Simulated in vitro gastrointestinal digestion of traditional Chinese Rushan and Naizha cheese: Peptidome profiles and bioactivity elucidation.

Chinese Rushan and Naizha, the traditional acid coagulated cheese types produced from cow and yak milk, respectively, have been consumed for more than thousands of years. In this study, we aimed to characterise peptides of Rushan and Naizha in simulated in vitro gastrointestinal digestion using label-free based peptidomic. The identified peptide sequences were subjected to BIOPEP database driven bioactivity search. In total, 309 and 225 peptides were identified from Rushan and Naizha cheese, respectively, corresponding to 20 protein annotations. Analysis of label-free quantification found different protein digestibility, where casein was the primary source of peptides in Rushan, among which 62% represented β-casein by peptide count. The release of peptides was concentrated in specific residues 145-155 of β-casein in Rushan. In contrast, κ-casein and 7 minor milk proteins were dominant in digestion of Naizha cheese (p < 0.05). In particular, there were 11 peptides from digestion that were exact matches in databases to sequences with immunomodulatory, antibacterial, ACE-inhibition, DPP IV inhibition and antioxidant activities. Four novel angiotensin I-converting enzyme inhibitory (ACEI) activities peptides (YPFPGPIH, LKNWGEGW, RELEEIR, and HPHPHLS) were explored using molecular docking, chemically synthesized, and in vitro ACEI activity. The peptides had lower estimated free energy values (-5.34 to -7.66 kcal/mol), and exhibited the lowest IC value of 109.5, 77.7, 196.6, and 64.30 μM, respectively. Our study is the most comprehensive peptidomic analysis of Chinese Rushan and Naizha cheese to date.