Comprehensive analysis of peptide profiles in pasteurized milk: seven key peptides as biomarkers for storage-dependent proteolysis

We explored changes in the peptide profiles of pasteurized milk during its shelf life. Our hypothesis was that residual enzymatic activity after pasteurization would systematically alter the peptide profile, enabling the identification of single peptides as markers for proteolysis. Two independent storage experiments were conducted, and the peptide profiles were recorded with microLC-QTOF MS/MS. The first experiment resulted in the identification of 2464 peptides. During the ten-day storage, 30 peptides showed significant changes. The second experiment involved four samples that were analyzed at the beginning, middle, and end of their shelf lives. Combining the results of both experiments revealed that seven key peptides were strongly associated with storage. Five of these peptides were derived from β-casein, indicating its susceptibility to proteolysis by endogenous and microbial proteases. These findings confirm that single peptides reflect storage-related changes and could serve as markers for product stability, quality control, and storage optimization strategies.