Optimization of Factors Affecting the Antioxidant Activity of Fenugreek Seed’s Protein Hydrolysate by Response Surface Methodology

Background and Objectives: Bioactive peptides have different health benefits and functional characteristics and are produced by enzymatic hydrolysis, microbial fermentation and chemical synthesis methods. The purpose of this study was to optimize the enzymatic hydrolysis conditions of fenugreek seed protein with alcalase and to evaluate the antioxidant properties of the resulting peptides. Materials and Methods: The hydrolysis conditions of fenugreek seed protein (temperature 20-60 °C, time 30-270 minutes, and the enzyme to substrate ratio of 0.25-3.25%) were optimized using response surface methodology (RSM) to achieve the maximum Fe2+ chelating activity and reducing power. The antioxidant properties of optimum treatment (DPPH radical scavenging activity, reducing power, hydroxyl radical scavenging activity, Fe2+ chelating activity and total antioxidant activity) at concentrations of 10-50 mg/ml were compared with the antioxidant activity of non-hydrolyzed protein and vitamin C. Results: The optimum conditions were determined by the software as enzyme to substrate ratio of 2.32%, temperature of 47.04 °C and time of 198.21 minutes. Under these conditions, the Fe2+ chelating activity and reducing power were predicted to be 79.15 % and 1.29, respectively. The results showed that the maximum total antioxidant activity (1.20), hydroxyl radical scavenging activity (69.49 %) and reducing power (1.12) were achieved at the concentration of 40 (mg/ml), and the maximum DPPH radical scavenging activity (50.99 %) was obtained at the concentration of 50 (mg/ml). The maximum Fe2+ chelating activity (71.92%) of protein hydrolysate was 2.5 times more than the unhydrolyzed protein. Conclusion: The results showed that enzymatic hydrolysis increased the antioxidant activity of fenugreek seed protein significantly. Fenugreek seed protein hydrolysate with its significant antioxidant properties could be used as potential functional ingredient in food formulations or pharmaceutical purposes.