Background: The human gut microbiota develops in concordance with its host over a lifetime, resulting in age-related shifts in community structure and metabolic function. Little is known about whether these changes impact the community’s response to microbiome-targeted therapeutics. Providing critical information on this subject, faecal microbiomes of subjects from six age groups, spanning from infancy to 70-year-old adults (n = six per age group) were harvested. The responses of these divergent communities to treatment with the human milk oligosaccharide 2’-fucosyllactose (2’FL), fructo-oligosaccharides (FOS), and lactose was investigated using the Ex vivo SIFR^® technology that employs bioreactor fermentation and is validated to be predictive of clinical findings. Additionally, it was evaluated whether combining faecal microbiomes of a given age group into a single pooled microbiome produced similar results as the individual microbiomes. Results: First, marked age-dependent changes in community structure were identified. Bifidobacterium levels strongly declined as age increased, and Bifidobacterium species composition was age-dependent: B. longum, B. catenulatum/pseudocatenulatum, and B. adolescentis were most prevalent for breastfed infants, toddlers/children, and adults, respectively. Metabolomic analyses (LA-REIMS) demonstrated that these age-dependent differences particularly impacted treatment effects of 2’FL (more than FOS/lactose). Further analysis revealed that while 2’FL enhanced production of short-chain fatty acids (SCFAs) and exerted potent bifidogenic effects, regardless of age, the specific Bifidobacterium species enhanced by 2’FL, as well as subsequent cross-feeding interactions, were highly age-dependent. Furthermore, single-pooled microbiomes produced results that were indicative of the average treatment response for each age group. Nevertheless, pooled microbiomes had an artificially high diversity, thus overestimating treatment responses (especially for infants), did not recapitulate interindividual variation, and disallowed for the correlative analysis required to unravel mechanistic actions. Conclusions: Age is an important factor in shaping the gut microbiome, with the dominant taxa and their metabolites changing over a lifetime. This divergence affects the response of the microbiota to therapeutics, demonstrated in this study using 2’FL. These results evidence the importance of screening across multiple age groups separately to provide granularity of how therapeutics impact the microbiome and, consequently, human health.

Background: Using dietary interventions to steer the metabolic output of the gut microbiota towards specific health-promoting metabolites is often challenging due to interpersonal variation in treatment responses. Methods: In this study, we combined the ex vivo SIFR^® (Systemic Intestinal Fermentation Research) technology with untargeted metabolite profiling to investigate the impact of carrot-derived rhamnogalacturonan-I (cRG-I) on ex vivo metabolite production by the gut microbiota of 24 human adults. Results: The findings reveal that at a dose equivalent to 1.5 g/d, cRG-I consistently promoted indole-3-propionic acid (IPA) production (+45.8% increase) across all subjects. At a dose equivalent to 0.3 g/d, increased IPA production was also observed (+14.6%), which was comparable to the effect seen for 1.5 g/d inulin (10.6%). IPA has been shown to provide protection against diseases affecting the gut and multiple organs. The Pearson correlation analysis revealed a strong correlation (R = 0.65, p_adjusted = 6.1 × 10⁻¹⁶) between the increases in IPA levels and the absolute levels of Bifidobacterium longum, a producer of indole-3-lactic acid (ILA), an intermediate in IPA production. Finally, the community modulation score, a novel diversity index, demonstrated that cRG-I maintained a high α-diversity which has previously been linked to elevated IPA production. Conclusions: The results from the ex vivo SIFR^® experiment mirrored clinical outcomes and provided novel insights into the impact of cRG-I on the gut microbiome function. Importantly, we demonstrated that cRG-I promotes tryptophan conversion into IPA via gut microbiome modulation, thus conferring benefits via amino acid derived metabolites extending beyond those previously reported for short chain fatty acids (SCFA) resulting from carbohydrate fermentation.

The prevalence of pediatric inflammatory bowel disease (pIBD) has been increasing over the last two decades. Yet, treatment strategies are still limited, in part due to the multifactorial nature of the disease and the complex interplay between genetic, environmental, dietary, immune, and gut microbial factors in its etiology. With their direct and indirect anti-inflammatory properties, human milk oligosaccharides (HMOs) are a promising treatment and management strategy for IBD. However, to date there are no insights into how HMOs may affect pIBD microbiota. Here, we compared the effects of 2′fucosyllactose (2′FL), difucosyllactose (DFL), 3′sialyllactose (3′SL), and blends thereof with fructooligosaccharide (FOS) on microbiota functionality (short- and branched-chain fatty acids, pH, and gas production) and composition (quantitative shallow shotgun sequencing) using fecal material from eight different pediatric Crohn’s disease patients inoculated in the SIFR^® technology. In general, all HMO treatments significantly increased total short-chain fatty acid production when compared with FOS, despite equal gas production. We found that 2′FL, either alone or in combination with DFL and 3′SL, exhibited a strong acetogenic and propiogenic effect, and 3′SL an acetogenic effect that surpassed the effects observed with FOS. No differences in overall community diversity between HMO- and FOS-treated pIBD microbiota were observed. There was, however, a stronger bifidogenic effect of 2′FL, 3′SL, 2′FL/DFL, and 2′FL/DFL + 3′SL when compared with FOS. In general, 3′SL and HMO blends enriched a broader species profile, including taxa with potentially anti-inflammatory properties, such as Faecalibacterium prausnitzii and Blautia species. This study suggests HMOs as a promising strategy to beneficially alter the gut microbial profile in pIBD.