One of the most common causes of unacceptability in meat quality is toughness. Toughness is attributed to a range of factors including the amount of intramuscular connective tissue, intramuscular fat, and the length of the sarcomere. However, it is apparent that the extent of proteolysis of key proteins within muscle fibres is significant determinant of ultimate tenderness. The objective of this manuscript is to describe the main endogenous proteolytic enzyme systems that have the potential to be involved in muscle post-mortem proteolysis and whether the experimental evidence available supports this involvement.

No abstract

The objective of this study was to investigate the protease family caspases in skeletal muscle and their potential contribution to postmortem proteolysis and meat tenderization. Ten Large White gilts were slaughtered, and samples of LM were taken at 0, 2, 4, 8, 16, 32, and 192 h after slaughter and immediately snap frozen in liquid nitrogen. Samples were subsequently analyzed for caspase 3/7 and caspase 9 activity, protein levels of known caspase substrates, alpha II spectrin and poly (ADP-ribose) polymerase (PARP), as well as, at 192 h, shear force. Specific degradation products of alpha II spectrin and PARP, which are known indicators of caspase activity, and apoptosis were detected on immunoblots of muscle samples taken over the postmortem period. The relationships between the changes observed in caspase activities and protein levels of PARP and spectrin across the entire postmortem conditioning period were investigated (n = 70). Protein levels of alpha II spectrin cleavage products across the conditioning period were found to correlate positively to caspase 3/7 activity (r = 0.38, P = 0.003) and caspase 9 activity (r = 0.32, P = 0.012), indicating that caspase-mediated cleavage was occurring in situ. There was a negative relationship between shear force and the 0 to 32 h ratio of caspase 3/7 (r = -0.62, P = 0.053) and caspase 9 activities (r = -0.68, P = 0.044). In addition, there was also a negative relationship between shear force and the level of the caspase-generated alpha II spectrin 120 kDa degradation product (r = -0.75, P = 0.012). The findings of this study indicate that changes in caspase activity and caspase-mediated cleavage take place in muscle during the conditioning period, and this could be associated with the development of tender meat.

The objective of this study was to investigate the potential role of the caspase protease family in meat tenderisation by examining if caspase 3 was capable of causing myofibril protein degradation. Full-length human recombinant caspase 3 (rC3) was expressed in Escherichia coli and purified. The rC3 was active in the presence of myofibrils isolated from porcine longissimus dorsi muscle (LD) and retained activity in a buffer system closely mimicking post mortem conditions. The effect of increasing concentrations of rC3, incubation temperature, as well as incubation time on the degradation of isolated myofibril proteins were all investigated in this study. Myofibril protein degradation was determined by SDS-PAGE and Western blotting. There was a visible increase in myofibril degradation with a decrease in proteins identified as desmin and troponin I and the detection of protein degradation products at approximately 32, 28 and 18 kDa with increasing concentrations of rC3. These degradation products were analysed using MALDI-TOF mass spectrometry and identified to occur from the proteolysis of actin, troponin T and myosin light chain, respectively. The production of these degradation products was not inhibited by 5 mM EDTA or semi-purified calpastatin but was inhibited by the caspase-specific inhibitor Ac-DEVD-CHO. The temperature at which isolated myofibrils were incubated with rC3 was also found to affect degradation, with increasing incubation temperatures causing increased desmin degradation and cleavage of pro-caspase 3 into its active isoform. Incubation of isolated myofibrils at 48C for 5 days with rC3 resulted in the visible degradation of a number of myofibril proteins including desmin and troponin I. This study has shown that rC3 is capable of causing myofibril degradation, hydrolysing myofibril proteins under conditions that are similar to those found in muscle in the post mortem conditioning period.

Longissimus thoracis steaks from steers (n = 464) with 0 to 50% inheritance of Angus, Charolais, Gelbvieh, Hereford, Limousin, Red Angus, and Simmental were evaluated during 6 d of display to assess genetic contributions to color stability. Color space values [CIE L* (lightness), a* (redness), b* (yellowness)], chroma, color change (DeltaE), and surface metmyoglobin (K/S 572/525) were determined on d 0 and 6 of display. Myoglobin concentration was highly heritable (0.85), but ultimate pH was weakly heritable (0.06). Day 0 L* values were moderately heritable (0.24). Variation in metmyoglobin, L*, and DeltaE on d 6 was moderately explained by genetic factors (41, 40, and 29%, respectively). Change during display was moderately heritable for a* (0.31), b* (0.23), chroma (0.35), and surface metmyoglobin (0.29). At the start of display, Angus steaks had greater (P < 0.05) L* values than those from all breeds except Charolais. On d 6, Angus steaks had greater (P < 0.05) L* (50.0) values than Gelbvieh, Hereford, and Simmental steaks (46.1, 44.0, and 44.5, respectively). Day 0 values for a*, b*, chroma, and DeltaE were not affected by breed (P > 0.05). On d 6, a* values were greater (P < 0.05) for Charolais and Limousin steaks (31.1 and 30.5) than Angus, Hereford, and Red Angus steaks (27.4, 27.7, and 26.3, respectively). Thus, a* changed less (P < 0.05) in Charolais and Limousin steaks (1.8 and 2.6, respectively) vs. steaks from other breeds. Day 6 b* values were greater (P < 0.05) in Charolais (24.5) and Limousin steaks (24.0) vs. Gelbvieh (22.2), Hereford (21.9), and Red Angus steaks (21.4). Thus, b* values changed less (P< 0.05) in Charolais and Limousin steaks (1.5 and 1.7, respectively) than in Angus, Gelbvieh, Hereford, and Red Angus steaks (4.3, 3.8, 4.4, and 5.1, respectively). After 6 d of display, Charolais and Limousin steaks had greater chroma (P < 0.05; 39.5 and 38.8, respectively) compared with Angus, Hereford, and Red Angus steaks (35.4, 35.3, and 33.9, respectively). Less (P < 0.05) change in chroma occurred for Charolais and Limousin (2.1 and 2.8, respectively) than in Angus, Gelbvieh, Hereford, and Red Angus steaks (7.1, 6.6, 7.4, and 9.0, respectively). Myoglobin concentration was less for Charolais and Limousin (P < 0.05; 2.77 and 2.72, respectively) compared with Gelbvieh, Red Angus, and Simmental steaks (3.62, 3.43, and 3.71, respectively). Breeds did not differ in pH (P > 0.05). These data suggest Charolais- and Limousin-carcasses produced steaks with greater lean color stability than Angus, Hereford, and Red Angus carcasses. Furthermore, these findings suggest that genetics contribute substantially to animal-to-animal variation in lean color, particularly in maintaining color.

The objective of this experiment was to determine whether the caspase proteolytic system has a role in postmortem tenderization. Six ewes and 6 wethers that were noncarriers and 6 ewes and 6 wethers that were expressing the callipyge gene were used for this study. Caspase activities were determined in LM at 7 different time points during the postmortem storage period: 0 h, 4 h, 8 h, 24 h, 2 ci, 7 ci, and 21 cl and in semimembranosus (SM) and infraspinatus (IS) muscles at 0 h, 8 h. 24 h, and 7 d from callipyge and noncallipyge (normal) lambs. Calpastatin activity was determined at 0 h, 2 d. 7 d, and 21 d and slice shear force measured at 2, 7, and 21 d in the LM. Calpastatin activity and slice shear force were greater in LM from callipyge lambs than normal lambs at each time point (P < 0.001 and P < 0.0001, respectively). Caspases 3 and 7 are executioner caspases, and their combined activity was found to decrease during the postmortem storage period in LM, SM, and IS muscles from calhpyge and normal lambs. Similarly, activity of the initiator caspase (caspase 9) decreased (P < 0.05) in all 3 muscles across the postmortem storage period in callipyge and normal lambs, and its decrease in activity preceded that of the executioner caspases 3/7. A positive relationship also was detected between caspase 9 and caspase 3/7 in LM, SM. and IS muscles (P < 0.0001, r = 0.85, r = 0.86, r = 0.84, respectively), which is consistent with caspa.se 9 being responsible for the cleavage and activation of the executioner caspases (caspase 3/7) downstream. Caspase 3/7 and caspase 9 activities at 8 h in SM were greater in normal lamb than callipyge lamb (P < 0.05), with a trend for caspase 3/7 activity to be greater at 24 h postmortem (P = 0.0841). There also was a trend for caspase 3/7 activity to be greater in LM at 21 cliii nornial lamb than in callipyge lamb (P = 0.053), although there were no differences detected in caspase activities between genotypes in the IS muscle, which is not affected by the callipyge gene. A negative relationship also was detected between peak caspa.se 3/7 activity at 8 Ii in LM from normal lambs and calpastatin activity at 0 and 2 d (r =-0.65, r =-0.68, respectively, P < 0.05). This relationship was not observed in LM from callipyge lambs, suggesting that caspase 3/7 may be cleaving calpastatin in normal lambs but the level of calpastatin in callipyge lambs is such that caspase 3/7 cannot degrade it sufficiently to overcome the increased content of caipastatin, and thus, calpastatin activity is the overriding factor in postmortem proteolysis in these animals. There was no direct evidence from this study that ca.spases have a significant role in postmortem tenderization, but they may have some role through calpastatin degradation.

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Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H₂O₂) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H₂O₂, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H₂O₂, and recombinant caspase 3 increased (P < 0.05) proteolysis of myofibril proteins, whereas DEVD inhibited degradation (P < 0.05). Proteolysis of myofibrillar proteins increased with incubation time (P < 0.0001), and incubation time × treatment interactions (P < 0.05) indicated that the treatment effects did not all occur at the same rate. This study has shown that manipulation of the caspase system through induction or inhibition of activity can affect degradation of myofibrillar proteins, providing further evidence that the caspase system could be involved in postmortem proteolysis and tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs.