Bovine Heart Hydrolysates as a Source of Potential Antihypertensive Peptides Through the Inhibition of ACE-I and Neprilysin Activity

Abstract The valorisation of meat by-products as sources of functional ingredients is gaining attention due to interest in sustainable protein recovery. This study explored the generation of antihypertensive peptides from bovine heart hydrolysates produced using Novozym®, Protamex®, and their combinations with the exopeptidase Protana™ Prime. Sequential enzyme treatments increased hydrolysis and amino acid release, while single endopeptidases yielded higher angiotensin-converting enzyme-I (ACE-I) and neprilysin (NEP) inhibitory activities, reaching 68.72% in the Protamex hydrolysate and 58.48% in the Novozym hydrolysate, respectively. Peptide profiling by LC–MS/MS identified 1251 peptides in the most bioactive fraction, mainly derived from muscle proteins. In silico analysis with PeptideRanker predicted 172 peptides with high bioactivity, whereas AHTpin supported antihypertensive activity for 109 peptides. MultiPep predicted ACE-inhibitory activity for NPPKFDK, VYPFPGPI, and VYPFPGPIPN, the latter showing the highest antihypertensive score (0.995). Additionally, KPPVVKW, LKPLPPK, and LKPRPPAR showed cell-penetrating peptide scores > 0.6, suggesting potential for cellular uptake. This study represents one of the first reports of NEP-inhibitory activity in bovine-derived peptides, highlighting heart hydrolysates as promising sources of multifunctional peptides for use in functional foods or nutraceuticals targeting cardiovascular health.