Action of serine, cysteine, and aspartate proteases during optimized chickpea germination improved the generation of bioactive peptides

Chickpea germination is a cost-effective method for producing bioactive peptides; however, the optimal temperature and pH conditions for maximizing proteolysis and bioactivity remain undefined. This study evaluated the effects of different pH and temperature conditions during germination on protease activity and the biological activity of resulting peptides. Using a 2 3 full-factorial experiment with surface response methodology, the optimal conditions were identified as 35 °C and pH 7.0, where the soluble protein content was 33.6% and proteolytic activity increased 1.4-fold after 48 h of germination. Enzyme inhibition assays revealed that serine, cysteine, and aspartic proteases were responsible for storage protein hydrolysis, exhibiting optimal activity at pH 6.0–7.0 and 35 °C. Electrophoresis of protein isolates revealed that albumin and glutelin were susceptible to hydrolysis, with globulin-derived peptides predominating. These peptides demonstrated potent in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory activity, with more than a fivefold increase in potency after complete colonic digestion (IC 50 reduced from 16.31 to 3.40 mg/mL). This was further supported by in silico analysis (A = 0.50–0.78), highlighting their strong potential for managing type 2 diabetes. In addition, total protein isolated extracts exhibited cyclooxygenase-2 (COX-2) inhibitory activity, with higher inhibition efficiency before digestion (IC 75 = 0.158 mg soluble protein/mL of prepared extract) than after digestion (IC 75 = 0.434–0.465 mg soluble protein/mL of prepared extract). These findings suggest that optimized germination efficiently releases peptides with potential anti-inflammatory and antidiabetic properties. The scalability of this process underscores its potential for industrial application in the production of functional food ingredients.