Abstract:The advantages of endogenous enzymes and commercial enzymes were combined to obtain earthworm protein peptides with efficient pancreatic lipase inhibition activity using a two-step enzymatic hydrolysis method to hydrolyze earthworm protein. The pancreatic lipase inhibition rate was used as the evaluation index, and single-factor experiments and orthogonal experiments were used to optimize the second-step enzymatic hydrolysis process. In addition, the hydrolysates were purified using ultrafiltration and Sephadex G-15 gel chromatography, and peptide segments with high pancreatic lipase inhibitory activity were screened by combining LC-MS/MS, molecular docking, and other technologies with the PeptideRanker database. The results showed that the optimal preparation process for the second-step enzymatic hydrolysis involved a solid: liquid ratio of 1:2 (50%, m/m), alkaline protease dosage of 6.0×104 U, temperature of 50 ℃, pH value of 9.5, and enzymatic hydrolysis time of 3 hours. Under these conditions, the pancreatic lipase inhibition activity reached 53.92%, the hydrolysis degree was 41.98%, and the soluble peptide yield reached 89.17%. A total of 10 peptide segments with high affinity, good biological activity, and non-toxicity were screened, namely GDAPPFFP, YPFDPGP, WGPDLP, FDFP, DDFFR, FLDF, SDDGFF, FDDF, SDDFF, and WDDFF. Small molecule peptides and amino acids are thought to be the primary substances causing the inhibitory effect of earthworm protein on pancreatic lipase. Overall, this study provides a new perspective for understanding the lipid-lowering activity of earthworm protein. It establishes a foundation for further research on the structure-activity relationship of earthworm protein functional peptides and the mechanisms of pancreatic lipase inhibition.
