ENHANCED BIOACTIVE PEPTIDE RELEASE FROM GERMINATED CHICKPEA THROUGH SYNERGISTIC PROTEASE ACTIVITY

Chickpea germination is a cost-effective method to produce bioactive peptides, but the effects of temperature and pH on proteolysis and peptide bioactivity remain unclear. This study optimized soaking (pH and time) and germination temperature to maximize protease activity and bioactive peptide yield using a 2³ full factorial design with response surface methodology. The independent variables and their respective levels were soaking pH (5.0 and 7.0), soaking time (11 and 15 h); and germination temperature (30 and 35 °C). Soluble protein content (%) was chosen as a response. At the optimal condition, enzymatic characterization was determined by total and specific proteolytic activity assays, pH/temperature profiles, and electrophoresis. Additionally, the inhibition profile against serine (PMSF), cysteine (E-64), aspartate (pepstatin A), and metalloprotease (EDTA) inhibitors was determined. Proteins from the optimized germination were extracted in their respective albumin, globulin, and glutelin fractions, followed by characterization as the electrophoretic profile and sequencing (HPLC-MS-MS). Parallelly, total protein extracts were also prepared and subjected to simulated digestion (INFOGEST 2.0), with digests analyzed for peptide profiles and cyclooxigenase-2 (COX-2) inhibition. Optimized germination conditions (11 h soaking, pH 7.0, 35°C) yielded 33.6% soluble protein content and a 1.4-fold increase in proteolytic activity after 48 h.  Enzymatic assays revealed that storage protein hydrolysis during germination is driven by the synergistic action of serine, cysteine, and aspartate proteases, with optimal activity at pH 6.0–7.0 and 35 °C. Electrophoresis showed pronounced degradation of albumin and glutelin, while globulin-derived peptides dominated pre- and post-digestion profiles. In silico analysis identified peptides with strong DPP-IV inhibitory activity (A = 0.50–0.78), indicating potential anti-diabetic properties. Additionally, chickpea protein isolates exhibited COX-2 inhibitory activity, with higher inhibition efficiency before digestion (IC₇₅ = 0.158 mg of soluble protein/mL of prepared extract) than after digestion (IC₇₅ = 0.434 – 0.465 mg of soluble protein /mL of prepared extract). These findings suggest that optimized germination conditions enhanced the release of bioactive peptides with potential anti-inflammatory and anti-diabetic properties. The scalability of this process underscores its potential for industrial application in the production of functional food ingredients.