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Major professor Date (Q 32¢ -' qR 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution _~ LIBRARY Mlchigan State g University PLACE IN RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE J51997] '.‘ "‘9 «:2 =7 MSU I. An Affirmative Action/Equal Opportunlty Institutlon cWMmS-M INTERACTIONS OF PARTIALLY INSOLUBILIZED WHEY PROTEIN CONCENTRATES AND CHICKEN BREAST SALT SOLUBLE PROTEIN IN MODEL GEL AND MEAT SYSTEMS BY Tan-Yi Hung A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Master of Science Department of Food Science and Human Nutrition 1992 ABSTRACT INTERACTIONS OF PARTIALLY INSOLUBILIZED WHEY PROTEIN CONCENTRATES AND CHICKEN BREAST SALT SOLUBLE PROTEIN IN MODEL GEL AND MEAT SYSTEMS by Tan-Yi Hung Viscoelasticityy microstructure, and protein composition of four ultrafiltered Parmessan cheese whey protein concentrates (WPCs) containing 62.4% protein with solubilities ranging from 98% to 27% in 0.1 M NaCl, pH 7.0 were evaluated. Storage (6') and loss (6") moduli of solutions containing 4% salt soluble protein (SSP), 16% WPC, or combinations of 4% SSP and 12% WPC (on protein basis) in 0.6 M NaCl, pH 7.0 buffer were determined while heating from 30 to 95°C or isothermally at 65 or 90°C. The WPCs altered the temperature and magnitude of G' and G" transitions of SSP during heating. At 65°C, insolubilized WPCs increased elasticity of SSP gels. Highly soluble WPC enhanced elasticity of combination gels more effectively at 90°C. Microstructure of combination gels containing highly soluble WPCs was composed of a fibrous network of SSP at 65°C, and globules of WPC at 90°C. For the combination gels containing highly insoluble WPCs, large denatured whey protein aggregates distorted the ordered SSP fibrous matrix. 3 gm /a- A ’7 69 DEDICATION To my mother, Wen-Chen Lo, and my whole family. iii ACKNOWLEDGMENTS The author expresses her deepest appreciation to major advisor Dr. Denise Smith for her guidance and support throughout the studyu Sincere appreciation is extended.to the committee members, Drs. Alden Booren and John Partridge, Department of Food Science and Human Nutrition, and Dr. Stanley Flegler, Pesticide Research Center. Special thanks are also extended to my friends, Chin-Gin Low, Virginia Vega, Cheng Hsin Wang, and Shuefung Wang for their friendship and help throughout this graduate program. iv TABLE OF CONTENTS P??? LIST OF TABLES...... ...... ..............................v111 LIST or FIGURES..... ..... ................................ xi CHAPTER I INTRODUCTION... ....................... ...... ...... ... 1 II LITERATURE REVIEW.............. ....... ............... 4 2.1 Whey Protein Concentrates....................... 4 2.1.1 Characteristics of Whey Proteins......... 4 2.1.2 Thermal Denaturation of Whey Proteins.... 6 2.2 Myofibrillar Proteins........................... 8 2.3 Theory of Protein Gelation...................... 9 2.4 Sinusoidal Oscillatory Testing.................. 11 2.5 Gelation of Muscle Proteins..................... 13 2.6 Gelation of Whey Protein Concentrates........... 19 2.7 Multicomponent Gels............................. 25 2.8 Nonmeat Ingredients in Meat Systems............. 30 III MATERIALS AND METHODS.............................. 35 3.1 Whey Protein Concentrate Treatments............. 35 3.2 Electrophoresis of Whey Protein Concentrate..... 36 3.2.1 Polyacrylamide Gel Electrophoresis....... 36 3.2.2 Sodium Dodecyl Sulfate Polyacrylamide Gel ElectrophoreSiSOOOOOO00......00...... 37 3.3 Whey Protein Concentrate Solution Preparation... 38 IV 3.4 Extraction of Chicken Breast Salt Soluble ProteinOO......OOOOOOOOOOOOOOCO00.000.000.000... 3.5 Combination of Whey Protein Concentrate and salt ProteinOO0....0..........OIOIOCOCOOOOOOO... 3 O 6 Dynamic Testing. 0 O O O O O O O I O O C O O O O O O O O O O O O O O O O O O O O 3.7 Scanning Electron Microscopy.................... 3 O 8 PrOduct Preparation 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 3.8.1 Silent Cutter Processed Low Fat Frankfurters............................. 3.8.2 Vacuum Processed Low Fat Frankfurters.... 3.8.3 Chicken Rolls.. ............. ............. 3.9 Product Evaluation.............................. 3.9.1 Chemical Analysis........................ 3.9.2 Cooked Yield............................. 3.9.3 Severe Reheat Yield...................... 3.9.4 Texture Profile Analysis................. 3.9.5 Tensile Strength......................... 3.9.6 Color.................................... 3.10 Statistical Analysis............................ RESULTS AND DISCUSSION............................... 4.1 Effect of Heat Treatment on Whey Protein Concentrates.................................... 4.1.1 Polyacrylamide Gel Electrophoresis (PAGE)0.000.0.I..........OOOOOOOOOOOOOOO. 4.1.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)........... 4.2 Dynamic Testing...’0.000.000.0000.........OOOOOC 4.2.1 Whey Protein Concentrate Gels............ 4.2.1.1 Thermal Scanning from 50 to 95°C....OOO......OOOOOOOOOOOOOOO vi 39 40 40 42 43 43 44 46 48 48 48 48 48 50 51 51 52 52 52 56 61 61 61 4.2.1.2 Isothermal Heating at 90°C...... 66 4.2.2 Chicken Salt Soluble Protein and Whey Protein Concentrate (SSP/WPC) Combination Gels.......OOOOOOOOOOOOOOOOO0.0.......... 71 4.2.2.1 Thermal Scanning from 30 to 95°C..........OOOIOOOOIOO0...... 71 4.2.2.2 Isothermal Heating at 65°C for 15 min.‘......OIOOOOOOOOOOOO.... 84 4.2.2.3 Isothermal Heating at 90°C for 15 min.’......OOOOOOOOOOIOOOOIO. 90 4.2.3 Effect of Heating Conditions on Rheological Properties of Gels........... 96 4.3 Scanning Electron Microstructure (SEM)..........100 4.3.1 Salt Soluble Protein (SSP) Gels..........100 4.3.2 Whey Protein Concentrate (WPC) Gels......100 4.3.3 Salt Soluble Protein/ Whey Protein Concentrate Combination Gels.............106 4.3.3.1 Combination Gels Heated at 65°C........106 4.3.3.2 Combination Gels Heated at 90°C.109 4.4 Effect of Whey Protein Concentrates in Meat Model Systems...................................115 4.4.1 Model System Low Fat Frankfurters........115 4.4.1.1 3.5% WPC-Supplemented Low Fat Frankfurters....................115 4.4.1.2 3.5% WPC-Supplemented Low Fat Frankfurters Processed Under VacuWOOOOOOOOOOOOO0.0.0.0000000119 4.4.1.3 7.0% WPC-Supplemented Low Fat Frankfurters....................126 4.4.2 Model System Chicken Rolls...............131 V CONCLUSIONS.........................................138 VI LIST OF REFERENCESOOOO........0......00.000.000.0000142 vii Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 LIST OF TABLES Examples of techniques used for protein salutionmeasurementOOOOOOOIlCO......00.0.00... 14 Conformational changes which may occur during the thermal denaturation of natural actomyosin (from Ziegler and Acton, 1984)................. 17 Formulation for low fat chicken frankfurters with 3.5% or 7.0% whey protein concentrate (WPC) substituted for meat on a weight basis......... 43 Formulation for vacuum processed low fat chicken frankfurters with 3.5% whey protein concentrate (WPC) substituted for meat on a weight basis................................... 45 Formulation for chicken rolls with the addition of 3.5% binders on a weight basis.............. 46 Cooking cycle for chicken rolls................ 47 Dynamic moduli of protein solutions containing 16% whey protein concentrates (WPC) with various solubilities heated isothermally at 90°C for 15 min......................................... 68 Thermal transition temperatures of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble protein and 12% whey protein concentrate (SSP/ WPC) heated from 30 to 95°C.................... 76 Dynamic moduli of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble protein and 12% whey protein concentrate (SSP/WPC) heated from 30 to 95°C... 81 Dynamic moduli of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble protein and 12% whey protein concentrate (SSP/WPC) heated isothermally at 65°C for 15 min................................ 89 viii Table Table Table Table Table Table Table Table Table Table Table 11 12 13 14 15 18 19 20 21 Dynamic moduli of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble protein and 12% whey protein concentrate (SSP/WPC) isothermally heated at 90°C for 15 min................................ 94 Effect of heating conditions on the storage moduli (G') of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble protein and 12% whey protein concentrate (SSP/WPC).................. 97 Chemical analysis of raw and cooked silent cutter processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis...116 Cooked yield and severe reheat yield of silent cutter processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis...117 Texture of silent cutter processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis.........................120 Chemical analysis of cooked vacuum processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis.....................121 Cooked yield and severe reheat yield of vacuum processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis...122 Texture of vacuum processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight baSiSO0......OO.......OCOOOOOOOOOOOOOOOO...... 124 Chemical analysis of raw and cooked silent cutter processed low fat frankfurters prepared by substituting 7.0% various whey protein concentrate (WPC) for meat on a weight basis...127 Cooked yield and severe reheat yield of silent cutter processed low fat frankfurters prepared by substituting 7.0% various whey protein concentrate (WPC) for meat on a weight basis...128 Texture of silent cutter processed low fat ix Table 22 Table 23 Table 24 frankfurters prepared by substituting 7.0% various whey protein concentrate (WPC) for meat on a weight basis.........................130 Chemical analysis of raw and cooked chicken rolls prepared with 3.5% whey protein concentrate (WPC), soy protein concentrate (SPC), or nonfat dry milk (NFDM) added on a weight basis...................................132 Yield, tensile strength, and Hunter color values of chicken rolls prepared with 3.5% whey protein concentrate (WPC), soy protein concentrate (SPC), or nonfat dry milk (NFDM) added on a weight basis........................134 Texture of chicken rolls prepared with 3.5% whey protein concentrate (WPC), soy protein concentrate (SPC), or nonfat dry milk (NFDM) added on a weight basis........................136 Figure Figure Figure Figure Figure LIST OF FIGURES Possible models for multicomponent gel systems composed of a gelling protein and a gelling or non-gelling coingredient (A) type I filled gel or single-phase gel; (B) type II filled gel or two-phase gel; (C) complex gel without continuous linkage; (D) interpenetrating network (from Ziegler and Foegeding, 1990)............................. 26 Polyacrylamide gel electrophoresis of total and soluble fraction of whey protein concentrates (WPC). Sixty ug protein were applied to each sample well. Lanes 1-4 represent WPC with a solubility of 98%, 80%, 41%, and 27%, respectively. Lanes 5-8 represent the soluble fraction of WPC with a solubility of 98%, 80%, 41% and 27%, respectively (Ig: immunoglobulin, BSA: bovine serum albumin, a-la: o-lactalbumin, B-lg: B-lactoglobulin)............................. 53 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and soluble fraction of whey protein concentrates (WPC). sixty ug protein were applied at each sample well. Lane 5 was molecular weight standard. Lanes 1—4 represent WPC with a solubility of 98%, 80%, 41%, and 27% respectively. Lanes 5-8 represent soluble fraction of WPC with a solubility of 98%, 80%, 41%, and 27%, respectively (BSA: bovine serum albumin, B-lg: B-lactoglobulin, a-la: o-lactalbumin)......... 57 Representative rheogram illustrating the storage moduli (G') of 16% whey protein concentrate (WPC) in 0.6 M NaCl, pH 7.0 buffer heated from 50 to 95°C................. 62 Representative rheogram illustrating the loss moduli (6") or 16% whey protein concentrate (WPC) in 0.6 M NaCl, pH 7.0 buffer heated from 50 to 95°C................. 63 xi Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10 11 12 13 14 15 Representative rheogram illustrating the loss tangent (tan 6) of 16% whey protein concentrate (WPC) in 0.6 M NaCl, pH 7.0 buffer heated from 50 to 95°C.................................... 65 Representative rheogram illustrating the storage moduli (G') of 16% whey protein concentrate (WPC) in 0.6M NaCl, pH 7.0 buffer heated at 90°C for 15 min.............. 67 Representative rheogram illustrating the loss moduli (G") of 16% whey protein concentrate (WPC) in 0.6M NaCl, pH 7.0 buffer heated at 90°C for 15 min.............. 70 Representative rheogram illustrating the loss tangent (tan 6) of 16% whey protein concentrate (WPC) in 0.6M NaCl, pH 7.0 buffer heated at 90°C for 15 min............................... 72 Representative rheogram illustrating the storage moduli (G') of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey protein concentrate (WPC) heated from 30 to 95°C.................................... 73 Representative rheogram illustrating the loss moduli (G") of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey protein concentrate (WPC) heated from 30 to 95°C.................................... 75 Definition for the terminology used in analyzing storage modulus (G') of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey protein concentrate (WPC) during thermal scanning test................................. 78 Definition for the terminology used in analyzing loss modulus (G") of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey protein concentrate (WPC) during thermal scanning test................................. 79 Representative rheogram illustrating the storage moduli (G') of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey protein concentrate (WPC) heated at 65°C for 15 min............................... 85 Representative rheogram illustrating the loss moduli (G") of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey xii Figure Figure Figure Figure Figure Figure Figure 16 17 18 19 20 21 22 protein concentrate (WPC) heated at 65°C for 15 minOOOOOO0...............OOOOOOOOOOOIOOOOI. 86 Representative rheogram illustrating the storage moduli (G') of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey protein concentrate (WPC) heated at 90°C for 15 min............................... 91 Representative rheogram illustrating the loss moduli (G") of 4% salt soluble protein (SSP) and combinations of 4% SSP and 12% whey protein concentrate (WPC) heated at 90°C for 15 min........................................ 92 Temperature-time profile for thermal scanning from 30 to 90°C at 2°C/min and isothermally heated at 90°C for 15 min..................... 98 Scanning electron micrographs of salt soluble protein (SSP) gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 65°C for 15 min. (A) bar length equals 5 um; (b) bar length equals 1 um............................101 Scanning electron micrographs of 16% whey protein concentrate (WPC) gels prepared in 0.6M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 90°C for 15 min. (A) 98% soluble WPC gel; (B) 80% soluble WPC; (C) 41% soluble WPC; (D) 27% soluble WPC. Bar length equals 5 umOOIOOOOOOOOOOOOOOO0.00.00.00.0000.000.000.103 Scanning electron micrographs of 4% salt soluble protein (SSP) and 12% whey protein concentrate (WPC) combination gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 65°C for 15 min. (A) 98% soluble WPC/SSP combination gel; (B) 80% soluble WPC/SSP combination gel; (C) 41% soluble WPC/SSP combination gel; (D) 27% soluble WPC/SSP combination gel. Bar length equals 1 um....................................107 Scanning electron micrographs of 4% SSP and 12% WPC combination gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 90°C for 15 min. (A) 98% soluble WPC/SSP combination gel; (B) 80% soluble WPC/SSP combination gel; (C) 41% soluble WPC/SSP combination gel; (D) 27% soluble WPC/SSP combination gel. Bar length equals 5 um....................................110 xiii Figure 23 Scanning electron micrographs of 4% SSP and 12% WPC combination gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 90°C for 15 min. (A) 98% soluble WPC/SSP combination gel; (B) 80% soluble WPC/SSP combination gel; (C) 41% soluble WPC/SSP combination gel; (D) 27% soluble WPC/SSP combination gel. Bar length equals 1 um....................................112 xiv INTRODUCTION The high price of meat products and large variations in the quality of meat proteins from different meat cuts are important stimuli for using non-meat proteins in both whole and comminuted meat products (deWit, 1984) . Whey protein concentrates (WPCs) have received considerable attention as potential food ingredients, because of their excellent nutritional value and wide range of functionality (Morr, 1979) . The high cost of disposal and the need to reduce environmental pollution also increased the use of whey (Delaney, 1976). The capacity to form heat-induced gels is one of the important functional properties of whey’proteins in many food systems (Mulvihill and Kinsella, 1988). The use of WPCs by the food industry has not reached its full potential, due in part to variations in product functionality (Harper, 1984), which result from wide differences in whey composition and processing conditions (deWit et a1., 1983; Morr and Foegeding, 1990). Mulvihill and Donovan (1987) stated that.heating”was used during whey processing and influenced both the structure and functionality of whey protein products. An understanding of the effects of heat on proteins, e.g., denaturation and aggregation, might permit preparation of products with 1 2 predictable functional properties. The effects of heat-insolubilized WPCs on chicken salt soluble protein (SSP) gels were determined by Beuschel (1990) . Results indicated that decreased WPC solubility decreased expressible moisture (EM) and increased gel strength for SSP/WPC gels at 65°C, but increased EM and decreased gel strength at 90°C (Beuschel, 1990). He suggested that WPCs with varying protein solubilities might be useful for improving yield and altering texture of processed meats under specific conditions. Beuschel's study evaluated the effects of heat- insolubilized WPCs on the final gel properties. The present study was conducted to further understand the changes in rheological characteristics during gel formation and the effects of insolubilized.WPCs on SSPIgel microstructure and in poultry products. Four WPCs with solubilities ranging from 98% to 27% (in 0.1 M NaCl, pH 7.0) were prepared using different time-temperature processing conditions. The objectives were: 1) to characterize the WPCs using both native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 2) to monitor the viscoelastic behavior of solutions containing SSP, WPC, and combination of SSP and.WPC*while heating from 30 to 95°C and isothermally at 65 and 90°C, using non-destructive dynamic testing. 3) to examine the microstructure of SSP gels, WPC gels, and SSP/WPC combination gels prepared either at 65 or 90°C. 3 4) to test the use of insolubilized WPCs in model system low fat frankfurters and chicken rolls. LITERATURE REVIEW 2.1 Whey Protein Concentrates 2.1.1 Characteristics of Whey Proteins Whey proteins are defined as those proteins remaining in solution after precipitation of the caseins from milk (Pearce, 1989) . Whey protein concentrate is one of the major whey products. A range of ultrafiltration-derived, spray-dried WPC powders containing 35 to 80% protein have been produced (Kinsella, 1984; Kinsella and Whitehead, 1989). More recently, whey protein isolates (WPI) containing >90% protein were prepared by ion exchange and gel filtration processes (Kinsella and Whitehead, 1989). Whey proteins include four gene products; B-lactoglobulin (B-lg), a-lactalbumin (a-la), bovine serum albumin (BSA), and immunoglobulins (Ig). other whey proteins, collectively termed proteose-peptones (PP) , are present as a result of post-translational proteolysis of caseins (Mulvihill and Donovan, 1987) . Whey proteins are compact globular proteins (deWit and Klarenbeek, 1984). Beta-lactoglobulin comprises up to 50% of the total whey protein (Pearce, 1989) . At room temperature and pH range from 5.5 to 7.0, 13-19 occurs as a dimer (Swaisgood, 1982), maintained largely by electrostatic forces (Pearce, 1989) , consisting of 2 identical subunits, each with a molecular 4 5 weight (MW) of 18.4 KDa (DeWit and Klarenbeek, 1984). Above 40°C, dimers dissociate into monomers (DeWit and Klarenbeek, 1984). Beta-lactoglobulin has a constrained secondary and tertiary structure containing 15% a-helix and 51% B-sheet structure (Creamer et al., 1983). Each monomer contains 5 cysteine residues, four of which are involved in internal disulfide bridges leaving one free thiol group (Pearce, 1989) 'which is inaccessible to solvent at or below neutral pH (Papiz et al., 1986; Kinsella and Whitehead, 1989). Beta- lactoglobulin exists as several genetic variants, of which A and B are most commonly cited in the literature (Eigel et al., 1984). Alpha-lactalbumin is the smallest whey protein with a MW of 14.2 KDa (DeWit and Klarenbeek, 1984). It is a compact Iglobular’ protein stabilized. by four' disulfide bonds and contains 30-40% a-helix (Pearce, 1989). It has been identified as a calcium-metalloprotein (Hiraoka et al., 1980; Pearce, 1989), binding one mole of calcium per mole of protein. Calcium binding may be essential to maintence of the native conformation of a-la (Bernal and Jelen, 1984; Pearce, 1989; Kinsella and Whitehead, 1989). Bovine serum albumin is a transport protein for insoluble fatty acids in the blood circulation system (Pearce, 1989; Kinsella and Whitehead, 1989). A calorimetric study revealed that fatty acids stabilize BSA against heat denaturation (DeWit and Klarenbeek, 1984). Bovine serum albumin is the largest monomeric protein (66 KDa) in whey (DeWit and 6 Klarenbeek, 1984), having 17 intra-chain disulfide bonds and one free thiol group (Mulvihill and Donovan, 1987; Kinsella and Whitehead, 1989). Immunoglobulins in milk and whey include IgM, IgA, IgE, and IgG (Eigel et al., 1984). Immunoglobulins are either monomers or polymers of a four-chain molecule, consisting of two light chains (22 KDa) and two heavy chains (50-70 KDa) (Mulvihill. and Donovan, 1987) joined. by disulfide bonds (Pearce, 1989). 2.1.2 Thermal Denaturation of Whey Proteins The conformation adopted by globular proteins is thermodynamically controlled to attain their most energetically favorable state (Mulvihill and Donovan, 1987). Upon heating (during denaturation) , most forces (hydrogen bonding, hydrophobic and electrostatic interactions, and disulfide bonds) stabilizing the native structure are ruptured, the protein conformation changes to a new identifiable but. predominantly random-coil configuration. Following unfolding of the proteins, binding sites within the native molecule are exposed, resulting in inter-molecular associations and aggregation, and a reduction in protein solubility. The reactivity of the side groups is also increased as a consequence of denaturation, especially the sulphydryl groups of 13-19 (Mulvihill and Donovan, 1987; deWit, 1981), which is thought to be responsible for cooked flavor development, reduced oxidation-reduction potential and anti- 7 oxidant properties. It is also responsible for some protein- protein interactions (Mulvihill and Donovan, 1987). Polyacrylamide gel electrophoresis was used to show that heat resistance of individual whey proteins decreased in the order: a-la, B-lg, BSA, Ig (Larson and Rolleri, 1955; Li- Chan, 1983; deWit, 1984; Donovan and Mulvihill, 1987, Mulvihill and Donovan, 1987). Heat resistance was confirmed by Donovan and Mulvihill (1987) using insolubility at pH 4.5 and reactive thiol content as indices of denaturation. The denaturation temperatures (Td) of whey proteins have been studied by DeWit and Klarenbeek (1984) using differential scanning calorimetry (DSC). They observed denaturation temperatures of 62, 78, 72, and 64°C for a-la, B-lg, BSA, and 196, respectively, in 0.07 M phosphate buffer at pH 6.0. Although a-la has the lowest Td, it is the most thermostable whey protein determined by electrophoresis and solubility measurements at pH 4.6. The discrepancy is explained by its ability to renature on cooling (Ruegg et al., 1977; DeWit and Klarenbeek, 1984; Pearce, 1989). The susceptibility of whey jproteins to denaturation in. whey or defined buffers is influenced by temperature, time, pH, calcium concentration, protein concentration, total solids and lactose concentration (Hillier and Lyster, 1979; DeWit, 1981; Li-Chan, 1983; DeWit and Klarenbeek, 1984; Donovan and Mulvihill, 1987). 2.2 Myofibrillar proteins The myofibrillar proteins constitute 50 to 55% of the total protein in mammalian skeletal tissue (Ziegler and Acton, 1984). Myosin and actin are the major myofibrillar proteins, constituting 50-55% and 20-25% of the myofibrillar proteins, respectively (Smith, 1988). Myosin consists of 2 heavy chains, each with a molecular weight of about 200 KDa, and 4 light chains in the molecular weight range of 16 to 30 KDa (Bandman, 1987). Two classes of light chains are present in skeletal muscle myosin, alkali light chains and DTNB [(s,s-dithiobis)-2-(nitrobenzoic acid)] light chains. Each myosin heavy chain is associated with one alkali light chain and one DTNB light chain. Each of the heavy chains is folded into an a-helical conformation for most of its length, and at the amino terminus forms a globular structure referred to as the myosin head. The myosin light chains are associated with the head portions of the molecule (Bandman, 1987) . Electron microscopy has shown that the myosin molecule is an assemmetric protein composed of a super- coiled a-helical tail region and a globular head region (Huxley, 1963). Myosin can be hydrolyzed by trypsin and/or chymotrypsin into two major fragments, heavy meromyosin (HMM) and light meromyosin (LMM). Further digestion with trypsin or chymotrypsin, HMM splits into subfragment 1 ($1) and subfragment 2 ($2). Myosin molecules are packed in the thick filaments of 9 myofibrils under physiological condition, but they are dissociated from the filaments and dispersed as monomers at an ionic strength above 0.3 (Huxley, 1963). Globular actin molecules (G-actin), of molecular weight 41.8 KDa, occur in muscle in long filamentous aggregates (F- actin) (Bandman, 1987). Actin is present in distilled water as a globular protein, G-actin. 0n addition of various salts, 0.1 M for monovalent cations and 0.1 mM for divalent cations, G-actin polymerizes to form F-actin (Leavis and Gergely, 1984) . Tropomyosin is an a-helical molecule. It consists of 2 subunit chains of approximate molecular weight 33 KDa. Troponin is a protein of 80 KDa molecular weight which confers Ca++ sensitivity to the actomyosin ATPase and has been shown to have a high affinity for calcium ions above a threshold concentration of 10‘6 M (Bandman, 1987) . Both tropomyosin and troponin are present in the grooves of the actin filament (Judge et a1. , 1989) and are related to the regulation of muscle contraction by calcium (Bandman, 1987). 2.3 Theory of Protein Gelation Protein gels may be defined as "three-dimensional matrices or networks in which polymer-polymer and polymer- solvent interactions occur in an ordered manner resulting in the immobilization of large amounts of water by a small proportion of protein" (Flory, 1974; Hermansson, 1979; Mulvihill and Kinsella, 1987; Morrissey et al., 1987). Ferry (1948) suggested the following mechanism for the 10 formation of protein gels: x Pn -> x Pd -> (Pd)x, where x is the number of protein molecules, Pn is the native protein, and Pd is denatured protein. Gelation is a two step process involving an initial unfolding of protein followed by an aggregation process. Hermansson (1978) suggested that protein denaturation prior to aggregation resulted in a finer gel structure, exhibiting a greater elasticity than if random aggregation occurred simultaneously or prior to denaturation. Denaturation is a continuous process of native protein structural changes involving' the secondary, tertiary, or quaternary structure. Alternations in hydrogen bonding, hydrophobic interactions, and ionic linkages occur during the transition to the denatured state (Ziegler and Acton, 1984). The ability of denatured protein to associate and coagulate, precipitate, or gel depends upon the protein; its amino acid composition, molecular weight, net hydrophobicity, and concentration, and critical balance between attractive and repulsive forces (Mulvihill and Kinsella, 1987). There are two basic types of heat-induced gel structures; thermoset (thermo-reversible) and thermoplastic (thermo- irreversible) gels. Thermoset gels, such as soya, conalbumin and gelatin, are transformed to a pro-gel condition on heating; solutions form a gel on cooling but may revert to the pro-gel state on subsequent reheating, suggesting that the aggregation step is reversible. Thermoplastic gels, such as whey protein gels, do not revert to the pro-gel state on reheating; they may soften or shrink (Hermanson, 1979; Shimada 11 and Matsushita, 1980; Schmidt, 1981; Morrissey et al., 1987; Mulvihill and Kinsella, 1987) 2.4 Sinusoidal Oscillatory Testing Rheology is defined as the study of material deformation and flow (Scott-Blair, 1969) and includes what is termed "small-strain" testing (deforming a small % of that required to break the sample) and "large-strain" testing (deforming to the point of permanent structural change) (Hamann, 1988). Small strain testing offers the possibility of monitoring transformations resulting in structure-texture changes because the forming structure is only minimally disturbed and not destroyed by the measurement process (Beveridge and Timbers, 1985). However, gel rigidity obtained from this test was not well correlated with sensory texture or rupture strength (Hamann and Webb, 1979; Montejano et al., 1985), therefore, Hamann (1987) stated that "Change in gel rigidity or elasticity is not producing food texture data but is monitoring physical property changes in the gel that relate to molecular changes (i.e. protein unfolding, bonding of molecules, etc)". The theory of dynamic testing has been review by Mitchell (1980), Sharma (1965) and Hamann (1991). Dynamic behavior of polymers can be studied by subjecting a sample to a sinusoidal strain and observing stress as a function of time (Sharma, 1965; Hamann, 1991). Suppose a specimen is subjected to a strain, 6, given by 12 e = 6 sin wt (1) 0 where so = strain amplitude, m = angular frequency, and t = time. The resulting shear stress, a, will be out of phase from the strain by an angle 6 and can be written as o = a0 sin (mt + 6) (2) where o = stress amplitude. The phase shift, 6, and the amplitude ratio, oo/eo, now characterize the rheological response of the sample at frequency, w (Bohlin et al., 1984). The ratio (00/60) is the absolute modulus, |G*|. The absolute modulus gives a measure of the total shear resistance to deformation (elastic + viscous) of the material and can be divided into two components, one in phase with the applied strain (storage modulus, G') and the other 90° out of the phase (loss modulus, G") (Hamann, 1991). The G' and G" represent elastic and viscous elements of a viscoelastic body, respectively. G'= 00 cos 6/60 (3) G"= oo sin 6/60 (4) The ratio G"/ G' is called the loss tangent and is equal to the tangent of the phase angle (tan 6). A.perfect elastic body would.exhibit 6=0°, whereas for an ideal viscous material 6=90° (Hamann, 1991). On heating the protein solution at high enough concentrations above the denaturation temperature, the sample begins to gel and G' increases (as does C" but to a more limited extent), and consequently tan 6 decreases (Ross-Murphy, 1988). The change in value of tan6 and that of the absolute magnitudes of G' and 13 G" with temperature help us to monitor the protein gelation (Ross-Murphy, 1988). A number of devices have been used to monitor structure formation in foods (Table 1). 2.5 Gelation of Muscle Proteins Actomyosin, and in particular myosin, is responsible for binding and gel formation in muscle food (Morrissey et al., 1987). When F-actin is heated it coagulates, exhibiting none of the viscoelastic properties of myosin or actomyosin gels (Samejima et al., 1969). However, actin exerts a synergistic effect which complements myosin, and maximum gel strength is obtained when the myosin to actin ratio is 2.7:1 (Yasui et al., 1980; Morrissey et al., 1987). The sarcoplasmiijroteins (Fukazawa et al., 1961), troponin, and tropomyosin (Samejima et al., 1982; Acton et al., 1983) did not influence gel formability of actomyosin. Nuckles et al. (1991) observed that substitution of 8.3% low ionic strength soluble (LIS) proteins for high ionic strength soluble (LIS) proteins reduced the expressible moisture and strength of the gels. When the substitution ratio increased to 16.6%, the expressible moisture of the HIS/LIS gels increased. They also reported that HIS proteins extracted from different muscles (Skeletal, cardiac, lung and spleen) exhibited different gelling abilities. Differential scanning calorimetry (DSC) has been used to study the thermal transitions of proteins during to denaturation. Transition temperatures (Tm) have been used 14 Table 1 Examples of techniques used for protein solution measurement Instrument Parameter Sample Reference thermal rigidity surimi Montejano et scanning viscosity a l . ( 1 9 8 3 ) rigidity " actomyosin Wu et al.(l985b) monitor (TSRM) " MF', wpcb, Burgarella et EWC al.(l985) rigidity solution from Montejano et energy damping comminuted meat al.(1984) " chicken myosin Wu et al.(1991) Rheomertics G', G", tan6 dialyzed WPId Rector et al. dynamic (1989) spectrometer (RDS) c' dialyzed WPId Rector et al. B-lg (1991) Rheometrics G" G”,tan6, chicken breast Wang et al. fluid I6 | salt soluble (1990) spectrometer protein Bohlin G; G", tan6 coagulating Bohlin et al. dynamic testing I6 I milk (1984) ” individual Paulsson at G. Weissenberh G' rheogoniometer wiener emulsion SAOTe output torque Rheograph sol G', G" whey proteins B-lg myosin beef myofibrils WPCb, egg albumin al.(l984) fish actomyosin, myosin, F-actin, myofibrils, myosin subfragments al.(l986) Paulsson et al. (1990) Egelandsdal et al.(l986) Samejima et al.(l985) Beveridge et Beveridge & Timbers(1985) Sano et al. (1988,1989a,b 1990) a. minced fish sol b. whey protein concentrate c. egg white d. whey protein isolate e. small amplitude oscillatory testing apparatus 15 extensively to identify points at which protein conformational changes occur upon absorption of thermal energy (Ziegler and Acton, 1984). Native lamb, pork, beef, and chicken ‘thigh. muscles exhibited three endothermic transitions which can be assigned to myosin (57-60°C), sarcoplasmic and connective tissue proteins (66-67°C) and actin (78-80°C) (Xiong et al. 1987). The Tm's were species and pH dependent (Xiong et al., 1987). Native chicken breast muscle (pH 5.6) showed five endothermic transitions at 57, 62, 67, 72, and 79°C (Xiong et al., 1987). The first (57°C) and last peaks (79°C) were due to myosin and actin, respectively, the third (67°C) and fourth (72°C) due to sarcoplasmic proteins, and the second peak (62°C) was from the combined effects of sarcoplasmic proteins, connective tissue and myosin or actomyosin denaturation. Using DSC, Xiong and Brekke (1990) observed that both chicken breast and leg salt soluble protein (SSP) at pH 6.0 showed a single endothermic peak at 60°C, suggesting the conformational change for individual SSP molecules from both muscles followed a similar mechanism. However, protein- protein association/interaction, as indicated by turbidity change, differed for breast and leg SSP. The tranisition temperatures of the protein-protein interactions were determined by calculating the differential change in optical density as a function of heating temperature (dA320/dT) . At pH 6.0-6.5, breast SSP showed three transitions. The first (43-45°C) and third (60-63°C) transitions were assigned to 16 myosin. and actin. association, respectively; The second transition might be attributed to myosin-actin interaction or other myofibrillar components that comprised the SSP. Twp (43-51°C, 53-63°C) transitions were observed for leg SSP. The results coincided with DSC results for chicken myofibril thermal denaturation reported by Xiong et al. (1987). They reported three transition peaks (53, 61, and 69°C) for chicken breast myofibrils but two (60 and 70°C) for chicken leg myofibrils at 0.1 M NaCl, pH 7.1. 'Wright et al. (1977) observed one to three Tm for myosin, depending on conditions of pH and ionic strength. Thermal denaturation of myosin appears to be associated with discrete regions of the myosin molecule (Wright and Wilding, 1984). Table 2 is a list of the events which may occur during heat denaturation of actomyosin adapted from Ziegler and Acton (1984). Samejima et al. (1981) concluded that the gelation of myosin involves two reactions: ( 1) aggregation of head portions (HMM 81) of the myosin molecules and (2) crosslinking of myosin rods. Aggregation of HMM 81 was closely associated with the oxidation of sulfhydryl groups and occurred upon heating at relatively low temperatures, e.g., at 35°C. The latter reaction involved the unfolding the helical tail (rod) at elevated temperature, which involved noncovalent interactions (Ziegler and Acton, 1984; Yamamoto et al. , 1988) . Ziegler and Acton (1984) stated that for myosin and actomyosin at temperatures below 50°C, aggregation was slow 17 Table 2 Conformational changes which may occur during the thermal denaturation of natural actomyosin (from Ziegler and Acton, 1984) Temperature Protein(s) or segment Description or events (°C) involved 30-35 Native tropomyosin Thermally dissociated from the F-actin backbone 3 8 F-act in "super"helix dissociates into single chains 4 0-4 5 Myos in Dissociates into light and heavy chains "Head" Possibly some conformational change "Hinge" Helix to random coil transformation 45-50 Actin-myosin Actin-myosin complex dissociates 50-55 Light meromyosin Helix to coil transformation and rapid aggregation > 70 Actin Major conformational changes in the G-actin 18 and the major events which occurred were changes in protein conformation (denaturation). It, appeared that rapid aggregation and subsequent gelation began at approximately 55°C brought about by changes in the native structure of the myosin rod. Mechanisms of myosin gel formation have been extensively studied. Gel properties are related to the length of native myosin filaments (Yamamoto et al., 1987, 1988). Long myosin filaments formed more rigid gels upon heating and showed a fine strand-like network structure when examined under the electron microscope. In contrast, short filaments formed a coarsely aggregated structure with lower rigidity. Studies have shown greater differences between red and white muscle myosin than between muscles from different animal species (Stabursvik and Martens, 1980) . The myofibrillar proteins isolated from poultry breast muscle formed stronger gels than those from leg muscle (Foegeding, 1987; Dudziak et al., 1988; Xiong and Brekke, 1989). One of the reasons to explain the difference was the specific isoforms of myosin subunits (heavy chain and light chains) present among white and red muscles (Asghar et al., 1984). Morita et al. (1987) suggested that strength of heat induced gels of chicken red and white muscle myosins was closely related to their morphological properties. They reported that chicken breast myosin had longer filamentous assemblies and formed stronger gels than leg myosin in 0.6 M KCl at pH 5.2 to 6.0. Samejima et al. (1989) reported that chicken breast and 19 leg myosin have different gelation characteristics, even though their filament lengths were similar, therefore, they suggested there were some factors other than the mode of filamentogenesis of myosin isomers contributing to differences in gel strength. Choe et al. (1991) mixed chicken leg myosin or leg myosin tail subfragment with breast myosin or breast myosin tail subfragment, and found that the rigidity of the mixed gel was higher than the sum of rigidity of the component gels. They suggested that difference in leg and breast myosin gels were caused by differences in filament-forming ability and in gel- forming ability of head and tail segments of both kinds of myosins. The effect of pH on the thermal transition temperatures of chicken breast SSP was investigated by Wang (1990). At neutral pH (6.5-7.5), four transitions (45-47, 53-55, 58-59, and 64-65°C) for G' and three transitions (42-43, 54-56, and 59-60°C) for G" were observed. As buffer pH decreased to pH 5.5, the transition temperature of the first peak, attributed to the denaturation of light meromyosin, increased, whereas the transition temperature of the second peak, attributed to the denaturation of subfragment 2, decreased. 2.6 Gelation of Whey Protein Concentrates One of the main functional applications of whey proteins is as a gelling agent“ Gelation of whey proteins is dependent on pH; at pH < 7.0, the proteins tend to coagulate due to 20 excessive electrostatic interactions (Kinsella, 1984). The mechanism of gelation of whey proteins is similar to that of other globular proteins, with an initial denaturation step followed by interaction to form a gel matrix (Mulvihill and Kinsella, 1987). Beta-lactoglobulin is the principle protein component of whey; hence its properties play a major role in determining the gelling properties of whey protein (Kinsella, 1984; Mulvihill and Kinsella, 1987; Mulvihill and Kinsella, 1988). Studies on denaturation of whey proteins are relevant to an understanding of their gelation properties (Mulvihill and Kinsella, 1987). A protein concentration of 7.5% was required for the formation of strong gels from WPC heated at 100°C for 10 min at pH 7.0. The opacity of gels increased with increasing protein concentration (Schmidt, 1981). Hillier and Cheeseman (1979) stated that gelling time decreased from 4'to 1 min with increasing protein concentrations from 8 to 12%. Gelling time is also dependent on temperature, with the time decreasing as heating temperature is increased (Hiller and Cheeseman, 1979) . The major compositional factors of WPC important to protein gel formation include calcium, free sulfhydryl content, and protein hydrophobicity (Fligner and Mangino, 1991) . The nonprotein composition, including lactose, lipids, and minerals, also has an influence on WPC gelation (Hagget, 1976; Mulvihill and Kinsella, 1987, deWit et al., 1988). Calcium effects appear to be concentration-dependent (Schmidt et al., 1979; Kohnhorst and.Mangino, 1985; Mulvihill 21 and Kinsella, 1988; Zirbel and Kinsella, 1988), with some calcium required for cross-linking (ionic bonds) but too much causing aggregation (Mulvihill and Kinsella, 1987). The sulfhydryl content of WPC has been related to the strength and textural characteristics of WPC gels. Langley and Green (1989) prepared whey protein gels with various whey protein powders. The composition of whey protein powders varied from 0-12% a-la, 44-87% B-lg, and 6-56% casein derived proteins. The results showed that increased compressive strength, elastic modulus and impact strength of whey protein gels was related to increased B-lg content of the whey protein powders. Since disulfide bonds can break and reform with heat denaturation, the high percentage of free sulfhydryl and disulfide bond content of B-lg might explain why B-lg contributed to gel strength more than a-la and casein. The effect of heat processing on WPC composition and functionality have been studied by Mangino et al. (1987). Whey protein concentrate was manufactured from milk, whey and ultra-filtration retentate that had either been pasteurized at 72°C for 15 sec or had no heat treatment. The correlation between functionality, hydrophobicity, and concentration of free sulfhydryl group of WPC was studied. They observed that pasteurization of milk caused a decrease in WPC hydrophobicity and gel strength at pH 6.5, but that heat treatment of whey had no significant effect. Pasteurization of ultra-filtration rententate resulted in decreased native B-lg which correlated well with observed decreases in free sulfhydryl content of the 22 resulting WPC. Gel strength at pH 8.0 was also decreased compared to gels prepared by undenatured WPC. They confirmed that undenatured whey proteins had good gelling properties (Zirbel and Kinsella, 1988) and heat processing generally led to decreases in protein functionality. More recently, Mangino et al. (1988) heated the retentate at 64 or 72°C for 15 sec and made WPC. The results indicated that heating at 72°C decreased retentate hydrophobicity and had a negative effect on WPC functionality (i.e., overrun, solubility, gel strength, cake height), while heating at 64°C did not. Beveridge et al. (1984) used dynamic shear measurement to monitor network development in heated WPC solutions. When temperature was increased stepwise to 90°C, elastic modulus increased markedly and the increase was modelled as a first order reaction. Upon cooling to 25°C, there was a further increase on elastic modulus. Gel formation between 85 to 90°C was attributed to the formation of hydrophobic interactions and disulfide interchange (Fukushima, 1980; Shimada and Matsushita, 1980). The increase of G' during cooling was reversible on reheating and was attributed to ionic interaction and hydrogen bonding (Catsimpoolas and Meyer, 1970). Rector et al. (1989) also observed that upon cooling, whey protein isolate (WPI) formed weak reversible thermoplastic gels after heating at 90°C for 15 min at pH 6.5 to 8.5 with protein concentration of 9.0 to 10.5%. Scanning electron microscopy indicated WPC gel networks 23 were made of spherical protein aggregates (Hermansson, 1979; Kalab and Harwalkar, 1973; Beveridge et al., 1984) with a coating or bridging material cementing the aggregates together (Beveridge et al. , 1984) . The spherical aggregates were probably formed.through hydrophobic interactions (Hermansson, 1979) at an early stage of gel formation and do not change despite the continuous development of elasticity during heating (Beveridge et al., 1984). The bridging material may represent the deposition of soluble protein between the particles, perhaps by disulfide interchange or sulfhydryl oxidation (Beveridge et al., 1984). These crosslinks within preformed spherical aggregates were believed to contribute to the slowly increasing elasticity during heating of the WPC (Beveridge et al., 1984; Beveridge and Timbers, 1985). The effect of NaCl concentration and heating rate on the gelation of WPC were investigated by Burgarella et al. (1985) , using a modified thermal scanning rigidity monitor (TSRM) . At a heating rate of 1°C/min, the transition from sol to gel began at approximately 70°C, regardless of NaCl content (0.0- 3.0%). Both the rate of rigidity development and final rigidity of the solution were lower in WPC samples containing added NaCl than in the control. .As the heating rate increased (0.5->1.5°C/min), the characteristic rigidity thermograms for WPC shifted to higher temperatures(65->70°C) and the final rigidity of gels decreased. They explained that a slower heating rate allowed proteins to attain a greater degree of "order" which was favorable to creating a fine, cohesive gel 24 structure (Lanier et al., 1982). Thermal gelation of individual whey proteins (BSA, B-lg, and a-la) in a 1% NaCl solution, pH 6.6, has been studied by a dynamic rheological method (Paulsson et al., 1986). The minimum concentration needed to obtain a gel from BSA and B-lg were 1% (w/v) and 2% (w/v), respectively. Alpha-lactalbumin did not gel below a protein concentration of 20% (w/v) . Thus, BSA was characterized as having good, B-lg intermediate, and a-la poor thermal gelation properties. The BSA gels were purely elastic, while the B-lg gels had viscous elements. Heat-induced gelation of whey proteins could be altered by mixing individual proteins at different combination, since the presence of other proteins might modify the gel characteristics observed in a single component system. Paulsson et al. (1990) investigated the rheological properties of B-lg during heat-induced gelation at pH 4.5, 5, and 7 and a concentration of 3, 4, and 5%. The temperature of the onset of gelation (84 to 88°C) for 4% B-lg was only slightly dependent on pH. The concentration effect on onset temperature was small at pH 4.5 and 5, but the effect was more pronounced at pH 7.0. A similar effect was observed by Paulsson et al. (1986) at pH 6.6. This may be explained by assuming that in gels at pH 7, aggregation is the rate determining step. During longer aging times at 90°C, the effect of concentration on gel stiffness was dramatic. However, at each concentration, gel stiffness was highest at pH 5. 25 The effects of temperature and WPC solubility on WPC gel jproperties were studied by‘ Beuschel (1990). Five ‘WPC- treatments with protein solubilities ranged from 98% to 27% (in 0.1 M NaCl, pH 7.0) were usedt The results indicated that as WPC solubility decreased to 41%, the temperature required to form a measurable gel increased. Gel strain decreased as WPC solubility decreased from 98% to 27%. At 70°C, gels prepared by insolubilized WPC expressed less moisture than gels prepared by high soluble WPC. At 80 and 90°C, highly soluble WPC produced a firmer gel with a higher water holding capacity than gels prepared with less soluble WPC. 2.7 Multicomponent Gels All food products are multicomponent systems where the high-molecular compounds are generally proteins and polysaccharides. In addition, the majority of the products were solids containing a large amount of water, normally 50- 90%, i.e. they are.gels. In terms of physical chemistry, most food products can be viewed as multicomponent gels (Tolstoguzov and Braudo, 1983). Ziegler' and. Foegeding (1990) proposed. five jpossible models for multicomponent gel systems composed of a gelling protein and a gelling or non-gelling coingredient (Figure 1): 1. Type I filled gel or single-phase gel (Figure 1A). The filler remains soluble in the interstitial fluid of the gel matrix. 2. Type II filled gel or two-phase gel (Figure 1B). The 26 Figure 1 Possible models for multicomponent gel systems composed of a gelling protein and a gelling or non-gelling coingredient. (A) type I filled gel or single-phase gel; (B) type II filled gel or two- phase gel; (C) complex gel without continuous linkage; (D) complex gel or coupled gel; (E) interpenetrating network. (from Ziegler and Foegeding, 1990) 27 filler exists as dispersed particles, unassociated with the gel matrix. These dispersed particles may be hydrated or unhydrated, and may consist of a secondary gel network. Thus, a "phase separated gel" as described by Morris (1986) where a matrix of one component gel enclosed within the matrix of another component gel can be included in this model. For a mixed gel formed from two polymers A and B there is a particular ratio (A/B) called the phase inversion point where the system changes from a matrix of gel A containing inclusions of gel B to a matrix of gel B containing inclusions of gel A. At the phase inversion point the system may consist of two continuous interpenetrating networks (Brownsey and Morris, 1988). Usually the concentration of protein fillers did not exceed 5-7%. Addition of larger amounts of protein might significantly distort the unique food structure and leaded to undesirable product modifications. The ability of protein filler to be distributed in the gel matrix without distorting it significantly is defined as "structural compatibility" (Tosltoguzov and Braudo, 1983). 3. Complex gel without continuous linkage (Figure 1C) . A nongelling component associates directly via non-specific interactions with the primary gelling component. 4. Complex gel (Figure 1D) . Two or more proteins may copolymerize to form a single heterogeneous network. This model of gel can be termed as "coupled gel" network as described by Morris (1986). 5. Interpenetrating network (Figure 1E) . Two polymers do not 28 directly interact with the other, but are structurally cooperative due to entwining of the two protein gel networks. Gel matrix development by myofibrillar proteins can be directly influenced by chemical interactions between the non- muscle proteins and myofibrillar proteins (Lanier, 1991). Peng and Nielsen (1986) has observed linkage formation between soy proteins and myosin when processed to a temperature near or above the denaturation of soy proteins. The non-muscle and myofibrillar protein linkages would enhance gel rigidity and strength when the cross-links extend or reinforce the gel matrix, but may have little positive and possibly a negative effect on gel strength when cross-linking leads only to attachment of particles to the matrix (Lanier, 1991). The presence of non-muscle proteins change the molecular environment (total protein concentration, water state and availability) and indirectly influence the gelation of myofibrillar proteins (Lanier, 1991). Foegeding and Lanier (1987) stated that most non-muscle proteins disperse in the gel matrix and act as a "sponge" to hold water and lipid. Water-binding and volume fraction displacement may be the most important roles of non-muscle proteins in certain processed meat applications (Lanier, 1991). Wu et al. (1985a,c) reported that the effects of starches on the textural properties of cooked surimi gels were dependent on their gelatinization characteristics. Transitions of fish proteins monitored by TSRM or DSC did not shift in temperature with the addition of starch, being essentially the same as in the 29 surimi system not containing starch, However, the absorption of water and swelling of starch.granules caused an increase in rigidity of starch-surimi system (Wu et al., 1985a). Burgarella et al. (1985) used TSRM and DSC to show the effect of WPC on cooking profiles of surimi (minced fish, MF). Both TSRM and DSC results indicated that MF and WPC exhibited little or no change in the temperatures at which transitions in rigidity development or endothermic peak occurred as a result of mixing prior to heat treatments .At low temperatures (40 to 60°C), MF proteins formed a gel whereas WPC remained soluble, therefore, a type I filled gel (Figure 1A) was formed. Above the gelation temperature of WPC, a markedly increase in rigidity of the mixed protein gel can be explained as resulting from either formation of a type II filled gel (Figure 1B), or an interpenetrating gel network (Figure 1E). It is also possible that direct chemical interactions:may’have occurred and formed a complex gel (Figure 1C) (Lanier, 1991). The final rigidity of MF/WPC gels have a lesser rigidity than an additive relationship would predict, indicating one protein seems to "dilute" the other, or interfere with its gelation in some way. Effects of heat-insolubilized WPC on chicken breast SSP gels were determined at 65 and 90°C by Beuschel (1990). Elasticity of SSP/WPC gels increased with decreased WPC solubility at 65° and 90°C. Decreased WPC solubility decreased expressible moisture and increased gel strength for SSP/WPC gels at 65°C, but increased expressible moisture and 30 decreased gel strength at 90°C. 2.8 Nonmeat Ingredients in Meat Systems The high price of meat products and large variation in the quality of meat proteins are important stimuli for using non-meat proteins in both whole and comminuted meat products (deWit, 1984). Non-meat materials are often referred to as binders or extenders. Use is generally restricted to 3.5% of the finished product weight with the exception of soy protein isolate with a 2% limit (Rust, 1987). Whey protein concentrates have received considerable attention as potential food ingredients, due to their high quality protein.and.availability at relatively lower'prices as compared. to currently ‘used binders and extenders. The utilization of a byproduct often discarded by the cheese industry also provides certain environmental benefits (Casella, 1983). DeWit (1984) stated that the main functional requirements of whey proteins in comminuted meat products were good emulsifying and fat-binding properties and the ability to support matrix formation. Comer and coworkers (1981, 1986) studied the functionality of fillers in comminuted meat products and suggested that water absorption and gelation characteristics played a greater role than emulsion formation in determining the stability and texture of a comminuted meat system. Hermansson and.Akesson (1975) obtained good correlations 31 between differences in functional properties (solubility, swelling, viscosity and gelation properties) of added proteins and moisture loss of model meat systems. They suggested that functional properties could be used as reliable predictors of changes in model meat systems. However, poor correlations were observed between functional properties in model systems and functional performance in comminuted meat systems by Comer (1979) . DeWit et al. (1988) suggested that information derived from basic protein research would be the most useful tool for predicting functional properties of food products. Unfortunately, the interactions between the different components of a food product under various processing conditions were still too complex for theoretical explanations. . The functional behavior of a number of binder ingredients (flours, starches, milk products, and non-meat protein extenders) have been studied in comminuted meat systems and products. In general, meat fillers have beneficial effects upon emulsion stability and yield, but negative effects upon product firmness (Comer, 1979). Lauck (1975) used partially delactosed whey as a binder in beef frankfurters and compared this to different whey products as well as nonfat dry milk (NFDM) and soy. The data indicated that partially delactosed whey equaled the control in fat stability. Regular whey was less effective, but was still a reasonably good replacement for beef. The frankfurters made with whey were tougher and less juicy than 32 those made with delactosed whey. Soy isolate and WPC bound more water as indicated by reduced shrinkage. The only frankfurters with a definite off-flavor were those made with soy isolate. Jelen (1975) used lactalbumin (heat-precipitated whey proteins) as extenders for meat balls and hamburgers and reported that they caused a softening effect with reduced cooking losses. IKarmas and.Turk (1976) reported that.addition of up to 5% sodium and calcium WPC increased water binding properties of fish proteins. Morr (1979) proposed that whey proteins could be utilized in processed meat products to improve their water and fat binding properties without adversely affecting flavor or textural properties. Lee et al. (1980) found that use of WPC resulted in equal bind, increased juiciness and improved flavor in a meat loaf when compared to an equal level of nonfat dry milk. Ensor et al. (1987) observed that WPC produced.similar stability, textural.and.sensory attributes in specific emulsion-type meat products when compared to equal levels of soy protein isolate and calcium-reduced nonfat dry milk. In addition, all binders increased product hardness in comparison to the control. Thompson et al. (1982) found succinylated WPC (SWC) was useful as an extender for meat patties. The SWC-extended meat patties had increased cooking yield, higher fat and moisture retention, and decreased shrinkage as compared to all-beef or WPC-extended patties. 33 Comer et al. (1986) examined the functional and microstructural effects on wieners from the addition of wheat flour, modified corn starch, skim milk powder, soy concentrate and wheat gluten at levels of 6.8% solids. All fillers improved stability and textural firmness. The firmness was primarily attributed to water binding and gelation characteristics of the ingredients. Starch fillers produced the firmest texture. However, the overall textural quality of the wieners containing fillers were similar. DeWit (1988) compared the effect of heat treatment on frankfurter-type meat products prepared at either 80 or 110°C, using formulations with and without a 30% exchange of lean meat proteins by various milk proteins (meat proteins were replaced by the milk proteins). The results indicate that substitution of meat proteins by whey proteins improved water- retention, but not fat-retention at both temperatures. All products revealed a significantly increased separation of water and fat after a heat treatment at 110°C as compared to 80°C. Beuschel (1990) examined the effects of insolubilized WPC on yield and texture of chicken frankfurters. Frankfurters were prepared with or without 3.5% WPCs (substituted on a protein basis), processed at either 70°C or 90°C. Results indicated that the use of highly soluble WPC increased firmness and yield of frankfurters when prepared at 90°C, at which whey proteins could form gels. However, WPC with reduced solubility might increase yield and firmness of 34 frankfurters processed at 70°C. The effects of incorporation of Dquiber (sugar beet pulp fiber), oat fiber, pea fiber, wheat starch, firm-tex (National starch and chemical Corp., Birgewater, NJ), and isolated soy jprotein.into 10% fat and 30% added.water bologna were examined by Claus and Hunt (1991). Test bolognas were less firm than the high-fat control but more firm than the low-fat control. Nonmeat ingredients, by absorbing some of the moisture available to the meat proteins, may help produce a firmer product (Comer et al., 1986) . Dquiber and oat fiber had greater cooking losses than the low-fat control, but purge was reduced by all test ingredients, particularly Firm-tex. Selected combinations of ingredients might be more effective in improving texture and processing characteristics of low-fat bologna. MATERIALS AND METHODS 3.1 Whey Protein Concentrate Treatments Whey protein concentrates (WPCs) were prepared and characterized by Beuschel (1990) . Defatted liquid whey protein concentrate from ultrafiltration of parmesan cheese whey was obtained from Foremost Whey Products (Clayton, WI 54004). Liquid whey protein concentrate was heat treated to produce four treatments. The extent of protein insolubilization was measured and represented by percentage protein solubility in 0.1M NaCl, pH 7.0 (Morr et al., 1985). One portion of the WPC was not heat treated and served as control with a solubility of 98%. Whey protein concentrates with solubilities of 41% and 27% were obtained by heat treatments of 92.2°C/305 and 126.7°C/30min respectively. A mixture of the 98% soluble WPC and 47% soluble WPC (78.2°C/305ec) was used to prepare a treatment with a solubility of 80%. Heat treatments were carried out in triplicate. Whey protein concentrates were freeze dried and stored in low density polyethylene freezer bags at -12°C. The average composition of the WPC was 62.4% protein, 27.8% lactose, 5.7% fat, 5.4% moisture 4.7% nonprotein nitrogen, 3.8% ash, and 0.47% calcium (Beuschel, 1990). 35 36 3.2 Electrophoresis of Whey Protein Concentrate Electrophoresis was conducted by the method of Laemmli (1970) with modifications. Both regular and sodium dodecyl sulfate polyacrylamide gel electrophoresis were performed on the total protein and soluble protein fractions of the four WPC treatments using a SE 600 series vertical slab unit (Hoefer Scientific Instruments, San Francisco, CA 94107), equipped with a Bio-Rad power supply (Model 1000/500, Richmond, CA 94804). 3.2.1 Polyacrylamide Gel Electrophoresis Total protein samples for polyacrylamide gel electrophoresis (PAGE) were prepared by dissolving WPC with 0. 0625 M Tris (Tris[hydroxymethyl]aminomethane) sample buffer, pH 6.8, containing 10% glycerol and 0.1% Bromophenol blue to give a protein concentration of 5 mg/ml. Soluble protein fractions of WPCs were prepared as described by Beuschel (1990). An appropriate mass of WPC was blended in 40 ml 0.1 M NaCl, and pH was adjusted to 7.0 with 0.1 M HCl or NaOH. Protein solutions (5 mg/ml) were stirred by a magnetic stirrer for one hour, while the pH was checked every 10 to 15 min. To prevent heating, the beaker which contained the protein solution was placed on a weight boat filled with ice. The protein solution was transferred quantitatively to a 50 ml volumetric flask, and made to the mark with 0.1 M NaCl to achieve a final protein concentration of 5 mg/ml. After inverting several times, the protein 37 solution.was centrifuged at 20,000 x g for 30 min at 2°C. The supernatant was filtered through Whatman No.1 filter paper and the aliquot was obtained as the soluble protein fraction. Samples for PAGE were prepared by dialyzing 9 ml soluble protein fraction 18 hr against 1 1 0.0625 M Tris sample buffer with one change of dialysis buffer. The slab gel system included a resolving gel containing 14% acrylamide in 0.75 M Tris buffer (pH 8.8), and a stacking gel containing 4.5% acrylamide in 0.25 M‘Tris buffer (pH 6.8). Sixty ug total protein or soluble protein fraction were added to each sample well and electrophoresed using TTis-Glycine electrode buffer (0.025 M Tris, 0.192 M glycine), pH 8.3. The current was held constant at 30 mA until the tracking dye reached the resolving gel, then increased to 50 mA for the remainder of the run. The gels were stained overnight with 0.4% Coomassie Brilliant Blhe in acetic acid-methanol-water (9:45:45), quickly‘ destained. one Ihour’ with acetic acid- methanol-water (1:5:4), followed by several changes of a destaining solution with acetic acid-methanol-water (6:4:7). Gels were stored in 7.5% (v:v) acetic acid solution. 3.2.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sample preparation for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was similar to that described above for PAGE, except: 1) the total protein samples were incubated with 2.0% SDS and 5.0% B- 38 mercaptoethanol in 0.0625 M Tris sample buffer, pH 6.8, for 10 min, and 2) the soluble protein samples (9 ml each) were combined with 1 ml 10% SDS solution and 3 drops of B- mercaptoethanol, boiled for 10 min before dialysizing against 0.0625 M Tris sample buffer, pH 6.8, containing 2.0% SDS and 5.0% B-mercaptoethanol. The resolving gel was composed of 12% acrylamide in pH 8.8 Tris-SDS solution (0.75 M Tris, 2.0% SDS), and the stacking gel was composed of 4% acrylamide in pH 6.8 Tris-SDS solution (0.25 M Tris, 2.0% SDS). The pH 8.3 Tris-Glycine electrode buffer contained 0.025 M Tris, 0.192 M glycine, and 0.1% SDS. Protein (60 ug) was loaded on each sample well, and run under the same conditions as PAGE. Standard proteins (MW- SDS-70L) were purchased from Sigma Chemical Company (St. Louis, MO 63178-9916). Molecular weights of samples were calculated by comparing the relative mobility of the samples to that of the standard proteins. 3.3 Whey Protein Concentrate Solution Preparation Whey protein concentrate solutions contained 24% protein (w/w) were prepared as described by Beuschel (1990). An appropriate amount of WPC was dispersed in 0.6 M NaCl, 0.05 M Na-phosphate buffer, pH 7.0, and was gently stirred with a glass rod to break the clumps. The pH was adjusted to 7.0 before the solution with buffer was transferred quantitatively to a volumetric flask to give the final concentration. After standing in a 4°C cold room overnight, the pH was checked 39 again and adjusted if necessary. Sixteen percent WPC solution was diluted from 24% WPC solution with buffer addition. 3.4 Extraction of Chicken Breast Salt Soluble Protein Salt soluble protein was extracted from fresh chicken breast muscle according to the method of Wang (1990) with.some modifications. Fresh chicken breast muscle purchased from a local grocery store was hand-skinned, deboned, defatted, and ground twice through a Hobart Kitchen Aid food grinder (Model KS-A, Troy, Ohio 45374) with a 4 mm plate. The ground meat was blended with 4 volumes of low salt buffer (0.1 M NaCl, 0.05 M Na-phosphate buffer, pH 7.0) for 90 sec in a waring Blender (Model 1120, Winsted, Conn 06057), stirred with a :motorized.propeller stirrer for*1 hr without foaming in a 4 °C cold room, and centrifuged at 8800 x:g for 10 min at 4°C. The pellet was resuspended in 4 volumes low salt buffer, stirred and centrifuged as above. The pellet was solubilized in one- third volume of 2.4 M NaCl, 0.05 M Na-phosphate buffer, pH 7.0, to bring the salt concentration to 0.6 M. Two volumes 0.6 M NaCl, 0.05 M Na-phosphate buffer, pH 7.0, were added and the solution was stirred for 1 hr followed by centrifugation at 21500 x g for 20 min. The supernatant was diluted with 5 volumes of deionized water, followed by centrifugation at 21500 x g for 30 min. The pellet which was designed as salt soluble protein was collected and centrifuged 2 more times to further increase the protein concentration. Protein concentration was determined by Micro Kjeldahl 4O (AOAC 24.038-24.039, 1984). The pellet was dissolved in one third volume of 2.4 M NaCl, 0.2 M Na-phosphate buffer, pH 7.0. The protein solution was then diluted to contain 80 or 40 mg protein per milliliter with 0.6 M NaCl, 0.05 M Na-phosphate buffer, pH 7.0. 3.5 Combination of Whey Protein Concentrate and Salt Soluble Protein Solutions composed of 12% protein (w/w) from WPC and 4% SSP (w/w) were prepared by mixing 24% protein (w/w) WPC solution with 8% (w/w) SSP at a 1:1 ratio. The protein solutions were blended with a Polytron Homogenizer (Model PT 10/35, Kinimatica, Switzerland) with. a ‘model PTA 10 T8 generator for three 3 sec periods at a setting of 4, and allowed to stand overnight at 4°C before using. 3.6 Dynamic Testing Continuous measurement of the dynamic moduli (G'and G") during thermal processing of the protein solution was carried out with a Rheometrics Fluid Spectrometer (RFS-8400, Rheometrics, Inc., Piscataway, NJ 08854). A programmable circulating oil bath (MTP-6 Microprocessor, Neslab Instruments, Inc., Newington, NH 03801) connected to RFS was used to control the temperature. About 7 mg of protein solution was loaded in a sample cup which was preheated to the desired temperature. A 25 mm diameter parallel plate was lowered to the sample surface and 41 the gap between the upper and lower plate was set at 1.5 mm. A few drops of corn oil (Mazola, Best Foods, CPC International Inc., Engelwood Cliffs, NJ 07632) were used to cover the top of the protein solution to avoid dehydration. In the preliminary test, strain sweeps from 0.1% to 100% were run at a fixed frequency of 10 rad/s, using solution of 4% SSP, 16% WPC (98%, 80%, 41%, and 27% soluble WPC), or a combination of 4% SSP and 12% WPC (98% and 27% soluble WPC) in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0. Three isothermal temperatures, 30°, 65°, and 90°C were used for the strain sweeps. At 30° and 65°C, 98%, 80%, and 41% soluble WPCs formed very thin solutions and generated torque below instrument sensitivity. A strain amplitude of 2% was within the linear range when running strain sweeps for all the other samples. When the viscoelasitc behavior is linear, the ratio of stress to strain is a function of time (or frequency) only, and not of the applied strain magnitude (Ziegler and Foegeding, 1990). Therefore, a strain of 2% and a frequency of 10 rad/s were selected for evaluating the gelation of WPC and chicken SSP. Protein solutions containing 4% SSP, 16% WPC or a combination of 4% SSP and 12% WPC in 0.6M NaCl, 0.05 M pH 7.0 buffer were either heated isothermally at 65 or 90 °C for 15 min or heated from 30 (or 50) to 95°C at a rate of 2°C/min. Data parameters were recorded every 0.3 min. All measurements were finished within 4 days after the protein was extracted. The dynamic measurements of each treatment were replicated 3 42 times, each from a batch of protein. 3.7 Scanning Electron Microscopy Protein gels containing 4% SSP, 16% WPC or a combination of 4% SSP and 12% WPC were prepared according to the method described by Beuschel (1990) . Seven grams of protein solution were placed into 1.0 cm (ID) x 13.0 cm (Length) stoppered glass tubes, centrifuged at 1,000 x g for 6 min to remove air bubbles, covered.with plastic caps, and.heated in.a water bath at an internal temperature of 65°C or 90°C for 15 min, then immediately cooled in ice water. Gel specimens were prepared for scanning electron microscopy as described by Klomparens et al. (1986) with some modifications. After standing in the cold room overnight, gels were carefully pushed out of the glass tubes, cut into 3mm cubes, and fixed at room temperature with 4.0% glutaraldehyde in 0.1 M Na-phosphate buffer, pH 7.0, for 1 hr. The fixed specimens were rinsed once in the same buffer for 30 min, and 4 times in deionized. water for 30 min each. Dehydration of fixed specimens was performed in a graded ethanol series (25, 50, 75, 95, 100%) for 15 min each, and stored in 100% ethanol overnight. The following day, after one more change of fresh 100% ethanol, critical point drying was performed with liquid C02. The dried specimens were mounted on aluminum stubs with double sided tape and conductive colloidal graphite, then coated with gold in a Emscope Sputter Coater (Model SC 500, Kent, England). 43 Microstructure was observed with a JEOL Scanning Microscope (Model JSM-35CF, Osaka, Japan) at a voltage of 10-15 KV. 3.8 Product Preparation 3.8.1 Silent Cutter Processed Low Fat Frankfurters Mechanically deboned chicken meat (MDC) (Ottawa Gardens, Athens, MI 49011), pork fat (Meat Lab, Michigan State University, MI 48824), water, salt and WPC were used in the production of frankfurters. The control frankfurters were formulated to contain 12% protein, 15% fat. 69% moisture, and 2% salt" Whey protein concentrate was substituted.for 3.5% or 7.0% chicken meat on a weight basis. Frankfurter formulations are listed in Table 3. Table 3 Formulation for low fat chicken frankfurters with 3.5 or 7.0% whey protein concentrate (WPC) substituted for meat on a weight basis Ingredient Control With 3.5% WPC With 7.0% WPC (9) (9) (9) Mechanically deboned 1417.6 1365.2 1312.8 chicken meat Water 352.0 352.0 352.0 Fat 80.4 80.4 80.4 Salt 37.0 37.0 37.0 WPC 0.0 52.4 104.9 Mechanically deboned chicken was thawed overnight in a 44 4 ° C cooler. Pork fat was ground through a 4 mm diameter grinder plate using a Kitchen Aid (Model K5-A, Troy, Ohio 45 :374) . Mechanically deboned chicken, half of the cold water, sa 1t, and WPC were chopped in a Hobart bowl chopper (Model 84 181 D, Troy, OH 45374) at a speed of 3450 RPM for 3 min in a 2°C cooler. Ground pork fat and the remaining water were added and the batter was chopped for another 6 min. The final batter temperature did not exceed 40°F. Batter was stuffed with a hand stuffer into ten 50 ml pre-weighed polycarbonate centrifuge tubes, weighed and capped. Batters were centrifuged 10 min at 1550 x 9 before cooking to remove air bubbles for frankfurters containing 7.0% WPC, but not for the 3 - 5% WPC-supplemented frankfurters. Products were heated in a 75°C water bath until an internal temperature of 72°C, then immediately cooled in ice. Cooked frankfurters were removed from tubes and weighed again when the internal temperature reached 4°C. Three replicate batters were made for each treatment. Chemical analysis of the raw and cooked products, cooked Yield, severe heat yield, and the texture profile analysis were conducted as described in section 3.9. 3 - 8.2 Vacuum Processed Low Fat Frankfurters To prevent air incorporation and improve reproducibility, a vertical cutting machine with vacuum was used instead of silent cutter. High fat MDC was obtained from Ottawa Gardens (Athens, MI 49011) and pork fat was eliminated from the 45 formulation. The control frankfurter formulation contained 12% protein, 15% fat, 69% moisture, and 2% salt. Three and ha. 1f percent of the meat was replaced by the WPC on a weight basis. Formulations are given in Table 4. Table 4 Formulation for vacuum processed low fat chicken frankfurters with 3. 5% whey protein concentrate (WPC) substituted for meat on a weight basis Ingredient Control With 3.5% WPC (9) (9) Mechanically deboned 908 . 0 876 . 2 Chicken meat Ice Water 181.6 181.6 8a It 21. 8 21.8 WPC 0. 0 31.9 ¥ Whey protein concentrate was hydrated with 100 ml water and stored in a 4°C cooler overnight. Thawed MDC, remaining ice water, salt, and hydrated WPC were placed into a vertical cutting machine (Stephan Uniiversalmaschine, UMC 5 electronic, c3°Zl.umbus, OH 43228) and chopped at a speed of 900 rpm/min, with a vacuum at 11.6 psi for 6 min. The temperature of the batter was maintained under 4°C by circulating cold water through the double jacket mixing bowl. The mixed batter was stuffed and cooked the same way as described earlier. Chemical analysis of the cooked products, cooked yield, severe heat yield, and texture profile analysis were 46 evaluated . 3 - 8.3 Chicken Rolls Raw materials for the chicken rolls were MDC, chicken breast meat (both from Ottawa Garden, Athens, MI 49011) , water, salt, sodium triphosphate (Rhone-Poulenc Inc. , Cranbury, NJ 08512-7500), and binders [WPCs, SPC (PromosoyR Plus soy protein concentrate, Fort Wayne, Indiana 46801-1400) , or NFDM (Food Club Natural Nonfat Dry Milk, Topco Associates, Inc. , Skokie, IL 60076) ] . Three and half percent binders were added on a weight basis. Formulations for chicken rolls are Presented in Table 5. Table 5 Formulation for chicken rolls with the addition of 3.5% binders on a weight basis k Ingredient Control With 3.5% binders (9) (9) Ch :‘lcken Breast Chunks 774.3 774.3 Mechanically deboned 516.2 516.2 Chicken meat ater 232.3 232.3 Salt (1.25%) 16.1 16.1 S<>g portion of frankfurters or chicken.roll was placed in 100 ml distilled water and.heated at 95°C for 10 min, cooled at room temperature for 5 min, and reweighed. Severe reheat yield was obtained by dividing the reheated weight by the initial weight and multiplying by 100. Determinations were performed in duplicate for each replicate. 3.9.4 Texture Profile Analysis Texture profile analysis was performed on an Instron Universal Testing Machine (Model 4202, Canton, Massachusetts 49 02021) equipped with a 500 N compression load cell using the method of Bourne (1978) . Texture was measured on chilled samples (4°C). Cylindrical specimens (15 x 15 mm for frankfurters, 20 x 20 mm for chicken rolls) were uniaxially compressed to 80% of original height for two cycles at a crosshead speed of 50 mm/min to determine the apparent stress and strain at failure, hardness, springiness, and cohesiveness. Apparent stress and apparent strain at failure were calculated following Hamann's (1983) equations. Stress was the force that the crosshead achieved before the sample fractured. Strain was the distance the crosshead traveled before the sample fractured divided by the initial sample length. Strain (e) was calculated as: e = 1 - L (5) L 0 where L = length of core at failure (cm) L0 = original length (cm) Apparent strain (6 ) was calculated as: aPP eapP=-ln(e) (6) Apparent stress (a, kPa) was calculated as: o = F +1000 (7) Pi r2 (1+fl62) where F = force at failure from chart (N) Pi = 3.14159 r = radius of core (m) Pi = Poisson's ratio (0.48) Hardness, springiness, and cohesiveness were calculated 50 according to definitions of Bourne (1978). Hardness was measured as the peak force (N) during the first compression cycle. Springiness was the height (mm) that the sample core recovered between the first and second compression. Cohesiveness (dimensionless) was calculated by dividing the positive force area during the second compression by the area under the first compression. 3.9.5 Tensile Strength Tensile strength was measured with a Model T-2100-C Texture-Test system (Food Technology Corp, Rockville, MD 20852) including a hydraulic system (or Texturepress, Model TP-6), a force transducer (Model PTA-3000) a thin slice tensile test cell (Model ST) and a Texturecorder (Model TR 5, Food Technology Corp, Rockville, MD 20852). Chicken rolls were cut using a Hobart meat slicer (Model 1512, Troy, Ohio 45374) to 1.3 cm thickness. Three slices of each replicate were cut and stored at 4°C prior to measurement. Pre-weighed meat slices were placed on the test cell. The force produced by meat sample to resist being pulling apart was recorded as a force-time curve on the chart by the Texturecorder. Tensile strength was calculated from the peak height of the curve: 501b*(reading/100)*selected load Tensile Strength = cell range * 4.4482lblN (8) (W9) sample weight(g) 51 3.9.6 Color Color measurements of cooked chicken rolls were made with a Hunter colorimeter (Model D25, HunterLab, Fairfax, Virginia 22090). Chicken rolls were cut using a Hobart meat slicer (Model 1512, Troy, Ohio 45374) to 1.3 cm thickness. L, a, and b values were determined using a white standard plate as the reference. Determinations were made in triplicate for each replicate. 3.10 Statistical Analysis A completely randomized block design was used for this study. A software program (MSTAT-c, 1989, Michigan State University, E. Lansing, MI) was used to compute the error of mean square, and analyze the variance. Various of each tests was computed using a two-way analysis of variance (replicate x treatments). Tukey's honestly significant difference test was used to determine significant differences among treatment means, using the mean square error as the error term. RESULTS AND DISCUSSION 4.1 Effect of Heat Treatment on Whey Protein Concentrates 4.1.1 Polyacrylamide Gel Electrophoresis (PAGE) Electrophoresis profiles of 98% soluble WPC exhibited typical protein components (Figure 2, Lane 1): the slower moving band.was immunoglobulin (Ig), followed by bovine serum albumin (BSA) , o-lactalbumin (oz-la) , and B-lactoglobulin B and A (B-lg) as reported by Hillier (1976) and Pallavicini et al. (1988) . Some high molecular weight proteins or protein aggregates did not enter the acrylamide gel matrix and were observed on top of the stacking or running acrylamide gel. The composition and intensity of protein bands present in 80% and 98% soluble WPC were similar. In 41% soluble WPC, 19 and BSA were absent showing their heat instability at 92.2°C/303ec. Only a small amount of B-lg B and A were observed, Band.density of B-lgA.was slightly higher than that of B-lgB. The quantity of o-la was similar in 41%, 80% and 98% soluble WPC, indicating a-la maintained its native structure during heating to 92.2°C. No protein bands were observed in the 27% soluble WPC suggesting denaturation of all the whey proteins. The quantity of native proteins decreased with increasing severity of heat treatment. The protein composition of 41% 52 Figure 2 53 Polyacrylamide gel electrophoresis of total and soluble fraction of whey protein concentrates (WPC) . Sixty ug protein were applied to each sample well. Lanes 1-4 represent WPC with a solubility of 98%, 80%, 41%, and 27%, respectively. Lanes 5-8 represent the soluble fraction of WPC with a solubility of 98%, 80%, 41%, and 27%, respectively. (Ig: immunoglobulin, BSA: bovine serum albumin, a- .la: a-lactalbumin, B-lg: B-lactoglobulin) 54 55 soluble‘WPC (92.2°C‘for 30 sec) was different from the 98% and 80% soluble WPCs. Results agreed with Pearce (1989) who reported.that.the majority of the whey proteins were denatured when heated from 75 to 90°C. The slightly higher density of B-lgA than B-lgB in 41% soluble WPC was in agreement with Hillier and Lyster (1979) who demonstrated that below 100°C, B-lgA was more thermostable than B-lgB in cheese whey. Denaturation as indicated by a decrease in band density showed heat resistance of whey proteins decreased as follows: a-la, B-lg, BSA and Ig. This trend agreed with previous studies (Li-Chan, 1983; deWit, 1984; Donovan and Mulvihill, 1987; .Mulvihill and Donovan, 1987). Heat stability of individual whey proteins is method dependent, and there is a discrepancy between results obtained from.electrophoresis and calorimetric studies. Electrophoresis results showed a-la (Li-Chan, 1983) was the most heat resistant fraction of whey protein. Using differential scanning calorimetry (DSC) , deWit and Klarenbeek (1984) reported a-la had the lowest (63°C) denaturation temperature (Td), however, it was 80-90% renatured after’ cooling' (Ruegg' et al., 1977; deWit and Klarenbeek, 1984; Pearce, 1989). The enthalpy change at 63°C was associated primarily with a conformational change due to the loss of Ca (Bernal and Jelen, 1984; Pearce, 1989). The thermostability of a-la has been interpreted in terms of a high degree of renaturation rather than a high temperature for denaturation (Donovan and Mulvihill, 1987; Mulvihill and Donovan, 1987). Studies using DSC showed that denaturation of 56 a-la was irreversible in a complex whey protein system (Donovan and Mulvihill, 1987) , probably due to heat-induced interactions with B-lg and BSA (deWit and Klarenbeek, 1984). Lanes 5-8 in Figure 2 represent the soluble protein components of four WPC treatments. Bovine serum albumin and Ig were absent in 41% soluble WPC (Figure 2, Lane 7), indicating their heat lability. B-lgA and B were denatured and only slight amounts remained in the soluble fraction of 41% soluble WPC. In contrast, the a-la band increased in intensity from 98%, 80% to 41% soluble WPC, indicating that 0:- la was the most heat stable whey protein. The quantity of native proteins decreased to negligible amounts in 27% soluble WPC, suggesting total whey protein denaturation. The thermostability of whey proteins observed in soluble WPC fractions coincided with what we found in total proteins. 4.1.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) By comparison with the relative mobilities of standard proteins, five major protein bands with molecular weights (MW) of 67.5K, 63.1K, 56K, 17.9K, and 14.1K daltons were identified in 98% soluble WPC (Figure 3). The 63.1K, 17.9K, and 14.1K protein subunits were identified as BSA, B-lg, and a-la, which have reported MW of 66.0K, 18.4K, and 14.2K, respectively (deWit and klarenbeek, 1984). The proteins with MW of 67.5K and 56.0K were identified as heavy chains of Ig. Immunoglobulins are either monomers or polymers of a four Figure 3 57 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and soluble fraction of whey protein concentrates (WPC). Sixty ug protein were applied at each sample well. Lane 5 was molecular weight standard Lanes 1-4 represent WPC with a solubility of 98%, 80%, 41%, and 27%, respectively. Lanes 5-8 represent soluble fraction of WPC with a solubility of 98%, 80%, 41%, and 27%, respectively. (BSA: bovine serum albumin, B-lg: B-lactoglobulin, a-la: a-lactalbumin) 58 MOLECULAR 7 WEIGHT ‘ (x1000) ”p", Polymer 59 chain molecule consisting of two light polypeptide chains (MW 22K) and two heavy chains (MW 50-70K) (Eigel et al., 1984; Mulvihill and Donovan 1987). The composition and intensity of the major protein bands separated by SDS-PAGE were similar for 98%, 80%, and 41% soluble WPCs (Figure 3, Lanes 1-3) . Results were in contrast to the native PAGE in which Ig, BSA, and B-lg decreased or disappeared from 41% soluble WPC (Figure 2, Lane 3). Additionally, the protein aggregates observed on top of the native acrylamide gel decreased when SDS and B-mercaptoethanol were used for SDS-PAGE. Thus, polymerization in the 98%, 80%, and 41% soluble WPC was mostly via noncovalent or disulfide bonds because the polymers were solubilized and dispersed into individual components in the presence of SDS and B- mercaptoethanol. DeWit (1981) reported that B-lg and BSA aggregated through thiol/disulfide (SH-SS) interchange and oxidation of sulphydryls to disulfide bonds during heat treatment. The 27% soluble WPC (Figure 3, Lane 4) indicated a different SDS-PAGE electrophoretic pattern from other WPC treatments (Figure 3, Lanes 1-3) . The intensity of BSA and a- la was decreased, but a much denser protein band was observed on top of the acrylamide gel, indicating that under severe heating conditions (126.7°C, 30 min), polymerization of protein molecules through non-reducible covalent bonds occurred. Zittle et al. (1962) and Pearce (1989) reported that o-la had no free-SH groups and did not undergo SH-SS 60 exchange reactions. The soluble fraction of 98% and 80% soluble WPC treatments had similar protein patterns on SDS-PAGE to those of total protein, indicating most of the proteins were soluble. Except for a-la and a small amount of B-lg, the other proteins from the soluble fraction of 41% soluble WPC (Figure 3, Lane 7) were insolubilized and absent from the electrophoretogram. Only a light band of B-lg was observed in the soluble fraction of 27% soluble WPC indicating most proteins were insolublized. In general, whey proteins that were insoluble as characterized by a decrease of band intensity in the soluble fraction compared to that in the total fraction on SDS-PAGE were also denatured when characterized by PAGE. This was in agreement with Id-Chan (1983) that denaturation generally precedes aggregation and loss of solubility. However, it is important to realize they are distinct and separate processes (Li-Chan, 1983). The light band.of B-lg present in the soluble fraction of 27% soluble WPC (Figure 3, Lane 8) was also observed by Beuschel (1990). However, B-lg was already denatured in 27% soluble WPC under this heating condition as shown in PAGE (Figure 2, Lane 8). Thus, there might be some denatured B-lg which did not form large enough aggregates to be precipitated during centrifugation but was converted to its monomeric form by SDS and. B-mercaptoethanol. This was in agreement with Singh and Creamer (1991) that although heating caused protein denaturation, only part of the heat-denatured whey proteins 61 were lost from the centrifugal supernatant. 4.2 Dynamic Testing 4.2.1 whey Protein Concentrate Gels 4.2.1.1 Thermal Scanning from 50 to 95°C Changes in storage moduli (G') and loss moduli (G") of 16% WPC in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0 were observed during heating from 50 to 95°C at a rate of 2°C/min (Figures 4 and.5). The 98% soluble WPC solutions did not form detectable structure, as defined by a G' above 5 Pa, below 91.8°C; only some scattered points were shown on the rheogram (Figure 4). .As the temperature reached 91.8°C, elasticity within the network was detected and G' increased with increased temperature, indicating heat induced formation of gel structure. The 41% and 27% soluble WPC solutions had detectable elastic character at 50°C. The G' of 41% soluble WPC solution was lower than 10 Pa at 50°C, and only increased to 20 Pa during heating to 95°C. The 27% soluble WPC solution had a G' of 7598 Pa at 50°C which decreased slightly to 6349 Pa during heating. The 98%, 80%, or 41% soluble WPC exhibited a G" value lower than instrument sensitivity at 50°C (Figure 5) . A slight increase in G" was observed when heating neared 94°C. The rheogram of G" for 27% soluble WPC was similar to that of G' when heated from 50°C to 95°C. The onset of gelation, characterized by a G' of 5 Pa or above, occurred at 91.8°C for 98% soluble WPC, which was 62 Comm 0» om Eon“ coucon hmuucn o.b mm .Homz z o.o CH Aom3v mucuucoocoo cflwuoua >023 won no A.UV wadUOE mmcuoum mcu mcwucuumoaaw acumomsu m>wumucmmwummm e whamflm A8 8388th om cm on om on _ _ t . t II. t h L D 4 a a an a a 4 c n o 4 D D D a a "no a o n o a D D 4 4 G D D 44 D d DOD d 00 d 4 Dd < D n¢5D a Do Q 4 a a D c nflmfiamam. mw o a. a 4 .6 4 444 o o a o n o o 8 090000 000000 .. 06% oo oooo oo 0 009000 0 coco 0000 098 o o m... 800 00000 0 0 0 0 ‘0. o 00 4 o a a on; o_b:_om NNN p on; o_b3_om N; o 0&2, o_n:_om Now a n. D >353 BEE>§>>§>E o p» B E p B E E: onB pawnEOprnwflergflmm: p p "£711? 1 'urn1fi “1* [1111111 7 AUF «.0— now .0.. (Dd) sngnpow 9601013 63 Comm ou om Eon“ 609cm; homusn o.h an .Hucz : m.o :fl Aomzv muchucoocoo sampoua awz3 wma no A=Uv waaooa mmoH on» ocflucuumsaafi Emuwomnu m>fiucucmmmummm m whamwm 8V EBotanfl. om cm on 00 on .[I L t h u h l- b L h Jdl 7.0.. o a o o a G D n— D M an O D a a 4 46 D 0 OF nod nonoaaua cam“ acne. «nonwoww aw833wcuao 00603 econ a oaoeaenefio % o%$.< figmmoooooocooowoooooo 0 can? om ooo 30$ ooovcooo eaoeo oo « 4 o .0? r n W Puow a 0&3 oEEom N; o h 013 o_n:_om NNN b m. figgg ggEbgfibg>E b 95 DD E h a E E9? D393 ED9EBO BF bfi bbP >9 F 9 D "O F 0&3 oiéom Now a 013 OED—om Nmm D .0— (Dd) snlnpow $901 64 higher than the:denaturation temperature (Td) of WPC (62-78°C, deWit and Klarenbeek, 1984) as determined by differential scanning calorimetry. This finding is in agreement with Paulsson et al. (1986) that measurable gel formation occurred at the same or higher temperature than the Td. Beveridge et al. (1984) used dynamic shear measurement to monitor network development in heated WPC solutions. They reported that when temperature was increased stepwise to 90°C, elastic modulus increased markedly and the increase was modelled as a first order reaction. Burgarella et al. (1985), using thermal scanning rigidity monitor (TSRM), determined the initial gelling transition for WPC solutions to be 65-70°C, but environmental conditions were different. Paulsson et al. (1990) suggested that the initiation of gelation was dependent on the protein, protein concentration, processing conditions (heating and cooling rates) , and environmental factors (pl-I and ionic strength) (Paulsson et al., 1990). After reaching the onset temperature of gelation, the 98% and 80% soluble WPCs exhibited a more extensive increase in G' as compared to G", resulting in a decrease of tan 6 as shown in Figure 6. The tan 6 is equal to the ratio G"/G'. In principle, as gel networks form, the sample becomes more elastic in nature and G' will rise while tan 6 decreases (Beveridge et a1. .1984). Thus, the decrease of tan 6 indicated gel networks were formed during heating (Beveridge et al, 1934). In 41% and 27% soluble WPC, most proteins were denatured 65 . comm 0» om sown cwummn umuusn 0.5 mm Hucz z 6.0 c“ Aomzv mucuucmocoo samuoum hmn3 woa mo «4 ccuv usmmcwu mmoa on» mcwuouumaaaw Bouoomnu m>fiumucmmmummm o ouscfim A8 838388. om om on om om r _ L _ L L i. _ I «nor . . on; 632% no a 0&5 @328 New 4 1 on; 6328 NS. 0 T o om? 0328 me D 1 o 4 w o n o o P 70 P o o a o o o o 6 9 0 4 339238 :5» BE: Kappppp» Waive Eébpvpgpg 4 $46 033 £4 5 :9 pa @ O 00 00 O 0 DD 3‘ ‘0 o D g o ‘ 0 00° 0 0° 0 0 o o o a? 4 4 4 4 40 9 0 00° 0 4 4 00 D 4 o o oo 4 4 o o a 4 4 4 4 4 4o 4 4 o 4 fi D 4 4 4404 4 4 4 4 4 4D 0 ... D4 44 4 44 D 4 '00—. o 4 4 o 44 4 4 o c a a4 44 D D D 4 T 4 D 4 4 4 o o o T o 04 1 D 1 T 4 w o o I.O_. 01190 um, 66 (unfolded) during preparation as indicated by decreased solubility, resulting in protein aggregation (Mulvihill and Donovan, 1987) and a viscous solution which displayed some characteristics of an elastic solid. The 27% soluble WPC had a higher G' than 41% soluble WPC due to more extensive aggregation. The relatively constant tan 6 throughout the heating period (Figure 6) indicated there was no development of gel structure during heating for 27% soluble WPC. 4.2.1.2 Isothermal Heating at 90°C Gel formation of 16% WPCs in.0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, as a function of time at 90°C was measured by monitoring changes in G' (Figure 7). For 98% and 80% soluble WPCs, gel structure with a G' of 5 Pa was detected after heating at 90°C for 6.3 and.5.4 min, respectively. Elasticity continued to develop throughout the 15 min heating period, and reached a final G' of 3149.0 Pa for 98% soluble WPC gel and 1023.7 Pa for 80% soluble WPC gel (Table 7). In contrast to 98% and 80% soluble WPCs, 41% and 27% soluble WPC solutions had detectable elasticity in the beginning of heating at 90°C, but the increase of G' was very limited for 41% soluble WPC and G' even decreased for 27% soluble WPC after 15 min of heating (Figure 7). At the end point, 27% soluble WPC gel had the highest G' value (5494.3 Pa), which should be attributed to aggregation rather than heat-induced gelation, due to a lack of increase of G' during heating. 67 c . . cwa ma non Doom M 60940: Hmmmsn o 5 :0 H042 Sw.o :« Aumzv mucuucmocoo cwmuoum >053 mod no «.00 «45008 mocuoum on» onwucuumaaafl smumomnu m>fiucucmmwummm h madman AEEV 0E5. mp OF m o F L- L b L— b h L h b L LI In! .P OP on; oBBOm Nmm 0 o 4 .... 0&3 min—om Now 4 o o 4 0&3 oBBom N; o 4 on; 6328 RR 9 a. «a 4 .9 4 4 4 no 400 4 one o o 4 4 a 4 000044 0 @4444 00 .Or oooooooooumaoooooooooooooooooooooooo oooooooooooo 44a 44 o 44 00 «OF 444 no 444 o 4444 con 44444 DD can now ooo DD Dfibbbbbb>>b>>b>>>bbbbbD9D§D>>>DDDDDDDDDDD>§>DD>DDD 40F (Dd) smnpow 950.1015 68 Table 7 Dynamic moduli of protein solutions containing 16% whey protein concentrates (WPCs) with various solubilities heated isothermally at 90°C for 15 min Dynamic Moduli (Pa) Treatmentsa Initial Point End Point at 0 min at 15 min Storage Modulus (Pa) wpc 98 ------ b 3149.0d wpc so ------ b 1023.7° WPC 41 32.5d 48.59 WPC 27 6532.3° 5464.3° Loss Modulus (Pa) WPC 98 ------ b 453.5d WPC so ------ b 151.9° WPC 41 6.9d 8.4° wpc 27 1373.7° 1130.0c a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b Sample did not form detectable structure and had a torque below instrument sensitivity. c-e Means within the same column with a different superscript letter are significantly different (P < 0.05). 69 Changes in G" of WPCs during heating at 90°C for 15 min (Figure 8) followed the same trend as that of G'. It took 7.3, 6.7, and 5.1 min to reach a G" of 5 Pa for 98%, 80%, and 41% soluble WPC, respectively, which was longer than for G'. The value of G" was smaller than 6' in all treatments, demonstrating that elasticity played a major role during gel formation (Table 7). The elasticity developed over the entire heating period (the difference between the initial and the end point) was greatest for 98% soluble WPC, followed by 80% soluble WPC. The 41% soluble WPC showed a very limited increase in G'. Elasticity decreased after heating 15 min at 90°C for 27% soluble WPC. Results agree with Zirbel and Kinsella (1988) that undenatured whey proteins have good gelling properties. The increased elasticity observed in highly soluble WPC during heating was due to crosslink formation within spherical aggregates of proteins by formation of disulfide bonds and hydrophobic bonds (Beveridge et al. , 1984; Beveridge and Timbers, 1985) . When examined by scanning electron microscopy (SEM) , the spherical whey protein aggregates formed at an early stage of gel formation did not change despite the continuous development of elasticity during heating (Beveridge et al., 1984). Beveridge et al. (1984) reported that elasticity resulted from energy storage within close-packed spherical aggregates forced to move relative to each other. The decrease of G' of 27% soluble WPC during heating at 90°C could be explained by 70 awe ma non Uoom um pound: homusn 0.5 m0 .Hocz Zw.o :fi Aomzv mucuucmocoo samuoum >0c3 «64 no A=UV flasooa mmoH on» mcfluouumSHHH Ecumomcu m>wucucmmmummm m musvflm AEEV 08:. MP OP m 0 —| b b h b h 1P L h I? L— L! L In I? b 'IIOF 0&3 o_n:_ow Nmm 0 013 o320w New 4 4 m 4 013 Baa—om N; o c a on; 23.8 RR 8 a a. a a 4. .3 0 0 0 0 4O 4 0 4 0 4 03 044 4 4444 o o o o 040 4momoooo oooo owoooooooo 00 0000000000 0000 0 4¢D «OP 4 o 444 a 1 44 o n 444 00 n 4444 on W.O— 444444 soon a can 1 con 1 0000 n op ppbbppppppbpp>>>>>>>>>>>>>>>>p>>>>>>>>>>> >>>>>>> now .0— (od) snlnpow $301 71 deformation of aggregates and breaking of interaggregate cement resulting from imposed oscillatory strain. This cementing material might regresent the deposition of soluble protein between the particles, perhaps by disulfide interchange (Beveridge et al., 1984). Changes in tan 6 of WPCs when heated at 90°C revealed heat-induced gelation of 98% and 80% soluble WPCs as indicated by a decrease of tan 6 during heating (Figure 9). Whereas, the relatively constant tan 6 throughout the entire heating period of 41% and 27% soluble WPCs (Figure 9) indicated the lack of gelling ability of the denatured whey proteins. 4.2.2 Chicken Salt soluble Protein and they Protein Concentrate (SSP/WPC) Combination Gels 4.2.2.1 Thermal Scanning from 30 to 95°C Changes in. G' of SSP and SSP/WPC combination. gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0 heated from 30 to 95°C at a heating rate of 2°C/min were observed (Figure 10). Beginning at 55.1°C, SSP gels exhibited an increase in G' which reached a peak at 63.2°C. On further heating, 6' decreased, reaching’a minimum.of 142 Pa at 69.1°C, which was lower than that before heating (852 Pa). The G' increased again until 78.4°C, then leveled off though 95°C. The SSP G" increased after the first transition point at 50.9°C, formed a broad peak at 64.0°C, then sharply decreased and showed a minimum at 69.6°C. On further rise in temperature, the G" increased slightly, and remained stable w“! I? ."' 72 WP I ..qDI‘ cfia ma you Uoom no 60940: noumsn o.h :0 .H042 So.o CH Aomzv mucuucmucoo cflououm moss ~44 mo A4 2400 ucomccu mmoH ecu mcwuouumsaaw Eouwomcu m>fluoucmmmunom m whamwm EEV 9:: or m o —r L b L- LF '1— } In L L I— L L L b ‘ mmaammmaumaa a 4 a n mowuoowow>~>pmmwalaolu>>o>ebpow>>> o o o o p o o oooo oo oow4e4wawo oooo oo ooo o 1 D 4 n40 4m 00 D Ofi D U D 4 1 0 4 0 n 0 0 n: 4 D4 4 4 D4 4 4 w j WI 1 4 n 4 m. 013 oBEom NBN 0&3 0320.01 RS. on; oiEom Now on; oiBom N80 0409 W [1111 1 1 1 ...O P a CD Ava «or 03,190 up; '73 comm on on Scum venom: Aumzv muouucoocoo samuoua >0s3 wma can mmm *4 mo mcofluccfinaoo can Ammmv cfiououm mansaom pawn «4 mo «.00 «H3003 ovououm on» ocaucuumsaafi Ecuooonu o>flumucmmoummm oH ouaufim 3V otswotanoH om om on om cm 04 on r F b i? L b b L P h F k r «O F 1mm o $30.13 6328 Now a 1 mmm\on§ 6328 Now a 1 133...; 6328 NS. 0 fl mmmhis 6328 RR 9 1.. 1 Tao F 1 pififluflnuunnflnnuunu 4 ...-00000 o $4003““44444444444 .. . ....1 o— ogc} 1.1.13.4... ................... n no o...... 44444444444488.3444: .. ::.:.:..o8o888888oooooooooo ml: _$2; > p p.399. cum :, 3:5FESREEESKEBESBkEEEKBkFEK2:592: onvw (Dd) sngnpow 960.1013 74 after 78.8°C (Figure 11). A similar dynamic rheogram of SSP was reported by Wang (1990). However, the transition temperatures were about 10°C higher in the present study, probably due to the faster heating rate. Wu et al. (1991) reported that increasing heating rate increased the unfolding rate of the protein but did not promote the aggregation. When a faster heating rate was used, the relatively slower aggregation step might not respond quickly enough and resulted in a shift of the dynamic rheogram to a higher temperatures (Wu et al., 1991). In addition, the decrease in total processing time which was inherent with a more rapid heating rate might be another interpretation (Burgarella et al., 1985). The first G" transition occurred earlier than the first G' transition (Table 8), probably because the proteins unfolded before subsequent network formation. The unfolded proteins caused an initial increase in viscosity or G" (Wang, 1990). Major SSP network formation occurred at 55.1°C, as indicated by a rapid increase in G'. The increase of G' stabilized after 78.4°C, suggesting the gelation of SSP was complete. Results agreed with Ziegler and Acton (1984) that rapid aggregation and subsequent gelation began at approximately 55°C brought about by changes in the native structure of the myosin rod. At temperatures below 50°C, aggregation was slow and the major events which occurred were changes in protein configuration (denaturation) (Ziegler and Acton, 1984). The SSP/WPC combination gels exhibited a different 75 comm on on scum cmummn Aomzc mumuucmucoo :wououm >053 wma can mmm we no mcofiuccwnaoo can Ammmv :Hmuoum mansaom pawn *4 mo A:uv flaccoa mmoH on» mcfipwuumsaafi amumoocu 0>fiumvcomouaom Ha musmfim A8 9.3anth om on on on on 04 on P L r \— I? F P [F {F LP L b L 1mm 3...... 1&3“; 13291. Nmm .. . 163%) 6328 Row 1&3“; 238 54 mmmba; 6328 RR D'<>‘4 £310 1 F1111‘1 '1 Au— nor 40F (Dd) snlnpow 390') 76 Table 8 Thermal transition temperatures of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble protein and 12% whey protein concentrate (SSP/WPC) heated from 30 to 95°C Transition Temperature (°C) Treatmentsa 1 st 1'st 2nd 3rd 4th 5th Storage Modulus (Pa) SSP 55.1” ----- 63.2” 69.1” 78.4” (Maximum)(Minimum) SSP/WPC 98 56.0”” 62.5” 64.9” 71.8” ----- SSP/WPC 80 57.3”” 62.3” 66.2” 72.0” ----- SSP/WPC 41 58.5” 62.0” 65.5” 71.2” 82.3” SSP/WPC 27 ---------- 65.3” 70.7” 77.7” Loss Modulus (Pa) ----- 64.0” 69.6d 78.8” (Maximum)(Minimum) SSP 50.9d ----- ZSSP/WPC 98 52.9”d 58.2” 64.4” 67.0” 72.5” 89.0” SSP/WPC 80 52.5””1 59.5” 64.7” 67.4” 72.2” 86.0”” SSP/WPC 41 53.7”” 60.2” 65.9”” 65.9”” 71.3”” 78.9” SSP/WPC 27 55.8” ------ ---- 64.7” 70.7”d 81.2” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript letter are significantly different (P < 0.05). 77 dynamic rheogram from SSP gels. The terminology used to examine the rheograms is described in Figures 12 and 13. ‘When comparing G' (Figure 10), the combination gel containing 98% soluble WPC showed a decrease after the first transition (1st Tr) at 56.0 to 62.5°C (2nd Tr), then increased and formed a Shoulder at 64.9°C (3rd.Tr). .After the shoulder, 6' decreased again and reached. a 'minimum at 71.8°C (4th Tr). The combination gel containing 80% or 41% soluble WPC exhibited a similar G' curve, but G' decreased after 82.3°C for 41% soluble WPC/SSP combination gel, which was the 5th transition point1 The addition of 27% soluble WPC to SSP gel resulted in an unique curve for G' . The G' remained constant until 65.3°C which was identical to the 3rd transition for the other combination gels, then G' decreased to a minimum at 70.7°C (4th Tr). Unlike other combination gels which showed a continuous increase of G' with increasing the temperature, G' decreased after 77.7°C (5th Tr) for the 27% soluble WPC/SSP combination gel (Figure 10). The 98% soluble WPC/SSP combination gel exhibited a very slight increase of G" after the first transition at 52.9°C (1st Tr) to 58.2°C (1'st Tr) (Figure 11). This increase was larger as the solubility of WPC decreased. The G" decreased to 64.4°C (2nd Tr) and formed a shoulder at 67.0°C (3rd Tr). The G" reached a minimum at 72.5°C (4th Tr), then increased on further rise in temperature. Similar rheograms were observed for the combination gels containing 98%, 80% and 41% soluble WPCs. No shoulder was observed for the SSP and 27% soluble 78 Aucflom :ofluflmcmuu “Hay ummu ocflccmom Hmfiuocu mcwusu Aumzv oumuucmocoo :flwuoum >053 wma 6:6 6mm 41 no mcoflumcfinsoo 8:6 Ammmc cflmuouo mansaom 046m we 06 1.00 MSHSUOE womuoum ocfln>amcm :4 00m: zuoHocflfiuou on» you newuflcfluwo NH 005040 A8 oLBotanmH om om on 8 on O1 om _ P h [F L h u L h L L — 0 r0 F 0mm 0 6mm\on§ q 1 T as :04 m ssswcflz n luor Qow oo oo o 09 sum oo o 1 O O 4.. 1 .66 o 1 n§§§§889§82§§000 MB :uv o 1 3 n 4&t44 4&6 0 MB UCN MB HMH n .... .144... ”1.1 : is .2 «444 .HH. MYHM 0° 4 4444444 5800 3834343344444444444344? 444444121444 um. sum ssfifixwz MB and 0500 as HmwuflcH1 0:400 cam m 1.13 (Dd) snlnpow 9601013 79 AUGwoa :oflufimccuu "Nev ummu wcficcoom Hmfiuwcu UCHHDU Aomzv oucuucmocoo cwmuoum >053 «NH 0:0 mmm *4 no wcoflumcflneoo can Ammmv :flmuoum mannaom uamm *4 no «:00 05H500E wmoH mcflnhamcc CM 000: amoHocwEumu on» you sawuficfiumo ma munmflm 8V 01.3900th om cm on om cm 04 on P L F 1F {Ir Pl h h L F L L lb '0 F MB 204 5:55: mmm O 8. 51 44...... $306; a 1 00000 0 1 Awbzxrnxmybsntaa oo 1 o I 09 :1: o as and m or asazaiz 4914434 6 .082698 a 3444 444 4444444844 44 0 MB 0.4m 303g 4 05.86 can as 5m 4 682568: a {$0.00 HE 90H 1. 09 40m 1 4 g3: 08 can 444 44444 ...2434444444444‘SSW 48.. 0566 1 .49 um; H6345 mlnor 1 1 1 m .2 (Dd) snlnpow SSO'] 80 WPC combination gel, therefore, the 1'st and 2nd transitions were missing. The addition of 98% soluble WPC increased the viscosity (G") and decreased the elasticity (G') of SSP at 30°C (Table 9) suggesting the 98% soluble WPC diluted the SSP and the protein combination behaved more like a viscous liquid than elastic solid. The combination gels containing insolubilized WPCs (80%, 41%, and 27% soluble WPC) had a higher initial value of G' and G" than the SSP alone, and the value increased significantly as the WPC solubility decreased (Table 9) . Since the unfolded and aggregated proteins had more surface and hydrophilic groups exposed (Beuschel, 1990) , the addition of the insolubilized WPCs (80%, 41%, and 27% soluble WPC) could reduce the effective moisture content by binding water and increase the SSP concentration in the continuous phase (Lanier, 1991). This resulted in a very viscous solution Which displayed some characteristics of an elastic solid. The first transition of G' and G" for SSP occurred at 55.1 and 50.9°C, respectively (Table 8) . The addition of WPCs generally shifted the first transition to a higher temperature. Lanier (1991) suggested that water dispersed the Itilrofibrillar protein molecules, allowing an expanded network to develop as protein-protein bonds formed during heating. when WPC was included in the system, it might compete with SSP for water, and reduce the available water to hydrate and Suspend the SSP molecules (Lanier, 1991). Therefore, the 81 frable 9 Dynamic moduli of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble and 12% whey protein concentrate (SSP/WPC) heated from 30 to 95°C Dynamic Moduli (Pa) Treatments” Initial Point 1'th Tr 3rd Tr End Point 30°C 95°C Storage Modulus (Pa) SSP 852.2” 1788.3” 940.5” (Maximum) SSP/WPC 98 533.6” 133.7” 2043.3”” SSP/WPC 80 1205.9” 519.9” 2705.0”” SSP/WPC 41 1915.3” 1390.9” 3123.0” SSP/WPC 27 10537.7” 822.1” 2664.0”” Loss Modulus (Pa) SSP 119.9” ------ 344.5” 44.4” (Maximum) SSP/WPC 98 207.9” 243.6” 61.5” 256.1” SSP/WPC 80 336.4” 435.6” 148.0” 277.8” SSP/WPC 41 456.1” 693.6” 332.3” 148.8” SSP/WPC 27 2229.0” ------ 3205.0” 577.2” \ iEl Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript letter are significantly different (P < 0.05) 82 gelling properties of the SSP was impaired, and resulted in a higher temperature for the first transition, and a higher thermostability of the combination gels. After the first transition, the G' of SSP increased indicating the formation of a SSP gel network (Figure 10). In contrast, the combination gels containing 98%, 80%, or 41% soluble WPC exhibited a decrease of G' indicating these WPCs interfered with gel network formation of SSP. At the 3rd Tr, a shoulder occurred approximately at 65°C for the combination gels containing 98%, 80% or 41% soluble WPC probably due to the formation of SSP gel network which eventually overcome the interference from WPCs. The 3rd Tr for the combination gels occurred at an insignificantly higher (2°C) temperature compared to the temperature where SSP formed its maximum peak. However, unlike SSP which formed a peak with a value higher than its initial G' value, the shoulder (3rd Tr) formed in the combination gel containing 98%, 80% or 41% soluble WPC never exceeded its initial 6' value (Table 9) . The G' of combination gel containing 27% soluble WPC remained constant until 65.3°C. It was probable that, because the high initial G ' , the increase of 0' due to development of SSP gel network was hidden. SSP formed its maximum peak at 63.2°C, which was identical to the 3rd Tr (63-65°C) in the combination gels. when comparing G' at this temperature, the elasticity of the Q<>mbination gel containing 98% or 80% soluble WPC was s:‘Lgnificantly lower than that of the SSP gel; the elasticity 83 of the combination gel containing 27% soluble WPC was significantly higher than that of the SSP gel (Table 9) . This suggested the addition of soluble WPC weakened the SSP gel network at this temperature (63-65°C); in contrast, the insolubilized whey proteins improved elasticity due to WPC ability to hold water. When comparing G" (Figure 11), the addition of 98%, 80% or 41% soluble WPC flattened or narrowed the transition peak of SSP, resulted in a 1'st Tr at 58.2-60.2°C, followed by a rapid decrease of G". A shoulder (3rd Tr) was formed at 65.9- 67.0°C. The addition of 98%, 80%, and 41% soluble WPCs might have interfered with SSP gelation and resulted in the decrease of G" between the 1'st and 2nd Tr in combination gels. The addition of 27% soluble WPC exhibited a flattened peak at an identical temperature where SSP formed its maximum peak. The G' value of SSP above 78.4°C did not change (Figure 10) . The G' of the combination gels containing 98%, 80%, or 4 1% soluble WPC increased throughout heating from 78.4 to 95°C, This indicated the presence of 98%, 80% or 41% soluble WPC enhanced the elasticity of the combination gels above 7 8.4°C. In contrast, the G' of the combination gel containing 2 7% soluble WPC gradually decreased at temperatures above .7 7.8°C. The decrease of elasticity in the 27% soluble WPC/SSP gel could be explained by two probable interpretations. F first, the denatured whey protein aggregates were incompatible with the SSP gel network. The whey proteins might have tiistorted the SSP gel structure leading to undesirable 84 modifications of gel properties. Tolstoguzov and Braudo (1983) reported "structural compatibility" was the ability of macromolecules or dispersed particles of the protein filler to be distributed in the gel network without distortion. Second, the denatured whey protein aggregates interfered with the crosslinking of SSP at its optimum gelation temperature. The number of crosslinkages were associated with the elastic moduli during thermal processing (Hamann, 1983; Nossal, 1988) . On further heating, the.continuous input.of oscillatory strain broke chemical bonds and decreased the elasticity of the combination gel. At the end point of 95°C, WPCs increased the G' of combination gels. However, due to the large experimental error, only 41% soluble WPC showed a significant influence (Table 9). After the 5th Tr, the increase of G" was not as great as that of G' and reached a much smaller value at 95°C for SSP and all the combination gels (Table 9). 4.2.2.2 Isothermal Heating at 65°C for 15 min The changes in G' and G" of SSP and SSP/WPC combination gels heated isothermally at 65°C for 15 min are observed (Figures 14 and 15). The G' of SSP increased from 633.4 to 1281.0 Pa during the first 1.6 min of heating, then decreased to 195.3 Pa which was lower than the initial G' value after 6.2 min of heating. The G' increased again and reached 600.8 Pa after 15 min of heating (Figure 14). The G" of SSP increased from 112.9 to 259.5 Pa during the first 2.1 min of ‘F: 85 0w8 ma Mom 06mm us 00000: A0m3v 0vmuv000000 cw0uoum >053 wad 0:4 0mm *4 no mcowuocwnaoo 0:0 sw0uoum 0HQSH00 pawn «4 no A.UV wasvofi 0000000 0:» mcfiumuumsaafl Smuoo0nu 0>auouc0m0un0m 4H 000040 38V 66: m? or m Pt L T L L L L 0! b 1? u 1? 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O TJKV? o m oooooooooooooooooooooo owmc oo oooooooo ooooooo oa ooo QMM\OA_>» 003200 NEW 9 QWM\0&; 2320” NT? 0 1 amm\on§ 6328 New 4 fl ammbas 6328 .88 a p>>>>>>1flflopw>>>> >9>D§>9D DDDDD>§D§§ fibfibbpbbfibfifi9bb 40F (Dd) sngnpow 8501015 86 :00 00 you 0.00 pm 00000: 00030 00000000000 0000000 >003 w~0 000 000 «4 mo 000000000000 000 0000000 0000000 0000 «4 00 “:00 000000 0000 000 000000000000 00000000 0>00000000000m 00 000000 AEEV 00:0 9 00 m o —l b r b L b - h L IF L In b - IIP FOP mmonuanmmammoommmmmnmmmmmmmoooomammma 1 00 o 0 1 o 000 .. o T 444444 0 444444 444 0 0 0 444444444 44 4444 4444 0 0 «O— 4 0 4 O 4 oooooooooooooooooooooooooooo OOG044 00000 0 >>>>>>>>>>>pp>>>>>>>>>>>>>>>>>>p>>>> 440 now 000%, «328 0R. 9 p p b p b p p p p p p “030%, «328 N z. “000%., 0328 08 0000.0, 0328 «mm amm O D 4 O 400 (Dd) snlnpow 930'] 87 heating, decreased to 22.1 after 8.9 min of heating, then remained relatively constant, with a final G" of 33.1 Pa (Figure 15). The transitions of G' and G" which appeared in the first 6 min of isothermal heating at 65°C were also observed in the 55-69°C temperature range in the SSP thermal scanning experiments. The addition of WPCs eliminated the transition peak, and resulted.in.a:re1atively small increase in.G' during the heating period, indicating WPC interfered with the gelation of SSP. The initial and final dynamic moduli of the combination gels were not very different. Except for a small transition during the first 2 (for G')- 4 (for G") min of heating, the G' or G" curve of all the combination gels remained constant, and paralleled each other (Figures.14 and 15). This suggested.the dynamic properties of combination gels was determined by the original properties of WPCs prior to heating. The dynamic moduli (G' and G") of SSP and combination gels after 15 min of isothermal heating at 65°C are shown in Table 10. The G' and G" of combination gels followed the same order after 15 min of heating: the dynamic moduli of the combination gel containing 27% soluble WPC was significantly higher than the combination gels containing 41%, 80%, and 98% soluble WPC. As the solubility of WPC decreased, the G' and G" of combination gels increased. The combination gels containing 98% and 80% soluble WPCs had a lower 6' but higher G" than SSP after completion of 88 heating (Table 10), indicating the addition of highly soluble WPC caused the SSP gel to behave more like a viscous liquid than an elastic solid" The addition of 41% or 27% soluble WPC increased both the G' and G" value of SSP gel at the end point. At 65°C, whey proteins were unable to form a gel, thus, SSP was.probably solely responsible for the network.of SSP/WPC combination gels. This type of combination gel matches the model of a "filled. gel" (TolstoguzOV' and Braudo, 1983; Oakenfull, 1987; Ziegler and Foegeding, 1990; Lanier, 1991) with WPC acting as the filler. The combination gels containing highly solubilized WPCs (98% or 80% soluble WPCs) are like a type I filled gel, in which the filler (WPC) remains soluble in the interstitial fluid of the SSP gel matrix (Ziegler and Foegeding, 1990; Lanier, 1991) . The addition of highly solubilized WPCs seemed to "dilute" the SSP or interfere with SSP gelation, resulting in mixtures of a lower 6' than an additive relationship would predict (Burgarella et al., 1985). Lanier (1991) suggested that since the filler was soluble, it should add to the viscosity of the interstitial fluid.and increase the:G" value. Hermansson and Akesson (1975) reported that whey proteins were highly soluble nonswelling proteins with very low viscosities. This could explain the limited increase of G" contributed by the addition of 98% soluble WPC (3Pa) to the combination.gels. The combination gels containing highly insolubilized WPCs (41% or 27% soluble WPC) are more like a type II filled gel, 89 Table 10 Dynamic moduli of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% salt soluble protein and 12% whey protein concentrate (SSP/WPC) heated isothermally at 65°C for 15 min Dynamic Moduli (Pa) Treatmentsa Initial Point End Point at 0 min at 15 min Storage Modulus (Pa) SSP 633.4c 600.8c SSP/WPC 98 416.5c 320.3c SSP/WPC 80 693.6c 574.7c SSP/WPC 41 1593.7c 1839.7c SSP/WPC 27 8725.3” 8023.7” Loss Modulus (Pa) SSP 112.9d 33.1c SSP/WPC 98 167.7d 36.3” SSP/WPC 80 243.1d 116.0c SSP/WPC 41 606.2c 236.7c SSP/WPC 27 1623.7” 811.6” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript letter are significantly different (P < 0.05). 90 in which the filler (insolubilized WPC) exists as dispersed particles, presumably unassociated with the gel matrix (Ziegler and Foegeding,1990; Lanier,1991). The insolubilized whey proteins absorbed water, reduced available water and concentrated the continuous protein phase (SSP) . In addition, whey proteins occupied the interstitial spaces within the gel matrix and reinforced the gel. Therefore, an increase in the dynamic moduli of these combination gels occurred. The results of dynamic tests coincided with that of failure compression tests (Beuschel, 1990); at temperatures below gelation of whey proteins, insolubilized WPC had a positive effect on the strength of SSP/WPC combination gels. 4.2.2.3 Isothermal Hosting at 90°C for 15 min When SSP was heated isothermally at 90°C, 6' decreased in the first 30 sec of heating, following by a rapid increase, then, the rate decreased after 2.5 min of heating (Figure 16). The SSP G" decreased during the first min of heating, then remained constant during the rest of the heating process (Figure 17). The typical transitions of G' and G" for SSP which occurred at 55 to 69°C during the thermal scanning experiment (Figures 10 and 11), which were also have been observed at the first six min of isothermal heating at 65°C (Figures 14 and 15), did not appear when SSP was heated at 90°C (Figures 16 and 17) . This was probably due to SSP going through its transitions much quicker at 90°C, resulting in.on1y'a decrease 91 000 m0 000 ooom 00 000000 00030 00000000000 0000000 0003 «N0 000 000 04 00 000000000000 000 0000000 0000000 0000 «4 00 0.00 000000 0000000 000 000000000000 00000000 0>000000000000 00 000000 3.5 0&0 m, 0 m 1.. 0mm 0 . t o .00 “000.0, 22.8 Now a o 0mm\0nt$ 0.03.00 Now 4 o o .0001; 4328 0:. 0 coupon m “000.13 2928 08 9 ocean IS 000 00 00000000 00 T ooooooooooooooo 00 1 000000000000000 0000 44MQOI 00 4 o 00 44 o cmmnooooaamwuwuooooo ooooooooooooommmmmmu444«¢ opp >>>>>wmpolumm>>>>>>>>>>>>>>>>>>>po>>>>>>>>>>>> b 400 mmmmm 400 (Dd) snlnpow 9501013 92 I . ---}.L Jar..- . 000 00 now ooom 00 000000 00030 00000000000 0000000 0003 0~0 000 000 04 mo 000000000000 000 0000000 0000000 0000 04 00 0:00 000000 0000 000 000000000000 00000000 0>000000000000 00 000000 905 9:0 PL 0 . Jr mmm 0003...; 2828 08 “000%, 22.8 Now 000%, 2828 NS “000%, 0328 «R 90600 00 O 0 oo o on one a 000 00000000 oo oo a j 1 1- 1 m VI 0 noun 000 O D 000°00000OOOOOOOWDOWOQ‘fiamwoooooo 0 0440444444 00 44 .0— (od) sngnpow $901 93 of G' and G" shown during the first 30 sec of heating. Combination gels containing 98%, 80%, or 41% soluble WPC showed a decrease of G' during the first 30 sec of heating, following by a more rapid and extensive increase of 6' than the gel prepared by SSP alone. The increase of G' was more extensive as the solubility of WPC increased. The G' of 27% soluble WPC/SSP combination gel decreased during heating. Similar trends were observed.for G" under the same conditions, but the effect upon G" was smaller (Figure 17). After heated isothermally at 90°C for 15 min, SSP had.the lowest 6' and G" (Table 11). The combination gels containing WPC had a greater G' and G" than an additive relationship would have predicted when WPC and SSP were heated alone at 90°C. The higher the solubility of WPC in the combination gels, the greater the dynamic moduli increased during the 15 min heating period (Table 11), suggesting soluble whey proteins were responsible for the increased dynamic moduli of combination gels. As the soluble whey proteins formed a gel on isothermal heating at 90°C, they could interact with SSP to form a "coupled network" as described by Oakenfull (1987) and Brownsey and Morris (1988) . Alternatively, whey proteins might gel within the interstitial spaces of the already-formed SSP gel network, and form a "phase separated network" as described by Oakenfull (1987) and Brownsey and Morris (1988). This kind of gel also matched the "type II filled gel" as described by Ziegler and Foegeding (1990) and.Lanier (1991) in 94 Table 11 Dynamic moduli of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% soluble soluble protein and 12% whey protein concentrate (SSP/WPC) isothermally heated at 90°C for 15 min Treatmentsa Dynamic Moduli (Pa) Initial Point End Point at 0 min at 15 min Storage Modulus (Pa) SSP 41.4C 660.7d SSP/WPC 98 124.9c 15490.0” SSP/WPC 80 350.2c 9976.0”c SSP/WPC 41 1056.0c 6688.0Cd SSP/WPC 27 8701.0” 8553.1”c Loss Modulus (Pa) SSP 16.9c 41.9c ssp/wpc 98 145.5c 2645.0” SSP/WPC 8o 338.8c 1455.0”c SSP/WPC 41 318.7c 766.3c SSP/WPC 27 2303.0” 805.7c a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d.Means within the same column with a different superscript letter are significantly different (P < 0.05). 95 which gelled whey proteins were the filler entrapped by the SSP network. The marked increase in dynamic moduli of the combination gel containing 98% soluble WPC could be explained as resulting from either of the gel models described above. The insolubilized whey proteins competed for available water and increased the SSP concentration in continuous phase (Lanier, 1991). Therefore, the combination gels containing less soluble WPC had a higher initial G' and G" (Table 11). The insolubilized whey proteins could precipitate as part of the entrapped particulate fraction within the SSP gel network to form a "type II filled gel" (Ziegler and Foegeding, 1990; Lanier, 1991). The 27% soluble WPC/SSP'gel primarily belonged to this kind of gel model. The decreased dynamic moduli of 27% soluble WPC/SSP gel after approximately 2.5 min of heating was probably due to the structural incompatibility (Tolstoguzov and Braudo, 1983) or the interference of SSP crosslinking from denatured whey protein aggregates. In conclusion, the combination gels tended to have a higher elasticity and viscosity than the SSP gel. Dynamic moduli of combination gels for solubilized and insolubilized whey proteins increased by different mechanisms. The dynamic moduli of combination gels containing more solubilized whey proteins increased more during heating above its gelation temperature. In contrast, the combination gel containing more insolubilized whey proteins had higher initial dynamic moduli, but lack of increase during heating. -r-IPUP 96 4.2.3 Effect of nesting Conditions on Rheological Properties of Gels When 4% SSP in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, was heated from 30 to 95°C at 2°C/min, 6' increased until 78.4°C then leveled off, suggesting the gelation of SSP was almost complete at this temperature. The G' of SSP reached 852.2 Pa at a temperature of 90°C during thermal scanning experiments, which was 191.4 KPa higher than the 6' produced by isothermal heating at 90°C for 15 min (660.8 Pa) (Table 12). However, both heating treatments gave protein solutions the same heat input which could be calculated by the area under the heating curve (Figure 18). It has been suggested that if aggregation occurred at a slower rate than denaturation, a finer and more ordered gel network with higher degree of elasticity is produced (Ferry, 1948; Hermansson, 1978). When SSP was heated isothermally at 90°C, denaturation and aggregation would be expected to occur almost simultaneously. In comparison, during thermal scanning, the protein molecules could unfold at relatively lower temperatures, followed by progressive aggregation as the temperature was increased. Therefore, there was more time for the denatured proteins to orient themselves and form an ordered gel with higher elasticity. Wu et al. (1991) reported that 1% myosin gels formed at low temperatures (44-56°C) were generally stronger and more elastic than those formed at higher temperatures (SB-70°C). Since crosslinking produces the shear modulus and elasticity 97 Table 12 Effect of heating conditions on the storage moduli (G') of protein solutions containing 4% salt soluble protein (SSP) or combinations of 4% SSP and 12% whey protein concentrate (SSP/WPC) Treatmentsa Thermal Scanningb Isothermalc experiment experiment SSP 852.2e 660.7f SSP/WPC 98 899.58 15490.0d ~~ SSP/WPC 8o 1577.0de 9976.0de SSP/WPC 41 2747.7de 6688.0ef SSP/WPC 27 3128.3d 8553.1de a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0 b Protein solutions heated from 30 to 90°C at 2°C/min c Protein solutions isothermally heated at 90°C for 15 min d-f Means within the same column with a different superscript letter are significantly different (P<0.05) 98 .1 -—- Thermal Scanning 90 H Isothermal Heating / 60-4 Temperature (C) “0-------—-------- \ a... m““""""““"" ....--...._..._......... Time (min.) Figure 18 Temperature-time profile for thermal scanning from 30 to 90°C at 2°C/min and isothermally heated at 90°C for 15 min 99 (Nossal, 1988), they suggested that low temperatures favored the crosslinking (aggregation) process. However, in the present study, isothermal heating of SSP at 90°C produced a gel with higher elastic modulus (660.7) than that at 65°C (600.8). Effects of isothermal heating at 90°C and thermal scanning at 2°C/min to 90°C on 4%SSP/12%WPC combination gels were compared. Storage moduli of combination gels formed during a thermal scanning experiment were lower than those produced by isothermal heating (Table 6). Since soluble whey proteins enhanced elasticity of combination gels more effectively in isothermal experiments, the heating conditions on WPC gelation should be considered. According to differential scanning calorimetry results reported by deWit and Klarenbeek (1984), denaturation temperatures (Td) of whey proteins in 0.07 M phosphate buffer at pH 6.0 are 62-78°C. That means, WPC was not able to form gel until the heating temperature increased above 62-78°C during thermal scanning. In comparison, when isothermally heated at 90°C, WPC start unfolding and aggregating immediately. Therefore, under isothermal heating at 90°C, whey proteins had more time to form a gel within the SSP gel network or to interact with SSP, which resulted in a combination gel with higher dynamic moduli. 100 4.3 Scanning Electron Microstructure (GEM) 4.3.1 Salt Soluble Protein (SSP) Gels The microstructure of 4% SSP gels, in 0.6 M NaCl, 0.05 M phosphate buffer, at pH 7.0, heated isothermally at 65°C for 15 min showed a fine three dimensional filamentous network, consisting primarily of beaded strands although some aggregated areas were present (Figure 19A). Examination at higher magnification (Figure 198) revealed some gel strands were made up of globular’ particles which were linearly arranged. When the particles clumped together, protein aggregates were formed. A similar filamentous matrix was also observed by‘Wang (1990) for SSP gel at neutral pH. IHermansson et al. (1986) suggested that myosin could form two different gel structures, strand-like or globular aggregates, depending on pH and ionic strength. 4.3.2 Whey Protein Concentrate (WPC) Gels The 16% WPC gels, in the same buffer solution, heated at 90°C for 15 min exhibited typical grape-like globular structures (Figure 20), similar to those observed by Beveridge et al. (1984), Hermansson (1986), and Aguilera and Kessler (1989). The globular particles aggregated randomly, without ordered network formation. Beveridge et al. (1984) observed that, in both.WPC and egg albumin gels, there was a coating or bridging material cementing aggregates together, and suggested the bridging material may represent the deposition of soluble protein between the particles, or perhaps as an artifact 101 Figure 19 Scanning electron micrographs of 4% salt soluble protein (SSP) gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 65°C for 15 min. (A) bar length equals 5 um; (b) bar length equals 1 um. 102 .. _ . . : 1., . i . . V a , n . 0, . _ . .v n .. , . . p . 0 I i. I . . .... .. J v .V T . .0 . . . , ... n y . . r .1 n i , : OI . . 8 . . . . 4. .. n. 0 a 0.0%} Figure 20 103 Scanning electron micrographs of 16% whey protein concentrate (WPC) gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 90°C for 15 min. (A) 98% soluble WPC gel; (B) 80% soluble WPC; (C) 41% soluble WPC; (D) 27% soluble WPC. Bar length equals 5 um. 104 105 generated by glutaraldehyde gelation of soluble protein. The 98%, 80%, and 27% soluble WPC gels (Figures 20A, B, and D) contained globular aggregates of approximately the same size within each treatment, and some small open.pockets evenly distributed on the gel surface. The 98% soluble WPC gel (Figure 20A) has globular aggregates with a diameter range from 0.6 to 1.0 um. 'The 80% and 27% soluble WPC’gels (Figures 208 and D) have the globular aggregates in a smaller diameter of 0.3 um. The 41% soluble WPC gel (Figure 20C) exhibited a distinct feature: the gel structure contained variable size particles, and many large voids existed within the gel. The small aggregates in the 41% soluble WPC gel were similar to those in the 98% soluble WPC gel, and the larger, granular surface aggragates were with a diameter range from 5.0 to 7.5 um. The denser microstructure of 98%, 80% and 27% soluble WPC gels was compatible with their higher elasticity compared to that of 41% soluble WPC gel. In addition, their dense proteins surrounding the evenly distributed open pockets were believed to be suitable for trapping water and resulted in higher water holding capacity as studied by Beuschel (1990). The coarser microstructure with big holes in the 41% soluble WPC gel was compatible with its lower elasticity and water holding capacity. 106 4.3.3 Salt Soluble Protein] Whey protein Concentrate (SSP/WPC) combination Gels 4.3.3.1 Combination Gels Heated at 65°C The finely defined filamentous beaded strand SSP gel network was evident in the 98% soluble WPC/SSP combination gel, yet no whey protein globular aggregates were observed (Figure 21A). Since whey proteins did not gel at 65°C, they might be soluble, and were washed out when gels were fixed for SEN. Both the SSP criss-crossed fibrous network and the small whey protein globules were visible in the 80% soluble WPC/SSP gel (Figure 21B). The whey protein globules were evenly distributed and were embedded within the SSP gel matrix. In the 41% soluble WPC/SSP combination gel (Figure 21C), some small whey protein globules with a diameter of 1.0 um were embedded in the paralleled oriented SSP network. However, some globules appeared to have aggregated into larger globules with diameter of approximately 5.0 umiandjbroken away from SSP structure to form two separate networks. In the 27% soluble WPC/SSP combination gel (Figure 21D), large whey protein assemblages were separated from the SSP network. The SSP fibrous weave pattern was replaced by oriented strand-like fibers. All strands were elongated and oriented in the same direction. Salt soluble protein maintained its ordered criss-cross fibrous weave pattern microstructure, and acted as the principle component to support the structure of the Figure 21 107 Scanning electron micrographs of 4% salt soluble protein (SSP) and 12% whey protein concentrate (WPC) combination gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 65°C for 15 min. (A) 98% soluble WPC/SSP combination gel; (B) 80% soluble WPC/SSP combination gel; (C) 41% soluble WPC/SSP combination gel; (D) 27% soluble WPC/SSP combination gel. Bar length equals 1 um. 108 5 o e 0. 5 1 0t rup- "o_ a. V.. ., .0... . £er .0». ..3 0 1% ile um. a. 0“ I... r f? 3 r ... h 14 , 109 combination gels containing 98% and 80% soluble WPCs heated at 65°C. These microstructures are consistent with the identical elasticity for the SSP gel and the combination gels containing 98% or 80% soluble WPC. The typical SSP fibrous network was partially destroyed when the 41% soluble WPC was added. The concentrated SSP matrix caused by the addition of big denatured whey protein aggregates might be responsible for the increased elasticity of 41% soluble/SSP combination gel. In the 27% soluble WPC/SSP combination gel, the SSP matrix was distorted by the denatured whey protein aggregates. The SSP strands were thinner, longer, and contained fewer cross-links indicating the interference by denatured WPC. The high gel elasticity can probably be attributed to the large denatured whey protein aggregates. 4.3.3.2 Combination Gels Heated at 90°C Globular structures, characteristic of whey protein gels, predominated the microstructure of combination gels containing 98% or 80% soluble WPC (Figures 22A and 223). The SSP's fine filamentous network which was observed in the same combination gels at 65°C was not observed. Some web-like fibrous threads interspersed throughout the gel structure were observed at higher magnification in the 80% soluble WPC/SSP combination gel (Figure 233) . The loosely-weaved fibrous SSP pattern indicated heat might have destroyed some of the crosslinking within the SSP gel matrix. Hermansson (1982, 1983) suggested Figure 22 110 Scanning electron micrographs of 4% salt soluble protein (SSP) and 12% whey protein concentrate (WPC) combination gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 90°C for 15 min. (A) 98% soluble WPC/SSP combination gel; (B) 80% soluble WPC/SSP combination gel; (C) 41% soluble WPC/SSP combination gel; (D) 27% soluble WPC/SSP combination gel. Bar length equals 5 um. 0.x. 03 n. 1'. wayward»? «0 . . ... .. .wh. 0.0.3“. . . .... .... . . 0.... ... ruin.“ a; ..H ...... .. .. .. a. by}? 0V. Figure 23 112 Scanning electron micrographs of 4% salt soluble protein (SSP) and 12% whey protein concentrate (WPC) combination gels prepared in 0.6 M NaCl, 0.05 M phosphate buffer, pH 7.0, heated at 90°C for 15 min. (A) 98% soluble WPC/SSP combination gel; (B) 80% soluble WPC/SSP combination gel; (C) 41% soluble WPC/SSP combination gel; (D) 27% soluble WPC/SSP combination gel. Bar length equals 1 um. 113 e 1.. be nu 5L IILII 55 01 AU 7L 0 I... B I.\ 0' .uble 11m. .1: ‘ ._ «ah; . a O .0”. 2. 114 that heating above the gelation temperature would increase tendency for protein-protein interaction and cause shrinkage or phase separation. In the 98% soluble WPC/SSP combination gel (Figure 22A), the mixture of threads and globules was homogeneous and more compact than the 80% soluble WPC/SSP combination gel, resulting in much higher gel elasticity. The microstructure of 41% soluble WPC/SSP combination gel heated at 90°C was similar to that at 65°C (Figure 23C vs 21C). Both the SSP fibrous structure and the whey protein globules were observed (Figures 22C and 23C). At 90°C, the SSP strands seem. to be enmeshed 'with the whey protein aggregates to a greater extent, and the SSP strands were in a twisted rather than a parallel conformation (Figure 23C) . The increased aggregation of the protein strands may also be responsible for the higher elasticity observed in the 90°C treatment. However, its microstructure was not as compact as the combination gels containing 98% soluble WPC (Figure 23A), resulting in significantly lower gel elasticity when compared to 98% soluble WPC/SSP combination gel. In the 27% soluble WPC/SSP combination gel (Figure 220), the individual globules merged to form large aggregates. There is still some parallel alignment of SSP strands, as observed in that at 65°C (Figure 210). At 90°C, whey proteins gelled and a globular structure predominated the combination gels containing highly soluble WPC (eg. 98% and 80% soluble WPCs). Both the SSP filamentous matrix and whey protein globular structure were present in the 115 combination gels containing highly insolubilized WPC. The existence of denatured whey protein aggregates before gelation of SSP seems to distort the SSP network, interfere with crosslinking of SSP and cause the strands to become oriented in a parallel direction. 4 - 4 Effect of whey Protein Concentrates in Meet Hodel systems 4 - 4.1 Model System Low Fat Frankfurters 4 - 4.1.1 3.5% WPC-Supplemented Low Fat Frankfurters The raw control frankfurters contained 9.3% protein, 14 .1% fat, and 70.5% moisture (Table 13) . Frankfurters containing 41% soluble WPC were higher (P < 0.05) in protein content in comparison to all-meat control. Frankfurters containing 27% soluble WPC had lower (P < 0.05) moisture content than the all-meat control (Table 13) . After cooking, control frankfurters contained 12.8% Protein, 15.3% fat, and 68.3% moisture (Table 13) which were similar to target values of 12.0% protein, 15.0% fat, and 69% moisture. Protein, fat, and moisture contents did not differ (P > 0.05) among treatments (Table 13) . Thompson et al. ( 1982) observed the protein, fat and moisture contents of raw meat patties containing 5% or 10% WPC were altered by cooking, and suggested that changes in composition during cooking were e)ercted from variation in cooked yield and water and fat retention. Cooked yield (Table 14) of model system frankfurters with 3 ~ 5% substituted WPC did not differ (P > 0.05) from the 116 Table 13 Chemical analysis of raw and cooked silent cutter processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis Raw Product Cooked Product WPC Treatmenta Protein Fat Moisture Protein Fat Moisture (%) (%) (%) (%) (%) (%) Control 9.3” 14.1” 70.5” 12.8” 15.3” 68.3” WPC 98 10.7”” 13.4” 69.7”” 13.5” 14.4” 68.2” WPC 80 10.6”” 14.4” 68.7”” 13.3” 15.3” 67.0” WPC 41 11.1” 14.5” 68.2”” 13.5” 15.4” 66.4” WPC 27 10.0”” 14.5” 67.9” 13.4” 15.6” 66.2” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-c Means within the same column with a different superscript are significantly different (P<0.05). 117 Table 14 Cooked yield and severe reheat yield of silent cutter processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis WPC Treatmenta Cooked Yield Reheat Yield (%) (%) Control 90.2” 79.4” WPC 98 93.3” 81.3” WPC 80 93.4” 81.8” 5 WPC 41 92.7” 80.3” WPC 27 91.2” 74.2” . a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-c Means within the same column with a different superscript are significantly different (P<0.05). 118 control. Ensor et al. (1987) added 1.75%, 2.0% and 3.5% WPC to knockwurst cooked to an internal temperature of 68°C. They reported no significant difference in cooked yield, but the fat binding ability increased compared to an all-meat control. Thompson et al. (1982) also found that 10% WPC-supplemented frankfurters and an all-meat control did not differ in cooked yield when heat processed to 89°C. Morr (1979) proposed that whey proteins could be utilized in processed meat products to improve water and fat binding properties without adversely affecting flavor or textural properties. Lauck ( 1975) reported that addition of WPC to frankfurters enhanced water- and fat-retention when heated between 70 and 80°C. However, deWit (1988) observed that substitution of meat proteins by whey proteins improved the water-retention, but not the fat- retention for frankfurter-type meat, when heated to both 80 and 100°C. Lauck ( 1975) stated that an active binder can be used to compensate for reduced functionality when a nutritionally equivalent meat source of lower cost and reduced functionality is used. Reheat yield for the 3.5% WPC-supplemented frankfurters did not differ (P > 0.05) from the all-meat control except for frankfurters containing 27% soluble WPC which had a lower (P < 0.05) reheat yield (Table 14). However, Beuschel (1990) reported no significant difference in reheat yield when 3.5% WPCs were added to frankfurters, regardless of the WPC Solubility. In his study, frankfurters were formulated to ccantain 12.0% protein, 30% fat, and 56% moisture which was 119 higher in fat and lower in moisture contents, compared to the present study. Formulation differences may have caused the different results. Apparent stress, apparent strain, hardness, and springiness did not differ between treatments (P > 0.05) (Table 15) . Differences between cohesiveness, although significant, were slight. The 27% soluble WPC-containing frankfurters were the only product with higher (P < 0.05) cohesiveness than the control. 4.4.1.2 3.5% WPC-Supplemented Frankfurters Processed Under Vacuum A vacuum mixer was used to eliminate the air bubbles which were incorporated into the batters during chopping with a silent cutter and was considered to be one of the sources of variation between the samples in the previous study. Frankfurters were formulated to contain similar protein, fat, and moisture contents. The proximate analysis showed the all-meat control contained 12.3% protein, 14.4% fat, and 66.9% moisture (Table 16) . The WPC-supplemented frankfurters had higher (P < 0.05) protein, and lower (P < 0.05) moisture contents than the control. Frankfurters processed under vacuum had improved cooked yield and reheat yield as compared to those processed without vacuum (Table 17 vs 14) . Identical cooked yield was observed for the control and frankfurters containing 98%, 80% and 41% soluble WPCs (P > 0.05) (Table 17). The addition of 27% 120 Table 15 Texture of silent cutter processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis At Failure Point WPC Hardness Springiness Cohesiveness Treatmenta Apparent Apparent stress strain (N) (mm) (kPa) Control 46.8” 0.64” 38.5” 5.58” 0.24” WPC 98 43.6” 0.62” 35.1” 5.49” 0.22” WPC 80 48.1” 0.63” 40.1” 5.20” 0.23d WPC 41 45.6” 0.63” 43.2” 5.50” 0.24” WPC 27 41.5” 0.67” 36.2” 5.70” 0.25” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-e Means within the same column with a different superscript are significantly different (P<0.05). 121 Table 16 Chemical analysis of cooked vacuum processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis WPC Protein Fat Moisture Treatment” ( %) ( %) ( %) Control 12.3” 14.4” 66.9” WPC 98 13.5” 14.3” 65.9” WPC 80 13.6” 14.4” 65.7”” WPC 41 13.7” 14.7” 65.6”” i WPC 27 13.8” 14.5” 65.1” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript are significantly different (P<0.05). 122 Table 17 Cooked yield and severe reheat yield of vacuum processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis VVPC Treatment” Cooked Yield Reheat Yield (%) (2) Control 98.4” 82.2”” WPC 98 98.8” 85.6” WPC 80 98.8” 85.2”” WPC 41 98.0” 83.4”” WPC 27 95.3” 80.1” :1 Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. Ib-d Means within the same column with a different superscript are significantly different (P<0.05). 123 soluble WPC decreased (P < 0.05) the cooked yield compared to the control. Reheat yield of frankfurters containing 27% soluble WPC was the lowest among WPC treatments, but did not differ (P > 0.05) from the control. The frankfurters containing 98% soluble WPC had a higher (P < 0.05) reheat yield than the control (Table 17). Apparent stress at failure and hardness.did.not differ (P > 0.05) among WPC-supplemented frankfurters (Table 18). When compared to the control, frankfurters containing 98% or 80% soluble WPC had higher (P < 0.05) apparent stress at failure. Hardness of frankfurters were increased (P < 0.05) upon the addition of 98% or 27% soluble WPC (Table 18). The results agreed with those of Swartz (1983) who stated that the addition of ‘WPC to 'comminuted. meat systems resulted in increased firmness, when compared to an all-meat control. Ensor et al. (1987) also reported an increase of hardness when WPC (1.75%, 2.0% and 3.5%) was added to knockwursts. Comer et al. (1986) suggested that. the firmness ‘was primarily a function of water binding and gelation properties of the ingredients. In low-fat products, extra water was usually added to substitute for fat and effectively prevented undesirable sensory changes associated with low-fat products (Claus et al., 1989) However, problems of water binding, soft texture and purge accumulation occurred. Rust and Olson (1988) stated that water binding capacity would replace fat binding capacity 124 Table 18 Texture of vacuum processed low fat frankfurters prepared by substituting 3.5% various whey protein concentrate (WPC) for meat on a weight basis At Failure Point WPC Hardness Springiness Cohesiveness Treatmenta Apparent Apparent stress strain (N) (mm) (kPa) Control 54.7” 0.86” 27.5” 6.07” 0.22” WPC 98 62.2” 0.82” 36.8” 5.87” 0.22” WPC 80 63.1” 0.83”” 32.7”” 6.20” 0.23”” WPC 41 60.8”” 0.85”” 30.6”” 6.43” 0.24”” WPC 27 57.0”” 0.78” 36.8” 6.17” 0.25” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript are significantly different (P<0.05). 125 as the critical property in products containing less fat and more added water. Since meat proteins can absorb more water and become softer than desirable for optimum firmness (Comer et al., 1986; Claus and Hunt, 1991), nonmeat ingredients, by absorbing some of the moisture available to the meat proteins, may help produce a firmer product (Comer et al., 1986; Claus and Hunt, 1991). Claus and Hunt (1991) successfully increased hardness of 10% fat, 30% added water bologna by addition of fiber, starch, and soy protein, processed at 72°C. Our results also suggest that firmness could be improved by the addition of WPC as indicated by the increased apparent stress at failure and hardness. However, results obtained by Sofos et al. (1977) indicated that addition of soy protein isolates to wieners resulted in a softening compared to nonsupplemented wieners. Thompson et al. (1982) also found that addition of succinylated whey protein concentrate or whey protein concentrate decreased firmness of wieners over controls when processed at 89°C. Frankfurters containing 27% soluble WPC had the lowest apparent strain at failure, followed by the 98% soluble WPC (Table 18). Both treatments were lower in apparent strain at failure than the control. The addition of 80% or 41% soluble WPC produced equally apparent strain at failure in comparison to all-meat control (P > 0.05) (Table 18). Springiness was the same (P > 0.05) in all frankfurter treatments (Table 18) . Thus, the addition of WPCs had no 126 effect on the recovery of the product between successive compressions. The addition of 98%, 80%, or 41% soluble WPC produced equally cohesive frankfurters in comparison to the control (Table 18) . Cohesiveness of frankfurters containing 27% soluble WPC did not differ (P > 0.05) from the frankfurters containing 41% or 80% soluble WPC, but was higher (P < 0.05) than the frankfurters containing 98% soluble WPC and the control (Table 18). Ensor et al. (1987) reported that the addition of WPC (1.75, 2.0, 3.5%) resulted in a more cohesive product than the control knockwurst. 4.4.1.3 7.0% ch-supplemented Low Pet Frankfurters The raw 7.0% WPC-supplemented frankfurters had higher protein, but lower fat and moisture contents than the all meat control (P < 0.05) (Table 19). This might be attributed to the higher protein and lower fat and moisture contents of WPCs. After cooking, the higher (P < 0.05) protein and lower moisture contents were still observed in WPC-supplemented frankfurters, however, the fat content of WPC-supplemented frankfurters did not differ (P > 0.05) from the control except the 41% soluble WPC-supplemented frankfurters (Table 19). Cooked yield was higher (P < 0.05) in 7.0% WPC- supplemented frankfurters than the all-meat control except for the addition of 41% soluble WPC (Table 20). The significant improvement on cooked yield by addition of WPC was not observed when frankfurters were prepared by substituting 3.5% 127 Table 19 Chemical analysis of raw and cooked silent cutter processed low fat frankfurters prepared by substituting 7.0% various whey protein concentrate (WPC) for meat on a weight basis Raw Product Cooked Product WPC Treatmenta Protein Fat Moisture Protein Fat Moisture (%) (%) (%) (%) (%) (%) Control 12.4” 12.2” 72.5” 14.5” 13.3” 70.1” WPC 98 14.6” 12.0”” 69.2” 16.3” 12.7”” 67.4” WPC 80 15.0” 11.6” 69.7” 16.0” 12.5”” 67.4” WPC 41 14.7” 11.6” 69.4”” 16.3” 12.3” 67.0” WPC 27 14.9” 11.6” 69.5”” 16.6” 12.7”” 66.9” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript are significantly different (P<0.05). 128 frable 20 Cooked yield and severe reheat yield of silent cutter processed low fat frankfurters prepared by substituting 7.0% various whey protein concentrate (WPC) for meat on a weight basis ‘WPC Treatmenta Cooked Yield Reheat Yield (%) (%) Control 88.1” 76.9” WPC 98 91.3” 81.2” WPC 80 91.2” 80.5”” WPC 41 90.9”” 77.7”” WPC 27 91.0” 75.2” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript are significantly different (P<0.05). 129 WPC (Table 14 and 17). Reheat yield was higher (P < 0.05) in frankfurters containing 98% or 80% soluble WPC in comparison to all-meat control (Table 20). Within the WPC treatments, reheat yield increased as WPC solubility increased and was observed in both 3.5% (Table 14 and 17) and 7.0% (Table 20) WPC-supplemented frankfurters. DeWit and deBoer (1975) reported that heat treated WPC had a better water uptake ability. They suggested the porosity of the aggregates as well as the enhanced water- binding of the denatured proteins might be the main causes of increased water uptake. According to their theory, the insolubilized WPC should have higher water uptake ability and result in less cook loss and a firmer product at temperatures below the WPC gelation point. However, the silent cutter processed, WPC-supplemented frankfurters had an identical cooked yield, regardless of the WPC solubility (Table 14 and 20). The vacuum processed frankfurters with 3.5% WPC was even lower in cooked yield for frankfurters containing 27% soluble WPC than the other WPC-supplemented frankfurters. Results were contrary to what we expected. The apparent stress at failure did not differ (P > 0.05) among the treatments (Table 21). Frankfurters containing 98% or 80% soluble WPC had lower (P < 0.05) apparent strain at failure than the frankfurters containing 41% or 27% soluble WPC and the all-meat control (Table 21). Hardness, springiness and cohesiveness did not differ (P 130 Table 21 Texture of silent cutter processed low fat frankfurters prepared by substituting 7.0% various whey protein concentrate (WPC) for meat on a weight basis At Failure Point WPC Hardness Springiness Cohesiveness Treatment” Apparent Apparent stress strain (N) (mm) (kPa) Control 50.2” 0.55” 49.8” 4.80” 0.23” WPC 98 55.0” 0.48” 35.8” 4.04” 0.21” WPC 80 56.1” 0.50” 50.0” 4.38” 0.21” WPC 41 65.9” 0.54” 54.0” 4.63” 0.20” WPC 27 59.8” 0.56” 53.6” 4.63” 0.21” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-c Means within the same column with a different superscript are significantly different (P<0.05). 131 > 0.05) among model system frankfurters with 7.0% added WPC. Large variability between samples was a major problem in the low fat frankfurter model system. Big air bubbles and visible fat particles were considered to be possible causes for poor reproducibility. These errors could be improved by using vacuum mixer and eliminating pork fat from the formula. Stuffing was another potential source of variability. Hand stuffing may result in nonuniformity in speed and pressure applied to the batter when the tubes were stuffed. 4.4.2 Model System Chicken Rolls Effects of WPCs, soy protein concentrate (SPC) , and nonfat dry milk (NFDM) as binders in chicken rolls were examined at 3.5% addition. Binders were directly added to the product on a weight basis. Batters containing 3.5% binders did not differ (P > 0.05) from the control in fat and moisture contents (Table 22). The addition of 80% soluble WPC and SPC increased (P < 0.05) the protein content of chicken rolls. The SPC-chicken rolls had the highest protein content probably due to the higher protein concentration (67%) in SPC than in WPC (62%). After cooking, the moisture content of supplemented chicken rolls was lower (P < 0.05) than the control (Table 22). The protein and fat contents were the same (P > 0.05) in all-meat control and in the rolls containing WPCs or SPC (Table 22). The chicken rolls containing NFDM had a lower (P < 0.05) protein content than the rolls containing 98%, 80% or 132 Table 22 Chemical analysis of raw and cooked chicken rolls with 3.5% whey protein concentrate (WPC), soy protein concentrate (SPC) , or nonfat dry milk (NFDM) added on a weight basis Raw Product Cooked Product WPC Treatment” Protein Fat Moisture Protein Fat Moisture (%) (%) (%) (%) (%) (%) Control 16.3” 5.2” 75.3” 17.6”” 6.5”” 74.4” WPC 98 17.0”” 5.6” 74.2” 18.5” 6.5”” 73.2” WPC 80 17.3” 5.7” 74.6” 18.4” 6.6”” 73.0” ‘WPC 41. 16.9”” 5.3” 74.2” 18.5” 6.8” ‘72.7” WPC 27 17.1”” 5.9” 74.5” 17.9”” 6.4”” 73.1” SPC 17.5” 5.6” 74.2” 18.2”” 6.5”” '73.1” NFDMI 16.2” 5.6” 74.3” 17.3” 6.1” '73.4” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-c Means within the same column with a different superscript are significantly different (P<0.05). 133 41% soluble WPC, and a lower (P < 0.05) fat content than the rolls containing 41% soluble WPC (Table 22). The addition of NFDM or WPCs did not affect the cooked yield of chicken rolls (Table 23). No differences (P > 0.05) were observed among WPC treatments, regardless of the WPC solubility. Only the SPC-added chicken rolls had higher (P < 0.05) cooked yield than the control. Tensile testing was done to determine the binding strength between the chicken pieces. The addition of WPCs, SPC, or NFDM did not affect the tensile strength when compared to the control (P > 0.05) (Table 23). .No differences (P > 0.05) were observed among WPC treatments. The SPC-added chicken rolls had a higher (P < 0.05) tensile strength than the chicken rolls containing 98% soluble WPC (Table 23). The 27% soluble WPC produced a lighter colored product than the all-meat control, indicated by a higher (P < 0.05) Hunter L value (Table 23). Thompson et a1. (1982) reported that WPC or succinylated WPC-supplemented wieners were lighter in color than all-meat wieners due to dilution of the meat block by the white WPC. The 98% and 80% soluble WPC- supplemented chicken rolls were significantly higher in "a" values (higher positive values are more red) and lower in "b" value (higher values are more yellow) than the all-meat control and other treatments (P < 0.05) (Table 23) . The addition of SPC and NFDM had no influence (P > 0.05) on the color evaluation of chicken rolls. The apparent stress at failure of chicken rolls with WPC, 134 Table 23 Yield, tensile strength, and Hunter color values of chicken rolls prepared with 3.5% whey protein concentrate (WPC) , soy protein concentrate (SPC) , or nonfat dry milk (NFDM) added on a weight basis Cooked Tensile Color Evaluationb WPC Yield Strength Treatment” (35) (W9) L a 10 Control 96 . 6” 0 . 39”” 49 . 1” 2 . 71”” 9.71” WPC 98 96.7”” 0.37” 49.7”” 5.33” 9.17” WPC 80 96.8”” 0.38”” 49.9”” 5.43” 9.27” WPC 41 96.8”” 0.40”” 50.0”” 2.46”” 9.96” WPC 27 97.2”” 0.44”” 50.5” 2.30”” rid? SPC 98.4” 0.47” 48.8” 3.15” 9.90” NFDM 97.4”” 0.41”” 48.8” 2.16” n10? a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b Hunter color values: L value: a value: b value: black=0, white=100; red=positive to green=negative yellow=positive to blue=negative c-e Means within the same column with a different superscript are significantly different (P<0.05). 135 and NFDM were not different from the all-meat control (P > 0.05) (Table 24). The addition of SPC increased (P < 0.05) the apparent stress at failure over the all-meat control and 98%, 80%, and 41% soluble WPC-supplemented chicken rolls. Among WPC treatments, the 41% soluble WPC-supplemented chicken rolls had the lowest apparent stress at failure which was higher (P < 0.05) than the 27% soluble WPC-supplemented chicken rolls, but did not differ (P > 0.05) from the rolls containing 80% or 98%soluble WPC (Table 24). The chicken rolls containing 98% or 80% soluble WPC had decreased (P < 0.05) apparent strain at failure in comparison to the control (Table 24). The 98% soluble WPC-added chicken rolls had.the lowest apparent strain at failure, which did.not differ (P > 0.05) from rolls containing 80% or 41% soluble WPC, but was lower (P < 0.05) than the rolls containing 27% soluble WPC, SPC, or NFDM (Table 24). No differences in hardness of Chicken rolls were observed among treatments (P > 0.05) (Table 24) . However, the addition of 98% soluble WPC, 27% soluble WPC, and SPC increased the hardness value by 19.0, 15.3, and 20.3 N, respectively, compared to the control. The insignificant differences might be due to the large variability between samples. The springiness of chicken rolls containing WPCs, SPC, or NFDM did not differ from the all-meat control (P > 0.05) (Table 24). Among WPC treatments, the springiness of rolls containing 98% soluble WPC was the lowest, but did not differ (P > 0.05) from the rolls containing 80% or 27% soluble WPC. 136 Table 24 Texture of chicken rolls prepared with 3.5% whey protein concentrate (WPC), soy protein concentrate (SPC), or nonfat dry milk (NFDM) added on a weight basis At Failure Point WPC Hardness Springiness Cohesiveness Treatment” Apparent Apparent stress strain (N) (mm) (kPa) Control 72 . 6”” 0 . 69” 83 . 0” 9 . 33”” 0. 24”” WPC 98 68.5”” 0.53” 102.0” 8.25” 0.23” WPC 80 67.4”” 0.59”” 84.1” 9.23”” 0.23” WPC 41 64.1” 0.60””” 86.0” 9.46” 0.25”” WPC 27 87.2”” 0.63”” 98.4” 9.22”” 0.23” SPC 96.7” 0.65”” 103.3” 10.23” 0.26” NFDM 83.6””” 0.68”” 86.8” 10.01” 0.24”” a Numbers indicate percentage protein solubility in 0.1 M NaCl, pH 7.0. b-d Means within the same column with a different superscript are significantly different (P<0.05). .1 137 The rolls containing SPC had the highest springiness, however, the value did not differ (P > 0.05) from all other treatments except for the rolls containing 98% soluble WPC (Table 24). The cohesiveness of Chicken rolls with WPCs, SPC, or NFDM were not different from the all-meat control (P > 0.05) (Table 24). No differences (P > 0.05) were observed among the WPC treatments. Chicken rolls containing SPC had the highest cohesiveness. Specifically, while not differing (P > 0.05) from those containing NFDM or 41% soluble WPC, cohesiveness of SPC-added chicken rolls were higher (P < 0.05) than the rolls containing 98%, 80%, or 27% soluble WPC (Table 24). CONCLUSIONS Electrophoresis of whey' protein concentrates (WPCs) showed that the quantity of native proteins decreased with increasing severity of heat treatment. Heat resistance of the individual whey proteins.decreased.as.follows: a-la, B-lg, BSA and Ig. The polymerization in WPC treatments with solubilities of 98%, 80%, and 41% was mostly via noncovalent or disulfide bonds, since the polymers which aggregated on top of the native acrylamide gel were solubilized and dispersed into individual components in the presence of sodium dodecyl sulfate (SDS) and B-mercaptoethanol. However, some polymers in 27% soluble WPC could not be converted to monomers by SDS and B-mercaptoethanol which indicated the presence of non- reduCible covalent bonds occurred. Protein solutions containing 4% SSP, 16% WPC, and a combination of 4% SSP and 12% WPC were either heated from 30° (or 50”) to 95°C at a rate of 2°C/min or heated isothermally at 65° or 90°C for 15 min. Changes in storage modulus (G') and loss modulus (G") were continuously monitored. The 98% soluble WPC did not form detectable structure, characterized by a storage modulus (G') of 5 Pa or above, until 91.8°C. When isothermally heated at 90°C, the WPC with higher solubility had a better gelling ability, indicated by an 138 139 increased 6' and decreased tan 6 during heating. The high dynamic moduli of 27% soluble WPC were attributed to aggregation rather than heat-induced gelation, due to lack of increase of G' and relatively constant tan 6 throughout the entire heating period. Four transitions for G' and G" occurred when SSP was heated from 30 to 95°C. The addition of WPCs shifted the first transition of G' and G" of SSP to a higher temperature. At temperatures below whey protein gelation point, the insolubilized WPCs absorbed water, occupied the interstitial spaces within the SSP gel matrix and therefore, increased gel elasticity. In contrast, highly soluble WPC enhanced the elasticity of combination gels more effectively when heated above its gelation temperature, by forming a "coupled network" or "phase separated network" with SSP. Microstructure of combination gels containing highly soluble WPCs (98% and 80% soluble WPCs) was composed primarily of a fibrous network of SSP at 65°C, and globules of WPC at 90°C. For the combination gels containing highly insolubilized WPC (41% and 27% soluble WPCs), large denatured whey protein. aggregates. appeared ‘to interfere 'with crosslinking of SSP and cause the SSP strands to become oriented in a parallel direction. Low fat frankfurters were prepared by substituting 3.5% or 7.0% WPCs for meat on a weight basis. The addition of 7.0% WPCs (98%, 80%, and 27% soluble WPCs) increased cooked yield of low fat frankfurters. Reheat yield increased as WPC 140 solubility increased in both 3.5% and 7.0% WPC-supplemented frankfurters. Most textural properties did not differ significantly among treatments, due to the large variability between samples. When processed under vacuum, the 98% and 80% soluble WPC-supplemented frankfurters had a higher apparent stress than the control. Hardness of frankfurters increased on the addition of 98% or 27% soluble WPC in comparison to the control. Chicken rolls were prepared with 3.5% WPC, soy protein concentrate (SPC), or nonfat dry milk (NFDM) added on.a*weight basis. The SPC-supplemented chicken rolls had the highest cooked yield, tensile strength and apparent stress at failure among all treatments. Addition of NFDM produced chicken rolls with similar cooked yield and texture as SPC-supplemented chicken rolls. The 27% soluble WPC might be a better binder than the other WPC treatments. The 27% soluble WPC- supplemented chicken rolls had the highest apparent stress at failure. Inn addition, cooked yield, tensile strength, and apparent strain at failure increased. with increased. WPC insolubilization. Results from the model gel system evaluated by dynamic testing were coincidence with Beuschel's study (1990) that WPCs with varying protein solubilities might be useful for improving texture of processed meats under certain conditions. The use of highly soluble WPC might produce firmer products when processed at high processing temperatures, at which whey protein forms a gel. Whey protein concentrate with reduced 141 solubility may increase firmness of meats processed around 65°C. Although frankfurters and chicken rolls were processed at 72-74°C, temperatures below the gelation temperature of whey proteins, the addition of 27% soluble WPC did not always produce a harder (higher apparent stress) product when compared to the other WPC treatments. Except for the large experimental error, the small amount of WPC (3.5%) in meat products in comparison to that in the model gel system (12%) might.mask.the effect of WPC treatments in meat.model systems. 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