Predicting tenderness from proteolytic enzyme activity in the live animal and on-line carcase.

Summary

Although the role of protease enzymes is generally considered fundamental to post mortem tenderisation, the role of differences in substrate susceptibility to protease activity has not been previously considered within meat science. In the physiology research literature, differences in rates of degradation of cell proteins are recognised to be affected by protein denaturation and post translational modifications such as phosphorylation and glycosylation. 
The service provider, Carne, developed a methodology for measuring substrate susceptibility in meat homogenates. This methodology was based on measuring the total protease activity in situ using a fluorescent calpain specific substrate (CalS1). To measure protein susceptibility, CalS1 was added to the homogenate at two concentrations: the first used CalS1 in excess, which determined the total calpain activity in the sample. 
​​The second used a constant but submaximal concentration, which created a competitive condition for the enzyme: if the affinity for the endogenous substrate was high, the rate of CalS1 degradation was reduced compared to a condition where the affinity for the endogenous substrate was low. A previous investigation of this assay identified that, in samples measured at kill or at rigor, the substrate susceptibility assay was highly correlated with the rate of tenderisation in the samples, and these results provided the justification for some further investigation of the underlying mechanisms responsible for differences in susceptibility. 
In this work, Carne investigated the effects of different pre-rigor temperatures on substrate susceptibility: high pre-rigor temperatures are known to cause denaturation of both myofibrillar and soluble cytoplasmic proteins, and these effects could be expected to engender changes in myofibrillar susceptibility. In addition to denaturation, changes in susceptibility could be expected during the ageing period. The likely reason for such changes would be due to degradation of substrate proteins, which might be expected to reduce susceptibility as the total number of binding sites decrease. 
Alternatively, as the number of breakdown products increase (and many myofibrillar protein breakdown products are soluble) susceptibility might increase as more binding sites become available. From the results of the current stage, it did not appear that calpain activity alone can explain the increased ultimate toughness of meat incubated at high temperatures during the pre-rigor period. ​
However, the present assay appeared to provide an additional marker for meat that has undergone denaturing conditions that in turn led to increased toughness. The overall conclusions of this work were :

The level of phosphorylation in the myofibrillar and supernatant fraction of muscle tissue varies between animals. This difference is evident both at kill and in post rigor samples. 

Although the experimental numbers were small and the comparisons did not reach statistical significance, the results suggested that the level of phosphorylation in the myofibrillar and supernatant fractions offer potential markers for tenderness 

These results, if confirmed, support the results of in-vitro manipulation of muscle protein phoshorylation, with demonstrated effects on calpain activity.