Evaluation of the antioxidant activities

The ability of the hydrolysates to scavenge the DPPH free radicals was measured by the slightly modified method of Bersuder et al. (1998). The fermented protein hydrolysates dissolved in distilled water at varying concentrations (1–5 mg/mL) were dispersed in an equal volume of an ethanol solution of DPPH (0.1 mM) and the following dispersions were stored at 25 °C for 60 min in the absence of light. The absorbance of the resulting mixtures was then measured at 517 nm (A_sample). The control reaction comprised of distilled water substituting the sample (A_control). The ability of the fermented hydrolysates to inhibit DPPH was compared to that of ascorbic acid and determined as follows:

A slightly modified method of Chen et al. (2012) was used for determining the reducing power ability of the hydrolysates. The protein hydrolysates with varying concentrations (1–5 mg/mL) were blended with 0.2 mol/L phosphate buffer (pH 6.6) and potassium ferricyanide solution. The resulting mixture was heated at 50 °C for 30 min and then cooled down. Trichloroacetic acid (10 g/100 mL) was then incorporated and resulting solution was centrifuged at 3000×g for 10 min. Following centrifugation, the supernatant obtained was blended in distilled water and ferric chloride solution (1 g/L) and their absorbance was measured at 700 nm.

The ability of the fermented protein hydrolysate to inhibit lipid peroxidation was determined following the method of Naqash and Nazeer (2013) with some modifications. Fermented ESM protein hydrolysate (5 mg) was dispersed in 50 mM phosphate buffer (pH 7.0; 5 mL) and 50 mM linoleic acid solution (5 mL). Eventually, they were reconstituted in distilled water to obtain a final volume of 12.5 mL. Following their exposure to dark at 45 °C, their level of oxidation was determined by measuring their ferric thiocyanate values every day for 6 days at 500 nm by UV–Vis Spectrophotometer (UNICAM, Alva, U.K).