanding of the manner in which the treatments affected emulsification. For the regression involving emulsification activity, solubility and the square of solubility as well as interaction effects between solubility and SQ, zeta potential and flow behavior index (n) accounted for 89.3% of the varia-bility of EA (Table 2.13). The quadratic effect of solubility indicated that EA increased rapidly at low levels of solubility but more slowly as solubility increased. The presence of solubility along with the interaction of solu-bi l i t y with S Q and zeta potential as significant independent variables underscored the importance of a balance between hydrophile and lipophile in emulsion formation (Aoki et al., 1981) as well as the involvement of elec-trical repulsion forces between droplets in retarding coalescence during emulsification. The importance of a hydrophilic-lipophilic balance was emphasized by Voutsinas et al. (1983b) who reported that regardless of protein solubility, as surface hydrophobicity increased, emulsification activity and emulsion stability initially increased and then decreased, and Li-Chan et al. (1984) who showed the importance of solubility, hydrophobicity, and their interaction in emulsifying properties of salt-soluble meat protein. The interaction between protein solubility and flow behavior index in the present study was also probably related to resistance to coalescence during emul-sification. The overall effect of the flow behavior index was to increase EA with a decrease in n, indicating that as the continuous phase of the disper-sions became more pseudoplastic, a greater interfacial area in the emulsions became stabilized. The physical meaning of a flow behavior index less than 1.0 is that the apparent viscosity of the dispersion decreases with an 119 Table 2.13. M u l t i p l e regression models f o r p r e d i c t i o n of e m u l s i f i c a t i o n a c t i v i t y and emulsion s t a b i l i t y (n=54). Dependent Variable Independent Variable C o e f f i c i e n t F-prob. E m u l s i f i c a t i o n A c t i v i t y S o l u b i l i t y 0.141 X 10" -1 0.0000 R 2 = 0.893 S o l u b i l i t y 2 -0.490 X 10" -4 0.0000 S.E.a = 0.0433 S 0 x S o l u b i l i t y -0.264 X 10" -4 0.0000 F-prob. = 0.0000 Z.P. x S o l u b i l i t y 0.409 X 10" -4 0.0004 n b x S o l u b i l i t y -0.572 X 10" -2 0.0000 Constant 0.239 Emulsion S t a b i l i t y S o l u b i l i t y x ^ JOOO 0.690 X 10" -2 0.0000 R 2 = 0.900 Zeta P o t e n t i a l -0.268 0.0032 S.E. = 4.430 Zeta P o t e n t i a l 2 -0.590 X 10" -2 0.0026 F-prob. = 0.0000 ADensity -212.790 0.0077 Constant 21.674 aStandard er r o r of estimate. bPower-law flow behavior Index. 120 increase in shear rate. At the high shear rates encountered at the blades of the Oranimixer used to create the emulsions, the dispersions would have had a low apparent viscosity, thereby facilitating breakup of the oil phase and emulsion formation. As the oil droplets flowed away from the blades into the bulk dispersion, oil droplets in dispersions with greater pseudoplasticity would have encountered a higher apparent viscosity at the low rates of shear in the bulk dispersion than if the dispersion was less pseudoplastic. Droplets in a highly viscous dispersion would tend to have fewer collisions and therefore less coalescence and demulsification than in a dispersion of lower viscosity, and this would be reflected in the EA. The multiple regression model for emulsion stability is presented in Table 2.13. In addition to protein solubility, hydrophobicity, zeta potential and rheological parameters, the difference in density between the oil phase and the aqueous phase at each NaCl concentration was included as an indepen-dent variable in the regression. Zeta potential and the square of zeta potential, along with the density difference between the dispersed and continuous phases, and the interaction between protein solubility and apparent viscosity at 1000 s" l of aqueous dispersions accounted for 90% of the vari-ation in emulsion stability. In general, emulsions became more stable as both protein solubility and apparent viscosity of the continuous phase increased, as the difference in density between the two phases decreased, and as the charge repulsion between protein molecules at the interface increased. The nonlinear effect of charge repulsion indicated that changes in zeta potential had a greater effect on stability when charge repulsion was high compared to when it was low. 121 ' Emulsions may d e s t a b i l i z e by creaming (the movement of d r o p l e t s under the i n f l u e n c e of a g r a v i t a t i o n a l f i e l d ) , f l o c c u l a t i o n (the c l u s t e r i n g together of d r o p l e t s ) , and/or coalescence (the merging of small d r o p l e t s into l a r g e r ones). Examination of the s i g n i f i c a n t independent v a r i a b l e s i n d i c a t e d that emulsion s t a b i l i t y as determined i n t h i s study was mainly a measure of r e s i s t a n c e to creaming. Creaming occ u r s as a r e s u l t of the d e n s i t y d i f f e r e n c e between the d i s p e r s e d and continuous phases as a f f e c t e d by such f a c t o r s as the v i s c o s i t y of the continuous phase, d r o p l e t s i z e , and the charge on the sur f a c t a n t molecules ( D i c k i n s o n and Stainsby, 1982; Powrie and Tung, 1976). Creaming rate is r e l a t e d to Stokes' equation: = 2 r 2 g ( A d ) m 977 1 ' where V is the v e l o c i t y of dro p l e t movement, r i s dro p l e t r a d i u s , g is the g r a v i t a t i o n a l f o r c e , A d is the d e n s i t y d i f f e r e n c e between the two phases, and 77 is the apparent v i s c o s i t y of the continuous phase (Powrie and Tung, 1976). Thus, emulsion s t a b i l i t y with re s p e c t to creaming is favored by a small d r o p l e t r a d i u s , a small d e n s i t y d i f f e r e n c e between the d i s p e r s e d and con-tinuous phases, and h i g h v i s c o s i t y of the continuous phase. Creaming, f l o c c u l a t i o n and coalescence are not independent processes, however. For example, the he t e r o g e n e i t y of the dro p l e t d i s t r i b u t i o n may a f f e c t not o n l y creaming rate but also f l o c c u l a t i o n and coalescence as a r e s u l t of c o l l i s i o n s between fast moving large d r o p l e t s and slow moving small d r o p l e t s . If f l o c c u l a t i o n and/or coalescence do occur, creaming rate would be enhanced since f l o c c u l a t i o n increases the e f f e c t i v e droplet size while coalescence in c r e a s e s a c t u a l d r o p l e t s i z e . With r e s p e c t to pH, emulsions tend to have greatest s t a b i l i t y to coalescence at the i s o e l e c t r i c point. Cohesiveness and 122 rigidity of protein surface films are usually greatest at the isoelectric point where electrostatic repulsions between different parts of a single protein molecule as well as between protein molecules are minimized. The cohesiveness and rigidity imparted by the compact protein molecules opposes deformation and rupture of the interfacial lamella, thus providing stability against coalescence (Hailing, 1981). There is a tendency for droplet diameter to be highest at the isoelectric point (Mita et al., 1973) and the rate of flocculation increases as the pH is adjusted closer to the isoelectric point (Hailing, 1981). Ionized surfactants help to stabilize o/w emulsions by means of an electric double layer in the aqueous phase. For protein emulsifiers this becomes more important as the pH goes away from the isoelectric point. Surface charges can originate from ionization of groups on ionic emulsifiers, adsorption of ions on nonionic emulsifier layers, or frictional contact between droplet surfaces and the continuous phase (Powrie and Tung, 1976). The electrical charge is proportional to the surface area; the smaller the droplet, the larger is the relative surface area and the higher is the relative charge. The repulsive energy is a function of kH Q where H 0 is the distance between the droplets and k""1 is the effective radius of the double layer (Powrie and Tung, 1976). Charged oil droplets can interact with one another via their electrical double layers which leads to a reduction in the mean creaming speed due in part to drag from the "ionic atmosphere" and a microelectrophoresis effect (Dickinson and Stainsby, 1982). The surface activity of a protein is a function of the ease with which a protein can migrate to, adsorb at, unfold and rearrange at an interface, and therefore the aqueous solubility of a protein is closely related to its 123 surface activity (Kinsella, 1976). As protein molecules are relatively hydrophilic, the bulk of the adsorbed molecule is probably located on the aqueous side of the oil-water interface with the hydrophilic groups directed toward the aqueous phase and hydrophobic groups toward the oil phase. The importance of solubility is related to several aspects of emulsion formation and stability. The ease of emulsion formation is affected by the rate at which protein is transferred from the bulk phase and penetrates into the o/w interface, irrespective of whether or not the protein stays at the interface to help stabilize the emulsion (Canella et al., 1979). It is possible then for a protein to have a high emulsification activity but poor stability, and this was indeed the case for several of the treatment combinations of the present study. However, since proteins adsorb relatively slowly at newly created oil-water interfaces, high protein solubility facilitates this process. Pearce and Kinsella (1978) noted that emulsion droplet size de-creased as protein concentration increased, probably as a result of an increased rate of adsorption at the. freshly formed o/w interfaces. Smaller oil droplets should enhance stability with respect to creaming. Proteins may cause flocculation of emulsions, however, by acting as "polymer bridges" between molecules (Hailing, 1981), and excessive emulsification may lead to depletion of surfactant and emulsion instability. The role of insoluble protein is not clear. Franzen and Kinsella (1976b) suggested that granular, insoluble proteins separate from the oil phase or just float on the oil surface where they remain inert and contribute little toward emulsification. Others, however, have suggested that insoluble proteins may provide a steric hindrance to coalescence by adsorbing at oil-water interfaces and reducing inter-droplet contact (Smith et al., 1973; Powrie and Tung, 1976; Paulson et al., 1984; Kitchener and Mussellwhite, 1968). 124 SUMMARY AND CONCLUSIONS Thermally induced gelation and oil emulsification properties of unmodi-fied and succinylated canola protein isolate were examined over a wide range of pH values (pH 3.5-11.0) and sodium chloride concentrations (0.0-0.7M). Reaction of the isolate with succinic anhydride at 5.2% and 14.2% of the protein weight resulted in 54% and 84% modification of free amino groups, respectively. Succinylation markedly enhanced protein solubility at alkaline and low acid pH, but exhaustive succinylation was not required to produce a large increase in solubility. Surface hydrophobicity decreased, and electro-negativity (zeta potential) increased as succinylation increased. Succiny-lation also appeared to cause a slight shift in the isoelectric region to lower pH values, which may have implications for the successful employment of succinylated protein in low-acid foods such as comminuted meat products. Protein dispersions followed power-law or power-law plastic flow beha-vior. The rheological properties appeared to reflect the microstructure of the dispersions; the succinylated isolates had higher apparent viscosities than the unmodified isolate under nearly all conditions of pH and ionic strength. Thermally induced gels were formed by heating 11.4% dispersions of the iso-lates at 72°C for 30 min. Succinylation improved the gelation ability of canola protein. For the unmodified isolate, gels formed at only 4 out of 18 combina-tions of pH and NaCl concentration, while 12 gels formed for each of the succiny-lated isolates under the same conditions. The unmodified isolate formed gels at pH 9.5 and 11.0 while the succinylated isolates formed gels from pH 5.0 to 11.0, but above pH 6.5 only in the presence of NaCl. In general, the firmest gels were obtained with the moderate level of succinylation. 125 Overall, the gels were divided into two types based on visual observa-tion, opaque and translucent, and each type appeared to respond in a different manner to rheological tests, and were related in different ways to the physicochemical and rheological properties of protein dispersions. The viscoelastic properties of the translucent gels reflected the properties of the gel matrix, and multiple regression analyses indicated that the visco-elastic parameters were governed mainly by protein solubility and hydro-phobicity. The viscoelastic properties of the opaque gels appeared to be influenced by both the gel matrix and insoluble aggregates, and were related to solubility, hydrophobicity, zeta potential and apparent viscosity of protein dispersions. The types of bonds involved in gel formation and stability were tentatively identified as hydrophobic interactions and hydrogen bonds. With the succinylated isolates, gels were formed in the presence of calcium ions at a concentration an order of magnitude less than was required for similar gel strengths with NaCl, which has implications for exploiting the gelation ability of succinylated protein in products where high concen-trations of NaCl are undesirable. Both emulsification activity and emulsion stability were improved by succinylation. Emulsification activity was related to protein solubility, hydrophobicity, zeta potential and flow behavior of protein dispersions, while emulsion stability appeared to be mainly a measure of resistance to creaming and was related to protein solubility, zeta potential, apparent viscosity of protein dispersions, and the difference in density between the aqueous and oil phases. Succinylation of 54% and 84% of the free amino groups improved the gelation and emulsification properties of canola protein isolate under 126 environmental and processing condi t ions simi lar to those employed in com-minuted meat products . Exhaustive succ iny la t ion was not requ i red to b r i ng about improvements in emuls i f i ca t ion a c t i v i t y and emulsion s t a b i l i t y , whi le the v iscoelast ic proper t ies of thermal ly induced gels appeared to be best w i th moderate succ iny la t ion . 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Chem. 36:719. \ 138 CHAPTER 3 DYNAMIC SHEAR VERSUS PUNCTURE PROBE MEASUREMENTS OF GEL TEXTURE INTRODUCTION R h e o l o g i c a l test methods for gels g e n e r a l l y f a l l into three categories: (1) fundamental te s t s that measure well d e f i n e d r h e o l o g i c a l parameters, (2) em p i r i c a l tests that depend on the geometry of the instrument used and do not provide fundamental r h e o l o g i c a l q u a n t i t i e s , and (3) imitative tests that s i m u l a t e c o n d i t i o n s t h a t a m a t e r i a l w i l l be subjected to i n p r a c t i c e ( M i t c h e l l , 1976). Fundamental tests, which a i d i n e l u c i d a t i n g gel s t r u c t u r e , are u s u a l l y performed at small s t r a i n s on the sample using r e l a t i v e l y s o p h i s t i c a t e d i n strumentation with complex c a l c u l a t i o n s . The test procedures can be v e r y time consuming and the r e s u l t s may not be well c o r r e l a t e d with sensory panel tests on the same ma t e r i a l (Mohsenin and M i t t a l , 1977). E m p i r i c a l and imitative tests are u s u a l l y of the large deformation or f a i l u r e type, are performed r a p i d l y with r e l a t i v e l y simple computation of r e s u l t s , and u s u a l l y are better c o r r e l a t e d with panel tests than are fundamental tests (Mohsenin and M i t t a l , 1977; Wood, 1979). However, sing l e point measurements of "gel s t r e n g t h " based on r u p t u r e tests w i l l not always rank a se r i e s of gels i n the same order as tests which involve small deformations without r u p t u r e ( M i t c h e l l , 1976). E m p i r i c a l test methods are commonly used for q u a l i t y c o n t r o l because of t h e i r s i m p l i c i t y , r a p i d i t y and r e p r o d u c i b i l i t y , although r e s u l t s may be dependent on the type of test employed. Wood (1979) measured the peak forc e and i n i t i a l slope from a compression test performed with an 139 Instron Universal Testing Machine, and the Bloom number for a series of seven gels prepared from agar, xanthan gum plus locust bean gum and carrageenan plus locust bean gum and found that the ranking order for the seven gels using the three different parameters was not the same. Penetration or puncture testing is among the simplest to perform and most widely used methods for the measurement of textural characteristics of food products. Although few studies have looked at the relationship between measurements obtained from empirical test methods and fundamental rheological studies, empirical tests are often used in situations where fundamental tests would be more appropriate but limitations on time, expertise and availability of instrumentation dictate that an empirical test be used. It would be desirable then, for fundamental rheological properties to be approximated or predicted from an empirical test method. The objective of this study was to examine the relationships between textural measurements of canola isolate gels obtained by means of a puncture test with an Instron Universal Testing Machine, and fundamental rheological parameters obtained from dynamic shear measurements with a Weissenberg Rheogoniometer. 140 LITERATURE REVIEW -All polymer dispersions, including gels, possess both fluid-like viscous behavior and solid-like elastic behavior, and are thus termed "viscoelastic". Information about the nature and rates of configurational rearrangements and the disposition and interaction of macromolecules in their short-range and long-range interrelations can be obtained from the measurement of viscoelastic properties (Ferry, 1980). Methods used to study viscoelasticity usually employ very low stress or strain so as to be nondestructive and hence minimize alteration of internal structure. Such methods include creep compliance testing where a small constant stress is applied to a sample and the resulting strain is followed with time, stress relaxation tests where a predetermined strain is suddenly imposed on a sample and kept constant while the relaxation of stress with time is measured, and dynamic testing where small sinusoidal strains are imposed on a sample and the resulting stress and difference in phase between the strain and stress waves are measured (Tung, 1978). Dynamic testing has been performed on a number of food materials including ovalbumen gels (Van Kleef et al., 1978), carrageenan gels (Elliott and Ganz, 1975), alginate gels (Segeren et al., 1974), rapeseed protein gels (Gill and Tung, 1978; Jones, 1980) and gelatin gels (Nijenhuis, 1974). Mitchell (1976, 1980) has extensively reviewed the testing and rheological properties of gels, while Tung (1978) reviewed the rheology of protein dispersions including protein gels. The puncture test is one of the most widely used techniques for measuring food texture and consists of measuring the force (and often the resulting deformation) required to push a probe into a food material. Some puncture tests merely deform the material without rupture while others are performed to 141 a depth that causes irreversible crushing and flow of the food material. Although puncture tests are mechanically simple, they are theoretically complex. Bourne (1979) reviewed the theory and application of puncture testing of food materials. The punch is usually a cylindrical metal probe but numerous other shapes have been used. Bourne (1966) showed that the yield-point force from a puncture test was directly proportional to both the area and perimeter of the punch. Kamel and deMan (1977) reported that the force readings for penetration of gelatin gels were related to the probe size and shape, but there was no relationship between the force readings and pene-tration speed in the range of speeds tested. Mohsenin and Gohlich (1962) reported no significant difference in the shape of force-deformation curves with penetration speeds of 0.05 to 2.5 cm/min. Rasmussen (1974) studied the texture of gels made from gelatin, agar, carrageenans and combinations of these with locust bean gum and guar gum, with a puncture test using an Instron. The author defined several characteristics of the gels from the force-deformation curves. Hermansson (1982) examined the texture of blood plasma gels by puncture and compression tests using an Instron and reported that although the information obtained by each test generally agreed, the compression test better indicated gel structural differences as a function of pH, while the puncture test was better for assessing the effects of temperature and salt induced changes at a constant pH. The initial slope of the stress-strain curve of compression tests is a common index of gel texture. Hermansson (1982) however, found that in spite of large differences in gel structure due to variation of pH, there were no differences in the initial slopes of the stress-strain curves. Mitchell and Blanshard (1976a) found a poor relationship between initial slopes and rupture 142 forces from compression tests of a lg inate gels and concluded that the pr imary molecular weight of the ge l l i ng mater ia l was a governing fac tor i n the t e x t u r a l response. The i n t e r p r e t a t i o n of the s t ress -s t ra in curves from compression or puncture tests is not s t ra igh t fo rward , however. Calzada and Peleg (1978) noted that an apparent ly l inear reg ion of a s t ress -s t ra in curve may be the resu l t of two antagonist ic e f fec ts such as f rac tu re and compression ra ther than being due to an e last ic reg ion . 143 MATERIALS AND METHODS Thermally-induced gels from unmodified or succinylated canola isolate were prepared as described in Chapter 2. Canola isolate was succinylated with 5.2 percent or 14.2 percent succinic anhydride (on a protein basis) which resulted in 54 percent and 84 percent modification of amino groups, respec-tively. Dispersions of the unmodified and succinylated isolates (11.4% w/w) at pH 3.5, 5.0, 6.5, 8.0, 9.5, or 11.0 containing 0.0, 0.35, or 0.7M NaCl were heated at 72°C for 30 min. After cooling in an ice bath and equilibration to room temperature, the samples that formed self-supporting gels were evaluated for texture with a puncture test and for viscoelastic properties with a Weissenberg Rheogoniometer. A. Puncture Test The Puncture test was conducted with a Model 1122 Instron Universal Testing Machine using an 8 ram diameter cylindrical steel probe with a rounded end (Magness-Taylor puncture probe; Bourne, 1968). The probe was driven into each gel to a depth of 12 mm at a crosshead speed of 50 mm/min and a recording chart speed of 200 mm/min. Tests were conducted in duplicate for each sample and the results averaged. The force-deformation curves were analyzed for rupture force (the force at which gel rupture occurred) and the distance the probe had travelled to the point of rupture (Figure 3.1). From these measure-ments were calculated the rupture slope (rupture force/rupture distance), and rupture area (1/2 rupture force x rupture distance) which approximated the area under the force-deformation curve to the point of rupture. 144 0) P Rupture Force (RF) Rupture Slope = Rl/RD Rupture Area = (RFxRD)/2 Rupture Distance (RD)-Distance (mm) Figure 3.1. Force-deformation curve from Instron puncture test for protein gels. 145 B. Dynamic V i s c o e l a s t i c Properties Dynamic shear behavior of the gels was assessed as described in Chapter 2. After the puncture tests were completed, the gels were carefully removed from the containers in which they were formed and the bottom portion of each gel cylinder, which was undisturbed by the puncture test, was used for dynamic testing by a Weissenberg Rheogoniometer equipped with 5 cm diameter parallel plate fixtures. The storage modulus, loss modulus and loss tangent of each gel were calculated for a frequency of 10 s -* from dynamic shear rheograms obtained over a frequency range of 0.19 to 19 s -*. Simple linear, multiple linear and polynomial regressions using the dynamic shear parameters as dependent variables and the puncture test measure-ments as potential independent variables were performed with the MIDAS statistical computer program (Fox and Guire, 1976) on the U.B.C. Amdahl 470/V8 mainframe computer. 146 RESULTS AND DISCUSSION Twenty-seven self-supporting gels that were formed under various con-ditions of pH, sodium chloride concentration and extent of protein succiny-lation were tested. The physical appearance of the gels ranged from opaque and pasty to translucent and springy as described in Chapter 2. The relationship between rupture force of the puncture test and storage modulus at 10 s -* is shown in Figure 3.2. Although significant (p<0.01, Table 3.1), rupture force alone accounted for less than thirty percent of the varia-tion in storage modulus. Of the twenty-seven gels tested, seventeen were classified as translucent and ten were opaque, although one of the opaque gels had a great deal of translucent character and seemed to be intermediate between the two types. Rupture force appeared to be related in a curvilinear manner to the nine gels with the most opaque character, but there was a poor relationship between rupture force and storage modulus for the translucent gels (Figure 3.2). The relationship between rupture slope and storage modulus is shown in Figure 3.3. For all gels, a highly significant correlation was observed between the two variables ( r 2 = 0.857, Table 3.1). For the translucent gels, rupture slope accounted for nearly ninety-five percent of the variation in storage modulus (Table 3.1). For the opaque gels, storage modulus was also well described by rupture slope (R 2 = 0.857, p<0.001), but the slope of the linear relationship between the two variables was greater than for the translucent gels. Thus for a given storage modulus, the rupture slope of the translucent gels was higher than for the opaque gels, which indicated a difference in response of opaque and translucent gels to the two types of gel texture measurement. The storage modulus of the opaque gels also followed a 147 900 800-700-.p-, 600 V) D "5 500H T5 O © 400 H g) O 300-(7) 200 100 0 • 00 A • • o Legend A Unmodified O 5.2% Succ. An. • 14.2% Succ. An. i 50 100 150 200 250 300 Rupture Force (mN) Figure 3.2. Rupture force vs. storage modulus for canola isolate gels (solid symbols indicate opaque gels). & in 3 TJ O 0) O) O i_ o tn 900 800-700-600-500-400-300-200-100-• 9 CO o Legend A Unmodified O 5.2% Succ. An. • 14.2% Succ. An. 10 20 30 40 50 60 70 Rupture Slope (mN/mm) 80 90 Figure 3.3. Rupture slope vs. storage modulus for canola isolate gels (solid symbols indicate opaque gels). Table 3.1. Rheogoniometer vs. Instron measurements of g e l texture. 148 Dependent Variable Independent Variable Coefficient F-prob. A. A l l gels (n=27) Storage Modulus r 2 = 0.296** S.E.a - 224.2 Storage Modulus r 2 = 0.857*** S.E. = 101.0 Loss Modulus R2 - 0.840*** S.E. • 28.19 Rupture Force Constant Rupture Slope Constant Rupture Slope Rupture Area Constant 2.275 x IO3 104.03 12.12 x 103 -4.260 2.912 x 103 •133.34 27.964 0.0033 0.0000 0.0000 0.0001 Loss Tangent R2 = 0.800*** S.E. = 0.029 Rupture Area Rupture Area2 Constant -0.536 0.389 0.321 0.0000 0.0085 B. Translucent gels (n=17) Storage Modulus r 2 = 0.946*** S.E. = 54.11 Rupture Slope Constant 10.94 x 103 -4.992 0.0000 Loss Modulus m R2 = 0.940*** S.E. = 13.19 Rupture Slope Rupture Area Constant 2.433 x 103 -80.123 16.229 0.0000 0.0038 C. Opaque Gels (n=10) Storage Modulus r 2 = 0.857*** S.E. = 126.0 Storage Modulus R2 = 0.958*** S.E. = 78.44 Rupture Slope Constant Rupture Slope Rupture Area Rupture Area2 Constant 13.83 x 103 4.156 7.961 x 103 40.321 0.192 -52.134 0.0001 0.0058 0.0091 0.0087 Loss Modulus r 2 = 0.779*** S.E. - 41.30 Rupture Slope Constant 3.469 x 103 1.552 0.0007 **, p<0.01; ***, p<0.001 aStandard error of estimate. 149 curvilinear relationship with rupture area, where storage modulus first increased and then decreased as rupture area increased. Rupture slope and rupture area (quadratic) together accounted for nearly ninety-six percent of the variation in storage modulus for these gels (Table 3.1). The relationship between loss modulus and rupture slope is shown in Figure 3.4. For all gels, loss modulus was well predicted by rupture slope and rupture area which together accounted for eighty-four percent of the variation in loss modulus (Table 3.1). Loss modulus increased as rupture slope increased and rupture area decreased. For the translucent gels, rupture slope and rupture area together accounted for ninety-four percent of the variation in loss modulus, while for the opaque gels rupture slope alone accounted for nearly seventy-eight percent of the variation in loss modulus. The loss tangent of the gels was well described by the rupture area of the puncture test (Figure 3.5, Table 3.1). As rupture area increased, loss tangent decreased in a curvilinear manner. The quadratic polynomial of rupture area accounted for eighty percent of the variation in loss tangent. No improvement in predictive ability was obtained by subdividing the gels into translucent and opaque for the analysis of loss tangent. In dynamic testing, the storage modulus is a measure of the energy stored per cycle of sinusoidal deformation, the loss modulus is a measure of the energy dissipated as heat, while the loss tangent is the proportion of energy dissipated to energy recovered per cycle (Ferry, 1980). The force-deformation (stress-strain) relationship obtained from a puncture or compression test indicates how a material deforms under load. The force-deformation curve represents the force required to obtain a given deformation at any point in the test while the area under the curve to the point of rupture is a measure of the energy required to rupture the sample. 150 CD Z3 o CO to o 250 200-150 100-50-cO 1 B Legend A Unmodified O 5.2% S u c c . An. • 14.2% S u c c . An. - T — 10 - T — 20 30 40 50 60 Rupture Slope (mN/mm) 70 - i — 80 90 Figure 3.4. Rupture slope vs. loss modulus for canola isolate gels ( s o l i d symbols indicate opaque ge l s ) . 0.4-1 0.3-c V o> c tn tn o 0.2 0.1 100 ° O D ° O  AD 0 ^ Legend A Unmodified O 5.2% S u c c . An. • 14.2% S u c c . An. i i i i i i r 200 300 400 500 600 700 800 Rupture Area (mN mm) Figure 3.5. Rupture area vs. loss tangent for canola isolate gels ( s o l i d symbols indicate opaque g e l s ) . 151 The initial slope of the force-deformation curve has been variously described as the elasticity modulus, apparent elasticity, rigidity, stiffness, deformability, and initial tangent (Hermansson, 1982; Mohsenin and Mittal, 1977). Mitchell (1976, 1980) and Mitchell and Blanshard (1976a,b) stated that the rigidity modulus of a gel is not necessarily related to its rupture strength, as the rigidity modulus is influenced primarily by short, stiff chains present in the gel whereas the rupture strength is governed by the number of long, flexible chains remaining after the cross-links joined by the short, stiff chains have ruptured. They suggested that this may explain why the rigidity modulus was independent of molecular weight above a certain critical value, while the rupture strength appeared to be strongly dependent on molecular weight (Mitchell and Blanshard, 1976a,b). Mohsenin (1970) and Mohsenin and Mittal (1977), however, stated that elasticity as defined by Hooke's Law does not really exist in food materials, as even some very hard materials show some unrecoverable deformation upon unloading. Hermansson (1982) and Mohsenin and Mittal (1977) suggested that the use of the term "elasticity modulus" be avoided in the case of non-linear stress-strain behavior or where the upper limit of the elastic region is unknown. Calzada and Peleg (1978) also cautioned that an apparently linear region of a stress-strain curve may not be due to an elastic region but may be a result of two antagonistic effects such as fracture and compression. Mohsenin (1970) suggested that a "modulus of deformability" be used instead, where the total elastic and plastic deformation at some selected point on the force-deforma-tion curve is taken for computing the strain. Using this concept, Mohsenin and Mittal (1977) computed a modulus of deformability for apple tissue at one-half the total deformation to the point of inflection, and as expected found no correlation between it and the force of deformation at the bioyield point. 152 They explained this by pointing out that the bioyield point is a failure phenomenon whereas the modulus of deformability is an indication of the stiffness of the tissue when small forces are applied, so the two measurements should not be expected to be correlated. In the present study, the rupture slope was highly correlated with the storage and loss moduli of the gels, and was faster and simpler to obtain than either the initial slope of the force-deformation curve or the modulus of deformability as defined by Mohsenin (1970). In addition, rupture area as calculated from the force and deformation at rupture, although an approxi-mation of the true area, was simpler to calculate and was well correlated with the loss tangent of the gels, and along with rupture slope was a significant predictor of storage modulus for the opaque gels and loss modulus for all gels. The different relationship between the dynamic viscoelastic parameters and the puncture test measurements for the translucent and opaque gels was probably the result of differences in microstructure between the two types of gels. Although the microstructure of the gels per se was not examined, light micrographs of the unheated dispersions revealed the presence of numerous insoluble aggregates in the dispersions that formed opaque gels, while the dispersions that formed translucent gels were relatively homogeneous (see Chapter 2). Under the small deformation, nondestructive conditions employed in dynamic testing, the moduli obtained would be the result of contributions from both the insoluble aggregates and the interaggregate gel matrix in the case of the opaque gels, but from the homogeneous gel matrix alone in the case of translucent gels. With the large deformations employed by the puncture test, however, the force-deformation curves for the opaque gels would reflect mainly the strength of the interaggregate matrix and the friction 153 between aggregates as they move with respect to one another. Thus for a given storage modulus, opaque gels would be expected to have a smaller rupture slope than translucent gels where the rupture slope and dynamic viscoelastic parameters reflect the microstructure of the homogeneous matrix, and this was indeed the case. Gill and Tung (1978) found a similar response to dynamic and steady shearing conditions of gels from the 12S fraction of rapeseed protein that contained aggregates. They hypothesized that the aggregates gave highly elastic recoveries under dynamic shear, but under steady shear the forces between the aggregates would be broken and the aggregates would be able to move with respect to one another resulting in lower apparent viscosities than from gels with an homogeneous matrix. 154 SUMMARY AND CONCLUSIONS The relationships between a large deformation puncture test and a non-destructive dynamic test method for assessing the rheological properties of canola protein gels were examined. Although the force required to rupture the gels, as measured by the puncture test, was poorly correlated with the viscoelastic parameters, the slope of the force-deformation curves to the point of rupture was well correlated with the storage and loss moduli of the gels. In addition, the area under the force-deformation curves followed a curvilinear relationship with the loss tangent of the gels. Of the 27 gels tested, 17 were translucent and 10 were opaque. The responses of the tran-slucent and opaque gels to the two types of rheological tests were not iden-tical, which indicated that gel microstructure may be an influential factor when evaluating gel texture by destructive or nondestructive methods. Although excellent correlations between puncture test measurements and viscoelastic parameters for canola protein gels were demonstrated, it is not certain that similar results would be obtained with gels from other gelation conditions, protein sources or concentrations, or with non-protein gels where the types of bonds involved in gel formation and stabilization may influence the results. Although more work is required to clarify these relationships, the present data provide a framework on which to base future research in this area. 155 REFERENCES Bourne, M.C. 1966. Measurement of shear and compression components of puncture tests. J. Food Sci. 31:282. Bourne, M.C. 1968. Texture profile of ripening pears. J. Food Sci. 33:223. Bourne, M.C. 1979. Theory and application of the puncture test in food texture measurement. In: Food Texture and Rheology. Sherman, P. (Ed.). Academic Press, New York, NY. Calzada, J.F. and Peleg, M. 1978. Mechanical interpretation of compressive stress-strain relationships of solid foods. J. Food Sci. 43:1087. Elliott, J.H. and Ganz, A.J. 1975. Gel characterization with the Weissenberg rheogoniometer: application to carrageenan gels. J. Food Sci. 40:394. Ferry, J.D. 1980. Viscoelastic Properties of Polymers, 3rd ed. Wiley, New York, NY. Fox, D.J. and Guire, K.E. 1976. Documentation for MIDAS, 3rd ed. The Statistical Research Laboratory, The University of Michigan, Ann Arbor, MI. Gi l l , T.A. and Tung, M.A. 1978. Thermally induced gelation of the 12S rapeseed glycoprotein. J. Food Sci. 43:1481. Hermansson, A.-M. 1982. Gel characteristics - compression and penetration of blood plasma gels. J. Food Sci. 47:1960. Jones, L.J. 1980. Functional properties of modified oilseed protein con-centrates and isolates. M.Sc. Thesis, University of British Columbia, Vancouver, B.C. Kamel, B.S. and deMan, J.M. 1977. Some factors affecting gelatin gel texture evaluation by penetration testing. J. Texture Stud. 8:327. Mitchell, J.R. 1976. Rheology of gels. J. Texture Stud. 7:313. Mitchell, J.R. 1980. The rheology of gels. J. Texture Stud. 11:315. Mitchell, J.R. and Blanshard, J.M.V. 1976a. Rheological properties of alginate gels. J. Texture Stud. 7:219. Mitchell, J.R. and Blanshard, J.M.V. 1976b. Rheological properties of pectate gels. J. Texture Stud. 7:341. Mohsenin, N.N. 1970. Physical Properties of Plant and Animal Materials -Structure, Physical Characteristics and Mechanical Properties. Gordon and Breach Science Publishers, New York, NY. 156 Mohsenin, N.N. and Goehlich, H. 1962. Techniques for determination of mech-anical properties of fruits and vegetables as related to design and development of harvesting and processing machinery. J. Agric. Eng. Res. 7:300. Mohsenin, N.N. and Mittal, J.P. 1977. Use of rheological terms and cor-relation of compatible measurements in food texture research. J. Texture Stud. 8:395. Nijenhuis, K. te 1974. Investigation into the ageing process of gel systems by the measurement of their dynamic moduli. Dechema-Monogr. 77:177. Rasraussen, J. 1974. Gel texture in foods - its relationship to choice of gelling agent, formulae and processing conditions. Dechema-Monogr. 77:187. Segeren, A.J.M., Boskamp, J.V. and van den Tempel, M. 1974. Rheological and swelling properties of alginate gels. Faraday. Diss. Chem. Soc. 57:255. Tung, M.A. 1978. Rheology of protein dispersions. J. Texture Stud. 9:3. Van Kleef, F., Boskamp, J. and van den Tempel, M. 1978. Determination of the number of cross-links in a protein gel from its mechanical and swelling properties. Biopolymers 17:225. Wood, F.W. 1979. Psychophysical studies on liquid foods and gels. In: Food Texture and Rheology. Sherman, P. (Ed.), p.21. Academic Press, New York, NY. Publications (cont.) G i l l , T.A. and Paulson, A.T. 1982. Localization, characterization and partial purification of TMAD-ase. Comp. Biochem. Physiol. 71B:49. Paulson, A.T., Vanderstoep, J. and Porritt, S.W. 1980. Enzymatic brcwning of peaches: effects of gibberellic acid and ethephon on phenolic compounds and polyphenoloxidase activity. J. Food Sci. 45:341. Paulson, A.T., Vanderstoep, J. and Eaton, G.W. 1979. Effects of gibberellic acid and ethephon on enzymatic browning of "Redhaven" peaches. HortScience 14:711. Douglas, M., Vanderstoep, J. and Paulson, A.T. 1977. Effect of gibb-erellic acid and ethephon on ascorbic acid content and ascorbic acid oxidase activity of Redhaven peaches. Can. Inst. Food Sci. Technol. J. 10:233. 

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