Purification and identification of novel antioxidant peptides from enzymatic hydrolysate of chickpea (Cicer arietinum L.) protein concentrate

• Chickpea protein hydrolysate was fractionated by gel filtration on Sephadex G-25.
• Four CPH fractions were identified and evaluated for antioxidant activity.
• Fra.III possessed high free radical scavenging activity.
• Fra.III was fractionated into 11 major sub-fractions by HPLC.
• Molecular masses and amino acid sequences were identified by ESI-MS and ESI-MS/MS.

Enzymatic hydrolysis of chickpea protein concentrate (CP) by Alcalase® and some physiochemical and antioxidant properties of the resulting hydrolysate (CPH) were characterised. CPH displayed higher antioxidant activity than CP. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into four major fractions (Fra.I, Fra.II, Fra.III, and Fra.IV). Fraction III, which exhibited the highest DPPH scavenging activity (54% at 1 mg/ml), was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Eleven antioxidant fractions were isolated and two peptide sub-fractions show antioxidant activity (P3 and P8). The P8 displayed the highest DPPH radical-scavenging activity (67%; at 200 µg/ml) among these peptides subfractions. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESIMS/MS, respectively. Their structures were identified as Asp-His-Gly and Val-Gly-Asp-Ile. These peptides did not show haemolytic activity towards bovine erythrocytes. The results suggest that CPH are good source of natural antioxidants.