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.     

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.     

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.     

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.     

Identification of biomarkers of meat tenderisation and its use for early classification of Asturian beef into fast and late tenderising meat

BACKGROUND: The objective of this work was to study the post‐mortem evolution of potential biomarkers (µ‐calpain activity and proteolytic profile) of meat tenderisation in bovine longissimus dorsi (LD) muscle from several biotypes coming from two beef breeds (‘Asturiana de los Valles’ and ‘Asturiana de la Montaña’) and showing different levels of muscular hypertrophy (mh/mh, mh/+, + /+). RESULTS: LD samples were taken at 2, 12, 24 and 48 h and 3, 7, 14 and 21 days post‐mortem. The presence of muscular hypertrophy produced a faster rate of pH decline, faster exhaustion of µ‐calpain activity and earlier occurrence of proteolytic changes. Changes in the electrophoretic pattern of some peptides from sarcoplasmic (glyceraldehyde‐3‐phosphate dehydrogenase) and myofibrillar (troponin T and troponin I) muscle extracts within the first 24 h significantly correlated with meat toughness and allowed accurate discrimination of meat products into two groups: (1) fast tenderising meat, coming from mh‐biotypes, and (2) late tenderising meat, from normal (+/+) biotypes. CONCLUSION: Early monitoring (within 24 h after slaughter) of selected biomarkers in LD muscle allowed accurate prediction of ultimate meat toughness and could be used in the meat industry as a tool for early classification of beef into fast and late tenderising meat. Copyright © 2012 Society of Chemical Industry     

Inhibition of protease activity. Part 1. The effect on tenderness and indicators of proteolysis in ovine muscle

To examine the effect of particular enzyme groups on tenderness specific cysteine protease inhibitors were injected into muscle early post-mortem. The protease enzyme inhibitor E-64 was injected into the m. longissimus thoracis et lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe-Ala-CHN(2). The left LTL (control) was injected with saline (0.25 M NaCl). To create variation in the rate of pH decline alternate carcasses were electrically stimulated (low voltage). The LTL was divided into cranial and caudal portions and aged for 1 or 2 days. Muscle samples at 1 day post-mortem were used for measurement of osmolality and sarcomere length (n=48), and others at 1 and 2 days post-mortem for shear force determination (n=96). The myofibrillar fragmentation index (MFI) was determined on samples taken at pH 6.2 and 1 and 2 days post-mortem (n=144). Other muscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=215). These samples were used for determination of protein solubility and the concentration of free amino acids. Stimulation caused a faster (P<0.05) decline in pH. There was no effect of stimulation (P>0.05) on shear force values, but injection of inhibitor and ageing both had effects (P<0.001). The inhibitor E-64 prevented any improvement in tenderness with ageing, whereas the inhibitor Z-Phe-Ala-CHN(2) and the control samples showed a similar ageing response. In the latter two treatments there was an average reduction of 1 kg in shear between 1 and 2 days post-mortem, whilst the inhibitor E-64 maintained shear force on average 2 kg higher than control samples. Injection and ageing had an effect on MFI (P<0.001) and there was an interaction (P<0.05) between stimulation and ageing for MFI, such that as stimulated muscle aged the rate of change of MFI was greater. There was an interaction between injection and ageing (P<0.05) for protein solubility such that samples treated with E-64 showed a minimal increase in protein solubility with ageing, whereas in samples treated with Z-Phe-Ala-CHN(2) and the control samples there was a significant increase. There was also an interaction between stimulation and ageing such that between sampling at pH 6.0 and 2 days post-mortem, stimulated muscle exhibited greater solubility (P<0.05). There were no effects (P>0.05) on the concentration of free amino acids. The evidence indicated that the cysteine proteases were responsible for post-mortem proteolysis and tenderisation, in particular the calpains, whereas the cathepsins (B and L) were unlikely to contribute to proteolysis and subsequent tenderisation in meat.     

CALCIUM CHELATORS AND SALT EXTRACTED MYOFIBRILLAR PROTEIN INJECTION INTO LEAN PRERIGOR BEEF MUSCLE: EFFECTS ON TENDERNESS INDICES

Prerigor beef longissimus muscles were removed within 40 min postmortem and injected 10% (within 2 h postmortem) with either a chelator (alginate, 3%; haametaphosphate, 3%; EDTA, 0.13%; citrate, 0.13%), meat slurry (45%), or a myofibrillar meat extract (3.7% protein) to evaluate the effects of each injection on the physical and chemical properties compared to a control and a water only injected treatment. Gross muscle shortening was not inhibited The water only, meat extract and citrate treatments had shorter (P<0.05) sarcomeres than the control. The meat shy, alginate, phosphate, and EDTA treatments produced sarcomere lengths equivalent to the control. The control had the lowest (P<0. 05) pH and percentage moisture. However, there were no differences in percentage cooking losses. Although variations in sarcomere length were apparent, these treatments did not improve tenderness (P>0.05) as measured by Warner-Bratzler shear.     

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.     

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.     

Pelvic suspension and fast post-mortem chilling: Effects on technological and sensory quality of pork - A combined NMR and sensory study

The effects of pre-rigor excision, which results in a fast post-mortem chilling; pelvic suspension, which results in a muscular stretching; control treatment of m. longissimus dorsi on water distribution measured by (1)H NMR relaxometry and on sensory properties were investigated in two sub-studies including a total of 16 pigs. Determination of sarcomere length revealed significant effects of the treatments on the degree of contraction, as pre-rigor excision and pelvic suspension resulted in shorter and longer sarcomere length, respectively. In addition, an effect of treatment on pH measured at 24h post-mortem was found, as pre-rigor excision was associated with a higher ultimate pH. NMR measurements revealed that water distribution in the meat was affected to a minor degree by the various treatments. However, an interaction between treatment and ageing period was observed, as the data demonstrated an effect of pre-rigor excision on water distribution in the cooked samples when meat was only aged for 2 days, but this effect was eliminated when meat was aged for 5 days. The effect of pre-rigor excision on water distribution in cooked meat after 2 days of ageing is suggested to reflect structural constraints in the meat that are eliminated during ageing. Sensory analyses demonstrated strong interactions between treatment and position on the muscle and between treatment and ageing period. However, in general pre-rigor-excised meat was sensed as significantly juicier compared with the other two treatments. Accordingly, the study demonstrates that under the present conditions the ultimate pH is more important for juiciness than the sarcomere length.     

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.     

Effects of Rigor Status during High‐Pressure Processing on the Physical Qualities of Farm‐Raised Abalone (Haliotis rufescens)

High‐pressure processing (HPP) is used to increase meat safety and shelf‐life, with conflicting quality effects depending on rigor status during HPP. In the seafood industry, HPP is used to shuck and pasteurize oysters, but its use on abalones has only been minimally evaluated and the effect of rigor status during HPP on abalone quality has not been reported. Farm‐raised abalones (Haliotis rufescens) were divided into 12 HPP treatments and 1 unprocessed control treatment. Treatments were processed pre‐rigor or post‐rigor at 2 pressures (100 and 300 MPa) and 3 processing times (1, 3, and 5 min). The control was analyzed post‐rigor. Uniform plugs were cut from adductor and foot meat for texture profile analysis, shear force, and color analysis. Subsamples were used for scanning electron microscopy of muscle ultrastructure. Texture profile analysis revealed that post‐rigor processed abalone was significantly (P < 0.05) less firm and chewy than pre‐rigor processed irrespective of muscle type, processing time, or pressure. L values increased with pressure to 68.9 at 300 MPa for pre‐rigor processed foot, 73.8 for post‐rigor processed foot, 90.9 for pre‐rigor processed adductor, and 89.0 for post‐rigor processed adductor. Scanning electron microscopy images showed fraying of collagen fibers in processed adductor, but did not show pressure‐induced compaction of the foot myofibrils. Post‐rigor processed abalone meat was more tender than pre‐rigor processed meat, and post‐rigor processed foot meat was lighter in color than pre‐rigor processed foot meat, suggesting that waiting for rigor to resolve prior to processing abalones may improve consumer perceptions of quality and market value.     

Changes in degradation and phosphorylation level of titin in three ovine muscles during postmortem

The objective of this study was to investigate the degradation of titin and its phosphorylation level in three muscles from sheep. The MFI and pH from the longissimus lumborum (LL), semimembranosus (SM) and psoas major (PM) muscles were measured at 30 min, 1, 2, 7, 14, 21 and 28 days postmortem. Myofibrillar proteins were extracted, separated by SDS‐PAGE and quantified by phosphor‐specific staining. Phosphorylation of titin was predicted by Pro‐Q Diamond‐SYPRO Ruby staining. Two days after exsanguination, the pH and MFI of the PM were higher than those of the LL and SM muscles (P < 0.05). The sarcomere length of the PM muscle was also longer than that of the LL and SM muscle (P < 0.05). PM muscles had a highest phosphorylation level (P < 0.05) at 0.5 h postmortem and showed the greatest degree of titin degradation over 28 days. This suggests that phosphorylation of titin might accelerate its degradation.     

Muscle fiber properties and thermal stability of intramuscular connective tissue in porcine M. semimembranosus

BACKGROUND: Strips can be easily peeled from raw destructured pork (M. semimembranosus, SM muscle) by hand but in normal meat these strips break. In general, destructured meat is pale in color. Porcine SM muscles have thick muscle fibers which could predispose them to destructuration. This study investigated whether the onset and peak temperatures of thermal shrinkage (T_o and T_p) of intramuscular connective tissue from SM muscles were associated with muscle fiber thickness, capillary density or extracellular space. We also investigated whether these muscle fiber properties of destructured muscles differed from those of normal muscles. RESULTS: The destructured and normal muscles were similar in muscle fiber cross‐sectional area, capillary density, extracellular space and sarcomere length. T_o correlated negatively with sarcomere length. The water content of differential scanning calorimetry samples consisting of intramuscular connective tissue was higher in destructured muscles than in normal muscles. CONCLUSION: Muscle fiber properties (muscle fiber cross‐sectional area and sarcomere length) and capillary density are similar in destructured and normal SM muscles. T_o and T_p of intramuscular connective tissue are similar in destructured and normal muscles. Muscle fiber properties show no association with the thermal shrinkage properties of intramuscular connective tissue. Copyright © 2009 Society of Chemical Industry     

The roles of the proteasome, and cathepsins B, L, H and D, in ostrich meat tenderisation

As very little research has been conducted on ostrich meat tenderisation, this study aims at investigating the roles of the proteasome and cathepsins B, L, H, and D in the tenderisation process. The enzyme activities in meat from eight ostriches during a 12-day ageing period and the corresponding physical characteristics (e.g. pH, shear force) and myofibril patterns were determined. After 12 days, substantial high remaining activities were found, especially of the proteasome, thus implicating their possible roles in the tenderisation process. The mean shear force values, however, showed no improvement in tenderness, but the myofibril patterns showed the appearance of a M(r) 32 K component. Myofibril degradation studies of the proteasome, analysed electrophoretically, also revealed a possible role of the proteasome, but under activating conditions. This study provides further insights into the tenderisation process, particularly of ostrich meat, which may ultimately be used for the advantageous manipulation of the process.     

低压电刺激对不同部位宰后牦牛肉肌原纤维蛋白水解的影响

研究电刺激对不同部位牦牛肉宰后成熟过程中微观结构及肌原纤维蛋白水解的影响。将牦牛宰后5min内进行低压电刺激（21V,50Hz,90s）并冷却排酸,于宰后0、1、3、5d取前部肱三头肌（TB）中部背最长肌（ML）和后部半膜肌（SM）,分析其肌节长度、肌纤维直径、MFI指数、肌原纤维超微结构和全肌肉蛋白电泳图谱。结果表明:电刺激使不同部位牦牛肉肌节长度显著变化（p<0.05）,肌纤维直径平均缩小6%、5%和9%;TB、ML和SM宰后0⁵d MFI值分别上升54%、55%和92%,SM嫩度显著增大（p<0.01）;不同部位牦牛肉肌原纤维超微结构中挛缩带占有率分别为23.9%、26.3%和31.2%,肌原纤维结构形变溶解;SDS-PAGE电泳图谱显示,TB、ML和SM部肌原纤维蛋白均在48⁷5ku之间降解彻底。本研究证实了电刺激缩短牦牛肉宰后成熟时间,对不同部位牦牛肉嫩度改善,特别是对肉质较差的SM效果显著。      

Effect of high-oxygen atmosphere packaging on mechanical properties of single muscle fibres from bovine and porcine <Emphasis Type="Italic">longissimus dorsi</Emphasis>

The effect of storage in high-oxygen atmosphere packaging for 48 h at 4 °C on the myofibrillar component of meat toughness was studied performing tensile tests on single muscle fibres, isolated from beef and pork longissimus dorsi (LD). Storage of bovine LD for 48 h in the presence of oxygen significantly increased the breaking strength of single muscle fibres when compared with storage in a 100% nitrogen atmosphere. In contrast, the breaking strength of porcine LD stored for 2 days post-mortem in the presence of oxygen in the packages was not influenced.     

Effect of electrical stimulation,delayed chilling and post‐mortem aging on the quality of M. longissimus dorsi and M. biceps femoris of grass‐fed steers

BACKGROUND: Roughage‐based low‐input beef production systems are gaining increasing interest owing to the perceived ecological advantages and potential health benefits associated with the favourable fatty acid composition of such beef. The low plane of nutrition may on the other hand yield less tender beef by affecting growth, carcass weight and fatness and therefore, indirectly, early post‐mortem (p.m.) proteolytic enzyme activity and sarcomere shortening. This study aimed to examine delayed chilling and electrical stimulation as promising techniques to control early p.m. muscle metabolism in a way that improves the tenderness of beef from purely grass‐fed steers in comparison with that from steers receiving a finishing diet with concentrates. RESULTS: Electrical stimulation decreased the pH at 1.5 and 3 h p.m. in the M. longissimus dorsi (LD) and M. biceps femoris (BF) of the treated carcass sides as well as the maximum shear force in the LD, while delayed chilling had no effect on pH or texture. The interactions of carcass fatness with electrical stimulation (P = 0.025) and delayed chilling (P = 0.089) indicated more pronounced effects of the p.m. treatments on beef texture in leaner carcasses. CONCLUSION : Electrical stimulation, but not delayed chilling, could markedly improve pasture beef texture and reduce the aging period needed for proper tenderisation. Copyright © 2008 Society of Chemical Industry     

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.     

Effect of electrical stimulation on quality of tenderstretched chevon sides

Thirty male Black Bengal goats 5-8 years of age, slaughtered at the Kolkata slaughter house, India, by the traditional 'halal' method, were used to assess the effect of electrical stimulation on tenderstretched sides of chevon carcasses. Five groups (T1, T2, T3, T4 and T5) were treated with variable voltages of 35, 110, 330, 550, 1100 with fixed 50Hz and 10pulses/s for a duration of 3min. Different quality parameters, such as, fibre diameter, sarcomere length, water holding capacity (WHC), pH, microbial content and taste panel score were studied at 24 and 48h post-stimulation. pH, WHC and fibre diameter decreased whereas sarcomere length showed an increasing trend in electrically stimulated meat, which was also microbiologically more stable. The non-stimulated control meat was tougher than the electrically stimulated chevon as evident through taste panel scoring. Treatment 3 (T3), i.e. 330V, 50Hz and 10pulses/s, showed superiority over the other four treatments in the majority of the important meat quality parameters.