The hydrolytic activity of 3 commercially available protease preparations (bacterial protease G, fungal 31000, and fungal 60000) were examined using fluorescent-labeled casein, azo dye-impregnated collagen, and meat protein extracts from bovine M. semimembranosus and Achilles tendon, and compared to that of papain. Assays showed that all proteases exhibited little activity at low temperature (5 °C), and maximal activity at 45 °C. The pH, at which optimal activity was observed for each of the protease preparations, differed and ranged from pH 5.0 to 8.0. Kinetic parameters (KM and Vmax) were also different between protease preparations, with the bacterial protease G and papain exhibiting significantly higher Vmax values (P < 0.001) and lower KM values (P < 0.01) for the casein substrate than the 2 fungal protease preparations. Meat protein hydrolysis was displayed on SDS-PAGE and proteins analyzed with mass spectrometry. The protease preparations were shown to have varying affinity toward different meat proteins. The bacterial protease G preparation was efficient at hydrolyzing most myofibril and collagen proteins, and appeared to be more efficient than papain at hydrolyzing collagen proteins. On the other hand the 2 fungal protease preparations showed a selective specificity toward meat myofibrillar proteins, and the fungal 60000 protease preparation exhibited high affinity toward collagen γ and collagen type I chain B proteins. The results generated in this study demonstrated that these commercial proteases have good potential for use in meat tenderization applications due to their mild and complementary effects on different meat proteins.
Practical Application: Bacterial and fungal protease preparations exhibited varying affinities for hydrolyzing meat proteins. This selective moderate capability of microbial proteases compared to papain is potentially an advantage in avoiding over-tenderization in meat. On the other hand, the bacterial protease G preparation, which appeared to be more efficient at hydrolyzing connective tissue proteins than papain, could be beneficial in tenderizing meat with high connective tissue content. The synergistic effect of these protease preparations could be incorporated into a meat tenderizing formula to give the tenderizer a broad activity spectrum, thus able to target different cuts of meat.