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Functional properties of modified oilseed protein concentrates and isolates Jones, Linda Jean 1980

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FUNCTIONAL PROPERTIES OF MODIFIED OILSEED PROTEIN CONCENTRATES AND ISOLATES by LINDA JEAN JONES B.Sc. (Agr.) Honours, University of B r i t i s h Columbia, 1977 A THESIS SUBMITTED LN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE 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 December, 1980 © Linda Jean Jones, 1980 In presenting th i s thesis in par t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It i s understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my writ ten permission. Department of frc^ vS-j'ps^re. The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i i i -ABSTRACT Compositional, s t r u c t u r a l and functional properties of nine o i l s e e d concentrates and i s o l a t e s were evaluated and compared. Three product sources, canola (rapeseed), sunflower and soybean were investigated, each treated during processing by enzyme-hydrolysis or l i n o l e i c acid addition, or l e f t untreated as controls. Protein, carbohydrate and moisture contents of the products were measured, and examination of the gross structure of 10% aqueous dispersions was car r i e d out using l i g h t microscopy techniques. Several f u n c t i o n a l i t y tests were performed, including determinations of water holding and water hydration c a p a c i t i e s , protein s o l u b i l i t y , steady shear flow behavior and v i s c o e l a s t i c i t y . Scanning electron microscopy was used to examine the microstructure of the g e l - l i k e materials produced when 10% aqueous dispersions of the products were heated i n a b o i l i n g water bath. Both t r y p s i n and l i n o l e a t e treatment were observed to s i g n i f i c a n t l y a f f e c t the s t r u c t u r a l and functional properties of the products, although the treatment effects d i f f e r e d among sources i n some cases. In general, trypsin-treated products contained smaller p a r t i c l e s i n dispersion, formed weaker gels on heating and exhibited higher s o l u b i l i t y and water hydration ca p a c i t i e s , and lower apparent v i s c o s i t i e s i n 10% dispersion. A trend toward larger p a r t i c l e s i n li n o l e a t e - t r e a t e d product dispersions was observed along with stronger gel-forming a b i l i t i e s , increased s o l u b i l i t i e s , water hydration capacities and apparent v i s c o s i t i e s i n 10% dispersion. Simple c o r r e l a t i o n c o e f f i c i e n t s between several measured functional and compositional variables were computed to i d e n t i f y r e l a t i o n s h i p s among the physico-chemical properties. Both the size of the dispersed phase p a r t i c l e s and the degree of i n t e r a c t i o n of the protein and carbohydrate components with water were determined to be important i n governing functional properties. - i v -TABLE OF CONTENTS Page ABSTRACT i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS ix INTRODUCTION 1 LITERATURE REVIEW 3 A. General 3 B. Enzyme and Surfactant Treatments 6 C. Protein S o l u b i l i t y 7 D. Water Hydration Properties 9 E. Rheological Properties 11 F. Gelation 15 G. Microstructure 17 MATERIALS AND METHODS 19 A. Source of Products 19 B. Chemical Composition 22 1. Crude protein 22 2. Carbohydrate 23 3. Moisture 24 C. S t r u c t u r a l and Functional Properties 24 1. S t r u c t u r a l properties 25 a) Light microscopy 25 b) Scanning electron microscopy 26 2. Functional properties 27 a) Protein s o l u b i l i t y 27 b) Water holding capacity 28 c) Water hydration capacity 28 d) Steady shear flow behavior 30 e) Dynamic shear flow behavior 32 - v -• Page D. S t a t i s t i c a l Analysis 33 1. F a c t o r i a l analysis of variance 33 2. Single factor analysis of variance 33 3. Simple correlations 34 RESULTS AND DISCUSSION 35 A. Composition 35 1. Crude protein 35 2. Carbohydrate 37 3. Moisture 38 4. Other components 38 B. S t r u c t u r a l Properties 38 1. Light microscopy - dispersions 38 2. Scanning electron microscopy - thermal aggregation 46 C. Functional Properties 63 1. pH 63 2. Conductance 63 3. Protein s o l u b i l i t y 65 4. Water hydration and water holding capacities 67 5. Rheological properties 70 a) Steady shear 70 b) Dynamic shear 75 D. S t a t i s t i c a l Analysis 83 1. Analysis of variance 83 2. Simple correlations among variables 83 CONCLUSIONS 92 LITERATURE CITED 96 - v i -LIST OF TABLES Page Table I Example of water hydration capacity determination. 29 Table II Mean values of protein, carbohydrate and moisture 36 contents of canola, sunflower, and soybean products. Table I II Mean values of pH and conductance for 10% dispersions 64 of canola, sunflower and soybean products (n = 2). Table IV Proportion of protein i n 10% aqueous dispersions present 66 i n soluble state (n = 2). Table V Mean values of water hydration and water holding capacities 68 of canola, sunflower and soybean products (n = 2). Table VI Mean values of steady shear Power-law flow parameters of 73 10% aqueous dispersions of canola, sunflower and soybean products (n = 2). Table VII Mean values of v i s c o e l a s t i c properties of 10% aqueous 79 dispersions of canola, sunflower and soybean products at 20°C (n = 2). Table VIII Levels of si g n i f i c a n c e of F values calculated from data 84 co l l e c t e d for each measured variable using a 3 x 3 (source x treatment) f a c t o r i a l analysis of variance. Table IX Results of Duncan's multiple range tests for variables 85 for which the source x treatment i n t e r a c t i o n was not s i g n i f i c a n t (p>0.05). Table X Correlation c o e f f i c i e n t s calculated for l i n e a r 86 cor r e l a t i o n s among a l l measured variables (n = 9). - v i i -LIST OF FIGURES Page Figure 1 Preparation of protein concentrates and i s o l a t e s . 20 Figure 2 Procedure employed for treating the products with 21 try p s i n and l i n o l e a t e . Figures 3-11 Light micrographs of 10% aqueous dispersions 40 (pw = 500 ym) Figure 3 Canola c o n t r o l . 41 Figure 4 Canola t r y p s i n . 41 Figure 5 Canola l i n o l e a t e . 41 Figure 6 Sunflower c o n t r o l . 43 Figure 7 Sunflower t r y p s i n . 43 Figure 8 Sunflower l i n o l e a t e . 43 Figure 9 Soybean co n t r o l . 45 Figure 10 Soybean try p s i n . 45 Figure 11 Soybean l i n o l e a t e . 45 Figures 12-25 Scanning electron micrographs of heated 10% aqueous 47 dispersions. Figure 12 Canola control (pw = 60 Um). 48 Figure 13 Canola control (pw = 20 ym). 48 Figure 14 Canola t r y p s i n (pw = 60 Um). 50 Figure 15 Canola trypsin (pw = 20 ym). 50 Figure 16 Canola l i n o l e a t e (pw = 60 ym). 52 Figure 17 Canola l i n o l e a t e (pw = 20 ym). 52 Figure 18 Sunflower control (pw = 60 ym). 54 Figure 19 Sunflower control (pw = 300 ym). 54 Figure 20 Sunflower t r y p s i n (pw = 60 ym). 56 - v i i i -Page Figure 21 Sunflower l i n o l e a t e (pw = 60 Um). 56 Figure 22 Soybean control (pw = 150 Um). 58 Figure 23 Soybean l i n o l e a t e (pw = 300 Um). 58 Figure 24 Soybean l i n o l e a t e (pw = 60 Um). 60 Figure 25 Soybean l i n o l e a t e (pw = 20 Um). 60 Figure 26 Flow behavior rheograms of untreated (c ) , t r y p s i n (T) 71 and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products i n 10% aqueous dispersions at 20°C. Figure 27 E f f e c t of frequency on storage moduli of 10% product 76 dispersions at 45°C. Figure 28 E f f e c t of frequency on loss tangents of 10% product 77 dispersions at 45°C. Figure 29 E f f e c t of temperature on storage moduli of 10% product 81 dispersions at a frequency of 6 s _ i . Figure 30 E f f e c t of temperature on loss tangents of 10% product 82 dispersions at a frequency of 6 s - i . Figure 31 Correlation between apparent v i s c o s i t y (100 s - i ) of 10% 88 product dispersions at 20°C and protein content of untreated (C), trypsin (T) and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products. Figure 32 C o r r e l a t i o n between water hydration capacity and protein 89 content of untreated (c ) , t r y p s i n (T) and l i n o l e a t e (L) * treated canola (Ca), sunflower (Su) and soybean (So) products. Figure 33 Correlation between apparent v i s c o s i t y (100 s ~ l ) of 10% 90 product dispersions at 20°C and water hydration capacity of untreated (C), trypsin (T) and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products. Figure 34 C o r r e l a t i o n between consistency c o e f f i c i e n t (m) and flow 91 behavior index (n) for 10% dispersions of untreated ( c ) , trypsin (T) and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products at 20°C. - ix -ACKNOWLEDGEMENT S The author wishes to express her appreciation to Dr. M.A. Tung, Department of Food Science, U.B.C, for h i s support of this research project, and h i s review of the manuscript. Thanks are also extended to Dr. W.D. Powrie and Dr. S. Nakai of the Department of Food Science and Dr. J . Gosline of the Department of Zoology for th e i r suggestions and comments. - 1 -INTRODUCTION Oilseeds have become important a g r i c u l t u r a l crops throughout the world. Although grown p r i m a r i l y as a source of o i l , considerable i n t e r e s t has been focussed recently on the protein fractions of these commodities. Following removal of the o i l s by pressing and solvent extraction, the defatted meals may contain over 50% protein. The meals are extensively u t i l i z e d i n animal feeds, while a small proportion i s incorporated into foods for human consumption. Processing of the meals to remove the solvent and varying amounts of soluble and insoluble carbohydrate must be c a r r i e d out on products destined for use i n foods. The end product may contain 50 to 95% protein, depending on the processing techniques employed. Soybean ranks f i r s t i n world oilseed production followed by cottonseed, peanut, sunflower and rapeseed (canola) (FAO, 1978). Of these, only canola and sunflower can be produced i n large quantities i n Canada. The n u t r i t i o n a l value of these high-protein o i l s e e d products i s high, with Protein E f f i c i e n c y Ratios ranging as high as 1.8 (Wolf and Cowan, 1971). However, often i t is the functional and not the n u t r i t i o n a l q u a l i t i e s that determine the a c c e p t a b i l i t y of the product to the consumer. Important functional properties are water hydration, e m u l s i f i c a t i o n , v i s c o s i t y , gel formation, foaming and s o l u b i l i t y . Evaluations of these properties provide indications of the s t r u c t u r a l and textural q u a l i t i e s of foods into which the oils e e d products may be incorporated. Improvements i n f u n c t i o n a l i t y of an oilseed protein product may be achieved by manipulating processing conditions. High protein s o l u b i l i t y can be attained - 2 -by maintaining low temperatures. The addition of modifying agents, such as enzymes or surfactants, i s another method of c o n t r o l l i n g f u n c t i o n a l i t y . The research described i n this thesis was undertaken to investigate the e f f e c t s of enzymatic and fat t y acid treatment on the functional properties of soybean and sunflower concentrates and a canola (rapeseed) i s o l a t e . Several functional properties were evaluated including protein s o l u b i l i t y , water hydration capacity and flow behavior. The composition of the products and th e i r s t r u c t u r a l properties were also investigated. - 3 -LITERATURE REVIEW A. General The production and processing of oi l s e e d crops has expanded over the past 40 years to such an extent that oilseeds now rank among the most important a g r i c u l t u r a l cash crops throughout the world. Soybean is the most dominant o i l s e e d on the world scene. Total world production of t h i s crop i n 1978 amounted to over 80 m i l l i o n metric tonnes (FAO, 1978). Although the o i l content is r e l a t i v e l y low, the crop is e a s i l y c u l t i v a t e d and provides a high crop y i e l d (Seal, 1978). Processing of soybeans to separate the o i l from the meal fracti o n s began i n the 1930's. The r e s u l t i n g o i l i s used for salad and cooking o i l s , and is hydrogenated to produce margarines and shortenings. The defatted meal i s used predominantly as a pro t e i n source for animal feeds, however a small but growing proportion ( 3-4%) ( K i n s e l l a , 1979) is being used as a food ingredient. For food uses, the defatted meal i s further processed into three classes of protein products. Soy flours are defatted, desolventized, deodorized and ground flakes which are used extensively i n bakery and cereal products. Concentrates are prepared by removing soluble components from defatted soy flour using a c i d i c , aqueous ethanol or hot water leaching agents. These products have better flavor and color and are higher i n protein than the flours and can therefore be used i n greater quantities i n many of the same foods. Soy is o l a t e s are produced from f l o u r that has been subjected to a minimum of heat treatment. The protein i s dissolved i n d i l u t e a l k a l i (pH 8.0), insolubles are removed by cen t r i f u g a t i o n or f i l t r a t i o n and the protein is p r e c i p i t a t e d out by lowering the pH to the i s o e l e c t r i c range (near pH 4.5). Isolates are used i n the manufacture of comminuted meats and - 4 -dairy foods where good emulsifying, thickening and g e l l i n g properties are important (Wolf and Cowan, 1971). Soybean proteins are mainly globulins, ranging i n molecular weight from 8,000 to 600,000 daltons, with minimum s o l u b i l i t i e s near pH 4.5. The major globulins are described by their sedimentation c o e f f i c i e n t s , 7 S and 11 S, and have complex quaternary structures (Wolf, 1970). The 7 S f r a c t i o n contains at least four d i f f e r e n t proteins, and has a carbohydrate content of approximately 6% (Wolf, 1977). Soy proteins are r e l a t i v e l y high i n the amino acid l y s i n e , as compared to other cereals; however, they are low i n methionine. Other o i l s e e d crops have been processed to produce p r o t e i n - r i c h products s i m i l a r to soy f l o u r s , concentrates and i s o l a t e s . Two oilseeds which are suitable for c u l t i v a t i o n i n Canadian a g r i c u l t u r a l and c l i m a t i c conditions are rapeseed (canola) and sunflower. Sunflower ranks fourth in importance among world vegetable o i l crops, and the defatted meal has considerable p o t e n t i a l as a food protein source. Although d e f i c i e n t i n ly s i n e , the protein is highly d i g e s t i b l e and contains no known toxic or a n t i - n u t r i t i v e f a c t o r s . Sosulski et a l . (1973) devised a procedure for extracting low molecular weight compounds that were found to be responsible for previously reported disagreeable dark brown and green colors. Globulins constitute 70 to 79% of the proteins from sunflower. The major g l o b u l i n has a sedimentation c o e f f i c i e n t of 12.1 S and a molecular weight of about 330,000 daltons. The next most predominant g l o b u l i n has a molecular weight of 20,000 daltons (Sabir et a l . , 1973). Rapeseed ranks f i f t h i n t o t a l world o i l s e e d production. Food uses of the meal have been limited u n t i l recently, due to high fi b e r l e v e l s and the presence of glucosinolates which have a n t i t h y r o i d a c t i v i t y . Low glucosinolate - 5 -v a r i e t i e s of rapeseed, safe for human consumption, are now being grown i n Canada and these new v a r i e t i e s are ca l l e d canola, rather than rapeseed, to d i s t i n g u i s h them from e a r l i e r v a r i e t i e s (Biely, 1980). A high molecular weight g l o b u l i n (12 S, 134,000 daltons) ( G i l l and Tung, 1976) comprises the major rapeseed protein f r a c t i o n (35%). Finlayson et a l . , (1969) reported that approximately 10% of the t o t a l seed nitrogen exists as a strongly basic g l o b u l i n with a molecular weight of 13,500 daltons and a sedimentation c o e f f i c i e n t of 1.7 S. Hydrophobic residues dominate the amino acid p r o f i l e of rapeseed proteins, while basic and a c i d i c amino acids are i n r e l a t i v e l y low concentration (Sosulski and Sarwar, 1973). The role of o i l s e e d protein products as food ingredients can be n u t r i t i o n a l , functional or i n many cases, both. Although oils e e d proteins are not n u t r i t i o n a l l y "complete", they can serve as excellent sources of many es s e n t i a l amino acids (Sosulski and Sarwar, 1973). In many cases, however, i t is functional and not n u t r i t i o n a l q u a l i t i e s that determine the a c c e p t a b i l i t y of the oilseed protein product to the consumer. As defined by K i n s e l l a (1979), functional properties are "the i n t r i n s i c physicochemical character-i s t i c s which a f f e c t the behavior of protein i n food systems during processing, manufacturing, storage and preparation". Functional properties are controlled by the composition and structure of the proteins and the interactions of proteins with one another and with other substances. The c h a r a c t e r i s t i c s can be modified or improved by d e l i b e r a t e l y a l t e r i n g the proteins. Wall (1979) found that denaturation of protein was b e n e f i c i a l i n improving protein hydration, r e s u l t i n g i n products with more desirable texture. Dalek et a l . (1970), through work on v i t a l wheat gluten, showed that drying methods strongly affected s o l u b i l i t y . Spray drying was - 6 -found to cause mechanical damage to the secondary and quaternary protein structure, r e s u l t i n g i n increased s o l u b i l i t y . Decreased s o l u b i l i t y due to thermal denaturation was reported by these authors for drum dried samples. The remaining sections of this l i t e r a t u r e review deal with research reported by other authors i n the f i e l d of protein modification for improved f u n c t i o n a l i t y . In addition, several functional properties w i l l be discussed i n d i v i d u a l l y , o u t l i n i n g t h e i r t h e o r e t i c a l bases and describing methodologies employed i n t h e i r evaluation. B. Enzyme and Surfactant Treatments Enzyme hydrolysis of soy protein was investigated by Puski (1975) using a neutral protease preparation from Aspergillus oryzae. Protein hydrolyzed by this enzyme was found to have higher s o l u b i l i t y , lower v i s c o s i t y i n aqueous dispersion and decreased gel-forming a b i l i t i e s . Pepsin hydrolyzates of soy proteins are used as foaming agents i n confections and bakery products (Wolf and Cowan, 1971). The s o l u b i l i t y of these hydrolyzates i n the i s o e l e c t r i c range (pH 4 to 5) is much higher than unmodified proteins. Thus, i n a c i d i c foods such as c i t r u s j u i c e s , these modified proteins could be incorporated without protein p r e c i p i t a t i o n and sedimentation. Generally, the foaming power of the protein is increased by enzymatic treatment, however the foam s t a b i l i t y i s poor (Horiuchi et a l . , 1978). Nash and Wolf (1967) reported that small amounts of unhydrolyzed protein, added to the hydrolyzates, produce stable foams. Soy protein hydrolyzates have b i t t e r and beany flavors which can be removed by u l t r a f i l t r a t i o n (Roozen and P i l n i k , 1973). The use of enzyme hydrolysis to a l t e r the properties of f i s h protein concentrate (Cheftel et a l . , 1971), cottonseed protein (Arzu et a l . , 1972) and egg albumen (Grunden et a l . , 1974) are also reported i n the l i t e r a t u r e . - 7 -Catsimpoolas and Meyer (1971b) prepared dispersions of soy i s o l a t e con-taini n g various types and concentrations of l i p i d . They found that the v i s c o s i t y of the dispersions increased with the concentration of t r i g l y c e r i d e added. This e f f e c t was attributed to the arrangement of protein molecules around the l i p i d to form micelles. Polar l i p i d s i n flour have been used for many years to improve the mixing c h a r a c t e r i s t i c s and strength of doughs. Wall (1979) c i t e d enhanced aggregation and cohesion of the wheat gluten protein as responsible for the improvement. Kobrehel and Bushuk (1977) found s a l t s of f a t t y acids to be e f f e c t i v e in s o l u b l i z i n g freeze-dried glutenin. Canella et a l . (1979) reported that surfactants disrupt hydrophobic bonds between proteins. Nakai et a l . (1980b) at t r i b u t e d the s o l u b l i z i n g action of anionic surfactants on o i l s e e d proteins to the i n t e r a c t i o n between hydrophobic groups on the protein and the surfactant. These interactions would have the e f f e c t of increasing negative charges on the protein due to the anionic t a i l (carboxyl group), r e s u l t i n g i n increased protein-protein repulsion. C. Protein S o l u b i l i t y Most globulins are insoluble i n the range of pH near the i s o e l e c t r i c point, but are soluble i n water or d i l u t e s a l t solutions above or below the i s o e l e c t r i c point. It i s generally assumed that association of protein molecules decreases s o l u b i l i t y and d i s s o c i a t i o n increases s o l u b i l i t y (Hermansson, 1973a). Ionizable amino acids play an important r o l e in determining the e l e c t r o s t a t i c interactions between protein molecules, and therefore pH, s a l t s and temperature influence the balance between a t t r a c t i v e and repulsive forces that controls s o l u b i l i t y (Wall, 1979). - 8 -The s o l u b i l i t i e s of complex protein systems d i f f e r considerably from the s o l u b i l i t y behavior of native proteins. Processing techniques strongly a f f e c t s o l u b i l i t y (Hutton and Campbell, 1977). After i s o e l e c t r i c p r e c i p i t a t i o n , the proteins i n a soy i s o l a t e are no longer completely soluble in a pH 7.6, 0.5 M phosphate buffer (Nash and Wolf, 1967). Thermal treatment during processing disorders the protein, permitting unfolding and the p o s s i b i l i t y of new interactions which may cause i n s o l u b i l i t y through aggregation. Hydrogen and hydrophobic interactions are reported by Hermansson (1973a) to be the major bonds a f f e c t i n g s o l u b i l i t y . The s o l u b i l i t y behavior of a p r o t e i n - r i c h product is often used as an index of the p o t e n t i a l or l i m i t a t i o n s of the material as a food component ( K i n s e l l a , 1976). A high s o l u b i l i t y is required, for example, i n proteins which are to be added to beverages (Wall, 1979). Protein s o l u b i l i t y is often used as an index of denaturation. Nash et a l . (1971) observed a rapid decrease i n s o l u b i l i t y when soy proteins were denatured. However, Hermansson (1979a), using d i f f e r e n t i a l scanning calorimetry techniques to follow denaturation, observed that high s o l u b i l i t y could be obtained from completely denatured soy proteins. The techniques used to determine protein s o l u b i l i t y are empirical, not e a s i l y duplicated, and often have l i t t l e relevance to the behavior of the protein in a food system. The most commonly used procedure is c a l l e d the Nitrogen S o l u b i l i t y Index (AOCS, 1969). Betschart (1974) defined the Nitrogen S o l u b i l i t y Index as "the proportion of nitrogen of a protein concentrate which is determined as soluble a f t e r a s p e c i f i c a l l y defined procedure". The basic procedure has been described by Betschart (1974), Hermansson (1973a, 1973b) and Lawhon and Cater (1971). The protein i s dispersed i n water and the pH i s - 9 -adjusted to some desired l e v e l . Centrifugation of the dispersion i s followed by a determination of the nitrogen content of the supernatant. The deter-mination i s af f e c t e d by the source, processing h i s t o r y , conditions of the s o l u b i l i t y determination and the presence of other components (Shen, 1976). Hermansson (1973a) reported that s o l u b i l i t y decreased as c e n t r i f u g a l forces increased. C e n t r i f u g a l forces ranging from 1400 x g (Paulsen et a l . , 1960) to 40,000 x g (van Megan, 1974) have been employed. Increased protein concen-t r a t i o n was found by Betschart (1974) to decrease s o l u b i l i t y . Hermansson (1973a), however, found s o l u b i l i t y differences of less than 1% between soy protein i s o l a t e concentrations of 1, 3 and 5%. Modifications of the basic procedure have been suggested. K i n s e l l a (1976) reported that measurement of s o l u b i l i t y at several standard pH values might be used to obtain Nitrogen S o l u b i l i t y P r o f i l e s of proteins, which would be of greater value i n p r e d i c t i n g the s o l u b i l i t y behavior i n food systems. Lawhon and Cater (1971) recommended a heating step i n the blending of the protein dispersion p r i o r to centrifugation. F i l t r a t i o n of the supernatant rather than simply decanting i s employed i n the AOCS (1969) standard procedure and by other researchers (Betschart, 1974; Lawhon and Cater, 1971). D. Water Hydration Properties Bound water is defined by Kuntz and Kauzman (1974) as "that water i n the v i c i n i t y of a macromolecule whose properties d i f f e r detectably from those of the 'bulk water' i n the same system". Proteins are capable of binding large quantities of water due to t h e i r a b i l i t y to form hydrogen bonds between water molecules and polar groups on the polypeptide chains. The degree of ass o c i a t i o n with water depends to a great extent on the s t e r i c a v a i l a b i l i t y of - 10 -the polar groups. Therefore, denaturation of protein should increase the water binding a b i l i t i e s of proteins as compared to the native globular state. Chou and Morr (1979) reported that aggregation can reduce the a b i l i t y of the protein to bind water by decreasing the a v a i l a b i l i t y of amino acids. However, they also stated that aggregation may increase water-protein interactions by forming a s t r u c t u r a l network capable of imbibing water. Water i n t e r a c t i n g with the protein i n t h i s manner can be described as "held" rather than "bound" water. The process of hydration of a dry protein powder has been described by Chou and Morr (1979). Upon exposure to water vapor, water i s adsorbed at polar s i t e s , forming a monolayer of water molecules. This monolayer comprises the only t r u l y "bound" water i n the t o t a l system, and may range from 0.3 to 0.5 g H^O/g protein (Fennema, 1977). Further association of water with the p r o t e i n takes the form of multilayer adsorption, with the water molecules closest to the monolayer most strongly attracted. The proteins swell, and i f they are soluble, swelling increases u n t i l there is s u f f i c i e n t water present to surround i n d i v i d u a l molecules and s o l u b i l i z e them. Excellent comprehensive reviews of the subject of protein hydration have been published by Fennema (1977) and Lumry (1973). Methods for studying protein hydration reported i n the l i t e r a t u r e are abundant, but diverse. Hermansson (1977) used moisture sorption isotherms to follow the swelling and solvation processes i n a soy protein i s o l a t e and other p r o t e i n powders. Hagenmaier (1972), using a s i m i l a r technique, found a p o s i t i v e r e l a t i o n s h i p between water binding a b i l i t i e s of several proteins and the number of hydrophilic amino acid groups minus the amide groups. He suggested that deamidation of o i l s e e d proteins, which generally contain large amounts of amide nitrogen, might improve t h e i r water binding a b i l i t i e s . Hansen (1978) found NMR to be a better method of following protein hydration than moisture sorption isotherms. Fleming et a l . (1974) measured the water absorption of sunflower and soy f l o u r s , concentrates and i s o l a t e s by centrifuging 10% dispersions at 2500 RPM for 15 min. The water retained per gram of product was calculated a f t e r removal of the released volume of water. This method provides a measure of the amount of water "held" by the product. Balmaceda et a l . (1974) recommended a s i m i l a r technique involving lower product concentrations, higher c e n t r i -fugation forces (18,000 x g) and a heating step i n the mixing procedure. After experiencing some d i f f i c u l t i e s with the more t r a d i t i o n a l methods of water hydration measurement, Quinn and Paton (1979) devised a simple, highly reproducible method that simulates more r e a l i s t i c a l l y actual food product applications i n which water supply is l i m i t i n g (eg. doughs). Using this new technique, they found a negative c o r r e l a t i o n between protein s o l u b i l i t y and water hydration capacity values, which has also been reported by Hermansson and Akesson (1975) and L i n et a l . (1974) using other techniques. Johnson (1970), however, found that this r e l a t i o n s h i p held only for s o l u b i l i t i e s between 70% and 90%, and that below 70% s o l u b i l i t y , water absorption decreased with decreasing s o l u b i l i t y . Other factors which have been reported to a f f e c t water hydration of protein products are carbohydrate content and p a r t i c l e size d i s t r i b u t i o n , which influences the rate of water absorption (Duerte, 1976; Johnson, 1970). E. Rheological Properties In dispersions, interactions of the dispersed phase with the solvent influence the flow behavior. A solution i s more viscous than i t s solvent alone because of the additional energy needed to overcome solute-solvent and solute-solute interactions ( F r i s c h and Simha, 1956). The extent to which v i s c o s i t y i s increased by adding protein solutes to water, depends p r i m a r i l y on hydrodynamic properties (Lee and Rha, 1979). Very low concentrations of r i g i d spheres suspended i n a solution do not p a r t i c i p a t e i n any hydrodynamic in t e r a c t i o n s , and the v i s c o s i t y of the suspension i s described by ri s = Tlo (1 + 2.5 q), where r i g is the suspension v i s c o s i t y , i s the l i q u i d phase v i s c o s i t y and q represents the volume r a t i o of the dispersed phase ( L a z t i t y , 1974). However, most proteins are not spherical and may be f l e x i b l e , so that not only volume f r a c t i o n but shape and f l e x i b i l i t y also contribute to v i s c o s i t y . Swelling of proteins increases the f l e x i b i l i t y , e f f e c t i v e hydrodynamic volume and a x i a l r a t i o of the macromolecules, r e s u l t i n g in increased v i s c o s i t y (Hermansson, 1972). Heating of proteins i n dispersion w i l l produce a s i m i l a r v i s c o s i t y -increasing e f f e c t due to d i s s o c i a t i o n ( K i n s e l l a , 1979). During processing of o i l s e e d protein products, conditions favoring denaturation may be encountered, often r e s u l t i n g i n agglomeration of protein molecules to form aggregates. When placed i n water, the size of the dispersed phase units can range from uni-molecular to large agglomerates. The p a r t i c l e s i z e d i s t r i b u t i o n a f f e c t s the hydrodynamic volume and v i s c o s i t y of the dispersion. Lee and Rha (1979) subjected soy protein dispersions to mild or vigorous blending regimes to produce dispersions with large or small sized aggregates, r e s p e c t i v e l y . The dispersions with larger p a r t i c l e s were found to be higher i n v i s c o s i t y than the small p a r t i c l e dispersions. These researchers also reported that hydrophobicity was extremely important i n determining the s i z e and shape of the dispersed protein. - 13 -Hermansson (1975) compared the flow behavior properties of dispersions of soy protein isolates processed under varying conditions. Isolates that had been produced under very mild extraction conditions exhibited flow behavior similar to that associated with dispersions of native protein, with low values of consistency coefficient (Power-law m value) and no yield values. Processes employing extremes of pH and/or temperature produced isolates which exhibited non-Newtonian flow in dispersion. She concluded that processing causes the formation of swelling aggregates that are responsible for the complex flow behavior. Lefebvre and Sherman (1977) measured the flow behavior of disper-sions of sunflower protein products and found a negative semi-logarithmic relationship between Power-law coefficients m (consistency coefficient) and n (flow behavior index). The viscosity of soy protein dispersions has been reported to increase exponentially with protein concentration (Circle et al., 1964). Deviations from this relationship at higher concentrations have been demonstrated by Lee and Rha (1979) who suggested that hydrophobic intermolecular interactions may be responsible. The effect of pH on viscosity has been studied by several researchers (Ehninger and Pratt, 1974; Ishino and Okamoto, 1975). Generally, as the pH of a soy protein dispersion is increased, the viscosity increases, up to pH 10-11. Above pH 11, alkali-denaturation occurs and the protein gels. Ehninger and Pratt (1974) found that the effect of pH was dependent upon protein concentration. Heating effects on the viscosities of 10% soy protein dispersions were found by Hermansson (1978) to be influenced by ionic strength. Reversible time-dependence (thixotropy) was observed by Hermansson (1975) in 20% Promine-D soy dispersions. Lefebvre and Sherman (1977) found thixo-tropic behavior i n sunflower protein dispersions and reported that ageing of the dispersions resulted i n a higher degree of thixotropy, due to the formation of stronger entanglements and linkages which required longer shearing times to disrupt. Viscous behavior is exhibited as a r e s u l t of the material d i s s i p a t i n g applied mechanical energy, through flow. The a b i l i t y to store energy r e v e r s i b l y during deformation is manifested as e l a s t i c i t y . Most f l u i d food materials exhibit behavior that is p a r t i a l l y viscous and p a r t i a l l y e l a s t i c and they are said to be v i s c o e l a s t i c materials (Mewis and Spaull, 1976). Even very d i l u t e polymer solutions exhibit some degree of e l a s t i c i t y . Visco-e l a s t i c i t y i s imparted by an i n t e r l i n k e d network of dispersed molecules. In f l u i d foods, these interactions take the form of weak van der Waals forces while i n s o l i d materials, much stronger forces are involved. Rheological measurements of v i s c o e l a s t i c i t y must involve very small stresses and strains in order to minimize a l t e r a t i o n of the i n t e r n a l network structure of the material during t e s t i n g (Tung, 1978). Fundamental studies of the v i s c o e l a s t i c properties of oi l s e e d protein dispersions are r e l a t i v e l y scarce. Isozaki et a l . (1976) used creep compliance tests to measure the v i s c o e l a s t i c i t y of agar, egg albumen and soy i s o l a t e gels. In th i s method of testing, a small constant stress i s applied to a sample and the r e s u l t i n g s t a i n followed over time. The stress relaxation c h a r a c t e r i s t i c s of 20% sunflower protein dispersions were measured by Lefebvre and Sherman (1977) using a Weissenberg Rheogoniometer. By imposing a pre-determined constant s t r a i n on the sample and following the stress relaxation as a function of time, they concluded that the dispersions exhibited v i s c o -e l a s t i c behavior. Dynamic testing i s another method of determining v i s c o e l a s t i c properties. Small, s i n u s o i d a l l y o s c i l l a t i n g shearing deformations are applied to the sample and the v i s c o e l a s t i c moduli are determined from the amplitude r a t i o and phase s h i f t between the stress and s t r a i n waves (Ferry, 1973). The v i s c o -e l a s t i c properties of hydrated wheat gluten (Cumming and Tung, 1977) and rapeseed protein dispersions and gels ( G i l l and Tung, 1976, 1978) have been measured using dynamic te s t i n g . Although considerable attention has been focussed on the gelation of soybean proteins, much of the rh e o l o g i c a l work i n th i s area has been done by measuring gel strengths and Brookfield consistencies, as opposed to the fundamental methods described above. F. Gelation Many food products have tex t u r a l properties which are strongly dependent upon the a b i l i t y of the components to form network structures (frankfurters, doughs, j e l l i e s ) . Although the gelation mechanisms of some food ingredients (polysaccharides and gelatin) have been comprehensively characterized ( M i t c h e l l , 1976), network formation by globular proteins i s s t i l l not completely understood. F l o r y (1974) described a gel as having s o l i d - l i k e behavior, involving a c e r t a i n degree of e l a s t i c i t y . For globular proteins, a gel can be i d e n t i f i e d as a state intermediate between a protein s o l u t i o n and a protein p r e c i p i t a t e (Hermansson, 1978). Globular proteins must be denatured before g e l a t i o n can occur (Ferry, 1948). The denatured protein then aggregates, e s t a b l i s h i n g protein-protein i n t e r a c t i o n s . I f the protein concentration exceeds some c r i t i c a l l e v e l , a balance between protein-protein and protein-solvent interactions i s achieved, and a gel i s formed (Hermansson, 1978). The rate of aggregation influences - 16 -the randomness of the intermolecular linkages. Highly ordered interactions produced by slow aggregation r e s u l t s i n a gel with low opacity and high e l a s t i c i t y (Tombs, 1974). Studies on gel-formation by oilseed proteins have been almost excl u s i v e l y confined to soy proteins. C i r c l e et a l . (1964) used Promine-D to determine that gel s t a b i l i t y depended primarily on protein concentration. Catsimpoolas and Meyer (1970) found that a minimum protein concentration of 8% was necessary i n order to form a self-supporting gel network at pH 7 and room temperature. They also reported that the heating process disrupted the quaternary structure of the soybean globulins, a c t i v a t i n g the protein sol to the progel state (Catsimpoolas and Meyer, 1971a). Studies on the e f f e c t s of pH and ionic strength on gel formation have been carried out by several researchers (Aoki, 1965; Catsimpoolas and Meyer, 1970; Hermansson, 1972; Ehninger and P r a t t , 1974). The e f f e c t s were found to be strongly dependent on the protein source and/or heating temperature. Gelation of the 12 S rapeseed protein f r a c t i o n has been studied by G i l l and Tung (1978). Gels could be formed at the 4.5% protein l e v e l and the strongest gels were created under conditions of high pH and i o n i c strength. G e l l i n g c h a r a c t e r i s t i c s of fababean, field-pea and sunflower protein products were compared to the gel-forming a b i l i t i e s of soy products by Fleming et a l . (1975). Isolate dispersions (10% protein) from a l l sources except sunflower formed gels of varying firmness at pH 7 a f t e r heating at 90°C for 45 minutes and cooling i n an i c e bath. Heating dispersions (10% protein) of soy and sunflower p r o t e i n flours resulted i n increases i n Brookfield consistency but did not produce gels. - 17 -G. Microstructure Wolf and Baker (1975) used scanning electron microscopy (SEM) to study freeze-fractured soybean cotyledons and commercial soy f l o u r s , concentrates and i s o l a t e s . They observed that some of the s t r u c t u r a l elements of the i n t a c t soy cotyledon (protein bodies and spherosomes) survived commercial processing. G i l l and Tung (1976) examined rapeseed cotyledons and aqueous pastes of the 12 S protein f r a c t i o n using both l i g h t microscopy (LM) and transmission electron microscopy (TEM). Evidence of protein agglomeration and a subunit structure were found by these researchers i n samples of the 12 S protein f r a c t i o n . Badley et a l . (1975) studied the subunit structure of g l y c i n i n , the major storage protein i n soybeans, using TEM. Hermansson (1972) employed techniques i n l i g h t microscopy to follow the swelling phenomena in Promine-D, caseinate and whey protein concentrate. Texture-structure r e l a t i o n s h i p s were investigated using SEM by Stanley et a l . (1972) i n rehydrated soy protein f i b e r s and by Cumming et a l . (1972) i n extruded soy products. Thermal aggregation and gelation of proteins have been studied using microscopy by several researchers. Tombs (1974), using TEM, found that the mesh structure produced by heating then cooling bovine serum albumin, was composed of protein strands a r i s i n g from the aggregation of i n d i v i d u a l protein molecules. He compared gel pore size data calculated from t h e o r e t i c a l considerations to measurements made from electron micrographs and found s a t i s f a c t o r y agreement. TEM studies published by Saio et a l . (1968) revealed the presence of numerous spheres, approximately 0.05 ym i n diameter i n an extract of soy proteins i n the presence of CaC^. Upon heating, these spheres were observed to aggregate. Lee and Rha (1978) employed LM and SEM to examine texture-structure relationships of soy proteins. They also observed destruction of the native protein structure on heating, and hypothesized that these s t r u c t u r a l changes were necessary to produce a gel network. Gels of the 12 S rapeseed protein f r a c t i o n were examined using SEM by G i l l and Tung (1978). These researchers reported that the gel pore size decreased, as the pH i n -creased from 6 to 10. Hermansson (1979b) observed whey protein gels under the SEM and found that the addition of s a l t gave r i s e to a coarser structured g e l . Powrie and Tung (1975) reviewed the use of electron microscopy in the study of immobilized water and discussed the microstructure of gels of milk proteins, g e l a t i n and alginates. - 19 -MATERIALS AND METHODS A. Source of Products Samples of control and treated canola (rapeseed), soybean and sunflower pr o t e i n products were available following completion of a two-year Agr i c u l t u r e Canada research contract awarded to Dr. S. Nakai and Dr. M.A. Tung of the Department of Food Science, U.B.C. As part of t h i s contract research, canola meal (var. Tower), a commercially available soybean protein concentrate (Promosoy 100, Central Soya Co.) and sunflower protein concentrate supplied by Dr. F. Sosulski, Department of Crop Science, University of Saskatchewan were studied to determine the factors involved i n protein i n s o l u b i l i t y . After i n v e s t i g a t i n g more than 30 d i f f e r e n t protein treatments for use during production of these high-protein products, three s o l u b i l i z i n g agents were selected for p i l o t - s c a l e treatment: 1. SDS (sodium dodecyl sulfate) 2. t r y p s i n hydrolysis 3. potassium l i n o l e a t e The addition of 5% SDS was found to leave excessive le v e l s of the agent bound to the protein i n spite of exhaustive washing procedures. As the l e v e l s of r e s i d u a l SDS were higher than the l e v e l currently permitted by the Food and Drug Directorate, further studies on SDS-treated proteins were not conducted as part of this thesis. Control, t r y p s i n hydrolyzed and potassium l i n o l e a t e treated protein products used i n t h i s study were prepared for the contract research studies according to the procedures outlined i n Figures 1 and 2. Approximately 2 kg - 20 -Canola Meal (var. Tower) 30 mesh Soy Concentrate (PR0M0S0Y 100) and Sunflower Concentrate - add 7 volumes water I - adjust pH to 10 with NaOH - s t i r for 2 hours - centrifuge 5000 x g at a feed rate of 6.82 1/min i n a Mercobowl centrifuge (Dorr-Oliver, Inc.) add 9 volumes water adjust pH to 8.2 with NaOH Residue (discard) Supernatant - adjust pH to 4.2 with HC1 with s t i r r i n g - centrifuge at 5000 x g (3.41 1/min) I Supernatant P r e c i p i t a t e \ - add d i s t i l l e d water to make 10% dispersion - adjust pH to 8.2 with NaOH Treatments Protein Dispersion spray dry (99°-104°C i n l e t temperature, 36°C outlet temperature, 34 ml/min) Niro Atomizer (Copenhagen) Figure 1. Preparation of protein concentrates and i s o l a t e s . - 2 1 -Trypsin - add tr y p s i n (1% vs concentrate/isolate) (Fisher S c i e n t i f i c Co., Ltd.) - heat at 60°C for'90 minutes - heat at 90°C for 10 minutes - quick cool - readjust pH to 8.2 with NaOH - spray dry next day Linoleate add l i n o l e i c acid (8.7% vs concentrate/isolate) (Fisher S c i e n t i f i c Co., Ltd.) adjust pH to 8.0 with KOH adjust pH to 10.0 with NaOH heat at 65°C for 10 minutes readjust pH to 8.2 spray dry next day Figure 2. Procedure employed for treating the products with t r y p s i n and l i n o l e a t e . of each of the nine d i f f e r e n t protein products were produced. After spray drying, the products were stored i n opaque bottles at 4°C u n t i l required for the various experiments performed i n th i s study. B. Chemical Composition The nine products were analyzed for crude protein, t o t a l carbohydrate (as starch) and moisture content, to determine the e f f e c t s of source and treatment on these parameters. 1. Crude protein Protein analysis was car r i e d out using a micro-Kjeldahl technique of Bradstreet (1965) and Concon and Soltess (1973). Using a Mettler a n a l y t i c a l balance, 0.05 g samples of the protein products were weighed accurately into clean Kjeldahl f l a s k s . A c a t a l y s t , 2.3 g of a K2SO^-HgO mixture (190:4, w/w), was added, followed by 2.3 ml R^SO^ (cone). The samples were digested by heating with periodic addition of 1^02 u n t i l a l l traces of organic material had been oxidized and the digest was r e f l u x i n g halfway up the neck of the f l a s k . When cooled s l i g h t l y , the digest was d i l u t e d with d i s t i l l e d deionized water to 100 ml. An aliquot of th i s d i l u t e d solution was analyzed for % N using a Technicon Autoanalyzer II (Technicon I n d u s t r i a l Systems, Tarrytown, NY). The % N values were converted to grams of N i n the sample. M u l t i p l i c a t i o n by 6.25 resulted i n values for crude protein content which were expressed as percentages of the sample weights. Although i t i s known that the factor 6.25 overestimates the amount of protein i n soy products (Wolf and Cowan, 1971; Balmaceda, 1974) th i s figure seems to be used for most oilsee d protein analyses reported i n the l i t e r a t u r e . The protein products - 23 -were analyzed i n random order and each analysis was repeated twice to obtain three estimates of protein content for each sample. 2. Carbohydrate The phenol-sulfuric acid method (Dubois et a l . , 1956; Pomeranz and Meloan, 1971) was used to estimate the percentage of t o t a l carbohydrates i n the nine i s o l a t e s and concentrates. S u l f u r i c acid was used to hydrolyze carbohydrate components i n the sample to y i e l d hexoses and pentoses, which reacted with phenol to produce a stable orange-yellow compound. The concentration of this compound was measured spectrophotometrically and related to t o t a l carbohydrate content by means of a standard curve. In this analysis, 50 mg (+_ 0.1 mg) samples of the canola, soybean and sunflower products were d i l u t e d to 500 ml to y i e l d suspensions containing approximately 20-240 yg/ml carbohydrate. Each suspension was prepared i n duplicate, and 2 ml aliquots of each were trans-ferred to 18 test tubes. To each test tube, 50 u l of 80% (w/v) phenol were added followed by the rapid addition of 5 ml H^SO^ (cone.). Immediately following t h i s addition, the contents of the test tubes were agitated vigorously for 10 s using a vortex mixer. When cooled to room temperature, absorbance readings were taken at 485 nm using a Beckman Model DB Spectro-photometer (Beckman Instruments, Inc., F u l l e r t o n ) . A standard curve was constructed by preparing suspensions of soluble starch containing 0, 20, 40, 60, 80, and 120 Jig/ml carbohydrate, reacting 2 ml aliquots with 5 ml I^SO^ (cone.) and 50 u l of 80% phenol. Absorbance was measured at 485 nm. - 24 -3. Moisture The AOAC vacuum oven method (AOAC, 1975, Section 14.002) was used to determine the moisture contents of the protein products. Samples (2 g) of each product were weighed accurately into nine pre-dried, desiccator cooled, weighed aluminum pans (70 mm diameter x 20 mm deep). The pans containing the samples were placed i n a vacuum oven at 100°C and 84.4 kPa for 6 h. Cooling of the samples was accomplished i n desiccators containing s i l i c a g e l . A f t e r opening the desiccator following cooling, the samples were weighed as quickly as possible to prevent any appreciable gain of moisture on exposure to the room a i r . The samples were returned to the oven for an a d d i t i o n a l 12 h period, cooled and reweighed. Moisture contents were calculated from the dry weights recorded a f t e r this second drying period, although there did not appear to be any s i g n i f i c a n t difference between the dry weights a f t e r 6 or 17 h of drying. The procedure was repeated, to obtain duplicate moisture determinations for each sample. C. S t r u c t u r a l and Functional Properties. With the exception of water hydration capacity, a l l s t r u c t u r a l and o f u n c t i o n a l properties of the protein concentrates and i s o l a t e s were measured on 10% (w/w) dispersions of the products i n d i s t i l l e d deionized water (7.5g product/75 g d i s p e r s i o n ) . The dispersions were prepared by hand mixing with a spatula for 1-2 min followed by blending/sonication for 1 h with a Polytron PT 10-35 blender (Brinkman Instruments, Inc., Westbury, NY) at a low speed (5) to minimize foaming. The blender was stopped two or three times during the mixing period in order to submerge and hydrate clumps of product f l o a t i n g on the surface of the dispersion or adhering to the blender shaft. The - 25 -dispersions were allowed to remain undisturbed for at least one hour a f t e r mixing to allow time for hydration of the dispersed s o l i d s . Functional testing of the dispersions was c a r r i e d out on the day of preparation i n order to minimize any ageing e f f e c t s or b a c t e r i a l degradation. Duplicate dispersions were prepared for each functional test performed. A Fisher Accumet Model 420 D i g i t a l pH/lon Meter (Fisher S c i e n t i f i c Company, Pittsbu r g , PA) was used to measure pH values of the dispersions. Conductance measurements on the samples were also taken to estimate the r e l a t i v e i o n i c strengths of the d i f f e r e n t dispersions. A YSI Model 31 Conductivity Bridge (Yellow Springs Instrument Co., Inc., Yellow Springs, OH) was used for this purpose. A 25 cm long 2.5 cm diameter Pyrex c e l l containing a platinum-i r i d i u m electrode was immersed in the sample dispersions. A 117 V l i n e voltage was used for the bridge supply. The reference c e l l resistance was adjusted to match that of the test solution. Conductance measurements were also recorded for several standard NaCl solutions, covering a range of i o n i c strengths from 0.01 to 1. 1. S t r u c t u r a l properties a) Light microscopy - dispersions Sample dispersions were observed under a L e i t z Labrolux microscope equipped with a Pentax ME 35mm camera. Unstained aliquots of the dispersions were contained on s l i d e s using cover s l i p s sealed with petrolatum to prevent dehydration. Bright f i e l d , Kohler i l l u m i n a t i o n was used and photographic images were recorded on I l f o r d FP4 black and white f i l m . Dispersion samples stained with Lugol's iodine (Cook, 1974) were also examined to test for the presence of starch. - 26 -b) Scanning electron microscopy - thermal aggregation S t r u c t u r a l examination of the g e l - l i k e materials produced by heating the dispersions was accomplished using scanning electron microscopy techniques. Dispersion samples, approximately 5 ml, were heated i n covered test tubes i n a b o i l i n g water bath for 30 min and subsequently cooled i n i c e water. No s i g n i f i c a n t moisture loss was noted by weighing the test tubes before and a f t e r the heating/cooling procedure. Ten ml of 5% glutaraldehyde f i x a t i v e i n 0.05 M phosphate buffer (pH 7.6) was c a r e f u l l y added to the cooled contents of each test tube. At room temperature the primary f i x a t i o n was c a r r i e d out for 20 h, at which time the f i x a t i v e appeared to have penetrated h a l f the depth of the semi-solid dispersion material. This procedure was adopted to achieve some degree of f i x a t i o n of the material before any mechanical action was i n i t i a t e d to remove the dispersions from the test tubes. The p a r t i a l l y - f i x e d material was cut into pieces, approximately 0.5 cm i n length, and immersed into fresh 5% glutaraldehyde f i x a t i v e overnight at room temperature. Two 15 min rinses i n phosphate buffer were followed by secondary f i x a t i o n i n 1% osmium tetroxide i n phosphate buffer for 90 minutes. Rinses of phosphate buffer (2 x 15 min) preceeded alcohol dehydration through a series of increasing ethanol concentrations (30 min each i n 30, 50, 70, 95 and 2 x 100%). Replacement of ethanol with amyl acetate was accomplished through 15 min changes of 25, 50, and 75% amyl acetate i n absolute ethanol, followed by 1 h i n 100% amyl acetate. The samples of dispersion material were then dried i n a Parr 5770 C r i t i c a l Point Drying Bomb (Parr Instrument Co., Moline, IL) using carbon dioxide as the t r a n s i t i o n a l f l u i d ( c r i t i c a l temperature and pressure: 30°C 7468 kPa). The dried fragments were fractured to expose an inner surface and mounted with s i l v e r paste and paint on aluminum stubs. A gold-palladium a l l o y coating was applied to the samples with a Hummer Ion Sputtering Device (Technics, Inc., Alexandria, VA). An Etec Autoscan Scanning Electron Microscope (Perkin Elmer Etec, Inc., Hawyard, CA) equipped with a Polaroid camera was used to observe the s t r u c t u r a l d e t a i l of the samples and photo-graphic images were recorded on Polaroid P/N 55 f i l m . An operating voltage of 20 kV was employed. 2. Functional properties F u n c t i o n a l i t y tests used in this study were selected and adapted from the numerous tests reported i n the l i t e r a t u r e . Those functional properties which appeared to measure the interactions of the concentrates and i s o l a t e s i n aqueous systems were of most i n t e r e s t . a) Protein s o l u b i l i t y The amount of protein i n the 10% dispersions that was "soluble" as opposed to simply dispersed was measured by cent r i f u g i n g 20 ml of the dispersions at 10,000 x g for 30 min (Sorval Superspeed RC2-B, Ian Sorval, Inc., Norwalk, CT) and analyzing a 2 ml a l i q u o t of the r e s u l t i n g supernatant for protein content using the micro-Kjeldahl technique of Concon and Soltess (1973). Although th i s technique i s referred to as protein s o l u b i l i t y i n the s c i e n t i f i c l i t e r a t u r e on f u n c t i o n a l i t y , the procedure i n r e a l i t y does not separate soluble from insoluble proteins. Rather a separation of sol p a r t i c l e s i s achieved, defined by a c r i t i c a l p a r t i c l e s i z e . In this case, the technique provides measurement of the amount of protein i n the dispersions present i n small p a r t i c l e s as compared to protein present i n large aggregates. A c e n t r i f u g a l force of 10,000 x g was a r b i t r a r i l y selected as an intermediate value of those reported i n the l i t e r a t u r e (4,000 x g to 40,000 x g) (Briskey, - 28 -1968). From Stokes law, i t was determined that a 10,000 x g force would cause p a r t i c l e s greater than approximately 0.05 ym to sediment during the 30 minute ce n t r i f u g a t i o n . Protein s o l u b i l i t y was expressed as a percentage, g protein i n 1 ml x g water added x 100% (1.0 ml-g protein/1.36 g ml~l) g protein in dispersion b) Water holding capacity The centrifuged dispersions used for protein s o l u b i l i t y determinations could be used to obtain an estimate of water holding capacity of the protein products. By weighing the empty centrifuge tube and the centrifuge tube containing protein dispersion both before and a f t e r c e n t r i f u g a t i o n and removal of supernatant, the weight of hydrated p e l l e t was calculated by difference and related back to the weight of product i n each dispersion. The water holding capacity was expressed as g R^O held/g product. Although, as e a r l i e r discussed, there are some fundamental problems with t h i s f u n c t i o n a l i t y test, these values provided an i n t e r e s t i n g comparison of the recently described water hydration capacity method of Quinn and Paton (1979) with the more t r a d i t i o n a l technique of water holding capacity. Water holding capacity = hydrated p e l l e t (g) - product (g) (g water/g product) product (g) c) Water hydration capacity (WHC) The method of Quinn and Paton (1979) was used to estimate the uptake of water by the products using a d i f f e r e n t technique than that used for water holding capacity. Samples of i s o l a t e or concentrate were weighed (+ .01 g) into weighed 50 ml polypropylene centrifuge tubes. Approximately 25 ml of d i s t i l l e d , deionized water was added i n increments, followed by mixing with a spatula to form an homogeneous paste. The tubes were centrifuged i n the SS-34 Rotor of a Sorval Superspeed RC2-B centrifuge for 10 min at 2,000 x g. A f t e r - 29 -cent r i f u g a t i o n , the supernatant was c a r e f u l l y removed and the tubes were weighed to determine the weight of the hydrated p e l l e t . The approximate water hydration capacity was then determined as Approximate WHC = hydrated p e l l e t (g) - product (g) (g water/g product) product (g) A series of 4 centrifuge tubes was then prepared for each product containing a constant amount of product i n each, and varying amounts of water to encompass a range of water hydration capacities around the approximate water hydration capacity calculated (Table I ) . The weights and volumes of product and water, re s p e c t i v e l y , were calculated to y i e l d a t o t a l weight of approx. 15 g. The centrifuge tube contents were mixed vigorously with a glass s t i r r i n g rod for two min and a l l tubes were centrifuged for 10 min at 2,000 x g. In the series of tubes, some contained supernatant and others d i d not. The water hydration capacity was defined as the mean of the water hydration capacities represented by two adjacent tubes, one containing supernatant and the next without. The products were tested i n duplicate in random order. Table I. Example of water hydration capacity determination. (Sunflower-Trypsin: approximate WHC = 2.9 g H20/g product) Sample Water WHC Tube # weight (g) added (ml) (g^O/g product) Supernatant 1 3.50 11.5 3.29 Yes 2 3.50 11.0 3.14 No 3 3.50 10.5 3.00 No 4 3.50 10.0 2.86 No Water hydration capacity =3.2 g H20/g product - 30 -d) Steady shear flow behavior Sample dispersions (10% w/w) were subjected to rh e o l o g i c a l evaluation i n steady shear using both a Brabender Rheotron coaxial cylinder viscometer (CW. Brabender Instruments Inc., South Hackensack, NJ) and a Weissenberg Rheogonio-meter cone/plate rheometer (Sangamo Controls Ltd., Bognor Regis, England). Duplicate dispersions were evaluated. For flow measurements with the Rheotron, 58 g dispersion samples were weighed into cup #A1 (radius =2.8 cm) which was loaded into the instrument around the #A1 tor s i o n sensing spindle (radius = 2.7 cm, length =8.0 cm). Temperature control was maintained at 22°C with a thermostatically controlled water supply c i r c u l a t i n g i n a water jacket around the sample cup. The cup was rotated through a programmed loop of l i n e a r l y increasing and then decreasing RPM for 200 s, representing a shear rate range of approximately 0 to 600 s 1 . Some of the samples were of such low v i s c o s i t y that shear stress readings could be recorded only at shear rates greater than 30 s \ Shear stress and shear rate readings were recorded as voltages on a Digitec multichannel recorder (United Systems Corp., Dayton, OH), recording continuously through the shear rate program. Approximately ten shear stress-shear rate voltage data points were taken from each recorded output at l o g a r i t h m i c a l l y spaced shear rate i n t e r v a l s to calculate flow parameters. C a l i b r a t i o n of the instrument was accomplished by measuring the shear stress-shear rate r e l a t i o n s h i p of d i s t i l l e d water at 22°C. Shear stress (a,Pa), shear rate (y,s *) and v i s c o s i t y (n,Pa.s) values were calculated from c a l i b r a t i o n data using a computer program written for this purpose for execution using the UBC Amdahl 470 V/6 computer. Also by computer, least squares l i n e a r regression was used to examine the f i t of a Power-law model to the transformed data. - 31 -l o g a = l o g m + n l o g Y or log n = log m + (n-1) log y where m i s the consistency c o e f f i c i e n t (Pa s 1 1) and n is the flow behavior index. The Krieger-Maron correction for non-Newtonian behavior was applied to r e c a l c u l a t e each shear rate value a f t e r an estimate of the flow behavior index 2 (n) was evaluated from the data, assuming n = 1. Values of r were calculated as a measure of the goodness of f i t . The transformed data were plotted to v i s u a l l y examine for c u r v i l i n e a r i t y . P l o t t i n g also provided for detection of time-dependent flow i n the sample which would be evident as a hysteresis loop formed by d i f f e r e n t shear stress values at a given shear rate when the shear rate was increasing as compared to values recorded as the shear rate was decreasing. The Weissenberg Rheogoniometer was f i t t e d with a #7 t o r s i o n bar (94 Pa 3 cm /urn) and 7.5 cm diameter platens. The top platen had a nominal cone angle of 1° and a gap set value of 44 Um. The f l a t bottom platen used was of the reservoir type, to contain excess f l u i d forced out of the gap during loading of the samples. C a l i b r a t i o n of the instrument was checked using o i l v i s c o s i t y standard S6 (Cannon Instrument Co., State College, PA). The bottom platen was rotated i n both forward and reverse di r e c t i o n s at several d i f f e r e n t speeds and the torque sensed by the torsion head transducer was recorded on a s t r i p chart recorder. Tests for y i e l d stress values were performed by stopping the rotation instantaneously with the brake and monitoring the de c l i n i n g shear stress readings as the sample relaxed. Calculations of shear stress, shear rate and v i s c o s i t y were c a r r i e d out by 2 computer. Power-law parameters m and n and r values and plots of the transformed data were obtained by computer using techniques s i m i l a r to those - 32 -previously described for analysis of the data c o l l e c t e d using the Brabender Rheotron. The dispersions prepared and tested on the Rheotron were also evaluated with the Rheogoniometer that same day. Thus, a pooling of the Rheotron and Rheogoniometer r e s u l t s for each dispersion was possible to obtain 18 sets of m and n values for the duplicate tests of nine product dispersions. This was c a r r i e d out using a computer program and the UBC computer. Values of apparent v i s c o s i t y at 100 s 1 were calculated from the Power-law parameters. e) Dynamic shear flow behavior The Weissenberg Rheogoniometer was used to measure the v i s c o e l a s t i c properties of duplicate dispersion samples. The samples were loaded between a bottom f l a t plate which was o s c i l l a t e d through a small s i n u s o i d a l l y varying s t r a i n (amplitude 500 ym) and a top 1° cone, 7.5 cm diameter platen supported by a #7 t o r s i o n bar. The frequency of the input s t r a i n was varied to assess the dependence of the v i s c o e l e s t i c parameters on frequency. A Tronotec Model 703A d i g i t a l phase meter (Tronotec, Inc., F r a n k l i n , NJ) was used to monitor the amplitudes of the input ( s t r a i n ) and the output (stress) voltage signals, and the phase differences between the two s i g n a l s . From these data, values of storage modulus (Pa), r e l a t i n g shear stress to shear s t r a i n and values of loss tangent, r e f l e c t i n g the r e l a t i v e proportions of e l a s t i c to viscous natures of the samples were calculated by computer. The v i s c o e l a s t i c parameters were measured at 20, 45, 70 and 95°C i n an attempt to follow rheological changes i n the dispersions due to thermally induced aggregation. Temperature control was achieved by using a water jacketed lower platen assembly connected to a c i r c u l a t i n g water bath. Evaporation of the sample from the gap was minimized by applying a thin layer of 10 mPa s s i l i c o n o i l to the exposed edge of the sample. - 33 -D. S t a t i s t i c a l Analysis 1. F a c t o r i a l analysis of variance Data c o l l e c t e d for a l l of the measured variables were coded for "source" (3 l e v e l s ) and "treatment" (3 l e v e l s ) and analyzed using an analysis of variance (ANOVA) program package (*MFAV) available for use on the UBC computer. Duplicate values were available for a l l v a r i a b l e s , with the exception of the protein determination which was c a r r i e d out i n t r i p l i c a t e . Log transformations were performed on consistency c o e f f i c i e n t and apparent v i s c o s i t y (100 s ^) data to achieve better homogeneity of variance, r e s u l t i n g i n more meaningful s t a t i s t i c a l analysis. Data for variables for which the source treatment i n t e r a c t i o n term was non-significant (p>0.05) were subjected to further s t a t i s t i c a l analysis using Duncan's multiple range test, available with the MFAV program. Duncan's multiple range test (Walpole, 1974) i s a commonly used method for performing multiple comparisons among means. These analyses determined which sources and/or treatments were s i g n i f i c a n t l y d i f f e r e n t from each other i n terms of each p a r t i c u l a r v a r i a b l e . 2. Single factor analysis of variance The same data analyzed i n the f a c t o r i a l analysis of variance were also treated as single factor "products" (9 l e v e l s ) and analyzed i n a one-way analysis of variance, using the *MFAV program. As there are no i n t e r a c t i o n terms i n a one-way ANOVA, Duncan's mutliple range tests could be used to perform multiple comparisons among means for a l l v a r i a b l e s . - 34 -3. Simple c o r r e l a t i o n s A c o r r e l a t i o n matrix involving means of 12 variables was generated using the s t a t i s t i c a l computer program MIDAS (Fox and Guire, 1976). Nine data points were av a i l a b l e for each c o r r e l a t i o n . Scatter plots of several s i g n i f i c a n t c o r r e l a t i o n s were obtained by computer for v i s u a l examination. - 35 -RESULTS AND DISCUSSION A. Composition Mean protein, carbohydrate and moisture contents of the nine oilseed products are presented with t h e i r standard errors in Table I I . 1. Crude protein Results of the crude protein determinations (Table II) revealed that the canola products were higher in protein content than the soy and sunflower samples. The term " i s o l a t e " can be used to describe the canola control product, as the dry basis (db) protein content is greater than 90%. Soy and sunflower control products, containing at least 70% db protein, can be referred to as protein concentrates. The value of 70.0% db protein reported for the soy-control product shows good agreement with data supplied in a technical brochure for t h i s product, Promosoy 100. The manufacturers of t h i s product reported a range of protein content from 70.1 to 70.8% db (Central Soya, 1979). The protein content of the sunflower concentrate has been reported by Sosulski (1978) to be 73.5% db. A mean value of 74.6% db protein, obtained in t h i s study shows s a t i s f a c t o r y agreement. Trypsin hydrolysis of the products during processing did not bring about a s i g n i f i c a n t change i n the measured protein contents as compared to control products. An increase in non-protein nitrogen would be expected as a r e s u l t of hy d r o l y s i s , however the t o t a l amount of nitrogen i n the product would remain unchanged. Nakai et a l . (1980a) reported an increase in non-protein nitrogen from 2% db i n control canola products to 42% db i n canola product that had been hydrolyzed by tr y p s i n at a l e v e l of 0.5% at pH 8.0 and 65°C for 30 min. - 36 -Table II. Mean values of protein, carbohydrate and moisture contents of canola, sunflower and soybean products. Product Crude protein (% db) Total carbohydrate (% db) Moisture (% db) Canola Control 91.3 a 1 (1.07) 2 7.1 a (0.015) 5.7 a b (0.0951) - Trypsin . 91.0 a (1.84) 7.1 a (0.365) 5.4 a (0.0645) - Linoleate 84.0 b (1.28) 5.9 a (0.004) 7.2d (0.0703) Sunflower - Control 74.6 C (1.17) 2 3 .7 b d e (0.380) 7.2d (0.110) - Trypsin 74.6 C (0.841) 22 2 b d (2.65) 6.4C (0.0955) - Linoleate 69 .8 d (1.24) 20. l b (0.590) 6.1 b c (0.220) Soybean Control 70.0 d (1.71) 27 .3 c e (1.095) 6.5C (0.257) - Trypsin 69 .7 d (1.73) 28.0 C (1.855) 6.5C (0.0588) - Linoleate 60.2 e (0.841) 2 5 . 8 c d e (0.950) 5.7 a (0.00504) n = 3 n = 2 n = 2 1-Means i n a column sharing the same superscript are not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) as determined by Duncan's multiple range test 2standard error of the mean - 37 -The addition of l i n o l e i c acid at a l e v e l of 8.7%, brought about s i g n i f -icant decreases i n protein content over controls for a l l three sources. This decrease could be attributed to displacement of some weight of protein by the fa t t y a c i d . Nakai et a l . (1980a) measured the amount of l i n o l e a t e bound to soy and sunflower proteins following bench-top treatment of the oilsee d products. They found that only 75 to 95% of the added l i n o l e a t e was bound to the protein when the surfactant was added at levels of 8 to 10%. In p i l o t -scale products, such as those used i n this study, the binding of l i n o l e a t e to the protein may be even less complete, due to less e f f i c i e n t mixing techniques. 2. Carbohydrate The standard curve derived from absorbance data c o l l e c t e d for starch solutions was found to be well described by the regression equation: A = (.0809 x ug CHO) + .0601 r = .943 The carbohydrate contents of the samples were calculated using t h i s equation. The canola i s o l a t e s contained much less t o t a l carbohydrate than the sun-flower or soybean concentrates (Table I I ) . Glycoproteins present i n the samples would contribute to the t o t a l carbohydrate measured i n this a n a lysis. The 12 S gl o b u l i n of canola i s a glycoprotein containing approximately 8% carbohydrate ( G i l l and Tung, 1976).• Wolf (1970) reported that the 7 S protein f r a c t i o n i n soy contains 6% carbohydrate. Treatment of the products with tr y p s i n or l i n o l e a t e did not s i g n i f i c a n t l y a l t e r the carbohydrate contents as compared to controls. There was, however, a trend towards lower carbohydrate contents i n li n o l e a t e - t r e a t e d samples, a re s u l t which would be expected due to displacement of some carbohydrate by the l i n o l e i c acid. - 38 -3. Moisture The range of moisture contents determined for a l l samples was found to be 5.4 to 7.2% db (Table I I ) . Although there were s i g n i f i c a n t differences among products, no trends were noted. During spray drying of the products, the target moisture content ranged from 3 to 5% wet basis as determined using a moisture balance. The observed differences i n moisture content may not be related to treatment e f f e c t s . 4. Other components Components not accounted for by the above analyses would be l i p i d ( i ncluding l i n o l e a t e i n the l i n o l e a t e treated samples) and ash. B. S t r u c t u r a l Properties I. Light microscopy - dispersions Micrographs depicting the nine dispersions are presented i n Figures 3 to II. In the canola control dispersion (Figure 3), aggregates were observed which were approximately spherical i n shape and which ranged i n diameter from 1 to 75 ym. As over 90% of the dry solids i n this product was found to be protein, these aggregates were assumed to be of proteinaceous material. The trypsin-treated canola dispersion (Figure 4) is observed to contain aggregates of smaller mean p a r t i c l e s i z e (maximum diameter approx. 25 ym). The breakdown of large polypeptides to smaller molecules as a res u l t of enzymatic hydrolysis may reduce protein-protein interactions responsible for aggregation. Treatment with l i n o l e i c acid brought about an increase i n mean aggregate size (Figure 5). The long chain f a t t y acids may have i n i t i a t e d the formation of m i c e l l e - l i k e structures, with the l i n o l e a t e buried within a s h e l l of protein. The sunflower control dispersion (Figure 6) contained fragments of c e l l wall material which were i r r e g u l a r l y shaped. These fragments ranged in size from less than 10 ym in length to over 100 ym. Smaller p a r t i c l e s (<5 ym) in the dispersion were assumed to be aggregates of protein and simple poly-saccharides. The presence of starch was indicated by the purple-blue color of some small p a r t i c l e s following staining of dispersion samples with Lugol's iodine. Treatment of the sunflower concentrate with t r y p s i n or l i n o l e i c acid did not a l t e r the s t r u c t u r a l properties of the dispersions to any great extent (Figures 7 and 8). The c e l l wall fragments would not be expected to be affected by the treatments, and the protein aggregates were too small to permit observation of any changes i n s i z e . -40-Figures 3 to 5. Light micrographs of 10% aqueous dispersions (photo width (pw) = 500 ym) -42-gures 6 to 8. Light micrographs of 10% aqueous dispersions (pw = 500 um) Figure 8 Sunflower l i n o l e a t e -44-Figures 9 to 11. Light micrographs of 10% aqueous dispersions (pw = 500 um) - 46 -The soy control dispersion (Figure 9) contained some fragments of c e l l wall material, however some of the larger p a r t i c l e s (approx. 100 Um) did not have the c h a r a c t e r i s t i c c e l l structure and were assumed to be aggregates cons i s t i n g predominantly of protein. Hydrolysis by tr y p s i n reduced the size of the protein aggregates (Figure 10). The large p a r t i c l e s appeared to be c e l l fragments. The l i n o l e a t e - t r e a t e d soy dispersion (Figure 11) contained large aggregates s i m i l a r to those observed i n the control dispersion. The i n t e r a c t i o n of starch with l i n o l e a t e and protein may be contributing to the formation of these large p a r t i c l e s . Pomeranz and Chung (1978) studied the interactions of l i p i d with soy protein and carbohydrate i n breadmaking and found that polar l i p i d s serve as an adhesive, e s t a b l i s h i n g hydrogen bonds with the starch and hydrophobic or hydrogen bonds with the protein. E f f o r t s to measure 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 by a number of d i f f e r e n t methods ( f i l t r a f u g a t i o n , Coulter counter, sieving and micromensuration) were unsuccessful. Clumping and d i l u t i o n of the p a r t i c l e s which occurred during these measurement procedures was believed to influence the size d i s t r i b u t i o n s , thus providing inaccurate estimates of the p a r t i c l e si z e d i s t r i b u t i o n s i n the 10% dispersions. 2. Scanning electron microscopy - thermal aggregation Scanning electron micrographs of the g e l - l i k e materials produced by heating the sample dispersions i n a b o i l i n g water bath, are presented i n Figures 12 to 25. Examination of the canola-control samples (Figures 12 and 13) revealed the presence of small protein aggregates (< 1 um diameter) i n t e r a c t i n g to form the larger aggregates observed in the aqueous dispersions under the l i g h t microscope. In trypsin-hydrolyzed canola samples (Figure 14), -47-Figures 12 and 13. Scanning electron micrographs of heated 10% aqueous dispersions. - 4 G -Figure 12. Canola control (pw = 60 um). Figure 13. Canola control (pw = 20 ym) -49-Figures 14 and 15. Scanning electron micrographs of heated 10% aqueous dispersions. Figure 15. Canola t r y p s i n (pw = 20 ym). -51-Figures 16 and 17. Scanning electron micrographs of heated 10% aqueous dispersions. - 52 -Figure 16. Canola l i n o l e a t e (pw = 60 um). Figure 17. Canola l i n o l e a t e (pw = 20 um). -53-Figures 18 and 19. Scanning electron micrographs of heated 10% aqueous dispersions. - 54 -Figure 18. Sunflower control (pw = 60 um). Figure 19. Sunflower co n t r o l (pw = 300 ym). -55-gures 20 and 21. Scanning electron micrographs of heated 10% aqueous dispersions. Figure 21. Sunflower l i n o l e a t e (pw = 60 ym). -57-Figures 22 and 23. Scanning electron micrographs of heated 10% aqueous dispersions. -59-gures 24 and 25. Scanning electron micrographs of heated 10% aqueous d i sp er s ion s. - 60 -Figure 25. Soybean l i n o l e a t e (pw = 20 um). - 61 -a fin e r texture was observed as a r e s u l t of a more uniform d i s t r i b u t i o n of i n d i v i d u a l aggregates throughout the matrix. This structure would suggest more protein-protein i n t e r a c t i o n s . Observation of the samples at higher magnification (Figure 15) indicated the presence of fine filamentous f i b e r s l i n k i n g protein aggregates, i n the trypsin-treated canola material. It i s possible that s i m i l a r f i b e r s were present i n the canola-control samples, but due to the strong nature of the protein-protein i n t e r a c t i o n , were fewer i n number and hidden from view . The l i n o l e a t e - t r e a t e d samples (Figure 16 and 17) contained linkages between protein aggregates that appeared stronger than those observed i n the tryspin-hydrolyzed samples. A more e l a s t i c texture might be expected. The canola gel samples had s i m i l a r s t r u c t u r a l properties to soy protein i s o l a t e gels examined by Lee and Rha (1978). In samples of heated control sunflower material, a g e l - l i k e substance was found to be d i s t r i b u t e d throughout the c e l l wall fragments (Figure 18). This g e l - l i k e material may be composed of protein or carbohydrate, or both. However, the major non-protein constituents of o i l s e e d concentrates are insoluble carbohydrates which are u n l i k e l y to form a network structure. Therefore the components involved i n the formation of the matrix were assumed to be predominantly protein. Figure 19 shows a large piece of c e l l wall material, probably a fragment of the sunflower seed h u l l . A finer-textured protein matrix was observed throughout the t r y p s i n -hydrolyzed sunflower samples (Figure 20). Linoleate treatment produced sunflower gels with strong interactions (Figure 21) s i m i l a r to those observed i n l i n o l e a t e - t r e a t e d canola samples (Figure 16). A g e l - l i k e matrix could not be located i n the soybean-control samples examined (Figure 22). Some degree of gelation must have occurred in order to - 62 -produce the semi-solid textured material on heating, however, much of the protein in this product is insoluble and would not be expected to form the c h a r a c t e r i s t i c gel matrix observed i n the canola and sunflower products. Possibly, polysaccharides are p a r t i c i p a t i n g in the structure-forming process through g e l a t i n i z a t i o n , exerting a s t a b i l i z i n g e f f e c t on a weak protein matrix. The soybean-trypsin dispersion did not form a s u f f i c i e n t l y s o l i d structure on heating to withstand mechanical damage on transfer from g e l l i n g tube to f i x a t i v e , and therefore examination of s t r u c t u r a l d e t a i l s was not possible for t h i s sample. Examination of soybean-linoleate samples indicated the presence of a g e l - l i k e substance d i s t r i b u t e d throughout the c e l l wall fragments (Figure 23). At higher magnification (Figures 24 and 25) the matrix was observed to have a fine texture, s i m i l a r to that observed i n the canola-trypsin samples. Visual examination of the samples a f t e r heating revealed that the l i n o l e a t e - t r e a t e d materials had more rubbery textures and shiny, translucent appearances as compared to corresponding control and trypsin-treated samples. These observations lend support to the results obtained from SEM examination which suggest that stronger protein-protein interactions were i n i t i a t e d by l i n o l e a t e treatment. The p a r t i c i p a t i o n of the long chain f a t t y acids in e s t a b l i s h i n g and maintaining linkages between protein aggregates may be at least p a r t i a l l y responsible. Tryspin hydrolysis might be expected to increase the opportunity for the proteins to i n t e r a c t , by exposing more amino acid side chains. However, re s u l t s of both SEM and v i s u a l examination indicated that the i n t e r a c t i o n was f a i r l y weak, and s e n s i t i v e to mechanical d i s r u p t i o n . - 63 -C. Functional Properties 1. pH Measurements of dispersion pH were taken to determine whether differences observed i n other functional properties could be a t t r i b u t e d to a pH e f f e c t . During processing of the products, the pH was adjusted to 8.2 just before spray drying. Due to the strong buffering capacity of the protein, a stable pH value was d i f f i c u l t to a t t a i n . When the spray dried products were re-dispersed i n t h i s study, and pH measured, values ranging from 7.3 to 8.4 were recorded. Means and th e i r standard errors are reported i n Table I I I . Although there were s i g n i f i c a n t differences among some of the means reported, the differences could be a t t r i b u t e d to deviations i n pH adjustment during processing, rather than to treatment e f f e c t s . 2. Conductance Conductance values for the 10% dispersions were measured as an index of ionic strength, to determine i f the observed e f f e c t s of treatment on other functional properties could be attributed to the e f f e c t of i o n i c strength. The r e s u l t s of the analyses are presented i n Table I I I . Comparing the control dispersions, canola and soy samples have si m i l a r conductance values, while sunflower ranks lower i n conductance. Conductance measurements were also recorded for several NaCl solutions of d i f f e r e n t i o n i c strength (0.01-1.00). The values recorded for the control samples, correspond to ionic strengths of approximately 0.04M NaCl (canola and soy) and 0.01M NaCl (sunflower). Treatment of the products with trypsin or l i n o l e i c acid produced dispersions with s i g n i f i c a n t l y higher values of conductance than corresponding - 64 -Table I I I . Mean values of pH and conductance for 10% aqueous dispersions of canola, sunflower and soybean products (n = 2). pH Conductance Product (umho) Canola - Control 8.4a 1 3370 a (0.0251) 2 (7.01) - Trypsin 7.6b 4180 b (0.212) (12.0) - Linoleate 8.2a 4745 c (0.00499) (3.49) Sunflower - Control 7.6b 1030 d (0.0150) (6.89) - Trypsin 7 . 9 b c 2145e (0.0395) (9.49) - Linoleate 8.1 a c 2755 f (0.00997) (7.50) Soybean - Control 7.7b 3450 a (0.0200) (18.0) - Trypsin 7.5 b 3870 b (0.0349) (7.01) - Linoleate 7.6 b 5590S (0.0399) (9.01) ^Means i n a column sharing the same superscript are not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) as determined by Duncan's multiple range test. Standard error of the mean - 65 -control dispersions. In the case of t r y p s i n hydrolysis, this would be caused by the increase i n hydrogen ion concentration as peptide bonds were cleaved. Linoleate treatment involved adjustment of pH to 10.0. In both treatments, therefore, more acid or base would be required to readjust the pH to 8.2, r e s u l t i n g i n higher i o n i c strength than controls. 3. Protein s o l u b i l i t y Protein s o l u b i l i t y determinations on the 10% aqueous dispersions were per-formed to assess the amount of protein i n the dispersions that was present in small c o l l o i d a l p a r t i c l e s (<0.05 um) as opposed to large protein aggregates. S o l u b i l i t y values (Table IV) were calculated on the basis that a l l of the water added to the dispersion would be available for s o l u b l i z i n g protein. This assumption w i l l lead to an overestimate of s o l u b i l i t y , as some of the added water is chemically bound to insoluble protein and carbohydrate and is unavailable for s o l u b l i z a t i o n . The three control products had s i g n i f i c a n t l y d i f f e r i n g s o l u b i l i t i e s . The low s o l u b i l i t y of the soy control product might suggest the presence of strong a t t r a c t i v e forces, r e s u l t i n g i n considerable aggregation of protein and carbohydrate components. Aggregation would be expected to decrease s o l u b i l i t y through sedimentation and reduced a v a i l a b i l i t y of solvation s i t e s . Trypsin hydrolysis was found to be e f f e c t i v e i n increasing the s o l u b l i t y of canola, sunflower and soybean proteins. This p r o t e o l y t i c enzyme cleaves peptide bonds adjacent to basic amino acids. This action could d i s s o c i a t e protein structure within the aggregates i n such a way as to increase i n t e r -f a c i a l area, exposing more charged and polar s i t e s on the proteins to the surrounding water. - 66 -Table IV. Proportion of protein i n 10% aqueous dispersion present i n soluble state as p a r t i c l e s <0.05 ym i n size (n = 2). Mean Standard Product (g protein soluble/100 g t o t a l protein) Error Canola - Control 56.6 a 1 .514 - Trypsin 70.4° .308 - Linoleate 55.2a 1.35 Sunflower - Control 79.3 b 5.31 - Trypsin 90. i c .462 - Linoleate 100.9 d 5.38 Soybean - Control 18.3 e 1.11 - Trypsin 58. 7 a 1.76 - Linoleate 9 1 . l c 4.29 1-Means sharing the same superscript are not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) as determined by Duncan's multiple range test. - 67 -The e f f e c t of l i n o l e a t e treatment on s o l u b i l i t y was found to vary with source. Sunflower and soy products treated with l i n o l e i c acid exhibited s i g n i f i c a n t increases i n s o l u b i l i t y over controls. Salts of f a t t y acids have been reported to disrupt protein-protein hydrophobic bonds (Canella et a l . , 1979; Wall, 1979), which may a f f e c t s o l u b i l i t y by increasing i n t e r f a c i a l area. Nakai et a l . (1980b) attributed the s o l u b i l i z i n g e f f e c t s of surfactants to the surrounding of proteins by negatively charged f a t t y acid carboxyl groups. The s o l u b i l i t y of the canola product, however, remained unchanged following l i n o l e a t e treatment. 4. Water hydration and water holding capacities The water hydration capacity (WHC) method of Quinn and Paton (19 79) was found to be an e f f e c t i v e method for assessing the a b i l i t i e s of the products to hold water. Errors associated with the conventional water holding technique due to the discard of protein i n the decanted supernatant and poor separation of phases, are avoided i n this new procedure. The water hydration measurement technique was more time-consuming; however i t provided a much more accurate comparison of the r e l a t i v e degree of i n t e r a c t i o n with water of a wide range of products. The water hydration and water holding capacities of the nine products are presented i n Table V. I t is of i n t e r e s t to note that agreement between water holding and water hydration capacities was s a t i s f a c t o r y only for soybean c o n t r o l . In this sample, the protein s o l u b i l i t y was low and phase separation was good, thus the errors i n the water holding measurement technique were minimized. For a l l other products, water holding capacity s u b s t a n t i a l l y under-estimated the true i n t e r a c t i o n of water with the product, more accurately described by the water hydration capacity. - 68 -Table V. Mean values of water hydration and water holding capacities of canola, sunflower and soybean products (n = 2). Product Water hydration capacity (g H2 n/g product) Water holding capacity (g H^O/g product) Canola - Control 1.8 a 1 0. ,60a (.100) 2 (. 117) - Trypsin 2.3 b 0. ,67a (.080) (. .0095) - Linoleate 3.0 C 2. ,02c (.140) (. .050) Sunflower - Control 2.9 d 1, ,5lb (.190) (. .040) - Trypsin 3.2 e 1, .30° (.075) (. .030) - Linoleate 3.8 f 0, ,84a (.125) (. .0065) Soybean - Control 3.4c 3.31 d (.105) (. .105) - Trypsin 2.78 2, .04c (.125) (. .150) - Linoleate 4.9 n 2, .42e (.100) ( .015) ^Means i n a column sharing the same superscript are not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) as determined by Duncan's multiple range test. ^Standard error of the mean - 69 -To evaluate the e f f e c t s of source and treatment on the degree of i n t e r -action of water with the products, only water hydration capacity values were considered. The control products had s i g n i f i c a n t l y d i f f e r e n t water hydration c a p a c i t i e s , with canola having the lowest value (1.8 g H^O/g product) and soybean the highest (3.4 g R^O/g product). Quinn and Paton (1979) measured the water hydration capacity of Promosoy 100 and found i t to be 3.00 g R^O/g product. The higher pH of the aqueous dispersions prepared i n the present study (pH 7.7 vs pH 7.0) would r e s u l t i n stronger i n t e r a c t i o n of water with the product. The e f f e c t of carbohydrate content appeared to be playing a role i n determining WHC, with the high carbohydrate products (soy and sunflower) i n t e r a c t i n g more strongly with water than the low carbohydrate canola i s o l a t e . Fiber residues are well known to have very high water holding c a p a c i t i e s , up to 100 g H^O/g db (Lapsley, 1980). Therefore, small amounts of crude f i b e r i n the products would be expected to strongly influence t h e i r water hydration capac i t i e s . Trypsin treatment caused the water hydration capacities to increase in the canola and sunflower products and decrease i n the soy product as compared to controls. The increased exposure of charged and polar amino acid groups to the aqueous environment would be expected to increase the i n t e r a c t i o n of water with the product r e s u l t i n g i n increased water hydration capacity, si m i l a r to the e f f e c t observed on s o l u b i l i t y . However, the packing arrangement of protein aggregates and c e l l wall fragments i n the hydrated p e l l e t w i l l also influence the amount of water that can be entrapped by the product. Possibly, the soybean t r y p s i n p a r t i c l e s can assume a closer packing arrangement than i n the control sample, l i m i t i n g the water hydration capacity. - 70 -Treatment with l i n o l e a t e resulted i n increased water hydration capacity, regardless of product source. Several factors may be responsible for this e f f e c t . F i r s t , there is the disruption of protein-protein hydrophobic bonds described by Cane 11a et a l . (1979) which would expose more polar and ion i c groups to the water. Second, the interactions between protein and l i n o l e a t e would increase the number of negatively charged s i t e s on the protein for hydrogen bonding (Nakai et a l . , 1980b). Third, the e f f e c t of l i n o l e a t e on aggregation may also be important. Linoleate treatment was observed to increase the mean p a r t i c l e size i n the canola and soybean dispersions. Chou and Morr (1979) reported that aggregation may increase water-protein interactions by forming a s t r u c t u r a l network capable of imbibing water. 5. Rheological Properties a. Steady shear The flow behavior rheograms of the nine sample dispersions are presented i n Figure 26. A l l dispersions were found to behave as non-Newtonian pseudo-p l a s t i c f l u i d s as indicated by the negative rheogram slopes. Sunflower and soy dispersions were higher in v i s c o s i t y than canola dispersions. The insoluble i r r e g u l a r l y shaped carbohydrate and protein materials i n the concentrate dispersions offered considerable resistance to flow. As shear rate increased, the p a r t i c l e s aligned themselves with the shear planes, o f f e r i n g less resistance to flow and e x h i b i t i n g a decreased v i s c o s i t y . Canola dispersions were generally less pseudoplastic than soy and sunflower samples. The s p h e r i c a l l y shaped protein aggregates offered l i t t l e resistance to flow at low shear rates and d i d not exhibit as great a thinning e f f e c t as the shear rate was increased, due to the r e l a t i v e l y uniform shape. None of the disperions were found to exhibit measurable y i e l d stress values. - 71 -1000 Figure 26. Flow behavior rheograms of untreated (C), t r y p s i n (T) and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products i n 10% aqueous dispersions at 20°C. - 72 -The Power-law flow parameters are l i s t e d i n Table VI. The Power-law model f i t t e d the data very well, as indicated by the high values for the c o e f f i c i e n t of determination. The degree of pseudoplasticity was indicated by the amount of deviation from a value of 1.0 for the flow behavior index. Values of apparent v i s c o s i t y at 100 s 1 represent apparent v i s c o s i t i e s at an i n t e r -mediate shear rate i n the range used i n t h i s study. The samples were found to be not s i g n i f i c a n t l y t h i x o t r o p i c , as indicated by the lack of a hysteresis loop i n the rheograms. Linoleate treatment brought about an increase in apparent v i s c o s i t y at a shear rate of 100 s 1 and a decrease in flow behavior index i n a l l sources as compared to controls. The e f f e c t of tr y p s i n treatment on the viscous properties of the disperisons varied considerably with the source. Trypsin hydrolysis increased the apparent v i s c o s i t i e s at 100 s 1 and decreased the flow behavior indices of canola and sunflower products. Soy product which had been treated with the enzyme, formed dispersions with lower apparent v i s c o s i t i e s at 100 s 1 and higher flow behavior indices. These r e s u l t s are i n agreement with those of Lynch et a l . (19 77) who found a decrease i n apparent v i s c o s i t y of a soy protein i s o l a t e dispersion as a r e s u l t of pepsin hydrolys i s . Trypsin treatment was also found to a f f e c t the water hydration capacity of soy product d i f f e r e n t l y than canola and sunflower sources. These observations suggest that treatment with tr y p s i n was a l t e r i n g the basic physical and chemical properties of the products i n d i f f e r e n t ways. Changes i n the two phases of the dispersion can be related to the t r e a t -ment e f f e c t s on flow behavior. The properties of the aqueous phase may be influenced by the treatments. In pure protein solutions, both apparent - 73 -Table VI. Mean values of steady shear Power-law flow parameters of 10% aqueous dispersions of canola, sunflower and soybean products (n = 2). Flow behavior Consistency Apparent C o e f f i c i e n t of index, n c o e f f i c i e n t , m v i s c o s i t y determination, (Pa s n) at 100 s - 1 r 2 Product (mPa s) Canola - Control - Trypsin - Linoleate Sunflower - Control - Trypsin - Linoleate Soybean - Control - Trypsin - Linoleate .955 a 1 (.0365) 2 .822b (.0125) .788 b c (.0100) .729 c d (.0100) .584e (.0160) .663 d (.0200) .577e (.0428) .828b (.0195) .568e (.0045) 0.0385 3 (.00651) 0.0950b (.00699) 0.317 C (.0224) 1.21d (.0790) 3.45 e f (.517) 2.26 fg (.0080) 2.198 (.584) .320c (.0491 4.63e (.308) 3.09 a (.0120) 4.16 b (.065) 11.9 C (.300) 35.0 d (3.71) 50.3e (3.91) 48. ie (4.25) 29. l d (2.47) 14.3c (.919) 63.Of (2.90) .941 .993 .971 .989 .980 .977 .911 .969 .975 1-Means i n a column sharing the same superscript are not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) as determined by Duncan's multiple range test. 2standard error of the mean - 74 -v i s c o s i t y and pseudoplasticity increase as protein concentration increases (Pradipasena and Rha, 1977). Thus, treatments which increase the s o l u b i l i t y of the protein i n the products might be expected to produce dispersions with increased v i s c o s i t i e s and decreased flow behavior indices. Such was found to be the case for canola and sunflower sources, however r e s u l t s for soybean are inconsistent with this explanation. Although an increase in s o l u b i l i t y was achieved, e f f e c t s on flow behavior were observed to be opposite to those expected. Obviously some factor other than s o l u b i l i t y was playing a r o l e i n determining flow behavior. The properties of the dispersed phase are also important i n determining flow properties. The s i z e , shape and hydrodynamic properties of the c o l l o i d a l p a r t i c l e s influence v i s c o s i t y . Lee and Rha (1979) compared the flow behavior and p a r t i c l e size ranges of two soy protein i s o l a t e dispersions prepared using d i f f e r e n t mixing techniques. They found that the v i s c o s i t y of a dispersion containing large p a r t i c l e s (100 to 500 um) was higher than the v i s c o s i t y of a smaller p a r t i c l e (2 to 20 Um) dispersion. The shape of the p a r t i c l e s i s another important factor i n determining how e a s i l y the p a r t i c l e s can move past one another i n the dispersion. In general, at the molecular l e v e l , long polymers o f f e r greater resistance to flow than the same concentration of smaller, more symmetrical molecules. For larger complex aggregates, hydration and swelling become important factors, a f f e c t i n g p a r t i c l e s i z e and density and immobilizing some volume of free water. Inter-molecular in t e r a c t i o n s also play a role i n governing flow properties. Aggregation of p a r t i c l e s may increase to such an extent that the dispersed phase may sediment. However, i f the degree of i n t e r a c t i o n i s f a i r l y low, increases i n p a r t i c l e s i z e , hydration and swelling may lead to the formation of stable high v i s c o s i t y dispersions. The e f f e c t s of tr y p s i n hydrolysis on the flow behavior of the soy protein concentrate may be related to changes i n the properties of the dispersed phase, which could outweigh the e f f e c t of increased s o l u b i l i t y on the aqueous phase. In the case of l i n o l e a t e treatment, also, the e f f e c t s on flow behavior cannot be at t r i b u t e d e n t i r e l y to the e f f e c t of increased s o l u b i l i t y . Linoleate treatment of the canola i s o l a t e was unsuccessful i n increasing protein s o l u b i l i t y and therefore some other factor must be contributing to the observed increase i n apparent v i s c o s i t y at 100 s ^, and pseudoplasticity of the dispersion. The increase i n aggregate size, observed under the l i g h t microscope, accompanied by increased hydration, may be at least p a r t i a l l y responsible. b. Dynamic shear behavior ( v i s c o e l a s t i c i t y ) The v i s c o e l a s t i c properties of the nine product dispersions were evaluated at 20, 4 5 , 70 and 95°C. The e l a s t i c and viscous moduli of a l i n e a r l y v i s c o -e l a s t i c f l u i d are independent of the amplitude and duration of o s c i l l a t i o n and dependent on the o s c i l l a t o r y frequency. To determine the r e l a t i o n s h i p between the v i s c o e l a s t i c parameters and frequency, dynamic testing was c a r r i e d out at f i v e d i f f e r e n t frequencies i n the f i r s t experimental run. The r e s u l t s indicated that a very poor r e l a t i o n s h i p e x i s t s for these samples. Figures 27 and 28 show the recorded values of storage modulus and loss tangent as functions of frequency, at 45°C. Similar r e s u l t s were obtained at 20, 70 and 95°C. Generally, increases i n storage modulus and decreases i n loss tangent are expected to occur as frequency increases. - 76 -Figure 27. Effect of frequency on storage moduli of 10% product dispersions at 45°C. - 77 -2.8 2 .4 U 2.0 1.6 c CO S '- 2 o .8 » C a n o l a • - - • » S u n f l o w e r ^ — « — S o y b e a n # C o n t r o • T r y p i i n • L i n o l e a t e . 1 9 . 6 0 1.9 F r e q u e n c y , s 6 .0 19 Figure 28. E f f e c t of frequency on loss tangents of 10% product dispersions at A5°C. - 78 -The values of the v i s c o e l a s t i c parameters were also found to fluctuate over the duration of testing at a constant frequency. Some form of time-dependence was concluded to be i n t e r f e r i n g with the expected behavior. Therefore, only values recorded at a frequency of 6.0 s 1 , taken soon af t e r i n i t i a t i o n of o s c i l l a t o r y testing were examined to assess the e f f e c t s of treatment on the v i s c o e l a s t i c properties. For the r e p l i c a t i o n of the experiment, only a frequency of 6.0 s 1 was employed. Table VII presents the values of storage modulus and loss tangent obtained for the nine samples at 20°C and 6.0 s 1 . The high degree of v a r i a b i l i t y as indicated by the large values of standard error of the mean prevented meaningful s t a t i s t i c a l analysis of this data. The high v a r i a b i l i t y of the r e p l i c a t e samples can be att r i b u t e d to dehydration of the sample at the circumference of the fi x t u r e and interference by p a r t i c l e s that were r e l a t i v e l y large i n r e l a t i o n to the gap thickness. In future work these sources of error may be minimized by using plate/plate f i x t u r e s with a large separation rather than a cone/plate system, and enclosing the fix t u r e s i n a high humidity chamber. The e f f e c t s of treatment on the v i s c o e l a s t i c properties of the dispersions at 20°C can be interpreted from trends i n the data (Table VII). The soy control dispersion exhibited the highest value of storage modulus, l i k e l y as a r e s u l t of the large amount of insoluble carbohydrate and protein material. The storage modulus describes the e l a s t i c i t y of the sample. Results for a l l trypsin-hydrolyzed samples suggested a trend toward lower e l a s t i c i t y , due perhaps to increased s o l u b i l i t y and reduced aggregate s i z e . The l i n o l e a t e e f f e c t was less d i s t i n c t . For canola, an increase i n e l a s t i c i t y was observed, probably as a r e s u l t of larger aggregate s i z e . Soy and sunflower products, - 79 -Table VII. Mean values of v i s c o e l a s t i c properties of 10% aqueous dispersions of canola, sunflower and soybean products at 20°C (n = 2). Storage modulus Loss tangent Product (Pa) Canola - Control 1.39 1.12 (0.478)1 (0.160) - Trypsin 0.202 4.25 (0.116) (3.90) - Linoleate 4.60 1.14 (3.20) (0.670) Sunflower - Control 3.94 2.76 (2.79) (1.06) - Trypsin 2.07 2.51 (0.295) (1.65) - Linoleate 2.60 2.89 (1.39) (1.57) Soybean - Control 55.2 1.46 (20.7) (0.125) - Trypsin 1.85 2.60 (1.14) (2.19) - Linoleate 30.2 1.37 (21.4) (0.155) 1Standard error of the mean. - 80 -treated with l i n o l e a t e , exhibited a decrease i n e l a s t i c i t y over controls, possibly related to increased s o l u b i l i t y . The values of loss tangent (Table V i i ) r e f l e c t the r e l a t i v e proportions of the e l a s t i c and viscous natures of the material. Loss tangents greater than 1.00 indicate behavior that is predominantly viscous and values less than 1.00 describe predominantly e l a s t i c material. At 20°C, a l l dispersions exhibited v i s c o e l a s t i c behavior that was more viscous than e l a s t i c . The higher loss tangent values reported for trypsin-hydrolyzed soy and canola samples indicate v i s c o e l a s t i c behavior that i s more viscous i n nature than corresponding control and l i n o l e a t e dispersions. This behavior was also r e f l e c t e d i n the low values of storage modulus for these trypsin-treated samples. Linoleate treatment did not appear to a f f e c t the loss tangents measured for the dispersions, as compared to controls. The e f f e c t of temperature on the v i s c o e l a s t i c properties of the dispersions is shown in Figures 29 and 30. As the temperature increased, the storage moduli (Figure 29) of the dispersions generally increased. This would be caused by the formation of a g e l - l i k e matrix within the sample, capable of storing more of the deformation energy. The values of loss tangent (Figure 30) were observed to follow a decreasing trend as temperature increased. This r e f l e c t s a s h i f t i n the r a t i o of viscous to e l a s t i c natures, as the dispersions undergo network formation to produce semi-solid g e l - l i k e materials. The e f f e c t of source and treatment on the response of the dispersions to heating cannot be discerned from the res u l t s due to the high degree of v a r i a b i l i t y of the data. Figure 29. E f f e c t of temperature on storage moduli of 10% product dispersions at a frequency of 6 s ~ l . Figure 30. E f f e c t of temperature on loss tangents of 10% product dispersions at a frequency of 6 s ~ l . - 83 -D. S t a t i s t i c a l Analysis 1. Analysis of variance The data c o l l e c t e d for 10 measured variables were analyzed as a 3 x 3 f a c t o r i a l analysis of variance, to determine the s i g n i f i c a n c e of the observed e f f e c t s of "source" and "treatment". The r e s u l t s are presented i n Table VIII. Source and treatment were found to s i g n i f i c a n t l y a f f e c t most of the measured properties. In cases where the i n t e r a c t i o n between source and treatment i s not s i g n i f i c a n t , methods can be employed to determine the e f f e c t s of a p a r t i c u l a r source or treatment. Duncan's multiple range t e s t , at the 5% l e v e l of s i g n i f i c a n c e , was employed for this purpose. For example, soy and sunflower products have s i g n i f i c a n t l y d i f f e r e n t protein contents from canola products, regardless of the treatment (Table IX). Also the protein contents of control and trypsin-treated products d i f f e r s i g n i f i c a n t l y from those of products treated with l i n o l e a t e , i r r e s p e c t i v e of source. For the measured properties for which the i n t e r a c t i o n term was s i g n i f i c a n t , i t was impossible to separate the e f f e c t of source from the treatment e f f e c t . Therefore, single factor analyses of variance were also was performed on the data to determine the s i g n i f i c a n c e of the e f f e c t of "product" on the measured properties. Duncan's multiple range t e s t s , at the 5% l e v e l of s i g n i f i c a n c e were performed to determine differences among i n d i v i d u a l means, r e s u l t i n g i n the superscripts assigned to means recorded i n Tables II-VII. 2. Simple c o r r e l a t i o n s among variables Correlation c o e f f i c i e n t s calculated for correlations among 12 measured variables are reported i n Table X. It is of i n t e r e s t to note that s o l u b i l i t y , the functional property which is most commonly reported i n the l i t e r a t u r e , was - 84 -Table VIII. Levels of si g n i f i c a n c e of F-values calculated from data c o l l e c t e d for each measured variable by a 3 x 3 (source x treatment) f a c t o r i a l analysis of variance. Variable Source df = 2 Treatment df = 2 Interaction df = 4 Protein ** ** NS Carbohydrate ** NS NS Moisture ** ** pH ** ** ** Conductance ** ** ** S o l u b i l i t y ** ** Water hydration capacity ** ** ** Flow behavior index ** ** ** Consistency c o e f f i c i e n t (log) ** ** ** Apparent v i s c o s i t y at 100 s ^ (log) ** ** ** ** p<0.01 * p<0.05 NS p>0.05 - 85 -Table IX. Results of Duncan's Multiple Range testing for variables for which the source x treatment i n t e r a c t i o n term was not s i g n i f i c a n t (p>0.05). Variable P r o t e i n Canola Sunflower Soybean Control Trypsin Linoleate Carbohydrate Canola Sunflower Soybean Source or treatments sharing the same underscore are not s i g n i f i c a n t l y d i f f e r e n t (p>0.05). - 86 -Table X. Cor r e l a t i o n c o e f f i c i e n t s calculated for linear c o r r e l a t i o n s among a l l measured variables (n = 9) Protein Carbo-hydrate Solub i l i t y Water hydration Water holding n value Conductance -.059 -.249 .186 .301 .306 .040 pH .490 -.626 .015 -.295 -.418 .363 Loss tangent (20°C) .173 -.001 .387 -.215 -.493 .071 Storage modulus (20°C) -.502 .462 -.542 .503 .836 -.605 Apparent v i s c o s i t y (100 s" 1) -.819 .667 .573 .878 .268 -.865 m value -.756 .605 .429 .874 .356 -.886 n value .767 .670 -.194 -.836 -.553 Water holding capac i t y -.615 .566 -.545 .508 Water hydration capac i t y -.858 .573 .370 .950 Apparent v i s c o s i t y S o l u b i l i t y -.231 .084 .311 .456 Storage modulus Carbohydrate .893 -.442 -.086 -.192 Loss tangent -.465 -.285 -.235 -.230 pH -.025 -.317 .272 -.113 .076 Conduc-tance pH Loss tangent Storage modulus Apparent v i s c o s i t y m value r . 0 5 = '666 r.01 = -798 - 87 -found to be not s i g n i f i c a n t l y correlated (p>0.05) with any of the other measured parameters. The non-significant c o r r e l a t i o n between water holding and water hydration capacities confirmed the e a r l i e r observation that the water holding capacity technique is not a meaningful measure of the hydration a b i l i t i e s of a product. Some of the more i n t e r e s t i n g s i g n i f i c a n t c o r r e l a t i o n s were those involving water hydration capacity and apparent v i s c o s i t y (at 100 s as these functional properties relate c l o s e l y to the performance of the products in food systems. Figures 31 to 33 present gra p h i c a l l y , some of these c o r r e l a t i o n s . When plotted, the c o r r e l a t i o n between consistency c o e f f i c i e n t (m) and flow behavior index (n) appeared to follow a semi-logarithmic r e l a t i o n s h i p , i n spite of the high value of the c o r r e l a t i o n c o e f f i c i e n t calculated for the lin e a r model. The data were re-plotted as log m vs. n (Figure 34) and an even higher value of the c o r r e l a t i o n c o e f f i c i e n t (r = .958) was computed for this semi-log c o r r e l a t i o n . Lefebvre and Sherman (1977) also report a hig h l y s i g n i f i c a n t c o r r e l a t i o n (r = 0.958, p<0.01) between log m and n based on res u l t s from several d i f f e r e n t sunflower i s o l a t e dispersions. Neither pH nor conductance were found to be correlated with any of the other measured properties, i n d i c a t i n g that the d i f f e r e n t pH and io n i c strengths of the product dispersions was not sole l y responsible for the observed treatment e f f e c t s . - 88 -• S o L 6 0 m Pa s 5 0 - • SuT S u L « 4 0 i o o • S u C X 30 -• S o C </> O u l/l > 2 0 C Sol • 0) k_ • C a L D D_ 10 -D_ < r = - . i 8 1 9 3 ^ C a T • C a C 1 1 1 6 0 7 0 8 0 9 0 100 P r o te i n , % d b Figure 31. C o r r e l a t i o n between apparent v i s c o s i t y (100 s ) of 10% product dispersions at 20°C and protein content of untreated (C), t r y p s i n (T) and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products. - 89 u => ~ 0 O \ O CN X u O a o U c o TJ X D 4 r - 3 h 60 70 80 P r o t e i n , 9b d b Figure 32. C o r r e l a t i o n between water hydration capacity and protein content of untreated (C), t r y p s i n (T) and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products. - 90 -S o l • 6 0 m Pa s 5 0 S u T « • SuL - 4 0 -T o o S u C « X 30 • SoC V i s c o s i 2 0 -Apparent 10 C a C « 1 S o T « C a L • r = . 9 4 9 8 • C a T 1 1 1 2 W a t e r 9 3 4 5 H y d r a t i o n C a p a c i t y H 2 0 / g p r o d u c t Figure 33. C o r r e l a t i o n between apparent v i s c o s i t y (100 s~ ) of 10% product dispersions at 20°C and water hydration capacity of untreated (C), t r y p s i n (T) and l i n o l e a t e (L) treated . canola (Ca), sunflower (Su) and soybean (So) products. - 91 -• S o L 3 • S u T • S o C • S u L c in O a. 1 • S u C £ c i> C o e f f i c i . 3 C a L • • SoT i s t e n c y . 1 C o n s i r = . 9 5 8 2 1 1 1 • C a T C a C • . 6 . 7 . 8 . 9 1.0 F l o w B e h a v i o r I n d e x n Figure 34. C o r r e l a t i o n between consistency c o e f f i c i e n t (m) and flow behavior index (n) for 10% dispersions of untreated (C), t r y p s i n (T) and l i n o l e a t e (L) treated canola (Ca), sunflower (Su) and soybean (So) products at 20°C. - 92 -SUMMARY AND CONCLUSIONS Nine high-protein oilseed products were examined for compositional, s t r u c t u r a l and functional properties. Trypsin and l i n o l e a t e treated products d i f f e r e d s i g n i f i c a n t l y from untreated controls in several of the parameters measured. Trypsin hydrolysis did not a f f e c t the protein, carbohydrate and moisture contents of products prepared from canola, sunflower or soybean sources. Under the l i g h t microscope, 10% dispersions of trypsin-hydrolyzed canola and soybean products were observed to contain smaller dispersion p a r t i c l e s than were found i n controls. A change i n p a r t i c l e s i z e could not be detected i n the trypsin-treated sunflower samples. Treatment with the enzyme appeared to increase the number of s i t e s a v a i l a b l e for g e l network formation, as indicated by examining thermally treated 10% dispersions under the scanning electron microscope. However, the protein-protein interactions were observed to be weak i n nature. In the case of trypsin-treated soy product, the material produced by heating a 10% dispersion was very se n s i t i v e to mechanical destruction and did not possess the necessary s t r u c t u r a l i n t e g r i t y to permit microscopic observation. Measurements of conductance of 10% aqueous dispersions, recorded as an index of i o n i c strength, indicated that trypsin-hydrolyzed samples had higher i o n i c strengths than controls. Protein s o l u b i l i t y was found to increase as a r e s u l t of t r y p s i n treatment. Canola and sunflower products treated with t r y p s i n exhibited higher water hydration c a p a c i t i e s , while trypsin-treated soy samples were found to have lower hydration a b i l i t i e s . - 93 -Trypsin-treatment produced canola and sunflower dispersions with lower flow behavior indices and higher values of apparent viscosity at 100 s \ as compared to controls. Soy product which had been similarly treated demon-strated the opposite effect, with higher flow behavior indices and lower apparent viscosities at 100 s 1. Although significant differences could not be detected, trypsin-hydrolyzed products in 10% aqueous dispersion exhibited trends toward lower values of storage modulus and higher loss tangents. These trends reflected viscoelastic behavior that more closely describes ideal viscous fluids than did the behavior of corresponding controls. Treatment of the oilseed products with linoleic acid brought about signif-icant decreases in protein and carbohydrate contents, due to displacement of some weight of these components by the linoleic acid which was added at a level of 8 . 7%. Observed differences in moisture content were not attributed to treatment effects. Light microscopic observation of 10% dispersions of linoleate-treated products revealed the presence of larger protein aggregates in canola and soybean sources as compared to controls. The structural properties of the sunflower dispersions were found to be unaffected by linoleate treatment. Linoleic acid treatment was observed to increase protein-protein interactions in thermally treated 10% dispersions from a l l sources, resulting in stronger gel-like materials than were produced by heating control dispersions. The ionic strengths of 10% product dispersions were found to increase as a result of linoleate treatment, as indicated by conductance measurements. Protein in linoleate-treated sunflower and soy samples was more soluble than in untreated products, while canola products exhibited no change in protein - 94 -s o l u b i l i t y as a r e s u l t of l i n o l e a t e treatment. Higher values of water hydration capacity were recorded for a l l l i n o l e a t e - t r e a t e d products as compared to controls. Dispersions of l i n o l e a t e - t r e a t e d products were found to have s i g n i f i c a n t l y higher apparent v i s c o s i t i e s at 100 s ^ than control dispersions. The flow behavior index of the 10% canola product dispersion was reduced s i g n i f i c a n t l y as a r e s u l t of l i n o l e a t e treatment. S i m i l a r l y treated soy and sunflower product dispersions exhibited trends toward lower values of flow behavior index. E f f e c t s of l i n o l e a t e treatment on the v i s c o e l a s t i c properties of 10% dispersions could not be discerned from the data. The water hydration capacity technique proposed recently by Quinn and Paton (1979) was found to provide a meaningful measurement of the hydration a b i l i t i e s of dry powdered protein products. V i s c o e l a s t i c properties of 10% product dispersions were recorded at 20, 45, 70 and 95°C to follow the changes occurring during the gelation process. Trends toward increases i n storage moduli and decreases in loss tangents with increasing temperature were observed. This research has confirmed the p o t e n t i a l of modifying agents for use i n producing p r o t e i n - r i c h products with desired functional properties. Trypsin and l i n o l e i c acid treatments brought about f u n c t i o n a l i t y changes i n aqueous systems that could be attributed not only to the degree of i n t e r a c t i o n of the product with water, but also to the size and shape of the product p a r t i c l e s . It has been demonstrated that complex r e l a t i o n s h i p s exist among the f u n c t i o n a l , s t r u c t u r a l and compositional properties of oils e e d concentrates and i s o l a t e s . The r e s u l t s provide an important basis for further research to define c l e a r l y the physico-chemical bases of functional properties. As long as food - 95 s c i e n t i s t s u t i l i z e f u n c t i o n a l i t y tests to protein products i n food systems, i t w i l l our understanding of the basic p r i n c i p l e s evaluate the p o t e n t i a l behavior of be necessary to continue to improve governing these phenomena. - 96 -LITERATURE CITED AOCS (American O i l Chemists' Society). 1969. Nitrogen s o l u b i l i t y index (NSl). O f f i c i a l method Ba 11-65 in " O f f i c i a l and Tentative Methods of the AOCS", 3rd e d i t i o n , eds. Mecklenbacher, V.C., Hopper, T.M. and Sallee, E.M., Champaign, IL AOAC (Association of O f f i c i a l A n a l y t i c a l Chemists). 1975. " O f f i c i a l Methods of Analysis", 12th ed i t i o n , Section 14.002, ed. Horowitz, W., Washington, DC Aoki, H. 1965. The gelation of soybean protein. I I . 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