Identification and inhibition mechanisms of novel ACE inhibitory peptides from Tenebrio Molitor protein based on peptidomics combined with AI tools

To fully utilize Tenebrio molitor (TM) at a high value, Tenebrio molitor protein (TMP) hydrolysates were obtained from the novel insect food resource TM after extracting its proteins for compound protease hydrolysis. In vitro simulated digestion was conducted to increase the angiotensin I-converting enzyme (ACE) inhibitory ability of TMP. Based on the experimental data, peptidomics identification of TMP hydrolysates was performed using UHPLC-ESIHRMS/MS. Four novel ACE-inhibitory peptides (ACEIPs) were obtained using AI tools and in vitro activity validation, namely YPGLQ, PGLPQ, MFGPQ and NLGPR, with IC₅₀ values of 0.8560 ± 0.0090 mmol/L, 1.0300 ± 0.0356 mmol/L, 0.8558 ± 0.0204 mmol/L, and 0.4709 ± 0.0391 mmol/L, respectively. The inhibition patterns of ACEIPs were analyzed by inhibition kinetics as competitive, uncompetitive, mixed, and non-competitive inhibition patterns. The result of CD spectra revealed that the original structure of ACE has been loosened by ACEIPs. The bindings of ACEIPs to ACE were spontaneous, and they had fluorescence quenching effects on ACE. Molecular docking and MD simulation indicated hydrogen bonds, hydrophobic forces, and electrostatic forces are the main forces between them and ACE. In this study, we tried to extract ACEIPs from TMP using a three-stage enzymatic digestion process, elucidated their inhibition mechanism, and demonstrated their biosafety using AI tools, which provided a new strategy for the preparation of ACEIPs and guided the expansion of TMP application.