Application of Moringa oleifera Seed Rennet in the Preparation of Buffalo Cheese: Optimization of Processing Technology and Mining of Angiotensin Converting Enzyme Inhibitory Peptides

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.