.0.‘5‘.».¢-' .19. . z: .... :15. .. fl. Earn. v V .L IQIH. n9. t.l‘v \fl 1.. r.» zit... i ,ximfid l .. x. y L x n :1”)?! S... ‘1!)- I-| .. lik1~ 'H " 1 9.3.6.: Fun; 97“ . . v. t .L. (I \u E: Ari (,1. . 3.33.5.2. ‘t‘l-tr '- a “5.... pg: .fiolf (If)!!! . :. «1,101.! v. (.7111? >c0 Ir (I‘I‘).4x 1.31:3}..‘3 .rl 2 4 ran. 7.. .bufi:4r . .. . . . .v «rut. u’. ‘ :7 . . . . , £L... ..~..I‘.u....). . . 0.3 . . . , , ‘ _. :f..- . . .éfiam .Egfifi ”(.4 figs TYIL IRB RABIES lllllll \‘l lull ll‘ll lllll llllll 31 This is to certify that the thesis entitled EFFECT OF pH AND NaCl ON THE THERMAL-PHYSICAL BEHAVIOR OF CHICKEN BREAST SALT-SOLUBLE PROTEINS presented by Shuefung Wang has been accepted towards fulfillment of the requirements for M° 8° degree in Food §cience [fim ”7. M Major professor 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University 1' PLACE IN RETURN BOX to remove this checkout from your record. ‘ TO AVOID FINES return on or before date due. II DATE DUE DATE DUE DATE DUE l #——-——‘ fi—fim _J'%% lfil= MSU Is An Affirmative Action/Equal Opportunity lndttution emeritus EFPECT OF DH AND NaCl ON THE THERMAL-PHYSICAL BEHAVIOR OF CHICKEN BREAST SALT-SOLUBLE PROTEINS BY Shuefung Wang A THESIS Submitted to Michigan State University in partial fulfillment of the require-ents for the degree of MASTER OF SCIENCE Depart-cut of Food Science and Human Nutrition 1990 ABSTRACT EFFECT OF DH AND NaCl ON THE THERMAL-PHYSICAL BEHAVIOR OF CHICKEN BREAST SALT-SOLUBLE PROTEINS BY Shuefung Wang Effect of pH (4.5-7.5), NaCl concentration (0.15—D.6 H) and holding temperature (SS-85°C for 20 min) on gelation of chicken breast salt-soluble proteins (889) were investigated using nondestructive dynamic, rheological testing. Rigidity did not differ significantly in gels held for 20 min at 70, 80 and 85°C. Buffer pH influenced the viscoelastic properties nore than changes in NaCl concentration. No major transition was observed for proteins at pH 4.5, while four transitions for storage modulus and three for loss modulus occurred at pH 5.5, 6.5 and 7.5. The first transition temperature for loss modulus was lower than that for storage modulus at all conditions. Dynamic testing results agreed with results of back extrusion, waterholding capacity and microstructure of SSP protein gels. Protein gels with a filamentous micro- structure exhibited higher gel rigidity and smaller water loss. Aggregated gel structures showed poor gel strength and waterholding. ACKNOWLEDGHENTS The author expresses sincere appreciation to the major professor, Dr. D. M. Smith for her inspiration, counsel and encouragement during this study. Appreciation is also extended to Drs. J. F. Steffe, A. Booren and J. Wilson for their assistance and advice given as nembers of the guidance committee. Special thanks are expressed to Fernando 0sorio and Kevin Hackey for their expertise. Lastly, I would like to thank my parents for their love, encouragement and support throughout this graduate study. iii TABLE OF CONTENTS Page LIST OF TABLES .......................................... vi LIST OF FIGURES ......................................... viii CHAPTER 1. INTRODUCTION ................ ...................... 1 2. REVIEW OF LITERATURE .................... .......... 3 2.1. Myofibrillar Proteins ... .................... 4 2.2. Theory of Protein Gelation . ........ . ........ 5 2.3. Gelation of Muscle Proteins ............ ..... 8 2.4. Methods for Measuring Rheological Changes Caused by Thermal Processing .......... ...... 9 2.4.1. Steady Shear Testing ..... . .......... 10 2.4.2. Oscillatory Testing . ................ 11 2.5. Thermal Stability of Muscle Proteins . ....... 13 2.5.1. DSC Analysis .... ..... ............... 14 2.5.2. Rheological Analysis .......... ...... 19 2.5.3. Comparisons between Rheological and Thermal Transitions of Muscle Proteins ...... ..... . ................ 23 2.6. Factors Affecting Thermal Stability of "usele PIOteina 0.00.00.00.00.000.000.00.0000 25 3 O HATERIus AND HBTHODS O O O O O O O O O O O O O l O O O I 00000 O 00000 28 3.1. Isolation of Chicken Salt-Soluble Proteins .. 28 3.2. Electrophoresis ....... ............ ......... . 29 3.3. Nondestructive Rheological Analysis ......... 31 iv 3.4. Gel Preparation ............................. 32 3.5. Back Extrusion ................ .............. 36 3.6. Expressible Moisture of Gels ................ 36 3.7. Scanning Electron Microscopy ................ 37 ‘3.8. Statistical Design and Analysis ............. 38 4. RESULTS AND DISCUSSION . ..... ...... ................ 39 4.1. Composition of Salt-Soluble Proteins ........ 39 4.2. Rheological Dynamic Analysis................. 39 4.2.1. Strain Sweep and Frequency Sweep .... 39 4.2.2. Effect of Protein Concentration and Heating Rate .................. 42 4.2.3. Effect of Holding Temperature ..... 46 4.2.4. Effect of Salt Concentration ...... . 52 4.2.5. Effect of pH ....................... 62 4.3. Effect of pH on Gel Strength, Waterholding Capacity and Microstructure ................. 71 50 CONCLUSIONS 00000000000000.000000 000000000000000000 81 60 FUTURERESBARCH 00.0000000000000000000000000000...0 83 APPENDICES A. ELECTROPHORESIS STANDARDS .................... ..... 84 B. FIRST DERIVATIVE PLOTS ................. ..... . ..... 86 BIBLIOGRAPHY 0.0000000000000000 000000 00000000000000 0000000 94 Table 10. 11. LIST OF TABLES Conformational changes which may occur during the thermal denaturation of natural actomyosin .... Effect of holding temperature on complex modulus of 3% chicken breast salt-soluble proteins in 0.6 M NaCl, pH 6.5 heated at 1°C/min ...... ........ Effect of NaCl concentration on dynamic moduli of 3‘ chicken breast salt-soluble proteins (pH 6.5) heated at 1°C/min .................. ..... . Effect of NaCI concentration on thermal transi- tion temperatures of 3‘ chicken breast salt- soluble proteins (pH 6.5) heated at 1°C/min ....... Onset temperatures of the complex modulus of 3% chicken breast salt-soluble proteins on the differential plot at different NaCl concentrations ............................. ....... Effect of NaCl concentration on thermal transi- tion slopes of 3‘ chicken breast salt-soluble proteins COO0.000.00I.00OOOOOOOOOOOOOOOOOCOOOOOO000 Effect of NaCl concentration on loss tangent (tan 6) of 3‘ chicken breast salt-soluble proteins (pH 6.5) heated at 1°C/min ............... Effect of pH on dynamic moduli of 3% chicken breast salt-soluble proteins heated at 1°C/min .... Effect of pH on thermal transition temperatures of 3% chicken breast salt-soluble proteins ........ Onset temperature of the complex modulus of 3% chicken breast salt-soluble proteins on the differential plot at different pH's ............... Effect of pH on thermal transition slopes of 3% chicken breast salt-soluble proteins .. ...... . ..... vi Page 17 51 54 SS 58 59 61 64 65 66 Table Page 12. Effect of pH on loss tangent (tan 6) of 3% chicken breast salt-soluble proteins heated at 1°C/min .... 70 13. Molecular weight standards used for electrophoresis 84 vii Figure 1. 10. 11. LIST OF FIGURES Page Schematic representation of the myosin molecule ... 4 Definition of terminology used in analyzing the dynamic moduli of the thermal transitions ......... 33 Definition of terminology used in analyzing loss tangent (tan d) on the thermal transition curve ... 34 Definition of terminology used in analyzing differential plots of complex modulus (6*) vs temperaature (T) .................................. 35 Sodium dodecyl sulfate-polyacrylamide electro- phoresis gel (10%) of chicken breast salt-soluble proteins (a: molecular weight stardands; b,c and d: salt-soluble proteins) ......................... 40 Representative densitometric tracing of chicken breast salt-soluble proteins ...................... 41 Strain sweep of 3% salt~soluble proteins in 0.6 M NaCl, pH 6.5 at 10 rad/sec frequency, 30°C .. 43 Strain sweep of 3% salt-soluble proteins in 0.6 M NaCl, pH 6.5 at 10 rad/sec frequency, 85°C .. 44 Frequency sweep of 3% salt-soluble proteins in 0.6 M NaCl, pH 6.5 at 1% strain, 30°C .......... 45 Representative rheogram of 1, 2 and 3% salt- soluble proteins (SSP) in 0.6 M NaCl, pH 6.5 heatedat 1°C/-1n .0000.00.00000000.00.00.00...000 47 Effect of heating rate on the complex modulus of 3% salt-soluble proteins in 0.6 M NaCl, pH 6.5 .... 48 viii Figure 12. 130 14. 15. 16. 17. 180 19. 20. 210 22. 23. 24. Page Representative rheogram of 3% salt-soluble proteins heated at 1°C/min to 55 and 60°C and he1d£°r20.1n0 00000 00000000000000000 0000000000 00 49 Representative rheogram of 3% salt-soluble proteins heated at 1°C/min to 70, 80 and 85°C, and held for 20 .1“ 0000000000000000000000000000000 50 Effect of salt concentration on the rheogram of 3% salt-soluble proteins at pH 6. 5 heated at 1°C/‘1n0 000000 0000000000000000000 0000000 0 0000000 53 Effect of pH on the rheogram of 3% salt-soluble proteins in 0.6 M NaCl heated at l°C/min ... ....... 63 Effect of pH on viscosity index of 3% salt- soluble proteins heated to indicated endpoint temperatures ......... ........... . ...... .. ......... 72 Effect of pH on waterholding capacity of 3% salt- soluble proteins heated to indicated end- point temperatures .. ...... ... ....... . ............ 75 Microstructure of chicken breast salt-soluble protein in 0.6 M NaCl heated to 55°C at (a) pH 4.5; (b) pH 5.5; (c) pH 6.5; (d) pH 7.5 .... 78 Microstructure of chicken breast salt-soluble protein in 0.6 M NaCl heated to 65°C at (a) pH 4.5; (b) pH 5.5; (c) pH 6.5; (d) pH 7.5 .... 79 Microstructure of chicken breast salt-soluble protein in 0.6 M NaCl heated to 80°C at (a) pH 4.5; (b) pH 5.5; (c) pH 6.5; (d) pH 7.5 .... 80 Molecular weight standard curve for sodium dodecyl sulfate-polyacrylamide gel electrophoresis ........ 85 Differential plot of complex modulus (6*) of 3% salt-soluble proteins vs temperature (T) at p" 705’ 006HNac1 000000000000000000000000000 00000 87 Differential plot of complex modulus (6*) of 3% salt-soluble proteins vs temperature (T) at p" 605' 006 "flaCI 0000 00000 0000000000 0000000000000 88 Differential plot of complex modulus (6*) of 3% salt-soluble proteins vs temperature (T) at p“ 505’ 006 H ”aCI 0000000 0000000000 0 00000000000000 89 ix Figure 25. 26. 27. 28. Page Differential plot of complex modulus (6*) of 3% salt-soluble proteins vs temperature (T) at D" 405’ 006HNaC1 000000000000000000000000000 00000 90 Differential plot of complex modulus (6*) of 3% salt-soluble proteins vs temperature (T) at pH605’00‘SNNac1000000000000000000000000‘0000000 91 Differential plot of complex modulus (6*) of 3% salt-soluble proteins vs temperature (T) at p" 605’ 0030MNac1 0000000000000000000000000000000 92 Differential plot of complex modulus (6*) of 3% salt-soluble proteins vs temperature (T) at pH605,0015MNac1 0000000000000000000000000000000 93 INTRODUCTION Muscle proteins play an important role in processed meat products. Gelation of muscle proteins, a transformation from viscous sols to elastic gels, contributes to the development of desirable texture, and physical and chemical stabilization of fat and water. Any factors changing the physicochemical properties of proteins in food systems will influence the final appearance of these products. For example, using different meat sources or cuts will produce different sensory attributes. Inappropriate thermal processing and storage conditions will cause shrinkage, toughening or release of juice which decrease the palatability. Low salt and low fat meat products have been introduced and developed to meet consumer demands. A fundamental understanding of the physicochemical properties of muscle proteins is required to manipulate protein functionalities, optimize processing conditions, reduce energy costs and improve the final quality of processed meat products. The physical and biochemical changes of muscle proteins during thermal processing are currently receiving attention by researchers. Salt-soluble proteins (SSP) are the principal functional components in meat products. Protein gelation requires certain degrees of SSP denaturation and aggregation 2 to form a three-dimensional network. The gel structure and other macroscopic properties will be influenced by the nature of protein-protein interactons. In terms of the rheological properties, most of the thermal gelation studies were investigated by large deformation testing using discrete samples heated for certain time and temperatures. This type of testing is not sensitive in detecting protein transitions. Montejano et a1. (1984) stated the necessity for employing a non-destructive, dynamic testing capable of continuously monitoring the changes in rheological characteristics during thermal gelation. The objectives of this study were: (1) to determine the influence of pH and NaCl concentration on the thermal transitions of chicken breast SSP using non-destructive dynamic rheological testing; (2) to monitor the effect of holding temperature on the gel properties of chicken breast SSP; (3) to evaluate the effect of pH on gel strength by back extrusion, waterholding capacity of gels, and micro- structure. REVIE' 0' LITERATURE Protein-to-protein interaction during thermal processing is necessary for the binding ability of formed and sectioned meat, and the stabilization of fat and water in comminuted meat products. A sol-to-gel transformation occurs due to the increase in temperature during thermal processing which leads to the formation of a three-dimensional protein network. Heat stability of muscle proteins influences the gelling mechanism and gel structure in a meat system, and thus the development of desired functional properties. It is well known that salt- soluble proteins or myofibrillar proteins contribute the most functionality to processed meat products. Myosin is the func- tional protein predominant in pre-rigor meat, while actomyosin is predominant in post-rigor meat (Ziegler and Acton, 1984b). In order to predict functionality, Smith (1988) stated the necessity to "elucidate the molecular properties of myofibril- lar proteins and how they are influenced by environmental and processing factors“. The gelation properties of the final meat product are related to biochemical and microstructural changes in the proteins, while rheological characteristics can provide information on changes in physical properties during heating. Montejano et al.(l984) also emphasized the 4 importance of understanding the rheological changes during heating to determine the textural properties of finished products. 2.1 Myofibrillar Proteins The major components of myofibrillar proteins are con- tractile proteins (myosin, actin) and regulatory proteins (troponin, tropomyosin). Myosin is a fibrous protein with a globular conformation at one end referred to as the head region. The myosin molecule contains six polypeptide chains: two heavy chains with identical sequences, and two pairs of light chains: alkali and DTHB light chains (Smith et al., 1983). Hydrolysis of myosin heavy chain with trypsin yields light meromyosin (LMM) and heavy meromyosin (HMM). In contrast, hydrolysis with papain yields two single globular heads (HRH S-1) and one myosin rod (LMM + HHM S-2) as shown in Figure l. l"--—'lflun01JMW i4‘--3auwornmw Alkali light cha' 2 nm D‘I'NB light chain 115,000 1 s2 \ HMM-Sl T..— . / "W / 17531:: Trypsin Papain }‘--40nm|-:1 Myosin Rod iaumo lflhmn I Figure 1. Schematic representation of the myosin molecule (adapted from Smith et al., 1983). 5 Actin constitutes about 20 % of skeletal myofibrillar protein. It is a globular shaped molecule referred to as monomeric G-actin. Linking of G-actin monomers forms F-actin (fibrous actin). Two strands of F-actin are spirally coiled around one another to form a "super helix" actin filament. Tropomyosin and troponin each constitute approximately 5 % of the myofibrillar proteins and are present in the grooves of the actin filament (Judge et al., 1989). 2.2 Theory of Protein Gelation The mechanism of gel formation by proteins has been intensively investigated. Perry (1948) described heat-induced gelation of proteins as a two-step process involving partial unfolding of protein followed by aggregation into a three- dimensional, well-ordered network: xen -> xpd -> (95),, where x is the number of protein molecules P, with n denoting the native state and d the denatured state. Barbu and Joly (1953) reported two types of aggregates depending on the net charge of the native protein: linear aggregates occurred when repulsion was large, and globular random aggregates occurred when repulsion was small as, for example, when the isoelectric pH was approached. Hermansson (1978), elaborating on Perry's mechanism, stated that the slower the protein aggregation step relative to the unfolding, the better the denatured chains orient themselves and the finer the gel network. When random 6 aggregation is suppressed prior to denaturation, the resulting network exhibits a higher degree of elasticity than if aggregation and denaturation occur simultaneously, or if aggregation precedes denaturation. Ziegler and Acton (1984a) found that natural actomyosin aggregation in dilute solution followed a first order reaction, but the existence of two thermal transitions supported a two-step reaction mechanism in solutions of higher protein concentration. Stafford (1985) described the partial denaturation or partial unfolding of myosin, a multidomain protein: c132 k1 I§§§§§§t k2 1?‘a6:%> 162 H1C2 \\ H132 / where H; and C1 refer to the helical and coiled forms of domain 1. Hermansson et a1. (1986) also stated that the associated state of the native molecules required for certain gel structures are already present before heating. This property was thought to be important to the thermal processing of muscle foods that different denatured molecules could associate to produce textural effects (Foegeding, 1988). Formation of covalent bonds (principally the disulfide cross-links) as well as hydrophobic, hydrogen and probably electrostatic interaction were thought to be involved in protein denaturation and aggregation in cooked products (Voutsinas et al., 1974). It is interesting to characterize a gelling system in terms of the cross-linking mechanisms. 7 However, Clark and Lee-Tuffnell (1986) stated the difficulties in distinguishing the types of bonds involved in crosslinking because of their simultaneous action. Physicochemical properties of proteins, including surface charges, sulfhydryl group content, hydrophobicity, conforma- tional stability, and association/dissociation behavior have been used for studying protein gelation mechanisms (Kinsella, 1982; Wilding et al., 1984; Smith, 1988). Solubility and turbidity are used for studying protein denaturation (Lanier et al., 1982; Liu et al., 1982; Samejima et al., 1981, 1984; Ziegler and Acton, 1984a). Oxidation of sulfhydryl groups is thought to be involved in protein aggregation (Jacobson and Henderson, 1973; Ishioroshi et al., 1979; Lanier et al., 1982; Liu et al., 1982; Voutsinas et al., 1983). Fluorescence studies of changes in hydrophobicity (Nakai, 1983; Voutsinas et al., 1983; Li—Chan et al., 1984; wicker et al., 1986), helix-to-coil transitions measured by optical rotary dispersion and circular dichroism (Samejima et al., 1981, 1984) are used to follow unfolding and conformational changes in protein structure. Electron microscopy is a valuable tool for studying the microstructure of protein gels in meat or model systems (Ishioroshi et al., 1979; Yasui et al., 1979, 1980, 1982; Samejima, et al., 1981, 1982; Montejano et al., 1984; Schmidt, 1984; Hermansson et al., 1986; Hermansson and Langton, 1988). 2.3 Gelation of Muscle Proteins Several fundamental studies on the gelling properties of different myofibrillar proteins and isolated myosin subfrag- ments have been described. Early investigations with model systems indicated that myosin is required to develop high gel strength (Samejima et al., 1969). Regulatory proteins, tropo- myoin and troponin, contribute little to gel formation (Haga and Ohashi, 1982; Samejima et al., 1982; Yasui et al., 1982). F-actin did not exhibit any gelation ability (Yasui et al., 1979, 1980) and revealed thixotropic behavior (Brotschi et al., 1978). However, F-actin and myosin exert a "synergistic gelling effect”. Maximum gel strength in 0.6 M KCl, pH 6.0‘ was obtained at a free myosin to F-actin molar ratio of 2.7:1, which corresponds to a weight ratio of 15:1. At this ratio, 15—20% of the total protein existed as an actomyosin complex and the remainder was free myosin. It was suggested that small amounts of F-actomyosin (about 1: 4 ratio of free myosin) acted as a crosslinker with free myosin on heating and might be a prerequisite for actin-induced improvement in the gelation of myosin (Ishioroshi et al., 1980; Yasui et al., 1982; Asghar et al., 1985). Dudziak et a1. (1988) found that myosin to actomyosin weight ratios for postrigor turkey breast and thigh were 3.8:1 and 6.9:1, respectively. Turkey breast gels were more stable and exhibited a greater rigidity than thigh gels. The results coincide with myosin-to-actomyosin ratio data reported by Yasui et al. (1982). 9 Efforts have been made to understand the role of myosin subfragments in gelation. The gelling potential of myosin is confined to the myosin heavy chain. Light chains contribute little to gelation of myosin in model systems, but possibly provide some stability to the gel if pH is increased above 6.0 (Samejima et al., 1984). Samejima et al. (1981) reported that intact myosin and myosin rod formed firm gels, while S-l showed poor gelling ability upon heating. Mixing rod with S-l fragments failed to restore gel strength to that of the intact myosin. Intact myosin and myosin rods formed the maximum gel strength at pH 6.0 and ionic strength 0.1. In contrast, gelation of S-l was independent of pH and ionic strength. According to a succeeding investigation, Yasui et a1. (1982) found that even though S-1 and HMM possess actin-binding sites and can crosslink with F-actin to form F-actomyosin, they are devoid of the necessary segment of the tail portion for the production of framework. Only rod and LMM showed the potential to form a network. So, it was concluded that cross- linking between free and bound myosin molecules is initiated only through interactions between their tail portions. 2.4 Methods for Measuring Rheological Changes Caused By Thermal Processing Aggregation and structure formation by proteins during thermal processing are responsible for the development of texture and other functionalities. Research interests are 10 thus directed toward elucidating gelling mechanisms of muscle proteins during heating. Mechanical properties of proteins have been evaluated by rheological techniques which consist of static and oscillatory testing. 21311.31eadx_fihear_1sstlns A wide variety of steady shear rheological techniques have been used for rheological evaluation of meat products and protein gels. Flow behavior of meat homogenates was assessed using tube viscometry (Toledo et al., 1977), rotary viscometry (Bianchi et al., 1985; Barbut and Mittal, 1988) and extrusion testing (Kim et al., 1986). The textural properties of cooked meat products have also been measured by shear testing (Bouton et al., 1975; Booren et al., 1981; Kijowski et al., 1982), compression testing (Voisey et al., 1975; Huang and Robertson, 1977; Bourne, 1978; Lanier et al., 1982; Singh et al., 1985), creep analysis (Siripurapu et al., 1987 ) and torsion failure testing (Montejano et al., 1983, 1984, 1985; Kim et al., 1986; Saliba et al., 1987). A Brookfield rotary viscometer was used by Liu et a1. (1982) to analyze the changes in apparent viscosity of fish actomyosin during heating. Borderias et a1. (1985) reported a linear relationship between apparent viscosity and protein concentration of fish and chicken muscle extracts. Hu et a1. (1985) used Brookfield viscometer to study the gelation of fish actomyosin and compared the results with rigidity scanning techniques. 11 Another type of instrument widely used for measuring gel- ling properties of proteins is the back extrusion rheometer. The force required to penetrate the gel is measured from the force-deformation curve. Acton et a1. (1981) used this tech- nique to evaluate the gel strength of natural actomyosin and reported a least protein concentration end point (LCE). Hickson et a1. (1982) described the calculation of viscosity index and apparent elasticity using a back extrusion technique developed by Morgan et al. (1979). Foegeding et al. (1986) investigated the effect of heating rate on myosin gelation and Smith et a1. (1988) developed a generalized mathematical model for predicting the gel strength of myofibrillar protein gels using the back extrusion. The limitation of these methods is the destruction of gel structure as a result of measurement, and thus discrete samples heated for certain temperatures and times are required to investigate the progress of gelation. It is impossible to continuously monitor the structural changes during heating using steady shear techniques. However, large deformation measurements have greater correlation with sensory parameters in textural evaluation (Brennan et al., 1974; Moskowitz and Kapsalis, 1974; Voisey, 1975). ' WW For a better understanding of the structural changes during protein gelation, a nondestructive (small strain), oscillatory test is required to intermittently or continuously 12 monitor the changes in viscoelastic behavior during thermal processing. Small-strain rigidity has been investigated in several studies on polymer behavior to characterize glass transition and melting temperatures (Otsubo et al., 1984, 1986, 1987; Winter and Chambon, 1986). Several articles have reported the use of oscillatory testing for textural control of meat products (Webb et al., 1975; Hamann and Webb, 1979; Bistany and Kokini, 1983b). However, gel rigidity was not correlated well with sensory texture or rupture strength (Schweid and Toledo, 1981; Montejano et al., 1985). In dynamic testing, the gelation of muscle proteins during heating can be continuously monitored using the same sample. The real and imaginary components of the complex shear modulus (6*) are expressed as the storage modulus (6') and the loss modulus (6") which represent the elastic and viscous elements, respectively. The loss tangent (tan d) is calculated as a measure of the energy lost compared with the energy recovered in cyclic deformation. 6* - (6' a + G" .) 1/2 tan 8 a 6”/6' Hakayama and Haugen (1977) studied dynamic and steady shear rate behavior of chicken meat for low temperature "setting" phenomenon by using a leissenberg rheogoniometer. Montejano et a1. (1983, 1984) developed the Thermal Scan- ning Rigidity Monitor (TSRM) attached to an Instron Universal Testing Device which continuously evaluated the modulus of 13 rigidity and energy damping. The shear modulus or the modulus of rigidity is calculated as the ratio of maximum shear stress to maximum shear strain. Energy damping or hysteresis less, an expression of elasticity in a material, is determined as the ratio of the hysteresis area to the work of deformation. Patana-Anake and Foegeding (1985) examined rheological and stability transitions during heating of meat batters using TSRM. Wu et al. (1985) developed two other TSRM devices for studying rigidity or viscosity changes of liquid to semisolid materials. Samejima et a1. (1985) used a Bohlin Rheometer System to evaluate gel strength of beef myofibrils in terms of dynamic shear stress response to small amplitude oscillations. The results observed were compared with their previous work performed with a shear modulus tester (Yasui et al., 1979, 1980; Samejima et al., 1983). Beveridgc and Timbers (1985) also developed an instrument using small amplitude oscillatory testing (SAOT) to characterize the structure transformation of egg albumen, whey protein concentrate and beef emulsion during cooking. The results are given as torque amplitude and rota- tion amplitude as functions of time for a given temperature history. 2.5 Thermal Stability of Muscle Proteins Since muscle proteins exhibit different properties during heating, the thermal stability of proteins has been examined 14 to provide useful basic knowledge of protein characteristics. Thermal input energy to muscle protein systems leads to con- formational and structural changes. Rheological techniques and differential scanning calorimetry (DSC) are two useful techniques and have been used extensively to study the thermal properties of food and food components. DSC can monitor the thermodynamic properties of proteins in their native state by measuring enthalpy of thermal transitions and denaturation temperatures. Physical changes of proteins can be evaluated by rheological methods and the transitions are often specified as the temperature at which maxima occur (Tn). Walnuts Isolated myosin and its subfragments from rabbit were examined by DSC. Thermal transitions of myosin were pH- dependent. Single or multiple transitions ascribed to myosin were due to differences between pH, salt concentration or ionic strength (Stabursvik and Martens, 1980; Wright and A Wilding, 1984; Akahane et al., 1985). The thermal transitions appeared to be associated with discrete regions of the myosin molecule. Potekhin et al. (1979) used DSC to study the step- wise denaturation pattern of rabbit myosin at 0.5 M KCl, pH 6.5, and proposed a resolution of the denaturing curves into individual stages. Wright and Wilding (1984) assigned the middle transition (50.1°C) of myosin at pH 6.0, high ionic strength (I-l.0 or 0.962 M KCl) to HMM S-l, while the other two transitions (43.6 and 59.200) were associated with the 15 denaturation of myosin rod or LMM. One transition at 53°C was assigned to HMM S-2 domain by comparing the thermograms of HMM and HMM S-l. At low ionic strength (180.05 or 0.012 M KCl), HMM S-l exhibited the lowest transition temperature (52°C), and the helical region displayed an increase in thermal stability (61 and 65°C) which might result from an interaction with HMM S-2 of a neighboring molecule. Cross et a1. (1984) reported two major transitions at 43°C and 52°C for myosin rod in 0.6 M KCl, pH 7.0 buffer. The lower temperature transition was attributed to LMM and the second to HMH S-2. At physio- logical ionic strength (0.12 H KCl), LHM was stabilized 10°C so that LMM and S-2 had essentially identical conformational stability. Akahane et al. (1985) found only a single peak in rabbit myosin which reflected the denaturation of LMM. The hinge region of myosin rod within the LMM/S-2 junction was postulated to be the primary flexible site of force generation and shortening in a contracting muscle (Burke et al., 1973; Harrington, 1979; Rodgers and Harrington, 1987). Potekhin et a1. (1979) assigned the low temperature transition for LMM and rod fragments to the central region of the LMM fragment. The lower thermal stability of myosin hinge was supported by Swanson and Ritchie (1980). They found a transition at 41°C for long HMM S-2 (containing the "hinge" region) which was absent for short S-2 (lacking the "hinge" region) and therefore assigned this transition to the myosin hinge. Wright and Wilding (1984) also suggested that the low temperature transition was probably a composite of two 16 transition, one associated with the "hinge" region and the other with a melting of helical structure in LMM. Table 1 gives a summary of the events (adapted from Ziegler and Acton, 1984) which may occur during the heat denaturation of actomyosin. DSC studies on muscle proteins from other species have also been reported. Three endothermic transitions were gene- rally observed in native muscles from rabbit and beef which were ascribed to the denaturation of myosin (T, of 57-60°C), sarcoplasmic and connective tissue proteins (66-6700) and actin (78-80°C) (Martens and Vold, 1976; Wright et al., 1977; Stabursvik and Martens, 1980; Akahane et al., 1985; Xiong et . al., 1987). A single peak near 58°C was attributed to regula- tory proteins by comparing DSC thermograms of pure actin and crude actin (Akahane et al., 1985). Myosins derived from fish muscle were less stable than those from mammalian tissues, while actin transitions were less species-dependent (Akahane et al., 1985; Poulter et al., 1985; Rodgers et al., 1987). Thermostability of fish proteins was found to be related to fish habitat temperature. The more stable proteins were those from the species found in higher ambient temperature (Akahane et al., 1985; Hastings et al., 1985; Poulter et al., 1985; Rodgers et al., 1987; Davies et al., 1988). DSC studies on chicken myosin have been published. How- ever, there is little information available regarding the thermal denaturation of specific proteins in chicken muscles from either thigh or breast. Greater differences in thermal 17 Table 1. Conformational changes which may occur during the thermal denaturation of natural actomyosin (from Ziegler and Acton, 1984) Temperature Protein(s) or segment Description (06) involved of events 30-35 Native tropomyosin Thermally dissociated from the F-actin backbone 38 F-actin "Super" helix dissociates into single chains 40-45 Myosin Dissociates into light and heavy chains "Head” Possibly some comformational 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 comformationl changes in the G-actin 18 properties of proteins were found between red and white muscles of chicken than between muscles from different animal species (Stabursvik and Martens, 1980; Xiong et al., 1987). Fretheim et a1. (1986) observed different heat gelling proper- ties between chicken myosin and beef myosin. Xiong et al. (1987) also found chicken actin to be more readily denatured than mammalian actin. According to their DSC studies, native chicken thigh muscle, with a postrigor pH of 6.5, exhibited three endothermic transitions near 60, 66, and 78°C. Isolated chicken thigh myofibrils (0.1 M NaCl, pH 7.1-7.2) showed only two transitions at 60 and 70°C. The first peak of native thigh muscle was ascribed to myosin, the second to sarco- plasmic and connective tissue proteins, and the third to actin denaturation. Native chicken breast muscle, with a postrigor pH of 5.6, showed a complex thermogram with five endothermic transitions at 57, 62, 67, 72, and 79°C. The sarcoplasmic proteins exhibited three peaks at 62, 67 and 72°C, whereas connective tissue transitions appeared as a single peak at 63°C. Isolated chicken breast myofibrillar protein (0.1 M NaCl, pH 7.1-7.2) showed three transitions at 53, 61, and 69°C. The first (57°C) and last (79°C) peaks of native breast muscle were ascribed to myosin and actin, res-pectively, and the second peak probably resulted from the combined effects of sarcoplasmic proteins, connective tissue proteins and myosin denaturation. A similar investigation on the heat stability of chicken broiler salt-soluble proteins was reported by kijowski and Mast (1988). 19 Differences in thermal stability of red (thigh) and white (breast) muscles could result from differences in native pH and sarcoplasmic protein composition (Xiong et al., 1987). White muscle had more proteins of molecular weights of 300, 81, 60, and 15K. The high molecular weight proteins are the structural proteins, i.e. connectin, nebulin and filamin, which are believed to be important to the integrity of myo- fibrils. The protein with a molecular weight of 15K is myosin light chain 3 (Porzio and Pearson, 1977). Rapid heating rates are required for good resolution in DSC analysis (Montejano et al., 1983). Dudziak et al. (1988) also stated that the DSC transition is heating rate dependent. The use of rheological techniques to monitor structure formation of protein gels during heating was suggested as a complement to DSC analysis. WW Yasui et a1. (1979) investigated the gelation of rabbit myosin by measuring changes in shear modulus with a band-type Oviscometer. They concluded that the thermal transition of myosin from sol to gel started at 30°C with a stepwise elevation of temperature, reaching a maximum at about 60-70°C. Ishioroshi et al.(1979) also observed two transition peaks in derivative plots as a function of temperature (43°C for T.1 and 53°C for le). These values corresponded to the helix- coil transition of myosin rod as measured by optical rotatory dispersion, and implied that the unfolding of the helical tail 20 portion of myosin may be involved in heat-induced gelation (Samejima et al., 1981). Previous denaturation studies on myosin (Kawaami et al., 1971) indicated that aggregation was due to cross-linking of the head portion of the molecules which occurred at temperatures as low as 35°C. Samejima et al. (1981) studied the thermal stabilities of myosin rod and S-l. A single transition was observed at 43°C for S-1 and 53°C for myosin rod. The results coincided with the myosin transitions observed by Ishioroshi et al. (1979). Therefore, they proposed the lower transition peak involving aggregation of myosin heads in addition to unfolding of the myosin rod. Additionally, in sol state, myosin rod has 100 % helical conformation. Unfolding causes the protein to assume a random coil structure which was believed to be responsable for the second transition of the myosin molecule. Samejima et al. (1984) observed an almost equal gel strength between myosin heavy chain (HHC) and intact myosin. MHC formed a weak gel at 35°C and gel strength increased with temperature up to 60°C. Higher temperatures tended to weaken the gel structure. Differential plots of rigidity versus temperature showed two transitions at 42 and 55°C for MHC which were similar to the thermogelling reaction of myosin reported by Yasui et al. (1979). Efforts were also directed toward the viscoelastic properties of actomyosin and actin. The rigidity developed upon heating actomyosin at 1.5 or 2.7 myosin-to-actin mole ratio exhibited essentially the same rigidity: temperature 21 profile as myosin alone (Yasui et at., 1980). Sano et al. (1988) compared the dynamic viscoelastic behavior of natural actomyosin and myosin from fish muscle. Before heating, actomyosin was found to have higher initial storage and loss moduli than myosin, which was ascribed to the molecular structure of actomyosin. They stated that the actomyosin formed has myosin tails protruding; therefore, the actomyosin molecules have a tendency to interact with each other to form a sol with higher storage and loss moduli than myosin. However, myosin gels showed higher rigidity than actomyosin and exhibited more elastic properties when heated to 80°C. The changes in dynamic viscoelasticity of F-actin were also investigated by Sano et al. (1989). No development of storage or loss modulus was observed during heating which agreed with the results of Yasui et al. (1979, 1980). Influence of muscle type and species on the rheological properties of heat-induced gelation have been studied. White myosin from chicken (Asghar et al., 1984; Morita et al.,l987) and bovine muscle (Fretheim et al., 1986) generally exhibited superior gel formability at pH 6.0 than red myosin. Asghar et a1. (1984) reported the maximum rigidity of red and white chicken myosin occurred at pH 5.9 and pH 5.6, respectively. According to Morita et al. (1987), the maximum gel strength for red myosin was near pH 5.1 and that for white myosin was close to pH 5.4. Montejano et al. (1983) found four rigidity transitions of fish gels at 13, 37, 47 and 64°C when measured by TSRM. A plateau from 60 to 64°C followed by a continuous 22 increase in rigidity at higher temperature was observed and ascribed to the onset of proteolytic degradation or another conformational transition occurring in the gelling protein matrix. Wu et a1. (1985) found shear-thinning, thixotropic behavior in fish actomyosin heated at 35 and 40°C. Three transition peaks were observed at 38, 46, and 60°C at higher protein concentrations, while only one peak occurred near 36°C at lower protein concentrations. However, a decrease in rigidity was observed at temperatures above 60°C for acto- myosin instead of a plateau in the 60-64°C temperature range for surimi found by Montejano et al. (1983). The magnitude of this decrease and its onset temperature were found to be concentration dependent. The greater the protein concen- tration, the lower was the onset temperature and the greater was the magnitude of the decrease. Wu et al. (1985) reported that the decrease in rigidity above 60°C was probably due to the destruction of gel structure, a phenomenon called "modori' by the Japanese (Lanier et al., 1981; Shimizu et al., 1981; Wu et al., 1985). Montejano et a1. (1984) compared the changes in shear rigidity and mechanical energy damping of surimi, turkey, beef and pork sols during heating. Surimi exhibited major rigidity transitions at 40, 48 and 65°C. A rapid linear increase was observed in the range of 48-65°C which indicated the formation of a stiff protein matrix. Haximum rigidity was observed at 65°C, followed by a moderate decrease. Beef and pork muscles presented very similar rigidity-temperature profiles. Hajor 23 transitions were found at 43, 56 and 69°C for beef; 44, 53 and 69°C for pork. Beef had higher initial rigidity than pork but lower rigidity above 69°C. Turkey showed three major transi- tions at 50, 53 and 79°, and a linear increase in rigidity between 53 and 79°C. A high final strength of turkey sols was observed close to that of surimi and almost twice as large as those for beef and pork. An increase in energy loss indicated an increase in the viscous character of the system and was attributed to fat melting (Montejano et al., 1984). Previous articles investigating fat melting profiles in lean beef and pork trimmings (Acton et al., 1983; Quinn et al., 1980; Town- send et al., 1968) agreed with these findings. To understand the molecular changes during gelation, several researchers have compared the results obtained from rheological testing with those of DSC and/or fluorescence measurements. Wicker et al. (1986) compared the thermal transitions in myosin-ANS (l-anilino-naphthalene-8-sulfonate) fluorescence and gel rigidity measured by TSRH. Thermal transitions detected by myosin-ANS fluorescence were found prior to an increase in gel rigidity. It was concluded that an increase in effective hydrophobicity was a prerequisite for gelation. Dudziak et al. (1988) examined the thermal transitions of myosin/actomyosin suspensions (pH 7.0, 0.5 H NaCl) from post-rigor turkey breast and thigh using DSC, 24 fluorescent probe measurements and uniaxial compression. No significant variation in ANS fluorescence nor DSC transitions were detected, which indicated little variation in thermal transition between breast and thigh proteins. Large rheological differences were reported between these two different protein sources. Myosin and actomyosin gels from breast tissue were more stable at lower protein concentrations and required a greater stress to failure than thigh gels. It was reported that the variation in gelation was not associated with major differences in thermal transitions but the types of native protein-protein interactions and differences in association of denatured molecules. Morita et al. (1987) also observed that chicken breast myosin had higher gel strength and longer filamentous assemblies than leg myosin between pH 5.2 to 6.0 in 0.6 H KCl. These findings emphasized the importance of native protein association which might be varied with different myosin isoforms or peptide mapping patterns (Libera et al., 1980), myosin-to-actomyosin ratios (Dudziak et al., 1988), and/or the percentage of other minor proteins in myofibrils (Acton and Dick, 1986; Xiong et al., 1987). Hontejano et al. (1984) investigated beef paste using DSC and rigidity scanning evaluations. DSC studies showed three peaks at 43, 58 and 71°C and were in very close agreement with the rigidity transitions observed at 43, 56 and 69°C. Wu et al. (1985) and Sano et al. (1988) also reported good correla- tions between DSC results and rheological changes. 25 2.6 Factors affecting Thermal Stability of Huscle Proteins Several researchers have attempted to derive a relation- ship between extrinsic factors and the resulting gelling properties of muscle proteins. Thermostability and gel strength are influenced by heating rate, pH value, salt and protein concentration. Ishioroshi et al. (1979) reported that myosin gels held at 65°C showed the highest maximum shear modulus at pH 6.0, while those formed at lower pH exhibited more syneresis. Samejima et al. (1984) found a gradual weakening of myosin gels with increase pH above 6.0. Wicker et al. (1986) investigated the effect of pH on viscoelastic properties of myosin. Myosin exhibited higher initial rigidity at pH 5.0 due to increased protein aggregation. Final rigidity reached a maximum at pH 6.0, which agreed with the results of Ishio- roshi et al. (1979). Ishioroshi et al. (1979) evaluated the gel strength of myosin incubated at 65°C by varying the salt concentration from 0.1 to 0.6 M KCl, keeping pH constant at 6.0. Gels exhibited the highest shear modulus in 0.1 to 0.2 N HCl. Samejima et al. (1985) studied the effect of ionic strength on gelling properties of beef myofibrils. They found that gel strength decreased and the final protein concentration in the liquid phase of the gel (i.e. the amount of protein not included in the gel structure) increased with decreasing salt concentration. Wicker et al. (1986) examined the effect of salt on gelation of fish myosin and found that the gel formed 26 at 0.3 H KCl had the lowest rigidity. When salt was greater than 0.3 M, gel rigidity increased with salt concentration. Myosin gel in 0.1H KCl developed greater rigidity than in 0.6 and 0.8 H KCl during heating, but weaker than in 0.3 H KCl. Under the scanning electron microscope, a relatively finer gel network was observed in 0.2 M KCl than in 0.6 H KCl ,(Ishioroshi et al., 1979). It has been suggested that myosin molecules were soluble and existed as monomers at high ionic strength, while the myosin molecules assembled into filaments at low ionic strength (Huxley, 1963; Kaminer and Bell, 1966). Soluble myosins tend to produce head-to-head aggregates. Hyosin filaments form a finer network and produce greater rigidity than monomeric myosin. Thermostability of muscle proteins is affected by pH and salt concentration. Actin transitions monitored by DSC showed little pH dependence (Stabursvik and Hartens, 1980; Davies et al., 1988). Davies et a1. (1988) observed that the first myosin transition decreased with increased pH at low ionic strength (180.06 H). However, Wagner and Anon (1985) reported that the transition of actin decreased 1°C and that of myosin increased 3°C with increasing pH from 5.4 to 6.2. Salt decreased the heat stability of muscle proteins by shifting their transitions to lower temperatures (Goodno and Swanson, 1975; Quinn et al., 1980; Wright and Wilding, 1984). Goodno and Swanson (1975) explained the effect of pH and nonvalent cations (Na*, K’) on protein denaturation which alter the charges on the myosin molecule and disturb the 27 balance between electrostatic forces. Once the balance of forces is disturbed, less thermal energy is required for protein denaturation and the transition temperature becomes lower. Ishioroshi et al. (1979) reported that the shear modulus of the myosin gel increased proportionally to the square of myosin concentration. Wright and Wilding (1984) found no significant effect of protein concentration on either DSC transition temperature or denaturation enthalpy. An increase in heating rate increased the DSC transition temperatures, as well as the denaturation enthalpy of rabbit skeletal myosin (Wright and Wilding, 1984). Hontejano et al. (1983) observed the rigidity of fish muscle gel measured by TSRH was heating rate dependent. Saliba et al. (1987) found the final gel strength of frankfurter batter measured by failure test increased linearly as heating rate decreased. However, slower heating rate increased the modulus of rigidity measured by TSRH only in the 58-65°C temperature range. Above 65°C, heating rate differences equilibrated. It was suggested that slower heating rates allowed more time for proteins to unfold and interactions to occur thus enabling a stronger gel matrix to form (Saliba et al., 1987). MATERIALS AND METHODS 3.1. Isolation of Chicken Salt-Soluble Proteins The isolation of salt-soluble proteins (SSP) followed the procedure described by Smith (1987) with modifications. Fresh chicken breast was obtained from a local retail store. Skin, visible connective tissue and bone were removed from the chicken muscle. Two low salt extractions (0.1 H NaCl, 0.05 H Na-phosphate buffer, pH 6.5) were followed by one high salt extraction (0.6 M NaCl, 0.05 H Ha-phosphate buffer, pH 6.5). Chicken breast was ground twice with a KitchenAid food grinder (Hodel FG-A, Troy, Ohio), blended with four volumes of low salt buffer for 90 sec in a Waring Blendor (Model 1120, Winsted, Conn), then stirred with a motorized propeller without foaming for 1 hr. The suspension was centrifuged at 5,860 x g for 10 min at 4°C to discard the supernatant containing fat and sarcoplasmic proteins. The pellet, resuspended in the original volume of fresh low salt buffer, was stirred for 1 hr followed by centrifugation and removal of the supernatant. The pellet obtained from the low-salt extraction was dissolved in one-third volume of 2.4 M NaCl, 0.05 H Na-phosphate buffer, pH 6.5 to a final concentration of 0.6 M NaCl, then suspended in three volumes of 0.6 M NaCl, 28 29 0.05 H Na-phosphate buffer for the high-salt extraction. The suspension was stirred for 1 hr and centrifuged at 20,000 x g for 20 min. The high salt extraction was performed to remove any low salt-soluble connective tissue and denatured myo- fibrillar proteins contained in the pellet. The supernatant was diluted in five volumes of water to precipitate SSP, then centrifuged at 20,000 x g for 30 min. The final pellet was solublized in one-third volume of 0.2 M phosphate buffer of the desired pH and NaCl concentration. The pH of the SSP solution was adjusted with 0.1 M HCl or 0.1 N NaOH if necessary. Protein concentration was determined by micro- kjeldahl (AOAC, 1980). Salt-soluble protein preparations were diluted to 3% (w/v) with desired buffer. The treatments selected were four different pH's (4.5, 5.5, 6.5 and 7.5) at 0.6 M NaCl and four salt concentrations (0.15, 0.3, 0.45 and 0.6 M) at pH 6.5. 3.2. Electrophoresis Samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared by diluting 3% SSP solutions below 1% with water. One-half milliliter of 10% SDS solution and 5 drops of a-mercaptoethanol were added to 4.5 ml of the diluted protein solution which was then heated in boiling water for 5 min. Protein solutions were dialyzed 18 hr against 25 mH Tris buffer, pH 7.25, containing 0.2% SDS and 30 0.2 mM EDTA with one change of dialysis buffer. Glycerol (20% by weight) and bromophenol blue tracking dye were added. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed with a SE 600 series vertical slab unit (Hoefer Scientific Instruments, San Francisco, CA) using a tris- glycine buffer system, pH 8.3, as described by Smith and Brekke (1985). Ten percent acrylamide in tris-SDS solution, pH 8.8, for resolving gel and 4% acrylamide in tris-SDS solution, pH 6.8, for stacking gel were employed. Fifty micrograms of protein solution and 15 ug of molecular weight standards (SDS-7 and SDS-6H, Sigma Chemical Co., St. Louis, MO) were injected into sample wells with a Hamilton syringe. A constant current of 30 mA was applied (H. V. power supply Hodel IP-l7, Heathkit, Benton Harbor, MI) until the protein migrated into the resolving gel, then the current was increased to 60 mA until the tracking dye reached the bottom of the resolving gel in about 6 to 8 hr. Gels were stained using 0.4% Coomassie Brilliant Blue (Sigma Chemical Co.) in acetic acid, methanol and water solution (with 9: 45: 45 volume ratio) for 6 hr. Destaining was performed in several changes of a 7.5% acetic acid, 25% methanol solution. Gels were stored in a 7.5% acetic acid solution. Molecular weights of the SSP were estimated by comparing the relative mobility of the proteins to that of the molecular weight standards (Appendix A). Protein bands were quantified 31 by scanning at 580 nm and integration using the Shimadzu Dual- Wave Length Thin-Layer Chromoto Scanner (Hodel CS-930, Kyoto, Japan). 3.3. Non-Destructive Rheological Analysis Oscillatory dynamic measurements were performed using a Rheometrics Fluid Spectrometer (RFS-8400, Rheometrics, Inc., Piscataway, NJ) fitted with a 50 mm diameter parallel plate apparatus. The transducer was 100 g-cm. Two to three milli- liters of protein solution were loaded in the sample cup heated with a water circulating system, and the temperature was controlled by a temperature programmer (HTP-6 Micro- processor, Neslab Instruments, Inc., Newington, NH). The gap between upper and lower plates was controlled within 1-1.5 mm. The changes in dynamic moduli with strain (0.01%-50%) and frequency (0.1-50 rad/s) were conducted to establish optimum instrumental parameters that dynamic moduli were independent on the strain and frequency selected (i.e. linear range). The effects of heating rate (1°C/min and 2°C/min) and protein concentration (1%, 2% and 3%) were investigated as preliminary studies. Three percent SSP solutions and a 1°C/min heating rate were selected for additional studies. Storage (6') and loss (6') moduli were measured at a fixed frequency of 10 rad/sec and strain of 1% during a controlled heating process. Effect of pH (4.5, 5.5, 6.5 and 7.5), NaCl concentration (0.15, 0.3, 32 0.45 and 0.6 H) and holding temperature (55, 60, 70, 80 and 85°C for 20 min) were analyzed in triplicate. The terminology used to examine the treatment effects is described in Figures 2, 3 and 4. The slopes of curves within each transition were determined using computerized best-fitting method (Neslab Instruments, Inc., Newington, NH). The intersection of two fitting lines was defined as the onset temperature. The first derivative was obtained by computing the first order difference of data. 3.4. Gel Preparation Gels were prepared as described by Smith et al. (1988). Eight grams of each 3% protein solution were pipetted into a 16 x 100 mm screw top test tube with an inner radius of 6.89 mm. Air bubbles were removed by low speed centrifugation (about 100 x g for 5 min) and all the tubes capped. Samples were heated in a water bath from 30°C to 80°C at a constant rate of 1°C/min using a temperature programmer (MTP-6 Microprocessor, Neslab Instruments, Inc., Newington, NH). Gels for each treatment were heated to nine different final temperatures (30, 40, 45, 50, 55, 60, 65, 70, and 80°C). Samples were put into ice immediately after reaching the desired temperatures which were measured in a blank tube (water) with a thermocouple. Dynamic Modulus (Pa) 33 1E+041 .l l ‘ 2nd Onset End Point 1E+03': . 4th Slope 1 e < lnitiol 3rd 51°” Point ,_ ...... ,' - 3rd Onset Initial Slope 1E+02-_ J 15+01 - . ' . ' ‘ ' ' ' 1 Temperature (’C) Figure 2. Definition of terminology used in analyzing the dynamic moduli of the thermal transitions. 34 15+oo-‘l l .l 4 initial . Point Peak 1 N 0953900 Peak 2 to owmoooi’db °o 683cm ° W s lE-Ol 1 ° +-' . o . °6> End 1 my Point Mm ‘ an“ 4 15"02 l T r F ' r ‘ t 30 4-0 50 60 7O 80 Temperature ('C) Figure 3. Definition of terminology used in analyzing loss tangent ( tan Delta ) on thermal transition curve. 35 300- ‘ 2nd Onset ZOO-J ' J ., “ ~ 4th Onset 100- n A - v" " K! n- 0 E O '1, SJ p s, U , V d lat Onset 1.... ‘0 \ -100~ 4:- (D .0 1 _200—4 ...300—1 . 3rd Onset “400 . r r l ' l t r T l 30 4O 50 60 7O 80 Temperature (’C) Figure 4. Definition of terminology used in analyzing differential plots of complex modulus (G*) vs temperature. 36 3.5. Back Extrusion Salt-soluble proteins fora weak gels when heated below 55°C, and weaker gels are not detectable if using conpression or failure tests. Back extrusion technique deternines gel strength in the tubes and, therefore, was selected for this study. An Instron Universal Testing Machine (Model 4202, Canton, MA) equipped with a 50 N load cell was used to deter-ine gel strength. Force-deformation curves of protein gels were recorded as a 8.5 an diaaeter plunger penetrated the gel at a constant rate of 50 an/nin to a distance 25 an below the gel surface. Viscosity index or work to penetrate the gel was obtained by calculating the area under the curve, as described by Hickson et al. (1982). Gels prepared at three different pfl's (5.5, 6.5 and 7.5) in 0.6 M NaCl and nine final teaperatures (30, 40, 45, 50, 55, 60, 65, 70 and 80°C) were analyzed in triplicate. 3.6. lxpressible Hoisture of Gels A low speed centrifugal aethod of Jauregui et al. (1981) with sons aodifications was used to determine the expressible aoisture of gels. Three pieces of lhatnan 02 filter paper, 9 cn in diaaeter, were folded into a 50 n1 centrifuge tube. A weighed 2 to 3 g sanple of protein gel was placed inside the filter paper and centrifuged at 755 x g for 5 ein in a refrigerated centrifuge at 2°C. The voluae of released fluid 37 in original tube was recorded. Expressible moisture of SSP gels was deter-ined by leasuring the weight gain of the filter paper plus the weight of released fluid divided by the weight of original sanple. Gel prepared at four different pH's (4.5, 5.5, 6.5 and 7.5) in 0.6 M NaCl and nine final temperatures (30, 40, 45, 50, 55, 60, 65, 70 and 80°C) were analyzed in triplicate. 3.7. Scanning Electron Microscopy Conditions selected for evaluating gels by scanning electron aicroscopy were four different pH's (4.5, 5.5, 6.5 and 7.5) and three final heating temperatures (55, 65 and 80°C) in 0.6 M NaCl. The procedure used was described by Kloaparens et al. (1986). The protein gels was cut about 3 an square, placed into vials and fixed with 2% glutaraldehyde in 0.1 H phosphate buffer (pH 7.0) for 1 hr at too. tenperature, then washed in the sane buffer solution to reaove excess fixative. Dehydration of fixed specinens was perforned in a graded ethanol series of 25, 50, 75, 95 and 100‘ for 10 nin per step and stored in 100‘ ethanol at least overnight for use. The carbon dioxide critical point drying nethod was used. Specinens were then aounted on silver stubs and coated with a thin layer of gold in a Eascope sputter coater (Hodel SCSOO, Kent, England). All coated specinens were observed with a JEOL scanning aicroscope (Hodel J8H-35CF, Osaka, Japan) at an accelerating voltage of 10 xv and condenser lens 600. 38 3.8. Statistical Design and Analysis A randomized complete block design (RCBD) was used to reduce the variation from chicken source and unexpectable external nuisance variables. The effects of pH and NaCl concentration were studied within each extraction of salt- soluble proteins. A software "STAT (Version 5.0) program was used to compute the mean square error ("83), analyze the variance and make multiple comparisons of means. Tukey's test (all pairwise comparisons) were utilized to test the differences among treatment means. RESULTS AND DISCUSSION 4.1 Composition of Salt-Soluble Proteins Electrophoregrams of salt-soluble proteins (SSP) isolated from chicken breast are shown in Figure 5. As compared with the relative mobility of standard proteins (Appendix A), two major protein bands appeared at about 186K x 13.6 and 44K t 0.5. The protein with higher molecular weight migrated close to standard rabbit myosin heavy chain (200K) and probably was chicken myosin heavy chain. The reason for the lower molecular weight of chicken myosin compared to rabbit myosin was not known, but could be due to the isoform of myosin. The protein with the lower molecular weight (about 44K) was identified as actin. The weight ratio between myosin and actin in the SSP extraction was 1.3 : l as determined by peak area under the curve (Fig. 6). The remaining protein bands of lower intensity were minor components of the SSP. 4.2 Rheological Dynamic Analysis AW To establish optimum instrumental and sample parameters for dynamic testing, strain and frequency sweeps were 39 Figure 5. Sodium dodecyl sulfate-polyacrylamide electrophoresis gel (10%) of chicken breast salt-soluble proteins (a: molecular weight standards; b, c and d: salt- soluble proteins). 40 Molecular Potein Weight Components 205 K- f! a" — llllllyosin ‘ eavy ‘ Chain 116 K- 97K- 66K - 45K— .‘.~- —Actin 29 K- 41 1.200 3 0.8004 5 .2: a: .5 l; 5 ii. m if e- {V c ii: >. 0.400 ‘lil: 3: ii E fig? ‘5 3:! /‘. "' :EE ‘ nil / l, ll J 0.000-4 .f i ....... I l l l l l I l I 00 50.0 1000 Relative Mobility Figure 6. Representative densitometric tracing of chicken breast salt-soluble proteins. 42 performed using 3% chicken breast SSP in 0.6 M NaCl, pH 6.5 buffer at 30° and 85°C. The rheogram (Fig. 7) exhibited less changes in storage modulus (G') and loss modulus (G") with strain varied from 0.6‘ to 2‘ (linear range) at 30°C, but some scatter for strain less than 0.6t due to the torque generated below instrument sensitivity. when strain increased beyond 2‘, the molecules were stretched apart that the elastic property of SSP depent on the strain selected. A decrease in G” was observed with strain above 10%. At 85°C, the linear viscoelasticity was observed over the range from 0.5\ to 10% strain (Fig. 8). When measurements are performed within the linear viscoelastic range, dynamic moduli of the material are not a function of strain amplitude. Strain can be changed to maintain the torque within the instrument sensitivity without affecting the results. Figure 9 shows the frequency sweep of chicken breast SSP at 30°C. Storage modulus increased with frequency, but tended to exhibit a plateau above 0.3 rad/sec. This observation indicated the SSP illustrate a liquid-like behavior at very low frequency, but, an elastic property storing energy were observed over wide range of frequency. No linear range for G” over frequency could be observed. WW Effects of protein concentration (1%, 2% and 3t) and heating rate (1°C/min and 2°C/min) on dynamic moduli of chicken breast SSP were determinded in 0.6 M NaCl, pH 6.5 buffer. The results indicated that complex modulus (G*) Dynamic Moduli (Pa) 43 1E+03- I 0 Storage modulus. G‘ « a Loss Modulus. G" . a . 0 ° W O a ‘ °8 ° °°° °°°°°a a J 0 ° 0 a a so a 1E+OZ- '3 ° 1 J 1 '3 a Donal? manmflagq -l D a a .l l O l a a 15+01 D. enmf’t . ”flaw . .....n, . -.. 0.01 0.10 1.00 10.00 Percent Strain Figure 7. Strain sweep of 3% salt-soluble proteins in 0.6 M NaCl, pH 6.5 at 10 rad/sec frequency, 30°C. 44 1E+03~ : 0 Storage modulus. G‘ . a Loss Modulus. G" ‘ ocean ‘ 850° oaaoaoflp °° °°° A o D. V 'fi 3 2 1E+02~ a ‘ a .9. ‘ g : 0 no a 1 D a a a Domain: can: a p a D Damn D U a i a l _ 1E+01 f . . .r..., .— . rum, re h"... r . ...... 0.01 0.10 1 .00 1 0.00 Percent Strain Figure 8. Strain sweep of 3% salt-soluble proteins in 0.6 M NaCl, pH 6.5 at 10 rad/sec frequency, 85°C. Dynamic Moduli (Pa) 45 154-05- ‘ 0 Storage Modulus. G‘ . a Loss Modulus. G“ a 000° " 000 a °°°° d co 00 9° 151—02- :1 3 a :1 #000001: :1 D 4 an n ‘3 ODD DD -4 Dan 0 an IE+O1 I I ITUTIIr f l IITI'T U 1'" fiirir] 0.1 1.0 10.0 100.0 Frequency (rad / see) Figure 9. Frequency sweep of 3% salt-soluble proteins in 0.6 M NaCl, pH 6.5 at 1% strain, 30°C. 46 increased by 1,000 Pa as protein concentration increased from 1 to 3‘ during heating (Fig. 10), and no significant differ- ences were observed in transition temperatures. Torque generated from a 1% protein solution was too low to be measured before formation of gel structure (<450C). An increase in heating rate from 1°C/min to 2°C/min decreased the G* at the end point, but increased the onset temperature of transitions by 2°C, that is, increased protein thermostability (Fig. 11). These results were similar to those found by Hontejano et al. (1983) and Foegeding et al. (1986). It has been suggested that slower heating rates allow more time for proteins to unfold and interaction to occur (Saliba et al., 1987). Decrease in onset temperature of transitions with slower heating rate agrees with this suggestion. W A holding test was performed to study the effect of holding temperature and holding time on SSP gelation. Figures 12 and 13 show the representative G* rheograms of 3‘ SSP heated to 55, 60, 70, 00 and 85°C at 1°C/min and held for 20 min. Salt-soluble proteins held at 55°C exhibited less of a decrease in 6* during the second transition, while proteins held at 60°C and higher decreased at the same magnitude. These results suggested that certain structural changes in SSP occurred between 55 to 60°C, which were dependent on the 47 153-041 a 1% SP D 2285? a 37655? 1 J LLJI 1 E+°° f l T l r I F l ' l 30 4-0 50 6O 7O 80 Temperature (‘0) Figure 10. Representative rheogram of 1, 2 and 3% salt-soluble proteins (SSP) in 0.6 M NaCl, pH 6.5 heated at 1C/min. Complex Modulus (0* Pa) 48 1E+031 : o 1°C/min 1 a 2°C/min 000 O 0 96 0° 0 ° mama o°° 0 154-021 g n °n a?” e ‘ n 1 {JD ° °°°f :13 a! D D 1?“ ° ° mu m 13 DD 1901-; “a :1 U0 1£+oo f . ,_ . I . r . 1 30 4-0 50 60 70 80 Temperature (°C) Figure 11. Effect of heating rate on the complex modulus of 3% salt-soluble proteins in 0.6 M NaCl, pH 6.5. 49 15+04- 0 55¢: . 0 mac 41 f\ 4 a Q. ... cl 3 {“0 O 0) a o 3 18-1-03 '3 ° '5 1 a o O «l 12 1 n x d 3 u 2' o ‘ ° 0 L) 154-02 .,.e ,.,. o 10 2'0 ' so 40 55 Time (min) Figure 12. Representative rheogram of 3% salt-solubled proteins heated at 1°C/min to 55 and 60° C, and held for 20 min. 50 15+041 o 700 a 80C )3 85C . _- . mI-’- . sm“-“ “...-III ' 2‘ IV" . . . Complex Modulus (Pa) 1E+O1 I I ' I w I w I w* r ”I ’FT r I w I 0 1 0 20 30 40 50 60 70 80 Time (min) Figure 13. Representative rheogram of 3% salt-soluble proteins heated at lC/min to 70, 80 and 85C, and held for 20 min. 51 thermal energy. According to Xiong et al. (1987), this temperature range was ascribed to myosin denaturation. However, how these changes influence the protein gelation could not be fully understood from this holding tests. No significant differences in 6* of SSP were observed as holding temperature was increased to 70°C or higher during heating (Table 2). Since the denaturation of actin occurs at 78°C (Xiong et al., 1987; Kijowski and Mast, 1988), this indicated that actin had little contribution to the texture of SSP gels. Results suggest that there is no benefit from a rheological perspective for cooking chicken breast above 70°C. Table 2. Effect of holding temperature on complex modulus of 3‘ chicken breast salt-soluble proteins in 0.6 M NaCl, pH 6.5 heated at 1°C/min 6* (Pa) at holding time Temperature (°C) 0 min 20 min 55 735.606b 370.53b so 93.04b 356.47b 70 473.03ab 2064.53a so 695.9oab 1738.90a as 1314.33a 1915.33a a,b any two means within the same column followed by different letters were significantly different at P<0.05. 52 Win The influence of NaCl concentration on SSP gel strength and thermostability was determined using dynamic testing, where the G* is plotted as a function of temperature (Fig. 14). Table 3 gives the statistical results of the effect of heating on the dynamic moduli of SSP as influenced by NaCl concentration. Dynamic moduli were almost identical for the four different NaCl concentrations at 30°C (P>0.05). The salt-soluble proteins in 0.45 M NaCl buffer tended to have lower, but nonsignificant dynamic moduli than the other treatments at the peak maximum point and end point of the gelling curve. Kaminer and Bell (1966) suggested that myosin exists as filaments in lower salt concentration ((0.3 M), while myosin is primarily monomeric and soluble in salt concentrations greater than 0.3 H. Wicker et al. (1986) also observed a greater gel rigidity for filamentous fish myosin. However, no significant differences in dynamic moduli were observed in this study for chicken SSP in 0.15 H and 0.30 H as compared with 0.60 M NaCl (P>0.05). Onset temperature of the SSP transitions were influenced by NaCl concentration. Different salt concentrations have no effect on the onset temperature of first transition for both 6' and G" (Table 4). It was noticed that the first onset point for G” occurred earlier than the first transition of 6' (P<0.05). The increase in G" at 42-44°C was thought due to the partial unfolding of protein structure which caused an initial increase in the viscous character of the SSP. The 53 0.15 M 0.30 M 0.45 M 0.60 M 0900 1 BO! . . . . . 1 30 4‘0 5'0 60 7b 80 Temperature (°C) Figure 14. Effect of NaCl concentration on the rheogram .of 3% salt-soluble proteins at pH 6.5 heated at 1 C/mm. 54 Table 3. Effect of NaCl concentration on dynamic moduli of 3% chicken breast salt-soluble proteins (pH 6.5) heated at 1°C/min NaCl Concentration (M) Parameters 0.15 0.30 0.45 0.60 W Initial Point 224.98 282.43 258.3a 212.3a Peak Maximum 1527.6a 1159.7a 811.1a 1277.0a End Point 1956.1a 1462.0a 642.7a 1529.3a WW Initial Point 222.2a 280.53 256.13 207.63 Peak Maximum 1519.7a 1151.0a 803.9a 1268.0a End Point 1947.3a 1458.8a 641.1a 1528.0a W Initial Point 33.4a 32.1a 33.6a 38.0a Peak Maximum ‘ 209.0a 149.5a 113.9a 152.2a End Point 170.4a 91.0a 38.9a 55.2a a any two means within the same row followed by different letters were significantly different at P<0.05. 55 Table 4. Effect of NaCl concentration on thermal transition temperatures of 3% chicken breast salt-soluble proteins (pH 6.5) heated at 1°C/min Onset Temperature (°C) NaCl (M) 1st 2nd 3rd 4th Stsrass_n9dulua 0.15 45.98b 54.6b 61.28 66.58 0.30 46.68 54.6b 60.58 65.98 0.45 46.48 54.9b 59.48 65.38 0.60 45.48bc 53.3b 56.08 63.98 o s od 0.15 42.3d 59.58 64.28 ---- 0.30 43.9bcd 56.68b 60.98 ---- 0.45 43.5cd 56.28b 59.68 ---- 0.60 42.1d 54.5b 59.18 ---- a,b,c,d any two means within the same column followed by different letters were significantly different at P<0.05. 56 subsequent increase in G', which indicated an increase in the elastic nature of material, suggested the SSP started to crosslink to form an elastic gel. According to Wicker et al. (1986), an increase in fluorescence intensity of chicken myosin-ANS (33°C) preceded the initiation of gelation measured by TSRM (47°C) at pH 6.5, 0.6 M KCl which agreed with this unfolding mechanism. No significant difference was found in the second onset temperature of G' between the four salt concentrations. The onset temperature of G” in 0.60 M NaCl was significantly different from that in 0.15 M NaCl (P<0.05), while no differences were observed between 0.30, 0.45 and 0.60 M NaCl. Protein solutions in 0.15, 0.30 and 0.45 M NaCl also exhibited almost identical second onset points of G”. The onset temperatures of G" in 0.15 M NaCl were found to be higher than G’ (P<0.05). In terms of the third transition, the onset temperatures of 6' increased with decreasing salt concentration, although it is not significantly different. The same trend was observed in the G", however, the experimental error was large and the differences were not significant. Again, the third onset temperatures for G" were almost identical to those for G'. Although the fourth onset temperature tended to increase with decreasing salt concentration, the changes in G' of SSP in the four salt concentrations were not significant. Most transition curves for SSP G" lacked the fourth transition. According to Cheftel, Cuq and Lorient (1985), some temporary 57 bonds stabilizing the secondary and tertiary structures are disrupted by the heating effect. Since the second onset points of G" varied with salt concentration, it is reasonable to suggest the involvement of electrostatic interactions. The ions of neutral salts react with the charges of proteins, decrease the electrostatic attraction between opposite charges of neighboring molecules and lead to a decrease in structural stability. Therefore, an increase in NaCl to 0.60 M would be expected to decrease the second onset temperatures in the transition curves of SSP. Table 5 represents the statistical results of the differential plot for SSP 6* vs temperature (rate of change) at different NaCl concentrations. Four transition temperatures were observed (see Appendix B). The positive rate started to increase from 45-46°C (first transition) for the four salt concentrations and reached a maximum at about 48-49°C. lithin this temperature region, myosin underwent a rapid conformational change which resulted in an increase of G' and G” (Table 4). Complex modulus still increased with temperature beyond 49°C, but the rate was slower and started to become negative at S3-55°C, which corresponded to the peak maximum for the original plot. A minimum point was observed at about 56-58°C followed by an increase in rate until 60- 62°C. Significant differences were observed between 0.6 M and 0.15 M, 0.3 M NaCl for the third and fourth transition temperatures. Table 5. Onset temperatures of the complex modulus of 3% chicken breast salt-soluble proteins on the differential plot at different NaCl concentrations Onset Temperature (°C) NaCl (M) 1st 2nd 3rd 4th 0.15 45.38 46.18 57.98 61.78 0.30 46.38 46.68 56.38 61.48 0.45 46.38 49.28 56.5b 60.78b 0.60 45.38 46.18 55.9b 59.6b a,b any two means within the same column followed by different letters were significantly different at P<0.05. The influence of salt concentration on the slopes of G' and G” transition curves were analyzed using a semilog plot (Table 6). Little change in G' was observed in the initial region as the slope was close to zero, while the G" slope decreased slightly, but non-significantly. In the first and third transitions, 6' had larger slopes than G". Both 6' and G" showed negative slopes on the second transition and no fouth transition was observed for G". Salt concentration did not change the slope of G' of SSP solutions (P>0.05), however, the first transition of G", SSP in 0.60 M NaCl exhibited the highest slope (P<0.05), and SSP in 0.45 and 0.15 M NaCl had the lowest slopes. 59 Table 6. Effect of NaCl concentration on thermal transition slopes of 3% chicken breast salt-soluble proteins Slope (Pa/0C) NaCl (M) Initial 1st 2nd 3rd 4th Modulus 0.15 0.01a 0.12a -0.14a 0.05a 0.02a 0.30 0.01a 0.10a -0.22a 0.09a 0.03a 0.45 0.00a 0.08a -0.26a 0.09a 0.03a 0.60 0.01a 0.12a -0.21a 0.08a 0.03a We. 0.15 -0.01a 0.05c -0.14a 0.01a ---- 0.30 -0.018 0.06b -0.168 0.028 ---- 0.45 -0.043 0.05c -0.29a 0.02a ---- 0.60 -0.02a 0.07a -0.18a 0.02a ---- a,b,c any two means within the same dynamic modulus in the same column followed by different letters were signi- ficantly different at P<0.05. 60 As discussed previously, the positive slope in the first transition was probably due to the unfolding of protein molecules. The succeeding molecular motion caused by heat, disrupted the bonds between protein molecules. This molecular motion leads to a decrease in resistance of flow, and thus negative slopes were observed. Since the dissociation or/and unfolding of protein molecules increased the exposure of hydrophobic groups, hydrophobic interactions were thought to be the main cause of subsequent aggregation and the positive slopes found in the third transition. A negative slope of G' on the second transition was also observed in natural fish actomyosin by Sana et al. (1988). They found that this phenomenon did not happen in fish myosin, and attributed this marked decrease to protein components other than myosin, such as actin or tropomyosin, and/or the interactions of myosin with other components. Tangent 8 represents the relative liquid to solid proper- ties of a material. Salt concentration strongly influenced the second tan 8 peak, although tan 0 tended to decrease non- significantly with an increase in salt concentration. No significant differences in peak temperature were observed. However, the tan d of SSP in 0.60 M NaCl was higher (more viscous character) than in 0.15 and 0.30 M at the first peak point (Table 7). Table 7. Effect of NaCl concentration on loss tangent (tan d) 61 of 3‘ chicken breast salt-soluble proteins (pH 6.5) heated at 1°C/min HaCl Concentration Transitions 0.15 0.30 0.45 0.60 Initial_221nt Temperature (°C) 30 30 30 30 tan d 0.14a 0.12a 0.12a 0.13a 1§£_E£ih Temperature (°C) 46a 47a 48a 47a tan 6 0.09b 0.11b 0.138b 0.168 an_E£ih Temperature (°C) 56a 56a 58a 58a tan d 0.11a 0.12a 0.17a 0.19a End_221nt Temperature (°C) 80 80 80 80 tan 6 0.05a 0.04a 0.04a 0.04a a,b any two means within the same row followed by different letters were significantly different at P<0.05. 62 W Figure 15 shows a representative rheogram of 6* vs temperature of SSP at different pH's. No major transition was observed for SSP at pH 4.5. The thermal transitions for SSP occurred earlier at pH 5.5 than at pH 6.5 and 7.5. The 6* transition peak at pH 5.5 was broader, showing a plateau in the 43-48°C temperature range and then became narrow with an increase in pH. The isoelectric point (IP) of the myofibril- lar protein system is pH 5, however, addition of NaCl results in a shift of the IP to lower pH due to the salts changing the protein conformation (Hamm, 1986). Salt-soluble proteins in pH 4.5, 0.6 M NaCl buffer were probably close to the IP. At this pH, all proteins coagulated and were unable to form gels. Salt-soluble proteins at pH 4.5 showed the lowest dynamic moduli at the initial point although it was not significantly different from SSP at pH 5.5 or 7.5. No differences in three dynamic moduli were observed among pH 5.5, 6.5 and 7.5 at initial point and peak maximum. At the end point, 6' were identical for SSP at pH 4.5 and 7.5, and G” was not different at pH 4.5, 6.5 and 7.5. Salt-soluble proteins at pH 5.5 and 6.5 tended to have higher G8 and 6' than at pH 7.5, while protein gels at pH 5.5 exhibited higher G" than at pH 6.5 (Table 8). When comparing 6', SSP underwent structural changes from 36°C to 64°C at pH 5.5, and from about 45°C to 65°C at pH 6.5 and 7.5 (Table 9). The first, second and third transition Complex Modulus (0*. Pa) 63 1E+04- 11 pH4$5 thifi leiS pH 7.5 ODDO 1E+01 . 30 r T I If ‘ U 50 60 70 6'0 Temperature (°C) 3-1 Figure 15. Effect of pH on the rheogram of 3% salt-soluble proteins in 0.6 M NaCl heated at 1°C/min. 64 Table 8. Effect of pH on dynamic moduli of 3% chicken breast salt-soluble proteins heated at 1°C/min pH Parameters 4.5 5.5 6.5 7.5 Gamnlex_fladulua_ifial Initial Point 35.1b 145.48b 201.08 169.78 Peak Maximum ---- 1004.9a 1197.03 621.33 End Point 217.7b 1730.08 1266.08 576.5b fitnzias_flgdnlua_iflal Initial Point 34.2b 141.68b 196.08 167.08 Peak Maximum ---- 1000.3a 1190.7a 614.7a End Point 216.3b 1725.78 1266.08 575.9b Loss_flmdulua_izal Initial Point 6.6b 32.68b 36.68 31.48b Peak Maximum ---- 116.6a 128.6a 82.8a End Point 24.4b 123.18 30.6b 24.9b a,b any two means within the same row followed by different letters were significantly different at P<0.05. 65 Table 9. Effect of pH on thermal transition temperatures of 3% chicken breast salt-soluble proteins Onset Temperature (°C) pH 1st 2nd 3rd 4th Storaae_noduiua 5.5 35.58 45.48 54.1b 63.98 6.5 45.48b 53.08b 56.38 64.28 7.5 47.08 54.98 59.28 64.68 Less_nodulns 5.5 3406d ‘709bc S‘elb ---- 6.5 42.18 53.98 59.08 ---- 7.5 42.7bc 55.68 59.48 ---- a,b,c,d any two means within the same column followed by different letters were significantly different at P<0.05. 66 temperatures of G' and G” were pH-dependent, while the fourth transition of G' was not affected by pH. The transitions of SSP at pH 5.5 occurred earlier than SSP at pH 6.5 and 7.5 (P<0.05) which indicated a lower structural thermostability of SSP at pH 5.5. As observed in the study on NaCl concentration effects, the first G" transition occurred earlier than the first 6' transition (P<0.05), which was thought due to the partial unfolding of protein structure. Fewer differences in onset temperatures between G' and G" were found for the second and third transitions. The SSP thermal transitions using differential plots of complex modulus against temperature (dG*/dT vs T) were determined (Table 10). The positive rate of change increased from 36° to 39°C, 45° to 48°C and 47°C to 50°C at pH 5.5, 6.5 and 7.5, respectively. After the second onset point, the rate of increase in G8 decreased. A minimum (the third onset Table 10. Onset temperature of the complex modulus of 3% chicken breast salt-soluble proteins on the differential plot at different pH's Onset Temperature (°C) pH 1st 2nd 3rd 4th 5.5 35.6b 39.3b 51.0b 55.2b 6.5 45.48 47.98 55.68 59.58 7.5 46.78 50.38 56.38 59.68 ‘cb any two means within the same column followed by different letters were significantly different at P<0.05. 67 temperature) was observed at 51°C at pH 5.5, and 56°C at both pH 6.5 and 7.5. When pH is decreased toward the IP of SSP, the net charges of the native protein become lower and smaller repulsive forces exist between protein molecules (Barbu and Joly, 1953). Random aggregation dominanted at the IP which led to a lower onset temperature of the first transition for SSP at pH 5.5. The pH dependence of myosin denaturation has also been observed in DSC studies (Goodno et al., 1976; Stabursvik and Martens, 1980; Wright and Wilding, 1984; Wagner and Anon, 1985). Two peaks found in DSC studies have been ascribed to light meromyosin for the lower transition temperature and subfragment 2 for the higher transition temperature (Cross et al., 1984). Stabursvik and Martens (1980) suggested that different subfragments of myosin exhibited dissimilar pH responses in DSC studies. The first peak was stabilized by increasing pH, while the second showed a considerable degree of destabilization. Therefore, the different pH responses of myosin subfragments caused the narrow transition peaks of SSP observed at pH 6.5 and 7.5. The effect of pH on the slopes of SSP thermal transition curves are shown by data in Table 11. The slopes within the initial heating range, i.e. before the first transition occurred, were close to zero at all pH's. Little difference was observed for SSP at pH 4.5 due to subsequent heating treatment. The first 6' transition slope decreased in the order of pH 6.5, 5.5 and 7.5, while no differences were 68 Table 11. Effect of pH on thermal transition slopes of 3% chicken breast salt-soluble proteins Slope (Pa/°C) pH Initial 1st 2nd 3rd 4th Storaas_nadulua 4.5 0.028b 0.008 --—- ---- ---- 5.5 0.038 0.1088 -0.14b 0.068 0.028 6.5 0.0188 0.138 -0.19b 0.078 0.028 7.5 0.008 0.06b -0.298 0.118 0.038 Less_ncdnlua 4.5 0.028 0.00b ---- —--- ---- 5.5 0.018b 0.058 -0.16b 0.038 ---- 6.5 0.0088 0.078 --o.21b 0.048 ---- 7.5 -0.018 0.058 -0.338 0.038 ---- a,b,c any two means within the same dynamic modulus in the same column followed by different letters were signi- ficantly different at P<0.05. 69 observed for G”. Salt-soluble proteins at pH 7.5 showed the highest negative slope for both 6' and G" in the second transition, followed by pH 6.5 and pH 5.5. Salt-soluble proteins at pH 7.5 had a higher positive slope in the third 6' transition than at pH 6.5 and 5.5, while little heating effect was observed for the fourth transition slope. The slopes in the third G” transition were identical at pH 5.5, 6.5 and 7.5. No change in slope was observed after the third G” transition. Table 12 shows the effect of pH on the tan d of SSP thermal transitions. Almost identical tan 0 were observed at initial point at all pH 's which implied similar rheological properties of the material. At pH 4.5, no second peak was observed which corresponded to the lack of transitions in both 6' and G” after the first peak. Among pH 5.5, 6.5 and 7.5, little difference for tan d was observed at the first and second peaks due to large experimental error. At the end point, protein gels at pH 4.5 tended to show higher viscous properties than at pH 6.5 and 7.5 which indicated a more elastic network in pH 6.5 and 7.5 gels. The peak temperatures of tan 0 did change with pH. The first and second peaks for SSP at pH 5.5 occurred earlier than at pH 6.5 and 7.5. No difference in peak temperatures was found between pH 6.5 and 7.5. The temperature range between two tan 6 peaks was larger at pH 5.5 (34-53°C) than at pH 6.5 (47-57°C) and 7.5 (49- 59°C). 70 Table 12. Effect of pH on loss tangent (tan d) of 3% chicken breast salt-soluble proteins heated at 1°C/min pH Transitions 4.5 5.5 6.5 7.5 Mini: Temperature (°C) 30 30 30 30 tan 6 0.22a 0.23a 0.20a 0.173 M Temperature (°C) 378 348 478 498 tan d 0.24a 0.20a 0.19a 0.173 M Temperature (°C) ---- 53b 57a 58a tan d ---- 0.15a 0.12a 0.15a End_Eaint Temperature (°C) 80 80 80 80 tan 6 0.128 0.0788 0.02b 0.04b a,b,c any two means within the same row followed by different letters were significantly different at P<0.05. 71 4.3. Effect of pH on Gel Strength, Haterholding Capacity and Microstructure Since buffer pH influenced the dynamic gel properties more than changes in NaCl concentration, the effect of pH on gel strength and water holding capacity were studied. Gel strength of discrete SSP samples heated to certain endpoint temperatures was investigated using back extrusion to measure the viscosity index at different pH's (Fig. 16). Salt-soluble proteins at pH 5.5 exhibited the highest gel strength from 45° to 80°C, followed by SSP at pH 6.5 and 7.5. The viscosity index of SSP at pH 5.5 started to increase from 45°C and reached a plateau at 65°C. Further heat treatment above 70°C tended to increase molecular motion and destroy the gel structure, as indicated by a slight decrease in viscosity index. At pH 6.5, the gel strength increased from 55°C and became stable at 70°C due to network formation. Little change in viscosity index was observed when SSP were heated above 70°C which agreed with the holding temperature results in dynamic testing. The viscosity index of SSP at pH 7.5 also increased at 55°C (same as pH 6.5), however, the final strength was lower than in pH 6.5. In the back extrusion study, it was found that the transition for SSP in pH 5.5 occurred earlier than in pH 6.5 and 7.5, which agreed with the dynamic testing results (Sec. 4.2.5), however, no decrease in transition curves was observed as in dynamic testing. This might be due to more bond formation during cooling of the protein samples prior to testing by back extrusion. The final Viscosity Index (poise) 72 1E+04q j o—o pH 5.5 . H pH 6.5 . a—a pH 7.5 J ,- : w ', fig fi 15+031 " 1 r 4 ‘ - ...—III“); 1E+02 1 . . . . 1 30 40 53 60 7B 60 Temperature (°C) Figure 16. Effect of pH on viscosity index of 3% salt-soluble proteins heated to indicated endpoint temperatures (SEM = 203.4). 73 texture of protein gels was examined in the back extrusion study rather than continuous structural changes monitored by dynamic testing. A maximum gel strength of rabbit myosin at 60 to 70°C has been reported by Yasui et al. (1979). The pH for maximum gel rigidity at 0.6 M KCl measured by a band-type viscometer was reported at pH 6.0 for rabbit myosin (Ishioroshi et al., 1979; Samejima et al., 1984) and pH 5.6 for chicken white myosin (Asghar et al., 1984) which agree with the results of visco- sity index in back extrusion study. Morita et al. (1987) compared the myosin gels of chicken breast and leg, and found the maximum gel rigidity for chicken breast and leg myosin was near pH 5.4 and pH 5.1, respectively. The breast myosin formed a stronger gel than leg myosin. Gel microstructure observed using transmission electron microscopy showed that breast myosin exhibited a filamentous assembly at pH 5.7, 0.6 M KCl. The filament length became longer at pH 5.4. Leg myosin had more dispersed particle-like filaments at pH 5.7 and assembled into filaments at pH 5.4, but the length still shorter than breast myosin filaments. Hermansson et a1. (1986) reported two types of myosin gels : stranded and aggregated gels, depending on certain combinations of pH and salt concentration. The fine stranded gel had a higher rigidity than the coarsely aggregated structure. It was then suggested that the longer filament found in breast myosin forms a finer network and leads to higher gel rigidity than leg myosin (Morita et al., 1987). 74 Gels prepared at pH 4.5 exhibited high expressible moisture values due to protein aggregation at IP (Fig. 17). Gels prepared at pH 6.5 and 7.5 had similar waterholding capacities. The expressible moisture slightly increased from 30 to 40°C, decreased to a minimum at 50°C, then increased until 65°C. At pH 6.5, the water loss of SSP gels decreased slightly then increased again, while at pH 7.5, a gradual increase was observed. Proteins prepared at pH 5.5 showed similar properties as SSP at pH 6.5 and 7.5 at heating temperatures below 40°C. An increase in heating temperature above 45°C caused a substantial increase in moisture loss until 65°C, which is coincident with the transition tempera- tures of viscosity index found in the back extrusion study. A further understanding of gel strength and waterholding of SSP at different pH's can be obtained with a knowledge of the protein microstructure. Representative scanning electron micrographs of 3% SSP heated to 55, 65 and 80°C at different pH's are presented at 10,000X magnification in Figures 18, 19 and 20, respectively. The heat-induced protein gel in pH 4.5 at three different temperatures showed a similar highly aggregated, globular structure with no network formation (Figs 18A, 19A and 20A). These microstructures are consistent with the poor gel strength and waterholding capacity observed. Comparing the microstructures of SSP heated to 55°C (Figs. 188,C and D), SSP at pH 5.5 was filamentous and exhibited an irregular network structure with large apen holes, while regular lacy networks were observed at pH 6.5 and 7.5. Gels 7S 80" : n 3 1 5 704 '- A N V 60-l 03 L ,1 :3 a...) .9. 50-« O .. 2 | 3 A: Q) .. I'c <3 :5 4'0 1‘ : c "a 4; ‘ c a) 7 -. ‘ 8 - .- Q 304 x LIJ -l 204 I H pH 4.5 10"1 B—a pH 5.5 . a—a pH 6.5 0 0—6 pH 7.5 ' I r I r I T I T I 30 40 50 60 7O 80 Temperature (°C) Figure 17. Effect of H on waterholding capacity of 3% salt-soluble proteins eated to indicated endpoint temperatures (SEM = 1.9). 76 prepared at pH 5.5 and heated to 65°C contained more globular, thicker protein filaments in a slightly irregular matrix resulting in higher gel strength (Fig. 198), but then changed to a highly aggregated structure when heated to 80°C (Fig. 208). Protein gels at pH 6.5 and 7.5 exhibited an open continuous filamentous matrix which is characteristic of good waterholding capacity (Figs. 19C and D). Firmer, regular filamentous networks were observed for SSP gels at pH 6.5 and 7.5 heated to 80°C (Figs. 20C and D), but contained smaller open spaces to immobilize water molecules than SSP heated to 65°C. This change in microstructure explains the decrease in waterholding capacity and increase in gel strength of SSP at temperatures above 70°C. Two types of heat-induced aggregation occur depending on the charge of the native protein : a linear aggregation at high repulsion and a globular ‘random' aggregation at low repulsion (Barbu and Joly 1953). Based on this fundamental theory, Hermansson (1986) suggested that as the repulsive forces between molecules increase, random aggregation is suppressed and the structure will change from an aggregated or phase-separated structure to an ordered strand structure. The micrographs for SSP heated at different endpoint temperatures and pH's agreed with both suggestions. Hermansson (1986) also stated that heating above the gelation point may result in an increased tendency for phase separation of aggregated gels. Phase separation results from locally strong interactions between protein molecules and causes a coarser structure which 77 leads to a decrease in waterholding capacity. The microstructure of SSP at pH 5.5 heated at 65° and 80°C illustrate this change in gel structure due to phase separation. However, temperatures above the gelation point may not have this effect on the ordered, filamentous gel structures because certain energy input is required to cause dissociation and reassociation of subunits into strands (Hermansson, 1986). Although the ‘gelation point' was not defined clearly, a relatively disordered SSP gel micro- structure at pH 6.5 and 7.5 was observed at 55°C (Figs. 18C and D). An increase in heating temperature to 65°C changed the structures to an ordered network (Figs. 19C and D). No phase separation was observed for further heating (Figs. 20C and D). However, in later work of Hermansson et al. (1986), a stranded-type bovine myosin gel was found at pH 4, 0.6 M KCl on dialysis. This structure did not change in character on heating. At the lower salt concentration, heating produced a gel structure composed of fine strands. At the higher salt concentration, globular aggregates dominated and no fine strand could be seen. All fine stranded gels were formed from Opaque solutions and the aggregate gels from transparent solutions. Variation of the heating temperature between 55 to 65°C had no effect on the type of structure formed. Therefore it was suggested that the conditions required for the forma- tion of strand-type myosin gels were already present before heat treatment. 78 .3 E. :3. "me E. E an :e 2: "m... E. E a uemm 6. 3.8.. 552 Z 0.: E 2:30.:— oEEcméam 3.3.5 53.25 ..c omsausbmcafiz .n— PEEK . 1A a e a...) Haste 79 .2 me :5 "me me .3 an E. 2: mm... ma 3 .n Dame 3 5.8.. 552 2 ed 5 2:29... 032825 83.5 coo—0.5 ..c eta—6:52:22 .3 Saw: 80 .3 E. .... .me .... .3 an .... .... .3 .... E ... was. 2 5.8.. 5.2 2 ed 5 ...—.35.... 2.3.823 «93.5 50320 ..c 9.22.38.22 .en 9...»: CONCLUSION The non-destructive dynamic testing was sensitive for detecting the structural transitions of chicken breast SSP during gelation. Four G' transitions and three G" transitions occurred at all conditions except the SSP prepared at pH 4.5. The first transition was ascribed to protein unfolding. The succeeding heating increased the molecular motion which disrupted the bonds between protein molecules and led to a decrease in resistance of flow (second transition). The third and fourth transitions were ascribed to protein reaggregation and network formation. The dynamic moduli of SSP were dependent on both heating rate and protein concentration. The 6* was not different for gels at pH 6.5, 0.6 M NaCl held for 20 min at 70, 80 and 85°C. According to the microstructure, the filamentous network has been formed at 65°C, where myosin denatured completely. No further changes in gel structure were observed with an increase in temperature to 80°C. These findings suggested little contribution of actin in gel network formation. No major changes in dynamic moduli, transition temperatures and transition slopes were found for SSP in 0.15 to 0.6 M NaCl at pH 6.5. Salt-soluble proteins at pH 4.5 had higher viscous properties (higher tan 0) than at pH 6.5 and at 81 82 pH 7.5 and no major transitions occurred. Gels at pH 4.5 showed globular structure and higher moisture loss due to protein aggregation near the IP. Salt-soluble proteins at pH 5.5 had lower transition temperatures than at pH 6.5 and 7.5 due to lower thermostability. Thicker protein filaments were observed for pH 5.5 gels heated at 65°C which resulted in higher rigidity. The succeeding heating of gels caused phase seperation and led to a decrease in waterholding capacity. Salt-soluble proteins prepared at pH 6.5 and 7.5 exhibited higher elastic properties (lower tan 0) compared with SSP at pH 4.5. A continuous filamentous matrix observed for SSP gels at pH 6.5 and 7.5 corresponded with the good waterholding capacity observed. The results of this study indicated that pH influenced the association of native protein molecules and thus the gel properties. Therefore, the pH of meat sources was expected to affect the performance of final products and may be an indicator of raw meat quality. By adjusting pH, the gel properties and waterholding capacity of meat products could be improved. According to the results of holding test, it was observed that there is no benefit to gel properties for cooking chicken breast above 70°C. This heating condition can improve product yields due to decreased drip loss. --J'n'..x."-X f m- '13 (1) (2) (3) (4) FUTURE RESEARCH Suggestions for future work include: determining the effects of myosin-to-actin ratios on the native protein association examined by scanning and/or transmission electron microscopy, and the rheological gel properties; determining how the extrinsic factors influence the thermal denaturation of myosin as well as its sub- fragments, and comparing to their rheological properties measured by dynamic testing; investigating myosin conformational changes during heating process such as hydrophobic interactions, and comparing to its rheological properties to evaluate the role of myosin subfragment in gel development; evaluating the effect of additives on gelation of salt- soluble proteins (changes in viscosity or elasticity), such as non-meat proteins or polysaccharides, and comparing to meat products. 83 APPENDICES 84 Appendix A. Electrophoresis Standards The molecular weight standards used for sodium dodecyl sulfate-polyacrylamide electrophoresis were Sigma SOS-6H and SDS-7 (Table 13). The molecular weights vs the relative mobilities are shown in Figure 21. Table 13. Molecular weight standards used for electrophorsis Standard mixtures Molecular Weight EDS-6H Myosin from rabbit muscle 205,000 B-Galactosidase 116,000 Phosphorylase B 97,400 Bovine albumin 66,000 Egg albumin 45,000 Carbonic anhydrase 29,000 SOS-7 Bovine albumin 66,000 Egg albumin 45,000 Glyceraldehyde-3-phosphate Dehydrogenase 36,000 Carbonic anhydrase 29,000 Trypsinogen, PMSF treated 20,100 e-Lactalbumin 14,200 85 1E+O3j Y = 1714(0.0587)X r = -0.987 A 35, .o .H .C. .E‘ 0 3 154-02- L . 2 8 oo 2 § 8‘ ‘ O O — .l 154-01 . , . r - , . , . , 0.0 0.2 0.4 0.6 0.8 1.0 Relative Mobility Figure 21. Molecular wcight standard curve for sodium dodecyl sulfate- polyacrylamide gel electrophoresis (K = 1000 daltons). _I -7 86 Appendix B. First Derivative Plots The differential plots of G* for 3% chicken breast SSP at different pH and NaCl concentration were used to determine the thermal transition temperatures. The representative plots were shown in Figures 22 to 28. 87 300- 200--l th/dT (Pa / C) 30 10'53'63'7‘0'6'0 Temperature (C) Figure 22. Differential plot of complex modulus (G') of 3% salt-soluble proteins vs temperature (T) at pH 7.5, 0.6 M NaCl. 88 300- 200-1 100- th/dT (Pa/C) 30 40 ' 5'0 ' 60 ' 7'0 6'0 Temperature (C) Figure 23. Differential plot of complex modulus (G') of 3% salt-soluble proteins vs temperature (T) at pH 6.5, 0.6 M NaCl. 89 500. 200~ 100- l 0-4 i -100-4 th/dT (Pa/C) r ' I ' I r 30 40 50 BO 70 Temperature (C) Figure 24. Differential plot of complex modulus (G’) of 3% salt-soluble proteins vs temperature (T) at pH 5.5, 0.6 M NaCl. 90 300- 200- 100- th/dT (Pa/C) I fi I ' l 30 ' 40 ' 5'0 ' 60 7o 60 Temperature (C) Figure 25. Differential plot of complex modulus (G‘) of 3% salt-soluble proteins vs temperature (T) at pH 4.5, 0.6 M NaCl. 91 300- 200- th/dT (Pa/C) I ' I I ' 30 40 50 60 70 80 Temperature (C) Figure 26. Differential plot of complex modulus (G‘) of 3% salt-soluble . proteins vs temperature (T) at pH 6.5, 0.45 M NaCl. 92 3001 200-4 100- 4 u B o— J J . CL V 1.— Q 4, -100- O ‘O -200. -300-4 -400 I I 8 I I I I T w I 30 4O 50 60 7O 80 Temperature (C) Figure 27. Differential plot of complex modulus (G‘) of 3% salt-soluble proteins vs temperature (T) at pH 6.5, 0.3 M NaCl. 93 300- 200- 100- -1004 th/dT (Pa/C) -200- I I r 7 30 4O 50 50 70 80 Temperature (C) Figure 28. Differential plot of complex modulus (G‘) of 3% salt-soluble proteins vs temperature (T) at pH 6.5, 0.15 M NaCl. BI BLI OGRAPHY BIBLIOGRAPHY Acton, J.C. and Dick, R.L. 1986. Thermal transitions of natural actomyosin from poultry breast and thigh tissues. Poultry Sci. 65: 2051. T Acton, J.C., Hanna, M.A., and Satterlee, L.D. 1981. 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