# Volatilome and Bioaccessible Phenolics Profiles in Lab-Scale Fermented Bee Pollen
## 3 Results and Discussion
### Volatilome Analysis
Data were subjected to one-way ANOVA; pair-comparison of treatment means was achieved by [PERSON]’s procedure at \(p\) < 0.05. For each compound, bars with different superscript letters differ significantly (\(p\) < 0.05). Terpenes were poorly represented in Raw-BCP, with only 4 compounds detected at low concentrations: d-limonene (16 \(\pm\) 1 ppb), cis-linalool oxide (34 \(\pm\) 3 ppb), \(\alpha\)-Linalool (15 \(\pm\) 1), and cis-geraniol (80 \(\pm\) 7 ppb). During fermentation, \(\alpha\)-linalool significantly increased in Started-BCP (ca. 400%), and, to a lesser extent, in Unstarted-BCP (ca. 200%). The amount of d-limonene increased in all fermented samples (ca. 100%). The increase of free terpene levels was attributable to the release of glycosylically conjugated precursors, as a consequence of enzymatically (\(\beta\)-glucosidase) or acid hydrolyses, resulting in enhanced flavoring and functional properties [54]. Terpenes are usually correlated to pine, citrus and spicy notes [37]. Natural monoterpenes as well as their synthetic derivatives were reported to have various health-promoting features. Antioxidant properties were attributed to limonene and linalool, which act as termination enhancing antioxidants. Their activity it is not linearly related to their concentration, and their effectiveness depends on the rate of chain-termination of the oxidizable substrate [34]. Anti-inflammatory, vaso-relaxation and sedative effects were also reported for linalool [55; 56; 57]. Anti-inflammatory activity of linalool was associated to the downregulation of pro-inflammatory mediators and to the modulation of NF-kB and MAPKs signaling pathways. Sedative and anesthetics effects were linked to the interference with nicotinic acetylcholine and \(\gamma\)-aminobutyric acid type A receptors by linalool. Vasco-relaxation activity was associated to the activation of soluble guanylyl cyclase and K\({}^{+}\) channels. Figure 5: Concentration (ppb) of esters identified in Raw- (black bars), Unstarted- (blue bars), and Started-BCP (red bars). Data are the means (\(\pm\) SD) of three independent experiments analyzed in triplicate. Data were subjected to one-way ANOVA; pair-comparisons of treatment means was achieved by [PERSON]’s procedure at \(p<0.05\). For each compound, bars with different superscript letters differ significantly (\(p<0.05\)). Several VOC, whose concentration increased during BCP fermentation, were previously shown to exert antimicrobial activities. For instance, limonene, phenyl ethyl alcohol, and 2-heptanone were shown to exert antibacterial or antifungal activities [32; 33; 58]. Focusing on antifungal activity, these compounds are able to damage the structure of fungal conidia, to prevent their germination, and to induce the down-regulation of gene expression involved in mycotoxins biosynthesis. Likely, such compounds contribute, together with organic acids, to the antimicrobial features recognized to lab scale-fermented BCP [13]. To our knowledge, few reports previously characterized the volatilome of bee bread or fermented BCP. Among the most complete studies, that of [PERSON] et al. [59] identified dimethyl sulphide, acetic acid, furfural, nonane, 1-heptadecene, dodecane, 2,4-dimethylheptane, and hexanoic acid as preeminent compounds in multifloreal bee bread. At a glance, only the high levels of some VFFA (acetic acid and hexanoic acid) and nonane were shared by both BCP fermented under the conditions of our study and beebread previously characterized. Our findings were partially discordant with that of [PERSON] et al. [60], who compared the fatty acid profiles of bee pollen and bee bread samples from the same bee hive. According to their report, the VFFA content in bee bread was not higher than found in fresh bee pollen, except for one sample showing higher level of octanoic acid. We have to point that a systematic and ultimate comparison between bee bread and lab-scale fermented BCP is difficult to achieve, because bee bread usually present traces of other interfering substances (e.g., honey).