2.8. Free Radical Scavenging Assay

DPPH radical (DPPH^•) scavenging activity of the peptide fractions was analysed according to Brand-Williams, Cuvelier, and Berset [30] with slight modification [12] regarding quantities of antioxidant solution. A 0.04 mL volume of the sample was mixed with 0.96 mL of a 6 µM solution of DPPH^• in 75% methanol. The absorbance was measured after 3 min of the reaction at 515 nm. 75% methanol was used as a blank. The scavenging effect was calculated according to Equation (1):

where Asample is the absorbance of the DPPH^• solution with the sample; and Acontrol is the absorbance of the DPPH^• solution without the sample.

The results were calculated as the IC₅₀ value (inhibitory concentration). The IC₅₀ was determined by assesment of the free radical scavenging activity of several dilutions of each sample and interpolating the peptide concentration at which the inhibition percentage reached 50%. The IC₅₀ value was calculated from the graph plotting scavenging activity for the four different contents of peptides.

ABTS radical cation (ABTS^•+) scavenging activity was determined according to Re et al. [31] with slight modification [12] regarding the quantities of the antioxidant solution. The radical solution was prepared with ABTS and potassium persulfate, diluted in water at a final concentration of 2.45 mM, and left in the dark for 16 h to allow for radical development. The solution was diluted to reach the absorbance measures around 0.7 at 734 nm. 0.96 mL of the ABTS^•+ solution was mixed with 0.04 mL of each sample. The absorbance was measured after 3 min of the reaction at 734 nm. Deionized water was used as a blank. The scavenging effect was calculated according to Equation (2):

where Asample is the absorbance of the ABTS^•+ solution with the sample; and Acontrol is the absorbance of the ABTS^•+ solution without the sample.

The results were calculated as the IC₅₀ value (inhibitory concentration). The IC₅₀ was determined by assessment of the free radical scavenging activity of several dilutions of each sample and interpolating the peptide concentration at which the inhibition percentage reached 50%. The IC₅₀ value was calculated from the graph plotting scavenging activity for the four different contents of peptides.