Abstract: To investigate the inhibitory effects of millet prolamin peptides on pancreatic lipase and cholesterol esterase activities, millet prolamin was prepared from three different millet varieties (Zhonggu 2, Zhaonong 21, and Hongmiaoyapoche) and subjected to enzymatic hydrolysis in this study. Peptides with potent inhibitory effects on lipid-digesting enzymes (pancreatic lipase and cholesterol esterase) were screened out from the prolamin hydrolysates, and their binding mechanisms were explored using molecular docking and molecular dynamics simulations. The results showed that the hydrolysate from Hongmiaoyapoche millet was rich in hydrophobic amino acids and exhibited the strongest lipid-lowering activity. From its < 3 kDa fraction, six peptides with low molecular mass (< 1 kDa), no toxicity or carcinogenicity, potential biological activity, resistance to gastrointestinal digestion, and strong binding affinity to the target enzymes were selected and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These peptides were WQHQY, YWTRPH, YWTARP, WQHMMP, FNPMFNPM, and ANPYWTRP. Among them, WQHQY and YWTRPH showed the highest in vitro inhibitory activity against both pancreatic lipase and cholesterol esterase. Molecular docking indicated that hydrophobic interactions and hydrogen bonds contributed to the stable binding of these peptides to the enzymes. Molecular dynamics simulations further supported the stability of the complexes, based on analyses of root mean square deviation, root mean square fluctuation, and radius of gyration. This study provides useful insights for developing novel peptide-based inhibitors of pancreatic lipase and cholesterol esterase, and offers a scientific foundation for the potential use of millet prolamin proteins in functional foods.

Key words: millet; prolamin protein peptide; lipid-digesting enzymes; molecular docking; molecular dynamics