Species dependence of fish myosin stability to heat and frozen storage

The thermal stabilities of the proteins of a range of fish muscles of different habitat temperatures were determined by differential scanning calorimetry before and after frozen storage at 20°C.
In whole muscle a clear relationship was seen between habitat temperature and the thermal denaturation of myosin, which persisted when isolated myosins were analysed under conditions close to physiological pH and ionic strength. the ionic environment of the myosin molecules in the whole tissue will, however, not be exactly the same as in the myosin solution.
No significant correlations were seen between habitat temperature, ultimate pH and other analytical parameters.
After frozen storage, the myosin transitions in red snapper, a warm water species, was markedly changed in whole muscle but not in isolated myosin, suggesting the post mortem development of an interaction with other muscle proteins. In contrast, in cod, a cold water species, changes in myosin transitions were very similar, both in whole muscle and isolated myosin.
The implications of species differences in the 'melting' of myosin domains and changes in textural quality during frozen storage are discussed briefly.     

Thermal stability of fish myofibrils: a differential scanning calorimetric study

The variation in myofibrillar protein thermostability was compared for various fish species, using differential scanning calorimetry. The tropical fish, catfish ( Clarius gariepinus ), carp ( Cyprinus carpio ), Nile perch ( Lates niloticus ), red snapper ( Lutianus sebae ), red mullet ( Parpeneus barberinus ), sea bream ( Gymnocranius rivalatus ), and cold-water reared trout ( Salmo gairdneri ) and cod ( Gadus morhua ) were analysed. Onset temperature of myofibrillar protein denaturation occurred at up to 11°C higher for tropical species (43.5°C, catfish), than cod (32.6°C) at pH 7 and low ionic strength (I). As pH (6.0-8.0) and I (0.05-1.00) were increased, thermal denaturation temperatures of myosins from tropical, but not cold-water, species decreased. Enthalpies of myofibrillar denaturation decreased for all species with increasing pH and I. Only one thermal transition was detected for myosin at pH 6 and low I, increasing to three as pH and I were increased. Changes in thermal characteristics of myosin subunits over iced and frozen storage suggest more rapid deterioration in cold-water than in tropical fish. The differences in myofibrillar stability of fish from different habitat temperatures have implications for the processing and storage of tropical fish.     

The effect of brine composition and pH on the yield and nature of water-soluble proteins extractable from brined muscle of cod (Gadus morhua)

Fillets of Atlantic cod (Gadus morhua) were held for 36 h in brines of pH 6.5 and 8.5, respectively, containing various combinations of NaCl, KCl, MgCl₂ and CaCl₂. Proximate analyses and chloride content were determined. Soluble muscle protein extracted in distilled water and protein released into the brine following salting was determined by SDS–PAGE. The soluble fraction was not affected by the presence of divalent salts or KCl in the brines, but it was affected by the initial pH. Both actin and myosin heavy chain (MHC) were released in pH 6.5 brines after immersion of the fish. On the other hand, an initial pH of 8.5 did not favour the release of these proteins. There was a smaller variety of remaining soluble proteins in salted muscle with an initial pH of 6.5, than with 8.5. However, this effect of the lower pH was offset to the extent that the ionic strength of the brine was higher.     

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.     

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     

鳕鱼和鲅鱼鱼肉蛋白酶解产物功能特性及抗氧化性

采用风味蛋白酶对鳕鱼和鲅鱼鱼肉进行酶解,研究水解度、pH值及酶解时间对酶解产物功能特性和抗氧化活性的影响.结果表明:随着酶解时间延长,鳕鱼和鲅鱼鱼肉酶解产物的水解度、亚铁离子螯合力逐渐增加,DPPH自由基清除能力逐渐下降;不同pH值下,鳕鱼和鲅鱼鱼肉蛋白酶解产物均具有良好的溶解性和热稳定性;鲅鱼酶解产物的水解度、溶解性、热稳定性和亚铁离子螯合力显著高于鳕鱼酶解产物.     

Differential Scanning Calorimetry of Fish Muscle: The Effect of Processing and Species Variation

Differential Scanning Calorimetry (DSC) has been used to study the thermal properties of fish muscle proteins and to measure the extent of their denaturation under various processing conditions. Fish myosin was susceptible to denaturation by frozen storage and dehydration. Denaturation of certain fish proteins was partially reversible. Although fish myosin was very unstable, its thermal stability was found to increase in species adapted, to higher environmental temperatures.     

Activity of Endogenous Muscle Proteases from 4 Australian Underutilized Fish Species as Affected by Ionic Strength,pH, and Temperature

Storage conditions may influence the hydrolytic activity of endogenous muscle enzymes postmortem, rate of autolysis of myofibrillar proteins, and biological properties of hydrolyzed end products. This study investigated the effect of ionic strength, pH, and temperature on the activity of endogenous calpain‐like, cathepsins B and B+L measured in crude extract obtained from deepwater flathead, silver warehou, ribaldo, and ribbonfish muscles. Activity of calpain‐like enzymes in 3 examined species was significantly higher at pH 6.5 than pH 6.0 or 5.5. Raising the reaction temperature increased (P < 0.05) calpain‐like activity in ribaldo. Endogenous activity of cathepsin B in ribbonfish and silver warehou declined significantly with increasing ionic strength at pH 6.5 to 6.0. The obtained results will further expand our understanding of the impact that postmortem storage conditions have on the activity of endogenous fish proteases with respect to quality and bioactivity of fish proteins and potentially diversify utilization of underutilized fish species.     

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.     

原子力显微镜研究L-半胱氨酸对鲢肌球蛋白溶液热聚集行为的影响

利用原子力显微镜技术分析L-半胱氨酸（L-cysteine,L-Cys）对鲢肌球蛋白热聚集行为的影响。在肌球蛋白溶液中添加5?mmol/L（pH?7.0）的L-Cys溶液,分别进行未加热（25?℃、30?min）、一段式加热（90?℃、30?min）、二段式加热（40?℃、60?min＋90?℃、30?min）处理,分别测定溶解度、表面疏水性、聚集行为表面形貌的变化。结果表明：3?种加热方式低盐条件下L-Cys均显著提高肌球蛋白的溶解度（P＜0.05）;一段式加热时L-Cys显著提高高/低盐条件下肌球蛋白的表面疏水性（P＜0.05）,二段式加热时高盐条件下表面疏水性显著提高（P＜0.05）,其他条件下表面疏水性无明显变化。高/低盐条件下添加L-Cys均能显著改变肌球蛋白聚集行为的表面形貌,使聚集体更加分散,抑制了肌球蛋白的聚集。L-Cys对肌球蛋白溶解度、表面疏水性的影响进一步影响了其聚集行为,改变其聚集形貌。     

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.     

Thermal denaturation of proteins in Post rigor muscle tissue as studied by differential scanning calorimetry

Post rigor bovine M. semimembranosus was analysed by differential scanning calorimetry (d.s.c.). After extractive removal of sarcoplasmic proteins, subsequent pH adjustment and manual connective tissue removal, d.s.c. yielded reproducible thermograms which permitted investigation of the individual major myofibrillar proteins in various pH and salt environments without prior isolation. The positions of two major peaks, interpreted as myosin transitions, proved to be strongly pH dependent. At pH 5.4, the peak maxima occurred at 58 and 65°C, respectively, at a heating rate of 10°C min-¹. Above pH 6.5 their order of denaturation was reversed. In the pH range 5.4–6.5 the peak ascribed to actin had its maximum near 80°C in intact muscle. Above this pH range it was displaced to lower temperature. The thermal stability of actin was studied after treatment of the muscle tissue with different salt solutions. At equal ionic strengths (μ = 0.15) at pH 5.5, calcium chloride and sodium chloride caused 6.5°C and 4°C displacement to lower temperature, respectively. The thermograms of bovine semimembranosus muscle were compared to those of two red and two white muscle types (bovine cardiac and rabbit soleus muscles, chicken breast and rabbit semimembranosus muscles, respectively) at two pH levels. Greater myosin differences were found between red and white muscles than between muscles from different animal species. All muscles gave similar actin transitions, with exception of the heart muscle where the actin peak appeared at 3 °C lower temperature. The necessity of a strict pH control in order to obtain reproducible muscle thermograms is demonstrated.     

Differential scanning calorimetric study of muscle and its proteins: Myosin and its subfragments

The thermal denaturation of rabbit skeletal muscle myosin and its subfragments was investigated by differential scanning calorimetry. The thermal denaturation of myosin was shown to occur via three (at least) partly independent cooperative endothermic processes. The temperatures at which these processes occurred (312, 317 and 323 K at pH 6.0 and I=1.0) were shown to vary with pH (5.5–8.0) and I (0.05–1.0). The apparent enthalpy of denaturation of myosin was also shown to be dependent on pH and I. By comparing thermograms of myosin with those of the isolated myosin subfragments, the three major processes associated with the thermal denaturation of rabbit myosin could be tentatively assigned to different regions of the myosin molecule, namely, the helical tail, the ‘hinge-region’ and the globular heads. The ‘hinge-region’ thermal denaturation was shown to be reversible at pH 6.0 and I=1.0. Investigations of the effects of ortho-, pyro-, and tripolyphosphate on the thermal denaturation of myosin showed that added pyrophosphate destabilised the myosin molecule by about 9 K compared to the effects of ortho and tripolyphosphate, even though the latter was probably hydrolysed to ortho and diphosphate.     

A Simplified Myosin Preparation from Marine Fish Species

A simple and rapid method for the purification of fish myosin with high yield was developed. The method consisted of washing chopped fish muscle with a 0.1M KCl solution, an extraction of myosin with a 0.45M KCl solution containing ATP, and steps of reprecipitation and redissolution by changes in ionic strength. Actin can easily be obtained as a component of the myosin preparation.     

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.     

Biochemical and thermo-mechanical analysis of collagen from the skin of Asian Sea bass (Lates calcarifer) and Australasian Snapper (Pagrus auratus), an alternative for mammalian collagen

Australasia has a large fish industry, and fish skin by-products from the processing industry could be used for the commercial production of fish collagen. The aim of this study was to characterize collagen extracted from the Asian sea bass (Australian barramundi) (Lates calcarifer) and snapper (Pagrus auratus) skin as an alternative to mammalian-derived collagen in gelatin products. The acid-soluble fractions of collagen from Asian sea bass and snapper skin were extracted and yielded about 8 and 7.5 % collagen (on a dry weight basis), respectively. The electrophoretic and chromatography patterns indicated that both collagens comprise of α1, α2, α3, and β chains, corresponding to the properties of calf skin collagen type I. Amino acid analysis and peptide mapping of digested collagen suggested differences in their amino acid sequences and collagen primary structure. Fourier transform infrared spectroscopy demonstrated that the helical structure of collagen was completely maintained in Asian sea bass and partially in snapper. Transition temperatures for the completion of the melting process in the two collagen networks were confirmed with differential scanning calorimetry and dynamic oscillatory rheology to be about 29 °C. Zeta potential analysis identified the isoelectric points (pI values) of collagen from Asian sea bass and snapper skin at pH 6.90 and 7.75, respectively. Thus, Asian sea bass and snapper skin could be an important alternative source of collagen to replace mammalian collagen for industrial applications.     

CHEMICAL COMPOSITION AND POSTMORTEM CHANGES IN SOFT TEXTURED MUSCLE FROM INTENSELY FEEDING ATLANTIC COD (GADIUS MORHUA, L)

Soft textured fillets from postspawning Atlantic cod caught during the influx of capelin exhibited significantly more drip than fillets from fish caught at other times of the 1983 inshore fishing season. The extent and rate of pH decline, protein, collagen and temperature of the muscle were not distinctive in fish caught during the influx of capelin. The muscle from these fish were unique in exhibiting a rapid and relatively low ultimate pH together with a stable pH after the onset of rigor mortis. The number of capelin present in the stomach of cod at the time of catch was positively related (r = 0.92) to the amount of free drip recovered from the fillets.     

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.     

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.     

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。