Dipeptidyl Peptidase 4 Inhibitory Activity of Protein Hydrolysate from Earthworms (Perionyx excavatus)

Diabetes is a complex metabolic syndrome, of which type 2 diabetes (T2DM), primarily caused by impaired insulin response, accounts for 95% of diabetes cases worldwide as of 2021. One treatment strategy to control T2DM includes inhibiting dipeptidyl peptidase-4 (DPP4), an enzyme that breaks down incretin hormones and impairs glucose regulation. This study explores the potential of hydrolysed proteins from earthworms (Perionyx excavatus) as natural DPP4 inhibitors, focusing on optimizing hydrolysis conditions, including enzyme type hydrolysis, earthworms:phosphate buffer (w/v), temperature (°C), pH, enzyme:substrate (E:S) ratio (U/g protein), and time hydrolysis (hour). Among the tested enzymes, Alcalase produced the most effective hydrolysate. The optimal conditions for hydrolysis were determined at an E:S ratio of 1:6 (w/v), a temperature of 55°C, pH 7, an enzyme: substrate ratio of 600 U/g protein, and a hydrolysis time of 4 hours. Under these conditions, the resulting hydrolysate exhibited vigorous DPP4 inhibitory activity (DPP4IA), with an IC50 value of 777.18 μg/mL. Further ultrafiltration fractionation revealed that the <1 kDa fraction had the highest inhibitory activity, with IC50 values of 395.03 and 323.07 μg/mL before and after digestion, respectively. Notably, the hydrolysate demonstrated stability across a broad pH range (1–11) and remained bioactive even after exposure to 100°C for up to 180 minutes. Interestingly, enzymatic hydrolysis and simulated digestion appeared to enhance bioactivity, likely due to the generation of smaller bioactive peptides. These findings suggest that peptides derived from Perionyx excavatus could be promising natural DPP4 inhibitors with potential applications in functional foods or pharmaceutical formulations for blood glucose management.