# Antioxidant Properties of Wafers with Added Pumpkin Seed Flour Subjected to In Vitro Digestion
## 4 Discussion
When analyzing the results obtained, it can be noted that the addition of pumpkin seed flour did not influence textural parameters significantly, which is positive from the point of view of potential consumers, because the texture of fortified wafers does not differ from that of traditional, well-known, and accepted wafers. [PERSON] et al. [32] observed that millet-flour-enriched wafers were characterized by lesser hardness than the control, while the addition of millet flour had no influence on the fracturability of wafers. Pumpkin seeds contain significant amounts of protein, which is a good source of biologically active peptides. Therefore, the peptide content was determined after each enzymatic hydrolysis step. Not every peptide chain fragment obtained from the protein by simulated digestion can be absorbed from the intestine into the bloodstream. The bioavailability of phytochemicals is determined by their molecular weight [33; 34]. Therefore, in this study, we conducted simulated absorption of peptide fractions with a molecular weight of 3.5 kDa. The content of peptides released during in vitro digestion of wafers depended on the percentage of pumpkin flour added. The highest peptides were obtained from wafers with a 3 wt. % or 4 wt. % addition of pumpkin seed flour. Wafers enriched with an appropriate amount of pumpkin seed flour can serve as a source of biologically active peptides released during the hydrolysis process. Similar conclusions were drawn by [PERSON] et al. (2014), who observed an increase in the peptide content in wheat bread enriched with pumpkin pulp [35]. From the point of view of human nutrition, what is important is not so much the content of bioactive compounds, but their potential bioavailability after consumption. Therefore, in the present study, the concentration of phenolic compounds was determined after hydrolyzing the wafers with enzymes present in the human digestive tract. The highest amount was recorded for wafers with a 4 wt. % pumpkin seed flour addition (0.429 mg GAE/mL). These results demonstrate the potential of pumpkin seed flour as a component rich in phenolic compounds, which may contribute to the improvement of wafers' healthiness. The total phenolic compounds in pumpkin seed powder were determined by [PERSON] et al. (2020) to be 25.62 \(\pm\) 1.86 mg GAE/100 g [31]. The liberation of dietary phenolics from the food matrix typically happens during intestinal digestion or through metabolism by the colonic microbiota. Additionally, the process of solubilizing released phenolics in the human body can be greatly influenced by interactions with the food matrix. These interactions can aid in facilitating the transportation of phenolics to the absorption site by impeding their degradation in the gastrointestinal tract. Regarding the antioxidant activity, both hydrolysates and \(\leq\)3.5 kDa fractions exhibited antiradical activity against DPPH* and ABTS*+. Potentially bioaccessible fractions (\(\leq\)3.5 kDa) showed stronger antiradical properties and Fe chelation ability (lower IC\({}_{50}\) values) than hydrolysates. In the case of hydrolysates, the greatest ability to neutralize free radicals was observed for the P4 sample with the greatest addition of pumpkin seed flour (4 wt. %). These results confirm the beneficial health properties of pumpkin seed flour. Similar studies were conducted by [PERSON] et al. [30]. The optimized cookies fortified with pumpkin flour had a higher total antioxidant activity (43.186%) than the control cookies (38.716%). Moreover, in our study, hydrolysates of wafers with pumpkin seed flour showed a very high capacity to reduce iron (III) ions compared to the control sample. However, fractions obtained after in vitro absorption exhibited a lower value of reduction power evaluated as the Trolox equivalent. Pumpkin seeds, which are usually discarded as a waste material during processing, are a very healthy source of protein and a storehouse of compounds with antioxidant properties, such as phenolic compounds.