Abstract
Although taste-active peptides have been isolated from enzymatic hydrolysates of defatted high-protein milk powder, their sensory properties, processing stability, and receptor-binding mechanisms remain largely unexplored. In this study, we synthesized milk-derived taste- active peptides in vitro and systematically characterized their taste profiles and underlying molecular mechanisms Eight key taste-activate peptides were identified from milk-derived protein hydrolysates. LSFD, LKPTPEGD (LD8), and TMKGLDIQ (TQ8) exhibited the lowest umami detection thresholds among all tested peptides. At a concentration of 1 mg/mL, the glutathione equivalent values of these peptides ranged from 0.66 to 1.48 mg/mL, with EDIKQME exhibiting the strongest kokumi-enhancing activity. All identified peptides remained stable under mildly acidic to near-neutral conditions (pH 4–6), and some exhibited high thermal stability at 75–85 °C, indicating their suitability for thermally processed foods. Furthermore, LSFD interacts with both CaSR and T1R1/T1R3 receptors via hydrogen bonds and hydrophobic interactions. Molecular dynamics simulations revealed conformational changes induced by LSFD binding to these receptors, providing novel insights into the dynamic mechanisms underlying peptide-receptor recognition. Collectively, these milk-derived taste-active peptides show considerable potential as natural flavor enhancers in food applications.