## 4 Discussion
Thus, since in vitro assays can only be considered predictive tools of the functional activity in vivo and testing substances directly on animals or human is not an easy approach, ex vivo assays offer the right compromise to better understand food functional properties. In our study, the MTT assay on the Caco2 cell line was used to determine the cytotoxicity of SD5, before and after fermentation, and none was found. Conversely, the expression of _TNF-\(\alpha\)_ and _IL-1B_ from Caco2 cells was investigated through RT-PCR after treatment with the extracts from SD0 and SD5. In vitro and in vivo antioxidant and anti-inflammatory properties of grape pomace, mainly ascribed to its phenolic compounds, have been already reported (for a review, see [PERSON] et al. [35]). Yet, in our study, fermentation led to a further down-regulation of the expression of the pro-inflammatory proteins, such as cytokines (_IL-1B_ and _TNF-\(\alpha\)_), showing a significant anti-inflammatory effect of SD5-T24 compared to the other sourdoughs (before fermentation or without GPP addition). This could be due to a series of factors, among which the liberation into a free form of GPP anthocyanins which were not in the SD5-T0 and the effect of the pH. Moreover, in the conditions of this study, only anthocyanins were detected, whereas, even though limited, a contribution of the phenolic compounds provided by the wheat flour used for sourdough, as well as the effect of _L. plantarum_ metabolism on them, cannot be excluded. Indeed, a series of hydroxcinamic and hydroxybenzoic acids constitute most of the free phenolic compounds of the flour [36] and lactic acid bacteria are known for the ability to decarboxylate phenolic acids to the corresponding phenol or vinyl derivatives or hydrogenated them through phenolic acid reductases [28]. Metabolites of phenolic acids conversion, compared to their precursors, have reduced antimicrobial activity, but higher biological activities than their precursors [28], thus explaining the overall higher functional features of SD5-T24 compared to SD5-T0. The functionality and health benefits of foods, especially fermented ones, are becoming increasingly important for consumers and in tourn food industries, which is why more research should focus on these aspects, particularly if a step towards the overall sustainability of the process is to be taken [29]. Therefore, SD5 was used as ingredient in bread making. In terms of nutritional composition, the main difference among breads was the higher fiber content in SD5-B compared to SD0-B and Cy-B. It should be noted that the increase in fiber is not only due to the addition of pomace, but we can hypothesize a contribution from resistant starch, which is generated following the biological acidification [37]. During sourdough fermentation, although negatively affected by GPP addition, a copious increase of GABA (up to 50-fold) was found compared to Cy-B. Such increase is of particular interest since \(\gamma\)-aminobutyric acid, produced by LAB as response mechanism to the low pH, is a functional compound with hypotensive, diuretic, and tranquilizing effects [38] which could further enhance bread functionality. The addition of GPP in the bread formulation, as expected, slightly impacted its technological properties; nevertheless, due to the small fortification and sourdough fermentation, known for its ability to modify rheology attributes [39], such effects were mitigated. These rheological attributes mainly rely on the physicochemical changes of the protein network, as a consequence of the acidic pH, which facilitate the larger dough expansion during fermentation [39]. Finally, the addition of SD5 to bread broadened its aroma profile with herbaceous and acid tastes due to the GPP itself and the sourdough fermentation, respectively.