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.

Spray-dried whey protein powders were produced from whey obtained as a by-product in the production of milk cake and milk fan as ethnic dairy products in Yunnan, and their main components and processing characteristics were analyzed. The results showed that the main components of whey protein powder obtained from the by-product of milk cake were protein 39.0%, fat 6.0%, moisture 4.1%, total ash 7.7%, acidity 23.2 °T, and pH 5.61, and the processing characteristics were 99.52% solubility, less than 3.2% precipitation at different temperatures, and emulsifying activity index (EAI) and emulsion stability index (ESI) both greater than 42.7% at different pH values. The main components in whey protein powder obtained from the by-product of milk fan were protein 72.0%, fat 19.8%, moisture 4.6%, total ash 8.1%, acidity 58.3 °T, and pH 5.20, and its processing characteristics were 98.70% solubility, less than 3.5% precipitation at different temperatures, and EAI and ESI both higher than 50% at different pH values. The sensory scores of yogurts with the two whey protein powders were 70.7 and 72.7 out of 100 points, respectively, indicating that both can be used as an ingredient in yogurt.

In order to extend the shelf life of milk cake, a Pickering emulsion containing antimicrobial peptide MOp2 from Moringa oleifera seeds was applied as preservative to milk cake. In this experiment, the preservative effect of the Pickering emulsion on milk cake was determined by measuring peroxide value, texture properties, total viable count, coliform count, color difference and sensory attributes. The results showed that Escherichia coli was detected in the vacuum packaging group (control) after 28 days of storage, and the count of E. coli exceeded the safety limit after 35 days. The total viable count, peroxide value (POV), and color difference was significantly higher than those of the experimental group coated with the Pickering emulsion. The experimental group remained unspoiled for 42 days. Sensory quality remained stable during the first 35 days of storage, total viable count increased slowly, and E. coli was not detected in the experimental group. Significant differences in texture properties between the two groups were observed starting from the 21th day of storage. Sensory and physicochemical evaluation showed that compared with the control group, the Pickering emulsion prolonged the storage life of milk cake by one week, indicating its good preservative effect.

In this study, angiotensin-converting enzyme (ACE) inhibitory peptides from an enzymatic hydrolysate of Moringa oleifera seeds were separated by sequential ultrafiltration and ion exchange chromatography. The peptide sequences were identified by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and potential ACE inhibitory peptides were selected by bioinformatics and molecular docking; their secondary structure was analyzed by Fourier transform infrared (FTIR) spectroscopy and their in vitro activity was evaluated by enzymatic inhibition kinetics and the methyl thiazolyl tetrazolium (MTT) method. The results showed that peptide fraction F-b had a good antihypertensive effect. A total of 11 peptide sequences were identified. Peptide QGPRPQ was identified as a potential ACE inhibitory peptide with a half-maximum inhibitory concentration (IC₅₀) (1.15 ± 0.3) mmol/L. Molecular docking showed that QGPRPQ could better bind to ACE through hydrogen bond and hydrophobic interaction. Secondary structure analysis showed that QGPRPQ was composed of 22.8% α-helix, 33.3% β-fold and 43.9% β-turn. The mode of inhibition of QGPRPQ was mixed type, and it had no toxic effect on HepG2 cells at a concentration lower than 0.01 mg/mL. This study can provide an important theoretical basis for the development and utilization of hypotensive peptides derived from M. oleifera seed protein.

In this study, buffalo cheese was prepared with rennet from Moringa oleifera seeds. The process was optimized based on sensory characteristics and hydrolysis degree. The composition of water-soluble peptides from the cheese was analyzed. Furthermore, bioactive peptides with angiotensin converting enzyme (ACE) inhibitory activity were identified and their potential molecular mechanisms of action were explored. The optimal conditions determined were as follows: pH 5.56, rennet dose 0.14%, incubation time 32 min, and stretching temperature 80 ℃. Under these conditions, the hydrolysis degree of cheese was 12.56% with a uniform and elastic texture, a soft and delicate taste, a uniform and glossy color, and a rich aroma. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), 1600 peptides with molecular mass less than 3 kDa were detected in the cheese, 613 of which demonstrated good bioactivity, predominately derived from β-casein and α_S1-casein. Notably, 37.29% of the peptides exhibited ACE inhibitory activity. By comparing with the bioactive peptide database and using bioinformatics, four peptides proven to have ACE inhibitory activity and four novel peptides with potential ACE inhibitory activity were identified. Molecular docking showed that the four novel peptides could effectively bind to ACE. Among them, FGGL and FSPL bound tightly to the S2 active pocket, thus exhibiting strong ACE inhibitory activity. This study provides a scientific basis for the development and utilization of new plant rennet resources and characteristic buffalo cheese.