Targeted Analysis of Bacterial Catabolism End-Products of Cacao Polyphenols by the Human Gut Microbiota

Objectives: Identify and quantify the microbial catabolites of cocoa flavanols generated through anaerobic fermentation by the human gut microbiota, using both an in vitro and a human intervention study. Methods: The in vitro fermentation experiments were carried out in bioreactors using a fecal inoculum from a pool of healthy volunteers. The intervention was conducted on eight volunteers aged 21-43 who consumed 40 g/day of dark chocolate for five days. Feces and urine samples were collected on the first day and at the end. Microbial catabolites were analyzed in fecal slurries and urine samples by GC/MS, while gut microbiota compositions (16S rRNA sequences) were measured in feces and fecal slurries. This study was registered on the WHO International Clinical Trials Registry Platform, Primary Registry RPCEC00000389. Results: Catabolites of dihydroferulic, 3- and 4-propionic, 3-(3-hydroxyphenyl) propionic, 4-hydroxyphenylacetic, 3-phenylpropionic, 4-hydroxybenzoic, 3-hydroxybenzoic, and benzoic acids, and catechol were detected both in vitro and in vivo models. In vitro, the 3-(3-hydroxyphenyl) propionic acid decreased after 6 hours of (+)-catechin fermentation, suggesting its conversion to 3-hydroxybenzoic acid. Conversely, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, and catechol increased after (+)-catechin fermentation. In the in vivo model, 24 hours after chocolate intake, several urinary catabolites previously detected and quantified in vitro showed increased levels: 3-hydroxy benzoic acid, 2-(3-hydroxyphenyl) acetic acid, 3-(3-hydroxyphenyl) propionic acid, vanillic acid, 3-(4-hydroxyphenyl)-(2E) propenoic acid and, sinapic acid. The urinary elimination of these catabolites accounted for their absorption and distribution. Finally, the microbiota analysis in vitro suggests that Eggerthella, Lachnoclostridium B, and Bacteroides eggerthii might play a role in the observed chemical transformations, while in vivo, Flavonifractor and Lachnoclostridium B seem to have changed between the beginning and the end of the intervention. Conclusions: The human gut microbiota's complete catabolism of cocoa flavanols. We identify bacterial species that might be responsible for their biotransformation and summarize the main end products formed during this interaction. Funding Sources: MinCiencias, CNC S.A.S, Vidarium, and University CES.