Effect of meat ultimate pH on rate of titin and nebulin degradation

The processes involved in the tenderisation of meat were studied on muscles with a range of ultimate pH values (5.4–7.0), produced by subcutaneous injection of various doses of adrenaline and exercise. The m. longissimus thoracicum et lumborum (LD) was removed from carcasses stored at 10 °C and held for 1, 3 or 6 days after slaughter, then frozen until tenderness assessment. The tenderness of meat cooked from the frozen state was determined as the force to shear samples of 10 mm × 10 mm cross-section using a MIRINZ tenderometer. The maximum toughness of 15 kgF occurred at an ultimate pH (pH_u) of about 6.0, resulting in a curvilinear relationship between tenderness and pH_u at 1 day post-slaughter. By 6 days post-slaughter, all meat had reached the same low shear value of approximately 3 kgF. SDS-PAGE patterns obtained from samples at 12, 24 and 48 hr post-slaughter showed increasing titin and nebulin degradation over time, with the slowest rate of degradation occurring at pH_u values 6.0–6.3. Titin and nebulin are known to play an important role in the stabilisation of myofibril structure, and it is suggested that the curvilinear relationship results when pH-dependent titin and nebulin degradation occurs.     

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.     

The effect of active caspase-3 on degradation of chicken myofibrillar proteins and structure of myofibrils

The objective of this study was to investigate the potential contribution of caspase-3 to meat postmortem tenderisation by examining the role of caspase-3 in the degradation of myofibrillar proteins and disruption of myofibril structure in vitro. Myofibrillar protein prepared from chicken muscle was incubated with EDTA or EDTA plus caspase-3 at 25 °C for 16 h and used for detecting muscle protein degradation and ultrastructure of myofibril. Results revealed that caspase-3 reproduced the degradation patterns of titin, nebulin and α-actinin during postmortem storage of meat, but caused little proteolysis of desmin and no appearance of 28–30 kDa peptides. Meanwhile, caspase-3 also induced the weakening in the I band adjacent to Z-lines, which occurred during meat postmortem ageing. Therefore, caspase-3 could account only for a part of the myofibrillar protein degradation observed in naturally aged meat and is likely involved in postmortem tenderisation of meat together with other endogenous proteases.     

The effects of Ca ions, EGTA and storage time on myofibrillar protein degradation, levels of Ca(2+)-dependent proteases and cathepsins B, H, L and D of ostrich skeletal muscle

The effect of Ca ions and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) on myofibrillar protein degradation showed that when ostrich iliotibialis lateralis muscle was incubated with 10 mM EGTA at 2–4 °C for 24 hr, the activity of extracted cathepsin H was unchanged compared with a buffer-incubated sample. Ca⁺⁺ had no effect on extracted cathepsin H activity, while that of Ca²⁺-dependent protease (CDP) decreased significantly (p < 0.05). Ca²⁺-treatment enhanced post-mortem changes observed in myofibrillar protein patterns (production of fragments around 30 K) that were not observed in EGTA-incubated myofibrils. The effect of storage time on shear force, CDP activity, cathepsin B, D, H and L activities and the SDS-PAGE pattern of myofibrils showed a time-dependent reduction in CDP activity. Of the cathepsins studied only cathepsin H showed a reduction (40%) in activity. The most prominent component appearing on storage at 2–4 °C had a M_r of 27 K. The incubation of myofibrils with CDP mimicked the post-mortem changes. CDP may be responsible for some of the post-mortem changes observed, although shear force measurements suggest these changes do not lead to significant 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.     

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.     

Chemical composition and structural characteristics of Arabian camel (Camelus dromedarius) m. longissimus thoracis

Saudi Arabian camels of four breeds (6 animals per breed) were used to evaluate characteristics and quality of their meat. Chemical composition, fibre cross sectional area, collagen content, muscle metabolism, cooking loss, pH at 24 h post mortem, colour values (except redness) and shear force of Longissimus thoracis (LT) muscle did not differ between the breeds. Elevated pH values and short sarcomeres reduced overall tenderisation, with a difference between myofibril fragmentation index (P < 0.001) and sarcomere length (P < 0.05) between breeds. A positive correlation was observed between the activities of the mitochondrial enzymes (r > 0.49), between the glycolytic activities (PFK and LDH) (r = 0.61) and between Myosin Heavy Chain IIa and LDH activity. The intramuscular fat content was positively associated with redness and muscle oxidative metabolism, whereas shear force had a slight positive association with collagen content and muscle glycolytic metabolism and a negative association with muscle oxidative metabolism and muscle fibre area.     

Effect of gamma irradiation on the texture and microstructure of chicken breast meat

This study investigated the textural and microstructural properties of chicken breast meat exposed to gamma irradiation ((60)Cobalt) and stored at 4?°C. Textural properties of the irradiated (n=27) and unirradiated chicken breasts (control) were determined by measuring shear force and cooking loss at day 0 and at 2-day intervals for a 14-day refrigerated storage period. In addition, microstructural changes of the irradiated and unirradiated chicken breasts were compared. Irradiated chicken breasts had more cooking loss and higher (P<0.0001) shear force than unirradiated chicken breasts. Transmission electron microscopy showed significant differences (P<0.0001) in size of myofibril units (sarcomeres) between irradiated and unirradiated breast. Shrinkage in sarcomere width (myofibril diameter) and disruption of myofibrils in irradiated breast meat were also noticed when compared with unirradiated breast meat.     

Can calcium chloride injection facilitate the ageing-derived improvement in the quality of meat from culled dairy cows?

This study investigated whether the positive effects of ageing on tenderness of meat from culled dairy cows can be facilitated by CaCl₂. Injections of 250 mM CaCl₂ solution (10% wt/wt) were performed on Longissimus dorsi samples from 32 7-yrs old cows. Samples were vacuum packaged and aged for 0, 1, 3, 5 and 7 days. Ageing alone produced lighter and less red meat with lower shear force, higher myofibrillar fragmentation and tenderness scores but also elevated lipid oxidation. For most traits investigated, CaCl₂-injected meat exhibited similar ageing effects, but drip loss increased with age. The CaCl₂-injected meat had a lower shear force and myofibrillar fragmentation increased more rapidly, but drip loss, off-flavour scores, colour stability and oxidative stability were inferior to untreated meat. Overall, it was found possible to accelerate tenderisation of such meat with CaCl₂, but only at the cost of adverse effects in some other quality traits.     

牦牛肉成熟过程中肉用品质及结构变化特点

以牦牛肉为研究对象,在宰后1、2、3、4、5、6、7、8d分别测定剪切力、PH值、失水率、钙激活酶（calpains）活性、肌原纤维超微结构,研究牦牛肉成熟过程中肉用品质及结构的变化。结果表明：在0-4℃条件下,牦牛肉的初始pH值为6．12,第4天降到最低点（极限pH值）为5．48;失水率在第4天达到最大值34．38％;Calpains活性呈逐步增高趋势,在第3-4天显著增加;成熟过程中,肌原纤维结构发生了明显的变化,第5天M线和H线消失,Z线破碎情况严重,此变化与嫩度变化特点相吻合。剪切力在第4天达到最大值55．95N,此时肉的嫩度最差,随后剪切力值开始下降,达到食用口感的适宜嫩度范围,牦牛肉完成成熟。     

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.     

Comparison of Proteolytic Enzyme Levels in Chicken,Pig, Lamb and Rabbit Muscle at Point of Slaughter: Role in Meat Tenderisation post mortem

In order to develop a clearer understanding of the biochemical mechanism underlying the process of meat tenderisation, a comparison was made of the levels of a comprehensive range of protein catabolising proteolytic enzymes in muscle from animal species known to differ markedly in rates of meat tenderisation, ie chicken (breast and thigh muscles), pig, lamb and rabbit ( longissimus dorsi muscles). Proteolytic enzyme activities were determined at point of slaughter: (i) for the following protease types using optimized specific fluorimetric assays: alanyl-, arginyl-, leucyl-, and pyroglutamyl aminopeptidases, dipeptidyl aminopeptidases III and IV, tripeptidyl aminopeptidase, proline endopeptidase, calpain and macropain (cytoplasmic proteases); dipeptidyl aminopeptidases I and II, cathepsins B, D H and L (lysosomal proteases)); (ii) using tissue homoge-nate time course assays to measure rates of structural protein degradation (via analytical electrophoresis) by endogenous cytoplasmic or lysosomal proteases. Although chicken (the most rapidly tenderising) muscles contained the highest levels of activity for most proteases measured, there was no general relationship between muscle proteolytic capacity and previously published meat tenderisation rates for the other species investigated. It would therefore appear that known differences in the rate of tenderisation in different species cannot be rationalised solely in terms of species specific differences in muscle proteolytic capacity and, whilst the latter is of importance in the tenderisation process, other potential factors should be taken into consideration.     

Lipolysis, proteolysis, physicochemical and sensory characteristics of different types of Spanish ostrich salchichon

The objective of this study was to compare three different types of salchichon: made of ostrich meat, made of ostrich meat and lean pork, and made of ostrich meat and pork belly, from physicochemical and sensory viewpoints. To evaluate the intensity of lipolysis and proteolysis produced during the ripening process, the profile and content of free fatty acids, the degree of rancidity, the non-protein, water-soluble and aminoacidic nitrogen content were determined. In addition, the composition of the fermented sausages (pH, a_w, moisture, fat, protein, ash, sodium chloride and sodium nitrite content) was analysed. From a sensory viewpoint, the organoleptic characteristics of the different types of salchichon were studied using free choice profiling. The fermented sausages had varying characteristics depending on their formulation (ostrich meat or ostrich meat plus pork) and all of them were well accepted by the panelists. This study helps characterise the different types of ostrich salchichon made in Spain.     

High and low rigor temperature effects on sheep meat tenderness and ageing

Immediately after electrical stimulation, the paired m. longissimus thoracis et lumborum (LT) of 40 sheep were boned out and wrapped tightly with a polyethylene cling film. One of the paired LT's was chilled in 15°C air to reach a rigor mortis (rigor) temperature of 18°C and the other side was placed in a water bath at 35°C and achieved rigor at this temperature. Wrapping reduced rigor shortening and mimicked meat left on the carcass. After rigor, the meat was aged at 15°C for 0, 8, 26 and 72 h and then frozen. The frozen meat was cooked to 75°C in an 85°C water bath and shear force values obtained from a 1×1 cm cross-section. The shear force values of meat for 18 and 35°C rigor were similar at zero ageing, but as ageing progressed, the 18 rigor meat aged faster and became more tender than meat that went into rigor at 35°C (P<0.001). The mean sarcomere length values of meat samples for 18 and 35°C rigor at each ageing time were significantly different (P<0.001), the samples at 35°C being shorter. When the short sarcomere length values and corresponding shear force values were removed for further data analysis, the shear force values for the 35°C rigor were still significantly greater. Thus the toughness of 35°C meat was not a consequence of muscle shortening and appears to be due to both a faster rate of tenderisation and the meat tenderising to a greater extent at the lower temperature. The cook loss at 35°C rigor (30.5%) was greater than that at 18°C rigor (28.4%) (P<0.01) and the colour Hunter L values were higher at 35°C (P<0.01) compared with 18°C, but there were no significant differences in a or b values.     

Hind-limb protein metabolism and calpain system activity influence post-mortem change in meat quality in lamb

This study is concerned with the rate of protein turnover in the hind limb muscle bed of intact lambs, the activity of calpain proteolytic system in the M. biceps femoris, and subsequent rates of myofibre breakdown and tenderisation in the M. longissimus dorsi. Feed restriction increased protein degradation in hind-limb muscle of lambs (p<0.1), with a concominant decrease in the extractable activity of calpastatin (p<0.01), the endogenous inhibitor of calpain. IGF-1 analog treatment decreased both protein degradation and assayed μ-calpain activity (p<0.05) with no effect on the activity of calpastatin. β-Agonist treatment increased hind-limb protein synthesis (p<0.01), calpastatin activity (p<0.1) and decreased (p<0.01) μ-calpain activity, but did not effect protein degradation. Significant correlations were observed between Myofibril Fragmentation Index (MFI) values during post-mortem storage and initial post-slaughter calpastatin activity at days 3 (r=-0.34, p<0.1), 5 (r=-0.58, p<0.01) and 9 (r=-0.58, p<0.1), and μ-calpain activity at days 5 (r=0.35, p<0.1) and 9 (r=0.41, p<0.05). However, stronger correlations were observed between the ratio of μ-calpain to calpastatin, an estimate of potential μ-calpain proteolytic activity, and the rate of myofibril fragmentation (r=0.75, p<0.001) and tenderisation (r=-0.64, p<0.01) during aging. These results are consistent with the calpain system being the major proteolytic system involved in myofibril fragmentation and hence aging-related tenderisation of meat.     

Purification and partial characterisation of a matrix metalloproteinase from ostrich skeletal muscle, and its activity during meat maturation

The matrix metalloproteinases (MMPs) are a homologous family of zinc proteinases that are collectively capable of catabolising the various macromolecular components of the extracellular matrix including collagens. In this study an MMP was successfully isolated and purified from ostrich skeletal muscle using Toyopearl Super Q-650S, hydroxylapatite and zinc-chelate chromatographies. The purified molecule had a molecular weight of 55K and a total of 467 amino acid residues. Purified ostrich MMP showed a pH optimum of 7 and a temperature optimum of 45°C. The activity of purified ostrich MMP was shown to be inhibited by metal chelators (1,10 phenanthroline and EDTA) and partially inhibited by soy bean trypsin inhibitor. All the functional properties of ostrich MMP were compared to previously reported values for MMPs from other sources. The MMP activities in ostrich meat during a 21-day ageing period were determined and an overall increase in MMP activities was observed.     

Optimisation of tenderisation, ageing and tenderness

Tenderness is an important part of meat acceptability and is affected by variations in production and processing. The tenderisation process was modelled on the activity of calpain proteinases. The extent of tenderisation is proportional to the level of calpains which accounts for the toughness of meat from β-adrenergic agonists. The rate of tenderisation increases with higher temperature and faster rigor development. These are responsible for the faster tenderisation in chicken, in meats following the use of electrical stimulation and in meats of high ultimate pH. Knowledge of these mechanisms of tenderisation afforded processes for optimisation of tenderness.     

Gelation of Turkey Breast and Thigh Myofibrils: Changes During Isolation of Myofibrils

The relationship between functional properties of meat and myofibrils was examined for turkey breast and thigh by determining gel-forming ability of proteins at each step in a myofibril isolation procedure. Physical properties of thermally induced gels (70°C for 30 min) were determined by torsional fracture analysis. Maximum shear stress (force) was obtained after removal of water-soluble components, whereas filtration was required to achieve maximum shear strain (deformability). Shear stress increased 150% and shear strain 59%. Changes in fracture properties were similar between meat types, and therefore, not the cause of meat type-associated differences in comminuted turkey.     

The postmortem μ‐calpain activity,protein degradation and tenderness of sheep meat from Duolang and Hu breeds

This study aimed to investigate the differences in meat tenderisation between two Chinese native sheep breeds, Duolang and Hu. The tenderness of longissimus thoracis (LT) muscle, μ‐calpain autolysis and proteolysis of myofibrillar protein was measured at 1‐ and 7‐days postmortem storage at 4 °C. At 7‐days postmortem ageing, meat from Duolang sheep was more tender compared to that from Hu sheep. The Warner–Bratzler shear force of Duolang and Hu sheep was reduced by 55.20% and 41.51% at 7 days compared with 1 day, respectively. In Duolang LT, a higher proportion of 80‐kDa μ‐calpain was autolyzed than in the Hu samples at 1‐day postmortem ageing, but they did not differ significantly at 7 days. More titin, desmin and troponin‐T were degraded in Duolang sheep compared to Hu sheep. Additionally, pH values at the different ageing time were not significantly different, indicating that pH may be not a determinant factor contributing to the tenderness difference between the two breeds. Our data suggest the earlier activation of μ‐calpain and a larger extent of proteolysis of key structural proteins may contribute to the higher rate of tenderisation of Duolang compared to Hu sheep during postmortem ageing.     

AMPK活性对宰后牛肉糖酵解、肌肉内环境及品质的影响

为探究牛肉宰后成熟过程中单磷酸腺苷活化蛋白激酶（adenosine monophosphate-activated protein kinase,AMPK）对糖酵解、肌肉内环境及品质的影响,以0.50 mol/L 5-氨基咪唑-4-甲酰胺核苷（5-amino-4-imidazolecarboxamide,AICAR）处理的西杂牛背最长肌为对象,于4 ℃进行成熟,测定宰后成熟期间肌肉AMPKα基因（PRKAA1、PRKAA2）转录量、P-AMPK表达量、AMPK活性、糖酵解水平及品质指标的变化情况。结果表明：宰后24～120?h,处理组AMPKα基因转录量、P-AMPK表达量及AMPK活力均显著高于对照组（P＜0.05）;72～168?h,处理组pH值和肌糖原含量显著低于对照组（P＜0.05）,乳酸含量显著高于对照组（P＜0.05）;12～168?h,处理组L*、b*值及ATP、ADP、AMP和IMP含量均显著高于对照组（P＜0.05）,a*值显著低于对照组（P＜0.05）;24～120?h,处理组蒸煮损失率和肌原纤维小片化指数显著高于对照组（P＜0.05）,剪切力显著低于对照组（P＜0.05）。AICAR通过激活AMPK并加快宰后糖酵解影响肌肉内环境、肉色、剪切力及肌纤维微观结构变化,加快宰后肌肉成熟进程,说明AMPK活性对宰后肌肉糖酵解及品质变化具有重要影响,且可通过调控宰后肌肉AMPK活性来调节肌肉品质。