*2.5. Trypan Blue Staining for Cell Viability*

The viability of PBLs was measured with 0.4% trypan blue staining. A total of 100 μL of cell suspension (1 × 106 cells/mL) was incubated with 100 <sup>μ</sup>L of 0.4% trypan blue. After 15 min of incubation at room temperature, the viability of the cells was measured in a Bürker chamber with the use of light microscope (Olympus CX31). Dead cells were labeled with navy-blue, and live cells remained unstained.

#### *2.6. Determination of Antioxidant Activity as the Ability to Scavenge DPPH Free Radicals*

The antioxidant activity of the obtained hydrolysates was assessed on the basis of the radical scavenging effect of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH (Sigma, St. Louis, MO, USA, D21140-0)) free radical activity according to Yen and Chen with minor modifications [12]. The tested samples were dissolved in water to a final volume of 1 mL and mixed with 1 mL of ethanol (98%). The reaction was started by adding 0.5 mL of 0.3 M DPPH in ethanol. The mixtures were left for 30 min at room temperature, and the absorbance of the resulting solutions was measured at 517 nm. For calibration, aqueous solutions of known Trolox concentrations ranging from 2 to 20 μg (able to scavenge 500 μL

of 0.3 mM DPPH radical solution) were used. Radical scavenging activity of the peptides was expressed as μM Troloxeq/mg protein.

#### *2.7. FRAP Method*

The FRAP method (ferric-reducing antioxidant power) was used to determine the antioxidative capacity of hydrolysates according to Benzie and Strain [13]. A total of 3 mL of FRAP working solution (300 mM acetate buffer pH 3.6; 10 mM 2,4,6,tripyridyl-s-triazine (TPTZ) (Fluka, 93285) and 20 mM FeCl3 × 6 H2O (10:1:1 *v*/*v*)) was mixed with 1 mL of the sample. After 10 min of reaction, the absorbance was measured at λ = 593 nm. An aqueous solution of known Fe (II) concentration was used for calibration (in the range from 100 to 1000 μg). Results were expressed as μg Fe2+/mg protein.
