Two Novel Antihypertensive Peptides Identified in Millet Bran Glutelin-2 Hydrolysates: Purification, In Silico Characterization, Molecular Docking with ACE and Stability in Various Food Processing Conditions

The addition of food-derived antihypertensive peptides to the diet is considered a reasonable antihypertension strategy. However, data about the stability of antihypertensive peptides in different food processing conditions are limited. In this study, through Sephadex G-15 gel chromatography and RP-HPLC separation, UPLC–ESI–MS/MS analysis and in silico screening, two novel ACE-inhibitory peptides, Pro-Leu-Leu-Lys (IC(50): 549.87 μmol/L) and Pro-Pro-Met-Trp-Pro-Phe-Val (IC(50): 364.62 μmol/L), were identified in millet bran glutelin-2 hydrolysates. The inhibition of angiotensin-I converting enzyme and the potential safety of PLLK and PPMWPFV were studied using molecular docking and in silico prediction, respectively. The results demonstrated that PLLK and PPMWPFV could non-competitively bind to one and seven binding sites of ACE through short hydrogen bonds, respectively. Both PLLK and PPMWPFV were resistant to different pH values (2.0–10.0), pasteurization conditions, addition of Na(+), Mg(2+) or K(+) and simulated gastrointestinal digestion. However, PLLK and PPMWPFV were unstable upon heat treatment at 100 °C for more than 20 min or treatment with Fe(3+) or Zn(2+). In fact, treatment with Fe(3+) or Zn(2+) induced the formation of PLLK–iron or PLLK–zinc chelates and reduced the ACE-inhibitory activity of PLLK. These results indicate that peptides derived from millet bran could be added to foods as antihypertension agents.