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

<i>In vitro</i> 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 <i>in vitro</i> 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.