A number of studies have suggested that Z-disk degradation is a major factor contributing to postmortem tenderization. These conclusions seem to have been based largely on experimental findings showing that the calpain system has a major role in postmortem tenderization, and that when incubated with myofibrils or muscle strips, purified calpain removes Z-disks. Approximately 65 to 80% of all postmortem tenderization occurs during the first 3 or 4 d postmortem, however, and there is little or no ultrastructurally detectable Z-disk degradation during this period. Electron microscope studies described in this paper show that, during the first 3 or 4 d of postmortem storage at 4 degrees C, both costameres and N2 lines are degraded. Costameres link myofibrils to the sarcolemma, and N2 lines have been reported to be areas where titin and nebulin filaments, which form a cytoskeletal network linking thick and thin filaments, respectively, to the Z-disk, coalesce. Filamentous structures linking adjacent myofibrils laterally at the level of each Z-disk are also degraded during the first 3 or 4 d of postmortem storage at 4 degrees C, resulting in gaps between myofibrils in postmortem muscle. Degradation of these structures would have important effects on tenderness. The proteins constituting these structures, nebulin and titin (N2 lines); vinculin, desmin, and dystrophin (three of the six to eight proteins constituting costameres); and desmin (filaments linking adjacent myofibrils) are all excellent substrates for the calpains, and nebulin, titin, vinculin, and desmin are largely degraded within 3 d postmortem in semimembranosus muscle. Electron micrographs of myofibrils used in the myofibril fragmentation index assay show that these myofibrils, which have been assumed to be broken at their Z-disks, in fact have intact Z-disks and are broken in their I-bands.

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.

Relationships between temperament and a range of performance, carcass, and meat quality traits in young cattle were studied in 2 experiments conducted in New South Wales (NSW) and Western Australia (WA), Australia. In both experiments, growth rates of cattle were assessed during backgrounding on pasture and grain finishing in a feedlot. Carcass and objective meat quality characteristics were measured after slaughter. Feed intake and efficiency during grain finishing were also determined in NSW. Brahman (n = 82 steers and 82 heifers) and Angus (n = 25 steers and 24 heifers) cattle were used in the NSW experiment. In NSW, temperament was assessed by measuring flight speed [FS, m/s on exit from the chute (crush)] on 14 occasions, and by assessing agitation score during confinement in the crush (CS; 1 = calm to 5 = highly agitated) on 17 occasions over the course of the experiment. Brahman (n = 173) and Angus (n = 20) steers were used in the WA experiment. In WA, temperament was assessed by measuring FS on 2 occasions during backgrounding and on 2 occasions during grain feeding. At both sites, a hormonal growth promotant (Revalor-H, Virbac, Milperra, New South Wales, Australia) was applied to one-half of the cattle at feedlot entry, and the Brahman cattle were polymorphic for 2 calpain-system markers for beef tenderness. Temperament was not related (most P > 0.05) to tenderness gene marker status in Brahman cattle and was not (all P > 0.26) modified by the growth promotant treatment in either breed. The Brahman cattle had greater individual variation in, and greater correlations within and between, repeated assessments of FS and CS than did the Angus cattle. Correlations for repeated measures of FS were greater than for repeated assessments of CS, and the strength of correlations for both declined over time. Average FS or CS for each experiment and location (NSW or WA × backgrounding or finishing) were more highly correlated than individual measurements, indicating that the average values were a more reliable assessment of cattle temperament than any single measure. In Brahman cattle, increased average FS and CS were associated with significant (P < 0.05) reductions in backgrounding and feedlot growth rates, feed intake and time spent eating, carcass weight, and objective measures of meat quality. In Angus cattle, the associations between temperament and growth rates, feed intake, and carcass traits were weaker than in Brahmans, although the strength of relationships with meat quality were similar.

The objective of this investigation was to test the hypothesis that -calpain is largely responsible for postmortem proteolysis of muscle proteins. To accomplish this objective, we compared proteolysis of known muscle proteins in muscles of wild type and micro-calpain knockout mice during postmortem storage. Knockout mice (n = 6) were killed along with control mice (n = 6). Hind limbs were removed and stored at 4 degrees C. Muscles were dissected at 0, 1, and 3d postmortem and subsequently analyzed for degradation of nebulin, dystrophin, metavinculin, vinculin, desmin, and troponin T. In a separate experiment, hind limb muscles from knockout (n = 4) and control mice (n = 4) were analyzed at 0, 1, and 3 d postmortem using casein zymography to confirm that mu-calpain activity was knocked out in muscle and to determine whether or not m-calpain is activated in murine postmortem muscle. Cumulatively, the results of the first experiment indicated that postmortem proteolysis was largely inhibited in micro-calpain knockout mice. The results of the second experiment established the absence of micro-calpain in the muscle tissue of knockout mice and confirmed the results of an earlier study that m-calpain is active in postmortem murine muscle. The results of the current study show that even in a species in which m-calpain is activated to some extent postmortem, micro-calpain is largely responsible for postmortem proteolysis. This observation excludes a major role for any of the other members of the calpain family or any other proteolytic system in postmortem proteolysis of muscle proteins. Therefore, understanding the regulation of micro-calpain in postmortem muscle should be the focus of further research on postmortem proteolysis and tenderization of meat.

The present experiment was conducted to determine whether calpastatin inhibits only the rate, or both the rate and extent, of calpain-induced postmortem proteolysis. Biceps femoris from normal (n = 6) and callipyge (n = 6) lamb was stored for 56 d at 4 degrees C. Calpastatin activity was higher (P < .05) in the callipyge muscle at 0 and 14 d postmortem, but not at 56 d postmortem. The activity of mu-calpain did not differ between normal and callipyge biceps femoris at 0 and 56 d postmortem (P > .05), but was higher at 14 d postmortem in the callipyge muscle (P < 0.05). The activity of m-calpain was higher in the callipyge muscle (P < 0.05). Western blot analyses of titin, nebulin, dystrophin, myosin heavy chain, vinculin, alpha-actinin, desmin, and troponin-T indicated that postmortem proteolysis was less extensive in callipyge than in normal biceps femoris at all postmortem times. The results of this experiment indicate that calpastatin inhibits both the rate and extent of postmortem proteolysis.

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.

No abstract

The present experiment was conducted to determine the effect of muscle temperature during the prerigor and early postrigor period on meat tenderness, postmortem proteolysis, calpain system activity, water-holding capacity, and color. Lamb longissimus muscle (n = 14) from the right and left carcass sides was excised immediately after dressing, divided into an anterior and posterior sample, vacuum-packaged, and stored overnight at 5 to 35 degrees C. Further storage, up to 14 d postmortem, was at 2 degrees C. Tenderness at 1 d postmortem, tenderization during further storage, and postmortem proteolysis were negatively affected by overnight incubation above 25 degrees C. This effect could be explained by an effect of temperature on muscle contraction and activity of the calpain system. Muscle contraction was at a minimum after incubation at 15 degrees C. Water-holding capacity was negatively affected by incubation above 25 degrees C. Color scores improved with increasing incubation temperature at 1 d postmortem. However, after 14 d of postmortem storage, no differences in color scores were observed. Based on the present results and results of other groups, a temperature around 15 degrees C at the onset of rigor seems optimal to maximize tenderness without having detrimental effects on water-holding capacity or color.