Identification and Screening of Xanthine Oxidase Inhibitory Peptides from Yunnan Distinctive Buffalo Milk Cheese and Their Action Mechanisms

To screen xanthine oxidase (XOD) inhibitory peptides with high activity and clarify their action mechanisms, water-soluble peptide fractions with good in vitro XOD inhibitory activity from Mozzarella cheese prepared with Yunnan distinctive betel nut buffalo milk were used as raw material. The peptide sequences in the water-soluble peptide fractions were identified by liquid chromatography-tandem mass spectrometry, and the peptides with good XOD inhibitory activity were selected by combining molecular docking binding energy scoring and amino acid composition characteristics. The in vitro XOD inhibitory activity of the peptides was verified by chemical synthesis, and the secondary structures were characterized by infrared spectroscopy. The binding stability and action mechanism of the peptides with XOD were further investigated via molecular docking and molecular dynamics simulation. The results showed that a total of 2500 peptides were identified from the water-soluble peptide fractions with molecular weight less than 3.0 kDa by mass spectrometry, which were mainly derived from α_S1-casein, α_S2-casein, β-casein, β-lactoglobulin and κ-casein, among which the peptides with molecular weight less than 1.5 kDa accounted for 62.86%. Three novel XOD inhibitory peptides, FVAPFPEMF, YPFPGPIPK and KFAWPQYLK, were screened out based on the results of active peptide database retrieval, relative content of peptides, biological activity prediction score and molecular docking binding energy, with IC₅₀ of 0.46, 1.15 and 1.18 mg/mL, respectively. Infrared spectroscopy analysis showed that the secondary structures of the three peptides were mainly β-turn and β-sheet, which could enhance the binding efficiency with the XOD active center and interfere with the catalytic conformation of the enzyme. Molecular docking and molecular dynamics simulation analysis showed that the three peptides could bind to XOD active sites through hydrogen bonds and hydrophobic interactions, and FVAPFPEMF could form a more stable complex with XOD. The study aimed to provide a theoretical basis for the development of high value-added buffalo milk cheese and milk-derived uric acid-lowering functional products.