# Volatilome and Bioaccessible Phenolics Profiles in Lab-Scale Fermented Bee Pollen
## 3 Results and Discussion  
### Volatilome Analysis  
BCP samples were characterized for the profile of VOC by HS-SPME-GC-MS. VOC (106) were identified and grouped according to the following chemical groups (Figure 1, Table S3): volatile free fatty acids (11 compounds identified), alkanes (9), alkenes (8), alcohols (23), aldehydes (6), ketones (12), esters (27), furrans (3), lactons (1), terpenes (4), and sulphur compounds (1). At a glance, the levels of VOC differentiated the BCP samples. Total VOC in Raw-BCP were estimated at 17,751 \(\pm\) 628 ppb, with aldehydes as predominant group (30% of the total VOC), followed by alcohols (21%), ketones (16%), and volatile free fatty acids (VFFA) (12%). The other chemical groups accounted for the remaining 21% of the total VOC. Few data on BCP volatiles are available in literature. Furthermore, it is not simple to compare our results with any previously obtained, because quantitative and qualitative compositions of VOC are mainly linked to the floral species, and, to a lesser extent, to climatic conditions and geographical locations. Thus, each pollen has its own specific VOC profile. VOC profiles approaching ours were previously found in bee pollen and honey samples [28; 29; 30; 31]. Volatiles are strongly related to some BCP properties. They confer a characteristic basic odor and aroma to BCP, which may impact on consumers acceptability. However, some VOC may exert antimicrobial or antioxidant activities [32; 33; 34] or act as signaling molecules for insects [35; 36]. Under the conditions of our study, Started-BCP underwent controlled fermentation driven by the selected starters composed by three _A. kunkeei_ strains and _H. uvarum_ AN8Y27B [13]. On the contrary, Unstarted-BCP was subjected to spontaneous fermentation carried out by the autochthonous microbiota naturally associated to BCP. Fermentation significantly (_p_ < 0.05) increased the amount of total VOC in both Unstarted-(90,014 \(\pm\) 4012 ppb) and Started-BCP (88,105 \(\pm\) 2285 ppb), as well as modifying the relative proportions among the chemical groups (Figure 1). VFFA were the predominant VOC in Unstarted-BCP (61% of total VOC) followed by esters (17%) and alcohols (9%). Similarly, VFFA (56% of total VOC), esters (20%) and alcohols (11%) prevailed in Started-BCP. Comparing BCP samples, the highest (_p_ < 0.05) level of aldehydes (5352 \(\pm\) 60 ppb) distinguished Raw-BCP. Unstarted-BCP showed by the highest (_p_ < 0.05) amount of VFFA (55,229 \(\pm\) 2508 ppb). Esters and alcohols were detected at the highest (_p_ < 0.05) concentrations in Started-BCP (17,906 \(\pm\) 577 and 9260 \(\pm\) 175 ppb, respectively) (Figure 1). Main aldehydes identified in Raw-BCP were hexanal (3650 \(\pm\) 91 ppb), 2,4-heptadienal (E,E) (813 \(\pm\) 31 ppb), nonanal (633 \(\pm\) 37 ppb), and propanal (230 \(\pm\) 11 ppb) (Table S3). Such compounds were totally or mostly depleted during fermentation of BCP (Table S3). It was likely due to the instability of aldehydes, which in fermented matrices frequently undergo reduction to alcohols, or oxidation to carboxylic acids [37]. This could explain the lowest levels (_p_ < 0.05) of aldehydes and the highest (_p_ < 0.05) amounts of VFFA and alcohols found in Unstarted- and Started-BCP (Figure 1). The levels of VFFA were more than twenty fold higher in Unstarted- and Started-BCP compared to Raw-BCP (Figure 1). Fatty acids are present in pollen both as triacylglycerlevels and in free form [38]. Acetic acid (1246 \(\pm\) 122 ppb), hexanoic acid (204 \(\pm\) 13 ppb), 2-methyldecanoic acid (173 \(\pm\) 11 ppb), and propionic acid (150 \(\pm\) 18 ppb) were the VFFA most represented in Raw-BCP (Figure 2). Accumulation of VFFA during BCP fermentation was one of the main microbiota dependent trend observed in our study (Figure 2). The increase was mainly due to the release of acetic acid (Figure 2), which represents, along with lactic acid, one of the main end metabolites of _A. kunkeei_[20]. This is in agreement with the inoculum of _A.