Peptide Profile Changes in Buffalo Milk Cheese during Different Storage Periods and Characterization of Novel Bioactive Peptides through Peptidomics

This study aimed to investigate the changes in the bioactive peptides (BPs) of buffalo milk cheese (BMC) within 15 days of storage. A total of 3605 peptides were identified in the BMC, with 260 peptides remaining stable for 15 days. Among these, the peak intensities of all reported BPs (9 peptides) increased on the 15th day. Additionally, two novel antioxidant peptides, AYF (IC₅₀ = 160.5 μM) and YPFPGPIPK (IC₅₀ = 108.6 μM), and two novel angiotensin-converting enzyme (ACE) inhibitory peptides, LRF (IC₅₀ = 214.9 μM) and APFPEVFGK (IC₅₀ = 880.0 μM), were identified. The molecular docking results implied that the active sites on ECH-associated protein 1 (Arg 415, Arg 483, Arg 380, and Asn 382) and the active sites in the three active pockets of ACE (S1, S2, and S'1) are crucial for peptide activity. This study demonstrates that BMC is a stable resource of BPs and has the potential to be used in functional foods.