Dynamic Viscoelastic Behavior of Natural Actomyosin and Myosin during Thermal Gelation

The changes in viscoelasticity of natural actomyosin and myosin during thermal gelation were investigated by dynamic viscoelasticity measurements. Thermal gelation of natural actomyosin could be divided into four characteristic temperature ranges. The storage modulus increased considerably in the 32–43°C range, decreased sharply in the 43–52°C range, and then increased again in the 52–80°C range. For the thermal gelation of myosin, the storage modulus increased in two steps at two temperature ranges, i.e., 30–41°C and 51–80°C. An increase in the loss modulus was observed at an early stage of each of the two ranges.     

HEAT-INDUCED GELATION OF MYOSIN IN THE PRESENCE OF ACTIN

ABSTRACT The rabbit muscle contractile proteins, myosin, actin and reconstituted actomyosin were mixed in 0.1–1.0 M KCl, 20 mM buffers, pH 5.0–8.0, and were tested quantitatively for thermally induced gelation properties by measuring the rigidity (shear modulus) of the system at 20–70°. Scanning electronmicroscopy (SEM) was also used to study the structure of the gels formed by gelation of myosin in the presence of F-actin. Under the standard condition, i.e. at 0.6 M KCl, pH 6.0 and 65°, decrease of the myosin/actin mole ratio to about 1.5–2.0 in the reconstituted acto-myosin system resulted in substantial augmentation of the rigidity of the gel formed. Further decreases in the myosin ratio relative to F-actin reduced the rigidity value of the gel to close to the level of myosin alone. Gel-formability of the reconstituted actomyosin was maximal at pH 5.5–6.0 and between 0.5 and 0.8 M KCl and decreased considerably at other pH values and KCl concentrations. The SEM studies revealed progressive changes in three dimensional ordering as actin concentration in the actomyosin varied. These were in concordance with the results of gel strength.     

Thermal Gelation Characteristics of Myosin Subfragments

To elucidate the roles of the head and the tail portions of the molecule in the thermal gelation of myosin, the gelation characteristics of heavy meromyosin (HMM) and of light meromyosin (LMM) were investigated. The aggregation process of HMM corresponded to that of myosin alone in the temperature range above 50°C. Both the dynamic viscoelastic behavior and the aggregation process of LMM agreed fairly well with those of myosin alone in the temperature range up to 45 °C. Therefore, the first development of gel elasticity of myosin was attributable mainly to the tail portion of the molecule and the second was to its head portion.     

Gelation and Thermal Transitions in Post-Rigor Turkey Myosin/Actomyosin Suspensions

A myosin/actomyosin mixture (MAM), actomyosin and myosin were isolated from post-rigor turkey. Rheological properties of MAM gels were determined by uniaxial compression. Breast MAM formed gels which were stable at lower protein concentrations (10 and 15 mg/mL) than thigh MAM (20 and 25 mg/mL). Differential scanning calorimetry (DSC) and a fluorescent hydrophobic probe, 8-anilino-l-napthalene sulfonate (ANS), were used to study thermal transitions. One DSC peak was observed in breast and thigh MAM. ANS thermograms showed that thigh actomyosin had a greater transition temperature (Tr) (51.8°C) than breast actomyosin (49.7°C). The ANS Tr was 48°C for both myosins. The difference in gelation appeared to be associated with protein-protein interactions.     

Rigidity and Viscosity Changes of Croaker Actomyosin During Thermal Gelation

Two types of thermal scanning rigidity monitors (TSRM) were developed which are nondestructive and capable of monitoring rigidity or viscosity changes during heating of proteins over a wide range of concentrations. Thermal transitions which occur during gelation of croaker actomyosin were studied by these TSRM devices and the Brookfield viscometer during constant rate heating (1°C/min). Gelation of actomyosin occurred only at protein concentrations above 5.5% under these conditions. In plots of rigidity versus temperature, three transitions were observed during gelation, occurring near 38°C 46°C and 60°C. At lower concentrations, only one peak was observed, occurring near 36°C. A relationship between the 36–38°C transition in rheological properties and the high temperature “setting” phenomenon of fish proteins is postulated.     

Thermal Denaturation and Aggregation of Proteins in Minced Fish as Studied by a Thermomechanical Method

A thermomechanical method of heating and mixing, was developed to study directly protein denaturation and aggregation in minced fish. It is based on simultaneous and continuous measurement of torque and temperature in a meat sample mixed while being heated. The torque and temperature were continuously recorded. From curves thus obtained, thermal denaturation temperatures of minced samples from 6 marine fish species were determined. The curves showed 4-6 peaks at 32-38°C, 44-46°C, 52-56°C, and 62-75°C, presumably corresponding to denaturation of myosin, actomyosin, sarcoplasmic proteins, and actin, respectively. The temperature range of peak 1 was 4-5°C higher in fatty fish (herring, mackerel) than in lean fish (cod, blue whiting).     

Temperature and pH Affect Transglutaminase-Catalyzed "Setting" of Crude Fish Actomyosin

Dynamic rheological properties of actomyosin from two species (Alaska pollock, Atlantic croaker) were monitored during preincubation (setting) at 25°C and 37°C. followed bv nronrammed (l°C/min) cookinn to 77°C. Added guinea pig liver transgluianase enhanced gelation, as indicated by increases in both storage modulus (G′) and percentage of polymerized myosin heavy chain (MHC). In the presence of added transglutaminase maximum G′ and MHC polymerization occurred at the same conditions of pH and temperature which were optimum for setting of surimi pastes. This suggested that the transglutaminase-mediated setting reaction in surimi was constrained more by the conformation of the substrate (i.e., myosin) than by that of the enzyme.     

Myofibrillar Protein Gelation: Viscoelastic Changes Related to Heating Procedures

Dynamic Theological properties were investigated during gelation of chicken myofibrillar protein as influenced by heating procedures, Thermal scan (1°C/min) of myofibril suspensions in 0.6M NaCl (pH 6.0) induced a major transition in storage modulus (G′, peak 48°C), preceded by a transition in protein-protein aggregation (46°C) and accompanied by a marked reduction in actomyosin solubility. Preheating at 50°C diminished the transition and resulted in increased final G′value. Isothermal heating produced complex, temperature-dependent Theological changes (G′and phase angles), particularly within 43–58°C. The rheological transitions of myofibrillar protein were probably related to kinetic changes during formation of elastic gel networks. Such Theological data on gel formation can help predict and control muscle food responses to specific thermal processes.     

Dynamic rheological behaviour and biochemical properties of rabbit skeletal actomyosin during storage at 0° C

The relationship between the dynamic rheological properties of heat induced gels of actomyosin (natural actomyosin) and the denaturation of actin in actomyosin during storage without ATP at pH 6.0 and 0°C was investigated using biochemical and dynamic rheological measurements. The complex modulus of gels after heating actomyosin containing 0.5 or 1.5 M KCl (pH 6.0) at 80°C increased with increasing storage time. The dynamic rheological behaviour during heat gelation of actomyosin in 1.5 M KCl indicated that the first rheological transition peak in the 50–53°C range induced by the presence of F-actin gradually disappeared with increasing storage time. However, in 0.5 M KCl, this transition peak clearly remained even after 15 days. The time course of denaturation of actin in actomyosin treated with 1.5 M KCl at pH 6.0 showed an increase in the percent denaturation after the storage was started, and about 100% of the actin became denatured after 21 days. In the case of 0.5 M KCl, unlike 1.5 M, the denaturation of actin occurred quickly within the first 5 days and then did not proceed. A sigmoidal relationship was found between the percent denaturation of actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.5 and 1.0 M. The data indicated that the change in the property of actin in actomyosin during storage at low temperature exerts a great influence on the viscoelasticity of heat-induced gels of actomyosin.     

Thermal Activation of Actomyosin Mg-ATPases from Ordinary and Dark Muscles of Tuna and Sardine

Changes in activities of actomyosin, acto-heavy meromyosin (acto-HMM), and acto-subfragment-I (acto-S-I) ATPases from tuna and sardine due to heat treatment (20°, 25°, 30°, 35°, 40°C) were compared for ordinary muscle and dark muscle. Activation of ordinary muscle actomyosin Mg-ATPases was more than doubled for tuna and tripled for sardine by heating at 35°C, while activation of dark muscle actomyosin was not observed at any temperature. The occurrence of thermal activation corresponded to a rapid loss of the EDTA-ATPase activity. Activation of hybrid actomyosins from dark and ordinary muscles was dependent upon myosin. For acto-HMM and acto-S-I thermal activation was not observed. The role of myosin tail fragments in thermal activation is discussed.     

Polymerization of Beef Actomyosin Induced by Transglutaminase

Polymerization of beef actomyosin was induced by addition of transglutaminase. The relative intensity analyzed by densitometry after sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that bands containing the polymerized myosin increased from 10.1 ± 2.2% to 20.7 ± 3.5% while the myosin monomer band decreased from 20.9 ± 3.4% to 13.0 ± 2.7% as the reaction time extended from 10 to 120 min at 35°C. Polymerization of actomyosin induced by transglutaminase resulted in gelation of the actomyosin that was visualized by confocal laser scanning microscopy.     

Effect of Ionic Strength on Dynamic Viscoelastic Behavior of Myosin during Thermal Gelation

The effect of ionic strength on the thermal gelation process of myosin was investigated by dynamic viscoelasticity measurements. The dynamic viscoelastic behavior of myosin was divided into three ionic strength groups. Each ionic strength group was closely related to the state of myosin molecules before the rise in temperature. Both the head and the tail portions of the molecule participated in the gel formation of myosin, but the temperature ranges differed. It was proposed that the first development of gel elasticity of myosin (30–45 °C) was attributed many to the tail portions of the molecules and that the second development (above 50 °C) was mainly to the head portions.     

Thermal Transitions in Myosin-ANS Fluorescence and Gel Rigidity

Thermal transitions (Tr) in myosin were monitored during constant rate heating with a thermal scanning rigidity monitor (TSRM) and a fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS). The Tr values from fluorescent probe measurements were 37°C, 44°C, and 44°C for tilapia, rabbit, and chicken myosin-ANS, respectively. Three Tr values at 43°, 49°C, and 55°C were observed in TSRM measurements of tilapia myosin gelation, whereas a single Tr was observed in rabbit and chicken gelation at 48°C and 49°C, respecitvely. In tilapia myosin, KCl concentration and pH significantly influenced the TSRM but not the fluorescence thermograms. These results indicated that a prerequisite change occurred in the hydrophobic character of myosin just prior to the onset of gelation.     

GELATION KINETICS of MEAT EMULSIONS CONTAINING VARIOUS FILLERS DURING COOKING

A cylindrical shaped thermal scanning rigidity monitor (TSRM) was developed to determine shear rigidity modulus of meat batters during cooking. A meat fatprotein ratio of 1.8, moisture content of 62% and 8.6% filler were used. the fillers were buttermilk powder, corn starch, micro-crystalline cellulose, modified corn starch, modified wheat flour, soy-protein concentrate and whey-protein concentrate. Plots of rigidity modulus versus product-temperature showed two major thermal transitions. the first and most important transition (53 to 61°C) was due to myosin gelation. the second transition (64 to 69°C) was ascribed to the collagen softening. the maximum rigidity-temperature slopes of 0.60 to 1.02 kPa/°C occurred after the first transition.     

Gelling Properties of Actomyosin from Pre- and Post-Spawning Hake (Merluccius hubbsi)

The biochemical properties and the characteristics of heat-induced gelation of natural actomyosin (NAM) from pre- and post-spawning hake were studied. Mg²⁺ ATPase activity, reduced viscosity and myosin/actin mole ratio of NAM from post-spawning fish were higher than those of pre-spawning ones. Gelation of both actomyosin at 10 mg mL^?1 of protein concentration was optimal at 60°C and pH 6.0. The highest rigidity was reached at 0.40M and 0.44M KCl with NAM from pre and post-spawning hake, respectively. Irrespective of heating temperature, ionic strength conditions and at pH range 5.5–7.5, rigidity of post-spawning hake NAM gels was higher than those of pre-spawning fish. Scanning electron micrographs of pre- and post-spawning hake NAM showed “actin-type” and “myosin-type” ultrastructures, respectively.     

Thermal Effects on Fast Skeletal Myosins from Alaska Pollock, White Croaker, and Rabbit in Relation to Gel Formation

Thermodynamic properties in differential scanning calorimetry (DSC) and changes in viscoelasticity upon heating of myosins from white croaker, Alaska pollock, and rabbit fast muscles were investigated in relation to their thermal gel formation abilities. Alaska pollock myosin unfolded in a wide temperature range of 19 to 69°C as revealed by DSC, whereas rabbit myosin unfolded in very narrow range of 32 to 56°C. Thermal unfolding of white croaker myosin occurred in an intermediate temperature range of 30 to 60°C. Viscoelastic properties determined as storage modulus, G′, and loss modulus, G″, reflected differences observed in DSC for the 3 myosins.     

Heat-induced Gelation of Myofibrillar Proteins and Sausages: Effect of Blood Plasma and Globin

The gelation of meat myofibrils and sausages was monitored during heating with and without added blood globin and plasma by the dynamic rheological method. During heating of beef myofibrils (pH 6.5; prot conc 6.0%) the storage modulus (G') reached a minimum at 47.7°C, decreased on further heating, and increased above 53°C. At higher pH (6.9) the G’value was lower and the magnitude of tanδ increased substantially between 37 and 42°C. Addition of blood globin and plasma changed the thermal gelation of myofibrils. During heating of control and protein-supplemented sausages, we observed two minor peaks in storage modulus in the range 40–55°C and a steep increase at 55–80°C. The storage modulus of low fat sausages, where 25% of meat was replaced by globin or plasma, was greater than that of control sausages.     

Curtailing Oxidation‐Induced Loss of Myosin Gelling Potential by Pyrophosphate Through Shielding the S1 Subfragment

In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron‐redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl₂ at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross‐linking was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation‐initiated cross‐linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G′) of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation‐induced G′ by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats.     

Bending strength and modulus of elasticity of particleboards at various temperatures

Bending strength and modulus of elasticity of phenol-formaldehyde (PF)-bonded particleboards were studied at temperatures between -40 °C and +40 °C. These conditions may occur, for example, during exterior use of boards. Bending strength and modulus of elasticity are significantly affected by the temperature. Moreover, positive temperatures cause higher reduction of strength properties than negative temperatures. The bending strength of boards in the temperature range from -40 °C to 0 °C, for example, is reduced by 15% and in the temperature range from 0 °C to 40 °C by 28%.     

Bending strength and modulus of elasticity of particleboards at various temperatures

Bending strength and modulus of elasticity of phenol-formaldehyde (PF)-bonded particleboards were studied at temperatures between -40 °C and +40 °C. These conditions may occur, for example, during exterior use of boards. Bending strength and modulus of elasticity are significantly affected by the temperature. Moreover, positive temperatures cause higher reduction of strength properties than negative temperatures. The bending strength of boards in the temperature range from -40 °C to 0 °C, for example, is reduced by 15% and in the temperature range from 0 °C to 40 °C by 28%.