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.     

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.     

Myosins from red and white bovine muscles: Differences measured by turbidimetric,calorimetric and rheological techniques

The aim of this study was to elucidate the functional performance of the most abundant protein component in meat, ie myosin, which is recognised as important for binding in meat products. As several genetic variants of skeletal myosin exist, myosins from two bovine muscles of different fibre type composition, M masseter and M cutaneus trunci were compared with respect to filament forming properties and denaturation characteristics. The principal methods used were turbidimetric measurements, which were used to monitor filament formation, calorimetry and rheology. The myosin systems were examined at two different salt levels (0.2 and 0.6 M NaCl) and at pH 5.5–7.0. The method of preparing myosin suspensions/solutions was also examined. Differences in the filament-forming process for the two myosins were detected. Measurements of turbidity revealed that at conditions of low pH and low ionic strength white myosin had a higher ultimate turbidity compared with red myosin. Early in the transition from low to high turbidity, red myosin had a higher turbidity than white myosin corresponding to reduced solubility. The turbidity increased with time of storing the myosin suspensions/solutions. This change was attributed to formation of filaments and further association of filaments. White myosin had a smaller apparent enthalpy of denaturation than red myosin. The calorimetric measurements recorded in 0.2 M NaCl suggested that the head and the rod of white myosin were less stable than the corresponding parts of red myosin. However, exceptions to this rule were found at pH 6.0. In 0.6 M NaCl the identification of the transitions for red myosin was more difficult. The method of preparing myosin suspensions affected calorimetric and rheological measurements. In 0.6 M NaCl and pH 6.0 calorimetric thermograms of both myosins were affected by the preparation method. At pH 5.5 this change was interpreted as caused by denaturation promoted by the dilution/rapid titration technique compared with dialysing the systems to pH 5.5 from pH 7. Differences in the filaments formed might, however, also contribute to the variations seen in the calorimetric ther-mograms. The gelling properties of white myosin were most sensitive to the preparation method used. Systems prepared by dialysis gave stronger heat-induced gels than those prepared by ‘dilution’. White myosin always produced stronger gels than red myosin independent of the preparation technique. The rheological properties (at 80°C) of red myosin were less affected by the preparation method than were those of white myosin. At lower temperatures, however, there was more variation in the shapes of the rheological thermograms (? versus temperature) for red myosin than in the corresponding thermograms of white myosin.     

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.     

Thermal gelation of brown trout myofibrils from white and red muscles: effect of pH and ionic strength

The effects of pH and ionic strength on the thermal gelation of brown trout myofibrils from white and red muscles were analysed by thermal scanning rheometry. The highest gelation ability was obtained at low pH (around 5.6) whatever the ionic strength. No effect of ionic strength was observed at pH 5.6; however, at pH 6.0, lowering the salt (KCl) concentration to 0.3 M or less improved the characteristics of the gels formed. The effects of pH and ionic strength on myofibrils from both muscle types appeared to be similar, but red muscle proteins were less sensitive to changes in their physicochemical environment. Consequently, the differences between muscle types appeared to be dependent on pH and ionic strength. Solubility measurements revealed large differences between muscle types and between different pH values. Ultrastructural observations confirmed that different kinds of gels were formed depending on the physicochemical conditions and muscle type origin. © 2002 Society of Chemical Industry     

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.     

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。     

咸味肽（鸟胺牛磺酸）添加量对低钠肉糜热凝胶特性的影响

研究咸味肽（鸟胺牛磺酸）添加量对低钠肉糜热凝胶特性的影响。采用单因素试验研究pH值（6.0～7.2）对50%咸味肽替代后的肉糜热凝胶质构特性和保水性能的影响。在单因素试验的基础上,利用完全随机排列试验,研究咸味肽替代比例（30%、40%、50%）和pH值（6.0、6.5、7.0）对肉糜热凝胶质构特性和保水性能的影响。结果表明：在2.5%的离子强度和50%咸味肽替代比例下,pH值的变化（6.0～7.2）对肉糜热凝胶的质构有显著影响,对保水性影响不显著。咸味肽使用会对肉糜的质构及保水性质产生不利的影响,但调节pH值至偏中性条件在一定程度上可降低咸味肽的不利作用。     

Thermal Gelation of Brown Trout Myofibrils: Effect of Muscle Type, Heating Rate and Protein Concentration

The thermal gelation properties of myofibril solutions (KCl 0.6M; pH 6.0) from reared brown trout white and red muscles were analyzed by thermal scanning rheometry. With a heating rate of 1°C/min, red muscle myofibrils exhibited a lower gelation capacity than white muscle myofibrils at low temperatures. No difference was observed above 60°C where solid gels were formed from the two myofibril types. Increasing protein concentration or reducing heating rate increased the values of the rheological parameters at 80°C for the two muscle type myofibrils. With a low heating rate (0.25°C/min), white muscle myofibrils formed stronger gels whatever the temperature.     

Heat-induced Gelation of Chicken Gizzard Myosin

Chicken gizzard myosin solution formed a gel when heated above 40°C. The rigidity of the gel was constant above 65°C. Maximum pH for gel formation was 5.9 at 0.6M and 5.7 at 0.15M KCl. Higher rigidity of the myosin gel was observed at low ionic strength than at high ionic strength. Rigidities of myosin at 0.6M KCl increased by (mg/mL)^2.5 and at 0.15M (mg/mL)^{1, 4} myosin concentration. The strength of gizzard myosin gels was comparable to that of myosin gels from chicken breast muscle under similar conditions.     

影响猪血浆蛋白热诱导凝胶质构特性及持水性因素的研究

本实验研究在一定的加热条件下猪血浆蛋白质量浓度、加热温度、加热时间、离子种类、离子强度和pH 值对猪血浆蛋白热诱导凝胶的质构、持水性等性质的影响。利用质构仪测定猪血浆蛋白热诱导凝胶的硬度和黏附性,利用离心的方法测定凝胶的持水性。结果表明,在80℃下加热45min,猪血浆蛋白质量浓度超过6g/100mL可以形成凝胶,并且随蛋白质量浓度的增大,凝胶强度和持水性也增大;凝胶强度随pH 值(3～9)增加而增大,pH5 时凝胶的持水性最小,pH3 时最大;NaCl 浓度0.2mol/L,CaCl2 浓度0.6mol/L 时,凝胶硬度最大。实验得出,猪血浆蛋白热诱导凝胶的质构特性及持水性受许多因素影响,在实际生产中应该控制加热条件,以获得高质量的凝胶。     

Protein extractability and thermal gel formability of myofibrils isolated from skeletal and cardiac muscles at different post-mortem periods

The effects of the post-mortem ageing period on the extractability of myofibrillar proteins from pork cardiac and rabbit skeletal muscles under various conditions of pH and ionic strength were studied with particular reference to the changes in the solubility of individual myofibrillar proteins and their denaturation characteristics. The ultimate influence of these changes on the heat-induced gel forming ability of myofibrils isolated from cardiac and skeletal muscles at different post-mortem stages was also investigated. Results showed that pork cardiac myofibrils always exhibited lower solubility than those from rabbit skeletal muscles under identical conditions of pH, ionic strength and temperature. SDS-PAGE profiles indicated several quantitative differences in the relative proportion of individual protein species present in cardiac and skeletal myofibrils. The solubility of various proteins present in myofibrils was also affected differently on heating in 0-1 and 0-6 _M NaCl solution at various pH values. Thermal denaturation of cardiac myofibrils occurred at about 10°C higher than that of skeletal myofibrils as revealed by differential scanning calorimetry. Cardiac myofibrils formed much weaker heat-induced gels than those produced by skeletal myofibrils under identical conditions of temperature, pH, ionic strength and protein content.     

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.     

The effect of pH on gel structures produced using protein–polysaccharide phase separation and network inversion

Forming heat-induced gels through combined effects of micro-phase separation of whey protein isolate (WPI; 5%, w/v, 100 mm NaCl) by pH change (5.5, 6.0, and 6.5), and addition of κ-carrageenan (0–0.3%, w/w), were evaluated. The microstructure of WPI gels was homogeneous at pH 6.0 and 6.5 and micro-phase separated at pH 5.5. Addition of 0.075% κ-carrageenan to WPI solutions caused the microstructure of the gel to switch from homogeneous (pH 6.0 and 6.5) to micro-phase separated; and higher concentrations led to inversion of the continuous network from protein to κ-carrageenan. Protein solutions containing 0.075% (w/w) κ-carrageenan produced gels with increased storage modulus (G′) at pH 6.5 and decreased G′ at pH 5.5. All gels containing 0.3% (w/w) κ-carrageenan had κ-carrageenan-continuous networks. It was shown that microstructural and rheological changes were different in WPI and κ-carrageenan mixed gels when micro-phase separation was caused by pH rather than ionic strength.     

Heat-induced gelation of myosin in a low ionic strength solution containing L-histidine

Binding properties are important for meat products and are substantially derived from the heat-induced gelation of myosin. We have shown that myosin is solubilized in a low ionic strength solution containing l-histidine. To clarify its processing characteristics, we investigated properties and structures of heat-induced gels of myosin solubilized in a low ionic strength solution containing l-histidine. Myosin in a low ionic strength solution formed transparent gels at 40-50 °C, while myosin in a high ionic strength solution formed opaque gels at 60-70 °C. The gel of myosin in a low ionic strength solution with l-histidine showed a fine network consisting of thin strands and its viscosity was lower than that of myosin in a high ionic strength solution at 40-50 °C. The rheological properties of heat-induced gels of myosin at low ionic strength are different from those at high ionic strength. This difference might be caused by structural changes in the rod region of myosin in a low ionic strength solution containing l-histidine.     

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.     

Effect of feed texture, meal frequency and pre-slaughter fasting on carcass and meat quality, and urinary cortisol in pigs

The gelation characteristics of myofibrillar proteins are indicative of meat product texture. Defining the performance of myofibrillar proteins during gelation is beneficial in maintaining quality and developing processed meat products and processes. This study investigates the impact of pH on viscoelastic properties of porcine myofibrillar proteins prepared from different muscles (semimembranosus (SM), longissimus dorsi (LD) and psoas major (PM)) during heat-induced gelation. Dynamic rheological properties were measured while heating at 1 °C/min from 20 to 85 °C, followed by a holding phase at 85 °C for 3 min and a cooling phase from 85 to 5 °C at a rate of 5 °C/min. Storage modulus (G′, the elastic response of the gelling material) increased as gel formation occurred, but decreased after reaching the temperature of myosin denaturation (52 °C) until approximately 60 °C when the gel strength increased again. This resulted in a peak and depression in the thermogram. Following 60 °C, the treatments maintained observed trends in gel strength, showing SM myofibrils produced the strongest gels. Myofibrillar protein from SM and PM formed stronger gels at pH 6.0 than at pH 6.5. Differences may be attributed to subtle variations in their protein profile related to muscle type or postmortem metabolism. Significant correlations were determined between G′ at 57, 72, 85 and 5 °C, indicating that changes affecting gel strength took effect prior to 57 °C. Muscle type was found to influence water-holding capacity to a greater degree than pH.     

Protein Components Precipitated from Egg White by Removal of Salt

A precipitate that formed in egg white at low ionic strength affected the transparency of heat-induced gels prepared at pH 2-4 and low ionic strength. The precipitate solubilized with SDS solution containing 2-mercaptoethanol and urea included major proteins of the egg white, as shown by SDS-polyacrylamide gel electrophoresis. The bands of ovalbumin, conalbumin and other protein were smaller when the precipitate was washed with water, but lysozyme and ovomacroglobulin probably remained in the precipitate fraction. It seemed that ovalbumin, conalbumin, and other proteins co-precipitated with these two kinds of proteins. Lysozyme added to the supernatant of egg white after dialysis, did not appear to produce turbidity upon heating at pH 2.5, but whole egg white was converted to a turbid gel upon being heated at this pH.     

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.