## 3 Results and Discussion
### Changes of Phenolic Bioaccessibility during the In Vitro Digestion
[26], the gut microbiota has a pivotal role in the metabolism of anthocyanins reaching the colon because of its ability to produce lower molecular weight and more bioavailable compounds, such as caffeic acid, protocatechuric acid and 4-hydroxybenzoic acid [27]. Therefore, looking at our findings, we could speculate that the higher dietary fiber content of GPP-fortified breads determined a major carrier effect of anthocyanins in the large intestine, because of interactions between fiber and anthocyanins (mainly those glycosylated) [28], thus enhancing their potential bioaccessibility in the colon. Regarding the other subclasses of phenolic compounds under investigation, the inclusion of GPP determined a higher bioaccessibility of flavones after the small intestine phase for GP5 and GP10 when compared with the control (on average: 14.5% vs. 2.7%, respectively), whilst GP5 showed the highest bioaccessibility of the remaining flavonoids (i.e., other flavonoids), being 29.2% after the small intestine phase of the in vitro static digestion. It was also interesting to notice that the GP5 sample showed a high bioaccessibility value for lower-molecular-weight compounds, being 61.7% at the end of the small intestine phase (Table 1); in this regard, the most abundant compounds detected by UHPLC-QTOF (Table S1) were hydroxytyrosol, phlorin and 2,3-dihydroxy-1-guaiacylpropanone. Finally, similar bioaccessibility values were recorded for the phenolic acids when considering the small intestinal phase, ranging from 17% (GP0) up to 23.3% (GP10). Taken together, these findings showed that the relatively high percentage bioaccessibility values observed at the end of the small intestine phase for some phenolic classes (e.g., anthocyanins, flavones, and tyrosol equivalents) could promote an antioxidant environment in the digestive tract. No comprehensive work based on untargeted phenolic profiling coupled with in vitro digestion exists in literature for GPP-fortified breads. Regarding recent existing literature based on similar experimental designs, [PERSON] et al. [29] evaluated the impact of berry polyphenols on starch digestion, testing both the co-digestion of a blueberry extract with bread and by fortifying bread with the same blueberry extract (considering 0%, 2.5%, and 5% inclusion levels). The authors reported a greater starch digestion inhibition effect for co-digesting berry extracts and bread than that obtained by digesting berry-fortified breads. Also, the interactions of polyphenols (mainly anthocyanins and procyanidins) with the food matrix reduced the polyphenols bioaccessibility, thus reducing the number of polyphenols potentially available for enzyme inhibition [29]. In another work, [PERSON] et al. [30] showed that the enrichment of rye breads with the 3% of a berry fruit powder determined a strong increase in the bioaccessibility of anthocyanins following an in vitro digestion process, whereas the least bioaccessible fraction was that of flavan-3-ols. Thereafter, multivariate statistics based on a supervised OPLS-DA approach was used to discriminate the different experimental breads during the in vitro digestion process, thus extrapolating the most discriminant compounds of the trends observed. To this aim, two different OPLS-DA score plots were produced and reported in Figure 2.  
Interestingly, both OPLS-DA models allowed the modification of phenolic profiles during the in vitro digestion process to be clearly observed, independently from the experimental bread (Figure 2A), thus revealing a strong impact of the gastrointestinal conditions in providing the detected modifications. On the other hand, the second OPLS-DA score plot (Figure 2B) showed a great difference between the free and the bound phenolic profiles of the experimental breads before the in vitro digestion (T0), with GP10 possessing the most characteristic phenolic profile.