Influence of cow-associated variables, protein content, and pasteurization on protease activities in bovine milk

Activities of plasmin, cathepsin D, cathepsin B, and tripeptide aminopeptidase were measured in milk from 41 individual cows, with raw milk and corresponding pasteurized aliquots incubated at 37°C for 0, 1, and 7 d. Associations between individual protease activities and somatic cell count (SCC), days in milk (DIM), lactation number (LN), and protein content were also examined. In fresh raw milk, SCC was positively correlated with tripeptide aminopeptidase and cathepsin B activities but not with plasmin or cathepsin D activities. Plasmin activity, in contrast, was positively correlated with LN, while no clear association was observed with DIM. Protein content was positively associated with tripeptide aminopeptidase activity. During incubation of raw milk at 37°C, all 4 proteases exhibited a significant decrease in activity over the 7-d period. High-temperature short-time pasteurization (HTST, 72°C for 15 s) significantly reduced the activity of all 4 proteases at d 0, although the extent of the reduction differed substantially between enzymes. Tripeptide aminopeptidase and cathepsin B were highly heat-sensitive, with activities reduced by 97.9% and 94.0%, respectively. Cathepsin D displayed intermediate heat resistance, with a reduction of 77.0%, whereas plasmin showed only moderate heat sensitivity, with activity decreasing by 45.9% following pasteurization. The behavior of the enzymes during subsequent incubation of pasteurized milk also differed. Tripeptide aminopeptidase activity remained negligible throughout the entire incubation period, indicating almost complete inactivation by heat treatment, and activities of cathepsins B and D continued to decline further during incubation. In contrast, plasmin activity increased significantly during incubation of pasteurized milk, rising by approximately 131% by d 7. Overall, these findings highlight the complexity of proteolytic systems in bovine milk and demonstrate that both cow-associated factors and thermal processing influence individual proteases in markedly different ways, with shifts in proteolytic balance having potential consequences for dairy product quality and shelf life.