Thermal scanning rheology of myofibrillar proteins from muscles of defined fibre type

Emulsion meat products are made with little regard to rheological properties of different muscles. Here the rheology of gels made from three classes of muscle defined by myosin type (fast twitch, slow twitch and heart) are compared. Myofibrillar fractions were prepared from representative bovine muscles-cutaneus trunci, masseter and heart-by a procedure that removed connective tissue, fat and sarcoplasmic proteins. Complicating effects of nongelling agents were thus minimized. Fractions were mixed with NaCl and pyrophosphate at concentrations typical of those used in processed meats. Rigidity and elasticity of the gelling mixtures were monitored during heating from 10 to 84°C over a range of pH values. Several indices of gelation showed that masseter and heart (slow muscle group) gelations were similar to each other but distinct from cutaneus trunci (fast) gelation. Gelation temperature was 10°C lower for fast than slow, perhaps explainable by one of two hypotheses: differential salt extraction or lower thermal stability of fast myosin rod. Cutaneus trunci gels were also more rigid at all pH values. Slow group gels suffered from proteolysis, which was combated with cathepsin inhibitors. For both fast and slow groups, rigidity increased as pH decreased, analogous to results reported for purified myosin in dilute solution. Texture also changed. Below pH 5·7 slow group gels were brittle and granular, with low water-holding capacity. Above that pH, gels were elastic and smooth. The equivalent point for cutaneus trunci gels was pH 5·5. Around pH 7, pyrophosphate had a fluidizing effect at pregelation temperatures, but elasticity data indicated that this ion plays no part in final gelation.     

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.     

Influence of pH on rheological properties of porcine myofibrillar protein during heat induced gelation

Texture of meat products is dependent on the gelation characteristics of myofibrillar protein. Gaining an understanding of the gelation mechanism of meat gel systems is beneficial for the development of processed meat products as well as maintaining quality in meat products. The aim of this study was to investigate the impact of pH (5.6, 6.0, 6.5, and 7.0) on heat-induced gelation properties of myofibrillar proteins from porcine semimembranosus muscle. Dynamic rheological measurements were taken as the temperature increased by 1°C/min from 20 to 85°C, followed by a holding phase at 85°C for 3min to ensure complete gelation and during a subsequent cooling where the temperature dropped from 85 to 5°C at a rate of 5°C/min. Storage modulus (G') 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 subsequent depression in the data. This depression in the curve was more pronounced with increasing pH. Results indicate protein denaturation and gel formation are pH dependent. Furthermore, rate of gelation appears to influence water-holding capacity.     

Myofibrillar protein from different muscle fiber types: Implications of biochemical and functional properties in meat processing∗

Texture, moisture retention, and tenderness of processed muscle foods are influenced by the functionality of myofibrillar protein. Recent studies have revealed large variations in processing quality between red and white muscle groups that can be attributed to differences in the functional properties of myofibrillar protein associated with the type of fiber. Myofibrillar proteins from fast‐ and slow‐twitch fibers exhibit different biochemical and rheological characteristics and form gels with distinctly different viscoelastic properties and microstructures. The existence and wide distribution of the numerous myosin isoforms in different muscle and fiber types contribute to the various functional behaviors of myofibrillar protein. The different sensitivities of fast and slow myofibrillar proteins to pH, ionic environment, temperature, and other external factors have been well documented and illustrate the importance of adjusting meat processing conditions, according to fiber type profile to achieve maximum protein functionalities, and hence, uniform quality of the final muscle foods.     

鸡胸肉肌原纤维蛋白的提取及凝胶特性的研究

研究从鸡胸肉中提取肌原纤维蛋白，探讨不同pH值对肌原纤维蛋白含量的影响。研究结果：在pH7．0时，蛋白含量最大为69．74％，通过SDS—PAGE凝胶电泳分析表明，其主要成分为肌球蛋白、肌动蛋白和肌动球蛋白及其他一些小分子肌原纤维蛋白碎片；在离子强度0．6、pH7．0时提取的肌原纤维蛋白所制备的凝胶的保水性、硬度、胶粘性最大。     

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.     

养殖暹罗鳄肉肌原纤维蛋白的理化性质

筛选养殖暹罗鳄肌原纤维蛋白含量最高部位,提取并分析离子强度、pH值和温度对肌原纤维蛋白溶液的溶解性、乳化性和热诱导凝胶特性的影响。结果表明：暹罗鳄尾部肌原纤维蛋白所占比例最高（（7.95±0.12） g/100 g,以湿质量计）,暹罗鳄尾肉中肌原纤维蛋白主要为肌球蛋白重链、副肌球蛋白、肌动蛋白和原肌球蛋白。在低离子强度条件下,肌原纤维蛋白的溶解度和乳化性较低,但有良好的凝胶特性,随着离子强度的升高,肌原纤维蛋白的溶解度和乳化性升高,凝胶特性则呈现下降趋势。随着pH值升高,肌原纤维蛋白溶解度呈现先迅速下降后升高的趋势,乳化性和凝胶特性则呈现持续缓慢下降的趋势,其中溶解度和保水性在pH 5.5达到最低点。随着热变温度升高,其凝胶特性显著增加,保水性先下降后略有升高,在低温（40 ℃）下有较好的保水性,在80 ℃保水性升高至又一峰值随后下降。结论：肌原纤维蛋白在NaCl浓度0.2 mol/L进行调配,并在80 ℃条件下加热处理,暹罗鳄肉类产品将具有较好的质构特性及保水性。     

Influence of muscle type on rheological properties of porcine myofibrillar protein during heat-induced gelation

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.     

Ultrastructural changes of myofibrillar protein gelled with 1,5-glucono-δ-lactone at chilled temperatures

Changes in the ultrastructure of myofibrillar protein as a result of 1,5-glucono-δ-lactone-induced gelation at chilled temperatures were investigated using transmission electron microscopy. The myofibril structure appeared to have completely disintegrated at pH 4.0 resulting in a granular, amorphous appearance. It was suggested that as the pH was lowered to about 4.5, partial extraction of the A-band proteins occurred. A composite system of a myosin gel network reinforced by the residual myofibrillar structure was proposed to have formed. As the pH was lowered further, complete depolymerisation of actomyosin was suggested to have resulted in the formation of a predominantly myosin gel. The inclusion of sodium chloride resulted in swelling of the myofibrillar protein and retention of the myofibrillar structure to pH 3.8.     

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.     

Gelling properties of myofibrillar protein from abalone (Haliotis Discus Hannai Ino) muscle

Texture of muscle food is dependent on the gelation properties of myofibrillar protein. Defining the performance of myofibrillar protein during gelation is beneficial for maintaining quality and developing muscle food. The myofibrillar protein from abalone muscle (AMP) was extracted and the gel-forming ability was investigated. The lowest protein solubility of AMP in distilled water was obtained at pH 5.5, but shifted to a lower pH value by ionic strength. The breaking force and deformation of AMP gel formed at pH 7.0 were 48.00 g and 27.70 mm, respectively, but they could not be detected at pH 5.0 and pH 5.5 due to the low gel-forming ability. Scanning electron microscopy data showed that a coarse and disorder gel containing clusters of agglomerates was observed at pH 5.0 and pH 5.5, but gradual compact network structure was observed in the gel formed at increasing pH. The glass transition temperature (Tg) of AMP gel formed at pH 5.5 was higher than that of AMP, and the Tg of AMP gel was increased with increasing the pH of gel-forming solution. It was found that the unfolding of tertiary structure of AMP at pH 7.0 was easier than that at pH 5.5 through the result of infrared spectra. We therefore conclude that both the solubility and gel-forming ability of AMP are pH dependent.     

Studies on fish and pork paste gelation by dynamic rheology and circular dichroism

ABSTRACT:  The muscle paste of fish, pork, and their mixtures were prepared to study the gelling characteristics by dynamic rheological measurement. The gelation mechanisms of muscle paste were also investigated by circular dichroism. Gel formation of fish paste occurred in 2 steps of 5 to 35 and 51 to 90 °C respectively, while pork paste mainly in 1 step of 49 to 72 °C. Gel formation was relative to the α-helix unfolding of myosin, which responded the melting temperatures of 40 and 50 °C for fish myosin and 50 and 60 °C for pork myosin, respectively. Alpha-helix unfolding of myosin was beneficial for gel formation. During gel formation, G ' of muscle paste was linearly related to α-helical content of myosin. The interactions of fish and pork proteins at high temperature (>35 °C) could change the gel forming characteristics of muscle paste. Mixed paste exhibited a similar gelation pattern to individual fish paste with 2 visible increases in G '. Addition of pork could suppress the breakdown of fish gel structure at approximately 50 °C. Mixing pork and silver carp in a certain ratio could improve the gel properties of silver carp products.     

Effect of frozen storage on protein denaturation in bovine muscle 1. Myofibrillar ATPase activity and differential scanning calorimetric studies

The effect of frozen storage on myofibrillar ATPase activity and thermal transitions in bovine muscle was investigated. Myofibrillar ATPase activity and total enthalpy of denaturation (ΔH) decreased with time of storage. The rate of decrease was lower at −20°C than at −5°C or −10°C. Differences in behaviour during storage of muscle after fast or slow freezing could be attributed to differences in ice recrystallization. The observed decreases in area of the first peak seen in the thermograms and Ca²⁺-myo-fibrillar ATPase activity show that the myosin head denaturated progressively during storage. The myosin tail also denaturated during storage but the thin filament remained unaltered. Kinetic analysis suggested that the denaturation of the myofibrillar proteins took place through two consecutive first order reactions; an initial rapid reaction followed by a slower one.     

Influence of salt and pyrophosphate on bovine fast and slow myosin S1 dissociation from actin

The kinetics of myosin dissociation from actin was investigated and also the impact of salt, MgPPi, and myosin heavy chain isoform on myosin subfragment 1 (S1) dissociation from actin using purified proteins and fluorescence spectroscopy. Both NaCl and MgPPi increased myosin S1 dissociation rate. When salt concentrations increased from 0.1 to 1.0 M, the dissociation rate of S1 from bovine masseter (slow) and cutaneous trunci (fast) muscle increased 38 and 78-fold, respectively. MgPPi had an even greater effect on S1 dissociation from actin. With the addition of MgPPi to the mixture of pyrene actin and S1, the fluorescence increased about 85% within the dead time of the mixing approach. Unlike salt, MgPPi had no apparent difference in its ability to dissociate slow or fast S1 isoforms from actin. The results reveal that salt and MgPPi increase myosin extraction and functionality in meat by weakening the actomyosin interaction and that some of the difference in the functionality of red and white muscle may be related to actomyosin dissociation.     

Contribution of sarcoplasmic proteins to myofibrillar proteins gelation

Surimi, a refined protein extract, is produced by solubilizing myofibrillar proteins during the comminuting and salting stages of manufacturing. The resulting paste gels on heating to produce kamaboko or a range of analog shellfish such as crab claw, filament sticks, fish mushroom, and so on. The myosin molecule is the major myofibrillar protein in gelation. It is believed that washing steps during the traditional surimi process play an important role in enhancing the gel properties of the resultant kamaboko by removing water-soluble (sarcoplasmic, Sp-P) proteins. By contrast, some researchers claim that retaining Sp-P or adding it into the surimi gel network not only does not interfere with the action of myofibrillar proteins during the sol-gel transition step but also improves the gel characteristics of the resultant kamaboko. It seems that retention of Sp-P or their addition into raw surimi does enhance the textural properties of kamaboko gel perhaps by functioning as a proteinase inhibitor, particularly against trypsin and trypsin-like proteinases but this depends on the type of applied surimi process. Among different types of Sp-P, it has been claimed that some proteins such as endogenous transglutaminase (TGase) play a more important role than other Sp-P in bond formation, by catalyzing the cross-linking of myosin heavy chain (MHC) molecules during low-temperature setting of surimi, resulting a more elastic kamaboko gel.     

SDS-PAGE of salted anchovies (Engraulis encrasicholus L) during the ripening process

 Changes in the muscle proteins during the ripening of salted anchovies have been examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis. The chemical changes occurring in the muscle proteins of anchovy during the ripening process were related to the degradation of the myofibrillar structure of the muscle. In spite of a considerable protein degradation the ripened salted anchovy maintained its structure and was easily cut into fillets. Hydrolysis of muscle proteins was significant during the first 6 weeks. Proteins of molecular weight higher than 35 kDa were more likely to be hydrolysed. Myosin heavy chains were the most sensitive myofibrillar protein. α-Actinin, actin and tropomyosin were more resistant to enzymatic degradation. Due to protein build-up of peptides resulting from the breakdown of the higher molecular proteins, it was difficult to measure changes in the myosin light chains and troponin sub-units. Received: 28 December 1999 / Revised version: 9 March 2000     

Characterization of Actin and Myosin Extracts Obtained Using Two Improved Laboratory Methods

Actin and myosin are the most important contractile proteins in the muscle. Numerous authors developed different methods for isolation and purification of myofibrillar proteins. The aim of this study was to obtain suitable actin and myosin extracts from post-rigor porcine muscle to be used in further studies, such as testing the proteolytic activity of microbial cultures. Actin and myosin were quantified in the extracts using spectrophotometric methods, yielding 0.17 and 0.22 mg/mL, respectively. The isolation methods proposed in this study provided low contaminated extracts, showing purity percentages of 74.36 % in the case of actin and 65.43 % for myosin, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Subsequently, these extracts were sterilized through a 0.22-μm polyvinylidene difluoride filter with no significant retention observed. In conclusion, the procedures described in this work for actin and myosin isolation can be recommended for microbiological studies requiring sterilized pure muscle proteins extracts.     

超声波辅助酸/碱溶解等电点沉淀法提取鸡架分离蛋白的组成及其凝胶特性

利用高强度超声（high intensity ultrasound,HIU）辅助酸/碱溶解等电点沉淀（isoelectric solubilization/precipitation,ISP）法提取鸡架分离蛋白（protein isolate,PI）,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳研究PI组成,并分析PI凝胶特性。结果表明：PI主要由肌原纤维蛋白组成;酸溶解会导致肌球蛋白重链降解,HIU可减弱降解程度;碱溶PI凝胶的硬度、蒸煮损失率、离心损失率与对照（鸡胸肉糜凝胶,后同）无显著差异,显著高于酸溶PI凝胶（P＜0.05）;HIU显著提高了酸溶PI凝胶的硬度和弹性（P＜0.05）,降低了蒸煮损失率及离心损失率（P＜0.05）;PI凝胶白度均显著低于对照（P＜0.05）。碱溶PI凝胶对水分子的结合力和束缚力均优于酸溶PI凝胶;碱溶PI凝胶与对照凝胶均具有均匀致密微观结构,而酸溶PI凝胶微观结构明显粗糙、不均匀。结论：HIU辅助碱溶ISP法可高效提取鸡架中肌原纤维蛋白并保持其凝胶特性,有助于鸡架增值利用。     

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.     

Changes in phosphorylation of myofibrillar proteins during postmortem development of porcine muscle

A gel-based phosphoproteomic study was performed to investigate the postmortem (PM) changes in protein phosphorylation of the myofibrillar proteins in three groups of pigs with different pH decline rates, from PM 1 to 24 h. The global phosphorylation level in the group with a fast pH decline rate was higher than that in the slow and intermediate groups at early PM time, but became the lowest at 24 h. The protein phosphorylation level of seven individual protein bands was only significantly (p < 0.05) affected by PM time, and two protein bands were subjected to a synergy effect between PM time and pH decline rate. A total of 35 non-redundant highly abundant proteins were identified from 19 protein bands; most of the identified proteins were sarcomeric function-related proteins. Myosin-binding protein C, troponin T, tropomyosin and myosin regulatory light chain 2 were identified in the highly phosphorylated protein bands with the highest scores. The results indicate that the phosphorylation pattern of myofibrillar proteins in PM muscle is mainly changed with PM time, but only to a minor extent influenced by the rate of pH decline, suggesting that the phosphorylation of myofibrillar proteins may be related to the meat rigor mortis and quality development.