Exploration and molecular mechanism of novel ACE inhibitory peptides from goat milk protein: A combined in silico and in vitro study

Hypertension poses a significant threat to human health, and the efficient exploration of food-derived angiotensin I-converting enzyme inhibitory (ACEI) peptides as milder and safer alternatives to ACEI drugs has garnered increasing attention. Therefore, a combined in silico and in vitro study was performed to investigate novel ACEI peptides from goat milk proteins. First, the papain plus proteinase K was identified as the best enzymatic combination for generating ACEI peptides using BIOPEP. Then the enzymatic conditions of the papain/proteinase, the input order of the enzymes, and the molecular weight (MW) of the hydrolysate were further optimized to obtain the hydrolysate with the highest ACEI activity. Under the optimized condition described, 698 peptides (<25 A A) were successfully identified from the milk hydrolysate of goat (MW < 3 KDa). Among them, six potential novel ACEI peptides were efficiently screened virtually. Verification studies using synthesized peptides partially confirmed the in silico results, with four out of the six selected peptides (FKF, FRY, RWL, and WKP) exhibiting ACEI activity. Additionally, the blood pressure-lowering effects of the top two ACEI peptides (FRY and FKF) were further validated in spontaneously hypertensive rats (SHRs), and FRY treatment significantly lowered the blood pressure of SHRs. To summarize, this study established a rapid enzymatic hydrolysis system for screening novel ACEI peptides and identified a novel antihypertensive peptide, FRY, which provides a new method for research on bioactive peptides research and high-value utilization of goat milk.