Discovery and characterization of protease activities in acidified bovine milk

Protease activities present in acidified bovine milk were investigated using a peptide digestion assay coupled to mass spectrometry analysis. Results from this assay showed considerable overlap of cleavages at pH 3.5 and 5.5, suggesting shared protease activities at these pH values; however, overall activity at pH 5.5 was significantly lower. Two main proteases were identified at pH 3.5: cathepsin D (endoprotease), and a previously unreported tripeptide aminopeptidase. Fluorimetric assays for measurement of activity of both proteases across the pH range 3.0-6.7 were developed. Cathepsin D activity was highest pH 3.0, and declined progressively with increasing pH; in contrast, the tripeptide aminopeptidase activity was restricted to pH 3.0-4.0. Both proteases were detected in a set of 26 individual milk samples, indicating that they might be part of a proteolytic system in milk. This study provides insights about the proteases present in milk and highlights the role of pH in their regulation.