# Volatilome and Bioaccessible Phenolics Profiles in Lab-Scale Fermented Bee Pollen
## 3 Results and Discussion
### In Vitro Gastrointestinal Batch Digestion of BCP and Phenolics Bioaccessibility Assay  
BCP represents a valuable source of phenolic compounds, with flavonoids as the most represented class, followed by phenolic acids [61]. A plethora of biological activities have been recognized to BCP phenolics [62], but their bioaccessibility is strongly limited by the pollen grain wall, that is not easily digestible by mono-gastric organisms, such as human [3; 4; 5]. Furthermore, bioaccessibility of phenolic compounds depends on their chemodiversity. Low bioaccessibility of phenolics may represent a critical point for using BCP as dietary supplement. Since fermentation was previously shown to improve the digestibility of BCP [13], we investigated how controlled or spontaneous fermentation may affect the profile of bioaccessible phenolic. In addition to the changes in the integrity of the pollen wall, fermentation may lead to structural and chemical changes of phenolics, which may result in modified bioaccessibility and bioactivity. Even though evidences resulting from metabolomic approaches are lacking, some authors previously reported the increase of total free phenolics level and antioxidant activity in bee pollen having undergone fermentation under controlled conditions [13; 63; 64]. Similarly, structural modification of the pollen grain with a consequent improved extractability of phenolics was also achieved after high pressure treatments [65]. The literature appears in agreement about the potential of lab-scale fermentation to promote the release of phenolic compounds from BCP [13; 63; 64]. On the other hand, the evaluations regarding the increase of free phenolics level during bee bread maturation are conflicting. According to [PERSON] et al. [66], bee bread presents higher level of phenolics compared to raw BCP. On the contrary, [PERSON] & [PERSON] [63] found comparable phenolic content in bee bread and raw BCP, whereas [PERSON] et al. [60] found lower levels of phenolics in bee bread compared to raw BCP. To our opinion, such discrepancies are due to the variability of bee bread maturation process. Under the conditions of our study, nine compounds were identified into the bioaccessible fraction obtained from Raw-BCP, including phenolic acids and flavonoids that are known to be present in bee pollen (Figure 7) [67]. The bioaccessibility of many of these compounds increased following BCP fermentation. With respect to Raw-BCP, rutin and \(p\)-coumaric acid were found at higher level (\(p<0.05\)) into the bioaccessible fraction of Unstarted- and Started-BCP (Figure 7). Luteolin and kampferol were also more bioaccessible (\(p<0.05\)) after digestion of Started-BCP, and to a lesser extent, of Unstarted-BCP (Figure 7). An increase of caffe acid bioaccessibility was found for Started-BCP, and especially, Unstarted-BCP. On the other side, quercetin, 4-ethyl catechol, dihydrocaffeic acid, and dihydroraulic acid were only found after digestion of fermented BCP samples, in particular of Started-BCP (Figure 7). Although the profile and the bioaccessibility of phenolics compounds is strongly dependent on the botanical origin of BCP, fermentation clearly enhanced the release of phenolics associated to carbohydrates, proteins, or to the pollen wall, and converted phenolics into different forms. Accordingly, the highest increase into the bioaccessible fraction was observed for \(p\)-coumaric acid, which is a structural component of sporopolenin, the principal matrices comprising the outer wall of pollen [68; 69]. In particular, yeasts may have a prominent role in degradation of the plant cell wall and in release of non-extractable phenolics [13; 26]. Yeast and/or lactic acid bacteria contributed also to increase bio-accessibility of non-extractable phenolics like proanthocyanidins, rutin, or luteolin by the degradation of associated proteins and carbohydrates [70; 71].