Human milk oligosaccharides (HMOs) shape the gut microbiota in infants by selectively stimulating the growth of bifidobacteria. Here, we investigated the impact of HMOs on adult gut microbiota and gut barrier function using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME^®), Caco2 cell lines, and human intestinal gut organoid-on-chips. We showed that fermentation of 2’-O-fucosyllactose (2’FL), lacto-N-neotetraose (LNnT), and combinations thereof (MIX) led to an increase of bifidobacteria, accompanied by an increase of short chain fatty acid (SCFA), in particular butyrate with 2’FL. A significant reduction in paracellular permeability of FITC-dextran probe was observed using Caco2 cell monolayers with fermented 2’FL and MIX, which was accompanied by an increase in claudin-8 gene expression as shown by qPCR, and a reduction in IL-6 as determined by multiplex ELISA. Using gut-on-chips generated from human organoids derived from proximal, transverse, and distal colon biopsies (Colon Intestine-Chips), we showed that claudin-5 was significantly upregulated across all three gut-on-chips following treatment with fermented 2’FL under microfluidic conditions. Taken together, these data show that, in addition to their bifidogenic activity, HMOs have the capacity to modulate immune function and the gut barrier, supporting the potential of HMOs to provide health benefits in adults.

Functional amino acids supplementation to farm animals is considered to not only be beneficial by regulating intestinal barrier, oxidative stress, and immunity, but potentially also by impacting the gut microbiota. The impact of amino acids on a piglet-derived colonic microbiota was evaluated using a 48-h in vitro batch incubation strategy. The combination of 16S rRNA gene profiling with flow cytometry demonstrated that specific microbial taxa were involved in the fermentation of each of the amino acids resulting in the production of specific metabolites. Branched chain amino acids (leucine, isoleucine, valine) strongly increased branched-chain fatty acids (+23.0 mM) and valerate levels (+3.0 mM), coincided with a marked increase of Peptostreptococcaceae. Further, glutamine and glutamate specifically stimulated acetate (~20 mM) and butyrate (~10 mM) production, relating to a stimulation of a range of families containing known butyrate-producing species (Ruminococcaceae, Oscillospiraceae, and Christensenellaceae). Finally, while tryptophan was only fermented to a minor extent, arginine and lysine specifically increased propionate levels (~2 mM), likely produced by Muribaculaceae members. Overall, amino acids were thus shown to be selectively utilized by microbes originating from the porcine colonic microbiota, resulting in the production of health-related short-chain fatty acids, thus confirming the prebiotic potential of specific functional amino acids.

Introduction: While modulation of the human adult gut microbiota is a trending strategy to improve health, the underlying mechanisms are poorly understood.Methods: This study aimed to assess the predictive value of the ex vivo, reactor-based, high-throughput SIFR^® (Systemic Intestinal Fermentation Research) technology for clinical findings using three structurally different prebiotics [inulin (IN), resistant dextrin (RD) and 2′-fucosyllactose (2′FL)].Results: The key finding was that data obtained within 1–2 days were predictive for clinical findings upon repeated prebiotic intake over weeks: among hundreds of microbes, IN stimulated Bifidobacteriaceae, RD boosted Parabacteroides distasonis, while 2′FL specifically increased Bifidobacterium adolescentis and Anaerobutyricum hallii. In line with metabolic capabilities of these taxa, specific SCFA (short-chain fatty acids) were produced thus providing insights that cannot be obtained in vivo where such metabolites are rapidly absorbed. Further, in contrast to using single or pooled fecal microbiota (approaches used to circumvent low throughput of conventional models), working with 6 individual fecal microbiota enabled correlations that support mechanistic insights. Moreover, quantitative sequencing removed the noise caused by markedly increased cell densities upon prebiotic treatment, thus allowing to even rectify conclusions of previous clinical trials related to the tentative selectivity by which prebiotics modulate the gut microbiota. Counterintuitively, not the high but rather the low selectivity of IN caused only a limited number of taxa to be significantly affected. Finally, while a mucosal microbiota (enriched with Lachnospiraceae) can be integrated, other technical aspects of the SIFR^® technology are a high technical reproducibility, and most importantly, a sustained similarity between the ex vivo and original in vivo microbiota.Discussion: By accurately predicting in vivo results within days, the SIFR^® technology can help bridge the so-called “Valley of Death” between preclinical and clinical research. Facilitating development of test products with better understanding of their mode of action could dramatically increase success rate of microbiome modulating clinical trials. Graphical Abstract

Benefits associated with probiotic use have been reported; however, the mechanisms behind these benefits are poorly understood. The effects of a probiotic formulation (MegaDuo™) containing Bacillus coagulans SC208 and Bacillus subtilis HU58 on intestinal permeability and immune markers was assessed using a combination of the in vitro gut model, the mucosal simulator of the human intestinal microbial ecosystem (M-SHIME^®), and an in vitro inflammatory bowel disease-like Caco-2/THP1 co-culture model in both healthy and antibiotic-induced dysbiosis conditions. Established M-SHIME^® proximal colon vessels were treated with/without clindamycin (1 week) and then with/without daily MegaDuo™ treatment (2 weeks). The mucosal and luminal microbial communities were sampled weekly. Suspensions were removed from the proximal colon vessels after 1 and 2 weeks of MegaDuo™ treatment and added to the co-culture system. Transepithelial resistance (membrane barrier function), cytokine/chemokine release, and NFκB activity were then measured. Under conditions of antibiotic-induced dysbiosis, suspensions from MegaDuo™ treated vessels showed reduced gut membrane barrier damage and decreased levels of TNFα and IL-6 compared with suspensions from untreated vessels; no appreciable differences were observed under healthy conditions. MegaDuo™ treatment had no effect on NFκB activity of THP1-Blue™ cells. The potential benefits of MegaDuo™ treatment appeared most evident after 2 weeks of treatment.

Background: The infant gut microbiota establishes during a critical window of opportunity when metabolic and immune functions are highly susceptible to environmental changes, such as diet. Human milk oligosaccharides (HMOs) for instance are suggested to be beneficial for infant health and gut microbiota. Infant formulas supplemented with the HMOs 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) reduce infant morbidity and medication use and promote beneficial bacteria in the infant gut ecosystem. To further improve infant formula and achieve closer proximity to human milk composition, more complex HMO mixtures could be added. However, we currently lack knowledge about their effects on infants’ gut ecosystems. Method: We assessed the effect of lactose, 2′-FL, 2′-FL + LNnT, and a mixture of six HMOs (HMO6: consisting of 2′-FL, LNnT, difucosyllactose, lacto-N-tetraose, 3′- and 6′-sialyllactose) on infant gut microbiota and intestinal barrier integrity using a combination of in vitro models to mimic the microbial ecosystem (baby M-SHIME^®) and the intestinal epithelium (Caco-2/HT29-MTX co-culture). Results: All the tested products had bifidogenic potential and increased SCFA levels; however, only the HMOs’ fermented media protected against inflammatory intestinal barrier disruption. 2′-FL/LNnT and HMO6 promoted the highest diversification of OTUs within the Bifidobactericeae family, whereas beneficial butyrate-producers were specifically enriched by HMO6. Conclusion: These results suggest that increased complexity in HMO mixture composition may benefit the infant gut ecosystem, promoting different bifidobacterial communities and protecting the gut barrier against pro-inflammatory imbalances.

Modulation of the gut microbiome as a means to improve human health has recently gained increasing interest. In this study, it was investigated whether cRG-I, a carrot-derived pectic polysaccharide, enriched in rhamnogalacturonan-I (RG-I) classifies as a potential prebiotic ingredient using novel in vitro models. First, digestion methods involving α-amylase/brush border enzymes demonstrated the non-digestibility of cRG-I by host-derived enzymes versus digestible (starch/maltose) and non-digestible controls (inulin). Then, a recently developed short-term (48 h) colonic incubation strategy was applied and revealed that cRG-I fermentation increased levels of health-promoting short-chain fatty acids (SCFA; mainly acetate and propionate) and lactate comparable but not identical to the reference prebiotic inulin. Upon upgrading this fermentation model by inclusion of a simulated mucosal environment while applying quantitative 16S-targeted Illumina sequencing, cRG-I was additionally shown to specifically stimulate operational taxonomic units (OTUs) related to health-associated species such as Bifidobacterium longum, Bifidobacterium adolescentis, Bacteroides dorei, Bacteroides ovatus, Roseburia hominis, Faecalibacterium prausnitzii, and Eubacterium hallii. Finally, in a novel model to assess host–microbe interactions (Caco-2/peripheral blood mononuclear cells (PBMC) co-culture) fermented cRG-I increased barrier integrity while decreasing markers for inflammation. In conclusion, by using novel in vitro models, cRG-I was identified as a promising prebiotic candidate to proceed to clinical studies.