# Antioxidant Properties of Wafers with Added Pumpkin Seed Flour Subjected to In Vitro Digestion
## 3 Results
### Antioxidant Properties  
As regards antioxidant capacity, DPPH, ABTS, and Fe\({}^{2+}\)chelation assays were used, and the results were expressed as IC\({}_{50}\), which is the maximum concentration required to obtain a 50% antioxidant effect. The lower the IC\({}_{50}\) values, the higher the antioxidant capacity. This study showed that free radicals generated from ABTS and DPPH were scavenged by hydrolysates and \(\leq\)3.5 fractions obtained after in vitro digestion of control wafers and wafers with pumpkin seed addition. The best results were obtained for wafers fortified with 3 and 4 wt. % pumpkin seed flour in the context of neutralizing free radicals. The reduction power increased proportionally with the increasing addition of pumpkin seed meal, but statistically significant differences were noted for samples P2 to P4 compared to the control wafers. This may be because some peptides and amino acids with high antioxidant activity are released from pumpkin seed proteins after in vitro digestion, which converts free radicals into more stable products and terminates radical chain reactions. Similarly, some phenolic compounds, known as strong antioxidants, are released from the food matrix after enzymatic hydrolysis. However, the ability to chelate iron ions was similar for all analyzed samples and did not differ statistically (Table 4). Hydrolysates obtained after in vitro wafer digestion showed a lesser ability to neutralize ABTS, DPPH free radicals, and Fe\({}^{2+}\) chelation compared to possible bioaccessible fractions with a molecular weight \(\leq\) 3.5 kDa (Tables 4 and 5). The highest antiradical activity was noted for the fraction obtained from P4 (0.491 \(\pm\) 0.037 for ABTS\({}^{**}\) and 3.843 \(\pm\) 0.291 for DPPH\({}^{**}\)). All low-molecular-fraction samples from fortified wafers had lower IC\({}_{50}\) values \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Hydrolysate Samples**} & \multicolumn{4}{c}{**Antioxidant Properties**} \\ \cline{2-4}  & **ABTS\({}^{**}\)**+** & **DPPH\({}^{*}\)** & **Fe\({}^{2+}\) Chelation** & **Reducing Power** \\  & **[IC\({}_{50}\) mg/mL]** & **[IC\({}_{50}\) mg/mL]** & **[IC\({}_{50}\) mg/mL]** & **[\(\mu\)mol Trolox/mL]** \\ \hline C & 4.452 \(\pm\) 0.080 \({}^{\rm c}\) & 83.08 \(\pm\) 9.968 \({}^{\rm c}\) & 2.222 \(\pm\) 0.133 \({}^{\rm a}\) & 2.493 \(\pm\) 0.086 \({}^{\rm c}\) \\ P1 & 4.449 \(\pm\) 0.099 \({}^{\rm b}\) & 90.39 \(\pm\) 7.382 \({}^{\rm bc}\) & 2.105 \(\pm\) 0.105 \({}^{\rm a}\) & 2.591 \(\pm\) 0.067 \({}^{\rm bc}\) \\ P2 & 3.240 \(\pm\) 0.100 \({}^{\rm b}\) & 90.21 \(\pm\) 3.475 \({}^{\rm c}\) & 2.083 \(\pm\) 0.115 \({}^{\rm a}\) & 2.811 \(\pm\) 0.059 \({}^{\rm ab}\) \\ P3 & 2.983 \(\pm\) 0.073 \({}^{\rm b}\) & 69.59 \(\pm\) 6.727 \({}^{\rm b}\) & 2.062 \(\pm\) 0.113 \({}^{\rm a}\) & 2.855 \(\pm\) 0.206 \({}^{\rm ab}\) \\ P4 & 2.653 \(\pm\) 0.026 \({}^{\rm a}\) & 53.86 \(\pm\) 2.603 \({}^{\rm a}\) & 2.041 \(\pm\) 0.100 \({}^{\rm a}\) & 3.033 \(\pm\) 0.081 \({}^{\rm a}\) \\ \hline \end{tabular} Different letters indicate statistically significant differences between samples (\(p\leq\) 0.05, Tukey’s test).  
\end{table} 
Table 4: Antioxidant properties of hydrolysates obtained after in vitro digestion of wafers. Figure 5: The phenolic contents in hydrolysates obtained after in vitro digestion of wafers without (C) and with the addition of pumpkin flour (P1–P4). a, b—the means labeled with different letters differ significantly, with a significance level of \(p<\) 0.05.  
than control wafers, but the differences in ABTS values were not statistically significant (Table 5). Unlike hydrolyses, peptide fractions with a mass \(\leq\) 3.5 kDa obtained from wafers enriched with pumpkin seed flour showed a greater ability to chelate iron ions compared to the control. The increased Fe\({}^{2+}\) chelating ability may be due to the presence of low-molecular-weight functional components, such as amino acids, peptides, and phenolic acids from pumpkin seeds, which could block the chain reaction of free radicals by chelating metal ions.