Optimization of an In Vitro Colonic Fermentation: Insights into Flavan-3-ol Catabolism and Microbiota Modulation

In vitro fecal fermentation models are essential for studying gut microbiota-mediated metabolism of dietary flavan-3-ols. Current methodologies typically limit fermentation periods to 24 h, potentially overlooking the complete kinetics of catabolites. This study aims to extend in vitro fecal fermentation of dietary (poly)phenols up to 48 h to improve the physiological relevance of the model. Fermentation dynamics were assessed through the simultaneous monitoring of polyphenol catabolites, pH, and microbiota composition. One flavan-3-ol monomer ((-)-epicatechin) and two oligomers (procyanidin B2 and procyanidin A2) were fermented using human fecal slurry. Fourteen catabolites were quantified at five time points, revealing that flavan-3-ol polymerization and procyanidin linkage influence bioaccessibility and catabolism. The extended fermentation provided a more complete view of flavan-3-ol metabolism, with stable pH (5-6) and unaffected microbial composition. Substrate-specific effects on microbial alpha diversity suggest distinct resilience patterns, and putative associations between microbial taxa and phenolic catabolites were identified. This study demonstrates that 48 h incubation maintains physiological relevance, capturing late-stage catabolites, making the colonic model more reliable, with significant implications for understanding the colonic fate of undigested dietary (poly)phenols and the microorganisms possibly involved in their transformation.