Bovine muscle 20S proteasome. III: Quantification in tissue crude extracts using ELISA and radial immunodiffusion techniques and practical applications

The 20S proteasome is a large complex (700kDa) that exhibits endo- and exo-peptidase activities with wide specificity. In postmortem muscles, several sets of evidence suggest a possible significant contribution of proteasome to meat tenderisation. Hence, an accurate and rapid quantification procedure is needed to attest that new function during the ageing of meat. In the present work, we developed an ELISA test enabling the quantification of nM concentrations of the 20S proteasome. We further tested the radial immunodiffusion (RID) technique described as a more simple method that can quantitatively determine the concentration of an antigen in a complex mixture. The ELISA test allowed us to quantify the 20S protesome in tissue homogenates and fluids with a recovery of 100%, a coefficient of variation lower than 5% and a detection limit of 9ng/ml. Quantification of the 20S proteasome in various bovine tissue by ELISA showed the highest concentration in liver followed by spleen and kidney, with muscles exhibiting the lowest concentrations. In addition, measurement of the proteasome concentration in eight different bovine muscles with various metabolic profiles led to the conclusion that the relationship between muscle metabolic properties and proteasome concentration is rather complex. Nevertheless, heart muscle exhibited the highest proteasome content (331μg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213μg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209μg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203μg/g wet tissue), an intermediate muscle. As compared to other endogenous peptidases, muscle tissue contains relatively high amounts of proteasome. Hence this complex can be quantified using the RID, which allows quantification of protein in the μg range. Plotting the concentration values determined with both methods for all bovine tissues tested gave a straight line with a correlation coefficient of 0.99.     

Bovine muscle 20S proteasome. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.     

MEAT TENDERIZATION: POSSIBLE CAUSES AND MECHANISMS. A REVIEW.

The postmortem meat tenderizing process is complex and not fully understood. The nature of changes associated with the improvement in tenderness and the exact mechanisms involved are still unknown. Based on relevant evidence, old and new, this review attempts to clarify the statement of our knowledge of these aspects. Of the different biochemical and ultrastructural changes occurring in meat, a key role of myofibril disruption taking place at the N₂-line level in meat tenderization has been emphasized. This may be ascribed to the action of lyosomal enzymes, especially cathespin B and L. However, all the changes thus far identified can be only explained by a synergistic action of lysosomal and calcium-dependent proteinases. Besides or together with proteolytic enzymes, weakening of myofibrils may also be mediated by the high ionic strength achieved in postmortem muscles. Both mechanisms possibly involved in the meat tenderizing process have been tentatively tested in relation with the large muscle variability in aging rate. It appears that some concepts are in conflict with the results presented. For instance, no direct relationship was found between aging rate and proteinase content of muscles.     

宰后牦牛肉成熟过程中钙激活酶与嫩度指标的相关性分析

以10头甘南牦牛为研究对象,对宰后8d成熟期间肌原纤维小片化指数、剪切力、肌纤维直径、μ-钙蛋白酶(μ-calpain)、m-钙蛋白酶(m-calpain)、钙蛋白酶抑素(calpastatin)的活力进行了测定,同时研究了3种酶活力与肌原纤维小片化指数、剪切力、肌纤维直径3个嫩度指标之间的相关性.结果表明:μ-calpain、m-calpain、calpastatin 3种酶与剪切力值及肌纤维直径均呈正相关;3种酶与肌原纤维小片化指数呈负相关,其中μ-calpain与calpastatin呈显著负相关(P＜0.05).因此,钙激活酶活力的变化可能导致了肌原纤维小片化指数的增加,肌原纤维的弱化和肉的嫩化,μ-calpain可能是牦牛肉嫩化的主要贡献者.     

极限pH对羊肉宰后成熟过程中肌原纤维蛋白特型的影响

为了研究羊肉宰后成熟过程中极限pH对肌原纤维蛋白特型即肌联蛋白、伴肌动蛋白、肌间线蛋白和肌钙蛋白-T降解及肌原纤维小片化指数的影响。本文选取50只羊的右侧背最长肌,贮存于4 ℃条件下,在宰后时间点分别为1 h、1、2、3、5 d和7 d时,测定其pH。按照宰后2 d的pH将肉样分成三组:高极限pH组(5.72±0.03),中极限pH组(5.54±0.01)和低极限pH组(5.40±0.02)。在每个宰后时间点,测定肌联蛋白、伴肌动蛋白、肌间线蛋白、肌钙蛋白-T降解程度和肌原纤维小片化指数(MFI)。结果表明:肌联蛋白在高极限pH组中宰后1 d开始降解;在宰后1 d时,高极限pH组肌间线蛋白相对灰度值显著低于中极限pH组和低极限pH组(p<0.05);肌钙蛋白-T在高极限pH组中,宰后1 d已出现降解条带。而伴肌动蛋白在中极限pH组中降解较快,在宰后1 d开始降解。另外在宰后1、2、3、5、7 d时,高极限pH组和中极限pH组的肌原纤维小片化指数显著高于低极限pH组的肌原纤维小片化指数(p<0.05)。极限pH通过影响这些肌原纤维蛋白降解来促进宰后肌肉成熟过程并且肌联蛋白、肌间线蛋白和肌钙蛋白-T的降解,加快了宰后前期嫩化过程。这为揭示宰后肉嫩度形成机理提供理论基础。     

Comparison of fresh beef and camel meat proteolysis during cold storage

The objective of this research was to determine the difference in myofibrillar fragmentation of camel meat and beef during postmortem aging. Semitendinosus muscle was excised at slaughter and muscle pH was measured at 6, 12, 24, 48, and 72h postmortem. Myofibril fragmentation index was measured on 1, 3, 5, and 7 days postmortem. Also, myofibrils isolated from semitendinosus muscles of camel and cattle at 1, 3, 5 and 7 days postmortem storage were analyzed using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results showed that the camel semitendinosus muscle had significantly higher myofibril degradation values compared to that in beef which was supported by a difference in troponin-T degradation and appearance of a 30kDa band. Postmortem pH decline of camel meat was significantly slower than that of beef. This study demonstrated that the semitendinosus protease activity of camel meat was superior to that of beef, which may have been due to the difference in pH decline.     

CHANGES IN THE CALPAIN-CALPASTATIN AND CATHEPSIN (B, B+L, H AND D) DURING POSTMORTEM STORAGE OF GOAT MUSCLES

The objective of this study was to elucidate a relationship between some endogenous proteinases and their inhibitors in four different goat muscles during postmortem storage. Samples were taken from the longissimus dorsi (LD), biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles stored up to 20 days postmortem at 5C. Activities of calpain-I, calpain-II, calpastatin, cathepsins (B, B+L, H and D) and cystatin(s) were determined. Decreases in calpain-I and calpastatin activities were significantly more than that of calpain-II activity. The cathepsin B, B+L, H and cystatin level were found to fall by 9–35 % after 20 days, whereas the cathepsin D showed 11–17% decline in all the muscles. Thus changes in muscle proteinases and their inhibitors during postmortem storage differ and the results may shed light on their role in myofibrillar proteolysis and goat meat tenderization.     

Muscle Characteristics and Meat Tenderness of Cimaterol-fed Lambs

Shear values of loineye muscle from (cimaterol) CIM-fed lambs were 32 and 28% greater than those of controls at 1 and 8 days postmortem. The CIM-fed animals showed lower (p<0.01) cytochrome oxidase activity, lower (p<0.01) initial glycogen, and higher (p<0.05) 24 hr pH in the longissimus and semimembranosus muscles. Intramuscular fat of treated lambs (2%) was lower (p<0.01) than that of controls (5.1%), whereas muscle protein concentration was greater (p<0.05). The decreased aerobic capacity, lower intramuscular fat, lower initial glycogen, higher ultimate muscle pH (5.8–6.2) with subsequently reduced postmortem proteolysis and higher protein concentration all appeared to contribute to greater meat toughness of CIM-fed lambs.     

Effects of cortisol on muscle proteolysis and meat quality in piglets

The present study was conducted to examine the effects of cortisol on muscle proteolysis and meat quality. Male piglets (n=14) were assigned to one of two treatment groups at 28 days of age. After 7 days adaptation period, each group was fed a commercial diet (86% total digestible nutrients, 21.5% crude protein) or the same commercial diet containing cortisol (120mg/kg diet) for 7 days from 35 days of age. All piglets were slaughtered at 42 days of age. The serum triiodothyronine (T3) concentration, μ- and m-calpain and proteasome activities and the content of easily releasable myofilament, which contains intermediates of the breakdown of myofibrils in the m. longissimus dorsi (LD) at slaughter were measured as parameters of muscle proteolysis. Serum T3 levels and μ-calpain activity were increased (P<0.01), as was the amount of easily releasable myofilament and m-calpain and proteasome activities were higher (P<0.05) in LD from cortisol-treated piglets than from non-treated controls. At 24 h postmortem, LD of cortisol-treated piglets showed higher (P<0.01) drip loss and lighter (P<0.05) color than those of the control. The results clearly show that the administration of cortisol increases serum T3 concentration and muscle proteolysis and reduces productivity and meat quality.     

Effects of Postmortem pH and Temperature Muscle Structure and Meat Tenderness

Bovine longissimus muscles with postmortem pH in the range 5.5 - 7.0 were subjected to different postmortem temperatures of 1°, 4°, 25° and 37°C. Intact beef sides with different postmortem pH were also subjected to two different environmental temperatures of 1° and 25°C. High pH muscles exhibited an extensive degradation of Z-lines, whereas low pH muscles showed a preferential degradation of M-lines and myosin heavy chains. Intermediate pH muscles did not show much degradation of muscle proteins, resulting in tougher meat than either low or high pH muscles. High postmortem temperatures enhanced the degradation of muscle proteins in excised and incubated muscle strips, but the delayed chilling of intact beef sides at 25°C for 8-hr did not affect either the structural changes or meat tenderness.     

Characterisation of proteolytic enzymes from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii)

BACKGROUND: Fresh water prawn in Thailand is widely consumed due to its delicacy. During postmortem handling and storage, prawn meat becomes soft and mushy, probably as a result of indigenous proteases. Therefore, an understanding of prawn proteases associated with the degradation of muscle proteins from fresh water prawn could pave the way for prevention of such a phenomenon during extended storage. RESULTS: Proteolytic enzymes in the crude extract (CE) from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) were characterised. CE from muscle exhibited the highest hydrolytic activities towards haemoglobin at pH 5 and 50 °C, while that from hepatopancreas had the highest activity on casein at pH 7 and 60 °C. Based on inhibitor study, cysteine protease and serine protease were dominant in CE from muscle and hepatopancreas, respectively. CE from muscle rarely hydrolysed natural actomyosin (NAM), but could not degrade pepsin‐soluble collagen (PSC). Conversely, NAM and PSC were susceptible to hydrolysis by CE from hepatopancreas as evidenced by the marked decreases in band intensity. Activity staining using haemoglobin, casein and gelatin as substrates revealed that no proteolytic or gelatinolytic activity was observed in CE from prawn muscle, while CE from hepatopancreas exhibited pronounced hydrolytic activities towards all substrates. CE from muscle showed calpain and cathepsin L activities but CE from hepatopancreas mainly exhibited tryptic and chymotryptic activities. CONCLUSION: Serine proteases, mainly trypsin‐like or chymotrypsin‐like, from hepatopancreas were probably responsible for the softening of prawn meat during postmortem storage via the degradation of both muscle and connective tissues. Copyright © 2010 Society of Chemical Industry     

冷却猪肉pH值变化与肉汁渗出率的关系研究

为了解决冷却肉流汁过多的问题,本项研究分别选取不同部位的猪肉作实验材料,在同等处理条件下检测了不同时点猪肉的pH值和肉汁渗出率。结果表明:不同部位猪肉的pH值下降速率和极限pH值存在一定的差异,有的存在极显著差异(p<0.01);较高的pH值下降速率和较低的极限pH值会导致较高的肉汁渗出率。     

Effects of dietary α-lipoic acid on glycolysis of postmortem muscle

The effects of dietary α-lipoic acid (α-LA) on the pH value, AMP-activated protein kinase (AMPK) activation, and the activities of glycogen phosphorylase and pyruvate kinase in postmortem muscle were investigated. Eighteen C57BL/6J mice were fed diets containing 0%, 0.5%, and 1.0% α-LA. At the end of 2-week feeding trial, the mice were killed and longissimus muscles were sampled at 0-, 1-, and 24-h postmortem for pH determination and enzyme assay. The results showed that dietary α-LA treatment significantly slowed down the decrease of pH values in postmortem muscle. The ultimate pH values in postmortem muscle of mice receiving 0.5% and 1.0% α-LA treatments were 6.40 and 6.63, respectively, significantly higher than that (6.21) of no α-LA treatment (p<0.05). AMPK was activated at the early postmortem stage. Dietary α-LA can suppress the activation of AMPK in postmortem muscle. At 1- and 24-h postmortem, activities of AMPK were much lower in postmortem muscle of mice receiving 0.5% and 1.0% α-LA treatments than that with no α-LA treatment. Between these two dietary α-LA treatments, however, no difference in AMPK activity was observed, indicating that 0.5% dietary α-LA is enough to suppress AMPK in postmortem muscle. Similar to AMPK, glycogen phosphorylase activity was higher in the treatment without dietary α-LA than those with 0.5% and 1.0% dietary α-LA supplements. No difference in the activity of glycogen phosphorylase was observed between the 0.5% and 1.0% dietary α-LA treatments. Dietary α-LA had no significant influence on the activity of pyruvate kinase in postmortem muscle. All these results indicate that AMPK plays a role in glycolysis in postmortem muscle. Dietary α-LA supplementation can suppress the activation of AMPK in postmortem muscle, down-regulate the activity of glycogen phosphorylase, resulting in a higher ultimate pH values in postmortem muscle. Therefore, dietary α-LA supplementation is a potential way to reduce the incidence of pale, soft, exudative (PSE) meat.     

Purification and properties of rabbit muscle proteasome, and its effect on myofibrillar structure

This paper describes the purification and properties of a multicatalytic proteinase complex, proteasome, from rabbit skeletal muscle, and its effect on myofibrillar structure. The purified proteasome gave a single band on polyacrylamide gel electrophoresis under non-denaturing conditions and gave eight bands under denaturing conditions, indicating that this enzyme comprises multiple hetero-subunits with low molecular mass. The purified proteasome was not activated by ATP and ubiquitin, and was markedly inhibited by Z-Leu-Leu-Leu-H (aldehyde). These data indicate that the purified proteasome is not 26S, but 20S. The proteasome degraded synthetic peptides maximally at pH 8.0. Relative to pH 8.0, activities were gradually decreased with the lowering of pH, but the degree of decrease was substrate-dependent, and the activity at pH 5.0 still retained about 30˜60% of the activity at pH 8.0, indicating the possibility that the proteasome is active in the muscle during conditioning. When the proteasome was heated at 60 °C for 20 min and treated in the presence of 0.005% SDS, the activity increased over 1.5 and 4.5 times, respectively. SDS remarkably increased the V_max value of the enzyme at pH 8.0. The proteasome was also activated by high hydrostatic pressure up to 100˜150 M Pa and gradually decreased at 200 MPa or higher. Electron microscopic observation revealed that obvious gaps between filamentous structure, the complete loss of M-line and partial loss of Z-line structure were caused by proteasome.     

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.     

超声波与菠萝蛋白酶协同作用对鸭肉嫩化的影响

采用单因素和响应面设计优化了超声波辅助菠萝蛋白酶嫩化鸭肉的最佳工艺参数,并对嫩化机理进行探讨。得到优化条件为处理温度45?℃、pH?7.2、菠萝蛋白酶添加量350?U/g、超声波功率80?W、嫩化时间15?min,在该条件下剪切力的预测值为20.208?N,实测值为21.110?N。相对于未处理的鸭肉,嫩化组鸭肉的pH值、持水力和肌原纤维小片化指数显著增加（P＜0.05）,剪切力、肌原纤维溶解度和不溶性胶原蛋白的含量显著下降（P＜0.05）,而色差无明显变化（P＞0.05）。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱显示嫩化组鸭肉中大分子蛋白被水解成小分子肽类或氨基酸。透射电镜结果显示嫩化组鸭肉肌原纤维Z线断裂溶解,肌节变形缩短,I带和A带模糊不清,出现了肌原纤维小片化现象。超声波与菠萝蛋白酶协同作用对鸭肉的嫩化具有显著的效果,大大缩短了嫩化时间,使鸭肉更加多汁并富有弹性,鸭肉的品质得到了极大的改善。     

Electrical Stimulation and 48 Hours Aging of Bull and Steer Carcasses

Bull (n = 30) and steer (n = 30) carcasses were measured for meat quality in the longissimus muscle after 48 hr on electrically stimulated (ES) sides or after 6 days on their unstimulated counterparts. Electrical stimulation and 48 hr aging had the same tenderizing effect as a 6 day aging period on both bulls and steers. Compared to meat from steers, that from bulls was tougher and had higher ultimate pH values and collagen contents. On an overall quality basis however, ES meat aged for 48 hrs was equivalent to nonstimulated meat aged for 6 days.     

Liberation of peptides during meat storage and their interaction with proteinase activity

The content of nitrogenous material extracted in perchloric acid from kid longissimus lumborum (LL), semimembranosus (Sm) and triceps brachii (TB) muscles increased 30% during storage for three weeks at 4 °C. The extract of fresh muscle contained a range of molecular sizes up to 13 kDa, the majority less than 4 kDa. The amount of the larger polypeptides (> 4 kDa) extracted from meat decreased to 50% of its initial level in meat stored for five days and to 20% after 25 days. The amount of intermediate sized peptides (1.9 to 4 kDa) remained largely constant throughout storage of meat of normal pH, whilst in meat of high ultimate pH, they showed a transitory increase during the first two weeks storage. Smaller size peptides increased with storage time; increasing more slowly in meat of high ultimate pH. This time-dependent pH effect indicates a sequential autolysis involving neutral followed by acidic proteolytic and peptidic activity. Calpain from beef muscle degraded peptides in the extract and the extract acted as a competitor against a synthetic substrate. This implies that the in-situ autolysis and tenderisation of meat postmortem could be reduced by peptide products.     

Postmortem muscle glycolysis and meat quality characteristics of intact male Korean native (Hanwoo) cattle

Five intact Korean bulls weighing about 550 kg were slaughtered to investigate postmortem glycolysis. Histochemical and meat quality characteristics of longissimus dorsi (LD) and psoas major (PM) muscles were made. Postmortem changes in ATP, glucose-6-phosphate and pH demonstrated that the rate of postmortem glycolysis in the PM was significantly faster than in the LD. The shear force to cut cooked PM was significantly lower than that of LD in 1 and 3 day aged samples, but no difference was observed between the two muscles in 7, 15 and 21 day aged samples. During the 21 days of aging, the rate of lipid oxidation, as measured by TBA value, was significantly faster in the PM than in the LD. The result suggests that PM muscle needs less aging time than LD muscle for optimum meat quality.     

Difference in tenderness and pH decline between water buffalo meat and beef during postmortem aging

The objective of this research was to determine the difference in tenderness and some characteristics of water buffalo meat and beef during postmortem aging. Five female crossbred water-buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native), were finished on the same diet for 6 months and slaughtered at 30 months of age. The muscle pH was measured at 40min, 3h, 7h, 24h, and 48h postmortem. Longissimus thoracis (LT) and semimembranosus (SM) muscles were excised at 2d postmortem, and shear force was measured at 2, 4, 7, and 14d postmortem. Glycogen and lactate concentrations were determined from 0, 2, and 4d LT samples, and myosin heavy chain type of buffalo and cattle LT was determined by ELISA methods. Myofibrillar protein degradation was also observed by SDS-PAGE and Western blotting of fast-type troponin T. Results showed that the buffalo meat had significantly lower shear force values compared to beef for LT and SM muscles, which was supported by a difference in troponin T degradation. Postmortem pH decline of buffalo meat was significantly slower than that of beef, which was confirmed by lactic acid concentrations, but was not explained by glycogen content. In addition, there was no significant difference in the ratio of slow to fast type muscle fibers in buffalo and cattle, indicating that myosin heavy chain type was not responsible for the difference in pH decline and tenderness between the buffalo meat and beef. This study demonstrated that the tenderness of water buffalo meat was superior to that of Brahman beef, which may have been due to the difference in pH decline and the subsequent effect on muscle protease activity.