## 3 Results and Discussion
### The Digestive Effect on Antioxidant Activity  
The antioxidant activity in frozen and freeze-dried samples subjected to the gastrointestinal digestion process was measured by ferric reducing antioxidant power (FRAP), 2\({}^{\prime}\)-azino-bis-(3-ethylbenzothazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC). The three antioxidant assays measure different mechanisms, at different conditions such as pH and temperature. As the human body has different processes, it is recommended to use different antioxidant assays to obtain the total overview of antioxidant activity in the raw material [68]. At five different time points (Figure 8), the FRAP activity in the frozen samples (dry weight) remained unchanged up until the near end of the digestive process (105 min), with a significant increase (\(p<0.05\)) at the end of digestion (165 min). The FRAP activity in freeze-dried _P. tremulus_ remained mostly stable. However, during digestion (75 min) of freeze-dried samples, the FRAP activity significantly decreased (\(p<0.05\)), before significantly increasing (\(p<0.05\)) throughout the rest of the digestive process. In addition, throughout the digestive process, no significant differences in FRAP activity were found between the frozen and freeze-dried samples, except at the end of the digestive process (165 min) where the FRAP activity of the frozen samples was significantly higher (\(p<0.05\)) compared to the freeze-dried samples. Figure 8: The antioxidant activity measured by ferric reducing antioxidant power (FRAP) during the simulated gastrointestinal digestion in frozen (\(n=10\)) and freeze-dried (\(n=5\)) red sea cucumber (_Parastichopus tremulus_). The frozen samples have theoretically been calculated from wet weight to dry weight. The results are presented as mean \(\pm\) standard error in mmol Trolox equivalents/100 g dry weight sea cucumber. * Denotes significant differences (\(p<0.05\)) between frozen and freeze-dried samples at the specific time point. x and a denote significant differences (\(p<0.05\)) in frozen (x) or freeze-dried (a) samples through the digestive process. The exhibited ABTS (Figure 9) and ORAC (Figure 10) activity in both frozen and freeze-dried samples showed the same trend as the FRAP activity. Frozen samples showed a significantly higher (\(p<0.05\)) ABTS activity at the end of the digestive process (165 min) compared to the previous time points. In addition, the ABTS activity in freeze-dried samples showed a significant decrease (\(p<0.05\)) in ABTS activity during the digestive process (75 min) before a significantly increase (\(p<0.05\)) in ABTS activity throughout the rest of the digestive process. Both frozen and freeze-dried samples showed a significantly higher (\(p<0.05\)) ORAC activity at the end of digestion compared to the beginning of the digestive process. Freeze-dried samples showed a significantly higher (\(p<0.05\)) ORAC activity at the beginning (0 min) of digestive process compared to frozen samples. However, the frozen samples exhibited higher ORAC activity during digestion and at the end of the digestive process (\(p<0.05\)). It has been shown previously that the antioxidant activity of _P. tremulus_ increased during the digestive process [27; 69]. The total antioxidant activity of proteins can be increased by breaking down the tertiary structure of the proteins to increase availability of amino acids with antioxidant activity [69]. This can be performed by enzymes, fermentation, or natural digestion [70]. The antioxidant activity measured by ORAC assay showed high variation between each replicate with high standard of error. One factor could be the measurement of ORAC activity, as a sensitive and unstable method with high dilution factors. Another factor could be the homogenization before the analysis, especially frozen samples during the digestive process.