V 650

The measurement of superoxide radical scavenging was carried out according to Yang et al. [24] protocol with minor modi cation. Brie y, different concentrations of test samples (5-1500 µg/ml) were mixed with 3 ml of Tris-HCl buffer (50 mM, pH 8.2) and incubated at room temperature, for 10 min. Then, 50 µL of pyrogallol solution (30 mM) were added and after 4 min of incubation at room temperature, the reaction was stopped by adding 300 µl HCl (8M). The OD was measured at 325 nm using an UV/VIS spectrophotometer (Jasco V650, Japan). The blank was prepared by replacing pyrogallol solution with buffer. BHT and ascorbic acid were used as controls. The percentage of superoxide radical scavenging and IC50 values were calculated as in DPPH assay.

Carbonyl groups content of untreated and HPH treated BSA and WPI samples was determined according to the method reported in Levine et al. (1990) , with some modifications as described in a previous paper (Siddique et al., 2016) . An aliquot of a protein sample, corresponding to 2 mg of protein, was incubated with 10 mmol/L 2, 4-Dinitrophenylhydrazin (DNPH) in 2 mol/L HCl (1 mL), for 30 min at room temperature (25 °C). Afterward, 1 mL of a 10% (w/v) trichloroacetic acid solution was used to precipitate proteins, which were recovered by centrifugation at 6500×g for 5 min (ALC PK130, Cologno Monzese (MI), Italy). Protein pellets were washed three times with 1 mL of ethanol/ethyl acetate 50:50 (v/v) to remove residual unreacted molecules of DNPH and subsequently dissolved in 1 mL of 6 mol/L urea (pH = 2.3). The concentration of carbonyl groups was determined by spectrophotometry at 370 nm (V-650, Jasco Inc. Easton, MD, USA) using an extinction coefficient of 2.2 × 10 4 L mol -1 cm -1 (Scheidegger, Pecora, Radici, & Kivatinitz, 2010) .