## 3 Results and Discussion
### Antioxidant Activity (AOA)  
One of the most popular types of biologically active compounds that intrigues researchers is antioxidants. The importance of oxidative stress and the protective functions of antioxidants encourage researchers to create different techniques to evaluate the antioxidant potential of various functional components in food. Recent studies have focused on proteins that can be enzymatically hydrolyzed to produce peptide antioxidants. As there is no universal method for its measuring, several methods are normally used to calculate the number of antioxidant molecules present in diverse food products and their potential to prevent oxidative food deterioration [64], including chemical techniques, in vitro cell biology techniques, and in vivo animal investigations. In our study, the AOA of the released peptides was measured by four generally recognized spectrophotometric methods, and the corresponding results are presented in Figure 1. Two-way ANOVA was performed in order to study the effect of the E:S ratio and the duration of hydrolysis on the AOA. For CUPRAC, FRAP, and ABTS, the single effect of both factors had impact (\(p<0.05\), results in Supplementary file). For these methods, the combined effect of both factors did not influence the AOA activity. For the DPPH method, only the effect of the E:S ratio mattered. \begin{table}
\begin{tabular}{c c} \hline
**Sample \(\mathbf{\mathscr{M}}\)** & **DH, \%** \\ \hline P1 & 9.06 \(\pm\) 0.20 \(\lx@math@degree\) \\ P2 & 14.20 \(\pm\) 0.18 \(\lx@math@degree\) \\ P3 & 18.83 \(\pm\) 0.11 \(\lx@math@degree\) \\ P4 & 13.18 \(\pm\) 0.11 \(\lx@math@degree\) \\ P5 & 17.01 \(\pm\) 0.11 \(\lx@math@degree\) \\ P6 & 23.09 \(\pm\) 0.48 \(\lx@math@degree\) \\ P7 & 16.97 \(\pm\) 0.04 \(\lx@math@degree\) \\ P8 & 22.80 \(\pm\) 0.08 \(\lx@math@degree\) \\ P9 & 27.97 \(\pm\) 0.37 \(\lx@math@degree\) \\ \hline \end{tabular} Values are means \(\pm\) SEM, n = 3 per treatment group. Means in a column without a common superscript letter differ (\(p<0.05\)) as analyzed by one-way ANOVA and the TUKEY test.  
\end{table} 
Table 2: Degree of hydrolysis of lupin protein hydrolysates. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline
**Band/MW** & **P1** & **P2** & **P3** & **P4** & **P5** & **P6** & **P7** & **P8** & **P9** \\ \hline
1 & 15,660 & 16,686 & 16,509 & 15,433 & 16,910 & 16,606 & 16,166 & 15,625 & 14,135 \\
2 & 9387 & 10,310 & 11,127 & 10,208 & 14,646 & 11,742 & 11,438 & 11,700 & 2301 \\
3 & 2308 & 2506 & 2365 & 2488 & 2758 & 2750 & 2572 & 2531 & 753 \\
4 & 843 & 1088 & 857 & 940 & 1109 & 1151 & 956 & 909 & \textless{}500 \\
5 & - & 835 & - & \textless{}500 & \textless{}500 & \textless{}500 & \textless{}500 & \textless{}500 & - \\ \hline \end{tabular}
\end{table} 
Table 3: Molecular weights (in Daltons) of identified fractions by SDS-PAGE. According to the results, presented in Figure 1, FRAP and ABTS displayed the most pronounced data. However, depending on the type of antioxidant molecule, each assay employed to evaluate antioxidant capacity should be considered as well [65]. When tested with ABTS\({}^{\bullet+}\) scavenging assay, a thousand times higher AOA was measured for all the hydrolyses, compared to the other three techniques. P2, corresponding to an E:S ratio of 0.5% showed the highest antioxidant activity (1.15 MTE/100 g protein). Until the first hour, we see a slow increase in the activity, and the last three protein hydrolyses (P7; P8 and P9) show a reduction in the free radical scavenging potential. The measurement with FRAP methodology appeared to exhibit significant reducing power. The activity varied between 5.25 and 6.45 mMTE/100 g protein, samples P2 and P6 being the most active. Similar results were obtained when CUPRAC and DPPH methods were applied. Values below 1.0 mMTE/100 g protein were measured. According to earlier research, protein hydrolyses or peptides with significant DPPH or other radical scavenging capabilities were obtained.