In vitro proteolysis mirrors intact muscle maturation in beef carcasses

An in vitro assay was developed to study protease activity during the maturation of beef postmortem. Myofibrils were purified from the semitendinosus and used as a sentinel for assessing the activity of endogenous proteases in longissimus thoracis et lumborum (LTL) and the extensor carpi radialis (ER) over time postmortem in beef carcasses. Samples were collected from each muscle at 0, 1, 2, 7, and 14 d of aging and snap frozen. Samples were powdered and added to an in vitro proteolysis assay containing buffer and purified myofibrils. Aliquots were collected at 0, 2, 120, 480, and 1440 min of incubation, and intact desmin and troponin-T were quantified. Digestions at 0 and 1 d using either muscle had little desmin degradation during the entire digestion period. In contrast, LTL muscle collected at 2, 7, and 14 d had the greatest proteolytic capacity as indicated by disappearance of intact desmin by 480 and 1440 min incubation. Though degradation ensued using powdered ER muscle, disappearance of intact proteins was limited. Degradation in vitro paralleled that observed in intact muscle. Addition of ethylene glycol tetra-acetic acid (EGTA), a cysteine protease inhibitor, and calpastatin inhibited proteolysis and suggest proteolytic activity observed in muscles and detected in our proteolysis assays is due to an active calpain protease. Collectively, our data show an active protease is minimal in bovine muscle until 48 h postmortem in the LTL muscle and suggest an in vitro assay containing purified myofibrils is a potential tool for studying temporal changes in proteolysis during the maturation and tenderization of beef across muscles. •Muscle-derived myofibril digestion reflects in vivo proteolysis.•Minimal proteolysis was observed with 0 and 24-h LTL muscle.•Proteolysis observed after 24 h postmortem is likely due to the proteolytic activity of an active calpain.