Contribution of major structural changes in myofibrils to rabbit meat tenderisation during ageing

The contribution of major structural (myofibrillar fragmentation upon mechanical treatment) and ultra-structural (Z-line degradation, loss of electron density of M-line, transversal disruption of sarcomeres at N(2)-line level, longitudinal fissure of myofibrils, and loss of transversal alignments of Z- and M-lines) changes in myofibrils to rabbit (Oryctolagus cuniculus L.) meat tenderisation, during the ageing period (9 days at +4?°C), was studied for different types of muscle (type I, semimembranosus proprius; type IIB, semimembranosus accessorius; and type IID, psoas major). The results strongly suggest that myofibrillar structure weakening at N(2)-line level (evaluated by myofibrillar fragmentation upon mechanical homogenisation and observed by transversal disruption of sarcomeres), which is very likely mediated by cysteine endopeptidases, might be the major structural change responsible for rabbit meat tenderisation during ageing. Both myofibrillar fragmentation and transversal disruption of sarcomeres are good ageing indices for rabbit meat. The other major ultra-structural changes in myofibrils appear to have no major role in rabbit meat tenderisation at refrigeration temperatures. Finally, it is proposed that meat tenderisation during ageing depends mainly on the specific cleavage of titin molecules/filaments and nebulin molecules, at their susceptible sites located at or very close to the N(2)-line region (extensible segment and near C-terminus, respectively), mediated by cysteine endopeptidases (possibly calpains).     

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.     

Alteration of post-mortem ageing in beef by the addition of enzyme inhibitors and activators

Twenty-four hours after stunning, slices of beef M. semitendinosus were soaked in buffers at 10 °C at different pH values containing enzyme inhibitors or calcium and magnesium salts and their enzyme levels and toughness determined up to 9 days. High pH meat was tender but appeared to have the same rate of ageing as meat of normal pH. Calcium chloride accelerated ageing and produced, after completion of conditioning, more tender meat than controls. In tenderising, sodium and potassium chlorides were 43% and MgCl(2) 73% as effective as Ca salts. Cysteine proteinase inhibitors were more effective in preventing ageing than serine or aspartate inhibitors. Cysteine and aspartate inhibitors together were the most effective in preventing ageing. The inhibition of ageing by cysteine inhibitors was overcome in the presence of 30 mM CaCl(2). The results suggest a main, but not exclusive, role for calpains in meat ageing and showed a synergistic non-enzymic tenderisation by the addition of high concentrations of salts.     

Comparative action of cathepsins D, B, H, L and of a new lysosomal cysteine proteinase on rabbit myofibrils

Specific action of cathespins D, B, H, L, and of a new high Mr (molecular weight relative to hydrogen) cysteine proteinase, on rabbit muscle myofibrils was studied at pH 5·7 by following changes affecting their ATPase activities, their calcium sensitivity, their effect on the ultrastructure, as well as the electrophoretic pattern of the contractile proteins in the presence of SDS. With regard to the MgCa-enhanced ATPase activity, all these proteinases had a very similar effect. A decrease in this activity was thus noted concomitantly with a shift of the straight-line graph obtained when plotting the present acto-myosin ATPase activity versus KCl concentrations. Cathepsins D, B, L and the high Mr cysteine proteinase induced a decrease in both the calcium ATPase activity of myosin and the calcium sensitivity of myofibrils. On the contrary, the Mg-EGTA-dependent ATpase activity was increased. Except for cathepsin H, extensive hydrolysis of proteins occurred in myofibrils treated with each of the lysosomal proteinases tested. However, different specificities could be distinguished. On the one hand, cathepsins D and B affected mainly myofibrillar protein running above and below actin, respectively, on SDS-polyacrylamide gel electrophoresis; on the other hand, the high Mr cysteine proteinase exhibited broader specificity since most of the proteins were hydrolyzed irrespective of their Mr. Myofibrils incubated with cathepsins B and the high Mr cysteine proteinase showed ultrastructural modifications at the level of Z-line, M-bands and A-bands. Myofibrils treated with cathepsin D and cathepsin H appeared almost unaltered. On the basis of these characteristics, cathepsin H hardly affected myofibrils. These results provide evidence for the involvement of the lysosomal proteinases in the meat ageing process and are discussed in regard to the changes occurring at the myofibrillar level during conversion of muscle into meat.     

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.     

Modelling post-mortem tenderisation-V: Inactivation of calpains

The calpain-activity model, which allows computation of the in-situ activities of calpains, was used to predict tenderisation. Tenderisation results from the net proteolysis which is governed by the relative activities and the intramolecular inactivation of calpains.

The activity increases non-interactively with increase in pH and increase in temperature. The rate of inactivation depends interactively upon pH and temperature. At high temperature, inactivation is high and almost independent of pH. The rate of inactivation decreases with decrease in temperature, but below about 10°C it increases at low pH. Rapid rigor development produces rapid activation and tenderisation but it may be short-lived, particularly in slowly-chilled meat, producing tough meat. Rapid cooling causes rapid inactivation of calpains and can give rise to very tough meat.

Therefore, the calpain-activity model predicts the toughness often observed in PSE meats and rapidly-chilled meats without evoking structural changes dependent upon water-holding capacity or the degree of overlap of actin and myosin. Furthermore, the model demonstrates the known interactions of ageing with these conditions, interactions which cannot be explained by those structural changes alone. Variations in post-mortem activity of calpains therefore provide a single concept accounting for the variations in texture arising from variations in animal production, chilling and ageing and their interactions.     

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.     

EFFECTS OF MUSCLE PROTEASES, ENDOGENOUS PROTEASE INHIBITORS AND MYOFIBRIL FRAGMENTATION ON POSTMORTEM AGING OF GOAT MEAT

The present study was conducted to evaluate the extent of postmortem proteolysis in longissimus dorsi, biceps femoris, semimembranosus and semitendinosus goat muscles on postmortem aging at an ambient (27C) temperature. The activities of calpains and calpastatin were determined after separation on a (diethylamino)ethyl–Sephacel column (Sigma, St. Louis, MO) and cathepsin (B, B + L and H) by carboxymethyl–Sepharose column (Sigma). The results showed that the decrease in calpain I and calpastatin activities was significantly higher than that of calpain II. Cathepsin B, B + L, H and cystatin were found to fall by 30–80% after 12 h, whereas cathepsin D decreased significantly in all the muscles. The disappearance of titin 1 and nebulin, and the appearance of a 30‐kDa component were confirmed by Western blot analysis. The appearance of the 30‐kDa component reported here explains the time‐induced structural changes of myofibrils. The Z‐line degradation had occurred by 6 h postmortem. Cathepsins are not stable compared to calpains during postmortem aging, and both enzymes may play a significant role in the proteolysis of myofibrillar proteins at ambient temperature.     

Modelling post-mortem tenderisation-II: Enzyme changes during storage of electrically stimulated and non-stimulated beef

Levels of calpains I and II, cathepsins B and L and β-glucuronidase were determined in extracts of electrically stimulated and control beef M. Pectoralis profundus stored at temperatures between 0 and 30°C and varied to avoid muscle shortening. The level of lysosomal enzymes remained essentially unchanged throughout storage. The levels of calpain II were largely unaffected by the early treatments and decreased slightly throughout ageing. The level of calpain I, in both stimulated and control meats, was unaffected by temperature prior to the attainment of about pH 6·2 and thereafter the loss was accelerated at higher temperatures. In the extreme case studied, that of stimulated meat held at 15°C, 73% of the activity was lost in the first 24 h. After ageing, the level was about 11% of the initial when stored at 1°C and 25% when stored at 15°C. The exponential decay constants for the decrease in the levels of calpain I were 0·01 h(-1) at 1°C and 0·06 h(-1) at 15°C, and were the same as those for the previously determined rate of tenderisation. This suggested that the rate of proteolysis by calpain I was linked to the rate of tenderisation.     

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.     

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.     

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.     

Does the newly discovered calpain 10 play a role in meat tenderization during post-mortem storage?

The objective was to study calpain 10 during meat tenderization. Western assays were developed and changes in calpain 10, tenderization level and desmin were determined in Longissimui (LTL) during post-mortem storage. A comparison between some characteristics of calpains 1, 2 and 10 indicated differences in the pI and sub-cellular distribution. Tenderness improved gradually during post-mortem storage and was accompanied by a decline in intact desmin level. Western analysis indicated that calpain 10 is present in sheep LTL probably as two proteins (MW 73.6 and 70.7 kDa). Post-mortem storage caused a decline in the level of the 73.6 and 70.7 kDa proteins in the sarcoplasmic fraction and a concomitant increase in these proteins in the myofibrillar fraction. Changes in the myofibrillar calpain 10 proteins were strongly correlated with the rate of tenderization. To conclude, calpain 10 proteins are present in sheep LTL and the sub-cellular distribution is dynamic during post-mortem storage.     

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.     

氧化对兔肉肌原纤维蛋白结构、乳化性和凝胶性的影响研究

为探讨氧化对兔肉肌原纤维蛋白结构的氧化修饰及对肌原纤维蛋白乳化性质和凝胶质构特性的影响,本文利用0~10.0 mmol/L 2,2'-盐酸脒基丙烷(2,2'-azobis(2-amidinopropane)dihydrochloride,AAPH)热解产生的烷过氧自由基对兔肉肌原纤维蛋白进行氧化处理。结果表明,随着AAPH浓度的增加,兔肉肌原纤维蛋白游离巯基含量、内源性荧光强度和蛋白乳化稳定性不断减小,而羰基含量、二聚酪氨酸含量、蛋白粒径、乳化活性显著增加(P<0.05),表面疏水性则呈现先增后减再增的趋势。聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,SDS-PAGE)结果显示,低浓度AAPH(0~1.0 mmol/L)和高浓度(10.0 mmol/L)分别诱导了蛋白的聚集和裂解,这些结构的改变引起了肌原纤维蛋白凝胶性质的改变。适度的氧化改性(AAPH 0~1.0 mmol/L)能提高兔肉肌原纤维蛋白凝胶的质构特性,而进一步氧化(AAPH 1.0~10.0 mmol/L)则显著降低了凝胶的性能(P<0.05)。因此,烷过氧自由基的氧化修饰能显著影响兔肉肌原纤维蛋白的结构和乳化凝胶性质(P<0.05)。     

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.     

Structural changes in meat during ageing

Summary. During the ageing of bovine muscle the most notable changes in the pattern of myofibrillar cross-striations are the complete disappearance of the Z lines and the lengthening of the A bands at the expense of the I zones. It is suggested that with the disintegration of the Z lines, the actin filaments of the I zones collapse onto the myosin rods, leading to apparent A-band lengthening. It is concluded that the weakening of the normally refractory Z lines of the myofibrillar structures is an event closely related to meat ageing.     

Relationship between proteolytic changes and tenderness in prerigor lactic acid marinated beef

A meat tenderising procedure involving injection of a lactic acid solution into prerigor muscle was investigated using beef M pectoralis profundus. The distribution of lysosomal enzymes in subcellular fractions, densities of myofibrillar protein bands after SDS‐PAGE and shear force were measured in non‐injected, 0.5 M and 1.0 M lactic‐acid‐injected samples during a 21 days ageing period. The activities of cathepsin B + L and β‐glucuronidase in the soluble fraction increased with level of lactic acid and with time post‐mortem (P < 0.001). Lactic acid and storage decreased densities of SDS‐PAGE bands migrating at the position of myosin heavy chain (MHC) and α‐actinin and increased densities of a 150 kDa band (P < 0.01). SDS‐PAGE of isolated perimysium cleaved with CNBr showed proteolytic cleavage of collagen after prolonged storage. Lactic acid injection significantly reduced shear force (P < 0.001). The cathepsin B + L activity in the soluble fraction correlated to shear force (r = −0.8), the degradation of MHC and α‐actinin (r = −0.88 and −0.90) and the generation of the 150 kDa fragment (r = 0.90) but not to the generation of a 31 kDa fragment (r = 0.05). A major part of the tenderness improvement after lactic acid injection was complete at 24 h post‐mortem, and was therefore due to a rapid process, eg pH‐induced swelling of the muscle structure. The data on enzyme activities and protein degradation, however, suggested that the action of lysosomal cathepsins also contributed to textural changes. © 1999 Society of Chemical Industry     

Early post-mortem conditions and the calpain/calpastatin system in relation to tenderness of double-muscled beef

Early post mortem temperature, pH, sarcomere length, colour, water holding capacity, calpain/calpastatin activities and myofibrillar protein concentrations (semi-quantative SDS-PAGE), measured at different times post mortem on 153 double-muscled Belgian Blue White bulls, were related to shear force (SF) measurements. The M. longissimus thoracis tenderised up to 8 days post mortem. SF was slightly correlated with temperature at 3 h post mortem (r = 0.20, p = 0.015), but not with pH early post mortem. Higher ultimate pH values measured at 24 h post mortem were related to darker meat, lower cooking losses and shorter sarcomere lengths. Sarcomere length was significantly related to SF, even after 12 days of ageing, suggesting that proteolytic activity was not able to overcome shortening. Up to 12 days post mortem, calpastatin and m-calpain activities were significantly correlated with SF suggesting a considerable role of calpains in meat tenderization of double-muscled Belgian Blue White bulls, although at 24 h post mortem, μ-calpain could no longer be detected. Titin, nebulin, filamin and troponin-T were degraded during the ageing period. Troponin-T was degraded by 80% between day 1 and 8. At 8 days post mortem its concentration was significantly correlated with 30 kDa (r = 0.63, p = 0.00), shear force (r = 0.39, p = 0.00), calpastatin activity at 1 day post mortem (r = 0.29, p = 0.01) and with the m-calpain/ calpastatin activity ratio at 1 day post mortem (r = -0.43, p = 0.00).     

Studies in Meat Tenderness. 6. The Nature of Myofibrillar Proteins Extracted from Meat During Aging

SUMMARY– A study has been made of the myofibrillar proteins extracted from beef and rabbit meat by a buffer that dissociates the actomyosin complex of the muscle cell. Myosin, which constitutes 50-52 per cent of the myofibrillar protein, can be wholly extracted throughout aging, whereas actin can be extracted in increasing amounts as aging proceeds. In contrast, tropomyosin cannot be extracted and remains firmly held within the myofibrillar structures throughout aging. A complex mixture of extra protein, soluble at low ionic strength, is also released in increasing quantity during aging. It is proposed that in meat aging there is a progressive loss of the tensile strength of the myofibrillar component of muscle brought about by the weakening and final dissolution of the Z-band structures. Such a disintegration would lead to the observed changes in the extractabilities of the myofibrillar proteins.