Peptidomic Profiling Analysis of Endogenous Peptides in Buffalo Milk During Lactation Stages

Buffalo milk is a rich source of various nutritional components and bioactive peptides, offering significant health benefits. Endogenous peptides, which occur naturally in milk, represent a valuable source of bioactive peptides with potential nutraceutical applications. However, research on endogenous peptides in buffalo milk remains limited. This study employed a quantitative peptidomic approach to characterize endogenous peptides across different lactation stages. A total of 2099, 2946, and 4418 peptides were identified in colostrum, transitional milk, and mature milk, respectively. The majority of these peptides were derived from β-casein, followed by α_S1-casein, κ-casein, and other proteins. Notably, variations in precursor proteins contributing to peptide production were observed throughout lactation. Phosphorylation levels of endogenous peptides were highest in mature milk, with serine residues predominating. Enzymatic cleavage analysis identified cathepsin D as the key enzyme involved in endogenous peptide production, while proline endopeptidase and plasmin exhibited stage-specific activities. Bioinformatics analysis revealed differentially expressed precursor proteins linked to complement cascades and NF-κB signaling, emphasizing the immune protective role of colostrum. Furthermore, 54 potentially bioactive peptides with favorable water solubility were identified in colostrum, of which 17 were predicted to possess anti-inflammatory properties. These findings contribute to a deeper understanding of the molecular basis of buffalo milk's functional properties, highlighting its potential as a source of bioactive peptides for both nutritional and pharmaceutical applications.