RELATIONSHIP OF pH AND TEMPERATURE TO DISRUPTION OF SPECIFIC MUSCLE PROTEINS AND ACTIVITY OF LYSOSOMAL PROTEASES

From a review of the literature, and from specific data presented in this paper, it was concluded that both postmortem temperature and pH have effects on meat tenderness and on disruption of specific myofibrillar proteins. Increased postmortem temperature porduces more tender muscles and increases the disruption of troponin-T, myosin, Z-lines, connectin and gap filaments. Elevated postmortem temperature also increases the activity of enzymes which cause the disruption of myofibrillar proteins. Higher ultimate postmortem pH (above 6.0) produces more tender muscle, but also produces dark-cutting meat Except for one experiment, lower pH in the first few hours postmortem (in muscle with normal ultimate pH; i.e., 5.8 or below) improves meat tenderness. High pH increases the activity of CAF and low pH increases the activity of lsosomal cathepsins. Both high and low pH increase the degradation of troponin-T, Z-lines, gap filaments and connectin, but the degradation of these proteins (except for Z-lines) is greater at a low pH. Low pH increases the degradation of myosin; conversely, high pH retards it degradation.     

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.     

Chicken muscle homogenate gelation properties: effect of pH and muscle fiber type

Gelation properties of chicken breast and thigh muscle homogenates at a protein concentration of 4.5% under different pH conditions (5.80–6.60) and those of myofibrillar proteins at a protein concentration of 2% were compared to determine the influence of muscle fibre types on gelation. The optimal gelling pH for breast muscle homogenates (pH 6.30) was slightly higher than that for thigh muscle homogenates (pH 5.80–6.30), a similar trend was found for the isolated chicken myofibrillar proteins (pH 6.00 for breast and 5.50 for leg). Similarly, the pH values at which breast muscle homogenate gels were weaker (pH<6.20) or stronger (pH6.20) than thigh muscle homogenate gels were higher when compared with chicken breast and leg myofibrillar protein gels (pH<5.80 and pH>5.90, respectively).     

SOLUBILITY OF THE PROTEINS OF MACKEREL LIGHT MUSCLE AT LOW IONIC STRENGTH

Over 90% of the proteins of mackerel light muscle were soluble in solutions of physiological ionic strength or less. To accomplish this solublization, it was necessary to extract certain proteins at moderate ionic strength and neutral pH before extracting the rest of the myofibrillar and cytoskeletal proteins in water. Six proteins were favorably solubilized by sodium chloride solutions of moderate ionic strength at neutral pH under conditions that allowed later dissolution of myofibrillar and cytoskeletal proteins in water. The possibility is suggested that three of these proteins were involved in preventing the solubilization in water of other myofibrillar and cytoskeletal proteins of mackerel light muscle. Based on molecular masses and relative abundance, these proteins could possibly be M-protein (166 kDa), α-actinin (95 kDa) and desmin (56 kDa).     

转谷氨酰胺酶对猪肉肌原纤维蛋白凝胶性质的影响

以猪背最长肌为材料,研究转谷氨酰胺酶(TG)浓度对肌原纤维蛋白质(MP)热诱导凝胶强度和持水力的影响,探讨转谷氨酰胺酶在不同pH 值条件下对肌原纤维蛋白质凝聚性和交联的作用以及对热诱导凝胶强度、持水力的影响,比较TG 处理对凝胶结构的微观影响。结果表明：TG 处理会显著提高肌原纤维蛋白质的凝胶强度,TG最佳使用量为0.3%,NaCl 浓度为0.6mol/L,pH 值在5.8～6.4 之间时添加TG 会增加蛋白质的蒸煮损失,而pH 值在6.4～6.7 范围内添加TG 可降低蒸煮损失;电镜扫描结果表明TG 处理可改善热诱导凝胶的网络结构;TG 处理可改善肌原纤维蛋白的质构性质。     

pH条件对肌原纤维蛋白乳液微凝胶性质的影响

肌原纤维蛋白乳液微凝胶是在加热肌原纤维蛋白乳状液过程中同时施加剪切,导致变性蛋白质聚集在乳状液滴上所形成的离散球形颗粒。通过测定不同pH条件下制备的肌原纤维蛋白乳液微凝胶流变行为及微观结构,研究pH对其流变性质的影响。结果表明：在强酸性条件及接近其等电点时,乳液微凝胶的粒径大于其他pH范围。不同pH条件下的乳液微凝胶均为非牛顿流体,具有假塑性流体特征,pH的变化不会改变其流体类型。其黏度随pH的升高呈现先减小后增大的趋势,pH为6时,在低频率扫描时呈现最高的黏弹性,触变性也最好。剪切恢复力测试中,微凝胶颗粒的结构受到一定程度的破坏,其中pH为5时恢复性最好。     

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.     

Extracellular Protease from Pseudomonas marinoglutinosa: Some Properties and Its Action on Fish Actomyosin

A bacterium isolated from Indian mackerel (Rastrelliger kanagurta) and identified as Pseudomonas marinoglutinosa, was found to produce appreciable amounts of extracellular protease when grown in nutrient medium. This enzyme which degraded several proteins, was found to be most active against mackerel myofibrillar proteins. The optimum temperature and pH range for enzyme activity were 50°C and 7–8 respectively. Treatment of mackerel actomyosin with the protease at 0–2°C for 4 days resulted in degradation of the protein as assessed by release of tyrosine, loss in Mg⁺⁺-dependent ATPase activity and changes in SDS-polyacrylamide gel electrophoretic patterns.     

CATHEPSIN D AND ITS EFFECTS ON MYOFIBRILLAR PROTEINS: A REVIEW1

The molecular and enzymatic properties of the extensively studied enzyme cathepsin D are reviewed and additional information concerning its activity presented. Cathepsin D at pH 5.5 (37°C) degraded several myofibrillar proteins. The most rapidly hydrolyzed included titin and perhaps nebulin, myosin heavy chain, and M and C-proteins. The effects of cathepsin D on myofibrillar structure under these conditions included reduction in A band width, cleared central region in the A band, and dislocation of the Z line. Temperature was found to exert a strong influence on activity of cathepsin D and maximum activity was observed at 45°C with both muscle and hemoglobin substrates. Activity was evident at even higher temperatures and approximately 49% remained at 55°C (hemoglobin assay). Low temperature (i.e., < 15°C) however, has been observed to result in almost complete inactivity of the enzyme. The implications of this information for involvement of cathepsin D in postmortem proteolysis and tenderization were discussed.     

Solubilization of fish muscle proteins with buffers containing sodium dodecyl sulfate

The extraction of fish muscle protein using SDS containing solubilization buffers was studied varying the time and the temperature of solubilization, as well as pH and SDS concentration of the buffer. At pH less than 6 the myofibrillar proteins were incompletely solubilized; temperatures of 80-100 degrees C resulted in protein degradation observable in the SDS-PAGE. Samples of fish muscle containing high amounts of formaldehyde (50 mmoles FA/kg wet weight) could only be solubilised at 100 degrees C; on the other hand it was possible to solubilize cooked and/or canned products under mild conditions (2% SDS, 1% 2-ME, pH 8.9, shaking for 2 h at 60 degrees C).     

Effect of Low-Calcium-Requiring Calcium Activated Factor on Myofibrils under Varying pH and Temperature Conditions

The effect of low-calcium-requiring calcium-activated factor (μM CAF) on the myofibrils under varying pH at 5°C and 25°C was examined spectrophotometrically (absorbance at 278 nm), electrophoretically (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and microscopically (phase microscopy and transmission electron microscopy). Results indicated that at conditions similar to those of postmortem storage (i.e., pH 5.5–5.8 and 5°C), μM CAF retained 24–28% of its maximum activity (pH 7.5 at 25°C). This 24–28% of maximum activity was sufficient to reproduce most of the known changes associated with the tenderization process during postmortem aging. It was concluded that because of the activity of μM CAF under postmortem conditions, it seems reasonable to suggest that μM CAF may be responsible, in part, for some of the postmortem changes observed.     

CHARACTERISTICS OF SARCOPLASMIC PROTEINS AND THEIR INTERACTION WITH MYOFIBRILLAR PROTEINS

The protein solubility and molecular‐weight distribution of freeze‐dried sarcoplasmic proteins (SPs) from rockfish treated under low and high pH as well as various NaCl concentrations were elucidated. The solubility of SPs was significantly suppressed at an acidic pH (2.0–4.0) and in the presence of high salt concentration (0.5 M NaCl). The least amount of protein was lost when SPs were treated at pH 2.0 or 3.0 followed by precipitation at pH 5.5. The interaction of SPs with Alaska pollock surimi (myofibrillar proteins) was also investigated. The addition of SPs appeared to delay the thermal denaturation of myosin and actin. The SPs positively contributed to the gelation of myofibrillar proteins as judged by breaking force.     

Effect of animal (lamb) diet and meat storage on myofibrillar protein oxidation and in vitro digestibility

Effect of pasture- or concentrate-diet on myofibrillar protein oxidation and in vitro digestibility was measured in lamb meat (M. longissimus dorsi) during a refrigerated storage of 7days under gas permeable film. Protein oxidation was measured by the carbonyl content determined chemically using 2,4-dinitrophenylhydrazine (DNPH) and specific targets of oxidation were identified by immunoblotting. Carbonyl content significantly increased during storage and diet affected protein oxidation where animals fed concentrate showed higher carbonyl group levels than animals fed pasture. To evaluate effect of diet and storage time on protein digestibility, myofibrillar proteins were exposed to proteases of the digestive tract (pepsin, and a mixture of trypsin and α-chymotrypsin) in conditions of pH and temperature which mimic digestive process. The myofibrillar protein digestibility was not influenced by the diet. Storage time had no significant effect on myofibrillar protein susceptibility to pepsin while an important increase in digestibility by trypsin and α-chymotrypsin was detected during storage.     

Myofibrillar protein degradation of carp (Labeo rohita (Hamilton)) muscle after post‐mortem unfrozen and frozen storage

The degradation of myofibrillar proteins of rohu carp (Labeo rohita (Hamilton)) muscle was analysed after post‐mortem storage. Muscle fillets were kept either unfrozen at 2 °C for up to 15 days or frozen at ?8 °C or ?20 °C for up to 6 months. A co‐ordinated histochemical, biochemical and electrophoretic study showed a differential response of the carp muscle, revealing clear degenerative/degradative changes specific to the post‐mortem storage temperatures. The myofibrillar protein fractions, namely myosin light chains and α‐actinin, showed degradative changes during the above storage conditions, whereas other protein fractions in the high‐molecular‐weight range fragmented to give lower‐molecular‐weight proteins. The importance of the post‐mortem storage temperature for controlling the degradation of the myofibrillar proteins was emphasised. This is the first report on this popular fish species, known for its culinary importance, showing that specific protein fractions of the myofibrils degrade during post‐mortem storage. © 2001 Society of Chemical Industry     

A Generalized Model for Predicting Heat-Induced Chicken Myofibrillar Protein Gel Strength

A generalized mathematical model was developed for predicting effects of temperature-time history (TTH), protein concentration, and pH on the strength of heat-induced chicken myofibrillar protein gels. A pseudo first-order kinetic model was used to describe integral TTH effects of protein denaturation on an apparent viscosity index. A reaction threshold temperature of 30°C and a denaturation activation energy of 20 kcal/mole were calculated. No significant increase in viscosity index was observed at TTH values above 1.0 × 10^10oK-sec. Maximum viscosity index was observed at pH 6.0. The effect of protein concentration on viscosity index was modeled as a logarithmic function. An a priori modeling approach based on fundamental theories can be used to predict the strength of gels under various processing conditions.     

响应面法优化鸡胸肉肌原纤维蛋白酶法水解工艺条件

以鸡胸肉为原料,提取肌原纤维蛋白,测定其蛋白质浓度及氨基酸组成;以水解度为测定指标,利用响应面法优化该肌原纤维蛋白酶解工艺条件,并考察其产物肽分子量分布。结果表明:提取的肌原纤维蛋白浓度为59.27%;该蛋白氨基酸种类齐全,组成比例均衡,必需氨基酸含量高达40.90%;碱性蛋白酶是肌原纤维蛋白酶解的最佳用酶;在单因素实验基础上,经Box-Behnken实验优化得到碱性蛋白酶水解肌原纤维蛋白最佳工艺条件为:加酶量3500 U/g、p H7.8、温度44℃,此条件下经6 h酶解,水解度达33.17%;肽分子量分布分析知,最佳水解条件下酶解液中<1000 u的小肽含量高达61.5%,说明碱性蛋白酶能较高程度的水解肌原纤维蛋白。      

Thermostable Water Dispersions of Myofibrillar Proteins from Atlantic Mackerel (Scomber scombrus)

A process for preparation of a thermostable, low viscosity water dispersion of myofibrillar proteins from Atlantic mackerel (Scomber scombrus) was developed. The comminuted meat was washed sequentially with cold water, a bicarbonate solution, and cold water. It was then homogenized in ice-cold water. Apparent viscosity of the dispersion depended on protein concentration and temperature. Lowering pH to 3.7 by acetic acid reduced viscosity of the dispersion which remained unchanged irrespective of temperature. The proteins did not precipitate upon heating to 100° followed by centrifugation at 5000 xg for 15 min, even in the presence of 50 mM NaCl solution. However, increasing pH of the acidified solution resulted in precipitation of proteins. Acetic acid also prevented separation of water upon centrifugation of heated dispersion.     

离子强度和pH值对肌原纤维蛋白热诱导凝胶特性的影响

以肌原纤维热诱导凝胶的保水性和质构特性,包括硬度、弹性、内聚性、胶黏性、回复性为指标,考察离子强度和pH值对猪背最长肌肌原纤维热诱导凝胶特性的影响.结果表明,离子强度和pH对凝胶不同特性的影响存在差异,综合各种指标,离子强度为0.5,pH值在6.5和7.0之间有利于形成较好的凝胶.不同条件下凝胶形成的机制可能存在差异.凝胶的硬度与弹性、胶黏性和保水性相关性显著(P＜0.05),可以作为重点考察的指标.     

The effect of acidification on myofibrillar proteins

Isolated myofibrillar proteins of mutton, beef and chicken were treated with an acidulent to give various pH values and stored at 5°C for 20 h before analysing the proteins using sodium dodecylsulphate polyacrylamide gel electrophoresis. It was found that protein degradation occurred below pH 4·5, with a decrease in band intensity of all major myofibrillar proteins, particularly myosin heavy chain, and the appearance of new bands at approximately 140 and 70 kd. The degradation had an optimum around pH 3·0 and was inhibited by a high temperature pre-treatment or the presence of the endopeptidase inhibitors pepstatin A and leupeptin. Results are discussed in terms of the action of acid proteinase enzymes.     

High post-mortem temperature combined with rapid glycolysis induces phosphorylase denaturation and produces pale and exudative characteristics in broiler Pectoralis major muscles

This study investigated the effect of early post-mortem temperature on broiler protein characteristics and meat quality. Muscles were kept at different temperatures (0, 20 and 40 °C) until 4h post-mortem and then stored at 4 °C. Rapid degradation of ATP and glycogen, thus inducing a high rate of lactate formation and pH drop, were found in the 40 °C group during incubation. When extracting proteins, a lower protein content of the sarcoplasmic fraction and a higher protein content of the myofibrillar fraction were found in the 40 °C group at 24h post-mortem; SDS-PAGE and western-blotting results revealed that phosphorylase was associated with the myofibrillar fraction. Furthermore, the 40 °C group had paler surfaces, higher drip loss and lower processing properties. These data suggest that elevated temperature during early post-mortem period, resulting in rapid glycolysis, induced phosphorylase denaturation and association with myofibrillar proteins thus generating pale and exudative characteristics.