*2.4. DPP-IV Inhibitory Activity*

A DPP-IV drug discovery kit was used to measure the ability of hydrolysates to inhibit DPP-IV activity (Enzo Life Sciences Inc., Farmingdale, New York, NY, USA). The assays were conducted according to the manufacturer's instructions. Briefly, the kit contained human recombinant DPP-IV enzyme, a chromogenic substrate (H-Gly-Pro-pNA, MW = 328.8, 10 mM in DMSO), a calibration standard (p-nitroaniline, MW = 138, in assay buffer), an inhibitor as positive control (P32/98, MW = 260.4, 1 mM in DMSO), and an assay buffer (50 mM Tris, pH 7.5). All the reagents of the kit were stored at −70 ◦C; the analyses were conducted at room temperature. The freeze-dried protein hydrolysates were dispersed in ultrapure water in concentrations from 0.78 to 12.5 mg mL−1. Assays were performed at 37 ◦C, in a 96-well microplate provided by the manufacturer and the reading was performed in a microplate reader every minute for a total of 30 min at λ 405 nm. Finally, absorbance values were plotted against time, and the "best fit" lines for data points and slope of the curves were obtained. Two technical replicates for each sample were performed. The % of inhibition was calculated with the formula:

% activity remaining (with inhibitor) = (slope of inhibitor sample/control slope) × 100

The obtained data were analyzed by a one-way analysis of variance (ANOVA) using a Statgraphics Centurion XVI for Windows software package (version 16.2.04; Statpoint Technologies, Inc. Warrenton, Virginia, VA, USA). Fisher's least significant differences (LSD) test was applied to assess the difference between each pair of means (*p* < 0.05).

### *2.5. ABTS Radical Scavenging Activity*

Antioxidant capacity was evaluated by colorimetric assay measuring the activity of the sample to scavenge the radical ABTS according to the method described by Petretto et al. [38]. The ABTS radical scavenging activity is based on the production of the radical cation (ABTS·+), prepared by reacting ABTS and potassium persulfate (2.45 mM) to reach a final concentration of 7 mM. Briefly, the solution obtained was kept in the dark at 25 ◦C for 12–16 h before the analysis. The ABTS radical solution was properly diluted with ethanol 70% to obtain an absorbance (λ = 734 nm) of 0.7 ± 0.02. The freeze-dried protein hydrolysates were dispersed in ultrapure water at concentrations ranging from 0.78 to 12.5 mg mL−1. The reduction of radical ABTS was monitored at the start and after 50 min from the beginning of the reaction. Two technical replicates for each sample were performed. The antioxidant power of samples was expressed as a percentage of inhibition, and an IC50 value was calculated from the regression curve plotting different concentrations of hydrolysates against the percentage of activity, and expressed as the mean ± SD. Data were analyzed as described in Section 2.4.
