MAIN CONCLUSION Contrarily to Bacteroides fragilis and Bifidobacterium longum, Lactobacillus reuteri, a gut bacterium, has shown the capacity to biotransform 5-O-caffeoylquinic acid (5-CQA), via different pathways, among them an oxidative one that has not been described so far. INTRODUCTION Polyphenols are known for their broad range of positive bioactivities in human. These molecules have shown the ability to lower oxidative stress involved in many pathologies and positively modulate redox cellular signalling pathways. However, given the pivotal role of microbiota to generate bioavailable and bioactive metabolites [1] and considering their superiority in terms of presence in systemic system compared to parent polyphenols, further insights are focused nowadays on their gut derived metabolites as promising approach to prevent and attenuate neurodegenerative diseases [2]. We have investigated the biotransformation of 5-O-caffeoylquinic acid, known as chlorogenic acid, by three gut-bacteria species: Lactobacillus reuteri (Firmicutes), Bacteroides fragilis (Bacteroidetes) and Bifidobacterium longum (Actinobacterium), belonging to the dominant bacterial phyla of the gut microbiota. The biotransformed extracts were analysed by LC-MS/MS and the data were subject to MzMine 2.53 and GNPS treatment for subsequent molecular networking analysis. It is interesting to note that gut microbiota polyphenol biotransformation such as hydrolysis, cleavage and reduction are well known but there is a lack of information on possible oxidative pathways [3]. Hence, an electrochemical strategy was adopted to generate oxidized compounds of chlorogenic acid (5-CQA) and caffeic acid (CA), a gut-derivative of 5-CQA, in order to characterize and compare their mass profiles to the extract ones. MATERIALS & METHODS Bacteria were grown in BHI broth (Brain Heart Infusion) under anaerobic conditions using an anaerobic chamber. Resting cells biotransformation studies were carried out, when bacteria reached the stationary phase, by adding the solution of 5-CQA (1 mM) in phosphate buffer (pH 5.8) on biomass of each bacterial culture. They were incubated under anaerobic condition using sealed jars and anaerobic bags. After 24 h of incubation the extraction process was performed on pellet and on supernatant using ethyl acetate. After evaporation of organic solvents, the resulting supernatant and pellet extracts were analyzed by LC-MS/MS. Additionally, electrooxidation of 5-CQA 2mM and CA 2mM were performed in phosphate buffer 0.1 M at pH = 5.8. A chronoamperometry technique i = f (t) was conducted at a fixed potential during 20 min using a platinum grid working electrode, in the absence of oxygen. The UV-Vis spectra were simultaneously recorded over a spectral range of 200 to 600 nm. RESULTS & DISCUSSION Some metabolites were annotated into extracts obtained after biotransformation of 5-CQA by L. reuteri, of which caffeic acid (CA) and 3-hydroxybenzoic acid (3-HBA). Interestingly, our study highlighted the formation of caffeic acid quinone generated by an oxidative pathway, which are unexpected in anaerobic gut environment. Mortele et al., in 2019 have already identified caffeic acid quinone in fecal biotransformation studies [4]. However, to our knowledge, the oxidative biotransformation reactions of cinnammate esters by gut microbiota has not been discussed so far. This finding highlighted the particular enzymatic machinery of L. reuteri strain. Compounds arising from the electrooxidation of chlorogenic acid at pH 5.8 were not found in the extracts. However, the electrolyzed solution of caffeic acid, has also shown the presence of caffeic acid quinone, although it was not the major compound, as its stability is probably low. So far, we have no explanation why the stability of the caffeic acid quinone seems higher in the biotransformed extracts. Moreover, we did not find metabolites after bioconversion by B. fragilis and B. longum, suggesting that they are not able to biotransform 5-CQA. Therefore, in this project, we aim to give more information on the oxidized derivatives issuing from the biotransformation of 5-CQA, which may show a higher activity that the native compounds. REFERENCES [1] Pasinetti, G. M. et al. 2018. J. Alzheimers Dis. 63: 409–421. [2] Carregosa, D. et al. 2020. Agric. Food Chem. 68: 1790–1807 [3] Espín, J. C., González-Sarrías, A. & Tomás-Barberán, F. A. 2017. Biochem. Pharmacol. 139: 82–93. [4] Mortelé, O. et al. 2019. J. Pharm. Biomed. Anal. 175: 112768.