HMOs Impact the Gut Microbiome of Children and Adults Starting from Low Predicted Daily Doses.

Recent studies suggest that the dietary intake of human milk oligosaccharides (HMOs) provides health benefits from infancy up to adulthood. Thus far, beneficial changes in the adult gut microbiome have been observed at oral doses of 5-20 g/day of HMOs. Efficacy of lower doses has rarely been tested. We assessed four HMO molecular species-2'Fucosyllactose (2'FL), Lacto-N-neotetraose (LNnT), 3'Sialyllactose (3'SL), and 6'Sialyllactose (6'SL)-at predicted doses from 0.3 to 5 g/day for 6-year-old children and adults (n = 6 each), using ex vivo SIFR® technology (Cryptobiotix, Ghent, Belgium). This technology employing bioreactor fermentation on fecal samples enables us to investigate microbial fermentation products that are intractable in vivo given their rapid absorption/consumption in the human gut. We found that HMOs significantly increased short-chain fatty acids (SCFAs), acetate, propionate (in children/adults), and butyrate (in adults) from predicted doses of 0.3-0.5 g/day onwards, with stronger effects as dosing increased. The fermentation of 6'SL had the greatest effect on propionate, LNnT most strongly increased butyrate, and 2'FL and 3'SL most strongly increased acetate. An untargeted metabolomic analysis revealed that HMOs enhanced immune-related metabolites beyond SCFAs, such as aromatic lactic acids (indole-3-lactic acid/3-phenyllactic acid) and 2-hydroxyisocaproic acid, as well as gut-brain-axis-related metabolites (γ-aminobutyric acid/3-hydroxybutyric acid/acetylcholine) and vitamins. The effects of low doses of HMOs potentially originate from the highly specific stimulation of keystone species belonging to, for example, the Bifidobacteriaceae family, which had already significantly increased at doses of only 0.5 g/day LNnT (adults) and 1 g/day 2'FL (children/adults).

Tomato seed extract promotes health of the gut microbiota and demonstrates a potential new way to valorize tomato waste.

The current effort to valorize waste byproducts to increase sustainability and reduce agricultural loss has stimulated interest in potential utilization of waste components as health-promoting supplements. Tomato seeds are often discarded in tomato pomace, a byproduct of tomato processing, yet these seeds are known to contain an array of compounds with biological activity and prebiotic potential. Here, extract from tomato seeds (TSE), acquired from pomace, was evaluated for their ability to effect changes on the gut microbiota using an ex vivo strategy. The results found that TSE significantly increased levels of the beneficial taxa Bifidobacteriaceae in a donor-independent manner, from a range of 18.6-24.0% to 27.0-51.6% relative abundance following treatment, yet the specific strain of Bifidobacteriaceae enhanced was inter-individually variable. These structural changes corresponded with a significant increase in total short-chain fatty acids, specifically acetate and propionate, from an average of 13.3 to 22.8 mmol/L and 4.6 to 7.4 mmol/L, respectively. Together, these results demonstrated that TSE has prebiotic potential by shaping the gut microbiota in a donor-independent manner that may be beneficial to human health. These findings provide a novel application for TSE harvested from tomato pomace and demonstrate the potential to further valorize tomato waste products.

Targeted remodeling of the human gut microbiome using Juemingzi (Senna seed extracts).

The genus Senna contains globally distributed plant species of which the leaves, roots, and seeds have multiple traditional medicinal and nutritional uses. Notable chemical compounds derived from Senna spp. include sennosides and emodin which have been tested for antimicrobial effects in addition to their known laxative functions. However, studies of the effects of the combined chemical components on intact human gut microbiome communities are lacking. This study evaluated the effects of Juemingzi (Senna sp.) extract on the human gut microbiome using SIFR® (Systemic Intestinal Fermentation Research) technology. After a 48-hour human fecal incubation, we measured total bacterial cell density and fermentation products including pH, gas production and concentrations of short chain fatty acids (SCFAs). The initial and post-incubation microbial community structure and functional potential were characterized using shotgun metagenomic sequencing. Juemingzi (Senna seed) extracts displayed strong, taxon-specific anti-microbial effects as indicated by significant reductions in cell density (40%) and intra-sample community diversity. Members of the Bacteroidota were nearly eliminated over the 48-hour incubation. While generally part of a healthy gut microbiome, specific species of Bacteroides can be pathogenic. The active persistence of the members of the Enterobacteriaceae and selected Actinomycetota despite the reduction in overall cell numbers was demonstrated by increased fermentative outputs including high concentrations of gas and acetate with correspondingly reduced pH. These large-scale shifts in microbial community structure indicate the need for further evaluation of dosages and potential administration with prebiotic or synbiotic supplements. Overall, the very specific effects of these extracts may offer the potential for targeted antimicrobial uses or as a tool in the targeted remodeling of the gut microbiome.

Metagenomes and metagenome-assembled genomes from ex vivo fecal incubations of six unique donors.

We present a donor-specific collection of 78 metagenomes (13/donor) and 143 metagenome-assembled genomes (MAGs), representing the gut microbiomes of six healthy adult human donors. In addition to adding to the catalog of publicly available human gut MAGs, this resource permits a genome-resolved look into microbial co-occurrence across six individuals.

A Long-Chain Dextran Produced by Weissella cibaria Boosts the Diversity of Health-Related Gut Microbes Ex Vivo.

Long-chain dextrans are α-glucans that can be produced by lactic acid bacteria. NextDextTM, a specific long-chain dextran with a high degree of polymerisation, produced using Weissella cibaria, was recently shown to exert prebiotic potential in vitro. In this study, the ex vivo SIFR® technology, recently validated to provide predictive insights into gut microbiome modulation down to the species level, was used to investigate the effects of this long-chain dextran on the gut microbiota of six human adults that altogether covered different enterotypes. A novel community modulation score (CMS) was introduced based on the strength of quantitative 16S rRNA gene sequencing and the highly controlled ex vivo conditions. This CMS overcomes the limitations of traditional α-diversity indices and its application in the current study revealed that dextran is a potent booster of microbial diversity compared to the reference prebiotic inulin (IN). Long-chain dextran not only exerted bifidogenic effects but also consistently promoted Bacteroides spp., Parabacteroides distasonis and butyrate-producing species like Faecalibacterium prausnitzii and Anaerobutyricum hallii. Further, long-chain dextran treatment resulted in lower gas production compared to IN, suggesting that long-chain dextran could be better tolerated. The additional increase in Bacteroides for dextran compared to IN is likely related to the higher propionate:acetate ratio, attributing potential to long-chain dextran for improving metabolic health and weight management. Moreover, the stimulation of butyrate by dextran suggests its potential for improving gut barrier function and inflammation. Overall, this study provides a novel tool for assessing gut microbial diversity ex vivo and positions long-chain dextran as a substrate that has unique microbial diversity enhancing properties.

Impact of Baizhu, Daqingye, and Hehuanhua extracts on the human gut microbiome.

Introduction: In traditional Chinese medicine, the rhizome of Atractylodes macrocephala (Baizhu), the leaves of Isatis indigotica (Daqingye), and the flowers of Albizia julibrissin (Hehuanhua) have been used to treat gastrointestinal illnesses, epidemics, and mental health issues. Modern researchers are now exploring the underlying mechanisms responsible for their efficacy. Previous studies often focused on the impact of purified chemicals or mixed extracts from these plants on cells in tissue culture or in rodent models. Methods: As modulation of the human gut microbiome has been linked to host health status both within the gastrointestinal tract and in distant tissues, the effects of lipid-free ethanol extracts of Baizhu, Daqingye, and Hehuanhua on the human adult gut microbiome were assessed using Systemic Intestinal Fermentation Research (SIFR®) technology (n=6). Results and discussion: Baizhu and Daqingye extracts similarly impacted microbial community structure and function, with the extent of effects being more pronounced for Baizhu. These effects included decreases in the Bacteroidetes phylum and increases in health-related Bifidobacterium spp. and short chain fatty acids which may contribute to Baizhu's efficacy against gastrointestinal ailments. The changes upon Hehuanhua treatment were larger and included increases in multiple bacterial species, including Agathobaculum butyriciproducens, Adlercreutzia equolifaciens, and Gordonibacter pamelaeae, known to produce secondary metabolites beneficial to mental health. In addition, many of the changes induced by Hehuanhua correlated with a rise in Enterobacteriaceae spp., which may make the tested dose of this herb contraindicated for some individuals. Overall, there is some evidence to suggest that the palliative effect of these herbs may be mediated, in part, by their impact on the gut microbiome, but more research is needed to elucidate the exact mechanisms.

Low-no-calorie sweeteners exert marked compound-specific impact on the human gut microbiota ex vivo.

Low-no-calorie sweeteners (LNCS) are used as sugar substitutes as part of strategies to reduce the risk of chronic diseases related to high sugar intake (e.g. type 2 diabetes (T2D)). This study investigated how a range of sweeteners [tagatose (TA)/maltitol (MA)/sorbitol (SO)/stevia (ST)/sucralose (SU)/acesulfame K (ACK)] impact the gut microbiota of T2D subjects and healthy human adults using the ex vivo SIFR® technology (n = 12). The cohort covered clinically relevant interpersonal and T2D-related differences. ACK/SU remained intact while not impacting microbial composition and metabolite production. In contrast, TA/SO and ST/MA were respectively readily and gradually fermented. ST and particularly TA/SO/MA increased bacterial density and SCFA production product-specifically: SO increased acetate (∼Bifidobacterium adolescentis), whilst MA/ST increased propionate (∼Parabacteroides distasonis). TA exerted low specificity as it increased butyrate for healthy subjects, yet propionate for T2D subjects. Overall, LNCS exerted highly compound-specific effects stressing that results obtained for one LNCS cannot be generalised to other LNCS.

Carrot RG-I Reduces Interindividual Differences between 24 Adults through Consistent Effects on Gut Microbiota Composition and Function Ex Vivo.

The human gut microbiota is characterized by large interpersonal differences, which are not only linked to health and disease but also determine the outcome of nutritional interventions. In line with the growing interest for developing targeted gut microbiota modulators, the selectivity of a carrot-derived rhamnogalacturonan I (cRG-I) was compared to substrates with demonstrated low (inulin, IN) and high selectivity (xanthan, XA), at a human equivalent dose (HED) of 1.5 g/d. The high throughput of the ex vivo SIFR® technology, validated to generate predictive insights for clinical findings, enabled the inclusion of 24 human adults. Such an unprecedented high number of samples in the context of in vitro gut microbiota modelling allowed a coverage of clinically relevant interpersonal differences in gut microbiota composition and function. A key finding was that cRG-I supplementation (already at an HED of 0.3 g/d) lowered interpersonal compositional differences due to the selective stimulation of taxa that were consistently present among human adults, including OTUs related to Bacteroides dorei/vulgatus and Bifidobacterium longum (suspected keystone species), Bacteroides thetaiotaomicron, Bifidobacterium adolescentis and butyrate-producing taxa such as Blautia sp., Anaerobutyricum hallii, and Faecalibacterium prausnitzii. In contrast, both IN and XA treatments increased interpersonal compositional differences. For IN, this followed from its low specificity. For XA, it was rather the extremely high selectivity of XA fermentation that caused large differences between 15 responders and 9 nonresponders, caused by the presence/absence of highly specific XA-fermenting taxa. While all test compounds significantly enhanced acetate, propionate, butyrate, and gas production, cRG-I resulted in a significantly higher acetate (+40%), propionate (+22%), yet a lower gas production (-44%) compared to IN. cRG-I could thus result in overall more robust beneficial effects, while also being better tolerated. Moreover, owing to its remarkable homogenization effect on microbial composition and metabolite production, cRG-I could lead to more predictable outcomes compared to substrates that are less specific or overly specific.

Bridging preclinical and clinical gut microbiota research using the ex vivo SIFR® technology.

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.

HMOs Exert Marked Bifidogenic Effects on Children's Gut Microbiota Ex Vivo, Due to Age-Related Bifidobacterium Species Composition.

Prebiotics are substrates that are selectively utilized by host microorganisms, thus conferring a health benefit. There is a growing awareness that interpersonal and age-dependent differences in gut microbiota composition impact prebiotic effects. Due to the interest in using human milk oligosaccharides (HMOs) beyond infancy, this study evaluated how HMOs [2'Fucosyllactose (2'FL), Lacto-N-neotetraose (LNnT), 3'Sialyllactose (3'SL), 6'Sialyllactose (6'SL)] and blends thereof affect the microbiota of 6-year-old children (n = 6) and adults (n = 6), compared to prebiotics inulin (IN) and fructooligosaccharides (FOS). The ex vivo SIFR® technology was used, given its demonstrated predictivity in clinical findings. First, HMOs and HMO blends seemed to maintain a higher α-diversity compared to FOS/IN. Further, while 2'FL/LNnT were bifidogenic for both age groups, 3'SL/6'SL and FOS/IN were exclusively bifidogenic for children and adults, respectively. This originated from age-related differences in microbiota composition because while 3'SL/6'SL stimulated B. pseudocatenulatum (abundant in children), FOS/IN enhanced B. adolescentis (abundant in adults). Moreover, all treatments significantly increased acetate, propionate and butyrate (only in adults) with product- and age-dependent differences. Among the HMOs, 6'SL specifically stimulated propionate (linked to Bacteroides fragilis in children and Phocaeicola massiliensis in adults), while LNnT stimulated butyrate (linked to Anaerobutyricum hallii in adults). Indole-3-lactic acid and 3-phenyllactic acid (linked to immune health) and gamma-aminobutyric acid (linked to gut-brain axis) were most profoundly stimulated by 2'FL and HMO blends in both children and adults, correlating with specific Bifidobacteriaceae. Finally, 2'FL/LNnT increased melatonin in children, while 3'SL remarkably increased folic acid in adults. Overall, age-dependent differences in microbiota composition greatly impacted prebiotic outcomes, advocating for the development of age-specific nutritional supplements. HMOs were shown to be promising modulators in the adult, and particularly the children's microbiota. The observed HMO-specific effects, likely originating from their structural heterogeneity, suggest that blends of different HMOs could maximize treatment effects.

Lacticaseibacillus rhamnosus ATCC 53103 and Limosilactobacillus reuteri ATCC 53608 Synergistically Boost Butyrate Levels upon Tributyrin Administration Ex Vivo.

Modulation of the gut microbiota is a trending strategy to improve health. While butyrate has been identified as a key health-related microbial metabolite, managing its supply to the host remains challenging. Therefore, this study investigated the potential to manage butyrate supply via tributyrin oil supplementation (TB; glycerol with three butyrate molecules) using the ex vivo SIFR® (Systemic Intestinal Fermentation Research) technology, a highly reproducible, in vivo predictive gut model that accurately preserves in vivo-derived microbiota and enables addressing interpersonal differences. Dosing 1 g TB/L significantly increased butyrate with 4.1 (±0.3) mM, corresponding with 83 ± 6% of the theoretical butyrate content of TB. Interestingly, co-administration of Limosilactobacillus reuteri ATCC 53608 (REU) and Lacticaseibacillus rhamnosus ATCC 53103 (LGG) markedly enhanced butyrate to levels that exceeded the theoretical butyrate content of TB (138 ± 11% for REU; 126 ± 8% for LGG). Both TB + REU and TB + LGG stimulated Coprococcus catus, a lactate-utilizing, butyrate-producing species. The stimulation of C. catus with TB + REU was remarkably consistent across the six human adults tested. It is hypothesized that LGG and REU ferment the glycerol backbone of TB to produce lactate, a precursor of butyrate. TB + REU also significantly stimulated the butyrate-producing Eubacterium rectale and Gemmiger formicilis and promoted microbial diversity. The more potent effects of REU could be due to its ability to convert glycerol to reuterin, an antimicrobial compound. Overall, both the direct butyrate release from TB and the additional butyrate production via REU/LGG-mediated cross-feeding were highly consistent. This contrasts with the large interpersonal differences in butyrate production that are often observed upon prebiotic treatment. Combining TB with LGG and especially REU is thus a promising strategy to consistently supply butyrate to the host, potentially resulting in more predictable health benefits.

Serum-Derived Bovine Immunoglobulin Stimulates SCFA Production by Specific Microbes in the Ex Vivo SIFR® Technology.

Serum-derived bovine immunoglobulins (SBI) exert health benefits mediated by their ability to bind microbial components, thereby preventing translocation and subsequent inflammation. While in vivo studies have shown that a fraction of SBI also reaches the colon, little is known about the impact of SBI on the dense colonic microbiota that has great potential to impact human health. This study, therefore, investigated the impact of three bovine plasma protein fractions (SBI, bovine plasma (BP) and albumin-enriched bovine plasma (ABP)) on the gut microbiota of six human adults using the novel ex vivo SIFR® technology, recently demonstrated to generate predictive findings for clinical studies. When dosed at an equivalent of 5 g/day, all protein fractions significantly increased health-related metabolites-acetate, propionate, and butyrate. Upon simulating small intestinal absorption, SBI still markedly increased acetate and propionate, demonstrating that SBI is more resistant to small intestinal digestion and absorption compared to the other protein sources. Despite noticeable interindividual differences in microbiota composition among human adults, SBI consistently stimulated a narrow spectrum of gut microbes, which largely differed from the ones that are typically involved in carbohydrate fermentation. The SBI-fermenting consortium included B. vulgatus and L. edouardi (correlating with acetate and propionate) along with Dorea longicatena, Coprococcus comes and the butyrate-producing bacterium SS3/4 (correlating with butyrate). Overall, this study revealed that protein bovine fractions can contribute to health benefits by specifically modulating the human gut microbiota. While health benefits could follow from the production of SCFA, a broader range of protein-derived metabolites could also be produced. This study also confirms that the concept of prebiotics (substrates selectively utilized by host microorganisms conferring a health benefit) could go beyond the use of ingestible carbohydrates and extend to partially indigestible proteins.