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II I 'I‘ I 'I I II II [II II.I I ‘ 'I h "_ III. .I‘.‘ ‘: "’ 1‘1‘::I ‘ [1.11:1 IIZI‘I'I I I I I. . II ‘ I ‘ I ‘1' “p ‘ . ‘ ‘I ' ' y 1‘ ‘ ‘ III I I II I I II III I II’I. III I‘ [LII I I .II I I I I I I I II - I .I I I II III‘. III‘ I I II IIIII III I III" I 4‘ I‘I“‘III ‘III I” ' “I . IIIII’ IIIIII. ’ II- III'IIII 'II‘I‘ II ‘1 IIII I III I I I' ‘ ‘ \ IIII IIII II I \III ”II N I [IL IIIII M I VI" II III' " . ‘I IIIII,IIIII|I'I‘II IIIH IIlIILIIIII III ““I ”IIII‘II 4 ‘III II I‘ .I 1 .I I'IIIII IIIIIIII LIII II IIIIIII I'IIIIII'I‘..I LI. "mum III?!I1 I'I-I II I . .4 I LIMA a Y Michigan State University This is to certify that the thesis entitled Functional Characterization and Heat Induced Interactions Between Milk Products and Soy Isolate presented by Bruce R. Harte has been accepted towards fulfillment of the requirements for Ph. D. degreein Food Science and Human Nutrition W Major professor Date December 21. 1978 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. 3&0 A00? z .- an” m“ W64» - ‘ “q“ 6 2005 " AUG2.080310] FUNCTIONAL CHARACTERIZATION AND HEAT INDUCED INTERACTIONS BETWEEN MILK PRODUCTS AND SOY ISOLATE By Bruce R. Harte A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1978 ABSTRACT FUNCTIONAL CHARACTERIZATION AND HEAT INDUCED INTERACTIONS BETNEEN MILK PRODUCTS AND SOY ISOLATE By Bruce R. Harte The functionality of milk productzsoy isolate blends was evaluated. The commercial milk components included: 1) sweet whey powder, 2) electrodialyzed whey powder, 3) whey protein concentrate (UPC), 4) nonfat dry milk (NFDM) and 5) sodium caseinate. A commercial soy isolate was selected based upon ease of dispersibility, soluble protein and flavor. Five functional properties were exam- ined: emulsion capacity, whipping ability, viscosity, solubility and sensory evaluation. Dispersions were pre- pared at protein levels of 3.2, 5.0 and 8.0% in ratios of 25:75, 50:50 and 75:25. The samples were subjected to various treatments including: heating, pH variation and addition of salts. A second set of treatments was designed after evaluation of the first trials. Additional treat- ments were selected to further improve functionality. Many of the treatments included variants from the first study in addition to the inclusion of chemical modifiers, gums, emulsifiers and an enzymatic digestion. Milk products demonstrated the highest solubility, with milk productzsoy isolate blends having appreciably Bruce R. Harte higher solubility than the soy isolate. Specific treatments resulted in widely varying solubility. The viscosities of the milk products, soy isolate and their blends were quite close regardless of the blend ratio. Viscosities rose moderately as the percent protein increased. The majority of treatments had little effect upon viscosities. In general, the soy isolate had less emulsion capacity than the milk products. Blends Often had nearly the same emul- sion capacity as the milk product. Emulsion capacities were affected by various treatments. Stable foams were produced from NFDM, sodium caseinate, electrodialyzed whey, and their soy isolate blends. WPC whipped into stable foams after subjection to Specific treatments. Blends produced foams which had Similar Specific volumes with Slightly less foam stability than the respective milk pro- duct. It was possible to substantially improve the whip— ping properties of the samples by utilization of specific treatments. The milk products, particularly NFDM and sodium caseinate had the highest flavor scores. The flavor characteristics of soy were considered unacceptable. How- ever, when used in combination with milk products the flavor properties were only slightly less than the respective milk product. Chemical modification, heating, pH variation, enzymatic hydrolysis and change in the ionic environment improved the functionality of many samples. Utilization of different Bruce R. Harte treatments made it possible to maintain the generally higher functionality associated with the milk products. In many instances, replacement of as much as 5 % of the milk protein product by soy isolate was achieved with little loss of functionality. Milkzsoy protein blends were examined for heat-induced interaction. The milk proteins were prepared from sweet whey, acid whey, colloidal casein and sodium caseinate. Samples of these proteins were heated with soy protein. The heat treatments included: unheated-control, 680C/30 min, 77°C/2O sec, 94°C/TO sec and l2lOC/5 sec. After heat- ing, the proteins were fractionated by gel filtration and resolved by gel electrophoresis. Whey protein suffered substantial denaturation at temperatures greater than 77°C/ 20 sec. Casein was stable to the heat treatments. Soy protein began to dissociate into minor components at 77°C/ 20 sec. Further heating resulted in the disappearance of the major bands. There did not appear to be any interaction between whey or casein and the major soy proteins. The possibility exists of hydrophobic interaction between casein and soy protein. An in_yitrg_enzymatic procedure was utilized to exam- ine release of total amino nitrogen from intact proteins as affected by: processing treatments and possible interactions between protein systems. Soy isolate, NFDM and their blend were subjected to 24 different treatments. In general, the Bruce R. Harte liberation of amino nitrogen was not significantly reduced by use of the process variants. Specific cases (such as the addition of chemical modifiers or extremely high pH) lowered the released amino nitrogen. Protein interaction detrimental to the release of amino nitrogen was minimal. ACKNOWLEDGMENTS The author expresses his sincere gratitude to Dr. C.M. Stine for his help and counsel during the course of this research and preparation of the manuscript. Grateful acknowledgment is due members of the guidance committee: Dr. J.R. Brunner, Department of Food Science and Human Nutrition, Dr. H.A. Lillevik, Department of Biochemis- try, Dr. N.G. Bergen, Department of Animal Husbandry, and Dr. J.w. Allen, Department of Marketing. Special thanks are also extended to Dairy Research, Inc. whose grant made this work possible. The financial assistance and laboratory Space provided by the Department of Food Science and Human Nutrition is also warmly acknowledged. The author is most grateful to his wife, Janice, who kept him going during the rough spots and who Shares in this with him. ii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES INTRODUCTION. LITERATURE REVIEW Functionality. Solubility . Viscosity and Gelation Emulsion Capacity. Whipping Ability Sensory Evaluation Heat Stability Soy Protein. Milk Proteins. . In Vitro Estimation of PrOtein Quality Assay Procedures . Evaluation of Processing Effects by In Vitro Enzymatic Procedures . . EXPERIMENTAL PROCEDURES Functional Studies . Preparation of Samples Whipping . . . Emulsion Capacity. Soluble Protein. Viscosity. . Solubility Index Gelation . Sensory Subjective Analysis. Examination for Heat Induced Interactions. Preparation of Products. Soy Protein. Whole Casein Sodium Caseinate . . Whey Proteins from Acid Whey Whey Proteins from Sweet Whey. Page ix Gel Filtration. . . Polyacrylamide Gel ElectrophOresis. Gel Scanning. . Nonprotein Nitrogen . . In Vitro Enzymatic Hydrolysis Preparation of Samples. Hydrolysis Procedure. Protein Determinations. . . Lowry Procedure for Soluble PrOtein Lowry Procedure for Insoluble Protein Kjeldahl Analysis RESULTS AND DISCUSSION Investigation of Functional Properties. Solubility. . . Solubility Index. Viscosity Gelation. . Emulsion Capacity Whipping Ability. Sensory Evaluation. . . Examination of Milk and Soy PrOtein Blends fOr. Heat Induced Interactions . Whey: Soy Protein Blends Casein: Soy Protein Blends Ln Vitro Estimation of Protein Quality. SUMMARY. APPENDIX Lowry Procedure for Soluble Protein Lowry Procedure for Insoluble Protein Kjeldahl Nitrogen PAGE Solutions. . Malik- Berrie Staining PrOcedure Ninhydrin Solution. Acid Hydrolysis Statistical Procedures Used in the Evaluation of. Protein Quality . BIBLIOGRAPHY iv Page OOOOOOOOOOOOOOOOOOOOOO kOkoooooucnmmbw—a KO ._1 —J—J—.l——J—l—J wwNNH-HKOKO NWV-DWON-J Table 10 11 12 - LIST OF TABLES Dispersible protein (%) of several soy isolates at 4.0% protein in water. . . . Protein solubility (%) of samples of NFDM, NAC, SI, WPC, EW and SW at a protein concentration of 3.2%. . . . . . . . . . . . . . . . . . . . . Protein solubility (%) of blends Of NAC and SI at a protein concentration of 5.0% . . . . . Protein solubility (%) Of blends of NFDM and SI at a protein concentration of 3.2%. . . Protein solubility (%) of blenda of NAC and $1 at a protein concentration of 5.0% . . . . . Protein solubility (%) of blends of EW and SI at a protein concentration of 3. 2% . . . Protein solubility (%) of blends of SW and $1 at a protein concentration of 5. 0% . . . . Protein solubility (%) of WPC, WPC/75, SW and EW/75 at a protein concentration of 3.2%. Protein solubility (%) of NFDM, NFDM/50, SI, NAC, and NAC/SO at a protein concentration of 3.2% Solubility index of NFDM, NFDM/50, NAC, NAC/SO, EW, EW/75, WPC, WPC/75, SW, SW/SO and SI at a protein concentration of 3.2% . . . . Viscosity (cp) for selected samples of NFDM, NFDM/50, NAC, NAG/50 and 51 at 3.2, 5.0 and 8.0 percent protein . . . . . . . . . . . Viscosity (cp) for selected samples of EW, EW/75, WPC, WPC/75, SW and SW/SO at 3. 2, 5.0 and 8.0 percent protein . . . . . . . . . Page 95 102 103 104 105 106 102 108 109 111 120 121 Table Page 13 Viscosity (cp) for selected samples of NFDM, NFDM/50, NAC, NAC/50 and SI at a protein concen- tration of 3.2%. . . . . . l22 l4 Viscosity (cp) for selected samples of EW, EW/75, WPC, WPC/75, SW and SW/SO at a protein concentration of 3.2%. . . . . . . . . . . . . l23 15 Gelation strength, measured with a Plummet*, was determined for SI, NFDM, WPC, WPC/50 and EW/25 . l26 l6 Emulsion capacity measured in g oil/mg protein for NFDM, NFDM/50, SI, NAC/50 and NAC at a pro- tein concentration of 3.2% . . . . . l3l l7 Emulsion capacity measured in g oil/mg protein for EW, EW/75, WPC, WPC/75, SW and SW/50 at a protein concentration of 3.2%. . . . . . . . . . 132 l8 Whipping ability as specific volume (SV) in ml/g and g stability time (at) in min for WPC at protein levels of 3.2, 5.0 and 8.0%. . . . . . . l43 l9 Whipping ability as Specific volume (SV) in ml/g and a stability time (at) in min for EW at protein levels of 3.2, 5.0 and 8.0% protein. . . l44 20 Whipping ability as specific volume (SV) in ml/g and a stability time (kt) in min for EW/25, EW/SO and EW/75 at a protein level of 5.0% . . . . . . I45 21 Whipping ability as specific volume (SV) in ml/g and a stability time (at) in min for NAC at protein levels of 3.2, 5.0 and 8.0%. . . . . . . 146 22 Whipping ability as specific volume (SV) in ml/g and % stability time (at) in min for NAC/25, NAC/50 and NAC/75 at a protein level of 3.2% . . 147 23 Whipping ability as specific volume (SV) in ml/g and g stability time (at) in min for NFDM at protein levels of 3.2, 5.0 and 8.0%. . . . . . . 148 24 Whipping ability as specific volume (SV) in ml/g and % stability time (at) in min for NFDM/25, NFDM/50. and NFDM/75 at a protein level of 3. %. l49 25 Whipping ability as specifiv volume (SV) in ml/g and % stability time (at) in min for NFDM, NFDM/50, NAC, NAC/50 and SI at a protein level of 3.2%. . . . . . . . . . . . . . . . . . . . 150 vi Table Page 26 Whipping ability as specific volume (SV) in ml/g and a stability time (at) in min for WPC, WPC/75, SW, SW/SO, EW and EW/75 at a protein level of 3.2%. . . . . . . . . . . . . . 151 27 Flavor scores* of the whips from samples of EW, EW/75, WPC, WPC/75, SW and SW/SO at a protein level of 3.2%. . . . . . . . . . . . . . . . . . 158 28 Flavor scores* of the whips from samples of NFDM, NFDM/50, NAC, NAC/50 and SI at a protein level of 3.2%. . . . . . . . . . . . . . . . 159 29 Texture scores* of the whips from samples of NFDM, NFDM/50, NAC, NAC/50, EW, EW/75, WPC, WPC/75, SW, SW/SO and SI at a protein level of 3.2% . . . . . . . . . . . . . . . . . . . 160 30 Color scores* of the whips from samples of NFDM, NFDM/50, NAC, NAC/50, EW, EW/75, WPC, WPC/75, SW, SW/SO and SI at a protein level of 3.2%; . . 161 31 Solubility (%) of water soluble soy protein in selected buffers at 1.0% protein . . . . . . . . 163 32 Solubility (%) of NAC, WC, AC, and SW in phos- phate buffer pH 7.0, p=0.1 at 1.0% protein . . . 163 33 Solubility of AC, AS, SP, SS and SW in phos— phate buffer pH 7.0, p=0.1 at a protein level of 1.0%. . . . . . . . . . . . . . . . . . . . . 165 34 Solubility of NAC, NS, WC and WS in phosphate buffer pH 7.0, p-0.l at a protein level of 1. %. 172 35 Percent nitrogen in each fraction of whey pro- tein separated by gel filtration . . . . . . . . 178 36 Percent nitrogen in each fraction of soy protein separated by gel filtration. . . . . . . . . . . 183 37 Percent nitrogen in each fraction of the whey: soy protein blend separated by gel filtration. . 188 38 Percent nitrogen in each fraction of whey pro- tein, soy protein, and their blend separated by gel filtration with sepharose 4B. . . . . . . 201 39 Percent nitrogen in each fraction of whole casein separated by gel filtration . . . . . . . 212 vii Table 40 41 42 Percent nitrogen in each fraction of the casein: soy protein blend separated by gel fraction. Nonprotein nitrogen of samples of AC, AS, SP, CS and WC heated at 94°C/1o sec and 121°C/5 sec Percent nitrogen in each fraction of whole casein and the caseinzsoy protein blend separa- ted by gel filtration with sepharose 4B. viii Page 216 231 232 LIST OF FIGURES Figure 1 Disc gels of whey protein, soy protein and casein heated at (from left to right): unheated- control6 68°C/3O min, 77°C/20 sec, 94°C/10. sec and 121 C/5 sec. . . . Gel filtration chromatograms of whey protein heated at: unheated-control (a), 68°C/30 min (b),77°C/20 sec (c), 94°C/10 sec (d), and 121°C/5 sec (e). . . . . . . . . . . . . Gel filtration chromatograms of soyO protein heated at: unheated- control (a), 680 C/30 min (b), 77°C/20 sec (c), 94°C/10 sec (d), and 121°C/5 sec (e). . . . . . . . . . . . . Gel filtration chromatograms of the wheyzsoy protein blend heated at: unheated-control (a), 680C/3O min O(b),7 77°C/20 sec (c), 94°C/10 sec (d) and 1210 0/5 Sec. . . . . . . . . . . Gel diagrams of unheated whey protein (AC), soy protein (SP) and their blend (AS). The gel filtration fraction is denoted by the number in the upper right hand corner. Gel diagrams of whey protein (AC), soy protein (SP) and their blend (AS) heated at 68°C/30 min. The gel filtration fraction is denoted by the number in the upper right hand corner. . Gel diagrams of whey protein (AC), soyO protein (SP) and their blend (AS) heated at 94°C/10 sec. The gel filtration fraction is denoted by the number in the upper right hand corner. . . Disc gels of whey protein, soy protein and their blend heated at: unheated control, 680 C/30 min and 94°C/10 sec. The gel bands represent pro- tein present in gel filtration fractions 1-6 (from left to right) ix Page 168 175 181 186 190 191 192 194 Figure 9 10 11 12 13 14 15 16 17 Gel filtration chromatograms of whey protein fraction 1, concentrated and reapplied over Sepharose 4B. The heat treatments included: unheated-control (a), 68°C/30 min (b) and 94°C/10 sec (d). . . . . . . . . . . . . Gel filtration chromatograms of soy protein fraction 1, concentrated and reapplied over Sepharose 4B. The heat treatments included: unheated-control (a), 68°C/30 min (b) and 94°C/10 sec (d). . . . . . . . . . . . . . Gel filtration chromatograms of the whey:soy protein blend fraction 1, concentrated and reapplied over Sepharose 4B. The heat treat- ments included: unheated-control (a), 68°C/30 min (b), 77°C/2O sec (c), 94°C/10 sec (d) and 121°C/5 sec (e). . . . . . . . . . . . . . . Disc gels of casein, whey and soy proteins and the milk:soy protein blends fraction 1, concen- trated and reapplied over Sepharose 4B. The heat treatments included: unheated control, 68°C/3O min and 94°C/1o sec ..... Gel filtration chromatograms of who e casein heated at: unheated-control (a), 68 C/30 min (b) 77°C/2O sec (c), 94°C/lO sec (d) and 121°C/5 sec (e) . . . . . . . . . . . . . . . . . Gel filtration chromatograms of the casein: soy protein blend heate at: unheated-control (a), 680C/30 min (b), 77 C/20 sec (c), 94°C/10 sec (d) and 121°C/5 sec (d). . . . . . . . . . . . Gel diagrams of unheated whole casein (WC), soy protein (SP), and their blend (CS). The gel filtration fraction is denoted by the number in the upper right hand corner. . Gel diagrams of whole casein (WC), soy protein (SP) and their blend (CS) heated at 68°C/3O min. The gel filtration fraction is denoted by the number in the upper right hand corner. . Gel diagrams of whole casein (WC), soy protein (SP) and their blend (CS) heated at 94°C/lo sec. The gel filtration fraction is denoted by the number in the upper right hand corner. . . ‘ Page 199 203 205 208 211 214 218 219 220 Figure 18 19 20 21 Disc gels of whole casein, soy protein and their blend, heated at: unheated-control, 68OC/30 min and 94°C/10 sec. The gel bands represent protein present in gel filtration fractions 1-4 (from left to right). Densitometric trace of the gels (unheated-con~ trol) of soy protein (SP), whole casein (WC) and their blend (CS). . . . . . . . . . Gel filtration chromatograms of whole casein concentrated and reapplied over Sepharose 4B. The heat treatments include : unheated-control (a), 68°C/30 min (b) and 94 C/lO sec (d). Gel filtration chromatograms of the caseinzsoy protein blend fraction 1, concentrated and reapplied over Sepharose 4B. The heat treatments included: unheated-control (a), 68°C/3O min (b), and 94°C/10 sec (d) . . . . . . . . . . . xi Page 222 225 227 230 INTRODUCTION A great deal of effort is being directed toward finding cheaper protein sources for the domestic food industry. Currently, animal derived ingredients and wheat are the major sources for human food, though soybean protein is becoming more popular. Animal protein foods are expensive in terms of land requirements and market price. Vegetable protein foods can supply more protein per acre of land (Childers, 1972). Proteins which are to be used in food products must satisfy three criteria. They must be nutri- tionally adequate, economically feasible and possess the necessary functional properties. That protein which comes closest to fulfilling these needs will probably have the highest utilization. Sufficient information is available to formulate a food composition which contains the required amounts of known nutrients, including vitamins, minerals, proteins, fats and carbohydrates in proper balance to supply the calories and chemicals needed by humans (Johnson, 1971). This can be accomplished by combining plant and animal products with synthetic chemicals. The problem is to pro- duce finished products that have the necessary functional and sensory characteristics which make them acceptable to the consumer. The common protein foods (meat, fish, eggs and dairy products) owe their widespread appeal to the gas— tronomic pleasure derived from their consumption (Mattil, 1971). The proteins in these foods are structural compo- nents that contribute Specific functional properties. If vegetable proteins are to be incorporated into the products of today's technology they must possess certain functional characteristics. Acceptable sensory properties are essen- tial in the development of new food products. New protein sources Should maintain or improve the quality and accepta- bility of food products. The functionality of food proteins denotes any physico- chemical characteristic which affects the processing and behavior of the protein in food systems. Functionality is a reflection of complex interactions between the composi- tion, structure and conformation of a protein which may also be influenced by reaction with other food components. In the food research laboratory, many types of protein systems are subjected to measurements in an attempt to quantitate functionality. Typical tests include flavor analysis, dispersibility, gelation, emulsification, foaming, viscosity and water absorption. Burrows g3 31. (1972) estimated that by 1980 the demand for functional protein in the United States will exceed three billion pounds. This will be approximately 15-20% of the total protein need. The actual demand for new functional proteins will depend on many factors including the availability and price of animal proteins and the apparent functionality of non- animal proteins. Many vegetable, cereal and seed proteins are being used or investigated for use in roles normally reserved for animal proteins. However, these proteins often lack functionality and nutritional adequacy. Both deficiencies can be improved by combining in one system the proper blend of animal and nonanimal proteins. The objective of this research was to provide an in depth analysis of the functional (whipping, emulsion capa- city, gelation, etc.) properties of milk productzsoy iso- late blends. AS adjuncts to this study two additional projects were undertaken. Protein interaction between different protein systems could have substantial impact upon the functionality of such systems. Milk proteins were combined with soy protein and examined for heat induced interactions. Manipulation of or prevention of such inter- actions would have appreciable importance in achieving the most functionality from proteins. The functionality of protein blends can be improved by modification with the appropriate treatment. However, if such treatment is detri- mental to protein quality the improved functionality will be of little importance. Therefore, the treatments employed in the prior analysis of functionality were assayed regar- ding their effect on the quality of the proteins used in this research. These three studies were designed to complement each other. Separate aspects of protein research were brought together to more fully complete the picture. The commercial milk products used in the study of functionality included: 1) sweet whey powder, 2) electro- dialyzed whey powder, 3) whey protein concentrate, 4) non- fat dry milk, and 5) sodium caseinate. A commercial soy isolate was selected, based upon ease of dispersibility, total soluble protein, color and flavor. The materials were blended in liquid systems at various ratios and protein concentrations. The functionality of the blends was evalu- ated by several different tests as affected by various treatments. The experimental design to evaluate potential heat induced interactions between the water soluble protein from soy isolate and milk proteins consisted of several heat treatments. These approximated those used in the pasteurization of liquid systems. lg_yj££g_enzymatic hydrolysis was used to assay: 1) the effect of processing treatments on the availability of total amino acids and 2) whether or not potential interaction between protein systems would influence release of amino acids. lfl.li££9 enzymatic digests offer a viable alterna- tive to animal assays as an option for monitoring due to their low cost, speed, and high correlation to animal assays. The ultimate Objective of any study dealing with the functionality of protein systems is to provide the infor- mation which may some day make it possible to create any type of protein food from low cost raw materials. This research provides information to those researchers working toward development of dairyzsoy protein foods. LITERATURE REVIEW Wolf (1970) reported that soybean proteins are com- mercially available as flours, grits, concentrates and iso- lates. The most refined form of soybean proteins are the isolates which contain not less than 90 percent protein and have a minimal of nonprotein components. Soy protein isolate is defined as the major proteinaceous fraction of soybeans prepared from high quality, sound, clean dehulled soybeans (Smith and Circle, 1973). Isolates are almost 100 percent protein and are essentially void of fiber, carbohydrate and other flavor components (Johnson, 1970). Cogan g£_al. (1967) observed that isolated soy protein has considerable advantages over soy flour from the viewpoint of human consumption. These include blander flavor, whiter color and excellent keeping qualities. ~In addition, the functional behavior can be modified to satisfy the necessary requirements. Ziemba (1966) stated that soy isolates are generally superior to flours with respect to color, texture, flavor, fiber content, ease and versatility of use. Iso- lates are the most expensive form of commercial soy protein. In the past the major interest in soybeans in this country has been to Obtain oil for edible purposes, with the meal being primarily used for animal feed. Soy products have been used for edible purposes for thousands of years but mainly limited to production of edible products in the orient (Johnson, 1970). Wolf (1970) felt that the availa- bility of a greater variety and of more refined forms would (in addition to the increasing cost of animal protein) re- sult in greater interest in soy products. Smith and Circle (1973) described the general proce- dure for manufacture of soy isolate. The usual starting material is defatted soy meal or flour of high nitrogen solubility. The flour is extracted in aqueous or mildly alkaline media under certain conditions with respect to temperature, liquid to solids ratio, pH and alkaline rea- gents. For edible, isolated protein production, aqueous alkaline extraction is preferably carried out at pH below 9 to avoid undue hydrolytic or rheological changes. The extract is separated from the insoluble residue by various screening, centrifuging or filtering devices. A food grade acid is then used to precipitate the protein. The curd- whey mixture is separated by means of centrifugation and several washing steps. The curd can then be either dried as the isoelectric product or neutralized with food grade alkali to form the sodium proteinate. It is usually spray dried. Nonfat dry milk (NFDM) is the most popular dairy based ingredient (Hugunin, 1977). It imparts excellent flavor, functional properties and nutrition to products. NFDM is manufactured by separating the fat and nonfat portions of milk by centrifugation. The Skimmilk fraction is heat treat- ed to meet the desired standards, pasteurized, evaporated to 45-50% solids and dried. Caseinates are salts of casein. They are manufactured by isoelectric precipitation from Skimmilk. After washing several times an aqueous colloidal suspension is made by the addition of alkali to pH 6.7. The suspension is pasteurized and spray dried (Hugunin, 1977). The caseinate produced is dependent upon the alkali used, although sodium and calcium caseinates are most commonly manufactured. Whey is that portion of Skimmilk remaining after the coagulation and separation of casein. The processing of whey involves centrifugal separation and clarification to remove fine curd and fat, pasteurization, concentration to 45-50% solids and spray drying. Most processed whey in the United States is identified as sweet whey, a byproduct of ripened cheese production (Hugunin, 1977). The term whey protein concentrate (WPC) identifies all whey products which contain higher than normal concentra- tions of protein (Hugunin, 1977). The composition of WPC is largely a function of the process used in their preparation. The protein concentration of most WPC is 35-60% (Morr, 1976). Today, such processes as membrane filtration, re- verse osmosis, gel filtration, metaphosphate complexing, CMC complexing, electrodialysis and ion exchange are used to produce WPC. Richert (1975) discussed the principles of gel filtration to produce a WPC. The first step involves remo— val Of insoluble proteins and lipids through a procedure requiring divalent cations. The material then undergoes evaporation and crystallization to remove lactose. The partially delactosed whey concentrate is now subjected to gel filtration to fractionate the high and low molecular weight solutes. Large scale gel filtration may utilize either a centrifugal or column process. After gel filtra- tion the protein fraction is dried. Richert (1975) discussed the principles of electro- dialysis to remove salts during the processing of fluid whey. In electrodialysis, ion selective membranes allow passage of ionic species Of one charge but not the other. The ions migrate in response to an electrical potential. Commercial operations usually employ repeating pairs Of cation and anion membranes which are arranged in stacks of 100 or more cell pairs. Product is circulated through alternating pairs and an electrically conductive brine is circulated between the remaining pairs. The feed material is usually partially delactosed whey concentrate but can be either unconcentrated sweet or acid whey. After removal of charged species, the remainder is dried. 10 Functionality Solubility Many of the important functional properties dealing with proteins relate to water-protein interactions, i.e., solubility, viscosity, gelation, foaming and emulsification. The investigation of the functional properties of proteins can be made more efficient if a systematic study is first made of the solubility properties of that protein in a vari- ety of environments (Mattil, 1971). Solubility behavior provides a good index Of the potential and limitations of proteins (Kinsella, 1976). The pH-Solubility profile is Often the first property measured and can provide useful information regarding the optimization of processing proce- dures. Solubility is affected by a multitude of factors such as protein source and concentration, processing his- tory, heat, ionic strength and interaction with other ingre- dients. The solubility profile is an excellent index of protein functionality (Kinsella, 1976). Paulsen g; _l. (1960) determined water dispersible protein (WDP) in soy products. The results were affected by blending time and speed, pH, sample size and blade arrangement. At low pH the soy products had low WDP. It was reported by Yasumatsu g: 31- (1972) that the WDP did not correspond directly to the amount of undenatured, native soy protein. This was because some denatured protein re- mained soluble. Divalent salts, Such as calcium or barium chloride markedly reduced the WDP of soy protein, even at low levels (Paulsen and Horan, 1965). Wolf (1961) found that the solubility of tne major soybean protein, glycinin, was increased by addition of reducing agents and alkaline pH. Fukushima and Van Buren (1970) demonstrated that disul- fide splitters such as mercaptoethanol and cysteine increased the solubility of soy milk products as did alkaline pH treat- ment. Chelating agents such as EDTA had little effect. The solubility Ofifscybeanproteins was at a minimum near pH 4.5 (Wolf, 1970). Solubility increased markedly with alkali treatment and after addition of disulfide reducing agents. Nash and Wolf (1967) studied the solubility of commercial and laboratory prepared soy proteins. Measurements were made in 0.5 ionic strength buffer, pH 7.6, potassium phos- phate-sodium chloride buffer with and without mercaptoetha- nol. Five laboratory soybean globulins prepared by isoelec- tric precipitation had solubilities of 37-73% in buffer and 66-78% in buffer containing mercaptoethanol. Five commer- cial samples (isoelectric form) had solubilities of 6.0- 5 % in buffer and 13-83% in mercaptoethanol buffer. Seven commercial soy proteinates had solubilities ranging from 13-83% and 6.0-81% with and without mercaptoethanol in the buffer. Addition of mercaptoethanol to the buffer increased the solubility of all samples. The solubility of WPC, soy isolate and sodium caseinate was examined by Hermansson and Akesson (1975). Solubility 12 was determined on 1% sols in water at 250C. The dispersions were subjected to heat treatments of 70, 80, 90 and 100°C for 30 min. Unheated samples of soy isolate, caseinate and WPC had solubilities of 52.9, 80.8 and 78.3 respectively. Moderate heating temperatures increased the solubility of the soy isolate but had much less effect upon sodium casein- ate and WPC. The solubility of these materials in 0.2 M- 1.0 M NaCl was also studied by Hermansson and Akesson (1975). Addition of NaCl decreased the solubility of soy isolate but increased the solubility of sodium caseinate. _Heat treated dispersions of WPC which were subjected to addition of NaCl had decreased solubilities, though nonheated samples were only slightly affected. Kelly and Pressey (1966) exposed acid precipitated soy protein to high pH levels by addition of sodium hydroxide. Solubility at pH 12 was markedly reduced. Addition Of urea and mercaptoethanol increased solubility. Amiliar _L _l. (1977) determined the total dispersible protein in reconstituted soy milk. The amount of dispersible protein increased as the pH rose, with addi- tion of sodium bisulfite and homogenization. The amount of soluble soy protein in soy flour was minimal near its iso- electric point but much higher in an alkaline pH range (McWatters and Cherry, 1977). Franzen and Kinsella (1976) reported that the solubility of soy isolate was increased 20, 71 and 8 % according to the level of succinylation. As the amount of succinylation increased so did Solubility. 13 Treatment of the soy isolate at alkaline pH also resulted in higher solubility. The nitrogen solubility index of soy protein isolate (as a function of pH and temperature) was determined by Hutton and Campbell (1977). Solubility was minimal at the isoelectric point while increasing rapidly as the pH diverged from that point. At high pH solubility increases were very pronounced though this was temperature dependent. The pH slope was roughly linear in the mid-tem- perature range and curvilinear at the extremes (4-900C). Bau g; _l. (1978) examined the solubility of cold, acid and salt precipitated soy protein. The soy fractions (10% w/v) were prepared in dispersions at pH 1.5-9.5. Minimum solubility was at pH 4.4-4.6. As the pH was brought into the alkaline region, solubility increased by 25 to 3 %. Heating at 1000C for 7 min decreased solubility approximately 50%. Continued heating had no additional effect on the rate of insolubilization. Howat and Wright (1933) examined the effect of heating temperature upon the solubility of commercial milk powders. The samples were reconstituted in water and subjected to heating. Solubility was determined by protein analysis of the supernatant. There was an increase in solubility as the temperature rose from 20 to 50°C. However, as the tempera- ture increased from 50 to 100°C solubility decreased. The solubility of caseins near their isoelectric point was investigated by Bingham (1971). AS the temperature was 14 lowered, the solubility of the caseins was greater than at ambient temperature. Aoki and Imamura (1976) determined the solubility of casein after addition of EDTA and heating. The solubility was highest in those samples receiving both heat treatment and 5-15 mM of EDTA. The solubility of un- heated casein samples containing EDTA also increased but to a lesser extent. Samples prepared in alkaline pH and heated were much more soluble than those samples receiving only the alkaline treatment. The solubility of soy isolate and milk products dispersed in Koop's buffer was studied by Hoffman (1974). “As casein was replaced by soy protein, an almost linear decrease was noted in the amount of soluble protein. There was little decrease in the amount of soluble protein as the percent of soy protein increased in whey protein con- centrate-soy isolate blends. Shen (1976) examined the effects of a wide range of experimental conditions on the solubility of three commercial soy isolates and a commercial sodium caseinate. Protein was quantitated by the Biuret method. The solubility of Edi-Pro N (a general purpose soy isolate) did not vary with increase in percent protein (1- 10%). The solubility of this isolate was reported to be 54% compared to the 80% solubility of sodium caseinate. The solubility of sodium caseinate did not change as the equili- bration temperature rose from 25-620C while the solubility of Edi-Pro N increased 7%. Blending speed had an appreciable effect on protein solubility. Marvopoulous and Kosikowski 15 (1973) reported on the composition, solubility and stability of whey powders. Eight spray dried powders and three freeze- dried, laboratory samples were analyzed. The solubility of whey powders ranged from 91.4 to 99. %, with the freeze- dried samples most soluble. The solubility of the 11 pow- ders was lower in 5% NaCl, ranging from 72.4 to 98.2%. Flavor evaluation by a trained panel showed the powders to have good flavor characteristics. The pasteurization of WPC solutions at temperatures of 78.20C/15 sec and 62.40C/30 min resulted in approximately 20% denaturation of the protein (McDonough g3 31., 1974). Enzymatic hydrolysis with trypsin was used by Jost (1977) to increase the solubility of WPC. Hydrolysis was approxi- mately 8% complete. The functional properties of electro- dialyzed whey, Enerpro 50, CMC bound WPC and metaphosphate complexed WPC were examined by Morr 33 _l; (1973). Protein solubility, whipping and emulSion capacity were determined. At pH 4, 6 and 8, electrodialyzed wheyhad solubilities of 92.2, 91.1 and 93.6 percent respectively. The solubilities of Enerpro 50 at the same pH values were 89.5, 90.2 and 91.2. Hidalgo and Gamper (1977) produced a WPC from sweet whey having a protein content of 88%. This product was examined for thermal stability both in the presence and absence of 0.03 M CaCl Solubility decreased rapidly as the samples 2. were heated at temperatures of either 80 or 1340C. When the / RWPC/Was heated at high temperatures coagulation occurred, 16 especially at pH values near the isoelectric point. In the presence Of CaCl2 heat stability was reduced over the pH (f. range 2-12. Tryptic hydrolysis of the WPC imprcved thermal stability considerably. Viscosity and Gelation Many proteins absorb water and swell, thereby causing changes in hydrodynamic properties that are reflected in thickening and increased viscosity (Kinsella, 1976). Pro- tein gels are composed of three dimensional matrices or networks of intertwined, partially associated, polypeptides in which water is entrapped (Kinsella, 1976). Gels are characterized by a relatively high viscosity, plasticity and elasticity. Viscosity and rheological properties related to flow are usually measured on dispersions, slurries or pastes using the Brookfield viscometer. Circle 33 _l. (1964) examined the viscosity of soy pro- tein dispersions. Results showed that in the unheated dis- persions viscosity rose exponentially with increasing con- centration. At pH 6 the viscosity of 10% sols dropped considerably below that at pH 7 or above. At pH 8 and 9 the viscosities rose slightly. A 1 % dispersion of isolated soy protein had a low initial viscosity but thickened notice- ably when heated one hour (Hafner, 1964). Kelley and Pressey (1966) examined the effect of pH and chemical additives on the viscosity of soy protein dispersions. AS alkali was added, viscosity increased rapidly. After a minimum was l7 reached viscosity slowly decreased, presumably due to pro- tein degradation. Addition of low amounts of bromate and iodate significantly decreased viscosity. Disulfide cleav- ing agents also reduced viscosity. The viscosity of casein- soy sols dispersed in Koop's buffer was investigated by Hoffman (1974). In general, the viscosity of the suspen- sions decreased as casein was replaced by soy protein. Whey protein-soy sols were slightly lower in viscosity than casein-soy sols. Replacement of whey protein with soy pro- tein slightly increased the viscosities. Fleming e3 _l. (1974) examined the viscosities of soy isolate and soy con- centrate slurries. Viscosities increased as the concentra- tion of protein increased and as the pH was raised. Hutton (1977) studied the effect of pH and heat treatment on the viscosity of soy isolate and concentrate. Apparent visco- sities increased dramatically as the temperature was raised to 900C. The flow properties of soy isolate, caseinate and WPC were studied by Hermansson (1975). The viscosity of protein dispersions (4-20%) were determined in a Haake Rotovisco instrument model RVI. The flow properties of soy isolate (Promine D) were characterized by low viscosities below concentrations of 8.0% protein. At 10% protein there was an enormous increase in viscosity. Reducing agents and addition of NaCl decreased the viscosity while heat, oxida- tion and pH treatments above 7.0 were responsible for Substantial increases. The viscosity Of sodium caseinate 18 was very concentration dependent with nearly logarithmic increase over a broad protein range. Addition of NaCl caused an increase in viscosity as did pH adjustment to pH 9.8-10. At higher pH values the viscosity rapidly declined. WPC had the lowest viscosities of all the materials studied. How- ever, at high protein concentrations (18-20%) the material became pseudoplastic which resulted in tremendous viscosity measurements. Factors Such as pH and ionic strength had little effect on viscosity. The viscosity of untreated WPC dispersions did not change appreciably until a solids level of 45% was reached. At concentrations of 10-20% the viscosi- ties were 10 cp or less. Neither succinylation nor heat treatment significantly altered the viscosities of soy iso- late at pH 7.0. Only slight changes in viscosity were ob- served upon addition of CaC12 (0.2-5.0%) to soy isolate at pH 7.0 and 10.0. Guy 33 _l. (1969) prepared a whey-soy flour mixture suitable for beverages. The product reconstituted in water to yield a milk with a cereal flavor. The viscosities Of the soy flour-whey mixtures were relatively insensitive to heat treatment. When the total solids were greater than 45%, significant changes were associated with high temperature treatment. Homogenization reduced the viscosity and solu- bility index. The viscosity and thickening of several pro- teins were correlated with the water binding properties of model meat systems by Hermansson and Akesson (1975). WPC. 19 soy isolate and sodium caseinate were examined in unheated and heated dispersions. Heat (70-1000C/30 min) caused gela- tion of 10% dispersions of soy isolate and WPC but did not cause gelation of caseinate. 0f the three proteins, soy isolate had the highest viscosity, swelling ability and formed the strongest gels. Caseinate had greater swelling ability and higher viscosity than WPC. WPC had the lowest viscosity and swelling ability of the three proteins but did form firm gels upon heating. In a further examination, Her- mansson and Akesson (1975) investigated the effect Of salt on dispersions of soy isolate, sodium caseinate and WPC. AS the amount of NaCl increased (0.2 M to 1.0 M) there was a marked decrease in the gelation of soy isolate. The viscosi- ty of the caseinate sols increased as the concentration of NaCl increased. Swelling and gelation were not affected. Nonheated WPC dispersions were little affected by addition of salt. However, when heated to 800C gel strength increased rapidly as NaCl was added. Sosulski 31 31. (1976) compared the functional proper- ties of rapeseed and soybean protein products. Oil emulsi- fication, whippability, viscosity and gelation were examined. Viscosity increased with increase in protein concentration for soy isolate. When soy isolate was subjected to heat treatment at 100°C, Smooth, firm gels were formed. The oil emulsification properties of the soy isolate were much less than that of the corresponding rapeseed product. Most of 20 the soy isolates did not exhibit good foam properties. The swelling ability of several protein systems was studied by Hermansson (1972). The soy isolate had excellent swelling properties while sodium caseinate demonstrated much less and WPC had vitrually none. The viscosity Of both soy iso- late and sodium caseinate was much higher when examined over a broad concentration range than WPC. The swelling ability of soy isolate decreased as the ionic strength increased. WPC was essentially unaffected by increase in ionic strength. The swelling ability of caseinate increased rapidly as the pH went up or down. Ehninger and Pratt (1974) studied the effect Of sugars, NaCl, and freeze thaw cycles on the gelation and stability of soy protein dispersions. Gelation was also studied at low protein levels and at pH ranges feasible in a food system. Gelation was measured with a Brookfield RVD visco- meter. Viscosity tended to increase exponentially as the concentration of protein increased, though it was pH depen- dent. Viscosity was much higher at elevated pH. Addition of 5 or 10% sucrose to the dispersions decreased the visco- sity of the gels while addition of dextrose at these same levels resulted in slight increase. Dispersions containing 0.2 M NaCl at pH 6.5 had decreased viscosities. Modler and Emmons (1977) prepared a soluble WPC by adjusting the pH of sweet whey to 2.5-3.5 prior to heating at 900C for 15 min. Concentration of the whey prior to heating or addition of 21 iron increased protein recoveries but decreased solubili- ties. The viscosities of the reconstituted samples (33% total solids) ranged from 4,000 to 36,800 cp. At protein concentrations of 2, 4, 6 and 8%, gelation Occurred when this WPC was heated at 95°C for 20 min. The samples retained excellent color and flavor. Kalab 33 _1. (1971a) reported on gels produced from NFDM. The gels were prepared by slur- rying the material (50% NFDM) into sausage casings and heating for 10 min in boiling water. A penetrometer was used to measure firmness. This study was continued by Kalab 33 _1. (1971b) when he examined various physical factors influencing the firmness of heat induced milk gels. Aqueous dispersions of 40-60% NFDM were heated at temperatures of 80-1000C for 10 to 30 min. Temperatures below 80°C did not induce gelation of 50% NFDM dispersions. Gel firmness was maximum at 1000C and high milk solids concentration. Kalab and Emmons (1972) investigated the effect of chemical addi- tives upon the firmness of heat coagulated milk gels. Several additives (0.25 M) were Screened for effectiveness. Divalent cations, especially Ca++ promoted gelation as did the addition of oxidizing and crosslinking agents. Addition of reducing agents and sulfhydryl blockers to the Skimmilk resulted in soft, sticky gels. Neutral salts such as NaCl and KCl had no effect on gel firmness. Catsimpoolas and Meyer (1970) examined the gelation phenomenon of soybean globulins. Aqueous dispersions of the proteins (8-10%) were 22 prepared and heated at Specified temperatures. A Brookfield viscometer equipped with a helipath stand was used to measure apparent viscosity. The minimum protein concentration neces- sary to Obtain a self supporting gel structure was 8 percent. Highest viscosity values were obtained at neutral or mildly alkaline pH. Both high and low pH decreased gel strength. Gel firmness was greatest when the samples (pH 7.0) were heated at 800C for approximately 30 min. Continued heating at this temperature had no detrimental effect though excess heating at higher temperatures substantially reduced gela- tion. The viscosity Of globulin dispersions in NaCl de- creased as the amount Of NaCl increased. Gels were prepared by Catsimpoolas and Meyer (1971a) from dispersions of soy- bean globulins in water-alcohol and water-glycol mixtures. The viscosities of the heated slurries were determined after cooling with a viscometer. Rheological data demonstrated that these mixtures formed gels of higher viscosity than water dispersions. Apparent viscosities increased as the length of the aliphatic chain increased. Solvents such as acetone, dioxan and dimethyl formamide also markedly enhanced gel viscosity. The role of lipids in the gelation of soybean globulins was examined by Catsimpoolas and Meyer (1971b). Protein-lipid dispersions were prepared by addition of pro- tein to the water-lipid mixture and heating. Gels produced from mixtures containing saturated fats had higher viscosi- ties than those produced with unsaturated fat. AS the length 23 Of the fatty acid chain length decreased, viscosities in- creased. The role of sulfhydryl groups on the physical characteristics of tofu like gel products was investigated by Saio 31 _1. (1971). The texture of the tofu became softer and more adhesive when sulfhydryl blocking agents were used. Saio (1973) found that the strength of soy protein gels was increased by the addition of calcium salts. Gels made from the 11S protein were firmer than those made from 75 protein. Heat coagulated gels from soy protein isolate were hard and elastic (Yasumatsu 31 _1., 1972a). Denatured soy isolates were more desirable because partial denaturation was believed to be conducive to preparation of firm gels. Soy isolates with a nitrogen solubility index (NSI) of 50 had better gel formation qualities than one with a NSI of 80 (Yasumatsu t 1., 1972). Circle 31 l. (1964) reported that soy protein dispersions readily gelled when heated. At protein concentrations of 10% gelation was best when the dispersions were heated at 100°C for 45 min. Addi- tion of sodium sulfite and free cysteine substantially reduced gelation while salts had much less effect. At pro- tein concentration greater than 10% heating could be reduced without loss of gelation. The effect of heat, pH, ionic strength and protein fraction on the expansion characteris- tics Of soy gels were examined by Saio _3 _1. (1974). Expansion increased as the temperature of heating rose from 100 to 1320C. Calcium gels had greater expansion and were 24 more elastic than acid gels. Fleming and Sosulski (1975) evaluated the gelling ability of aqueous dispersions of 10% protein from soybean flours, concentrates and isolates. Gelation was induced by heating the slurries in sealed con- tainers at 900C for 45 min in a water bath. After cooling in an ice bath, viscosities were determined. Uniform firm gels were produced from the soy isolate. A pH cycling treat- ment, 7 to 12 to 7, increased gelation strength Of the gels. The viscosities of soy proteins prepared by cold, acid and salt precipitation were compared by Bau 31 _1. (1978). Viscosities were determined on 1 % protein slurries. Moist heating at 1000C increased the viscosity of all fractions. Prolonged heating at this temperature resulted in sharply decreased values. Viscosities were at a minimum near the pH of their isoelectric point (4.5). As the pH diverged from this point in either direction, viscosities increased. McDonough 31 _1. (1974) formed firm, resilent gels from 10% WPC solutions which did not leak after standing for several hours. Heat denaturation resulted in what the author described as classical gel structure. Schmidt 31 31. (1978) studied the heat induced gelation of peanut/whey pro- tein blends. Heating at 700C or less resulted in no gel formation. Heating temperatures of 1000C-45 min were neces- sary to obtain satisfactory gelation. Qualitative techniques showed no difference in gel strength as the pH was raised from 7 to 10. At pH 11 the gels were very soft with 25 measurable browning. Gels formed satisfactorily when 25% of the protein was from peanut flour. Sodium chloride (up to 0.5 M) increased the gel strength of the whey protein gels. Addition of CaCl at levels of 10 and 30 mM resulted in 2 increased gel firmness. Emulsion Capaci1y The emulsifying properties of food proteins are a pri- mary functional characteristic. Three different procedures, i.e. emulsifying capacity, emulsion stability and emulsify- ing activity are used in the investigation of this property (Kinsella, 1976). Emulsion or emulsifying capacity (EC) is the method most commonly used, and is defined as the volume of Oil (ml) that can be emulsified by protein (gm) before phase inversion occurs. Swift 31__1, (1961) developed a method to determine EC. This procedure, known as the viscosity drop method, utilized a high speed mixer and melted lard to prepare the emulsions. The mixer speed, rate of fat addition and temperature affec- ted EC. Carpenter and Saffle (1964) developed a method to determine EC subjectively. An ”Osterizer“ was modified to accompany an inverted ball jar. Formation and collapse of the emulsion was viewed through the glass jar. Blender speed, mix temperature, protein aliquot and rate of oil addition affected the final volume. A method (using sau- sages) was described by Inklaar (1969) as being capable of 26 accurately evaluating protein emulsion capacity and stability. Using this procedure, soya isolate had better EC than sodium caseinate. Both products had better EC at higher pH. Becher (1966) suggested electrical conductivity as a method for determining types of emulsions. Electrical resistance was used by Webb 31 31. (1970) to measure EC of protein. Corn oil was delivered in close proximity to the propeller blades and mixed with the protein extract at high speed. Emulsion formation and collapse were monitored by a ohm recorder measuring resistance. Precise control of blender speed, rate of oil delivery, and location of delivery tube were critical in obtaining repeatability. The validity of using electrical a-c impedance and d-c resistance methods in the evaluation of EC was investigated by Haq 31 _1. (1973). For dilute, aqueous systems undergoing agitation, Webb’s _1 _1. (1970) procedure was Shown to be satisfactory. In highly viscous systems, a-c impedance measurements had greater accuracy. Marshall 31 31. (1975) used a red colored dye (oil-red 0) to determine the EC Of protein slurries, including soy isolate. The author claimed greater accuracy for this method due to the increased visibility of the inversion point. Morr 31 31. (1973) investigated the EC of several dif- ferent WPC and found similiar results for all samples tested. The EC ranged from 32-40 9 corn oil for a 0.1% solution except for CMC complexed WPC which was double that of the 27 other WPC. The effect of enzymatic hydrolysis on the EC of whey protein was studied by Kuehler and Stine (1974). EC was not significantly affected by proteolysis with prolase. Emulsion capacities were approximately 3.0, 1.9 and 2.2 g/ Oil/mg protein for dried whey, whole casein and nonfat dry milk. Pearson 31 31. (1965) compared the EC of soy sodium proteinate, potassium caseinate and NFDM by the viscosity drop procedure. The EC of soy was greatest at pH 10.7 and p=0.05. At higher ionic strength (0.3) EC decreased slight- ly. At pH 5.1 EC was much lower. Potassium caseinate had satisfactory emulsifying properties in water at pH 6.9. Emulsion capacity was highest at pH 10.4 and ionic strength 0.05 while lowest at pH 5.4. NFDM had slightly less EC than potassium caseinate at high pH. The EC of NFDM was greatest at pH 5.6. At low concentration NFDM had good EC while at high concentration EC decreased. Several protein additives were assayed by Smith 31 31. (1973), including soy isolates, flours, concentrates and NFDM. EC and stability were evalu- ated in frankfurter emulsions. In this system animal pro- teins had better EC than the soy products. Hoffman (1974) reported that the EC of casein-soy blends decreased as the amount of soy protein increased. In addition, the ability Of WPC to emulsify Oil decreased significantly as soy protein was substituted for whey protein. Crenweldge 31 l. (1974) investigated the EC Of NFDM, soybean concentrate, cottonseed 28 flour and bovine hemoglobin. A viscosity drop procedure was utilized in determining EC. Both soy and NFDM had emulsion capacities resembling pH solubility profiles. At pH values near the isoelectric point NFDM and soy exhibited minimal EC. As the pH increased to 7.0 and above, EC increased rapidly. NFDM had greater EC than the soy concentrate. Various proteins were compared by Lauck (1975) as fat bin- ders in sausage products. The proteins included Enerpro 50 (WPC), dried sweet whey, NFDM and promine D (a soy isolate). Binders were added either as dry ingredients or predispersed in water. Following laboratory testing, pilot plant produc- tion of sausage products containing the binders was initiated with subsequent evaluation. The laboratory tests indicated that, as a class, protein containing binders might be better than meat proteins in binding fat. The pilot plant evalua- tion revealed that the frankfurters produced with the various binders were of good quality but did differ in several aspects. The whey product sausages had thin emulsions while the soy isolate frankfurters had a definite off-flavor. Yasumatsu 31 31. (1972) examined the emulsion activity of soy products. Systems containing both Skim milk and soy flour had the highest emulsion activity. Soy protein extract had more emulsifying activity than soy protein iso- late in a sodium chloride brine. High correlation coeffi- cients were Observed between dispersible nitrogen and emul- sifying properties. 29 Succinylation enhanced the emulsifying capacity of soy isolate dispersion (Franzen and Kinsella, 1976). When suc- cinic anhydride was added on a g to 9 basis with protein EC doubled. McWatterS and Cherry (1977) reported on the EC of defatted soybean flour. Suspensions, prepared in distilled water were evaluated at several pH values. Maximum emulsi- fication was at pH 6.5. The emulsion properties of a soy concentrate and isolate were examined by Hutton and Campbell (1977). The interdependent influence of pH and temperature on EC was reviewed. Soy dispersions were studied at pH 5.0, 6.0 and 7.0 with temperature treatments of 40C and 900C. Emulsification was significantly affected by such treatment. At low pH emulsification was substantially less, probably due to the loss of solubility. Whippinngbility Foaming or whipping, i.e. the capacity to form stable foams with air, is an important functional property Of many foams (Kinsella, 1976). Foaming properties include whippa- bility or foamability, both of which are used interchange- ably. These properties are measured by foam capacity, foam expansion or overrun, all of which refer to the volume in- crease Of a protein dispersion following incorporation of air by agitation or whipping. Foam stability refers to the ability of foam to retain its volume over time. Webb (1941) examined the foaming capacity of reconsti- tuted Skimmilk by whipping at 1000 rpm's. Foam stabilities 30 and specific volumes were determined for samples containing 10-30 percent solids. AS the concentration of solids in- creased to 20%, specific volumes decreased but stability times increased. Whipping properties were reduced when severe heat treatments were employed. Peter and Bell (1930) investigated the foaming characteristics of normal and modified whey protein solutions. Untreated whey had poor foaming ability, though heating to 60°C resulted in some improvement. Addition of Ca(OH)2 had a pronounced effect, significantly improving the whipping quality of the whey protein. When the solution was neutralized, the improvement in whipping was still evident. Addition of NaOH or calcium salts failed to have the same effect. However, when calcium salts were added in an alkaline medium an excellent foam was produced. The addition of sodium sulfite considerable im- proved whipping characteristics. Tamsma 31 31. (1969) described the increased foaming from Skimmilk, homogenized prior to drying. NFDM produced in this manner was whipped to form a stable foam after re- constitution in water. The percent overrun and the stability of the resultant foams was dependent upon homogenization pressure and percent total solids. Foam stability was op- timal at 30% total solids. The whipping properties of unheated WPC were studied by Morr 31 l. (1973). Metaphosphate complexed WPC failed to form stable foams while CMC complexed WPC had the most 31 stable foams of any of the materials tested. None of the WPC had as high an overrun as sodium caseinate. The whip- ping properties of spray-dried whey protein/CMC complexes were examined by Hansen and Black (1972). The powder was resuspended in water and whipped to foam resembling egg white. A 4% protein sol whipped for 15 min proved optimal. Heat treatment and homogenization decreased both specific volume and foam stability. Foam development increased rapidly as the pH went from 5 to 9. Addition of Ca(OH)2 resulted in the greatest improvement in whipping quality. Addition of H202 increased foaming with the best results achieved at a concentration of 0.1%. At higher levels of H202 foam development deteriorated. Addition of 3% sucrose after whipping improved foam stability. Foam stability was reduced when soy isolate was added to the sols. Jelen (1973) conducted whipping studies with delactosed cheese whey. The concentrated product was whipped for 10 min. The unheated whey protein had poor whippability, poor foam sta- bility and low overrun. Heating to 90°C improved all whip- ping properties, especially after the heat denaturable, acid precipitable proteins were removed. Overruns greater than 2000% were achieved with the acid supernatant at 34% total solids. Addition of 5-40 9 soluble starch per 100 g of the whey concentrate increased the foam stability but resulted in lower overruns. Devilbiss 31 l. (1974) examined WPC as a possible replacement for egg white in angel food cakes. 32 Commercial samples of WPC were obtained and dispersed in water at a concentration of 30%. The samples were whipped for 10 min prior to baking. Samples heated at 550C for 60 min had increased foam stabilities which did not collapse when heated. The whipping properties of WPC were studied by McDonough _1 31. (1974). Untreated solutions did not form stable foams unless the sols contained at least 25% protein. Heat treatment significantly improved the whipping characteristics, apparently due to partial denaturation of the protein. Adjustment of the pH upward with.Ca(0H)2 resulted in excellent foams which were not duplicated by adjustment with NaOH. Samples subjected to both heat and alkaline pH treatment had excellent whips with stability times of several hours. The effect of enzymatic hydrolysis on the whipping properties of whey protein was studied by Kuehler and Stine (1974). Heat treatment was a necessary prerequisite to insure foam production and stability. Spe- cific volumes (SV) ranged from 10-15 ml/g with % stability times, up to a temperature of 850C. The data indicated that the greater the net Charge on the protein, the greater was the tendency of the sols to foam. Thus, at pH 9 and 2, foams were produced with the largest SV, though acid foams had low stability times. Stability increased with alkalinity above pH 6.0. Addition of 0.1% CMC and heating to 85°C resulted in doubled stability times. Addition of 0.5% CMC increased stability times additionally. Four percent casein sols had 33 foams of poor stability. Enzymatic hydrolysis (prolase) for one hour increased SV. Additional digestion slightly lowered SV. Stability times decreased through the first hour of digestion and then leveled off. A limited amount of hydrol- ysis was desirable to increase foaming properties, though excessive digestion decreased SV and stability of the foams. The foaming capacity (FC) of 10% WPC sols was determined by bubbling N2 through the solution at a constant rate and noting the expansion in a given time (Cooney, 1976). Foam stability (FS) was measured by recording the time required for k the liquid volume to drain from the foam. FC was en- hanced by removing triglycerides via ultracentrifugation. Instantaneous heating to 60°C overcame the inhibitory effect triglycerides had on FC. FS was inversely dependent on the ionic strength of the WPC. Calcium and barium ions decreased FC more than monovalent cations at pH 4.3. Phosphates and tripolyphosphates improved foaming at pH 4.3 but not at pH 7.0. Foam overrun was optimum at pH 8.0. Foaming was im- proved by addition of 0.50 mM $05 but not by Tween 20 or Triton X-100. Trypsin hydrolysis of WPC increased the over- run but Slightly decreased the FS. Min and Thomas (1977) investigated several variables which affected the physical properties of dairy whipped toppings. These included: sta- bilizer and emulsifier concentration, milk protein fraction, homogenization pressure and interaction among selected in- gredients. Stabilizer level markedly affected overrun and 34 firmness. Toppings made with a stabilizer concentration of 0.3% had maximum overrun. Emulsifier concentration and type had a positive influence on overrun and firmness. The milk protein fraction (Skim milk colloid, whey protein, CMC pre- cipitated milk protein, sodium caseinate and NFDM) had a very significant effect on the foaming capacity. Toppings made with sodium caseinate and whey protein were unsatisfac- tory. Calcium (50 mg/ml) was added prior to whipping of some topping formulations. Increased overrun and firmness were noted. Tripling the Ca++ concentration substantially increased overrun. Hoffman (1974) examined the whipping properties of soy isolate and milk products at low protein levels. Replacement of casein with soy isolate had little effect on SV but drastically reduced FS. Whey and soy mix- tures maintained their SV but were so unstable that drip could not be measured. Watts (1937) investigated the whipping ability of com- mercial soy flours and a solvent extracted laboratory pre- pared soy flour. All commercial products tested had little or no whipping ability. Petroleum ether, dry heat and pressure were used to produce the soy flour. This product, reconstituted in water, whipped to a stiff white foam resem- bling egg white. The material whipped best at concentrations of 7-8 percent and at pH values far removed from its isoelec- tric point. Salt (NaCl) at concentrations up to 2 percent increased the whipping ability. The extraction Of a 35 substance responsible for the whipping ability of soy flour was reported by Watts and Ulrich (1939). It was apparent that neither the glycinin nor the legumelin were the active substance. Stable foams were produced by water dispersing 3-10% purified soy protein. The dispersions were heated prior to whipping. Stable foams were produced at all pH's except 3-6. Franzen and Kinsella (1976) found that Succiny- lation markedly enhanced the FC and FS of soy isolate dis- persions. The values were approximately double that of the unmodified isolate. Addition of NaCl also increased the FC of native soy isolate while addition of sucrose decreased FS. Soy isolate was Subjected to an alcohol wash prior to whipping (Eldrige 31 _1., 1963). The samples were heated for 15 min in a boiling water bath prior to whipping. The low density foams had stability times of approximately 200 min. The alcohol washed protein had improved color and flavor. Stable foams were produced below pH 3.0 and above 6.5. Minimum foam stability (in addition to the lowest volume foams) occurred in the isoelectric area of protein. Heating the protein enhanced foam expansion. Addition of 5.0% NaCl resulted in foams of low stability. The addition of tripoly and hexametaphosphate had little effect on the whips. Yasumatsu 31 _1. (1972) measured foam expansion and stability in model systems containing soybean proteins. Native soy flour had high foam expansion and stability. Denatured forms of soy flour had poor whipping 36 characteristics. The highest whipping values were from systems containing both soy flour and Skimmilk. The whip- ping properties of soy flour were maximum at pH 6.5 (McWat- ters and Cherry, 1977). Sensory Evaluation Flavor is perhaps the most important property in deter- mining food acceptability and where flavor is the less dominant trait, mouth feel or texture assumes greater importance (Kinsella, 1976). Color, odor, flavor and texture are key attributes in deciding whether or not a new protein will be used in a food product. Proteins affect texture in sols, gels, foams, emulsions and extruded foods. The flavor of a food product can be affected by proteins due to browning reactions, sulfide elimination, proteolysis and by entrapment and binding. Color can be Significantly affected by browning reactions. Altering a protein source may result in flavor changes in the food. These flavors may be contaminants of the protein per se, or they may be gen- erated during processing and storage. The astringency in milk products was thought to be caused primarily by heat altered whey proteins and milk salts (Josephson, 1967). Both of these groups were associ- ated, by adsorption or by interaction, with the casein micelles. Lang t al. (1976) examined the influence of compositional variations and processing on the sensory 37 properties Of Skimmilk. Pasteurization at 79.4°C and 85.00C produced a heated flavor noticeable to trained panelists. Variations ln homogenization pressure and fat content did not result in detectable flavor or mouthfeel differences. Judges detected differences between samples with and without 200-300 ppm of stabilizer when the samples were heated at 850C. The panelists were not able to detect 400 ppm of emulsifier unless fat contents were below 0.5%. The addi- tion of stabilizers increased viscosity while addition of emulsifiers did not. Sensory evaluation of casein-soy sols revealed a sig- nificant decrease in flavor scores as casein was replaced with soy protein (Hoffman, 1974). The odor of soy isolate was described as beany. No color change was noted as soy isolate replaced WPC in sols though the blends were des- cribed as having a beany odor. Maga and Lorenz (1973) con- ducted a sensory and analytical flavor evaluation of NFDM, sodium caseinate, isolated soy proteinate and various other protein supplements. Odor intensities and flavor evaluations were repeated randomly by 20 taste panelists using a scale of l to 10. A product with a completely bland character received a score of 10 while a strong characteristic was scored 1. 0f the 12 products examined, NFDM had the most bland odor and flavor when tested in a rehydrated system. As a group, the milk protein supplements were the most bland products. The soy products had lower flavor and odor 38 scores. In another study Maga and Lorenz (1972) examined the flavor and odor intensities of milk, marine and vegetable protein supplements. Sensory panel scores were recorded for odor and flavor properties. Milk products included NFDM, sodium caseinate, whey powder and demineralized whey powder. Soy products included soy isolate, soy concentrate and soy flour. There were no statistical differences between odor intensities of any of the milk products. Many of the vegeta- ble products had less bland odor properties. Reconstituted NFDM was judged to have the most bland flavor. The flavor of soy isolate was judged to be statistically inferior. Demineralized whey powder was considered to be more bland than standard whey powder. As a group, the milk products had a definite flavor advantage over the vegetable products. Kalbrener 31 31. (1971) reported on taste panel studies on commercial soy flours, concentrates and isolates. Sam- ples were evaluated in 2% dispersions in water. A raw defatted flour prepared in the laboratory received an odor score of 5.8, a flavor score of 4.1 and was described as beany and bitter. Commercial flours generally were rated higher, ranging from 4.2 to 6.7 for flavor. Concentrates had flavor scores ranging from 5.6 to 7.0. Isolates scored from 5.9 to 6.4. It was generally agreed that beany and bitter flavors persisted in isolates, though at low levels. An inverse relationship was found between flavor score and solubility. Raw flour had the highest solubility but lowest 39 flavor scores. Maga (1970) compared the flavor profile of commercial isolate and raw soybeans Of the same source. Overall intensity scores revealed that the isolate had the blandest flavor and odor properties. Processing decreased the green and bitter flavors, probably due to the sweet like flavor which appeared. Repeated washings with water and solvents removed some but not all of the objectionable soy flavor. Bitterness was found in a partial proteolytic hydroly— zate of soybean protein (Yamashita 31 1., 1969). Arai 31 _1. (1970) pointed out that enzymatic proteolysis of soy resulted in the formation of bitter compounds. This bitter- ness was caused by peptides containing leucine at the C- terminal.‘ In an earlier study Fujumaki 31 _1. (1968) found that the beany flavor generally decreased during the early stages of enzymatic digestion, though the bitter flavor increased. Bitterness was found in soybean digests after partial hydrolysis with pepsin (Fujumaki 31 31., 1970). Johnson (1975) reported that enzymatically modified soy proteins could be used as whipping agents, though there might be problems with flavor due to bitterness. Mattick and Hand (1969) investigated the raw, green bean like flavor of soybeans which they described as the major sensory defect of soybean products. A compound was isolated and identified as ethyl vinyl ketone which had a green bean like flavor. Pelissier (1976) compared the bitter 4O taste Of enzymic hydrolysates from cow, ewe and goat caseins. Caseins from bovine milk were generally more bitter than the other caseins. The total hydrophobicity Of the protein and the nature of natural proteases were important in the devel- opment of bitterness. Sensory evaluation by a trained panel showed that alkali pretreated soy had better flavor properties than the water soaked control (Badenhop and Hackler, 1975). However, this treatment did result in partial destruction Of the amino acid cystine. The use of alkali treatment to improve the flavor of soymilk was explored by Bourne 31 31. (1976). Addition of NaOH caused a rapid increase in pH while addition of Na2C03 or NaHCO3 caused much less. An experienced taste panel demonstrated a greater acceptability for soymilk adjusted to pH 7-7.5 with NaOH but noted a soapy flavor with the other compounds. Soymilks were then prepared with sodium salts at the same sodium ion concentration that was used in the original tests with NaOH. These samples were given approximately the same scores by the panel as the NaOH treated group, even though the pH was not in the same range. This led the authors to conclude that the sodium ion con- centration rather than the pH was the effective mechanism. The effect of chemical modification on flavor was investigated by Franzen and Kinsella (1976) who examined the role of acetylation and succinylation on the sensory proper- ties of soy protein. The color of the sols was lightened 41 and no flavor problems were reported by these treatments. Cowan 31 31. (1973) reviewed the flavor components of soybean products. Steam treatment in combination with alco- hol extraction produced soybean flakes with flavor scores of 7.0 (1—10 scale, 10 most desirable). Beany and bitter fla- vors were still detectable even at low concentrations. The effect of hexane:ethanol azeotropic extraction on the organoleptic qualities of defatted soy isolates was inves- tigated by Honig 31 31. (1976). Flavor and odor scores were based on a ten point system (1 strong, 10 bland). Following azeotropic extraction the isolate had a flavor score of 7.2 which was an improvement over the original score of 6.2. Odor intensities were lower and were judged to be comparable to sodium caseinate. Eldridge 31 31. (1977) examined alco- hol treatment as a mechanism for reducing the beany flavor Of soybean protein products. Raw soybeans and soybean products were steeped or wet milled with ethyl alcohol to inactivate enzymes in situ or with disruption of cellular disruption. When aqueous alcohol was used enzymatic acti- vities were reduced and flavor properties were improved. Flavor scores improved from 6.1 (control) to 7.7 for a soy isolate. Yasumatsu 31 _1. (1972) observed that almost all types of soy bean products have some undesirable flavor charac- teristics. These defects have proven to be the greatest barriers to increased use of soybean products in food. 42 Principle component analysis was used to analyze the flavor profile of soy isolate. Even though soy isolate was the most purified form of soy protein, it still had flavor prob- lems. Johnson (1970) made the Observation that the use of soy products in human food has largely been limited due to flavor problems. Heat Stability Soy Protein The nomenclature system based on sedimentation coeffi- cients has been used extensively for soybean proteins (Smith and Circle, 1973). Four main fractions have been isolated with sedimentation coefficients of 25, 7S, 118 and 15S. The 2S fraction is a multiprotein component with molecular weights ranging from 8,000-24,000. The 73 globulin has a molecular weight of approximately 180,000 and is composed of many Subunits. The 115 fraction has a molecular weight of approximately 350,000 and is also composed of many sub- units. Together the 7S and 118 comprise between 65 and 7 % of the total protein. The 15S component is a large, molecu- lar weight species having a molecular weight of approximately 8 million. I A Mann and Briggs (1955) examined the effect of heat on soybean proteins using electrophoretic analysis with a Tiselius apparatus. Protein sols were heated by immersing in an oil bath for the desired time and temperature. 43 Temperatures ranged from 45°C to 90°C with heating times of 2 to 30 hr. Electrophoretic patterns varied widely with heat treatment with some apparent aggregation. The quantity of protein precipitated increased with increased temperature and length of heating. Watanbe and Nakayama (1962) des- cribed the formation of soluble aggregates during heating of water extracts from defatted soy meal. Ultracentrifugal analyses indicated that the 11S, 75 and 15S fractions par- tially disappeared after 10 min at 800C or higher tempera- tures. The major reserve protein of soybeans, glycinin was heated by Catsimpoolas 31 _1. (1969) at temperatures of 35-9OOC for 1 hr. No significant changes were observed to occur from 35-500C and only Slight change was noted between 50-700C. When the protein was heated at tempera- tures above 70°C a sudden alteration occurred. Examination by disc electrophoresis revealed significant dissociation of the protein into subunits. The protein was not com- pletely dissociated as some undissociated glycine could still be detected. Some precipitation of the protein occurred at 900C. Wolf (1970) reported that heating dilute solutions of 11S globulin caused about one half of the protein to precipitate while the remainder was converted into a 3-4S form that remained soluble. Wolf and Tamura (1969) heated 0.5% solutions of 115 fraction at 100°C in a pH 7.6, p=0.5 buffer. The solution 44 became turbid and precipitation Occurred. The 118 protein disappeared in less than 5 min and a soluble aggregate of 80-1OOS appeared. With continued heating the aggregate grew and precipitation OCCUrred in 7 min. Disappearance of the 115 was accompanied by appearance of a 3-4S component. This fraction reached its maximum in 5-7 min and was stable to heat for more than 30 min. Saio _1__1. (1971) investi- gated the effect Of heating on the 7S and 118 fractions of soybean protein. The proteins were heated at temperatures of O-lOOOC for l min. After heating at the higher tempera- tures (700C and above) several fast moving bands appeared on polyacrylamide gels. The less mobile 7S and 115 bands disappeared. The appearance of the fast moving bands seemed to coincide with an increase in sulfhydryl groups. The antigenic changes involved in the thermal denatu- ration Of glycinin, the major reserve protein of soybeans were reported by Catsimpoolas 31 31. (1971). The protein, consisting of multiple subunits was dissociated by heating into both soluble and insoluble complexes. Glycinin was heated at temperatures of 30-900C for 30 min. Disc electro- phoresis was used to demonstrate the changes occurring to the native protein. The gel patterns from samples heated to 70-750C were almost identical to the unheated sample. As the temperature rose to 800C and above, the intensity of the glycinin band gradually declined and disappeared entirely at 900C. Concurrent with the disappearance of 45 glycinin, several faster migrating components appeared in the gels. In addition, the amount of glycinin not entering the gels increased. The fate of water soluble soy protein during thermo- plastic extrusion was examined by Cumming 31 _1. (1973). The extruded material consisted of commercial defatted soy- bean meal which had been cooked to temperatures of 93, 121, 149, 177 and 204°C. After processing, a portion was extrac- ted with distilled water and subjected to PAGE in an alka- line system on 7% gels. As the cooking temperature rose the concentration of those components close to the origin Slowly disappeared and bands with faster migration rates appeared. Six major fractions were found in these gels, though differing in intensity. Fractions A and 8 decreased with increasing temperature while fractions D and E in- creased. In the unprocessed meal, dense bands were iden- tified as 75, 113 and 158 fractions. At elevated cooking temperatures there was a reduction in all three fractions, accompanied by a noticeable increase in breakdown products. Saio _1 31. (1975) induced soy 7S and 118 proteins to gel by heat treatment at 100-1700C. After heating the samples, the material was solubilized with a solution con- taining 0.075 M sodium dodecyl sulphate (SDS) and 0.025 M mercaptoethanol (ME). After dialysis, a portion was sub- mitted to both disc and SDS polyacrylamide gel electrophore- sis (PAGE). Disc PAGE was difficult to interpret because 46 of the large number of minor components which resulted in substantial diffusion. With 508 electrophoresis, the unheated, cold insoluble fraction (CIF) resolved into two principal bands and several minor ones. Similar patterns were obtained by heating to temperatures of 130°C. No bands were observed at heating temperatures above 150°C. The effect of heat on the PAGE patterns of crude 78 gel was similar to that observed for the CIF proteins. In a fur- ther study, Saio _1 31. (1975) investigated the breakdown of protein subjected to heat treatments greater than 1500C. Sodium dodecyl sulfate and disc electrophoresis with o- phthalaldehyde as the stain were employed. The authors concluded that the gross structure of soybean subunits degraded to form lower molecular weight substances by heating at temperatures above 150°C. Aldrick (1977) examined selected milk and soy fractions for specific heat induced interaction. Soy 7S and 113 proteins were prepared from whole soybeans. Sodium casein- ate and B lactoglobulin were prepared from fresh whole milk. Soy-milk protein combinations were heated in a 1:1 ratio at three temperatures; 63, 74 and 121°C. After cooling and centrifugation, aliquots of the protein were electrophoresed in the appropriate gel system. The gel bands corresponding to the 7S fraction diminished in intensity at 63°C/30 min and vanished at higher temperature. The soy 7S fraction was found to move independently of either sodium caseinate or 47 B lactoglobulin. The soy llS fraction was more stable to heat treatment at 740C than the 7S fraction. The 11S frac- tion also moved independent of either 8 lactoglobulin or sodium caseinate. Milk Proteins Caseins are those phosphoproteins precipitated from raw Skimmilk by acidification to pH 4.6 at 20°C (Whitney 31 31., 1976). Caseins can be divided into four main groups based upon their electrophoretic mobilities: as caseins, B caseins, k-caseins and y-caseins. The oS-caseins consist of one major and several minor components. Approximately 45-55% of the Skimmilk protein is in this fraction. The molecular weight of ds-casein is about 23,000 (Rose 31 _1., 1970). The milk protein present in the second largest amount is B casein which accounts for 25-35% of the total Skimmilk protein and has a molecular weight of 19,000. The remaining two fractions k and y represent 8-15 and 3-7% of the total Skimmilk protein. The whey proteins are those remaining in the serum after precipitation of casein by addition of acid (Whitney _1 _1., 1976). The major whey protein, B lactoglobulin is .responsible for 7-12% of the total Skimmilk protein and has a molecular weight of 18,000. The whey protein present in the second greatest amount is d lactalbumin which has a molecular weight of about 14,000 and represents 2-5% of the total Skimmilk protein. The immunoglobulins are a 48 heterogeneous group of proteins with molecular weights ranging from 160,000 to 1,000,000. They represent 1.9-3.3% of the total Skimmilk protein. Bovine serum albumin accounts for 0.7-1.3% of the Skimmilk protein and has a molecular weight of approximately 66,000. The proteose peptone fraction is a multicomponent group with molecular weights ranging from 4,000 to 41,000. They represent approximately 2-6% of the total Skimmilk protein. Pasteurization at 67OC/30 min produced no appreciable change in the nitrogen distribution of whole milk (Shahani and Sommer, 1951). Pasteurization at 73OC/30 min resulted in a decrease in the globular nitrogen and an increase in the NPN. Sullivan 31 31. (1957) examined the changes, occurring in the centrifugable nitrogen fraction of skim- milk as a function of time following several heat treat- ments. Portions of Skimmilk were heated for one min at 75, 104, and 132°C, cooled and centrifuged. All samples had a marked decrease in protein content after heat treatment. The decrease in nitrogen was roughly proportional to the increase in heat treatment. Josephson _1 31. (1967) heated selected milk systems at various temperatures to determine astingency flavor response. Sephadex gel filtration, PAGE, electron micro- scopy and chemical analyses were used to determine changes in the size, shape and composition of protein-salt particles. The astringent components in rennet whey, heated at 63-900C 49 for 30 and 10 min were quite large as evidenced by their sedimentation patterns. The proteins in heated rennet whey (9OOC/10 min) were present as aggregates of sufficient size to prevent their migration into the gel (PAGE). Heating for 2 hr was required to produce distinct astringency in caseinate ultrafiltrate systems. Morr (1965) investigated the effect of heat upon the composition of the protein aggregates in normal and concen- trated Skimmilk systems. A portion of unheated Skimmilk was used as the control with the remaining milk heated to 88°C for 10 min. After heating and cooling aliquots were ‘fractionated by ultracentrifugation. The centrifugal Super- natant and sediment fractions were then examined for compo- sitional makeup. Heating Skimmilk at 880C for 10 min had no Significant effect upon the protein particle Size dis- tribution or chemical composition of the protein-sediment fractions. In another study, Morr (1969) investigated the extent of protein aggregation-disaggregation produced by heating Skimmilk at 90°C for 10 and 30 min. An additional series of high temperature-short time treatments (UHT) extending to 149°C for up to 16 sec were also examined. Heating at 900C caused formation of 3 75 5 500$ protein particles whereas UHT treatment favored formation of larger size particles. It appeared that UHT treatments in excess of 1270C for 16 sec favored formation of nonsedimenting 50 nitrogen compounds, presumably by disaggregating casein micelles. Similar amounts of whey protein disappeared from the 900C heated Skimmilk ultracentrifugate as from com- prable pH 4.6 whey systems. Whey proteins heated at 900C for 10 and 30 min Showed extensive denaturation and Subse- quent aggregation when subjected to zonal electrophoresis. Progressively higher UHT treatments produced gradual reduc- tion in the quantity of all whey proteins. Heat treatment at 900C for 10 and 30 min did not substantially alter the zonal electropherograms of casein. Heating milk to 900C caused substantial aggregation of the whey proteins but only minor changes in the physical dimensions of the casein micelles (Hostettler 31 31., 1965). Ultra-high temperature heating of whey protein systems was responsible for only small amounts of denaturation compared to conventional sterilization. Conventional and UHT sterilization processes produced gross aggregation of casein micelles. However, with continued heating at high temperatures the casein micelles began to break down into soluble casein. Hansen and Melo (1977) examined the effect of ultra- high temperature steam injection upon the constituents of Skimmilk. Alterations occurring to the native protein systems were determined by two techniques: 1. isolation procedures for specific proteins and 2. electrophoretic and densitometric methods. Processing Skimmilk at temperatures of 138 to 1490C for 8-10 sec denatured many of the milk proteins. Whey proteins were most susceptible to denatura- tion while caseins were least affected. 8 lactoglobulin and o lactalbumin decreased Substantially. Wilson (1971) examined the large protein particles formed during sterilization and storage of concentrated Skimmilk. Concentrated Skimmilk was sterilized in a heat exchanger at 155°C with no holding time and canned asepti- cally. Protein particles from the sterilized concentrate were fractionated in a centrifuge and Subjected to gel electrophoresis and Kjeldahl nitrogen determinations. After heating and storage the percent of sedimenting material had substantially increased. Particles with a diameter greater- than 0.75 u were primarily composed of casein. The objective of research conducted by Fox 31 31. (1966) was to ascertain the factors responsible for the Presence of nonsedimentable nitrogen (NSN) in sterile milk concentrates. Milk samples were heated in a thermostated bath at temperatures of 70-117OC for 30 min. The NSN de- creased as the heating temperature increased to 900C. The NSN began to increase as the temperature rose to 1100C and above. Disc electrophoretic techniques were used to esta- blish the identity of the nonsedimenting species. Below 103°C the NSN consisted principally of whey proteins while at temperatures of 1100C and above it was predominantly casein. 52 Rowland (1937) examined the heat denaturation of the albumin and globulin fractions in milk heated at tempera- tures of 75-1000C. Denaturation of albumin and globulin took place rapidly in samples of milk heated at tempera- tures of 750C and above and was complete in approximately 60, 30, 10-15, and 5-10 min at 80, 90, 95 and 100°C, respectively. There was no change in the nonprotein nitro- gen (NPN) content of milk heated at temperatures up to 1000C. With continued heating at 95 and 1000C small amounts of NPN developed. At 115 and 120°C, appreciable hydrolysis of protein followed denaturation of the protein. The heat stability of whey proteins separated from Skimmilk by treat- ment with sodium chloride and hydrochloric acid was studied by Harland and Ashworth (1945). Treatment at 80°C for 45 min or at higher temperatures for shorter times resulted in the denaturation of 93-95 percent of the whey proteins. Hetrick (1950) reported the effect of high temperature short time heat treatment on the denaturation of albumin and globulin in milk. Heat treatments of 81°C to 152°C with holding times of 0.03 to 64 sec were used. Heat treatment sufficient to cause the first noticeable cooked flavor re- sulted in the denaturation of approximately 58% of the pro- tein. Milk was subjected to heat treatments of 65-96°C for 30 min by Larson and Rolleri (1955). Heat treatment of 70°C for 30 min denatured 2 % of the total serum proteins 53 due to the Specific denaturation of 89% of the immune globulins, 32% of the B lactoglobulin, 52% of the serum albumin and 6% of the o lactalbumin. The denaturation curves indicated that the immune globulins were the least and d lactalbumin the most heat resistant. Harland 31 31. (1955) reported on the quantitative changes which occurred during heat treatment of Skimmilk at temperatures ranging from 80-1410C. A high temperature tubular heater was used in these experiments. At 1410C a heating time of 6 sec was sufficient to denature 67% of the serum proteins. Ninety seven seconds at 1000C denatured 84% of the serum proteins. AS the heating temperature increased, a secreased holding time was necessary to denature approximately the same amount of protein. The role of colloidal phosphate and pH on the heat stability of milk was examined by Rose (1962). Heat stability was determined after treatment at 140°C over a wide range of pH values. The denaturation of B lactoglobu- lin was highly correlated to the heat stability of the milk. The heat denaturation of the milk proteins 8 lacto- globulins A and B were compared by Gough and Jenness (1962). Five ml aliquots of 1% solutions were heated at 730C for 7-97 min. After heating for 30 min there was a 46. % de- crease in 8 lactoglobulin A and 71.4% decline in d lacto- globulin B. Melachouris and Tuckey (1966) investigated the denatu- ration of the whey proteins when milk was heated to high 54 temperatures for short times. Whole milk was divided into separate lots which received heat treatments of 61.7OC for 30 min, 93.3, 110, 126.7 and l43.3°C for 2.08 sec. The denaturation markedly increased as the heat treatment in- creased. The B lactoglobulin fraction was very sensitive to heat (7 % denatured at 143°C). Albumin also exhibited an appreciable degree of denaturation when the milk was heated at 110°C or higher. The proteose-peptone fraction decreased as temperature of heating rose. The globulins were rapidly denatured even at the lower temperatures. The serum protein denaturation in Skimmilk which resulted from direct steam injection was assessed by Dill 31 31. (1964). Seven temperatures, ranging from 75 to 1410C and four hol- ding times of 8 to 190 sec were employed. The amount of denaturation varied from 10% for the least severe treatment to greater than 80% for the most severe. As the holding time increased, the percent of denatured protein increased. Kenkare 31 31. (1964) examined the heat induced aggregation of Skimmilk serum proteins. Samples were heated in Erlen- meyer flasks by immersing in a boiling water bath or in a steam autoclave to temperatures of 1200C. Heating desta- bilized the serum proteins in acid-prepared serum to a greater extent than protein from Ultracentrifugal serum. The 90.5°C treatment denatured 55 and 40% of the protein, respectively. Gel filtration with sephadex G-lOO indicated that heating acid-prepared serum protein caused aggregation 55 and destabilization of each of the protein fractions. Josephson 11‘_1. (1967) examined several milk systems for heat induced changes in the protein-salt balance. Sephadex gel filtration, PAGE, electron microscopy, chemi- cal analyses and flavor evaluation were employed. Gel filtration elution patterns from heated milk revealed measurable losses of whey protein. Similarly, results from PAGE indicated that the characteristic whey protein bands were absent from the heated (900C-10 min) Skimmilk super- natant. Patterns Of heated rennet whey demonstrated that the proteins were present as aggregates of sufficient Size to prevent their migration into the gel. The nature and extent of protein aggregation in heated whey systems were studied by Morr and Josephson (1968). Heating at 900C for 10 min caused drastic reduction in the amount and resolu- tion of B lactoglobulin, a lactalbumin, serum albumin and globulin fractions. Gel filtratiOn patterns indicated appreciable aggregation of the proteins. The calcium con- tent and thiol-disulfide reactions were of major importance in these interactions. Approximately 80% of the cottage cheese whey proteins were denatured when heated at 910C for 30 min (Guy 31 31., 1967). Denaturation increased dramati- cally in the temperature range of 85-900C. Nielson 31 _1. (1973) used response surface experimental design to evaluate the role of four factors, e.g. temperature (60-900C), heating period (1-30 min), pH (4.5-8.5) and total solids 56 (6-60%) on the denaturation of cottage and Colby cheese whey proteins. The most important of the four different para- meters was Shown to by heating temperature. Minimum protein denaturation occurred in the intermediate pH region (6-7). The whey proteins were least Susceptible to denaturation at 20% total solids. PAGE patterns revealed denaturation of the proteins when the whey systems were heated at 85.6°C for 17.2 min. This heat treatment resulted in aggregation of the denatured whey protein. Schafer and Olson (1975) heated milk at ultrahigh temperatures (UHT) prior to manufacture into Mozzarella cheese. Raw Skimmilk was heated in a tubular heat exchanger at temperatures of 80, 90, 100, 110, 120 and 130°C for 2 sec. There was a linear increase in the percentage of denatured whey protein as the heating tem- perature rose to 120°C. Approximately 27% Of the whey pro- tein was denatured at this temperature while at 80°C only 2-3% was denatured. Whey was heated in a helically coiled tube heat exchan- ger at temperatures of 65.6-148.9°C (Senter _1 _1., 1973). The heating, holding and cooling times were 4.8, 0.18 and 1.9 sec, respectively. Proteins and disulfide and sulfhy- dryl groups were quantified fluorometrically and electro- phoretically. The a lactalbumin component was the most heat resistant. None of the heat treatments (65.6-148.9°C) decreased the electrophoretic area of this protein. B lac- toglobulin was sensitive to temperatures of 87.8-148.9°C 57 with denaturation increasing with length of holding time. Calcium caseinate was found to be very stable to heat (White and Davies, 1958). Temperatures greater than 130°C for 30 min were required to precipitate the complexes. Kresheck 31 a1. (1964) studied the behavior and properties Of the major milk caseins at temperatures of 30, 50, 70 and 90° C. Light scattering techniques were used to examine the heated samples for changes in particle size. Heating times of 30 min to 2.5 hr were employed. The summation of values from the light scattering experiments revealed little change in molecular weight or radius of gyration for whole casein at the temperatures studied. Turbidity increases were Slight even when heated at the higher temperatures for 2.5 hr. Heated casein sols were examined for protein degrada- tion by Alais 31 _1. (1967). Casein dispersions were heated to 120°C for 10-80 min and analyzed for peptide content by electrophoresis, peptide mapping and NPN. The amount of NPN increased from 0.45% for unheated whole casein to 2.25% for casein heated 80 min. Electrophoresis of heated sam- ples showed PAGE patterns to be only slightly altered. Fractions were not as distinct with Slightly more material failing to enter the gel. Peptide mapping revealed the presence of high molecular weight peptides in the heated casein samples. Cheeseman and Knight (1974) examined the nature of casein aggregates in heated milk. Gel filtration on sepharose 6B was used to demonstrate the size distribution 58 of casein aggregates in fresh, heated and UHT treated milks. The elution profile of the nonheated samples were separated into five areas. The first peak eluted at the void volume and was primarily composed of large casein aggregates. Heat treatment at 100°C for 40 min or 1430C for 3 sec resul- ted in slight changes in the Size distribution of casein micelles. The changes which occurred to casein after steriliza- tion were reported by Aoki and Imamura (1974a). Samples of milk were heated at 135°C for 45 sec prior to storage. This heat treatment resulted in an increase in the amount of nonsedimenting, soluble casein. This was further demon- strated by Aoki _1 _1. (1974b). The amount of soluble casein increased when whey protein free milk (WPFM) was heated at temperatures of 135-140°C for 15 sec. Soluble nitrogen was determined on the Ultracentrifugal Supernatant from the superheated WPFM. Soluble casein began to increase at 105-110°C. Soluble casein further increased as the heating temperature rose. The amount of soluble casein was greatest in concentrated samples. The cold disaggregation of casein micelles in heated, concentrated WPF milks was studied by Aoki and Imamura (1975). The amount of soluble casein was much greater in the heated product compared to the unheated sample. Heating at temperatures above 105°C may have caused formation of soluble casein by disruption of hydrogen and hydrophobic bonds. Aoki 31 a1. (1977) 59 heated WPF milk at 135-140°C for 15 sec to further examine Changes occuring to casein micelles. By means of electron microscopy and differential centrifugation it was confirmed that casein micelles aggregated when concentrated WPF milk was heated at these temperatures. The amount of micelles which sedimented at 3000 x g nearly doubled after heating. El-Negoumy (1978) described the changes occurring in sodium caseinate model systems heated to 100°C for 30 min in the presence of various ionic Species. The sols, con- taining 3% sodium caseinate were dispersed in deionized water with one of the following species: 1) 0.02M CaClZ, 4) 0.01 M 2) % lactose monohydrate, 3) 0.011 M KH PO 2 4’ Na Citrate, 5) 0.039 M NaCl 6) 0.02 M CaCl2 + 5% lactose + 3 0.011 M KH2P04 3 7) milk dialyzate. All model systems (except an unheated 2, + 0.01 M Na Citrate + 0.039 M NaCl and control) were heated to 100°C for 30 min prior to dialysis and freeze drying. A portion of each sample was chromato- graphed on ion-exchange diethylaminoethylcellUlose. Elec- trophoretic analysis of chromatographic fractions was done on starch gels. Sodium caseinate sols heated in the presence of these ionic species underwent change in composition for most of the model systems. Formation of aggregates varied with the composition of the dispersing medium. 60 In Vitro Estimation of Protein Quality Assay Procedures Block and Mitchell (1946) demonstrated a workable system for the quantitative evaluation of a protein's nutritive value. The amino acid composition of a protein was determined by chemical means. Using whole egg protein as the standard, the nutritive value of protein was expressed as a chemical score equal to the greatest deficit in an essential amino acid in the test protein. An 13_11113_technique was used by Melnick 31 _1. (1946) to explain various factors affecting the nutritive value of soybean products. Following enzymatic digestion, the amount of hydrolysis was measured by a modified fOrmal procedure. 13 11133 digestibility increased for soy pro- ducts subjected to autoclaving when compared to the raw material. This data was supported by animal bioassays. Sheffner 31 _1. (1956) examined the relationship between the amino acid profile released by digestive enzymes and the biological value (BV) of food proteins. The ultimate purpose of their work was to develop an 13 11133 procedure which could adequately estimate the nutritional value of proteins. Pepsin digests were prepared by incubating 1 g of protein with 25 mg of pepsin and incubating at 37°C for 24 hr. Amino acid analyses were performed by microbiologi- cal procedures. After analysis of the digest and residue material, a pepsin-digest—residue (PDRI) index was 61 calculated by comparing the ratio of 11 amino acids in a test protein to that in a reference protein (egg). This index incorporated both the essential amino acid pattern released by 13 11133 pepsin digestion and the amino acid pattern of the residue. The PDRI had high correlation with the net protein utilization (NPU) value for a variety of proteins. Division of the PDRI by the coefficient of diges- tibility yielded values which accurately predicted the bio- logical values of the proteins studied. I vit33 methods ————¢ of protein evaluation were useful in Screening new protein foods and processing methods because of their rapidity (Akeson and Stahman, 1964). These researchers devised a rapid, accurate 13 31133 procedure using a pepsin-pancreatin digest. An index of protein quality was calculated from the amino acids released during digestion with pepsin and pancreatin. Using whole egg as a standard, excellent cor- relation was observed between the pepsin-pancreatin index for 12 proteins and their biological values determined from feeding trials. Van Buren 31 31. (1964) examined several indices used in the determination of protein quality. The methods in- vestigated included soluble nitrogen, urease inactivation, available lysine, free amino groups and the Hunter "L" color procedure. There was no significant correlation between soluble nitrogen and protein efficiency ratio, but the free amino groups, Hunter ”L" values and available lysine had 62 satisfactory correlation with PER at the 99% level. The protein damage which resulted from overheating of soymilk was measured with the greatest accuracy through determina- tion of the available lysine. 13 31133 enzymatic studies have demonstrated that amino acid availability and amino acid content of foods may differ markedly (Morrison and Rao, 1966). Acid hydrolysis proce- dures release all amino acids regardless of their availa- bility. lfl £1113 enzymatic procedures only release those amino acids ”available” thus providing much meaningful information on the inactivation of amino acids due to pro- cessing effects. A comparison was made of several 13 vivo and 13 11133 methods by Buchanan (1969). Several leaf pro- tein concentrates were employed as sample material. Papain solubility and true digestibility were highly correlated. Pepsin-pancreatin solubility was less well correlated. Microbiological estimations of available amino acids, invol- ving predigestion with pepsin did not Show good correlation with true digestibility. Pepsin and trypsin 1 1jtro enzymatic digestions were _— Used by Yamashita 31 31. (1970) to estimate the digestibili- ty of soy plastein. Pepsin and trypsin 13 11133 digesti- bilities were 84.8 and 76.5%, respectively. These values were almost identical to those of the undenatured soy pro- tein. Evaluating protein digestibility by 13 11133 methods are important because of their rapidity and sensitivity 63 (Saunders 31 _1., 1973). This researcher used several dif- ferent 13 31133 techniques in arriving at the protein diges- tibility of alfalfa protein concentrates. The enzymatic procedures included hydrolysis with papain, pepsin-pancre— atin and pepsin-trypsin. After digestion, the residue protein was determined with subsequent calculation of percent digestible protein. This was in contrast to Akeson and Stahmann (1964) who measured free amino acids following enzymatic hydrolysis. The values obtained from the systems containing pepsin had a high correlation with values from rat feeding studies. Poor correlation was found with the papain digestion. Maga 31 _1. (1973) measured the initial rate of proteolysis with trypsin as a simple 13 31133 means of gastronomic acceptability. The 13 31133 enzyme hydroly- sis was a modification of Sheffner's (1963) procedure. The products tested included: commercial sodium caseinate, peanut flour, cottonseed flour, fish protein concentrate and soy isolate. There were significant differences between rates of proteolysis for the different products. Sodium caseinate was by far the most easily digestible protein source. Soy isolate was the least digested material. Steaming resulted in faster hydrolysis for all samples tested. Osner and Johnson (1975) used several methods to pre- dict the nutritional and chemical Changes occurring to heated casein. The relative nutritive value (RNV), 64 available amino acids, net protein utilization (NPU) and pepsin 13 31133 digestibility were determined. The NPU and 8V of casein processed at 120°C for 8 hr was lower than the control. The availability of seven amino acids fell uni- formily and correlated with RNV. The pepsin-pancreatin digest index, pepsin digest residue index and NPU were used to monitor the effect of heat processing on casein (Stahmann and Woldegiorgis, 1975). It was observed that all three methods predicted similar values for protein quality. An enzyme score, which was calculated like the chemical score, compared the essential amino acids released from the test protein (heat processed casein) and those released from egg protein. The chemical score and enzyme score compared well with the enzyme indexes. Several procedures possibly having importance in rapidly estimating protein quality were reviewed by Satter- lee (1977). The model incorporated the essential amino acid (EAA) profiles of a sample and reference protein with their 13_31133 digestibilities. An 13 31133 PER could be deter- mined with this procedure in 72 hr. Another method which was reviewed included the use of the Protozoan, Tetrahymena pyriformis W, to measure food protein quality. Using an enzyme predigestion the subsequent Tetrahymena growth was highly correlated to the PER of the protein. This method was inexpensive and could predict a calculated PER within 72-96 hr. 65 Hsu 31 31. (1977) developed a multienzyme technique for estimating protein digestibility. This procedure was suffi- ciently sensitive to detect the effects of processing and protease inhibitors on the enzyme hydrolysis of proteins. The multienzyme system consisted of trypsin, chymotrypsin and peptidase. It was found that the pH of a protein sus- pension after 10 min of digestion was Significantly corre- lated with 13 3133 apparent digestibility. The correlation coefficient was 0.09 with a standard error of estimation (of 13 31133 or 1 v1v3) of i 1.72. The effect of processing and heat treatment on protein digestibility were satisfac- torily explained using this technique. The presence of fat in the samples had no apparent effect on the 13 31133 diges- tibilities. It was suggested that substances containing strong buffering capacities might affect the results though the food systems and protein sources investigated had no effect on the 13 vitro protein digestibilities. Evaluation of Processing Effects by In Vitro Enzymatic Procedures Valaris and Harper (1973a) investigated the effect of carboxymethylcellulose (CMC) on the rate of peptic diges- tion of as casein. Pepsin was immobilized on glass beads. At concentrations of 0.02% and 0.03% CMC inhibited proteo- lysis of 0 casein by immobilized pepsin. The inhibitory S mechanism could not be satisfactorily explained. The effect 66 of CMC on the proteolysis of a casein by immobilized trypsin s was also studied by Valaris and Harper (1973b). CMC inhibi- ted activity on intact as casein. However, CMC failed to inhibit tryptic degradation of OS casein partially hydrolyzed by pepsin. They suggested that CMC would have no significant effect on the tryptic digestion of casein and that any nutritional significance would relate only to its effect on pepsin digestion. McCune (1977) determined protein quality using an 13 31133 enzymatic digestion and a rat bioassay (PER). The model systems were prepared from a casein-glucose mixture and a casein-safflower blend. Thermal and chemical treat- ments included: 1) incubation of the casein-safflower oil mix at 55°C to promote oxidation of the oil, 2) autoclaving of casein and 3) autoclaving of a casein-glucose mixture. Protein quality was most accurately predicted by use of an 13 31133 pepsin-pancreatin sequential digestion to release amino acids. The enzyme index was calculated by computing the geometric means of the ratios of the released essential amino acids of the test protein to a reference protein. This index correlated best with the bioassay results. Casein exposed to lipid oxidation had Substantially reduced PER. Casein and casein-glucose mixtures subjected to autoclaving had Slightly lower PER. Evans and Butts (1949) investigated the heat inactiva- tion of methionine in soybean meal. No inactivation occurred 67 when soybean protein was autoclaved by itself, but 46 to 97% was inactivated when glucose was added to the samples. An enzymatic 13 31133 digestion was used. Because biological experiments are expensive and time consuming a faster method was sought by Menden and Creamer (1966) to evaluate protein quality. Casein was used as the model system and was subjected to severe heat treatment both in the presence and absence of glucose. Enzymatic hydrolysis with pancreatin led to the conclusion that heat treatment of casein did not decrease the availability Of essential amino acids. Heat treatment in the presence of glucose significantly diminished the availability of amino acids. Rao and Rao (1972) examined the effect of non-enzymatic browning on the nutritive value of casein-Sugar complexes. The autoclaving of casein with arabinose, glucose or lactose resulted in a reduction of amino nitrogen, partial destruc- tion of lysine, arginine, methionine and leucine. '13_31133 digestibility of the protein also decreased. Loss of available lysine, due to the binding of the 8 amino groups with the aldehyde group of the sugars occurred most rapidly with arabinose, glucose and lactose. Acid and enzymatic hydrolysis was Used by Hankes 31 _1. (1948) to examine the effect of autoclaving on the liberation of amino acids from casein. Pepsin, whole pancreatin and erepsin were selected as the enzymes to be used in the 13 vitro digestions. Amino 68 acids were quantitated by microbial procedures. Samples were prepared by autoclaving casein for 4 min and for 20 hr at 15 psi. Autoclaving casein for 4 min had no effect on the amino acid composition while autoclaving for 20 hr Slightly reduced cystine. The release of amino acids by pepsin, pancreatin or erepsin digests was higher in the casein autoclaved for 4 min than in unheated casein. Pan- creatin digestion for 2 hr released 19% of the 3 amino nitro- gen. An additional 2 hr digestion with erepsin released 27% of the total. Continued digestion for 5 days released 50% of the 3 amino nitrogen. An investigation into the liberation of essential amino acids from raw, properly heated and overheated soybean oil meal was made by Riesen 31 31. (1947). Acid, alkaline and pancreatic hydrolyses were performed on each sample. The results indicated that the essential amino acids liberated by acid hydrolysis were unaffected by heat treatment, with the exception of lysine, arginine and tryptophan. Prolonged autoclaving (4 hr) decreased the liberation of these amino acids. Liberation of the essential amino acids was increased by proper heat- ing when Subjected to pancreatic digestion. Excessive heating decreased liberation Of essential amino acids. Cys- tine destruction and cystine inactivation Of autoclaved soybean oil meal were studied by Evans 31 _1 (1951). Cystine destruction was determined by acid hydrolyzing a sample and quantitating the amino acids. Cystine 69 inactivation was estimated by an 13 vitro enzymatic hy- drolysis. Autoclaving soybean oil meal for 4 hr at 15 psi destroyed 31-36% of the cystine. An 13 31133 enzymatic hydrolysis with trypsin and erepsin revealed that 82-87% of the cystine had been inactivated. No cystine remained available after cystine was autoclaved in the presence of glucose. The nature and extent of amino acid inactivation in milk processed by four widely used methods was studied by Mauron 31 _1. (1955). Destruction and inactivation of the amino acids, tryptophan, tyrosine, methionine and lysine were determined in fresh and boiled milk, in roller and spray dried milk powders, and in evaporated and sweetened condensed milk. The enzymatic liberation of amino acids was followed by a dynamic 13 31133 digestion procedure using pepsin and pancreatin. NO differences were detected between the amino acid compositions of fresh and boiled milk. Tryptophan, tyrosine and methionine were not affected by any of the processing treatments. Destruction of amino groups was noted in a slightly scorched milk powder, with 13% inactivated as measured by 13_31133_digestion. Lysine suffered the most damage of any of the amino acids. Slight destruction took place in spray dried milk (4%) but rose to 26% in slightly Scorched roller dried milk. Ford and Salter (1966) used both static and dynamic digestions to estimate protein quality. Portions of freeze-dried fish 70 were subjected to different heat treatments prior to 13 31133_digestion with pronase or successively with pepsin, pancreatin and erepsin or sequentially with pepsin and papain. The digests were analyzed for soluble protein, peptide content and free amino acids. Broadly similiar results were achieved with the different enzyme digestions and correlated satisfactorily with rat feeding studies. AS the severity of heating increased, the proportion of certain amino acids in the digests diminished. Severe heating retarded the 13 31133 enzymic release of several amino acids which was consistent with animal growth studies. Dimler (1975) examined the protein quality of milk proteins using an 13 31133 enzymatic approach. Three milk proteins were studied: whole casein, as casein and B lacto- globulin. Enzymatic hydrolysis with pepsin for 20 hr was followed by a second digestion with pancreatin for 6.5 hr. After digestion, TCA was added to precipitate the unhy- drolyzed protein. The clear supernatant was analyzed for total nitrogen, alpha amino nitrogen and free amino acids. The milk proteins were subjected to four different treat- ments prior to analysis; these included 1) untreated, 2) autoclaved for 30 min at 15 psi, 3) autoclaved with glucose and 4) autoclaved with 5x the amount of glucose. After digestion of the treated samples the total free amino acids remained unchanged. Several essential amino acids suffered some losses. Both o5 casein and 8 lactoglobulin 71 digest fractions had progressive reduction in several amino acids. EXPERIMENTAL PROCEDURES Functional Studies Mixtures of milk products and soy isolate were com- bined to determine which functional characteristics could be demonstrated. The materials utilized in this study were: Nonfat dry milk (NFDM) was Obtained from Land 0' Lakes, Inc., Minneapolis, Minnesota. Edi—ProN, a soy isolate, was obtained from Ralston Purina, Inc., St. Louis, Missouri. Sodium caseinate was obtained from Milk Proteins, Inc., Troy, Michigan. Enerpro 50, a dehydrated whey protein concentrate (WPC) was obtained from Stauffer Chemical, Inc., Westport, Connecticut. Spray dried sweet whey was obtained from Land 0' Lakes, Inc., Minneapolis, Minnesota. Electrodialyzed whey powder was obtained from Foremost Foods, Inc., Appleton, Wisconsin. Promine D, a soy isolate, was obtained from Cen- tral Soya, Inc., Chicago, Illinois. Preparation of Sam31es In order to properly assess the functional value of the blends, dispersions were prepared at protein levels of 3.2, 5.0 and 8.0% (w/w) in ratios of 0:100, 25:75, 50:50, 75:25 and 100:0 (milk product to soya isolate). The blends 72 73 were prepared in distilled water and mixed for 10 minutes at 2500 rpm by a Fisher variable Speed mixer. The disper- sions were subjected to the following treatments: 1. control 9. addition of 0.1% NaZHPO4 2. heating 68°C/30 min 10. addition of 0.2% “ 3. heating 770C/no hold 11. addition of 0.3% ”. 4. heating 77°C/30 min 12. addition of 0.1% Na3C1tYat€ 5. heating 94°C/4 sec 13. addition of 0.2% " 6. heating 121°C/4 sec 14. addition of 0.3% " 7. adjustment to pH 8.5 15. addition of 0.1M CaClZ 8. adjustment to pH 4.5 16. homogenization 2250 psi Six tests were chosen as indicators of the functional value of the samples. These were emulsion capacity, whip- ping ability, viscosity, solubility, solubility index and sensory evaluation. After the first series of functional tests was completed and evaluated an additional set was designed to further improve functionality. Eleven protein systems were chosen as representative of the samples studied in part I. A protein concentration of 3.2% was used in these samples. The blends included: NFDM-100, NFDM 50:50 soy isolate, electrodialyzed whey-100, elec- trodialyzed whey 75:25 soy isolate, WPC-100, WPC-75:25 soy isolate and soy isolate-100. These samples were subjected to the following conditions: .1M CaClZ with 0.1% Na3Citrate .1M CaClz with 0.1% NaZHP04 .1M CaC12 with 0.1% EDTA .1M CaC12 and heating 680C/30 min .1M CaC12 and 0.1M EDTA .1M CaC12 and 1.0% EDTA addition of 0.1% EDTA addition of 0.1% NaZHP04 and heating 68°C/30 min addition of 0.1% Na3Citrate and heating 68°C/30 min adjusting the pH to 8.5 and heating 68 C/30 min OKOCDNmm-DWN—J OOOOOO .——.l 74 11 addition of 12. addition of 13. addition of 14 addition of catalase 15 addition of 16. addition of 0. % sodium hexametaphosphate (SHMP) l7. addition of 123 ppm sodium dodecyl sulfate (SDS) 18 addition of succinic anhydride one 9 per g protein 19. addition of maleic anhydride one 9 per g protein 20. addition of 5% sucrose 21. addition of 5% sodium chloride 22. addition of 0.1% carboxymethylcellulose (CMC) 23. addition of 0.1% CMC and heating 68°C/30 min 24. addition of 0.5% CMC 25. addition of 0.2% monoglycerides 26. addition of glycan and homogenization at 2250 psi .06% H202 and catalase treatment .06% H202-680C/30 min and catalase .01% L cysteine .01% L cysteine—0.06% H202 and 0 0000 .01% mercaptoethanol The final experimental treatment consisted of an enzy- matic hydrolysis with a protease (prolase). The enzyme was added to the samples which were then adjusted to pH 7.5 and incubated for one hour at 50°C. Aliquots of material were removed, heated to 770C and tested for functionality. After 3 hr of incubation, additional aliquots were removed and retested. One additional test, gelation, was assessed for spe- cific samples under selected conditions. Gelation studies were made on aqueous dispersions of 10% protein in the same ratios as used for other functional tests. The variants included: 1) no treatment, 2) pH 8.5, 3) 0.1% Na2HP04, 4) 0.1% Na3Citrate, 5) 0.1M CaC12, 6) pH 4.5 and 7) pH activated (Fleming, 1974). The experimental techniques which were used for each functional test are described in the following section. 75 Whipping The whipping ability of the samples were obtained by taking a quantity of the protein sol which upon whipping did not result in clogging of the blades. For blends com— posed of 3.2, 5.0 and 8.0% protein this amounted to 75, 50 and 40 ml, respectively. The mixtures were whipped at speed 8 in a Kitchen Aid Model 3-C mixer, equipped with a wire whip. A whipping time of 6 min proved optimal. Specific volumes (ml/g) and % stability times (min) were determined. After whipping was completed, the material was transferred to a tared beaker of known volume and reweighed to determine specific volume. To determine % stability the beaker was covered with g in stainless steel mesh screen and inverted over a funnel which collected liquid draining from the foam. The time required for col- lection of liquid equal to 8 of the weight of the original foam was recorded. Stability times of less than 5 min were reported as zero. Emulsion Capacity Emulsion capacity was determined by a procedure Similar to that of Webb 31 31. (1970). An aliquot of the protein sol, equivalent to 10 mg of protein was taken from each sample. This was added to 100 ml of 1.0M NaCl solution in a 600 m1 beaker at room temperature. This mixture was then weighed. The beaker was covered with a rubber stopper 76 fitted with holes for a propeller, two electrodes and a delivery tube. The propeller (resting in the sample) was attached to a Talboy 1 line variable speed, laboratory stirrer. Refined corn oil was added from a 500 ml separa- tory funnel and delivered via tubing into the beaker at a constant rate of 0.1 ml/sec. Stirring was maintained at 3000 rpm during delivery of the oil until the emulsion inverted. The inversion point was monitored with a volt- meter which was set to record electrical resistance. After the break point was reached, the beaker was detached from the apparatus and reweighed to determine the amount of corn oil added. The emulsifying capacity was calculated as the g of oil required to reach an infinite electrical resistance minus a blank (100 ml of a 1.0M NaCl solution) divided by the amount of protein. Soluble Protein The percent soluble protein in each sample was deter- mined by filtering several ml through Whatman no. 42 filter paper. An appropriate portion of filtrate was removed for analysis by a Lowry protein assay (Lowry _1 _1., 1951). Bovine serum albumin dissolved in distilled water was used to prepare the standard curve. Absorbance was read at 540 nanometers on a Beckman DKU Spectrophotometer. 77 Viscosity Viscosity was measured at room temperature with a Brookfield RVF rotary viscometer. The rotor speed was kept constant at 50 rpm using spindle no. 4 (except in those circumstances where the viscosity exceeded the capa- city of the spindle). Measurement was made in a 400 m1 beaker filled with the appropriate amount of sample. Values were recorded in centipoise. Solubility Index The solubility index was determined according to a procedure from the American Dry Milk Institute, Inc. (1954). Conical graduated centrifuge tubes were filled to the 50 m1 mark and centrifuged for 5 min at 1000 rpm. Immediately after centrifugation, the supernatant liquid was poured off leaving approximately 5 m1 above the sediment. Twenty five ml of distilled water were added to the tubes and the sediment dispersed. The tubes were then filled to the 50 ml mark and centrifuged again. After 5 min the tubes were removed from the centrifuge and the m1 of sediment in the tube reported to the nearest graduation. Gelation In evaluating the gelation capacity of the blends, the procedure followed was that of Fleming 31 31. (1975). Aqueous dispersions of 1 % protein were prepared by mixing at 2500 rpm for 10 min. Gelation experiments were conducted 78 by heating the slurries in sealed glass containers to 90°C for 45 min in a water bath. After heating, the containers were quickly cooled in an ice water bath to 25°C. The firmness of the gel was measured with a plummet dropped from a height of ten inches. With this device a score of 10 indicated firmness while 1 indicated softness. Sensory Subjective Analysis In this study subjective analysis was carried out on the foam produced during whipping of the samples. A total of thirty points was assigned, 10 each for color, flavor and texture. High scores indicated greater acceptability while samples with less than 6 % of the total were unsatis- factory. Examination for Heat Induced Interactions A set of experiments was designed to examine for heat induced interactions between the water soluble protein of soy isolate and milk proteins. The milk proteins were prepared from sweet whey, acid whey, ammonium sulfate precipitated casein and sodium caseinate. Samples were prepared and heated separately and in a l to l combination with soy for each of the milk protein materials. Five heat treatments were used: no treatment, 68°C/30 min, 770C/ 20 sec, 940C710 sec and 121°C/5 sec. After treatment, the sols were centrifuged at 25,000 x g for 30 minutes and filtered through Whatman no. 42 filter 79 paper. Approximately 8 ml of filtrate were pumped upward through a Pharmacia column packed with sephacryl S-200 superfine. After resolution of the proteins, the fractions were collected and polyacrylamide disc gel electrophoresis (PAGE) and nitrogen analysis performed on each. Proteins that eluted in the void volume of the sephacryl column were concentrated 10 to l and reapplied over a column packed with sepharose 4B. The analyses previously des- cribed were repeated. Preparation of Products Soy Protein Soy soluble protein was prepared from soy isolate according to a modified technique from Soybeans: Chemistry and Technology. The soy isolate was suspended in distilled water at a protein concentration of 3.2% (w/w). The dis- persion was stirred at 2500 rpm for 15 min followed by centrifugation at 1500 rpm for 10 min. After centrifuga- tion the supernatant was filtered through Whatman no. 42 filter paper. The filtrate was dialyzed for 48 hours at 4°C followed by freeze drying at 7 u absolute pressure. The freeze-dried material was stored at -20°C. Whole Casein Colloidal casein was prepared according to the method described by McKenzie, 1972. Ammonium sulfate (26.4 g) was added with stirring to 100 m1 of Skimmilk over a period 80 of 40 min at 2°C. After the ammonium sulfate was added the milk was stirred for an additional 90 min. The precipitate was collected after centrifugation at 14,600 x g for 35 min. The precipitate was redissolved in distilled water making certain the total volume did not exceed 90 ml. With mechanical agitation and stirring this was accomplished in approximately 50 min. The volume was increased to 100 ml, holding the temperature at 3°C. The casein was reprecipi- tated with 24.0 g ammonium sulfate and stirred for 90 min after addition of the salt. The casein was separated by centrifugation as before and redissolved in water to a final volume of 60 ml. This was dialyzed against distilled water for 48 hr at 4°C and freeze-dried. The material was stored at ~20°C. Sodium Caseinate Whole, raw milk was obtained from the Michigan State dairy farm. The cream was removed by centrifugal separa- tion at 30°C. The casein was precipitated by addition of 1N HCl to a pH of 4.6. The casein was collected with cheese cloth, washed with water, and redissolved in water by adjusting the pH to 7.0 with 1N NaOH. This procedure was repeated 3 times. The final solution was dialyzed against distilled water for 48 hr at 4°C and freeze-dried. The dried material was stored at -20°C. 81 Whey Proteins from Acid Whey After precipitation of the casein from Skimmilk by addition of 1N HCl the whey was gathered and centrifuged at 2000 rpm for 20 min. This was followed by filtration through Whatman no. 42 filter paper. The filtrate was dialyzed for 96 hr at 40C and freeze-dried. The whey pro- teins were stored at -20°C. Whey Proteins from Sweet Whey Whole raw milk was obtained from the MSU dairy farm. The milk was warmed to 30°C and incubated with 1% Cheese starter. After the acidity had risen 0.03% rennet was added and the milk allowed to coagulate. In approximately 45 min the curd was cut. After cooking to 39°C (following a 15 min resting period), the whey was drained and the casein removed by filtration through cheese cloth. The whey was centrifuged at 2000 rpm for 20 min and filtered throuth Whatman no. 42 filter paper. The filtrate was dialyzed for 96 hr at 4°C and freeze—dried. The whey pro- teins were stored at -20°C. Gel Filtration A 2.5 cm x 100 cm Pharmacia column was packed with sephacryl S-200 superfine. Sephacryl S-200 superfine (Phar- macia, Inc.) is a gel filtration media which can withstand high flow rates. The exclusion limit is approximately 200,000 daltons. This material purchased as a slurry was 82 washed with phosphate buffer, defined 3 times and degassed for five hours. After degassing was completed, the material was slurried into the column and packed in a downward flow at 200 ml/hr using a Pharmacia peristalic 3-P pump. After several volumes of buffer had passed through the material, the flow was reversed and the procedure repeated. For sample separation, flow was in the upward direction at a rate Of 125 ml/hr. An Isco Model UA-2 ultraviolet analyzer and recorder were employed to monitor the proteins at 280 nm. An Isco Model 326 fraction collector, equipped with automatic actuator was used to Simplify collection of the fractions. Approximately 70 mg of protein was applied to the column and eluted with phosphate buffer, pH 7.0, u = 0.1. Mercaptoethanol and human serum were used to determine total volume and void volume, respectively. Proteins that eluted in the void volume were concentrated approximately 10 to 1 with a millipore immersible separator. Approxi— mately 4 ml of this material were pumped upward through a 2.5 cm x 45 cm Pharmacia column packed with sepharose 4B (Pharmacid, Inc.). Sepharose 48, a gel filtration material with an effective range of 100,000-10,000,000 daltons was prepared and packed at 60 ml/hr. The proteins were eluted from the column using upward flow at a rate of 50 ml/hr. Phosphate buffer was used as the mobile phase. The UV analyzer and fraction collector used were those previously described. Mercaptoethanol and Blue dextran G—2000 were 83 used to determine total volume and void volume, respec- tively. Polyacrylamide Gel Electrophoresis A11 electrophoretic analyses were made using a Bio-Rad model 400 power supply and model 150 gel electrophoresis cell. Disc gel electrophoresis was carried out by modifi- cation of Melachouris (1969) procedure. For systems con- taining whey, soy and whey-soy blends the procedure consis- ted of preparing gel buffer pH 8.9 (see appendix) and adding to it the proper amount of cyanogum to give either 5.0 or 7.0 percent gels. Five percent gels were run on all fractions eluted from the sepharose 48 column while 7.0 percent gels were run on those eluted from the seph- acryl S-200 column. To initiate polymerization 0.05 ml of a 5 percent ammonium persulfate solution was added to the mixture. Temed (N,N,N,N-tetramethylethylenediamine) was added (10 pl/ml of gel solution) to accelerate polymeri- zation. The gel solution was deposited in glass tubes to a depth of 6.5 to 7.0 cm. Water was layered on top to prevent a meniscus and to prevent inhibition of polymeri- zation. The electrode buffer, pH 8.3 was composed of trisglycine (see appendix). Reagent grade sucrose was added to the samples to increase the density. One drop of Bromophenol Blue (1% in phosphate buffer) was added as the marker dye. After polymerization was completed and the 84 tubes assembled in the cell, sample was layered onto the top Of the gel. It was found that satisfactory results could be obtained without switching to tris-HCl buffer, pH 6.7, from the phosphate buffer, pH 7.0 (Aldrich, 1977). Constant current was applied to the gels at the rate of 2.5 mA/tube for approximately 60 min. The gels were stained according to the Malik-Berrie (1974) staining pro— cedure (see appendix) using Coomassie Brilliant Blue R-250. Samples containing either casein, soy or casein—soy blends were examined in a system containing 7M urea. The gel buffer, tris—HCl pH 8.9 was made to contain 7M urea. Five percent and seven percent gels were prepared for the purpose previously discussed. All other conditions and buffers remained as previously mentioned. Gel Scanning Casein, soy and casein-soy gels from no treatment and 94°C/5 sec samples were analyzed densitometrically. The gels were scanned at 2 cm/min by a Gilford gel scanner attached to a Beckman DU Spectrophotometer at 550 nm. The bands were monitored by a Hewlett-Packard automatic inte- grator, model 33808. Chart Speed was set at 2 cm/min, with slope sensitivity at 0.3 mV/min and an attentuation of 32. 85 Nonprotein Nitrogen Casein, whey, soy and their blends were separated by gel filtration into many fractions. Nonprotein nitrogen (NPN) was determined on the fraction eluting closest to the experimental total volume (determined with 2 mercap- toethanol). NPN was determined by taking 10 m1 of the fraction and mixing with it an equal amount of 2 % tri- chloroacetic acid (TCA). Blank determinations were made by adding 10 ml of TCA (2 % w/w) to phosphate buffer. After standing 30 min the S01 was filtered through Whatman no. 42 filter paper. Five ml of the filtrate was then examined for nitrogen content by a semi—micro Kjeldahl procedure. In Vitro Enzymatic Hydrolysis Samples of NFDM, soy isolate and their blend were prepared, treated, dialyzed and freeze—dried. The purpose of which was to examine release of total amino nitrogen from intact proteins as affected by various processing treatments and possible interactions between protein systems. The soy isolate used in this study was identical to that utilized in the functional tests. Nonfat dry milk was chosen as the milk protein product over sodium caseinate and the whey products because of its extensive use in the food industry and because it contains both protein systems found in the other products. The procedure used to assess 86 release of o amino nitrogen was the enzymatic approach as modified by Dimmler (1974). With this method both pepsin, pH optimum 1.8 and pancreatin, pH optimum 7-8 were added to the digestion mixture. Preparation of Samples All samples were made up to 500 g in distilled water with a protein concentration of 3.2% (w/w). Samples were divided into three groups, which included: 1) samples made entirely from soy isolate, 2) samples made entirely from NFDM and 3) samples made from a l to 1 combination of each. The mixtures were subjected to 24 different treatments which totaled 72 separate samples. The treatments were designed to Simulate various processes, which included: 1) no treatment (control), 2) heated at 68°C/30 min, 4) heated at 94OC/4 sec, 5) heated at 121°C/4 sec, 6) heated at 1130C/15 min, 7) heated (microwave) 77°C/no hold, 8) sunlight oxidized 2 hr, 9) pH adjusted to 8.5 and heated at 68°C/30 min, 10) pH adjusted to 11.5, 11) addition of 0. % Na3Citrate and heated at 68°C/30 min, 14) addition of 5.0% NaCl and heated at 68°C/30 min, 15) addition of 0.1% CMC and heated at 6800/30 min, 15) addition of 0.1M CaCl 2 and heated at 680C/30 min, 17) addition of 0.05% H202, followed by catalase treatment and heating at 68°C/30 min, 18) homogenization at 2000 psi and heated 68°C/30 min, 19) addition of 3.5% glycan (yeast cell wall polysaccharide), 87 homogenized at 2000 psi and heated at 68°C/30 min, 20) addition of 3.5% partially hydrogenated soybean oil, homogenized at 2000 psi and heated at 68°C/30 min, 21) addition of 0.01% L cysteine, followed by 0.0 % H202 and catalase, 22) addition of maleic anhydride at one gram per gram protein, 23) addition of succinic anhydride at one gram per gram protein, 24) protein mixed with safflower oil and held at 450C for four days to allow oxidation to occur. After preparation the samples were dialyzed for 72 hours, freeze-dried and stored at -20°C. Hydrolysis Procedure Samples for enzymatic hydrolysis were prepared by dissolving 300 mg of the freeze-dried material in 25 ml of deionized distilled water. The pH of the mixtures was lowered to 1.8 with 1N HCl. Additional water was added, which increased the volume to 30 ml, representing a sample concentration of 1.0%. Twenty-five mg of pepsin was added to the solution. After swirling, the flask was placed in a 37°C oven for 20 hours of incubation. Peptic digestion was terminated by the addition of 0.5M NaHCO3 buffer, and 1.0N NaOH to a final pH of 8.0. A final incremental addi- tion of water brought the total volume to 35.0 ml. Thirty mg of pancreatin was dissolved into the reaction mixture. The samples were reincubated at 37°C for 3 hours followed by a second addition of 30 mg of pancreatin and an 88 additional 3.5 hours of incubation at 37°C. After incuba- tion was completed, a 30% TCA solution was added to the reaction mixtures to a final concentration of 15%. Enzyme blanks were treated identically except for the absence of the Specimen protein. Protein blanks were treated identi- cally except for the absence of the enzymes. After addi- tion of the TCA, the samples were centrifuged and filtered to yield a crystal clear supernatant. When the proper dilution was made the samples were assayed for total alpha amino nitrogen by the ninhydrin test (Clark, 1964). The assay consisted of adding ninhydrin solution (see appendix) to the amino acid mixture and boiling in a water bath for 20 minutes. After cooling, 8 ml of 50 percent aqueous n-propanol were added. After ten minutes, absorbances were read at 570 nm against a blank. Glycine was used to pre- pare the standard curve. Acid hydrolysis (see appendix) was used to correlate percent enzymatic hydrolysis with total hydrolysis on the control samples. Protein Determinations Lowry Procedure for Soluble Protein Percent soluble protein was determined by the method of Lowry _1 _1. (1951) with one modification. Reagent C was prepared daily from a mixture of 50 parts reagent A and 1 part reagent 8 (see appendix). A standard curve, ranging in protein concentration from 50-500 ug/ml was prepared 89 from crystallized bovine serum albumin (BSA). The protein content of BSA was determined by Kjeldahl analysis. Absor- bance was measured at 540 nm. Lowry Procedure for Insoluble Protein Percent total protein in the freeze-dried samples used in the 13 31133 enzymatic hydrolyses was determined accor- ding to the method Of Lowry 31 31. (1951). Reagent E was prepared daily from a mixture of 50 parts reagent 0 and 1 part reagent B (see appendix). A standard curve was pre- pared with BSA and absorbance read at 540 nm. flieldahl Analysis Total nitrogen was determined using a semimicro Kjel- dahl method (Swaisgood, 1963). Samples containing approxi- mately 15 mg of nitrogen were digested by adding 5 ml of digestion mixture (see appendix) and heating for 90 min. After the initial digestion, the samples were cooled and 2 ml of 30% H202 added. Digestion was continued for an additional 60 min. Following heating the flasks were cooled and rinsed down with 10 ml of water. The flasks were then connected to a steam distillation apparatus. The mixture was made alkaline by addition of approximately 25 m1 of 4 % NaOH. The released ammonia was steam distilled into 15 ml of 4% boric acid containing 4 drops of indicator (see appendix). The ammonium borate complex was titrated with 0.02 N HCl to an olive green endpoint. Factors of 90 6.25 and 6.38 were used to convert nitrogen to protein for samples containing soybean and milk protein respectively. Recovery of nitrogen was checked with DL tryptophan. RESULTS AND DISCUSSION Investig31ion of Functional Properties The results and discussion pertaining to examination of functional properties will be presented in the same manner as outlined in the experimental section pages 68. Each property will be discussed relevant to the treatments described in Functionality Part I. Immediately following, the results of Functionality Part II (pertinent to the second set of treatments) will be presented. In Part I, dispersions were prepared at protein levels of 3.2, 5.0 and 8.0% (w/w) in ratios of 0:100, 25:75, 50:50, 75:25 and 100:0 milk product to soy isolate. The products included: nonfat dry milk (NFDM), sodium caseinate (NAC), electrodialyzed whey (EW), sweet whey powder (SW), whey protein concentrate (WPC) and soy isolate (SI). In Part II eleven protein systems were chosen as representative of the samples studied in Part I. A protein concentration of 3.2% was employed. The samples included: NFDM, NFDM 50:50 SI (NFDM/50), NAC; NAC 50:50 SI (NAC/50), EW, EW 75:25 SI (EM/75), WPC, WPC 75:25 SI (WPC/75), SW, SW 50:50 SI (SW/50) and SI. The soy isolate used in this study was Edi-ProN from Ralston Purina. This isolate was chosen because of ease of 91 92 dispersion, protein solubility (Table l) and flavor. Solubility The solubility of a protein is affected by pH, tempera- ture, ionic strength, compounds which change their dielec- tric constant and by interaction with other substances. Solubility is an important physical parameter to measure because of its substantial effect on the many other func- tional properties of proteins. Measurement of solubility provides the researcher with important information concer- ning the effect of various processing treatments on proteins. Data pertaining to solubility are shown in Tables 2-9. SI dispersions had the lowest solubilities of any of the products tested. The solubility of untreated samples ranged from 57.8 to 62.5% for dispersions containing 3.2 and 8.0% protein respectively. Shen (1976) reported that the solu- bility of this 'solate was 54.0% and did not vary appreciably as the protein concentration changed. Heating at moderate temperatures slightly increased solubilities except for those samples subjected to Ultra High Temperature (UHT) heating. Solubilities decreased Significantly at 121°C/ 4 sec. Shen (1976) and Hermannson and Akesson (1975a) both reported that low temperature heating slightly improved solubilities probably due to increased hydration or disso- ciation of the protein into subunits. Aldrich (1977), Mann and Briggs (1955), Catsimpoolas t 1. (1969), Wolf (1970) and Catsimpoolas 31 a1. (1971) found that the amount of 93 protein insolubilized increased with increased heat treat- ment. Loss of solubility was due to aggregation of the proteins. Adjustments of the pH of SI sols to 8.5 increased their solubility. Wolf (1961), Wolf (1970), Fukushima and Van Buren (1970), Franzen and Kinsella (1976) and Amilara 31 31. (1977) all reported that alkaline pH treatment improved the solubility of soy protein. AS the pH of the sols was raised into the alkaline region the net negative charge of the proteins increased thus increasing their relative attrac- tion toward water molecules. This increased the hydration of the water molecules and hence their solubility. Wolf (1970) suggested that alkaline pH treatment of soy protein involved dissociation through rupture of weak secondary forces such as hydrogen bonds and van der Waals forces. This in turn lead to electrostatic repulsion between posi- tively and negatively charged subunits. Lowering the pH of soy sols to 4.5 practically eliminated their solubility. Paulsen and Horan (1965), McWatters and Cherry (1977) and Hutton and Campbell (1977a) found that the solubility of soy protein was minimal near its isoelectric point. At pH 4.5 (the isoelectric point of soy protein) there was maxi- mum attraction of the positive and negative forces of adjacent protein molecules which resulted in precipitation and loss of protein solubility._ Addition of CaClZ to the SI sols also markedly reduced their solubilities. Paulsen and Horan (1955), Aldrich (1977), Mann and Briggs (1950) 94 and Smith and Circle (1972) reported that divalent cations such as calcium significantly reduce the solubility of soy protein. Heavy metal ions such as calcium, probably func- tion to precipitate proteins by neutralization of charges or by forming crosslinkages with the protein molecules (Whitaker, 1972). The addition of either NaZHP04 or NaBCitrate Slightly increased the solubilities of SI sam- ples, especially at the higher salt levels. This may have been due to a salting in effect. Homogenization increased the solubility of SI dispersions at all three protein con- centrations. Amilari _1 31. (1977) also reported that homogenization increased the solubility of soy proteins. NFDM had the lowest solubility of any milk product at 3.2% protein. Heating increased solubilities to a small extent. Raising the pH of the samples to 8.5 elevated solubilities while lowering the pH to 4.5 markedly reduced this property due to isoelectric precipitation of the casein. Addition of CaC12 had little effect upon the solu- bility of NFDM dispersions probably because of the influence of whey proteins and the ionic environment of NFDM sols. The addition of NaZHPO4 or Na3Citrate, at low concentrations, increased the solubility of NFDM dispersions probably by increasing the net charge of the protein and thus their hydration. Homogenization increased the solubility of these samples. Untreated NAC redispersed in distilled water had high solubility at the protein concentrations studied. Solubilities were close to those found for EW and WPC. 95 Table l. Dispersible protein (%) of several soy isolates at 4.0% protein in water Soy Isolate pH 3.0 7.0 9.0 Edi-ProN1 86.9 88.7 92.1 Supro 6101 24.1 23.6 29.5 Promine D2 49.1 55.9 58.6 Promine F2 39.1 41.3 51.8 Pro-FAM 90 H53 27.9 73.6 74.1 Pro-FAM 90 LS3 6.8 14.2 19.3 Protein Max 904 3.1 11.1 11.7 1Edi-ProN and Supro 610 are products of Ralston Purina. 2Primine D and F are products of Central Soya, Inc. 3Pro-FAM 90 HS and LS are products of Grain Processing Corp. 4Protein Max 90 is a product of Worthington Foods. 96 Heat treatment and adjustment of the pH to 8.5 had little effect upon solubility of NAC sols. Lowering the pH to the isoelectric region of casein precipitated nearly 100% of the protein. Addition of CaCl2 also reduced the solu— bility though not to the same extent as found for SI dis- persions. The major casein components bind calcium exten- sively at the normal pH of milk. The instability of caseinate sols at high calcium concentrations has been attributed either to their zeta potential or to cross- links established by the divalent cation (El-Negoumy, 1971). Sweet whey powder (SW) had the lowest solubilities of any of the whey products, while EW had the highest. The solubility of WPC was slightly less than that recorded for EW. Marvopoulous and Kosikowski (1973) reported that the solubility of whey powders ranged from 91.4 to 99. %. The solubilities of EW were 92.2, 91.1 and 93.6% at pH 4, 6 and 8 (Morr 31 31., 1973). The solubilities of WPC at the same pH values were 89.5, 90.2 and 91.2. Heating generally decreased the solubility of the whey products, especially 31 higher temperatures. WPC was the most vulnerable to heat 3enaturation. Howat and Wright (1933), Dill and Roberts (1964), Guy 31 _1. (1967) and Morr (1969) reported that the denaturation of whey protein increased dramatically as heating temperature increased. McDonough _1 31. (1974) found that pasteurization of WPC solutions decreased their solu- bilities and Hidalgo and Gamper (1977) reported that the solubility of WPC sols decreased rapidly when they were 97 heated from 80-134°C. Adjustment of the pH to either 8.5 or 4.5 had little effect upon the solubilities of WPC or EW sols. This concurred with data reported by Morr 31 31. (1973). The solubility of SW samples increased Slightly when the pH was raised and decreased to a small extent when the pH was lowered. The solubility of SW also decreased when dispersed in 3311 Addition of Na HPO orhNawCitrate 2‘ 2 4 3 had little effect upon the solubilities of any of the whey products. At the protein concentrations studied the solu— bilities of the different whey products remained similar. NAC:SI blends had solubilities (Table 3) intermediate between SI and NAC. Samples containing more SI had solu- bilities very similar to SI, while blends containing more NAC had solubilities close to those of NAC. Hoffman (1974) reported that the solubility of caseinzsoy blends decreased as soy protein displaced casein. At 5.0 and 8.0% protein solubilities differed only Slightly compared to samples containing 3. % protein. Heating had little effect upon the solubilities of these samples. Raising the pH to 8.5 Slightly increased, while lowering the pH to 4.5 drasti- cally decreased their solubilities. Adjustment of the samples to 0.1M CaCl decreased solubilities markedly. The 2 addition of stabilizing salts or homogenization only slightly affected solubilities. NFDM:SI blends had lower solubilities (Table 4) than NAC SI blends. Solubility increased as the amount of NFDM in these blends increased. As protein concen- tration rose, solubilities decreased. Heating had little 98 effect upon the solubility of these blends, while alkaline treatment at pH 8.5 resulted in higher values. Lowering the pH to 4.5 or addition of CaC12 to the samples substantially decreased their solubility. EM and WPC:SI blends had higher solubilities (Tables 5 and 6) than SWzSI (Table 7) blends. Solubilities increased as the proportion of whey product in the blend increased. The amount of soluble protein remained approximately the same at all three protein concentrations. Heating in general, decreased the solubility of the samples, especially for those blends having ratios composed of 75% whey product. Raising the pH of the blends to 8.5, generally increased their solu- bilities, particularly for samples containing 75% SI. Lowering the pH to 4.5 decreased the solubility of the blends especially for samples containing 75% SI. Addition of CaCl2 also reduced solubilities in much the same manner. These same effects were seen regardless of the whey product. Addition of stabilizer salts did not appreciably change their solubilities while homogenization was responsible for slight improvement. The solubilities of the samples exposed to the treat- ments from functionality Part II are shown in Tables 8 and 9. The addition of ethylenediamine tetracetic acid (EDTA). Na HP04or Na 2 3 the drastic loss of solubility noted for many of the samples. Citrate to samples containing CaCl2 prevented In general, solubilities were slightly lower than the 99 controls. Addition of EDTA increased the solubility of NFDM and NFDM/50 dispersions. Aoki and Imamura (1976) and Morr and Josephson (1968) found that EDTA increased the solubility of caseinate sols by removal Of calcium from the micelle. This 16ad to disaggregation of the micelle which increased the amount of soluble casein. El-Negoumy (1974), Sommer (1952) and Morr (1975) reported that EDTA and citrate complexed calcium and thus prevented itscmstabifizing effect. -.-~. -~— - r .-- The solubility of the whey products was reduced by heating samples dispersed in 0.1M CaClZ. The solubility of the blends was also decreased. Richert (1975), Monica 31 31. (1958) and Townsend and Gyuricsek (1974) repOrted that heating whey proteins in the presence of CaCl2 caused Sub- SEantial precipitation of the protein. This was due to the cembinationeffect of heat-salt denaturation. The solubili- ties of NFDM, NAC, NFDM/50, NAC/50 and'SI were all markedly reduced by this treatment. The addition of either NaZHPO4 or Na3Citrate prior to heating at 68°C/30 min had minimal effect upon the solubili- ties of the whey products or their SI blends. The solubility of NFDM, SI and their blend was increased by this treatment. Sommer (1952) reported that heating calcium caseinate sols in the presence of Na3Citrate or NazHPO4 increased their solubilities probably by substituting sodium for calcium. Sodium caseinate was more soluble than calcium caseinate. Raising the pH to 8.5 prior to heating at 68°C/30 min had negligible effect upon the solubilities of the whey 100 products or their SI blends. This treatment improved the solubility of NFDM and SI. Addition of H202 (followed by catalase) increased the solubility of NFDM and SI. Fish and Michelsen (1967) found that Skimmilk treated with catalase before heating caused a decrease in the denaturation of the whey proteins. Fukushima and Van Buren (1970) found that H202 increased the dispersi- bility of soy milk due to its effect upon disulfides. The addition of redUcing agents, such as L-cysteine or mercaptoethanol increased the solubilities of NFDM, NAC and S1. The whey products maintained their high solubility. Fukushima and Van Buren (1970), Wolf (1970), Wolf (1961), Nash and Wolf (1967), Kelley and Pressey (1967) and Amilari 31 31. (1977) reported that the addition of reducing agents to soy protein sols increased their solubilities. This was found to be due to the disruption of disulfide bonds which resulted in the depolymerization of soy protein. The addi- tion of SDS to the samples improved the solubilities of SI and its milk product blends to a small extent. Kelley and Pressey (1966), Catsimpoolas (1969), Wolf and Tamura (1969), and Koshiyama (1970) observed that detergents disrupted the quaternary structure of soy protein which increased their solubility. Addition of succinic or maleic anhydride to the samples increased the solubilities of NFDM, SI and their blend. The whey products maintained their high solubilities while the solubilities of their SI blends increased. Franzen and 101 Kinsella (1976) found that succinylation improved the solu- bility of soy sodium proteinate. Succinylation of a protein converts the cationic amino groups to anionic residues. The increase in net negative charge alters the physicochemical character of the protein resulting in enhanced aqueous solubility. The ammonium cations of lysine are replaced by succinate ions which due to their negative charge repel native carboxyl groups thus reducing protein—protein inter- action and increase protein-water interaction. The addition of NaCl to the samples reduced the solu- bility of NAC, SI and the whey products. Hermannson and Akeson (1975b) reported that NaCl markedly reduced the solu- bility of soy sodium proteinate. They ascribed the reduced solubility to changes in the quaternary structure of the protein. WPC became Slightly less soluble as the concentra- tion Of NaCl increased in the samples, though the solubility of MAC was not affected. Enzymatic hydrolysis markedly improved the solubility of both SI and its milk product blends. Digestion for three hours did not appreciably alter the results. Jost (1977) and Monti and Jost (1978) increased the solubility of proteins by enzymatic digestion. The remaining treatments had little affect upon the solubilities of the samples. In general, milk products demonstrated the highest degree of solubility, with milk productzsoy isolate blends having appreciably higher solubility than SI. Specific treatments resulted in widely varying solubilities. This 102 cowuechmmoEoz 3.33 3.33 3.33 3.33 3.33 N._3 N.N3 3.33 3.33 3.33 3.33 3.3N 3333333332 3_.3 3.33 3.33 3._3 3.33 3.33 3.33 3333332 3_.3 3.33 3.33 3.33 3.3 _.33 3.33 33333 z_.3 3.33 3.33 3.33 3.3 3.3 3.33 3.3 :3 3.33 3.33 3.33 3.33 3.33 _.3e 3.3 :3 3.33 3.33 3.33 _.33 3.33 3.33 .33\3333 3.33 3.33 3.33 3.33 3.33 3.33 3323333 33 33 333 33 332 2332 3333333 o_aacm xm.m co coeumcpcmocou cwmpoca m um 3m vcm 3m .uaz .Hm .u 3N.m mo cowpmgpcmucou :333033 3 pm Hm 3:3 om\2m .33 .33\333 .333 .33333 .33 .33\333 .333 .3333333 .3333 33 33333 3333333333 .3_ 3_333 112 The SBI of soy iso1ate were substantia11y higher than those of the mi1k products. Heat treatment 1owered the $81 due to the greater dispersibi1ity of the protein. Homogeni- zation marked1y reduced 881. This was a1so found by Guy gt _1. (1969) who used homogenization to reduce the 881 of soy-whey systems. As the amount of soy protein increased to 8.0% SBI increased a1most 1inear1y. Addition of stabi1i— zing sa1ts had 1itt1e effect upon these va1ues. B1ends of NFDM and SI had va1ues intermediate (Tab1e 10) between the unb1ended materia1s. As the amount of NFDM in the b1ends increased 881 decreased. So1ubi1ity index in- creased with increasing protein concentration. Processing treatments had approximate1y the same effect upon the b1ends as was indicated for the unb1ended materia1s. B1ends between NAC and SI had resu1ts very simi1ar to those recorded for NFDM:SI b1ends. Scores increased as protein concentration increased. B1ends having the greater percentage NAC had 1ower SBI. B1ends between WPC and SI had higher index va1ues than the unb1ended WPC. This was true for 811 whey proteinzsoy iso1ate b1ends. SBI decreased as the amount of whey product in the samp1es increased. Proportiona11y the va1ues were s1ight1y 1ower than the same b1end ratios of NFDM and NAC. This may have been due to the high so1ubi1ity maintained by the whey product soy iso1ate b1ends. SBI va1ues increased as percent protein rose. 113 Many of the processing variants emp1oyed in function- a1ity Part II, did not substantia11y affect the 881. How- ever, the treatments invoIving added CaC12 did appreciab1y affect SBI. These treatments genera11y resu1ted in higher measurements for a1most a11 samp1es. Many of the samp1es subjected to these treatments suffered substantia1 denatu- ration which resu1ted in measurab1e precipitation. In these instances so1ubi1ities decreased marked1y. Addition of 3.5% g1ycan to the dispersions resu1ted in very high measurements. This was because the g1ycan sett1ed out. After homogeniza- tion of these samp1es scores were 1owered to approximate1y their former va1ues. Very few of the remaining treatments had any significant affect upon the S81 va1ues of the sam- p1es. So1ubi1ity index is a usefu1 parameter for re1ating the amount of sedimentab1e materia1 to dispersibi1ity. SBI is affected by protein composition and processing treatments. In this study, certain treatments did affect SBI though va1ues obtained were more re1ated to the amount of SI in the samp1e. Viscosity Viscosity is re1ated to the degree of protein hydration which is affected by pH, ionic strength, temperature and by agents which affect the water of hydration. Viscosity is an important functiona1 property of many products in addition to its obvious effect upon processing equipment and techniques. 114 Viscosity measurements are presented in Tab1es 11-14. As protein concentration increased the viscosity (Tab1e 11) of SI 5015 rose. Va1ues more than doub1ed as the protein concentration went from 3.2 to 8.0%. Sosu1ski ”t a1. (1976), ~ Circ1e gt 1. (1964), F1eming gt 1. (1975) and Hermannson (1975) reported that increasing the soy protein concentration caused substantia11y higher viscosities. Hermannson (1975c) fe1t this increase was due to protein~protein interaction which 1ed to the formation of a protein network at e1evated protein 1eve1s. Heating the soy so1s further increased the viscosity measurements. Hermannson (1975), Circ1e gt gt. (1964), Hutton (1977a) and Bau gt _t, (1978) found that heating dramatica11y increased the viscosities of soy pro- tein, especia11y at temperatures of 900C or more. The in- creases noted were probab1y due to increased swe11ing of the proteins. Circ1e gt gt. (1964), Ke11ey and Pressey (1966), F1eming gt _t. (1975), Ehninger and Pratt (1974) and Hermann- son (1975c) observed that a1ka1i treatment of soy protein (pH 8-9) resu1ted in higher viscosities. This effect was confirmed in research reported in this thesis. The greater viscosities were probab1y due to the greater net charge and increased swe11ing of the proteins. Homogenization of the samp1es reduced viscosities s1ight1y whi1e addition of either NaZHPO4 or Na3Citrate had 1itt1e effect. Guy gt gt. (1969), who examined soy f1our-whey b1ends,a150 found that homogeni- zation reduced viscosity. 115 Viscosities of NFDM dispersions increased as protein concentration increased to 8.0%, though this increase was much 1ess than that observed with SI. Viscosities were aTSo s1ight1y higher as the pH of 5015 were adjusted to 8.5. The remaining treatments had 1itt1e effect upon the viscosity measurements of NFDM. At 3.2% protein NAC had viscosities approximate1y the same as other mi1k products. Substantia1 increases were noted as the protein concentration rose to 8.0%. The samp1es were s1ight1y syrupy at this protein 1eve1, with va1ues simi1ar to those recorded for SI. Her- mannson (1975c) reported that the viscosity of NAC was very concentration dependent with near1y 1ogarithmic increase over a broad protein range. This researcher fe1t that the increased viscosities were due to an increase in the number of so1vated caseinate partic1es. Adjustment of the pH to 8.5 resu1ted in higher measurements as was a1$o reported by Hermannson (1975c). The addition of stabi1izer sa1ts had 1itt1e effect. WPC 5015 had the 1owest viscosities (Tab1e 12) of any of the products tested. This was a1so observed by Hermann- son (1972) who reported that WPC had 1ower viscosities than either SI or NAC when examined over a broad protein concen- tration. Increasing the amount of protein in the samp1es had very 1itt1e effect upon viscosity measurements. At 8.0% protein, heating increased viscosities to a sma11 extent. None of the other treatments emp1oyed had appreciab1e effect upon the viscosities of HPC soIs. Hermannson (1975c) demonstrated that WPC dispersions maintained 10w viscosity unti1 a protein concentration of 18-20% was reached. Fac— tors such as pH and ionic strength had 1itt1e effect upon viscosities. Viscosity measurements of EN and SN increased tremendous1y as protein content was raised to 8.0%. This was because of the paste—1ike products which resu1ted due to the high tota1 so1ids 1eve15. At 3.2% protein SN had the highest viscosities of any of the products tested, whi1e EH had va1ues simi1ar to the other mi1k products. Addition of CaC12 substantia11y increased viscosities at 8.0% protein for both products, possib1y due to interaction between pro— tein and ca1cium ions. Heating a1so increased the viscosity measurements. NFDM:SI b1ends had viscosities on1y s1ight1y different from the unb1ended systems. At 3.2% protein, viscosities were very c1ose regard1ess of the b1end ratio. At 5.0% protein, viscosities rose s1ight1y, with no major differences found due to b1end ratio. At 8. % protein viscosities were higher for those samp1es containing the greater percent SI. The viscosities of samp1es composed primari1y of NFDM or SI had va1ues c10se to those recorded for the unb1ended protein systems. The viscosity of the b1ends was affected by the treatments emp1oyed in much the same manner as the individua1 products. The viscosities of NAC:SI b1ends rose as tota1 protein increased. At 3.2 and 5.0% protein s1ight differen- ces were noted due to b1end ratio. At 8.0% protein the samp1es containing the greater percent NAC had inght1y 117 higher viscosities, but were not substantia11y different than those measurements recorded for the individua1 proteins. Heating and pH adjustment increased the viscosity of the b1ends as previous1y reported for the unb1ended products. There were on1y sma11 differences in the viscosities of the various b1end ratios of WPC:SI. Viscosities rose inght1y as the percent protein increased. At 8.0% protein, b1ends containing 75% of the tota1 as SI had s1ight1y higher viscosities. HPC:SI b1ends had Iower viscosities than NAC:SI b1ends which was in agreement with Hoffman (1974). Hoffman (1974) 8150 found that as soy protein rep1aced whey protein in b1ends, viscosities rose s1ight1y. There were s1ight differences in the viscosities of EW:SI b1ends at 3.2 and 5.0% protein. At 8.0%, b1ends containing 75% EN had higher viscosities due to the high tota1 so1ids content. Addition of CaC12 further enhanced these higher va1ues. The viscosity of these b1ends (at a11 ratios) increased s1ight1y with increased protein. At the protein concentrations studied, SH:SI b1ends which contained 75% SW had higher viscosities. Viscosity rose with increased protein content due to the high tota1 so1ids. B1ending SI and SH 1owered the viscosities of the samp1es as compared to unb1ended SW systems. Addition of CaCI2 to the samp1es resu1ted in higher viscosities. The viscosities of the samp1es exposed to the treatments from Functiona1ity Part II are shown in Tab1es 13 and 14. Treatments which invoIved the uti1ization of CaC12 with either NaZHPO or Na Citrate resu1ted in higher viscosities 4 3 118 for SW and EH samp1es. The remaining samp1es had viscosities approximate1y the same. Addition of Cac12 and heating to 6800/30 min increased the viscosity of SW. The viscosity of SW a1so increased when either NaZHPO4 or NaBCitrate were added to the samp1es prior to heating at 6800/30 min. Addition of ma1eic anhydride to the samp1es increased the viscosities of NFDM, NFDM/50, NAC, NAC/50 and SI, though Franzen and Kinse11 (1976) reported that succiny1a- tion did not significant1y a1ter the viscosity of soy pro- teins. The viscosity of whey products was much 1ess affected by this treatment. Addition of 0.1% CMC increased the viscosities of a11 samp1es approximate1y by the same magnitude. Heating at 680C/30 min had no appreciab1e effect upon the measurements. Addition of 0.5% CMC to the samp1es marked1y increased the viscosities. The increase in viscosity caused by addition of CMC was due to the stabi1izing and thickening character- istics of this hydroco11oid. Lang _t _t. (1976) reported that addition of stabi1izer to skimmiIk increased its visco- sity. Addition of g1ycan was ineffectua1 because it sett1ed out. Homogenization fo11owing addition of g1ycan dispersed the materia1 and resu1ted in higher viscosities. This was probab1y due to the po1ysaccharide nature of this materia1. The addition of 5. % NaC1 was responsib1e for causing a s1ight decrease in the viscosity of SI. This a1so was found 119 by Hermannson (1975c) and Ehninger and Pratt (1974). They postu1ated that sa1t must have a genera1 effect on the struc- ture of soy proteins. Swe11ing and soTubiIity were reduced. The presence of 1ess swo11en, more rigid aggregates and 1ess so1vated protein mo1ecu1es cou1d 1ead to 1ower viscosities. NAC 5015 were s1ight1y more viscous in 5.0% NaC1 dispersions. Hermannson (1975c) fe1t that the effect of sa1t might be due to dehydration, resu1ting in changes in the repu1sive ba1ance or the mice11ar structure of the proteins. Addition of mercaptoethano1 or cysteine as reducing agents were reSponsi- b1e for s1ight viscosity reductions. Hermannson (1975c) and Ke11ey and Pressey (1966) a1so reported that reducing agents 1owered the viscosity of soy protein dispersions. This reduction was probab1y due to the breaking of disu1fide bonds resu1ting in some dissociation. Enzymatic hydro1ysis of SI 1owered its viscosity to a sma11 extent probab1y due to re1ease of peptides. The remaining treatments had 1itt1e or no affect upon the viscosities of the samp1es. The viscosity of protein dispersions is of prime impor- tance because 1) information is obtained re1ating to qua1ity contro1; 2) insight concerning changes in the mo1ecu1ar structure is provided; 3) data may be obtained regarding suitab1e fie1ds of app1ication for a new product; 4) infor- mation necessary for the optima1 design of unit processes may resu1t; and 5) information re1evant to mouthfee1 and hence acceptabi1ity characteristics may be obtained. In genera1, viscosities of mi1k products, soy iso1ates and mi1k 120 Tab1e 11. Viscosity (cp) for se1ected samp1es of NFDM, NFDM/50, NAC, NAC/50 and SI at 3.2, 5.0 and 8.0 percent protein Samp1e Variant _—HFDM NFDM/50 51 NAC/50 NAC 3.2% Contro1 10 10 9 10 11 680C/30' 10 10 9 11 12 pH 8.5 12 12 12 11 12 4‘ 5.0% ControI 12 12 11 12 15 680C/30' 12 12 12 11 14 pH 8.5 13 12 14 15 19 8.0% Contro1 14 17 19 . 19 28 6806/30' 14 19 25 22 26 pH 8.5 19 23 28 25 ' 35 *— 121 333 333.. 33 33 33 333 3333333 33.3 333.3 333.3 33 33 333 333.3 33333 33.3 33 333 33 33 333 333 3333333 33.3 3 3 . . 33 33 3 3 33 33 333 33 3_ 3 33 333 3 3 33 33 33333 33.3 33 33 3 3 33 33 3333333 33.3 3 33 . . 33 33 3 3 33 33 333 .3 33 3 33 33 3 3 33 33 33333 33.3 33 33 3 3 33 33 3333333 - 11 33.3 - - .1 om\3m 3m mm\um3 0&3 mm\3m 3m ucwwLm> 33333m 3333033 3233333 o.w 3:3 o.m .m.m 33 om\3m 3:3 3m .333333 .332 .m3\33 .23 30 3333533 33333333 303 333v 333303333 .m_ 33333 122 Tab1e 13. Viscosity (cp) for se1ected samp1es of NFDM, NFDM/50, NAC, NAC/50 and SI at a protein concen- tration of 3.2% Samp1e Variant NFDM NFDM/50 SI NAC/50 NAC Homogenization 9 1O 8 1O 11 pH 8.5-680C/30' 11 1o 10 11 11 Ma1eic anhydride 13 14 15 15 12 0.1% CMC 17 19 24 22 22 0.5% CMC 114 126 170 125 105 3.5% g1ycan-Homog. 189 241 258 237 230 0.1M CaC1 -1.0% EDTA 15 14 9 8 9 2 Enzyme hydro1ysis 10 8 7 8 8 123 Tab1e 14. Viscosity (cp) for se1ected samp1es of EN, EW/75, WPC, WPC/75, tion of 3.2% SN and SW/SO at a protein concentra- Samp1e variant Ew EN/75 ch WPC/75 sw sw/so 680C/3O' 12 11 8 8 23 1o 0.1M CaC12-O.1% Na Cit. 12 10 9 10 41 11 0.1M CaC12—680C/3O' 13 10 1o 9 28 1o Ma1eic anhydride 13 12 9 9 18 15 0.1% CMC 23 24 15 15 29 27 0.5% CMC 134 143 79 99 215 150 3.5% g1ycan-homog. 327 310 275 241 471 433 Enzyme hydro1ysis 1O 11 8 8 19 10 124 product:soy isoTate b1ends were quite c1ose regardTess of the b1end ratio, with the exception of the whey products at 8.0% protein. Viscosities increased moderate1y as the percent protein increased. Specific treatments resu1ted in higher (or 1ower) viscosity, but the majority had 1itt1e effect. Ge1ation Ge1ation is an important functiona1 property pertaining to many food products. The objective of this study was to eva1uate the geTation capabi1ity of soy iso1atezmi1k product b1ends. Edi-ProN, the soy isoTate uti1ized through out this research did not demonstrate any ge11ing abi1ity under the conditions used. Ya5umatsu at al. (1972) reported that dena- tured soy iso1ates were more amenab1e to ge1ation because partiaT denaturation was be1ieved to be conducive to prepa- ration of firm geTs. Soy iso1ates with a nitrogen soTubiTity index (NSI) of 50 had better ge1 formation qua1ities than one of 80. Edi-ProN with high nitrogen so1ubi1ity did not form firm geTS, therefore, a substitution was made. Promine D, a SI manufactured by CentraT Soya, Inc., was used in 811 experiments dea1ing with ge1ation. The data pertaining to ge1ation are presented in Tab1e 15. SI formed firm ge1s except when the dispersion was adjusted to pH 8.5 prior to heating. Under the inf1uence of this treatment the samp1e turned b1ack and remained f1uid. The decrease in ge1ation at this pH may have been caused by hydro1ytic action (Circ1e and Meyer, 1964). Circ1e and 125 Meyer (1964) formed soy geTS by heating 10% dispersions at 1000C for 45 min. These researchers described the ge1atin phenomenon as being primari1y dependent on heat denatura- tion. Ge1 rigidity was principaTTy due to protein concen— tration. Ge1s of denatured proteins invo1ve first an unfo1ding or extension of the g1obu1ar protein into a more unsymmetric shape, exposing reactive groups and nonpo1ar amino acids. Association of the chains by cross1inks and by 10ca1ized and non1oca1ized attractive forces 1ead to the formation of a three dimensionaT network. The irreversi— bi1ity of the soy ge1s indicated that primary cova1ent bonds were invo1ved in cross1inking. The addition of NaZHPO4, Na3Citrate or CaC12 increased 981 strength. Saio (1975b) reported that addition of ca1cium to soy increased strength of 9815 perhaps due to greater cross1inking. Hermannson and Akesson (1975a), Sosu1ski _t .l- (1976), Catsimpoo1as and Meyer (1970) and F1eming and Sosu1ski (1975) a11 demonstrated the ge1ation capabi1ity of soy protein. Untreated dispersions of NFDM demonstrated weak ge1 properties. Addition of CaC12 substantia11y increased ge1 strength. Ka1ab and Emmons (1972) reported that ca1cium promoted ge1ation. Firm ge1s were produced by heating NFDM at 1000c (Ka1ab _g___1_., 1971) though 50% dispersions were used. High tota1 so1ids content were necessary in the deve10pment of these ge1s. Sodium caseinate 5015 did not demonstrate any ge1ation abi1ity in this study. Hermannson 126 Tab1e 15. Ge1ation strength, measured with a P1ummett*, was determined for SI, NFDM, WPC, WPC/50 and EH/25 Variant samp19 Contro, 0.1% 0.1% pH pH ‘NaZHPO4 CaC12 8.5 _ activ. SI 4.5 7.0 6.8 NG 4.8 NFDM 2.0 NGa 7.5 NG NG WPC 10.0 10.0 8.8 10.0 9.0 WPC/50 6.5 10.0 7.5 9.0 9.0 Ew/ZS 7.0 7.5 6.2 8.0 7.1 *A score of 10 = very firm and a score of 1 = very soft. a; N8 = no ge1 formation. 127 and Akesson (1975a) a1so reported that NAC fai1ed to ge1 when heated at 1000C. Sweet and e1ectrodia1yzed whey dis- persions fai1ed to show any ge1 formation in this research. The remaining mi1k product WPC, did form very strong ge1s when subjected to the conditions used. The ge1s were firm, (a1most rubbery) trans1ucent and possessed good sensory qua1ities. McDonough gt al. (1974) prepared firm, resi1ent ge1s from 10% JPC so1utions which did not whey off. Mod1er and Emmons (1977) reported ge1ation of a WPC fo110wing heating at 950C for 20 min. Hermannson and Akesson (1975a) aTso showed that NPC formed firm ge1s when heated. Schmidt _3 _1. (1978), in examining the ge1ation characteristics of peanut/whey protein b1ends, observed that whey protein formed firm ge1s when heated. Very 1imited success was achieved when b1ends between SI and the mi1k products were examined for ge1ation abi1ity. B1ends containing 25% EN formed ge1s of moderate strength though wheying off occurred. HPC:SI b1ends demonstrated substantia1 ge11ing capabi1ity. A sma11 amount of wheying off did occur, with partia1 visua1 separation of the two ge1 systems. Of the mi1k products examined on1y WPC, in asso— ciation with or without SI, appeared to have substantia1 ge1ation characteristics. Emu15ion Capacity The abi1ity of a protein to emu1sify fat is an important functiona1 characteristic of that protein. This property is 128 extreme1y important in meat products, sa1ad dressings, spreads, etc.,vfimre ever emu1sification is required. Kin- se11a (1976) described emu1sification as a surfacant pro- perty reTated to the capacity of proteins to Tower the interfacia1 tensions between the hydrophobic and hydrophi1ic components in foods. Genera11y, surfactant properties are re1ated to the aqueous so1ubi1ity of proteins. Emu1sion capacities (EC) were determined for the samp1es at 3.2% protein. The resu1ts are shown in Tab1es 16 and 17. NFDM and WPC had the highest EC of the products tested. SH and SI had the 1owest. The apparent discrepancy between the EC of the whey products was probab1y due to compositiona1 and processing differences. Sodium caseinate had s1ight1y 1ess EC than NFDM. Kueh1er and Stine (1974) reported that WPC had greater EC than casein with NFDM having EC interme- diate between the two. Smith t 1. (1973) and Crenwe1dge “t _1. (1974) found that NFDM had more emu1sifying capacity than SI at neutra1 pH. NFDM SI b1ends which were composed of not more than 5 % NFDM had 1ower EC than NFDM. B1ends containing 75% NFDM had EC resemb1ing those found for NFDM. Sodium caseinate: soy iso1ate b1ends had 1ess EC than NAC except for those b1ends containing 75% NAC. Hoffman (1974) reported that the EC of casein~soy b1ends decreased as the amount of soy pro- tein increased. B1ends of EN:SI had a1most identica1 EC regard1ess of the b1end ratio. This a1$o was found to be true with SN:SI b1ends. B1ends of HPCzSI had 1ower EC than 129 WPC regard1ess of the b1end ratio, though EC increased as the percent WPC in the b1end increased. Hoffmann (1974) found that the abi1ity of WPC to emu1sify oi1 decreased as SI was substituted into the samp1e. Heating had on1y a s1ight effect on the EC of SI, NFDM, NAC and there b1ends. Emu1sion capacity increased to a smaT1 extent perhaps due to the s1ight1y higher so1ubi11ties. The effect of heat on the EC of the whey products was opposite to that observed for the other samp1es. As the heating tem- perature rose the EC decreased probab1y due to decreased so1ubi1ity. The EC of whey product:soy iso1ate b1ends de- creased, but 1ess so, when heated at the higher temperatures. Lowering the pH to 4.5 substantia11y decreased the EC of SI, NFDM, NAC and their b1ends. Crenwe1dge gt gt. (1974) and Hutton and Campbe11 (1977b) found that the EC of NFDM and SI were minima1 near their isoe1ectric points due to 1oss of so1ubi1ity. WPC and its SI b1ends a1so had 1ower EC. Hhey product:SI b1ends had 1ower EC proportiona1 to the amount of SI in the b1end. Raising the pH of the samp1es to 8.5 increased the EC of NFDM, NAC and SI. Ink1aar (1969) and Pearson gt _1. (1965) reported that soy protein, NFDM and sodium caseinate had greater EC at higher pH probab1y due to increased so1u- bi1ity. The whey products were much 1ess affected. Addition of CaCTZ to samp1es of SI, NAC and NAC:SI b1ends marked1y reduced their EC due to 1oss of so1ubi1ity. The EC of NFDM a1so decreased (though not near1y to the 130 same extent as for the previous samp1es). B1ends of NFDM:SI had decreased EC proportiona1 to the amount of SI in the samp1e. This treatment had 1itt1e effect upon the EC of the whey products but did decrease the EC of their SI b1ends. Homogenization of the samp1es resu1ted in on1y minor changes in EC. Addition of stabi1izing sa1ts did not appre- ciab1y a1ter the emu1sion capacities. The emu1sion capacities of the samp1es exposed to the treatments from FunctionaTity Part II are summarized in Tab1es 16 and 17. Addition of 0.2% monog1ycerides increased the EC of a11 samp1es. Increased EC may have been due to the emu1sification activity of monog1ycerides. Addition of 125 ppm 808 may have a1so improved the EC of the samp1es due to the surfactant nature of this compound. Addition of succinic or ma1eic anhydride increased the EC of the samp1es. Franzen and Kinse11a (1976) observed that succiny1ation enhanced the EC of soy iso1ate disper- sions. They conc1uded that it was due to the increased so1ubi1ity of the protein. The EC of many of the samp1es was improved by the addi- tion of 5.0% NaC1. Addition of anions may increase EC by enhancing the unfo1ding of protein mo1ecu1es, thereby en- 1arging their effect surface area avai1ab1e for interfaciaT membranes (Kinse11a, 1976). Soy protein is known to be dissociated by high concentrations of NaC1. Enzymatic hydro1ysis did not significant1y affect the EC of the majority of samp1es. Digestion for 1 hr did 131 Tab1e 16. Emu1sion capacity measured in g oi1/mg protein for NFDM, NFDM/50, SI, NAC/50 and MAC at a pro- tein concentration of 3.2% Samp1e NFDM NFDM/50 SI NAC/50 NAC Contro1 3.5 3.0 2.4 3.0 3.2 7700/30' 3.9 3.1 2.5 2.9 3.4 pH 4.5 1.7 1.4 1.0 0.4 0.1 0.1M CaC12 2.7 2.5 0.8 1.0 1.2 0.1M CaC12~0.1% NaZHPO4 5.1 3.1 2.1 2.1 1.7 Homogenization 3.1 2.9 2.1 2.3 2.8 0.1% NaZHP04-68OC/30' 5.3 4.0 2.2 3.9 5.1 pH 8.5-680C/3O' 4.9 3.4 2.1 3.6 5.1 Ma1eic anhydride 5.1 4.5 3.0 4.6 5.2 0.5% SHMP 2.8 2.4 2.3 2.6 3.0 125 ppm SDS 3.6 3.1 3.0 3.2 3.4 0.2% monog1yceride 4.6 3.5 2.7 3.6 4.1 5.0% NaC1 4.3 3.8 3.0 2.9 2.4 Enzymatic hydro1ysis 3.5 3.0 3.1 3.1 3.5 132 4.4 4.4 4.4 4.4 4.4 4.4 4444444444 424444 4.4 4.4 4.4 4.4 4.4 4.4 4244 44.4 4.4 4.4 4.4 4.4 4.4 4.4 4442 44.4 4.4 4.4 4.4 4.4 4.4 4.4 444444444 444442 4.4 4.4 4.4 4.4 4.4 4.4 4444444444445 44.4 4.4 4.4 4.4 4.4 4.4 4.4 444 E44 444 4.4 4.4 4.4 4.4 4.4 4.4 44444444 3 44.4 4.4 4.4 4.4 4.4 4.4 4.4 .4444444-4443442 44.4 4.4 4.4 4.4 4.4 4.4 4.4 4444 44.4-44444 24.4 4.4 4.4 4.4 4.4 4.4 4.4 .444443 44.4-44444 24.4 4.4 4.4 4.4 4.4 4.4 4.4 44444 34.4 4.4 4.4 4.4 4.4 4.4 4.4 4.4 :4 4.4 4.4 4.4 4.4 4.4 4.4 .4444444 4.4 4.4 4.4 4.4 4.4 4.4 4444444 44434 4 34 4mw443 . 443 44434 34 4444443 myaamm 4N.m 4o :owpmgpcmucou cwmpoga 4 44 om\3m new 34 .444443 .443 .44434 .34 444 4444444 454444 4 :4 44444445 44444444 44444454 .44 44444. 133 increase the EC of SI probab1y due to the increase in squ- bi1ity. After 3 hr of digestion EC decreased s1ight1y. Kueh1er and Stine (1974) reported that enzymatic digestion (Pro1ase) did not significant1y affect the EC of whey pro- tein, NFDM or NAC. Addition of either NaZHPO4 or Na3Citrate prior to heating at 680C/3O min increased the EC of the samp1es. Adjustment of the pH to 8.5 and heating a1so increased the EC of most of the samp1es. Increased so1ubi1ity may have been responsib1e for the improvement noted. The EC of SI, NAC and their b1end remained the same or decreased when any of the treatments emp1oying CaC12 were tested. The EC of NFDM and NFDM/50 increased when these materia1s were dispersed in 0.1M CaC12 containing either NaZHPO4 or Na3Citrate. The EC of the whey products a1so increased when subjected to these treatments. Emu1sification is a primary functiona1 requirement in severa1 food proteins. In genera1, the soy iso1ate had 1ess EC than the mi1k products. B1ends between the two often had near1y as much EC as the mi1k product and usua11y a1ways more than the SI. By se1ecting the treatment corres- ponding to a particu1ar protein it was possib1e to obtain the higher EC associated with the mi1k products. Whipping Abi1ity Foaming or whipping, i.e. the capacity to form stab1e foams with air, is an important functiona1 property of 134 proteins. This property is important in ange1 food cakes, confections, candy, meringues, souff1es and toppings. Foaming properties inc1ude whippabiIity (or foamabi1ity) and foam stabi1ity. Food foams usua11y consist of air drop1ets dispersed in and enve1oped by a 1iquid containing a so1ub1e surfactant. The surfactant 1owers the surface tension of the 1iquid, thereby faci1itating deformation of the 1iquid and the marked expansion in its tota1 surface area against its own surface tension (Kinse11a, 1976). In this study, specific vo1ume (SV) in m1/g was used to assess the increase in vo1ume f011owing whipping. Foam stabi1ity was measured by recording the one-ha1f stabi1ity time (%t) in min. Stabi1ity times of 1ess than 5 min were reported as 0. SI foams had 1ow SV with zero stabi1ity times at a11 three protein concentrations. Watts (1937) reported that severa1 commercia1 soy f1ours had 1itt1e or no whipping abi1ity and Sosu15ki gt a1. (1976) found that severa1 soy isoIates did not exhibit good foaming properties. Samp1es which were dispersed in 0.1M CaC1 were the on1y SI 5015 to 2 have stab1e foams. Substantia1 1055 of so1ubi1ity was suffered by these samp1es. Improved foam stabi1ity may have been due to a charge effect, cross1inking or partia1 denaturation. Kinse11a (1976) observed that proteins for- ming foams must exhibit a critica1 ba1ance between their abi1ity to engage in 1imited intermo1ecu1ar cohesion re- quired to form a stab1e e1astic membrane and the tendency 135 to se1f—associate excessiver, which wou1d resu1t in aggre- gation and breakdown of the foam. Soy protein in the pre- sence of ca1cium may have had sufficient proteineprotein interaction to stabi1ize a foam structure. Reconstituted SN powder exhibited minima1 foam produc— tion and stabi1ity. $015 of sweet whey did not whip into stab1e foams, regard1ess of the treatment, unti1 the protein concentration was 8.0%. At this concentration the tota1 soIids 1eve1 was very high which resu1ted in very viscous samp1es. When these samp1es were whipped paste 1ike foams were produced with 1ow SV but 1ong stabi1ity times. B1ends of SH:SI did not whip into stab1e foams except at 8.0% protein where paste 1ike foams were produced. These high1y viscous foams were not observed un1ess 7 % of the b1end ratio was derived from SW. Heating was necessary to produce stab1e foams (Tab1e 18) from HPC so1s. Some heat denaturation appeared to be a prerequisite to whipping. PartiaI heat denaturation may be necessary to increase the degree of cohesion, i.e. protein- protein interaction necessary in the formation of a protein fi1m (Kinse11a, 1976). The optimum time/temperature re1a- tionship appeared to be 68-770C/30 min. Increased heat treatment reduced stabi1ity times. Stabi1ity times increased as the percent protein increased. Peter and Be11 (1930), JeIen (1973), Devi1biss gt gt. (1974), Kueh1er and Stine (1974) and McDonough gt 1. (1974) reported that heat treat- a.— ment significant1y improved the whipping properties of whey 136 and HPC. McDonough gt gt. (1974) observed that partia1 denaturation of the protein was apparent1y necessary to produce stab1e foams from WPC. B1ends of WPC:SI did not whip into stab1e foams at 3.2% protein un1ess 75% of the protein was derived from WPC. From these samp1es stab1e foams were prepared when the sam- p1es were heated at temperatures not higher than 77°C. The stabi1ity of the foams diminished with increased heat treat— ment. At 5.0% protein, stab1e foams were produced from those b1ends containing at 1east 50% WPC which were heated. Specific vqume and at increased as the HPC in the b1ends increased. Foam stabi1ity (FS) dec1ined when the samp1es were subjected to UHT heating. At 8.0% protein SV and %t stabi1ity increased. Foam stabi1ity and specific vo1Ume increased with increased WPC in the b1ends. Hoffman (1974) reported that WPC-soy isoIate b1ends maintained the SV associated with WPC but were so unstabIe that drip cou1d not be measured. McDonough gt gt. (1974) found that the stabi1ity of WPC 5015 increased as protein concentration increased. Samp1es of EN whipped into stab1e foams (Tab1e 19) at a11 three protein concentrations. Heating had much 1ess effect upon the whipping properties of this whey product compared to WPC. Adjustment of the pH to 8.5 or 4.5 in- creased both SV and kt. Hansen and B1ack (1972) and Kueh1er and Stine (1974) observed that the greater the net charge, the greater was the tendency to whip. Peter and Be11 (1930) 137 and Je1en (1973) found that acid whey so1s had improved whipping properties. Acidification enhanced the vu1nera- bi1ity of whey proteins to denaturation during the whipping process. This may have accentuated protein-protein inter- action, thus increasing the cohesiveness of the proteins during membrane formation. Addition of CaC12 to EN 5015 increased the stabi1ity of the foams. This may have been due to denaturation, cross1inking or charge distribution. At 6.0% protein F8 substantia11y improved. At 8.0% pro- tein, untreated EH foams had viscous, paste 1ike properties. These foams had very 1ong stabi1ity times though SV were marked1y reduced. BIends of Ew:SI whipped into stab1e foams (Tab1e 20), but had much shorter stabi1ity times than EN foams. At a b1end ratio of EM 25:75 SI stab1e foams were produced from those samp1es which were modified by addition of stabi1izer sa1ts, CaC12, or which were acidified to pH 4.5. As the percent Ew in the b1end increased so did the specific vo1ume and stabi1ity of the foam. Litt1e improvement was noted in the whipping properties as the concentration of protein increased. In genera1, disp1acement of EH for SI substan- tia11y depressed both SV and gt of the whips. Sodium caseinate whipped into stab1e foams (Tab1e 21) at a11 protein 1eve1s. Untreated caseinate 5015 had shorter stabi1ity times than NFDM. Neither SV or gt changed appreciab1y as the percent protein increased from 3.2-8.0% protein. Heating had 1itt1e effect upon the whips though 138 UHT heating did depress both SV and FS. Lowering the pH to 4.5 destroyed a11 foaming tendencies whi1e addition of CaC1 to the $015 resu1ted in marked1y improved whipping 2 properties. Since both treatments significant1y 1owered the so1ubi1ities of the samp1es the improvement noted for CaC12 treated 3015 may have been due to crosinnking of proteins. Min and Thomas (1977) reported that addition of ca1cium to NAC 5015 increased foam overrun and firmness. Homogenization s1ight1y decreased FS. Sodium caseinatezsoy iso1ate b1ends whipped into foams (Tab1e 22) having poor stabi1ity. Specific vo1umes were 1ower than those recorded for NAC. Specific vo1umes and stabi1ity times were simi1ar regard1ess of the b1end ratio. Stabi1ity times remained approximate1y the same as the amount of protein increased whi1e SV decreased s1ight1y. Heating had 1itt1e effect upon the whipping properties of these b1ends. Lowering the pH to 4.5 e1iminated the whip- ping properties of the b1ends whi1e addition of CaC12 to the dispersions marked1y improved their whippabi1ity. Hoffman (1974) reported that rep1acement of casein with SI had 1itt1e effect upon specific vo1ume but drastica11y reduced foam stabi1ity. Untreated NFDM soIs whipped into foams (Tab1e 23) having the 1ongest stabi1ity times of any of the products tested. The specific vo1umes of these foams were second on1y to those of NAC. Foam stabi1ity increased as the percent protein increased, though SV decreased. webb (1941) 139 reported that SV decreased but stabi1ity times increased as the concentration of NFDM so1ids increased. Whipping properties were reduced when severe heat treatments were emp1oyed. Tamsma gt _t. (1969) found that foam stabi1ity of NFDM 5015 was optima1 at 30% tota1 so1ids. Lowering the pH of the $015 to 4.5 drastica11y reduced whipping proper- ties. The addition of CaC1 to the samp1es fai1ed to have 2 the same affect on the whipping properties of NFDM that it did on NAC foams. Addition of stabi1izing sa1ts increased both SV and 4t possib1y due to their effect on so1ubi1ities. Homogenization reduced foam stabi1ity s1ight1y. B1ends of NFDM:SI had decreased foam stabi1ity compared to NFDM foams (Tab1e 24). Specific vo1umes were approxi- mate1y the same. Foam stabi1ity improved as the percent NFDM in the b1ends increased. The stabi1ity of NFDM:SI foams was greater than the foams from the other product b1ends. Addition of stabi1izer sa1ts increased FS. The whipping properties of the samp1es exposed to the treatments from Functiona1ity Part II are shown in Tab1es 25 and 26. Addition of Na Citrate (or Na HPO to WPC $015 3 2 4) dispersed in 0.1M CaC1 improved both the SV and at of its 2 whips. This may have been due to the partia1 denaturation which resu1ted or cross1inking of the proteins. The whipping properties of the remaining samp1es were either unaffected or s1ight1y reduced. Addition of EDTA to WPC and WPC/75 so1s dispersed in 0.1M CaC1 marked1y improved both the SV and gt of these 2 140 whips. The whipping properties of the other samp1es were reduced. Addition of either NaZHPO4 or Na3Citrate to the sam- p1es prior to heating resu1ted in the production of stab1e foams from SW and SW/SO. The SV and %t of WPC and WPC/75 whips a1so increased. Raising the pH of the $015 to 8.5 prior to heating resu1ted in the formation of stab1e whips from SW and SW/SO. The stabi1ity and specific vo1umes of NFDM foams a1so increased. McDonough gt _1. (1974) reported that samp1es subjected to both heat and a1ka1ine pH treatment had exce1- 1ent whips. Increased whippabi1ity may have been due to greater so1ubi1ity. Heating fo110wing addition of H 02 to the samp1es 2 increased the whipping properties of SW and SW/SO. The foam stabi1ity of EW foams a1so increased. This may have been due to oxidation of the heated (partia11y denatured) pro- teins. Hansen and B1ack (1972) reported that addition of H202 to whey protein so1s improved their whipping proper- ties. These researchers reported that H202 caused definite changes in the e1ectrophoretic patterns of the proteins and that these changes were probab1y important in the improved whipping properties. Addition of reducing agents such as L-cysteine or mercaptoethano1 to the samp1es resu1ted in greater foam stabi1ity and expanded specific vqumes for many of the $015. The reducing agents may have affected the whipping 141 properties of these products by their direct effect upon disu1fides or indirect1y through increased so1ubi1ity. Peter and Be11 (1930) found that reducing agents improved the whipping characteristics of whey proteins. The addition of succinic or ma1eic anhydride marked1y improved the foamabi1ity of SI 5015. The specific vo1umes of NFDM and NAC 5015 increased though the FS of NAC foams decreased. The whipping properties of EW were practica11y e1iminated. Franzen and Kinse11a (1976) found that succiny- 1ation marked1y enhanced the FS and SV of soy protein dis- persions, probab1y due to the increase in squbi1ity of these 5015. The foams from NAC and NAC/50 were more stab1e when 5.0% NaC1 was added to these samp1es. The squbi1ities of these diSpersions were reduced, thereby possib1y en1arging their effective surface area. Addition of 0. % sodium hexametaphosphate (SHMP) marked1y improved the SV and gt of WPC and WPC/75 so1s. Me1achouris (1972) observed that whey protein acts as a cation whi1e SHMP acts as an anion. Under the right condi- tions they bind and form sizab1e aggregates. These aggre- gates may be of sufficient size to increase the strength of the protein membrane. Addition of g1ycan to the samp1es was ineffectua1 because the materia1 sett1ed out. Homogenization was necessary to disperse the g1ycan. This treatment improved the whipping properties of many of the $015. Foams produced 142 from these samp1es had Iong stabi1ity times but reduced SV. The addition of CMC to the samp1es increased the FS of practica11y a11 the samp1es (except SI at.1% CMC). Specific vo1umes were essentia11y unchanged. Heating fo11owing addition of CMC further increased the stabi1ity of the whey products and their SI b1ends. Foams with 1ong stabi1ity times were noted after addition of 0.5% CMC. The whips had paste—1ike properties with 10w overrun. SI produced foams of the 1east stabi1ity. Morr gt gt. (1973), Kueh1er and Stine (1974) and Hansen and B1ack (1972) reported that uti- 1ization of CMC in protein so1s resu1ted in proIonged stabi1ity times. Enzymatic hydro1ysis (pro1ase) for 1 hr increased the FS of the materiaIS except for NAC and its SI b1end. 'Spe- cific vqumes aISo increased. After digestion for 3 hr FS decreased inghtIy. Cooney (1976) and Kueh1er and Stine (1974) used enzymatic hydro1ysis to improve the whipping properties of proteins. These researchers postu1ated that the increased foamabi1ity was probab1y due to the greater po1ypeptide content which increased the avai1ab1e surface of the protein. This a11owed more air to be incorporated into the foam structure. Extensive hydro1ysis resu1ted in a greater number of sma11 peptides which 1acked the strength to maintain the protein membrane. In genera1, stab1e foams were produced from NFDM, NAC. EW and their SI b1ends. Whey protein concentrate subjected to specific treatments a1so produced stab1e foams. The 143 Tab1e 18. Whipping abi1ity as specific vo1ume (SV) in m1/g and % stabi1ity time (kt) in min for WPC at protein 1eve1s of 3.2, 5.0 and 8.0% Samp1e Variant ‘*‘ 3.2 5.0 8.0 SV %t SV %t SV %t Contro1 2.6 0 2.3 o 5.4 o 680C/3O' 10.3 19 7.0 23 7.9 45 77OC/NH 7.7 20 6.9 25 7.1 43 77OC/3O‘ 6.9 18 6.9 22 6.7 68 94OC/4” 4.6 o 6.0 11 6.6 11 1210C/4” 5.1 o 7.2 7 5.6 10 144 Tab1e 19. Whipping abi1ity as specific vo1ume (SV) in m1/g and % stabi1ity time (%t) in min for EW at pro- tein 1eve15 of 3.2, 5.0 and 8.0% protein Samp1e Variant 3.2 5.0 8.0 sv at sv at ,sv at Contro1 6.4 30 7.3 28 3.1 150* 680C/30' 6.6 28 6.3 24 3.3 150* 7700/30' 5.1 32 5.7 30 3.5 150* pH 8.5 7.0 40 7.3 70 4.5 150* pH 4.5 9.9 36 9.5 120 5.6 150* 0.1M CaC12 6.2 50 8.3 94 3.1 150* 0.1% 1462111304 7.0 29 6.6 52 2.5 150* 0.1% Na3Citrate 6.8 30 6.6 65 3.6 150* *did not breakdown 145 Tab1e 20. Whipping abi1ity as specific vqume (SV) in m1/g and % stabi1ity time (%t) in min for EW/25, EW/SO and EW/75 at a protein 1eve1 of 5.0% Samp1e Variant EW/25 EW/SO EW/75 sv 4t sv at sv at Contro1 3.5 0 5.1 22 6.4 27 680C/30' 3.6 0 4.7 20 5.4 23 77OC/NH 3.8 0 5 2 16 6.0 24 pH 8.5 3.6 0 4.7 13 4.1 45 pH 4.5 5.6 5 7.3 19 5.1 6 0.1M CaC12 5.5 18 6.5 38 6.8 41 0. % NaZHPO4 4.1 5 5.6 23 6.6 44 0.2% NaZHPO4 4.3 6 5.6 24 6.6 41 0.1% Na3Citrate 3.9 7 5.1 19 6.4 40 0. % Na Citrate 3.8 5 5.3 25 6.4 37 3 146 Tab1e 21. Whipping abi1ity as specific vo1ume (SV) in m1/g and P stabi1ity time (4t) in min for NAC at protein 1eve1s of 3.2, 5.0 and 8.0% Samp1es variant 3.2 5.0 8.0 sv at sv at sv %t Contr01 8.7 17 9.3 16 8.5 15 9400/4" 8.4 15 8.2 10 8.1 8 121°C/4" 8.1 9 8.1 10 8.1 8 pH 4.5 2.4 0 2.3 0 2.1 0 0.1M CaC12 16.8 50 21.2 49 19.5 45 0.1% NaZCitrate 9.0 20 8.4 18 9.1 16 Homogenization 8.7 13 8.5 13 8.1 12 147 Tab1e 22. Whipping abi1ity as specific vo1ume (SV) in m1/g and 8 stabi1ity time (at) in min for NAC/25, NAC/50 and NAC/75 at a protein 1eve1 of 3. % Samp1e variant NAC/25 NAC/50 NAC/75 sv at, sv at sv 8t Contr01 7.2 7 8.2 8 9.3 10 770C/NH 6.3 6 8.2 10 8.0 _15 pH 8.5 7.3 6 8.4 7 7.7 12 pH 4.5 2.8 0 1.9 0 3.3 0 0.1M CaC12 10.2 25 9.5 28 15.6 17 0.1% Na2HP04 8.1 16 8.5 12 8.6 10 0. % Na Citrate 7.8 12 8.3 10 8.9 11 3 148 Tab1e 23. Whipping abi1ity as specific vqume (SV) in m1/g and % stabi1ity time (at) in min for NFDM at protein 1eve1s of 3.2, 5.0 and 8.0% Samp1e variant 3.2 5.0 8.0 sv at sv at sv gt Contr01 7.5 35 7.1 48 6.8 65 68OC/3O' 7.2 32 7.2 46 7.2 58 7700/30' 7.2 22 6.3 37 5.8 48 pH 4.5 3.0 5 3.1 5 3.1 5 0.1M CaC12 6.4 32 6.6 53 6.7 60 0.2% NaZHPO4 7.7 40 7.8 63 6.9 97 0.3% 14211904 7.7 39 7.9 60 6.8 90 0.2% Na3Citrate 8.8 32 8.0 61 7.4 85 0.3% Na3Citrate 9.3 35 8.8 63 8.7 83 Homogenization 8.1 33 7.3 36 6.7 61 149 Tab1e 24. Whipping abi1ity as specific vo1ume (SV) in m1/g and % stabi1ity time (at) in min for NFDM/25, NFDM/50, and NFDM/75 at a protein 1eve1 of 3.2% Samp1e Variant NFDM/25 NFDM/50 NFDM/75 SV 8t SV at sv gt Contr01 8.0 22 8.7 27 7.6 30 770C/NH 8.0 20 8.6 25 7.9 30 pH 8.5 7.8 20 7.2 25 10.6 57 pH 4.5 2.8 0 2.0 0 3.2 5 0.1M CaC12 4.6 5 5.3 12 6.5 15 0.1% Na Citrate 8.3 22 8.9 32 9.6 46 3 150 Tab1e 25. Whipping abi1ity as specific vo1ume (SV) in m1/g and a stabi1ity time (%t) in min for NFDM, NFDM/50, NAC, NAC/50 and SI at a protein 1eve1 of 3.2% Samp1e Variant SV gt sv gt NFDM NFDM/50 pH 8.5—680C/30' 9.2 60 8.0 29 0.01% L cysteine 7,7 40 7.9 38 0.01% mercaptoethano1 6.9 32 7.2 23 Succinic anhydride 11.4 27 8.8 21 Ma1eic anhydride 12.8 17 8.6 20 0.5% SHMP 7.8 45 8.9 33 0.1% CMC 7.6 81 8.8 45 0.5% CMC 8.4 360 7.7 260 3.5% 91ycan-hom09. 4.3 150 5.1 145 Enzyme hydr01. 14,4 50 10.1 18 NAC NAC/50 SI Succinic anhydride 10.9 17 .6 13 4.5 20 Ma1eic anhydride 9.7 16 .3 13 6.0 41 5.0% NaC1 10.3 35 .7 37 3.7 0 0.5% CMC 9.7 60 .2 68 4.1 20 Enzyme Hydro1. 8.2 15 .6 10 9.7 19 151 Tab1e 26. Whipping abi1ity as specific vo1ume (SV) in m1/g and stabi1ity time (%t) in min for WPC, WPC/75, sw, sw/50, EW and EW/75 at a protein 1eve1 of 3.2% __t Samp1e Variant sv at sv at sv at sv gt ch WPC/75 sw sw/75 0.1M CaC12-0.1% Na3Cit. 7.7 17 7.1 14 2.2 0 3.4 0 0.1M CaC12-0.1% NaZHP04 7.6 17 6.5 14 3.0 0 3.6 0 0.1M CaC12-0.1M EDTA 10.7 35 8.8 22 4.6 0 2. 0 0.1M CaC12-1.0% EDTA 8.4 22 8.2 9 2.8 0 2. 0 0.1% NaZHP04-68OC/30' 7.2 47 8.4 43 5.7 37 4. 5 0.1% Na3Cit.-68OC/30' 9.1 40 8.2 32 6.3 51 4. 0 pH 8.5-68OC/30' 9.0 16 9.0 36 6.3 35 5. 12 H202-680C/30'-Cat, 8.2 18 7.2 18 5.6 40 4. 5 0.01% L cysteine 4.8 0 5.7 0 5.7 39 5. 6 0.5% SHMP .8 30 6.5 22 4.9 0 4. 0 Ma1eic anhydride 6.5 0 2.2 0 5.7 6 4. O 0.1% CMC 5.3 5 3.5 0 5.2 24 4. 12 0. % CMC-68°c/3o 6.3 17 5.5 22 4.4 140 4. 20 0.5% CMC 4.0 38 4.9 78 5.8 300 3. 28 3.5% egcan-hom, 4.6 5 4.0 O 3.4 0 2. 0 Enzyme Hydro], 10.6 47 10.1 19 4.8 5 5. 5 EW ‘ EW/75 H202 7.1 35 5.8 14 0.1% CMC 5.7 47 5.3 43 0.5% CMC 3.0 338 4.5 140- 3.5% 91ycan homog. 4.1 240 4.1 65 152 b1ends of these products with SI genera11y had simiTar SV va1ues with s1ight1y shorter at. By utiTization of the appropriate treatment it was possibTe to substantia11y improve the whipping properties of the samp1e. Sensory Eva1uation Subjective sensory anaiysis is an important property to measure in any food system. The product shou1d be appeaTing, attractive and pa1atab1e. One of the major probTems pre- venting greater utiTization of soy iso1ates has been their offensive f1avor and odor (Johnson, 1970). Therefore, in any system emp10ying soy isoTates, sensory evaTuation is imperative. For the purposes of this study the foam produced during whipping was used as the test material. A possibie 10 points was awarded to each characteristic, f1avor, co1or and tex— ture. The stiffest, whitest and most b1and foam received maximum scores of 10. The scores presented in this study were not taste pane] averages but the opinion of a 1imited group of paneTists. A score of 6 was considered acceptabie. The data in Tab1es 29-30 present the sensory eva1uation scores for the products tested in this study. In genera1, fiavor, co1or and texture scores were highest for the mi1k products, particu1ar1y NFDM and NAC. Foams made from NFDM and NAC received high f1avor, c010r and texture scores at a11 three protein concentrations. The on1y exceptions were for samp1es adjusted to pH 4.5 or to which CaC12 was added. 153 Foams produced from b1ends of NAC and SI received acceptabIe f1avor, coIor and texture scores at a11 protein concentrations. F1avor acceptabi1ity increased inghtIy as tota1 protein increased. As the amount of NAC in the b1ends increased, sensory properties improved. Hoffmann (1974) a150 reported that sensory evaIuation of casein—soy 5015 showed f1avor acceptabi1ity to decrease as casein was re— p1aced with soy protein. The odor was described as beany. Foams produced from NFDM:SI b1ends received simi1ar sensory scores. Coior, f1avor and texture scores were sti1I accep- tabie when as much as 75% of the tota1 b1end protein was from SI. Scores rose sTight1y as the amount of NFDM in- creased in the b1ends and as tota1 protein increased. There were on1y inght differences in the sensory properties of samp1es subjected to the different treatments. Lowering the pH to 4.5 imparted a bitter-acid f1avor to the samp1es whi1e addition of CaC12 resu1ted in very bitterly f1avored samp1es due to the nature of this sa1t. NFDM foams had greater acceptabi1ity, though the sensory characteristics of NFDM:SI b1ends were satisfactory. The SI foams received 10w f1avor, coTor and texture scores regard1ess of the protein concentration. KaIbrener g£,al. (1971) reported that in test paneT resu1ts commercia1 SI had f1avor scores ranging from 5.9-6.4. It was agreed that beany and bitter fiavors persisted in isoIates. Cowan gt 1. (1973) reported that beany and bitter f1avors were stiII detectab1e in soybean products after an initiai 154 extraction. Yasumatsu gg g1. (1972) observed that a1most aTT types of soybean products have some undesirabTe fTavor characteristics. Even though SI was the most purified form of soy protein, the characteristic soy f1avor was present in the isoTate. The SI foams had poor texture characteristics and were definiteTy off-white. Some improvement in texture was noted for those samp1es having stabTe whips. In genera1, the processing variants had TittTe effect though aTkaTi treatment sTightTy improved f1avor. Bourne gg gl. (1970) reported that a1kaTi treatment increased fTavor scores of soymi1k. In practicaTTy aTT cases 51 had unacceptabTe fTavor properties which were a1ways Tower than NFDM and NAC. Maga and Lorenz (1973a) reported that in major sensory studies of NFDM, NAC and SI, NFDM and NAC had the most bTand fTavor and odor characteristics. The mi1k group was sub- stantia11y more bTand than the vegetabTe proteins. ETectrodiaTyzed whey foams had very acceptabTe sensory properties. CoTor, fTavor and texture scores were high, though sTightTy Tess than those recorded for NFDM. The whips were Tess stiff and faintTy yeTTow. Texture scores rose as totaT protein increased. The sensory properties of these foams were not substantia11y affected by any of the processing treatments. SW foams received much Tower fTavor scores because of the saTtiness associated with this product. CoTor scores were aTso Tower due to yeTTowness of the whips. Most samp1es had Tow texture scores because they did not whip into stabTe foams. Treatment variation had negTigibTe 155 effect upon the sensory properties of SN. ch foams re- ceived moderate1y high f1avor and coTor scores for practica11y aTT variants (except pH 4.5 and CaC12 samp1es). Texture scores were Tow except for samp1es subjected to heating. Heat treatment was necessary for the production of stabTe foams. Increasing the protein concentration did not affect sensory properties. The whey products had good sensory characteris- tics; this was aTso noted by MarvopouTous and Kosikowski (T973). Maga and Lorenz (1972) examined the f1avor and odor intensities of mi1k and soy protein suppTements. MiTk pro- ducts incTuded NFDM, NAC, whey powder and demineraTized whey powder. There were no statisticaT differences between odor intensities of the mi1k products. NFDM was judged to be the most b1and. The fTavor of SI was statisticaTTy inferiOr. DemineraTized whey powder was more b1and than standard whey powder. BTends prepared between SN and SI produced foams of Tow qua1ity. CoTor and texture scores were unacceptabTe regard- Tess of protein content or b1end ratio. BTends of WPC:SI whipped into foams receiving medium to high scores for coTor and fTavor at the protein 1eve1s studied. Foams were given Tow texture scores except for those samp1es heated. Texture, fTavor and coTor improved as the amount of WPC increased in the samp1es and as totaT protein increased. Variants other than heating had TittTe effect upon the sensory properties of WPC:SI b1ends. Foams prepared from Ew:SI b1ends scored higher than any other whey product b1ends. FTavor scores 156 were higher probab1y because the sweetness from the EN masked the beany f1avor of the SI. Sensory improvement was noted as the b1end ratio changed to contain more EN. The subjective anaTyses of samp1es exposed to the treat— ments from FunctionaT characterization Part II are shown in Tab1es 27-30. Many of the treatments did not affect the sensory properties of the samp1es. However, any of the treatments which empToyed CaCTZ resuTted in Tow f1avor scores due to the bitter nature of this sa1t. ‘SeveraT treatments improved the fTavor scores of SI, these inc1uded: raising the pH to 8.5 and heating, addition of H202, addition of CMC and heating, addition of NaZHPO and heat- 4 ing, addition of NaCT and addition of gTycan. However, the f1avor characteristics of these SI foams were stiTT c0n- sidered unacceptabTe. These same treatments genera11y increased the f1avor scores of foams produced from mi1k product:SI bTends. Sucrose raised the fTavor scores of SI foams to an acceptabTe TeveT. Addition of ma1eic anhydride to the sampTes reduced the fTavor scores of many foams due to the saTty—bitter f1avor‘ properties of this compound. This was in contrast to that reported by Franzen and KinseTTa (1976) who submitted that there were no fTavor probTems associated with the succiny- Tation of soy protein. Enzymatic hydroTysis marked1y decreased the fTavor scores of NFDM, NAC and their SI b1ends. Foams produced from these samp1es were very bitter. Arai t 1. (1970), Yamashita t T. (1969), Fujimaki g; T. 157 (1968), Johnson (T975) and PeTissier and Manchon (T976) aTT reported bitterness in protein hydroTysates of soy and casein. The whey products and their SI b1ends did not have bitter f1avored foams. CoTor and texture scores of SN and SW/SO foams were improved by treatments such as addition of CMC and heating, addition of stabi1izer sa1ts and heat- ing, and pH adjustment to 8.5. CoTor and texture scores of SI foams were improved by many of these treatments inc1uding enzymatic hydroTysis. FTavor scores of NFDM and NAC were the highest of any mi1k products. E1ectrodia1yzed whey and NFDM bTended with soy isoTate had the most favorabTe sensory properties of any of the b1ends. The sensory characteristics of SI foams were unacceptabTe. However, when 51 was bTended with the mi1k products the fTavor scores were on1y sTightTy Tess than those recorded for the respective mi1k product. MiTk pro- ducts were used to effectiveTy improve the coTor, fTavor and texture of SI soTs. Many treatments did not substan- tia11y improve the sensory properties of the sampTes. SeveraT treatments whi1e markedTy improving functionaTity resu1ted in Tow fTavor scores for many of the samp1es. This may in fact Timit or modify the use of such treatments in actuaT food products. 158 Tab1e 27. FTavor scores* of the whips from samp1es of Ew, EN/75, WPC, WPC/75, SN and SN/SO at a protein TeveT of 3.2% Variant samp1e EH EW/75 WPC WPC/75 SN SN/SO Contr01 8 8 8 7 6 6 0.1M CaC12 2 2 3 2 3 '4 pH 4,5 7 8 6 7 5 3 pH 8.5—68°C/3o' 9 8 9 7 8 8 Ma1eic anhydride 4 4 3 3 4 4 0.1% CMC-68°C/3o' TO 8 8 9 7 8 Enzyme hydr01. 9 8 9 8 7 7 o. % SHMP 8 6 8 8 6 4 5.0% NaCT 7 8 5 6 6 6 3.5% gTycan-Hom. 8 8 9 7 8 7 *A scaTe of T-TO was used; TO=most desirabTe 159 Tab1e 28. FTavor scores* of the whips from sampTes of NFDM, NFDM/50, NAC, NAC/50 and SI at a protein TeveT of 3.2% Variant Samp1e NFDM NFDM/50 SI NAC/50 NAC ControT TO 8 4 6 TO 0.1M CaC12 T T T 3 3 pH 4.5 4 4 4 2 3 Ma1eic anhydride 4 3 4 3 3 o. % CMC-68°C/30' 1o 8 5 7 10 pH 8.5—6806/30' 1o 8 5 7 1o Enzyme hydr01. 3 2 3 2 2 0.1% L cysteine 9 8 5 6 10 5.0% Sucrose TO T0 6 8 T0 3.5% gTycan-Hom. 7 7 5 6 6 *A scaTe of 1-10 was used; TO=most desirabTe 160 m—ch—wmmv HmOEHIF ”Nam: mm; OPT? mo NNNQN <« N N N N N N HN N N N N N N NNNNN N N N N N N 3N N N N N N N NNNNNz N N N N N N NNz N N N N N N NNNNN N N N N N N 3N N N N N N N NNNNNZ N N N N NN N NNZ N N N N N N NNNZNNZ N NN N N N N zNNz .NNNNNNN .NNNNNNN . .NoNNNN .NNNNN NNNNN NNNINNZ NN.N NZN N_.o NENNNN UNNNNz 2N.o NNNNNON NNNENN ycmmgm> NN.N No NN>NN :NNNNLN N 88 NN Nee NNN3N .3N .NNNNNz .NNz .NNN3N .NN .NNNNNZ .qu .NNNZNNZ .zNNz No NNNNENN ENLN NNNNz 8:8 No Nmagoum NNNNXNN .mm mpnmh NNDNNNNNN pmosuoN ”New: mm; opuN No m—NUN <8 161 N N N N N N HN N N N N N N NNN3N N N N N N N 3N N N N N N N NNNNNz N N N N N N NNz N N N N N N NNNNN N N N N N N 3N N N N ,N _ N N NNNNNZ OF N N N ON ON NNZ N N N . N N N NNNNNNZ ON ON N_ N ON. ON ZNNZ .NNNNONN .NNNNNNN .NoLNNN .NNNNN NNNNN NNNINNZ NN.N NZN NN.N NENNNN NNNNNZ z_.N NONNNON NNNENN pcmwgm> . . NN.N No NN>NN CNNNNNN N 88 NN NNN NNNZN .3N .NNNNNz .NNz .NNNzN 3N NNNNNZ .NNZ .NNNzNNz .ZNNZ No NNNNENN soeN NNNgz 8:8 No NNNNNNN NNNNN .NN NNNNN 162 Examination of MiTk and Soy Protein BTends for Heat Induced Interaction The purpose of this study was to examine mi1k and soy protein b1ends for heat induced interactions. GeT fiTtra- tion, disc poTyacryTamide ge1 eTectrophoresis (PAGE) and densitometry were used as the primary anaTyticaT tooTs. After preparation and freeze-drying of the materia1s used in this study, portions were taken and anaTyzed for nitrogen content, by a semimicro KjerahT procedure. Fac- tors of 6.38 and 6.25 were used for mi1k and soy protein respectiveTy. SeveraT buffers were prepared to determine in which medium the proteins shoqu be resuspended. These incTuded: 1) phosphate buffer, pH 7.0, u=O.T, 2) mi1k uTtrafiTtrate prepared from fresh skimmiTk, 3) Koop's buffer and 4) deionized distiTTed water. Soy protein was used as the test protein because of its vuTnerabiTity to ca1cium ions. One percent suspensions were made by stirring the protein into each buffer for 10 min at 1200 rpm. ATi- quots of each were then centrifuged at 14,000 x g for 30 min. FoTTowing centrifugation portions were taken from the supernatant of each buffer-protein system and anaTyzed for nitrogen. The resu1ts are shown in TabTe 3T. Soy protein was the most soTubTe in the phosphate buffer system. SoTu- biTity was substantiaTTy reduced in both Koop's buffer and mi1k uTtrafiTtrate probabTy due to the presence of ca1cium ions. The other test proteins were then resuspended in phosphate buffer and examined for totaT soTubTe protein. 163 TabTe 3T. SoTubiTity (%) of water soTubTe soy protein in se1ected buffers at 1.0% protein Samp1e Buffer D.D. water Koop's UTtrafiTtrate phosphate Soy protein 85.3 63.4 64.3 97.9 Tab1e 32. SoTubiTity (%) of NAC, NC, AC, and SW in phos- phate buffer pH 7.0, u=0.T at T.O% protein Buffer b Prote1n NACa wc AcC sud phosphate 99.8 99.6 TO0.0 99.1 a = sodium caseinate b = whoTe casein c = whey protein from acid whey d = whey protein from sweet whey 164 The resu1ts are shown in TabTe 32. Due to the exceTTent soTubiTity of a11 the test proteins in phosphate buffer (pH 7.0, u=O.T) it was chosen as the buffer system. The proteins inc1uded: 1) sodium caseinate (NAC), 2) whoTe casein (WC), 3) whey protein from acid whey (AC), 4) whey protein from sweet whey (SW) and 5) soy protein from a water extract of soy isoTate. The proteins were resuspen— ded in phosphate buffer at a concentration of 1.0%. MiTk and soy protein bTends were prepared by combining the respective proteins at a T to 1 ratio ( % totaT protein). The samp1es were heated at temperatures of: T) controT- unheated, 2) 6896/30 min, 3) 770C/20 sec, 4) 9406/10 sec and 5) TZTOC/S sec. These temperatures were chosen because they approximated simi1ar treatments used in the pasteuri- zation and UHT heating of fTuid miTk products. Heating was accompTished by immersing a test tube (containing the sam- pTe) into a siTicone oiT bath adjusted to the appropriate temperature. A deTay time of approximate1y one and a han min cou1d not be avoided in bringing the sampTe to the desired temperature. After heating, cooTing, centrifugation and fiTtration, the nitrogen soTubiTity was determined for each sampTe. The resu1ts for AC (acid whey protein), AS (the acid whey- soy protein combination), SW (sweet whey protein), SS (the sweet whey-soy protein combination) and soy protein (SP) are shown in TabTe 33. The initiaT soTubiTities were near1y 165 TabTe 33. SoTubiTity of AC, AS, SP, SS and SN in phosphate buffer pH 7.0, u=O.T at a protein TeveT of 1.0% SoTubTe protein % Treatment 46a 45b SPC ssd swE ControT TO0.0 98.5 97.9 98.T 99.T 680C/30' 98.9 99.3 99.8 98.3 97.4 77°c/20" 98.0 96.7 99.9 94.9 94.9 940c/10" 94.0 92.8 94.T 86.2 83.6 121°c/5" 89.1 86.7 85.8 8T.6 80.3 aAC - whey protein from acid whey bAS - acid whey soy protein combination CSP - soy protein dSS - sweet whey:soy protein combination eSW = whey protein from sweet whey 166 100 percent. The high soTubiTities of the bTended systems indicated apparent compatibiTity of the different proteins. Acid whey protein retained high soTubiTity as the heat treatment increased to 7700/20 sec. At 94OC/T0 sec and 12106/5 sec there was a decrease in the soTubiTity of this protein system. Sweet whey protein was not as stab1e to heat treatment as AC. Loss of soTubiTity was more drastic at the higher temperatures. PreviousTy in this research it was shown (soTubiTity data pertaining to functionaTity) that heating generaTTy decreased the soTubiTity of whey protein, especiaTTy at eTevated temperatures. Howat and Wright (1933), DiTT and Roberts (1964), Guy g3 g1. (T967) and Morr (1969) reported that the denaturation of whey pro— tein increased dramaticaTTy as heating temperature increased. Disc ge1s of whey protein are presented in Figure T. The geTs are ordered from right to Teft in terms of Teast to most severe heat treatment. The whey proteins were identi- fied according to their migration rates. Heating the protein at 68°C/3o min did not substantia11y affect band prominence. This was aTso found when the protein $01 was heated at 77oC/20 sec. The immunogTobuTins and serum aTbumin bands were sTightTy Tess intense. It appeared that Tess materia1 entered the ge1. Major changes occurred when the $01 was heated to 940C/TO sec. The band represen- ting serum aTbumin had compTeteTy disappeared. B-Tacto- gTobuTin was much Tess distinct though recognizabTe. The Teast affected whey protein was a-TactaTbumin. It appeared Figure 1. Disc gels of whey protein, soy protein and casein heated at (ficnuleft to right): unheated-control, 680C/30 min, 770C/20 sec, 94OC/lO sec and lZlOC/S sec. . 01:68 ZHmEHumm — 8 TM _1: _ H .I L I 833 . . ZHEQE . . New ' o ___..J N ZHmmNQU “Ill—H IIIT‘ ‘f 213:“ - __.__.ILII 169 that more protein had fai1ed to enter the ge1. Protein resoTution and band intensities were further aTtered when whey protein was heated at TZTOC/S sec. Extensive smearing was noted as weTT as an increase in the amount of protein faiTing to enter the ge1. Josephson t T. (1967), Morr “—— (1969) and Kenkare _g _l. (1964) reported that progressi- veTy higher heat treatments resuTted in greater denaturation of whey protein which resuTted in the formation of aggre- gates of sufficient size as to prevent their migration into PAGE ge1s. Hetrick (T950), Me1achouris and Tuckey (1966) and Senter gg‘gl. (1973) found that d-TactaTbumin was the most heat resistant of the major whey proteins. Beta- TactogTobuTin, the immune gTobuTins and serum aTbumin were much more sensitive to heating. - The soTubiTity of soy protein increased when heated at 6806/30 min.. Heating at 94oC/TO sec and TZTOC/S sec decreased the soTubiTity of the soy protein appreciab1y. PreviousTy in this research it was shown that (soTubiTity data pertaining to functionaTity) Tow temperature heating increased the soTubiTity whiTe UHT heating decreased the soTubiTity of soy protein. Shen (T976) and Hermannson and Akesson (T975a) reported that moderate heating sTightTy improved the soTubiTity of soy protein. Mann and Briggs (T955), CatsimpooTas g3 g1. (1969), Non (1970) and Cat— simpooTas _t‘_l. (T97Ta) found that the amount of protein insoTubiTized increased with increased heat treatment. Disc geTS of soy protein are presented in Figure 1. The 170 geTs are ordered from right to Teft in terms of Teast to most severe heat treatment. The major soy proteins, the 7S and TTS, were identified according to their migration rates. SeveraT faster migrating species were aTso present incTuding a prominant component near the marker dye. Heating at 6806/30 min had very TittTe effect upon the resoTution or prominence of the proteins. Some change was noted in the e1ectropherogram of soy protein foTTowing heating at 77°C/20 sec. The 75 fraction was partiaTTy dissociated whiTe the intensity of the TTS band was dimi- nished. These changes were accompanied by an increase in the number and intensity of minor bands. These new compo- nents were presumabTy breakdown products from the 7S and TTS proteins. In addition, the very distinct band Tocated near the marker dye had disintegrated into a smear. The 75 fraction compTeteTy disappeared when soy protein was heated at 9406/10 sec. The TTS fraction had aTso Tost much of its identity and was Tess concise. The breakdown products were more distinct and intense. The smear Tocated near the marker dye (from the ge1 representing protein heated at 770C/20 sec) was Tess prominant. Heating the protein at TZTOC/S sec resuTted in very TittTe change from those ob- served when the protein was heated at 94OC/TO sec. The species resu1ting from dissociation of 7S and TTS proteins were stabTe to this heat treatment. CatsimpooTas g; g1. (1969) reported that soy protein was stabTe to temperatures up to 70°C. At temperatures higher than this CatsimpooTas 171 gt_aT. (T969), Watanbe and Nakayama (T962), Won (T970), ai (f) 0 g; T. (1971), Won and Tamura (1969), CatsimpooTas (D t T. (1971a), Cumming t T. (1973), Saio gt gl. (T975a) and ATdrich (T977) found that soy 7S and TTS proteins suffered significant dissociation. Many of these same researchers reported that the disappearance of these frac- tions was accompanied by the appearance of faster migrating components. The soTubiTity of soy isoTatezwhey protein bTends de- creased as the heating temperature rose reTative to the reduction in the soTubiTity of the individuaT protein sys- tems. This was aTso found when these sampTes were studied during examination of their functiona1 properties. The acid whey:soy protein bTend was more stabTe to heat than the sweet whey:soy sampTe. The soTubiTities of NAC, NS (the sodium caseinate-soy protein b1end), WC and WS (the whoTe casein-soy protein bTend) are presented in TabTe 34. The initiaT soTubiTities were near1y 100%. The soTubiTity of both NAC and WC soTs remained stab1e as the heat treatment increased. Heating was aTso found to have affected the soTubiTity of sodium caseinate dispersions very TittTe during examination of its functiona1 properties. White and Davies (T958), Kresheck g£’_l. (1964) and ATais _g _l. (1967) studied the behavior of casein and concTuded that it was very resistant to heat denaturation. 172 TabTe 34. SoTubiTity of NAC, NS, WC and HS in phosphate buffer pH 7.0, u=0.T at a protein TeveT of 1.0% % SoTubTe protein Treatment NACa N5b wcc wsd Contr01 99.8 99.5 99.6 100.0 6806/3o'- 99.9 99.9 99.4 99.3 77OC/20" 99.0 100.0 98.8 99.1 94°C/1o" 98.1 99.1 98.3 98.7 121°C/5" 98.1 99.0 97.8 98.T a = sodium caseinate b = sodium caseinatezsoy protein b1end c = whoTe casein d = whoTe casein:soy protein b1end 173 Disc geTs of whoTe casein and soy protein are presented in Figure 1. These are 7% geTs in 7M urea. The geTs are arranged from Teft to right in terms of Teast to most severe heat treatment. Soy protein underwent substantiaT dissociation when the heat treatment rose to 94°C and higher. There was an appreciab1e decrease in the intensity of the primary components which was accompanied by a sub- stantia1 increase in the number of minor constituents. There was simiTarity between urea-soy geTs and nonurea-soy geTs. The e1ectropherograms of whoTe casein depict what other researchers have shown (Kresheck t 1., 1964; Hos- “non—~— tettTer g; _1., 1965; ATais _g gl., 1967; Morr, T969; and Hensen and MeTo, T977). Casein was very stabTe to heat treatment. Very TittTe change was noted as the heat treat- ment rose. The geT representing casein heated at TZTOC/ 5 sec was inadvertentTy inverted during photography. Whey:Soy_Protein BTends After heating, centrifugation and fiTtration, approxi— mate1y 7 m1 of the cTear fiTtrate was pumped upward through a coTumn packed with sephacryT S-200 superfine. GeT fi1- tration patterns were obtained for each samp1e. The geT fiTtration patterns of acid and sweet whey proteins were aTmost identicaT. ETution voTumes and peak patterns were extremeTy simi1ar. For these reasons on1y the ge1 fiTtration chromatograms of AC protein are shown (Figure 2). Each peak pattern is indicative of one of the 174 Figure 2. Gel filtration chromatograms of whey protein heated at; unheated-control(a), 680C/30 min (b), 77°C/2o sec (c), 940C/1o sec (a) and lZlOC/S sec (e). 175 .02 eZOEb ocooq __ 1339?):- ~°~°~ N .3 o c c 90?: v-2: n; c N... A My “‘ (Z: '— i— : —-— ”W y/lml _— ‘tyxu 3:33 i h—A M —.J CS 8 P W C CS 8 P 3 ‘ ’ T T 1 4 non aux: m u u 1 - _1., L_. N. CS S P WC CS 8 P GeT diagrams of unheated whoTe casein (WC), soy protein (SP), and their b1end (CS). The ge1 fiTtration fraction is denoted by the number in the upper right hand corner. 219 1 1 M, 2 “”1 75!? L332” eé' “0" 1—1 A- -- 1:! fife/1‘ “ L—T -:- 813 C53 VVC S13 (IS VVC 2} 7 m S P CS WC 8 P CS WC Figure 16. GeT diagrams of whoTe casein (WC), soy protein (SP) and their b1end (CS) heated at 680C/30 min. The geT fiTtration fraction is denoted by the number in the upper right hand corner. bbbbbbb MIC IUUU ”‘-1 Figure 17. 220 1 3 O O DQCCSCS .— EOOCSCE 9000:: 33:3: DOOCOCC .575; honoree) _ t: : (:22: WA' 1——-I L C : = 1 m A n n 4 h‘ 5 9 c -—-—1 CS wc cs SP 1 1 1 .‘,_"_,"i 1.... cs 3 P we cs 3 p GeT diagrams of whoTe casein (WC), soy prote1n (SP) and their b1end (CS) heated at 94°C/TO sec. The ge1 fiTtration fraction 15 denoted by the number in the upper right hand corner. Figure 18 . P.) Disc gels of whole casein, soy protein and t‘nsir blend, heated at: unheated-control, 68 C/30 min and 94°C/10 sec. The gel bands represent protein present in gel filtration fractions 1-4 (fran left to right). ZHmpOmm wow .. .. ' mmr ozmqm 2 2 1r}- -flq...- I szmac 4H . 48.528 ZHMHONE Mow 2.ng Q l. - 1. ~—-‘ . ‘f 3.1 .l ZHmbHOmm Mow szqm ZHmm/NU '33. com NNNooNN 223 component. The casein geTs remained approximateTy the same regard1ess of the heat treatment empToyed. Soy protein, fraction 1, was composed of the 7S and TTS fractions primari1y. Fraction 2, contained (in addition to residuaT 7S and TTS proteins) two species which migrated farther down the geTs than the 7S and TTS fractions. Frac- tions 3 and 4 were aTso composed of faster migrating species. These patterns were simi1ar to the soy nonurea geTs. The ge1 diagrams of samp1e heated to 68OC/3O min remained about the same. Disc PAGE of the samp1es heated to 940C/TO sec demonstrated the heat susceptibiTity of soy 7S and TTS pro- teins. The breakdown of these fractions resuTted in a greater number of 1ess intense bands. VisuaT examination and ca1cu1ation of RF va1ues (com- paring the bTended and unb1ended systems) were used to investigate the possibiTity of casein:soy protein interac- tion. Soy 7S and TTS proteins did correspond to zones found on geTs of the CS b1end, fraction 1. SeveraT minor soy protein bands aTso had the same RF va1ues. In the b1ended system the major caseins were eTuted in the first two frac- tions. The casein geTs from a11 three fractions were examined and compared primari1y against CS ge1s T and 2. The Targe number of minor soy and casein bands made it extremeTy difficuTt to reTate a11 of the bands. In addi- tion, the smearing of soy 7S and TTS proteins (caused by heating at the higher temperatures) compTicated the situ— ation. A heat treatment of 68°C/30 min did not substantia11y 224 a1ter those resu1ts recorded for the unheated samp1es. Heating the soTs at 94°C/TO sec resu1ted in the partia1 breakdown of soy 7S and 11S proteins. These same proteins were not as dissociated when heated in the presence of casein at 94oC/TO sec. The bands retained greater inten- sity. There appeared to be some mechanism protecting the soy protein from heat dissociation. The casein portion of the b1end geTs remained approximate1y the same. The major casein and soy protein bands were accounted for by visuaT examination and ca1cu1ation of the RF va1ues. It was dif- ficuTt to reTate aTT of the minor bands because of their number and Tocation. A densitometric scan was performed upon geTs of WC, SP and the CS b1end which had been subjected to heat treat- ments of: T) unheated—controT and 2) 94°C/TO sec. Migration distances and traces were obtained. The densitometric traces of the geTs (unheated—controT) of WC, SP and CS are shown in Figure 19. Soy and casein had many minor components. By combining the traces obtained from soy and casein it was observed that its pattern was quite cTose to that found for CS. Retention times were very simi1ar. This work confirmed the data gathered by visuaT examination of the protein systems. The ge1 fiTtration chromatogram for casein (void voTume from the sephacryT coTumn concentrated TO to 1) reappTied over sepharose is shown in Figure 20. OnTy patterns for samp1es heated at: T) unheated-controT, 2) 68°C/3O min and 225 .NNNN NeNNN NNNNN Nee Nuzv Newman m_o;3 .Aamv :Nmboca New No ANocucooieprmsczv MNNN New No muecp NNNNNEONNNCNQ .NN mezmwd mQZ_ NN \hi N : mm mo ABSORBANCE 226 Figure 20. Gel filtration chromatograms of whole casein concentrated and reapplied over Sepharose 4B. The heat treatments included: unheated-control (a), 680C/30 min (b) and 9400/10 sec (d). 227 .02 20:069.... . OO O? N }./11111\\111 T _ EONVBBOSBV 228 3) 94°C/TO sec are shown because samp1es heated at 770C/ 20 sec and 121°C/5 sec did not show appreciab1e differences from those presented. GeT fiTtration of casein soTs resuT- ted in the resoTution of on1y one fraction. This pattern changed very 1itt1e as the heat treatment varied. The geT fiTtration chromatogram of the CS bTend is presented in Figure 21. GeT fiTtration resu1ted in the separation of two fractions. The first, eTuting near the tota1 voTume of the coTumn was composed of both casein and soy protein whi1e the other was principaTTy soy protein. The nitrogen distribution (Tab1e 42) shifted towards the second peak as the heating temperature rose. This shift foTTowed that observed for soy protein. The disc PAGE geTs ( % in 7M urea) of the materia1 reappTied over sepharose are shown in Figure 12. The casein ge1s contained severa1 bands, many of which had RF va1ues cTose to those observed for bands found in the CS geTs. The soy 7S and TTS proteins were aTso observed on the geTs of the CS b1end. There was no evidence that any interaction (disu1fide) ' had occurred between casein and soy proteins. There was no indication that new bands had been formed or that on bands had disappeared. The possibiTity remains that there may be substantia1 hydr0phobic interaction between casein and soy proteins. ATdrich (1977) demonstrated that soy 7S and TTS proteins owe much of their stabi1ity to intramoTecuTar hydrophobic bonding. Ribadeau-Dumas t 1. (1972) and 229 Figure 21. Gel filtration chrcrnatograms of the casein: soy protein blend fraction 1 , concentrated and reapplied over Sepharose 4B. The heat treatments included: unheated-control (a), 68°C/30 min (b) and 94°C/10 sec (d). 230 GO . .OZ ZO§U<~E O? \\ SONY/8808817 231 Tab1e 41. Nonprotein nitrogen of samp1es of AC, AS, SP, CS and WC heated at 94°C/TO sec and TZTOC/S sec Treatment Samp1e o o 94 C/TO sec 121 C/5 sec 409 5.0 6.3 Asb 4.3 6.0 SPC 4.4 6.6 csd 2.8 3.0 woe 0.9 13 a = whey protein from acid whey b = whey soy protein combination c = soy protein d = casein:soy protein combination e = whoTe casein Tab1e 42. 232 Percent nitrogen in each fraction of whoTe casein and the casein:soy protein bTend sepa- rated by geT fiTtration with sepharose 4B Treatment Fraction No. WhoTe casein 1 2 Contr01 100.0 - 680C/3O' 100.0 - 9400/10" 100 o - Caseinzsoy protein b1end Contr01 38.2 61.8 680C/30' 37.1 62.9 94OC/TO” 17.2 82.8 233 Mercier g3 g1. (1973) reported that the casein constituents possess higher than average hydrophobicity. The stabi1ity of soy protein in the presence of casein may be due to this bonding. In Vitro Estimation of Protein QuaTity In vitro enzymatic digestions are rapid, sensitive t001s which have importance in the nutritionaT evaTuation of protein qua1ity. Many researchers have used these pro- cedures and concTuded that the resu1ts observed were com- parabTe to animaT feeding studies (Sheffner _g gl., T956; Akesson and Stahmann, 1964; Stahmann and Wonegiorgis, T975; SatterTee, T977; and McCune, 1977). In vitro enzy- matic hydroTyses have been used to estimate the differences in qua1ity between proteins and between treatments affec- ting one or more proteins. Various techniques (incTuding quantitation of amino acids) have been empToyed for the purpose of measuring and re1ating enzymatic hydroTyses to protein qua1ity. MeTnick (1964) used a modified formaT titration to estimate the extent of hydroTysis. Van Buren t T. (1964) found significant correTation between free amino groups and PER (at the 99% TeveT). Yamashita _g‘gl. (1970) caTcuTated the pepsin-pancreatin digestibiTity of soy pTastein whi1e Saunders g3 g1. (1973) estimated the digestibiTity of anana protein by determination of the residue protein. Ford and SaTter (1966) used both static 234 and dynamic digestions to estimate protein quaTity. Digests were anaTyzed for soTubTe protein, peptide content and free amino acids. BroadTy simiTar resu1ts were obtained for a11 the methods. Maga g3 g1. (1973) measured the initiaT rate of proteoTysis and was abTe to correTate this to protein quaTity. Hsu _g _l. (1977) used a muTtienzyme technique to estimate protein digestibiTity. This method was sensitive enough to detect processing and protease inhibitor effects. In this research, nonfat dry mi1k, soy isoTate and their b1end were reconstituted in distiTTed water and sub- jected to 24 different treatments. Portions of each samp1e were then exposed to a sequentiaT pepsin-pancreatin diges- tion. FoTTowing precipitation and removaT of the residue protein, the amino nitrogen was determined in the supErna- tant of each samp1e. The purpose of this study was twofon: firstTy, was there significant interaction between proteins which affected Tiberation of amino nitrogen and secondTy, did any of the treatments increase or decrease the amount of nitrogen re1eased. The statisticaT procedures empToyed (appendix) consisted of 2 factor anaTysis, Dunnett's t test for comparison of treatment affects and a f test for con- trast among materiaTs. The amino nitrogen of the in vitro digested samp1es were compared to va1ues from acid hydroTy- ses of the same untreated materiaTs. A percent of the totaT (acid hydroTysis) was then computed for each samp1e. The data in Tab1e 43 are representative of the va1ues gathered in this study. Two controTs were used to compare 235 treatment effects. No treatment (NT) samp1es were empToyed as controTs against the samp1es not heated to 68OC/30 min. The samp1es heated at 680C/3O min were used as controTs for a11 samp1es empToying this heating process as part of their treatment. ATT samp1es were prepared and anaTyzed in tripTicate. In vitro enzymatic hydroTysis re1eased approximateTy 59% of the amino nitrogen from each of the untreated samp1es. The va1ues were very cTose regard1ess of the protein source. None of the heat treatments empToyed (680C/30 min, 770C/ 30 min, 9400/4 sec, 1210C/4 sec, 11300/15 min, microwave 770C/f1ash) significant1y aTtered the re1ease of amino nitrogen from the samp1es. The amount Tiberated from soy increased sTightTy, though the va1ues were not significant. 1. (T965), HackTer g3 g1. (T965) and Fritz g: T. Taira gg (1947) reported that heating soybean products at high tem- peratures (TOO-1150C) for one hr or 1ess had no detrimentaT effect on the growth rates of animaTs fed these products. Hankes g3 1. (1948), Menden and Creamer (T966) and Mauron gg a1. (1955) found that autocTaving had no effect on the BV of casein. Cook _3 _1. (1951) reported that there was no change in the PER of a commerciaTTy prepared TactaTbumin heated at 100°C. NormaT heat processing does TittTe nutri- tionaT damage to the protein in miTk products and often enhances the qua1ity of Tegume proteins (Bender, 1972). Addition of either gTycan or CMC prior to heating at 680C/30 min did not significant1y a1ter re1ease of amino 236 nitrogen. Addition of reducing and oxidizing agents Tikewise did not substantia11y modify Tiberation of nitrogen. Patton (1954) suggested that the sunTight fTavor in miTk products might be caused by degradation of methionine to methionaT. In this study samp1es were exposed to sunTight sufficient to cause this fTavor probTem. However, no significant decrease was noted in the amount of re1eased nitrogen. Addition of soybean oiT to the samp1es prior to heating did not signifi- cant1y a1ter Tiberation of amino nitrogen. Hsu (1977) found that the presence of fat had no effect on the proteoTytic digestion of severa1 samp1es. Addition of gTucose prior to heating did not resu1t in decreased Tiberation of amino nitrogen. Various researchers have shown that autocTaving casein in the presence of gTu- cose resu1ted in an appreciab1e Toss of avai1abTe Tysine. DimTer (1975) observed that the totaT amino acids remained approximate1y the same (when casein was autocTaved in the presence of gTucose) even though the TeveTs of certain amino acids decreased. In this study the materia1 was not auto- cTaved but heated at the much Tower temperature of 68°C. Thus the temperature enhancing effect may have been Tess. SecondTy, this technique may not be sensitive enough to compensate for partia1 Toss of one amino acid reTative to the totaT. Of the 24 treatments examined onTy 4 were found to significant1y affect the Tiberation of amino nitrogen from intact proteins (Tab1e 44). UtiTizing the Dunnet t test 237 these 4 treatments were found to have significance at the 99% TeveT. Adjustment of the pH to 11.5 substantia11y re- duced the Tiberation of amino nitrogen. Badenhop (1970) reported that high pH reduced the PER of soy protein, which was mainTy due to the destruction of cystine. Degroot and STump (T969) evaTuated the treatment of food proteins at pH 12.2. The TeveTs of cystine, Tysine, serine and arginine decreased which correTated to the reduction in NPU of the protein. Osner and Johnson observed that high pH treatment resuTted in the destruction and avai1abi1ity of amino acids. The Toss of amino nitrogen Tiberated by in vitro digestion was partiaTTy due to the decomposition of squur containing amino acids. The succinyTation of the samp1es resu1ted in marked1y Tower amino nitrogen va1ues. Succinic anhydride reacts with the 8 amino group of Tysine and renders this bond unavaiTabTe to tryptic digestion. With tryptic hydroTysis being 1imited to those bonds invoTving arginine, the tota1 amino nitrogen re1eased cou1d conceivabTy be reduced. MaTeyTation with ma1eic anhydride did not resu1t in the same reduction. This is possib1y due to the fact that ma1eic anhydride is debTocked when her at Tow pH for severa1 hrs (conditions encountered in the digestion pro- cess) whereas succinic anhydride is more difficuTt to de- bTock. SuccinyTation to improve the functionaTity of proteins cou1d depress the nutritive vaTue of the protein. 238 The incubation of oxidizing saffTower oi1 with protein significant1y Towered the amount of re1eased amino nitrogen. The Tiberated nitrogen was reduced by about 45%. Osner and Johnson (1968), Horigome and Miura (1974) and Yanagita and Sugano (1973) reported that oxidized fats Towered both the digestibiTity and bioTogicaT vaTue of the protein. Lewis and WiTTs (1962) found that exposing protein to oi1 under- going oxidation resuTted in rapid destruction of the SH groups associated with the amino acids.' RoubaT and TappeT (1966) described the damage to proteins by peroxidizing Tipids. Transient free radicaTs were produced in the pro- tein-Tipid system. The damage to proteins inc1uded Toss of soTubiTity and destruction of such amino acids as methi- onine, histidine, cystine and Tysine. McCune (T977) repor- ted that oxidizing Tipid reduced the PER of proteins incu- bated in its presence. Homogenization of the samp1es significant1y increased the Tiberation of amino nitrogen. This may have in part been due to the increased soTubiTity of the samp1es (see Functiona1ity page87). This treatment may have physicaTTy disrupted the structure of the proteins, possib1y increas- ing their vuTnerabiTity to enzymatic digestion. The examination for materia1 interactions was conducted using a f test for contrast among materiaTs. AnaTysis of the data reveaTed that there were no materia1 interactions which resuTted in significant1y higher or Tower amounts of re1eased amino nitrogen. No va1ues were obtained from 239 hydroTysis of the b1end samp1es (regard1ess of the treat— ment) which cou1d not be accounted for by examination of the measurements from the unb1ended samp1es. MateriaT interactions were not significant at the 95% TeveT. Processing damage can resu1t from four different types of reaction, nameTy: T) destruction of amino acids by oxi- dation, 2) Toss of paTatabiTity, 3) modification of Tinkages or functiona1 groups and 4) formation of Tinkages that are not bioTogicaT avai1ab1e. Loss of avai1abi1ity can occur through: 1) reaction between the amino group of the amino acid and a reducing substance, 2) reaction between the terminaT group of Tysine and the carbonyT Secondary decom- position products of autoxidizing Tipids and 3) protein- protein interaction independent of the presence of reducing substances (Bender, 1973). In this study there were no interactions between the protein systems that were respon- sibTe for significant1y different TeveTs of Tiberated amino nitrogen. Four of the treatments tested significant1y affected the re1ease of amino nitrogen from intact proteins. In genera1, most of the processing treatments empToyed had TittTe effect. 240 TabTe 43. The percent of amino nitrogen re1eased by in vitro hydroTysis of NFDM, soy isoTate and their bTend as affected by various treatments * Treatment Samp1e NFDM NFDM:Soy IsoTate Soy IsoTate NT 59.8 58.3 59.1 680C/30 min 58.4 57.3 59.0 121°0/4 sec 56.7 60.4 60.2 CMC-68°C/30 min 55.4 63.0 60.9 gTycan-68°C/30 min 62.3 59.7 60.3 Cys.-H202-Cat. 61.0 62.6 58.1 gTucose-68°C/3O min 60.1 60.2 58.5 SunTight oxid, 58.4 59.8 57.1 *Means of tripTicates 241 Tab1e 44. The percent of amino nitrogen re1eased by in vitro hydroTysis of NFDM, soy isoTate and their b1end as affected by various treatments Treatment Samp1e* NFDM NFDM:Soy IsoTate Soy IsoTate pH 11.5 45.3 41.3 33.7 Succinic Anhyd. 37.7 40.1 41.4 Oxid. OiT 32.7 32.4 32.4 Homog.-680C/3O min 59.1 63.2 66.0 ___. *Means of tripTicates SUMMARY The functiona1 properties of miTk products, soy iso~ Tate and their b1ends were examined at three protein con- centrations and severaT bTend ratios. The sampTes were subjected to forty five different treatments prior to evaTuation of their functionaTity. MiTk products were the most soTubTe of the materiaTS studied. Sodium caseinate, WPC and eTectrodiaTyzed whey had soTubiTities greater than 90%. Soy isoTate had the Towest soTubiTity of any of the materiaTs examined. MiTk product:soy isoTate b1ends were found to have appreciab1y higher soTubiTities than soy isoTate. Specific treatments resu1ted in wideTy varying soTubiTities which made it pos- sibTe to achieve greater soTubiTity by se1ecting the appropriate treatment for the protein. At 3.2% protein the viscosities of the miTk products, soy isoTate and their b1ends were simi1ar regard1ess of the treatment. The viscosities of either sodium caseinate or soy isoTate increased with increasing concentration. At 8.0% protein the viscosities of reconstituted eTectrodia- Tyzed and sweet whey powders were very high due to the high totaT soTids content. Specific treatments resu1ted in higher (or Tower) viscosities but the majority had TittTe 242 243 effect. Very 1imited success was achieved when the miTk pro- ducts were subjected to a heat process designed to encourage ge1ation. OnTy WPC demonstrated appreciab1e potentiaT. Edi-ProN, the soy isoTate used throughout this research, did not geT when subjected to the estabTished conditions. Promine D, a soy isoTate, which demonstrated substantia1 ge1ation abi1ity was used in Tieu of Edi-ProN. WPC:Promine D b1ends formed firm geTs though sTight wheying off occurred with partia1 visuaT separation of the two protein systems. Soy isoTate had sTightTy Tess emuTsion capacity than the mi1k products. BTends of soya and miTk components often had emu1sion capacities approaching those of pure mi1k products. Specific treatments improved (or Towered) the emuTSion capabi1ity of the samp1es. By se1ecting the treat- ment corresponding to a particuTar protein it was possibTe to obtain substantia11y improved emuTsion capacity. StabTe foams were produced from 5015 of NFDM, sodium caseinate, eTectrodiaTyzed whey and their soy isoTate bTends. WPC 5015 were whipped into stab1e foams after subjection to the appropriate treatment. Soy isoTate soTs whipped into stab1e foams in onTy a few instances. MiTk product:soy isoTate foams had approximate1y the same specific voTumes but shorter stabi1ity times than the respective miTk pro- duct. Many treatments improved the whipping properties of the samp1es. 244 The sensory properties of soy isoTate were unaccep- tabTe. However, the fTavor scores of miTk product:soy isoTate b1ends were on1y sTightTy 1ess than those recorded for the particuTar mi1k product. Many treatments faiTed to improve the sensory qua1ities of the samp1es. SeveraT treatments whiTe marked1y improving functionaTity resu1ted in unsatisfactory fTavor characteristics. This may Timit or modify the use of such treatments in actuaT food pro- ducts. FunctionaT testing of proteins provides the technoTo- gist with information important to his understanding of food ingredients. The data gathered in this study shoqu not be interpreted as having direct appTication to food products. The resu1ts shoqu be used as guideTines from which to make reasonabTe decisions. ModeT systems provide the basic information necessary to incorporate ingredients, in a TogicaT order, into food products. The second part of this research invoTved the exami- nation of miTkzsoy protein b1ends for heat induced inter- actions. The miTk proteins inc1uded whey proteins and caseinates. The soy protein was extracted from soy isoTate. GeT fiTtration resoTved whey protein into five dis- tinct fractions none of which were homogenous but a11 of which were composed of one major species. GeT e1ectrophore- sis identified these proteins as immunogTobuTins, serum aTbumin, B-TactogTobuTin and o-TactaTbumin. These proteins were stab1e to heat treatment through 77OC/20 sec. Heating 245 at greater temperatures re5u1ted in substantia1 denatura— tion. Serum aTbumin, the immunogTobuTins and B~TactogTo- buTin were the most heat sensitive whi1e a-TactaTbumin was the most heat resistant. GeT fiTtration resoTved casein into three major and one minor fraction. The fractions were heterogenous. ATT of the casein components were stab1e to the heat treatments empToyed in this study. GeT fiTtration resoTved soy protein into four fractions. Fraction 1 was composed of the soy 7S and TTS proteins. These proteins were stabTe to the Tow temperature pasteuri- zation processes normaTTy encountered during the heating of fTuid miTk products. Heating soy protein at UHT tempera— tures resu1ted in partia1 denaturation and Toss of soTubiTity. The 7S and TTS proteins underwent substantia1 dissociation into other components. At 94°C/TO sec and 121°C/5 sec the 7S and TTS proteins near1y disappeared. UtiTizing geT e1ectrophoresis, no interaction cou1d be detected between the major whey and soy proteins. The pro- teins of both systems moved independentTy of each other. The addition of urea to samp1es containing casein precTuded the possibiTity of detecting other than disu1fide interchange between soy and casein. There did not appear to be any interaction of this type taking p1ace. The DOSSTbTTTtY exists that hydrophobic interaction between the two systems may haveoccurred.Interaction between proteins from different sources coqu conceivabTy affect the functionaTity of protein 246 bTends. Examination of these interactions is of importance because of the possibTe benefit derived from the modifica- tion or manipuTation of them. The finaT segment of this research was concerned with examining the protein qua1ity of products used in this study. Samp1es of NFDM, soy isoTate and their b1end were exposed to 24 different treatments prior to in vitro enzy- matic hydroTysis. The amino nitrogen Tiberated by this digestion was quantitated and compared to the amino nitrogen Tiberated by acid hydroTysis. Two factor anaTysis, Dunnett's t test and a f test were empToyed to determine if materia1 interactions had significant1y affected the re1ease of amino nitrogen and to determine whether or not the processes had aTtered Tiberation of amino nitrogen. Of the treatments examined onTy four (succinic anhydride, oxidized oiT, pH 11.5 and homogenization) significant1y modified the Tibera- tion of amino nitrogen. No materia1 interactions took pTace which significant1y affected the re1ease of amino nitrogen. Most of the treatments empToyed in this study did not resu1t in nutritionaT damage to the proteins. APPENDIX APPENDIX Lowry Procedure for SoTubTe Protein Reagent A was 2% Na2C03 and 0.02% sodium tartrate in a 0.1N NaOH soTution. Reagent B was a 0.5% CuSO -5 H O soTu- 4 2 tion, Reagent C was prepared from 50 parts A + 1 part B. To samp1es containing SO-SOO pg of protein per m1, 5.0 m1 of reagent D was added. FoTTowing 10 min of incubation, 0.5 m1 of phenoT reagent (FoTin-CiocaTteau reagent-Fischer) was added in Tess than 2 sec with a bTow out pipet. CoTor was aTTowed to deveTop 30 min before absorbance was read.. Lowry Procedure for InsoTubTe Protein Reagent D was 2% Na2C03 and 0.0 % sodium tartrate in H20. Reagent B was discussed previousTy. Reagent E was prepared from 50 parts 0 and 1 part B. The sampTes contain- ing 50-500 ug/mT were made up in T.ON NaOH and p1aced in a boiTing water bath for 10 min. The standards were treated in an identicaT manner. After cooTing the samp1es were handed in the same way as described previous1y for Lowry soTubTe protein. KjerahT Nitrogen The digestion mixture was prepared by adding 5.0 g CuSO4°5 H20 and 5.0 g SeO2 in 500 m1 of concentrated H2S04. 247 248 The indicator consisted of 400 mg bromocresoT green and 40 mg of methyT red in 100 m1 of 95% ethanoT. PAGE SoTutions For systems not containing mi1k caseins as a component, a stock soTution of 10% Cyanogum 41 (Fischer) in Tris-HC1 buffer, pH 8.9 (4.6 g tris/TOO m1 made to pH 8.9 with HCT) was uti1ized. The geTs, 5 and 7% were made by taking the proper amount of stock cyanogum 4T soTution and adding to it additionaT Tris-HC1 buffer, pH 8.9 to a tota1 of 16 m1. To this was added 20 p1 of TEMED, (N, N, N', N' tetramethyT- enediamine, Bio-Rad Laboratories). To initiate poTymeriza- tion, 0.1 m1 of a 5% soTution of ammonium persquate was added. The samp1es containing casein were examined in 7 M urea ge1s. The ge1 buffer (Tris-HCT, mentioned previousTy) was made to contain 7 M urea (42 g/TOO m1 of stock soTution). The geTs, 5 and 7% were prepared and run as before. The running buffer was prepared by adding 11.2 g tris and 57.6 g gTycine to 500 m1 of deionized distiTTed water and adjus- ting the pH to 8.3 with 2M gTycine. MaTik-Berrie Staining Procedure GeTs stained with this procedure did not require any destaining. The position of the tracking dye remained visibTe after staining. EquaT voTumes of 0.2% Coomassie b1ue G 250 and 2N H2S04 were mixed together. After standing overnight in the dark the soTution was fiTtered. Ten normaT KOH was added untiT the cTear brown soTution turned dark 249 purpTe-bTue. This required approximate1y 10 m1 of 10 N KOH per 90 m1 of cTear brown soTution. The purpTe-bTue soTution was then made 12% in TCA. Ninhydrin SoTution Four hundred mg of stannous chTOride dehydrate were dissoTved in 250 m1 of 0.2M citrate buffer at pH 5.0. The above soTution was added to 250 m1 methyT ceTTosoTve (ethy- Tene gTycoT monomethyT ether) containing 10 g of dissoTved ninhydrin (CaTbiochem). The soTution was f1ushed with nitro- gen and stored in a brown gTass bottTe at 40C. Acid HydroTysis Acid hydroTysis was performed by adding 5 m1 of 6 N HCT to the dried samp1e and freezing the mixture in a dry ice-ethanoT bath. The ampouTes were evacuated, aTTowed to meTt under vacuum to remove gasses, refrozen, seaTed with a propane torch and p1aced in an oi1 bath maintained at 1100C. After 22 hours the ampouTes were removed and aTTowed to cooT to room temperature. StatisticaT Procedures Used in the EvaTuation of Protein QuaTity 2-factor anaTysis of variance degrees of freedom (df) Summ. Treatments 23 sst = (trt. tot.)2/9 - CF MateriaTs 2 55m = (mat. tot.)2/2 - CF T X M 46 SStm = (comb. tot.)2/3 - CF Error 144 sse = ssy-(sst+sstm) 250 CF - (grand tota1)2/216 ssy = (216 oosv)2 MSt = SSt/23’ MSm = SSm/Z, MStm f = MSt/MSe, f = MSm/MSe, f = Mstm/Mse -CF = 46, sse/144 f test for contrast among materiaTs _ 2 f — (meanNFDM + MeanSI — 2 MeanComb.) /6 (MSe/3) Dunnett's t test for treatment effects t = treatment meanx - treatment meancontr01/ 2MSe/3 BIBLIOGRAPHY BIBLIOGRAPHY Akeson, W.A. and Stahmann, M.A. 1964. A pepsin-pancreatin digest index of protein qua1ity evaTuation. J. Nutri- tion 83:257. ATais, C., N. Kiger and P. JoTTes. 1967. Action of heat on cow K-casein. Heat caseino-gTycopeptide. J. Dairy Sci. 50:1738. ATdrich, L.C. 1977. Heat induced interactions between soy (7S and TTS) and mi1k proteins. Thesis, Michigan State University. AmiTari, M., L.K. Ferrier and A.I. NeTson. 1977. 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