Optimization of Enzymatic Hydrolysis for Preparing Cassava Leaf Hydrolysate with Antioxidant Activity

Introduction: Cassava leaves are the agro-industrial by-product considered to be one of the most vegetable resource protein-richis. Methods: This study aimed to optimize the enzymatic hydrolysis of CL proteins using Alcalase 2.4 L and studied the antioxidant properties of the hydrolysate. Effects of pH (7.0–10), temperature (40–65 °C), and enzyme/substrate (E/S) ratio (0.015–0.1 EU/ g protein) on the efficiency of protein extraction (E) (%), protein content (P) (%), and antioxidant activity (TEAC) (µmol TE/g) of the CL hydrolysate (CLH) were conducted. The models obtained were optimized through response surface methodology to maximize the value of P, E, and TEAC, respectively. Degree of hydrolysis, antioxidant activity (TEAC, ORAC, and FRAP), total phenol content (FT), and total protein content of CLH obtained under the optimal condition were measured. Amino acid composition of CLH with higher antioxidant activity was determined. CLH was fractionated by ultrafiltration into four fractions, whose antioxidant activities were then assessed in rat aortic vascular smooth muscle A7r5 cells. The fraction with the greatest activity was further fractionated based on hydrophobicity. Results: The results showed that response variables depended on pH, E/S, and time used in the hydrolysis reaction. The fractionation process enriched antioxidant peptides, as reflected in the enhanced antioxidant activity of fractions in VSMCs. The antioxidant capacity of CLH or its derived fractions, as determined by chemical assays, corresponded with their cellular antioxidant capacity in VSMCs. Conclusions: Our study shows the potential of cassava leaf as a source of producing antioxidant peptides.