Formation of two types of gels from bovine myosin

Myosin was isolated from bovine m. semimembranosus and gels were formed by heat treatment at different pH values and ionic strengths. The gels were subjected to rigidity measurements and their microstructure was studied by scanning electron microscopy. This article provides evidence that myosin can form two completely different gel structures in the pH range 5.5–6.0, depending on ionic strength. Fine stranded gel structures were formed at low ionic strength (0.25M KCl), whereas coarsely aggregated gel structures were formed at high ionic strength (0.6M KCl). The fine stranded structure had a higher rigidity than the coarsely aggregated structure. It was found that all fine strand myosin gels were formed from turbid solutions and the aggregate gels from clear solutions. When the pH was lowered to 4 in 0.6M KCl a strand-type gel structure formed spontaneously on dialysis, even without heat treatment. This structure did not change in character on heating. It was concluded that the conditions required for the formation of strand-type myosin gels were already present before the heat treatment and that the strands were made up of myosin filaments at certain pH and ionic strength combinations, which produced a turbid solution. The strand-type structures were considered specific with regard to myosin interactions which was not the case for the aggregated structures. Variation of the heating temperature in the range 55 to 65°C had no major effect on the type of structure formed.     

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.     

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.     

Ionic Strength Effects on Heat-induced Gelation of Myofibrils and Myosin from Fast- and Slow-twitch Rabbit Muscles

The effects of ionic strength on myofibrils and myosin from rabbit fast-twitch Psoas major (PM) and slow-twitch Semimembranosus proprius (SMp) muscles before and after heating were studied by electron microscopy and thermal scanning rheometry. The direct suspension of proteins in low ionic strength (0.2M KCl; pH 6.0) led to very weak gels, whereas a gradual lowering of the ionic strength (by dialysis against 0.2M KCl; pH 6.0) of 0.6M KCl protein solutions induced strand-type networks at low temperature and strong heat-induced gels. As shown by transmission and scanning electron micrographs, in low ionic strength, SMp myosins formed shorter filaments before heating and thinner and shorter structures in heat-induced gels, as well as a lower gel porosity than PM myosins.     

组氨酸与氯化钾混合液对兔肉肌球蛋白特性的影响

目的：研究组氨酸与氯化钾混合液对肌球蛋白溶出率、聚集特性和热凝胶特性的影响。方法：提取纯化兔腰大肌肌球蛋白,并用含有组氨酸的盐溶液透析处理,测定不同离子强度条件下蛋白溶出率、浊度以及热诱导凝胶的硬度和保水性（water holding capacity,WHC）。结果：经组氨酸处理后,在低离子强度（1 mmol/L KCl）条件下肌球蛋白的溶出率从17.2%提高到64.4%,聚集程度显著下降,热凝胶的硬度和保水性显著提高（P＜0.05）;而在生理离子强度（0.15 mol/L KCl）和高离子强度（0.6 mol/L KCl）条件下肌球蛋白的溶出率和聚集特性均未受组氨酸处理的影响,但其热凝胶硬度值显著降低（P＜0.05）;虽然在高离子强度条件下肌球蛋白热凝胶的保水性显著降低（P＜0.05）,但是在生理离子强度条件下凝胶保水性没有发生变化。结论：组氨酸处理可以显著增强低离子强度条件下肌球蛋白溶出率和其热凝胶形成能力,是低钠凝胶类肉制品生产和研发的一个新思路。     

Heat-Induced Gelation of Myofibrillar Proteins and Myosin from Fast- and Slow-Twitch Rabbit Muscles

Heat-induced gelation of myofibrillar proteins and myosin (0.6M; pH 6.0) from rabbit fast- and slow-twitch muscles was analyzed by thermal scanning rheometry. Proteins from slow-twitch muscle exhibited higher thermostability and lower gel strength than those from fast-twitch muscle. Purifying myosin from myofibrillar proteins changed heat-gelation profiles and generally increased gel rigidity at 80°C. However, the effect of some proteins on the gelation of myosin was muscle dependent. Complete elimination of actin decreased the heat-gelling ability of slow myosin and increased that of fast myosin. Also, elimination of C-protein led to a greater increase in rigidity of gels from slow myosin than from fast myosin. The heat-behavior of the different protein fractions was related to the degree and type of aggregation in the gel.     

Further Studies on the Roles of the Head and Tall Regions of the Myosin Molecule in Heat-induced Gelation

Heat induced gelation properties of the two proteolytic fragments of myosin, heavy (HMM) and light meromyosin (LMM), were studied by rigidity measurement in a band type viscometer and by a direct examination using a scanning electron microscope. A heat induced network forming ability for both LMM and HMM was found in 0.6M KCl at a pH 6.0. LMM produced gels corresponding to a reversible helix-coil transition at temperatures ranging from 40–70°C, with little evidence of aggregation as assessed from a turbidity change of the system. Contrary, HMM associated irreversibly producing a gel with increased rigidity at pH 5.0 and a salt concentration of 0.1 M. Oxidation of SH-groups appeared to be involved only in HMM and not in LMM gelation process.     

Protein Oxidation at Different Salt Concentrations Affects the Cross‐Linking and Gelation of Pork Myofibrillar Protein Catalyzed by Microbial Transglutaminase

In a fabricated then restructured meat product, protein gelation plays an essential role in producing desirable binding and fat‐immobilization properties. In the present study, myofibrillar protein (MFP) suspended in 0.15, 0.45, and 0.6 M NaCl was subjected to hydroxyl radical stress for 2 or 24 h and then treated with microbial transglutaminase (MTGase) in 0.6 M NaCl (E : S = 1 : 20) at 4 and 15 °C for 2 h. Protein cross‐linking and dynamic rheological tests were performed to assess the efficacy of MTGase for mediating the gelation of oxidized MFP. MTGase treatments affected more remarkable polymerization of myosin in oxidized MFP than in nonoxidized, especially for samples oxidized at 0.6 M NaCl. Notably, the extent of MTGase‐induced myosin cross‐linking at 15 °C in oxidized MFP improved up to 46.8%, compared to 31.6% in nonoxidized MFP. MTGase treatment at 4 °C for MFP oxidized in 0.6 M NaCl, but not MFP oxidized in 0.15 M NaCl, produced stronger gels than nonoxidized MFP (P < 0.05). The final (75 °C) storage modulus (G′) of oxidized MFP gels was significantly greater than that of nonoxidized, although the G′ of the transient peak (～44.5 °C) showed the opposite trend. Overall, oxidation at high salt concentrations significantly improved MTGase‐mediated myosin cross‐linking and MFP gelation. This might be because under this condition, MTGase had an increased accessibility to glutamine and lysine residues to effectively initiate protein–protein interactions and gel network formation.     

Heat-induced Gelation of Myosins/Subfragments from Chicken Leg and Breast Muscles at High Ionic Strength and Low pH

Heat-induced gelation was studied to examine the reason for marked differences in gel strength of myosins at 0.6M KCl and pH 5–6. When leg myosin (L-myosin) or L-myosin tail subfragment was mixed with breast myosin (B-myosin) or B-myosin tail subfragment, rigidity of the mixture gel was higher than the sum of rigidities of the component gels. Difference in strength of heat-induced gel between L- and B-myosin seemed to be caused by differences in filament-forming ability and in gel-forming ability of head and tail segments of both kinds of myosins.     

肌球蛋白-琼脂混合凝胶的特性

比较肌球蛋白、琼脂、肌球蛋白-琼脂3 种凝胶样品的质构特性、流变特性和超微结构,并研究不同pH值（pH 5、6、7）和不同离子强度（0.2、0.4、0.6）条件下肌球蛋白-琼脂混合凝胶的特性变化。结果表明：肌球蛋白-琼脂混合凝胶的硬度和弹性明显大于纯肌球蛋白凝胶或纯琼脂凝胶（P＜0.05）,且均在pH 6时获得最大值;肌球蛋白-琼脂混合凝胶的硬度和弹性随离子强度的增加而增加,在离子强度为0.6时出现最大值;在肌球蛋白溶液中加入琼脂,无论在加热过程还是降温过程中,混合凝胶的储能模量（G’）和损耗模量（G’’）均显著高于相同温度条件下的纯肌球蛋白凝胶或纯琼脂凝胶（P＜0.05）;在冷却过程中,琼脂对G’和G’’的贡献超过肌球蛋白;用扫描电子显微镜观察肌球蛋白-琼脂混合凝胶（肌球蛋白、琼脂质量浓度分别为10、4 mg/mL）的超微结构时发现,混合凝胶为相分离型凝胶,琼脂为连续相,肌球蛋白为分散相。     

Dynamic rheological measurements on heat-induced myosin gels: Effect of ionic strength,protein concentration and addition of adenosine triphosphate or pyrophosphate

Myosin solutions and suspensions have been monitored during heating at pH 6.0 by using dynamic rheological measurements. The storage modulus (G′), the loss modulus (G) and the phase angle (δ) all showed a marked dependence on ionic strength in the temperature range 25–75°C. The filamentous gels (ionic strength <0.34) displayed a temporary reduction in G′ at temperatures between 50 and 60°C, presumably due to denaturation in parts of the rod portion of the myosin molecule. In the same temperature region the concentration dependence of G′ changed by a power of 2. The loss modulus also showed a marked concentration dependence, while the phase angle varied with concentration primarily at low (<50°C) temperatures. For the final gels, heated to 75°C, only G′ indicated marked differences due to different protein concentrations and ionic strengths; all gels were almost completely elastic (δ?1°). Adenosine triphosphate was shown to have a pronounced temporary effect on the filamentous gel formed at low temperatures, i.e. on the gel with the highest concentration dependence, while pyrophosphate had no such effect. However, both adenosine triphosphate (or rather its hydrolysis product: adenosine diphosphate) and pyrophosphate appeared to have a small, lasting effect on the heat-gelling ability of myosin: the former a detrimental effect, the latter an improvement.     

Slow lowering of pH induces gel formation of myosin

It has been found that solutions of myosin (10 mg/ml) form gels at 5°C if the pH is decreased slowly, by dialysis, to a value in the region of 2.5 to 5.5. Gel strength displays strong dependence on final pH, having a maximum at about pH 4.5. Salt (KCl) concentration was found to affect gel strength positively and linearly. Differential scanning calorimetry revealed that the myosin of pH-induced gels absorbed no thermal energy when heated, implicating acid-induced denaturation as the basis of gel formation. By comparison with heat-induced gelation of myosin and from the fact that low pH is conducive to filament formation, it is suggested that filaments may also be involved in the gelation process.     

Filamentous structures of bovine myosin in diluted suspensions and gels

Turbid solutions and fine-stranded gels of myosin from bovine semi-membranous muscle were investigated by transmission and scanning electron microscopy. Evidence is given that the turbidity was caused by filament formation upon dialysis to pH 5.5 and 0.25 M KCl and to pH 4.0 and 0.6 m KCl at 4°C. The filaments formed at pH5.5 and 0.25 m KCl had a backbone with a diameter of c. 25 nm with the myosin heads located close to the filament backbone. The total width of these filaments was c.45nm. The filaments were prepared for electron microscopy by adsorption on various substrates, negative staining, or freeze drying and rotary shadowing. Variations in the preparation technique did not affect the appearance of the filaments. The filaments formed at pH 4.0 and 0.6 m KCl had a more irregular appearance, and the total filament width varied between 20 and 45 nm. Fringes of globular material surrounding the filament backbone were seen but also clusters of myosin molecules protruding further out from the backbone and from the filament ends. Comparison of heat-treated filaments in dilute solutions with strands of the gel network confirmed that the gel strands originated from filaments formed upon dialysis prior to gelation. Typical features of the network structure were junction zones formed by parallel alignments of filaments in pairs and by end-to-side interactions forming so-called Y-junctions. At pH5.5 and 0.25 m KCl these interactions resulted in a rather loose and open network structure. At pH 4.0 and 0.6 M KCl the filaments often interacted approximately at right angles, which resulted in a denser network than that observed atpH5.5 and 0.25 m KCl. The efficient network formation at pH 4.0 gave rise to spontaneous gel formation upon dialysis without any heat treatment. Additional heating did not change the character of the network, and no differences could be observed between unheated and heat-treated gels at low magnifications. At higher magnifications it could be seen that heating resulted in loss of details of the filaments at both pH values and ionic strengths. The shape of the myosin heads was lost, and the heads fused together on the filament backbone.     

Dynamic rheological measurements on heat-induced myosin gels: An evaluation of the method's suitability for the filamentous gels

Formation of bovine myosin gels (10 mg ml^?1) by heat treatment at pH 6 and an ionic strength of 0.24 M has been monitored by using the Bohlin Rheometer System in the oscillatory mode. Rheological thermograms were determined with a general repeatability of about 2% for a given suspension. A pronounced maximum and an accompanying minimum in storage modulus (G′) were found at about 50 and 55°C, respectively. The thermograms for the loss modulus (G″) and the phase angle (δ) displayed complex behaviour as well, suggesting a multitransition process. Presumably, denaturational events in parts of the molecule are responsible for the complex rheology observed. This complexity is not related to trivial wall slippage as data obtained from cells with different gap sizes were highly reproducible and consistent with other measurements. A decrease in heating rate from 2.5 to 0.1°C min^?1 had a large effect on G′; it increased from 905 to 1600 N m^?2 for gels at 75°C. The phase angle was also affected by the heating rate, especially at about 55°C. The effect of increasing the strain from 0.003 to about 0.1 was significant in two temperature regions; G′ at temperatures higher than 65°C and δ at temperatures lower than 54°C increased with increasing strain.     

Urea disc-gel electrophoresis of rabbit and bovine myosin

Numerous procedures for improving the entry of aggregates and of large myosin subunits into disc gels were investigated. Both rabbit and bovine myosin behaved essentially the same during electrophoresis in urea. Aggregates of myosin and large and small subunits were found to enter and migrate in a 2·5% acrylamide gel in 7^m urea on using a continuous buffer system. Washing with low ionic strength buffer (0·04m KCl prior to the addition of urea increased the proportion of migrating myosin. Storage of myosin aggregates in urea for periods up to three weeks increased the yield of electrophoretically migrating protein. The possible significance of these findings with regard to the properties of meat is discussed.     

Effect of microbial transglutaminase and setting condition on gel properties of blend fish protein isolate recovered by alkaline solubilisation/isoelectric precipitation

The effect of microbial transglutaminase (M-TGase) (0–0.6 units g⁻¹ sample) and setting condition (25 °C/180 min, 30 °C/120 min, 35 °C/60 min and 40 °C/30 min) on gel properties of blend protein isolate of gutted kilka and silver carp was studied. The protein isolate provided a good substrate for M-TGase activity so that a low amount of M-TGase (0.2 unit g⁻¹ sample) substantially improved textural properties and water holding capacity (WHC) of the gels. Breaking force of the gels was positively affected by M-TGase up to 0.6 unit g⁻¹ sample, but it negatively affected their WHC. Prior setting at 25–35 °C increased the breaking force of proteins compared to directly heated gel, resulting in maximum breaking force at 35 °C/60 min. However, the setting at 40 °C/30 min caused proteolysis, which was reflected in higher amounts of TCA-soluble peptides and gel weakening. Denser microstructure and higher myosin heavy chain polymerisation observed in the gels which experienced the setting was well correlated with improvement in textural properties.     

Differences in properties of myofibrillar proteins from bovine semitendinosus muscle after hydrostatic pressure or heat treatment

Hydrostatic pressure (HP) and heat treatments of myofibrillar proteins have both been shown to induce protein denaturation, but different gel formation properties result from these treatments. To characterise differences in the properties of proteins resulting from HP or heat treatment, Ca‐ and Mg‐ATPase activities (ATP, adenosine triphosphate) and protein solubility in 0.1 and 0.6 mol L^?1 KCl buffers (pH 7) were evaluated in this study. The inactivation rate of Ca‐ATPase of myofibrillar proteins (Mf) induced by HP was slower than that of Mg‐ATPase at each of the tested pressures. However, the inactivation rate of Ca‐ATPase induced by heating was faster than that of Mg‐ATPase at each of the tested temperatures. The level of soluble proteins in Mf suspension induced by HP in 0.1 mol L^?1 KCl buffer increased with increasing pressure up to 400 MPa and then decreased slightly at 500 MPa. However, the level of soluble proteins in Mf suspension induced by heat treatment in 0.1 mol L^?1 KCl buffer increased with increasing temperature up to 55°C. According to the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the levels of soluble myosin heavy chain and actin in Mf suspension induced by HP in 0.6 mol L^?1 KCl buffer decreased simultaneously at pressures higher than 300 MPa. The level of soluble MHC in 0.6 mol L^?1 KCl buffer decreased gradually with increasing temperature, but there were no changes in the level of soluble actin in 0.6 mol L^?1 KCl buffer with increasing temperature up to 50°C. These results showed that the mechanism of HP‐induced protein denaturation was different from the mechanism underlying heat‐induced protein denaturation. Copyright © 2006 Society of Chemical Industry     

Factors Influencing Gel Formation by Myofibrillar Proteins in Muscle Foods

Abstract: Considerable research has been done to better understand the basis for gel formation by myofibrillar proteins (MPs) in effort to manufacture acceptable processed meats with lower cost and more desirable nutritional characteristics. Results from research available indicate that there is no substitute for the myofibrillar protein myosin in gel formation by proteins from a wide variety of animal and fish species. This report consolidates information on determinants of protein gel formation, examining types of muscles and fibers, the species influence, and interactions of the MPs actin and myosin with each other and with fat, gelatin, starch, hydrocolloids, some protein soy, whey, and nonprotein additives such as phosphates and acidifiers, and the influences of pH, ionic strength, rates of heating, and its absence, protein oxidation, as well as the use of transglutaminase and high hydrostatic pressure. It is of interest that myosin alone will form acceptable gels. Gel formation by MPs is optimized at pH 6, an ionic strength of 0.6 M, and at 60 to 70 °C. The observations that collagen‐derived gelatin can reduce the rubbery texture of low‐fat products and that solubilization of MPs is not always essential for gel formation, and the observation that good gels can be formed in the absence of salt, are exciting developments that should be considered as pressure mounts to continue to reduce fat and salt in the diet.     

Effect of Heating Rate on Thermally Formed Myosin, Fibrinogen and Albumin Gels

Myosin, fibrinogen and albumin gels were formed by heating in pH 6.0 phosphate buffer at three heating rates. Turbidity (A_660nm) and solubility were monitored along with gel strength, as measured with an annular pump. Myosin and fibrinogen suspensions became turbid and solubility decreased as temperatures preceding the development of gel strength. Linearly increasing heating rates of 12°C/hr and 50°C/hr produced the strongest myosin and fibrinogen gels at 70°C, whereas albumin gels formed at 95°C by heating at 12°C/hr or constant heating for 20 min did not differ in strength.     

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.