Effect of heat shock protein 27 on the in vitro degradation of myofibrils by caspase‐3 and μ‐calpain

This study was designed to investigate the effect of heat shock protein 27 (HSP27) on the in vitro degradation of myofibrils induced by caspase‐3 or μ‐calpain. Myofibrillar proteins were prepared from at‐death beef muscles and incubated with caspase‐3 or μ‐calpain with and without HSP27, or with HSP27 alone, at 30 °C for 2 h, and protein degradation was assessed. Results showed that caspase‐3 promoted the degradation of titin, nebulin and troponin‐T, and μ‐calpain promoted the degradation of nebulin, desmin and troponin‐T, observed during normal PM ageing. Moreover, the addition of HSP27 restricted the degradation of troponin‐T in μ‐calpain‐ and caspase‐3‐treated myofilaments, and restricted the degradation of desmin in μ‐calpain‐treated myofilaments. Therefore, HSP27 may indirectly or directly interact with caspase‐3 and μ‐calpain, reducing their activity and mediating PM proteolysis of muscle proteins to affect meat tenderisation.     

Role of the ubiquitin‐proteasome pathway on proteolytic activity in postmortem proteolysis and tenderisation of sheep skeletal muscle

The objective of this study was to investigate the effects of ubiquitin‐proteasome pathway on meat tenderisation. The sheep muscle longissimus lumborum was injected with or without PYR‐41 (inhibitor of ubiquitination) or MG‐132 (inhibitor of proteasome). Muscle samples were collected at 6, 15, 24 and 48 h after injection. Myofibrillar protein degradation, muscle ultrastructure and sarcomere length were determined. Results showed that inhibition of proteasome or ubiquitination affected sarcomere length at 48 h after treatments. Destruction of muscle ultrastructure in both treatments was reduced when compared to control. Inhibition of proteasome produced different fragments of myofibrillar proteins in comparison with control at 48 h. In conclusion, ubiquitin‐proteasome plays a role in postmortem proteolysis and might contribute to meat tenderisation.     

不同屠宰方式对新疆多浪羊肉品质的影响

以新疆多浪羊为研究对象,对比分析传统屠宰（对照）和3 个不同电压（90、127 V和220 V）击晕屠宰 （分别计作EST 90、EST 127、EST 220）对多浪羊肉品质的影响。结果表明：EST 90处理组成熟过程中pH值下降 速率最快,血液中的皮质醇浓度、肌酸激酶和乳酸脱氢酶活力最高,羊应激反应最剧烈,宰后1、7 d的蒸煮损失率 最高（P＜0.05）;EST 127处理组的持水力最好;电击晕对多浪羊肉嫩度有一定影响,宰后7 d各处理组嫩度显著 改善,而EST 127处理组表现最佳;EST 220处理组放血不充分,且胴体表皮有多处血斑形成;电击晕屠宰对多浪羊 肉色泽无显著影响（P＞0.05）。综合分析实验结果,EST 127处理组持水力与嫩度较优,羊应激反应较小,肉品质 相对较好,因此EST 127处理适合应用于多浪羊的屠宰加工。     

Postmortem proteome degradation profiles of longissimus muscle in Yorkshire and Duroc pigs and their relationship with pork quality traits

Conversion of muscle to meat is regulated by complex interactions of biochemical processes that take place during postmortem storage of the carcass. Enzymatic proteolysis, among other postmortem biochemical phenomena; e.g. glycolysis; changes tough intact muscle tissue into more tender meat. Knowledge on proteome-wide proteolysis of muscle tissue in relation to meat quality is limited and potential breed-specific differences have received little attention. Therefore, we investigated meat quality traits and proteolysis profiles of the longissimus proteome of five Yorkshire and five Duroc pigs at slaughter and after 1, 2, 3, 7, and 10 days of ageing. Drip loss increased with ageing while cooking loss was unchanged in both breeds. Shear force varied between animals and decreased with ageing. Analysis of the proteomes showed four types of temporal expression profiles. Association analysis suggested several potential protein biomarkers for drip loss and shear force in both breeds, but none for cooking loss.     

Postmortem proteome degradation profiles of longissimus muscle in Yorkshire and Duroc pigs and their relationship with pork quality traits

Conversion of muscle to meat is regulated by complex interactions of biochemical processes that take place during postmortem storage of the carcass. Enzymatic proteolysis, among other postmortem biochemical phenomena; e.g. glycolysis; changes tough intact muscle tissue into more tender meat. Knowledge on proteome-wide proteolysis of muscle tissue in relation to meat quality is limited and potential breed-specific differences have received little attention. Therefore, we investigated meat quality traits and proteolysis profiles of the longissimus proteome of five Yorkshire and five Duroc pigs at slaughter and after 1, 2, 3, 7, and 10 days of ageing. Drip loss increased with ageing while cooking loss was unchanged in both breeds. Shear force varied between animals and decreased with ageing. Analysis of the proteomes showed four types of temporal expression profiles. Association analysis suggested several potential protein biomarkers for drip loss and shear force in both breeds, but none for cooking loss.     

Effects of nitric oxide and oxidation in vivo and postmortem on meat tenderness

Metabolic processes in muscle tissue in vivo result in the production of reactive oxygen species and oxidative compounds including superoxide anions and nitric oxide (NO). Reactive oxygen species can react with both lipids and proteins and often have deleterious effects, contributing to the onset of ageing and senescence as well as cell death. Nitric oxide (NO) is a free radical that is constantly produced or released throughout the body by diverse tissues and is known to influence proteolytic activity in human and rodent skeletal muscle as well as being involved in regulation of calcium homeostasis in the muscle cell. The influence of nitric oxide on development of meat tenderness has been studied through postmortem manipulation and also through in vivo studies. The effect of NO on meat tenderness is postulated to be via its regulatory effects on the proteins calpain, cathepsins, ryanodine receptor channel in the sarcoplasmic reticulum (SR) and the sarcoplasmic-endoplasmic release calcium ATPase in the SR. NO is an oxidant although the effects of NO on effector proteins can be distinguished from a direct oxidation reaction. The onset of oxidation in meat postmortem is well known to produce off-odours, discolouration and unacceptable flavours associated with rancidity. Oxidation during the immediate postmortem period appears to inhibit tenderisation during ageing, probably through an inhibitory effect of oxidation on the calpain enzyme. Oxidation of muscle tissue occurring as a result of availability of oxygen during modified atmosphere packaging may also have deleterious consequences for tenderness development during storage of meat prior to retail display. In conclusion, it is proposed that postmortem meat tenderisation is influenced by skeletal muscle's release of NO pre-slaughter and the oxidation of proteases postmortem. This proposal is compatible with the existing tenderness model and will hopefully assist in increasing the accuracy of prediction of meat tenderness. Future directions for research are discussed.     

饲养方式对新疆多浪羊肉品质的影响

分别以戈壁滩放养及工厂集约化饲养的6 月龄多浪羊背最长肌为对象,分析测定不同饲养条件下羊肉的屠 宰性能、pH值、持水力、嫩度、色泽、抗氧化能力、脂肪酸含量、肌原纤维超微结构等指标。结果表明：工厂集 约化饲养组多浪羊肉的嫩度、成熟7 d的亮度值和脂肪酸总量优于戈壁滩放养组（P＜0.05）,而戈壁滩放养组多浪 羊肉的持水力、抗氧化能力、宰后1 d的红度值及多不饱和脂肪酸含量优于工厂集约化饲养组（P＜0.05）;工厂集 约化饲养组多浪羊肉肌原纤维密度较大、直径小,肌原纤维之间排列紧密;而戈壁滩放养组的多浪羊肉肌原纤维密 度小、直径大,肌原纤维之间空隙较大。两种饲养方式各有优势,在生产中可考虑结合已有的饲养条件加以完善, 以生产出更优质的羊肉。     

宰后电刺激对新疆多浪羊肉品质的影响

以新疆地方品种多浪羊为研究对象,探讨电刺激对其宰后羊肉品质的影响。结果表明：电刺激显著加快多浪羊宰后肌肉pH值的下降速率（P＜0.05）,显著降低多浪羊肉贮藏损失和宰后1?d的蒸煮损失率（P＜0.05）;电刺激组肌原纤维小片化指数显著高于对照组（0?s）（P＜0.05）,宰后1?d多浪羊肉剪切力显著低于对照组（P＜0.05）。电刺激可以显著降低宰后3?d羊肉的L*值和b*值（P＜0.05）;电刺激能够显著加快宰后初期（14?h内）肌肉无氧糖酵解速率,糖原、三磷酸腺苷和二磷酸腺苷降解速率以及乳酸、一磷酸腺苷和肌苷酸生成速率（P＜0.05）。综合分析3?个不同电刺激时间处理组（30、60、90?s）,其中电刺激60?s对多浪羊肉品质改善效果最佳,可以将电压70?V、频率50?Hz、60?s的电刺激处理作为新疆多浪羊宰后电刺激的参数推广应用。     

新疆和田地区10个不同品种羊肉品质特性差异性分析

通过研究新疆和田地区10个不同品种羊肉品质特性,探寻羊肉品质特性与其理化指标的关联性。对10种不同的羊肉原料（湖羊、和田羊、澳湖羊、皮山红羊、策勒黑羊、杜泊羊、寒羊、多浪羊、杜寒羊及哈萨克羊）进行屠宰性能及理化指标测定,揭示显著影响羊肉品质特征的因素。结果表明：杜泊羊屠宰率达到57.27%,显著高于其他品种,具有较好的产肉性能和生产竞争力;多浪羊肉的脂肪含量最高;哈萨克羊肉的蛋白质含量最高,同时游离氨基酸总量最多,鲜味氨基酸含量高于其他品种,风味较佳;杜泊羊肉微量元素钙和铁含量高于其他品种,而蛋白质含量低于其他品种,策勒黑羊肉中富含锌元素,最具特色;因此,不同遗传背景的羊肉营养成分和品质特性具有差异性。     

Advances in apoptotic mediated proteolysis in meat tenderisation

Meat tenderness is considered to be one of the most important attributes of meat quality; however it is also one of the most variable. Ultimate meat tenderness is influenced by the amount of intramuscular connective tissue, the length of the sarcomere and also the proteolytic potential of the muscle. Post-mortem proteolysis by endogenous proteases causes the weakening of myofibril structures and associated proteins, which results in tenderisation. The caspase proteolytic system was first identified to be a potential contributor to post-mortem proteolysis and tenderisation in 2002. Since then research has both supported and challenged this hypothesis. The purpose of this review is to examine the experimental evidence available for caspases' involvement in post-mortem proteolysis, and to highlight cross-talk between this proteolytic system and the calpain system, a known contributor to meat tenderisation.     

Calpain-calpastatin and toughness in M. longissimus from electrically stimulated lamb and beef carcasses

Shear strength, pH, temperature, μ-calpain, m-calpain and calpastatin levels were measured over a two-week post-slaughter period in Longissimus lumborum et thoracis (LD) from six lamb and six beef carcasses. All carcasses were subjected to high voltage electrical stimulation. The toughness of the beef LD determined by a MIRINZ tenderometer at 24 h post-slaughter showed a strong correlation (r=0.91) with pH of the LD at 3 h. Beef LD toughness at 14 days was correlated (r=0.84) with initial m-calpain levels. In both lamb and beef, LD toughness at 4 and 14 days respectively was also correlated with initial levels of calpastatin (r=0.85, 0.83, respectively). The strong correlation between calpastatin and the rate of tenderisation indicates that the calpain system is closely linked to the proteolytic breakdown of myofibrillar proteins. There is also evidence of an interaction between pH and μ-calpain activity. The μ-calpain, m-calpain, calpastatin, pH and temperature kinetic changes which occurred during the post-mortem ageing of beef and lamb LD were applied to a computer program which predicted rate of meat tenderisation by calculating in situ calpain activity. The closeness of fit between the predicted rate of meat tenderisation and the observed tenderness values of beef and lamb LD indicates that the post-mortem activity of μ-calpain is the major determinant of variations in tenderness. However, application of the meat tenderisation predictive program to LD from individual animals revealed that the program was not sufficiently robust for this use.     

Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system

Tenderness has been repeatedly reported as the most important quality aspect of meat. However, a number of studies have shown that a significant portion of retail meat can be considered tough. As a consequence, a significant consumer segment is willing to pay a premium for guaranteed tender meat. However, apart from measuring the shear force, there is no reliable method to predict tenderness. Most of the branded meat programs therefore attempt to ensure eating quality by controlling some of the factors that affect tenderness. Meat tenderness is determined by the amount and solubility of connective tissue, sarcomere shortening during rigor development, and postmortem proteolysis of myofibrillar and myofibrillar-associated proteins. Given the effect of postmortem proteolysis on the muscle ultrastructure, titin and desmin are likely key substrates that determine meat tenderness. A large number of studies have shown that the calpain proteolytic system plays a central role in postmortem proteolysis and tenderization. In skeletal muscle, the calpain system consists of at least three proteases, μ-calpain, m-calpain and calpain 3, and an inhibitor of μ- and m-calpain, calpastatin. When activated by calcium, the calpains not only degrade subtrates, but also autolyze, leading to loss of activity. m-Calpain does not autolyze in postmortem muscle and is therefore not involved in postmortem tenderization. Results from a number of studies, including a study on calpain 3 knockout mice, have shown that calpain 3 is also not involved in postmortem proteolysis. However, a large number of studies, including a study on μ-calpain knockout mice, have shown that μ-calpain is largely, if not solely, responsible for postmortem tenderization. Research efforts in this area should, therefore, focus on elucidation of regulation of μ-calpain activity in postmortem muscle. Discovering the mechanisms of μ-calpain activity regulation and methods to promote μ-calpain activity should have a dramatic effect on the ability of researchers to develop reliable methods to predict meat tenderness and on the meat industry to produce a consistently tender product.     

Modelling post-mortem tenderisation-III: Role of calpain I in conditioning

A simple model is developed to show how proteolysis by calpain I can account for the variations in tenderness in electrically stimulated and nonstimulated beef pectoralis profundus muscles stored between 0°C and 30°C. As the pH of the muscle falls to about 6·1, calpain I is activated and causes proteolysis and tenderisation. The rate of tenderisation is then proportional to the concentration of calpain I which is autolysed slowly reducing its concentration and the rate of tenderisation. The activation energy for the inactivation (autolysis) of calpain I is slightly higher than that for its activity in tenderisation (proteolysis) and therefore, at higher temperatures, less tenderisation occurs. Proteolysis and tenderisation continue at a rate governed by the concentration of calpain I and the temperature until calpain I is depleted when tenderisation stops. Parameters for the activity and inactivation of calpain I were derived and were shown to predict 68% of the variation in toughness.     

Should electrical stimulation be applied when cold shortening is not a risk?

This experiment was designed to show whether delayed high voltage stimulation (ES) is more beneficial than no stimulation (NS) to secure tenderness under circumstances where rigor conditions are difficult to control due to variations in carcass size, fatness and/or chilling capacity. Ten Charolais carcasses were split during slaughter, the left sides were stimulated at 45 min post-mortem for 45 s, and the right sides were left unstimulated. The carcass sides were then chilled at a medium chilling rate. Sarcomere length measurements confirm that there was neither cold nor heat shortening in the M. longissimus (LD). LD from ES sides aged for 2 days was more tender than non-stimulated LD (NES), although prolonged ageing eroded the advantage of ES to a non-significant advantage after 14 days. Initial tenderness differences coincided with lower 24 h calpain activity, suggesting an early onset of proteolysis and ageing (tenderisation). In contrast to conventional early ES, delayed ES, appears to be beneficial for the early development of tenderness without too much interference with enzyme. Myofibril fragment length (MFL) was a good indicator of the development of tenderness during prolonged ageing but not for the early post-mortem variation in tenderness. No colour (L*, a*, b*) differences, occurred due to stimulation treatment, while drip loss was slightly higher at 24 h post-mortem for ES meat.     

Proteolytic pattern of myofibrillar protein and meat tenderness as affected by breed and aging time

The effects of breed and aging time (1, 7, 14, 21 days) were evaluated on meat tenderness and on proteolysis in 24 young bulls from Romagnola × Podolian crossbreed, Podolian and Friesian breed. Shear force decreased with aging in all breeds and showed the highest values at 1 and 7 days in Podolian meat. Myofibrillar fragmentation index significantly increased in Podolian meat throughout aging whereas in Friesian and in Crossbreed meat it increased only in the first week. Proteolysis was investigated by SDS-PAGE and 2-dimensional electrophoresis showing a different quantity and expression profile of myofibrillar proteins among breeds. In all breeds a decrease of troponin-T and an increase of troponin-T derived polypeptides during aging were observed. The highest decrease of troponin-T together with the presence of fragments of MHC in Podolian meat during aging was an outcome of a more extensive proteolysis in this breed. Data suggest that tenderness and proteolytic changes during aging are related to animal's breed.     

Muscle proteome and meat eating qualities of Longissimus thoracis of "Blonde d'Aquitaine" young bulls: A central role of HSP27 isoforms

Longissimus thoracis (LT) of 10 Blonde d'Aquitaine young bulls were sampled at slaughter. Protein composition of fresh muscle and of meat aged for 14 days was investigated by two-dimensional electrophoresis. Cooked meat properties were also evaluated by sensory analysis. When searching for early predictors of tenderness, abundance of succinate dehydrogenase (SDH) was the best common predictor of initial and overall tenderness, explaining 65.6% and 57.8% of variation of these palatability traits. Study of the evolution of the protein content during ageing allowed to identify targets of postmortem proteolysis. They were mainly structural (actin, MyBPH) and chaperone (HSP27, α-crystallin) proteins. Furthermore, in a regression analysis modelling sensory tenderness, levels of HSP27 in fresh muscle and levels of HSP27 fragments in aged meat explained up to 91% of variation in sensory scores. Data suggest the existence of an underlying HSP27-related cellular mechanism, with consequences on tenderness development.     

Up- and down-regulation of longissimus tenderness parallels changes in the myofibril-bound calpain 3 protein

The objective of this study was to utilize Ca(2+) and Zn(2+) treatments of meat to critically explore the possible role of calpain 3 in meat tenderisation. Calpains 1 and 2 were also examined for comparative purpose. Control animals plus animals infused with CaCl(2), ZnCl(2) or H(2)O were used (six lambs per treatment) to determine the temporal changes in muscle calpain 3 protein in the Longissimus thoracis et lumborum (LTL) during post-mortem storage. Concurrently, the temporal changes of; (1) shear force, (2) sarcomere length, (3) proteolysis of titin and nebulin and (4) calpains 1 and 2 proteins were also determined. Infusing LTL with Ca(2+) or Zn(2+) caused significant up- and down-regulation of LTL tenderisation, respectively, compared to water infusion and the control animals. Furthermore, the rate of breakdown of calpain 3, the rate of proteolysis of titin and nebulin and the rate of meat tenderisation during post-mortem storage of LTL in the various treatments were highly correlated. These studies suggest that calpain 3, like calpain 1, may be involved in the tenderisation of meat through limited proteolysis of specific muscle structural proteins such as titin and nebulin.