Bioactive peptides refer to specific protein fragments that can have a positive impact on human health, and have attracted much attention in the development of biomedicine and health food due to their advantages such as easy cell transport, high absorption and utilization, toxicity and low immunogenicity.This research selected the microbial fermentation method to prepare pine nut active peptides.Firstly, 14 strains were pre-screened and re-screened, and the strain N-LYS-20 with the highest protease activity was selected and identified as Bacillus subtilis by physiological and biochemical characteristics and molecular biology.The enzyme production type, degree of hydrolysis, peptide content, and amino acid content of strain N-LYS-20 were explored by protease inhibitor, phthalatedehyde method, Coomassie brilliant blue method, and ninhydrin method.This study showed that strain N-LYS-20 mainly secreted serine protease and metalloproteinase, and the protease activity reached the highest value (325.62 U/mL) at 40 h.The degree of hydrolysis showed an upward trend in fermentation, reaching the highest value (27.6%) at 48 h.The content of peptide (>3 kDa) increased from 622.26 mg/mL at 4 h to 1 456.23 mg/mL at 28 h, and then decreased to 712.83 mg/mL at 48 h.The amino acid content increased from 0.067 mg/mL at 4 h to 0.589 mg/mL at 44 h, indicating that strain N-LYS-20 had the ability to hydrolyze pine nut protein into peptides (<3 kDa) and free amino acids in a good and sustainable manner.In conclusion, this study discussed the screening of fermentation strains and the analysis of protease-producing types, which was expected to provide a reference for the enzyme-producing ability and enzyme-producing types of fermented strains for related research on fermented pine nut protein.This study mainly explored the dynamic changes of hydrolysis degree, peptide content, and amino acid content during the fermentation process of pine nut protein, which was expected to provide data references for the study of hydrolysis efficiency and yield of pine nut protein hydrolyzed by microorganisms.
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