Protease activities and casein proteolysis in raw and pasteurized bovine milk under neutral and acidic conditions

This study elucidated the role of specific milk proteases in generating distinct proteolytic patterns and peptide profiles in bovine milk. Specifically, the activities of plasmin and the lysosomal proteases cathepsin D and cathepsin B were analyzed in both raw and pasteurized bulk bovine milk. Samples were incubated at their enzymes' optimal pH levels (6.7 for plasmin; 5.0 for lysosomal proteases) at 37°C. Protease activities, together with casein breakdown monitored by urea-PAGE and UPLC, were followed over 3 d, with urea-PAGE used to assess breakdown of intact caseins and larger proteolytic fragments, and UPLC to detect the accumulation of soluble low-molecular-weight peptides. Peptide profiles were further characterized by LC-MS after 7 d to allow greater peptide accumulation and improved resolution of protease-specific hydrolysis patterns. Overall, pH had a dramatic effect on proteolysis; both UPLC and LC-MS analyses revealed a marked reduction in total peptide abundance under acidic conditions compared with neutral pH. However, the impact of pH was not limited to a simple decrease in proteolytic extent. Rather, acidification induced a shift in the balance among active proteases, which translated directly into changes in substrate preference and casein breakdown patterns. In raw milk at neutral pH, plasmin activity declined gradually over time, whereas lysosomal protease activity decreased sharply after 24 h at 37°C. Under these conditions, α_s1- and β-caseins were the primary substrates. 
At neutral pH, lasmin predominantly hydrolyzed β-casein to generate γ-caseins, while cathepsin D was likely associated with the formation of α_s1-I-casein. At acidic pH, despite lower overall peptide yields, the proteolytic profile shifted toward preferential breakdown of α_s2- and κ-caseins, driven mainly by cathepsin D and potentially by AprX, a bacterial metalloprotease. In contrast, cathepsin B exhibited minimal involvement in intact casein hydrolysis across both pH conditions, suggesting a more specialized functional role. Heat treatment further modulated protease dynamics. Plasmin activity was not significantly reduced following high-temperature short-time pasteurization (72°C for 15 s) and increased during incubation, whereas lysosomal protease activities were markedly reduced by pasteurization and further declined throughout the incubation period. Collectively, these findings demonstrate that both pH and heat treatment regulate milk proteolysis not only by affecting total enzymatic activity but also by altering protease balance, thereby redefining casein substrate utilization and peptide generation patterns.