Ming-Hsu Chen

Dietary fibers promote positive health outcomes that are generally attributed to large bowel bacterial fermentation and associated bioactive metabolites. Historically, studies of the latter have focused on short-chain fatty acids such as butyrate. The gastrointestinal microbiota generate thousands of xenometabolites (microbe-derived, “non-host” metabolites). Most remain uncharacterized for composition and potential bioactivity, and little is known about the impact of fiber structure on the xenometabolome. Using LC/MS, we characterized culture supernatant metabolite profiles in human stool lineages derived from three healthy adult donors and six inulins covering a range of degrees of polymerization (DPs): chicory-derived Frutalose L90 (L90; DP ~ 3), Alfa Aesar (AA; DP ~ 5), Frutafit CLR (CLR; DP ~ 8), Frutafit IQ (IQ; DP ~ 12), Frutafit TEX (TEX; DP > 23), and the highly branched Frutafit agave inulin (AGA). Of the 1,219 LC/MS detected metabolites included in the final data analysis, concentrations of 704 were statistically significant (FDR < 0.1; Kruskal-Wallis test). Of these, 15 metabolites had a structural annotation, highlighting the large number of “unknown” xenometabolites associated with inulin substrates. Each fiber type led to distinct metabolite signatures, despite lineages displaying highly disparate microbial community structures within and across donors. This illustrates that fiber-specific metabolic functional guilds manifest despite highly diverse human gut bacteria communities. While speculative, these metabolic functional guilds could help explain why health effects of dietary fibers are prevalent across the population despite highly disparate gut microbiota patterns. The results also reinforce that fiber structures have a profound effect on the xenometabolome. IMPORTANCE: Dietary fibers can convey positive health effects, but the full suite of mechanisms and fiber type differences remains to be elaborated. Historically, most discussions have focused on the impact of fibers in promoting lower gut bacterial fermentation, leading to the generation of short-chain fatty acids (especially butyrate) and promoting growth of specific microbes. That said, health effects associated with dietary fiber are generally shared across diverse individuals harboring disparate gut microbial species, and it is increasingly appreciated that xenometabolites derived from microbial metabolism number in the thousands. In the current report, we applied metabolomics characterization to human stool cultures incubated with six structurally distinct inulin fibers. The results indicate that distinct, fiber-specific metabolite signatures manifest despite quite diverse bacterial community structures across donors. Such outcomes point to the existence of metabolic functional guilds that shape the metabolite landscape—and likely the unique bioactive characteristics—across dietary fiber types.

Butyrate, a physiologically active molecule, can be synthesized through metabolic interactions among colonic microorganisms. Previously, in a fermenting trial of human fecal microbiota, we observed that the butyrogenic effect positively correlated with the increasing Bifidobacterium population and an unidentified Megasphaera species. Therefore, we hypothesized that a cross-feeding phenomenon exists between Bifidobacterium and Megasphaera , where Megasphaera is the butyrate producer, and its growth relies on the metabolites generated by Bifidobacterium . To validate this hypothesis, three bacterial species ( B. longum , B. pseudocatenulatum , and M. indica ) were isolated from fecal cultures fermenting hydrolyzed xylan; pairwise cocultures were conducted between the Bifidobacterium and M. indica isolates; the microbial interactions were determined based on bacterial genome information, cell growth, substrate consumption, metabolite quantification, and metatranscriptomics. The results indicated that two Bifidobacterium isolates contained distinct gene clusters for xylan utilization and expressed varying substrate preferences. In contrast, M. indica alone scarcely grew on the xylose-based substrates. The growth of M. indica was significantly elevated by coculturing it with bifidobacteria, while the two Bifidobacterium species responded differently in the kinetics of cell growth and substrate consumption. Coculturing led to the depletion of lactate and increased the formation of butyrate. An RNA-seq analysis further revealed the upregulation of M. indica genes involved in the lactate utilization and butyrate formation pathways. We concluded that lactate generated by Bifidobacterium through catabolizing xylose fueled the growth of M. indica and triggered the synthesis of butyrate. Our findings demonstrated a novel cross-feeding mechanism to generate butyrate in the human colon. IMPORTANCE: Butyrate is an important short-chain fatty acid that is produced in the human colon through microbial fermentation. Although many butyrate-producing bacteria exhibit a limited capacity to degrade nondigestible food materials, butyrate can be formed through cross-feeding microbial metabolites, such as acetate or lactate. Previously, the literature has explicated the butyrate-forming links between Bifidobacterium and Faecalibacterium prausnitzii and between Bifidobacterium and Eubacterium rectale . In this study, we provided an alternative butyrate synthetic pathway through the interaction between Bifidobacterium and Megasphaera indica. M. indica is a species named in 2014 and is indigenous to the human intestinal tract. Scientific studies explaining the function of M. indica in the human colon are still limited. Our results show that M. indica proliferated based on the lactate generated by bifidobacteria and produced butyrate as its end metabolic product. The pathways identified here may contribute to understanding butyrate formation in the gut microbiota.